Using capillary electrophoresis to identify Anopheline species in routine sampling sites

In the Anopheles genus, various mosquito species are able to transmit the Plasmodium parasites responsible for malaria, while others are non-vectors. In an effort to better understand the biology of Anopheles species and to quantify transmission risk in an area, the identification of mosquito species collected in the field is an essential but problematic task. Morphological identification requires expertise and cannot be checked after processing samples in a destructive treatment, while sequencing of numerous samples is costly. Here, we introduce a method of Species identification via Simple Observation Coupled with Capillary Electrophoresis Technology (SOCCET). This molecular technique of species identification is based on precise determination of ITS2 length combined with a simple visual observation, the colour of mosquito hindleg tip. DNA extracted from field-collected Anopheles mosquitoes was amplified with universal Anopheles ITS2 primers and analysed with a capillary electrophoresis device, which precisely determines the size of the fragments. We defined windows of amplicon sizes combined with fifth hind tarsus colour, which allows discrimination of the major Anopheles species found in our collections. We validated our parameters via Sanger sequencing of ITS2 amplicons. Using the SOCCET method, we characterised the composition of Anopheles populations in five locations of French Guiana, where we detected a total of nine species. Anopheles braziliensis and Anopheles darlingi were detected in four locations each and represented 13 and 67% of our samples, respectively. The SOCCET method can be particularly useful when working with routine sampling sites with a moderate species diversity, that is, when the number of local species is too high to define species-specific primers but low enough to avoid individual ITS2 sequencing. This tool will be of interest to evaluate local malaria transmission risk and this approach may be further implemented for other mosquito genera.