Effectiveness of cutaneous antifungal microbe bioaugmentation for disease prevention in the Cascades frog, Rana cascadae, of northern California

Graduation Date

2016

Document Type

Thesis

Program

Other

Program

Thesis (M.S.)--Humboldt State University, Biology, 2016

Committee Chair Name

John Reiss

Committee Chair Affiliation

Cal Poly Humboldt Faculty or Staff

Keywords

Southern Cascades, Cutaneous bacterial flora, Klamath Mountains, Cascades frog, Semi-quantitative spectrophotometric challenge assay, Amphibian decline, Antifungal bacteria, Rana cascadaerthern, Bd prevalence, Quantitative PCR, Bd load, Inhibitory potential, Mountain ranges, Bioaugmentation, Bd zoospores, Pseudomonas, Polymerase chain reaction, Humboldt State University -- Theses -- Biology, Chytrid fungus (Bd), Northern California

Abstract

The disease chytridiomycosis has caused population declines in hundreds of amphibian species worldwide, and is caused by the pathogenic fungus Batrachochytrium dendrobatidis (Bd). One promising method to combat the disease is to augment the amphibian's naturally occurring cutaneous antifungal bacterial fauna. I surveyed frogs for cutaneous bacteria across 13 sites among declining populations of Cascades frogs (Rana cascadae) in the Klamath and southern Cascades Mountains of northern California. In vitro challenge assays were used to analyze 182 bacterial supernatants for inhibitory properties against Bd, with 70.33% classified as highly inhibitory, 21.98% slightly/non-inhibitory, and 7.69% as Bd enhancing. The two most effective anti-Bd bacterial isolates were identified as Pseudomonas species by 16S rRNA gene sequencing, and tested for anti-Bd properties in vivo on lab-reared recently metamorphosed R. cascadae. Treatments were as follows: Control, Bd only, Bacteria only, Bacteria + Bd, and Bd + Bacteria. During the 70-day lab trial, frogs were weighed, measured for snout-vent-length, and swabbed weekly for Bd zoospore presence/load. Surprisingly, overall mortality was low. Mortality was highest in the Bd only treatment (36.4%) and lowest in the Bd + Bacteria (0%). No mortality differences were seen between the treatments with the two strains of Pseudomonas tested. Zoospore loads revealed frogs were clearing the infection, even in the absence of bacterial treatment. The Bd only treatment had the highest initial zoospore load (30 zoospore equivalents/frog on average). However, the Bacteria + Bd group maintained a higher proportion of Bd-infected individuals than the Bd + Bacteria group. Differences were also found in weight gain/loss, with the Bd + Bacteria treatment being the only group to lose weight (0.07 ± 0.02 g on average). The results from antifungal bioaugmentation suggest that addition of antifungal bacteria to Bd-exposed frogs has a positive impact on survival and reduction in Bd load. A future direction would be to conduct a field trial at Shimmy Lake in the Klamath Mountains using the Pseudomonas sp. identified in this research and collected from the same field site to determine the effectiveness of antifungal bioaugmentation applications on wild populations.

https://scholarworks.calstate.edu/concern/theses/0z708z52k

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