Laura A. Brannelly

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.

Clinical trials with itraconazole as a treatment for chytrid fungal infections in amphibians

Due in large part to recent global declines and extinctions, amphibians are the most threatened vertebrate group. Captive assurance colonies may be the only lifeline for some rapidly disappearing species. Maintaining these colonies free of disease represents a challenge to effective amphibian conservation. The fungal disease chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis (Bd), is one of the major contributors to global amphibian declines and also poses a serious threat to captive assurance colonies. Many treatment options for Bd infection have not been experimentally tested and the commonly administered dosages of some drugs are known to have negative side effects, highlighting a need for clinical trials. The objective of this study was to clinically test the drug itraconazole as a method for curing Bd infection. We bathed Bd-positive juveniles of 2 anuran amphibian species, Litoria caerulea and Incilius nebulifer, in aqueous itraconazole, varying the concentration and duration of treatment, to find the combination that caused the fewest side effects while also reliably ridding animals of Bd. Our results suggest that a bath in 0.0025% itraconazole for 5 min d-1 for 6 d reliably cures Bd infection and causes fewer side effects than the longer treatment times and higher concentrations of this drug that are commonly administered.

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

Dynamics of Chytridiomycosis during the Breeding Season in an Australian Alpine Amphibian.

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.

Batrachochytrium dendrobatidis in natural and farmed Louisiana crayfish populations: prevalence and implications.

Understanding disease dynamics during the breeding season of declining amphibian species will improve our understanding of how remnant populations persist with endemic infection, and will assist the development of management techniques to protect disease-threatened species from extinction. We monitored the endangered Litoria verreauxii alpina (alpine treefrog) during the breeding season through capture-mark-recapture (CMR) studies in which we investigated the dynamics of chytridiomycosis in relation to population size in two populations. We found that infection prevalence and intensity increased throughout the breeding season in both populations, but infection prevalence and intensity was higher (3.49 and 2.02 times higher prevalence and intensity, respectively) at the site that had a 90-fold higher population density. This suggests that Bd transmission is density-dependent. Weekly survival probability was related to disease state, with heavily infected animals having the lowest survival. There was low recovery from infection, especially when animals were heavily infected with Bd. Sympatric amphibian species are likely to be reservoir hosts for the disease and can play an important role in the disease ecology of Bd. Although we found 0% prevalence in crayfish (Cherax destructor), we found that a sympatric amphibian (Crinia signifera) maintained 100% infection prevalence at a high intensity throughout the season. Our results demonstrate the importance of including infection intensity into CMR disease analysis in order to fully understand the implications of disease on the amphibian community. We recommend a combined management approach to promote lower population densities and ensure consistent progeny survival. The most effective management strategy to safeguard the persistence of this susceptible species might be to increase habitat area while maintaining a similar sized suitable breeding zone and to increase water flow and area to reduce drought.

Reservoir-host amplification of disease impact in an endangered amphibian.

The pathogenic chytrid fungus Batrachochytrium dendrobatidis (Bd) has been linked to global declines and extinctions of amphibians, making it one of the most devastating wildlife pathogens known. Understanding the factors that affect disease dynamics in this system is critical for mitigating infection and protecting threatened species. Crayfish are hosts of this pathogen and can transmit Bd to amphibians. Because they co-occur with susceptible amphibian communities, crayfish may be important alternative hosts for Bd. Understanding the prevalence and seasonal dynamics of crayfish infections is of agricultural and ecological interest in areas where crayfish are farmed and traded for human consumption. We conducted a survey of Bd in farmed and natural crayfish (Procambarus spp.) populations in Louisiana, USA. We found that Bd prevalence and infection intensity was low in both farmed and native populations and that prevalence varied seasonally in wild Louisiana crayfish. This seasonal pattern mirrors that seen in local amphibians. As crayfish are an important globally traded freshwater taxon, even with low prevalence, they could be an important vector in the spread of Bd.

Amphibians with infectious disease increase their reproductive effort: evidence for the terminal investment hypothesis.

Emerging wildlife pathogens are an increasing threat to biodiversity. One of the most serious wildlife diseases is chytridiomycosis, caused by the fungal pathogen, Batrachochytrium dendrobatidis (Bd), which has been documented in over 500 amphibian species. Amphibians vary greatly in their susceptibility to Bd; some species tolerate infection, whereas others experience rapid mortality. Reservoir hosts-species that carry infection while maintaining high abundance but are rarely killed by disease-can increase extinction risk in highly susceptible, sympatric species. However, whether reservoir hosts amplify Bd in declining amphibian species has not been examined. We investigated the role of reservoir hosts in the decline of the threatened northern corroboree frog (Pseudophryne pengilleyi) in an amphibian community in southeastern Australia. In the laboratory, we characterized the response of a potential reservoir host, the (nondeclining) common eastern froglet (Crinia signifera), to Bd infection. In the field, we conducted frog abundance surveys and Bd sampling for both P. pengilleyi and C. signifera. We built multinomial logistic regression models to test whether Crinia signifera and environmental factors were associated with P. pengilleyi decline. C. signifera was a reservoir host for Bd. In the laboratory, many individuals maintained intense infections (>1000 zoospore equivalents) over 12 weeks without mortality, and 79% of individuals sampled in the wild also carried infections. The presence of C. signifera at a site was strongly associated with increased Bd prevalence in sympatric P. pengilleyi. Consistent with disease amplification by a reservoir host, P. pengilleyi declined at sites with high C. signifera abundance. Our results suggest that when reservoir hosts are present, population declines of susceptible species may continue long after the initial emergence of Bd, highlighting an urgent need to assess extinction risk in remnant populations of other declined amphibian species.

Low humidity is a failed treatment option for chytridiomycosis in the critically endangered southern corroboree frog

Mounting an immune response to fight disease is costly for an organism and can reduce investment in another life-history trait, such as reproduction. The terminal investment hypothesis predicts that an organism will increase reproductive effort when threatened by disease. The reproductive fitness of amphibians infected with the deadly fungal pathogen Batrachochytrium dendrobatidis (Bd) is largely unknown. In this study, we explored gametogenesis in two endangered and susceptible frog species, Pseudophryne corroboree and Litoria verreauxii alpina. Gametogenesis, both oogenesis and spermatogenesis, increased when animals were experimentally infected with Bd. In P. corroboree, infected males have thicker germinal epithelium, and a larger proportion of spermatocytes. In L. v. alpina, infected males had more spermatic cell bundles in total, and a larger proportion of spermatozoa bundles. In female L. v. alpina, ovaries and oviducts were larger in infected animals, and there were more cells present within the ovaries. Terminal investment has consequences for the evolution of disease resistance in declining species. If infected animals are increasing reproductive efforts and producing more offspring before succumbing to disease, it is possible that population-level selection for disease resistance will be minimized.

Characterization of MHC class IA in the endangered southern corroboree frog

Abstract Context. One of the major drivers of the current worldwide amphibian decline and extinction crisis is the spread of the amphibian chytrid fungus, Batrachochytrium dendrobatidis (Bd). Captive assurance colonies may be the only lifeline for some species. Current antifungal chemotherapies can be effective for clearing infection but may have detrimental side effects. The only non-chemotherapeutic treatment that has been clinically tested is heat, which is impractical for cold-adapted species. The fungus does not survive desiccation, and therefore a plausible alternative non-chemotherapeutic treatment would be a low-humidity regimen. Aims. We tested the efficacy of a low-humidity treatment regimen for treating Bd-infected animals of the critically endangered species Pseudophryne corroboree. This species has high tolerance of dry environments, and is thus an ideal candidate for this treatment trial. Methods. Forty frogs were exposed to 1 × 106 zoospores. At three weeks after exposure access to water was reduced to once daily for 10 days for 20 animals. Ten of these animals were also rinsed daily with the aim of removing zoospores. Key results. The drying regimen neither increased survival nor decreased infection load, and treatment may have hastened mortality. All infected frogs died between 21 and 80 days after exposure, except one untreated frog. Conclusions. Our results suggest that a drying regimen is not a viable treatment for chytridiomycosis. Infection may decrease the host’s ability to cope with water stress. Implications. The failed drying treatment in P. corroboree suggests that drying is not an appropriate treatment for chytridiomycosis and treatment efforts should be focussed on chemotherapies for cold-adapted species. P. corroboree is a critically endangered species for which chytridiomycosis is the primary cause of decline, yet few experiments have been carried out on this species with regards to chytridiomycosis. All experimental information about this species and disease will benefit the management and protection of the species.

Reduced Itraconazole Concentration and Durations Are Successful in Treating Batrachochytrium dendrobatidis Infection in Amphibians

Southern corroboree frogs (Pseudophryne corroboree) have declined to near extinction in the wild after the emergence of the amphibian chytrid fungus Batrachochytrium dendrobatidis in southeastern Australia in the 1980s. A major captive breeding and reintroduction program is underway to preserve this iconic species, but improving resistance to B. dendrobatidis would help the wild population to be self-sustaining. Using 3′ and 5′ rapid amplification of complementary DNA ends (RACE), we characterized the major histocompatibility complex (MHC) class IA locus in this species. We then used sequences generated from RACE to design primers to amplify the peptide-binding region (PBR) of this functional genetic marker. Finally, we analysed the diversity, phylogeny, and selection patterns of PBR sequences from four P. corroboree populations and compared this with other amphibian species. We found moderately high MHC class IA genetic diversity in this species and evidence of strong positive and purifying selection at sites that are associated with putative PBR pockets in other species, indicating that this gene region may be under selection for resistance to Bd. Future studies should focus on identifying alleles associated with Bd resistance in P. corroboree by performing a Bd laboratory challenge study to confirm the functional importance of our genetic findings and explore their use in artificial selection or genetic engineering to increase resistance to chytridiomycosis.