Morphological and molecular differentiation between Culicoides oxystoma and Culicoides kingi (Diptera: Ceratopogonidae) in Tunisia

Culicoides kingi and Culicoides oxystoma belong to the Schultzei group of biting midges. These two species are vectors of disease in livestock of economic importance. As described in the literature, morphological identification for discrimination between them is still unclear. However, species-specific identification is necessary to solve taxonomic challenges between species and to understand their roles in disease transmission and epidemiology. This study aims to develop accurate tools to discriminate C. oxystoma from C. kingi using traditional morphometry and polymerase chain reaction-restriction fragment length polymorphism (PCR RFLP) assays for use in developing countries. Specimens were collected from the region of Kairouan in central Tunisia. A total of 446 C. oxystoma/C. kingi individuals were identified using traditional morphometric analyses combined with PCR-RFLP of the cytochrome c oxidase subunit I gene. Thirteen morphometric measurements were performed from the head, wings, and abdomen of slide-mounted specimens, and six ratios were calculated between these measurements. Multivariate analyses of the morphometric measurements were explored to identify which variables could lead to accurate species identification. Four variables, namely antennae, wings, spermathecae, and palpus length, were suitable morphometric characteristics to differentiate between the species. Digestion with the SspI restriction enzyme of the PCR product led to good discriminative ability. Molecular procedures and phylogenetic analysis confirmed the efficiency of this simple and rapid PCR-RFLP method. This study highlights for the first time in Tunisia the presence of C. oxystoma and its discrimination from C. kingi using abdominal measurements and the PCR-RFLP method. This approach could be applied in future epidemiological studies at the national and international levels.