Molecular characterisation of Swiss Ceratopogonidae (Diptera) and evaluation of real-time PCR assays for the identification of Culicoides biting midges

Abstract

Biting midges of the genus Culicoides (Diptera, Ceratopogonidae) are vectors of several viruses of veterinary relevance, and they can cause insect bite hypersensitivity. As the morphological identification of these tiny insects is difficult, we aimed at developing real-time PCRs. Partial mitochondrial cytochrome oxidase I gene (mtCOI) sequences were determined from 380 Culicoides midges from three regions of Switzerland (Alps, Midland, Ticino) and from non-biting midges. Sequence variability (haplotypes) was observed in all 21 morphologically identified species. For each of C. grisescens and C. obsoletus, a cryptic species was identified. Further, a sister taxon to C. pulicaris was determined (Culicoides sp.). Alignments of available mtCOI sequences from Ceratopogonidae (GenBank, this study) were used to design real-time PCRs to distinguish C. chiopterus, C. deltus, C. dewulfi, C. grisescens, C. imicola, C. lupicaris, C. obsoletus (both species), C. pulicaris, C. scoticus and Culicoides sp. Specificities of the assays were tested with cloned targets from 1 to 4 haplotypes of 18 Culicoides spp. and 4 other Ceratopogonidae revealing no cross-reactivity when testing 5x106 gene copies. The sensitivities of two assays were tested by spiking single insects into pools of 99 or 999 randomly selected non-target Ceratopogonidae. Primers and probes of this study were devised to be suitable for multiplexed assays but these evaluations await to be performed.