PCR identification of culicoid biting midges (Diptera, Ceratopogonidae) of the Obsoletus complex including putative vectors of bluetongue and Schmallenberg viruses

Background: Biting midges of the Obsoletus species complex of the ceratopogonid genus Culicoides were assumed to be the major vectors of bluetongue virus (BTV) in northern and central Europe during the 2006 outbreak of bluetongue disease (BT). Most recently, field specimens of the same group of species have also been shown to be infected with the newly emerged Schmallenberg virus (SBV) in Europe. A reliable identification of the cryptic species of this group is fundamental for both understanding the epidemiology of the diseases and for targeted vector control. In the absence of classical morphological characters unambiguously identifying the species, DNA sequence-based tests have been established for the distinction of selected species in some parts of Europe. Since specificity and sensitivity of these tests have been shown to be in need of improvement, an alternative PCR assay targeting the mitochondrial cytochrome oxidase subunit I (COI) gene was developed for the identification of the three Obsoletus complex species endemic to Germany (C. obsoletus, C. scoticus, C. chiopterus) plus the isomorphic species C. dewulfi. Methods: Biting midges of the genus Culicoides caught by UV light traps all over Germany were morphologically pre-identified to species or complex level. The COI region was amplified from their extracted DNA and sequenced. Final species assignment was done by sequence comparison to GenBank entries and to morphologically identified males. Species-specific consensus sequences were aligned and polymorphisms were utilized to design species-specific primers to PCR-identify specimens when combined with a universal primer. Results: The newly developed multiplex PCR assay was successfully tested on genetically defined Obsoletus complex material as well as on morphologically pre-identified field material. The intended major advantage of the assay as compared to other PCR approaches, namely the production of only one single characteristic band for each species, could be realized with high specificity and sensitivity. Conclusion: To elucidate the biological characteristics of potential vectors of disease agents, such as ecology, behaviour and vector competence, and the role of these haematophagous arthropods in the epidemiology of the diseases, simple, cost-effective and, most importantly, reliable identification techniques are necessary. The PCR assay presented will help to identify culicoid vector species and therefore add to bluetongue and Schmallenberg disease research including vector control and monitoring.