Advances in Invertebrate

(Neuro)Endocrinology-A Collection of
Reviews in the Post-Genomic Era
Volume 1: Phyla Other Than
Anthropoda 1st Edition Saber
Saleuddin (Editor)
Visit to download the full and correct content document:
https://textbookfull.com/product/advances-in-invertebrate-neuroendocrinology-a-colle
ction-of-reviews-in-the-post-genomic-era-volume-1-phyla-other-than-anthropoda-1st-e
dition-saber-saleuddin-editor/
More products digital (pdf, epub, mobi) instant
download maybe you interests ...

Advances in Invertebrate (Neuro)Endocrinology-A

Collection of Reviews in the Post-Genomic Era, Volume
2: Arthropoda 1st Edition Saber Saleuddin (Editor)

https://textbookfull.com/product/advances-in-invertebrate-
neuroendocrinology-a-collection-of-reviews-in-the-post-genomic-
era-volume-2-arthropoda-1st-edition-saber-saleuddin-editor/

Like a Thief in Broad Daylight Power in the Era of Post

Humanity Slavoj Žižek

https://textbookfull.com/product/like-a-thief-in-broad-daylight-
power-in-the-era-of-post-humanity-slavoj-zizek/

Managing Health in the Genomic Era: A Guide to Family

Health History and Disease Risk 1st Edition Vincent
Henrich

https://textbookfull.com/product/managing-health-in-the-genomic-
era-a-guide-to-family-health-history-and-disease-risk-1st-
edition-vincent-henrich/

Recent Advances in Medicinal Chemistry Volume 1 1st

Edition Choudhary

https://textbookfull.com/product/recent-advances-in-medicinal-
chemistry-volume-1-1st-edition-choudhary/
Politics And Technology In The Post Truth Era Anna
Visvizi

https://textbookfull.com/product/politics-and-technology-in-the-
post-truth-era-anna-visvizi/

Advances in Psychology and Law Volume 1 1st Edition

Monica K. Miller

https://textbookfull.com/product/advances-in-psychology-and-law-
volume-1-1st-edition-monica-k-miller/

Advances in food security and sustainability. Volume 1

First Edition Barling

https://textbookfull.com/product/advances-in-food-security-and-
sustainability-volume-1-first-edition-barling/

Plant Nanobionics Volume 1 Advances in the

Understanding of Nanomaterials Research and
Applications Ram Prasad

https://textbookfull.com/product/plant-nanobionics-
volume-1-advances-in-the-understanding-of-nanomaterials-research-
and-applications-ram-prasad/

Advances in Mathematical Chemistry and Applications

Volume 1 1st Edition Subhash C. Basak

https://textbookfull.com/product/advances-in-mathematical-
chemistry-and-applications-volume-1-1st-edition-subhash-c-basak/
 ADVANCES IN INVERTEBRATE
(NEURO)ENDOCRINOLOGY
A Collection of Reviews in the Post-Genomic Era

VOLUME 1: Phyla Other Than Arthropoda

 ADVANCES IN INVERTEBRATE
(NEURO)ENDOCRINOLOGY
A Collection of Reviews in the Post-Genomic Era

VOLUME 1: Phyla Other Than Arthropoda

Edited by
Saber Saleuddin, PhD
Angela B. Lange, PhD
Ian Orchard, DSc, PhD
Apple Academic Press Inc. Apple Academic Press Inc.
4164 Lakeshore Road 1265 Goldenrod Circle NE
Burlington ON L7L 1A4 Palm Bay, Florida 32905
Canada USA
© 2020 by Apple Academic Press, Inc.
Exclusive worldwide distribution by CRC Press, a member of Taylor & Francis Group
No claim to original U.S. Government works
Advances in Invertebrate (Neuro)Endocrinology: A Collection of Reviews in the Post-Genomic Era
International Standard Book Number-13: 978-1-77188-809-7 (Hardcover)
International Standard Book Number-13: 978-0-42926-445-0 (eBook)
Volume 1: Phyla Other Than Arthropoda
International Standard Book Number-13: 978-1-77188-892-9 (Hardcover)
All rights reserved. N1nformation obtained from authentic and highly regarded sources. Reprinted material is quoted with permission and
sources are indicated. Copyright for individual articles remains with the authors as indicated. A wide variety of references are listed. Reasonable
efforts have been made to publish reliable data and information, but the authors, editors, and the publisher cannot assume responsibility for the
validity of all materials or the consequences of their use. The authors, editors, and the publisher have attempted to trace the copyright holders of
all material reproduced in this publication and apologize to copyright holders if permission to publish in this form has not been obtained. If any
copyright material has not been acknowledged, please write and let us know so we may rectify in any future reprint.
Trademark Notice: Registered trademark of products or corporate names are used only for explanation and identification without intent to
infringe.

Library and Archives Canada Cataloguing in Publication

Title: Advances in invertebrate (neuro)endocrinology, two volumes : a collection of reviews in the post-genomic era /
edited by Saber Saleuddin, PhD, Angela Lange, PhD, Ian Orchard, DSc, PhD.
Names: Saleuddin, Saber, editor. | Lange, Angela, 1957- editor. | Orchard, Ian, 1951- editor.
Description: Includes bibliographical references and indexes. | Contents: Volume 1. Phyla other than arthropoda.
Identifiers: Canadiana (print) 2019018860X | Canadiana (ebook) 20190188677 | ISBN 9781771888097 (set ; hardcover) |
ISBN 9781771888929 (v. 1 ; hardcover) | ISBN 9780429264450 (set ; ebook)
Subjects: LCSH: Invertebrates—Endocrinology. | LCSH: Neuroendocrinology.
Classification: LCC QP356.4 .A38 2020 | DDC 573.412—dc23

Library of Congress Cataloging-in-Publication Data

Names: Saleuddin, Saber, editor. | Lange, Angela, 1957- editor. | Orchard, Ian, 1951- editor.
Title: Advances in invertebrate (neuro)endocrinology : a collection of reviews in the post-genomic era / edited by
Saber Saleuddin, Angela Lange, Ian Orchard.
Description: Oakville, ON ; Palm Bay, Florida : Apple Academic Press, [2020] | Includes bibliographical references and index.
| Contents: v. 1. Phyla other than Arthropoda -- v. 2. Arthropoda. | Summary: “Advances in Invertebrate (Neuro)Endocrinology:
A Collection of Reviews in the Post-Genomic Era (2-volume set) provides an informative series of reviews from expert
scientists who are at the forefront of their research into the endocrinology of invertebrates. These two volumes are timely
and appropriate in this post-genomic era because of the rapid pace of change brought about by genome projects, functional
genomics, and genetics (omics technologies). The volumes show the rich history and strong tradition of cutting-edge research
using invertebrates that has opened up our broader understanding of comparative endocrinology and the evolution of regulatory
pathways and systems. These reviews set the scene and context for this exciting new era of understanding that has come
from this post-genomic revolution. This book undertakes the daunting task of covering most of the diverse endocrine systems
that exist among invertebrates. The papers in this book will advance our knowledge of invertebrate endocrinology but also of
endocrinology in general, making the book valuable to researchers and students. Key features: Looks at the enormous diversity
of species involved and the variety of hormonal pathways covers the diverse endocrine systems that exist among invertebrates
makes relevant comparisons of molecular, cellular, and behavioral aspects of invertebrate endocrinology Explores the
molecular genetics techniques are now allowing exploitation of these genomes through specific interference with genes, and
thereby interference with their phenotypic expression”-- Provided by publisher.
Identifiers: LCCN 2019042135 | ISBN 9781771888097 (set ; hardcover) | ISBN 9781771888929 (v. 1 ; hardcover) |
ISBN 9781771888936 (v. 2 ; hardcover) | ISBN 9780429264450 (ebook)
Subjects: MESH: Neurosecretory Systems--physiology | Invertebrates--physiology | Invertebrates--genetics |
Hormones--physiology | Neuropeptides--physiology | Neuroendocrinology
Classification: LCC QP356.4 | NLM WL 102 | DDC 612.8--dc23
LC record available at https://lccn.loc.gov/2019042135
Apple Academic Press also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in
electronic format. For information about Apple Academic Press products, visit our website at www.appleacademicpress.com and the CRC Press
website at www.crcpress.com
 DEDICATION
“Knowledge advances progress, whereas ignorance impedes it.”
This book is dedicated to Dr. Berta Scharrer for her pioneering
work in (neuro)endocrinology and to our parents for giving us the
encouragements and opportunities to become biologists.
About the Editors

Saber Saleuddin, PhD

Saber Saleuddin, PhD, is a University Professor Emeritus in the Department
of Biology, York University in Toronto, Ontario, Canada. He received his
early education in Bangladesh and his doctorate from the University of
Reading in the UK. His studies on biomineralization in mollusks started
at the University of Alberta in Edmonton, Canada, and continued at Duke
University, Durham, North Carolina, in the laboratory of Karl Wilbur. Though
offered a teaching position at Duke University, he accepted a faculty position
at York University, where he still teaches. His outstanding contributions in
teaching, research, and administration were recognized by York University.
He has published extensively in international journals and has co-edited five
books on molluscan physiology. He served as co-editor of the Canadian
Journal of Zoology for 18 years and was president of the Canadian Society of
Zoologists, from whom he received a Distinguished Service Medal.

Angela B. Lange, PhD

Angela B. Lange, PhD, is a world leader in the field of insect neuroendocri-
nology, with over 175 research publications and numerous invited research
talks at international and national conferences. Professor Lange also demon-
strates leadership in the research community, being a Council Member of
the North American Society of Comparative Endocrinology, the European
Society of Comparative Endocrinologists, and the International Federation
of Comparative Endocrinology Societies. In addition to her international
stature in research, Professor Lange was Chair of the Department of Biology,
Acting Vice-Principal and Dean, and is currently Vice-Dean, Faculty, at the
University of Toronto Mississauga, Ontario, Canada. She obtained her BSc
and PhD degrees from York University, Canada.

Ian Orchard, DSc, PhD

Ian Orchard, DSc, PhD, is a Professor Emeritus, Biology, of the University of
Toronto, Ontario, Canada. An expert in insect neuroendocrinology with over
200 research publications, Professor Orchard has been funded by the Natural
Sciences and Engineering Council of Canada since 1980, has chaired the
viii About the Editors

NSERC Animal Physiology Grant Selection Committee, and has presented

numerous invited keynote and plenary research talks at international
conferences. Professor Orchard served as a Vice President of the University
of Toronto and Principal of the University of Toronto Mississauga (2002–
2010) and Vice President Academic and Provost, University of Waterloo
(2014–2017), Canada. He earned a BSc (1972), PhD (1975), and DSc
(1988), all from the University of Birmingham, UK.
Contents

.....................................................................................................
Abbreviations.................................................................................................. xiii
Preface ............................................................................................................xix

1. Cnidarian Peptide Signaling Molecules......................................................... 1

Toshio Takahashi

2. Sex-Inducing Substances Terminate Dormancy in Planarian

Postembryonic Reproductive Development................................................ 25
Kiyono Sekii and Kazuya Kobayashi

3. Fine Tuning of Behaviors Through Neuropeptide Signaling in

Caenorhabditis elegans................................................................................... 63
William G. Bendena and Ian D. Chin-Sang

4. Annelids Neuro-Endrocrino-Immune Response......................................... 93

Michel Salzet

5. Neuropeptide Signaling in Echinoderms: From “Physiologic

Activity of Nerve Extracts” to Neuropeptidomics and Beyond .............. 125
Maurice R. Elphick

6. Endocrine Control of Gametogenesis and Spawning in Bivalves........... 173

Makoto Osada and Toshie Matsumoto

7. Peptidergic Systems in the Pond Snail Lymnaea:

From Genes to Hormones and Behavior................................................... 213
Paul R. Benjamin and Ildikó Kemenes

8. A Critical Review of Sex Steroid Hormones and the Induction

Mechanisms of Imposex in Gastropod Mollusks...................................... 255
Toshihiro Horiguchi and Yasuhiko Ohta

9. Hormones May Shape Sexual Behavior in Cephalopods ........................ 285

Anna Di Cosmo, Marina Paolucci, and Valeria Maselli

10. Physiological Functions of Gastropod Peptides and

Neurotransmitters.........................................................................................311
Spencer T. Mukai and Fumihiro Morishita
x Contents

11. Ascidian Neuropeptides and Peptide Hormones...................................... 409

Honoo Satake

Index .................................................................................................................... 437

Contributors

William G. Bendena
Department of Biology, Queen’s University, Kingston, ON, K7L 3N6, Canada

Paul R. Benjamin
Sussex Neuroscience, School of Life Sciences, University of Sussex, Brighton BN1 9QG, UK

Ian D. Chin-Sang
Department of Biology, Queen’s University, Kingston, ON, K7L 3N6, Canada

Anna Di Cosmo
Department of Biology, University of Naples Federico II, Italy

Maurice R. Elphick
Professor, Maurice Elphick, School of Biological & Chemical Sciences, Queen Mary University of
London, Mile End Road, London E1 4NS, UK, Tel.: +44(0) 20 7882 6664, Fax: +44(0) 20 7882 7732,
E-mail: m.r.elphick@qmul.ac.uk

Toshihiro Horiguchi
Ecosystem Impact Research Section, Center for Health and Environmental Risk Research, National
Institute for Environmental Studies, Tsukuba, Ibaraki 305-8506, Japan, E-mail: thorigu@nies.go.jp
Ildikó Kemenes
Sussex Neuroscience, School of Life Sciences, University of Sussex, Brighton BN1 9QG, UK

Kazuya Kobayashi
Department of Biology, Faculty of Agriculture and Life Science, Hirosaki University, 3 Bunkyo-Cho,
Hirosaki, Aomori 036-8561, Japan, E-mail: kobkyram@hirosaki-u.ac.jp

Valeria Maselli
Department of Biology, University of Naples Federico II, Italy

Toshie Matsumoto
National Research Institute of Aquaculture, 422-1 Nakatsuhamaura, Minami-ise, Mie 516-0193, Japan

Fumihiro Morishita
Department of Biological Science, Graduate School of Science, Hiroshima University,
1-3-1 Kagamiyama, Higashi-Hiroshima 739-8526, Hiroshima, Japan.
E-mail: fumi425@hiroshima-u.ac.jp

Spencer T. Mukai
Department of Multidisciplinary Studies, Glendon College, 2275 Bayview Avenue, Toronto, ON,
Canada M4N 3M6. E-mail: smukai@yorku.ca

Yasuhiko Ohta
Laboratory of Experimental Animals, Department of Veterinary Medicine, Faculty of Agriculture,
Tottori University, Tottori, Tottori 680-8553, Japan, E-mail: yohta1022@gmail.com

Makoto Osada
Graduate School of Agricultural Science, Tohoku University, 1-1Tsutsumidori-Amamiyamachi,
Sendai 981-8555, Japan, Tel.: +81227178725, Fax: +81227178727.
E-mail: makoto.osada.a8@tohoku.ac.jp
xii Contributors

Marina Paolucci
Department of Science and Technology, University of Sannio, Italy

Michel Salzet
INSERM U1192–Laboratoire Protéomique, Réponse Inflammatoire et Spectrométrie de Masse
(PRISM), Université de Lille 1, F-59000 Lille , France
Honoo Satake
Suntory Foundation for Life Sciences, Bioorganic Research Institute, 8-1-1 Seikadai,
Seika-Cho, Soraku-gun, Kyoto 619-0284, Japan, E-mail: satake@sunbor.or.jp
Kiyono Sekii
Department of Biology, Faculty of Agriculture and Life Science, Hirosaki University, 3 Bunkyo-Cho,
Hirosaki, Aomori 036-8561, Japan

Toshio Takahashi
Suntory Foundation for Life Sciences, Bioorganic Research Institute, 8-1-1 Seikadai, Seika-Cho,
Soraku-gun, Kyoto 619-0284, Japan
Abbreviations

ABFs anti-bacterial factors

ACE angiotensin-converting like enzyme
ACh acetylcholine
AII angiotensin II
AKH adipokinetic hormone
AL anterior lobe
AM adrenomedullin
AMY amylin
AR androgen receptor
ATRA all-trans retinoic acid
BWM body wall muscles
CaFl calfluxin
CBs chromatoid bodies
CC canopy cell
CCAP crustacean cardioactive peptide
CCK cholecystokinin
CDCH caudodorsal cell hormone
CDCs caudodorsal cells
CGRP CT gene-related peptide
CHO Chinese hamster ovary
CKR-2 cholecystokinin receptor 2
CNCs caenacins
CNS central nervous system
CO2 carbon dioxide
CPEB cytoplasmic polyadenylation element-binding protein
CPG cerebral and pedal ganglia
CREB cAMP response element-binding protein
CRF corticotrophin-releasing factor
CRH corticotropin-releasing hormones
CRSP CT receptor-stimulating peptide
CT calcitonin
DAG diacylglycerol
DAO d-amino acid oxidase
DBD DNA-binding domain
xiv Abbreviations

DBL-1 transforming growth factor-β homolog

DBs dorsal bodies
DEG degenerin
Deg/ENaC degenerin/epithelial sodium channel
DMSR dromyosuppressin-like receptor
DTS developmentally timed sleep
EEA1 early endosome antigen-1
EGF epidermal growth factor
EH eclosion hormone
ELFamide thyrotropin-releasing hormone-like peptides
ELH egg-laying hormone
ELISAs enzyme-linked immunosorbent assays
ENaC epithelial Na+ channel
ERR estrogen-related receptor
ERs estrogen receptors
FARPS FMRFamide-related peptides
FLP FMRFamide-like peptide
GABA γ-aminobutyric acid
GALP galanin and galanin-like peptide
GC-MS gas chromatography with mass spectrometry
GFP green fluorescent protein
GI gonad index
GPCRs G-protein coupled receptors
GSS gamete shedding substance
GSS gonad stimulating substance
GTH gonadotropin
GVBD germinal vesicle breakdown
GVs germinal vesicles
H3K5 histone H3 lysine 5
HA heart accessory
HK-1/EKs hemokinin-1/endokinins
HPG hypothalamus–pituitary-gonadal axis
HSNs hermaphrodite specific neurons
INS insulin
INS-1 insulin-1
INS-Ls insulin-like peptides
IP3 inositol triphosphate
LBD ligand-binding domain
LGC-55 tyramine gated channel
LH luteinizing hormone
Abbreviations xv

LIPs lymnaea inhibitory peptides

LL lateral lobes
LNPY lymnaea neuropeptide Y
LPS lipopolysaccharides
LRR leucine-rich repeat
LSCPR2 second Lys-conopressin receptor
LUQ left upper quadrant
LYC light yellow cell
LYCP light yellow cell protein
MAPK mitogen-activated protein kinase
MCH melanin-concentrating hormone
MCT mutable collagenous tissue
ME methionine-enkephalin
MERF methionine-enkephalin arginine phenylalanine
MIP molluscan insulin-related peptide
MIPR molluscan insulin-related receptor
MIPs molluscan insulin-related peptides
MIS meiosis-inducing substance
MLN median lip nerves
MMA metamorphosin A
MPF maturation promoting factor
MS mass spectrometry
NKA neurokinin A
NKB neurokinin B
NLP neuropeptide-like peptide
NO nitric oxide
NPF neuropeptide F
NPR neuropeptide receptor
NPS neuropeptide-S
NPY neuropeptide Y
NR nuclear receptor
NT neurotensin
NTLPs neurotensin-like peptides
NTR nematocin receptor
ODS octadecylsilane
OKs orcokinins
OL olfactory lobe
OMP olfactory marker protein
ORNs olfactory receptor neurons
OT oxytocin
xvi Abbreviations

OTR OT receptor
PCs prohormone convertases
PDF pigment dispersing factor
PDFR pigment dispersing factor receptor
PEA proenkephalin A
PER period protein
PGCs primordial germ cells
PGE2 prostaglandin E2
PGF2α prostaglandin F2α
PIP2 phosphatidylinositol-4,5 bisphosphate
PN penis nerve
PP/OK pedal peptide/orcokinin
PPAR peroxisome proliferator-activated receptor
PPs pedal peptides
PRR pattern recognition receptors
PTH parathyroid hormone
QMUL Queen Mary University of London
RAMPs receptor activity-modifying proteins
RAR retinoic acid receptor
RAS renin-angiotensin system
RGP relaxin-like gonadotropic peptide
RIA radio immune assay
RLM radial longitudinal muscle
RN ring neuron
RNAi RNA interference
RNP ribonucleoprotein
RXR retinoid X receptor
SCPA SGYLAFPRMamide
SG segmental ganglia
SIS stress-induced sleep
SMP starfish myorelaxant peptide
SP substance P
SRs steroid receptors
TBT tributyltin
TcHT tricyclohexyltin
TeBT tetrabutyltin
TIR toll/IL-1 receptor
TKRPs tachykinin-related peptides
TLRs toll-like receptors
TPhT triphenyltin
Abbreviations xvii

TPrT tripropyltin
TRH thyrotropin-releasing hormone
TRHR thyrotropin-releasing hormone receptor
VDS vas deferens sequence
VP vasopressin
VPF vitellogenesis promoting factor
YCs yellow cells
Preface

Neuroendocrine control of cellular activities first evolved in invertebrates,

and the presence of endocrine cells was first reported in a mollusk. Both
peptidergic and lipid-derived hormones have been found in invertebrates,
and a few hormones are unique to invertebrates. Berta Scharrer, along with
her husband, Ernst, were the first to coin the term “neuroendocrinology.”
Later, Berta, based on her work on Aplysia (a mollusk) and Nereis (an
annelid) in the early 1930s, introduced the term “neurosecretion.” As a result
of her subsequent work on arthropods, the concept (neuroendocrinology/
neurosecretion) became accepted by the scientific community.
This series of reviews bring together expert scientists who are at the
forefront of their research into the endocrinology of invertebrates. These
two volumes are timely and appropriate in this so-called post-genomic era.
Timely, because of the pace of change brought about by genome projects,
functional genomics, and genetics (‘omics technologies), including tran-
scriptomics, peptidomics, proteomics, metabolomics, gene microarrays, and
sophisticated mass spectrometry techniques. Appropriate, because of the rich
history and strong tradition of cutting edge research using invertebrates that
have opened up our broader understanding of comparative endocrinology
and the evolution of regulatory pathways and systems. These reviews set
the scene and context for this exciting new era we find ourselves in, and the
depth of understanding that has come from this post-genomic revolution.
Studies broadly defined as invertebrate endocrinology have been trans-
formed over the last two decades since the original sequencing of the Drosophila
genome. Sequenced genomes are now available for many invertebrates, and a
bold initiative is underway to sequence genomes from 5,000 arthropod species
(the i5k project; http://i5k.github.io/). This project, along with other inverte-
brate projects, is transformative and is consolidating the discipline in this 21st
century. These projects have global implications, since invertebrates are so
important in; for example, agriculture (pollination, crop destruction), aqua-
culture and other food sources, medicine (toxins, analgesics), and industry
(silk). Researchers are now able to approach questions concerned with evolu-
tion and phylogeny, and make relevant comparisons of molecular, cellular, and
behavioral aspects of invertebrate endocrinology. These comparisons can be
made between invertebrates, but also between invertebrates and vertebrates,
xx Preface

since another bold initiative, the Genome 10K project, aims to assemble the
genomes of 10,000 vertebrate species (https://genome10k.soe.ucsc.edu/).
Developments in molecular genetics techniques are now allowing
exploitation of these genomes through specific interference with genes, and
thereby interference with their phenotypic expression. Functional genomics
and genetics are, therefore, helping to unravel complex regulatory processes.
The genes and proteins at all levels of the endocrine signaling pathway are
being defined. Gene and peptide expression can be determined for specific
tissues and individual cells, and experimental manipulation in their expres-
sion can aid in an understanding of endocrine regulation and physiology.
Editing a review series on invertebrate endocrinology is a difficult task
because of the enormous diversity of species involved and the variety of
hormonal pathways. Though we are aware it is a daunting task, this book
covers the diverse endocrine systems that exist among invertebrates. In
spite of our sincere desire, we were unable to find contributions in certain
groups because of the unavailability of contributors. Thus, the list of papers
in this book is based on expert colleagues willing and able to write within the
allotted time frame. In order to compensate for the absence of certain topics/
fields, we have reproduced two already published papers.
We are indeed indebted to those colleagues who contributed to this
book and gratefully acknowledged the role of reviewers who, with their
thoughtful comments, made our work easier. We hope that the papers in this
book will advance our knowledge of invertebrate endocrinology but also of
endocrinology in general and that the book will be valuable to researchers
and students.
Finally, we are grateful to Sandra Jones Sickels, Ashish Kumar and
Rakesh Kumar of Apple Academic Press for their support in every step
involving planning, editing, printing, and marketing.
CHAPTER 1

Cnidarian Peptide Signaling Molecules

TOSHIO TAKAHASHI
Suntory Foundation for Life Sciences, Bioorganic Research Institute,
8-1-1 Seikadai, Seika-Cho, Soraku-gun, Kyoto 619-0284, Japan

1.1 INTRODUCTION

The Cnidaria are a group of animals that branched early in the eukaryotic
tree of life, and they can be divided into five classes: Scyphozoa (true jelly-
fish), Cubozoa (box jellyfish), Hydrozoa (the species Hydra and Hydrac-
tinia), Anthozoa (sea anemones, corals, and sea pens), and Staurozoa
(stalked jellyfish). Cnidarians have a diffuse nervous system that includes a
nerve net in which the sensory and ganglionic neurons, and their processes
are interspersed among the epithelial cells of the ectodermal and endodermal
layers. Although the cnidarian nervous system is strongly peptidergic, it is
not solely peptidergic [1]. Classical molecules such as the biogenic amines
that have long been studied as major neurotransmitters in higher eukaryotes
are also involved in cnidarian neurotransmission [2]. The prevailing view
of the nervous system in the freshwater polyp Hydra (Hydrozoa) is that the
neuronal network is simple and diffused throughout the animal’s body. As to
the hydrozoan medusae, marginal nerve rings and ganglion-like structures
associated with sensory organs are observed.
Cnidaria such as Hydra is composed of multiple cell types that represent
the fundamental architecture of multicellular organisms. Hydra exhibits a
simple body plan with a single body axis. A head composed of tentacles and
hypostome is located at one end of the body axis, with a foot composed of
a peduncle and a basal disk at the other end. The gastric region is located
between the head and foot. The epithelium of Hydra consists of two layers,
the ectodermal and endodermal epithelial cell layers, which are separated by
a basement membrane known as the mesoglea. The cells of both epithelial
layers also function as muscles cells. Hydra also has multipotent interstitial
stem cells from which the nerve cells [3], nematocytes [3], gland cells [4],
2 Advances in Invertebrate (Neuro)Endocrinology, Volume 1

and germ cells [5] are derived. The capability of Hydra for regeneration and
asexual reproduction by budding are well known. Indeed, a good deal of our
understanding of axial pattern formation into head-and foot-specific tissues
has been gained through the exploitation of these features.
Peptides play important roles in signaling in developmental and physi-
ologic processes. For example, a wide variety of peptides function in inter-
cellular communication, neurotransmission, and signaling pathways that
spatially and temporally control axis formation and cell differentiation.
Recent discoveries in the cnidarian Hydra [6]—one of the most basic meta-
zoans and a key model system for studying the peptides involved in these
processes—have shown that the Hydra peptides act directly on muscle cells
to induce contraction and relaxation and also are involved in cell differentia-
tion and morphogenesis. Moreover, epitheliopeptides produced by epithelial
cells in Hydra exhibit morphogen-like activities and play roles in regu-
lating neuron differentiation through neuron-epithelial cell interactions. The
primary aim of the present overview is to describe the structure and func-
tions of peptide signaling molecules such as neuropeptides and epitheliopep-
tides in cnidarians. The importance of these peptide-signaling molecules in
the development and physiology of cnidarians is also discussed.

1.2 CNIDARIAN NEUROPEPTIDES

1.2.1 FLPS (FMR-FAMIDE-LIKE PEPTIDES)

The peptide FMRFamide, which is composed of four amino acid residues

with C-terminal amidation, was first isolated from the cerebral ganglion of
the clam Macrocallista nimbosa [7, 8]. To date, peptides sharing a similar
sequence have been isolated from other mollusks and from members of most
other phyla. These peptides are now divided into two groups according to
the level of structural similarity compared with FMRFamide. One group is
known as FMRFamide-related peptides (FaRPs), which contain N-terminal
extensions of the C-terminal FMRFamide or FLRFamide core sequence
[9]. The other group is the FLPs, which include all peptides with only the
RFamide sequence [10]. Thus, FLPs include FaRPs and all other RFamide
peptides. An excellent review has examined FaRPs from invertebrates [11].
This overview primarily focuses on cnidarian FLPs.
The evolutionarily ancient nervous system of cnidarians expresses a
variety of FLPs (Table 1.1). Peptides with GRFamide at the C-terminus
have been found in a scyphozoan [12], three hydrozoans [13–18], and an
Cnidarian Peptide Signaling Molecules 3

anthozoan [19], whereas peptides with TRFamide and/or RRFamide at the

C-terminus have been described in another anthozoan [20].

TABLE 1.1 FLPs in Cnidaria

Name Peptide Sequence Species References
Antho-RFamide pQGRFamide Anthopleura elegantissima [19]
Cyanea-RFamide I pQWLRGRFamide
Cyanea-RFamide II pQPLWSGRFamide Cyanea lamarckii [12]
Cyanea-RFamide III GRFamide
Pol-RFamide I pQLLGGRFamide Polyorchis penicillatus [14]
Pol-RFamide II pQWLKGRFamide [15]
Hydra-RFamide I pQWLGGRFamide Hydra magnipapillata [13]
Hydra-RFamide II pQWFNGRFamide
Hydra-RFamide III KPHLRGRFamide
Hydra-RFamide IV HLRGRFamide
Hydra-RFamide V pQLMSGRFamide Hydra magnipapillata [16]
Hydra-RFamide VI pQLMRGRFamide
Hydra-RFamide VII pQLLRGRFamide
Hydra-RFamide VIII KPHYRGRFamide
Hydra-RFamide IX HYRGRFamide
Hydra-RFamide X KPHLIGRFamide Hydra magnipapillata [17]
Hydra-RFamide XI pQLMTGRFamide
He-RFamide pQWLKGRFamide Hydractinia echinata [18]
Nv-RFamide I pQITRFamide Nematostella vectensis [20]
Nv-RFamide II VVPRRFamide
Abbreviations: pQ – pyroglutamate.

All neuropeptides are produced and secreted by highly regulated secretion

pathways. In general, the precursors of neuropeptides are incorporated as
preprohormones in the endoplasmic reticulum, where they are converted into
prohormones. The prohormones are then transported to the Golgi apparatus,
where they undergo post-translational modification such as endoproteolysis
and C-terminal amidation before assuming their final active peptide forms.
Cnidarian FLP cDNAs have been identified in several different animals.
A cDNA from Calliactis parasitica contains 19 copies of Antho-RFamide
(Table 1.1), two copies of FQGRFamide, and one copy of YVPGRYamide
[21]. In Anthopleura elegantissima, two cDNAs have been isolated, one of
which has 13 copies of Antho-RFamide (Table 1.1) and nine other FLPs,
whereas the other has 14 copies of Antho-RFamide and eight other FLPs [22].
4 Advances in Invertebrate (Neuro)Endocrinology, Volume 1

In Renilla koellikeri, 36 copies of Antho-RFamide are present [23]. A cDNA

from Polyorchis penicillatus has one copy of Pol-RFamide I (Table 1.1) and
11 copies of Pol-RFamide II (Table 1.1), along with another predicted FLP
[24]. The Hydra-RFamides are derived from three different preprohormones.
Preprohormone-A contains all four Hydra-RFamides (Hydra-RFamide I-IV)
(Table 1.1) [16]. Preprohormone-B contains one copy of Hydra-RFamide
I, one copy of Hydra-RFamide II, and two putative Hydra-RFamides [16].
Preprohormone-C contains one copy of Hydra-RFamide I and seven copies of
putative neuropeptide sequences [16]. In Hydractinia echinata, one copy of
He-RFamide is present (Table 1.1) [18]. In Nematostella vectensis, two FLPs
(Nv-RFamide I and II) are present (Table 1.1) [20]. Collectively, precursor-
encoding cnidarian FLP cDNAs yield many neuropeptides of great structural
diversity, indicating that they have great functional diversity as well.
Cnidarian FLPs mediate a variety of functions, including control of
muscle contraction, feeding, sensory activity, reproduction, metamorphosis,
and larval movement. In the sea anemone Calliactis parasitica, application
of Antho-RFamide causes increases in the tone, contraction amplitude, and
frequency of slow muscle contraction [25]. The same peptide induces tonic
contractions in the rachis and peduncle of the colony in individual autozooid
polyps of Renilla koellikeri, with a threshold of 5 nM in summer colonies
and 1 μM in winter colonies [26]. In Hydra, Hydra-RFamide III exhibits a
dose-dependent effect on the pumping activity of the peduncle [27]. Because
the gastrovascular cavity not only digests food but also delivers nutrients
throughout the body, it has been suggested that the contractility of the
peduncle is akin to cardiac activity in higher organisms.
A peptide-gated ion channel in snails is regulated by FMRFamide [28, 29].
Three ion channel subunits of the degenerin (DEG)/epithelial Na+ channel
(ENaC) gene family were cloned from Hydra and designated Hydra Na+
channel (HyNaC) 2–4 [30]. Subsequently, a new subunit, designatedHyNaC5,
was cloned, and expression of the gene was shown to be co-localized with
HyNaC2 and HyNaC3 at the base of the tentacles [31]. Co-expression of
HyNaC5 with NyNaC2 and HyNaC3 in Xenopus oocytes greatly increases
current amplitude after peptide stimulation and increases the affinity of the
channel for Hydra-RFamide I and II [31]. A combination of HyNaC2/3/5 forms
a peptide-gated ion channel of the DEG/ENaC gene family that contributes to
fast neurotransmission in cnidarians. Analysis of a HyNaC and ENaC chimera
revealed intriguing insights regarding the evolutionary aspects of the ion
channel. From analyses of HyNaCs, researchers have speculated that release
of Hydra-RFamide I and/or II leads to tentacle contraction, possibly when the
animals are feeding [30, 31]. Assmann and co-workers reported the molecular
Cnidarian Peptide Signaling Molecules 5

cloning of seven more HyNaC subunits, HyNaC6-HyNaC12, all of which

are members of the DEG/ENaC gene family [32]. In Xenopus oocytes, these
subunits assemble together with the four previously described subunits into 13
different ion channels that are directly gated by Hydra-RFamide I and II with
high affinity. An inhibitor of HyNaCs, diminazene, delays tentacle movement
in live Hydra. It has been shown that Hydra has a large variety of peptide-
gated ion channels that are activated by a restricted number of FLPs [32].
Thus, Hydra may select FLPs for fast neuromuscular transmission. However,
the possible function of Hydra-RFamide IV in Hydra remains unclear.
In addition to nerve cells, cnidarians have differentiated and highly
specialized mechanoreceptor cells that play a pivotal role in prey capture
and defense [33]. These are phylum-specific stinging cells known as nema-
tocytes. Ultrastructural studies demonstrated the presence of two- and three-
cell synaptic pathways in the tentacle epidermis of the sea anemone, including
synaptic connections between nematocytes surrounding nerve cells [34, 35].
FLPs likely play a role in the function of cnidarian sensory structures. The
presence of immunoreactivity for FMRFamide and RFamide in the tentacles
of our classes suggests that FLPs are involved in the chemosensory regula-
tion of cnidocyte discharge [36]. FMRFamide immunoreactivity has also
been observed in the epidermal sensory cells of the spot ocellus in Aurelia
[37]. This neuronal control likely decreases the spontaneous firing activity
of nematocytes.
FLPs are also involved in cnidarian reproduction, larval movement,
and metamorphosis. Colonial octocorals such as Renilla koellikeri repro-
duce via a two-step spawning and exfoliation process. During spawning,
intact gamete follicles are released into the environment, and during exfo-
liation, the follicles rupture, freeing the gametes. Antho-RFamide, which is
expressed in ciliated neurons within the follicle epithelium of Renilla koel-
likeri, induces exfoliation of the follicle epithelium, releasing the gametes
into the surrounding medium [38]. Furthermore, the potency of the peptide
is enhanced by light [38].
Hydractinia echinata is a colonial marine hydroid closely related to the
freshwater hydra. Fertilized eggs of this species undergo rapid cleavage divi-
sions for about 1day and develop into spindle-shaped planula larvae in about
3days [39]. The planula larvae are capable of migrating toward light [40],
and they metamorphose into adult polyps when they receive appropriate
environmental stimuli [41, 42]. Hydra-RFamide I inhibits the migration
of planula larvae, thus modulating phototaxis by inhibiting myomodula-
tion [40]. Metamorphosis is also inhibited by the peptide, leading to the
suggestion that the endogenous FLPs function in stabilizing the larval stage
6 Advances in Invertebrate (Neuro)Endocrinology, Volume 1

[43]. Thus, FLPs may play a role in regulating the movement of the planula
larvae prior to metamorphosis, possibly linking movement to chemotactic or
phototactic processes [44]. The presence of FLP-expressing sensory neurons
in the planula larvae indicate that planula migration and metamorphosis may
be regulated by the release of endogenous neuropeptides in response to envi-
ronmental cues.

1.2.2 GLWAMIDES

GLWamides have characteristic structural features in their N- and C-terminal

regions. For example, most of the peptides share a GLWamide motif at their
C-terminus (Table 1.2). In Hydra, seven GLWamide peptides were isolated
and found to have a proline residue at the second position (X-Pro) or at the
second and third positions (X-Pro-Pro) of their N-terminal region (Table 1.2)
[6, 45]. Metamorphosin A (MMA), which was isolated from the anthozoan
Anthopleura elegantissima, has a pyroglutamyl residue at the N-terminus
(Table 1.2) [46]. Both of these N-terminal structures confer resistance to
aminopeptidase digestion [47].
Cnidarian GLWamide cDNAs have also been identified in several
different animals. Leviev et al., [48] cloned a cDNA encoding the prepro-
hormone from Hydra magnipapillata containing 11 (eight different) imma-
ture peptide sequences. The cDNA encodes one copy each of Hym-53
(NPYPGLWamide), Hym-54 (GPMTGLWamide), and Hym-249 (KPIPGL-
Wamide), two copies of Hym-248 (EPLPIGLWamide), and three copies of
Hym-331 (GPPPGLWamide) along with three other predicted GLWamides
(Table 1.2). One of the predicted peptides, designated Hydra-LWamide
VIII, likely includes GMWamide at the C-terminus [48]. In H. echinata,
one cDNA encoding GLWamides was cloned [49]. The cDNA encodes
one copy of He-LWamide I and 17 copies of He-LWamide II (Table 1.2).
Compared with the preprohormones of GLWamides, two distinct cDNAs
were cloned from the anthozoans Actinia equine and Anemonia sulcata
[49]. The cDNA from the Actinia species encodes one copy each of MMA,
Ae-LWamide IV, Ae-LWamide V, Ae-LWamide VI, and Ae-MWamide,
two copies each of Ae-LWamide I and Ae-LWamide III, and four copies of
Ae-LWamide II (Table 1.2). By contrast, the cDNA from the Anemoniaspe-
cies encodes one copy each of MMA, Ae-LWamide II, and As-IWamide,
two copies of As-LWamide II, and four copies of As-LWamide I (Table 1.2)
[49]. The original MMA is contained in anthozoan preprohormones but
not hydrozoan preprohormones. Thus, MMA is a species-specific peptide.
Cnidarian Peptide Signaling Molecules 7

In addition, the peptide may be a prototype of the family with the protec-
tion of the N-terminus by pyroglutamate [50]. Two other peptides that are
possibly encoded in the preprohormones of Actinia and Anemonia are likely
processed into -GMWamide (Ae-MWamide) and -GIWamide (As-IWamide)
at their C-terminus (Table 1.2). Whether these two peptides and Hydra-
LWamide VIII belong to the GLWamide family or not is uncertain, as it has
been reported that substitution of the Leu residue in GLWamide with Met or
Ile results in complete or almost-complete loss of contractile activity in the
retractor muscle of the anthozoan Anthopleura fuscoviridis [51]. It is prob-
able that other novel neuropeptide families exist.
Species of the genus Hydractinia are colonial and usually live on snail
shells inhabited by hermit crabs. In the Hydractinia life cycle, only a
planula larval stage exists, with no medusa stage. Upon settling on snail
shells, the planula larvae undergo metamorphosis and develop into polyps
after approximately 1week [52]. MMA induces this metamorphosis [46].
This finding demonstrates that cnidarian neuropeptides function as neuro-
hormones and control developmental processes in addition to playing roles
as neurotransmitters and neuromodulators. All Hydra GLWamide peptides
also induce the metamorphosis of Hydractinia serrata planula larvae into
polyps [6, 45]. Studies involving an N-terminal deletion series revealed that
a common GLWamide sequence is necessary for the induction of metamor-
phosis in Hydractiona, indicating that induction of metamorphosis is very
specific for the GLWamide terminus and that amidation is essential [53].
Furthermore, displacement of the Gly residue of GLWamide with another
common amino acid (with the exception of Cys) resulted in either a decrease
or complete loss of potency, and displacement of the Leu or Trp residues
of GLWamide with another common amino acid except Cys resulted in
complete or almost-complete loss of potency in muscle contraction of Antho-
pleura fuscoviridis [51]. However, the precise mechanism of the actions of
GLWamide peptides in the induction of metamorphosis is not yet clearly
understood. Interestingly, larvae can be induced to undergo metamorphosis
in response to a chemical signal secreted by environmental bacteria [46].
This chemical signal is most likely received by the sensory neurons of the
planula larvae, which then release endogenous GLWamide peptides that
act on the surrounding epithelial cells, resulting in a change in phenotype.
Because hydra develops directly from embryos into adult polyps and has no
intermediate larval stage, the precise function of the GLWamide peptides in
early embryogenesis in Hydra remains an open question.
8 Advances in Invertebrate (Neuro)Endocrinology, Volume 1

TABLE 1.2 GLWAmide-Family Peptides in Cnidaria

Name Peptide Sequence Species References
MMA pQQPGLWamide Anthopleura elegantissima [48]
Hym-53 NPYPGLWamide Hydra magnipapillata [6]
Hym-54 GPMTGLWamide [45]
Hym-248 EPLPIGLWamide
Hym-249 KPIPGLWamide
Hym-331 GPPPGLWamide
Hym-338 GPPhPGLWamide
Hym-370 KPNAYKGKLPIGLWamide
He-LWamide I pQRPPGLWamide Hydractinia echinata [49]
He-LWamide II KPPGLWamide
Ae-LWamide I pQQHGLWamide Actinia equine [49]
Ae-LWamide II pQNPGLWamide
Ae-LWamide III pQPGLWamide
Ae-LWamide IV pQKAGLWamide
Ae-LWamide V pQLGLWamide
Ae-LWamide VI RSRIGLWamide
Ae-MWamide pQDLDIGMWamide
MMA pQQPGLWamide
As-LWamide I pQQAGLWamide Anemonia sulcata [49]
As-LWamide II pQHPGLWamide
As-IWamide pQERIGIWamide
Ae-LWamide II pQNPGLWamide
MMA pQQPGLWamide
Abbreviations: pQ – pyroglutamate; hP – hydroxyproline.

Sexual reproduction in reef-building corals also involves motile planula

larvae, which undergo complex metamorphosis after location of an appropriate
substrate, resulting in the founding of a juvenile coral colony. In the coral genus
Acropora, Iwao, and co-workers found that Hym-248 induces metamorphosis
of Acropora planula larvae into polyps at high rates (approximately 100%)
and that Acropora planula larvae respond to the peptide in a dose-dependent
manner [54]. Interestingly, however, Hym-248 cannot induce metamorphosis
in other coral genera [54, 55]. Therefore, the specificity of ligand recognition
by receptors appears to depend on the extent to which peptide(s) with partic-
ular structures are recognized by corals. In Hydractinia, the specificity is less
Another random document with
no related content on Scribd:
 — Te olette vaikutusvaltainen mies, herra kauppaneuvos. Teillä on
paljon tuttavia sanomalehtimiespiireissäkin.

Päivä pääsi paistamaan herra kauppaneuvoksen kasvoille. Niin,

niin, niinhän se oli. Kun sitä oli ennättänyt tehdä jotain tässä
elämässä, niin tiesivät muutkin, mihin sitä pystyi.

Hän siirsi tyytyväisenä syrjään pienen hopeatarjottimen, jolla

nähtävästi hänen aamupäiväkahvinsa oli tuotu tänne
työhuoneeseen, sitte keikautti hän kirjoitustuolinsa enemmän
Saimaan päin ja katsoi kelloaan.

— Oikeastaan on minulla kova kiire, mutta kun auttaminen on

kysymyksessä, täytyy olla valmis uhraamaan vähän aikaakin. —
Olkaa hyvä, puhukaa vapaasti asianne.

— Olen kauan hakenut työtä, — jos jonkinlaista. Hiukan olen

saanut, mutta siitä on ansiota liian vähän…

— Mutta neiti, — kauppaneuvos ei voi olla keskeyttämättä, —

oletteko pyrkinyt kotiopettajattareksi? Tai rouvan apulaiseksi? Kun on
nuori kuten te, ja kun lisäksi on tuollaiset kauniit kasvot…

Saima ei huomaa sanoja tai ei tahdo huomata. Hänkin keskeyttää.

— Herra kauppaneuvos, minulla on vanha ja kivuloinen äiti.

— Ai, ai, — ai, ai, se oli hullumpaa. — Hän näpäyttää sormiaan. —

Vanhoja ei pitäisi olla muuta kuin varakkaissa kodeissa. Eikä
sielläkään liiemmästi, ei liiemmästi. Se on minun kokemukseni.

Veri syöksähtää Saiman kasvoille.

 — Suurinkin rikkaus olisi minulle sulaa köyhyyttä ilman… Hän ei
saa lausetta lopetetuksi.

— Ymmärrän, ymmärrän! "Nicht so hitzig", neitiseni!

— Olen vapaahetkinäni vähän kirjoitellut, vain omaksi huvikseni,

en ansaitakseni. Sitä en koskaan ajatellut enkä olisi uskaltanutkaan,
mutta nyt täytyy —. Jos tämä voisi kelvata johonkin sanomalehteen?
Itse en uskaltaisi tarjota tätä, eikä kai kukaan silloin viitsisi asiaa
ajatellakaan. Mutta jos te…

Hän laskee pakettinsa pöydälle.

— Ahaa, vai tällainen teidän asianne olikin! — Kauppaneuvos

irroittaa paperit kääröstä ja rupeaa selailemaan lehtiä.

Saima tuntee vapisevansa. On kuin olisi hänen sisimpänsä siinä

pengottavana. Ja penkomassa on mies, jonka huone on lämmin ja
valoisa, ja joka juo kahvinsa hopeiselta tarjottimelta sillä aikaa, kun
äiti ja muut,— joiden ei pitäisi elää, kun ei ole varaa…

— Hyvästi, sanoo hän hätäisesti. — Minä tulen joskus perimään

vastausta. — Ja ennenkuin kauppaneuvos on ennättänyt tointua
hämmästyksestään, on Saima jo kadonnut huoneesta.

Hitaasti astuu hän kotiin päin. Kohdalle tultuaan jatkaa hän

matkaansa, kääntyy sitte takaisin kotiportille ja sieltä uudelleen pois
päin. Kotioven salpana on pelko, sydäntäkouristava pelko, ettei äiti
jaksa kestää tätä iskua. Eihän hän paljon ole puhunut tuosta
paikasta eikä näyttänyt uskovan sen saantiin, mutta jos sittekin on
odottanut ehkä hartaammin kuin konsanaan Saima.
 Ja vaikka äiti sen vielä jaksaisi kestääkin, oli se sittekin niin
armottoman lohdutonta, kun eivät olot ottaneet valjetakseen. Nyt oli
apu tarpeen. Nyt tulisi se aikanaan. Nyt ei sitä annettu. Jos sitte
kerran päästäisiin myötämäkeen, ei ehkä enää olisi häntä, jonka
voimat vastamäessä olivat ennenaikojaan loppuun kuluneet.

Hämärsi jo kun Saima avasi kodin oven. Keittiö oli tyhjä. Pimeässä
huoneessa ei hän voinut nähdä mitään.

— Äiti, missä sinä olet?

— Täällä. Minä lepäilen vähän.

— Oletko sairas?

— En, en. Muuten vaan rupesin tähän, kun ei ollut mitään

tekemistä ja kun tässä on lämpöisempikin.

Se vihlasi kuin veitsen terä. He tunsivat sen kumpikin ja puhe

katkesi siihen. Toinen ei rohjennut kertoa, toinen ei kysyä.

Vasta jouluaaton edellisenä iltana se vihdoin purkautui. Sininen

maito, nurkka, joka oli niin hatara, että siihen Saiman rääsytukoista
huolimatta kerääntyi jäätä ja lunta, ja ne kengät, ne lämpimät,
ostamattomat joululahjakengät, kävivät Saimalle viimein
ylivoimaisiksi. Yöt oli hän itkenyt, päivät vaiennut. Nyt puhkesi hän
viimein nauruun.

Hän nauroi pahaa, ilkkuvaa naurua sille lämpimälle ja mukavalle

huoneelle, jossa hänelle hauskaa joulua toivotettiin samalla kun
tingittiin vähän pois hänen vaivalla ansaituista penneistään. Hän
nauroi ja ivasi niitä, jotka joivat herkkujaan hopeisilta tarjottimilta,
samalla kun valittivat köyhien vanhusten olemassa-oloa.
 Äiti koetti rauhoittaa, mutta silloin suuttui Saima. Oli äitikin
sellainen raukkamainen nahjus! Olisi kai osaansa tyytynyt, vaikka
olisi kuollut nälkään!

— Sanoisit edes jotain, suuttuisit, olisit suunniltasi niinkuin minä,

silloin ehkä helpottaisi. Mutta kun sinä aina vaan kärsit, kärsit, käy
kuorma minulle sietämättömäksi.

Saima ei ollut nukkunut viikkoon, mutta sinä iltana nukkui hän

itkuunsa ja mielenliikutukseensa.

Ensi kertaa ei hän viime työkseen peittänyt äitiä ja saanut

osakseen äidin tavanmukaisia iltahyväilyjä.

Aamuyöstä hän heräsi. Hän oli nähtävästi nukkunut vaatteet

päällään ja äiti oli peittänyt hänet. Hän rupesi muistuttelemaan
itselleen edellistä iltaa. Kurkkuun nousi henkeä salpaava pala.
Sydäntä kirveli kirpeän kipeästi.

Eikö köyhyys itsessään ollut tarpeeksi raskas taakka? Vieläkö sen

piti kasvaa ja monistua sen kautta, että se synnytti katkeraa mieltä ja
kovia, haavoittavia sanoja.

Tai siitäkö katkeruus johtui, ettei hän ottanut surua vastaan

oikealla tavalla, ei niinkuin äiti?

Kuinka hän oli saattanut sanoa sellaista kuin sanoi äidille, juuri
hänelle, jota hän käsin oli tahtonut kantaa läpi kaiken raskaan ja
kovan? Vai olikohan hän juuri siksi suuttunut, että hän rakasti niin
paljon ja kärsi niin pohjattomasti.

Äidin vuoteelta kuului samassa hiljaista valitusta.

 — Oma äiti-kultani! — Saima kumartuu äidin puoleen.

— Tästä minä en enää nouse. Minä tunnen sen.

Hymy sammuu Saiman huulilta. Hyväilyyn kohonnut käsi painuu

puutuneena.
— Mitä voin tehdä, kysyy hän. Ja hän säikähtää oman äänensä
koleutta.

*****

Ne kengät, ne kengät, ne kauniit joululahjakengät, niitä ei enää

tarvittu. —

*****

Tammikuun viimepäivinä, pitkän piilossaolon jälkeen, rupesi päivä

yht'äkkiä paistamaan kevättä ennustavan iloisesti. Suurin osa kodin
irtaimistoa oli jo silloin panttilaitoksella ja kylmilleen jätetyssä
huoneessa makasi kuollut laudoilla.

Saima asui keittiössä. Hänen täytyi olla lähellä äitiä ja jakaa kaikki
hänen kanssaan niinkauan kuin mahdollista.

Ja hän sai paljon jaettavaa.

Lähettäjän kultaisilla alkukirjaimilla kaunistetulla, muodikkaalla

paperilla kauppaneuvos ilmotti hänelle, että käsikirjoitus tuli
julaistavaksi, ei sanomalehdessä, vaan eri kirjana. Hyvä palkkio oli
myöskin tiedossa. Kauppaneuvos onnitteli tämän johdosta, kauniisti
ja monisanaisesti lupautuen uraansa alkavan suojelijaksi. Hän oli
aina koettanut auttaa nuoria, eteenpäin pyrkiviä ihmisiä. Sen neiti
Särkkä kyllä tiesi.
 Saima näkee yhtäkkiä edessään kuvan nuoresta tytöstä, joka
jännityksestä vavisten repäisee auki tämän kirjeen, ahmii sen
sisällön ja kiiruhtaa sitte viereiseen huoneeseen, jossa viskautuu
polvilleen, painaa päänsä äidin syliin ja toistaa riemu joka äänen
väreessä: Äiti, äiti, nyt se tuli, nyt minä voin pitää Sinua hyvänä.

Mutta tuo kuva onkin vain haihtuva harhanäky, kuva siitä, mikä
olisi voinut tapahtua, jos…

Itse teossa nousee hän, kääntää välinpitämättömänä kirjeen

kokoon ja astuu hitain askelin kuolleen luo. Hänen katseensa on
yhtä eloton kuin kuolleen käsi on kylmä.

— Äiti, hän sanoo hiljaa ja kuiskaamalla, — se tuli nyt — mutta tuli

liian myöhään.
 *** END OF THE PROJECT GUTENBERG EBOOK PAKENEVIEN
PARISSA ***

Updated editions will replace the previous one—the old editions will
be renamed.

Creating the works from print editions not protected by U.S.

copyright law means that no one owns a United States copyright in
these works, so the Foundation (and you!) can copy and distribute it
in the United States without permission and without paying copyright
royalties. Special rules, set forth in the General Terms of Use part of
this license, apply to copying and distributing Project Gutenberg™
electronic works to protect the PROJECT GUTENBERG™ concept
and trademark. Project Gutenberg is a registered trademark, and
may not be used if you charge for an eBook, except by following the
terms of the trademark license, including paying royalties for use of
the Project Gutenberg trademark. If you do not charge anything for
copies of this eBook, complying with the trademark license is very
easy. You may use this eBook for nearly any purpose such as
creation of derivative works, reports, performances and research.
Project Gutenberg eBooks may be modified and printed and given
away—you may do practically ANYTHING in the United States with
eBooks not protected by U.S. copyright law. Redistribution is subject
to the trademark license, especially commercial redistribution.

START: FULL LICENSE

THE FULL PROJECT GUTENBERG LICENSE
 PLEASE READ THIS BEFORE YOU DISTRIBUTE OR USE THIS WORK

To protect the Project Gutenberg™ mission of promoting the free

distribution of electronic works, by using or distributing this work (or
any other work associated in any way with the phrase “Project
Gutenberg”), you agree to comply with all the terms of the Full
Project Gutenberg™ License available with this file or online at
www.gutenberg.org/license.

Section 1. General Terms of Use and

Redistributing Project Gutenberg™
electronic works
1.A. By reading or using any part of this Project Gutenberg™
electronic work, you indicate that you have read, understand, agree
to and accept all the terms of this license and intellectual property
(trademark/copyright) agreement. If you do not agree to abide by all
the terms of this agreement, you must cease using and return or
destroy all copies of Project Gutenberg™ electronic works in your
possession. If you paid a fee for obtaining a copy of or access to a
Project Gutenberg™ electronic work and you do not agree to be
bound by the terms of this agreement, you may obtain a refund from
the person or entity to whom you paid the fee as set forth in
paragraph 1.E.8.

1.B. “Project Gutenberg” is a registered trademark. It may only be

used on or associated in any way with an electronic work by people
who agree to be bound by the terms of this agreement. There are a
few things that you can do with most Project Gutenberg™ electronic
works even without complying with the full terms of this agreement.
See paragraph 1.C below. There are a lot of things you can do with
Project Gutenberg™ electronic works if you follow the terms of this
agreement and help preserve free future access to Project
Gutenberg™ electronic works. See paragraph 1.E below.
1.C. The Project Gutenberg Literary Archive Foundation (“the
Foundation” or PGLAF), owns a compilation copyright in the
collection of Project Gutenberg™ electronic works. Nearly all the
individual works in the collection are in the public domain in the
United States. If an individual work is unprotected by copyright law in
the United States and you are located in the United States, we do
not claim a right to prevent you from copying, distributing,
performing, displaying or creating derivative works based on the
work as long as all references to Project Gutenberg are removed. Of
course, we hope that you will support the Project Gutenberg™
mission of promoting free access to electronic works by freely
sharing Project Gutenberg™ works in compliance with the terms of
this agreement for keeping the Project Gutenberg™ name
associated with the work. You can easily comply with the terms of
this agreement by keeping this work in the same format with its
attached full Project Gutenberg™ License when you share it without
charge with others.

1.D. The copyright laws of the place where you are located also
govern what you can do with this work. Copyright laws in most
countries are in a constant state of change. If you are outside the
United States, check the laws of your country in addition to the terms
of this agreement before downloading, copying, displaying,
performing, distributing or creating derivative works based on this
work or any other Project Gutenberg™ work. The Foundation makes
no representations concerning the copyright status of any work in
any country other than the United States.

1.E. Unless you have removed all references to Project Gutenberg:

1.E.1. The following sentence, with active links to, or other

immediate access to, the full Project Gutenberg™ License must
appear prominently whenever any copy of a Project Gutenberg™
work (any work on which the phrase “Project Gutenberg” appears, or
with which the phrase “Project Gutenberg” is associated) is
accessed, displayed, performed, viewed, copied or distributed:
 This eBook is for the use of anyone anywhere in the United
States and most other parts of the world at no cost and with
almost no restrictions whatsoever. You may copy it, give it away
or re-use it under the terms of the Project Gutenberg License
included with this eBook or online at www.gutenberg.org. If you
are not located in the United States, you will have to check the
laws of the country where you are located before using this
eBook.

1.E.2. If an individual Project Gutenberg™ electronic work is derived

from texts not protected by U.S. copyright law (does not contain a
notice indicating that it is posted with permission of the copyright
holder), the work can be copied and distributed to anyone in the
United States without paying any fees or charges. If you are
redistributing or providing access to a work with the phrase “Project
Gutenberg” associated with or appearing on the work, you must
comply either with the requirements of paragraphs 1.E.1 through
1.E.7 or obtain permission for the use of the work and the Project
Gutenberg™ trademark as set forth in paragraphs 1.E.8 or 1.E.9.

1.E.3. If an individual Project Gutenberg™ electronic work is posted

with the permission of the copyright holder, your use and distribution
must comply with both paragraphs 1.E.1 through 1.E.7 and any
additional terms imposed by the copyright holder. Additional terms
will be linked to the Project Gutenberg™ License for all works posted
with the permission of the copyright holder found at the beginning of
this work.

1.E.4. Do not unlink or detach or remove the full Project

Gutenberg™ License terms from this work, or any files containing a
part of this work or any other work associated with Project
Gutenberg™.

1.E.5. Do not copy, display, perform, distribute or redistribute this

electronic work, or any part of this electronic work, without
prominently displaying the sentence set forth in paragraph 1.E.1 with
active links or immediate access to the full terms of the Project
Gutenberg™ License.
1.E.6. You may convert to and distribute this work in any binary,
compressed, marked up, nonproprietary or proprietary form,
including any word processing or hypertext form. However, if you
provide access to or distribute copies of a Project Gutenberg™ work
in a format other than “Plain Vanilla ASCII” or other format used in
the official version posted on the official Project Gutenberg™ website
(www.gutenberg.org), you must, at no additional cost, fee or expense
to the user, provide a copy, a means of exporting a copy, or a means
of obtaining a copy upon request, of the work in its original “Plain
Vanilla ASCII” or other form. Any alternate format must include the
full Project Gutenberg™ License as specified in paragraph 1.E.1.

1.E.7. Do not charge a fee for access to, viewing, displaying,

performing, copying or distributing any Project Gutenberg™ works
unless you comply with paragraph 1.E.8 or 1.E.9.

1.E.8. You may charge a reasonable fee for copies of or providing

access to or distributing Project Gutenberg™ electronic works
provided that:

• You pay a royalty fee of 20% of the gross profits you derive from
the use of Project Gutenberg™ works calculated using the
method you already use to calculate your applicable taxes. The
fee is owed to the owner of the Project Gutenberg™ trademark,
but he has agreed to donate royalties under this paragraph to
the Project Gutenberg Literary Archive Foundation. Royalty
payments must be paid within 60 days following each date on
which you prepare (or are legally required to prepare) your
periodic tax returns. Royalty payments should be clearly marked
as such and sent to the Project Gutenberg Literary Archive
Foundation at the address specified in Section 4, “Information
about donations to the Project Gutenberg Literary Archive
Foundation.”

• You provide a full refund of any money paid by a user who

notifies you in writing (or by e-mail) within 30 days of receipt that
s/he does not agree to the terms of the full Project Gutenberg™
 License. You must require such a user to return or destroy all
copies of the works possessed in a physical medium and
discontinue all use of and all access to other copies of Project
Gutenberg™ works.

• You provide, in accordance with paragraph 1.F.3, a full refund of

any money paid for a work or a replacement copy, if a defect in
the electronic work is discovered and reported to you within 90
days of receipt of the work.

• You comply with all other terms of this agreement for free
distribution of Project Gutenberg™ works.

1.E.9. If you wish to charge a fee or distribute a Project Gutenberg™

electronic work or group of works on different terms than are set
forth in this agreement, you must obtain permission in writing from
the Project Gutenberg Literary Archive Foundation, the manager of
the Project Gutenberg™ trademark. Contact the Foundation as set
forth in Section 3 below.

1.F.

1.F.1. Project Gutenberg volunteers and employees expend

considerable effort to identify, do copyright research on, transcribe
and proofread works not protected by U.S. copyright law in creating
the Project Gutenberg™ collection. Despite these efforts, Project
Gutenberg™ electronic works, and the medium on which they may
be stored, may contain “Defects,” such as, but not limited to,
incomplete, inaccurate or corrupt data, transcription errors, a
copyright or other intellectual property infringement, a defective or
damaged disk or other medium, a computer virus, or computer
codes that damage or cannot be read by your equipment.

1.F.2. LIMITED WARRANTY, DISCLAIMER OF DAMAGES - Except

for the “Right of Replacement or Refund” described in paragraph
1.F.3, the Project Gutenberg Literary Archive Foundation, the owner
of the Project Gutenberg™ trademark, and any other party
distributing a Project Gutenberg™ electronic work under this
agreement, disclaim all liability to you for damages, costs and
expenses, including legal fees. YOU AGREE THAT YOU HAVE NO
REMEDIES FOR NEGLIGENCE, STRICT LIABILITY, BREACH OF
WARRANTY OR BREACH OF CONTRACT EXCEPT THOSE
PROVIDED IN PARAGRAPH 1.F.3. YOU AGREE THAT THE
FOUNDATION, THE TRADEMARK OWNER, AND ANY
DISTRIBUTOR UNDER THIS AGREEMENT WILL NOT BE LIABLE
TO YOU FOR ACTUAL, DIRECT, INDIRECT, CONSEQUENTIAL,
PUNITIVE OR INCIDENTAL DAMAGES EVEN IF YOU GIVE
NOTICE OF THE POSSIBILITY OF SUCH DAMAGE.

1.F.3. LIMITED RIGHT OF REPLACEMENT OR REFUND - If you

discover a defect in this electronic work within 90 days of receiving it,
you can receive a refund of the money (if any) you paid for it by
sending a written explanation to the person you received the work
from. If you received the work on a physical medium, you must
return the medium with your written explanation. The person or entity
that provided you with the defective work may elect to provide a
replacement copy in lieu of a refund. If you received the work
electronically, the person or entity providing it to you may choose to
give you a second opportunity to receive the work electronically in
lieu of a refund. If the second copy is also defective, you may
demand a refund in writing without further opportunities to fix the
problem.

1.F.4. Except for the limited right of replacement or refund set forth in
paragraph 1.F.3, this work is provided to you ‘AS-IS’, WITH NO
OTHER WARRANTIES OF ANY KIND, EXPRESS OR IMPLIED,
INCLUDING BUT NOT LIMITED TO WARRANTIES OF
MERCHANTABILITY OR FITNESS FOR ANY PURPOSE.

1.F.5. Some states do not allow disclaimers of certain implied

warranties or the exclusion or limitation of certain types of damages.
If any disclaimer or limitation set forth in this agreement violates the
law of the state applicable to this agreement, the agreement shall be
interpreted to make the maximum disclaimer or limitation permitted
by the applicable state law. The invalidity or unenforceability of any
provision of this agreement shall not void the remaining provisions.

1.F.6. INDEMNITY - You agree to indemnify and hold the

Foundation, the trademark owner, any agent or employee of the
Foundation, anyone providing copies of Project Gutenberg™
electronic works in accordance with this agreement, and any
volunteers associated with the production, promotion and distribution
of Project Gutenberg™ electronic works, harmless from all liability,
costs and expenses, including legal fees, that arise directly or
indirectly from any of the following which you do or cause to occur:
(a) distribution of this or any Project Gutenberg™ work, (b)
alteration, modification, or additions or deletions to any Project
Gutenberg™ work, and (c) any Defect you cause.

Section 2. Information about the Mission of

Project Gutenberg™
Project Gutenberg™ is synonymous with the free distribution of
electronic works in formats readable by the widest variety of
computers including obsolete, old, middle-aged and new computers.
It exists because of the efforts of hundreds of volunteers and
donations from people in all walks of life.

Volunteers and financial support to provide volunteers with the

assistance they need are critical to reaching Project Gutenberg™’s
goals and ensuring that the Project Gutenberg™ collection will
remain freely available for generations to come. In 2001, the Project
Gutenberg Literary Archive Foundation was created to provide a
secure and permanent future for Project Gutenberg™ and future
generations. To learn more about the Project Gutenberg Literary
Archive Foundation and how your efforts and donations can help,
see Sections 3 and 4 and the Foundation information page at
www.gutenberg.org.
 Section 3. Information about the Project
Gutenberg Literary Archive Foundation
The Project Gutenberg Literary Archive Foundation is a non-profit
501(c)(3) educational corporation organized under the laws of the
state of Mississippi and granted tax exempt status by the Internal
Revenue Service. The Foundation’s EIN or federal tax identification
number is 64-6221541. Contributions to the Project Gutenberg
Literary Archive Foundation are tax deductible to the full extent
permitted by U.S. federal laws and your state’s laws.

The Foundation’s business office is located at 809 North 1500 West,

Salt Lake City, UT 84116, (801) 596-1887. Email contact links and up
to date contact information can be found at the Foundation’s website
and official page at www.gutenberg.org/contact

Section 4. Information about Donations to

the Project Gutenberg Literary Archive
Foundation
Project Gutenberg™ depends upon and cannot survive without
widespread public support and donations to carry out its mission of
increasing the number of public domain and licensed works that can
be freely distributed in machine-readable form accessible by the
widest array of equipment including outdated equipment. Many small
donations ($1 to $5,000) are particularly important to maintaining tax
exempt status with the IRS.

The Foundation is committed to complying with the laws regulating

charities and charitable donations in all 50 states of the United
States. Compliance requirements are not uniform and it takes a
considerable effort, much paperwork and many fees to meet and
keep up with these requirements. We do not solicit donations in
locations where we have not received written confirmation of
compliance. To SEND DONATIONS or determine the status of
compliance for any particular state visit www.gutenberg.org/donate.

While we cannot and do not solicit contributions from states where

we have not met the solicitation requirements, we know of no
prohibition against accepting unsolicited donations from donors in
such states who approach us with offers to donate.

International donations are gratefully accepted, but we cannot make

any statements concerning tax treatment of donations received from
outside the United States. U.S. laws alone swamp our small staff.

Please check the Project Gutenberg web pages for current donation
methods and addresses. Donations are accepted in a number of
other ways including checks, online payments and credit card
donations. To donate, please visit: www.gutenberg.org/donate.

Section 5. General Information About Project

Gutenberg™ electronic works
Professor Michael S. Hart was the originator of the Project
Gutenberg™ concept of a library of electronic works that could be
freely shared with anyone. For forty years, he produced and
distributed Project Gutenberg™ eBooks with only a loose network of
volunteer support.

Project Gutenberg™ eBooks are often created from several printed

editions, all of which are confirmed as not protected by copyright in
the U.S. unless a copyright notice is included. Thus, we do not
necessarily keep eBooks in compliance with any particular paper
edition.

Most people start at our website which has the main PG search
facility: www.gutenberg.org.

This website includes information about Project Gutenberg™,

including how to make donations to the Project Gutenberg Literary
Archive Foundation, how to help produce our new eBooks, and how
to subscribe to our email newsletter to hear about new eBooks.