Amphibian diversity has declined, in part, due to the infectious disease chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd). Andean water frogs in the genus Telmatobius are particularly vulnerable to the disease and the genus has been extirpated from Ecuador and in Andean cloud forests, yet populations of species persist in the high Andes of Peru and Bolivia. The Alpaca Water Frog (Telmatobius intermedius), endemic to the Peruvian Andes, can be found infected with Bd. Alpaca Water Frogs inhabit high elevation open canopy freshwater systems. My overall goal was to study the effect of chytrid infection on the trophic ecology of Telmatobius tadpoles. I used stable isotope ratios of carbon (δ13C) and nitrogen (δ15N) to characterize the the trophic structure and energy flow in this system. I observed the values of δ15N were higher for tadpoles than algal material (t-test, t= -8.60, df= 34, p< 0.01), mayfly nymphs (t-test, t= 5.25, df= 30, p< 0.01), and predatory aquatic invertebrates (t-test, t= -4.18, df= 47, p< 0.01). In regard to the δ15N values of tadpoles and frogs, tadpoles had a lower value (t-test, t= -3.0, df= 31, p< 0.01). Values of δ15N in tadpoles were relatively high, signaling the presence of animal tissue in their diet. I also investigated changes in tadpole diet associated with mouthpart deformities caused by the fungal pathogen Bd. There was a positive association between the extent of mouthpart deformity and Bd infection (Fisher’s Exact test, p<0.001). The relative proportions of diatom morphotaxa groups found in the foregut of T. intermedius tadpoles varied in association with the degree of mouthpart deformity, as indicated by an ANOSIM analysis (R=0.875, p<0.001). Finally, in addition to Bd prevalence in adult aquatic frogs, I investigated whether Alpaca Water Frogs and other Andean Water Frogs tested positive for the antifungal bacterium Janthinobacterium lividum (Jliv). My results show that 57% of the sampled frogs were infected with Bd, 12.5% of frogs hosted both Jliv and Bd, while 7.2% hosted just Jliv. We found that the probability of an individual being infected with Bd was independent of the presence of Jliv; however, we did detect a protective effect of Jliv with respect to intensity of infection. My findings demonstrate that the fungal pathogen Bd influences the trophic ecology of tadpoles of Andean water frogs.

Communities are a critical link that impact how species-level population dynamics translate into ecosystem functions, and thus, understanding community assembly is an important goal of ecology. Variation in the relative importance of the four processes of drift, selection, speciation, and dispersal likely govern much of the variation that is observed in community structure across landscapes. Microbial communities provide critical functions across an array of environments, but only recently have technological advances in DNA sequencing allowed us to study these communities with higher resolution. My dissertation research has investigated community assembly in host-associated microbial communities, with a focus on understanding how stochasticity in dispersal that leads to priority affects can impact bacterial community assembly in amphibian embryos. In chapter 1, I experimentally show that priority effects resulting from stochastic dispersal can be observed in the microbiome of newly-hatched hourglass treefrog (Dendropsophus ebraccatus) tadpoles. Changes in microbiome composition due to priority effects could be observed in a simple two bacteria system and when the inoculation by the initial bacteria is followed by a more diverse community inoculum. Outcomes of my two taxa system in co-culture do not strictly mirror those observed in treefrog embryos, highlighting that priority effect outcomes are context dependent. Additionally, these results provide support that priority effects do not benefit all bacterial species equally and the magnitude of these effects will be dependent on the traits of individual colonists. In chapter 2 I demonstrate that priority effects are not unique to the hourglass treefrog system but can be observed in spring peeper (Pseudacris crucifer) tadpoles as well. This study demonstrates the applicability of priority effects in increasing the abundance of target probiotic taxa; a benefit to amphibian populations facing threats by a lethal fungal pathogen. By treating embryos with a priority inoculation of Janthinobacterium lividum, a bacterial species known to inhibit fungal pathogen growth, I increased the relative abundance of J. lividum on newly hatched tadpoles. I also provide evidence that closely-related species of bacteria can effectively co-exist regardless of priority inoculation. An understanding of variation in the amphibian microbiome across life stages in the wild is required to better understand the long-term impacts of priority effects in embryos. My final chapter, therefore, examined compositional changes in the microbiomes of locally occurring amphibians in Virginia across the egg, tadpole, and juvenile developmental stages. In this study, I show characterize the initial egg microbiome across amphibian species and demonstrate that egg microbiomes, are distinct between species but are more similar across species than tadpole or juvenile microbiomes. Additionally, I show that minor differences in host environment can lead to differences in the microbiome structure of conspecific tadpoles. Overall, my dissertation empirically demonstrates the role of dispersal, and more specifically priority effects, in the assembly of the vertebrate microbiome. / Doctor of Philosophy / An ecological community is a set of species that occur at a given site. Communities have been a fundamental focus of ecological research, as communities serve to link the population dynamics of individual species to ecosystem level processes provided by species. Microbial communities, in particular, are of interest because of the wide range of important functions they provide across a variety of systems, yet relatively little is known about how these communities initially come together and are maintained. This is particularly true for the microbial communities that live in and on plants and animals, which are called "host-associated" communities. Host-associated microbial communities contribute many important functions to their hosts, including guiding host development, assisting with nutrient assimilation, and providing disease resistance. Four processes are thought to govern how ecological communities assemble across landscapes at local sites or habitat patches: selection, dispersal, speciation, and drift. Variation in the relative importance of these processes is thought to drive the variation in community composition across sites, or in the case of host-associated microbial communities, across hosts. Selection occurs at a local level when environmental variables or the presence of other species impact where a species occurs. Dispersal of individuals among habitat patches can also impact what species occur at a local site, and speciation gives rise to new species in communities over time. Drift is the stochastic, or random, element of species abundance that is driven by variation in the birth and death rates of a population at a site. I have investigated the assembly of host-associated microbial communities using amphibians as a study system. In chapter 1, I experimentally demonstrate that stochasticity in dispersal that impacts which species arrive first to a site (priority effects) can be observed in the host-associated bacterial communities of newly-hatched treefrog (Dendropsophus ebraccatus) tadpoles. This can be observed in a simplified system where only two bacterial species are used, and also when a single bacterial species arrives and is followed by a more diverse community of bacteria. However, not every bacterial species is able to take advantage of priority, and these results seem to be context dependent, as the outcomes in treefrog embryos do not exactly mirror the outcomes when the bacteria are grown in a nutrient broth together. In chapter 2, I show that priority effects are not unique to the hourglass treefrog system; priority effects can also be observed in spring peeper (Pseudacris crucifer) tadpoles. In this study, I also demonstrated that we may be able to apply our knowledge of priority effects to benefit amphibian populations threatened by a potentially lethal fungal pathogen by manipulating the abundances of bacteria on the skin during development. Priority treatment of embryos with Janthinobacterium lividum, a bacterial species known for its ability to inhibit growth of this fungal pathogen, resulted in increased relative abundance of J. lividum in the tadpoles following hatching. Additionally, I found that even closely-related bacterial species can have differing abilities to take advantage of priority effects and can co-exist on tadpoles. To determine long-term impacts of priority effects in embryos requires an understanding of the variation associated with amphibians in the wild across different life stages. My final chapter, therefore, focused on examining changes in the bacterial communities associated with locally occurring amphibians in Virginia across the egg, tadpole, and juvenile stages of development. Specifically, I characterize the initial communities associated with eggs across different species, including predicted associations with algal symbionts, and examine patterns of host-associated communities among species and across development. Overall, my dissertation showcases the role that dispersal, but more specifically priority effects, can play in the development of the vertebrate microbiome.

The bacteria in host-associated microbial communities influence host health through various mechanisms, such as immune stimulation or the release of metabolites. However, viruses that target bacteria, called bacteriophages (phages), may also shape the animal microbiome. Most phage lifecycles can be classified as either lytic or temperate. Lytic phages infect and directly kill bacterial hosts and can directly regulate bacterial population size. Temperate phages, in contrast, have the potential to undergo either a lytic cycle or integrate into the bacterial genome as a prophage. As a prophage, the phage may alter bacterial host phenotypes by carrying novel genes associated with auxiliary metabolic functions, virulence-enhancing toxins, or resistance to other phage infections. Lytic phages may also carry certain auxiliary metabolic genes, which are instead used to takeover bacterial host functions to better accommodate the lytic lifecycle. In either case, the ability to alter bacterial phenotypes may have important ramifications on host-associated communities. This dissertation focused on the genetic contributions that phages, and particularly prophages, provide to the bacterial members of two separate host-associated communities: the honey bee (Apis mellifera) gut microbiome and the amphibian skin microbiome. My second chapter surveyed publicly available whole genome sequences of common honey bee gut bacterial species for prophages. It revealed that prophage distribution varied by bacterial host, and that the most common auxiliary metabolic genes were associated with carbohydrate metabolism. In chapter three, this bioinformatic pipeline was applied to the amphibian skin microbiome. Prophages were identified in whole genome bacterial sequences of bacteria isolated from the skin of American bullfrogs (Lithobates catesbeianus), eastern newts (Notophthalmus viridescens), Spring peepers (Pseudacris crucifer) and American toads (Anaxyrus americanus). Prophages were additionally identified in publicly available genomes of non-amphibian isolates of Janthinobacterium lividum, a bacteria found both on amphibian skin and broadly in the environment. In addition to a diverse set of predicted prophages across amphibian bacterial isolates, several Janthinobacterium lividum prophages from both amphibian and environmental isolates appear to encode a chitinase-like gene undergoing strong purifying selection within the bacterial host. While identifying the specific function of this gene would require in vitro isolation and testing, its high homology to chitinase and endolysins suggest it may be involved in the breakdown of either fungal or bacterial cellular wall components. Finally, my fourth chapter revisits the honey bee gut system by investigating the role of geographic distance in bacteriophage community similarity. A total of 12 apiaries across a transect of the United States, from Virginia to Washington, were sampled and honey bee viromes were sequenced, focusing on the lytic and actively lysing temperate community of phages. Although each apiary possessed many unique bacteriophages, apiaries that were closer together did have more similar communities. Each bacteriophage community also carried auxiliary carbohydrate genes, especially those associated with sucrose degradation, and antimicrobial resistance genes. Combined, the results of these three studies suggest that bacteriophages, and particularly prophages, may be contributing to the genetic diversity of the bacterial community through nuanced relationships with their bacterial hosts. / Doctor of Philosophy / The microbial communities of animals, called "microbiomes", play important roles in the health of animals. The bacteria in these microbiomes can help strengthen the immune system, provide resistance to dangerous pathogens, and break down nutrients. However, bacteria are not alone in the microbiome; viruses are also present. Surprisingly, the vast majority of the world's viruses, even those living inside animals, infect bacteria. These viruses, called "bacteriophages" or "phages", can impact the bacterial communities in a microbiome. Phages can be grouped in to two broad categories based on lifecycle. Lytic phages kill the bacterial host directly after infection. Temperate phages, on the other hand, can either immediately kill the host like lytic phages or alternatively, become a part of the bacterial genome and live as prophages. Phages with both lifecycles can sometimes carry genes that, although not essential to the phage, may change the traits of the bacteria during infection. For example, some phages carry toxin genes, which bacteria use to cause disease in animals. Other phages might carry genes that provide antibiotic resistance or alter the metabolism of the infected bacteria. If a phage gene benefits the infected bacteria, the bacteria may begin interacting with its environment in a new way or may even become more abundant. Alternatively, phages that directly kill infected bacteria may have a negative effect on bacterial population sizes. To begin unraveling how phages influence bacterial species in microbiomes, I investigated two different animal systems: the Western honey bee (Apis mellifera) gut microbiome and the amphibian skin microbiome. I first identified prophages of several common bacterial species that reside in the honey bee gut (Chapter 2). Prophages were more common in certain bacterial species than others, and some possessed genes associated with the breakdown of sugars or pollen, suggesting they help honey bees process their food. Using similar techniques, I then identified prophages in bacteria isolated from the skin microbiomes of several amphibian species common in the eastern United States (American bullfrogs, Eastern newts, Spring peepers, and American toads) (Chapter 3). Most notably, the bacteria Janthinobacterium lividum may benefit from prophages that carry genes for potentially antifungal chitinase enzymes that destroy the fungal cell wall. Finally, I returned to the honey bee gut microbiome system by investigating how honey bee bacteriophage communities change over large geographic distances (Chapter 4). This study, which examined honey bees from 12 apiaries sampled from the east to west coast of the United States, looks primarily at lytic phage and temperate phage that are not integrated as prophage, but are instead seeking a bacterial host to infect. I found that nearby apiaries tended to have more similar communities of bacteriophages, compared to apiaries far away. Additionally, most bacteriophage communities carry genes associated with the breakdown of sugars like sucrose. Overall, these three studies show that phages, and especially prophages, contribute to the genetic landscape of the microbiome by broadly providing bacterial hosts with access to a diverse set of genes.