A novel mosquito species identification method based on PCR and capillary electrophoresis

Abstract

In the Anopheles genus, various mosquito species are able to transmit 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 on the field is an essential but problematic task. Morphological identification requires expertise, well-preserved specimens and high-quality equipment, and it does not allow any subsequent verification when samples are later used in a destructive treatment. Moreover, it involves physical manipulations that are not compatible with experiments requiring fast sampling and processing of specimens, hence species identification is often based on DNA sequencing of reference genes or region such as the Internal Transcribed Spacer 2 (ITS2) region of nuclear ribosomal DNA. Sequencing ITS2 for numerous samples is costly, but the design of species-specific PCR primers is not always possible when local species diversity is high. Here, we introduce a molecular technique of species identification based on precise determination of ITS2 length combined with a simple visual observation, the color of mosquito hindleg tip. DNA extracted from field-collected Anopheles mosquitoes was amplified with universal Anopheles ITS2 primers and analyzed with a capillary electrophoresis device, which precisely determines the size of the fragments. We defined windows of amplicon sizes combined with fifth hind tarsus color, which allow to discriminate the major Anopheles species found in our collections. We validated our parameters via Sanger sequencing of the ITS2 amplicons. This method can be particularly useful in situations with a moderate species diversity, i.e. 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 further be implemented for other mosquito genera.

Using capillary electrophoresis to identify Anopheline species in routine sampling sites

“…Our data consolidate previous knowledge on Anopheles distribution in French Guiana, which may be helpful to prioritise sampling sites for future studies, whether focusing on a species of interest or on locations with high species diversity. Regarding variations in ITS2 sequences, most of our consensus sequences perfectly or almost perfectly matched previous French Guianese sequences of the same species (Chabanol et al., 2023; Vezenegho et al., 2014). Intraspecies variations were rare, mostly observed between An.…”

“…We checked that intervals were explained by imprecisions of capillary electrophoresis rather than by sequence variations within each species. Firstly, we built consensus sequences of forward and reverse sequences for 42 samples spanning all nine species (Chabanol et al., 2023). We saw no or negligible difference in size between samples taken from the beginning, middle and end of their species intervals.…”

“…triannulatus . The sequences are accessible in GenBank (NCBI, NIH) database with accession numbers from OR149213 to OR149254 (Chabanol et al., 2023). Consensus sequences were aligned against sequences retrieved from the NCBI Nucleotide database (listed in Table S1) using Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/; Data S2).…”

“…42 consensus sequences are accessible in the GenBank (NCBI, NIH) database with accession numbers from OR149213 to OR149254 (Chabanol et al., 2023). All forward and reverse sequences are provided in Data S1, and consensus sequence alignment can be found in Data S2 file.…”