Amphibian Chytridiomycosis Research Articles

A Simplified, CRISPR-Based Method for the Detection of Batrachochytrium salamandrivorans.

The fungal pathogen Batrachochytrium salamandrivorans (Bsal) is one of two species (the other, B. dendrobatidis/Bd) that cause amphibian chytridiomycosis, an emerging infectious disease that has been indicated in the declines of hundreds of amphibian species worldwide. While Bd has been near globally distributed for decades, Bsal is a more recently emerged pathogen, having been identified just over a decade ago with current impacts localized to salamandrids in parts of Europe. However, because there is concern that Bsal will cause widespread declines if introduced to naïve regions-such as the Americas where the greatest diversity of salamandrids exist-it is imperative that widespread monitoring strategies be implemented to mitigate the spread of Bsal. As standard molecular diagnostic approaches-such as qPCR-tend to be expensive, time-consuming, or require specialized instrumentation and training, we have developed a simplified, rapid, CRISPR-based approach for Bsal-DNA detection. Here, we demonstrate this approach-termed FINDeM (Field-deployable, Isothermal, Nucleotide-based Detection Method)-and show that it can detect clinically relevant concentrations of Bsal DNA in under an hour using only inexpensive supplies and body-heat inducible reactions. Further, we highlight drawbacks and limitations associated with FINDeM-such as decreased DNA extraction yields and detection sensitivity when compared to more traditional approaches-and provide suggestions for additional development and future application of this method.

Drosophila melanogaster as a model arthropod carrier for the amphibian chytrid fungus Batrachochytrium dendrobatidis.

The fungal pathogen Batrachochytrium dendrobatidis (Bd) causes the disease amphibian chytridiomycosis, which has contributed to population declines in many species of amphibians throughout the world. Previous observational studies have shown that nematodes, waterfowl, lizards, other dipterans, and crayfish have properties which may allow them to harbor and spread Bd; therefore, we sought to determine the carrier capabilities of invertebrates to a further extent in a laboratory setting. We use the insect Drosophila melanogaster as a model organism to quantify the potential relationship between insects and Bd. Our findings show that D. melanogaster can test positive for Bd for up to five days post-exposure and can transmit Bd to conspecifics without suffering mortality. Insects of various types interact with the amphibian habitat and amphibians themselves, making this a potentially important route of transmission between amphibians and of dispersal across the environment.

Cutaneous shedding in amphibians causes shifts in bacterial microbiomes.

Considerable research has focused on microbes on amphibian skin, as they act as the first line of defense against invading pathogens. This effort has generated substantial data on patterns across species, space, time, and ontogeny, alongside a growing list of beneficial antifungal symbionts. Though there is evidence of stability in amphibian skin microbial communities, there is also an indication that regular skin shedding reduces cultivable bacteria, with regrowth and recolonization in the period between sheds. This suggests that skin communities are in constant flux, and we lack an understanding of how the membership and structure of those communities are affected by shedding events. In this study, we conducted experiments on cane toads (Rhinella marina) to investigate the influence of shedding on skin microbiomes. We first used quantitative PCR to verify a positive correlation between bacterial loads and time in the days after shedding. We then resampled individuals over time to describe changes in community composition in the 38 h after shedding using amplicon sequencing. Similar to trends of bacterial loads, we found increases in alpha diversity over time after shedding, suggesting that shedding reduces bacterial diversity as it knocks down bacterial loads. During the 38-h period, community structure became similar to pre-shed communities in some individuals, but there was no consistent pattern in structural changes among individuals. In light of the amphibian chytridiomycosis pandemic, understanding how physiological events such as skin shedding affect beneficial bacteria and communities on amphibians would provide important insight into amphibian ecology.

The complement system and its involvement in inhibition of Batrachochytriym dendrobatidis, a lethal fungal pathogen of amphibians

The field of ecological immunology, or ecoimmunology, has provided valuable insights on the immune responses of diverse host organisms threatened by infectious diseases in many different environments. One infectious disease that has been particularly notable for its impacts on host populations is amphibian chytridiomycosis, which has been linked with amphibian declines around the world. Amphibian immune responses to the pathogen that causes chytridiomycosis (Batrachochytriym dendrobatidis) are not well understood but thought to involve innate immune factors, including the complement system. In this study, we tested the ability of complement proteins to inhibit B. dendrobatidis in in vitro challenge assays. We found that complement proteins from amphibian plasma that were not heat inactivated reduced the viability and growth of B. dendrobatidis. The inhibitory efficacy was similar to effects on Pseudomonas fluorescens, a bacterium that is known to be inhibited by complement protein activation. These findings suggest inhibition of B. dendrobatidis that is consistent with the involvement of the complement system. In addition, we provide methods for standardizing pathogen killing assays, and set a foundation for further investigations on the amphibian complement system and other immune responses to amphibian chytridiomycosis.

Stable in vitro fluorescence for enhanced live imaging of infection models for Batrachochytrium dendrobatidis.

Realistic and modifiable infection models are required to study the pathogenesis of amphibian chytridiomycosis. Understanding the mechanism by which Batrachochytrium dendrobatidis (Bd) can infect and kill diverse amphibians is key to mitigating this pathogen and preventing further loss of biodiversity. In vitro studies of Bd typically rely on a tryptone based growth media, whereas the recent development of a kidney cell-line infection model has provided a more realistic alternative, without the need for live animals. Here we use expression of a fluorescent reporter to enhance the in vitro cell-line based growth assay, and show that transformed Bd cells are able to invade and grow in an amphibian kidney epithelial cell line (A6) as well as in a new system using a lung fibroblast cell line (DWJ). Both Bd and host cells were modified to express reporter fluorescent proteins, enabling immediate and continuous observation of the infection process without the need for destructive sampling for fixation and staining. Plasmid DNA conferring hygromycin resistance and TdTomato (RFP) expression was delivered to Bd zoospores via electroporation, and continuous antibiotic selection after recovery produced stable fluorescent Bd transformants. Host cells (A6 and DWJ) were transfected before each assay using lipofection to deliver plasmid DNA conferring green fluorescent protein (GFP) and containing an empty shRNA expression cassette. Bd RFP expression allowed easy localisation of fungal cells and identification of endobiotic growth was assisted by host GFP expression, by allowing visualization of the space in the host cell occupied by the invading fungal body. In addition to enabling enhanced live imaging, these methods will facilitate future genetic modification and characterisation of specific genes and their effect on Bd virulence.

Batrachochytrium dendrobatidis in the Arid and Thermally Extreme Sonoran Desert.

Batrachochytrium dendrobatidis (Bd), the causative agent of the devastating global amphibian disease chytridiomycosis, was not projected to threaten amphibians in hot and arid regions due to its sensitivity to heat and desiccation. However, Bd is being detected more frequently than ever in hot and arid regions of Australia and the USA, challenging our current understanding of the environmental tolerances of the pathogen under natural conditions. We surveyed for Bd in an extremely hot and arid portion of the Sonoran Desert, where the pathogen is not projected to occur, and related presence and prevalence of the pathogen to local environmental conditions. We collected eDNA samples from isolated desert water sites including six tinajas and 13 catchments in June and August of 2020 and swabbed a total of 281 anurans of three species (red-spotted toad Anaxyrus punctatus, Couch's spadefoot Scaphiopus couchii, and the Sonoran Desert toad Incillius alvarius) across five catchments and six tinajas from June to September of 2020. Overall, Bd occurred at 68.4% of sites, despite extreme heat and aridity routinely exceeding tolerances established in laboratory studies. Average summer maximum air and water temperatures were 40.7°C and 30.7°C, respectively, and sites received an average of just 16.9mm of precipitation throughout the summer monsoon season. Prevalence was low (5.7%) across species and life stage. Our results demonstrate that Bd is capable of persisting and infecting amphibians beyond its projected range, indicating a need to account for higher thermal tolerances when quantifying risk of Bd presence and infection.

Genetic mechanisms and biological processes underlying host response to ophidiomycosis (snake fungal disease) inferred from tissue-specific transcriptome analyses.

Emerging infectious diseases in wildlife species caused by pathogenic fungi are of growing concern, yet crucial knowledge gaps remain for diseases with potentially large impacts. For example, there is detailed knowledge about host pathology and mechanisms underlying response for chytridiomycosis in amphibians and white-nose syndrome in bats, but such information is lacking for other more recently described fungal infections. One such disease is ophidiomycosis, caused by the fungus Ophidiomyces ophidiicola, which has been identified in many species of snakes, yet the biological mechanisms and molecular changes occurring during infection are unknown. To gain this information, we performed a controlled experimental infection in captive Prairie rattlesnakes (Crotalus viridis) with O. ophidiicola at two different temperatures: 20 and 26°C. We then compared liver, kidney, and skin transcriptomes to assess tissue-specific genetic responses to O. ophidiicola infection. Given previous histopathological studies and the fact that snakes are ectotherms, we expected highest fungal activity on skin and a significant impact of temperature on host response. Although we found fungal activity to be localized on skin, most of the differential gene expression occurred in internal tissues. Infected snakes at the lower temperature had the highest host mortality whereas two-thirds of the infected snakes at the higher temperature survived. Our results suggest that ophidiomycosis is likely a systemic disease with long-term effects on host response. Our analysis also identified candidate protein coding genes that are potentially involved in host response, providing genetic tools for studies of host response to ophidiomycosis in natural populations.

FINDeM: A CRISPR‐based, molecular method for rapid, inexpensive and field‐deployable organism detection

Abstract The field of ecology has undergone a molecular revolution, with researchers increasingly relying on DNA‐based methods for organism detection. Unfortunately, these techniques often require expensive equipment, dedicated laboratory spaces and specialized training in molecular and computational techniques; limitations that may exclude field researchers, underfunded programmes and citizen scientists from contributing to cutting‐edge science. It is for these reasons that we have designed a simplified, inexpensive method for field‐based molecular organism detection—FINDeM (Field‐deployable Isothermal Nucleotide‐based Detection Method). In this approach, DNA is extracted using chemical cell lysis and a cellulose filter disc, followed by two body‐heat inducible reactions—recombinase polymerase amplification and a CRISPR‐Cas12a fluorescent reporter assay—to amplify and detect target DNA, respectively. Here, we introduce and validate FINDeM in detecting Batrachochytrium dendrobatidis, the causative agent of amphibian chytridiomycosis, and show that this approach can identify single‐digit DNA copies from epidermal swabs in under 1 h using low‐cost supplies and field‐friendly equipment. This research signifies a breakthrough in ecology, as we demonstrate a field‐deployable platform that requires only basic supplies (i.e. micropipettes, plastic consumables and a UV flashlight), inexpensive reagents (~$1.29 USD/sample) and emanated body heat for highly sensitive, DNA‐based organism detection. By presenting FINDeM in an ecological system with pressing, global biodiversity implications, we aim to not only highlight how CRISPR‐based applications promise to revolutionize organism detection but also how the continued development of such techniques will allow for additional, more diversely trained researchers to answer the most pressing questions in ecology.

Predominant prevalence of Ranavirus in southern Brazil, a region with widespread occurrence of the amphibian chytrid

AbstractEmerging infectious diseases are a major cause of amphibian declines worldwide. Two of these diseases are amphibian chytridiomycosis caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd) and a viral disease caused by different ranaviruses (Rv). Whereas Bd is known to be widespread in Brazil, Rv was only recently detected in natural habitats. We therefore investigated the co‐occurrence of Bd and Rv in wild tadpoles in the southern Brazilian Atlantic Forest from 21 ponds. Because infection by Bd might cause immune suppression in the host, we hypothesized that tadpoles infected with Bd would be more prone to infection with Rv than Bd‐free individuals, hence presenting higher Rv loads. Surprisingly, co‐infection was rare (less than 6% of the sampled specimens), and we did not find evidence that Bd would facilitate Rv infection. On the other hand, we detected Rv in 38% of tadpoles (125 out of 324), representing twice the prevalence of Bd in the same populations. We tested the influence of biotic and abiotic factors on infection by each pathogen on tadpoles, and we detected a positive relationship between forest cover and infection by Rv, whereas no relationship was found for Bd infection. Based on the putatively low rate of co‐infection and high prevalence of Rv in wild anuran populations, we advocate for the inclusion of Rv screening and monitoring in addition to Bd in conservation‐oriented amphibian surveys. Only surveying both diseases will provide a complete picture of amphibian disease‐related declines.

United States amphibian imports pose a disease risk to salamanders despite Lacey Act regulations

Batrachochytrium salamandrivorans (Bsal), one of two fungal pathogens that cause the deadly amphibian disease chytridiomycosis, is a major impending threat to salamander biodiversity in North America, where it is not yet known to occur. In the United States, a 2016 wildlife trade policy restricted trade in 20 salamander genera in attempts to prevent Bsal introduction. However, little comprehensive data is available to evaluate the impact of this policy action. Here we collate a dataset of United States amphibian imports from 1999 to 2021 using Law Enforcement Management Information System (LEMIS) data and show that reported legal trade in the targeted taxa was effectively reduced by the ban. Unfortunately, amphibian trade into the United States continues to risk Bsal introduction given that other species and genera now known to carry Bsal are still traded in large quantities (millions of live individuals annually). Additional policy responses focused on Bsal carrier taxa, especially frogs in the genus Rana, could help mitigate the impact of Bsal on North American salamanders.

Identification of major histocompatibility complex genotypes associated with resistance to an amphibian emerging infectious disease.

Amphibian chytridiomycosis, caused by Batrachochytrium dendrobatidis (Bd), emerged from Asia and spread globally. By comparing functional MHC IIß1 alleles from an Asian Bd-resistant anuran species (Bufo gargarizans) with those of an Australasian Bd-susceptible species (Litoria caerulea), we identified MHC genotypes with a glycine deletion (G90β1) and adjacent motifs in the deepest pathogen-derived peptide-binding groove associated with Bd resistance. Every Bd-resistant individual, but no susceptible individuals, possessed at least one allele encoding the variant. We detected trans-species polymorphism at the end of the MHC IIβ1 sequences in both species. The G90β1 deletion was encoded by different alleles in the two species, suggesting it may have evolved independently in the two species rather than having been derived from a common ancestor. It is possible that MHC adaptations to chytridiomycosis evolved independently among amphibian species. These results are consistent with a scenario by which MHC adaptations that confer resistance to the pathogen have evolved by convergent evolution. Immunogenetic studies such as this are critical to ongoing conservation efforts.

Widespread amphibian Perkinsea infections associated with Ranidae hosts, cooler months and Ranavirus co-infection.

Amphibians suffer from large-scale population declines globally, and emerging infectious diseases contribute heavily to these declines. Amphibian Perkinsea (Pr) is a worldwide anuran pathogen associated with mass mortality events, yet little is known about its epidemiological patterns, especially in comparison to the body of literature on amphibian chytridiomycosis and ranavirosis. Here, we establish Pr infection patterns in natural anuran populations and identify important covariates including climate, host attributes and co-infection with Ranavirus (Rv). We used quantitative (q)PCR to determine the presence and intensity of Pr and Rv across 1234 individuals sampled throughout central Florida in 2017-2019. We then implemented random forest ensemble learning models to predict infection with both pathogens based on physiological and environmental characteristics. Perkinsea infected 32% of all sampled anurans, and Pr prevalence was significantly elevated in Ranidae frogs, cooler months, metamorphosed individuals and frogs co-infected with Rv, while Pr intensity was significantly higher in ranid frogs and individuals collected dead. Ranavirus prevalence was 17% overall and was significantly higher in Ranidae frogs, metamorphosed individuals, locations with higher average temperatures, and individuals co-infected with Pr. Perkinsea prevalence was significantly higher than Rv prevalence across months, regions, life stages and species. Among locations, Pr prevalence was negatively associated with crayfish prevalence and positively associated with relative abundance of microhylids, but Rv prevalence did not associate with any tested co-variates. Co-infections were significantly more common than single infections for both pathogens, and we propose that Pr infections may propel Rv infections because seasonal Rv infection peaks followed Pr infection peaks and random forest models found Pr intensity was a leading factor explaining Rv infections. Our study elucidates epidemiological patterns of Pr in Florida and suggests that Pr may be under-recognized as a cause of anuran declines, especially in the context of pathogen co-infection.

Accounting for bias in prevalence estimation: The case of a globally emerging pathogen

Abstract Accurate quantification of infection parameters is necessary to ensure effective surveillance, investigation and mitigation of infectious diseases. However, hosts and pathogens are often imperfectly observed and key epidemiological parameters, such as infection prevalence, can be biased if this observational uncertainty is not properly accounted for. Here, we evaluated the combined effects of imperfect pathogen detection and host pseudoreplication on the estimation of infection prevalence of the pathogen Batrachochytrium dendrobatidis (Bd) in the southern Darwin's frog (Rhinoderma darwinii). This pathogen causes amphibian chytridiomycosis, a panzootic disease responsible for the greatest documented loss of biodiversity due to an infectious disease. From November 2018 to March 2019, we made 1085 captures of 641 R. darwinii individuals in two areas of Southern Chile. Captured frogs were individually identified to eliminate host pseudoreplication, skin‐swabbed twice in sequence, and each swab was analysed in duplicate using a real‐time polymerase chain reaction (qPCR) assay to detect Bd. To provide a robust estimate of period prevalence, we used a Bayesian multiscale occupancy model that considers pathogen imperfect detection arising from both sampling and diagnostic testing processes. Finally, using a deterministic matrix population model, we illustrated how the method chosen to estimate prevalence influenced our conclusions regarding the impact of Bd infection on host population trajectories. Our results showed that Bd prevalence could be underestimated by 55% if false negatives and host pseudoreplication were not accounted for. Host pseudoreplication had a greater impact on prevalence underestimation than pathogen imperfect detection in our study. This underestimation in prevalence changed our interpretation of the impacts of Bd infections on our model species, from a nearly stable population using the naïve period prevalence to a declining one using our robust estimate. Synthesis and applications. These results highlight the importance of using robust inferences to inform disease risk assessments and to efficiently allocate limited resources during mitigation strategies of infectious diseases. The methods used here can be applied to a wide range of host–pathogen systems, and will be of interest to both researchers and practitioners aiming to investigate and mitigate the impacts of infectious diseases on free‐ranging populations.

Selection of an anti-pathogen skin microbiome following prophylaxis treatment in an amphibian model system.

With emerging diseases on the rise, there is an urgent need to identify and understand novel mechanisms of prophylactic protection in vertebrate hosts. Inducing resistance against emerging pathogens through prophylaxis is an ideal management strategy that may impact pathogens and their host-associated microbiome. The host microbiome is recognized as a critical component of immunity, but the effects of prophylactic inoculation on the microbiome are unknown. In this study, we investigate the effects of prophylaxis on host microbiome composition, focusing on the selection of anti-pathogenic microbes contributing to host acquired immunity in a model host-fungal disease system, amphibian chytridiomycosis. We inoculated larval Pseudacris regilla against the fungal pathogen Batrachochytrium dendrobatidis (Bd) with a Bd metabolite-based prophylactic. Increased prophylactic concentration and exposure duration were associated with significant increases in proportions of putatively Bd-inhibitory host-associated bacterial taxa, indicating a protective prophylactic-induced shift towards microbiome members that are antagonistic to Bd. Our findings are in accordance with the adaptive microbiome hypothesis, where exposure to a pathogen alters the microbiome to better cope with subsequent pathogen encounters. Our study advances research on the temporal dynamics of microbiome memory and the role of prophylaxis-induced shifts in microbiomes contributing to prophylaxis effectiveness. This article is part of the theme issue 'Amphibian immunity: stress, disease and ecoimmunology'.

A new species of barred frog, Mixophyes (Anura: Myobatrachidae) from south-eastern Australia identified by molecular genetic analyses.

Mixophyes are large ground-dwelling myobatrachid frogs from eastern Australia and New Guinea. Several of the species found in mid-eastern and south-eastern Australia are listed as threatened, due largely to declines presumably caused by the amphibian disease chytridiomycosis. Given the wide distribution of several of these species and that their distributions cross well-known biogeographic boundaries that often correspond to deep genetic breaks or species boundaries among closely related vertebrates, we undertook a molecular genetic assessment of population structure across the range of each species to determine the presence of undescribed species. Of the four species of Mixophyes subject to molecular population genetic analyses, one, the Stuttering Frog (Mixophyes balbus), showed a level of diversity consistent with the presence of two species. Morphometric, meristic and bioacoustic analyses corroborate these distinctions, and a new species is described for the populations south of the Macleay River valley in mid-eastern New South Wales to east Gippsland in Victoria. Applying the IUCN Red List threat criteria the new species meets the conservation status assessment criteria for Endangered 2B1a,b because its extent of occupancy and area of occupancy are below the threshold value and it has declined and disappeared from the southern two thirds of its distribution over the past 30 years.

Amphibian survival compromised by long-term effects of chytrid fungus

Chytridiomycosis, the disease caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), has been unambiguously implicated in the decline of amphibian populations worldwide. However, the impact of this devastating infectious disease can be difficult to gauge without empirical data on the population-level effects of Bd. Often, assessments of the amphibian chytridiomycosis panzootic are based primarily on expert opinions; as a result, declines in tropical areas are promptly attributed to Bd while its impact on temperate species not suffering from adult mass mortalities is frequently overlooked. Here, we investigated the survival probability in an amphibian species from a temperate area that until now has not been considered to be severely impacted by the disease. Specifically, we related individual survival to Bd infection status using long-term capture-mark-recapture data of male spiny common toads (Bufo spinosus) in Sierra de Guadarrama National Park in central Spain. Even though the study population has demonstrated potential for adaptation to Bd and die-offs of adult individuals have not been recorded, our results clearly indicated that the probability of survival was lower for Bd-positive individuals. Moreover, the probability of becoming Bd-positive was higher than the probability of clearance, driving the population to a slow but certain decline. These results are consistent with other indicators of a negative population trend and suggest that the impact of Bd on temperate species of less concern may be greater than previously thought.

Predicted Batrachochytrium dendrobatidis infection sites in Guyana, Suriname, and French Guiana using the species distribution model maxent.

The fungal pathogen Batrachochytrium dendrobatidis (Bd) which causes that amphibian disease chytridiomycosis is expanding its worldwide range from an Asian origin, infecting amphibians in a growing number of countries. Modelling the potential range of this amphibian pathogen using environmental variables and presence data could advance our understanding of at-risk areas and species in locations with limited surveillance to date. We used a species distribution model to assess Bd habitat suitability in the three Guiana’s (Guyana, Suriname, and French Guiana) in South America. The model output showed that all three countries have substantial areas where Bd could grow and proliferate, and maximum temperature of the warmest month was the top predictor of suitable Bd habitat, inversely correlated with modeled Bd occurrence. Predicted Bd infection areas in Guyana and French Guiana were large and localized whereas possible sites in Suriname were more scattered throughout the country. The areas projected as potential suitable in Suriname were mostly high elevation regions. These results could help inform efficiencies for development of a proactive monitoring program that could alert managers of novel Bd outbreaks for focused mitigation actions to forestall the spread of this amphibian disease.

Synthesis ofBatrachochytrium dendrobatidisinfection in South America: amphibian species under risk and areas to focus research and disease mitigation

Amphibian chytridiomycosis, caused byBatrachochytrium dendrobatidis(Bd), has been recognized as the infectious disease causing the most catastrophic loss of biodiversity known to science, with South America being the most impacted region. We tested whetherBdprevalence is distributed among host taxonomy, ecoregion, conservation status and habitat preference in South America. Here we provide a synthesis on the extent ofBdinfection across South America based on 21 648 molecular diagnostic assays, roles of certain species in the epidemiology ofBdand explore its association with the reported amphibian catastrophic declines in the region. We show thatBdis widespread, with a continental prevalence of 23.2%. Its occurrence in the region shows a phylogenetic signal and the probability of infection is determined by ecoregion, preferred habitat and extinction risk hosts' traits. The taxa exhibiting highestBdoccurrence were mostly aquatic amphibians, including Ranidae, Telmatobiidae, Hylodidae, Calyptocephalellidae and Pipidae. Surprisingly, families exhibiting unusually lowBdprevalence included species in which lethal chytridiomycosis and population declines have been described (generaAtelopus,RhinodermaandEleutherodactylus). Higher than expected prevalence ofBdoccurred mainly in amphibians living in association with mountain environments in the Andes and Atlantic forests, reflecting highly favourableBdhabitats in these areas. Invasive amphibian species (e.g.Lithobates catesbeianusandXenopus laevis) exhibited highBdprevalence; thus we suggest using these as sentinels to understand their potential role as reservoirs, vectors or spreaders ofBdthat can be subjected to management. Our results guide on the prioritization of conservation actions to prevent further biodiversity loss due to chytridiomycosis in the world's most amphibian diverse region.

A new species of Philoria (Anura: Limnodynastidae) from the uplands of the Gondwana Rainforests World Heritage Area of eastern Australia.

The six species of mountain frogs (Philoria: Limnodynastidae: Anura) are endemic to south-eastern Australia. Five species occur in headwater systems in mountainous north-eastern New South Wales (NSW) and south-eastern Queensland (Qld), centred on the Gondwana Rainforests of Australia World Heritage Area. A previous molecular genetic analysis identified divergent genetic lineages in the central and western McPherson Ranges region of Qld and NSW, but sampling was inadequate to test the species status of these lineages. With more comprehensive geographic sampling and examination of the nuclear genome using SNP analysis, we show that an undescribed species, P. knowlesi sp. nov., occurs in the central and western McPherson Ranges (Levers Plateau and Mount Barney complex). The new species is not phylogenetically closely related to P. loveridgei in the nuclear data but is related to one of two divergent lineages within P. loveridgei in the mtDNA data. We postulate that the discordance between the nuclear and mtDNA outcomes is due to ancient introgression of the mtDNA genome from P. loveridgei into the new species. Male advertisement calls and multivariate morphological analyses do not reliably distinguish P. knowlesi sp. nov. from any of the Philoria species in northeast NSW and southeast Qld. The genetic comparisons also enable us to define further the distributions of P. loveridgei and P. kundagungan. Samples from the Lamington Plateau, Springbrook Plateau, Wollumbin (Mt Warning National Park), and the Nightcap Range, are all P. loveridgei, and its distribution is now defined as the eastern McPherson Ranges and Tweed caldera. Philoria kundagungan is distributed from the Mistake Mountains in south-eastern Qld to the Tooloom Scrub on the Koreelah Range, southwest of Woodenbong, in NSW, with two subpopulations identified by SNP analysis. We therefore assessed the IUCN threat category of P. loveridgei and P. kundagungan and undertook new assessments for each of its two subpopulations and for the new taxon P. knowlesi sp. nov., using IUCN Red List criteria. Philoria loveridgei, P. kundagungan (entire range and northern subpopulation separately) and P. knowlesi sp. nov. each meet criteria for Endangered (EN B2(a)(b)[i, iii]). The southern subpopulation of P. kundagungan, in the Koreelah Range, meets criteria for Critically Endangered (CE B2(a)(b)[i, iii]). These taxa are all highly threatened due to the small number of known locations, the restricted nature of their breeding habitat, and direct and indirect threats from climate change, and the potential impact of the amphibian disease chytridiomycosis. Feral pigs are an emerging threat, with significant impacts now observed in Philoria breeding habitat in the Mistake Mountains.

Impact of heavy metal exposure on biological control of a deadly amphibian pathogen by zooplankton

Despite devastating effects on global biodiversity, efficient mitigation strategies against amphibian chytridiomycosis are lacking. Since the free-living pathogenic zoospores of Batrachochytrium dendrobatidis (Bd), the infective stage of this disease, can serve as a nutritious food source for components of zooplankton communities, these groups may act as biological control agents by eliminating zoospores from the aquatic environment. Such pathogen-predator interaction is, however, embedded in the aquatic food web structure and is therefore affected by abiotic factors interfering with these networks. Heavy metals, released from both natural and anthropogenic sources, are widespread contaminants of aquatic ecosystems and may interfere with planktonic communities and thus pathogen elimination rates. We investigated the interaction between zooplankton communities and chytridiomycosis infections in a Flemish agricultural region. Moreover, we also investigated the impact of heavy metal contamination, that was previously investigated in the region and presented in recent work, on zooplankton assemblages and chytridiomycosis infections. Finally, we tested the effect of sublethal concentrations of copper and zinc on Bd removal rates by Daphnia magna in a laboratory assay. Although zinc, copper, nickel and chromium were widely abundant pollutants, heavy metals were no driving force for zooplankton assemblages at our study locations. Moreover, our field survey did not reveal indirect effects of zooplankton assemblages on chytridiomycosis infections. However, sampling occasions testing negative for Bd showed a higher degree of copper contamination compared to positive sampling occasions, indicating a potential inhibitory effect of copper on Bd prevalence. Finally, whereas D. magna significantly reduced zoospore densities in its environment, sublethal concentrations of copper and zinc showed no interference with pathogen removal in the laboratory assay. Our results provide perspectives for further research on such a biological control strategy against chytridiomycosis by optimizing environmental conditions for pathogen predation.