Antifungal treatment of wild amphibian populations caused a transient reduction in the prevalence of the fungal pathogen, Batrachochytrium dendrobatidis.
ABSTRACT: Emerging infectious diseases can drive host populations to extinction and are a major driver of biodiversity loss. Controlling diseases and mitigating their impacts is therefore a priority for conservation science and practice. Chytridiomycosis is a devastating disease of amphibians that is caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), and for which there is an urgent need to develop mitigation methods. We treated tadpoles of the common midwife toad (Alytes obstetricans) with antifungal agents using a capture-treat-release approach in the field. Antifungal treatment during the spring reduced the prevalence of Bd in the cohort of tadpoles that had overwintered and reduced transmission of Bd from this cohort to the uninfected young-of-the-year cohort. Unfortunately, the mitigation was only transient, and the antifungal treatment was unable to prevent the rapid spread of Bd through the young-of-the year cohort. During the winter, Bd prevalence reached 100% in both the control and treated ponds. In the following spring, no effects of treatment were detectable anymore. We conclude that the sporadic application of antifungal agents in the present study was not sufficient for the long-term and large-scale control of Bd in this amphibian system.
Project description:Climate change and infectious disease by the chytrid fungus Batrachochytrium dendrobatidis (Bd) are major drivers of amphibian extinctions, but the potential interactions of these two factors are not fully understood. Temperature is known to influence (1) the infectivity, pathogenicity and virulence of Bd; (2) host-parasite dynamics, especially when both hosts and parasites are ectothermic organisms exhibiting thermal sensitivities that may or may not differ; and (3) amphibian vulnerability to extinction depending on their heat tolerance, which may decrease with infection. Thus, in a global warming scenario, with rising temperatures and more frequent and extreme weather events, amphibians infected by Bd could be expected to be more vulnerable if temperatures approach their critical thermal maximum (CTmax). However, it is also possible that predicted high temperatures could clear the Bd infection, thus enhancing amphibian survival. We tested these hypotheses by measuring CTmax values of Bd-infected and Bd-free aquatic tadpoles and terrestrial toadlets/juveniles of the common midwife toad (Alytes obstetricans) and examining whether exposure of A. obstetricans individuals to peak temperatures reaching their CTmax clears them from Bd infection. We show that (1) Bd has a wide thermal tolerance range; (2) Bd is capable of altering the thermal physiology of A. obstetricans, which is stage-dependent, lowering CTmax in tadpoles but not in toadlets; and (3) Bd infection is not cleared after exposure of tadpoles or toadlets to CTmax. Living under climatic change with rising temperatures, the effect of Bd infection might tip the balance and lead some already threatened amphibian communities towards extinction.
Project description:Real-time quantitative PCR studies largely depend on reference genes for the normalization of gene expression. Stable reference genes should be accurately selected in order to obtain reliable results. We here present a study screening commonly used reference genes (TEF1F, ?-centractin, Ctsyn1, GAPDH, R6046, APRT and TUB) in the chytrid fungi Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal), which cause the lethal amphibian skin disease chytridiomycosis. We evaluated the stability of the reference gene candidates during different growth stages of the fungi, using different statistical software packages: ?CT, BestKeeper, GeNorm, NormFinder and RefFinder. In order to reflect the in vivo situation, the stability of the candidates was assessed when taking all growth stages into account. Using an ex-vivo approach, we tested whether the expression of GAPDH, TUB, R6046 and APRT (Bd) and GAPDH, TUB, R6046 and ?-centractin (Bsal) remained stable when these fungi came in contact with host tissue. Finally, their role as in vivo reference genes was examined in skin tissue of experimentally infected midwife toads (Alytes obstetricans) (Bd) and fire salamanders (Salamandra salamandra) (Bsal). Summarized, the present study provides guidance for selecting appropriate reference genes when analyzing expression patterns of these fungal organisms during different growth stages and in Bd- or Bsal-infected tissues.
Project description:To prevent transmission of the pathogenic chytrid fungi Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal), hygiene protocols prescribe the single use of disposable gloves for handling amphibians. We discovered that rinse water from nitrile gloves instantly kills 99% of Bd and Bsal zoospores. Transmission experiments using midwife toads (Alytes obstetricans) and Bd, and Alpine newts (Ichthyosaura alpestris) and Bsal, show that the use of the same pair of gloves for 2 subsequent individuals does not result in significant transmission of any chytrid fungus. In contrast, handling infected amphibians bare-handed caused transmission of Bsal in 4 out of 10 replicates, but did not result in transmission of Bd. Based on the manufacturer's information, high resolution mass spectrometry (HRMS) and colorimetric tests, calcium lactate and calcium nitrate were identified as compounds with antifungal activity against both Bd and Bsal. These findings corroborate the importance of wearing gloves as an important sanitary measure in amphibian disease prevention. If the highly recommended single use of gloves is not possible, handling multiple post-metamorphic amphibians with the same pair of nitrile gloves should still be preferred above bare-handed manipulation.
Project description: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'.
Project description:Batrachochytrium dendrobatidis (Bd), a chytrid fungus, has increasingly been implicated as a major factor in the worldwide decline of amphibian populations. The fungus causes chytridiomycosis in susceptible species leading to massive die-offs of adult amphibians. Although Bd infects the keratinized mouthparts of tadpoles and negatively affects foraging behavior, these infections are non-lethal. An important morphogen controlling amphibian metamorphosis is thyroid hormone (T3). Tadpoles may be infected with Bd and the fungus may be exposed to T3 during metamorphosis. We hypothesize that exposure of Bd to T3 may induce the expression of factors associated with host colonization and pathogenicity. We utilized a proteomics approach to better understand the dynamics of the Bd-T3 interaction. Using liquid chromatography-mass spectrometry (LC-MS), we generated a data set of a large number of cytoplasmic and membrane proteins following exposure of Bd to T3. From these data, we identified a total of 263 proteins whose expression was significantly changed following T3 exposure. We provide evidence for expression of an array of proteins that may play key roles in both genomic and non-genomic actions of T3 in Bd. Additionally, our proteomics study shows an increase in several proteins including proteases and a class of uncommon crinkler and crinkler-like effector proteins suggesting their importance in Bd pathogenicity as well as those involved in metabolism and energy transfer, protein fate, transport and stress responses. This approach provides insights into the mechanistic basis of the Bd-amphibian interaction following T3 exposure.
Project description:Batrachochytrium dendrobatidis (Bd) is the causative agent of chytridiomycosis, a fungal skin disease in amphibians and driver of worldwide amphibian declines.We focussed on the early stages of infection by Bd in 3 amphibian species with a differential susceptibility to chytridiomycosis. Skin explants of Alytes muletensis, Litoria caerulea and Xenopus leavis were exposed to Bd in an Ussing chamber for 3 to 5 days. Early interactions of Bd with amphibian skin were observed using light microscopy and transmission electron microscopy. To validate the observations in vitro, comparison was made with skin from experimentally infected frogs. Additional in vitro experiments were performed to elucidate the process of intracellular colonization in L. caerulea. Early interactions of Bd with amphibian skin are: attachment of zoospores to host skin, zoospore germination, germ tube development, penetration into skin cells, invasive growth in the host skin, resulting in the loss of host cell cytoplasm. Inoculation of A. muletensis and L. caerulea skin was followed within 24 h by endobiotic development, with sporangia located intracellularly in the skin. Evidence is provided of how intracellular colonization is established and how colonization by Bd proceeds to deeper skin layers. Older thalli develop rhizoid-like structures that spread to deeper skin layers, form a swelling inside the host cell to finally give rise to a new thallus. In X. laevis, interaction of Bd with skin was limited to an epibiotic state, with sporangia developing upon the skin. Only the superficial epidermis was affected. Epidermal cells seemed to be used as a nutrient source without development of intracellular thalli. The in vitro data agreed with the results obtained after experimental infection of the studied frog species. These data suggest that the colonization strategy of B. dendrobatidis is host dependent, with the extent of colonization most likely determined by inherent characteristics of the host epidermis.
Project description:Pathogenesis is strongly dependent on microbial context, but development of probiotic therapies has neglected the impact of ecological interactions. Dynamics among microbial communities, host immune responses, and environmental conditions may alter the effect of probiotics in human and veterinary medicine, agriculture and aquaculture, and the proposed treatment of emerging wildlife and zoonotic diseases such as those occurring on amphibians or vectored by mosquitoes. Here we use a holistic measure of amphibian mucosal defenses to test the effects of probiotic treatments and to assess disease risk under different ecological contexts. We developed a non-invasive assay for antifungal function of the skin mucosal ecosystem (mucosome function) integrating host immune factors and the microbial community as an alternative to pathogen exposure experiments. From approximately 8500 amphibians sampled across Europe, we compared field infection prevalence with mucosome function against the emerging fungal pathogen Batrachochytrium dendrobatidis. Four species were tested with laboratory exposure experiments, and a highly susceptible species, Alytes obstetricans, was treated with a variety of temperature and microbial conditions to test the effects of probiotic therapies and environmental conditions on mucosome function. We found that antifungal function of the amphibian skin mucosome predicts the prevalence of infection with the fungal pathogen in natural populations, and is linked to survival in laboratory exposure experiments. When altered by probiotic therapy, the mucosome increased antifungal capacity, while previous exposure to the pathogen was suppressive. In culture, antifungal properties of probiotics depended strongly on immunological and environmental context including temperature, competition, and pathogen presence. Functional changes in microbiota with shifts in temperature provide an alternative mechanistic explanation for patterns of disease susceptibility related to climate beyond direct impact on host or pathogen. This nonlethal management tool can be used to optimize and quickly assess the relative benefits of probiotic therapies under different climatic, microbial, or host conditions.
Project description:Variation and population structure play key roles in the speciation process, but adaptive intraspecific genetic variation is commonly ignored when forecasting species niches. Amphibians serve as excellent models for testing how climate and local adaptations shape species distributions due to physiological and dispersal constraints and long generational times. In this study, we analysed the climatic factors driving the evolution of the genus Alytes at inter- and intraspecific levels that may limit realized niches. We tested for both differences among the five recognized species and among intraspecific clades for three of the species (Alytes obstetricans, A. cisternasii, and A. dickhilleni). We employed ecological niche models with an ordination approach to perform niche overlap analyses and test hypotheses of niche conservatism or divergence. Our results showed strong differences in the environmental variables affecting species climatic requirements. At the interspecific level, tests of equivalence and similarity revealed that sister species were non-identical in their environmental niches, although they neither were entirely dissimilar. This pattern was also consistent at the intraspecific level, with the exception of A. cisternasii, whose clades appeared to have experienced a lower degree of niche divergence than clades of the other species. In conclusion, our results support that Alytes toads, examined at both the intra- and interspecific levels, tend to occupy similar, if not identical, climatic environments.
Project description:Methods to mitigate the impacts of emerging infectious diseases affecting wildlife are urgently needed to combat loss of biodiversity. However, the successful mitigation of wildlife pathogens in situ has rarely occurred. Indeed, most strategies for combating wildlife diseases remain theoretical, despite the wealth of information available for combating infections in livestock and crops. Here, we report the outcome of a 5-year effort to eliminate infection with Batrachochytrium dendrobatidis affecting an island system with a single amphibian host. Our initial efforts to eliminate infection in the larval reservoir using a direct application of an antifungal were successful ex situ but infection returned to previous levels when tadpoles with cleared infections were returned to their natal sites. We subsequently combined antifungal treatment of tadpoles with environmental chemical disinfection. Infection at four of the five pools where infection had previously been recorded was eradicated, and remained so for 2 years post-application.
Project description:Anthropogenic and natural stressors often interact to affect organisms. Amphibian populations are undergoing unprecedented declines and extinctions with pesticides and emerging infectious diseases implicated as causal factors. Although these factors often co-occur, their effects on amphibians are usually examined in isolation. We hypothesized that exposure of larval and metamorphic amphibians to ecologically relevant concentrations of pesticide mixtures would increase their post-metamorphic susceptibility to the fungus Batrachochytrium dendrobatidis (Bd), a pathogen that has contributed to amphibian population declines worldwide. We exposed five anuran species (Pacific treefrog, Pseudacris regilla; spring peeper, Pseudacris crucifer; Cascades frog, Rana cascadae; northern leopard frog, Lithobates pipiens; and western toad, Anaxyrus boreas) from three families to mixtures of four common insecticides (chlorpyrifos, carbaryl, permethrin, and endosulfan) or herbicides (glyphosate, acetochlor, atrazine, and 2,4-D) or a control treatment, either as tadpoles or as newly metamorphic individuals (metamorphs). Subsequently, we exposed animals to Bd or a control inoculate after metamorphosis and compared survival and Bd load. Bd exposure significantly increased mortality in Pacific treefrogs, spring peepers, and western toads, but not in Cascades frogs or northern leopard frogs. However, the effects of pesticide exposure on mortality were negligible, regardless of the timing of exposure. Bd load varied considerably across species; Pacific treefrogs, spring peepers, and western toads had the highest loads, whereas Cascades frogs and northern leopard frogs had the lowest loads. The influence of pesticide exposure on Bd load depended on the amphibian species, timing of pesticide exposure, and the particular pesticide treatment. Our results suggest that exposure to realistic pesticide concentrations has minimal effects on Bd-induced mortality, but can alter Bd load. This result could have broad implications for risk assessment of amphibians; the outcome of exposure to multiple stressors may be unpredictable and can differ between species and life stages.