The AalNix3&4 isoform is required and sufficient to convert Aedes albopictus females into males

Abstract

Aedes albopictus is one of the most invasive insect species in the world and an effective vector for many important arboviruses. We reported previously that Ae. albopictus Nix (AalNix) is the male-determining factor of this species. However, whether AalNix alone is sufficient to initiate male development is unknown. Transgenic lines that express each of the three AalNix isoforms from the native promoter were obtained using piggyBac transformation. We verified the stable expression of AalNix isoforms in the transgenic lines and confirm that one isoform, AalNix3&4, is sufficient to convert females into fertile males (pseudo-males) that are indistinguishable from wild-type males. We also established a stable sex-converted female mosquito strain, AalNix3&4-♂4-pseudo-male. The pseudo-male mosquitoes can fly and mate normally with wild-type female, although their mating competitiveness is lower than wild-type. This work further clarifies the role of AalNix in the sex determination pathway and will facilitate the development of Ae. albopictus control strategies that rely on male-only releases such as SIT and sex-ratio distortion. Nix serves as a conserved male-determining factor in the two most important mosquito arboviral vectors, Ae. aegypti and Ae. albopictus. AaeNix alone can convert Ae. aegypti females into fertile but flightless males. AalNix has four alternative splice isoforms whereas AaeNix has one. Little was known previously about which AalNix isoform(s) serve as the primary signal for sex determination. We cloned the promoter region of AalNix gene and constructed piggybac-based AalNix overexpression constructs with different isoform variants. Following transformation and recovery of transgenic lines, we observed that expression of the AalNix3&4 isoform could shift the alternative splicing of the sex determination genes, doublesex and fruitless, from female to male isoforms, and phenotypically masculinize females or completely convert females into males. Importantly, the sex-converted pseudo-males are fertile and capable of flight. Thus, AalNix is the primary signal for male sex determination in Aedes albopictus and provides a basis for sex segregation and further Cas9-mediated gene-drive population suppression.