Hotspot shelters stimulate frog resistance to chytridiomycosis.

Many threats to biodiversity cannot be eliminated; for example, invasive pathogens may be ubiquitous. Chytridiomycosis is a fungal disease that has spread worldwide, driving at least 90 amphibian species to extinction, and severely affecting hundreds of others1-4. Once the disease spreads to a new environment, it is likely to become a permanent part of that ecosystem. To enable coexistence with chytridiomycosis in the field, we devised an intervention that exploits host defences and pathogen vulnerabilities. Here we show that sunlight-heated artificial refugia attract endangered frogs and enable body temperatures high enough to clear infections, and that having recovered in this way, frogs are subsequently resistant to chytridiomycosis even under cool conditions that are optimal for fungal growth. Our results provide a simple, inexpensive and widely applicable strategy to buffer frogs against chytridiomycosis in nature. The refugia are immediately useful for the endangered species we tested and will have broader utility for amphibian species with similar ecologies. Furthermore, our concept could be applied to other wildlife diseases in which differences in host and pathogen physiologies can be exploited. The refugia are made from cheap and readily available materials and therefore could be rapidly adopted by wildlife managers and the public. In summary, habitat protection alone cannot protect species that are affected by invasive diseases, but simple manipulations to microhabitat structure could spell the difference between the extinction and the persistence of endangered amphibians.

Baseline haematological parameters in three common Australian frog species.

Amphibians are experiencing declines globally, with emerging infectious diseases as one of the main causes. Haematological parameters present a useful method for determining the health status of animals and the effects of particular diseases, but the interpretation of differential cell counts relies on knowing the normal ranges for the species and factors that can affect these counts. However, there is very little data on either normal haematological parameters or guides for blood cell types for free-ranging frog species across the world. This study aims to 1) create a visual guide for three different Australian frog species: Litoria paraewingi, Limnodynastes dumerilii, and Crinia signifera, 2) determine the proportions of erythrocytes to leukocytes and 3) differential leukocytes within blood smears from these three species and 4) assess the association between parasites and differential counts. We collected blood samples from free-ranging frogs and analysed blood smears. We also looked for ectoparasites and tested for the fungal disease chytridiomycosis. Overall, we found that the differentials of erythrocytes to leukocytes were not affected by species, but the proportions of different leukocytes did vary across species. For example, while lymphocytes were the most common type of leukocyte across the three species, eosinophils were relatively common in Limnodynastes dumerilii but rarely present in the other two species. We noted chytridiomycosis infection as well as ectoparasites present in some individuals but found no effect of parasites on blood parameters. Our results add baseline haematological parameters for three Australian frog species and provide an example of how different frog species can vary in their differential blood cell counts. More information is needed on frog haematological data before these parameters can be used to determine the health status of wild or captive frogs.

Pioneer access of the foam nest bacterial community of Leptodactylidae frogs and its biotechnological potential.

Many anuran amphibians deposit their eggs in foam nests, biostructures that help protect the eggs and tadpoles from predators. Currently, there are no other identification and description studies of the cultivable microbiota role in the nests of the Leptodactylid frogs such as Physalaemus cuvieri, Leptodactylus vastus and Adenomera hylaedactyla. This study aimed to isolate and identify the culturable bacteria from these three anuran species' nests, as well as to prospect enzymes produced by this microbiota. Foam nests samples and environmental samples were diluted and viable cell count was determined. Bacterial morphotypes from foam nest samples were isolated through spread plate technique. Isolates' DNAs were extracted followed by rRNA 16S gene amplification and Sanger sequencing. To evaluate their enzymatic potential, the isolates were cultured in ATGE medium supplemented with starch (0.1% w/v), gelatin (3% w/v) and skimmed milk (1% w/v), to verify amylase and protease activity. A total of 183 bacterial morphotypes were isolated, comprising 33 bacterial genera. Proteobacteria phylum was the most abundant in all the three nests (79%). The genera Pseudomonas and Aeromonas were the most abundant taxon in P. cuvieri and L. vastus. In A. Hylaedactyla, were Enterobacter and Bacillus. Regarding enzymatic activities, 130 isolates displayed protease activity and 45 isolates were positive for amylase activity. Our results provide unprecedented information concerning culturable bacterial microbiota of the foam nests of the Leptodactylid frogs, as well as their potential for biomolecules of biotechnological interest.

Priority effects alter microbiome composition and increase abundance of probiotic taxa in treefrog tadpoles.

Host-associated microbial communities, like other ecological communities, may be impacted by the colonization order of taxa through priority effects. Developing embryos and their associated microbiomes are subject to stochasticity during colonization by bacteria. For amphibian embryos, often developing externally in bacteria-rich environments, this stochasticity may be particularly impactful. For example, the amphibian microbiome can mitigate lethal outcomes from disease for their hosts; however, this may depend on microbiome composition. Here, we examined the assembly of the bacterial community in spring peeper (Pseudacris crucifer) embryos and tadpoles. First, we reared embryos from identified mating pairs in either lab or field environments to examine the relative impact of environment and parentage on embryo and tadpole bacterial communities. Second, we experimentally inoculated embryos to determine if priority effects (i) could be used to increase the relative abundance of Janthinobacterium lividum, an amphibian-associated bacteria capable of preventing fungal infection, and (ii) would lead to observed differences in the relative abundances of two closely related bacteria from the genus Pseudomonas. Using 16S rRNA gene amplicon sequencing, we observed differences in community composition based on rearing location and parentage in embryos and tadpoles. In the inoculation experiment, we found that priority inoculation could increase the relative abundance of J. lividum, but did not find that either Pseudomonas isolate was able to prevent colonization by the other when given priority. These results highlight the importance of environmental source pools and parentage in determining microbiome composition, while also providing novel methods for the administration of a known amphibian probiotic. IMPORTANCE: Harnessing the functions of host-associated bacteria is a promising mechanism for managing disease outcomes across different host species. In the case of amphibians, certain frog-associated bacteria can mitigate lethal outcomes of infection by the fungal pathogen Batrachochytrium dendrobatidis. Successful probiotic applications require knowledge of community assembly and an understanding of the ecological mechanisms that structure these symbiotic bacterial communities. In our study, we show the importance of environment and parentage in determining bacterial community composition and that community composition can be influenced by priority effects. Further, we provide support for the use of bacterial priority effects as a mechanism to increase the relative abundance of target probiotic taxa in a developing host. While our results show that priority effects are not universally effective across all host-associated bacteria, our ability to increase the relative abundance of specific probiotic taxa may enhance conservation strategies that rely on captive rearing of endangered vertebrates.

Widespread occurrence of the amphibian chytrid panzootic lineage in Uruguay is constrained by climate.

The amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) causes chytridiomycosis, a disease among the main causes of amphibian declines worldwide. However, Bd studies on Neotropical amphibians from temperate areas are scarce. We present a comprehensive survey of Bd in Uruguay, in temperate central eastern South America, carried out between 2006 and 2014. Skin swabs of 535 specimens of 21 native and exotic frogs were tested by PCR. We used individual-level data to examine the relationship between infection, climatic variables, and their effects on body condition and the number of prey items found in stomach contents. Infection was widespread in free-ranging anurans with an overall prevalence of 41.9%, detected in 15 native species, wild American bullfrogs Aquarana catesbeiana, and captive specimens of Ceratophrys ornata and Xenopus laevis. Three haplotypes of the Bd ITS region were identified in native amphibians, all belonging to the global panzootic lineage (BdGPL), of which only one was present in exotic hosts. Despite high infection frequencies in different anurans, we found no evidence of morbidity or mortality attributable to chytridiomycosis, and we observed no discernible impact on body condition or consumed prey. Climatic conditions at the time of our surveys suggested that the chance of infection is associated with monthly mean temperature, mean humidity, and total precipitation. Temperatures below 21°C combined with moderate humidity and pronounced rainfall may increase the likelihood of infection. Multiple haplotypes of BdGPL combined with high frequencies of infection suggest an enzootic pattern in native species, underscoring the need for continued monitoring.

Are novel or locally adapted pathogens more devastating and why? Resolving opposing hypotheses.

There is a rich literature highlighting that pathogens are generally better adapted to infect local than novel hosts, and a separate seemingly contradictory literature indicating that novel pathogens pose the greatest threat to biodiversity and public health. Here, using Batrachochytrium dendrobatidis, the fungus associated with worldwide amphibian declines, we test the hypothesis that there is enough variance in "novel" (quantified by geographic and phylogenetic distance) host-pathogen outcomes to pose substantial risk of pathogen introductions despite local adaptation being common. Our continental-scale common garden experiment and global-scale meta-analysis demonstrate that local amphibian-fungal interactions result in higher pathogen prevalence, pathogen growth, and host mortality, but novel interactions led to variable consequences with especially virulent host-pathogen combinations still occurring. Thus, while most pathogen introductions are benign, enough variance exists in novel host-pathogen outcomes that moving organisms around the planet greatly increases the chance of pathogen introductions causing profound harm.

Combined effect of seasons and life history in an anuran strengthens the response and relationship between their physiology and gut microbiota.

Gut microbiota impact host physiology, though simultaneous investigations in ectothermic vertebrates are rare. Particularly, amphibians may exhibit more complex interactions between host physiology and the effects of gut microbiota due to the combination of seasonal changes and complex life histories. In this study, we assessed the relationships among food resources, gut bacterial communities, and host physiology in frogs (Phelophylax nigromaculatus), taking into account seasonal and life history variations. We found that food sources were not correlated with physiological parameters but had some relationships with the gut bacterial community. Variations in gut bacterial community and host physiology were influenced by the combined effects of seasonal differences and life history, though mostly driven by seasonal differences. An increase in Firmicutes was associated with higher fat content, reflecting potential fat storage in frogs during the non-breeding season. The increase in Bacteroidetes resulted in lower fat content in adult frogs and decreased immunity in juvenile frogs during the breeding season, demonstrating a direct link. Our results suggest that the gut microbiome may act as a link between food conditions and physiological status, and that the combined effect of seasons and life history could reinforce the relationship between gut microbiota and physiological status in ectothermic animals. While food sources may influence the gut microbiota of ectotherms, we contend that temperature-correlated seasonal variation, which predominately influences most ectotherms, is a significant factor.

High-mortality epizootic Mycobacterium ulcerans ecovar Liflandii in a colony of Zaire Dwarf Clawed Frogs (Hymenochirus boettgeri).

Mycobacterium ulcerans ecovar Liflandii (MuLiflandii) was identified as the causative agent of mycobacteriosis in a research colony of Zaire dwarf clawed frogs (Hymenochirus boettgeri) at the University of Michigan. Clinical presentation included lethargy, generalized septicemia, cutaneous granulomas, coelomic effusion, and acute mortality. Identification of the mycobacterial species was based on molecular, microbiological, and histopathologic characteristics. These findings indicate that MuLiflandii is a primary cause of morbidity and mortality in Zaire dwarf clawed frogs and should be considered in the differential diagnosis of sepsis and coelomic effusion in amphibians. Mycobacterial speciation is important given the variability in pathogenesis within the family Mycobacteriaceae and the implications for both animal and human health as potential zoonoses. The Zaire dwarf clawed frog is a species common in the pet trade, and these findings provide consideration for this pathogen as a potentially important public health concern. This is the first report of MuLiflandii infection in the genus Hymenochirus and illustrates the diagnostic challenges of differentiating among both mycolactone-producing mycobacteria and Mycobacterium marinum. Furthermore, we demonstrate the utility of environmental sampling for this pathogen within the tank system, suggesting this mode of sampling could replace the need for direct frog surveillance.

The difference and variation of gut bacterial community and host physiology can support adaptation during and after overwintering in frog population.

The hibernation of amphibians can offer a unique window into overwintering adaptation processes and host-gut microbiota interactions through changes in metabolic availability and homeostasis. We attempted to identify differences in the physiology and gut microbiome during and after hibernation in Japanese wrinkled frogs (Glandirana rugosa), an aquatic overwintering amphibian. After hibernation, the high alpha and beta diversity of the gut bacterial community appears to reflect the more diverse and complex environmental conditions. During winter, Proteobacteria dominated the majority of the gut bacterial community, likely due to high oxygen saturation. After hibernation, Firmicutes and Bacteroidetes increased, which are supportive of host metabolism by gut microbiota. Corticosterone also showed high values and variances after hibernation, presumably allowing the population to remain adaptable across a broad range of environmental gradients. Innate immunity was high after hibernation but exhibited low variation among populations, which supports the idea of a prioritized investment in immunity after hibernation. Blood biochemistry suggests that aquatic overwintering frogs have a mechanism to adapt through overhydration and regulate homeostasis through water excretion associated with the kidney and urine after hibernation. Frog populations exhibit variations and adaptability in gut microbiota and physiology during and after hibernation: Through this, they may demonstrate an adaptive response that regulates metabolic availability in preparation for unpredictable environmental changes. We also propose that the maintenance of Proteobacteria during hibernation can support the colonization of Firmicutes and Bacteroidetes after hibernation, underscoring the need to study the complex effects of gut microbiota across multiple life stages.

Chytridiomycosis causes high amphibian mortality prior to the completion of metamorphosis.

Amphibian populations are undergoing extensive declines globally. The fungal disease chytridiomycosis, caused by the pathogenic fungus Batrachochytrium dendrobatidis (Bd), is a primary contributor to these declines. The amphibian metamorphic stages (Gosner stages 42-46) are particularly vulnerable to a range of stressors, including Bd. Despite this, studies that explicitly examine host response to chytridiomycosis throughout the metamorphic stages are lacking. We aimed to determine how Bd exposure during the larval stages impacts metamorphic development and infection progression in the endangered Fleay's barred frog (Mixophyes fleayi). We exposed M. fleayi to Bd during pro-metamorphosis (Gosner stages 35-38) and monitored infection dynamics throughout metamorphosis. We took weekly morphological measurements (weight, total body length, snout-vent-length and Gosner stage) and quantified Bd load using qPCR. While we observed minimal impact of Bd infection on animal growth and development, Bd load varied throughout ontogeny, with an infection load plateau during the tadpole stages (Gosner stages 35-41) and temporary infection clearance at Gosner stage 42. Bd load increased exponentially between Gosner stages 42 and 45, with most exposed animals becoming moribund at Gosner stage 45, prior to the completion of metamorphosis. There was variability in infection outcome of exposed individuals, with a subgroup of animals (n = 5/29) apparently clearing their infection while the majority (n = 21/29) became moribund with high infection burdens. This study demonstrates the role that metamorphic restructuring plays in shaping Bd infection dynamics and raises the concern that substantial Bd-associated mortality could be overlooked in the field due to the often cryptic nature of these latter metamorphic stages. We recommend future studies that directly examine the host immune response to Bd infection throughout metamorphosis, incorporating histological and molecular methods to elucidate the mechanisms responsible for the observed trends.

Body size mediates latitudinal population differences in the response to chytrid fungus infection in two amphibians.

Factors behind intraspecific variation in sensitivity to pathogens remain poorly understood. We investigated how geographical origin in two North European amphibians affects tolerance to infection by the chytrid fungus Batrachochytrium dendrobatidis (Bd), a generalist pathogen which has caused amphibian population declines worldwide. We exposed newly metamorphosed individuals of moor frog Rana arvalis and common toad Bufo bufo from two latitudinal regions to two different BdGPL strains. We measured survival and growth as infections may cause sub-lethal effects in fitness components even in the absence of mortality. Infection loads were higher in B. bufo than in R. arvalis, and smaller individuals had generally higher infection loads. B. bufo had high mortality in response to Bd infection, whereas there was little mortality in R. arvalis. Bd-mediated mortality was size-dependent and high-latitude individuals were smaller leading to high mortality in the northern B. bufo. Bd exposure led to sub-lethal effects in terms of reduced growth suggesting that individuals surviving the infection may have reduced fitness mediated by smaller body size. In both host species, the Swedish Bd strain caused stronger sublethal effects than the British strain. We suggest that high-latitude populations can be more vulnerable to chytrids than those from lower latitudes and discuss the possible mechanisms how body size and host geographical origin contribute to the present results.

Toxicities of Legacy and Current-Use PFAS in an Anuran: Do Larval Exposures Influence Responses to a Terrestrial Pathogen Challenge?

Legacy polyfluoroalkyl substances (PFAS) [perfluorooctanesulfonate (PFOS) and perfluorooctanoic acid (PFOA)] are being replaced by various other fluorinated compounds, such as hexafluoropropylene oxide dimer acid (GenX). These alternatives are thought to be less bioaccumulative and, therefore, less toxic than legacy PFAS. Contaminant exposures occur concurrently with exposure to natural stressors, including the fungal pathogen Batrachocytrium dendrobatidis (Bd). Despite evidence that other pollutants can increase the adverse effects of Bd on anurans, no studies have examined the interactive effects of Bd and PFAS. This study tested the growth and developmental effects of PFOS, PFOA, and GenX on gray treefrog (Hyla versicolor) tadpoles, followed by a Bd challenge after metamorphosis. Despite PFAS exposure only occurring during the larval stage, carry-over effects on growth were observed post metamorphosis. Further, PFAS interacted with Bd exposure to influence growth; Bd-exposed animals had significantly shorter SVL [snout-vent length (mm)] with significantly increased body condition, among other time-dependent effects. Our data suggest that larval exposure to PFAS can continue to impact growth in the juvenile stage after exposure has ended. Contrary to predictions, GenX affected terrestrial performance more consistently than its legacy congener, PFOA. Given the role of Bd in amphibian declines, further investigation of interactions of PFAS with Bd and other environmentally relevant pathogens is warranted.

Tadpole growth rates and gut bacterial community: Dominance of developmental stages over temperature variations.

Tadpoles present an intriguing model system for studying the regulation and selection of gut microbiota. They offer a unique perspective to enhance our understanding of host-microbiota interactions, given their capacity to alter the dynamics of the gut microbial community by interacting with multiple environmental factors within a complex life cycle. In this study, we comprehensively investigated variations in growth rate and gut bacterial community in relation to temperature differences during the complex process of amphibian metamorphosis. Higher temperatures prompted tadpoles to metamorphose more rapidly than at lower temperatures, but the impact on size and weight was minimal. Differences in temperature were not associated with gut bacterial diversity, but they did affect certain aspects of beta diversity and bacterial composition. However, the developmental stage invoked greater heterogeneity than temperature in gut bacterial diversity, composition, and functional groups. These findings suggest that inherent biological systems exert stronger control over an organism's homeostasis and variation than the external environment. Although results may vary based on the magnitude or type of environmental factors, metamorphosis in tadpoles greatly influences their biology, potentially dominating microbial interactions.

Limited impact of chytridiomycosis on juvenile frogs in a recovered species.

The amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) has caused catastrophic frog declines on several continents, but disease outcome is mediated by a number of factors. Host life stage is an important consideration and many studies have highlighted the vulnerability of recently metamorphosed or juvenile frogs compared to adults. The majority of these studies have taken place in a laboratory setting, and there is a general paucity of longitudinal field studies investigating the influence of life stage on disease outcome. In this study, we assessed the effect of endemic Bd on juvenile Mixophyes fleayi (Fleay's barred frog) in subtropical eastern Australian rainforest. Using photographic mark-recapture, we made 386 captures of 116 individuals and investigated the effect of Bd infection intensity on the apparent mortality rates of frogs using a multievent model correcting for infection state misclassification. We found that neither Bd infection status nor infection intensity predicted mortality in juvenile frogs, counter to the expectation that early life stages are more vulnerable to disease, despite average high infection prevalence (0.35, 95% HDPI [0.14, 0.52]). Additionally, we found that observed infection prevalence and intensity were somewhat lower for juveniles than adults. Our results indicate that in this Bd-recovered species, the realized impacts of chytridiomycosis on juveniles were apparently low, likely resulting in high recruitment contributing to population stability. We highlight the importance of investigating factors relating to disease outcome in a field setting and make recommendations for future studies.

Divergent allele advantage in the MHC and amphibian emerging infectious disease.

Genetic variation in the major histocompatibility complex (MHC) may be associated with resistance to the amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd). The pathogen originated in Asia, then spread worldwide, causing amphibian population declines and species extinctions. We compared the expressed MHC IIß1 alleles of a Bd-resistant species, Bufo gargarizans, from South Korea with those of a Bd-susceptible Australasian species, Litoria caerulea. We found at least six expressed MHC IIß1 loci in each of the two species. Amino acid diversity encoded by these MHC alleles was similar between species, but the genetic distance of those alleles known for potential broader pathogen-derived peptide binding was greater in the Bd-resistant species. In addition, we found a potentially rare allele in one resistant individual from the Bd-susceptible species. Deep next-generation sequencing recovered approximately triple the genetic resolution accessible from traditional cloning-based genotyping. Targeting the full MHC IIß1 enables us to better understand how host MHC may adapt to emerging infectious diseases.

Host immune responses to enzootic and invasive pathogen lineages vary in magnitude, timing, and efficacy.

Infectious diseases of wildlife continue to pose a threat to biodiversity worldwide, yet pathogens are far from uniform in virulence or host disease outcome. Within the same pathogen species, virulence can vary considerably depending on strain or lineage, in turn eliciting variable host responses. One pathogen that has caused extensive biodiversity loss is the amphibian-killing fungus, Batrachochytrium dendrobatidis (Bd), which is comprised of a globally widespread hypervirulent lineage (Bd-GPL), and multiple geographically restricted, enzootic lineages. Whereas host immunogenomic responses to Bd-GPL have been characterized in a number of amphibian species, immunogenomic responses to geographically restricted, enzootic Bd lineages are less clear. To examine lineage-specific host immune responses to Bd, we exposed a species of pumpkin toadlet, Brachycephalus pitanga, which is endemic to Brazil's Southern Atlantic Forest, to either the Bd-GPL or the enzootic Bd-Asia-2/Brazil (hereafter Bd-Brazil) lineage. Using temporal samples from early, mid, and late infection stages, we quantified functional immunogenomic responses over the course of infection using differential gene expression tests and coexpression network analyses. Host immune responses varied significantly with Bd lineage. Relative to controls, toadlet responses to Bd-Brazil were weak at early infection (25 genes significantly differentially expressed), peaked by mid-stage infection (414 genes), and were nearly fully resolved by late-stage infection (nine genes). In contrast, responses to Bd-GPL were magnified and delayed; toadlets significantly differentially expressed 111 genes early, 87 genes at mid-stage infection, and 726 genes by late-stage infection relative to controls. Given that infection intensity did not vary between mid- and late-stage disease in either Bd-Brazil or Bd-GPL treatments, this suggests that pumpkin toadlets may be at least partially tolerant to the enzootic Bd-Brazil lineage. In contrast, late-stage immune activation against Bd-GPL was consistent with immune dysregulation previously observed in other species. Our results demonstrate that both the timing of immune response and the particular immune pathways activated are specific to Bd lineage. Within regions where multiple Bd lineages co-occur, and given continued global Bd movement, these differential host responses may influence not only individual disease outcome, but transmission dynamics at the population and community levels.

First evidence of ranavirus in native and invasive amphibians in Colombia.

Ranaviruses can cause mass mortality events in amphibians, thereby becoming a threat to populations that are already facing dramatic declines. Ranaviruses affect all life stages and persist in multiple amphibian hosts. The detrimental effects of ranavirus infections to amphibian populations have already been observed in the UK and in North America. In Central and South America, the virus has been reported in several countries, but the presence of the genus Ranavirus (Rv) in Colombia is unknown. To help fill this knowledge gap, we surveyed for Rv in 60 species of frogs (including one invasive species) in Colombia. We also tested for co-infection with Batrachochytrium dendrobatidis (Bd) in a subset of individuals. For Rv, we sampled 274 vouchered liver tissue samples collected between 2014 and 2019 from 41 localities covering lowlands to mountaintop páramo habitat across the country. Using quantitative polymerase chain reaction (qPCR) and end-point PCR, we detected Rv in 14 individuals from 8 localities, representing 6 species, including 5 native frogs of the genera Osornophryne, Pristimantis and Leptodactylus, and the invasive American bullfrog Rana catesbeiana. Bd was detected in 7 of 140 individuals, with 1 co-infection of Rv and Bd in an R. catesbeiana specimen collected in 2018. This constitutes the first report of ranavirus in Colombia and should set off alarms about this new emerging threat to amphibian populations in the country. Our findings provide some preliminary clues about how and when Rv may have spread and contribute to understanding how the pathogen is distributed globally.

Constant-temperature predictions underestimate growth of a fungal amphibian pathogen under individual host thermal profiles.

Ectotherm body temperatures fluctuate with environmental variability and host behavior, which may influence host-pathogen interactions. Fungal pathogens are a major threat to ectotherms and may be highly responsive to the fluctuating thermal profiles of individual hosts, especially cool-loving fungi exposed to high host temperatures. However, most studies estimate pathogen thermal performance based on averages of host or surrogate environmental temperatures, potentially missing effects of short-term host temperature shifts such as daily or hourly heat spikes. We recorded individual thermal profiles of Australian rainforest frogs using temperature-sensitive radio-transmitters. We then reproduced a subset of individual thermal profiles in growth chambers containing cultures of the near-global amphibian pathogen Batrachochytrium dendrobatidis (Bd) to investigate how realistic host temperature profiles affect Bd growth. We focused on thermal profiles that exceed the thermal optimum of Bd because the effects of realistic heat spikes on Bd growth are unresolved. Our laboratory incubation experiment revealed that Bd growth varied in response to relatively small differences in heat spike characteristics of individual frog thermal profiles, such as a single degree or a few hours, highlighting the importance of individual host behaviors in predicting population-level disease dynamics. The fungus also grew better than predicted under the most extreme and unpredictable frog temperature profile, recovering from two days of extreme (nearly 32 °C) heat spikes without negative effects on overall growth, suggesting we are underestimating the growth potential of the pathogen in nature. Combined with the previous finding that Bd reduces host heat tolerance, our study suggests that this pathogen may carry a competitive edge over hosts in the face of anthropogenic climate change.

Rapid detection of amphibian chytrid fungus Batrachochytrium dendrobatidis using in situ DNA extraction and a handheld mobile thermocycler.

The amphibian chytrid fungus (Bd) has caused declines and some extinctions of amphibian populations worldwide. Early and accurate Bd detection is essential for management of susceptible anurans. We analyzed the effectiveness of in situ DNA extraction with a handheld mobile quantitative PCR (qPCR) thermocycler to detect Bd on frog skin swabs and in water samples using environmental DNA (eDNA). We collected duplicate eDNA samples and skin swabs from 3 Bd-positive Rana sierrae populations. We processed one set of samples using a field protocol (a handheld thermocycler) and the other half using a standard lab protocol. We detected Bd DNA in all R. sierrae swabbed using both the field and lab protocols. We also detected Bd DNA in eDNA samples at all sites, although the field and lab protocols failed to detect Bd eDNA at separate singular sites; results from the field and lab eDNA protocol did not match. The probability of detecting Bd DNA in the technical replicates was lower for the field protocol than the lab protocol, suggesting the field protocol has lower sensitivity and may not detect low quantities of DNA. Our results suggest that the field extraction protocol using a handheld qPCR platform is a promising tool for rapid detection of Bd in susceptible amphibian populations, yielding accurate results in less than 60 min. However, the applied field protocol may be prone to false negatives when analyzing low-quantity DNA samples such as eDNA water samples or frog swabs with low pathogen loads.