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Abstract Ongoing amphibian population declines are caused by factors such as climate change, habitat destruction, pollution and infectious diseases not limited to chytridiomycosis. Unfortunately, action is taken against these factors once population collapses are underway. To avoid these post hoc responses, wildlife endocrinology aims to analyse physiological mediators that predict future population declines to inform wildlife management. Mediators typically investigated are stress hormones known as glucocorticoids, which are produced by the Hypothalamus—Pituitary—Interrenal axis (HPI axis). The HPI axis is the part of the endocrine system that helps amphibians cope with stress. Chronic increases in glucocorticoids due to stress can lead to immune dysfunction, which makes amphibians more susceptible to infectious diseases. Despite this predictive potential of glucocorticoids, interpretation of glucocorticoid data is confounded by sampling design and type. Glucocorticoid monitoring classically involves blood sampling, which is not widely applicable in amphibians as some are too small or delicate to sample, and repeated samples are often valued. To address this, we tried to validate skin swabbing via corticosterone (CORT) and adrenocorticotropin hormone (ACTH) injections in adults of two amphibian species: Eastern red-spotted newts, Notophthalmus viridescens viridescens, with natural skin infections with Batrachochytrium dendrobatidis (Bd) upon collection in the field, and Northern leopard frogs, Rana (Lithobates) pipiens, raised in captivity and naïve to Bd exposure. Further, we determined the predictive potential of skin glucocorticoids on Bd load in the field via correlations in Eastern red-spotted newts. We found that hormones present in the skin are not related to the HPI axis and poorly predict infection load; however, skin hormone levels strongly predicted survival in captivity. Although skin swabbing is not a valid method to monitor HPI axis function in these species, the hormones present in the skin still play important roles in organismal physiology under stressful conditions relevant to wildlife managers. 

A diverse metabolome exists on amphibian skin that mediates interactions between hosts and skin microbiomes. Tetrodotoxin is one such metabolite that occurs across a variety of taxa, and is particularly well studied in newts of the genusTarichathat are susceptible to infection with chytrid fungi. The interaction of tetrodotoxin with the skin microbiome, including pathogenic fungi, is not well understood, and here we describe these patterns across 12 populations ofTaricha granulosaandT. torosain Washington, Oregon, and California. We found no correlation of TTX andBatrachochytrium dendrobatidis(Bd) infection in eitherT. granulosaorT. torosa, a pattern inconsistent with a previous study. In addition, TTX, but not Bd, was significantly correlated with the skin microbiome composition inT. granulosa. InT. torosa, however, Bd, but not TTX, was correlated with the skin microbiome structure. The relationship between TTX and skin microbiome composition differed between species, with significant correlations observed only inT. granulosa, which exhibited higher TTX concentrations. We also detected significantly higher abundances of bacterial taxa (e.g., Pseudomonadaceae) associated with TTX production in newts with higher skin TTX. These taxa (ASVs matchingAeromonas, Pseudomonas, Shewanella, andSphingopyxis) were associated with all body sites of previously sampledT. granulosa, but not found in soil samples. Our results suggest that toxins can shape the newt skin microbiome and may influence pathogen infection through indirect mechanisms, as TTX showed no direct inhibition of Bd orB. salamandrivoransgrowth. 

Some of the amphibian populations in Panama are demonstrating slow recovery decades after severe declines caused by the invasion of the fungal pathogenBatrachochytrium dendrobatidis(Bd). However, new species remain to be described and assessed for the mechanisms of disease resilience. We identified seven skin defense peptides from a presumably novel leopard frog species in the Tabasará range, at Buäbti (Llano Tugrí), Ngäbe-Buglé Comarca, and Santa Fe, Veraguas, Panama, herein called the Ngäbe-Buglé leopard frog. Two of the peptides were previously known: brevinin-1BLb fromRana (Lithobates) blairiand a previously hypothesized “ancestral” peptide, ranatuerin-2BPa. We hypothesized that the peptides are active againstBdand shape the microbiome such that the skin bacterial communities are more similar to those of other leopard frogs than of co-occurring host species. Natural mixtures of the collected skin peptides showed a minimum inhibitory concentration againstBdof 100 μg/ml, which was similar to that of other leopard frogs that have been tested. All sampled individuals hosted high intensity of infection withBd. We sampled nine other amphibian species in nearby habitats and found lower prevalence and intensities ofBdinfection. In addition to the pathogen load, the skin microbiomes were examined using 16S rRNA gene targeted amplicon sequencing. When compared to nine co-occurring amphibians, the Ngäbe-Buglé leopard frog had similar skin bacterial richness and anti-Bdfunction, but the skin microbiome structure differed significantly among species. The community composition of the bacterial skin communities was strongly associated with theBdinfection load. In contrast, the skin microbiome composition of the Ngäbe-Buglé leopard frog was similar to that of five North American leopard frog populations and the sympatric and congenericRana (Lithobates) warszewitschii, with 29 of the 46 core bacteria all demonstrating anti-Bdactivity in culture. Because of the highBdinfection load and prevalence in the Ngäbe-Buglé leopard frog, we suggest that treatment to reduce theBdload in this species might reduce the chytridiomycosis risk in the co-occurring amphibian community, but could potentially disrupt the evolution of skin defenses that provide a mechanism for disease resilience in this species. 

The emerging fungal pathogenBatrachochytrium salamandrivorans(Bsal) threatens the diversity of amphibians, particularly in North America where it is projected to invade. Amphibian skin defenses include a mucosal layer containing microorganisms that can potentially modulate host response to pathogens such asBsal. In this study, we focused on the composition of the skin microbiome across life stages of spotted salamanders (Ambystoma maculatum). We also evaluated the stress hormone corticosterone and skin microbiome response to inoculations withBsaland probiotics at both the larval and juvenile developmental stages, and the response to different environmental conditions. Results indicated that both bacterial and fungal communities found on the skin significantly differed in structure and diversity between life stages ofA. maculatum. Exposure to three different probiotics (Bacillus thuringiensis,Chryseobacterium rhizoplanae, andPenicilliumsp.) andBsalevoked shifts in the microbiome of larvae and juveniles, and the metabolite profile of the larval mucosal layer ofA. maculatum. Despite changes in the microbiome, all tested probiotics andBsalwere unable to persist on the skin. Larval bacterial microbiomes shifted in response toBsalandB. thuringiensiswith no significant impacts on antifungal function or bacteria richness, however fungi strongly responded toBsalandB. thuringiensisapplication. This indicates that developmental shifts in the microbiome can be initiated by microbial applications such asB. thuringiensis, a widely used mosquito larvicide. Overall, experimental results indicate that life stage, growth and development, and environmental conditions appeared to be the main factors driving changes in the amphibian skin microbiome and potential anti-Batrachochytriumfunction. 

Abstract Background 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 in Herpele squalostoma, an oviparous direct-developing caecilian in which females obligately attend juveniles that feed on their mother’s skin (dermatophagy). Results We used 16S rRNA amplicon-sequencing of the skin and gut of wild caught H. squalostoma individuals (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 of H. squalostoma and that of many frogs and salamanders, which warrants further investigation. Conclusion 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. 

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. 

Bats are widespread mammals that play key roles in ecosystems as pollinators and insectivores. However, there is a paucity of information about bat-associated microbes, in particular their fungal communities, despite the important role microbes play in host health and overall host function. The emerging fungal disease, white-nose syndrome, presents a potential challenge to the bat microbiome and understanding healthy bat-associated taxa will provide valuable information about potential microbiome-pathogen interactions. To address this knowledge gap, we collected 174 bat fur/skin swabs from 14 species of bats captured in five locations in New Mexico and Arizona and used high-throughput sequencing of the fungal internal transcribed (ITS) region to characterize bat-associated fungal communities. Our results revealed a highly heterogeneous bat mycobiome that was structured by geography and bat species. Furthermore, our data suggest that bat-associated fungal communities are affected by bat foraging, indicating the bat skin microbiota is dynamic on short time scales. Finally, despite the strong effects of site and species, we found widespread and abundant taxa from several taxonomic groups including 

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’. 

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’.