HEALTH MONITORING: AMPHIBIANS
FROGS AND TOADS
Kerby J. L., Schieffer A., Brown J. R., Whitfield S. (2013): Utilization of fast qPCR techniques to detect the amphibian chytrid fungus: a cheaper and more efficient alternative method. Methods in Ecology and Evolution 4: 162-166.
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A pathogen of great significance to amphibian populations is the chytrid fungus, Batrachochytrium dendrobatidis (Bd). It has been demonstrated as causing recent epizootics in wild populations and is also widely found in captive animals. It is listed as a notifiable disease within the pet, bait and food trade because of its risk of introduction into wild populations. Due to this status, there has been much emphasis on reliably identifying and quantifying the pathogens in amphibians. Quantitative polymerase chain reaction (qPCR) has served as the recent standard for identifying this pathogen’s presence. Newer technologies have greatly improved these reactions enabling researchers to use smaller volumes and run the reactions in less time. These ‘fast’ qPCR chemistries are gaining popularity because the reduced volumes required to run the reactions can save funding resources and reduce the time to data acquisition. In this study, we compare the results from differing reaction methodologies using the same DNA extracts from pathogens collected from wild sampled amphibians. In addition to comparing the standard methodology and fast methodology for both pathogens, we also conducted a reduced volume methodology using the standard TaqMan chemistry for Bd. Estimated pathogen loads from 114 field swab samples were compared among methodologies. We found that for Bd, all three methodologies produced similar results for prevalence (presence/absence) estimates. In terms of estimating pathogen loads in the samples, both the standard and fast methodologies produced comparable estimates but the reduced volume methodology exhibited significantly lower values. Therefore, it appears that the fast methodology is adequate for use with Bd, and potentially several other wildlife pathogens, in estimating both prevalence and quantity, but the reduced volume methodology is inadequate and not recommended for use in quantifying samples.
Woodhams D. C., Brandt H., Baumgartner S., Kielgast J., Küpfer E., Tobler U., Davis L. R., Schmidt B. R., Bel C., Hodel S., McKenzie V. (2014): Interacting symbionts and immunity in the amphibian skin mucosome predict disease risk and probiotic effectiveness. Plos One 9: e96375.
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Pathogenesis is strongly dependent on microbial context, but development of probiotic therapies has neglected the impact of ecological interactions. Dynamics among microbial communities, host immune responses, and environmental conditions may alter the effect of probiotics in human and veterinary medicine, agriculture and aquaculture, and the proposed treatment of emerging wildlife and zoonotic diseases such as those occurring on amphibians or vectored by mosquitoes. Here we use a holistic measure of amphibian mucosal defenses to test the effects of probiotic treatments and to assess disease risk under different ecological contexts. We developed a non-invasive assay for antifungal function of the skin mucosal ecosystem (mucosome function) integrating host immune factors and the microbial community as an alternative to pathogen exposure experiments. From approximately 8500 amphibians sampled across Europe, we compared field infection prevalence with mucosome function against the emerging fungal pathogen Batrachochytrium dendrobatidis. Four species were tested with laboratory exposure experiments, and a highly susceptible species, Alytes obstetricans, was treated with a variety of temperature and microbial conditions to test the effects of probiotic therapies and environmental conditions on mucosome function. We found that antifungal function of the amphibian skin mucosome predicts the prevalence of infection with the fungal pathogen in natural populations, and is linked to survival in laboratory exposure experiments. When altered by probiotic therapy, the mucosome increased antifungal capacity, while previous exposure to the pathogen was suppressive. In culture, antifungal properties of probiotics depended strongly on immunological and environmental context including temperature, competition, and pathogen presence. Functional changes in microbiota with shifts in temperature provide an alternative mechanistic explanation for patterns of disease susceptibility related to climate beyond direct impact on host or pathogen. This nonlethal management tool can be used to optimize and quickly assess the relative benefits of probiotic therapies under different climatic, microbial, or host conditions.
Huver J. R., Koprivnikar J., Johnson P. T. J., Whyard S. (2015): Development and application of an eDNA method to detect and quantify a pathogenic parasite in aquatic ecosystems. Ecological Applications 25: 991-1002.
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Approaches based on organismal DNA found in the environment (eDNA) have become increasingly utilized for ecological studies and biodiversity inventories as an alternative to traditional field survey methods. Such DNA-based techniques have largely been used to establish the presence of free-living organisms, but have much potential for detecting and quantifying infectious agents in the environment, which is necessary to evaluate disease risk. We developed an eDNA method to examine the distribution and abundance of the trematode Ribeiroia ondatrae, a pathogenic parasite known to cause malformations in North American amphibians. In addition to comparing this eDNA approach to classical host necropsy, we examined the detectability of R. ondatrae in water samples subject to different degradation conditions (time and temperature). Our test exhibited high specificity and sensitivity to R. ondatrae, capable of detecting as little as 14 fg (femtograms) of this parasite’s DNA (1/2500th of a single infectious stage) from field water samples. Compared to our results from amphibian host necropsy, quantitative PCR was ~90% concordant with respect to R. ondatrae detection from 15 field sites and was also a significant predictor of host infection abundance. DNA was still detectable in lab samples after 21 days at 25°C, indicating that our method is robust to field conditions. By comparing the advantages and disadvantages of eDNA vs. traditional survey methods for determining pathogen presence and abundance in the field, we found that the lower cost and effort associated with eDNA approaches provide many advantages. The development of alternative tools is critical for disease ecology, as wildlife management and conservation efforts require reliable establishment and monitoring of pathogens.
Longo A. V., Savage A. E., Hewson I., Zamudio K. R. (2015): Seasonal and ontogenetic variation of skin microbial communities and relationships to natural disease dynamics in declining amphibians. Royal Society Open Science 2: 140377.
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Recently, microbiologists have focused on characterizing the probiotic role of skin bacteria for amphibians threatened by the fungal disease chytridiomycosis. However, the specific characteristics of microbial diversity required to maintain health or trigger disease are still not well understood in natural populations. We hypothesized that seasonal and developmental transitions affecting susceptibility to chytridiomycosis could also alter the stability of microbial assemblages. To test our hypothesis, we examined patterns of skin bacterial diversity in two species of declining amphibians (Lithobates yavapaiensis and Eleutherodactylus coqui) affected by the pathogenic fungus Batrachochytrium dendrobatidis (Bd). We focused on two important transitions that affect Bd susceptibility: ontogenetic (from juvenile to adult) shifts in E. coqui and seasonal (from summer to winter) shifts in L. yavapaiensis. We used a combination of community-fingerprinting analyses and 16S rRNA amplicon sequencing to quantify changes in bacterial diversity and assemblage composition between seasons and developmental stages, and to investigate the relationship between bacterial diversity and pathogen load. We found that winter-sampled frogs and juveniles, two states associated with increased Bd susceptibility, exhibited higher diversity compared with summer-sampled frogs and adult individuals. Our findings also revealed that hosts harbouring higher bacterial diversity carried lower Bd infections, providing support for the protective role of bacterial communities. Ongoing work to understand skin microbiome resilience after pathogen disturbance has the potential to identify key taxa involved in disease resistance.
Hall E. M., Crespi E. J., Goldberg C. S., Brunner J. L. (2016): Evaluating environmental DNA‐based quantification of ranavirus infection in wood frog populations. Molecular Ecology Resources 16: 423-433.
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A variety of challenges arise when monitoring wildlife populations for disease. Sampling tissues can be invasive to hosts, and obtaining sufficient sample sizes can be expensive and time-consuming, particularly for rare species and when pathogen prevalence is low. Environmental DNA (eDNA)-based detection of pathogens is an alternative approach to surveillance for aquatic communities that circumvents many of these issues. Ranaviruses are emerging pathogens of ectothermic vertebrates linked to die-offs of amphibian populations. Detecting ranavirus infections is critical, but nonlethal methods have the above issues and are prone to false negatives. We report on the feasibility and effectiveness of eDNA-based ranavirus detection in the field. We compared ranavirus titres in eDNA samples collected from pond water to titres in wood frog (Lithobates sylvaticus; n = 5) tadpoles in sites dominated by this one species (n = 20 pond visits). We examined whether ranavirus DNA can be detected in eDNA from pond water when infections are present in the pond and if viral titres detected in eDNA samples correlate with the prevalence or intensity of ranavirus infections in tadpoles. With three 250 mL water samples, we were able to detect the virus in all visits with infected larvae (0.92 diagnostic sensitivity). Also, we found a strong relationship between the viral eDNA titres and titres in larval tissues. eDNA titres increased prior to observed die-offs and declined afterwards, and were two orders of magnitude higher in ponds with a die-off. Our results suggest that eDNA is useful for detecting ranavirus infections in wildlife and aquaculture.
Huggins L. G., Michaels C. J., Cruickshank S. M., Preziosi R. F., Else K. J. (2017): A novel copro-diagnostic molecular method for qualitative detection and identification of parasitic nematodes in amphibians and reptiles. Plos One 12: e0185151.
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Anthropogenic disturbance via resource acquisition, habitat fragmentation and climate change, amongst other factors, has led to catastrophic global biodiversity losses and species extinctions at an accelerating rate. Amphibians are currently one of the worst affected classes with at least a third of species categorised as being threatened with extinction. At the same time, they are also critically important for many habitats and provide man with a powerful proxy for ecosystem health by acting as a bioindicator group. Whilst the causes of synchronised amphibian losses are varied recent research has begun to highlight a growing role that macroparasites are playing in amphibian declines. However, diagnosing parasite infection in the field can be problematic, principally relying on collection and euthanasia of hosts, followed by necropsy and morphological identification of parasites in situ. The current study developed a non-invasive PCR-based methodology for sensitive detection and identification of parasitic nematode DNA released in the faeces of infected amphibians as egg or tissue fragments (environmental DNA). A DNA extraction protocol optimised for liberation of DNA from resilient parasite eggs was developed alongside the design of a novel, nematode universal, degenerate primer pair, thus avoiding the difficulties of using species specific primers in situations where common parasite species are unknown. Used in conjunction this protocol and primer pair was tested on a wide range of faecal samples from captive and wild amphibians. The primers and protocol were validated and detected infections, including a Railletnema nematode infection in poison dart frogs from ZSL London Zoo and Mantella cowani frogs in the wild. Furthermore, we demonstrate the efficacy of our PCR-based protocol for detecting nematode infection in other hosts, such as the presence of pinworm (Aspiculuris) in two tortoise species and whipworm (Trichuris muris) in mice. Our environmental DNA approach mitigates problems associated with microscopic identification and can be applied to detect nematode parasitoses in wild and captive hosts for infection surveillance and maintenance of healthy populations.
Spitzen-van der Sluijs A., Canessa S., Martel A., Pasmans F. (2017): Fragile coexistence of a global chytrid pathogen with amphibian populations is mediated by environment and demography. Proceedings of the Royal Society B: Biological Sciences 284: 20171444.
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Unravelling the multiple interacting drivers of host–pathogen coexistence is crucial in understanding how an apparently stable state of endemism may shift towards an epidemic and lead to biodiversity loss. Here, we investigate the apparent coexistence of the global amphibian pathogen Batrachochytrium dendrobatidis (Bd) with Bombina variegata populations in The Netherlands over a 7-year period. We used a multi-season mark–recapture dataset and assessed potential drivers of coexistence (individual condition, environmental mediation and demographic compensation) at the individual and population levels. We show that even in a situation with a clear cost incurred by endemic Bd, population sizes remain largely stable. Current environmental conditions and an over-dispersed pathogen load probably stabilize disease dynamics, but as higher temperatures increase infection probability, changing environmental conditions, for example a climate-change-driven rise in temperature, could unbalance the current fragile host–pathogen equilibrium. Understanding the proximate mechanisms of such environmental mediation and of site-specific differences in infection dynamics can provide vital information for mitigation actions.
Aivelo T., Harris K., Cadle J. E., Wright P. (2018): Exploring non-invasive sampling of parasites by metabarcoding gastrointestinal nematodes in Madagascar frog species. Basic and Applied Herpetology 32: 29-40.
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The diversity of Anuran parasites is poorly surveyed, despite arguably being one of the most important threats to anuran populations worldwide. Additionally, parasites also interact with a number of other stressors, such as invasive species, pollution, sedimentation and changing light conditions, caused by anthropogenic disturbance in natural habitats. We aimed to explore the use of metabarcoding, a new, non-invasive tool to survey the parasite assemblages in frogs in different environments facing different levels of anthropogenic pressure. We collected fecal samples from frogs across three different transects in Ranomafana National Park, located in southeastern Madagascar, and then used the 18S metabarcoding technique to identify nematode species from the collected fecal samples. We were able to find four different putative species, which were all identified to the genus level. In comparison to the literature on previous surveys done with traditional methods, the metabarcoding approach seems to provide similar diversity estimates and taxonomical accuracy. Our results suggest that non-invasive sampling and metabarcoding can provide a suitable tool for intestinal parasite surveys in anuran host populations.
Christian K., Weitzman C., Rose A., Kaestli M., Gibb K. (2018): Ecological patterns in the skin microbiota of frogs from tropical Australia. Ecology and Evolution 8: 10510-10519.
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The microbiota of frog skin can play an important role in protecting against diseases and parasites. The frog skin microbial community represents a complex mix of microbes that are promoted by the chemical environment of the frog skin and influenced by the animal’s immediate past environment. The microbial communities of six species of frogs sampled from the campus of Charles Darwin University (CDU) were more similar within species than between species. The microbiota of the introduced cane toad (Rhinella marina) was most dissimilar among the species. Pairwise comparisons showed that the microbial communities of each species were different, except for the terrestrial Litoria nasuta and the arboreal L. rothii. The microbial communities of the six species were not related to ecological habit (arboreal or terrestrial), and neither was the alpha diversity of the microbes. The core microbes (defined as being on ≥90% of individuals of a species or group) were significantly different among all species, although 89 microbial operational taxonomic units (OTUs) were core microbes for all six species at CDU. Two species, Rhinella marina and Litoria rothii, were sampled at additional sites approximately 10 and 30 km from CDU. The microbial communities and the core OTU composition were different among the sites, but there were nevertheless 194 (R. marina) and 181 (L. rothii) core OTUs present at all three sites. Thus, the core microbiota varied with respect to geographic range and sample size.
DiRenzo G. V., Campbell Grant E. H., Longo A. V., Che‐Castaldo C., Zamudio K. R., Lips K. R. (2018): Imperfect pathogen detection from non‐invasive skin swabs biases disease inference. Methods in Ecology and Evolution 9: 380-389.
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Conservation managers rely on accurate estimates of disease parameters, such as pathogen prevalence and infection intensity, to assess disease status of a host population. However, these disease metrics may be biased if low-level infection intensities are missed by sampling methods or laboratory diagnostic tests. These false negatives underestimate pathogen prevalence and overestimate mean infection intensity of infected individuals. Our objectives were two-fold. First, we quantified false negative error rates of Batrachochytrium dendrobatidis (Bd) on non-invasive skin swabs collected from an amphibian community in El Copé, Panama. We swabbed amphibians twice in sequence, and we used a recently developed hierarchical Bayesian estimator to assess disease status of the population. Second, we developed a novel hierarchical Bayesian model to simultaneously account for imperfect pathogen detection from field sampling and laboratory diagnostic testing. We evaluated the performance of the model, using simulations and varying sampling design to quantify the magnitude of bias in estimates of pathogen prevalence and infection intensity. We show that Bd detection probability from skin swabs was related to host infection intensity, where Bd infections <10 zoospores have <95% probability of being detected. If imperfect Bd detection was not considered, then Bd prevalence was underestimated by as much as 71%. In the Bd-amphibian system, this indicates a need to correct for imperfect pathogen detection in enzootic host populations persisting with low-level infections. More generally, our results have implications for study designs in other disease systems, particularly those with similar objectives, biology, and sampling decisions. Uncertainty in pathogen detection is an inherent property of most sampling protocols and diagnostic tests, where the magnitude of bias depends on the study system, type of infection, and false negative error rates. Given that it may be difficult to know this information in advance, we advocate that the most cautious approach is to assume all errors are possible and to accommodate them by adjusting sampling designs. The modelling framework presented here improves the accuracy in estimating pathogen prevalence and infection intensity.
Miaud C., Arnal V., Poulain M., Valentini A., Dejean T. (2019): eDNA increases the detectability of Ranavirus infection in an alpine amphibian population. Viruses 11: 526.
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The early detection and identification of pathogenic microorganisms is essential in order to deploy appropriate mitigation measures. Viruses in the Iridoviridae family, such as those in the Ranavirus genus, can infect amphibian species without resulting in mortality or clinical signs, and they can also infect other hosts than amphibian species. Diagnostic techniques allowing the detection of the pathogen outside the period of host die-off would thus be of particular use. In this study, we tested a method using environmental DNA (eDNA) on a population of common frogs (Rana temporaria) known to be affected by a Ranavirus in the southern Alps in France. In six sampling sessions between June and September (the species’ activity period), we collected tissue samples from dead and live frogs (adults and tadpoles), as well as insects (aquatic and terrestrial), sediment, and water. At the beginning of the breeding season in June, one adult was found dead; at the end of July, a mass mortality of tadpoles was observed. The viral DNA was detected in both adults and tadpoles (dead or alive) and in water samples, but it was not detected in insects or sediment. In live frog specimens, the virus was detected from June to September and in water samples from August to September. Dead tadpoles that tested positive for Ranavirus were observed only on one date (at the end of July). Our results indicate that eDNA can be an effective alternative to tissue/specimen sampling and can detect Ranavirus presence outside die-offs. Another advantage is that the collection of water samples can be performed by most field technicians. This study confirms that the use of eDNA can increase the performance and accuracy of wildlife health status monitoring and thus contribute to more effective surveillance programs.
Sabino-Pinto J., Krause E. T., Bletz M. C., Martel A., Pasmans F., Steinfartz S., Vences M. (2019): Detectability vs. time and costs in pooled DNA extraction of cutaneous swabs: a study on the amphibian chytrid fungi. Amphibia-Reptilia 40: 29-39.
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Epidemiology relies on understanding the distribution of pathogens which often can be detected through DNA-based techniques, such as quantitative Polymerase Chain Reaction (qPCR). Typically, the DNA of each individual sample is separately extracted and undergoes qPCR analysis. However, when performing field surveys and long-term monitoring, a large fraction of the samples is generally expected to be negative, especially in geographical areas still considered free of the pathogen. If pathogen detection within a population – rather than determining its individual prevalence – is the focus, work load and monetary costs can be reduced by pooling samples for DNA extraction. We test and refine a user-friendly technique where skin swabs can be pooled during DNA extraction to detect the amphibian chytrid fungi, Batrachochytrium dendrobatidis and B. salamandrivorans (Bsal). We extracted pools with different numbers of samples (from one to four swabs), without increasing reaction volumes, and each pool had one sample inoculated with a predetermined zoospore amount. Pool size did not reduce the ability to detect the two fungi, except if inoculated with extremely low zoospore amounts (one zoospore). We confirm that pooled DNA extraction of cutaneous swabs can substantially reduce processing time and costs without minimizing detection sensitivity. This is of relevance especially for the new emerging pathogen Bsal, for which pooled DNA extraction had so far not been tested and massive monitoring efforts in putatively unaffected regions are underway.
Goff C. B., Walls S. C., Rodriguez D., Gabor C. R. (2020): Changes in physiology and microbial diversity in larval ornate chorus frogs are associated with habitat quality. Conservation Physiology 8: coaa047.
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Environmental change associated with anthropogenic disturbance can lower habitat quality, especially for sensitive species such as many amphibians. Variation in environmental quality may affect an organism’s physiological health and, ultimately, survival and fitness. Using multiple health measures can aid in identifying populations at increased risk of declines. Our objective was to measure environmental variables at multiple spatial scales and their effect on three indicators of health in ornate chorus frog (Pseudacris ornata) tadpoles to identify potential correlates of population declines. To accomplish this, we measured a glucocorticoid hormone (corticosterone; CORT) profile associated with the stress response, as well as the skin mucosal immune function (combined function of skin secretions and skin bacterial community) and bacterial communities of tadpoles from multiple ponds. We found that water quality characteristics associated with environmental variation, including higher water temperature, conductivity and total dissolved solids, as well as percent developed land nearby, were associated with elevated CORT release rates. However, mucosal immune function, although highly variable, was not significantly associated with water quality or environmental factors. Finally, we examined skin bacterial diversity as it aids in immunity and is affected by environmental variation. We found that skin bacterial diversity differed between ponds and was affected by land cover type, canopy cover and pond proximity. Our results indicate that both local water quality and land cover characteristics are important determinants of population health for ornate chorus frogs. Moreover, using these proactive measures of health over time may aid in early identification of at-risk populations that could prevent further declines and aid in management decisions.
Preuss J. F., Greenspan S. E., Rossi E. M., Lucas Gonsales E. M., Neely W. J., Valiati V. H., Woodhams D. C., Becker C. G., Tozetti A. M. (2020): Widespread pig farming practice linked to shifts in skin microbiomes and disease in pond-breeding amphibians. Environmental Science & Technology 54: 11301-11312.
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Farming practices may reshape the structure of watersheds, water quality, and the health of aquatic organisms. Nutrient enrichment from agricultural pollution increases disease pressure in many host–pathogen systems, but the mechanisms underlying this pattern are not always resolved. For example, nutrient enrichment should strongly influence pools of aquatic environmental bacteria, which has the potential to alter microbiome composition of aquatic animals and their vulnerability to disease. However, shifts in the host microbiome have received little attention as a link between nutrient enrichment and diseases of aquatic organisms. We examined nutrient enrichment through the widespread practice of integrated pig–fish farming and its effects on microbiome composition of Brazilian amphibians and prevalence of the globally distributed amphibian skin pathogen Batrachochytrium dendrobatidis (Bd). This farming system drove surges in fecal coliform bacteria, disturbing amphibian skin bacterial communities such that hosts recruited higher proportions of Bd-facilitative bacteria and carried higher Bd prevalence. Our results highlight previously overlooked connections between global trends in land use change, microbiome dysbiosis, and wildlife disease. These interactions may be particularly important for disease management in the tropics, a region with both high biodiversity and continually intensifying anthropogenic pressures on aquatic wildlife habitats.
Zhou J., Nelson T. M., Rodriguez Lopez C., Sarma R. R., Zhou S. J., Rollins L. A. (2020): A comparison of nonlethal sampling methods for amphibian gut microbiome analyses. Molecular Ecology Resources 20: 844-855.
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Noninvasive sampling methods for studying intestinal microbiomes are widely applied in studies of endangered species and in those conducting temporal monitoring during manipulative experiments. Although existing studies show that noninvasive sampling methods among different taxa vary in their accuracy, no studies have yet been published comparing nonlethal sampling methods in adult amphibians. In this study, we compare microbiomes from two noninvasive sample types (faeces and cloacal swabs) to that of the large intestine in adult cane toads, Rhinella marina. We use 16S rRNA gene sequencing to investigate how microbial communities change along the digestive tract and which nonlethal sampling method better represents large intestinal microbiota. We found that cane toads’ intestinal microbiota was dominated by Bacteroidetes, Proteobacteria and Firmicutes and, interestingly, we also saw a high proportion of Fusobacteria, which has previously been associated with marine species and changes in frog immunity. The large and small intestine of cane toads had a similar microbial composition, but the large intestine showed higher diversity. Our results indicate that cloacal swabs were more similar to large intestine samples than were faecal samples, and small intestine samples were significantly different from both nonlethal sample types. Our study provides valuable information for future investigations of the cane toad gut microbiome and validates the use of cloacal swabs as a nonlethal method to study changes in the large intestine microbiome. These data provide insights for future studies requiring nonlethal sampling of amphibian gut microbiota.
Douglas A. J., Hug L. A., Katzenback B. A. (2021): Composition of the North American wood frog (Rana sylvatica) bacterial skin microbiome and seasonal variation in community structure. Microbial Ecology 81: 78-92.
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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.
Neely W. J., Greenspan S. E., Stahl L. M., Heraghty S. D., Marshall V. M., Atkinson C. L., Becker C. G. (2021): Habitat disturbance linked with host microbiome dispersion and Bd dynamics in temperate amphibians. Microbial Ecology 84: 901-910.
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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.
Proença D. N., Fasola E., Lopes I., Morais P. V. (2021): Characterization of the skin cultivable microbiota composition of the frog Pelophylax perezi inhabiting different environments. International Journal of Environmental Research and Public Health 18: 2585.
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Microorganisms that live in association with amphibian skin can play important roles in protecting their host. Within the scenarios of global change, it is important to understand how environmental disturbances, namely, metal pollution, can affect this microbiota. The aim of this study is to recognize core bacteria in the skin cultivable microbiota of the Perez frog (Pelophylax perezi) that are preserved regardless of the environmental conditions in which the frogs live. The characterization of these isolates revealed characteristics that can support their contributions to the ability of frogs to use metal impacted environments. Frog’s skin swabs were collected from P. perezi populations that inhabit a metal-polluted site and three reference (non-metal polluted) sites. Bacterial strains were isolated, identified, and subjected to an acid mine drainage tolerance (AMD) test, collected upstream from a site heavily contaminated with metals, and tested to produce extracellular polymeric substances (exopolysaccharide, EPS). All frog populations had Acinetobacter in their cutaneous cultivable microbiota. Significant growth inhibition was observed in all bacterial isolates exposed to 75% of AMD. EPS production was considered a characteristic of several isolates. The data obtained is a preliminary step but crucial to sustain that the cultivable microbiota is a mechanism for protecting frogs against environmental contamination.
SALAMANDERS AND NEWTS
Bletz M. C., Kelly M., Sabino-Pinto J., Bales E., Van Praet S., Bert W., Boyen F., Vences M., Steinfartz S., Pasmans F., Martel A. (2018): Disruption of skin microbiota contributes to salamander disease. Proceedings of the Royal Society B 285: 20180758.
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Escalating occurrences of emerging infectious diseases underscore the importance of understanding microbiome–pathogen interactions. The amphibian cutaneous microbiome is widely studied for its potential to mitigate disease-mediated amphibian declines. Other microbial interactions in this system, however, have been largely neglected in the context of disease outbreaks. European fire salamanders have suffered dramatic population crashes as a result of the newly emerged Batrachochytrium salamandrivorans (Bsal). In this paper, we investigate microbial interactions on multiple fronts within this system. We show that wild, healthy fire salamanders maintain complex skin microbiotas containing Bsal-inhibitory members, but these community are present at a remarkably low abundance. Through experimentation, we show that increasing bacterial densities of Bsal-inhibiting bacteria via daily addition slowed disease progression in fire salamanders. Additionally, we find that experimental-Bsal infection elicited subtle changes in the skin microbiome, with selected opportunistic bacteria increasing in relative abundance resulting in septicemic events that coincide with extensive destruction of the epidermis. These results suggest that fire salamander skin, in natural settings, maintains bacterial communities at numbers too low to confer sufficient protection against Bsal, and, in fact, the native skin microbiota can constitute a source of opportunistic bacterial pathogens that contribute to pathogenesis. By shedding light on the complex interaction between the microbiome and a lethal pathogen, these data put the interplay between skin microbiomes and a wildlife disease into a new perspective.
Smith H. K., Pasmans F., Dhaenens M., Deforce D., Bonte D., Verheyen K., Lens L., Martel A. (2018): Skin mucosome activity as an indicator of Batrachochytrium salamandrivorans susceptibility in salamanders. Plos One 13: e0199295.
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Recently emerged fungal diseases, Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal) are an increasing threat to amphibians worldwide. In Europe, the threat of Bsal to salamander populations is demonstrated by the rapid decline of fire salamander populations in Germany, the Netherlands and Belgium. Although most European urodelans are susceptible to infection in infection trials, recent evidence suggests marked interspecific differences in the course of infection, with potentially far reaching implications for salamander conservation. As a salamander’s skin is the first line of defense against such pathogens, interspecific differences in innate immune function of the skin may explain differential susceptibility. Here we investigate if compounds present on a salamander’s skin can kill Bsal spores and if there is variation among species. We used a non-invasive assay to compare killing ability of salamander mucosomes of four different species (captive and wild Salamandra salamandra and captive Ichtyosaura alpestris, Cynops pyrrhogaster and Lissotriton helveticus) by exposing Bsal zoospores to salamander mucosomes and determining spore survival. In all samples, zoospores were killed when exposed to mucosomes. Moreover, we saw a significant variation in this Bsal killing ability of mucosomes between different salamander host species. Our results indicate that mucosomes of salamanders might provide crucial skin protection against Bsal, and could explain why some species are more susceptible than others. This study represents a step towards better understanding host species variation in innate immune function and disease susceptibility in amphibians.
Riepe T. B., Calhoun D. M., Johnson P. T. (2019): Comparison of direct and indirect techniques for evaluating endoparasite infections in wild-caught newts (Taricha torosa and T. granulosa). Diseases of Aquatic Organisms 134: 137-146.
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Studies of amphibian parasites have increased over the past 20 yr, in part because of their role in amphibian population declines and deformities. Such patterns underscore the importance of non-lethal methods for detecting and quantifying endoparasitic infections. The goal of this study was to compare results of indirect methods (fecal smears and fecal floats) with quantitative necropsies to detect endoparasitic infections in adult newts. In 2015, we collected fecal samples from 68 adult newts (Taricha granulosa and T. torosa) in the East Bay region of California and used fecal smears, sodium nitrate fecal flotation solution, and Sheather’s sugar flotation solution to assess infection (i.e. the presence and/or abundance of a parasite). Across all methods, we detected 3 protozoans (Eimeria tarichae, Tritrichomonas sp., and Balantidium sp.) and 3 nematodes (Rhabdias tarichae, Cosmocercoides variabilis, and Chabaudgolvania sp.). Based on generalized linear mixed models, the likelihood of detection varied between hosts (with T. torosa showing more overall infection relative to T. granulosa) and by assessment method: while fecal smears were more sensitive in detecting protozoans, comprehensive necropsies were the most reliable for quantifying infections of R. tarichae. Nonetheless, both the likelihood of R. tarichae detection within fecal samples as well as the number of infectious stages observed correlated strongly with infection intensity from necropsy, highlighting the utility of non-lethal assessment methods. The overall congruence between indirect methods and gross necropsy helps to validate the use of less-invasive methods for parasite detection and abundance, especially for sensitive or protected host taxa such as amphibians.
Spitzen‐van der Sluijs A., Stark T., DeJean T., Verbrugghe E., Herder J., Gilbert M., Janse J., Martel A., Pasmans F., Valentini A. (2020): Using environmental DNA for detection of Batrachochytrium salamandrivorans in natural water. Environmental DNA 2: 565-571.
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Rapid, early, and reliable detection of invasive pathogenic microorganisms is essential in order to either predict or delineate an outbreak, and monitor appropriate mitigation measures. The chytrid fungus Batrachochytrium salamandrivorans is expanding in Europe, and infection with this fungus may cause massive mortality in urodelans (salamanders and newts). In this study, we designed and validated species-specific primers and a probe for detection of B. salamandrivorans in water. In a garden pond in close proximity to the B. salamandrivorans index site in the Netherlands, B. salamandrivorans-infected newts had been detected in 2015 and have been monitored since. In 2016 and 2017, no B. salamandrivorans was detected at this site, but in 2018 B. salamandrivorans flared up in this isolated pond which allowed validation of the technique in situ. We here present the development of an environmental DNA technique that successfully detects B. salamandrivorans DNA in natural waterbodies even at low concentrations. This technique may be further validated to play a role in B. salamandrivorans range delineation and surveillance in both natural waterbodies and in captive collections.
Pereira K. E., Woodley S. K. (2021): Skin defenses of North American salamanders against a deadly salamander fungus. Animal Conservation 24: 552-567.
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The recently emerged fungal pathogen, Batrachochytrium salamandrivorans (Bsal) causes the lethal skin disease chytridiomycosis in susceptible salamander species and is predicted to emerge within the Americas with devastating consequences. Host responses to Bsal are variable but the factors underlying these differences are unknown. To investigate the role of skin-associated immune defenses against Bsal and the closely related, B. dendrobatidis (Bd), we sampled skin peptides from wild and captive North American salamanders (spotted salamanders [Ambystoma maculatum], n = 10; hellbenders [Cryptobranchus alleganiensis], n = 2; red-legged salamanders [Plethodon shermani], n = 18; and Ocoee salamanders [Desmognathus ocoee], n = 15) and conducted in vitro experimental assays to test whether salamander skin peptides inhibit chytrid growth. Interspecies differences in skin defenses against Bsal and Bd, and peptide mixtures were also assessed using a standardized measure and RP-HPLC, respectively. For A. maculatum, skin peptides inhibited Bsal and Bd growth, consistent with known Bsal resistance. Cryptobranchus alleganiensis skin peptides inhibited the growth of Bsal but not Bd. Plethodon shermani and D. ocoee skin peptides facilitated Bsal growth and had either no effect or inconsistent effects on Bd growth. With the exception of A. maculatum, most species had relatively weak skin defenses against both chytrid pathogens. Collectively, we demonstrate that salamander skin peptide defenses against chytrid pathogens are highly variable and not always equally effective against Bsal and Bd. By advancing knowledge about the factors underlying chytrid susceptibility, particularly Bsal, our findings will help inform conservation initiatives aimed at reducing disease impacts and biodiversity loss.
Wang Y., Smith H. K., Goossens E., Hertzog L., Bletz M. C., Bonte D., Verheyen K., Lens L., Vences M., Pasmans F., Martel A. (2021): Diet diversity and environment determine the intestinal microbiome and bacterial pathogen load of fire salamanders. Scientific Reports 11: 20493.
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Diverse communities of symbiotic microbes inhabit the digestive systems of vertebrates and play a crucial role in animal health, and host diet plays a major role in shaping the composition and diversity of these communities. Here, we characterized diet and gut microbiome of fire salamander populations from three Belgian forests. We carried out DNA metabarcoding on fecal samples, targeting eukaryotic 18S rRNA of potential dietary prey items, and bacterial 16S rRNA of the concomitant gut microbiome. Our results demonstrated an abundance of soft-bodied prey in the diet of fire salamanders, and a significant difference in the diet composition between males and females. This sex-dependent effect on diet was also reflected in the gut microbiome diversity, which is higher in males than female animals. Proximity to human activities was associated with increased intestinal pathogen loads. Collectively, the data supports a relationship between diet, environment and intestinal microbiome in fire salamanders, with potential health implications.
Wuerthner V. P., Hua J., Hernández‐Gómez O. (2022): Life stage and proximity to roads shape the skin microbiota of eastern newts (Notophthalmus viridescens). Environmental Microbiology 24: 3954-3965.
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Host-associated microbiomes play an essential role in the health of organisms, including immune system activation, metabolism and energy uptake. It is well established that microbial communities differ depending on the life stage and natural history of the organism. However, the effects of life stage and natural history on microbial communities may also be influenced by human activities. We investigated the effects of amphibian life stage (terrestrial eft vs. aquatic adult) and proximity to roadways on newt skin bacterial communities. We found that the eft and adult life stages differed in bacterial community composition; however, the effects of roads on community composition were more evident in the terrestrial eft stage compared to the aquatic adult stage. Terrestrial efts sampled close to roads possessed richer communities than those living further away from the influence of roads. When accounting for amplicon sequence variants with predicted antifungal capabilities, in the adult life stage, we observed a decrease in anti-fungal bacteria with distance to roads. In contrast, in the eft stage, we found an increase in anti-fungal bacteria with distance to roads. Our results highlight the need to consider the effects of human activities when evaluating how host-associated microbiomes differ across life stages of wildlife.
Fieschi‐Méric L., Van Leeuwen P., Denoël M., Lesbarrères D. (2023): Encouraging news for in situ conservation: translocation of salamander larvae has limited impacts on their skin microbiota. Molecular Ecology 32: 3276-3289.
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The key role of symbiotic skin bacteria communities in amphibian resistance to emerging pathogens is well recognized, but factors leading to their dysbiosis are not fully understood. In particular, the potential effects of population translocations on the composition and diversity of hosts’ skin microbiota have received little attention, although such transfers are widely carried out as a strategy for amphibian conservation. To characterize the potential reorganization of the microbiota over such a sudden environmental change, we conducted a common-garden experiment simulating reciprocal translocations of yellow-spotted salamander larvae across three lakes. We sequenced skin microbiota samples collected before and 15 days after the transfer. Using a database of antifungal isolates, we identified symbionts with known function against the pathogen Batrachochytrium dendrobatidis, a major driver of amphibian declines. Our results indicate an important reorganization of bacterial assemblages throughout ontogeny, with strong changes in composition, diversity and structure of the skin microbiota in both control and translocated individuals over the 15 days of monitoring. Unexpectedly, the diversity and community structure of the microbiota were not significantly affected by the translocation event, thus suggesting a strong resilience of skin bacterial communities to environmental change—at least across the time-window studied here. A few phylotypes were more abundant in the microbiota of translocated larvae, but no differences were found among pathogen-inhibiting symbionts. Taken together, our results support amphibian translocations as a promising strategy for this endangered animal class, with limited impact on their skin microbiota.
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McKenzie V. J., Bowers R. M., Fierer N., Knight R., Lauber C. L. (2012): Co-habiting amphibian species harbor unique skin bacterial communities in wild populations. The ISME Journal 6: 588-596.
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Although all plant and animal species harbor microbial symbionts, we know surprisingly little about the specificity of microbial communities to their hosts. Few studies have compared the microbiomes of different species of animals, and fewer still have examined animals in the wild. We sampled four pond habitats in Colorado, USA, where multiple amphibian species were present. In total, 32 amphibian individuals were sampled from three different species including northern leopard frogs (Lithobates pipiens), western chorus frogs (Pseudacris triseriata) and tiger salamanders (Ambystoma tigrinum). We compared the diversity and composition of the bacterial communities on the skin of the collected individuals via barcoded pyrosequencing of the 16S rRNA gene. Dominant bacterial phyla included Acidobacteria, Actinobacteria, Bacteriodetes, Cyanobacteria, Firmicutes and Proteobacteria. In total, we found members of 18 bacterial phyla, comparable to the taxonomic diversity typically found on human skin. Levels of bacterial diversity varied strongly across species: L. pipiens had the highest diversity; A. tigrinum the lowest. Host species was a highly significant predictor of bacterial community similarity, and co-habitation within the same pond was not significant, highlighting that the skin-associated bacterial communities do not simply reflect those bacterial communities found in their surrounding environments. Innate species differences thus appear to regulate the structure of skin bacterial communities on amphibians. In light of recent discoveries that some bacteria on amphibian skin have antifungal activity, our finding suggests that host-specific bacteria may have a role in the species-specific resistance to fungal pathogens.
Brannelly L. A., Wetzel D. P., Ohmer M. E., Zimmerman L., Saenz V., Richards-Zawacki C. L. (2020): Evaluating environmental DNA as a tool for detecting an amphibian pathogen using an optimized extraction method. Oecologia 194: 267-281.
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Environmental DNA (eDNA) detection is a valuable conservation tool that can be used to identify and monitor imperiled or invasive species and wildlife pathogens. Batrachochytrium pathogens are of global conservation concern because they are a leading cause of amphibian decline. While eDNA techniques have been used to detect Batrachochytrium DNA in the environment, a systematic comparison of extraction methods across environmental samples is lacking. In this study, we first compared eDNA extraction methods and found that a soil extraction kit (Qiagen PowerSoil) was the most effective for detecting Batrachochytrium dendrobatidis in water samples. The PowerSoil extraction had a minimum detection level of 100 zoospores and had a two- to four-fold higher detection probability than other commonly used extraction methods (e.g., QIAamp extraction, DNeasy+Qiashredder extraction method, respectively). Next, we used this extraction method on field-collected water and sediment samples and were able to detect pathogen DNA in both. While field-collected water filters were equivalent to amphibian skin swab samples in detecting the presence of pathogen DNA, the seasonal patterns in pathogen quantity were different between skin swabs and water samples. Detection rate was lowest in sediment samples. We also found that detection probability increases with the volume of water filtered. Our results indicate that water filter eDNA samples can be accurate in detecting pathogen presence at the habitat scale but their utility for quantifying pathogen loads in the environment appears limited. We suggest that eDNA techniques be used for early warning detection to guide animal sampling efforts.