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Date of Award

Spring 2022

Rights

Access is available to all users

Document Type

Thesis: EWU Only

Degree Name

Master of Science (MS) in Biology

Department

Biology

Abstract

The fungal pathogen, Batrachochytrium dendrobatidis (Bd), has led to the decline and extinction of many amphibian populations, but some bacteria in the skin microbiome can inhibit its growth. In Turnbull National Wildlife Refuge (TNWR) in eastern Washington, Bd is highly prevalent, but the role of the skin microbiome in Bd infection dynamics have not been examined in this region. We hypothesized that frogs with lower Bd infection intensities would have higher skin bacterial diversity and more abundant anti-Bd bacteria, indicative of a more protective function. Our study combined cultureindependent and culture-dependent methods to assess the relationship between Bd and the microbiome of the Columbia Spotted Frog (Rana luteiventris, N=46) and the Pacific Chorus Frog (Pseudacris regilla, N=72) in TNWR. We characterized skin bacterial diversity with 16S rRNA gene amplicon sequencing on Illumina MiSeq, and quantified Bd infection intensity with qPCR. P. regilla had significantly higher Bd infection intensities (14,480 zoospore equivalents, number of zoospores per swab) and prevalence (91.43%) compared to R. luteiventris (intensity: 1,647.36 zoospore equivalents, prevalence: 67.74%). To evaluate whether these infection differences correlate with the skin microbiome, a culture-dependent method was used to determine which bacterial isolates produce anti-Bd metabolites in in vitro co-culture assays, followed by a comparison of culture and culture-independent DNA sequences to determine relative abundance of anti-Bd bacteria on wild frogs. PCF and CSF had significantly different skin microbiomes (p=0.001, pseudo-F=19.07, PERMANOVA), and the two species varied in the interaction between the microbiome and Bd. Bd infection intensity was significantly correlated with the skin microbiome in P. regilla (Mantel test, r=0.43, p=0.02), which had higher Bd levels, but this pattern was not observed in the less infected R. luv (Mantel test, r=-0.0086, p=0.93). Lastly, for P. regilla only, skin microbiomes varied across wetland sites (PERMANOVA, p=0.004), which could explain the variation in Bd infection intensities observed across sites in this species (p=0.038, Kruskal-Wallis). These results are a basis of understanding for the Bd-microbiome relationship and for frog conservation in this area.

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