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

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 a difficult task in many cases, alternative approaches are expedient. With the aim to develop real-time PCRs, we determined partial mitochondrial cytochrome oxidase I gene (mt COI) sequences from 380 Culicoides midges representing three regions of Switzerland, namely the Alps, Midland north of the Alps (Atlantic climate), and South of the Alps (Mediterranean climate). The same region was also sequenced from non-biting midges of the genera Atrichopogon, Brachypogon, Dasyhelea, Forcipomyia and Serromyia. A total of 21 Culicoides species were identified by morphology. Sequence variability (haplotypes) was observed in all species. For each of C. grisescens and C. obsoletus, a novel cryptic species was identified. Whereas all individuals of C. grisescens and of the cryptic C. obsoletus species (O2) originated only from Alpine sites, the known C. obsoletus (O1) species was found in all three regions. Further, a sister taxon to C. pulicaris was identified based on the mt COI sequences and named Culicoides sp. Alignments of available mtCOI sequences from Ceratopogonidae (GenBank, this study) were used to design real-time PCR primers and probes to distinguish C. chiopterus, C. deltus, C. dewulfi, C. grisescens (including the cryptic species), C. imicola, C. lupicaris, C. obsoletus O1, C. obsoletus O2, C. pulicaris, C. scoticus and Culicoides sp. Specificities of primers and probes was tested with cloned targets representing 1 to 4 haplotypes of 18 Culicoides spp. and 1 haplotype each from 4 other Ceratopogonidae. No cross-reactivity was observed when plasmid template representing 5 × 10(6) gene copies was tested, but it was evident (Ct values ≤ 30) in few instances when plasmid template representing 5 × 10(9) gene copies was utilized, the latter corresponding to the total gene copy number (as determined in this study) in 20 insects. The sensitivities of two assays (C. imicola, C. grisescens) were tested by spiking single insects into pools of 99 or 999, randomly selected non-target Ceratopogonidae (with approx. 90% Culicoides specimens). In the pools of 100, Ct values were in the range of those obtained with single insects when employing 1% of the isolated DNA, whereas the sensitivity with the pools of 1000 was low, presumably due to the low DNA concentrations obtained with a protocol that seems inadequate for these larger pools. Thus, the assays as described are applicable for the specific identification of biting midges in small pools. Primers and probes of this study were devised to be suitable for multiplexed assays but these evaluations await to be performed.