Discrimination of Culicoides Midge Larvae Using Multiplex Polymerase Chain Reaction Assays Based on DNA Sequence Variation at the Mitochondrial Cytochrome C Oxidase I Gene

The recent spread of Bluetongue disease in northwestern Europe has indicated the ability of Palaearctic Culicoides species to vector the disease. Because the different midge species vary in their ability to harbor and transmit the Bluetongue virus, quick and reliable identification is necessary to resolve the species composition of midge communities, both adult and larval, at any place at any given time point. Given that morphological identification of Culicoides species is problematic, we developed three multiplex polymerase chain reaction (PCR) assays that facilitate high-throughput analysis of midge specimens. One assay distinguishes between species of the so-called Culicoides obsoletus s.l. complex (including C. dewulfi), whereas two assays facilitate differentiation of species of the Culicoides pulicaris s.l. complex. These assays yield two PCR products: one species-specific and one generic band. We show the application of the assays in the analysis of Culicoides larvae from three different farms in northeast Scotland.