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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.

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Climate change has led to an alarming increase in the frequency and severity of wildfires worldwide. While it is known that amphibians have physiological characteristics that make them highly susceptible to fire, the specific impacts of wildfires on their symbiotic skin bacterial communities (i.e., bacteriomes) and infection by the deadly chytrid fungus, Batrachochytrium dendrobatidis, remain poorly understood. Here, we address this research gap by evaluating the effects of fire on the amphibian skin bacteriome and the subsequent risk of chytridiomycosis. We sampled the skin bacteriome of the Neotropical species Scinax squalirostris and Boana leptolineata in fire and control plots before and after experimental burnings. Fire was linked with a marked increase in bacteriome beta dispersion, a proxy for skin microbial dysbiosis, alongside a trend of increased pathogen loads. By shedding light on the effects of fire on amphibian skin bacteriomes, this study contributes to our broader understanding of the impacts of wildfires on vulnerable vertebrate species.

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By altering the abundance, diversity, and distribution of species-and their pathogens-globalization may inadvertently select for more virulent pathogens. In Brazil's Atlantic Forest, a hotspot of amphibian biodiversity, the global amphibian trade has facilitated the co-occurrence of previously isolated enzootic and panzootic lineages of the pathogenic amphibian-chytrid (Batrachochytrium dendrobatidis, 'Bd') and generated new virulent recombinant genotypes ('hybrids'). Epidemiological data indicate that amphibian declines are most severe in hybrid zones, suggesting that coinfections are causing more severe infections or selecting for higher virulence. We investigated how coinfections involving these genotypes shapes virulence and transmission. Overall, coinfection favored the more virulent and competitively superior panzootic genotype, despite dampening its transmission potential and overall virulence. However, for the least virulent and least competitive genotype, coinfection increased both overall virulence and transmission. Thus, by integrating experimental and epidemiological data, our results provide mechanistic insight into how globalization can select for, and propel, the emergence of introduced hypervirulent lineages, such as the globally distributed panzootic lineage of Bd.

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BACKGROUND: Host microbiomes may differ under the same environmental conditions and these differences may influence susceptibility to infection. Amphibians are ideal for comparing microbiomes in the context of disease defense because hundreds of species face infection with the skin-invading microbe Batrachochytrium dendrobatidis (Bd), and species richness of host communities, including their skin bacteria (bacteriome), may be exceptionally high. We conducted a landscape-scale Bd survey of six co-occurring amphibian species in Brazil's Atlantic Forest. To test the bacteriome as a driver of differential Bd prevalence, we compared bacteriome composition and co-occurrence network structure among the six focal host species. RESULTS: Intensive sampling yielded divergent Bd prevalence in two ecologically similar terrestrial-breeding species, a group with historically low Bd resistance. Specifically, we detected the highest Bd prevalence in Ischnocnema henselii but no Bd detections in Haddadus binotatus. Haddadus binotatus carried the highest bacteriome alpha and common core diversity, and a modular network partitioned by negative co-occurrences, characteristics associated with community stability and competitive interactions that could inhibit Bd colonization. CONCLUSIONS: Our findings suggest that community structure of the bacteriome might drive Bd resistance in H. binotatus, which could guide microbiome manipulation as a conservation strategy to protect diverse radiations of direct-developing species from Bd-induced population collapses.

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Amphibians breeding in aquatic environments may encounter a myriad of threats during their life cycle. One species known to prey on native amphibians in aquatic habitats is the invasive North American bullfrog Lithobates catesbeianus, which, besides being a voracious predator and competitor, often acts as a pathogen carrier and disease superspreader because it tolerates high infection loads of the frog-killing fungus Batrachochytrium dendrobatidis (Bd). Here, we hypothesized that the presence of the bullfrogs in microcosms should either (1) decrease Bd disease severity in native frog species by discouraging them from using the aquatic environment, or (2) increase the mortality of the native species. We tested these 2 mutually exclusive hypotheses by co-housing the snouted treefrog Scinax x-signatus (native to our study area) with L. catesbeianus in the laboratory, exposing them to Bd, and using qPCR analysis to quantify the resulting Bd infection loads in the native frogs. Our experiment had the following replicated treatments: (1) native-only treatment (3 individuals of S. x-signatus), (2) native-predominant treatment (2 S. x-signatus + 1 L. catesbeianus), and (3) exotic-predominant treatment (1 S. x-signatus + 2 L. catesbeianus). We found that Bd infection loads in the native S. x-signatus were highest in the native-only treatment, and lowest in the exotic-predominant treatment, indicating that bullfrogs may discourage native frogs from occupying the aquatic habitat, thus reducing encounter rates between native frogs and the waterborne pathogen. This effect could be driven by the bullfrogs' predatory behavior and their high philopatry to aquatic habitats. Our results highlight that predation risk adds to the complexity of host-species interactions in Bd epidemiology.

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Habitat fragmentation and infectious diseases threaten wildlife globally, but the interactions of these threats are poorly understood. For instance, while habitat fragmentation can impact genetic diversity at neutral loci, the impacts on disease-relevant loci are less well-studied. We examined the effects of habitat fragmentation in Brazil's Atlantic Forest on amphibian genetic diversity at an immune locus related to antigen presentation and detection (MHC IIB Exon 2). We used a custom high-throughput assay to sequence a fragment of MHC IIB and quantified Batrachochytrium dendrobatidis (Bd) infections in six frog species in two Atlantic Forest regions. Habitat fragmentation was associated with genetic erosion at MHC IIB Exon 2. This erosion was most severe in forest specialists. Significant Bd infections were detected only in one Atlantic Forest region, potentially due to relatively higher elevation. In this region, forest specialists showed an increase in both Bd prevalence and infection loads in fragmented habitats. Reduced population-level MHC IIB diversity was associated with increased Bd infection risk. On the individual level, MHC IIB heterozygotes exhibited a trend toward reduced Bd infection risk, although this was marginally non-significant. Our results suggest that habitat fragmentation increases Bd infection susceptibility in amphibians, mediated at least in part through erosion of immunogenetic diversity. Our findings have implications for management of fragmented populations in the face of emerging infectious diseases.

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The effects of exercise training on oxidative stress in gastrocnemius of rats with pulmonary hypertension were studied. Four groups were established: sedentary control (SC), sedentary monocrotaline (SM), trained control (TC), trained monocrotaline (TM). Exercise was applied for 4 weeks, 5 days/week, 50-60 min/session, at 60% of VO2 max. Right ventricular (RV) pressures were measured, heart and gastrocnemius were removed for morphometric/biochemical analysis. Lipid peroxidation (LPO), H2O2, GSH/GSSG, and activity/expression of antioxidant enzymes were evaluated. Increased RV hypertrophy, systolic and end-diastolic pressures (RVEDP) were observed in SM animals, and the RVEDP was decreased in TM vs. SM. H2O2, SOD-1, and LPO were higher in the SM group than in SC. In TM, H2O2 was further increased when compared to SM, with a rise in antioxidant defences and a decrease in LPO. GSH/GSSG was higher only in the TC group. Exercise induced an efficient antioxidant adaptation, preventing oxidative damage to lipids.

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Abstract Here, we summarize examples of significant advances in amphibian research supported by the São Paulo Research Foundation (FAPESP), focusing on recent discoveries in the fields of community ecology, habitat change, infection diseases, and multipurpose DNA sequencing. We demonstrated that FAPESP has been fundamental not only by directly funding research projects and scholarships, but also through its science training policy, fostering international collaborations with world-class research institutions, improving and consolidating new lines of research that often depended on a synergetic combination of different knowledge and complex tools. We emphasized that future studies will continue to focus on basic questions, such as description of new species, as well as taxonomic and systematic corrections. Furthermore, we also expect that there will be a strong integration among different disciplines using novel bioinformatics tools and modeling approaches, such as machine learning. These new approaches will be critical to further develop our understanding of foundational questions of amphibian life-history trait variation, disease transmission, community assembly, biogeography, and population forecasts under different global change scenarios such as agricultural expansion, agrochemical use, habitat loss, and climate change.

Resumo No presente estudo apresentamos exemplos de avanços significativos nas pesquisas com anfíbios financiadas pela Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), focando em descobertas recentes nos campos de ecologia de comunidades, modificação do habitat, doenças infecciosas e o sequenciamento de DNA com múltiplos propósitos. Demonstramos que a FAPESP tem sido fundamental não somente pelo financiamento direto de projetos de pesquisa e bolsas de estudo, mas também através de sua política de formação científica, fomentando colaborações internacionais com instituições de pesquisa de excelência mundial, melhorando e consolidando novas linhas de pesquisa que frequentemente dependem da combinação sinérgica entre diferentes linhas de conhecimento e ferramentas complexas. Enfatizamos que futuros estudos continuem com foco em questões básicas, como a descrição de novas espécies, bem como correções taxonômicas e sistemáticas. Além disso, esperamos uma forte integração entre diferentes disciplinas usando novas ferramentas de bioinformática e abordagens de modelagem, como o aprendizado de máquina. Essas novas abordagens serão críticas para desenvolver ainda mais nossa compreensão a respeito de questões fundamentais sobre as características da história de vida dos anfíbios, transmissão de doenças, estrutura de comunidades, biogeografia e previsões populacionais em diferentes cenários de mudanças globais, como a expansão da agricultura, uso de agrotóxicos, perda de habitat e mudanças climáticas.

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Amphibian skin bacteria may confer protection against the fungus Batrachochytrium dendrobatidis (Bd), but responses of skin bacteria to different Bd lineages are poorly understood. The global panzootic lineage (Bd-GPL) has caused amphibian declines and extinctions globally. However, other lineages are enzootic (Bd-Asia-2/Brazil). Increased contact rates between Bd-GPL and enzootic lineages via globalization pose unknown consequences for host-microbiome-pathogen dynamics. We conducted a laboratory experiment and used 16S rRNA amplicon-sequencing to assess: (i) whether two lineages (Bd-Asia-2/Brazil and Bd-GPL) and their recombinant, in single and mixed infections, differentially affect amphibian skin bacteria; (ii) and the changes associated with the transition to laboratory conditions. We determined no clear differences in bacterial diversity among Bd treatments, despite differences in infection intensity. However, we observed an additive effect of mixed infections on bacterial alpha diversity and a potentially antagonistic interaction between Bd genotypes. Additionally, observed changes in community composition suggest a higher ability of Bd-GPL to alter skin bacteria. Lastly, we observed a drastic reduction in bacterial diversity and a change in community structure in laboratory conditions. We provide evidence for complex interactions between Bd genotypes and amphibian skin bacteria during coinfections, and expand on the implications of experimental conditions in ecological studies.

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