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