The thesis brings new phylogenetic data on the rapidly growing bacterial genus Arsenophonus, and summarizes the current evolutionary picture of its symbiotic association with insects and other hosts. The study is prepared in form of a regular publication and will be submitted in microbiological journal.

Arsenophonus is vertically and horizontally transmitted parasitic bacteria and strengthens its transfer through phenomenon called son killing. Arsenophonus has been detected in the arthropod hosts, insects, ticks and the garden spider Araneus diadematus. The aim of this study is detection parasitic bacteria Arsenophonus in honeybees and its parasite Varroa destructor. We find out that bacteria Arsenophonus is present in both hosts and it is identified as Arsenophonus nasoniae. Detection of bacteria Arsenophonus is discovered for the first time in Varroa destructor mites. We proved that the frequency of bacteria Arsenophonus in mites Varroa destructor is significantly higher than in honeybees. This study shows that the mite Varroa destructor could act as a vector for transmission parasitic bacteria Arsenophonus among honeybees. Results of this study could lead to the future application of Arsenophonus as a biological control for the mite Varroa destructor.

Bacterial endosymbionts can have profound impacts on their host’s ecology. Notably, endosymbionts can protect their hosts against natural enemies and influence host plant interactions. The endosymbiont Candidatus Arsenophonus infects a wide taxonomic range of arthropod hosts, and is suspected of an uncharacterized mutualistic role in hemipterous insects. In the soybean aphid, Aphis glycines, an introduced pest of soybeans in the United States, Arsenophonus is the sole facultative endosymbiont. The focus of this dissertation is to characterize the role of Arsenophonus in the aphid, with an overall emphasis on its impact on aphid management strategies. I first used diagnostic PCR to determine Arsenophonus infection frequency and strain diversity for native and introduced soybean aphids. I found that Arsenophonus infection is a uniform strain that is highly prevalent in soybean aphid. I then determined if Arsenophonus was a defense symbiont by curing two genotypes of soybean aphid of their natural Arsenophonus infection, resulting in infected and uninfected isolines within the same genetic background. I subjected these isolines to assays with three parasitoid species and a common aphid fungal pathogen, Pandora neoaphidis. I did not find differences in parasitism or fungal infections within the treatments. These results indicate that, although Arsenophonus is widespread, the symbiont should not interfere with biological control efforts. I next examined the influence of Arsenophonus on the ability of soybean aphid “biotypes” to colonize resistant Rag plants. I cured three additional soybean aphid biotypes. All isolines were subjected to growth rate assays on resistant Rag versus susceptible soybean. My results indicate that Arsenophonus infected soybean aphids have an increased population growth compared to uninfected aphids regardless of soybean plant type Finally, I induced soybean plants with jasmonic acid (JA) or salicylic acid (SA) to determine the effective plant defense against soybean aphid feeding. I also used Arsenophonus infected and uninfected aphids to determine any interaction between Arsenophonus and plant defense. I found SA treatment decreased soybean aphid population growth for one experiment, but had no effect when replicated. JA treatment had no effect, and there were no interactions between Arsenophonus infection and plant treatments.

Solitary bees are important pollinators of crops, with species in the family Megachilidae (mason bees) being used for orchard pollination. Commercial movement of these bees also moves their microbiota, including bacterial endosymbionts capable of reproductive manipulation. To test for presence of these bacteria, I screened commercially available species of US orchard pollinators and locally captured solitary bees from Kentucky. I also set up mason bee boxes in five apple orchards to examine recruitment of local pollinators. I conducted 454-pyrosequencing to determine bacterial diversity within four species followed by diagnostic PCR of 30 collected species (184 individuals) to determine infection frequency of selected endosymbionts. Consistent with literature, Wolbachia was abundant in these bees. I also found two other endosymbiotic bacteria, Sodalis (previously undetected in Hymenoptera), and Arsenophonus. Diagnostic screening demonstrated that Sodalis was present at moderate frequency in Osmia aglaia, whereas Arsenophonus was present at low frequency in Lasioglossum pilosum. Neither was found in other bees, but three bee species were infected with Sodalis-like endosymbionts. Although recruitment of bees to bee boxes was ineffective, I was able to independently collect native orchard pollinating Andrenidae species. My results demonstrate that other endosymbionts capable of reproductive manipulation, besides Wolbachia, are present in bees.

Obligately blood-sucking parasites harbour symbiotic bacteria providing them B-vitamins and cofactors missing from their blood diet. Within Hippoboscoidea group (parasites of birds and mammals), tsetse flies as medically important vectors have been studied extensively while bat flies and louse flies tend to be neglected. This thesis is composed of two complementary manuscripts focused on phylogeny and origin of bacterial symbionts in Hippoboscidae family (manuscript 1) and their genome evolution (manuscript 2). First, phylogenetic approach was employed to determine lineages of obligate and facultative symbionts present in this group. Second, genomic and phylogenomic analyses were carried out to better understand evolution of obligate endosymbionts from the Arsenophonus genus in this group. Results of the two studies indicate that relationships between Hippoboscoidea and their symbionts are extremely dynamic with frequent replacements of obligate symbionts. This hypothesis is supported by both phylogenetic and genomic evidence, in particular, Arsenophonus endosymbionts of Hippoboscidae represent several distinct lineages (of likely different ages) with noticeable differences in genome features and metabolic capabilities. The data presented in this thesis thus greatly extend our knowledge about evolution and genomics of symbiotic bacteria in Hippoboscidae and bloodsucking hosts in general.

Since the introduction of advanced molecular methods the research on insect bacterial symbioses underwent a major focus shift towards large scale phylogenetics and comparative genomics. These new fields provided answers to several fundamental questions of symbiont evolution, functional capabilities of the host-associated bacteria, and the role of symbionts in the host?s biology. However, the vast diversity and complexity of symbiotic relationships still leaves gaps in our understanding to a rich mosaic of various symbiont types, effects and transitions from facultative association to obligate mutualism. The presented study focuses on distribution, diversity, phylogenetic patterns, evolutionary transitions and genome evolution of two less known but ecologically diverse bacterial genera, Arsenophonus and Sodalis. The thesis also takes advantage of the knowledge on a well established symbiotic model between aphids and Buchnera and reveals several evolutionary patterns in the host and symbiont.

Many insects form close relationships with microbial symbionts. Insect symbionts can provide novel phenotypes to their hosts, including influencing dietary breadth. In the polyphagous cowpea aphid, Aphis craccivora, the facultative symbiont Arsenophonus improves aphid performance on one host plant (locust), but decreases performance on other plants. The goal of my thesis was to investigate the mechanism by which Arsenophonus facilitates use of locust. First, I assembled an Aphis craccivora-Arsenophonus-Buchnera reference transcriptome to conduct RNAseq analysis, comparing gene expression in aphids feeding on locust and fava, with and without Arsenophonus infection. Overall, few transcripts were differentially expressed. However, genes that were differentially expressed mapped to a variety of processes, including metabolism of glucose, cytoskeleton regulation, cold and drought regulation, and B-vitamin synthesis. These results imply that Arsenophonus is producing B-vitamins, which might be deficient in locust. In a second set of experiments, I used qPCR to test whether symbiont function across host plants might be mediated by bacterial titer. I measured relative Arsenophonus abundance across plants, and found Arsenophonus titer was variable, but generally greater on locust than fava. In summary, my results suggest that Arsenophonus synthesis of B-vitamins should be further investigated and may be mediated by bacterial titer.

The gammaproteobacterium Arsenophonus nasoniae infects Nasonia vitripennis (Hymenoptera: Pteromalidae), a parasitic wasp that attacks filth flies. This bacterium kills virtually all male offspring of infected females. Female wasps transmit A. nasoniae both vertically (from mother to offspring) and horizontally (to unrelated Nasonia developing in the same fly). This latter mode may enable the bacterium to colonize novel species and spread throughout a filth fly-parasitoid guild. This spread may be important for maintenance of the bacterium. The ecology of novel hosts may be significantly impacted by infection. The actual and potential host range of A. nasoniae was assessed. I used Arsenophonus-specific primers to screen a large sample of filth flies and their parasitoids. The bacterium infects a wide range of wasp species in the environment. The potential host range was determined by inoculating three wasp and one fly species with an isolate of A. nasoniae from Lethbridge, AB. The bacterium successfully infected all insects and was transmitted by two wasp species. It reduced host longevity, but did not kill males, in Trichomalopsis sarcophagae. It also caused pupal mortality in Musca domestica.