Many insects and other arthropods have symbiotic microorganisms that may influence key facets of their biology. Cardinium hertigii is an intracellular bacterial symbiont, (phylum Bacteroidetes) of arthropods and nematodes. This versatile symbiont has been shown to cause three of four reproductive manipulations of their arthropod hosts known to be caused by symbionts: parthenogenesis induction (PI), where genetic males are converted into genetic females; feminization, where genetic males become functional females; and cytoplasmic incompatibility (CI), the symbiont-induced death of offspring from matings of infected males and uninfected females. Here, I explored the evolution of this symbiont and its reproductive manipulations, and found that closely related Cardinium strains have a tendency to associate with closely related hosts and the reproductive manipulations do not display a clear phylogenetic signal. To further understand the possible genes underlying these reproductive manipulations, I sequenced four Cardinium genomes and compared these with the two genomes analyzed in the literature. In these comparisons, I found that, although closely related Cardinium strains tend to reside in closely related hosts, there is no evidence for a suite of genes associated with host specificity, as few differences separate two strains residing in different host orders, suggesting that ecological opportunity for horizontal transmission may be more limiting to Cardinium than genomic capability. I additionally identify some genes that may be associated with the Cardinium’s ability to induce PI and CI in its wasp host. Overall, this dissertation has led to a better understanding of Cardinium and its effects on its hosts.

Reciprocal and homologous crosses were carried out using pairs or groups of twenty males and females for three biotypes of the whitefly Bemisia tabaci complex. Crosses were undertaken for the A biotype-Arizona (AzA), the B biotype-Africa (AzB), and the monophagous, Jatropha (Jat) biotype-Puerto Rico. The maternal haplotype pedigree of parents and selected offspring (F₁, F₂) was determined using the mitochondria cytochome oxidase I sequence. All reciprocal crosses yielded viable female offspring, indicating reproductively compatibility, except for AzB♀ x AzA♂, or AzB♀ x Jat♂ crosses, which yielded females unidirectionally. As an unidirectional pattern was reminiscent of cytoplasmic-mediated incompatibility (CI), the possibility was investigated that the phenotype might be caused by CI-bacteria, instead of a germ line barrier. Using the 16S rRNA sequence three prospective CI-bacterial species were identified in whitefly colonies. A Cardinium spp. (Bacteroidetes) was present in the A biotype (isolates AzA, CulA, RivA, SalA), whereas the B biotype (isolates AzB, FlB1, FlB2) was infected with Rickettsia bellii (Proteobacteria), and a Wolbachia spp. (Proteobacteria) was associated with the Jat biotype. The unidirectional incompatible phenotypes were consistent with CI-bacterial infection of AzA (Cardinium) and Jat (Wolbachia), but no such association was apparent for B biotype-Rickettsia infections. The bidirectional compatibility for Jat x AzA suggested a CI-bacteria-mediated reciprocal rescue. However, that Wolbachia-infected Jat♀ and Cardinium-infected AzA♂ crosses yielded fewer females, compared to AzA-Cardinium♀ and Jat- Wolbachia♂ crosses, suggested that Cardinium could better counter Wolbachia-induced female mortality than Wolbachia. This suggested the possibility that these phylogenetically divergent bacteria might utilize similar CI-mechanisms. In this study, the suspect CI-bacteria were strongly associated with complete or partial obstruction of gene flow in certain crosses, and with sex bias in the AzA x Jat crosses. This is the first evidence that female offspring can be produced between phylogeographically divergent, and polyphagous and monophagous B. tabaci, for which gene flow barriers are widely reported, suggesting that hybridization is utilized as a means of diversification in B. tabaci. The inability to rid colonies of CI-bacteria has necessitated introgression experiments to investigate direct CI-causality over CI-association.

Cytoplasmic incompatibility (CI) is an intriguing, widespread, symbiont-induced reproductive failure that decreases offspring production of arthropods through crossing incompatibility of infected males with uninfected females or with females infected with a distinct symbiont genotype. For years, the molecular mechanism of CI remained unknown. Recent genomic, proteomic, biochemical, and cell biological studies have contributed to understanding of CI in the alphaproteobacterium Wolbachia and implicate genes associated with the WO prophage. Besides a recently discovered additional lineage of alphaproteobacterial symbionts only moderately related to Wolbachia, Cardinium (Bacteroidetes) is the only other symbiont known to cause CI, and genomic evidence suggests that it has very little homology with Wolbachia and evolved this phenotype independently. Here, we present the first transcriptomic study of the CI Cardinium strain cEper1, in its natural host, Encarsia suzannae, to detect important CI candidates and genes involved in the insect-Cardinium symbiosis. Highly expressed transcripts included genes involved in manipulating ubiquitination, apoptosis, and host DNA. Female-biased genes encoding ribosomal proteins suggest an increase in general translational activity of Cardinium in female wasps. The results confirm previous genomic analyses that indicated that Wolbachia and Cardinium utilize different genes to induce CI, and transcriptome patterns further highlight expression of some common pathways that these bacteria use to interact with the host and potentially cause this enigmatic and fundamental manipulation of host reproduction. IMPORTANCE The majority of insects carry maternally inherited intracellular bacteria that are important in their hosts' biology, ecology, and evolution. Some of these bacterial symbionts cause a reproductive failure known as cytoplasmic incompatibility (CI). In CI, the mating of symbiont-infected males and uninfected females produces few or no daughters. The CI symbiont then spreads and can have a significant impact on the insect host population. Cardinium, a bacterial endosymbiont of the parasitoid wasp Encarsia in the Bacteroidetes, is the only bacterial lineage known to cause CI outside the Alphaproteobacteria, where Wolbachia and another recently discovered CI symbiont reside. Here, we sought insight into the gene expression of a CI-inducing Cardinium strain in its natural host, Encarsia suzannae. Our study provides the first insights into the Cardinium transcriptome and provides support for the hypothesis that Wolbachia and Cardinium target similar host pathways with distinct and largely unrelated sets of genes.