title: Rapid diagnostic PCR assays for members of the Culicoides obsoletus and Culicoides pulicaris species complexes, implicated vectors of bluetongue virus in Europe.
abstract: Biting midges of the Culicoides obsoletus Meigen and Culicoides pulicaris L. species complexes (Diptera: Ceratopogonidae) are increasingly implicated as vectors of bluetongue virus in Palearctic regions. However, predicting epidemiological risk and the spread of disease is hampered because whilst vector competence of Culicoides is expressed only in adult females, morphological identification of constituent species is only readily applicable to adult males and some species distinguishing traits have overlapping character states. Furthermore, adult males are typically rare in field collections, making characterisation of Culicoides communities impossible. Here we highlight the utility of mitochondrial cytochrome oxidase subunit I (COI) DNA sequences for taxonomic resolution and species identification of all species within C. obsoletus and C. pulicarus complexes. Culicoides were collected from 18 sites in the UK and Continental Europe, and identified to species level, or species complex level, based on morphological characters. The sample comprised four species from the C. obsoletus complex (n = 88) and five species from the C. pulicaris complex (n = 39). The DNA sequence of the 5' end of the COI gene was obtained from all individuals. Each member species formed a well-supported reciprocally monophyletic clade in a maximum likelihood phylogeny. Levels of DNA sequence divergence were sufficiently high between species to allow the design of species-specific PCR primers that can be used in PCR for identification of members of the C. pulicaris complex or in a multiplex PCR to identify members of the C. obsoletus complex. This approach provides a valuable diagnostic tool for monitoring species composition in mixed field collections of Culicoides.
authors.affiliation: School of Biological Sciences, University of Aberdeen, Zoology Building, Tillydrone Avenue, Aberdeen AB24 2TZ, Scotland, UK.
authors.forename: Damien V
authors.initials: DV
authors.lastname: Nolan
authors.forename: Simon
authors.initials: S
authors.lastname: Carpenter
authors.forename: James
authors.initials: J
authors.lastname: Barber
authors.forename: Philip S
authors.initials: PS
authors.lastname: Mellor
authors.forename: John F
authors.initials: JF
authors.lastname: Dallas
authors.forename: A Jennifer
authors.initials: AJ
authors.lastname: Mordue Luntz
authors.forename: Stuart B
authors.initials: SB
authors.lastname: Piertney
mesh_terms: D000818:Animals; D001819:Bluetongue / Q000635:transmission*; D001820:Bluetongue virus; D002519:Ceratopogonidae / Q000145:classification* / Q000235:genetics / Q000821:virology; D003576:Electron Transport Complex IV / Q000235:genetics*; D005260:Female; D007303:Insect Vectors / Q000145:classification* / Q000235:genetics / Q000821:virology; D016013:Likelihood Functions; D008297:Male; D008928:Mitochondria / Q000201:enzymology; D008969:Molecular Sequence Data; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary; D016415:Sequence Alignment / Q000662:veterinary; D013045:Species Specificity
chemical_list: D003576:Electron Transport Complex IV