DOI: 10.5061/dryad.p2ngf1vsf
title: Seasonality of host immunity in a tropical disease system

description: 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.
description: We captured adult individuals of Panamanian rocket frog
 (<em>Colostethus panamansis</em>) 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 (<em>Colostethus panamansis</em>) 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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