Frances C. Clare

Multiple emergences of genetically diverse amphibian-infecting chytrids include a globalized hypervirulent recombinant lineage

Batrachochytrium dendrobatidis (Bd) is a globally ubiquitous fungal infection that has emerged to become a primary driver of amphibian biodiversity loss. Despite widespread effort to understand the emergence of this panzootic, the origins of the infection, its patterns of global spread, and principle mode of evolution remain largely unknown. Using comparative population genomics, we discovered three deeply diverged lineages of Bd associated with amphibians. Two of these lineages were found in multiple continents and are associated with known introductions by the amphibian trade. We found that isolates belonging to one clade, the global panzootic lineage (BdGPL) have emerged across at least five continents during the 20th century and are associated with the onset of epizootics in North America, Central America, the Caribbean, Australia, and Europe. The two newly identified divergent lineages, Cape lineage (BdCAPE) and Swiss lineage (BdCH), were found to differ in morphological traits when compared against one another and BdGPL, and we show that BdGPL is hypervirulent. BdGPL uniquely bears the hallmarks of genomic recombination, manifested as extensive intergenomic phylogenetic conflict and patchily distributed heterozygosity. We postulate that contact between previously genetically isolated allopatric populations of Bd may have allowed recombination to occur, resulting in the generation, spread, and invasion of the hypervirulent BdGPL leading to contemporary disease-driven losses in amphibian biodiversity.

Microscopic Aquatic Predators Strongly Affect Infection Dynamics of a Globally Emerged Pathogen

Research on emerging infectious wildlife diseases has placed particular emphasis on host-derived barriers to infection and disease. This focus neglects important extrinsic determinants of the host/pathogen dynamic, where all barriers to infection should be considered when ascertaining the determinants of infectivity and pathogenicity of wildlife pathogens. Those pathogens with free-living stages, such as fungi causing catastrophic wildlife declines on a global scale, must confront lengthy exposure to environmental barriers before contact with an uninfected host. Hostile environmental conditions therefore have the ability to decrease the density of infectious particles, reducing the force of infection and ameliorating the impact as well as the probability of establishing an infection. Here we show that, in nature, the risk of infection and infectious burden of amphibians infected by the chytrid fungus Batrachochytrium dendrobatidis (Bd) have a significant, site-specific component, and that these correlate with the microfauna present at a site. Experimental infections show that aquatic microfauna can rapidly lower the abundance and density of infectious stages by consuming Bd zoospores, resulting in a significantly reduced probability of infection in anuran tadpoles. Our findings offer new perspectives for explaining the divergent impacts of Bd infection in amphibian assemblages and contribute to our understanding of ecosystem resilience to colonization by novel pathogens.

Assessing Risk and Guidance on Monitoring of Batrachochytrium dendrobatidis in Europe through Identification of Taxonomic Selectivity of Infection

Amphibians are globally threatened, but not all species are affected equally by different threatening processes. This is true for the threat posed by the chytridiomycete fungus (Batrachochytrium dendrobatidis). We compiled a European data set for B. dendrobatidis to analyze the trends of infection in European amphibians. The risk of infection was not randomly distributed geographically or taxonomically across Europe. Within countries with different prevalence, infection was nonrandom in certain amphibian taxa. Brown frogs of the genus Rana were unlikely to be infected, whereas frogs in the families Alytidae and Bombinatoridae were significantly more likely to be infected than predicted by chance. Frogs in the 2 families susceptible to B. dendrobatidis should form the core of attempts to develop spatial surveillance studies of chytridiomycosis in Europe. Ideally, surveys for B. dendrobatidis should be augmented by sampling the widespread genus Pelophylax because this taxon exhibits geographically inconsistent overinfection with B. dendrobatidis and surveillance of it may facilitate recognition of factors causing spatial variability of infection intensity. Several European amphibian taxa were not represented in our data set; however, surveillance of unsampled species should also occur when warranted.

Fatal chytridiomycosis in the tyrrhenian painted frog.

Batrachochytrium dendrobatidis (Bd), the causative agent of the amphibian disease chytridiomycosis, is an important factor in the global decline of amphibians. Within Europe, animals that exhibit clinical signs of the disease have only been reported in Spain despite the pathogen’s wide, but patchy, distribution on the continent. Recently, another occurrence of chytridiomycosis was reported in Euproctus platycephalus, the Sardinian brook newt, on the Mediterranean island of Sardinia, but without any evidence of fatal disease. We report further evidence of the emergence of Bd on Sardinia and the first evidence of lethal chytridiomycosis outside of Spain. Unusual mortalities of the Tyrrhenian painted frog (Discoglossus sardus) were found at three sites in the Limbara mountains of northern Sardinia. Molecular and histological screens of corpses, frogs, and tadpoles from these sites revealed infection with Bd. Infection and mortality occurred at locations that are unusual in terms of the published habitat requirements of the pathogen. Given the endemicity, the IUCN Red List status of the amphibian species on Sardinia, and the occurrence of infection and mortality caused by chytridiomycosis, there is serious reason for concern for the impact that disease emergence may have on the conservation of the amphibians of the island.

Climate forcing of an emerging pathogenic fungus across a montane multi-host community

Changes in the timings of seasonality as a result of anthropogenic climate change are predicted to occur over the coming decades. While this is expected to have widespread impacts on the dynamics of infectious disease through environmental forcing, empirical data are lacking. Here, we investigated whether seasonality, specifically the timing of spring ice-thaw, affected susceptibility to infection by the emerging pathogenic fungus Batrachochytrium dendrobatidis (Bd) across a montane community of amphibians that are suffering declines and extirpations as a consequence of this infection. We found a robust temporal association between the timing of the spring thaw and Bd infection in two host species, where we show that an early onset of spring forced high prevalences of infection. A third highly susceptible species (the midwife toad, Alytes obstetricans) maintained a high prevalence of infection independent of time of spring thaw. Our data show that perennially overwintering midwife toad larvae may act as a year-round reservoir of infection with variation in time of spring thaw determining the extent to which infection spills over into sympatric species. We used future temperature projections based on global climate models to demonstrate that the timing of spring thaw in this region will advance markedly by the 2050s, indicating that climate change will further force the severity of infection. Our findings on the effect of annual variability on multi-host infection dynamics show that the community-level impact of fungal infectious disease on biodiversity will need to be re-evaluated in the face of climate change. This article is part of the themed issue ‘Tackling emerging fungal threats to animal health, food security and ecosystem resilience’.

Host species vary in infection probability, sub-lethal effects, and costs of immune response when exposed to an amphibian parasite

The amphibian parasite Batrachochytrium dendrobatidis (Bd) is regarded as an extreme generalist, infecting over 500 species, but amongst these hosts there exists a great deal of variation in the susceptibility to and the costs of parasite exposure. We use two infection experiments to determine whether inter-specific variation in the sublethal and lethal effects of parasite exposure exist in two host species. We then tested the relative roles of host density and diversity on infection probability of a focal susceptible host. Our results show significant heterogeneity in host species response to parasite exposure, and that both lethal and sub-lethal costs exist in individuals that are able to resist infection, indicating that successful immune response to infection comes at a cost. Further, we show that increasing host density significantly increased the likelihood of susceptible individuals becoming infected with Bd irrespective of host diversity and variation in host susceptibility. These results suggest that populations of resistant species are likely to suffer ill-effects of exposure to Bd regardless of their infection status, and that at the stage of initial infection there was no support for the dilution of transmission events, in contrast to other studies that focus on subsequent transmission of infection.

What Caused the UK's Largest Common Dolphin (Delphinus delphis) Mass Stranding Event?

On 9 June 2008, the UKs largest mass stranding event (MSE) of short-beaked common dolphins (Delphinus delphis) occurred in Falmouth Bay, Cornwall. At least 26 dolphins died, and a similar number was refloated/herded back to sea. On necropsy, all dolphins were in good nutritive status with empty stomachs and no evidence of known infectious disease or acute physical injury. Auditory tissues were grossly normal (26/26) but had microscopic haemorrhages (5/5) and mild otitis media (1/5) in the freshest cases. Five lactating adult dolphins, one immature male, and one immature female tested were free of harmful algal toxins and had low chemical pollutant levels. Pathological evidence of mud/seawater inhalation (11/26), local tide cycle, and the relative lack of renal myoglobinuria (26/26) suggested MSE onset on a rising tide between 06∶30 and 08∶21 hrs (9 June). Potential causes excluded or considered highly unlikely included infectious disease, gas/fat embolism, boat strike, by-catch, predator attack, foraging unusually close to shore, chemical or algal toxin exposure, abnormal weather/climatic conditions, and high-intensity acoustic inputs from seismic airgun arrays or natural sources (e.g., earthquakes). International naval exercises did occur in close proximity to the MSE with the most intense part of the exercises (including mid-frequency sonars) occurring four days before the MSE and resuming with helicopter exercises on the morning of the MSE. The MSE may therefore have been a “two-stage process” where a group of normally pelagic dolphins entered Falmouth Bay and, after 3–4 days in/around the Bay, a second acoustic/disturbance event occurred causing them to strand en masse. This spatial and temporal association with the MSE, previous associations between naval activities and cetacean MSEs, and an absence of other identifiable factors known to cause cetacean MSEs, indicates naval activity to be the most probable cause of the Falmouth Bay MSE.

Assessing the ability of swab data to determine the true burden of infection for the amphibian pathogen Batrachochytrium dendrobatidis

AbstractBatrachochytrium dendrobatidis (Bd) is a pathogenic fungus which causes the disease chytridiomycosis in amphibians by infecting the animals’ epidermis. The most commonly applied method for the detection of Bd is the use of a sterile swab, rubbed over the keratinized areas of an amphibian and then processed to yield DNA for detection by qPCR. This method has been used to infer a threshold of lethal infection in some species; however, how reliable and reproducible the swabbing method is at detecting the true burden of infection suffered by individuals is not known. European midwife toads, Alytes obstetricans, are susceptible to chytridiomycosis and are highly parasitised by Bd across Europe. By quantifying Bd-load throughout the entire skin and comparing this to swab results taken from the same individual, we determined whether epidermal swabs provide a quantifiable and accurate indication of the true fungal burden suffered. Further, we examined whether we could infer a threshold for lethal infection based on comparison of swab data taken from infected A. obstetricans exhibiting different clinical states. From swab data, we detected significantly higher fungal burdens from moribund metamorphs compared to visually healthy individuals; however, the ability of these swab data to provide an accurate indication of the true fungal burden was not reliable. These data suggest that fungal load dynamics play an important role in disease-induced mortality in A. obstetricans at these sites, but that using swab data to infer an exact threshold for Bd-associated mortality might be inappropriate and misleading.

Amphibian chytridiomycosis outbreak dynamics are linked with host skin bacterial community structure

Host-associated microbes are vital for combatting infections and maintaining health. In amphibians, certain skin-associated bacteria inhibit the fungal pathogen Batrachochytrium dendrobatidis (Bd), yet our understanding of host microbial ecology and its role in disease outbreaks is limited. We sampled skin-associated bacteria and Bd from Pyrenean midwife toad populations exhibiting enzootic or epizootic disease dynamics. We demonstrate that bacterial communities differ between life stages with few shared taxa, indicative of restructuring at metamorphosis. We detected a significant effect of infection history on metamorph skin microbiota, with reduced bacterial diversity in epizootic populations and differences in community structure and predicted function. Genome sequencing of Bd isolates supports a single introduction to the Pyrenees and reveals no association between pathogen genetics and epidemiological trends. Our findings provide an ecologically relevant insight into the microbial ecology of amphibian skin and highlight the relative importance of host microbiota and pathogen genetics in predicting disease outcome.Amphibian skin microbe communities have been putatively associated with the severity of chytrid fungal disease. Here, the authors show that different types of disease dynamics (enzootic versus epizootic) are associated with different microbiota in the host populations.

Development and worldwide use of a non-lethal and minimal population-level impact protocols for the isolation of chytrids from amphibians

Parasitic chytrid fungi have emerged as a significant threat to amphibian species worldwide, necessitating the development of techniques to isolate these pathogens into sterile culture for research purposes. However, early methods of isolating chytrids from their hosts relied on killing amphibians. We modified a pre-existing protocol for isolating chytrids from infected animals to use toe clips and biopsies from toe webbing rather than euthanizing hosts, and distributed the protocol to interested researchers worldwide as part of the BiodivERsA project RACE – here called the RML protocol. In tandem, we developed a lethal procedure for isolating chytrids from tadpole mouthparts. Reviewing a database of use a decade after their inception, we find that these methods have been widely applied across at least 5 continents, 23 countries and in 62 amphibian species, and have been successfully used to isolate chytrids in remote field locations. Isolation of chytrids by the non-lethal RML protocol occured in 18% of attempts with 207 fungal isolates and three species of chytrid being recovered. Isolation of chytrids from tadpoles occured in 43% of attempts with 334 fungal isolates of one species (Batrachochytrium dendrobatidis) being recovered. Together, these methods have resulted in a significant reduction and refinement of our use of threatened amphibian species and have improved our ability to work with this important group of emerging fungal pathogens.