Development of Conventional Multiplex PCR Assays for the Identification of 21 West Palaearctic Biting Midge Taxa (Diptera: Ceratopogonidae) Belonging to the Culicoides Subgenus Culicoides, including Recently Discovered Species and Genetic Variants

In 2006 and 2011, two biting-midge-borne arboviruses of high veterinary importance emerged for the first time in Central Europe: bluetongue virus (BTV) and Schmallenberg virus (SBV). Members of the native Obsoletus and Pulicaris Groups were soon identified as the potential vectors. However, despite several years of extensive taxonomic research on these groups, correct species identification and differentiation from closely related species are still challenging due to isomorphic features, the existence of cryptic species and obsolete PCR identification assays. At present, 17 valid West Palaearctic biting midge species of the Culicoides subgenus Culicoides, including the Pulicaris Group, are known, and additional genetic variants have been described. For many of them, no identification tests are available, and their roles in disease transmission have remained unknown. In this study, 465 GenBank DNA sequence entries of the mitochondrial cytochrome c oxidase subunit I (COI) gene were used to design PCR primers as specific genetic markers for 21 West Palaearctic biting midge taxa of the Culicoides subgenus Culicoides. During their validation with DNA from field-collected biting midges and synthetic DNA from biting midge genotypes not available from the field, all primers detected their target taxa, while few showed cross-reactions. Our results indicate the great potential of the new primers in PCR assays and clearly demonstrate the suitability of the COI gene as an excellent marker for the identification of different biting midge species and genetic variants of the Culicoides subgenus Culicoides.