Violacein is a purple pigment typically produced by species of Chromobacterium and Janthinobacterium. A soil isolate, identified as Janthinobacterium, was studied. Maximal pigmentation occurred at 250C under aerobic conditions in the Keeble and Cross medium. Intracellular pigment was shown to be located in the cell membrane. Comparision of pigment production and growth curves indicated that violacein is synthesized in the cell and released into the environment possibly as a result of cell lysis. Extracellular pigment is water soluble, makes up 60% of the total pigment and shows a blue shift when compared to solvent extracted pigment. Results from purification indicated that the pigment is non-covalently bound to a small protein and aggregated into a larger molecule.

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.

Underexplored environments such as soil samples continue to be an untapped source of bacterial strains with great potential to produce secondary metabolites for medicinal applications. As a result, these microorganisms represent a broad and yet unknown reservoir of new strains capable of producing these novel compounds. The current research primarily seeks to perform the isolation, purification, and characterization of secondary metabolites from a soil bacterium (Janthinobacterium lividum TAJX1901). The isolated soil bacterium was successfully cultured on rich media agar plates, followed by extraction using methanol and chloroform. The purification methods utilized include flash column chromatography, preparative thin-layer chromatography, and high-performance liquid chromatography. For structural elucidation, UV-Vis analysis, infrared spectroscopy, and nuclear magnetic resonance spectroscopy were employed. The extraction resulted in a dominant violet pigment soluble in methanol. Results revealed the presence of highly conjugated, polar, and aromatic compounds (violacein or relatives of violacein) and dioctyl phthalate (a contaminant).

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.