All species of plants and animals harbor microbes (bacteria and fungi) that live symbiotically in and on them. How those microbes are related to the health of their host organisms is largely undetermined and, in particular, there is a limited understanding of how symbiotic microbes influence a host's ability to cope with disease-causing pathogens. The proposed research will use amphibians as a model system and a fungal skin pathogen that is linked to the decline of amphibian populations around the world. Different amphibian species are either highly susceptible or tolerant to disease caused by the fungal pathogen, and there is reasonable evidence to suggest that naturally occurring skin microbes may play an important role. This research will use both field observations from wild amphibian populations and a series of specific experiments that will be conducted in the laboratory. In the field, amphibians will be sampled for microbes to compare differences among species in pond habitats. In the lab, controlled experiments will examine how microbes on the skin respond to the pathogen over time and also how they respond to probiotic treatments (beneficial bacteria) in an effort to understand the ecology of the microorganisms that live on the skin. Novel high-throughput DNA sequencing techniques and innovative computer-based analysis tools will be used to examine the identity and composition of different bacterial and fungal groups in each sample.
Broader Impacts: This research aims to advance our knowledge about the role of symbiotic skin microbes and how probiotic treatments can be developed for use in conservation of amphibians as well as a broad range of human and wildlife disease systems. The project will advance the research program of an early career scientist (PI) and support the training of a graduate student, a postdoctoral researcher, and undergraduates from under-represented backgrounds. All DNA sequence data and analytic tools produced during this project will be made available for use by other researchers. Boreal toads, an endangered amphibian species in Colorado, will serve as a focal species in this project and the PIs will build collaborations with the Boreal Toad Recovery Program in Colorado.
Intellectual Merit Our project has greatly expanded what we know about the amphibian skin microbiome (the bacteria, fungi and micro eukaryotic organisms that live in close association with the host.) Amphibians are a diverse and important group of vertebrate animals that are threatened globally, and one of the threats that looms large is an infectious fungal pathogen that is causing amphibian declines. We have been studying how the naturally occurring bacteria that live on amphibians plays a role in helping to protect the host from this fungal pathogen, using both field collected samples as well as manipulative lab experiments. Some amphibian species are highly susceptible to disease caused by the fungus, while others are tolerant. Some bacteria hosted by amphibians can have very strong inhibition against fungal pathogens. Specifically, we have learned that across multiple datasets ranging from Colorado, California, Costa Rica, and Madagascar, that different species of amphibians harbor distinct communities of bacteria on their skin. Species differences in in the skin microbiome appear to be particularly different across species that are either susceptible versus tolerant to the fungal pathogen, indicating a strong role of the microbiome. We have found that the microbiome does not act alone on the skin mucosal surface. Peptides produced by the host amphibian immune system from glands on the skin, interact with metabolites produced by the bacteria, and that the combination of these compounds provides a strong predictive assay as to whether an amphibian is highly susceptible to disease caused by the fungus. This further suggests that amphibians employ a combination of immune and bacterial symbiont factors to protect from pathogens. Additionally, using a non-destructive, field-collected dataset from endangered, wild boreal toads in Colorado combined with microbial bioinformatics, we demonstrate that the skin microbial communities on toads shift in a repeatable pattern across distinct developmental stages of the host. Our findings show that early lifestage boreal toads have skin bacterial communities that are dominated by taxa with pathogen defense properties, and that these negatively co-occur with fungi that inhabit later lifestage toads. This study is unique compared to all previous studies of amphibian skin-associated microbiota for the following novel contributions: 1) This is the first study to examine shifts in amphibian skin microbial communities through development. 2) In addition to the bacteria on the toads, we also provide the first next-gen sequencing based study of the micro-eukaryotic organisms present in the amphibian skin microbiome (e.g., fungi, protozoans etc.), through the use of universal 18s primers. 3) We use a novel database of bacteria isolated from amphibians globally and proven to inhibit fungal pathogens, developed in parallel, to examine the distribution of fungal inhibitory bacteria present on wild boreal toads. 4) We include analytical approaches such as network analyses to detect significant interactions between members of bacteria and fungi the boreal toad skin microbiome. Overall, this project has directly produced 6 publications thus far (Molecular Ecology, ISME Journal, 2 in PLOS One, Biological Invasions, and a data paper in Ecology), two other manuscripts are currently in review, and seven more are in preparation. Broader Impacts This project has provided a successful launch of PI McKenzie's research program as a pre-tenured academic researcher. It has also provided training opportunities for 4 graduate students, 1 postdoctoral associate, many undergraduates including an intern from Puerto Rico, and a highschool student. The McKenzie lab has played a large role in merging science into the Americas Latinos Festival in Boulder, Colorado, as well as integration of the project with the Boreal Toad Recovery Program in Colorado. PI McKenzie has presented this work in invited talks around the globe, featured this research in a piece in High Country News, and was the lead organizer of an Organized Oral Session at the Ecological Society of America conference in 2014 focused on amphibian microbes that featured the work of this project along with 10 speakers from the USA, Colombia, Puerto Rico, and Australia. Postdoc Woodhams led an effort to merge the research outcomes of this project with an international group of collaborators that has resulted in a published open database the will facilitate further collaborative work on amphibian microbes broadly.
