Distribution of Culicoides biting midges (Diptera: Ceratopogonidae) in southern Australia and insight into the Culicoides victoriae morpho-variants.
Certain Culicoides species (biting midges) are important vectors globally of a range of viruses, protozoa and filarial parasites, imposing a significant economic and health burden. In a changing climate, understanding which Culicoides species occur in a region is important for biosecurity risk management. We examined the occurrence of predominant Culicoides species in south-east Australia and provide insight into five Culicoides victoriae morpho-variants. Culicoides were surveyed using Centre for Disease Control light traps and Yellow Sticky traps, with identification performed morphologically and molecularly. Two polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) assays based on mitochondrial cytochrome c oxidase subunit I were developed to differentiate morphologically similar C. victoriae, with a subset sequenced for nuclear carbomoylphosphate synthase. The structure of the five C. victoriae morpho-variants was investigated through phylogenetic trees, haplotype networks and the Barcode Index Number system (BINs) in the Barcode of Life Data (BOLD) database. Twenty-five different Culicoides species were identified in the sampled region with Culicoides austropalpalis, C. victoriae, C. marksi and C. molestus Gp sp No 2 being the most common. Two PCR-RFLP assays were developed using either one or three enzymes, with a 90% or 99% success rate, respectively, of being able to differentiate the five C. victoriae morpho-variants. Mitochondrial and nuclear sequence divergence supported by wing patterning allowed the recognition of multiple potentially new species of C. victoriae and identified species in uncharacterised regions.