Chytridiomycosis causes catastrophic organism-wide metabolic dysregulation including profound failure of cellular energy pathways

Chytridiomycosis is among several recently emerged fungal diseases of wildlife that have caused decline or extinction of naive populations. Despite recent advances in understanding pathogenesis, host response to infection remains poorly understood. Here we modelled a total of 162 metabolites across skin and liver tissues of 61 frogs from four populations (three long-exposed and one naive to the fungus) of the Australian alpine tree frog (Litoria verreauxii alpina) throughout a longitudinal exposure experiment involving both infected and negative control individuals. We found that chytridiomycosis dramatically altered the organism-wide metabolism of clinically diseased frogs. Chytridiomycosis caused catastrophic failure of normal homeostatic mechanisms (interruption of biosynthetic and degradation metabolic pathways), and pronounced dysregulation of cellular energy metabolism. Key intermediates of the tricarboxylic acid cycle were markedly depleted, including in particular a-ketoglutarate and glutamate that together constitute a key nutrient pathway for immune processes. This study was the first to apply a non-targeted metabolomics approach to a fungal wildlife disease and specifically to dissect the host-pathogen interface of Bd-infected frogs. The patterns of metabolite accumulation we have identified reveal whole-body metabolic dysfunction induced by a fungal skin infection, and these findings have broad relevance for other fungal diseases

Evolution of resistance to chytridiomycosis is associated with a robust early immune response

Potentiating the evolution of immunity is a promising strategy for addressing biodiversity diseases. Assisted selection for infection resistance may enable the recovery and persistence of amphibians threatened by chytridiomycosis, a devastating fungal skin disease threatening hundreds of species globally. However, knowledge of the mechanisms involved in the natural evolution of immunity to chytridiomycosis is limited. Understanding the mechanisms of such resistance may help speed-assisted selection. Using a transcriptomics approach, we examined gene expression responses of endangered alpine tree frogs (Litoria verreauxii alpina) to subclinical infection, comparing two long-exposed populations with a naïve population. We performed a blinded, randomized and controlled exposure experiment, collecting skin, liver and spleen tissues at 4, 8 and 14\ua0days postexposure from 51 wild-caught captively reared infection-naïve adult frogs for transcriptome assembly and differential gene expression analyses. We analysed our results in conjunction with infection intensity data, and the results of a large clinical survival experiment run concurrently with individuals from the same clutches. Here, we show that frogs from an evolutionarily long-exposed and phenotypically more resistant population of the highly susceptible alpine tree frog demonstrate a more robust innate and adaptive immune response at the critical early subclinical stage of infection when compared with two more susceptible populations. These results are consistent with the occurrence of evolution of resistance against chytridiomycosis, help to explain underlying resistance mechanisms, and provide genes of potential interest and sequence data for future research. We recommend further investigation of cell-mediated immunity pathways, the role of interferons and mechanisms of lymphocyte suppression

Using site-occupancy models to prepare for the spread of chytridiomyosis and identify factors affecting detectability of a cryptic susceptible species, the Tasmanian tree frog

Context: The global reduction of amphibian biodiversity as a result of the disease chytridiomycosis (caused by the fungus Batrachochytrium dendrobatidis; Bd) has highlighted the need to accurately detect local population declines in association with Bd presence. Although Bd has spread globally, some remote regions such as the Tasmanian Wilderness World Heritage Area (1.40 million ha; TWWHA) in Australia, remain largely, but not entirely, disease free. The Tasmanian tree frog (Litoria burrowsae) resides primarily within TWWHA boundaries, and is believed to be susceptible to chytridiomycosis.\ud \ud Aims: In the absence of historical abundance data, we used a single-season multi-state site-occupancy model to investigate the impact of Bd on L. burrowsae populations, on factors affecting species detection and to inform ongoing surveillance and conservation.\ud \ud Methods: We recorded frog detection and ranked call intensity (estimation of population size) from repeated independent surveys within a season to estimate the role of covariates, such as presence of Bd and environmental variables, on species occupancy and detection probability.\ud \ud Key results: Modelling revealed large frog populations are more likely to be present at naturally formed than human-formed ponds, strong winds negatively affect detection of populations, and time after sunset affects detection of large populations. Large frog populations were more likely to be Bd-negative; however, models including Bd presence were not well supported, in part a result of the small number of Bd-positive sites recorded.\ud \ud Conclusions and Implications: The utility of site-occupancy modelling in understanding the impact of disease on populations is little known, but has the potential to improve the accuracy and efficiency of many conservation programs

Sodium hypochlorite denatures the DNA of the amphibian chytrid fungus Batrachochytrium dendrobatidis

Batrachochytrium dendrobatidis, an aquatic amphibian fungus, has been implicated in many amphibian declines and extinctions. A real-time polymerase chain reaction (PCR) TaqMan® assay is now used to detect and quantify B. dendrobatidis on amphibians and other substrates via tissue samples, swabbing and filtration. The extreme sensitivity of this diagnostic test makes it necessary to rigorously avoid cross-contamination of samples, which can produce false positives. One technique used to eliminate contamination is to destroy the contaminating DNA by chemical means. We tested 3 concentrations of sodium hypochlorite (NaOCl) (1, 6 and12%) over 4 time periods (1, 6, 15 and 24 h) to determine if NaOCl denatures B. dendrobatidis DNA sufficiently to prevent its recognition and amplification in PCR tests for the fungus. Soaking in 12% NaOCl denatured 100% of DNA within 1 h. Six percent NaOCl was on average 99.999% effective across all exposure periods, with only very low numbers of zoospores detected following treatment. One percent NaOCl was ineffective across all treatment periods. Under ideal, clean conditions treatment with 6% NaOCl may be sufficient to destroy DNA and prevent cross-contamination of samples; however, we recommend treatment with 12% NaOCl for 1 h to be confident all B. dendrobatidis DNA is destroyed

Lethal effect of latex, nitrile, and vinyl gloves on tadpoles

Tadpoles are studied in a variety of fi elds including husbandry, developmental physiology, toxicity testing, and basic biological and ecological research. In many instances it is necessary to use gloves when handling tadpoles or during water changes to protect the experimenter (e.g. teratology research) or to promote hygiene and prevent the transfer of pathogens between tadpoles (Retallick et al. 2006; Sobotka and Rahwan 1999). While investigating aspects\ud of the virulent amphibian fungal pathogen Batrachochytrium\ud dendrobatidis, we discovered that a variety of gloves can be\ud lethal to tadpoles. We present here two case studies, one in the lab and one in the fi eld, and two experiments, all demonstrating the lethal effect of gloves on tadpoles. Following exposure to the various glove treatments, all tadpoles were categorized as either fine, listless, or dead

Preserving pathogens for wildlife conservation: a case for action on amphibian declines

Infectious disease is an important driver in biological systems but its importance in conservation has historically been underestimated. Recently, however, researchers have increasingly recognized the impact of diseases on wildlife populations and have grappled with disease-related conservation challenges. For example, the phenomenon of worldwide amphibian declines caused by the fungal disease chytridiomycosis has contributed to the creation of a global Amphibian Conservation Action Plan. The sense of urgency in the protection of amphibians and mitigation of the effects of chytridiomycosis is well-warranted but determining the best way to respond to chytridiomycosis is challenging. Current conservation strategies focus on the preservation of the amphibian hosts, their habitats and their genetic materials. However, we suggest that to confront disease threats fully, particularly in the case of amphibian declines, insight into host–pathogen coevolution \ud may be critical and we must therefore also preserve the pathogen for basic disease research. Here we outline priority targets for virulence research and urge researchers and managers to isolate and archive the pathogen Batrachochytrium dendrobatidis to ensure viable long-term amphibian conservation

Surveillance for emerging biodiversity diseases of wildlife

[Extract] Effective surveillance is crucial for early detection and successful mitigation of emerging diseases [1]. The current global approach to surveillance for wildlife diseases affecting biodiversity ("biodiversity diseases") is still inadequate as demonstrated by the slow characterization and response to the two recent devastating epidemics, chytridiomycosis and white-nose syndrome [2]–[5]. Current surveillance for wildlife disease usually targets diseases that affect humans or livestock, not those impacting wildlife populations. Barriers to effective surveillance for biodiversity diseases include a relative lack of social and political will and the inherent complexity and cost of implementing surveillance for multiple and diverse free-ranging populations. Here we evaluate these challenges and the inadequacies of current surveillance techniques, and we suggest an integrated approach for effective surveillance

Chytridiomycosis and seasonal mortality of tropical stream-associated frogs 15 years after introduction of Batrachochytrium dendrobatidis

Assessing the effects of diseases on wildlife populations can be difficult in the absence of observed mortalities, but it is crucial for threat assessment and conservation. We performed an intensive capture-mark-recapture study across seasons and years to investigate the effect of chytridiomycosis on demographics in 2 populations of the threatened common mist frog (Litoria rheocola) in the lowland wet tropics of Queensland, Australia. Infection prevalence was the best predictor for apparent survival probability in adult males and varied widely with season (0–65%). Infection prevalence was highest in winter months when monthly survival probabilities were low (approximately 70%). Populations at both sites exhibited very low annual survival probabilities (12–15%) but high recruitment (71–91%), which resulted in population growth rates that fluctuated seasonally. Our results suggest that even in the absence of observed mortalities and continued declines, and despite host–pathogen co-existence for multiple host generations over almost 2 decades, chytridiomycosis continues to have substantial seasonally fluctuating population-level effects on amphibian survival, which necessitates increased recruitment for population persistence. Similarly infected populations may thus be under continued threat from chytridiomycosis which may render them vulnerable to other threatening processes, particularly those affecting recruitment success

Batrachochytrium dendrobatidis: requirement for further isolate collection and archiving

The fungal pathogen Batrachochytrium dendrobatidis (Bd) causes the disease chytridiomycosis, which is lethal to many species of amphibians worldwide. Many studies have investigated the epidemiology of chytridiomycosis in amphibian populations, but few have considered possible host–pathogen coevolution. More specifically, investigations focused on the evolution of Bd, and the link with Bd virulence, are needed. Such studies, which may be important for conservation management of amphibians, depend on access to Bd isolates. Here we provide a summary of known Bd isolates that have been collected and archived in various locations around the world. Of 257 Bd isolates, we found that 53% originate from ranids in the United States. In many cases, detailed information on isolate origin is unavailable, and it is unknown how many isolates are cryo-archived. We suggest the creation of a centralized database of isolate information, and we urge researchers and managers to isolate and archive Bd to facilitate future research on chytridiomycosis