Wolbachia pipientis are maternally transmitted endosymbiotic bacteria commonly found in arthropods and nematodes. These bacteria manipulate reproduction of the host to increase their transmission using mechanisms, such as cytoplasmic incompatibility, that favor infected female offspring. The underlying mechanisms of reproductive manipulation by W. pipientis remain unresolved. Interestingly, W. pipientis infection partially rescues female fertility in flies containing hypomorphic mutations of bag of marbles (bam) in Drosophila melanogaster, which plays a key role in germline stem cell daughter differentiation. Using RNA-seq, we find that W. pipientis infection in bam hypomorphic females results in differential expression of many of bam's genetic and physical interactors and enrichment of ubiquitination and histone lysine methylation genes. We find that W. pipientis also rescues the fertility and germline stem cell functions of a subset of these genes when knocked down with RNAi in a wild-type bam genotype. Our results show that W. pipientis interacts with ubiquitination and histone lysine methylation genes which could be integral to the mechanism by which W. pipientis modulates germline stem cell gene function.

The protein encoded by the Drosophila melanogaster gene bag of marbles (bam) plays an essential role in early gametogenesis by complexing with the gene product of benign gonial cell neoplasm (bgcn) to promote germline stem cell daughter differentiation in males and females. Here, we compared the AlphaFold2 and AlphaFold Multimer predicted structures of Bam protein and the Bam:Bgcn protein complex between D. melanogaster, D. simulans, and D. yakuba, where bam is necessary in gametogenesis to that in D. teissieri, where it is not. Despite significant sequence divergence, we find very little evidence of significant structural differences in high confidence regions of the structures across the four species. This suggests that Bam structure is unlikely to be a direct cause of its functional differences between species and that Bam may simply not be integrated in an essential manner for GSC differentiation in D. teissieri. Patterns of positive selection and significant amino acid diversification across species is consistent with the Selection, Pleiotropy, and Compensation (SPC) model, where detected selection at bam is consistent with adaptive change in one major trait followed by positively selected compensatory changes for pleiotropic effects (in this case perhaps preserving structure). In the case of bam, we suggest that the major trait could be genetic interaction with the endosymbiotic bacteria Wolbachia pipientis. Following up on detected signals of positive selection and comparative structural analysis could provide insight into the distribution of a primary adaptive change versus compensatory changes following a primary change.

In Drosophila melanogaster, bag of marbles (bam) encodes a protein essential for germline stem cell (GSC) daughter differentiation in early gametogenesis. Despite its essential role in D. melanogaster, direct functional evaluation of bam in other closely related Drosophila species reveal this essential function is not necessarily conserved. In Drosophila teissieri, for example, bam is not essential for GSC daughter differentiation. Here, we generated bam null alleles using CRISPR-Cas9 in a species more distantly related to D. melanogaster, Drosophila americana, to interrogate whether bam's essential GSC differentiation function is novel to the melanogaster species group or a function more ancestral to the Drosophila genus. To further characterize the extent of the functional flexibility of other GSC-regulating genes, we generated a gene ortholog dataset for 366 GSC-regulating genes essential in D. melanogaster across 15 additional Drosophila and two outgroup species. We find that bam's essential GSC function is conserved between D. melanogaster and D. americana and therefore originated prior to the formation of the melanogaster species group. Additionally, we find that ∼8% of the 366 GSC genes essential in D. melanogaster are absent in at least one of the 17 species in our ortholog dataset. These results indicate that developmental systems drift, in which the specific genes regulating a function may change, but the final phenotype is retained, occurs in stem cell regulation and the production of gametes across Drosophila species.

In Drosophila melanogaster, a key germline stem cell (GSC) differentiation factor, bag of marbles (bam) shows rapid bursts of amino acid fixations between sibling species D. melanogaster and Drosophila simulans, but not in the outgroup species Drosophila ananassae. Here, we test the null hypothesis that bam's differentiation function is conserved between D. melanogaster and four additional Drosophila species in the melanogaster species group spanning approximately 30 million years of divergence. Surprisingly, we demonstrate that bam is not necessary for oogenesis or spermatogenesis in Drosophila teissieri nor is bam necessary for spermatogenesis in D. ananassae. Remarkably bam function may change on a relatively short time scale. We further report tests of neutral sequence evolution at bam in additional species of Drosophila and find a positive, but not perfect, correlation between evidence for positive selection at bam and its essential role in GSC regulation and fertility for both males and females. Further characterization of bam function in more divergent lineages will be necessary to distinguish between bam's critical gametogenesis role being newly derived in D. melanogaster, D. simulans, Drosophila yakuba, and D. ananassae females or it being basal to the genus and subsequently lost in numerous lineages.

In Drosophila melanogaster, the maternally inherited endosymbiont Wolbachia pipientis interacts with germline stem cell genes during oogenesis. One such gene, bag of marbles ( bam ) is the key switch for differentiation and also shows signals of adaptive evolution for protein diversification. These observations have led us to hypothesize that W. pipientis could be driving the adaptive evolution of bam for control of oogenesis. To test this hypothesis, we must understand the specificity of the genetic interaction between bam and W. pipientis . Previously, we documented that the W. pipientis variant, w Mel, rescued the fertility of the bam BW hypomorphic mutant as a transheterozygote over a bam null. However, bam BW was generated more than 20 years ago in an uncontrolled genetic background and maintained over a balancer chromosome. Consequently, the chromosome carrying bam BW accumulated mutations that have prevented controlled experiments to further assess the interaction. Here, we used CRISPR/Cas9 to engineer the same single amino acid bam hypomorphic mutation ( bam L255F ) and a new bam null disruption mutation into the w 1118 isogenic background. We assess the fertility of wildtype bam , bam L255F /bam null hypomorphic, and bam L255F / bam L255F mutant females, each infected individually with ten W. pipientis w Mel variants representing three phylogenetic clades. Overall, we find that all of the W. pipientis variants tested here rescue bam hypomorphic fertility defects with w MelCS-like variants exhibiting the strongest rescue effects. Additionally, these variants did not increase wildtype bam female fertility. Therefore, both bam and W. pipientis interact in genotype-specific ways to modulate female fertility, a critical fitness phenotype.

Jaclyn E. Bubnell1*, Paula Fernandez-Begne1, Cynthia K. Ulbing1, and Charles F. Aquadro1* 5 6 7 1 Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA 8 9 10 *Correspondence: Jaclyn E. Bubnell, jeb486@cornell.edu and Charles F. Aquadro, 11 cfa1@cornell.edu 12 13 14

We have found evidence of recurrent low-abundance infections of Wolbachia in our laboratory Drosophila fly stocks. These fly lines were previously “cleared” of infection by the conventional method of tetracycline treatment and should not display evidence of the bacteria. Regardless, sporadic positive results have been consistently produced when screening fly stocks for infection via endpoint PCR. Follow up analyses with qPCR confirmed these results. Extra care was taken to ensure that positive-testing samples were not a result of amplicon contamination. “Clean” PCR techniques were performed, yet positive results persisted. Water samples tested negative. We were confident there was an actual infection that was not being eliminated by tetracycline, so we wanted to characterize it. We tried to increase the titer of Wolbachia in these lowly-detectible fly lines so that it could be more easily characterized. Based on the literature, we tried raising flies on high sucrose food, which has been previously been shown to increase Wolbachia titer, but it failed in our flies. We also tried raising flies on food containing cycloheximide, which has been hypothesized to affect Wolbachia titer through translational repression, with no success. Finally, we attempted to rear gnotobiotic flies based on a previous observation in our lab that this may cause Wolbachia titer to increase or become more easily detectable, however this attempt also failed.We have begun analysis of our low-titer Wolbachia strain using multilocus sequence typing. In this poster we describe our efforts to characterize this Wolbachia infection as well as our recommendations for screening Drosophila lines for low Wolbachia titer. We advise against the use of endpoint PCR for low titer strains and suggest qPCR and cytological imaging as alternatives.

Many reproductive proteins from diverse taxa evolve rapidly and adaptively. These proteins are typically involved in late stages of reproduction such as sperm development and fertilization, and are more often functional in males than females. Surprisingly, many germline stem cell (GSC) regulatory genes, which are essential for the earliest stages of reproduction, also evolve adaptively in Drosophila. One example is the bag of marbles (bam) gene, which is required for GSC differentiation and germline cyst development in females and for regulating mitotic divisions and entry to spermatocyte differentiation in males. Here we show that the extensive divergence of bam between Drosophila melanogaster and D. simulans affects bam function in females but has no apparent effect in males. We further find that infection with Wolbachia pipientis, an endosymbiotic bacterium that can affect host reproduction through various mechanisms, partially suppresses female sterility caused by bam mutations in D. melanogaster and interacts differentially with bam orthologs from D. melanogaster and D. simulans. We propose that the adaptive evolution of bam has been driven at least in part by the long-term interactions between Drosophila species and Wolbachia. More generally, we suggest that microbial infections of the germline may explain the unexpected pattern of evolution of several GSC regulatory genes.

Coevolution between Drosophila and its endosymbiont Wolbachia pipientis has many intriguing aspects. For example, Drosophila ananassae hosts two forms of W. pipientis genomes: One being the infectious bacterial genome and the other integrated into the host nuclear genome. Here, we characterize the infectious and integrated genomes of W. pipientis infecting D. ananassae (wAna), by genome sequencing 15 strains of D. ananassae that have either the infectious or integrated wAna genomes. Results indicate evolutionarily stable maternal transmission for the infectious wAna genome suggesting a relatively long-term coevolution with its host. In contrast, the integrated wAna genome showed pseudogene-like characteristics accumulating many variants that are predicted to have deleterious effects if present in an infectious bacterial genome. Phylogenomic analysis of sequence variation together with genotyping by polymerase chain reaction of large structural variations indicated several wAna variants among the eight infectious wAna genomes. In contrast, only a single wAna variant was found among the seven integrated wAna genomes examined in lines from Africa, south Asia, and south Pacific islands suggesting that the integration occurred once from a single infectious wAna genome and then spread geographically. Further analysis revealed that for all D. ananassae we examined with the integrated wAna genomes, the majority of the integrated wAna genomic regions is represented in at least two copies suggesting a double integration or single integration followed by an integrated genome duplication. The possible evolutionary mechanism underlying the widespread geographical presence of the duplicate integration of the wAna genome is an intriguing question remaining to be answered.