Cell-based assays and comparative genomics revealed the conserved and hidden effects of Wolbachia on insect sex determination.

It is advantageous for maternally transmitted endosymbionts to skew the sex ratio of their hosts toward females. Some endosymbiotic bacteria, such as Wolbachia , cause their insect hosts to exclusively produce female offspring through male killing (MK) or feminization. In some lepidopteran insects, MK is achieved by affecting the sex-determining process in males, and a unique mechanism of MK and its functional link with feminization have been implicated. However, comparative analysis of these phenotypes is often difficult because they have been analyzed in different host-symbiont systems, and transinfection of Wolbachia across different hosts is often challenging. In this study, we demonstrated the effects of nine Wolbachia strains on the splicing of sex-determining genes in Lepidoptera by fixing the host genetic background using a cell culture system. Cell transinfection assays confirmed that three MK-inducing Wolbachia strains and one feminization-inducing Wolbachia strain increased the female-type splicing products of the core sex-determining genes doublesex , masculinizer , and zinc finger protein 2 . Regarding Wolbachia strains that do not induce MK/feminization, three had no effect on these sex-determining genes, whereas two strains induced female-type splicing of masculinizer and doublesex but not zinc finger protein 2 . Comparative genomics confirmed that homologs of oscar , the Wolbachia gene responsible for MK in Ostrinia , were encoded by four MK/feminizing Wolbachia strains, but not by five non-MK/nonfeminizing strains. These results support the conserved effects underlying MK and feminization induced by oscar -bearing Wolbachia and suggested other potential mechanisms that Wolbachia might employ to manipulate host sex.