Infection, Genetics and Evolution 30 (2015) 249–266

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Infection, Genetics and Evolution

journal homepage: www.elsevier.com/locate/meegid

Culicoides Latreille (Diptera: Ceratopogonidae) taxonomy: Current

challenges and future directions
L.E. Harrup a,⇑, G.A. Bellis b, T. Balenghien c,d, C. Garros c,d
a
Vector-borne Viral Diseases Programme, The Pirbright Institute, Ash Road, Pirbright, Surrey GU24 0NF, UK
b
University of Queensland, St Lucia, Brisbane, Qld, Australia
c
Cirad, UMR15 CMAEE, 34398 Montpellier, France
d
INRA, UMR1309 CMAEE, 34398 Montpellier, France

a r t i c l e i n f o a b s t r a c t

Article history: Culicoides Latreille biting midges (Diptera: Ceratopogonidae) cause a significant biting nuisance to
Received 12 November 2014 humans, livestock and equines, and are the biological vectors of a range of internationally important
Received in revised form 12 December 2014 pathogens of both veterinary and medical importance. Despite their economic significance, the delimita-
Accepted 13 December 2014
tion and identification of species and evolutionary relationships between species within this genus
Available online 20 December 2014
remains at best problematic. To date no phylogenetic study has attempted to validate the subgeneric
classification of the genus and the monophyly of many of the subgenera remains doubtful. Many informal
Keywords:
species groupings are also known to exist but few are adequately described, further complicating accu-
Culicoides
Biting midge
rate identification. Recent contributions to Culicoides taxonomy at the species level have revealed a high
Arbovirus correlation between morphological and molecular analyses although molecular analyses are revealing
Taxonomy the existence of cryptic species. This review considers the methods for studying the systematics of
Molecular entomology Culicoides using both morphological and genetic techniques, with a view to understanding the factors
DNA barcode limiting our current understanding of Culicoides biology and hence arbovirus epidemiology. In addition,
we examine the global status of Culicoides identification, highlighting areas that are poorly addressed,
including the potential implementation of emerging technologies.
Ó 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://
creativecommons.org/licenses/by/4.0/).

1. Introduction are of significant importance in ruminants, deer and equines,

Biting midges of the genus Culicoides Latreille, 1809 (Diptera:
Ceratopogonidae) are among the smallest haematophagous flies
described, measuring from 1 to 3 mm in body length (Mellor
et al., 2000). The genus receives considerable attention through
the role of several species as biological vectors of pathogens of
medical and veterinary importance. In addition to several nema-
tode and protozoan species, over 50 arboviruses have been isolated
from species of Culicoides and their role in the transmission of vet-
erinary (Borkent, 2004; Meiswinkel et al., 2004b; Mellor et al.,
2000) and human (Carpenter et al., 2013; Linley, 1985) pathogens
has been reviewed. Opportunistic feeding of Culicoides species on
humans can additionally impact upon tourism, forestry and agri-
cultural industries (Mellor et al., 2000). Currently, the greatest eco-
nomic impact of Culicoides lies in their ability to transmit
bluetongue virus (BTV), epizootic haemorrhagic disease virus
(EHDV) and African horse sickness virus (AHSV). These arboviruses
⇑ Corresponding author. Tel.: +44 01483 232441; fax: +44 01483 232448.
E-mail address: lara.harrup@pirbright.ac.uk (L.E. Harrup).
respectively and outbreaks are notifiable to the Office International
des Épizooties (OIE) (OIE, 2014). Culicoides have also recently been
identified as the vector of the novel Orthobunyavirus, Schmallen-
berg virus (Elbers et al., 2013).
In biologically-transmitted, vector-borne pathogens, the pheno-
typic and genetic traits of the vector(s) play a key role in determin-
ing the epidemiology of transmission. Subtle differences in the
biology and ecology of closely related species can exert significant
effects on the probability of transmission, the most important of
which are the ability to become infected with and transmit patho-
gens (‘vector competence’) and the degree of association with a
specific host (‘host preference’). Accurate separation of species is
therefore basic to understanding the epidemiology of disease
transmission.
The vast majority of taxonomic studies of Culicoides rely on
morphological analyses although in the nearly thirty years since
the classification of the Culicoides genus was described as being
in disarray (Jones et al., 1985), a substantial decline in the availabil-
ity of ‘classical’ morphological taxonomic expertise and infrastruc-
ture has occurred. The advent of ‘molecular entomology’ for

http://dx.doi.org/10.1016/j.meegid.2014.12.018
1567-1348/Ó 2014 The Authors. Published by Elsevier B.V.
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
250 L.E. Harrup et al. / Infection, Genetics and Evolution 30 (2015) 249–266
systematics is perceived by many as providing a rapid alternative
to the development of classical taxonomic expertise (Tautz et al.,
2003) and the genetic characterisation and delimination of species
of Culicoides via the phylogenetic species concept are increasing in
importance. This process has been accelerated by the unprece-
dented emergence of arboviruses in new regions, requiring rapid
characterisation of the local fauna to answer epidemiological ques-
tions in the absence of classical taxonomic expertise (Carpenter
et al., 2009).
This review considers the taxonomic methods used to identify
Culicoides from the perspective of both morphological and genetic
techniques, with a view to understanding the factors limiting our
current understanding of their biology and role in pathogen epide-
miology. We additionally examine the global status of Culicoides
identification, highlighting areas that are poorly addressed includ-
ing the implementation of emerging technologies that may assist
in this process.
2. Systematic classification of Culicoides
Early checklists of Culicoides species, for example Kieffer (1906),
were hampered by a lack of clarity regarding generic limits and
contained numerous species that have since been removed from
the genus and omitted others now regarded as valid members of
Culicoides. The genera of Ceratopogonidae were reassessed in sub-
sequent papers by Kieffer (1919, 1925) and Goetghebuer (1920)
and adopted by Edwards (1926) and this forms the basis of the
modern concept of Culicoides.
The first comprehensive worldwide checklist for species of
Culicoides following stabilisation of the genus, was produced by
Arnaud (1956) and subsequently updated by Arnaud and Wirth
(1964). A later list provided by Boorman and Hagan (1996) did
not identify synonyms, so their total number did not reflect valid
species but Borkent and Wirth (1997) provided a comprehensive
list of valid names which has been maintained online by Borkent
(2014b) to date. Linked to the latter publication, Borkent (2014a)
provides the only comprehensive list of subgeneric placements of
species of Culicoides which, despite not strictly following the most
recent valid combinations from the literature nor listing where
transfers were actioned, is an invaluable resource.
2.1. Subgeneric classification
The current subgeneric classification of Culicoides consists of 31
subgenera (Table 1) containing 63% of extant species, 38 unplaced
groups of species containing 24% of extant species and a further
13% of extant species that workers have not been able to place into
any of these groupings (Borkent, 2014a). Species of economic
importance are placed into a wide variety of these subgeneric
groupings although some, for example C. subg. Avaritia, appear to
contain a larger proportion of vector species than most other
groupings (Meiswinkel et al., 2004a; Wirth and Dyce, 1985). The
lack of vector competence data for most species of Culicoides does
not, however, allow conclusions to be drawn on how vector com-
petence for the different Culicoides-borne viruses have evolved
within the genus.
Contributions to the subgeneric classification of Culicoides have
in general been based on regional assessments with limited
attempts to rationalise groupings with those from other regions
(Fox, 1948, 1955; Khalaf, 1954; Root and Hoffman, 1937). There
is mounting evidence to suggest that at least some of the current
subgenera are polyphyletic i.e. derived from more than one com-
mon ancestor (Gomulski et al., 2006; Linton et al., 2002; Perrin
et al., 2006; Schwenkenbecher et al., 2009a) while others are prob-
ably synonymous. A major consequence of such poorly defined
subgenera is the frequent disagreement on the placement of some
species. For example, Yu et al. (2005) placed C. nudipalpis
Delfinado, 1961 into C. subg. Culicoides based on the absence of a
palpal pit while other workers, using a suite of other characters,
have consistently placed this species into C. subg. Avaritia (Bellis
et al., 2014a; Dyce et al., 2007; Meiswinkel and Baylis, 1998;
Meiswinkel et al., 2004a; Wirth and Hubert, 1989). Similarly,
C. subg. Oecacta is often used as a dumping ground for species
authors have trouble placing elsewhere (Boorman, 1988; Wirth
and Dyce, 1985).
Borkent (2014a) noted that the subgeneric classification of
Culicoides is almost entirely phenetic i.e. based on morphological
similarity, generally of adult specimens, although a very limited
number of studies have also included characteristics of immature
stages in their assessments but with variable success (Glukhova
1977; Nevill and Dyce, 1994; Nevill et al., 2009). Cladistic studies,
using morphological comparison with outgroups to identify taxa
with shared synapomorphies, have been used with some success
to validate the status of several genera of Ceratopogonidae
(Borkent, 2000), but the subgeneric groupings within Culicoides
are largely untested (Borkent 2014a) leading to the instability
noted above.
Few workers have attempted to utilise genetic data and phylo-
genetic analysis to test subgeneric groupings of Culicoides (Bellis
et al., 2014a; Pagès et al., 2009). At a higher level, only a single
study has used genetic data to test the higher classification of Cera-
topogonidae, however, support for many of the resulting clades
was low and did not support the monophyly of several recognised
taxa, including the genus Culicoides (Beckenbach and Borkent,
2003).
While the International Code of Zoological Nomenclature (ICZN)
provides guidelines for the use and format of formal nomenclature
terms including species name and subgenus (ICZN, 2012), the
infrasubgeneric categories ‘species complex’ and ‘species group’
have no formal status under the ICZN yet their use is prevalent
within the Culicoides literature. Borkent (2014a) lists species
groups which are currently unplaced to formal subgenera but not
those that are placed within a formal subgenus, for example the
10 groupings of species of C. subg. Avaritia recognised by
Meiswinkel et al. (2004a) and Dyce et al. (2007). While many of
the species groups listed by Borkent (2014a) are likely to eventu-
ally be raised as valid subgenera, as recently occurred with C. subg.
Marksomyia Bellis and Dyce (2011), the groups currently placed
within existing subgenera are not likely to be elevated to a formal
taxon and are currently referred to as either species groups or spe-
cies complexes, often interchangeably.
The adoption for Culicoides of the standardised use of the infor-
mal vernacular names ‘group’ and ‘complex’ as currently used in
the Culicidae literature (Harbach, 1994) would go some way to
alleviating this problem. Under Harbach’s (1994) scheme, the term
‘species group’ is used to include a collection of related/similar spe-
cies which exhibit a notable degree of morphological differentia-
tion and the term ‘species complex’ defines a collection of
seemingly isomorphic species, which cannot currently be differen-
tiated morphologically in one or both sexes. In contrast to, and in
order to differentiate them from species names, names for species
complexes and species groups are printed in roman letters with the
first letter capitalised i.e. ‘Obsoletus complex’ rather than
‘C. obsoletus complex’. For example, the Imicola group consists of
14 morphologically similar and closely related species (Nevill
et al., 2007; Bellis et al., 2014a), two of which, C. brevitarsis Kieffer,
1917 and C. bolitinos Meiswinkel, 1989 are genetically distinct but
morphologically indistinguishable as adults (Bellis et al., 2014a)
and as pupae (Nevill et al., 2007) and these two species conceivably
form the Brevitarsis complex, named after the first described
species in the complex.
 L.E. Harrup et al. / Infection, Genetics and Evolution 30 (2015) 249–266 251

Table 1
Subgenera of Culicoides as recognised by Borkent (2014a) with wing pattern of representative species morphological characteristics.

Subgenus Type species Number of extant species Wing pattern of representative species 
Amossovia Glukhova in Glukhova C. dendrophilus Amosova, 1957 12 C. dendrophilus Amosova, 1957 $
and Leningrad (1989)

Anilomyia Vargas (1960) C. covagarciai Ortiz, 1950 19 C. decor (Williston), 1896 $

Avaritia Fox (1955) C. obsoletus (Meigen), 1818 103 C. obsoletus (Meigen), 1818 $

Beltranmyia Vargas (1953) C. crepuscularis Malloch, 1915 43 C. crepuscularis Malloch, 1915 $

Cotocripus Brèthes (1912) C. caridei (Brèthes), 1912 6 C. caridei (Brèthes), 1912 $

(=Cotocripus caridei Brèthes)

Culicoides Latreille in Mirzaeva and C. punctatus (Meigen), 1804 56 C. punctatus (Meigen), 1804 $
Isaev (1990)

Diphaomyia Vargas (1960) C. baueri Hoffman, 1925 22 C. baueri Hoffman, 1925 $

Drymodesmyia Vargas (1960) C. copiosus Root and 25 C. loughnani Edwards, 1922 $

Hoffman, 1937

(continued on next page)

252
Table 1 (continued)
Subgenus
Fastus Liu in Yu and Huang (2006)
Glaphiromyia Vargas (1960)
Haemophoructus Macfie (1925)
Haematomyidium Goeldi (1905)
Hoffmania Fox (1948)
Jilinocoides Chu (1983)
Macfiella Fox (1955)
Marksomyia Bellis and Dyce (2011))
L.E. Harrup et al. / Infection, Genetics and Evolution 30 (2015) 249–266
Type species Number of extant species Wing pattern of representative species 
C. alpigenus Yu and Liu, 2006 11 C. erairai Kono and Takahasi, 1940 $
C. scopus Root and Hoffman, 3 C. scopus Root and Hoffman, 1937
1937
C. maculipennis (Macfie), 1925 16 C. maculipennis (Macfie), 1925 $
(= Haemophoructus maculipennis
Macfie)
C. paraensis (Goeldi), 1905 39 C. debilipalpis Lutz, 1913 $
(= Haematomyidium paraensis
Goeldi)
C. insignis Lutz,1913 82 C. peregrinus Kieffer, 1910 $
(= C. inamollae Fox and
Hoffman 1944)

C. dunhuaensis Chu, 1983 4
C. phlebotomus (Williston), 1896 2 C. phlebotomus (Williston), 1896 $
(= Ceratopogon phlebotomus
Williston)
C. marksi Lee and Reye, 1953 6 C. marksi Lee and Reye, 1953 $

Table 1 (continued)
Subgenus
Mataemyia Vargas (1960)
Meijerehelea Wirth and Hubert
(1961)
Monoculicoides Khalaf (1954)
Nullicella Lee (1982)
Oecacta Poey (1853)
Pontoculicoides Remm in Remm
and Zhogolev (1968)
Psychophaena Philippi (1865)
Remmia Glukhova (1977)
Selfia Khalaf (1954)
L.E. Harrup et al. / Infection, Genetics and Evolution 30 (2015) 249–266 253
Type species Number of extant species Wing pattern of representative species 
C. mojingaensis Wirth and 19 C. mojingaensis Wirth and Blanton, 1953
Blanton, 1953
C. guttifer (de Meijere), 1907 11 C. guttifer (de Meijere), 1907 $
(= Ceratopogon guttifer de
Meijere)
C. nubeculosus (Meigen), 23 C. nubeculosus (Meigen), 1830 $
1830 (= Ceratopogon
nubeculosus Meigen)

C. lasaensis Lee, 1978 1
C. furens (Poey), 1853 178 C. furens (Poey), 1853 $
(= Oecacta furens Poey)
D
C. tauricus Gutsevich, 1959 14
C. venezuelensis Ortiz and Mirsa, 2 C. venezuelensis Ortiz and Mirsa, 1950 #
1950 (= Psychophaena
pictipennis Philippi)
C. schultzei (Enderlein), 1908 8 C. oxystoma Kieffer, 1910 $
(= Ceratopogon schultzei
Enderlein)
C. hieroglyphicus, Malloch, 1915 7 C. hieroglyphicus Malloch, 1915 $
(continued on next page)
254 L.E. Harrup et al. / Infection, Genetics and Evolution 30 (2015) 249–266

Table 1 (continued)

Subgenus Type species Number of extant species Wing pattern of representative species 
Silvaticulicoides Glukhova (1977) C. fascipennis (Staeger) 1839 (= 12 C. fascipennis Kieffer, 1919 $
Ceratopogon fascipennis Staeger)


Sinocoides Chu (1983) C. hamiensis Chu, Qian and Ma, 8
1982
Synhelea Kieffer (1925) C. tropicalis Kieffer, 1913 34 C. tropicalis Kieffer, 1913 $

Trithecoides Wirth and Hubert C. flaviscutatus Wirth and 60 C. fulvithorax (Austen), 1912 $
(1959) Hubert, 1959

Tokunagahelea Dyce and C. mikros Dyce and Meiswinkel, 3 C. mikros Dyce and Meiswinkel, 1995 $
Meiswinkel (1995) 1995

Wirthomyia Vargas (1973b) C. segnis Campbell and Pelham- 8 C. segnis Campbell and Pelham-Clinton, 1960 $
Clinton, 1960

⁄
An additional 324 extant species are currently placed within 38 species groups that are unplaced within the current subgeneric classification of Culicoides, with an additional
173 extant species simply placed within a miscellaneous group and 5 unplaced extant species listed as nomina dubia (Borkent, 2014a).
 
Wing images not shown to scale.
D
No wing photograph available see Mathieu et al. (2012) for photograph of wing patterns characteristic of this subgenus.

No wing photograph available see Yu et al. (2005) for drawings of wing patterns characteristic of this subgenus.

Where possible, the phylogenetic validity of any species group
or complex should be assessed before being proposed to help clar-
ify the placement of species within groups and avoid later confu-
sion. The majority of cryptic species groups/complexes described
to date are based on the inability to separate adult, often female,
specimens morphologically. Additional diagnostic characteristics
are however, often present in immature stages and these may
assist in clarifying the status of species with similar adult morphol-
ogy (Nevill et al., 2007, 2009; Bellis and Dyce, 2011). Harbach
(1994) and earlier proponents of the standardised use of the terms
species group and species complex (Peyton, 1990), however, do not
discuss how these terms should be applied when for example adult
forms are isomorphic but diagnostic characteristics are present in
larval and/or pupal forms. Ideally all life stages should be studied
to reconstruct phyletic lines and grouping (Belkin, 1962), where
this is not possible clear statements on which life stages have been
considered when proposing new or revising existing groups should
be made.
 L.E. Harrup et al. / Infection, Genetics and Evolution 30 (2015) 249–266 255

2.2. Species recognition
Culicoides currently contains 1343 extant and 44 extinct species
(Borkent, 2014b), representing the largest genus of the Ceratopog-
onidae and comprising 21.5% of Ceratopogonid species. As in other
Dipteran vector groups, species classification of Culicoides has tra-
ditionally been dominated by morphological analyses and remains
heavily reliant upon the work of individual specialists. Perhaps the
best known contributor was Willis Wirth whom, during a prolific
career of over 55 years, authored or co-authored more than 400
publications on Dipteran taxonomy. The studies of Wirth and his
contemporaries have resulted in a patchy but relatively standard-
ised reporting of the Culicoides fauna across a broad global range
and major studies addressing specific regions have been produced
(Table 2).
In addition to these major taxonomic works are a huge number
of other studies of Culicoides often with relatively poor descriptions
of single or small numbers of species (Boorman, 1988; Crosskey,
1988) many of which do not provide the information required to
assess the accuracy of identification. Additionally, many of these
studies do not make reference to original type specimens, either
due to poor attention to detail on the part of the worker or the loss
of type specimens. The latter is a significant problem with older
specimens, for example the majority of species described by
Jean-Jacques Kieffer (1857–1925), were destroyed by a fire at the
Hungarian Natural History Museum (Boros, 1957). An example of
the difficulties this creates is C. oxystoma Kieffer, 1910 whose ori-
ginal description does not contain sufficient detail to allow separa-
tion of this species from others in the Schultzei group, and whose
type specimen has been lost (Wirth and Hubert, 1989). This has
resulted in the reporting of at least 19 misidentifications and 6 syn-
onymies (Wirth and Hubert, 1989) with these problems continuing
to this day e.g. Morag et al. (2012). In these cases the ICZN
recommends the designation of neotypes, preferably from the type
locality of the species. The increasing number of cryptic species
being revealed by genetic analysis (see Section 3.2) emphasises
the importance of obtaining material from the type locality as
specimens from other regions may eventually be shown to belong
to a different species. The loss of type specimens coupled with
inadequate descriptions of several other species has led Borkent
(2014a) to suggest these are likely nomen dubia (ICZN, 2012).
3. Tools for Culicoides species identification
Species definition of Culicoides has traditionally been based on
the morphology of adults. Other approaches including the mor-
phology of immatures and genetic characterisation are, however,
becoming increasingly common in taxonomic studies and are
beginning to reveal deficiencies in the traditional reliance on adult
morphology.
3.1. Morphological characterisation
3.1.1. Immature Culicoides
The morphology of immature specimens has received less
attention than that of adults with only 13% of species known as lar-
vae and 17% as pupae (Borkent, 2014a), probably due to the rela-
tive difficulty in collecting immatures compared to adults.
Immatures are most commonly identified by rearing field-col-
lected specimens to adult and the relative ease of rearing pupae
compared to larvae has probably contributed to the higher number
of species known as pupae than as larvae.
There are no examples of a species described solely from imma-
ture specimens although Nevill et al. (2009) recently provided a
description of the pupae of two African species whose adults are

Table 2
Identification aids to the Culicoides fauna by biogeographical region as defined by Holt et al. (2013).

Biogeographical region Keys to the Culicoides fauna

Afrotropical and Boorman and Dipeolu (1979, Nigeria), Cornet and Brunhes (1994, Schulzei group, Africa), Glick (1990, Kenya), Khamala and Kettle (1971,
Madagascan eastern Africa), Meiswinkel (1995, Imicola group of C. subg. Avaritia), Meiswinkel and Dyce (1989, C. subg. Synhelea, Africa), Nevill and Dyce
(1994, Simils group pupae, Africa), Nevill et al. (2007, Imicola group of C. subg. Avaritia pupae, Africa)
Australasian and Oceania Bellis et al. (2013b, Immaculatus group, Australasia; 2014a, Imicola group of C. subg. Avaritia, Australasia; 2014b, Northern Territory,
Western Australia and South Australia), Bellis and Dyce (2011, C. subg. Marksomyia, Australasia; 2012, Bancrofti group, Australasia), Dyce
et al. (2007, Australasia and Oceania), Dyce and Meiswinkel (1995, C. subg. Tokunagahelea, New Guinea), Dyce and Wirth (1997, Purus
group, Australasia, Indonesia), Kettle and Elson (1976, Australian pupae), Lee and Reye (1953, Australia), Tokunaga (1959, 1962a, 1963,
1976, New Guinea), Tokunaga and Murachi (1959, Micronesia), Wirth and Arnaud (1969, Polynesia), Elson-Harris and Murray (1992,
Australian pupae)
Nearctic Atchley (1967) (New Mexico, USA; 1970, C. subg. Selfia, Nearctic), Atchley and Wirth (1979) (Haematopotus group, Nearctic), Blanton and
Wirth (1979, Florida, USA), Downes and Wirth (1981, Nearctic), Foote and Pratte (1954, eastern USA), Fox (1955, Nearctic), Holbrook et al.
(2000, Variipennis complex of C. subg. Monoculicoides, Nearctic), Jamnback (1965, New York, USA), Jones and Wirth (1978, Stonei group,
Nearctic), Macfie (1948, Chiapas, Mexico), Root and Hoffman (1937, North America), Wirth (1952, California, USA), Wirth et al. (1985,
Nearctic), Wirth and Blanton (1967, Guttipennis group, Nearctic; New World; 1969, Pulicaris group, Nearctic; 1974, Caribbean), Wirth and
Hubert (1962, Piliferus group, New World), Wirth and Rowley (1971, Palmerae group, New World)
Neotropical and Aitken et al. (1975, Trinidad and Tobago), Brickle and Hagan (1999, Belize), Felippe-Bauer et al. (2003, Paraensis group, Amazonian region;
Panamanian 2013, C. subg. Mataemyia, Neotropical), Matta (1967, El Salvador and Honduras), Rodriguez and Wirth (1986, Columbia), Santarém et al.
(2014, Reticulatus group, Neotropical), Spinelli et al. (1993, Guttatus group of C. subg. Hoffmania, Neotropical; 2005, Argentina), Spinelli and
Martinez (1991, Uruguay), Williams (1956, Bermuda), Wirth and Blanton (1959, Panama; 1968, Hylas group; 1974, Caribbean), Wirth et al.
(1988, Neotropical)
Oriental and Sino- Arnaud (1956, Japan and Korea), Bellis (2014, Indian C. subg. Avaritia), Delfinado (1961, Philippines), Howarth (1985, Lao PDR adults and
Japanese pupae), Hubert and Wirth (1961, Okinawa, Japan), Kitaoka (1977, Nansei Islands, Japan; 1985a,b, Japan), Lien et al. (1997, 1998, Taiwan),
Sen and Das Gupta (1959, India), Takahashi (1941, 1958, Japan), Tokunaga (1940, Japan, China, Micronesia, Mongolia; 1962b, Ryukyu
Islands, Japan), Wada (1986, Claggi group, Japan; 1990, Verbosus group, Japan; 1999 Japan), Wirth and Hubert (1959, C. subg. Trithecoides,
Oriental and Afrotropical; 1961, Taiwan; 1989, Southeast Asia), Yu et al. (2005, China)
Palaearctic and Saharo- Boorman (1989, Arabian Peninsula), Boorman and van Harten (2003, Yeman), Campbell and Pelham-Clinton (1960, United Kingdom),
Arabian Delécolle (1985, Northeastern France), Dzhafarov (1964, Transcaucasus), Edwards et al. (1926, 1939, United Kingdom), Glukhova (2005,
Russia and adjacents lands), Glukhova and Braverman (1999, Langeroni group), Glukhova and Leningrad (1989, USSR), Goffredo and
Meiswinkel (2004, Italy), González de Heredia and Lafuente (2011, Spain), Gutsevich (1966, Central Asia), Kettle and Lawson (1952, United
Kingdon pupae), Kremer (1965, France), Mathieu et al. (2012, Western Palaearctic), Rawlings (1996, Iberian Peninsula, Europe), Shahin
(1977, Southwestern Asia)
256 L.E. Harrup et al. / Infection, Genetics and Evolution 30 (2015) 249–266

yet to be described and are difficult to separate morphologically. provide new insights into Culicoides systematics. In practice, how-
Although keys to immature stages of Culicoides have been pro- ever, the requirement for specimens to be slide mounted,
duced for some regional faunas (e.g. Kettle and Lawson, 1952; image-captured, measured and analysed precludes the use of mor-
Howarth, 1985; Elson-Harris and Murray, 1992) the bulk of identi- phometrics for identification purposes in high-throughput systems
fication aids for Culicoides are based on adult morphology, partially such as surveillance programs, even though some aspects of image
because this is the only means of recognising the bulk of species analysis can be automated (Weeks et al., 1999).
but also because this is the life stage most commonly encountered
either biting hosts or in monitoring programs using light traps.
3.2. Genetic characterisation
3.1.2. Adult Culicoides
3.2.1. Application of genetic studies to Culicoides systematics
Adult Culicoides are notable for their characteristic wing pig-
The use of genetic data in studies of Culicoides systematics is
mentation pattern and distribution of wing macrotrichia (Table 1),
increasing but the scientific rigor with which these studies have
which in certain species can be used as the principle diagnostic fea-
been conducted and the phylogenetic analysis interpreted is vari-
ture. Indeed, identification aids based primarily on wing patterns
able (Garros et al., 2010). Only a small proportion of studies have
have been published for Nearctic (Wirth et al., 1985), Neotropical
used multiple molecular markers for species delineation and
(Wirth et al., 1988), Australasian (Bellis et al., 2014b; Dyce et al.,
assessed concordance between the resulting gene trees as per-
2007), Austro-Oriental (Bellis et al., 2013b) and western Palaearctic
formed by Bellis et al. (2014a). Similarly, few studies have com-
faunas (González de Heredia and Lafuente, 2011; Rawlings, 1996).
pared specimens collected from a sufficiently wide geographic
Atypical variants in wing patterns are commonly observed in sev-
area to confirm that species identifications were conspecific
eral species e.g. C. newsteadi Austen, 1921 variants N1, N2 and N3
between countries/regions, an exception being the examination
(Pagès et al., 2009), but whether these represent distinct species
of C. imicola Kieffer, 1913 by Dallas et al. (2003). In addition, stud-
has yet to be fully resolved. A broad range of other morphological
ies have seldom attempted to correlate phylogenetic clades with
characters of adult specimens are also commonly used including
detailed morphological analysis (Bellis et al., 2013b).
the body and wing size, the distribution, type and number of
Despite the challenges discussed, the morphological identifica-
antennal sensilla, the shape, size and sensilla arrangement of the
tions of Culicoides by taxonomists have commonly been shown to
palpal segments, the number and form of the hind tibial comb,
be concordant with later studies utilising genetic characterisation
presence of interfacetal hairs, leg colour patterns, the shape and
(Gomulski et al., 2005, 2006; Linton et al., 2002). There are, how-
form of the male genitalia and the shape, size and number of the
ever, exceptions and the status of some species regarded as con-
female spermatheca (Cornet, 1974; Wirth and Hubert, 1989).
specific by morphologists may require reassessment given the
While the terminology of morphological characters was stable
widely divergent genetics of different populations. An example of
for many years, the relatively recent inclusion in descriptions of
this is the presence of cryptic species within the subgenus
multiple forms of antennal sensilla (Cornet, 1974) led to inconsis-
Culicoides in Europe (Gomulski et al., 2006; Pagès et al., 2009). Con-
tencies in the terminology of these structures, later standardised
versely, some species regarded by morphologists as valid have
by Wirth and Navai (1978). More recently, changes to the naming
been unable to be distinguished by genetic analysis e.g. C. circum-
of some characters, particularly the gonocoxite and gonostylus of
scriptus Kieffer, 1918, C. manchuriensis Tokunaga, 1941 and
the male genitalia and the numbering of antennal flagellar seg-
C. salinarius Kieffer, 1914 (Ander et al., 2013; Bellis et al., 2013a),
ments, have been adopted by Culicoides taxonomists to conform
while, the specific status of C. deltus Edwards, 1939 and C. lupicaris
with terminology used in other Diptera (McAlpine, 1981; Teskey,
Downes and Kettle (1952) is still controversial and needs to be
1981; Merz and Haenni, 2000; Sinclair, 2000).
resolved (Meiswinkel et al., 2004a; Nielsen and Kristensen, 2011;
The largely Wirth-inspired systematic standardisation of char-
Ramilo et al., 2013).
acters used to describe Culicoides species allowed direct compari-
The presence of ‘isomorphic’ species i.e. genetically distinct, mor-
son between species and led to the production of identification
phologically indistinguishable ‘sibling’ species which frequently
aids and dichotomous keys to species. These have been success-
occur sympatrically, also precludes identification on the basis of
fully applied to a range of regional faunas (Table 2) although little
morphological characteristics and requires the use of molecular
attempt has so far been made to develop keys applicable across
methods for separation. With regards to vector-borne diseases, fail-
classical biogeographic boundaries. More recently, the first open-
ure to recognise cryptic species may confound epidemiological
access interactive key to a regional fauna was developed
investigations due to potential variation in vector competence
(Mathieu et al., 2012) heralding a new era in Culicoides identifica-
and/or host preference between sibling species and may complicate
tion aids. These keys offer a significant improvement on traditional
control efforts due to variation in the bionomics of the sibling spe-
dichotomous keys as they typically provide a suite of illustrations
cies, for example as observed in the Anopheles Gambiae complex
of the important characters for each species and allow the user to
in relation to malaria transmission and control (Collins et al., 2000).
select the characters used to progress through the key. Their main
disadvantage is the requirement to have a computer alongside the
microscope. The lack of agreement on the morphological charac- 3.2.2. Molecular identification assays in Culicoides
ters important in defining subgeneric groupings has so far impeded To a greater extent than in morphological studies, genetic anal-
the production of identification aids to these groupings and few yses of Culicoides have centred upon putative or confirmed arbovi-
keys to regional subgenera have been developed, exceptions rus vector species. Initial cytotaxonomy studies of Culicoides
include Vargas (1960, 1973a) and Yu et al. (2005). (Hagan and Hartberg, 1986; Isaev, 1982, 1983) were followed by
Traditional morphometrics (Augot et al., 2010; Delécolle, 1985; the use of isozyme-based assays in preliminary studies in Europe
Pagès et al., 2009) and more recent landmark-based geometric (Waller et al., 1987) and then addressed cryptic species of the
morphometrics (Henni et al., 2014; Muñoz-Muñoz et al., 2011, Variipennis complex in the USA (Nunamaker and McKinnon,
2014) have been shown to be useful in enabling the separation 1989; Tabachnick, 1992). The latter studies contributed to an even-
of previously intractable specimens. The move from qualitative tual systematic reassessment and the raising of several sub-species
to quantitative assessments of morphological characters in the lat- to full species rank (Holbrook et al., 2000). While isozyme tech-
ter technique has only been applied to a handful of species but has niques are now less commonly used, they were recently employed
the potential, in combination with phylogenetic analysis, to in Sardinia (Pili et al., 2010) to investigate population genetic
 L.E. Harrup et al. / Infection, Genetics and Evolution 30 (2015) 249–266 257

structure in C. obsoletus (Meigen) 1818 and C. scoticus Downes and
Kettle (1952).
Alternative identification methodologies have recently been
proposed, including Matrix-Assisted Laser Desorption/Ionization-
Time of Flight (MALDI–TOF) analysis (Kaufmann et al., 2012a,b;
Kaufmann et al., 2011; Steinmann et al., 2013), however, the ben-
efits of these techniques in reduced cost or labour in comparison to
PCR-based assays are yet to be demonstrated. In particular, unlike
PCR-base methods, identification profiles for the different life-
stages of Culicoides are not transferable between life-stages using
MALDI–TOF (Kaufmann et al., 2011). While the MALDI–TOF
method may have a place within Culicoides identification systems,
the results produced do not infer any evolutionary information and
as such cannot easily be used to further resolve the phylogenetic
relationship of the genus.
By far the most commonly employed molecular techniques
used to identify Culicoides species are those based on polymerase
chain reaction (PCR) amplification of DNA. Following pioneering
studies (Raich et al., 1993; Sebastiani et al., 2001), sequence anal-
ysis of a wide range of mitochondrial DNA (mtDNA) markers has
become commonplace and are now the focus of the majority of
Culicoides phylogenetic studies (Table 3). Recently the fused Car-
bamoyl phosphate synthetase, Aspartate transcarbamylase and
Dihydroorotase (CAD) nuclear marker has also been assessed for
its utility in distinguishing species (Table 3). In addition, a micro-
array hybridization based method based on ITS1 sequence poly-
morphism has also been utilised for the identification of
Palaearctic C. subgen. Avaritia species (Deblauwe et al., 2012;
Augot et al., 2013a) have also developed a Restriction Length Frag-
ment Polymorphism (RLFP) assay for the identification of the mor-
phologically similar species C. stigma (Meigen), 1818 and C. parroti
(Kieffer), 1922.
Direct sequence analysis of molecular markers for Culicoides
species identification is becoming more common; however logisti-
cal and financial constraints still limit their application, particu-
larly in a surveillance role that may require the processing of
thousands of individuals. While the use of medium-throughput
multiplex PCR assays for the identification of Culicoides species
has become routine in small to medium sized research projects
investigating Culicoides abundance, distribution and bionomics in
Europe e.g. Harrup et al. (2013) and Viennet et al. (2013), these
techniques remain logistically and financially beyond the reach
of small, non-specialist research groups. A solution to this bottle
neck may lie in the use of quantitative real-time PCR assays for
identification of pooled specimens. Four assays of this type have
been developed for Culicoides. Three of these are based on sequence
polymorphism of ITS, the first targeting C. imicola (Cêtre-Sossah
et al., 2008), the second targeting differentiation of C. obsoletus
and C. scoticus (Mathieu et al., 2011), and a third targeting
C. obsoletus, C. scoticus and C. montanus Shakirzjanova, 1962
(Monaco et al., 2010). The fourth assay was based on COI polymor-
phism and was able to distinguish at least 11 Culicoides species or
variants (Wenk et al., 2012). While commonly used for quantifica-
tion and typing of pathogen burdens in both hosts (Mens et al.,
2006) and vectors (Hatta et al., 2013). Mathieu et al. (2011) is
the first to attempt the use of real-time quantitative PCR tech-
niques for quantification of the relative proportion of species con-
tained within pooled samples for insect species identification
purposes and once optimised, this technique should make analysis
of large Culicoides collections to species-level viable.
While the relative merits of the DNA extraction techniques for a
variety of sample types has been previously examined for many
types of insect e.g. Wang and Eang (2012), only one study has
investigated the impact of DNA extraction technique selection on
the sensitivity and specificity of multiplex PCR assay based identi-
fication success in Culicoides (Garros et al., 2014). Overall, commer-
cial kits where found to produce more consistent results across
DNA samples than in-house DNA extraction methods, but the abil-
ity for the techniques to be scaled up to at least medium through-
put was variable (Garros et al., 2014).
3.2.3. COI and DNA barcoding
There have been several high-profile cases made for a universal
DNA-based taxonomic identification system for eukaryotic organ-
isms (Blaxter, 2004; Godfray, 2007; Hebert et al., 2003; Tautz
et al., 2002, 2003). In response, the use of sequence diversity in
the 5’ section of the COI gene region (defined relative to the mouse
mitochondrial genome as the 648 bp region that starts at position
58 and stops at positive 705) has been selected as the focus of a
‘DNA barcoding’ system for animal life (Hebert et al., 2003).
A major advantage of the Consortium for the Barcode of Life
(CBOL) initiative (Ratnasingham and Hebert, 2007) lies in the
requirement to maintain a set of metadata standards (Hanner,
2012; Ratnasingham and Hebert, 2007). The requirement to upload
trace files for sequence data records in particular allows external
validation of sequence data quality. This is a prerequisite for
records submitted to the Barcode of Life Database (BOLD) to
achieve Barcode status (Ratnasingham and Hebert, 2007), but not
yet a requirement of GenBank (Federhen et al., 2009). The increas-
ing emphasis on producing at least partially validated sequence
data is a step towards reference sequences for species and to a
degree combats the proliferation of unauthenticated sequence data

Table 3
Molecular markers used for phylogenetic analysis within Culicoides.

Genomic Molecular References

region marker
Mitochondrial COI Ander et al. (2013), Augot et al. (2010, 2013a,b), Bakoum et al. (2013), Bellis (2013), Bellis et al. (2013a,b, 2014a), Cêtre-Sossah et al.
(2008), Dallas et al. (2003), Dik et al. (2014), Henni et al. (2014), Jan Debila (2010), Lassen et al. (2012), Lehmann et al. (2012), Linton
et al. (2002), Matsumoto et al. (2009a), Meiswinkel and Linton (2003), Morag et al. (2012), Muñoz-Muñoz et al. (2014), Nolan et al.
(2007), Pagès et al. (2009), Ramilo et al. (2013), Schwenkenbecher et al. (2009a), Swanson (2011), Wenk et al. (2012), Yanase et al.
(2013)
COII Beckenbach and Borkent (2003), Hey et al. (2003), Matsumoto et al. (2009a)
28S Henni et al. (2014)
18S rRNA Kiehl et al. (2009)
16s rRNA Jan Debila (2010), Meiswinkel and Linton (2003)
Cytb Augot et al. (2013a)
Ribosomal ITS1 Augot et al. (2013a), Cêtre-Sossah et al. (2004), Li et al. (2003), Mathieu et al. (2007), Matsumoto et al. (2009b), Morag et al. (2012),
Nielsen and Kristensen (2011), Perrin et al. (2006), Ritchie et al. (2004), Schwenkenbecher et al. (2009a), Stephan et al. (2009), Yanase
et al. (2013)
ITS2 Gomulski et al. (2005, 2006), Matsumoto et al. (2009b), Monaco et al. (2010), Schwenkenbecher et al. (2009a)
Nuclear CAD Bellis (2013), Bellis et al. (2013b, 2014a)
258 L.E. Harrup et al. / Infection, Genetics and Evolution 30 (2015) 249–266
held on GenBank which, where investigated, has been found to be
unreliable (James Harris, 2003; Nilsson et al., 2006). Conflict
between species assignments and genetic similarities have already
been identified for several Culicoides entries in GenBank (Ander
et al., 2013; Bellis et al., 2013a), which could be the result of either
misidentifications, evidence of synonymous species or an inability
of COI analyses to separate these species. The CBOL initiative also
emphasises the importance of retaining morphological ‘vouchers’
for all sequenced specimens as these constitute an essential link
between the genetic data and the taxonomic classification enabling
verification of identifications and cross-referencing of any subse-
quent specimens (Pleijel et al., 2008).
Several attempts have been made to clarify relationships and
develop identification tools for species of Culicoides based on the
COI marker, for which both universal (Folmer et al., 1994) and
genus-specific (Bellis et al., 2013b; Dallas et al., 2003) primers have
been designed and utilised in population and genetic studies e.g.
Nolan et al. (2004). Many of these studies, however, have utilised
primers which produce truncated sequences shorter than the
500 bp required to achieve Barcode status (Hanner, 2012;
Ratnasingham and Hebert, 2007). While there is good evidence
to suggest that the COI barcode region is suitable for the discrimi-
nation of species of C. subg. Avaritia (Ander et al., 2013; Bellis et al.,
2014a; Jan Debila, 2010; Linton et al., 2002), C. subg. Culicoides
(Ander et al., 2013; Lassen et al., 2012), the Immaculatus group
(Bellis et al., 2013a), the Schultzei group (Bakoum et al., 2013;
Morag et al., 2012) and the Coronalis, Molestus and Kusaiensis
groups (Bellis et al., 2013a), there are indications that variation
in this region is insufficient for delimitations of species within at
least some other subgenera e.g. C. subg. Beltranmyia (Ander et al.,
2013; Bellis, 2013).
3.2.4. ITS
Ribosomal DNA markers have been used to investigate phylog-
enies (ITS1 and ITS2: Gomulski et al., 2005, 2006; Perrin et al.,
2006; 28S: Henni et al., 2014), interspecific genetic distances
(ITS1: Nielsen and Kristensen, 2011) and population structure
(ITS1: Ritchie et al., 2004) within Culicoides. Sequence diversity in
the ITS regions has also been used to develop both conventional
(Mathieu et al., 2007; Stephan et al., 2009) and real-time (Cêtre-
Sossah et al., 2008; Mathieu et al., 2011; Monaco et al., 2010)
PCR assays for the identification of BTV vector species from indi-
vidual specimens and pooled samples.
3.2.5. CAD
An alternative marker which is gaining popularity within insect
phylogenetics is the nuclear CAD gene, which is present within
Diptera as a single copy (Adams et al., 2000; Freund and Jarry,
1987; Holt et al., 2002). Previous studies in other insect genera
have suggested that parts of this gene contain a high number of
parsimony informative sites (Moulton and Wiegmann, 2004;
Sharanowski et al., 2011; Swaffield and Purugganan, 1997). Por-
tions of the CAD region have increasingly been used to aid resolv-
ing of both shallow and deep evolutionary relationships of insect
genera e.g. Wiegmann et al. (2009) and Moulton and Wiegmann
(2004), and has shown promise as a valuable secondary marker
for Culicoides studies utilising the COI Barcode region marker
(Bellis et al., 2014a; Bellis et al., 2013b).
3.2.6. Other molecular markers
In addition to COI, CAD, ITS1 and ITS2, several additional mark-
ers have been utilised on a small-scale in Culicoides studies
(Table 3). In comparison to other vector groups there has, however,
been little investigation into other marker regions. Several markers
which have previously been utilised for Dipteran phylogenetics but
which have not yet been utilised for Culicoides studies include the
small ribosomal subunit (12S), elongation factor-1a (EF-1a), NADH
dehydrogenase subunit 2 (ND2), NADH dehydrogenase subunit 4
(ND4), NADH dehydrogenase subunit 5 (ND5), NADH dehydroge-
nase subunit 6 (ND6), 6-phosphogluconate dehydrogenase (PGD),
triose phosphate isomerase (TPI) and the white, wingless, cacophony
and period genes (Barr and McPheron, 2006; Dyer et al., 2008;
Gibson et al., 2011; Lins et al., 2002). Further assessment into rel-
ative performance for these markers for the systematics of
Culicoides, as performed for other insect genera (Baker et al.,
2001; Gibson et al., 2010; Wild and Maddison, 2008), and an
assessment of the ease of amplification and sequencing of these
regions is required before firm recommendations on their utility
for Culicoides systematics can be made.
4. Phylogenetics to phylogenomics
With increasing technical capabilities to produce sequence data
and corresponding increases in computational capacity, phyloge-
netic analysis is beginning to enter a new era, moving from the
analysis of evolutionary relationships on the basis of single genes
or small numbers of genes towards the use of genome-scale
(Stencel and Crespi, 2013) datasets to infer evolutionary relation-
ships (Barrett et al., 2013; Brito and Edwards, 2009; Narum et al.,
2013; Xi et al., 2012), so called ‘phylogenomics’ (Delsuc et al.,
2005). Following on from initial preliminary physical mapping of
the Culicoides genome (Nunamaker et al., 1999) progress began in
2013 on producing the first annotated whole genome dataset for
a species of Culicoides (Fife et al., 2013; Nayduch et al., 2014), target-
ing the American BTV vector C. sonorensis Wirth and Jones, 1957. In
addition to aiding the design of appropriate primers for other gene
regions not yet investigated in Culicoides, the publication of this first
Culicoides genome represents a vital step to increasing the ease in
which additional Culicoides species genomes can be assembled,
annotated and incorporated into future phylogenomic studies.
The usefulness of these datasets for taxonomic purposes was
recently demonstrated with the production and analysis of refer-
ence genome assemblies for 16 species of Anopheles (Neafsey
et al., 2013) which have revealed discord between the relative
placement of Anopheles complex species based on phylogenomic
data (Besansky, 2014) in comparison to relationships based on
more limited numbers of genetic markers (Obbard et al., 2009).
5. Combining morphological and genetic resources
The use of molecular data has rejuvenated interest and activity
in systematics (Pires and Marinoni, 2010). Although the use of
morphological data remains prevalent within insect systematics
(Bybee et al., 2010), the common misconception that molecular
data can replace the role of classical taxonomy has been repeatedly
refuted in the literature (Carvalho et al., 2007; Ebach and Holdrege,
2005; Will et al., 2005; Will and Rubinoff, 2004). It is therefore
essential that ‘classical’ morphological taxonomy be integrated
with high-quality phylogenetic analysis, at least in initial
instances.
Dayrat (2005) sets out guidelines for the level of support
required prior to the creation of new species including the incorpo-
ration of genetic information in the taxa description. The existence
of isomorphic species, however, questions the requirement for a
morphological description for every new species. Cook et al.
(2010) argued that new species descriptions need not be accompa-
nied by morphological confirmation and proposed a protocol for
describing new species based solely on genetic data. While this
has not yet occurred for any species of Culicoides, Bellis et al.
(2014a) recently confirmed the existence of two isomorphic spe-
cies in this genus, C. brevitarsis and C. bolitinos, indicating that
 L.E. Harrup et al. / Infection, Genetics and Evolution 30 (2015) 249–266
the protocols offered by Cook et al. (2010) may eventually be
required.
Obtaining genetic data from holotype specimens would seem a
logical progression in defining a species and should be encouraged
whenever new species are described. While the retrieval of viable
genetic material retrospectively from slide-mounted Culicoides
type specimens may prove impossible, in these cases, the next best
approach is to utilise specimens from the type locality of the spe-
cies (Bellis et al., 2013b).
The recent increase in availability of relatively low-cost digital
imaging systems for stereo- and compound microscopes, in combi-
nation with software for the analysis of images, has greatly
enhanced the acquisition of high-quality images of key diagnostic
characteristics from voucher specimens. The cost of good quality
imaging systems, however, is still prohibitive for some laboratories
and despite the increase in the ease with which high-quality digital
images of diagnostic characteristics can be acquired, the benefits of
traditional high-quality drawings to illustrate morphological vari-
ation should also not be over looked, for example see Bellis and
Dyce (2011). The widespread availability of internet connectivity
in addition to digital imaging facilities also enables an unprece-
dented level of connectivity between ecologists, taxonomists and
any other workers with an interest in Culicoides, enabling second
opinions to be readily sought and material compared without the
need to physically move specimens between labs (Ang et al.,
2013; Beaman and Cellinese, 2012; Smith and Blagoderov, 2012),
which is particularly important where movement of specimens is
problematic due to biodiversity legislation (Schindel, 2010). Bioin-
formatics tools for the free online dissemination of linked morpho-
logical and genetic data exist via BOLD (Ratnasingham and Hebert,
2007), and data from several Culicoides studies have already been
deposited and made publicly available via BOLD (Bellis et al.,
2013a, 2013b, 2014a). GenBank also provides the utility, although
more limited than that of BOLD, to link morphological specimen
data to sequence data records (Federhen et al., 2009).
5.1. Progress in integrative taxonomy in Culicoides
Datasets of sequences for national checklists of Culicoides mor-
phospecies are starting to be published (Ander et al., 2013;
Matsumoto et al., 2009b; Slama et al., 2014), although often with
a limited number of specimens per morphospecies. A dramatic
increase in the number of specimens analysed per morphospecies
and the range of geographic areas from which these samples are
collected is required in order to investigate within-species haplo-
type diversity and reassess species deliminations using cladistic
analysis.
There is clearly a need for combining morphological and genetic
analyses in taxonomic studies of Culicoides with the assessment of
congruence between morphological and molecular phylogenetic
data, so called ‘integrative taxonomy’ (Will et al., 2005), providing
independent confirmation of species definitions. Studies integrat-
ing detailed morphological examinations with phylogenetic analy-
sis have already yielded important information regarding the
subgeneric classification of Culicoides and the identification of
new species (Bellis et al., 2014a; Bellis et al., 2013b).
The association of sex and life history stages is a prerequisite to
understanding the bionomics of a species, but as a result of the dif-
ficulties in sampling and defining diagnostic morphological charac-
teristics of the juvenile life-stages of Culicoides, integrative
taxonomic efforts have nearly entirely been driven by examination
of adult specimens e.g. Bellis et al. (2014a) and Pagès and Sarto i
Monteys (2005). To date, only a handful of studies have utilised
molecular techniques to aid identification of immature Culicoides
specimens (Schwenkenbecher et al., 2009b; Swanson, 2011;
Yanase et al., 2013). It is this area, however, that molecular tools
259
perhaps have the most to offer with regard to identifying conspe-
cific life-stages and sexes of specimens for which morphological
identification is either difficult or impossible (Bellis et al., 2013b).
The use of molecular data has also proven useful for the identifica-
tion of Culicoides specimens which have been damaged during col-
lection preventing conclusive morphological identification (Bellis
et al., 2013a).
5.2. Specimen selection for integrative taxonomic studies
Selection of specimens in integrative taxonomic studies is
almost always based initially on morphological examination. The
development of comprehensive identification aids based on mor-
phology should therefore be the first step in embarking on these
studies. After compiling a checklist of species likely to be present,
a key designed to assist with sorting unmounted specimens such
as those produced by Goffredo and Meiswinkel (2004), Rawlings
(1996) and Bellis et al. (2014b) will help workers to assign tenta-
tive names to the specimens they encounter. While taxonomic lit-
erature can still be challenging to obtain, the increasing availability
of electronic resources now enables rapid collating of documents
(Table 4). Alternative sampling strategies for the collection of spec-
imens may also be required in addition to collections made via
light trapping e.g. Capela et al. (2003), Goffredo and Meiswinkel
(2004) and Venail et al. (2012). Of paramount importance is to col-
lect specimens from as wide a geographic and ecological range as
possible to maximise the chances of sampling the full range of
genetic variability within a species. Simply increasing sampling
size will not necessarily increase the percentage of total haplotypes
discovered (Zhang et al., 2010), as haplotype diversity may vary
across a species’ geographic range and between ecosystems uti-
lised by the species (Frézal and Leblois, 2008; Sperling, 2003). This
was recently demonstrated when Bellis et al. (2014a) investigated
the genetic differences between populations of C. brevitarsis at the
extremities of this species’ distribution which proved to belong to
different species.
5.3. Vouchering of selected specimens
Definitive identification of species using morphology requires
that specimens be slide-mounted in permanent media (Boorman,
1988; Borkent et al., 2009). Although previously popular, pin
mounting of Culicoides is not suitable for specimen preservation
due to their fragile nature (Foote and Pratte, 1954). Standardised
methods for slide mounting dissected adult Culicoides are available
(Boorman, 1988; Boorman and Rowland, 1988; Clastrier, 1984;
Wirth and Marston, 1968) and preparation of immature specimens
for slide mounting are discussed by Kettle and Lawson (1952).
Culicoides specimens mounted whole are not suitable for detailed
study of morphological characteristics and should be avoided
wherever possible (Boorman, 1988). While gum/chloral media
may provide rapid mounts, methods such as that described by
Campbell and Pelham-Clinton (1960) should not be used for Culi-
coides voucher specimens as these mounts can darken with age
(Boorman, 1986; Boorman, 1988). Care must be taken not to intro-
duce artefactural variation into specimens as a result of the slide
mounting process (Arnqvist and Martensson, 1998; Dujardin
et al., 2010).
The recent development of a non-destructive DNA extraction
technique for use with Culicoides (Bellis et al., 2013b) has the
advantage of producing entire, cleared specimens ready for slide
mounting and has made it possible to develop a library of sequence
data with matched high-resolution digital images of slide-
mounted morphological voucher specimens. Importantly, it also
allows for the deposition of mounted holotype specimens along-
side corresponding DNA data (Bellis et al., 2014a). Previous studies
260 L.E. Harrup et al. / Infection, Genetics and Evolution 30 (2015) 249–266

Table 4
A compilation of online taxonomic tools and resources available for use by the Culicoides worker.

Name Description Available from

Armed Forces Pest Management An extensive online collection of scientific papers relevant to pathogen vectors http://www.afpmb.org/content/
Board Literature Retrieval including many older and out-of-print journal articles, reports and book sections welcome-literature-retrieval-
System system/
AVAbase (Arthropod Vector of Interest A multilingual online database of information of arthropod vectors of medical and http://avabase.cirad.fr/
for Animal Health Database) veterinary importance maintained by Cirad
Culicoides.net A resource maintained by The Pirbright Institute, aims to collate and update information http://www.culicoides.net
on Culicoides species recorded in northern and western Europe and other areas with
ongoing collaborations
DrawWing A free software package for the description and illustration of insect wings, the associate http://www.drawwing.org/
website also hosts a gallery of wing images for various genera
FLYTREE Official project webpage of the NSF Assembling the Tree of Life Project EF-0334948 http://wwx.inhs.illinois.edu/
Building the Dipteran Tree of Life: Cooperative Research in Phylogenetic & research/flytree/
Bioinformatics of True Flies (Insecta: Diptera), checklists of Ceratopogonid taxa and
Culicoides subgeneric affiliations
IIKC An interactive identification key for female Culicoides (Diptera: Ceratopogonidae) from http://www.iikculicoides.net/
the West Palaearctic region (Mathieu et al., 2012)
India Bluetongue Vector Network Official project website of the India Bluetongue Vector Network (IBVNet), hosts http://www.ibvnet.com
standardised protocols for the production of matched morphological voucher
specimens and COI Barcode data for Culicoides
The Ceratopogonidae Information A bi-annual newsletter and website hosted by Belmont University providing a directory http://campus.belmont.edu/
Exchange (CIE) of Ceratopogonid Workers, brief articles and links to recently published literature of cienews/cie.html/
relevance to the Ceratopogonidae fauna
The Dipterists Forum A website and forum affiliated to the British Entomological and Natural History Society http://
(BENHS) hosting a photo gallery and copies/links to literature of relevance to the www.dipteristsforum.org.uk/
Diptera fauna.
VectorBase Bioinformatics resource for invertebrate vectors of human pathogens https://www.vectorbase.org/

have required the use of destructive (Augot et al., 2010) or
semi-destructive (Stephan et al., 2009; Slama et al., 2014) DNA
extraction protocols with only partial specimens then available
for morphological examination. Where semi-destructive protocols
are followed there is a need to preserve the important morpholog-
ical features of voucher specimens; most of the important body
parts needed for identification are on the head, abdomen, wings
and legs and these should, where possible, be retained. Standard-
ised methodology for the collection of matched morphological
and COI Barcode data are now also available online (Harrup, 2014).
6. The current global status of Culicoides taxonomic knowledge
The biological sciences are facing a tremendous loss of classical
morphological taxonomic expertise (Wilson, 1985), the so called
‘taxonomic impediment’ (Tautz et al., 2003). Developments in
molecular species delimination and identification methods are,
however, progressing at such a pace that there is a danger that
the link to expertise in field-based bionomics investigations and
morphological taxonomy will be lost. Contributing to this is a lack
of support for traditional morphological studies by funding bodies
who tend to favour research using new technologies. It is as a
response to this that an ‘integrative’ future for Culicoides taxonomy
should be promoted, where molecular, ecological and morpholog-
ical investigations are closely linked e.g. Holbrook et al. (2000) and
Meiswinkel and Linton (2003).
The major challenges facing Culicoides taxonomists include the
small size of specimens, a poorly defined subgeneric classification,
the lack of descriptions for conspecific life-stages and sexes, the
lack of phylogenetic data, intra-specific variation in diagnostic
morphological characters, the identification and resolution of
potentially synonymous species and a lack of consensus on defin-
ing appropriate intraspecific genetic distances. Without a resolu-
tion to these problems we are unable to accurately delineate the
geographical range of many species and have limited information
on the regional occurrence and temporal abundance patterns of
the majority of species. Currently the effort expended on Culicoides
taxonomy globally is uneven with areas serviced by taxonomists or
which suffer veterinary/public health implications exhibiting high
species richness while other areas where high levels of biodiversity
would be expected, have relatively few Culicoides species recorded,
for example in the Neotropics. This is highly likely to be as a result
of sampling bias rather than a true measure of biodiversity. For
example the Culicoides fauna of Madagascar is probably largely
underestimated given its size and habitat diversity, having been
subject to only limited study (Augot et al., 2013b; De Meillon,
1961; Kremer et al., 1972).
The ability to accurately identify specimens from large-scale
surveillance projects would enable investigations into relation-
ships between species richness and climate, latitude, landcover,
topography, host availability and seasonality (Andrew et al.,
2013). International collaboration for knowledge exchange and
technology transfer, in addition to the utilisation of online open-
access systems for both morphological and molecular taxonomic
information, is essential if the regional variations in species delim-
itations and identification are to advance and coalesce (Ang et al.,
2013). This is particularly the case in response to the proliferation
of species which has occurred as a result of recent research in the
oriental region (Yu et al., 2005).
Specific taxonomic questions which still require addressing
include the species C. oxystoma and members of the Schultzei group
(Bakoum et al., 2013; Boorman, 1988), C. lupicaris and C. deltus
(Downes and Kettle, 1952; Glukhova and Leningrad, 1989;
Gomulski et al., 2006; Kettle and Lawson, 1952), and whether spe-
cies with wide geographic ranges are in fact conspecific. Examples
of the latter include C. montanus described both in Europe and Cen-
tral Asia (Gomulski et al., 2006; Shakirzjanova, 1962), C. obsoletus
recorded in the Palaearctic, Nearctic and Oriental regions (Arnaud,
1956; Boorman, 1988), C. circumscriptus in the Palaearctic and Orien-
tal regions (Arnaud, 1956; Kremer and Delécolle, 1974) and
C. punctatus (Meigen), 1804 which exhibits a >12% sequence diver-
gence between European and Japanese specimens (Matsumoto
et al., 2009a). Whether differences such as those evident in C. punct-
atus represent the existence of multiple species or a gradation in
genetic diversity across a geographical gradient remains to be
 L.E. Harrup et al. / Infection, Genetics and Evolution 30 (2015) 249–266
seen. In contrast, specimens of C. imicola from the two extremities of
its distribution i.e. the Republic of South Africa and eastern China,
have an identical COI haplotype (Bellis et al., 2014a).
7. Conclusions and prospects for the future
DNA characterisation provides a new means and impetus to
understand morphological and functional disparity within the
genus Culicoides. Previous studies have demonstrated
congruence between morphological and molecular analysis
(Gomulski et al., 2005; Pagès et al., 2009; Pagès and Sarto i
Monteys, 2005) indicating that when used in tandem these tech-
niques can help resolve species complexes and investigate pheno-
typic plasticity. Their use in attempts to unravel morphologically
similar species has begun, for example within the subgenus Avari-
tia (Bellis et al., 2014a; Mathieu et al., 2007; Pagès and Sarto i
Monteys, 2005).
This review has summarised the concepts and techniques that
underlie Culicoides taxonomy and concludes with the following
wish-list for methodologies and resources for integrative taxon-
omy within the genus: (i) the proliferation of validated multi-mar-
ker phylogenetic information with sound phylogenetic analysis of
the resulting data; (ii) the continued maintenance of a catalogue
of Culicoides species, their synonyms and subgeneric placement,
which accurately reflects the latest valid treatment in the literature
and provides detail on the history of subgeneric placements; (iii)
real-time multiplex PCR assays for isomorphic species identifica-
tions; (iv) cost-effective next generation sequence analysis pipe-
lines suitable for use with Culicoides data; (v) standardisation of
morphological terminology and characters used for species and
subgeneric descriptions; (vi) user-friendly, well-illustrated taxo-
nomic keys with an evolution away from the traditional dichoto-
mous keys towards interactive Bayesian keys; (vii) landmark-
based geometric morphometrics; (viii) testing of the current sub-
generic classification system using molecular and/or morphologi-
cal cladistic analyses; and (ix) the integration of novel taxonomic
techniques into large-scale surveillance schemes. Support and rec-
ognition by funding bodies and institutions of the importance of
having a stable and well resolved taxonomy for Culicoides and
other significant pathogen vectors is also utmost if research into
vector-borne diseases is to progress beyond the use of model spe-
cies (Agnarsson and Kuntner, 2007; Besansky et al., 2003; Godfray,
2007; Mallet and Willmott, 2003; Wilson, 2003).
A cladistic reinterpretation of the subgeneric classification of
Culicoides and species delimitations based on molecular phyloge-
netic analyses with phenotypic and bionomic data overlaying the
cladograms, should be the ultimate goal of Culicoides taxonomy.
While this may seem a distant target, the increasing use of molec-
ular techniques to investigate the evolutionary history of Culicoides
will allow some aspects of Culicoides species delimination and rela-
tionships to be resolved. Such resolution will allow for the investi-
gation of co-evolution of vector-host and vector-pathogen systems
to begin (Jaafar et al., 2014), an aspect of Culicoides biology that has
to date received little attention and for which a stable and well
resolved classification of the Culicoides genus is indispensable.
Acknowledgements
The authors would like to thank Eric Denison, Ian Roper and Craig
O’Brien for their contribution to the preparation and production of
the Culicoides wing images, and Simon Carpenter for his valuable
comments during the preparation of this manuscript. The work of
LEH is supported by funding from the Horserace Betting Levy Board
(HBLB) (Vet/PRJ/766). GAB acknowledges support received from Lyn
261
Cook and Alan Dyce. The authors are grateful to the anonymous ref-
erees whose comments greatly improved this manuscript.
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