Presence of invasive American bullfrogs may reduce infectious disease in a native frog species.

Amphibians breeding in aquatic environments may encounter a myriad of threats during their life cycle. One species known to prey on native amphibians in aquatic habitats is the invasive North American bullfrog Lithobates catesbeianus, which, besides being a voracious predator and competitor, often acts as a pathogen carrier and disease superspreader because it tolerates high infection loads of the frog-killing fungus Batrachochytrium dendrobatidis (Bd). Here, we hypothesized that the presence of the bullfrogs in microcosms should either (1) decrease Bd disease severity in native frog species by discouraging them from using the aquatic environment, or (2) increase the mortality of the native species. We tested these 2 mutually exclusive hypotheses by co-housing the snouted treefrog Scinax x-signatus (native to our study area) with L. catesbeianus in the laboratory, exposing them to Bd, and using qPCR analysis to quantify the resulting Bd infection loads in the native frogs. Our experiment had the following replicated treatments: (1) native-only treatment (3 individuals of S. x-signatus), (2) native-predominant treatment (2 S. x-signatus + 1 L. catesbeianus), and (3) exotic-predominant treatment (1 S. x-signatus + 2 L. catesbeianus). We found that Bd infection loads in the native S. x-signatus were highest in the native-only treatment, and lowest in the exotic-predominant treatment, indicating that bullfrogs may discourage native frogs from occupying the aquatic habitat, thus reducing encounter rates between native frogs and the waterborne pathogen. This effect could be driven by the bullfrogs' predatory behavior and their high philopatry to aquatic habitats. Our results highlight that predation risk adds to the complexity of host-species interactions in Bd epidemiology.

Is overwintering mortality driving enigmatic declines? Evaluating the impacts of trematodes and the amphibian chytrid fungus on an anuran from hatching through overwintering.

Emerging infectious diseases are increasing globally and are an additional challenge to species dealing with native parasites and pathogens. Therefore, understanding the combined effects of infectious agents on hosts is important for species' conservation and population management. Amphibians are hosts to many parasites and pathogens, including endemic trematode flatworms (e.g., Echinostoma spp.) and the novel pathogenic amphibian chytrid fungus (Batrachochytrium dendrobatidis [Bd]). Our study examined how exposure to trematodes during larval development influenced the consequences of Bd pathogen exposure through critical life events. We found that prior exposure to trematode parasites negatively impacted metamorphosis but did not influence the effect of Bd infection on terrestrial growth and survival. Bd infection alone, however, resulted in significant mortality during overwintering-an annual occurrence for most temperate amphibians. The results of our study indicated overwintering mortality from Bd could provide an explanation for enigmatic declines and highlights the importance of examining the long-term consequences of novel parasite exposure.

Epidermal galactose spurs chytrid virulence and predicts amphibian colonization.

The chytrid fungal pathogens Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans cause the skin disease chytridiomycosis in amphibians, which is driving a substantial proportion of an entire vertebrate class to extinction. Mitigation of its impact is largely unsuccessful and requires a thorough understanding of the mechanisms underpinning the disease ecology. By identifying skin factors that mediate key events during the early interaction with B. salamandrivorans zoospores, we discovered a marker for host colonization. Amphibian skin associated beta-galactose mediated fungal chemotaxis and adhesion to the skin and initiated a virulent fungal response. Fungal colonization correlated with the skin glycosylation pattern, with cutaneous galactose content effectively predicting variation in host susceptibility to fungal colonization between amphibian species. Ontogenetic galactose patterns correlated with low level and asymptomatic infections in salamander larvae that were carried over through metamorphosis, resulting in juvenile mortality. Pronounced variation of galactose content within some, but not all species, may promote the selection for more colonization resistant host lineages, opening new avenues for disease mitigation.

Niche Contraction of an Endangered Frog Driven by the Amphibian Chytrid Fungus.

Introduced pathogens can alter the geographic distribution of susceptible host species. For example, Batrachochytrium dendrobatidis (Bd) is a fungal pathogen that has been linked to the global decline and extinction of numerous amphibian species during the last four decades. A growing number of studies have described the distribution of Bd and susceptible hosts across the globe; however, knowledge on how Bd may shape the climatic niche of susceptible species is still missing. We estimated the effect of Bd on the geographic distribution and niche dynamics of the critically endangered lowland robber frog (Craugastor ranoides) in Costa Rica. We found a reduction of 98% in the geographic range of this species by 1995, following the epizootic outbreaks of Bd that affected Costa Rica in the 1980 and early 1990s. We also quantified niche contraction and found that the species is currently restricted to dry and warm environments that have been considered unsuitable for Bd. Our results contribute to the understanding of how emerging pathogens shape the climatic niches and geographic distribution of susceptible species.

Winter is coming-Temperature affects immune defenses and susceptibility to Batrachochytrium salamandrivorans.

Environmental temperature is a key factor driving various biological processes, including immune defenses and host-pathogen interactions. Here, we evaluated the effects of environmental temperature on the pathogenicity of the emerging fungal pathogen, Batrachochytrium salamandrivorans (Bsal), using controlled laboratory experiments, and measured components of host immune defense to identify regulating mechanisms. We found that adult and juvenile Notophthalmus viridescens died faster due to Bsal chytridiomycosis at 14°C than at 6 and 22°C. Pathogen replication rates, total available proteins on the skin, and microbiome composition likely drove these relationships. Temperature-dependent skin microbiome composition in our laboratory experiments matched seasonal trends in wild N. viridescens, adding validity to these results. We also found that hydrophobic peptide production after two months post-exposure to Bsal was reduced in infected animals compared to controls, perhaps due to peptide release earlier in infection or impaired granular gland function in diseased animals. Using our temperature-dependent susceptibility results, we performed a geographic analysis that revealed N. viridescens populations in the northeastern United States and southeastern Canada are at greatest risk for Bsal invasion, which shifted risk north compared to previous assessments. Our results indicate that environmental temperature will play a key role in the epidemiology of Bsal and provide evidence that temperature manipulations may be a viable disease management strategy.

Discriminating lineages of Batrachochytrium dendrobatidis using quantitative PCR.

The ability to detect and monitor infectious disease in a phylogenetically informative manner is critical for their management. Phylogenetically informative diagnostic tests enable patterns of pathogen introduction or changes in the distribution of genotypes to be measured, enabling research into the ecology of the pathogen. Batrachochytrium dendrobatidis (Bd), a causative agent of chytridiomycosis in amphibian populations, emerged worldwide in the 21st century and is composed of six lineages which are display varying levels of virulence in their hosts. Research into the distribution, ecology and pathogenicity of these lineages has been hampered by an inability to type lineage efficiently. Here, we describe a lineage-specific TaqMan qPCR assay that differentiates the two lineages of Bd most commonly associated with chytridiomycosis: BdGPL and BdCAPE. We demonstrate how this assay can be used for the surveillance of wild populations of amphibians in Southern Africa using skin swabs, tissue samples and cultured isolates.

Skin microbiome correlates with bioclimate and Batrachochytrium dendrobatidis infection intensity in Brazil's Atlantic Forest treefrogs.

In Brazil's Atlantic Forest (AF) biodiversity conservation is of key importance since the fungal pathogen Batrachochytrium dendrobatidis (Bd) has led to the rapid loss of amphibian populations here and worldwide. The impact of Bd on amphibians is determined by the host's immune system, of which the skin microbiome is a critical component. The richness and diversity of such cutaneous bacterial communities are known to be shaped by abiotic factors which thus may indirectly modulate host susceptibility to Bd. This study aimed to contribute to understanding the environment-host-pathogen interaction determining skin bacterial communities in 819 treefrogs (Anura: Hylidae and Phyllomedusidae) from 71 species sampled across the AF. We investigated whether abiotic factors influence the bacterial community richness and structure on the amphibian skin. We further tested for an association between skin bacterial community structure and Bd co-occurrence. Our data revealed that temperature, precipitation, and elevation consistently correlate with richness and diversity of the skin microbiome and also predict Bd infection status. Surprisingly, our data suggest a weak but significant positive correlation of Bd infection intensity and bacterial richness. We highlight the prospect of future experimental studies on the impact of changing environmental conditions associated with global change on environment-host-pathogen interactions in the AF.

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.

Chytridiomycosis-induced mortality in a threatened anuran.

Effectively planning conservation introductions involves assessing the suitability of both donor and recipient populations, including the landscape of disease risk. Chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), has caused extensive amphibian declines globally and may hamper reintroduction attempts. To determine Bd dynamics in potential source populations for conservation translocations of the threatened California red-legged frog (Rana draytonii) to Yosemite National Park, we conducted Bd sampling in two populations in the foothills of the Sierra Nevada Mountains, California, U.S.A. At one of two sites, we observed lethally high Bd loads in early post-metamorphic life stages and confirmed one chytridiomycosis-induced mortality, the first such report for this species. These results informed source population site selection for subsequent R. draytonii conservation translocations. Conservation efforts aimed at establishing new populations of R. draytonii in a landscape where Bd is ubiquitous can benefit from an improved understanding of risk through disease monitoring and ex situ infection studies.

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.

Dampened virulence and limited proliferation of Batrachochytrium salamandrivorans during subclinical infection of the troglobiont olm (Proteus anguinus).

Emerging infections add to existing threats to the survival of amphibians worldwide. The olm (Proteus anguinus) is a vulnerable, troglobiont urodele species with a small European range and restricted to underground karstic systems. Population declines to emerging threats like the chytrid fungus Batrachochytrium salamandrivorans, are likely to go unnoticed due to inaccessibility of the species' habitat. We here studied the interaction between olms and B. salamandrivorans. Experimental inoculation of olms resulted in low-level, asymptomatic but persistent infections, with limbs as predilection sites. The lack of exponential fungal growth in the olms' epidermis correlated with limited fungal proliferation and dampened virulence gene expression after exposure to olm skin compounds. The olm is one of few western Palearctic urodeles that is tolerant to B. salamandrivorans infection and may act as a subterranean disease reservoir, yet costs of subclinical infection may compromise olm fitness on the long term.

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

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

Gene expression varies within and between enzootic and epizootic lineages of Batrachochytrium dendrobatidis (Bd) in the Americas.

While much research focus is paid to hypervirulent fungal lineages during emerging infectious disease outbreaks, examining enzootic pathogen isolates can be equally fruitful in delineating infection dynamics and determining pathogenesis. The fungal pathogen of amphibians, Batrachochytrium dendrobatidis (Bd), exhibits markedly different patterns of disease in natural populations, where it has caused massive amphibian declines in some regions, yet persists enzootically in others. Here we compare in vitro gene expression profiles of a panel of Bd isolates representing both the enzootic Bd-Brazil lineage, and the more recently diverged, panzootic lineage, Bd-GPL. We document significantly different lineage-specific and intralineage gene expression patterns, with Bd-Brazil upregulating genes with aspartic-type peptidase activity, and Bd-GPL upregulating CBM18 chitin-binding genes, among others. We also find pronounced intralineage variation in membrane integrity and transmembrane transport ability within our Bd-GPL isolates. Finally, we highlight unexpectedly divergent expression profiles in sympatric panzootic isolates, underscoring microgeographic functional variation in a largely clonal lineage. This variation in gene expression likely plays an important role in the relative pathogenesis and host range of Bd-Brazil and Bd-GPL isolates. Together, our results demonstrate that functional genomics approaches can provide information relevant to studies of virulence evolution within the Bd clade.

Temperature-mediated shifts in salamander transcriptomic responses to the amphibian-killing fungus.

Life processes of ectothermic vertebrates are intimately linked to the temperature of their environment, influencing their metabolism, reproduction, behaviour and immune responses. In amphibians infected by the generalist chytrid pathogen Batrachochytrium dendrobatidis (Bd), host survival, infection prevalence and infection intensity are often temperature- and/or seasonally dependent. However, the transcriptional underpinnings of thermal differences in infection responses remain unknown. Measuring the impact of temperature on host responses to infection is a key component for understanding climatic influences on chytrid disease dynamics. The Bd-responsive gene pathways in frogs are well documented, but our understanding of salamander immune expression profiles during infection with chytrids remains limited. Here we characterize the transcriptomic responses of Plethodon cinereus using RNA sequencing by comparing skin and splenic gene expression of individuals uninfected, succumbing to Bd infection and naturally cleared of Bd infection at three temperatures. We suggest that amphibian temperature-dependent susceptibility to Bd is probably driven by shifts in expression of the innate and adaptive immune axes. Our study shows increased expression of transcripts associated with inflammation at lower temperatures and a shift towards increased expression of adaptive immune genes, including MHC (major histocompatibility complex), at higher temperatures. In the face of climate change, and as concerns for the spread of emergent chytrid pathogens increase, our results provide important functional genomic resources to help understand how these pathogenic fungi may continue to affect amphibian communities globally in the future.

Bullfrog farms release virulent zoospores of the frog-killing fungus into the natural environment.

Bullfrog farming and trade practices are well-established, globally distributed, and economically valuable, but pose risks for biodiversity conservation. Besides their negative impacts on native amphibian populations as an invasive species, bullfrogs play a key role in spreading the frog-killing fungus Batrachochytrium dendrobatidis (Bd) in the natural environment. Bullfrogs are tolerant to Bd, meaning that they can carry high infection loads without developing chytridiomycosis. To test the potential of bullfrog farms as reservoirs for diverse and virulent chytrid genotypes, we quantified Bd presence, prevalence and infection loads across approximately 1,500 farmed bullfrogs and in the water that is released from farms into the environment. We also described Bd genotypic diversity within frog farms by isolating Bd from dozens of infected tadpoles. We observed individuals infected with Bd in all sampled farms, with high prevalence (reaching 100%) and high infection loads (average 71,029 zoospore genomic equivalents). Average outflow water volume from farms was high (60,000 L/day), with Bd zoospore concentration reaching approximately 50 million zoospores/L. Because virulent pathogen strains are often selected when growing in tolerant hosts, we experimentally tested whether Bd genotypes isolated from bullfrogs are more virulent in native anuran hosts compared to genotypes isolated from native host species. We genotyped 36 Bd isolates from two genetic lineages and found that Bd genotypes cultured from bullfrogs showed similar virulence in native toads when compared to genotypes isolated from native hosts. Our results indicate that bullfrog farms can harbor high Bd genotypic diversity and virulence and may be contributing to the spread of virulent genotypes in the natural environment. We highlight the urgent need to implement Bd monitoring and mitigation strategies in bullfrog farms to aid in the conservation of native amphibians.