Cell-based assays and comparative genomics revealed the conserved and hidden effects ofWolbachiaon insect sex determination

Abstract

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.

Prophage-encodedHm-oscargene recapitulatesWolbachia-induced male killing in the tea tortrix mothHomona magnanima

“…These results strongly suggested that Hm-Oscar underlies the wHm-t-induced MK function in H. magnanima. The Wolbachia-encoded Oscar homologs identified so far are classified as type I and type II (Arai et al, 2024a). Although long ankyrin repeat sequences are postulated to be critical for the function of wFur-encoded Oscar (1830 aa, type I) (Katsuma et al, 2022a), our study revealed that the functions of Hm-Oscar (1181 aa, type II), which encodes fewer ankyrin repeats, were comparable with those of Oscar carried by wFur.…”

“…Although long ankyrin repeat sequences are postulated to be critical for the function of wFur-encoded Oscar (1830 aa, type I) (Katsuma et al, 2022a), our study revealed that the functions of Hm-Oscar (1181 aa, type II), which encodes fewer ankyrin repeats, were comparable with those of Oscar carried by wFur. Interestingly, type II Oscar is also present in the feminizing Wolbachia wFem in butterflies belonging to genus Eurema (Arai et al, 2024a). Oscar homologs, which inhibit the masculinizing function and induce female sex determination, may have a conserved function in Wolbachia-induced MK and feminization in Lepidoptera.…”

“…Although Oscar homologs have been identified in several MK-inducing Wolbachia strains in Lepidoptera, some MK Wolbachia strains in Diptera (Drosophila) and Lepidoptera do not carry this protein (Arai et al, 2023b;. Furthermore, Oscar homologs are evolutionarily dynamic, with highly variable sequences and structures (classified as Type I and Type II Oscar) (Arai et al, 2024a), making it difficult to assess their functional relevance. In addition to Oscar, the helix-turn-helix domain-containing putative transcriptional regulator Wmk (approximately 300 aa) is widely conserved among Wolbachia strains and induces various toxicities in Drosophila flies (i.e., no effect, weak male lethality [30% of males die], strong male lethality [90%-100% of males die], and death of all males and females) (Perlmutter et al, 2019(Perlmutter et al, , 2020(Perlmutter et al, , 2021Arai et al, 2023b).…”