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 or feminization. In some lepidopteran insects, male killing is achieved by affecting the sex-determining process in males, and a unique mechanism of male killing 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 male killing-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 male killing/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 male killing in Ostrinia, were encoded by male killing/feminizing Wolbachia strains, but not by non-male killing/non-feminizing strains. These results demonstrated the conserved effects underlying male killing and feminization induced by oscar-bearing Wolbachia, and suggested other potential mechanisms that Wolbachia might employ to manipulate host sex. IMPORTANCEArthropods commonly carry maternally transmitted microbial symbionts such as Wolbachia. The lack of paternal transmission frequently led to the evolution of reproductive parasitism traits, namely the manipulation of host reproduction in favor of female hosts, substantiated by male killing and feminization. Although Wolbachia induces these phenotypes in a wide range of insects, the underlying mechanisms, diversity, and commonality remain largely unclear. In this study, we used a combination of transinfection assays and comparative genomics to reveal the conserved effects of male killing and feminizing Wolbachia strains on lepidopteran sex determination. Furthermore, we demonstrated that some non-male killing/non-feminizing Wolbachia strains also have an inherent ability to influence sex determination, albeit in a different manner, suggesting the potential for multiple mechanisms to manipulate host sex. This study also implied the frequent evolution of host suppressors against Wolbachia-induced reproductive manipulation.