DISSERTATION RESEARCH: The Genetic Basis of Cytoplasmic Incompatibility

Symbiotic bacteria are extremely widespread in nature and interest in the way that they manipulate animal evolution has risen rapidly in recent years. Discoveries have ranged from bacteria that affect nutrition, immunity, and reproduction to bacteria that cause the splitting of one species into two. An evolutionary enigma surrounds these adaptations - how do bacteria evolve to contribute to these complex animal processes? One of the preeminent bacteria involved in these interactions, Wolbachia pipientis, occurs worldwide in ~40% of all insect species and other invertebrates. Previous work has shown that Wolbachia's ability to alter animal reproduction affects animal populations by spurring the evolution of new insect species, reproductive strategies, and offspring production. Despite these evolutionary outcomes of animal-Wolbachia symbioses, we do not yet know the Wolbachia genes that evolved to control their animal hosts. This study begins the first in-depth analysis of two Wolbachia genes that are likely involved in the symbiosis.

Cytoplasmic incompatibility (CI) is Wolbachia's main alteration of host sex ratios. CI drastically decreases fertility of uninfected females in crosses to Wolbachia-infected males and gives infected females a distinct advantage within the population. Preliminary work discovered two Wolbachia genes, with conserved protein and DNA binding domains, putatively involved in CI. To study their interactions, tagged versions of each gene will be expressed in Wolbachia-free Drosophila melanogaster. Immunofluorescent microscopy will then be utilized to determine whether expression of these genes in uninfected males crossed with uninfected females causes explicit CI defects in developing embryos. Next, after dissecting the larval testes, immunoprecipitation of the tagged proteins using commercially available antibodies will be used to isolate any bound host factors. Samples will be prepared for mass spectrometry to determine what host proteins may be bound. Additional samples using chromatin immunoprecipitation, coupled with next generation sequencing, will determine what regions of host DNA are targeted. Finally, candidate Drosophila pathways will be verified for their role in CI through a combination of genetic mutants and RNAi knockdown.