Hereditary bacterial symbiosis is a common mechanism by which eukaryotic hosts can acquire traits beneficial for their fitness. Many insects have symbiotic associations with bacteria that trace back millions of years, whose function and evolution are well characterized. Insects can also possess more recently derived symbionts that are closely related to free-living bacteria, and often play a role in host defense. Serratia symbiotica is a recently derived symbiont that infects aphids and provides protection against heat stress, and possibly also plays a nutritional role. Many aspects of the biology of recent symbionts are less well studied, including the diversity of functional roles and evolution among hosts for single lineages of symbionts, the molecular mechanisms that contribute to defense, the early stages of symbiont genome evolution, and interactions with hosts. This dissertation focuses upon S. symbiotica to contribute research addressing each of these themes. Functional studies revealed that S. symbiotica lysis during heat-shock is correlated with protection of the nutritional symbiont Buchnera , and that S. symbiotica has a large effect upon aphid metabolite pools. Despite this large metabolic effect, S. symbiotica does not seem to dramatically influence expression of aphid genes, including those involved in immunity. Analysis of the evolution of S. symbiotica lineages in different aphid hosts revealed this symbiont is common in the aphid subfamily Lachninae, but did not support the obligate nutritional role hypothesized in the literature for this group. Finally, comparison of the S. symbiotica genome to close free-living relatives revealed a genome undergoing massive decay, and provided a rare opportunity to examine the evolution of a recently acquired symbiont.

This dissertation comprises a series of studies designed to explore the associations between plants and the endophytic fungi they harbor in their above-ground tissues. By viewing endophyte diversity in ecologically and economically important hosts through the lenses of phylogenetic biology, microbiology, and biotechnology, this body of work links plant ecology with newly discovered symbiotic units comprised of endophytic fungi and the bacteria that inhabit them.This work begins with a large-scale survey of endophytic fungi from native and non-native Cupressaceae in Arizona and North Carolina. After isolating over 400 strains of endophytes, I inferred the evolutionary relationships among these fungi using both Bayesian and parsimony analyses. In addition to showing that native and introduced plants contained different endophytes, I found that the endophytes themselves harbor additional microbial symbionts, recovering members of the beta- and gamma-proteobacterial orders Burkholderiales, Xanthomonadales, and Enterobacteriales and numerous novel, previously uncultured bacteria. This work finds that phylogenetically diverse bacterial endosymbionts occur within living hyphae of multiple major lineages of ascomycetous endophytes.A focus on 29 fungal/bacterial associations revealed that bacterial and fungal phylogenies are incongruent with each other and did not reflect the phylogenetic relationships of host plants. Instead, both endophyte and bacterial assemblages were strongly structured by geography, consistent with local horizontal transmission. Endophytes could be cured of their bacterial endosymbionts using antibiotics, providing a tractable experimental system for comparisons of growth and metabolite production under varying conditions. Studies of seven focal fungal/bacterial pairs showed that bacteria could significantly alter growth of fungi at different nutrient and temperature levels in vitro, and that different members of the same bacterial lineages interact with different fungi in different ways.Focusing on one isolate, I then describe for the first time the production of indole-3-acetic acid (IAA) by a non-pathogenic, foliar endophytic fungus (Pestalotiopsis neglecta), suggesting a potential benefit to the host plant harboring this fungus. I show that this fungus is inhabited by an endohyphal bacterium (Luteibacter sp.) and demonstrate that mycelium containing this bacterium produces significantly more IAA in vitro than the fungus alone. I predict that the general biochemical pathway used by the fungal-endohyphal complex is L-tryptophan-dependent and measure effects of IAA production in vivo, focusing on root and shoot growth in tomato seedlings.

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Every year over two hundred million cases of malaria occur worldwide causing human death and suffering often in the poorest countries. Most people who die from malaria are children under five years of age. Malaria is caused by parasites spread by mosquitoes when they feed on human blood. Currently prevention methods include insecticides and anti-malarial drugs. The problem with both is the increasing resistance towards them by mosquitoes and parasites, respectively. Therefore other approaches need to be investigated to find new solutions to this problem. One such research area is paratransgenesis, the genetic modification of symbiotic microorganisms in the mosquitoes to produce anti-malaria parasite molecules. One bacterium identified as a potential candidate for paratransgenesis is Thorsellia anophelis. When this study started, only one Thorsellia isolate existed in the world. The aim of this study was therefore to retrieve more Thorsellia isolates from Kenyan mosquito and water samples. The samples were screened by PCR followed by bacterial culturing of positive samples, which resulted in 38 new Thorsellia isolates confirmed by DNA sequencing. The isolation of new Thorsellia species enables further investigation of the potential for their use in paratransgenesis with the aim of contributing to the prevention of malaria transmission.

This thesis analyses the interaction between missionary medicine and Ndyuka medicine from the perspective of a Baptist missionary nurse operating a clinic in the village of Lantiwei in Suriname. Based on two months of anthropological fieldwork conducted in the summer of 2014, this thesis argues that in her everyday practice of medicine, the Baptist nurse is dependent on the cooperation of a local Ndyuka healer and his family to such an extent that their working together can be regarded as a social form of symbiosis. This thesis furthermore demonstrates that the Baptist nurse has incorporated Ndyuka ideas to her understanding of illness and death, and that even though she continues to abhor and reject the practice of Ndyuka medicine as a form of devil worship, she recognizes its spiritual powers. Taken together, these findings show that an opposition between missionary medicine as a—perhaps unwitting—agent of modernity and local Ndyuka medicine as a ‘traditional’ form of medicine increasingly superseded by ‘modern’ medicine, is fictitious, and that the lack of scholarly attention to the interaction of missionary medicine with local practices of medicine demontrates anthropology’s obsession with the ‘traditional’ ‘Other’.

Biologists have taken the concept of organism largely for granted. However, advances in the study of chimerism, symbiosis, bacterial-eukaryote associations, and microbial behavior have prompted a redefinition of organisms as biological entities exhibiting low conflict and high cooperation among their parts. This expanded view identifies organisms in evolutionary time. However, the ecological processes, mechanisms, and traits that drive the formation of organisms remain poorly understood. Recognizing that organismality can be context dependent, we advocate elucidating the ecological contexts under which entities do or do not act as organisms. Here we develop a "contextual organismality" framework and provide examples of entities, such as honey bee colonies, tumors, and bacterial swarms, that can act as organisms under specific life history, resource, or other ecological circumstances. We suggest that context dependence may be a stepping stone to the development of increased organismal unification, as the most integrated biological entities generally show little context dependence. Recognizing that organismality is contextual can identify common patterns and testable hypotheses across different entities. The contextual organismality framework can illuminate timeless as well as pressing issues in biology, including topics as disparate as cancer emergence, genomic conflict, evolution of symbiosis, and the role of the microbiota in impacting host phenotype.

Background: Steinernematid nematodes form obligate symbioses with bacteria from the genus Xenorhabdus. Together Steinernema nematodes and their bacterial symbionts successfully infect, kill, utilize, and exit their insect hosts. During this process the nematodes and bacteria disassociate requiring them to re-associate before emerging from the host. This interaction can be complicated when two different nematodes co-infect an insect host. Results: Non-cognate nematode-bacteria pairings result in reductions for multiple measures of success, including total progeny production and virulence. Additionally, nematode infective juveniles carry fewer bacterial cells when colonized by a non-cognate symbiont. Finally, we show that Steinernema nematodes can distinguish heterospecific and some conspecific non-cognate symbionts in behavioral choice assays. Conclusions: Steinernema-Xenorhabdus symbioses are tightly governed by partner recognition and fidelity. Association with non-cognates resulted in decreased fitness, virulence, and bacterial carriage of the nematode-bacterial pairings. Entomopathogenic nematodes and their bacterial symbionts are a useful, tractable, and reliable model for testing hypotheses regarding the evolution, maintenance, persistence, and fate of mutualisms.

I examined the effect of Frankia spp. and ectomycorrhizal fungi on green alder (Alnus viridis ssp. crispa), growing in nutrient-poor soil and saline conditions. The first experiment involved inoculating green alder growing in low fertility soil with three species of ectomycorrhizal fungi (Lactarius torminosus, Lactarius theiogalus, Hebeloma crustuliniforme) alone or in combination, with and without Frankia spp. on. Frankia spp. inoculation significantly increased plant performance compared to non-Frankia treatments. However, nodulated plant total biomass decreased with an increasing number of fungi. The second experiment examined the effect of Hebeloma crustuliniforme and Frankia spp. on green alder exposed to 0, 50 and 100 mM NaCl. Frankia spp. inoculation showed significant increase on plant performance but Hebeloma crustuliniforme did not. Plant mass, root:shoot ratio, nodule allocation and total nitrogen fixation decreased with NaCl exposure. A decrease in root:shoot ratio caused by salt was more moderate in nodulated plants compared to non-nodulated plants. / October 2016

Background: Symbioses throughout the animal kingdom are known to extend physiological and ecological capabilities to hosts. Insect-microbe associations are extremely common and are often related to novel niche exploitation, fitness advantages, and even speciation events. These phenomena include expansions in host diet, detoxification of insecticides and toxins, and increased defense against pathogens. However, dissecting the contributions of individual groups of symbionts at the molecular level is often underexplored due to methodological and analytical limitations. Termites are one of the best studied systems for physiological collaborations between host and symbiota; however, most work in lower termites (those with bacterial and protist symbionts) focuses on the eukaryotic members of this symbiotic consortium. Here we present a metatranscriptomic analysis which provides novel insights into bacterial contributions to the holobiont of the eastern subterranean termite, Reticulitermes flavipes, in the presence and absence of a fungal pathogen. Results: Using a customized ribodepletion strategy, a metatranscriptome assembly was obtained representing the host termite as well as bacterial and protist symbiota. Sequence data provide new insights into biosynthesis, catabolism, and transport of major organic molecules and ions by the gut consortium, and corroborate previous findings suggesting that bacteria play direct roles in nitrogen fixation, amino acid biosynthesis, and lignocellulose digestion. With regard to fungal pathogen challenge, a total of 563 differentially expressed candidate host and symbiont contigs were identified (162 up-and 401 downregulated; a/FDR = 0.05) including an upregulated bacterial amidohydrolase. Conclusions: This study presents the most complete bacterial metatranscriptome from a lower termite and provides a framework on which to build a more complete model of termite-symbiont interactions including, but not limited to, digestion and pathogen defense.

Wolbachia are maternally transmitted endosymbionts that infect a large proportion of arthropods and reduce vector competency of mosquitoes carrying deadly diseases such as Dengue, Zika, West Nile Virus, and Chikungunya. Wolbachia preferentially infect specific host cells, a phenomenon termed tissue tropism. Wolbachia tissue tropism has consequences in proper vertical and horizontal transmission. Although Wolbachia tropism to multiple Drosophila gonadal cell types has been identified, the mechanisms of tropism during development have not been characterized, in part because Wolbachia infected cell types are challenging to study during morphogenesis. Here we describe a novel Wolbachia tropism to polar cells (PCs) of the Drosophila ovary, a developmentally well characterized system. We show that Wolbachia intracellular accumulation is triggered by specific events of PC morphogenesis including differentiation from progenitors, and during stage 8 to 9 transition. Using genetic tools, we also demonstrate that induction of ectopic PC fate is sufficient to drive Wolbachia tropism. These findings implicate a tight coordination of host developmental events with Wolbachia tropism. Wolbachia tropism to multiple host cell types suggest that host pathways common to these would be conducive to intracellular Wolbachia growth. Indeed, we found that Wnt signaling, is active in gonadal cell types with Wolbachia tropism. Wnt signaling, first characterized in embryonic development and patterning, has novel functions in immunity and intracellular pathogen survival. Using RNAi mediated gene knockdowns, we studied the effect of Wnt signaling on Wolbachia in various infected cell types, including the testis stem cell niche, PCs and germline. Reduction of Wnt signaling caused a decrease in Wolbachia density and increased signaling led to higher density suggesting the reliance of Wolbachia on host Wnt signaling for its tropism. Moreover, expression of ectopic Wnt signaling was sufficient to drive Wolbachia tropism to previously uninfected tissues such as the D. melanogaster female germline stem cell niche. Finally, small molecule Wnt signaling agonists were sufficient to drive high Wolbachia titers in mosquito cell lines. These findings describe the effect of host signaling on Wolbachia tropism and provide an approach to affect Wolbachia levels in disease-causing vectors, thereby contributing to Wolbachia based vector control strategies.