Nix alone is sufficient to convert female Aedes aegypti into fertile males and myo-sex is needed for male flight

Significance The presence of a dominant male-determining locus (M-locus) in one of a pair of autosomes establishes the male sex in the dengue fever mosquito Aedes aegypti. The Ae. aegypti M-locus contains 30 genes, including Nix, a previously reported male-determining factor. Here we show that the Nix transgene alone was sufficient to convert females into fertile males, which continued to produce sex-converted progeny. We also show that a second M-locus gene named myo-sex was needed for male flight. Nix-mediated sex conversion was 100% penetrant, heritable, and stable, indicating great potential for developing mosquito-control strategies to reduce vector populations by female-to-male conversion. This work also sheds lights into the molecular basis of the function of the M-locus. A dominant male-determining locus (M-locus) establishes the male sex (M/m) in the yellow fever mosquito, Aedes aegypti. Nix, a gene in the M-locus, was shown to be a male-determining factor (M factor) as somatic knockout of Nix led to feminized males (M/m) while transient expression of Nix resulted in partially masculinized females (m/m), with male reproductive organs but retained female antennae. It was not clear whether any of the other 29 genes in the 1.3-Mb M-locus are also needed for complete sex-conversion. Here, we report the generation of multiple transgenic lines that express Nix under the control of its own promoter. Genetic and molecular analyses of these lines provided insights unattainable from previous transient experiments. We show that the Nix transgene alone, in the absence of the M-locus, was sufficient to convert females into males with all male-specific sexually dimorphic features and male-like gene expression. The converted m/m males are flightless, unable to perform the nuptial flight required for mating. However, they were able to father sex-converted progeny when presented with cold-anesthetized wild-type females. We show that myo-sex, a myosin heavy-chain gene also in the M-locus, was required for male flight as knockout of myo-sex rendered wild-type males flightless. We also show that Nix-mediated female-to-male conversion was 100% penetrant and stable over many generations. Therefore, Nix has great potential for developing mosquito control strategies to reduce vector populations by female-to-male sex conversion, or to aid in a sterile insect technique that requires releasing only non-biting males.