Browse Subject Areas

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

First Record of Culicoides oxystoma Kieffer and Diversity of Species within the Schultzei Group of Culicoides Latreille (Diptera: Ceratopogonidae) Biting Midges in Senegal

  • Mame T. Bakhoum , (MTB); (CG)

    Affiliation Institut Sénégalais de Recherches Agricoles, Laboratoire National de l’Elevage et de Recherches Vétérinaires, Dakar, Sénégal

  • Moussa Fall,

    Affiliation Institut Sénégalais de Recherches Agricoles, Laboratoire National de l’Elevage et de Recherches Vétérinaires, Dakar, Sénégal

  • Assane G. Fall,

    Affiliation Institut Sénégalais de Recherches Agricoles, Laboratoire National de l’Elevage et de Recherches Vétérinaires, Dakar, Sénégal

  • Glenn A. Bellis,

    Affiliation Department of Agriculture, Winnellie, Australia

  • Yuval Gottlieb,

    Affiliation Koret School of Veterinary Medicine, Robert H. Smith Faculty of Agriculture, Food, and Environment, The Hebrew University of Jerusalem, Rehovot, Israel

  • Karien Labuschagne,

    Affiliations Parasites, Vectors and Vector-borne Diseases, Agricultural Research Council-Onderstepoort Veterinary Institute, Onderstepoort, South Africa, Department of Zoology & Entomology, University of Pretoria, Hatfield, Pretoria, South Africa

  • Gert J. Venter,

    Affiliations Parasites, Vectors and Vector-borne Diseases, Agricultural Research Council-Onderstepoort Veterinary Institute, Onderstepoort, South Africa, Department of Veterinary Tropical Diseases, University of Pretoria, Onderstepoort, South Africa

  • Mariame Diop,

    Affiliation Institut Sénégalais de Recherches Agricoles, Laboratoire National de l’Elevage et de Recherches Vétérinaires, Dakar, Sénégal

  • Iba Mall,

    Affiliation Institut Sénégalais de Recherches Agricoles, Laboratoire National de l’Elevage et de Recherches Vétérinaires, Dakar, Sénégal

  • Momar T. Seck,

    Affiliation Institut Sénégalais de Recherches Agricoles, Laboratoire National de l’Elevage et de Recherches Vétérinaires, Dakar, Sénégal

  • Xavier Allène,

    Affiliation Cirad, UMR15 Contrôle des Maladies; INRA, UMR1309 Contrôle des Maladies, Montpellier, France

  • Maryam Diarra,

    Affiliation Institut Sénégalais de Recherches Agricoles, Laboratoire National de l’Elevage et de Recherches Vétérinaires, Dakar, Sénégal

  • Laëtitia Gardès,

    Affiliation Cirad, UMR15 Contrôle des Maladies; INRA, UMR1309 Contrôle des Maladies, Montpellier, France

  • Jérémy Bouyer,

    Affiliations Institut Sénégalais de Recherches Agricoles, Laboratoire National de l’Elevage et de Recherches Vétérinaires, Dakar, Sénégal, Cirad, UMR15 Contrôle des Maladies; INRA, UMR1309 Contrôle des Maladies, Montpellier, France

  • Jean-Claude Delécolle,

    Affiliation Institut de Parasitologie et de Pathologie Tropicale de Strasbourg, Faculté de Médecine, Strasbourg, France

  • Thomas Balenghien,

    Affiliation Cirad, UMR15 Contrôle des Maladies; INRA, UMR1309 Contrôle des Maladies, Montpellier, France

  •  [ ... ],
  • Claire Garros (MTB); (CG)

    Affiliation Cirad, UMR15 Contrôle des Maladies; INRA, UMR1309 Contrôle des Maladies, Montpellier, France

  • [ view all ]
  • [ view less ]

First Record of Culicoides oxystoma Kieffer and Diversity of Species within the Schultzei Group of Culicoides Latreille (Diptera: Ceratopogonidae) Biting Midges in Senegal

  • Mame T. Bakhoum, 
  • Moussa Fall, 
  • Assane G. Fall, 
  • Glenn A. Bellis, 
  • Yuval Gottlieb, 
  • Karien Labuschagne, 
  • Gert J. Venter, 
  • Mariame Diop, 
  • Iba Mall, 
  • Momar T. Seck


The Schultzei group of Culicoides Latreille (Diptera: Ceratopogonidae) is distributed throughout Africa to northern Asia and Australasia and includes several potential vector species of livestock pathogens. The taxonomy of the species belonging to this species group is confounded by the wide geographical distribution and morphological variation exhibited by many species. In this work, morphological and molecular approaches were combined to assess the taxonomic validity of the species and morphological variants of the Schultzei group found in Senegal by comparing their genetic diversity with that of specimens from other geographical regions. The species list for Senegal was updated with four species: Culicoides kingi, C. oxystoma, C. enderleini and C. nevilli being recorded. This is the first record of C. oxystoma from Africa south of Sahara, and its genetic relationship with samples from Israel, Japan and Australia is presented. This work provides a basis for ecological studies of the seasonal and spatial dynamics of species of this species group that will contribute to better understanding of the epidemiology of the viruses they transmit.


The recent introduction and expansion of bluetongue virus (BTV) [1], [2] and emergence of Schmallenberg virus [3] in Europe has emphasis the importance of accurate species identification and taxonomy of the genus Culicoides Latreille (Diptera: Ceratopogonidae) responsible for transmitting these viruses. Currently approximately 1,400 species placed into 29 formal subgenera and 39 informal species groups are recognized worldwide [4], [5]. In the Afrotropical region, some 156 species have been described but many more await description [6] and most of the literature on the taxonomy of Afrotropical species are in the need for revision [7][10].

The Afrotropical region is endemic for most of the known Culicoides-borne economical important diseases of livestock, like bluetongue virus and epizootic haemorrhagic disease virus (EHDV) [11]. While many local breeds of livestock appear to have achieved some level of tolerance to some of these diseases [12], others, for example African horse sickness virus (AHSV), still cause serious outbreaks in many parts of Africa [13] as recently illustrated with the epidemic recorded in Senegal in 2007 [14], [15].

In the Afrotropical region, Culicoides imicola Kieffer, subgenus Avaritia Fox, is regarded as the most important and proven orbivirus vector species of livestock diseases and this was recently reinforced when this species became an apparent invasive species throughout the Mediterranean basin [16], [17]. Other groupings of Culicoides which have been implicated in the transmission of some of these viruses is the Schultzei species group. Species belonging to this group have been associated with BTV [18], [19], AHSV [20] and EHDV [21], [22]. In especially the Australian and Oriental subregions, C. oxystoma Kieffer is a well-known vector of bovine arboviruses such as Akabane virus [23], [24] and is suspected of being vector of EHDV in Israel [25].

The status of the Schultzei group and its subgeneric affiliation has been disputed. Several authors placed this species group within C. subg. Remmia Glukhova [4], [6], [26] with regarding this group as a valid subgenus, while others maintain that C. subg. Remmia is a junior synonym of C. subg. Oecacta (Poey) [8], [27] or leave the group as unplaced to subgenus [28]. The status of the species within this group is equally contentious with widespread use of the name C. schultzei for members of the group [29]. The revision of Afrotropical species by Cornet & Brunhes [8] resolved most of the issues associated with the African fauna and Borkent [4] currently places eight species in C. subg. Remmia ( =  Schultzei group): C. schultzei (Enderlein), C. oxystoma, C. kingi Austen, C. rhizophorensis Khamala and Kettle, C. neoschultzei Boorman and Meiswinkel, C. subschultzei Cornet and Brunhes, C. enderleini Cornet and Brunhes and C. nevilli Cornet and Brunhes.

Culicoides oxystoma is known from the Oriental and Australasian regions [28], whereas all the other species within the Schultzei group are confined to the afrotropical region with some species extending north to the Mediterranean and Middle East [8], [18], [25], [28], [30][34]. The identity of the most widespread species, currently referred to as C. oxystoma, is unclear as many of the records listed by Wirth & Hubert [28] are based on misidentifications of C. schultzei and no taxonomic revisions of the Schultzei group have included this species. One barrier to the inclusion of C. oxystoma in a revision is that the original description by Kieffer is very brief [35] and the type specimen of C. oxystoma from Kolkata ( =  Calcutta) in India, has been lost [28].

Cornet & Brunhes [8] listed three species belonging to the Schultzei group from Senegal: C. enderleini, C. nevilli and two morphological forms of C. kingi termed a Kenya form and a Senegal form. They separated these latter two forms by the degree of posterior expansion of the basal pale spot basally in cell m1 of the wing with this spot crossing vein M2 in the Kenya form but not crossing the vein in the Senegal form (Figure 1). This morphological difference appeared to be associated with a difference in breeding habitat but whether these two forms constitute a single species or not remains unclear. Similarly, the morphological similarity between Oriental C. oxystoma and the Afrotropical C. subschultzei makes differentiation of these species problematic [36]. Culicoides schultzei ( = C. irroratus) is recorded in southern, eastern and central Africa [8], although it has not been recorded in western Africa [9].

Figure 1. Female wing pattern and male genitalia of the Schultzei species group collected in Senegal (samples from the study and Cornet’s collection) and South Africa.

The slide reference number refers to the Cornet collection reference numbers [8].

Using molecular analysis of the Cytochrome Oxidase I (COI) gene, Morag et al. [25] recently established that two distinct lineages of the Schultzei group are present in Israel. Not only does this suggest the presence of two species in Israel, one of which is synonymous with the species in Japan, it also suggests that analysis of the COI gene may be useful in addressing some of the taxonomic problems associated with this group, for example the status of the Kenya and Senegal forms of C. kingi and that of C. oxystoma and C. subschultzei. In this work, we combined morphological and molecular analyses to assess the diversity of the species of the Schultzei group present in Senegal in relation to that found in other regions. The specific status of morphological variants of C. kingi and C. oxystoma present in Senegal was established and an updated species list of Senegal for this group is provided.

Materials and Methods

Specimen Collection

Adult midges were collected using black light/suction traps placed near horses within stud farms at five sites in Senegal (Figure 2) between September and October 2011 (3 nightly trappings/site/month). The owners of stud farms gave permission to conduct the study on their sites. Field workers did not have any contact with the horses. Specimens were preserved in 70% ethanol and identified and sexed under a binocular microscope using the identification keys of Glick [7], Cornet & Brunhes [8] and Boorman [37]. For each species, specimens representing all available morphological variants were included in the analysis. Wings from females and male genitalia were dissected prior to processing and slide-mounted to record these morphological variations. Samples from La Reunion Island (C. enderleini), South Africa (C. enderleini, C. subschultzei, C. schultzei), and Australia (C. oxystoma) were used to represent the geographic variation of these species. COI sequences submitted by Morag et al [25], Matsumoto et al [38] and Augot et al [39] were added to the dataset (Table 1).

Figure 2. Location of the study sites in Senegal and abundance of species from the Schultzei group.

Table 1. Female samples used for the molecular analysis, localization and Genbank accession numbers for the COI sequences.

Extraction of Genomic DNA

Genomic DNA was individually extracted following an extraction protocol with Chelex resin in 5% (Resin Chelex100 ®, Chelating Ion Exchange Resin, Bio-Rad, France ) as described in Viennet et al [40] and Solano et al [41]. A volume of 500 µl of Chelex solution was dispensed into each tube of 1.5 ml. Each individual was removed from ethanol and dried on blotting paper. The individual was retrieved and transferred to a tube with Chelex solution and ground using a piston. The tubes were incubated at 56°C for 60 minutes and then at 95°C for 30 minutes (for thermal lysis). Immediately after heating, the tubes were centrifuged at 13,000 revs/min for 1 minute to pellet the Chelex resin with inhibitor ions and cellular debris.

Polymerase Chain Reaction Amplification and Sequencing of COI

Cytochrome Oxidase I (COI) amplification of the gene was carried out using primers C1J1718 (Forward) 5′- GGA- GGA-TTT-GGA-AAT-TGA-ATT-GT-3′ and C1N2191 (Reverse) 5′ -CAG-GTA-TTA-AAA-AAA-AAA-TAT-CTT-CTG-G-3′ to obtain an approximately 600 bp product as described previously [42]. Amplification reactions by Polymerase Chain Reaction (PCR) were performed in 25 µl of reaction volume with 5 µl of buffer 5X, 0.5 µl of dNTP (10 mM), 2 µl of MgCl2 (25 mM), 0.5 ml of each primer; 0.5 µl of Taq polymerase (5 U/µl), 15 µl of sterile water and 1 µl of DNA. Touch-up PCR amplification was used to reduce non-specific amplifications and optimize the quality of amplification for better sequencing. The cycling profile of the COI gene consisted of an initial denaturation stage of 1 minute at 94°C, followed by 5 cycles of 40 seconds at 94°C, 40 seconds at 45°C and 1 minute at 72°C, then 35 cycles of 40 seconds at 94°C, 40 seconds at 51°C, 1 minute at 72°C and a final elongation of 5 minutes at 72°C. The PCR products were visualized on 1% agarose gel with a Gel Red staining after migration of 90 minutes at 100 volts by electrophoresis, before being sent to Cogenics (Grenoble, France) for sequencing.

Sequence Alignment, Phylogenetic and Genetic Distance Analyses

Multiple alignments of the sequences were generated using the CLUSTALW algorithm in BioEdit [43]. Molecular evolutionary analyses were conducted using DAMBE [44] and MEGA version 5 [45]. The phylogenetic reconstructions were performed by Maximum Likelihood (ML) and Bayesian analyses (BA). The ML analyses were carried out with MEGA v5 [45], incorporating best fit models of sequence evolution determined using the Akaike Information Criterion (AIC) and employing 1,000 bootstrap replications to determine node reliabilities. The AIC implemented within jModelTest was used to determine the most suitable evolutionary model(s) for the Bayesian and ML analyses [46]. The AIC model selected for the COI was T92+G, followed by T92+G+I and HKY+G. The best fit model (T92+G) was used for both ML and BA analysis. Summary sequence statistics were generated using MEGA v5. Bayesian analyses was performed using MrBayes [47] with the following settings: the ML model employed two substitution types (“nst = 2”), with rate variation across sites modeled using a gamma distribution (rates = ”gamma”); Markov Chain Monte Carlo searches were done with four chains for 500,000 generations, with trees sampled every 100 generations (the first 1,000 trees were discarded as “burn in”). The appropriate burn-in fraction and convergence of the Markov Chain Monte Carlo chains were graphically assessed by evaluating the stationary phase of the chains using Tracer v1.5 [48]. Convergence metrics provided by MrBayes were checked to ensure that the maximum standard deviation of split frequencies of any of the runs was under 0.05 and that the potential scale reduction factor for all parameters approached 1.0. Culicoides imicola was used as an out-group. Estimates of average evolutionary divergence over sequence pairs within and between groups were made using the Maximum Composite Likelihood model for the COI sequences. The average genetic distances between the clades inferred by phylogenetic analyses were computed by Tamura-3 parameter or Jukes-Cantor model with the program Mega v5. The two models gave the same genetic distance matrix (data shown for JC model).


Morphological Identification

Morphological examination of 82,506 individuals from the 5 study sites revealed the presence of four species belonging to the Schultzei group (Figure 2). These species were referable to C. kingi (both the Kenya and Senegal forms sensu Cornet & Bruhnes), C. oxystoma (showing large phenotypic variation) (Figure 3), C. enderleini and C. nevilli. Although C. nevilli was present at three sites, the other species were abundant and equally present on the five sampled sites.

Figure 3. Variation in the wing patterns of C. subschultzei and C. oxystoma found in Senegal: Culicoides subschultzei typical form (A), Culicoides oxystoma typical form (B) and morphological variants: absence of pale spots in the second median cell (C) and presence of two pale spots in cubital cell (D).

Phylogenetic Analysis

A total of 63 COI sequences were obtained referable to six species including C. imicola used as out-group (Table 1). Twenty COI sequences were added to the dataset from the literature (Table 1). Sequencing of COI sequences of C. schultzei was not successful. Four individuals of each C. kingi form were obtained (Kenya form: KF682484–KF682485, KF682491–KF682492); Senegal form: KF682487, KF682489–KF682490, KF682494). The ML and BA trees depicted the same topology. Six distinct and well-supported phylogenetic lineages were clearly differentiated (Figure 4). One clade grouped the individuals identified as C. kingi, including both the Senegal and Kenya forms, and the three unidentified sequences from Israel (JN545050–51, JN545053). Another included all specimens of C. enderleini from all different location sites. Specimens of C. oxystoma formed two separate clades, one including all specimens from the Palaearctic and Australasian regions (Japan, Israel, and Australia) and the other including all individuals from Senegal. The mean genetic distance between these two clades was two times higher than the mean variation within a clade. The specimens from Senegal identified as C. oxystoma and representing all of the morphological variations illustrated in Figure 2 were clustered in the same clade. The maximum variation within a clade was 2.8% within the C. nevilli clade, and the minimum distance between clades being 1.5% between C. oxystoma from Australia and Japan (Table 2).

Figure 4. Phylogenetic analysis of species of the Schultzei group using COI sequence data.

The topology shows Bayesian inference tree using program MrBayes. Culicoides imicola was designated as an outgroup taxon. Numbers indicate bootstrap values from ML (bottom) analyses, and posterior probabilities from Bayesian analysis (top). Only bootstrap values >60% and Bayesian posterior probabilities >0.5 are shown. Branch lengths represent nucleotide substitutions per site.

Table 2. Estimates of evolutionary divergence of sequence pairs between and within populations and species.


At least four species belonging to the Schultzei group are now known from Senegal: C. enderleini, C. kingi, C. nevilli, and C. oxystoma. Our phylogenetic analyses have identified the three unidentified individuals (JN545050–51, JN545053) from Morag et al. (2012) as C. kingi which confirms the presence of this species in Israel. Our study confirmed that the Kenya and Senegal forms of this species, described by Cornet & Brunhes (1994) partially based on differing larval ecology, do not appear to be distinct species.

The relatively high genetic diversity observed within C. nevilli and C. enderleini probably reflects historically independent populations of these species on Madagascar and on mainland Africa as recently observed within Australian populations of C. immaculatus [49]. Overall, the Culicoides fauna of Madagascar has been understudied [39], [50] and remains to be investigated.

The genetic similarity between Oriental and Australian specimens of C. oxystoma and those from Senegal confirms the identity of this species and is the first record of C. oxystoma in Africa south of Sahara. There has been a great deal of confusion in the literature between this species, C. schultzei and C. subschultzei [8], [25], [37]. Boorman (1989) suggested that most of the records of C. schultzei or the Schultzei group from north of the Sahara and eastwards through India refer to C. oxystoma and its presence in Senegal indicates that it is also present south of the Sahara. Taken together, Boorman et al (1989) and our results showed that multiple species of the Schultzei group are sympatric in the Middle East (for instance at least C. kingi and C. oxystoma in Israel), and we recommend use of the most updated keys (Boorman 1989; Cornet and Brunhes 1994) when identifying material collecting from this area.

Culicoides oxystoma is a widespread species with a wide phenotypic variation that warrants further exploration. Specimens exhibiting the full range of morphological variation observed in Senegal (Figure 3) were shown to be conspecific but also showed strong support for two lineages within C. oxystoma indicating some level of reproductive isolation within this species. This might confirm what Boorman suspected as a differentiation within C. oxystoma, creating an Afrotropical lineage and a Palearctic and oriental lineage [36]. Despite the morphological similarity between C. oxystoma and C. subschultzei [36], COI analysis indicates that these two species are distinct with C. subschultzei being more closely related to C. nevilli than to C. oxystoma. Further investigations need to be implemented including C. schultzei. Unfortunately, attempts to amplify DNA from the C. schultzei samples from South Africa were unsuccessful, probably because they were too old (collected in 1996). Further works will also need to include other known members of the group, C. rhizophorensis and C. neoschultzei, and probably look at other species showing morphological similarity [51].

Up to date no phylogenetic study has ever successfully validated the subgeneric classification of the genus Culicoides [4], [5] and the validity of the Schultzei group, C. subg. Remmia, C. subg. Oecacta, and many other subgeneric groupings of Culicoides, remains doubtful. Augot et al. (2013) recently analysed the COI gene to test the monophyly of the Schultzei group and C. subg. Oecacta but their analysis were confounded by homoplasy of this gene and no conclusions could be drawn. They suggested the use of alternative genes to explore these relationships and it is likely that an integrated approach using a combination of morphological and molecular analyses might clarify this situation.

The wide distribution and economic importance of species of the Schultzei group highlight the need to re-evaluate the status of these species and a molecular approach might be an appropriate means of achieving that goal. Based on the numbers collected and wide distribution of the suspected vector species based on previous virus studies, these species can indeed play an important role in the epidemiology of AHSV in Senegal. In particular, future studies should focus on the differences between African and Oriental populations of C. oxystoma and establish the morphological and genetic limits of this species. Inclusion of material from the type locality of C. oxystoma in India would help to confirm the identity of specimens currently referred to this species.


The authors are particularly grateful to all the people who gave assistance in operating traps on several nights (technicians, animal keepers, shepherds) in Senegal. The authors would like to thank Amélie Desvars (Cirad, CRVOI, La Réunion) and Thierry Baldet (Cirad, Montpellier, France) for the Culicoides samples collected in La Réunion Island, France. The authors would like to thank also Hélène Guis (Cirad) for helping with the map.

Author Contributions

Conceived and designed the experiments: MTB MF AGF MTS JB CG. Performed the experiments: MTB MF MB IM XA MD LG. Analyzed the data: MTB MF GAB YG KL GJV JCD CG. Contributed reagents/materials/analysis tools: GAB KL GJV JCD. Wrote the paper: MTB MF AGF GAB YG KL GJV MTS JB JCD TB CG.


  1. 1. Purse BV, Mellor PS, Rogers DJ, Samuel AR, Mertens PPC, et al. (2005) Climate change and the recent emergence of bluetongue in Europe. Nat Rev Microbiol 3: 171–181. doi: 10.1038/nrmicro1090
  2. 2. Carpenter S, Wilson AJ, Mellor PS (2009) Culicoides and the emergence of bluetongue virus in northern Europe. Trends Microbiol 17: 172–178. doi: 10.1016/j.tim.2009.01.001
  3. 3. Hoffmann B, Scheuch M, Höper D, Jungblut R, Holsteg M, et al. (2011) Novel orthobunyavirus in cattle, Europe, 2011. Emerging Infect Dis 18: 469–472. doi: 10.3201/eid1803.111905
  4. 4. Borkent A (2012) Catalog of the Ceratopogonids. Available: Accessed 2013 Sept 1.
  5. 5. Borkent A (2012) The subgeneric classification of species of Culicoides - thoughts and a warning February 1.
  6. 6. Meiswinkel R, Venter GJ, Nevill EM (2004) Vectors: Culicoides spp; Coetzer JAW, Tustin RC, editors. Cape Town: Oxford University Press, Infectious diseases of livestock.
  7. 7. Glick JI (1990) Culicoides biting midges (Diptera : Ceratopogonidae) of Kenya. J Med Entomol 27: 85–195. doi: 10.1007/springerreference_85541
  8. 8. Cornet M, Brunhes J (1994) Révision des espèces de Culicoides apparentées à C. schultzei (Enderlein, 1908) dans la région afrotropicale (Diptera, Ceratopogonidae). Bull Soc Entomol Fr 99: 149–164.
  9. 9. Rawlings P, Snow WF, Boorman J, Denison E, Hamblin C, et al. (1998) Culicoides in relation to transmission of African Horse Sickness virus in The Gambia. Med Vet Entomol 12: 155–159. doi: 10.1046/j.1365-2915.1998.00094.x
  10. 10. Khamala CPM, Kettle DS (1971) The Culicoides Latreille (Diptera : Ceratopogonidae) of East Africa. Transaction of the Royal Entomological Society, London 123: 1–95. doi: 10.1111/j.1365-2311.1971.tb00840.x
  11. 11. Mellor PS, Baylis M, Mertens PPC (2009) Bluetongue; Pastoret PP, editor. London: Elsevier. 483 p.
  12. 12. Gerdes GH (2004) A South African overview of the virus, vectors, surveillance and unique features of bluetongue. Vet Ital 40: 39–42.
  13. 13. Mellor PS (1993) African horse sickness: transmission and epidemiology. Vet Res 24: 199–212.
  14. 14. Diouf ND, Etter E, Lo MM, Lo M, Akakpo AJ (2013) Outbreaks of African horse sickness in Senegal, and methods of control of the 2007 epidemic. Vet Rec 172: 152. doi: 10.1136/vr.101083
  15. 15. Akakpo AJ, Wombou Toukam CM, Mankor A, Ly C (2011) Economic impact of African horse sickness outbreak in Senegal in 2007. Bull Animal Health Prod Africa 59: 1–16. doi: 10.4314/bahpa.v59i1.68403
  16. 16. Venail R, Balenghien T, Guis H, Tran A, Setier-Rio ML, et al. (2012) Assessing diversity and abundance of vector populations at a national scale: example of Culicoides surveillance in France after bluetongue virus emergence. In: Mehlhorn H, editor. Arthropods as Vectors of Emerging Diseases: Springer Berlin Heidelberg. 77–102.
  17. 17. Mardulyn P, Goffredo M, Conte A, Hendrickx G, Meiswinkel R, et al. (2013) Climate change and the spread of vector-borne diseases: using approximate Bayesian computation to compare invasion scenarios for the bluetongue virus vector Culicoides imicola in Italy. Mol Ecol 22: 2456–2466. doi: 10.1111/mec.12264
  18. 18. Standfast HA, Dyce AL, Muller MJ (1985) Vectors of bluetongue virus in Australia. Prog Clinical Biol Res 178.
  19. 19. Venter GJ, Mellor PS, Wright I, Paweska JT (2007) Replication of live attenuated vaccine strains of bluetongue virus in orally infected South African Culicoides species Med Vet Entomol. 21: 239–247. doi: 10.1111/j.1365-2915.2007.00687.x
  20. 20. Venter GJ, Wright IM, van der Linde TC, Paweska JT (2009) The oral susceptibility of South African field populations of Culicoides to African horse sickness virus. Med Vet Entomol 23: 367–378. doi: 10.1111/j.1365-2915.2009.00829.x
  21. 21. Barnard BJ, Gerbes GH, Meiswinkel R (1998) Some epidemiological and economic aspects of a bluetongue-like disease in cattle in South Africa–1995/96 and 1997. Onderstepoort J Vet Res 65.
  22. 22. Mellor PS, Osborne R, Jennings DM (1984) Isolation of bluetongue and related viruses from Culicoides spp. in the Sudan. J Hyg 93.
  23. 23. Oem JK, Chung JY, Kwon MS, Kim TK, Lee TU, et al. (2013) Abundance of biting midge species (Diptera : Ceratopogonidae, Culicoides spp.) on cattle farms in Korea. J Vet Science 14: 91–94. doi: 10.4142/jvs.2013.14.1.91
  24. 24. Yanase T, Kato T, Kubo T, Yoshida K, Ohashi S, et al. (2005) Isolation of bovine arboviruses from Culicoides biting midges (Diptera : Ceratopogonidae) in southern Japan : 1985–2002. J Med Entomol 42: 63–67. doi: 10.1603/0022-2585(2005)042[0063:iobafc];2
  25. 25. Morag N, Saroya Y, Bravermann Y, Klement E, Gottlieb Y (2012) Molecular identification, phylogenetic status and geographic distribution of Culicoides oxystoma (Diptera : Ceratopogonidae) in Israel. PLoS ONE 7: e33610. doi: 10.1371/journal.pone.0033610
  26. 26. Dyce AL, Bellis GA, Muller MJ (2007) Pictorial atlas of Australasian Culicoides wings (Diptera: Ceratopogonidae); Australian Biological Resources Study, editor. Canberra. 88 p.
  27. 27. Yu YX, Liu JH, Liu GP, Liu ZJ, Hao BS, et al. (2005) Ceratopogonidae of China, Insecta, Diptera; Military Medical Science Press, editor. Beijing. 1699 p.
  28. 28. Wirth WW, Hubert AA (1989) The Culicoides of southeast Asia (Diptera : Ceratopogonidae). Mem Am Entomol Inst 44: 1–509.
  29. 29. Wirth WW, Dyce AL (1985) The current taxonomic status of the Culicoides vectors of bluetongue viruses. Prog Clinical Biol Res 178: 151–164.
  30. 30. Hilali M, Abu-Elzein ET, Al-Afaleq A, Mellor PS, Boorman J, et al. (2003) Culicoides midges in some localities of Saudi Arabia and their veterinary significance. Veterinarski Arhiv 73: 285–294.
  31. 31. Dik B, Yagci S, Linton YM (2006) A review of species diversity and distribution of Culicoides Latreille, 1809 (Diptera: Ceratopogonidae) in Turkey. J Nat Hist 40: 1947–1967. doi: 10.1080/00222930601046634
  32. 32. Alahmed AM, Kheir SM, Al Khereijii MA (2010) Distribution of Culicoides Latreille (Diptera : Ceratopogonidae) in Saudi Arabia. J Entomol 7: 221–234. doi: 10.3923/je.2010.227.234
  33. 33. Tsutsui T, Hayama Y, Yamakawa M, Shirafuji H, Yanase T (2011) Flight behavior of adult Culicoides oxystoma and Culicoides maculatus under different temperatures in the laboratory. Parasitol Res 108: 1575–1578. doi: 10.1007/s00436-010-2048-y
  34. 34. Labuschagne K, Gerber LJ, Espie I, Carpenter S (2007) Culicoides biting midges at the National Zoological Gardens of South Africa. Onderstepoort J Vet Res 74: 343–347. doi: 10.4102/ojvr.v74i4.120
  35. 35. Sen P, Das Gupta SK (1959) Studies on indian Culicoides (Ceratopogonidae : Diptera). Ann Entomol Soc America 52: 617–630.
  36. 36. The Pirbright Institute (2010) Available: Accessed 2013 July 1.
  37. 37. Boorman J (1989) Culicoides (Diptera : Ceratopogonidae) of the Arabian peninsula with notes on their medical and veterinary importance. Fauna Saudi Arabia 10: 160–224.
  38. 38. Matsumoto Y, Yanase T, Tsuda T, Noda H (2009) Characterization of internal transcribed spacer (ITS1–ITS2) region of ribosomal RNA gene from 25 species of Culicoides biting midges (Diptera : Ceratopogonidae) in Japan. J Med Entomol 46: 1099–1108. doi: 10.1603/033.046.0517
  39. 39. Augot D, Randrianambinintsoa FJ, Gasser A, Depaquit J (2013) Record of two species of Culicoides (Diptera : Ceratopogonidae) new for Madagascar and molecular study showing the paraphylies of the subgenus Oecacta and the Schultzei group. Bull Soc Pathol Exotique.
  40. 40. Viennet E, Garros C, Lancelot R, Allene X, Gardès L, et al. (2011) Assessment of vector/host contact : comparison of animal-baited traps and UV-light/suction trap for collecting Culicoides biting midges (Diptera : Ceratopogonidae), vectors of orbiviruses. Parasites and Vectors 4.
  41. 41. Solano P, Duvallet G, Dumas V, Cuisance D, Cuny G (1997) Microsatellite markers for genetic population studies in Glossina palpalis (Diptera : Glossinidae). Acta Trop 65: 175–180. doi: 10.1016/s0001-706x(97)00663-3
  42. 42. Simon C, Frati A, Beckenbach B, Crespi H, Lui H, et al. (1994) Evolution, weighting and phylogenetic unity of mitochondrial gene sequence sand a compilation of conserved polymerase chain reaction primers. Ann Entomol Soc America 87: 651–701.
  43. 43. Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series 41: 95–98.
  44. 44. Xia X, Xie Z (2001) DAMBE: data analysis in molecular biology and evolution. J Heredity 92: 371–373. doi: 10.1093/jhered/92.4.371
  45. 45. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, et al. (2011) MEGA5: Molecular Evolutionary Genetics Analysis using Maximum Likelihood, Evolutionary Distance and Maximum Parsimony Methods. Mol Biol Evol 28: 2731–2739. doi: 10.1093/molbev/msr121
  46. 46. Darriba D, Taboada GL, Doallo R, Posada D (2012) jModelTest 2 : more models, new heuritics and parallel computing. Nat Methods 9: 772. doi: 10.1038/nmeth.2109
  47. 47. Ronquist F, Teslenko M, van der Mark P, Ayres D, Darling A, et al. (2012) MrBayes 3.2: Efficient Bayesian phylogenetic inference and model choice across a large model space. Syst Biol 61: 539–542.
  48. 48. Rambaut A, Drummond AJ (2007) Tracer v1.4 Available: 2013 Jan 1.
  49. 49. Bellis GA, Dyce AL, Gopurenko D, Mitchell A (2013) Revision of the Immaculatus group of Culicoides Latreille (Diptera: Ceratopogonidae) from the Australasian biogeographic region with description of two new species. Zootaxa 3680: 15–37. doi: 10.11646/zootaxa.3680.1.4
  50. 50. De Meillon B (1961) The Madagascan Ceratopogonidae. Rev Entomol Mocambaise 4: 34–64.
  51. 51. The Pirbright Institute (2010) Available: 2013 Accessed 2013 Sept 1.