Prediction of species composition ratios from the pooled Anopheles Hyrcanus Group specimens

Vivax malaria is transmitted by members of the six Anopheles Hyrcanus Group (Anopheles sinensis, Anopheles pullus, Anopheles kleini, Anopheles belenrae, Anopheles lesteri, and Anopheles sineroides) in the Republic of Korea. Individual Anopheles species within the Hyrcanus Group demonstrate differences in their geographical distributions, vector competence and insecticide resistance, making it crucial for accurate species identification. Conventional individual species identification using species-specific markers requires extensive time and resources. Using species- or group-specific loci of the cytochrome C oxidase subunit I (COI) and the internal transcribed spacer 2 (ITS2) region, a rapid two-step quantitative sequencing (QS) based prediction protocol was devised. DNA fragments of COI and ITS2 genes were amplified from pooled gDNA specimens to predict species-specific nucleotide frequencies at each locus. Species composition ratios between An. sineroides, An. pullus and An. lesteri were estimated from QS of the COI amplicons based on the mC.260A, mC.122C and mC.525C alleles, followed by the prediction of species composition ratios between An. sinensis, An. kleini and An. belenrae based on QS of the ITS2 amplicons using the rI.370G and rI.389T alleles. A validation test proved that predicted species composition ratios from pooled DNA specimens and pooled mosquito specimens were not statistically different from the actual values. This prediction protocol will facilitate rapid estimation of the species composition ratios in field-collected Anopheles Hyrcanus Group populations and is particularly useful for studying the vector ecology of Anopheles population and epidemiology of malaria.