Sex determination is a fundamental developmental pathway governing male and female differentiation, with profound implications for morphology, reproductive strategies, and behavior. In animals, sex differences between males and females are generally determined by genetic factors carried by sex chromosomes. Sex chromosomes are remarkably variable in origin and can differ even between closely related species, indicating that transitions occur frequently and independently in different groups of organisms. The evolutionary causes underlying sex chromosome turnover are poorly understood, however. Here we provide evidence indicating that Wolbachia bacterial endosymbionts triggered the evolution of new sex chromosomes in the common pillbug Armadillidium vulgare. We identified a 3-Mb insert of a feminizing Wolbachia genome that was recently transferred into the pillbug nuclear genome. The Wolbachia insert shows perfect linkage to the female sex, occurs in a male genetic background (i.e., lacking the ancestral W female sex chromosome), and is hemizygous. Our results support the conclusion that the Wolbachia insert is now acting as a female sex-determining region in pillbugs, and that the chromosome carrying the insert is a new W sex chromosome. Thus, bacteria-to-animal horizontal genome transfer represents a remarkable mechanism underpinning the birth of sex chromosomes. We conclude that sex ratio distorters, such as Wolbachia endosymbionts, can be powerful agents of evolutionary transitions in sex determination systems in animals.sex chromosome | horizontal transfer | Wolbachia | endosymbiont | isopod crustacean

Bracoviruses are domesticated viruses found in parasitic wasp genomes. They are composed of genes of nudiviral origin involved in particle production and proviral segments encoding virulence genes necessary for parasitism success. During particle production, proviral segments are amplified and individually packaged as DNA circles in nucleocapsids. These particles are injected by parasitic waspstogether with their eggs into host larvae. Bracovirus circles of two wasp species were reported toundergo chromosomal integration in parasitized host hemocytes, through a conserved sequence named Host Integration Motif (HIM). Here, we used bulk Illumina sequencing to survey integrations of Cotesia typhae bracovirus circles in the DNA of its host, the maize corn borer ( Sesamia nonagrioides ) seven days after parasitism. First, assembly and annotation of a high-quality genome for C. typhae enabled us to characterize 27 proviral segments clustered in proviral loci. Using these data, we characterized large numbers of chromosomal integrations (from 12 to 85 events per host haploid genome) for all 16 bracovirus circles containing a HIM. Integrations were found in four S. nonagrioides tissues and in the body of a caterpillar in which parasitism had failed. The 12 remaining circles do not integrate but are maintained at high levels in host tissues. Surprisingly, we found that HIM-mediated chromosomal integration has occurred at least six times accidentally in thewasp germline during evolution. Overall, our study furthers our understanding of wasp-host genome interactions and supports HIM-mediated chromosomal integration as a possible mechanism ofhorizontal transfer from wasps to their hosts. Importance Bracoviruses are endogenous domesticated viruses of parasitoid wasps that are injected together with wasp eggs into wasp host larvae during parasitism. Several studies have shown that some DNA circles packaged into bracovirus particles become integrated into host somatic genomes during parasitism, but the phenomenon has never been studied using non-targeted approaches. Here we use bulk Illumina sequencing to systematically characterize and quantify bracovirus circle integrations that occur in four tissues of the Mediterranean corn borer ( Sesamia nonagrioides ) during parasitism by the Cotesia typhae wasp. Our analysis reveals that all circles containing a host integration motif (HIM) integrate at substantial levels (from 12 to 85 integrations per host cell in total) in all tissues while other circles do not integrate. In addition to shedding new light on wasp-bracovirus-host interaction, our study supports HIM-mediated chromosomal integration of bracovirus as a possible source of wasp-to-host horizontal transfer with long term evolutionary consequences.