PROJECT SUMMARY Animals contain a small number of germline stem cells (GSCs) that are essential for making sperm or eggs. The decision for a dividing stem cell to renew itself or to differentiate, and the regulation of early GSC divisions are some of the most critical processes in development. Studies in the fruit fly, Drosophila melanogaster, have identified many of the key genes involved in the cellular signaling for GSC maintenance and differentiation. We have shown that the gene bag of marbles (bam), the key “switch” for GSC differentiation, is adaptively evolving with a dramatic excess of amino acid substitutions in D. melanogaster, D. simulans, D. yakuba but not in six other related species. During our current funding period we have generated new bam null mutations in sequenced genomes of D. melanogaster, D. simulans, D. yakuba, D. teissieri, and D. ananassae to test for conservation of function. These have allowed us to demonstrate that surprisingly unlike in D. melanogaster, bam is not required for female GSC differentiation in D. teissieri nor is it required for male GSC differentiation in D. teissieri and D. ananassae. We have shown that infection with different strains of the intracellular endosymbiont Wolbachia pipientis vary in their degree of rescue of the fertility defects characteristic of the bam hypomorph. This proposal focuses on identifying the functional and evolutionary consequences of observed sequence diversification at bam and discriminating between the hypothesis that bam’s role in GSC function has been selected for new functions, vs the hypothesis that interactions between bam and W. pipientis has been the evolutionary force driving changes at bam. This includes generating and analyzing novel bam hypomorphs in D. melanogaster and D. simulans, and testing interactions of these new hypomorphs with intra and interspecific Wolbachia strains. We will carry out RNA-seq studies of the bam rescue by W. pipientis. We are developing a computational model to generate experimentally testable predictions of sequence diversification under several mechanisms of interaction between W. pipientis and GSC genes. Finally, we will functionally evaluate evolutionary diversification of the bam protein sequence between D. melanogaster and D. simulans, and the loss of the requirement for bam for female and male gametogenesis and fertility in D. teissieri compared to its close relative D. yakuba. What we learn will contribute significantly to our understanding of how GSCs are regulated during development in diverse species, how such critical developmental functions can evolve, and how their mis-regulation due to mutation and interaction with germline parasites can lead to infertility, germline cancers, and reproductive isolation.