cifB- transcript levels largely explain cytoplasmic incompatibility variation across divergent Wolbachia .

Divergent hosts often associate with intracellular microbes that influence their fitness. Maternally transmitted bacteria are the most common of these endosymbionts, due largely to cytoplasmic incompatibility (CI) that kills uninfected embryos fertilized by -infected males. Closely related infections in females rescue CI, providing a relative fitness advantage that drives to high frequencies. One prophage-associated gene () governs rescue, and two contribute to CI ( and ), but CI strength ranges from very strong to very weak for unknown reasons. Here, we investigate CI-strength variation and its mechanistic underpinnings in a phylogenetic context across 20 million years (MY) of evolution in hosts diverged up to 50 MY. These encode diverse Cif proteins (100% to 7.4% pairwise similarity), and AlphaFold structural analyses suggest that CifB sequence similarities do not predict structural similarities. We demonstrate that -transcript levels in testes explain CI strength across all but two focal systems. Despite phylogenetic discordance among and the bulk of the genome, closely related tend to cause similar CI strengths and transcribe at similar levels. This indicates that other non- regions of the genome modulate -transcript levels. CI strength also increases with the length of the host's larval life stage, presumably due to prolonged action. Our findings reveal that transcript levels largely explain CI strength, while highlighting other covariates. Elucidating CI's mechanism contributes to our understanding of spread in natural systems and to improving the efficacy of CI-based biocontrol of arboviruses and agricultural pests globally.