On the origin and evolution of the mosquito male-determining factorNix

The mosquito family Culicidae is divided into two subfamilies named the Culicinae and Anophelinae.Nix, the dominant male-determining factor, has only been found in the culicinesAedes aegyptiandAe. albopictus, two important arboviral vectors that belong to the subgenus Stegomyia. Here we performed sex-specific whole-genome sequencing and RNAseq of divergent mosquito species and explored additional male-inclusive datasets to investigate the distribution ofNix. Except for the Culex genus,Nixhomologs were found in all species surveyed from the Culicinae subfamily, including 12 additional species from three highly divergent tribes comprising 4 genera, suggestingNixoriginated at least 133-165 MYA. Heterologous expression of one of three divergentNixORFs inAe. aegyptiresulted in partial masculinization of genetic females as evidenced by morphology anddoublesexsplicing. It is not clear whether insufficient transgene expression or sequence divergence or both are responsible for the lack of phenotype for the other two. Phylogenetic analysis suggestsNixis related tofemaleless(fle), a recently described intermediate sex-determining factor found exclusively in anopheline mosquitoes.Nixfrom all species has a conserved structure, including three RNA-recognition motifs (RRMs), as doesfle. However,Nixhas evolved at a much faster rate thanfle. The RRM3 of bothNixandfleare related to the single RRM of a widely distributed and conserved splicing factortransformer-2(tra2). RRM3-based phylogenetic analysis suggests this domain inNixandflemay have evolved fromtra2in a common ancestor of mosquitoes. Our results provide insights into the evolution of sex-determination and sex chromosomes in mosquitoes, and will inform broad applications of mosquito-control strategies based on manipulating sex ratios towards the non-biting males.