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 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 MK presence is the spread of suppressor mutations that restore male survival. In this study, we demonstrate 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 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 wBol1Ys unique prophage insert - was sufficient to disrupt the male sex determination pathway. Our study demonstrates the diversity of male-killing mechanisms is a product both of interaction with varying insect sex determination systems and evolution of male-killing within a host species. Our data indicate male-killer and host may be involved in escalating arms races, where spread male-killing suppression drives evolution of additional systems that reestablish male killing by the symbiont.