DNA barcoding for molecular identification of Japanese mosquitoes

Abstract

A total of 512 mosquito specimens, collected in 2013 and 2014 from 18 study sites from Hokkaido to Kagoshima, Japan, were analyzed for DNA barcoding based on the nucleotide sequence of cytochrome c oxidase subunit I (COI) region, following which the gene sequences of 240 mosquitoes from 45 species in 11 genera were registered in GenBank. Aedes mosquitoes with black legs that belong to the punctor-subgroup, such as Ae. punctor and Ae. communis, found in northern Japan, were difficult to identify morphologically, but the specimens separated into two genetically distinct populations by COI gene sequence analysis. Morphological differences between the two populations were examined and the presence of postprocoxal scales was suggested as a key characteristic for identifying female specimens. The intraspecific divergence of nucleotide sequence was examined using Kimura two-parameter distance model for each species; 44 species showed an average sequence divergence at less than 2%, except for Anopheles lindesayi japonicus. Intraspecific variation in nucleotide sequence and neighbor-joining phylogenetic analyses indicated the existence of genetically heterogeneous populations in the following Japanese mosquito species: An. lindesayi japonicus, Culex rubensis, Cx. hayashii hayashii, Cx. kyotoensis, Ae. esoensis, and Toxorhynchites towadensis.