Impact of wildfire ash on skin and gut microbiomes and survival of Rana dybowskii.

Climate change and human activities escalate the frequency and intensity of wildfires, threatening amphibian habitats and survival; yet, research on these impacts remains limited. Wildfire ash alters water quality, introduces contaminants, and may disrupt microbial communities, impacting gut and skin microbiota; however, the effects on gut and skin microbiota remain unclear. Rana dybowskii were exposed to five concentrations (0 g L-1, 1.25 g L-1, 2.5 g L-1, 5 g L-1, and 10 g L-1) of aqueous extracts of wildfire ashes (AEAs) for 30 days to assess AEAs' metal content, survival, and microbiota diversity via Illumina sequencing. Our results showed that the major elements in ash were Ca > K > Mg > Al > Fe > Na > Mn, while in AEA they were K > Ca > Na > Mg > As > Al > Cu. A significant decrease in amphibian survival rates with increased AEA concentration was shown. The beta diversity analysis revealed distinct shifts in microbiota composition. Notably, bacterial genera associated with potential health risks showed increased abundance in skin microbiota, emphasising the potential for ash exposure to affect amphibian health. Functional prediction analyses revealed significant shifts in metabolic pathways related to health and disease, indicating that wildfire ash exposure may influence amphibian health through changes in microbial functions. This study highlights the urgent need for strategies to mitigate wildfire ash impacts on amphibians, as it significantly alters microbiota and affects their survival and health.

Gut microbiome diversity and function during hibernation and spring emergence in an aquatic frog.

The gut microbiota maintains a deeply symbiotic relationship with host physiology, intricately engaging with both internal (endogenous) and external (exogenous) factors. Anurans, especially those in temperate regions, face the dual challenges of significant external influences like hibernation and complex internal variances tied to different life histories. In our research, we sought to determine whether different life stages (juvenile versus adult) of the Japanese wrinkled frog (Glandirana rugosa) lead to distinct shifts in gut bacterial communities during winter (hibernation) and its subsequent transition to spring. As hypothesized, we observed a more pronounced variability in the gut bacterial diversity and abundance in juvenile frogs compared to their adult counterparts. This suggests that the gut environment may be more resilient or stable in adult frogs during their hibernation period. However, this pronounced difference was confined to the winter season; by spring, the diversity and abundance of gut bacteria in both juvenile and adult frogs aligned closely. Specifically, the variance in gut bacterial diversity and composition between winter and spring appears to mirror the frogs' ecological adaptations. During the hibernation period, a dominance of Proteobacteria suggests an emphasis on supporting intracellular transport and maintaining homeostasis, as opposed to active metabolism in the frogs. Conversely, come spring, an uptick in bacterial diversity coupled with a dominance of Firmicutes and Bacteroidetes points to an upsurge in metabolic activity post-hibernation, favoring enhanced nutrient assimilation and energy metabolism. Our findings highlight that the relationship between the gut microbiome and its host is dynamic and bidirectional. However, the extent to which changes in gut bacterial diversity and composition contribute to enhancing hibernation physiology in frogs remains an open question, warranting further investigation.

Culturable bacterial flora of juveniles of Pelophylax ridibundus (Pallas, 1771) and influence of abiotic factors on diversity.

This study aims to determine the bacterial flora on the skin surface of the juvenile forms of Pelophylax ridibundus inhabiting three different altitudes and examine potential correlations between bacterial diversity, ecological location, and factors. It was attempted to characterize thirty-two bacteria isolated from the Melet River, Sülük Lake, and Çambasi Pond through combined biochemical and molecular methods. Canonical correspondence analysis showed that the most important ecological factors for microorganisms to settle on frog skin were determined as water conductivity and dissolved oxygen amount. The most frequently isolated bacteria belonged to the genera Erwinia and Pseudomonas. Altitude positively affected Exiguobacterium. This first report of skin cultivable bacteria from P. ridibundus juvenile forms natural population improves our knowledge of amphibian skin bacterial flora. This study contributes to a better understanding of their ecology and how this species has survived in an environment modulated by altitude.

Co-infections of Klebsiella pneumoniae and Elizabethkingia miricola in black-spotted frogs (Pelophylax nigromaculatus).

Pelophylax nigromaculatus is a common commercial specie of frogs that generally cultured throughout China. With the application of high-density culture, P. nigromaculatus can be co-infected by two or more pathogens, which thereby induce synergistic influence on the virulence of the infection. In this study, two bacterial strains were simultaneously isolated from diseased frogs by incubating on Luria-Bertani (LB) agar. Isolates were identified as Klebsiella pneumoniae and Elizabethkingia miricola by morphological, physiological and biochemical features, as well as 16S rRNA sequencing and phylogenetic analysis. The whole genome of K. pneumoniae and E. miricola isolates consist single circular chromosome of 5,419,557 bp and 4,215,349 bp, respectively. The genomic sequence analysis further indicated that K. pneumoniae isolate conserved 172 virulent and 349 antibiotic-resistance genes, whereas E. miricola contained 24 virulent and 168 antibiotic resistance genes. In LB broth, both isolates could grow well at 0%-1% NaCl concentration and pH 5-7. Antibiotic susceptibility testing revealed that both K. pneumoniae and E. miricola were resistant to kanamycin, neomycin, ampicillin, piperacillin, carbenicillin, enrofloxacin, norfloxacin and sulfisoxazole. Histopathological studies showed that co-infection caused considerable lesions in the tissues of brain, eye, muscle, spleen, kidney and liver, including cell degeneration, necrosis, hemorrhage and inflammatory cell infiltration. The LD50 of K. pneumoniae and E. miricola isolates were 6.31 × 105 CFU/g and 3.98 × 105 CFU/g frog weight, respectively. Moreover, experimentally infected frogs exhibited quick and higher mortality under coinfection with K. pneumoniae and E. miricola than those single challenge of each bacterium. To date, no natural co-infection by these two bacteria has been reported from frogs and even amphibians. The results will not only shed light on the feature and pathogenesis of K. pneumoniae and E. miricola, but also highlight that co-infection of these two pathogen is a potential threat to black-spotted frog farming.

A Variety of Fungal Species on the Green Frogs' Skin (Pelophylax esculentus complex) in South Banat.

In the last several decades, amphibian populations have been declining worldwide. Many factors have been linked to global amphibian decline, including habitat destruction, pollution, introduced species, global environmental changes, and emerging infectious diseases. Recent studies of amphibian skin infections were mainly focused on the presence of chytridiomycosis, neglecting other members of the frogs' skin communities. The diversity pattern of fungal dwellers on the skin of green frogs (Pelophylax esculentus complex) was investigated. A total of 100 adults were sampled from three localities in South Banat (northern Serbia) over three consecutive years and detected fungal dwellers were identified using light microscopy and ITS and BenA gene sequencing. Structures belonging to fungi and fungus-like organisms including a variety of spores and different mycelia types were documented in the biofilm formed on amphibian skin, and are classified into 10 groups. In total, 42 fungal isolates were identified to species, section, or genus level. The difference in mycobiota composition between sampling points (localities and green frog taxa) was documented. The highest number of fungal structures and isolates was recorded on the hybrid taxon P. esculentus and locality Stevanove ravnice. Parental species showed a markedly lower diversity than the hybrid taxon and were more similar in diversity patterns and were placed in the same homogenous group. The locality Stevanove ravnice exhibited more pronounced differences in diversity pattern than the other two localities and was placed in a distinct and separate homogenous group. Among the fungal isolates, the highest isolation frequency was documented for Alternaria alternata, Aspergillus sp. sect. Nigri, Epicoccum nigrum, Fusarium proliferatum, and Trichoderma atroviride. Among the documented species, dematiaceous fungi, causative agents of chromomycosis in amphibians, were also recorded in this research with high isolation frequency. Also, some rare fungal species such as Quambalaria cyanescens and Pseudoteniolina globosa are documented for the first time in this research as microbial inhabitants of amphibian skin.

Habitat Disturbance Linked with Host Microbiome Dispersion and Bd Dynamics in Temperate Amphibians.

Anthropogenic habitat disturbances can dramatically alter ecological community interactions, including host-pathogen dynamics. Recent work has highlighted the potential for habitat disturbances to alter host-associated microbial communities, but the associations between anthropogenic disturbance, host microbiomes, and pathogens are unresolved. Amphibian skin microbial communities are particularly responsive to factors like temperature, physiochemistry, pathogen infection, and environmental microbial reservoirs. Through a field survey on wild populations of Acris crepitans (Hylidae) and Lithobates catesbeianus (Ranidae), we assessed the effects of habitat disturbance and connectivity on environmental bacterial reservoirs, Batrachochytrium dendrobatidis (Bd) infection, and skin microbiome composition. We found higher measures of microbiome dispersion (a measure of community variability) in A. crepitans from more disturbed ponds, supporting the hypothesis that disturbance increases stochasticity in biological communities. We also found that habitat disturbance limited microbiome similarity between locations for both species, suggesting greater isolation of bacterial assemblages in more disturbed areas. Higher disturbance was associated with lower Bd prevalence for A. crepitans, which could signify suboptimal microclimates for Bd in disturbed habitats. Combined, our findings show that reduced microbiome stability stemming from habitat disturbance could compromise population health, even in the absence of pathogenic infection.

Changes in the gut microbiota diversity of brown frogs (Rana dybowskii) after an antibiotic bath.

BACKGROUND: Captive amphibians frequently receive antibiotic baths to control bacterial diseases. The potential collateral effect of these antibiotics on the microbiota of frogs is largely unknown. To date, studies have mainly relied on oral administration to examine the effects of antibiotics on the gut microbiota; in contrast, little is known regarding the effects of bath-applied antibiotics on the gut microbiota. The gut microbiota compositions of the gentamicin, recovery, and control groups were compared by Illumina high-throughput sequencing, and the functional profiles were analysed using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt). Furthermore, the relationship between the structure and predicted functional composition of the gut microbiota was determined. RESULTS: The alpha diversity indices were significantly reduced by the gentamicin bath, illustrating that this treatment significantly changed the composition of the gut microbiota. After 7 days, the gut microbiota of the recovery group was not significantly different from that of the gentamicin group. Forty-four indicator taxa were selected at the genus level, comprising 42 indicators representing the control group and 2 indicators representing the gentamicin and recovery groups. Potential pathogenic bacteria of the genera Aeromonas, Citrobacter, and Chryseobacterium were significantly depleted after the gentamicin bath. There was no significant positive association between the community composition and functional composition of the gut microbiota in the gentamicin or control frogs, indicating that the functional redundancy of the gut bacterial community was high. CONCLUSIONS: Gentamicin significantly changed the structure of the gut microbiota of R. dybowskii, and the gut microbiota exhibited weak resilience. However, the gentamicin bath did not change the functional composition of the gut microbiota of R. dybowskii, and there was no significant correlation between the structural composition and the functional composition of the gut microbiota.

Composition of the North American Wood Frog (Rana sylvatica) Bacterial Skin Microbiome and Seasonal Variation in Community Structure.

While a number of amphibian skin microbiomes have been characterized, it is unclear how these communities might vary in response to seasonal changes in the environment and the corresponding behaviors that many amphibians exhibit. Given recent studies demonstrating the importance of the skin microbiome in frog innate immune defense against pathogens, investigating how changes in the environment impact the microbial species present will provide a better understanding of conditions that may alter host susceptibility to pathogens in their environment. We sampled the bacterial skin microbiome of North American wood frogs (Rana sylvatica) from two breeding ponds in the spring, along with the bacterial community present in their vernal breeding pools, and frogs from the nearby forest floor in the summer and fall to determine whether community composition differs by sex, vernal pond site, or temporally across season (spring, summer, fall). Taxon relative abundance data reveals a profile of bacterial phyla similar to those previously described on anuran skin, with Proteobacteria, Bacteroidetes, and Actinobacteria dominating the wood frog skin microbiome. Our results indicate that sex had no significant effect on skin microbiota diversity; however, this may be due to our limited female frog sample size. Vernal pool site had a small but significant effect on skin microbiota, but skin-associated communities were more similar to each other than to the communities observed in the frogs' respective pond water. Across seasons, diversity analyses suggest that there are significant differences between the bacterial skin microbiome of frogs from spring and summer/fall groups while the average α-diversity per frog remained consistent. These results illustrate seasonal variation in wood frog skin microbiome structure and highlight the importance of considering temporal trends in an amphibian microbiome, particularly for species whose life history requires recurrent shifts in habitat and behavior.

Habitat disturbance influences the skin microbiome of a rediscovered neotropical-montane frog.

BACKGROUND: The skin microbiome serves as a first line defense against pathogens in vertebrates. In amphibians, it has the potential to protect against the chytrid fungus Batrachochytrium dendrobatis (Bd), a likely agent of amphibian declines. Alteration of the microbiome associated with unfavorable environmental changes produced by anthropogenic activities may make the host more susceptible to pathogens. Some amphibian species that were thought to be "extinct" have been rediscovered years after population declines in the late 1980s probably due to evolved Bd-resistance and are now threatened by anthropogenic land-use changes. Understanding the effects of habitat disturbance on the host skin microbiome is relevant for understanding the health of these species, along with its susceptibility to pathogens such as Bd. Here, we investigate the influence of habitat alteration on the skin bacterial communities as well as specifically the putative Bd-inhibitory bacterial communities of the montane frog Lithobates vibicarius. This species, after years of not being observed, was rediscovered in small populations inhabiting undisturbed and disturbed landscapes, and with continuous presence of Bd. RESULTS: We found that cutaneous bacterial communities of tadpoles and adults differed between undisturbed and disturbed habitats. The adults from disturbed habitats exhibited greater community dispersion than those from undisturbed habitats. We observed a higher richness of putative Bd-inhibitory bacterial strains in adults from disturbed habitats than in those from undisturbed habitats, as well as a greater number of these potential protective bacteria with a high relative abundance. CONCLUSIONS: Our findings support the microbial "Anna Karenina principle", in which disturbance is hypothesized to cause greater microbial dispersion in communities, a so-called dysbiosis, which is a response of animal microbiomes to stress factors that decrease the ability of the host or its microbiome to regulate community composition. On the positive side, the high richness and relative abundance of putative Bd-inhibitory bacteria may indicate the development of a defense mechanism that enhances Bd-protection, attributed to a co-occurrence of more than 30-years of host and pathogen in these disturbed habitats. Our results provide important insight into the influence of human-modified landscapes on the skin microbiome and health implications of Bd-survivor species.

[Expression of MHCâ genes in different tissues of Rana dybowskii under the stress of Aeromonas hydrophila].

The aim of this study was to investigate the expression of MHCⅠ gene in different tissues of Rana dybowskii under the stress of Aeromonas hydrophila (Ah), and to provide evidence for revealing the anti-infective immune response mechanism of amphibians. The experimental animal model of Aeromonas hydrophila infection was first constructed, and the pathological changes were observed by HE staining. The MHCⅠ gene α1+α2 peptide binding region of Rana dybowskii was cloned by RT-PCR and analyzed by bioinformatics. Real-time PCR was used to detect the transcription level of MHCⅠ in different tissues under Ah stress. After Ah infection, the skin, liver and muscle tissues showed signs of cell structure disappearance and texture disorder. The MHCⅠ gene α1+α2 peptide binding region fragment was 494 bp, encoding 164 amino acids, and homology with amphibians. Above 77%, the homology with mammals was as low as 14.96%, indicating that the α1+α2 region of MHC gene was less conserved among different species. The results of real-time PCR show that the liver, spleen and kidney of the experimental group were under Ah stress. The transcript levels of MHCⅠ gene in skin and muscle tissues were higher than those in the control group at 72 h, but the time to peak of each tissue was different (P<0.01), indicating that the response time of MHCⅠ gene in different tissues was different under Ah stress. This study provides a reference for further exploring the immune function of MHC molecules in anti-infection.

Changes in the community structure of the symbiotic microbes of wild amphibians from the eastern edge of the Tibetan Plateau.

Environment has a potential effect on the animal symbiotic microbiome. Here, to study the potential relationship of the symbiotic microbiomes of wild amphibians with altitude, we collected the gut and skin samples from frogs (nine species) and the environmental samples (water and soil samples) from the Leshan Mountains (altitude: 360-410 m) and Gongga Mountains (altitude: 3340-3989 m) on the eastern edge of the Tibetan Plateau. Bufo gargarizans (Bg) samples were collected from both the Leshan and Gongga mountain regions (Bg was the only species sampled on both mountains). The DNA extracted from each sample was performed high-throughput sequencing (MiSeq) of bacterial 16S rRNA gene amplicons. High relative abundance of Caulobacteraceae and Sphingomonadaceae was found in skin samples from both Bg and the other high-altitude amphibians (nine species combined). High relative abundance of Coxiellaceae and Mycoplasmataceae was found in gut samples from both Bg and the other high-altitude amphibians. Furthermore, the alpha and beta diversities of skin and gut samples from Bg and the other amphibian species (nine species combined) were similar. In terms of the symbiotic microbial community, the low-altitude samples were less diverse and more similar to each other than the high-altitude samples were. We speculated that extreme high-altitude environments and host phylogeny may affect the amphibian microbiome. Despite the distinct microbial community differences between the skin and gut microbiomes, some functions were similar in the Bg and combined high-altitude samples. The Bg and high-altitude skin samples had higher oxidative stress tolerance and biofilm formation than the low-altitude skin samples. However, the opposite results were observed for the Bg and high-altitude gut samples. Further study is required to determine whether these characteristics favor high-altitude amphibian adaptation to extreme environments.

Probiotics Modulate a Novel Amphibian Skin Defense Peptide That Is Antifungal and Facilitates Growth of Antifungal Bacteria.

Probiotics can ameliorate diseases of humans and wildlife, but the mechanisms remain unclear. Host responses to interventions that change their microbiota are largely uncharacterized. We applied a consortium of four natural antifungal bacteria to the skin of endangered Sierra Nevada yellow-legged frogs, Rana sierrae, before experimental exposure to the pathogenic fungus Batrachochytrium dendrobatidis (Bd). The probiotic microbes did not persist, nor did they protect hosts, and skin peptide sampling indicated immune modulation. We characterized a novel skin defense peptide brevinin-1Ma (FLPILAGLAANLVPKLICSITKKC) that was downregulated by the probiotic treatment. Brevinin-1Ma was tested against a range of amphibian skin cultures and found to inhibit growth of fungal pathogens Bd and B. salamandrivorans, but enhanced the growth of probiotic bacteria including Janthinobacterium lividum, Chryseobacterium ureilyticum, Serratia grimesii, and Pseudomonas sp. While commonly thought of as antimicrobial peptides, here brevinin-1Ma showed promicrobial function, facilitating microbial growth. Thus, skin exposure to probiotic bacterial cultures induced a shift in skin defense peptide profiles that appeared to act as an immune response functioning to regulate the microbiome. In addition to direct microbial antagonism, probiotic-host interactions may be a critical mechanism affecting disease resistance.

Effects of Seasonal Hibernation on the Similarities Between the Skin Microbiota and Gut Microbiota of an Amphibian (Rana dybowskii).

Both the gut and skin microbiotas have important functions for amphibians. The gut microbiota plays an important role in both the health and evolution of the host species, whereas the role of skin microbiota in disease resistance is particularly important for amphibians. Many studies have examined the effects of environmental factors on the skin and gut microbiotas, but no study has yet explored the similarities between the skin and gut microbiotas. In this study, the gut and skin microbiotas of Rana dybowskii in summer and winter were investigated via high-throughput Illumina sequencing. The results showed that the alpha diversity of gut and skin microbiotas decreased significantly from summer to winter. In both seasons, the microbial composition and structure differed significantly between the gut and skin, and the similarities between these microbiotas differed between seasons. The pairwise distances between the gut and skin microbiotas were greater in winter than in summer. The ratio of core OTUs and shared OTUs to the sum of the OTUs in the gut and skin microbiotas in summer was significantly higher than that in winter. The similarities between the gut and skin microbiotas are important for understanding amphibian ecology and life history.

Brucella microti-like prevalence in French farms producing frogs.

In the last 10 years, many atypical novel members of Brucella species have been reported, including several Brucella inopinata-like strains in wild-caught and "exotic" amphibians from various continents. In 2017, a strain of Brucella was isolated for the first time in animals from a French farm producing frogs-Pelophylax ridibundus-for human consumption and identified as B. microti-like. Following this first isolation, investigations were performed in this farm as well as in the farm of the research unit that provided the domestic frog strain to estimate the prevalence of B. microti-like infection and its presence in the surrounding environment. Farming practices were investigated and samples including frogs at different development stages, surface tank swabs, water, feed and soil were analysed by real-time PCR and bacteriological methods. High B. microti-like prevalence values (higher than 90%) were obtained in frog samples in the commercial farm, and its presence was highlighted in the environmental samples except feed. In the research unit farm, B. microti-like species was also isolated and detected in frog and environmental samples. These results show that B. microti-like organisms are able to colonize amphibians and persist in their environment. Its presence could constitute a possible risk for consumers and workers proving the importance of assessing the zoonotic and pathogenic potentials of these new and atypical Brucella species.

Comparison of the gut microbiota of Rana amurensis and Rana dybowskii under natural winter fasting conditions.

Rana amurensis and R. dybowskii occupy similar habitats. As temperatures decrease with the onset of winter, both species migrate to ponds for hibernation. Our goal was to determine whether different species possess different intestinal microbiota under natural winter fasting conditions. We used high-throughput Illumina sequencing of 16S rRNA gene sequences to analyse the diversity of intestinal microbes in the two species. The dominant gut bacterial phyla in both species were Bacteroidetes, Proteobacteria and Firmicutes. Linear discriminant analysis (LDA) effect size revealed significant enrichment of Proteobacteria in R. amurensis and Firmicutes in R. dybowskii. There were significant differences in the gut microbiota composition between the species. The core operational taxonomic unit numbers in R. amurensis and R. dybowskii shared by the two species were 106, 100 and 36. This study indicates that the intestinal bacterial communities of the two frog species are clearly different. Phylum-level analysis showed that R. amurensis was more abundant in Proteobacteria and Verrucomicrobia than R. dybowskii was This is the first study of the composition and diversity of the gut microbiota of these two species, providing important insights for future research on the gut microbiota and the role of these bacterial communities in frogs.

Candidate Animal Disease Model of Elizabethkingia Spp. Infection in Humans, Based on the Systematic Pathology and Oxidative Damage Caused by E. miricola in Pelophylax nigromaculatus.

Most species of the genus Elizabethkingia are pathogenic to humans and animals, most commonly causing meningitis. However, our understanding of the pathogenic mechanisms involved is poor and there have been few pathological studies of Elizabethkingia spp. in animals. To understand the host injury induced by Elizabethkingia spp., we established a model of E. miricola infection in the black-spotted frog (Pelophylax nigromaculatus). The systematic pathology in and oxidative damage in the infection model were investigated. Our results show that recently isolated E. miricola is a bacterium that mainly parasitizes the host brain and that neurogenic organs are the predominant sites of damage. Infection mainly manifested as severe brain abscesses, meningoencephalitis, necrotic spondylitis, and necrotic retinitis. The liver, spleen, kidney, gastrointestinal tract, and lung were also affected to varying degrees, with bacterial necrotic inflammation. P. nigromaculatus also suffered enormous damage to its oxidative system during E. miricola infection, which may have further aggravated its disease state. Our results provide a preliminary reference for the study and treatment of Elizabethkingia spp.-induced neurological diseases in animals.

Population-Level Resistance to Chytridiomycosis is Life-Stage Dependent in an Imperiled Anuran.

Amphibian declines caused by chytridiomycosis have been severe, but some susceptible populations have persisted or even recovered. Resistance to the causal agent Batrachochytrium dendrobatidis (Bd) could result from alleles of the adaptive immune system. During metamorphosis, however, immune systems may not be fully functional, implying that an effective immune response to Bd may be life-stage dependent. We evaluated the susceptibility of the relict leopard frog (Rana onca) sourced from two areas where Bd was present or absent, and where the populations appeared to show differences in pathogen resistance. We evaluated whether population-level resistance manifested across life stages using challenge experiments with late-stage tadpoles (Gosner stage 31-38), metamorphs (stage 45-46), and juvenile frogs. We used three different Bd isolates including one from wild R. onca to challenge juvenile frogs and focused on the isolate from R. onca to challenge tadpoles and resulting metamorphs. We found that juveniles from the Bd exposed population were 5.5 times more likely to survive Bd infection and 10 times more likely to clear infections than those from the area without Bd. In contrast, and regardless of the source area, we observed 98% survivorship of tadpoles, but only 19% survivorship of resulting metamorphs following re-exposure. Given the low survivorship of exposed metamorphs in the laboratory, we speculate on how resistance characteristics, whether adaptive or innate, that do not manifest at each life stage could develop in the wild. We suggest that seasonal high temperatures during times when metamorphosis appears common may modulate the effects of the pathogen during this most susceptible life stage.

Out in the cold and sick: low temperatures and fungal infections impair a frog's skin defenses.

Amphibians worldwide continue to battle an emerging infectious disease, chytridiomycosis, caused by Batrachochytrium dendrobatidis (Bd). Southern leopard frogs, Rana sphenocephala, are known to become infected with this pathogen, yet they are considered 'of least concern' for declines due to chytridiomycosis. Previous studies have shown that R. sphenocephala secretes four antimicrobial peptides (AMPs) onto their skin which may play an important role in limiting susceptibility to chytridiomycosis. Here, we examined (1) the effects of temperature and AMP depletion on infections with Bd and (2) the effects of temperature and Bd infection on the capacity to secrete AMPs in juvenile leopard frogs. Pathogen burden and mortality were greater in frogs exposed to Bd at low temperature but did not increase following monthly AMP depletion. Both low temperature and Bd exposure reduced the capacity of juvenile frogs to restore peptides after monthly depletions. Frogs held at 14°C were poorly able to restore peptides in comparison with those at 26°C. Frogs held at 26°C were better able to restore their peptides, but when exposed to Bd, this capacity was significantly reduced. These results strongly support the hypothesis that both colder temperatures and Bd infection impair the capacity of juvenile frogs to produce and secrete AMPs, an important component of their innate defense against chytrid fungi and other pathogens. Thus, in the face of unpredictable climate changes and enzootic pathogens, assessments of disease risk should consider the potential for effects of environmental variation and pathogen exposure on the quality of host defenses.

Host age alters amphibian susceptibility to Batrachochytrium dendrobatidis, an emerging infectious fungal pathogen.

Parasites and pathogens are often aggregated in a minority of susceptible hosts within a population, with a majority of individuals harboring low infection intensities. However, determining the relative importance of host traits to explain this heterogeneity is a challenge. One ecologically important pathogen is Batrachochytrium dendrobatidis (Bd), which causes the disease chytridiomycosis and has been associated with many amphibian population declines worldwide. For many hosts, post-metamorphic stages are generally more susceptible than the larval stage. Yet, examination of the effects of Bd infection at different ages within a life stage, has received little attention. This study investigated the hypothesis that recently-post-metamorphic frogs were more sensitive to chytridiomycosis than older frogs, and that sensitivity to Bd infection decreased as frogs aged. We examined this relationship with Pacific treefrogs (Pseudacris regilla) and red legged frogs (Rana aurora). Age had a strong effect on susceptibility to infection, infection intensity, and survival-but not in the directions we had predicted. In both host species, an increase in age was associated with frogs becoming more susceptible to Bd infection, harboring larger infection intensities, and greater risk of mortality. This suggests that the timing of Bd exposure may influence amphibian population dynamics.

Shifts in temperature influence how Batrachochytrium dendrobatidis infects amphibian larvae.

Many climate change models predict increases in frequency and magnitude of temperature fluctuations that might impact how ectotherms are affected by disease. Shifts in temperature might especially affect amphibians, a group with populations that have been challenged by several pathogens. Because amphibian hosts invest more in immunity at warmer than cooler temperatures and parasites might acclimate to temperature shifts faster than hosts (creating lags in optimal host immunity), researchers have hypothesized that a temperature shift from cold-to-warm might result in increased amphibian sensitivity to pathogens, whereas a shift from warm-to-cold might result in decreased sensitivity. Support for components of this climate-variability based hypothesis have been provided by prior studies of the fungus Batrachochytrium dendrobatidis (Bd) that causes the disease chytridiomycosis in amphibians. We experimentally tested whether temperature shifts before exposure to Batrachochytrium dendrobatidis (Bd) alters susceptibility to the disease chytridiomycosis in the larval stage of two amphibian species-western toads (Anaxyrus boreas) and northern red legged frogs (Rana aurora). Both host species harbored elevated Bd infection intensities under constant cold (15° C) temperature in comparison to constant warm (20° C) temperature. Additionally, both species experienced an increase in Bd infection abundance after shifted from 15° C to 20° C, compared to a constant 20° C but they experienced a decrease in Bd after shifted from 20° C to 15° C, compared to a constant 15° C. These results are in contrast to prior studies of adult amphibians highlighting the potential for species and stage differences in the temperature-dependence of chytridiomycosis.