skip to main content

Title: Inhibitory Bacterial Diversity and Mucosome Function Differentiate Susceptibility of Appalachian Salamanders to Chytrid Fungal Infection
ABSTRACT Mucosal defenses are crucial in animals for protection against pathogens and predators. Host defense peptides (antimicrobial peptides, AMPs) as well as skin-associated microbes are key components of mucosal immunity, particularly in amphibians. We integrate microbiology, molecular biology, network-thinking, and proteomics to understand how host and microbially derived products on amphibian skin (referred to as the mucosome) serve as pathogen defenses. We studied defense mechanisms against chytrid pathogens, Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal), in four salamander species with different Batrachochytrium susceptibilities. Bd infection was quantified using qPCR, mucosome function (i.e., ability to kill Bd or Bsal zoospores in vitro ), skin bacterial communities using 16S rRNA gene amplicon sequencing, and the role of Bd-inhibitory bacteria in microbial networks across all species. We explored the presence of candidate-AMPs in eastern newts and red-backed salamanders. Eastern newts had the highest Bd prevalence and mucosome function, while red-back salamanders had the lowest Bd prevalence and mucosome function, and two-lined salamanders and seal salamanders were intermediates. Salamanders with highest Bd infection intensity showed greater mucosome function. Bd infection prevalence significantly decreased as putative Bd-inhibitory bacterial richness and relative abundance increased on hosts. In co-occurrence networks, some putative Bd-inhibitory bacteria were found as more » hub-taxa, with red-backs having the highest proportion of protective hubs and positive associations related to putative Bd-inhibitory hub bacteria. We found more AMP candidates on salamanders with lower Bd susceptibility. These findings suggest that salamanders possess distinct innate mechanisms that affect chytrid fungi. IMPORTANCE How host mucosal defenses interact, and influence disease outcome is critical in understanding host defenses against pathogens. A more detailed understanding is needed of the interactions between the host and the functioning of its mucosal defenses in pathogen defense. This study investigates the variability of chytrid susceptibility in salamanders and the innate defenses each species possesses to mediate pathogens, thus advancing the knowledge toward a deeper understanding of the microbial ecology of skin-associated bacteria and contributing to the development of bioaugmentation strategies to mediate pathogen infection and disease. This study improves the understanding of complex immune defense mechanisms in salamanders and highlights the potential role of the mucosome to reduce the probability of Bd disease development and that putative protective bacteria may reduce likelihood of Bd infecting skin. « less
; ; ; ; ; ; ; ;
Reguera, Gemma
Award ID(s):
1845634 2131060 2131062 1814520
Publication Date:
Journal Name:
Applied and Environmental Microbiology
Sponsoring Org:
National Science Foundation
More Like this
  1. Abstract Hellbenders ( Cryptobranchus alleganiensis ) are large, aquatic salamanders from the eastern United States. Both subspecies, eastern and Ozark hellbenders, have experienced declines resulting in federal listing of Ozark hellbenders. The globally distributed chytrid fungus, Batrachochytrium dendrobatidis (Bd) has been detected in both subspecies, and Batrachochytrium salamandrivorans ( Bsal ) poses a new threat if introduced into North America. Ozark hellbenders also suffer a high prevalence of toe lesions of unknown etiology, with changes in host immunocompetence hypothesized to contribute. Antimicrobial peptides (AMPs) secreted from dermal granular glands may play a role in hellbender health. We collected skin secretions from free-ranging hellbenders and enriched them for small cationic peptides used for growth inhibition assays against Bd and Bsal . Generalized linear mixed models revealed the presence of active toe lesions as the strongest and only significant predictor of decreased Bd inhibition by skin peptides. We also found skin secretions were more inhibitory of Bsal than Bd . MALDI-TOF mass spectrometry revealed candidate peptides responsible for anti-chytrid activity. Results support the hypothesis that hellbender skin secretions are important for innate immunity against chytrid pathogens, and decreased production or release of skin peptides may be linked to other sub-lethal effects ofmore »disease associated with toe lesions.« less
  2. Host-associated microbiomes play important roles in host health and pathogen defense. In amphibians, the skin-associated microbiota can contribute to innate immunity with potential implications for disease management. Few studies have examined season-long temporal variation in the amphibian skin-associated microbiome, and the interactions between bacteria and fungi on amphibian skin remain poorly understood. We characterize season-long temporal variation in the skin-associated microbiome of the western tiger salamander ( Ambystoma mavortium ) for both bacteria and fungi between sites and across salamander life stages. Two hundred seven skin-associated microbiome samples were collected from salamanders at two Rocky Mountain lakes throughout the summer and fall of 2018, and 127 additional microbiome samples were collected from lake water and lake substrate. We used 16S rRNA and ITS amplicon sequencing with Bayesian Dirichlet-multinomial regression to estimate the relative abundances of bacterial and fungal taxa, test for differential abundance, examine microbial selection, and derive alpha diversity. We predicted the ability of bacterial communities to inhibit the amphibian chytrid fungus Batrachochytrium dendrobatidis ( Bd ), a cutaneous fungal pathogen, using stochastic character mapping and a database of Bd -inhibitory bacterial isolates. For both bacteria and fungi, we observed variation in community composition through time, between sites, andmore »with salamander age and life stage. We further found that temporal trends in community composition were specific to each combination of salamander age, life stage, and lake. We found salamander skin to be selective for microbes, with many taxa disproportionately represented relative to the environment. Salamander skin appeared to select for predicted Bd -inhibitory bacteria, and we found a negative relationship between the relative abundances of predicted Bd -inhibitory bacteria and Bd . We hope these findings will assist in the conservation of amphibian species threatened by chytridiomycosis and other emerging diseases.« less
  3. Abstract

    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 againstBdcorrespond 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 ofBdgrowth inhibition in vitro. The diversity and proportion of bacterial reads with a 99% match to sequences of isolates known to inhibitBdgrowth in vitro were used as an estimate of predicted anti‐Bdfunction of the skin microbiome.

    Batrachochytrium dendrobatidisdynamics followed the expected seasonal fluctuations—peaks in cooler months—which coincided with when host mucosal defences were most potent againstBd. Specifically, the concentration and expression of stored AMPs cycled synchronously withBddynamics. Although microbiome changes followed more linearmore »trends over time, the proportion of bacteria that can function to inhibitBdgrowth was greatest when risk ofBdinfection was highest.

    We interpret the increase in peptide storage in the fall and the shift to a more anti‐Bdmicrobiome 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 ofBdin this host may be in part regulated by annual immune rhythms, and dominated by the effects of temperature.

    « less
  4. Abstract

    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 inmore »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.

    « less
  5. Abstract

    Life processes of ectothermic vertebrates are intimately linked to the temperature of their environment, influencing their metabolism, reproduction, behaviour and immune responses. In amphibians infected by the generalist chytrid pathogenBatrachochytrium dendrobatidis(Bd), host survival, infection prevalence and infection intensity are often temperature‐ and/or seasonally dependent. However, the transcriptional underpinnings of thermal differences in infection responses remain unknown. Measuring the impact of temperature on host responses to infection is a key component for understanding climatic influences on chytrid disease dynamics. TheBd‐responsive gene pathways in frogs are well documented, but our understanding of salamander immune expression profiles during infection with chytrids remains limited. Here we characterize the transcriptomic responses ofPlethodon cinereususing RNA sequencing by comparing skin and splenic gene expression of individuals uninfected, succumbing toBdinfection and naturally cleared ofBdinfection at three temperatures. We suggest that amphibian temperature‐dependent susceptibility toBdis probably driven by shifts in expression of the innate and adaptive immune axes. Our study shows increased expression of transcripts associated with inflammation at lower temperatures and a shift towards increased expression of adaptive immune genes, including MHC (major histocompatibility complex), at higher temperatures. In the face of climate change, and as concerns for the spread of emergent chytrid pathogens increase, our resultsmore »provide important functional genomic resources to help understand how these pathogenic fungi may continue to affect amphibian communities globally in the future.

    « less