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Our current understanding of vertebrate skin and gut microbiomes, and their vertical transmission, remains incomplete as major lineages and varied forms of parental care remain unexplored. The diverse and elaborate forms of parental care exhibited by amphibians constitute an ideal system to study microbe transmission, yet investigations of vertical transmission among frogs and salamanders have been inconclusive. In this study, we assess bacteria transmission inHerpele squalostoma,an oviparous direct-developing caecilian in which females obligately attend juveniles that feed on their mother’s skin (dermatophagy).

We used 16S rRNA amplicon-sequencing of the skin and gut of wild caughtH. squalostomaindividuals (males, females, including those attending juveniles) as well as environmental samples. Sourcetracker analyses revealed that juveniles obtain an important portion of their skin and gut bacteria communities from their mother. The contribution of a mother’s skin to the skin and gut of her respective juveniles was much larger than that of any other bacteria source. In contrast to males and females not attending juveniles, only the skins of juveniles and their mothers were colonized by bacteria taxa Verrucomicrobiaceae, Nocardioidaceae, and Erysipelotrichaceae. In addition to providing indirect evidence for microbiome transmission linked to parental care among amphibians, our study also points to noticeable differences between the skin and gut communities ofH. squalostomaand that of many frogs and salamanders, which warrants further investigation.

Our study is the first to find strong support for vertical bacteria transmission attributed to parental care in a direct-developing amphibian species. This suggests that obligate parental care may promote microbiome transmission in caecilians.

 

Anthropogenic habitat disturbance is fundamentally altering patterns of disease transmission and immunity across the vertebrate tree of life. Most studies linking anthropogenic habitat change and disease focus on habitat loss and fragmentation, but these processes often lead to a third process that is equally important:habitat split. Defined as spatial separation between the multiple classes of natural habitat that many vertebrate species require to complete their life cycles, habitat split has been linked to population declines in vertebrates, e.g. amphibians breeding in lowland aquatic habitats and overwintering in fragments of upland terrestrial vegetation. Here, we link habitat split to enhanced disease risk in amphibians (i) by reviewing the biotic and abiotic forces shaping elements of immunity and (ii) through a spatially oriented field study focused on tropical frogs. We propose a framework to investigate mechanisms by which habitat split influences disease risk in amphibians, focusing on three broad host factors linked to immunity: (i) composition of symbiotic microbial communities, (ii) immunogenetic variation, and (iii) stress hormone levels. Our review highlights the potential for habitat split to contribute to host‐associated microbiome dysbiosis, reductions in immunogenetic repertoire, and chronic stress, that often facilitate pathogenic infections and disease in amphibians and other classes of vertebrates. We highlight that targeted habitat‐restoration strategies aiming to connect multiple classes of natural habitats (e.g. terrestrial–freshwater, terrestrial–marine, marine–freshwater) could enhance priming of the vertebrate immune system through repeated low‐load exposure to enzootic pathogens and reduced stress‐induced immunosuppression.

 

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 linear 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.

 

Abstract Batrachochytrium salamandrivorans ( Bsal ) is a fungal pathogen of amphibians that is emerging in Europe and could be introduced to North America through international trade or other pathways. To evaluate the risk of Bsal invasion to amphibian biodiversity, we performed dose-response experiments on 35 North American species from 10 families, including larvae from five species. We discovered that Bsal caused infection in 74% and mortality in 35% of species tested. Both salamanders and frogs became infected and developed Bsal chytridiomycosis. Based on our host susceptibility results, environmental suitability conditions for Bsal , and geographic ranges of salamanders in the United States, predicted biodiversity loss is expected to be greatest in the Appalachian Region and along the West Coast. Indices of infection and disease susceptibility suggest that North American amphibian species span a spectrum of vulnerability to Bsal chytridiomycosis and most amphibian communities will include an assemblage of resistant, carrier, and amplification species. Predicted salamander losses could exceed 80 species in the United States and 140 species in North America. 

The immune equilibrium model suggests that exposure to microbes during early life primes immune responses for pathogen exposure later in life. While recent studies using a range of gnotobiotic (germ-free) model organisms offer support for this theory, we currently lack a tractable model system for investigating the influence of the microbiome on immune system development. Here, we used an amphibian species ( Xenopus laevis ) to investigate the importance of the microbiome in larval development and susceptibility to infectious disease later in life. We found that experimental reductions of the microbiome during embryonic and larval stages effectively reduced microbial richness, diversity and altered community composition in tadpoles prior to metamorphosis. In addition, our antimicrobial treatments resulted in few negative effects on larval development, body condition, or survival to metamorphosis. However, contrary to our predictions, our antimicrobial treatments did not alter susceptibility to the lethal fungal pathogen Batrachochytrium dendrobatidis ( Bd ) in the adult life stage. While our treatments to reduce the microbiome during early development did not play a critical role in determining susceptibility to disease caused by Bd in X. laevis , they nevertheless indicate that developing a gnotobiotic amphibian model system may be highly useful for future immunological investigations. This article is part of the theme issue ‘Amphibian immunity: stress, disease and ecoimmunology’. 

The amphibian chytrid fungus, Batrachochytrium salamandrivorans ( Bsal ) threatens salamander biodiversity. The factors underlying Bsal susceptibility may include glucocorticoid hormones (GCs). The effects of GCs on immunity and disease susceptibility are well studied in mammals, but less is known in other groups, including salamanders. We used Notophthalmus viridescens (eastern newts) to test the hypothesis that GCs modulate salamander immunity. We first determined the dose required to elevate corticosterone (CORT; primary GC in amphibians) to physiologically relevant levels. We then measured immunity (neutrophil lymphocyte ratios, plasma bacterial killing ability (BKA), skin microbiome, splenocytes, melanomacrophage centres (MMCs)) and overall health in newts following treatment with CORT or an oil vehicle control. Treatments were repeated for a short (two treatments over 5 days) or long (18 treatments over 26 days) time period. Contrary to our predictions, most immune and health parameters were similar for CORT and oil-treated newts. Surprisingly, differences in BKA, skin microbiome and MMCs were observed between newts subjected to short- and long-term treatments, regardless of treatment type (CORT, oil vehicle). Taken together, CORT does not appear to be a major factor contributing to immunity in eastern newts, although more studies examining additional immune factors are necessary. This article is part of the theme issue ‘Amphibian immunity: stress, disease and ecoimmunology’. 

With emerging diseases on the rise, there is an urgent need to identify and understand novel mechanisms of prophylactic protection in vertebrate hosts. Inducing resistance against emerging pathogens through prophylaxis is an ideal management strategy that may impact pathogens and their host-associated microbiome. The host microbiome is recognized as a critical component of immunity, but the effects of prophylactic inoculation on the microbiome are unknown. In this study, we investigate the effects of prophylaxis on host microbiome composition, focusing on the selection of anti-pathogenic microbes contributing to host acquired immunity in a model host–fungal disease system, amphibian chytridiomycosis. We inoculated larval Pseudacris regilla against the fungal pathogen Batrachochytrium dendrobatidis ( Bd ) with a Bd metabolite-based prophylactic. Increased prophylactic concentration and exposure duration were associated with significant increases in proportions of putatively Bd -inhibitory host-associated bacterial taxa, indicating a protective prophylactic-induced shift towards microbiome members that are antagonistic to Bd. Our findings are in accordance with the adaptive microbiome hypothesis, where exposure to a pathogen alters the microbiome to better cope with subsequent pathogen encounters. Our study advances research on the temporal dynamics of microbiome memory and the role of prophylaxis-induced shifts in microbiomes contributing to prophylaxis effectiveness. This article is part of the theme issue ‘Amphibian immunity: stress, disease and ecoimmunology’. 

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.