Infectious disease systems frequently exhibit strong seasonal patterns, yet the mechanisms that underpin intra-annual cycles are unclear, particularly in tropical regions. We hypothesized that host immune function fluctuates seasonally, contributing to oscillations in infection patterns in a tropical disease system. To test this hypothesis, we investigated a key host defense of amphibians against a lethal fungal pathogen, Batrachochytrium dendrobatidis (Bd). We integrated two field experiments in which we perturbed amphibian skin secretions, a critical host immune mechanism, in Panamanian rocket frogs (Colostethus panamansis). We found that this immunosuppressive technique of reducing skin secretions in wild frog populations increased Bd prevalence and infection intensity, indicating that this immune defense contributes to resistance to Bd in wild frog populations. We also found that the chemical composition and anti-Bd effectiveness of frog skin secretions varied across seasons, with greater pathogen inhibition during the dry season relative to the wet season. These results suggest that the effectiveness of this host defense mechanism shifts across seasons, likely contributing to seasonal infection patterns in a lethal disease system. More broadly, our findings indicate that host immune defenses can fluctuate across seasons, even in tropical regions where temperatures are relatively stable, which advances our understanding of intra-annual cycles of infectious disease dynamics.

We captured adult individuals of Panamanian rocket frog (Colostethus panamansis) using clean, inverted plastic freezer bags. 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 Pesola 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 used capture-mark-recapture (CMR) to identify frogs and collect additional diagnostic samples to test for Bd infection. This approach allowed us to track infection status over time. We used a quantitative polymerase chain reaction (qPCR) assay to assess prevalence and intensity of Bd infection in our diagnostic samples that were collected over the course of the field experiment.

We also collected skin secretions samples from common rocket frogs (Colostethus panamansis) at two sites (OTNP1, OTNP2), across seasons (wet, dry). To collect frog skin secretions, we used the same method described above. In total 50 samples were analyzed using MALDI-TOF (Biosystems Voyager Elite mass spectrometer (Applied Biosystems, Foster City, California, USA) operated in reflector, delayed extraction, and positive ion mode. We prepared peptide samples by spotting peptides onto a MALDI plate in a 1:1 ratio with matrix [10 mg/ml α-cyano-4-hydroxycinnamic acid (Fluka, Sigma, St. Louis, MO), 60% acetonitrile, 39.6% HPLC-grade water, and 0.4% trifluoroacetic acid (v/v/v)]. Each sample was analyzed by averaging signals from 256 consecutive laser shots. The MS data was acquired in the m/z range 500 to 7,000, truncated at m/z 4,500 and baseline-corrected with Data Explorer v4.4 (Applied Biosystems). 

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