Preparatory immunity: Seasonality of mucosal skin defences and Batrachochytrium infections in Southern leopard frogs.

Accurately predicting the impacts of climate change on wildlife health requires a deeper understanding of seasonal rhythms in host-pathogen interactions. The amphibian pathogen, Batrachochytrium dendrobatidis (Bd), exhibits seasonality in incidence; however, the role that biological rhythms in host defences play in defining this pattern remains largely unknown. The aim of this study was to examine whether host immune and microbiome defences against Bd correspond with infection risk and seasonal fluctuations in temperature and humidity. Over the course of a year, five populations of Southern leopard frogs (Rana [Lithobates] sphenocephala) in Tennessee, United States, were surveyed for host immunity, microbiome and pathogen dynamics. Frogs were swabbed for pathogen load and skin bacterial diversity and stimulated to release stored antimicrobial peptides (AMPs). Secretions were analysed to estimate total hydrophobic peptide concentrations, presence of known AMPs and effectiveness of Bd growth inhibition in vitro. The diversity and proportion of bacterial reads with a 99% match to sequences of isolates known to inhibit Bd growth in vitro were used as an estimate of predicted anti-Bd function of the skin microbiome. Batrachochytrium dendrobatidis dynamics followed the expected seasonal fluctuations-peaks in cooler months-which coincided with when host mucosal defences were most potent against Bd. Specifically, the concentration and expression of stored AMPs cycled synchronously with Bd dynamics. Although microbiome changes followed more linear trends over time, the proportion of bacteria that can function to inhibit Bd growth was greatest when risk of Bd infection was highest. We interpret the increase in peptide storage in the fall and the shift to a more anti-Bd microbiome over winter as a preparatory response for subsequent infection risk during the colder periods when AMP synthesis and bacterial growth is slow and pathogen pressure from this cool-adapted fungus is high. Given that a decrease in stored AMP concentrations as temperatures warm in spring likely means greater secretion rates, the subsequent decrease in prevalence suggests seasonality of Bd in this host may be in part regulated by annual immune rhythms, and dominated by the effects of temperature.