The Antifungal Arsenal in Amphibian Skin: Innate Immune Defenses Against Batrachochytrium dendrobatidis in Southern Leopard Frogs

Abstract

The fungus Batrachochytrium dendrobatidis (Bd) causes chytridiomycosis and has played a role in at least 90 amphibian extinctions since 1980. However, some amphibian species are more resistant to Bd than others, which may be due to one or more innate defenses in the skin, including antimicrobial peptides (AMPs) and symbiotic bacteria that produce antifungal metabolites. In this dissertation, I explored the contributions of these defenses to resistance against Bd in southern leopard frogs, Rana sphenocephala, which required developing methods to reduce or eliminate these defenses in laboratory animals. I examined the ability of skin bacteria to defend R. sphenocephala prior to the development of a substantial AMP defense and identified new symbiotic bacteria capable of inhibiting or enhancing Bd growth in vitro. I more fully characterized a known peptide, Peptide C, in R. sphenocephala, quantified the inhibitory activity of four known R. sphenocephala AMPs in vitro, elucidated the AMP defense ontogeny in this species, and determined the impact of induced AMPs on viability of skin bacteria. For some endangered species, captive colonies are the best intermediate solution for long-term survival, and effective antifungal treatments are needed to treat chytridiomycosis and limit Bd spread in captive colonies. Unfortunately, the only currently accepted treatment uses itraconazole, which can be toxic. In studying alternative anti-chytrid drugs, I quantified the minimum inhibitory concentrations of nikkomycin Z, chloramphenicol, and amphotericin B and undertook the first analysis of the interactions and effects of these drugs on amphibian innate immune defenses. In total, my studies shed light on two important immune defenses against Bd and contribute to the search for new drugs against a significant fungal pathogen endangering amphibians worldwide.