Lack of variations in the salamander chytrid fungus, Batrachochytrium salamandrivorans, at its alleged origin: Updating its Japanese distribution with new evidence.

The chytrid fungus Batrachochytrium salamandrivorans [Bsal] is causing declines in the amphibian populations. After a decade of mapping the pathogen in Europe, where it is causing dramatic outbreaks, and North America, where its arrival would affect to the salamander's biodiversity hotspot, little is known about its current status in Asia, from presumably is native. Japan has several species considered as potential carriers, but no regulation is implemented against Bsal spreading. Previous Bsal known presence detected various cases on the Okinawa Island, southwestern Japan. Previous studies on its sister species, B. dendrobatidis presented a high genomic variation in this area and particularly on Cynops ensicauda. Here, we have done the largest monitoring to date in Japan on the Cynops genus, focusing on Okinawa Island and updating its distribution and providing more information to unravel the still unknown origin of Bsal. Interestingly, we have provided revealing facts about different detectability depending on the used molecular techniques and changes in its Japanese distribution. All in all, the Bsal presence in Japan, together with its low variability in the sequenced amplicons, and the lack of apparent mortalities, may indicate that this part of Asia has a high diversity of chytrids.

Phylosymbiosis shapes skin bacterial communities and pathogen-protective function in Appalachian salamanders.

Phylosymbiosis is an association between host-associated microbiome composition and host phylogeny. This pattern can arise via the evolution of host traits, habitat preferences, diets, and the co-diversification of hosts and microbes. Understanding the drivers of phylosymbiosis is vital for modelling disease-microbiome interactions and manipulating microbiomes in multi-host systems. This study quantifies phylosymbiosis in Appalachian salamander skin in the context of infection by the fungal pathogen Batrachochytrium dendrobatidis (Bd), while accounting for environmental microbiome exposure. We sampled ten salamander species representing >150M years of divergence, assessed their Bd infection status, and analysed their skin and environmental microbiomes. Our results reveal a significant signal of phylosymbiosis, whereas the local environmental pool of microbes, climate, geography, and Bd infection load had a smaller impact. Host-microbe co-speciation was not evident, indicating that the effect stems from the evolution of host traits influencing microbiome assembly. Bd infection is correlated with host phylogeny and the abundance of Bd-inhibitory bacterial strains, suggesting that the long-term evolutionary dynamics between salamander hosts and their skin microbiomes affect the present-day distribution of the pathogen, along with habitat-linked exposure risk. Five Bd-inhibitory bacterial strains showed unusual generalism: occurring in most host species and habitats. These generalist strains may enhance the likelihood of probiotic manipulations colonising and persisting on hosts. Our results underscore the substantial influence of host-microbiome eco-evolutionary dynamics on environmental health and disease outcomes.

Alpine salamanders at risk? The current status of an emerging fungal pathogen.

Amphibians globally suffer from emerging infectious diseases like chytridiomycosis caused by the continuously spreading chytrid fungi. One is Batrachochytrium salamandrivorans (Bsal) and its disease ‒ the 'salamander plague' ‒ which is lethal to several caudate taxa. Recently introduced into Western Europe, long distance dispersal of Bsal, likely through human mediation, has been reported. Herein we study if Alpine salamanders (Salamandra atra and S. lanzai) are yet affected by the salamander plague in the wild. Members of the genus Salamandra are highly susceptible to Bsal leading to the lethal disease. Moreover, ecological modelling has shown that the Alps and Dinarides, where Alpine salamanders occur, are generally suitable for Bsal. We analysed skin swabs of 818 individuals of Alpine salamanders and syntopic amphibians at 40 sites between 2017 to 2022. Further, we compiled those with published data from 319 individuals from 13 sites concluding that Bsal infections were not detected. Our results suggest that the salamander plague so far is absent from the geographic ranges of Alpine salamanders. That means that there is still a chance to timely implement surveillance strategies. Among others, we recommend prevention measures, citizen science approaches, and ex situ conservation breeding of endemic salamandrid lineages.

Metagenomic and transcriptomic analysis revealing the impact of oxytetracycline and ciprofloxacin on gut microbiota and gene expression in the Chinese giant salamander (Andrias davidianus).

Excessive antibiotic use has led to the spread of antibiotic resistance genes (ARGs), impacting gut microbiota and host health. However, the effects of antibiotics on amphibian populations remain unclear. We investigated the impact of oxytetracycline (OTC) and ciprofloxacin (CIP) on Chinese giant salamanders (Andrias davidianus), focusing on gut microbiota, ARGs, and gene expression by performing metagenome and transcriptome sequencing. A. davidianus were given OTC (20 or 40 mg/kg) or CIP (50 or 100 mg/kg) orally for 7 days. The results revealed that oral administration of OTC and CIP led to distinct changes in microbial composition and functional potential, with CIP treatment having a greater impact than OTC. Antibiotic treatment also influenced the abundance of ARGs, with an increase in fluoroquinolone and multi-drug resistance genes observed post-treatment. The construction of metagenome-assembled genomes (MAGs) accurately validated that CIP intervention enriched fish-associated potential pathogens Aeromonas hydrophila carrying an increased number of ARGs. Additionally, mobile genetic elements (MGEs), such as phages and plasmids, were implicated in the dissemination of ARGs. Transcriptomic analysis of the gut revealed significant alterations in gene expression, particularly in immune-related pathways, with differential effects observed between OTC and CIP treatments. Integration of metagenomic and transcriptomic data highlighted potential correlations between gut gene expression and microbial composition, suggesting complex interactions between the host gut and its gut microbiota in response to antibiotic exposure. These findings underscore the importance of understanding the impact of antibiotic intervention on the gut microbiome and host health in amphibians, particularly in the context of antibiotic resistance and immune function.

Ontogeny drives shifts in skin bacterial communities in facultatively paedomorphic salamanders.

Microbiomes are major determinants of host growth, development and survival. In amphibians, host-associated bacteria in the skin can inhibit pathogen infection, but many processes can influence the structure and composition of the community. Here we quantified the shifts in skin-associated bacteria across developmental stages in the striped newt (Notophthalmus perstriatus), a threatened salamander species with a complex life history and vulnerable to infection by the amphibian chytrid fungus Batrachochytrium dendrobatidis and ranavirus. Our analyses show that pre-metamorphic larval and paedomorphic stages share similar bacterial compositions, and that the changes in the microbiome coincided with physiological restructuring during metamorphosis. Newts undergoing metamorphosis exhibited microbiome compositions that were intermediate between paedomorphic and post-metamorphic stages, further supporting the idea that metamorphosis is a major driver of host-associated microbes in amphibians. We did not find support for infection-related disruption of the microbiome, though infection replicates were small for each respective life stage.

Broad host susceptibility of North American amphibian species to Batrachochytrium salamandrivorans suggests high invasion potential and biodiversity risk.

Batrachochytrium salamandrivorans (Bsal) is a fungal pathogen of amphibians that is emerging in Europe and could be introduced to North America through international trade or other pathways. To evaluate the risk of Bsal invasion to amphibian biodiversity, we performed dose-response experiments on 35 North American species from 10 families, including larvae from five species. We discovered that Bsal caused infection in 74% and mortality in 35% of species tested. Both salamanders and frogs became infected and developed Bsal chytridiomycosis. Based on our host susceptibility results, environmental suitability conditions for Bsal, and geographic ranges of salamanders in the United States, predicted biodiversity loss is expected to be greatest in the Appalachian Region and along the West Coast. Indices of infection and disease susceptibility suggest that North American amphibian species span a spectrum of vulnerability to Bsal chytridiomycosis and most amphibian communities will include an assemblage of resistant, carrier, and amplification species. Predicted salamander losses could exceed 80 species in the United States and 140 species in North America.

Encouraging news for in situ conservation: Translocation of salamander larvae has limited impacts on their skin microbiota.

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.

Metabolite compositions on skins of eastern hellbenders Cryptobranchus alleganiensis alleganiensis differ with location and captivity.

Eastern hellbenders Cryptobranchus alleganiensis alleganiensis, large aquatic salamanders, are declining over most of their range. The amphibian-killing fungus Batrachochytrium dendrobatidis (Bd) has contributed to global amphibian declines and has been detected on eastern hellbenders, but infection intensities were lower than those of species that are more susceptible to Bd. The factors limiting Bd on hellbenders may include antifungal metabolites produced by their skin microbiota. We used a metabolite fingerprinting technique to noninvasively identify the presence, but not identity, of metabolites associated with eastern hellbenders. We surveyed the skin of wild eastern hellbenders to test whether the composition and richness (i.e. number of metabolites) of their metabolites are explained by Bd status or location. Furthermore, we surveyed for metabolites on captive eastern hellbenders to test whether metabolite compositions were different between captive and wild eastern hellbenders. Bd detection was not associated with either metabolite richness or composition. Both metabolite composition and richness differed significantly on hellbenders from different locations (i.e. states). For metabolite composition, there was a statistical interaction between location and Bd status. Metabolite richness was greater on captive eastern hellbenders compared to wild hellbenders, and metabolite compositions differed between wild and captive eastern hellbenders. The methods we employed to detect metabolite profiles effectively grouped individuals by location even though metabolite composition and richness have high levels of intraspecific variation. Understanding the drivers and functional consequences of assemblages of skin metabolites on amphibian health will be an important step toward understanding the mechanisms that result in disease vulnerability.

Factors Influencing Bacterial and Fungal Skin Communities of Montane Salamanders of Central Mexico.

Host microbial communities are increasingly seen as an important component of host health. In amphibians, the first land vertebrates that are threatened by a fungal skin disease globally, our understanding of the factors influencing the microbiome of amphibian skin remains incomplete because recent studies have focused almost exclusively on bacteria, and little information exists on fungal communities associated with wild amphibian species. In this study, we describe the effects of host phylogeny, climate, geographic distance, and infection with a fungal pathogen on the composition and structure of bacterial and fungal communities in seven tropical salamander species that occur in the Trans-Mexican Volcanic Belt of Central Mexico. We find that host phylogenetic relatedness is correlated with bacterial community composition while a composite climatic variable of temperature seasonality and precipitation is significantly associated with fungal community composition. We also estimated co-occurrence networks for bacterial and fungal taxa and found differences in the degree of connectivity and the distribution of negative associations between the two networks. Our results suggest that different factors may be responsible for structuring the bacterial and fungal communities of amphibian skin and that the inclusion of fungi in future studies could shed light on important functional interactions within the microbiome.

Bacterial Skin Assemblages of Sympatric Salamanders Are Primarily Shaped by Host Genus.

Bacterial assemblages on the skins of amphibians are known to influence pathogen resistance and other important physiological functions in the host. Host-specific factors and the environment play significant roles in structuring skin assemblages. This study used high-throughput 16S rRNA sequencing and multivariate analyses to examine differences in skin-bacterial assemblages from 246 salamanders belonging to three genera in the lungless family Plethodontidae along multiple spatial gradients. Composition and α- and ß-diversity of bacterial assemblages were defined, indicator species were identified for each host group, and the relative influences of host- versus environment-specific ecological factors were evaluated. At the broadest spatial scale, host genus, host species, and sampling site were predictive of skin assemblage structure, but host genus and species were more influential after controlling for the marginal effects of site, as well as nestedness of site. Furthermore, assemblage similarity within each host genus did not change with increasing geographic distance. At the smallest spatial scale, site-specific climate analyses revealed different relationships to climatic variables for each of the three genera, and these relationships were determined by host ecomode. Variation in bacterial assemblages of terrestrial hosts correlated with landscape-level climatic variability, and this pattern decayed with increasing water dependence of the host. Results from this study highlight host-specific considerations for researchers studying wildlife diseases in co-occurring, yet ecologically divergent, species.

The fungal pathogen Batrachochytrium salamandrivorans is not detected in wild and captive amphibians from Mexico.

The recent emergence of the pathogen Batrachochytrium salamandrivorans (Bsal) is associated with rapid population declines of salamanders in Europe and its arrival to new areas could cause dramatic negative effects on other amphibian populations and species. Amphibian species, present in areas with high amphibian diversity such as Mexico, could be highly threatened due to the arrival of Bsal, particularly salamander species which are more vulnerable to chytridiomycosis caused by this pathogen. Thus, immediate surveillance is needed as a strategy to efficiently contend with this emerging infectious disease. In this study, we analyzed 490 wild and captive amphibians from 48 species across 76 sites in the North, Central, and South of Mexico to evaluate the presence of Bsal. Amphibians were sampled in sites with variable degrees of amphibian richness and suitability for Bsal according to previous studies. From the 76 sampling sites, 10 of them were located in areas with high amphibian richness and potential moderate to high Bsal habitat suitability. We did not detect Bsal in any of the samples, and no signs of the disease were observed in any individual at the time of sampling. Our results suggest that Bsal has not yet arrived at the sampled sites or could be at low prevalence within populations with low occurrence probability. This is the first study that evaluates the presence of Bsal in different regions and amphibian species in Mexico, which is the second most diverse country in salamander species in the world. We highlight the risk and the importance of continuing surveillance of Bsal in Mexico and discuss control strategies to avoid the introduction and spread of Bsal in the country.

Inhibitory Bacterial Diversity and Mucosome Function Differentiate Susceptibility of Appalachian Salamanders to Chytrid Fungal Infection.

Mucosal defenses are crucial in animals for protection against pathogens and predators. Host defense peptides (antimicrobial peptides, AMPs) as well as skin-associated microbes are key components of mucosal immunity, particularly in amphibians. We integrate microbiology, molecular biology, network-thinking, and proteomics to understand how host and microbially derived products on amphibian skin (referred to as the mucosome) serve as pathogen defenses. We studied defense mechanisms against chytrid pathogens, Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal), in four salamander species with different Batrachochytrium susceptibilities. Bd infection was quantified using qPCR, mucosome function (i.e., ability to kill Bd or Bsal zoospores in vitro), skin bacterial communities using 16S rRNA gene amplicon sequencing, and the role of Bd-inhibitory bacteria in microbial networks across all species. We explored the presence of candidate-AMPs in eastern newts and red-backed salamanders. Eastern newts had the highest Bd prevalence and mucosome function, while red-back salamanders had the lowest Bd prevalence and mucosome function, and two-lined salamanders and seal salamanders were intermediates. Salamanders with highest Bd infection intensity showed greater mucosome function. Bd infection prevalence significantly decreased as putative Bd-inhibitory bacterial richness and relative abundance increased on hosts. In co-occurrence networks, some putative Bd-inhibitory bacteria were found as hub-taxa, with red-backs having the highest proportion of protective hubs and positive associations related to putative Bd-inhibitory hub bacteria. We found more AMP candidates on salamanders with lower Bd susceptibility. These findings suggest that salamanders possess distinct innate mechanisms that affect chytrid fungi. IMPORTANCE How host mucosal defenses interact, and influence disease outcome is critical in understanding host defenses against pathogens. A more detailed understanding is needed of the interactions between the host and the functioning of its mucosal defenses in pathogen defense. This study investigates the variability of chytrid susceptibility in salamanders and the innate defenses each species possesses to mediate pathogens, thus advancing the knowledge toward a deeper understanding of the microbial ecology of skin-associated bacteria and contributing to the development of bioaugmentation strategies to mediate pathogen infection and disease. This study improves the understanding of complex immune defense mechanisms in salamanders and highlights the potential role of the mucosome to reduce the probability of Bd disease development and that putative protective bacteria may reduce likelihood of Bd infecting skin.

Does the thermal mismatch hypothesis predict disease outcomes in different morphs of a terrestrial salamander?

Many aspects of ectotherm physiology are temperature-dependent. The immune system of temperate-dwelling ectothermic host species is no exception and their immune function is often downregulated in cold temperatures. Likewise, species of ectothermic pathogens experience temperature-mediated effects on rates of transmission and/or virulence. Although seemingly straightforward, predicting the outcomes of ectothermic host-pathogen interactions is quite challenging. A recent hypothesis termed the thermal mismatch hypothesis posits that cool-adapted host species should be most susceptible to pathogen infection during warm temperature periods whereas warm-adapted host species should be most susceptible to pathogens during periods of cool temperatures. We explore this hypothesis using two ecologically and physiologically differentiated color morphs of the Eastern Red-backed Salamander (Plethodon cinereus) and a pathogenic chytrid fungus (Batrachochytrium dendrobatidis; hereafter "Bd") using a fully factorial laboratory experiment. At cool temperatures, unstriped salamanders (i.e., those that are tolerant of warm temperatures) had a significantly higher probability of Bd infection compared with cool-tolerant striped salamanders, consistent with the thermal mismatch hypothesis. However, we found no support for this hypothesis when salamanders were exposed to Bd at warm temperatures: the probability of Bd infection in the cool-tolerant striped salamanders was nearly identical in both cool and warm temperatures, opposite the predictions of the thermal mismatch hypothesis. Our results are most consistent with the fact that Bd grows poorly at warm temperatures. Alternatively, our data could indicate that the two color morphs do not differ in their tolerance to warm temperatures but that striped salamanders are more tolerant to cool temperatures than unstriped salamanders.

Infection with Batrachochytrium dendrobatidis reduces salamander capacity to mount a cell-mediated immune response.

The vertebrate immune system is a costly defense system that is responsible for preventing and eliminating parasites and pathogens. Theory predicts that hosts experience tradeoffs associated with immune deployment and other physiological functions. Although empirical evidence for immune-physiology tradeoffs are well documented in the literature, fewer studies have examined tradeoffs within the immune system in wild vertebrates. We explored the topic of concomitant immune challenges in amphibians by exposing salamanders (Plethodon cinereus) to a fungal pathogen Batrachochytrium dendrobatidis (hereafter "Bd") and then to phytohemagglutinin (hereafter "PHA"). We measured Bd infection using quantitative PCR and used measurements of the tail thickness at the PHA injection site as an estimate of skin swelling. We tested whether Bd reduced the salamander's capacity to mount an immune response towards PHA or whether Bd would stimulate immune activity and thereby increase the response towards PHA. Salamanders that were infected with Bd had a reduced skin-swelling when injected with PHA compared to noninfected salamanders, a result that is consistent with the hypothesis that Bd-infected salamanders have lower immunocompetence than noninfected salamanders. We also found that PHA-induced swelling response was negatively associated with Bd infection abundance (i.e., the infection burden of all exposed salamanders, including those that were exposed but not infected), indicating that salamanders with a higher infection abundance had the lowest swelling response to PHA. Our results suggest that individuals of P. cinereus might experience an energetic tradeoff between successfully fighting off Bd and mounting an immune response towards PHA.

Diversity, multifaceted evolution, and facultative saprotrophism in the European Batrachochytrium salamandrivorans epidemic.

While emerging fungi threaten global biodiversity, the paucity of fungal genome assemblies impedes thoroughly characterizing epidemics and developing effective mitigation strategies. Here, we generate de novo genomic assemblies for six outbreaks of the emerging pathogen Batrachochytrium salamandrivorans (Bsal). We reveal the European epidemic currently damaging amphibian populations to comprise multiple, highly divergent lineages demonstrating isolate-specific adaptations and metabolic capacities. In particular, we show extensive gene family expansions and acquisitions, through a variety of evolutionary mechanisms, and an isolate-specific saprotrophic lifecycle. This finding both explains the chytrid's ability to divorce transmission from host density, producing Bsal's enigmatic host population declines, and is a key consideration in developing successful mitigation measures.

Batrachochytrium salamandrivorans Can Devour More than Salamanders.

Batrachochytrium salamandrivorans is an emerging fungus that is causing salamander declines in Europe. We evaluated whether an invasive frog species (Cuban treefrog, Osteopilus septentrionalis) that is found in international trade could be an asymptomatic carrier when exposed to zoospore doses known to infect salamanders. We discovered that Cuban treefrogs could be infected with B. salamandrivorans and, surprisingly, that chytridiomycosis developed in animals at the two highest zoospore doses. To fulfill Koch's postulates, we isolated B. salamandrivorans from infected frogs, exposed eastern newts (Notophthalmus viridescens) to the isolate, and verified infection and disease by histopathology. This experiment represents the first documentation of B. salamandrivorans chytridiomycosis in a frog species and substantially expands the conservation threat and possible mobilization of this pathogen in trade.

Preparing for invasion: Assessing risk of infection by chytrid fungi in southeastern plethodontid salamanders.

Understanding the responses of naïve communities to the invasion of multihost pathogens requires accurate estimates of susceptibility across taxa. In the Americas, the likely emergence of a second amphibian pathogenic fungus (Batrachochytrium salamandrivorans, Bsal) calls for new ways of prioritizing disease mitigation among species due to the high diversity of naïve hosts with prior B. dendrobatidis (Bd) infections. Here, we applied the concept of pathogenic potential to quantify the virulence of chytrid fungi on naïve amphibians and evaluate species for conservation efforts in the event of an outbreak. The benefit of this measure is that it combines and summarizes the variation in disease effects into a single numerical index, allowing for comparisons across species, populations or groups of individuals that may inherently exhibit differences in susceptibility. As a proof of concept, we obtained standardized responses of disease severity by performing experimental infections with Bsal on five plethodontid salamanders from southeastern United States. Four out of five species carried natural infections of Bd at the start of the experiments. We showed that Bsal exhibited its highest value of pathogenic potential in a species that is already declining (Desmognathus auriculatus). We find that this index provides additional information beyond the standard measures of disease prevalence, intensity, and mortality, because it leveraged these disease parameters within each categorical group. Scientists and practitioners could use this measure to justify research, funding, trade, or conservation measures.

Significant reductions of host abundance weakly impact infection intensity of Batrachochytrium dendrobatidis.

Infectious diseases are considered major threats to biodiversity, however strategies to mitigate their impacts in the natural world are scarce and largely unsuccessful. Chytridiomycosis is responsible for the decline of hundreds of amphibian species worldwide, but an effective disease management strategy that could be applied across natural habitats is still lacking. In general amphibian larvae can be easily captured, offering opportunities to ascertain the impact of altering the abundance of hosts, considered to be a key parameter affecting the severity of the disease. Here, we report the results of two experiments to investigate how altering host abundance affects infection intensity in amphibian populations of a montane area of Central Spain suffering from lethal amphibian chytridiomycosis. Our laboratory-based experiment supported the conclusion that varying density had a significant effect on infection intensity when salamander larvae were housed at low densities. Our field experiment showed that reducing the abundance of salamander larvae in the field also had a significant, but weak, impact on infection the following year, but only when removals were extreme. While this suggests adjusting host abundance as a mitigation strategy to reduce infection intensity could be useful, our evidence suggests only heavy culling efforts will succeed, which may run contrary to objectives for conservation.

Presence of low virulence chytrid fungi could protect European amphibians from more deadly strains.

Wildlife diseases are contributing to the current Earth's sixth mass extinction; one disease, chytridiomycosis, has caused mass amphibian die-offs. While global spread of a hypervirulent lineage of the fungus Batrachochytrium dendrobatidis (BdGPL) causes unprecedented loss of vertebrate diversity by decimating amphibian populations, its impact on amphibian communities is highly variable across regions. Here, we combine field data with in vitro and in vivo trials that demonstrate the presence of a markedly diverse variety of low virulence isolates of BdGPL in northern European amphibian communities. Pre-exposure to some of these low virulence isolates protects against disease following subsequent exposure to highly virulent BdGPL in midwife toads (Alytes obstetricans) and alters infection dynamics of its sister species B. salamandrivorans in newts (Triturus marmoratus), but not in salamanders (Salamandra salamandra). The key role of pathogen virulence in the complex host-pathogen-environment interaction supports efforts to limit pathogen pollution in a globalized world.

Experimental methodologies can affect pathogenicity of Batrachochytrium salamandrivorans infections.

Controlled experiments are one approach to understanding the pathogenicity of etiologic agents to susceptible hosts. The recently discovered fungal pathogen, Batrachochytrium salamandrivorans (Bsal), has resulted in a surge of experimental investigations because of its potential to impact global salamander biodiversity. However, variation in experimental methodologies could thwart knowledge advancement by introducing confounding factors that make comparisons difficult among studies. Thus, our objective was to evaluate if variation in experimental methods changed inferences made on the pathogenicity of Bsal. We tested whether passage duration of Bsal culture, exposure method of the host to Bsal (water bath vs. skin inoculation), Bsal culturing method (liquid vs. plated), host husbandry conditions (aquatic vs. terrestrial), and skin swabbing frequency influenced diseased-induced mortality in a susceptible host species, the eastern newt (Notophthalmus viridescens). We found that disease-induced mortality was faster for eastern newts when exposed to a low passage isolate, when newts were housed in terrestrial environments, and if exposure to zoospores occurred via water bath. We did not detect differences in disease-induced mortality between culturing methods or swabbing frequencies. Our results illustrate the need to standardize methods among Bsal experiments. We provide suggestions for future Bsal experiments in the context of hypothesis testing and discuss the ecological implications of our results.