Cell-based assays and comparative genomics revealed the conserved and hidden effects of <i>Wolbachia</i> 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 <i>Wolbachia</i>, 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 <i>Wolbachia</i> across different hosts is often challenging. In this study, we demonstrated the effects of nine <i>Wolbachia</i> 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 <i>Wolbachia</i> strains and one feminization-inducing <i>Wolbachia</i> strain increased the female-type splicing products of the core sex-determining genes <i>doublesex</i>, <i>masculinizer</i>, and <i>zinc finger protein 2</i>. Regarding <i>Wolbachia</i> strains that do not induce MK/feminization, three had no effect on these sex-determining genes, whereas two strains induced female-type splicing of <i>masculinizer</i> and <i>doublesex</i> but not <i>zinc finger protein 2</i>. Comparative genomics confirmed that homologs of <i>oscar</i>, the <i>Wolbachia</i> gene responsible for MK in <i>Ostrinia</i>, were encoded by four MK/feminizing <i>Wolbachia</i> strains, but not by five non-MK/nonfeminizing strains. These results support the conserved effects underlying MK and feminization induced by <i>oscar</i>-bearing <i>Wolbachia</i> and suggested other potential mechanisms that <i>Wolbachia</i> might employ to manipulate host sex.