Michael McFadden

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.

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.

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

A review of the Green and Golden Bell Frog Litoria aurea breeding program at Taronga Zoo

15 million over 5 years to be allocated to recovery actions as determined by a National Chytridiomycosis Working Group of amphibian managers and scientists. Procrastination on this issue will likely result in additional extinction of Australia’s amphibians in the near future.

Optimal release strategies for cost-effective reintroductions

The Green and Golden Bell Frog Litoria aurea is a threatened species, having declined greatly in abundance throughout its range in recent decades. In 1994, Taronga Zoo, Sydney, established a captiv...

Survival, gene and metabolite responses of Litoria verreauxii alpina frogs to fungal disease chytridiomycosis

Summary Ex situ programmes for endangered species commonly focus on two main objectives: insurance against immediate risk of extinction and reintroduction. Releases influence the size of captive and wild populations and may present managers with a trade-off between the two objectives. This can be further complicated when considering the costs of the captive population and the possible release of different life stages. We approached this decision problem by combining population models and decision-analytic methods, using the reintroduction programme for the southern corroboree frog Pseudophryne corroboree in Australia as an example. We identified the optimal release rates of eggs and subadults which maximized the size of the captive and reintroduced populations while meeting constraints. We explored two scenarios: a long-term programme for a stable age-distributed captive population and a short-term programme with non-stable age distribution and limited budget. We accounted for uncertainty in the estimated vital rates and demographic stochasticity. Assuming a stable age distribution, large proportions of individuals could be released without decreasing the captive population below its initial size. The optimal strategy was sensitive to the post-release survival of both life stages, but subadult releases were generally most cost-effective, producing a large wild population and requiring a cheaper captive population. Egg releases were optimal for high expected juvenile survival, whereas mixed releases of both life stages were never optimal. In the short-term realistic scenario, subadult releases also produced the largest wild population, but they required a large increase in the size and cost of the captive population that exceeded the available budget. Egg releases were cheaper but yielded smaller numbers in the wild, whereas joint releases of both life stages provided more wild individuals, meeting budget constraints without depleting the captive population. Synthesis and applications. Optimal release strategies for endangered species reflect the trade-offs between insurance and reintroduction objectives and depend on the vital rates of the released individuals. Although focusing on a single life stage may have practical advantages, mixed strategies can maximize cost-effectiveness by combining the relative advantages of releasing early and late life stages.

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

The fungal skin disease chytridiomycosis has caused the devastating decline and extinction of hundreds of amphibian species globally, yet the potential for evolving resistance, and the underlying pathophysiological mechanisms remain poorly understood. We exposed 406 naïve, captive-raised alpine tree frogs (Litoria verreauxii alpina) from multiple populations (one evolutionarily naïve to chytridiomycosis) to the aetiological agent Batrachochytrium dendrobatidis in two concurrent and controlled infection experiments. We investigated (A) survival outcomes and clinical pathogen burdens between populations and clutches, and (B) individual host tissue responses to chytridiomycosis. Here we present multiple interrelated datasets associated with these exposure experiments, including animal signalment, survival and pathogen burden of 355 animals from Experiment A, and the following datasets related to 61 animals from Experiment B: animal signalment and pathogen burden; raw RNA-Seq reads from skin, liver and spleen tissues; de novo assembled transcriptomes for each tissue type; raw gene expression data; annotation data for each gene; and raw metabolite expression data from skin and liver tissues. These data provide an extensive baseline for future analyses.

The efficacy and pharmacokinetics of terbinafine against the frog-killing fungus (Batrachochytrium dendrobatidis)

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 days 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.

Hormone-induced spawning of the critically endangered northern corroboree frog Pseudophryne pengilleyi

Captive and wild amphibians are under threat of extinction from the deadly fungal pathogen Batrachochytrium dendrobatidis (Bd). The antifungal drug terbinafine (TBF) is used by pet owners to treat Bd-infected frogs; however, it is not widely used in academic or zoological institutions due to limited veterinary clinical trials. To assess TBFs efficacy, we undertook treatment trials and pharmacokinetic studies to investigate drug absorption and persistence in frog skin; and then we correlated these data to the minimal lethal concentrations (MLC) against Bd. Despite an initial reduction in zoospore load, the recommended treatment (five daily 5 min 0.01% TBF baths) was unable to cure experimentally infected alpine tree frogs and naturally infected common eastern froglets. In vitro and in vivo pharmacokinetics showed that absorbed TBF accumulates in frog skin with increased exposure, indicating its suitability for treating cutaneous pathogens via direct application. The MLC of TBF for zoosporangia was 100 μg/ml for 2 h, while the minimal inhibitory concentration was 2 μg/ml, suggesting that the drug concentration absorbed during 5 min treatments is not sufficient to cure high Bd burdens. With longer treatments of five daily 30 min baths, Bd clearance improved from 12.5% to 50%. A higher dose of 0.02% TBF resulted in 78% of animals cured; however, clearance was not achieved in all individuals due to low TBF skin persistence, as the half-life was less than 2 h. Therefore, the current TBF regime is not recommended as a universal treatment against Bd until protocols are optimized, such as with increased exposure frequency.

Dose and life stage-dependent effects of dietary beta-carotene supplementation on the growth and development of the Booroolong frog

Fundamental knowledge of the optimal hormone concentrations required to stimulate amplexus and spawning in breeding pairs of amphibians is currently lacking, hindering our understanding of the proximate mechanisms underpinning mating behaviour. The present study investigated the effects of: (1) the dose of a gonadotropin-releasing hormone analogue (GnRH-a) administered; (2) male-female hormone administration interval; and (3) topical application of GnRH-a, on spawning success in the northern corroboree frog. Administration of GnRH-a at doses of 0.5, 1.0 and 2.0μgg-1 were highly successful, with a significantly greater proportion of hormone-treated pairs ovipositing (89-100%) compared with the 0μgg-1 treatment (22%). Of the hormone-treated pairs, those receiving 0.5μgg-1 GnRH-a exhibited the highest fertilisation success (61%). Administration of GnRH-a to males and females simultaneously (0h) was more effective than injecting males either 48 or 24h before the injection of females. Overall, administration of GnRH-a was highly successful at inducing spawning in northern corroboree frogs. For the first time, we also effectively induced spawning following the topical application of GnRH-a to the ventral pelvic region. Topical application of GnRH-a eliminates the need for specialised training in amphibian injection, and will allow assisted reproductive technologies to be adopted by a greater number of captive facilities globally.