DOI: 10.5061/dryad.h44j0zpnz
title: Atelopus zeteki skin secretions not protective against Bd

description: 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.
description: We obtained Panamanian golden frogs
 (<em>Atelopus zeteki</em>)
 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
 <em>Bd</em> 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 <em>Bd</em> isolate Rio
 Maria. We included a negative control group of frogs, which received
 injections of sterile saline and were exposed to heat-killed
 <em>Bd</em>. 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
 <em>Bd</em> 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 <em>Bd
 in vitro</em> using an MTT assay (Lindauer et al.
 2019).
description: Please see README file.