Large-Scale Cytochrome C Oxidase Subunit I Gene Data Analysis for the Development of a Multiplex Polymerase Chain Reaction Test Capable of Identifying Biting Midge Vector Species and Haplotypes (Diptera: Ceratopogonidae) of the Subgenus Fox, 1955.

The emergence of culicoid-transmitted bluetongue and Schmallenberg viruses in several European countries demonstrated the ability of indigenous biting midge species to transmit pathogens. Entomologic research programs identified members of the Obsoletus Group ( subgenus ) as keyplayers in disease epidemiology in Europe. However, morphological identification of potential vectors is challenging due to the recent discovery of new genetic variants (haplotypes) of sensu stricto (s.s.), forming distinct clades. In this study, 4422 GenBank entries of the mitochondrial cytochrome c oxidase subunit I (COI) gene of subgenus members of the genus were analyzed to develop a conventional multiplex PCR, capable of detecting all vector species and clades of the Western Palearctic in this subgenus. Numerous GenBank entries incorrectly assigned to a species were identified, analyzed and reassigned. The results suggest that the three clades represent independent species, whereas should rather be regarded as a genetic variant of s.s. Based on these findings, specific primers were designed and validated with DNA material from field-caught biting midges which achieved very high diagnostic sensitivity (100%) when compared to an established reference PCR (82.6%).