The genus Wolbachia is an archetype of maternally inherited intracellular bacteria that infect the germline of numerous invertebrate species worldwide. They can selfishly alter arthropod sex ratios and reproductive strategies to increase the proportion of the infected matriline in the population. The most common reproductive manipulation is cytoplasmic incompatibility (CI), which results in embryonic lethality in crosses between infected males and uninfected females. Females infected with the same Wolbachia strain rescue this lethality. Despite more than 40 years of research1 and relevance to symbiont-induced speciation2,3, as well as control of arbovirus vectors4,5,6 and agricultural pests7, the bacterial genes underlying CI remain unknown. Here, we use comparative and transgenic approaches to demonstrate that two differentially transcribed, codiverging genes in the eukaryotic association module of prophage WO8 from Wolbachia strain wMel recapitulate and enhance CI. Dual expression in transgenic, uninfected males of Drosophila melanogaster crossed to uninfected females causes embryonic lethality. Each gene additively augments embryonic lethality in infected males crossed to uninfected females. Lethality associates with embryonic defects that parallel those of wild type CI and is notably rescued by wMel-infected embryos in all cases. The discovery of cytoplasmic incompatibility factor genes cifA and cifB pioneers genetic studies of prophage WO-induced reproductive manipulations and informs Wolbachia’s ongoing utility to control dengue and Zika transmission to humans.

SignificanceThe World Health Organization recommended pilot deployment of Wolbachia-infected mosquitoes to curb viral transmission to humans. Releases of mosquitoes are underway worldwide because Wolbachia can block replication of these pathogenic viruses and deterministically spread by a drive system termed cytoplasmic incompatibility (CI). Despite extensive research, the underlying genetic basis of CI remains only half-solved. We recently reported that two prophage WO genes recapitulate the modification component of CI in a released strain for vector control. Here we show that one of these genes underpins rescue of CI. Together, our results reveal the complete genetic basis of this selfish trait and pave the way for future studies exploring WO prophage genes as adjuncts or alternatives to current control efforts.

Unidirectional cytoplasmic incompatibility (CI) results in a postfertilization incompatibility between Wolbachia-infected males and uninfected females. CI contributes to reproductive isolation between closely related species and is used in worldwide vector control programs to drastically lower arboviral vector population sizes or to replace populations that transmit arboviruses with those resistant to transmission. Despite decades of research on the factors that influence CI, penetrance is often variable under controlled laboratory conditions in various arthropods, suggesting that additional variables influence CI strength. Here, we demonstrate that paternal D. melanogaster grandmother age influences the strength of CI induced by their sons. Older D. melanogaster females have higher Wolbachia densities and produce offspring with higher Wolbachia densities that associate with stronger CI. This work reveals a multigenerational impact of age on CI and expands our understanding of host-Wolbachia interactions and the biology of CI induced by the Wolbachia strain infecting the most widely used arthropod model, D. melanogaster.

Wolbachia are maternally-inherited, intracellular bacteria at the forefront of vector control 22 efforts to curb arbovirus transmission. In international field trials, the cytoplasmic incompatibility (CI) 23 drive system of wMel Wolbachia is deployed to replace target vector populations, whereby a Wolbachia-24 induced modification of the sperm genome kills embryos. However, Wolbachia in the embryo rescue the 25 sperm genome impairment, and therefore CI results in a strong fitness advantage for infected females 26 that transmit the bacteria to offspring. The two genes responsible for the wMel-induced sperm 27 modification of CI, cifA and cifB, were recently identified in the eukaryotic association module of prophage 28 WO, but the genetic basis of rescue is unresolved. Here we use transgenic and cytological approaches to 29 demonstrate that cifA independently rescues CI and nullifies embryonic death caused by wMel Wolbachia 30 in Drosophila melanogaster. Discovery of cifA as the rescue gene and previously one of two CI induction 31 genes establishes a new 'Two-by-One' model that underpins the genetic basis of CI. Results highlight the 32 central role of prophage WO in shaping Wolbachia phenotypes that are significant to arthropod evolution 33 and vector control. 34 35Significance Statement: The World Health Organization recommended pilot deployment of Wolbachia-36 infected mosquitoes to curb viral transmission to humans. Releases of mosquitoes are underway 37 worldwide because Wolbachia can block replication of these pathogenic viruses and deterministically 38 spread by a drive system termed cytoplasmic incompatibility (CI). Despite extensive research, the 39 underlying genetic basis of CI remains only half-solved. We recently reported that two prophage WO 40 genes recapitulate the modification component of CI in a released strain for vector control. Here we show 41 that one of these genes underpins rescue of CI. Together, our results reveal the complete genetic basis of 42 this selfish trait and pave the way for future studies exploring WO prophage genes as adjuncts or 43 alternatives to current control efforts. 44 \body 45 46Introduction: 47Wolbachia are an archetype of maternally-inherited, intracellular bacteria. They occur in an 48 estimated 40-52% of arthropod species (1, 2) and 47% of the Onchocercidae family of filarial nematodes 49(3), making them the most widespread bacterial symbiont in the animal kingdom (2). In arthropods, 50Wolbachia mainly reside in the cells of the reproductive tissues, transmit transovarially (4), and often 51 commandeer host fertility, sex ratios, and sex determination to enhance their maternal transmission via 52 male-killing, feminization, parthenogenesis, or cytoplasmic incompatibility (CI) (5, 6). 53Discovered nearly half a century ago (7), Wolbachia-induced CI is the most common reproductive 54 modification and results in embryonic lethality when an infected male mates with an uninfected female, 55 but this lethality is rescued when the female is likewise infected (8). As su...

Wolbachia pipientis is an incredibly widespread bacterial symbiont of insects, present in an estimated 25 to 52% of species worldwide. Wolbachia is faithfully maternally transmitted both in a laboratory setting and in the wild. In an established infection, Wolbachia is primarily intracellular, residing within host-derived vacuoles that are associated with the endoplasmic reticulum.