Genetic Diversity and Phylogenetic Relationships of Mosquitoes (Diptera: Culicidae) in Central Nepal

Mosquitoes (family: Culicidae) include several species that act as vectors of major human diseases by transmitting pathogens such as Plasmodium spp. (malaria), dengue virus (dengue), filarial worms (filariasis), and various other arboviruses. Species from the subfamilies Anophelinae and Culicinae are key transmitters of pathogens. This research aimed to assess genetic diversity and phylogenetic relationships among different species of mosquitoes collected from five districts of central Nepal. The mitochondrial cytochrome c oxidase I (COI) and Internal Transcribed Spacer 2 (ITS2) genes were employed for genetic diversity. A total of 7223 mosquitoes were gathered, encompassing 18 species from eight genera across an altitudinal range from 62 to 3840 m above sea level. From the collected mosquitoes, 114 high-quality sequences were obtained. Ae. aegypti exhibited lower diversity, with a haplotype diversity (Hd) of 0.46 ± 0.20 and a nucleotide diversity (π) of 0.006. The significantly negative Tajima's D value (-1.76, p < 0.05) suggests the possibility of a recent population expansion or the effect of purifying selection on this species. Phylogenetic analysis revealed distinct clades for most genera, while Aedes exhibited paraphyly. Intraspecific genetic divergence (K2P distances) was below 2.39%, while interspecific divergence exceeded 8%. The highest interspecific divergence was observed between Tx. splendens and An. subpictus (25.28%), and the lowest between Cx. pipiens and Cx. quinquefasciatus (0.43%). Haplotype network analysis revealed that Ae. aegypti exhibited a star-like pattern with a widely shared central haplotype and low-frequency variants, whereas Ae. albopictus showed a more structured network with two main clusters. These findings provide valuable information on the genetic diversity and evolutionary relationships among mosquito species in Nepal.