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

Abstract

A dominant male-determining locus (M-locus) establishes the male sex (M/m) in the yellow fever mosquito, <i>Aedes aegypti</i><i>Nix</i>, a gene in the M-locus, was shown to be a male-determining factor (M factor) as somatic knockout of <i>Nix</i> led to feminized males (M/m) while transient expression of <i>Nix</i> 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 <i>Nix</i> 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 <i>Nix</i> 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 <i>myo-sex</i>, a myosin heavy-chain gene also in the M-locus, was required for male flight as knockout of <i>myo-sex</i> rendered wild-type males flightless. We also show that <i>Nix</i>-mediated female-to-male conversion was 100% penetrant and stable over many generations. Therefore, <i>Nix</i> 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.