Molecular Identification of Species Belonging to Culex vishnui Subgroup (Diptera: Culicidae), Vectors of Japanese Encephalitis Virus, in Taiwan

Classification of mosquitoes with overlapping features remains problematic when using traditional morphological identification alone. In this study, we used molecular methods to elucidate the taxonomic status of Culex tritaeniorhynchus, Culex annulus, and Culex pseudovishnui species as vectors of the Japanese encephalitis virus belonging to the Culex vishnui subgroup and gene flow among them. In this study, 76, 59, and 3 samples of Cx. annulus, Cx. tritaeniorhynchus, and Cx. pseudovishnui, respectively, were collected around Taiwan. Phylogenetic analysis and genetic divergence were based on genomic sequence variations in ribosomal DNA and the internal transcribed spacer (rDNA) and cytochrome c oxidase subunit I (COI). Our results revealed that Cx. annulus and Cx. vishnui are genetically similar and share a gene pool among the species from Taiwan and other Asian countries. However, two hidden taxa of Cx. tritaeniorhynchus, which clustered together according to the rDNA sequences, were discovered based on the COI sequences. In addition, Cx. pseudovishnui has different gene pools from those of the strains from other countries, implying that the population from Taiwan is probably either a unique strain or a sibling species. This study provides molecular information on the taxonomic status of the species in the Cx. vishnui subgroup in Taiwan and gene flow between these species, providing valuable information for vector control operations and the delineation of the evolutionary process.