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.

The bacterial endosymbiont Wolbachia manipulates arthropod reproduction to facilitate its maternal spread through host populations. The most common manipulation is cytoplasmic incompatibility (CI): Wolbachia-infected males produce modified sperm that cause embryonic mortality, unless rescued by embryos harboring the same Wolbachia. The genes underlying CI, cifA and cifB, were recently identified in the eukaryotic association module of Wolbachia’s prophage WO. Here, we use transcriptomic and genomic approaches to address three important evolutionary facets of the cif genes. First, we assess whether or not cifA and cifB comprise a classic toxin–antitoxin operon in wMel and show that the two genes exhibit striking, transcriptional differences across host development. They can produce a bicistronic message despite a predicted hairpin termination element in their intergenic region. Second, cifA and cifB strongly coevolve across the diversity of phage WO. Third, we provide new domain and functional predictions across homologs within Wolbachia, and show that amino acid sequences vary substantially across the genus. Finally, we investigate conservation of cifA and cifB and find frequent degradation and loss of the genes in strains that no longer induce CI. Taken together, we demonstrate that cifA and cifB exhibit complex transcriptional regulation in wMel, provide functional annotations that broaden the potential mechanisms of CI induction, and report recurrent erosion of cifA and cifB in non-CI strains, thus expanding our understanding of the most widespread form of reproductive parasitism.

The most widespread intracellular bacteria in the animal kingdom are maternally-inherited endosymbionts of the genus Wolbachia. Their prevalence in arthropods and nematodes worldwide and stunning arsenal of parasitic and mutualistic adaptations make these bacteria a biological archetype for basic studies of symbiosis and applied outcomes for curbing human and agricultural diseases.Here, we conduct a summative, centennial analysis of living in the Wolbachia world.We synthesize literature on Wolbachia's host range, phylogenetic diversity, genomics, cell biology, and applications to filarial, arboviral, and agricultural diseases. We also review the mobilome of Wolbachia including phage WO and its essentiality to hallmark phenotypes in arthropods. Finally, the Wolbachia system is an exemplar for discovery-based science education using biodiversity, biotechnology, and bioinformatics lessons. As we approach a century of Wolbachia research, applications, and education, the interdisciplinary science and knowledge

Viruses are trifurcated into eukaryotic, archaeal and bacterial categories. This domain-specific ecology underscores why eukaryotic viruses typically co-opt eukaryotic genes and bacteriophages commonly harbour bacterial genes. However, the presence of bacteriophages in obligate intracellular bacteria of eukaryotes may promote DNA transfers between eukaryotes and bacteriophages. Here we report a metagenomic analysis of purified bacteriophage WO particles of Wolbachia and uncover a eukaryotic association module in the complete WO genome. It harbours predicted domains, such as the black widow latrotoxin C-terminal domain, that are uninterrupted in bacteriophage genomes, enriched with eukaryotic protease cleavage sites and combined with additional domains to forge one of the largest bacteriophage genes to date (14,256 bp). To the best of our knowledge, these eukaryotic-like domains have never before been reported in packaged bacteriophages and their phylogeny, distribution and sequence diversity imply lateral transfers between bacteriophage/prophage and animal genomes. Finally, the WO genome sequences and identification of attachment sites will potentially advance genetic manipulation of Wolbachia.

Wolbachia are the most widespread maternally-transmitted bacteria in the animal kingdom. Their global spread in arthropods and varied impacts on animal physiology, evolution, and vector control are in part due to parasitic drive systems that enhance the fitness of infected females, the transmitting sex of Wolbachia. Male killing is one common drive mechanism wherein the sons of infected females are selectively killed. Despite decades of research, the gene(s) underlying Wolbachia-induced male killing remain unknown. Here using comparative genomic, transgenic, and cytological approaches in fruit flies, we identify a candidate gene in the eukaryotic association module of Wolbachia prophage WO, termed WO-mediated killing (wmk), which transgenically causes male-specific lethality during early embryogenesis and cytological defects typical of the pathology of male killing. The discovery of wmk establishes new hypotheses for the potential role of phage genes in sex-specific lethality, including the control of arthropod pests and vectors.