To combat the threat of emerging infectious diseases in wildlife, ecoimmunologists seek to understand the complex interactions among pathogens, their hosts, and their shared environments. The cutaneous fungal pathogen Batrachochytrium dendrobatidis (Bd), has led to the decline of innumerable amphibian species, including the Panamanian golden frog (Atelopus zeteki). Given that Bd can evade or dampen the acquired immune responses of some amphibians, nonspecific immune defenses are thought to be especially important for amphibian defenses against Bd. In particular, skin secretions constitute a vital component of amphibian innate immunity against skin infections, but their role in protecting A. zeteki from Bd is unknown. We investigated the importance of this innate immune component by reducing the skin secretions from A. zeteki and evaluating their effectiveness against Bd in vitro and in vivo. Following exposure to Bd in a controlled inoculation experiment, we compared key disease characteristics (e.g., changes in body condition, prevalence, pathogen loads, and survival) among groups of frogs that had their skin secretions reduced and control frogs that maintained their skin secretions. Surprisingly, we found that the skin secretions collected from A. zeteki increased Bd growth in vitro. This finding was further supported by infection and survival patterns in the in vivo experiment where frogs with reduced skin secretions tended to have lower pathogen loads and survive longer compared to frogs that maintained their secretions. These results suggest that the skin secretions of A. zeteki are not only ineffective at inhibiting Bd but may enhance Bd growth, possibly leading to greater severity of disease and higher mortality in this highly vulnerable species. These results differ from those of previous studies in other amphibian host species that suggest that skin secretions are a key defense in protecting amphibians from developing severe chytridiomycosis. Therefore, we suggest that the importance of immune components cannot be generalized across all amphibian species or over time. Moreover, the finding that skin secretions may be enhancing Bd growth emphasizes the importance of investigating these immune components in detail, especially for species that are a conservation priority.

We obtained Panamanian golden frogs (Atelopus zeteki) from the Omaha zoo as part of the captive breeding program 'Project Golden Frog'. For each frog, we measured mass to the nearest 0.01 g, and snout-vent length (SVL) to the nearest 0.1 mm using calipers and scales. We also collected skin swab samples to test for Bd presence and infection intensity using standardized swabbing techniques. To collect frog skin secretions, we induced each frog to secrete its store of secretions from cutaneous granular glands by administering a stimulatory injection of norepinephrine (NE) below the skin surface (Rollins-Smith et al. 2005, Woodhams et al. 2006). We randomly assigned frogs to one of three NE treatment groups: a high-dose treatment, hereafter referred to as the "Reduced" group, a low-dose treatment, hereafter referred to as the "Partially Reduced" group, and a "Control" group that received an injection of sterile saline solution. Following reduction of skin secretions, we exposed frogs to the Bd isolate Rio Maria. We included a negative control group of frogs, which received injections of sterile saline and were exposed to heat-killed Bd. We measured mass and SVL weekly to calculate frog body condition (mass/SVL). We swabbed frogs weekly and used a quantitative polymerase chain reaction (qPCR) assay to assess prevalence and intensity of Bd infection in our diagnostic samples. We analyzed the quantity of skin secretions collected from each group of frogs using a MicroBCA assay using bradykinin to generate a standard curve. We assessed the inhibitory effectivenss of these skin secretions against Bd in vitro using an MTT assay (Lindauer et al. 2019).

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