Evolution of Wolbachia male-killing mechanism within a host species

Male-killing bacterial symbionts, prevalent in arthropods, skew population sex ratios by selectively killing male progeny, profoundly impacting ecology and the evolution of their hosts. Male killing is a convergently evolved trait, with microbes evolving diverse male-killing mechanisms across host species with widely divergent sex determination pathways. A common evolutionary response to male-killing presence is the spread of suppressor mutations that restore male survival. In this study, we demonstrate the evolution of a novel male-killing mechanism that is insensitive to an existing male-killing suppressor. Hypolimnas bolina butterflies from Yogyakarta, Indonesia, showed extreme female-biased population sex ratio associated with high prevalence of a male-killing Wolbachia. This strain, wBol1Y, shared a very recent common ancestor with the previously characterized "suppressed" male-killing strain in the species, wBol1, but it retained its male-killing ability in the presence of the male-killing suppressor. The genome of wBol1Y differed from the suppressed wBol1 in carrying an additional prophage that included strong candidate genes for male killing. In vitro and in vivo data demonstrated that wBol1Y feminized splicing and expression of lepidopteran sex determination pathway genes and that the gene Hb-oscar-present on wBol1Y's unique prophage insert-was sufficient to disrupt the male sex determination pathway. Our study demonstrates that the diversity of male-killing mechanisms is a product both of interaction with varying insect sex determination systems and the evolution of male killing within a host species. Our data indicate that the male killer and host may be involved in escalating arms races, where spreading male-killing suppression drives the evolution of additional systems that reestablish male killing by the symbiont.