Ongoing declines for the world's amphibians in the face of emerging threats.

Systematic assessments of species extinction risk at regular intervals are necessary for informing conservation action1,2. Ongoing developments in taxonomy, threatening processes and research further underscore the need for reassessment3,4. Here we report the findings of the second Global Amphibian Assessment, evaluating 8,011 species for the International Union for Conservation of Nature Red List of Threatened Species. We find that amphibians are the most threatened vertebrate class (40.7% of species are globally threatened). The updated Red List Index shows that the status of amphibians is deteriorating globally, particularly for salamanders and in the Neotropics. Disease and habitat loss drove 91% of status deteriorations between 1980 and 2004. Ongoing and projected climate change effects are now of increasing concern, driving 39% of status deteriorations since 2004, followed by habitat loss (37%). Although signs of species recoveries incentivize immediate conservation action, scaled-up investment is urgently needed to reverse the current trends.

Sequence capture identifies fastidious chytrid fungi directly from host tissue.

The chytrid fungus Batrachochytrium dendrobatidis (Bd) was discovered in 1998 as the cause of chytridiomycosis, an emerging infectious disease causing mass declines in amphibian populations worldwide. The rapid population declines of the 1970s-1990s were likely caused by the spread of a highly virulent lineage belonging to the Bd-GPL clade that was introduced to naïve susceptible populations. Multiple genetically distinct and regional lineages of Bd have since been isolated and sequenced, greatly expanding the known biological diversity within this fungal pathogen. To date, most Bd research has been restricted to the limited number of samples that could be isolated using culturing techniques, potentially causing a selection bias for strains that can grow on media and missing other unculturable or fastidious strains that are also present on amphibians. We thus attempted to characterize potentially non-culturable genetic lineages of Bd from distinct amphibian taxa using sequence capture technology on DNA extracted from host tissue and swabs. We focused our efforts on host taxa from two different regions that likely harbored distinct Bd clades: (1) wild-caught leopard frogs (Rana) from North America, and (2) a Japanese Giant Salamander (Andrias japonicus) at the Smithsonian Institution's National Zoological Park that exhibited signs of disease and tested positive for Bd using qPCR, but multiple attempts failed to isolate and culture the strain for physiological and genetic characterization. We successfully enriched for and sequenced thousands of fungal genes from both host clades, and Bd load was positively associated with number of recovered Bd sequences. Phylogenetic reconstruction placed all the Rana-derived strains in the Bd-GPL clade. In contrast, the A. japonicus strain fell within the Bd-Asia3 clade, expanding the range of this clade and generating additional genomic data to confirm its placement. The retrieved ITS locus matched public barcoding data from wild A. japonicus and Bd infections found on other amphibians in India and China, suggesting that this uncultured clade is widespread across Asia. Our study underscores the importance of recognizing and characterizing the hidden diversity of fastidious strains in order to reconstruct the spatiotemporal and evolutionary history of Bd. The success of the sequence capture approach highlights the utility of directly sequencing pathogen DNA from host tissue to characterize cryptic diversity that is missed by culture-reliant approaches.

Body condition, skin bacterial communities and disease status: insights from the first release trial of the limosa harlequin frog, Atelopus limosus.

Many endangered amphibian species survive in captive breeding facilities, but there have been few attempts to reintroduce captive-born individuals to rebuild wild populations. We conducted a soft-release trial of limosa harlequin frogs, Atelopus limosus, which are highly susceptible to the amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd), to understand changes associated with the transition from captivity to the wild. Specifically, we assessed changes in body condition, skin-associated bacterial communities and disease status after release. Frogs were housed individually in field mesocosms and monitored for 27 days. Body condition did not significantly change in the mesocosms, and was similar to, or higher than, that of wild conspecifics at day 27. The skin bacteria of captive-born frogs, based on 16S rRNA gene amplicons, became similar to that of wild conspecifics after 27 days in mesocosms. Prevalence of Bd in wild conspecifics was 13-27%, and 15% of the A. limosus in mesocosms became infected with Bd, but no mortality of infected frogs was observed. We conclude that mesocosms are suitable for systematically and repeatedly monitoring amphibians during release trials, and that body condition, the skin microbiome, and Bd status can all change within one month of placement of captive-born individuals back into the wild.

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.

Expanded host and geographic range of tadpole associations with the Severe Perkinsea Infection group.

Severe Perkinsea infection is an emerging disease of amphibians, specifically tadpoles. Disease presentation correlates with liver infections of a subclade of Perkinsea (Alveolata) protists, named Pathogenic Perkinsea Clade (PPC). Tadpole mortality events associated with PPC infections have been reported across North America, from Alaska to Florida. Here, we investigate the geographic and host range of PPC associations in seemingly healthy tadpoles sampled from Panama, a biogeographic provenance critically affected by amphibian decline. To complement this work, we also investigate a mortality event among Hyla arborea tadpoles in captive-bred UK specimens. PPC SSU rDNA was detected in 10 of 81 Panama tadpoles tested, and H. arborea tadpoles from the UK. Phylogenies of the Perkinsea SSU rDNA sequences demonstrate they are highly similar to PPC sequences sampled from mortality events in the USA, and phylogenetic analysis of tadpole mitochondrial SSU rDNA demonstrates, for the first time, PPC associations in diverse hylids. These data provide further understanding of the biogeography and host range of this putative pathogenic group, factors likely to be important for conservation planning.

Sustained immune activation is associated with susceptibility to the amphibian chytrid fungus.

The disease chytridiomycosis caused by the fungus Bd has devastated amphibian populations worldwide. Functional genomic contributions to host susceptibility remain enigmatic and vary between species and populations. We conducted experimental Bd infections in Rana yavapaiensis, a species with intraspecific variation in chytridiomycosis susceptibility, to assess the skin and spleen transcriptomic response to infection over time. We predicted that increased immune gene expression would be associated with a positive disease outcome, but we instead found that surviving frogs had significantly reduced immune gene expression compared to susceptible frogs and to uninfected controls. MHC class IIß gene expression was also significantly higher in susceptible frogs compared to surviving frogs. Furthermore, susceptible frogs expressed a significantly larger number of distinct class IIß alleles, demonstrating a negative correlation between class IIß expression, functional diversity, and survival. Expression of the MHC class IIß locus previously associated with Bd disease outcomes was a significant predictor of Bd infection intensity at early infection stages but not at late infection stages, suggesting initial MHC-linked immune processes are important for ultimate disease outcomes. We infer through disease association and phylogenetic analysis that certain MHC variants are linked to the immune expression that was negatively associated with survival, and we hypothesize that frogs that did not express these alleles could better survive infections. Our study finds that MHC expression at early and late infection stages predicts Bd infection intensity, and suggests that generating a sustained immune response against Bd may be counterproductive for surviving chytridiomycosis in this partially susceptible species.

Prodigiosin, Violacein, and Volatile Organic Compounds Produced by Widespread Cutaneous Bacteria of Amphibians Can Inhibit Two Batrachochytrium Fungal Pathogens.

Symbiotic bacteria can produce secondary metabolites and volatile compounds that contribute to amphibian skin defense. Some of these symbionts have been used as probiotics to treat or prevent the emerging disease chytridiomycosis. We examined 20 amphibian cutaneous bacteria for the production of prodigiosin or violacein, brightly colored defense compounds that pigment the bacteria and have characteristic spectroscopic properties making them readily detectable, and evaluated the antifungal activity of these compounds. We detected violacein from all six isolates of Janthinobacterium lividum on frogs from the USA, Switzerland, and on captive frogs originally from Panama. We detected prodigiosin from five isolates of Serratia plymuthica or S. marcescens, but not from four isolates of S. fonticola or S. liquefaciens. All J. lividum isolates produced violacein when visibly purple, while prodigiosin was only detected on visibly red Serratia isolates. When applied to cultures of chytrid fungi Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal), prodigiosin caused significant growth inhibition, with minimal inhibitory concentrations (MIC) of 10 and 50 µM, respectively. Violacein showed a MIC of 15 µM against both fungi and was slightly more active against Bsal than Bd at lower concentrations. Although neither violacein nor prodigiosin showed aerosol activity and is not considered a volatile organic compound (VOC), J. lividum and several Serratia isolates did produce antifungal VOCs. White Serratia isolates with undetectable prodigiosin levels could still inhibit Bd growth indicating additional antifungal compounds in their chemical arsenals. Similarly, J. lividum can produce antifungal compounds such as indole-3-carboxaldehyde in addition to violacein, and isolates are not always purple, or turn purple under certain growth conditions. When Serratia isolates were grown in the presence of cell-free supernatant (CFS) from the fungi, CFS from Bd inhibited growth of the prodigiosin-producing isolates, perhaps indicative of an evolutionary arms race; Bsal CFS did not inhibit bacterial growth. In contrast, growth of one J. lividum isolate was facilitated by CFS from both fungi. Isolates that grow and continue to produce antifungal compounds in the presence of pathogens may represent promising probiotics for amphibians infected or at risk of chytridiomycosis. In a global analysis, 89% of tested Serratia isolates and 82% of J. lividum isolates were capable of inhibiting Bd and these have been reported from anurans and caudates from five continents, indicating their widespread distribution and potential for host benefit.

BATRACHOCHYTRIUM DENDROBATIDIS IN A CAPTIVE COLLECTION OF GREEN SALAMANDERS ( ANEIDES AENEUS), LONG-TAILED SALAMANDERS ( EURYCEA LONGICAUDA), AND TWO-LINED SALAMANDERS ( EURYCEA BISLINEATA).

A chytridiomycosis outbreak from Batrachochytrium dendrobatidis ( Bd) in a mixed-species plethodontid salamander exhibit resulted in four green salamander ( Aneides aeneus) deaths. One green salamander died before treatment, and three died during treatment with daily 0.005% itraconazole baths. All salamanders had evidence of severe Bd infections via cytology, histopathology, and/or polymerase chain reaction (PCR) at the time of death. Ten long-tailed salamanders ( Eurycea longicauda) and one two-lined salamander ( Eurycea bislineata) that shared the enclosure were initially negative for Bd on quantitative PCR but were prophylactically treated with daily 0.01% itraconazole baths for 11 days. Posttreatment testing yielded eight long-tailed salamanders and one two-lined salamander positive for Bd with low gene equivalents. All salamanders were negative after two to three treatment courses, and there were no additional mortalities. The difference in mortality and fungal load suggested that genus Aneides salamanders may be more susceptible to Bd than genus Eurycea salamanders.

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

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

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.

Cryptic impacts of temperature variability on amphibian immune function.

Ectothermic species living in temperate regions can experience rapid and potentially stressful changes in body temperature driven by abrupt weather changes. Yet, among amphibians, the physiological impacts of short-term temperature variation are largely unknown. Using an ex situ population of Cryptobranchus alleganiensis, an aquatic North American salamander, we tested the hypothesis that naturally occurring periods of temperature variation negatively impact amphibian health, either through direct effects on immune function or by increasing physiological stress. We exposed captive salamanders to repeated cycles of temperature fluctuations recorded in the population's natal stream and evaluated behavioral and physiological responses, including plasma complement activity (i.e. bacteria killing) against Pseudomonas aeruginosa, Escherichia coli and Aeromonas hydrophila. The best-fit model (ΔAICc=0, wi=0.9992) revealed 70% greater P. aeruginosa killing after exposure to variable temperatures and no evidence of thermal acclimation. The same model predicted 50% increased E. coli killing, but had weaker support (ΔAICc=1.8, wi=0.2882). In contrast, plasma defenses were ineffective against A. hydrophila, and other health indicators (leukocyte ratios, growth rates and behavioral patterns) were maintained at baseline values. Our data suggest that amphibians can tolerate, and even benefit from, natural patterns of rapid warming/cooling. Specifically, temperature variation can elicit increased activity of the innate immune system. This immune response may be adaptive in an unpredictable environment, and is undetectable by conventional health indicators (and hence considered cryptic). Our findings highlight the need to consider naturalistic patterns of temperature variation when predicting species' susceptibility to climate change.

Immune priming prior to pathogen exposure sheds light on the relationship between host, microbiome and pathogen in disease.

Dynamic interactions between host, pathogen and host-associated microbiome dictate infection outcomes. Pathogens including Batrachochytrium dendrobatidis (Bd) threaten global biodiversity, but conservation efforts are hindered by limited understanding of amphibian host, Bd and microbiome interactions. We conducted a vaccination and infection experiment using Eastern hellbender salamanders (Cryptobranchus alleganiensis alleganiensis) challenged with Bd to observe infection, skin microbial communities and gene expression of host skin, pathogen and microbiome throughout the experiment. Most animals survived high Bd loads regardless of their vaccination status and vaccination did not affect pathogen load, but host gene expression differed based on vaccination. Oral vaccination (exposure to killed Bd) stimulated immune gene upregulation while topically and sham-vaccinated animals did not significantly upregulate immune genes. In early infection, topically vaccinated animals upregulated immune genes but orally and sham-vaccinated animals downregulated immune genes. Bd increased pathogenicity-associated gene expression in late infection when Bd loads were highest. The microbiome was altered by Bd, but there was no correlation between anti-Bd microbe abundance or richness and pathogen burden. Our observations suggest that hellbenders initially generate a vigorous immune response to Bd, which is ineffective at controlling disease and is subsequently modulated. Interactions with antifungal skin microbiota did not influence disease progression.

Physiological impacts of temperature variability and climate warming in hellbenders (Cryptobranchus alleganiensis).

Cold-adapted hellbender salamanders that inhabit cool mountain streams are expected to fare poorly under warmer projected climate scenarios. This study investigated the physiological consequences of long-term, naturalistic temperature variation on juvenile hellbenders under simulated current and warmer (+1.6 C) climates vs. controlled steady temperatures. Mean temperature and temperature variability were both important predictors of growth as indicated by monthly body mass change (%), stress as indicated by neutrophil:lymphocyte (N:L) ratio and bacteria-killing ability of blood. Cold exposure in hellbenders was associated with weight loss, increased N:L ratios and reduced Escherichia coli killing ability of blood, and these effects were less pronounced under a warmer climate scenario. These observations suggest that cold periods may be more stressful for hellbenders than previously understood. Growth rates peaked in late spring and late fall around 14-17°C. Hellbenders experiencing warmer simulated climates retained body condition better in winter, but this was counter-balanced by a prolonged lack of growth in the 3-month summer period leading up to the fall breeding season where warmer simulated conditions resulted in an average loss of -0.6% body mass/month, compared to a gain +1.5% body mass/month under current climate scenario. Hellbenders can physiologically tolerate projected warmer temperatures and temperature fluctuations, but warmer summers may cause animals to enter the fall breeding season with a caloric deficit that may have population-level consequences.

Genetically modifying skin microbe to produce violacein and augmenting microbiome did not defend Panamanian golden frogs from disease.

We designed two probiotic treatments to control chytridiomycosis caused by Batrachochytrium dendrobatidis (Bd) on infected Panamanian golden frogs (Atelopus zeteki), a species that is thought to be extinct in the wild due to Bd. The first approach disrupted the existing skin microbe community with antibiotics then exposed the frogs to a core golden frog skin microbe (Diaphorobacter sp.) that we genetically modified to produce high titers of violacein, a known antifungal compound. One day following probiotic treatment, the engineered Diaphorobacter and the violacein-producing pathway could be detected on the frogs but the treatment failed to improve frog survival when exposed to Bd. The second approach exposed frogs to the genetically modified bacterium mixed into a consortium with six other known anti-Bd bacteria isolated from captive A. zeteki, with no preliminary antibiotic treatment. The consortium treatment increased the frequency and abundance of three probiotic isolates (Janthinobacterium, Chryseobacterium, and Stenotrophomonas) and these persisted on the skin 4 weeks after probiotic treatment. There was a temporary increase in the frequency and abundance of three other probiotics isolates (Masillia, Serratia, and Pseudomonas) and the engineered Diaphorobacter isolate, but they subsequently disappeared from the skin. This treatment also failed to reduce frog mortality upon exposure.

Spindly leg syndrome in Atelopus varius is linked to environmental calcium and phosphate availability.

Spindly leg syndrome (SLS) is a relatively common musculoskeletal abnormality associated with captive-rearing of amphibians with aquatic larvae. We conducted an experiment to investigate the role of environmental calcium and phosphate in causing SLS in tadpoles. Our 600-tadpole experiment used a fully-factorial design, rearing Atelopus varius tadpoles in water with either high (80mg/l CaCO3), medium (50mg/l CaCO3), or low calcium hardness (20mg/l CaCO3), each was combined with high (1.74 mg/l PO4) or low (0.36 mg/l PO4) phosphate levels. We found that calcium supplementation significantly improved tadpole survival from 19% to 49% and that low calcium treatments had 60% SLS that was reduced to about 15% at the medium and high calcium treatments. Phosphate supplementation significantly reduced SLS prevalence in low calcium treatments. This experimental research clearly links SLS to the calcium: phosphate homeostatic system, but we were unable to completely eliminate the issue, suggesting an interactive role of other unidentified factors.

Response to Comment on "Amphibian fungal panzootic causes catastrophic and ongoing loss of biodiversity".

Lambert et al question our retrospective and holistic epidemiological assessment of the role of chytridiomycosis in amphibian declines. Their alternative assessment is narrow and provides an incomplete evaluation of evidence. Adopting this approach limits understanding of infectious disease impacts and hampers conservation efforts. We reaffirm that our study provides unambiguous evidence that chytridiomycosis has affected at least 501 amphibian species.

Amphibian fungal panzootic causes catastrophic and ongoing loss of biodiversity.

Anthropogenic trade and development have broken down dispersal barriers, facilitating the spread of diseases that threaten Earth's biodiversity. We present a global, quantitative assessment of the amphibian chytridiomycosis panzootic, one of the most impactful examples of disease spread, and demonstrate its role in the decline of at least 501 amphibian species over the past half-century, including 90 presumed extinctions. The effects of chytridiomycosis have been greatest in large-bodied, range-restricted anurans in wet climates in the Americas and Australia. Declines peaked in the 1980s, and only 12% of declined species show signs of recovery, whereas 39% are experiencing ongoing decline. There is risk of further chytridiomycosis outbreaks in new areas. The chytridiomycosis panzootic represents the greatest recorded loss of biodiversity attributable to a disease.