The relationship between plants and rhizosphere bacteria collected from a 45 year old permanent pasture was investigated. Several methods of strain identification within Rhizobium trifolii were evaluated. Separation of bacterial isolates based on differences in intrinsic antibiotic resistance was not appropriate because strains developed hybrid resistance patterns when grown in a common broth. Serological analyses of bacterial antigens using polyclonal antiserum yielded two reliable methods for identifying R. trifolii isolates. Agglutination and immunofluorescence procedures were not useful in distinguishing these strains but immunodiffusion and the enzyme-linked immunosorbent assay (ELISA) were highly suitable. Adaptation of the ELISA allowed isolates to be identified directly from individual root nodules without first subculturing the bacteria. A strain of Bacillus polymyxa isolated from the same pasture was shown to stimulate growth of crested wheatgrass (Agropyron cristatum L.) and white clover (Trifolium repens L.). The primary manifestation of the effect was increased root weight (P < 0.05), but shoot responses were also observed. Perennial ryegrass (Lolium perenne L.) generally reacted negatively to inoculation with this bacterium. Further stimulation of growth was noted when ramets of the white clover genotype homologous to (sharing a common origin) B. polymyxa were inoculated in pure stands (P < 0.05). Clones of the homologous perennial ryegrass genotype also showed a yield increase from slightly below control levels to slightly above them when tested in a similar manner. Detailed analysis of the crested wheatgrass response to inoculation revealed that bacterial production of indole acetic acid was the most likely cause of the growth stimulation. Other bacterial characteristics such as the ability to fix atmospheric nitrogen or to solubilize organic phosphorus were concluded to be unrelated to the growth response. Co-adaptive compatibility between genotypes of L. perenne and T. repens was not apparent when the effect of R. trifolii was ignored. However, when clones of pasture plants that had been neighbours in the field were inoculated with R. trifolii isolated from root nodules of the "parental" clover genotype, biotic specialization between the pasture plants became evident. The magnitude of the effect, which was characterized by superior white clover yields (P < 0.05), could be largely accounted for by the presence of the adapted L. perenne/R. trifolii combinations, regardless of the white clover genotype. Since T. repens was the dominant component in the species mixture, these trends were also apparent when total forage biomass was analyzed (P < 0.05). However, ecological combining ability was found to be lowest in these associations (P < 0.05). Similar experimentation with isolates of B. polymyxa (or B. polymyxa-like organisms) was performed. Again the grass/bacteria combination was shown to be influential in the growth response as the presence of homologous L. perenne/B. polymyxa combinations resulted in superior white clover and perennial ryegrass performance (P < 0.05). When T. repens was inoculated with a mixture of R. trifolii strains, unrelated isolates formed more root nodules than did homologous ones (P < 0.05). The presence of perennial ryegrass did not mitigate this effect. However, when homologous R. trifolii was administered as a single strain inoculum, yield advantages in white clover were observed (P < 0.05). If B. pol ymyxa was present, homologous strains of R. trifolii tended to form most of the root nodules regardless of the T. repens or L. perenne genotypes. The significance of the yield advantages observed in various two and three-way plant/microbe genotype combinations is discussed with respect to above ground plant performance. / Land and Food Systems, Faculty of / Graduate

Coral reefs are in global decline due to ocean warming and ocean acidification. While these stressors are commonly studied in climate change predictions, salinity, although being an important environmental factor, is not well understood. The response of the coral holobiont (the association of the coral host, its algal endosymbiont and a suit of other microbes) to changes in salinity and the contribution of each holobiont compartment underlying the necessary osmoadaptation remain especially elusive. Interestingly, we find some of the most thermotolerant corals in some of the most saline seas, e.g. the Red Sea and the Persian Arabian Gulf. This observation sparked the hypothesis of a link between osmoadaptation and coral thermotolerance. Here, we set out to elucidate the putative effects of high salinity on conveying thermotolerance and thereby a possible link to bleaching in the context of the coral holobiont. For this, we conducted a series of heat stress experiments at different salinities in the coral model Aiptasia and subsequently validated our findings in corals from the central Red Sea. We confirm a role of osmoadaptation in increased thermotolerance and reduced bleaching in Aiptasia and Red Sea corals. This salinity-conveyed thermotolerance was characterized by a reduction in algal endosymbiont loss, photosystem damage and leakage of damaging reactive oxygen species (ROS) in high salinity. Further analysis of the osmoadaptation response using targeted GC-MS uncovered high levels of the sugar floridoside at high salinity only in holobionts that show the salinity-conveyed thermotolerance. The increase of floridoside, an osmolyte capable of scavenging ROS, and the concurrent reduction of ROS argues for a mechanistic link of increased thermotolerance and reduced bleaching in high salinities. In addition, the restructuring of the microbiome at high salinity that aligned with the difference in thermotolerance in Aiptasia may be indicative of a microbial contribution towards a more beneficial holobiont composition. Hence, emphasizing the potential cumulative contribution of each holobiont compartment during stress-resilience, as well as highlighting the overall role of osmoadaptation in the thermotolerance of corals.

Biodiversity of coral reefs is higher than in any other marine ecosystem, and significant research has focused on studying coral taxonomy, physiology, ecology, and coral-associated fauna. Yet little is known about symbiotic copepods, abundant and numerous microscopic crustaceans inhabiting almost every living coral colony. In this thesis, I investigate the genetic diversity of different groups of copepods associated with reef-building corals in distinct parts of the Indo-Pacific; determine species boundaries; and reveal patterns of biogeography, endemism, and host-specificity in these symbiotic systems. A non-destructive method of DNA extraction allowed me to use an integrated approach to conduct a diversity assessment of different groups of copepods and to determine species boundaries using molecular and taxonomical methods. Overall, for this thesis, I processed and analyzed 1850 copepod specimens, representing 269 MOTUs collected from 125 colonies of 43 species of scleractinian corals from 11 locations in the Indo-Pacific. The genetic assessment of the most abundant copepod morphotypes associated with hermatypic corals in Lizard Island (Great Barrier Reef) revealed a large number of species previously unknown for this region. Analyses of diversity and patterns of biogeographical distribution of copepods associated with Galaxea corals throughout the Indo-Pacific showed that the species diversity of this group is high and appears to be regionally specific, an uncommon pattern in most coral reef-associated invertebrates. Results for the symbiotic copepod fauna of Red Sea pocilloporid corals, a family of corals with a high level of morphological variability within and among its members, showed that the majority of the discovered poecilostomatoid copepods belong to the genus Spaniomolgus, which demonstrated a significant genetic diversity of morphologically-similar species. Assessment of the diversity of copepods associated with the Red Sea mushroom corals revealed several undescribed species and showed no evidence of specificity to the hosts neither on species nor on the family level, which contradicts a modern assumption of high host-specificity of copepods. Overall, this dissertation is a first study of genetic diversity of copepods associated with invertebrates, and it provides substantial insight into the diversity of coral-associated microcrustaceans and insight to patterns of their host-specificity as well as distribution around the Indo-Pacific.

The symbiotic relationship between cnidarians and their photosynthetic dinoflagellate symbionts underpins the success of coral reef communities in oligotrophic, tropical seas. Despite several decades of study, the cellular and molecular mechanisms that regulate the symbiotic relationship between the dinoflagellate algae and the coral hosts are still not clear. One of the hypotheses on the metabolic interactions between the host and the symbiont suggests that ammonium assimilation by the host can be the underlying mechanism of this endosymbiosis regulation. An essential intermediate of the ammonium assimilation pathway is glutamate, which is also known for its glutamatergic signaling function. Interestingly, recent transcriptomic level and DNA methylation studies on sea anemone Aiptasia showed differences in metabotropic glutamate signaling components when comparing symbiotic and non-symbiotic animals. The changes in this process on transcriptional and epigenetic levels indicate the importance of glutamate signaling in regard to cnidarian symbiosis. In this study, I tested glutamatergic signaling effect on symbiosis in sea anemone Aiptasia using a broad-spectrum glutamate receptor inhibitor 7- CKA and glutamate. Significantly decreased cell density was observed in animals with inhibitor treatment suggesting a possible correlation between glutamate signaling and the establishment or maintenance of symbiosis. Using RNA-Seq, I was able to obtain transcriptional profiles of the animals under inhibitor and glutamate treatment. Differential gene expression and gene ontology analyses indicated changes in amino acid metabolism, lipid metabolism and such signaling pathways as MAPK, NF-kappa B and phospholipase C. Although amino acid and lipid metabolism could be a result of the reduced symbiotic state of inhibitor treated Aiptasia, the signaling pathways which are related to apoptosis and immune response provide an exciting venue for direct regulatory interaction between symbiosis and glutamatergic signaling. However, as these signaling pathways mainly act via signal transduction through protein phosphorylation, further studies looking at changes on a post-translational level might provide further insight into the mechanisms underlying the observed phenotype.

No description available.

Local factors have the potential to generate genetic structure within species if populations respond differently to varying environmental conditions across their geographic range. In this project, spatial genetic structure was examined in the legume, Chamaecrista fasciculata, its symbiotic nitrogen fixing bacteria, and rhizosphere microbiomes. In the first chapter, the aim was to test for genetic structure among populations of C. fasciculata in the Southeast and to evaluate whether phenotypic variation in leaf pubescence is associated with genetic divergence among populations, which would be consistent with local adaptation. My results did not detect a significant association between genetic structure and phenotypic variation in leaf pubescence, but the role of environmental variables in generating the observed patterns of spatial genetic variation in C. fasciculata was demonstrated. In the second project, I analyzed genetic structure within a single population to test for the presence of fine-scale genetic structure of the host plant and to determine if genetic structure of symbiotic nitrogenixing rhizobia is influenced by host plants. Neighboring plants are expected to be more genetically similar than distant plants. If this expectation is supported and genotype x genotype interactions are important in this system, then I anticipated that spatially close host plants would show more genetically similar microbiota in their nodules and rhizospheric soil than distant host plants. The results indicated fine-scale genetic structure for both host plants and nodulating rhizobia, suggesting that the both organisms are influenced by similar mechanisms structuring genetic diversity or shared habitat preferences. In the third project, I characterized fine-scale structure and diversity of microbial communities of the rhizosphere of host plants within a single population. The results revealed significant differences in bacterial and fungal communities among host plants and a significant association between genetic distance of both microbial communities and spatial and plant genetic distances. These data confirm the importance of plant genotype and physical distance in shaping the genetic structure and diversity of bacterial and fungal communities of the rhizosphere. Overall, this study enhances our understanding of the degree to which intraspecific genetic variation in plants influences the diversity and structure of soil microbial communities.

Termites are known to harbor within their gut a diverse assemblage of symbiotic microorganisms. Little work has been done, however, to describe the diversity and function of the bacteria in the economically important eastern subterranean termite, Reticulitermes flavipes. The first object of this study was to characterize the bacterial diversity in the gut of R. flavipes using amplified rDNA restriction analysis (ARDRA) and 16S rRNA gene sequencing. It was determined that ARDRA was an effective technique for characterizing the diversity of the termite gut microbiota. Of the 512 clones analyzed in the ARDRA study, 261 different ARDRA profiles were found. Forty-two 16S rRNA gene sequences were also analyzed, resulting in 33 different ribotypes. Representatives from six major bacterial phyla, Proteobacteria, Spirochaetes, Bacteroidetes, Firmicutes, Actinobacteria, and the newly proposed "Endomicrobia," were discovered. Further analysis indicated that the gut of R. flavipes may harbor as many as 1,318 ribotypes per termite. The second objective was to determine if the gut bacterial diversity could be manipulated by changing the termite's food source. Using ARDRA analysis, I found no evidence that changing the food source affected the termite gut bacterial diversity. In addition, changing the food source did not induce aggression in nestmates fed on different food sources. The third objective was to search for patterns of coevolution between termites and their gut symbiotic bacteria. Using rRNA gene sequences from this study and sequences from public databases (1,450 sequences total), a neighbor-joining tree demonstrated strong evidence for coevolution of termites and their symbiotic bacteria, especially in the phyla Bacteroidetes, Actinobacteria, Spirochaetes, and "Endomicrobia." Many monophyletic clusters were entirely composed of phylotypes specific to Isoptera. / Ph. D.

Crayfish throughout the holarctic are found in association with an order of worms known as branchiobdellidans. This relationship has been confirmed as a cleaning symbiosis in several species. The Conhaway crayfish,​ Cambarus appalachiensis, is a species of crayfish endemic to the New River Basin in Virginia and West Virginia. We studied a population of ​ C. appalachiensis​ in Sinking Creek in Newport, VA from March 2017 until February 2018. We collected morphological data and quantified the branchiobdellidan communities on 986 individuals, and kept note of egg brooding and young of year throughout the study period. The life cycle of C. appalachiensis was found to be similar to other large-bodied species of Cambarus crayfish. Molting occurred throughout the year, peaking in the months of April and September. This molting served as a disturbance effect to the symbiotic branchiobdellidan community and reset community assembly. The worm communities on larger, recently molted crayfish more closely resembled the less diverse communities on smaller crayfish. Most worms on recently molted crayfish were ones that we know are early colonizers. This thesis work provides the first life history information on a newly described species of Cambarus crayfish and provides both seasonal data on its branchiobdellidan associates and one of the first empirical examples of host ontogeny acting as a disturbance on a symbiotic community. / Master of Science / Crayfish throughout North America and Eurasia are the symbiotic partners to a number of small worms. This relationship has been confirmed as a cleaning symbiosis for several crayfish, similar to the cleaning stations at a coral reef. The Conhaway crayfish is a species of crayfish found in the New River Basin in Virginia and West Virginia. We studied a population of Conhaway crayfish in Sinking Creek in Newport, VA from March 2017 until February 2018. We collected data on the physical attributes of the crayfish, quantified the worms present on 986 individuals, and kept note of female crayfish with eggs and baby crayfish throughout the study period. The life cycle of the Conhaway crayfish was found to be similar to other large-bodied species of closely related crayfish. Molting, where the crayfish loses its shell and grows a new one, occurred throughout the year, peaking in the months of April and September. This molting served as a disturbance effect to the symbiotic worms, similar to how a wildfire might displace animals in a grassland ecosystem. The type and number of worms found on larger, recently molted crayfish more closely resemble the type and number of worms found on smaller crayfish. This thesis work provides the first information on the physical and reproductive attributes of a newly described species of crayfish and provides both seasonal data on its symbiotic worms and one of the first empirical examples of host growth and aging acting as a disturbance to symbiotic organisms living on that host.

Amphibian populations have undergone unprecedented declines in recent decades. Many of these declines are due to the spread of the cutaneous fungal pathogen Batrachochytrium dendrobatidis (Bd), which causes the disease chytridiomycosis. The Panamanian golden frog (Atelopus zeteki) has not been seen in the wild since Bd spread through western Panama. In response to initial declines, golden frogs were collected from wild populations and placed in captive colonies with the goal of future reintroductions. An understanding of this species' natural defense mechanisms against Bd is needed for reintroduction to be successful. Previous studies indicate that cutaneous bacteria are an important defense mechanism for some amphibians and applying antifungal bacteria to the skin of Bd-susceptible amphibians (probiotic therapy) can prevent chytridiomycosis. Therefore, the goals of my dissertation were to characterize the bacterial community of A. zeteki and determine if probiotic therapy could be used to prevent chytridiomycosis in this species. I initially characterized the bacterial community of wild and captive golden frogs using samples collected prior to the initial declines and after approximately eight years in captivity. I found that the community structure of the microbiota was significantly different between wild and captive frogs; however, the offspring of the original captive frogs still shared 70% of their microbial community with wild frogs. Then, I characterized the Bd-inhibitory properties of 484 bacteria isolated from 11 species of free-living Panamanian amphibians. I found a large proportion of bacteria (75.2%) had the ability to inhibit Bd and this trait was widely distributed among bacterial taxa, although there was also significant variation within bacterial genera in their ability to inhibit Bd growth. I then experimentally tested the ability of four of these isolates to prevent chytridiomycosis in captive golden frogs. None of them successfully prevented infection; however, there were several frogs that cleared infection and this was correlated with composition of the bacteria initially present on their skin. Overall these results demonstrate that the structure of microbial communities of A. zeteki are important to host health and building on this might provide the best hope for reintroducing this iconic species back to its native habitat. / Ph. D.

Annually, it is estimated that 82 million tons of global plastic waste is either mismanaged or littered, bypassing waste management practices. This mismanagement causes the permeation of plastic debris into the environment, which then undergoes natural degradation processes. These degradation processes result in the proliferation of miniscule plastic particles known as microplastics. Due to the inherent proximity to sources of anthropogenic waste, concerns of microplastic pollution and its impact on freshwater ecosystems have recently increased. Until recently, microplastic research has primarily been focused on the toxicological affects felt by an individual organism rather than the intricate interactions that occur between taxa. Only focusing on the individual toxicological impact turns a blind eye on the communities that maintain ecosystem health and stability. To that end, our experiment was unique as it will be the first study assessing the impact of a freshwater symbiosis, as symbioses in the scope of toxicokinetic studies have primarily been dominated by that of terrestrial and marine relationships. This knowledge gap is a serious concern as its argued freshwater systems are more contaminated, than that of other aquatic habitats. To address this knowledge gap, we conducted mesocosm-based exposure-response assays, exposing the crayfish-branchiobdellidan symbiosis to microplastics of fibrous, microsphere, and tire wear particle morphologies while varying symbiont densities. We used the crayfish-branchiobdellidan model system in our study due to its amenability to laboratory monitoring and manipulation. The crayfish C. appalachiensis, common in the Virginia New River Basin, served as hosts to obligate ectosymbiotic annelids in the order Branchiobdellida. Previous research, using the crayfish-branchiobdellidan symbiosis demonstrated that the interaction is a cleaning symbiosis, where hosts benefit from reduced gill fouling while symbionts benefit from increased resource availability. We observed the physical and behavioral changes of the crayfish-branchiobdellidan symbiosis over a 172-day chronic exposure assay. Our results show, crayfish hosts with higher symbiont densities experienced decreased physical growth when exposed to microplastics compared to the control. This alteration in host growth was the result of increased antagonistic symbiont behavior in the form of gill tissue consumption. Our results suggest microplastics caused a reduction in epibiont abundance, thus decreasing symbiotic resource availability. This reduction in resources resulted in a shift of context dependency, thus increasing parasitic symbiont behavior. This study demonstrates microplastics have the capability to shift symbiotic context from a mutualism to a parasitism. / Master of Science / Every year, due to improper waste management, approximately 82 million tons of global plastic waste ends up in the environment. Once in the environment, this plastic waste naturally breaks down into tiny particles known as microplastics. Recently, there's been growing concern about how these microplastics affect freshwater habitats. One particular worry is the impact that this contaminant can have on the relationships between different organisms living in freshwater environments. Currently, multiple scientific experiments have studied the effects of microplastics on marine and land-based organismal relationships, however, none have studied the effect of microplastics on freshwater organismal relationships. To explore this, we used the relationship of crayfish and a type of worm called Branchiobdellida, due to their close, prolonged relationship commonly found in nature. These Branchiobdellida worms live on the outside of crayfish and keep the gills of the crayfish clean by consuming any matter that has grown or lodged itself on the gill filaments. As such, the worms rely on the crayfish as a home and means of collecting food. Depending on food availability, this relationship can be mutually beneficial for both the crayfish and worm. Due to the worm's reliance of the crayfish and the mutually beneficial relationship they facilitate, the crayfish-branchiobdellidan relationship is known as a symbiosis. To best understand how microplastics affect the crayfish-branchiobdellidan symbiosis, we conducted experiments where we exposed crayfish with varying numbers of worms to a controlled dosage of microplastics for a prolonged period of time within artificial environments called mesocosms. Each microplastic dosage contained different types of microplastics, that mimicked the type and amount commonly found in nature. We found that crayfish with higher worm densities grew less when exposed to microplastics compared to those not exposed to microplastics. We also noticed that when microplastics were present, the worms exhibited more aggressive behavior, like consuming the gills of the crayfish. Based on assays which measured the number of resources accessible to the symbionts, our findings suggest that microplastics reduce the resource availability to the worms, which then triggers a change in their relationship with the crayfish host. Instead of being helpful, the worms start behaving more like parasites when exposed to microplastic. Ultimately, this study shows that microplastics can change the way different organisms interact, turning a beneficial symbiosis into a harmful one.