Amphibian Skin Research Articles

Harnessing gene-encoded antioxidant peptides from amphibian skin secretions as a novel therapeutic strategy against influenza a virus

Harnessing gene-encoded antioxidant peptides from amphibian skin secretions as a novel therapeutic strategy against influenza a virus

Seasonality influences skin bacterial community structure and anti-Bd function in two anuran species

Microorganisms on amphibian skin reduce disease susceptibility and play an important role in pathogen defense. We hypothesized that anuran skin bacterial communities would change in response to seasonal variation and host species. To test this hypothesis, we used 16S rRNA amplicon sequencing to identify cutaneous bacterial communities of two frogs from the Qinling Mountains of China, Pelophylax nigromaculatus and Nanorana quadranus. We matched the amplicon sequence variants (ASVs) of microbes exhibiting protective effects against the pathogen Batrachochytrium dendrobatidis (Bd), using a database containing over 1900 16S rRNA gene sequences from amphibian skin bacteria. The results showed that seasonal variation had a stronger effect than host species on the structure (alpha-diversity, beta-diversity, species composition and abundance, and biomarkers) and anti-Bd function of cutaneous bacterial communities. These communities were highly dynamic but varied similarly between hosts. Their structural similarities were more consistent at the phylum level, but markedly less so at finer taxonomic levels. The highest relative abundance of anti-Bd reads was observed in P. nigromaculatus during summer, but anti-Bd reads were present in both frog species during different seasons. Therefore, the protective function of cutaneous microbial communities appears to be continuous despite between-species differences in anti-Bd ASV abundance. This observation does not directly explain why Bd infections have not been recorded in the region, butprovides important insight on anuran pathogen defense mechanisms. Our findings also suggest that specific seasons can be periods of high infection risk, with major implications for research on amphibian ecology and conservation.

Micromanagement: conditions influencing antipathogen function of the skin microbiome in spotted salamanders, Ambystoma maculatum

The emerging fungal pathogen Batrachochytrium 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 as Bsal. 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 with Bsal and 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 of A. maculatum. Exposure to three different probiotics (Bacillus thuringiensis, Chryseobacterium rhizoplanae, and Penicillium sp.) and Bsal evoked shifts in the microbiome of larvae and juveniles, and the metabolite profile of the larval mucosal layer of A. maculatum. Despite changes in the microbiome, all tested probiotics and Bsal were unable to persist on the skin. Larval bacterial microbiomes shifted in response to Bsal and B. thuringiensis with no significant impacts on antifungal function or bacteria richness, however fungi strongly responded to Bsal and B. thuringiensis application. This indicates that developmental shifts in the microbiome can be initiated by microbial applications such as B. 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-Batrachochytrium function.

Adaptation of skin structures to environmental variations in anurans from southern and southwestern China.

Variations in skin structures can possibly reflect local adaptation to distinct environmental factors. As the primary interface with the surrounding environment, amphibian skin undergoes phenotypic innovations that play a key role in protection, water absorption, and respiration. However, the effects of environmental factors on skin structures have been examined in only a limited number of species. Here, we conducted a comparative analysis of the skin structures of 102 Chinese anuran species across varying geographical distributions and habitat types. Our results revealed that the total volume of granular glands and capillary density in the dorsal skin significantly increased with increasing latitude. We also found that the thickness of calcified layers in both dorsal and ventral skin was positively correlated with annual temperature and negatively correlated with humidity. Additionally, terrestrial species exhibited the largest dorsal granular gland, whereas arboreal species had the smallest one. Likewise, the largest dorsal mucous gland was observed in aquatic species, while the smallest was found in terrestrial species. These results highlighted the importance of understanding the relationship between skin phenotypes and environmental variables and thus providing conservation strategies based on the evolutionary adaptations in anurans. Our study can contribute to the broader knowledge of evolutionary biology in anurans by demonstrating how specific skin traits are linked to survival and fitness across various ecological contexts.

Diverse Relationships between Batrachochytrium Infections and Antimicrobial Peptide Defenses Across Leopard Frog Populations.

Antimicrobial peptides (AMPs) play a fundamental role in the innate defense against microbial pathogens, as well as other immune and non-immune functions. Their role in amphibian skin defense against the pathogenic fungus Batrachochytrium dendrobatidis (Bd) is exemplified by experiments in which depletion of host's stored AMPs increases mortality from infection. Yet, the question remains whether there are generalizable patterns of negative or positive correlations between stored AMP defenses and the probability of infection or infection intensity across populations and species. This study aims to expand on prior field studies of AMP quantities and compositions by correlating stored defenses with an estimated risk of Bd exposure (prevalence and mean infection intensity in each survey) in five locations across the United States and a total of three species. In all locations, known AMPs correlated with the ability of recovered secretions to inhibit Bd in vitro. We found that stored AMP defenses were generally unrelated to Bd infection except in one location where the relative intensity of known AMPs was lower in secretions from infected frogs. In all other locations, known AMP relative intensities were higher in infected frogs. Stored peptide quantity was either positively or negatively correlated with Bd exposure risk. Thus, future experiments coupled with organismal modeling can elucidate whether Bd infection affects secretion/synthesis and will provide insight into how to interpret amphibian ecoimmunology studies of AMPs. We also demonstrate that future AMP isolating and sequencing studies can focus efforts by correlating mass spectrometry peaks to inhibitory capacity using linear decomposition modeling.

Assessing the Efficacy of Protease Inactivation for the Preservation of Bioactive Amphibian Skin Peptides.

The skin of amphibians is a rich source of peptides with a wide range of biological activities. They are stored in secretory granules in an inactive form. Upon stimulation, they are secreted together with proteases into the skin. Once activated, they rapidly exert their biological effects, including fighting microorganisms and predators, while their excess is immediately destroyed by the released proteases. To keep bioactive peptides in their initial form, it is necessary to inhibit these enzymes. Several inhibitors for this purpose have previously been mentioned; however, there has not been any reliable comparison of their efficiency so far. Here, we studied the efficiency of methanol and hydrochloric and formic acids, as well as phenylmethylsulfonyl fluoride, in the inhibition of nine frog peptides with the known sequence, belonging to five families in the secretion of Pelophylax esculentus. The results demonstrated that methanol had the highest inhibitory efficiency, while phenylmethylsulfonyl fluoride was the least efficient, probably due to its instability in aqueous media. Possible cleavages between certain amino acid residues in the sequence were established for each of the inhibitors. These results may be helpful for future studies on the nature of proteases and on prediction of the possible cleavage sites in novel peptides.

Bioactivity of synthetic peptides from Ecuadorian frog skin secretions against Leishmania mexicana, Plasmodium falciparum, and Trypanosoma cruzi.

Chagas disease, leishmaniasis, and malaria are major parasitic diseases disproportionately affecting the underprivileged population in developing nations. Finding new, alternative anti-parasitic compounds to treat these diseases is crucial because of the limited number of options currently available, the side effects they cause, the need for long treatment courses, and the emergence of drug-resistant parasites. Anti-microbial peptides (AMPs) derived from amphibian skin secretions are small bioactive molecules capable of lysing the cell membrane of pathogens while having low toxicity against human cells. Here, we report the anti-parasitic activity of five AMPs derived from skin secretions of three Ecuadorian frogs: cruzioseptin-1, cruzioseptin-4 (CZS-4), and cruzioseptin-16 from Cruziohyla calcarifer; dermaseptin-SP2 from Agalychnis spurrelli; and pictuseptin-1 from Boana picturata. These five AMPs were chemically synthesized. Initially, the hemolytic activity of CZS-4 and its minimal inhibitory concentration against Escherichia coli, Staphylococcus aureus, and Candida albicans were determined. Subsequently, the cytotoxicity of the synthetic AMPs against mammalian cells and their anti-parasitic activity against Leishmania mexicana promastigotes, erythrocytic stages of Plasmodium falciparum and mammalian stages of Trypanosoma cruzi were evaluated in vitro. The five AMPs displayed activity against the pathogens studied, with different levels of cytotoxicity against mammalian cells. In silico molecular docking analysis suggests this bioactivity may occur via pore formation in the plasma membrane, resulting in microbial lysis. CZS-4 displayed anti-bacterial, anti-fungal, and anti-parasitic activities with low cytotoxicity against mammalian cells. Further studies about this promising AMP are required to gain a better understanding of its activity.IMPORTANCEChagas disease, malaria, and leishmaniasis are major tropical diseases that cause extensive morbidity and mortality, for which available treatment options are unsatisfactory because of limited efficacy and side effects. Frog skin secretions contain molecules with anti-microbial properties known as anti-microbial peptides. We synthesized five peptides derived from the skin secretions of different species of tropical frogs and tested them against cultures of the causative agents of these three diseases, parasites known as Trypanosoma cruzi, Plasmodium falciparum, and Leishmania mexicana. All the different synthetic peptides studied showed activity against one of more of the parasites. Peptide cruzioseptin-4 is of special interest since it displayed intense activity against parasites while being innocuous against cultured mammalian cells, which indicates it does not simply hold general toxic properties; rather, its activity is specific against the parasites.

Bacillus thuringiensis volatile organic compounds (VOCs) inhibit the growth of the pathogenic amphibian fungus, Batrachochytrium dendrobatidis while autoclaved secondary metabolites promote fungal growth

Environmental microorganisms have a complex, dynamic relationship with flora and fauna, with consequences for the host organism that range from immunogenic to immunoprotective. The pathogenic chytrid fungus, Batrachochytrium dendrobatidis (Bd), can infect the skin of amphibians and may lead to the deadly disease, Chytridiomycosis. Some skin-associated microorganisms of amphibians have the potential to prevent or mitigate Bd infection by producing anti-fungal metabolites. Yet, the majority of environmentally derived bacteria that may inhabit amphibian skin have not been investigated for their interaction with Bd. The soil-dwelling bacteria, Bacillus thuringiensis (Bt), is a ubiquitous species that is widely applied as a natural insecticide and produces anti-fungal metabolites as a byproduct of metabolism. With the increased use of Bt-based insecticides and the potential benefit of antifungal properties, we investigated the effects of Bt against Bd. This study investigates (1) the impact of Bt volatile metabolites on the growth of Bd and (2) the impact of autoclaved Bt products on the growth of Bd in vitro. Our findings showed growth inhibition of Bd (p < 0.001) by Bt volatile organic compounds (VOC) when cultured in split petri plates, relative to the control. Yet, in a liquid culture of Bd, autoclaved secondary metabolites from Bt demonstrated a significant increase in Bd growth (p < 2.2e-16). Together these results suggest that Bt may produce metabolites that beneficially inhibit Bd in some conditions while negatively enhancing the growth of the Bd in others.

Expression patterns of melanin-related genes are linked to crypsis and conspicuousness in a pumpkin toadlet.

Colour signals play pivotal roles in different communication systems, and the evolution of these characters has been associated with behavioural ecology, integumentary production processes and perceptual mechanisms of the species involved. Here, we present the first insight into the molecular and histological basis of skin colour polymorphism within a miniaturized species of pumpkin toadlet, potentially representing the lowest size threshold for colour polytypism in tetrapods. Brachycephalus actaeus exhibits a coloration ranging from cryptic green to conspicuous orange skin, and our findings suggest that colour morphs differ in their capability to be detected by potential predators. We also found that the distribution and abundance of chromatophores are variable in the different colour morphs. The expression pattern of coloration related genes was predominantly associated with melanin synthesis (including dct, edn1, mlana, oca2, pmel, slc24a5, tyrp1 and wnt9a). Up-regulation of melanin genes in grey, green and brown skin was associated with higher melanophore abundance than in orange skin, where xanthophores predominate. Our findings provide a significant foundation for comparing and understanding the diverse pathways that contribute to the evolution of pigment production in the skin of amphibians.

Cutaneous shedding in amphibians causes shifts in bacterial microbiomes.

Considerable research has focused on microbes on amphibian skin, as they act as the first line of defense against invading pathogens. This effort has generated substantial data on patterns across species, space, time, and ontogeny, alongside a growing list of beneficial antifungal symbionts. Though there is evidence of stability in amphibian skin microbial communities, there is also an indication that regular skin shedding reduces cultivable bacteria, with regrowth and recolonization in the period between sheds. This suggests that skin communities are in constant flux, and we lack an understanding of how the membership and structure of those communities are affected by shedding events. In this study, we conducted experiments on cane toads (Rhinella marina) to investigate the influence of shedding on skin microbiomes. We first used quantitative PCR to verify a positive correlation between bacterial loads and time in the days after shedding. We then resampled individuals over time to describe changes in community composition in the 38 h after shedding using amplicon sequencing. Similar to trends of bacterial loads, we found increases in alpha diversity over time after shedding, suggesting that shedding reduces bacterial diversity as it knocks down bacterial loads. During the 38-h period, community structure became similar to pre-shed communities in some individuals, but there was no consistent pattern in structural changes among individuals. In light of the amphibian chytridiomycosis pandemic, understanding how physiological events such as skin shedding affect beneficial bacteria and communities on amphibians would provide important insight into amphibian ecology.

Screening the antimicrobial, cytotoxic and hemolytic effects of skin-parotoid gland secretions of 13 taxa from six species and one hybrid population of Anatolian endemic Lycian salamanders (Genus: Lyciasalamandra)

Background and purpose: The diverse bioactive content in the skin and parotoid glands of amphibians makes them unique and highly potential sources for pharmacological developments. The main aim of the study is to determine the total protein amounts, in vitro cytotoxicities, antimicrobial activities and hemolytic effects of skin-parotoid secretions of these endemic Lycian salamanders (Genus: Lyciasalamandra). Materials and methods: Lycian salamander specimens of 13 taxa from six species and one hybrid population are collected from southwestern Anatolia. Skin-parotoid secretions were obtained, clarified, supernatants snapfrozen then lyophilized. Total protein amounts were determined by BCA assay kit. The cytotoxicity was determined on eight different cell lines (one healthy and seven cancerous) using MTT assay. The antimicrobial activity was assesed using 11 different microorganisms (nine bacteria and two yeast) from six genus by MIC method. Hemolytic effects were measured on rabbit red blood cells. Results: Lycian salamanders’ skin-parotoid gland secretions showed variable cytotoxic effects on all cell lines with IC50 values from 5.41 ± 0.17 μg/ml to ineffective. MIC results of antimicrobial activity tests were also variable and from 3.9 μg/mL to non-effective. Studied skin-parotoid secretions have no hemolytic activities on rabbit red blood cells at concentrations of 0.5, 5 and 50 μg/mL. Conclusions: Skin-parotoid gland secretions of Anatolian endemic Lycian salamanders have sufficient and remarkable therapeutic potential to be candidate sources of natural and biologically active substances for novel drug discoveries.

Оцінка можливості та перспективності використання гістологічних та морфометричних методів в учнівських іхтіологічних та батрахологічних дослідженнях

Resume. The methods used by the students of the Zoology Section of the Volyn Regional Junior Academy of Sciences in 1996–2023 and 48 participants of the II (regional) stage of the All-Ukrainian competition-defense of research works of students-members of the Junior Academy of Sciences in the study of fish and amphibians were analyzed. It has been established that morphological studies make up a small proportion of the work, which is due to both the lack of necessary equipment in general secondary education institutions and the lack of methodological recommendations that would ensure the successful implementation of scientific work on the available equipment. Promising areas of student research are the study of fish and amphibian blood, amphibian skin, and fish respiratory organs using histological and morphometric methods. It is recommended that morphometric methods be more widely used in ecological and faunal studies. Conducting such research by students with the use of morphometric methods promotes the development of a scientific outlook, clear creative thinking, and facilitates the realization of interdisciplinary connections.

Environmental factors and host sex influence the skin microbiota structure of Hong Kong newt (Paramesotriton hongkongensis) in a coldspot of chytridiomycosis in subtropical East Asia.

Chytridiomycosis, an infectious skin disease caused by the chytrid fungi, Batrachochytrium dendrobatidis and B. salamandrivorans, poses a significant threat to amphibian biodiversity worldwide. Antifungal bacteria found on the skin of chytrid-resistant amphibians could potentially provide defense against chytridiomycosis and lower mortality rates among resistant individuals. The Hong Kong newt (Paramesotriton hongkongensis) is native to East Asia, a region suspected to be the origin of chytrids, and has exhibited asymptomatic infection, suggesting a long-term coexistence with the chytrids. Therefore, the skin microbiota of this resistant species warrant investigation, along with other factors that can affect the microbiota. Among the 149 newts sampled in their natural habitats in Hong Kong, China, putative antifungal bacteria were found in all individuals. There were 314 amplicon sequence variants distributed over 25 genera of putative antifungal bacteria; abundant ones included Acinetobacter, Flavobacterium, and Novosphingobium spp. The skin microbiota compositions were strongly influenced by the inter-site geographical distances. Despite inter-site differences, we identified some core skin microbes across sites that could be vital to P. hongkongensis. The dominant cores included the family Comamonadaceae, family Chitinophagaceae, and class Betaproteobacteria. Moreover, habitat elevation and host sex also exhibited significant effects on skin microbiota compositions. The antifungal bacteria found on these newts offer an important resource for conservation against chytridiomycosis, such as developing probiotic treatments for susceptible species.

Glove decontamination procedures to prevent pathogen and DNA cross-contamination among frogs.

Working with aquatic organisms often requires handling multiple individuals in a single session, potentially resulting in cross-contamination by live pathogens or DNA. Most researchers address this problem by disposing of gloves between animals. However, this generates excessive waste and may be impractical for processing very slippery animals that might be easier to handle with cotton gloves. We tested methods to decontaminate cotton or nitrile gloves after contamination with cultured Batrachochytrium dendrobatidis (Bd) or after handling heavily Bd-infected Xenopus laevis with layered cotton and nitrile gloves. Bleach eliminated detectable Bd DNA from culture-contaminated nitrile gloves, but gloves retained detectable Bd DNA following ethanol disinfection. After handling a Bd-infected frog, Bd DNA contamination was greatly reduced by removal of the outer cotton glove, after which either bleach decontamination or ethanol decontamination followed by drying hands with a paper towel lowered Bd DNA below the detection threshold of our assay. These results provide new options to prevent pathogen or DNA cross-contamination, especially when handling slippery aquatic organisms. However, tradeoffs should be considered when selecting an animal handling procedure, such as the potential for cotton gloves to abrade amphibian skin or disrupt skin mucus. Disposing of gloves between animals should remain the gold standard for maintaining biosecurity in sensitive situations.

Insights into caudate amphibian skin secretions with a focus on the chemistry and bioactivity of derived peptides.

Amphibians are well-known for their ability to produce and secrete a mixture of bioactive substances in specialized skin glands for the purpose of antibiotic self-protection and defense against predators. Some of these secretions contain various small molecules, such as the highly toxic batrachotoxin, tetrodotoxin, and samandarine. For some time, the presence of peptides in amphibian skin secretions has attracted researchers, consisting of a diverse collection of- to the current state of knowledge- three to 104 amino acid long sequences. From these more than 2000 peptides many are known to exert antimicrobial effects. In addition, there are some reports on amphibian skin peptides that can promote wound healing, regulate immunoreactions, and may serve as antiparasitic and antioxidative substances. So far, the focus has mainly been on skin peptides from frogs and toads (Anura), eclipsing the research on skin peptides of the ca. 700 salamanders and newts (Caudata). Just recently, several novel observations dealing with caudate peptides and their structure-function relationships were reported. This review focuses on the chemistry and bioactivity of caudate amphibian skin peptides and their potential as novel agents for clinical applications.

Amphibian tolerance to arsenic: microbiome-mediated insights

Amphibians are often recognized as bioindicators of healthy ecosystems. The persistence of amphibian populations in heavily contaminated environments provides an excellent opportunity to investigate rapid vertebrate adaptations to harmful contaminants. Using a combination of culture-based challenge assays and a skin permeability assay, we tested whether the skin-associated microbiota may confer adaptive tolerance to tropical amphibians in regions heavily contaminated with arsenic, thus supporting the adaptive microbiome principle and immune interactions of the amphibian mucus. At lower arsenic concentrations (1 and 5 mM As3+), we found a significantly higher number of bacterial isolates tolerant to arsenic from amphibians sampled at an arsenic contaminated region (TES) than from amphibians sampled at an arsenic free region (JN). Strikingly, none of the bacterial isolates from our arsenic free region tolerated high concentrations of arsenic. In our skin permeability experiment, where we tested whether a subset of arsenic-tolerant bacterial isolates could reduce skin permeability to arsenic, we found that isolates known to tolerate high concentrations of arsenic significantly reduced amphibian skin permeability to this metalloid. This pattern did not hold true for bacterial isolates with low arsenic tolerance. Our results describe a pattern of environmental selection of arsenic-tolerant skin bacteria capable of protecting amphibians from intoxication, which helps explain the persistence of amphibian populations in water bodies heavily contaminated with arsenic.

Bacteria from the skin of amphibians promote growth of Arabidopsis thaliana and Solanum lycopersicum by modifying hormone-related transcriptome response

Plants and microorganisms establish beneficial associations that can improve their development and growth. Recently, it has been demonstrated that bacteria isolated from the skin of amphibians can contribute to plant growth and defense. However, the molecular mechanisms involved in the beneficial effect for the host are still unclear. In this work, we explored whether bacteria isolated from three tropical frogs species can contribute to plant growth. After a wide screening, we identified three bacterial strains with high biostimulant potential, capable of modifying the root structure of Arabidopsis thaliana plants. In addition, applying individual bacterial cultures to Solanum lycopersicum plants induced an increase in their growth. To understand the effect that these microorganisms have over the host plant, we analysed the transcriptomic profile of A. thaliana during the interaction with the C32I bacterium, demonstrating that the presence of the bacteria elicits a transcriptional response associated to plant hormone biosynthesis. Our results show that amphibian skin bacteria can function as biostimulants to improve agricultural crops growth and development by modifying the plant transcriptomic responses.

Amphibian skin bacteria display antifungal activity and induce plant defense mechanisms against Botrytis cinerea.

Botrytis cinerea is the causal agent of gray mold, which affects a wide variety of plant species. Chemical agents have been used to prevent the disease caused by this pathogenic fungus. However, their toxicity and reduced efficacy have encouraged the development of new biological control alternatives. Recent studies have shown that bacteria isolated from amphibian skin display antifungal activity against plant pathogens. However, the mechanisms by which these bacteria act to reduce the effects of B. cinerea are still unclear. From a diverse collection of amphibian skin bacteria, three proved effective in inhibiting the development of B. cinerea under in vitro conditions. Additionally, the individual application of each bacterium on the model plant Arabidopsis thaliana, Solanum lycopersicum and post-harvest blueberries significantly reduced the disease caused by B. cinerea. To understand the effect of bacteria on the host plant, we analyzed the transcriptomic profile of A. thaliana in the presence of the bacterium C32I and the fungus B. cinerea, revealing transcriptional regulation of defense-related hormonal pathways. Our study shows that bacteria from the amphibian skin can counteract the activity of B. cinerea by regulating the plant transcriptional responses.

Batrachochytrium dendrobatidis and Hannemania mite’s relationships with Mexican amphibians in disturbed environments

The rapid transformation and pollution of ecosystems have severely impacted biodiversity. Specifically, anthropogenic activities have imposed adverse effects on amphibians, with evidence suggesting that these activities alter parasite and pathogen interactions within their hosts. To investigate these interactions in areas affected by different anthropogenic activities, our study focused on analyzing a pathogen and a parasite known to interact within the amphibian skin (spongy epidermis layer) and both compromising amphibian health: Batrachochytrium dendrobatidis (Bd), a fungus responsible for chytridiomycosis, a disease associated with massive population declines in amphibians and the Hannemania sp. mite in Mexico. Four sampling areas along the Sonora River were selected, representing different human activities: mining, livestock, wastewater discharge, agriculture, and one in an urban zone. We analyzed 135 amphibians across 10 anuran species. Among these, the most abundant species (Lithobates yavapaiensis) exhibited the highest prevalence of both pathogen and parasite (90.1% and 27.3%, respectively) and was significantly associated with the intensity of Bd-infection. The prevalence of Hannemania mites varied significantly across sampling sites as did Bd prevalence and infection load, with the highest Bd load found at the wastewater discharge site. A significant association between the intensity of Bd-infection and both mite abundance and amphibian species was observed when the sampling site was considered. Additionally, sites with Bd-positive individuals and Hannemania parasitism coincide with refractory elements characterized by mechanical or corrosion resistance. The persistence of these elements in the environment, along with the small particle size (&amp;lt;850 nm) found in sediments, poses a potential risk of internalization, bioaccumulation (e.g., Fe, Co, and Ti), and their transfer through the food chain. It is thus essential to consider monitoring environmental and biotic factors that modulate the relationships between parasites, pathogens, and amphibians if we are to propose conservation strategies adapted to disturbed environments.

Nor climate nor human impact factors: Chytrid infection shapes the skin bacterial communities of an endemic amphibian in a biodiversity hotspot.

The bacterial communities of the amphibian skin (i.e., the bacteriome) are critical to the host's innate immune system. However, it is unclear how different drivers can alter this function by modulating the bacteriome's structure. Our aim was to assess the extent to which different host attributes and extrinsic factors influence the structure of the bacterial communities of the skin. Skin bacterial diversity was examined in 148 individuals of the four-eyed frog (Pleurodema thaul) from 16 localities spanning almost 1800 km in latitude. The richness and beta diversity of bacterial families and the richness and abundance of Bd-inhibitory bacterial genera were used to describe their structure. Predictors associated with the host (developmental stage, genetic lineage, individual Batrachochytrium dendrobatidis [Bd] infection status) and the landscape (current climate, degree of anthropogenic disturbance) were used in the statistical modeling in an information theoretical approach. Bd infection and host developmental stage were the only predictors affecting bacteriome richness, with Bd+ individuals and postmetamorphic stages (adults and juveniles) having higher richness than Bd- ones and tadpoles. High diversity in Bd+ individuals is not driven by bacterial genera with known anti-Bd properties. Beta diversity was not affected by Bd infection and was mainly a consequence of bacterial family turnover rather than nestedness. Finally, for those bacterial genera known to have inhibitory effects on chytrid, Bd+ individuals had a slightly higher diversity than Bd- ones. Our study confirms an association between Bd infection and the host developmental stage with the bacterial communities of the skin of P. thaul. Unexpectedly, macroclimate and human impact factors do not seem to play a role in shaping the amphibian skin microbiome. Our study exemplifies that focusing on a single host-parasite system over a large geographic scale can provide essential insights into the factors driving host-parasite-bacteriome interactions.