Fungal disease and temperature alter skin microbiome structure in an experimental salamander system

Abstract

Pathogens compete with host microbiomes for space and resources. Their shared environment impacts pathogen-microbiome-host interactions, which can lead to variation in disease outcome. The skin microbiome of red-backed salamanders (Plethodon cinereus) can reduce infection by the pathogen Batrachochytrium dendrobatidis (Bd) at moderate infection loads, with high species richness and high abundance of competitors as putative mechanisms. However, it is unclear if the skin microbiome can reduce epizootic Bd loads across temperatures. We conducted a laboratory experiment to quantify skin microbiome and host responses (P. cinereus: n = 87) to Bd at mimicked epizootic loads across temperatures (13, 17 and 21°C). We quantified skin microbiomes using 16S rRNA gene metabarcoding and identified operational taxonomic units (OTUs) taxonomically similar to culturable bacteria known to kill Bd (anti-Bd OTUs). Prior to pathogen exposure, temperature changed the microbiome (OTU richness decreased by 12% and the abundance of anti-Bd OTUs increased by 18% per degree increase in temperature), but these changes were not predictive of disease outcome. After exposure, Bd changed the microbiome (OTU richness decreased by 0.1% and the abundance of anti-Bd OTUs increased by 0.2% per 1% increase in Bd load) and caused high host mortality across temperatures (35/45: 78%). Temperature indirectly impacted microbiome change and mortality through its direct effect on pathogen load. We did not find support for the microbiome impacting Bd load or host survival. Our research reveals complex host, pathogen, microbiome and environmental interactions to demonstrate that during epizootic events the microbiome will be unlikely to reduce pathogen invasion, even for putatively Bd-resistant species. K E Y W O R D Santifungal, Batrachochytrium dendrobatidis, climate, epidemic, invasion, microbiota, symbiosis

Temperature‐mediated shifts in salamander transcriptomic responses to the amphibian‐killing fungus

“…In the larger survival study, from which we sampled individuals for this study, survival of uninfected salamanders was 100%, 100% and 86% at 13, 17 and 21°C, respectively. Survival of Bd ‐challenged salamanders was 6.6%, 26.7% and 33.3% at 13, 17 and 21°C, respectively (Muletz‐Wolz, Barnett, et al, ; Muletz‐Wolz, Fleischer, et al, ). We found a significant effect of Bd exposure on survival rate for Bd ‐exposed vs. Bd ‐control; exposed individuals had lower survival rates.…”

“…The salamanders used in this study represent a subset of a larger study investigating temperature‐dependent mortality in response to Bd infection (Muletz‐Wolz, Barnett, et al, ; Muletz‐Wolz, Fleischer, et al, ). Briefly, adult Plethodon cinereus (>35 mm snout–vent length) were acclimatized for 47 days to either 13 ( n = 29), 17 ( n = 29), or 21°C ( n = 29).…”

“…We used qPCR (quantitative polymerase chain reaction) to confirm that individuals euthanized due to clinical signs of chytridiomycosis were “infected,” control individuals were “uninfected” and Bd‐ exposed individuals that survived had “cleared” their infections. Because the individuals used in this experiment were part of a larger Bd ‐survival study (Muletz‐Wolz, Barnett, et al, ; Muletz‐Wolz, Fleischer, et al, ) and because progression of chytridiomycosis varies widely among individuals even under controlled inoculation doses (Carey et al, ), we opted for a tissue sampling regime that maximized the opportunity to capture expression responses in a broadly comparable “mature” stage of infection (i.e., when hosts were actively shedding zoospores). Thus we sampled hosts over a range of days post‐inoculation (Figure , Appendix ).…”

“…Indeed, expression profiling of Xenopus tropicalis revealed differential activation of innate immune genes in response to infection at two temperatures (Ribas et al, ). However, despite evidence of temperature‐dependent survival in salamanders (Muletz‐Wolz, Barnett, et al, ; Muletz‐Wolz, Fleischer, et al, ; Vazquez et al, ), we do not yet have any study of temperature‐dependent gene expression responses of salamanders to Bd . More broadly, we have limited knowledge of how salamander immune expression responses to Bd infection — regardless of temperature—compare to those in anurans (Farrer et al, ).…”

“…In some species, underlying individual thermal preferences may be a predictor of infection susceptibility (Sauer et al, ), while in others ectotherm behavioural fever (active preference for warmer environments) appears to be important in enhancing survival during infections (Boltana et al, ). Widespread thermal‐dependence of parasite and pathogen life‐history traits (Bakke, Cable, & Harris, ; Muletz‐Wolz, Barnett, et al, ; Muletz‐Wolz, Fleischer, & Lips, ; Voyles et al, ) indicates that temperature is key to understanding infection processes and disease dynamics in ectotherm hosts.…”