PROJECT SUMMARY Across the tree of life species interact. This includes interactions between animals and microorganisms that colonize them. Many animals harbor microbes inside their cells and form endosymbiotic associations. Endosymbionts can have profound effects on host biology and fitness, but the consequences of these effects depend on endosymbiont prevalence in host populations. While some endosymbionts evolve obligate relationships, many associations are facultative, with both infected and uninfected host individuals. Wolbachia bacteria play both roles, but they usually form facultative relationships with arthropods. Indeed, facultative Wolbachia variants infect most insects making them the most common endosymbionts in nature. Despite this taxonomic prevalence, Wolbachia frequencies vary widely within and among host systems. The mechanisms underlying this variation remain unknown. Filling this gap in knowledge is crucial to understand the abundance of Wolbachia in nature and to improve the efficacy of Wolbachia biocontrol, where vector-control groups are attempting to establish pathogen-blocking Wolbachia variants in mosquito populations to reduce human disease transmission (particularly dengue). To achieve a comprehensive understanding of the mechanisms that govern Wolbachia spread, research in our lab will advance in three directions. First, many Wolbachia cause cytoplasmic incompatibility (CI) that kills embryos when infected males mate with uninfected females. This promotes Wolbachia spread in natural and in vector systems. However, CI-inducing males rarely kill all offspring when mated with uninfected females such that CI strength varies from very weak (most eggs hatch) to complete (no eggs hatch). We will determine the molecular mechanisms responsible for this variation. Second, while Wolbachia are maternally transmitted, transmission rates vary significantly. We recently discovered that cold temperatures disrupt maternal Wolbachia transmission. We will leverage this discovery to dissect the cellular-genetic basis of transmission rate variation. Third, our research has demonstrated rapid Wolbachia host switching with Wolbachia that diverged thousands of years ago infecting hosts that diverged many millions of years ago. This implies that Wolbachia regularly occur at initially rare frequencies in novel host species and must increase host fitness to spread and ultimately establish. We will compare effects of “old” and “young” infections on host fitness—in both natural and divergent host backgrounds—to quantify how much Wolbachia may diverge and still successfully spread in novel hosts. Together, these projects will leverage nearly 50 million years of Wolbachia divergence distributed across the Drosophila genus to understand the mechanisms that govern Wolbachia spread. More broadly, this research will promote a deeper understanding of the causes and consequences of endosymbiont prevalence in nature.