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BIOLOGY OF TICKS
Volume 1
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BIOLOGY OF TICKS
Volume 1
S E CO N D E D IT IO N
Edited by Daniel E. Sonenshine

AND
R. Michael Roe
1
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Library of Congress Cataloging-in-Publication Data

Biology of ticks / edited by Daniel E. Sonenshine and R. Michael Roe.—2nd ed.
p. cm.
ISBN 978-0-19-974405-3 (alk. paper)—ISBN 978-0-19-974406-0 (alk. paper)
1. Ticks. 2. Ticks as carriers of disease. 3. Ticks—Control. I. Sonenshine,
Daniel E. II. Roe, R. Michael.
QL458.15.P37B55 2013
571.9′86—dc23 2012045386
1 3 5 7 9 8 6 4 2
Printed in the United States of America
on acid-free paper
CONTENTS
Preface ix
Contributors xiii
1. Overview: Ticks, People, and Animals 3
Daniel E. Sonenshine and R. Michael Roe
2. Modern Tick Systematics 17
Lance A. Durden and Lorenza Beati
3. Life Cycles and Natural History of Ticks 59
Dmitry A. Apanaskevich and James H. Oliver, Jr.
4. External and Internal Anatomy of Ticks 74
Daniel E. Sonenshine and R. Michael Roe
5. Integument and Ecdysis 99
W. Reuben Kaufman
6. Mouthparts and Digestive System: Anatomy and Molecular Biology
of Feeding and Digestion 122
Daniel E. Sonenshine and Jennifer M. Anderson
7. Salivary Glands: Structure, Physiology, and Molecular Biology 163
Francisco J. Alarcon-Chaidez
8. Excretion and Water Balance: Hindgut, Malpighian Tubules, and Coxal Glands 206
Daniel E. Sonenshine
9. Heme Processing and the Evolution of Hematophagy 220
Ben J. Mans
vi Contents
10. Respiratory System: Structure and Function 240

Laura J. Fielden and Frances D. Duncan
11. Circulatory System and Hemolymph: Structure, Physiology, and
Molecular Biology 258
Libor Grubhoffer, Nataliia Rudenko, Marie Vancova, Maryna Golovchenko,
and Jan Sterba
12. Fat Body and Nephrocytes: Structure and Function 287
Lewis B. Coons
13. Nervous and Sensory Systems: Structure, Function, Genomics, and
Proteomics 309
Ladislav Šimo, Daniel E. Sonenshine, Yoonseong Park, and Dušan Žitňan
14. Molecular Biology and Physiology of Chemical Communication 368
Albert Mulenga
15. Heme-binding Lipoglyco-storage Proteins 398
Sayed M. S. Khalil, Kevin V. Donohue, R. Michael Roe, and
Daniel E. Sonenshine
16. Hormonal Regulation of Metamorphosis and Reproduction in Ticks 416
R. Michael Roe, Kevin V. Donohue, Sayed M. S. Khalil, Brooke W. Bissinger,
Jiwei Zhu, and Daniel E. Sonenshine
17. Female Reproductive System: Anatomy, Physiology, and Molecular Biology 449
Mari H. Ogihara and DeMar Taylor
18. Male Reproductive System: Anatomy, Physiology, and Molecular Biology 484
Daniel E. Sonenshine and Lewis B. Coons
Index 519
To view the book’s supplementary materials, please go to www.oup.com/us/biology

ofticks2e
1. Volume 1, Supplementary Fig. S18.1: Lobe of Dermacentor variabilis male accessory

gland periodic acid schiff stain.
2. Volume 1, Supplementary Fig. S18.2: Lobe of Dermacentor variabilis male accessory
gland mucicarmine stain.
3. Volume 1, Supplementary Table S18.1: Proteomics of the Dermacentor variabilis male
reproductive system and spermatophore.
Contents vii
Volume 2. Ecology, Genomics, Disease and Control
Contributors ix
1. Ecology of Non-nidicolous Ticks 3
Sarah E. Randolph
2. Ecology of Nidicolous Ticks 39
Jeremy S. Gray, Agustín Estrada-Peña, and Laurence Vial
3. Tick Genetics, Genomics, and Transformation 61
Jason M. Meyer and Catherine A. Hill
4. Tick–Host Interactions 88
Stephen K. Wikel
5. How Ticks Control Microbes: Innate Immune Responses 129
Wayne L. Hynes
6. Tick-borne Protozoa 147
Adalberto A. Pérez de León, Edouard Vannier, Consuelo Almazán,
and Peter J. Krause
7. Tick-borne Viruses 180
Patricia A. Nuttall
8. Tick-borne Spotted Fever Group Rickettsioses and Rickettsia Species 211
Kevin R. Macaluso and Christopher D. Paddock
9. Tick-borne Rickettsioses II (Anaplasmataceae) 251
Holly D. Gaff, Katherine M. Kocan, and Daniel E. Sonenshine
10. Non-rickettsial Tick-borne Bacteria and the Diseases They Cause 278
Nick H. Ogden, Harvey Artsob, Gabriele Margos, and Jean Tsao
11. Tick-induced Paralysis and Toxicoses 313
Agustín Estrada-Peña and Ben J. Mans
12. Development of Vaccines for Control of Tick Infestations and Interruption of
Pathogen Transmission 333
José de la Fuente and Katherine M. Kocan
13. Acaricide Research and Development, Resistance, and Resistance
Monitoring 353
Felix D. Guerrero, Adalberto A. Pérez de León, Roger I. Rodriguez-Vivas,
Nick Jonsson, Robert J. Miller, and Renato Andreotti
viii Contents
14. Tick Repellent Research, Methods, and Development 382

Brooke W. Bissinger and R. Michael Roe
15. Tick Control: Trapping, Biocontrol, Host Management, and Other
Alternative Strategies 409
Howard S. Ginsberg
16. Tick Rearing and in Vitro Feeding 445
Sandra A. Allan
Index 475
Supplementary Materials (www.oup.com/us/biologyofticks2e)
1. Volume 2, Supplementary Table 3.1: Summary of Published Complete Genome Sequences

for Selected Tick-borne Pathogens
2. Volume 2, Supplementary Table 3.2: Summary of RNAi-based Functional Studies in
Species of Ixodid Ticks
3. Volume 2, supplementary references for Supplementary Tables 3.1 and 3.2.
4. Volume 2, Supplementary Table 7.1: Tick-borne Viruses.
PREFACE
T
icks transmit a greater variety of pathogenic agents than any other arthropod group, even
mosquitoes. In humans, the diseases caused by these agents include Lyme disease, Rocky
Mountain spotted fever, human granulocytic anaplasmosis, human monocytic ehrlichio-
sis, tularemia, Colorado Tick Fever, tick-borne encephalitis, and many others. The illnesses
involve tens of thousands of cases every year. In domestic animals, the numerous tick-borne
disease agents cost the livestock industry billions of dollars. Moreover, many of these diseases have
emerged (or re-emerged) within the past 2 or 3 decades (e.g., Lyme disease, human granulocytic
anaplasmosis, and others). Despite the use of modern medical and veterinary treatments, the
widespread use of pesticides, and sophisticated novel methods for their control, ticks continue to
flourish. Clearly, detailed and up-to-date knowledge of tick biology and the diseases ticks transmit
must be included in medical and veterinary school programs, in the curricula of advanced under-
graduate and graduate programs in the biological sciences, and in public health curricula.
There is an unquestioned need for a single comprehensive, authoritative, and up-to-date
book on the biology of ticks and tick-borne diseases. The first edition of the 2-volume Biology of
Ticks, published in 1991 and 1993, was written to address this need. However, more than 20 years
have elapsed since its publication. The first edition met with widespread success, so much so that
it is now out of print. In the meantime, numerous scientists, physicians, veterinarians, and other
professionals have contacted us to urge the creation of a second edition of Biology of Ticks. How-
ever, other treatises on ticks and tick-borne diseases have been published in recent years, espe-
cially (1) Ticks: Biology, Disease and Control (2008), A. S. Bowman and P. A. Nuttall (Eds.),
published by Cambridge University Press, and (2) Tick-borne Diseases of Humans (2005),
J. L. Goodman, D. T. Dennis, and D. E. Sonenshine (Eds.), published by ASM Press. Although
there are many excellent chapters on tick biology and diseases, these works are not and never
were intended to be fully comprehensive. Many modern aspects of tick biology, such as molec-
ular systematics; tick genetics and genomics; or the molecular basis of blood feeding, digestion,
development, reproduction, and innate immune responses, as well as many other aspects of tick
biology, ecology, and control, are missing or covered only briefly.
Although much that was included in the first edition of Biology of Ticks, especially basic tick
morphology and life cycle concepts, is still relevant today, tremendous changes in methodology
x Preface
have occurred since its publication. Real time polymerase chain reaction studies for measuring
gene expression; massive high-throughput genetic sequencing leading to the entire genome of
the Lyme disease vector Ixodes scapularis, as well as transcriptomes and proteomes of different
body organs; and bioinformatics, along with the availability of databases of sequence informa-
tion, are among the most prominent changes that have revolutionized biological and medical
research. Equally important is the digital revolution that has affected virtually all aspects of
modern science. Consequently, it is now possible to ask questions about molecular, chemical,
and physiological processes that were inconceivable decades ago. Enormous databases of DNA
and protein sequences for molecules from tens of thousands of species, both prokaryotes and
eukaryotes, are maintained in government institutions. Using bioinformatics techniques, sci-
entists and students can submit new molecular or protein sequences, annotate and characterize
their findings, and compare them to similar molecules in other species. Clearly, any new trea-
tise would have to include the rapidly accumulating knowledge made possible by these new
technologies.
Convinced that the need for a second edition was genuine, we, the editors, considered how
the book should be structured and what it should contain. We determined that it should contain
mostly new information, although we agreed to retain limited descriptions of tick morphology
and ultrastructure as needed as a framework for understanding the tick organs and/or tissues
reviewed in the book chapters. We consulted with numerous colleagues in arriving at our plan,
and the result is shown in the following Table of Contents. We would produce a multi-authored
book, written by recognized experts from virtually all disciplines and addressing all of the im-
portant tick-borne diseases. We would seek the participation of experts from different regions
of the world, so as to reflect the book’s multi-disciplined character.
This 2-volume book covers the evolution, modern systematics (including molecular system-
atics), biology, life cycles, molecular biology of different biological and physiological processes,
physiology, biochemistry, and ecology of ticks, as well as the diseases caused by the microbial
agents or toxic compounds that they transmit to their hosts.
Volume 1, comprising 18 chapters, is focused entirely on the tick. Following an overview of
all aspects of tick biology and disease relationships, 2 chapters review tick systematics, including
a molecular-based understanding of tick evolution and the life cycles of ticks. Next, we review
the external and internal anatomy of ticks so as to provide the reader with a single, well-
integrated overview of all aspects of tick body structure. Most of the remaining chapters are
dedicated to specific organ systems, specifically, the tick integument, mouthparts and digestive
system, salivary glands, organs of waste removal, respiratory system, circulatory system and
hemolymph, fat body, nervous and sensory systems, and male and female reproductive systems.
In order to provide space for the most up-to-date information from molecular studies,
genetics, and high-throughput global insights (e.g., transcriptomes), descriptions of the gross
anatomical and ultrastructural features were reduced or omitted, as they were described in the
previous edition. However, readers may access these anatomical and ultrastructural descriptions
by visiting the Vectorbase website (www.vectorbase.org) and following the instructions given in
the chapters for finding specific structures of interest. Finally, several chapters are devoted to
major biological/physiological processes essential for tick host finding, blood feeding and diges-
tion, and hormonal regulation of the tick’s development and reproduction.
Volume 2, comprising 16 chapters, covers tick ecology, genetics and genomics, immunity,
tick-associated diseases, tick control, and tick rearing. Chapter 1 deals with the ecology of non-
nidicolous ticks (i.e., ticks dispersed widely in the landscape, surviving in soil and vegetation
Pref ace xi
and feeding by attacking passing hosts). Chapter 2 covers the ecology of nidicolous ticks, which
survive in caves, burrows, nests, or other sheltered environments where they wait, often for
months or even years, for the return of the nesting hosts. Chapter 3 covers the most up-to-date
information on tick genetics and genomics, including a review of the landmark genome of the
Lyme disease vector Ixodes scapularis. The next two chapters deal with immunity. Chapter 4 covers
tick–host interactions, especially the host immune responses to tick feeding and how ticks modu-
late the host response to enhance their feeding success. Chapter 5 covers the innate immune re-
sponses of ticks to microbes, which provide protection against most microbial encounters.
Subsequent chapters, from Chapter 6 through Chapter 10, cover the many diseases caused by the
major tick-borne pathogens, including tick-borne protozoa, viruses, rickettsiae of all types, other
types of bacteria (e.g., the Lyme disease agent), and diseases related to tick paralytic agents and
toxins. The next 4 chapters, Chapters 12–15, are devoted to tick control through vaccines, acari-
cides, repellents, trapping, biocontrol, and most of the more novel alternative strategies. Finally, we
devote the last chapter, Chapter 16, to tick rearing and research on the methods for feeding ticks on
suitable laboratory hosts or, alternatively, in vitro feeding in order to develop tick colonies for sci-
entific study.
No work of this magnitude could be done without the assistance and encouragement of
others. First and foremost, we thank Ms. Phyllis Cohen, Senior Editor, Oxford University Press,
for her thoughtful guidance, encouragement, and assistance during the many processes and
procedures involved in preparing this book for publication. We also appreciate the encourage-
ment given by Dr. Olaf Kahl, Senior Editor, Ticks and Tick-borne Diseases, and the guidance
offered by Dr. Ernest Hodgson, North Carolina State University. We thank the many graduate
students who assisted in various ways, including Noble Egekwu, Department of Biological Sci-
ences at Old Dominion University, and Jiwei Zhu and Anirudh Dhammi, Department of Ento-
mology, North Carolina State University. Finally, we thank our wives, Helen and Janet, for their
sympathetic understanding and patience.
This book is dedicated to the memory of Dr. Harry Hoogstraal, whose enthusiasm, dedica-
tion, and immense contributions to our knowledge of ticks and tick-borne disease have inspired
us in our scientific endeavors.
CONTRIBUTORS
Francisco J. Alarcon-Chaidez Brooke W. Bissinger

Department of Pathology TyraTech, Inc.
University of Texas Medical Branch Morrisville, NC 27560 USA
301 University Boulevard
Galveston, TX 77555 USA Lewis B. Coons
University of Memphis
Jennifer M. Anderson Memphis, TN 38152 USA
Vector Molecular Biology Unit 1
Laboratory of Malaria and Vector Kevin V. Donohue
Research Department of Entomology
National Institutes of Allergy and Infectious North Carolina State University,
Diseases Raleigh, NC 27695 USA
National Institutes of Health
Rockville, MD 20852 USA Frances D. Duncan
School of Animal, Plant and Environmental
Dmitry A. Apanaskevich Sciences
The James H. Oliver, Jr. Institute of University of the Witwatersrand
Arthropodology and Parasitology Wits 2050, South Africa
Georgia Southern University
Statesboro, GA 30460 USA Lance A. Durden
Department of Biology
Lorenza Beati Georgia Southern University
U.S. National Tick Collection P.O. Box 8042
Department of Biology Statesboro, GA 30460 USA
Statesboro, GA 30460 USA
xiv Contributors
Laura J. Fielden Mari H. Ogihara

Department of Biology Department of Integrated Bioscience
Truman State University Graduate School of Frontier Sciences
Kirksville, MO 63501 USA University of Tokyo
Kashiwa, Chiba, Japan
Maryna Golovchenko
James H. Oliver, Jr.
Biology Centre of the AS CR and Faculty of
Science
P.O. Box 8056
University of South Bohemia
Statesboro, GA 30460 USA
Ceske Budejovice, Branisovska 31, 370 05,
Czech Republic Yoonseong Park
Department of Entomology
Libor Grubhoffer Kansas State University
Biology Centre of the AS CR Manhattan, KS 66506 USA
Faculty of Science
University of South Bohemia R. Michael Roe
Ceske Budejovice, Branisovska 31, 370 05, Department of Entomology
Czech Republic North Carolina State University
Raleigh, NC 27695 USA
W. Reuben Kaufman
Nataliia Rudenko
Z606 Department of Biological
Biology Centre of the AS CR
Sciences
Faculty of Science
University of Alberta
Edmonton, Alberta T6G 2E9, Canada
Czech Republic
Sayed M. S. Khalil
Department of Entomology Ladislav Šimo
North Carolina State University Department of Entomology
Raleigh, NC 27695 USA Kansas State University
Manhattan, KS 66506 USA
Ben J. Mans
Parasites, Vectors and Vector-Borne Daniel E. Sonenshine
Diseases Department of Biological Sciences
Onderstepoort Veterinary Institute Old Dominion University
Onderstepoort 0110, South Africa Norfolk, VA 23529 USA
Jan Sterba
Albert Mulenga Biology Centre of the AS CR
Department of Entomology Faculty of Science
Texas A & M University University of South Bohemia
AgriLife Research Ceske Budejovice, Branisovska 31, 370 05,
College Station, TX 77843 USA Czech Republic
Contributors xv
DeMar Taylor Jiwei Zhu

Graduate School of Life and Environmental Department of Entomology
Sciences North Carolina State University
University of Tsukuba Raleigh, NC 27695 USA
Tsukuba, Ibaraki 305-8577, Japan
Dušan Žitňan
Marie Vancova Institute of Zoology
Biology Centre of the AS CR Slovak Academy of Sciences
Faculty of Science Dúbravská Cesta 9
84506 Bratislava, Slovakia
Czech Republic
BIOLOGY OF TICKS
Volume 1
C H A P T E R 1
OVERVIEW
Ticks, People, and Animals
DANI EL E. S ONENSHINE AND R. MICHAEL ROE
1. INTRODUCTION
Ticks are familiar to most people in tropical and temperate regions of the world. They are widely
feared because of their role in the transmission of human and animal diseases. Ticks are believed
to surpass all other arthropods in the variety of infectious agents that they transmit. Examples
of human diseases caused by these tick-borne pathogens include Lyme disease, Rocky Mountain
spotted fever, Mediterranean spotted fever, human granulocytic anaplasmosis, human mono-
cytic anaplasmosis, tularemia, Colorado tick fever, and tick-borne encephalitis. Examples of
tick-borne diseases of livestock and companion animals include babesiosis, theileriosis, heart-
water, anaplasmosis, Lyme disease, and ehrlichiosis. In addition to transmitting dangerous in-
fectious agents, tick bites may lead to severe toxic reactions, allergic responses, or even deadly
paralytic symptoms (tick paralysis). Ticks are also important as pests, even when they do not
transmit harmful pathogens. Severe tick infestations are injurious to livestock and companion
animals, often injuring their hides and leaving open wounds that can become infected. Heavy
tick burdens can also lead to reduced weight gain, lost milk production, and/or abortion. In
some regions of the world, livestock production is all but impossible without major investments
in tick control. Although precise figures are unavailable, the worldwide economic loss due to
tick-borne diseases and/or severe tick infestations, as well as the costs of vaccination and acari-
cide treatment, is estimated to be in the billions of dollars annually (Jongejan and Uilenberg
2004). Examples include an estimated $281 per patient for the treatment of human Lyme disease
(Zhang et al. 2006) and an estimated $384 million loss due to tick-borne diseases of livestock in
Tanzania (Kivaria 2006). Heavy tick infestations in the natural environment may impact the
recreational value of parks, campgrounds, and other natural areas or the willingness of home
owners to use their own yards and gardens during the seasons when ticks are active. Despite the
4 BIOLOGY OF TICKS
many monumental advances in the control of ticks and tick-borne disease during the past 100
years, ticks continue to flourish, and tick-borne diseases continue to threaten human and animal
health throughout the world.
In this 2-volume book, we examine the biology, structure, ecological adaptations, evolution,
genomics, and molecular processes that underpin the growth, development, and survival of
these parasites. In addition, we describe the remarkable array of diseases transmitted (or caused)
by ticks, as well as the most up-to-date methods for their control. Finally, in contrast to the first
edition, this new edition of Biology of Ticks is multi-authored, with chapters written by many of
the best known experts in the field. There have been many advances in chemistry, molecular
biology, proteomics, genomics, imaging, computational biology, and other fields of science since
the publication of the first edition of Biology of Ticks (Sonenshine 1991, 1993), and these are in-
cluded in this second edition. This work should serve as a modern reference for students, scien-
tists, physicians, veterinarians, and other specialists.
2. SYSTEMATICS AND EVOLUTIONARY

RELATIONSHIPS
Ticks are obligate blood-sucking arthropods found throughout most regions of the world.
Ticks are chelicerates (i.e., as a group they did not evolve true biting mandibles like those of
the advanced Crustacea and insects). They are also distinct from the Mandibulata in their lack
of antennae, and they are unique within the Arthropoda because of their reduction in seg-
mentation. The chelicerate subphylum is believed to have evolved more than 500 million years
ago, having diverged from the ancestral stem line and then commenced the first adaptive ra-
diation during the Cambrian (Weygoldt 1998). More recent studies using both fossil evidence
and detailed molecular analysis suggest that the divergence time for the Atlantic horseshoe
crab, Limulus polyphemus, can be estimated as 475 ± 53 million years ago (mya), and that for
ticks also occurred during the late Paleozoic era. The spider-scorpion clades (class Arachnida)
are estimated to have diverged 397 ± 23 mya (Jeyaprakash and Hoy 2009). Presumably, the
Acari, the subclass that contains the mites and ticks, evolved around that time. Estimates of
the time and geographic region where ticks evolved are controversial. Barker and Murrell
(2008) cite reports that ticks evolved on ancient amphibians in that part of Gondwanaland
that eventually became Australia around 390 mya. Other authors place the origin of ticks
much later, in the mid-Cretaceous period, with primeval hosts being either amphibians or
reptiles (Nava et al. 2009).
At present, ticks are classified as a subgroup of the subclass Acari, namely, the order Ix-
odida, of the superorder Parasitiformes (Krantz and Walter 2009; Nicholson et al. 2009).
Ticks can be recognized by their flattened body shape (when unfed), their 4 pairs of walking
legs (3 pairs in larvae), and the presence of a hypostome with numerous recurved teeth. Al-
most 900 species have been described (Barker and Murrell 2008). They are subdivided into 3
families: Ixodidae (692 species), Argasidae (186 species), and Nuttallielidae (1 species) (Nava
et al. 2009). The Ixodidae (with 12 genera) are known as hard ticks because of their sclerotized
dorsal scutal plate, whereas the Argasidae (with 6 genera) are referred to as soft ticks because
of their flexible, leathery cuticle. The Ixodidae are further subdivided into 2 groups: the Pros-
triata, comprising a single genus, Ixodes, and the Metastriata, comprising the remaining 11
Ticks, People, and Animals 5
genera. The Nuttalliellidae, with only 1 species, Nuttalliela namaqua, shares some features of
both Ixodidae and Argasidae but also has other features that are unique, such as ball and
socket joints in the leg segments that provide a wider range of movement than that seen in the
other tick groups. For a more detailed review of tick systematics and evolution, see Chapters
2 and 11.
3. TICKS AND DISEASE
Ticks infest every class of terrestrial vertebrates, including mammals, birds, reptiles, and (in a
few cases) even amphibians. Ticks transmit a greater variety of disease-causing agents than any
other group of arthropods. Although mosquitoes transmit pathogens that infect far more people
and cause more severe diseases of humans and animals (e.g., malaria, dengue fever, yellow fever,
heartworm, etc.), ticks transmit a greater variety of pathogenic organisms, including fungi, vi-
ruses, bacteria (including rickettsiae), and protozoa. There are even reports of tick transmission
of pathogenic nematodes (Londoño 1976). In addition, ticks are the direct cause of deadly tick
paralysis, allergic reactions, and tick-caused toxicoses. All of the tick-borne infectious diseases
are zoonoses (i.e., diseases of animals transmissible to humans). Despite the many impressive
advances in medicine and pest control that have curtailed most vector-borne diseases, virtually
all of the tick-borne diseases caused by these pathogens have persisted and even increased their
geographic range in recent years.
3.1. TICK-BORNE DISEASES

At present, more than 16 specific tick-borne (or tick-caused) diseases of humans and more
than 19 tick-borne diseases of livestock and companion animals have been described (Nichol-
son et al. 2009). A representative list of these diseases is shown in Table 1.1. A few examples are
discussed briefly below. A much more detailed review of these and other diseases is given in
Volume 2.
Lyme disease was first recognized as a reportable human disease in the United States in the
1980s, with only a few hundred cases per year. Its incidence has since increased to more than
28,000 cases per year in 2008 (28,921 confirmed and 6,277 probable cases) (Anonymous 2008);
thousands of cases per year also occur in Europe and northern Asia (i.e., throughout much of
the holoarctic region of the world). Moreover, rather than a single genotype, Borrelia burgdor-
feri (s.l.), a number of different genotypes (e.g., B. garinii, B. bissettii) are now known to cause
this disease, with significant differences in clinical symptoms in different regions of its vast
zoogeographic range. Also increasing in frequency is Rocky Mountain spotted fever, a severe,
life-threatening rickettsial disease (caused by the intracellular bacterium Rickettsia rickettsii)
that is widespread throughout most of the United States and much of Latin America. Mediter-
ranean spotted fever (i.e., boutonneuse fever), a similar rickettsial disease (caused by R. cono-
rii), occurs throughout large areas of the Near East, central and northern Asia, Africa, and
southern Europe. Several other tick-borne rickettsial diseases have emerged or re-emerged, all
caused by intracellular bacteria that invade white blood cells. Among the most important of
these are human granulocytic anaplasmosis (caused by Anaplasma phagocytophilum), human
6 BIOLOGY OF TICKS
Table 1.1: A summary of the different categories of tick-borne diseases of humans

and domestic or companion animals, with some representative examples.
Category Representative disease Causative agent Major tick vector(s) Primary host(s)a
of causative
agent
Protozoa Babesiosis Babesia bigemina Rhipicephalus spp. Cattle, deer
B. microti, etc.
East Coast fever Theileria parva Rhipicephalus Cattle, cape buffalo
appendiculatus
Theileriosis T. annulata Hyalomma spp. Cattle, water buffalo
Human babesiosis B. microti Ixodes scapularis, Humans, mice
I. ricinus
Bacteria RMSFb Rickettsia rickettsii Dermacentor spp. Humans, dogs, small
mammals
Mediterranean R. conorii Rhipicephalus Humans, hedgehogs,
spotted feverc sanguineus small mammals
Anaplasmosis Anaplasma I. scapularis, Humans, deer, dogs,
phagocytophilium I. ricinus, others others
A. marginale, Dermacentor spp., Cattle, sheep, other
A. centrale others ruminants
Human ehrlichiosis Ehrlichia chafeenesis Amblyomma Humans, deer
americanum
Canine ehrlichiosis E. canis, E. ewingii R. sanguineus, others Dogs
Heartwater E. ruminantium A. hebraeum, Cattle, other
A. variegatum ruminants
Q fever Coxiella burnetti Various species Cattle, humans,
other mammals
Lyme disease/lyme Borrelia burgdorferi, I. scapularis, Humans, diverse
borreliosis other Borrelia spp. I. ricinus, others mammals, birds
Tick-borne Borrelia spp. Various argasid spp. Humans, other
relapsing feverd mammals
Tularemia Francisella tularense Haemaphysalis Lagomorphs,
leporis-palustris, humans, other
Dermacentor mammals
spp., others
Virus Tick-borne Flavivirus I. ricinus, other Rodents, humans,
encephalitis Ixodes spp. other mammals
Colorado tick fever Coltivirus D. andersoni Humans, various
mammals
Crimean-Congo Nairovirus Hyalomma spp. Humans,
hemorrhagic lagomorphs,
fever hedgehogs, etc.
African swine fever Iridovirus Ornithodoros Pigs, warthogs
porcinus
Fungus Dermatophilosis Dermatophilosus Tick-associated; Cattle, other
(Actinomy- congolensis no proven domestic animals
cetales) transmission
Table 1.1: (continued)
Category Representative disease Causative agent Major tick vector(s) Primary host(s)a
of causative
agent
Tick Tick paralysis Tick-transmitted Many tick species Humans, cattle,
proteins other domestic
animals, birds, etc.
Tick toxicoses, Tick-transmitted Many tick species Humans, cattle,
tick-bite proteins sheep, other
allergies mammals, birds
a
Host experiencing illness or death.
b
RMSF = Rocky Mountain spotted fever.
c
Synonym for boutonneuse fever.
d
A similar disease is caused by different species, B. recurrentis, which is transmitted by human body lice, Pediculus
humanus.
monocytotropic ehrlichiosis (caused by Ehrlichia chafeensis), and Ehrlichiosis (caused by E.

ewingii); some 1,300 cases had been confirmed by the U.S. National Centers for Disease Control
in 2006. In Europe and broad regions of northern Asia, a tick-borne virus causes tick-borne
encephalitis, a dangerous viral disease that occurs over much of Europe and northern Asia,
where it infects unsuspecting woodsmen, livestock workers, farmers, campers, hikers, and
others who enter tick-infested habitats. Symptoms are often severe, with high fever, headache,
muscle aches, and nausea. Approximately 10,000 to 12,000 cases occur annually (Nuttall and
Labuda 2005). Some of these same pathogens also cause similar diseases in companion and
domestic animals (e.g., Lyme disease, which is common in dogs).
Other tick-borne diseases affect livestock. Diseases such as heartwater; babesiosis of
cattle, sheep, and other livestock; East Coast fever; and louping ill all kill large numbers of
these animals, curtailing exports and destroying vitally needed protein resources for local
populations. Bovine babesiosis is the most important arthropod-borne disease of cattle
worldwide; it is believed to cause billions of dollars in losses. In Zimbabwe, the cessation of
tick control measures during that country’s 7-year war resulted in the death of an estimated
1 million cattle from tick-borne disease (Lawrence et al. 1980). Tick feeding alone can also be
a direct cause of illness or death in animals, aside from the pathogens that they transmit
(e.g., tick paralysis).
3.2. TICK-CAUSED DISEASES

Perhaps the most notorious of these tick-caused diseases is tick paralysis, characterized by
flaccid, ascending paralytic symptoms often ending in death unless the tick is discovered and
removed from the patient. The bites of some tick species have also been reported to cause severe
toxemias (although rarely leading to death). Blood loss is another important cause of injury, as
ixodid ticks consume large blood meals. According to Balashov (1972), Hyalomma asiaticum
females were estimated to consume more than 8 ml per tick. In some instances, infestations of
8 BIOLOGY OF TICKS
ticks may reach such extreme magnitude that the host animals die of exsanguination or become
susceptible to other illnesses as a result of their weakened condition.
4. CHARACTERISTICS OF TICKS
4.1. BODY STRUCTURE AND COMPARISON WITH

OTHER ARTHROPODS
4.1.1. External anatomy

There are 3 major regions: the capitulum (comparable to the gnathosoma of other acarines),
the body (the idiosoma), and the legs. The capitulum contains the basis capituli that attaches the
capitulum to the body, the chelicerae (i.e., appendages for cutting, ripping, and tearing skin), the
leg-like palps, and the hypostome with rows of recurved teeth for attachment to the host skin.
The capitulum should not be mistaken for a head, as there is no true brain located therein. The body
is subdivided into an anterior region, the podosoma, bearing the 4 pairs of walking legs and the
genital pore, and a posterior region, the opisthosoma, bearing the spiracular plate and the anal ap-
erture. Extreme fusion has masked all other divisions. The legs are divided into 6 segments and ar-
ticulate with the body via the coxae. The tarsus of leg I contains Haller’s organ, an important sensory
apparatus that includes sensilla for detecting odors, heat, and possibly other external factors.
Hard ticks (Ixodidae) are recognized by a hard, sclerotized plate on the dorsal body surface, the
scutum, which covers the entire dorsum in adult males but only the anterior half in adult females,
nymphs, and larvae. Thus, males exhibit only slight growth in body size during feeding. In the
other life stages, the remainder of the body cuticle can expand greatly. This is accomplished via
synthesis of new cuticle rather than simple expansion and occurs during the lengthy blood-feeding
period. Soft ticks (Argasidae) do not have a scutum or any other hard plates. Instead, they have a
leathery, folded cuticle, which facilitates limited expansion during feeding. The cuticle unfolds
during blood feeding, which enables soft ticks to swell and consume their blood meals rapidly,
usually within minutes and at most within 1 to 2 hours (see Chapters 2 and 4 for images of ticks).
A detailed description of tick morphology (both external and internal anatomy) is presented
in the subsequent chapters of this volume. For a hierarchical approach to the anatomy of ticks
(electronic version), see OBO-EDIT v. 2.1, an open source ontology editor that links all of the
tick body structures (more than 650 structures) in a hierarchical arrangement. An ontology is a
controlled vocabulary that uses preselected and predefined terms (concepts) that are linked to
one another through a limited set of statements termed “relations” (see Topalis et al. [2008] for
a discussion of ontology and access to the tick anatomical ontology). The editor for viewing the
ontology can be downloaded from the Open Source Java tool OBO-Edit, available at http://
sourceforge.net/project/showfiles.php?group_id=36855&package_id=192411. To browse the tick
anatomy hierarchy without saving it to a computer, the reader may view it at www.Vectorbase.
org and follow the instructions in the footnote below.1
4.1.2. Internal anatomy

Internally, the organs of the tick body are bathed in a freely circulating fluid, hemolymph. The
most prominent of the major internal organs are the digestive tract (comprising the pharynx,
esophagus, midgut, and rectal sac), the salivary glands, the reproductive organs, the synganglion
(the fused central nervous system), the Malpighian tubules, and the tracheae. The hemo-
lymph contains hemocytes and numerous proteins in solution. It serves to distribute nutri-
ents and remove wastes and is important in innate immunity. The midgut is the largest organ
in the body. When fully engorged, the midgut fills most of the body interior, crowding out
all of the other organs. It consists of a central stomach and many large, prominent diverticu-
lae. In addition to its role in blood digestion, it also functions as a food storage organ, be-
cause ticks digest their blood meal slowly (except in mated females). The structure,
physiology, and molecular biology of the midgut are described further in Chapter 6. The
second largest organ (except for the ovaries of egg-laying females) is the pair of salivary
glands. These large glands, which resemble clusters of grapes, are located in the anterolateral
region of the body. Saliva is secreted via the salivary ducts connected to the mouthparts.
These complex glands contain multifunctional lobes that secrete a rich and highly diverse
mixture of antihemostatic factors, digestive enzymes, and proteins that facilitate wound for-
mation and dilate blood vessels in the host skin They even secrete salty solutions to imbibe
moisture from subsaturated atmospheres. The structure, physiology, and molecular biology
of the salivary glands are described further in Chapter 7. In the adults, the reproductive
organs occupy much of the body’s interior. In fed males, which consume only small amounts
of blood, the testis and the white, multi-lobed male accessory gland are the most prominent
internal organs. In feeding virgin females, the most prominent reproductive organs are the
ovary, filled with numerous white oocytes of varying size, the oviducts, and the vagina. Fol-
lowing mating, the oocytes expand greatly and fill with innumerable brown yolk globules
(vitellin). When the fully engorged female drops from its host, the brown ovary occupies
most of the interior of the body.
4.2. DEVELOPMENT AND REPRODUCTION

4.2.1. Development
Ticks possess many unusual features that distinguish them from other arachnids, insects, and
crustaceans. These characteristics are summarized briefly in Table 1.2. Tick life cycles exhibit
hemimetabolous development in which the general body form of immature individuals resem-
bles that of the adults, a pattern also found in the hemimetabola of insects. There are 4 life stages
in tick development: egg, larva, nymph, and adult (obvious sexual characteristics such as a genital
pore are evident only in the adult stage). Ixodid ticks do not have multiple nymphal instars, but
argasid ticks might have 2 or more nymphal instars. In most species, larvae find hosts, feed, de-
tach, and molt to nymphs. In ixodid ticks, the nymphs feed, detach, and molt to adults; in a few
species, larvae and nymphs remain and molt to adults on the same host. In argasid ticks, each of
the nymphal instars must find hosts, feed, detach, and molt again until the final molt to the adult
stage. Adults seek hosts, feed, and, in the case of engorged females, drop off to lay their eggs. Ix-
odid females feed only once, lay thousands of eggs, and die (a single gonotrophic cycle). In con-
trast, most argasid females feed, lay a small clutch of eggs, and then feed and oviposit several
additional times (multiple gonotrophic cycle).
One of the most outstanding characteristics of ticks is their long life cycle. Most ixodid tick
species have life spans that last at least 1 year; many live for 2 or even 3 years. The life cycles of
argasid ticks are usually much longer (up to 20 years in some instances) because there are mul-
tiple nymphal instars, each requiring a separate blood meal. These ticks are uniquely adapted for
10 BIOLOGY OF TICKS
Table 1.2: Some biological features used to distinguish ticks from

other blood-sucking arthropods.
Biological characteristics Ticks Insects

Mouthparts: Hypostome with Hypostome with recurved teeth, No anchoring feature, mandibles
recurved teeth, no anchoring chelicerae for cutting/tearing for cutting/piercing skin
feature, teeth, chelicerae, skin
mandibles
Legs Four pairs (except larvae) Three pairs
Body regions Single fused Multiple: head, thorax, and
abdomen
Duration of life cycle Long, months to years Short, weeks or months
Blood volume ingested Large, up to 8 ml/tick Small, <1 ml
Cuticle expansion during Fresh growth in ixodids, Limited or none
feeding unfolding in argasids
Egg mass Large, up to 23,000/female Small, only hundreds/female
Blood meal digestion Intracellular within midgut Extracellular within midgut
digestive cells lumen
Heme-binding storage proteins Yes No
in hemolymph
Vector competency Protozoan, bacterial (including No other arthropod group
rickettsial), viral, fungi, and transmits as many pathogens
even nematode pathogens
Excretory waste Guanine—little water loss Uric acid—more water loss
Genome size From 1,281 to 2,671 Mbpa
Fat body Dispersed, alongside tracheae Compact
Juvenile hormone Absent Present
Antennae Absent Present
Compound eyes Absent Present
Host complementation Present (Ixodidae) Absent for long-term feeding
a
Mbp = megabase pair (Geraci, N.S., Johnston, S.J., Robinson, J.P., Wikel, S.K., and Hill, C.A [2007] Variation in genome
size of argasid and ixodid ticks. Insect Biochem. Mol. Biol. 37:399–408).
long periods of starvation between blood meals. Their exceptional longevity also perpetuates the
pathogens they carry (e.g., relapsing fever bacteria).
Ticks differ from all other blood-feeding arthropods in the manner in which they obtain
blood. Mosquitoes or biting flies pierce or tear the host skin to draw small quantities of blood
quickly before the host can respond. In contrast, ticks cut into the skin, insert the anchoring
hypostome, and remain attached for extended periods (many minutes, hours, or even days)
while consuming large quantities of blood. In ixodid species, the ticks cement themselves to the
skin and are extremely difficult to dislodge. Mated females may increase to approximately 100
times their original body weight before dropping off to lay their eggs.
Another unusual feature of ticks is their digestive process. This is almost entirely intracellular
and, except for that of the egg-laying female, is a relatively slow process. Blood taken into the tick’s
midgut remains largely undigested for long periods. Aside from hemolytic enzymes that lyse blood
cells and release hemoglobin, virtually no proteins are secreted into the midgut lumen. Hemoglo-
bin digestion is entirely intracellular. Consequently, the blood meal remains as a food reserve and,
except in ovipositing females, is consumed gradually over many months or years. This feature of
blood meal digestion also insulates any pathogens that have been ingested against rapid destruc-
tion, allowing time for the penetration of disease-causing organisms into the body tissues.
4.2.2. Reproduction
Ticks exhibit several different strategies for regulating their reproductive activity. In the Prostriata,
gametogenesis is initiated during the nymphal ecdysial period and is advanced or even completed
when the adults emerge from the nymphal exuviae. Consequently, mating may occur before or
during blood feeding. However, in the Metastriata, newly molted adults are sexually immature.
Gametogenesis is initiated during blood feeding, and mating occurs only while feeding on a verte-
brate host. In metastriate ticks, copulation and insemination induce profound changes in the recip-
ient females, including rapid engorgement of the blood meal, synthesis and secretion of vitellogenins
(vg) by the fat body, incorporation of vg into the oocytes and its conversion into vitellin, and enor-
mous oocyte enlargement. Other profound changes also occur, such as reduction and resorption of
the salivary glands, expansion of the vagina (vestibular region) to facilitate egg passage, and growth
of the egg waxing organ (Gené’s organ). Oviposition occurs only following mating, engorgement,
and drop off from the host. Mated females lay thousands of eggs in a single clutch, and the female
dies. Typical egg masses range from 2,000–10,000 eggs. The most ever recorded was from an Am-
blyomma nuttalli female that laid more than 22,000 eggs (Arthur 1962). Males, however, may feed
and mate multiple times. Mating is regulated by multiple sex pheromones secreted by the females
that attract fed males, identify the conspecific females as suitable partners, and guide the males to
the female genital pore (Sonenshine et al. 1989). Following copulation, hormones, especially 20-
hydroxyecdysone, stimulate synthesis, secretion, and uptake of vg (Thompson et al. 2005); the
messages for peptide hormones (Donohue et al. 2010) in the synganglion or associated tissues are
also hypothesized to regulate important developmental and reproductive events, but their exact
functions remain to be discovered. In argasid ticks, gametogenesis also begins during nymphal
ecdysis, and the adults are sexually mature soon after emerging from the nymphal molt. Conse-
quently, mating may occur before, during, or after blood feeding. However, with few exceptions,
vitellogenesis, oocyte growth, fertilization, and oviposition occur only following a fresh blood
meal, and only in mated females. Female argasid ticks lay only a few hundred eggs after each blood
meal but are capable of several cycles of feeding, mating, and oviposition. The only exceptions to
these reproductive strategies are the few instances of autogenous egg production and parthenogen-
esis. In most cases, however, reproduction coincides with periods when hosts are available, and
benign environmental conditions favor rapid tick population expansion.
4.3. BLOOD FEEDING AND DIGESTION

In contrast to most other hematophagous arthropods, which suck blood from vessels or torn,
injured skin, ticks are long-term pool feeders. Upon reaching the host skin, the tick inserts its
chelicerae, cutting and tearing through the corneal layer, cellular epidermis, and outer layers of
the dermis, and then embeds itself with its barbed hypostome. Blood soon flows from torn ves-
sels into the wound site, forming a feeding pool. In argasid ticks, blood is consumed rapidly;
they may complete their meal within minutes or, at most, a few hours (or up to several days for
larvae), expanding to about 5 to 10 times their pre-fed body weight. To concentrate the blood
12 BIOLOGY OF TICKS
meal, a clear, colorless watery fluid is excreted from the tick’s coxal glands during or shortly after
the completion of feeding. Finally, the engorged tick departs from the host to seek shelter in a
suitable microenvironment where it can digest its meal. In contrast, ixodid ticks attach as de-
scribed above but then secrete copious quantities of cement from their salivary glands. As the
cement hardens, it binds the attached tick tightly to the host skin, allowing for an extended
feeding period ranging from a few days to several weeks. These ticks feed slowly, synthesizing
new cuticle to accommodate the enormous blood meal, another unique feature that distin-
guishes ticks from blood-feeding insects. As ticks acquire blood, excess water is extracted from
the hemolymph by the salivary glands and secreted back into the host. Additional water may be
lost through the cuticle and the passage of fecal wastes, but this is inefficient. Thus, the true vol-
ume of blood consumed is 2 to 3 times greater than that of the fully engorged tick (at the time of
drop off ). For animals infested with hundreds or perhaps even thousands of ticks, the aggregate
blood loss may cause severe injury and even result in death due to exsanguination.
For successful hematophagy (blood feeding), ticks have evolved mechanisms to disrupt the
natural hemostatic mechanisms of the host. The ticks must also maintain blood flow and prevent
or minimize pain, inflammation, itching, and immune rejection. Several recent reviews summa-
rize the remarkable molecular biology of tick saliva and its function in accomplishing these tasks
(Francisschetti et al. 2009; Mans and Neitz 2004). Ticks have evolved a veritable pharmacopeia of
bioactive lipids and proteins to suppress the hemostatic system and enhance blood flow in the
host. Recent discoveries concerning the molecular biology of tick salivary gland function have
revealed hundreds of different proteins, including many that have no known function and many
that are unique to ticks. Especially prominent are those adapted to prevent blood coagulation,
inhibit platelet aggregation (e.g., lipocalins) and histamine secretion, suppress inflammation, and
bind host immunoglobulins. In species in which a long evolutionary association with a narrow
spectrum of vertebrates has occurred, the ticks are able to suppress most host immune responses,
thereby allowing repeated utilization of the same hosts. Lastly, and often overlooked, is the ability
of tick saliva to shelter microbes from host rejection in the wound site (Nuttall et al. 2000).
4.4. ECOLOGY
Most ixodid ticks are non-nidicolous (exophiles) (i.e., living in open environments such as forests,
brush lands, savannahs, meadows, and even semi-deserts). They shelter in leaf litter, duff, rotting
vegetation, or buried in sand and under gravel and stones. Almost all exophiles are species of the
Metastriata subgroup of the Ixodidae. Most argasid and many prostriate ticks are nidicolous (endo-
philes), living in nests, burrows, caves, or other shelters used by their hosts or hiding close by. Here
we address only briefly the strategies used by ticks for survival in the natural environment, for finding
hosts, for adapting to seasonal changes, and for recognizing preferred hosts. A more detailed, com-
prehensive description of this aspect of tick biology is presented in Chapters 1 and 2 of Volume 2.
4.4.1. Tick survival in the natural environment

Ticks have evolved several different strategies to facilitate their survival while waiting for verte-
brate hosts. In non-nidicolous species, the ticks crawl up emergent vegetation (e.g., blades of
grass or woody stems along roadsides or trails in meadows and woodlands), where they wait for
a passing host (the ambush strategy). In many species, the ticks are able to survive for long pe-
riods because they can minimize evaporative water loss. This ability is facilitated by the lipid
coat on the cuticular surface, by the infrequent opening of the paired respiratory spiracles, and
by the use of guanine as an excretory waste product, as it requires little water for its elimination.
However, after many days or weeks without a host, the ticks become desiccated and may de-
scend down into the mass of leaf litter, dead or decaying grass, or similar materials. In this more
humid microclimate, they can replenish their body water. Ticks are able to absorb water directly
from sub-saturated atmospheres, using hygroscopic solutions secreted by the salivary glands on
the hypostome. Hydrated ticks then return to the emergent vegetation or exposed ground sur-
faces to resume questing for hosts. In some species (e.g., the sheep tick, Ixodes ricinus), repeated
cycles of descent from the exposed open environment, rehydration, and return to the questing
perch enable the ticks to extend their search for hosts for many weeks or even months. Some
ticks, attracted by CO2, ammonia, and other odorants, emerge from their shelters and walk or
run toward their hosts (the hunter strategy). An example is the camel tick, Hyalomma dromeda-
rii, specimens of which are often found hiding in sand or under stones and duff near barns,
corrals, and caravanserais. They remain hidden until stimulated by the arrival of animals and
their human handlers, whereupon they emerge and run in search of a blood meal. In many spe-
cies that inhabit temperate or boreal regions of the world, the ticks cease host seeking and over-
winter deep in rotting vegetation, sand, or cracks and crevices in the soil where they can shelter
below the frost level. Surviving individuals emerge the following spring to resume host seeking.
This phenomenon (diapause) extends the life cycle for at least one and sometimes several years.
Host detection is facilitated by sensory systems for detecting host odors, vibrations, temper-
ature changes, and other external cues. Ticks use their forelegs in a manner similar to how in-
sects use antennae, exposing the sensilla of Haller’s organ on each tarsus to the air stream for the
detection of sensory information. Sensilla in this organ respond to thermal energy and to ele-
vated levels of CO2, NH3, H2S, and other odorants from warm-blooded animals that come within
range. These adaptations facilitate host location and contribute to successful attachment. Other
sensilla enable ticks to recognize when they are in cracks or crevices or under leaf litter or soil
where they can shelter from environmental extremes. Using subtractive hybridization and other
molecular techniques, Mulenga et al. (2007) showed that as many as 40 genes were upregulated
in Amblyomma americanum ticks that had attained appetence (i.e., willingness to feed) and were
exposed to feeding stimuli even before they began to penetrate the host skin. Expression of these
particular genes was believed to facilitate readiness to commence attachment and feeding.
Nidicolous ticks exhibit very different behavioral patterns than the non-nidicolous species. Ni-
dicolous ticks, most of which are argasid and prostriate species, shelter in the nests or burrows of their
preferred hosts (or close by). There they are exposed to greater humidity and less extreme environ-
mental conditions than their non-nidicolous relatives. Assembly pheromones induce clustering at
the most favorable sites, thereby enhancing survival and the likelihood of finding hosts. Most argasid
ticks do not exhibit patterns of seasonal or reproductive activity; exceptions are found in certain
species that feed exclusively on migratory hosts (e.g., Carios kelleyi, which feed on migratory bats).
4.4.2. Host specificity

Most argasid and ixodid ticks are host specific to varying degrees. Many specialize in feeding on
a specific group of vertebrates (e.g., argasid ticks that feed only on bats, or ixodids such as R. [B.]
microplus that feed only on large ruminants). Most of the nidicolous species are highly specific.
14 BIOLOGY OF TICKS
Other species are opportunistic, such as the black-legged tick, I. scapularis, which feeds readily
on lizards, birds, small mammals, and larger hosts like sheep and humans. The molecular basis
of host specificity and its evolutionary origins represent a fascinating study of evolutionary ad-
aptations to changing vertebrate hosts that have occurred over tens of millions of years; this is
explored further in the succeeding chapters of this book.
5. FUTURE PERSPECTIVES
The study of ticks and tick-borne diseases has contributed greatly to improvements in human
and animal health. Beginning with the pioneering work of Smith and Kilbourne (1893), ticks
have been identified as the vectors or causative agents of at least 100 different human and animal
diseases, discoveries that enable scientists to help find treatments to control and even cure these
illnesses and prevent their reoccurrence. A detailed summary of these accomplishments is be-
yond the scope of this book.
The future of research on ticks and tick-borne diseases is largely being driven by the molec-
ular biology revolution and new technologies to rapidly sequence the transcriptomes and ge-
nomes. The sequence of the genome of I. scapularis is now available (Volume 2, Chapter 3) and
is currently being annotated. Plans to sequence the genomes of other ticks and even mites are in
progress. These advances offer outstanding opportunities to understand the molecular basis for
innumerable biological functions. Transcriptomes of the salivary glands, midgut, synganglion,
ovaries, female genital track, male reproductive system, and other tick organs or tissues have
been reported (see Chapters 7, 16, and 18), making it possible to characterize tissue-specific gene
expression. The development of solid surface nucleic acid synthesis, as well as the use of micro-
arrays and quantitative polymerase chain reaction, is providing us with the ability to measure
gene expression at different developmental times and under varying experimental conditions.
Advances in techniques in proteomics allow us to look at all gene products simultaneously. RNA
interference experiments have become a useful tool for determining the function of the specific
messages (see Vol. 2, Chapter 3). Computer-assisted bioinformatics and computational analysis
are expanding faster than ever. With all of these advances, and certainly with more to come,
future studies can examine how infection with diverse microbes influences molecular expres-
sion in terms of both protein synthesis and genetic control, how these pathogens are controlled,
and how the regulation of the host–pathogen interface facilitates the ability of ticks to harbor
and transmit disease-causing microbes. These same approaches are currently having a signifi-
cant impact on every aspect of tick biology discussed in this book and will certainly change the
field even more in the near future. It is also predicted that the rate of increase in our knowledge
of ticks will only increase as it is driven by new technology.
ACKNOWLEDGMENTS
This book is dedicated to the memory of Dr. Harry Hoogstraal (Fig. 1.1). Dr. Hoogstraal published
over 500 works on the systematics, biology, ecology, and disease relationships of hundreds of tick
species. He remains one of the giants among the great scholar-scientists of the 20th century.
This work was supported in part by Grant No. 0723692 from the National Science Foundation.
FIGURE 1.1: Dr. Harry Hoogstraal, when he was Head, United States Naval Medical Research Unit No. 3,
Cairo, Egypt. Photo credit: Dr. W. S. Bailey, Auburn University, Auburn, Alabama (photo provided
courtesy of Ms. Katherine Walker Hoogstraal).
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Smith, T. and Kilbourne, F.L. (1893) Investigations into the nature, causation and prevention of Texas or
southern cattle fever. Bur. Anim. Ind. Bull. 1:301–324.
Sonenshine, D.E. (1991) Biology of Ticks, Vol. 1. New York: Oxford University Press.
Sonenshine, D.E., Taylor, D., Phillips, J.S., Hamilton, J.G.C., and Allan, S.A. (1989) Sex pheromones in
ixodid ticks: identification and role in species discrimination. In M. Hoshi and O. Yamashita (Eds.),
Advances in Invertebrate Reproduction, Vol. 5. Amsterdam: Elsevier Science Publishers, 545–551.
Thompson, D.M., Khalil, S.M., Jeffers, L.A., Ananthapadmanaban, L.A.U., Sonenshine, D.E., Mitchell,
R.D., Osgood, C.J., Apperson, C., and Roe, R.M. (2005) In vivo role of 20-hydroxyecdysone and
juvenile hormone in the regulation of the vitellogenin message and egg development in the Ameri-
can dog tick, Dermacentor variabilis (Say). J. Insect Physiol. 51: 11050–11116.
Topalis, P., Tzavlaki, C., Vestaki, K., Dialynas, E., Sonenshine, D.E., Butler, R., Bruggner, R.V., Stinson,
E.O., Collins, F.H., and Louis, C. (2008) Anatomical ontologies of mosquitoes and ticks, and their
web browsers in VectorBase. Insect Mol. Biol. 17:87–89.
Weygoldt, P. (1998) Evolution and systematics of the Chelicerata. Exp. Appl. Acarol. 22: 63–79.
Zhang, X., Meltzer, M.I., Peria, C.A., Hopkins, A.B., Wroth, L., and Fix, A.D. (2006) Economic impact
of Lyme disease. Emerg. Infect. Dis. 12:653–660.
NOTE
1. Link to www.Vectorbase.org (if available, select the “old version”). In the task bar near the top of
the screen, select “Tools.” Under “Available Tools,” select “Controlled Vocabulary Search.” In the
next screen, use the down arrow to open the drop-down box and click on “Tick Anatomy.” The
user may then browse the anatomical anatomy by clicking on “Material Anatomical Entity” and
then on “Anatomical Structure.” The greatest number of terms will be found by clicking on “Organ-
ism Subdivision.” The definition of the term will be shown in the upper right-hand corner. Clicking
on other terms will reveal other hierarchical relationships at varying levels. In this manner, the user
can find all of the anatomical components linked to a particular body structure and their relation-
ships. In many instances, a figure will be shown to illustrate the structure. The user may also insert
a term in the search box and find the information desired without searching through the ontology.
C H A P T E R 2
MODERN TICK SYSTEMATICS

LANC E A. DURDEN AND LORENZA BEATI
1. INTRODUCTION
Ticks are arachnids belonging to the subclass Acari (mites), the superorder Parasitiformes,
the order Ixodida (Metastigmata according to some authors), and the superfamily Ixodoidea
(Keirans 2009). Modern tick systematics is arguably in a state of current flux as increasing
numbers of researchers endeavor to identify, classify, and phylogenetically interpret tick bio-
diversity using molecular methods. Despite this trend, morphology-based interpretations
and especially identifications provide the cornerstone of tick systematics. In many parts of
the world, especially under field conditions, a light microscope and printed identification
keys or comparative illustrations are the only readily available tools that can consistently be
used to identify ticks. Also, currently available computer-based tick identification keys are
based on morphological characters. Nevertheless, as molecular methods continue to be
refined and the required laboratory equipment becomes more widespread, molecular tick
identification techniques will become more commonplace. However, morphological tick
identifications will always be important, and we anticipate that competent morphology-
based tick taxonomists will remain in demand. Some treatments of tick identification, sys-
tematics, phylogenetics, or biogeography have combined morphological and molecular
datasets in a type of “total evidence” approach (Klompen et al. 2000; Beati and Keirans 2001;
Beati et al. 2008; Nava et al. 2009).
In this chapter, we cover aspects of both morphological and molecular tick systematics
and highlight some areas of contention. We also include morphology-based dichotomous
keys for identifying adult ticks to genus and provide images of the immature and adult stages
of representative argasid (soft) ticks and ixodid (hard) ticks and of an adult of the only known
species of nuttalliellid tick. Figures 2.1–2.22 are ordered by stage and taxonomic group.
FIGURE 2.1: An ixodid larva (Ixodes woodi) (ventral aspect). Note the 6 legs and the absence of
spiracular plates. Scale bar, 100 μm. Scanning electron microscopy photo courtesy of R. G. Robbins.
FIGURE 2.2: An argasid larva (Ornithodoros sp.) (dorsal aspect). Note the 6 legs and the dorsal plate on
the idiosoma. Scanning electron microscopy photo courtesy of R. G. Robbins.
18
FIGURE 2.3: An argasid larva (Ornithodoros sp.) (ventral aspect). Note the 6 legs and the long, almost
leg-like palps. Scanning electron microscopy photo courtesy of R. G. Robbins.
FIGURE 2.4: An ixodid nymph (Ixodes woodi) (ventral aspect). Note the 8 legs and the absence of a
genital aperture. Scale bar, 100 μm. Scanning electron microscopy photo courtesy of R. G. Robbins.
19
FIGURE 2.5: Adult Ornithodoros capensis (dorsal aspect) (Argasidae). Note the mamillate idiosomal
integument and the fact that the capitulum and mouthparts are not visible (because they are situated
ventrally). Scale bar, 500 μm. Scanning electron microscopy photo courtesy of R. G. Robbins.
FIGURE 2.6: Adult female Ornithodoros capensis (ventral aspect) (Argasidae). Note the mamillate
integument and the ventrally situated capitulum and mouthparts, which are recessed in a cavity, the
camerostome. Scale bar, 500 μm. Scanning electron microscopy photo courtesy of R. G. Robbins.
20
FIGURE 2.7: Adult Argas cucumerinus (dorsal aspect) (Argasidae). Note the mamillate idiosomal
integument and the fact that the capitulum and mouthparts are not visible (because they are situated
ventrally). Scale bar, 500 μm. Scanning electron microscopy photo courtesy of R. G. Robbins.
FIGURE 2.8: Adult Argas brumpti (lateral aspect) (Argasidae). Note the distinct sutural line that
separates the dorsal and ventral portions of the idiosoma. Scale bar, 500 μm. Scanning electron
microscopy photo courtesy of R. G. Robbins.
21
FIGURE 2.9: Otobius megnini (spinose ear tick), nymph (dorsal aspect) (Argasidae). Note the spiny
integument. Scale bar, 200 μm. Scanning electron microscopy photo courtesy of R. G. Robbins.
FIGURE 2.10: Adult male Antricola marginatus (dorsal aspect) (Argasidae). Note the prominent, narrow,
funnel-shaped anterior idiosoma and the tuft-like setal clusters. Scale bar, 100 μm. Scanning electron
22
FIGURE 2.11: Adult female Antricola marginatus capitulum (Argasidae). Note the broad scoop-like shape
of the hypostome. Scale bar, 200 μm. Scanning electron microscopy photo courtesy of R. G. Robbins.
FIGURE 2.12: Adult Nothoaspis reddelli (dorsal aspect) (Argasidae). Note the false scutum. Scale bar,
100 μm. Scanning electron microscopy photo courtesy of R. G. Robbins.
23
FIGURE 2.13: Adult female Nuttalliella namaqua (dorsal aspect) (Nuttalliellidae). Note the unique
pseudoscutum, the body surface integument, and the ball-and-socket leg joints. Scanning electron
microscopy (SEM) photo courtesy of Dan Corwin; SEM reproduced from Keirans, J.E., Clifford, C.M.,
Hoogstraal, H., and Easton, E.R. (1976) Discovery of Nuttalliella namaqua (Acarina: Ixodoidea:
Nuttalliellidae) in Tanzania and redescription of the female based on scanning electron microscopy.
Ann. Entomol. Soc. Am. 69:926–932.
FIGURE 2.14: Adult female Nuttalliella namaqua (ventral aspect) (Nuttalliellidae). Note the unique
ball-and-socket leg joints, the body surface integument, and the capitulum. Scanning electron
microscopy (SEM) photo courtesy of Dan Corwin; SEM reproduced from Keirans, J.E., Clifford, C.M.,
Hoogstraal, H., and Easton, E.R. (1976) Discovery of Nuttalliella namaqua (Acarina: Ixodoidea:
Nuttalliellidae) in Tanzania and redescription of the female based on scanning electron microscopy.
Ann. Entomol. Soc. Am. 69:926–932.
24
FIGURE 2.15: Adult male Ixodes cookei (ventral aspect) (Ixodidae). Note the ventral plates and anal
groove extending anteriorly around the anus. Scale bar, 200 μm. Scanning electron microscopy photo
courtesy of R. G. Robbins.
FIGURE 2.16: Adult male Amblyomma americanum (ventral aspect) (Ixodidae). Note long palps and
hypostome and anal groove posterior to anus. Scale bar, 500 μm. Scanning electron microscopy photo
courtesy of R. G. Robbins.
25
FIGURE 2.17: Adult female Haemaphysalis leporispalustris (dorsal aspect) (Ixodidae). Note the laterally
extended portion of palpal article 2. Scale bar, 200 μm. Scanning electron microscopy photo courtesy of
R. G. Robbins.
FIGURE 2.18: Adult male Dermacentor andersoni (ventral aspect) (Ixodidae). Note the fl ap-like genital
cover and the greatly expanded plate-like coxa IV. Scale bar, 500 μm. Scanning electron microscopy
photo courtesy of R. G. Robbins.
26
FIGURE 2.19: Adult female Dermacentor andersoni (ventral aspect) (Ixodidae). Note the relatively short
palps and hypostome and the anal groove posterior to the anus. Scale bar, 500 μm. Scanning electron
FIGURE 2.20: Adult male Hyalomma dromedarii (ventral aspect) (Ixodidae). Note the protruding adanal
(arrow) and accessory (arrowhead) plates/shields and the anal groove posterior to the anus. Scale bar,
500 μm. Scanning electron microscopy photo courtesy of R. G. Robbins.
27
FIGURE 2.21: Adult male Rhipicephalus (Boophilus) microplus (ventral aspect) (Ixodidae). Note the very
short palps and hypostome, the distinct adanal (arrow) and accessory (arrowhead) plates/shields, and
the anal groove posterior to the anus. Scale bar, 200 μm. Scanning electron microscopy photo courtesy
of R. G. Robbins.
FIGURE 2.22: Adult male Rhipicephalus (Rhipicephalus) sanguineus complex (dorsal aspect) (Ixodidae).
Note the hexagonal basis capituli and the relatively short palps. Scale bar, 200 μm. Scanning electron
28
Modern Tick Systematics 29
2. MORPHOLOGY-BASED TICK SYSTEMATICS
2.1. CHOICE OF MORPHOLOGICAL CHARACTERS

External tick morphology is detailed in Chapter 4. Essentially, ixodid ticks have a dorsal scutum
(see Figs. 4.2B, 4.2D, 4.2F, 4.2H) of hardened cuticle and anteriorly projecting mouthparts (see
Figs. 4.14, 4.15). The scutum is entire (covers most of the dorsal idiosomal body surface) in adult
male ixodids (Fig. 2.22; also see Figs. 4.2B, 4.2D, 4.2F, 4.2H) (in which it can be referred to as a
conscutum) but partial (covering only the anterior dorsal body surface) in larvae, nymphs, and
adult females (Fig. 2.17; also see Figs. 4.2A, 4.2B, 4.2C, 4.2F), all of which engorge during blood
feeding as the partially expandible softer posterior body section (the alloscutum) enlarges. Ar-
gasid ticks lack a scutum in all life stages (although a “false scutum” is present in both known
species of Nothoaspis [Fig. 2.12], placed in the genus Carios by some authors), and nymphs and
adults have ventral mouthparts situated inside a depression, the camerostome (Figs. 2.6, 2.11;
also see Figs. 4.3B, 4.3D). Argasid larvae (Figs. 2.2, 2.3) have anteriorly directed mouthparts and
therefore resemble ixodid larvae, but typically they have a centralized dorsal idiosomal region of
harder cuticle (the dorsal plate). Argasid nymphs and adults of both sexes are morphologically
similar (Figs. 2.5–2.12; also see Fig. 4.3), but nymphs lack the ventral genital opening of adults.
Adult females have a smile-shaped genital slit, and adult males have a more or less horizontal slit
with a small almost semi-circular protrusion that represents the external male genitalia. Adults
of the only known nuttalliellid tick species, Nuttalliella namaqua, are morphologically interme-
diate between ixodids and argasids but also have some unique characters such as ball-and-socket
leg articulations and a pseudoscutum with a ridged surface (Keirans et al. 1976) (Figs. 2.13, 2.14).
The immature stages of N. namaqua have now been collected but have yet to be formally
described (Mans et al. 2011). All of these characters are typically used for identifying ticks and
interpreting phylogenies, but additional characters are also used for different tick stages, as
discussed below. For the definition of anatomical terms, see Section 2.3, “Morphological
Terminology.”
2.1.1. Larvae
The larval stage of virtually all species of argasid ticks (Figs. 2.2, 2.3) differs from conspecific
nymphal and adult stages in that it attaches and feeds on vertebrate hosts for an extended pe-
riod (days) rather then the shorter attachment period (minutes to a few hours) of the nymph
and adult (N.B., a few specialized argasids do not feed as nymphs and/or adults) (Oliver 1989;
Durden 2006). Because larval argasids attach to hosts for extended periods, this life stage is
typically collected more commonly than conspecfic post-larval stages, and many argasid spe-
cies are still known only from the larval stage. For this reason, the choice of morphological
characters for larval argasids is important and has been fairly well refined (Klompen 1992;
Klompen and Oliver 1993). Morphology-based identification guides to argasid larvae be-
longing to the subfamily Ornithodorinae are available for both the Western Hemisphere
(Kohls et al. 1965) and the Eastern Hemisphere (Sonenshine et al. 1966), with recent additions
by other authors.
Chaetotaxy (interpretation of the number and arrangement of setae) of the ventral and
dorsal body surfaces (idiosoma and capitulum) has taxonomic importance for both argasid
30 BIOLOGY OF TICKS
(Klompen, 1992; Klompen and Oliver 1993) and ixodid (Clifford et al. 1961; Klompen et al.
1996b) larvae (Fig. 2.1). Setae inserted in different body positions are abbreviated and num-
bered from anterior to posterior, including, for example, marginal dorsals (MD1, MD2, etc.),
central dorsals (CD1, CD2, etc.), and others on the dorsal surface of the idiosoma following
the terminology of Clifford et al. (1961) and Klompen et al. (1996b). Larvae of different tick
genera have different numbers and exact positions of these setae. The number and arrange-
ment of pores (lyrifissures), including wax gland openings (large wax glands have been re-
ferred to as sensilla sagitiformia by some authors), relative to various setae are also important
taxonomic characters for assigning larval ticks to genera and species. Klompen (1992) and
Klompen and Oliver (1993) detailed taxonomically important morphological structures of
larval argasids. Similarly, Klompen et al. (1996b) added to the taxonomic utility of idiosomal
setae and lyrifissures in ixodid larvae and proposed a revised system of nomenclature for
these structures that corresponds better with the nomenclatural system used for these struc-
tures in mesostigmatid mites, thus facilitating more direct comparisons between these 2
groups of acarines.
Additional morphological characters useful for identifying larval ticks are generally those
that are also used for nymphs and adults, including hypostomal dentition; scutal shape; number,
size, and arrangement of coxal spurs; shape of the basis capituli; and position of the anal groove.
Also, the number and arrangement of festoons and the shape and length of the palpal articles
can be important characters for ixodid larvae, and the shape and size of the dorsal plate are of-
ten diagnostic for argasid larvae. Few morphology-based keys to larval ixodids are currently
available; one that is widely used to identify larval ixodids of the eastern United States is that by
Clifford et al. (1961).
A few tick genera are more reliably determined using larval rather than adult morphological
characters. For example, the generic assignments for argasids recognized by Klompen and Oli-
ver (1993) are mostly based on larval characters. Similarly, the ixodid genus Bothriocroton is
most easily distinguished from the genus Amblyomma (including species formerly placed in
Aponomma) by the presence of 3 large wax glands on the idiosoma (as well as via molecular
analysis) (Klompen et al. 2002).
2.1.2. Nymphs
While chaetotaxy and lyrifissures are important morphological characters used for identifying
larval ticks and assessing phylogenetic relationships between taxa, these characters are used far
less frequently for identifying and assessing nymphal and adult ticks. Many nymphal argasids
closely resemble conspecific adults except that they are slightly smaller (usually), lack a genital
aperture, and, in some genera, have a different hypostomal morphology (e.g., Otobius nymphs
have distinct hypostomal denticles that are absent in Otobius adults). Other nymphal argasid
characters are discussed with adult argasid characters in the next section.
Characters useful for identifying nymphal ixodids (Fig. 2.4) are also quite similar to
those of conspecific adults, again with the exception that nymphs lack a genital aperture and
also lack porose areas on the dorsal surface of the basis capituli (present in adult females)
and accessory plates on the ventral idiosoma (present in adult males in some genera) (Dur-
den and Keirans 1996). Important nymphal ixodid characters include the number, size, and
arrangement of coxal spurs (if present) and trochanteral or tarsal spurs (if present); the
shape and length of the palpal articles (and whether palpal spurs or flanges are present on
them); the hypostomal dentition; the shape of the basis capituli, including its posterior dor-
sal margin; whether ventral protuberances are present on the ventral basis capituli; the
shape and size of cornua and auriculae (if present); the shape of the scutum; the presence
and position of scutal punctations (if present); the shape of the spiracular plates and the
arrangement and size of goblets; the presence or absence of cervical grooves; the position of
the anal groove; and the number and arrangement of festoons (if present). As with ixodid
larvae, few morphology-based keys or illustrated guides to ixodid nymphs are available; one
that is widely used to identify Ixodes species nymphs of North America is that by Durden
and Keirans (1996).
2.1.3. Adults
Important morphological characters useful for identifying adult and nymphal argasids include
the number, shape, and arrangement of mammillae and/or discs on the dorsal idiosoma; the
shape of the hood and anterior projection (acuminate or pointed in some genera and species),
camerostome, cheeks, basis capituli, and genital aperture; the shape and dental formula (if den-
ticles are present) of the hypostome; and the contours of various idiosomal grooves such as the
supracoxal fold and the transverse postanal groove. The arrangement of eyes (along the supra-
coxal fold) and the shape of dorsal humps on various leg segments can also be important taxo-
nomic characters for assessing postlarval argasids.
As well as the characters listed for ixodid nymphs in the preceding section, several addi-
tional characters are frequently used for assessing adult ixodids. These include the shape of the
genital aperture, the texture of the scutum (smooth, rough, shagreened, etc.), the shape of the
scapulae for both sexes, the size and shape of porose areas in females, the presence or absence of
a caudal process, and the presence (and shape) or absence of various plates on the ventral idio-
soma in males. Depending on the genus, the male plates that might be present are flat anal,
median, and epimeral plates (in Ixodes) or protruding accessory plates (in Hyalomma and Rhipi-
cephalus, including members of the subgenus Boophilus). Adanal plates may also be present in
adult males and are flattened in Ixodes but projecting in Hyalomma and Rhipicephalus, including
members of the subgenus Boophilus. Keirans and Durden (2005) provide a list of identification
guides for ticks (mostly for adult ixodids) for different parts of the world.
2.2. IDENTIFYING ADULT TICKS TO GENUS

In this section we provide a list and definitions of morphological terms commonly used for
identifying ticks and for phylogenetic interpretations based on morphology. Following this
list, we provide a dichotomous key for identifying extant ticks to family and genus. Tick gen-
era recognized in the key are those reported in the latest compilation of valid tick names by
Guglielmone et al. (2010), in which 896 species are listed (193 argasids, 1 nuttalliellid, 702 ixo-
dids). This list of valid tick names builds on prior compilations of world tick names by Keirans
(1992), Camicas et al. (1998), Horak et al. (2002), and Barker and Murrell (2004) and of lists
of world Ixodidae (Kolonin 2009), tick taxa described between 1973 and 1997 (Keirans and
Robbins 1999), and controversial tick names (Guglielmone et al. 2009). Although there is
32 BIOLOGY OF TICKS
typically close consensus, none of these lists completely agree with each other, and in some
cases authors on the same paper do not entirely agree on the recognition of certain tick names
(Guglielmone et al. 2010). Given this fact, and allowing for future interpretations of tick
classification, particularly with respect to molecular data, revised lists of tick names will likely
be forthcoming.
Two monotypic fossil (amber-preserved) ixodid genera listed by Guglielmone et al. (2010),
Cornupalpatum and Compluriscutula (Poinar and Brown 2003; Poinar and Buckley 2008), are
not included in the identification key we provide. The few other known fossil ticks all belong to
extant genera and are therefore included in the key. However, the fossil soft tick Carios jerseyi
described by Klompen and Grimaldi (2001) is treated as Ornithodoros jerseyi by Guglielmone et
al. (2010), and we follow that classification because it represents the most recent interpretation.
The contentious nature of and lack of consensus on argasid classification are discussed under the
appropriate heading later in this chapter (see also Table 2.1).
Table 2.1: Classification of argasid (soft) ticks by different researchers.
Russian Schoola American Schoolb Klompen and Oliver Schoolc

Subfamily Argasinae Subfamily Argasinae Subfamily Argasinae
Tribe Argasini
Genus Argas Genus Argas Subgenera Argas, Genus Argas Subgenera Argas
Subgenera Argas, Persicargas, Persicargas, Microargas, (incorporating Persicargas,
Carios, Chiropterargas, Carios, Chiropterargas, Secretargas, Ogadenus,
Secretargas Secretargas, Ogadenus Proknekalia, Alveonasus)
Subfamily Ornithdorinae Subfamily Ornithdorinae Subfamily Ornithdorinae
Tribe Otobiini
Genus Otobius Genus Otobius Genus Otobius
Genus Alveonasus Genus Ornithodoros Subgenera Genus Ornithodoros
Subgenera Ogadenus, Proknekalia, Alveonasus, (incorporating Ornithodoros,
Proknekalia, Alveonasus Ornithodoros, Pavlovskyella Pavlovskyella, Theriodoros,
Tribe Ornithodorini (incorporating Theriodoros), Ornamentum, Microargas)
Genus Ornithodoros Ornamentum, Alectorobius, Genus Carios (incorporating
Subgenera Ornithodoros, Reticulinasus, Subparmatus Carios, Chiropterargas,
Pavlovskyella, Theriodoros, Genus Nothoaspis Alectorobius, Subparmatus,
Ornamentum, Alectorobius, Reticulinasus, Parantricola,
Reticulinasus, Subparmatus Antricola, Nothoaspis)
Genus Antricola Genus Antricola Subgenera
Subgenera Parantricola, Parantricola, Antricola
Antricola
a
Based on Filippova, N.A. (1966) Argasid Ticks (Argasidae). Leningrad: Zoolologicheskogo Institut Akademii Nauk USSR
[in Russian]; and Pospelova-Shtrom, M.V. (1969) On the system of classification of ticks of the subfamily Argasidae Can.,
1890. Acarolgia, Paris 11:1–22. Note that the subgenus Argas (Microargas) was described in 1966 after the first of these
Russian papers was published, and the genus Nothoaspis was described in 1975, after both Russian papers were published.
b
Based on Clifford, C.M., Kohls, G.M., and Sonenshine, D.E. (1964) The systematics of the subfamily Ornithodorinae
(Acarina: Argasidae). I. The genera and subgenera. Ann. Entomol. Soc. Am. 57:429–437; and Hoogstraal, H. (1985)
Argasid and nuttalliellid ticks as parasites and vectors. Adv. Parasitol. 24:135–238.
c
Based on Klompen, J.S.H. and Oliver, J.H., Jr. (1993) Systematic relationships in the soft ticks (Acari: Ixodida:
Argasidae). Syst. Entomol. 18:313–331.
2.3. MORPHOLOGICAL TERMINOLOGY

The following is an alphabetical list of terms relevant to tick morphology and their definitions.
Many of these terms pertain to structures that have taxonomic significance and are thus useful
for tick identification. These terms are used throughout this book, but especially in this chapter
with respect to tick systematics and in Chapter 4 with respect to external anatomy.
Accessory plates: Paired projecting, sclerotized structures (Figs. 2.20, 2.21) on the ventral idio-
soma, lateral to the projecting adanal plates in males of Hyalomma, and Rhipicephalus, including
members of the subgenus Boophilus.
Adanal plates: Paired posteriorly directed sclerotized plates (Figs. 2.20, 2.21) on the posterior
ventral surface in males of some ixodid genera. These plates are flattened in male Ixodes (Fig. 2.15)
but projecting in male Hyalomma (Fig. 2.20) and Rhipicephalus, including members of the subge-
nus Boophilus (Fig. 2.21).
Alloscutum: The dorsal body region posterior to the scutum in ixodids (see Fig. 4.2).
Anal aperture/anal plates: The posterior terminus of the alimentary canal (see Fig. 4.12). The
anal aperture is surrounded by a cuticular ring and, in males belonging to some ixodid genera,
bordered by anal plates.
Anal groove: In ixodids, a groove adjacent to the anus. In prostriate ticks (genus Ixodes), the
anal groove curves around the anus anteriorly (Figs. 2.1, 2.4, 2.15); in metastriate ticks (all other
ixodid genera), the anal groove is smaller and situated posteriorly to the anus (Figs. 2.16, 2.19, 2.20, 2.21).
Anterior projection: The dorsal anterior projection of the body beyond the capitulum in
argasids, so that the capitulum is not visible from the dorsal aspect (Figs. 2.5, 2.7, 2.10, 2.12).
Sometimes the projection is narrow and almost pointed (Fig. 2.10).
Articles: Segments of the palps (see Fig. 4.13) (rarely, also of the legs). Article I is proximal
(connected) to the capitulum or body.
Auriculae (singular: auricula): Paired extensions of the ventral posterolateral margins of the
basis capituli in ixodids (see Fig. 4.13); can be large, small, or absent.
Basis capituli: The basal ring of cuticle to which the palps, chelicerae, and hypostome are at-
tached (see Figs. 4.13, 4.14, 4.16). The basis capituli can be moved dorsoventrally and articulates
with the body proper (idiosoma).
Camerostome: In nymphal and adult argasids, a cavity in which the capitulum is situated (Fig.
2.6; also see Fig. 4.3B).
Capitulum: The movable anterior extension of the body that includes the palps and mouth-
parts; homologous to the gnathosoma of other acarines (see Figs. 4.13, 4.14, 4.16).
Caudal process: A distinct projection arising from the median posterior idiosoma in males of
some species of Margaropus and Rhipicephalus (subgenus Boophilus) (Fig. 2.21).
Cervical grooves: Paired grooves on the scutum extending posteriorly from the inner angles
of the scapulae; can be deep, shallow, faint, or absent.
Chaetotaxy: Arrangement and nomenclature of setae.
Cheeks: In nymphs and adults of some argasids, paired, movable, flap-like structures that can
close medially to cover and protect the capitulum.
Chelicerae (singular: chelicera): The first pair of appendages of arachnids, including ticks (see
Fig. 4.16). Tick chelicerae have 3 segments: the cheliceral bases, which are located within the basis
capituli; the cheliceral shafts, which originate within the basis capituli and extend anteriorly; and
the cheliceral digits that bear denticles.
34 BIOLOGY OF TICKS
Conscutum: The scutum of an adult male ixodid tick, which covers almost the entire dorsal
surface of the idiosoma (Fig. 2.22).
Cornua (singular: cornu): Paired posterolateral projections on the dorsum of the capitulum in
many ixodids (see Fig. 4.14); can be large, small, or absent.
Corona: A field of tiny denticles at the tip of the hypostome (see Fig. 4.15).
Coxae (singular: coxa): The basal (first) segment of each leg that articulates with the body and
to which body muscles attach (see Fig. 4.17).
Coxal spurs: Large or small projections extending posteriorly from coxae in many ixodids
(absent in argasids). Often, 2 spurs are present on a single coxa, in which case the medial spur is
called the internal spur and the lateral spur is called the external spur.
Crenulations: Small hypostomal ridges or denticle-like structures present in some species,
usually in non-feeding males; can also be present beyond the denticulate zone in species in which
the hypostome bears prominent denticles.
Denticles: “Teeth” or small recurved projections on the ventral surface of the hypostome (see
Fig. 4.15) and also apically on the cheliceral digits (see Fig. 4.16).
Dentition (sometimes referred to as the dental formula): The arrangement of denticles on
either side of the midline of the ventral hypostome (see Fig. 4.15) (usually for the central or sub-
apical hypostomal area) (e.g., 2/2 refers to 2 longitudinal files of denticles on each side).
Discs: In argasids, flattened depressions, usually subcircular, with a smooth or mottled surface
(see Figs. 4.3A, 4.3C; Figs. 2.5, 2.7, 2.10, 2.12), representing sites of dorso-ventral muscle attach-
ments (often arranged in distinct patterns).
Dorsal humps: Prominent elevations on the dorsal surfaces of some leg segments of some
ticks.
Dorsal plate: A more or less centralized smooth area, usually elongate or subcircular, on the
dorsum of many argasid larvae (Fig. 2.2).
Emargination: In ixodids, the anterior indentation in the scutum between the scapulae, in
which the basis capituli is situated.
Epimeral plates: Paired latero-ventral plates in males of the genus Ixodes, located posteriorly
to the spiracular plates (Fig. 2.15).
Festoons: In some ixodid ticks, small portions of the ventral posterior idiosomal margin,
marked by grooves (Figs. 2.16, 2.22), usually rectangular (absent in members of the genera Ixodes,
Haemaphysalis, and Anomalohimalaya and in the subgenus Boophilus).
Folds: Prominent cuticular ridges on the ventral or marginal body surface of argasid nymphs
and adults.
Foveae dorsalis: Paired pheromone gland openings (see Fig. 4.6) on the dorsum of ixodids;
located just posterior to the scutum in females, nymphs, and larvae and one-half to two-thirds
posterior on the scutum of males.
Goblets: Tiny cavities within the cuticle of spiracular plates (see Fig. 4.10), appearing as
subcircular structures when viewed from the external surface.
Granulations: Irregular bumps or elevations on the body surface of post-larval argasids.
Grooves: Linear depressions in the body cuticle, usually on the ventral surface.
Haller’s organ: A highly sensory structure situated on the dorsal surface of the tarsus of leg
I (see Fig. 4.18), present in all post-embryonic stages of all tick species.
Hood: In argasids, the anterior projection of the camerostome, anterior to the capitulum (not
to be confused with the cheliceral digit hood).
Hypostome: The median ventral extension of the basis capituli, between the palps, covered
with recurved teeth (denticles) on its ventral surface (see Fig. 4.15) in most species and having a
pronounced preoral canal (the hypostomal gutter) on its dorsal surface (see Fig. 6.1).
Idiosoma: The main body region of ticks and other mites (excluding the capitulum/gnathosoma).
Lateral carinae: Ridges along the lateral margins of the scutum in some ixodids.
Lyrifissures: Small pores or depressions on the idiosoma of ticks (and other mites). These can
be used for distinguishing different groups (and sometimes species) of ticks.
Macula: A prominent sclerotized fold or hump within part of the externally visible spiracular
plate (see Fig. 4.10).
Mammillae: Minute conical or truncated elevations covering the body surface and legs in
Ornithodoros ticks (Fig. 2.5), distinct from tubercles and granulations found in some other
argasids.
Median plate: In male Ixodes, the sclerotized ventral plate between the coxae, posterior to the
genital orifice (Fig. 2.15).
Ornate: Symmetrical color patterns of the sclerotized cuticle of many ixodids (see Figs. 4.2F,
4.2H), usually confined to the scutum but rarely also on the dorsal basis capituli or palps.
Palps: The second pair of appendages, normally with 4 segments (articles) (see Fig. 4.16).
Article I articulates with the basis capituli. In argasids, article I is elongate, about as long as ar-
ticles II and III, and movable. In ixodids, article I is usually immobile and shorter than articles
II and III. Articles II and III are almost always the longest palpal segments and are more flexible.
Article IV is terminal in argasids but recessed in a cavity on the ventral surface of article III in
most ixodids.
Porose areas: Two distinct clusters of tiny depressions on the dorsal basis capituli in female
ixodids (see Figs. 4.2B, 4.14, 4.16) (absent in 1 Asian species, Ixodes kopsteini).
Pulvillus (plural: pulvilli): In ixodids, a pad-like structure situated terminally on each tarsus,
adjacent to the claws (also present in the larvae of most argasids).
Punctations: Tiny depressions or pits on the surface of sclerotized cuticle, not to be confused
with the bases of setae, which they sometimes resemble.
Scapulae: Small projections on the antero-lateral edges of the scutum of many ixodids.
Scutum: The dorsal sclerotized plate covering the anterior body in larval, nymphal, and female
ixodids (see Figs. 4.2B, 4.2F) and almost the entire dorsal surface of males (see Figs. 4.2D, 4.2H;
Fig. 2.22), not to be confused with the dorsal plate of most argasid larvae, which is located more
centrally on the dorsal body surface.
Sensilla sagittiformia: Four paired glandular structures located ventrally behind each coxa; 1
dorsal pair is also present on the posterolateral idiosoma of some ixodid larvae (also called large
wax glands). Despite the name, these are not true sensilla.
Spiracles (spiracular apertures): External openings of the respiratory system of many arthro-
pods. In ticks (see Fig. 4.10), each spiracle opens into an atrium, a large sinus-like structure from
which the tracheal trunks emanate.
Spiracular plates: Modified plate-like structures on the ventral idiosomal surface of
nymphal and adult ticks (see Figs. 4.10, 4.11) that contain the spiracles. In argasids, each plate
consists of a small, simple, elevated plate termed the macula and the spiracular opening next to
a ridge of cuticle, all on the supracoxal fold. In ixodids, 2 large plates are present, each with
numerous ovoid air spaces (or goblets), which are visible externally to give each plate a distinc-
tive appearance.
36 BIOLOGY OF TICKS
Spurs: Projections (usually pointed, sometimes blunt) present on various parts of the body
(e.g., coxae, trochanters, or palpal segments) in some ticks.
Supracoxal fold: In argasids, a prominent fold on the lateral ventral margin of the idiosoma,
lateral to the legs. This fold bears the spiracles and, if present, the eyes.
Sutural line: A distinct cleavage along the lateral margin of argasids belonging to the genus
Argas that separates the dorsal and ventral idiosomal surfaces (Fig. 2.8). Typically, this line is
marked by marginal discs.
Tarsus: The terminal segment of each leg (see Fig. 4.17).
Transverse post-anal groove: In argasids, a horizontal groove on the ventral idiosomal surface,
perpendicular to the post-anal groove.
Wax glands: Glands with an opening on the dorsal idiosoma of some ticks; these can be dis-
tinguished as either large wax glands (sensilla sagittiformia) or small glands (sensilla hastiformia).
The presence of 3 large wax glands on each side of the idiosoma in larvae of Bothriocroton distin-
guishes members of this genus from all other metastriate ticks (which have 1 in that region) and
from prostriate ticks (which have none).
2.4. MORPHOLOGICAL KEY FOR IDENTIFYING EXTANT TICKS TO

FAMILY AND GENUS
The numbers of species listed in this section are world figures from Guglielmone et al. (2010)
with updates. The recognition of genera in the family Argasidae is currently contentious, with 3
main “schools” (see Table 2.1). In this key, we follow the “American School” of Clifford et al.
(1964) and Guglielmone et al. (2010) because genera are easier to recognize in that system for
adult argasids using morphological characters, and because this is the system followed in the
most recent list of world ticks (Guglielmone et al. 2010). Also see Klompen and Oliver (1993) for
more comparisons between the 3 systems.
1A. Scutum or pseodoscutum present on dorsal body surface (covering the anterior half of
the dorsum in unfed females, nymphs, and larvae and almost the entire dorsum in
males); capitulum anterior and visible from dorsal aspect; ventral spiracular plates
prominent in adults and nymphs (absent in larvae), situated posterior to coxa IV; palpal
articles all about equal in size, or article IV reduced and recessed into article
III. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1B. Integument leathery, lacking a true scutum (but a leathery, poorly sclerotized
pseudoscutum is present in Nothoaspis); capitulum ventral or subterminal, not visible
from dorsal aspect; spiracular plates inconspicuous, situated on supracoxal fold between
coxae III and IV; palpal articles about equal in size; article IV never subterminal or
recessed into article III. . . . . .Family Argasidae. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2A. Dorsal body surface convoluted and highly folded with a leathery, papillated
pseudoscutum; 3 palpal articles (segments) with terminal article not notably reduced or
recessed. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Family Nuttalliellidae (genus Nuttalliella)
(Figs. 2.13, 2.14) (1 known species, Afrotropical).
2B. Dorsal body surface with a d..istinct, sclerotized (hard) scutum; integument covered with
minute wrinkles or folds except on sclerotized plates; palpal article IV reduced and recessed
in a cavity on article III. . . . . . . . .Family Ixodidae. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
3A. Distinct sutural line separating dorsal and ventral surfaces . . . . . . . . . . . . . . . . . . . . . genus Argas
(Figs. 2.7, 2.8) (61 species, cosmopolitan)
3B. Lacking a sutural line between dorsal and ventral surfaces . . . . . . . . . . . . . . . . . . . . . . . . 4
4A. Nymphs with spiny integument and well developed hypostome; adults with granular
integument and vestigial hypostome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . genus Otobius
(Fig. 2.9) (2 species, Nearctic; however, 1 pest species, O. megnini, has been introduced
to some other parts of the world)
4B. Nymphal and adult integument lacking spines; adult hypostome of various forms, but
never vestigial . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
5A. Dorsal surface of adult and nymph with false scutum; palpal article I with a large flange
partially obscuring the hypostome . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . genus Nothoaspis
(Fig. 2.12) (2 species, Neotropical)
5B. Dorsal surface of adult and nymph lacking a false scutum; palpal article I lacking a
flange. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
6A. Hypostome broad at base and scoop-like; associated with bats . . . . . . . . . genus Antricola
(Figs. 2.10, 2.11) (17 species, Neotropical and Nearctic)
6B. Hypostome variously shaped, but with denticles and never scoop-like; associated with
various vertebrates, including bats. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .genus Ornithodoros
(Figs. 2.5, 2.6) (112 species, cosmopolitan)
7A. Eyes absent. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8
7B. Eyes present. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12
8A. Festoons absent; anal groove curving anteriorly around anus. . . . . . . . . . . . . . . genus Ixodes
(see Figs. 4.2A–4.2D; Fig. 2.15) (254 species, cosmopolitan)
8B. Festoons present; anal groove not extending anteriorly around anus. . . . . . . . . . . . . . . . 9
9A. Scutum ornate (or rarely inornate); palps elongate and subcylindrical. . . . . . . . . . . . . . . .10
9B. Scutum inornate; palps long or short and conical (not subcylindrical). . . . . . . . . . . . . . . . . . .11
10A. Subterminal spur present on trochanters. . . . . . . . . . . . . . . . . . . genus Bothriocroton
(7 species, Australasian)
10B. Subterminal spur absent on trochanters. . . . . . . . . . . . . . . . . . . .genus Amblyomma
(subgenus Aponomma) (formerly genus Aponomma) (20 species; widespread except in
Australasia, Europe, South America, and Antarctica)
11A. Basis capituli rectangular dorsally; palps short and conical with article II at least as broad
as long and extended laterally in most species. . . . . . . . . . . . . . . . . . . . . genus Haemaphysalis
(Fig. 2.17) (166 species, cosmopolitan)
11B. Basis capituli of males quadrangular dorsally with diverging anterolateral margins; basis
capituli of females hexagonal dorsally; palps long and conical with article II at least twice
as long as broad and not extended laterally. . . . . . . . . . . . . . . . . . . . . . . . genus Anomalohimalaya
(3 species, Palaearctic)
12A. Spiracular plate with irregular ridges and partially ornamented with ivory color; 9
festoons. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .genus Cosmiomma (1 species, Afrotropical)
12B. Spiracular plate without irregular ridges or coloration; festoons present or absent (if
present, never 9 in number). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13
13A. Palps much longer than basis capituli; palpal article II much longer than broad
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14
13B. Palps about as long as basis capituli; palpal article II about as long as broad
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .16
38 BIOLOGY OF TICKS
14A. Scutum and palps ornamented; palpal article III with dorsal and ventral flange;
male with paired adanal, accessory, and subanal plates . . . . . . . . . . . . . .genus Nosomma
(1 or 2 species [see text], India)
14B. Scutum ornate or inornate; palpal article III lacking dorsal and ventral flanges; male
with or without ventral plates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15
15A. Scutum inornate; males with adanal and subanal plates; festoons irregular, partially
coalesced. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . .genus Hyalomma (Fig. 2.20)
(27 species, Afrotropical, Palaearctic, and Oriental)
15B. Scutum usually ornate; males lacking adanal and subanal plates; festoons regular, not
coalesced. . . . . . . . . .genus Amblyomma (Fig. 2.16) (130 species, mostly pan-tropical and
pan-subtropical; 110 species if subgenus Aponomma is excluded)
16A. Palps extremely short (shorter than hypostome), ridged dorsally and laterally; anal
groove indistinct; festoons absent. . . . . . . . . . . .genus Rhipicephalus (subgenus Boophilus
[previously genus Boophilus]) (Fig. 2.21) (6 species, almost Cosmopolitan)
16B. Palps not extremely short (at least as long as hypostome), not ridged dorsally and
laterally; festoons present or absent. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17
17A. Festoons absent; leg IV of males greatly enlarged . . . . . . . . . . . . . . . . . .genus Margaropus
(3 species, Afrotropical)
17B. Festoons present; leg IV of males normal. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .18
18A. Seven festoons; spiracular plate round with few large goblet cells. . . . . . . . . . . . . . . .genus
Dermacentor (subgenus Anocentor) (placed in either Dermacentor or Anocentor by
different authors) (1 species, neotropical)
18B. Eleven festoons; spiracular plate round or with dorsal prolongation and with numerous
goblet cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19
19A. Basis capituli rectangular dorsally; usually ornate. . . . . . . . . . . . . . . . . . .genus Dermacentor
(see Figs. 4.2E–4.2H; Figs. 2.18, 2.19) (33 species, cosmopolitan; 34 species if Anocentor
is included)
19B. Basis capituli hexagonal dorsally; usually inornate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20
20A. Scutum usually inornate (4 ornate species); anal and accessory plates present in
males; coxa IV of males not much larger than coxae I–III and lacking 2 long
spurs. . . . . . . . . . . . . . . . . . . . . . . . . . . .genus Rhipicephalus (subgenus Rhipicephalus)
(Fig. 2.22) (77 species, cosmopolitan; 83 species if subgenus Boophilus is included).
20B. Scutum inornate; anal and accessory plates absent in males; coxa IV of males much
larger than coxae I–III, with 2 long spurs. . . . . . . . . . . . . . . . . . . . . . . .genus Rhipicentor
(2 species, Afrotropical).
3. MOLECULAR-BASED TICK SYSTEMATICS
3.1. TICK EVOLUTION AND PHYLOGENETICS

Early efforts at classifying ticks into cohesive groups based on morphology, life history, host
associations, chromosomal organization, or biogeography resulted in a number of somewhat
contradictory hypotheses about tick relationships and, more significantly, about the age of the
Ixodida and of its main lineages (Warburton 1907; Pospelova-Shtrom 1946; Filippova 1966;
Morel 1969; Camicas and Morel 1977; Hoogstraal 1978; Hoogstraal and Aeschlimann 1982;
Hoogstraal and Kim 1985; Oliver 1989; Klompen 1992; Klompen and Oliver 1993). Molecular
tools have helped to resolve a number of such controversial issues while revealing additional
taxonomic problems (Black and Piesman 1994; Black et al. 1997; Mangold et al. 1998a; Klompen
et al. 2000, 2002, 2007; Beati and Keirans 2001; Beati et al. 2008; Jeyaprakash and Hoy 2009;
Mans et al. 2011). In this section, we review the knowledge of tick systematics from an evolu-
tionary perspective.
3.1.1. Early hypotheses

A number of authors (Hoogstraal 1978; Hoogstraal and Aeschlimann 1982; Hoogstraal and
Kim 1985) placed the origin of the Ixodida in the late Paleozoic as obligate parasites of reptiles,
diverging in the Mesozoic into 2 lineages, the ancestors of the soft and hard ticks, respectively.
They proposed that Amblyomma, Aponomma, and Haemaphysalis had evolved in the Creta-
ceous period, and that the Rhipicephalinae was the youngest group, with an origin in the
mammal- and bird-rich Tertiary period. In their reconstructions, ticks were separated into 3
lineages corresponding to the 3 families (Argasidae, Ixodidae, and Nuttalliellidae). Nuttalliel-
lidae was an intermediate branch located between the 2 other families. The argasids were
monophyletic and subdivided in 2 sister clades, the Argasinae (Argas) and the Ornithodori-
nae (Ornithodoros, Otobius, Antricola, and Nothoaspis). The genus Ixodes was basal to all Ix-
odidae, followed by the Amblyomminae (Amblyomma and Aponomma), the monogeneric
Haemaphysalinae, the monogeneric Hyalomminae, and the most recently diverging Rhipi-
cephalinae (Dermacentor, Cosmiomma, Rhipicentor, Rhipicephalus, Anocentor, Anomalohima-
laya, Nosomma, and the sister lineages Boophilus and Margaropus). Few main elements
differentiated this classification from previously published hypotheses on tick relationships
(Warburton 1907; Pospelova-Shtrom 1946; Morel 1969; Camicas and Morel 1977). In these
reports, the Argasinae and the Ornithodorinae contained additional genera (Table 2.1), and
within the Metastriata, Hyalomma was included in the Rhipicephalinae. Oliver (1989) argued
that, as acariform mites were abundant in the Devonian, their sister group, the Parasitiformes
(including ticks), had also originated in the same period, and he proposed that ticks first ap-
peared as parasites of amphibians. Balashov (1994) used biogeographical patterns of conti-
nental drift to date tick evolutionary steps. He placed the origin of ticks before the breakup of
Pangea and the origins of Ixodes, Amblyomma, and Aponomma around 180 to 190 million
years ago (mya), in the early Mesozoic. He hypothesized that Haemaphysalis and Dermacentor
arose during the late Jurassic and Cretaceous, at the separation between Gondwana and Lau-
rasia, whereas Rhipicephalus and Hyalomma were treated as recent lineages originating during
the Paleocene in Africa and Asia, respectively.
3.1.2. Fossil record

Because of a low fossilization rate, fossil records are extremely sparse for the Ixodida, and they
mostly represent recognizable modern extant genera. They were all collected from relatively
young fossil deposits no older than the mid-Cretaceous. As explained by Keirans et al. (2002), it
is unclear whether Scudder (1885) really reported the finding of a fossil tick from the Oligocene
deposits of Wyoming. Schille (1916) described Dermacentor near reticulatus from a Pliocene
40 BIOLOGY OF TICKS
woolly rhinoceros (5.3 to 2.5 mya), and Weidner (1964) described Ixodes succineus from Baltic
amber (35 to 40 mya). Miocene Dominican amber (15 to 20 mya) has yielded a male Ambly-
omma specimen reported to be similar to Amblyomma testudinis (which is currently treated as
a synonym of Amblyomma argentinae) (Lane and Poinar 1986), several larvae of an Amblyomma
species close to A. dissimile (Keirans et al. 2002), and an argasid tick, Ornithodoros antiquus
(Poinar 1995). Klompen and Grimaldi (2001) reported the oldest known fossil argasid tick,
Carios jerseyi, from New Jersey amber deposits (90 to 94 mya). That specimen raised a number
of interesting questions about tick dispersal, as the genus Carios had previously been thought to
have arisen after the isolation of South America. Grimaldi et al. (2002) reported an Amblyomma
specimen from Cretaceous Burmese amber (90 to 100 mya). Two new genera of ticks, Cornupal-
patum and Compluriscutula, described by Poinar and Brown (2003) and Poinar and Buckley
(2008), respectively, were also recovered from Burmese amber. The most recently collected tick
fossil was a specimen recognizable as Ixodes sigelos from a Holocene owl pellet found in Las
Máscaras, Argentina (Sanchez et al. 2010). Although the tick fossil record is gradually increasing
in taxonomic breadth, the available information is only marginally contributing to a better un-
derstanding of tick evolution. Nevertheless, assuming that the sediments in which fossil ticks
have been found were accurately dated, it appears that ticks recognizable as Amblyomma and
Carios occurred in the late Cretaceous, placing the age of the Ixodida and its basal radiations
much earlier (see next section).
3.1.3. Phylogenetic molecular studies

Much interest was generated by molecular techniques when they became widely available for
the resolution of controversial aspects of tick evolutionary systematics. The first molecular
analysis of tick systematics based on mitochondrial 16S rDNA gene sequence analysis was by
Black and Piesman (1994). Although the chosen gene did not provide good resolution at su-
prageneric levels, Black and Piesman’s work raised a number of additional questions about
traditional morphology-based tick taxonomy, in particular about the monophyly of the arga-
sids and of the genus Amblyomma. Their work showed weak support for the Ornithodorinae
but was consistent with some of the earlier reconstructions placing Hyalomma within the
Rhipicephalinae. In their study, Haemaphysalis was embedded in Amblyomma. Based on their
reconstruction, the origin of the Ixodidae was placed no later than the late Jurassic (140 mya),
an estimate closer to Filippova’s (1977) hypothesis based on host associations placing the or-
igin of the Prostriata (the genus Ixodes) in the Cretaceous. Because 16S rRNA did not appear
to be informative at the generic level, Black et al. (1997) analyzed representatives of all tick
subfamilies using more conserved 18S rDNA gene sequences. This work revealed that argasids,
Argasinae and Amblyomma, were monophyletic; that the Argasinae was the sister group of the
Ornithodorinae; that Amblyomma did not include Haemaphysalis; and that Hyalomma origi-
nated within the Rhipicephalinae. As branch lengths within soft ticks were found to be twice
as long as in the Ixodidae, the 18S rRNA reconstruction supported the hypothesis that hard
ticks arose more recently (Hoogstraal and Aeschlimann 1982). A total evidence analysis
(Klompen et al. 2000), integrating morphological, nuclear, and mitochondrial datasets,
reached similar conclusions and showed that the Argasidae, Ixodidae, and Metastriata are
monophyletic. Another study also confirmed the paraphyly of the genus Aponomma (Dobson
and Barker 1999). The genus Ixodes was also not monophyletic, and the placement of the basal
metastriate lineages remained unresolved. This study provided evidence for the occurrence of
exclusively Australian clades. One of them later became the genus Bothriocroton and included
all former Aponomma strictly associated with the Australian continent (and New Guinea)
(Klompen et al. 2002; Beati et al. 2008). The genus Ixodes was found to be basal within the Ix-
odidae but paraphyletic. The first diverging Ixodes lineage was strictly Australian (with the ex-
ception of the widespread Ixodes uriae), whereas the second encompassed taxa from the rest of
the world. Similarly, the basal lineages within Amblyomma were Australian. These findings
and the fact that holothyrid mites (Klompen et al. 2007), a typical Gondwanan group, were
found to be more closely related to Ixodida than any other group of parasitiform mites sub-
stantiated the hypothesis of an early Cretaceous origin for the Ixodidae (Klompen et al. 2002).
Barker and Murrell (2002) used the same arguments to suggest that ticks originated in the area
of Gondwana that is now Australia. Jeyaprakash and Hoy (2009) analyzed the relationships
between the main chelicerate groups based on a large number of gene sequences and placed
the origin of the Ixodida in the late Paleozoic, at the end of the Carboniferous. Their estimate
is closer to that of Hoogstraal and Aeschlimann (1982). Mans et al. (2011) analyzed all 18S
rRNA tick sequences from Genbank and concluded that all main tick families and subfamilies
are monophyletic. Their analysis did not, however, include any of the taxa that have been dif-
ficult to classify and which are likely to disrupt group monophyly. They also argued for a South
African origin (Karoo Basin) of ticks in the Permian (260–270 mya), based on the fact that
Nuttalliella namaqua is the sister lineage of a clade that contains all other Ixodida and on the
fact that diapsid reptiles radiated from that region at the end of the Permian after a massive
extinction event (PermoTriassic Boundary, 251 mya). However, N. namaqua might constitute
the only remaining species of a larger group of taxa that might have occurred on larger areas
of the Gondwana subcontinent and which might have been feeding on different hosts.
Molecular phylogenetic analyses have greatly enhanced our confidence in several aspects of
tick systematics. The use of a set of markers characterized by mutation rates that are adapted to
different taxonomic hierarchical levels has resulted in the resolution of most of the initial main
questions. In general, nuclear ribosomal gene sequences are more informative at the family and
subfamily levels (Black et al. 1997; Dobson and Barker 1999; Fukunaga et al. 2000; Klompen
et al. 2000; Beati et al. 2008; Mans et al. 2011), whereas mitochondrial genes and intergenic tran-
scribed spacers (ITS1 and ITS2) provide better resolution at the intrageneric and intraspecific
levels (McLain et al. 1995; Norris et al. 1996; Barker 1998; Kain et al. 1999; Klompen et al. 2000;
Beati and Keirans 2001; Hlinka et al. 2002; Shaw et al. 2002; Xu et al. 2003; Casati et al. 2008;
Reichard et al. 2005; Szabó et al. 2005; Teglas et al. 2005; Mixon et al. 2006; Marrelli et al. 2007;
Nava et al. 2008, 2010a; Trout et al. 2010; Mastropaolo et al. 2011; Beati et al. 2012). For a sum-
mary of the most frequently used molecular markers in acarology, refer to Navajas and Fenton
(2001) and Cruickshank (2002).
3.2. POPULATION GENETICS

Investigations of taxonomic status, population geographical structure and dispersal, reproduc-
tive strategies, demographic histories, or population genetics parameters (population size, mi-
gration rates, and gene flow) are the common main goals of population genetic studies in
general, and this has applied also to the study of tick intraspecific relationships.
42 BIOLOGY OF TICKS
3.2.1. Mitochondrial and nuclear genes

As higher-level taxonomy has progressively been unraveled by a combination of morphological
and molecular approaches, the number of mitochondrial and nuclear datasets has steadily in-
creased, providing data for the study of ticks at the intraspecific level. Some of these studies have
revealed discrepancies between morphological and molecular taxonomies. For example, mor-
phologically distinct species sometimes cannot be differentiated via molecular methods. This is
the case for some taxa belonging to the most recently evolved lineages within the Rhipicephalinae
(Zahler et al. 1997; Beati and Keirans 2001; Santos-Silva et al. 2008), in which mitochondrial in-
trogression may be detected (Rees et al. 2003). In contrast, homogeneous morphology sometimes
hides important genetic variability (Norris et al. 1996; Labruna et al. 2009a; Beati 2011). Norris et
al. (1997) used 16S rDNA analysis to establish that Ixodes neotomae and Ixodes spinipalpis are
conspecific. Taxonomic issues have also been investigated by analyzing different mitochondrial
markers within the genus Amblyomma (Nava et al. 2008; Labruna et al. 2009b; Nava et al. 2010a).
Kain et al. (1999) used cytochrome oxidase III sequences to analyze geographical genetic
structure in Ixodes pacificus and reported relatively little nucleotide diversity, particularly in ticks
from Utah, and this was therefore considered to be the most recently diverging population. Casati
et al. (2008) detected very little mitochondrial genetic diversity within European populations of
Ixodes ricinus when analyzing 5 mitochondrial markers in ticks from 6 countries. Similarly, the
analysis of ITS2 gene sequences, used to study the population structure of Ixodes holocyclus along
the eastern Australian coast, revealed that this tick is genetically very homogeneous (Shaw et al.
2002). In contrast, a number of studies on Ixodes scapularis have revealed important genetic var-
iability between southern and northern populations (Norris et al. 1996; Qiu et al. 2002). More
recently, Humphrey et al. (2010) used 16S rDNA to develop demographic hypotheses on the dis-
persal of I. scapularis from the South to the Northeast and from there to the Midwestern United
States. McLain et al. (2001) used a variable portion of the 28S rDNA to establish that within the
Ixodes ricinus/persulcatus complex, I. persulcatus is characterized by large levels of divergence,
even more significant than those reported for I. scapularis. Also, significant variability was re-
ported for populations of Amblyomma americanum from Georgia (Mixon et al. 2006) and from
Arkansas (Trout et al. 2010), which contrasts with the relative genetic homogeneity found in Am-
blyomma variegatum throughout a much larger distributional range (Beati et al. 2012), indicating
that these ticks have very different demographic histories.
3.2.2. Microsatellite markers

The use of isoenzyme profiles has now definitively been replaced by that of microsatellite
markers (Delaye et al. 1998; McCoy and Tirard 2000; Fagerberg et al. 2001; Guzinski et al. 2008;
Dharmarajan et al. 2008, 2009; Labruna et al. 2009a), whereas single nucleotide polymorphism
(SNP) analysis has not yet been implemented in this field. Given the availability of SNP informa-
tive markers in the I. scapularis genome (see Vol. 2, Chapter 3), their use for population genetics
studies of the blacklegged tick is just a matter of time.
Microsatellite markers have been successfully used for exploring reproductive strategies in Ixo-
des uriae (McCoy and Tirard 2002) and Ixodes ricinus (de Meeûs et al. 2004; Hasle et al. 2008). In
both cases, multiple paternity resulting from multiple matings was detected. Therefore, it appears
that, at least in the genus Ixodes, female ticks not only mate with different males but also success-
fully produce multiparental offspring. Because this is valid for ticks belonging to evolutionarily
distant Ixodes subgenera, one may deduce that multiple paternity is common within the genus.
Similarly, multiple paternity has also been reported in Rhipicephalus (B.) microplus (Cutullé et al.
2010). For a more detailed description of multiple paternity, see Kaufman (2008).
Host race formation has been extensively investigated in Ixodes uriae where spatial genetic
structure was found within colonies of the bird hosts (McCoy et al. 2003a, 2003b). The evolution
of host races in this tick species appears to have been rapid and fairly recent (McCoy et al. 2005;
Kempf et al. 2009a). The evolution of preferential host associations in R. (B.) microplus was also
extremely rapid (de Meeûs et al. 2010). Some populations of this cattle tick became specifically
associated with the introduced rusa deer in New Caledonia within fewer than 244 generations
and have recently been recognized as a distinct species, R. (B.) australis, together with popula-
tions from Australia and some other regions (Estrada-Peña et al. 2012). Whereas host race for-
mation is easily conceived of in nest-dwelling I. uriae and in a 1-host tick (R. [B.] microplus),
significant evidence of host-race formation in Ixodes ricinus, an exophilic and generalist tick
species, was more unexpected (Kempf et al. 2011).
An analysis of mating pairs of R. (B.) microplus showed that there is no correlation between
genetic relatedness and mating status (genetically closely related specimens are not more likely
to mate with each other than more distantly related individuals) (Chevillon et al. 2007). In
contrast, Kempf et al. (2009b) detected significant assortative mating in wild-caught pairs of
Ixodes ricinus.
The study of geographical genetic structure in I. ricinus using microsatellite markers showed
little diversity within Swiss populations and a more important structure between Swiss and
Tunisian populations (de Meeûs et al. 2002). Local diversity appeared to be sex biased, indi-
cating that sex might have an impact on dispersal ability (de Meeûs et al. 2002; Kempf et al.
2011). Geographical genetic structure and dispersal mechanisms have also been investigated in
R. (B.) microplus (southern cattle tick) in New Caledonia (Koffi et al. 2006), in Australia (Cutullé
et al. 2009), and at a worldwide scale (Labruna et al. 2009a). The latter study suggests that the
southern cattle tick is probably a complex of species.
The study of the genetic microsatellite variability in Dermacentor variabilis (American dog
tick) from raccoons collected in a relatively small geographical area revealed that spatial and
temporal structures have an important influence on encounters between hosts and ticks
(Dharmarajan et al. 2010). A genetic “ripple effect” in population dynamics was detected in an
Australian reptile tick, Bothriocroton hydrosauri; larvae were found to be more genetically re-
lated than nymphs and adults on the same host (Guzinski et al. 2009).
To date, a single microsatellite marker has been used to confirm the fact that little gene
flow is maintained between northern and southern populations of Ixodes scapularis (Rosenthal
and Spielman 2004). Additional markers have been made available through the I. scapularis
genome project, and it has become clear that, contrary to previous assumptions (Fagerberg et al.
2001), microsatellite loci are abundant in the blacklegged tick genome (see Vol. 2, Chapter 3).
3.3. MOLECULAR BARCODING AND MOLECULAR KEYS

The main purpose of barcoding of life projects is to be able to identify individual animals
at the species level by comparing a 658 bp fragment of their cytochrome c oxidase subunit
1 (CO1) gene sequences to thousands of homologous sequences inventoried in ad hoc
44 BIOLOGY OF TICKS
databases (Hebert et al. 2003). After determining the intraspecific versus interspecific CO1
sequence divergence in recognized taxa of any given group, upper and lower cut-off diver-
gence levels are established, respectively, at the interspecific and the specific level. The
intra- and interspecific cut-off levels are ideally separated by a large barcoding gap. Natu-
rally, if such a barcoding method would work for ticks, it would allow tick identification to
be undertaken by anyone without any need for formal training in tick morphological sys-
tematics. It would also facilitate the linking of unknown immature stages with the corre-
sponding recognized adult stages. Although the method has been used to successfully
identify known species, to discover new species, and to unravel cryptic speciation in some
groups of taxa (Hebert et al. 2004a, 2004b; Ward et al. 2005), it should not be applied blindly
to ticks. In general, a multilocus approach is preferable because mitochondrial genomes are
known to contain pseudogenes (nonfunctional gene fragments that have not evolved in
concert with the homologous maternally inherited mitochondrial gene). Similarly, intro-
gression through interspecific accidental hybridization also can muddle the interpretation
of barcoding data. Finally, divergence values based on pairwise comparisons do not cor-
rectly reflect the evolutionary relationships between taxa, and it is advisable to analyze phy-
logenetically aligned datasets before drawing any conclusion at the species level. The
mutation rate of mitochondrial gene sequences varies at different hierarchical levels within
the Ixodida (Black and Piesman 1994). Cut-off divergence values applicable to a basal
lineage, for example, cannot be applied to identify/differentiate ticks belonging to more
recent lineages. Natural hybridization and introgression were revealed in analyses of Hya-
lomma CO1 mitochondrial genes (Rees et al. 2003), indicating that mitochondrial gene se-
quences should be considered with the utmost caution when used for tick identification. In
conclusion, the validity of DNA barcoding in ticks needs first to be validated within each
major lineage in order to determine whether or not it can be safely applied to any unidenti-
fied tick specimen. The barcoding method should not be dismissed, however, and if the
method can be validated in some tick lineages, it should be considered as a complementary
method for the study of tick systematics.
Other methods have been developed for the molecular identification of ticks at least at the
genus level. Poucher et al. (1999) used polymerase chain reaction (PCR) restriction fragment
length polymorphism to identify several North American Ixodes species. Shone et al. (2006)
described a real-time PCR assay for distinguishing among 4 important species of vector ticks in
North America, namely, Amblyomma americanum, Dermacentor variabilis, Ixodes pacificus, and
Ixodes scapularis. Similarly, Anderson et al. (2004) developed a multiplex PCR diagnostic assay
for the identification of mostly North American immature ticks. The applicability of such tech-
niques for wider taxonomic undertaking still needs to be tested.
4. RECENT DEVELOPMENTS IN MORPHOLOGICAL

AND MOLECULAR-BASED TICK SYSTEMATICS
In this section, we review some recent developments in the morphological and molecular taxo-
nomic and phylogenetic interpretations of the various tick genera by family (for Nuttalliellidae
and soft ticks) or subfamily (for hard ticks). For ixodids, we follow the subfamilial assignments
used by Nava et al. (2009). In order to conserve space, we do not list all recently described or
redescribed ticks. However, we do mention some important papers containing new taxonomic
keys or identification guides and papers in which morphological descriptions are accompanied
by molecular data.
4.1. NUTTALLIELLIDAE
Fresh material of the only member of the genus Nuttalliella, N. namaqua, has recently been
collected (Mans et al. 2011) and its systematic position established by comparing its 18S
rDNA and 16S rDNA gene sequences to homologous sequences from Genbank. After the
inclusion of this tick species in a phylogenetic analysis, the monophyly of the Ixodida
(Klompen et al. 2000) was confirmed. However, the long-held hypothesis that the Nuttalliel-
lidae represent an intermediate evolutionary form between Ixodidae and Argasidae was not
corroborated, as N. namaqua was found to occupy a basal position within the clade Ixodida
(Mans et al. 2011).
4.2. ARGASIDAE
Unfortunately, there is currently considerable disagreement among tick workers regarding the
higher classification of argasid ticks. In general, there are 3 main schools of thought on argasid
classification, which can be treated as the Russian (Filippova 1966; Pospelova-Shtrom 1969),
American (Clifford et al. 1964; Hoogstraal 1985), and Klompen–Oliver (Klompen and Oliver
1993) schools, respectively. The basic generic assignments and rankings within these 3 systems
are shown in Table 2.1. More details on these 3 classification systems are provided in Klompen
and Oliver (1993). Although each of these systems has merit, here we follow the argasid
classification that is used in the most recent compilation of valid tick names of the world, that
of Guglielmone et al. (2010), which uses the American system. This system also provides easy
morphological identification of adult argasids to genus and is followed in the tick key included
in this chapter.
Both the American school and the work of Guglielmone et al. (2010) recognize the argasid
genera Antricola, Argas, Nothoaspis, Ornithodoros, and Otobius (Table 2.1). The other schools
also recognize some of these genera but include some additional genera as well. For example,
based mostly on phylogenetic analyses of larval characters, Klompen and Oliver (1993) assigned
most bat-associated argasids to the genus Carios, including ticks placed in Antricola or Nothoas-
pis by some other authors; Klompen and Oliver (1993) did not recognize these latter 2 genera.
Klompen et al. (1996a) also argued that biogeography and ecological specificity have had as
much (or greater) influence in the evolution of ticks as has host specificity, particularly for arga-
sids. For example, a given species of bat-associated argasid might be more likely to parasitize a
suite of bat species sharing the same habitat than it would be to specialize on parasitizing a single
species of bat.
As an example of the disparate interpretations of argasid generic assignments by different
researchers, Nava et al. (2010b) described the second known species of Nothoaspis from bat
caves in the Brazilian Amazon basin. This genus had not been described when Filippova (1966)
46 BIOLOGY OF TICKS
and Pospelova-Shtrom (1969) proposed their “Russian-school” argasid classification; further,

the Klompen–Oliver school does not recognize this genus, and Keirans (1992, 2009), Gugliel-
mone et al. (2009), and several other tick researchers have all recognized Nothoaspis as a valid
genus. Hopefully, future molecular and morphology-based studies of argasids will provide fur-
ther insights into the evolution and phylogenetics of soft ticks with the aim of better standard-
izing generic boundaries recognized by tick taxonomists. To date, the molecular phylogenetic
analysis of argasid tick subfamilies and genera has not been based on sufficiently informative
characters (16S rRNA) or does not include the most controversial taxonomic units (Nava et al.
2009, 2010b; Labruna et al. 2011). Based on 16S rDNA analysis, the genus Argas is consistently
well supported, but within the subfamily Ornithodorinae, the main taxonomic questions have
yet to be resolved. 16S rDNA gene sequences have, however, been successfully used to study the
genetic structure of Carios capensis (Ushijima et al. 2003), Ornithodoros coriaceus (Teglas et al.
2005), and Argas monachus (Mastropaolo et al. 2011). A total evidence analysis including mor-
phological and multiple molecular markers characterized by different mutation rates should
help to resolve these issues.
4.3. IXODINAE
This ixodid subfamily encompasses the Prostriata and just 1 tick genus, Ixodes, which cur-
rently includes 244 recognized species (Guglielmone et al. 2010; Apanaskevich et al. 2011).
Prostriata and Ixodes are characterized by an anal groove that extends anteriorly around the
anus. Based on morphological and/or molecular data, Klompen (1999), Klompen et al. (2000,
2007), and Shao et al. (2004) showed that Ixodes is paraphyletic. However, data reported by
Murrell et al. (2003), based on tRNA sequences, provided support for monophyly of the
Prostriata.
Because of the large number of species included in Ixodes and the wide variation in mor-
phology within this genus, Clifford et al. (1973) listed and defined 14 subgenera of Ixodes
based on morphology. With minor modifications, such as the adoption of the subgenus Ixodi-
opsis as designated by Filippova (1977), this subgeneric classification is still followed by most
tick researchers. Nevertheless, with the advent of molecular techniques, a few challenges to
Ixodes subgeneric limits have been forthcoming, as reported by Nava et al. (2009). For ex-
ample, based on molecular data, the subgenus Ixodes does not appear to be monophyletic, and
several species from the southern neotropics previously designated as Ixodes (Ixodes) evi-
dently do not belong to this subgenus either. Reevaluation of the subgenera of Ixodes using
multiple molecular markers combined with morphological data (including immature stages)
should resolve these issues.
Within the genus Ixodes, Keirans et al. (1999) defined the medically important Ixodes rici-
nus complex (also referred to as the Ixodes persulcatus complex, Ixodes ricinus/persulcatus
complex, or “ricinus-like ticks” by some authors) based on morphological characters. This
group includes vectors of the agents of several zoonotic pathogens, including those that cause
tick-borne encephalitis, Lyme disease (Lyme borreliosis), human granulocytic anaplasmosis,
and human babesiosis, mostly in the Northern Hemisphere. Despite the morphological simi-
larity of members of the I. ricinus complex, mitochondrial 16S rDNA sequence data for 11
species in this complex reported by Xu et al. (2003) suggest that this group is paraphyletic.
Clearly current morphological and molecular data are in conflict in this case, and the contra-
dicting results need to be tested with more informative molecular markers. Recent papers on
this genus include an illustrated identification guide to neotropical ticks of medical/veterinary
importance (Barros-Battesti et al. 2006), an illustrated guide to the Ixodes species of Brazil
(Onofrio et al. 2009), and an identification guide to the larval Ixodes of California (Kleinjan
and Lane 2008).
4.4. AMBLYOMMINAE
Members of the Metastriata are characterized by an anal groove that does not pass anteriorly
around the anus; this group includes all of the ixodid genera except Ixodes (i.e., the Prostriata).
The first metastriate subfamily we consider is Amblyomminae, which includes the genus Ambly-
omma as currently recognized (Guglielmone et al. 2010). Prior to 2002, Aponomma was recog-
nized as a separate genus, distinct from Amblyomma. However, most species formerly assigned
to Aponomma are currently treated as members of the genus Amblyomma, although the subge-
neric assignment (Aponomma) is typically retained (Klompen et al. 2002; Guglielmone et al.
2010). Members of the former genus Aponomma were previously distinguished from members
of Amblyomma by the lack of eyes on their scutum, a character that is evidently not useful for
generic assignment within Amblyomminae.
Three species formerly assigned to Aponomma remain systematically problematic and
await confirmation of their generic placement. These ticks are currently treated as Ambly-
omma elaphense (a parasite of the Trans-Pecos rat snake in the western United States), Am-
blyomma sphenodonti (a parasite of the tuatara in New Zealand), and Amblyomma transversale
(a parasite of Afrotropical reptiles) (Nava et al. 2009). Miller et al. (2007) showed that 18S
rRNA sequences of A. sphenodonti do not place this species in a monophyletic clade with
either Amblyomma or Bothriocroton, suggesting that it should be placed in a separate genus
and possibly in its own subfamily. Recent systematic works on this genus include an illus-
trated identification guide to neotropical ticks of medical/veterinary importance (Barros-
Battesti et al. 2006), an analysis of the molecular taxonomy and phylogenetics of neotropical
Amblyomma species (Marrelli et al. 2007), and a morphological (scanning electron micro-
graph) identification guide to the nymphs of Brazilian Amblyomma species (Martins et al.
2010).
4.5. BOTHRIOCROTONINAE
Dobson and Barker (1999) first noted that the former genus Aponomma was paraphyletic based
on 18S rDNA sequences, thereby also showing that Amblyomminae, as recognized at that time,
was paraphyletic. Based on 18S nuclear rDNA sequences, cytogenetics, host associations, and
morphological characters (especially of larvae), Klompen et al. (2002) also recognized poly-
phyly in the (former) genus Aponomma and reassigned Australian species from this group by
elevating them to their own genus, Bothriocroton. Klompen et al. (2002) also erected the sub-
family Bothriocrotoninae to accommodate Bothriocroton. Bothriocroton was recognized as a
subgenus of Aponomma when it was described by Keirans et al. (1994). Based on 18S rDNA
partial sequences and morphology, Beati et al. (2008) also reassigned Aponomma oudemansi, an
48 BIOLOGY OF TICKS
ectoparasite of the western long-beaked echidna in New Guinea, to the genus Bothriocroton,
thereby adding New Guinea to the known distribution of this genus and subfamily.
4.6. HAEMAPHYSALINAE
This speciose subfamily includes just 1 genus, Haemaphysalis, with 166 currently recognized
species. Although the genus Haemaphysalis is almost cosmopolitan in distribution, it is most
speciose in southeast Asia and is poorly represented in the Americas, where only 3 species occur.
Members of this genus were extensively studied by the late Harry Hoogstraal and colleagues,
who described many new taxa from the 1960s to the 1980s. Since that time, relatively little atten-
tion has been given to taxonomic studies for this genus.
Phylogenetically, the relationships of Haemaphysalinae to the other ixodid subfamilies and
of Haemaphysalis to other ixodid genera remain incompletely resolved (Klompen et al. 1997,
2000). Based on certain shared morphological characters such as leg and palpal spurs, Hoog-
straal and Aeschlimann (1982) placed the Haemaphysalinae on a common branch with the Hya-
lomminae and Rhipicephalinae. Future studies analyzing multiple genes in combination with
morphological reinterpretations should help to resolve the relationships of Haemaphysalis and
Haemaphysalinae with other ixodid taxa.
4.7. RHIPICEPHALINAE
This is a large subfamily that includes the genera Anomalohimalaya, Cosmiomma, Dermacentor
(including the subgenus Anocentor), Hyalomma, Margaropus, Nosomma, Rhipicentor, and Rhipi-
cephalus (including the subgenus Boophilus). Little recent systematic work has been completed
for Anomalohimalaya, Cosmiomma, Margaropus, or Rhipicentor; therefore, our discussion of
this subfamily will address the other genera. The Indian rhipicephaline genus Nosomma was
long considered as monotypic; a second species was described in 2007, but because the original
description was poor, Guglielmone et al. (2010) doubt its validity.
The almost cosmopolitan genus Dermacentor is most speciose in the northern hemisphere
and consists of up to 34 currently recognized species as listed and discussed by Guglielmone
et al. (2010). This genus includes important vectors of zoonotic rickettsial agents such as Rickettsia
rickettsii, which causes Rocky Mountain spotted fever and is transmitted mainly by D. andersoni
and D. variabilis, but also by a Rhipicephalus of the R. sanguineus complex (Eremeeva et al. 2011)
in North America. Based on morphology, Filippova (1994) included Dermacentor in the sub-
family Amblyomminae and tribe Amblyommini; within this tribe, she recognized the subtribe
Dermacentorini, consisting of the genera Dermacentor and Rhipicentor. Conversely, Hoogstraal
and Aeschlimann (1982), Horak et al. (2002), and Barker and Murrell (2004) all placed Derma-
centor within the Rhipicephalinae.
One area of contention with respect to the classification of Dermacentor has been the place-
ment of the monotypic Anocentor. Adults of Anocentor nitens have some unique morphological
characters, such as a small number of large goblets in the spiracular plates, whereas larvae share
certain morphological characters with larvae of Dermacentor. Although Anocentor has been
recognized as a separate genus in the past by several authors, analysis of 16S rDNA sequences by
Crosbie et al. (1998) showed that A. nitens and Dermacentor albipictus are closely related. Fur-
ther, combined morphological and molecular phylogenetic analyses by Murrell et al. (2001) re-
vealed that Anocentor is embedded in the Dermacentor clade. Thus, based on current evidence,
Anocentor should be included within the genus Dermacentor.
The Old World genus Hyalomma includes several species that have medical importance
as vectors of Crimean-Congo hemorrhagic fever virus in parts of Europe, Asia, and Africa.
Also, some Hyalomma species can be pests of livestock animals such as camels, cattle, sheep,
and goats. Previously, the genus Hyalomma was considered to constitute a separate ixodid
subfamily, the Hyalomminae, which was considered primitive (because of elongated mouth-
parts, i.e., the palps and hypostome) and basal to the Rhipicephalinae (Hoogstraal and
Aeschlimann 1982). However, several molecular analyses strongly support the placement of
the Hyalomminae within the Rhipicephalinae (Black and Piesman 1994; Black et al. 1997;
Klompen et al. 2000). Many members of this genus present problems for tick taxonomists
because of the significant amount of intraspecfic morphological variation. Molecular sys-
tematic studies directed at this genus should be forthcoming and might enhance and so-
lidify knowledge of species limits. A molecular study of the cytochrome oxidase I gene
sequences (Rees et al. 2003) revealed introgression or natural hybridization within Hya-
lomma marginatum rufipes, Hyalomma truncatum, and Hyalomma dromedari, ticks that are
arguably easy to differentiate morphologically within this genus. Therefore, it could be con-
ceived that these ticks never differentiated sufficiently from their common ancestor to be-
come separate species, or that a vicariant event separated them into morphologically
distinct species, after which their geographical distributions re-converged, promoting
mating along hybrid zones.
A comprehensive guide to the taxonomy, identification, distribution, and host associations
of the approximately 77 species of ticks belonging to the subgenus Rhipicephalus (Rhipicepha-
lus) is available (Walker et al. 2000). Since the publication of that work, the 6 currently recog-
nized species in the former genus Boophilus have been transferred to Rhipicephalus and are now
treated as belonging to the subgenus Rhipicephalus (Boophilus) as followed by Guglielmone
et al. (2010) and others. Guglielmone et al. (2010) therefore recognize 82 valid species of Rhipi-
cephalus, 77 in the subgenus Rhipicephalus and 5 in the subgenus Boophilus (note that a sixth
species has recently been reinstated and redescribed by Estrada-Peña et al. [2012]). Based on
DNA sequence data of CO1, 12S rDNA, 16S rDNA, and ITS2 rDNA, Rhipicephalus becomes
paraphyletic when Boophilus taxa are included in the analysis (Murrell et al. 2000; Beati and
Keirans 2001). Also, based on 16S rDNA sequences (Mangold et al. 1998b) and total evidence
analysis (Beati and Keirans 2001), Rhipicephalus of the R. evertsi and R. pravus groups cluster
with R. (B.) annulatus and R. (B.) microplus and not with other Rhipicephalus (Rhipicephalus)
species included in their analyses. Some morphological characters shared by members of these
subgenera (Murrell et al. 2000; Beati and Keirans 2001), including the presence of projecting
adanal and accessory plates in males, validate the synonymy. For these reasons, it was taxonom-
ically more parsimonious to synonymize Boophilus with Rhipicephalus (Murrell and Barker
2003) than to create a number of new subgenera within Rhipicephalus. Because of the huge
amount of previous literature and the veterinary importance of ticks treated as Boophilus, this
name is retained as a subgeneric rank.
50 BIOLOGY OF TICKS
Several species in the genus Rhipicephalus have considerable economic importance as ecto-
parasites and pathogen vectors for dogs (the brown dog tick, R. (R.) sanguineus), cattle (all 6
species in the subgenus Boophilus), and other mammals, especially ungulates. Some of these
species have widespread distributions across much of the globe, such as R. sanguineus, which
has been stated to be the most widely distributed species of tick in the world; the brown ear tick,
R. appendiculatus, in Africa; and the cattle ticks R. (B.) annulatus and R. (B.) microplus. How-
ever, some recent molecular data suggest that the biogeographical and taxonomic relationships
within these taxa (and some others) should be reevaluated. Based on ITS2 sequence analysis,
Zahler et al. (1997) provided some evidence for the conspecificity of R. sanguineus and R. turani-
cus and of R. pumilio and R. rossicus, which was later partially substantiated by total evidence
analysis of 12S rDNA and morphology (Beati and Keirans 2001). Long thought to be confined to
the Old World, ticks identifiable as R. turanicus based on 12S rDNA sequences have been re-
ported from the New World, so that now the term “R. sanguineus-like ticks” is sometimes ap-
plied to ticks within this group (Szabó et al. 2005; Moraes-Filho et al. 2011). Similarly, based on
microsatellite data, intriguing faunal connections are apparent between various world regions
for R. (B.) microplus, and some analyses suggest that populations in Australia are distinct from
populations in the Americas and Africa (Labruna et al. 2009a). Also, based on 12S rDNA and
ITS2 sequences, the morphologically similar Afrotropical species R. appendiculatus and R. zam-
beziensis have been shown to be distinct species (Mtambo et al. 2006), a finding that is also sup-
ported by enzyme electrophoresis data reported by other researchers. Further molecular analyses
combined with morphological data should serve to clarify these issues and elucidate other spe-
cies relationships within the genus Rhipicephalus as it is currently treated.
With the explosive and pervasive advent of molecular techniques in biology, we anticipate in-
creased research activity in the molecular biology, population genetics, genomic analysis, and
DNA gene sequence–based identification of ticks in the future. Nevertheless, we also anticipate
that additional important user-friendly hard copy and web-based guides to morphology-based
tick identification will be forthcoming, especially for taxa, geographical regions, and tick stages
(larvae and nymphs) that have previously received little attention. One possible future applica-
tion of molecular tick systematic studies would be the development of commercial tick identifi-
cation kits to evaluate risks posed by tick-borne pathogens transmitted by certain tick taxa.
Overall, combined morphological and molecular analyses of tick systematics should continue to
prosper and provide answers to questions concerning systematics and tick-borne disease
ecology and epidemiology.
ACKNOWLEDGMENTS
This work was supported by NSF Grant Nos. DEB-REVSYS-1026146 and EF0914390. We thank
Daniel Sonenshine for making several of the figures available for reproduction and Daniel
Sonenshine and R. Michael Roe for editorial guidance.
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C H A P T E R 3
LIFE CYCLES AND NATURAL

HISTORY OF TICKS
DM I TRY A. APANASKEVICH AND JAMES H. OLIVER, J R.
1. INTRODUCTION
The life cycle of ticks consists of 4 developmental stages, the egg and 3 active parasitic stages
(larva, nymph, and adult [male and female]). Below we describe the life cycles of ticks of the
families Ixodidae and Argasidae separately because there are considerable differences in their
life cycle strategies. The third family of ticks, the Nuttalliellidae, consists of only 1 described
species, Nuttalliella namaqua, and little is known about its life cycle.
2. IXODIDAE
The life cycle of all hard ticks (family Ixodidae) consists of the egg and 3 active stages (larva,
single nymph, and adult). Each active stage feeds only once in its life, although females forcibly
detached from their hosts are able to reattach and continue feeding. For instance, females of
Ixodes ricinus were able to reattach to another host and finish their blood meal after 5 days of
feeding and attaining a weight of 50 mg; Dermacentor reticulatus and Hyalomma asiaticum were
able to do this after 6 or 7 days of feeding at weights of 122 or 206 mg, respectively. However,
after reaching a critical mass, detached females were unable to reattach to the host, although
they were able to produce some eggs. It is interesting to note that after a particular time period
of feeding and the consumption of a certain amount of blood, detached females were able to
either reattach or lay eggs. For example, Ixodes ricinus females removed from the host that had
attained a weight of 23 to 50 mg on a fifth day of feeding were able to either produce eggs or
reattach to the host (Balashov 1998).
Another obligatory factor determining a female’s ability to feed to completion is mating.
Normally, females cannot feed to repletion without mating (Pappas and Oliver 1971). A few
60 BIOLOGY OF TICKS
species are parthenogenetic (i.e., the females reproduce without breeding with males) (Oliver
1971, 1981), but among the vast majority of ixodid ticks under normal circumstances, mated fe-
males consume large amounts of blood and increase their body mass to up to 100 times their
prefeeding weight. After attaining complete engorgement, females drop off their host and lay
their eggs in suitable microenvironments such as burrows, crevices, or leaf litter.
The number of eggs varies depending on the tick species and the degree of engorgement. The
average number of eggs laid by ixodid females is a few thousand. The maximum recorded egg
batch is 36,206 in Amblyomma variegatum (Dipeolu and Ogunji 1980), whereas females of Hae-
maphysalis inermis usually produce a low number of eggs (a few hundred) (Filippova 1997).
Partially engorged females can lay eggs as well after reaching a critical weight, but in much lower
numbers than those achieving full engorgement. For example, after 6 days of feeding, females of
Hyalomma asiaticum attain an average body mass of 109 to 136 mg and lay 69 to 214 eggs,
whereas after 9 days of feeding they can reach an average of 1,711 mg and lay 12,263 to 21,204 eggs
(Balashov 1998). After depositing the eggs, females stay alive for several days and then die. Thus,
the ixodid ticks have only 1 gonotrophic cycle. Depending on whether larvae and nymphs molt
to the nymphal or adult stage, respectively, off or on the host, ixodid ticks are classified as 3-, 2-,
or 1-host ticks. However, it should be noted that some species can behave as 3-, 2-, or even 1-host
ticks depending on various factors discussed below in this chapter.
2.1. THREE-HOST LIFE CYCLES

The vast majority of ixodid ticks are characterized by a 3-host life cycle (Fig. 3.1). In a 3-host life
cycle, the larvae drop off from the host after feeding and molt to the nymphal stage. Subse-
quently, the unfed nymphs quest for a host and may attach to the same or different hosts, feed,
drop off, and molt to the adult stage. The adults then find their host; then females mate and feed,
drop off the host, lay eggs, and die. All ixodid ticks have only a single gonotrophic cycle; that is,
engorged, mated females lay all of their eggs continuously over a period of several weeks, after
which the exhausted females die.
Among adults, there is considerable difference between the Metastriata and the Prostriata.
Males of most Metastriata need to feed on hosts in order to promote the maturation of their
sperm (Oliver 1982), but there are exceptions, such as Bothriocroton hydrosauri and B. concolor
(Oliver and Stone 1983). Mating and copulation in most Metastriata occur on the host after the
male feeds. The amount of blood meal consumed by the males is considerably less than that
consumed by the females, and the body mass of males increases only about 1.5 to 2 times. In the
majority of the prostriate ticks, males do not require a blood meal and are able to copulate with
the females off or on the host. The male hypostome of many Ixodes species (Prostriata) may be
reduced, especially in its dentitions. The longevity of Ixodes males is much shorter than that of
the male metastriate ticks.
2.1.1. Representative example of 3-host ticks among ixodids

Dermacentor andersoni is a widely distributed tick species in western North America; adults of
this species parasitize mostly larger mammals such as ungulates, whereas larvae and nymphs feed
on rodents and leporids. In laboratory experiments at 21°C –26°C, adult Dermacentor andersoni
Lif e Cycles and Natural History 61
FIGURE 3.1: Three-host life cycle of Ixodidae ticks.
feeding on rabbits needed 7 to 9 days after nymphal eclosion before they were ready to feed. The
duration of feeding lasted 7 to 12 days. Following repletion and drop off, the preoviposition pe-
riod was 2 to 6 days. Oviposition continued for 8 to 22 days, and the incubation period of the eggs
needed for hatching was 24 to 28 days. Larvae were ready to feed after 14 to 21 days; larvae fed for
3 to 6 days, after which they dropped from the host. Larvae molted to the nymphal stage after 16
to 27 days, and nymphs were ready to feed after 7 to 10 days. The duration of nymphal feeding was
6 to 8 days, after which they dropped off the host; nymphs molted to the adult stage after 18 to 34
days (Loomis 1961). Under these controlled conditions, the generation time from freshly depos-
ited egg to adult ranged from 88 to 134 days. However, much longer development periods may be
expected under natural conditions.
2.1.2. Species distribution of the 3-host life cycle

Most, if not all, species of the genera Amblyomma, Anomalohimalaya, Bothriocroton, Hae-
maphysalis, and Ixodes are obligate 3-host ticks. The majority of studied species of Rhipi-
cephalus and Dermacentor are also obligate 3-host ticks. Many Hyalomma species are
obligate 3-host ticks, but several species can use 3 hosts facultatively and usually develop on
2 hosts or 1 host.
62 BIOLOGY OF TICKS
FIGURE 3.2: Two-host life cycle of Ixodidae ticks.
2.2. TWO- AND 1-HOST LIFE CYCLES

A number of ixodid ticks from the Metastriata group are characterized by 2- or 1-host life cycles
(Figs. 3.2, 3.3). In the case of the 2-host life cycle, engorged larvae remain and molt to nymphs
on the host; thereafter, nymphs feed, engorge, drop off the host, and molt to the adult stage in
the natural environment. In the case of the 1-host life cycle, engorged larvae and nymphs remain
on the same host, and only engorged females drop off the host.
2.2.1. Examples of 2-host and 1-host life cycles in ixodids

Hyalomma isaaci, as are other species of the Hyalomma marginatum group, is an obligate 2-host
tick. The major hosts of the adults of this species are various ungulates, whereas larvae and
nymphs parasitize hares and birds. In laboratory experiments on Hyalomma isaaci, when
feeding on various mammals, adults required a 7- to 13-day prefeeding period before they were
ready to feed. The duration of feeding was 6 to 24 days, a preoviposition period was 3 to 10 days
at 30°C, oviposition continued for 3 to 31 days at 30°C, and the egg incubation period needed for
hatching was 24 to 33 days. The larvae were ready to feed after 7 days and, after engorgement,
molted on the host body 7 to 8 days after the attachment of unfed larvae. The nymphs reattached
to the host within 6 to 12 hours. The engorged nymphs dropped off the host 13 to 25 days after
the release of the unfed larvae; nymphs molted to adults within 14 to 20 days at 30°C (Geevarghese
FIGURE 3.3: One-host life cycle of Ixodidae ticks.
and Dhanda 1987). Dermacentor nitens (= Anocentor nitens) is an obligate 1-host tick species
parasitizing mainly horses in southern North America, Central America, and northern parts of
South America. The length of the parasitic period of this species feeding on horses under labo-
ratory conditions was 25 days; preoviposition and incubation periods were 4 and 21 days on
average, respectively (Drummond et al. 1969).
2.2.2. Species distribution of different life cycle types in ixodid ticks

Some species of Rhipicephalus (e.g., R. bursa and R. evertsi), Dermacentor (e.g., D. auratus), and
Hyalomma (e.g., H. marginatum, H. rufipes, H. isaaci, H. turanicum, H. anatolicum, H. excava-
tum, H. dromedarii, H. scupense, and others) are obligatory or facultative 2-host ticks. Several
species of the genus Rhipicephalus (R. microplus and other species from the formerly valid Bo-
ophilus genus), Dermacentor (D. albipictus and D. nitens), and Margaropus (M. whintemi) are
characterized by an obligate 1-host life cycle. Two species of Hyalomma (H. scupense and
H. dromedarii) are facultative 1-host ticks. The major hosts of all 1-host ticks are larger ungulates.
Many 2-host tick species can have different hosts for the adult and immature stages, whereas
others can use the same host for all life stages. Adults and the immature stages of Rhipicephalus
bursa, R. evertsi, H. anatolicum, and H. scupense feed on the same hosts, which are larger
64 BIOLOGY OF TICKS
ungulates. Adults of Hyalomma marginatum, H. rufipes, and H. excavatum parasitize larger un-
gulates also, but their immature stages use smaller mammals (e.g., hares, rodents, hedgehogs,
etc.) and birds as their hosts. It is interesting to note that many species of Hyalomma are unique
in their ability to develop by utilizing 2 or even 3 hosts. For example, H. anatolicum and H. ex-
cavatum can develop via either 3- or 2-host life cycles, H. scupense can develop via 2- or 1-host
life cycles, and H. dromedarii can develop by any of the 3 types of life cycles.
Climatic conditions (mostly temperature) and the type of host are the main factors influ-
encing how many hosts these ticks will use for their development. For instance, Hyalomma drom-
edarii in Turkmenistan can develop via 3- or 2-host cycles on camels in the summer, whereas in
the spring and autumn, when temperatures are lower, they are 1-host ticks (Berdyev 1980). Similar
observations are recorded for H. anatolicum, for which the probability of a 3-host life cycle is
raised with increasing temperature (Balashov 1972). In the case of H. scupense (= H. detritum), the
northern populations of the species are characterized by a 1-host life cycle, whereas southern
populations have a 2-host life cycle. This was the main criterion underlying the consideration of
southern populations as an independent species, H. detritum (Apanaskevich et al. 2010). Appar-
ently, the type of host also has a part in determining the type of life cycle. When 1- or 2-host ticks
feed on unusual hosts, their life cycle can shift to the 3-host cycle (Berdyev 1980; Balashov 1998).
2.3. EVOLUTION OF LIFE CYCLES IN THE HARD TICKS

It is considered that the 3-host life cycle is ancestral for the ixodid ticks. Indirectly, this assump-
tion is supported by the fact that all studied species of Ixodes and basal lineages of the Meta-
striata, such as the genera Haemaphysalis and Amblyomma, are exclusively 3-host ticks. Two- and
1-host life cycles appeared independently in several genera of the Rhipicephaline lineage only.
One of the possible factors that might have led to the adoption of 2- and 1-host cycles is the
parasitism of ticks on highly mobile mammals such as ungulates. This hypothesis may be true
for 1-host tick species that are specific to ungulates, but it does not adequately explain the advan-
tages of 2-host ticks whose larvae and nymphs parasitize small mammals and birds. In any case,
tick species with 2- and 1-host life cycles have more chances to successfully finish their develop-
ment because the time spent host questing is shortened, and thus the chance of death due to lack
of a suitable host is decreased. Another factor that possibly influenced the appearance of short-
ened life cycles is the adaptation of some species to colder climates. Some 1-host species even
adapted to overwinter on the host’s body (e.g., H. scupense) (Balashov 1998). Another example
is the winter tick, Dermacentor albipictus, the larvae and nymphs of which develop on moose
and other large ungulates during the winter season (Drew and Samuel 1989).
3. ARGASIDAE
3.1. DESCRIPTION OF NUMEROUS LIFE CYCLE VARIATIONS

IN ARGASIDS (WITH EXAMPLES)
Life cycles of the soft ticks (family Argasidae) are more variable than those of Ixodidae. All argasid
ticks have the same life cycle stages (i.e., egg, larva, nymph, and adult) (males and females)
(Fig. 3.4). However, the number of nymphal instars in argasid ticks is always more than 1, and this
FIGURE 3.4: The marked misspelling has been corrected, but a space has been introduced before the
hyphen in “Multi-Host” and should be deleted.
number is not constant in many species. For example, species of the genus Otobius have only 2
nymphal instars, whereas Ornithodoros (Alveonasus) lahorensis has 3 obligatory nymphal instars.
In some species of Ornithodoros, the number of nymphal instars varies from 3 to 8. The number
of instars depends on various factors. First of all, this number can be due to the species, degree of
engorgement of previous stages or instars, and temperature. For instance, at 20°C, 37% of Argas
persicus stage II nymphs molt to adults, but the rest of these nymphs molt to stage III nymphs;
then 17% of these stage III nymphs molt to stage IV nymphs, and then to females. At 25°C, 90%
of stage II nymphs molt to males and females, and only 10% molt to stage III nymphs, which sub-
sequently molt to adults. At 30°C, 3% of ticks had only 1 nymphal instar, most of the specimens
had 2 nymphal instars, and only 1% had 3 instars (Filippova 1966). For the majority of argasid
ticks, 1 blood meal is enough for the development of larvae and nymphs to the next stage or instar.
However, there are numerous exceptions. In some species of Ornithodoros, larvae do not feed at
all and molt to the nymphal stage using resources accumulated in the eggs, and in other Orni-
thodoros species (e.g., O. capensis), stage I nymphs do not take blood meals and directly molt to
the next stage using nutrients accumulated during the larval stage. Adults of the majority of soft
ticks can feed several times (up to 6) and oviposit after each blood meal (i.e., they have several
gonotrophic cycles). The number of eggs deposited by each female usually is no more than several
hundred. Again, there are deviations from this rule. Adults of the genus Otobius do not feed at all.
Females of some Ornithodoros species (e.g., O. lahorensis) may lay the first or even a second batch
66 BIOLOGY OF TICKS
of eggs without prior feeding. Remarkably, unfed females of O. papillipes can oviposit up to 5.5
years after molting to the adult stage (Filippova 1966). All stages of most argasid tick species drop
off the host after engorgement (i.e., they use multihost developmental patterns analogous to the
3-host life cycle of the ixodid ticks). Again, there are some exceptions, such as O. (Alveonasus)
lahorensis, which usually has 3 nymphal instars and a life cycle similar to that of the 2-host ixo-
dids. Unfed larvae attack the host, and only engorged stage III nymphs drop off the host and molt
to the adult stage in the natural environment. Otobius megnini has a 1-host life cycle, with the
larva and 2 nymphal instars feeding on a single host (Hoogstraal 1985). This life cycle is almost
analogous to that of 1-host ixodid ticks, with the exception that the adults do not feed.
Another biological characteristic of argasid ticks that makes them drastically different from the
ixodid ticks is the time spent feeding. All active stages of the majority of soft ticks feed rapidly (15
to 30 minutes). Nevertheless, larvae of many Ornithodoros species (e.g., Ornithodoros amblus) feed
similarly to ixodid ticks for a period of several days. Females lay considerably fewer eggs (usually a
few hundred). The females compensate for this low number of eggs by having several gonotrophic
cycles. Females of the 1-host argasid O. megnini deposit as many as 1,500 eggs (Hoogstraal 1985).
3.2. EVOLUTION OF LIFE CYCLES IN SOFT TICKS

Because the phylogenetic relationships among the argasid ticks are still far from being resolved,
it is difficult to draw definite evolutionary trends regarding their life cycles. Apparently, originally
all stages of argasid ticks required blood meals. The examples described above of nonfeeding
stages or instars are probably secondary. A possible explanation for the appearance of the non-
feeding stages or instars is the need to decrease dependence on the host. As in ixodid ticks, some
of the argasid ticks shorten their life cycle by becoming 2- or even 1-host ticks, thus guaranteeing
blood meals for most of the stages. Another direction in the evolution of the life cycles in argasid
ticks is to increase the number of nymphal instars. As noted previously, this number is highly
adaptable. In suitable climatic conditions and with full engorgement of the preceding stages, the
cycle becomes shorter. In unfavorable circumstances, the number of nymphal instars can be
greater, which guarantees the appearance of normally developed adults having maximal egg pro-
duction, a greater number of gonotrophic cycles, and greater longevity.
4. OVERVIEW OF HOST-SEEKING BEHAVIOR
The host-seeking behavior of ticks is a complex of specific behavioral reactions that contribute
to finding a host. Ticks become aggressive (i.e., react to host stimuli after a short period of post-
molting development). The periods of questing for hosts are seasonally related and can be inter-
rupted by unfavorable conditions.
4.1. DIFFERENT TYPES OF QUESTING STRATEGIES IN TICKS

Generally, ticks can be divided into 2 major groups according to the locations in which they
quest for their hosts, molt, and lay eggs. There are nidicolous (nest or burrow) and non-
nidicolous (so-called pasture) ticks. However, it should be noted that in many cases, there is no
clear border between these 2 types. In the case of nidicolous ticks such as Ixodes crenulatus and
I. lividus, their contact with the host occurs in the host’s burrow or nest. These ticks rarely, if
ever, are found questing outside of the host’s dwelling. Most of the argasid ticks are nidicolous,
with many species feeding on bats in caves. The day–night drop-off rhythms of nidicolous ticks
are strictly coordinated with the activity of the host. The ticks will drop off only in the shelter
occupied by the host (see below). All stages of most of the nidicolous ticks feed on the same type
of host. Often the nidicolous lifestyle leads to host specificity, as it does to some degree in many
species of ixodid ticks. Interestingly, synchronization between the life cycles of ticks and their
host is observed in Ixodes lividus, a parasite of the sand martin (Glashchinskaya-Babenko 1956).
Unfed larvae feed on adult sand martins that have recently arrived from their overwintering
sites. Unfed nymphs then emerge by the time hatchling birds appear and use these hatchlings as
hosts. Female adults also feed on these growing hatchlings, and unfed larvae appear by the time
the birds leave their nest for migration to their southern overwintering regions.
The “pasture” questing ixodid ticks seek their hosts in open spaces of their forest, brushland,
or grassland habitats. The general behavioral strategy of such ixodid ticks is to climb vegetation,
spread their first pair of legs containing their sensory organs (Haller’s organ), and wait for the
host. Typical examples of ticks with this behavior are Ixodes ricinus, I. scapularis, and I. persul-
catus. In these species, moisture and temperature are the main factors determining the ticks’
daily rhythm of climbing vegetation and descending back to the leaf litter to restore lost water.
The height to which ticks climb depends mainly on 2 major factors: moisture and the size of the
host. Thus adults and immature stages of ticks feeding on large ungulates might climb more than
1 m in height, whereas ticks feeding on small hosts climb only several centimeters above the
ground or quest for their host on the soil surface.
Many species of Hyalomma ticks (e.g., H. asiaticum) that live in harsh arid environments are
characterized by mixed questing strategies. Their larvae and nymphs parasitize burrowing ani-
mals such as gerbils and thus become nidicolous parasites. But the adults who feed on large
ungulates such as camels or cattle behave as pasture ticks (non-nidicolous). The tactic of the
adults of these Hyalomma species in the hot desert environment is usually active pursuit of their
host, which they identify by sight and chemoreception using Haller’s organ. According to Balas-
hov (1960), in April, when daytime temperatures are about 25°C, the adults of Hyalomma ac-
tively search for suitable hosts throughout the day. At the end of April and in the beginning of
May, when temperatures rise higher than 25°C and the humidity drops, ticks are active only in
the morning and evening. The speed at which the adults of Hyalomma can run is remarkable. At
24°C to 26°C, unfed adult H. asiaticum can run toward a host at a speed of 1 m/min (Balashov
1998). Another interesting modification of the pasture questing strategy in some 1- and 2-host
Hyalomma that parasitize domestic cattle is their questing for hosts in stables. This mechanism
provides a protected environment and always-available hosts. This strategy somewhat resembles
that of the nidicolous ticks, but on a macro scale.
4.2. EVOLUTION OF QUESTING STRATEGIES

With regard to its evolutionary origin, some scientists believe that the pasture type of questing
is basal for the ixodid ticks. The nidicolous type of life strategy is thought to be derived and ap-
parently appeared independently in various groups of ticks (Balashov 1998). According to Bal-
ashov (1998), this is supported by the fact that the morphologically basal subgenera of Ixodes
68 BIOLOGY OF TICKS
behave as pasture parasites. Unfortunately, phylogenetic relationships among the groups of Ixo-
des are far from being resolved and therefore do not strongly support this hypothesis. Filippova
(1977) hypothesized that one of the most primitive questing strategies occurs in Ixodes triangu-
liceps. Adults of I. trianguliceps have limited vertical migration activities between soil, litter, and
vegetation (up to 10 cm). Their immature stages apparently quest only in leaf litter. This small
vertical range of movement correlates with the hosts of this tick, such as shrews and voles. It is
easy to imagine that from this type of questing in leaf litter, some other species evolved to invade
burrows, and others for questing at the tips of grasses. Apparently, the “burrow-pasture” and
“stable” parasitism behaviors of the Hyalomma described above are adaptations to unfavorable
arid environments.
4.3. MORPHOLOGICAL ADAPTATIONS IN RELATION

TO QUESTING STRATEGIES
The most prominent morphological adaptation used to identify a host is Haller’s organ (see
Chapter 13 for a detailed description of this structure). This organ contains capsules with mul-
tiple sensory sensillae composed of distant chemo- and thermoreceptors. Haller’s organ is situ-
ated on the dorsal side of the first pair of legs on the tarsi. In the questing position, ticks extend
the reach of the first pair of legs. The strongest stimulus for this behavior is direct contact with
the host. This promotes an immediate reaction to attach with their claws. If stimuli released
by the host are consistent but there is no direct contact between the host and the tick for an ex-
tended period of time, the tick can descend off the grass and walk to the host. The adults of
Hyalomma ticks that can actively quest for their hosts have well-developed eyes. They are able to
identify objects with angle sizes of 15° to 20° and react to different intensities of light. These ticks
start to run in a zigzagging pattern toward a potential host that has been identified visually as
well as by olfactory cues, and they walk or run with the first pair of legs extended. At distances
of 1 to 2 m, Haller’s organ chemoreceptors are able to locate the host precisely, and ticks run
straight to the host (Leonovich 1986).
5. OVERVIEW OF F EEDING BEHAVIOR
5.1. DURATION OF FEEDING AND FACTORS INFLUENCING DURATION

One of the essential behavioral characteristics providing easier access to the host, especially for
the nidicolous ticks, is their day–night rhythms of drop-off. The majority of ixodid tick larvae
feed during a 3- to 6-day period, nymphs during a 3- to 10-day period, and females during a 6- to
12-day period. The exceptions are several species of Haemaphysalis (e.g., H. inermis) larvae that
can complete feeding in 2 to 3 hours (Nuttall and Warburton 1915). The duration of feeding of
1- or 2-host ticks is generally the same as that of 3-host ticks. The extension of the feeding period
of ixodid females is dependent on whether they copulate with a male or not. Unmated females
can stay attached to hosts for up to several weeks. The longevity of feeding also can depend on
the host. For example, ticks feed longer on reptile hosts; larvae of Amblyomma marmoreum fed
for 30 days, nymphs for 51 days, and females for 60 days on tortoises (Norval 1975). The longevity
of feeding on reptiles can depend on the temperature; for example, at 20°C, larvae of Ixodes rici-
nus complete feeding on lizards in 8 to 12 days, and nymphs in 11 to 15 days; at 25°C, larvae
needed 6 to 11 days and nymphs required 6 to 10 days; and at 30°C, larvae engorged in 3 to 4 days
and nymphs in 4 to 7 days (Balashov 1998). Other factors such as locality on the host and im-
mune status of the host are also important for the duration of feeding. The majority of the argasid
ticks, with few exceptions, are fast feeders as described earlier in this chapter.
5.2. DAY–NIGHT RHYTHMS OF DROP-OFF AND

LOCATION ON HOST’S BODY
Fed ixodid ticks have limited mobility, so the place of drop-off from their host is of utmost impor-
tance. The mechanism of day–night rhythms of drop-off allows fed ticks to detach in favorable
natural environments for successful development, oviposition, and embryogenesis, as well as for a
location appropriate for hosts. For instance, drop-off of engorged female Ixodes ricinus occurs when
their hosts are active. The day–night rhythm can change depending on the circumstances. For ex-
ample, when cattle graze on pastures during the day, fed females drop at that time. If cattle grazing
activity is shifted to the night, the ticks also change their drop-off rhythm and fall during the scoto-
phase (Balashov 1954). Generally, species of ticks with pasture questing behavior drop off their host
during the time of their host’s activity. In nidicolous ticks, day–night rhythm depends on whether
their hosts are in their nests. The drop-off rhythms of these ticks are regulated by the activity of host
behavior as well. If the host is active at night (i.e., leaves its shelter), then ticks drop off during the
day when the host is in the nest at rest. The opposite is true if hosts are active during the day, in
which case the ticks drop at night. For example, engorged Ixodes lividus, a parasite of sand martins,
drop off during the night when all the birds are in their burrows (Glashchinskaya-Babenko 1956).
Presumably, the photoperiod is responsible for the regulation of such day–night rhythms of drop-
off. It is also interesting to note that many 2- or 1-host ticks (e.g., H. scupense) that feed on the same
host, such as cattle, in hot arid areas at all stages drop off their hosts in stables at night in the sum-
mertime. Such a rhythm resembles that of the nidicolous ticks, although in spring and fall these
ticks can drop off either at night in stables or during the day in pastures (Berdyev 1980).
Most ticks select particular locations on their host’s body for attachment. For example, 60% to
70% of female Ixodes persulcatus in southwestern Russia and western Siberia attach to the neck
and dewlap of cattle, 20% to 24% of the ticks are found on the groin and head, and 10% attach at
other locations (Babenko and Leonovich 1985). The choice of feeding location on the host’s body
can be different not only among tick species but also among different stages of the same species.
In India, the majority of adult H. anatolicum attach on the groin (48%), udder (31%), or axilla
(14%) of cattle. The majority of larvae (98%) and nymphs (90%) of this species are found on the
ears of the same host (Geevarghese and Dhanda 1987). In cases when hosts are smaller (mammals
such as hares and rodents), the ticks mostly attach to the ears and head. For instance, 75% of Ixodes
persulcatus larvae are found on the ears, 24% on the head, and 3% on the body of small mammals
(Vasilyeva 1964). The majority of ticks feeding on birds attach to the head, as well as around the
eyes, the base of the beak, and the ears. Ticks are found in different locations on adult birds and on
nestlings; Ixodes lividus and I. subterraneus mostly attach to the head of adult birds but to the body
of hatchlings (Balashov 1998). Particular locations on reptiles are also noted. Mostly these ticks
attach around the ears, around the eyes, and at the base of the front and hind legs. There are at least
several factors determining the location of ticks on their host. Feeding tends to be in places where
70 BIOLOGY OF TICKS
it is difficult for the host to groom and remove the tick. The low number of ticks on primates can
be partially explained by their exceptional individual and collective grooming activities. Sec-
ondary factors regulating the attachment of ticks to a restricted place on the host’s body concern
the structure of the host’s skin and the density of hairs or fur. Other factors responsible for the
specialization of attachment locations are the various microclimatic conditions found on different
parts of the body. In hot, arid areas of Asia, adults of H. scupense attach mostly to the bottom part
of the bodies of cattle that is protected from direct sunlight and excess temperatures. Also, the
density of ticks can influence their distribution on the host; the more ticks on the host, the less
specific the location where feeding occurs (Balashov 1998).
6. NATURAL LONGEVITY
Ticks are relatively long-lived arthropods. Recall that the life cycle of ixodid ticks from egg to
adult is a process of morphological and physiological changes in the same individual. Thus,
eventually, the longevity of an individual tick’s life equals the longevity of its life cycle. The life
spans of individual stages, especially that of the adults, are remarkable in some cases. The ability
to stay alive for a long period of time at each stage adds to the adaptability needed for long sur-
vival in unfavorable environmental and host-availability conditions.
6.1. LONGEVITY OF LIFE CYCLES

The longevity of life cycles (i.e., the time from eggs hatching in one generation to the hatching
of eggs in the next generation) varies from a few months to several years. Mostly, this time
depends on climatic conditions and the availability of hosts. Thus, in tropical climates, the life
cycles of the 1-host ticks Rhipicephalus (Boophilus) microplus and Dermacentor nitens can take
as little as 8 weeks. In the constant high temperatures and humidity of the tropics, preoviposi-
tion of R. (B.) microplus takes 2 to 4 days in the summer, oviposition requires 13 to 25 days, and
on average eggs are incubated for 22 days. Thus, to finish its nonparasitic life (i.e., from drop-off
of engorged female to active larvae), this species needs a minimum of approximately 37 days.
Of course, this period can be extended up to the duration of the larval life span (an average of
109 days) in nature because of the time needed for unfed larvae to find their hosts. The parasitic
life of R. (B.) microplus can take as little as 20.5 days under favorable climatic conditions (Nu-
ñez et al. 1985). Because the extremely short longevity of the above-described life cycle in the
tropics under favorable climatic conditions, this 1-host tick species can develop without inter-
ruptions and produce multiple generations per year. In contrast, in temperate or polar climates,
the 3-host ticks Ixodes ricinus and I. uriae require from 2 to 7 years to finish 1 generation. Kar-
povich (1973) studied the life cycle of Ixodes uriae (a parasite of sea birds) under natural condi-
tions, and the following data were reported: larvae hatched in the autumn of 1967 from eggs
laid in 1965 and 1966; in the spring of 1968, 100% of the larvae fed; 10% of the larvae emerged
from eggs in the summer of 1969; nymphs fed in the summer of 1970 and molted to adults in
the autumn of the same year; and females fed in the spring of 1971 and oviposited in the au-
tumn of the same year. Thus, based on these data, the life cycle of Ixodes uriae can take 5 to 6
years to finish.
Another example illustrating varying longevity is that of Ixodes persulcatus. The longevity of
this species not only depends on the geographical locality of the tick population but also varies
in the same population under varying climatic conditions. There are 3 possible ways to complete
1 generation within 2 years. (i) Larvae may emerge from the eggs in the first year and overwinter
in diapause, after which they feed and molt to nymphs, and the nymphs feed and molt to adults
in the second year; unfed adults overwinter, and females feed and lay eggs in the third year.
(ii) Larvae may emerge from eggs in the first year and feed in the same year; thereafter, engorged
larvae overwinter in diapause. Fed larvae molt into nymphs, and the nymphs feed and molt to
adults in the second year. Unfed adults overwinter in diapause, and females feed and lay eggs in
the third year. (iii) Larvae hatch, feed, and molt into nymphs in the first year; unfed nymphs
overwinter. Nymphs feed and molt to adults in the second year. Unfed adults overwinter, and
females feed and lay eggs in the third year. There are 6 variants of development for 3 years and 4
possible ways to finish the life cycle in 4 years. The maximum longevity for this species would be
5 years: larvae emerge in the first year, unfed larvae overwinter, larvae feed in the second year,
fed larvae overwinter, larvae molt to nymphs in the third year, unfed nymphs overwinter,
nymphs feed in the fourth year, fed nymphs overwinter, nymphs molt to adults in the fifth year,
unfed adults overwinter, and females feed and lay eggs in the sixth year (Shashina 1985).
The life cycle of the argasid ticks lasts from 6 months to 2 years under favorable conditions. In
unfavorable conditions (e.g., in the absence of hosts), the life cycle can extend for years or even
decades (Filippova 1966). In fact, the longevity of life cycles can be dependent on the number of
nymphal stages, which in turn is partially dependent on climatic conditions. For instance, Orni-
thodoros coniceps requires 5 to 10 months to finish its life cycle when only 3 nymphal stages are
developed, 10 to 12 months if there are 4 nymphal stages, 17 months if there are 5 nymphal stages,
and more than 2 years in the case of development involving 6 nymphal stages (Filippova 1966). It
has been reported that in the laboratory, if there are lengthy delays in the feeding of nymphal in-
stars, the life cycle of Ornithodoros papillipes can take up to 20 years to finish (Filippova 1966).
6.2. INDIVIDUAL LONGEVITY OF LIFE STAGES

Unfed ticks are remarkable even among the long-lived arthropods. The life span of an individual
stage (i.e., larva, nymph, or adult) can vary from a few months to 3 years. Based on compiled data,
it appears that the life cycle of adult ticks of the Metastriata is longer than that of larvae and nymphs.
In contrast, adult females of Ixodes (Prostriata) have longevity comparable to that of the immature
stages (Balashov 1998). This is especially true for male ticks. Apparently males of the majority of
prostriate ticks do not feed and survive on resources accumulated during nymphal feeding, but
males of the metastriate ticks feed and stay on their host for long periods, waiting for females.
Many argasid ticks are also characterized by extended durations for each life stage (Filippova 1966).
6.3. EXAMPLES OF LONG-LIVED TICKS

Adults of Ixodes ricinus have been reported to live up to 1.5 years in nature, and those of Ambly-
omma americanum lived up to 3 years (Jaworski et al. 1984; Balashov 1998). Stages of some argasid
ticks appear to be longer lived than those of the ixodids. For instance, females of Ornithodoros
papillipes survived 6 to 10 years without a blood meal under laboratory conditions (Filippova 1966).
72 BIOLOGY OF TICKS
6.4. ADAPTATIONS FOR LONGEVITY

The major adaptation utilized by ticks in order to survive long periods of time is the develop-
mental pause. According to Belozerov (1981), ticks have 2 types of diapause, morphogenetical
and behavioral diapause. Behavioral diapause is expressed by the absence of specific behaviors
of ticks related to host finding. This type of diapause is observed in unfed ticks. Many Palearctic
species of Ixodes and Haemaphysalis may have this type of diapause during all active parasitic
stages of their life cycle (i.e., larva, nymph, and adult). Morphogenetic diapause is a delay in
metamorphosis in fed larvae and nymphs, ovogenesis in engorged females, and embryogenesis
in egg. Also, there is an unusual form of diapause that causes a delay in engorgement in some
winter parasitizing tick species (e.g., H. scupense). Obviously, one of the major functions of dia-
pause is to enable the ticks to survive unfavorable conditions such as low or high temperatures
or an absence of hosts. The ability to stay inactive at any particular stage of the life cycle for ei-
ther unfed or fed ticks adds enormously to the longevity of their life span.
First of all, it is necessary to obtain data on the life cycles of as many tick species as possible.
Some unsuspected discoveries might come from this, and this definitely will give us a clearer
picture of the distribution of various life cycle strategies throughout the Ixodoidea. In conjunc-
tion with completely reconstructed phylogenetic relationships among various groups of ticks,
this will possibly provide some insights regarding the possible evolution of life cycles. Addi-
tional studies on the questing strategies of unstudied (in this respect) species of ticks might help
us understand better the possible evolution of those strategies. More in-depth research of exo-
genic and endogenic mechanisms forcing the same species of Hyalomma to change from 3-host
to 2- or 1-host tick species is needed. Discoveries in this topic might help to explain the appear-
ance of shortened life cycles in other groups of ticks.
ACKNOWLEDGMENTS
We are greatly thankful to Mrs. Maria A. Apanaskevich for her kind help in the preparation of
the illustrations for this chapter.
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tologiya 7:128–134.
Leonovich, S.A. (1986) Orientation behavior of ixodid tick Hyalomma asiaticum under conditions of
desert. Parazitologiya 20:431–440.
Loomis, E.C. (1961) Life histories of ticks under laboratory conditions (Acarina: Ixodidae and Argasi-
dae). J. Parasitol. 47:91–99.
Norval, R.A.I. (1975) Studies on the ecology of Amblyomma marmoreum. J. Parasitol. 61:737–742.
Nuñez, J.L., Muñoz-Cobeñas, M.E., and Moltedo, H.L. (1985) Boophilus microplus, the Common Cattle
Tick. Berlin: Springer-Verlag.
Nuttall, G.H.F. and Warburton, C. (1915) Ticks. A Monograph of the Ixodoidea. Part III. The Genus
Haemaphysalis. Cambridge, UK: Cambridge University Press.
Oliver, J.H., Jr. (1971) Parthenogenesis in mites and ticks (Arachnida: Acari). Am. Zool. 11:283–299.
Oliver, J.H., Jr. (1981) Sex chromosomes, parthenogenesis, and polyploidy in ticks. In W.R. Atchley and
D.C. Woodruff (Eds.), Evolution and Speciation: Essay in Honor of M.J.D. White. New York: Cam-
bridge University Press, 66–77.
Oliver, J.H., Jr. (1982) Tick reproduction: sperm development and cytogenetics. In F.D. Obenchain and
R. Galun (Eds.), The Physiology of Ticks. New York: Pergamon Press, 245–275.
Oliver, J.H., Jr. and Stone, B.F. (1983) Spermatid production in unfed, Metastriata ticks. J. Parasitol. 69:420–421.
Pappas, P.J. and Oliver, J.H., Jr. (1971) Mating necessary for complete feeding of female Dermacentor
variabilis (Acari: Ixodidae). J. Georgia Entomol. Soc. 6:122–124.
Shashina, N.I. (1985) Total duration of life cycle. In N.A. Filippova (Ed.), Taiga Tick Ixodes persulcatus Schulze
(Acarina, Ixodidae). Morphology, Systematic, Ecology, Medical Importance. Leningrad: Nauka, 275–277.
Vasilyeva, I.S. (1964) On localization of ixodid ticks on body of small mammals. Ektoparazity 4:28–30.
C H A P T E R 4
EXTERNAL AND INTERNAL

ANATOMY OF TICKS
DANIEL E. S ONENS HINE AND R. MICHAEL ROE
1. INTRODUCTION
Externally, ticks are divided into 3 regions: the idiosoma or body region, the capitulum, and the
legs. A prominent sclerotized plate, the scutum, occurs on the dorsal surface in ixodid ticks, but
this structure is absent in argasid ticks. The capitulum bears the mouthparts. The capitulum and
podosoma constitute the prosoma, a primitive body region characteristic of all arachnids and
still evident in the developing embryo of ticks.
The arthropod ground plan, including that for ticks, is of a multisegmented organism; the
basic ground plan for an individual segment is shown in Fig. 4.1. The number of segments and
the prominence of this segmentation that remains either externally or internally in contempo-
rary arthropods are variable. Internally, the tick body is consistent with this ground plan, with
an open circulatory system filled with hemolymph that bathes the various internal organs.
Within the body cavity, there are prominent dorso-ventral muscles that extend from insertions
on the dorsal and ventral regions of the integument. In addition, there are modifications of ven-
tral and dorsal promoter and remoter muscles for moving the coxae, which in turn moves the
rest of the leg (or telopodite). Other specialized muscles that are important for retracting the
chelicerae, controlling pharyngeal action, walking, sensory action, digestion, reproduction, and
many other functions are discussed throughout this book. Each segment in the hypothetical
ground plan contains a pair of ganglia internally that innervate a single pair of appendages and
which are connected from front to back by a ventral nerve cord of variable length (Fig. 4.1).
However, during the evolution of ticks, extreme fusion has occurred that obscures this ancestral
ground plan. Thus, instead of a separate brain and ventral nerve chord, the entire central nervous
system of ticks appears to be fused into a single structure, the synganglion. Also present is a
complex system of anastomosing tracheae for gas exchange and peripheral nerves from the syn-
ganglion (central nervous system) to the various internal organs and appendages. Tracheae also
serve as connective tissue, holding organs in place. The hemolymph is circulated by a dorsal
External and Internal Anatomy 75
FIGURE 4.1: Back-to-front view of a single segment of the hypothetical ground plan for the Arthropoda
(provided by B. Boudreaux). Each segment ventrally contains a pair of ganglia and ventral nerve cord, a
dorsal blood vessel, and pair of appendages. The beginning and end of each segment are marked by
ventral and dorsal transverse tendons with ventral and dorsal longitudinal muscles attached to each,
respectively. The tergum is sclerotized, and the pleuron and sternum are membranous. There is a single
site of articulation for the coxa with the tergum and vertical axis of rotation for the coxa; the appendages
move via promotion and remotion typical for walking. DBV, dorsal blood vessel; DLM, dorsal longitudinal
muscles; DPM, dorsal promotor muscle; DRM, dorsal remotor muscle; DTT, dorsal transverse tendon;
DVM, dorsal longitudinal muscle; TLP, telepodite; VLM, ventral longitudinal muscle; VPAM, ventral
promoter (abductor) muscle; VRAM, ventral remotor (adductor) muscle; VTT, ventral transverse tendon.
blood vessel (Fig. 4.1) and is important in nutrient and nitrogen waste transport, water balance,
and immunity, but not oxygen/carbon dioxide exchange.
For an electronic version of the anatomy of ticks, the reader may browse the tick anatomical
ontology (illustrations and a description of structure) at www.Vectorbase.org by following the
instructions in the footnote below.1 Additional illustrations of the anatomy of ticks not included
in this edition may be found at this site.
2. EXTERNAL ANATOMY
2.1. THE BODY (IDIOSOMA)
The idiosoma consists of an anterior podosoma and a posterior opisthosoma (Figs. 4.2–4.12).
2.1.1. Podosoma
The podosoma bears the walking legs and the genital pore (in adults). In the Ixodidae, the
scutum is the most important feature distinguishing this family from the Argasidae. The scutum
is located on the anterior dorsal surface in females and immatures but covers the entire dorsal
76 BIOLOGY OF TICKS
FIGURE 4.2: External morphology of representative ixodid ticks. Photographs of adult female ticks:
(A) Ixodes scapularis female, ventral view; (B) same, dorsal view. (C) Ixodes scapularis male, ventral
view; (D) same, dorsal view. (E) Dermacentor variabilis female, ventral view; (F) same, dorsal view.
(G) Dermacentor variabilis male, ventral view; (H) same, dorsal view. A.G., anal groove (note groove
anterior to anus, diagnostic feature of genus); As, alloscutum; B.C., basis capituli; G.A., genital
aperture; Sp., spiracular plate with spiracle. Photo courtesy of J. Graham Snodgrass, U.S. Army Public
Health Command, Aberdeen Proving Ground, MD; used with permission.
body surface in males. The scutum bears cervical grooves near the midline and lateral ridges
(lateral carinae) along its lateral margins. The surface may be smooth or rugose and is covered
with numerous setae and tiny pits (punctate pattern) (Fig. 4.4). A simple eye is found along the
lateral margins on each side (absent in some genera) (Fig. 4.7). On the ventral surface, a gen-
ital groove is located medial to the coxae and extends from the genital pore to the posterior
body margin (Figs. 4.2A and 4.3). In males, the genital pore is covered by a movable plate that
can be elevated during copulation (Fig. 4.9). In males of the genus Ixodes, several hard sclero-
tized plates occur on the ventral body surface, namely, the sternal, epimeral, and median plates
(Fig. 4.2C). In females, the genital aperture (Figs. 4.2A, 4.2E, and 4.8) appears as a posteriorly
FIGURE 4.2: (continued)
directed U- or V-shaped groove with prominent marginal folds. Also in females, a paired pro-
trusible organ, Gené’s organ (egg waxing organ), may be visible in the camerostomal or emargi-
nation cavity between the scutum and the basis capituli (not shown).
2.1.2. Opithosoma
Posterior to the scutum is the alloscutum, characterized by innumerable fine striations that repre-
sent superficial folds of the cuticular surface (Figs. 4.4, 4.5). Minute pores, sensilla auriformia, are
abundant throughout this body region (they also are found on the scutum) (Fig. 4.5). Immediately
posterior to the scutum are the paired foveal pores (Fig. 4.6), used for secretion of the volatile at-
tractant, the sex pheromone (2,6-dichlorophenol). Although most evident in females, they also
occur in males (they are absent in the genus Ixodes). The opisthosoma also bears the spiracles and
the anus, as well as grooves, plates, and other body structures characteristic of different genera. In
Ixodes spp. males, sclerotized plates cover most of the opisthosomal surface (Fig. 4.2C). Prominent
marginal grooves occur along the lateral and posterior edges of the body in many species. On the
ventral surface, the prominent body features include the genital pore (in adults), the spiracular
plates, and the anal aperture. Ticks have only a single pair of spiracles (absent in the larval stage).
In ixodid ticks, the spiracular plate (Fig. 4.10) is a large, elongated, suboval or oval structure
that contains the macula adjacent to the small ostium, the opening to the respiratory system. The
FIGURE 4.3: External morphology of a female argasid tick. (A) Photograph of Carios kelleyi, dorsal view;
(B) same, ventral view. (C) Drawing of the dorsal surface of a generalized argasid tick (Ornithodoros
spp.) showing prominent body structures; (D) same, ventral surface. BC, basis capituli; Cam,
camerostome; Co.P, coxal pore; GP, genital pore (genital aperture); Pr.G, pre-anal groove; POG,
postanal groove; TPOG, transverse post-anal groove. Redrawn from Mullen and Durden (Eds.), Medical
and Veterinary Entomology (2nd Edition), with permission from Elsevier, Ltd.
78
FIGURE 4.4: Scutum and alloscutum of an ixodid tick, Ixodes affinis. Note the relatively smooth
appearance of the scutum bearing minute punctations and occasional setae versus the fi nely folded
alloscutum. Measurement bar, 200 μm.
FIGURE 4.5: Pore of a sensillum auriformae on the alloscutum of an ixodid tick. Note the adjacent
external folds. Measurement bar, 10 μm.
FIGURE 4.6: Fovea of the paired foveae dorsales of Dermacentor variabilis. The pore cluster consists of
approximately 25 to 30 slit-like pores. Measurement bar, 50 μm.
FIGURE 4.7: Eye of D. variabilis. Measurement bar, 50 μm.
FIGURE 4.8: Female genital groove of the ixodid tick, Aponomma elaphense. Measurement bar, 50 μm.
FIGURE 4.9: Male genital plate of the ixodid tick, A. elaphense. Measurement bar, 50 μm.
FIGURE 4.10: Spiracle of Dermacentor latus. Note the numerous pore-like goblets on the spiracular plate
and the curved slit-like ostium on the macula. Measurement bar, 100 μm. Reproduced from Yunker, C.E.,
Keirans, J.E., Clifford, C.M., and Easton, E.R. (1986) Dermacentor ticks (Acari: Ixodoidea: Ixodidae) of
the new world: a scanning electron microscope atlas. Proc. Entomol. Soc. Wash. 88:609–627, with
permission from the Proceedings of the Entomological Society of Washington.
FIGURE 4.11: Spiracle of an argasid tick, Ornithodoros puertoricensis. Measurement bar, 10 μm.
FIGURE 4.12: Anal aperture and anal valves of A. elaphense. Each anal valve has 4 setae. The valves are
separated to reveal the anus. Measurement bar, 50 μm. Als, alloscutum; E, eye; O, ostium; Sc, scutum;
Spp, spiracular plate.
79
80 BIOLOGY OF TICKS
spiracular plates are located on the lateral body margins posterior to the coxae of legs IV. In all
ixodid species, the spiracular plate is covered by distinct semitransparent goblets representing
internal spaces (aeropyles) within the plate structure (see Chapter 10). Posterior to the spiracular
plates is the anus, the opening of which is covered by a pair of movable plates (Fig. 4.12). The entire
body is covered by numerous setae (except in larvae, on which there are very few) and pore-like
sensilla. The latter are ear-like in appearance (sensilla auriformia), but other types also occur. On
the ventral opisthosoma of prostriate ticks, a prominent U-shaped anal groove curves anterior to
the anus and extends to the posterior body margins. However, in metastriate ticks, the anal groove
is posterior to the anus and does not extend to the body margin. Also present are disk-like fes-
toons located along the posterior ventral margin in most metastriate Ixodidae (absent in Ixodes).
Ventral plates, commonly found on the ventral opisthosoma in males of Ixodes spp., are generally
lacking in males of metastriate Ixodidae (males of Hyalomma species have anal and adanal plates).
The argasid tick body presents a very different appearance from that of the ixodid ticks (Fig.
4.3); the body lacks a scutum. In most species, the body is rounded, and the cuticular surface is
covered by tiny elevations termed mammillae. In Argas, the lateral edges of the body are flattened
and marked by rectangular marginal discs; mammillae are absent, and the cuticular surface is
covered by numerous sinuous wrinkles and tiny discs. On the ventral surface, genital grooves
(median grooves) are evident medial to the coxae, and prominent supracoxal folds occur lateral
to the coxae near the body margin. Eyes (ocelli), when present, occur on the supracoxal folds. In
addition, the paired coxal pores (i.e., openings of the coxal glands) also occur in this region, lo-
cated between coxae I and II. Each spiracle consists of a raised macula adjacent to a small, pitted
plate (Fig. 4.11) located on the lateral body surface between legs III and IV. In females, the genital
pore appears as a horizontal slit surrounded by a fusiform cuticular fold (Figs. 4.3B, 4.3D). In
males, the genital pore and cuticular fold are subtriangular or suboval in appearance; there is no
genital apron. Foveal pores are absent. There is a short pre-anal groove. Posterior to the anus, the
median and transverse post-anal grooves are arranged in a T-shaped formation.
2.2. CAPITULUM
The capitulum articulates with the idiosoma via a cavity, the emargination (ixodids) or camero-
stome (argasids), and can be flexed ventrally or extended (i.e., returned to the horizontal axis).
The horns of Gené’s organ protrude over the dorsal surface of the capitulum through this cavity
during oviposition. The capitulum consists of (1) the basis capituli, where the mouthparts are
anchored, (2) the paired 2-segmented chelicerae, (3) the toothed hypostome, and (4) the paired
4-segmented palps.
The basis capituli is a large ring of integument that encloses the pharynx and the basal re-
gions of the chelicerae (Figs. 4.13, 4.14). The palps are attached to its anterior lateral margins, and
the hypostome emerges from its midsection. In ixodid females, there is a pair of depressions
bearing numerous tiny pores, the porose areas (“Pa” in Figs. 4.14 and 4.16), covering almost the
entire dorsal surface in some species. Complex glands with secretory cells arranged in rosettes
are located below the pores, but little is known about the function of their secretions (Goethe et
al. 1987). The capitulum of argasid ticks is similar in structure to that of the ixodids; the most
striking difference is that it is located ventrally and is not visible from the dorsal aspect. In many
argasid species, it is covered by flap-like cheeks. Porose areas are absent in the argasid females.
FIGURE 4.13: Capitulum of D. andersoni, ventral aspect. Measurement bar, 100 μm.
FIGURE 4.14: Capitulum of D. andersoni, dorsal aspect. Measurement bar, 200 μm.
FIGURE 4.15: Hypostome and terminal segments of the palps of D. andersoni. Note the small terminal
fi eld of tiny denticles near the anterior tip of the broad, blunt hypostome (arrow points to these
denticles). Measurement bar, 100 μm.
FIGURE 4.16: Capitulum of Ixodes cookei (dorsal aspect) showing the dorsal surface of the hypostome
with its narrow hypostomal gutter. Measurement bar, 200 μm. Bc, basis capituli; Ch, chelicera; Hyp,
hypostome; P, palp; Pa, porose area; P-IV, palpal article 4; Sc, scutum. Note the cluster of setae forming
a sensory fi eld on the terminal (4th article) segment of the palp.
81
82 BIOLOGY OF TICKS
The chelicerae, situated dorsally on the capitulum, protrude beyond the basis capituli and lie
medial to the palps; their shafts are surrounded by the spinose cheliceral sheaths (Fig. 4.16).
A delicate, transparent cheliceral hood covers the digits in most species. Protruding anteriorly
from the shafts are the 2-segmented cheliceral digits, which bear the laterally directed cutting
spines. Their movements result in a ripping and tearing action against the host skin. The medial
digit (internal article) is movable and is directed from side to side by tendons attached to the
powerful muscle masses in the bulbous cheliceral bases (not visible externally). The outer digit
(external article) resides in a cavity of the inner digit and moves with it. Both digits have sharp
denticles. The inner digit bears mechanosensory and chemosensory sensilla that provide infor-
mation on shear forces, as well as the chemical composition of host fluids and the genital sex
pheromone (Sonenshine et al. 1984).
The tick hypostome is located ventrally on the capitulum between the palps (Fig. 4.15). The
ventral side of the hypostome is covered by rows of recurved denticles in a 2/2 or 3/3 arrange-
ment that help to anchor the tick in the host skin during blood feeding. A narrow food canal
(preoral canal) is located on the dorsal surface in the midline (Fig. 4.15). Blood is drawn from
the host through this narrow channel into the mouth and pharynx. The prominent, denticulate
hypostome is one of the major distinguishing characteristics used to distinguish ticks from other
arthropods (although males of some Ixodes species lack denticles).
In both Argasidae and Ixodidae, the palps comprise 4 distinct segments (articles) (Figs.
4.13–4.16). Although leg-like in structure, there is no terminal apotele with claws or a pulvillus,
as are found in the walking legs. In argasids, the individual segments of the palps are flexible
and similar in size. In ixodids, however, the terminal segment (article IV) is reduced and re-
cessed within a cavity of article III; it can be retracted or protruded as needed (Fig. 4.15, arrow).
Numerous fine chemo-/mechanosensory setae occur on the tip of this segment and form a
sensory field; they are discussed later in this chapter (Section 3.8.3). Other setae, often long and
stout, occur on the ventral and medial surfaces of the palps and serve to shield the delicate
mouthparts.
2.3. LEGS
Ticks have 4 pairs of walking legs (only 3 pairs in larvae) that are divided into 6 podomeres,
namely, the coxa, trochanter, femur, patella, tibia, and tarsus (Fig. 4.17). The coxae are inserted
on the ventral surface of the body. They can rotate slightly in the antero-posterior and dorsal-
ventral planes. This allows the ticks to right themselves when they land on their dorsal surface.
In the males of some species, the coxae are greatly enlarged and cover most of the ventral surface
of the body. The other leg segments are highly flexible but can move only in 1 plane (i.e., exten-
sion and flexion). Thus, the legs can be extended for walking or folded against the body for
protection. These segments are connected to one another by soft articulation cuticle. The tarsus
of each leg bears an apotele that includes the claws and the pulvillus (footpad), which facilitates
walking and climbing on smooth surfaces; the latter is absent in nymphs and adults of most ar-
gasid species. A complex sensory apparatus, Haller’s organ, occurs on the dorsal surface of the
tarsus of leg I (Fig. 4.18). It consists of an anterior pit and a posterior capsule, both with multi-
porose and single-pore sensilla. Haller’s organ is immensely important as the primary organ for
determining host location, determining host odors, recognizing pheromones, and other sensory
functions.
FIGURE 4.17: Diagram illustrating the structure of the leg in an argasid tick, Carios kelleyi. The femur is
superfi cially subdivided into basal and distal portions; similarly, the tarsus is superfi cially subdivided
into a metatarsus and the tarsus proper (telotarsus). Mtar., metatarsus; Pat., patella; Prt., pretarsus;
Tro., trochanter. Modifi ed from Sonenshine, D.E. (1962) External anatomy of the bat tick, Ornithodoros
kelleyi Cooley & Kohls (Acarina: Argasidae). J. Parasitol. 48: 470–485, with permission from the Journal
of Parasitology.
FIGURE 4.18: Scanning electron micrograph illustrating the dorsal surface and terminal organ (apotele)
of tarsus I of an ixodid tick, Dermacentor variabilis. Ap, anterior pit of Haller’s organ; Pc, posterior
capsule of Haller’s organ; Pv, pulvillus.
3. INTERNAL ANATOMY
This section is intended to provide the reader with a brief overview of the tick’s internal organs.
More detailed descriptions and discussions of the physiology and molecular biology of these
organs are provided in later chapters. The interior of the tick body is an open cavity with inter-
nal organs bathed in a freely circulating fluid, hemolymph. Also present are numerous muscles
modified from the basic arthropod ground plan (Fig. 4.1, discussed earlier). In nymphs and
adults, a complex system of anastomosing tracheal trunks and tracheal tubes is present, and
these are linked to smaller tracheae providing gas exchange. The central nervous system is
fused into a compact synganglion, from which peripheral nerves extend to all regions of the
body and appendages. Figures 4.19–4.21 illustrate the organization of the internal organs in
ixodid and argasid ticks. These figures should be consulted in part while reading the descrip-
tions that follow.
84 BIOLOGY OF TICKS
FIGURE 4.19: Drawing illustrating details of the internal anatomy of a female ixodid tick. The left-hand
side of the figure shows the internal organs as they appear when the midgut is removed; the right-hand
side shows the midgut cut (jagged area) along the midline of the body, with the midgut diverticula
overlaying most of the other organs. LG, longitudinal groove (of ovary); MD, midgut diverticulum; MS,
midgut-stomach (ventriculus); Mal. T, Malpighian tubule; O, ovary; Ov, oviduct; PnT, pedal nerve trunks;
Rec. S, rectal sac; SGA, salivary gland, zone of agranular (type I) acini; SGG, salivary gland, zone of
granular acini (types II and III); SD, salivary duct; Syn, synganglion; TAG, tubular accessory gland; TrT,
tracheal trunks; Tr, trachea.
3.1. ALIMENTARY SYSTEM

The alimentary system is divided into 3 primary regions: (1) the preoral canal and foregut,
(2) the midgut, and (3) the hindgut.
3.1.1. Preoral canal and foregut

The preoral canal2 (hypostomal gutter) is situated on the dorsal-medial part of the hypostome,
where it functions as an extension of the mouth. Blood ingested during feeding is drawn into the
preoral canal by the action of a powerful sucking pharynx and passed into the midgut via the
esophagus. In ixodid ticks, this same channel also serves to pass saliva from the salivary ducts in
the opposite direction, into the feeding lesion in the host’s skin. Blood uptake alternates with
saliva secretion. The cheliceral shafts situated immediately above the hypostome provide a roof-
like covering, effectively enclosing this channel and enhancing fluid flow. A tiny, delicate flap-like
labrum is situated at the base of the preoral canal in ixodid ticks, where it opens into the mouth
(at the junction with the pharynx). Here also is the salivarium formed by the fusion of the paired
FIGURE 4.20: Drawing illustrating details of the internal anatomy of male ixodid ticks. The left-hand side
of the figure shows the internal organs as they appear when the midgut is removed; the right-hand side
shows the midgut cut (jagged area) along the midline of the body, with the midgut diverticula overlaying
most of the other organs. Acg, accessory gland; ED, ejaculatory duct; MD, midgut diverticulum; MS,
midgut (stomach); Mal. T, Malpighian tubule; PnT, pedal nerve trunks; Rec. S, rectal sac; SGA, salivary
gland, zone of agranular (type I) acini; SGG, salivary gland, zone of granular acini (types II and III); SD,
salivary duct; Spc, spermatocysts indicated by irregular dotted lines; Syn, synganglion; T, testis; TrT,
tracheal trunks; Tr, tracheae; V.D., vas deferens.
salivary ducts. The alternation of sucking and salivary secretion separates the flow of fluids in the
preoral canal. A complex pharyngeal valve controls the influx of blood from the preoral canal
and the mouth into the pharynx. It consists of a movable dorsal wedge and movable walls that
form a groove-shaped channel. The valve can be closed when the pharynx contracts, preventing
regurgitation. In argasid ticks, the preoral canal is physically separated from the overlying sali-
varium by an elongated, movable, V-shaped labrum that extends the full length of the hypo-
stome. During blood uptake, the labrum is elevated, opening the preoral canal; during salivation,
it is depressed and seals the preoral canal. Thus the labrum acts like a valve, effectively preventing
the mixing of blood and saliva. It also prevents the regurgitation of ingested blood from the
pharynx. There is no separate pharyngeal valve, because the labrum serves this function.
The pharynx is an elongated (~500 μm) fusiform organ located entirely within the basis ca-
pituli. It consists of a delicate cuticular lining surrounded by multiple layers of smooth muscle
86 BIOLOGY OF TICKS
FIGURE 4.21: Drawing illustrating details of the internal anatomy of a male argasid tick. Acg, accessory
gland of the male; ANT. C. ANAST., anterior cerebral anastomosis; ANT. DIV., anterior diverticulum;
ANT. DOR. T., antero-dorsal tracheal trunk; ANT. LAT. DIV., antero-lateral diverticulum; ANT. MED.
DIV., antero-median diverticulum; CH. TR., cheliceral trachea; GN. TR., gnathosomal trachea; LAT. DIV.,
lateral diverticulum; Mal. T., Malpighian tubule; MED. DOR. T., median dorsal tracheal trunk; MED. T.,
median tracheal trunk; P.A.M., posterior accessory muscles; P. C. ANAST., posterior central
anastomosis (to synganglion); P. DOR. T., posterior dorsal tracheal trunk; P. LAT. DIV., postero-lateral
diverticulum; P. MED. DIV., postero-median diverticulum; P. MED. M., postero-median muscles; PR. M.,
preanal muscles; PRTRS., pre-transversal muscles; P. TRS., post-transversal muscles; Rec. S., rectal
sac; R.T., rectal tube (intestine); SP., spiracle; STOM., stomach; Syn., synganglion; T., testis; TRANS.
P.A.G., transverse post-anal groove; V.D., vas deferens.
cells. Externally, dilator muscles extend from its surface to the ventral and lateral walls of the
basis capituli and dorsally to the epistome (subcheliceral plate). The pharynx narrows at its pos-
terior end to join the cuticle-lined esophagus. The esophagus penetrates the synganglion, en-
tering at the antero-dorsal end and emerging at the postero-ventral end. Beyond the synganglion,
it turns dorsally to join the central portion of the midgut (ventriculus). A proventricular valve at
the junction of the esophagus and the midgut prevents the regurgitation of host fluids back into
the pharynx.
3.1.2. Midgut
The midgut is the largest organ in the body in the fed or feeding tick. Unlike in other blood-
feeding arthropods, the tick’s midgut also serves as a storage organ, allowing the tick to gradu-
ally digest its contents over many months or even years. It consists of a central ventriculus
(stomach) from which the numerous caeca (diverticula) emerge to penetrate virtually all parts
of the body cavity. The midgut wall consists of an epithelium and a thin outer layer of elongated
smooth muscle cells. In the epithelium, 2 major cell types occur: undifferentiated and digestive.
In some species, secretory cells have also been described. Following lysis of the erythrocytes and
other blood cells in the lumen, digestion of the hemoglobin (and occasionally other nutrients)
is entirely intracellular. The digestive cells develop from the undifferentiated cells during feeding,
whereupon they hypertrophy and ingest hemoglobin, which binds to clathrin-coated pits in the
luminal surfaces of these cells (receptor-mediated endocytosis). Hemoglobin molecules bound
to these digestive cells are internalized, incorporated into phagolysosomes, and digested (Lara et
al. 2005). Gradually, the distal portions of the large, flask-shaped digestive cells fill with black
droplets (almost entirely hematin waste). Subsequently, these parts of the cells break off, float in
the lumen, and are passed to the exterior via the rectal sac and anus. In addition to the heme
moiety’s being detoxified to hematin, some heme is transferred to the hemolymph and bound to
storage proteins for later use, especially during vitellogenesis and oocyte development. Free
amino acids, monosaccharides, free fatty acids, and large amounts of water and salts (but few
proteins) may be absorbed directly from the lumen. The molecular basis for the complex blood
digestion process is discussed in Chapter 6.
The midgut is connected (except in a few Ornithodoros spp.) to the rectal sac by a narrow
tubular intestine histologically similar to the midgut lining that transports wastes to the rectal
sac, where they mix with guanine from the Malpighian tubules. These wastes include dead or
dying digestive cells, cell fragments, hematin, undigested hemoglobin crystals, and fluids from
the midgut lumen.
3.1.3. Hindgut
The hindgut consists of the intestine, the rectal sac, and the rectum. The rectal sac is a large,
bulb-like structure located in the posterior region of the body. It may appear white when filled
with masses of guanine crystals or red (during feeding) when it contains hematin and undi-
gested blood. The sac walls are composed of a simple epithelium surrounded by bands of smooth
muscle. Extending ventrally from the rectal sac is the rectum (anal canal), a short, narrow, cuti-
cle-lined duct that connects the rectal sac with the anal aperture. The latter is surrounded exter-
nally by paired cuticular flaps, the movement of which is controlled by muscles inserted on the
flaps and the rectum. Periodic filling of the rectal sac leads to opening of the anus via contraction
of the flap muscles, with consequent voiding of feces.
3.2. SALIVARY GLANDS

In both ixodid and argasid ticks, the paired salivary glands comprise large clusters of acini
(alveoli) located in the antero-lateral regions of the body cavity. Saliva from these glands is ex-
pelled anteriorly via the salivary ducts, salivarium, and preoral canal, whereupon it is injected
into the feeding lesion. Almost all tick-borne pathogens enter the host body via this route.
88 BIOLOGY OF TICKS
The salivary glands expand greatly during feeding. They increase to up to 25 times their
original size and deplete most of their cellular contents during this period, although new protein
and lipid synthesis also occurs (Bowman et al. 2008). Each salivary gland consists of 2 basic
types of acini, agranular and the more numerous granular acini.
The agranular acini (type I) are located in the anterior and middle regions of the gland. Most
are attached directly to the main salivary duct. Each acinus has a large vacuolated central cell
surrounded by pyramidal cells with innumerable lamellae and peritubular cells surrounding the
short acinar duct (Bowman et al. 2008). Type I acini secrete hygroscopic solutions onto the
mouthparts that enable ticks to take up moisture from subsaturated atmospheres (Kahl and
Knulle 1988; Needham and Teel 1991). The granular acini constitute the bulk of the salivary
glands. They occur in clusters attached to ramifying secondary ducts, although some also com-
municate directly with the main duct. Each acinus has an acinar duct draining its interior that
connects to the lobular duct (a branch from the main salivary duct). Peritubular cells (neck
cells) surround the acinar duct. Most of the remaining cells are granular, but small ablumenal
cells occur around the margin, and adlumenal cells occur between the more prominent granule-
containing cells. Three different types of granular acini have been described in ixodid ticks:
types II, III, and IV (type IV acini are found only in males). Most ixodid species have types II
and III, which are distinguished by differences in the cell types within these lobes. Type II and
type III acini secrete the cement that binds the tick’s mouthparts to the host skin. Type II acini
have 3 different cell types (A, B, and C) characterized by differences in their granular composi-
tions and reactions to histochemical stains. Type A cells have complex granules composed of
numerous subunits, whereas the B and C cells have more homogeneous granules. Type III acini
are the most numerous of the acinar types. They also have 3 cell types, termed D, E, and F. The
D and E cell types contain granules similar in composition to the A and B cell types. However,
the F cell is distinct and undergoes a remarkable transformation during feeding. In females, a
tremendous proliferation of membranous folds formed from the basal labyrinth occurs, and
these folds also interdigitate with the highly convoluted contiguous membranes of the adjacent
ablumenal cells. The interior of the acinus becomes highly vacuolated and filled with watery
fluid. The transformation of these cells enables the ixodid ticks to eliminate much of the water
and salts (~70%) taken up during feeding and thereby concentrate the blood meal. In effect, the
salivary glands are the primary osmoregulatory organs in ixodid ticks (Bowman and Sauer
2004). In males, which take up very little blood (or none at all in some species), the F cells are
also transformed, but to a much more limited degree than in the females. Types II and type III
acini are dispersed throughout the salivary glands but are concentrated primarily in the middle
and posterior regions. Type IV acini have been found only in males of several ixodid species. In
argasid ticks, only 1 type of granular acinus (type II) was reported to occur. However, a more
recent ultrastructural and functional assessment suggests that the salivary glands of these ticks
might be considerably more complex than previously thought and not readily amenable to
classification solely on the basis of morphological features (Mans et al. 2004). Other details of
the acinar granular structure and ultrastructure and their various functions are described in
Chapter 7.
Salivary secretion is controlled by neurosecretory transmitters and an intricate system of
intracellular second messengers and modulators, which are discussed further in Chapter 13. The
secretory products are antihemostatic, thereby maintaining blood and tissue fluid intake into
the wound site. The secretory products are also anti-inflammatory and in some cases immuno-
suppressive, minimizing host awareness of the attached and feeding tick. The molecular basis for
the expression of this remarkable dominance of the host tissue environment has been described
elsewhere (Francischetti et al. 2009) and is explored in more detail in Chapter 7.
Following a blood meal, the salivary glands degenerate in response to elevated titers of
20-hydroxyecdysone (Kaufman 1991). In mated, replete ixodid females, a protein factor from the
male reproductive organs has been reported to promote gland degeneration, but the mode of
action remains to be explored (Bowman et al. 2008).
3.3. MALPIGHIAN TUBULES

In ticks, as in most other arthropods, the Malpighian tubules are connected to the rectal sac at
its junction with the intestine. The lining of the tubules is histologically similar to that of the
rectal sac, with simple cuboidal epithelium. Only a single pair of tubules occurs in ticks, in con-
trast with the multiple-tubule system found in many insects. The long, narrow tubules are in
close proximity to virtually every organ in the hemocoel. Nitrogenous waste products collected
in these tubules are accumulated and consolidated primarily in the form of guanine crystals,
which are passed to the rectal sac and voided with other wastes from the gut to the exterior.
3.4. COXAL GLANDS

In argasid ticks, small paired coxal glands are situated dorsal to the coxae of legs I and II. These
organs contain a filtration chamber surrounded by a distensible sac serving as an ultrafiltration
membrane. The glands are connected to coiled ducts leading to the coxal pores located on the
ventral body cuticle between coxae I and II. This system, described in more detail elsewhere
(Chapter 8), provides the primary route for the rapid elimination of excess blood meal water
ingested during feeding by nymphs and adults. Coxal glands are absent in argasid larvae and in
all ixodid ticks (in some ixodid ticks, a tiny secretory gland connected to the coxal pores also
occurs in the same region). The causative agents of tick-borne relapsing fever often can be pre-
sent and transmitted via coxal fluid to infect unsuspecting humans (Barbour 2005).
3.5. THE CIRCULATORY SYSTEM AND HEMOLYMPH

Ticks, like other arthropods, have an open circulatory system (Fig. 4.1). Circulation is aided by a
heart, an aorta, short arterial vessels, and several sinuses. The heart is a relatively simple, elon-
gated tube located in the dorsal midline of the body and is a basic feature of the ground plan for
arthropods (Fig. 4.1; see also Figs. 11.1–11.7). It is surrounded by a pericardial sinus. Hemolymph
from the body cavity enters the sinus, which acts as a filtration system, and then the heart via
paired ostia. Muscle bands around the organ provide the pulsatile action. Hemolymph flows from
the heart via the anterior aorta to the periganglionic sinus surrounding the synganglion. From
there it is distributed via pedal arteries to the legs and to anterior and ventral sinuses. Tiny arteries
from these sinuses carry hemolymph to the mouthparts, the basis capituli, and the body cavity.
Hemolymph fills the body cavity, capitulum, and legs. All organs and tissues are bathed in
this fluid. It remains relatively constant as a percentage of tick body weight regardless of physio-
logical activity (e.g., feeding). Hemolymph consists of plasma and a variety of cells (hemocytes).
Considerable controversy exists regarding the classification of tick hemocytes. In ixodid ticks, in
90 BIOLOGY OF TICKS
addition to prohemocytes (small undifferentiated cells that give rise to the other types of hemo-
cytes), there are granulocytes and hemocytes with few or no granulocytes (plasmatocytes). Two
categories of granulocytes occur: GRI granulocytes with oval or spindle-shaped, diffuse, electron-
lucent granules containing tubular structures, and GRII granulocytes with numerous, mostly
rounded electron-dense granules (Borovickova and Hypsa 2005). Both plasmatocytes and GRI
granulocytes function as phagocytes, whereas the GRII granulocytes are believed to function in
the clotting of hemolymph and also to release granules that coagulate and encapsulate large par-
ticles or microbes. The presence of antimicrobial peptides such as defensin (Ceraul et al. 2003),
lysozyme, and lectins has been reported in the hemocytes of ticks, although it is not known
whether they are stored in the granules or elsewhere in these cells. Soft ticks (e.g., Ornithodoros
moubata) also have a fourth type of hemocyte resembling the spherulocytes of insects.
In addition to hemocytes, hemolymph is rich in proteins. Among the most abundant are
hemelipoglycoprotein, a large protein (354 kDa) also called common or carrier protein (CP)
(Donohue et al. 2008), and, in females at the time of developing eggs, vitellogenin (Vg). Ticks
studied so far are unusual among eukaryotes in that they lack the ability to synthesize heme and
therefore depend upon heme extracted from blood meal digestion. CP and Vg store and trans-
port heme to tissues and the egg (Maya-Monteiro et al. 2000; Donohue et al. 2008). Heme
binding also serves to prevent oxidative stress and tissue damage and consequently is an impor-
tant adaptation to hematophagy. A more detailed description of the circulatory system and he-
molymph of ticks is presented in Chapter 11.
3.6. RESPIRATORY SYSTEM

In adults and nymphs of all tick species, a system of tracheal tubes is used for oxygen uptake and
CO2 elimination. Ticks have only a single pair of respiratory pores (ostia) located on the spirac-
ular plates on the lateral body surface. Each spiracular plate contains numerous enclosed air sacs
separated by pedicels and visible externally as tiny goblets. Below each ostium is a sac-like
atrium, connected by up to 8 large tracheal trunks to a system of ramifying tracheae. The tra-
cheae anastomose throughout the body cavity to supply all organs and tissues. In ixodid ticks,
muscles attached to the spiracular plate and the atrial walls dilate the ostia and open the system
to the external air. Hydrostatic pressure is believed to be responsible for closing the system. The
tracheae are lined with cuticle with a spiral thickening (taenidium). Tracheal branches to the
internal organs often ramify over the surfaces of these structures, enmeshing them in a basket-
like network of tracheal tubes with tiny tracheoles penetrating into the tissues themselves. Tra-
cheal systems are absent in larval ticks. A more detailed description of respiratory structure and
physiology is provided in Chapter 10. In addition to enabling gas exchange, the tracheal system
serves as connective tissue, holding the different organs of the tick in place.
3.7. FAT BODY AND NEPHROCYTES

In ticks, the fat body consists of chords or membranes of cells found adhering to the tracheae and
as occasional clusters below the epidermis (peripheral fat body) or surrounding the reproductive
and other major internal organs (internal fat body). Fat body cells are especially abundant around
the tracheal trunks but are also distributed throughout the body as chords of cuboidal cells along
the numerous tracheae. The primary cell type is the trophocyte, although small groups of
free-floating nephrocytes often are found attached to the fat body chords. The fat body is one
source of Vg in ovipositing female ticks. Vg synthesis is initiated in the fat body of the replete
pre-ovipositing female in response to elevated 20-hydroxyecdysone (Thompson et al. 2005).
Subsequently, Vg is secreted into the hemolymph and absorbed with the aid of Vg receptors on
the surface of maturing oocytes (Mitchell et al. 2007). The fat body is also one source of the pre-
dominant hemolymph storage protein CP, which is found in both males and females throughout
development.
The nephrocytes (so named because of their resemblance to similar cells in some insect
species) (Denholm and Skaer 2009) are believed to be functionally related to the fat body.
Whereas insect nephrocytes sequester and store materials from the hemolymph, their role in
ticks is uncertain. A more detailed description of fat body ultrastructure, function, and molec-
ular biology is discussed in Chapters 12, 15, and 16.
3.8. NERVOUS SYSTEM (INCLUDING SENSORY ORGANS)

3.8.1. Synganglion
In contrast to insects, ticks lack an obvious separate brain and ventral nerve cord. Rather, the gan-
glia of the central nervous system (CNS) have coalesced to form a synganglion (often misnamed
“brain”), which is located in the anterior ventral region of the body. Externally, there is no obvious
head region containing the brain. The synganglion is subdivided into supra- and subesophageal
regions by the esophagus, which passes diagonally between these 2 regions (Fig. 4.22). This posi-
tioning of the esophagus is an important landmark in possibly understanding the origin of the
different paired ganglia that make up the tick CNS, as discussed in more detail later in this chapter.
The synganglion is surrounded by a thick, amorphous perineurium. Histological examination
reveals a peripheral mass of neuron cell bodies (perikarya), evident from their intensely staining,
closely packed nuclei, which form the cortex. In the central portion of the organ, perikarya are
virtually absent, and the great mass of closely packed, branching axonal and dendritic nerve fibers
forms the neuropile.
Specialized nerve ganglia have been identified in different regions of the synganglion. Paired
protocerebral, cheliceral, and palpal ganglia with associated paired protrusions or nerves
(antero-dorsal glomerus, cheliceral nerve, and palpal nerve, respectively) make up the majority
of the supraesophageal region of the synganlion. Nerves from this region of the synganglion also
innervate the salivary glands, pharynx, esophagus, and eyes. A retrocerebral organ complex on
the dorsal side of the supraesophageal region is believed to serve as a neurohaemal organ and,
interestingly, is in the same location as parts of the stomatogastic nervous system of insects,
which might suggest similar functions. The subesophageal region of the synganglion contains the
4 pairs of pedal ganglia, each bearing a trunk-like pedal nerve extending laterally. This is followed
by the unpaired opisthosomal ganglion and the associated opisthosomal nerves on each side that
innervate most of the internal organs. Olfactory lobes on the ventral surface of the synganglion
are believed to receive axons from Haller’s organ (Hummel et al. 2007) (Fig. 4.23). Ticks lack
antennae, and one site of olfaction is the Haller’s organs on the distal ends of the first pair of
walking legs. Association-motor neurons are numerous in the subesophageal region, suggesting
a primary role in motor coordination, which would be typical of the ventral nerve cord in insects.
FIGURE 4.22: Diagram (lateral view) of the synganglion, with the anterior region on the left, from the tick
Ornithodoros parkeri, showing the major ganglia, nerves, and other regions. The esophagus penetrates
(ventral–dorsal) through the synganglion separating the structure into anterior supraesophageal and
posterior subesophageal regions. ADgl, antero-dorsal glomerus; CC, commissures and connecting fiber
tracts; CHg, cheliceral ganglion; CHn, cheliceral nerve; ES, esophagus; ESn, esophageal nerve; OL,
olfactory ganglion; OPg, opisthosomal ganglion; OPn, opisthosomal nerve; Pg, palpal ganglion; PDg
I–IV, pedal ganglia I–IV; Pn, palpal nerve; PnT I–IV, pedal nerve trunks I–IV; Prt, protocerebrum; PRV,
proventriculus; ROC, retrocerebral organ complex; STP, stomodeal pons; SUPRA-E, supraesophageal
region; SUB-E, subesophageal region. Black circles with adjacent numbers indicate the neurosecretory
centers. Figure redrawn from Pound, J.M. and Oliver, J.H., Jr. (1982) Synganglial and neurosecretory
morphology of female Ornithodoros parkeri (cooley) (acari: Argasidae). J. Morphol. 173:159–177, with
permission from Wiley Periodicals, Inc.
FIGURE 4.23: Diagrams (dorsal and ventral views) of the synganglion showing the major regions,
ganglia, and lobes (excluding nerves and lateral segmental organs). Ch, cheliceral ganglion; ES,
esophagus; Ol, olfactory lobes; Os, opisthosomal lobe; Pa, palpal ganglion; Pd 1–4, pedal ganglia; Pc,
protocerebral ganglion; PgS, periganglionic sheath; St, stomodeal lobe; Sub-E, subesophageal region;
Supra-E, supraesophageal region. The dotted line marks the approximate delineation between the two
major regions. Redrawn from Simo, L., Slovák, M., Park, Y., and Zitnan, D. (2009) Identifi cation of a
complex peptidergic neuroendocrine network in the hard tick, Rhipicephalus appendiculatus. Cell Tissue
Res. 335:639–655, with permission from Springer-Verlag, GMBH, Heidelberg, Germany.
92
Small nerves extend between the pedal trunks on either side of the synganglion and form
a lateral plexus containing the lateral segmental organs. The function of these tiny organs is
unknown, although recent evidence supports their role as a source of neuropeptide activity. Eigh-
teen neurosecretory centers associated with 1 or more neuropilar ganglia occur in the synganglion
(Pound and Oliver, 1982). The neurosecretory cells are the source of numerous neuropeptides that
are secreted or transported via axons to tissue sites throughout the body (Simo et al. 2009).
3.8.2. Evolution of the tick CNS

Each segment in the ground plan for the Arthropoda contains a pair of appendages (Fig. 4.1),
which, in living organisms today, are modified for different functions, reduced to vestigial struc-
tures, or completely eliminated. Each metamere also contains a ventral pair of ganglia connected
to those before and behind (when present) the ventral nerve cord; the ganglia each bear nerves
laterally to the appendages in the same segment. The paired ganglia internally and/or the ap-
pendages externally are excellent landmarks for identifying segmentation in arthropods that
otherwise might be visually obtuse as a result of tagmosis and/or segmental specialization.
In insects, the brain is anterior to the mouth (and esophagus) and composed of 3 pairs of
ganglia (the proto-, deuto-, and tritocerebrum), with the front-to-back interganglionic connec-
tives visually absent when viewing the intact brain. The paired ganglia represent segments 1–3 of
the hypothetical ancestral ground plan for the Arthropoda, with the paired preoral ganglia for
segment 3 actually representing the first postoral ganglia; this shift appears to be clear, as the
tritocerebrum is clearly anterior of the esophagus, but a postesophageal commissure connecting
both sides of the tritocerebrum runs behind the mouth in some insects. Interestingly, in ticks,
the supraesophageal region of the synganglion also contains what appear to be 3 pairs of ganglia
(the protocerebrum, cheliceral ganglion, and palpal ganglion; Figs. 4.22, 4.23), suggesting that
this region is homologous to the insect brain. If this is correct, this structure of the brain is a
common feature of both Chelicerata and Mandibulata, 2 of the major groups within the Ar-
thropoda. Posterior to the esophagus in ticks, apparently the 4 pairs of pedal ganglia, followed
by the fused opisthosomal ganglia and their associated nerves (Figs. 4.22, 4.23), most probably
represent body segments 4–8, respectively, with the ventral nerve cord absent when the poste-
sophageal region is viewed externally. If this analysis is correct, the tick body consists of a total
of 8 body segments, as compared to 20 segments in most insects (excluding the prostonium).
3.8.2. Peripheral nerves

Paired cheliceral, palpal, and optic nerves extend from the supraesophageal region of the syngan-
glion to the chelicerae, palps, and eyes, respectively. An unpaired esophageal nerve extends from
the stomadeal pons to innervate the pharynx and esophagus. The remaining nerves originate in the
subesophageal region of the brain. The largest are the 4 pairs of pedal nerve trunks that emerge
from the lateral sides of the synganglion and extend into each of the corresponding legs, where they
branch to innervate the striated muscle and integumental sensilla. Small nerves that emerge from
either side of this same part of the synganglion form a lateral plexus that contains the tiny lateral
segmental organs. Posteriorly, a pair of opisthosomal nerves extend from the synganglion into the
hemocoel and branch to innervate most of the internal organs. The homology of the stomodeal
pons and olfactory lobe relative to segmentation is undetermined and needs further analysis.
94 BIOLOGY OF TICKS
3.8.3. Peripheral sensilla

Ticks have a variety of peripheral sensory organs that provide information on the state of the
external environment. Each consists of 1 or more neuron cell bodies within the epidermis,
plus supporting cells (trichogen and tormogen cells) adjacent to them. Dendrites extend
from the neuron cell bodies into the external structure of the sensilla (e.g., the seta). The
most common types of sensilla are the hair-like setae that occur all over the body, legs, and
mouthparts. These nonporous setae serve as tactile sensilla (mechanosensory sensilla) that
sense the substratum, vibrations, or other mechanical changes. Porose setae detect chemicals
in the tick’s environment. Multiporose sensilla serve as odorant receptors (olfactosensilla) to
detect odorant molecules. Tip pore sensilla (gustatory sensilla) detect chemicals in aqueous
solutions or in lipids. Haller’s organ, located on the dorsal surface of the tarsi of leg I, con-
tains both olfactory and gustatory sensilla. Ticks wave their forelegs much like insect
antennae to detect odorant molecules. Gustatory sensilla are numerous on the terminal seg-
ments of the palps, where they may serve a role in host recognition by detecting chemicals in
hair or on the skin. Gustatory sensilla even occur on the cheliceral digits. Numerous tiny,
pore-like structures (sensilla auriformia) occur abundantly all over the body. They are be-
lieved to serve as sensory organs (Walker et al. 1996). The variety of sensory organs, their
structure and physiology, and the molecular biology and biochemistry of chemoreception
are discussed in Chapter 13.
3.9. REPRODUCTIVE SYSTEM

3.9.1. Female reproductive system
This system consists of a single ovary, paired oviducts, a single uterus, the vagina (subdivided
into cervical and vestibular regions), and the seminal receptacle. The U-shaped ovary is lo-
cated in the posterior region of the body. In unfed females, the ovary appears as a thin band
of cells; in fed females, it appears as a large organ with numerous oocytes of varying size pro-
truding from the walls of the ovary. In metastriate Ixodidae, long, folded oviducts connect the
vagina to the short, inconspicuous uterus, followed by a short connecting tube linking the
uterus to the cervical vagina. The latter is a short (thick-walled) muscular tube. The cuticle-
lined vestibular vagina, the longest part of this organ, connects the reproductive system with
the external genital pore. A lobular accessory gland surrounds most of the vestibular vagina,
and a pair of tubular accessory glands occurs at its base near the junction with the cervical
vagina. The seminal receptacle is collapsed and virtually indistinguishable in unfed or fed
virgin females but greatly enlarged in mated females. Argasid and prostriate females lack a
seminal receptacle. In these species, the uterus is enlarged distally where it joins the vagina;
in argasids, it is also bilobed, further increasing its size and facilitating its role as a sperm
storage organ. In many argasid species, the ovary may appear paired, connected by a slender
isthmus. In prostriate ticks, the cervical vagina is enlarged and can also serve as a sperm
storage organ.
External to the genital tract is the paired egg waxing organ, Gené’s organ, located in the
anterior region of the body adjacent to the emargination or camerostomal fold. The organ
comprises between 2 and 4 saccular structures that are lined with cuticle. The saccules evert
as paired horns through the capitular foramen during oviposition and coat each egg with
waxy material as it emerges (Schöl et al. 2001). This waxy coating protects the eggs against
desiccation and also provides limited protection against microbial infection (Arrieta et al.
2006). Also important for the tick’s reproductive success are the pheromone glands. The
paired foveal glands are located in the opisthosomal region of the body. They are attached
to the foveal pores, located on the dorsal alloscutum. These glands secrete a phenolic sex
pheromone (2,6-dichlorophenol) that initiates mating behavior in most ixodid ticks. Non-
volatile sex pheromones also occur on the external body surface and, in some species, even
in the vaginal tract. A more detailed description of the female reproductive system, as well
as the biochemistry and molecular biology of this system, is discussed elsewhere (Chapters
17 and 18).
3.9.2. Male reproductive system

In adult males, this system consists of the paired testes, the vasa deferentia, the seminal ves-
icle, the ejaculatory duct, and the large, complex, multilobed male accessory gland. The
paired testes, which appear fused into a single gland joined by a narrow septum, are located
in the posterior part of the body. A pair of short, coiled vasa deferentia extend from the tes-
tes to the common seminal vesicle located just below the male accessory gland in the ante-
rior region of the body cavity. During feeding, both the testes and the vasa deferentia enlarge,
and the latter fill with masses of spermatids. Extending anteriorly from the seminal vesicle is
the short, cuticle-lined ejaculatory duct that connects the seminal vesicle with the genital
pore. The accessory gland is the largest organ in the male body. It comprises a central body
and at least 7 or 8 lobes of varying size and shape, depending upon the species. The accessory
gland secretes the proteins that form the spermatophore and the seminal fluid that sustain
the spermatids and facilitate fertilization. It lies dorsal to the ejaculatory duct and slightly
posterior to the synganglion. A more detailed description of the male reproductive system
and of the physiology, biochemistry, and molecular biology of this system is presented in
Chapter 18.
3.10. MUSCULATURE
The most common muscles are the bundles of striated muscle fibers in the body cavity, the ca-
pitulum, and the legs. Smooth muscle fibers form the external walls of the midgut, certain
organs of the reproductive system, and other internal organs. In the body cavity, dorso-ventral
muscles extend between the inner surfaces of the ventral and dorsal body walls; this is a basic
feature of the ground plan for arthropods (Fig. 4.1). In argasid ticks, their attachment sites are
marked by the external discs visible on the outer surface of the cuticle. In ixodid ticks, the dorso-
ventral muscles of the alloscutal region are arranged in longitudinal rows attached to the major
grooves. Contraction of the dorso-ventral body muscles generates hydrostatic pressure involved
in hemolymph circulation and facilitates walking and climbing activity. Muscle groups located
in the anterior body region flex or extend the capitulum, while others retract the chelicerae and
Gené’s organ. In the podosomal region, bundles of muscles extend from the dorsal body wall
and insert on the inner surfaces of the coxae, where they serve promoters and remotors of these
leg segments.
96 BIOLOGY OF TICKS
The capitulum contains striated muscles that extend from the ventral and lateral surfaces of the
basis capituli and insert on the walls of the pharynx; other muscles extend from the subcheliceral
plate and insert on the dorsal surface of this organ. Contraction of these muscles dilates the phar-
ynx. Concentrated rings of striated muscles constrict the organ when the dilator muscles relax.
Coordinated contraction and relaxation of these dilator and constrictor muscles synchronized
with the opening and closing of the pharyngeal valves enables this organ to serve as a powerful
muscular pump for ingesting blood.
The leg segments contain flexor and extensor muscles that originate in one segment and pass
through the intersegmental cavity to insert on the inner face of the next distal joint. Flexor and
extensor muscles from the body wall insert on each of the coxae. The action of these muscles
allows rotary movements of the entire leg, whereas the muscles of the other segments permit
movement in only 1 plane (i.e., folding or straightening of the legs). At the distal end, tendons
from the tarsal muscles are inserted at the base of the apotele, thereby enabling up and down
movements of the claws.
Much remains to be learned about the external and internal anatomy of ticks. More detailed
knowledge of the ultrastructure of the cuticle, its development, its re-absorption during molt-
ing, and especially its growth during blood feeding is critical to understanding the novel
adaptations used by ticks for their successful hematophagous lifestyle. Understanding the
hormonal regulation of cuticle development and associated behaviors has recently been made
possible because of discoveries from tick transcriptomics and genomics, and great advances
are expected in the near future. Further study of the mouth, labrum, and anatomical separa-
tions that allow alternating cycles of blood uptake and salivary outflow is needed. Also, the
evolution of segmentation and structure/function in the context of other arthropods has re-
ceived minimal attention but is especially interesting because of the unique body structure of
the Acari, the adaption to obligatory blood feeding in the ticks, and the significant enlarge-
ment of genome size in hard ticks discussed elsewhere in this text. Also fascinating are the
unique features of ticks in contrast to the better-studied insects in areas of olfaction, such as
Haller’s organs on the front legs of ticks, the centralization and condensation of the CNS,
regulation of growth and development, and cuticle growth without molting that occurs in
some ticks during adulthood. The understanding of the structure of the tick neuroendocrine
and endocrine systems is in its infancy, along with that of chemical communication. Under-
standing of the morphology and histology of these regulatory systems, including the nervous
system, is expected to expand because of advances in molecular biology, proteomics, and
microscopy.
ACKNOWLEDGMENTS
The figure of the hypothetical ancestral arthropod (Fig. 4.1) was provided by Dr. Bruce Boudreaux
(deceased), formerly of Louisiana State University, Baton Rouge, LA.
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NOTES
2. See www.Vectorbase.org for descriptions and illustrations of most of the structures described in
this section.
C H A P T E R 5
INTEGUMENT AND ECDYSIS

W. REUBEN KAUFMAN
1. INTRODUCTION
“Mention the words ‘arthropod cuticle’ to most biologists and they usually provoke a glazed
expression. This is because the cuticle is commonly regarded as an inert substance. It is hoped
that this book will dispel this fallacy.”
So begins the preface to A. C. Neville’s Biology of the Arthropod Cuticle (1975). The micro-
scopic structure and chemical composition of the integument are based on a common plan
throughout the arthropod phylum that reflects a system inherited from the earliest arthropod
ancestors (Neville 1975). Although this book focuses on the biology of ticks, one must often al-
lude to information available for insects. Most of the assumptions based on other arthropods,
however, are likely to be reasonable because so many aspects of the arthropod cuticle are highly
conserved (Cohen 2010; Moussian 2010).
2. FUNCTIONAL MORPHOLOGY OF THE INTEGUMENT
2.1. GENERAL CONSIDERATIONS

The integument comprises the epidermis (hypodermis) and its secreted cuticle (Fig. 5.1). The 2
basic cuticular layers are an epicuticle and a procuticle. The epicuticle is a thin structure (1 μm
or less) coated by a layer of wax. In the Argasidae only, a cement layer is also present. Details on
the fine structure and timing of secretion of these layers have been presented by Hackman
(1982), Hackman and Filshie (1982), and Coons and Alberti (1999). The procuticle comprises
inner and outer endocuticular regions. In insects, the external portion of the procuticle is usu-
ally sclerotized, and in such regions it is termed the exocuticle. The insect and tick cuticle
comprises microfibrils of α-chitin embedded in a protein matrix, but mixed to varying degrees
in different parts of the body.
100 BIOLOGY OF TICKS
2.2. ARCHITECTURE OF THE CUTICLE

Chitin is a polymer of β-(1-4)-linked N-acetyl-D-glucosamine that assumes a helicoidal
form with 6 residues per turn. Microfibrils of chitin consist of ~20 molecules aligned anti-
parallel to each other. This tightly packed molecular arrangement likely contributes to the
mechanical strength and chemical stability of arthropod cuticle (Cohen 2010). Chitin is se-
creted as filaments from the apical microvilli of the hypodermal cells, and the proteins are
secreted independently via exocytosis. Proteins that include a Rebers-Riddiford consensus
sequence (Gx8Gx6YxAxExGYx7Px2P) bind to chitin directly. The ultimate pattern that the
chitin fibrils assume likely depends on which proteins cover the surface of the fibers (Ander-
sen 1998). The chitin pattern that is laid down in locust cuticle varies with the day–night
rhythm, a phenomenon that might be explained by a diurnal rhythm of varying protein
secretion (Neville 1975).
The inner endocuticle, shown in Fig. 5.2A, is subdivided into lamellae. The lamellae in in-
sects result from the progressively changing “rotation of [the] planes of the chitin crystallites”
(Neville 1975). But as new layers of cuticle are secreted, the hypodermal cells become ever far-
ther removed from the older layers. How the hypodermis regulates the relative amounts of
secreted protein and chitin and the control for rotation of microfibrils within the matrix to
FIGURE 5.1: A diagrammatic representation of the integument of Rhipicephalus (Boophilus) microplus.

(A) Scutal region. ec, epicuticle; dz, deposition zone for secretion of new cuticle; pc, pore canal. (B)
Alloscutal region. en, endocuticle; ex, exocuticle; Ep, epidermis; GLC, glandular cell; prc, procuticle; TR,
trichogen cell (creates the shaft of the sensory hair). The ovoid cell between the GLC and the TR is a
putative oenocyte. From Hackman, R.H. (1982) Structure and function in tick cuticle. Ann. Rev. Entomol.
27:75–94. Reprinted with permission from the Annual Review of Entomology. ©1982 by Annual Reviews,
www.annualreviews.org.
Integument and Ecdysis 101
form distinct lamellae within some cuticular layers but not others is of special interest. Neville
(1975) favors a process of “self-assembly.” Locke (1998) states that self-assembly may pertain to
some specialized cases but is not likely to be the general case. He has proposed instead that the
specific orientation of chitin microfibrils is influenced by the actin-generated movements of
the microvilli themselves. Alternatively, Moussian (2010) speculates that the rotation of la-
mellae is triggered by a yet unknown biological, chemical, or physical signal (p. 367). Moussian
further posits, “Hence, the construction of the procuticle and its stabilization, including com-
munication with the above epicuticle, is an enormous task that the epidermal cell has to ac-
complish during cuticle differentiation. The concerted underlying molecular mechanisms are
awaiting characterization.”
The layering of chitin fibrils might not depend only on when they are initially secreted. It has
been shown in x-ray diffraction experiments that during the formation of the puparium in
FIGURE 5.2: Transmission electron micrographs of the alloscutal cuticle of female Rhipicephalus
(Boophilus) microplus (A) at the end of the slow feeding phase (maximum thickness) and (B) at full
engorgement. Scale bar in both cases = 1 μm. (A) Border between inner and outer endocuticle is
distinct. The inner, but not the outer, endocuticle is laminate; pore canals (pc) can be seen in both
regions, though they are more numerous, branched, and anastomosed in the inner region. The
subcuticle (sub) is equivalent to the deposition zone labeled in Fig. 5.1. The apical surface of the
cuticle is pleated (see also Fig. 5.5) to allow for complete expansion at engorgement. (B) In the fully
engorged state, the cuticle is about half the thickness of that shown in (A), and the apical folds are
almost completely fl attened. The border between inner and outer endocuticle is indistinct, though it is
imagined to be in the vicinity of the dotted line. The laminae seen earlier in the inner endocuticle are no
longer apparent, and the pore canals are much distorted because of the stretching. Figure taken from
Hackman, R.H. and Filshie, B.K. (1982) The tick cuticle. In F.D Obenchain and R. Galun (Eds.), The
Physiology of Ticks. Oxford, UK: Pergamon Press, 1–42. Reproduced with the kind permission of
CSIRO and Elsevier.
houseflies and blowflies, there is a significant reorientation of the chitin fibrils and a marked
drop in water content of the cuticle 36 hours post-pupation (Fraenkel and Rudall 1940). Tick
alloscutal cuticle has a high water content during the first day after molting (~49% wet weight)
and then declines within 3 weeks to ~31% wet weight (W. R. Kaufman and P. C. Flynn, unpub-
lished data). Water content also increases substantially during the feeding period when new
endocuticle is laid down (Kaufman et al. 2010; see also Section 7.3.3).
2.3. CELLULAR COMPONENTS OF THE INTEGUMENT

The arthropod epidermis normally comprises at least 4 cell types: epithelial cells, oenocytes,
dermal gland cells, and sensory receptor cells.
2.3.1. Epithelial cells

In unfed ticks (female Ixodes ricinus), the epidermal cells are diminutive, staining only feebly with
pyronin (indicating very little RNA). After 7 days of feeding (just prior to the rapid phase of en-
gorgement), epidermal cell volume increases 100-fold, and pyronin staining is marked. The cor-
relation between an astounding degree of epidermal cell hypertrophy and an intensive bout of
endocuticle synthesis underlines the essential role of the epidermal cells in cuticle synthesis (Lees
1952). The role of the epidermis in the secretion of ecdysteroids is discussed in Section 5.2.1.
2.3.2. Oenocytes
The oenocytes of insects constitute a population of large, usually polyploid, cells that are distrib-
uted among the hypodermal cells. They can also be found in the hemolymph or clustered in the
abdominal fat body (Fan et al. 2003). Several reviews of tick integument illustrate oenocytes in
the diagrams, but no further mention of them is made in the text (e.g., Hackman 1982). Coons
and Alberti (1999) reproduce the diagram from Hackman that figures an oenocyte, though,
tellingly, it is left unlabelled (Fig. 5.1B, this volume) and is not mentioned further. This was not
an oversight. “Everyone wants to believe [oenocytes] are present, but we have looked for them
for decades and cannot locate them” (L. B. Coons, University of Memphis, personal communi-
cation, September 21, 2010). Consequently, when I refer to ticks here, I use the term “putative
oenocytes.” Oenocytes have been identified in other arachnids, however. Romer and Gnatzy
(1981) show cells in the legs of Opilionidae (harvestmen) that have all the typical features of
oenocytes (i.e., large size [130 μm], highly polyploid, and abundant agranular endoplasmic retic-
ulum showing tubular and vesicular profiles).
Oenocytes in insects synthesize hydrocarbons (paraffins) destined for the cuticular surface.
In the locust, isolated oenocyte-rich peripheral fat body incorporates radiolabeled acetate into
hydrocarbons and releases these hydrocarbons into the bathing medium when exposed to he-
molymph of locusts, but not when exposed to fetal calf serum (Romer 1990). Wigglesworth
(1988), working with Rhodnius prolixus larvae, provided strong evidence that oenocytes are
the source of precursors of structural lipids for the epicuticle (cuticulin layer), based on the
osmiophilic nature of the cytoplasm, the abundance of smooth endoplasmic reticulum, and the
presence of free lipid spheres.
The formation of the cuticulin layer of insect epicuticle requires polyphenol in addition to
protein and lipid. The lipid and polyphenol are transported to the epicuticle via the pore canals.
Although the cuticulin layer is deposited soon after apolysis, the epicuticle must be permeable
so that the digested products of the old cuticle can be absorbed through it (Wigglesworth
1988). Fan et al. (2003) prepared oenocyte-enriched and epidermal-cell-enriched fractions
from the cockroach Blatella germanica. Using a radiochemical assay with sodium-14C-propio-
nate as a precursor, they demonstrated that it was only the oenocyte fraction that synthesized
hydrocarbons.
In insects, the oenocytes, in addition to the prothoracic gland, are involved in ecdysteroid
synthesis. Locke (1969) notes that the most conspicuous utrastructural feature of oenocytes
in the lepodopteran Calpodes ethlius is the tubular endoplasmic reticulum, which resembles
that of cells known to secrete vertebrate steroids. It has been suggested for the mealworm,
Tenebrio molitor, that in vitro prothoracic glands synthesize ecdysone (E), and that this is
converted to 20-hydroxyecdysone (20-OHE) by the abdominal oenocytes (Romer 1990). Be-
cause oenocytes are abundant in the femora of harvestmen (Arachnida: Opiliones) and iso-
lated femora synthesize both E and 20-OHE, Romer and Gnatzy (1981) suggested that the
source of these ecdysteroids is the oenocytes. Ecdysteroid synthesis in ticks is discussed in
Section 5.2.1.
2.3.3. Dermal gland cells

In insects, the dermal glands secrete the cement that protects the wax overlying the epicuticle
(Wigglesworth 1965). Both argasid and ixodid ticks possess dermal glands, but only the argasid
ticks secrete a cement layer; the dermal glands are assumed to be the source of the cement
(Hackman 1982).
In ticks, there are at least 6 types of dermal glands, the names of which are variable and the
functions of which are somewhat in dispute (Coons and Alberti 1999). Most of these glands are
covered in other chapters. The dermal gland secretions discharge to the surface of the cuticle,
but the nature of the secretion varies according to the tick species, and the glands might not be
active in all life cycle stages (Hackman and Filshie 1982). In engorged female I. ricinus, the der-
mal glands produce an oily secretion that spreads over the surface of the cuticle. When handled,
Rhipicephalus (B.) microplus females deposit a clear fluid onto the cuticle; between the periods
of apolysis and ecdysis, the dermal glands of larval and nymphal I. holocyclus (Australian
paralysis tick) secrete a creamy exudate. In the engorged nymphs and adults of the latter, the
dermal glands secrete a paralysis toxin with properties similar to its salivary toxin (Hackman
and Filshie 1982).
The dermal gland secretion apparently does not contribute to the water impermeability
of the tick integument. Yoder and Peterson (1998) tested this assumption on Amblyomma
americanum by comparing to controls the rate of water loss and the critical transition tem-
perature of ticks induced to produce dermal gland secretion. They found that there was no
significant difference in these parameters between the 2 groups. Moreover, the dermal gland
secretion had no inhibitory effect on the ability of the ticks to absorb water vapor directly
from the atmosphere.
Pavis et al. (1994) comment on the resistance of A. variegatum to various pathogenic agents.
When ticks are handled roughly, the dermal glands secrete droplets over the cuticle. Pavis et al.
tested whether this material might constitute a defensive secretion protecting against predators
and pathogens by collecting the secretions of various life stages of ticks. The fire ant, Solenopsis
geminate, will normally prey on larvae of the lepidopteran Galleria mellonella, but when the
larvae were coated with extract of dermal gland secretion, predation was reduced significantly.
Although the dermal secretion extracts of A. variegatum could repel fire ants, the material
showed no overt toxicity to the Galleria larvae and no antifungal or antinematode activity; there
was, however, antibiotic activity of dermal gland secretions from engorged females against Ba-
cillus thuringiensis and Serratia marcescens (Pavis et al. 1994).
During the blood meal, ixodid ticks spend numerous days at relatively high tempera-
tures. One-host ticks (e.g., Rh. [B.] microplus and Dermacentor albipictus) pass through the
larval, nymphal, and adult stages on the same host and thus spend many weeks at an ele-
vated temperature. Yoder et al. (2009) demonstrated that the dermal gland secretions of Rh.
sangineus confer resistance to heat exposure during the feeding period. Dermal gland secre-
tion was induced by pinching the legs with forceps. Controls were handled in a similar way,
but without pinching the legs, and the absence of secretion was confirmed. Heat tolerance
was then tested in all ticks (adult females) maintained at 97% relative humidity (RH). Tem-
peratures of ≥60°C were lethal to all unfed ticks, but at 58°C, 100% of the controls died fol-
lowing 1 hour of exposure, whereas only 50% of the pinched ticks died. At 56°C, 70% of the
unpinched ticks, but only 4% of the pinched ticks, died. Although a major component of the
dermal gland secretion in several (though not all) species of tick consists of squalene (Yoder
et al. 1993), exogenous application of squalene to ticks did not enhance temperature toler-
ance. Finally, Yoder et al. (2009) exposed ticks to 37°C (100% RH) for up to a month and
compared the survivorship curves of ticks that secreted with those that did not secrete der-
mal fluid. The curves spread apart within only a few days, and the deviation between the
curves broadened steadily, with the secreting ticks surviving significantly longer than the
non-secreting group.
2.3.4. Sensory receptor cells

In insects and ticks, cuticular sensory hairs (setae) are ubiquitous on the body surface. The hair
communicates directly with a sensory neuron that is surrounded by a number of other cells, all
sitting among the epidermal cells (Chapman 1998; Gillott 2005). Sensory function is covered in
Chapter 13, but the question arises of how sensory continuity might be maintained during the
pharate stage of the molting process. A detailed discussion is beyond the scope of this chapter,
but in the caterpillar of the noctuid moth, Barathra brassicae, sensitivity is completely lost for
only about 30 minutes at ecdysis (Chapman 1998).
3. SCLEROTIZATION
Sclerotization is the main process whereby most arthropods convert the flexible cuticle into a
rigid structure. Wherever flexibility is required (e.g., arthrodial membranes), the cuticle remains
non-sclerotized. Although arthrodial membranes are flexible, they are not inherently extensible.
Ticks and blood-sucking insects expand enormously during the blood meal, so in these crea-
tures, much of the cuticle is not sclerotized and has the further property of being extensible. This
is discussed in Section 7.
3.1. THE CHEMISTRY AND CONTROL OF SCLEROTIZATION

Although very little work has been conducted on ticks, the basic pathways are assumed to be
similar to those described for insects. Sclerotization involves the cross-linking of cuticular
matrix proteins and chitin by o-quinones. The sites at which cross-linking occurs include the
imidazol group and terminal amino groups of cuticular proteins, and probably the hydroxyl
groups of the N-acetylglucosamine residues in chitin (Andersen 2005). The consequences of
these reactions are increased hydrophobicity, greater rigidity of the peptide chain, reduced
movement between proteins and reduced movement between protein and the chitin filament
system (Andersen 2005). The detailed biochemical pathways producing the precursors of o-
quinones have been presented by Andersen (2005).
O-quinones are highly reactive molecules, so most of the animal’s proteins must be shielded
from them. Thus, quinones themselves are formed only at the sites of sclerotization, from pre-
cursors synthesized elsewhere and transported by the epithelial cells to the subcuticular space.
This process is described in some detail by Andersen (2005). Bursicon is the hormone in insects
that triggers the process of sclerotization soon after emergence of each instar (Reynolds 1983).
The status of bursicon in ticks is considered in Section 4.2.
4. MOLECULAR STUDIES RELATING

TO THE INTEGUMENT
In recent years, molecular studies have identified proteins in ticks that relate directly to integu-
mental function.
4.1. CUTICULAR PROTEINS IN INSECTS AND

THEIR HOMOLOGUES IN TICKS
Cuticular proteins (CPs) have been categorized into 12 families based on some defining motifs
(Willis 2010). Assembled genomic sequence data reveal over 100 CP genes in I. scapularis. Ten of
these have 2 or 3 consensus regions well documented in Crustacea (Willis 2010), but there is still
no expressed sequence tag support for these multiple occurrences in Ixodes (Judy Willis, Uni-
versity of Georgia, personal communication, December 3, 2010).
4.2. BURSICON
Two cDNA partial sequences for bursicon (bur-α and bur-β) have been detected in the syngan-
glion (including lateral segmental organs and lengths of pedal nerves) of female D. variabilis
(unfed, partially fed virgin, partially fed mated, and engorged) (Donohue et al. 2010). It is puz-
zling that transcripts for a hormone that normally appears only during a brief period following
ecdysis (Reynolds 1983) can be found in the synganglion of an ixodid female long after ecdysis.
One possibility relates to another known function of bursicon in insects. Shortly after ecdysis,
but prior to sclerotization, the integument of some insects becomes more compliant, a process
referred to as plasticization. Post-ecdysial plasticization in adult blowflies is triggered by bursi-

con (Reynolds 1983), as is the plasticization event occurring in the wings of newly emerged adult
Manduca sexta (Reynolds 1977). The possibility of cuticular plasticization in female ixodid ticks
is explored in Section 7. Bissinger et al. (2011) report on the expression of numerous genes, in-
cluding that of bursicon, in the synganglion of Dermacentor variabilis at various stages in the life
cycle, including in the unfed and feeding female. It is certainly conceivable that bursicon might
be involved in post-ecdysial cuticle plasticization in ticks (though a plasticization function has
not yet been demonstrated even in pharate nymphs or adults) and/or associated with the me-
chanical properties of cuticle during feeding. In the latter case, there is evidence that dopamine
(DA) plays a role (Section 7), but this does not preclude the possibility that bursicon might also
participate.
4.3. ECLOSION HORMONE

Donohue et al. (2010) also revealed the presence of a cDNA partial sequence homologous to an
insect (Aedes aegypti) eclosion hormone (EH). The same question that arises regarding bursicon
above also arises here regarding the presence of transcripts for a hormone that, in insects, is se-
creted only briefly around the period of ecdysis. However, EH, like bursicon, contributes to the
signal for cuticle plasticization in the wings of newly molted M. sexta (Reynolds 1977) and con-
ceivably could be involved in the plasticization that occurs in ticks during feeding, although, as
for bursicon, no experimental evidence is available.
4.4. CHITINASES
Chitinases are a widely distributed group of enzymes that hydrolyze chitin to oligosaccharides
(Arakane and Muthukrishnan 2010). In addition to their well-known role in cuticle digestion
during molting, chitinases also serve other functions: (i) as digestive enzymes in organisms that
have chitin in their diet, (ii) in immunity against pathogens containing chitin, (iii) in the regu-
lation of cell proliferation and remodeling in insect and mammalian cells, (iv) in insect eclosion,
(v) as a component of some insect venoms, and (vi) to facilitate the transmission of some path-
ogens by disrupting the peritrophic matrix or other chitin-containing structures. In insects, the
induction of chitinase at the time of molting is induced by ecdysteroids (Arakane and Muth-
ukrishnan 2010).
Chitinase has been identified and characterized in 1 ixodid tick, Haemaphysalis longicornis,
from a cDNA library and designated as CHT1 cDNA (You et al. 2003). Recombinant CHT1 ex-
hibits chitinolytic activity in a biochemical assay, and allosamidin, a potent chitinase inhibitor
of the 18-chitinase family (Arakane and Muthukrishnan 2010), is likewise a potent inhibitor of
recombinant CHT1 of H. longicornis (You et al. 2003). Although the deduced amino acid
sequence of CHT1 is only 31% homologous to chitinase of Aedes aegypti, the sequence of the
catalytic center region II (indispensable for enzyme activity) is highly conserved in all family 18
chitinases characterized so far (You et al. 2003). Immunohistochemical localization of CHT1 in
H. longicornis was confined to the epidermis and the midgut of engorged nymphal ticks 10 days
post-engorgement. The epidermal location is consistent with the presumed role of chitinase in
digesting the old cuticle.
5. MOLTING
5.1. OVERVIEW OF MOLTING IN TICKS

Argasid ticks achieve the adult stage via a number of larval and nymphal instars, whereas ixodid
ticks proceed to the adult stage via a single larval and single nymphal stage. Diehl et al. (1982),
using radio-immunoassay/high-performance liquid chromatography, documented the hemo-
lymph ecdysteroid titer and total ecdysteroid content of nymphal A. hebraeum from the begin-
ning of the blood meal through ecdysis to the adult stage; ticks were maintained at 26°C while
off the host. Ecdysteroid levels rose slowly during the 7 days of feeding and the initial 10 days
post-engorgement, attaining about 1 ng/tick (50 ng/ml hemolymph) at that time. By the 18th
day, ecdysteroid levels had begun rising more steeply, peaking at around day 22 (14 ng/tick; 500
ng/ml). Ecdysteroid levels fell precipitously thereafter, attaining the low values characteristic of
the unfed state (0.5 ng/tick; <20 ng/ml) by day 35. Diehl et al. mapped the various stages of cu-
ticle growth (deposition of nymphal endocuticle, epidermal mitoses, apolysis, deposition of
pharate adult epicuticle and exocuticle, and final ecdysis) onto the ecdysteroid profile. They also
provided useful information on the ultrastructure of the integument during the whole process.
A study parallel to that described above was conducted on fifth-stage nymphs of Ornithodo-
ros moubata (Germond et al. 1982). In this case, the total period between feeding and ecdysis
was only about 10 days, but the rising/falling profile of total ecdysteroid content and hemolymph
ecdysteroid titer were astonishingly similar over this time to what was observed in A. hebraeum
over 35 days, and the same predominance of E and 20-OHE was observed. Moreover, the story
is similar for third instar nymphs of O. parkeri (Zhu et al. 1994) and for the production of em-
bryonic and hexapod larval cuticle in O. moubata (Dotson et al. 1991). In brief, the role of ecdys-
teroids in molting is similar in both families of ticks, and it is generally similar to what is
observed in other arthropods (Diehl et al. 1982; Germond et al. 1982). In Ixodidae, ecdysteroids
also trigger salivary gland degeneration following the adult female blood meal (see Chapter 7)
and serve as the vitellogenic hormone in both families of ticks (see Chapters 16 and 17).
5.2.1. Site and control of ecdysteroid synthesis

The major source of the molting hormone precursor, E, in insects is the prothoracic gland
(PTG), a tissue that is of ectodermal origin (Gillott 2005). Ticks produce E and 20-OHE, but
they do not have PTGs. In insects, several tissues are known to be sources of E (i.e., the PTG,
oenocytes, epidermis, testes, and ovaries) (Delbecque et al. 1990). The source of E in argasid
ticks is the epidermis; E is converted to 20-OHE by the fat body (Zhu et al. 1991). The epidermis
is also the source of ecdysteroids in ixodid ticks. When isolated fragments of A. hebraeum integ-
ument taken from females 4 days post-engorgement are cultured in vitro, ecdysteroid synthesis
does not occur unless the culture includes an extract of synganglion taken from similar females.
Other tissues (ovary and fat body) do not show steroidogenic activity when incubated with syn-
ganglion. Treatment of synganglion extracts with trypsin abolishes the steroidogenic activity,
indicating that the active factor in the synganglion is a peptide or protein (Lomas et al. 1997).
Lomas et al. also demonstrated that ecdysteroid synthesis by fragments of integument is medi-
ated by a cyclic adenosine monophosphate second messenger system. See Chapter 16 for a more
detailed analysis of the ecdysteroid systems in ticks.
A major gap in our knowledge is the specific cell population in the epidermis responsible for
ecdysteroid secretion. However, as mentioned earlier (Section 2.3.2), the oenocytes have also
been proposed to secrete ecdysteroids in some insects and arachnids, so it remains an open ques-
tion whether the putative oenocytes of ticks might also be the specific site of E synthesis in ticks.
6. INTEGUMENT DURING THE

POST- MOLT AND F EEDING PERIODS
6.1. OVERVIEW OF CHANGES IN CUTICLE STRUCTURE POST-MOLT

In insects, cuticle synthesis begins around the time of apolysis. Following a short period of body
volume expansion, much of the cuticle becomes sclerotized and at that point consists of the
epicuticle, exocuticle, and a small amount of outer endocuticle. To synthesize more endocuticle
during the intermolt stage, the epithelial cells secrete apically a mixture of proteins and chitin,
which ultimately layers itself in the complex manner already discussed (Section 2.2). Morpho-
genetic hormones probably play an important role in regulating the changing pattern of protein
and chitin secretion over time. Wolfgang and Riddiford (1986) demonstrate this for M. sexta, for
which species we know the detailed temporal pattern for the secretion of prothoracicotropic
hormone, 20-OHE, and juvenile hormone (JH). They correlate the changing texture and thick-
ness of the cuticular lamellae with the appearance and disappearance of specific larval cuticular
proteins throughout the fourth and fifth instars. Exogenous application of the JH analogue me-
thoprene affected the pattern of protein synthesis, as well as the lamellar structure of the cuticle.
Finally, exogenous application of 20-OHE was able to induce the changes that are inhibited by
JH. In brief, the pattern of the cuticle laid down by the epidermal cells is controlled hormonally.
6.2. GROWTH OF ENDOCUTICLE DURING FEEDING

IN FEMALE IXODID TICKS
In insects generally, the laying down of endocuticle occurs during the intermolt period (Gillott
2005). In adult female A. hebraeum, endocuticle is laid down for about 3 weeks after ecdysis and
before the taking of the blood meal. During this time, the dry weight of the cuticle dissected from
the dorsal alloscutum increases almost 3-fold (W. R. Kaufman and P. C. Flynn, unpublished data).
In blood-sucking insects and in argasid ticks, one assumes that there is sufficient endocuticle
available at the beginning of the instar to accommodate the 5- to 10-fold increase in body size. But
the enormous size of the blood meal in female ixodid ticks raises the question of whether there
could be enough cuticle at ecdysis to cover the surface area of a fully engorged tick. Rhodnius larvae
also lay down more endocuticle immediately following the blood meal (Hillerton 1978), which
seems odd considering that by this time it has already expanded as much as it will during the sta-
dium. Hillerton speculates on the physiological rationale for synthesizing endocuticle that will be
reabsorbed again within a matter of days, but there is little experimental data relating to the matter.
Lees (1952) investigated endocuticle synthesis during the blood meal using female I. ricinus.
Lees concluded that cuticle synthesis in the alloscutum occurs during the slow phase of engorge-
ment only (the thickness increasing from ~50 μm in unfed ticks to ~105 μm in partially
fed ticks). During the rapid phase of engorgement, the cuticle thins out, reaching ~50 μm in
fully engorged ticks.
Okura et al. (1997a) examined the histological and ultrastructural changes occurring in the
alloscutal integument of H. longicornis during feeding. The epidermal cell layer, flat and incon-
spicuous in unfed specimens, grows enormously during the early days of feeding. The outer
endocuticle thickens about 1.5-fold, and the inner endocuticle about 5-fold, during the first 4
days of feeding. It is interesting that these authors report no signs of epidermal cell division in
the adult female accompanying the marked increase in cuticular surface area during feeding.
This is in contrast to what occurs in the nymph-to-adult molt in both A. hebraeum (Diehl et al.
1982) and in the fifth instar-to-adult molt in O. moubata (Germond et al. 1982) (Section 5.2).
Electron micrographs of hypodermal cells in H. longicornis during the slow feeding phase show
an abundance of rough endoplasmic reticulum (RER) and small secretory vesicles near the
apical border of the cell; this is presumably cuticular material destined for exocytosis. Following
copulation, large vesicles are also seen and have been interpreted as fusion products of the small
vesicles. The amount of RER is also reduced later during the rapid expansion phase (Okura et al.
1997a). Okura et al. are somewhat equivocal about whether or not endocuticle synthesis occurs
during the rapid phase of feeding.
A few authors have reported that cuticle synthesis might continue into the rapid phase of
feeding (Kitaoka et al. 1958; Cherry 1973). This view has been supported recently. In A. he-
braeum, the cuticle thickness of unfed ticks increases by ~31% through the slow phase of
engorgement (from ~133 μm to ~174 μm) and then thins to ~50% of the maximum value
(~90 μm in large engorged ticks) (Kaufman et al. 2010). Flynn and Kaufman (2010) con-
ducted a dimensional analysis of volume data from a large sample of ticks throughout the
feeding period, modeling the body as an ellipsoid. From these measurements, both the vol-
ume and the surface area of the body throughout the feeding period could be calculated. The
results of this modeling exercise are shown in Fig. 5.3, and the basic conclusion is that 37%
to 43% of the total final mass of cuticle is synthesized during the rapid phase of engorge-
ment, but synthesis ceases at a fed-to-unfed weight ratio of ~60. Flynn and Kaufman (2010)
conducted a similar analysis of the data provided by Lees (1952), Dillinger and Kesel (2002),
and Andersen and Roepstorff (2005) for I. ricinus and concluded that approximately 46% to
53% of the total cuticle synthesis in the latter species also occurs during the rapid phase of
engorgement. The signaling processes that trigger cuticle synthesis have not been investi-
gated. Okura et al. (1997a) suggest that the act of copulation might be a signal resulting in the
shut-down of cuticle synthesis in H. longicornis. In our laboratory colony of A. hebraeum,
however, ~70% of females will have copulated by the fifth day of feeding, when the weight
range of the ticks is only 50–75 mg (Mao and Kaufman 1999). This is equivalent to a fed-to-
unfed weight ratio of only about 2 to 5, well below the weight ratio (~13) at which cuticle
thickness peaks (Kaufman et al. 2010), so copulation cannot be the signal to cease endocu-
ticle synthesis in A. hebraeum.
7. MECHANICAL PROPERTIES OF THE CUTICLE
The difference in size between an unfed and a fully engorged female ixodid tick never fails to as-
tound the person who sees it for the first time (Fig. 5.4). The fed/unfed weight ratio can exceed
100-fold, which is greater than what is achieved by any other blood-sucking animal (Kaufman
FIGURE 5.3: Model calculation of total cuticle weight of female A. hebraeum as a function of tick weight,
and measured wet and dry weights for dorsal cuticle samples. The model shows that cuticle continues to
be synthesized partway through the rapid engorgement phase. The measured weights of the dorsal
cuticle samples (wet and dry) taken during the rapid phase are greater than those taken during the slow
phase. Figure reproduced from Flynn, P.C. and Kaufman, W.R. (2010) Female ixodid ticks grow
endocuticle during the rapid phase of engorgement. Exp. Appl. Acarol. 53:167–178 (Fig. 5) with kind
permission from Springer Science+Business Media B.V.
2007).1 How can the integument expand to such an enormous degree? Certainly one part of this is
the highly pleated nature of the external surface of the cuticle, presented in Fig. 5.5. Another part
might be the presence of resilin, the “rubber-like” protein found in the cuticle of many insects in
structures that require great flexibility (e.g., arthrodial membrane) or elasticity (e.g., the catapult
mechanisms and flight mechanisms in jumping and flying insects). Good descriptions of these
systems have been provided by Neville (1975) and Alexander (1983). The most convenient way to
detect resilin is to measure its pH-dependent blue fluorescence under ultraviolet illumination.
Fluorescence (due to the abundance of dityrosine cross-links in resilin) is apparent under neutral
and strong alkali conditions and is inhibited by strong acid (Neff et al. 2000; Lyons et al. 2011).
Resilin has been identified via such a fluorescence assay in the alloscutum of I. ricinus (Dillinger
and Kesel 2002). In Section 7.3, I consider the mechanical properties of the alloscutal cuticle.
7.1. CHANGING MECHANICAL PROPERTIES OF THE ALLOSCUTAL

CUTICLE DURING THE POST-MOLT PERIOD
In blood-sucking insects and ticks for which specific regions of the cuticle must expand substan-
tially during feeding, the cuticle must supply skeletal support without being sclerotized. We have
recently monitored cuticle growth and water content in A. hebraeum during the first few months
FIGURE 5.4: An unfed and a fully engorged A. hebraeum female to show the incredible difference in size.
Photo reproduced by kind permission of Mr. Alex Smith, Department of Biological Sciences, University
of Alberta and of the Company of Biologists Ltd; cover photo of the Journal of Experimental Biology,
Volume 213(16), August 2010 (reprinted with permission).
post-molt, with the ticks maintained at a nominal 99% relative humidity. The water content of
the cuticle fell from a mean value of ~49% of cuticle wet weight during the first day post-molt
to ~32% between days 7 and 10, and it then remained at that level (~30%–32%) for at least 21
weeks. During this period, the mean cuticle weight of the dorsal alloscutum rose from ~22 μg/
mg body weight (bw) to ~59 μg/mg bw by week 21 (W. R. Kaufman and P. C. Flynn, unpublished
data). Apparently the reduced water content and the addition of endocuticle during this time
provide sufficient skeletal support without the need for sclerotization.
7.2. CUTICULAR PLASTICIZATION IN INSECTS

The few studies to date on plasticization in female ixodid tick cuticle have taken their lead
from a number of classic studies on Rhodnius, so it is convenient at this point to summarize
the conclusions of those studies. Maddrell (1966) demonstrated the following in fifth instars
of Rhodnius: (i) complete plasticization of the abdominal integument occurs within only a
minute or two of commencing the blood meal; (ii) plasticization in any segment is prevented
by surgical denervation of that segment; (iii) the normal occurrence of plasticization is not
triggered by a component of the blood meal; (iv) the sensory system that triggers the process
probably resides somewhere in the pharynx, rather than in the midgut; and (v) the plasticiza-
tion effect is completely reversed within 7 hours after engorgement. The story for Rhodnius
was further advanced by Reynolds (1974, 1975) as follows: (i) micromolar concentrations of
5-hydroxytryptamine (5-HT) trigger plasticization when injected into unfed fifth instars of
Rhodnius; (ii) plasticized cuticle is about 20% more hydrated than non-plasticized cuticle; and
(iii) the transport of protons into the cuticle (i.e., a reduction in pH) is likely the intracuticular
FIGURE 5.5: Scanning electron micrographs of the cuticle of I. ricinus showing the pleated apical
surface at 3 stages of feeding: (A), (B) unfed, (C), (D) partially fed, and (E), (F) fully engorged. The
pleats consist of outer endocuticle coated by a thin epicuticle. The panels illustrate clearly the
progressive fl attening of the pleats with increasing tick size. Figure is taken from Dillinger, S.C.G. and
Kesel, A.B. (2002) Changes in the structure of the cuticle of Ixodes ricinus L. 1758 (Acari, Ixodidae)
during feeding. Arthropod Struct. Dev. 31:95–101 and reproduced with the kind permission of Elsevier.
mechanism for plasticization. With a decrease in intra-cuticular pH, the consequent increase
in fixed positive charges would cause electrostatic repulsion between the protein molecules,
making their close approach and the formation of van der Waals bonds more difficult. The
excess of positive charges represents a Donnan osmotic excess within the cuticle, and this will
tend to draw water in, further reducing the extent of secondary interactions among the mac-
romolecules. The result is a marked reduction in the force needed to stretch the cuticle.
7.3. CUTICULAR PLASTICIZATION IN TICKS

7.3.1. The foundation study
Okura et al. (1996) reported that copulated females (H. longicornis) expanded more than
virgin females when “inflated” for 30 to 50 h with a saline solution at a pressure of 150 mmHg
(20 kPa). Cuticle loops were stiff at pH 7 and 8 but became progressively more extensible at
pH 6, 5, and 4 (Okura et al. 1997b). An extract of the synganglion from virgin females stim-
ulated cuticular plasticization when injected into partially fed virgin females, as did an ex-
tract of hemolymph from copulated females (Okura et al. 1997c). Octopamine, noradrenaline,
and DA also stimulated plasticization when injected into virgin females; 5-HT had no plas-
ticizing effect in amounts of up to 10 mM (Okura et al. 1997c). Okura et al. proposed that
copulatory stimuli trigger the release from the synganglion of a factor, perhaps a biogenic
amine, that results in plasticization. It has been proposed that a mitochondria-rich acido-
philic epidermal cell type found in the hypodermis of H. longicornis secretes into the sub-
cuticular space the protons ultimately accounting for the descent of intracuticular pH (Okura
et al. 1997b).
7.3.2. Feeding-induced plasticization in A. hebraeum and O. moubata

We have begun to explore the question of cuticular plasticization in A. hebraeum and have made
limited observations on O. moubata. Using a technique based on the one developed by Reynolds
for Rhodnius, we measure the creep of the loop (in millimeters) when it is subjected to a defined
force, and we calculate the creep per unit loop length as a function of time (“rate of creep,” in
units of μm min−1 mm−1 loop length). As argasid ticks have a soft, flexible cuticle and feed to a
modest size relative to ixodid ticks, we hypothesized that the cuticle would not experience a
significant degree of plasticization during engorgement, and this is what we observed (Kaufman
et al. 2010).
A more elaborate data analysis was conducted for cuticle loops of A. hebraeum. Our exper-
imental protocol permitted the determination of a so-called Maxwell viscosity of the loops,
calculated from the slope of the creep curve, the units for which are gigaPascal seconds (GPa s).
Plasticization according to this measure is indicated by a decrease in Maxwell viscosity. We
had expected the process of cuticular plasticization to begin early in the slow feeding phase,
but we also hypothesized that the degree of plasticization would be markedly enhanced during
the rapid phase, because of the very high rate of cuticular expansion occurring at that time.
The mean Maxwell viscosity fell markedly from no less than ~720 GPa s for unfed cuticle
to ~108 GPa s for cuticle during the slow phase of engorgement. Thereafter, there was no sig-
nificant further reduction in Maxwell viscosity, even for the smaller engorged ticks (fed-to-
unfed ratio < 60; 85 GPa s). For the very largest engorged ticks, however (fed-to-unfed ratio >
60), Maxwell viscosity fell significantly further to about 42 GPa s. In A. hebraeum females that
engorged to >60 times their unfed weight, the mean Maxwell viscosity rose from 42 GPa s on
the day of engorgement to 106 GPa s 24 h later, but with no further reversal toward the vis-
cosity characteristic of unfed ticks (>720 GPa s) over the next 3 days. Because of their small
meal size and sclerotized dorsum, males do not plasticize their cuticle during feeding (Kaufman
et al. 2010).
Recent, unpublished work in our laboratory calls into question some of the results reported
by Kaufman et al. (2010). I elaborate on these in Section 9.7 (“Future Perspectives”).
7.3.3. Proton transport and hydration of cuticle as the

mechanism of cuticle plasticization
Similar to what had been shown for H. longicornis (Okura et al. 1997b), the Maxwell viscosity of
A. hebraeum cuticle loops is highly influenced by the prevailing pH. Mean Maxwell viscosity fell
from close to 400 GPa s at pH 8.0 to 2.2 GPa s at pH 5.7. Although these extreme pH values are
unlikely to be within the physiological range (we have not yet been able to measure the endogenous
pH of cuticle from unfed and feeding ticks), a cuticular pH of ~6.5 would be sufficient to account for
the lowest viscosity observed under physiological conditions (42 GPa s for large engorged ticks on
the drop-off day) (Kaufman et al. 2010). DA stimulates cuticular plasticization in A. hebraeum (see
Section 7.3.4). Concanamycin A (ConA) is a well-established H+-ion-transport inhibitor in many
tissues. Consistent with the hypothesis of H+-ion transport being the mechanism for plasticization,
ConA significantly attenuates the low Maxwell viscosity induced by DA (Kaufman et al. 2010).
As mentioned above, in Rhodnius, the water content of plasticized cuticle is ~20% greater
than that of unplasticized cuticle (Reynolds 1975). In A. hebraeum females, the water content of
the cuticle rose progressively from a mean of ~23.4% (wet weight of cuticle) in unfed ticks to
about 34% in ticks at a fed-to-unfed weight ratio of 3; there was no further hydration thereafter
to full engorgement. This is consistent with the observation that the major drop in Maxwell
viscosity occurs in the weight ratio range of 2–10. There was no reduction in Maxwell viscosity
correlating to the observed fluctuations in the cation content of tick cuticle (Na+, K+, Ca2+, and
Mg2+) during the feeding cycle (Kaufman et al. 2010).
7.3.4. Pharmacology of cuticle plasticization

Kaufman et al. (2010) tested a number of biogenic amines and related drugs on small partially
fed ticks (all at 1 mmol kg−1 bw). The response in each case was compared to the Maxwell vis-
cosity pertaining to saline-injected controls (~75 GPa s). Maxwell viscosity following injection
of 5-HT was ~114 GPa s. Unlike the case for 5-HT, DA reduced Maxwell viscosity significantly
relative to the control (~29 GPa s). We next tested 2 drugs that are structurally related to DA:
octopamine and tyramine. Both of these drugs augmented Maxwell viscosity significantly rela-
tive to the control (~210 GPa s for octopamine and ~146 GPa s for tyramine). It is not yet known
whether these drugs act as competitive antagonists at the putative DA receptor. Two of the drugs
that did not induce plasticization (5-HT and octopamine) were tested as potential antagonists of
DA-induced plasticization; both were tested at 1 mmol kg−1 bw. Neither, however, significantly
reversed the effect of DA. Because of the relatively small sample sizes, the high variability, and
the fact that only a single dose was tested, all these results must be considered as preliminary for
the moment, but at least they point to the likelihood of elucidating the pharmacological control
pathway of cuticular plasticization.
The pharmacological results with biogenic amines suggest that cuticle plasticization in ix-
odid ticks might be under neural control similar to what was shown for Rhodnius (Maddrell
1966), and indeed there are a series of opisthosomal nerves that terminate in the integument of
both families of ticks (Binnington 1981; Shoukrey and Sweatman 1984).
8. WHAT DETERMINES THE SIZE OF EACH INSTAR?
The ultimate size of the engorged female determines the number of eggs that she can produce,
and so understanding what determines tick instar size has implications for biological control.
The egg mass laid is directly related to the engorged weight, and engorged weight is directly re-
lated to unfed tick size (Obenchain et al. 1980). Unfortunately, many studies on ticks use abso-
lute weight or, even less satisfactory, the number of days feeding as a proxy for “size.” But unfed
individuals of a given species can vary enormously in size; for example, in my current colony of
A. hebraeum, unfed females can range from ~10 mg to >40 mg. It thus seems obvious that the
physiological state of a partially fed female will be better gauged by the fed-to-unfed weight ratio
than by absolute weight. Obenchain et al. (1980) introduced the concept of “relative engorge-
ment state” (RES) based on a “scutal index” (SI), a methodology that has not been adequately
exploited among tick biologists, in my opinion. A distinct advantage of the SI measure is that
one can calculate the RES in wild engorged or partially fed ticks for which the unfed weight
cannot be known directly.
In the conclusion to his review article on tick cuticle, Hackman (1982, p. 92) makes the following
statement that remains pertinent today:
Literature on tick cuticle contains its share of speculations as explanations of mechanisms and
reaction pathways. These speculations serve a useful purpose by stimulating further research, even
though they lack sound supportive evidence, but they become counterproductive if they reach the
stage of “proof by repeated affirmation.” As has been stated before in reference to work on insect
cuticle, steady progress demands an uncompromising methodology.
In the spirit of Hackman’s admonition, I offer the following suggestions.
9.1. THE CELLS COMPRISING THE INTEGUMENT

Whereas the function of the hypodermis in secreting the cuticle is obvious, the functions of
other cells (in particular the putative oenocytes) have not been adequately studied. Ultrastruc-
tural and cytochemical techniques applied to the integument of larval or nymphal ticks during
the days following feeding are needed in order to firmly establish the roles of these cells in cu-
ticle synthesis.
9.2. SCLEROTIZATION
Nothing is known about the control and mechanism of sclerotization in ticks, at either hor-
monal or biochemical levels, or about what determines the detailed regional differences. What
accounts for the argasids having only small islands of sclerotization on the idiosoma in addition
to the capitulum and appendages? Although the functional significance of maintaining an un-
sclerotized alloscutum in ixodids is to permit enormous expansion, why should the scutum be
sclerotized? Sclerotized capitulum and appendages are also obvious (the penetration of skin and
strength to support body weight), but why the gonopore, spiracular plates, and anal pore? In
addition to these questions on the adaptive significance, how does each region of the hypo-
dermis know what type of cuticle to produce? Standard cell biological techniques for answering
these questions could easily be adapted to ticks.
9.3. THE HORMONAL INFLUENCE ON CUTICULAR ARCHITECTURE

How can a “simple” epithelium, secreting new layers of chitin and protein under old, manage to
achieve such complexity? As described earlier for M. sexta, the changing hormonal environment
throughout the first stadium is a major component of this control mechanism (Wolfgang and
Riddiford 1986). The experimental design is straightforward, and at least we know something
about the changing ecdysteroid levels throughout the feeding and post-engorgement periods in
nymphal and adult ticks. Experiments on ticks similar to those of Wolfgang and Riddiford
should be attempted, perhaps using hemolymph from various stages of the feeding cycle as a
source of the putative signaling molecules controlling cuticle development. An unidentified
hemolymph-borne factor is believed to be responsible for the incredible growth and develop-
ment of the salivary glands occurring in ixodid females during the feeding period (Coons and
Kaufman 1988). It seems reasonable to hypothesize that this factor may well act more generally
than on the salivary glands alone, and the cuticle is an obvious possibility.
9.4. WHICH SPECIFIC CELL TYPES SECRETE ECDYSTEROIDS?

Although it has been established that the source of ecdysteroids in ticks is the integument, which
of the specific cell types are involved has not been explored. One way to pursue this would be to
produce (putative) oenocyte-rich or epidermal-cell-rich cultures (Fan et al. 2003) and look for
the release of ecdysteroids in the culture medium using radioimmunoassay (e.g., Mao and
Kaufman 1999). Another possibility would be to look for specific enzymes involved in the met-
abolic pathway for E synthesis.
9.5. ENDOCUTICULAR SYNTHESIS: CONTROL

OF ONSET AND TERMINATION
The literature regarding the time course of feeding-induced endocuticle synthesis is still contro-
versial, with some authors supporting the view that synthesis ceases at the onset of the rapid
phase of engorgement (Lees 1952; Dillinger and Kesel 2002; Andersen and Roepstorff 2005).
One suggestion is that spermatophore transfer might be the signal for termination (Okura et al.
1997a). Other authors suggest that endocuticle synthesis continues well into the rapid phase of
engorgement (Kitaoka and Yajima 1958; Cherry 1973; Flynn and Kaufman 2010). The issue
should be resolved by measuring cuticle synthesis directly using the incorporation of radiolabeled
amino acids into cuticular protein or of radiolabeled N-acetylglucosamine into chitin. These
precursors could be injected at various stages of feeding, cuticle samples would be taken within
a fixed time post-injection, and the amount of labeling specifically incorporated into the macro-
molecular fabric of the cuticle could be determined. Such experiments would not only resolve
the issue definitively but also give a direct measure of the rate of synthesis at various stages of
feeding. Perhaps more difficult to establish, but equally important, is the signaling process that
triggers cuticle growth initially and that which stops it prior to the end of engorgement.
9.6. CUTICULAR PLASTICIZATION: PHARMACOLOGY

AND THE ROLE OF OPISTHOSOMAL NERVES
The limited pharmacological evidence we have to date on cuticle plasticization is in accord with
the idea that the process may be triggered and controlled by branches of the opisthosomal nerves
terminating at the epidermis; this is how cuticle plasticization is controlled in Rhodnius (Mad-
drell 1966). Maddrell showed that it is possible to denervate the abdomen without inhibiting
feeding, and that the integument failed to plasticize. Might such a surgical approach be possible
in an ixodid tick?
Our pharmacological data are still woefully incomplete, with all effects supported by only a
single high dose of drug. Full dose-response curves in vivo should be generated for the drugs of
interest in order to determine relative potencies. Once these are available, putative antagonists
should likewise be tested. A better handle on the pharmacological properties of the plasticiza-
tion phenomenon could suggest drugs that one might test as inhibitors of plasticization. If one
could inhibit plasticization pharmacologically, would that attenuate blood meal size, and might
that in turn be a potential tool for inhibiting pathogen transmission?
Are the opisthosomal integumental nerves dopaminergic, as the initial pharmacological re-
sults suggest? There are well-established histochemical techniques that would be relevant here.
One is the classical Falk–Hillarp technique, which lights up tissues containing catecholamines.
Another is based on the immunohistochemical localization of one of the enzymes (tyrosine
hydroxylase) that acts at an early part of the metabolic pathway for DA synthesis and which has
been used successfully in many systems, including ticks (Kaufman et al. 1999).
9.7. CUTICULAR PLASTICIZATION: A CURRENT ENIGMA

We have continued to explore the mechanical properties of the female alloscutal cuticle, but
using a far more robust mechanical set-up and a more comprehensive system of collecting and
analyzing data (P. C. Flynn and W. R. Kaufman, unpublished data). Our current results are in
accord with what was reported by Kaufman et al. (2010) for feeding and engorged ticks, with the
Maxwell viscosity being in the vicinity of 100 GPa s. However, whereas Kaufman et al. reported
a Maxwell viscosity of ≥720 GPa s for the alloscutal cuticle of unfed females, we are now finding
values in the same vicinity as those pertaining to feeding and engorged ticks; because of im-
provements to our experimental technique since 2010, we have greater confidence in our current
values. We are now also monitoring several cuticular properties in addition to viscosity—for
example, compliance, which is a measure of how much the cuticle expands under a given stress.
The range of values we are observing (in the vicinity of 0.05–0.15 MPa−1) for applied stresses in
the range of 0.3–3 MPa also does not seem to change between the unfed state and full engorge-
ment (P. C. Flynn and W. R. Kaufman, unpublished data).
We are left with a number of unanswered questions about how the cuticle is modified as the
female progresses to engorgement. As already discussed (Section 6.2), a great deal of alloscutal
endocuticle is synthesized during the feeding period, because there is not a sufficient amount of
cuticle in the unfed tick to contain the 60- to 100-fold increase in volume that occurs at engorge-
ment (Flynn and Kaufman 2010). We suspect that this newly synthesized endocuticle possesses
properties rather similar to those of the endocuticle laid down during the first few weeks post-
molt (Section 7.1). During the rapid phase of engorgement, the cuticle thins to approximately
half its peak value, but the mechanism by which thinning occurs has not been determined. Ac-
cording to Laplace’s law, the tension in a vessel under pressure is proportional to the radius of the
vessel, and stress within the wall is inversely proportional to thickness. If the peak hydrostatic
pressure of 13 kPa (100 Torr) observed in the hemocoel of O. moubata (Kaufman et al. 1982) also
applied to A. hebraeum, the body wall stress in the engorged tick would be in the vicinity of 1 to
2 MPa. However, in our recent work, stretching cuticle loops at stresses of up to 2.5 MPa has re-
sulted in no appreciable thinning of the cuticle. This suggests 2 areas of future focus. First, the
importance of determining the hydrostatic pressure within the hemocoel due to the action of the
pharyngeal pump cannot be overestimated, and we are currently in the process of attempting to
measure this (P. C. Flynn, R. L. Jacobs, and W. R. Kaufman, unpublished data). Second, we know
that DA causes a dramatic reduction in cuticular viscosity, an effect that is markedly reduced by
the proton-transport inhibitor ConA (Kaufman et al. 2010). This raises the question of whether,
at moderate hemocoelic hydrostatic pressures, a transient plasticization effect might be occur-
ring to facilitate thinning of the cuticle. We are currently designing experiments to explore the
drug regime and stress levels under which cuticular thinning may be observed.
The foregoing is only a sampling of the many unresolved questions relating to the integu-
ment. Trite as it sounds, there is much left in integumental studies to occupy the attention of the
next generation at least of tick biologists!
ACKNOWLEDGMENTS
Research in my laboratory has been funded most generously over the years by NSERC Canada.
I thank my doctoral student, Mr. Alex Smith, for permission to reproduce the image of an unfed
and an engorged A. hebraeum female in Fig 5.4. I am grateful to Professor Emeritus Svend Olav
Andersen, of the Royal Danish Academy of Science and Letters, and Professor Emeritus Stuart
Reynolds, University of Bath, for much enlightenment on the complexities of arthropod cuticle.
Professor Judith Willis, Department of Cellular Biology, University of Georgia, likewise enlight-
ened me about modern ideas on cuticular proteins in insects and ticks.
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NOTE
1. As a graduate student, I remember checking the daily progress of my very first tick feeding,
D. andersoni. On what turned out to be the last day, I was gobstopped to see how huge the detached
engorged females appeared compared to what I had seen the day before! This confirms that when
we read a phrase like “100 times the unfed weight,” we do not get an intuitive feeling for what it
really means until we actually see it!
C H A P T E R 6
MOUTHPARTS AND
DIGESTIVE SYSTEM
Anatomy and Molecular Biology of Feeding and Digestion
DANIEL E. S ONENS H INE AND J ENNIFER M. ANDERSON
1. INTRODUCTION
Ticks are obligate parasites that feed entirely on vertebrate blood. Ticks have evolved novel
methods for acquiring and processing blood; instead of biting their hosts to draw blood quickly
as all other blood-sucking arthropods do, ticks attach to the host skin. They use their hypostome
as a holdfast and create a feeding lesion from which they imbibe blood and other fluids. Argasid
ticks use their chelicerae to cut and tear into the host skin, then feed rapidly from the simple
lesion, often completing their meal within minutes or hours. Their flexible, expandable body
cuticle enables them to increase their body size many-fold as they feed. Ixodid ticks also begin
attachment by cutting into the host skin, but then they secrete cement into and above the grad-
ually expanding lesion, a process that may take from 1 to 2 days. When completely affixed to the
wound site, these ticks feed slowly from the pooled blood there for several additional days, de-
pending upon the life stage. Synthesizing fresh cuticle as they feed, they expand their bodies
greatly and consume enormous blood meals.
Hematophagy is believed to have evolved independently several times during the course of
arthropod evolution (Ribeiro 1995). According to Mans (2011), blood-feeding lifestyles have
evolved independently across the various arthropod groups more than 20 different times. How-
ever, the tick blood-feeding model has no known parallel among the many thousands of hema-
tophagous arthropods. For example, no species of blood-sucking insect remains attached to its
host for such long periods. The unique adaptations developed by ticks are likely the result of a
long evolutionary history, believed to have begun during the late Cretaceous period (Mans and
Neitz 2004). Utilizing this remarkably effective attachment system, ticks create a feeding lesion,
Mouthparts and Digestive System 123
facilitating the flow of blood and other fluids. The salivary glands contribute to the success of the
feeding process, facilitating the flow of blood by means of anticoagulants, immunodulatory
proteins, anti-inflammatory compounds, and numerous other pharmacologically active anti-
hemostatic agents. This enables feeding ticks to avoid recognition and rejection by the parasit-
ized host, although repeated infestations can compromise further feeding success (Trager 1939).
As a result, ticks are able to imbibe enough blood to increase greatly in size—to between 5 and
10 times their pre-feeding weight in the fast-feeding argasids and as much as 100 times in the
slow-feeding ixodid ticks. In addition to their highly efficient system for acquiring host blood,
ticks also have evolved biochemical pathways for hemoglobin uptake, digestion, and protection
against heme toxicity (Graca-Souza et al. 2006). Hemoglobin is their primary food source, so
much so that virtually all other nutrients are excluded.
Blood feeding involves the 3 different regions of the alimentary canal: (i) the mouthparts,
pharynx, and esophagus for fluid acquisition; (ii) the midgut for blood digestion; and (iii) the
rectal sac/anal canal for waste storage and elimination. The salivary glands also contribute to the
blood-feeding process but are external to the alimentary canal.
Blood meal digestion in almost all hematophagous arthropods occurs in the lumen of the
midgut (i.e., it is extracellular). In blood-feeding ticks, however, digestion is different. Except for
lysis of the blood cells in the midgut lumen, digestion of the blood proteins and other molecules
is entirely intracellular within the epithelial cells of the midgut (Sonenshine 1991; Lara et al.
2005; Anderson et al. 2008). Intracellular digestion of the blood meal is an unusual phenom-
enon, unknown except in coelenterates and perhaps other lower invertebrates. Intracellular di-
gestion is also comparatively slow. Consequently, the gradual process of digesting large volumes
of blood enables the fed tick to hold large quantities of the original meal for many weeks, months,
or even years in its digestive tract. Thus, in addition to serving its digestive functions, the midgut
also serves as the tick’s primary food storage organ.
In this chapter, we review the structure of the appendages that constitute the mouthparts
and the organs of the alimentary system. Following a brief review of the mouthparts, the foregut,
the dynamics of tick attachment, and methods for acquiring large volumes of host blood, most
of this chapter is dedicated to the midgut. Here we review its ultrastructure, as well as the bio-
chemistry and molecular biology of hemoglobin absorption, digestion, and dispersal of the end
products to the body tissues for growth and development. This description is generic for both
ixodid and argasid ticks.
For an electronic version of the anatomy and ultrastructure of these structures, the reader
may browse the tick anatomical ontology at www.Vectorbase.org, following the instructions in
the footnote.1 Additional illustrations of the anatomy of ticks not included in this edition may be
found there.
2. MORPHOLOGY OF THE MOUTHPARTS

AND THE FOREGUT
The mouthparts and foregut comprise the structures for accessing the host skin, preparing the
feeding lesion in the host skin, and blood uptake from the host into the midgut. The mouthparts
consist of the unpaired hypostome, paired chelicerae, and paired palps, all anchored on the basis
capituli. The latter encloses the shafts of the chelicerae, the salivary ducts, and the powerful
sucking pharynx (Fig. 6.1). For an overview of the external organization of these structures, see
Section 2 of Chapter 4 in this book.
2.1. HYPOSTOME, FOOD CANAL, AND ASSOCIATED STRUCTURES

The hypostome is situated on the ventral side of the capitulum (gnathosoma) (Fig. 6.2). Internally,
it is hollow except at its distal end. The hypostome is the primary organ used to attach the tick to
the host skin. The ventral side is armed with rows of recurved teeth, but the dorsal side is flat ex-
cept for a narrow V-shaped channel, the preoral canal, in the middle. This channel functions as an
extension of the mouth. During feeding, blood is drawn into this food channel by the powerful
sucking action of the pharynx and passed into the midgut, while saliva from the salivary ducts is
passed out into the host in an alternating pattern of sucking and secretion. In ixodid ticks, blood
uptake alternates with the expulsion of saliva, with long intervals between each action (Gregson
1960). Apparently, the organization of the ixodid mouthparts does not allow for the separation of
fluids in the preoral and salivary channels, and some mixing of blood and saliva may occur. How-
ever, recent evidence suggests that the lining cuticle of the preoral canal is hydrophilic, whereas
the thin, delicate membrane overlying it is hydrophobic, thereby retaining water in the canal when
it is drawn up during sorption from nearly saturated air (Gaede and Knulle 1997). In ixodids, a few
FIGURE 6.1: Diagram illustrating the mouthparts of a representative ixodid tick (Dermacentor andersoni)
as seen in tangential and cross-section. Tangential section through the capitulum. ala, Alae (indicated
by the heavy black line; alar muscles indicated by fine parallel lines on either side of the alae); b. cav.,
buccal cavity (mouth); ch, chelicera; c.s., cheliceral sheath (inner sheath or cone sheath in Gregson
[1960]); di, cheliceral digits; G.o., Gene’s organ; hd, hood; hyp., hypostome; i.c.b., inter-coxal bridge; lab,
labrum; mb, membrane over preoral canal; o.c.s., outer cheliceral sheath; o.m., oval membrane-covered
area of chelicera; P. II, palpal segment II; p.a., porose areas (indicated by lines through the cuticle);
p.c.s., posterior cheliceral sheath; Pr.c., preoral canal; ph., pharynx; p.o., pharyngeal orifice; p.v.,
pharyngeal valve (indicated by curved semicircle); R.ch., retractor muscle; sal., salivarium; s.d., salivary
duct; s.ch.p., subcheliceral plate (epistome); Te, tectum. Redrawn and labeled from Gregson, J.D. (1960)
Morphology and functioning of the mouthparts of Dermacentor andersoni Stiles. Part I. The feeding
mechanism in relation to the tick. Acta Tropica 17:48–72, with permission from Elsevier.
small dilator muscles within the hypostomal cavity, near its junction with the capitulum, help
widen the preoral canal and enhance fluid uptake. In argasids, however, dilator muscles occur
along most of the length of the preoral canal, providing a powerful supplement to the sucking
pharynx for rapid fluid uptake. In ixodids, the food channel is closed (temporarily) on its dorsal
side by the cheliceral sheaths; periodic raising and lowering of chelicerae alternate with lengthy
periods of blood sucking and salivation. A tiny, flap-like labral membrane at the posterior end of
the preoral canal assists in regulating fluid flow into the mouth (Coons and Alberti 1999). Also
present at that junction is a complex groove-shaped pharyngeal valve (Fig. 6.3), which controls the
influx of blood into the mouth and pharynx (see Section 2.3 for a detailed description). The valve
can be closed when the pharynx contracts, preventing regurgitation (Sonenshine 1991).
In argasid ticks, the preoral canal is covered by an elongated, movable, V-shaped labrum that
extends the full length of the hypostome (Fig. 6.4). The labrum flutters up and down during the
short bursts of rapid blood sucking, a feature characteristic of soft ticks. When the labrum is
elevated, blood is sucked up into the preoral canal, mouth, and pharynx. When the labrum is
depressed, the preoral canal is sealed, shutting off blood uptake while the salivarium (located
above the hypostome) fills with fluid and saliva flows into the feeding lesion. These actions pre-
vent the mixing of blood and saliva and also prevent regurgitation. The exact mechanism by
which the hollow labrum is elevated or depressed is unclear, but these actions are believed to be
controlled by changes in hydrostatic pressure from the body hemolymph (with which it is con-
tiguous). Thus, the morphology of the food channel allows for slow feeding in the ixodid ticks
but is adapted for rapid feeding in the argasids.
2.2. CHELICERAE AND PALPS

The paired chelicerae (Figs. 6.5, 6.6) are used to cut the skin and gain access to the host’s blood.
Each comprises 3 parts: (i) a bulbous muscular cheliceral base, (ii) an elongated hollow shaft that
contains the flexor and extensor tendons that move the digits from side to side, and (iii) the
FIGURE 6.2: Serial cross-sections through the anterior part of the capitulum showing the relationships
among the hypostome, the membrane over the preoral canal, the chelicerae, the mouth, and the pharyngeal
valve region of a representative ixodid tick (Dermacentor andersoni). A, Section through the denticulate
part of the hypostome and shafts of the chelicerae. B, Section through the hypostome showing the
denticle cavity and folded state of the membrane. C, Section through the hypostome, membrane
collapsed. Ch.r., cheliceral ridge; D.c., denticle cavity; Hyp., hypostome; Mb., membrane over preoral
canal; T.g., tongue and groove-like ridges along adjacent faces of chelicerae. Redrawn and labeled from
Gregson, J.D. (1960) Morphology and functioning of the mouthparts of Dermacentor andersoni Stiles. Part I.
The feeding mechanism in relation to the tick. Acta Tropica 17:48–72, with permission from Elsevier.
FIGURE 6.3: Diagrammatic reconstruction of the pharyngeal valve region in ixodid ticks. A–D,
Cross-sections illustrating the pharyngeal valve and pharynx in an ixodid tick, Dermacentor
andersoni (A, cross-section at the entrance to the pharynx; B, cross-section showing the pharyngeal
orifi ce; C, cross-section in the vicinity of the pharyngeal valve and sublabial area; and D,
cross-section through the valve at the vicinity of the pharyngeal teeth). E–G, Cross-sections
illustrating the pharyngeal valve from the point of entrance (E) through its center (F) and opening
into the pharynx (G) in an ixodid tick, Rhipicephalus evertsi. Note the wedge and alar plate. Ala, alar
plate; Cav., Cavity; Ch, chelicera; Gl. st., globular structure (non-sclerotized); Lab., labrum; Lab.m.,
labral membrane; Ph., pharynx; P.o., pharyngeal orifi ce; Sal., salivarium; Sub.ph.m., subpharyngeal
muscles. Panels A–D from Gregson, J.D. (1960) Morphology and functioning of the mouthparts of
Dermacentor andersoni Stiles. Part I. The feeding mechanism in relation to the tick. Acta Tropica
17:48–72, with permission from Elsevier; E–G from Arthur, D.R. (1962) Ticks and Disease. Oxford, UK:
Pergamon Press.
cutting digits. Each is covered by a delicate inner sheath and a thick outer sheath made up of
thickened, sclerotized layers of cuticle bearing innumerable minute denticles. On the ventral
margins, the outer sheaths fuse with the labial plate and the walls of the basis capituli to form the
intercoxal bridge, which is continued posteriorly as the subcheliceral plate. The cheliceral digits
are mounted on the terminal ends of the cheliceral shafts. Finally, at their anterior ends, the
chelicerae are modified to form the cheliceral digits. These structures contain heavily sclerotized
spines (teeth), mostly oriented in the horizontal plane. Thus, almost all of the cutting action is
directed laterally; tick chelicerae can cut but cannot grasp. The outer digit (external article) is
FIGURE 6.4: Diagrammatic reconstruction of the basis capituli and associated mouthparts of an argasid
tick, Carios kelleyi. B.cap., Basis capituli; Ch.b., cheliceral base; Ch.dig., cheliceral digit; Ch.sh.,
cheliceral shaft; Con.ph., constrictor pharyngeal muscles; Dil.ph., dilator pharyngeal muscles; Dil.pr.c.,
dilator preoral canal; E, esophagus; Epist., epistome (subcheliceral plate); Ext.ch.di., extensor cheliceral
digit; Fl.ch.di., fl exor cheliceral digit; Hyp., hypostome; Hyp.cav., hypostomal cavity; I.c.s., inner
cheliceral sheath; Lab., labium; Lab.pl., labial plate; Lab.sac., labial sac; O.c.s., outer cheliceral sheath;
Ph., pharynx; Ph.o., pharyngeal orifi ce; Post-hyp.s., posthypostomal seta; Pr.c., preoral canal; R.Ch.,
retractor cheliceral muscles; Sal., salivarium; Te., tectum. From Sonenshine, D.E. and Gregson, J.D.
(1970) A contribution to the internal anatomy and histology of the bat tick Ornithodoros kelleyi Cooley
and Kohls, 1941. I. The alimentary system, with notes on the food channel in Ornithodoros denmarki
Kohls, Sonenshine, and Clifford, 1965. J. Med. Entomol. 7:46–64, with permission from the Entomological
Society of America.
FIGURE 6.5: Detail of the digits in the metastriate tick D. variabilis showing the external article (outer
digit) and the internal article (inner digit) protruding from the spinose outer cheliceral sheaths (arrow).
Measurement bar, 100 μm. Ch. D., cheliceral denticles; Ch. S., cheliceral shaft.
FIGURE 6.6: Detail of the digits in the prostriate tick Ixodes cookei. Measurement bar, 50 um. Ch. D.,
cheliceral denticles; Ch. S., cheliceral shaft.
127
reduced and positioned in a cavity of the more prominent inner digit (internal article). The
flexor and extensor tendons attach to the posterior base of the inner digit. Their action results in
a rocking motion, moving both digits in the horizontal plane but not in any other direction (i.e.,
the digits cannot rotate or pronate). In ixodid ticks, a delicate, translucent hood that can be
withdrawn when the digits are exposed surrounds the digits and is attached to the cheliceral
sheaths (Gregson 1960).
Except when a tick is feeding, the chelicerae are withdrawn. When ticks attach to host skin,
the chelicerae are protruded (by hydrostatic pressure) to expose the digits. The spines on the
digits bear sensory pores, especially on the inner digit. One pore is innervated by chemosen-
sory neurons; other pores appear to act as mechanosensilla and possibly in a thermosensory
role. A prominent mechanosensory placoid sensillum occurs on the outer face of the inner
cheliceral digit, near its junction with the shaft (Sonenshine et al. 1984). Together, these sensilla
provide information on the biochemical characteristics of the wound site environment; for
example, they detect adenosine triphosphate, glutathione, or other phagostimulants, as well as
minute differences in skin tissue temperatures. Proprioceptive responses from mechanosen-
sory sensilla located on the spines and basal regions of the cheliceral digits are believed to en-
able the tick to sense the direction and shear forces needed for tissue cutting activity (Sonenshine
1991). Such sensory information facilitates tick attachment and subsequent blood-sucking be-
havior. In males, the cheliceral digits are used to probe the female’s vulva and, in certain species
(e.g., Dermacentor variabilis and D. andersoni), detect the female genital sex pheromone.
If male ticks’ digits are excised, those that commence mating withdraw without copulating
(Sonenshine 1991).
The paired palps are located on the anterolateral portions of the capitulum. Each consists of
4 segments (articles). In ixodids, the terminal segment (article IV) is greatly reduced, recessed
in a cavity of the third segment, and invisible when viewed from the dorsal side. This small ter-
minal segment bears a field of sensory sensilla used to probe the host skin and identify chemical
compounds. In argasid ticks, all 4 segments are more or less equal in length. In the ixodids,
however, palpal article I is short, broad, and more or less immobile. The palps are splayed out on
the skin surface after the ticks attach and play no role in the feeding process. The chelicerae of
ticks resemble similar structures in mites of the suborder Holothyroidea, suggesting a close
evolutionary relationship between the 2 groups (Coons and Alberti 1999).
2.3. PHARYNX
In ixodid ticks, a complex pharyngeal valve is located just behind the mouth at its junction with
the pharynx (Fig. 6.3). The valve consists of L-shaped struts adjacent to its inner walls and a
dorsal V-shaped wedge that protrudes ventrally from its roof. The struts and walls are flexible
and can be forced against the V-shaped wedge, compressing them together and closing the
valve. In some species, small teeth appear on the inner walls of the pharyngeal valve, apposed
by sharp ridges on the V-shaped wedge. This forms a sort of “grinding organ” that might be
useful for degrading particulates; however, its actual function is unknown (Kemp et al. 1982).
Inside the basis capituli, muscle bundles extend from wing-like sclerotized plates adjacent to
the valve (alae) to the ventral surface of the basis capituli. The action of these muscles helps to
open the valve. Operation of the pharyngeal valve is accomplished via contraction of the di-
lator muscles inserted on the wing-like alae; this raises or lowers the V-shaped wedge, which
can be elevated or depressed in a manner similar to that of the labrum (and, apparently, coor-
dinated with its movements). When the wedge is raised, the lateral walls of the valve are also
dilated, allowing fluid uptake. Evidence of its role in fluid uptake was demonstrated in a study
by Gaede and Knulle (1997), who showed the accumulation of water at the mouth opening
when atmospheric moisture is collected. They noted that “a clear fluid can be observed in the
buccal channel during absorption” assisted by powerful contractions of the sucking pharynx.
When the wedge is lowered, the lateral walls collapse, and the valve is closed, stopping the flow
of blood into the pharynx. When the valve is closed, the salivarium (located above it) expands,
allowing saliva to be ejected. The pharyngeal valve is believed to be an effective barrier prevent-
ing regurgitation of ingested fluids back into the host during blood feeding. However, this
subject remains controversial (see below). The interior of the pharynx is lined with cuticle (and
a thin epithelium), which might appear to have collapsed as a tri-radiate structure during the
intervals between sucking actions. This internal lining is surrounded by layers of circular
muscles interspersed with dilator muscles; the latter extend to and insert on the inner walls of
the basis capituli. The contractions of the circular and dilator muscles alternate with each
other, resulting in the periodic contraction and relaxation of the pharynx that facilitates the
sucking activity.
During most of the period when ixodid ticks are attached, the sucking of blood is spas-
modic, with periods of rapid pulsation alternating with single pulses; in other words, blood
feeding is not a continuous process. Rather, there are long periods of quiescence followed by
periodic spurts of salivation alternating with periods of fluid uptake. According to Gregson
(1960), these pulsations may occur at rates of up to 1 per second. Opening and closing of the
pharyngeal valve was thought to prevent regurgitation from the pharynx back into the feeding
lesion. In adult females that have mated, blood uptake accelerates rapidly and is more or less
continuous, resulting in a huge increase in fluid volume, as well as in body size.
At the end of the pharynx is the narrow, cuticle-lined esophagus, which extends from the
posterior end of the pharynx through the synganglion to the midgut. At its junction with the
midgut, there is a small muscular unidirectional valve, the proventriculus (see www.Vector-
base.org, controlled vocabulary search, TADS 0000179, for a description). Considerable con-
troversy remains as to whether the proventriculus can prevent regurgitation completely or
might allow some of the midgut contents to flow back into the host. Several authors have de-
scribed evidence of regurgitation as a means of pathogen transmission (Connat 1991; Hum-
phrey-Smith et al. 1993; Miyamoto and Hashimoto 1998). According to Brown (1988, p. 335),
this would explain the “ability of gut-derived preparations to induce immunity and may help to
understand the mechanism of transmission of several tick-borne pathogens that have been
only clearly demonstrated to infect the gut of ticks.” None of these findings allows for an un-
equivocal conclusion that regurgitation is a regular phenomenon in ticks, and the anatomy of
the valves at the oral–pharyngeal junction and proventriculus clearly argues against it. Never-
theless, the possibility that regurgitation occurs cannot be excluded. This phenomenon should
be investigated further.
In argasid nymphs and adults, the pumping action of the pharynx is continuous with rapid
bursts of salivary secretion alternating with blood uptake. This is facilitated by the prominent,
well-developed labrum, which fully separates the preoral food canal from the salivarium as it
moves up and down (Fig. 6.4). When specimens are viewed with a stereoscopic microscope,
numerous regularly spaced spurts—as many as 125 to 140 per minute (D. E. Sonenshine, unpub-
lished data)—can be seen flowing in the preoral canal and into the pharynx.
3. MORPHOLOGY OF THE MIDGUT
The midgut is the largest organ in the tick body. It is the sole digestive organ of the animal and
is functionally analogous to the vertebrate intestine, even though the mode of digestion is dif-
ferent. The midgut consists of a central ventriculus (stomach) and numerous branching diver-
ticula (caeca). In ixodids and some argasid ticks, a small intestine (absent in Ornithodoros
savignyi and O. moubata) is connected to the hindgut, allowing the passage of hematin and
other waste residues to the hindgut. The main mass of the midgut is formed by the diverticula,
which radiate in all directions. In unfed ticks and feeding males, the caeca appear narrow and
tube-like (Fig. 6.7). In feeding females, however, the blood-filled caeca appear as large, broad
sacs that subdivide further as tube-like extensions. Thus, the midgut penetrates into all regions
of the tick body. A proventricular valve at the junction of the ventriculus and the esophagus
prevents (or at least minimizes) regurgitation.
The midgut consists of a delicate epithelium surrounded by external layers of circular and
longitudinally oriented muscle fibers. A thin basal lamina forms the outer part of the epithelial
cells and separates them from the muscle layer. The periodic contractions of the latter provide a
type of peristaltic flow that circulates the fluids within this multi-lobed organ. (For additional
information about the midgut, see Chapter 4, Figs. 4.19–4.21.)
3.1. STRUCTURE OF THE MIDGUT IN UNFED IXODID TICKS

In unfed ixodids, the midgut epithelium of unfed, hungry ticks is characterized by a monolayer of
large degenerating digestive cells (DDCs) interspersed with undifferentiated reserve cells (UDCs)
surrounding the thin, barely recognizable lumen (Figs. 6.8, 6.9). According to Tarnowski and
Coons (1989), the UDCs are believed to represent only a single type. The large DDCs contain in-
numerable endosomes (inclusion bodies), most of which contain very dark-staining hematin-like
granules; others are believed to contain partially digested or undigested hemoglobin. Granules
range from minute, almost submicroscopic particles to very large spherical structures, together
filling most of the cytoplasm. The nuclei are relatively large. Residual bodies containing the rem-
nants of lysosomal digestion (see below) are also common. The epithelial cells at this stage are re-
ported to contain relatively few cytoplasmic organelles—mostly glycogen granules, lipid droplets,
and residual bodies—all reflecting the remnants of the nymphal blood meal (Tarnowski and
Coons 1989). In addition, numerous cells show evidence of degeneration, with peroxisomes and
myelininc figures (Caperucci et al. 2010a). In some species (e.g., Amblyomma americanum), the
midgut epithelial cells of recently molted adults contain large quantities of inclusion bodies,
presumably rich in hemoglobin or remnants of hemoglobin digestion, known as hemosomes
(i.e., inclusion bodies rich in organically bound iron), located apically. Numerous lipid droplets
occur in the basal regions of the cells. Chemical tests (Prussian Blue) showed that the abundant
hemoglobin-containing droplets gradually declined and eventually disappeared in ticks subjected
to long periods of starvation (e.g., 6 to 12 months). Moreover, as the ticks age, the siderosomes re-
semble residual bodies containing hematin and myelin figures (myelinosiderosomes). The cell
lipid content, initially abundant, also gradually declines as food reserves are utilized. The molec-
ular events involved in these digestive processes are described elsewhere in this chapter (see Sec-
tion 4.2.2, “Digestion of Hemoglobin and Other Blood Meal Proteins”). Thus, the process of
FIGURE 6.7: Light micrograph of a dissection of a female ixodid tick, Dermacentor variabilis, showing the
multiple lobes of the midgut (black) within the interior of the tick’s body.
gradual digestion enables the tick to store much of the blood meal undigested for long periods of
time. In effect, the midgut lumen, rather than the epithelium, serves as the major nutrient storage
organ in these blood-feeding parasites (summarized by Sonenshine [1991]).
3.2. STRUCTURE OF THE MIDGUT IN FEEDING IXODID TICKS

3.2.1. Cellular organization in virgin females
The midgut undergoes profound changes following the onset of feeding and the influx of host
blood. The large hematin-filled DDCs begin to slough off and float freely in the fluid-filled
lumen (Fig. 6.10). Some disintegrate, liberating innumerable black-colored hematin granules.
FIGURE 6.8: Light micrograph illustrating the histologic structure of the midgut in an unfed female ixodid
tick, Dermacentor variabilis. The lumen is collapsed and virtually obliterated by the elongated epithelial
cells. The digestive cells are fi lled with numerous inclusion bodies. Midgut diverticulum, longitudinal
section. Measurement bar, 100 μm.
FIGURE 6.9: Light micrograph illustrating the histologic structure of the midgut in an unfed female ixodid
tick, Dermacentor variabilis. The lumen is collapsed and virtually obliterated by the elongated epithelial
cells. The digestive cells are fi lled with numerous inclusion bodies. Midgut diverticulum, cross-section.
Measurement bar, 100 μm.
Stem-cell-like reserve cells, mostly adjacent to the outer wall, proliferate and extend toward the
lumen. The feeding process and the changes that occur among the differentiating cells consist of
2 major phases, namely, (i) the slow feeding and digestion phase, during which cuticle synthesis
occurs and the tick integument gradually expands to accommodate the increasing blood volume,
and (ii) the rapid feeding phase that follows copulation, when the ticks imbibe to repletion. In
feeding females, the slow phase continues up until the time when the tick is inseminated, after
which the females feed to repletion. The descriptions that follow apply only to the feeding
females.
During the first 1 to 2 days following attachment to the host and formation of the feeding
lesion, little ingestion of blood takes place. During this period, the predominant activity by the
tick is the preparation of the feeding lesion, insertion of the mouthparts, and secretion of cement
to secure the positioning of the tick for feeding. Subsequently, the attached ticks gradually im-
bibe blood and other fluids for several days and expand slowly. In D. variabilis females, a repre-
sentative ixodid tick, the body weight of the attached tick increases from approximately 6 to 7
mg to approximately 125 to 150 mg after 5 days. If mating is delayed, most D. variabilis females
will continue to imbibe blood slowly until they reach a weight of approximately 200 to 250 mg
FIGURE 6.10: Light micrograph illustrating the histologic structure of the midgut in a feeding virgin
female metastriate ixodid tick, Dermacentor variabilis (forcibly detached after 3 days). The lumen
contains fragments of hematin-rich lysed cells and free particulates. The midgut epithelium consists of
greatly enlarged digestive cells fi lled with masses of hematin-rich inclusion bodies, mostly
concentrated near their apical (luminal) sides. Measurement bar, 200× magnifi cation. Photo courtesy of
Lewis Coons, University of Memphis, Memphis, TN.
(rarely, up to 300 mg), whereupon little further increase is observed. Blood digestion proceeds
gradually during this period and continues at this rate until mating occurs. The midgut epithe-
lium responds with the growth and differentiation of digestive cells (DGCs) from the pool of
UDCs (Coons et al. 1986). As blood and other fluids enter the midgut lumen, the UDC cells
begin to proliferate, and many differentiate into secretory or digestive cells at that time. During
the first few days of feeding, cell growth is gradual, and the midgut lumen enlarges only slightly.
By the fifth or sixth day of continuous feeding, the lumen has enlarged greatly and all of the in-
gested blood cells have lysed, forming large masses containing fragments of dead cells, heme
aggregates, and other excreta from the disintegrating remnants of the nymphal blood meal.
Profiles of the midgut in feeding females of the prostriate tick I. scapularis show a similar
response, with greatly enlarged digestive cells filled with masses of granular material presumed
to be hematin particles and/or partially digested hemoglobin aggregates (Fig. 6.11). The
cytoplasmic inclusions appear much finer than in comparable metastriate feeding females
FIGURE 6.11: Light micrograph illustrating the histologic structure of the midgut in a feeding female
prostriate ixodid tick, Ixodes scapularis (forcibly detached after 6 days). The lumen contains numerous
lysed or intact erythrocytes (red) and free-floating cell fragments from lysed epithelial cells. The midgut
epithelium consists of greatly enlarged, fl ask-shaped digestive cells fi lled with masses of fine
particulates (mostly near their luminal sides), presumably iron-rich hematin or similar remnants of
hemoglobin digestion. DGC, differentiated digestive cell; L, lumen. Measurement bar, 100 μm.
(e.g., D. variabilis). Some cells have disintegrated, and whole cellular fragments are floating free
in the lumen, presumably via the holocrine method in which the apical region of the cell dis-
lodges to release its mass of stored endosomes. In contrast to what is seen in D. variabilis, many
of the ingested erythrocytes have not been lysed (the luminal contents appear red in histological
preparations), even though as many as 6 days of continuous blood feeding have passed.
3.2.2. Cellular organization of the midgut in feeding males

In feeding metastriate ixodid males, the midgut appears similar, but with far fewer cells and a
small lumen (Fig. 6.12). Hematin granules are scattered throughout the cytoplasm. Some cells
show evidence of apocrine secretion (large cell fragments ready to break off and float in the
lumen). The lumen contains small masses of digested remnants, presumably hematin.
3.2.3. Ultrastructure of midgut cells in virgin females

When the ultrastructure of the midgut of feeding virgin females is examined, transmission elec-
tron microscopy profiles show the enormously enlarged DGCs filled with numerous endosomes
and lipid inclusions of varying size and shape, as well as accumulated residual bodies. Mito-
chondria have also increased in abundance, and microvilli have proliferated and become
prominent (Figs. 6.13, 6.14). Coated pits, coated vesicles, and tubular elements, all structures
characteristic of absorptive cells, are also evident in the DGCs at this time (Tarnowski and
Coons 1989). Numerous endosomes are visible, especially near the luminal side of the cells.
Some degenerating cells from the previous meal have been sloughed off. The digestive cells of
the midgut take up blood components, especially hemoglobin, via receptor mediated pinocytosis,
FIGURE 6.12: Light micrograph illustrating the histologic structure of the midgut in a fed male ixodid
tick, Dermacentor variabilis (forcibly detached after 6 days). The small lumen contains fragments of
hematin-rich lysed cells and free particulates. The midgut epithelium consists of a small number of
digestive cells fi lled with masses of hematin-rich inclusion bodies. Measurement bar, 100 μm. Photo
courtesy of Lewis Coons, University of Memphis, Memphis, TN.
as well as through phagocytosis. Lipid droplets form in certain cells but are absent in others.
Glycogen, in the form of rosette-like clusters or loosely organized individual granules, is also
apparent in some cells. Some cells may have much more glycogen than lipid. Intracellular diges-
tion is accomplished via heterophagy (see below). As digestion of the accumulating blood meal
proceeds, waste products are generated and excreted from the digestive cells. Heme aggregates
(previously known as hematin; see below), resulting from intracellular lysosomal digestion, ac-
cumulate as distinctive crystals within residual bodies; this has been confirmed via x-ray micro-
analysis (Sonenshine 1991). Enlargements of the DGCs show numerous small, electron-dense
inclusions adjacent to and/or near the luminal surface, along with other, larger electron-dense
inclusions in the center of the cells (Fig. 6.15). In some species (e.g., I. scapularis), an amorphous,
non-cellular structure, the peritrophic membrane, forms in the lumen approximately 0.5 to 1.0
μm from the plasma membrane (Fig. 6.16). This chitin-rich membranous barrier divides the
midgut lumen into 2 regions: the endoperitrophic space, encompassing most of the lumen, and
the narrow ectoperitrophic space adjacent to the epithelial cells. It is not known whether the
peritrophic membrane is produced in all tick species.
Little is known about the morphological and functional changes that occur in the tick
midgut during feeding on a “tick resistant” host (i.e., a host that has been exposed to ticks several
times). Caperucci et al. (2010a, 2010b) examined the ultrastructure and histology of midguts
from Amblyomma cajennense females fed on a rabbit during a second and third infestation. They
FIGURE 6.13: Transmission electron micrograph illustrating the ultrastructure of the midgut in a feeding
virgin female tick, Dermacentor variabilis. Profi le of the midgut from the outer wall to the lumen.
Undifferentiated and differentiated digestive cells (DGC) occur adjacent to one another. The
differentiated cell extends from the basal lamina to the lumen and has inclusions (endosomes, also
called phagolysosomes) near the luminal margin. Measurement bar, 4.0 μm.
FIGURE 6.14: Transmission electron micrograph illustrating the ultrastructure of the midgut in a feeding
virgin female tick, Dermacentor variabilis. Enlargement illustrating cellular details of a differentiated
digestive cell. Numerous endosomes of varying size occur near the luminal surface. Lipid inclusion
bodies are numerous, often clustered in large groups. Measurement bar, 3.0 μm. Bl, basal lamina; DGC,
digestive cell (differentiated); L, lumen; Li, lipid inclusion body; Mu, muscle cell; Mv, microvilli; Nu,
nucleus; Rd, residual dense bodies (fi lled with electron-dense material); Sv, smooth (uncoated)
vesicles; UDC, undifferentiated digestive cell.
136
FIGURE 6.15: Transmission electron micrograph illustrating the ultrastructure of a representative
differentiated digestive cell in the midgut in a feeding virgin female tick, Dermacentor variabilis.
Numerous intensely electron-dense endosomes (hemosomes) are evident and fi ll most of the apical
region of the cell. Mv, microvilli. Asterisks indicate probable hemosomes. Measurement bar, 2.5 μm.
FIGURE 6.16: Transmission electron micrograph illustrating the digestive cells in the midgut of a feeding
Ixodes scapularis nymph. A peritrophic membrane is evident adjacent to the luminal surface of these
cells, clearly separating the cells from the lumen of the midgut. Photo courtesy of Star Dunham Ems,
Department of Medicine, University of Connecticut Health Center, Hartford, CT. P.M., peritrophic
membrane. Measurement bar, 2 μm.
137
found that the gut epithelium was highly disorganized and speculated that this might be due to
the immunologic response of the host in the form of antibodies against tick gut and salivary
components. Considering that the midgut is a potential target for anti-tick vaccines, a better
understanding of how the midgut responds (or not) to a blood meal on a tick-resistant host
might be useful and warrants further investigation.
3.2.4. The peritrophic membrane in virgin females

In insects, the peritrophic membrane (PM) occurs in the midgut lumen as “a network of chitin
microfibrils within a matrix of carbohydrate and protein” and protects the mucosal surface from
abrasion by gut contents (Klowden 2002, p. 172). In fluid-feeding insects, the PM forms a com-
partment (endoperitrophic space) that is permeable to some digestive enzymes and digested
metabolites and permits a countercurrent fluid flow between it and the ectoperitrophic space.
The PM also might protect the insect against invasion by some microbes and some toxins to
which the PM is relatively impermeable (Klowden 2002, p. 172). However, numerous blood-
sucking arthropods serve as vectors of various pathogens, and it is not clear how these patho-
gens can cross the PM. One suggestion is that they use chitinase to disrupt the PM.
Rudzinska et al. (1982) provide what they believe to have been the first report of a PM in
I. scapularis in a study of how Babesia microti crosses the PM and invades the tissues of the
vector. The PM in this tick is formed de novo only during blood meal ingestion in each instar.
Although blood cells and ribosomes from lysed cells are found in the endoperitrophic space,
they are absent from the ectoperitrophic space, indicating that the PM constitutes an effective
mechanical barrier to particulate matter. At least some macromolecules, such as hemoglobin,
appear in both ecto- and endoperitrophic spaces, indicating that the PM also functions as a
micro-filter. However, about 60 hours after the start of feeding, the cytoplasm of B. microti ga-
metocytes develops an organelle resembling an arrowhead that appears to be essential for pene-
trating the PM (because only parasites possessing the arrowhead organelle were seen crossing
the PM into the ectoperitrophic space) (Rudzinska et al. 1982). Rudzinska and her colleagues
hypothesize that penetration might depend on, or be facilitated by, some enzyme(s) associated
with the arrowhead organelle, because its structure changes during the course of penetration.
One obvious candidate for the putative enzyme(s) would be a chitinase. For example, the Plas-
modium ookinete produces a chitinase (family-18-type but distinguishable from that of the mos-
quito vector). Two chitinase genes from Plasmodium gallinaceum have been characterized, and
the chitinase activity of their respective proteins has been demonstrated (Langer and Vinetz
2001). Blocking this chitinase activity inhibits penetration of the PM and subsequent invasion of
the midgut by Plasmodium, confirming the functional role of this enzyme activity and suggest-
ing that various interventions to block chitinase activity might constitute a potent strategy for
controlling malaria (Langer and Vinetz 2001).
It is interesting that Borrelia burgdorferi, the etiologic agent of Lyme disease, utilizes chitobi-
ose as a nutrient source (Tilly et al. 2004). Chitobiose is the dimer subunit of chitin. The mono-
mer N-acetylglucosamine is an essential metabolite for spirochete growth in vitro (and
presumably in vivo); the obvious source of chitobiose/N-acetylglucosamine for the spirochete
would be the PM. The hypothesis is that spirochetes require chitobiose/N-acetylglucosamine for
cell wall synthesis. A mutation of the chbC gene (predicted to be the trans-membrane compo-
nent of a chitobiose transporter) in an infectious clone of B. burgdorferi inhibited the ability of
the spirochete to transport chitobiose (although ultimately it did not reduce the infectivity of the
spirochete). The chitobiose is believed to arise from chitin biosynthesis and degradation pro-
cesses by the tick (Tilly et al. 2004), as they could not uncover genomic evidence for a chitinase
in the B. burgdorferi genome. You et al. (2003, p. 8562) suggested that the occurrence of chitinase
in the midgut of ticks is important “for the control of turnover and porosity of the PM.”
The existence of a PM has been established for several other species of tick. In Haemaphysa-
lis longicornis, it was clearly noted in engorged larvae, but its form in the female is substantially
different from that in larvae (Matsuo et al. 2003). It also is present in I. ricinus (Zhu et al. 1991)
and in Ornithodoros moubata (Grandjean 1984).
Finally, with chitin being such an essential substance in the fabric of the tick, chitinase has
received some attention as a potential component of a bioacaricide. Assenga et al. (2006)
prepared a recombinant baculovirus expressing a chitinase gene (AcMNPV-CHT1) from
H. longicornis and applied it topically to nymphal ticks. They demonstrated that the intact bac-
ulovirus-chitinase system killed ticks significantly more rapidly than the pure chitinase or bacu-
lovirus alone. Moreover, when ticks were treated with baculovirus and chitinase together with
the synthetic pyrethroid flumethrin, a clear synergistic effect was observed, demonstrating the
potential for ultimately reducing, if not eliminating altogether, dependence on toxic chemicals
for the control of ticks. The effect of the chitinase in this system is assumed to take place via in-
terference with the molting process and disruption of the PM (You et al. 2003).
3.2.5. Absorption and digestion of ingested blood

Absorption and digestion are accomplished by the digestive cells of the midgut epithelium.
These cells have a highly modified plasma membrane with numerous microvilli, as well as nu-
merous clathrin-coated and uncoated pits, believed to be the sites for uptake of the blood meal
constituents. Hemoglobin released from lysed erythrocytes (by extracellular hemolysins) binds
to specific (but as yet unidentified) receptors in the clathrin-coated pits. The epithelial cell mem-
brane extends out and around the pit to enclose it (via pinocytosis), forming an endosome. The
entire process is termed receptor mediated endocytosis. Clathrin is important in facilitating re-
ceptor transport at the plasma membrane, as well as at membrane surfaces within the cyto-
plasm. Albumin is also absorbed from the midgut lumen, but this is believed to occur in
uncoated pits, a process termed fluid phase endocytosis. Phagocytosis is also believed to occur
and allows the cells to capture larger particles (e.g., large hemoglobin crystals). Pinocytosis is a
phase of phagocytosis but is limited to minute particulate materials. In both cases, pseudopod-
like extensions of the cell membrane extend out into the lumen and enclose the particles or cells.
Regardless of how the cells take up the meal, the material is internalized; the plasma membrane
reforms, pits become vesicles, and clusters of vesicles fuse to form endosomes. Lysosomes fuse
with these endosomes to form heterolysosomes (phagolysosomes or secondary lysosomes),
whereupon digestion begins. These cells also develop a highly infolded basal lamina forming a
labyrinth system, a feature characteristic of transporting cells. This feature probably facilitates
fluid transport, which is essential for concentration of the blood meal. The digestive cells hyper-
trophy and fill with large numbers of these inclusions, which are slowly transformed into iron-
rich residual bodies. This process of endocytosis and intracellular lysis is termed “heterophagy.”
Gradually, the digestive cells fill with iron-rich lamellate residual bodies (also known as hemo-
somes; see below), presumably storing detoxified heme in the form of heme aggregates (hema-
tin-like masses). In some cells, these masses are eliminated via exocytosis. In this process, the
residual bodies accumulate adjacent to the plasma membrane. Eventually, the vesicular and
plasma membranes rupture, releasing their contents into the lumen. Subsequently, the plasma
membrane reforms. In others cases, parts of the large digestive cells detach from the basal lamina
to float free in the lumen. Undifferentiated cells begin to differentiate from the reserve pool to
provide a second cycle of growth and to replace exhausted digestive cells from the preceding
cycle. Moreover, this pattern of cyclical development is not controlled precisely; rather, different
cells commence activity and accumulate wastes at different rates, in addition to eliminating
them via the 2 different processes described above. Naturally, such variability in cellular devel-
opment and activity has led to considerable confusion and controversy among workers inter-
preting these events.
Hemoglobin digestion liberates heme, which is highly toxic because of its ability to stimulate
lipid peroxidation, leading to the formation of reactive oxygen species (ROS) and free radicals,
all of which can promote damage to the tissues (Lara et al. 2005; Citelli et al. 2007). Conse-
quently, blood-feeding arthropods, as well as their eukaryotic pathogens, must detoxify the
heme moiety in order to continue digesting hemoglobin without injury. In blood-feeding in-
sects such as the malaria mosquito, Anopheles gambiae, or the kissing bugs, Rhodnius prolixus,
heme is detoxified via its conversion to hemozoin (Graca-Souza et al. 2006). A similar conver-
sion mechanism occurs in the malaria parasites (e.g., Plasmodium spp.) (Pandey et al. 2003).
Malaria hemozoin (also known as malaria pigment) is an aggregate of heme moieties linked
together (i.e., “linked into dimers through reciprocal iron-carboxylate bonds”) to form insoluble
crystals (Pagola et al. 2000, p. 307; Stiebler et al. 2011). In mosquitoes and other blood-feeding
insects, blood digestion takes place entirely in the midgut lumen. However, in ticks, hemoglobin
absorbed by the midgut epithelial cells via the processes described above is hydrolyzed within
the intracellular vesicles through the action of various proteases and other enzymes as described
later in this chapter (see Section 4), a process that also releases heme. Until recently, heme liber-
ated as a result of hemoglobin digestion was reported to be detoxified into hematin. However,
the tick heme aggregates have a characteristic Fourier transform infrared spectrum distinct
from that of vertebrate hematin (Graca-Souza et al. 2006). As described by Lara et al. (2005),
heme liberated during these digestive processes is converted to a non-toxic heme aggregate,
whereupon it accumulates within specialized vesicles known as hemosomes. Heme sequestra-
tion in these vesicles is an effective detoxification method. In a novel study to investigate the
process, Lara and colleagues cultured midgut epithelial cells and incubated them in the presence
of rhodamine-labeled bovine hemoglobin and palladium-mesoporphyrin (Pd-mP)-labeled
globin or bovine albumin in order to trace their fates during the digestive process. The process
was also investigated in vivo using capillary artificial feeding with the same labeled molecules.
After binding to specific cell surface receptors (i.e., the clathrin-coated receptors), hemoglobin
was incorporated into large (3–12 μm) endosomes, whereas albumin (one of the few other
proteins absorbed from the blood meal) was taken up in much smaller (1–2 μm) endosomes.
Within as little as 2 hours, the large endosomes filled with hemoglobin (i.e., digestive vesicles)
were abundant near the luminal side of the cell. As hemoglobin uptake and digestion progressed,
numerous small, dark vesicles known as hemosomes accumulated, predominantly near the
basal side of the cell. Analysis of the Pd-mP label during digestion showed color changes consis-
tent with heme transport by heme-binding proteins migrating through the cytosol and eventu-
ally accumulating entirely within the hemosomes. The authors suggest that heme sequestration
is accomplished by an “intracellular shuttle from the digestive vesicle to the hemosomes” and
serves as a novel detoxification mechanism. If the hypothesis put forth by Lara et al. (2005) is
accepted, then ticks (R. microplus) detoxify heme not to hematin but to a novel form of heme
aggregate. However, this interpretation of the process is not universally accepted. Moreover, it is
not known whether this mode of heme detoxification is unique to R. microplus or applies to
other tick species. Heme is also sequestered by ferritin, a heme-trapping molecule highly con-
served throughout the Eukaryota. The molecular biology of blood meal digestion is described
elsewhere in this chapter (see Section 4); also see Chapter 9 for a brief overview of these
processes.
Hemolysis, hemoglobin, and albumin absorption and intracellular digestion are all contin-
uous processes that occur throughout the midgut epithelium. Consequently, new vesicles repre-
senting each type are continuously being formed. There is no evidence of phased digestive
activity in different regions of the ixodid midgut (although this does occur in argasid ticks; see
Section 3.2.8). At the same time that the midgut epithelial cells are engaged in blood digestion,
other cells expel their digested hemoglobin fragments or non-toxic heme aggregates. In some
digestive cells, these crystals are expelled via exocytosis; in others, the cell might fragment, or
the entire cell or its distal sections might detach and disintegrate (holocrine secretion), adding
to the wastes accumulating in the midgut lumen. Waves of peristaltic contractions drive these
luminal wastes posterior, where they accumulate in the rectal sac. Defecation begins at this time
(within the first 2 to 3 days after attachment), and in some species, large quantities of heme ag-
gregates mixed with undigested hemoglobin, other nutrients, and guanine excreta are passed
out of the body. These dark red or black semi-solid wastes accumulate around the feeding ticks,
contaminating the host’s fur. Thus, blood meal ingestion, digestion, and waste elimination all
occur during this slow growth period in a more or less continuous process.
An important, unresolved question is whether the midgut epithelial cells differentiate into a
single cell type that performs all of the functions involved in processing of the blood meal or
into discrete cell types, namely, secretory or digestive. Secretory cells were reported to occur in
the midgut of feeding female B. microplus ticks. The secretory cells were reported to resemble
digestive cells but had undergone much greater hypertrophy; some cells were reported to expel
their secretory granules via exocytosis or cell rupture (reviewed by Sonenshine [1991]). How-
ever, molecular studies and histochemical tests showed the appearance of peroxidase and alka-
line phosphatase activity in the same midgut cells during hemoglobin uptake and digestion
(Agyei et al. 1992). According to those authors, the presence of these enzymes suggests that the
midgut digestive cell is a multifunctional cell capable of both secretory and digestive activities.
3.2.6. Midgut of mated females

In mated females, the gradual release of the sperm and semen contents into the reproductive
tract provides the signal (mechanism unknown) for the commencement of rapid blood sucking
and engorgement, sometimes known by the colloquial name “the big sip.” Very large quantities
of blood are taken within as little as 12 to 36 hours, during which time the tick expands enor-
mously. Final body weights vary greatly with the different species and genera. In (unpublished)
studies by Sonenshine on the American dog tick, D. variabilis (fed on rabbits), the average final
body weight is 625 ± 69 mg, mostly as a result of fluids taken within the last 24 hours of feeding.
Of course, the type of host species and other variables can affect the body weight. At repletion,
the tick has increased in size to approximately 100 times the unfed body weight of a young adult
female (Sonenshine 1991). In certain African Hyalomma and Amblyomma spp. parasitic on large
herbivores, final body weights might exceed 1,200 mg. This is actually several times larger than
the measured final body weight because of the secretion of excess water back into the host
(see below). During this rapid engorgement period, the midgut lumen acts as a blood reservoir.
The digestive cells absorb the blood meal contents, but the rate of intracellular digestion is re-
duced during this period of rapid blood sucking (Tarnowski and Coons 1989). Coons and his
co-workers showed evidence that vitellogenin-producing cells form at this time (Coons et al.
1989). Although most vitellogenin (Vg1 and Vg2) is produced in the fat body, it is likely that the
midgut is a secondary synthesis site for vitellogenin (both Vg1 and Vg2), which is expressed in
replete females after mating. Northern blots localized Vg2 in the midgut and in the fat body
(Khalil et al. 2011). The role of the midgut as a secondary site of vitellogenin production is dis-
cussed elsewhere (see Chapter 15 of this book).
3.2.7. Post-feeding digestion

Following engorgement to repletion and drop-off from the host, digestion of the blood meal
continues gradually. Digestive cells begin to show accelerated lysis of stored intracellular blood
components, primarily hemoglobin and albumin. The digestive cells reactivate the lysosomal
digestion of the ingested hemoglobin within the endosomes, especially by means of acid prote-
ases and ROS compounds important in the digestion of hemoglobin (see Section 4). The basal
labyrinth and extracellular spaces gradually decline as the digestive cells shrink and fill with
great masses of hemosomes (Figs. 6.10, 6.11) and residual bodies. Waste elimination appears to
be accomplished via exocytosis rather than through cell detachment or breakup. Eventually the
exhausted digestive cells die and are sloughed off after the larvae or nymphs molt and begin
feeding again or when the replete females oviposit. Similar processes appear to occur in the
midguts of feeding male ticks (Fig. 6.12).
3.2.8. Structure and function of the midgut in argasid ticks

Overall, the dynamics of blood meal digestion in argasid ticks is similar to that in ixodids, with
certain notable exceptions, as discussed below. No digestive activity occurs during the feeding
process itself, which is accomplished within minutes or, at most, several hours (except in the
larvae of some argasid species that feed for several days). Digestion in the Argasidae is accom-
plished in 3 phases, namely, (i) hemolysis, from 2 to 3 days post-feeding to as long as 10 to 15 days
in some species; (ii) “rapid digestion,” beginning during or immediately after hemolysis and
continuing for several weeks to as long as 2 to 3 months after feeding; and (iii) “slow” digestion,
lasting months or even years in some species.
During the first phase of blood meal digestion, most of the large, heme-aggregate-filled di-
gestive cells are sloughed off, and the epithelium is represented by a layer of small cuboidal or
even squamous cells. In the central regions of the gut, these lining cells differentiate and com-
mence secretory or absorptive activity. Elsewhere, in most of the lateral branches, the midgut
epithelium remains in the undifferentiated state (see Chapter 8, Fig. 8.7). This is an important
difference between the Argasidae and the Ixodidae. Hemolysis is intense at this stage, as it is in
ixodid ticks. Most argasids hemolyze the erythrocytes within the first few days after engorge-
ment (Balashov 1972). In Ornithodoros concanensis fed on bovine blood, erythrocytes show a
gradual disruption of their surfaces, expressing large spikes and then becoming spherical before
they are fully hemolyzed after several days (Kirsch et al. 1991). Hemolysis may result from dis-
ruption of phosphatidylcholine in the erythrocyte cell membrane by phospholipase A secreted
in the saliva during blood ingestion, as occurs in ixodid ticks (Zhu et al. 1997). Whether other
enzymes, especially hemolytic enzymes from the midgut, also function in this capacity (as is the
case in ixodid ticks) is unknown (see also Section 4.2.1). In Argas persicus, most erythrocytes
agglutinate soon after engorgement and are lysed rapidly. Some erythrocytes, thrombocytes,
and most leucocytes may be ingested directly by the digestive cells via phagocytosis, forming
large vacuoles in which the form of the ingested blood cells might remain evident for several
days. During this period, a periodic acid Schiff positive colloid is secreted by midgut epithelial
cells. This material is secreted within 24 hours after feeding and accumulates around aggluti-
nated masses of erythrocytes. Apparently, this colloid contains a hemolysin, as the blood cells in
the vicinity gradually disintegrate. Hemolysis is completed within 6 days in this species. The
midgut then enters the second phase, so-called rapid digestion, in which the digestive cells en-
large and ingest protein and cellular elements through the same endocytotic processes described
previously. Periods of accelerated or reduced rates of digestion may occur in response to changes
in the physiological state of the animal (e.g., preparation for molting, oviposition, etc.).
Comparison of the blood digestion processes in argasid and ixodid ticks suggests that the
methods of nutrient absorption by the digestive cell are fundamentally similar, although the
relative importance of the processes may differ (see below). In O. moubata, the apical cell mem-
branes are differentiated into microvilli that are covered externally by a dense coat of glycocalyx,
as well as numerous coated and uncoated pits and tubules. Whether a peritrophic membrane
exists is unknown; no evidence of this membrane has been reported for argasid ticks. Endocy-
totic vesicles formed after internalization of the food material can fuse to form larger inclusions,
which in turn fuse with lysosomes to form heterolysosomes similar to that seen previously in
ixodid ticks. Phagocytosis is prominent, with the extrusion of pseudopod-like extensions of the
apical cell membrane to engulf blood cells, large hemoglobin crystals, or other formed elements.
This suggests an important difference from the Ixodidae, in which both phagocytosis and the
various endocytotic processes play a major role in absorption. Consequently, considerable con-
troversy exists as to the relative importance of the different types of absorptive mechanisms in
ixodid and argasid ticks. It is not clear at the time of this writing (i) whether certain digestive
cells are specialized for ingestion via phagocytosis and others for receptor-mediated or fluid
phase endocytosis or (ii) whether phagocytosis is a property of all digestive cells and is merely
expressed at different times (as advocated by Coons et al. [1986]). Studies on O. moubata reveal
profiles of hypertrophied digestive cells containing large phagosomes with intact blood cells or
hemoglobin crystals, as well as numerous endosomes of varying sizes internalized through
other methods. The ingestion of leukocytes and thrombocytes appears to be accomplished en-
tirely via phagocytosis. As digestion proceeds, the cells fill with phagosomes of varying types.
The basal membrane becomes highly infolded and forms an extensive basal labyrinth, a feature
characteristic of transport epithelia. The basal lamina is unusually thick where it is not infolded
and is much thicker than in ixodid ticks. Communication with adjacent epithelial cells is evi-
dent and is accomplished by the zonula adherens and septate desmosomes. Within the digestive
cells, intracellular digestion begins with the fusion of the lysosomes with the phagosomes and
the release of enzymes. The lytic processes appear to be similar to those described previously for
the ixodid ticks, resulting in the breakdown of ingested cellular elements and metabolism of the
globin moiety. Whether heme is detoxified to heme aggregates within hemosomes as occurs in
feeding ixodid ticks (see Section 3.2) or to some other form is unknown. Residual bodies filled
with heme aggregates, as well as others filled with membrane fragments and particulates, accu-
mulate in the swollen apical regions of these cells. Eventually, the spent digestive cells are dis-
rupted, releasing hematin-like granules or detached cell fragments into the lumen.
The third phase, slow digestion, occurs primarily in the diverticula, especially in the lateral
and posterior branches. This is the single most important difference between the mechanics of
blood meal digestion in the 2 tick families. Histological sections of the midguts of starving arga-
sid ticks reveal little or no digestive activity in the peripheral regions of the gut diverticula, in
contrast to the very advanced digestion in the ventriculus and medial portions of the diverticula.
During this period, waves of digestive activity proceed centrifugally until eventually the con-
tents of the outermost diverticula are consumed and the tick dies, a process that can extend over
several years. Thus, in argasid ticks, as digestive activity progresses from the ventriculus to the
lateral lobes, it initiates the third phase, or slow digestion, in the peripheral regions. As a result,
argasids can survive on a single feeding for exceptionally long periods. In ixodids, however, di-
gestion progresses more or less uniformly throughout the midgut, and survival is not as great
once digestion has commenced.
The regulatory events described above allow for gradual consumption of the stored blood
meal. In starving argasid ticks (e.g., Carios kelleyi), the digestive epithelial cells hypertrophy and
fill with masses of heme aggregates and hemoglobin inclusions, especially in the swollen, apical
portions of the cells. As digestion is completed, these cells detach from the basal lamina or frag-
ment, some floating free in the fluid-filled lumen, others liberating masses of heme granules into
the lumen (Sonenshine 1991). In contrast, digestion in ixodid ticks proceeds more or less uni-
formly in all diverticula and regions of the midgut. Rates of digestion may also vary within in-
dividual cells. For example, in some cells in O. moubata, the fusion of the phagosomes with the
lysosomes may be delayed, enabling the ticks to store hemoglobin intracellularly in the intact
state.
3.2.9. Elimination of blood meal water

In ixodid ticks, excess water in the blood meal is transferred to the salivary glands and expelled
by specialized granular acini during the feeding process (see Chapter 7). In most argasid ticks,
excess blood meal water is excreted via the coxal glands (see Chapter 8). According to Mans et
al. (2011), in Nuttalliella namaqua, the sole surviving species of the ancient tick family Nuttal-
liellidae, blood meal water is eliminated by means of excretion via the malpighian tubules and
the rectal ampulla. This mode of excess fluid elimination also occurs in the argasid tick
O. moubata (in most other argasid ticks, excess blood meal water and salts are eliminated via
the coxal glands). No evidence of coxal glands was found in N. namaqua. Excretion of excess
blood meal water via the coxal glands and salivary glands is believed to have evolved separately
(Mans et al. 2011).
4. MOLECULAR BIOLOGY OF BLOOD MEAL

DIGESTION AND MIDGUT FUNCTION
The midgut is the largest organ in the tick body and the first site for microbes ingested with the
blood meal to make contact with the tick’s body tissues. It is also the organ where freshly in-
gested blood is processed for digestion (the preoral canal, pharynx, and esophagus are lined
with cuticle, preventing absorption or digestion). Consequently, it has been the target of intense
study in order to determine the genes that are expressed in response to blood feeding and the
proteins involved in these processes. In recent years, new molecular tools have become available
that make it possible to examine the global gene expression in the tick midgut and to compare the
gut response to absorption and digestion of the blood components. Among the most important
of these new tools is the transcriptome, essentially a cDNA library of the mRNA messages (tran-
scripts) coding for proteins expressed in that organ, including those that are involved in process-
ing the blood meal. Transcriptomes provide an opportunity to examine at high resolution the
entire repertoire of mRNA molecules expressed in a particular organ or tissue at a particular
moment in time. Very few analyses of this nature have been reported. Those that have been done
include a transcriptomic analysis of 2 cDNA libraries of 1,679 high quality expressed sequence
tags (ESTs) from Dermacentor variabilis midguts fed for 2 or 6 days on a rabbit (Anderson et al.
2008), 4 cDNA libraries of 4,702 ESTs from the midguts of Ixodes scapularis female ticks
(uninfected or infected with Borrelia burgdorferi) fed on rabbits for 2 or 6 days (J. M. Anderson,
D. E. Sonenshine, M. Figeroa, and J. G. Valenzuela, unpublished data), and a proteomic analysis of
R. microplus (Kongsuwan et al. 2010). Various expressed proteins from a midgut-specific cDNA
library from fed Haemaphysalis longicornis have been reported in multiple publications (Alim et al.
2007), yet a transcriptome-wide analysis has not been published. Additionally, there have been
many reports of individual proteins isolated or cloned from midgut tissue of various tick species.
4.1. PROTEIN EXPRESSION IN THE TICK MIDGUT

It is unclear exactly how many different proteins are expressed in the tick midgut during feeding
and blood meal digestion. As with the salivary glands, it is assumed that midgut proteins are
temporally expressed, but the diversity of proteins and the timing of expression have yet to be
fully unraveled. Comparison of the 2 most extensive midgut-specific transcriptome analyses to
date representing the metastriate (D. variabilis) and prostriate (I. scapularis) subfamilies of Ix-
odidae has revealed an array of functional proteins classified into 20 categories (Fig. 6.17A). The
most abundant functional class identified in both ticks was protein synthesis machinery com-
prising housekeeping genes involved in protein synthesis, followed next by proteins involved in
the energy functions of metabolism (Fig. 6.17A). These findings are consistent with the cell
growth and digestion of the blood meal. Other important categories include oxidative stress,
peptidases, peptidase inhibitors, lipases, carbohydrate digestive enzymes, immunity-related
peptides, iron/heme metabolism, heme sequestration and transport, and others. A notable dif-
ference between the 2 transcriptomes was the number of transcripts categorized as transposable
elements, with only 1 transcript found in D. variabilis and 148 in I. scapularis. Other notable
differences were found in the energy metabolism, protein modification machinery, and signal
transduction categories. In comparisons of the functional groups primarily associated with
blood meal digestion (Fig. 6.17B), major differences were found in the expression of metallopep-
tidases (5 transcripts found in D. variabilis, versus only 1 in I. scapularis), cysteine peptidases,
serine peptidases, aspartyl peptidases, and peptidase inhibitors. In contrast to D. variabilis, no
transcripts for aspartyl peptidases were found in the midgut of I. scapularis, which suggests a
possible difference in hemoglobin digestion between this species and I. ricinus, where aspartyl
peptidases participate in the initial stages of hemoglobin digestion (see Section 4.2.2). Serine
peptidases were much less numerous, whereas cysteine peptidases were much more numerous,
in the D. variabilis midgut than in that of I. scapularis. However, 2 cathepsin L transcripts and 1
cathepsin B transcript were found in the I. scapularis midgut, suggesting a possible role in
hemoglobin digestion similar to that described for I. ricinus (see Section 4.2.2). As expected, few
transcripts containing a secretion signal were found (only 6.9% in D. variabilis and 4.5% in I.
scapularis), consistent with their purported role in intracellular processes. In contrast, examina-
tion of the transcriptomes of tick salivary glands showed a much higher proportion (29%) of
secreted proteins (Ribeiro et al. 2006). Furthermore, the midgut transcriptomes from both tick
species fed for 2 days and 6 days revealed a similar temporal pattern, with a greater number and
variety of proteins expressed in 6-day-fed ticks. In the succeeding sections we describe the major
classes of proteins involved in midgut function and blood meal digestion.
A cytoskeletal
extracellular matrix
Immunity
metabolism, carbohydrate
metabolism, energy
metabolism, heme
Putave Biological Funcon
metabolism, lipid
metabolism, nuc. D. variabilis
nuclear regulaon I. scapularis
pepdase
pepdase inhibitor
proteasome machinery
protein export machinery
protein modiﬁcaton machinery
protein synthesis machinery
signal transducon
transcripon factor
transcripon machinery
transporter
transposable element
0 20 40 60 80 100 120 140 160
Number of Transcripts
B
Iron/heme Metabolism
Immunity
Putave biological funcon in
blood meal digeson
Lipids
Metallopepdase I. scapularis
D. variabilis
Cysteine Pepdase
Aspartyl Pepdase
Serine Pepdase
Pepdase Inhibitors
Oxidave Stress
0 2 4 6 8 10 12 14 16 18
Number of Transcripts
FIGURE 6.17: Comparison of categories of putative biological functions (A) and the biological functions
associated with blood meal digestion (B) identifi ed in the midgut-specifi c transcriptomes from
Dermacentor variabilis and Ixodes scapularis.
4.1.1. Peptidases expressed in the tick midgut

Peptidases constitute the largest single category of genes important in blood meal digestion in
the tick midgut. Members of 4 major groups of peptidases (serine, aspartic, cysteine, and metal-
lopeptidase) have been found in the tick midgut. The most important of these are cysteine and
aspartic proteases, which together form a cascade of peptidases that have been shown to be in-
tricately involved in tick hemoglobin digestion (discussed in Section 4.2.2). Several cysteine
peptidases—specifically, cathepsin-like proteases and asparaginyl endopeptidases—have been
identified and functionally characterized from the midgut of various tick species and include a
cathepsin L from R. microplus (BmCL1) (Renard et al. 2000), H. longicornis (H1CPL-A) (Yamaji
et al. 2009b), and I. ricinus (IrCL1) (Sojka et al. 2008); a cathepsin B termed longipain from
H. longicornis (Tsuji et al. 2008) and I. ricinus (IrCB1) (Sojka et al. 2008); a cathepsin C from
I. ricinus (IrCC) (Sojka et al. 2008); a legumain/AE from I. ricinus (IrAE) (Sojka et al. 2008); and
2 from H. longicornis (HlLgm1 and HlLgm2) (Alim et al. 2007, 2008). An aspartic peptidase,
cathepsin D, implicated in both blood meal digestion and antimicrobial function, was identified
originally from R. microplus (Mendiola et al. 1996) and more recently from H. longicornis
(termed longepsin) (Boldbaatar et al. 2006), I. ricinus (IrCD) (Sojka et al. 2008), and R. microplus
(BmCL1) (Cruz et al. 2010).
Serine proteinases are known to play an important role in various physiological processes of
hematophagous insects such as defense responses (Paskewitz et al. 2006) and the digestion of
blood (Lehane et al. 1996). Trypsin and chymotrypsin are the most extensively studied groups
of digestive serine peptidases in hematophagous insects. Three midgut-specific serine proteases
(HlSP, HlSP2, and HlSP3) have been identified from the hard tick H. longicornis (Miyoshi et al.
2004, 2008). Distinct from other digestive serine proteases, HlSP was shown to contain potent
hemolytic activity and might be involved in the hemolysis of ingested red blood cells (discussed
in Section 4.2.1) (Miyoshi et al. 2007). Structurally distinct from HlSP and more similar to diges-
tive serine peptidases found in other hematophagous insects, HlSP2 and HlSP3 are expressed
much later than H1SP, with a significant increase in expression >96 hours after blood feeding.
Because of this expression profile, HlSP2 and HlSP3 are thought to be involved in blood meal
digestion, although their exact function has not been characterized (Miyoshi et al. 2007). Three
serine peptidases (RAMSP-1 to -3) have also been cloned and characterized from Rhipicephalus
appendiculatus (Mulenga et al. 2003). The authors could not conclusively determine the func-
tion of the 3 chymotrypsin-like polypeptides, but based on the expression pattern, they suggest
involvement in regulating blood meal uptake or digestion. A serine carboxypeptidase (SCP) has
been characterized from H. longicornis (HlSCP1) (Motobu et al. 2007) and is involved in blood
meal digestion (Horn et al. 2009). SCPs are proteolytic enzymes that cleave the carboxyl termi-
nus of its substrate at acidic pH levels and exploit serine in their catalytic activity (Remington
and Breddam 1994). A metallopeptidase characterized from R. microplus (BmMP) (Barnard
et al. 2012) was found to be a member of the astacin metzincin family of metallopeptidases and
might be involved in reproduction and blood meal digestion. Finally, a leucine aminopeptidase
involved in midgut digestion has been found in H. longicornis (HlLAP) (Hatta et al. 2006) and
might play a role in protein biosynthesis and degradation.
Midgut-specific cDNA library analyses such as the one conducted by Anderson et al (2008)
for D. variabilis and I. scapularis allow for a global view of the various peptidases expressed
during blood feeding. In D. variabilis, peptidases were the largest single category of genes found
to be important in blood meal digestion (26 transcripts). Similar results were found in the
I. scapularis midgut library, in which 19 transcripts were identified. In D. variabilis, 4 different

peptidase families were found—namely, 14 cysteine peptidases, 5 serine peptidases, 3 aspartyl
peptidases, and 4 metallopeptidases (Fig. 6.17B)—whereas in I. scapularis, only 3 cysteine pep-
tidases and 1 metallopeptidase were found, yet 15 transcripts related to serine peptidases (in-
cluding chymotrypsin and trypsin-like peptidases) were identified. Cathepsins B and L were
found in both D. variabilis and I. scapularis, yet, interestingly, no cathepsin D (an aspartic pep-
tidase) was found in the I. scapularis midgut-specific libraries, and 1 transcript was found in the
D. variabilis library. In both cDNA libraries, several highly homologous genes were found to be
expressed at the same time. This duplication of genes with similar functions is of great func-
tional value for the blood-feeding process and is an example of the remarkable redundancy that
is characteristic of highly specialized parasitic life styles, “consistent with the hypothesis that
gene duplication contributed to the successful adaptation of ticks to hematophagy” (Anderson
et al. 2008, p. 552). This phenomenon was noted previously by Ribeiro et al. (2006) in their
study of the salivary glands.
Comparing D. variabilis females fed for only 2 days to those fed for 6 days, Anderson
et al. (2008) found that some peptidases were expressed only during the initial attachment
period and were downregulated later, whereas others were expressed only in those imbibing
and digesting blood (fed for 6 days). This is consistent with the initiation of attachment and
formation of the feeding lesion, during which little blood is imbibed. Notably, no serine or
aspartyl peptidases and only 5 cysteine peptidases were expressed in the 2-day attached
ticks. In contrast, all 4 of the metallopeptidases were expressed in the 2-day-fed females, but
none were noted in the 6-day-fed females. It is possible that the expression of metallopepti-
dase at this early stage of tick attachment might be more closely related to cellular immune
defense (Willot and Tran 2002) than to blood digestion. In hookworms and other blood-
feeding helminths, metallopeptidases function in the digestion of the intermediate products
of the hemoglobin-digestive cascade (Williamson et al. 2004), which would not be expected
to occur in the midguts of the 2-day-fed females. By the sixth day of D. variabilis feeding, all
5 serine peptidases, all 3 aspartic peptidases, and 12 of the 14 cysteine proteases were
expressed (Fig. 6.18). Two of the serine peptidases were further characterized as carboxy-
peptidases. The 3 aspartic peptidases found are representative of the family of proteins that
includes pepsins, cathepsin D, cathepsin E, and renins and are reported to be important in
hemoglobin proteolysis (Alim et al. 2007; Sojka et al. 2007). One transcript, DvM 254, has a
signal peptide indicating it is a secreted protein. This transcript matched a cathepsin D
found in H. longicornis (Anderson et al. 2008) that is believed to be critical for the final
stages of hemoglobin digestion (Alim et al. 2007). Interestingly, no peptidases were found in
the I. scapularis 2-day-fed midgut library, and all 19 transcripts were found in the 6-day-fed
library (J. M. Anderson, D. E. Sonenshine, M. Figeroa, and J. G. Valenzuela, unpublished
data).
4.1.2. Peptidase inhibitors expressed in the tick midgut

Peptidase inhibitors, particularly cysteine peptidase inhibitors (cystatins) and serine peptidase
inhibitors (serpins), play an important role in regulating digestive activity. Cystatins are inhibi-
tors of papain-like cysteine proteases and are divided into secreted and intracellular forms. Cys-
tatins have been implicated in the regulation of protein turnover and defense against pathogens.
Three cystatins have been identified from the midgut of H. longicornis: an intracellular cystatin,
Hlcyst-1 (Zhou et al. 2009), and 2 secreted cystatins, Hlcyst-2 (Zhou et al. 2009) and Hlcyst-3
Secreted
Putative biological function in blood meal digestion
Iron/heme Metabolism
Immunity
Carbohydrate digestion
Lipids
Unfed/2 d fed
Metallopeptidase 6 d fed
Cysteine Peptidase
Aspartyl Peptidase
Serine Peptidase
Peptidase Inhibitors
Oxidative Stress
0 5 10 15 20 25 30
Number of Sequences
FIGURE 6.18: Differential display of proteins associated with Dermacentor variabilis midgut function
from unfed/2 days post–host attachment (unfed/2 d fed) versus proteins from 6 days post–host
attachment (6 d fed). From Anderson, J.M., Sonenshine, D.E., and Valenzuela, J.G. (2008) Exploring the
mialome of ticks: an annotated catalogue of midgut transcripts from the hard tick, Dermacentor variabilis
(Acari: Ixodidae). BMC Genomics 9:552, with permission from BioMed Central.
(Zhou et al. 2010). All 3 are induced by blood feeding and inhibit a cysteine peptidase, cathepsin L,
a component of the blood meal degradation cascade (Yamaji et al. 2010). Hlcyst2 and (possibly)
Hlcyst1 have been shown to be involved in tick innate immunity and midgut physiology. Ser-
pins, including inhibitors of trypsin and thrombin, regulate serine peptidases involved in blood
coagulation, fibrinolysis, and complement activation (Rubin 1996). One type of serpin, a kunitz-
type serine protease inhibitor (KPI), is normally associated with the anticoagulant activity of
hematophagous insect saliva, yet Ceraul et al. (2008) found increased expression of a KPI iso-
lated from the midgut of D. variabilis (DvKPI). DvKPI is induced upon feeding and has antico-
agulant and trypsin inhibitory properties. DvKPI was also shown to exhibit bacteriostatic
properties in the midgut. A kunitz-type thrombin inhibitor was identified from R. microplus
(boophilin) (Ricci et al. 2007; Macedo-Ribeiro et al. 2008) and H. longicornis (hemalin) (Liao
et al. 2009). Both are expressed in the tick midgut, and their expression levels are highest during
the rapid feeding phase, when it is critical to keep the large amount of blood being imbibed fluid
for subsequent digestion. Because of their anticoagulant properties, thrombin inhibitors are es-
sential for feeding success and survival in hematophagous insects. A KPI has been identified
from H. longicornis (HlMKI) (Miyoshi et al. 2010). Specifically expressed in the midgut, HlMK1
contains a single kunitz domain. HlMK1 co-localizes with an H. longicornis midgut-derived
serine proteinase (H1SP) in the gut epithelial cells and appears to inhibit trypsin.
Several putative peptidase inhibitors were identified in the cDNA libraries constructed
from 2-day- and 6-day-fed D. variabilis and I. scapularis midguts (Anderson et al. 2008;
J. M. Anderson, D. E. Sonenshine, M. Figeroa, and J. G. Valenzuela, unpublished data). Nine
peptidase inhibitors were identified in the D. variabilis midgut-specific cDNA library. Three
were expressed only in the unfed/2-day-fed midguts, including 2 peptides that showed a
match to serine protease inhibitors in other species of ticks (i.e., boophilin) and a third
that was identified as a cysteine protease inhibitor. The remaining 6 peptidase inhibitors
were expressed in the midguts of 6-day-fed females, including 3 identified as serpins,
2 as cystatins, and another as a zinc-binding protein of unknown function. All were found to be
non-secreted, cytoplasmic peptides, with 1 exception, a secreted serpin (Anderson et al. 2008).
Seventeen transcripts homologous to peptidase inhibitors were found in the midgut-specific cDNA
libraries from I. scapularis and include 1 cystatin, 3 serine peptidase inhibitors, and 13 transcripts
with homology to the trypsin inhibitor-like cysteine-rich domain. Interestingly, all of the tran-
scripts were found in the 6-day-fed tick midguts, and none were found in the 2-day-fed ticks
(J. M. Anderson, D. E. Sonenshine, M. Figeroa, and J. G. Valenzuela, unpublished data). Multiple
copies of serine protease inhibitors, described as lopsins, were also identified in A. americanum;
most were ubiquitously expressed in different tissues. However, at least 6 lopsins were predomi-
nantly expressed in the midgut (Mulenga et al. 2007). Most are slow, tight-binding inhibitors of the
kunitz family, a single bovine pancreatic trypsin inhibitor (BPTI)-like domain or 2 BPTI-like do-
mains. Multiple copies of serpins have also been reported to be expressed at various times during
feeding in the midgut of I. scapularis (Mulenga et al. 2009), during the first several days post-attach-
ment in the midguts of H. longicornis (Sugino et al. 2003), and in the midguts of other species of
ticks. Expression of the specific protease inhibitors during different phases of blood digestion might
provide an important mechanism for suppressing each of the various peptidases, enabling them to
act sequentially during the course of the digestive cascade. Midgut peptidases and their inhibitors
are believed to function in a cascade of enzymatic steps that function sequentially in the digestion
of the hemoglobin molecule and its protein moieties, as discussed below (see Section 4.2.2).
4.1.3. Oxidative stress and heme sequestration

Blood digestion may contribute to significant oxidative stress resulting from the release of heme,
free iron radicals, H2O2, and other stress-inducing molecules. At least 16 contigs were identified
in the D. variabilis midgut with functional roles involving oxidative stress. Most were found in the
6-day-fed females. Among the most numerous were copies of glutathione-S-transferase; others
found included thioredoxin, glutaredoxin, phospholipid glutathione perioxidase, superoxide dis-
mutase (SOD), metallothionein, quinoid dihydropteridine reductase, aldehyde dehydrogenase,
and selenoprotein M precursor. SODs scavenge free radicals, converting the superoxide anion
into oxygen and hydrogen peroxide; metallothionein chelates heavy metals liberated from the
metal co-factors, and aldehyde dehydrogenase “may function to detoxify aldehydes such as the
toxic byproducts resulting from lipid peroxidation” (Anderson et al. 2008, p. 561). In some species
(e.g., R. microplus), H2O2 (a highly oxidative compound) liberated in the midgut digestive cells is
controlled by catalase, which decomposes it to water and oxygen (Citelli et al. 2007).
Heme liberated from hemoglobin digestion and its detoxification into heme aggregates have
already been reviewed (see Sections 3.2.1 and 3.2.5). In addition to heme, large amounts of free
iron in the form of ferric iron (Fe+++) are also liberated as a byproduct of hemoglobin digestion
and are toxic unless removed. Blood-feeding arthropods express ferritin, which captures ionic
iron, serving “as an iron storage reservoir as well as for protection against iron overload” (An-
derson et al. 2008, p. 577). Two transcripts for ferritin were found in the cDNA library of the
D. variabilis midgut, both showing excellent matches to similar sequences in other ticks, as well
as to the conserved ferritin domain. These molecules were among the most frequently sequenced
ESTs, further supporting the importance of their role in iron sequestration.
Ferritins are highly conserved among many different tick species. In I. ricinus, a secreted
ferritin (designated FER2) functions as the primary transporter of non-heme iron between the
tick gut and the peripheral tissues, and an intracellular ferritin (FER1) regulates transcriptional
control of the secreted ferritin (Hajdusek et al. 2009).
Although most heme is detoxified as described previously, small amounts are transported
from the midgut by means of a heme-binding protein, hemelipoglycoprotein (also known as
HeLp or CP), which is believed to sequester heme and transfer it to the hemocoel for subsequent
transfer to other heme-binding proteins for use in body tissues. Heme can be transported across
cell membranes for uptake by heme-binding proteins. CP is highly expressed in the fat body and
salivary glands in response to blood feeding. The protein is especially abundant in the hemo-
lymph, causing the brown color characteristic of this fluid in ixodid ticks (Donohue et al. 2008).
The role of heme-containing storage proteins in the physiology of ticks is discussed in detail
elsewhere in this book (see Chapter 15).
4.1.4. Antimicrobial defenses in the tick midgut

As obligate blood feeders, ticks are at risk of ingesting foreign organisms, including potentially
harmful microbes. Consequently, protection against invasive microorganisms is important for
successful blood feeding. Contrary to previous assumptions, the tick midgut lumen is a hostile
environment for ingested microbes, mainly as a result of the antimicrobial activity of fragments
of digested hemoglobin (Sonenshine et al. 2005), antimicrobial peptides, and antioxidants. Sev-
eral immunopeptides were identified in the D. variabilis midgut (Anderson et al. 2008). Among
the most important were the metabolic lipid domain protein (also found in Niemann-Pick type
C2 [Npc2] proteins), allergen-like proteins and surface antigens important in pathogen recogni-
tion, serine protease inhibitors, and lectins similar to Dorin-M from the soft tick O. moubata
(Kovár et al. 2000) and Ixoderin from I. ricinus (Rego et al. 2005). Defensins were found in the
midguts of soft ticks (e.g., O. moubata) (Nakajima et al. 2002) and several species of hard ticks
(Sonenshine and Hynes 2008). One of the most effective antimicrobial peptides is the KPI, sim-
ilar to anticoagulants recognized in the salivary glands. In their studies with the rickettsial sym-
biont Rickettsia montanensis, Ceraul et al. (2008) showed that challenging the tick with cultures
of this microbe resulted in sustained KPI gene expression in the midgut, indicating its function
as a bacteriostatic protein, presumably by inhibiting bacterial trypsin. Some oxidative stress
proteins (e.g., glutathione-S-transferase) may also function to inhibit microbial growth. For a
more in-depth review of the role of the antimicrobial defenses of the tick midgut, see Chapter 5
of Volume 2.
4.2. MOLECULAR PROCESSES FOR HEMOLYSIS

AND HEMOGLOBIN DIGESTION
Enough molecular information has now been accumulated for us to address the most basic
questions concerning the digestion of the tick’s blood meal. When and how are the blood cells
lysed and the proteins (especially hemoglobin) released? What are the specific receptors that
bind the hemoglobin molecules and facilitate their absorption into the digestive cells? Why are
so many different enzymes expressed in the midgut epithelial (digestive) cells? Which enzymes
are secreted into the midgut lumen to lyse the erythrocytes? Which ones bind hemoglobin for
incorporation into the digestive cells? Which enzymes digest hemoglobin? Do different enzymes
target different regions of the globin moieties, acting in concert as a catalytic cascade to degrade
the molecules and transport the amino acids and/or small peptides to the hemolymph for use
elsewhere in the body (anabolic processes)? Which molecules catalyze the formation of heme
aggregates in the hemosomes? How and when are the various peptidase inhibitors (e.g., cys-
tatins and serpins) expressed to suppress the activity of selected peptidases, thereby allowing
others to function more efficiently? These and other questions concerning the molecular basis
of blood digestion are addressed below.
4.2.1. Hemolysis of ingested blood cells

The first step in blood meal digestion is hemolysis of the formed elements (e.g., the erythro-
cytes), a process that begins soon after these cells are ingested. Several enzymes have been re-
ported to function as hemolytic agents in different tick species. Secreted phospholipase A2,
found in the saliva of the hard tick A. americanum, was reported to promote hemolysis of the
ingested red cells (Zhu et al. 1997). Evidence of a salivary phospholipase was also reported for
the soft tick O. parkeri (summarized by Francischetti et al. 2008).
Hemolysins are also secreted into the midgut lumen by the digestive cells. A trypsin-like
serine protease induced in the first several days of the feeding process was identified as the he-
molytic enzyme in H. longicornis and was identified in the lumen and the midgut epithelial cells
of this tick. This enzyme has a pH optimum of pH 6.0, similar to the natural pH of the midgut
lumen (Miyoshi et al. 2007), but it is active over a wide pH range. According to Miyoshi et al.
(2008), HlSP, a cubulin-related serine protease, attacks a specific protein or group of proteins on
the erythrocyte membrane, leading to hemolysis of these cells. The expression peak for this en-
zyme occurs at day 3 post-infestation, after which it is virtually undetectable. A similar serine
protease (AY078095) was also found in R. appendiculatus (e-value: 2 × 10−174). Serine proteases
were also reported from the midgut of D. variabilis, but only in 6-day-fed ticks. However, no
evidence of serine protease expression was observed in the midguts of 2-day-fed or unfed
D. variabilis females (Anderson et al. 2008), so it is possible that host blood cell hemolysis might
not occur until later in the feeding process or that salivary enzymes had already hemolyzed the
blood cells in this species (of course, it is also possible that they were not detected because of the
type of library construction used in the study; studies using 454 pyrosequencing or Illumina
sequencing generate much greater coverage and may reveal the presence of transcripts that were
not detected by earlier-generation library construction kits). This is consistent with a report by
Ribeiro (1988), who could not detect in vitro erythrocyte lysis activity using midguts of unfed or
2-day-fed I. (dammini) scapularis.
In contrast to ticks, different patterns of erythrocyte lysis occur in blood feeding insects. Lice
(e.g., Pediculus humanus) and fleas (e.g., Ctenocephalides felis) hemolyze their ingested red cells
rapidly. In contrast, bed bugs, sand flies, and various mosquitoes delay this process for many
hours (Vaughan and Azad 1993).
4.2.2. Digestion of hemoglobin and other blood meal proteins

Following the dissolution of the blood cells (hemolysis) in the midgut lumen, hemoglobin is
absorbed via receptor-mediated endocytosis in clathrin-coated vesicles (CCVs) at the lu-
minal surfaces of the midgut epithelial cells as described previously (Section 3.2.5). This
requires the expression of molecules to bind hemoglobin liberated from the lysed erythro-
cytes. Studies of the proteome of the R. microplus midgut revealed the presence of clathrin-
adaptor protein, a critically important protein involved in the assembly of CCVs (Kongsuwan
et al. 2010). The clathrin coating serves as a mechanical scaffold aided by clathrin-binding
adaptors that link the clathrin lattice to the membrane (Di Pietro et al. 2010). Precisely how
hemoglobin is recognized by the CCVs in ticks is unknown. Hemoglobin-binding proteins
were recognized in the cDNA library of the midgut of feeding D. variabilis females, but their
precise molecular structure and mode of action were not elucidated. However, in R. microplus,
certain membrane-associated trafficking proteins such as syntaxin 6 and surfeit 4 (Kongsu-
wan et al. 2010) are believed to be involved in hemoglobin transport within the cytosol of the
absorbing cell. Thus, it is likely that a similar mechanism may occur in the midgut of other
ticks. More research is needed in order for us to completely understand the molecular pro-
cesses involved in hemoglobin uptake and intracellular transport during blood feeding in
ticks.
Hemoglobin molecules absorbed by the digestive cells accumulate in heterolysosomes,
where they are digested intracellularly. Contrary to previous expectations of a single “hemoglo-
binase,” hemoglobin digestion is carried out by a cascade of proteolytic enzymes and peptidase
inhibitors exquisitely synchronized in the timing of their expression to target specific sites on
the globin moieties, most functioning at acidic pH levels. In I. ricinus, these proteolytic enzymes
comprise an evolutionarily conserved network of aspartic and cysteine peptidases (Sojka et al.
2008). However, the composition of this digestive network may differ in different tick species. As
described by Horn et al. (2009) using imaging with specific activity-based probes, 5 significant
endo- and exopeptidase proteolytic processes are believed to occur, involving 3 cysteine pepti-
dases: (i) cathepsin B, (ii) cathepsin L, and (iii) cathepsin C of the papain-type peptidase group
(CA clan); in addition to the aforementioned enzymes, the process also uses (iv) an asparaginyl
endopeptidase, namely, a legumain (CD clan), and (iv) an aspartic endopeptidase, specifically,
cathepsin D (a member of the AA clan). A model illustrating hemoglobin digestion in the
I. ricinus midgut is shown in Fig. 6.19. The first steps in the degradation pathway are initiated by
the aspartic and cysteine class endopeptidases (cathepsins D and L and legumain). Legumains,
also known as asparaginyl endopeptidases (AEs), are a class of cysteine endopeptidases found in
the lysosomes. They are members of the peptidase family C13 that show strict specificity for the
hydrolysis of asparaginyl bonds. Cathepsin D is the most dynamic enzyme in the initial degra-
dation process, with the highest turnover efficiency. Together, as shown in Fig. 6.19, these 3 en-
zymes (D, AE, and L) cleave hemoglobin into large globin fragments. Following digestion by
these enzymes, the large fragments are further degraded by cysteine amino- and cysteine car-
boxydipeptidases (cathepsin B and cathepsin L) into smaller fragments. Finally, the smaller
fragments are degraded further by cathepsins C and B, liberating dipeptides. According to Sojka
et al. (2011), free amino groups are released from the resulting dipeptides through the action of
leucine aminopeptidase (LAP) and serine proteases (serine monopeptidases). It is possible that
LAP (a type of metallopeptidase) might function in a different, less acidic intracellular compart-
ment closer to its pH optimum of ~6.5. In I. ricinus, hemoglobinolysis increases rapidly toward
the end of the slow feeding period, increasing to the maximum extent during full engorgement
(Franta et al. 2010).
Comparisons with blood digestion in argasid ticks are reviewed elsewhere (Chapter 9).
It is not clear whether the same network of digestive enzymes is widespread throughout the
suborder Ixodida or whether they function in identical roles. The evidence available to date
FIGURE 6.19: A model illustrating the proteolytic pathway of hemoglobin digestion that occurs in the
heterolysosomes of the midgut epithelial cells of a typical tick, Ixodes ricinus. In this model, cathepsins
D and L plus legumain initiate the primary cleavage that begins the digestive process. Next, the large
fragments resulting from these enzymatic steps are further digested, primarily by cathepsin B
(secondarily by cathepsin L) in smaller fragments. Next, cathepsins C and B, with secondary digestion
by leucine amino peptidase (LAP) and serine carboxypeptidase (SCP), act on the small fragments,
cleaving them further into dipeptides and free amino acids. From Horn, M., Nussbaumerova, M., Sanda,
M., Kovarava, Z., Srba, J., Franta, Z., Sojka, D., Bogyo, M., Caffrey, C.R., Kopacek, P., and Mares, M.
(2009) Hemoglobin digestion in blood-feeding ticks: mapping a multipeptidase pathway by functional
proteomics. Chem. Biol. 16:1053–1063, with permission from Cell Press.
suggests that the composition of the digestive network is fundamentally similar in other ticks,
especially among the metastriate tick species. Among those ticks, the most extensive studies
have been done with H. longicornis. In this species, a cathepsin-L-type cysteine peptidase was
found in feeding ticks. It is active in a pH range of 3.2–5.6 and has been shown to digest hemo-
globin (Yamaji et al. 2009b). A cathepsin L (cysteine peptidase) also was reported from the
midgut endosomes of feeding R. microplus (Renard et al. 2000). Cathepsin B, an exopeptidase,
also was found in the gut tissues of H. longicornis. Described as longipain, it was found to have
an essential role in the transmission of Babesia parasites. At least 2 different AEs were reported
from H. longicornis (HlLgm1 and HlLgm2). Both are believed to play important roles in host
blood meal digestion and might be critical for the final process of digestion of blood compo-
nents (summarized by Sojka et al. 2011; Alim et al. 2007, 2008). According to these authors,
HlLgm1 and HlLgm2 might also stimulate midgut cell proliferation and cell morphology, in
addition to having a possible role in hemoglobin digestion (summarized by Sojka et al. 2011).
Another possible difference between the I. ricinus model and the metastriate species is the role
of serine peptidases. Although a serine carboxypeptidase was found to function in the final steps
in the hemoglobinolytic process in I. ricinus, the role of serine peptidases might be more
prominent in the metastriate ticks. Five different serine carboxypeptidases and serine peptidases
were identified in the midguts of 6-day-fed females (i.e., late in the feeding and digestive process),
but no evidence of LAP was reported (Anderson et al. 2008). Serine peptidases were also iden-
tified in the midgut digestive cell in R. appendiculatus and H. longicornis (Mulenga et al. 2003;
Miyoshi et al. 2008). However, unlike in I. ricinus, LAP was not found in either the 2-day-fed or
the 6-day-fed D. variabilis midguts. Blood-feeding mites (e.g., A. siro and Tetranynchus urticae)
also are known to contain cysteine and aspartic peptidases and were found capable of hydro-
lyzing hemoglobin intracellularly in the lysosomal compartments of their midguts (Nesbitt and
Billingsley 2000).
The end products of hemoglobin digestion migrate via transcytosis to the hemolymph and
are dispersed to the body tissues for anabolic metabolism. Heme released as a byproduct of he-
moglobin digestion is detoxified through a unique mechanism described elsewhere (see Section
3.2.5).
Little is known about how the timing of expression (i.e., upregulation and downregulation)
of the different enzymes in the digestive network is regulated. Multiple gene copies of cysteine
and serine peptidase inhibitors (cystatins and serpins, respectively) occur in the midgut, several
of which are secreted (e.g., the Kunitz-type serpin described by Ceraul et al. [2008]). However,
the primary role of these inhibitors appears to be associated with antimicrobial rather than
digestive functions.
Most other blood meal proteins remain undigested in the midgut lumen. An important ex-
ception is albumin, which is also absorbed by the digestive cells, presumably via fluid phase
endocytosis rather than receptor-mediated endocytosis (Lara et al. 2005). Studies by Alim et al.
(2007) showed that digestion of this nutrient is accomplished by the asparaginyl legumain pep-
tidases. Most likely, this occurs in a separate type of lysosomal endosome in the digestive cells,
as the pH optimum for these enzymes is close to pH 7.0.
4.2.3. Blood meal digestion in hematophagous insects and

other blood-feeding invertebrates
Unlike in ticks, blood digestion, including of hemoglobin, takes place in the midgut lumen
rather than in the digestive cells in blood-feeding insects and other invertebrates. Neverthe-
less, many of the same enzymes found in ticks are also present. Cathepsin B- and L-like en-
zymes were found in the midgut of the bloodsucking bug Triatoma infestans (Kollien et al.
2004). Of interest is the finding of secreted zinc metalloprotease and secreted zinc carboxy-
peptidase in the midgut of the tsetse fly, Glossina morsitans, genes that were upregulated fol-
lowing the blood meal (Yan et al. 2002) (note that in the tick D. variabilis, metalloproteases
were expressed only early in the attachment phase). In addition to the blood-feeding insects,
many of the same enzymes classes, specifically, cathepsin B (Sm31), cathepsin L1, cathepsin
L2, cathepsin D, cathepsin C, and legumain (Sm32), are known or believed to function in
hemoglobin digestion in parasitic helminthes (e.g., the sheep liver fluke, Fasciola hepatica
[Tort et al. 1999], and hookworms [Williamson et al. 2004]). Leucine amino peptidase was
reported to digest the intermediate fragments of hemoglobin digestion to amino acids in
Schistosoma mansoni and S. japonicum (Dalton et al. 1997; McCarthy et al. 2004). Thus, except
for its novel intracellular location and minor differences in gene expression, hemoglobin di-
gestion in ticks appears to be fundamentally similar to that found in many other blood-feeding
invertebrates.
5. MIDGUT INF ECTION BY TICK- TRANSMITTED

PATHOGENS AND SYMBIONTS
As noted previously, the midgut is the first site of contact between ingested microbes and the tick’s
body tissues. Some pathogens (e.g., B. burgdorferi) colonize the luminal surface of the midgut
epithelial cells but do not penetrate the cells. However, most pathogenic microbes (e.g., Rickettsia
rickettsii, Babesia microti, etc.) and symbionts (e.g., R. montanensis) invade the digestive cells,
proliferate, and then disperse to the tissues. The modes of infection, proliferation, and dispersal
from the midgut of these different tick-borne microbes are described in detail in chapters on
these subjects (specifically, in Chapters 6–10 of Volume 2). Of special note is a recent study by
J. M. Anderson, D. E. Sonenshine, M. Figeroa, and J. G. Valenzuela, (unpublished data) comparing
midgut-specific cDNA libraries constructed from female I. scapularis infected with B. burgdorferi
or uninfected, fed for 2 or 6 days. An interesting finding of this analysis was the overall down-
regulation of expression (i.e., ~30% fewer ESTs in the infected libraries) among infected ticks at
both 2 and 6 days post-attachment. This was also reflected in the lack of transcripts from the in-
fected libraries associated with blood meal digestion, including those involved in immunity.
Despite the impressive growth in knowledge about blood feeding, digestion, protection against
oxidative stress, and microbial infections in ticks, many unanswered questions remain. For ex-
ample, how did the tick’s novel method of blood feeding and digestion evolve? Although this
topic is discussed in depth in Chapter 9, more studies are needed in order for us to fully under-
stand how this occurred. When ticks suck blood, does regurgitation occur, and, if so, does it
allow another mode of pathogen transmission to vertebrate hosts? This question has generated
considerable controversy among investigators. Conclusive evidence is needed to resolve this
question. Another issue concerns blood meal concentration, essential for all hematophagous
invertebrates. Are the methods for blood meal concentration and water elimination via the sal-
ivary glands in ixodids or the coxal glands in argasids ancestral for these groups? As noted pre-
viously, in Nuttallia namaqua, blood meal concentration occurs via malpighian tubule excretion
of water (Mans et al. 2011). Phylogenetic analysis of these different modes of concentration of the
blood meal using modern molecular tools should yield valuable insights into the evolution of
the novel blood-feeding habits adapted by ticks. How and where are the blood cells lysed? Are
erythrocytes and other blood cells lysed by saliva, by enzymes secreted by the midgut cells, or
both? Equally important is research into how hemoglobin and the few other blood meal proteins
(e.g., albumin) are recognized by the digestive cells. How is hemoglobin transported from the
primary heterolysomes to the hemosomes, and where is it digested?
A major issue concerns the processes involved in hemoglobin digestion. Is the model de-
scribed by Horn et al. (2009) specialized for the prostriate ticks, or is it highly conserved
throughout the Ixodida? The finding of similar endo- and exopeptidases in the midguts of di-
verse tick species argues for the latter, but additional research is needed to answer this question.
Is the catalytic network of digestive enzymes of ancient origin? This network is similar in so
many different blood-feeding invertebrates, and such an origin would place it ancestral to the
evolution of hematophagy in ticks. In addition, are the small fragments of hemoglobin digestion
transported to other, less acidic endosomes where different proteolytic enzymes complete the
process and liberate dipeptides and amino acids? How is albumin digested? Is legumain the sole
enzyme involved in the process?
Major questions also concern heme detoxification and heme sequestration. Is the novel
process of heme aggregate deposition in the hemosomes described by Lara et al. (2005) for
R. microplus common to all tick species? Clearly, it does not encompass all heme liberated
from the digestion of hemoglobin, as significant amounts of heme moieties are incorporated
into the hemolymph protein CP (HeLp) (Donohue et al. 2008) and vitellogenin (Thompson
et al. 2007).
New molecular tools (e.g., transcriptomes and proteomes) have revealed numerous novel
peptides and proteins in tick tissues. To date, only 1 transcriptome for the midgut of a tick has
been published. New transcriptomes and proteomes from the midguts of other species of ticks,
including unfed versus feeding or fed specimens, should be especially useful for comparing the
numerous molecules and molecular functions described in this review. In addition to the eluci-
dation of fundamental knowledge about the biology of tick feeding processes, knowledge of
these molecules might provide new targets for vaccine development and/or novel methods for
tick control.
ACKNOWLEDGMENTS
JMA is supported by the Division of Intramural Research, National Institute of Allergy and In-
fectious Diseases, National Institutes of Health. We thank Dr. Reuben Kaufman for providing
the information about the peritrophic membrane cited in this review. We are most grateful to
Dr. Lewis Coons for the provision of several of the micrographs of tick midguts used in this
chapter. We also thank Dr. Star Dunham Ems, Department of Medicine, University of Con-
necticut Health Center, Hartford, CT, for the figure of the midgut of an Ixodes scapularis nymph
showing the peritrophic membrane. Finally, we thank Dr. Paul J. Homsher, Old Dominion Uni-
versity, Norfolk, VA, for reviewing the manuscript.
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NOTE
C H A P T E R 7
SALIVARY GLANDS
Structure, Physiology, and Molecular Biology
FRANC I SCO J. ALARCON-CHAIDEZ
1. INTRODUCTION
Tick salivary glands perform a variety of complex functions that not only are vital to tick phys-
iology but also ensure survival and the development of tick-borne pathogens. In both argasid
and ixodid ticks, salivary glands have been described as an alveolar structure composed of
agranular and granular acini. In ixodids, the agranular acini play a key role in osmoregulation,
whereas the main function of granular alveoli is the secretion of numerous bioactive proteins
and lipid molecules with diverse pharmacological properties.
The salivary glands are instrumental in maintaining water balance by concentrating the
blood meal and eliminating not only excess water but also ions that otherwise would create
significant osmotic stress. During off-host periods and in unfed ticks, the salivary glands
secrete a hyperosmotic solution that enables the absorption of water vapor from unsaturated
air and allows ticks to survive long periods of water deprivation. Tick saliva is also a major
determinant of pathogen transmission and establishment because of the potent antihemostatic
and anti-inflammatory agents that are secreted to maintain blood flow in the feeding lesion.
These bioactive agents facilitate the dissemination of pathogens by making the tick–host inter-
face a less hostile environment during feeding. In addition, many tick species secrete cement-
like substances that allow them to remain secured to the host for prolonged feeding periods.
Within this framework, transcriptomic and proteomic analyses of tick saliva and salivary
glands have led to major advances in our understanding of the molecular basis of tick–host–
pathogen interactions.
The aim of this chapter is to summarize what is known about tick salivary gland structure,
physiology, and molecular biology and their contribution to the remarkable ability of these
ectoparasites to evolve sophisticated adaptive strategies to ensure survival. In addition, a brief
overview of the current status of genomics and proteomics in the field of tick biology is
summarized.
For an electronic version illustrating the structure of the salivary glands, the reader may
browse the tick anatomical ontology for a description of the structures and related illustrations
by visiting www.Vectorbase.org and following the instructions in the footnote below. Additional
illustrations of the anatomy of ticks not included in this edition may be found there.1
2. SALIVARY GLAND MORPHOLOGY
In female ticks, 3 morphologically distinct acini (alveoli) types are present. Type I has been as-
sociated with off-host osmoregulation, and types II and III are involved in the synthesis and
secretion of protein factors and water transport (Binnington 1978; Sauer et al. 1995).
2.1. AGRANULAR ALVEOLI (ACINUS I)

Type I acini are devoid of secretory granules and are found in all life stages of ixodid ticks (Bin-
nington and Kemp 1980). These acini are located adjacent to and open directly into the main
salivary duct. The overall cellular organization of cells contained in type I acini has shown at
least 4 distinct types of cells, including a single lamellate central cell that is in direct contact
with the acinus lumen, multiple pyramidal cells on the basal portion, and peritubular and con-
strictor cells surrounding the short duct leading to the main salivary duct (Fig. 7.1). The basal
region of type I acini is characterized by highly convoluted membrane infoldings extending
from the outer cell membrane and enclosing numerous mitochondria and vacuoles, which are
characteristic of epithelia involved in active transport and fluid regulation (Needham et al.
1990). These structures are believed to play key functions in tick water balance. Type I acinar
cells are also believed to contribute to the formation of attachment cement. Ultrastructural
studies of type I acini in Amblyomma americanum revealed a gradual depletion of lipid drop-
lets during desiccation/rehydration experiments; these droplets were thought to be a compo-
nent of the cement material during the early stages of attachment (Barker et al. 1984). The
presence of lipid inclusions in type I acini has also been associated with the generation of en-
ergy needed for the production of the hygroscopic saliva to maintain water balance (Needham
et al. 1990). In contrast, agranular acini from argasid ticks are devoid of lipid inclusions, and
energy is thought to be provided by the abundant deposits of glycogen. The salivary glands of
male ixodids also contain agranular acini; however, their rate of secretion is only a fraction of
that reported for females, indicating that male salivary glands are not involved in osmoregulation
(Sauer et al. 1995).
2.2. GRANULAR ACINI

Granular acini show a dense accumulation of secretion granules in the cytoplasm of their cells.
The morphology of their cytoplasm and granules based on histochemical staining and their
position in the alveolus allows their further classification into 7 to 9 cell types. These cell types
Salivary Glands 165
FIGURE 7.1: Schematic representation of a typical type I agranular acinus from an unfed Amblyomma
americanum female tick. This figure illustrates the organization and distribution of its 4 different cell
types. From Needham, G.R., Rosell, R., and Greenwald, L. (1990) Ultrastructure of type-I salivary-gland
acini in four species of ticks and the influence of hydration states on the type-I acini of Amblyomma
americanum. Exp. App. Acarol. 10:83–104, with kind permission from Springer Science + Business Media.
are found in 2 different types of acini in female ticks, types II and III, and in 3 different types in
males, types II, III, and IV.
2.2.1. Type II
In ixodids, type II acini consist of 6 distinct granular cell types (a, b, and c1–c4) (Coons and
Roshdy 1973). In unfed male and female ticks, large type a cells packed with membrane-bound
granules of varying sizes and densities are found at the base of the alveoli (Fig. 7.2). These cells
have been associated with the production of precursors of the cement secreted during ticks’
attachment to their hosts (Binnington 1978). Type b cells are believed to produce secretions
composed of glycoproteins possibly associated with manipulation of the host immune response
or, as with type a cells, the formation of the cement cone during attachment (Walker et al.
1985). During tick feeding, type a cells release and deplete their granules while b and c cells ex-
perience a dramatic increase in size and the number of secretory granules without changes in
cell numbers.
Morphological and histochemical studies have shown that the salivary glands of argasids are
less complex than those of ixodids. Earlier studies on the ultrastructural organization of type I
acini in Argas (Persicargas) persicus showed similarities to that in ixodids, whereas type II acini
appear to contain only 3 granular cell types, designated a, b, and c (Roshdy and Coons 1975).
However, a more recent study revealed a fourth type of granular cell that was designated as d in
FIGURE 7.2: Diagram showing detail of type II alveolus organization in unfed male Dermacentor
variabilis salivary glands. The 3 types of granular cells, a, b, and c, are depicted; c1, c2, and c3 appear to
be subdivisions of type c cells showing variable electron density. Reproduced with permission of the
Journal of Parasitology from Coons, L.B. and Roshdy, M.A. (1973) Fine structure of the salivary glands
of unfed male Dermacentor variabilis (Say) (Ixodoidea: Ixodidae). J. Parasitol. 59:900–912; permission
conveyed through Copyright Clearance Center, Inc.
the salivary glands of the argasid tick Ornithodoros savignyi (Mans et al. 2004). In argasids, fluid
secretion occurs through the coxal organs, and salivary gland acini undergo few morphological
changes during feeding.
2.2.2. Type III

Type III acini are found distributed in the distal region of the salivary glands and are the most
abundant of the 3 glandular types. The overall structural organization of the type III acinus is
similar to that of type II, but type III contains only 3 types of granular cells, d, e, and f (Fig. 7.3).
In addition, there is a single adlumenal and several ablumenal interstitial cells, the development
of which during feeding contributes to the formation of the basal labyrinth in both acinus types
(Fawcett et al. 1981a). The morphology of type d cells resembles that of type a cells of acinus II.
Together with type e cells, they are thought to contribute to the formation of the cement depos-
ited during tick attachment to the host (Jaworski et al. 1990). Type f cells are usually located at
Salivary Glands 167
FIGURE 7.3: Diagrammatic representation of type III acinus showing the organization of its cell types
(d, e, and f) in unfed female Rhipicephalus appendiculatus. Reproduced from Fawcett, D.W., Doxsey, S.,
and Buscher, G. (1981) Salivary gland of the tick vector (R. appendiculatus) of East Coast fever. I.
Ultrastructure of the type III acinus. Tissue Cell 13:209–230, with permission from Elsevier.
the base of the acinus, and they are flanked by d and e cells. These cells are agranular in unfed
ticks, but their granularity and size increase gradually as feeding progresses. In contrast, type f
cells in male ticks do not increase in size during feeding. In argasids, interstitial epithelial cells
also develop extensively during feeding, but to a lesser extent than that observed in type III acini
of ixodid ticks (Roshdy and Coons 1975).
2.2.3. Type IV
Type IV acini are present only in male ticks. In addition to adluminal and abluminal interstitial
cells, type IV acini contain 1 granular cell type, designated type g, which fills with secretion
granules during tick feeding (Binnington 1978; Walker et al. 1985). Male ticks exhibit intermit-
tent feeding on the host by attaching and detaching several times during the course of feeding,
but, unlike in females, no degeneration of the salivary gland cells has been detected, and the
glands appear to remain functional throughout the feeding period (Walker et al. 1985). Overall,
male salivary gland morphology is similar to that of the females, with the only difference being
the presence of type IV acini in the former. This additional acinus in males has been associated
with lubrication and transfer of the spermatophore to female ticks during mating, suggesting a
reproduction-specific function in the male tick (Sauer et al. 1995).
3. SALIVARY GLAND DEVELOPMENT
3.1. CHANGES IN GRANULAR SALIVARY GLANDS DURING FEEDING

Studies on the morphological and structural aspects of tick feeding have shown that the process
of attachment and feeding in ixodids induces remarkable physiological changes in their salivary
glands (Binnington 1978). In Ixodes ricinus, salivary gland cells of the 23-day-old embryo begin
to differentiate and form specialized structures resembling those of fully developed salivary
glands. Completion of this process is achieved after 28 days, at which point salivary glands are
thought to be active, as secretions appear to be present in the duct lumen (Jasik and Buczek
2004).
Acini types and cells in larval and nymphal ticks are similar in structural organization to
those found in adults, with the exception of type IV acini, which are not found in larval salivary
glands (Sauer et al. 1995). Initial studies on the ultrastructure of type I acini in ixodid and argasid
ticks revealed no changes in the numbers and types of cells during the course of feeding (Bin-
nington 1978; Walker et al. 1985). At the present time, however, it is generally believed that the
type I acini undergo significant structural changes in response to off-host osmoregulation. In
nymphal ixodid ticks, agranular acini are concentrated in the proximal and middle regions of
the gland and are easily recognized by their large nucleus and non-granular appearance. In un-
fed nymphs of I. ricinus, the agranular acini are slightly larger than the granular ones, and the
lumen is barely visible. In fully engorged and detached nymphal ticks, granular acini are consid-
erably larger, showing a distinct, large lumen, whereas agranular cells remain unchanged in
terms of gross morphology and size relative to those of unfed nymphs (Kahl et al. 1990). Soon
after the detachment of fully engorged larvae and nymphs, degeneration ensues, but the salivary
ducts give rise to branching ducts that terminate in small alveoli consisting of undifferentiated
cells. Further development of these cells results in a morphology similar to that of unfed nymphs
and adults. No distinct gross morphological changes of the agranular acini have been recog-
nized in this period (Kahl et al. 1990).
During feeding, the salivary glands of adult ixodid ticks undergo remarkable growth and
differentiation, with acini types II and III showing the greatest changes in morphology. These
dramatic changes are also accompanied by significant increases in the rate of protein synthe-
sis. For instance, major and sustained protein changes in the salivary glands of feeding A.
americanum were associated with the growth and development of acini types II and III, whereas
transient changes disappearing toward the end of feeding might have been related to secretion
during the feeding process (McSwain et al. 1982)
In unfed ticks, cell types a of type II acini and d and e of type III acini occupy most of the
acini volume, and they are packed with numerous complex secretory granules. At the onset of
feeding, granules are released, and they are mostly depleted by the end of the feeding process
(7 to 14 days) (Binnington 1978). In contrast, type f cells from type III acini become hypertro-
phied during feeding and, together with the surrounding ablumenal interstitial cells, contribute
Salivary Glands 169
to the formation of an extensive basal labyrinth that is involved in fluid transport and which
might play a key role in the elimination of excess water and salts during the concentration of a
blood meal. Granular materials released by both type II and type III acini are believed to be
components of bioactive molecules found in tick saliva. Finally, type g cells from type IV acini
are small and agranular in unfed male ticks, but they gradually increase in size and in the
number of granules during feeding. The secretions of these cells are released during mating and
have been associated with spermatophore transfer to female feeding ticks (Sauer et al. 1995).
3.2. SALIVARY GLAND DEGENERATION

Following a blood meal, tick salivary glands undergo degeneration and remodeling processes
that are likely to be under hormonal control (Sauer et al. 2000; Lamoreaux et al. 2003). Salivary
gland degeneration in detached ixodid ticks is thought to proceed through autophagy or type II
programmed cell death in the granular acini (Harris and Kaufman 1981). Tissue degeneration
results in a complete loss of secretory function, which is probably due to the loss of type II and
III acini. The type I acini remain functional, presumably for water vapor uptake during the ovi-
position period (Lomas et al. 1998). Mated female ticks detach from the host shortly after they
reach a “critical weight” (CW), which has been defined as the transition between the slow and
rapid phases of feeding; however, exceeding CW and detachment are not necessarily linked, as
unmated female ticks that feed beyond the CW continue to increase in size and do not sponta-
neously detach. Further, their secretory competence does not seem to be affected, indicating
that granular acini in these ticks are functional for as long as the ticks remain attached to the
host (Friesen and Kaufman 2009). Therefore, it is hypothesized that detachment from the host
might be the signal that triggers salivary gland degeneration and that mechanical or chemical
receptors might be involved in this process.
Salivary glands undergo autolysis within 4 days in mated ticks, whereas virgin ticks can take
up to 8 days from the time they are forcibly removed from the host. This delay, however, can be
reversed by the injection of a proteinaceous factor from the male reproductive tract (EF) into
the hemocoel of feeding unmated ticks; EF stimulates engorgement and the release of ecdyster-
oid (Lomas and Kaufman 1992). Two additional engorgements factors (MF) cloned from the
male gonad of A. hebraeum appear to work in concert to stimulate degeneration of the salivary
glands and partially restore ovary development; however, the site and mode of action of native
EF and MF are still unknown (Weiss and Kaufman 2004).
The process of salivary gland degeneration in fed mated female ticks begins 3 to 4 days post-
detachment, and it is during this period that vitellogenesis, oocyte maturation, and oviposition
take place, continuing for about a month until the tick dies (Lamoreaux et al. 2003). Extensive
studies on salivary gland (SG) degeneration have associated this process with increases in the
concentration of the ecdysteroid 20-hydroxyecdysone (20E) in the tick hemolymph in the days
following detachment (Kaufman 1991). Further evidence came from the recognition of homo-
logs of the ecdysone receptor (EcR) and ultraspiracle protein (USP) from Drosophila melanogas-
ter in A. hebraeum SG extracts, which suggested the existence of a potential tick EcR mediating
analogous functions (Mao and Kaufman 1998). Later investigations on A. hebraeum revealed
that increases in the titers of ecdysteroid in the hemolymph at the onset of SG degeneration
paralleled increases in ecdysteroid-binding activity, indicating the establishment of an EcR/USP
system in the tick SGs (Mao and Kaufman 1999). Similarly, an ecdysteroid receptor containing
conserved DNA and ligand binding domains and a retinoid X receptor that is known to form
heterodimers with multiple hormone receptors were cloned and shown to be expressed in the
SGs of A. americanum (Guo et al. 1997, 1998). In A. hebraeum male gonads, voraxin has been
shown to stimulate SG degeneration in vivo; however, its role in ecdysteroid synthesis remains
to be determined (Weiss and Kaufman 2004).
4. REGULATION AND MECHANISM OF

FLUID SECRETION
4.1. EXOCYTOSIS
Innervation of tick SGs indicates that fluid secretion is controlled by nerves from the syngan-
glion that terminate in synapses on gland cells near the lumen (see Chapter 13 for an in-depth
review). It is also well established that ixodid tick SGs are stimulated by the catecholamine neu-
rotransmitter dopamine. Dopamine released into the neuroeffector junction binds to a do-
pamine D1 receptor and activates adenylate cyclase, causing an increase in the intracellular
concentration of cyclic adenosine monophosphate (cAMP) in the tick SGs. Evidence in support
of this mechanism comes from studies showing that exogenous dopamine and cAMP are capable
of stimulating in vitro fluid secretion and protein phosphorylation in isolated tick SGs. The ex-
act function of phosphoproteins in tick fluid secretion is not known; however, a link between
fluid transport and phosphoproteins associated with acinus type III has been suggested (Sauer
et al. 2000). Inhibition of the ability of dopamine to induce SG secretion by okadaic acid, a spe-
cific inhibitor of phosphatases, and the fact that phosphatase I levels increase in the feeding tick
further support this hypothesis (Sauer et al. 1995). Another line of evidence in support of this
mechanism came from the identification of genes coding for the protein kinase catalytic sub-
units in the SGs of A. americanum (Palmer et al. 1999) and A. hebraeum (Tabish et al. 2006).
Furthermore, the involvement of acinus III in fluid secretion was reported by Bowman and
Sauer (2004); dopamine was shown to increase the weight of partially fed I. ricinus SGs, with a
concomitant increase in the lumen of type III acini, but not of type II. Findings from these
studies provide strong evidence supporting the role of acinus type III as a myoepithelial cell
directly involved in fluid secretion (Coons et al. 1994).
Dopamine is also involved in stimulating cytosolic phospholipase A2 (cPLA2) by making
intracellular Ca2+ available through the opening of voltage-dependent Ca2+ channels. PLA2
generates free arachidonic acid from membrane phospholipids that is then used by the cyclo-
oxygenase pathway to synthesize the series 2 prostaglandins prostaglandin-E2 (PGE2) and
prostaglandin-F2α (PGF2α), 2 of the most highly abundant prostaglandins found in tick sa-
liva. PGE2 interacts with and activates an EP1-like receptor, which in turn activates
phospholipase C (PLC) via a cholera-toxin-sensitive G-protein-coupled receptor. The activa-
tion of PLC results in the synthesis of the second messengers inositol 1,4,5-triphosphate (IP3)
and diacylglycerol (DAG). DAG remains bound to the plasma membrane, where it recruits
calcium-dependent protein kinase C (PKC). Soluble IP3, in contrast, is released into the cyto-
sol, mobilizing Ca2+ from endoplasmic reticulum stores. The increase in intracellular calcium
concentration activates PKC through DAG, leading to signaling that mediates a cascade of
cellular events and the subsequent exocytosis of proteins from the SG acini into the saliva
Salivary Glands 171
(Sauer et al. 2000). A similar mechanism of exocytosis has also been proposed for argasid SGs
(Maritz-Olivier et al. 2005).
Current evidence suggests that key soluble N-ethylmaleimide-sensitive factor attachment
protein receptor (SNARE) complex proteins might be important in the process of exocytotic
protein secretion in the SGs (Karim et al. 2002). SNAREs are membrane receptors that bind
soluble N-ethylmaleimide-sensitive factor attachment proteins (SNAPs) and N-ethylmaleimide-
sensitive factor (NSF) and are known to regulate exocytosis by driving intracellular membrane
fusion and membrane trafficking (Malsam et al. 2008). SNARE proteins assemble between
membranes into a remarkably stable complex called SNAREpin that catalyzes fusion by forcing
membranes closely together as it zippers up and bridges the exocytic vesicle to the plasma mem-
brane. Evidence of vesicle trafficking in tick SGs involving intracellular membrane fusion driven
by SNAREs was reported in A. americanum (Karim et al. 2002, 2004a, 2004b). Vertebrate-
derived antibodies to cytosolic α/β SNAP and NSF, Ca2+ sensitive synaptotagmin, vesicle-
associated synaptophysin, and the regulatory cell trafficking GTPases Rab3A and nSec1 were
shown to hybridize to A. americanum SG proteins of similar molecular mass. More important,
these antibodies were capable of inhibiting PGE2-stimulated secretion of anticoagulant
proteins in digitonin-permeabilized cells except α/β SNAP (Karim et al. 2002). Furthermore,
double-stranded RNA (dsRNA)-mediated RNA interference (RNAi) of the tick synaptobrevin
homolog was shown to decrease PGE2-stimulated protein secretion rates by approximately 50%
(Karim et al. 2004b). Similar mechanisms were confirmed for O. savignyi, although an inhibi-
tory effect of PEG2 was also reported (Maritz-Olivier et al. 2005).
4.2. ATTACHMENT CEMENT

During feeding, the cheliceral digits of hard ticks lacerate host tissues, allowing the hypostome
to be inserted and anchored to the host skin, and in most ixodid tick species, a cement-like sub-
stance is secreted by the tick into the feeding cavity (Sonenshine 1991). This substance, which is
secreted by the d and e cells of types II and III granular acini, is mostly composed of proteins and
lipids that congeal around the penetrating hypostome, forming a cone. Some of these compo-
nents are conserved in a number of ticks and appear to have antigenic properties. Seventy-
kilodalton polypeptides in the cement of Dermacentor variabilis, A. americanum, and
Rhipicephalus sanguineus were recognized by antiserum specific for a 90 kDa polypeptide from
the SGs of female D. variabilis and A. americanum. These polypeptides were localized to type d
and e cells in type III acini in unfed and partially fed ticks (Jaworski et al. 1992).
Cement is secreted by most slow-feeding ixodid ticks, enabling them to remain attached to
their hosts during the long periods of feeding. In contrast, argasid ticks, which are fast feeders, rely
on deep penetration of the host skin for secure attachment. Among ixodid ticks, the length of the
mouthparts determines the pattern of secretion of cement around the hypostome. For instance,
the secretion of an external cement cone compensates for the short mouthparts in Rhipicephalus
(Boophilus) microplus and Dermacentor andersoni. In contrast, in Amblyomma spp., cement is
secreted around the hypostome after full-length insertion into the host skin (Kemp et al. 1982).
The SG of the Australian paralysis tick Ixodes holocyclus, in contrast to that of most other
ixodids, does not secrete cement (Binnington and Stone 1981). A further difference between
I. holocyclus and many other ixodids is that the former secretes a paralyzing salivary toxin (Sauer
et al. 1995).
4.3. OSMOTIC BALANCE

Hard ticks must conserve water over periods ranging from a few weeks to many months before
obtaining a blood meal. Although having an integument that is relatively impermeable to water
contributes to reduced water loss, the SGs of ixodid ticks play a more vital role as the organs of
osmoregulation during extended periods off the host, as well as during feeding (Sauer et al. 1995).
Accumulated evidence suggests that the agranular acini (type I) aid in maintaining water balance
by secreting hygroscopic material that absorbs water vapor from a subsaturated atmosphere (<95%
relative humidity). This highly concentrated salt secretion is deposited onto the dorsal surface of
the tick hypostome, which is later internalized, providing hydration and enabling tick survival
during off-host activities (Yoder et al. 2006). Active water vapor absorption has been reported
for all tick life stages, with larvae capable of absorbing water from the lowest relative humidities
(80%–85%), followed by nymphs (85%–90%) and adults (90%–95%) (Benoit and Denlinger 2010).
Tick SGs are also known to play a role in hemolymph volume regulation by returning excess
ions and water back into the host via secreted saliva (Kaufman et al. 1980). During the concentra-
tion of a blood meal, excess ions and water are mobilized across the gut epithelium into the hemo-
coel and transported back into the host via the SGs. The major constituents of tick salivary secretions
are known to be Cl− and Na+ ions; therefore, active transport involving these ions appears to be the
driving force in fluid secretion (Sauer et al. 1995). In soft ticks (Argasidae), excess water is secreted
from the coxal gland, a specialized structure unique to these ticks (Araman and Said 1972).
4.4. WATER EXCRETION

Blood feeding causes drastic changes in tick physiology, with the mass of female adults increasing
to up to 100 times their original body weight following a blood meal (Sauer et al. 1995). The main
organs of fluid elimination in ixodid ticks are the SGs, which return most of the water and ions to
the host in the form of salivary secretions (Kaufman et al. 1980). This process facilitates the concen-
tration of a large blood meal and helps maintain constant levels of hemolymph solutes and water.
In feeding ticks, the type III acini experience remarkable changes in structural organization and
size (Fawcett et al. 1981b). Ultrastructural studies on the contribution of the SGs to fluid transport
suggest that type III acini expand during the movement of fluid into the lumen and contract during
secretion into the salivary ducts. Fluid movement into the duct system is controlled by a valve that
also functions to prevent fluid from returning back from the host into the duct (Coons and Roshdy
1973; Fawcett et al. 1981a). The localization of f-actin filaments in the adlumenal interstitial cell of
the acini bordering the lumen further indicates that acinus contraction may be driven by the con-
traction of this cell. Therefore, tick SG fluid secretion seems to be dependent on an intact cytoskel-
etal system, and the contractile mechanism is actin driven, consistent with the previous hypothesis
of the existence of a functional myoepithelial cell in the type III acini of tick SGs (Coons et al. 1994;
Lamoreaux et al. 1994). Further, SG ducts dilate only in response to the dopamine-dependent stim-
ulation of nitric oxide, which indicates dopaminergic innervation of the myoepithelial cell and the
probable presence of dopamine receptors (Bhattacharya et al. 2000; Lamoreaux et al. 2000).
In blood-feeding arthropods, the acquired blood meal not only represents a source of nutri-
ents but also contains excess Na+, Cl−, and water. Fluid uptake from the hemolymph of the tick
into the lumen of the SG acini has been shown to be dependent on a catecholamine-activated
cAMP system (Bowman and Sauer 2004). Water and solute transport through polarized epithelia
Salivary Glands 173
is provided by the integrated function of primary ion pumps, classically the ouabain-sensitive
Na+/K+-ATPase, carriers, and ion channels. In tick SGs, adenylate cyclase, Na+, K+-ATPase
activity, and fluid secretion rates increase with increased tick feeding, reaching a maximum in
the SGs of mating, rapidly feeding females (Sauer et al. 2000). The identification of a gene coding
for the C subunit of vacuolar-type proton pumps (V-ATPase) in the SGs of A. americanum
(McSwain et al. 1997) prompted investigations into the mechanisms of an active cation transport
potentially energized by V-ATPase, similar to that reported in insects. Findings from these
studies, however, revealed that specific inhibition of V-ATPase activity in dopamine-induced
fluid secretion was only marginally affected and that the Na+, K+-ATPase pump is the primary
energizer of epithelial transport in A. americanum SGs.
In argasids, the coxal glands are responsible for secreting excess water from the blood meal
during feeding. This process is believed to occur via a filtration-resorption mechanism that
serves to concentrate the blood meal prior to digestion and regulate the hemolymph chloride of
the tick (Kaufman 2010). Of note, however, coxal glands have not been identified in larvae of the
argasid tick Argas persicus, suggesting that argasid ticks at the larval stage might concentrate a
blood meal by means of secretion through the SGs in a manner similar to that of ixodid ticks.
The role of aquaporins (AQPs) in facilitating trans-epithelial fluid transport in the SGs of
ticks has been addressed recently (Ball et al. 2009; Campbell et al. 2010). AQPs are transport
channels that regulate the movement of water and small solutes across cell membranes and have
been implicated in various responses to osmotic challenge as occurs during urine concentration
and glandular fluid secretion (Hachez and Chaumont 2010).
The AQPs are a family of small (30 kDa/monomer), hydrophobic, integral membrane pro-
teins that are expressed widely in organisms ranging from vertebrates and insects to plants,
fungi, bacteria, and viruses. In invertebrates, 7 AQP homologues have been identified and char-
acterized, and their involvement in cell water homeostasis has been clearly demonstrated (Spring
et al. 2009; Hachez and Chaumont 2010). There is now compelling evidence indicating that
AQPs are critical to tick salivation and general osmoregulation, and perhaps a variety of other
physiological and cellular functions. The role of an AQP in the fluid secretion of tick SGs was
first investigated in I. ricinus; it was found that the treatment of isolated glands with the AQP
inhibitor mercury chloride disrupted dopamine-induced secretion, whereas treatment with the
reducing agent beta-mercaptoethanol partially reversed this inhibition (Bowman and Sauer
2004). Using a bioinformatics approach, Ball et al. (2009) identified the AQP RsAQP1 in the SGs
and gut tissue of unfed larval, nymph, and adult R. sanguineus ticks. The expression of recombi-
nant RsAQP1 in Xenopus oocytes demonstrated that the tick AQP had high permeability to water
but no other solutes and was reversibly inhibited by Hg2+, suggesting an active role in off- and
on-host osmoregulation. Recently, the AQP IrAQP1 was identified in the sheep tick I. ricinus and
was found to be essential for water transport through the gut and SGs. However, the disruption
of IrAQP1 did not seem to have any significant influence on tick feeding (Campbell et al. 2010).
5. BIOACTIVE FACTORS IN TICK SALIVA
During the acquisition of a blood meal, ticks alternate blood feeding with the secretion of
saliva into the wound. Tick saliva contains an array of pharmacological compounds that
include immunomodulators, inhibitors of pain/itch response, anticoagulants, inhibitors of
platelet aggregation, and vasodilatory molecules, all of which contribute to both successful
feeding and evasion of the host immune and hemostatic defenses (Ribeiro 1995; Ribeiro and
Francischetti 2003; Anderson and Valenzuela 2008).
5.1. ANTI-HEMOSTATIC ACTIVITIES

Hematophagous arthropods have evolved to overcome host hemostasis by secreting a myriad of
compounds in their saliva that act to ensure a continuous flow of blood to the bite sites during
the acquisition of a blood meal (Ribeiro 1995). Blood coagulation is initiated when tissue factor
is exposed to plasma proteases VII or VIIa as a result of vascular damage. Proteolytic activation
of factor X into Xa quickly follows, triggering a cascade of events that lead to the formation of
thrombin, the activation of platelet aggregation, and fibrin-clot formation at the site of injury.
Stages of this process known to be the target of anti-hemostatic factors in arthropod saliva in-
clude platelet adhesion/aggregation, activation of the intrinsic and extrinsic pathways of coagu-
lation, and thrombin formation (Maritz-Olivier et al. 2007). Molecules with anti-hemostatic
properties were first described for the ixodid tick Ixodes scapularis (Ribeiro et al. 1985b) and the
argasid tick Ornithodoros moubata, the vector of tick-borne relapsing fever (Waxman et al. 1990;
Keller et al. 1993). Ixolaris, a tissue factor pathway inhibitor (Francischetti et al. 2002), and a
group of related proteins with anticoagulant properties (Narasimhan et al. 2002) were purified
from the saliva of I. scapularis and characterized.
Ornithodoros moubata ticks possess a large array of molecules with anti-coagulant activity.
A 60-amino-acid peptide designated as tick anticoagulant peptide (TAP) was identified in the
tick SGs as a serine protease inhibitor (serpin) that showed limited homology to the family of
Kunitz-type protease inhibitors and was able to specifically inhibit factor Xa (Waxman et al.
1990). A similar, low, tight-binding competitive thrombin inhibitor, americanin, was later iden-
tified in the SGs of the lone star tick, A. americanum (Zhu et al. 1997).
Tick-encoded serpins are considered as important factors not only in blood coagulation but
also in complement activation and in inflammatory and immune responses (Mulenga et al.
2001). Moubatin, a 17 kDa lipocalin from O. moubata, was shown to inhibit the aggregation of
human platelets when stimulated with collagen, adenosine diphosphate (ADP), or thrombin
(Keller et al. 1993). The mechanism of inhibition by Moubatin resembled that of Rhodnius pro-
lixus aggregation inhibitor 1 (RPAI-1), a 19 kDa lipocalin purified from the SGs of R. prolixus
(Francischetti et al. 2000). Also in O. moubata, a 15 kDa tick-adhesion inhibitor has been shown
to inhibit platelet adhesion to collagen via glycoprotein integrin α2β1 (GPIa/IIa), but not to
fibrinogen, and only partially to fibronectin (Karczewski et al. 1995).
Once platelets are activated at the tick attachment site, they secrete factors such as throm-
boxane A2 and ADP that promote further platelet aggregation. Therefore, many hematophagous
arthropods, including ticks, counteract aggregation by secreting enzymes known as apryases,
which hydrolyze adenosine triphosphate (ATP) and ADP into adenosine monophosphate to
inhibit blood coagulation. ADP is a potent inducer of platelet aggregation, whereas ATP is pro-
inflammatory. Putative apyrases have been described for I. scapularis (Ribeiro et al. 1985a), O.
moubata (Ribeiro et al. 1991), and O. savignyi (Mans et al. 2000). Another inhibitor of platelet
aggregation is variabilin, an antagonist of the fibrinogen receptor purified from SGs of the ixodid
tick D. variabilis (Wang et al. 1996).
Thrombin is a serine protease that functions to cleave fibrinogen to fibrin, which forms the
fibrin gel of a hemostatic plug or a pathologic thrombus. In addition, thrombin potentiates the
Salivary Glands 175
procoagulant process by activating factors V, VIII, XI, and XIII (Eyre and Gamlin 2010). At least
4 different classes of thrombin inhibitors have been isolated from R. microplus: boophilin
(Macedo-Ribeiro et al. 2008), BmAP (Horn et al. 2000), microphilin (Ciprandi et al. 2006), and
the gut thrombin inhibitor BmGTI (Ricci et al. 2007). Madanin isoforms and chimadanin are
groups of 7 kDa proteins with anti-thrombin activity identified in the SGs of the hard tick Hae-
maphysalis longicornis (Iwanaga et al. 2003; Nakajima et al. 2006). Madanins appear to bind only
to thrombin exosite-I, whereas chimadanin inhibits the function of the thrombin active site. In
contrast with thrombin inhibitors from other ticks, the translated amino acid sequence of mada-
nins and chimadanin are devoid of cysteines and show low similarity to other proteins and to
each other.
Anti-thrombins from soft ticks include ornithodorin from O. moubata (van de Locht et al.
1996), savignin from O. savignyi (Nienaber et al. 1999; Mans et al. 2002a), and monobin from
Argas monolakensis (Mans et al. 2008b). These Kunitz-type thrombin inhibitors function by
blocking thrombin interactions in a non-canonical manner as a result of distorted configura-
tions in their Kunitz domains.
The kallikrein–kinin system comprises a complex of proteins that, when activated, lead to
the release of vasoactive kinins. The kinins are released from both high molecular weight
kininogen (HMWK) and low molecular weight kininogen as a result of the activation of either
tissue kallikrein or plasma kallikrein. Kinin action on endothelial cells leads to vasodilation,
increased vascular permeability, the release of tissue plasminogen activator, the production of
nitric oxide, and the mobilization of arachidonic acid, primarily resulting in prostacyclin pro-
duction by endothelial cells. Kunitz-type contact system inhibitor haemaphysalin from the hard
tick H. longicornis (Kato et al. 2005) disrupts the activation of fXII and prekallikrein by binding
to both fXII and HMWK, thus inhibiting initiation of the classical intrinsic pathway of coagula-
tion. Similarly, Kunitz-type inhibitors BmTI-A (Tanaka et al. 1999) and BmTI-2 (Sasaki et al.
2004) found in B. microplus inhibit the amidolytic activity of plasma kallikrein.
5.2. ANTI-INFLAMMATORY FACTORS

Initial studies on the characterization of tick-acquired resistance demonstrated that inflamma-
tory responses could be reduced by the concurrent administration of histamine type I and type
II receptor antagonists, which bound histamine and reduced tick rejection (Brossard 1982; Wikel
1982; Paine et al. 1983). Histamine is a mediator of inflammation that is secreted by mast cells
and infiltrating basophils in response to tissue damage. Therefore, ticks have developed strat-
egies to suppress inflammation by secreting histamine-binding proteins into the attachment
site. Earlier studies reported the presence of a number of histamine-binding proteins in the
saliva of the metastriate ticks R. appendiculatus and Dermacentor reticulatus (Paesen et al. 1999,
2000; Sangamnatdej et al. 2002) that, in addition to binding histamine, also might interfere with
hemostasis via a mechanisms similar to that of Moubatin and the lipocalin RPAI-1 (Keller et al.
1993; Francischetti et al. 2000). A significant number of lipocalins belonging to the family of tick
SG proteins from O. savignyi have shown a wide range of anti-inflammatory activities, including
scavenging of histamine, serotonin, cysteinyl leukotrienes, and thromboxane A2 (Mans and
Ribeiro 2008; Mans et al. 2008c). Recently, lipocalins from the soft ticks A. monolakensis, Argas
reflexus, and O. savignyi were also shown to bind histamine and 5-hydroxytryptamine (Mans
et al. 2008c).
Prostaglandins (PGs) are metabolites derived from arachidonic acid that normally are found
in high concentrations in tick salivary secretions (Sauer et al. 2000). Some of the activities attrib-
uted to tick saliva PGs are the inhibition of platelet aggregation through the inhibition of ADP
secretion, vasodilation, and the impairment of T-cell function via suppression of the production
of interleukin (IL)-2 and interferon (IFN)-γ (Bowman et al. 1996; Champagne and Valenzuela
1996; Sauer et al. 2000). The role of PGs in tick feeding and pathogen transmission has not been
properly established. Recently, however, PGE2 in the saliva of R. sanguineus was shown to mod-
ulate dendritic cell (DC) function by inhibiting the production of IL-12p40 and tumor necrosis
factor (TNF)-α and the expression of CD40 molecules on the surface of bone-marrow-derived
DCs (Sa-Nunes et al. 2007; Oliveira et al. 2011).
5.3. COMPLEMENT INHIBITORS

A critical component of host-acquired resistance subject to modulation by tick salivary antigens
is the alternate pathway of complement activation, which can be activated by a number of infec-
tious agents in the absence of specific antibody (Nielsen et al. 2000). This system is a major
effector of mechanisms of humoral and innate immunity. The most important event in comple-
ment activation is proteolysis of the C3 component, which results in the deposition of C3b on
antigen surfaces or antibody bound to the antigen, followed by phagocytosis of opsonized par-
ticles (Nielsen and Leslie 2002). In addition, proteolytic complement fragments c3a, c4a, and c5a
increase vascular permeability and mediate mast cell degranulation contributing to inflamma-
tion at the sites of complement activation, whereas the membrane attack complex c5b-9 causes
osmotic lysis of cells (Kohl 2001). An anti-complement protein (Isac) was isolated from the SGs
of I. scapularis, the vector of Lyme borreliosis (Ribeiro 1987; Valenzuela et al. 2000). Recombinant
Isac was able to inhibit the deposition of complement components C3b and C5b on activating
surfaces and the generation of anaphylatoxin C3a, which induces the degranulation of mast cells
and neutrophils localized at tick attachment sites with the release of vasoactive mediators. Tyson
et al. (2007) characterized the I. scapularis salivary protein 20 (Salp20), a 48 kDa inhibitor of the
alternative complement pathway that shares homology with the Isac protein family.
The inhibition of anaphylatoxin production has also been attributed to a carboxypeptidase-
like activity found in tick saliva (Ribeiro et al. 1985b; Ribeiro and Mather 1998). This enzymatic
activity is caused by kininase, a metalloenzyme that degrades bradykinin, a protein released in
response to vascular injury and a known mediator of pain (Ribeiro and Mather 1998). Similar
activity was also detected in chromatographic fractions from the SGs of the cattle tick R. micro-
plus. However, involvement of this enzyme in facilitating tick feeding was not established (Bastiani
et al. 2002). Anti-complement activity was also detected in saliva from I. ricinus, and it prevented
the hemolysis of sheep red blood cells via the human alternative pathway of complement (Mejri
et al. 2002). Similarly, anti-complement (Irac) proteins I and II co-expressed constitutively in I.
ricinus SGs but up-regulated during blood feeding were able to inhibit the alternative pathway
(Daix et al. 2007). Additional evidence supporting tick anti-complement was provided by the
work of Rathinavelu et al. (2003), who observed inhibition of C3 deposition on the B. burgdor-
feri spirochete after incubation with the contents of I. scapularis nymphal tick guts. Lastly, in the
soft tick O. moubata, the 17 kDa complement inhibitor OmCI binds directly and tightly to C5,
preventing convertase cleavage of C5 into C5b and C5a (Nunn et al. 2005).
Salivary Glands 177
The first line of defense against extracellular parasites resides in the alternative pathway of
complement activation, and findings from a number of studies suggest that ticks have evolved
mechanisms to impair this complement activity. Therefore, components of these mechanisms
represent potential candidates for the design of transmission-blocking vaccines.
5.4. MODULATORS OF HOST IMMUNITY

Based on studies involving a variety of tick species and vertebrate host species, elements of host
immune defenses modulated by bioactive compounds that have been characterized in tick saliva
include the diminished killing activity of natural killer cells (Kubes et al. 2002), inhibition of
T-lymphocyte in vitro proliferation (Schoeler and Wikel 2001), suppression of the production of
pro-inflammatory cytokines, and induction of a polarized Type 2 immune response (Brossard and
Wikel 2004). Earlier studies on tick salivary components employed mainly traditional biochemical
and molecular-biological methods for protein identification. This approach led to the identification
of a number of molecules, mainly proteins that are specifically induced by tick SGs during feeding.
For example, the recombinant form of I. ricinus, Iris, significantly suppressed in vitro proliferation
of ConA-stimulated naïve spleen cells (Leboulle et al. 2002a). Iris was also able to down-regulate
the production of IFN-γ by T-lymphocytes and antigen-presenting cells while leaving the levels of
IL-5 and IL-10 unchanged or reduced (Leboulle et al. 2002a). Also in I. ricinus, the native 18 kDa
B-cell inhibitory protein purified from tick saliva via liquid chromatography was shown to be a
potent inhibitor of mitogen-induced murine B-lymphocyte proliferation (Hannier et al. 2004).
SG extracts from feeding D. andersoni were screened for immunosuppressive activities,
resulting in the identification and purification of a 36 kDa protein named Da-p36 (Bergman et
al. 2000; Alarcon-Chaidez et al. 2003). In H. longicornis, a homologue of Da-p36 termed rHL-
p36 not only inhibited the proliferation of murine splenocytes but also reduced the expression
of IL-2 (Konnai et al. 2009). The 15 kDa cement-related protein 64P identified in female R. ap-
pendiculatus SGs was found to be capable of inducing humoral and cell-mediated immune
responses while showing cross-reactivity to antigens from R. sanguineus, I. ricinus, Amblyomma
variegatum, and R. microplus (Trimnell et al. 2005). Recently, Deruaz et al. (2008) identified
chemokine binders (Evasins) in the saliva of R. appendiculatus that possessed affinity for a wide
range of chemokines and potent anti-inflammatory activity. Recently, I. scapularis salivary PGE2
was found to be responsible for the inhibition of IL-12, IL-2, and TNF-α production via murine
DCs (Sa-Nunes et al. 2007).
6. ROLE OF TICK SALIVARY GLANDS IN PATHOGEN

TRANSMISSION
The public health importance of ticks resides in their role in the transmission of a significantly
greater number of disease-causing agents to humans and domestic animals than any other type
of blood-feeding arthropod (Gayle and Ringdahl 2001). A major goal of the study of the tick–
host–pathogen interface is the elucidation of the genetic determinants of compatibility between
tick vectors and the pathogens they transmit and their contribution to disease maintenance and
transmission.
6.1. TICK–HOST–PATHOGEN INTERACTIONS

Transcriptional profiling of SGs in tick vectors during the acquisition of a blood meal has demon-
strated differential gene expression in response to both host immune defenses and pathogen infec-
tion; however, a detailed functional analysis of these patterns of expression is still lacking. Although
a significant body of research has been directed to the study of the tick–host and host–pathogen
interfaces, there are still very few reports regarding the study of all 3 interacting organisms.
Tick-borne pathogens can be transmitted by ticks from stage to stage (transstadially) or
from one generation to the next (transovarially), allowing ticks to act as reservoirs for that path-
ogen. A number of studies are now focusing on identifying both vector and pathogen proteins
that might be important for not only pathogen transmission and dissemination but also vector
colonization. Most tick-borne pathogens are acquired when ticks ingest an infective blood meal;
therefore, the tick midgut serves as both barrier and gateway to pathogen invasion.
Spotted fever group (SFG) rickettsiae are primarily maintained in nature through trans-
ovarial transmission; however, the molecular mechanisms involved are largely unknown.
Earlier studies on the transcriptional response of D. variabilis ticks to infection with the SFG
rickettsia Rickettsia montanensis revealed up-regulation in the expression of a number of
transcripts in infected tick ovaries that presented homology to genes potentially involved in
rickettsial maintenance. A drastic reduction in transcript levels in the tick SGs and midgut
further confirmed that the differential gene expression observed was indeed induced by
rickettsial infection and possibly was a result of increased bacterial replication in the tick
ovaries (Macaluso et al. 2003). Subsequent studies targeting expression in rickettsia-infected
ovaries revealed differential expression of a significant number of proteins related to tick
immune response, adhesion molecules, and stress response, among others (Mulenga et al.
2003). Recently, studies using expressed sequence tag (EST)-based approaches provided
characterization of 3 different anti-microbial peptides and a Kunitz-type serine protease in-
hibitor in D. variabilis midgut, the in vitro expression of which was correlated with reduced
viability and impaired ability of R. montanensis to colonize mouse fibroblasts (Ceraul et al.
2007, 2008).
Human anaplasmosis (formerly human granulocytic ehrlichiosis) is caused by Anaplasma
phagocytophilum, an obligate intracellular pathogen closely related to organisms in the genera
Rickettsia and Ehrlichia that primarily resides within the neutrophils of its mammalian hosts
(Dumler et al. 2005). A. phagocytophilum is vectored by ticks in the I. ricinus complex, in-
cluding I. ricinus in Europe, Ixodes persulcatus in Asia, I. scapularis in eastern North America,
and I. pacificus in western North America, and they are able to transmit the infection as nymphs
or adults. A. phagocytophilum is present in the glands of unfed infected ticks at low levels, but
feeding appears to increase replication in preparation for transfer to the vertebrate host. The
infection of I. scapularis with A. phagocytophilum was shown to induce significant expression
of the SG tick protein Salp16, suggesting that this protein is required specifically in order for
this bacterium to establish residence in the tick SGs (Sukumaran et al. 2006). Also in I. scapu-
laris, Borrelia burgdorferi, the causative agent of Lyme disease, induces up-regulation of Salp15,
a homolog of Salp16 that binds to the outer surface protein C (OspC) in the bacterium and to
the CD4 receptor of T-cells in mammalian hosts (Anguita et al. 2002). The latter interaction
inhibits CD4+ T-cell activation and proliferation. Silencing expression of the OspC gene in
B. burgdorferi resulted in reduced numbers of bacteria in the tick SGs; however, once bacteria
Salivary Glands 179
were already established, the inhibition of Salp16 had no effect. Further, functional Salp16 was
not required for transmission to the vertebrate host. Recently, another surface protein from
B. burgdorferi, OspA, has been shown to interact with the tick gut receptor, tick receptor outer
surface protein A (TROSPA), presumably to assist the spirochete in surviving through off-host
periods (Pal et al. 2004).
Bovine anaplasmosis caused by the intracellular pathogen A. marginale is an infectious
disease of cattle transmitted by R. microplus. A. marginale invade tick gut epithelial cells but are
transmitted to susceptible hosts through the SGs during feeding (Aubry and Geale 2011). The
3-host tick Rhipicephalus appendiculatus is the most important vector of Theileria parva, an
apicomplexan pathogen of cattle that causes East Coast fever (Bishop et al. 2004). Transmission
of T. parva is strictly transstadial, as the parasite is transmitted only by the nymphal and adult
stages after they have acquired infections as feeding larvae or nymphs. The sporozoite develops
in e cells of type III acini of the SGs of the vector and is introduced into the mammalian host
during tick feeding (Binnington et al. 1983). Genomic resources for R. appendiculatus are cur-
rently being developed. Extensive cDNA databases derived from the SGs of uninfected and
T. parva-infected R. appendiculatus were recently generated; however, a comparative analysis of
both databases did not reveal any significant differences in tick mRNA expression profiles (Nene
et al. 2004). Recently, differential susceptibility to infection by T. parva was observed among
different strains, and even different instars within the same strain, of R. appendiculatus, sugge-
sting that genetic influence might be relevant to transmission efficiency (Ochanda et al. 1996;
Odongo et al. 2009). The mechanisms involved in these differences, however, await further
investigation.
Accumulated evidence indicates that tick-borne intracellular organisms are able to manipu-
late a diversity of both vector and host cell countermeasures to support their own growth and life
cycles. These contrasting and apparently species-specific strategies used by different tick-borne
pathogens illustrate the complex relationships among vectors, reservoir hosts, and pathogens
that exist in a tightly coevolved system. Revealing how they accomplish this will lead to a better
understanding of the dynamics of pathogen transmission, dissemination, and establishment.
6.2. VECTOR COMPETENCE

The complexity of SG fluid secretion mechanisms and the diversity of the secreted products not
only enable tick on-host and off-host survival for extended periods of time but also greatly en-
hance pathogen transmission and establishment in the hosts. Pharmacological agents secreted
in tick saliva are known to modulate inflammatory, immune, and hemostatic reactions at the
tick–host interface, and extensive studies on SG exocytosis and water transport suggest that
pathogen delivery could also be facilitated by these mechanisms. Efficient transmission mecha-
nisms are highly dependent on a variety of factors, including, among others, the ability of path-
ogens to replicate and persist in the vector prior to transmission into a susceptible host and the
species, life stages, and distribution of the vectors involved (Nuttall and Labuda 2003; Scoles et
al. 2007; Agnes et al. 2010). For a significant number of pathogens, the SGs are the preferred site
for transmission; however, most tick-borne pathogens are known to initially invade the tick
midgut epithelium, where they undergo several developmental stages prior to exiting through
the SGs. The intracellular digestion of blood (heterophagy) by tick midgut epithelial cells might
give pathogens ample time to diffuse into tick body tissues. The lack of proteolytic enzymes in
the midgut lumen might provide a non-hostile environment, potentially allowing pathogens
passage into host cells, where replication may continue undisturbed (Coons et al. 1986). On the
other hand, recent evidence suggests that Kunitz-type serine protease inhibitors secreted in the
tick midgut might be important in limiting the establishment of tick-borne pathogens (Ceraul
et al. 2008).
In Dermacentor and Rhipicephalus ticks, the replication of Anaplasma and Ehrlichia spp.
takes place intracellularly in the midgut epithelium and is followed by invasion of the SGs,
where they undergo a second round of replication during feeding before being released into the
saliva for transmission (Scoles et al. 2007; Ueti et al. 2007). The transmission efficiencies appear
to vary among genetically different strains, however, and evidence suggests that the transcrip-
tion of strain-specific genes is more relevant to establishment in the mammalian host than to
vector competence (Ueti et al. 2009; Agnes et al. 2010). Tick-transmitted protozoan parasite
Babesia and Theileria spp. also invade tick gut epithelial cells and develop into kinetes, which
are released into the hemocoel and then invade multiple tick tissues, including the SGs and, in
the case of Babesia, the ovary epithelial cells and oocytes (Bock et al. 2004). In R. microplus,
Babesia bigemina was shown to infect cell types a and d from acini II and III, respectively (Bin-
nington and Kemp 1980). Other Babesia strains such as Babesia bovis invade tick ovaries and
eggs and develop to infectious stages within the SGs of the next generation of ticks (trans-
ovarial transmission), whereas Theileria lack vertical transmission and have to transmit
the parasites to their respective mammalian host within the same tick generation (Florin-
Christensen and Schnittger 2009). Theileria is known to preferentially infect the e cells of the
type III acinus of ixodid ticks of the genera Rhipicephalus, Amblyomma, Hyalomma, and Hae-
maphysalis; however, infection of d cells and type II acini with Theileria annulata has been re-
ported (Bishop et al. 2004). Tick-borne encephalitis viruses follow a similar pattern of infection
by initially targeting cells of the midgut epithelium of I. ricinus and I. persulcatus, where they
replicate and spread to the SGs to be transmitted to a new host (Nuttall and Labuda 2003).
These observations indicate that the prevalence and transmission efficiency of tick-borne path-
ogens are highly dependent on the susceptibility of the host to infection and the ability of tick
vectors to acquire infection.
7. SALIVARY GLAND COMPARATIVE

TRANSCRIPTOMICS AND GENE DISCOVERY
Tick-saliva-induced changes to the host environment are important factors in successful blood
feeding, as well as in the transmission and establishment of pathogens (Brossard and Wikel
2004). Advances in genomics and proteomics have revolutionized the study of tick SG physi-
ology, resulting in the identification of an unimagined diversity of activities (Ribeiro and Fr-
ancischetti 2003; Anderson and Valenzuela 2008). Transcriptomic technologies have been
particularly valuable in providing a relatively quick way to gain experimental information from
comparative analyses of different tick tissues, cells, and even developmental stages. In addition,
the release of the I. scapularis genome is providing data that will further help to unravel tick–
host interactions and evolutionary relationships (Pagel Van Zee et al. 2007).
Salivary Glands 181
7.1. EXPRESSED SEQUENCE TAGS (ESTS)

Recent advances in transcriptomics and genome sequencing have resulted in a wealth of infor-
mation derived from a growing number of tick species, offering exciting new opportunities in
the search for genes that might encode vaccine candidates. However, the immense amount of
information now accumulated calls for more efficient, high-throughput methods to meet the
demands of the challenging task of characterizing the expression patterns of all genes and the
biological function of the proteins encoded by them. Thus, the combination of data mining of
available sequences with expression profiles from microarrays and digital transcriptomic tech-
nologies can be expected to increase the probability of identifying genes involved in host–
pathogen interactions.
Genomics resources for the study of ticks continue to grow, and the use of ESTs in gene dis-
covery projects continues to be one of the high-throughput methods of choice to obtain infor-
mation from cDNA libraries generated from tick SGs. ESTs are short tagged DNA sequences
that are generated from mRNA expressed during 1 or more stages of the life cycle of an organism
(Bashiardes and Lovett 2001). In studying tick–host pathogen interactions, the ultimate goal of
generating sequence data from tick EST databases is to identify genes whose products will help
in deducing important processes that take place during host evasion and pathogen transmis-
sion. Although information on the genomes of ticks of major veterinary and medical impor-
tance is still limited, a significant number of EST projects are now at a stage where enough data
exist to provide new insights into host–tick–pathogen relationships (Table 7.1).
Cluster analysis of gene expression data from DNA microarray hybridization studies has
proved to be a useful tool for identifying biologically relevant groupings of genes and samples.
Studies on the genome of the southern cattle tick R. microplus using EST approaches were aimed
at finding potential genes that could aid in the control of this important ectoparasite (Crampton
et al. 1998; Guerrero et al. 2005). Differences in developmental and feeding stages of the lone star
tick, A. americanum, were studied by analyzing sequence information from over 5,500 ESTs
collected from the mRNA of adult and larval stages (Hill and Gutierrez 2000; Aljamali et al.
2009a). Similarly, sequences from a cDNA library from the SGs of A. variegatum contributed to
Table 7.1: Partial list of tick EST databases currently available.
Tick species Sequences Reference

A. americanum 3,868 Aljamali et al. (2009a)
A. cajennense 1,234 Batista et al. (2008)
A. variegatum 2,109 Nene et al. (2002)
A. monolakensis 1,472 Mans et al. (2008a)
D. andersoni 762 Alarcon-Chaidez et al. (2007)
I. pacificus 557 Francischetti et al. (2005b)
I. ricinus 1,274 Chmelar et al. (2008)
I. scapularis 3,020 Ribeiro et al. (2006)
O. coriaceus 1,089 Francischetti et al. (2008b)
O. parkeri. 649 Francischetti et al. (2008a)
R. microplus 8,270 Guerrero et al. (2005)
R. appendiculatus 7,359 Nene et al. (2004)
R. sanguineus 2,034 Anatriello et al. (2010)
the identification of proteins with potential anti-hemostatic, anti-inflammatory, and immuno-

suppressive properties that could be involved in the ability of this ectoparasite to modulate the
host response (Nene et al. 2002). Other EST sequencing projects included work with the hard
ticks I. ricinus (Leboulle et al. 2002b; Chmelar et al. 2008), I. scapularis (Valenzuela et al. 2002),
I. pacificus (Francischetti et al. 2005b), D. andersoni (Alarcon-Chaidez et al. 2007), and R. san-
guineus (Anatriello et al. 2010) and the soft ticks A. monolakensis (Mans et al. 2008a), Orni-
thodoros coriaceus (vector of epizootic bovine abortion) (Francischetti et al. 2008b), and
Ornithodoros parkeri, the vector of the relapsing fever agent Borrelia parkeri (Francischetti et al.
2008a).
Findings from these and other tick sequencing projects stimulated the first large-scale ge-
nome sequencing project for the black-legged tick, I. scapularis, owing to its relevance to human
disease as the principal vector in North America of the causative agents of Lyme disease, babe-
siosis, and anaplasmosis. This project has been a major step forward in the study of tick biology
and to date has generated over 19 million trace reads and more than 200,000 EST clones, with
these numbers continuing to increase (Pagel Van Zee et al. 2007). Recently, a comprehensive
catalogue of combined cDNA libraries from I. scapularis SGs from different life and feeding
stages was compiled and deposited in the National Center for Biotechnology Information data-
bases (http://exon.niaid.nih.gov/transcriptome.html). An exhaustive analysis of over 8,000 ESTs
identified an estimate of 3,020 unique genes (Ribeiro et al. 2006). According to this study, 49%
of the I. scapularis sequences were putatively secreted by tick SGs, but there is currently no in-
formation regarding their function. In addition, 15% of the total sequences were placed in the
category of “unknown” proteins, because no matches were found in any of the publicly available
databases.
For tick species for which genomic data are not readily available, the preferred methods of
obtaining transcriptome data continue to be the construction of cDNA libraries, massively par-
allel (next-generation) sequencing, and, in some cases, the construction of cDNA arrays. The
use of microarray technologies to study tick–host interactions is still in the early stages; how-
ever, the expression profiles of mRNA obtained from this approach have already proved valuable
for the understanding of gene expression changes in the SGs of male and female A. americanum
(Aljamali et al. 2009b). These proven methods can be improved further by second-generation
sequencing approaches that significantly reduce costs, labor, and mistakes associated with the
handling of large numbers of clones. Over the past few years, massively parallel DNA sequencing
platforms, also known as next generation sequencing (NGS) platforms, have become widely
available, dramatically reducing the cost of DNA sequencing and increasing sequencing data
throughput by several orders of magnitude. In spite of the significant improvements in high-
throughput sequencing, these resources are still underutilized in the field of tick genomics.
Comparative genomics through the use of NGS approaches can be especially relevant for tick
species for which little or no genome information is available, allowing researchers to study a
particular biological process and/or identify candidate target genes for vaccine design.
7.2. TEMPORAL GENE EXPRESSION DURING TICK FEEDING

During tick feeding, the rate of SG secretion increases progressively, and a large number of mol-
ecules are newly synthesized to enable ticks to feed successfully on a host. Several studies have
demonstrated changes in SG gene expression accompanied by increases in protein mass and the
Salivary Glands 183
overall weight of the SGs (Sauer et al. 1995). Evidence from studies on the toxicity of I. holoyclus
saliva suggests that the synthesis and release of granules from type b cells in type II acini were
correlated with the level of toxicity of SG homogenates (Binnington and Stone 1981).
Using an in vitro rabbit reticulocyte translation system and polyadenylated RNA extracted
from the SGs of slow- and fast-feeding A. americanum adult ticks, Oaks et al. (1991) confirmed
the expression of newly synthesized polypeptides that were present only in the fast-feeding
ticks, demonstrating not only new synthesis of SG proteins but also different rates of expression
at different stages of tick feeding and SG development. This was also confirmed in a study by
Jaworski et al. (1990) in which the expression of SG antigens in A. americanum, D. variabilis,
and I. scapularis was shown to vary between unfed and partially fed ticks in all species ana-
lyzed. Some of the salivary antigens preferentially induced during feeding appeared to be con-
served among all 3 species, and they were potentially associated with components of the
attachment cement. In D. andersoni, a 36 kDa immunosuppressant protein (Da-p36) in the SGs
of feeding female ticks that was temporally regulated with maximum mRNA levels was
observed 6 days post-feeding. Ticks that were close to detachment from the host showed sig-
nificantly reduced expression of p36, suggesting the synthesis of different sets of proteins with
different functions for the final phase of feeding (Bergman et al. 2000). Similarly, an immuno-
suppressive protein from I. ricinus, designated as Iris, was identified from a subtractive library
generated from the SGs of unfed and 5-day-fed ticks. This protein was differentially expressed
during feeding and was shown to be responsible for Th2 polarization of the host response (Leb-
oulle et al. 2002a, 2002b). Using differential display by RNA arbitrarily primed polymerase
chain reaction (PCR), Bior et al. (2002) compared gene expression profiles in the SGs of fed and
unfed male A. americanum ticks and identified a number of differentially expressed genes with
potential relevance in SG development and physiology. This technique allows unbiased finger-
printing, which reduces both the amount of initial material needed and the complexity of the
tick SG transcriptome. Utilizing a similar strategy, this group also followed the dynamics of
gene expression in the SGs of male D. andersoni ticks fed in the presence or absence of females
and observed a significant effect on the patterns of gene expression induced by the presence
of female ticks, suggesting the involvement of unknown signals from females during feeding
(Anyomi et al. 2006).
As a response to invading microorganisms, the innate immune system of ticks utilizes a
number of constitutive or pathogen-inducible defensin-like antimicrobial peptides that are
essential components of the tick immune response (Sonenshine and Hynes 2008). For example,
expression of the antimicrobial peptide Hebraein from the synganglia of the hard tick A.
hebraeum, which is a homolog of microplusin from B. microplus, was expressed at the onset of
blood feeding and showed a gradual increase in expression not only during feeding but also 4
days after detachment from the host (Lai et al. 2004). The use of subtractive cDNA hybridization
has been a powerful approach to identify and isolate cDNAs of differentially expressed genes in
tick SGs. An improved variant of this method termed suppressive subtractive hybridization
(SSH) has been shown to selectively amplify differentially expressed transcripts while suppress-
ing the amplification of abundant, less relevant transcripts, thus eliminating the need to separate
single- and double-stranded molecules. In addition, SSH normalizes target transcripts to ap-
proximately equal abundance. Using this approach, Mulenga et al. (2007) identified genes in
A. americanum that were up-regulated during the first 24 hours in preparation for feeding on a
host. Approximately 38% of these genes seemed to be involved in host evasion, and the remain-
ing 62% did not show homology to any known protein.
7.3. GENE EXPRESSION PROFILING BETWEEN

DEVELOPMENTAL STAGES
In spite of the increasing number of published tick SG transcriptomes, comparative profiling of
SG gene expression between developmental stages of a given tick vector is still unavailable. No-
table exceptions have been the black-legged tick, I. scapularis, which is arguably the most exten-
sively studied tick vector of disease, and the cattle tick R. microplus (Nene 2009). Ribeiro et al.
(2006) carried out the most comprehensive study on the comparative transcriptomics of gene
expression in I. scapularis SGs, which revealed differential gene expression not only at different
times during feeding but also between life stages and in the presence or absence of an infection.
In that work, differential salivary gene expression between adult and nymphal ticks revealed a
predominance of putative anticoagulant proteins such as Salp9, a number of proteins from the
uncharacterized basic-tail family, and a histamine-binding protein.
Taking a more focused approach, Maruyama et al. (2010) demonstrated unique patterns of
expression of glycine-rich proteins (GRPs) in the metastriate ticks R. microplus, R. sanguineus,
and Amblyomma cajennense that proved to be not only species dependent but also host depen-
dent. In that work, transcripts coding for GRPs identified in the cDNA libraries from the SGs of
the single-host tick R. microplus were found in greater numbers than in those of the 2 representa-
tives of 3-host ticks, R. sanguineus and A. cajennense. These results suggest that different tick
species utilize different strategies for successful feeding on their hosts. In H. longicornis, putative
protein disulfide isomerase-like genes were differentially expressed not only between develop-
mental stages but also during feeding. Furthermore, expression seemed to increase up to 4-fold in
larvae in response to infection by Babesia parasites relative to uninfected ticks (Liao et al. 2007).
Given the complexity of gene expression in tick SGs, a natural first step in identifying key
biological processes and genes regulating SG development and physiology will require the com-
bination of genomic and proteomic profile studies performed over a time course during the
different developmental and feeding stages, many of which are already underway.
8. PROTEOMICS OF TICK SALIVA AND

SALIVARY GLANDS
Most tick SG transcriptome studies have revealed a large percentage of transcripts with no
sequence similarity to any known protein and therefore no known function (Ribeiro et al. 2006).
Although mRNA profiling is an important tool in gene expression analysis, transcript abun-
dance does not always correlate with protein expression levels. Furthermore, transcriptomics
data do not provide information regarding post-translational modifications, subcellular loca-
tion, or features involved in protein–protein interactions.
Proteomics is defined as the study of the function of all proteins expressed by the genome (Mal-
lick and Kuster 2010). Traditionally, the profiling of protein expression in tick vectors has involved
the resolution of soluble protein extracts from different tick tissues by means of either one-
dimensional (1D) or two-dimensional (2D) polyacrylamide gel electrophoresis (PAGE) followed by
N-terminal sequencing or high-pressure liquid chromatography (HPLC) coupled to mass spectrom-
etry (MS). In MS, 2 soft ionization methods, electrospray ionization (ESI) and matrix-assisted laser
desorption/ionization (MALDI), remain the tools of choice in the study of tick proteomics (Fig. 7.4).
Salivary Glands 185
FIGURE 7.4: Schematic overview of standard proteome analysis. Complex protein mixtures are resolved
via 1D- or 2D-PAGE. Individual spots are then excised and subjected to in-gel enzymatic digestion, and
the resulting peptides are analyzed via MS in ESI or MALDI mode. Peptide masses allow protein
identifi cation using peptide mass fingerprinting, whereby their spectrum is compared with predicted
tandem mass spectra generated from a sequence database. Alternatively, digested peptides can be
separated by means of on-line HPLC, ionized via ESI, and fragmented in the mass spectrometer to
collect sequence information (MS/MS).
8.1. 2D-POLYACRYLAMIDE GEL ELECTROPHORESIS

In recent years, there has been an explosion of reports devoted to the characterization of the
proteomes of both tick tissues and cells in pathogen-free or infected conditions. One of the first
large-scale proteomic analyses of tick SGs was reported for I. scapularis (Valenzuela et al. 2002).
In that study, both saliva and SG proteins were resolved using traditional 1D separation and
N-terminal sequencing for identification; however, the complexity of the tick proteome has
prompted the use of more efficient and sensitive methods of separation. In order to achieve the
level of resolution needed, one of the most popular and versatile high-throughput methods of
protein separation used in tick proteomics has been 2D-PAGE (O’Farrell 1975). This technique
has been the workhorse for protein resolution and relative quantification and has greatly con-
tributed to the study of the physiology of tick SGs. 2D-PAGE is capable of resolving up to several
thousand proteins in complex mixtures through a combination of isoelectric focusing to separate
proteins according to their native isoelectric point (pI) and denaturing sodium dodecyl sulfate
PAGE, which separates them further according to their molecular mass. 2D-PAGE utilizes im-
mobilized pH gradient strips, which form a fixed pH gradient providing greater reproducibility
and higher resolution within narrower pH ranges.
Some of the more recent tick proteomes analyzed via 2D-PAGE include those of the soft
ticks O. moubata, Ornithodoros erraticus (Oleaga et al. 2007), O. parkeri (Francischetti et al.
2008a), and O. coriaceus (Francischetti et al. 2008b). The proteome of A. monolakensis SGs was
initially resolved via 2D-PAGE, and protein spots of interest were further analyzed using liquid
chromatography and mass fingerprinting, revealing protein profiles similar to that observed in
hard ticks (Mans et al. 2008a). Some of the limitations encountered when using 2D-PAGE in
proteomics-based studies include gel-to-gel and run-to-run variability and the failure to detect
proteins in extreme conditions, such as proteins with extreme pI or those exceeding the upper
or lower size limits. Some of these disadvantages have been addressed with the development of
2D differential in-gel electrophoresis (DIGE) (Unlu et al. 1997). This variation of 2D-PAGE
allows up to 3 distinct protein mixtures to be separated within a single gel. In a typical 2D-DIGE
experiment, proteins extracted from 2 different samples (i.e., treatment and control) are cova-
lently labeled, each with a cyanine fluorescent dye that has a different excitation and emission
wavelength. This labeling takes place via interactions with the ε-amino group of lysine residues
under conditions that make the process compatible with in-gel digestion and MS analysis.
Because of its superior resolving power and ability to compare proteins from different sources in
a single gel, this technology remains the gold standard in quantitative differential proteomics.
Using this technology, Narasimhan et al. (2007) were able detect the differential expression
of salivary proteins in B. burgdorferi-infected I. scapularis within 24 hours of attachment to the
host. Immune responses directed against these proteins this early in the feeding process were
associated with the induction of acquired resistance to tick feeding and to impairments in the
transmission of B. burgdorferi. Similarly, Dai et al. (2010) identified a putative histamine release
factor (tHRF) in the saliva of I. scapularis that was up-regulated in response to infection by B.
burgdorferi and which appeared to be responsible for reduced efficiency of tick feeding and
pathogen transmission to the host. The use of saturation labeling 2D-DIGE was recently reported
by Villar et al. (2010). In that study, the proteome of questing and feeding Rhipicephalus spp ticks
was screened for differential expression of tick, host, and tick-borne pathogen proteins in re-
sponse to infection, demonstrating the potential of 2D-DIGE as a powerful tool in the study of
tick–host–pathogen interactions.
8.2. HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

Generally, proteins that are visualized in 2D gels via conventional staining methods are often
high-abundance proteins, such as housekeeping or structural proteins. Less abundant proteins
may include regulatory proteins, receptors, and other proteins that play key roles in cellular
processes that might be relevant for proteomic studies. In a standard 2D-PAGE analysis, between
3,000 and 10,000 proteins can be visualized using conventional methods; however, it has been
speculated that these spots represent a small fraction of the most abundant proteins, and as
much as 76% of the proteins expressed by a given cell will be below the detection limit of con-
ventional staining methods (Vuong et al. 2000). Other practical limitations include poor solu-
bility of hydrophobic and membrane proteins, narrow dynamic range, and difficulty in focusing
Salivary Glands 187
highly basic and acidic proteins; therefore, the enrichment of proteins of interest through pre-
fractionation techniques is routinely used to overcome these drawbacks.
Among the different techniques available for reducing the complexity of peptide mixtures,
chromatographic fractionation through the use of HPLC has been the preferred method of
choice. Different modes of HPLC including reversed-phase (RP), ion exchange (IEX), size ex-
clusion chromatography (SEC), affinity, and hydrophobic interaction chromatography (HIC)
are routinely combined in hyphenated multidimensional formats for enhanced fractionation
and/or separation of peptides. In tick proteomic research, RP-HPLC is the most widely used
method for protein or peptide separation. RP chromatography is based on the interaction be-
tween the sample with a hydrophobic stationary phase and a polar hydrophilic mobile phase.
The stationary phase in RP columns is typically made up of hydrophobic alkyl chains (–(CH2)n
CH3) that interact with the molecules; there are 3 common chain lengths, C4, C8, and C18. C8
and C18 columns can be used for more hydrophilic peptides and small proteins with molecular
weights of less than 5,000 Da. Larger proteins or small hydrophobic polypeptides are usually
resolved on C4 columns.
Using RP-HPLC, Ribeiro and Mather (1998) detected a dipeptidyl carboxypeptidase in frac-
tions from I. scapularis saliva and SG homogenates that was identified as bradykinin, a known
mediator of pain and vascular permeability. In a similar study, Narasimhan et al. (2002) de-
scribed the use of RP-HPLC to purify the 9.8 kDa anticoagulant protein Salp14 from the saliva
of I. scapularis. Riberio and coworkers (1992) utilized RP-HPLC to separate fractions from
A. americanum saliva that contained smooth muscle contracting activities consistent with the
presence of PGE2 and PGF2α. Koh et al. (2007) examined the salivary gland extract (SGE) of the
tropical bont tick, A. variegatum, via RP-HPLC and identified the thrombin inhibitor variegin.
In this strategy, tick SGEs were fractionated in 3 separate runs on C18 and C4 columns until
apparent homogeneity of a protein showing anti-thrombin activity was obtained.
The development of chromatographic strategies designed to resolve the tick SG proteome
has typically included off-line prefractionation methods including electrophoresis-based tech-
niques such as 2D- and 1D-PAGE or liquid chromatography using SEC, IEX, or a combination
of approaches. Off-line multidimensional liquid chromatography allows the use of different
separation columns that are not directly compatible with each other in terms of required sol-
vents and enables the re-analysis of collected fractions without the requirement of instrumental
modifications.
In a typical experiment, the complexity of protein mixtures in SGEs is reduced by means of
the initial separation of molecules based on size (SEC) or charge (IEX), followed by RP-HPLC
as the refining step to further separate proteins on the basis of their various hydrophobicities.
The active fractions separated via HPLC are then collected and subjected to identification via
MS techniques. Karczewski et al. (1994) employed SEC followed by RP-HPLC for the identifica-
tion of disagregin, the fibrinogen receptor agonist from O. moubata. Other proteins purified
following this strategy are variabilin, the inhibitor of platelet aggregation from D. variabilis
(Wang et al. 1996); the anticomplement proteins Isac from I. scapularis and OmCI from O. mou-
bata (Valenzuela et al. 2000; Nunn et al. 2005); and, recently, an antimicrobial peptide (Isamp)
from the saliva of I. scapularis (Pichu et al. 2009).
Further resolution of the tick proteome has been achieved by using multidimensional
fractionation strategies. For instance, a combination of SEC and IEX chromatography with
RP-HPLC was used to purify anticoagulant peptides from crude extracts of the soft tick O. mou-
bata (Waxman et al. 1990; Waxman and Connolly 1993). Mans and coworkers reported an offline
multidimensional separation scheme to identify several members of a lipocalin family in the

soft ticks O. savignyi (Mans et al. 2001, 2002b, 2003) and A. monolakensis (Mans et al. 2008a). In
this technique, tick SGEs were first applied to an anion exchange column and fractionated. The
flow-through was then applied to a cation exchange column and fractionated under the same
conditions. Fractions that represented major peaks were pooled and finally applied to RP chro-
matography. Individual peaks were then collected and analyzed via peptide mass fingerprinting.
In contrast, other approaches have used RP-HPLC to reduce the complexity of the peptide
mixture and followed it with IEX chromatography as the refining step (Gaspar et al. 1996; Zhu
et al. 1997).
8.3. PEPTIDE MASS FINGERPRINTING

Traditionally, tick SG proteins have been identified by means of de novo sequencing, most
frequently via the stepwise chemical degradation (Edman degradation) of proteins or peptide
fragments (Valenzuela 2002). Rapid advances in the area of high-throughput genomics and
transcriptomics, however, have accelerated the trend toward the use of MS-based proteomics,
making it the tool of choice for this purpose.
The most commonly used techniques to volatize and ionize the proteins or peptides for mass
spectrometric analysis are MALDI and ESI. An in-depth description of different types of mass
analyzers is beyond the scope of this chapter, and only those commonly used in tick proteome
research are briefly mentioned. For a more detailed description, the interested reader is referred
to the work of Walther and Mann (2010).
Proteomic studies involving the analysis of proteins in the saliva of A. americanum and A.
maculatum (Madden et al. 2002) via MALDI time-of-flight (TOF) MS led to the identification
of an abundant protein with a molecular weight of 95 kDa that was homologous to a subunit
from a major hemolymph lipoprotein found in a number of ticks (Donohue et al. 2008). A sim-
ilar approach was used to characterize the molecular mass of multiple lipocalin isoforms from
O. moubata (Mans et al. 2001; Oleaga et al. 2007). MALDI-TOF has been especially useful in the
identification of small bioactive peptides such as the thrombin inhibitors microphilin (1.77 kDa)
and variegin (3.77 kDa), found in the saliva of B. microplus and A. variegatum, respectively (Cip-
randi et al. 2006; Koh et al. 2007), and the anti-microbial peptide Isamp from I. scapularis, with
a confirmed molecular mass of 5.3 kDa (Pichu et al. 2009).
Whereas MALDI-MS is a standard technique used for simple peptide mixtures, liquid chro-
matography coupled to ESI-MS systems is preferred for the analysis of complex samples. Many
variants of this approach have been developed in which various sequential electrophoretic or
chromatographic separation methods are combined to achieve sufficient peak capacity to re-
solve complex samples. This is exemplified by a hybrid tandem MS strategy incorporating an ESI
source (ESI-Q-TOF) that was employed to determine the molecular masses of a thrombin in-
hibitor from O. savignyi (Nienaber et al. 1999) and several biogenic amine-binding proteins
from A. monolakensis (Mans et al. 2008c).
An important development in ESI techniques has been the implementation of nano-ESI, in
which the flow rates at the ESI emitter are significantly reduced (30 to 1,000 nl/min), leading to
improved ionization efficiency and greater sensitivity. Studying the SG proteome of A. monolak-
ensis, Mans and colleagues (2008) utilized a combination of 2D-PAGE, Edman sequencing
analysis, and peptide mass fingerprinting via nano-ESI-QTOF-MS to identify 35 unique proteins
Salivary Glands 189
out of a transcriptome of approximately 1,472 open reading frames (ORFs), demonstrating the
utility of this technique for the accurate mass quantification and identification of specific se-
quences from complex protein mixtures.
8.4. TANDEM MASS SPECTROMETRY

Tandem mass spectrometry (MS/MS) is an alternative technique for large-scale protein identi-
fication that bypasses the initial protein separation step. This technique is being used increas-
ingly for the identification of tick SG and saliva proteins because of its enhanced sensitivity
during the generation of fragment mass fingerprints. For example, a MALDI-TOF/TOF ap-
proach was applied to the study of differential expression profiles of the I. scapularis SG pro-
teome in response to infection by B. burgdorferi (Dai et al. 2010).
Currently, peptide sequencing is typically performed via nano-ESI-MS/MS analysis of
crude, concentrated peptide mixtures or via hyphenated techniques such as capillary HPLC
coupled to micro/nano-ESI-MS/MS. Microfluidics-based nanospray ionization coupled with
MS substantially increases the levels of detection in limited samples such as tick saliva.
A specific example of this application is provided by studies on the salivary proteome of the
soft ticks O. coriaceus and O. parkeri (Francischetti et al. 2008a, 2008b). Results from these re-
ports revealed gene families common to both ixodid and argasid ticks, as well as some proteins
unique to the soft ticks.
The high mass resolution, accuracy, and sensitivity of Fourier transform ion cyclotron reso-
nance (FTICR) can increase the confidence of protein identifications by a factor of more than
100 over that obtained with current MS technologies. Although not as popular as the current MS
technologies because of its relatively high operational and maintenance costs, FTICR holds a
great promise for the study of the tick SG proteome. Sa-Nunes et al. (2007) demonstrated the
power of FTICR-MS for the identification of PGE2 in the saliva of I. scapularis. These initial
findings indicate that FTICR is a powerful method with many potential applications in complex
tick proteomic studies.
8.5. RECOMBINANT PROTEIN EXPRESSION

Over the past decade, the expression of a spectrum of recombinant tick proteins in different
expression systems for a wide variety of purposes has been a major feature in the study of tick
biology (Anderson and Valenzuela 2008). An array of technologies has been employed to clone
and express tick proteins, including bacterial, yeast, baculovirus/insect cell, and mammalian
expression systems. Among these, bacterial host systems employing the gram-negative bacte-
rium Escherichia coli continue to be the primary expression system for producing recombinant
proteins because of their short doubling time and high rates of translation with yields of ex-
pressed proteins of up to 30% of the total biomass.
The use of recombinant baculovirus systems and insect cells in the expression of tick antigens
has also proven to be versatile for the expression of a number of tick recombinant proteins. In-
sect cells carry out many post-translational modifications of proteins comparable to those that
occur in mammalian cells. In addition, insect cells are easy to maintain in suspension culture.
Earlier work on the characterization of the gut glycoprotein Bm86 from B. microplus reported
expression of the recombinant protein in bacterial and baculovirus expression systems and
showed that it was able to induce an antibody response in cattle that dramatically impacted the
reproductive efficiency of ticks (Willadsen et al. 1989). Subsequently, the same group identified 2
more glycoproteins from B. microplus, the 86 kDa Bm91 (Riding et al. 1994) and the 63 kDa BmA7
(McKenna et al. 1998) antigens. After initial successful evaluation of the gut-antigen-based vac-
cine Bm86, the protective efficacy of a large number of antigens against tick feeding was deter-
mined from studies using recombinant proteins expressed in a variety of expression systems.
Other tick proteins expressed in baculovirus-based systems include the histamine-binding protein
from R. appendiculatus (Paesen et al. 1999), the engorgement factor voraxin from A. hebraeum
(Weiss and Kaufman 2004), and the anticoagulant fXaI from O. savignyi (Joubert et al. 1998).
Drosophila Schneider (S2) cells are also a proven experimental system for high-level tick
protein expression. In contrast to insect expression systems using recombinant baculovirus, S2
cells can be stably transformed to achieve continuous protein production. Among some of the
tick proteins that have been expressed in S2 cells are Salp15, Salp16, and tHRF from I. scapularis
(Das et al. 2000; Anguita et al. 2002; Dai et al. 2010) and voraxin-α from R. appendiculatus
(Yamada et al. 2009).
Another traditional tool for the expression of recombinant proteins is yeast, which is gradu-
ally becoming the preferred host for high-level tick protein production. Neeper et al. (1990)
successfully expressed TAP from O. moubata as a secreted protein using the fission yeast Sa-
ccharomyces cerevisiae. The recombinant protein was shown to be a potent inhibitor of factor Xa.
The methylotrophic yeast Pichia pastoris is also becoming the yeast of choice because it provides
higher levels of recombinant protein expression than S. cerevisiae. P. pastoris has been used to
express a number of tick genes, including Bm86 homologues from R. microplus (Jittapalapong et
al. 2008), Hyalomma anatolicum anatolicum (Azhahianambi et al. 2009), Rhipicephalus annula-
tus, and R. decoloratus (Canales et al. 2008) and a serine carboxypeptidase from H. longicornis
(Motobu et al. 2007). The lipocalin OmCI from O. moubata was expressed in Pichia methanolica,
another methylotrophic yeast also utilized for the expression of recombinant proteins.
Mammalian expression methods are not as efficient as those described above; however, they
are able to carry out proper protein folding and glycosylation, in addition to an extensive array
of post-translational modifications, which ensures the production of biologically active proteins.
The utility of mammalian cell expression systems in tick protein production was demonstrated
by Frauenschuh et al. (2007), who were able to express a glycosylated form of the chemokine-
binder Evasin-1 from R. sanguineus in human embryonic kidney cells (HEK293) (Deruaz et al.
2008). Similarly, lipocalin LiR6 from I. ricinus known to interact with Leukotriene B4 was
expressed in a HEK293-derived cell line called 293T (Beaufays et al. 2008).
9. FUNCTIONAL GENOMICS
9.1. TRANSCRIPTIONAL PROFILING IN RESPONSE

TO PATHOGEN EXPOSURE
Tick gene expression has long been studied effectively at the level of individual genes, but the
current trend is shifting to the level of the whole genome. The use of genome-wide transcrip-
tomic approaches to study pathogen-induced changes in gene expression is facilitating the rapid
Salivary Glands 191
discovery of determinants that play key roles in aspects of vector competence such as suscepti-
bility to infection, permissiveness of pathogen reproduction, extent of dissemination in ticks,
and comparative transmission efficiencies of different tick species of a particular pathogen.
Transcriptomic techniques based on cDNA subtraction or differential display have been
quite useful for comparing gene expression differences between 2 cDNA populations. Using
differential display PCR, Macaluso et al. (2003) identified 54 genes differentially regulated in
D. variabilis ovaries following infection with R. montanensis, and Mulenga et al. (2003) studied
the transcriptional response in the same tick–pathogen association using subtractive hybridiza-
tion (SH). This technique was also applied in studies on I. scapularis gene expression during
infection with B. burgdorferi (Rudenko et al. 2005) in which different sets of novel genes were
induced in the tick SGs after a B. burgdorferi-infected blood meal. De la Fuente et al. (2007) used
a modification of SH called suppression subtractive hybridization (SSH) to study differential
expression in both cultured I. scapularis IDE8 tick cells and ticks in response to infection with
A. marginale. Subsequent studies with A. marginale-infected R. microplus identified substantial
differences in the expression of genes that were implicated in infection and multiplication of this
important pathogen in tick tissues (Zivkovic et al. 2010).
The implementation of microarray analysis of tick SGs and other tissues has enormous po-
tential to impact the discovery of new control strategies against tick and tick-borne infectious
diseases. However, relatively few studies have been undertaken describing the use of microarray-
based methods for high-throughput analysis of gene expression at the tick–host–pathogen inter-
face. For example, Zivkovic et al. (2009) used low-density microarrays to characterize tick gene
expression profiles in I. scapularis ticks and cultured cells (ISE6) in response to infection with
A. phagocypthilum and A. marginale. These authors reported that A. phagocytophilum modula-
tion of nymphal tick gene expression was different from that observed in tick cells infected with
A. phagocytophilum and A. marginale, demonstrating that microarray-based studies can be valu-
able for dissecting out the mechanisms involved in tick–pathogen interactions. Similarly, Alja-
mali et al. (2009b) utilized a 1,145-gene microarray to profile gene expression during feeding of
both male and female A. americanum ticks. These results provide novel insights and can be used
to generate, for example, comprehensive lists of host- and pathogen-responsive tick genes that
can be used in the identification of new targets for the control of ticks and tick-borne diseases.
9.2. RNA INTERFERENCE

Since the discovery that dsRNA can trigger the silencing of homologous genes, RNAi has be-
come a widely used tool to knock down and analyze the function of genes, especially in non-
model organisms where classical genetics are not available. The initial observation that RNAi
could silence histamine-binding proteins in vivo in A. americanum (Aljamali et al. 2002) led to
the widespread use of this approach as an effective screening tool to determine the loss-of-
function phenotype of tick genes of interest (De la Fuente et al. 2008). The injection of dsRNA
into I. scapularis adult ticks was also used to silence expression of the anticoagulant protein
Sapl14, which resulted in a significant reduction in tick engorgement weight and anti-factor Xa
activities in tick saliva (Narasimhan et al. 2004).
The use of RNAi approaches in tick research continues to advance at a very fast pace, and the
number of studies continues to increase in this area. For an in-depth review of the current status
of RNAi in tick research, see Chapter 3, “Tick Genetics, Genomics, and Transformation,” in Vol.
2 of this series.
10. EMERGING AREAS OF RESEARCH
10.1. NEXT-GENERATION TRANSCRIPTOME SEQUENCING

Massively parallel or “next generation” sequencing (NSG) platforms offer a prime opportunity
to generate molecular resources for many non-model species. These technologies produce an
abundance of short reads at a much higher throughput than is achievable using the traditional
Sanger technique (MacLean et al. 2009).
A major advantage of NSG technologies is the elimination of in vivo cloning into bacterial
vectors, a step that is replaced by PCR amplification. In tick transcriptome research, NSG has
already been applied to characterize gene expression using the 454/Roche platforms for high-
throughput sequencing of cDNAs, a strategy known as RNA-seq (Wang et al. 2009).
Recently, Guerrero et al. (2010) combined Cot filtration and 454/Roche sequencing to in-
crease the length of EST reads from R. microplus. Cot filtration is a technique used to reduce the
complexity of a genome by filtering repetitive DNA and increasing the proportion of single and
low-copy-number genes. The pyrosequencing approach of the 454/Roche technology was also
used in a genome-wide association approach to characterize the response of D. variabilis to in-
fection by a number of bacteria and fungi (Jaworski et al. 2010). These studies demonstrate the
potential of 454 pyrosequencing to rapidly characterize tick-expressed genes that can be used to
address pertinent questions regarding physiology and evolution.
10.2. GENE EXPRESSION PROFILING AT THE TICK–HOST INTERFACE

In the past few years, the genome-wide analysis of tick vectors of disease has become an area of
considerable activity. Such studies have been made possible by several advances, including the
completion of large EST libraries for many medically important ticks, the publication of genome
sequences of the Lyme disease vector I. scapularis, the development of microarray and RNAi
technologies, and high-throughput proteomics. However, despite these advances, relatively little
effort has been expended in the study of transcriptomics at the tick–host interface.
The process of tick feeding activates a highly complex sequence of events at the bite site that
facilitate the acquisition of a blood meal and create a suitable microenvironment for pathogen
transmission and establishment (Brossard and Wikel 2004). Earlier studies demonstrated that
I. scapularis tick saliva is a potent inhibitor of endothelial cell proliferation and angiogenesis
(Francischetti et al. 2005a). Maxwell et al. (2005) established that tick salivary proteins exert a
significant effect on the in vitro expression pattern of murine endothelial cell adhesion mole-
cules after exposure to SG extracts. Using an in vitro wound healing assay, Kramer et al. (2008)
demonstrated that SGE and saliva from D. variabilis were able to inhibit fibroblast migration
by modulating platelet derived growth factor expression and extracellular signal-regulated
kinase signaling. Feeding of nymphal I. scapularis ticks on naïve BALB/cJ mice was shown to
induce localized immune modulation of cell function at the bite site adjacent to the tick hypo-
stome in the feeding lesion (Krause et al. 2009). Components of the saliva from R. sanguineus,
Salivary Glands 193
A. variegatum, R. appendiculatus, and D. reticulatus are known to inhibit the migration of cells
into the bite site by binding a number of chemokines, including CxCL1 and CxCL8, which in
turn modify the wound healing response (Vančová et al. 2010; Hajnicka et al. 2011). Similarly,
Salp15 from I. scapularis has been shown to inhibit in vitro keratinocyte inflammation by down-
regulating several chemokines and antimicrobial peptides induced in response to infection by B.
burgdorferi sensu stricto (Marchal et al. 2011), and I. ricinus ticks target both inflammation and
wound healing processes through the secretion of the inhibitory serpin IRS-2 (Chmelar et al.
2011). In summary, the modulation of host immunity by tick-secreted proteins provides the ap-
propriate microenvironment at the tick–host interface for pathogen transmission and establish-
ment, which will be combined with, or followed by, immune evasion mediated by the infectious
agent. The complex mechanisms behind this process, however, await further investigation.
The recent advances in high-throughput sequencing are facilitating the discovery of gene varia-
tion by enabling the generation of vast amounts of data across entire genomes. Third-generation
sequencing technologies, referred to as single-molecule sequencing (SMS), are capable of pro-
filing samples without the need for amplification (Schadt et al. 2010). Single-cell transcriptome
analysis via SMS has tremendous potential in the study of gene expression in tick SGs and can be
used in combination with RNAi to knock down genes of interest and determine their role in the
regulation of gene expression. For example, single-cell transcriptomics can be particularly useful
in the study of tick–pathogen interactions by facilitating the identification of molecules serving
as receptors for invading pathogens in permissive tick cell populations. Furthermore, quantitative
analysis of single-cell whole pathogen transcriptomes within infected tick cells will be extremely
useful in performing a more comprehensive examination of the tick–pathogen interface. This will
greatly improve our understanding of how the interactions between tick and tick-borne patho-
gens influence the establishment, dissemination, and transmission of these infectious agents.
dsRNA silencing of endogenous gene expression at transcriptional and post-transcriptional
levels continues to be a powerful tool for the analysis of gene function in a wide variety of tick
species. Advances in RNAi and massively parallel sequencing technologies combined with
sequence data from the recently released genome project of I. scapularis have unlocked myriad
possibilities for the systematic screening of genes of interest through the development of high-
throughput screening approaches. In addition, improvements in tick cell culture methods in the
past decade have resulted in the establishment of additional cell lines from different tick species,
including Amblyomma, Dermacentor, Hyalomma, Ixodes, and Rhipicephalus ticks (Bell-Sakyi et
al. 2007). Although sequence specificity, delivery, and off-target effects are some of the chal-
lenges of the RNAi technology that are still being addressed in tick research, it is now possible
to undertake systematic genome-wide functional screens utilizing cell-based approaches in
much the same way as in well-established genetic model systems such as Caenorhabditis elegans
and Drosophila (Mohr et al. 2010). The application of this technology is likely to have a profound
impact in tick SG functional transcriptomics by increasing the scope and pace at which gene
interrogation can proceed.
The ultimate goal of genome-wide screens is to develop a systems biology approach that will
integrate proteomic and genomic data sets into a comprehensive database of gene function. This
strategy should provide greater insight into the physiological processes occurring at the tick–
host interface that mediate host immunity and pathogen transmission.
12. CONCLUDING REMARKS
The current state of understanding in tick genomics and proteomics has reached a level at which
the application of systems biology approaches can be extremely valuable in the study of the ge-
netic and biochemical regulatory networks and pathways underlying tick–host–pathogen inter-
actions. The recent availability of the I. scapularis genome and the continuously increasing
number of tick transcriptome libraries have opened the door for the design of high-throughput
analyses that are currently been implemented for genome-wide analysis of numerous tick spe-
cies of medical importance.
Whereas single-protein characterization has provided a valuable baseline for the study of
various aspects of ticks and tick-borne pathogens, genome-wide association approaches are
likely to yield greater insight into the complex nature of this cross-talk by revealing how the
dynamic interplay in the microenvironment of the tick feeding lesion determines the final out-
come of an infection. More specifically, a systems biology approach based on the integration of
diverse and high-quality proteomic and genomic data is likely to reveal novel connections be-
tween physiological processes that will substantially increase our understanding of the complex
mechanisms occurring at the tick–host–pathogen interface.
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NOTE
C H A P T E R 8
EXCRETION AND WATER BALANCE

Hindgut, Malpighian Tubules, and Coxal Glands
DANIEL E. S ONENSH INE
1. INTRODUCTION
The excretion of metabolic wastes takes place in the Malpighian tubules and the hindgut, the
terminal region of the alimentary canal, which includes the rectal sac and the anus. In argasid
ticks, some metabolic wastes are also excreted via the coxal glands.
The hindgut consists of the small, tubular intestine (a narrow extension from the posterior end
of the midgut), the rectal sac, the anal canal, and the anus; the intestine is absent in some species
of argasid ticks. The paired Malpighian tubules are connected to the rectal sac close to its junction
with the intestine (see Chapter 4, Figs. 4.19 and 4.20). The Malpighian tubules act as an elementary
excretory system to extract nitrogenous wastes, primarily in the form of guanine, from the hemo-
lymph and deposit these materials in the rectal sac. Thus, the 2 organs function together to elimi-
nate most body wastes. The elimination of excess water and inorganic salts consumed during the
huge blood meal is accomplished by the paired salivary glands during feeding in ixodid ticks (see
Chapter 7) or by the coxal glands during feeding in argasid ticks. The roles of these different sys-
tems in maintaining water balance in ixodid and argasid ticks is reviewed in this chapter.
In general, the Malpighian tubules, rectal sac, and anal canal of ticks have many features in co-
mmon with such structures in other arachnids and insects. A pair of terminally closed Malpighian
tubules is a common feature of many acarines, although some mites have 2 pairs of tubules, and others
have none (Woolley 1988). In insects, a large number of tubules connect with the rectal sac. Coxal
glands are also commonly found in mites and other arachnids. In some mites, they are basically sim-
ilar to those of argasid ticks, with a sacculus and labyrinth. For more information about these systems
in ticks and related groups, the reader is referred to the descriptions put forth by Woolley (1988).
For an electronic version of the anatomy of ticks, including the anatomical structures de-
scribed below, the reader may browse the tick anatomical ontology (illustrations and a de-
scription of structure) at www.Vectorbase.org by following the instructions in the footnote
Excretion and Water Balance 207
below. Additional illustrations of the anatomy of ticks not included in this edition may be
found at this site.1
2. THE HINDGUT
The hindgut in ticks comprises the intestine, the rectal sac, the rectum, and the anus.
2.1. INTESTINE
The hindgut begins with the intestine, a short, narrow tube lined with a single layer of columnar or
cuboidal epithelial cells. Its sole function appears to be the transport of undigested wastes (primarily
hematin, undigested hemoglobin, and cellular debris) from the midgut to the rectal sac. It extends
from the so-called stomach (i.e., the median lobe, or ventriculus) of the midgut, from which all of
the other branches arise, and projects postero-ventrally to the rectal sac. When filled with waste
fluids, it becomes distended and can easily be mistaken for one of the midgut diverticula. Histolog-
ically, the intestine consists of a single layer of columnar or cuboidal epithelial cells on a fine base-
ment membrane. The outer wall is a thin layer of circular and longitudinal smooth muscle cells.
According to Balashov (1972), the circular muscles form a sphincter at the junction of the intestine
with the rectal sac, preventing backflow to the midgut. In contrast to that in insects, the intestine in
ticks is not lined with cuticle. Nevertheless, it is considered part of the hindgut because of its appar-
ently passive transport role, allowing the passage of hematin and other undigested residues from the
midgut to the rectal sac, as well as of excretory wastes from the Malpighian tubules. The intestine is
absent in certain argasid ticks, notably Ornithodoros moubata (sensu Murray 1877) and O. savignyi.
2.2. RECTAL SAC

The rectal sac is a large, thin-walled sac lying in the ventral posterior midline of the body. In some
species, especially argasid ticks, it appears bifurcated (see Chapter 4, Fig. 4.20; also see www.vec-
torbase.org). Histologically, it is lined with a single layer of epithelial cells which, depending upon
the degree of swelling of the sac, may be either cuboidal (when empty) or squamous (when filled).
A thin basement membrane separates the epithelium from a thin layer of mostly circular muscle
fibers arranged in a fine mesh over the sac. In contrast to the rectal sac of insects, it is not lined with
cuticle. Frequently, the rectal sac appears pink or red as a result of undigested hemoglobin and
hematin wastes from the midgut, especially when examined during or immediately after feeding.
At other times, it might appear white as a result of large masses of iridescent guanine crystals ac-
cumulated within. The Malpighian tubules enter into the rectal sac, 1 on either side. The rectal sac
also lacks a cuticular lining. The rectal sac is believed to play an important role in body water reg-
ulation (Campbell et al. 2010) by concentrating urine from the Malpighian tubules, leaving only a
semi-solid mass of excreta and other wastes for passage out of the body via the anus (other organs,
such as the salivary glands in ixodid ticks or the coxal glands in argasid ticks, also function in
water regulation, but their function is to expel excess water taken in during blood feeding). Study
of the ultrastructure of the rectal sac also supports this view (Raikhel 1983). The luminal lining of
the cells is covered by numerous microvilli that form a well-developed brush border.
2.3. MALPIGHIAN TUBULES

The Malpighian tubules are the primary organs in ticks responsible for nitrogenous waste elim-
ination. They also might play some role in water elimination (Ball et al. 2009). In contrast with
many of the insects (but similar to what is found in other acarines), only a single pair of these
tubules occurs in ticks. The Malpighian tubules are believed to be of endodermal origin, and
probably originated as a specialized extension of the intestine to which they are connected,
often near the junction with the posterior end of the midgut (similar to the structure of these
organs in insects). These tubules consist of a single-celled epithelium formed mostly of cu-
boidal cells surrounded by a thin layer of smooth muscle cells. Histologically, the Malpighian
tubules and the rectal sac appear similar. At the ultrastructural level, comparison of the rectal
sac and the Malpighian tubules, especially the proximal regions of the latter, does not reveal
any difference in organization (see below). Cells of both organs have a well-developed basal
labyrinth on the outer side facing the hemolymph and a brush-border-like layer of microvilli,
indicating a role in fluid transport, on the inner (luminal) side. Raikhel (1983) concludes that
FIGURE 8.1: Transmission electron micrograph illustrating the ultrastructure of the Malpighian tubules of
an ixodid tick, Dermacentor variabilis (partially fed virgin female). Section through the wall of the tubule.
The plasma membrane facing the lumen is bordered by numerous long microvilli. The cell cytoplasm is
highly vesiculated, suggesting lipid accumulations. The outer edge of the cells has a well-developed
basal lamina and labyrinthine infoldings of the plasma membrane (arrow). Measurement bar, 3.3 μm.
L, lumen; MV, microvilli.
the rectal sac is at least functionally analogous to the rectum of insects, despite its apparent
endodermal origin, and serves as an integral part of the tick’s system for the reabsorption of
water, ions, and other essential materials. These extremely long tubules loop around the inter-
nal body organs and extend virtually everywhere throughout the body interior. In the starved
tick, the lumen is virtually absent, and the cells appear small. According to Balashov (1972), the
tubules are about 50 to 70 μm in diameter at this stage. During and after feeding, the lumen
enlarges as it fills with white guanine crystals and the cells gradually hypertrophy. The tubules
increase many times in diameter. As many as 3 distinct regions of the Malpighian tubules have
been described (distal, main, and proximal) based on differences in the epithelial cell ultra-
structure. Ultrastructurally, epithelial cells of the main region and of the proximal region are
similar (Figs. 8.1–8.3). These are the primary cell type in the tubules and make up most of the
tubular wall; presumably, they are analogous to the principal cells in Drosophila and other
insects (see Section 2.5). The luminal surface is covered by microvilli, which form a brush bor-
der resembling that of the rectal sac cells. The basal region of the cell rests on a substantial basal
lamina with numerous infoldings forming a basal labyrinth (Fig. 8.1). As in the rectal sac, these
subcellular specializations greatly increase the available surface for fluid transport; indeed,
these features are characteristic of transporting epithelia. In addition, mitochondria increase
in abundance during feeding, especially in the vicinity of the basal labyrinth. Large amounts of
lipid-rich vesicles are also present in many of the epithelial cells. Glycogen granules abound
and increase during feeding. The cells hypertrophy, with bulbous apices (Fig. 8.2) protruding
into the lumen, which gradually fills with guanine crystals of varying size (Figs. 8.3, 8.4). These
FIGURE 8.2: Transmission electron micrograph illustrating the ultrastructure of the Malpighian tubules
of an ixodid tick, Dermacentor variabilis (partially fed virgin female). Section through a Malpighian
tubule showing masses of finely granular material accumulated adjacent to the luminal edge of the
epithelial cells. Measurement bar, 3.3 μm.
FIGURE 8.3: Transmission electron micrograph illustrating the ultrastructure of the Malpighian tubules
of an ixodid tick, Dermacentor variabilis (partially fed virgin female). Enlargement showing the luminal
edge of an epithelial cell of a Malpighian tubule and the tubule contents; note the masses of finely
granular particles and small guanine crystals of varying shapes and sizes. Measurement bar, 1.8 μm.
GnC = guanine crystal; Mv = microvilli.
FIGURE 8.4: Scanning electron micrograph illustrating a mass of large guanine crystals released by the
rupture of the rectal sac in a female ixodid tick (Dermacentor andersoni). Measurement bar, 5 μm. Photo
courtesy of J. Slusser, Logaras Electron Microscopy Facility, Eastern Virginia Medical School, Norfolk,
Virginia, USA.
210
crystals are usually white or iridescent in appearance; most are spheroidal, with a distinctly
laminate structure, and they can be as large as 80 μm in diameter. Other materials of unknown
composition also occur in the lumen. A common feature of these cells is the presence of sym-
biotic rickettsiae of the genus Wohlbachia, which occur in colony-like masses dispersed among
the cells. Intracellular clusters of Wohlbachia also occur in the oocytes, the foveal glands (pher-
omone glands), and perhaps other tissues. Cells of the distal region are less highly developed,
with few microvilli and weakly developed basal infoldings. These cells may be analogous to the
stellate cells found in similar locations in the Malpighian tubules of Drosophila and other in-
sects (see Section 2.5). The Malpighian tubules are similar in both families of ticks. In argasid
ticks, 2 types of epithelial cells have been described based on the degree of microvilli develop-
ment and the extent of infolding of the basal membrane (Raikhel 1983; El Shoura 1987). In the
soft tick A. arboreus, the 2 types are termed pyramidal and cuboidal cells. For further details of
the ultrastructure of the Malpighian tubules, the reader is referred to the excellent review by
Raikhel (1983).
2.4. RECTUM (ANAL CANAL) AND ANUS

The rectum (anal canal) is a thin-walled tube connecting the rectal sac with the anal aperture.
The rectal wall consists of a thin layer of epithelial cells on a delicate basement membrane. The
luminal surface is lined with a rather thick layer of cuticle. The rectal canal may be filled with
masses of guanine crystals and/or black semi-solid hematin residues. Smooth muscles from the
anal valves are inserted on either side of the rectum. According to Balashov (1972), the contrac-
tion of these muscles enlarges the rectum and even causes it to evert slightly, thereby facilitating
defecation. At the same time, the anal valves separate, presumably forced open by the increased
hydrostatic pressure of the hemolymph.
2.5. COMPARISON WITH INSECTS

In insects, the hindgut consists of the ileum (anatomically similar to the tick intestine), the rec-
tum, and the anus. The Malpighian tubules join at the junction of the ileum and the posterior
end of the midgut. Unlike in ticks and other acarines, the entire region is lined with cuticle (Wall
et al. 1975). The number of Malpighian tubules is extremely variable, from only 4 tubules in
higher Diptera such as Drosophila spp. or blood-sucking bugs to hundreds in many of the Or-
thoptera (Bradley 2003). In insects, as in ticks and, presumably, other acarines, the tubules are
closed at their distal ends. This results in a build-up of hydrostatic pressure that drives the excre-
tory contents from the tubules into the hindgut for elimination.
Histologically, the Malpighian tubules consist of 2 cell types, analogous to the different
cell types observed in the Malpighian tubules of ticks. These are (1) principal cells and (2)
stellate cells. In Drosophila, principal cells are much more numerous than stellate cells
(Jung et al. 2005). In most insects, the histology of the rectum reflects structures organized
for urine concentration. In ants, principal cells also occur in the rectum, where they line
the luminal surface. The principal cells are surrounded by an outer layer of cuboidal sec-
ondary cells (Garayoa et al. 1999). The roles of these cells in urine formation are discussed
briefly below.
3. PHYSIOLOGY, BIOCHEMISTRY, AND

MOLECULAR BIOLOGY OF EXCRETION AND
WASTE ELIMINATION
3.1. INSECTS
Urine is formed in the Malpighian tubules and passed into the rectal sac. A hyperosmotic urine
solution is formed via the transport of water and ions (primarily potassium) from the hemo-
lymph across the tubular epithelium and into the lumen, driven largely by the osmotic pressure
gradient of the tubular lumen versus the hemolymph. In most insects, cation transport across
the epithelium is regulated by Na+-K+ ATPase, which transports sodium out of the cell and into
the lumen and potassium in the reverse direction. Also important is the Na+/K+/2Cl co-trans-
porter, which moves chloride ions, as well as sodium and potassium ions, from the hemolymph
into the cell cytoplasm, primarily via the stellate cells. The Malpighian tubules also function in
the removal of nitrogenous wastes from the hemolymph. In blood-feeding insects such as the
kissing bug, Rhodnius prolixus, uric acid is formed and bound to potassium, forming insoluble
crystals of potassium urate (at neutral or acid pH). Excess potassium ions are returned to the
hemolymph, but sodium is concentrated. Further concentration of the urine is completed in
the rectal sac (Bradley 2003). Once the urine solution passes into the rectal sac, it is concen-
trated further through the resorption of proportionately more water than solutes, moving
water from the rectum lumen toward the hemolymph. Water moves against an increasing os-
motic gradient, resulting in progressively more hyperosmotic excretory wastes. Two mecha-
nisms have been implicated in rectal fluid absorption: (1) local osmosis, followed by (2) solute
recycling.
3.2. IXODID TICKS

In contrast with insects, in which the primary nitrogenous waste product is uric acid, ticks are
able to conserve precious body water by eliminating their nitrogenous wastes in the form of
guanine-rich excreta rather than uric acid. Guanine (2-amino-6-oxypurine) is formed in the
Malpighian tubules and accumulates in crystalline form. According to Hamdy (1977), guanine
constituted between 1.4% and 9.3% of the excreta of feeding ixodid ticks that were studied, and
a second, unidentified purine constituted an additional 2% to 15%. Hematin was relatively
limited, accounting for between 0.2% and 1.5% of the total excreta. Proteins, including hemoglo-
bin and albumin, were an important constituent of the excreta of many species, representing as
much as 68% in one case.
Ticks have distinct cycles of waste elimination. In many ixodid ticks, guanine is excreted
soon after eclosion; some hematin wastes from the digestion of the previous blood meal also
might be passed at this time. Large amounts of hematin and undigested hemoglobin are passed
during feeding. In D. variabilis, elongate, coil-like masses of black semi-solid material appear
posterior to each feeding tick, accumulating rapidly as the tick engorges. This material consists
of the hematin from the digestion of the previous blood meal, as well as from fresh blood. In
addition, some ixodids might also pass undigested blood from the blood meal. According to
Hamdy and Sidrak (1982), replete D. andersoni females eliminated an average of 2.68 ± 0.2 mg
of hematin during feeding. In my own studies with D. variabilis, we observed totals of 4.64 and
1.84 mg/tick of excreta (only a small part of which consisted of hematin) from fed females and
fed males, respectively, during a 7-day attachment period (see Chapters 6, 9). Following feeding,
the ticks also excrete guanine. According to Bassal (1977), Hyalomma dromedarii females ex-
crete as much as 13% of their dry body weight in the form of guanine during oogenesis and
oviposition. Guanine excretion in D. andersoni, based on wet weight, averaged 3.97 mg/tick. In
some ixodids, protein, amino acids, and an unidentified purine are excreted in addition to gua-
nine. In contrast, argasid ticks are believed to excrete only guanine as their nitrogenous waste
product (Coons et al. 1986).
The biochemistry of the excretory processes in the rectal sac and Malpighian tubules of
ticks is understood poorly, if at all. Lysosomes and residual bodies have been tentatively
identified in the cells of the major region of the tubule (Raikhel 1983), suggesting the degra-
dation of macromolecules, presumably proteins. Ion resorption is believed to occur pri-
marily in the proximal region of the tubules, and this may be correlated with increased
guanine crystal deposition in this region. Using radiolabeled precursors, Hamdy and Sidrak
(1982) demonstrated that ticks, vertebrates, and insects synthesize guanine from similar pre-
cursors and via a similar metabolic pathway. Hypoxanthine was the most important of 4
different metabolites as a precursor of guanine synthesis; 91.3% and 92.8% of the hypoxan-
thine injected was converted into guanine in Argas arboreus and D. andersoni, respectively.
Formate and glycine ranked second and third, respectively, in importance after hypoxan-
thine as precursors of this excretory product. In I. scapularis, the purine content of the ex-
creta from nymphal ticks comprised a mixture of guanine and xanthine, in a consistent ratio
of 10.6:1. Both purines were also found in the excreta of fed larvae, but in almost equal
amounts (Sonenshine et al. 2003). Similar findings were noted for the European sheep tick,
I. ricinus, for which guanine constituted ~90% of the purines in the excreta, with hypoxan-
thine and xanthine making up the rest (Dusbábek et al. 1991). Much remains to be learned
about these very important processes.
Little else is known about the molecular processes responsible for excretion in these ticks.
Some studies have shown evidence of the presence of aquaporins, molecules that form pores in
the cell membrane that facilitate water secretion. Unlike in many insects, in which they occur in
the rectal sac and regulate rapid water elimination, in the ixodid tick aquaporins are most
strongly expressed in the salivary glands and midgut, consistent with their role in water elimi-
nation during feeding. An aquaporin was identified in the rectal sac of the sheep tick, Ixodes
ricinus, as well as in other tissues involved in mass water flux, namely, the salivary glands and
midgut (Campbell et al. 2010). An aquaporin was also expressed in the Malpighian tubules of the
brown dog tick, Rhipicephalus sanguineus, but much less so than in the salivary glands (Ball et
al. 2009). However, Holmes et al. (2008) did not find evidence of aquaporin expression in the
Malpighian tubules in engorged females of D. variabilis. Further studies are needed to determine
what role, if any, aquaporins play in the regulation of water elimination in the tick excretory
system.
Neuropeptides might play a role in regulating excretion in ticks. According to Neupert et al.
(2005), diuresis in insects is regulated by neuropeptides that modulate secretion and/or reab-
sorption processes in the Malpighian tubules and the rectum. In the case of ticks, these authors
demonstrated the presence of periviscerokinin by means of mass spectrometry studies of single
cells in the tick synganglion, suggesting a role in the regulation of diuresis similar to that seen in
insects.
3.3. ARGASID TICKS

In contrast to the Ixodidae, in which the salivary glands play the primary role in water elimination
during feeding, argasid ticks eliminate blood meal water via the coxal glands. Coxal glands are
commonly found in many arachnids, including horseshoe crabs, whip spiders, and spiders, as
well as in mites and argasid ticks (Little 1983). In some arachnids, such as whip spiders, 2 pairs of
coxal glands occur. The structure of the first pair of coxal glands is similar to that found in other
chelicerates, with a large sacculus and a coiled duct. The sacculus contains numerous podocytes
and probably serves as the ultrafiltration site, whereas the duct has a highly differentiated epithe-
lium believed to serve as the transporting site (Weygoldt 2000). In argasid ticks, coxal glands
occur in the nymphal and adult stages, but not in the larvae. These glands are located in the ven-
tral anterior region of the body, below the salivary glands, and adjoining the epidermis and deli-
cate membranes between the coxae of legs I and III. The paired orifices of these glands are located
in the soft articulatory cuticle between the coxae of legs I and II. During or soon after feeding,
copious quantities of a clear, watery fluid are excreted by the tick, serving to concentrate the blood
meal and eliminate excess water and ions. The coxal gland functions as an ultrafiltration system,
FIGURE 8.5: Diagram illustrating the coxal gland of argasid ticks and details of its structure. Detail of
the coxal gland showing the arrangement of the fi ltration membrane around the collecting tubule and
the proximal and distal parts of the collecting tubule. The largest arrowhead (top) shows the junction of
the fi ltration membrane and the collecting tubule. Smaller arrows indicate the direction of flow. Acg =
accessory gland; Dt = distal tubule (of collecting tubule); Pt = proximal tubule (of collecting tubule);
Fm = fi ltration membrane. After Hecker, H., Diehl, P.A., and Aeschlimann, A. (1969) Recherches sur
l’ultrastructure et l’histochimie de l’organe coxal d’Ornithodoros moubata Murray (Ixodoidea;
Argasidae) [Research on the ultrastructure and histochemistry of the coxal organ of Ornithodoros
moubata Murray (Ixodoidea: Argasidae)]. Acta Tropica 26:346–360, copyright Elsevier, with kind
permission from Elsevier Publishing Co. (Note: Some labels are unique to this figure and should not be
confused with similar letters used to label other organs.)
retaining most hemolymph proteins and other organic compounds while allowing the removal of
salts and water. Figures for the quantity of fluid eliminated vary greatly for different species and
life stages. For example, in a study of the argasid tick Ornithodoros papillipes, between 17% and
48% of the engorged blood meal weight was eliminated in the coxal fluid (Balashov 1972). In my
own studies with O. tholozani, I observed an average of 41.3% of the blood meal water and salts
excreted as coxal fluid, based on a gravimetric analysis. When measured using a radiotracer (36Cl,
as NaCl), the estimate was 57% (Sonenshine, unpublished data).
Each coxal gland terminates in a fine duct connected to pores in the ventral cuticle between
coxae I and II. When viewed from the inside of the tick’s body, each gland is found to comprise
a flask-shaped structure consisting of an external, membranous, sac-like filtration chamber and
an internal, coiled tubular system, the coxal tubule (Figs. 8.5–8.7), located near the coxae of legs
I and II. In O. moubata, the gland is approximately 1 to 2 mm long by 0.5 to 1 mm wide (Hecker
et al. 1969). A small aciniform accessory gland is attached to the coxal tubule near its junction
FIGURE 8.6: Diagram illustrating the 2 types of cells found in the collecting tubule: A, columnar cells
characteristic of the distal collecting tubule; B, cuboidal cells characteristic of the proximal collecting
tubule. Bl = basal lamina; BLb = basal labyrinthe; Gly = glycogen; Gc = golgi complex; Hl = hemocoel;
Lu = lumen of tubule; Ly = lysosome; Mt = mitochondria; Mtb = microtubules; Mv1 = microvilli, short;
Mv2 = microvilli, long; Nu = nucleus; RER = rough endoplasmic reticulum; Sds = septate desmosomes;
SER = smooth endoplasmic reticulum. After Hecker, H., Diehl, P.A., and Aeschlimann, A. (1969)
Recherches sur l’ultrastructure et l’histochimie de l’organe coxal d’Ornithodoros moubata Murray
(Ixodoidea; Argasidae) [Research on the ultrastructure and histochemistry of the coxal organ of
Ornithodoros moubata Murray (Ixodoidea: Argasidae)]. Acta Tropica 26:346–360, copyright Elsevier, with
kind permission from Elsevier Publishing Co. (Note: Some labels are unique to this figure and should
not be confused with similar letters used to label other organs.)
with the coxal pore (Fig. 8.5). Its function is unknown, although Schlein and Gunders (1981)
suggest that these structures might be the source of the argasid sex pheromone. Muscle bundles
attach to the membranous filtration chamber. The sac-like filtration chamber is highly folded
into a labyrinthine network, enormously increasing the available surface. The filtration mem-
brane appears to be connected to an overlying sac surrounding the entire gland. The folds of the
filtration membrane invest the coils of the coxal tubule, thereby bringing the fluid contents into
close proximity to the tubule cells. The coxal tubule consists of a proximal part with radiator-like
coils and a distal tubule that proceeds directly to the external pore. Contraction of the numerous
fine muscle fibers is thought to distend the filtration chamber, allowing water and ions to enter
from the surrounding hemolymph (Fig. 8.7). Thus, the filtration chamber acts as an ultrafiltra-
tion system for the rapid accumulation of water and small, soluble molecules and ions (Lees
1946). The coiled coxal tubule accumulates these materials and selectively reabsorbs certain
ions, particularly potassium (Kaufman and Sauer 1982). The cells of the tubular system are of 2
types, hereinafter termed podocytes in view of their similarity to the excretory cells of other
animals, namely, (1) tall columnar and (2) cuboidal (Fig. 8.6). The 2 cell types are similar, with
FIGURE 8.7: Histological section of the body of an Ornithodoros hermsi fed female tick 3 weeks after
engorgement showing the location of the coxal glands. The arrow on the left-hand side of the figure
points to the coxal gland sac. Fine muscles are evident immediately to the left of the gland that might
function in dilating the gland sac. The arrow on the right-hand side points to a structure believed to be
granular component of the coxal gland. Figure courtesy of Dr. Thomas Schwann, NIAID, NIH, Rocky
Mountain Laboratories, Hamilton, Montana, USA.
extensive infoldings of the basal lamina, modest amounts of endoplasmic reticulum, lysosomes,
and numerous microtubules. However, the columnar cells have a few short microvilli, whereas
the cuboidal cells have numerous, very long microvilli. The extensive, deep infoldings of the
basal labyrinth are similar to those found in the cells of insect Malpighian tubules or vertebrate
kidney glomeruli. In the vicinity of the proximal tubule, these cells are especially rich in mito-
chondria, indicating energetic metabolism supporting the active transport of selected ions and
compounds. Energy appears to be supplied by stored glycogen, which is also abundant in the
podocytes, and probably from stored lipids, which are especially abundant in the cuboidal cells.
Although coxal glands are absent in ixodid ticks, paired multi-lobed glands similar to the
argasid accessory coxal glands have been found in pharate nymphs and pharate adults. Each
“coxal organ” is connected via a duct to an opening in the body wall between coxae I and II.
These glands are active only during apolysis. Their function is unknown (Binnington 1975).
The molecular biology of excretion in ticks is largely a black box. Virtually nothing is known
about the synthesis of guanine, xanthine, and other purines that enable ticks to excrete their
nitrogenous wastes with little loss of body water (guanine and xanthine are largely insoluble in
water). Some work on guanine biosynthesis was done by Hamdy and Sidrak (1982), but the spe-
cific enzymes in the pathway remain to be identified. In contrast, much has been learned about
the physiology, biochemistry, and molecular biology of excretion in insects. In the latter (e.g., in
Drosophila), urate oxidase expressed primarily in the Malpighian tubules converts nitrogenous
wastes absorbed from the hemolymph into uric acid, the most common form of excretory waste
in insects. In the mosquito Aedes aegypti, an alternate uricolytic pathway involving three
enzymes—urate oxidase, allantoinase, and allantoicase—metabolizes uric acid further to urea,
which is excreted via the excretory system (Scaraffia et al. 2008). Clearly, further effort is re-
quired in order to elucidate the full pathways for nitrogenous waste elimination, ultrafiltration,
and water resorption and the tissue sites where these processes occur. Aside from the discovery
of periviscerokinin (Neupert et al. 2005), a putative regulator of diuresis in insects, virtually
nothing is known about the hormonal regulation of urine formation in ticks.
Knowledge of the molecular events regulating tick urine secretion and water resorption
could lead to the development of novel, highly targeted approaches to tick control, as any dis-
ruption of metabolic waste elimination would be a fast and effective way to kill these parasites
without causing harm to their hosts.
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Ball, A., Campbell, E.M., Jacob, J., Hoppler, S., and Bowman, A.S. (2009) Identification, functional
characterization and expression patterns of a water-specific aquaporin in the brown dog tick,
Rhipicephalus sanguineus. Insect Biochem. Mol. Biol. 39:105–112.
Bassal, T.T.M. (1977) Demonstration of guanine biosynthesis in Hyalomma dromedarii ticks using
[14C-] labeled glycine and glyoxylate. J. Parasitol. 63:758–759.
Binnington, K.C. (1975) Secretory coxal gland, active during apolysis in ixodid and argasid ticks (Aca-
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Bradley, T.J. (2003) Excretion. In V.H. Resh and R.T. Cardé (Eds.), Encyclopedia of Insects. Orlando, FL:
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Campbell, E.M., Burdin, M., Hopple, S., and Bowman, A.S. (2010) Role of an aquaporin in the sheep tick
Ixodes ricinus: assessment as a potential control target. Int. J. Parasitol. 40:15–23.
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Dusbábek, F., Simek, P., Jegorov, A., and Tríska, J. (1991) Identification of xanthine and hypoxanthine as
components of assembly pheromone in excreta of argasid ticks. Exp. Appl. Acarol. 11:307–316.
El Shoura, S. (1987) Malpighian tubule ultrastructure of female Argas (Persicargas) arboreus (Acari:
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hindgut of the ant (Formica nigricans, Hymenoptera): II. Structure of the rectum. J. Morphol. 242:205–228.
Hamdy, B.H. (1977) Biochemical and physiological studies of certain ticks (Ixodoidea). Excretion dur-
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Hamdy, B.H. and Sidrak, W. (1982) Guanine biosynthesis in the ticks (Acari) Dermacentor andersoni
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ture and histochemistry of the coxal organ of Ornithodoros moubata Murray (Ixodoidea: Argasi-
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Holmes, S.P., Li, D., Ceraul, S.M., and Azad, A.F. (2008) An aquaporin-like protein from the ovaries and
gut of American dog tick (Acari: Ixodidae). J. Med. Entomol. 45:68–74.
Jung, A.C., Denholm, B., Skaer, H., and Affolter, M. (2005) Renal tubule development in Drosophila: a
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NOTE
C H A P T E R 9
HEME PROCESSING AND THE

EVOLUTION OF HEMATOPHAGY
BEN J. MANS
1. INTRODUCTION
Ticks (Ixodida) are monophyletic obligate blood-feeding ectoparasites comprising 2 major

families, the Argasidae (soft ticks) and the Ixodidae (hard ticks), and the monotypic Nuttalliel-
lidae (Klompen et al. 2000, 2007; Barker and Murrell 2004; Mans et al. 2011, 2012). Parsimo-
nious arguments would indicate that the ancestral lineage of these families adapted to a
hematophagous lifestyle and was argasid-like in behavior (Mans et al. 2011, 2012). However,
comparison of vector–host interaction strategies, especially the modulation of host defense
mechanisms such as the inflammatory and hemostatic systems by salivary-gland-derived
proteins, has indicated significant differences among the tick families (Mans et al. 2002, 2008;
Mans and Neitz 2004; Mans 2011). This suggests that the different families evolved tick–host
modulatory mechanisms independently, with the larger implication being that blood-feeding
lifestyles evolved in the tick families after their divergence (Mans et al. 2002; Mans 2011). The
extensive differences observed in the host modulatory mechanisms of salivary gland proteins
from the different families have been attributed to pressure by the host immune systems (Fran-
cischetti et al. 2009). The argument can therefore be made that many or most homologous
characters involved at the blood-feeding interface could have been lost after the divergence of
the main tick families, making salivary gland proteins a poor measure of common hematoph-
agous ancestry. Processes in blood feeding that are not directly involved at the vector–host in-
terface, and which are therefore not in contact with the host’s immune system, would be less
prone to change and could thus have maintained homologous characters from the ancestral
blood-feeding lineage.
Heme Processing and Hematophagy 221
2. FOUR STAGES IN THE EVOLUTION OF

A BLOOD -F EEDING LIF ESTYLE
In order to define processes in blood feeding not directly involved at the vector–host interface, the
general strategies needed for the evolution of blood-feeding behavior need to be considered. Hard
and soft ticks have different lifestyle strategies, with important implications for their biological dif-
ferences. The feeding of hard ticks takes place over extended periods of several days in all life stages,
whereas adult and nymphal soft ticks and Nuttalliella namaqua generally feed within minutes to
hours (Nuttall 1911; Sonenshine 1991; Mans et al. 2011, 2012). This leads to hard ticks’ being exposed
to the host’s immune system for much longer periods than soft ticks. It allows for the development
and growth of ixodids on the host while feeding, leading to increases in body weight by more than
100 times as a result of the volume of blood meal that is ingested (Balashov 1972; Sonenshine 1991).
In adult hard ticks, this ingested blood mass is efficiently converted into egg mass so that females
may once lay several thousands of eggs before dying. Soft ticks generally engorge to only 5 to 10
times their body weight and consequently lay small egg batches of only a few hundred eggs at a time
(Balashov 1972). Adult females can, however, feed multiple times between egg batches and can
therefore lay a significant amount of eggs over a lifetime that could be comparable to that of a hard
tick. Nuttall (1911) linked the reproductive strategies of the different families directly to the specific
ecological niches that they occupy. Argasids generally occupy the burrow of their host, thereby
having constant access to a blood meal for themselves and their offspring. This results in their laying
smaller egg batches, because the probability of the survival of progeny is higher. Hard ticks are not
as permanently associated with their hosts and need to find a host within a larger ecological niche
that depends to a large extent on the stochastic movement of that host. This leads to higher mor-
tality rates and the subsequent necessity of laying large egg batches, as only a small number of
progeny will eventually complete the life cycle. This in turn requires longer feeding periods, which
result in the typical prolonged association that ixodids have with their hosts during feeding.
Using Tinbergian analysis, Waladde and Rice (1982) analyzed tick feeding as a complex of
behavioral processes that resulted in 9 main events: appetence, engagement, exploration, penetra-
tion, attachment, ingestion, engorgement, detachment, and disengagement. However, from a ho-
listic perspective, tick feeding might be broken down to only 4 main stages that can be considered
as major milestones that need to be addressed when evolving a blood-feeding lifestyle. These are
(1) host detection, (2) host attachment, (3) tick–host interaction, and (4) blood meal processing
(Fig. 9.1). An analysis of these 4 stages allows one to determine which are host independent and
therefore likely to have retained homologous characters from the ancestral blood-feeding lineage.
2.1. HOST DETECTION

This stage would be similar to the appetence phase as defined by Waladde and Rice (1982), which
includes locomotory hunting for a host or seeking from a vantage point. In both cases, ticks need
to detect their hosts within their respective ecological niches and do so using a combination of
gustatory, mechano-, olfactory, thermo-, and visual sensation (Hess and Vlimant 1986). The latter
FIGURE 9.1: The 4 stages in the evolution of a blood-feeding lifestyle. (1) Host detection. The host needs
to be detected, and ticks use specialized organs such as Haller’s organ (a), which contains gustatory and
chemo-reception sensilla (b), or mechano-sensory hairs (c). (2) Host attachment. Attachment to the host
requires specialized mouthparts such as the hypostome and chelicerae (d) to cut into the dermis, and
cement (e) is used by metastriate ticks for anchoring. (3) Tick–host interaction. The tick needs to interact
with the host at the feeding site, where it encounters red blood cells (f), platelets (g), antibodies (h), mast
cells, basophils (i), and neutrophils (j). (4) Blood meal processing. After engorgement, the blood meal
needs to be processed; this includes red blood cell lysis in the gut (k), the uptake of hemoglobin into
endosomes by the digestive cells (l), proteolytic processing of the hemoglobin (m), the capturing of excess
heme in residual bodies (n), and the excretion of residual bodies and old digestive cells (o). Blood meal
concentration occurs via the transport of excess water from the gut into the hemolymph (p), from which
point it is secreted back into the host via the salivary glands in the case of ixodids (q), via the coxal organs
in argasids (r), or via the Malphigian tubules in N. namaqua (s). Heme need to be transported across the
gut into the hemolymph and is bound by either carrier proteins (CP) or vitellogenin (Vg) (t). Vittelogenin is
transported to ovaries and processed into vitellin in the eggs (u). Ronel Pienaar is thanked for the picture.
is accomplished by ticks with eyes, although this is considered to be a derived characteristic

(Klompen et al. 1997). Vibrations are detected via mechanosensilla distributed across the legs and
bodies of ticks, which are an ancient adaptation found in most arthropods (Alberti and Coons
1999). Haller’s organ is a set of clusterized sensilla located on the foreleg tarsi of all ticks and is
involved mainly in gustatory and olfactory sensing (Sonenshine 1991). This organ is not unique
to ticks; it also occurs in the Holothyrida, and as such has established sensory functions associ-
ated with a non-blood-feeding lifestyle (Alberti and Coons 1999).
Systematic analysis of the sensilla found within Haller’s organ has provided some support for
the homology of sensory setae among the tick families (Klompen and Oliver 1993; Klompen
et al. 1997). However, morphological analysis of the receptor types in soft and hard ticks showed
that the 2 families differ in the number and types of receptors found, as well as in their degree
of innervation (Hess and Vlimant 1986). Significant differences also exist in the numbers of
intracuticular mechanosensory sensilla and neurons found on the legs of hard and soft ticks
(Hess and Vlimant 1986). Although these studies were done on a small subset of ticks and it is
not known whether these are generalizations, it would seem that soft and hard ticks detect their
hosts to different extents. As yet, no molecular data exist regarding receptors involved in the
detection of host-derived semiochemicals. Physiological data are also relatively scarce for pur-
poses of comparison between the tick families. Thus far, the only common host-derived mole-
cule detected by both soft and hard ticks is carbon dioxide (Garcia 1962; Nevill 1964; Sonenshine
2004). In this regard, the only carbon dioxide receptor homologues discovered in arthropods
thus far were found in insects including Drosophila, mosquitoes, Bombyx, and Tribolium (Rob-
ertson and Kent 2009). Receptor homologues were not detected in the genomes of the honey-
bee, the parasitoid wasp, the human louse, the pea aphid, the waterflea, or the hard tick Ixodes
scapularis. It was therefore concluded that the detection of carbon dioxide evolved multiple
times within various arthropod lineages and, based on parsimony arguments, once within the
tick lineage (Robertson and Kent 2009). However, reports exist that indicate that the ability to
detect carbon dioxide varies or is absent in various hard and soft tick species (Estrada-Peña
et al. 1991; Fourie et al. 1993; Oorebeek et al. 2009). It has been suggested that this variation is due
to host locating strategy (i.e., hunting versus ambush) and would therefore be host related
(Oorebeek et al. 2009). Whether such adaptations were ancestral, were lost among certain line-
ages, or evolved numerous times as a result of convergence cannot yet be determined. The diver-
sity of attachment sites found for various ticks on various hosts would also suggest that
host-specific adaptations in regard to host kairomone detection occurred (Sonenshine 2004). It
is therefore expected that few conserved receptor orthologs will be found between tick families,
or even between genera and species. The study of host detection mechanisms as a means to find
evidence of shared characteristics involved in blood feeding in the last common ancestral lineage
is therefore unlikely to be a good model. However, using the same line of reasoning, finding re-
ceptor orthologs with conserved functions (e.g., carbon dioxide detection) would be considered
definitive proof of common derived vertebrate-host-finding capabilities in hard and soft ticks.
Additional details on general chemical communication in ticks are discussed in Chapter 14.
2.2. ATTACHMENT TO THE HOST

The engagement (physical contact with and adherence to the host pelage), exploration (search-
ing for an attachment site on the host), and penetration (insertion of the mouthparts into host
integument) phases described by Waladde and Rice (1982) would correspond with the stage of
attachment to the host. The only molecular study that probed into this area described the up-
regulation of certain genes in the hard tick Amblyomma americanum when it was exposed to
feeding stimuli on the host in the exploration phase (Mulenga et al. 2007). This stage presents
the most convincing morphological evidence of common characteristics in blood-feeding be-
havior, in that all ticks possess a hypostome with 2 distinct features (i.e., sclerotized recurved
teeth on the ventral surface and chelicerae) (Sonenshine 1991; Coons and Alberti 1999). Chelic-
erae have been modified as cutting organs and have been considered unique in the Acari. Hard
and soft ticks possess a gustatory pore on the internal article of the cheliceral cutting digit that
is probably involved in tasting the blood meal and the detection of shear forces (Waladde and
Rice 1977). The hypostomal teeth of various ticks can also vary considerably; this variation has
been associated with the feeding durations of various life stages or genders. When ticks feed for
short periods or not at all, dentition will be reduced or absent, whereas teeth are generally well
developed in ticks associated with hosts for prolonged periods (Nuttall 1911; Snodgrass 1948;
Arthur 1962; Sonenshine 1991). In argasid ticks, the digits are heavier and their musculature
more extensive, presumably for more rapid penetration and feeding (Balashov 1972). However,
particularly in predatory or parasitic gamasid mites, the hypostomal morphological structures
are diverse, and hypostomes with dentition as well as cutting chelicerae are present (Alberti and
Coons 1999). Balashov (1972) considered that although the capitula of hard and soft ticks show
conserved features, significant differences also exist with regard to their particular lifestyles.
Argasid mouthparts are considered as less specialized than those of ixodids; their capitulum is
shorter, and a labrum divides the pre-oral cavity into a dorsal salivary and a ventral food channel.
In ixodids, the labrum is absent, and blood and saliva are mixed during feeding (Gregson 1960;
Sonenshine 1991). Conversely, ixodid ticks have a well-developed pharyngeal valve that blocks
blood uptake during saliva secretion, a feature that is absent in argasids. Argasids perform this
function using the moveable labrum (Sonenshine 1991). Significant morphological and mecha-
nistic differences therefore exist, such that convergent evolution of hypostomal structure cannot
be ruled out if it is considered that both tick families needed to penetrate a common obstacle
(i.e., the vertebrate dermis). Initial adaptation of the ancestral tick lineage as vertebrate lymph
feeders has also been proposed and could also explain those homologous features of the hypo-
stome involved in tick feeding (Mans and Neitz 2004). An adaptation directly linked with at-
tachment occurred exclusively within the ixodid lineage, namely, the secretion of proteins that
polymerize and harden to form a cement cone that enhances the tick’s attachment to the host
(Kemp et al. 1982). All metastriate ticks secrete cement, and the size of the cement cone or plug
can be related to the size of the mouthparts. In ticks with short mouthparts that do not penetrate
deeply into the epidermis (Dermacentor, Haemaphysalis, and Rhipicephalus), the cement cone
extends significantly into the feeding site and is anchored to the external hypostome and palps
and the surrounding the skin of the host. Ticks with long hypostomes that penetrate deeply into
the host’s dermis (Amblyomma, Aponomma, and Hyalomma) have cement cones that encase the
full length of the hypostome in addition to being anchored to the external mouthparts and sur-
rounding skin area (Kemp et al. 1982). The proteins involved in cement formation exhibit char-
acteristic regions of low complexity; they are described as glycine-rich proteins with suggested
similarity to spider silk proteins (Maruyama et al. 2010). An inverse correlation between the
transcript levels and types of glycine-rich proteins was found for metastriate ticks with short
versus long mouthparts, suggesting the specific co-evolution of mouthparts and cement produc-
tion (Maruyama et al. 2010). In the Prostriata (Ixodes spp.), cement formation is not a conserved
feature, but it is nonetheless present in some species (Kemp et al. 1982). Peptides with similarity
to the glycine-rich cement proteins of metastriates have also been found in Ixodes salivary tran-
scriptomes (Francischetti et al. 2009). It is therefore likely that cement formation developed
within the ancestral ixodid lineage, with significant expansion in the metastriates.
2.3. THE TICK–HOST INTERFACE

The tick–host interface corresponds with the ingestion (uptake of host body fluids), engorgement
(uptake of a full meal), and detachment (withdrawal of mouthparts from the host’s integument)
phases described by Waladde and Rice (1982). Soft ticks’ method of feeding and feeding sites are
considered as similar to those of other rapid blood-feeding insects in that argasids feed from a
pool of blood or hematoma generated by mechanical damage to blood and capillary vessels
(Lavoipierre and Riek 1955; Balashov 1972). Conversely, ixodids establish a considerably remod-
eled feeding site over a prolonged period that includes the uptake of mainly lymphatic fluid and
lysed tissues in the first few days and a mixture of inflammatory and blood infiltrate during the
final days of rapid engorgement (Balashov 1972; Binnington and Kemp 1980). Ixodid ticks there-
fore have to deal with a completely different host interface than soft ticks and, because of their
prolonged feeding stages, with host wound healing processes such as inflammation, proliferation,
and wound contraction (Francischetti et al. 2009). The interaction at the tick–host interface oc-
curs mainly via salivary gland secretions, and the differences between ixodid and argasid salivary
protein family repertoires have been extensively discussed in regard to their evolutionary impli-
cations (Mans and Neitz 2004; Mans et al. 2008; Mans 2011). The tick–host interface is the only
point at which ticks and their host’s defense mechanisms interact in a direct manner, and it is the
site where acquired host resistance to tick invasion occurs. The effect that host processes have on
the evolution of blood-feeding mechanisms could therefore have been significant, as mentioned
in the Introduction. Therefore, this stage is not dealt with in more detail in this chapter.
2.4. BLOOD MEAL PROCESSING

Ticks depend on consecutive blood meals in order to molt from larvae to nymphs to adults, as
well as to lay eggs (Oliver 1989). Exceptions to this include some soft ticks from the genus Orni-
thodoros that molt directly from larvae to nymphs without feeding and some adult stages (soft
tick genera Otobius, Antricola, and Nothoaspis) that do not feed at all and are considered to be
obligatorily autogenous (i.e., they have the ability to produce egg batches without a cycle of
feeding) (Oliver 1989). However, even these exceptions depend on blood meals from former life
stages, and it could be argued that adequate nutrition is derived, processed, and stored from
previous blood meals to enable their functional biology. The relative scarcity of species that dis-
play these deviant behaviors and their terminal phylogenetic relationships would suggest that
these are mostly characteristics derived after the evolution of blood feeding, with little impact on
the broad generalization that ticks are obligate blood feeders.
The actual phase of blood meal processing or digestion is not part of the feeding scheme
proposed by Waladde and Rice (1982), but it is an integral part of a blood-feeding arthropod’s
lifestyle. Blood meal processing uses organs and molecules not in contact with or exposed to the
host’s immune system and is considered to represent concealed antigens (Nuttall et al. 2006).
Although cross-reactivity might occur with salivary gland antigens, it would be expected that
this would be minimal for most proteins found in non-salivary gland tissues. Blood meal pro-
cessing is therefore largely a host-independent process that should not be affected by mamma-
lian defenses. It would thus be expected that homologous characteristics that have evolved for
blood meal digestion should be more conserved than factors involved in the modulation of the
host’s defenses. Blood meal processing should thus be a host-independent model for the evolu-
tion of a blood-feeding lifestyle in ticks. The processing of the blood meal can be divided up into
several stages that include (1) the elimination of excess blood-meal-derived water, (2) the mor-
phology of the midgut and its development in relation to blood digestion, and (3) the processing,
transport, sequestration, and excretion of heme. The latter stage also would include all mole-
cules that interact with heme.
Ticks ingest enormous amounts of blood in relation to their body size during a single meal. For
soft ticks, the maximum expansion limit of their integument determines the amount of blood that
can be engorged in a single feeding event, which can be as much as 2 to 10 times their body weight
(Balashov 1972). To a limited extent, the secretion of blood-meal-derived water occurs during actual
feeding, whereas excess water is secreted after detachment to allow time to concentrate the blood
meal. Water secretion takes place via the coxal organs, which are absent in hard ticks, and ~40% of
blood-meal-derived water may be secreted to concentrate the blood meal by almost 2-fold (Balas-
hov 1972). In hard ticks, 60% to 70% of the blood-meal-derived water is actively secreted via the
salivary glands during the rapid engorgement phase of feeding (Tatchell 1967; Kaufman 1983). Prior
to this, the f cells in the type III granular salivary gland acini need to develop the labyrinthine struc-
ture needed to transport water from the hemolymph across the salivary membranes (Meredith and
Kaufman 1973; Fawcett et al. 1981; Kaufman 1983). Water secretion via the salivary glands is depen-
dent on the synthesis of prostaglandin E2 (PGE2), a chemical synthesized and secreted by most hard
ticks that also acts as an anti-inflammatory molecule at the feeding site (Bowman et al. 1996). In soft
ticks, PGE2 does not induce secretion in glands, and it is absent from salivary gland secretions, sug-
gesting that argasids lack the ability to synthesize this molecule (Astigarraga et al. 1997; Maritz-
Olivier et al. 2005). It also suggests that the functional role of PGE2 in ixodid tick biology evolved
after divergence of the tick families. Nuttalliella namaqua can ingest up to 14 times its body weight
and can concentrate the blood meal approximately twice by secreting excess water via the Malpi-
ghian tubules (Mans et al. 2011, 2012). This latter mode has been considered as ancestral.
3. BLOOD MEAL PROCESSING AS A MODEL FOR

THE EVOLUTION OF BLOOD-F EEDING BEHAVIOR
IN TICKS
Although the digestion and processing of a blood meal would certainly be accommodated to a
certain extent by any non-hematophagous arthropod, blood is no trivial food source, and spe-
cialized adaptations are necessary for animals such as ticks that rely exclusively on blood as a
source of energy. Hemoglobin comprises almost 60% of all blood proteins, occurs at levels of 150
mg/ml, and is thus a major component of the blood meal (Graça-Souza et al. 2006). In its native
form, hemoglobin binds heme on a 1:1 molar ratio, which will result in the accumulation of high
concentrations of heme (3.38% of hemoglobin) during proteolytic digestion (Balashov 1972).
Heme in its free form has been shown to be highly toxic, inducing oxidative stress and causing
damage to tissues and proteins as a result of the formation of reactive oxygen species (Vincent
1989; Citelli et al. 2007). Mechanisms for dealing with heme toxicity would thus have been a
prerequisite for ticks’ adaptation to a blood-feeding lifestyle, and this has been found to be a
universal phenomenon for all hematophagous arthropods (Graça-Souza et al. 2006).
It was shown that the hard tick Boophilus microplus lacks a functional heme biosynthetic path-
way and is therefore exclusively dependent on host-derived hemoglobin (Braz et al. 1999). Vitel-
lin, the major egg yolk protein, and its precursor, vitellogenin, are heme-binding storage proteins,
a feature thus far unique to ticks. It was therefore suggested that the evolution of vitellogenin
to bind host-derived heme was critical for the evolution of blood-feeding behavior in ticks (Roe
et al. 2008; Cabrera et al. 2009; Donohue et al. 2009). Additional details on tick storage proteins in
general are discussed in Chapter 15. The biology of ticks and their evolution as hematophagous
parasites are thus intricately linked with the acquisition of a blood meal and the downstream
processing necessary for the completion of their life cycle.
4. BLOOD MEAL PROCESSING IN HARD

AND SOFT TICKS
Overall, the argasid and ixodid mechanisms for blood meal processing are similar. They differ from
that of blood-feeding insects in which proteolytic digestion occurs in the gut lumen (Akov 1982). In
the case of ticks, red blood cells are lysed in the gut lumen, but released hemoglobin is taken up by
digestive cells before intracellular digestion. Intracellular digestion has been considered as a phe-
nomenon unique to ticks (Akov 1982; Sonenshine 1991). However, all mites possess both secretory
and digestive cells, and intracellular digestion has been indicated to occur in other Acari (Alberti and
Coons 1999; Nisbet and Billingsley 2000; Šobotník et al. 2008). It is, as such, probably an ancestral
function that was conserved before ticks evolved blood-feeding lifestyles. In ixodids, red blood cells
are rapidly lysed, some even at the feeding site, whereas in soft ticks and N. namaqua lysis is slower,
and intact red blood cells might be found in the midgut lumen and caecae for weeks or months after
feeding (Balashov 1972; Coons et al. 1986; Mans et al. 2011, 2012). Hard ticks take up soluble hemo-
globin rapidly and store it in the endosomes until it is digested (Balashov 1972; Coons et al. 1986;
Coons and Alberti 1999). Hemoglobin also crystallizes in the gut lumen, and in hard ticks it is ex-
creted, whereas soft ticks can store these crystals for prolonged periods, after which whole crystals
can be absorbed via phagocytosis (Balashov 1972; Coons et al. 1986; Coons and Alberti 1999).
4.1. DEVELOPMENT OF THE MIDGUT DURING

BLOOD MEAL DIGESTION
Blood meal processing mainly occurs in the midgut and midgut caeca or diverticulae (Coons
et al. 1986). The morphology of the alimentary system of ticks is described in detail in Chapter 4.
In argasids, the midgut is central, with numerous secondary branches of the primary caeca. Di-
gestion takes place primarily in the midgut and median and proximal parts of the posterolateral
caeca. The latter is distended and serves as the main reservoir of undigested blood (Balashov
1972). Ixodids have a shorter and thinner midgut with longer caeca with loops and limited sec-
ondary branches (Balashov 1972). The basic cellular composition of the midgut and caeca is
similar to that observed in other arachnids such as spiders and scorpions and is composed of
digestive, secretory, and undifferentiated reserve cells (Balashov 1972). Recent literature pro-
poses that for hard ticks, only 2 cell types exist, namely, undifferentiated reserve cells and diges-
tive cells (Coons et al. 1986). In ixodids, a third type, the vitellogenesis cell, is proposed as a
digestive cell that differentiates at different feeding phases. If this assessment is correct, then ar-
gasid ticks also probably have only reserve and digestive cells, implying that only 1 gut epithelium
cell exists that differentiates in response to feeding stimuli (Grandjean and Aeschlimann 1973).
However, there is evidence that gut epithelium cells secrete components that are important for
the lysis of red blood cells (Balashov 1972). Gut epithelium from unfed hard and soft ticks pos-
sesses reserve cells with relatively few digestive cells present (Grandjean 1983; Coons et al. 1986).
Blood digestion in argasids can be divided into 3 phases: (1) preparation, (2) rapid digestion,
and (3) slow digestion (Grandjean 1983; Coons et al. 1986). Engorgement leads to stretching of the
midgut followed by the secretion of excess water via the coxal organs. The first phase occurs after
engorgement and involves initial agglutination (2 to 3 days) and hemolysis (10 to 15 days). Aggluti-
nation and hemolysis generally occur after excess water has been secreted. Secretory cells release
their contents soon after feeding and are replaced by digestive cells. Hemolysis is due to secretions
from the secretory or digestive cells (Grandjean 1983). Released hemoglobin is therefore in a super-
saturated solution and crystallizes to form oxyhemoglobin/methemoglobin deposits in the midgut
lumen (Balashov 1972). In the second phase, the hemoglobin mass is assimilated; this can take sev-
eral weeks to months. The second phase is associated with rapid digestion and continues until ovi-
position (Grandjean 1983; Coons et al. 1986). Reserve cells are replaced by active digestive cells that
take up hemoglobin, mostly via pinocytosis, into food vacuoles, forming hemoglobin inclusions;
these hemoglobin inclusions are digested to form hematin granules ranging from 1 to 4 μm in size
(Balashov 1972). Digestive cells also contain lipid and carbohydrate granules assimilated from the
blood meal. Mature cells are shed into the lumen and degenerate, releasing hematin and digested
products. There is a correlation between the decrease in hemoglobin content and an increase in
alkaline hematin, which is maximal after oviposition in adults or molting in nymphs (Balashov
1972). In the third phase, few digestive cells remain, and the hemoglobin is slowly digested until the
next blood meal (Balashov 1972; Coons et al. 1986). In ticks that have not fed for prolonged periods,
less than 1% of the hemoglobin remains in the gut, and minute spherical hematin granules impart
the dark black color observed in the gut. In cases in which host red blood cells are resistant to he-
molysis, whole cells are phagocytized by digestive cells and digested intracellularly (Balashov 1972).
In hard ticks, 4 main phases are recognized during feeding: (1) attachment, (2) slow feeding,
(3) rapid feeding, and (4) detachment that can be related to blood meal processing events in the
midgut (Coons et al. 1986; Sonenshine 1991). The attachment phase relates to the initial piercing
of the skin and establishment of the feeding site and can take up to 1 day. The midgut mor-
phology indicates mainly replacement cells, with a few digestive cells present (Coons et al. 1986).
Proteolytic activity in this phase is at a minimum (Franta et al. 2010). The slow feeding phase
takes 3 to 5 days, depending on the species, and significant morphological changes occur during
this phase. Replacement cells progressively differentiate into digestive cells, which start to digest
the ingested blood meal (Coons et al. 1986). During this phase, proteolytic transcription and
activity progressively increase, and toward the end of this phase, spent digestive cells start to
detach (Coons et al. 1986; Franta et al. 2010). During the rapid engorgement phase, which can
last for 1 to 2 days, the shedding of spent cells stops, and the gut epithelium is composed mainly
of digestive cells (Coons et al. 1986). The proteolytic activity reaches a maximum and remains at
that level after detachment (Franta et al. 2010). After detachment, an increase in shed digestive
cells is observed that indicates an increase in active digestion.
4.2. HEME PROCESSING, SEQUESTRATION, AND EXCRETION

In both argasids and ixodids, hemoglobin is taken up into digestive cells via heterophagy at clath-
rin-coated pits by means of receptor-mediated endocytosis, phagocytosis, or pinocytosis (Coons et
al. 1986). Hemoglobin vesicles fuse to form endosomes, which fuse with primary lysosomes to form
secondary lysosomes where proteolytic degradation takes place. In the hard tick Ixodes ricinus,
proteolytic digestion of hemoglobin occurs via a sequential cascade of protease enzymes (Sojka
et al. 2008; Horn et al. 2009). The levels of these enzymes increase steadily over the progression of
feeding (Franta et al. 2010). Hemoglobin is first degraded into large fragments by aspartic and cys-
teine peptidases of the cathepsin D and L types, respectively, as well as legumain-type asparaginyl
endopeptidase. The large hemoglobin fragments are further proteolyzed by cysteine endopeptidase
cathepsin B and the amino and carboxydipetidase activities of cathepsin C and cathepsin B, respec-
tively (Horn et al. 2009). Cathepsins B, C, D, and L and asparaginyl endopeptidase activity has been
shown to be present in the midgut of B. microplus (Mendiola et al. 1996; Renard et al. 2002; Ber-
gamo Estrela et al. 2010). Transcriptome analysis of the midgut of Dermacentor variabilis indicated
the presence of similar proteases (Anderson et al. 2008). For a more detailed description of the
enzymatic processes involved in hemoglobin digestion, see Section 4 of Chapter 6.
The series of proteolytic events involved in the degradation of hemoglobin in soft ticks is not
known. However, the presence of various proteolytic enzymes, including cathepsin B, cathepsin
C, and legumain asparaginyl endopeptidase, was detected in gut extracts of Ornithodoros mou-
bata (Grunclová et al. 2006). The absence of cathepsins D and L in soft ticks has not yet been
confirmed but would indicate some differences in the proteolytic digestive capabilities of the
different tick families. Even so, cysteine and aspartic protease activity capable of hemoglobin
proteolysis has been found in free-living and ectoparasitic mites (Nisbet and Billingsley 2000).
The intracellular proteolytic digestive systems necessary to degrade hemoglobin are therefore
ancestral and did not evolve specifically for a blood-feeding lifestyle.
Secondary lysosomes eventually become residual bodies, also known as hemosomes, that
contain hematin crystals (Coons et al. 1986; Lara et al. 2003). It has been suggested that residual
bodies or hemosomes are not merely aged secondary lysosomes but specific organelles to which
heme is transported after the degradation of hemoglobin via specific transport proteins for de-
toxification (Lara et al. 2005). The molecular nature of these heme-transporting proteins has not
yet been elucidated, nor is it known whether soft ticks perform this same feat. The possibility
also exists that these transporters are in reality carrier proteins or vitellogenins destined for se-
cretion. Residual bodies have been shown to have high concentrations of iron (Sonenshine
1991). In the hard tick B. microplus, hematin is composed of aggregated iron-protoporphirin IX
(Lara et al. 2003). Its Fourier transform infrared spectrum differs significantly from that of
heme, lacking the characteristic 1,704 cm−1 carboxylate peak, which indicates interaction with
other non-heme products in the aggregate. Interestingly, the hemosome also lacks the 1,660 cm−1
peak characteristic of β-hematin, the heme component in hemozoin from Plasmodium falci-
parum or Rhodnius prolixus, suggesting a unique heme-based supramolecular structure (Lara
et al. 2003). Whether this unique structure is found in all ticks is not yet known.
Digestive cells cannot rid themselves of residual bodies, which leads to the cells’ being filled
up until they become non-functional. Spent digestive cells filled with residual bodies are shed
into the gut lumen before disintegration, thereby releasing hematin crystals (Coons et al. 1986;
Sonenshine 1991). Exocytosis of residual bodies into the lumen or hemocoel has not been ob-
served. Most of the hematin is excreted via defecation or remains in the midgut lumen.
4.3. HEME TRANSPORT AND STORAGE PROTEINS

It has been shown that B. microplus lacks δ-aminolevulinate dehydratase (ALA-D), the rate-
limiting factor in the formation of porphyrin, and therefore cannot synthesize heme, making it
dependent on host-derived heme for its heme-related needs (Braz et al. 1999). A September 2011
BLAST analysis of the non-redundant protein and nucleotide sequence databases, as well as of
the expressed sequence tag (EST) databases, indicated that no potential homologues for ALA-D
can be found in ticks. This analysis included data for the draft genome sequence of I. scapularis
and the impressive number of EST sequences available for hard ticks, including the midgut
transcriptome of D. variabilis (Anderson et al. 2008). It is therefore possible that the inability to
synthesize heme is a conserved feature of ixodid ticks. Homologues for ALA-D exist for the two-
spotted spider mite, Tetranychus urticae (GW034806.1, GW034807.1), and the scab mite, Pso-
roptes ovis (BQ835077.1), suggesting that the ability to synthesize heme exists in other mites. It
is not known whether soft ticks lack the ability to synthesize heme.
Ixodid ticks are thus far the only arthropods unable to synthesize heme (Braz et al. 1999;
Donohue et al. 2009). Heme plays important functions in oxidative phosphorylation, a process
universal in all metazoans (Hosler et al. 2006). Given the toxicity and relative insolubility of free
heme, the dependence on host-derived heme necessitates mechanisms for heme transport from
the midgut to tick tissues that need heme. In this regard, specific transporters have been identified
in ticks that include carrier proteins (CP) and vitellogenins (Donohue et al. 2008, 2009). All CP
belong to the vitellogenin family, with the difference being that vitellogenin’s main function is as
a storage protein in eggs in the form of vitellin. CP and vitellogenin are general transporters of
carbohydrates and lipids and are known as lipoglycoheme proteins (Gudderra et al. 2001). CP
have been attributed functions as scavengers of heme to prevent oxidative damage (Maya-
Monteiro et al. 2000, 2004). CP have been identified in the hard ticks A. americanum, B. micro-
plus, and D. variabilis (Maya-Monteiro et al. 2000; Gudderra et al. 2001; Donohue et al. 2008).
When hemolymph from the soft tick O. parkeri was subjected to electrophoresis, the heme sepa-
rated from CP (Gudderra et al. 2002); this does not occur with hemolymph from hard ticks. It was
not clear whether the separation was electrophoretic or occurred because CP do not bind heme
in this species. However, it has been shown that vitellogenins from the soft tick O. moubata bind
heme (Chinzei et al. 1983).
Vitellogenin and CP are synthesized in the fat body of hard and soft ticks, but they are also
found in the midgut, salivary glands, and ovaries (Chinzei and Yano 1985; Taylor et al. 1991;
James et al. 1999; Gudderra et al. 2002; Donohue et al. 2008; Boldbaatar et al. 2010; Horigane
et al. 2010). Localization to the midgut provides a possible mechanism for heme loading. It is not
clear whether vitellogenin synthesized in the fat body binds heme before or after secretion into
the hemolymph. Tick vitellogenins are the only members of this family known to be able to bind
and transport heme, and this would seem to indicate a unique adaptation of this protein family
in response to blood feeding (Donohue et al. 2009). Analysis of egg extracts from the two-
spotted spider mite, Tetranychus urticae, has shown that no heme is bound by vitellogenin in its
native form (Cabrera et al. 2009). However, it was not determined whether heme could poten-
tially be bound in vitro. It was suggested that 3 specific motifs found in ixodid carrier proteins
and vitellogenin were not shared by other heme-binding proteins studied thus far (Donohue et
al. 2008). These motifs are an N-terminal lipoprotein domain, a central domain of unknown
function (DUF1943), and a C-terminal von Willebrand factor type D domain. It should be
noted, however, that these domains are common for vitellogenins described from all arthropods
thus far and would not in themselves be significant enough to designate heme-binding function.
The degradation and processing of vitellin pose problems similar to those of hemoglobin, as
heme would again be released. An aspartic acid protease capable of targeting vitellin by binding
to heme has been identified in B. microplus (Sorgine et al. 2000; Pohl et al. 2008). Although
heme-binding serine and cysteine proteases involved in the degradation of hemoglobin have
been identified in bacteria, this is the first report for a blood-feeding arthropod. Whether this
specific heme-binding protease is also present in soft ticks remains to be determined.
5. EVOLUTIONARY PERSPECTIVES ON
BLOOD MEAL DIGESTION
Parsimonious arguments would suggest that the unique mechanisms that have evolved in ticks
to allow them to deal with blood meal digestion should be conserved between hard and soft
ticks. However, significant physiological differences exist and suggest that the different families
did, at least in part, develop different mechanisms to deal with specific aspects of blood meal
processing (Table 9.1). The most intriguing variation is that in the mechanisms for water elimi-
nation from the blood meal. Whole organ systems evolved or were adapted for this specific
purpose. Coxal organs in soft ticks are homologous to structures in other arthropods, as coxal
organs are used for osmoregulation to eliminate water during feeding (Alberti and Coons 1999).
It is therefore possible that a coxal organ was present in the ancestral tick lineage but was subse-
quently lost in hard ticks. In the case of hard ticks, this demanded the complete duplication of
the type II acini to yield type III acini capable of transporting water via the f cells. PGE2 synthe-
sis capability also developed. In this regard, it should be mentioned that no homologs for verte-
brate cyclooxygenase or PGE2 synthase have been found in the EST database for ticks or in the
I. scapularis genome assembly. Protistans have been shown to synthesize prostaglandins via in-
dependent pathways, and no homologues to any of the vertebrate enzymes involved in the PGE2
pathway have yet been discovered (Kubata et al. 2007). Furthermore, prostaglandin H/cyclooxy-
genase homologues were recently discovered in crustaceans and the genome of the body louse
Pediculus humanus, but not in any of the other insect genomes sequenced (Varvas et al. 2009).
It is therefore possible that PGE2 synthesis by ixodids has a unique origin in the hard tick lineage.
Some interesting questions arise as to whether hard ticks evolved PGE2 synthesis capability in
order to inhibit the host’s inflammatory systems and whether this led to the evolution of its en-
docrine properties, or whether it was exapted for use at the blood-feeding interface after evolving
its endocrine role in fluid secretion in ixodid salivary glands.
The comparative analysis of blood digestion in ticks and the derivation of homologous char-
acters that specifically evolved during adaptation to a blood-feeding environment are hampered
by the ancient nature of the digestive system in mites (Table 9.1). All proteolytic enzyme types
involved in digestion would have been present in the non-hematophagous ancestor to ticks, and
they would have performed very similar functions (i.e., intracellular protein digestion) (Nisbet
and Billingsley 2000). These enzymes and their previous digestive housekeeping functions
would have been co-opted (exapted) during the evolution of blood-feeding behavior. Their ho-
mology cannot as such be taken as evidence of a common blood-feeding ancestry in ticks, unless
their absence in non-hematophagous mites can be established.
The formation of hematin in digestive cells or the gut lumen is also a phenomenon that
seems to be universal among blood-feeding arthropods (Graça-Souza et al. 2006). This can most
probably be related to the insolublility of free heme under acidic conditions as found in the ly-
sosomes. However, the supramolecular structure of the hematin aggregates would be deter-
mined by specific molecular modifications to the porphyrin moiety or interactions with
non-heme components (Lara et al. 2003). The formation of hemosomes via specific proteins
involved in intracellular transport should also be considered as a distinct adaptation (Lara et al.
2005). Comparative analysis could therefore give insights into whether the molecular mecha-
nisms for hematin formation are unique and conserved among ticks.
As yet, no comparative data exist for heme carrier proteins that would allow for the estab-
lishment of homology in hard and soft ticks (Gudderra et al. 2002; Donohue et al. 2009). The
data thus far rather suggest that heme is transported by different molecules in the different tick
families. However, if vitellogenin is considered as a transport protein as well as a precursor to
the storage protein vitellin, then both tick families do have homologous proteins that can be
considered as heme-transporting proteins. Recently, a vitellogenin from a marine annelid was
shown to be able to transport biliverdin and bind heme (Schenk and Hoeger 2011). Vitello-
genins are part of the large lipid protein transfer superfamily that includes apolipophorin II/I,
Table 9.1: A comparison of blood meal processing in soft and hard ticks
from an evolutionary perspective.
Process/organs/molecules Ixodids Argasids Comment

Concentration of blood Salivary glands Coxal organs Coxal organs ancestral in
meal via water mites Independent
secretion evolution in ixodids
Gut epithelium cells Reserve and digestive Reserve and digestive Ancestral in arachnida
cells cells
Lysis of red blood cells Feeding site, gut lumen, Gut lumen, controlled, Ancestral in arachnida
rapid gradual
Uptake of blood meal Pinocytosis, endocytosis Pinocytosis, endocytosis Ancestral in mites
Digestion locality Intracellular lysosomal Intracellular lysosomal Ancestral in mites
Digestive enzymes Cysteine/aspartic Cysteine/aspartic Ancestral in mites
cathepsins cathepsins
Heme sequestration Intracellular transport Residual bodies Derived in ancestral tick
to hemosomes Transport?a lineage?a
Molecular structure? Molecular structure?
Excretion Shedding/rupture of Shedding/rupture of Ancestral in mites?a
cells in gut lumen cells in gut lumen Derived in ancestral
release hematin release hematin tick lineage?
Heme carrier proteins CP homologues of CP homologues do not Derived in ixodids?a
(CP) vitellogenin bind heme?a Derived in ancestral
tick lineage?a
Heme binding/storage Vitellogenin/vitellin Vitellogenin/vitellin Convergent evolution?a
by vitellogenin/vitellin binds heme binds heme Derived in ancestral
tick lineage?a
Heme biosynthesis Lacks ALA-Db/host ALA-D?b Derived in ixodids?a
dependent Host dependent?a Derived in ancestral
tick lineage?a
Vitellin processing Heme-binding aspartic Heme-binding aspartic Derived in ixodids?a
protease protease?a Derived in ancestral
tick lineage?a
a
A postulated state or information that is not currently certain.
b
ALA-D: δ-aminolevulinate dehydratase.
apolipoprotein B, vitellogenins, and microsomal triglyceride transfer protein (Babin et al. 1999).
An apolipophorin from Drosophila was shown to bind heme, and apolipoprotein B binds
hemin, a heme derivative (Duncan et al. 1999; Seki et al. 2008). It is therefore possible that vitel-
logenin and related carrier proteins can bind heme because of their natural propensity to bind
lipids (Smolenaars et al. 2007). The convergent evolution of heme binding in vitellogenins from
hard and soft ticks therefore cannot be excluded if it is considered that this is the major egg yolk
protein of ticks (Rosell and Coons 1991; Chinzei 1983). In this regard, the convergent evolution
of function from conserved protein folds is a central theme in the evolution of blood-feeding
behavior in arthropods (Mans et al. 2011). Even so, if the binding sites and molecular mecha-
nisms of binding for heme are structurally conserved in soft and hard tick vitellogenin-related
proteins, it will constitute a significant argument for common blood-feeding origins in the an-
cestral tick lineage. This argument will be further strengthened if heme biosynthesis capability
is found to have been lost before divergence of the tick families.
An analysis of the vitellogenin family shows some interesting features (Fig. 9.2). It is clear
that gene duplications occurred in multiple vitellogenin lineages. In ticks, at least 3 major clades
FIGURE 9.2: Phylogenetic analysis of vitellogenin-related proteins. Major animal groups other than
Ixodida are indicated (e.g., Insecta, Crustacea, etc.). Vitellogenins from the soft tick Ornithodoros
moubata and the honeybee parasitic mite Verroa destructor are shaded in grey. Black dots indicate
sequences with CP signature sequences. Vitellogenin-related sequences were retrieved from the
sequence database using PSI-BLAST and the sequence of O. moubata vitellogenin. Sequences were
aligned using ClustalX, trimmed and analyzed by a neighbor-joining analysis (Poisson correction,
pairwise deletion, 10,000 bootstraps). Numbers in brackets indicate Genbank accession numbers.
can be observed, of which only clade A thus far has hard and soft tick sequences. The hard tick
sequences from clade A have not yet been functionally confirmed as vitellogenins, but the
sequence from O. moubata has been characterized as a vitellogenin (Horigane et al. 2010). Tick
clade B is composed of vitellogenin-related proteins, of which at least 1 from B. microplus has
been shown to be a hemolymph glycoprotein GP80 related to vitellogenin (Tellam et al. 2002).
Clade C is composed of CP described from D. variabilis and A. americanum and vitellogenins
described from Haemaphysalis longicornis (Donohue et al. 2008; Boldbaatar et al. 2010). Clade
C also has a sequence for the mesostigmatid honeybee parasitic mite Verroa destructor (Cabrera
et al. 2011). Potential vitellogenin sequences from the predatory mite Phytoseiulus persimilis have
been grouped with tick sequences from clade A and clade B (Cabrera et al. 2011). Similarly, pu-
tative vitellogenin sequences from T. urticae have been grouped in clade C (Horigane et al.
2010). It would therefore seem that vitellogenin from the Acari is monophyletic to the exclusion
of all other arthropods but is paraphyletic within the Acari. If these analyses are correct, it would
suggest that the gene duplications for the various tick clades arose in the ancestral lineage to the
Parasitiformes or even the Acari. This implies that heme-binding capability was present in the
vitellogenins from mites or that heme binding evolved independently on several occasions
within ticks.
The distinction between CP and vitellogenin is therefore not clear from a phylogenetic per-
spective. It has been suggested that clear biological distinctions exist between vitellogenins and
CP (Khalil et al. 2011). For example, vitellogenins are expressed in adult females prior to ovipo-
sition, whereas CP are present in all developmental stages. In addition, CP possess a conserved
N-terminal sequence (FEVGKEYVY) not found in vitellogenins. They also possess only 1
cleavage site in DUF1943 prior to the signature sequence (DASAKERKEIED) that is not found
in vitellogenins, yielding 2 subunits. Vitellogenins, in contrast, have 1 or more cleavage sites
dispersed through their sequence (Khalil et al. 2011). All of the proteins that carry the CP
sequence signature can be grouped into a terminal branch of clade C and are from metastriate
ticks (Fig. 9.2). Other members of this clade have similar or unrelated motifs, indicating that
these signature motifs might not be conserved for all ixodid species. The well-supported
grouping of this clade, the fact that its structure recapitulates known ixodid tick phylogeny, and
the basal position of the verroa mite vitellogenin suggest that this clade is a cluster of ortholo-
gous genes composed of CP that are conserved in other mites. It also might imply that CP are
the ancestral vitellogenin orthologs, and that the vitellogenins from clade A and B evolved in
ticks after gene duplications in the ancestral tick lineage. How this relates to the biology of vitel-
logenins and CP in regard to differential expression and function cannot be determined until
the conservation of these aspects has been established in more genera and species. If CP were
the ancestral vitellogenins that evolved heme-binding capacity for the transport of host-derived
heme to tick tissues in general, the need to separate this function from their traditional function
as storage proteins during oogenesis could have led to the subsequent gene duplication and exa-
ptation of a new class of oocyte-specific heme-binding vitellogenins in ticks.
Finally, does blood meal processing, as a host-independent adaptation to a hematophagous
lifestyle, serve as a good model for the reconstruction of ancestral characters involved in the
evolution of blood feeding in the ancestral tick lineage? Several characteristics associated with
blood meal processing seem to have evolved independently in the hard and soft tick families
(i.e., secretion of excess water via coxal or salivary glands). Other characteristics deemed ances-
tral were probably exapted and therefore not useful for the inference of derived characteristics
(i.e., endocytosis and lysosomal digestion). For those characteristics that seem to be specifically
derived (i.e., heme binding by the vitellogenin family), too little information is available for any
meaningful evolutionary conclusions to be reached. Blood meal processing therefore does not
as yet give good support for common origins of hematophagous behavior in ticks. This parallels
to a large extent the findings for the other stages involved in blood feeding.
Several avenues are available for testing hypotheses on the origins of blood feeding in ticks using
blood meal processing as a model (Table 9.1). Are the unique structures of the heme aggregates
found in hemosomes conserved in all ticks? Do soft ticks also lack the ability to synthesize heme,
and can this be linked to an absence of ALA-D? It should be confirmed that the CP from soft ticks
do not bind heme, and if that is found to be the case, the heme transporter proteins should be char-
acterized to determine whether they are homologous to the CP of hard ticks. The mechanism of
heme binding by vitellogenin and vitellin in hard and soft ticks should be investigated. More vitel-
logenins from distantly related mites should be characterized to determine whether tick-derived
vitellogenins are monophyletic and whether heme binding evolved within the tick lineage.
In conclusion, our knowledge of the molecular mechanisms of blood meal digestion and
heme processing is still rudimentary. Significant basic research into these mechanisms will have
to be conducted before comparative biology can yield solid data that can be used to make mean-
ingful interpretations regarding the evolution of blood-feeding behavior in ticks. High-through-
put approaches for the transcriptomic and proteomic analysis of midgut- and hemolymph-derived
transcripts will obviously aid in this process. However, for meaningful comparative analysis, the
structural mechanisms responsible for function need to be solved, preferably for many tick
genera and species.
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C H A P T E R 1 0
RESPIRATORY SYSTEM
Structure and Function
LAURA J. FIELDE N AND FRANCES D. DUNCAN
1. INTRODUCTION: OVERVIEW OF THE IXODID

RESPIRATORY SYSTEM
Adult and nymphal ticks have a tracheal system to supply oxygen throughout their body and to
discharge carbon dioxide back into the atmosphere. The entrance to the tracheal system is a single
pair of spiracles situated on the ventro-lateral surface directly posterior to the hind leg (coxae IV).
Larvae of ixodid ticks and some argasids do not have trachea and rely entirely on diffusion across
the integument. There is great variation in the structure of the respiratory systems, in particular
in the structure of the spiracle. Thus the Ixodidae and the Argasidae are dealt with separately, with
a brief mention of the Nuttalliellidae. For an electronic version of the anatomy of ticks, the reader
may browse the tick anatomical ontology (illustrations and a description of structure) at www.
Vectorbase.org by following the instructions in the footnote below.1 Additional illustrations of the
anatomy of ticks not included in this edition may be found at this site.
2. STRUCTURE AND FUNCTIONAL MORPHOLOGY

OF THE RESPIRATORY SYSTEM
2.1. SPIRACULAR MORPHOLOGY IN THE IXODIDAE

Ixodid spiracles have large and distinctive spiracular plates (Fig. 10.1) that show species-specific
characteristics in adults and nymphs (Baker 1997; Pugh 1997) and are a taxonomic key characteristic
for species identification (Yunker et al. 1986). An understanding of the spiracular structure of ixodid
Respiratory System 241
ticks comes from numerous studies based on light microscopy (e.g., Arthur 1960; Roshdy and Hef-
nawy 1973), scanning electron microscopy (Hinton 1967; Woolley 1972; Pugh et al. 1988; Baker 1997),
and corrosion cast fracture (Schöl et al. 1995). Reviews of spiracular plate structure have been given
by Evans (1992) and Coons and Alberti (1999). Following Schöl et al. (1995), who used fraction and
corrosion casts of Hyalomma truncatum, we describe the spiracle structure as 3 main components
(Fig. 10.1A).
FIGURE 10.1: The spiracular structure of Ixodid ticks. A, Diagrammatic drawing of a longitudinal fracture
of an entire spiracle of adult Hyalomma truncatum. Ac, atrial chamber (tracheal atrium in text); Bp, base
plate; Ips, interpedicullar space (atrial chambers in text); L, lip of the macula; Lw, lateral wall of the atrial
chamber (tracheal atrium in text); M, macula; Mu, muscle; Mw, medial wall of the atrial chamber (tracheal
atrium in text); Na, non-porous area; O, ostium; P, pedicels; Pa, porous area; Sos, subostial space; Sp,
surface plate; T, trachea. From Schöl, H., Dongus, H., and Gothe, R. (1995) Morphology of spiracles in
adult Hyalomma truncatum ticks (Acari: Ixodidae). Exp. Appl. Acarol. 19:287–306, with permission from
Springer. B, Spiracular plate of female Amblyomma americanum. A, aeropyles; M, macula; O, ostium. C,
Spiracular plate of female Dermacentor variabilis. A, aeropyles; M, macula; O, ostium.
2.1.1. Spiracular plate

The spiracle opens to the outside via a comma-shaped or circular spiracular plate, which
varies in form between species (Fig. 10.1) (Hinton 1967, 1970; Woolley 1972; Baker 1997;
Pugh 1997). Numerous pores, termed aeropyles (Figs. 10.1B, 10.1C), open into a cuticular
labyrinth. This labyrinth has many internal air chambers that are separated by pedicels, ver-
tical cuticular struts with flanges (Figs. 10.2A, 10.2B). Pugh et al. (1988, 1990) refer to these
chambers as atrial chambers and have distinguished 3 types, viz., primary, secondary, and
peripheral (designated as type 1, type 2, and type 3 air chambers, respectively, by Schöl et al.
[1995]). Primary atrial chambers are found directly beneath each aeropyle and extend into
the basal plate. Secondary atrial chambers are not usually found beneath the aeropyle and
do not extend into the basal plate. The peripheral chambers are associated with the smaller
peripheral aeropyles on the outside of the plate. The atrial chambers communicate with each
other via fenestrations formed by slits between the pedicels (Figs. 10.2C, and 10.2D). The
primary atrial chambers that extend to the subplate may communicate with sub-spiracular
multicellular glands, variously termed hypodermal (Pugh et al. 1988), bicellular (Sixl and
Sixl-Voight 1974), or spiracular glands (Walker et al. 1996; Coons and Alberti 1999). These
glands are similar to type I dermal glands and likely have a glandular/secretory role, although
their exact function is unknown (Coons and Alberti 1999). Spiracular glands do not occur
in the argasids or the Nuttalliellidae (Roshdy et al. 1982). Spiracular glands might retard
water vapor transpiration (Pugh et al. 1992b). The ostium forms a crescentic slit-like opening
in the surface of the plate (Figs. 10.1A, 10.2B) and does not participate in gas exchange in the
adult tick as was originally thought (Roshdy and Hefnawy 1973). Rather, it represents the
exterior opening of a collapsed ecdysial tube resulting from the transition from nymph to
adult during molting (Hinton 1967b; Woolley 1972). The macula constitutes the upper lip of
the ostium (Baker 1997). In ixodids, the lip of the macula is immovable and does not func-
tion as an external sealing valve for the subostial space (Schöl et al. 1995). Thus, in ixodid
ticks, air travels in and out via the aeropyles of the sieve plate, and not via the ostium
(Fig. 10.1A).
2.1.2. Subostial space

The subostial space is a horseshoe-shaped chamber that surrounds the stalk of the macula
(Roshdy and Hefnawy 1973; Roshdy 1974; Pugh et al. 1988, 1990) (Figs. 10.1A, 10.2A). In
H. truncatum, the chamber was shown to be the largest part of the spiracle (Schöl et al. 1995).
The atrial chambers within the spiracular plate communicate with the subostial space via
fenestrations.
2.1.3. Tracheal atrium

The tracheal atrium (referred to as the atrial chamber by Schöl et al. [1995]) lies below the sub-
ostial space and should not be confused with the atrial chambers of the spiracular plate (Pugh
et al. 1988, 1990; Coons and Alberti 1999) (Figs 10.1A, 10.2A). At the base of the tracheal atrium
are the openings of the tracheal trunks, which number 9 to 12 in H. truncatum (Schöl et al.
1995) and 6 in Dermacentor andersoni (Arthur 1960). The wall of the tracheal atrium and the
associated musculature open and close the trachea. Based on a series of corrosion casts that
FIGURE 10.2: The spiracular plate morphology of ixodid ticks. A, Scanning electron microscope images
of a longitudinal fracture of a spiracle of adult female Dermacentor variabilis. M, macula; O, ostium; Sos,
subostial space; Sp, surface plate; T, trachea; Ta, tracheal atrium. B, Cuticular labyrinth of spiracle of
adult female Dermacentor variabilis. A, aeropyles; Ac, atrial chamber; Bp, basal plate; F, fenestration; P,
pedestal. A and B from Fielden, L.J., Knolhoff, L.M., Villarreal, S.M., and Ryan, P. (2011) Underwater
survival in the dog tick Dermacentor variabilis (Acari: Ixodidae). J. Insect Physiol. 57:21–26, with
permission from Elsevier. C and D, Differences in labyrinth morphology of ixodid ticks Aponomma latum
(C) and Dermacentor marginatus (D). A, aeropyle; Ap, primary atrial chamber; As, secondary atrial
chamber; Bp, basal plate; Sd, subatrial duct. From Pugh, P.J.A., King, P.E., and Fordy, M.R. (1990)
Spiracular transpiration in ticks: a passive diffusion barrier in three species of Ixodidae (Metastigmata:
Acarina). J. Zool. 221:63–75, with permission from John Wiley and Sons.
243
showed marked spatial variability in the tracheal atrium, Schöl et al. (1995) presented evidence
that a bellow-like mechanism ventilates the tracheal system in H. truncatum (Fig. 10.1A). The
tracheal atrium has a thick lateral wall and an opposite medial wall that is thin and flexible and
serves as the opening and closing device of the spiracle. The wall of the tracheal atrium is in-
verted into the tracheal atrium by increased hemolymph pressure. When inverted, the medial
wall seals the opening between the subostial space and the tracheal trunks and closes the spi-
racle. The atrium wall is everted via contraction of a muscle that extends from the center of the
medial wall to the dorsal wall of the integument. Subsequent expansion of the tracheal atrium
(i.e., spiracle opening) draws air in, the wall of the tracheal atrium is inverted again, and the air
is pressed into the main tracheal trunks. The number of atrial muscles involved shows interge-
neric variation (Evans 1992). One atrial muscle is present in H. truncatum (Schöl et al. 1995)
and Haemaphysalis longicornis (Roshdy and Hefnawy 1973), whereas 2 are found in D. ander-
soni (Arthur 1960).
In addition to interspecific variations, sex-dependent differences exist in the spiracular
structure in adult ticks. Baker (1997) showed that in 5 species of ticks, females exhibited larger
surface areas of spiracular plates and a greater number of aeropyles than males. Schöl et al.
(1995) reported that in H. truncatum, the height of the tracheal atrium in males was 70 μm, as
opposed to 92 μm in females, and of the 9 to 12 tracheal trunks opening into the tracheal atrium,
the diameter in males was 93 μm, compared to 150 μm in females. The larger spiracular compo-
nents of female ticks might be related to the considerably greater metabolic demand and gas
exchange associated with feeding (Fielden et al. 1999) (see Section 3.3.1).
2.2. SPIRACULAR MORPHOLOGY IN THE ARGASIDAE

The spiracles of the argasidae have a simpler structure than those of the ixodids (Fig. 10.3).
Differences between the 2 families have been highlighted by Evans (1992), Sonenshine (1991),
Pugh et al. (1992a), and Coons and Alberti (1999). The spiracle is posterolateral to coxae III or
IV (Evans 1992). The spiracular plate is much smaller than in the Ixodidae and in a different
position behind the coxa because of differences in the idiosomal musculature between the 2
groups (Pugh 1997). There are smaller and fewer aeropyles, and they do not open into indi-
vidual air chambers that communicate via fenestrations. Instead the labyrinth has simple, co-
lumnar pedicels forming an interpedicellar space that is not divided into chambers (except in
Argas transverses, the Galapagos tick [Roshdy et al. 1992]). Unlike in the ixodids, the ostium is
open and can be sealed by the lip of the macula, which inflates as a result of hemolymph pres-
sure. During exhalation, the ostium is sealed by the macula, and expired air escapes via the
sieve plate. During inhalation, the ostium is open, and air enters via the ostial opening (Pugh
et al. 1991). The ostium opens into a subostial space, which in turn transitions into a large atrial
chamber from which the tracheal trunks arise. Gothe and Schöl (1992) describe an additional
chamber in Argus walkerae, the vestibulum, that is situated between the subostial and atrial
chamber and is important in ventilating the trachea. The roof of the vestibulum is flexible and
can invert or evert with changes in hemolymph pressure and muscular action. When the roof
is inverted, the volume of the vestibulum is either partially or completely confined, and air is
driven out. When the roof is everted via contraction of a muscle inserting at its center, the
vestibule expands and air is drawn into the atrial chamber. The atrial valve described in earlier
histological and scanning electron microscopy studies of argasid spiracles might be an artifact
FIGURE 10.3: The spiracular structure of Argasid ticks. A, Diagrammatic drawing of a longitudinal
fracture of an entire spiracle of adult Argus walkerae. Ac, atrial chamber; Ips, interpedicellar space; M,
macula; Mu, roof muscle; O, ostium; P, pedicels; Sos, subostial space; Sp, spiracular plate; V, vestibulum;
Ve, roof of vestibulum everted; Vi, roof of vestibulum inverted; T, tracheae. From Gothe, R. and Schöl, H.
(1992) Morphology and structural organization of spiracles in female Argas (Persicargas) walkerae
(Acari: Ixodidae). Exp. Appl. Acarol. 14:151–163, with permission from Springer. B, Spiracular plate of
Ornithodorous savignyi. M, macula; O, ostium; aeropyles not visible, as they are covered by the macula.
of specimen preparation and subsequent interpretation (see Gothe and Schöl 1992; Coons and
Alberti 1999).
2.3. SPIRACULAR MORPHOLOGY IN THE NUTTALLIELLIDAE

This family consists of 1 species, Nuttalliella namaqua, collected from Southern Africa, and is
remarkable in that it has features characteristic of both ixodid and argasid ticks (Sonenshine
1991). As such, the respiratory system shares features of both Ixodidae and Argasidae (Roshdy et
al. 1983). The spiracles are very small and posterolateral to coxae IV. The spiracular plate has a
small macula and a fenestrated area made up of pedicels enclosing the crescent-shaped ostium.
The ostium leads into the subostial space, which in turn opens into an atrial chamber. Five tra-
cheal trunks arise from the base of the chamber. As in argasids, the ostium is the functional
opening of the spiracle.
2.4. FUNCTIONS OF THE SPIRACULAR PLATE

Apart from its role in gas exchange, the spiracular plate has several functions in ticks, none of
which are mutually exclusive. These functions include (i) protecting the spiracular opening
from particulate debris, (ii) reducing respiratory water loss, and (iii) serving as a plastron (see
Section 3.3.3) (Hinton 1967, 1970; Woolley 1972; Fielden et al. 2011).
2.4.1. Air filter

The spiracular plate in ixodids and argasids, in which there are numerous small holes or aero-
pyles, reduces the chance of any particular debris entirely blocking the opening of the spiracle
(Hinton 1967, 1970).
2.4.2. Reduction of respiratory water loss

The complex morphology of the spiracular plate might be important in limiting respiratory
water loss from the tracheal system by reducing the diffusion gradient between the trachea and
the atmosphere (Pugh et al. 1988, 1990; Hadley 1994). Ixodes ricinus has a circular sieve plate
with many closely spaced aeropyles arranged in concentric rings. This dense geometric arrange-
ment of aeropyles causes a water-vapor-saturated cloud to form immediately above the spiracle.
This cloud acts as a water barrier to transpiration from the tracheal system. A different mecha-
nism is found in 3 other species of ticks, Aponomma latum, D. marginatus, and Rhipicephalus
sanguineus. Here, numerous pedicels with well-developed flanges in conjunction with aeropyles
that are smaller and more widely spaced than in I. ricinus (Figs. 10.2C, 10.2D) generate a high
internal resistance to transpiration within, rather than above, the spiracular plate.
2.4.3. Plastron respiration

The spiracular plate functions as a plastron, a structure that extracts oxygen from water and enables
the survival of ticks under water (Fielden et al. 2011). The role of the spiracle as a plastron is covered
in more detail in the discussion of methods of respiration and gas exchange patterns (Section 3.3.3).
2.5. TRACHEAL SYSTEM

The tracheal system of argasids and ixodids has been reviewed by Sonenshine (1991) and Coons
and Alberti (1999). Gas exchange occurs via internal air-filled trachea that branch to every single
tissue and organ throughout the body (Fig. 10.4). The origin of the tracheal “tree” is at the base
of the atrial chambers, where several tracheal trunks arise. The number of trunks varies; there
are 9 to 12 in H. truncatum (Schöl et al. 1995), 6 in D. andersoni (Arthur 1960), 5 in Argas wal-
kerae (Gothe and Schöl 1992), and 8 in Carios (Ornithodoros) kelleyi (Sonenshine 1970). Tra-
cheae are slender tubes consisting of a single layer of epithelial cells (cuticular intima) lined with
a cuticle comprising a thin epicuticle overlying a procuticle (Coons and Alberti 1999). The cu-
ticle forms distinctive folds, each of which is called a taenidium. These taenidia prevent the
collapse of the trachea and are responsible for the ringed appearance of the trachea (Sonenshine
1991; Coons and Alberti 1999). Tracheal cells that surround the cuticular intima are sometimes
FIGURE 10.4: The tracheal system of ixodid ticks. S, spiracles; Ttr, tracheal trunks. Imaged acquired
using a high-energy photon source. Use of the Advanced Photon Source, an Offi ce of Science User
Facility operated for the U.S. Department of Energy (DOE) Offi ce of Science by Argonne National
Laboratory, was supported by the U.S. DOE under Contract No. DE-AC02-06CH11357.
folded around the cell to form a structure termed a mestracheon (Coons and Alberti 1999). The
trachea branch into fine tubes called tracheoles. Gas exchange occurs in the terminal parts of
these tracheoles, which penetrate into tissues and cells via invagination (Gullan and Cranston
2010). Coons and Alberti (1999) distinguish between the trachea and tracheoles as follows: tra-
cheoles do not branch further, do not have taenidia, and do not have a mestracheon.
Unlike in larval ixodids, which rely solely on cutaneous diffusion, tracheal systems have
been described for several species of argasid larvae, namely, Argas, Carios, and Ornithodoros
(Theodor and Costa 1960; Roshdy et al. 1982). The larval system lacks the spiracular plates found
in the adults and nymphs and instead opens as minute apertures between coxae I and II. In
Argus arboreus, this aperture opens into a narrow vertical vestibule and then a wider atrial
chamber, both of which have minute spiny projections. These interlocking projections might
regulate the flow of air into the trachea and/or prevent particles from entering the trachea
(Roshdy et al. 1982). A single tracheal trunk with a spiral taenedial lining arises from the atrium
before ramifying into many branches. Evidence of any kind of active atrial ventilation of the
larval respiratory system is lacking (Roshdy et al. 1982).
2.5.1. Molting of the tracheal system

In ticks, the process of molting involves the formation of new cuticle and subsequent ecdysis, the
shedding of the old cuticle. The mechanisms of spiracular and tracheal molting in argasids and
ixodids have been differentiated by Pugh et al. (1992a) into simple and elateroid, respectively,
based on the fate of the ostial aperture after ecdysis. Argasids exhibit simple spiracular molting
because the spiracular and tracheal lining is withdrawn through the functional respiratory
opening (the ostium), which remains open after ecdysis. In Ixodidae, spiracular molting is called
“elateroid” because the tracheal lining is removed through an ecdysial tube (the ostium) that is
formed to one side of the functional opening of the respiratory system (the aeropyles) and is
sealed at the completion of ecdysis (Hinton 1967; Roshdy 1974).
3. RESPIRATION AND GAS EXCHANGE PATTERNS
3.1. GENERAL PRINCIPLES OF GAS EXCHANGE

IN THE ARTHROPOD TRACHEAL SYSTEM
The tracheal system is remarkably efficient for gas exchange (Maina 2002). Air enters through
the spiracle, and O2 moves within the tracheal system to the target tissues along a concentration
gradient from the high O2 concentration in incurrent air to the low concentrations in the re-
spiring tissues. CO2 is retained in the tissues and hemolymph, where it is buffered as bicarbonate
until the concentration is too high to be maintained, at which point the CO2 enters the tracheal
system and is eliminated through the spiracles. During the process of gas exchange, water vapor
also leaves the tracheal system through the spiracles. Thus, in most terrestrial arthropods, there
is a compromise between obtaining sufficient oxygen to meet the metabolic requirements and
reducing water loss.
3.2. DISCONTINUOUS GAS EXCHANGE CYCLE

Discontinuous gas exchange is found in insects (Miller 1981; Kestler 1985; Lighton 1994) and in
other tracheate arthropods, including spiders (Lighton and Fielden 1995) and ticks (Fielden
et al. 1994). The discontinuous gas exchange cycle (DGC) described for insects (Fig 10.5A) com-
prises 3 sequential stages: (i) the closed phase, during which the spiracles are closed and there is
negligible external gas exchange or respiratory water loss; (ii) the flutter phase, triggered by en-
dotracheal hypoxia, during which rapid fluttering of the spiracles allows diffusive and/or con-
vective ingress of O2 but little egress of CO2 or water vapor; and (iii) the burst phase or open
phase, in which hypercapnia (caused by the accumulation of CO2 from respiring tissue) triggers
the spiracles to open and results in the rapid release of CO2 and water vapor.
3.3. GAS EXCHANGE PATTERNS IN TICKS

3.3.1. Ixodidae
Unfed adult and nymphal stages of 3-host ixodid ticks display discontinuous gas exchange with
short and clearly defined bursts of CO2 release alternating with long interburst periods of low
CO2 emission (Fig 10.5B and Table 10.1) (Fielden et al. 1993, 1994; Lighton et al. 1993). A major
difference between the discontinuous ventilation cycles of ixodid ticks and those of insects is
that the diffusive loss of CO2 through the spiracles is very low in ticks and is barely distinguish-
able from the closed phase, when the spiracles are completely closed. This might in part reflect
the spiracular plate morphology, which reduces not only water diffusion gradients (Pugh et al.
1988, 1991) within the spiracles but also CO2 diffusion gradients. The DGC shows much inter-
specific variation among tick species with regard to the frequency of spiracular openings, which
varies from several bursts per hour to a single burst every 1 to 3 hours (Table 10.1). Reasons for
this interspecific variation have not been investigated, but on an intraspecific basis, the rate of
CO2 emission (μl CO2 h−1) modulates the frequency of the ventilation cycle; that is, higher rates
FIGURE 10.5: Representative recordings of the discontinuous release of CO2 from an insect and from
ixodid and argasid ticks. A, A typical insect discontinuous gas exchange cycle obtained from the ant
Camponotus maculatus. C, closed phase; F, flutter phase; B, burst or open phase. B, Representative
recording of ixodid tick (Rhipicephalus evertsi evertsi) CO2 emission from an unfed female. Note the
absence of clear closed and flutter phases and the changes in CO2 cycle frequency due to activity. C,
Representative recording of argasid tick (Ornithodorous savignyi) CO2 emission from an unfed female.
Note that all 3 phases of the discontinuous gas exchange cycle are present.
of CO2 emission result in the spiracles opening more frequently, as occurs during activity (Fig
10.5B), active water uptake, and feeding (Rechav and Fielden 1995; Fielden and Lighton 1996;
Fielden et al. 1999). Relative to the unfed stages, the feeding stages are far less conservative in
terms of energy and water use (Rechav and Fielden 1995; Fielden et al. 1999). On attaching to the
host, female D. variabilis have a period of slow feeding during the first 6 to 7 days, during which
Table 10.1: Differences in the discontinuous gas exchange cycle (DGC) characteristics of adult ticks from different species and
between unfed and engorged ticks of the same species (values are means, with standard deviations in parentheses). The first
column describes the DGC characteristics measured; units of measurement are in parentheses.
Species A. heba A. marmoreumb D. anc D. variabilisd O. save
STATE UNFED UNFED ENGORGED ENGORGED, UNFED UNFED ENGORGED UNFED

DIAPAUSE
Mass (mg) 31.3 (9.9) 70.2 (1.9) 3273 (679) 2601 (657) 8.25 (1.51) 5.78 (1.05) 541.15 (18.6) 293.8 (76.0)
N 12 5 12 5 16 4 4 3
VCO2 (μl h−1) 0.416 (0.269) 1.334 (0.228) 257.9 (79.9) 141.9 (74.6) 0.280 (0.138) 0.179 (0.03) 87.32 (5.72) 10.53 (6.07)
VCO2 (μl mg−1 h−1) 0.0136 (0.008) 0.019 (0.003) 0.0789 (0.017) 0.0558 (0.0334) 0.0279 (0.129) 0.033 (0.008) 0.162 (0.131) 0.037 (0.02)
Burst phase No DGC No DGC
250
Duration (min) 5.16 (1.03) 6.78 (1.06) - - 3.95 (0.57) 3.69 (0.84) - 30.86 (8.18)
VCO2 (μl g−1) 26.2 (10.4) 50.2 (1.5) - - 55.7 (20.0) 14.2 (4.0) - 38.07 (8.50)
Interburst phase
Duration (min) 95.13 114.8 - - 100.02 (43.75) 29.25 (10.26) - C: 6.2 F:79.2
VCO2 (μl g−1) ** 5.5 (1.7) - - ** ** - C: ** F:14.3
Burst frequency (h−1) 0.74 (0.26) 0.49 (0.26) - - 0.70 1.99 (0.61) - 0.63 (0.3)
Notes: N, number of ticks; VCO2, rate of CO2 emission; A. Heb, Amblyomma hebraeum; D. an, Dermancentor andersoni; O. sav, Ornithodoros savignyi; C, closed phase; F, flutter phase.
a
From Fielden, L.J., Duncan, F.D., Rechav, Y., and Crewe, R.M. (1994) Respiratory gas exchange in the tick Amblyomma hebraeum (Acari: Ixodidae). J. Med. Entomol. 31:30–35.
b
From Lighton, J.R.B., Fielden, L.J., and Rechav, Y. (1993) Characterization of discontinuous ventilation in a non-insect, the tick Amblyomma marmoreum. J. Exp. Biol. 180:229–245.
c
From Fielden, L.J. and Lighton, J.R.B. (1996) Effects of water stress and relative humidity on ventilation in the tick Dermacentor andersoni (Acari: Ixodidae). Physiol. Zool. 69:599–617.
d
From Fielden, L.J., Jones, R.M., Goldberg, M., and Rechav, Y. (1999) Feeding and respiratory gas exchange in the American dog tick, Dermacentor variabilis. J. Insect Physiol. 45:297–304.
e
Unpublished data.
**
CO2 emission indistinguishable from baseline levels.
FIGURE 10.6: Representative recordings of CO2 emission for unfed (0 days, 6.5 mg), early (day 1, 6.1 mg),
slow (day 3, 7.6 mg), slow (day 6, 33.2 mg), and rapid (day 9, 565.1 mg) feeding stages of 5 individual
female Dermacentor variabilis. For days 0–6, CO2 release is discontinuous; for day 9, CO2 release is
continuous. From Fielden, L.J., Jones, R.M., Goldberg, M., and Rechav, Y. (1999) Feeding and respiratory
gas exchange in the American dog tick, Dermacentor variabilis. J. Insect Physiol. 45:297–304, with
permission from Elsevier.
251
there is a gradual increase in mass, metabolic expenditure, and frequency of the DGC (Fig. 10.6).
The period of rapid engorgement, when large quantities of blood are imbibed prior to detach-
ment, is characterized by a large increase in mass and metabolic expenditure accompanied by a
change from discontinuous to continuous gas exchange (Fig 10.6). This change in ventilation
pattern during feeding results from the increase in metabolic expenditure and consequent CO2
generation. An increased frequency of the spiracular opening with eventual continuous CO2
release results from an increase in CO2 concentration in the hemolymph in engorged females.
3.3.2. Argasidae
Studies on gas exchange in the argasids are more limited than in the ixodids and have focused
primarily on the effect of starvation, as this group is well known for its impressive longevity
(Belozerov 1966; Phillips et al. 1995). Results are conflicting. For example, respiratory rates in
starved adult female Ornithodoros papilles were higher than in engorged females (Belozerov
1966). However, rates in starved adult O. turicata were significantly lower than in fed cohorts,
although the reverse was true for immature O. turicata (Phillips et al. 1995). The higher meta-
bolic rates in immature unfed O. turicata might reflect increased host-seeking activity, whereas
the adults reduce their metabolic rate during starvation.
The DGC in argasids was first reported for unfed adult O. turicata, although the closed mi-
crorespirometry technique used did not allow the precise quantification of DGC frequency or
the duration of the burst phase (Phillips et al. 1995). Flow-through respirometry has since dem-
onstrated that unfed argasid ticks (i.e., O. savignyi) show DGC in the nymphal and adult stages.
The argasids, in contrast to ixodids, show a clear distinction between the closed and flutter
phases and a longer burst phase (Fig 10.5C), yet the DGC frequency is similar (Table 10.1). The
flutter phase is important for reducing respiratory water loss, as the outward movement of CO2
and water is inhibited by the inward convective movement of air, which replenishes the O2
supply without the spiracles opening fully (Kestler 1985). An increased flutter phase length en-
ables the tick to conserve water until the CO2 concentration is sufficiently high to cause the
spiracles to open fully and the CO2 to rapidly diffuse from the tick as a result of the increased
concentration gradient. Differences in the spiracle structure might account for the more evident
flutter phase in the argasid tick. The passing of CO2 through the atrial chambers in ixodid ticks
might slow the CO2 release so that it is not evident during the flutter phase. In argasid ticks, the
air chambers in the sieve plate are less complex, so CO2 passes through quickly in order to be
visible during the flutter phase, rather than retained and released during the burst phase.
3.3.3. Plastron respiration

One remarkable aspect of off-host tick survival is the ability to survive immersion in water for
long periods, which could be important after heavy rainfall or temporary flooding. Smith (1973)
reported survival times of several weeks to months in water for unfed larvae and nymphs of the
African ixodid species R. appendiculatus and A. variegatum, and Fielden et al. (2011) reported a
survival time of 15 days for unfed adult D. variabilis (Fig 10.7A). Underwater survival in ticks
appears to be facilitated by plastron respiration. A plastron is a thin film of air held by water-re-
pellent cuticular hairs or projections that can extract oxygen from the water, which then diffuses
into the internal respiratory system of the organism. Plastron respiration is seen in numerous
FIGURE 10.7: Underwater survival and plastron respiration in Dermacentor variabilis. A, Survivorship for
female Dermacentor variabilis submerged in normoxic (normal oxygen level) and hypoxic (oxygen
content depleted via nitrogen infusion) water. Results presented as mean survival per day, with the
uncertainty in survival represented as ±1 S.E. (n = 25 to 45 ticks per treatment). The total numbers of
ticks submerged were 671 for normoxic water and 344 for hypoxic water. B, Wetting of the spiracular
plate. Lower ventral side of a submerged female Dermacentor variabilis showing the spiracular plate
behind the last pair of legs. Note the characteristic darkening of the spiracular plate wetted with alcohol
(Sw) in contrast to an untreated spiracle with a silver sheen (Sd). From Fielden, L.J., Knolhoff, L.M.,
Villarreal, S.M., and Ryan, P. (2011) Underwater survival in the dog tick Dermacentor variabilis (Acari:
Ixodidae). J. Insect Physiol. 57:21–26, with permission from Elsevier.
arthropods (for review, see Withers 1992; Hoback and Stanley 2001). Fielden et al. (2011) pro-
posed that in ixodids, the spiracular plate functions as a plastron, with the aeropyles and sur-
rounding cuticle being hydrophobic and preventing the entry of water into the air chambers.
When the tick is submerged, an air–water interface forms across the aeropyles, and oxygen from
the water diffuses into the air chambers below and then into the tracheal system via the atrial
chambers (as a result of an oxygen gradient established by metabolizing tick tissues). The role of
the spiracular plate as a plastron was verified by “wetting” the spiracles (Fig. 10.7B) with alcohol.
The darkening of the perforated plate was a clear indication of wetting (i.e., destruction of the
hydrophobic properties of the plate). The resulting expulsion of air from the cuticular labyrinth
and associated airspaces underneath the plate incapacitated the plastron function. Ticks in
which 1 or both spiracular plates were wetted with alcohol showed significantly decreased sur-
vival relative to those with both spiracles intact (Fielden et al. 2011).
3.4. GAS EXCHANGE PATTERNS, METABOLIC RATE,

AND OFF-HOST SURVIVAL
Mechanisms that contribute to the maintenance of water and energy balance in ticks during
periods of starvation between blood meals have been reviewed by Needham and Teel (1986,
1991) and Randolph (2009). Metabolic rates are unusually low in the unfed stages of both ixodid
and argasid ticks (Belozerov 1964, 1966; Belozerov and Rechav 1993; Fielden et al. 1993, 1994;
Phillips et al. 1995). Indeed, ticks have a metabolic rate that is only 12% of that predicted on the
basis of body mass relative to other terrestrial arthropods (Lighton and Fielden 1995). A defini-
tive selection mechanism for the evolution of discontinuous gas exchange in arthropods is not
evident (for reviews, see Chown et al. 2006; Quinlan and Gibbs 2006), but the hygric hypothesis
suggests that it might have developed as a way to reduce respiratory water loss. In arthropods
such as ticks that have very low cuticle permeabilities (Hadley 1994), any reduction in respira-
tory water loss afforded by discontinuous gas exchange would help in the conservation of water
and be of great benefit during long periods off the host (Jaworski et al. 1984; Needham and Teel
1986, 1991). In several tick species, large increases in water loss (up to 17-fold) resulted when the
spiracles were forced to remain open by exposure to high CO2 concentrations. Blocking the
spiracles prevented this loss (Mellanby 1935; Hefnawy 1970; Rudolph and Knülle 1979).
4. FUTURE DIRECTIONS: METABOLIC DEPRESSION,

ANOXIA TOLERANCE, AND ANAEROBIC RESPIRATION
Evidence of hypoxia and anoxia tolerance in ticks is provided so far by accounts of their ability
to survive underwater in both oxygenated and deoxygenated water (Smith 1973; Fielden et al.
2011) (Fig 10.7A). Plastron respiration is important for prolonging survival under water, but it
appears that there is also some degree of metabolic depression in the ticks, depending on how
long they have been submerged or whether they were submerged in normoxic or hypoxic water.
This metabolic depression induced by submergence begs the question of whether ticks rely to
any extent on anaerobic metabolism or simply on the cessation of aerobic metabolism in order
to survive long periods of submergence. Metabolic depression and anaerobic respiration in ticks
warrant future investigation as means of prolonging off-host survival.
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NOTE
C H A P T E R 1 1
CIRCULATORY SYSTEM
AND HEMOLYMPH
Structure, Physiology, and Molecular Biology
LIBOR GRUBHOFFER , NATALIIA RUDENKO , MARIE VANCOVA ,

MARYNA GOLOVCHENKO , AND JAN STERBA
1. INTRODUCTION
Tick hemolymph is the primary means of nutrient and hormone transport, maintains os-
motic pressure, and fights injuries and pathogens with the help of hemocytes while allowing
the dissemination of successfully surviving pathogens. One would anticipate a tremendous
amount of available information about this system; however, there is still a lot of informa-
tion missing. This chapter tries to compile the most important knowledge on the mor-
phology of the circulatory system and its components, as well as on their composition and
function.
2. MORPHOLOGY AND ULTRASTRUCTURE OF THE

TICK CIRCULATORY SYSTEM
The morphology of the tick circulatory system (CS) of Argasidae and Ixodidae was de-
scribed in detail by Obenchain and Oliver (1976), and a comprehensive summary was given
by Sonenshine (1991).
The CS is composed of the heart suspended by muscles in the dorsal anterior region of the
body, the aorta, periganglionic and anterior sinuses, and arterial vessels (Figs. 11.1–11.6). Arteries
Circulatory System and Hemolymph 259
FIGURE 11.1: Schematic diagram of the circulatory system in ixodid ticks. From the pericardial sinus,
hemolymph enters the pulsatile part of the heart through paired ostia. The heart pumps the
hemolymph into the aorta and periganglionic sinus. The hemolymph is then directed by arterial
sinuses and arteries into other parts of tick body. A, aorta; Aomc, aortic myocardial cone; As, anterior
sinus; Dlsm, dorsolateral suspensory muscles; Hpp, pulsatile part of the heart; Lso, lateral segmental
organ; O, ostia; Pa 1–4, pedal arteries; Pcsp, pericardial septum; Pgs, periganglionic sinus; Syn,
synganglion; Vlsm, ventrolateral suspensory muscles. From Binnington, K.C. and Obenchain, F.D.
(1982) Structure and function of the circulatory, nervous, and neuroendocrine systems of ticks. In F.D.
Obenchain and R. Galun (Eds.), Physiology of Ticks. Oxford, UK: Pergamon Press, 277–350, with
permission from the author.
facilitate the directional transport of the hemolymph into different hemocoele spaces. The CS is
tightly connected with the synganglion, the central nervous system of the tick.
The morphology of the CSs of Argasidae and Ixodidae differs in several details, such as the
shape of the heart in diastole, the extent of tracheation, and the presence of an endosternum in
Argasidae. The dorsal anterior sinus is better developed in Ixodidae than in Argasidae (Oben-
chain and Oliver 1976).
FIGURE 11.2: Scanning electron micrograph illustrating the morphology of the circulatory system of the
Ixodes ricinus male tick. The circulatory system is located in the middle of the dorsal side of the tick
body. The main components of the circulatory system are the heart (H), the aorta (A), periganglionic
(Pgs) and arterial sinuses, and pedal vessels (Pa). The aorta directs hemolymph from the heart into the
periganglionic sinus that encloses the synganglion (Syn). Extensions of the sinus form pedal arteries.
Sg, salivary glands; Md, midgut diverticulum. Measurement bar: 100 μm.
2.1. STRUCTURE (AND FUNCTION) OF THE HEART

The principal organ of the CS, the heart, is localized in the space between the dorsal cuticle and
the midgut region (Fig. 11.2). It is anteriorly bordered by cheliceral retractor muscles and is pos-
teriorly surrounded by secretory foveal gland tissue in metastriate ticks (Obenchain and Oliver
1976). The heart is fastened into the dorsolateral (dlsm) and ventrolateral (vlsm) suspensory
muscle apparatus (Figs. 11.4, 11.6) and is surrounded by an extensive system of tracheae and
tracheolae.
The vlsm forms long branches that are connected to the ventral side of the heart (Fig. 11.6).
These muscles were found to be associated with nerve endings that provide motor innervation.
The dlsm is arranged in groups and probably determines the shape of the heart during the dias-
tole phase of the cardiac cycle, when the heart shape is pentagonal (Ixodidae) (Fig. 11.4) or
subtriangular (Argasidae). The dlsm and vlsm are highly branched in the proximity of the peri-
cardial septum (Fig. 11.4) and are associated with the connective tissue of the septum, which
contains numerous perforations through which the hemolymph flows. The septum creates a
connection between the heart and the dorsal part of the tick cavity. Connective tissues of the
septum function as a support membrane for pericardial cells (Figs. 11.4–11.5) (Obenchain and
Oliver 1976).
The heart is a dorsoventrally flattened sac-like structure that is composed of several layers.
Its outer sheet, the pericardial septum, consists of a layer of mesenchymal cells interwoven
densely with trachea (Fig. 11.7) (Obenchain and Oliver 1976). The layer is a continuation of the
Ixodes ricinus male tick. The median notch (white arrow) on the dorsal surface of the midgut diverticula
(Md) accommodates the aorta (missing here). The black arrow points out the entrance of the aorta to the
periganglionic sinus (Pgs). Measurement bar: 10 μm.
layer of the surrounding the synganglion (periganglionic sinus). The myocardium, an important
inner layer of the heart, is composed of striated muscle. Based on the orientation of the muscle
fibers, the heart is divided into 2 regions that differ in function: (i) the aortic myocardial cone is
formed from the longitudinal striated fibers in the anterior part of the heart, and (ii) the circu-
larly oriented muscles form a pulsatile part in the posterior region of the heart (Fig. 11.7)
(Binnington and Obenchain 1982). In the posterior part of the heart, the muscles are perforated
ventrolaterally by 2 pairs of openings, the ostia (according to Balashov [1972], there is only 1 pair
in the case of Argas persicus). Ostia allow the hemolymph to flow from the pericardial sinus to
the inside of the heart. The ostia are formed with muscular lips whose contraction during the
systolic phase leads to closing of the ostia and movement of the hemolymph forward to the ad-
jacent cardiac compartments (Obenchain and Oliver 1976).
FIGURE 11.4: Scanning electron micrograph of the dorsal myocardium (M), the pericardial septum
(Pcsp), and associated dorsolateral suspensory muscles (Dlsm) in an Ixodes ricinus male tick.
Measurement bar: 10 μm.
FIGURE 11.5: Scanning electron micrograph showing a pericardial cell attached to the pericardial
septum in an Ixodes ricinus male tick (enlarged view of Fig. 11.4). Measurement bar: 1 μm.
262
Ixodes ricinus male tick. Ventral view of the heart; branches of ventrolateral suspensory muscles (Vlsm)
attach to the pericardial septum (Pcsp). Pc, pericardial cells. Measurement bar: 10 μm.
Ixodes ricinus male tick. Ventral view of the inside of a damaged pericardial sinus (Pcsi). M, myocardium;
Pcsp, pericardial septum. Measurement bar: 10 μm.
2.2. STRUCTURE AND FUNCTION OF ARTERIAL VESSELS AND SINUSES

The main arterial vessel, the aorta, arises from the anterior part of the heart and opens into the
periganglionic sinus surrounding the synganglion. This arrangement enables sufficient perfu-
sion of the synganglion by hemolymph. The aorta is located below the cheliceral retractor mus-
cles in a median notch of the dorsal surface of the midgut (Fig. 11.3). The aortic valve lies between
the aortic-myocardial cone and the aorta.
The inner layer of the aorta is formed by longitudinal muscle fibers. The organization of the
muscle fibers differs from that in the pulsatile part of the heart. The aorta is covered with an
outer sheet composed of 1 layer of membrane-bound mesenchymal cells. Similar structures were
shown to form walls of pericardial, periganglionic, and anterior sinuses and pedal arteries. In
contrast to the aorta, the sinus wall does not contain muscle fibers (Obenchain and Oliver 1976).
A ventral extension of the periganglionic sinus surrounds the pedal nerve trunks and thus
encloses the spaces that function as pedal arteries. Four pedal arteries at each side of the tick
body direct hemolymph to the legs (Figs. 11.1, 11.2). Suspensory extrinsic muscles are associated
with the periganglionic sinus and pedal arteries and influence the arterial pressure. The perigan-
glionic sinus continues anteriorly and forms the ventral and dorsal anterior sinuses. Palpal and
stomodeal nerves are present in the center of the ventral (periesophageal) anterior sinus. The
dorsal anterior sinus surrounds the cheliceral and optic nerves and leads directly to the mouth-
part region. Increased hemolymph pressure in the dorsal anterior sinus allows the movement of
chelicerae and the hypostome during feeding (Obenchain and Oliver 1976).
3. HEMOLYMPH COMPOSITION AND FUNCTION
Tick hemolymph is a complex fluid composed of plasma (usually clear to brown in color) and
hemocytes (circulating cells).
Hemolymph surrounds all internal tissues and organs and serves as an exchange medium
for the transport of nutrient molecules, hormones, and products of cellular metabolism. Local
changes in hemolymph pressure are important in the ventilation of the tracheal system, in ther-
moregulation, and in molting (Gullan and Cranston 2010). It is involved in protection from
physical injury (e.g., the wound-healing process that involves hemocytes and plasma coagula-
tion) and pathogen invasion (the immune response). The amount of hemolymph in arthropods
varies among species and depends on the developmental and feeding stage. A total hemolymph
volume calculated for the land crab Cardisoma guanhumi was assumed to be 30% of its body
weight (Burggren et al. 1990). The volume of hemolymph in soft-bodied mosquito larvae ranges
from 20% to 40% of their body weight (Gullan and Cranston 2010). In ixodid ticks, the hemo-
lymph volume accounts for about 20% to 30% of the body weight (Kaufman and Phillips 1973).
Compared to vertebrate blood, tick hemolymph contains relatively high concentrations of
proteins, lipids, carbohydrates, amino acids, pigments, organic acids, and inorganic ions, which
make the major contribution to the total hemolymph osmolality (Araman 1979). Hemolymph
composition can be altered after the consumption of a blood meal or through the influence of
temperature changes or the presence of pathogens (Mullins 1985). Recently, mRNA-based and
proteomics approaches have been employed to reveal differentially expressed proteins in the
hemolymph of challenged ticks, allowing the investigation of tick innate immunity at the protein
level (Stopforth et al. 2010).
3.1. PLASMA PROTEINS IN TICKS

Plasma composes approximately 90% of tick hemolymph, and proteins constitute its major sol-
uble component (11.5%–14.3% by weight) (Gudderra et al. 2002). In spite of the importance of
ticks as disease vectors, our knowledge about tick hemolymph proteins is limited relative to sim-
ilar knowledge about other arthropods, particularly insects (Gudderra et al. 2002). For example,
a two-dimensional map of 160 hemolymph proteins of Drosophila melanogaster has been con-
structed for investigation of the changes that occur at the protein level in different developmental
stages, in different physiological conditions, or after infection (Vierstraete et al. 2003). In addi-
tion to enzymes and hormones, hemolymph proteins associated with tick immunity represent
a variety of proteins secreted into the plasma in response to physical injury, blood feeding, or
invasion of the tick body by pathogenic or nonpathogenic microorganisms. They are termed
“humoral factors” and represent a wide variety of antimicrobial peptides (AMPs), lysozymes,
defensins, histidine-rich peptides, coagulation factors, lectins, non-cationic peptides, alpha-
macroglobulin-like glycoproteins, proteases and their inhibitors, and other recognition mole-
cules (reviewed in Taylor 2006; Sonenshine and Hynes 2008).
3.1.1. Vitellogenins and other heme-lipoproteins

Vitellogenin, the precursor of vitellin, represents one of the major proteins in females at the time
of egg development (11% of the total hemolymph protein) (Thompson et al. 2007). Vitellogenin is
a hemelipoglycocarrier protein synthesized by the fat body (FB), gut cells, and, to a lesser extent,
ovaria of tick females (in some species) after mating and feeding. It is secreted into the hemo-
lymph and then is transported and incorporated into eggs as vitellin. The heme in vitellogenins is
derived from the digestion of host hemoglobin (Gudderra et al. 2002). Vitellogenins have been
studied and partially characterized in Ornithodoros moubata (Chinzei 1983; Chinzei and Yano
1985), O. parkeri (Taylor et al. 1991), Hyalomma dromedarii (Schriefer 1991), Dermacentor variabilis
(Thompson et al. 2007; Khalil et al. 2011), Ixodes scapularis (James and Oliver 1997), Haemaphysa-
lis longicornis (Boldbaatar et al. 2010), and Rhipicephalus microplus (Granjeno-Colin et al. 2008).
Other major heme-binding proteins are the carrier proteins (CPs) and hemelipoglycopro-
teins (HLGPs).
In contrast to vitellogenin, CPs are found in each developmental stage of female and male
ticks. These proteins were detected and named DvCP or HLGP in D. variabilis (Guddera et al.
2001; Donohue et al. 2008), HeLp in R. microplus (RmCP) (Maya-Monteiro et al. 2004), and
HLGP in D. marginatus (Dupejova et al. 2011).
CPs are high-molecular-weight dimeric proteins (approximately 340 to 400 kDa) that contain
3 motifs found in tick vitellogenins: (i) a lipoprotein N-terminal domain, (ii) a domain of unknown
function, and (iii) a C-terminal von Willebrand factor type D domain (Donohue et al. 2008).
Further information on vitellogenins, vitellin, and CPs can be found in Chapter 15.
3.1.2. Defensins and other antimicrobial peptides

The key element of innate immunity is the speed with which defense responses occur. One of the
facets of tick defense is the rapid and transient synthesis of a battery of potent AMPs following
infection or trauma. AMPs and other immune-related proteins are secreted proteins synthesized
within the FBs or hemocytes. The activity spectrum of the immune peptides is diverse. The most
studied and representative group of tick AMPs are the defensins, cationic peptides with 6 cyste-
ines that have been identified in the hemolymph of many insects and other invertebrates.
The defensins form the first line of host defense against pathogens. They are classified into 3
major groups: (i) peptides with an α-helical conformation (cecropins, magainins, etc.), (ii) cy-
clic and open-cyclic peptides with pairs of cysteine residues (defensins, protegrins, etc.), and
(iii) peptides with an overrepresentation of some amino acids (proline rich, histidine rich, etc.).
Despite extreme diversity in their primary and secondary structures, all natural AMPs are active
against gram-positive or gram-negative bacteria, fungi, yeast, viruses, and protozoa.
Arthropod defensins were first isolated from cultured cells of the flesh fly, Sarcophaga pere-
grine, by Matsuyama and Natori (1988). All invertebrate defensins have the same cysteine pair-
ings, Cys1-Cys4, Cys2-Cys5, and Cys3-Cys6, that stabilize the molecule and maintain the tertiary
structure, known as the “defensin fold” (Ganz 2003). Defensins are small peptides approximately
4 kDa in size. Together with signal peptide and prepro regions, which are separated by an “RVRR”
sequence from the mature region, defensins are about 67 to 92 amino acids long. Mature peptides
usually range from 36 to 46 amino acids in length (Bulet et al. 2004) and are highly conserved. To
date, tick defensins have been identified in nearly 20 hard and soft tick species (Rudenko et al.
2005, 2007; Sonenshine and Hynes 2008; Saito et al. 2009; Chrudimska et al. 2010).
Varisin, the major hemolymph defensin in D. variabilis, was initially identified in tick hemocytes
(Johns et al. 2001). Being active against gram-positive bacteria, varisin is active against B. burgdorferi.
In combination with chicken lysozymes, it killed 65% of the borrelia spirochetes in 1 hour, indicating
a possible synergism between varisin and lysozymes (Johns et al. 2001; Sonenshine et al. 2002).
The role of varisin in the innate immunity of D. variabilis was confirmed by RNAi. The ac-
tivity of varisin knock-down tick hemolymph against the gram-positive bacterium Microccocus
luteus was reduced by 50%. This fact suggests that varisin is important, but not vital, for the an-
timicrobial activity of D. variabilis hemolymph (Hynes et al. 2008). An unexpected effect of va-
risin silencing was the reduced infection of D. variabilis with the rickettsial pathogen Anaplasma
marginale (Kocan et al. 2008).
A second defensin, defensin 2, was recently isolated from D. variabilis midgut. It appears to
be phylogenetically distinct from most of other tick defensins. Its role in innate immunity is
unclear (Ceraul et al. 2007).
Two defensin isoforms, def1 and def2, were identified from another hard tick, I. ricinus
(Rudenko et al. 2005, 2007; Chrudimska et al. 2010). Both def1 and def2 inhibit the growth of
gram-positive bacteria (M. luteus, B. subtilis, S. aureus) in very low concentrations (minimum
inhibitory concentration [MIC] of 0.37–50 μM) but show no activity against borrelia. The defen-
sin isoforms differ by only 1 amino acid in their mature peptide sequence, but the antimicrobial
activity of the def2 isoform (found in each tick tissue) is at least twice as strong as that of def1
(midgut specific) (Chrudimska et al. 2010). Alignment of the mature region showed similarities
to defensins from other species of hard ticks ranging from 77% for I. scapularis to 56% for A.
hebraeum. The similarity to the 4 already described defensins (omdef-A to omdef-D) from the
soft tick O. moubata was 61% to 63% in the mature peptide. Analysis of the genomic structure of
I. ricinus defensin genes showed the presence of 3 exons and 2 introns; this was the first evidence
of an intron/exon defensin structure in the hard tick (Rudenko et al. 2007).
The partial sequence of a third I. ricinus defensin, def 3 (AAT46066), was described as well.
It contains 4 of the 6 essential Cys residues and is clearly different from def1 and def2 at both
nucleotide and amino acid levels. The nucleotide and deduced amino acid sequences of this
partial defensin show greater similarity to defensins from D. variabilis and H. longicornis than to
those from I. ricinus (Rego 2005).
A sequence matching the tick defensins was found in the salivary glands of I. scapularis (Va-
lenzuela et al. 2002). Later, a novel defensin, scapularisin, was identified in the midgut, hemocytes,
and FBs of I. scapularis (Hynes et al. 2005). Its mature peptide showed 78.9% similarity to the
mature hemolymph defensin from D. variabilis. The role of scapularisin as an AMP is unclear, as
it does not seem to contribute to the tick immune defense. B. burgdorferi incubated with I. scapu-
laris hemolymph remained viable and active. The finding that I. scapularis has a defensin that does
not control B. burgdorferi suggests that other unknown factors are important in the antimicrobial
defense in this tick (Hynes et al. 2005).
A sequence similar to scapularisin was found in the whole body cDNA library of I. persulca-
tus. Its expression was induced by blood feeding in all tick developmental stages. It is unknown
whether I. persulcatus defensin is secreted into hemolymph. Commercially synthesized putative
mature I. persulcatus defensin markedly inhibited the growth of gram-positive bacteria, inclu-
ding Staphylococcus aureus (MIC = 0.8 μM), Bacillus subtilis (MIC = 0.5 μM), and Corynebacte-
rium rendle (MIC = 0.3 μM), but not of gram-negative bacteria, with the exception of Escherichia
coli O157 (Saito et al. 2009).
Genes encoding 2 non-cationic defensin-like peptides were isolated from a differentially ex-
pressed cDNA library of A. hebraeum synganglia. In addition, an Amblyomma defensin peptide-2
was purified from the hemolymph of blood-fed tick females and showed antimicrobial activity
against E. coli and S. aureus, with MICs of 30 and 7.5 μM, respectively. No antifungal activity was
found (Lai et al. 2004a). Amblyomma defensin peptide-2 has a predicted net negative charge, a unique
feature among all other known defensin-like peptides. The mechanism of interaction between Am-
blyomma defensin 2 and bacteria is unclear (Lai et al. 2004a). One of the possibilities is the employ-
ment of an alternative mechanism, such as the stimulation of autolytic enzymes, the inhibition of
DNA synthesis, and interference with bacterial DNA and/or protein synthesis (Wu et al. 1999).
Recently, a defensin (americin) transcript was found in the hemocytes, midgut, FBs, and sali-
vary glands (SGs) of A. americanum (Todd et al. 2007). The mature americin peptide showed 44.7%
and 42.1% similarity to anionic Amblyomma defensin peptide-1 and peptide-2, respectively. But
deeper analysis of sequential similarities and tissue expression patterns revealed that americin is
much more closely related to the D. variabilis (varisin) and I. scapularis (scapularisin) defensins than
to defensins of A. hebraeum. Similar tissue distributions of defensins in non-competent and compe-
tent vectors indicate that anti-Borrelia activity in non-competent vectors may be triggered by either
post-transcriptional regulation of defensin or the activity of a different set of proteins (Ceraul 2005).
Defensin also is expressed in the hemocytes and FBs of R. microplus (Fogaca et al. 2004).
Containing 6 conserved cysteines, a 4.29 kDa peptide shows high similarity to the insect defen-
sin family members. The tissue expression pattern of R. microplus defensin is closest to that of
the Ornithodoros moubata defensin D isoform (Nakajima et al. 2003b).
Defensins have been identified in the soft tick O. moubata, in which 4 different isoforms
(A, B, C, and D) have been characterized (Nakajima et al. 2001, 2002). The 4 isoforms appear to be
divided into 2 types: 1 type shows primary expression in the midgut (A, B, and C), and the other
is primarily expressed in the FBs (D). Gene expression of defensin A, B, or C in the FBs is weak
relative to expression in the midgut. In contrast, defensin D gene expression in the FBs is stronger
than in the midgut. It seems that the 2 types of Ornithodoros defensins are involved at different
ratios in the immunity of the hemolymph and the midgut (Nakajima et al. 2003b). Analysis of the
active functions of synthetic Ornithodoros defensin showed the presence of bactericidal and anti-
bacterial activity against many gram-positive bacteria, but not against gram-negative bacteria,
and low hemolytic activity characteristic of invertebrate defensins (Nakajima et al. 2003a).
In addition to classical defensins, a large number of other peptides with antimicrobial ac-
tivity have been identified in arthropods.
Microplusin, a 10.2 Da polypeptide with 6 conservative cysteine residues, was isolated from
the hemolymph of R. microplus (Fogaca et al. 2004). It belongs to a new type of AMP family with
multiple histidines and a novel secondary structure composed of 4 to 6 α-helices. The recent
functional analysis showed that microplusin chelates copper ions by binding them, likely to
histidines. Microplusin was active against gram-positive bacteria, filamentous fungi like Asper-
gillus niger, and yeast C. neoformans, but not C. albicans. The detected bacteriostatic effect of
microplusin against Microccocus luteus was probably caused by its ability to bind copper ions,
which are vital for bacterial respiration (Silva et al. 2009). Almost simultaneously, another histi-
dine-rich AMP, hebraein, was isolated from the hemolymph of fed females of A. hebraeum. This
11 kDa anionic protein has 6 cysteine residues, an all-α-helical structure, and a histidine-rich
carboxyl-terminal region. Hebraein has the same cysteine motif as microplusin, and they are
62% identical and 73% similar. Both proteins possess the same antimicrobial pattern. Antifungal
activity was greatly reduced in a histidine-deficient mutant, indicating that the histidine resi-
dues are important for antifungal activity (Lai et al. 2004b).
Further information on AMPs can be found in Chapter 5 of Volume 2.
3.1.3. Lysozymes
The fact that some other antimicrobial activities are present in tick hemolymph was confirmed
by multiple studies. RNAi studies of D. variabilis defensin revealed that after its inactivation, tick
hemolymph still possessed significant activity against M. luteus. The most probable candidate in
tick hemolymph capable of inhibiting the growth of M. luteus was presumed to be lysozyme(s)
expressed by hemocytes (Simser et al. 2004a). The synergetic role of lysozymes in enhancing
varisin antimicrobial activity supported this assumption (Johns et al. 2001).
Lysozymes are ubiquitously expressed enzymes that lyse bacteria by cleaving the β-1,4 glyco-
sidic bonds between N-acetyl muramic acid and N-acetyl glucosamine in the peptidoglycan
layer of the bacterial cell walls. Ticks utilize host proteins as a defensive tool against invasive
pathogens. For example, rabbit lysozyme has been purified from the hemolymph of Ornithodo-
ros ticks (Hetru et al. 1997). Two hemolymph factors of D. variabilis showed activity against B.
subtilis (Johns et al. 1998). One of these factors, a 14.5 kDa peptide, was identified as a lysozyme.
Reports characterizing lysozymes in ticks have been based on circumstantial evidence such as
the lysozyme-like immunoreactivity of hemocytes of I. ricinus (Kuhn and Haug 1994). The out-
comes of these reports revealed that purified lysates from various tick species display lysozyme-
like activities in response to bacterial challenges (Johns et al. 1998). The lysozyme lytic activity
of hemolymph, gut contents, and homogenates from the soft ticks Alveonasus lahorensis and O.
papillipes was tested against that of M. lysodeikticus. Lysozyme activity increased 8- to 10-fold
only in ticks challenged with different bacteria (Podboronov and Berdyev 1991; for an English
review, see Podboronov 1991).
Recently, a c-type lysozyme from H. longicornis (HlLysozyme) was characterized (Tanaka
et al. 2010). HlLysozyme was detected in all tick developmental stages. Gene expression of Hl-
Lysozyme was up-regulated in the FBs, midgut, ovaries, and hemolymph of females after bacte-
rial challenge, suggesting a possible role in the tick immune response to bacterial invasion. The
facts presented support the theory that tick lysozyme is an enzyme with both immune and di-
gestive characteristics that plays an important role in the innate immune responses of both soft
and hard ticks (Kopacek et al. 1999; Grunclova et al. 2003).
Comparative analyses of the highly conserved lytic peptides from D. variabilis, O. moubata,
and H. longicornis identified them all as c-type lysozymes that share conserved common structural
features, namely, 8 cysteine residues that form 4 disulfide bridges and conserved catalytic sites
comprising glutamic acid and asparatic acid residues (Tanaka et al. 2010).
3.1.4. Lectins
Lectins are proteins capable of recognizing carbohydrates and are known to function as pattern-
recognition molecules in invertebrate immune mechanisms or as mediators of biological ac-
tivity in cells (Vasta et al. 1994). Most lectins that have been isolated from arthropods are from
the hemolymph (Mullins 1985; Vasta et al. 1999).
The history of lectin studies in ticks is relatively brief. The earliest studies were done on hemo-
lymph lectins of O. tartakovskyi, O. tholozani (papillipes), A. polonicus, and I. ricinus (Grubhoffer
et al. 1991). Partially characterized lectins revealed their affinity to sialic acid and N-acetyl-D-
glucosamine. Later, the binding activity of a novel 85 kDa lectin from I. ricinus with sialic acid,
D-galactose, and N-acetyl-D-glucosamine was revealed (Kuhn et al. 1996). These findings led to
the suggestion that lectins were the molecular factors of self- and non-self-recognition and defense
in ticks, mediating, for example, the coiling phagocytosis of B. burgdorferi (Kuhn et al. 1994).
No other lectin studies in ticks were recorded until the isolation of Dorin M, a sialic-acid-
binding lectin from the hemolymph of the tick O. moubata (Grubhoffer and Kovar 1998) that is
possibly involved in the tick immune system (Kovar et al. 2000). Dorin M shows sequence sim-
ilarity to lectins from the horseshoe crab Tachypleus tridentatus, Tachylectins 5A and 5B (Kawa-
bata and Tsuda 2002). Tachylectins are the major hemolymph lectins in the horseshoe crab and
belong to a family of fibrinogen-related domain-containing proteins involved in immunity
within vertebrates and invertebrates (Wang et al. 2004).
The use of a bioinformatics approach coupled with molecular techniques resulted in the
identification of a clearly different fibrinogen-related protein, OMFREP, in O. moubata hemo-
cytes that was 65% identical to Dorin M. Two newly identified related molecules, Ixoderin A
and Ixoderin B, were identified in I. ricinus. Both showed significant homology to Dorin M.
Ixoderin A shares a similar expression profile. Ixoderin B was shown to be constitutively ex-
pressed only in the SGs of I. ricinus, with very marginal expression in the hemocytes (Rego et al.
2005). Studies revealed high similarity at the protein level of OMFREP and Ixoderin A to verte-
brate immune proteins known as ficolins. Ficolins consist of a collagen-like domain and a
C-terminal fibrinogen-related domain and structurally resemble mannose-binding lectin (Mat-
sushita and Fujita 2001) that is known to have opsonic activity in serum, as well as the capacity
to activate complement. Immune sera directed against Dorin M and other tick lectins have been
used for the identification of novel lectins in other tick species (Sterba et al. 2011).
A novel galectin (OmGalec), a protein with galactose-binding properties, was isolated from
O. moubata. OmGalec has no significant similarity to OMFREP or Dorin M. It consists of tan-
dem repeated carbohydrate recognition domains, in which the typical motifs important for car-
bohydrate affinity are conserved (Huang et al. 2007). This might lead to the modulation of
hemocyte aggregation during infection or cross-linking with other cell types (Pace et al. 2002).
3.1.5. Macroblogulins
The expression pattern of Dorin M is very similar to that of another plasma protein, an α2-
macroglobulin from O. moubata (TAM) (Kopacek et al. 2000). It is produced by hemocytes and
is significantly expressed in the SGs after a blood meal. It was shown that TAM accounts for
about 3% of the total plasma proteins of O. moubata. The defense role of TAM via the inhibition
of microbial proteases (virulence factors of invading pathogens) is known to be an efficient
mechanism in innate immunity (Armstrong 2001). Because of its high expression in the SGs, the
role of TAM as a potential anti-coagulant should be considered.
Primary structure and functional analysis of α2-macroglobulin from I. ricinus (IrAM) showed
that it is closely related to TAM (Buresova et al. 2009). IrAM is expressed in all tick developmen-
tal stages and tissues (except the midgut), and the protein is mainly present in the hemolymph.
RNAi silencing of IrAM significantly reduces phagocytosis of the gram-negative bacteria Chrys-
eobacterium indologenes by tick hemocytes. This bacterium is highly pathogenic to the soft tick O.
moubata, but it is harmless to I. ricinus (Buresova et al. 2006). IrAM silencing had no effect on the
phagocytosis of B. burgdorferi or gram-positive Staphylococcus xylosus. Such strict specificity in
the reduction of phagocytosis might be mediated by the interaction of IrAM with C. indologenes
metalloprotease, the major virulence factor for this pathogen (Pan et al. 2000).
3.1.6. Proteases and protease inhibitors

The involvement of proteases in tick immunity is not clear, and knowledge of their role in tick
hemolymph is limited. The arthropod clip-domain family serine proteinases consist of both an
N-terminal disulfide knotted regulatory domain and a trypsinogen-like catalytic domain in the
C-terminus (Jiang and Kanost 2000). A clip-domain serine proteinase homologue was identi-
fied in D. variabilis (Simser et al. 2004b). It was most similar to T. tridentatus hemocyte antimi-
crobial factor D and highly homologous to a number of immune-responsive gene products of
arthropods, including insect prophenoloxidase-activating co-factors. Analysis of the expression
pattern revealed that hemocytes were the primary site of its expression, and the level of expres-
sion was slightly higher in fed adults than in unfed. The orthologue of the D. variabilis clip-
domain molecule was identified in D. andersoni embryonic cell line DAE100. It was shown that
both tick proteases are present at steady-state levels within granules of tick hemocytes as “stored”
immunity effectors and are released upon pathogen invasion (Simser et al. 2004b).
Tick hemolymph contains proteins with serine protease inhibitory activity (serpins), and
these inhibitors may exist in plasma or in hemocyte granules (Kanost and Jiang 1996). The serine
protease inhibitors in tick hemolymph are likely involved in protection from infection. Some of
them might inhibit fungal or bacterial proteases, whereas others probably contribute to the reg-
ulation of endogenous proteases involved in coagulation, prophenol oxidase activation, or cy-
tokine activation (Polanowski and Wilusz 1996). Serpins might represent some of the most
interesting antigens for the development of an anti-tick vaccine because of their potential func-
tions as inhibitors in arthropod homeostasis (Mulenga et al. 2000).
A novel cysteine-rich peptide (Ixodidin) was isolated from the hemocytes of R. microplus. In
addition to antimicrobial effects, this peptide exhibited proteolytic inhibitory activity against 2
exogenous serine proteinases, chymotrypsin and elastase. It is most likely that Ixodidin exerts
different actions in tick hemolymph, acting directly against invasive pathogens, mediating other
immune responses in the R. microplus hemocoel, and preserving its own integrity and the integ-
rity of other AMPs (Fogaca et al. 2006).
3.1.7. Oxidoreductases
Hemocytes of R. microplus have been shown to produce reactive oxygen species (ROS) upon
phagocytosis (Pereira et al. 2001). Two separate thioredoxin peroxidase (peroxiredoxin) genes
were strongly induced in the hemolymph, SGs, and midgut of I. ricinus after B. burgdorferi in-
fection (Rudenko et al. 2005). It is possible that the I. ricinus peroxiredoxin gene is induced in
response to oxidative stress as a consequence of B. burgdorferi infection. This could be a result

of ROS released by tick hemocytes as part of the antibacterial response (Pereira et al. 2001). In
addition, ticks encounter ROS generated by vertebrate immune cells such as neutrophils during
blood feeding (Tsuji et al. 2001). Another gene identified in I. ricinus after a B. burgdorferi–
infected blood meal with a possible role as an antioxidant is glutathione S-transferase (GST)
(Rudenko et al. 2005). GST belongs to a gene family involved in the detoxification of xenobiotic
compounds, including insecticides. It was observed that GST genes are often induced by ROS.
This represents an adaptive response, as these enzymes detoxify some of the toxic metabolites
produced within the cell by oxidative stress (Hayes and Strange 2000).
3.1.8. MD-2-related lipid-recognition domain-related proteins

Expression of the gene encoding Der-p2 allergen-like protein in I. ricinus is induced by a blood
meal (Horackova et al. 2010a). I. ricinus Der-p2 belongs to a diverse family of MD-2-related
lipid-recognition (ML) proteins that includes major allergens of house dust mites, human MD-
2, or similar proteins from D. melanogaster. Similar genes or their protein products have not yet
been characterized in any other tick species. Analysis of the expression pattern showed that Der-
p2 is strongly induced in the hemolymph and gut. The ability of Der-p2 to interact with IgE
might indicate its involvement in the tick immune response. The predicted three-dimensional
structure of I. ricinus Der-p2 showed the presence of a cavity between the plates formed by the
β-sheets for lipid binding. This feature was confirmed in several members of the ML protein
family. Although the characterization of Der-p2 is not yet complete, the protein most probably
participates in the recognition and transport of lipids from blood or in the recognition of surface
patterns created by lipidic molecules of microorganisms (Horackova et al. 2010a).
A second gene encoding ML-domain-containing protein (IrML) was strongly induced in I.
ricinus by a blood meal (Horackova et al. 2010b). Tick guts were defined as a primary site of
IrML expression, but it was detected in hemolymph and SGs in all tick developmental stages.
Analysis of the predicted structure of I. ricinus ML-domain-containing protein and its localiza-
tion in the tick body suggests that IrML is a secreted protein and is possibly involved in tick in-
nate immunity (Horackova et al. 2010b).
3.2. OTHER HEMOLYMPH COMPOUNDS REGULATING

OSMOLALITY AND pH
In addition to the wide range of proteins with diverse functions discussed above, other com-
pounds make up tick hemolymph (minerals, lipids, hormones, etc.).
The volume of hemolymph changes corresponding to each of the tick life stages, as well as
during feeding. However, the proportion of the hemolymph in the tick body does not change
significantly in the course of feeding. In A. arboreus, the hemolymph volume increases from 3.8
μl (28.5% of the whole body) in unfed females to 8.57 μl (25%) on engorgement day and then
decreases to 6.81 μl (22.5%) on oviposition day. In A. persicus, these values are slightly higher,
and the hemolymph represents 31%–38% of the whole body (Hefnawy 1972a). In D. andersonii,
the hemolymph volume constituted about 23% of the total body weight in unfed and feeding
ticks (Kaufman and Phillips 1973).
The content of the most abundant ions was studied in D. andersonii. Overall, hemolymph
osmotic pressure and ion concentration decreased during tick feeding. In unfed ticks, the ion
concentration was approximately 280 mM Na+, 20 mM K+, and 170 mM Cl−. The sodium con-
centration decreased on day 3 of tick feeding to 160 mM Na+ and remained stable throughout
the remainder of the feeding process. On feeding day 5, ion concentrations declined to 7.5 mM
for K+ and 125 mM for Cl−. The osmotic pressure decreased from approximately 527 mOsm/l
during the first day of feeding to about 375 mOsm/l on the fifth day of feeding (Kaufman and
Phillips 1973). Hemolymph osmotic pressure changes have been studied in detail in A. persicus
and A. arboreus. In both cases, the unfed tick hemolymph osmolality was 390 mOsm/l. On the
day of feeding, but before coxal fluid emission, the concentration decreased to 365 and 342
mOsm/l in the 2 species, respectively. Following coxal fluid emission, the osmolality declined to
310 and 322 mOsm/l, respectively. The osmotic pressure rose again after the completion of
feeding; 1 day after feeding, the values were 370 and 360 mOsm/l, respectively, and 1 day after
oviposition, the pressure exceeded the values prior to feeding and rose to 454 and 491 mOsm/l
for A. persicus and A. arboreus, respectively (Hefnawy 1972b).
The fatty acid content of hemolymph was studied in H. dromedarii and A. excavatum ticks.
In both ticks, the concentration patterns of fatty acids were similar. A total of 20 fatty acids were
identified with carbon chain lengths ranging from C2 to C24. Palmitic, stearic, oleic, and linoleic
acids were present in pre-molting nymphs and engorging and ovipositing females and con-
stituted 80% to 90% of the total fatty acids (Hajjar 1972). As for free amino acids, gamma-
aminobutyric acid, glycine, serine, glutamine, alanine, glutamate, aspartate, and taurine were
detected in tick hemolymph (Lucien et al. 1995).
Free sugars were detected in tick hemolymph, predominantly glucose, and the presence of
mannose, inositol, trehalose, and sorbitol was confirmed (Levenbook et al. 1980; Binnington
and Obenchain 1982; Hwang 2006). Glycerol is part of tick hemolymph, playing a protective role
against injuries induced by low or high temperatures. The normal concentration of glycerol in
D. variabilis is 19 mM. Similarly, sorbitol seems to play a protective role in ticks: its concentra-
tion under normal conditions is negligible, but it increases to 1.3 mM during cold hardening
(Hwang 2006).
Low levels of antioxidants were found in tick hemolymph; however, there has been minimal
research in this area (Graça-Souza et al. 2006). Antioxidants probably serve as protectants
against the oxidative damage caused by an excess of heme from a blood meal. Protoheme was
found in the hemolymph of fed D. andersonii females at a concentration of 0.46 μg/μl (Hamdy
et al. 1974). The information regarding the source of heme is confusing. Whereas heme synthetic
activity was reported in D. andersonii (Hamdy et al. 1974), the same pathway was shown to be
inactive in B. microplus, and this tick relies solely on host heme supplies (Braz et al. 1999). He-
moglobin fragments with possible antimicrobial activity are present in tick hemolymph (Fogaca
et al. 1999). Freely soluble hemocyanin was found in the hemolymph of the tick A. cajennense
(Carneiro and Daemon 2002).
3.3. HEMOLYMPH CLOTTING AND IMMUNE DEFENSE

Hemolymph clotting is a defense mechanism in arthropods that occurs in response to injury or
pathogen invasion. Hemolymph clotting was described in D. variabilis as a process accompa-
nied by granulocyte degranulation (Eggenberger et al. 1990). Several potential clotting factors
have been described (Mulenga et al. 2001, 2003). The presence of platelet-derived growth factor
and transforming growth factor, which are implicated in wound healing (Matsuo et al. 2007),
was confirmed in hemocytes and other tick tissues.
4. TICK HEMOCYTES
Hemocytes are freely circulating cells in the hemolymph. Attached hemocytes were found
in association with arterial vessels, fat bodies (FBs), nephrocytes (Kuhn and Haug 1994), and
SGs (Sterba et al. 2011).
4.1. CLASSIFICATION
Mature hemocytes are divided into several types based on their morphology and function
(phagocytic ability). The classification system for hemocytes is the subject of considerable con-
troversy. Three basic morphological types (i.e., prohemocytes, plasmatocytes, and granulocytes)
are present in hard and soft ticks (Figs. 11.8–11.12). So far, only putative proliferation sites of he-
mocytes or hemocyte reservoirs have been described in wounded or infected ticks. Mitotic and
FIGURE 11.8: Ultrastructure of the circulating hemocytes of the tick Ixodes ricinus. Granulocyte type I
with electron-dense granules (dg) and granules with a tubular structure (sg). Black arrows point out
invagination pits; secondary lysosomes are identifi ed by white arrows. Measurement bar: 1 μm. From
ricinus and Ornithodoros moubata. Exp. Appl. Acarol. 35:317–333, with permission from Experimental and
Applied Acarology.
FIGURE 11.9: Ultrastructure of the circulating hemocytes of the tick Ixodes ricinus. Granulocyte type II.
Measurement bar: 1 μm.
FIGURE 11.10: Ultrastructure of the circulating hemocytes of the tick Ixodes ricinus. Plasmatocytes.
Measurement bar: 1 μm. From Borovickova, B. and Hypsa, V. (2005) Ontogeny of tick hemocytes: a
comparative analysis of Ixodes ricinus and Ornithodoros moubata. Exp. Appl. Acarol. 35:317–333, with
permission from Experimental and Applied Acarology.
proliferative activities of mature hemocytes (as observed during the engorgement of I. ricinus
females) indicate that hemocytes can divide and be differentiated directly from the free circu-
lating population of hemocytes (Kuhn 1996; Borovickova and Hypsa 2005).
Granulocytes and plasmatocytes are 2 abundant, freely circulating hemocyte types in hard
ticks. Two morphologically different types of granulocytes (granulocyte types I and II) have
FIGURE 11.11: Ultrastructure of the circulating hemocytes of the tick Ornithodoros moubata.
Granulocyte. dg, electron-dense granules. Measurement bar: 1 μm. From Borovickova, B. and Hypsa, V.
(2005) Ontogeny of tick hemocytes: a comparative analysis of Ixodes ricinus and Ornithodoros moubata.
Exp. Appl. Acarol. 35:317–333, with permission from Experimental and Applied Acarology.
been distinguished, predominantly on the base of granule morphology (Eggenberger et al. 1990;
Kuhn and Haug 1994; Borovickova and Hypsa 2005; Habeeb and El-Hag 2008), in I. ricinus,
I. scaluparis, D. variabilis, H. dromedarii, and H. asiaticum. However, in D. andersoni, Brinton
and Burgdorfer (1971) distinguished 4 types of spherule cells (former name for granulocytes)
and, moreover, disputable oenocytes (oenocytoids). In soft ticks, only 1 type of granulocyte, with
morphological similarity to granulocyte type II of hard ticks, was described in O. moubata
(Inoue et al. 2001; Borovickova and Hypsa 2005), O. erraticus (El Shoura 1989), and Argas arbo-
reus (El Shoura 1986).
Aside from the 3 basic types of hemocytes, unique granular hemocytes were observed in
O. moubata and were classified as spherulocytes based on their morphology, which is similar to
the lepidopteran hemocytic type (Cook et al. 1985; Borovickova and Hypsa 2005). However, this
granulocyte type was not observed in previous studies that involved immature tick stages of
Ornithodoros and Argas genera (El Shoura 1986, 1989).
4.2. STRUCTURE
The important morphological features that help to distinguish the type of hemocytes are the
quantity, size, shape, presence of filopodia in, and nucleus/cytoplasm ratio of cells and the size,
shape, quantity, and substructure of inclusion bodies. However, differences exist between the
ultrastructures of immature and mature stages of hemocytes.
Plasmatocyte. m, mitochondria; dg, electron-dense granules; hb, heterogeneous bodies. Lysosomes are
identifi ed by white arrows, and multivesicular bodies by the black arrow. Measurement bar: 1 μm. From
ricinus and Ornithodoros moubata. Exp. Appl. Acarol. 35:317–333, with permission from Experimental and
Applied Acarology.
Prohemocyte-like cells were found occasionally in the hemolymph of engorged I. ricinus

females and more often in the hemolymph of both larval and nymphal ixodid ticks (Balashov
1972). Attached prohemocytes were found to be associated with connective tissues (Kuhn and
Haug 1994). The cells are small and round to oval (7 μm × 6 μm) with a large nucleus, numerous
mitochondria, and several small granules (Borovickova and Hypsa 2005).
Type I granulocytes in I. ricinus are large round or highly pleomorphic cells (6 μm in length)
that contain variable electron-dense granules with homogeneous or tubular substructures
(Fig. 11.8). A typical feature of the type I granulocytes is the presence of lysosomes and filopodia
(Kuhn and Haug 1994). Type II granulocytes of hard ticks (12 μm × 9 μm) contain numerous
electron-dense granules about 2 μm in diameter (Fig. 11.9). Different electron densities of central
and peripheral areas were observed in condensing immature granules (Borovickova and Hypsa
2005). Granulocytes of soft ticks (Fig. 11.11) resemble morphologically the type II granulocytes
of Ixodidae (Borovickova and Hypsa 2005).
Plasmatocytes are the predominant hemocytes in ixodid ticks (Fig. 11.10) and vary in
size (11 μm × 9 μm, along the long axis up to 30 μm) and shape. Granular inclusions are
absent; the presence of primary lysosomes and secondary lysosomes accommodating en-
gulfed material, lipid droplets, and numerous filopodia is typical. Plasmatocytes of O.
moubata (Fig. 11.12) and A. arboreaus (El Shoura 1986) are mostly rounded or ovoid cells (10
μm × 12.5 μm) that contain a few small electron-dense granules and vacuoles. Filopodia are
present infrequently.
Spherulocyte with characteristic large granules in different stages of development (stars).
Measurement bar: 1 μm. From Borovickova, B. and Hypsa, V. (2005) Ontogeny of tick hemocytes: a
comparative analysis of Ixodes ricinus and Ornithodoros moubata. Exp. Appl. Acarol. 35:317–333, with
permission from Experimental and Applied Acarology.
A large proportion of the hemocytes in O. moubata represent spherulocytes (11 μm × 14 μm)

(Fig. 11.13). These cells are characterized by numerous electron-lucent granules with a tubular
substructure that fill almost the entire cell content (Borovickova and Hypsa 2005).
Oenocytes were observed in limited numbers only in D. andersoni hemolymph. The cells
have an ovoid shape (18.3 μm × 11.9 μm) with numerous pronounced infoldings of the plasma
membrane and contain granules measuring 0.4 μm in diameter (Brinton and Burgdorfer 1971).
5. MOLECULAR BASIS OF HEMOCYTE FUNCTION
The immune system provides ticks with quite an efficient defense against invasive pathogens.
The defense responses of ticks to microorganisms are mediated via the immune effector cells
(hemocytes) and guided by regulatory mechanisms of gene expression.
The hemocyte populations in the hemolymph are dynamic and directly influenced by path-
ogen invasion. Pathogen invasion induces an increase in hemocyte number and their migration
toward the pathogen. The typical hemocyte count is approximately 103 to 104 cells/μl (Johns et al.
1998; Ceraul et al. 2002), but the number increases rapidly in response to bacterial challenge,
supporting the role of hemocytes in the phagocytosis of bacteria and yeasts (Loosova et al. 2001).
For example, challenge of D. variabilis with spore-forming B. subtilis led to a 6.4-fold increase in
hemocytes within 48 hours (Johns et al. 1998). The response of D. variabilis to infection with
B. burgdorferi was even faster, with the amount of hemocytes increasing 3.1 times during the first
hour after the challenge. Lysis and removal of spirochetes from the system resulted in a rapid
decrease of the hemocyte population; cell numbers dropped to normal levels within 24 hours
(Johns et al. 2000).
The cellular response to infection includes phagocytosis, encapsulation, and nodulation.
Phagocytosis by hemocytes is a primary cellular defense response to microbial infection in
several ticks (Kuhn and Haug 1994; Zhioua et al. 1996). In hard ticks, phagocytic activity has been
reported mainly for plasmatocytes and type I granulocytes, and rarely for type II granulocytes. It
seems that the diverse types of hemocytes that occur in the adult tick differ in their functional
contribution to the tick immune system (Kuhn and Haug 1994; Borovickova and Hypsa 2005).
The process of phagocytosis and the fate of the engulfed material are affected by many fac-
tors such as opsonization (opsonizing factors may originate from the tick or the host serum) or
the ability to recognize pathogens. Inoue et al. (2001) proved that the phagocytosis of polysty-
rene beads by tick hemocytes was enhanced by blood-meal components such as complement.
All these factors and the production of AMPs (Section 3.1.1) consequently influence the vector
competence of tick species to transmit pathogens (Johns et al. 2001; Sonenshine et al. 2005).
Hemocytes of the immunotolerant Ixodes ticks showed a slow phagocytic response to B. burg-
droferi, in contrast to hemocytes of D. variabilis, which possess bactericidal factors that influ-
ence the phagocytic process and lead to the killing of bacteria (Johns et al. 2000, 2001). Tick
(Ixodes spp.) hemocytes use 2 different mechanisms in the phagocytosis of B. burgdorferi: con-
ventional and coiling phagocytosis (Fig. 11.14) (Rittig et al. 1996; personal observation). In some
cases, cellular defense is diminished by the invading pathogen; for example, the intracellular
FIGURE 11.14: Transmission electron micrograph illustrating a coiling type of phagocytosis of Borrelia
burgdorferi B31 spirochetes by isolated tick hemocytes of Ixodes ricinus. Arrow shows the formation
of pseudopods around engulfed bacteria (asterisk). Measurement bar: 200 nm. Photo courtesy of
K. Zacharovová.
parasite Rickettsia ricketsii is engulfed but not destroyed by plasmatocytes, which then serve as
a route of dissemination from the gut to other tick tissues (Socolovschi et al. 2009).
Lysosomal compartments are well developed, especially in plasmatocytes, and to a lesser
degree in granulocytes of both types (Kuhn and Haug 1994; Inoue et al. 2001).
Expression of the c-type lysozyme in hemocytes of D. variabilis was shown to be upregulated
by gram-negative bacteria (E. coli) but was not influenced by blood meal (Simser et al. 2004a).
Similarly, blood meal did not increase the expression of lysozymes in O. moubata hemocytes
(Grunclova et al. 2003). Up-regulation of the c type lysozyme in hemolymph was observed in
H. longicornis after infection (Tanaka et al. 2010). Tick hemocytes produce a number of AMPs
such as defensins and cystatins, which help the tick to fight infection (Section 3.1.2). In addition
to lysozyme and protease, granulocytes and plasmatocytes of R. microplus produce ROS after
phagocytosis (Pereira et al. 2001), which leads to the destruction of pathogens.
Protein–carbohydrate interactions are believed to be involved in pathogen recognition. Lectin
molecules expressed on the surface of hemocytes can be utilized by ticks for this purpose (Grub-
hoffer et al. 1997, 2005). Several lectins expressed by hemocytes and other cells have been identified
in ticks to date (Grubhoffer and Kovar 1998; Kovar et al. 2000; Rego et al. 2005; Huang et al. 2007).
The ability of hemocytes to recognize lipopolysaccharides on a pathogen surface brings
up another mechanism of microbial control in ticks: nodulation. Upon exposure to bacteria,
plasmatocytes cluster, forming a bacteria-hemocyte microaggregate that prevents hemo-
lymph circulation and results in adhesion to different organs within the hemocoel (the
nodulation process). Nodulation is similar to encapsulation (see below), but without mela-
nization, and probably without the layering of flattened dead and dying plasmatocytes. Nod-
ule formation is the quantitatively predominant cellular defense reaction to bacterial
challenges, responsible for clearing the largest proportion of pathogens (up to 90%) from
circulation (Ceraul et al. 2002).
Encapsulation is another mechanism used by arthropods to clear the hemocoel, partic-
ularly for gram-negative bacteria or for organisms too large to be engulfed via phagocytosis
(Eggenberger et al. 1990; Ceraul et al. 2002). Encapsulation is a biphasic event that starts
with the degranulation of type I granulocytes and hemolymph coagulation and continues
with the formation of the capsule. The capsule is formed by both types of granulocytes and
flattened plasmatocytes (Eggenberger et al. 1990). In insects, hemocytes surround abiotic
objects with consecutive layers of dead and degranulating cells, with melanization as the
final step (Gillespie et al. 1997).
Considering the importance of hemolymph for tick survival, hemolymph’s involvement in vital
processes in all life stages and in blood feeding, and the role of hemocytes in pathogen survival
and transmission, the CS of ticks should attract far more interest from researchers in the future.
There is a significant gap between the information currently available about the morphology of
the CS and that about the function of its components.
The rapidly expanding employment of mass spectrometry, transcriptomics, and genomics should
provide a more thorough description of the tick proteome and other hemolymph components in
different life stages and in response to the environment, hosts, and infection. The identification of
molecules present only in ticks and not in their vertebrate hosts (e.g., ferritin-2) (Hajdusek et al. 2010)
might provide new methods for the prevention of tick-borne disease infections.
Moreover, research of (but not limited to) tick AMPs could provide new ways to fight bacte-
rial, viral, and protistan infections in the current dawn of the era of antibiotic-resistant bacteria.
ACKNOWLEDGMENTS
This work was supported by Grant Nos. MSM 6007665801 and LC06009 (Ministry of Educa-
tion), institutional research grants Z60220518 and KJB600960906 (Grant Agency of the ASCR),
Grant Nos. 206/09/1782 and P302/11/1901 (Czech Science Foundation), and Grant Nos. CZB1-
2963-CB-09 (M.G.) and CZB1-2966-CB-09 (N.R.) from the CRDF.
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C H A P T E R 1 2
FAT BODY AND NEPHROCYTES

Structure and Function
LEWIS B. COONS
1. INTRODUCTION
The fat body and nephrocytes are found in the hemocoel of arthropods, where they are often closely
associated with each other and with organs such as the epidermis, digestive system, trachea, and
ovary. Each has a distinct ultrastructure and function. The fat body has diverse functions that in-
clude intermediate metabolism, energy storage, heme sequestration, and reproduction (it is a major
source of the female specific yolk precursor vitellogenin [Vg]). It has been compared to the verte-
brate liver, although it is quite different in both structure and physiology. Nephrocytes have multiple
functions, maintaining homeostasis by removing specific compounds from the hemolymph.
2. FAT BODY
The fat body may contain a single type of cell or a variety of different cell types, depending on
the species of arthropod and its life stage. It is often composed of sheets of closely associated
cells, but in some species it occurs as isolated groups of cells, and in others the fat body is absent
or appears only at certain life stages.
2.1. INSECTS
The fat body of insects is a well-developed organ with major functions in metabolism, storing
and releasing energy reserves, producing many of the important constituents of the hemolymph,
and producing Vg (reviewed by Hagedorn and Kunkel 1979; Locke, 1980, 1984, 1998; Dean et al.
1985; Keeley 1985; Kanost et al. 1990; Telfer and Kunkel 1991: Haunerland and Shirk 1995;
Haunerland 1996; Arrese and Soulages 2010; Roma et al. 2010). Insect fat body occurs commonly
as a sheet of cells just under the epidermis of the integument and as ribbons of cells around
organs, especially the digestive and reproductive system. These positions allow the hemolymph
effective access to fat body cells. This is critical for a low-pressure, open circulatory system like
that found in all arthropods. The fat body has regions of specialization. Fat body located near the
intestines is often more metabolically active than that located just beneath the cuticle, which is
more commonly involved in storage.
The fat body is important in the metabolism and storage of lipids, carbohydrates, and
proteins. Triglycerides are the general storage form of lipids, whereas diglycerides are the mobile
form. Glycogen is the principal storage form of carbohydrates in fat body cells. Trehalose, the
major carbohydrate found in the hemolymph, is synthesized only by the fat body. The fat body
synthesizes several different families or groups of storage proteins. Most are hexameric proteins
in the range of 70 to 80 kDa and are evolutionarily related to hemocyanin. Structurally similar
hexameric proteins are termed heximerins. Although both holo- and hemi-metabolous insects
have been found to synthesize such proteins, the former have been more carefully studied.
Storage proteins are released into the larval hemolymph by the fat body and then taken up by the
fat body before pupation. They are stored in the pupal fat body and eventually broken down to
yield amino acids required for adult development. In the diamondback moth, Plutella xylostella,
a post-eclosion increase in the synthesis of hexameric storage proteins occurs (Wheeler et al.
2000). These are subsequently depleted during Vg synthesis and egg production. Biliverdin-
binding proteins also can act as amino acid storage proteins (Yoshiga et al. 1998). In the rice
moth, Corcyra cephalonica, hexamerin protein synthesis in the fat body is transcriptionally reg-
ulated by 20-hydroxyecdysone (Manohar et al. 2010).
The trophocyte (adipocyte) is the most common of several types of cells that make up the
insect fat body. Insect trophocytes are polyploid. They can continue to divide, producing dif-
ferent layers in the ribbons of cells. The most common feature of trophoctes is the presence of
large amounts of lipid inclusions and glycogen. The fat body trophocytes of some insects store
phenolics; these are depleted during the tanning of cuticle. Other cell types include the urocyte,
the mycetocyte (bacteriocyte), and chromatocytes. The oenocyte is sometimes found dispersed
among trophocytes, but it is not a fat body cell. Oenocytes found in the fat body are usually, but
not always, enclosed in their own basal lamina (basement membrane). Oenocytes have a
complex relationship with fat body cells. For example, in the German cockroach, hydrocarbons
produced by oenocytes are thought to be delivered by the hemolymph to the fat body and other
internal tissues (Fan et al. 2003). In Drosophila, a bidirectional coupling exists in lipid metabo-
lism between the fat body and oenocytes (Gutierrez et al. 2007). The urocytes found in the fat
bodies of cockroaches and locusts accumulate urate, a waste product produced by the metabo-
lism of amino acids and nucleic acids. The mycetocyte, found in the fat bodies of cockroaches
and some Hemiptera, contains symbiotic microorganisms with possible roles in insect nutri-
tion. The chromatocyte is believed to be a modified trophocyte. Chromatocytes that contain
colored pigments are found dispersed in the fat body of aquatic larvae of Simulidae and Thaum-
aleidae that have transparent integuments. The fat body is important in iron metabolism and is
a source of the ferritin that is found in the hemolymph. The fat body cells of chironomids syn-
thesize and secrete hemoglobin into the hemolymph. Cells of the fat body are in communication
though gap junctions that occur between trophocytes. Changes in the ultrastructural compo-
nents of fat body cells and in their physiology follow the intermolt/molt cycle of the insect,
Fat Body and Nephrocytes 289
which is under the control of ecdysone. No innervation of the insect fat body has been demon-
strated. The development and function of the fat body in Bombyx mori and Drosophila melano-
gaster appear to be regulated by a complex interaction involving insulin and ecdysteroids,
oncogenes and tumor suppressors, and nutritional signals (Liu et al. 2009). This suggests
that the developmental changes of the fat body are perhaps even more complex than previously
thought.
Vg is synthesized by the insect fat body and, in some insects, the ovary. Vg from the fat body is
secreted into the hemolymph and taken up by developing oocytes via receptor-mediated endocy-
tosis to become the yolk protein vitellin (Vn) in a process known as vitellogenesis. In most insects,
Vg production by the fat body is under the control of juvenile hormone (JH), with the exception of
some dipterans that utilize ecdysteroids (Hagedorn and Kunkel 1979; Koeppe et al. 1985).
2.2. OTHER ARTHROPODS

Fat body occurs in many species of Chelicerata. In spiders (Foelix 2010) and in whip spiders
(Weygoldt 2000), the fat body is suggested to function as a site of long- and short-term storage.
Fat body also is found in both of the major taxa of mites, the Parasitiformes and the Acari-
formes, (reviewed in Alberti and Coons 1999; Alberti et al. 2003). Not all species of mites have
fat body, and in some species the fat body appears only at certain stages of the life cycle. The fat
body of mites contains 1 type of cell, the trophocyte. These are found closely associated with
internal tissues such as the trachea and nephrocytes. Gap junctions sometimes occur between
the fat body and the midgut. This close association of the fat body and internal organs occurs in
other chelicerates and is thought to be a plesiomorphic feature in this group. In some mites,
trophocytes are suggested to have a storage function.
Fat body is found in representatives of all the major taxa of the Myriapoda. In the Chil-
opoda, a syncytial fat body covers the muscles and occurs between the internal organs (Lewis
1981). Fat body cells contain fat deposits and yellow, possibly proteinaceous inclusions that give
the fat body a characteristic color. The fat body of the centipede Lithobius variegatus stores zinc
(Hopkin and Martin 1983). In the Diplopoda, the fat body is a loosely packed tissue that is pre-
sent throughout the hemocoel (Hopkin and Read 1992; Fontanetti et al. 2006). It stores lipids,
glycogen, proteins, and uric acid and acts as a permanent site for storing excretory products. The
fat body of the diplopod Rhinocricus padbergi is sensitive to soil pollutants and might be a stress
biomarker (Souza et al. 2011). Fat body is present in the symphylan Hanseniella agilis (Tiegs
1945) and in the pauropod Pauropus silvaticus (Tiegs 1947).
Some crustaceans have a fat body, but many do not. A fat body is present in the post-larval
puerulus and postpuerulus stages of the rock lobster, in which it extends outward from the he-
patopancreas and is thought to be a storage site that contributes energy reserves for the long-
distance swim of the puerulus (Takahashi et al. 1994). In many crustaceans, vitellogenesis involves
the hepatopancreas and is regulated by methyl farnesoate and farnesolic acid (a precursor to the
insect JHs) produced by the mandibular organs (Laufer et al. 1987; Chan et al. 2005; Mak et al.
2005; Phiriyangkul and Utarabhand 2006; Tiu et al. 2006). In the land crab Potamon potamios,
both the fat body and the hepatopancreas are involved in the production of Vg (Pateraki and
Stratakis 2000). In the terrestrial isopod Armadillidium vulgare, the fat body is the site of Vg
production (Suzuki et al. 1989; Okuno et al. 2000), as is the fat body of the marine isopod Idotea
balthica basteri (Souty and Picaud 1981).
2.3. TICKS
The fat body is present in the larvae, nymphs, and adults of all ticks (see reviews by Balashov
1972; Amosova 1983; Sonenshine 1991; Coons and Alberti 1999). It is thought to be mesodermal
in origin. Tick fat body tends to occur in 2 places: closely associated with trachea and just be-
neath the epidermis, and around the internal organs, especially the reproductive system. The
former is sometimes referred to as the peripheral fat body, and the latter as the central fat body
(Obenchain and Oliver 1973). These were renamed the parietal and perivisceral fat body, respec-
tively, by Denardi et al. (2008). This division of the fat body is based on morphology and not on
functional specialization. In ticks, the fat body is not as well developed as in insects and occurs
as rather isolated anastomosing strands of closely attached cells; as a practical matter, is difficult
to separate it via dissection from other organs in the hemocoel. The fat body of both locations
expands during feeding, especially in ixodid females.
The trophocyte is the single type of cell found in the fat body of ticks (Coons et al. 1990;
Sonenshine 1991; Coons and Alberti 1999). Several subtypes have been described. El Shoura
(1989a) divided trophocytes into type I and type II cells in ovipositing Ornithodoros (Pavlovaky-
ella) erraticus based in part on the presence or absence of a Golgi system that is closely associ-
ated with the rough endoplasmic reticulum. Cuboidal and round trophocytes were described in
the fat body of Amblyomma cajennense (Denardi et al. 2008). One study at the light microscopic
level treated trophocytes and nephrocytes as part of the fat body of several species of ticks and
divided them into acidophils and basophils, respectively (Obenchain and Oliver 1973). How-
ever, nephrocytes have a different ultrastructure and function than trophocytes and are found in
other parts of the hemocoel in addition to being associated with the fat body. They also occur as
freely circulating cells in the hemolymph. Nephrocytes are described in Section 3 of this chapter.
2.3.1. Pre-vitellogenic fat body

Trophocytes become more abundant and exhibit changes in ultrastructure with each life stage
and with feeding (reviewed by Coons and Alberti [1999]). Histochemical studies on the tropho-
cytes of several species of argasid and ixodid ticks demonstrated lipid deposits. In unfed larval
trophocytes of Dermacentor variabilis, lipid inclusions are often prominent features (Fig. 12.1).
Trophocytes of unfed nymphs have lipid inclusions and often have larger profiles of rough endo-
plasmic reticulum and more mitochondria than trophocytes of unfed larva (compare Fig. 12.1 to
Fig. 12.2). Some trophocytes from unfed nymphs show dense inclusions of an unknown nature
(Coons et al. 1990). Dense inclusions are a prominent feature in trophocytes from unfed female
adults (Fig. 12.3). Possibly, these dense inclusions represent a method of sequestering toxic sub-
stances such as iron from the blood meal. Trophocytes from unfed females also show small fields
of glycogen and scant profiles of rough endoplasmic reticulum (Fig. 12.3). An unusual cell-to-
cell junction, the scalariform junction, is present (Fig. 12.3). This junction has periodic cross-
striations between the opposed plasma membranes that resemble pillars and is closely associated
with rough endoplasmic reticulum that lacks ribosomes on the side opposed to the junction
(Coons et al. 1986; Dallai et al. 1997). The scalariform junction is both a homocellular and a
heterocellular junction. It is more common in fed female trophocytes of D. variabilis than in
trophocytes of immatures. The function of the scalariform junction in trophocytes of ticks is
unknown. Feeding increases deposits of lipid and glycogen in trophocytes of immatures and
adults of both sexes relative to unfed ticks, suggesting that this is a dynamic organ with an
FIGURE 12.1: Fat body trophocyte (Tr) and nephrocyte (Ne) from an unfed Dermacentor variabilis larva.
Bm, basement membrane; Di, dense inclusions; Li, lipid inclusions; Va, vacuole. From Coons, L.B.,
Lamoreaux, W.J., Rosell-Davis, R., and Starr-Spires, L. (1990) Fine structure of the fat body and
nephrocytes in the life stages of Dermacentor variabilis (Say). Exp. Appl. Acarol. 8:125–142, with
permission from Springer.
FIGURE 12.2: Fat body trophocyte from an unfed Dermacentor variabilis nymph. Er, rough endoplasmic
reticulum; Li, lipid inclusion; Nu, nucleus of trophocyte; Mg, midgut; Ta, trachea.
291
FIGURE 12.3: Fat body trophocyte from an unfed adult Dermacentor variabilis female. Bm, basement
membrane; Di, dense inclusions; Er, rough endoplasmic reticulum; Gl, glycogen; Sj, scalarifom junction.
From Coons, L.B., Lamoreaux, W.J., Rosell-Davis, R., and Starr-Spires, L. (1990) Fine structure of the fat
body and nephrocytes in the life stages of Dermacentor variabilis (Say). Exp. Appl. Acarol. 8:125–142, with
energy-storage function used during the non-feeding stages of tick development, as is the case
for insects. The fat body of feeding females develops a different ultrastructure than that of males
or the immature life stages, as discussed in the following section on the vitellogenic fat body.
2.3.2. Vitellogenic fat body

The fat body was suggested as a source of Vg based on the ultrastructure of trophocytes in fed
female adults (Hecker and Aeschlimann 1970; Diehl et al. 1982; Coons et al. 1982, 1986, 1990;
Amosova 1983). During the blood meal, female trophocytes develop large arrays of rough endo-
plasmic reticulum, prominent Golgi bodies, and secretory products (Figs. 12.4 and 12.5). This
type of ultrastructure is characteristic of cells involved in the active synthesis and secretion of
proteins. Trophocytes reach their maximum development during oviposition when the rough
endoplasmic reticulum shows swollen cisternae and the foot processes develop around extracel-
lular spaces that contain a putative secretory product (Fig. 12.5). Trophocytes from fed males do
not show large arrays of rough endoplasmic reticulum, but they do show large fields of glycogen
deposits, electron-dense inclusions, and small foot processes (Fig. 12.6).
Fat body trophocytes have been identified as a source of Vg using different localization tech-
niques. Araman (1979) used [14C] amino acids to show that fat body from mated fed female adult
Rhipicephalus sanguineus secreted labeled proteins with electrophoretic characteristics similar
to those of hemolymph Vg. Diehl et al. (1982), using indirect immunofluorescence, localized an
antiserum developed from purified Vn from eggs of Rhipicephalus appendiculatus to the vitel-
logenic fat body. Using double-diffusion and immunoelectrophoretic techniques, Vg was local-
ized to the fat body in R. sanguinius (Coons et al. 1982). Rosell and Coons (1992) used a
monospecific polyclonal antiserum raised to Vn to localize Vg to fat body trophocytes in
D. variabilis females with immunolocalization at the electron microscope level. Chinzei and
FIGURE 12.4: Nucleus of fat body trophocyte (Tr) from a mated female Dermacentor variabilis following
completion of feeding. Bm, basement membrane; Er, rough endoplasmic reticulum; Fp, foot processes;
Gb, Golgi body; Li, lipid inclusion; Mi, mitochondria. From Coons, L.B., Lamoreaux, W.J., Rosell-Davis,
R., and Starr-Spires, L. (1990) Fine structure of the fat body and nephrocytes in the life stages of
Dermacentor variabilis (Say). Exp. Appl. Acarol. 8:125–142, with permission from Springer.
FIGURE 12.5: Fat body trophocyte from a fully fed mated female Dermacentor variabilis sampled during
oviposition. Bm, basement membrane; Er, rough endoplasmic reticulum (note swollen cisternae); Fp,
foot processes; Mi, mitochondrion; Sp, secretory product. From Coons, L.B., Lamoreaux, W.J., Rosell-
Davis, R., and Starr-Spires, L. (1990) Fine structure of the fat body and nephrocytes in the life stages of
Dermacentor variabilis (Say). Exp. Appl. Acarol. 8:125–142, with permission from Springer.
293
FIGURE 12.6: Fat body trophocyte (Tr) from a male Dermacentor variabilis fed for 3 days. Bm, basement
membrane; Di, dense inclusions; Fp, foot processes; Gl, glycogen. From Coons, L.B., Lamoreaux, W.J.,
Rosell-Davis, R., and Starr-Spires, L. (1990) Fine structure of the fat body and nephrocytes in the life
stages of Dermacentor variabilis (Say). Exp. Appl. Acarol. 8:125–142, with permission from Springer.
Yano (1985) incubated cultured organs from vitellogenic female adults of Ornithodoros moubata
with [35S] methionine, which was incorporated rapidly into a protein from fat body. Using elec-
trophoresis and fluorography, they showed that this protein was identical to Vg, and they local-
ized Vg to the fat body using immunofluorescence. Other studies demonstrated that Vg from
the fat body and hemolymph is immunologically similar to Vn from eggs (Coons et al. 1982;
Diehl et al. 1982; Chinzei et al. 1983; Rosell and Coons 1991; James and Oliver 1997). Vg tran-
scripts were localized to the fat body of mated and virgin O. moubata by means of whole mount
in situ hybridization (Horigane et al. 2010). In addition to fat body, the midgut and ovary are
putative sites of Vg production in argasid and ixodid ticks (Coons et al. 1982, 1989; Rosell and
Coons 1990; Roe et al. 2008; Boldbaatar et al. 2010a; Horigane et al. 2010); this is discussed in
Chapters 15 and 17. The fat body of Amblyomma cajennense was determined not to be involved
in Vg production through the use of polyacrylamide gel electrophoresis (Denardi et al. 2009).
Only partially fed ticks (not fully fed or ovipositing ticks) were used in that study; it was not
reported whether these ticks were mated. Furthermore, the localization of Vg by means of im-
munological methods or in situ hybridization, currently considered the most reliable techniques
for establishing Vg production in organs, was not reported in that study.
Vg synthesis in ticks was at first thought to be regulated by JH, based mostly on its role in
insects (reviewed by Sonenshine [1991]). This was supported by the results of some studies
using natural JH, JH mimics, and anti-JH compounds. Pound and Oliver (1979) found that the
anti-allotropic compound precocene 2 stopped vitellogenesis, as well as ovarian development
and embryogenesis, in Ornithodoros parkeri. This effect was partially reversed with JH III.
Obenchain and Mango (1980) induced oviposition in some virgin Ornithodoros porcinus porci-
nus using topical treatment with isomers of JH I and JH III. Connat et al. (1983) induced vitel-
logenesis and oviposition with different JH analogs or a mixture of JH I and III stereoisomers
topically applied to fed virgin female Ornithodoros moubata. Eggs produced in that study were
sterile and abnormally shaped. Other studies failed to find a role for JH in Vg synthesis. Vg
synthesis was not stimulated in unfed female O. parkeri (Taylor et al. 1991) or O. moubata
(Chinzei et al. 1991) when JH I, JH II, JH III, and methoprene were topically applied. Vg synthe-
sis was analyzed with fluorography using in vivo labeling with [35S] methroprene and immuno-
precipitation with anti-Vn serum. The injection of JH III into D. variabilis female adults did not
stimulate Vg expression (Thompson et al. 2005). Furthermore, JH and its precursors, as well as
the ability to biosynthesize JH, were shown to be absent from D. variabilis and O. parkeri (Neese
et al. 2000).
Ecdysone, a regulator of Vg production in some arthropod groups, as previously noted, be-
came another candidate for a possible role in the hormonal regulation of Vg synthesis in ticks.
Sankhon et al. (1999) found that the treatment of fat body organ cultures and backless explants
from unfed female adults of D. variabilis with 20-hydroxyecdysone (20E), but not with metho-
prene, increased Vg production in the culture media. The injection of 20E into unfed O. mou-
bata stimulated Vg synthesis (Taylor et al. 1997). Unfortunately, these ticks did not live long
enough for changes in ovary development to be evaluated. A positive correlation was shown to
exist between ecdysteroid concentration and Vg synthesis in female Ixodes scapularis (James
et al. 1997). Injections of 20E into partially fed, detached, non-vitellogenic Ambylomma he-
braeum led to an increase in hemolymph Vg (Friesen and Kaufman 2002, 2004). JH III did not
stimulate Vg production or the uptake of Vg by the ovary. Thompson et al. (2005) showed that
20E, but not JH III, injected into virgin feeding D. variabilis initiated expression of the Vg gene,
and Vg absorption occurred in the ovary. Studies at the molecular level showed that 20E caused
the expression of Vg genes in D. variabilis (Thompson et al. 2005, 2007; Khalil et al. 2010) and
Ornithodoros moubata (Taylor et al. 1997; Taylor and Chinzei 2002; Horigane et al. 2010). The Vg
message was localized to the female fat body and midgut via whole body in situ hybridization
(Horigane et al. 2010). These studies also showed that an increase in Vg production is concomi-
tant with an increase in the hemolymph concentration of ecdysone. Together, these studies sup-
port the conclusion that Vg is synthesized in the fat body and midgut of ticks, secreted into the
hemolymph, and taken up by the ovary to become yolk (Vn), and that the vitellogenic hormone
is 20E. A 2-phase regulation of vitellogenesis involving both engorgement and mating has been
proposed by Horigane et al. (2010). Vitellogenesis and its regulation are more fully discussed in
Chapters 16 and 17.
Vg genes from several ticks have been sequenced in full or in part and are listed in public
databases. These include a full-length Vg gene from D. variabilis, DvVg1(AY885250), a full le-
ngth gene from O. moubata (AB440159), 2 putative genes from I. scapularis (XM_002415179 and
XM_002403922), and 2 partial sequences from Rhipicephalus microplus (GP80, U49934 and the
EST, CV436305). Three putative Vgs have been sequenced from Haemaphysalis longicornis
(AB359900, AB359899, and AB359901) (Boldbaatar et al. 2010b). A GATA transcription factor
is a specific activator of the Vg gene in H. longicornis (Boldbaatar et al. 2010a). The full-length
sequence of a second Vg gene from D. variabilis has been published (Khalil et al. 2010). Tick Vg
genes are discussed in greater detail in Chapters 9, 15, 16, and 17.
Vg protein has been isolated and partially characterized from the hemolymph of several
ixodid and argasid ticks (reviewed in Sullivan et al. 1999; Gudderra et al. 2002a; Donohue et al.
2009). Tick Vg has heme, carbohydrate, lipid, and protein components. The heme in Vg is de-
rived from the digestion of host hemoglobin (O’Hagan 1974). The mechanism of incorporation
of heme into Vg is not known. Vg is further described in Chapters 15 and 17.
2.3.3. Other functions of fat body

Several antimicrobial compounds are produced by the fat body and secreted into the hemo-
lymph (see the reviews in Taylor 2006; Sonenshine and Hynes 2008). In Amblyomma america-
num, a transcript for the defensin, americin, occurs in several tissues, including the fat body.
Four defensin isoforms are expressed in different organs of O. moubata. Defensins A, B, and C
are strongly expressed in the midgut, whereas defensin D is strongly expressed in the fat body
(Nakajima et al. 2002). Varisin, the major defensin of D. variabilis, is produced mostly in the
hemocytes, but a transcript of the gene also occurs in the fat body. Two antimicrobial peptides
have been characterized in Rhipicephalus (Boophilus) microplus (Fogaça et al. 2004), a defensin
and an antimicrobial peptide named microplusin. Gene expression of both occurs in hemo-
cytes, fat body, and ovary, but the defensin expression is stronger in the fat body and hemocytes
than in the ovary. Scapularisin, a defensin from I. scapularis, was shown to be expressed in the
midgut, hemocytes, and fat body using a real-time polymerase chain reaction, but no evidence
of the defensin peptide could be found in ticks challenged with B. burgdorferi, B. subtilis, or
E. coli (Hynes et al. 2005). Defensins are further characterized in Chapter 11 of this volume and
in Chapter 5 of Volume 2.
Other proteins are also synthesized in the fat body. A follistatin-related protein was shown
to be expressed in the fat body and other organs of A. americanum (Zhou et al. 2006) and was
suggested to be involved in tick oviposition. A statin with an unknown function was expressed
in the fat body and other organs of R. microplus (Lima et al. 2006). The fat body is the site of the
conversion of ecdysone to 20E, but it is not the site of ecdysone production. Instead, ecdysone is
synthesized in the epidermis in argasid and ixodid ticks (Zhu et al. 1991; Oliver and Dotson
1993). A lipoglycoheme carrier protein (CP) similar to, but not the same as, Vg appears to be
synthesized in the fat body and salivary gland of the hard tick D. variabilis and the soft tick
Ornithodoros parkeri; the hypothesis is that the protein is then secreted into the hemolymph
(Gudderra et al. 2001, 2002b; Donohue et al. 2009). The amino acid sequences of CP and Vg
have similarities, but there are also significant differences and different expression profiles,
tissue sources, and post-translational modifications (Donohue et al. 2008, 2009). The CP of D.
variabilis is present in both sexes and in all life stages, and it is thought to be involved in the
sequestration of heme from the blood meal (Gudderra et al. 2001). CP is discussed in detail in
comparison with Vg in Chapters 9 and 15.
3. NEPHROCYTES
Nephrocytes occur in all major taxa of arthropods. They take up vital dyes such as trypan blue.
Nephrocytes are cells with a characteristic ultrastructure that includes a well-developed base-
ment membrane, a cortical region that consists of slit diaphragms, a network of irregular extra-
cellular channels, and an extensive system of vacuoles and lysosomes (see Figs. 12.7–12.9). The
slit diaphragm acts as a size and ionic filter. Slit diaphragms are located just beneath the base-
ment membrane and guard the entrance to the extracellular channels. Material filtered from the
hemolymph by the slit diaphragm is endocytosed by the membranes of the extracellular chan-
nels and then moved into the lysosomal system for digestion.
Nephrocytes regulate the composition of the hemolymph by removing toxic materials and
molecules via filtration and endocytosis of the filtrate. A few studies have examined the filtered
FIGURE 12.7: Nephrocyte (Ne) showing a central nucleus from an unfed Dermacentor variabilis larva.
Note the close association with fat body trophocytes (Tr). Bm, basement membrane; Di, dense
inclusions; Gl, glycogen; Va, vacuole.
FIGURE 12.8: Nephrocyte from an unmated female Dermacentor variabilis fed for 6 days. Note the 3
regions. The inner region is characterized by the presence of glycogen (Gl), lipid inclusions (Li), rough
endoplasmic reticulum (Er), and mitochondria (Mi). The middle region consists of dense inclusions (Di),
Golgi body (Gn), and vacuoles (Va). The outer or cortical region lies just beneath the basement
membrane (Bm) and is better illustrated at higher magnifi cation (see Fig. 12.9). From Coons, L.B.,
Lamoreaux, W.J., Rosell-Davis, R., and Starr-Spires, L. (1990) Fine structure of the fat body and
nephrocytes in the life stages of Dermacentor variabilis (Say). Exp. Appl. Acarol. 8:125–142, with
297
FIGURE 12.9: Cortical region of a nephrocyte from a fully fed female Dermacentor variabilis. Bm,
basement membrane; Cp, coated pits; Cv, coated vesicles; Ec, extracellular channel; Sd, slit diaphragm;
Te, tubular elements; Va, vacuoles. From Coons, L.B., Lamoreaux, W.J., Rosell-Davis, R., and Starr-
Spires, L. (1990) Fine structure of the fat body and nephrocytes in the life stages of Dermacentor
variabilis (Say). Exp. Appl. Acarol. 8:125–142, with permission from Springer.
material and molecules, but their exact chemical nature is unknown. Nephrocytes are found in
the hemocoel as wandering or disseminated cells, or they are closely associated with major
organs, especially the fat body, heart, and ovaries. They are commonly located at sites where
hemolymph flow is most heavy. Nephrocytes are some of the largest cells found in arthropods.
Structurally, nephrocytes appear to be 1 basic type of cell with a variety of names, such as garland
cells, guirlandzellen, wreath cells, diaphragm cells, circumoesophageal cells, suboesophageal
cells, or pericardial cells. Some workers have replaced the general term “nephrocyte” with “ath-
rocyte” (Locke and Russell 1998; Owa et al. 2006, 2008). Some nephrocytes carry out functions
other than endocytosing material from the hemolymph, as discussed below.
3.1. INSECTS
Insect nephrocytes have been extensively studied at the cell and molecular level (as reviewed
in Smith 1968; Crossley 1985; Locke and Russell 1998; Denholm and Skaer 2009). They are de-
rived from embryonic mesoderm and persist through all development stages to the adult
(Ward and Skeath 2000; Sellin et al. 2006). Pericardial cells (PCs), located dorsally along the
heart of many insects, are composed of subsets of cells. The Odd-pericardial subset develops
from heart progenitors in Drosophila (Ward and Skeath 2000). Drosophila PCs are not involved
in cardiac function but contribute to the maintenance of homeostasis in the organism via the
uptake and sequestering of toxins (Das et al. 2008b). However, Drosophila wing hearts origi-
nate from a subset of PCs (Tögel et al. 2008). Most PCs are cell doublets and are not binucleated
(Locke and Russell 1998). In Calpodes, they increase greatly in volume during larval instars,
becoming increasingly polyploid. PCs of Manduca sexta synthesize and secrete some proteins
into the hemolymph while taking up and degrading others (Brockhouse et al. 1999). Calliphora
PCs accumulate lysozyme from the hemolymph, especially after bacterial invasion, and then
release the lysozyme back into the hemolymph (reviewed by Locke and Russell [1998]). The
lysozyme is synthesized in the fat body, and the role of the pericardial cells is most likely to
control its presence in the hemolymph.
Nephrocytes have been suggested as the functional analogue of the reticular endothelial
system (RES) in vertebrates (Poll 1934; Wigglesworth 1970). Like the RES, nephrocytes filter out
and store waste products and toxic materials and help regulate hormones and enzymes (Das
et al. 2008a; Denholm and Skaer 2009; Weavers et al. 2009). Two examples of this RES-like func-
tion are the regulation of toxic compounds via the uptake of hemoglobin and its digested prod-
ucts in nephrocytes of Rhodnius prolixus (Wigglesworth 1943; Locke and Nichol 1992), and the
uptake of JH esterase by larval PCs of Manduca sexta via receptor-mediated endocytosis (Bon-
ning et al. 1997). Nephrocytes synthesize some proteins that are released into the hemolymph
(reviewed by Klowden [2007]).
Material taken up from the hemolymph by insect nephrocytes is restricted to a size range of
16–20 Å, and the process has been characterized as microphagocytic rather than macrophago-
cytic (reviewed by Das et al. [2008a]). This size range is a function of the slit diaphragms that
guard the extracellular channels in the cortical region of the nephrocytes. Filtered material is
then endocytosed and processed by the lysosomal system. Some compounds or their digested
remnants are thought to remain in the nephrocyte, making it a storage organ for waste products
(Denholm and Skaer 2009).
Insect nephrocytes have a dynamic, changing ultrastructure that reflects their diverse func-
tions, especially the uptake and cellular digestion of compounds from the hemolymph. Several
recent studies have elucidated the cell and molecular biology of insect nephrocytes as it relates to
their ultrastructure. A study of the cytoskeleton of nephrocytes from the larvae of the Mediter-
ranean fruit fly, Ceratitis capitata, suggests that the cytoskeleton is involved in maintaining the
shape of the cortical region, especially the channel system, which resembles a similar system in
podocytes of the vertebrate kidney (Dallai et al. 1994). The endosomal compartment of the Dro-
sophila melanogaster nephrocyte is located in its cortical cytoplasm and consists of a tubular/
vacuolar complex that constantly undergoes a transformal process requiring replenishment by
coated vesicles, as described by Koenig and Ikeda (1990). That study also suggests that the tubu-
lar component of the endosomal compartment transforms into vacuoles via expansion of the
tubular membrane. The vacuoles then invaginate into flattened cisternae. Genetic studies on
Drosophila garland cells have confirmed their endocytic activity (Chang et al. 2004). Vertebrate
Rudhira is a conserved WD40 protein. WD40 proteins are found in all eukaryotes and have a
variety of functions, including cell signal transduction, transcription regulation, autophagy, and
apoptosis. The Drosophila ortholog of Rudhia, Drudh, is a specific marker for pericardial cells
and garland cells (Das et al. 2008c). In addition, Drudh regulates macropinocytic uptake in peri-
cardial cells (Das et al 2008a). A gene expression study showed that silkworm peritracheal ath-
rocytes had lower expression of genes encoding hemolymph proteins than the fat body, whereas
those genes involved in protein degradation were highly expressed (Owa et al. 2008). The same
study used fluorescent probes to show that athrocytes always possess a lysosomal degradation
apparatus. The nephrocyte of D. melanogaster has a distinct anatomical, molecular, and func-
tional relationship to the slit diaphragm of the glomerular podocyte of the vertebrate kidney,
suggesting that the 2 cells are evolutionarily related (Weavers et al. 2009). Studies at the molec-
ular level support the idea of the nephrocyte as a functional analogue of the vertebrate RES, but
they also show that this cell is genetically related to the podocyte of the vertebrate kidney.
3.2. OTHER ARTHROPODS

Nephrocytes are found throughout the Chelicerata. In spiders, they are large cells with a diam-
eter of 30 to 80 μm that sequester waste products (Fahrenbach 1999; Foelix 2010). They occur in
the harvestmen Leiobunum limbatum and L. rotundum (Zanger et al. 1991). Nephrocytes of
L. rotundum contain lysosyzme either synthesized by the nephrocyte or obtained from hemo-
lymph (Zanger 1995). Nephrocytes occur in scorpions (Farley 1999) and in mites (Shatrov 1998;
Alberti and Coons 1999). They also have been described from larval pycnogonids (Thompson
1909).
In the Myriapoda, nephrocytes occur in the Chilopoda (Lewis 1981), the Diplopoda (Seifert
and Rosenberg 1976), and the Symphyla (Tiegs 1947). Nephrocytes of the centipede Lithobius
forficatius are involved in detoxification by accumulating heavy metals in cytosomes (Vanden-
bulcke et al. 1998).
Nephrocytes are present in many crustacean species (see the references cited by Doughtie
and Rao [1981] and Hessler and Elofsson [1995]). In the Crustacea, the terms “nephrocyte” and
“podocyte” are used interchangeably (Doughtie and Rao 1981; Hessler and Elofsson 1995). Podo-
cytes function in the uptake of material from hemolymph. Some podocytes are syncytial in na-
ture, with multiple nuclei, unlike nephrocytes in other arthropods. In the prawn Palaemonetes
argentinus, nephrocytes are found in the stem of the phyllobranchiate gill associated with ef-
ferent hemolymph (Sousa and Petriella 2005). These nephrocytes have a large vacuole and a
lateral nucleus. In some crustaceans, nephrocytes respond to increases in toxic metals. The
number of nephrocytes in the gill filaments of the shrimp Penaeus japonicus increased after ex-
posure to copper (Soegianto et al. 1999b) or cadmium (Soegianto et al. 1999a). In the lobster
Homarus americanus, nephrocytes line the efferent gill channels (Kimura et al. 1994). These are
characterized by a single large vacuole often making up over 50% of the cell and by the localiza-
tion of an antigen associated with the invertebrate electrogenic 2Na+/1H+ antiporter. This anti-
porter is a protein in the plasma membrane that catalyzes the net uptake of extracellular calcium
coupled with the net expulsion of protons from the cell (Grinstein 1988).
3.3. TICKS
The nephrocytes of ticks are relatively large cells surrounded by a conspicuous basement mem-
brane (Figs. 12.1, 12.7, and 12.8). Nephrocytes are present in larvae, nymphs, and adults of both
sexes of D. variabilis (Coons et al. 1990). Nephrocytes occur either unattached in the hemo-
lymph, where they are known as wandering cells (Fig. 12.1), or in close association with organs
or fat body trophocytes (Fig. 12.7). Nephrocytes can be roughly divided into 3 regions. The cor-
tical region next to the basement membrane contains slit diaphragms that guard the openings of
extracellular channels (Fig. 12.9). These channels often extend into the peripheral cytoplasm of
the cell. Coated pits indicative of receptor-mediated endocytosis occur along the extracellular
channels. These are taken up by the nephrocyte as coated vesicles (Fig. 12.9). Presumably, these
are involved in the uptake of unknown material from the hemolymph that has passed through
the slit diaphragms. Tubular elements are common in this region and are often associated
with developing vacuoles (Fig. 12.9). Coated vesicles indicate receptor-mediated endocytosis
(Fig. 12.9). The middle region is a transition area characterized by electron-dense inclusions of
varying sizes, Golgi bodies, and scattered lipid inclusions (Fig. 12.8). An inner region is charac-
terized by fields of glycogen and rough endoplasmic reticulum. Mitochondria are common.
When present, the nucleus is commonly found in this region (Fig. 12.7). Gap junctions are found
between nephrocytes and adjacent fat body trophocytes (Coons et al. 1990). Coons et al. (1990)
found that near the end of oviposition, nephrocytes had fewer organelles than nephrocytes of
ticks entering oviposition, and no tubular elements, coated pits, or coated vesicles. At this stage,
large vesicles with a granular deposit were the most common cellular inclusions.
Nephrocytes have been described from adult Argas (Persicargas) arboreus (El Shoura 1989a),
larval Ornithodoros (Pavlovskyella) erraticus (El Shoura 1989b), and Hyalomma asiaticum
(Amosova 1983). Tick nephrocytes mirror the major structural features of insect nephrocytes: a
prominent basement membrane, slit diaphragms, and a complex array of organelles. Nephro-
cytes were not observed in female Amblyomma cajennense (Denardi et al. 2008). There is no ex-
perimental evidence that nephrocytes in ticks filter the hemolymph as they do in insects or that
they are also related to the vertebrate podocyte; this is assumed based on structural similarities.
Compared to other arthropods, especially insects, little is known about the functions of the tick
fat body beyond its role in vitellogenesis. We need to determine whether the trophocyte is poly-
ploid and whether it divides prior to or during the fat body expansion during and after blood
feeding. The fat body of ticks has been divided into central and peripheral fat bodies, but we do
not know whether these have different functions. The possible role of the fat body in the forma-
tion and tanning of the cuticle in ticks after molts is not known. Studies on the ultrastructure of
tick fat bodies show that trophocytes are able to store lipids and glycogen reserves in both im-
matures and adults. This in turn suggests a role for this organ in the storage and utilization of
reserve materials for energy. Ticks, especially soft ticks, have the capability to live a long life, and
the fat body might be important in providing stored energy between blood meals for this pur-
pose. At the molecular level, we need information on the transcriptome of the fat body in all life
stages of argasid and ixodid ticks, including its expression pattern and regulation.
Likewise, we need to understand the function of the tick nephrocytes, especially in the re-
moval of materials from the hemolymph in all life stages, and specifically we need to learn what
these materials are. We do not know whether tick nephrocytes have more than 1 nucleus or more
than 1 set of chromosomes. We need to study the role, if any, that tick nephrocytes have in the
uptake of specific proteins from the hemolymph. We need to elucidate the role, if any, that neph-
rocytes play in iron metabolism. We need to study the nephrocyte transcriptome and any
changes in gene expression during all life stages of ticks. As a starting point, we should compare
the genome of Ixodes scapularis, now available online, to what is known about the genes of insect
fat body and nephrocytes. We should determine whether the tick nephrocyte is evolutionarily
related to the vertebrate podocyte, as is that of Drosophila.
What we know about the biology of the tick fat body and nephrocyte is decades behind what
we know about these organs in insects. However, we now have the cell and molecular tools to
elucidate how these important organs function in the lives of ticks, and we should make a major
effort to do so.
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C H A P T E R 1 3
NERVOUS AND SENSORY SYSTEMS
Structure, Function, Genomics, and Proteomics
LADI S LAV Š IM O , DANIEL E. SONENSHINE , YOONSEONG PARK ,

AND DUŠAN ŽIT ŇAN
1. INTRODUCTION
In this chapter, we review the neural systems of ticks, including the anatomy of the central
nervous system (synganglion) and the peripheral nervous system. We also discuss the signaling
molecules that regulate most body functions, such as neuropeptides, neurotransmitters, and
their receptors. This review also includes the external sensilla used for sensory perception of the
external environment.
The tick central nervous system (CNS) is a highly condensed, fused nerve mass in which the
ancestral cerebral ganglia and ventral nerve cord have coalesced into a synganglion (occasionally
misnamed “brain”). It comprises 2 regions: (i) the supraesophageal (also called pre-esophageal)
region, which contains the paired protocerebral, cheliceral, and palpal ganglia, as well associated
paired protrusions and nerves that innervate the salivary glands (SG), pharynx, esophagus, and
optic lobes (a retrocerebral organ complex is located on the dorsal side of the supraesophageal
region); and (ii) the subesophageal (also called post-esophageal) region, which contains the 4
pedal ganglia, each connected to its respective trunk-live pedal nerves, plus the unpaired poste-
rior opisthosomal ganglion and the associated opithosomal nerves that innervate most of the
internal organs and the paired olfactory lobes. The olfactory lobes are believed to receive axons
from Haller’s organ (Hummel et al. 2007). The peripheral nervous system comprises nerves that
innervate the appendages, muscles, sensory structures, integument, and various internal organs.
The synganglion also contains neurosecretory centers from which neurotransmitter substances
pass via a complex system of neurosecretory tracts to reach the target organs. For a review of the
evolution of the tick CNS and a comparison with that of insects, see Chapter 4.
In this chapter, we review the basic organizational structure of the nervous system, the neu-
rosecretory/neurohemal centers, the various cell types that are found in these systems, and the
molecular processes that define their functions. Special attention is given to the nervous system’s
complex peptidergic neuroendocrine network.
For an electronic version of the structure of the nervous system, the reader may browse the
tick anatomical ontology for a description of the structures and related illustrations by visiting
www.vectorbase.org and following the instructions in the footnote below.1 Additional illustra-
tions of the synganglion, peripheral nervous system, and various sensory organs not included in
this edition may be found there.
2. STRUCTURE OF THE NERVOUS SYSTEM
2.1. SYNGANGLION
The synganglion constitutes the entire CNS of the tick. There is no ventral nerve cord. The
synganglion is enclosed within a periganglionic sheath containing a periganglionic sinus,
which is continuous with the dorsal aorta, allowing it to be supplied with fresh, filtered hemo-
lymph from the heart (Fig. 13.1). The esophagus penetrates the synganglion, entering at the
antero-dorsal end and emerging at the postero-ventral end (Figs. 13.1C and 13.2). For detailed
reviews of synganglion anatomy and ultrastructure, the reader should see the descriptions in
Chapter 4, as well as the reviews by Sonenshine (1991), Woolley (1988), Coons and Alberti
(1999), Lees and Bowman (2007), Šimo et al. (2009a), Prullage et al. (1992), and Szlendak and
Oliver (1992).
2.1.1. Supraesophageal region

The supraesophageal region is the smaller part of the synganglion. It contains the cheliceral,
palpal, protocerebral, and optic lobes and the stomodeal pons (or bridge) (see also Figs. 4.22 and
4.23). Paired cheliceral, palpal, and optic nerves extend from the corresponding lobes (ganglia)
to innervate the target organs; branches of the palpal nerve also innervate parts of the SG. Nerves
from the stomodeal pons innervate the pharynx, the esophagus, and a hypoesophageal ganglion
adjacent to the retrocerebral organ complex (ROC). Pairs of glomeruli (areas of highly dense
neuropile) are also present and constitute an important section of this region. The ROC is situ-
ated in the periganglionic sinus on the dorsal surface of the synganglion. This complex organ
consists of 3 lobes, an unpaired dorsal lobe and paired ventral lobes, each with membranous
coverings that are continuous with the neurilemma. Although considered as a neurohemal
organ by earlier researchers, its function is unclear.
2.1.2. Subesophageal region

The subesophageal region is the largest part of the synganglion. It also contains numerous
paired glomeruli (areas of highly dense neuropile) connected by commissures with glomeruli
located elsewhere throughout the synganglion. This region contains the paired pedal lobes
where the 4 pedal nerves originate, the olfactory lobes, and the unpaired opisthosomal lobe
Nervous and Sensory Systems 311
FIGURE 13.1: Anatomy of the synganglion and associated peripheral nerves and organs.
A, Representations of various lobes in the synganglion. Ch, cheliceral lobe; Ol, olfactory lobe; Os,
opisthosomal lobe; Pa, palpal lobe; Pd1–4, pedal lobes 1–4; Pc, protocerebral lobe; St, stomodeal lobe;
Pgsh, periganglionic sheath. B, Dorsal view of peripheral nerves and lateral segmental organs (LSO).
ChN, cheliceral nerve; GeN, genital nerve; LN, lateral nerve; OsN, opisthosomal nerve; OpN, optical
nerve; PaN, palpal nerve; PsN, paraspiracular nerve; PeN1–4, pedal nerves 1–4; StN, stomodeal nerve;
SgN1–4, salivary gland nerves 1–4; E, esophagus; Nl, neurilemma (neural lamella). C, Lateral view of the
synganglion depicting positions of associated structures and organs (adapted from Obenchain, F.D.
[1974] Neurosecretory system of the American dog tick, Dermacentor variabilis [Acari: Ixodidae]. I.
Diversity of cell types. J. Morphol. 142:433–446). A, aorta; E, esophagus; N nerves; NL, neurilemma; PgS,
periganglionic sheath; RCO, retrocerebral organ; PE, pre-esophageal; PO, post-esophageal. From Šimo,
L., Slovák, M., Park, Y., and Žit{an, D. (2009) Identifi cation of a complex peptidergic neuroendocrine
network in the hard tick, Rhipicephalus appendiculatus. Cell Tissue Res. 335:639–655, with permission
from Springer-Verlag, GMBH, Heidelberg, Germany.
(see also Figs. 4.22 and 4.23); nerves (primarily the opisthosomal, genital, paraspiracular, and
salivary gland nerves) from the latter supply most of the internal body organs, especially the
reproductive organs, dorso-ventral body muscles, and anal and spiracular muscles. Nerve
branches from the pedal nerves also extend antero-posteriorly to form the lateral nerve plexus
containing the 2 to 4 lateral segmental organs (LSO), tiny neurohemal/endocrine organs
located along the lateral margins of the synganglion (Fig. 13.1B).
2.1.3. Cortical zone

The synganglion proper is covered by a 1.5 to 3.5 μm thick acellular neurilemma comprising
repetitive layers of fine lamellae and granular material located just below the periganglionic
sheath. The interior of the synganglion is subdivided into (i) an outer cortical zone (cortex)
FIGURE 13.2: Diagrammatic reconstruction of the synganglion as seen from the lateral aspect (anterior
left) in Ornithodoros parkeri illustrating the major regions and locations of the neurosecretory centers.
accN-ROC, accessory nerve to retrocerebral organ complex (ROC); Ao, anterior aorta; APnA, anterior
perineurial association; aTrT, anterior tracheal trunk; compn-ROC, compound nerve to ROC; Cor,
cortex; dPC-NST, dorsal protocephalic neurosecretory tract; ES, esophagus; L-NST, lateral
neurosecretory tract; Np, neuropile; p(II)ChPw, chiasmatic neurosecretory pathway of pedal ganglion;
peS, periesophageal sinus; Pgs, periganglionic sinus; Pgsh, periganglionic (vascular) sheath; Ph-NST,
pharyngeal neurosecretory tract; prin-ROC, primary nerve to ROC; PRPnA, periesophageal ridge/
perineurial association; SUB-E, subesophageal region; SUPRA-E, supraesophageal region; VM-NST,
ventro-medial neurosecretory tract; vPC-NST, ventral protocephalic neurosecretory tract. Black areas
with adjacent numbers indicate the neurosecretory centers. Redrawn after Binnington, K. C. and
Obenchain, F.D. (1982). Structure and function of the circulatory, nervous, and neuroendocrine system of
ticks. In F.D. Obenchain and R. Galum (Eds.), Physiology of Ticks. Oxford, UK: Pergamon press, 351–398,
with permission from Elsevier.
containing the neuron cell bodies and glial cells and (ii) an inner neuropile containing fiber
tracts formed of numerous axons and dendrites (Figs. 13.3, 13.4).
The cortical zone consists of a thin layer of glial cells, the perineum, located just below the neu-
rilemma, and a much thicker layer composed of neuronal perikarya (neuron cell bodies) intermixed
with glial cells. Glial cells are believed to assist in the metabolic regulation of neural activities. It is
often possible to distinguish them by mucopolysaccharides and dense glycogen deposits in their
cytoplasm. They vary in shape, often having elongated nuclei and membranous extensions that
surround the neuronal perikarya. A subperineurium of glial cells, tracheae, and tracheoles separates
the cortical zone from the neuropile. The cortical zone contains numerous cell bodies of motor and
motor-association neurons, as well as neurosecretory neurons, clusters of which form the many
neurosecretory centers that can be identified by their position relative to the adjacent lobes.
FIGURE 13.3: Transmission electron micrograph illustrating the ultrastructure of the synganglion of
Dermacentor variabilis. Section illustrating the characteristic ultrastructure of the cortical zone, perineurium,
and periganglionic sheath of the synganglion in type I neurons, which have relatively little cytoplasm
surrounding their nuclei, and type II neurons, identifi ed by their masses of neurosecretory substance. Glial
cells interdigitate between the neuron cell bodies.The glial cells have abundant endoplasmic reticulum with
swollen vesicles. Nl, neurilemma; Pgsh, periganglionic sheath; Prn, perineurium. Measurement bar, 1.5 μm.
All synganglion neurons in ticks and other acarines are believed to be unipolar; that is, there is
a single process that bifurcates distally in the neuropile into an axon and a dendrite (or dendrites).
Three neuron cell types, types I, II, and III, have been reported in the tick synganglion, all in the
cortical zone. Type I neurons are the most abundant. They comprise neuron cell bodies (perikarya)
that are small (about 6 to 9 μm in diameter) with a pyriform shape narrowing at one end to a fu-
nicle shape, from which the axon and the dendrite of the cell diverge. In some locations within the
synganglion, type I neurons may be considerably larger (e.g., about 27 to 35 μm in the vicinity of
abdominal ganglia). In transmission electron micrographs, the cells may appear subcircular or oval
depending upon the plane of the section. These cells have a characteristically low cytoplasmic:nuclear
ratio. Type I cells are believed to function as motor or motor/association neurons. True association
neurons have not been found in ticks. Type II neurons are neurosecretory (NS) cells, variable in
size and organized in small clusters, the NS centers. NS cells secrete neurohormones, typically
neuropeptides, which are transported via their axonal projections and terminate at neurohemal
organs. There, the neurohormones are released into the surrounding hemolymph.
When actively secreting, they tend to enlarge considerably and might be from 25 to 40 μm in di-
ameter. Type II cells are easily recognized by their much larger cytoplasmic:nuclear ratio and the oc-
currence of masses of NS vesicles within the cell cytoplasm. NS cells increase in size, and the amounts
Dermacentor variabilis. Enlargement showing the neural lamella with numerous minute fibrils and the
cellular structure of the perineurium surrounding the synganglion. Note the axons with large numbers of
electron-dense neurosecretory granules. Er, endoplasmic reticulum; Nu, nucleus of glial cell; Nl,
neurilemma; Prn, perineurium. Measurement bar, 2.5 μm.
of their NS vesicles change greatly during different phases of physiological activity. Type III cells (not
shown in the figures) are relatively small (about 6 μm) and are limited to the paired globuli on the
ventral surface of the synganglion anterior to the first pair of pedal ganglia. Their function is unknown.
2.1.4. Neuropile
The neuropile consists of fibrous tracts of axons and dendrites organized into distinct neuropilar
lobes, ganglia, bilaterally symmetrical glomeruli, and a system of commissures and connectives
(Fig. 13.5). According to Prullage et al. (1992), the arrangement of the neuropile structure facili-
tates the division of the synganglion into its various ganglia. The neuropilar ganglia are the sites
from which the paired optic, cheliceral, palpal, pedal, and opisthosomal nerves extend to the
various body structures (Szlendak and Oliver 1992).
2.1.5. Peptidergic neurons in the synganglion

As noted previously, the tick CNS represents a single synganglion that is composed of multiple fused
lobes or ganglia. Numerous neurons of various sizes form a neuronal cortex on the surface of these
lobes. Until recently, the function of these cells was unknown. However, immunohistochemical
Dermacentor variabilis. Section through the cortical/neuropile interface illustrating the neuropile and
the adjacent cortex. Note the neurosecretory cell in the cortex. Measurement bar, 1.0 μm. Cor, cortical
zone; Er, endoplasmic reticulum; Nu, nucleus of glial cell; Nl, neurilemma; Np, neuropile; Pgsh,
periganglionic sheath; Prn, perineurium; I, type I neuron; II, type II neuron. Arrowhead indicates mass of
neurosecretory granules.
staining with antibodies against insect or crustacean neuropeptides combined with fluorescence
microscopy revealed that many of these neurons are peptidergic (Figs. 13.6 and 13.7). These
studies have led to the discovery of a very complex peptidergic network innervating various
parts of the synganglion, as well as different peripheral endocrine and secretory organs. Some of
these antibodies react with tick neurons that resemble those identified in insects, but many neu-
rons and their arborizations appear to be unique to ticks (Figs. 13.7–13.9). The unique neuronal
networks (e.g., neurons innervating the SG and LSO) probably reflect adaptations to the ecto-
parasitic life style (i.e., specifically obligate blood feeding on various vertebrates and modulation
of the host hemostatic and immune responses). As most tick neurons and neuropeptides have
been identified only recently, further studies are necessary to reveal their physiological func-
tions and mechanisms of action.
This description of the neuronal network is based entirely on immunoreactivity (IR) analysis.
For a molecular identification and characterization of the neuropeptides expressed in the tick syn-
ganglion, see Sections 3.1.1 and 3.1.2 in this chapter and Chapter 16. Peptidergic neurons with iden-
tified axonal projections were found to fall into 3 groups: (i) interneurons (IN), usually smaller
central neurons arborizing exclusively within the synganglion in the neuropile; (ii) larger NS cells
that project axons into the peripheral regions of the body, with their axon terminals forming typical
varicosities on the surface of putative neurohemal organs (periganglionic sheath and LSO); and
FIGURE 13.6: Schematic representations of all peptidergic neurons described in unfed Rhipicephalus
appendiculatus females. A, Bombyxin through CRF/DH. B, CCAP through AT. PTTH, prothoracicotropic
hormone; CHH/ITP, crustacean hyperglycemic hormone-related ion transport peptide; PDF, pigment
dispersing factor; BMS, bombyx myosupressin; MIP, myoinhibitory peptide; CRF/DH, corticotropin-
releasing-factor-related diuretic hormone; CCAP, crustacean cardioactive peptide; TRP, tachykinin-
related peptides; FGLa/AST, FGLamide-related allatostatin; AT, allatotropin. Neuropeptide and peptide
hormone nomenclature has been modifi ed according to Coast and Schooley ([2011] Toward a consensus
nomenclature for insect neuropeptides and peptide hormones. Peptides 32:620–631). An explanation of
the abbreviations of labeled peptidergic neurons is given in Section 2.1.5. After Šimo, L., Slovák, M.,
Park, Y., and Žit{an, D. (2009) Identifi cation of a complex peptidergic neuroendocrine network in the hard
tick, Rhipicephalus appendiculatus. Cell Tissue Res. 335:639–655, with permission from Springer-Verlag.
(iii) visceral modulatory neurons, large cells projecting their axons through peripheral nerves and
innervating various internal organs including the SG and the alimentary system.
In order to distinguish among numerous stained cells in the synganglion, the nomenclature
of White et al. (1986) was applied, with modifications. The first 2 letters characterize the location
of each neuron in a specific lobe/region: cheliceral (Ch), palpal (Pa), stomodeal (St), protocere-
bral (Pc), pedal 1–4 (Pd1–4), opisthosomal (Os), pre-esophageal (Pe), or post-esophageal (Po).
Subsequent letters denote its dorsal (D), ventral (V), anterior (A), posterior (P), medial (M), or
lateral (L) position. The peripheral neurons (PN) are named according to their association with
an identified nerve (i.e., cheliceral [Ch] and paraspiracular [Ps]). The subscript numbers after
the last letter refer to the number of neuron pairs.
FIGURE 13.6: (continued)
Specific reactions with 19 neuropeptide antibodies (Table 13.1) revealed remarkable diversity
in the tick peptidergic network, comparable to that found in insects, crustaceans, and other
invertebrates. The neuropeptide IR described in the following list has been observed mostly in
the hard tick Rhipicephalus appendiculatus (Šimo et al. 2009a), although in some specific cases
it has been noted in several other ticks (Ixodes scapularis, Ixodes ricinus, Dermacentor reticula-
tus, Hyalomma anatolicum, and Amblyomma variegatum) (Šimo et al. 2009b, 2011b; L. Roller,
A. Mizoguchi, Y. Tanaka, M. Slovák , and D. Žitňan, unpublished data).
1. Bombyxins are insulin-like neuropeptides that function as growth factors and regulate
hemolymph sugars in the silkworm and in flies (Wu and Brown 2006; Okamoto et al. 2009a,
2009b). In ticks, bombyxin (insulin)-like IR is restricted to the dorsal synganglion, where sev-
eral small paired neurons are located in the protocerebrum (PcIN1-3, PcDM1-3) and the cheliceral
lobe (ChD) (Figs. 13.6A and 13.7A). Two giant neurons are present in the opisthosomal lobe
(OsDM), but their axonal projections have not been determined.
2. Prothoracicotropic hormone (PTTH) is an ecdysteroidogenic hormone secreted by the
insect brain that stimulates the prothoracic glands to synthesize and secrete ecdysone (Ishizaki
and Suzuki 1994). In ticks, PTTH-like IR is limited to 2 pairs of small lateral neurons in the
cheliceral lobes (ChD1,2; Figs. 13.6A and 13.7C). These neurons closely resemble 2 pairs of
contralateral NS cells producing PTTH in the insect brain.
3. Ion transport peptide (ITP) controls water balance (Coast et al. 2002; Dircksen 2009), and
a closely related family of crustacean hyperglycemic hormones (CHHs) is involved in hemo-
lymph sugar regulation and in molting and reproduction (Dominique and Van Herp 1995;
Chung et al. 1999). In the tick, CHH-related ITP (CHH/ITP)-like staining is found in small
neurons scattered on the dorsal surface of the protocerebrum (PcAM and PcDM1,2), cheliceral
lobe (ChD1-4), and post-esophageal lobes (PoDM and OsDM1-2) (Figs. 13.6A and 13.7D).
4. The pigment-dispersing factor (PDF) is a member of a family of closely related octadeca-
peptide neuropeptides in numerous species of insects and crustaceans. It has been identified as
FIGURE 13.7: Neuropeptide immunoreactivity (IR) in unfed Rhipicephalus appendiculatus female synganglia
and lateral segmental organs (LSO). A, B, Bombyxin-like IR in the synganglion and intrinsic cells of the
LSO. C, Prothoracicotropic hormone (PTTH)-like IR in the anterior synganglion. D, Crustacean
hyperglycemic hormone–related ion transport peptide (CHH/ITP)-like IR in the synganglion. E, F, Pigment
dispersing factor (PDF)-like IR in the synganglion, lateral nerves (LN), and intrinsic cells of the LSO.
318
a hormone controlling pigment dispersion in chromatophores and pigment movements in eye

cells of crustaceans. PDF produced by the brain IN serves as an important transmitter for the
regulation of circadian biological rhythms in insects, and probably in crustaceans as well (Renn
et al. 1999; Rao 2001; Wei et al. 2010). In ticks, PDF-like IR is found in small paired neurons in
the first pedal lobe (Pd1DL) and in 2 pairs of large visceral modulatory neurons in the opistho-
somal lobe that innervate the SG (OsSG1,2) (see Figs. 13.6A and 13.7E).
5. Orcokinins are myotropic neuromodulators that regulate muscle contractions in various
arthropods, including ticks, and ecdysteroid production in Bombyx (Donohue et al. 2010; Ya-
manaka et al. 2011). Orcokinin IR is distributed in several small neurons in the protocerebral, pedal,
and opisthosomal lobes in I. ricinus, H. anatolicum, A. variegatum, and R. appendiculatus (data not
shown; L. Roller, A. Mizoguchi, Y. Tanaka, M. Slovák, and D. Žitňan, unpublished data). The 2 most
prominent pairs of large neurons in the opisthosomal lobe innervate the SG (see below).
6. Corazonin regulates heart activity in cockroaches, cuticle melanization in locusts, and
ecdysis triggering hormone (ETH) release to initiate the ecdysis sequence in moths (Tawfik et al.
1999; Roller et al. 2003; Kim et al. 2004). Corazonin IR is found in small paired neurons in the
protocerebrum (PcAM and PcIN), the first pedal lobe (Pd1DL1-3), and 2 clusters of neurons in the
opisthosomal lobe (OsDM1-5) (Figs. 13.6A and 13.7G). Identified protocerebral interneurons
(PcIN) show co-localization of corazonin- and myoinhibitory peptide (MIP)-like IR.
7. PRVamides are neuropeptides with cardioactive and diuretic activities in insects. They are
derived from 2 genes and are referred to as CAPA or periviscerokinins (Predel and Wegener
2006). In the tick, PRVamide-like IR is detected in a pair of PcIN (Figs. 13.6A and 13.7H) and 3
pairs of larger NS cells (PcNS1-3) that produce elaborate arborizations in the periganglionic
sheath. In addition, paired small neurons are present in pedal lobes 3 and 4 (Pd3,4DM) and the
opisthosomal lobe (OsDM1,2).
8. RFamides are stimulatory or inhibitory peptides that are derived from multiple genes and
which control various functions including muscle contractions, feeding, and ecdysteroid produc-
tion (Yamanaka et al. 2005, 2006; Palasoon et al. 2011). RFamide-like IR is detected in a large
number of small neurons in the protocerebrum (PcAM, PcDM1-5, and PcVM1,2) (Figs. 13.6A and
13.7I), while single neurons are stained in each cheliceral (ChD), palpal (PaSG), and opisthosomal
lobe (OsDM). PaSG likely innervates the main salivary ducts (see below). Several neurons of var-
ious sizes are present in pedal lobes (Pd3VM1,2, Pd2DM, Pd3DL, and Pd3DM1,2). The latter neurons
show co-expression of RFamide-, SIFamide-, and MIP-like peptides (Šimo et al. 2009a, 2009b).
G, Corazonin-like IR in the synganglion. H, PRVamide-like IR in the synganglion. I, J, RFamide-like IR in the

synganglion and putative neurohaemal axon terminals containing varicosities in the LSO. K, Bombyx
myosupressin (BMS)-like IR in anterior neurons and putative neurohaemal areas in the dorsal
periganglionic sheath of the synganglion. L, Kinin-like IR in anterior synganglion neurons and the same
neurohaemal areas as in K. M, Composite view of myoinhibitory peptide (MIP)-like IR in most identifi ed
dorsal and ventral neurons. N, Composite confocal sections of the synganglion showing crustacean cardio
active peptide (CCAP)-like IR in all described neurons. O,Tachykinin-like IR in dorsal cell bodies and
elaborate axonal arborizations of Pd1SG neurons. P, FGLamide-related allatostatin (FGLa/AST, allatostatin
A)-like IR in selected dorsal and ventral neurons. R, S, Allatotropin (AT)-like IR in dorsal (R) and ventral
(S) sides of the synganglion. An explanation of the abbreviations of labeled peptidergic neurons is given in
Section 2.1.5. Measurement bars: 50 μm (A, C–E, G–I, K–S) and 20 μm (B, F, J). After Šimo, L., Slovák,
M., Park, Y., and Žit{an, D. (2009) Identification of a complex peptidergic neuroendocrine network in the hard
tick, Rhipicephalus appendiculatus. CellTissue Res. 335:639–655, with permission from Springer-Verlag.
FIGURE 13.8: Myoinhibitory peptide (MIP) and SIFamide in Ixodes scapularis synganglion and salivary
gland acini. Double staining with antibodies to MIP (red, A) and SIFamide (green, B) revealed different
types of neurons, including protocerebral salivary gland neurons (PcSG) coexpressing MIP and
SIFamide (shown as yellow in the merged image, C). Axon terminals in type II (D–F) and III (G–I) acini
from PcSG neurons in a 1 day post-attachment female; MIP (red), SIFamide (green), and merged
(yellow). Arrowheads indicate immunoreactive axon terminals. DAPI-stained nuclei are blue.
Measurement bars: 50 μm (A–C) and 10 μm (D–I). MALDI-TOF mass spectra of synganglia extract (J)
and salivary gland extract (K). Two large peaks, [M + H]+ 1,395.07 representing SIFamide and 1,320.94
representing MIP2, were commonly observed in both extracts. An explanation of the abbreviations of
labeled peptidergic neurons is given in Section 2.1.5. From Šimo, L., Žit{an, D., and Park, Y. (2009) Two
novel neuropeptides in innervation of the salivary glands of the black-legged tick, Ixodes scapularis:
myoinhibitory peptide and SIFamide. J. Comp. Neurol. 517:551–563 and Šimo, L., Žit{an, D., and Park, Y.
(2011) Neural control of salivary glands in ixodid ticks. J. Insect Physiol. 58(4):459–466, with permission
from John Wiley & Sons and Elsevier, respectively.
9. Myosuppressins belong to the RFamide family of peptides, but their N-terminal sequences
are unique. These decapeptides inhibit visceral muscle contraction and suppress the production
of ecdysteroids in insects (Nachman et al. 1993; Yamanaka et al. 2005). Bombyx myosuppressin
(BMS)-like IR is apparent in 3 distinct clusters of anterior neurons. Three pairs of IN in the pro-
tocerebrum (PcIN1-3) show co-localization of BMS- and crustacean cardioactive peptide (CCAP)-
like IR. A cluster of 4 NS cells (PcNS1-4) in each cheliceral lobe projects posterior-lateral axons
that arborize in the periganglionic sheath (Figs. 13.6A and 13.7K). Other paired neurons are lo-
cated in the palpal lobes (PaD1,2). ChNS1-4 and PaD1,2 show co-localization of BMS- and kinin-like
IR. Five additional pairs of ventral neurons are found in the pedal lobes (Pd1-4VM) (Fig. 13.6A).
FIGURE 13.9: Schematic diagram showing all peptidergic neurons reacting with myoinhibitory peptide
(MIP) and SIFamide antibodies in unfed Ixodes scapularis females. MIP immunoreactivity (A, dorsal; C,
ventral) and SIFamide immunoreactivity (B, dorsal; D, ventral; PaN, palpal nerve; SgN1, salivary nerve 1)
in cells and projections in synganglion for both dorsal and ventral views. An explanation of the
abbreviations of labeled peptidergic neurons is given in Section 2.1.5. Measurement bar, 50 μm. From
Šimo, L., Žit{an, D., and Park, Y. (2009) Two novel neuropeptides in innervation of the salivary glands of
the black-legged tick, Ixodes scapularis: myoinhibitory peptide and SIFamide. J. Comp. Neurol. 517:551–
563, with permission from John Wiley & Sons.
10. Kinins have been identified as myoactive and diuretic hormones in insects. Centrally re-
leased kinins control pre-ecdysis contractions (Kim et al. 2006), whereas peripherally secreted
kinins regulate diuresis (Coast 2007). In the kissing bug, Rhodnius prolixus, kinins are reported
to increase hindgut contraction, which might contribute to rapid post-feeding diuresis through
the mixing of hemolymph and/or hindgut contents and the removal of wastes (Te Brugge and
Orchad 2002). In ticks, kinin-like IR is limited to several cell types. Four very small neurons are
in the protocerebrum (PcAM). Four pairs of anterior NS cells are stained in the cheliceral lobes
(PcNS1-4). Numerous immunoreactive axon terminals in the periganglionic sheath, apparently
Table 13.1: List of neuropeptides antibodies. Rabbit polyclonal (R), mouse

polyclonal (Mp) and mouse monoclonal (Mm) antibodies.
Antibody to: Type Dilution Source/first characterization

Adipokinetic hormone (AKH) R 1:1,000 D. Kodrík, unpublished data
Arg-Phe-NH2 (RFa) R 1:1,000 Grimmelikhuijzen and Spencer,
1984
Bombyx bombyxin (insulin-like) Mm 1:300 Mizoguchi et al., 1987
Bombyx prothoracicotropic Mm 1:1,000 Mizoguchi et al., 1990
hormone (PTTH)
Bombyx myosupressin (BMS) Mm 1:1,000 Yamanaka et al., 2006
Bombyx orcokinin (OK) Mp 1:1,000 A. Mizoguchi, unpublished data
Bombyx calcitonin-like DH31 Mp 1:2,000 A. Mizoguchi, unpublished data
Bombyx allatostatin CC Mp 1:1,000 A. Mizoguchi, unpublished data
Bombyx CCHamide Mp 1:1,000 A. Mizoguchi, unpublished data
SIFamide R 1:1,000 Terhzaz et al., 2007
Bombyx myoinhibitory peptide I (MIP-I) Mm 1:1,000 Kim et al., 2006
Crustacean cardioactive peptide (CCAP) R 1:500 Stangier et al., 1988
Drome-pigment disperzing factor (PDF) R 1:1,000 Persson et al., 2001
Dippu-FGLa-related allatostatin Mm 1:100 Stay et al., 1992
(allatostatin A)
Leuma-kinin R 1:1,000 Nässel et al., 1992
Locmi-tachykinin R 1:2,000 Nässel et al., 1993
Locmi-tachykinin R 1:1,000 Schoofs et al., 1993
Manse-allatotropin R 1:1,000 Žitňan et al., 1995
Manse-PISCF-related allatostatin R 1:1,000 Žitňan et al., 1995
(allatostatin C)
Manse-CRF-related diuretic R 1:2,000 Žitňan et al., 1995
hormone (CRF/DH)
Manse-eclosion hormone (EH) R 1:1,000 Žitňan et al., 2002
Manse-ion transport peptide (ITP) R 1:10,000 Dai et al., 2007
Manse-pre-ecdysis triggering R 1:1,000 Žitňan et al., 1999
hormone (PETH)
Peram-corazonin (CRZ) R 1:5,000 Roller et al., 2003
originating from PcNS1-4, indicate their neurohemal function. Additional paired neurons are pre-
sent in the palpal lobes (PaD1,2) (Figs. 13.6A and 13.7L). As described above, PcNS1-4 and PaD1,2
produce kinin- and BMS-like substances.
11. In insects, corticotropin releasing factor (CRF)-like diuretic hormones (DH) control the
secretion of waste products from the Malpighian tubules and the hindgut, as well as pre-ecdysis
(Kim et al. 2006; Coast 2007). In the tick synganglion, CRF-related diuretic hormone (CRF/DH)
IR is observed in 3 pairs of dorso-medial neurons in the pedal region (Pd2DM1-3) and in a pair of
larger neurons in the opisthosomal lobe (OsDM1) (Fig. 13.6A). Double staining revealed co-
localization of CRF/DH- and FGLamide (allatostatin A)-like IR in OsDM1 neurons, which pro-
jected branching axons into the lateral, opisthosomal, and pedal nerves 4.
12. FGLamide allatostatins are neuropeptides that inhibit juvenile hormone biosynthesis in
cockroaches and exert myoinhibitory activity in other insects (Bendena et al. 1999). In ticks,
FGLamide-related allatostatin (allatostatin A) IR is expressed in a very large number of small

neurons on the dorsal (~55 pairs) and ventral (~90 pairs) regions of the synganglion (Figs. 13.6B
and 13.7P). About 50 neurons are in the protocerebrum (PcDM, PcVM), clusters of 3 and 6 neu-
rons are in each cheliceral lobe (ChD1-3 and ChV1-6), and ~6 neurons are in each palpal lobe
(PaD1-6). Each pedal lobe contains 15 to 20 lateral neurons (PdDL and PdVL), and 2 or 3 dorso-
medial neurons are present in pedal lobes 2 and 3 (Pd2DM1-3 and Pd3DM1,2). The opisthosomal
lobe contains ~50 neurons; the largest coupled dorso-medial cells (OsDM1) show FGLamide-
and DH-like IR.
13. In insects, calcitonin-like DH (CT/DH) regulates water balance by controlling fluid se-
cretion by the Malpighian tubules (Furuya et al. 2000; Coast 2007). In ticks, CT/DH-IR is pre-
sent in several small neurons in the protocerebrum, 2 to 3 cells in each pedal lobe, and 2 pairs of
large and small neurons in the opisthosomal lobe of I. ricinus, H. anatolicum, A. variegatum, and
R. appendiculatus (data not shown; L. Roller, A. Mizoguchi, Y. Tanaka, M. Slovák, and D. Žitňan,
unpublished data).
14. CCHamides are novel neuropeptides of unknown function (Roller et al. 2010). In ticks,
CCHamide IR is restricted to a single small cell in each palpal lobe and 4 large neurons in the
opisthosomal lobe of I. ricinus and H. anatolicum (data not shown; L. Roller, A. Mizoguchi,
Y. Tanaka, M. Slovák, and D. Žitňan, unpublished data).
15. In insects and crustaceans, CCAP increases cardiac output, but most important, it is a
central signal for the regulation of ecdysis behavior (Žitňan and Adams 2011). In ticks, CCAP-IR
is observed in various neurons in the synganglion. The most apparent is a cluster of 6 IN in the
anterior protocerebrum (PcIN1-3) that shows co-expression of CCAP- and BMS-like peptides.
Additional small neurons are in the protocerebrum (PcAM, PcDM1-3) and palpal lobes (PaD1,2
and PaV1,2). Each pedal lobe 2 contains a couple of small dorsal neurons (Pd2DM, Pd2DL), and
singly distributed ventral neurons are in pedal lobes 1–4 (Pd1VM, Pd1-4VL; Figs. 13.6B and 13.7N).
16. In insects, MIPs suppress gut contractions and ecdysteroid levels and participate in
the ecdysis process (Žitňan and Adams 2011). MIP IR was detected in numerous neurons of
R. appendiculatus and I. scapularis (Figs. 13.6A, 13.7M, 13.8A, 13.9A, and 13.9C). Overall, the
MIP staining pattern was similar in both species (Šimo et al. 2009a, 2009b). A cluster of very
small neurons is present in the anterior protocerebrum (PcAM and PcDM) (Fig. 13.6A). A pair
of larger IN in the central protocerebrum (PcIN) co-express MIP and corazonin IR. A pair of
giant neurons in the posterior protocerebrum (PcSG) innervate the SG (see below). Small paired
neurons are observed in cheliceral (ChD1,2) and palpal lobes (PaD and PaV1,2). The pedal region
contains several medial neurons (PoDM and Pd3DM1-2); the latter, smaller neurons show co-
expression of MIP- and RFamide-like peptides. Paired lateral neurons are also present in pedal
lobes 2–4 and the opisthosomal lobe (Pd2DL, Pd2-4VM, OsVM, and OsIN) (Fig. 13.6A). In
I. scapularis, several additional ventral neurons are present in the stomodeal lobe (StVM1-3) and
pedal lobes 1–4 (Pd1-4VL and Pd1VL1-2) (Fig. 13.9C).
17. SIFamides are a family of highly conserved arthropod neuropeptides that modulate sexual
behavior and the contractions of visceral organs (Terhzaz et al. 2007). In ticks, SIFamide IR is
detected in various neurons on the dorsal and ventral sides of the synganglion (Figs. 13.8B, 13.9B,
and 13.9D). Two pairs of small IN were found in the central region of the dorsal protocerebrum
(PcIN1,2). A single pair of giant neurons (PcSG) showing co-expression of SIFamide and MIP is
located in the posterior part of the protocerebrum (Figs. 13.8B, 13.8C). We also detected 4 SI-
Famide-immunoreactive medial neurons on the ventral side of the protocerebrum (PcVM1-4).
Those neurons projected posteriorly a loop of paired axons that arborized in the pedal lobe
regions within the synganglion (Fig. 13.9D). The cheliceral lobes included a pair of immunore-
active neurons on the dorsal side (ChD). The dorsal pedal region contained a pair of small
lateral neurons (Pd1DL1,2 and Pd2DL1,2) and medial neuronal cells (Pd3DM1,2) that showed co-
expression with MIP (Fig. 13.8C). Two small IN (OsIN) were detected in the posterior syngan-
glion, and a pair of small medial neurons was found in each pedal lobe 3 (Pd3VM; Figs. 13.8B,
13.9B, and 13.9D).
18. Tachykinin-related peptides (TRP) constitute a large family of related peptides found in
both invertebrates and vertebrates and are best known for their ability to stimulate gut contrac-
tions (Nässel 1999). In ticks, tachykinin-like IR is characterized by a very distinct staining pat-
tern. The most obvious is a pair of large dorso-lateral neurons in the first pedal lobe (Pd1SG)
(Figs. 13.6B and 13.7O). These neurons project multiple axonal branches to innervate several
major peripheral nerves (palpal, pedal, lateral, and opisthosomal nerves), as well as the lateral
periganglionic sheath. Immunoreactive axons in the palpal nerve terminate on the surface of the
salivary ducts (see below). Six other, very small neurons are clustered in the anterior protocer-
ebrum (PcAM), and a single larger cell is in each cheliceral lobe (ChD) (Figs. 13.6B and 13.7O).
Single small neurons are also in the ventral pedal lobes (Pd1-4VL and Pd2VM) (Fig. 13.6B).
19. Allatotropin has been identified as a factor stimulating the biosynthesis of juvenile
hormone (JH) in adult moths (Kataoka et al. 1989), but its physiological function is not clear.
Likewise, its function in ticks, which appear not to produce JH, is unknown. Allatotropin-like
IR is observed in numerous neurons all over the synganglion (Figs. 13.6B, 13.7R, and 13.7S).
Several groups of small neurons are found in the protocerebrum (PcDM1-5, PcDL1,2, and PcVM)
and palpal lobes (PaD1,2 and PaV1,2). The largest number of neurons is in the pedal lobes. The
lateral regions of each pedal lobe contain between 4 and 8 neurons (Pd1DL1-3, Pd1VL1-5, Pd2-4DL,
and Pd2-4VL1-3), whereas 2 to 8 medial neurons are found in the posterior pedal lobes (Pd2-4VM1,2
and Pd3DM1-6). The opisthosomal lobe contains 5 pairs of neurons (OsDM1-3 and OsVM1,2)
(Figs. 13.7R, 13.7S).
2.2. PERIPHERAL INNERVATION AND NEUROHEMAL

AND ENDOCRINE ORGANS
2.2.1. Peripheral nervous system
The peripheral nervous system of a tick is composed of multiple nerves originating from the
synganglion-paired cheliceral, palpal, optic, lateral, opisthosomal, genital, paraspiracular, and sal-
ivary gland nerves (Fig. 13.1B). Tick peripheral nerves are composed almost exclusively of axons
surrounded by glial cells, all enclosed in a thin, amorphous neurilemma. Dendrites are usually
short, extending only a short distance from the cell body to the sensilla or other innervated struc-
ture. A remarkable feature of tick peripheral nerves is the invagination and fusion of the neuri-
lemma with the glial cells to form sheaths around individual axons or small groups of axons.
These anastomo sing sheaths are termed mesaxons. The neurilemma invaginates to surround in-
dividual nerve fibers, a feature that differentiates ticks from insects. Electron-dense reaction
products in tests for acetylcholinesterase activity occur between the axons, indicating the perva-
sive extent of this enzyme and the concomitant cholinergic system. Profiles of sections of a pedal
nerve trunk reveal hundreds of axons of varying size. Each axon contains numerous neurotubules
resembling (at the magnification shown) numerous tiny dots. Membranous infoldings of glial
cells, often several layers thick, can be seen between adjacent axons. Profiles of smaller peripheral
nerves show mesaxons from the neurilemma and extensive infoldings of the glial cells sur-
rounding axons. Many axons show masses of NS vesicles (Figs. 13.10 and 13.11).
Immunohistochemical staining with allatotropin antibody revealed 2 clusters of bipolar PN
attached to cheliceral and paraspiracular nerves. Each of 3 to 6 cheliceral PN (ChPN1-3) projects
a single axonal process along the cheliceral nerve into the periphery and another branching
process into the synganglion (Fig. 13.12A). Five paraspiracular PN (PsPN1-5) project peripheral
axons into a side branch of the paraspiracular nerve and opposite axonal processes into the syn-
ganglion (Fig. 13.12B) (Šimo et al. 2009a). Similar PN producing several neuropeptides are
found in insects and crustaceans (Davis et al. 1993; Lu et al. 2002; Dai et al. 2007).
2.2.2. Innervation of the salivary glands

Tick salivary secretions are important for manipulation of the host immune system and the
transmission of various microbial pathogens. The tick SG are composed of numerous oval acini
(alveoli) clustered along branches of salivary secretory ducts. Female ticks contain 3 types of
acini: non-granular type I, attached mostly to the main salivary duct, and granular types II and
III, which are distributed more peripherally and differ in the number and structure of granular
secretory and epithelial cells (Sauer et al. 1995; reviewed in Sonenshine 1991).
FIGURE 13.10: Cross-section of pedal nerve trunk 1 in Dermacentor variabilis showing the large number
of axons and dendrites surrounded by a thick neurilemma. No mesoaxons are evident in this profi le. The
black outline of many of the axons represents acetylcholinesterase reaction product. The highly folded
membranes between many of the axons and dendrites probably represent glial cells. Measurement bar,
1.0 μm. From Carson, K.A., Sonenshine, D.S., Boland, L.M., and Taylor, D. (1987) Ultrastructural
localization of acetylcholinesterase in the synganglion of the tick, Dermacentor variabilis (Say). Cell
Tissue Res. 249:615–623, with permission from Springer-Verlag.
FIGURE 13.11: Transmission electron micrograph illustrating the foveal nerve, an example of a peripheral
nerve in Dermacentor variabilis. The section shows the entire nerve suspended in an amorphous fusiform
sac with narrow fascicles extending from either end. Note the mesaxons (single arrow) from the
neurilemma surrounding groups of axons. Measurement bar, 2.0 μm. From Sonenshine, D.E., Homsher,
P.J., VandeBerg, J.S., and Dawson, D. (1981) Fine structure of the foveal glands and foveae dorsales of
the American dog tick, Dermacentor variabilis (Say). J. Parasitol. 67:627–646, with permission from the
American Society of Parasitologists.
Classical histochemical and ultrastructural techniques revealed the innervation of SG by

different peripheral nerves originating from the synganglion. The innervation of SG by a pair of
pedal nerves was described for the first time in Haemaphysalis flava (Saito 1960). Later, several
authors reported that the SG of ticks are innervated by palpal, lateral, paraspiracular, and pedal
nerves 1–3 (Obenchain and Oliver 1976; Binnington 1978). The specific innervation of non-
granular and granular acini by axon terminals indicates direct neural control of salivary gland
activity, presumably through the paracrine release of catecholamines (see below) (Coons and
Alberti 1999).
Immunohistochemical staining with antibodies to MIP, SIFamide, orcokinin, PDF, RFamide,
and tachykinin revealed the peptidergic nature of this innervation (Šimo et al. 2009a, 2009b, 2013
[also see Šimo et al. 2011b for a review]; L. Roller, A. Mizoguchi, Y. Tanaka, M. Slovák, and
D. Žitňan, unpublished data). Thus far, axonal projections of 4 distinct types of central neurons
innervating SG have been traced. Each neuronal type innervates very specific regions or acini of
the SG. A pair of giant protocerebral neurons (PcSG) producing MIPs and SIFamide innervates
only the type II and III acini (Figs. 13.13, 13.14A, and 13.14B; also see Figs. 13.8A–13.8I). The anatomy
and innervation patterns of these neurons are very similar in most tick species described below.
FIGURE 13.12: Allatotropin-like immunoreactivity in the peripheral nervous system of an unfed

Rhipicephalus appendiculatus female. A, Cluster of 3 cheliceral peripheral neurons (ChPN1-3,
arrowheads) in each cheliceral nerve. These bipolar neurons project axons into the periphery and into
the synganglion (arrows). B, Group of 5 paraspiracular peripheral neurons (PsPN1-5, arrowheads)
projecting axons into the synganglion and opposite processes into the periphery through a side branch
of the paraspiracular nerve (arrows). Measurement bar, 5 μm. From Šimo, L., Slovák, M., Park, Y., and
Žit{an, D. (2009) Identifi cation of a complex peptidergic neuroendocrine network in the hard tick,
Rhipicephalus appendiculatus. Cell Tissue Res. 335:639–655, with permission from Springer-Verlag.
Each neuron projects a thick axon via the palpal nerve and salivary nerve 1 that forms an elabo-
rate branching network of fine axons believed to terminate on the luminal surfaces of secretory
cells in the basal region of the type II and III acini (Fig. 13.13). Both MIP and SIFamide were
detected in the synganglia and in salivary gland extracts via matrix-assisted laser desorption/
ionization–time of flight mass spectrometry (MALDI TOF/TOF) (Figs. 13.8J, 13.8K). This fi-
nding confirms the immunohistochemistry data suggesting that both of these highly conserved
arthropod neuropeptides are delivered via axonal projection to the salivary gland acini (Šimo
et al. 2009b). Comparative morphology has shown that in the evolutionarily older genus Ixodes,
MIP- and SIFamide-immunoreactive axons enter each acinus through the basal region and ar-
borize into 4 to 5 and 3 to 4 elongated axon terminals with varicosities in the basal luminal surface
of the type II and III acini, respectively (Figs. 13.8D–13.8I, 13.13B, 13.13C, 13.14B, 13.15A, and
13.15B). In contrast, phylogenetically younger tick genera such as Dermacentor, Amblyomma, and
Rhipicephalus show characteristic short bifurcating axon terminals in both type II and type III
acini (Figs. 13.14B, 13.15C–13.15F, and 13.16A–13.16D). In I. scapularis, three-dimensional recon-
structions of confocal images of MIP/SIFamide-immunoreactive axon terminals led to the iden-
tification of the target secretory cells in the SG. These studies showed that PcSG neurons innervate
4 to 5 and 3 to 4 basal granular cells in type II and III acini, respectively (Šimo et al. 2009b, 2013;
Figs. 13.8D–13.8I, 13.13B, and 13.13C). Both receptors for MIP and SIFamide have been recently
identified, and they are found to be expressed in I. scapularis SG and synganglion (Šimo et al.
2013). The localization of the SIFamide receptor in the SG suggests three potential target cell types
and their probable functions: myoepithelial cell that might function in the contraction of the acini
and/or the control of the valve; large, basally located dopaminergic granular cells for the regula-
tion of paracrine dopamine; and neck cells that might be involved in the control of the acinar duct
and its valve (Fig. 13.13D) (Šimo et al. 2013).
Antibodies to PDF and orcokinin have been used to describe axonal projections of 4 OsSG
neurons in the dorso-medial region of the opisthosomal lobe. These neurons are even more
specific and innervate only type II acini. A single axon from each OsSG neuron enters the lateral
nerve and projects 3 branches into salivary nerves 2–4, which run along major salivary ducts
and terminate within acini II. In all tick species examined, axon terminals of OsSG are charac-
terized by 3 to 7 branches containing varicosities (Figs. 13.14C, 13.14D, and 13.16A–13.16C) (Šimo
et al. 2009a, 2011b; L. Roller, A. Mizoguchi, Y. Tanaka, M. Slovák, and D. Žitňan, unpublished
data). A single identified RFamide-immunoreactive neuron in each palpal lobe innervates the
salivary ducts. This neuron, named PaSG, projects an immunoreactive axon through the palpal
nerve and salivary nerve 1 and forms a branching axonal network along the main ducts of the
SG. Numerous varicosities along these branching axons indicate that RFamide-like peptides are
released on the surface of the salivary ducts (Fig. 13.16D). These axons never terminate on secre-
tory acini of the SG (Šimo et al. 2009a). Finally, as described above, the most prominent pair of
neurons showing tachykinin-like IR (Pd1SG) projects several branching axons to multiple pe-
ripheral nerves. The anterior axonal branch of each of these neurons exits the synganglion via
the palpal nerve and projects fine arborizing axons along the salivary ducts. These axons contain
numerous varicosities and do not innervate salivary gland acini (Fig. 13.16E) (Šimo et al. 2009a).
The complex and very specific innervation of the SG by peptidergic neurons might represent
an adaptation for the ectoparasitic life of ticks. These data indicate that neuropeptides from
these axon terminals might control salivary duct contractions and the activity of specific cells in
acini types II and III during the feeding of ticks.
2.2.3. Periganglionic sheath

This sheath is composed of a thin layer of cells (approximately 1.5 to 5 μm in thickness) sur-
rounding the synganglion. It is considered to be a putative neurohemal organ, as indicated by
elaborate axonal arborizations and axon terminals containing varicosities that originate from sev-
eral identified NS cells in the synganglion. The periganglionic sheath probably serves as a release
site for neuropeptides into the periganglionic sinus and/or hemolymph (Šimo et al. 2009a). Im-
munohistochemical staining with antibodies to PRVamide, BMS, kinin, and tachykinin identified
FIGURE 13.13: Simplifi ed schematic diagram showing the myoinhibitory peptide (MIP) and SIFamide
axonal innervation of the unfed Ixodes scapularis female salivary gland. A, Axonal projection from the
PcSG neuron to the salivary glands. Different colors of nuclei in B and C indicate different types of cells
based on their size and shape in the confocal image. B, purple, neck cells; blue, basal granular cells;
green, apical granular cells; pink, epithelial cells. C, purple, neck cells; blue, basal granular cells; aqua
blue, basal cells; greenish gray, apical cells; pink, epithelial cells. Note that only acini II and III are
innervated; MIP/SIFamide innervation was never observed in acini I. Four or fi ve axonal branches
(arrowhead) in acini II and 3 or 4 branches in acini III are in contact with the basal cells that have large,
round nuclei (arrow—axon running along the salivary duct). D, Simplifi ed diagram of the acinus type III
basal region in a partially fed female. The nuclei of different cells are shown as circles. SIFamide
peptidergic axon terminals are closely associated with SIFamide receptors, as depicted. Three different
types of cells in the basal region are annotated, with the cell boundary indicated only for the
myoepithelial cell, as described by Krolak et al. (Krolak, J.M., Ownby, C.L., and Sauer, J.R. [1982] Alveolar
structure of salivary glands of the lonestar tick, Amblyomma americanum (L.): unfed females. J. Parasitol.
68:61–82). PcSG, protocerebral salivary gland neurons; PaN, palpal nerve; SgN1, salivary nerve 1. From
Šimo, L., Žit{an, D., and Park, Y. (2009) Two novel neuropeptides in innervation of the salivary glands of
the black-legged tick, Ixodes scapularis: myoinhibitory peptide and SIFamide. J. Comp. Neurol. 517:551–
563; and Šimo, L., Koči, J., and Park, Y. (2013) Receptors for the neuropeptides, myoinhibitory peptide and
SIFamide, in control of the salivary glands of the blacklegged tick Ixodes scapularis. Insect Biochem. Mol.
Biol. 43:376–387, with permission from John Wiley & Sons and Elsevier, respectively.
329
FIGURE 13.14: Representations of identifi ed central neurons innervating salivary glands (SG).
A, Myoinhibitory peptide (MIP)-like immunoreactive (IR) axon terminals from giant protocerebral
salivary gland neurons (PcSG) exit the synganglion through the palpal nerve and salivary nerve 1 (SgN1)
and innervate acini types II and III. B, I. ricinus and I. scapularis axon terminals of these neurons showed
4 to 5 and 3 to 4 branches containing varicosities in acini types II and III, respectively. In Dermacentor
reticulatus, Rhipicephalus appendiculatus, Rhipicephalus sanguineus, Amblyomma variegatum, and
Amblyomma americanum, these axon terminals lack varicosities and show typical bifurcating axon
terminals. C, Pigment dispersing factor (PDF)-like IR neurons (OsSG1, 2, opisthosomal salivary gland
neurons) exit the synganglion through lateral and salivary nerves 2–4 (SgN2–4) to innervate specifi c type
II acini. D, Detailed view of PDF-like IR axon terminals in acini type II in 7 different tick species.
Branching axon terminals contain varicosities. Note that immunostaining using orcokinin antibody has
revealed staining patterns in OsSG1,2 neurons and their projections that are similar to those in type II
acini (L. Roller, A. Mizoguchi, Y. Tanaka, M. Slovák and D. Žit{an, unpublished data). From Šimo, L.,
Slovák, M., Park, Y., and Žit{an, D. (2009) Identifi cation of a complex peptidergic neuroendocrine network
in the hard tick, Rhipicephalus appendiculatus. Cell Tissue Res. 335:639–655 and Šimo, L., Žit{an, D., and
Park, Y. (2011b) Neural control of salivary glands in ixodid ticks. J. Insect Physiol. 58(4):459–466, with
permission from John Wiley & Sons and Elsevier, respectively.
330
FIGURE 13.15: Myoinhibitory peptide (MIP) immunoreactivity in salivary gland innervation of 3 Ixodid
ticks. Ixodes ricinus (unfed female) whole salivary gland (A) and detailed view to acini III (B).
Dermacentor reticulatus (unfed female) whole salivary gland (C) and detailed view to acini III (D).
Amblyomma variegatum (4-day-fed larvae) whole salivary gland (E) and detailed view to acini II and III
(F). Note that 3 or 4 branches of axon terminals were found in I. ricinus, whereas bifurcated axon
terminals were observed in D. reticulatus and A. variegatum. Arrows in A indicate non-innervated acini
I. Arrowheads indicate MIP-immunoreactive axon terminals in acini II and III. Measurement bars: 50 μm
(A, C, E) and 30 μm (B, D, F). Based on unpublished data from L. Šimo and D. Žit{an.
distinct sets of NS cells in the supraesophageal region of the synganglion that project branching
axons containing numerous varicosities to the periganglionic sheath. These include 3 pairs of
large protocerebral NS cells showing PRVamide-like IR (PcNS1-3), 4 NS cells co-expressing kinin-
and BMS-like IR in each cheliceral lobe (PcNS1-4), and large pedal neurons (Pd1SG) producing
tachykinin-like substances (Figs. 13.6A, 13.6B, 13.7H, 13.7K, 13.7L, and 13.7O) (Šimo et al. 2009a).
FIGURE 13.16: Neuropeptide immunoreactivity (IR) in the unfed Rhipicephalus appendiculatus female salivary
gland innervation. A–C, Merged consecutive confocal sections of salivary glands showing double staining
with antibodies to pigment dispersing factor (PDF) (green, solid arrowheads) and myoinhibitory peptide
(MIP) (red, open arrowheads). A, Dorsal side. B, Ventral side. C, Higher magnification. Note the PDF-like IR
in axons containing varicosities along salivary ducts and multiple axon terminals exclusively on acini type II,
whereas axons containing MIP-like IR terminate on acini II and III. Acini I located along the main salivary
duct are not innervated (arrow). D, Double-staining with antibodies to RFamide (green, solid arrow) and MIP
(red, open arrowheads). Note that RFamide-like IR is restricted to axons running along salivary ducts,
whereas adjacent MIP-like immunopositive axons terminate on salivary acini II and III. E,Tachykinin-like IR
(green, solid arrows) and MIP-like IR (red, open arrowheads) in branching axonal processes innervating
ducts of salivary glands. Measurement bars: 100 μm (A, B) and 50 μm (C–E). From Šimo, L., Slovák, M., Park,
Y., and Žit{an, D. (2009) Identification of a complex peptidergic neuroendocrine network in the hard tick,
Rhipicephalus appendiculatus. CellTissue Res. 335:639–655, with permission from Springer-Verlag.
2.2.4. Lateral segmental organs

These small cell clusters are located in the lateral nerve plexus (LNP) (Fig. 13.1B). The LSO con-
sist of 2 to 4 pairs of globular glands in the LNP adjacent to the synganglion proper. Staining
with paraldehyde-fuchsin and vital dyes characterized the LSO as secretory glands (Obenchain
and Oliver 1975; Panfilova 1978), and ultrastructural studies suggested their ecdysteroidogenic
role (Binnington 1981). However, immunohistochemical staining with neuropeptide antibodies
revealed possible dual functions of these organs (i.e., neurohemal and endocrine). RFamide-
like IR in axons and their terminals on the surfaces of LSO indicate a possible neurohemal
function of these organs reminiscent of the corpora cardiaca in insects (Fig. 13.7J). Although
neurons projecting their axons into the LSO have not been identified, it is very probable that a
limited number of NS cells in the synganglion use the LSO as neurohemal organs. Their pos-
sible endocrine function is indicated by strong bombyxin- and PDF-like IR in intrinsic cells in
each of the LSO (Figs. 13. 7B, 13.7F) (Šimo et al. 2009a). Light microscopy studies suggest that
there are 6 to 8 cells in each of the LSO (Roshdy and Marzouk 1982) but the exact number of
these putative endocrine cells could not be determined using immunofluorescence techniques.
These data suggest that the LSO are composed of peptidergic endocrine cells and might not
produce ecdysteroids.
2.2.5. Retrocerebral organ complex

This organ is located on the posterior dorsal margin of the supraesophageal region, just below
the junction of the esophagus with the proventriculus. The ROC is supplied by a pair of com-
pound nerves formed by the fusion of the primary and accessory nerves to the ROC noted
previously. The NS axonal terminals from these 3 major NS tracts are located in this organ.
Other adjacent nerves, such as the recurrent nerve, which supplies the adjacent hyperesophageal
ganglion, are not involved in innervating the ROC and should not be confused with NS activity.
The ROC consists of an unpaired dorsal lobe and paired ventral lobes and is surrounded by an
extension of the neurilemma and perineurium. Extensions of this sheath form a neurohemal
sinus that opens into the periganglionic sinus, facilitating the passage of secretions directly into
the hemolymph. NS axons from the compound ROC nerve also extend to the proventriculus,
where they terminate in a complex proventricular nerve plexus (PvPlx). In O. parkeri, the ROC
is innervated by a retrocerebral branch of the esophageal nerve. In spite of the neuroanatomical
and electron microscopy studies indicating a putative neurohemal role of the ROC (Sonenshine
1991), no neuropeptide IR has been detected in these organs (Šimo et al. 2009a).
2.2.6. Gut innervation and endocrine cells

In R. appendiculatus, antibody to FGLamide-related allatostatin (allatostatin A) reacts with a
network of branching axon terminals on the surface of the rectum (Fig. 13.17A) (L. Šimo and
D. Žitňan, unpublished data). This innervation probably originates from the central OsDM1
neurons projecting axons into the opisthosomal nerves, although a direct connection between
these neurons and the gut needs to be confirmed. FGLamide IR is also detected in several endo-
crine cells in the anterior region of the midgut (Fig. 13.17B) (L. Šimo and D. Žitňan, unpublished
data). Although this is the first report of neuropeptide IR in the tick gut, it has been demon-
strated that the gut is a very rich source of regulatory peptides in most insects and other inver-
tebrates (Sehnal and Žitňan 1996; Veenstra et al. 2008).
2.2.7. Pedal endocrine cells

A peptide sequence closely related to insect pre-ecdysis triggering hormone (PETH) and ETH
was identified in the I. scapularis genome using a BLAST search. Insect PETHs and ETHs are
produced by endocrine Inka cells located on the surface of the tracheal system (Žitňan et al.
FIGURE 13.17: FGLa-related allatostatin (allatostatin A) immunoreactive responses in branching axon

terminals on surface of the rectum (A) and posterior gut endocrine cells (B) in unfed Rhipicephalus
appendiculatus females. Arrows indicate axons running along the gut surface. Arrowheads indicate axon
terminal network on the rectum surface. Measurement bar, 50 μm. Based on unpublished data from
L. Šimo and D. Žit{an.
FIGURE 13.18: Pre-ecdysis triggering hormone (PETH)-immunoreactive peripheral cells in pharate

nymphs of the ticks Ixodes ricinus (A) and Rhipicephalus appendiculatus (B). These paired cells are
located on lateral sides of each pedal segment. Measurement bar, 5 μm. From Roller, L., Žit{anová, I.,
Dai, L., Šimo, L., Park, Y., Satake, H., Tanaka, Y., Adams, M.E., and Žit{an, D. (2010) Ecdysis triggering
hormone signaling in arthropods. Peptides 31:429–441, with permission from Elsevier.
2003). This indicates the possible presence of Inka cell homologues that produce ETH in ticks.
The cross-reaction of PETH antiserum with a PRXamide (X = V, I, L, or M) was used to identify
cells producing this peptide in the tracheal system or peripheral organs of I. ricinus and
R. appendiculatus. No cells or structures were stained on the tracheae, but a novel system of
paired oval cells named “pedal endocrine cells” were observed on the lateral sides of each pedal
segment in nymphs 1 day before ecdysis (Fig. 13.18) (Roller et al. 2010). However, further studies
are necessary in order to determine whether these pedal endocrine cells represent tick homo-
logues of insect Inka cells.
3. CHARACTERIZATION OF TICK NEUROPEPTIDES

AND BIOGENIC AMINES
3.1. IDENTIFICATION OF NEUROPEPTIDES

Ixodid ticks are unique among blood-feeding arthropods in their ability to feed on vertebrate
hosts for prolonged periods. During feeding, in addition to compromising of the host defense
system, major physiological challenges include the need to increase body size to accommodate
the enormous amount of blood consumed, increased metabolic rate, and the removal of meta-
bolic waste and excessive water via the SG. Blood feeding occurs along with mating and egg
development in the adult stage and with molting in the larval and nymphal stages. Neuropep-
tides and protein hormones (collectively, neuropeptides) are among the most important
signaling molecules directly and indirectly involved in all of these biological processes.
The neuropeptide signaling system is believed to be of ancient lineage. It has been found in
the first appearance of metazoans and has largely diversified into several groups in various bio-
logical systems. Higher metazoan animals utilize a wide repertoire of peptidergic signaling
systems (Hökfelt 1991; Strand 1999; Hewes and Taghert 2001; Bouwman et al. 2006; Husson et al.
2009). Even sessile animals with relatively simple behaviors and physiology, such as Hydra, uti-
lize numerous neuropeptides in their signaling systems (Grimmelikhuijzen et al. 1996; Hansen
et al. 2002; Koizumi et al. 2004; Takahashi et al. 2008; Anctil 2009). In the genomes of insects
more closely related to ticks, about 40 to 45 genes encoding pre-pro neuropeptides have been
described (Hewes and Taghert 2001; Johnson 2006; Li et al. 2008; Roller et al. 2008), including
42 genes in Drosophila melanogaster and 41 genes in the red flour beetle, Tribolium castaneum.
As neuropeptide signaling systems in metazoan taxa are highly conserved, tick neuropep-
tides are closely related to those identified in insects and crustaceans. Most tick neuropeptides
have clear counterparts in other invertebrate species. Neuropeptides can be identified by their
conserved sequence motifs, as well as by the structure of pre-pro hormones presenting multiple
repeated mature peptide sequences separated by cleavage sites; these are typical structures found
in many ticks’ pre-pro peptides (L. Šimo and Y. Park, unpublished data). Some neuropeptide
precursor genes are predicted to encode single neuropeptides, whereas others often contain
tandemly repeated multiple peptides of identical, similar, or different peptides. Signatures of the
orthologies in insects, crustaceans, and even mollusks suggest that these signaling molecules
have an early evolutionary origin in arthropods.
3.1.1. Genomic approach

Several species of ticks have been the subject of transcriptome projects because of their importance
in disease transmission (e.g., Francischetti et al. 2005; Ribeiro et al. 2006; Alarcon-Chaidez et al.
2007; Wang et al. 2007). In total, 80 putative neuropeptides from different tick species have been
characterized from expressed sequence tags (ESTs) using homology searches (Christie 2008).
A recent transcriptome study of the Dermacentor variabilis synganglion resulted in the discovery
of 14 neuropeptide genes (Table 13.2) and 5 putative neuropeptide receptors (gonadotropin re-
ceptor, leucokinin receptor, sulfakinin receptor, calcitonin receptor, and pyrokinin receptor), iden-
tified via the pyrosequencing of unfed, partially fed, and replete female ticks (Donohue et al. 2010).
These EST analyses have greatly expanded the number of known/predicted ixodid neuropeptides.
For a description of the putative roles of these neuropeptides in regulating biological functions in
ticks, see Chapter 16.
The recently available I. scapularis genome (Hill and Wikel 2005) represents the largest data
set of putative neuropeptide genes to date (L. Šimo and Y. Park, unpublished data) and provides
an excellent opportunity to study the functions and evolution of the genes encoding neuropep-
tides. In the I. scapularis genome sequence, homology-based searches identified a total of 34
genes encoding more than 60 mature peptides (Table 13.2) (L. Šimo and Y. Park, unpublished
data). Nineteen of the 34 neuropeptide genes are also present in the EST dataset, and the pres-
ence of 18 mature peptide sequences was confirmed via MALDI TOF/TOF analyses (see Section
3.1.2 for more details) (Neupert et al. 2009; L. Šimo and Y. Park, unpublished data). The total
number of identified neuropeptide genes is lower than the number generally found in insects;
this is likely due to the difficulties experienced in obtaining the complete sequence and assembly
of this large tick genome. In addition, there might be novel neuropeptide genes that were missed
using homology-based searches. Although there is some risk of missing data in the homology-
based prediction of neuropeptide genes, the consensus between genome annotation and pro-
teomics strongly supports our belief that the majority of neuropeptides have been discovered
using neuropeptide annotation from the genome sequence.
The functions of most neuropeptides in ticks are not currently known; however, current
studies of the localization of neuropeptides using immunohistochemistry (Šimo et al. 2009a,
2009b) have provided insightful observations. Because there are only a few examples of studies
of tick neuropeptides, here we briefly discuss the recent highlights of these studies. For a de-
scription of the putative roles of these neuropeptides in regulating biological functions in ticks,
see Chapter 16.
Orcokinins were found to be important in regulating contractions of the digestive tract in
crustaceans (Stangier et al. 1992; Pascual et al. 2004) and in neuronal regulation of ecdysteroido-
genesis in the silkworm Bombyx mori (Yamanaka et al. 2011). Interestingly, there seem to be
multiple genes encoding orcokinins in ticks. To date, 4 orcokinin genes have been found in
D. variabilis (Donohue et al. 2010), 2 in Rhipicephalus (Boophilus) microplus (L. Šimo and Y.
Park, unpublished data), and 1 in each of I. scapularis and Amblyomma americanum (Christie
2008). Further study might determine whether this gene expansion in ticks, as opposed to the
case of single genes in insects, is the result of the tick’s hematophagous biology.
A G protein–coupled receptor (GPCR) for the kinin-like peptides has been cloned and func-
tionally characterized in the southern cattle tick, R. microplus (Taneja-Bageshwar et al. 2006).
Numerous kinin-like immunoreactive axon terminals in the periganglionic sheath of the
R. appendiculatus synganglion indicate a neurohormonal function of these neuropeptides (see
Section 2.1.5 of this chapter) (Šimo et al. 2009a). The partial sequence of a kinin gene has been
found in the I. scapularis genome. This gene encodes 16 tandemly repeated homologous mature
peptides, 6 of which are identical in their amino acid sequence (L. Šimo and Y. Park, unpub-
lished data). Most of the recent studies on insect kinins have described the diuretic action of
these peptides through their actions on the stellate cells of the renal tubules (Terhzaz et al. 1999;
Radford et al. 2002, 2004; Dow and Davies 2006; Coast 2007). The removal of excessive water
during blood feeding, which is performed through the SG in ticks (Kaufman and Phillips 1973),
is considered an important function. Thus, osmoregulatory peptides are an important area for
future studies. Whether kinin-like peptides regulate other functions in ticks as they do in insects
remains to be determined.
The recent discovery of MIPs (also called type B allatostatins) and SIFamide neuropeptides
in tick SG innervations (Šimo et al. 2009b) and the identification of their representative recep-
tors (Šimo et al. 2013) suggest an important role (or roles) for these 2 peptides in the control of
SG function (see Section 2.2.2 of this chapter). The function of SIFamide peptide in arthropods
is still unclear. In the lobster Homarus americanus, the injection of SIFamide elicited myostimu-
latory action on the pyloric valve, which regulates the flow of food particles from the foregut to
the midgut (Christie et al. 2006), whereas in Drosophila, SIFamide controls male and female
mating behaviors (Terhzaz et al. 2007). In contrast, various types of bioassays have been used to
study inhibitory functions of MIPs in insects, including the inhibition of rhythmic contractions
of the isolated hindgut and oviduct of Drosophila (Schoofs et al. 1991), inhibitory effects of JH
production in crickets (Lorenz et al. 1995a, 1995b), the suppression of ecdysteroid secretion from
prothoracic glands in B. mori (Hua et al. 1999), and the inhibition of spontaneous peristaltic
contractions in the adult Manduca sexta hindgut (Blackburn et al. 2001). We speculate that the
2 antagonistic activities—stimulation by SIFamide and inhibition by MIP—are important in the
neural control of tick SG.
Bursicon, corazonin, and eclosion hormone are known to be regulators of insect cuticle de-
velopment and molting (Truman and Copenhaver 1989; Dewey et al. 2004; Kim et al. 2004; Dai
et al. 2008). In ticks, molting does not occur in the adult stage. Consequently, differential tem-
poral expressions of bursicon, corazonin, and eclosion hormone during the different stages of
D. variabilis females might indicate an important role for these 3 classes of neuropeptides, pos-
sibly in relation to the massive cuticle growth needed for feeding and reproduction (Bissinger
et al. 2011). However, their real roles in ticks remain unknown.
Uncovering the functions of neuropeptides in ticks is an open area of study. Developing
molecular tools such as RNA interference and increasing the pool of resources including the
genome sequence of I. scapularis will assist new discoveries in this area of study. These discov-
eries will provide new insights into mechanisms for disrupting the tick life cycle and physiology
related to pathogen transmission.
3.1.2. Proteomic approach

The neuropeptide annotations obtained from a genome or transcriptome sequence also can be
confirmed via proteomic analysis. Indeed, the modern proteomic tool of MALDI TOF/TOF has
proven to be the simplest way to identify mature peptide molecules in ticks (Neupert et al. 2005,
2009; Šimo et al. 2009b). Because the predictions of the function of mature neuropeptides are
often unreliable, such proteomic identification of the mature peptides is essential. Total mass
(mass-to-charge, or m/z) and the spectra in tandem mass spectrometry allow the accurate iden-
tification of peptide sequences. This technique is often expanded to identify the tissue source of
particular neuropeptides through the use of MALDI imaging (DeKeyser and Li 2006) and for
the quantification of peptides (Brockmann et al. 2009).
CAPA peptide (molecular weight [MW] = 1,008.6 Da, PALIPFPRVamide) was the first tick
peptide neurohormone identified via MALDI TOF/TOF on a single neuronal cell isolated from
the synganglion of Ixodes ricinus (Neupert et al. 2005). This peptide shows high sequence ho-
mology to insect CAPAs such as periviscerokins/CAP2b, which are well known for their func-
tions in diuresis (Kean et al. 2002) and pheromone production (Rafaeli 2002). Two other
neuropeptides, MIP (MW = 1,321.6 Da, ASDWNRLSGMWamide) and SIFamide (MW =
1,395.7 Da, AYRKPPFNGSIFamide), were also identified in I. scapularis by means of MALDI
Table 13.2: Neuropeptides identified in various species of ticks.
Gene name/mature peptide(s) Amino acid sequence Evidence References

IXODES SCAPULARIS
Adipokinetic hormone (AKH)
AKH QITFSKNWQPa G 1
Allatostatin CC
Allatostatin CC GEGKMYWKCYFNAVSCF G 5
Allatotropin (AT)
AT-1 ..GFQKLRLSTARGFa G 1
AT-2 GFRKMKISTARGFa G 1
Arginine-vasopressin-like peptide (AVLP)
AVLP ..CFITNCPPGa G 1
Bursicon alpha subunit
Bursicon alpha subunit >ISCW004617-RA G 2
Bursicon beta subunit
Bursicon beta subunit >ISCW004618-RA G 2
Calcitonin-like diuretic hormone 34a (CT/DH)
CT/DH 34a AGGLLDFGLSRGASGAEAA- M, E, G 4, 7
KARLGLKLANDPYGPa
Calcitonin-like diuretic hormone 34b (CT/DH)
CT/DH 34b SRGMLDFGMTRGASGAKAAK- E, G 1
ARLGLKLANDPFGPa
CCHamide
CCHamide ..SCKMYGHSCLGGHa E, G 1
Corticotropin-releasing-factor-related diuretic hormone (CRF/DH)
CRF/DH >ISCW007845-RA E, G 7
Corazonin (CRZ)
CRZ pQTFQYSRGWTNa M, E, G 2, 4, 7
Crustacean cardioactive peptide (CCAP)
CCAP PFCNAFTGCGa G 1
Crustacean-hyperglycemic-hormone-related ion transport peptide
(CHH/ITP) a
CHH/ITPa >ISCW023228-RA E, G 2, 7
(CHH/ITP) b
CHH/ITPb >EW937910.1, >EW937909.1 E, G 1
Ecdysis triggering hormone (ETH)
ETH ..QGSWNGTIKMGAVFTSDA- G 6
QNIPRIa
Eclosion hormone (EH)
EH >ISCW001941-RA G 2
FGLa-related allatostatin (FGLa/AST)
FGLa/AST-1 RPPAAMYGFGLa G 2
FGLa/AST-2 GERPQHPLRYGFGLa G 2

FGLa/AST-3 LDRDGNYPGSIDHNRRERHR- G 2
FGFGLa
FGLa/AST-4 RYNFGLa G 2
Glycoprotein hormone alpha
Glycoproteine hormone alpha >CAR94694.2 E, G 2, 8
Glycoprotein hormone beta
Glycoproteine hormone beta >ISCW010926 E, G 2, 8
Insulin related peptide (Irp) 1
Irp >ISCW002549-RA E, G 1
Insulin-related peptide (Irp) 2
Irp >ISCW007400 G 1
Insulin-related peptide (Irp) 3
Irp >ISCW020331 G 2
Kinin
Kinin-1 ENDKDKELSFNPWGa G 1
Kinin-2 GSFSSWGa G 1
Kinin-3 (6 copies) DTFGSWGa G 1
Kinin-4 QDKESGFNPWGa G 1
Kinin-5 EDPFNPWGa G 1
Kinin-6 KEDKNAFSPWGa G 1
Kinin-7 DQNFNPWGa G 1
Kinin-8 TTKDSTFSPWGa G 1
Kinin-9 EGPFNPWGa G 1
Kinin-10 GDSDTAFAPWGa G 1
Kinin-11 DNNFNPWGa G 1
Myoinhibitory peptide (MIP)
MIP-1 ..WNALSGMWa M, G 3
MIP-2 ASDWNRLSGMWa G 3, 4
MIP-3 ..WNDLSGYWa G 3
Neuroparsin (NP)
NP >EW836547.1 G 1
Orcokinin (OK)
OK-1 WYGHGDFDEIDNVGWPGFT M, E, G 4, 7
OK-2 NFDEIDRTGFEGFY M, E, G 4, 7
PISCF-related allatostatin (PISCF/AST)
PISCF/AST SGWKQCSFNAVSCFa M, G 4, 5
Proctolin
Proctolin RYLPT M, E, G 4, 7
Prothoracicotropic hormone (PTTH)
PTTH >ISCW001809 G 1
(continued)

Pyrokinin diapause hormone pheromone biosynthesis-activating
neuropeptide (PK/PBAN)
PK/PBAN-1 pQGLIPFPRVa M, E, G 4, 7
PK/PBAN-2 RSNNFTPRIa M, E, G 4, 7
PK/PBAN-3 ..SQQMIPIPRNa E, G 1, 7
PK/PBAN-4 GSFVPRLa M, E, G 4, 7
PK/PBAN-5 GSFTPRIa M, E, G 4, 7
PK/PBAN-6 AAFTPRIa E, G 1
PK/PBAN-7 TPFTPRIa E, G 1
SIFamide
SIFa AYRKPPFNGSIFa M, E, G 3, 4, 7, 9
Short neuropeptide F (sNPF)
sNPF GGRSPSLRLRFa M, E, G 4, 7
sNPF 3-11 SPSLRLRFa M 4
Sulfakinin (SK)
SK-1 QDDDYGHMRFa M, E, G 2, 4, 7
SK-2 SDDYGHMRFa M, E, G 2, 4, 7
Tachykinin-related peptides (TRP)
TRP-1 (2 copies) AFHAMRa M, E, G 4, 7
TRP-2 GSGFFGMRa M, E, G 4, 7
DERMACENTOR VARIABILIS
Bursicon alpha subunit >EU574002 E 2
Bursicon beta subunit >EU616824 E 2
Corazonin (CRZ)
CRZ pQTFQYSRGWTNa E 2
(CHH/ITP)
CHH/ITP >EU620224 E 2
Eclosion hormone (EH)
Eclosion hormone (EH) >EU567122 E 2
FGLa/AST-1 GPREPLRYGFGLa E 2
FGLa/AST-2 ERHRFSFGLa E 2
FGLa/AST-3 RYNFGLa E 2
Glycoprotein hormone alpha
Glycoprotein hormone alpha >EU620226 E 2
Glycoprotein hormone beta
Glycoprotein hormone beta >EU620225 E 2
Insulin-related peptide (Irp)
Irp >EU616823 E 2

Orcokinin (OK) 1
OK-1-1 NFDEIDRSDFGGFY E 2
OK-1-2 NFDEIDRTGFEGFY E 2
OK-1-3 GYGHGEFDEIDNAGWPGFY E 2
Orcokinin (OK) 2
OK-2-1 GYGHGEFDEIDHAGWPGFY E 2
Orcokinin (OK) 3
OK-3-1 NFDEIDRTDFGEFR E 2
OK-3-2 NFDEIDRTGFEGFR E 2
OK-3-3 NFDEIDRTGFGGFY E 2
Orcokinin (OK) 4
OK-4-1 GYGHGDFDEIDHAGWPGFY E 2
OK-4-2 (2 copies) NFDEIDRSGFDGFY E 2
Sulfakinin (SK)
SK-1 QEDDYGHMRFa E 2
SK-2 SDDYGHMRFa E 2
RHIPICEPHALUS MICROPLUS
FGLa/AST-1 AGPAPLYSFGLa E 2
FGLa/AST-1 GPREPLRYGFGLa E 2
Orcokinin (OK) 1
OK-1-1 GYGHGEFDEIDHAGWPGFY E 7
OK-1-2 NFDEIDRNGFEGFT E 7
Orcokinin (OK) 2
OK-2-1 GYGHGEFDEIDNAGWPGFY E 7
OK-2-2 NFDEIDRSDFGGFY E 7
Proctolin
Proctolin RYLPT E
AMBLYOMMA AMERICANUM
Crustacean-hyperglycemic-hormone-related ion transport
peptide (CHH/ITP)
CHH/ITP >BF007944 E 1
Insulin-related peptide (Irp)
Irp >BM290637, >BM292347, E 1
>BM291472, >BM292589
Orcokinin (OK)
OK-1 GYGHGEFDEIDHAGWPGFY E 7
OK-2 NFDEIDRTGFEGFY E 7
(continued)

RHIPICEPHALUS SANGUINEUS
Pyrokinin diapause hormone pheromone biosynthesis-activating
neuropeptide (PK/PBAN)
PK/PBAN-1 QGLIPFPRVa E 1
PK/PBAN-2 RSNXFTPRIa E 1
AMBLYOMMA VARIEGATUM
PISCF-related allatostatin (PISCF/AST)
PISCF/AST GEGKMFWRCYFNAVSCF E 10
References: (1) Šimo and Park (unpublished data), (2) Donohue et al. (2010), (3) Šimo et al. (2009b), (4) Neupert et al.
(2009), (5) Veenstra (2009), (6) Roller et al. (2010), (7) Christie (2008), (8) Dos Santos et al. (2009), (9) Verleyen et al.
(2008), (10) Christie et al. (2011).
Neuropeptide and peptide hormone nomenclature is based on that of Coast and Schooley (2011).
M, mass spectrometry; E, EST; G, genome sequence; dots on N-terminal, unknown cleveage site; pQ, pyroglutamate on
N-terminal; a on C-terminal, putative amidation of C-terminal.
For longer peptide sequences, the accession number(s) assigned by the representative species project is given (Šimo and
Park, unpublished data).
TOF/TOF analysis. Both of these highly conserved arthropod neuropeptides were detected in
the synganglia and in salivary gland extracts, suggesting the involvement of these peptides in
the neural control of the SG (see Section 2.2.2 of this chapter; Figs. 13.8J, 13.8K) (Šimo et al.
2009b).
The first comprehensive proteomic analysis of I. scapularis using MALDI TOF/TOF was
provided by Neupert et al. (2009). In that study, direct tissue (synganglion) profiling yielded the
identification of 20 mature neuropeptides. The MALDI spectra of female synganglion from
I. scapularis showed a large variety of detectable peaks (potential peptides), but many of them
await further biochemical identification (Fig. 13.19) (L. Šimo and Y. Park, unpublished data).
Like the preceding examples in insects and other invertebrates, immunocytochemistry and
in situ hybridization have been helpful as tools to examine the localization of neuropeptides and
their transcripts. The anatomy of neuropeptidergic systems in ticks was described using dif-
ferent antibodies originally raised against insect and crustacean neuropeptides (see Section 2.1.5
of this chapter) (Šimo et al. 2009a). The application of developing proteomic techniques to the
study of tick neurobiology, such as MALDI imaging to show the spatial distribution of mass
spectra (DeKeyser and Li 2006) and quantitative MALDI (Brockmann et al. 2009), will expand
our knowledge when combined with molecular tools.
3.2. BIOGENIC AMINES

The metabolism of amino acids often leads to the formation of many small signaling molecules
used as hormones, neuromodulators, and neurotransmitters (NTs). NTs act through membrane
receptors, which are generally ion-gated channels or GPCRs, to activate, inhibit, or modulate
the excitability of the post-synaptic cell.
FIGURE 13.19: MALDI-TOF mass spectra of synganglia extract from Ixodes scapularis unfed females.
Spectra were measured using accumulation at 200 laser shots per second with conditions optimized for
the detection of m/z 500–3,500 Da. Note that synganglia extract was purifi ed using a ZipTip C18 column
(Millipore, Bedford, MA). TRP, tachykinin-related peptide; sNPF, short neuropeptide F; MIP,
myoinhibitory peptide; PISCF/AST, PISCF-related allatostatin/allatostatin C; CT/DH 34a, calcitonin-
like diuretic hormone. Based on unpublished data from L. Šimo and Y. Park.
Earlier reports have indicated the presence or biological activity of certain NTs in ticks. Do-
pamine, norepinephrine, serotonin, octopamine, γ-aminobutyric acid, glutamate, and acetyl-
choline represent molecules investigated at different levels in various tick species (see review by
Lees and Bowman [2007]). Additionally, nucleotide sequences encoding several enzymes (or
their activity) involved in the metabolism of NTs and putative NT receptors were identified.
Despite these findings, knowledge of the neurobiology of ticks has lagged behind studies in
other invertebrates. The recently assembled and annotated I. scapularis genome (Hill and Wikel
2005) has the potential to produce major progress in the field of tick physiology. Bioinformatics
combined with molecular techniques such as RNA interference can allow a reverse genetics
approach that could accelerate discoveries in tick neurobiology. These neurobiological studies
will likely result in the development of novel acaricides and vaccine targets.
3.2.1. Dopamine
Dopamine (DA) has been found in many arthropods, whereas DA in ticks has been studied al-
most exclusively with regard to salivary gland physiology. The functions of DA in arthropods
can be subdivided into 2 main categories: (i) neural and hormonal functions, and (ii) as the
source of catecholamine for sclerotization of the exoskeleton. In ticks, DA directly activates
salivary secretion following in vivo injection, as well as in isolated SG (for recent reviews, see
Sauer et al. 2000; Bowman and Sauer 2004). Pharmacological characterization of the DA re-
ceptor suggests that it is a D1-type receptor acting through an adenylcyclase to cause an increase
in cyclic adenosine monophosphate (cAMP).
A more comprehensive model of DA action on the SG in the partially fed tick has been pro-
posed (Sauer et al. 2000) in which DA activates 2 independent signaling pathways, cAMP-
dependent signal transduction leading to fluid secretion and a calcium-dependent signaling
pathway activating the release of prostaglandin E2 (PGE2). A high titer of PGE2 (average of 469
ng PGE2/ml [or ~14 ng] tick saliva), which is required in order to overcome the host defense
system, is found in tick salivary secretions (Ribeiro et al. 1992) and is also active in immunosup-
pression and possibly vasodilation (Oliveira et al. 2011). Additionally, PGE2 has an autocrine or
paracrine role within the SG, inducing the secretion of bioactive salivary proteins via intracel-
lular Ca2+ mobilization (Sauer et al. 2000).
The source of DA for salivary gland activation was not clearly defined until recently. In an
early study, histofluorescent methods (Falck and Torp 1962) for detecting catecholamines re-
vealed the presence of DA/noradrenaline in the synganglia and axons innervating SG in R. mi-
croplus (Binnington and Stone 1977), leading to a hypothetical pathway of neural dopaminergic
synapses controlling the SG. However, electrochemical and radioenzymatic assays found large
quantities of DA in the SG and synganglion of Amblyomma hebraeum, which does not corrobo-
rate the neural DA hypothesis (Kaufman et al. 1999). Furthermore, DA-immunoreactive large
vesicles were found in the basal cells of type II and type III acini, the cells innervated by the axon
terminals containing the neuropeptides (see above), SIFamide, and MIPs (Šimo et al. 2011a).
This DA IR was observed only between 12 and 40 hours post-attachment in I. scapularis females
(Figs. 13.20A–13.20F′). The large vesicles in the basal cells of acini were described by an indepen-
dent electron microscopy study showing the temporal dynamics of the vesicles released in the
first 2 days of feeding (Grigorieva and Amosova 2008). These findings strongly support Kaufman
et al.’s (1999) hypothesis that the major DA pool within the salivary gland is the source of the DA
activating the SG as a paracrine system. The paracrine DA hypothesis of salivary gland activa-
tion is further supported by the localization of the DA receptors. Homology-based searches
identified at least 3 DA receptors in the genome of I. scapularis; these are named for their or-
thologous relationship with the mammalian DA receptors D1, invertebrate specific D1-like
(InvD1L), and D2 (Šimo et al. 2011a). Two DA receptor transcripts were also confirmed in the
D. variabilis synganglion (Bissinger et al. 2011). Temporal and spatial expression patterns of the
I. scapularis D1 receptor examined via immunohistochemistry and reverse transcription poly-
merase chain reaction suggest that the D1 receptor is expressed in clustered patterns in the
junctions between cells on the luminal surface of type II and III acini (Figs. 13.20G–13.20L).
Reporter assays for the D1 receptor showed both DA-activated calcium mobilization and cAMP
elevation in heterologous expression systems (Fig. 13.21) (Šimo et al. 2011a). The D1 receptor
expressed in a mammalian cell line was also reactive to high doses of norepinephrine, with sen-
sitivity 20× lower than to DA (Meyer et al. 2011; Šimo et al. 2011a), but not to 10 μM octopamine
(Šimo et al. 2011a). Recently, the InvD1L receptor was functionally tested (Meyer et al. 2011) and
was found to be expressed in I. scapularis SG (L. Šimo, J. Koči, and Y. Park, unpublished data).
Summing up these findings, neural components including SIFamide and MIP neurons
might control the basal dopaminergic cells of type II and III acini, and the luminal secretion of
paracrine DA activates the D1 receptors on the luminal surface of these acini (Šimo et al. 2011a).
Further studies on other DA receptors might reveal diverging signal transduction pathways that
direct physiological changes in the SG. D1-receptor-immunoreactive neurons in various regions
FIGURE 13.20: Dopamine (A–F′) and dopamine D1 receptor (G–J) immunohistochemistry in the salivary
glands of female Ixodes scapularis during various feeding phases. A, A region of salivary glands with
clustered acini II (2) and III (3) 12 to 16 hours after attachment. B–F, Acini II 0–52 hours after attachment
to the host. B′–F′, Acini III at the same time points. Note that dopamine-positive staining (green marked
with arrowheads) was detected in acini II and III in the vesicles and their surrounding regions, but only
at 12–40 hours post-attachment. The images are z-stacks of multiple confocal layers with thicknesses of
20 μm (A) or 2 to 5 μm (B–F′). G, Whole acinus II of unfed female shown as a 27 μm thick composite
image. G′, Optical section (10 μm) of acinus II demonstrating the D1 receptor immunoreactivity. H,
Whole acinus III of an unfed female shown as a composite image of 34 μm thickness. H′, Optical section
(5 μm) of acinus III demonstrating the apical location of D1 receptor immunoreactivity. I, Acinus II. J,
Acinus III of 5 day post-attachment females. Confocal composite images (10 μm) overlaid with the
differential interference contrast images are shown. Schematic diagram showing the D1 (green),
dopamine (blue), and neuropeptidergic innervation (red) in acini II and III of females 12 to 16 hours
post-attachment (K) and 5 days post-attachment (L). Arrowheads in G–L indicate the scattered patches
of D1 receptor immunoreactivity on the luminal side of the acini. Dotted lines in G′, H′, I, and J indicate
the acini boundary. Blue in A–H′ is 40,6-diamidino-2-phenylindole staining for nuclei. Measurement bars,
10 μm. From Šimo, L., Koči, J., Žit{an, D., and Park, Y. (2011) Evidence for D1 dopamine receptor
activation by a paracrine signal of dopamine in tick salivary glands. PLoS One 6(1):e16158, with
permission from the Public Library of Science.
345
FIGURE 13.21: Reporter assays for Ixodes scapularis D1 dopamine receptor showing dopamine (DA)-
activated calcium mobilization and cAMP elevation. A, Luminescent reporter calcium assay dose-
response curves of Chinese hamster ovary cells transfected with the D1 and aequorin constructs and
treated with DA and norepinephrine. B, Luminescent reporter cAMP assay (GloSensor) showing
dose-response curves of human embryonic kidney cells transfected with the D1 and GloSensor
constructs for DA and norepinephrine. Measurement bars indicate standard errors for a minimum of 4
replicated plates. From Šimo, L., Koči, J., Žit{an, D., and Park, Y. (2011) Evidence for D1 dopamine
receptor activation by a paracrine signal of dopamine in tick salivary glands. PLoS One 6(1):e16158, with
permission from the Public Library of Science.
of the I. scapularis synganglion and their dynamic changes during blood feeding have also been
described (Fig. 13.22).
In addition to its role in stimulating salivary secretion, DA also has been found to stimulate
cuticle plasticization (Kaufman et al. 2010). The increase in tick size during blood feeding in-
volves expansion of the exoskeleton. In vivo injection of DA into partially fed A. hebraeum
showed that DA is the component inducing the plasticization of the cuticle. Pharmacological
data suggest that control of this plasticization is achieved via dopaminergic innervation of the
alloscutal integument (Kaufman et al. 2010).
Another catecholamine, norepinephrine (NE), has been described in ticks. Equal quantities
of DA and NE were isolated from the synganglion of R. microplus (Megaw and Robertson 1974).
Two NE transporters were reported in the D. variabilis synganglion (Bissinger et al. 2011).
NE has been shown to be the principal catecholamine in neurons of R. microplus (Stone et al.
1978). When injected, NE was shown to have high potential for triggering salivary secretion in
this species (Megaw 1974). The role of NE in ticks needs further investigation.
3.2.2. Serotonin
Serotonin (5-hydroxytryptamine) acts as an NT through multiple receptors and plays a key role
in regulating and modulating various physiological and behavioral processes. Serotonin-like IR
has been identified in the CNS of many species of invertebrates (reviewed by Nässel [1988]),
including spiders (Seyfarth et al. 1990a, 1990b), and is a well-documented NT/neurohormone in
insects (Coast et al. 2002; Lange 2004).
FIGURE 13.22: Schematic diagrams showing D1 receptor neurons in the synganglia of unfed females (A)
and females after repletion (F). Boxes with dotted lines on the diagrams represent regions of the
synganglion corresponding to immunohistochemical reactions shown in the right panel. B, Dorsal
protocerebral region. C, Pedal region. D, Opisthosomal neurons on the dorsal side. E, Periganglionic
sheath surface of the dorsal-pedal region showing the axon terminals arborization. G, Ventral
protocerebral, pedal, and opisthosomal region. H, Pedal and opisthosomal region containing rich axonal
arborization. Arrowheads in E indicate axonal arborization on the periganglionic sheath surface. The
fi rst 2 letters refer to the position of each D1-positive neuron in a specifi c lobe of the synganglion;
protocerebral (Pc), pedal 1–4 (Pd1–4), and opisthosomal (Os) lobes and the letters that follow refer to
the anatomical location of the neuron: dorsal (D), ventral (V), anterior (A), medial (M), or lateral (L) and
periganglionic sheath (PgS). An explanation of the abbreviations of labeled peptidergic neurons is
given in Section 2.1.5. Measurement bars: 50 μm (A–C, F–H) and 10 μm (D, E). From Šimo, L., Koči, J.,
Žit{an, D., and Park, Y. (2011) Evidence for D1 dopamine receptor activation by a paracrine signal of
dopamine in tick salivary glands. PLoS One 6(1):e16158, with permission from the Public Library of
Science.
347
A blood meal supplemented with serotonin and histamine in an artificial feeding system
resulted in a decrease in sucking and salivation, indicating an anti-feedant activity of serotonin
(Paine et al. 1983). However, in vitro application of serotonin to isolated SG did not show any
effect on salivary secretion (Needham and Sauer 1975; Kaufman 1977). A serotonin receptor was
isolated from a whole larvae extract of R. microplus, and its expression was found in all life stages
(i.e., eggs, larvae, nymphs, and adults) (Chen et al. 2004). Studies of the anatomical structures of
serotonin IR in the nervous systems of Dermacentor albipictus and A. americanum (Hummel et
al. 2007) found significant differences between species but not between sexes, highlighting
unique serotonin distributions in the synganglia depending on species (Fig. 13.23). The sero-
tonin IR in the glomeruli of the olfactory lobes implies a function of serotonin in olfactory sig-
nal transduction (Hummel et al. 2007).
3.2.3. γ-aminobutyric acid

γ-aminobutyric acid (GABA) is an NT that functions as a synaptic inhibitor, binding to
specific transmembrane ion-channel receptors. In ticks, GABA increased the membrane
FIGURE 13.23: Composite illustrations of the serotonin-like immunoreactive (5HT-IR) cells and
processes in the synganglion of male and female (A) D. albipictus and (B) A. americanum, drawn from
the dorsal perspective. These drawings were prepared after viewing many stacks of slices of whole
mounts of synganglion that were 5HT-IR labeled and imaged using a Zeiss LSM 510 laser scanning
confocal microscope. The esophagus appears twice in the drawings and is labeled as a marker to show
the locations of other regions with respect to the esophagus. The esophagus (Es) enters the synganglion
postero-dorsally and exits the synganglion antero-ventrally. I–IV, fi rst through fourth pedal neuromeres;
C, cheliceral neuromeres; Es, esophagus; Op, opisthosomal neuromere; OL, olfactory lobe; P,
protocerebrum; SB, stomadeal bridge; *, posterior dorsal neuromeres; open arrows, ventro-medial
neurosecretory tract; solid arrows, lateral neurosecretory tract; open arrowheads, 5HT-IR neurons at
base of pedal nerves; solid arrowheads, posterior 5HT-IR neurons; solid lines, regions of the synganglion
that contain an abundance of 5HT-IR neuronal processes; dashed lines, 5HT-IR neuronal processes
projecting from neurons to neuromeres or between neuromeres; spheres, 5HT-IR neurons. The figure
depicts 5HT-IR neurons in the ventral aspect, which would lie below the regions illustrated above these
5HT-IR neurons. Measurement bar, 100 μm. From Hummel, N.A., Li, A.Y., and Witt, C.M. (2007) Serotonin-
like immunoreactivity in the central nervous system of two ixodid ticks. Exp. Appl. Acarol. 43:265–278,
with permission from Springer-Verlag.
conductance associated with hyperpolarization when applied to the dorso-ventral body

muscle of A. hebraeum. This action was blocked by an antagonist of the GABA-A receptor (the
GABA-gated chloride channel), picrotoxin (Gration et al. 1986). High concentrations (>50
μM) of GABA applied to the synganglion of A. hebraeum caused a decrease in the frequency
of action potentials recorded from pedal nerve 4 (Gration et al. 1986). Measurements of the
free amino acid levels in the A. hebraeum synganglion showed a high quantity of GABA in the
synganglion relative to levels in other tissues, especially during feeding and early reproductive
periods (Lucien et al. 1995). GABA treatment of isolated A. hebraeum SG potentiated the ef-
fect of DA on fluid secretion (Lindsay and Kaufman 1986). The modulatory activity of GABA
on the SG was blocked by the GABA-A receptor antagonists picrotoxin and bicuculline. A
gene encoding the GABA-A receptor RdlDv was identified from D. variabilis. Heterologous
expression of RdlDv in Xenopus oocytes produced GABA-activated currents blocked by the
GABA antagonists fipronil and picrotoxin (Zheng et al. 2003). Because the GABA-A receptor
is a validated target of many compounds for anthelmintics and insecticides (ffrench-Constant
et al. 1993; Vassilatis et al. 1997; Dent et al. 2000; Kane et al. 2000), the receptor might provide
a target site for a novel acaricide. Another type of GABA receptor, the GABA-B receptor, a
member of the GPCR group, has not yet been studied in ticks. Two GABA receptors and 2
GABA transporters were found in the transcriptome of the D. variabilis synganglion (Bissinger
et al. 2011).
3.2.4. Octopamine
Octopamine has been reported to affect many functions in insects and other invertebrates, stim-
ulating muscle contraction, oviposition, and other energy-demanding activities.
Octopamine has been shown to stimulate salivary secretion in A. americanum, but with a
much lower activity than that of DA (Pannabecker and Needham 1982; Needham and Panna-
becker 1983). In addition, octopamine-stimulated adenylcyclase in the R. microplus synganglion
suggests the presence of an octopaminergic system in the tick CNS (Morton 1984), a hypothesis
supported further by the finding of a transcript for an octopamine receptor in the D. variabilis
synganglion (Bissinger et al. 2011). The involvement of octopamine was implied by the arrest-
ment of oviposition by an injection of octopamine in the same tick species. Octopamine also
caused an accumulation of egg wax around the mouthparts (Booth 1989). Activity of tyramine
β-hydroxylase, the enzyme involved in octopamine biosynthesis in R. microplus synganglion
homogenate, was also described (Kempton et al. 1992). The octopamine receptor is the target site
of formamidine ectoparasiticides (Nathanson 1985). Amitraz, a rapidly acting acaricide
formamidine group, exerts its toxic action on the octopamine receptor(s) (Taylor 2001; George
et al. 2004).
A GPCR orthologous to the mammalian octopamine receptor was first isolated from
amitraz-susceptible and -resistant strains of Australian R. microplus (Baxter and Barker 1999b).
Although this study did not find resistance-associated mutations in the octopamine receptor
gene, a later study on the same species in Brazilian amitraz-susceptible and Mexican amitraz-
resistant strains found coding polymorphisms that are suspected to be the mutations conferring
resistance to amitraz (Chen et al. 2007). Other amitraz-resistance mechanisms, including meta-
bolic resistance (Baxter and Barker 1999b) or the involvement of other octopamine receptor
genes (Jonsson and Hope 2007), need to be studied further.
3.2.5. Glutamate
Glutaminergic synapses have been demonstrated at the neuromuscular junctions of arthropods
(Otsuka et al. 1967; Usherwood et al. 1968; Cull-Candy 1976; Usherwood 1981). This NT was also
described in ticks. For example, in A. variegatum, L-glutamate causes depolarization when ap-
plied to the retractor muscles of Gené’s organ, the accessory glands that secrete waterproof wax
around each egg, suggesting the presence of glutaminergic synapses in this system (Booth et al.
1985). Using an assay based on high-performance liquid chromatography, Lucien et al. (1995)
detected glutamate in several tissues in A. hebraeum, including the synganglion. Two U.S. patent
applications have been submitted for 2 (RsGluCL1 and RsGluCL2) and 4 glutamate-gated
chloride channels (GluCls) from Rhipicephalus sanguineus (U.S. Patent No. 7202054) and D.
variabilis (U.S. Patent No. 7267964), respectively (Cully and Zheng 2007; Warmke et al. 2007).
The phylogenetics of the protein sequences of insect, nematode, copepod, and tick GluCls indi-
cate that tick GluCls are highly divergent from other invertebrate sequences, except for Rs-
GluCL1, which is clearly clustered with the insect taxa (see review by Lees and Bowman [2007]).
3.2.6. Acetylcholine
The first report on the presence of acetylcholine (ACh) in ticks was from a biological, chromato-
graphic, and electrophoretic study of R. microplus (Smallman and Schuntner 1972). Evidence of
the activity of choline acetyltransferase, the enzyme required for the biosynthesis of ACh, was
found in the homogenates of larvae and adult synganglia of the cattle tick, R. microplus (Small-
man and Riddles 1977). There are 2 main types of ACh receptors, ligand-gated cationic channels
(the nicotinic ACh receptor, nAChR) and GPCRs (the muscarinic ACh receptor, mAChR). Tran-
scripts for both types of ACh receptors were found in the D. variabilis synganglion (Bissinger et
al. 2011). The presence of putative mAChR and nAChR in the homogenate of whole R. microplus
larvae was examined via ligand-binding assays for various radioligands and nonlabeled cholin-
ergic compounds. [3H] quinuclidinyl benzilate (an mAChR agonist) and [3H] nicotine (an nAChR
agonist) showed binding ability without competition with each other in the whole-larvae homog-
enate, suggesting the presence of specific mAChRs and nAChRs (Turberg et al. 1996). Pilocar-
pine, an mAChR antagonist, produced an increase in action potential frequency in hemal nerve
4 and salivary nerves (from LNP) in R. microplus (Binnington and Rice 1982). Oviposition in R.
microplus was severely reduced when pilocarpine or arecoline was injected (Booth 1989). The
application of nicotine to the main pedal nerve 4 in the A. hebraeum synganglion increased spon-
taneous spike activity (Harrow et al. 1991). Although there is strong support for the presence of a
cholinergic system in ticks, mAChR and nAChR have not been described at the molecular level.
Pilocarpine has been used to induce tick oral secretion via in vivo injection or topical appli-
cation (Tatchell 1967; Ribeiro and Spielman 1986). Because pilocarpine failed to stimulate secre-
tion in isolated SG (Needham and Sauer 1975), mAChR is thought to be involved in the activation
of salivary secretion indirectly through the CNS (Kaufman 1978).
Acetylcholine esterase (AChE), the enzyme that degrades ACh to choline and acetate at the
synapse, was found in the homogenate of adult R. microplus synganglia (Stone 1968). Addition-
ally, the presence of AChE within the D. variabilis synganglion was confirmed via immunocyto-
chemistry (Carson et al. 1987). Different numbers of putative AChE genes have been described
in various tick species (see review by Lees and Bowman [2007]) as follows: AChE1 in R. microplus
(Baxter and Barker 1999a); AChE2 and AChE3 in R. microplus (Hernandez et al. 1999; Baxter
and Barker 2002; Temeyer et al. 2004); and AChE1 in R. (Boophilus) decoloratus, R. appendicu-
latus, D. variabilis, and R. sanguineus (Xu et al. 2003). Only AChE3 has been functionally tested
(Temeyer et al. 2006); however, the physiological roles that AChE genes play need to be investi-
gated. Considering that commonly used acaricides target cholinergic synapses (e.g., organo-
phosphate and carbamate pesticides that inhibit AChE and neonicotinoids that target nAChR),
this area of study needs to be further expanded in order to improve understanding of the selec-
tivity of the toxicants in different tick species.
4. SENSORY ORGANS (SENSILLA ) AND

SENSORY PHYSIOLOGY
Ticks have a variety of sensory organs for monitoring the status of their external and internal
environments. These sensilla may be classified by function (e.g., as chemosensilla, mechanosen-
silla, photosensilla, and thermosensilla) or by form (e.g., setiform sensilla), as well as by combi-
nations of these criteria. Some are multifunctional sensilla (e.g., mechano-chemosensory
sensilla). Many sensilla are dispersed over the body and appendages. Others are localized in
specialized organs such as Haller’s organ on the foreleg tarsi (Figs. 13.24, 13.25), the palpal organ
on article IV of the palps (Fig. 13.26), the cheliceral digits, and the eyes (Fig. 13.27). This section
of the chapter presents a brief description of the tick sensory organs and their role in tick behav-
ior. For more detailed information, see Chapters 14 and 16 of this volume, as well as in the pre-
vious edition of Biology of Ticks (Sonenshine 1991), and the articles cited therein.
FIGURE 13.24: Scanning electron micrograph of the dorsal aspect of the tarsus of leg I in a
representative ixodid tick, Dermacentor variabilis. Haller’s organ, consisting of an anterior pit and a
posterior capsule, is situated on a prominent elevation in the mid-dorsal region of the segment. Pre- and
post-capsular setae occur distal and proximal to this organ. The anterior pit contains 6 sensilla. The
capsular portion is evident as a dark slit-like opening in its roof. Ant. Pit, anterior pit of Haller’s organ;
P. cap, posterior capsule; Pocs, post-capsular setae; Prcs, pre-capsular setae. Measurement bar, 100
μm. From Sonenshine, D.E. (1991) Biology of Ticks, Vol. 1. New York: Oxford University Press, with
permission from Oxford University Press.
FIGURE 13.25: Scanning electron micrographs showing the anterior pit sensilla of Haller’s organ in the
ixodid tick Dermacentor variabilis. The very large, tall seta is a multiporose sensillum covered with
innumerable minute pores. The other, smaller setae are either smooth-walled or grooved (double-
walled) sensilla. Measurement bar, 20 μm.
4.1. CHEMOSENSILLA
Chemosensilla have pores in the cuticle of the seta (sensory hair) or in the setal base, in addi-
tion to other features characteristic of all sensilla. Chemosensory sensilla may appear as setae
or, less frequently, as pits. Two types of chemosensilla are known: olfactory and gustatory. Ol-
factosensilla detect molecules in the form of vapors (odorants), often in minute concentrations
(e.g., <10−9 molar). Gustatory sensilla detect molecules in solutions or solids and usually
require higher thresholds for detection.
Chemosensory sensilla consist of (i) the seta, a hollow, tubular shaft or hair-like structure
bearing branched or simple unbranched pores along its walls or at the tip; (ii) the socket, a
pore that traverses the cuticle and ends in an enlarged rounded or oval cavity on the dorsal
surface; (iii) bipolar neurons with the dendrite in the setal cavity and branching close to the
cuticular pores; and (iv) the scolopale, an amorphous sheath around the neuron cell body and
the base of dendrites. Specialized epidermal cells, the inner trichogen and outer tormogen
cells, which help form the sensillum, are known to occur in insect sensilla but have not been
confirmed in ticks.
4.1.1. Olfactosensilla
Ticks have 2 major types of olfactosensilla: (i) single-walled (SW) sensilla, the sensilla basi-
conicum, and (ii) double-walled (DW) (spoke wheel) sensilla or sensilla coeliconicum, sim-
ilar to those in insects. In the SW type, numerous pores (approximately 120 nm in diameter)
occur along the sensillar shaft. The stimulus-conducting dendrites may be branched or un-
branched. DW or spoke wheel sensilla have few pores. Externally, the pores appear partially
FIGURE 13.26: Scanning electron micrograph illustrating the appearance of the sensory fi eld found on
palpal article IV of the camel tick, Hyalomma dromedarii. A shallow depression at the tip of each
sensillum contains a pore. The 10-terminal sensilla at the terminal end of the segment are tip-pore
sensilla. Measurement bar, 20 μm. Photo courtesy of J.E. Keirans and R. Robbins.
plugged with a cap-like structure surrounded by a circular gap continuous with the internal
cavity of the pore (plugged pores). Plugged pore sensilla are common in tick olfactorecep-
tors, but olfactoreceptors with simple pores also might occur. These pores lead directly into
the cavity of the sensillum. The pores are filled with an unknown material believed to be
continuous with a similar substance over the surface. In olfactosensilla, the dendrites are sur-
rounded by scolopales near the base of the sensillum; distally, where the dendrites branch
and come in close proximity to numerous pores, scolopales are absent. The SW sensilla on
Haller’s organ (see below) of Amblyomma variegatum respond to short-chain fatty acids,
2,6-dichlorophenol, and a wide range of other substituted phenols (Sonenshine 1991). Recep-
tors sensitive to CO2, H2S, and other odorants are also known in this vicinity of tarsus I
(Steullet and Guerin 1992a, 1992b; McMahon and Guerin 2002) in this species, although they
are not always associated with multiporous olfactory sensilla. Olfactoreceptors on Haller’s
organs of other species (e.g., A. americanum) are very responsive to NH3 (Sonenshine 1991)
and other common host odorants (also see Chapter 14).
DW sensilla may have grooved or smooth surfaces. Grooved DW sensilla have tiny pores (10
to 20 nm) located only in the grooves. In cross-section, the walls resemble the spokes of a wheel.
In contrast to the SW sensilla, the dendrites of the grooved DW sensilla are unbranched. DW
smooth-walled sensilla usually have only 1 pore at the tip. Both types are commonly found in
Haller’s organ, but their functions are not known.
FIGURE 13.27: Schematic diagrams illustrating the eye in an ixodid tick, Amblyomma americanum.
A, Entire eye showing the dome-like lens and the underlying photoreceptor neurons. B, Enlargement
showing a single photoreceptor neuron and an adjacent glial cell. Ax, Axon; CV, coated vesicles;
E, epidermis; GC, glial cell; Gl, glycogen; GN, glial cell nucleus; Go, Golgi complex; GS, glial sheath;
L, lens; LPC, lenticular pore canals; M, mitochondria; Mv, microvilli of photoreceptor neurons;
N, nucleus; RER, rough endoplasmic reticulum; S, scutum; SPC, scutellar pore canals. In panel B, A is
a region characterized by numerous microvilli, B is a region containing numerous mitochondria and
intracellular channels, C is the soma with a nucleus, and D is the proximal part of an axon. From Phillis,
W.A., 3rd and Cromroy, H.L. (1977) The microanatomy of the eye of Amblyomma americanum (Acari:
Ixodidae) and resultant implications of its structure. J. Med. Entomol. 13:685–698, with permission from
the Entomological Society of America.
For an understanding of how odorant molecules reach the dendrites of the receptor cells in
insects, see the work of Benton (2006) and Sánchez-Gracia et al. (2009). Unlike in insects, virtu-
ally nothing is known about the physiology and molecular aspects of odorant detection in ticks.
4.1.2. Gustatory sensilla

These sensilla may be setiform and usually have only a single pore at the tip. Gustatory sensilla
detect soluble or other non-volatile molecules (e.g., adenosine triphosphate or glutathione) that
can stimulate feeding, sexual, and/or reproductive behaviors, as well as other responses. An ex-
ample is the sensory field of 10 to 20 setiform sensilla found on the tip of palpal article IV, situ-
ated in a cavity of article III. Article IV can be everted to expose the sensory field, which is useful
for recognizing stimulatory molecules on host skin surfaces. Gustatory sensilla also occur on the
distal end of leg tarsus I (Phillips and Sonenshine 1993), where they detect non-volatile mole-
cules important in mate recognition, and on the cheliceral digits in ixodid ticks, where they
detect soluble molecules important for feeding and mate finding as described elsewhere in this
chapter (Section 4.5). Molecular evidence of a gustatory receptor was found in the genome of
I. scapularis (ISCW012263, a 64.5 kDa GPCR transmembrane protein that might be associated
with taste; Gene Ontology term 0050909).
4.2. MECHANOSENSILLA
Mechanosensilla are sensilla with neurons that contain neurotubular cytoskeletons. The setal
socket may be innervated (sensilla chaetica) or not (sensilla basiconica or sensilla trichoidea). In
exclusively mechanosensory sensilla, the setal shaft lacks pores. Most common are the setiform
sensilla, which occur over the capitulum, body surface, and legs. Others are non-setiform sen-
silla with a simple socket (campaniform sensilla or sensilla auriformia) and pore or are disc
shaped (chordotonal sensilla). Occasionally, multifunctional sensilla occur (i.e., sensilla that
also include chemoreceptive dendrites). Mechanosensilla respond to physical contact. In addi-
tion, mechanosensilla may detect sounds, as in Ornithodoros concanensis, which responds to
sounds from nestling cliff swallows within the range of 3,000 to 8,000 Hz.
In ticks, mechanosensilla are of 4 types. (i) Setiform mechanosensilla occur over the body, the
capitulum, and the legs. (ii) Campaniform sensilla lack the seta but exhibit all of the remaining
structures. They occur throughout the body—even on the legs—and are also known as sensilla
auriformia (see Chapter 4, Fig. 4.4), sensilla hastiformia, sensilla lanterniformia, and other types.
The socket is closed by a dome or ridge of cuticle. (iii) Chordotonal sensilla are disc-shaped sensilla
that detect the movement of a body segment (e.g., the placoid sensillum at the base of the inner
cheliceral digit). (iv) Distal tarsal slit sense organs occur on walking legs II, III, and IV in Ambly-
omma variegatum, and presumably in many other tick species as well (also see Sonenshine 1991).
4.3. HALLER’S ORGAN

This unique structure on the dorsal surface of the leg I tarsi is the primary organ for detecting
odorants, heat, and other external stimuli. Haller’s organ consists of (i) a posterior capsule con-
taining numerous sensilla exposed to the exterior via a small aperture and (ii) an open anterior pit
containing a few sensilla located distal to the posterior capsule. Small groups of olfactory sensilla
occur nearby in many tick species and might also contribute to the tick’s sensory capabilities.
The posterior capsule contains numerous setae. In R. microplus, only 4 of the 12 or more
setae are innervated; these are SW multiporous olfactosensilla with plugged pores. In R. micro-
plus, 29 neurons are reported to innervate this cluster of capsular sensilla, suggesting a wide
array of sensory capabilities. A small glandular apparatus of unknown function is also present.
The composition of the anterior pit varies considerably between the 2 different families of
ticks. In ixodid ticks, the anterior pit contains 6 or 7 olfactory sensilla. These include the large
SW multiporous olfactosensillum covered by innumerable tiny pores and innervated by nu-
merous dendrites. Another multiporose sensillum occurs anterior to Haller’s organ in some
species. Also included in the anterior pit are the tip pore sensilla, which might serve gustatory,
mechanosensory, or other functions. Several other Haller’s organ sensilla are strictly mechano-
sensilla, including sound perception, or have unknown functions. In R. microplus, 3 of the ante-
rior pit sensilla are grooved DW (spoke wheel) porous sensilla, whereas the others lack pores. In
argasid ticks, the anterior pit contains 9 sensilla in most species. Two additional types, serrate
and setiform, also occur. In Argas transgariepinus, there are 2 SW multiporous sensilla, and the
very large setiform sensillum is also serrate.
4.4. PALPAL SENSORY ORGAN

The terminal segment of the palp, article IV, bears a sensory field that provides information
useful for host identification, sex recognition, and other functions. In adult ixodid ticks, this
segment can be everted or withdrawn into a cavity within segment III as needed; in argasid ticks
and ixodid larvae, it forms the terminal end of the appendage. A cluster of 10 to 15 setiform sen-
silla at the end of the segment forms the sensory field, functioning as gustatory, mechanosen-
sory, and possibly thermosensory receptors. They include 2 types, types A and B. Both types
have smooth walls and have pores at the tip. Type A sensilla are elongated, have smooth walls,
and are multifunctional (i.e., they have chemoreceptor and mechanoreceptor neurons). Type B
sensilla are shorter and thicker and are solely mechanoreceptors.
4.5. CHELICERAL DIGIT SENSILLA

Long regarded as simple cutting appendages, the cheliceral digits are now known to be inner-
vated and supplied with gustatory and mechanosensory sensilla. In ixodid ticks (e.g., D.
variabilis), sensory structures occur on both the inner and outer digits of the chelicerae. Certain
of these sensilla were found to have mechanosensory functions. Others, specifically the pore
sensilla on the inner digits, contain contact chemosensilla. Electrophysiological studies show
that the pore sensilla respond to NaCl in physiological concentrations, as well as to adenosine
triphosphate and glutathione. All of these compounds are normal plasma components of host
blood with documented roles as phagostimulants. In D. variabilis, a cluster of 3 pore-like struc-
tures is found on the cheliceral digits. When examined via transmission electron microscopy,
the middle pore-like structure is found to be a cone-shaped depression (i.e., not a true pore)
associated with a pair of mechanosensory neurons that connect to its inner surface. One of the
other pores is a chemosensillum with 11 neurons (Psd-2), and the remaining pore sensillum
contains a single neuron (PSd-1). Study of their ultrastructure suggests that the PSd-2 neurons
are chemosensory, whereas the PSd-1 neuron may be either chemosensory or thermosensory.
The organization of the latter, with its extensive glial sheath, is similar to that of thermosensory
sensilla found in insects. A large placoid sensillum, innervated by a branch of the cheliceral
nerve, occurs at the base of the inner digit, similar to the “pit” organ in R. microplus.
In addition to their role in the recognition of feeding stimulants, the chelicerae are also im-
portant in mating behavior. In D. variabilis and D. andersoni, the contact chemoreceptor (i.e.,
gustatory receptor) neurons also recognize the genital sex pheromone. Males in which the palps
or hypostome have been removed recognized conspecific females and copulated. However,
males in which the cheliceral digits were excised failed to form spermatophores even though
they probed the female’s genital pores. Apparently, they were unable to detect the genital sex
pheromone, a signal necessary for the initiation of spermatophore formation. Electrophysiolog-
ical studies of the male cheliceral digits showed highly significant responses to extracts of con-
specific anterior reproductive tract organs, including ecdysone, 20-hydroxyecdysone, or both,
depending upon the species (reviewed by Sonenshine [1991]).
4.6. EYES
Many tick species have eyes. In ixodid ticks, paired, simple eyes are located on the lateral mar-
gins of the scutum. In Hyalomma truncatum, the eye is situated in a circular depression and
represents orbited eyes consistent with their hunter host-finding strategy. Each eye consists of a
smooth, convex lens of clear cuticle. In the thin cuticle below the outer lens are numerous
narrow channels that converge uninterruptedly to the lens interior below a fine fibrillar layer.
The inner side of the lens is shaped like an oval plateau with a cone-like projection that focuses
the light onto the underlying cells (also present in Hyalomma dromedarii). It also screens out
harmful ultraviolet radiation while allowing visible light to pass. The hypodermis below the lens
consists of a single, thin layer of cells. Below this is a group of ~35 club-shaped, unipolar photo-
receptor neurons with large round or oval nuclei in a rosette-like arrangement. These ticks are
believed capable of recognizing objects according to shape, size, luminance, and contrast ratios
but are not believed capable of distinguishing colors (Bergermann et al. 1997). Some differences
in eye morphology and visual capabilities occur among the various tick species that have been
examined. In A. americanum, the pore canals of the lens are oriented in the dorso-ventral axis
and converge toward the center of the eye. Presumably, they may act as wave guides, directing
and intensifying the light impinging on the photoreceptor cells. These neurons are surrounded
by glial cells. At the base of the neurons, axons arise and fuse to form the optic nerve. The pho-
toreceptor neurons are unusual in that they have an enormous number of microvilli in each cell,
all oriented to the light path. In H. dromedarii, mesoaxons form around nerve fiber bundles and
connect with the surrounding perineurium. In argasid ticks, eyes (when present) are located on
the ventro-lateral surface, lateral to the legs. Although the eyes of ticks are broadly similar to the
simple eyes of many other arthropods, no evidence of true rhabdomes and screening pigment
has been reported in them.
Little else is known about the physiology of vision in ticks. In general, ticks respond to
shadows and variations in light intensity, and some species, especially those that employ the
hunter host-finding strategy, are believed capable of discriminating shapes. When questing,
D. variabilis respond to shadows by extending their legs, ready to attach to a host. Other ticks
recognize differences between light and dark areas and modify their behavior accordingly.
D. variabilis and the camel tick, H. dromedarii, respond to monochromatic light with peaks at
510 and 470 nm, respectively. This is similar to the peak spectral sensitivity found in other che-
licerates and in insects. The camel tick has a monochromatic receptor system. In studies with
both species, responses were obtained over a range from 350 to 650 nm, that is, including the
ultraviolet range but with very little activity in the near-infrared (red light) range.
Our knowledge of the physiology, biochemistry, and molecular biology of tick neural and sen-
sory functions is very limited relative to the more familiar and better-known insects. Of special
importance for the future is to fully catalog and sequence the numerous neuropeptides and
clearly identify their neurohormonal roles in regulating the tick’s body functions. Equally im-
portant is to identify the chemoreceptors that enable the ticks to recognize hosts and phero-
mones and identify blood components needed for feeding, digestion, development, and
reproduction. Also important are the odorant-binding proteins and enzymes that facilitate the
odorant-recognition process. These molecular components of tick biology also might be useful
as novel targets for tick control via vaccines or genetic manipulation to control ticks and tick-
borne diseases. New molecular tools have become available in recent years that offer opportu-
nities for examining large data sets of genes and proteins. Of special interest is the creation of
transcriptomes, which is done using instruments such as the Roche 454 and Illumina sequencing
machines. The results provide an important means of global analysis of the expressed genes in
tick organs such as the synganglion or Haller’s organ. Transcriptomes have already been pub-
lished for the synganglion from female D. variabilis. Transcriptomes have also been created for
the synganglion of I. scapularis females. Another transcriptome for Haller’s organ of D. variabi-
lis is in preparation (R.M. Roe, unpublished data). Following assembly of the contiguous se-
quences, BLAST matching against the NCBI database and the I. scapularis genome can be done
to identify and characterize the expressed genes, providing opportunities for further study to
confirm their functional roles. Further evidence of function can be obtained through real-time
polymerase chain reaction and microarray experiments at relatively modest cost. RNA interfer-
ence experiments have evolved as the method of choice for verifying the function of the target
molecules. Finally, improvements in proteomics methodology offer additional new tools for the
direct study of proteins and are especially useful when combined with transcriptomics and ge-
nomics to study the roles of different neuropeptides and NTs in the regulation of the tick’s fun-
damental biological processes.
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NOTE
1. Link to www.vectorbase.org (if available, select the “old version”). In the task bar near the top of the
screen, select “Tools.” Under “Available Tools,” select “Controlled Vocabulary Search.” In the next
screen, use the down arrow to open the drop-down box and click on “Tick Anatomy.” The user may
then browse the anatomical anatomy by clicking on “Material Anatomical Entity” and then on
“Anatomical Structure.” The greatest number of terms will be found by clicking on “Organism
Subdivision.” The definition of the term will be shown in the upper right-hand corner. Clicking on
other terms will reveal other hierarchical relationships at varying levels. In this manner, the user
C H A P T E R 1 4
MOLECULAR BIOLOGY AND

PHYSIOLOGY OF CHEMICAL
COMMUNICATION
ALBERT MULENGA
1. INTRODUCTION
Communication is an essential adaptation that is conserved in all organisms, from animals to

plants and microbes (Sonenshine 2006; Witzany 2010; Cocroft 2011; Hauser et al. 2011; McGrath
2011; Zhang 2011). All organisms use some form of communication to regulate behavioral adap-
tations that are essential for the existence of the species. Some of the most notable behavioral
adaptations that are dependent on communication are those that allow an organism to interact
with its surroundings, find mating partners for purposes of procreation, sense danger, recog-
nize kin, fend off intruders and attackers, and find suitable niches in the environment for its
survival. The overall additive effect of all this is success of the species. Thus, disruption of this
process for the purpose of controlling parasites is very attractive. The attractiveness of this idea
is in the simplicity and straightforwardness of potentially interfering with the process. In the
field of parasite research, one of the most aggressively pursued behavioral adaptations is com-
munication regulating the parasite’s host-finding behavior. The approach here is simply to find
out how a parasite does this and then block and starve the parasite. It is clear that determining
how a tick communicates with its environment is low-hanging fruit for the purpose of devel-
oping novel tick-control strategies. Despite this, very little is known about the molecular bi-
ology of tick communication. In the discussions that follow, we attempt to set up a framework
for future studies on the molecular bases of tick communication. The starting point for these
studies is going to be what is already known on the roles of communication in regulating tick
biological adaptations. Although the focus of this chapter is molecular biology, we cannot dis-
cuss this in isolation; the immense wealth of information on the regulation of tick behavioral
adaptations by different forms of communication must be considered. The regulation of tick
Chemical Communication 369
behavioral adaptations by different forms of communication is a huge topic. In the 1982 book
The Physiology of Ticks, edited by Obenchain and Galun, 4 of the 13 extensive chapters cover
aspects of tick biology that are dependent on communication. What I attempt to do here is
provide a synopsis of these studies to put our discussion in the context of tick communication.
This is intended to set a framework for the subsequent discussion on emerging studies on the
molecular biology and physiology of tick chemical communication.
Although the intent of this chapter is to highlight advances regarding the molecular biology
and physiology of chemical communication by ticks, I overlay organism-level studies to put
molecular and cellular data in the context of tick biology. As outlined in other chapters of this
book, there are 2 types of ticks, soft and hard ticks. On the basis of their effect on veterinary and
public health, hard ticks are considered most important, and thus they are the most studied.
This, coupled with my own interest in hard tick biology, biases this chapter, so that commentary
and discussion in this chapter is skewed toward hard tick biology.
1.1. BIOLOGY OF COMMUNICATION

The purpose of animal communication is to influence behaviors that promote the well-being of
a species. Animal communication is most often defined by the sensory signal that is being per-
ceived and which triggers the animal’s behavioral change. It is said to be chemical when the
sensing of chemicals or odors is involved, thermo in the case of temperature shifts, audio in the
case of sound cues, visual when involving the sense of sight, tactile when involving touch, photo
in the case of light changes, and hygro in the case of water or moisture detection. Communica-
tion occurs in a defined environment that is different for each animal species. Figure 14.1 sum-
marizes the principle interacting components of the tick’s communication environment. In the
tick’s environment, key interacting components include the tick’s macro- and micro-habitat, the
ticks themselves (males, females, and immatures), the tick’s blood meal sources (animal and/or
human hosts), and the tick’s predators. Within this environment, the tick feeds, reproduces, and
acquires and transmits multiple disease agents, and above all it has to survive in a hostile
environment. In tick biology, chemical communication and, to a lesser extent, other forms of
communication play a dominant role in regulating tick behavioral adaptations within the
communication environment (Hamilton 1992; Sonenshine 1985, 2004, 2006; Kiszewski et al.
2001). Some of the most notable behavioral adaptations regulated by chemical communication
include locating areas in the environment that are likely to be frequented by animals, being
alerted when the animal host is present, finding the feeding site on the animal, finding mating
partners, and fending off predators. Key behavioral adaptations that are not dependent on
chemical communication include finding cooler and moist niches for survival and questing
behavior in some species that involves sensing changes in moisture and temperature. A classic
example is the observation of how questing behavior in ticks such as Haemaphysalis leporis-
palustris larvae is guided by the differential or rolling sensitivity of hydrated and dehydrated
ticks to moisture and light (Camin and Drenner 1978). Unfed saturated larvae are photopositive
and respond negatively to moisture, and the opposite is true for dehydrated ticks. This guides
hydrated ticks to move up vegetation during cooler parts of the day. After a few hours on vege-
tation, ticks become dehydrated and respond negatively to light and positively to moisture and
move toward the moist mat. From the perspective of finding weak links in tick biology that can
FIGURE 14.1: Interacting components within the tick’s communication environment, including between
ticks, with the animal host during the parasitic phase, and with the tick’s micro-habitat during the
non-parasitic phase.
be targeted for the development of novel tick-control methods, tick behavioral adaptations
regulated by chemical communication are highly amenable to manipulation. In this regard,
chemical communication, the focus of this chapter, has received the most research attention.
1.2. BIOLOGY OF TICK CHEMICAL COMMUNICATION

All animals, particularly those with rudimentary or absent vision development, use chemicals to
interact with their environment (Hildebrand 1995). There are a considerable number of expert
reviews on the roles of chemical communication in tick biology (Obenchain and Galun 1982;
Sonenshine 1985, 2004, 2006; Gothe 1987; Hamilton 1992; Kiszewski et al. 2001; Tegoni et al.
2004), which the reader is advised to consult. This section attempts to provide a synthesis of
these reviews and studies in order to provide a context for the discussion of the molecular biology
of tick communication in subsequent sections. Chemical communication is a 2-component
system comprising the emitter and the perceiver of chemical stimuli. By simple definition,
chemical communication is a biological process in which information-bearing chemicals, also
known as semiochemicals, are emitted and/or perceived by an organism, resulting in behavioral
change that benefits the emitter and/or perceiver. Semiochemicals are classified into 4 cate-
gories: pheromones, allomones, kairomones, and synomones (Sonenshine 2006). Except for
synomones, all classes of semiochemicals have been identified and/or demonstrated to play
roles in the regulation of tick behavioral adaptations (Yoder 1995; Sonenshine 2006). These
classifications are based on the benefit that is accrued to the animal that is emitting or perceiving
the semiochemical.
Pheromones regulate conspecific behavioral adaptations that ultimately lead to the success
of a species. An individual of a species secretes pheromones to influence the behavior of another
individual within the same species, to the benefit of the species as a whole. Among the most
studied include female tick sex pheromones, which regulate the mating behavior of male ticks
(Hamilton 1992; Sonenshine 1985, 2004, 2006; Kiszewski et al. 2001). Sonenshine (2006) has
indicated that the secretion of sex pheromones appears to be tightly linked to developmental
processes, such as metastriate female ticks’ feeding on a vertebrate animal for at least 1 or 2 days
prior to mating. Likewise, the tick that perceives the pheromone has to be in a certain physio-
logical state. For instance, feeding male metastriate ticks, but not unfed males, respond to sex
pheromones.
Kairomones are semiochemicals that influence the behavior of individuals of a different spe-
cies. The emitter is an individual of one species, and the perceiver whose behavior is altered is a
member of a different species; the process benefits the perceiver. Some examples in tick biology
include animal host body secretions, different odors, and CO2 emissions that stimulate tick
host-seeking behavior (Obenchain and Galun 1982). As with pheromone sensing, the tick’s
physiological state is a factor in kairomone sensing. There is evidence that unfed ticks respond
to kairomones, particularly CO2, which is a sign of the availability of a blood meal source; the
ticks must have attained a physiological status of hunger before they become responsive (Glad-
ney et al. 1970; Tukahirwa 1976; Davey 1987; Anderson et al. 1998).
Allomones are semiochemicals involved in communication between different species of an-
imals in which the behavior of the perceiver is altered to the benefit of the emitter. Allomones
are the least studied among tick-produced semiochemicals, presumably because of the lack of
potential to utilize the resultant data in finding new ways to control ticks and tick-borne dis-
eases. Tick hydrocarbon secretions that are deterrents to ants have been described (Yoder 1995).
Lastly, synomones are semiochemicals that benefit both the emitter and the perceiver. The
roles of synomones are well documented in plants; they are part of plant volatiles that attract
pollinators to the plant, leading to enhanced crop production (Hilker et al. 2002). In ticks, syno-
mone activity has not been directly demonstrated.
2. BEHAVIORAL ADAPTATIONS REGULATED BY

CHEMICAL COMMUNICATION AND THEIR UTILITY
IN TICK CONTROL
There are obviously numerous biological adaptations that are under different control systems. In
this chapter we focus on those that are wholly or partially under chemical communication con-
trol, as summarized in Fig. 14.2. These adaptations represent the framework for designing studies
on the molecular biology of chemical communication in ticks. Thus, to put our subsequent dis-
cussion into the context of the biology of tick chemical communication, it is imperative that we
review tick behavioral adaptations that are regulated by chemical communication. The molec-
ular mechanisms underpinning these behavioral adaptations are part of the bigger picture of the
molecular biology of chemical communication. The prioritization plan for studying the molec-
ular basis of behavioral adaptations summarized in Fig. 14.2 will be influenced by the feasibility
of blocking the cognate behavioral response at the molecular level.
FIGURE 14.2: Tick behavioral adaptations that are controlled by chemical communication.
2.1. OFF-HOST CLUSTERING BEHAVIOR

In nature, ticks are found in close proximity to one another. Current evidence suggests that this
behavior, referred to as clustering, is potentially regulated via arrestment pheromone (Leahy
et al. 1973; Sonenshine 2004, 2006) and kairomones (animal sensing) (Carroll et al. 1995, 1996,
1998; Carroll 1998, 2002). The arrestment pheromone (formerly known as assembly pheromone)
has been identified in both soft and hard tick species (Leahy et al. 1975a, 1975b; Rechav et al.
1977; Hájková et al. 1980; Goethe and Neitz 1985; Goethe 1987; Taylor et al. 1987; Allan and So-
nenshine 2002). In laboratory studies, ticks that were exposed to arrestment pheromone ceased
all movement (Yoder et al. 2008). This cessation of movement causes ticks to aggregate in one
area. Arrestment pheromone has been demonstrated to be present in cast larval skins, fecal
droppings, and tick body exudates (Sonenshine et al. 2003). In an expert review article, Sonen-
shine (2004) indicates that arrestment pheromone, originally demonstrated in soft ticks, had
been demonstrated in 14 hard tick species by 2004. An interesting feature associated with arrest-
ment pheromone is its non-specific nature. Whereas by definition a pheromone regulates con-
specific behavioral adaptations, arrestment pheromone is functionally interspecies specific
(Sonenshine 2004). For instance, the Ornithodoros moubata arrestment pheromone induced the
cessation of movement in O. tholozani. This behavior was also observed between genera when
O. moubata arrestment pheromone induced the cessation response in Argas persicus (reviewed
in Sonenshine 2006).
In laboratory experiments, the cessation of tick movement was also demonstrated when
I. scapularis, A. americanum, and D. variabilis ticks were exposed to surfaces that had been in
repeated contact (surface to surface) with their respective principal hosts (i.e., whitetail deer
for the former two species and dogs for the latter) (Carroll 2002). This induction of akinetic
behavior under laboratory conditions is suggestive of the possibility of a tick response to an-
imal kairomones that participates in regulating the clustering behavior of ticks. Different tick
species feed on different preferred animal hosts. For instance, I. scapularis adults and all stages
of A. americanum will preferentially feed on white tailed deer, whereas D. variabilis feed on
dogs (Carroll 2002). However, in the study described above, it is interesting to note that, con-
sistent with the interspecies nature of arrestment pheromone, kairomone residues displayed
non-specificity in their ability to induce the cessation of tick movement. From the perspective
of the study referenced above, it appears that the tick’s ability to choose its preferred host is
independent of the tick’s ability to select a site in the environment that is likely to be fre-
quented by animals.
In the development of novel tick-control strategies targeting ticks, knowledge of the chemi-
cal structures and/or composition of the active ingredients in arrestment pheromone and in
animal host extracts that induce tick akinetic behavior will have broad relevance. There is evi-
dence that mixing arrestment pheromone with an acaricide in a slow-release system can in-
crease the number of ticks killed (Sonenshine 2006). It is clear that detailed knowledge of the
components of arrestment pheromone will have broad effects with regard to the development of
this technology. There have been studies that show that arrestment pheromone is composed of
a varying ratio of purines such as guanine, xanthine, and hypoxanthine that are commonly
found in tick droppings and tick cast skins (Dusbabek et al. 1991; Allan and Sonenshine 2002;
Sonenshine et al. 2003).
2.2. THE PRE-APPETENCE PHASE

The tick feeding process is characterized by a series of behavioral adaptations that can be
traced from the time when ticks hatch and/or molt to when the tick feeds to repletion and
spontaneously disengages from the host. Most workers have considered the tick feeding
process as beginning when the tick attains appetence (an increased desire to seek out the host
and start feeding). However, if one wishes to look at the tick feeding process in a holistic way,
one can make an argument that the tick starts to prepare for the feeding process when it is
molted or hatched (Mulenga et al. 2007a). In experiments to optimize protocols to feed ticks
on surrogate hosts, it was observed that young, newly hatched larval or newly molted nymphal
and adult ticks responded poorly to CO2 exposure. When introduced to a host animal, they
fed poorly or not at all (Gladney et al. 1970; Tukahirwa 1976; Davey 1987; Anderson et al.
1998). However, increased sensitivity to CO2 and improved feeding efficiency coincided with
young ticks undergoing a physical maturation known as “the hardening-off phase” (Anderson
et al. 1998). The hardening-off phase involves hardening and darkening of the tick cuticle ac-
companied by the deposition of whitish fecal excretory materials (Davey 1987). During the
hardening-off phase, nutrients derived from the egg or from the previous blood meal are uti-
lized, after which the host-seeking behavior (attainment of appetence or hunger) is initiated
(Davey 1987). In laboratory feeding experiments with A. americanum, feeding success pro-
gressively improved from as low as 2% in young 1-day-olds to more than 70% in 4- to 7-day-
old larvae and nymphs and 8- to 9-day-old adult ticks (Gladney et al. 1970). Similarly, the
ability of A. hebraeum to quest or seek out a CO2 source (the equivalent of seeking out a blood
meal source) was found to significantly increase with age (Anderson et al. 1998). Anderson
et al. (1998) observed that whereas newly emerged ticks infrequently quested in response to
CO2, the response of older ticks progressively increased with age, with responsiveness being
greatest in the oldest ticks tested, namely, those 6 weeks post-molting. It is interesting to note
that, comparable to ticks, older mosquitoes also are much more responsive to CO2 sensing
(Grant and O’Connell 2007).
It is important to note here that the pre-appetence phase appears to be under developmental
control. However, it provides a reference point for determining when the tick starts to be re-
sponsive to chemical communication. It raises important questions as to when the tick expresses
the necessary molecular machinery in preparation for host-seeking behavior and subsequently
interacting with the host to start the feeding process. This knowledge will be useful in experi-
ments looking to clone chemosensory receptors.
2.3. HOST-SEEKING/LOCATION BEHAVIOR

The increase in feeding success or responsiveness to feeding stimuli such as CO2 by older
ticks in laboratory settings could be viewed as the equivalent of attaining appetence in na-
ture, which is when the tick starts to quest or seek out a host for its blood meal. In ticks that
have attained appetence, 3 distinct host-seeking and/or locating behaviors (hunter versus
ambusher, 1-host versus multi-host, and host-specific versus host-divergent tick species) are
well described in the literature (Waladde and Rice 1982). Significant amounts of data have
been collected on sensory stimuli that evoke these behavioral adaptations. Waladde and Rice
(1982) classified these sensory stimuli into 3 categories on the basis of the type of information
they carry: “host is distant versus in close proximity,” “directional versus non-directional,”
and “general versus specific” stimuli. The “distant versus in close proximity” and “directional
versus non-directional” stimuli are of either a physical or a chemical nature. Notable exam-
ples of physical stimuli that guide host location/seeking behaviors include (i) touch sensa-
tion, an example of “close proximity” stimuli that is the basis for flagging, and (ii) warmth
and water vapor, which are probable examples of “directional” stimuli. Physical stimuli that
have been demonstrated to provoke host-seeking behavior from a considerable distance
include visual images, vibrations, shadows, and radiant heat (Waladde and Rice 1982).
Although the majority of physical stimuli are non-specific, there is evidence that vibrations
and visual images could be host specific. Laboratory experiments have shown that vibrations
ranging between 3000–8000 Hz provoked host-seeking behavior in Ornithodoros concanen-
sis, and Rhipicephalus (Boophilus) microplus is stimulated by sounds ranging between 80–800
Hz (reviewed in Waladde and Rice 1982). In ticks with developed eyes such as Hyalomma
asiaticum, hunting behavior is stimulated by visual images of the host (reviewed in Waladde
and Rice 1982).
The role of chemical stimuli in regulating host-seeking behavior has received tremendous
research attention. Major chemical stimuli components that regulate host-seeking/location be-
havior include body odors, volatile emissions, and CO2 (reviewed in Sonenshine 2008). Similar
to physical stimuli, these chemical stimuli can be considered as either specific/non-specific or
directional/non-directional. Body odors and variable compositions of different volatile emis-
sions such as butyric acid are specific. CO2 is the major generalist chemical stimulus that has
been studied. It is a universal regulator of host-finding behavior in all blood-feeding arthropods.
Interfering with ticks’ perception of chemical stimuli that provoke host-seeking/location behav-
ior is an interesting point for designing tick control. Despite the significance of body odor and
CO2 sensing in tick parasitism, the molecular basis of this system in tick biology has not been
explored. In later sections of this chapter, we examine emerging molecular biology data on CO2
signaling in blood-feeding arthropods.
Although not classically considered a form of chemical sensing, the sensing of moisture
in combination with temperature changes plays an important role in host-seeking behavior.
In certain tick species that ambush their host, such as R. microplus and Haemaphysalis lep-
orispalustris larvae, the thermal-hygro-reception system appears to play an important role
(Camin and Drenner 1978). Questing behavior has been studied in many tick species (e.g.,
I. persulcatus). Questing ticks display a repetitive behavioral pattern of periods of exposure
to dehydration when questing on vegetation alternating with periods of rehydration when
they move to the moist underbrush. Ticks normally begin questing during periods of the
day when temperatures are low, mostly in the morning. When temperatures start to rise,
the tick moves to the moist underbrush for rehydration. This behavior continues until
the tick finds a blood meal source. In laboratory studies on H. leporispalustris larvae, the
questing behavior was mediated by changes in humidity and light (Camin and Drenner
1978). Unfed hydrated larvae were shown to be photopositive and responded negatively to
moisture, and the opposite was true for unfed dehydrated larvae. This differential sensi-
tivity to light and moisture is suspected to guide ticks’ climbing up or down vegetation.
After locating a high-humidity area, ticks can compensate for any water loss through the
active absorption of water vapor via the hyperosmotic salivary gland secretions around
the hypostome (Gaede and Knulle 1997). In the context of discovering tick proteins that
regulate host-seeking behavior, one can ask whether the expression of CO2-binding proteins
and/or sensing receptors coincides with ticks’ being hydrated and photopositive. This
knowledge is going to be crucial in our quest to determine the molecular mechanisms
regulating chemical communication in ticks.
2.4. HOST DISCRIMINATION: THE HOST RANGE ADAPTATION

Tightly linked to host-seeking behavior is the tick’s adaptation for host specificity, or the range
of hosts on which it will feed. Different tick species have evolved and adapted to feed on different
animal hosts. Certain tick species such as A. americanum feed on a broad range of hosts, whereas
ticks such as R. microplus feed preferentially on cattle and very rarely on other ungulates. The
former may be described as a generalist, and the latter may be described as a specialist. A tick
will proceed with the feeding process when an appropriate animal host is encountered. The
sensory stimuli that regulate this behavior remain unknown. Speculatively, this behavior might
be regulated by the tick’s sensing signature blends of volatile body odors and/or body fluids that
are unique to different animal species.
Signature odor compositions are unique to animals (Dreumont-Boudreau et al. 2006;
Coureaud et al. 2008; Setchell et al. 2010). The signature scent of humans influences mosquito
feeding preferences (Verhulst et al. 2009, 2010; Smallegange et al. 2011). There is also evidence
that in humans, different microbiota on the skin affect individual odor blends that influence
mosquito feeding behavior (Verhulst et al. 2009). The discovery of sensory stimuli and receptors
regulating this behavior will have a far-reaching impact on tick biology research. For instance,
do generalist feeders such as A. americanum have non-specific receptors, or do they have mul-
tiple receptors that can sense multiple sensory signals from the different animal species on
which the tick is adapted to feed?
2.5. FEEDING SITE PREDILECTION

In nature, certain tick species display a predilection for specific feeding sites on the host body.
Although the physical and chemical stimuli used by ticks to locate and select an appropriate site
have received a great deal of interest, very little has been done to elucidate the molecular and
physiological basis of feeding site predilection. The ability of these ticks to feed on different parts
of the body suggests that different ticks perceive different, yet unknown, host-derived physical
and chemical stimuli, which they use to select their different feeding sites. Wanzala et al. (2004)
compared 2 sympatric tick species that feed on cattle, R. appendiculatus and R. evertsi, to dem-
onstrate that odor blends potentially emitted from their predilection sites controlled the behav-
ior of these ticks in locating their preferred attachment sites. R. appendiculatus and R. evertsi
predominantly feed on the inner ear and peri-anal regions of bovines, respectively.
Consistent with this observation, Wanzala et al. (2004) demonstrated that odor extracted
from cattle ears attracted R. appendiculatus but repelled R. evertsi, and vice versa with extracts
from the anal region. A deeper understanding of how a tick selects its feeding site will affect
efforts to develop artificial feeding systems on which ticks feed through artificial membranes.
Artificial feeding systems have been attempted with a variable amount of success (Waladde
et al. 1996; Young et al. 1996; de Moura et al. 1997; Barré et al., 1998; Abel et al. 2008; Tajeri and
Razmi 2011).
2.6. ON-HOST CLUSTERING BEHAVIOR AND TICK ATTACHMENT

The biology of on-host clustering behavior has been part of recent extensive expert reviews, to
which the reader is referred for further reading (Sonenshine 1985, 2004, 2006). Ticks display
on-host clustering behavior that is similar to off-host clustering behavior, in that ticks of the
same species are found feeding in close proximity to each other. This behavior is thought to
enhance tick feeding efficiency and pathogen transmission (Wang et al. 2001). As a result, mech-
anisms mediating this on-host clustering behavior are candidates for further study, as they offer
possibilities for blocking the key facets of tick parasitism. This on-host clustering behavior is
regulated by the aggregation-attraction-attachment (AAA) pheromone (Sonenshine 2006) in
the case of the tropical bont tick, Amblyomma variegatum, and the bont tick, A. hebraeum. The
AAA pheromone is produced by fed male ticks, and all unfed tick stages are sensitive to the
pheromone. However, replete fed A. variegatum ticks failed to respond to AAA, suggesting that
the reception system for this pheromone is turned off once ticks have attached to the animal.
The bulk of data on the AAA pheromone are predominantly based on studies in Amblyomma
tick species (Lusby et al. 1991; Norval et al. 1992a, 1992b, 1992c; Price et al. 1994; Rechav et al.
2000; Maranga et al. 2003; McMahon et al. 2003). However, given that the on-host clustering
behavior is present in other tick species, a pheromone system functionally similar to the AAA
pheromone might also occur in several other tick species. The active ingredients of the AAA
pheromone have been defined and validated as attracting unfed ticks. These include mixtures of
2 substituted phenols (methyl salicylate and O-nitrophenol), a nonanoic fatty acid, 2,6-dichlo-
rophenol, and 1-octen-3-ol (Hess and De Castro 1986; Lusby et al. 1991; Norval et al. 1992a,
1992b, 1992c; Yunker et al. 1992; Yoder et al. 1999; McMahon and Guerin 2000). Differences in
the relative compositions of the active ingredients apparently mediate the tick species specificity
of the AAA pheromone. However, geographical isolation of ticks of the same species did not
seem to affect their sensitivity. Sonenshine et al. (2000) showed that A. variegatum ticks from
wide geographical areas (i.e., from the Caribbean to East Africa) had no significant differences
in pheromone composition or biological responses to male tick pheromone secretions. The ap-
parent lack of geographical segregation means that targeting the AAA pheromone system for
tick control will be of global use.
2.7. TICK COURTSHIP AND MATING

More than 600 hard tick and 180 soft tick species have been described to date. In nature, these
different tick species co-exist and share hosts. As ticks co-exist with different tick species, the
challenge for each individual tick is to locate conspecific partners for mating purposes. Although
certain tick species can reproduce via parthenogenesis (i.e., mating is not required in order for
females to reproduce), the majority of tick species have to mate in order to feed to repletion and
subsequently lay eggs. Thus, understanding how ticks mate offers a lot of promise for finding
new ways to control ticks and tick-borne diseases. It is therefore fitting that mating is arguably
among the most studied systems in tick biology. Sonenshine (2006) has summarized the ritual-
istic nature of tick mating, which is uniform in all species. Mating begins with a male tick recog-
nizing and contacting a receptive female tick that is ready to mate. The male tick starts by
mounting the female tick dorsally and positioning itself to crawl over the posterior edge onto the
female tick’s ventral surface. The male tick then locates the female’s genital pore, inserts its
mouthparts into the female vulva, and begins to form a spermatophore. After the completion of
spermatogenesis, the copulating male deposits its spermatophore to inseminate the female tick.
Except in tick species that reproduce via parthenogenesis, insemination of the female also trig-
gers tick feeding to repletion. Although the tick mating ritual described above is common to all
ticks, there are key differences between the Prostriata and Metastriata, as well as between ixodid
and argasid ticks. Whereas metastriate ticks mate only on the host after the commencement of
the feeding process, prostriate ticks can mate as unfed ticks off the host during the host-seeking
phase or on the animal at the inception of the tick feeding process. Whereas unfed prostriate
ticks are sexually mature, unfed metastriate ticks are sexually immature, with oogenesis and
spermatogenesis starting to develop only after the ticks have started to feed (Sonenshine 2006).
Current knowledge of the tick mating process is based on studies predominantly conducted in
metastriate ticks; much less is known about the mating behavior of prostriate ticks.
Whereas in other, higher animals, courtship may be triggered by other forms of communi-
cation such as visual and vocal signals, the mating or courtship process in ticks is tightly con-
trolled by a well-choreographed sequence of female-tick-produced pheromones. Pheromone
control of the tick mating process has been the subject of a number of expert reviews (Sonen-
shine 1985, 2004, 2006; Kiszewski et al. 2001). Three tick sex pheromones have been described.
The first in the sequence is the attractant sex pheromone (ASP), which is secreted by female ticks
within 2 days of their starting to feed. Chemical analysis has revealed 2,6-dichlorophenol (DCP)
and 2,4-DCP as the principal active ingredients of ASP (Borges et al. 2002; Hanson et al. 2002;
Louly et al. 2008). These chemicals stimulate male ticks that are feeding nearby to detach and
search for the female tick that is emitting the pheromone. It is interesting that 2,6-DCP also at-
tracts immature stages of D. variabilis ticks (Yoder and Stevens 2000). Various concentrations of
2,6-DCP might stimulate different behavioral patterns. Further work is needed to define the
different levels of this chemical that regulate different behavioral patterns. Upon contacting the
female tick, the male detects the tick-species-specific mixtures or combinations of cholesteryl
esters that constitute the mounting sex pheromone (MSP) (Hamilton et al. 1994; Hamilton and
Sonenshine 1995; Sonenshine 2006). The MSP stimulates the male tick to crawl to the ventral
side of the female tick and search for the genital pore, into which it inserts its mouthparts in
order to start to form the spermatophore in preparation for copulation (as discussed earlier). In
certain tick species (e.g., D. variabilis and D. andersoni), a third pheromone, the genital sex
pheromone (GSP), is required in order for the male tick to start copulation (Allan et al. 1989,
1991; Sonenshine et al. 1982, 1984). Studies in D. variabilis have shown that GSP is composed of
a species-specific mixture of long chain saturated (C14–C20) fatty acids and the ecdysteroid
20-hydroxyecdysone (Allan et al. 1989). In species in which MSP is present, detection of the
correct composition of this mixture stimulates copulation behavior, whereas its absence induces
male ticks to leave (Sonenshine 2006).
3. MOLECULAR BASIS OF TICK CHEMICAL

COMMUNICATION
From the preceding sections of this chapter, it is clear that substantial organism-level data have
been collected on the biology of the regulation of tick behavioral adaptations by chemical com-
munication. We also know that in the few instances when knowledge of tick chemical commu-
nication has been applied toward the development of tick control devices, such as in a tick decoy
using the AAA pheromone, the resulting success has been considerable (Norval et al. 1991, 1996;
Allan et al. 1996, 1998; Sonenshine 2004, 2006). Given the paucity of data on the subject, the goal
of this section is to review and/or provide food for thought and some insights into the molecular
biology and physiology of chemical communication. We hope to be able to reveal salient vulner-
able points in tick biology that can be targeted for the development of novel tick control methods.
The molecular biology and physiology of tick chemical communication can be viewed as a
3-legged stool. The key components here are (i) semiochemical biosynthesis pathways, (ii) mo-
lecular bases of the chemosensory reception system, and (iii) molecular mechanisms regulating
tick behavioral adaptations that are controlled by chemical communication. In subsequent sec-
tions, these 3 segments are used as guiding umbrella topics under which our discussions have
been framed. Another important point of discussion is that these umbrella topics have the
potential to reveal weak points that could be manipulated for practical applications. This deter-
mination may be considered a prioritization plan based on relevance to the development of
novel tick control methods.
Given the vast amount of organism-level data on tick chemical communication already
available, we have opportunities to design both forward and reverse genetics studies. In forward
genetics studies, we will have opportunities to reproduce behavioral adaptations under labora-
tory conditions in order to generate study materials. Such an approach will produce a catalog of
candidate genes. We can then utilize RNAi silencing technology to validate the roles and signif-
icance of these candidate genes (see below for a review of studies of the molecular basis of the
tick attachment phase employing this strategy). With reverse genetics, we can take advantage of
advances in bioinformatics to find corresponding genes in other organisms and employ the
RNAi silencing technology to determine whether a behavioral adaptation can be aborted.
3.1. SEMIOCHEMICAL BIOSYNTHESIS PATHWAYS

Two types of semiochemicals, pheromones and kairomones, are central to the regulation of tick
chemical communication. Although knowledge of the pathways involved in kairomone biosynthe-
sis is important for a complete understanding of tick chemical communication, this section focuses
on tick pheromones. By definition, a pheromone regulates behavior between animals of the same
species; this means that ticks must have both production and sensing bio-pathways. Currently,
molecular cascades involved in the biosynthesis of tick pheromones are unknown. Needless to say,
an understanding of the molecular cascades involved in the biosynthesis of tick pheromones will
have a strong effect on the design of novel tick control strategies. Ideally, these data will lead to the
ability to manipulate all pheromone-regulated tick behavioral adaptations for the purpose of devel-
oping novel tick control methods. Despite the lack of data on genes involved in tick pheromone
biosynthesis, considerable data essential for carrying out studies on the molecular biology of tick
pheromone biosynthesis have been collected. The 2 sets of data include our knowledge of tick
organs involved in tick pheromone biosynthesis (Layton and Sonenshine 1975; Vernick et al. 1978;
Axtell and Lefurgey 1979; Sonenshine et al. 1981) and of the chemical structures of active ingredi-
ents in tick pheromones (Price et al. 1994; Sobbhy et al. 1994; Tkachev et al. 2000; Sonenshine et al.
2003). Complementary to these data are advances in other organisms that can serve as valid exper-
imental templates. To recap, tick behavioral adaptations are regulated by at least 3 pheromone
types: (i) the arrestment pheromone, which induces akinetic off-host clustering behavior that is
thought to promote survival during the non-parasitic phase, (ii) the AAA pheromone, which reg-
ulates the behavior of ticks of the same species and leads them to feed in clusters on a host animal,
and (iii) female-tick-produced sex pheromones that control the tick mating process. Chemical
mixtures that make up the arrestment pheromone in metastriate ticks—purines in almost all
cases—are produced in the Malpighian tubules and the rectal sac (Sullivan et al. 1979) (hematin is
also an arrestant in the prostriate tick Ixodes scapularis). The AAA pheromone has been shown to
be synthesized in type II dermal glands (Sonenshine 2006). Sex pheromones appear to be produced
by the foveal glands, the dermal type I gland, and possibly the lobular accessory gland surrounding
the vestibular region of the vagina (Sonenshine 2006). The ASP chemical constituent 2,6-DCP was
shown to be synthesized in the foveal glands of metastriate ticks, whereas cholesteryl esters that
constitute the MSP were shown to be produced in type I dermal glands (Sonenshine 2006).
Studies on determining candidates involved in tick pheromone biosynthesis can be exploited
to develop strategies for both forward and reverse genetics investigations. In the forward ge-
netics strategy, a simple approach will be to dissect pheromone-producing organs and conduct
transcriptome analyses to look for putative gene candidates that might be involved in phero-
mone biosynthesis. Successful transcriptome analyses of dissected tick organs have provided
insight into the molecular biology of multiple tick organs (Anatriello et al. 2010; Lees et al. 2010;
Bissinger et al. 2011; Francischetti et al. 2011; Sonenshine et al. 2011). Similar approaches can be
adopted for dissected pheromone-producing glands, given that the tick anatomy has been well
described. In the reverse genetics approach, the strategy will be to begin with genome-wide
mining for genes that are believed to be involved in the biosynthesis of chemical components of
tick pheromones. The second step will be the confirmation of gene expression in tick phero-
mone-producing organs under the appropriate tick physiological conditions. Here we can take
advantage of our knowledge of tick anatomy to dissect tick organs involved in pheromone bio-
synthesis. Messenger RNA of the dissected tick pheromone-producing organs could then be
subjected to RNA sequencing or microarray technologies to confirm the expression of candidate
genes. Validated genes can then be subjected to RNAi to confirm their roles in the production of
the pheromone using methods that have been validated for pheromone extraction and func-
tional analysis bioassays (Price et al. 1994; Sobbhy et al. 1994; Tkachev et al. 2000; Sonenshine et
al. 2003). Biosynthetic and metabolic pathways for purines (the constituents of the arrestment
pheromone), phenolics (constituents of AAA and ASP), and cholesteryl esters (the constituents
of MSP) are well known in other organisms. The starting point will be to find these gene homo-
logues in the I. scapularis genome, which at the time of this writing was the only tick genome
sequenced. For other tick organs, targeted RNA sequencing of mRNA from tick pheromone-
producing organs can be conducted. In enacting the approach suggested here, we can learn from
comparable data in insects. The lepidopteran moth pheromone constituents, namely, long car-
bon chains with added functional groups (acetates, alcohols, or aldehydes), are comparable to
what has been observed in ticks. The lepidopteran moth pheromone biosynthetic pathway in-
cludes 2 key enzymatic processes, desaturation and the long-chain shortening process (Arse-
quell et al. 1990; Choi et al. 2002; Knipple et al. 2002; Jeong et al. 2003). At the time of this
writing, scanning of the I. scapularis genome and the tick expressed sequence tag (EST) database
had revealed that the desaturase enzyme sequences are conserved in ticks (A. Mulenga, unpub-
lished data). The conservation of the desaturase enzyme in ticks points to the possibility that the
molecular interaction cascade is essential to pheromone production in ticks.
3.2. MOLECULAR MECHANISMS UNDERLYING THE TICK CHEMOSENSORY

RECEPTION SYSTEM
By simple definition, the sensory reception system converts sensory or energy signals such as
light, heat, sound, electricity, gravity, moisture, and chemicals into action potentials that can be
transmitted in the nervous system, where they are processed. The process of converting signals to
some form of energy is referred to as transduction, and the perceiving sensory receptors housed
in sensory organs are transducers. An understanding of the molecules involved in these processes
will help explain the molecular basis for communication signaling in tick biology. A signaling
molecule relays information between cells. In the same context, external sensory chemical stimuli
regulating chemical communication can be viewed as signaling molecules that relay information
between the environment and the tick. The binding of these molecules to receptors triggers a mo-
lecular cascade that controls a physiological change. The state of the chemical stimuli (e.g., liquids
or odorants) determines what types of molecules are involved in the reception of the chemical
stimuli. In the case of a signal being in liquid form, signaling occurs via gustatory receptors,
whereas in the case of odors, sensing of the chemical stimuli occurs via olfactory sensory receptors
(olfactosensilla). Odorant-binding proteins can bind odorant signals before they interact with a
receptor. The final result of stimuli’s binding to receptors is the initiation of a transduction cascade.
The gross anatomy of the tick sensory system is well documented and is expertly reviewed
elsewhere in this book. The most studied tick sensory organ is Haller’s organ, which is located
on the tarsi of the first pair of legs (forelegs). This organ is key in regulating essential tick be-
havioral adaptations such as host finding and temperature sensing, both of which are necessary
in order for ticks to find their blood meal source. Evidence of the involvement of Haller’s organ
in sensing myriad signals is the fact that it houses a number of receptors that are yet to be char-
acterized. There are interesting data in the literature that speak to the robust regenerative,
adaptive, and/or plastic nature of the tick’s Haller’s organ. In several tick species, amputation of
the foreleg together with Haller’s organ resulted in the foreleg’s being regenerated along with
an intact Haller’s organ in the next instar (Leonovich and Belozerov 1992, 2004; Belozerov and
Leonovich 1995). The ability of Haller’s organ to regenerate presents a robust system that we
can use to dissect the role(s) of the tick sensory system in regulating numerous tick behavioral
adaptations, such as differential host preferences of the different tick life stages. Although there
are a few 1-host ticks such as Rhipicephalus microplus, in which each of the 3 life stages feeds on
the same animal, the majority of ticks are 2-host or 3-host tick species, in which each life stage
feeds on a different animal. Most often, immature ticks feed on small animals, whereas adult
ticks feed on larger animals. The regenerative nature of Haller’s organ provides the opportunity
to track molecular changes within Haller’s organ that are unique to the different tick develop-
mental stages and which influence their differential preference for small or large animals. The
other important chemosensory organs are the chelicerae, which sense chemical signals in host
fluids (e.g., ATP), and the palps, which contain a sensory field on the terminal segment that
senses non-volatile chemicals (e.g., urea and lactic acid) found on host skin.
Chemical sensory receptors are classified as olfactory when they sense airborne chemicals
and gustatory when they detect chemical signals in fluids such as host blood. Haller’s organ is
thought to express both olfactory and gustatory receptors, whereas the chelicerae and palps ex-
press gustatory receptors only (Sonenshine 2006). These receptors have been shown to perceive
chemosensory signal components of animal host odors (Steullet and Guerin 1992a, 1992b, 1994;
Leonovich 2004). At the time of this writing, no cloning of receptors involved in regulating
chemical communication by ticks had been reported. Going forward, the availability of the de-
tailed anatomy of the tick sensory organ systems will offer opportunities to begin dissecting the
molecular basis of the tick sensory reception system. If we know the anatomy of the tick sensory
reception system, we can make targeted dissections of tick sensory organs and obtain material
with which to generate gene expression information for each organ.
Based on the spectrum of stimuli regulating tick behavioral adaptations to which ticks are
known to respond, the tick sensory reception system functionally includes, at the very least,
chemosensory, mechanosensory, photosensory, thermosensory, osmosensory, and hygrosen-
sory receptors. Presumably because the system is amenable to manipulation for purposes of de-
veloping novel tick control methods, at almost all levels, studies on tick chemical communication
have focused on how tick chemical communication regulates host-finding and mating behavior.
These studies have mainly focused on how the tick’s olfaction system perceives host breath
components, body odors, and CO2 for purposes of host location (Steullet and Guerin 1992a,
1992b, 1994; Leonovich 2004; Nana et al. 2010). Anatomically, the tick’s olfactory system resides
in some 20 wall-pore sensory organs found on the tick’s foreleg tarsi. Thus, we can take a forward
genetics approach to sequence Haller’s organ and generate sequence data on putative receptors.
3.3. THE MOLECULAR BASIS OF THE TICK OLFACTORY SYSTEM:

LESSONS FROM OTHER ARTHROPODS
Olfaction is a key adaptation that is important to many arthropods, particularly vectors of an-
imal and human disease agents. Without olfaction, vector arthropods would not be able to find
a vertebrate host for their blood meal and thus would not be able to acquire and transmit disease
agents. Studies on the molecular basis of olfaction in insects (Stance and Stowe 1999; Tegoni
et al. 2004; Kent et al. 2008; Li et al. 2008; Bohbot and Dickens 2009; Robertson and Kent 2009)
provide templates for validated approaches for dissecting the molecular basis of tick olfaction.
At the time of this writing, very little was known about the molecular basis of olfaction in ticks.
All blood-feeding arthropods sense host body odors, temperature, and CO2 in order to locate
their blood meal source. This, coupled with the fact that CO2 is the same molecule regardless of
its source, might suggest that the molecular structure of CO2-binding proteins or receptors is
similar or related across species. Thus we can logically take lessons from insects in which the
molecular identities of odor-binding proteins and CO2 receptors (Kent et al. 2008; Robertson
and Kent 2009) have been determined. Emerging molecular-level data suggest that olfactory
signal transduction is mediated by a complex family of G-protein-coupled-receptor (GPCR)-
mediated pathways (Dryer and Berghard 1999; Hill et al. 2002; Gaillard et al. 2004; Man et al.
2004; Tegoni et al. 2004). Some of the characterized mediators of olfaction include odorant-
binding receptor proteins (Ors), pheromone-binding proteins, olfactory arrestins (which regu-
late the function of GPCRs), G-proteins, and biotransformation enzymes (Tegoni et al. 2004).
At the time of this writing, multiple I. scapularis GPCRs were being manually annotated in prep-
aration for publication of the I. scapularis genome paper. Manual annotation approaches that
have been used in mosquitoes to discover putative Ors in insect genomes (Kent et al. 2008;
Robertson and Kent 2009) could be employed in ticks. A quick scanning of the public tick
sequence databases reveals that “arrestins” are among the auto-annotated protein families in the
tick genome. This indicates that at least the putative molecular structure of olfaction is present
in the tick genome. The next, much needed work should be the discovery and functional analysis
of molecular systems regulating olfaction in ticks.
As the tick community learns from advances in insects, will there be a limit to this approach?
In order to address this question, a limited comparative sequence analysis was conducted. Scan-
ning of the annotated Anopheles gambiae G-coupled protein odorant-binding receptor
(AY363725) (Pitts et al. 2004) against tick sequences showed identity to an auto-annotated pu-
tative I. scapularis invertebrate gustatory receptor (XP_002413910) at an e-value of 8-e−5. This
finding might suggest that XP_002413910 is a gustatory receptor and therefore a candidate for
reverse genetics to determine the possibility that this molecule is involved in animal odorant
reception in ticks. In a second case, the possibility that 2 CO2-sensing Drosophila gustatory re-
ceptors (Gr21a and Gr63a) conserved in mosquitoes (Robertson and Kent 2009) also might be
conserved in ticks is worth considering. Although this might be a case of limited available tick
sequences, this analysis revealed that these 2 receptors are apparently not encoded in ticks, as it
did not yield any sequences with significant matches. This is an interesting observation, given
that the CO2 reception system appears to be evolutionarily conserved. All blood feeders have to
sense CO2 to some extent in order to locate a host. Thus, the apparent lack of conservation of
Gr21a and Gr63a homologues in ticks might mean that ticks use very divergent CO2 receptors,
and thus data from insects might have limited utility in tick research. It is important to note here
that the comparative analysis conducted here was narrow and thus cannot be a basis for broad
conclusions. However, the lack of conservation in ticks of seemingly conserved genes in insects
may be explained by the fact that insects and ticks respond to different concentrations of CO2.
The universally conserved ability of terrestrial arthropods to sense moisture in the environ-
ment is an adaptation that is also used by ticks to locate suitable microhabitats, as well as in host
finding. To further test the utility of data from insects in research in ticks, sequences of mois-
ture- and dryness-sensing Drosophila melanogaster receptors, known as water witch (wtrw) and
nanchung (nan) genes (Liu et al. 2007), were scanned in the tick EST databases (A. Mulenga,
unpublished data). This analysis revealed that wtrw (CG31284) and nan (NP_648696) gene
sequences were conserved at least in I. scapularis and Rhipicephalus sanguineus. Among insects,
these receptors are conserved with e-values of e0.00. In ticks, however, conservation levels of the
wtrw and nan genes drop to e-values of between e1-25 and e1-12, which might signal differences in
the overall biology of the hygro-reception system. It is interesting to note that, like in insects,
wtrw and nan-like tick genes are highly conserved in ticks, with e-values of e0.00 (A. Mulenga,
unpublished data). Like the insect genes, tick wtrw and nan-like genes are both characterized by
ankyrin domains in the amino terminus region and the transient receptor potential channel
domain in the carboxy-terminal domain, which is a good indication that the humidity-sensing
system is conserved in all arthropods. The high conservation of this system might limit the
utility of this system as a target for tick control because of the potential unintended conse-
quences for beneficial arthropods.
The apparent diversity among arthropod CO2 receptors and the similarity among moisture-
sensing reception systems is interesting. Regarding our quest to advance our knowledge of tick
chemical communication by learning from advances with insects, the significance of the limited
analysis here is that the utility of advances in insect knowledge to tick research is going to be
mixed—helpful in some cases and without impact in other instances. Thus, although the tick
research community will be able to adopt experimental templates from advances in insect re-
search, necessary discovery research will have to be conducted in order to advance the field of
tick chemical communication.
3.4. MOLECULAR BASIS OF CHEMICAL-COMMUNICATION-

RESPONSIVE TICK BEHAVIORAL ADAPTATIONS
The end point of any form of communication is a behavioral change in the organism. We can
logically assume that when a tick that has attained appetence responds to CO2, a unique molec-
ular cascade that controls its host-seeking behavior is triggered. The questions are, what are
these cascades, and what happens if they are disrupted? We can also ask what happens to the
physiological status of the male tick when it responds to female tick sex pheromones. Does dis-
covery of the molecular response lead to opportunities to block tick mating, especially for meta-
striate ticks? Another interesting question pertains to the molecular basis of tick host range
adaptation. For generalist feeders such as A. americanum, does the sensing of kairomones
emitted by different animal species (e.g., chickens or cattle) lead to different molecular re-
sponses? Is there a convergence in molecular responses when ticks from different genera re-
spond to the same cues? These questions are classic candidates for forward genetics studies. The
strategy is to reproduce the behavioral adaptation and then perform transcriptome and/or pro-
teome analyses to discover candidate proteins. In conducting these studies, a major consider-
ation will be whether or not the generated data will be useful in efforts to discover novel methods
to control ticks and tick-borne diseases. This will serve as some form of a prioritization plan to
streamline the work.
One such behavioral adaptation that has been intensely studied is tick feeding behavior. The
rationale behind this effort is that tick proteins that regulate the tick feeding process will repre-
sent candidates for the development of anti-tick vaccines (Mulenga et al. 2000, 2003a, 2003b,
2007a, 2007b, 2008; Mulenga and Khumthong 2010a, 2010b; Chalaire et al. 2011). The majority
of reported studies on tick feeding regulation have focused on ticks that had fed for 3 to 5 days
(Narasimhan et al. 2006; Chalaire et al. 2011). These studies have resulted in the discovery of
multiple tick proteins ranging from anticoagulants to anti-complement proteins to proteases
and protease inhibitors (Das et al. 2001; Anguita et al. 2002; Ribeiro et al. 2006; Anderson et al.
2008). Over the years, the role of the tick salivary glands in tick feeding regulation has been a
subject of intense study (Sauer et al. 1995; Bowman and Sauer 2004; Francischetti et al. 2009;
Simo et al. 2012). Since the advent of DNA biotechnology, several EST libraries of the salivary
glands of partially fed ticks have been published that further our understanding the molecular
complexity of this organ (Santos et al. 2004; Francischetti et al. 2005; Nakajima et al. 2005;
Untalan et al. 2005; Alarcon-Chaidez et al. 2007). These studies have set the foundation for
in-depth studies on the roles of tick salivary proteins in tick feeding regulation.
The tick feeding cycle includes the preparatory feeding phase (attachment and creation of
the feeding lesion) during the first 24 to 36 hours; the slow feeding phase (moderate blood meal
uptake, pathogen transmission, increase in physical size), which may last up to 7 days; and the
rapid feeding phase (feeding to repletion), which may last for 24 hours and occurs after 7 or
more days of feeding. Given that most reports have focused on ticks during the slow feeding
phase, it is likely that those genes that are expressed at the start of feeding have been missed.
Mulenga et al. (2007a, 2008; Mulenga and Khumthong 2010a, 2010b) and others (Lew-Tabor et
al. 2010; Rodriguez-Valle et al. 2010) have focused on genes from unfed ticks that are differen-
tially expressed at the start of tick feeding. The assumption is that these genes encode for proteins
that potentially regulate events associated with the preparatory feeding phase or the tick attach-
ment phase. We believe these proteins play important roles in not only preparing the tick for
feeding but also conditioning the host to accept the transmitted tick-borne disease agents.
3.4.1. Molecular basis of the tick attachment phase: the case for
“molecular preparation to start feeding”
The Pavlovian reinforcement experiment, in which dogs that were conditioned to associate bell
sounds with being fed salivated at the sound of the bell, pointed to higher organisms’ under-
going enzymatic preparation for an impending task. Whether or not ticks have a system similar
to that of dogs is difficult to prove. However, several events during the tick feeding process point
to ticks’ potentially having an “enzymatic preparation” that prepares them to start feeding. One
line of evidence for this is that during the pre-appetence phase, newly molted or newly hatched
young ticks do not have the desire to feed until they have undergone the maturation phase
(Gladney et al. 1970; Tukahirwa 1976; Davey 1987; Anderson et al. 1998). After the maturation
phase, ticks become responsive to CO2, the signal that an animal host is present in the area. In
other experiments, ticks that had undergone the maturation phase fed efficiently when pre-
sented with a blood meal source (Tukahirwa 1976). This might suggest that after completion of
the maturation phase, genes that prepare the tick to interact with a host and start feeding are
expressed. A second source of support for enzymatic preparation is that the tick feeding style
and available experimental data strongly point to ticks’ having a set of proteins that control the
beginning steps of the tick feeding process, “the preparatory feeding phase.” The tick feeding
style of tearing host tissue and then sucking the blood that bleeds into the wounded area is ex-
pected to provoke a swift tissue-repair response by the host in order to stop any further blood
loss. Thus, one might expect that in order for the tick to feed successfully, it needs to swiftly
counter the host’s defense response to tick feeding. Given such, it is logical to assume that tick
proteins that regulate early stages of the tick feeding process are expressed before the tick inserts
its hypostome to start feeding.
There is experimental evidence that ticks are programmed to express proteins that are
unique to the beginning phase of the tick feeding process. Within 5 to 30 minutes of inserting
its hypostome, the tick injects an incompletely characterized cocktail of substances that are
important for tick cement formation into the host. This cement includes a protein secreted by
specific cell types of the salivary glands. Immunologic studies have highlighted the similarity
between the secreted cement and polypeptides in specific cell types in the type III acini of the
salivary glands of ticks (Jaworski et al. 1992; Bishop et al. 2002). In several reported studies and
personal unpublished observations, it was found that tick salivary gland protein profiles con-
stantly change during the tick feeding process, indicating that there are unique proteins that
regulate the different tick feeding phases (Wang et al. 1999; Rolníková et al. 2003). In a detailed
study, Wang et al. (1999) demonstrated that the tick R. appendiculatus expressed proteins that
were unique to unfed ticks and 24–72 hour tick salivary glands. In that study, R. appendiculatus
were allowed to feed on rabbits for 2 to 8 days, with the latter time point being when engorged
ticks normally detach. The authors observed that the majority of ticks that were partially fed
for 2 and 4 days were able to survive for 4 weeks in an incubator. Those ticks that survived were
able to attach to the second rabbit, feed to repletion, and lay eggs. The most interesting obser-
vation in this study was that the re-attachment of these ticks was accompanied by switching of
the salivary gland protein expression pattern back to that of the non-parasitic state, before the
ticks could re-attach (Wang et al. 1999). This study (Wang et al. 1999) clearly demonstrates that
unfed ticks express certain unique proteins that must be essential for survival off the host and/
or that are needed in order for the tick to engage with the host to initiate the feeding process.
In related studies, differential anti-mammalian cell proliferation activities were reported in
salivary gland protein extracts from unfed and partially fed ticks (Rolníková et al. 2003; Ka-
zimírová et al. 2006). Crude protein preparations of tick salivary glands that were dissected
from unfed and partially fed I. ricinus, A. variegatum, and R. appendiculatus differentially sup-
pressed PHA- and Concanavalin A-induced BALB/C mouse lymphocyte proliferation, with
more potency observed in unfed tick salivary gland extracts (Rolníková et al. 2003). The
studies reviewed here collectively suggest that ticks express unique proteins that regulate im-
portant functions during the different tick feeding phases. These proteins potentially regulate
tick feeding events that precede blood meal uptake and the acquisition and transmission of
tick-borne disease agents. Although there are exceptions, such as tick-borne encephalitis and
Powassan viruses that are transmitted within 15 minutes of the tick’s attaching to the host, the
majority of tick-borne disease agents are transmitted after the tick has been attached to host
skin for at least 48 hours (Piesman et al. 1987, 1991; Yeh et al. 1995; Sood et al. 1997; Katavolos
et al. 1998; des Vignes et al. 2001; Crippa et al. 2002; Ebel and Kramer 2004; Meiners et al.
2006; Konnai et al. 2007). Thus the appeal of these proteins is that blocking their functions will
protect the host from all facets of tick parasitism, blood loss, and tick-borne disease agent
transmission. It is logical to assume that tick saliva proteins that regulate tick feeding events at
the inception of the feeding process play significant roles in preparing the host for the trans-
mission of tick-borne disease agents. The question that remains is, at what point during the
tick feeding process are these proteins expressed? Is it before the ticks attach to host skin, or
is it post-attachment? In our lab, we are investigating the possibility of these proteins being
expressed prior to the tick’s attachment to host skin and whether the expression of these
proteins is potentially regulated by chemical communication.
3.4.2. Discovery of the tick’s molecular preparation to start feeding

From the foregoing, it is apparent that the preparatory feeding phase is preceded by defined
behavioral changes that are subject to chemical communication. The question is whether mim-
icking behavioral changes that precede the attachment phase will allow us to capture candidate
tick proteins that mediate tick–host interactions at the inception of the tick feeding process.
We developed an experimental strategy based on 3 key considerations of the tick feeding
process, as summarized in Fig. 14.3. Firstly, the newly hatched or molted ticks must undergo a
physical maturation phase before they can attain appetence (physiological readiness to feed),
respond to feeding stimuli, and seek a blood meal source (Gladney et al. 1970; Tukahirwa 1976;
Davey 1987; Anderson et al. 1998). Secondly, the tick feeding style of lacerating host tissue and
then sucking host blood from the hematoma that forms in the wounded area (i.e., the tick
feeding site) is thought to stimulate host tissue repair responses that are aimed at stopping
further blood loss. However, ticks ensure a full blood meal by secreting a cocktail of enzymes
that disarm the host’s tissue repair response (Ribeiro et al. 1985; Ribeiro 1989, 1995; Valenzuela
et al. 2005). As the host’s tissue repair response to tick feeding activity is expected to be rapid
(Edmonson et al. 2003), logically, it must be swiftly countered by the tick. On the basis of these
observations, one can argue that upon the attainment of appetence and/or exposure to feeding
stimuli, ticks must express genes that are required to regulate the preparatory feeding phase.
The term “tick feeding stimuli” is being used here to loosely mean host odorants, host contact,
and body temperature.
Studies have shown that newly molted young ticks less than 7 to 21 days of age respond
poorly to CO2 stimuli, which represent host breath; the ticks walk away from the source, indi-
cating that they are not ready to initiate feeding. However, ticks older than 21 days show a strong
response to CO2 stimuli; these ticks move toward the source, indicating physiological readiness
to start feeding (Gladney et al. 1970; Tukahirwa 1976; Davey 1987; Anderson et al. 1998). With
the assumption that the attainment of physiological readiness to feed is controlled by the differ-
ential expression of genes that regulate feeding, the subtractive hybridization methodology was
used to subtract the cDNA library of newly molted (~0 to 1 day) adult A. americanum female
ticks that were not stimulated to start feeding from the cDNA library of 5-week-old unfed ticks
that were exposed to feeding stimuli for ~7 hours (Mulenga et al. 2007a). The limiting factor in
this experiment was the generation of female ticks that were stimulated to start feeding. The
AAA pheromone, which is secreted by male ticks that have fed for at least 1 day, promotes fe-
male tick attachment (Sonenshine 2006). In our experiment, we hypothesized that the exposure
of female ticks to the AAA pheromone triggers the expression of some of the genes that prepare
the tick to start feeding. Thus, in our experiment, we exposed female A. americanum ticks to the
AAA pheromone by exposing them to male ticks that had been pre-attached to a calf for 3 days.
The female ticks were enclosed in a nylon mesh sachet that was then placed in a cell contain-
ment device (i.e., sealed capsule) on the back of the calf. The cell contained male ticks that had
been pre-fed for 3 days. These ticks were left there for 7 hours before being removed, after which
they were immediately pulverized in liquid nitrogen and processed for total RNA extraction.
A similar protocol was repeated for the control group of young newly molted female ticks.
As summarized in Fig. 14.3, the extracted total RNA was subjected to suppressive subtractive
FIGURE 14.3: Discovery of the tick’s molecular preparation to start feeding. A, The cDNA library of older
ticks (OT) that were stimulated to start feeding was subtracted from the cDNA library of newly molted
young ticks (YT) that were not stimulated to start feeding. B, Polymerase chain reaction (PCR)
amplifi cation of subtracted (S) and non-subtracted (NS) cDNA libraries using adapter primers with
PCR products resolved on 2% agarose gels containing 1% ethidium bromide and viewed in inverse
format to improve the contrast. Lane 1 = NSYT cDNA library of young ticks that were not stimulated to
start feeding; lane 2 = cDNA library of young ticks not stimulated to start feeding subtracted from the
cDNA library of older ticks that were stimulated to start feeding (SYT); lane 3 = cDNA library of older
ticks that were stimulated to start feeding subtracted from the cDNA library of young ticks that were
not stimulated to start feeding (SOT); lane 4 = non-subtracted cDNA library of older ticks that were
stimulated to start feeding (NSOT). C, D, Validation of differential gene expression via dot blot
hybridization analysis. Putatively differentially expressed genes (SOT library) were dot blotted onto
positively charged nylon membrane. Following appropriate treatment of the membranes, dot blots were
probed with radioisotope dATP (P32)-labeled SYT (C) or SOT (D). Asterisks (*) denote validated
differentially expressed genes in response to tick feeding stimulation. Adapted from Mulenga, A.,
Blandon, M., and Khumthong, R. (2007) The molecular basis of the Amblyomma americanum tick
attachment phase. Exp. Appl. Acarol. 41:267–287.
hybridization (SSH). Following SSH analysis, we further validated differential gene expression
via cDNA dot blot hybridization analysis. This approach allowed the identification of 40
A. americanum genes that were differentially up-regulated or induced in response to the attain-
ment of appetence and/or exposure to tick feeding stimuli (Mulenga et al. 2007a). Of the 40
genes, 38% were provisionally identified and classified into 6 groups: ligand binding, immune
responsive, stress response proteins, transporter polypeptides, enzymes/regulator, and extracel-
lular matrix-like proteins. Since the publication of the results in 2007, 2 other studies on
R. microplus employing SSH and microarray approaches have been published (Lew-Tabor et al.
2010; Rodriguez-Valle et al. 2010). Using protocols similar to that in Fig. 14.3, the authors cre-
ated cDNA libraries of unfed larvae, nymphs, and adult females that were exposed to a host
animal for 24 hours without being allowed to attach to the host. The next set of libraries in-
cluded male and partially fed female adult ticks. These libraries were subjected to SSH or micro-
array analyses. The most interesting observation from the 3 studies reviewed here is that
although these ticks were not of the same species, some of the differentially up-regulated genes
were functionally similar based on provisional identification. Most notable among these were
the GGY-domain-containing proteins, histamine binding proteins, carboxypeptidase A, and
heat shock proteins, which were found to be up-regulated in all 3 studies (Mulenga et al. 2007a;
Lew-Tabor et al. 2010; Rodriguez-Valle et al. 2010). This is encouraging, as it points to ticks’
using similar molecular pathways to regulate initial stages of tick feeding.
It is important to note that if the experiments reviewed here were to be repeated using more
robust transcriptome analysis methods such as RNA sequencing technologies (e.g., Illumina
and 454 sequencing), it might be possible to obtain a clearer picture of what molecular systems
are at play at the start of tick feeding. Regardless of these limitations, these data sets have pre-
sented an opportunity to dissect the molecular basis of the tick attachment phase.
Observations of the up-regulation of genes such as the histamine binding proteins, which
can inhibit the host’s inflammatory responses, are understandable. One line of defense for the
tick is to inhibit the inflammatory response, which is the core host immune response. Likewise,
the up-regulation of GGY-domain-containing proteins is interesting. These proteins are thought
to have antimicrobial functions. In addition to overcoming host defense reactions, the tick must
protect the feeding site from microbial contamination.
Experiments described here are not without limitations. Probably the most important lim-
itation is that more than 60% of the identified genes were novel, which raises the problem of
how to start functional characterization studies. As a strategic follow-up experiment, RNAi
silencing technology could be used to validate the role(s) and significance of candidate genes
in regulating the preparatory tick feeding phase. The expectation is that the RNAi silencing
methodology would narrow down the pool of important candidate genes. Validated genes
could then be subjected to a battery of assays in order to establish the roles of candidate genes
in tick feeding regulation. Given that the long-term practical goal of this type of research is
vaccine development, assays would be biased toward finding anti-tick vaccine target antigens.
RNAi mediated silencing is being used against A. americanum genes that are differentially up-
regulated in ticks that have attained appetence and/or that are exposed to tick feeding stimuli.
Some that have been validated as playing important roles in tick feeding success include 3
A. americanum insulin-like growth factor binding protein-related proteins (Mulenga and
Khumthong 2010a) and the CD147 receptor homolog (Mulenga and Khumthong 2010b), for
which silencing significantly reduced tick feeding efficiency. Most important, the silencing of
these genes blocked the tick’s ability to efficiently lay eggs. We also have silenced genes that
were differentially up-regulated with no effect on tick feeding efficiency. For instance, silencing
the A. americanum organic anion transporter polypeptide did not affect tick feeding efficiency
(Mulenga et al. 2008), despite its being up-regulated at the start of tick feeding. Whether the
fact that tick feeding efficiency was not affected by the silencing of certain genes means that
those genes are not essential is debatable. We have recently observed that despite the complete
disruption of the cognate transcript in RNAi silencing, the target protein that was translated
prior to RNAi silencing remains active (Chalaire et al. 2011). From the perspective of targeting
proteins that are functional at the start of tick feeding, the observations of Chalaire et al. (2011)
might mean that RNAi silencing will not necessarily affect the ability of the tick to start feeding.
The limitations of RNAi silencing in tick research will hamper progress toward understanding
the molecular mechanisms regulating tick feeding. One way to solve this problem is to vacci-
nate animals with recombinant proteins as a means of validating the significance of target
genes in tick feeding regulation. This approach, however, is limited by the prohibitive cost of
applying high-throughput screening methods.
4. FUTURE DIRECTIONS
Little is known about the molecular biology of chemical communication in ticks. The necessary
fundamental discovery research that will be required includes cloning and functional character-
ization of the receptors for the different chemosensory signals. Data on the identity and chemi-
cal structure of chemosensory signals will be necessary in order for the cloned receptors to be
functionally characterized. Regarding the 3 aspects of tick chemical communication discussed
earlier, the molecular mechanisms that underpin tick behavioral adaptations should be the eas-
iest to resolve and have the most promise for the development of novel tick control methods.
Most behavioral adaptations are reproducible under laboratory conditions. Forward genetics
strategies can be employed to discover candidate genes or pathways that might regulate these
behavioral adaptations.
There are other behavioral adaptations that are not controlled by chemical communica-
tion; the study of these adaptations should reveal important data with broader relevance to
the development of novel tick control methods. One such interesting biological adaptation
is the ability of ticks to undergo diapause, an adaptive arrestment in development and/or
behavioral activity (Loomis 2010; MacRae 2010). Synchronization of the tick’s life stages
with seasonal environmental changes is the basis for diapause. From the perspective of dis-
covering molecular pathways that regulate behavioral adaptations important to the biology
of the tick, the diapause system will be interesting. What is turned on and off during dia-
pause? What is turned on when diapause is broken? What is the molecular basis for breaking
diapause? The availability of technologies for the comparative analysis of genome-wide
DNA methylation profiles or micro-RNA expression, for example, can lead to discovery of
the important regulatory genes; those that are regulated when diapause is broken might
represent important candidates essential for tick parasitism.
Given the paucity of data on tick chemical communication in general, this chapter offers
much food for thought for future directions and discussion, some of which is likely to pro-
voke debate and research interest in the tick community. Hopefully, this discussion has set a
foundation and will spur interest in fundamental research on the biology of tick chemical
communication.
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Entomol. 104:1189–1194.
C H A P T E R 1 5
HEME-BINDING LIPOGLYCO-
STORAGE PROTEINS
SAYED M . S . KHALI L , KEVIN V. DONOHUE , R. MICH AEL ROE , AND
DANIEL E. S ONENSH INE
1. INTRODUCTION
Two major storage proteins are found in ticks: (i) the hemelipoglyco-carrier protein (CP) and
(ii) the female-specific yolk proteins, named vitellogenin (Vg) at their site of synthesis (and
when found in the hemolymph) and vitellin (Vn) when transferred into the egg. Our under-
standing of these storage proteins, especially at the molecular level, and especially of CP, has
developed only in about the past decade. Both Vg and CP are large-molecular-weight proteins
that in their native form in hemolymph are at least 200 kDa and which bind lipids, carbohy-
drates, and heme. CP is constitutively expressed throughout most of tick development, whereas
Vg is found only in females at the time of egg development. Vg and CP also appear to share a
common evolutionary origin belonging to a lipid transfer protein superfamily (Donohue et al.
2008, 2009). Because of the similarities between CP and Vg, the presence of multiple genes for
each, and the different life styles of ticks (e.g., sexual reproduction versus parthenogenesis), the
classification of these proteins can be problematic, especially when only the amino acid sequence
of the protein is considered. In addition, most of the research so far on storage proteins in ticks
has been on Vg. Further information on Vg and CP can be found in Chapters 9 and 17.
2. DEVELOPMENTAL CHANGES IN PROTEIN

LEVELS OF CP VERSUS V g
Figure 15.1 shows the developmental profiles of CP and Vg as analyzed via native polyacrylamide gel
electrophoresis (PAGE) over the entire development of the American dog tick, Dermacentor varia-
bilis. CP was the major protein in the hemolymph when it was evaluated on a total plasma protein
Heme-binding Lipoglyco-storage Proteins 399
basis or plasma volume basis (data not shown) in both unfed and blood-fed adult males and females.
In fact, even after ticks had gone without feeding for months, CP remained the dominant plasma
protein in males and females. Blood feeding had no obvious effect on the abundance of CP in males.
Males unfed for 3 months had approximately the same levels of CP, based on equal protein loading
of PAGE gels, as males that had fed and been detached from the host for 10 days (Fig. 15.1). However,
the same was not true for females. On an equal-protein basis, the abundance of CP declined in par-
tially fed females (10 days after attachment) and in replete females (14 days after detachment) relative
to unfed adult females (Fig. 15.1). On a per-unit-of-plasma basis, the highest levels of CP in blood-
fed females were found in partially fed ticks 11 and 13 days after attachment, relative to partially fed
ticks 17 days after attachment and replete females 9 to 17 days after detachment (data not shown). Vg
appeared in the hemolymph in these studies only in replete females and had the same electropho-
retic mobility as purified Vn (which was clearly different from that of CP).
CP constituted 60%–70% of the total plasma protein in virgin (unfed) females. Whereas CP is
the predominant hemolymph protein in replete mated females, only traces of CP were found in
1- and 9-day-old eggs (Fig. 15.1). The Vg receptor in this tick (Mitchell et al. 2007) apparently is effec-
tive in excluding CP, even though the proteins have a similar primary structure; the basis for this
FIGURE 15.1: Native-PAGE of CP based on equivalent protein (10 μg) per lane of D. variabilis whole body
homogenate from unfed larvae (unfed for 6 months), fed larvae (3 days after detachment), and fed
nymphs (10 days after detachment) and plasma from unfed adult males (unfed approximately 3 months),
fed adult males (10 days after detachment), unfed adult females (unfed for 3 months), partially fed virgin
adult females (10 days after attachment), and replete adult females (14 days after detachment). The
remaining lanes are 1-day-old eggs, 9-day-old eggs, purifi ed native CP from the plasma of partially fed
virgin females (10 days after attachment), and purifi ed native Vn from day 1 eggs. In fed nymphs and in
eggs, CP was visualized on the gels at low levels that could not be photographed. Gel was stained with
Coomassie Blue. CP, carrier protein; PF, partially fed; Vg, vitellogenin; Vn, vitellin. From Gudderra, N.P.,
Neese, P.A., Sonenshine, D.E., Apperson, C.S., and Roe, R.M. (2001) Developmental profi le, isolation,
and biochemical characterization of a novel lipoglycoheme-carrier protein from the American dog
tick, Dermacentor variabilis (Acari: Ixodidae) and observations on a similar protein in the soft tick,
Ornithodoros parkeri (Acari: Argasidae). Insect Biochem. Mol. Biol. 31:299–311, with permission
from Elsevier.
exclusion is unknown. In these studies, it was difficult to obtain hemolymph from larvae and nymphs
because of their small size. However, in 6-month-old unfed larvae, CP was the major protein found
in whole body homogenates. Considering the low levels of CP in 9-day-old eggs versus its predom-
inance in unfed larvae, CP must be synthesized de novo sometime during embryological and/or
larval development. CP was also found in the whole body homogenates of fed larvae and fed nymphs,
but at lower levels than in unfed larvae based on equal protein loading. This reduction is likely a re-
sult of the ingestion of large amounts of vertebrate blood protein, rather than changes in CP protein
levels in the tick. The assumption is that CP is a major hemolymph protein throughout tick develop-
ment, although this has not been confirmed in embryos, larvae, and nymphs.
3. TISSUE LEVELS OF CP AND Vg
CP was resolved by means of native-PAGE from 10 μg protein from fat body, salivary gland,
ovary, and muscle homogenates and was compared to hemolymph from partially fed females of
the American dog tick obtained 12 days after attachment (Fig. 15.2) (Gudderra et al. 2002). CP
in these studies was most abundant in salivary glands and plasma relative to fat body and muscle.
At a higher loading rate of 30 μg, CP was also more abundant in salivary glands than in fat body.
Northern analyses by Donohue et al. (2008) (Fig. 15.3A) showed that after attachment for 6 days,
the expression of CP messages in female D. variabilis was greater in the fat body and salivary
glands than in the ovary or midgut. Both Vg1 and Vg2 (Thompson et al. 2007; Khalil et al. 2011)
were found mostly in the midgut and ovary in Northern analyses (see Fig. 15.3B for Vg1). The
tissue distribution of Vg synthesis is discussed also in Chapters 16 and 17. The molecular biology
of CP and Vg is discussed in more detail later in this chapter, as well as in Chapters 9, 16, and 17.
It is interesting that we actually sequenced Vg2 and CP in the American dog tick from the con-
struction of a synganglion 454 transcriptome. In some cases, the assembly contained a consid-
erable number of reads, suggesting that the message was abundant. Further clarification is
needed as to whether this is fat body contamination, which is likely at least to some extent, or
whether these proteins are actually synthesized by the synganglion. Additional studies are
needed to localize the exact cellular locations of both CP and Vg synthesis in the different tissues
that have been collected from ticks.
4. CHEMICAL COMPOSITION OF STORAGE PROTEINS

AND THEIR FUNCTION
4.1. STORAGE PROTEINS IN ARTHROPODS OTHER THAN TICKS

Our knowledge of storage proteins and their function in insects is highly advanced relative to such
knowledge about ticks. For example, insect lipophorin (Lp) is a lipoglycoprotein that can trans-
port di- and tri-acylglycerols, phospholipids, and free fatty acids. Lps are divided into 2 classes:
high-density lipophorins (HDLp) with a molecular weight of 450 to 600 kDa and low-density li-
pophorins (LDLp) with a molecular weight of 1,500 kDa (Weers and Ryan 2006; Ziegler and An-
twerpen 2006). The lipid content of HDLp is 30% to 50%, and for LDLp it is up to 62%. Another
FIGURE 15.2: Native-PAGE of D. variabilis tissue homogenates and plasma from partially fed virgin
females 12 days after attachment. 1, purifi ed native CP (5 μg) from partially fed virgin female plasma (10
days after attachment); 9, purifi ed native vitellin (5 μg) from day 1 eggs. Lanes 2–6 were loaded with 10
μg of protein each; lanes 7 and 8 were loaded with 30 μg of protein each. Gels were stained with
Coomassie blue. CP, carrier protein; FB, fat body; MU, muscle; OV, ovary; PL, plasma; SG, salivary
gland; VN, vitellin. From Gudderra, N.P., Sonenshine, D.E., Apperson, C.S., and Roe, R.M. (2002)
Tissue distribution and characterization of predominant hemolymph carrier proteins from Dermacentor
variabilis and Ornithodoros parkeri. J. Insect Physiol. 48:161–170, with permission from Elsevier.
group of insect storage proteins is the hexameric family synthesized in the larval fat body and
stored in the hemolymph and fat body as an available amino acid reserve. Hexameric hemocya-
nins are found in crustaceans and chelicerates, where, unlike in insects, they have the ability to
bind elemental copper (Beintema et al. 1994). Another insect storage protein is Vg, the major egg
yolk precursor found in insect hemolymph. During vitellogenesis, Vg is synthesized by the fat
body in insects or by the hepatopancreas in crustaceans (Gudderra et al. 2001). Storage proteins
are important in insects because they provide reserves during periods of non-feeding, especially
of amino acids and lipids. Insects are not able to feed during embryogenesis, the period prior to
ecdysis (including apolysis), pupation, reproduction (in some species), migration, and diapause.
4.2. COMPOSITION OF STORAGE PROTEINS IN TICKS

Lipid, carbohydrate, and heme binding of Vg are discussed in detail in Chapter 17. CPs iso-
lated from hard ticks appear to bind the same compounds. Maya-Monteiro et al. (2000) re-
ported that carbohydrates constituted 3% (w/w) of the heme lipoprotein (HeLp or RmCP) in
FIGURE 15.3: Northern analysis of the tissue-specifi c expression of D. variabilis (A) CP in tissues
isolated from 6-day-fed females and (B) Vg1 in tissues isolated from pre-ovipositing vitellogenic
females. FB, fat body; M, RNA molecular weight marker; MG, midgut; OV, ovary; SG, salivary gland.
The size in nucleotides is shown. Panel A from Donohue, K.V., Khalil, S.M.S., Mitchell, R.D.,
Sonenshine, D.E., and Roe, R.M. (2008) Molecular characterization of the major hemelipoglycoprotein
in ixodid ticks. Insect Mol. Biol. 17:197–208, with permission from John Wiley and Sons. Panel B from
R.M. (2007) Sequence and the developmental and tissue-specifi c regulation of the fi rst complete
vitellogenin messenger RNA from ticks responsible for heme sequestration. Insect Biochem. Mol. Biol.
37:363–374, with permission from Elsevier.
Rhipicephalus (Boophilus) microplus, with mannose being the major sugar. Dupejova et al.
(2011) reported the presence of N-glycans (hybrid and complex types) in hemelipoglycopro-
tein (HLGP), another CP isolated from the ornate sheep tick, D. marginatus, with terminal
mannose, galactose, or sialic acid. They also suggested the presence of O-glycans. Lipids con-
stituted 33% of RmCP, with 55% being neutral lipids (cholesterol ester, triglycerides, free fatty
acids, and free cholesterol) and 44% being phospholipids (phosphatidylethanolamine and
phosphatidylcholine) (Maya-Monteiro et al 2000). One of the unique characteristics of HeLp
is the presence of cholesterol esters (~34% of the total lipid content), which had not been re-
ported before in invertebrate lipoproteins. CP from the American dog tick carried phospho-
lipids, free cholesterol, free fatty acids, triheptadecanoin, and monoacylglycerol (Gudderra
et al. 2001).
4.3. FUNCTION OF STORAGE PROTEINS IN TICK NUTRITION

The role of CP in ticks is not fully understood, whereas Vg is known to be the major yolk protein
supplying nutrients to growing embryos (discussed in more detail in Chapters 16 and 17). CP is
thought to play a role in the storage and transfer of carbohydrates and lipids (Fig. 15.4; CP
stained for lipids with Sudan black), and as a high-molecular-weight protein, it should serve as
FIGURE 15.4: Native-PAGE of tissue homogenates and plasma of D. variabilis replete females 3 days
after detachment. 1, purifi ed native CP from partially fed virgin female plasma (10 days after
attachment); 2, fat body (15 μg); 3, salivary gland (15 μg); 4, plasma. Gel was stained with Sudan black.
CP, carrier protein; FB, fat body; SG, salivary gland; PL, plasma. From Gudderra, N.P., Sonenshine,
D.E., Apperson, C.S., and Roe, R.M. (2002) Tissue distribution and characterization of predominant
hemolymph carrier proteins from Dermacentor variabilis and Ornithodoros parkeri. J. Insect Physiol.
48:161–170, with permission from Elsevier.
FIGURE 15.5: SDS-PAGE analysis of purifi ed native CP from plasma of partially fed (PF) virgin female
adults of D. variabilis (10 days after attachment). CP (purifi ed or from crude plasma) consists of 2 bands
(92 and 98 kDa). CP, carrier protein; Vn, vitellin. From Gudderra, N.P., Neese, P.A., Sonenshine, D.E.,
Apperson, C.S., and Roe, R.M. (2001) Developmental profi le, isolation, and biochemical characterization
of a novel lipoglycoheme-carrier protein from the American dog tick, Dermacentor variabilis (Acari:
Ixodidae) and observations on a similar protein in the soft tick, Ornithodoros parkeri (Acari: Argasidae).
Insect Biochem. Mol. Biol. 31:299–311, with permission from Elsevier.
a major source of amino acids needed for the synthesis of other proteins. Because of the
extended periods of non-feeding in ticks (sometimes years), other developmental processes
similar to those of insects (e.g., diapause and molting), and the requirement of finding a host
and blood meal before progressing to the next stage or laying eggs (which in some cases can take
months or longer), storage proteins like CP in ticks likely have the same function as storage
proteins in insects. This is a reasonable assumption, as CP levels appear to decrease in the hemo-
lymph in replete females during Vg synthesis and uptake by developing eggs, suggesting that CP
is used to some degree in the synthesis of the yolk protein. However, a decrease in hemolymph
CP levels in unfed ticks with time has not been demonstrated; such a decrease would suggest
that CP is a source of nutrients during non-feeding periods. More research is needed before the
role of CP in larval, nymphal, and adult development can be better understood.
4.4. FUNCTION OF STORAGE PROTEINS IN HEME PROCESSING

Adaptation to hematophagy has developed multiple times within the Arthropoda, and even within
particular groups such as the Diptera (Law et al. 1992; Mans and Neitz 2004). Important adapta-
tions that co-evolved with blood feeding are heme sequestration by heme-binding proteins and a
means of heme detoxification, both of which prevent oxidative stress and tissue damage (see Chap-
ter 6 for a more detailed description of this process). The cytotoxicity of free heme results from the
formation of reactive oxygen species, which leads to lipid peroxidation (Hamza et al. 1998). Heme
is also important as a prosthetic group for respiration, enzymatic detoxification, and oxygen trans-
port (Furuyama et al. 2007). In Rhodnius prolixus, for example, host hemoglobin is digested and
converted to free heme, which is then absorbed into the hemolymph and sequestered by a 15 kDa
heme-binding protein, RHBP. RHBP prevents lipid peroxidation and functions as an antioxidant
(Oliveira et al. 1995; Machado et al. 1998). Other heme-binding proteins present in Rho. prolixus
include nitrophorins for nitric oxide transport (Weichsel et al. 1998), which have been implicated
in host vasodilation during blood feeding. This suggests multiple uses for heme and heme-binding
proteins in blood-feeding insects, and possibly in other organisms such as ticks.
Despite the abundance of heme from the host hemoglobin, triatomines apparently have the
ability to synthesize heme, as evidenced by the expression of delta-aminolevulinic acid dehydra-
tase, the rate-limiting enzyme in the heme biosynthetic pathway (Braz et al. 1999). However, the
southern cattle tick, R. microplus (Braz et al. 1999), apparently has lost the ability to make heme.
More studies exploring this hypothesis are needed in light of the ongoing effort to annotate the
Ixodes scapularis genome and the discovery of at least several enzymes involved in heme synthe-
sis in both the genome and the transcriptome of different tick species. Because of the extended
periods between blood feeding in ticks and/or their apparent inability to synthesize heme based
on the work of Braz et al. (1999), it is reasonable to assume that ticks might have a mechanism
by which they sequester and store heme in a non-toxic form.
It appears that tick Vg and CP are unique within the Arthropoda in their ability to bind, store,
and transport heme, providing a potential source of heme during periods of non-feeding. Vg is
also involved in the transport of heme into the egg during embryogenesis. Vg and CP also might
be important in heme detoxification because of their ability to sequester heme and, if nothing else,
at least provide a safe method for the storage and supply of heme during periods of non-feeding.
For more details on blood meal digestion and heme processing, see Chapter 9.
Maya-Monteiro et al. (2000) reported that HeLp is the only heme-binding protein in the
hemolymph of R. microplus. One mole of HeLp in hemolymph bound 2 moles of heme, whereas
the binding capacity in vitro was 8 moles of heme. In the adult stage, before the initiation of
vitellogenesis, CP in the American dog tick is the major tick storage protein. Then this function
shifts to Vg in replete ticks as a result of mating and increased levels of ecdysteroids. Chinzei
(1983) could not detect CP in the eggs of the soft tick Ornithodoros moubata, although it was
detected as the most abundant protein in the hemolymph of adult males and females. Gudderra
et al. (2001) also detected only trace amounts of CP, in contrast to the large amounts of Vn, in the
eggs of D. variabilis. As heme is required for development, the fact that tick embryos cannot
synthesize heme means Vn must be the only source of heme in the egg. In newly oviposited eggs,
tick Vn binds heme at a molar ratio of 1:1, but in vitro, 1 Vn molecule can bind more than 30
heme molecules (Logullo et al. 2002). Based on these findings, Lugullo et al. (2002) suggested
that the ability of Vn to bind heme provides a safe mechanism for heme storage and provides this
essential nutrient to embryos with minimal toxic effects.
4.5. OTHER POSSIBLE FUNCTIONS OF CP

As with the insect storage proteins, there are likely multiple functional roles for heme-binding
proteins in ticks. Dupejova et al. (2011) reported that HLGP has the ability to bind immobilized
carbohydrates, suggesting a possible role in innate immunity. Also, the existence of CPs inside
gut cells and on their outer surface and the ability of CP to hemeagglutinate red blood cells sug-
gest that the protein might have a role in the concentration of the blood meal and in facilitating
water excretion after feeding.
Chinzei (1983) was the first to report the presence of CP in a soft tick and found 2 proteins in the
hemolymph of adult males and females of O. moubata. Later, Gudderra et al. (2001, 2002) reported
the presence of a predominant protein (OpCP) in the hemolymph of unfed and fed O. parkeri
males and females and hypothesized that this protein has the same function as CP in D. variabilis.
OpCP was detected in the hemolymph and coxal fluid but was absent in the female fat body, salivary
gland, and ovaries. Interestingly, the hemolymph and eggs of soft ticks were reported to contain
heme (Chinzei et al. 1983; Taylor et al. 1991; Gudderra et al. 2002), but heme was not detected in
purified OpCP or in coxal fluid that contained CP. More studies are needed in order to determine
whether heme in the soft tick is associated with CP and to determine the role of CP in the soft tick.
Dupejova et al. (2011) raised the question of the real role of CPs in hard ticks, noting that CP is the
major storage protein found in males, which ingest smaller amounts of host blood than females.
Also, CP was detected in the hard tick salivary glands, where no blood digestion or heme release
takes place (this is discussed in more detail later). Finally, the brown color of heme and its seques-
tration into eggs as Vn result in the production of brown eggs; this transition from white to brown
is shown in Chapter 16. This color transition makes the eggs more difficult to visualize in leaf litter,
a common ovipositional site for ticks, and might provide some adaptive advantage for egg survival.
5. MOLECULAR BIOLOGY OF STORAGE PROTEINS
5.1. Vg1 VERSUS Vg2

Two Vgs have now been characterized from the American dog tick. Boldbaatar et al. (2010b)
found multiple Vgs in Haemaphysalis longicornis, and 2 partial sequences were reported from
R. microplus (GP80, U49934 and the expressed sequence tag [EST] CV436305). All the full-
length Vgs available from ticks are in the range of 5–6 kb. Several Vg mRNAs have been isolated
from insects, and these are mostly 6–7 kb (Tufail et al. 2005; Tufail and Takeda 2008). Most
insect Vgs are synthesized as ~200 kDa precursors that undergo post-translational proteolytic
cleavage by endopeptidases and coupling with carbohydrates, lipids, and other moieties. Vgs
appear in the insect hemolymph as 400 to 600 kDa proteins (Tufail et al. 2005). Taylor and
Chinzei (2002) indicated that Vg in the tick O. moubata appears in the hemolymph as 2 bands
of 300 and 600 kDa. DvVg1 (Thompson et al. 2007) and DvVg2 (Khalil et al. 2011) have calcu-
lated molecular weights of 206 and 215 kDa, respectively. It is well known that insect Vgs gener-
ally exist in oligomeric forms (reviewed by Thompson et al. 2007; Roe et al. 2008). The molecular
weight of the 2 DvVgs is in the range of 412–430 kDa, depending on whether the dimer is 2X
Vg1/2X Vg2 or 1X Vg1/1X Vg2. This is within the range reported before for native Vg in the
American dog tick (Thompson et al. 2007; Roe et al. 2008) and suggests that the native protein
is dimeric, potentially consisting of different combinations of Vg1 and Vg2.
Vgs belong to the large lipid transfer protein superfamily with specific characteristics such
as the N-terminus lipid binding domain (LPD_N), the C-terminus von Willebrand type D do-
main (vWD), the GL/ICG domain, and the cleaving sites (R/KXXR/K). Vgs from insects con-
tain a conserved GL/ICG domain near the C-terminus (Sappington and Raikhel 1998; Tufail and
Takeda 2008), whereas Vgs from Cructacea contain a GLLG motif, and vertebrates a longer
TCGL/ICG motif (Mouchel et al. 1996). Examinations of the available Vgs from ticks revealed
that DvVg2, both IsVgs, OmVg, and the conceptual translation of the Rm EST CV436305 con-
tain a typical GLCG domain, whereas DvVg1 and Rm GP80 contain the mutated GLCS domain;
regardless, all of them are located near the C-terminus. Baker (1988) compared the vWD in Vgs
with the vW factor, which plays a role in blood clotting in mammals, and suggested that the
vWD of Vg might play a role in binding to the Vg receptors found on the surface of oocytes.
All known insect Vgs (with the exception of Apocrita) contain 1 or more R/KXXR/K cleavage
sites (Tufail and Takeda 2008). Such sites are recognized by subtilisin-like convertases (Rouille
et al. 1995), which cleave the Vgs into 2 or more subunits. Thompson et al. (2007) noticed that
DvVn resolved on sodium dodecyl sulfate (SDS)-PAGE as 7 subunits (210, 172, 157, 111, 76.2,
58.7, and 50.8 kD). They predicted that the 2 subunits with observed molecular weights of 50.8
and 157 kD resulted from cleavage at the only RXXR cleavage site found in DvVg1. In DvVg2,
one of the RXXR sites (RPLR, aa 1550–1553) gives 2 expected fragments of 173 and 42 kDa. The
expected 173 kDa fragment might contribute to the 172 kDa band resolved via SDS-PAGE,
whereas the small fragment (42 kDa) might be subjected to further degradation and is not de-
tected. All other cleavage sites give expected products of calculated molecular weights different
from those noted by Thompson et al. (2007). Understanding the exact cleavage pattern of Vgs in
the American dog tick has been problematic and will likely require N-terminal sequencing of
each subunit. We successfully used tryptic digest-mass fingerprinting to recognize some frag-
ments from both Vg and Vn bands that were predicted from the Vg1 and Vg2 messages and
which indicate that both DvVg1 and DvVg2 contribute to both Vg and Vn found in the hemo-
lymph and in the egg, respectively, in the American dog tick. Further discussions of the molec-
ular biology of tick Vgs can be found in Chapter 17.
5.2. CP
The first full-length CP sequence, from the tick D. variabilis, was published by Donohue et al.
(2008); soon after an Amblyomma americanum CP was released. Several CP partial sequences
were reported from EST libraries made from the salivary gland or the whole body of several
other ticks such as R. appendiculatus (Nene et al. 2004), R. microplus (Guerrero et al. 2005),
A. varieqatum (Nene et al. 2002), A. cajennens (Batista et al. 2008), and others. Nene et al. (2004)
were the first to indicate that multiple CP homologues were present in R. appendiculatus. This
finding was confirmed in D. variabilis by Donohue et al. (2009), who indicated the presence of a
second CP gene and released its partial sequence to GenBank. Searching the I. scapularis data-
base, we found several putative CP sequences, only one of which is full length with a start and
stop codon (IsCP, Fig. 15.6; discussed in more detail later). One more gene that was reported as
Vg from the tick H. longicornis (HlVg-2; Genbank accession number BAG12981) was analyzed
by Donohue et al. (2009) and Khalil et al. (2011), who showed that this gene is much closer to CP
than Vg based on protein architecture and a phylogenetic analysis. A recent phylogenetic analysis
of Vgs and Vg-related proteins across the major animal groups (see Chapter 9) showed that all 3
HlVgs are closer to CP than Vg; the 3 HlVgs were grouped with other confirmed or predicted
CPs from acarines, and not with Vgs from the same group. This is discussed in more detail later.
The isolated full-length CP cDNA sequences are about 5,000 nucleotides long, coding for a
protein of about 1,550 amino acids with a calculated molecular weight of 177 kDa. CPs appear in
the hemolymph as hemelipoglycoproteins of a higher molecular weight (~350 kDa). CP in
FIGURE 15.6: Schematic representation of the confi rmed and putative full-length and partial sequences
of CPs and Vgs isolated from ticks showing similarities and differences between the predicted amino
acid sequences. DUF1943, a domain of unknown function; LPD_N, lipid binding domain; SP, signal
peptide; vWD, von Willebrand type D domain. Arrows represent the RXXR location, and vertical solid
lines represent the GLCG domain location. It is not known whether AvCP and RaCP fragments belong
to the same gene or result from different genes.
D. variabilis consists of 2 subunits of 92 and 98 kDa (Fig 15.5). Comparison of the calculated and
native molecular weights of CP indicates that native CP in the hard tick is tetrameric and each
subunit is represented twice. Maya-Monteiro et al. (2000) indicated that the 2 subunits of HeLp
existed in equal molar ratio based on densitometry measurement of a polyacrylamide gel.
Because multiple CP messages have been found in the same tick, the possibility exists that the
protein could exist in the hemolymph as a mixture of proteins from multiple messages; whether
this occurs or not has not been determined.
Like Vgs, CPs share motifs that are characteristic of proteins in the large lipid transfer
protein superfamily such as LPD_N, vWD, the GL/ICG domain, and cleaving sites (R/KXXR/K)
(Fig 15.6). Storage proteins are glycosylated, and the main sugar is N-linked mannose (Chinzei
and Taylor 1990; Dupejova et al. 2011). Searches for the putative N-linked glycosylation sites
(NXS/T) confirmed the presence of several sites in both Vg and CP. Some reports on insects
indicated that glycosylation is needed for the secretion of Vgs from the insect fat body (reviewed
by Tufail and Takeda [2008]).
6. GENE REGULATION
As discussed before, Gudderra et al. (2001) found that CP protein in D. variabilis was present
throughout the life cycle of the American dog tick, from eggs to adult (Fig 15.1). Trace amounts of CP
were detected in 1- and 9-day-old eggs. The presence of large amounts of Vn in the tick egg but only
traces of CP indicates that the growing oocyte should have the ability to selectively take up Vg and
not CP from the hemolymph. Greater amounts of CP were detected in unfed larvae than in eggs.
Given the low level of CP in 9-day-old eggs and the high levels in unfed larvae, CP must be synthe-
sized de novo sometime during late embryonic and/or larval development. High levels of CP were
detected in the hemolymph of both fed and unfed adult males, and results indicated that feeding has
no effect on the CP level in males. Similar results were reported for HLGP in D. marginatus males;
however, the same was not true for females. After attachment, CP levels increased with the initiation
of blood feeding and then declined 10 days after attachment. CP levels declined even more in replete
females but remained detectable until 17 days after detachment from the host. In order to better
understand the regulation of CP during adult development, Donohue et al. (2008) examined the CP
gene developmental expression using Northern blot analysis. From their studies, it was apparent that
the expression of CP in the whole body samples of adult virgin D. variabilis females was initiated
after attachment to the host and with the initiation of blood feeding. CP mRNA was detected 6 hours
after attachment, and its levels increased gradually until day 6, results that agree with the protein data
obtained by Gudderra et al. (2001). No additional studies were conducted after the sixth day post-
attachment or after engorgement. The factors that regulate CP expression are not known and have
not been investigated. The changes in expression levels at different developmental stages (Fig. 15.1)
might suggest different physiological reasons for expression, as the role of CP might change from one
stage to the next, but the mechanism of regulation could be the same.
In the adult stage, the only factor that is known to stimulate CP expression is attachment to
the host and the initiation of blood feeding. It is not known whether regulatory factors (e.g.,
hormones or neuropeptides) that regulate the expression of CP are released after blood feeding.
Another alternative is nutritional regulation. The nutritional regulation of vitellogenesis was
studied in mosquitoes (reviewed by Attardo et al. [2005]), in which it is known that high levels
of amino acids after a blood meal or infusion can stimulate Vg synthesis by the fat body. A sim-
ilar mechanism was suggested by Horigane et al. (2010) for the soft tick O. moubata and by
Boldbaatar et al. (2010a) for the hard tick H. longicornis. The regulation of CP expression might
occur via a similar mechanism.
The regulation of CP in female adults of D. variabilis appears to be different from that of Vg,
even though they might share a common evolutionary origin and have similar functions in binding
lipids, carbohydrates, and heme. Reports from soft and hard ticks (James et al. 1997; Ogihara et al.
2007) indicated the presence of low or undetectable levels of ecdysteroids in adult female ticks after
attachment to the host for at least a few days. However, blood feeding in D. variabilis initiated the
synthesis of CP. Gudderra et al. (2001) found that mating and blood feeding to repletion reduced
the levels of CP, and it is now well established that mating and repletion or ecdysteriod injection in
virgin females can increase the levels of Vg gene expression and Vg in the hemolymph (see Chap-
ters 16 and 17 for more information on the regulation of Vg). Taken together, such evidence might
indicate that CP is regulated not by ecdysteroids but at least in part by nutrition signals.
Ferritin is another protein that is known to play a role in iron metabolism in all living organ-
isms (Arosio et al. 2009). Harrison and Arosio (1996) described in detail the regulation of fer-
ritin expression at the post-transcription level through the interaction between iron regulatory
proteins and iron responsive elements (IREs) found in the 5′ untranslated region (UTR) of the
ferritin mRNA. In this model, ferritin mRNA already exists in the cell, but in the absence of iron,
the iron regulatory proteins bind to the IREs, preventing mRNA translation. Mulenga et al.
(2004) isolated ferritin cDNA from D. variabilis and identified the putative IREs in the 5′ UTR
of the mRNA, indicating the same regulatory mechanism. Searches for the IREs in the 5′ UTRs
of different CP mRNAs from ticks were unsuccessful. Also, Donohue et al. (2008) were not able
to detect CP mRNA in unfed D. variabilis; CP messages appeared only after blood feeding.
These results suggest that CP is not regulated by IREs.
Vg gene expression and regulation in ticks has been studied in much greater depth than CP.
Several studies so far indicate that mating and feeding are the 2 main factors required in order
for a female tick to be able to reproduce. Also, 20-hydroxyecdysone (20E)—not insect juvenile
hormone—is the main hormone regulating the vitellogenesis process in ticks (for more details,
see Chapters 16 and 17). Khalil et al. (2011) studied the hormonal and developmental regulation
of a second Vg from D. variabilis. Similar to Vg1, Vg2 gene expression was initiated by the injec-
tion of 20E into partially fed virgin females. Both DvVgs are female specific and expressed in
adult females only after mating and feeding to repletion. Tryptic digest-mass fingerprinting
analysis of the putative Vg and Vn bands confirmed the presence of both DvVg1 and DvVg2
proteins in the hemolymph and eggs. The sources of DvVg2 were confirmed as the fat body and
the gut of the vitellogenic females. A study by Thompson et al. (2007) indicated that the ovary
might be a possible source of Vg1 based on Northern blot studies.
7. WHY IS CP IN THE SALIVARY GLAND ?
Some studies, although contradictory, have suggested that CP might occur in tick saliva and the
cement cone and might be involved in host complementation. This view is partially supported
by the fact that one of the major sites for CP and CP message in D. variabilis is the salivary gland
(Gudderra et al. 2002; Donohue et al. 2008). Shapiro et al. (1986) found a 94 kDa antigen in the
cement cone of 3- and 5-day-fed adult R. appendiculatus. In addition, Jaworski et al. (1992)
showed that a 90 kDa antigen present in salivary glands cross-reacted with a 70 kDa cement
antigen in D. variabilis, A. americanum, and R. sanguineus, but not with a 90 kDa antigen.
Jaworski et al. (1992) also could not find a 90 kDa protein in the cement proteins of D. varia-
bilis and A. americanum via SDS-PAGE. Wang and Nuttall (1994) found a 98 kDa protein
present in the salivary glands of unfed and 2-, 4-, 6-, and 8-day-fed female R. appendiculatus,
as well as in the saliva of 6- and 8-day-fed female ticks of the same species. The 98 kDa protein
in saliva appeared to be a dominant component of the saliva in addition to being the dominant
hemolymph protein. Madden et al. (2002) analyzed the saliva of A. americanum and A. macu-
latum and found that the dominant protein in each sample had an N-terminal sequence iden-
tical to that of RmCP (Maya-Monteiro et al. 2000) and nearly identical to that of DvCP
(Guddera et al. 2001).
In consideration of the conflicting literature evidence that CP might be important in host
complementation and of our own work showing that CP message was found in salivary glands,
repeated attempts were made to resolve CP from the saliva (via native-PAGE) and cement cones
(via SDS-PAGE) of adult American dog ticks (Donohue et al. 2008). Because of the high levels
of CP in hemolymph, and considering that injections into the hemolymph of dopamine and
pilocarpine were required in order to initiate salivation, special attention was given to the pre-
vention of any contamination of the saliva by hemolymph. In these studies, no bands were found
with mobility similar to that of hemolymph CP. In order to address whether CP exists in a mod-
ified form in the cement cone, a possibility suggested by the results of a cross-reactive serum in
studies by Jaworski et al. (1992), cement cone proteins were separated via SDS-PAGE, and
proteins with molecular weights of ≥66 kDa and <66 kDa were analyzed via liquid chromatog-
raphy coupled with tandem mass spectrometry (LC-MS/MS). A positive control was const-
ructed consisting of cement cone spiked with hemolymph containing CP. Again, in these studies,
no CP fragments could be found in the cement cone for either molecular weight range tested,
whereas CP was identified in the positive control sample of proteins ≥ 66 kDa (data not shown).
These results suggest that CP is not a component of tick saliva or the cement cone at the detec-
tion limit of silver staining or below the 50–75 ng detection limit of LC-MS/MS (for a ~100 kDa
protein for the latter).
The role of the CP message and CP in the salivary gland is not clear, but it is suggested that
the salivary gland might be a source of the hemolymph CP. Alternatively, the lack of CP in the
saliva might be a result of the artificial method used to collect the saliva. However, no CP was
found in the cement cone, as had been suggested by others. CP, which is the major hemelipo-
glyco-storage protein in ticks and the predominant tick protein throughout development, might
also be a critical component in host complementation. Further studies will be needed in order
to resolve these questions.
Despite our exhaustive attempts to find CP in saliva and in the cement cone of D. variabi-
lis using the methods described, the protein could not be found. However, an argument could
be made that the salivary gland tissue might be a good site for the synthesis of CP and its se-
cretion into the hemolymph, given that the salivary gland is at its largest size and most active
at the time of blood feeding, which is positively correlated with the time at which CP synthe-
sis is greatest. When the salivary gland degenerates after feeding to repletion in D. variabilis,
CP levels in the hemolymph also decrease. More studies are needed in order to determine
what cells in the salivary gland are making CP and whether the gland can secrete the protein
into the hemolymph.
8. HOW TO DISTINGUISH Vg FROM CP
As discussed before, 2 storage proteins are found in tick hemolymph, CP and Vg, which
share a common evolutionary origin (Donohue et al. 2008, 2009). These proteins share sim-
ilar structural motifs such as LPD_N, vWD, the DUF1943 domain of unknown function,
cleavage sites (RXXR), and the GL/ICG domain (Fig. 15.6). CP in hard ticks was found in
both sexes and in all developmental stages and tissues studied. All CPs studied are composed
of 2 subunits: a small subunit of 92 kDa, and a larger subunit of ~100 kDa. Donohue et al.
(2008) showed that the main sources of CP mRNA in D. variabilis are the fat body and the
salivary gland. They also showed that host attachment and blood feeding initiated CP ex-
pression in virgin females, whereas mating and feeding to repletion reduced the level of CP.
The regulation of full-length Vg genes was studied mainly in the hard tick D. variabilis and
the soft tick O. moubata. Studies on D. variabilis showed that DvVg1 and DvVg2 are exclu-
sively expressed in females after mating and feeding to repletion and are up-regulated by
ecdysteroids. Neither Vg is expressed in males (fed and unfed) or females before mating and
feeding to repletion. The main sources of DvVg1 and DvVg2 are the fat body and the gut
cells. In the soft tick O. moubata, Horigane et al. (2010) showed that the sources of OmVg are
the fat body and the gut, and that it is regulated by ecdysteroids, like in D. variabilis. They
noticed a major difference between D. variabilis and O. moubata: in the latter, Vg expression
was initiated by engorgement in both virgin and mated females, but it increased further in
mated females.
We examined the predicted amino acid sequences of the confirmed and putative full-length
and partial CP and Vg genes available from ticks (Fig. 15.6). All CPs are similar in amino acid
length and have the characteristic domains (LPD_N, DUF1943, vWD, RXXR, and GLCG) at
almost the same locations. The N-terminus sequence for the small subunit is FEVGKEYVY,
which is 100% identical to that determined by Maya-Monteiro et al. (2000) for HeLp. This
sequence is directly downstream from the secretion signal and marks the start of the LPD_N.
The N-terminus of the larger subunit is DASAKERKEIED, which is almost 100% identical to
HeLp and exists directly downstream from the only cleavage site in CP.
The amino acid sequence from the available tick Vg genes shows 3 domains (LPD_N,
DUF1943, and vWD) at locations that are similar but variable among Vgs and unlike that of the
functional domains for CP. Also, the RXXR cleavage site may be totally absent (e.g., for the I.
scapularis Vg) or variable in number and location, as in other available tick Vgs. In ticks, Vgs
usually consist of several subunits with variable N-terminus sequences, whereas CPs consist of
only 2 subunits produced by only 1 RXXR cleavage site. We also found that all Vgs have an
amino acid spacer (10 to 20 amino acids) between the secretion signal and the LPD_N (Fig. 15.6)
that does not exist in CPs.
The 3 Vgs isolated from the hard tick H. longicornis are controversial, as they show greater
similarity to CPs than other Vgs that have been studied. HlVg2 has a structure that is similar to
CP, with only 1 RXXR cleavage site and 2 subunits with an N-terminus amino acid sequence that
is 100% similar to CP. HlVg1 and HlVg3 have more than 1 RXXR cleavage site distributed ran-
domly over the protein molecule but still retain 1 RXXR location upstream, a sequence that is
highly similar to the N-terminus amino acid sequence of the other CP large subunit. Also, they
have a secretion signal that is directly upstream of a sequence similar to the small CP subunit.
The N-terminus of the mature HlVg1 and HlVg3 proteins is directly downstream of the secretion
signal with no spacer amino acids in between, a structure that is more similar to CP than Vg.
However, all HlVgs were detected only in females and not in males (unfed and fed), a character
that is exclusive to Vg in other tick studies.
Because of the similarities between CP and Vg in ticks, the assignment of function can be
problematic, especially when based only on BLAST results and not considering alignment
and expression levels during development and between sexes. As a result, we propose that the
following criteria be used for the assignment of function for these proteins in ticks: (i) CP
typically is present in both sexes throughout development, from the egg through the adult
stage, whereas Vg appears only in adult females at the time of egg development. (ii) Vg
protein in hemolymph and in the egg comprises more than 2 subunits, whereas CP consists
of only 2 subunits. (iii) As CP is found at low levels in the egg and Vn can usually be resolved
from CP via native gel electrophoresis, a putative Vg message should be validated via MS
fingerprinting of purified Vn (the same can be done for CP purified from hemolymph). (iv)
The CPs characterized so far consist of 2 subunits with highly conserved unique N-terminal
amino acid sequences predicted by a single cleavage site. Vgs contain 1 to several cleavage
sites and multiple subunits with N-terminal amino acid sequences that are not conserved
and which differ from those in CP. In general, it is recommended that the study of Vg always
be coupled with the characterization and study of CP; otherwise, experiments can be difficult
to interpret.
Although significant advances in understanding the structure of tick storage proteins have been
made, this information raises many new questions. Some of the obvious ones include (i) deter-
mining the role of CP in tick development during embryogenesis, larval and nymphal develop-
ment, and adult male and female reproduction; (ii) describing the mechanism for the regulation
of CP expression; (iii) determining whether ticks can synthesize heme and the significance of
CP and Vg as a heme source versus as a protective agent from heme toxicity; (iv) understanding
the mechanism of heme binding to CP and Vg; (v) determining whether CP binds heme in soft
ticks; (vi) determining the function of CP in the coxal fluid of soft ticks and in the salivary gland
of hard ticks; and (vii) the potential other roles of storage proteins relative to lipid transport, egg
coloration, immunity, host blood coagulation, or host complementation. It is also interesting
that although CP and Vg have similar functional domains, the Vg receptor effectively excludes
CP from eggs, even though both CP and Vg are found in hemolymph in high concentrations.
An understanding of Vg uptake will be important in understanding the physiology of egg devel-
opment, because this receptor might serve as a portal for the movement of biotic and abiotic
factors to the next generation.
ACKNOWLEDGMENTS
The laboratories of R.M.R. and D.E.S. are supported by grants from the National Science Foun-
dation (IBN-0315179 and IBN-0723692) and National Institutes of Health (1R21AI096268-01)
and research support to R.M.R. from the North Carolina Agricultural Research Service.
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C H A P T E R 1 6
HORMONAL REGULATION OF
METAMORPHOSIS AND
REPRODUCTION IN TICKS
R. M I C HAEL ROE , KEVI N V. DONOH UE , S AYED M. S. KHALIL ,

BROOKE W. BIS S ING ER , JIWEI ZH U , AND DANIEL E. SONENS HINE
1. INTRODUCTION
Much of what we know about the molecular endocrinology of the Arthropoda has been based
on studies of insects. Work on the Crustacea has contributed to a lesser, but still significant, ex-
tent, with the Mandibulata being by far the main research focus. The emphasis on the Insecta has
been based on a number of practical considerations, including their importance as a pest in ag-
riculture, their significance in the transmission of animal and human diseases, the ease in
working with insects in the laboratory as a result of their relatively large size and the separation
of the body into clearly delineated segments (i.e., head, thorax, and abdomen), and the avail-
ability of research funding. Insects are also interesting because they represent the apex of arthro-
pod phylogeny and are a highly diverse and highly successful group of animals that have
developed many different life strategies and which have established themselves in high numbers
in many different terrestrial ecosystems. Because of the advances that have been made in under-
standing the endocrinology of insect larval development, metamorphosis, and reproduction and
the apparent similarities in these mechanisms between insects and the Crustacea, the most
common working hypothesis has been that the regulatory mechanisms of arthropods in general,
including mites, ticks, and other Chelicerata, must be the same as those of the Insecta. This con-
clusion, however, was based on similarities in the morphology of the Arthropoda, namely, that
they feed, grow, and shed their cuticle in developmental stages and instars; they undergo meta-
morphosis from a juvenile to an adult stage; and adults typically mate and lay eggs. Genomic
data are increasing supporting this hypothesis to a degree, but as we discuss in this chapter, there
are also important and mostly unknown differences between insects and ticks. This chapter ad-
dresses what we know about the molecular endocrinology of tick development and reproduction
Hormonal Regulation 417
in the brief context of the well-studied Mandibulata. With the recent release of the first tick ge-
nome from Ixodes scapularis and the development of new, high-throughput sequencing technol-
ogies such as 454 and Illumina, our understanding of tick endocrinology is expanding rapidly;
as we demonstrate, however, we are uncovering more questions with these technologies faster
than we are providing answers.
Ticks are ancient acarines comprising 2 major families, the Ixodidae (hard ticks) and the Ar-
gasidae (soft ticks), which appear to have evolved during the Cretaceous period. The class Arach-
nida, which includes mites and ticks, diverged from the Insecta in the Paleozoic Era. Considering the
substantial evolutionary time after the divergence of insects, substantial differences might be ex-
pected between these 2 groups. Ticks also are obligate blood-feeding ectoparasites. In each case
where the evolution of hematophagy has been studied in the Animalia, it appears that the adaptation
to blood feeding has been a unique event peculiar to the group in which it developed. The evolution
of hematophagy in ticks might have occurred prior to the divergence of their chelicerate and man-
dibulate ancestors, which would suggest that ticks might have a number of atypical adaptations dis-
tinct from those of the better-known insects, maybe even for fundamental regulatory processes, and
of the more closely related mites. In contrast to blood-feeding insects, ticks also undergo long pe-
riods of starvation between blood meals, often lasting months or even years (discussed elsewhere in
this book). The ways in which ticks deal with these challenges appear to be unique (i.e., they are not
shared by blood-feeding insects). This chapter highlights these unique tick mechanisms and shows
that ticks are not merely “another kind of insect” in terms of how they regulate their development
and reproduction. We also believe that studies of the physiology of ticks can uncover mechanisms
not previously understood in the Mandibulata. The recent availability of the spider mite genome
(Grbić et al. 2011), which was published during the preparation of this chapter, will also facilitate the
understanding of ticks, but the study of this new genome in respect to ticks is obviously in its infancy.
Significant strides in the advancement of our knowledge of tick endocrinology have been made
recently and are represented by a relatively small number of studies. Although some aspects of devel-
opment, such as reproduction, appear to be understood with some certainty, although admittedly on
the basis of only a few select model systems, others such as the regulation of development and meta-
morphosis remain a black box, and we have much more to learn. Included in this discussion is
consideration of whether ticks have juvenile hormone and what we know about the regulation of tick
metamorphosis. Also, recent advances regarding the role of male protein pheromones, ecdysteroids,
and neuropeptides in the regulation of blood feeding and reproduction are discussed, including re-
cent research on the molecular biology of the tick vitellogenin receptor and storage proteins in gen-
eral, the latter of which appear to be critical to hematophagy and reproduction. Related chapters in
this book should also be examined (Chapter 4, Section 3.8.2; Chapters 9, 15, 17, and 18).
2. UNDERSTANDING OF MANDIBULATE ENDOCRINE

REGULATION OF DEVELOPMENT VERSUS THAT
IN TICKS
2.1. INSECTS
Our most complete understanding of the arthropod endocrine system and the role of hormones in
the regulation of growth, molting, metamorphosis, and reproduction is that for insects. Two major
hormones regulate these developmental processes: (i) the ecdysteroids, which are synthesized
from dietary steroids, and (ii) the juvenile hormones (JHs), which are represented by a number of
different types (JH0, JH I, JH II, JH III, JH bisepoxide, and others) and are synthesized de novo
(Kerkut and Gilbert 1985; Roe and Venkatesh 1990; Gilbert et al. 2000; Klowden 2007; Gilbert
2012a, 2012b).
Ecdysteroid titers are regulated by the peptidic hormone prothoracicotropic hormone
(PTTH). PTTH is synthesized by neurosecretory cells in the insect brain and released from the
corpora allata (CA) into circulating hemolymph. The CA is a neurohemal organ and part of the
stomatogastric nervous system; it is located posterior to the brain in the insect head. The appear-
ance of circulating PTTH in the hemolymph promotes the release of the penta-hydroxylated
prohormone, or ecdysone, from the insect prothoracic gland. Ecdysone is then further oxidized
to 20-hydroxyecdysone (20-HE) by peripheral tissues. 20-HE initiates molting and regulates a
variety of other insect functions.
JH is synthesized and released from the CA. The synthesis of JH is regulated by peptidic
hormones, the allatotropins and allatostatins, synthesized in the insect brain. Insect systems are
unique in that the concentration of JH in the hemolymph is regulated by dynamic changes in both
the rate of JH synthesis and release from the CA and hemolymph degradation by a specific JH es-
terase (Hammock 1985; Roe and Venkatesh 1990; Gilbert et al. 2000). The degradation process is
controlled by JH sequestration to highly specific hemolymph JH-binding protein in the Lepidop-
tera and other insects (Hammock 1985; Roe and Venkatesh 1990; Touhara and Prestwich 1993;
Touhara et al. 1993, 1995; Gilbert et al. 2000), which protects the hormone from degradation by non-
specific esterases, epoxide hydrolases, monooxygenases, and glutathione transferases. The balance
between the rates of synthesis and degradation of JH allows for the appearance and rapid disappear-
ance of the hormone at precise times relative to food availability, photoperiod, and changing sea-
sonal conditions. The precise regulation of development is critical to insect survival and reproduction,
but at the same time it provides maximum physiological and developmental plasticity.
Our best understanding of the role of hormones in the regulation of arthropod development
is without question in the Insecta, and especially within the Lepidoptera (the moths). This was
made possible because of research contributions by many different laboratories and at least a
generation and a half of scientific effort that continues today. JH is known as the “status quo”
hormone, because its presence during larval development prevents metamorphosis to the adult
stage. The regulation of larval-larval molting and the timing of metamorphosis is regulated by a
balance between the levels of JH and the concentration of ecdysteroids. Ecdysteroids in the
presence of JH trigger molting from one larval instar to the next (the maintenance of the “status
quo”), whereas ecdysteroids in the absence of JH during the last larval stadium trigger molting
to the adult stage in the case of hemimetabolous insects or molting to the pupa for the holome-
tabola. High levels of JH esterase activity during the first half of the last stadium and after the
cessation of JH synthesis remove all traces of JH from the insect; then the presence of ecdyster-
oids in the absence of JH initiates cellular reprogramming, which leads to metamorphosis.
In most insects that have been studied, JH also appears to be the female gonadotropic
hormone (Kerkut and Gilbert 1985; Klowden 2007; Gilbert 2012a, 2012b). The synthesis and re-
lease of JH in the female adult initiates the synthesis of vitellogenin, a glycolipostorage protein
that is secreted into the insect hemolymph from the fat body and absorbed as vitellin in devel-
oping oocytes. One exception to this model for the regulation of insect egg development is the
mosquito, in which vitellogenesis is regulated by ecdysteroids (Shapiro et al. 1986). After adult
eclosion, JH from the CA promotes host-seeking behavior in female mosquitoes, promotes
ovary development to the resting stage, and allows the fat body to be responsive to ecdysteroids.
Once a blood meal is taken, ovarian ecdysteroidogenic hormone (Brown et al. 1998) is released
from the brain, which stimulates the ovary to synthesize ecdysteroids. Ecdysteroid in turn
initiates the production of vitellogenin by the fat body.
2.2. CRUSTACEA
The Crustacea in many ways are like insects with respect to the regulation of their development
and reproduction, but with a few exceptions. For example, it appears that the Crustacea are
unable to synthesize the C10,11 epoxide found in JH (Charmantier et al. 1991; Chang 1993; Chang
et al. 1993; Laufer et al. 1993; Homola and Chang 1997; Nagaraju 2007). In this case, methyl
farnesoate (MF) has the approximate function of JH in the Crustacea. MF promotes the produc-
tion of ecdysone, reduces the duration of the intermolt period, and might be involved in the
regulation of metamorphosis in the Crustacea, although the exact understanding of this process
is not as advanced as is the case for insects. MF, like in insects, regulates the process of vitello-
genin synthesis and yolk deposition.
2.3. TICKS
Our knowledge of the acarine endocrine system is in its infancy relative to that for the Man-
dibulata, and we know even less about mites than we do about ticks. However, recent advances
in the genomics of both ticks and mites should change this paradigm in the near future; some of
these advances are discussed later in this chapter. One of the historical problems with tick endo-
crinology research—and this is also true for mites—is that research often has been misdirected
by the assumption that ticks regulate their larval, nymphal, and adult development with the
same hormones and the same mechanisms as insects. This hypothesis is a reasonable starting
point for research but should be only that; it is more reasonable to assume that they share a
common heritage with insects, but with significant differences as well, especially considering the
evolution to obligatory hematophagy in ticks.
Until recently, our knowledge of the neuroendocrine system of ticks was based mostly on
histological investigations (reviewed by Sonenshine 1991; also see Chapters 4, [Section 3.8.2] and
13). The prominent aspect of the central nervous system of the tick is the synganglion, homolo-
gous to the brain and ventral nerve cord of insects. The synganglion is located in the center of
the body in the tick hemocoel. Pedal nerves from the synganglion innervate the walking legs.
The retrocerebral organ complex (RCO) and associated synganglion neurosecretory cells of the
tick are most likely the counterpart to the corpora cardiaca (CC)–CA complex of the insect
stomatogastric nervous system. However, the function of the RCO in the regulation of molting,
metamorphosis, and reproduction is not clear. It has also been hypothesized that the syngan-
glion perineurium can serve as a neurohemal organ, but currently this is uncertain (see Chapter
13). In addition, lateral segmental organs located in the lateral nerve plexus between the pedal
nerves have been assumed to be the site of JH biosynthesis, primarily based on the abundance
of smooth endoplasmic reticulum in these glands and the histological similarity of these organs
to the insect CA. The recent discovery of a possible farnesyl or MF biosynthesis pathway in the
synganglion—the same pathway that makes JH—should provide an opportunity in the future to
determine the location of the enzymes in this pathway. The evidence is clear that ticks can syn-
thesize 20-HE in the epidermis (Dees et al. 1984; Sonenshine et al. 1985; Zhu et al. 1991; Lomas
et al. 1997), and this hormone, like in insects, can regulate molting. It also appears that the tick
synganglion, like the insect brain, produces a peptidic hormone that regulates ecdysteroid bio-
synthesis in the epidermis, although the most definitive proof of this is limited to a single sci-
entific paper and tick species, and the protein sequence has not been determined (Lomas et al.
1997). Recently, a number of neuropeptides and their receptors were identified from ticks using
bioinformatics (Christie 2008), immunohistochemistry (Šimo et al. 2009), matrix-assisted
laser desorption ionization (MALDI)–time of flight (TOF)/TOF mass spectrometry (Neupert
et al. 2009), expressed sequence tags (Lees et al. 2010), and 454 pyrosequencing (Donohue et al.
2010; Bissinger et al. 2011). The role of these neurohormones in reproduction is discussed later
in this chapter. Chapter 13 of this book examines the distribution of neurohormones in the tick
synganglion.
3. DO TICKS HAVE JH?
The general assumption for many years was that ticks have JH and regulate their development,
metamorphosis, and reproduction with JH, like in insects (reviewed by Obenchain and Galun
[1982] and Sonenshine [1991]). Venkatesh et al. (1990) and Roe et al. (1993) measured low levels
of JH esterase and JH epoxide hydrolase activity during D. variabilis nymphal and adult devel-
opment, and Kulcsár et al. (1989), Sonenshine et al. (1989), and Lomas et al. (1996) described
proteins in D. variabilis and Amblyomma hebraeum that bound JH. However, these studies were
equivocal in answering the question of whether ticks have JH. Enzymes that are capable of me-
tabolizing the epoxide or methyl ester of JH or of binding JH but which are not necessarily JH
specific can be found in most, if not all, biological systems. What was needed in order to prove
that ticks have JH was the direct identification of the hormone.
Connat (1987) discovered JH-positive immunoreactive material in ticks but was unable to
definitively identify JH using high-performance liquid chromatography (HPLC)-coupled radio-
immunoassays in hemolymph from Rhipicephalus (Boophilus) microplus. Neese et al. (2000)
conducted the most definitive study to date on whether ticks make the insect JHs in hard and
soft ticks (D. variabilis and Ornithodorus parkeri, respectively) using 3 different direct detection
methods.
3.1. JH BIOSYNTHESIS IN TICKS

Pratt and Tobe (1974) developed a highly sensitive assay to detect JH synthesis in insects. The
assay monitors the addition of a radiolabeled methyl group from methionine to farnesoic acid
(a precursor of JH), which produces a tritium-labeled methyl ester on MF. MF is further me-
tabolized by a P450 (Cyp15 in insects so far studied) to produce JH. This assay is typically con-
ducted by incubating the CA in tissue culture media with radiolabeled methionine, the media
extracted with an organic solvent, and the radiolabeled products resolved via chromatography.
Using high-specific-activity tritium-labeled methionine, biosynthesis can be detected at ex-
tremely low levels. Neese et al. (2000) were the first to apply this approach to determine whether
ticks could synthesize JH. When synganglia, salivary gland, midgut, ovary, fat body, and muscle
from D. variabilis and O. parkeri are incubated, alone and in different combinations, in vitro in
separate experiments with high-specific-activity L-[methyl-3H]methionine and farnesoic acid
or with [1-14C]acetate (another JH precursor) and the hexane-soluble products are analyzed via
radioHPLC, any biosynthesis of JH or its synthesis from farnesoic acid or acetate should be
detectable by the appearance of radiolabled farnesol, MF, or JH. Multiple life stages were exam-
ined in these studies, including, for D. variabilis, 3- and 72-hour-old (after ecdysis) unfed
nymphs, partially fed nymphs (18 and 72 hours after attachment to the host), fully engorged
nymphs (2 days after detachment from the host), 3- and 72-hour-old (after eclosion) unfed fe-
males, partially fed unmated females (12 to 168 hours after attachment to the host), and mated
replete females (2 days after detachment from the host). The life stages from O. parkeri were
third and fourth stadium nymphs and females, 1 to 2 days after detachment. Positive controls
were also conducted using the CA from Diploptera punctata, Periplaneta americana, or
Gromphadorina portentosa. In order to prevent any possible degradation of the tritiated methyl
ester of JH by either non-specific or JH-specific esterases, the assays were conducted in the
presence of the highly potent JH esterase inhibitor octylthio-1,1,1-trifluoropropan-2-one. In
these studies by Neese et al. (2000), no synthesis of farnesol, MF, JH I, JH II, JH III, or JH III
bisepoxide was detected in any tissues or at any developmental life stage examined in either tick
species studied, whereas JH III, MF, and farnesol were detected in the positive controls. In
many of the tick incubations, farnesoic acid was also provided, so ticks had to synthesize only
the methyl ester and/or the C10,11 epoxide, and yet no MF or JH was found. The synganglion
preparations in all of the studies included the lateral segmental organs that were hypothesized
to be the site of JH biosynthesis in ticks (reviewed by Sonenshine [1991]). The lower limit of
detection for [methyl-3H]-JH and MF in these assays was 1.27 fmol per 10 tick equivalents of
tissue after a 3-hour incubation.
3.2. CAN TICK EXTRACTS JUVENILIZE INSECTS?

DeLoof and Van Loon (1980) developed a sensitive insect bioassay for JH known as the Gal-
leria pupal cuticle bioassay. In this assay, an organic insect extract is mixed into oil and molten
wax and then applied to a small area of the pupal epidermis of the wax moth after removal of
the cuticle. The wax is allowed to harden, and the insect is allowed to develop to the pharate
adult stage. As discussed before, JH in insects is a “status quo” hormone. Therefore, the pres-
ence of JH on the epidermis of the pupa should result in the retention of darkened, wrinkled
pupal cuticle in the pharate adult. The interesting aspect of this assay is that any hormone that
might produce a juvenilizing effect on insect cuticle, irrespective of the structure, would be
detected via this method. Using the Galleria bioassay, no compounds with juvenilizing activity
were detected in 10- and 15-day-old eggs, unfed larvae, unfed nymphs, or partially fed adult
females of the American dog tick, D. variabilis (Neese et al. 2000). The lower limits of detec-
tion were 28 pg for the detection of JH I, 28 pg for JH II, and 980 pg for JH III per gram of tick
tissue. Neese et al. (2000) also found that synganglion from blood-fed second instar nymphs
did not inhibit tick metamorphosis when transplanted into fed last instar nymphs of O. parkeri,
further suggesting that regardless of the structure, ticks do not have a “status quo” hormone.
The ticks molted normally to the adult stage, and the adult females bore a genital pore not
found in nymphs.
3.3. ELECTRON IMPACT (SELECTIVE ION) CAPILLARY GAS

CHROMATOGRAPHY–COUPLED MASS SPECTROMETRY
SEARCH FOR JH IN TICKS
One of the most definitive methods used in insects to indentify JH has been electron impact
selective ion (EI-SI) capillary gas chromatography (GC) coupled with mass spectrometry (MS).
In this method, JH is usually partially purified from insect hemolymph and chemically deriva-
tized to produce an ethyl-d3-methoxyhydrin. Using the separation power of capillary GC cou-
pled with SI monitoring, the insect JHs can be identified with a high degree of certainty and with
a lower detection limit that permits the detection of JH at levels normally found in insect hemo-
lymph or other tissues. This same approach was used to determine whether the common insect
JHs were present in the hemolymph of partially fed, unmated (forcibly detached 7 days after
attachment) D. variabilis; mated, replete (allowed to drop naturally) D. variabilis females; and
mated, replete (1 to 2 days after detachment) O. parkeri females. These life stages were chosen
because ticks are developing eggs at this time, and the hypothesis was that egg development, like
in insects, was initiated by JH. Nymphal ticks were too small for enough hemolymph to be col-
lected from them for this analysis. The internal standard, JH III-ethyl-d3-methoxyhydrin (m/z
76 and 239), was detected in the tick assays at a retention time of 10.32 min. In the analyses of
tick hemolymph, no JH I, JH II, JH III, JH III bisepoxide, farnesol, or MF was detected. None of
the tick samples contained any ions diagnostic of the common insect JHs, farnesol, or MF when
the MS sensitivity was 1.6 pg in the scan mode from 40 to 300 AMU and 750 fg in the SIM mode
for fragments with m/z ratios of 76 and 225 (Neese et al. 2000).
3.4. CONCLUSION: DO TICKS SYNTHESIZE JH?

It is difficult to prove the absence of a compound, and the number of studies conducted in order
to look for insect JHs in ticks is minimal, with only a single comprehensive effort using direct
detection. However, Neese et al. (2000) were unable to find any of the common insect JHs or MF
in either hard or soft ticks using radiobiosynthesis and EI SI GC-MS. In addition, the Galleria
bioassay showed that regardless of structure, there were no compounds in the American dog
tick that would juvenilize insect epidermis. Given that JH so far has been identified only in in-
sects (and not in any other arthropods) and MF is found in the Crustacea, the existence of the
insect JHs in ticks is doubtful. However, more research is needed to address this question in ticks
and mites, as the conclusion is based mostly on a single study of the direct detection of JH/MF
and radiobiosynthesis of these hormones. Also, earlier steps in the pathway for the synthesis of
JH might be present that would not have been detected in the studies of Neese et al. (2000); this
is discussed in more detail later in this chapter.
4. REGULATION OF TICK METAMORPHOSIS
As discussed before, the regulation of larval-larval molting in insects and the initiation of meta-
morphosis are well understood and involve simply a balance between 2 hormones. Ecdysteroids
in the presence of JH initiate molting from one larval instar to the next, whereas the presence of
ecdysteroids in the absence of JH initiates metamorphosis. The most convincing evidence in in-
sects of the larval “status quo” function of JH involves the topical application of JH or JH ana-
logues to demonstrate delayed metamorphosis and/or supernumerary molting and the rescue
with JH or JH analogues of anti-JH effects on molting and metamorphosis. These same experi-
ments have been conducted by a number of investigators (McDaniel and Oliver 1978; Leahy and
Booth 1980; Khalil et al. 1984; Abdelmonem et al. 1986) in 3 species of ticks. The results have
mostly been negative. The only study to show any effect of JH treatments was that by Khalil et al.
(1984) in which JH I delayed nymphal molting by 9% to 13% but did not produce supernumerary
molts when applied on the day of engorgement in the camel tick, Hyalomma dromedarii. These
results are far from compelling in arguing for a role for a “status quo” hormone or JH in the
regulation of tick metamorphosis. Leahy and Booth (1980) could not rescue precocene-induced
nymphal molt mortality with JH in the tick Argas persicus. As discussed before, Neese et al.
(2000) also could not juvenilize last instar nymphs of O. parkeri when they were the recipient of
transplanted synganglia including putative endocrine glands from next to last instar nymphs.
With no direct evidence of JH in ticks in general and no clear indication of a “status quo” effect
from the topical application of JH, the possible role of hormones in the regulation of larval/
nymphal development and metamorphosis is unknown. It also is difficult to develop a plausible
hypothesis for how ticks might regulate their larval and nymphal development without falling
back on the insect model as a beginning hypothesis. Other ideas might include fixed genetic
programming triggered simply by the presence of ecdysteroids and without the plasticity and
developmental polymorphisms demonstrated in larval insects. Development could also be reg-
ulated by different levels of ecdysteroids or “status quo” hormones in ticks that have not yet been
identified and which might or might not be produced by the same synthetic pathway as for the
insect JHs. Because of the small size of larval and nymphal ticks and the central location of the
synganglion, delineating the developmental mechanism for growth and metamorphosis using
ligation and transplantation, parabiosis, and homogenate injection experiments at best can be
described as challenging. However, with recent advances in tick genomics and techniques in
global gene expression, in addition to the discovery of many neuropeptides and possible early
steps in the JH pathway in adult ticks (discussed later in this chapter), the tools to begin uncov-
ering the regulatory elements of tick larval and nymphal development and metamorphosis are
on the horizon.
5. REGULATION OF TICK F EMALE REPRODUCTION
5.1. HISTORICAL PERSPECTIVE

Prior to the work of Neese et al. (2000) discussed earlier, the strongest evidence suggesting that
ticks might have JH was the studies of Pound and Oliver (1979). In their research, the application
of precocene, an anti-JH compound, inhibited egg production in adult females of the soft tick
O. parkeri. When the ticks were subsequently treated with 1 μg of JH III, the effects of the preco-
cene were reversed and egg development was re-started. These results suggested that JH regu-
lated egg production in ticks. However, the experiments were not entirely clear. For example,
the application of JH in amounts greater than 1 μg in a dose-dependent manner in these same
rescue experiments decreased egg production. JH is not an especially toxic compound, and
increasing levels of JH are usually positively correlated with increased levels of yolk deposition
in insects, not a decrease. Also, when similar experiments were conducted in 3 other tick spe-
cies, Argas persicus (Leahy and Booth 1980), R. microplus (Connat 1988), and another species of
Ornithodoros, O. moubata (Connat and Nepa 1990; Taylor et al. 1992), JH was unsuccessful in
rescuing anti-JH effects using both precocene and another anti-JH, fluoromevalonate.
There was also other evidence that JH was not involved in female reproduction in ticks. Taylor
et al. (1991) and Chinzei et al. (1991) found that the topical application in acetone and the injection
in mineral oil of JH homologues, JH acid, and JH analogues did not initiate vitellogenesis more
strongly than the carrier, whereas the pyrethroid cypermethrin induced yolk synthesis. It appeared
that the synthetic pyrethroid insecticide in some way was able to initiate female reproduction,
presumably via action on the nervous system, and not because it mimics a tick gonadotrophic
hormone. Khalil et al. (1984) also were unable to initiate reproduction with JH I, but Connat et al.
(1983) found that JH and JH analogues increased egg laying. Dees et al (1982), Khalil et al. (1984),
and Abdelmonem et al. (1986) found that JH, JH analogues, and precocenes had no effect on
sexual attraction. Solomon and Evans (1977), Mansingh and Rawlins (1977), and Teel et al. (1996)
found that JH analogues inhibited vitellogenesis or oviposition. These contradictory results, com-
bined with the lack of definitive reports of the direct detection of JH in ticks or evidence of a “status
quo” function for JH, suggest that egg development must be controlled by another hormone or
mechanism.
5.2. PROOF OF THE ROLE OF ECDYSTEROIDS IN THE REGULATION

OF EGG DEVELOPMENT IN THE AMERICAN DOG TICK
At about the same time that Neese et al. (2000) were reporting that JH could not be found in ticks,
Sankhon et al. (1999) found that ecdysteroids added to fat body organ culture from the American
dog tick were able to increase the levels of the yolk protein vitellogenin (Vg). Although ecdysteroid
regulation of vitellogenesis is not common in insects, there is one well-studied example in mosqui-
toes where ecdysteroids have been shown to initiate yolk protein synthesis and egg maturation
(Shapiro et al. 1986). Friesen and Kaufman (2002) later found that ecdysteroids injected into non-
vitellogenic fed female adults of Amblyomma hebraeum that had been forcibly detached from the
host caused the release of yolk protein into the hemolymph, and further evidence of ecdysteroid
regulation in other ticks is discussed in Chapter 17 of this book. Recent advances have been made
in understanding the molecular biology of tick storage proteins in D. variabilis, including the fe-
male specific yolk protein Vg (Thompson et al. 2005, 2007; Khalil et al. 2011), which has led to the
most convincing argument yet that ticks regulate egg development with ecdysteroids and not JH.
Thompson et al. (2005, 2007) were able to sequence Vg from the American dog tick and developed
methods to monitor the expression of the Vg gene. In addition, Thompson et al. (2005) developed
a novel bioassay system whereby they could inject partially fed ticks still attached to a rabbit host
and examine the effect of hormone injections on blood feeding, Vg gene expression, Vg protein
synthesis and secretion into the hemolymph, and the development of oocytes in the ovary. In these
studies, they found that the injection of 20-HE at physiological levels of the hormone into partially
fed (virgin) non-vitellogenic females of D. variabilis attached to a rabbit host initiated the expres-
sion of the Vg gene 1 to 3 days after treatment. This was noted as an increase in Vg message in
whole tick preparations (Fig. 16.1), increased levels of Vg protein in the hemolymph as determined
by electrophoresis (data not shown), and Vg uptake by the ovary producing fully developed eggs
(Fig. 16.2). The injection of 20-HE into part-fed virgin females also produced ovaries similar in
weight to that of a mated, replete female. Microscopic examination of the vitellogenic ovaries indi-
cated that almost all of the eggs could be classified as category 5 on the Balashov scale (large and
brown). Other eggs were category 3 or 4 and filled with yolk granules. In other experiments, they
found the Vg message appeared only after mating and feeding to repletion and during oviposition
FIGURE 16.1: Northern analysis for vitellogenin (Vg) from the whole body of the American dog tick,
Dermacentor variabilis, on different days after injection with 20-hydroxyecdysone. The major band in the
Pre-Ov lane is the Vg message. Note that the Vg message is not found in partially fed female adults or
in the solvent-injected controls, but it was present each day after the injection of ecdysteroid. MWM,
molecular weight markers (units in number of nucleotides); Part-fed, RNA from partially fed virgin
females; Pre-Ov, RNA from pre-ovipositional, mated, replete (vitellogenic) females; 20E, RNA from
partially fed (virgin) females injected with 20-hydroxyecdysone.
FIGURE 16.2: Ovaries from part-fed virgin females of the American dog tick, Dermacentor variabilis,
after the ticks were injected with solvent (A) or 20-hydroxyecdysone (B) 4 days after attachment to the
host and then examined 4 days after injection. Note that fully developed, brown (vitellogenic) eggs
(45× magnifi cation) are present in the ticks injected with ecdysteroid (B), whereas those injected with
solvent only (A, 37.5× magnifi cation) showed no development.
and was positively correlated with increased hemolymph Vg protein levels, hemolymph ecdyster-
oid levels, and egg maturation (Dees et al. 1984); this has since been shown for other ticks (Connat
et al. 1985; Kaufman 1991; see Chapter 17 as well). The injection of partially fed (virgin) females of
the American dog tick with 1,000 ng of JH III per tick in these studies had no effect on oocyte
development. This was the first, most complete work showing that in the American dog tick, ec-
dysteroids, and not JH, are directly responsible for the initiation of Vg gene expression and the
appearance of the Vg message, Vg protein synthesis and secretion into the hemolymph, and the
uptake of Vg in the ovary to produce fully developed eggs and ovaries. Khalil et al. (2011)
sequenced a second Vg from the American dog tick and showed that this Vg was up-regulated by
ecdysteroids using the same bioassay approach as described by Thompson et al. (2005). These
studies in toto showed that ecdysteroids alone could produce fully developed eggs in partially fed
virgin females of D. variabilis still attached to the host; however, 20-HE would not initiate blood
feeding to repletion, suggesting that other factors might be controlling the progression to repletion
(discussed in more detail later).
Gudderra et al. (2001, 2002a, 2002b) describe the protein chemistry of another storage protein,
the carrier protein (CP), in the American dog tick and in the soft tick Ornithodoros parkeri. This
protein, like Vg, is a heme-glycolipoprotein found in hemolymph and other tissues and was first
sequenced by Donohue et al. (2008, 2009a). CP, along with Vg, is discussed in additional detail in
Chapters 9, 15, and 17 of this book. Khalil et al. (2011) discuss the difficulty of differentiating be-
tween Vg and CP (each of which is produced by multiple genes). The study of the regulation of
both proteins (Vg and CP) is critical to understanding the regulation of reproduction because both
are important to adult feeding and development, but they are not always easy to distinguish by
means of immunological or protein electrophoretic techniques. The potential misidentification of
these proteins (or the study of one but not the other) in the current published literature complicates
the understanding of the regulation of reproduction in ticks. In addition, the different reproductive
strategies of ticks, including parthenogenesis, make it difficult to completely extrapolate the study
of one tick model to the next (Chapter 17 of this book does a nice job of addressing these issues).
6. WORKING MODEL FOR THE REGULATION OF

FEMALE REPRODUCTION IN THE HARD TICK
D. VARIABILIS
The regulation of female reproduction in the American dog tick, and probably in most tick spe-
cies, concerns the regulation of 2 classes of hemolymph storage proteins: (i) the heme-binding
lipo-CPs (also known as HeLp), which are coded by multiple genes, have their own unique post-
translational processing, and are found in both males and females throughout larval, nymphal,
and adult development; and (ii) Vg, another heme-binding protein. Vg is coded by multiple
genes, with post-translational processing different from that of CP, and is found only in adult
females at the time of egg development. Female reproduction in D. variabilis is also a multistep
feeding process (Figs. 16.3A, 16.3B) in which (i) the unfed female and male virgins find the same
host; (ii) both sexes attach to the host (the female part-feeds and the male feeds to completion);
(iii) the male detaches from the host, finds the part-fed female, inserts its mouthparts into the
genital pore, and ultimately transfers a spermatophore into the female genital tract; (iv) the
FIGURE 16.3: A, Step 1, results for unfed virgin females of D. variabilis feeding to the part-fed condition
on a rabbit host on carrier proteins (CP) in the fat body, salivary gland, and hemolymph (blood). Blood
feeding results in an increase (up) in CP message and CP protein. B, Step 1, results for part-fed virgin
females of D. variabilis that are mated and fed to the replete condition and then drop from this rabbit
host. See text for details. 20-E, 20-hydroxyecdysone; JH, juvenile hormone; Vg, vitellogenin; VgR,
vitellogenin receptor; Vn, vitellin.
female, after mating, feeds to repletion and then drops from the host; and (v) the female lays a
single batch of eggs and then dies.
In the first step in female reproduction (Fig. 16.3A), host cues, detected most likely by
Haller’s organ and other sensory systems and interpreted within the synganglion, result in host
attachment and the regulation of feeding to the partially fed condition. This stage of feeding
results in an increase in the message(s) for CP in the fat body and salivary glands (Donohue
et al. 2008, 2009a) and an increase in CP protein levels in these tissues and hemolymph. Because
CP is a storage protein, an increase in CP levels would be expected for blood meal processing
each time the tick feeds, regardless of the developmental stage and sex. The different tissue
sources of CP (fat body versus salivary gland) are discussed in Chapter 15 of this text; the relative
importance of each in terms of its contribution to hemolymph CP is unclear. Little research has
been conducted on the hormonal factors, if any, that regulate CP. During the period of female
adult feeding to the part-fed condition, the yolk protein messages are not expressed.
The second step in female reproduction in the American dog tick (Fig. 16.3B) appears to be
associated with mating and a result of the transfer of a male pheromone to the female genital
tract. Male courtship alone, the male’s insertion of its mouthparts into the female genital pore
alone, and the injection of Sephadex beads into the part-fed virgin female’s genital track (to
mimic expansion from the insertion of the spermatophore) did not initiate feeding to repletion
or vitellogenesis (Donohue et al. 2009b). The injection of replete female synganglia, fed male
accessory glands alone, or fed male testes/vas deferens alone into the hemolymph also failed to
initiate feeding to repletion or egg development (Donohue et al. 2009b). A small percentage of
females injected with replete female vagina/seminal receptacle fed beyond 300 mg (suggesting
feeding to repletion was initiated) and produced brown eggs (an indication of Vg uptake by the
oocytes). However, the greatest effect was observed in female ticks injected with a suspension of
the male accessory gland and testes/vas deferens combined; 50% fed to repletion, and all of these
dropped from the host and laid brown eggs. The effect was abolished if the centrifuged superna-
tant only was injected, suggesting that the male pheromone is membrane bound or intracellular.
6.1. IDENTITY OF THE MALE PHEROMONE AND ITS MODE OF ACTION

Weiss and Kaufman (2004) suggested that 2 proteins from male Amblyomma hebraeum were
transferred to the female during mating and were responsible for the transition to rapid engorge-
ment. These 2 genes (identified from a male-specific cDNA library) were termed engorgement
factors alpha (EF-alpha) and beta (EF-beta) and were found to be 800 and 600 bp, respectively,
by means of Northern blot. Only the injection of recombinant EF-alpha and EF-beta protein to-
gether stimulated feeding to repletion. Previous studies (Kaufman and Lomas 1996) had sug-
gested that the “engorgement factor” in ticks was within the size range of 20–100 kDa.
In the American dog tick, Donohue et al. (2009b), using degenerate primers based on EF-
alpha and EF-beta from A. hebraeum, were able to obtain sequences via real-time polymerase
chain reaction (RT-PCR) from the reproductive system of blood-fed males that were similar to
EF-alpha; however, the primers for EF-beta did not produce any products similar to the sequence
of this protein. Further analysis of a D. variabilis male reproductive system 454 transcriptome
assembled from a total of 563,093 reads (constructed from blood-fed males) produced a match
with EF-alpha but not with EF-beta. The EF-alpha sequence (EF203418) was 69.5% and 78.9%
identical at the nucleotide and amino acid levels, respectively, to EF-alpha from A. hebraeum. EF-
alpha message was found only in fed males (not in unfed males) of D. variabilis, as would be ex-
pected. The first 30 amino acids at the N-terminus are 80.0% identical to those from A. hebraeum;
however, the 5′ UTR was 335 bp, in comparison to 128 bp for A. hebraeum EF-alpha. The
D. variabilis EF-alpha also contains an AU(3)A element in the 3′ UTR of the message that was not
present in A. hebraeum; these elements have been shown to decrease mRNA stability (Shaw and
Kamen 1986). Further attempts to obtain a homologue to EF-beta in other ticks also were unsuc-
cessful. A BLAST search with the EF-beta nucleotide sequence from A. hebraeum against the
Genbank databases (omitting the first 24 nucleotides of the published sequence, which corre-
sponds to a 12-mer Stratagene EcoRI adapter in the forward and reverse orientations) did not re-
turn results with a significant expect value (≤1 × 10−10). Northern blots showed that the message in
D. variabilis was 7 kb in size, much larger than what was reported for A. hebraeum. EF-alpha was
also found in public databases for the Southern cattle tick, Rhipicephalus microplus, and the deer
tick, Ixodes scapularis, with the latter including the deer tick genome; no evidence of EF-beta
could be found in these searches. Finally, silencing EF-alpha by the injection of dsRNA into unfed
male ticks of D. variabilis and then allowing the ticks to blood feed and mate with part-fed females
failed to significantly affect female engorgement to repletion and egg development relative to
controls. Although the bioassay evidence discussed earlier suggests the presence of a pheromone
in D. variabilis that initiates feeding to repletion and vitellogenesis, there is no evidence so far that
EF-alpha in the American dog tick (which appears in only the reproductive system of the blood-
fed adult male) is acting as a male pheromone that regulates female reproduction, and there has
been no evidence of the presence of EF-beta (Sonenshine et al. 2011).
The structure of the D. variabilis pheromone (Fig. 16.3B) is unknown. Whether this pheromone
is acting directly on the female reproductive system and/or traveling from this site via the hemo-
lymph to affect the synganglion or other tissues is also unknown (Fig. 16.3B). The working hypothesis
is that the synganglion is controlling blood feeding to repletion and the production of 20-HE; it is
possible that the male factor is affecting both of these processes via the synganglion or through other
systems not yet identified. Furthermore, the pheromone could be multiple factors acting together or
independently (with different functions). We also do not know whether blood feeding to repletion
itself is what initiates vitellogenesis (see Chapter 17 for more discussion on this point) or whether each
is regulated independently by the transfer of male factors to the female reproductive system. In addi-
tion, there is essentially nothing known about the influence of the ovary and female genital tract on
the overall function of the female tick during female adult development and reproduction (Fig.
16.3B). Most of the work so far has been aimed at how systems outside of the female reproductive
system regulate its function; however, there is also precedent for the reverse in some insects.
6.2. ECDYSTEROID INDUCTION OF VITELLOGENESIS

BUT NOT ENGORGEMENT
Substantial evidence now exists that ticks regulate reproduction through increases in the level of
circulating ecdysteroids, and not that of JH as was once thought. Studies currently suggest that
ecdysteroids regulate the transcription of Vg mRNA, Vg protein synthesis, Vg secretion into he-
molymph, and Vg uptake into the oocytes via a Vg receptor. The lack of physical and bioassay
evidence of JH in ticks; the developmental expression of the Vg message correlated with the ap-
pearance of increased hemolymph ecdysteroid titers and Vg protein levels; and the induction of
Vg mRNA, Vg protein synthesis, and egg development in partially fed virgin females of D. varia-
bilis by 20-HE support this perspective. Other factors in the hemolymph (Seixas et al. 2008) also
might influence Vg synthesis and/or uptake by oocytes as well as the Vg receptor (VgR); this is
discussed in more detail later. Although ecdysteroid injections into part-fed virgin adult females
attached to the host can initiate vitellogenesis and the growth of ovaries to weights similar to those
in mated females, 20-HE had no effect on the initiation of blood feeding to repletion. This sug-
gests that an unknown regulatory pathway is present, starting with the transfer of the male pher-
omone into the female genital track and ending with blood feeding to repletion, but excluding a
direct effect of ecdysteroids on this initiation of the second phase of feeding (discussed earlier).
6.3. EPIDERMALTROPHIC HORMONE

The ecdysteroids that regulate vitellogenesis are synthesized by the tick epidermis, which is ap-
parently regulated by a peptidic hormone from the tick synganglion, as discussed earlier in this
chapter. The identity of this hormone in ticks has not been determined, although a number of
neuropeptides from the tick synganglion have now been identified and should be examined in
this respect (discussed later in this chapter). K. Donohue, S. M. S. Khalil, and M. Roe, with the
assistance of Dr. Larry Gilbert at the University of North Carolina (Chapel Hill, NC), found that
synganglia from replete females of D. variabilis co-incubated in organ culture with epidermis
from part-fed females increased the level of ecdysteroids in the incubation medium (unpub-
lished data). This supports the hypothesis that a hormone originating from the synganglion
specifically of the American dog tick at the time of vitellogenesis triggers ecdysteroid synthesis.
It is assumed that (i) the epidermatrophic hormone is released from the synganglion and, via the
hemolymph, increases 20-HE synthesis in the epidermis, and (ii) the male pheromone is not
acting directly on the epidermis to regulate 20-HE synthesis (Fig. 16.3B). At this juncture, this is
not confirmed.
6.4. SYNGANGLION FARNESYL PYROPHOSPHATE PATHWAY

Although there is evidence that ticks do not make or have the typical insect JHs or MF (dis-
cussed earlier in this chapter), there is some evidence that the pathway is present in the syngan-
glion of adult D. variabilis for the de novo synthesis of farnesyl pyrophosphate (PP) from
acetate (Fig. 16.4). Although there is less evidence of the presence of the JH branch, and espe-
cially of the presence of P450 CYP15A, which in insects adds the C10,11 epoxide to MF, there is
evidence in the JH branch of a methyl transferase (MT) with matches to insect JH MTs (Fig.
16.4). Whether this synganglion MT is actually a JH MT is yet to be determined. MTs have been
found in other tick tissues in adult females of D. variabilis. Furthermore, matches with the JH
MTs in insects would be expected, given that MTs in insects in general have been described
mostly in relation to their role in JH synthesis, and MTs that would be expected to have the
same enzymatic mechanism of action would be expected to share similar motifs. MTs have a
variety of other functions in addition to the synthesis of JH in insects, and likely also in ticks.
The other enzymes involved in the earlier steps in the insect JH branch before JH-MT have not
been studied in detail (at the molecular level) in insects, which makes their comparison to our
tick synganglion transcriptomes more problematic. As discussed later in this chapter, there
appears to be a neuropeptide, allatostatin, found in the synganglion of D. variabilis that, among
its different functions in insects, is involved in the regulation of JH biosynthesis. Regardless of
the function of this neuropeptide, and even though there is no evidence of a role for JH in the
regulation of reproduction in ticks, a role for the farnesyl PP pathway or other JH precursors
must still be considered.
6.5. REGULATION OF CP VERSUS Vg

Multiple putative tissue sources for the synthesis of Vg found in tick hemolymph have been
identified through detection of the Vg message in the fat body, midgut, synganglion, and ovary.
Some of these discoveries could be fat body contamination. The relative importance of each
tissue in contributing to hemolymph and egg Vg levels is uncertain at this time and will be dif-
ficult to quantify. There also appear to be multiple Vgs per tick, and the expression of both ap-
pears to be controlled by elevated levels of 20-HE in D. variabilis (Khalil et al. 2011).
Juvenile Hormone Bio-synthesis pathway from Demacentor variabilis

Top match from Genebank
Acetyl-CoA Organism e - value % Identity
Acetoacetyl-CoA thiolase M. hirsutus 4e-22 61%

(mealybug)
Acetoacetyl-CoA
A. gambiae 1e-13 57%
HMG-S (mosquito)
Hydroxymethylglutharyl-CoA
I. paraconfusus 5e-42 77%
HMG-R (beetle)
Mevalonate
Mevalonate kinase NF
Mevalonate-5-P
B. mori 5e-14 46%
Phosphomevalonate kinase (moth)
Mevalonate-5-PP
D. mojavensis 2.8 56%
Diphsphomevalonate decarboxylase
(fruit fly)
Isopentenyl Ademine Isopentenyl-PP
Dimethylallyl-PP
(t-RNA) Geranyl-PP
D. jeffreyi 7e-11 57%
Farnesyl diphosphate synthase (beetle)
Sterol Branch Farnesyl-PP JH Branch

Sqaulene synthase Farnesyl diphosphate pyrophosphatase NF
Sqaulene Farnesol
Sqaulene monoxygenase Farnesol oxidase NF
Squalene epoxide Farnesal
Lanosterol synthase Farnesal dehydrogenase NF
Lanosterol Farnesoic acid
JH methyltransferase A. pisum 1e-18 35%

(aphid)
Cholesterol Methyl Farnesoate
JH epoxidase NF
Juvenile hormone III
FIGURE 16.4: Juvenile hormone (JH) biosynthesis in insects from acetyl-CoA to JH III, also showing
the sterol branch. Proteins found in a 454 transcriptome constructed from unfed, part-fed, and replete
female syngnalia from D. variabilis with signifi cant matches to the enzymes involved in JH biosynthesis
in insects are shown. Matches shown in boxes were taken from the synganglia of I. scapularis and were
not found in D. variabilis. Anopheles gambiae; Acyrthosiphon pisum; Bombyx mori; Drosophila
mojavensis; Dendroctonus jeffreyi; HMG-R, hydroxymethylglutaryl-CoA reductase; HMG-S,
hydroxymethylglutaryl-CoA synthase; Ips paraconfusus; Maconellicoccus hirsutus; NF, not found.
Vg is the major egg storage protein, and it also appears to be important in the transport of
heme to developing eggs. The latter is especially significant because the hypothesis is that ticks have
lost the ability to synthesize heme. The heme also changes the color of the eggs to brown, which
might be important after oviposition, as it allows the eggs to appear as part of the leaf litter. The
synthesis of CP, which is the major larval, nymphal, and adult storage protein prior to commitment
to egg laying in female ticks, also appears to be important in heme sequestration; its levels are low
in the egg. Even though CP and Vg might share a common evolutionary origin and have similar
functions in binding lipids, carbohydrates, and heme, the whole body tissue sources and regulation
of their messages are different. In the adult stage before the initiation of vitellogenesis, CP is the
major tick storage protein, with increased synthesis from the time of blood feeding to the part-fed
condition (Fig. 16.3A). Then this function shifts to Vg (Fig. 16.3B) in replete ticks (CP regulation is
down-regulated at this time) as a result of mating and increased levels of ecdysteroids.
6.6. VgR
Northern analyses in the American dog tick show that the VgR message occurs only in the ovary
of female adults, as would be expected based on its function of transferring yolk protein from
the hemolymph to the egg. The message is also found only in mated, replete vitellogenic females,
as would be expected. The VgR is absolutely essential for egg development. When the VgR mes-
sage was silenced by RNAi, Vg accumulated in the hemolymph, no absorbance of Vg by eggs
occurred, and no eggs were oviposited (Mitchell et al. 2007). Therefore, the timing for the ap-
pearance of the VgR message during vitellogenesis is critical to female tick reproduction. Chap-
ter 17 provides more information about the structure of VgR.
It was discussed earlier in this review that the injection of 20-HE into part-fed virgin Amer-
ican dog tick females still attached to the host initiated the expression of the Vg message, Vg
protein synthesis and secretion into the hemolymph, and the absorption of Vg in developing
eggs. Part-fed females do not have VgR message (Mitchell et al. 2007) and presumably do not
have the VgR protein. If the latter assumption is correct, it appears that 20-HE either directly or
indirectly is responsible for the expression of the VgR message and the regulation of the uptake
of Vg by the ovaries. Based on RNAi experiments, VgR appears to be essential for yolk uptake
into the egg. Although further studies are needed to validate this hypothesis, there is some evi-
dence in the mosquito Aedes aegypti that the upstream region of the VgR gene has binding sites
for the ecdysone regulatory gene products E74 and BR-C, along with the necessary transcription
factors (Cho et al. 2006), and in this insect, ecdysteroids initiate vitellogenesis, as is also the case
in ticks.
7. GLOBAL DIFF ERENTIAL GENE EXPRESSION

DURING TICK REPRODUCTION
Bissinger et al. (2011) constructed Agilent microarrays from a 454 transcriptome first described
by Donohue et al. (2010) from the synganglion of unfed, part-fed, and replete female adults of
D. variabilis. Global gene expression was examined at these same stages. From this analysis, 602
contigs were differentially expressed, of which 28% had no match to the nr database (e-value ≤
1e−06). Of the remaining 436 contigs, 391 had significant gene ontology (GO) matches (1e−06)
(Conesa et al. 2005; Götz et al. 2008). When using microarrays to examine gene expression in
the salivary glands of different feeding stages of A. americanum, Aljamali et al. (2009) hypothe-
sized that the expression of housekeeping genes in blood-feeding ticks would increase relative to
unfed ticks because of the increase in general metabolic activity. On our arrays, 125 contigs were
significantly up-regulated with the initiation of blood feeding (unfed versus part-fed), many
of which were housekeeping genes. Twelve of these contigs had no match in the nr database
(e-value ≤ 1e−06), 21 had matches with unknown functions, and 59 had GO term assignments.
Aljamali et al. (2009) found that few genes decreased significantly in the salivary gland from
unfed to part-fed ticks. This was not the case in the D. variabilis synganglion, where 101 contigs
were significantly down-regulated with the initiation of blood feeding. Differences between our
results and those of Aljamali et al. (2009) might reflect differences in the tissue examined and/
or could be the result of species differences. Of the down-regulated contigs, 46 had no match in
the nr database (e-value ≤ 1e−06), 10 had unknown functions, and 29 had GO term assignments.
Only 50 contigs were significantly up-regulated following mating and feeding to repletion
(part-fed versus replete), 35 of which had matches to the nr database (e-value ≤ 1e−06). Four of
these contigs had unknown functions, and 18 had GO term assignments. A total of 144 contigs
were significantly down-regulated following mating and feeding to repletion. Of these contigs,
27 had no significant match to the nr database (e-value ≤ 1e−06), 28 had unknown functions,
and 66 had GO term assignments.
It is not surprising that comparatively fewer genes are up-regulated following mating and
engorgement and that more genes are down-regulated at that time. Unlike argasids, metastriate
ixodid ticks exhibit a single gonadotropic cycle (Oliver 1986). Degeneration of the salivary
glands and endocuticle occurs during vitellogenesis and egg deposition (Kaufman 1986). This is
followed by death of the female within weeks after drop-off from the host. At the molecular
level, the decline in gene expression after feeding to repletion could be related to the shift from
host seeking, feeding, and mating to oviposition, senescence, and death.
Hierarchical clustering was conducted to determine whether specific underlying expression
patterns were present among the significantly regulated genes across the 3 different developmental
stages of reproduction (Fig. 16.5). Hierarchical clustering grouped part-fed and replete ticks as
more similar in terms of differentially expressed genes, with unfed ticks as the outgroup (Fig. 16.5).
This again indicates that the majority of changes in gene expression in these ticks occur with the
initiation of blood feeding in unfeds, rather than with mating and engorgement to repletion.
Microarrays to expressed sequence tags (ESTs) from the salivary glands of A. americanum similarly
showed that the greatest change in gene expression occurred with the initiation of blood feeding in
unfeds (Aljamali et al. 2009). In general, more research is needed on whole organismal and tissue-
specific global gene expression in almost every area of tick vector biology and physiology.
8. NEUROPEPTIDE EXPRESSION DURING

REPRODUCTION
Donohue et al. (2010) described 14 neuropeptides (allatostatin, bursicon-α/β, preprocorazonin,

eclosion hormone, glycoprotein hormone-α/β, insulin-like peptide, ion-transport peptide, 4
orcokinins, and preprosulfakinin) and 5 neuropeptide receptors (calcitonin receptor, gonado-
tropin receptor, leucokinin-like receptor, pyrokinin receptor, and sulfakinin receptor) from a
D. variabilis synganglion 454 library constructed from unfed, part-fed, and replete females. Biss-
inger et al. (2011) recently examined the developmental regulation of these proteins in virgin
unfed, virgin part-fed, and mated replete females of D. variabilis and their potential function
in adult female development and reproduction. Nine of these neuropeptides (allatostatin;
bursicon-β; preprocorazonin; glycoprotein hormone-α; insulin-like peptide; orcokinins 1, 3, and
4; and preprosulfakinin) and the gonadotropin receptor were significantly differentially regu-
lated during adult female blood feeding and reproduction as determined via microarray and/or
qRT-PCR analyses (Table 16.1). Three of these neuropeptides (bursicon-α, eclosion hormone,
and glycoprotein hormone-β) were not found to be significantly differentially expressed by
either of our assay approaches. Interestingly, for the 7 out of 8 significantly regulated genes in
which the directionality of the change was the same on both the arrays and in qRT-PCR, all
except bursicon-β, glycoprotein-β, and the gonadotropin-hormone-releasing receptor were
down-regulated with the initiation of blood feeding in unfeds (Table 16.1). For the entire
-2.453
-1.962
-1.472
-0.981
-0.491
0
0.4906
0.9811
1.4717
1.9623
2.4529
Part-fed Replete Unfed
FIGURE 16.5: Hierarchical clustering of standardized least-squares means clustered by treatment and
gene for signifi cant contigs (analysis of variance, false discovery rate correction, P = 0.01; 16 clusters
shown) for the differentially regulated transcripts from a Dermacentor variabilis synganglia
transcriptome (Bissinger, B.W., Donohue, K.V., Khalil, S.M.S., Grozinger, C.M., Sonenshine, D.E., Zhu, J.,
and Roe, R.M. [2011] Synganglion transcriptome and developmental global gene expression in adult
females of the American dog tick, Dermacentor variabilis [Acari: Ixodidae] Insect Mol. Biol. 20:465–491).
D. variabilis synganglion transcriptome, 52.3% of transcripts were significantly down-regulated

in part-feds relative to unfeds, and 62.8% of transcripts were down-regulated in unfeds relative to
repletes. Only 33.7% of transcripts were up-regulated following mating and feeding to repletion
(unfeds versus repletes). This again points to a decrease in gene expression with the shift from
host seeking, feeding, and mating to oviposition, senescence, and death, as discussed earlier.
8.1. ALLATOSTATIN
As discussed earlier in this chapter, Neese et al. (2000) were unable to find the common insect
JHs or the crustacean MF in the hard tick D. variabilis or in the soft tick O. parkeri, and Thomp-
son et al. (2005) showed that ecdysteroids, and not JH, regulated vitellogenesis in the former.
Table 16.1: Fold change in gene expression of neuropeptides from the synganglia
of unfed, part-fed, and replete female Dermacentor variabilis as determined via
microarray and qRT-PCR analyses.
Microarrays qRT-PCR
Protein (known functions) Accession U vs. P P vs. R U vs. R U vs. P P vs. R U vs. R
number
Bursicon-α (insect ecdysis ACC99596 NS NS NS −1.05 1.39 1.33
behavior, molting)
Bursicon-β (insect ecdysis ACC99598 3.87* 2.17 4.53* 4.22* 3.03* 12.80*
behavior, molting)
Eclosion hormone (insect ACC99595 NS NS NS −10.65 8.14 −1.31
ecdysis behavior,
molting)
Glycoprotein hormone-α ACC99601 −2.74* −1.67 −3.04* −4.26* −1.08 −4.62*
precursor (invertebrate
function unknown)
Glycoprotein hormone-β ACC99600 NS NS NS 1.66 −1.45 1.15
(invertebrate function
unknown)
Gonadotropin-releasing ACC99628 −2.04 2.28* 2.23* −3.18* 1.01 −3.15*
hormone receptor
(controls reproduction
in mammals)
Insulin-like peptide (insect ACC99597 −2.65* −2.18 −2.99* −2.73* −2.20 −5.99*
ecdysteroidgenesis,
metabolism, and
reproduction)
Orcokinin precursor 1 ACC99605 2.26 2.02 2.29* −3.39* −1.05 −3.57*
(affects crayfish
hindgut contraction;
neuromodulator in
decapods; insect
circadian locomotor
activity)
Orcokinin precursor 3 ACC99607 −2.27* 2.09 −2.16 −2.51* −1.41 −3.54*
(affects crayfish hindgut
contraction; neuromod-
ulator in decapods;
insect circadian
locomotor activity)
Orcokinin precursor 4 ACC99608 −3.15* 2.13 −2.86* −5.24* −1.18 −6.17*
(affects crayfish hindgut
contraction; neuromod-
ulator in decapods;
insect circadian
locomotor activity)
(continued)
Microarrays qRT-PCR
Preproallatostatin ACC99603 −4.12* 2.98* −2.45 −3.21* 1.32 −2.44*

(down-regulation of
insect JH and other
functions)
Preprocorazonin (insect ACC99609 NS NS NS −1.25 −1.89 −2.36*
ecdysis behavior,
molting)
Preprosulfakinin (in ACC99604 −2.82* −2.17 −3.20* −3.22* −1.73 −5.58*
insects, elicits gut
myotrophic activity;
affects feeding, odor
preference, and
locomotion)
Proprotein convertase type ACD63025 −3.12* 3.04* −2.03 −16.17* 5.83 −2.77
2 precursor (activation
of prohormones)
*
Indicates significance between pairs by Tuckey’s test (pairwise comparison, P ≤ 0.1 for microarrays; pairwise compar-
ison, P ≤ 0.05 for qRT-PCR) From Bissinger, B.W., Donohue, K.V., Khalil, S.M.S., Grozinger, C.M., Sonenshine, D.E.,
Zhu, J., and Roe, R.M. (2011) Synganglion transcriptome and developmental global gene expression in adult females of
the American dog tick, Dermacentor variabilis (Acari: Ixodidae) Insect Mol. Biol. 20:465–491.
Interestingly, allatostatin, which down-regulates JH biosynthesis in insects, was present in a

D. variabilis synganglion transcriptome (Donohue et al. 2010). Allatostatin-C was also identi-
fied in the central nervous system (CNS) of I. scapularis by Neupert et al. (2009) using MALDI-
TOF/TOF mass spectrometry. Much earlier, Zhu and Oliver (2001) identified allatostatin ac-
tivity in the synganglion of D. variabilis using a monoclonal antibody against Diploptera
punctata allatostatin I and showed increased immunoreactivity in unfed 1-month-old females
relative to newly molted females. Šimo et al. (2009) conducted more extensive mapping studies
of this and other neuropeptides in the brain of Rhipicephalus appendiculatus. Zhu and Oliver
(2001) hypothesized that allatostatin was synthesized in females prior to blood feeding and was
depleted during molting from the nymphal to the adult stage, and they noted that immunos-
taining was similar in unfed, part-fed, and replete females 6 days after drop-off from the host.
Our microarray and qRT-PCR results showed that allatostatin was expressed at significantly
lower levels in part-fed (microarrays, −4.12-fold; qRT-PCR, −3.21-fold; Table 16.1) and replete
(qRT-PCR only, −2.44-fold) than in unfed female ticks. Subsequently, allatostatin expression
increased from part-fed to replete ticks (microarrays only, 2.98-fold), indicating that alla-
tostatin is produced after mating and feeding to repletion. The function of allatostatin in these
ticks is unknown. Although the evidence so far argues that ticks (at least the American dog
tick), unlike crustaceans and insects, do not make MF and JH and do not use these hormones
to initiate vitellogenesis, it would be reasonable to assume that earlier parts of this pathway
from acetyl Co-A to farnesyl PP might be present in the synganglion of D. variabilis (Fig. 16.4).
It is interesting that allatostatin levels decreased in part-feds, which might suggest an increase
in the activity of this pathway prior to mating, and then the reverse occurred in repletes at the
start of vitellogenesis. Currently, we do not know the reason for these changes, but we must
assume that they play some role in tick reproduction. Allatostatins in insects have other func-
tions in addition to the regulation of JH synthesis that might be applicable to ticks as well
(Donohue et al. 2010).
8.2. ECLOSION HORMONE, CORAZONIN, AND BURSICON

Pre-ecdysis, ecdysis, and post-ecdysis behavior and cuticle sclerotization in insects are regulated
by eclosion hormone, corazonin, pre-ecdysis and ecdysis-triggering hormone, crustacean cardio-
active peptide, and bursicon (Klowden 2007). Interestingly, 3 of these hormones (i.e., eclosion
hormone, corazonin, and bursicon [α and β]) were found in the synganglion of female American
dog ticks, which do not molt as adults. In unfed hard ticks, the outer epicuticle is folded exten-
sively, and as engorgement occurs, this cuticle unfolds, causing the appearance of an increased
area; in actuality, there is no new growth of epicuticle as reported by Lees (1952) and Hackman
and Filshie (1982). Visual observation of feeding ticks also shows that the cuticle expands and is
flexible and colored. In contrast, the endocuticle of the adult female grows during reproduction,
increasing approximately 10-fold in weight 6 days after the initiation of feeding (Lees 1952). This
increase is the result of the deposition of new endocuticle. The amino acid dityrosine is believed
to be involved in the cross-linking of proteins during this cuticle formation (Hopkins and Kramer
1992). In the early period of feeding, levels of dityrosine in the cuticle rise (Andersen and Roep-
storff 2005). During the period of rapid engorgement after mating, the cuticle thins as it stretches,
but no additional cuticle is added (Lees 1952). Correspondingly, dityrosine levels do not increase
(Andersen and Roepstorff 2005).
The identification of eclosion hormone, corazonin, and bursicon in the synganglion tran-
scriptome was surprising, because ticks do not molt as adults. No statistically significant
changes in eclosion hormone expression were noted via either microarray or qRT-PCR be-
tween unfed, part-fed, and replete females (Table 16.1). However, the trend with qRT-PCR was
a 10.65-fold decrease between unfed and part-fed females and an 8.14-fold increase between
part-fed and replete females. Microarray analysis also did not reveal any significant change in
corazonin concentrations during development, but qRT-PCR showed a 2.36-fold decrease
among unfeds and repletes. Bursicon-α levels were also constant. However, this was not the
case for bursicon-β, which was significantly up-regulated in part-fed and replete ticks relative
to unfed ticks (Table 16.1). The trend in expression was the same for the 2 methods used to
measure transcript levels, though the amount of the increase differed. The qRT-PCR results
showed a 12.80-fold increase in expression from unfed to replete ticks, whereas the microar-
rays showed only a 4.53-fold increase. Bursicon-β was also up-regulated in part-fed females
relative to unfed females (microarrays, 3.87-fold; qRT-PCR, 4.22-fold). With the presence of
eclosion hormone, preprocorazonin, and bursicon (α and β) in the synganglion of adult fe-
male D. variabilis and the developmental regulation of at least bursicon-β related to tick blood
feeding and reproduction, and considering the role of these hormones in the regulation of
insect attachment prior to ecdysis, cuticle expansion during and after ecdysis, and sclerotiza-
tion and melanization, one hypothesis is that these hormones in ticks might be regulating
adult tick behavior and cuticle development related to female feeding and reproduction, or at
least some aspect of female reproduction in general. These hormones, and possibly others not
yet identified in D. variabilis, such as the insect ecdysis-triggering hormone and the crusta-
cean cardioactive peptide, might be controlling the process of attachment, the expansion of
cuticle during the process of blood feeding to repletion, sclerotization and melanization, or
other processes critical to egg production and oviposition.
Bursicon is highly conserved in insects, and homologues have been identified in echino-
derms and other arthropod classes aside from the Insecta (Van Loy et al. 2007; Wilcockson and
Webster 2008). In insects, bursicon is the final hormone to be expressed during ecdysis. How-
ever, in the green shore crab, Carcinus maenas, bursicon-α and -β are constitutively expressed
throughout the adult molting cycle, with bursicon-α being more highly expressed than
bursicon-β (Wilcockson and Webster 2008). In addition to cuticle tanning, bursicon also func-
tions in wing inflation in Drosophila melanogaster (Dewey et al. 2004) and Bombyx mori (Huang
et al. 2007). Additionally, microarray analysis showed that bursicon regulated 87 genes in
D. melanogaster, 30 of which have unknown functions. The proteins encoded by these genes
exhibit a wide array of functions, including cell adhesion, cell signaling, cytoskeleton formation,
DNA/RNA binding, gene transcription, immune response, ion trafficking, and proteolysis-
peptidolysis metabolism (Shiheng et al. 2008). Bursicon also appears to be involved in the apo-
ptosis of wing epidermal cells in D. melanogaster following wing expansion (Kimura et al. 2004).
The function of bursicon homologues in ticks has yet to be elucidated. Apoptosis occurs in the
ovaries, salivary glands, and synganglion of ticks after engorgement (Freitas et al. 2007). It could
be possible that bursicon plays a role in programmed cell death in these organs, and bursicon-β
might have functions other than cuticle tanning in ticks. It is unclear why the expression of one
bursicon subunit changes with feeding and mating but not the other.
8.3. ECDYSONE RECEPTOR

Other important hormones involved in molting and metamorphosis are the ecdysteroids. In
ticks, these hormones also appear to be involved in the degeneration of the salivary glands in
replete females (Harris and Kaufman 1985; Kaufman 1986, 1990), and they have been shown to
regulate vitellogenesis (Friesen and Kaufman 2002; Thompson et al. 2005) and to be transported
to developing eggs (Diehl et al. 1982). Ecdysteroids have been well characterized in insects,
where they play multiple roles in growth and development (Rees 2004); however, much less is
known about their function in the Acari (Diehl et al. 1986; Sonenshine 1991). Ecdysteroid action
is mediated by a heterodimeric receptor made up of an ultraspiracle and an ecdysone receptor
(EcR). All nuclear receptors are characterized by a variable domain at the N-terminus and highly
conserved DNA and ligand binding domains separated by a variable hinge region (Rees 2004).
A putative EcR with highest homology to an EcR (AamECRA1; AAB94566) from A. america-
num (e-value = 4e−79) was identified in the synganglion transcriptome. The D. variabilis
sequence appears to be truncated at both the 5′ and 3′ ends but contains 124 of the 220 amino
acids found in the ligand-binding domain of EcRs and other members of the nuclear receptor
superfamily (Guo et al. 1997). cDNAs that encode 3 isoforms of EcR were isolated by Guo et al.
(1997) from A. americanum. During blood feeding, cells in the acini of the salivary glands dra-
matically increase in size and undergo differentiation. Consistent with this cell growth, Guo
et al. (1997) found higher expression in 2 of the EcR isoforms (AamEcRA1 and AamEcRA3) via
Northern blot analysis of the salivary glands of adult female ticks during the late stages of
feeding; however, the expression of a third EcR isoform (AamECRA2) remained constant.
Expression of the putative D. variabilis EcR in the synganglion was not significantly differen-
tially expressed based on microarray analysis (qRT-PCR was not conducted for this transcript).
Interestingly, the transcript was most similar to AamEcRA1, which was differentially expressed
in the salivary glands in the aforementioned study. This indicates that whereas expression of this
receptor varies with feeding in the salivary glands, the same is not true for expression in the
synganglion. Expression of EcR has been observed in the arthropod CNS before. In insects that
undergo complete metamorphosis, 2 different isoforms of EcR are expressed. Maturation occurs
in neurons exposed to ecdysone that contain the EcR-A receptor, whereas neurons bearing the
EcR-B1 receptor regress and experience loss of the synapse (Truman et al. 1994). In the hemime-
tabolous blood-sucking bug Rhodnius prolixus, EcR expression was stage- and tissue-specific in
the brain, epidermis, fat body, and spermatocytes. In the brain, strong EcR immunofluorescence
was observed in multiple neurons and in the large neurosecretory cells of the dorsal protocere-
brum of nymphs 12 days after a blood meal (Vafopoulou et al. 2005). Gene expression in nymphal
ticks has not been examined yet, and it is unknown why EcR appears to be constitutively ex-
pressed in adult female D. variabilis, but this does suggest that ecdysteroids have some role in the
regulation of synganglion function during female reproduction in ticks.
8.4. GLYCOPROTEIN HORMONES

Glycoprotein hormones comprise 2 cysteine-knot-containing proteins. For a given species, the
α subunit is identical, and the β subunit determines the functional characteristics of the
hormone. The combined α and β proteins act to control facets of reproduction and metabolism
(Pierce and Parson 1981). Vertebrate glycoprotein hormone homologues have been identified in
invertebrates; however, their function is not well understood (Park et al. 2005). A glycoprotein
hormone-α precursor found in the 454 library of D. variabilis adult females (Bissinger et al. 2011)
was expressed at significantly lower levels in part-fed (microarrays, −2.74-fold; qRT-PCR, −4.26-
fold) and replete (microarrays, −3.04-fold; qRT-PCR, −4.62-fold) ticks than in unfed ticks; how-
ever, the β subunit was not significantly differentially expressed according to microarray and
qRT-PCR analyses (Table 16.1). The presence and the developmental regulation of this hormone
suggest that it must play some role in female development in D. variabilis that has not yet been
identified.
8.5. GONADOTROPIN-RELEASING HORMONE RECEPTOR

In mammals, reproduction is controlled by gonadotropin-releasing hormone, which stimulates
the release of glycoproteins, follicle-stimulating hormone, and luteinizing hormone after binding
to receptors in the anterior pituitary (Conn and Crowley 1994). Gonadotropin-releasing
hormone receptors (GRHRs) are members of the G protein–coupled receptor family (Millar
et al. 2004). Results of expression analyses are conflicting with regard to the GRHR in the
D. variabilis synganglion that has been studied. The receptor was expressed at a higher level in
unfed than in part-fed ticks, although this was significant only according to qRT-PCR analysis
(−3.18-fold) (Table 16.1). Microarray analysis showed significantly greater (2.28-fold) expression
in replete than in part-fed ticks, whereas qRT-PCR analysis showed no change in expression for
the same comparison. According to microarrays, the GRHR was expressed at 2.23-fold greater
levels in replete ticks relative to unfed ticks; however, the opposite was found via qRT-PCR
analysis, which revealed lower expression in repletes than in unfed ticks (−3.15-fold) (Table 16.1).
Because of the disparity in the results between the 2 methods of analysis, Bissinger et al. (2011)
were unable to speculate about the role of mating in expression of the receptor.
8.6. INSULIN-LIKE PEPTIDE

Insulin plays a key role in growth and metabolism in vertebrates. In insects, insulin-like peptides
are involved in ecdysteroidogenesis (Gu et al. 2009), metabolism (Rulifson et al. 2002), and
reproduction (Nagasawa et al. 1984). Insulin-like immunoreactivity has been shown in the
synganglia of the ticks D. variabilis (Davis et al. 1994), O. parkeri (Zhu and Oliver 1991), and
R. sanguineus (Šimo et al. 2009) and appears to play a similar role. Using RNAi, Mulenga and
Khumthong (2010) showed that silencing insulin-like growth factor binding proteins in Ambly-
omma americanum led to an inability of females to complete a blood meal, and approximately
half of the ticks died. Bombyxin from the moth Bombyx mori has high amino acid similarity to
bovine insulin (Iwami et al. 1989), and Šimo et al. (2009) observed that antibodies to bombyxin
bound to the same regions in R. sanguineus synganglion as in D. variabilis synganglia immunos-
tained with insulin antibody as described by Davis et al. (1994). Previously it was believed that
bombyxin was a PTTH; however, this is no longer the case, and the role of bombyxin in insects
remains undetermined (Klowden 2007). Davis et al. (1994) observed decreased insulin-like im-
munoreactivity after blood feeding and mating in male and female D. variabilis. This was also
the case in the studies of Bissinger et al. (2011), in which an insulin-like peptide was expressed at
lower levels in part-fed (microarrays, −2.65-fold; qRT-PCR, −2.73-fold) and replete (microar-
rays, −2.99-fold; qRT-PCR, −5.99-fold) ticks than in unfed ticks (Table 16.1).
8.7. ORCOKININS
Orcokinin was first isolated from and named for a myotropic neuropeptide that increased the
frequency and amplitude of contractions in the hindgut of the crayfish Orconectes limosus
(Stangier et al. 1992). Orcokinins were also implicated as neuromodulators in decapods (Li et al.
2002). Orcokinins have been identified from A. americanum, I. scapularis, and R. sanguineus
using in silico searches of publicly available EST databases (Christie 2008) and MALDI-TOF/
TOF mass spectrometry (Neupert et al. 2009). In the cockroach Leucophaea maderae, orcokinin
caused changes in circadian locomotor activity (Hofer and Homberg 2006). Four orcokinins
were identified by Donohue et al. (2010) from a D. variabilis synganglia transcriptome
(DervaO1–4, named in the order in which they were identified). Bissinger et al. (2011) found that
DervaO1, -3, and -4 were differentially expressed during female reproduction. DervaO3 was
expressed at a significantly lower level in part-fed than in unfed ticks (microarrays, −2.27-fold;
qRT-PCR, −2.51-fold) and in replete than in unfed ticks (qRT-PCR only: −3.54-fold). Expression
profiles for DervaO1 contrasted in microarray and qRT-PCR analyses: levels increased more
than 2-fold from unfed to part-fed to replete ticks in microarrays, whereas the trend levels of the
transcript were seen to decrease for the same comparisons when using qRT-PCR (Table 16.1).
Similarly, discrepancies were observed between the microarrays and qRT-PCR for DervaO4:
expression of the transcript was 2.13-fold higher in repletes than in part-feds when using micro-
arrays, but expression was lower in repletes than in part-feds when using qRT-PCR (although
neither was significantly differentially expressed) (Table 16.1). Further studies are needed to de-
termine the function of orcokinins in ticks and to verify the directionality of expression levels
between feeding stages.
8.8. PROPROTEIN CONVERTASE PRECURSOR

Gene expression of proprotein convertase in the tick synganglion might provide information
on the regulation of cleavage of prohormones to their active forms. Expression levels in the syn-
ganglion of part-fed female ticks were lower than in unfed ticks (microarrays, −3.12-fold;
qRT-PCR, −16.17-fold) but were not significantly higher than in replete ticks as determined via
qRT-PCR. Levels in replete ticks were higher than in part-fed ticks when determined via micro-
arrays only (3.04-fold; Table 16.1).
8.9. SULFAKININ
Arthropod sulfakinins were originally isolated from the cockroach L. maderae (Nachman et al.
1986a, 1986b), in which they elicit myotropic activity on the gut. Sulfakinins also decrease
feeding activity in the German cockroach, Blatella germanica, in a dose-dependent manner
(Maestro et al. 2001), and feeding was stimulated in the Mediterranean field cricket, Gryllus
bimaculatus, when sulfakinin was suppressed following ingestion or injection of double-
stranded RNA (Meyering-Vos and Müller 2007). In larval D. melanogaster, sulfakinins play a
role in odor preference and locomotion (Nichols et al. 2008). In G. bimaculatus, sulfakinin ex-
pression decreases in the adult brain with age (Meyering-Vos and Müller 2007). Sulfakinins
have been detected in the synganglion of I. scapularis using MALDI-TOF/TOF mass spectrom-
etry (Neupert et al. 2009). Bissinger et al. (2011) found that preprosulfakinin expression was
significantly lower in part-fed (microarrays, −2.82-fold; qRT-PCR, −3.20-fold) and replete (mi-
croarrays, −3.20-fold; qRT-PCR, −1.73-fold) than in unfed D. variabilis (Table 16.1). This finding
is not surprising, because lower levels of sulfakinin have been correlated with increased feeding
(Meyering-Vos and Müller 2007).
Significant advances have been made in the past decade in understanding tick storage proteins
(CP and Vg), the Vg receptor, male pheromones, the respective roles of JH and ecdysteroids in
female reproduction, and neuropeptides (also see Chapter 13 on the tick nervous system and
neuropeptides). Our understanding of the endocrine regulation of larval and nymphal tick
development and metamorphosis is essentially a black box. The good news is that the many
advances in studies of the regulation of female reproduction and the identification of factors
involved in this process can be used to at least begin the study of immature development. There
are also many additional challenges in understanding female reproduction in ticks, which in-
clude (i) the role of Haller’s organ and other sensory systems, along with the synganglion, in the
regulation of host detection, adult development, and reproduction; (ii) identification of the reg-
ulatory mechanisms for blood meal digestion and CP synthesis; (iii) identification of the male
pheromone(s) regulating female blood feeding and vitellogenesis; (iv) elucidation of the path-
way by which male pheromone(s) regulates blood feeding, ecdysteroid biosynthesis, and vitello-
genesis, including the role of the synganglion and other tissues in this process; (v) the role of the
ovary in the regulation of other tissues during reproduction; (vi) identification of the epidermal-
trophic hormone that regulates ecdysteroid biosynthesis; (vii) understanding the function of the
early JH pathway; and (viii) determination of the function of the many neuropeptides that now
have been identified in the tick synganglion relative to adult female development. New tools for
measuring global gene expression should be more widely used in all aspects of this work and
should be extended to all aspects of the study of tick physiology. It should also be noted that little
work has been conducted so far in the regulation of male reproduction and in the regulation of
development and reproduction in soft ticks. With the current availability of the first mite ge-
nome and the additional mite and tick genomes that likely will be available in the next decade,
the pace of progress in tick endocrinology will only be increasing. There appear to be many
discoveries in tick endocrinology that will also impact our understanding of the physiology of
other arthropods.
One of the greatest impediments to advancements in our understanding of tick physiology
is the difficulty of raising ticks in the laboratory and the necessity of using animals as hosts.
A significant effort is needed to develop methods to feed and breed ticks, especially ixodid ticks,
on artificial membranes and, if possible, artificial media. Considering the different life strategies
of ticks and how little we know about other regulatory processes associated with host–tick inter-
actions, vector biology, ecology and population dynamics, diapause, migration, etc., more re-
search is needed in these other areas, and multiple species should be used in order to better
determine basic regulatory pathways. It is also our hope that researchers will not only advance
our basic understanding of tick biology but extend this knowledge to new applied technologies
relative to both the acarines and applications in other areas.
ACKNOWLEDGMENTS
The laboratories of R.M.R. and D.E.S. are supported by grants from the National Science Foun-
dation (IBN-0315179 and IBN-0723692) and National Institutes of Health (1R21AI096268-01)
and research support to R.M.R. from the North Carolina Agricultural Research Service.
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C H A P T E R 1 7
FEMALE REPRODUCTIVE SYSTEM

Anatomy, Physiology, and Molecular Biology
M ARI H. OGIHARA AND DEMAR TAYLOR
1. INTRODUCTION
In this chapter, the anatomy, ultrastructure, proteomics, genomics, and physiology of the female
reproductive system are described, as well as the developmental changes that occur during fe-
male tick feeding, mating, oogenesis, and oviposition. The endocrine mechanisms regulating
reproduction are referred to when needed to explain the physiological processes. A detailed
explanation of the hormonal regulation of reproduction can be found in Chapter 16 of this vol-
ume. In addition, the gonotrophic cycles of the 2 major tick families, Ixodidae and Argasidae,
are compared, with focus on differences in engorgement and mating.
Reproduction in ticks has been the subject of intense interest for many decades, and nu-
merous excellent descriptions of these systems can be found in the literature. Here we attempt to
bring much of this information together in a very concise description of the structure and func-
tions of the female reproductive system. For more detailed information about the tick female re-
productive system, we recommend that the reader consult excellent reviews by Balashov (1972),
Oliver (1974, 1986), Diehl et al. (1982a, 1982b), Raikhel (1983), Chinzei and Taylor (1990, 1994),
Sonenshine (1991), Taylor and Chinzei (2002), Rees (2004), and Roe et al. (2008). The VectorBase
website also may serve as an excellent reference, particularly for recent information on the
anatomy and ultrastructure of specific anatomical structures and their relationships in ticks.1
2. ANATOMY AND ULTRASTRUCTURE
2.1. OVERVIEW
The female genital system is basically similar in the Ixodidae and Argasidae, but noteworthy dif-
ferences have been reported. In the Ixodidae, this system consists primarily of a single tubular
band or U-shaped ovary in the posterior region of the body; paired, coiled, or folded oviducts; a
FIGURE 17.1: Diagram illustrating the reproductive system in representative female ixodid ticks. The
diagram illustrates a dorsal view of the reproductive system of a mated, replete (72 hours after
engorgement) female Dermacentor andersoni. Details of the genital aperture are not shown. GA, genital
aperture; LAG, lobular accessory gland; LG, longitudinal groove; O, ovary; Oc, oocyte; Ov, oviduct; Rs,
receptaculum seminis (seminal receptacle); TAG, tubular accessory gland; VV, vestibular vagina.
Modifi ed from Brinton, L.P. and Oliver, J.H., Jr. (1971) Gross anatomical, histological and cytological
aspects of ovarian development in Dermacentor andersoni Stiles (Acari: Ixodidae). J. Parasitol.
57:708–719, with permission from the American Society of Parasitologists (Journal of Parasitology/
Alliance Communications).
single uterus (common oviduct); a muscular connecting tube (absent in argasid ticks); a vagina;
paired tubular accessory glands; and a genital aperture (Fig. 17.1). The vagina is subdivided into a
posterior muscular, thick-walled cervical region and a cuticle-lined vestibular vagina. In the
Metastriata, a large seminal receptacle (receptaculum seminis) lies above the cervical vagina, to
which it is connected; this receptacle serves as the storage site for sperm introduced during cop-
ulation. In addition to a pair of tubular accessory glands near the junction of the vestibular vagina
and the cervical vagina, ixodid females have a 3-lobed lobular accessory gland (LAG) that sur-
rounds the vestibular vagina. Externally, porose areas (PA) occur on the dorsal surface of the
basis capituli of only ixodid female ticks (see Chapter 4, Figs. 4.1, 4.14, in Sonenshine 1991). These
pores are openings for accessory glands to release secretions that function as a lubricant for
movement of Gené’s organ (Kakuda et al. 1995a, 1995b). The glandular Gené’s organ is remote
from the genital tract but vital for reproduction because of the finger-like extensions that pro-
trude through the camerostomal cavity and wax the eggs during egg laying. Gené’s organ occurs
in females of both ixodid and argasid ticks. Dramatic changes occur in the structures of the fe-
male genital system during feeding. Epithelial cells lining the vestibular cuticle become the LAGs,
cuticle of the cervical vagina unfolds for longitudinal enlargement, tubular accessory glands in-
crease in size, the epithelial cells of the connecting tube become irregularly shaped and unfold as
the cells enlarge, oviduct cells become columnar, numerous vesicles appear in the cytoplasm,
oocytes protrude into the hemocoel, and spermatozoa ascend into the ovarian lumen (Kakuda
et al. 1995c).
Generally, the female reproductive system is similar in argasid ticks (Fig. 17.2). The ovary is
smaller, typically occupying only a transverse region in the posterior region of the body. The
longitudinal groove, a common feature of the ovary in Ixodidae, is apparently absent in the Ar-
gasidae. The oviducts are long and coiled as in the ixodid ticks. In addition, there is an enlarge-
ment, the ampulla, near the anterior end. The oviducts enlarge greatly near their junction with
the uterus. A pair of tubular accessory glands occurs near the junction of the vestibular and
Female Reproductive System 451
FIGURE 17.2: Diagram illustrating the reproductive organs of female argasid ticks. A, Dorsal view of a
fed, mated female Carios kelleyi. The large uterus has a common anterior region and a large bifurcate
posterior region (uterine horns). There is a prominent ampulla in the oviducts. From Sonenshine, D.E.
(1970) A contribution to the internal anatomy and histology of the bat tick, Ornithodoros kelleyi Cooley
and Kohls, 1941. II. J. Med. Entomol. 7:289–312, with permission from the Entomological Society of
America. B, Dorsal view of a fed, mated female Ornithodoros moubata. Ova are shown migrating through
the oviducts and accumulating in the uterus. The cervical vagina and the tubular accessory
glands are omitted. Amp, Ampulla; CV, cervical vagina; O, ovary; Ov, oviducts; TAG, tubular accessory
glands; U, uterus; Uh, uterine horns (bifurcate region of the uterus); VV, vestibular vagina. From
Aeschlimann, A. (1958) Developpment enbryonnaire d’Ornithodoros moubata (Murrary) et transmission
transovarriene de Borrelia duttoni [Embryonic development of Ornithodoros moubata (Murray) and
transovarial transmission of Borrelia duttoni]. Acta Trop. 15:15–64, with permission from Elsevier.
cervical regions of the vagina, but the LAG and PA are absent in argasid ticks. In mites, the fe-
male reproductive system seems to be generally similar to that seen in ticks, but recent research
indicates important differences between the numerous species of mites.
2.2. OVARY
The ovary is a simple tube-like structure in the juvenile stages and the unfed female. This organ is
evident in the unfed female nymph, where it appears as a crescent tube lying in front of the rectal
sac. Paired tubular oviducts extend to an undifferentiated cell mass that is destined to form the
organs of the anterior reproductive tract. The ovary at this stage consists of oogonia, undifferenti-
ated interstitial cells, and a simple external epithelium with occasional smooth muscle cells. The
interstitial cells line the lumen of the ovary and occur between the oogonia (Brinton and Oliver
1971b). Following nymphal feeding and emergence of the adult female, further differentiation oc-
curs. The wall of the ovary then contains small, developing primary oocytes in addition to the in-
terstitial cells and oogonia. The ovary is surrounded by (i) a single cell layer of epithelial cells and
occasional smooth muscle cells and (ii) the acellular tunica propria (TP), a connective tissue sheath
comprising 4 or 5 layers of finely fibrillar material (Raikhel 1983). In Dermacentor variabilis, the
lamellate TP is approximately 1 μm thick. The TP extends around each of the oocytes and forms
their outer coverings as they enlarge. The TP is believed to be permeable to vitellogenin and other
hemolymph proteins that are taken up by the oocytes. Internally, there is a narrow, slit-like lumen
that expands during feeding. When viewed in cross-section, the ovary is similar in unfed or feeding
virgin females; narrow bulges and folds lined by interstitial cells are apparent, and undeveloped
oogonia and primary oocytes project from the ovary wall. Histologically, the ovary is similar in
ixodid and argasid ticks. In the unfed female D. andersoni, the ovary is approximately 6 mm long
and 64 to 110 μm in diameter. In ixodid ticks, a longitudinal groove occurs along one side of the
ovary. Germinal cells in the longitudinal groove are generally less developed than elsewhere in this
organ (i.e., this appears to be a region of slower developmental activity). Symbiotic rickettsia-like
intracellular microbes (Wohlbachia sp.) (Rickettsiales) occur in the interstitial cells and in the oo-
cytes. The oocytes in the unfed female ovary, according to the classification system of Balashov
(1972) and Denardi et al. (2004), are stage I oocytes (see “Oogenesis” below [Section 3] for a de-
scription of oocyte development). The oocytes and oogonia are predominantly round in shape and
communicate with one another via intercellular bridges (Brinton and Oliver 1971a). The cytoplasm
is dense and filled with numerous free ribosomes. In contrast to that of insects, the tick ovary is not
segmented into separate zones such as the germarium or vitellarium, all with differing rates of re-
productive development, as occurs in the different ovarioles of the insect ovary. Figure 17.3 sum-
marizes the changes in the ovarian wall and the different stages of oogenesis in the cattle tick
Rhipicephalus (Boophilus) microplus (Saito et al. 2005). This description of changes in the oocytes
is similar to descriptions of other ixodid tick species reported by Balashov (1972), Brinton and
Oliver (1971a), and Diehl et al. (1982a).
FIGURE 17.3: Diagram of Rhipichepalus (Boophilus) microplus ovary showing details of the ovary wall,
pedicel cells, and oocytes.The stages of oocyte development from the earliest period to ovulation (I to V) are
shown. I, oocyte I; II, oocyte II; III, oocyte III; IV, oocyte IV; V, oocyte V; ep, epithelium; lu, lumen; ovd, oviduct;
gv, germinal vesicle. From Saito, K.C., Bechara, G.H., Nunes, E.T., Oliveira, P.R., Denardi, S.E., and Mathias,
M.I. (2005) Morphological, histological, and ultrastructural studies of the ovary of the cattle-tick Boophilus
microplus (Canestrini, 1887) (Acari: Ixodidae). Vet. Parasitol. 129:299–311, with permission from Elsevier.
During feeding in ixodid ticks (or after feeding in argasid ticks), the oocytes enlarge and
transform into more advanced stages, migrating to a position above the formerly flat ovarian
wall. When viewed with a scanning electron microscope or stereoscopic microscope, the ovary
appears covered with innumerable spherical and subspherical eggs. Oocytes along one side of
the ovary develop more slowly and remain relatively undeveloped. The ovary with its numerous
bulging oocytes resembles an elongated cluster of grapes. The rapidly enlarging ooctyes appear
to bulge from the wall of the ovary and expand into the surrounding hemolymph. Oocytes are
easily recognized in histological sections by their comparatively large size and very large nuclei;
the latter often contain large granular nucleoli. Large, swollen cells remain connected to the
ovarian wall by a thin layer of pedicular cells that form a short hollow pedicel, previously called
the funiculus. Each oocyte is in contact with the ovarian lumen via the narrow channel in the
center of the pedicel. As feeding progresses, the ovary increases in size to about 27 mm in length
and about 0.40 mm in diameter in D. andersoni. Following mating and repletion, the ovary
reaches its greatest size, increasing to as much as 70 mm in length and 0.37 mm in diameter
(Brinton and Oliver 1971b). Enormous increases occur in the size of the oocytes; this is discussed
later (see Section 3). The individual oocytes fill with amber or brown-colored yolk granules.
These vitellogenic oocytes are easily recognized even when viewed at low magnification with a
dissecting microscope. The argasid tick ovary is similar to that of ixodid ticks, except for the
absence of a longitudinal groove.
2.3. OVIDUCTS
In the unfed state, the walls of the oviducts consist of a thin layer of epithelial cells about 24 to
40 μm thick throughout most of their length. The lumen is small. The epithelial cells are cu-
boidal with large nuclei. Few organelles are apparent in the cytoplasm. The luminal side of the
cells contains numerous microvilli that virtually fill the narrow lumen. Externally, there is a
thick basal lamina and a thin connective tissue membrane with occasional smooth muscle cells
arranged circumferentially around the duct. In argasid ticks, the oviducts expand near the
junction with the uterus, forming bulbous ampullae, and the walls are considerably thicker in
this region. Following feeding, the epithelial cells divide and also expand to several times their
original size. The cytoplasm fills with cisternae of rough endoplasmic reticulum (RER) and nu-
merous dense bodies believed to be lysosomes (Raikhel 1983). Extensive development of the
RER and the Golgi complexes (GC) suggests high levels of synthetic activity.
2.4. CONNECTING TUBE

In the ixodid tick D. variabilis, the connecting tube (CT) is a narrow duct lined with a layer of
thin cuticle (2 to 3 μm). In unfed or feeding virgin females, the lumen is collapsed, and the sur-
rounding walls are somewhat folded, forming a labyrinth. A thin layer of epithelial cells sur-
rounded by bands of smooth muscle cells constitutes the remainder of the wall of this tube.
Electron micrographs show a deeply folded cuticular lining bordered by cells with dense cyto-
plasm. No evidence of secretory activity is found in this region. According to Raikhel (1983),
profiles of the CT in Hyalomma asiaticum show long, dense, tubular invaginations (1 to 2 μm
long) that extend from the apical plasma membrane into the cytoplasm. Microtubules from
these structures traverse the cytoplasm and attach to hemidesmosomes on the basal plasma
membrane, providing an anchoring system that serves to protect the CT membrane when the
wall stretches during oviposition. During feeding, the epithelium expands. The ultrastructure of
the epithelium of the CT resembles that of the epithelium of the cervical vagina.
2.5. CERVICAL VAGINA

In D. variabilis, the cervical vagina is the most highly muscularized section of the female reproduc-
tive tract (Fig. 17.1). In unfed and feeding virgin females, it is lined with a highly folded, labyrinthine
cuticle about 13 to 25 μm thick. The epithelium is many cells thick, and the cells appear to be highly
interdigitated. Microvilli appear at the luminal boundaries of the proximal cells. The cell cytoplasm
reveals moderate numbers of mitochondria and ribosomes, but secretory vesicles are not apparent
at this stage. The basal plasma membrane is highly folded. In Hy. asiaticum, bundles of microtu-
bules attach to hemidesmosomes on the basal plasma membranes (Raikhel 1983). Beyond the epi-
thelium is a thick layer of smooth muscle cells. The muscle mass is thicker here than in any other
part of the reproductive system, suggesting that this organ is especially important for forcing the
eggs out of the system during oviposition. In contrast to insects, ticks lack a separate ovipositor.
Anteroventrally, the cervical vagina opens into the vestibular vagina, which extends ventrally to the
genital pore. On its posterodorsal side, the cervical vagina opens into the seminal receptacle.
2.6. SEMINAL RECEPTACLE

In D. variablis, the seminal receptacle (RS) is a large, folded sac-like organ lying dorsal to the cer-
vical vagina and the connecting tube. In the unfed tick, it has a collapsed lumen and highly folded
walls. In the feeding female, the lumen is large and is lined by a thin layer of cuticle about 1 to 2 μm
thick. The epithelium is multilayered with many large cells containing huge nuclei, some in excess
of 30 μm. The external surface appears lobed. The few smooth muscle cells that occur do not form
an organized layer. After mating, the endospermatophores fill the RS and distend it greatly.
Observations of this region in fed mated Hy. asiaticum revealed epithelial cell hypertrophy and
showed ultrastructural features characteristic of secretory activity with numerous microvilli, RER,
and secretory vacuoles. In Haemaphysalis longicornis, secretory products are released into the
lumen and attach to the wall of the endospermatophore, where they appear to act as destructive
chemicals to break down the wall of the endospermatophore (Kakuda et al. 1997b). The basal
plasma membrane forms an extensive labyrinth suggesting intense fluid transport. Dissection of
the RS from a fed mated ixodid female reveals innumerable spermatozoa filling this organ.
2.7. TUBULAR ACCESSORY GLANDS

These tubes are located at the junction of the cervical and vestibular regions of the vagina (Figs. 17.1
and 17.2). They are present in both ixodid and argasid ticks. In D. variabilis, they each consist of a
narrow neck-like section and a broad, vermiform glandular region that may be folded. When
viewed with a transmission electron microscope, the proximal duct is found to contain a thin layer
of cuticle (0.3 μm) surrounding a subcircular lumen. The remainder of the duct wall in this region
consists of a thin layer of relatively inactive epithelial cells, each with a thick basal lamina. In the
glandular region, there is a multilayered epithelium of large secretory cells and stellate supporting
cells. In Ha. longicornis, the supporting cells were clearly shown to contain abundant microtubules
traversing the cytoplasm, indicating that these cells function for support and in maintaining the
shape of the glands during the release of secretions into the genital tract (Kakuda et al. 1994). The
supporting cells are relatively difficult to distinguish from the secretory cells in unfed females, but
they greatly increase in size after feeding. Masses of secretory vesicles fill the apical regions of the
secretory cells. Many secretory vesicles contain electron-dense material, but others are much less
dense or reticulate and lack granular material. The apical cell surfaces contain masses of microvilli
that protrude into and obscure the lumen. A thin connective tissue layer with few smooth muscle
cells surrounds the epithelial wall of the tubular accessory gland (TAG). The microvilli and muscle
cells might contribute to the smooth transport of the secretions into the lumen (Kakuda et al.
1994). According to Chinery (1965) and Balashov (1972), the TAG secretes colloidal material that
contains basic proteins. The nature of these secretions and their functions are still unknown.
2.8. VESTIBULAR VAGINA AND LAG

In D. variabilis, the vestibular vagina is an elongated, slightly rounded tube approximately 320 to
360 μm long (Fig. 17.1). The cuticular lining is folded into deep trabeculae forming a thickened
layer about 15 to 20 μm thick. A thin layer of epithelial cells surrounds the cuticle. Surrounding
the vestibular vagina is a glandular epithelium, the LAG, found only in ixodid ticks. Although
thin and undeveloped in the unfed female, the gland expands greatly during feeding as a result
of cell growth and cell proliferation. The LAG forms a bulbous trilobed gland about 150 μm thick
surrounding the vestibular vagina. Histological sections show extensive channels penetrating
into the glandular lobes and fusing with a sinus surrounding the vestibular vagina. The channels
and sinus appear to be filled with an amorphous material, evidently an unknown fluid secretion.
When examined via transmission electron microscope, the cells of the lobular accessory gland
in feeding virgins are found to have numerous mitochondria and extensive development of RER
and ribosomes. The luminal plasma membrane bears a layer of microvilli. The cell profiles at this
stage show features characteristic of secretory cells, although the nature of the secretory sub-
stances is unknown. Sonenshine et al. (1985) did not observe detachment of the LAG from
the vestibular vagina, contrary to Balashov’s (1972) description of separation of the LAG from
the vestibular vagina (the latter might have been an artifact of the histological procedures). The
function of the LAG is still not well known. According to Lees and Beament (1948), these struc-
tures produce a lipid-rich secretion that coats the eggs as they pass through the vagina during
oviposition. Sonenshine et al. (1985) considered the LAG as the most likely source of the genital
sex pheromone. Kakuda et al. (1997a) report a lack of epicuticular pores and an ultrastructure of
the LAGs that has the characteristics of transport epithelia. This indicates that the LAG might
secrete low-molecular-weight compounds that lubricate the vestibular vagina for the passage of
the eggs. Following repletion and preparation for oviposition, the lining of the vestibular vagina
softens, allowing it to expand so as to accommodate the large ovum that must pass through this
duct. As the ovum nears the genital pore, the vestibular vagina prolapses through the opening,
serving as an ovipositor to eject the eggs from the genital tract.
2.9. GENÉ’S ORGAN

An unusual paired organ found only in ticks (Arthur 1962), Gené’s organ is located in the cranio-
dorsal region of the podosoma immediately beneath the anterior part of the scutum (Fig. 17.4).
Studies on Gené’s organ in ixodid species have shown that the morphology and structural orga-
nization are basically the same for R. microplus (Booth et al. 1984; Booth 1989), Hy. dromedarii
(El Shoura 1987), Ha. longicornis (Kakuda et al. 1992), and D. reticulatus (Schöl et al. 2001b). It
consists of a double-sac structure with outer epithelial sacs (ES) and inner cuticular sacs (CS).
The ES and CS in unfed and ovipositing females are strictly separated, forming a lumen between
them. The camerostomal aperture is located between the posterior edge of the basis capituli and
the anterior edge of the scutum. In unfed ticks, Gené’s organ consists of a corpus, 2 posterior
horns, and finger-like undeveloped glands on each side; 4 glands are present in prostriate ticks
(Fig. 17.4A). In ovipositing ticks, the morphology and structural organization are basically the
same as in unfed ticks, but the corpus and horns are longer and broader, and the finger-like lobes
differentiate to form complex branched tubular glands that open into the lumen between the ES
and the CS (Fig. 17.4B). In unfed ticks, the organ is retracted within the anterior region of the
body near the capitulum, lying just below the dorsal cuticle (Fig. 17.4C), whereas in ovipositing
females the CS is evertable (Fig. 17.4D). The actual mechanism of eversion and retraction of the
CS during egg laying is still unknown, but it is probably due to an increase in hemolymph pres-
sure caused by the contraction of the dorso-ventral muscles. The simultaneous relaxation of the
retractor muscles originating from the scutum and inserting into the CS allows the CS to turn
inside out and pass through the camerostomal aperture to the exterior. Secretions are also
FIGURE 17.4: Diagram of Gené’s organ during female reproduction. Gené’s organ of unfed (A) and
ovipositing (B) Dermacentor reticulatus. After oviposition, Gené’s organ shows retraction (C) and eversion
of cuticular sac (D). A, aperture of Gené’s organ; AP, porose area; BC, basis capituli; C, corpus; CL,
cuticular layer; CS, cuticular sac; ES, epithelial sac; G, genial aperture; H, horn; L, lumen between epithelial
and cuticular sac; MD, main efferent duct of the glands; Pe, pedipalps; R, retractor muscles; S, scutum;TE,
tubular endpieces;TG, tubular glands; UG, undeveloped glands. Modifi ed from Schöl, H., Sieberz, J., Gobel,
E., and Gothe, R. (2001b) Morphology and structural organization of gene’s organ in Dermacentor reticulatus
(Acari: Ixodidae). Exp. Appl. Acarol. 25:327–352, with permission from Springer.
squeezed through the tubuli and pores of the pits to the surface of the everted CS by this same
increase in hemolymph pressure. The distribution of the pores corresponds with the surface area
that comes in contact with the eggs, allowing for the coating of eggs with secretions from the
long tubuli. The balloon-like everted CS also likely acts to grip the eggs as they are being ex-
pelled from the vestibular vagina, resulting in turning of the eggs so secretions can be com-
pletely spread over their surface. The coating of eggs with these secretions is indispensable,
because eggs without contact to the CS dry up and shrivel immediately after deposition. The
secretions are believed to be wax or wax precursors, but the chemical nature of the secretions
from both Gené’s organ and the glands of the porose areas are still unknown (Sieberz and Gothe
2000). The morphology of Gené’s organ in argasid species has almost exclusively been investi-
gated via light microscopy, with the anatomical or histological structure only briefly described
by schematic drawings. However, Schöl et al. (2001a) investigated the morphology and struc-
tural organization of Gené’s organ in Argas walkerae and showed that the structures and
mechanisms appear to be basically the same as in the ixodid ticks, but with some obvious
differences—for instance, circular cribrate pits, ledges, and cone-like structures that occur in the
CS of D. reticulates appear to be lacking in this argasid species. For a more detailed review of the
structures and functions of this unusual organ, the reader is referred to the work of Balashov
(1972), Lees and Beament (1948), Kakuda et al. (1992), and Schöl et al. (2001a, 2001b).
3. OOGENESIS
3.1. OOCYTE DEVELOPMENT

During tick oogenesis, the development of oocytes is accompanied by morphological and phys-
iological changes. The morphological changes were formally classified by Balashov (1972). In
recent years, a modified classification proposed by Denardi et al. (2004) is often used to define
oocyte development. Because tick ovaries are panoistic, there are no nurse and follicle cells sur-
rounding the oocytes, and the oocytes are attached to the ovarian wall by pedicel cells that face
the hemocoel before deposition into the ovarian lumen. The region between the pedicel cells
and oocytes contains an infolding of plasma membrane between the cells for the exchange of
cellular components (Denardi et al. 2004; Oliveira et al. 2007). Pedicel cells are proposed to play
a role similar to that of follicle or nurse cells of insect ovaries. Oocytes attached to the ovarian
walls are surrounded by the basal lamella, except in the region where the pedicel cells attach to
the ovarian wall. In tick oogenesis, the developing oocytes are primary oocytes that undergo
cytoplasmic changes during oocyte development. These cytoplasmic changes are classified into
5 stages (Stages I to V) based on the work of Denardi et al. (2004) (Fig. 17.5). From Stage I to
Stage III (and sometimes in Stages IV and V), oocytes face the hemocoel and are attached to the
ovarian wall by the pedicel. Oocytes are surrounded by the plasma membrane in all stages. The
plasma membrane (oolemma) lies on a thick basal lamina separated by 2 regions, a thicker re-
gion in direct contact with the plasma membrane of the oocytes and a thinner, more external
region. Oocytes include a germinal vesicle, the nucleus of the oocytes that fills more than half of
the oocyte cellular space. Organelles contained in the oocyte include mitochondria, GC, ribo-
somes, lamellar RER, and a small number of peroxisomes. At the initial stage (Stage I), the
oocytes are small and rounded or elliptical, and the cytoplasm of Stage I oocytes is homoge-
neous. In Stage II, the oocytes become larger with a thicker plasma membrane but are still
attached to the ovarian wall by the pedicel cell. In the cytoplasm, the germ vesicle is still ob-
served. The cytoplasm appears to contain fine dispersed yolk granulation when acid lipids are
stained with Nile blue (Denardi et al. 2004). From Stage III, oocytes clearly develop yolk gran-
ules and are defined as vitellogenic oocytes (Denardi et al. 2004). The plasma membrane appears
thick, and a chorion begins to form. In the cytoplasm of the Stage III oocyte, the secretion gran-
ules (yolk granules) are smaller than in Stage IV, and germ granules are difficult to detect. In
Stage IV, oocyte cytoplasm includes numerous yolk granules of various sizes. In the peripheral
region of the oocytes, the largest granules appear, whereas small granules appear in the central
region. A large number of electron-dense protein granules also appear in the central region of
these oocytes. This central region also contains numerous GC and a large amount of endoplas-
mic reticulum, indicating that the oocyte itself synthesizes these components. However, the
germ vesicle is no longer observed and the chorion is nearly mature. The Stage IV oocytes are
deposited into the ovarian lumen. Stage V oocytes have reached a maximum size and are filled
with large, dense yolk granules. The chorion becomes thick with 2 distinct layers. The chorion
has a micropyle-like structure to facilitate spermiophore penetration (see Section 3.2, “Chorio-
genesis”) that may appear around ovulation. The final-stage oocytes are fertilized in the ovary or
oviduct and then oviposited. The above scheme of oocyte development defined in Ambylomma
cajennense by Denardi et al. (2004) has also been used in recent years to describe oocyte devel-
opment in R. sanguineus (Oliveira et al. 2005), R. microplus (Saito et al. 2005), A. triste (Oliveira
et al. 2006), A. brasilliense (Sanches et al. 2010), and A. rotundatum (Sanches et al. 2012).
Tick oogenesis progresses after blood feeding in nymphs and adult females. If normal blood
feeding or mating is disrupted, inhibition of oogenesis and reabsorption of yolk from the oocytes
occurs (Connat et al. 1986). Tick oogonia appear in the germinal primordium of the larval stage,
FIGURE 17.5: Development of oocytes in Amblyomma cajennense tick ovaries. Modifi ed from Denardi,
S.E., Bechara, G.H., Oliveira, P.R., Nunes, E.T., Saito, K.C., and Mathias, M.I. (2004) Morphological
characterization of the ovary and vitellogenesis dynamics in the tick Amblyomma cajennense (Acari:
Ixodidae). Vet. Parasitol. 125:379–395, with permission from Elsevier.
and transformation of the oogonia into primary oocytes occurs after the last nymphal blood
meal. Subsequently, the development of primary oocytes is arrested until female feeding in most
ticks. Many soft tick species finish blood feeding within several hours or less, so oocyte develop-
ment occurs after detachment from the host animal. Hard tick species require several days of
feeding, and oocyte development proceeds during blood feeding. Oocyte development in hard
ticks is correlated with the status of blood feeding and mating. The primary oocytes of feeding
females become larger (Stage II or previtellogenic oocytes) soon after the attachment of female
hard ticks. Oocyte growth does not proceed uniformly, so that the ovary appears studded with
spherical or ovoid ova of varying sizes. In D. andersoni, the previtellogenic oocytes are large cells,
spherical or subspherical in appearance and 49 to 60 μm in diameter, with large nuclei. Following
mating, the oocytes progress to the vitellogenic stages (Stages III and IV).
In the hard tick, all of the changes described below occur following female repletion and
dropping from the host. The main component of yolk is vitellin (Vn), a large hemoglycolipo-
phosphoprotein. The precursor of Vn, vitellogenin (Vg), is synthesized mainly in extraovarian
sites such as the fat body and midgut (see Section 4, “Vitellogenesis,” for more detail) and taken
into the oocytes via endocytosis. At the vitellogenic stage, microvilli developing on the oocyte
surface provide an enlarged surface area for protein transport. The pinocytotic vesicles fuse into
larger bodies and eventually into massive homogeneous yolk granules. Once inside the oocytes,
Vg is deposited directly into developing yolk bodies instead of being processed through lyso-
somes (Coons et al. 1989). Vesicles including the yolk protein fuse and form huge yolk granules.
Lipid droplets and glycogen granules are also stored in the cytoplasm between the yolk granules.
During the course of these events, the maturing oocyte enlarges into a huge cell (600 to more
than 1,200 μm in some tick species) with a large nucleus and nucleolus. In hard ticks, mating
appears to provide the signal for repletion and initiates vitellogenic oocyte development. In
some argasid ticks, the blood meal is a stimulus for the onset of vitellogenesis, but feeding alone
is not sufficient for completion of the gonotrophic cycle. Seminal fluid is believed to contain
unknown substances that initiate oogenesis in some argasid and ixodid ticks (Oliver 1986; Weiss
and Kaufman 2004; Donohue et al. 2009a).
The oocyte enters Stage IV, the period of maximum growth, after mating. During this period,
the nuclear and nucleoli membranes disintegrate, and the chromatin coalesces into a karyosphere
(Diehl et al. 1982a). The mature, vitellogenic oocytes expand into the hemocoel (Stage IV
oocytes) and are soon ovulated, passing into the ovarian lumen (Stage V oocytes). Once in the
lumen of the ovary or in the oviducts, the spermiophores (i.e., mature spermatozoa) penetrate
into the ova, but syngamy is delayed and the male and female nuclei remain apart. The binucleate
oocytes pass through the reproductive tract and undergo the first meiotic divisions to become
secondary oocytes. Oogenesis is completed after oviposition, when the final reduction division
(second meiotic division) takes place and the nuclei fuse (syngamy) (Balashov 1972; Diehl et al.
1982a). This in situ oocyte development of ticks is greatly different from the insect system.
3.2. CHORIOGENESIS
The egg shell (Vn envelope) is also synthesized during oocyte maturation. The deposition of
cuticular materials for the egg shell occurs initially around the base of the microvilli. Ultrastruc-
tural studies indicate that the egg shell is formed by the oocyte itself, rather than by other ovarian
cells or extra-ovarian tissues (Brinton and Oliver 1971a; Yano et al. 1989). Egg shell formation
accelerates greatly in vitellogenic oocytes (Stage III) and is completed by the end of Stage IV.
Shell precursors are formed in vesicles budded from the GC, passed to the plasma membrane,
and expelled via exocytosis (Diehl et al. 1982a). These precursors polymerize in the extracellular
space below the basal lamina to form a homogeneous layer. This secretion occurs around the
numerous microvilli on the oocyte cell surface so that narrow channels extend across the shell
envelope as it develops. When completed, the shell is 2 to 4 μm thick.
During maturation, the egg shell is believed to be permeable to most substances, so proteins
as large as Vg can enter the oocytes by means of direct passage. Although tick eggs lack a dis-
tinct micropyle (Diehl et al. 1982a), a micropyle-like zone in the oocyte surface has been re-
ported (Aeschlimann and Hecker 1967; Brinton and Oliver 1971a; El Shoura et al. 1989;
Montasser 2010). At the vitellogenic stages of oocytes, the nucleus migrates to a position adja-
cent to the pedicel cell (Diehl et al. 1982a; Saito et al. 2005). The vicinity of the pedicel cell in the
oocytes seems to be a micropyle, and fertilization (i.e., sperm penetration) likely occurs when
sperm penetrate into the ovary at this location. The shell is thinner in the vicinity of the pedicel
cell than elsewhere. The early development of the egg shell has important implications re-
garding the penetration of tick-borne pathogens. Transovarial infections are reported from
various species and pathogens, and transovarial passage induces high infection rates in off-
spring, as reported with Borrelia burgdorferi in I. pacificus (Lane and Burgdorfer 1987) and
Rickettsia rickettsii in R. sanquineus (Silva Costa et al. 2011). Although evidence is limited, it
appears that only the less developed oocytes become infected by pathogens such as B. duttoni,
Coxiella burnetu, R. rickettsii, and Babesia major (reviewed by Diehl et al. 1982a). See Volume 2
for further information on tick-borne diseases.
3.3. OVULATION AND FERTILIZATION

Ovulation begins when the mature oocyte passes into the ovarian lumen. The pedicel cells en-
large during oocyte maturation and appear to communicate with the oocytes. Subsequently, the
oocyte is ejected via the pedicel into the ovarian lumen. The mechanics of the ovulation process
are obscure. Once in the lumen, peristaltic activity of the ovary increases pressure against the
ovarian wall, forcing the oocytes into the oviducts (El Said 1992), from which they pass to the
uterus and vagina. The walls of the oviducts become greatly extended following mating and re-
pletion, and the cells fill with masses of secretory material of unknown function. These se-
cretions are thought to facilitate dissolution of the spermatophore and subsequent sperm
capacitation (Diehl et al. 1982a). The oocytes accumulate in great numbers in the coils of the
oviduct and the distended uterus. The reproductive tract, including the lumen of the ovary, is
also filled with masses of spermatozoa. As noted previously, sperm penetration is believed to
occur soon after ovulation, although the actual site of this crucial event is unknown. According
to Balashov (1972), sperm penetration occurs in the anterior part of the oviducts in ixodid ticks
or in the ampullate regions of the oviducts in argasid ticks. However, there is considerable con-
troversy regarding the site where the spermiophores enter the oocytes. Studies by Brinton and
Oliver (1971a) and others suggest that it occurs in the ovary or while the oocytes are still attached
to the wall of the ovary (i.e., the spermiophores pass into the pedicel to enter the eggs). Histolog-
ical analysis of Ha. longicornis showed that spermiophores appear in the ovarian lumen and the
pedicel cells (Yano et al. 1989), confirming that the sperm encounters the oocytes in the ovary.
It is believed that the sperm penetrates the oocytes by dissolving a portion of the egg shell at the
delicate micropyle-like zone. Most elongate spermiophores disintegrate during this process, and
the sperm nucleus penetrates into the oocytes. Only the sperm nuclei penetrate, and most of the
other parts of the spermatozoa remain outside of the ovum and are digested. This sketchy de-
scription illustrates the limited extent of our knowledge on the important events of fertilization
and the need for additional investigations.
Following entry of the sperm nucleus, the shell of the oocyte hardens. Within the oocyte,
meiotic divisions occur, producing the haploid pronucleus. However, fusion of the male and
female pronuclei does not occur until after oviposition, which terminates the period of oogen-
esis. Embryogenesis progresses outside the female body (see Chapter 3).
3.4. OVIPOSITION
The time from the drop-off of fed mated females to the commencement of oviposition consti-
tutes the preoviposition stage. During this period, which may last from only 3 or 4 days to as long
as several weeks, dramatic changes occur within the female genital tract as a female prepares for
oviposition. Fertilized oocytes in which syngamy has not yet occurred pass from the oviducts via
the uterus and the expanded connecting tube into the vagina. The cuticular lining of the vestib-
ular vagina has innumerable trabeculae that might facilitate this expansion and enable the pas-
sage of the large eggs (Sonenshine et al. 1985). Peristaltic contractions of the muscular cervical
vagina propel the eggs into the vestibular vagina. As the eggs pass through the vagina, secretions
from the swollen LAGs are deposited onto the shell. The lobular glands in fed mated females
show swelling and well-developed organelles, probably for protein synthesis, but the swelling
and organelles are not seen in unfed and fed virgin females of Ha. longicornis (Kakuda et al.
1994). Unfortunately, nothing is known about the chemistry of these secretions and the roles, if
any, they play in protecting the passing ovum. The cuticle surrounding the vestibular vagina and
the genital aperture also softens considerably during the preoviposition period. With an egg
within its cavity, the softened, flexible vestibular vagina expands and prolapses slightly through
the genital pore, thereby acting as an ovipositor to deposit the egg (a true ovipositor is absent in
ticks). Once outside the genital tract, the eggs are captured by the mouthparts and Gené’s organ.
Gené’s organ has a double-sac structure with an outer epithelial and an inner cuticular sac, and
it everts the cuticular sacs through the camerostomal aperture to coat eggs with its components
(see Section 2.9, “Gené’s Organ,” as well as Figs. 17.4 and 17.6) (Schöl et al. 2001a, 2001b).
Gené’s organ develops after blood feeding and synthesizes components including a mixture
of long chain hydrocarbons, wax esters, fatty acids, and steroids (a detailed list is given by Diehl
et al. [1982a]). Following wax deposition, Gené’s organ is retracted, the capitulum is extended
to its normal anterior position, and the egg is projected posteriorly and dorsally, where it comes
to lie above the basis capituli. The process of gland protrusion, waxing, and retraction is re-
peated up to several hundred times per day until the female appears to be embedded in the egg
mass. In ixodids, antioxidants from the PAs on the dorsal surface of the capitulum (absent in
argasid ticks) are mixed with these waxy secretions and inhibit degradation of the unsaturated
lipids in the secretions from Gené’s organ (Atkinson and Binnington 1973). Oocytes without
coating from Gené’s organ show considerably decreased hatching rates, but paraffin coating
partially restores the hatching rates (Kakuda et al. 1992). This indicates that these secretions
provide the primary protection against desiccation for the ova. Another function ascribed to
the PA secretions is to lubricate the movements of Gené’s organ, facilitating its normal eversion
FIGURE 17.6: Major stages of oviposition in a representative ixodid tick. A, Flexing of capitulum to
receive emerging egg. B, Eversion of Gené’s organ and prolapse of vagina through the genital pore.
C, Emergence of egg and its capture by the horns of Gené’s organ. D, Passage of egg above Gene’s
organ and capitulum. E., egg; G.O., Gené’s organ; Ga., genital aperture; Vag., vagina prolapsed through
the genital pore. Figure from Sonenshine, D.E. (1991) Biology of Ticks, Vol. 1. New York: Oxford University
Press, 280–304.
and retraction. If the PAs are obstructed or ablated, Gené’s organ does not function properly
(Feldman-Muhsam 1963). In addition to protecting the eggs from desiccation, the egg wax is
important for protecting eggs from microorganisms. Antimicrobial activity was reported in the
egg wax of A. hebraeum and increased in Gené’s organ at the time of oviposition (Arrieta et al.
2006). In addition, an antimicrobial peptide was detected from the genital tract and egg surface
of R. microplus (Esteves et al. 2009), indicating that ticks have several sources of antimicrobial
molecules to protect their eggs.
4. VITELLOGENESIS
4.1. CHARACTERIZATION OF Vg
Tick eggs include large amounts of yolk proteins, and the major component of these yolk
proteins is Vn. Vn is synthesized from its precursor, Vg. Characteristics of Vg have been re-
ported from numerous ticks in the past and, more recently, with the identification of Vg genes.
The discovery of large hemoglycolipoproteins in both eggs and hemolymph of female ticks was
the subject of the first reports on vitellogenesis. Vn and its precursor Vg are included in this
category of proteins and incorporate lipids, sugars, and heme molecules into their structure. The
main conjugated sugar is mannose, and the key lipid is triacylglycerol (Chinzei and Taylor 1990).
An interesting feature of tick Vg is the inclusion of heme. Tick Vgs can bind over 30 heme mol-
ecules in vitro, but the number of heme molecules bound in vivo is lower (Logullo et al. 2002).
Embryos require heme for development, but heme itself can cause heme-induced lipid peroxi-
dation, so the heme-binding capacity of Vn/Vg is thought to be important in balancing the
availability of heme for embryonic development without causing heme toxicity (Logullo et al.
2002). The color of heme contributes to the amber color of tick eggs. Native Vgs are synthesized
in different tissues and secreted into the hemolymph so that large quantities of Vgs tend to be
found in the hemolymph of vitellogenic females. However, some species, such as metastriate
ixodid ticks, rapidly incorporate Vg proteins into the oocytes, so Vg concentrations in the he-
molymph are not large in those ticks. In the hemolymph of O. moubata, 2 different sizes of Vgs
are detected, 300 kDa and 600 kDa (Chinzei et al. 1983). The larger protein is similar in size to
Vn extracted from eggs and is thought to be a dimer of 2 identical 300 kDa Vg molecules.
Dimerization of Vg also has been reported in several other species of ticks (reviewed in Taylor
and Chinzei 2002). In addition, Vg molecules are composed of several subunits. The size and
number of subunits differ between tick species (Table 17.1). Vgs are synthesized in secreting
tissues, expelled into the hemolymph, and finally taken up by oocytes as a nutritional source for
embryo development. During this process, the precursor Vg molecules are cleaved into subunits
that combine to form the mature Vn proteins that are stored in the eggs as a nutrient source.
In recent years, the identification of complete sequence and molecular structure Vg messages
from several ticks has greatly increased our understanding of tick vitellogenesis. The full sequences
of tick Vg messages were described for the first time from the hard tick D. variabilis (DvVg1)
(Thompson et al. 2007) and next for the soft tick O. moubata (Horigane et al. 2010). A second Vg
gene from D. variabilis (DsVg2) was recently reported (Khalil et al. 2011). In Ha. longicornis, 3 Vgs
were identified (HlVg1–3) (Boldbaatar et al. 2010), but HlVg2 has also been classified as a carrier
protein (CP), and HlVg3 has sequences more similar to CPs than Vg (Khalil et al. 2011; discussed
below). In addition, partial Vg sequences have been reported from A. americanum (Bior et al.
2002) and R. microplus (GP80) (Tellam et al. 2002). Vg genes were also described for the mite
Tetranychus urticae (Kawakami et al. 2009). Tick Vg genes have several conserved motifs that are
similar to those that occur in insect Vgs. Vgs have signal peptide sequences in the N-terminus
composed of 15 to 22 amino acid residues (Thompson et al. 2007; Boldbaatar et al. 2010; Horigane
et al. 2010; Khalil et al. 2011) that enable secretion from the cite of synthesis into the hemolymph.
In addition, tick Vgs contain an N-terminal lipoprotein domain common to most arthropod Vg
genes that follows the N-terminus signal peptides. Tick Vg genes also have a DUF1943 domain, of
which the function is unknown, in the middle region of the gene (over 600 aa residues). In the
C-terminus, the von Willebrand factor type D domain and GL/ICG motifs are encoded. The GL/
ICG motif is common in arthropod Vgs, and this site is often used for Vg gene isolation (Lee et al.
2000). Both hard and soft tick Vgs, as well as mite Vgs, have this conserved region.
The sizes of the Vg genes differ between species and encode 1,400 to 1,900 amino acid resi-
dues (Table 17.1). The predicted weight of these Vgs is approximately 200 kDa, and this matches
the molecular weights of tick Vg and Vn proteins as determined previously by proteomics (Table
17.1). Mature Vgs are composed of several subunits and often form a dimer in the hemolymph
and oocytes. Tick Vg genes are thought to contain several cleavage sites for the formation of
these subunits. The RXXR motif is a conserved cleavage site that is cut by peptidases. Analyses
of tick Vgs reveal they have several RXXR sequences (Thompson et al. 2007; Boldbaatar et al.
Table 17.1: Summary of vitellogenin (Vg) and vitellin (Vn) protein subunits and gene sequences reported from the Acari.
Species Molecular weights of protein References of Number of Vg Accession number Length of Vg Speculated References for Vg
subunits (kDa) proteins sequencesa of Vg sequenceb sequence (nt) molecular weight sequences
(kDa)c
Hard tick
A. americanum Vg 1 BI273562d Bior et al. (2002)
Vn
A. hermanni Vg 170, 150, 116, 108, 78, 66, Shanbaky et al.
16.5, 14.4 (1990)
Vn 170, 108, 98.5, 90, 78, 74, Shanbaky et al.
25, 16.5, 14.4 (1990)
D. andersoni Vg 305, 210, 175, 110, 101, Schriefer (1991)
51, 44
Vn 210, 175, 110, 101, 51, 44 Schriefer (1991)
464
D. variabilis Vg 200, 170, 155, 98, 93, 80, Rosell-Davis and 2 DvVg1 1,844 206.4 Thompson et al.
67, 50, Coons (1989) (AY885250) 1,925 207.2 (2007)
or 7 subunits Sullivan et al. DvVg2 Khalil et al. (2011)
(22–215 kDa)e (1999) (EU204907)
VnA 135, 110, 98, 80, 67, 50,
45, 35 Rosell and Coons
VnB 135, 110, 98, 93, 80, 67, (1991)
50, 45, 35
H. dromedarii Vg 326, 310, 212, 169, 114, Schriefer (1991)
101, 85, 71, 56, 41, 35
Vn 212, 169, 114, 101, 85, 71, Schriefer (1991)
56, 35
H. longicornis Vg 3 HlVg1 1,694 194.6 Boldbaatar et al.
Vn (AB359899) 1,545 174.9 (2010)
HlVg2 1,463 163.2 Boldbaatar et al.
(AB359901) (2010)
HlVg3 Boldbaatar et al.
(AB359902) (2010)
I. scapularis Vg 8 subunits (48 to 145 James and Oliver 2 XM_002415179d Genome
kDa)f (1999) XM_002403922d annotation
Genome
annotation
Vn 154, 135, 87, 78, 67, 64, 35 James and Oliver
(1997)
R. microplus Vg 1 GP80 (U49934)d,g Tellam et al.
(2002)
Vn ~200, 107, 102, 87, 67, 65, Tellam et al.
44, 35, 33, 18 (2002)
465
Soft tick
O. moubata Vg 215, 210, 160, 140, 125, 100 Chinzei et al. 1 AB440159 1,834 203.6 Horigane et al.
(1983) (2010)
Vn 160, 140, 125, 100, Chinzei et al.
64, 50 (1983)
O. parkeri Vg 215, 210, 160, 140, 125, Taylor et al. (1991)
100, 64
Vn 160, 140, 125, 100, 50g Taylor et al. (1991)
(continued)
Species Molecular weights of protein References of Number of Vg Accession number Length of Vg Speculated References for Vg
subunits (kDa) proteins sequencesa of Vg sequenceb sequence (nt) molecular weight sequences
(kDa)c
Mite
T. urticae Vg 4 TuVg1 Kawakami et al.
Vg (AB455063)d (2009)
TuVg2 Kawakami et al.
(AB455064)d (2009)
(AB455065)d (2009)
(AB455066)d (2009)
466
a
Number of Vg sequences (including partial sequences of Vg mRNA) found in public database.
b
Accession numbers from GenBank.
c
Speculated weight calculated from native weight without signal peptides.
d
Partial sequence(s).
e
Molecular weight range of Vg subunits for D. variabilis described by Sullivan et al. (1999) (specific band sizes not available).
f
Molecular weight range of Vg subunits for I. scapularis described by James and Oliver (1999) (specific band sizes not available).
g
Sequence (EU086096) shorter than U49934 also reported.
2010; Horigane et al. 2010). In addition, the KXXK motif, a cleavage site reported in bean bugs,
occurs as a cleavage site in Ha. longicornis (Boldbaatar et al. 2010). Most tick Vgs are composed
of 6 to 8 subunits, so these cleavage sites appear important for post-translational processing of
Vg to the respective subunits for the formation of the mature Vn proteins. Processing occurs via
proteolysis, and sequential modification may occur in the organ of synthesis, the hemolymph,
and the oocytes. Further studies are needed to clarify the actually processing of Vg into mature
Vn proteins and to determine where it occurs.
As mentioned above, some species have several Vg genes, but few genes have been com-
pletely characterized. Some Vg genes show structures similar to those of storage proteins (CP)
that have the same origin as Vg genes (Donohue et al. 2009b; Khalil et al. 2011; see Chapter 15).
Vgs show low homology between species and variations in the length and number of cleavage
sites. Khalil et al. (2011) report that HlVg2 of Ha. longicornis has sequences conserved in CPs and
propose that this is a CP (not Vg) gene, and HlVg3 also appears to be more similar to CP than
Vg. CP proteins are usually common in all stages of ticks, whereas Vgs are female specific and
always associated with Vg deposition for egg production. HlVg2 expression is seen only in fe-
male ticks, with the exception of very low expression in male ticks immediately after blood
feeding (Boldbaatar et al. 2010). The HlVg3 gene is similarly expressed at only low levels in
males. In addition, Boldbaatar et al. (2010) showed that HlVg2 and HlVg3 knockdown with
RNAi lowered yolk accumulation in the oocytes of Ha. longicornis, indicating these HlVgs also
function in oocyte formation and as yolk proteins in this species. Further studies on the func-
tional analysis of the Vg and CP genes of hard ticks are needed for better understanding of the
different roles of these genes in tick vitellogenesis and egg production.
4.2. SITE OF Vg SYNTHESIS

There appear to be multiple sites of Vg synthesis in ticks. Vg proteins have been observed in the
trophocytes of the fat body (see Chapter 12; Araman 1979) and basophilic cells in the midgut
epithelium (see Chapter 6; Coons et al. 1982, 1986, 1989; Agbede et al. 1986). Vg mRNA expres-
sion has been reported in the midgut and fat bodies of D. variabilis and Ha. longicornis (Thomp-
son et al. 2007; Boldbaatar et al. 2010). These studies indicate that the midgut and fat body are
sites of Vg expression and synthesis in hard ticks. However, in addition to these 2 sources of Vg,
ovarian expression of Vg mRNA has also been reported (Thompson et al. 2007; Boldbaatar et al.
2010). HlVg1 and HlVg3 of Ha. longicornis are expressed in the fat body and midgut, respectively,
but HlVg2 expression was observed in the ovary in real-time polymerase chain reaction (RT-
PCR) and Western blot studies (Boldbaatar et al. 2010; see Chapter 15). Weak Vg expression in the
ovary of vitellogenic females was also reported for D. variabilis (Thompson et al. 2007). Ovarian
Vg expression and synthesis also have been reported in insects, and the follicle or nurse cells of
the egg chamber are sources of this Vg (Brennan et al. 1982; Isaac and Bownes 1982; Melo et al.
2000). Similarly Vg synthesis was also reported in the crustacean Panaeus japonicas (Tsutsui et al.
2000). The tick ovary is panoistic and does not have functional follicle or nurse cells, but several
authors suggest that the pedicel cells or oocytes might synthesize Vg (Diehl et al. 1982a). More
careful analyses are needed to confirm the ovary as a site for the synthesis of Vgs in hard ticks.
The site of Vg protein synthesis in soft ticks was reported to be the fat body because only the
fat body showed Vg synthesis during in vitro culture (Chinzei and Yano 1985). However, Vg
proteins were detected in the midgut of O. parkeri (Taylor et al. 1991), and mRNA expression was
FIGURE 17.7: Vitellogenin (Vg) mRNA expression in fed females of the argasid tick Ornithodoros moubata.
Stained (black) areas indicate Vg expression detected via whole mount in situ hybridization. A, Vg expression
in the midgut of a mated female after engorgement. B, Vg expression in the enlarged fat bodies associated
with the trachea of a mated female after engorgement. C, Vg mRNA expression in the fat body associated
with the ovary and uterus of mated females after engorgement. D, magnification of the region marked by the
asterisk in C showing that the stained fibrous tissues are fat body. Ov: ovary; Ut: uterus. Scale bar: 1 mm.
Modified from Horigane, M., Shinoda,T., Honda, H., andTaylor, D. (2010) Characterization of a vitellogenin
gene reveals two phase regulation of vitellogenesis by engorgement and mating in the soft tick Ornithodoros
moubata (Acari: Argasidae). Insect Mol. Biol. 19:501–515, with permission from John Wiley & Sons.
detected in the midgut of O. moubata (Fig. 17.7) (Horigane et al. 2010), indicating that the
midgut also contributes to Vg synthesis in soft ticks. Vg messenger expression was not detected
in ovarian cells of O. moubata via whole mount in situ hybridization (Fig. 17.7) (Horigane 2010;
Horigane et al. 2010). Interestingly, the timing of Vg mRNA expression is different depending on
tissue and mating status. In O. moubata, strong Vg mRNA expression first appears in the midgut
and fat body associated with the more centrally located trachea of both mated and virgin fe-
males (Horigane et al. 2010). As time progresses, the fat bodies enlarge and show strong expres-
sion of Vg throughout the body in only mated fed females; this enlargement and expression of
Vg is not observed in virgin females. Vg expression and synthesis in distinct tissues might be
under different regulatory mechanisms (discussed below). The identification of Vg genes from
other species of both hard and soft ticks, as well as from other mite species, is necessary in order
to clarify the similarities and differences in Vg synthesis between the different groups of acari.
4.3. Vg SYNTHESIS
The synthesis of Vg differs among tick species, but nutritional conditions and mating status are
important keys for opening the vitellogenesis gates. Although the roles of nutrition and mating
in the coordination of tick vitellogenesis are difficult to separate, numerous studies indicate that
both nutrition and mating are essential to drive the regulatory mechanisms. The first key for the
regulation of tick vitellogenesis is nutritional, as shown in several tick species of both hard and
soft ticks. Ticks require blood feeding that allows them to surpass a threshold weight (critical
weight). In A. hebraeum, the critical weight is approximately 12-fold the unfed weight (Weiss
and Kaufman 2001). A. hebraeum mated females that surpass this critical weight carry out vitel-
logenesis and oocyte development, whereas females under the critical weight are unable to pro-
duce Vg and eggs (Weiss and Kaufman 2001). Although A. hebraeum requires mating for egg
production, some virgin females can exceed the critical weight with a long period of feeding
(Kaufman and Lomas 1996) and show Vg synthesis and oocyte development (Friesen and
Kaufman 2009). In D. variabilis, Vg appears after blood feeding (Thompson et al. 2005, 2007;
Khalil et al. 2011) even in females that are prematurely knocked of the host and have not fully
engorged. The expression of Vg without mating in both parthenogenetic and bisexual strains of
Ha. longicornis also indicates that stimulation by nutrition is essential for egg production (Bold-
baatar et al. 2010). The soft tick O. moubata can engorge whether it has mated or not and shows
Vg expression and synthesis in both mated and virgin females after engorgement (Figs. 17.8,
17.9) (Ogihara et al. 2007; Horigane 2010; Horigane et al. 2010). Therefore, obtaining sufficient
nutrition is the first essential key for tick reproduction.
The regulation of Vg synthesis by nutritional signals has been reported in the blood-feeding
mosquito Aedes aegypti (Park et al. 2006), and similar mechanisms for the regulation of Vg
transcription are reported for Ha. longicornis (Boldbaatar et al. 2010; Umemiya-Shirafuji et al.
2012). A transcriptional factor GATA and an S6 kinase (S6K) are components of the target of the
rapamycin (TOR) nutritional responsive signaling pathway. RNAi of GATA and S6K results in
low Vg expression and synthesis (Boldbaatar et al. 2010), and the inhibition of TOR by RNAi
also reduces Vg expression and egg production in Ha. longicornis (Umemiya-Shirafuji et al.
2012). These studies indicate that a nutritional signaling pathway, likely the TOR pathway,
is essential for the regulation of Vg transcription in ticks. In addition, factors transferred to
females during mating and insemination, such as male engorgement factors, also appear to be
necessary to stimulate engorgement in some species of hard ticks (Weiss and Kaufman 2004;
Donohue et al. 2009a). These studies indicate that mating in many hard ticks is important as a
check point for critical weight leading to sufficient Vg synthesis. Further studies are needed to
determine the importance of nutrition and the connection of these regulatory mechanisms to
mating in the direct stimulation of vitellogenesis in hard and soft ticks.
Although engorgement to an excess critical weight is required for hard tick vitellogenesis,
mating is the second important key necessary for sufficient Vg synthesis for the formation of
mature eggs leading to the production of offspring. However, the mechanisms by which mating
regulates vitellogenesis may differ between hard and soft ticks, and also between species. In most
hard ticks, mating is required for complete engorgement. For example, factors transferred from
males to females during mating and insemination are associated with engorgement in D. varia-
bilis (Thompson et al. 2005, 2007; Khalil et al. 2011). Detailed comparisons of mated and virgin
female hard ticks have not been carried out to date and are needed to elucidate the roles of
mating in the vitellogenesis of hard ticks.
Soft ticks can mate before and after blood feeding, and a host blood meal is unnecessary for
mating. Vg expression and synthesis also are different than in the hard ticks: both virgin and
mated females express and synthesize Vg when fully fed. Soft ticks provide an excellent model
for studies on the separate roles of engorgement and mating in the regulation of reproduction.
In the soft tick O. moubata, mated females show high levels of Vg expression and Vg protein
FIGURE 17.8: Relative Vitellogenin (OmVg) mRNA expression in mated and virgin females of
Ornithodoros moubata. A, Relative Vg mRNA expression in mated and virgin females from unfed
condition (U) to 20 days after engorgement. B, Effect of mating on Vg expression in females 21 days
after engorgement. Females 21 days after engorgement (D21) were mated or unmated (control), and
Vg mRNA expression was determined at 1 and 3 days after mating (equal to 22 and 24 days after
engorgement), when virgin females normally show low levels of Vg mRNA expression. Modifi ed from
Horigane, M., Shinoda, T., Honda, H., and Taylor, D. (2010) Characterization of a vitellogenin gene
reveals two phase regulation of vitellogenesis by engorgement and mating in the soft tick
Ornithodoros moubata (Acari: Argasidae). Insect Mol. Biol. 19:501–515, with permission from John
Wiley & Sons.
titers (Figs. 17.8, 17.9) (Ogihara et al. 2007; Horigane et al. 2010). High levels of Vg expression are
observed from 4 days to at least 20 days after engorgement in mated females, and expression
continues even after oviposition begins on approximately day 10 after engorgement. Virgin fe-
males of O. moubata initiate Vg expression similar to mated females at the onset of vitellogen-
esis, but the expression levels decrease with time, and virgin females do not produce mature
oocytes or oviposit eggs (Figs. 17.8A, 17.10). However, if virgin females of O. moubata are mated
even 22 days after feeding, Vg expression resumes, and they can oviposit mature eggs (Fig.
17.8B). Mated and virgin O. moubata females likely show differences in Vg mRNA expression
FIGURE 17.9: Summary of factors regulating vitellogenin (Vg) synthesis and expression in virgin and
mated Ornithodoros moubata females. Ecdysteroid and Vg titers in the hemolymph and ovarian
development were modifi ed from Ogihara, K., Horigane, M., Nakajima, Y., Moribayashi, A., and Taylor,
D. (2007) Ecdysteroid hormone titer and its relationship to vitellogenesis in the soft tick, Ornithodoros
moubata (Acari: Argasidae). Gen. Comp. Endocrinol. 150:371–380, with permission from Elsevier.
Ecdysteroid receptor (OmEcR) and retinoid X receptor (OmRXR) of O. moubata expression patterns
were modifi ed from Horigane, M., Ogihara, K., Nakajima, Y., Shinoda, T., and Taylor, D. (2007) Cloning and
expression of the ecdysteroid receptor during ecdysis and reproduction in females of the soft tick,
Ornithodoros moubata (Acari: Argasidae). Insect Mol. Biol. 16:601–612; and Horigane, M., Ogihara,
K., Nakajima, Y., and Taylor, D. (2008) Isolation and expression of the retinoid X receptor from last instar
nymphs and adult females of the soft tick Ornithodoros moubata (Acari: Argasidae). Gen. Comp.
Endocrinol. 156:298–311, with permission from John Wiley & Sons and Elsevier, respectively. Vitellogenin
of O. moubata (OmVg) expression was modifi ed from Horigane, M., Shinoda, T., Honda, H., and Taylor,
D. (2010) Characterization of a vitellogenin gene reveals two phase regulation of vitellogenesis by
engorgement and mating in the soft tick Ornithodoros moubata (Acari: Argasidae). Insect Mol. Biol.
19:501–515, with permission from Elsevier. Solid lines represent mated females, and dashed lines
represent virgin females. U, unfed; E, engorgement.
and synthesis because these processes are regulated by vitellogenin-inducing factors (VIF, dis-
cussed below) secreted from the synganglion after mating (Taylor and Chinzei 2002). Uptake of
sufficient nutrition by blood feeding (this might be equivalent to excess critical weight in hard
ticks) functions as the first check point for Vg synthesis, and mating subsequently upregulates
Vg expression and synthesis to levels sufficient for the production of mature eggs.
Tick vitellogenesis is under hormonal regulation, and the key regulatory hormone is thought
to be an ecdysteroid. Vitellogenesis differs between hard and soft ticks, but the hormonal regu-
lation is thought to be similar in both families of ticks. Early studies reported that another im-
portant insect hormone that regulates vitellogenesis in many insects, juvenile hormone (JH),
FIGURE 17.10: Two-phase model of vitellogenesis (Vg) in the soft tick Ornithodoros moubata. Both
mated and virgin females of O. moubata showed Vg mRNA expression (in early phase) in the midgut
(MG) and fat body (FB) associated with the main trachea (T) expanded from spiracles. Both mated and
virgin females have this phase, so it might be dependent on nutrition via the target of rapamycin (TOR)
pathway. Following increased Vg expression in the early phase, a second phase induced by mating
progresses only in mated females. Only mated females showed high ecdysteroid (E) synthesis/
secretion and enlarged peripheral fat bodies throughout the body cavity with Vg expression during the
late phase. Modifi ed from Horigane, M., Shinoda, T., Honda, H., and Taylor, D. (2010) Characterization of
a vitellogenin gene reveals two phase regulation of vitellogenesis by engorgement and mating in the
soft tick Ornithodoros moubata (Acari: Argasidae). Insect Mol. Biol. 19:501–515, with permission from
John Wiley & Sons.
also regulates Vg synthesis and egg production in ticks (Pound and Oliver 1979). However, no
direct evidence of JH or its immediate precursors has been found, even with extensive studies in
several species and stages of ticks (Neese et al. 2000). This indicates that JH is unlikely to func-
tion in the regulation of vitellogenesis in ticks (see Chapter 16). Ecdysteroids have been identi-
fied and shown to increase in fed females of several tick species (Dees et al. 1984; Kaufman 1991;
James et al. 1997; Friesen and Kaufman 2002; Ogihara et al. 2007). In addition to hormonal in-
creases, the application of ecdysteroids to unfed females induces Vg expression and synthesis
(Sankhon et al. 1999; Friesen and Kaufman 2004; Thompson et al. 2005; Seixas et al. 2008; Khalil
et al. 2011). Generally, the active form of ecdysone is 20-hydroxyecdysone (20E), and it has been
shown to upregulate target genes by binding with a heterodimer receptor. The ecdysteroid
receptor (EcR) and the retinoid X receptor (RXR) form a heterodimer, and ecdysteroids bind
to the receptor to form the functional ecdysteroid complex. This complex recognizes specific
sequences on the regulatory region of target genes and regulates gene transcription. In O. mou-
bata, both mated and virgin females simultaneously express EcR and RXR immediately after
engorgement (Horigane et al. 2007, 2008). Mated females show increases in ecdysteroids when
these receptors are present, after which mated females express Vg and secrete increased levels of
Vg into the hemolymph (Fig. 17.9) (Ogihara et al. 2007; Horigane et al. 2010). Only mated
females secrete high titers of ecdysteroids and have continuously high expression of Vg. There-
fore, ecdysteroids are essential for the stimulation of vitellogenesis after mating and are thought
to be the hormones that regulate Vg expression and synthesis.
Several models summarize the regulation of Vg synthesis in ticks. In hard ticks, the model of
vitellogenesis starts with mating (Roe et al. 2008; Cabrera et al. 2009). During mating, materials
from the male testes or vas deferens are transferred into the female genital tract. These materials,
called engorgement factors, are essential for repletion and oocyte development (Weiss and
Kaufman 2004; Donohue et al. 2009a). Signals from these factors may induce both blood feeding
to repletion and the synthesis of ecdysone. The synthesis of ecdysteroids is regulated by the ec-
dysiotropic hormone, which stimulates ecdysteroidogenesis, after which Vg synthesis occurs in
the fat body and midgut (Roe et al. 2008; Cabrera et al. 2009). Synthesized Vg is secreted into the
hemolymph and moves into the ovary via receptor-mediated endocytosis. A similar model is also
proposed for mite vitellogenesis (Cabrera et al. 2009) based on the model by Roe et al. (2008).
In soft ticks, a model of Vg regulation was developed by Chinzei and Taylor (1990). In this model,
2 important factors were defined, a VIF and a fat body stimulating factor (FSF). Based on ligation
experiments, VIF was hypothesized to be a neuropeptide secreted from the synganglion (Chinzei
et al. 1992; Taylor and Chinzei 2002). The second factor, FSF, appears to be an ecdysteroid and
stimulates tissues (e.g., fat body and midgut) to synthesize Vg (Chinzei and Yano 1985; Taylor et
al. 1991). Therefore, blood feeding and mating appear to stimulate the secretion of VIF, which
stimulates FSF secretion that in turn induces vitellogenesis in the midgut and fat body.
In order to understand the processes regulating vitellogenesis, it is necessary to divide the
roles of engorgement and mating in the regulation of tick vitellogenesis. In the soft ticks, regula-
tion requires 2 different phases, a nutrition-dependent phase and a mating-dependent phase (Fig.
17.10). During the first phase, engorgement can stimulate Vg messenger expression in the midgut
and the fat body associated with the main trachea extending from the spiracle (main fat body) in
both mated and virgin females (Horigane et al. 2010). Based on studies of Ha. longicornis, regula-
tion by nutrients likely requires signals included in the TOR signaling pathway such as the GATA
factor (Boldbaatar et al. 2010; Umemiya-Shirafuji et al. 2012). In the second phase, the stimulant
is mating. Mated females after engorgement have higher ecdysteroid titers in the presence of the
nuclear receptors EcR and RXR (Ogihara et al. 2007; Horigane et al. 2007, 2008). The complex of
ecdysteroid and the 2 nuclear receptors appears to induce higher levels of Vg expression in en-
gorged mated females during a late phase. Therefore, this phase might be under ecdysteroid reg-
ulation. Vg mRNA expression in mated O. moubata females occurs mainly in the developed fat
bodies throughout the body cavity (Horigane et al. 2010). The enlarged fat body synthesizes suffi-
cient Vg proteins for egg maturation, so that fertilization and oviposition can follow.
In the soft tick, female ticks do not require mating for engorgement, so this 2-phase model
easily describes the regulation in this family of ticks. In contrast, hard ticks require mating during
feeding on the host to induce repletion, so it is difficult to separate the roles of nutrition and
mating in those species. Particularly, stimulation by factors delivered from male ticks at copula-
tion (male engorgement factors) that are essential in order to induce egg production has been
reported for A. hebraeum and D. variabilis (Weiss and Kaufman 2004; Donohue et al. 2009a),
suggesting mating is also necessary for repletion. However, studies on Ha. longicornis with both
parthenogenetic and bisexual strains show that increases in Vg expression depend on blood
feeding, indicating that nutritional stimulation might be enough for vitellogenesis in this species
(Boldbaatar et al. 2010; Umemiya-Shirafuji et al. 2012). Although variation in the effects of mating
occur among hard and soft tick species, all species share the important nutritional requirement,
so that the early phase of the model for O. moubata is also applicable to the nutrient regulation of
vitellogenesis in hard ticks. In order for the initiation of vitellogenesis in ticks (and in other ar-
thropods as well) to be fully understood, the pathways for the regulation of feeding and egg de-
velopment must be delineated. However, based on the current evidence, we can conclude that
nutrition and mating are essential factors for the regulation of reproduction in ticks.
4.4. Vg UPTAKE
When Vg synthesis occurs in extraovarian tissues, mature Vg secreted into the hemolymph is
carried to the ovary and taken up by the oocytes. In engorged females of I. scapularis, increases
in Vg protein titer in the hemolymph are synchronized with increases in Vg titers in the ovaries
of these females (James and Oliver 1996) and Vg thought to be immediately uptaken from the
hemolymph by the oocytes. The absorption of Vg into the oocytes occurs via receptor-mediated
endocytosis, and vitellogenin receptors (VgRs) have been identified in 2 hard ticks, D. variabilis
(Mitchell et al. 2007) and Ha. longicornis (Boldbaatar et al. 2008). In addition, the tick genome
project has revealed the presence of VgR in I. scapularis. VgR is a member of the low-density
lipoprotein receptor superfamily and has a structure with 5 commonly conserved elements (Fig.
17.11): a ligand-binding domain including class A cysteine-rich repeats, an epidermal growth
factor precursor homology domain including class B cysteine-rich repeats and Tyr-Trp-X-Asp
(YWXD) repeats, an O-linked carbohydrate domain, a transmembrane domain, and a cytoplas-
mic tail (Sappington and Raikhel 1998; Tufail and Takeda 2005). VgRs of arthropod species are
generally larger than vertebrate VgRs (they have a predicted molecular weight of approximately
194 kDa) and encode approximately 1,800 residues. The homology between the 2 published tick
VgRs shows 79% identity, whereas homology with insects is lower than 40%. Both VgRs of ticks
have all the domains common to VgR elements.
VgR expression observed in the plasma membrane of oocytes was initiated after blood
feeding and showed high expression during the previtellogenic stage in Ha. longicornis (Bold-
baatar et al. 2008). In the same study, the suppression of VgR resulted in immature oocytes and
low oviposition. In addition, VgR expression was observed in D. variabilis after mating. Gene
knockdown by RNAi in D. variabilis eliminated Vg uptake in dsRNA-VgR injected females, re-
sulting in non-vitellogenic oocytes and no oviposition (Mitchell et al. 2007). Both of these find-
ings indicate that VgR is necessary in order for Vg secreted from extraovarian tissues to be taken
up by oocytes via endocytosis. In addition, VgR also has been shown to also function in the
transovarial infection of eggs with Babesia parasites (Boldbaatar et al. 2008).
In insects, most yolk uptake depends on the presence of JH, but JH and its analogues have
not been detected in ticks, and although genomic analyses indicate that ticks might be able to
synthesize JH, they are thought to not have JH (Neese et al. 2000). Increases in VgR expression
induced by 20E application indicate that 20E functions in yolk protein uptake as well as Vg syn-
thesis in ticks (Boldbaatar et al. 2008). As seen above, tick egg production, including Vg uptake,
is regulated by hormones through most steps, but the detailed processes still remain unknown.
Needless to say, better understanding of each step is necessary. Composite and multidirectional
analyses to connect hormonal and nutritional signaling to egg protein uptake and other pro-
cesses are required in order for tick egg production to be comprehensively understood.
FIGURE 17.11: Schematic comparison of Haemaphysalis longicornis vitellogenin receptor (HlVgR)

protein with proteins from other ticks and insects. HlVgR compared with Dermacentor variabilis VgR
(DvVgR), Aedes aegypti VgR (AaVgR), and Drosophila melanogaster yolk protein receptor (DmYPR).
Numbers 1–8 indicate the cysteine-rich repeats in the ligand-binding domains (LBD). Roman numerals
I–IV indicate the cysteine-rich repeats in the epidermal growth factor (EGF)-like domains, whereas
letters a–f indicate repeats containing Tyr-Trp-X-Asp (YWXD) motifs. Percent identities relative to the
HlVgR protein in the specifi c domains are shown below each respective domain. Ratios (%) on the right
represent the full sequence identities as compared with the full length HlVgR protein. SP, putative
signal peptide; O, O-linked sugar domain; TM, transmembrane region; C, cytoplasmic tail. Sequences
were taken from the GenBank database (accession numbers in parentheses): HlVgR (AB299015),
DmVgR (DQ103506), AaVgR (L77800), and DmYPR (U13637). From Boldbaatar, D., Battsetseg,
B., Matsuo, T., Hatta, T., Umemiya-Shirafuji, R., Xuan, X., and Fujisaki, K. (2008) Tick vitellogenin receptor
reveals critical role in oocyte development and transovarial transmission of Babesia parasite. Biochem.
Cell Biol. 86:331–344, with permission from NRC Research Press.
5. GONOTROPHIC CYCLE
In all ixodid ticks except the genus Ixodes, all events described above are completed in a single
gonotrophic cycle. Thousands of ooctyes become mature and are ovulated and oviposited until the
exhausted female dies. An enormous blood meal provides the energy reserves, especially protein,
for yolk formation on a massive scale, and well over 50% of the replete female body weight is con-
verted into eggs. Ixodes species appear to be phylogenetic intermediates between argasids and the
other ixodids, because they have retained the ability to mate without blood feeding.
The argasid strategy is very different from that seen in the ixodids. Argasid ticks have mul-
tiple gonotrophic cycles, often distributed over a period of years. For example, O. moubata fe-
males might have as many as 8 gonotrophic cycles. Each cycle requires a blood meal, but only a
single mating is necessary to provide spermatozoa for all of the female’s subsequent reproduc-
tive activity (although mated females can accept additional copulations). It is difficult to separate
the processes of mating and engorgement in describing the regulation of vitellogenesis, but re-
cent research on vitellogenesis in O. moubata shows that this species provides an excellent model
for better understanding the separate roles of engorgement and mating in the regulation of tick
reproduction (see Section 4 for the regulation of vitellogenesis).
5.1. ENGORGEMENT
Ixodid ticks attach to a host and feed for several days to months, depending on the stage and
species, but partial feeding must occur in metastriate ixodids before the male is attracted to a
female and mating takes place. After mating, females enter a rapid feeding stage wherein they
feed to repletion. Female ixodid feeding is usually divided into 3 phases (Balashov 1972) as
follows: (i) a preparatory phase (about 1 day) for the female to establish a feeding lesion, (ii) a
slow phase (7 to 9 days) for the tick to grow to about 10 times its unfed body weight, and (iii) a
rapid phase (about 1 day) in which the tick increases its weight a further 10 times. This rapid
feeding phase allows females to reach the critical weight necessary as a prerequisite for vitello-
genesis (Friesen and Kaufman 2009). Finally, the females must detach from the host to trigger
vitellogenesis and the production of mature eggs (Friesen and Kaufman 2009). Therefore, not
only must ixodid ticks obtain sufficient nutrients to synthesize the massive amounts of Vg
proteins needed to produce eggs, but it appears they must also detach from the host to initiate
the reproductive processes.
In argasids, engorgement is essential for oviposition in almost all instances (only a few au-
togenous species occur). Argasid females and I. scapularis can mate before or after feeding, and
both mated and virgin females imbibe similar amounts of blood. If unmated, fed virgins digest
their blood meals slowly. When mating occurs, digestion is accelerated (see Chapter 6). Fed
mated females digest their blood meals rapidly until the first cycle of oogenesis and oviposition
is completed. These females mature only a small number of ova (about 50 to 300), depending
upon the species, with development proceeding at unequal rates in ova in the same egg clutch.
The number of eggs deposited is clearly related to the volume of blood imbibed or remaining.
Large numbers of oogonia and primary oocytes remain in the ovary. The development of these
remaining oocytes is arrested, and mature ova that were not oviposited are resorbed. If virgin
females are mated even a long time after feeding, they can quickly convert to the mated female
strategy and oviposit viable eggs (Horigane et al. 2010). The factors that terminate oogenesis and
oviposition are unknown. Following oviposition, the argasid female is ready for another cycle of
feeding, digestion, and oviposition. The capability for many gonotrophic cycles ensures the ex-
tended survival of these ticks. This strategy is especially valuable for nidiculous parasites depen-
dent for their survival upon an extremely scarce host population and infrequent host visits.
Engorgement is usually required in order to initiate reproduction in adult ticks, but females
of a few species, if they have mated, can lay eggs before engorgement and are thus considered
autogenous (Feldman-Muhsam 1973; Feldman-Muhsam and Havivi 1973; Oliver 1974; Oliver
et al. 1984). Even some anautogenous species have low rates of autogeny. For example, a low rate
(3.6%) of autogeny has been observed in O. moubata females (Chinzei et al. 1989). Ticks are able
to take in much larger quantities of blood than the capacity of their guts by excreting water and
electrolytes from the salivary or coxal glands and concentrating the blood during feeding. The
concentrated blood is stored in the gut, incorporated into the gut epithelial cells as needed, grad-
ually digested in the cells, and absorbed to provide nutrients for development or reproduction
(Tarnowski and Coons 1989). Engorgement also occurs in the immature stages, and blood re-
mains in the gut through subsequent stages, possibly providing a nutrient source for autogenous
reproduction.
5.2. MATING
Mating must take place in order for oviposition to occur, and it differs between the ixodid and
argasid ticks. As described above, most ixodid ticks must mate on the host during feeding. Fac-
tors delivered from male ticks at mating (male engorgement factors) appear to be essential in
order to induce egg production in ixodid ticks (Weiss and Kaufman 2004; Donohue et al. 2009a).
These factors also appear to be essential for stimulating the rapid feeding phase, suggesting
mating is also necessary for repletion.
Unlike most hard ticks, soft tick species can mate before and after blood feeding. Vitellogen-
esis also differs in argasid ticks in that both virgin and mated argasid females express and syn-
thesize Vg after engorgement (Ogihara et al. 2007; Horigane et al. 2010). However, only mated
females show continuously high levels of Vg expression, maturation of oocytes, and oviposition
of eggs. Argasid virgin females initiate Vg expression similarly to mated females at the onset of
vitellogenesis, but the expression level decreases as time proceeds, and virgin females cannot
produce mature eggs. However, if virgin females of O. moubata are mated even a long time after
feeding, Vg expression increases and they can oviposit mature eggs. Therefore, mating appears
to be necessary to stimulate an increase in ecdysteroid titers (Ogihara et al. 2007) that stimulates
Vg expression in the fat body and leads to the development of mature eggs in argasid ticks
(Horigane et al. 2010). Mating is essential in both ixodid and argasid ticks, but how mating can
regulate vitellogenesis remains to be elucidated.
Early studies on the ultrastructure and morphology of the female tick reproductive system by
Balashov (1972), Raikhel (1983), and other researchers have provided an excellent reference
point for understanding the functions of the female reproductive processes. However, some
discrepancies and the need for better understanding of some processes such as the ovarian
transmission of diseases will require further studies involving the morphology and ultrastruc-
ture of the female reproductive system. The use of improved technology should help in devel-
oping knowledge of the processes involved in oocyte development and the identification of sites
for Vg synthesis, ecdysteroidogenesis, and the synthesis of other regulatory factors. Chapters 13
and 16 discuss some of these factors, and the former raises the possibility of a role for the early
part of the insect JH pathway in the tick synganglion for the regulation of reproduction. There
is also a need for additional investigations to help increase our limited knowledge of the impor-
tant events that occur during ovulation and fertilization. In addition, studies are needed to
clarify the processes by which ooctyes initially begin to develop and become vitellogenic. The
structure and function of the pedicel cell also must be clarified.
Molecular and biochemical techniques have made recent advances in the understanding of
vitellogenesis and its regulation possible, but there are still numerous areas that need to be studied
in more detail. Studies need to be conducted to confirm the Vg gene numbers in different species
and to analyze the subunits, and their correlation with the Vg genes needs to be elucidated. Fur-
ther studies are also needed on the regulation of Vg synthesis, especially in terms of the nutrient
pathway, which must play an essential role in tick reproduction. Factors from males and their
functions also must be identified and elucidated. The epidermis has been shown to be a site of
ecdysteroid synthesis (Zhu et al. 1991; Lomas et al. 1997), but ligation experiments (Chinzei et al.
1992) indicate that ecdysteroids are synthesized in a tissue only in the posterior portion of argasid
ticks, and high concentrations of ecdysteroids in the ovary of hard ticks (Kaufman 1991) indicate
that the ovary might synthesize ecdysteroids. Therefore, studies are needed to determine whether
the ovary or other tissues can synthesize ecdysteroids. In addition, the link between the male
factors and hormonal regulation of vitellogenesis must be clarified. The role of the synganglion
and, especially, the identification of possible neuropeptides that regulate vitellogenesis are other
critical areas needing further investigation. Further advances in these areas will provide a better
understanding of reproduction in ticks and might lead to improved methods of control for these
important disease vectors. These studies will also contribute to a better understanding of repro-
duction in general in the Arthropoda, including in insects.
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NOTE
user may then browse the anatomical structure by clicking on “Material Anatomical Entity” and
C H A P T E R 1 8
MALE REPRODUCTIVE SYSTEM

Anatomy, Physiology, and Molecular Biology
DANIEL E. SONENSHINE AND LEWIS B. COONS
I. INTRODUCTION
The male reproductive system of ticks consists of paired tubular testes, paired vas deferens, a
seminal vesicle surrounded by the accessory gland, and an ejaculatory duct connected to the
genital pore. The male accessory gland is a complex, multi-lobed organ situated immediately
posterior to the synganglion. It produces the spermatophore and contributes to the seminal
fluids. These same features are found in other acarines and show many similarities to the
male reproductive system of insects. However, several important differences occur. Male
ticks lack an intromittent organ such as the aedeagus of insects or a spermatodactyl, a sperm-
delivery organ found on the chelicerae of many mites. Instead, spermatozoa are transferred
within a spermatophore created during copulation, which the male inserts into the vulva of
the female with its chelicerae. Reproductive development varies in the different types of
ticks. Spermatogenesis commences in the nymphal stage. In prostriate ixodids and all argasid
species, spermatogenesis is completed following molting to the adult stage, and the sper-
matocytes elongate into spermatids without the adults having to feed on a vertebrate host.
Males may commence mating and copulation soon after molting from the nymphal stage
(Kiszewski et al. 2001). In metastriate ticks, spermatogenesis is arrested at the primary sper-
matocyte phase after males molt to adults. Males must feed prior to mating in order to
complete spermatogenesis and become sexually mature. Blood feeding stimulates the
differentiation and growth of the spermatids into the greatly elongated, non-motile prosper-
mia (characterized as spermiogenesis by Coons and Alberti [1999]). Subsequently, when the
ticks mate, spermatophore formation and insemination occur rapidly during the brief copu-
latory period. Spermiogenesis continues within the endospermatophore after the sperm-
filled spermatophore is inserted into the vulva of the female (Kiszewski et al. 2001).
Male Reproductive System 485
Capacitation, the transformation of the spermatids into elongated spermatozoa, also begins
at this time. Capacitation is a complex process in which the sperm elongates after detach-
ment of the operculum and eversion of the acrosomal canal. The sperm’s fertilizing ability is
not fully realized until after the spermatids are transferred to the recipient female (Oliver
1982; White 2011). Tick spermatozoa are remarkable because of their exceptional length and
morphological characteristics. In addition, they exhibit an unusual mode of locomotion
(gliding) (Resler et al. 2009). In some species (e.g., Ixodes scapularis), male ticks mate only
once, often prior to feeding. In many or most metastriate ticks, males feed and mate repeat-
edly, occasionally as often as 25 to 35 times during their adult life.
Although much has been learned about the anatomical organization and fine structure of
the male reproductive systems in ticks, little is known about the proteomics or molecular
biology of male reproduction. In this chapter, we review the male reproductive system, paying
special attention to the molecular biology of the male reproductive process.
For an electronic version of the structure of the male reproductive system, the reader may
browse the tick anatomical ontology for a description of the structures and related illustra-
tions by visiting www.Vectorbase.org and following the instructions in the footnote below.1
Additional illustrations of the male reproductive system not included in this edition may be
found there.
2. MORPHOLOGY
2.1. ANATOMY OF REPRODUCTIVE SYSTEM IN IXODID

AND ARGASID TICKS
Figure 18.1 illustrates the reproductive system in a representative ixodid tick. The male
system consists of (1) a pair of tubular testes (T), U-shaped or convoluted in appearance,
which in many species are connected posteriorly by a variously broad or thin isthmus
(giving it the appearance of a single organ); (2) paired, highly convoluted vasa deferentia;
(3) a single seminal vesicle (not shown); (4) the ejaculatory duct; and (5) a large, multi-
lobed male accessory gland (MAG).
Figure 18.2 illustrates the system in a representative fed male. The testes, vasa deferentia, and
MAG are greatly enlarged. The MAG is located just posterior to the synganglion. It surrounds
the seminal vesicle and ejaculatory duct on their dorsal and lateral surfaces, obscuring these
structures when viewed from the dorsal aspect. In general, the male reproductive system is sim-
ilar in both families of the Ixodida. The main body of the MAG is the conspicuous dorsal me-
dian lobe (DML), lying above the seminal vesicle. Four groups of paired lobes extend from the
DML, namely, the dorsal lateral lobes, anterior ventral lobes, posterior lateral lobes, and poste-
rior ventral lobes.
Figure 18.3 illustrates the reproductive system in a representative argasid tick. The system is
similar to that of ixodids, but with several noteworthy differences. Each vas deferens has an en-
largement (ampulla) nears its distal (anterior) end, where it joins with the seminal vesicle. The
MAG contains a dorsal granular lobe, below which is the unpaired ventral granular lobe and the
spongy lobe (not shown in the figure). Three pairs of prominent lateral granular lobes are con-
nected to the posterior region of the gland.
FIGURE 18.1: Structure of the male reproductive system in an unfed male ixodid tick, based on
Dermacentor andersoni. Acg, male accessory gland; E.D., ejaculatory duct; L.a.d., antero-dorsal lobe; L.d.l.,
dorso-lateral lobe; L.p.d., postero-dorsal lobe; L.p.l., postero-lateral lobe; L.p.v., postero-ventral lobe;
T, testis; V.D., vas deferens. After Douglas, J.R. (1943) The Internal Anatomy of Dermacentor andersoni
Stiles. Berkeley, CA: University of California Press, 207–282, with permission from the University of
California Press.
FIGURE 18.2: Structure of the reproductive system of a fed male ixodid tick based on Rhipicephalus
(Boophilus) decoloratus. The greatly thickened testis is fi lled for most of its length with numerous
primordial cells and spermatocysts fi lled with spermatogonia and spermatids. The long, coiled vasa
deferentia fi lled with elongated spermatids extend from the testis to the seminal vesicle (obscured by
the male accessory gland). The massive male accessory gland (MAG) is one of the largest organs in
the body of the fed male. It consists of a dorsal median lobe and multiple secondary lobes. The
ejaculatory duct extends from the seminal vesicle below the MAG and the synganglion to the genital
pore. AVL, anterior ventral lobe; DLL, dorsal lateral lobe; DML, dorsal median lobe; ED, ejaculatory
duct; PLL, posterior lateral lobe; PVL, posterior ventral lobe; Syn, synganglion; T, testis; VD, vas
deferens. Modifi ed from Londt, J.G.H. and Spickett, A.M. (1976) Gonad development and
gametogenesis in Rhipicephalus (Boophilus) decoloratus Koch, 1844 (Acarina: Metastriata: Ixodidae).
Onderstepoort J. Vet. Res. 43:79–96, with permission from the Onderstepoort Journal of Veterinary
Entomology.
FIGURE 18.3: Structure of the reproductive system in an unfed male argasid tick (Carios kelleyi), dorsal
aspect. D.G.L., dorsal granular lobe; E.D., ejaculatory duct; P.L.G.L. 1–3, postero-granular lobes 1–3;
T., testis; V.D., vas deferens; V.G.L., ventral granular lobe. From Sonenshine, D.E. (1970) A contribution
to the internal anatomy and histology of the bat tick, Ornithodoros kelleyi Cooley & Kohls, 1941. J. Med.
Entomol. 7:289–312, with permission of the Journal of Medical Entomology.
486
2.2. HISTOLOGY AND ULTRASTRUCTURE

2.2.1. Testes
The paired tubular testes consist of layers of germinal cells surrounded by a thin connective
tissue membrane and loosely organized smooth muscle cells. The narrow central lumen is
surrounded by interstitial cells with numerous microvilli on their luminal borders. In unfed
males, the testes are very small, narrow, and often difficult to find; the majority of the cells
consist of undifferentiated cells, spermatogonia, and a few primary spermatocytes (Fig.
18.4). In males fed at least 5 to 7 days, the testes are much thicker, and there are numerous
primary and secondary spermatocytes, spermatids in varying stages of development, and
residual spermatogonia. In the distal regions of the testes, numerous spermatogonia are pre-
sent, many with nuclei showing mitotic (or meiotic) figures. The cells are starting to organize
into spermatocysts (clusters of spermatids). Few elongated spermatids are evident (Fig.
18.5). In the middle regions of the testes, most of the developing early stage spermatids (pri-
mary spermatocytes) have clustered together into spermatocysts (Fig. 18.6). In more anterior
regions of the testes, near the junction with the vas deferens, the spermatids (now secondary
spermatocytes) have undergone their great growth cycle but remain within the spermato-
cysts. When this region of the testis is viewed in cross-section, each of the spermatids is
found to contain a ring of tiny vesicles (subplasmalemmal cisternae) lining the inner edges
of the cells so that they resemble thick-walled tubes. Each cluster contains between 32 and
62 secondary spermatocytes (Fig. 18.7).
Transmission electron micrographs of the posterior region of the testis from a fed male
show the primary and/or secondary spermatids as they appear at the beginning of their
growth phase and elongation. Small numbers of minute tubular structures, the subplasma-
lemmal cisternae, have begun to form and accumulate adjacent to the plasma membrane.
Numerous Golgi complexes are evident throughout these developing spermatids (Fig. 18.8).
As the spermatids develop and elongate, the subplasmalemmal cisternae also elongate, their
numbers increase, and they accumulate in multiple layers (Fig. 18.9). The nuclei are still large
and more or less central, although the chromosomes do not appear to have condensed (or
have just barely commenced doing so); some also show nucleoli. Abundant Golgi complexes,
rough endoplasmic reticulum, and mitochondria suggest that vigorous protein synthesis and
metabolic activity have occurred in these rapidly growing spermatocytes and spermatids
(Fig. 18.10). No evidence of an acrosome partially or completely enclosing the spermatid nu-
cleus appears at this stage such as that found in certain species of mites (Alberti et al. 2007).
Subsequently, in the terminal ends of the testes, near or at the junction with the vasa defer-
entia, the spermatids become mature, and many exhibit a large acrosome along one side of
each of these cells (Fig. 18.11).
2.2.2. Vasa deferentia, seminal vesicle, and ejaculatory duct

The paired vasa deferentia are coiled, folded, or convoluted tubes, each consisting of a thin-
walled epithelium of thin squamous cells. The ducts are surrounded by a thin connective tissue
layer with occasional smooth muscle cells. In the unfed male, they are difficult to find. The sem-
inal vesicle is underneath the MAG. The wall of the ejaculatory duct is lined with a thin, folded
cuticle covered with a layer of cuboidal epithelium and elongated muscle cells (Fig. 18.12). Pairs
of muscles from the body wall insert on its dorsal and ventral surfaces. The ejaculatory duct
FIGURE 18.4: Light micrograph illustrating the histology of the testis and early phase of
spermatogenesis in a partially fed virgin male Dermacentor variabilis. Section of the anterior region of
the testis. There is little or no evidence of spermatogenesis. The nuclei are vesicular and, in most cells,
indistinguishable because the chromosomes have not condensed at this stage. A thin squamous
epithelium lines the narrow lumen, and a similar epithelium forms the outer boundary of this organ.
MAG, male accessory gland; T, testis. Bar = 50 μm.
FIGURE 18.5: Light micrograph illustrating the histology of the testis and early phase of
spermatogenesis in a partially fed virgin male Dermacentor variabilis. Profi le of a section through the
anterior/middle region of the testes showing the zone of spermatogonia. Many of the nuclei in the
spermatogonial cells appear in various stages of mitosis, with well-defi ned chromosomes
(arrowheads). The spermatogonia are starting to organize into clusters (spermatocysts), although
the separations between the clusters are not always discernible at this magnifi cation. Several
elongated spermatids are visible in the lumen. S., spermiophores (spermatids); Spg =
spermatogonial cells. Bar = 50 μm.
appears buried within the lobes of the MAG. Anteriorly, the ejaculatory duct fuses with the
genital aperture (see Chapter 4).
In the fed male, the vasa deferentia are enlarged and easily identified. They are filled with
masses of tightly packed, elongated spermatids. Many appear elongated, whereas others are cut
in cross-section and appear as small tubes (Fig. 18.13).
FIGURE 18.6: Light micrograph illustrating the histology of the testis in a fed male Dermacentor variabilis
(fed 6 days). Longitudinal section near the anterior region of the testis (near the vas deferens). Most of
the early stage spermatids are organized into spermatocysts (some near the left end are still
undergoing development and beginning to cluster into spermatocysts). Bar = 200 μm.
FIGURE 18.7: Light micrograph illustrating the histology of the testis in a fed male Dermacentor variabilis
(fed 6 days). Cross-section showing elongated spermatids (after completing their great growth cycle)
grouped into spermatocysts. The dark black structures are the nucleoli. Bar = 100 μm.
2.2.3. Male accessory gland

Aside from the midgut, this is the largest organ in the male tick’s body. The structure of this
multi-lobed gland varies greatly in the different species of ticks. In the American dog tick,
D. variabilis, it consists of a prominent dorsal median lobe, a large ventral lobe, and at least
3 pairs of finger-like lobes projecting laterally and posteriorly, similar to the pattern found in
D. andersoni (Fig. 18.1). In the bat tick, Carios kelleyi, 9 lobes were observed, 8 of which were
granular, and the other spongy (Sonenshine 1970). The unpaired ventral granular lobe is the
largest; other lobes, in outline form, are as shown in the Fig. 18.3. The structure of this organ is
quite similar in the various species examined. In ixodid ticks, the lobes are more elongated and
tubular in appearance.
In the unfed male, histological sections show that the MAG consists largely of multiple
layers of undifferentiated cells. The vast majority of cells appear inactive. However, small
numbers of cells adjacent to the large lumen show small amounts of granule formation and
accumulation, presumably in preparation for spermatophore or semen formation (Fig. 18.14,
arrows).
During blood feeding and just prior to copulation, the MAG undergoes a remarkable trans-
formation. With the exception of the spongy lobe, all lobes show tremendous cellular activity. In
FIGURE 18.8: Transmission electron micrograph illustrating the fine structure of the testes of a fed
virgin male ixodid tick, Dermacentor variabilis (fed 6 days). The ultrastructural characteristics of several
primary spermatocytes early in the great growth phase of their development can be noted. No
chromosomes are apparent in the vesicular nuclei, and some nuclei still have nucleoli (dense mass).
Minute tube-like subplasmalemmal cisternae have begun to accumulate adjacent to the cell membranes.
GC, Golgi complexes; Nu, nucleus; Nucl, nucleolus; Spc, subplasmalemmal cisternae. Bar = 2 μm.
Photo courtesy of Ms. Lou Boykins, Integrated Microscopy Center, University of Memphis, Memphis, TN.
the thin, finger-like peripheral lobes, the glandular epithelium expands greatly. Numerous tall
columnar cells, some as long as 50 to 100 μm, each filled with masses of secretory granules, are
evident. The outer wall of the lobe comprises a single layer of cuboidal cells and an outer layer of
muscle cells. However, little or no secretory activity has taken place by this point, and the lumen
is narrow and quite small (Figs. 18.15, 18.16). In other lobes of the gland, extensive secretory ac-
tivity is evident; many cells have depleted their contents, and the large lumen is filled with abun-
dant secretory globules (Fig. 18.17). Secretion appears to be primarily apocrine. Large globules,
each filled with numerous small stained granules, are evident at the luminal (apical) ends of
many cells and appear to have broken off. Other large globules are still intracellular. In the
lumen, numerous secreted globules are evident. Some of these globules have ruptured, liber-
ating masses of free granules, whereas others are still intact. Other cells appear to have released
their entire mass of secretory material (i.e., via the holocrine pattern of secretion in which the
cell disintegrates and releases its accumulated secretory vesicles). The epithelial covering sur-
rounding these very active lobes is reduced to a thin layer of squamous cells with small fusiform
nuclei. Below the epithelium are smaller cells containing numerous granules. Toward the inte-
rior, the cells are more densely packed and irregularly shaped. As noted by other workers who
have studied the MAG in different tick species (Garcia-Fernandez et al. 1998), histochemical
analysis of the glands indicates that the secretions (but not the cytoplasm or nuclei) comprise
strongly acidic glycoproteins and amyloid proteins. Other tests show a strong response to muci-
carmine staining, indicating the inclusion of mucins, presumably as mucopolysaccharides, in
FIGURE 18.9: Transmission electron micrographs illustrating the fine structure of the testes of a fed
virgin male ixodid tick, Dermacentor andersoni (fed 7 days), and the ultrastructural characteristics of the
later stages of development of the rapidly enlarging secondary spermatocytes. Profi le of developing
cells showing accumulation of subplasmalemmal cisternae below the cell membrane; many of these
cisternae have elongated. The dense, curved lamellate structures in each of the spermatocytes are
Golgi complexes. G.C., Golgi complex; Hc, hemocoel; Nu, nucleus of primary spermatocyte; Spc,
subplasmalemmal cisternae. Bar = 2.8 μm. Inset (left): Enlargement illustrating details of two Golgi
complexes. Bar = 0.75 μm.
FIGURE 18.10: Transmission electron micrograph illustrating the fi ne structure of the testes of a fed
virgin male ixodid tick, Dermacentor andersoni (fed 7 days), and the ultrastructural characteristics
of the later stages of development of the rapidly enlarging secondary spermatocytes. Higher
magnifi cation profi le illustrating several secondary spermatocytes adjacent to the testicular
lumen. Chromosomes appear to be forming in the nucleus of the lower cell. A thin layer of
squamous epithelium forms a lining adjacent to the lumen. Ep, epithelial cell of the testicular lining;
L, lumen; Rer, rough endoplasmic reticulum; S, spermiophore; Spc, subplasmalemmal cisternae.
Bar = 2.5 μm.
the granules (see Supplementary Figs. S18.1 and S18.2 at www.oup.com/us/biologyofticks2e).

Proteins with disulfide or phenolic moieties are also believed to be present. Transmission elec-
tron microscope images show accumulations of electron-dense vesicles characteristic of neu-
tral lipids (see below). Finally, one lobe resembles the spongy lobe described by other authors
who have studied the MAG in ticks and insects, which is largely devoid of secretory material
FIGURE 18.11: Transmission electron micrograph of a section of the testis from a fed virgin male
D. variabilis illustrating numerous mature spermatids (spermiophores or prospermia). An acrosome
(black structure) is visible in many of the spermatids. Bar = 2.0 μm.
FIGURE 18.12: Light micrograph of a histological section of the reproductive organs from an unfed virgin
male D. variabilis. The seminal vesicle is surrounded by components of the male accessory gland and
testis. The ejaculatory duct is lined with a thin layer of cuticle. ED, (presumed) ejaculatory duct; SV,
(presumed) seminal vesicle. Bar = 50 μm.
492
FIGURE 18.13: Light micrograph of a histological section from a fed virgin D. variabilis male showing the
vas deferens (VD) fi lled with elongated spermatids. Bar = 200 μm.
FIGURE 18.14: Light micrograph of a histological section from an unfed D. variabilis male showing the
male accessory gland. A few cells adjacent to the lumen have begun to accumulate granules (arrows),
but the remainder of the tissues show no differentiation or granule formation. Bar = 100 μm.
(Fig. 18.18). It is not clear whether the spongy lobe is a specific functional structure or merely a
reflection of different stages of glandular activity. El Shoura (1987) noted that certain lobes of the
gland in Argas arboreus always remain in the spongy condition.
When the MAG is viewed with a transmission electron microscope, it is apparent that the
numerous vesicles visible in these lobes are of 2 major types, namely, (1) simple electron-dense
vesicles and (2) complex vesicles containing granular material dispersed in a reticulate pattern
(Figs. 18.19, 18.20). The electron-dense vesicles contain lipids, including neutral lipids (less dense
FIGURE 18.15: Light micrograph illustrating the differentiation of the cells in a representative lobe of
male accessory gland from a copulating fed D. variabilis male. Note the extensive accumulation of
granules in the elongated cells. The lumen is small and without any secretory material. Methylene blue
stain. Ep/Mu, external epithelial muscle layer; L, lumen. Bar = 100 μm.
FIGURE 18.16: Light micrograph illustrating the differentiation of the cells in a representative lobe of
male accessory gland from a copulating fed D. variabilis male. Note the extensive accumulation of
granules in the elongated cells. The lumen is small and without any secretory material. Methylene blue
stain. Enlargement showing the tall columnar cells and granule accumulation in these cells. Ep/Mu,
external epithelial muscle layer. Bar = 20 μm.
FIGURE 18.17: Light micrograph of a histological section of a lobe of the male accessory gland from a
copulating fed D. variabilis male. Trichrome stain. The greatly enlarged lumen is fi lled with masses of
secreted material and cell fragments, suggesting both apocrine and holocrine secretion. Bar = 100 μm.
vesicles) and polyunsaturated lipids. The particulate material in the complex vesicles varies in
density, from a dispersed, fine network of granules to a condensed, closely packed mass, and is
believed to comprise glycoproteins, lipoproteins, and mucins. Abundant cisternae of rough
endoplasmic reticulum fill the cytoplasm between the granules (Fig. 18.21).
The MAGs provide the secretions needed to form the spermatophore and the numerous
proteins in the seminal fluid that nourish the spermatids, enhance sperm motility, protect
against microbial infection and oxidative and environmental stress, and likely facilitate the sper-
matids’ capacitation after they have passed into the recipient female (see below). Despite their
FIGURE 18.18: Light micrograph of a histological section of the spongy lobe of the male accessory gland
from a copulating fed D. variabilis male. Bar = 100 μm.
obvious importance, little else is known of the ultrastructure of these glands or of their physio-
logical and biochemical activity. However, several recent reports might provide new insights
into the functions of this system (see Sections 3 and 4).
2.2.4. Comparison with the male reproductive system of mites

The male genital system of mites consists of testes, genital ducts or vasa deferentia, an accessory
gland or glands, and an ejaculatory duct leading to an external genital pore. This comparison of
male mites’ genital systems is taken from the reviews of Alberti and Coons (1999), Walter and
Proctor (1999), and Krantz (2009). The reader is referred to these reviews for detailed access to
the literature. Male genital structures vary greatly within the mite taxa, especially between the 2
major superorders, the Anactinotrichida (Parasitiformes) and the Actinotrichida (Acariformes).
The Anactinotrichida have the least diversity among male genital systems in mites. Testes
are paired structures in the Opilioacarida and Holothyrida. In the Opilioacarida, developing
spermatozoa occur in spermatocysts surrounded by somatic cells. All germ cells in each sper-
matocyst are in the same developmental stage. Mature spermatozoa are released into a lumen in
the testis that leads to the vasa deferentia. Nothing is known about spermatogenesis in the Ho-
lothyrida. In the Gamasida, paired testes occur in the Uropodina, whereas a partially fused or
FIGURE 18.19: Transmission electron micrograph illustrating the ultrastructure of a granular lobe of the
male accessory gland from a fed copulating D. variabilis male. The image shows the dense accumulation
of polyunsaturated lipid vesicles in the cells. Clear vesicles probably represent saturated lipids. Li(s),
saturated lipids; Li(un), unsaturated lipids. Bar = 2 μm.
male accessory gland from a fed copulating D. variabilis male. The image shows the dense accumulation
of large, complex granular inclusion bodies with unknown contents dispersed in a reticulate pattern.
These vesicles are believed to contain glycoproteins and other non-lipid-rich molecules. Adjacent cells
contain numerous dense-staining lipid droplets. In. bod., inclusion body, unknown content. Bar = 2 μm.
single testis is found in the Parasitina and Dermanyssina. Development of spermatozoa occurs
in spermatocysts surrounded by somatic cells in a manner similar to that described for the
Opilioacarida.
The greatest variation in the structure of the testis occurs in the Actinotrichida. Paired or
multi-lobed testes connected by bridges of tissue are common in the Actinedida. Testes with
lateral lobes connected by tissue bridges are common in the Oribatida and snout mites (Actin-
edida: Bdellidae). In some very small mites such as in the prostigmatic taxa (Actinedida), the
testes are fused or single. The type of testis found in the Oribatida and snout mites is thought to
be plesiomorphic in the Actinotrichida. All other forms of the testis are believed to have devel-
oped from this type. The plesiomorphic testis is divided into a germinal region and a glandular
region. Spermatogenesis occurs in the germinal portion. Here, germ cells are closely associated
with one or more somatic cells during their development. Secretions from cells in the glandular
region are thought to be involved in spermatophore formation and possibly make up part of the
male accessory gland from a fed copulating D. variabilis male. The cells in this profi le show extensive
elaboration of the rough endoplasmic reticulum, indicating intense synthesis activity. RER, rough
endoplasmic reticulum. Bar = 2.0 μm.
fluid accompanying sperm. Very little is known about capacitation in mites. It is assumed to
occur in the female.
Almost all mites have paired vasa deferentia that lead distally to the single ejaculatory duct
and genital atrium. The histology of the ducts and atrium differs from that of the testis. A muscle
layer surrounds the ducts. In mites producing complex spermatophores, the ejaculatory duct is
often a complex structure.
Accessory glands are found in most species of mites. However, little is known about their
function. It is assumed that their secretions are involved in the formation of the spermatophore.
A complex set of accessory glands occurs in the Opilioacarida. In the Mesostigmata, a single
multi-lobed accessory gland opens into the seminal vesicle near its junction with the vasa defer-
entia. Accessory glands are poorly developed or absent in Oribatida and in some Acaridida.
Spider mites (Actinedida: Tetranychidae) transfer sperm directly and do not have accessory
glands. The most complex set of accessory glands occurs in snout mites.
Spermatozoa of mites are aflagellate cells that differ greatly in fine structure between the 2
superorders. No symbiotic microorganisms have been found in the spermatozoa of mites. Sper-
matozoa of Parasitiformes are larger with a complex ultrastructure and a large vacuole that de-
velops during spermatogenesis. Spermatozoa of many species of Parasitina and Dermanyssina
lack the vacuole but have a characteristic elongate ribbed structure in the periphery of the cell
that is referred to as a “ribbon” in the literature. Spermatozoa of Actinotrichida are mostly
smaller than those of the Anactinotrichida, with a less complex ultrastructure. Spermatozoa of
Actinotrichida have developed the most diversity of the 2 superorders.
Most mites transfer sperm in a packet referred to as a spermatophore. These range in com-
plexity from sperm packets with a simple covering of secretions to complex stalked spermato-
phores with several internal layers covering the sperm. Spermatophores are transmitted either
directly by the male into the female genital opening or indirectly via deposition on the substrate.
Both methods are widely used in the Acari. Direct transfer involves the mouthparts of the male,
which are often modified to hold the spermatophore, as in some species of Parasitina and Der-
manyssina, or the use of a penis (aedeagus) as found in some Actinedida and all Acaridida. In
some families of Oribatida, the female has an elongated intromittent organ that is inserted into
the male’s genital opening to extract sperm. Indirect transfer of a spermatophore occurs in the
Actinedida and Oribatida. Spermatophores transmitted in this manner can be divided into 2
groups: the first and simplest type is found in a eupodoid mite (Actinedida) and consists of
threads containing sperm droplets; the second and more complex type is the stalked spermato-
phore found in the Oribatida. At least 5 morphological types occur. Nothing is known about the
method of sperm transfer in the Opiloacarida or Holothyrida.
The genome of the spider mite, Tetranynchus urticae, has been published (Grbić et al. 2011).
In silico analysis of this genome might uncover many genes associated with spermatogenesis
and spermiogenesis in this mite and facilitate an understanding of mite reproduction function.
Comparisons with the genome of Ixodes scapularis and the transcriptome of Dermacentor varia-
bilis also might prove useful.
2.2.5. Comparison with the male reproductive system of insects

Although generally similar, there are many differences between the male tick reproductive
system and that of insects. In insects, the basic anatomical plan comprises paired testes con-
taining testicular tubules or follicles bound together by a mesodermal sheath, paired vasa def-
erentia, and a common ejaculatory duct that terminates in the gonopore. The vasa deferentia
may be expanded to form a seminal vesicle. The MAGs vary from simple tubules to masses of
mesodermal tubules surrounding the seminal vesicles (mushroom bodies) (Davey 1985). For
further information about these structures in insects, the reader is referred to the work of
Marchini et al. (2008).
3. SPERMATOGENESIS AND SPERMIOGENESIS
In male ticks, spermatogenesis usually begins in fed nymphs during the ecdysial process. In
prostriate ticks and most argasids, spermatogenesis is completed without interruption, and
young adults are ready to mate. However, in metastriate ticks, spermatogenesis is arrested after
adult emergence, and further development does not continue until the males feed. Sperm devel-
opment occurs in 2 stages: (1) spermatogenesis, or the sequence of mitotic and meiotic divisions
leading to the haploid spermatids, and (2) spermiogenesis, or the growth and differentiation
that leads to the elongated prospermia. Spermiogenesis is further subdivided into 2 phases: (1)
growth and elongation to form the very long prospermia (these elongated spermatids are trans-
ported to the ejaculatory duct and, eventually, to the spermatophore), and (2) capacitation, the
process of sperm maturation that precedes migration and fertilization. Capacitation of the
sperm (i.e., maturation of the elongated spermatids to mature spermatozoa) begins in the endo-
spermatophore and is completed in the reproductive tract of the female (Kiszewski et al. 2001).
Tick spermatozoa lack flagella and move by gliding; this motion is believed to be due to move-
ments of actin filaments in the subplasmalemmal cytoplasm (Witaliński and Dallai 1994).
Sperm development differs in the major taxa within the Ixodida. Balashov (1956) reported
that spermatogenesis is completed during nymphal development in the Prostriata and that cop-
ulation can take place immediately after ecdysis. However, subsequent research suggests that
this is not universal and that spermatogenesis can also occur in the young, newly emerged adults
of some Ixodes species (Oliver 1974). In the metastriate Ixodidae, the early stages of spermato-
genesis occur in the nymphal instar. During the ecdysial process in the fed ixodid nymph (which
has only 1 nymphal instar), waves of differentiation proceed toward the anterior parts of the
testes. Spermatocyte accumulation is biphasic, at least in the Dermacentor variabilis nymphal
testis, occurring as 2 distinct waves during the developmental period (Dumser and Oliver 1981).
Thereafter, maturation of the testes and spermatogenesis are arrested in the ecdysing metastriate
nymph (anautogeny) at the level of the late-prophase primary spermatocytes.
In the unfed nymph of D. variabilis, the testis contains a small number of germ cells sur-
rounded by somatic cells. During nymphal feeding, the germ cells develop, forming cysts of
spermatogonial cells. In many cysts, the cells divide again, forming primary spermatocytes. At
this stage, the elongated testes are filled with an orderly, linear arrangement of spermatogonial
and primary spermatocyte cysts. The latter contain many nuclei in various stages of prophase,
but no later mitotic stages occur. Somatic cells also proliferate during the nymphal ecdysial pe-
riod. These increases in the germinal cell population suggest a steady doubling of the germ cell
population through synchronous divisions of the secondary spermatogonia within each cyst.
The germ cell population increases from fewer than 100 cells prior to nymphal feeding to about
46,000 cells at the end of the ecdysial period, when the males emerge from the nymphal molt
(Dumser and Oliver 1981). No further development beyond the primary spermatocyte stage
occurs at this time, in contrast to the Prostriata and the Argasidae, in which spermatogenesis
progresses without such restraint.
Following eclosion of the unfed male of the Metastriata, primary spermatocytes in special-
ized spermatocysts occupy most of the testis; spermatogonia occur only near the proximal end,
which is the germinal zone (Dumser and Oliver 1981). Spermatogenesis proceeds toward the
posterior parts of the testes. This unusual arrangement is opposite that found in most other ar-
thropods. However, the cell population is not entirely inactive; some spermatogonial “turnover”
does occur, as shown by labeling with 3H-thymidine. At any given time, approximately 400 cells
are in active DNA synthesis in the testis of an unfed D. variabilis male. Spermatogonial cells at
this stage are relatively small. In D. variabilis, nuclear diameters were reported to average 15.1 μm
(Homsher and Sonenshine 1972). The continuation of spermatogenesis in these metastriate ticks
is initiated following the commencement of feeding. In argasids and prostriate ixodids, develop-
ment is continuous, and mature, elongated (but pre-capacitated) prospermia are formed with-
out the need for a blood meal. Regardless of when development is initiated, in this stage, the
great growth phase, the primary spermatocytes grow rapidly and become elongated. The cells
enlarge 5- to 6-fold, and certain cytoplasmic organelles (e.g., Golgi and rough endoplasmic re-
ticulum) become prominent. Gradually, the chromosomal bivalents assume their characteristic
shapes, and nucleoli appear. The first meiotic division is reductional, the second equational (Ol-
iver 1982). In D. variabilis, anaphase I was first detected in males that had fed for 64 hours, and
anaphase II in males fed for 75 hours (Homsher and Sonenshine 1972). Reduction in cell size
may occur following each division, leading to substantially smaller spermatids. After telophase,
the chromosomal vesicles merge, the nucleolus becomes indistinct, and the nucleus appears as
an intensely dark mass. In feeding D. variabilis, prospermia were first seen in males attached to
hosts for 130 hours (Homsher and Sonenshine 1972). In the Prostriata and the Argasidae, as
noted above, spermatogenesis occurs prior to adult feeding and is more advanced than in the
Metastriata, in which it is delayed until feeding; the spermatocysts contain secondary spermato-
cytes and elongated (but not capacitated) spermatids. Thus, argasid and prostriate males are able
to mate before feeding, whereas, as noted above, males of almost all metastriate species require
a blood meal in order to initiate sperm development (reported exceptions include 2 Aponomma
and 1 Amblyomma species in Australia) (Guglielmone and Moorhouse 1983; Oliver and Stone
1983). During spermatogenesis, a pool of pre-spermatogenetic germ cells is retained (or recon-
stituted) and remains available for recruitment as spermatocytes during subsequent blood
meals. This germ cell pool is a continuing source for repeated waves of spermatogenesis fol-
lowing each new blood meal, enabling the ticks to mate many times (Oliver 1986). Analogous
stem cells are believed to occur in parts of the testes of many insects and function in a similar
manner.
Hormones are believed to regulate the precise timing of the different waves of spermatogen-
esis and their correlation with feeding. Injections of synganglion extracts made from fed males
or 20-hydroxyecdysone (20-E) accelerated DNA synthesis in the testes of unfed males (D. varia-
bilis); injections of as little as 0.2 μg 20-E/tick led to an increase in the number of cells synthe-
sizing DNA to an amount similar to that in naturally feeding ticks. The response of the adult
testis to 20-E is not surprising, given that the adult males naturally make this compound (Dees
et al. 1984). However, injections of 20-E did not induce further development of spermatogenesis
(Oliver 1986). It is likely that the regulation of this process is complex and might involve other
hormones and/or neurosecretory substances. Hormonal regulation of reproduction is discussed
further in Chapter 16, but in general, the hormonal regulation of male reproduction is under-
studied, especially relative to that of female reproduction.
Following the series of cell divisions leading to the formation of the spermatids, the sperma-
tids continue their differentiation into elongated spermatids (prospermia) (Figs. 18.9, 18.10).
At first, each primary spermatocyte is rounded, and there are numerous tiny vesicles below
the plasmalemma, the subplasmalemmal cisternae, believed to be derived from infoldings of
the plasma membrane. After the meiotic divisions (not shown) leading to the formation of the
spermatid, these tiny vesicles enlarge, the nucleus moves toward one pole, and the cisternae fuse
to form a single cisternal cavity, or cisternum. The cells soon elongate, and subcellular organ-
elles, especially the unusual tubular endoplasmic reticulum, become apparent. The cisternum
moves to one pole of the cell, and the nucleus to the other. As these events occur, an acrosomal
vesicle forms at the nuclear pole via the fusion of the Golgi complexes. The cell continues to
elongate, including the cisternum and the acrosomal vesicle. At first, one pole appears distinctly
broader than the other. The acrosomal vesicle soon fills with densely staining material and is
gradually transformed into a dense body (Fig. 18.11). As the spermatid elongates, densely stain-
ing material accumulates at one end of the cisternal cavity and gradually spreads along its sides;
this is the location where the “inner core” will invaginate into the cavity. The nucleus, which
until now has remained at a terminal location, elongates and comes to lie along one side of the
cell, between the cisternal cavity and the plasma membrane, fixed to the acrosome. The nucleus
now changes to a broadly fusiform shape. Often, a protruding cap-like or spine-like body ap-
pears at the broader end of the spermatid (operculum) (not shown). The cisternum also changes.
An invagination occurs as an inner cord is formed and is folded into the cisternal cavity, form-
ing a tube within a tube. Externally, there is a smooth outer membrane, below which are the
innumerable spiral subplasmalemmal cisternae that give the spermatid its characteristic ridged
appearance. The outer membrane is shed when the spermatid matures (Wuest et al. 1978).
The greatly elongated spermatids (often 500 μm or longer), termed prospermia, are now stored
in the vasa deferentia and seminal vesicle, ready for insemination (Fig. 18.13). The prospermia
are often termed spermiophores, because after capacitation (see below), only the nucleus will
enter the oocyte, while the remainder of the cell eventually disintegrates (Balashov 1972). Pros-
permia are usually much longer in argasid than in ixodid ticks—perhaps the longest of any ar-
thropods—occasionally exceeding 1 mm in length (Balashov 1972). Spermatogenesis in
Argasidae is similar to that of the Ixodidae. The spermatids are stored mostly in the vasa defer-
entia and/or seminal vesicle, where they remain immotile and presumably less active metabol-
ically. Here they remain for months or even years (Oliver 1982, 1989). For a more detailed review
of the morphological changes accompanying spermatogenesis in ticks, see the work of Oliver
(1982, 1989) and El Said et al. (1981).
4. COPULATION AND SPERM CAPACITATION
4.1. COPULATION
In ticks, as in many other arachnids, sperm transfer is accomplished via a spermatophore.
Males that are attracted by female sex pheromones locate, contact, and mount females. In the
metastriate Ixodidae, mating takes place during the parasitic phase on the host. A male en-
countering a conspecific female will investigate further and, guided by specific pheromones
(Sonenshine et al. 1989; Sonenshine 2008), will locate the gonopore and probe the female’s
genital pore with its mouthparts. Although most metastriate males respond to the female sex
pheromone, 2,6-dichlorophenol, differences in cholesteryl esters (mounting sex pheromone)
on the female body surfaces help guide the males to their conspecific mates. Copulation fol-
lows when males identify the conspecific genital sex pheromones (GSP) during probing of the
female genital pore. Perception of the conspecific GSP stimulates spermatophore formation
and the transfer of semen.
The pear- or flask-shaped spermatophore is formed by male ticks after a period of genital
probing ranging from as little as 10 to 15 minutes in D. variabilis (Sonenshine et al. 1982) to as
long as 3 hours in Ixodes holocyclus (Feldman-Muhsam 1986). Subsequently, the spermatophore
is formed externally from the seminal fluids ejaculated by the copulating male. At first, a clear,
viscous droplet containing mucopolysaccharides, proteins, and other molecules exudes from
the male genital pore. These materials coagulate into membranes to form the sac-like ectosper-
matophore. Next, prospermia and seminal fluid flow into the droplet, causing it to balloon and
turn opaque. Finally, a second droplet, the endospermatophore, is introduced, sealing the sper-
matophore. The process is rapid, requiring only 30 seconds (Feldman-Muhsam 1986). Only 1
endospermatophore occurs in each ectospermatophore in ixodids, but 2 occur in each ectosper-
matophore in argasids. The endospermatophore is bilobed and filled with an amorphous mass
of proteinaceous material (seminal peptides) and innumerable prospermia (elongated sperma-
tids). The molecular contents of the spermatophore are reviewed in Section 5.2. The amount of
semen secreted by male ticks is unknown. In insects (e.g., honeybees), approximately 1 μl of
semen is secreted, ca. 50% of which is seminal plasma. In general, arthropods, including ticks,
ejaculate very small amounts of semen with very high sperm contents (Davey 1985). When for-
mation of the spermatophore is completed, it emerges from the male’s genital pore. The male
seizes it with its chelicerae and, using these same appendages, deposits it in the female’s genital
pore. In ixodids, extensive contortions of the body are required in order to force the sper-
matophore forward to where it can be grasped by the chelicerae and implanted into the fe-
male. The male does not remove its mouthparts from the female’s genital pore until the
spermatophore has arrived near this aperture. In contrast, argasid ticks use their mouthparts
to transfer the spermatophore the entire distance from the male to the female genital pore.
Copulating males also salivate at this time, and this fluid is used to moisten the spermato-
phore and prevent its adhesion to either the male’s or the female’s cuticle (Feldman-Muhsam
1986). The spermatophore is not a simple, passive sac. Rather, it is an active partner in copu-
lation and itself accomplishes the process of sperm transfer. Placed (rather imprecisely) on the
female genital pore, the spermatophore’s neck elongates and evaginates into the female’s ves-
tibular vagina. CO2 generated within the ectospermatophore creates pressure that forces the
entire mass of sperm, seminal fluid, and symbionts into the everting endospermatophore
(Feldman-Muhsam 1986). The latter expands as a pair of bilobed capsular sacs (Argasidae) or
a single lobed structure (Ixodidae). This sequence of events occurs even if the spermatophore
is removed from the ticks. In argasids, each lobe of the endospermatophore detaches (or is
held by narrow strands) to form sperm-filled capsules that proceed into the bifurcate uterus.
In ixodids, the sperm-filled single capsule is stored in the seminal receptacle. Once inside the
female genital tract, sperm probably leave the capsules through their tube-like openings and
pass into the oviducts and ovary. The ectospermatophore remains on the external surface of
the female, attached to the vulva by its narrow neck. However, it soon dries, shrivels, collapses,
and falls off.
In the Prostriata and the Argasidae, mating occurs off the host as well as during feeding.
This process of sperm transfer by means of an external sperm sac, the spermatophore, en-
sures that tick spermatozoa are conveyed via a closed system, with the passage of sperm and
seminal fluid orchestrated precisely by the contents of the spermatophore itself.
4.2. CAPACITATION AND FERTILIZATION

In ticks, mating and copulation stimulate profound changes in the recipient females. In ixodid
ticks, mated females enter rapid engorgement when they suck rapidly, often swelling to 2 or 3
times their pre-mated weight (so-called “big sip”) within 1 or 2 days, and feeding is followed by
drop-off and oviposition. The final stages of spermiogenesis, known as capacitation (spermate-
leosis), occur in spermatids after transfer to the female reproductive tract. According to Oliver
(1989), all ticks transfer uncapacitated spermatids via their spermatophores. This process is not
unique to ticks. It is widespread, and probably occurs in most animals. Following the introduc-
tion of the endospermatophore into the seminal receptacle, the sperm mature, the endosper-
matophore dissolves, and the elongated, fully capacitated spermatozoa migrate to the oviducts.
These events may be triggered by 1 or more polypeptides in the male seminal fluid (Shepherd et
al. 1982). Capacitation is initiated within the spermatophore as a result of secretions from the
MAG (seminal fluid) and continues as the spermatozoa enter the female genital system. The
accessory gland secretions induce opening of the cap-like operculum. The specific compound in
these secretions believed to initiate this process is a polypeptide with a molecular weight of ap-
proximately 12,500 Da, the characteristics of which vary in different tick families (Oliver 1986).
To date, the molecular structure of this polypeptide has not been determined. After the oper-
culum opens, the inner chord of the sperm cell elongates while the outer sheath slides over it,
carrying the nucleus with it. The greatly elongated spermatozoan now has the nucleus and the
acrosome at its “posterior” end, next to the invaginated acrosomal canal. A long internal canal
extends from the posterior end. The clavate anterior end becomes covered with numerous
ridges. When fully mature, the elongated spermatozoa may exceed 1,000 μm. Most spermatozoa
are stored in the seminal receptacle in ticks of this family.
In addition to the morphological changes that occur, capacitation also enables the sperma-
tozoa to become motile. Fibrils located beneath the plasma membrane may represent myofibrils;
waves of fibrillar contractions are thought to occur, producing a gliding motion. The spermato-
zoa travel along the oviducts of the female reproductive system to reach the oocytes. Gliding is
reported to be accomplished via movements throughout the entire length of the spermatozoan
and contortions of the anterior tip of the sperm head that allow the mature sperm to move
through the female genital tract to fertilize the ova without any assistance from the oviducts
and/or ovary (Resler et al. 2009; White 2011).
Capacitation and the morphology of the spermatozoa in argasid ticks are similar to that of
ixodids. The anterior part of the sperm cell is covered by longitudinal ridges of cellular pro-
cesses, beneath which are bundles of microfibrils. El Shoura (1987) suggests that the microfibrils
correspond to actin and myosin fibers that, through their contractile activity, produce the
gliding motion of these cells. In the argasid tick Ornithodoros tholozani, the anterior end is head-
like and separated from the body by a narrow collar (Feldman-Muhsam and Filshie 1976). In
argasids, the capacitated spermatozoa are stored in the uterus (there is no seminal receptacle).
From this supply, which might last for years in the case of the long-lived argasids, oocytes gen-
erated over the course of several gonotrophic cycles may be fertilized without the need for addi-
tional inseminations. It should be noted that in many argasids, spermatophores from newly
emerged, very young, or starved males might be sterile. In O. tholozani, 70% of the first copula-
tions involving starved males were unsuccessful (Feldman-Muhsam 1986).
5. MOLECULAR BASIS OF SPERMATOGENESIS,

SPERMIOGENESIS, AND CAPACITATION
5.1. SPERMATOGENESIS AND SPERMIOGENESIS IN INSECTS

In insects, molecular studies on male reproduction have identified several molecules that
regulate specific components of sperm development, motility, and passage to females during
mating. Early growth and development from the primordial stem cell line in Drosophila spp.
(and, presumably, in many other arthropod species) is promoted by insulin-like peptides.
Insulin signaling is a major factor leading to sperm production in this species (Ueishi et al.
2009). Other classes of proteins are also involved in spermatogenesis. Proteases (e.g., angio-
tensin-converting enzyme [ACE]) are among the proteins known to be important for male
fertility. In Drosophila, ACE is an important regulator of spermiogenesis, presumably because
it processes a regulatory peptide required for spermatid differentiation. ACE is also found in
the secondary cells of the MAG and is secreted during copulation. However, its role in stim-
ulating female reproduction has not been determined (Rylett et al. 2007). Another important
regulator of spermatogenesis is poly(A) polymerase GlD2, which was found to be essential
for post-meiotic development of the spermatids in Drosophila. GlD2 is believed to regulate
F-actin assembly and chromosomal reorganization during late-stage spermatogenesis, essen-

tial for spermatid elongation (Sartain et al. 2011). Nieman-Pick C1, which controls sterol traf-
ficking, was also found to be important in spermatogenesis, particularly in membrane
remodeling, which is dependent upon sterol utilization (Wang et al. 2010). Another impor-
tant gene class includes the tubulins, particularly β-tubulin, which forms stable heterodimers
with α-tubulin that are essential for the formation of the sperm tail axoneme in Drosophila
(Popodi et al. 2005). Many other regulatory proteins also contribute to spermatid elongation
and cellular remodeling, such as myoV, a member of the myosin class, which regulates actin
and microtubule development required for these processes (Mermall et al. 2005). Some cate-
gories of sperm proteins have only recently been discovered and have no known function,
such as the leucine amino peptidases that are present in extraordinary concentrations in Dro-
sophila sperm (Dorus et al. 2011).
Transcriptomes have proven useful for the study of the molecular expression of male repro-
ductive gland proteins in Drosophila and mosquitoes, as well as in other animal groups.
In the mosquito Aedes aegypti, 101 sperm-associated proteins were identified in the testes
and seminal vesicles via proteomic analysis (tryptic digestion/liquid chromatography–tandem
mass spectrometry [LC-MS/MS]), of which 49 were believed to play some role in spermatogen-
esis and/or sperm maintenance (Sirot et al. 2008).
Dottorini et al. (2007) identified 46 proteins from the MAGs of Anopheles gambiae mosquitoes,
including 25 specific for the male reproductive tract. Among the latter was Acp 70, the sex peptide
known to function as the principal modulator of female post-mating reproductive behavior. Nu-
merous other homologues to Drosophila accessory gland proteins were also reported. Studies by
Sirot et al. (2008) identified 63 putative male reproductive gland proteins in the male reproductive
organs of Aedes aegypti, 21 of which were also found in the reproductive organs of mated females;
most of the A. aegypti male proteins were similar to male reproductive gland proteins found in other
arthropods. The classes of proteins found included proteases, protease inhibitors, chaperones, in-
nate immune peptides, oxireductases, lipases, antimicrobial peptides, and others; notably, 25% were
proteases and 11% were protease inhibitors. Proteolysis regulation by proteases and protease inhibi-
tors is believed to be important in regulating post-coital sperm activity or mated female responses.
In a more recent study, Sirot et al. (2011) used stable isotope labeling coupled with pro-
teomics to distinguish seminal fluid from sperm-associated proteins that were transferred to the
female genital tract during copulation. Among the seminal fluid proteins were several predicted
membrane-bound and intracellular proteins believed to have been released via apocrine secre-
tion from the accessory glands. These findings, as well as the others cited previously, suggest that
accessory gland proteins contribute to stimulating female reproductive activity.
Proteomic methods have revealed an even greater complexity of the Drosophila sperm pro-
teome, and presumably of those of many other arthropods, than previously believed. Recent studies
by Wasbrough et al. (2010) revealed a total of 1,108 proteins, more than half of which are novel.
5.2. SPERMATOGENESIS AND SPERMIOGENESIS IN TICKS

Relative to that in insects, much less is known about the molecular biology of male reproduction
in ticks. Early studies by Weiss et al. (2002) using a cDNA library made from the testis and vas
deferens of fed Amblyomma hebraeum detected 35 genes that were putatively up-regulated in tis-
sues of fed animals relative to those of unfed animals. Included were 2, an adenosine triphosphate
(ATP) synthase and acylphosphatase (9.8 and 10.4 kDa basic proteins, respectively), with homol-
ogies to similar genes in D. melanogaster. Subsequent studies using RNA interference showed
that these genes were essential for sperm development in this tick species (Guo and Kaufman
2008). Subsequently, a recombinant protein, voraxin (see below for a description), based on 2
proteins derived from the original male tick cDNA library, was found to stimulate virgin females
to engorge to repletion and lay eggs (Weiss and Kaufman 2004). Further studies showed that
when normal, mated females fed on a rabbit immunized with the voraxin proteins, most failed to
fully engorge and oviposit. However, it is possible that at least 1 of the voraxin proteins, engorge-
ment factor alpha (EFα), might also play an important role in spermatogenesis. EFα is a male-
specific protein; in D. variabilis, it is found only in fed males. According to records published in
GenBank, EFα (but not EFβ) has also been reported in other ixodid ticks, namely, R. microplus,
I. scapularis, Haemaphysalis longicornis, and Dermacentor silvarum. Partial sequencing of ex-
tracts from D. variabilis males revealed a 2,704 bp transcript (much larger than that reported in
A. hebraeum); although the full-length molecule has not been sequenced, no evidence of a secre-
tion signal could be found in the partial sequence known thus far. The transcript for EFα was also
identified in a cDNA library created via 454 pyrosequencing of RNA from the male reproductive
system (discussed below). Males injected with dsRNA EFα (RNA interference) appeared to de-
velop normally and were able to mate and inseminate females without disruption of normal re-
productive development (Donohue et al. 2009). Thus, although widespread in different tick
species, the true role of EFα in male reproductive activity is enigmatic. That, along with the ab-
sence of EFβ, does not provide compelling support for a role in stimulating female engorgement
and post-mating reproduction.
Another protein believed to be important in tick spermatogenesis is subolesin, a regulatory
protein highly conserved in numerous different tick species. Although its specific mode of ac-
tion is unknown, silencing subolesin via RNA interference disrupted tick feeding success and
salivary gland development (de la Fuente et al. 2006). Studies by Smith et al. (2009) showed that
the silencing of subolesin led to disruption of spermatogenesis, resulting in sterile males. Histo-
logical observations of the testis showed deformed or fragmented prospermia along with
cellular debris.
In order to better understand the molecular basis of sperm development and spermatophore
synthesis, a transcriptome of the cDNA library of the combined MAGs and testis/vas deferens
(TVD) of D. variabilis was created via 454 pyrosequencing (Sonenshine et al. 2011). Of the more
than 12,800 contigs assembled from the raw reads, Gene Ontology (biological processes) as-
signed 3,866 to 73 different level III categories, including categories of greatest importance for
spermatogenesis, spermiogenesis, and copulation: reproductive processes and sexual reproduc-
tion (0.6%), cell adhesion (2.8%), innate immunity (0.03%), oxidation reduction (3.5%), actin-
filament-based processes (0.29%), developmental processes (1.6%), and catabolic process,
primarily proteases (3.1%). Of special interest for tick spermatogenesis were serine/threonine
kinase, ADAM-metalloproteases, ferritins, cathepsin L-like cysteine proteases, and acylphos-
phatases that were found only in the MAG/TVD. Also present were heat shock proteins HSP70
and HSP90, glutathione-S-transferase, thioredoxin, quinone oxireductases, serine protease,
phospholipase C gamma, actin, alpha and beta tubulins, and calreticulin, all found in both the
MAG/TVD and the spermatophore. Additional spermatogenesis-related proteins recognized
since publication of Sonenshine et al.’s (2011) work include HSP60 and myosin, found only in the
MAG/TVD, and dynein, found in both the reproductive system and spermatophore proteomes.
Some of these proteins are similar to those known to have important roles in spermatogenesis in
Table 18.1: Expression of selected genes of interest from the Dermacentor variabilis
male transcriptome in response to feeding/courtship as determined via
quantitative real-time PCR.
Category Gene Contig Folda P-value Significance

Reproduction Astacin metalloprotease 03261 7.06:1 1.91 P = 0.20
Serine/threonine kinase 11582 1.61:1 0.49 P = 0.48
Zinc metalloprotease 00843 5.01:1 6.23 P = 0.016
Proteases Cathepsin B 00689 1.03:1 0.14 P = 0.90
Cysteine proteaseb 05033 0.21:1 3.08 P = 0.05
Serine protease 12380 22.0:1 12.39 P = 0.003
Trypsin 03705 13.5:1 5.45 P = 0.003
Protease inhibitor Serine protease inhibitor 11029 0.10:1 7.31 P = 0.003
Oxidase stress Thioredoxin 09407 1.13:1 0.49 P = 0.63
Structure/adhesion Calreticulin 10500 0.39:1 2.29 × 10−7 P = 0.001
Keratin 01860 0.89:1 5.14 × 10−12 P = 0.001
Laminin 12680 0.45:1 0.0005 P = 0.001
Tetraspanin 10467 2.93:1 0.002 P = 0.001
Lipases Phospholipase C 06555 1.20:1 0.64 P = 0.58
Neuropep receptorc G protein–coupled receptor 08424 2.97:1 3.54 P = 0.002
Notes: Transcriptome of extract made from fed Dermacentor variabilis male accessory glands, testis, and vas deferens
(MAG/TVD). Males were exposed to females (defined as courtship) but were not allowed to copulate. Expression com-
pares fed versus unfed males for the MAG/TVD.
From Sonenshine, D.E., Bissinger, B.W., Egekwu, N., Donohue, K.V., Khalil, S.M., and Roe, RM. (2011) Transcriptome
of the testis-vas deferens-male accessory gland and proteome of the spermatophore from Dermacentor variabilis (Acari:
Ixodidae). PloS ONE 6:e24711.
a
Change in fed male relative to unfed male MAG/TVD.
b
Equals Cathepsin L.
c
Neuropeptide receptor.
insects (e.g., α and β tubulins, myosin, dynein, and others). Studies by Wasbrough et al. (2010)
identified 40 proteins in the Drosophila melanogaster proteome that fall in this category. Most
affect sperm development and motility. Especially abundant were actin and tubulins, which are
important components of the sperm axoneme. Others play important roles in sperm capacita-
tion, motility, and other functions related to copulation and fertilization, which are discussed
further below (Section 5.2).
Although a detailed review of the functions of each of these numerous proteins is beyond
the scope of this chapter, several do merit special consideration because of their high abun-
dance, extent of up-regulation, or novelty. Serine/threonine kinases, among the most frequently
sequenced transcripts in the male transcriptome, are believed to function in the reorganization
of sperm chromatin during spermiogenesis, a function that facilitates capacitation (Sabeur
et al. 2008). In D. variabilis, this protein was also identified via LC-MS/MS in the male repro-
ductive organs, but not in the spermatophore. Transmembrane proteins are also important for
sperm development, as well as for cell–cell fusion (e.g., the distintegrin-metalloprotease family
of proteins [ADAM]). ADAMs may also disassociate and execute protease functions later
during sperm transport (Primakoff and Myles 2000). Astacin- and zinc-metalloproteases were
also strongly up-regulated as determined via real-time quantitative polymerase chain reaction
(qPCR) (Table 18.1). These and other non-secretory proteins and peptides may be liberated via
holocrine secretion from MAG cells during spermatophore formation (see Sections 2.2.3 and
5.3.2). Peptidases constitute another class of proteins important for sperm development. Tran-
scripts for at least 18 different peptidases were found, including trypsin protease, carboxypepti-
dase, dipeptidyl carboxypeptidase, serine protease, leucine aminopeptidase, and cathepsin
L-like cysteine protease. Particularly noteworthy for a possible role in spermiogenesis was the
high redundancy of transcripts for serine protease, trypsin, and carboxypeptidase. Serine pro-
tease and trypsin were strongly up-regulated during feeding (Table 18.1). Sonenshine et al.
(2011) also found transcripts for acylphosphatases, specifically transcripts that matched similar
peptides (9.8 and 10.4 kDa proteins) identified in the male reproductive organs of A. hebraeum
that were reported to be important in spermatogenesis in that species. Males in which these
genes were knocked down via RNAi demonstrated abnormal spermiogenesis, and females
mated to these males demonstrated reproductive failure (Guo and Kaufman 2008). Also im-
portant were the ferritins, expressed with high abundance in the MAG and testis and believed
to be an important indicator of the high metabolic activity of these organs during reproduction.
Ferritins also might function as antimicrobials. Their loss has been shown to disrupt reproduc-
tive activity in Ixodes ricinus females (Hajdusek et al. 2009). Presumably, this would also affect
reproduction in the males.
5.3. SPERM CAPACITATION AND STIMULATION

OF FEMALE REPRODUCTION
5.3.1. Insects
In insects, copulation stimulates the mated females to begin reproductive activity. The sem-
inal fluid contains numerous proteins, mostly from the MAGs, known as accessory gland
proteins (Acps). Several of these seminal fluid peptides, especially male sex peptide, have
been identified and shown to be the primary factors in stimulating female reproductive ac-
tivity (summarized in Gillott 2003; Wolfner 2007). Others are important for nourishing the
spermatids and facilitating their motility. In Drosophila, male sex peptide was found to
stimulate increased food uptake in females (Carvalho et al. 2006), a finding of particular
interest with regard to ticks, as the switch from slow to rapid feeding in mated females that
initiates the full sequence of reproductive processes is unknown. In the mosquito Aedes ae-
gypti, different classes of seminal fluid proteins included proteolysis proteins and regulators,
lectins, lipases, oxireductases, and others (Sirot et al. 2011). At least 93 male-derived seminal
fluid proteins were identified that were released into the semen from the cells of the acces-
sory glands via apocrine secretion. Seminal fluid proteins were distinguished from proteins
from testes, seminal vesicles, or the spermatids through separate analyses. Those that were
transferred to females were determined by comparing the proteome of the reproductive
tracts of mated females with the seminal fluid proteins. Especially noteworthy was the re-
port of numerous intracellular or membrane-bound proteins, ATPases, dipeptidyl pepti-
dases, glutathione S-transferase, ACE, and others (Sirot et al. 2011). The authors suggest that
these proteins are evidence of secretion (via either apocrine or holocrine processes) by the
MAGs, rather than just contaminants resulting from cell rupture. Other seminal fluid
proteins in the males of this mosquito species included the proteolysis regulators trypsin,
zinc carboxypeptidase, a metalloprotease, serine protease inhibitors, and a sterol carrier

(Niemann-Pick C2) that stimulates ecdysteroidogenesis in the recipient females. Many of
these seminal peptides were found in the females following mating. This is especially rele-
vant to reproduction in ticks, as the injection of physiological concentrations of the ecdys-
teroid 20-E into feeding virgin females was sufficient to stimulate vitellogenesis, oocyte
development, and vitellogenin uptake independent of feeding to full engorgement (Thomp-
son et al. 2005).
Seminal fluids of many insects also contain high concentrations of prostaglandins (PGs),
usually in the form of PGE2. PGs are long-chain fatty acids widely distributed in many organs
and tissues. PGs exhibit a diverse array of pharmacological effects, among the most important
being stimulation of the contraction and relaxation of smooth muscle. In crickets, PG synthetase
has been reported from the testes, seminal vesicles, vas deferens, and spermatophore. Subse-
quently, PG synthetase was found in the mated females. Similar findings have been reported for
ticks, and it was believed that PG or the enzyme PG synthetase transferred with the seminal
fluid served as the oviposition stimulating male factor by inducing the production of PG in the
female reproductive organs (summarized by Kaufman [2008]). However, other studies (Mur-
taugh and Denlinger 1987) showed that the process is much more complicated and depends
primarily on sperm-associated factors for continued, long-term oviposition. The possible role of
PGs in tick reproduction is discussed below.
5.3.2. Ticks
Sonenshine et al. (2011) described the proteins in tick semen based on the fed male transcrip-
tome and spermatophore proteome. In order to identify the seminal proteins and peptides in
the tick reproductive organs and spermatophore, freshly extruded spermatophores and male
reproductive organs from copulating males were collected and separated via protein gel elec-
trophoresis. Analysis of the samples showed distinct differences in protein/peptide expression
(Fig. 18.22). Among the numerous bands expressed in the spermatophore extract were 4 that
were either absent in or much more strongly expressed than in the MAG/TVD. These bands
were selected for protein analysis via LC-MS/MS. Proteins of similar molecular weights were
also reported to occur in the MAGs of Drosophila spp. and other insects (Ravi-Ram and Ra-
mesh 2007). Differences in protein expression were also noted between extracts of tissues
from unfed and fed males, and bands unique to the latter were also submitted for analysis via
LC-MS/MS.
Analysis of the proteome of the spermatophore revealed a total of 461 proteins and pep-
tides. Classification of the different categories was based on Gene Ontology, as well as com-
parison with similar studies in insects (Avila et al. 2010; Wasbrough et al. 2010; Sirot et al.
2011). Among these numerous tick seminal fluid proteins/peptides were several known or
believed to be important in sperm motility, mating, and copulation in other arthropods,
notably the following:
1. Neprilysins, sperm-associated metalloprotease membrane-bound enzymes that

hydrolyze signaling peptides.
2. ACE (dipeptidyl carboxypeptidase II), which is expressed in the MAGs in D.
melanogaster but is lost during mating, consistent with transfer to the female via the
seminal fluid (Rylett et al. 2007).
FIGURE 18.22: Photographs of protein gels of extracts from the spermatophore and male accessory
gland/testis vas deferens (MAG/TVD) from D. variabilis fed males. A, Coomassie Blue–stained gel of
the proteins from the male reproductive system and spermatophore. B, Silver-stained gel comparing
protein bands present in the MAG/TVD of fed and unfed males. For a detailed discussion of these
findings, see Sonenshine, D.E., Bissinger, B.W., Egekwu, N., Donohue, K.V., Khalil, S.M., and Roe, RM.
(2011) Transcriptome of the testis-vas deferens-male accessory gland and proteome of the
spermatophore from Dermacentor variabilis (Acari: Ixodidae). PloS ONE 6:e24711. MW, molecular
weight; Sharp, molecular weight stain (Invitrogen); Sph, spermatophore.
3. Numerous peptidases reported to function in various aspects of male reproduction, including

at least 7 digestion-associated peptidases, including trypsin protease, carboxypeptidase,
serine protease, and leucine aminopeptidase. qPCR showed significant up-regulation
of serine protease and trypsin, supporting the importance of these seminal peptides
(Table 18.1).
4. Protease inhibitors, including cystatin (cysteine protease inhibitor), aprotinin, and
trypsin inhibitor.
5. Hydrolases, including mannosyl-3-phosphoglycerate phosphatase, lactate dehydrogenase,
and lactamase domain protein.
6. Lipases, including phospholipase C and other phospholipases.
7. Oxidative stress proteins. These proteins are highly conserved throughout the Metazoa
and are believed to be essential for protecting sperm DNA from oxidative damage.
Among the most important in this category is spermine (Khan et al. 1992). Spermine
has long been thought to play a vital role in fertilization and semen coagulation, and
perhaps even as a bacteriostatic agent (El Shoura 1987). A transcript for spermine
(peroxisomal N(1)-acetyl-spermine/spermidine oxidase) was found in the trans-
criptome (contig 01448, e value = 4.1 × 10−4), as well as in the I. scapularis genome
(gene ISCW005123; GB EEC05394). The corresponding peptide was found in the
male reproductive organs. Other antioxidant proteins found in the proteome of
the spermatophore include quinone oxireductase, pyridine nucleotide disulphide
oxidreductase, superoxide dismutase, glutathione S-transferase, thioredoxin, and

others.
8. Environmental stress proteins, all of which were heat shock proteins (HSP70, HSP90,
and others).
9. Immune proteins including lysozyme, macrophage migration inhibitory factor, and
ML-domain protein.
10. Cell-adhesion-related proteins including actin, alpha- and beta-tubulin, cytokeratin,
keratin, calreticulin, and others.
11. Vesicle transport proteins and receptors (synaptosomal-associated proteins [SNAPs]
and target SNAP receptors). Transcripts for these proteins (contig 10966, e-value =
1.6E−97) and their receptors were found in the male transcriptome, and a synaptic
vesicle-associated membrane protein similar to I. scapularis synaptobrevin was found
in the MAG extract but not in the spermatophore. Vesicle transport proteins (SNAPs)
mediate vesicle movement to and fusion with the plasma membrane for secretion of the
vesicular contents. It is likely that this protein system is responsible for the apocrine
and/or holocrine secretory activity described previously in the MAGs.
12. Dynein. This protein was found in both MAG/TVD extract and the spermatophore.
This is believed to be a sperm protein that facilitates sperm motility. Dynein is
an intracellular motor protein (also called a molecular motor or motor molecule)
that converts the chemical energy contained in ATP into the mechanical energy of
movement.
For a more detailed description of the spermatophore proteome, see Supplementary Table
S18.1 (the URL for supplementary material can be found at the end of the Table of Contents).
PGs are potent excitants that are found in the semen of some insects, as well as that of mam-
mals and various other animals (Davey 1985). Although PGs have not been reported in the
semen of ticks, Sonenshine et al. (2011) found that the transcript for PGE2 synthase was present
in the transcriptome of the D. variabilis male reproductive organs. As noted above (Section
5.2.1), PGE2 synthase was found in the male reproductive organs and spermatophores of several
insect species, as well as in the reproductive tracts of mated tick females (but not in virgin fe-
males) (summarized by Kaufman [2008]). However, PGs occur in high concentrations in tick
saliva, and it is important to note that male ticks salivate while implanting their spermatophores
into the females. Thus, it is possible that the saliva of copulating males, which contains high
concentrations of PGE2 (Sá-Nunes et al. 2007), might introduce this important stimulant into
the vulvas of mated females.
An unexpected discovery was the finding of alpha and beta hemoglobin in both the sper-
matophore and the proteome of the MAG/TVD. It is possible their presence in the MAG/TVD
resulted from host blood contamination during dissection or from hemolymph contamination.
However, the presence of hemoglobin in the spermatophore suggests that the proteins are se-
questered and secreted by the MAG/TVD either for a specific function or as a host contami-
nant incorporated during the formation of this structure. Fragments resulting from hemoglobin
digestion have been shown to have antimicrobial activity in ticks (Nakajima et al. 2002; Sonen-
shine et al. 2005) and could have a similar role in the spermatophore and female genital tract,
which might explain their presence in the MAG and spermatophore.
In ticks, as in insects, copulation induces profound physiological changes in the repro-
ductive biology of the mating females. However, the specific molecule, the so-called male
engorgement factor (EF) that stimulates female feeding to repletion, has remained an
enigma. Studies summarized by Oliver (1989) implicated catecholamines, PGs, and possibly
a juvenile-hormone-like gonadotrophic hormone in inducing female reproductive activity.
Rather than a single molecule, Oliver (1989, p. 416) suggested that “a complicated stimuli-
feedback relationship exists among the nervous, endocrine, and reproductive systems prior
to and during egg maturation and oviposition.” More recently, Weiss et al. (2002) showed
evidence of an EF based on the screening of 35 fed-male-specific sequences from the testis
and vas deferens of the African bont tick, Amblyomma hebraeum. In subsequent studies, as
noted above (Section 5.2), they showed that the male EF comprised 2 small peptides, EFα
and EFβ, which they termed voraxin. Injection of the recombinant forms of these peptides
stimulated most virgin females to feed to repletion and initiate reproductive activity (Weiss
and Kaufman 2004). However, attempts to replicate these initially encouraging findings in
other tick species have not been successful thus far. In the dog tick, D. variabilis, Donohue
et al. (2009) found an Efα-like transcript that was expressed only in fed males (GB: ABM92922),
but no evidence of EFβ. The absence of the latter suggests that voraxin might not be the long-
sought EF. Moreover, silencing EFα via RNAi in male ticks failed to significantly reduce the
engorgement weight of females allowed to mate with dsRNA-EFα-treated males relative to
controls. Finally, the absence of the EFα protein in the D. variabilis spermatophore (Sonen-
shine et al. 2011) indicates that it is not a secreted peptide.
If voraxin is not the male EF, what is? In an effort to address this question, Donohue
et al. (2009) injected extracts of reproductive tissues from fed males or mated females into
feeding virgin females to determine whether these extracts would stimulate the females to
engorge to repletion. Significant evidence of a male EF was found using suspensions of the
combined fed MAG/TVD, which stimulated 50% of feeding virgin females to fully engorge
and oviposit brown eggs (Table 18.2). However, the effect was abolished when only the
aqueous fraction of the homogenates was injected, which suggests that the male EF is a non-
secreted membrane-bound or intracellular protein. Tests with aqueous extracts of other tis-
sues (e.g., fed MAG alone, fed TVD alone), mated female synganglia, and even Sephadex
beads all failed to stimulate full female engorgement, although an exception was found with
extracts made from the vagina/seminal receptacles of inseminated females, which did stim-
ulate rapid engorgement and oviposition of vitellogenic eggs. Based on these findings, it
may be hypothesized that the male EF is bound to the spermatozoa and transferred to the
recipient females during copulation. However, it is likely that the male EF is a complex of
many proteins and peptides, rather than a single molecule or pair of closely related mole-
cules, a possibility recognized by Oliver (1989). A noteworthy finding in support of this
hypothesis was reported by Thompson et al. (2005), who observed that in ticks, the injection
of physiological concentrations of the ecdysteroid 20-E stimulates vitellogenesis and vitel-
logenin uptake into the ovary. Clearly, female ixodid ticks have an ecdysteroidogenic path-
way, believed to be regulated by unknown factors from the synganglion (Lomas and Rees
1998), which we may hypothesize is more intensely up-regulated by the male EF introduced
during mating.
Little is known about the events surrounding fertilization. Even the site of fertilization,
whether in the oviducts or the ovary, is unclear, although there is considerable evidence for the
latter (see Chapter 17). Having contacted an oocyte, the spermatozoan penetrates the cell. How-
ever, only the sperm nucleus enters the oocyte. The mechanism by which the sperm locate and
penetrate the oocytes is unknown.
Table 18.2: Effect of the injection of combined male Dermacentor variabilis male accessory glands and testis
into feeding virgin females.
Percent repletea and mean weight (mg) of females after feeding

Experiment MAG/TVD Solvent (PBS) only Untreated ♂♂ absent Untreated ♂♂ present
% replete (n) Wt ± SD % replete (n) Wt ± SD % replete (n) Wt ± SD % replete (n) Wt ± SD
Suspension injected into 50.0 (24) 341.3 ± 161.4b 0 (27) 143.5 ± 69b 16.7 (12) 252.0 ± 53.9c 88.2 (17) 528.5 ± 61.4c
body cavity
Aqueous extract injected 0 (14) 30.3 ± 20.8b 0 (15) 152.4 ± 85.3b ND ND ND ND
into body cavity during
feeding
Suspension injected into 9.8 (61) 159.9 ± 137.4d 0 (52) 53.2 ± 20.9d ND ND ND ND
512
gonopore
Aqueous extract < 100 kDa 0 (10) 157.8 ± 103.7c 0 (25) 96.4 ± 48.3c ND ND ND ND
injected into gonopore
a
Replete is defined as an adult female that has reached an engorgement weight ≥ 300 mg.
b
Two replicates.
c
One replicate.
d
Three replicates.
MAG, male accessory gland; ND, not done; PBS, phosphate-buffered saline; SD, standard deviation; TVD, testis/vas deferens; Wt, weight (mg).
Reprinted from Donohue, K.V., Khalil, S.M., Sonenshine, D.E., Ross, E., Mitchell, R.D., and Roe, R.M. (2009) Male engorgement factor: role in stimulating engorgement to repletion in the
ixodid tick Dermacentor variabilis. J. Insect Physiol. 55:909–918, with permission from Elsevier.
Further studies are needed to determine the function of the numerous proteins that are known
or believed to be associated with male reproduction. Special attention should be aimed at the
function of these proteins in the different phases of male reproductive activity, including which
proteins are responsible for spermatogenesis and spermiogenesis and which of the many
proteins in the semen are transferred to the female. The latter can be determined in part using
methods similar to those applied to mosquitoes (Sirot et al. 2008). Obvious questions include
which of the numerous semen proteins contribute to the formation of the spermatophore, facil-
itate spermatid survival (e.g., immune peptides, antioxidants, etc.) and motility, trigger capaci-
tation after passage into the reproductive tract of the recipient females, and, finally, stimulate
female engorgement and post-mating reproduction. Studies also are needed to separate the
spermatids from the rest of the semen in order to identify and determine the function of the
seminal proteins versus that of the spermatid proteins and peptides.
Understanding the role of male pheromones in the regulation of female behavior, blood
feeding, and the development of eggs, including the process of vitellogenesis, has been difficult.
The hypotheses have ranged from simple ones involving only 2 proteins, EFα and EFβ, to some-
thing more complex involving many male factors that might be acting alone or in conjunction
with processes in the reproductive tract (or even outside of this system) in adult females. It
should be cautioned that the factors and/or pheromones are not necessarily limited to proteins.
Understanding this male regulation of female function is expected to be challenging. A global
examination of gene expression in the different components of both the male reproductive
system and the female reproductive system will be needed during the course of adult develop-
ment in order to achieve this goal. In addition, an understanding of how females regulate their
feeding behavior, ecdysteroid biosynthesis, egg development, and the movement of materials
from the female to the egg will be critical. Unfortunately, the work so far suggests that the de-
tailed knowledge of male reproduction in insects might not be useful in finding the same factors
in ticks. However, the general insect strategies will continue to be a useful starting point for tick
research in the future, and the general strategies in insect reproduction might still apply to ticks.
Although significant advances have been made in the past decade in understanding the
regulation of female adult development, much less is known about the regulation of males.
More research is needed in order for the roles of male feeding and nutrition, host and female
interactions, sensory systems, and the synganglion in this process to be understood. For ex-
ample, what hormones, neurohormones, and receptors are at play in the development of the
male reproductive system and copulation? We know that physiologically significant increases
in the concentration of 20-E in post-mated females are essential for stimulating vitellogenesis
and the uptake of vitellogenin into the oocytes (Thompson et al. 2005), calling into question the
existence of a male ecdysteroidogenic factor similar to that described in the seminal peptides of
mosquitoes (Sirot et al. 2011). What changes occur in the expression of male reproductive
proteins and peptides during courtship and the transfer of the spermatophore? The variability
of the different life strategies among ticks in terms of reproduction suggests that several model
systems will be needed.
There are some significant applied advantages to the study of tick male reproduction and its
regulation of female reproduction, for many of the same reasons that this question is under in-
vestigation in insects. Knowledge of the identity and function of male factors regulating sperm
development, motility, nourishment, and the male EF might lead to novel methods for disrupt-
ing tick reproduction, perhaps even allowing the sterilization of male ticks in the field. Com-
bined with conventional tick control procedures, they could be used as part of an overall
integrated pest management approach for tick control. Also, understanding how the male con-
trols female reproduction could provide new leads for controlling reproduction in ticks.
ACKNOWLEDGMENTS
The research described in this chapter was supported in part by grants from the National
Science Foundation (IBN-0315179 and IBN-0723692). The authors are most grateful to
Ms. Lou G. Boykins, Integrated Microscopy Center, University of Memphis, Memphis, TN,
for her assistance in preparing tick specimens for transmission electron microscopy (TEM)
and light microscopy examination. The TEM images were taken with an AMT JEOL 1200
EX transmission electron microscope. The light micrographs were taken with an Olympus
BX 51 microscope and DP70 digital camera. We also thank Dr. Paul J. Homsher for his crit-
ical review of the manuscript.
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NOTE
INDEX
Page numbers in italics indicate figures and tables.
A Aeropyles, 80, 241, 242, 244, 246

AAA pheromone. See Agglutination, 228
Aggregation-attraction-attachment Aggregation-attraction-attachment (AAA)
pheromone pheromone, 376–377, 379, 386
Absorption, in midgut, 139–141 Agranular acini, 88, 164, 165
Acari, 4 Air filtration, 246
Accessory glands. See also Male accessory gland ALA-D (δ-aminolevulinate dehydratase), 229
anatomy of, 86, 95, 454–455, 489–495 Albumin, 139
lobular, 450, 451, 455, 461 Aldehyde dehydrogenase, 150
mites and, 497 Allantoicase, 217
tubular, 454–455 Allantoinase, 217
ACE. See Angiotensin-converting enzyme Allatostatin B. See Myoinhibitory peptides
Acetylcholine (ACh), 350–351 Allatostatins, 316, 318, 322–323, 333, 334, 418,
Acetylcholine esterase (AChE), 350–351 430, 434–437
Acini Allatotropins, 316, 318, 324, 327, 418
agranular (type I), 88, 164, 165 Allomones, overview of, 370, 371
granular Alloscutum, 77, 79, 116
anatomy of, 144–145, 165–168, 166, 167 Alveoli. See Acini
changes in during feeding, 168–169 Amblyomma spp.
overview of, 88 feeding behavior of, 68
type II, 165–166, 166 feeding-induced plasticization in, 114
type III, 166–167, 167 fossil record and, 40
type IV, 167–168 life cycle of, 61
Acp 70, 504 molecular phylogenetics of, 40–41
Acrosomal canal, 503 morphologic identification of, 30
Acrosomal vesicle, 500 neuropeptides of, 341, 342
Acylphosphatases, 507 population genetics of, 42–43
ADAMs, 506 recent developments in classification of, 47
Adipocytes. See Trophocytes Ambush strategy, 13
Aedeagus, 484 Americanin, 174
Aedes aegypti, 217 Americin, 267, 296
520 Index
γ-Aminobutyric acid (GABA), 348 of ostia, 90, 242, 244, 261

g-Aminobutyric acid (GABA), 348–349 overview of, 8–9, 74–75, 75, 76–77, 78,
δ-Aminolevulinate dehydratase (ALA-D), 229 79, 83, 84
Amitraz, 349 of podosoma, 75–77
Ampulla, 450, 485 of preoral canal and foregut, 84–87
Anaerobic respiration, 254 of rectal sac, 207
Anal canal. See Rectum of rectum, 87, 211
Anal groove, 80 of reproductive system, 94–95
Anaphylatoxin, 176 of reproductive system (female)
Anaplasma spp., 5, 178, 180 cervical vagina, 454
Anaplasmosis, 5, 6, 178–179 connecting tube, 453–454
Anastomosing tracheae. See Tracheae Gené’s organ, 456, 456–457
Anatomy ovary, 451, 451–453, 452
of anus, 211 overview, 449–451, 450
of arterial vessels and sinuses, 263–264 oviducts, 453
of capitulum, 80–82, 81 seminal receptacle, 454
of circulatory system and hemolymph, 89–90, tubular accessory glands, 454–455
258–259, 259 vestibular vagina and LAG, 455
of coxal glands, 89, 214, 214–216, 216 of reproductive system (male)
of eyes, 80, 354 male accessory gland, 489–495
of fat body, 90–91, 290–296 overview, 485, 486
of heart, 260–261 testes, 487
of hemolymph, 8, 9, 87, 89–90, 264 vas deferentia, seminal vesicle, and
hierarchical approach to, 8 ejaculatory duct, 487–488
of hindgut of respiratory system, 90, 240–247, 241,
anus, 211 243, 245
comparison with insects, 211 of salivary glands
intestine, 207 agranular (type I) acini, 164, 165
Malpighian tubules, 208, 208–211, 209, 210 granular acini, 164–168, 166, 167
overview, 87 overview, 87–89
rectal sac, 87, 141, 207 of spiracles
rectum, 87, 211 of hard ticks, 240–241, 241
of idiosoma, 75–80, 76, 77, 78, 79 of Nuttalliella namaqua, 245
of integument, 99–102, 100 of soft ticks, 244–245, 245
of intestine, 207 of spiracular plate, 241, 242, 243
of legs, 82–83, 83 of subostial space, 241, 242
of Malpighian tubules, 89, 208, 208–211, 209, of synganglion, 91–93, 92, 310–314, 311, 312
210 systematics and, 4–5
of midgut of tracheal atrium, 241, 242–243
in feeding ixodid ticks, 131–144, 133, 135, of tracheal system, 246–247, 247
136, 137 Angiotensin-converting enzyme (ACE), 503, 508
overview of, 87, 130, 131 Anocentor nitens. See Dermacentor nitens
in unfed ixodid ticks, 130–131, 132 Anomalohimalaya spp., 61
of mouthparts and foregut Anoxia, 254
chelicerae and palps, 125–128, 127 Anterior pit, 355
hypostome, food canal, and associated Antero-dorsal glomerus, 91
structures, 124–125, 125 Antibodies, polyclonal. See Polyclonal antibodies
overview, 123–124, 124 Anticoagulants, 174, 270
pharynx, 126, 128–129 Antifungal peptides, 267–268
of musculature, 74, 95–96 Anti-hemostatic compounds, 174–175
of nephrocytes, 90–91, 296–298, 297 Anti-inflammatory factors, 175–176
of nervous system, 91–94, 92 Anti-microbial compounds, 265–268, 296. See
of opithosoma, 77–80 also Specific compounds
Index 521
Antioxidants, 272 Atrial chambers. See Tracheal atrium

Anus, 80, 211 Attachment, 69–70, 222, 223–224, 384–385
Aorta, 260, 263–264 Attachment cement. See Cement
Aortic myocardial cone, 261 Attractant sex pheromone (ASP), 377, 379
Apolipophorins, 231–232 Autogeny, 476
Aponomma spp., 47 Autophagy, 169
Apoptosis, 169
Apotele, 82 B
Appetence, 221 Babesia spp., 180
Aprotinin, 509 Babesiosis, 7
Apryases, 174 Bacteria, tick-borne diseases and, 6
Aquaporins (AQP), 173, 213 Bacteriocytes. See Mycetocytes
Arachidonic acid, 170 Barcoding and molecular keys, 44–45
Arachnida, 4, 10, 214 Basis capituli, 80
Arecoline, 350 Behavior patterns
Argasidae (soft ticks) chemical-communication-responsive,
anatomy of, 8 molecular basis of, 383–384
blood feeding and, 11–12 feeding
classification of by different researchers, 32, day-night rhythms and, 69–70
45–46 duration, 68–69
development of, 9 host-seeking
excretion and waste elimination in, 214, evolution of, 67–68
214–217, 215, 216 morphological adaptations with, 68
gas exchange in, 252 strategies for, 66–67
gonotrophic cycles in, 475–477 overview of, 12–14
molecular phylogenetics of, 41 regulation of by chemical communication
morphologic identification of, 29 courtship and mating, 377–378
overview of, 4 feeding site predilection, 376
recent developments in classification of, on-host clustering and attachment,
45–46 376–377
reproduction of, 11 host seeking/location, 374–375
reproductive systems of, 450–451, 451, host specificity, 375
485, 486 off-host clustering behavior, 372–373
respiratory system of, 240–245, 245 overview of, 371, 372
spiracles of, 244–245, 245 pre-appetance phase, 373–374
spiracular plate of, 241, 242, 246 Bicellular glands, 242
structure and function of midgut in, 142–144 Bicuculline, 349
Argas monachus, 46 Big sip, 141, 501
Argas persicus, 65 Biliverdin, 288
Arrestins, 382 Bioacaricides, 139
Arrestment pheromone, 372–373, 379 Biogenic amines. See also Specific molecules
Arrowhead organelle, 138 acetylcholine, 350–351
ASP. See Attractant sex pheromone dopamine
Asparaginyl endopeptidases (legumains), 147, cuticular viscosity and, 118
153, 229 overview of, 343–346, 345, 346
Aspartic acid proteases, 230 plasticization and, 106, 113, 114
Aspartic peptidases, 147–148, 155, 229 salivary glands and, 170
Aspartyl peptidases, 145, 148, 149, 229 SIFamide and, 328
Assembly (arrestment) pheromone, 372–373, 379 γ-aminobutyric acid, 348, 348–349
Association neurons, 313 glutamate, 350
Astacin metalloprotease, 506, 506–507 octopamine, 113, 114, 349
Athrocytes. See Nephrocytes overview of, 342–343
Atria, 90 serotonin, 346–348, 348
522 Index
Blood feeding (hematophagy). See also Feeding Carinae, 76

behavior Carios spp., 32, 78, 83
male accessory gland and, 489–490 Carrier proteins (CP, hemelipoglycoproteins)
oogenesis and, 458–459 anatomy of, 90
overview of, 11–12, 122–123, 220 developmental changes in protein levels of,
spermatogenesis and, 484 398–400, 399
stages in evolution of endocrine regulation of, 430–432
attachment, 222, 223–224 evolution of blood meal digestion and, 231–232
blood meal processing, 222, 225–226 fat body and, 296
host-seeking, 221–223, 222 genetic regulation of, 408–409
tick-host interface, 222, 224–225 heme processing and, 404–405
unusual nature of, 10–11 heme transport and, 151, 230
vitellogenesis and, 469 molecular biology of, 406–408, 407
Blood meal, elimination of, 144 nutrition and, 402–404
Blood vessel, dorsal, 75, 75 overview of, 265, 398
Bm86 protein, 190 reproduction and, 426, 427, 427
Bm91 protein, 190 role of in salivary gland, 400, 409–410
BmA7 protein, 190 sources of, 91
BmAP, 175 tissue levels of, 400
BmGTI, 175 vitellogenins vs., 231–234, 411–412
BMS. See Bombyx myosupressin Catecholamines, 117, 170, 172, 326, 343–346, 511
BmTI-2, 175 Cathepsin-like proteases, 147
BmTI-A, 175 Cathepsins, 145–146, 147–148, 152–153, 229
Body odors, 374–375 Cattle, tick-borne diseases of, 7
Body region. See Idiosoma Caudal processes, 31
Bombyxins, 316, 317, 318, 440 CCAP. See Crustacean cardioactive peptide
Bombyx myosupressin (BMS), 316, 318, 322, CCHamides, 323
328–331 CCV. See Clathrin-coated vesicles
Boophilin, 149, 175 CDNA subtraction, 191
Borrelia burgdorferi, 5, 138–139 Cecropins, 265
Bothriocroton spp., 30, 41, 44, 47, 61 Cell culture, 193
Bradykinins, 187 Cement
Brain. See Synganglion attachment and, 224
Bursicon, 105–106, 336, 437–438 blood feeding and, 12
carrier proteins and, 409–410
C composition of, 385
C3 component, 176 overview of, 171
Caeca, anatomy of, 130 skin morphology and, 99, 103
Calcitonin-like DH (CT/DH), 323 Central fat body, 289. See also Peripheral fat body
Calcitonin receptor, 335 Central nervous system. See Synganglion
Calcium, exocytosis and, 170 Cervical vagina, anatomy of, 454
Calreticulin, 510 Chaetotaxy, 29–30
Camerostomal fold, 94 Chelicerae, 8, 80, 82, 125–128, 127
Camerostome, 80 Cheliceral digits, 126, 127, 354, 356
Campaniform sensilla. See Sensilla auriformia Cheliceral ganglia, 91, 93
Capacitation, 485, 498, 502–503, 507–511 Cheliceral hood, 82
CAPA peptide, 336 Cheliceral sheath, 82
Capitulum, 8, 80–82, 81, 95–96 Chelicerates, overview of, 4
Carbohydrates, fat body and, 288 Chemical communication. See Communication,
Carbon dioxide, 371, 374–375, 382–383 chemical
Carbon dioxide receptors, 223, 382–383 Chemosensilla, 352–355, 380–381
Carboxypeptidases, 147, 148, 154, 154–155, 190, Chitin, 99, 100–102, 138
388, 507–509 Chitinases, 106, 138–139
Index 523
Chitobiose, 138–139 Complement inhibitors, overview of, 176–177

Chordotonal sensilla, 355 Compluriscutula spp., 32, 40
Choriogenesis, 459–460 Concanamycin A, 114
Chromatocytes, 288 Concentration, 155
CHT1 cDNA, 106 Connecting tube, 453–454
Chymotrypsin, 147, 270 Copper ions, microplusin and, 267–268
Circulatory system. See also Hemolymph Copulation. See Mating
arterial vessels and sinuses, 263–264 Corazonin, 316, 318, 319, 336, 437–438
heart, 260–261 Cornupalpatum spp., 32, 40
overview, 89–90, 258–259, 259 Corpora allata, 418
Circumesophageal cells, 298 Cortical zone, 311–314
Cisternum, 500 Corticotropin-releasing-factor-related diuretic
Classification. See Systematics hormone, 316, 322
Clathrin, 139, 140 Cot filtration, 192
Clathrin-adaptor protein, 152 Courtship, 377–378
Clathrin-coated pits, 139, 228 Coxal glands
Clathrin-coated vesicles (CCV), 152–153 anatomy of, 89, 214, 214–216, 216, 217
Cleavage sites function of, 144, 206, 226, 230
in carrier proteins vs. vitellogenins, 234, 411–412 Coxal pores, 89
neuropeptides and, 335 Coxal spurs, 30
in vitellogenins, 406, 463, 467 Coxal tubule, 215, 215–216
Climate, 64, 66, 67, 70 CP. See Carrier protein
Clotting, 272–273 CPLA2. See Cytosolic phospholipase A2
Clustering, 372–373, 376–377 Creep. See Rate of creep
Coated pits, 134, 139 Critical weight (CW), 169
Coated vesicles, 134, 299, 301 Crustacean cardioactive peptide (CCAP), 316,
Coiling phagocytosis, 278, 278 318, 323
Colloids, 143 Crustacean hyperglycemic hormone-related ion
Columnar cells, 216–217 transport peptide, 316, 318
Common protein. See Carrier protein Crustaceans, ticks vs., 10
Communication, chemical CT/DH. See Calcitonin-like DH
behavioral adaptations regulated by Cuboidal cells, 211
courtship and mating, 377–378 Cuticle
feeding site predilection, 376 anatomy of, 8
on-host clustering and attachment, 376–377 changes in after molt, 108–109
host seeking/location, 374–375 dopamine and, 346
host specificity, 375 functional morphology of, 100–102, 101
off-host clustering behavior, 372–373 hormonal influence on architecture of,
overview of, 371, 372 116, 337
pre-appetance phase, 373–374 mechanical properties of, 109–111, 110
biology of, 369–371, 370 molting and, 107–108
defined, 370 plasticization of in insects, 111–112
future research directions, 389 plasticization of in ticks, 113–114, 117–118
molecular basis of sclerotization and, 104–105
chemical-communication-responsive tick of tracheal system, 246
behavioral adaptations, 383–384 Cuticular proteins (CP), 105
chemosensory receptor system, 380–381 Cuticular sacs, 456–457
feeding, 384–389 Cuticulin, 102–103
olfactory system, 381–383 CW. See Critical weight
overview, 378 Cyclooxygenases, 230
semiochemical biosynthetic pathways, Cystatins, 148–150, 509
379–380 Cysteine peptidases, 145, 147–149, 149,
overview of, 368–369 153–154, 229
524 Index
Cytochrome C Oxidase subunit 1 (CO1), 44 peptidase expression and, 147–148

Cytochrome oxidase III, 42 peptidase inhibitor expression and, 148–150
Cytokeratin, 510 protein expression and, 145–147
Cytosolic phospholipase A2 (cPLA2), 170 chitinases and, 106
evolution of, 231–235, 232, 233
D evolution of hematophagy and, 222,
DAG. See Diacylglycerol 225–226
Da-p36 protein, 177, 183 future perspectives on, 156–157
Day-night rhythms, 68–70 in hematophagous insects and other
DDC. See Degenerating digestive cells blood-feeding invertebrates, 155
Defecation, 141 in ixodid ticks, 131–142, 152–154, 153
Defensin fold, 266 midgut development during, 227–228
Defensins, 90, 151, 265–267, 296 overview of, 11–12
Degenerating digestive cells (DDC), 130, unusual nature of, 10–11
131–133, 132, 133, 134–135 Digestive cells, 227–229
Degeneration of salivary glands, 169 Digestive tract, anatomy of, 8
Dendrites, 94 Dilator muscles, 125
Dense inclusions, 290 Directional stimuli, 374
Denticulate hypostome. See Hypostome Discontinuous gas exchange, 248, 249, 250, 251
Dermacentor albipictus, 49 Diseases, 5–8, 6–7, 47
Dermacentor andersoni, 60–61 Distal tarsal slit sense organs, 355
Dermacentor nitens, 49, 63, 70 Diuresis, 213, 217
Dermacentor spp. Diverticula, 86, 87, 130
classification of, 48–49 Dlsm. See Dorsolateral suspensory muscle
life cycles of, 61, 63 apparatus
vector competence and, 180 DNA barcoding. See Molecular barcoding
Dermacentor variabilis, 44, 76, 145–146, 146, Dopamine
340–341 cuticular viscosity and, 118
Dermal gland cells, 103–104 overview of, 343–346, 345, 346
Der-p2, 271 plasticization and, 106, 113, 114
Detachment, 59, 69–70 salivary glands and, 170
Development SIFamide and, 328
endocrine system in Dorin M, 269
crustacean, 419 Dorsal blood vessel, 75, 75
insect, 417–419 Dorsal plate, 30
tick, 419–420 Dorsolateral suspensory muscle apparatus
of midgut during blood meal digestion, 227–228 (dlsm), 260, 262
overview of, 9–11, 10 Double-walled (DW) sensilla, 352, 355
Diacylglycerol (DAG), 170 Drop-off. See Detachment
Diapause, 72 Drosophila S2 cells, 190
Diaphragm cells, 298 Drudh, 299
Differential display analysis, 191 DW sensilla. See Double-walled sensilla
Differentiation, 141, 142 Dynein, 510
DIGE. See 2D differential in-gel electrophoresis
Digestion E
in argasid ticks, 142–144 East Coast fever, 7, 179
of blood meal in midgut Ecdoperitrophic space, 135, 137, 138
antimicrobial defenses during, 151 Ecdysiotropic hormone, 473
hemolysis and hemoglobin digestion in, Ecdysis, 247, 323
151–155 Ecdysis triggering hormones (ETH), 333
overview of, 139–141, 144–145 Ecdysone, 103, 295. See also 20-Hydroxyecdysone
oxidative stress, heme sequestration and, Ecdysone receptor (EcR), 169–170, 438–439,
150–151 472–473
Index 525
Ecdysteroids (E) orcokinins, 440–441

cells secreting, 116 overview, 433–434, 435–436
insect development and, 417–419 proprotein convertase precursor, 441
molting and, 107–108, 438 spermatogenesis, 500
oenocytes and, 103 sulfakinin, 441
regulation of egg development by, 424–426 synganglion farnesyl pyrophosphate pathway
vitellogenesis and, 429, 471–472 and, 430
Eclosion hormone (EH), 106, 336, 437–438 Endocuticle, 99, 100, 101, 108–109, 116–118
Ecology, 12–14 Endocytosis, 139, 143, 296–298
Economic impacts of ticks, 3 Endosomes. See Inclusion bodies
EcR. See Ecdysone receptor Endospermatophores, 501
Ectoperitrophic space, 135, 137, 138 Engorgement, 429, 472, 473–474, 476–477
EDG. See Ecdysis triggering hormones Engorgement factors (EF), 169, 428–429, 505, 511
Egg masses, sizes of, 11, 60 Environment, tick survival in, 12–13
Eggs, 424–426. See also Oogenesis; Oviposition EP1-like receptor, 170
Egg waxing organ. See Gené’s organ Epicuticle, 99, 100
Ehrlichia spp., 7, 180 Epidermal trophic hormone, 429–430
Ehrlichiosis, 6, 7 Epidermis, 102–104, 107, 109
EI-SI GC MS. See Electron-impact selective Epithelial cells, 102
ion capillary gas chromatography mass Epithelial sacs, 456
spectrometry Erythrocytes, 142–143, 152
Ejaculatory duct, 95, 487–488, 497 ESI-MS, 188–189
Elastase, 270 Esophageal nerve, 93
Elateroid molting, 247 Esophagus, 129
Electron-impact selective ion capillary gas EST. See Expressed sequence tags
chromatography mass spectrometry Evaporative water loss, minimization of, 13
(EI-SI GC MS), 422 Evasins, 177, 190
Emargination, 80, 94 Evolution
Encapsulation, as response to infection, 278, 279 of blood feeding
Encephalitis, overview of, 7 attachment stage, 222, 223–224
Endocrine system. See also Lateral segmental blood meal processing as model for,
organs; Neuropeptides; Specific hormones 226–227
in development blood meal processing stage, 222, 225–226
of crustacea, 419 host-seeking stage, 67–68, 221–223, 222
of insects, 417–419 tick-host interface and, 222, 224–225
of ticks, 419–420 of blood meal digestion, 231–235, 232, 233
ecdysteroids and, 429 of life cycles in hard ticks, 64
epidermal trophic hormone and, 429–430 of life cycles in soft ticks, 66
future research directions, 441–442 of questing strategies, 67–68, 221–223, 222
male pheromone and, 428–429 of tick central nervous system, 93
overview of, 416–417 Evolutionary relationships, 4–5. See also
regulation of carrier proteins vs. vitellogenins Systematics
by, 430–432 Excretion and waste elimination
regulation of metamorphosis by, 422–423 in argasid ticks, 214, 214–217, 215, 216, 226
in reproduction heme processing and, 228–229
allostatin, 434–437 in insects, 212
differential gene expression during, 432–433 in ixodid ticks, 212–213, 226
ecdysone receptor, 438–439 Exocuticle, 99
eclosion hormone, corazonin, bursicon, 437–438 Exocytosis, 139–140, 141, 170–171, 229
female, 423–428 Expressed sequence tags (EST), 181,
glycoprotein hormones, 439 181–182, 192–193, 229–230. See also
gonadotropin-releasing hormone receptor, Transcriptome analysis
439–440 Eyes, 80, 354, 357
526 Index
F regulation of
Falk-Hillarp technique, 117 ecdysteroids and, 424–426
Families, key for identification to, 36–39 historical perspective, 423–424
Farnesyl pyrophosphate pathway, 430 working model for, 426–427, 427
Fat body sperm capacitation and, 507–511, 512
anatomy of, 90–91, 290–296 vitellogenesis and
arthropod, 289 ecdysteroids and, 429
functions of, 296 initiation of, 429
insect, 287–289 in insects, 289
pre-vitellogenic, 290–292 juvenile hormone and, 418–419
vitellogenic, 292–295 overview of, 468–474, 470, 471, 472
vitellogenin synthesis in, 467–468, 468 salivary gland degeneration and, 169
Fat body stimulating factor (FSF), 473 site of, 467–468, 468
Fatty acids, 272 Ferritins, 141, 150–151, 288, 409, 507
Feeding Fertilization, 460–461, 502–503, 511–513
discontinuous gas exchange and, 248–251, Festoons, 30
250, 251 FGLamide-related allostatins, 316, 318, 322–323,
salivary gland gene expression during, 182–183 333, 334
spermatogenesis and, 499–500 Ficolins, 269
Feeding behavior Fingerprinting, 188–189
blood meal processing as model for evolution Fire ants, 104
of, 226–227 Fluid phase endocytosis, 139
chemical communication and, 373–374, Follistatin-related protein, 296
384–389 Food canal, 124–125, 125
day-night rhythms and, 69–70 Foregut, 84–87. See also Mouthparts and foregut
duration, 66, 68–69 Formate, 213
growth of endocuticle during, 108–109 Fossil record, 32, 40
of hard vs. soft ticks, 221, 225 Fourier transform ion cyclotron resonance
molecular preparation for initiation of, (FTICR), 189
386–389 Foveal glands, 95
overview of, 11–12, 122–123 Foveal nerve, 326
plasticization and, 113–114, 116–117 Foveal pores, 77, 79, 95
role of male pheromones in, 513 Free radicals, 140
site section, 376 FSF. See Fat body stimulating factor
Female reproductive system FTICR. See Fourier transform ion cyclotron
anatomy of resonance
cervical vagina, 454 Functional genomics, 191–192
connecting tube, 453–454 Fungi, tick-borne diseases and, 6
Gené’s organ, 456, 456–457 FXaI, 190
ovary, 451–453, 452
overview, 449–451, 450, 451 G
oviducts, 453 GABA (γ-Aminobutyric acid), 348, 348–349
seminal receptacle, 454 GABA-gated chloride channel, 349
tubular accessory glands, 454–455 Galectins, 269
vestibular vagina and LAG, 455 Galleria pupal cuticle bioassay, 421
future research directions, 477–478 Gametogenesis, different strategies for, 11
gonotrophic cycle in, 433, 475–477 γ-Aminobutyric acid (GABA), 348, 348–349
juvenile hormone and, 418–419 Ganglia, 93
oogenesis in Gap junctions, 301
choriogenesis, 459–460 Garland cells, 298, 299
oocyte development, 457–459, 458 Gas exchange
oviposition, 461–462 in argasid ticks, 252
ovulation and fertilization, 460–461 discontinuous, 248, 249, 250, 251
Index 527
general principles in arthropods, 248 Grinding organ, 128

in ixodid ticks, 248–252 GRP. See Glycine-rich proteins
metabolic rate, off-host survival and, 254 GSP. See Genital sex pheromone
plastron respiration and, 252–254, 253 GST. See Glutathione-S-transferase
tracheae and, 74, 90 Guanine, 212–213
Gene expression. See Transcriptome analysis Guanine crystals, 209–211, 210
Gené’s organ Guirlandzellen, 298
anatomy of, 77, 80, 94–95, 450, 456, 456–457 Gustatory sensilla, 94, 354–355
oviposition and, 461–462 Gut, innervation of, 333
Genital aperture, morphologic identification
and, 31 H
Genital groove, 76, 79, 80 H1SP, 147, 149, 152
Genital pore, 8, 76, 77, 79 Haemaphysalin, 175
Genital sex pheromone (GSP), 378, 501 Haemaphysalis spp., 40, 41, 48, 61, 68, 145
Genome sequences, 14, 180–181, 182, 336 Haller’s organ
Genus, key for identification to, 36–39 anatomy of, 8, 82, 94, 352, 355–356
Germinal vesicle, 457 chemosensory reception and, 380–381
Germinarium, 452 olfactory lobes and, 91, 309
GID2, 503–504 overview of, 13, 355–356
Glandular cells, 100 questing behavior and, 68, 222
Glial cells, 312 Hardening-off phase, 373
Glossina morsitans (tsetse fly), 155 Hard ticks. See Ixodidae
Glucose, 272 HDLp. See High-density lipophorins
Glutamate, 350 Heart, 89, 260–261
Glutathione-S-transferase (GST), 150, 151, 271, 510 Heartwater, 7
Glycerol, 272 Heat shock proteins, 505, 510
Glycine, 213 Hebraein, 183, 268
Glycine-rich proteins (GRP), 184 Helminthes, 155
Glycocalyx, 143 HeLp. See Heme lipoprotein
Glycogen, 135, 164, 209, 217, 288 Hemalin, 149
Glycoprotein hormones, 439 Hematin, 87, 212–213, 229, 230–231
Gnathosoma. See Capitulum Hematin-like masses, 139–140
Gonadotropin receptor, 335 Hematophagy. See Blood feeding
Gonadotropin-releasing hormone receptors Heme
(GRHR), 439–440 evolution of digestion of, 226, 231–235,
Gonotrophic cycle, 433, 475–477 232, 233
G-protein coupled receptors (GPCR), 382 formation and detoxification of, 140–141, 143, 157
G-proteins, 382 inability to synthesize, 229–230
Granular acini processing, sequestration, and excretion of,
anatomy of, 144–145, 165–168, 166, 167 228–229
changes in during feeding, 168–169 sequestration in midgut during digestion, 150–151
overview of, 88 source of, 272
type II, 165–166, 166 transport and storage proteins of, 229–231,
type III, 166–167, 167 404–405
type IV, 167–168 in vitellogenins, 463
Granular lobes, 485, 496, 497 Heme aggregates, 135, 141
Granules, 130. See also Yolk granules Hemelipoglycoproteins. See Carrier proteins
Granulocytes, 90, 273, 274–275, 276 Heme lipoprotein (HeLp), 401–402, 404, 411, 426
Granulocytic anaplasmosis, 5, 6, 178–179 Hemocyanins, 272, 401
GRHR. See Gonadotropin-releasing hormone Hemocytes
receptors classification of, 89–90, 273–275
GRI granulocytes, 90 molecular basis of function of, 277–279
GRII granulocytes, 90 structure of, 273, 274, 275, 275–277, 276, 277
528 Index
Hemoglobin Hoogstraal, Harry, 48

digestion of, 10–11, 140, 152–155, 154 Hookworms, 155
nephrocytes and, 299 Hormones. See also Endocrine system; Specific
as primary food source, 123, 226 hormones
Hemolymph bursicon, 105–106, 336, 437–438
anatomy of, 8, 9, 87, 89–90, 264 eclosion hormone, 106, 336, 437–438
carrier proteins and, 151 sclerotization and, 105
clotting of, immune defense and, 272–273 spermatogenesis and, 500
compounds regulating osmolality and pH, Horses, 1-host ticks of, 63
271–272 Horseshoe crab. See Limulus polyphemus
overview of, 264 Host complementation, carrier proteins and,
plasma proteins of 409–410
defensins and other antimicrobial peptides, Host race formation, microsatellite markers
265–268 and, 42
lectins, 90, 151, 269, 279 Host resistance, 135–138, 225
lysozymes, 90, 266, 268, 279, 510 Hosts, 135–138. See also Tick-host interface
macroglobulins, 269–270 Host-seeking behavior
MD-2-related lipid-recognition chemical communication and, 369–370,
domain-related proteins, 271 374–375
overview, 264–265 evolution of, 67–68
oxidoreductases, 270–271 evolution of hematophagy and, 221–223, 222
proteases and protease inhibitors, 270 morphological adaptations with, 68
vitellogenins and other heme-lipoproteins, strategies for, 66–67
265 Host specificity, 13–14, 375
salivary glands and, 172 HPLC. See High-performance liquid
Hemolysins, 143, 152 chromatography
Hemolysis HRF. See Histone release factor
in argasid ticks, 142–143 5-HT. See 5-Hydroxytryptamine
in blood meal digestion, 228 Humoral factors, 265
in ixodid ticks, 135, 141 Hunter strategy, 13
molecular process, 151–155 Hyalomma asiaticum, 7–8
Hemosomes. See Residual bodies Hyalomma dromedarii, 13
Hemostasis, 12, 174–175 Hyalomma isaaci, two-host life cycle of, 62
Hemozoin, 140, 229 Hyalomma spp.
Heterolysosomes, 139, 143, 152 classification of, 49
Heterophagy, 135, 139–140, 228 life cycles of, 61, 62, 63–64
Heximerins, 288 molecular phylogenetics of, 41
High-density lipophorins (HDLp), 400 questing behavior of, 67
High molecular weight kininogen (HMWK), 175 Hydration, 13, 114, 369–370, 375. See also Water
High-performance liquid chromatography content
(HPLC), 186–188 Hydrolases, 509
Hindgut Hydrostatic pressure, 118
anus, 80, 211 20-Hydroxyecdysone
comparison with insects, 211 genital sex pheromone and, 378
intestine, 207 reproduction and, 11
overview, 87 salivary gland degeneration and, 89, 169–170
rectal sac, 87, 141, 207 spermatogenesis and, 500
rectum, 87, 211 synthesis of, 107–108
Histamines, 175 vitellogenesis and, 91, 295, 472
Histone release factor (tHRF), 186 5-Hydroxytryptamine (5-HT), 111, 114
HLGP. See Hemelipoglycoproteins Hygric hypothesis, 254
HlLysozyme, 268 Hypodermis, 100–101
HMWK. See High molecular weight kininogen Hypostomal gutter. See Preoral canal
Index 529
Hypostome during post-molt and feeding periods,

anatomy of, 8, 80, 81, 82, 124–125, 125 108–109
attachment and, 223–224 sclerotization and, 104–105, 115–116
morphologic identification and, 30, 31 size determination and, 115, 226
Hypoxanthine, 213 Interface. See Tick-host interface
Hypoxia, tolerance of, 254 Internal fat body, 90
Interneurons (IN), 315
I Intestine, anatomy of, 87, 207
Idiosoma Intrapedicellar space, 244
opithosoma, 76, 77, 77–80, 78, 79 Invagination pits, 273
podosoma, 75–77 Ion transport peptide (ITP), 317
Ileum, 211 IrAM, 270
Immune system (host), 221 IRE. See Iron responsive elements
Immune system (tick), 277–279 Iris, 177
Immunity, 106, 265, 265–267 IrML, 271
Immunomodulators, 151, 165, 177, 192–193 Iron, 150, 229, 288
IN. See Interneurons Iron responsive elements (IRE), 409
Inclusion bodies (endosomes), 130, 134, 136, 137 Isac, 176, 187
Inka cells, 333, 334 Isamp, 187
Innate immunity, defensins and, 265–267 ITP. See Ion transport peptide
Inositol, 272 ITS2 gene sequences, 42
Insects Ixoderins, 269
blood meal digestion in, 155 Ixodes persulcatus, 71
excretion and waste elimination in, 212 Ixodes ricinus, 13, 59, 69, 71
fat body of, 287–289 Ixodes ricinus complex, 47
hindgut anatomy of, 211 Ixodes scapularis
male reproductive system of, 498 anatomy of, 76
nephrocytes of, 298–300 genome sequence of, 180, 182
ticks vs., 10 neuropeptides of, 338–340, 342
Insulin-like growth factor binding protein-related population genetics of, 42
proteins, 388 transcriptome analysis of, 145–146
Integument transcriptome of, 146
cellular components of Ixodes spp.
dermal gland cells, 103–104 feeding behavior of, 69–70
future perspectives on, 115 longevity of, 70–72
oenocytes, 102–103 microsatellite markers of, 42
sensory receptor cells, 104 molecular phylogenetics of, 41
cuticle population genetics of, 42
functional morphology of, 100–102, 101 questing behavior of, 67–68
hormonal influence on architecture of, 116 recent developments in classification of, 46–47
mechanical properties of, 109–111, 110 Ixodes trianguliceps, 68
plasticization of in insects, 111–112 Ixodidae (hard ticks)
plasticization of in ticks, 113–114, 117–118 anatomy of, 8
functional morphology of, cuticle, 100–102, blood feeding and, 12
101 development of, 9
future perspectives on, 115–118 digestion in, 131–142, 152–154, 153
molecular studies relating to excretion and waste elimination in, 212–213
bursicon, 105–106 gas exchange in, 248–252
chitinases, 106–107 life cycles of
cuticular proteins, 105 evolution of, 64
eclosion hormone, 106 one host, 62–63, 63
molting and, 107–108 overview, 59–64
overview of, 99, 100 species distribution of, 63–64
530 Index
Ixodidae (hard ticks) (continued) Lectins, 90, 151, 269, 279

three-host, 60–62, 61 Legs, 8, 82–83, 83, 96
two-host, 61, 62–63 Legumains (asparaginyl endopeptidases), 147,
midgut structure in, 130–144 153, 229
molecular phylogenetics of, 40–41 Leucine aminopeptidase (LAP), 147, 153, 155,
morphologic identification of, 29 504
overview of, 4 Leukokinin receptor, 335
reproductive systems of, 449–450, 450, 485, Life cycles
486 of hard ticks
respiratory system of, 240–244 evolution of, 64
spiracles of, 240–241, 241 one host, 62–63, 63
spiracular plate of, 241, 242, 243, 246 overview, 59–60
Ixodidin, 270 species distribution of, 63–64
Ixolaris, 174 three-host, 60–62, 61
two-host, 62, 62–63
J longevity of, 70–72
Juvenile hormones (JH) overview of, 9
biosynthesis of, 431 of soft ticks, 64–66, 65
cuticle structure and, 108 synchronization between ticks and hosts, 67
insect development and, 418 Life spans, 9–10
juvenilization of insects by tick extracts of, 421 Limulus polyphemus (horseshoe crab), 4
nephrocytes and, 299 Lipases, 509
presence/absence of in ticks, 420–422, Lipids, 102–103, 135, 164
471–472, 474 Lipocalins, 174, 175, 188, 190
tick reproduction and, 423–424 Lipophorins (Lp), 400
vitellogenesis and, 289, 294–295 Lipopolysaccharides, 279
Livestock, 7, 50, 63
K Lobular accessory gland (LAG), 450, 451, 455,
Kairomones 461
biosynthesis of, 379–380 Longevity, overview of, 70–72
clustering behavior and, 372–373 Longipain, 147, 154
overview of, 370, 371 Lopsins, 150
sensing of, 383 Louping ill, 7
Kallikrein-kinin system, 175 Low-density lipophorins (LDLp), 400
Keratin, 510 Lp. See Lipophorins
Kininase, 176 LSO. See Lateral segmental organs
Kinin-like peptides, 336 Lyme disease, 5, 6
Kininogen, 175 Lyrifissures, 30
Kinins, 316, 318, 321–322, 328–331, 336 Lysosomes, 213
KPI. See Kunitz-type serine protease inhibitor Lysozymes, 90, 266, 268, 279, 510
Kunitz-type serine protease inhibitor (KPI), 149,
150, 151, 174 M
MAChR. See Muscarinic ACh receptor
L Macroglobulins, overview of, 269–270
Labrum, 84–85, 125, 127, 129, 224 Macrophage migration inhibitory factor, 510
Labyrinth system, 88, 139, 142–143, 166, Macula, 242
208–209, 242, 244 Madanins, 175
LAG. See Lobular accessory gland MAG. See Male accessory gland
Lamellae, 100–101 Magainins, 265
Larvae, 29–30 Malaria hemozoin, 140
Lateral segmental organs (LSO), 311, 311, MALDI-MS analysis, 188
332–333 MALDI-TOF analysis, 188–189
LDLp. See Low-density lipophorins MALDI TOF/TOF analysis, 336
Index 531
Male accessory gland (MAG), 485, 486, 487, Microphilin, 175

489–495, 492, 494, 502, 504 Microplusin, 183, 267–268, 296
Male reproductive system Micropyle, 460
anatomy of, 485, 486 Microsatellite markers, systematics and, 43–44
capacitation, fertilization and, 485, 502–503 Midgut
copulation and, 501–502 anatomy of, 9, 87
future research directions, 513–514 antimicrobial defenses in, 151
histology and ultrastructure of development of during blood meal digestion,
male accessory gland, 489–495 227–228
testes, 487 heme loading and, 230
vas deferentia, seminal vesicle, and infection by pathogens and symbionts, 156
ejaculatory duct, 487–488 oxidative stress, heme sequestration in,
mite system vs., 495–498 150–151
overview of, 484–485 peptidase expression and, 147–148
spermatogenesis/spermiogenesis in, 498–501, peptidase inhibitor expression in, 148–150
503–507 protein expression in, 145–147, 146
Malpighian tubules structure in feeding ixodid ticks, 131–144
anatomy of, 9, 86, 89, 208, 208–211, 209, 210 structure in unfed ixodid ticks, 130–131
excretion and, 144, 226 vitellogenin synthesis in, 467–468, 468
role of, 206 Midgut diverticula, 261
Mammillae, 31 MIP. See Myoinhibitory peptides
Mannose, 272, 463 Mites, male reproductive system of, 495–498
Margaropus spp., life cycles of, 63 Mitochondrial genes, 42–43
Mass spectroscopy, 188–189 ML proteins, 271
Mating. See also Reproduction Molecular barcoding, 44
blood feeding and, 141–142 Molecular-based systematics
chemical communication and, 377–378 barcoding and molecular keys, 44–45
life cycle and, 59–60 evolution and phylogenetics
overview of, 501–502 early hypotheses, 39–40
oviposition and, 477 fossil record, 32, 40
regulation of, 11 phylogenetic molecular studies, 40–42
vitellogenesis and, 468–471, 470, 471, 473, population genetics
473–474 microsatellite markers, 43–44
MD-2-related lipid-recognition domain-related mitochondrial and nuclear genes, 42–43
(ML) proteins, 271 recent developments in, 45–50
Mechanosensilla, 222–223, 355 Molecular keys, 45
Median notch, 261 Molecular phylogenetics, 40–42
Mediterranean spotted fever, 5, 6 Molting, 107–108, 247, 337
Mesaxons, 324 Monobin, 175
Mestracheon, 247 Monocytotropic ehrlichiosis, 6, 7
Metabolic lipid domain protein, 151 Morphological tick identification
Metabolism, 254, 288 of adults, 30–32
Metallopeptidases, 145, 147–148, 149. See also basis of, 29
Leucine aminopeptidase identification of adults to genus, 31–32, 32
Metalloproteases, 155, 270, 506, 506–508 key for identifying to family and genus, 36–39
Metamorphosis, regulation of, 422–423 of larvae, 29–30
Metastriata, 4–5, 11 of nymphs, 30–31
Methallothioneins, 150 recent developments in, 45–50
Methemoglobin, 228 terminology of, 33–36
Methyl farnesoate (MF), 169, 419–420 Mosquitoes, ticks vs., 10
Methyl transferases, 430 Motility, of spermatozoa, 485, 498, 503
MF. See Methyl farnesoate Moubatin, 174
Microarrays, 191 Mounting sex pheromone (MSP), 378
532 Index
Mouthparts and foregut neuropile, 314

chelicerae and palps, 125–128, 127 peptidergic neurons of, 314–324, 316–317,
hypostome, food canal, and associated 318, 319, 320, 321
structures, 124–125, 125 subesophageal region, 310–311
overview, 123–124, 124 supraesophageal region, 310
pharynx, 126, 128–129 Neurilemma, 312
MS/MS. See Tandem mass spectrometry Neurohormones, 313, 314–324. See also Specific
MSP. See Mounting sex pheromone neurohormones
Multiporose sensilla, 94 Neurons, 313–324
Muscarinic ACh receptor (mAChR), 350 Neuropeptides. See also Specific neuropeptides
Musculature, anatomy of, 74, 95–96 excretion and, 213
Mycetocytes, 288 expression during reproduction
Myelinic figures, 130 allostatin, 434–437
Myocardium, 260, 262, 263 ecdysone receptor, 438–439
Myoinhibitory peptides (MIP) eclosion hormone, corazonin, bursicon,
genomic analysis of, 336 437–438
morphology of, 316, 318, 320, 321 glycoprotein hormones, 439
overview of, 323 gonadotropin-releasing hormone receptor,
proteomic analysis of, 337, 342 439–440
salivary gland innervation and, 326–328, 329, orcokinins, 440–441
330, 331, 332 sulfakinin, 441
Myosuppressins, 320 identification of
MyoV protein, 504 genomic approach to, 335–337
overview, 335
N proteomic approach to, 337–342, 338–342
Na+-K+ /2Cl co-transporter, 212 in specific tick species, 338–342
Na+-K+ ATPase, 212 type II neurons and, 313
NAChR. See Nicotinic ACh receptor Neuropile, 314
Nanchung (nan) genes, 383 Neurosecretory (NS) cells, 313–315
Nephrocytes Next generation sequencing, 182, 192
arthropod, 300 Nicotinic ACh receptor (nAChR), 350
insect, 298–300 Nidicolous ticks, 13, 66–67
overview of, 300–301 Niemann-Pick C1, 504
structure of, 91, 296–298, 297, 300–301 Niemann-Pick C2, 508
Neprilysins, 508 Nodulation, as response to infection, 278, 279
Nervous system Non-nidicolous ticks, 66–67
anatomy of Noradrenaline, 113
peripheral nerves, 93 Norepinephrine (NE), 346
peripheral sensilla, 94 Nothoaspis spp., 46
synganglion, 91–93, 92, 310–314 NS cells. See Neurosecretory cells
evolution of, 93 Nuclear genes, population genetics and, 42–43
overview of, 309–310 Nutrition, 402–404, 468–470, 473
peripheral Nuttalliela namaqua, 5, 29, 41, 45, 245
gut innervation and endocrine cells, 333 Nuttallielidae, overview of, 4, 5
lateral segmental organs, 332–333
overview of, 324–325 O
pedal endocrine cells, 333–334 Octopamine, 113, 114, 349
periganglionic sheath, 328–331 Odorant-binding receptor proteins (Ors), 382
retrocerebral organ complex, 333 Oenocytes, 102–103, 108, 276, 277, 288
salivary gland innervation, 325–328 20-OHE. See 20-Hydroxyecdysone
synganglion Okadaic acid, 170
anatomy of, 91–93, 92, 310–324, 311, 312 Olfactory lobes, 91, 93, 309, 310, 311
cortical zone, 311–314 Olfactory system, 352–354, 381–383
Index 533
Olfactosensilla, 352–354, 355 Pathogens

OmCI protein, 187 chitinases and, 106
OMFREP, 269 defenses against, 103–104
One-host life cycles, 62–63, 63 infection of midgut by, 156
Ontologies, 8 peritrophic membrane and, 138
Oocytes, development of, 452–453, 457–459, 458 regurgitation and, 129
Oogenesis salivary glands and, 177–180
choriogenesis, 459–460 tick-host-pathogen interactions, 178–179
oocyte development, 457–459, 458 transcriptional profiling during exposure to,
overview of, 452, 452 190–191
oviposition, 461–462 PCR (polymerase chain reaction), 45
ovulation and fertilization, 460–461 PDF. See Pigment dispersing factors
Oogonia, 451, 452 Pedal arteries, 260, 264
Oolemma, 457 Pedal endocrine cells, 333–334
Operculum, 502 Pedal lobes, 310, 311
Opisthosoma, 8, 93 Pedal nerves, 93, 326
Opisthosomal ganglion, 91 Pedicels, 242, 457–458, 460
Opisthosomal lobe, 310, 311, 328 Peptidase inhibitors, 145, 146, 148–155
Opisthosomal nerves, 117 Peptidases. See also Specific peptidases
Opsonization, 278 expressed in tick midgut, 145, 146, 147–148
Orcokinins, 319, 326, 328, 330, 336, 440–441 heme digestion and, 152–155, 229
Ornithodorin, 175 reproduction and, 504, 507, 509
Ornithodoros spp., 32, 46, 65–66, 71, 92, 114 vitellogenins and, 406, 463–464
Ors. See Odorant-binding receptor proteins Peptide mass fingerprinting, 188–189
Osmoregulation, 88, 172, 230, 271–272 Peptidergic neurons. See Neuropeptides
Ostia, 90, 242, 244, 261 Peptidic prothoracicotropic hormone (PTTH),
Otobius spp., 65–66 418
Ovary Pericardial cells, 260, 262, 298–299
anatomy of, 94, 451, 451–453, 452 Pericardial septum, 260, 262, 263
vitellogenin synthesis in, 467, 468 Pericardial sinus, 89, 263
Oviducts, 94, 453 Periganglionic sheath, 328–331
Oviposition, 169, 461–462, 462, 476 Periganglionic sinus, 260, 260, 261, 264
Ovulation, 460–461 Perikarya, 91, 312, 313
Oxidative phosphorylation, 230 Perineum, 312
Oxidative stress, 150–151 Perineurium, 91
Oxidoreductases, 270–271, 509–510 Peripheral fat body, 90, 289. See also Parietal fat
Oxyhemoglobin, 228 body
Peripheral nerves, anatomy of, 93
P Peripheral nervous system. See under Nervous
P36 protein. See Da-p36 protein system
PA. See Porose areas Peripheral sensilla, 94
Palpal articles, 30, 31, 354 Peristaltic contractions, 141
Palpal ganglia, 91, 93 Peritrophic membrane (PM), 135, 137, 138–139,
Palpal sensory organ, 356 143
Palps, 82, 127, 128 Peritubular cells, 88
Paracrine dopamine hypothesis, 344, 345, 346 Perivisceral fat body, 289. See also Central fat
Paraffins, 102 body
Paralysis toxin, 103 Periviscerokinins, 213, 217, 336
Parasitiformes, 4 Peroxiredoxins, 270–271
Paraspiracular peripheral nerves, 325 Peroxisomes, 130
Parietal fat body, 289. See also Peripheral fat body PETH. See Pre-ecdysis triggering hormones
Parthenogenesis, 60 PG. See Prostaglandins
Pasture ticks, 66–67 pH, hemolymph compounds regulating, 271–272
534 Index
Phagocytosis Podocytes, 216–217, 300

digestion in midgut and, 135, 139, 143 Podosoma, 8
hemocyte classification and, 273 Polyclonal antibodies, 322
hemoglobin uptake and, 228 Polyphenols, 103
as response to infection, 278, 278–279 Population genetics, systematics and, 42–43
Phagolysosomes. See Inclusion bodies Pore canals, 100, 101
Phagosomes, 143 Porose areas (PA)
Pharyngeal valve, 85, 125, 126, 129, 224 anatomy of, 80, 81, 450
Pharynx, anatomy of, 85–86, 96, 126, 128–129 morphologic identification and, 30, 31
Phenols, 353, 376, 380 oviposition and, 461–462
Pheromone-binding proteins, 382 Porose setae, 94
Pheromones. See also Specific pheromones Potassium urate, 212
biosynthesis of, 379–380 Precocene, 423
foveal glands and, 95 Pre-ecdysis triggering hormones (PETH), 333, 334
mating regulation and, 11 Prekallikrein, 175
overview of, 370, 371 Preoral canal, 82, 84–87, 124
Phosphatases, salivary glands and, 170 Preparatory feeding phase, 384–389, 387
Phosphatidylcholine, 142 Principal cell stellate cells, 211
Phospholipases, 142, 152, 170 Procuticle, 99, 100, 246
Phylogenetic studies, 40–42, 233, 233–234 Programmed cell death, 169
Pichia pastoris, 190 Prohemocytes, 90, 273, 276
Picrotoxin, 349 Proprotein convertase precursor, 441
Pigment dispersing factors (PDF), 316, 317–319, Prospermia, 500–501
318, 326, 328, 332 Prostaglandin E2 (PGE2)
Pilocarpine, 350 dopamine and, 344
Pinocytic vesicles, 459 male reproductive system and, 508, 510
Pinocytosis, 134, 139, 228 tandem mass spectrometry for identification
Placoid sensillum, 128 of, 189
Plasma proteins. See also Vitellogenins water secretion and, 170, 226
defensins and other antimicrobial peptides, Prostaglandin F2α (PGF2α), 170, 187
265–268 Prostaglandin H, 230
lectins, 90, 151, 269, 279 Prostaglandins, 176, 230, 508, 510
lysozymes, 90, 266, 268, 279, 510 Prostriata, 4, 11
macroglobulins, 269–270 Protease inhibitors, 270, 504, 509
MD-2-related lipid-recognition Proteases. See also Specific proteases
domain-related proteins, 271 blood coagulation and, 174, 269
overview, 264–265 expressed in tick midgut, 147–148
oxidoreductases, 270–271 heme digestion and, 140, 229, 230–231
proteases and protease inhibitors, 270 immunity and, 270
Plasmatocytes, 273, 274–275, 276, 276, 279 male fertility and, 503–507, 506
Plasticization overview of, 270
feeding-induced, 113–114, 116–117 Protegrins, 265
hormones and, 105–106 Proteomic analysis
in insects, 111–112 of male reproductive gland proteins, 504, 505–506
mechanism of, 114 for neuropeptide identification, 337–342,
pharmacology of, 114, 117 338–342
unanswered questions about, 117–118 of salivary glands
Plastron, 246, 253 2-D polyacrylamide gel electrophoresis,
Plastron respiration, 252–254, 253 185–186
Platelets, 174, 176 high-performance liquid chromatography,
Plates, 31, 76, 79. See also Scutum 186–188
Plugged pore sensilla, 353 overview, 184, 185
PM. See Peritrophic membrane peptide mass fingerprinting, 188–189
Index 535
recombinant protein expression, 189–190 ecdysone receptor, 438–439

tandem mass spectrometry, 189 eclosion hormone, corazonin, bursicon,
of spermatophore, 508–510 437–438
of vitellogenins and vitellin, 464–466 glycoprotein hormones, 439
Prothoracic gland, 107 gonadotropin-releasing hormone receptor,
Prothoracicotropic hormone (PTTH), 316, 317, 318 439–440
Protocerebrum, 93 orcokinins, 440–441
Protoheme, 272 overview, 433–434, 435–436
Proton transport, 114 proprotein convertase precursor, 441
Protozoa, tick-borne diseases and, 6 sulfakinin, 441
Proventricular nerve plexus (PvPlx), 333 overview of, 11. see also Mating
Proventricular valve, 130 Reptile hosts, 68–69
Proventriculus, 129 RES. See Relative engorgement state
PRVamides, 316, 318, 319, 328–329, 331 Reserve cells, 132, 227–228
PRXamides, 334 Residual bodies (hemosomes), 130, 137,
PTTH. See Prothoracicotropic hormone 139–143, 213, 229–231
Pupal cuticle bioassay. See Galleria pupal cuticle Resilin, 110
bioassay Resistance. See Host resistance
PvPlx. See Proventricular nerve plexus Respiration, 254. See also Gas exchange
Pyramidal cells, 211 Respiratory system. See also Gas exchange
Pyridine nucleotide disulphide oxidoreductase, anatomy of
509–510 overview, 90
Pyrokinin receptor, 335 spiracles, 240–241, 241, 244–245, 245
Pyrosequencing, 192 spiracular plate, 241, 242, 243
subostial space, 241, 242
Q tracheal atrium, 241, 242–243
Questing. See Host-seeking tracheal system, 246–247, 247
Quinone oxidoreductase, 509 tracheal system
o-Quinones, 105 anatomy of, 246–247, 247
function of, 246–247
R Restriction fragment length polymorphisms
Radiant heat, 374 (RFLP), 45
Rapamycin (TOR) nutritional responsive Reticular endothelial system, 299–300
signaling pathway, 469, 473 Retinoid X receptor (RXR), 472–473
Rapid-digestion stage, 143–144 Retrocerebral organ complex (ROC), 91, 309,
Rate of creep, 113 333, 419
Reactive oxygen species (ROS), 140, 142, 226, RFamides, 318, 319, 326, 328, 332, 333
270–271 RFPL. See Restriction fragment length
Reattachment, life cycle and, 59 polymorphisms
Rebers-Riddiford consensus sequences, 100 RHBP, 404
Receptor mediated endocytosis, 139, 228 Rhipicephalinae, 48–49
Recombinant protein expression, 189–190 Rhipicephalus appendiculatus, 179
Rectal sac, 87, 141, 207 Rhipicephalus microplus
Rectum, 87, 211 integument of, 100, 101
Regurgitation, 129, 130 longevity of, 70
Relative engorgement state (RES), 115 neuropeptides of, 341
Remodeling, 106 transcriptome of, 145
Reproduction, 477–478. See also Female reproductive Rhipicephalus sanguineus, 342
system; Male reproductive system Rhipicephalus sanguineus-like ticks, 50
anatomy of, 8, 9, 94–95 Rhipicephalus spp.
differential gene expression during, 432–433 classification of, 49–50
neuropeptide expression during life cycles of, 63–64
allostatin, 434–437 vector competence and, 180
536 Index
RHL-p36, 177 recombinant protein expression,

Ricinus-like ticks. See Ixodes ricinus complex 189–190
Rickettsiales, 452 tandem mass spectrometry, 189
Rickettsia spp., 5, 178–179 regulation and mechanism of fluid secretion by
RNA interference, 171, 191–192, 193 attachment cement, 171
RNA-seq, 192 exoctytosis, 170–171
ROC. See Retrocerebral organ complex osmotic balance, 172
Rocky Mountain spotted fever, 5, 6 water excretion, 172–173, 226
RPAI-1, 175 Salivary toxin, 103
RXR. See Retinoid X receptor Salp9 protein, 184
Salp14 protein, 187
S Salp15 protein, 190
Saccharomyces cerevisiae, 190 Salp16 protein, 178–179, 190
Saliva, bioactive factors in Scalariform junction, 290–292, 292
anti-hemostatic, 174–175 Scapulae, 31
anti-inflammatory factors, 175–176 Scapularisin, 266–267, 296
complement inhibitors, 176–177 Sclerotization, 99, 104–105, 115–116
immunomodulators, 177 Scolopales, 352, 353
overview, 173–174 Scutae, 8, 30
Salivarium, 84–85, 87, 129 Scutal index (SI), 115
Salivary glands Scutum, anatomy of, 74, 79–80, 100
anatomy of, 8, 9, 87–89 Secretory cells, 141
carrier proteins and, 400, 409–410 Secretory granules, 164–165, 168–169, 490–491,
comparative transcriptomics and gene 496
discovery in Segmentation, 74, 75, 82, 93
expressed sequence tags, 181, 181–182 Seminal fluid, 95, 459, 501, 507–508
gene expression during feeding, 182–183 Seminal peptides, 501, 507–508
gene expression profiling in developmental Seminal receptacle, 94, 454
stages, 184 Seminal vesicle, 95, 487, 492
overview, 180 Semiochemicals, 370–371, 379–380. See also
degeneration of, 169–170 Communication, chemical; Specific
development of, 168–170, 184 chemicals
dopamine and, 343–344, 345, 346 Sensilla
emerging areas of research, 192–193 chelicerae and, 128, 356
functional genomics of chemosensilla, 352–355
RNA interference, 191–192 eyes, 357
transcriptional profiling during pathogen Haller’s organ, 352, 355–356
exposure, 190–191 mechanosensilla, 355
hydration and, 13 overview of, 351, 351
innervation of, 325–328, 329, 330 palpal sensory organ, 356
molecular biology of, 12 Sensilla auriformia, 77, 79, 80, 94, 355
morphology Sensilla basiconica, 352, 355
agranular (type I) acini, 164, 165 Sensilla chaetica, 355
granular acini, 164–168, 166, 167 Sensilla coeliconicum, 352, 355
overview of, 163–164 Sensilla hastiformia, 355
pathogen transmission and, 177–180 Sensilla lanterniformia, 355
proteomic analysis of Sensilla trichoidea, 355
2-D polyacrylamide gel electrophoresis, Sensory pores, 128
185–186 Sensory receptor cells, 104
high-performance liquid chromatography, Sensory system, 309, 351. See also Nervous
186–188 system; Sensilla
overview, 184, 185 Septate desmosomes, 143
peptide mass fingerprinting, 188–189 Septum, 260
Index 537
Sequestration, 228–229 Spherules. See Granulocytes

Serine carboxypeptidases, 147, 154, Spherulocytes, 277, 277
155, 190 Spiracles
Serine proteases, 147–155, 174, 178, 180, 270, of hard ticks, 240–241, 241
505–509, 506 of Nuttalliella namaqua, 245
Serine/threonine kinases, 505, 506, 506 overview of, 77, 79
Serotonin, 346–348, 348 of soft ticks, 244–245, 245
Serpins, 148–150, 174, 270 Spiracular glands, 242
Serrate sensilla, 355–356 Spiracular plate
Setae anatomy of, 241, 242, 243
anatomy of, 76, 79, 80, 94 functions of, 246
morphologic identification and, 29–30 overview of, 77–80, 79
sensory receptor cells and, 104, 352 as plastron, 246, 253
Setal socket, 355 Spoke-wheel sensilla, 352, 355
Setiform sensilla, 355, 356 Spotted fever group (SFG) rickettsiae, 178
SFG rickettsiae. See Spotted fever group Squalene, 104
rickettsiae SSH. See Suppression subtractive
Shadows, 374 hybridization
SI. See Scutal index Starvation, 252, 254
Sieve plates, 246 Statins, 296
SIFamides, 320, 321, 323–324, 326–328, 329, Status quo hormones, 418, 421, 423
336–337, 342 Steroids. See Ecdysteroids
Single-molecule sequencing (SMS), 193 Stomodeal pons, 93
Single nucleotide polymorphisms (SNP), 43 Storage proteins. See also Carrier protein;
Single-walled (SW) sensilla, 352, 355 Vitellin; Vitellogenins
Sinuses, 263–264 in arthropods other than ticks, 400–401
Size, 109–110, 110, 111, 115 developmental changes in protein levels of,
Slow-digestion stage, 144 398–400, 399
SMS. See Single-molecule sequencing distinguishing between different types of,
SNAP. See Synaptosomal-associated 411–412
proteins functions of, 402–405
SNAREpin, 171 genetic regulation of, 408–409
SNARE proteins, 171 molecular biology of
SNP. See Single nucleotide polymorphisms carrier proteins, 406–408, 407
Socket, 352 vitellogenins, 405–406, 407
SOD. See Superoxide dismutases overview of, 229–231, 398
Soft ticks. See Argasidae structure of, 400–402
Soluble N-ethylmaleimide-sensitive factor tissue levels of, 400
attachment protein receptor (SNARE) Subcuticle, 101
complex proteins, 171 Subesophageal cells, 298
Sorbitol, 272 Subolesin, 505
Spermateleosis. See Capacitation Subostial space, 241, 242
Spermatids, 492 Subplasmalemmal cisternae, 500
Spermatocysts, 488, 499 Sub-spiracular glands, 242
Spermatodactyl, 484 Subtractive cDNA hybridization, 183
Spermatogenesis, 484, 498–501, 503–507 Sucking, 129
Spermatogonia, 488, 499 Sulfakinin receptor, 335
Spermatophores, 95, 168, 169, 497–498, Sulfakinins, 441
501–502 Superoxide dismutases (SOD), 150, 510
Spermatozoa, 485, 497, 498, 503 Suppression subtractive hybridization
Spermine, 509 (SSH), 191
Spermiogenesis, 484–485, 498–501, 503–507 SW sensilla. See Single-walled sensilla
Spermiophores, 459, 460–461, 488, 491 Symbionts, 156, 211
538 Index
Synaptosomal-associated proteins (SNAP), 510 tHRF. See Histone release factor

Syngamy, 459 Thrombin, 174–175
Synganglion Thrombin inhibitors, 175
anatomy of, 8–9, 86, 86, 91–93, 92, 310–324, Tick anticoagulant peptide (TAP), 174
311, 312 Tick-host interface, 192–193, 222, 224–225
circulatory system and, 260, 260 Tick paralysis, overview of, 7, 7–8
cortical zone, 311–314 Tip pore sensilla, 353, 353, 355
farnesyl pyrophosphate pathway of, 430 Tormogen cells, 94, 352
neuropeptide gene expression in, 435–436 TOR pathway, 469, 473
neuropile, 314 Total evidence approach, 17
octopamine and, 349 Touch sensation, 374
peptidergic neurons of, 314–324, 316–317, Toxins, 103, 176, 349
318, 319, 320, 321 TP. See Tunica propria
in regulation of molting, development, and Tracheae, 9, 74, 83, 90, 246–247
reproduction, 419–420 Tracheal atrium, anatomy of, 241, 242–243
subesophageal region, 310–311 Tracheal system
supraesophageal region, 310 anatomy of, 246–247, 247
Synomones, 370, 371 function of, 246–247
Systematics gas exchange in, 248
molecular-based molting of, 247
barcoding and molecular keys, 44–45 Tracheal trunks, 242, 246
microsatellite markers, 43–44 Tracheoles, 247
phylogenetic studies, 39–42 Transcriptome analysis
population genetics, 42–43 during feeding, 145, 182–183
recent developments in, 45–50 of male reproductive gland proteins, 504,
morphology-based 505–506, 506
adults, 30–31 for neuropeptide identification, 335–337
basis of, 29 next-generation, 192
identification of adults to genus, 31–32, 32 of salivary glands, 181–184, 192
key for identifying to family and genus, at tick-host interface, 192–193
36–39 transcriptional profiling, 190–191
larvae, 29–30 Trehalose, 272, 288
nymphs, 30–31 Triacylglycerol, 463
recent developments in, 45–50 Triatoma infestans, 155
terminology of, 33–36 Triatomines, 404
overview of, 17 Trichogen cells, 94, 100, 352
Trochanteral spurs, 30
T Trophocytes, 91, 288, 290–292, 291, 292
Tachykinin-related peptides, 316, 318, 324, 326, Trypsin, 147, 507
328–331, 332 Tubular accessory glands (TAG), 454–455
Tachylectins, 269 Tubular elements, 134
Taenidium, 90, 246, 247 Tubulins, 504, 510
TAG. See Tubular accessory glands Tunica propria (TP), 451
TAM, 269–270 2D differential in-gel electrophoresis
Tandem mass spectrometry, 189 (DIGE), 186
TAP. See Tick anticoagulant peptide 2-D polyacrylamide gel electrophoresis
Tarsal spurs, 30 (2D-PAGE), 185–186
Tarsus, 82, 83 Two-host life cycles, 62, 62–63
Temperature resistance, 104 Type I (agranular) acini, 88, 164, 165
Testes, 95, 487, 488, 489, 495–496, 499 Type II acini, 165–166, 166
Theileria spp., 179, 180 Type III acini, 166–167, 167
Thioredoxin, 510 Type IV acini, 167–168
Three-host life cycles, 60–62, 61 Tyramine, 114
Index 539
U Vitellogenesis cells, 227

UDC. See Undifferentiated reserve cells Vitellogenic oocytes, 458
Ultraspiracle protein (UP), 169–170 Vitellogenin-inducing factors (VIF),
Undifferentiated reserve cells (UDC), 130, 132, 133 471, 473
UP. See Ultraspiracle protein Vitellogenin receptors (VgR), 432,
Urate oxidase, 217 474, 475
Urea, 217 Vitellogenins (Vg)
Uric acids, 212, 217 anatomy of, 90
Urine formation, 212 in arthropods other than ticks, 401
Urocytes, 288 carrier proteins vs., 231–234, 411–412
Uterus, 94, 503 developmental changes in protein levels of,
398–400, 399
V endocrine regulation of, 430–432
Vaccines, 138 evolution of blood meal digestion and,
Vacuolar-type proton pumps (V-ATPase), 173 231–234, 233
Vagina, 94, 454, 455 fat body and, 289, 292–295
Variabilin, 174, 187 genetic regulation of, 408–409
Variegin, 187 as heme-binding storage protein, 226
Varisin, 266, 296 heme processing and, 404–405
Vas deferens, 95, 485, 487–488, 493, 497 heme transport and, 230
V-ATPase. See Vacuolar-type proton pumps molecular biology of, 405–406, 407
Vectorbase, 8 nutrition and, 402–403
Vector competence, 179–180 overview of, 265, 398, 462–467,
Venoms, 106 464–466
Ventral plates, 80 protein sequences and gene sequences,
Ventriculus, 86, 87, 130 464–466
Ventrolateral suspensory muscle apparatus reproduction and, 11, 424–427, 425, 427
(vlsm), 260, 263 sources of, 91
Vestibular vagina and LAG, 455 synthesis in midgut, 142
Vestibulum, 244 tissue levels of, 400
Vg. See Vitellogenins uptake of, 474, 475
Vibrations, 222–223, 374 Vlsm. See Ventrolateral suspensory muscle
VIF. See Vitellogenin-inducing factors apparatus
Virgin females, 131–138, 136, 137 Vn. See Vitellin
Viruses, 6 Voraxin, 170, 190, 505, 511
Visual images, 374
Vitellarium, 452 W
Vitellin (Vn) Wandering cells, 300
degradation and processing of, 230–231 Warmth, host-seeking behavior and, 374
formation of, 289, 459 Waste elimination. See Excretion and waste
heme processing and, 404 elimination
overview of, 226, 398 Water, survival in, 252–254, 253
protein sequences and gene sequences, Water content
464–466 coxal glands and, 206, 226
in tick eggs, 462–463 cuticle properties and, 110–111
Vitellogenesis Malpighian tubules and, 208, 226
ecdysteroids and, 429 plasticization and, 114
initiation of, 429 rectal sac and, 207
in insects, 289 salivary glands and, 172–173, 226
juvenile hormone and, 418–419 spiracular plate and, 246
overview of, 468–474, 470, 471, 472 Water elimination, 226, 230
salivary gland degeneration and, 169 Water vapor, 374
site of, 467–468, 468 Water witch (wtrw) genes, 382–383
540 Index
Wax glands, 30 Y
WD40 proteins, 299 Yeast, protein expression in, 190
Wohlbachia spp., 211, 452 Yolk granules, 452, 458, 459
Wound healing assays, 192 Yolk protein receptor (YPR), 475
Wreath cells, 298 YPR. See Yolk protein receptor
Wtrw genes. See Water witch genes
Z
X Zinc metalloprotease, 506, 506–507
Xanthine, 213 Zonula adherens, 143

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BIOLOGY OF TICKS

Edited by Daniel E. Sonenshine

Oxford New York

© Oxford University Press 2014

All rights reserved. No part of this publication may be reproduced, stored in a

You must not circulate this work in any other form

Library of Congress Cataloging-in-Publication Data

10. Respiratory System: Structure and Function 240

To view the book’s supplementary materials, please go to www.oup.com/us/biology

1. Volume 1, Supplementary Fig. S18.1: Lobe of Dermacentor variabilis male accessory

Volume 2. Ecology, Genomics, Disease and Control

14. Tick Repellent Research, Methods, and Development 382

Supplementary Materials (www.oup.com/us/biologyofticks2e)

1. Volume 2, Supplementary Table 3.1: Summary of Published Complete Genome Sequences

Francisco J. Alarcon-Chaidez Brooke W. Bissinger

Laura J. Fielden Mari H. Ogihara

DeMar Taylor Jiwei Zhu

DANI EL E. S ONENSHINE AND R. MICHAEL ROE

2. SYSTEMATICS AND EVOLUTIONARY

3. TICKS AND DISEASE

3.1. TICK-BORNE DISEASES

Table 1.1: A summary of the different categories of tick-borne diseases of humans

Table 1.1: (continued)

monocytotropic ehrlichiosis (caused by Ehrlichia chafeensis), and Ehrlichiosis (caused by E.

3.2. TICK-CAUSED DISEASES

4.1. BODY STRUCTURE AND COMPARISON WITH

4.1.1. External anatomy

4.1.2. Internal anatomy

4.2. DEVELOPMENT AND REPRODUCTION

Table 1.2: Some biological features used to distinguish ticks from

Biological characteristics Ticks Insects

4.3. BLOOD FEEDING AND DIGESTION

4.4.1. Tick survival in the natural environment

4.4.2. Host specificity

REF ERENCES CITED

MODERN TICK SYSTEMATICS

2. MORPHOLOGY-BASED TICK SYSTEMATICS

2.1. CHOICE OF MORPHOLOGICAL CHARACTERS

2.2. IDENTIFYING ADULT TICKS TO GENUS

Table 2.1: Classification of argasid (soft) ticks by different researchers.

Russian Schoola American Schoolb Klompen and Oliver Schoolc

2.3. MORPHOLOGICAL TERMINOLOGY

2.4. MORPHOLOGICAL KEY FOR IDENTIFYING EXTANT TICKS TO

3. MOLECULAR-BASED TICK SYSTEMATICS

3.1. TICK EVOLUTION AND PHYLOGENETICS

3.1.1. Early hypotheses

3.1.2. Fossil record

3.1.3. Phylogenetic molecular studies

3.2. POPULATION GENETICS

3.2.1. Mitochondrial and nuclear genes

3.2.2. Microsatellite markers

3.3. MOLECULAR BARCODING AND MOLECULAR KEYS

4. RECENT DEVELOPMENTS IN MORPHOLOGICAL

and Pospelova-Shtrom (1969) proposed their “Russian-school” argasid classification; further,

REF ERENCES CITED

LIFE CYCLES AND NATURAL

2.1. THREE-HOST LIFE CYCLES

2.1.1. Representative example of 3-host ticks among ixodids

FIGURE 3.1: Three-host life cycle of Ixodidae ticks.

2.1.2. Species distribution of the 3-host life cycle

FIGURE 3.2: Two-host life cycle of Ixodidae ticks.

2.2. TWO- AND 1-HOST LIFE CYCLES

2.2.1. Examples of 2-host and 1-host life cycles in ixodids

FIGURE 3.3: One-host life cycle of Ixodidae ticks.