Host size influences the effects of four isolates of an amphibian chytrid fungus

Understanding factors that influence host–pathogen interactions is key to predicting outbreaks in natural systems experiencing environmental change. Many amphibian population declines have been attributed to an amphibian chytrid fungus, Batrachochytrium dendrobatidis (Bd). While this fungus is widespread, not all Bd‐positive populations have been associated with declines, which could be attributed to differences in pathogen virulence or host susceptibility. In a laboratory experiment, we examined the effects of Bd isolate origin, two from areas with Bd‐associated amphibian population declines (El Copé, Panama, and California, USA) and two from areas without Bd‐related population declines (Ohio and Maine, USA), on the terrestrial growth and survival of American toad (Anaxyrus americanus) metamorphs reared in larval environments with low or high intraspecific density. We predicted that (1) Bd isolates from areas experiencing declines would have greater negative effects than Bd isolates from areas without declines, and (2) across all isolates, growth and survival of smaller toads from high‐density larval conditions would be reduced by Bd exposure compared to larger toads from low‐density larval conditions. Our results showed that terrestrial survival was reduced for smaller toads exposed to Bd with variation in the response to different isolates, suggesting that smaller size increased susceptibility to Bd. Toads exposed to Bd gained less mass, which varied by isolate. Bd isolates from areas with population declines, however, did not have more negative effects than isolates from areas without recorded declines. Most strikingly, our study supports that host condition, measured by size, can be indicative of the negative effects of Bd exposure. Further, Bd isolates’ impact may vary in ways not predictable from place of origin or occurrence of disease‐related population declines. This research suggests that amphibian populations outside of areas experiencing Bd‐associated declines could be impacted by this pathogen and that the size of individuals could influence the magnitude of Bd's impact.     

Is Chytridiomycosis Driving Darwin’s Frogs to Extinction?

Darwin’s frogs (Rhinoderma darwinii and R. rufum) are two species of mouth brooding frogs from Chile and Argentina that have experienced marked population declines. Rhinoderma rufum has not been found in the wild since 1980. We investigated historical and current evidence of Batrachochytrium dendrobatidis (Bd) infection in Rhinoderma spp. to determine whether chytridiomycosis is implicated in the population declines of these species. Archived and live specimens of Rhinoderma spp., sympatric amphibians and amphibians at sites where Rhinoderma sp. had recently gone extinct were examined for Bd infection using quantitative real-time PCR. Six (0.9%) of 662 archived anurans tested positive for Bd (4/289 R. darwinii; 1/266 R. rufum and 1/107 other anurans), all of which had been collected between 1970 and 1978. An overall Bd-infection prevalence of 12.5% was obtained from 797 swabs taken from 369 extant individuals of R. darwinii and 428 individuals representing 18 other species of anurans found at sites with current and recent presence of the two Rhinoderma species. In extant R. darwinii, Bd-infection prevalence (1.9%) was significantly lower than that found in other anurans (7.3%). The prevalence of infection (30%) in other amphibian species was significantly higher in sites where either Rhinoderma spp. had become extinct or was experiencing severe population declines than in sites where there had been no apparent decline (3.0%; x ² = 106.407, P<0.001). This is the first report of widespread Bd presence in Chile and our results are consistent with Rhinoderma spp. declines being due to Bd infection, although additional field and laboratory investigations are required to investigate this further.     

Recent Emergence of a Chytrid Fungal Pathogen in California Cascades Frogs (<Emphasis Type="Italic">Rana cascadae</Emphasis>)

The pathogenic fungus Batrachochytrium dendrobatidis (Bd) has been associated with global amphibian declines, but it is often difficult to discern the relative importance of Bd as a causal agent in declines that have already occurred. Retrospective analyses of museum specimens have allowed researchers to associate the timing of Bd arrival with the timing of past amphibian declines. Cascades frogs (Rana cascadae) have experienced dramatic declines in northern California, but it is not clear whether the onset of these declines corresponds to the arrival of Bd. We used quantitative real-time PCR assays of samples collected from museum specimens to determine historical Bd prevalence in the northern California range of Cascades frogs. We detected Bd in 13 of 364 (3.5%) Cascades frog specimens collected between 1907 and 2003, with the first positive result from 1978. A Bayesian analysis suggested that Bd arrived in the region between 1973 and 1978, which corresponds well with the first observations of declines in the 1980s.     

The Lethal Fungus Batrachochytrium dendrobatidis Is Present in Lowland Tropical Forests of Far Eastern Panamá

The fungal disease chytridiomycosis, caused by Batrachochytrium dendrobatidis (Bd), is one of the main causes of amphibian population declines and extinctions all over the world. In the Neotropics, this fungal disease has caused catastrophic declines in the highlands as it has spread throughout Central America down to Panamá. In this study, we determined the prevalence and intensity of Bd infection in three species of frogs in one highland and four lowland tropical forests, including two lowland regions in eastern Panamá in which the pathogen had not been detected previously. Bd was present in all the sites sampled with a prevalence ranging from 15–34%, similar to other Neotropical lowland sites. The intensity of Bd infection on individual frogs was low, ranging from average values of 0.11–24 zoospore equivalents per site. Our work indicates that Bd is present in anuran communities in lowland Panamá, including the Darién province, and that the intensity of the infection may vary among species from different habitats and with different life histories. The population-level consequences of Bd infection in amphibian communities from the lowlands remain to be determined. Detailed studies of amphibian species from the lowlands will be essential to determine the reason why these species are persisting despite the presence of the pathogen.     

Exposure to a fungal pathogen increases the critical thermal minimum of two frog species

The ability of an organism to tolerate seasonal temperature changes, such as extremely cold temperatures during the winter, can be influenced by their pathogens. We tested how exposure to a virulent fungal pathogen, Batrachochytrium dendrobatidis (Bd), affected the critical thermal minimum (CT_min) of two frog species, Hyla versicolor (gray treefrog) and Lithobates palustris (pickerel frog). The CT_min is the minimum thermal performance point of an organism, which we estimated via righting response trials. For both frog species, we compared the righting response of Bd‐exposed and Bd‐unexposed individuals in either a constant (15ºC) environment or with decreasing temperatures (−1°C/2.5 min) starting from 15°C. The CT_min for both species was higher for Bd‐exposed frogs than unexposed frogs, and the CT_min of H. versicolor was higher than L. palustris. We also found that Bd‐exposed frogs of both species righted themselves significantly fewer times in both decreasing and constant temperature trials. Our findings show that pathogen exposure can reduce cold tolerance and limit the thermal performance range of hosts, which may lead to increased overwintering mortality.     

Defects in Host Immune Function in Tree Frogs with Chronic Chytridiomycosis

The amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) has caused mass mortality leading to population declines and extinctions in many frog species worldwide. The lack of host resistance may be due to fungal immunosuppressive effects that have been observed when Bd is incubated with cultured lymphocytes, but whether in vivo host immunosuppression occurs is unknown. We used a broad range of hematologic and protein electrophoresis biomarkers, along with various functional tests, to assess immune competence in common green (Litoria caerulea) and white-lipped (L. infrafrenata) tree frogs experimentally infected with Bd. Compared with uninfected frogs, Bd infection in L. caerulea caused a reduction in immunoglobulin and splenic lymphocyte responses to antigenic stimulation with sheep red blood cells, along with decreased white blood cell and serum protein concentrations, indicating possible impaired immune response capability of Bd-infected frogs. This is the first in vivo study suggesting that infection with Bd causes multiple defects in systemic host immune function, and this may contribute to disease development in susceptible host species. Although L. infrafrenata failed to maintain Bd infection after exposure, white blood cell and serum globulin concentrations were lower in recovered frogs compared with unexposed frogs, but antigen-specific serum and splenic antibody, and splenic cellular, responses were similar in both recovered and unexposed frogs. This may indicate potential systemic costs associated with infection clearance and/or redirection of host resources towards more effective mechanisms to overcome infection. No clear mechanism for resistance was identified in L. infrafrenata, suggesting that localized and/or innate immune defense mechanisms may be important factors involved in disease resistance in this species.     

Genetic characterization of chytrids isolated from larval amphibians collected in central and east Texas

Chytridiomycosis, an emerging infectious disease caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), has caused amphibian population declines worldwide. Bd was first described in the 1990s and there are still geographic gaps in the genetic analysis of this globally distributed pathogen. Relatively few genetic studies have focused on regions where Bd exhibits low virulence, potentially creating a bias in our current knowledge of the pathogen's genetic diversity. Disease-associated declines have not been recorded in Texas (USA), yet Bd has been detected on amphibians in the state. These strains have not been isolated and characterized genetically; therefore, we isolated, cultured, and genotyped Bd from central Texas and compared isolates to a panel of previously genotyped strains distributed across the Western Hemisphere. We also isolated other chytrids from east Texas not known to infect amphibians. To identify larval amphibian hosts, we sequenced part of the COI gene. Among 37 Bd isolates from Texas, we detected 19 unique multi-locus genotypes, but found no genetic structure associated with host species, Texas localities, or across North America. Isolates from central Texas exhibit high diversity and genetically cluster with BdGPL isolates from the western U.S. that have caused amphibian population declines. This study genetically characterizes isolates of Bd from the south central U.S. and adds to the global knowledge of Bd genotypes.     

A Non-Invasive Stress Assay Shows That Tadpole Populations Infected with Batrachochytrium dendrobatidis Have Elevated Corticosterone Levels

Batrachochytrium dendrobatidis (Bd) is a fungus that causes the disease chytridiomycosis and is associated with widespread amphibian declines. Populations vary in their susceptibility to Bd infections, and the virulence of the infecting lineage can also vary. Both of these factors may manifest as a differential physiological stress response. In addition, variation in disease susceptibility across amphibian populations may be influenced by immunosuppression caused by chronic stress imposed by environmental factors. Here, we use a non-invasive water-borne hormone technique to assess stress levels (corticosterone) of free-living tadpole populations that are infected by Bd. We found that corticosterone release rates were higher in infected populations of two species of tadpoles (Alytes obstetricans and A. muletensis) than in an uninfected population for both species. The relationship between corticosterone and the intensity of infection differed between species, with only the infected A. obstetricans population showing a significant positive correlation. The higher corticosterone release rates found in A. obstetricans may be an outcome of infection by a highly virulent lineage of Bd (BdGPL), whereas A. muletensis is infected with a less virulent lineage (BdCAPE). These results suggest that different lineages of Bd impose different levels of stress on the infected animals, and that this may influence survival. The next step is to determine whether higher corticosterone levels make individuals more susceptible to Bd or if Bd infections drive the higher corticosterone levels.     

Cutaneous Bacteria of the Redback Salamander Prevent Morbidity Associated with a Lethal Disease

Chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), is an infectious disease that causes population declines of many amphibians. Cutaneous bacteria isolated from redback salamanders, Plethodon cinereus, and mountain yellow-legged frogs, Rana muscosa, inhibit the growth of Bd in vitro. In this study, the bacterial community present on the skin of P. cinereus individuals was investigated to determine if it provides protection to salamanders from the lethal and sub-lethal effects of chytridiomycosis. When the cutaneous bacterial community was reduced prior to Bd exposure, salamanders experienced a significantly greater decrease in body mass, which is a symptom of the disease, when compared to infected individuals with a normal bacterial community. In addition, a greater proportion of infected individuals with a reduced bacterial community experienced limb-lifting, a behavior seen only in infected individuals. Overall, these results demonstrate that the cutaneous bacterial community of P. cinereus provides protection to the salamander from Bd and that alteration of this community can change disease resistance. Therefore, symbiotic microbes associated with this species appear to be an important component of its innate skin defenses.     

Host‐specific thermal profiles affect fitness of a widespread pathogen

Host behavior can interact with environmental context to influence outcomes of pathogen exposure and the impact of disease on species and populations. Determining whether the thermal behaviors of individual species influence susceptibility to disease can help enhance our ability to explain and predict how and when disease outbreaks are likely to occur. The widespread disease chytridiomycosis (caused by the fungal pathogen Batrachochytrium dendrobatidis, Bd) often has species‐specific impacts on amphibian communities; some host species are asymptomatic, whereas others experience mass mortalities and population extirpation. We determined whether the average natural thermal regimes experienced by sympatric frog species in nature, in and of themselves, can account for differences in vulnerability to disease. We did this by growing Bd under temperatures mimicking those experienced by frogs in the wild. At low and high elevations, the rainforest frogs Litoria nannotis, L. rheocola, and L. serrata maintained mean thermal regimes within the optimal range for pathogen growth (15–25°C). Thermal regimes for L. serrata, which has recovered from Bd‐related declines, resulted in slower pathogen growth than the cooler and less variable thermal regimes for the other two species, which have experienced more long‐lasting declines. For L. rheocola and L. serrata, pathogen growth was faster in thermal regimes corresponding to high elevations than in those corresponding to low elevations, where temperatures were warmer. For L. nannotis, which prefers moist and thermally stable microenvironments, pathogen growth was fastest for low‐elevation thermal regimes. All of the thermal regimes we tested resulted in pathogen growth rates equivalent to, or significantly faster than, rates expected from constant‐temperature experiments. The effects of host body temperature on Bd can explain many of the broad ecological patterns of population declines in our focal species, via direct effects on pathogen fitness. Understanding the functional response of pathogens to conditions experienced by the host is important for determining the ecological drivers of disease outbreaks.     

External Reinfection of a Fungal Pathogen Does not Contribute to Pathogen Growth

Chytridiomycosis is an emerging infectious disease of amphibians caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), which has led to devastating declines in amphibian populations worldwide. Current theory predicts that Bd infections are maintained through both reproduction on the host’s skin and reinfection from sources outside of the host. To investigate the importance of external reinfection on pathogen burden, we infected captive-bred individuals of the highly susceptible Panamanian Golden Frog, Atelopus glyphus, and wild-caught glass frogs, Espadarana prosoblepon, with Bd. We housed the animals in one of three treatments: individually, in heterospecific pairs, and in conspecific pairs. For 8 weeks, we measured the Bd load and shedding rate of all frogs. We found that Atelopus had high rates of increase in both Bd load and shedding rate, but pathogen growth rates did not differ among treatments. The infection intensity of Espadarana co-housed with Atelopus was indistinguishable from those housed singly and those in conspecific pairs, despite being exposed to a large external source of Bd zoospores. Our results indicate that Bd load in both species is driven by pathogen replication within an individual, with reinfection from outside the host contributing little to the amplification of host fungal load.     

The Influence of Temperature on Chytridiomycosis In Vivo

Chytridiomycosis, an amphibian disease caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), is an ideal system for studying the influence of temperature on host–pathogen relationships because both host and pathogen are ectothermic. Studies of Bd in culture suggest that optimal growth occurs between 17 and 23°C, and death of the fungus occurs above 29 or below 0°C. Amphibian immune systems, however, are also temperature dependent and often more effective at higher temperatures. We therefore hypothesized that pathogen load, probability of infection and mortality in Bd-exposed frogs would peak at a lower temperature than that at which Bd grows best in vitro. To test this, we conducted a study where Bd- and sham-exposed Northern cricket frogs (Acris crepitans) were incubated at six temperatures between 11 and 26°C. While probability of infection did not differ across temperatures, pathogen load and mortality were inversely related to temperature. Survival of infected hosts was greatest between 20 and 26°C, temperatures where Bd grows well in culture. These results demonstrate that the conditions under which a pathogen grows best in culture do not necessarily reflect patterns of pathogenicity, an important consideration for predicting the threat of this and other wildlife pathogens.     

Susceptibility of frogs to chytridiomycosis correlates with increased levels of immunomodulatory serotonin in the skin

Chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis (Bd), is a skin disease responsible for the global decline of amphibians. Frog species and populations can vary in susceptibility, but this phenomenon remains poorly understood. Here, we investigated serotonin in the skin of infected and uninfected frogs. In more susceptible frog populations, skin serotonin rose with increasing infection intensity, but decreased in later stages of the disease. The more resistant population maintained a basal level of skin serotonin. Serotonin inhibited both Bd sporangial growth and Jurkat lymphocyte proliferation in vitro. However, serotonin accumulates in skin granular glands, and this compartmentalisation may prevent inhibition of Bd growth in vivo. We suggest that skin serotonin increases in susceptible frogs due to pathogen excretion of precursor tryptophan, but that resistant frogs are able to control the levels of serotonin. Overall, the immunosuppressive effects of serotonin may contribute to the susceptibility of frogs to chytridiomycosis.     

Variation in phenotype and virulence among enzootic and panzootic amphibian chytrid lineages

The Global Panzootic Lineage of the fungus Batrachochytrium dendrobatidis (Bd-GPL) is threatening amphibians worldwide. In contrast, four lineages (Bd-Brazil, Bd-CH, Bd-Cape, and Bd-Korea) that diverged early in the history of Bd have not yet been directly linked to amphibian declines. Bd likely evolves in response to strong selective pressure imposed by hosts and the environment, leading to differences among pathogen phenotypes and genotypes that may directly affect virulence. Here, we report on variation in phenotype, genotype, and virulence of Bd-Brazil and Bd-GPL. Specifically, we (i) used a controlled infection experiment to compare virulence between one Bd-Brazil and three Bd-GPL isolates on a North American amphibian host (Lithobates sylvaticus), (ii) tested for relative phenotypic and genotypic differentiation among Bd isolates from Brazil, and (iii) tested for possible correlations between environmental variables and Bd phenotypes. We found substantial variation in virulence among Bd-GPL isolates and found that our Bd-Brazil isolate showed virulence comparable to an average North American Bd-GPL. North American hosts infected with a Bd-GPL isolate from Panama did not show significant mortality. Bd phenotypes varied significantly across sampling locations; these phenotypes were neither spatially clustered nor correlated with any environmental variables. Additionally, we found a surprising lack of correlation between genotypic divergence and zoospore and zoosporangium sizes in our sample. Although Bd-Brazil was less virulent infecting L. sylvaticus than one Bd-GPL isolate, this endemic lineage still caused ∼50% mortality in our experimental North American hosts. This indicates that Bd-Brazil has the potential to kill amphibians if introduced to naïve wild populations. Our findings underscore that characterizing virulence of multiple Bd isolates and lineages is important for understanding the evolutionary history and diversity of Bd.     

Reduced Itraconazole Concentration and Durations Are Successful in Treating Batrachochytrium dendrobatidis Infection in Amphibians

Amphibians are experiencing the greatest decline of any vertebrate class and a leading cause of these declines is a fungal pathogen, Batrachochytrium dendrobatidis (Bd), which causes the disease chytridiomycosis. Captive assurance colonies are important worldwide for threatened amphibian species and may be the only lifeline for those in critical threat of extinction. Maintaining disease free colonies is a priority of captive managers, yet safe and effective treatments for all species and across life stages have not been identified. The most widely used chemotherapeutic treatment is itraconazole, although the dosage commonly used can be harmful to some individuals and species. We performed a clinical treatment trial to assess whether a lower and safer but effective dose of itraconazole could be found to cure Bd infections. We found that by reducing the treatment concentration from 0.01-0.0025% and reducing the treatment duration from 11-6 days of 5 min baths, frogs could be cured of Bd infection with fewer side effects and less treatment-associated mortality.     

Persistence with Chytridiomycosis Does Not Assure Survival of Direct-developing Frogs

The chytrid fungus, Batrachochytrium dendrobatidis (Bd) has been linked to extinction and decline of numerous amphibians. We studied the population-level effects of Bd in two post-decline anuran species, Eleutherodactylus coqui and E. portoricensis, at El Yunque National Forest, Puerto Rico. Data on amphibian abundance was updated to report long-term population trends. Mark–recapture data was used to monitor Bd-infection status and estimate survival probabilities of infected versus uninfected adults. Prevalence of Bd (number of infected/total sampled) and individual infection level (number of zoospores) were compared among age classes at Palo Colorado Forest (661 m) and Elfin Forest (850 m). Results revealed that both species continued to decrease in Palo Colorado Forest, while in the Elfin Forest, E. portoricensis recuperated from drastic declines. Age class, season, and locality significantly predicted zoospore load. Age was also significantly associated with high zoospores loads among Bd-positive frogs, and the prevalence of Bd was higher in juveniles than adults in all populations studied. We suggest that early age represents a critical life stage in the survival of direct-developing frogs infected by this fungus. Survival probability was always higher for uninfected frogs, but recapture rates of infected versus uninfected adults were significantly different only in Palo Colorado, alerting that the negative effect of Bd infection under enzootic conditions is greater at mid-elevations. This work contributes to our understanding of how direct-developing amphibians persist with Bd, pointing to critical life stages and synergistic interactions that may induce fluctuations and/or declines in the wild.     

Retrospective Survey of Museum Specimens Reveals Historically Widespread Presence of Batrachochytrium dendrobatidis in China

Chytridiomycosis, caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd), has been implicated in amphibian population declines worldwide. However, no amphibian declines or extinctions associated with Bd have been reported in Asia. To investigate the history of this pathogen in China, we examined 1,007 museum-preserved amphibian specimens of 80 species collected between 1933 and 2009. Bd was detected in 60 individuals (6.0%), with the earliest case of Bd infection occurring in one specimen of Bufo gargarizans and two Fejervarya limnocharis, all collected in 1933 from Chongqing, southwest China. Although mainly detected in non-threatened native amphibians, Bd was also found in four endangered species. We report the first evidence of Bd for Taiwan and the first detection of Bd in the critically endangered Chinese giant salamander (Andrias davidianus). Bd appears to have been present at a low rate of infection since at least the 1930s in China, and no significant differences in prevalence were detected between decades or provinces, suggesting that a historical steady endemic relationship between Bd and Chinese amphibians has occurred. Our results add new insights on the global emergence of Bd and suggest that this pathogen has been more widely distributed in the last century than previously believed.     

<Emphasis Type="Italic">Batrachochytrium dendrobatidis</Emphasis> and the Decline and Survival of the Relict Leopard Frog

Epizootic disease caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd) is a major driver of amphibian declines, yet many amphibians declined before the pathogen was described. The Relict Leopard Frog, Rana onca (=Lithobates onca), was nearly extinct, with the exception of populations within a few geothermal springs. Growth of Bd, however, is limited by high water temperature, and geothermal springs may have provided refuge during outbreaks of chytridiomycosis. We conducted field surveys and laboratory experiments to assess the susceptibility of R. onca to Bd. In the field, we found Bd at one of the two areas where remnant populations of R. onca still occur, but not in the other. In the laboratory, we infected juvenile frogs from these two areas with two hypervirulent Bd isolates associated with declines in other ranid species. In our experiments, these Bd isolates did not affect survivorship of R. onca and most infections (64%) were cleared by the end of the experiments. We propose that R. onca either has inherent resistance to Bd or has recently evolved such resistance. These results may be important for conservation efforts aimed at establishing new populations of R. onca across a landscape where Bd exists. Resistance, however, varies among life stages, and we also did not assess Bd from the local environment. We caution that the resistance we observed for young frogs under laboratory conditions may not translate to the situation for R. onca in the wild.