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 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.

Keywords: chytrid; microbiome networks; skin bacterial communities; skin mucus; skin peptides.

FIG 1

Hypothetical model of innate immune defenses against the chytrid pathogen Bd linked to differential Bd susceptibility in Appalachian salamanders. Illustration elaborated by Nina McDonnell.

FIG 2

Mucosome function (measure of the pathogen-killing ability of skin mucus samples) against the chytrid pathogens Bd and Bsal among four salamander species. Each gray point represents an individual sample.

FIG 3

Relationship between Bd infection intensity and mucosome function (measure of the pathogen-killing ability of skin mucus samples) on salamanders. Species samples were pooled to performed hurdle model. The continuous lines indicate the predicted fit and the shaded areas are 95% confidence intervals. Samples were coded by color and shape to observed species samples: green triangles = E. bislineata, blue squares = D. monticola and purple cross = N. viridescens.

FIG 4

Non-Metric Multidimensional Scaling plots (NMDS) based on the (a) unweighted UniFrac and (b) weighted UniFrac showing bacterial beta diversity across salamander species sampled at two localities. Each point represents the bacterial skin community of an individual; point color indicates host species and shape indicates sampling locality. Ellipses show 95% confidence intervals (95% CIs) of each host species.

FIG 5

Venn diagram showing putative Bd-inhibitory ASVs across four salamander species.

FIG 6

Relationship of Bd infection prevalence with putative Bd-inhibitory bacterial richness (a) and relative abundance (b) on salamanders. The continuous lines indicate the predicted fit and the shaded areas are 95% confidence intervals. Samples were coded by color and shape to observed species samples: red circles = P. cinereus, green triangles = E. bislineata, blue squares = D. monticola and purple cross = N. viridescens.

FIG 7

Co-occurrence networks of the skin bacterial communities per salamander species. Nodes represent ASVs. Edges represent significant correlations. Networks on the left side (a, b, c, d) show all nodes colored by bacterial phylum and the nodes that represent hub and nonhub ASVs are shape coded. Networks on the right side (e, f, g, h) show the nodes that represent hub and nonhub ASVs and putative Bd inhibitory ASVs and are color and shape coded. Only positive and negative correlations for Bd inhibitory hubs are highlighted in networks on the right side (e-h).

FIG 8

Features with mass ≥ 1000 and gas-phase charge between +3 to +8 detected in (a) all salamander samples, (b) P. cinereus (POSH samples) and (c) N. viridescens (LEEP samples). To better visualize shared features, features with detection in only 1 sample are not included in plots.