Large-Scale COI Gene Data Analysis for the Development of a Gel-Based Multiplex PCR Test Capable of Identifying Biting Midge Vector Species and Haplotypes (Diptera: Ceratopogonidae) of theCulicoidessubgenusAvaritiaFox, 1955

Abstract

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 programmes identified members of the Obsoletus Group (CulicoidessubgenusAvaritia) as keyplayers in disease epidemiology in Europe. However, morphological identification of potential vectors to species level is challenging due to the existence of isomorphic species. PCR tests developed to facilitate genetic species determination have been dismantled by the discovery of new genetic variants (haplotypes) ofC. obsoletussensu stricto (s.s.), forming distinct clades. In this study, 4,422 GenBank entries of the mitochondrial cytochrome c oxidase subunit I (COI) gene of subgenusAvaritiamembers of the genusCulicoideswere analyzed to develop a conventional multiplex PCR, capable of detecting all vector species and recently described clades of the western Palaearctic in this subgenus. Numerous GenBank entries incorrectly assigned to a species were identified, analyzed and reassigned. The results suggest that the three clades ofC. obsoletusrepresent independent species, whereasC. montanusshould rather be regarded as a genetic variant ofC. obsoletuss.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%). Hence, the newly developed multiplex PCR represents the first molecular tool which enables both the identification of accepted species and of the three clades ofC. obsoletuss.s. and could provide new insights into the ecology of the latter.