Investigating Differences across Host Species and Scales to Explain the Distribution of the Amphibian Pathogen Batrachochytrium dendrobatidis

Many pathogens infect more than one host species, and clarifying how these different hosts contribute to pathogen dynamics can facilitate the management of pathogens and can lend insight into the functioning of pathogens in ecosystems. In this study, we investigated a suite of native and non-native amphibian hosts of the pathogen Batrachochytrium dendrobatidis (Bd) across multiple scales to identify potential mechanisms that may drive infection patterns in the Colorado study system. Specifically, we aimed to determine if: 1) amphibian populations vary in Bd infection across the landscape, 2) amphibian community composition predicts infection (e.g., does the presence or abundance of any particular species influence infection in others?), 3) amphibian species vary in their ability to produce infectious zoospores in a laboratory infection, 4) heterogeneity in host ability observed in the laboratory scales to predict patterns of Bd prevalence in the landscape. We found that non-native North American bullfrogs (Lithobates catesbeianus) are widespread and have the highest prevalence of Bd infection relative to the other native species in the landscape. Additionally, infection in some native species appears to be related to the density of sympatric L. catesbeianus populations. At the smaller host scale, we found that L. catesbeianus produces more of the infective zoospore stage relative to some native species, but that this zoospore output does not scale to predict infection in sympatric wild populations of native species. Rather, landscape level infection relates most strongly to density of hosts at a wetland as well as abiotic factors. While non-native L. catesbeianus have high levels of Bd infection in the Colorado Front Range system, we also identified Bd infection in a number of native amphibian populations allopatric with L. catesbeianus, suggesting that multiple host species are important contributors to the dynamics of the Bd pathogen in this landscape.     

Distribution and risk factors for spread of amphibian chytrid fungus Batrachochytrium dendrobatidis in the Tasmanian Wilderness World Heritage Area, Australia

Chytridiomycosis is an emerging infectious disease caused by the pathogen Batrachochytrium dendrobatidis (Bd) and is the cause of the decline and extinction of amphibian species throughout the world. We surveyed the distribution of Bd within and around the Tasmanian Wilderness World Heritage Area (TWWHA), a 1.38 million ha area of significant fauna conservation value, which provides the majority of habitat for Tasmania's 3 endemic frog species (Litoria burrowsae, Bryobatrachus nimbus and Crinia tasmaniensis). Bd was detected at only 1 (3%) of the 33 sites surveyed within the TWWHA and at 15 (52%) of the 29 sites surveyed surrounding the TWWHA. The relatively low incidence of the disease within the TWWHA suggests that the majority of the TWWHA is currently free of the pathogen despite the fact that the region provides what appears to be optimal conditions for the persistence of Bd. For all survey sites within and around the TWWHA, the presence of Bd was strongly associated with the presence of gravel roads, forest and < 1000 m altitude--factors that in this study were associated with human-disturbed landscapes around the TWWHA. Conversely, the presence of walking tracks was strongly associated with the absence of Bd, suggesting an association of absence with relatively remote locations. The wide distribution of Bd in areas of Tasmania with high levels of human disturbance and its very limited occurrence in remote wilderness areas suggests that anthropogenic activities may facilitate the dissemination of the pathogen on a landscape scale in Tasmania. Because the majority of the TWWHA is not readily accessible and appears to be largely free of Bd, and because Tasmanian frogs reproduce in ponds rather than streams, it may be feasible to control the spread of the disease in the TWWHA.     

Spatially explicit conservation issues for threatened bird species in Mediterranean farmland landscapes

Coupling habitat models based on GIS and on ground variables could help identify suitable areas (by means of landscape models obtained by GIS variables) to concentrate management actions for species’ conservation. In this study, the habitat requirements of Lesser Greys (LGS) and Woodchat Shrikes (WS), two threatened farmland bird species declining in Europe, were assessed in Apulia (south-eastern Italy) by means of binary logistic regression at two different levels: landscape (using GIS-measured variables); and, territory (using ground-measured variables) scales. The LGS occurrence at landscape scale was correlated to steppe-like areas and cereal crops. At the territory level, significant effects were detected for deciduous forests and the presence of isolated trees and shrubs. The WS occurrence at landscape scale was promoted by steppe-like areas and cereal crops, whereas, at the territory level significant effects were detected for steppe-like areas positively and suburban areas negatively. The landscape model was extrapolated to the entire region. Within highly suitable areas (occurrence probability higher than 0.66 according to the landscape model), we measured average habitat features and compared them with the optimal mosaic depicted by the territory level models. This allowed us to give spatially explicit and site-specific management recommendations for these two threatened species. LGS will mostly benefit from an increase in isolated shrubs and trees; whereas for WS, the most widespread recommendations are to increase steppe-like habitat and to prevent further urbanisation.Coupling “coarse” landscape models with the species ecology provided by fine-scaled models can integrate relevant information on species potential distribution and territory level requirements, making planning fine-tuned habitat management (within potentially suitable landscapes) in a spatially explicit way possible.     

Selecting for extinction: nonrandom disease-associated extinction homogenizes amphibian biotas

Studying the patterns in which local extinctions occur is critical to understanding how extinctions affect biodiversity at local, regional and global spatial scales. To understand the importance of patterns of extinction at a regional spatial scale, we use data from extirpations associated with a widespread pathogenic agent of amphibian decline, Batrachochytrium dendrobatidis ( Bd ) as a model system. We apply novel null model analyses to these data to determine whether recent extirpations associated with Bd have resulted in selective extinction and homogenization of diverse tropical American amphibian biotas. We find that Bd -associated extinctions in this region were nonrandom and disproportionately, but not exclusively, affected low-occupancy and endemic species, resulting in homogenization of the remnant amphibian fauna. The pattern of extirpations also resulted in phylogenetic homogenization at the family level and ecological homogenization of reproductive mode and habitat association. Additionally, many more species were extirpated from the region than would be expected if extirpations occurred randomly. Our results indicate that amphibian declines in this region are an extinction filter, reducing regional amphibian biodiversity to highly similar relict assemblages and ultimately causing amplified biodiversity loss at regional and global scales.     

Effects of nutrient supplementation on host‐pathogen dynamics of the amphibian chytrid fungus: a community approach

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Infection with Batrachochytrium dendrobatidis is common in tropical lowland habitats: Implications for amphibian conservation

Numerous species of amphibians declined in Central America during the 1980s and 1990s. These declines mostly affected highland stream amphibians and have been primarily linked to chytridiomycosis, a deadly disease caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd). Since then, the majority of field studies on Bd in the Tropics have been conducted in midland and highland environments (>800 m) mainly because the environmental conditions of mountain ranges match the range of ideal abiotic conditions for Bd in the laboratory. This unbalanced sampling has led researchers to largely overlook host–pathogen dynamics in lowlands, where other amphibian species declined during the same period. We conducted a survey testing for Bd in 47 species (n = 348) in four lowland sites in Costa Rica to identify local host–pathogen dynamics and to describe the abiotic environment of these sites. We detected Bd in three sampling sites and 70% of the surveyed species. We found evidence that lowland study sites exhibit enzootic dynamics with low infection intensity and moderate to high prevalence (55% overall prevalence). Additionally, we found evidence that every study site represents an independent climatic zone, where local climatic differences may explain variations in Bd disease dynamics. We recommend more detection surveys across lowlands and other sites that have been historically considered unsuitable for Bd occurrence. These data can be used to identify sites for potential disease outbreaks and amphibian rediscoveries.     

Batrachochytrium dendrobatidis: requirement for further isolate collection and archiving

The fungal pathogen Batrachochytrium dendrobatidis (Bd) causes the disease chytridiomycosis, which is lethal to many species of amphibians worldwide. Many studies have investigated the epidemiology of chytridiomycosis in amphibian populations, but few have considered possible host-pathogen coevolution. More specifically, investigations focused on the evolution of Bd, and the link with Bd virulence, are needed. Such studies, which may be important for conservation management of amphibians, depend on access to Bd isolates. Here we provide a summary of known Bd isolates that have been collected and archived in various locations around the world. Of 257 Bd isolates, we found that 53% originate from ranids in the United States. In many cases, detailed information on isolate origin is unavailable, and it is unknown how many isolates are cryo-archived. We suggest the creation of a centralized database of isolate information, and we urge researchers and managers to isolate and archive Bd to facilitate future research on chytridiomycosis.     

Composition of symbiotic bacteria predicts survival in Panamanian golden frogs infected with a lethal fungus

Symbiotic microbes can dramatically impact host health and fitness, and recent research in a diversity of systems suggests that different symbiont community structures may result in distinct outcomes for the host. In amphibians, some symbiotic skin bacteria produce metabolites that inhibit the growth of Batrachochytrium dendrobatidis (Bd), a cutaneous fungal pathogen that has caused many amphibian population declines and extinctions. Treatment with beneficial bacteria (probiotics) prevents Bd infection in some amphibian species and creates optimism for conservation of species that are highly susceptible to chytridiomycosis, the disease caused by Bd. In a laboratory experiment, we used Bd-inhibitory bacteria from Bd-tolerant Panamanian amphibians in a probiotic development trial with Panamanian golden frogs, Atelopus zeteki, a species currently surviving only in captive assurance colonies. Approximately 30% of infected golden frogs survived Bd exposure by either clearing infection or maintaining low Bd loads, but this was not associated with probiotic treatment. Survival was instead related to initial composition of the skin bacterial community and metabolites present on the skin. These results suggest a strong link between the structure of these symbiotic microbial communities and amphibian host health in the face of Bd exposure and also suggest a new approach for developing amphibian probiotics.     

Anuran site occupancy and species richness as tools for evaluating restoration of a hydrologically-modified landscape

A fundamental goal of wetland restoration is to reinstate pre-disturbance hydrological conditions to degraded landscapes, facilitating recolonization by native species and the production of resilient, functional ecosystems. To evaluate restoration success, baseline conditions need to be determined and a reference target needs to be established that will serve as an ecological blueprint in the restoration process. During the summer wet seasons of 2010 and 2011, we used automated recording units to monitor a community of calling anuran amphibians in the Picayune Strand State Forest of Southwest Florida, USA. This area is undergoing hydrological restoration as part of the Comprehensive Everglades Restoration Plan. We compared occurrence of anurans at sites in the restoration area, to nearby locations in relatively undisturbed habitat (reference sites). We assessed the utility of the latter as restoration targets, using a hierarchical model of community species occupancy to estimate the probability of occurrence of anurans in restoration and reference locations. We detected 14 species, 13 of which were significantly more likely to occur in reference areas. All 14 species were estimated by our model to occur at these sites but, across both years, only 8–13 species were estimated to occur at restoration sites. The composition and structure of these habitats within and adjacent to the Picayune Strand State Forest indicate that they are suitable targets for habitat restoration, as measured by amphibian occurrence and species richness. These areas are important sources for recolonization of anuran amphibians as the hydrologically degraded Picayune Strand undergoes restoration to mitigate the effects of overdrainage and habitat loss.     

Higher temperature variability increases the impact of Batrachochytrium dendrobatidis and shifts interspecific interactions in tadpole mesocosms

The emergence of amphibian chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis (Bd) has led to the decline and extinction of numerous amphibian species. Multiple studies have observed links between climatic factors and amphibian declines apparently caused by Bd. Using outdoor experimental mesocosms, we tested the response of red‐legged frog (Rana aurora) tadpoles to increased variation in temperature, a component of climate linked to amphibian declines, and Bd exposure. We included tadpoles of a sympatric competitor species, Pacific chorus frog (Pseudacris regilla), in a fully factorial design to test the effects of Bd and temperature on interspecific interactions. We found that higher variation in temperature had numerous effects in mesocosms, including interacting with Bd presence to decrease the condition of R. aurora, shifting the relative performance of competing P. regilla and R. aurora, and accelerating the development of P. regilla relative to R. aurora. Our results demonstrate that increased variation in temperature can affect amphibians in multiple ways that will be contingent on ecological context, including the presence of Bd and competing species.     

Mapping the Global Emergence of Batrachochytrium dendrobatidis,the Amphibian Chytrid Fungus

The rapid worldwide emergence of the amphibian pathogen Batrachochytrium dendrobatidis (Bd) is having a profound negative impact on biodiversity. However, global research efforts are fragmented and an overarching synthesis of global infection data is lacking. Here, we provide results from a community tool for the compilation of worldwide Bd presence and report on the analyses of data collated over a four-year period. Using this online database, we analysed: 1) spatial and taxonomic patterns of infection, including amphibian families that appear over- and under-infected; 2) relationships between Bd occurrence and declining amphibian species, including associations among Bd occurrence, species richness, and enigmatic population declines; and 3) patterns of environmental correlates with Bd, including climate metrics for all species combined and three families (Hylidae, Bufonidae, Ranidae) separately, at both a global scale and regional (U.S.A.) scale. These associations provide new insights for downscaled hypothesis testing. The pathogen has been detected in 52 of 82 countries in which sampling was reported, and it has been detected in 516 of 1240 (42%) amphibian species. We show that detected Bd infections are related to amphibian biodiversity and locations experiencing rapid enigmatic declines, supporting the hypothesis that greater complexity of amphibian communities increases the likelihood of emergence of infection and transmission of Bd. Using a global model including all sampled species, the odds of Bd detection decreased with increasing temperature range at a site. Further consideration of temperature range, rather than maximum or minimum temperatures, may provide new insights into Bd-host ecology. Whereas caution is necessary when interpreting such a broad global dataset, the use of our pathogen database is helping to inform studies of the epidemiology of Bd, as well as enabling regional, national, and international prioritization of conservation efforts. We provide recommendations for adaptive management to enhance the database utility and relevance.     

Unlocking the story in the swab: A new genotyping assay for the amphibian chytrid fungus Batrachochytrium dendrobatidis

One of the most devastating emerging pathogens of wildlife is the chytrid fungus, Batrachochytrium dendrobatidis (Bd), which affects hundreds of amphibian species around the world. Genomic data from pure Bd cultures have advanced our understanding of Bd phylogenetics, genomic architecture and mechanisms of virulence. However, pure cultures are laborious to obtain and whole‐genome sequencing is comparatively expensive, so relatively few isolates have been genetically characterized. Thus, we still know little about the genetic diversity of Bd in natural systems. The most common noninvasive method of sampling Bd from natural populations is to swab amphibian skin. Hundreds of thousands of swabs have been collected from amphibians around the world, but Bd DNA collected via swabs is often low in quality and/or quantity. In this study, we developed a custom Bd genotyping assay using the Fluidigm Access Array platform to amplify 192 carefully selected regions of the Bd genome. We obtained robust sequence data for pure Bd cultures and field‐collected skin swabs. This new assay has the power to accurately discriminate among the major Bd clades, recovering the basic tree topology previously revealed using whole‐genome data. Additionally, we established a critical value for initial Bd load for swab samples (150 Bd genomic equivalents) above which our assay performs well. By leveraging advances in microfluidic multiplex PCR technology and the globally distributed resource of amphibian swab samples, noninvasive skin swabs can now be used to address critical spatial and temporal questions about Bd and its effects on declining amphibian populations.     

Amphibian skin fungal communities vary across host species and do not correlate with infection by a pathogenic fungus

Amphibian population declines caused by the fungus Batrachochytrium dendrobatidis (Bd) have prompted studies on the bacterial community that resides on amphibian skin. However, studies addressing the fungal portion of these symbiont communities have lagged behind. Using ITS1 amplicon sequencing, we examined the fungal portion of the skin microbiome of temperate and tropical amphibian species currently coexisting with Bd in nature. We assessed cooccurrence patterns between bacterial and fungal OTUs using a subset of samples for which bacterial 16S rRNA gene amplicon data were also available. We determined that fungal communities were dominated by members of the phyla Ascomycota and Basidiomycota, and also by Chytridiomycota in the most aquatic amphibian species. Alpha diversity of the fungal communities differed across host species, and fungal community structure differed across species and regions. However, we did not find a correlation between fungal diversity/community structure and Bd infection, though we did identify significant correlations between Bd and specific OTUs. Moreover, positive bacterial–fungal cooccurrences suggest that positive interactions between these organisms occur in the skin microbiome. Understanding the ecology of amphibian skin fungi, and their interactions with bacteria will complement our knowledge of the factors influencing community assembly and the overall function of these symbiont communities.     

Susceptibility of amphibians to chytridiomycosis is associated with MHC class II conformation

The pathogenic chytrid fungus Batrachochytrium dendrobatidis (Bd) can cause precipitous population declines in its amphibian hosts. Responses of individuals to infection vary greatly with the capacity of their immune system to respond to the pathogen. We used a combination of comparative and experimental approaches to identify major histocompatibility complex class II (MHC-II) alleles encoding molecules that foster the survival of Bd-infected amphibians. We found that Bd-resistant amphibians across four continents share common amino acids in three binding pockets of the MHC-II antigen-binding groove. Moreover, strong signals of selection acting on these specific sites were evident among all species co-existing with the pathogen. In the laboratory, we experimentally inoculated Australian tree frogs with Bd to test how each binding pocket conformation influences disease resistance. Only the conformation of MHC-II pocket 9 of surviving subjects matched those of Bd-resistant species. This MHC-II conformation thus may determine amphibian resistance to Bd, although other MHC-II binding pockets also may contribute to resistance. Rescuing amphibian biodiversity will depend on our understanding of amphibian immune defence mechanisms against Bd. The identification of adaptive genetic markers for Bd resistance represents an important step forward towards that goal.     

Treatment of chytridiomycosis with reduced-dose itraconazole

Effective treatment methods to eliminate infection with Batrachochytrium dendrobatidis (Bd) are required for development of sustainable captive survival assurance populations of amphibians and to reduce the risk of introducing Bd to new locations as part of amphibian trade or reintroduction programs. Treatment with itraconazole baths at 100 mg l-1 is commonly used in captive amphibians, but side effects are observed in some amphibian species and life stages. Naturally occurring outbreaks of chytridiomycosis in Wyoming toads Anaxyrus baxteri and White's tree frogs Litoria caerulea were treated with lower-dose itraconazole baths (e.g. 50 mg l-1 for White's tree frogs) and followed post-treatment with serial Taqman PCR testing to confirm elimination of Bd infection. Post-treatment PCR tests were consistently negative for the presence of Bd and treatment was deemed successful. Although this was not a controlled clinical trial, results suggest that lower doses of itraconazole may be effective for treatment of chytridiomycosis with resulting cost savings to amphibian conservation programs and a potential for a reduction in dose-related side effects from itraconazole treatment. Prospective clinical trials of alternative itraconazole treatment protocols are encouraged.     

Survey protocol for detecting chytridiomycosis in all Australian frog populations

Spread of the amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) has caused the decline and extinction of frogs, but the distribution of Bd is not completely known. This information is crucial to implementing appropriate quarantine strategies, preparing for outbreaks of chytridiomycosis due to introduction of Bd, and for directing conservation actions towards affected species. This survey protocol provides a simple and standard method for sampling all frog populations in Australia to maximise the chances of detecting Bd. In order to structure and prioritise the protocol, areas are divided by bioregion and frog species are allocated depending on the water bodies they utilize into 3 groups representing different levels of risk of exposure to Bd. Sixty individuals per population need to be tested to achieve 95% certainty of detecting 1 positive frog, based on the minimum apparent prevalence of > or =5% in infected Australian frog populations and using a quantitative real-time TaqMan PCR test. The appropriate season to sample varies among bioregions and will ideally incorporate temperatures favourable for chytridiomycosis (e.g. maximum air temperatures generally <27 degrees C). Opportunistic collection and testing of sick frogs and tadpoles with abnormal mouth-parts should also be done to increase the probability of detecting Bd. The survey priorities in order are (1) threatened species that may have been exposed to Bd, (2) bioregions surrounding infected bioregions/ecological groups, and (3) species of frogs of unknown infection status in infected bioregions. Within these priority groups, sampling should first target ecological groups and species likely to be exposed to Bd, such as those associated with permanent water, and areas within bioregions that have high risk for Bd as indicated by climatic modelling. This protocol can be adapted for use in other countries and a standard protocol will enable comparison among amphibian populations globally.     

Pond-Breeding Amphibian Species Richness and Habitat Selection in a Beaver-Modified Landscape

ABSTRACT Beaver (Castor canadensis) activity creates wetland habitats with varying hydroperiods important in maintaining habitat diversity for pond-breeding amphibians with significantly different breeding habitat requirements. We documented pond-breeding amphibian assemblages in 71 freshwater wetlands in Acadia National Park, Maine, USA. Using 15 variables describing local pond conditions and wetland landscape characteristics, we developed a priori models to predict sites with high amphibian species richness and used model selection with Akaike's Information Criterion to judge the strength of evidence supporting each model. We developed single-species models to predict wood frog (Rana sylvatica), bullfrog (R. catesbeiana), and pickerel frog (R. palustris) breeding site selection. Sites with high species richness were best predicted by 1) connectivity of wetlands in the landscape through stream corridors and 2) wetland modification by beaver. Wood frog breeding habitat was best predicted by temporary hydroperiod, lack of fish, and absence of current beaver activity. Wood frog breeding was present in abandoned beaver wetlands nearly as often as in nonbeaver wetlands. Bullfrog breeding was limited to active beaver wetlands with fish and permanent water. Pickerel frog breeding sites were best predicted by connectivity through stream corridors within the landscape. As beavers have recolonized areas of their former range in North America, they have increased the number and diversity of available breeding sites in the landscape for pond-breeding amphibians. The resulting mosaic of active and abandoned beaver wetlands both supports rich amphibian assemblages and provides suitable breeding habitat for species with differing habitat requirements. Land managers should consider the potential benefits of minimal management of beavers in promoting and conserving amphibian and wetland diversity at a landscape scale.     

High Prevalence of the Amphibian Chytrid Pathogen in Gabon

Amphibian chytridiomycosis is an infectious disease caused by the fungus Batrachochytrium dendrobatidis (Bd) that is implicated in the worldwide decline and extinction of amphibians. Africa has been proposed as a potential source for the global expansion of Bd, yet the distribution of Bd across the continent remains largely unexplored. Using quantitative polymerase chain reaction (qPCR), we screened for the presence of Bd in 166 adult anurans from two national parks in Gabon (Monts de Cristal and Ivindo). Bd was detected in 20 of the 42 species and was present at all three sites surveyed (two in Monts de Cristal, and one in Ivindo) with high prevalence (19.6%-36.0%). Both national parks were Bd-positive at all elevations and across habitat types, though no dead or dying frogs were encountered. To our knowledge, this study presents the first evidence of Bd in Gabon and the first record of infection for 19 of the 20 species that were Bd-positive. Documenting the distribution and virulence of Bd across Africa will be essential for understanding the dynamics of amphibian chytridiomycosis across the globe.     

Populations of a susceptible amphibian species can grow despite the presence of a pathogenic chytrid fungus

Disease can be an important driver of host population dynamics and epizootics can cause severe host population declines. Batrachochytrium dendrobatidis (Bd), the pathogen causing amphibian chytridiomycosis, may occur epizootically or enzootically and can harm amphibian populations in many ways. While effects of Bd epizootics are well documented, the effects of enzootic Bd have rarely been described. We used a state-space model that accounts for observation error to test whether population trends of a species highly susceptible to Bd, the midwife toad Alytes obstetricans, are negatively affected by the enzootic presence of the pathogen. Unexpectedly, Bd had no negative effect on population growth rates from 2002-2008. This suggests that negative effects of disease on individuals do not necessarily translate into negative effects at the population level. Populations of amphibian species that are susceptible to the emerging disease chytridiomycosis can persist despite the enzootic presence of the pathogen under current environmental conditions.     

A reservoir species for the emerging Amphibian pathogen Batrachochytrium dendrobatidis thrives in a landscape decimated by disease

Chytridiomycosis, a disease caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), is driving amphibian declines and extinctions in protected areas globally. The introduction of invasive reservoir species has been implicated in the spread of Bd but does not explain the appearance of the pathogen in remote protected areas. In the high elevation (>1500 m) Sierra Nevada of California, the native Pacific chorus frog, Pseudacris regilla, appears unaffected by chytridiomycosis while sympatric species experience catastrophic declines. We investigated whether P. regilla is a reservoir of Bd by comparing habitat occupancy before and after a major Bd outbreak and measuring infection in P. regilla in the field, monitoring susceptibility of P. regilla to Bd in the laboratory, examining tissues with histology to determine patterns of infection, and using an innovative soak technique to determine individual output of Bd zoospores in water. Pseudacris regilla persists at 100% of sites where a sympatric species has been extirpated from 72% in synchrony with a wave of Bd. In the laboratory, P. regilla carried loads of Bd as much as an order of magnitude higher than loads found lethal to sympatric species. Histology shows heavy Bd infection in patchy areas next to normal skin, a possible mechanism for tolerance. The soak technique was 77.8% effective at detecting Bd in water and showed an average output of 68 zoospores per minute per individual. The results of this study suggest P. regilla should act as a Bd reservoir and provide evidence of a tolerance mechanism in a reservoir species.