Scott D. Cashins

Spread of chytridiomycosis has caused the rapid global decline and extinction of frogs

The global emergence and spread of the pathogenic, virulent, and highly transmissible fungus Batrachochytrium dendrobatidis, resulting in the disease chytridiomycosis, has caused the decline or extinction of up to about 200 species of frogs. Key postulates for this theory have been completely or partially fulfilled. In the absence of supportive evidence for alternative theories despite decades of research, it is important for the scientific community and conservation agencies to recognize and manage the threat of chytridiomycosis to remaining species of frogs, especially those that are naive to the pathogen. The impact of chytridiomycosis on frogs is the most spectacular loss of vertebrate biodiversity due to disease in recorded history.

Mitigating amphibian disease: strategies to maintain wild populations and control chytridiomycosis

BackgroundRescuing amphibian diversity is an achievable conservation challenge. Disease mitigation is one essential component of population management. Here we assess existing disease mitigation strategies, some in early experimental stages, which focus on the globally emerging chytrid fungus Batrachochytrium dendrobatidis. We discuss the precedent for each strategy in systems ranging from agriculture to human medicine, and the outlook for each strategy in terms of research needs and long-term potential.ResultsWe find that the effects of exposure to Batrachochytrium dendrobatidis occur on a spectrum from transient commensal to lethal pathogen. Management priorities are divided between (1) halting pathogen spread and developing survival assurance colonies, and (2) prophylactic or remedial disease treatment. Epidemiological models of chytridiomycosis suggest that mitigation strategies can control disease without eliminating the pathogen. Ecological ethics guide wildlife disease research, but several ethical questions remain for managing disease in the field.ConclusionsBecause sustainable conservation of amphibians in nature is dependent on long-term population persistence and co-evolution with potentially lethal pathogens, we suggest that disease mitigation not focus exclusively on the elimination or containment of the pathogen, or on the captive breeding of amphibian hosts. Rather, successful disease mitigation must be context specific with epidemiologically informed strategies to manage already infected populations by decreasing pathogenicity and host susceptibility. We propose population level treatments based on three steps: first, identify mechanisms of disease suppression; second, parameterize epizootiological models of disease and population dynamics for testing under semi-natural conditions; and third, begin a process of adaptive management in field trials with natural populations.

Environmental refuge from disease-driven amphibian extinction.

Species that are tolerant of broad environmental gradients may be less vulnerable to epizootic outbreaks of disease. Chytridriomycosis, caused by the fungus Batrachochytrium dendrobatidis, has been linked to extirpations and extinctions of amphibian species in many regions. The pathogen thrives in cool, moist environments, and high amphibian mortality rates have commonly occurred during chytridiomycosis outbreaks in amphibian populations in high-elevation tropical rainforests. In Australia several high-elevation species, including the armored mist frog (Litoria lorica), which is designated as critically endangered by the International Union for the Conservation of Nature (IUCN), were believed to have gone extinct during chytridiomycosis outbreaks in the 1980s and early 1990s. Species with greater elevational ranges disappeared from higher elevations, but remained common in the lowlands. In June 2008, we surveyed a stream in a high-elevation dry sclerophyll forest and discovered a previously unknown population of L. lorica and a population of the waterfall frog (Litoria nannotis). We conducted 6 additional surveys in June 2008, September 2008, March 2009, and August 2009. Prevalences of B. dendrobatidis infection (number infected per total sampled) were consistently high in frogs (mean 82.5%, minimum 69%) of both species and in tadpoles (100%) during both winter (starting July) and summer (starting February). However, no individuals of either species showed clinical signs of disease, and they remained abundant (3.25 - 8.75 individuals of L. lorica and 6.5-12.5 individuals of L. nannotis found/person/100 m over 13 months). The high-elevation dry sclerophyll site had little canopy cover, low annual precipitation, and a more defined dry season than a nearby rainforest site, where L. nannotis was more negatively affected by chytridiomycosis. We hypothesize this lack of canopy cover allowed the rocks on which frogs perched to warm up, thereby slowing growth and reproduction of the pathogen on the hosts. In addition, we suggest surveys for apparently extinct or rare species should not be limited to core environments.

Susceptibility of amphibians to chytridiomycosis is associated with MHC class II conformation

The pathogenic chytrid fungus Batrachochytrium dendrobatidis (Bd) can cause precipitous population declines in its amphibian hosts. Responses of individuals to infection vary greatly with the capacity of their immune system to respond to the pathogen. We used a combination of comparative and experimental approaches to identify major histocompatibility complex class II (MHC-II) alleles encoding molecules that foster the survival of Bd-infected amphibians. We found that Bd-resistant amphibians across four continents share common amino acids in three binding pockets of the MHC-II antigen-binding groove. Moreover, strong signals of selection acting on these specific sites were evident among all species co-existing with the pathogen. In the laboratory, we experimentally inoculated Australian tree frogs with Bd to test how each binding pocket conformation influences disease resistance. Only the conformation of MHC-II pocket 9 of surviving subjects matched those of Bd-resistant species. This MHC-II conformation thus may determine amphibian resistance to Bd, although other MHC-II binding pockets also may contribute to resistance. Rescuing amphibian biodiversity will depend on our understanding of amphibian immune defence mechanisms against Bd. The identification of adaptive genetic markers for Bd resistance represents an important step forward towards that goal.

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.

Temperature alters reproductive life history patterns in Batrachochytrium dendrobatidis, a lethal pathogen associated with the global loss of amphibians

Understanding how pathogens respond to changing environmental conditions is a central challenge in disease ecology. The environmentally sensitive fungal pathogen Batrachochytrium dendrobatidis (Bd), which causes the amphibian disease chytridiomycosis, has spread globally causing amphibian extirpations in a wide variety of climatic regions. To gain an in-depth understanding of Bds responses to temperature, we used an integrative approach, combining empirical laboratory experiments with mathematical modeling. First, we selected a single Bd isolate and serially propagated two lineages of the isolate for multiple generations in two stable thermal conditions: 4°C (cold-adapted lineage) and 23°C (warm-adapted lineage). We quantified the production of infectious zoospores (fecundity), the timing of zoospore release, and zoospore activity in reciprocal temperature transplant experiments in which both Bd lineages were grown in either high or low temperature conditions. We then developed population growth models for the Bd lineages under each set of temperature conditions. We found that Bd had lower population growth rates, but longer periods of zoospore activity in the low temperature treatment (4°C) compared to the high temperature treatment (23°C). This effect was more pronounced in Bd lineages that were propagated in the low temperature treatment (4°C), suggesting a shift in Bds response to low temperature conditions. Our results provide novel insights into the mechanisms by which Bd can thrive in a wide variety of temperature conditions, potentially altering the dynamics of chytridiomycosis and thus, the propensity for Bd to cause amphibian population collapse. We also suggest that the adaptive responses of Bd to thermal conditions warrant further investigation, especially in the face of global climate change.

Minimising exposure of amphibians to pathogens during field studies

Many of the recent global amphibian mass mortalities, declines and extinctions have been attributed to the emerging infectious disease chytridiomycosis. There have been mass mortalities due to ranaviral disease but no major declines or extinctions. Controlling the transmission and spread of disease is of utmost importance, especially where there is the potential for human involvement. We have reviewed current hygiene guidelines for working with wild frogs, identified potential flaws and recommended those most suitable and effective for the field environment. Our within-site hygiene measures aim to reduce the risk of transmission among individuals. These measures encompass the capture, handling and holding of amphibians, skin disinfection before and after invasive procedures, marking frogs, sealing open wounds and treatment of accessory equipment. Our between-site hygiene measures aim to mitigate the risk of pathogen spread among populations. We have designed a risk calculator to help simplify and standardise the decision-making process for determining the level of risk and appropriate risk mitigation strategies to reduce the risk of increasing pathogen spread above background levels. Calculation of an overall risk score for pathogen spread takes into account the prior activity of field workers, the proposed activity, remoteness of the site, presence of known pathogens and the consequences of increased pathogen spread for amphibians in a given area.

Prior infection does not improve survival against the amphibian disease chytridiomycosis

Many amphibians have declined globally due to introduction of the pathogenic fungus Batrachochytrium dendrobatidis (Bd). Hundreds of species, many in well-protected habitats, remain as small populations at risk of extinction. Currently the only proven conservation strategy is to maintain species in captivity to be reintroduced at a later date. However, methods to abate the disease in the wild are urgently needed so that reintroduced and wild animals can survive in the presence of Bd. Vaccination has been widely suggested as a potential strategy to improve survival. We used captive-bred offspring of critically endangered booroolong frogs (Litoria booroolongensis) to test if vaccination in the form of prior infection improves survival following re exposure. We infected frogs with a local Bd isolate, cleared infection after 30 days (d) using itraconazole just prior to the onset of clinical signs, and then re-exposed animals to Bd at 110 d. We found prior exposure had no effect on survival or infection intensities, clearly showing that real infections do not stimulate a protective adaptive immune response in this species. This result supports recent studies suggesting Bd may evade or suppress host immune functions. Our results suggest vaccination is unlikely to be useful in mitigating chytridiomycosis. However, survival of some individuals from all experimental groups indicates existence of protective innate immunity. Understanding and promoting this innate resistance holds potential for enabling species recovery.

Application of the survey protocol for chytridiomycosis to Queensland, Australia

Spread of the amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd), which causes chytridiomycosis, has resulted in the extinction of frogs, but the distribution of Bd is incompletely known. We trialled the survey protocol for Bd by attempting to systematically map its distribution in Queensland, Australia. Bd was easily detected in known infected areas, such as the Wet Tropics and South East Queensland. It was not detected in bioregions adjacent to, but inland from or to the north of, infected regions: Einasleigh Uplands and Cape York adjacent to the infected Wet Tropics; and Brigalow Belt South adjacent to the infected South East Queensland bioregion. These regions where Bd was not detected have bordered infected regions for between 15 yr (in northern Queensland) and 30 yr (in southern Queensland), and so they define the geographical limits of Bd with regard to the long-term environmental conditions in Queensland. The Gulf Plains, a bioregion distant from infected bioregions, was also negative. Bd was confined to rainforest and bordering habitats, such as wet eucalypt forests. Infections were largely confined to permanent water-associated species, consistent with this being an important cause of this group having the greatest declines. Our data supports biogeographic climatic models that show much of inland and northern Australia to be too hot and dry to support Bd. As there is limited opportunity for Bd to spread further in Queensland, the priority for management is reducing the impact of Bd in affected populations and assisting frogs to disperse into their former distributions. Given that the survey protocol has been applied successfully in Australia it may be useful for mapping the distribution of Bd in other parts of the world.

Priorities for management of chytridiomycosis in Australia: saving frogs from extinction

Abstract. To protect Australian amphibian biodiversity, we have identified and prioritised frog species at an imminent risk of extinction from chytridiomycosis, and devised national management and research priorities for disease mitigation. Six Australian frogs have not been observed in the wild since the initial emergence of chytridiomycosis and may be extinct. Seven extant frog species were assessed as needing urgent conservation interventions because of (1) their small populations and/or ongoing declines throughout their ranges (southern corroboree frog (Pseudophryne corroboree, New South Wales), northern corroboree frog (Pseudophryne pengilleyi, Australian Capital Territory, New South Wales), Baw Baw frog (Philoria frosti, Victoria), Litoria spenceri (spotted tree frog, Victoria, New South Wales), Kroombit tinkerfrog (Taudactylus pleione, Queensland), armoured mist frog (Litoria lorica, Queensland)) or (2) predicted severe decline associated with the spread of chytridiomycosis in the case of Tasmanian tree frog (Litoria burrowsae, Tasmania). For these species, the risk of extinction is high, but can be mitigated. They require increased survey effort to define their distributional limits and to monitor and detect further population changes, as well as well-resourced management strategies that include captive assurance populations. A further 22 frog species were considered at a moderate to lower risk of extinction from chytridiomycosis. Management actions that identify and create or maintain habitat refugia from chytridiomycosis and target other threatening processes such as habitat loss and degradation may be effective in promoting their recovery. Our assessments for some of these species remain uncertain and further taxonomical clarification is needed to determine their conservation importance. Management actions are currently being developed and trialled to mitigate the threat posed by chytridiomycosis. However, proven solutions to facilitate population recovery in the wild are lacking; hence, we prioritise research topics to achieve this aim. Importantly, the effectiveness of novel management solutions will likely differ among species due to variation in disease ecology, highlighting the need for species-specific research. We call for an independent management and research fund of AU