Disturbance is one of the central concepts explaining how diversity arises and is perpetuated in ecological time. A good model system for testing hypotheses related to disturbance is the longleaf pine ecosystem in the southeastern U.S. because in this ecosystem frequent, low-severity fires acts as a disturbance that maintains a unique vegetation structure and high species richness. Vegetation structure influences animal distributions; in fire-dependent ecosystems many animals rely on open-structured, fire-maintained vegetation but shrubs and trees encroach into fire-dependent ecosystems where fire has been excluded. Prescribed burning and mechanical removal are commonly used as restoration tools to control encroachment. To better assess and compare the restoration potential of these tools, a more thorough understanding of how they change vegetation structure and habitat suitability for animals is necessary. The southeastern U.S. is a diversity hot-spot for amphibians, many of which require ephemeral wetlands embedded in longleaf pine uplands for the aquatic phase of their life cycle. Amphibian diversity has been declining in recent decades and habitat loss/degradation has been cited as one of the leading causes. Although often overlooked in studies of fire ecology, the ephemeral wetlands required by many amphibians are also fire-dependent habitats that have been negatively impacted by lack of fire. To understand how disturbance interacts with wetland vegetation and aquatic-phase amphibians, three disturbance treatments meant to mimic the effects of natural disturbance on vegetation structure were applied randomly to 28 dry ephemeral iii wetlands in the Lower Coastal Plain of South Carolina, U.S. The treatments consisted of early growing-season prescribed fire, mechanical vegetation removal (a proposed fire surrogate), and a combination of mechanical removal plus fire; some sites were left untreated for reference. Vegetation structure was quantified and amphibian assemblages were monitored before and after treatments. In addition, one species of amphibian was used in a tadpole survival experiment to examine differences in performance among treatments. Other factors that could be affected by treatments and in turn influence amphibians were measured, including water chemistry, wetland depth, quantity and quality of epilithon, and leaf litter composition. Amphibian survival was lowest, and species depauperateness highest in untreated wetlands. Depauperateness of species whose range was restricted to the range of longleaf pine was lowest in sites that had mechanical treatment plus fire. The mechanical plus fire treatment created the most open vegetation structure with lowest leaf litter accumulation, especially of hardwood litter, conditions correlated with high amphibian survival and diversity. When data from this study was combined with data from a previous study of similar nearby wetlands, a pattern emerged in which one suite of species was absent from recently burned sites, while an entirely different suite of species was absent from long-unburned sites. This evidence suggests that disturbance is related to a shift in amphibian assemblage possibly due to changes in vegetation structure and perhaps wetland ecology in general, from an algal-based system maintained by frequent fire to a detrital-based system that develops in the absence of fire

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Mosquitoes are considered to be the deadliest animals in the world due to the diseases they spread. Naturally, most research centers around anthropophilic disease vector mosquitoes such as Aedes aegypti or Anopheles gambiae. However, mosquitoes can feed on, and pose a threat to, more than humans. In fact, mosquitoes can specialize in feeding on a wide variety of hosts, including ectotherms such as amphibians and reptiles. Culex territans is a mosquito that feeds primarily on amphibians and reptiles and is known to transmit parasites to its hosts. However, little else is known about this mosquito. This work dives into the biology, physiology, and vector potential of this mosquito species to provide critical insights into understanding the evolution of feeding on endotherms as well as the importance of Cx. territans to amphibian health. The first study investigates activity patterns and feeding behaviors as well as the potential cues that may attract this mosquito to its hosts. We also compare transcriptomics and anatomy of the heads of three Culex species. The second study compares three Culex species' thermopreference and host-seeking to understand the effects of the differences of geographical distribution and preferred hosts. The last three studies determine the presence of ranaviruses, giant anuran trypanosomes, and the fungus Batrachochytrium dendrobatidis at Mountain Lake Biological Station and explore the potential role of Cx. territans in transmitting these pathogens. Overall, this work provides insights into the biology of Cx. territans and the implications it may have for understanding endotherm-feeding mosquitoes and amphibian disease epidemiology. / Doctor of Philosophy / We are all familiar with the annoying buzzing sound and itching bite of a mosquito. But mosquitoes are more than irritating: they are considered the deadliest animal in the world because of their ability to spread deadly diseases such as malaria and yellow fever to humans. However, not all mosquitoes bite humans. In fact, mosquitoes can feed on the blood of just about any animal, including worms. This work focuses on Culex territans, the northern frog-biting mosquito, which feeds mostly on the blood of amphibians, like frogs and toads. This fascinating mosquito species is present around the world, living in freshwater ponds alongside frogs, but we know very little about it. For example, how does a frog-biting mosquito find a frog host to bite? Once the mosquito bites, how is it able to feed on cold, thick blood through a tiny straw that makes up its mouthparts? While it is feeding, is it spreading diseases that could harm the frog? Because amphibians are experiencing extinctions worldwide, it is important to know if mosquitoes could be involved in their demise. These questions inspired this work to better understand the biology of Cx. territans and to shed light on the biology of other disease-spreading mosquitoes.

Chytridiomycosis is a fungal skin disease in amphibians that is primarily caused by Batrachochytrium dendrobatidis (Bd). We analyzed whole genome sequences of 40 bacterial isolates that had been previously cultured from the skin of four amphibian species from Virginia, USA, and tested for their ability to inhibit Bd growth via an in vitro challenge assay. These 40 isolates spanned 11 families and 13 genera. The aim of this study was to identify genomic differences among the amphibian skin bacterial isolates and generate hypotheses about possible differences that could contribute to variation in their ability to inhibit the growth of Bd. We identified sixty-five gene families that were present in all 40 isolates. We also looked for the presence of biosynthetic gene clusters. While this set of isolates contained a wide variety of biosynthetic gene clusters, the two most abundant clusters with potential anti-fungal activity were siderophores (N=17) and Type III polyketide synthases (N=20). We then analyzed the isolates belonging to the phylum Proteobacteria in more detail. We identified 197 gene families that were present in all 22 Proteobacteria. We examined various subsets of the Proteobacteria for genes for specific compounds with known activity against fungi, including chitinase and violacein. We identified a difference in the number, as well as amino acid sequences, of predicted chitinases found in two isolates belonging to the genus Agrobacterium that varied in their inhibition of Bd. After examining the annotated genomes, we identified a predicted chitinase in a Sphingomonas isolate that inhibited the growth of Bd that was absent from the five Sphingomonas isolates that did not inhibit Bd growth. The genes vioA, vioB, vioC, vioD and vioE are necessary to produce violacein, a compound which inhibits the growth of Bd. Differences in these genes were identified in three out of the four Janthinobacterium isolates. Of these three isolates, two showed strong inhibition of Bd growth, while the third inhibited Bd growth to a lesser extent. Using comparative genomics, we generated several testable hypotheses about differences among bacterial isolates that could contribute to variation in ability to inhibit Bd growth. Further work is necessary to test the various mechanisms utilized by amphibian skin bacterial isolates to inhibit Bd. / Master of Science / Many amphibian population declines around the world have been caused by chytridiomycosis, a skin disease. This disease is caused by the fungus Batrachochytrium dendrobatidis (Bd). The skin of amphibians is also home to many bacteria that can provide important functions for the amphibian host, like preventing infection by Bd. To understand how these bacteria might provide protection, we examined the entire genomes of 40 bacterial isolates that reside on the skin of four amphibian species from Virginia, USA. These bacteria were previously tested for their ability to prevent Bd growth and 40 of them were chosen for sequencing based on selecting closely related isolates that varied in their ability to inhibit Bd growth. This allowed us to compare their genomes and generate hypotheses about possible genomic differences that could contribute to the variation in Bd growth inhibition. We identified sixty-five gene families that were present in all 40 bacteria. We also looked for sets of genes (biosynthetic gene clusters) that are known to produce secondary metabolites, which are compounds that can include antifungals. The two most abundant clusters we identified that had the potential to produce compounds that inhibit fungal growth were siderophores and Type III polyketide synthases. We then looked for genes that were not part of biosynthetic gene clusters that could produce specific compounds that can inhibit Bd growth, such as chitinase and violacein. We found variation in chitinase genes in several isolates that seemed to be associated with the ability to inhibit Bd growth. In addition, there were some differences in violacein genes that should be examined more in future studies. Overall, we suggest that using comparative genomic approaches can be valuable for identifying key bacterial functions in the microbiome.

Mercury (Hg) is an important environmental contaminant due to its global distribution, tendency to bioaccumulate, and toxicity to wildlife. However, Hg has received little attention in amphibians compared to other vertebrates, despite the fact that amphibian population declines have been documented worldwide and environmental contaminants are believed to contribute to some declines. During my dissertation research, I used a pluralistic approach which combined field studies and manipulative laboratory and mesocosm experiments to examine the bioaccumulation and ecological effects of environmentally relevant Hg exposure routes acting at various early life stages in amphibians. By collecting amphibians in the field at the Hg-contaminated South River, VA, I confirmed that amphibians exhibiting different life histories and occupying different ecological niches (Plethodon cinereus, Eurycea bislineata, and Bufo americanus) can bioaccumulate sufficient levels of Hg to warrant concern (Chapter 2) and female Bufo americanus transfer accumulated Hg to their eggs (Chapter 3). Maternal transfer of contaminants is a parental effect which typically has negative consequences for offspring because early development is a critical organizational period in the ontogeny of vertebrates. Through laboratory observations and mesocosm experiments, I examined the short and long-term effects of maternal contaminant exposure on offspring, and found the negative effects of maternal Hg exposure manifested either immediately at the embryonic stage or later during the larval stage, depending on the year in which the study was conducted (Chapters 4 and 5). Lastly, using a factorial laboratory experiment, I examined whether the latent effects of maternal transfer of contaminants manifests differently depending on the environment in which offspring develop, and found both maternal and dietary Hg exposure independently produced negative, but different, sublethal effects on larval development. Most importantly, maternal exposure to Hg combined with high dietary Hg exposure later in ontogeny had a lethal effect in larvae (Chapter 6). This study is one of the first to demonstrate that the latent effects of maternally transferred contaminants may be exacerbated by further exposure later in ontogeny, findings that may have important implications for both wildlife and human health. / Ph. D.

As part of the ongoing loss of global biodiversity, amphibian populations are experiencing declines and extinctions. A primary factor in these declines is the skin disease chytridiomycosis, which is caused by the fungus Batrachochytrium dendrobatidis (Bd). Recent research suggests that the amphibian skin microbiota has anti-Bd activity and may be an important factor in host disease resistance. However, little is known about the basic ecology of this host-microbe symbiosis, such as how much variation there is in microbial symbionts among host species and populations, and the nature of symbiont transmission, culturability, and function. My dissertation research addressed these basic questions in microbial ecology, as well as used a novel system to examine the long-standing ecological theory of community structure-function relationships. First, host-specificity, population-level variation and potential environmental transmission of the microbiota were examined by conducting a field survey of bacterial communities from bullfrogs, newts, pond water, and pond substrate at a single pond, and newts from multiple ponds. There was variation among amphibian host species and populations in their skin symbionts, and, in a host species-specific manner, amphibian skin may select for microbes that are generally in low abundance in the environment. Second, the culturability of amphibian skin bacteria was assessed by directly comparing culture-dependent and -independent bacterial sequences from the same individuals. Although less than 7% of the amphibian skin microbes were captured using R2A medium, most of the dominant bacteria were represented in our cultures, and similar patterns of diversity among four amphibian species were captured with both approaches. Third, the relationship between microbial community structure and function and selective forces shaping structure and function were examined in bullfrogs by tracking microbial community structure and function following experimental manipulation of the skin microbiota and pathogen exposure. Results of this study demonstrated that Bd is a selective force on cutaneous bacterial community structure and function, and suggest that beneficial states of bacterial structure and function may serve to limit infection and negative fitness consequences of Bd exposure. Using a combination of observational and experimental approaches, my dissertation contributes to understanding structure-function relationships of these complex symbiotic communities of vertebrates. / Ph. D.

Artificial shelters show great promise as novel, non-invasive tools for studying hellbenders, but their use thus far has faced several challenges. During initial trials in multiple river networks, artificial shelters routinely became blocked by sediment and dislodged during high stream discharge events, and were rarely used by hellbenders. We sought to determine whether these complications could be overcome via alternative shelter design, placement, and maintenance. Between 2013 and 2018, we deployed 438 artificial shelters of two different designs across ten stream reaches and three rivers in the upper Tennessee River Basin. We assessed evidence for several hypotheses, postulating broadly that the availability, stability, and use of artificial shelters by hellbenders would depend on how shelters were constructed, deployed, and/or maintained. We found that maintaining shelters at least once every 40 days limited sediment blockage, and building ~ 40 kg shelters with 3-4 cm thick walls and recessed lids improved their stability during high discharge events. Additionally, we found that hellbenders most frequently occupied and nested in artificial shelters when they were deployed in deeper (~50+ cm) portions of reaches with high adult hellbender densities. Our results suggest that artificial shelters can serve as effective tools for studying hellbenders when designed, deployed, and maintained with these advancements, but also highlight some limitations of their use. / Master of Science / Hellbenders are large, fully-aquatic salamanders that live primarily in cool, rocky, swift-flowing streams in portions of Appalachia and the lower Midwest. They are imperiled across most of their native range due to human-caused habitat degradation, but their declines, conservation needs, and population status have historically been difficult to study due to the fact that they spend the majority of their lives beneath large, often inaccessible boulders. While these boulders are sometimes possible to lift, doing so can disturb critical hellbender habitat. Therefore, alternate, less invasive hellbender sampling methods are necessary in order to improve knowledge about their conservation status and needs. Artificial shelters, which are large, hollow, concrete structures that mimic natural boulder crevices and feature removable lids, show promise as a novel, innovative tool for non-invasively studying hellbenders. However, initial trials of these shelters have yielded mixed results, with shelters often becoming swept away and destroyed during floods, becoming blocked by sand and sediment and thus inaccessible to hellbenders, or simply not being used by hellbenders when accessible. We sought to determine whether these complications could be overcome by optimizing the way that shelters were constructed, deployed, and maintained in streams inhabited by hellbenders. Between 2013 and 2018, we deployed 438 artificial shelters of two different designs across ten stream reaches and three rivers in the upper Tennessee River Basin. Using multiple analyses, we tested one broad overall hypothesis: that the efficacy of using artificial shelters to study hellbenders would depend on how they were constructed, how frequently they were maintained, and where they were placed in the stream. We found that maintaining shelters at least once every 40 days limited sediment blockage, and building ~ 40 kg shelters with 3-4 cm thick walls and recessed lids improved their stability during flood events. Additionally, we found that hellbenders most frequently occupied and nested in artificial shelters when they were deployed in deeper (~50+ cm) portions of reaches with high adult hellbender densities. Our results suggest that artificial shelters are effective tools for studying hellbenders when designed optimally, maintained frequently enough, and placed in appropriate locations. However, exceptions to these findings may exist in certain heavily degraded stream reaches.

The global amphibian population is rapidly declining. Although many threats that affect amphibians are known, there are many contributing factors which are not fully understood. For adult amphibians, the largest part of the mortality comes from predation. We know of many predators which prey upon amphibians, but to what extent they affect the populations has not been subject to much investigation. Neither has the effects of specific taxa or species of predators. Birds are reputed amphibian-eaters, and they often occur in the same habitats. Both birds and amphibians often select for wetlands and other water bodies with high biodiversity, that often support a high diversity and/or density of birds as well as amphibians. The fact that a prey might flourish in an area that one of its strongest predators also inhabits might seem controversial. Especially when taking into account that it is often the density of predators that best explains the population growth of prey species. In this review, I investigate what effects large amounts of birds might have on amphibian populations as a result of their predation on adults. I consider how the effects might differ with high vs low densities of both prey and predator, and whether an eventual effect might impact the survival of a metapopulation differently depending on its size and spatial distribution. As an example of a habitat where both bird and amphibian species occur in large numbers, I use Trönninge ängar - a bird conservation area just outside the city of Halmstad, where populations of both amphibians and birds have been increasing over the last years. My results show that high densities of predatory birds in such communities could potentially cause declines in amphibian populations, but that this impact is softened when the population is more widely distributed in the area. There are also possibilities that birds do not only affect amphibians by predation, but also might facilitate their distribution by reducing other predators. Thus, bird predation in itself might not be a sufficient predictor for their effect on amphibian populations, as the relationship might be more complex than simple predator-prey interactions.