Rupinder Kaur

Symbiont-mediated male killing (MK) is a mechanism that selectively eliminates male offspring, often by disrupting sex-specific developmental processes. In Drosophila melanogaster, the WO-mediated killing gene wmk from Wolbachia prophage WO transgenically reproduces the MK phenotype, yet how the gene evolves and functions across diverse Wolbachia has not been systematically investigated. We analyzed 32 Wolbachia genomes available in the NCBI database to study wmk homologs across different arthropod hosts, reproductive parasitism functions, and Wolbachia supergroups. First, we report at least five distinct wmk phylogenetic clusters (Type I-V), often organized in multigenic dyads or triads. Second, among MK Wolbachia, there is a significantly higher number of wmk genes and diversity in Lepidoptera strains than in Drosophila strains, which exclusively harbor wmk Types I and III. Third, there are three patterns of wmk sequence and genomic organizational changes in Drosophila MK strains that associate with different evolutionary trajectories underpinning the MK phenotype. Fourth, single and combinatory transgenic expression of Types I and III in D. melanogaster uncovers male-biased lethality associated with Type I; however, dual expression of the Types together elicits a major reduction in offspring number. Fifth, wmk genes have low expression level across D. melanogaster developmental stages relative to the cifA and cifB genes, which could explain why cytoplasmic incompatibility is expressed in this system. These findings establish a complex and phylogenetically-informed genetic basis of wmk-induced lethality, highlighting the role of gene copy number and expression, wmk Types, and host background in shaping the phenotype.

The symbiosis between arthropods and Wolbachia bacteria is globally widespread, largely due to selfish-drive systems that favour the fitness of symbiont-transmitting females. The most common drive, cytoplasmic incompatibility (CI), is central to arboviral control efforts. In Drosophila melanogaster carrying w Mel Wolbachia deployed in mosquito control, two prophage genes in Wolbachia, cifA and cifB , cause CI that results in a paternal-effect lethality of embryos in crosses between Wolbachia -bearing males and aposymbiotic females. While the CI mechanism by which Cif proteins alter sperm development has recently been elucidated in D. melanogaster and Aedes aegypti mosquitoes, the Cifs’ extended impact on male reproductive fitness such as sperm morphology and quantity remains unclear. Here, using cytochemical, microscopic and transgenic assays in D. melanogaster, we demonstrate that both CifA and CifB cause a significant portion of defects in elongating spermatids, culminating in malformed mature sperm nuclei. Males expressing Cifs have reduced spermatid bundles and sperm counts, and transgenic expression of Cifs can occasionally result in no mature sperm formation. We reflect on Cifs’ varied functional impacts on the Host Modification model of CI as well as host evolution, behaviour and vector control strategies.

The rising interest and success in deploying inherited microorganisms and cytoplasmic incompatibility (CI) for vector control strategies necessitate an explanation of the CI mechanism. Wolbachia-induced CI manifests in the form of embryonic lethality when sperm from Wolbachia-bearing testes fertilize eggs from uninfected females. Embryos from infected females however survive to sustain the maternally inherited symbiont. Previously in Drosophila melanogaster flies, we demonstrated that CI modifies chromatin integrity in developing sperm to bestow the embryonic lethality. Here, we validate these findings using wMel-transinfected Aedes aegypti mosquitoes released to control vector-borne diseases. Once again, the prophage WO CI proteins, CifA and CifB, target male gametic nuclei to modify chromatin integrity via an aberrant histone-to-protamine transition. Cifs are not detected in the embryo, and thus elicit CI via the nucleoprotein modifications established pre-fertilization. The rescue protein CifA in oogenesis localizes to stem cell, nurse cell, and oocyte nuclei, as well as embryonic DNA during embryogenesis. Discovery of the nuclear targeting Cifs and altered histone-to-protamine transition in both Aedes aegypti mosquitoes and D. melanogaster flies affirm the Host Modification Model of CI is conserved across these host species. The study also newly uncovers the cell biology of Cif proteins in the ovaries, CifA localization in the embryos, and an impaired histone-to-protamine transition during spermiogenesis of any mosquito species. Overall, these sperm modification findings may enable future optimization of CI efficacy in vectors or pests that are refractory to Wolbachia transinfections.

The extent to which prophage proteins interact with eukaryotic macromolecules is largely unknown. In this work, we show that cytoplasmic incompatibility factor A (CifA) and B (CifB) proteins, encoded by prophage WO of the endosymbiont Wolbachia, alter long noncoding RNA (lncRNA) and DNA during Drosophila sperm development to establish a paternal-effect embryonic lethality known as cytoplasmic incompatibility (CI). CifA is a ribonuclease (RNase) that depletes a spermatocyte lncRNA important for the histone-to-protamine transition of spermiogenesis. Both CifA and CifB are deoxyribonucleases (DNases) that elevate DNA damage in late spermiogenesis. lncRNA knockdown enhances CI, and mutagenesis links lncRNA depletion and subsequent sperm chromatin integrity changes to embryonic DNA damage and CI. Hence, prophage proteins interact with eukaryotic macromolecules during gametogenesis to create a symbiosis that is fundamental to insect evolution and vector control.

Inherited microorganisms can selfishly manipulate host reproduction to drive through populations. In Drosophila melanogaster, germline expression of the native Wolbachia prophage WO proteins CifA and CifB cause cytoplasmic incompatibility (CI) in which embryos from infected males and uninfected females suffer catastrophic mitotic defects and lethality; however, in infected females, CifA expression rescues the embryonic lethality and thus imparts a fitness advantage to the maternally transmitted Wolbachia. Despite widespread relevance to sex determination, evolution, and vector control, the mechanisms underlying when and how CI impairs male reproduction remain unknown and a topic of debate. Here, we use cytochemical, microscopic, and transgenic assays in D. melanogaster to demonstrate that CifA and CifB proteins of wMel localize to nuclear DNA throughout the process of spermatogenesis. Cif proteins cause abnormal histone retention in elongating spermatids and protamine deficiency in mature sperms that travel to the female reproductive tract with Cif proteins. Notably, protamine gene knockouts enhance wild-type CI. In ovaries, CifA localizes to germ cell nuclei and cytoplasm of early-stage egg chambers; however, Cifs are absent in late-stage oocytes and subsequently in fertilized embryos. Finally, CI and rescue are contingent upon a newly annotated CifA bipartite nuclear localization sequence. Together, our results strongly support the Host modification model of CI in which Cifs initially modify the paternal and maternal gametes to bestow CI-defining embryonic lethality and rescue.

Summary: The worldwide endosymbiosis between arthropods and Wolbachia bacteria is an archetype for reproductive parasitism. This parasitic strategy rapidly increases the proportion of symbiont-transmitting mothers, and the most common form, cytoplasmic incompatibility (CI), impacts insect evolution and arboviral control strategies. During CI, sperms from symbiotic males kill embryos of aposymbiotic females via two nuclear-targeting proteins, CifA and CifB, that alter sperm chromatin organization in Drosophila melanogaster . Here we hypothesize that Cif proteins metabolize nucleic acids of developing sperm to initiate genome integrity changes. Using in vitro and in situ transgenic, mutant, enzymatic, and cytochemical assays, we show that CifA is a previously-unrecognized DNase and RNase, and CifB is a DNase. Notably, in vitro nuclease activity translates to in situ spermatid DNA damage at the canoe stage of spermiogenesis. Evolution-guided mutations ablate Cif enzymatic activity. Nucleic acid metabolism by Cif enzymes expands a fundamental understanding of the mechanism of symbiont-mediated reproductive parasitism.

Inherited microorganisms can selfishly manipulate host reproduction to drive through populations. In Drosophila melanogaster , germline expression of the native Wolbachia prophage WO proteins CifA and CifB cause cytoplasmic incompatibility (CI) in which embryos from infected males and uninfected females suffer catastrophic mitotic defects and lethality; however, in infected females, CifA expression rescues the embryonic lethality and thus imparts a fitness advantage to the maternally-transmitted Wolbachia . Despite widespread relevance to sex determination, evolution, and vector control, the mechanisms underlying when and how CI impairs male reproduction remain unknown and a topic of debate. Here we use cytochemical, microscopic, and transgenic assays in D. melanogaster to demonstrate that CifA and CifB proteins of w Mel localize to nuclear DNA throughout the process of spermatogenesis. Cif proteins cause abnormal histone retention in elongating spermatids and protamine deficiency in mature sperms that travel to the female reproductive tract with Cif proteins. Notably, protamine gene knockouts enhance wild type CI. In ovaries, CifA localizes to germ cell nuclei and cytoplasm of early-stage egg chambers, however Cifs are absent in late-stage oocytes and subsequently in fertilized embryos. Finally, CI and rescue are contingent upon a newly annotated CifA bipartite nuclear localization sequence. Together, our results strongly support the Host Modification model of CI in which Cifs initially modify the paternal and maternal gametes to bestow CI-defining embryonic lethality and rescue.

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 a 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 from this symbiosis stand as a model for consolidating and teaching the integrative rules of endosymbiotic life.