Chytridiomycosis causes high amphibian mortality prior to the completion of metamorphosis.

Amphibian populations are undergoing extensive declines globally. The fungal disease chytridiomycosis, caused by the pathogenic fungus Batrachochytrium dendrobatidis (Bd), is a primary contributor to these declines. The amphibian metamorphic stages (Gosner stages 42-46) are particularly vulnerable to a range of stressors, including Bd. Despite this, studies that explicitly examine host response to chytridiomycosis throughout the metamorphic stages are lacking. We aimed to determine how Bd exposure during the larval stages impacts metamorphic development and infection progression in the endangered Fleay's barred frog (Mixophyes fleayi). We exposed M. fleayi to Bd during pro-metamorphosis (Gosner stages 35-38) and monitored infection dynamics throughout metamorphosis. We took weekly morphological measurements (weight, total body length, snout-vent-length and Gosner stage) and quantified Bd load using qPCR. While we observed minimal impact of Bd infection on animal growth and development, Bd load varied throughout ontogeny, with an infection load plateau during the tadpole stages (Gosner stages 35-41) and temporary infection clearance at Gosner stage 42. Bd load increased exponentially between Gosner stages 42 and 45, with most exposed animals becoming moribund at Gosner stage 45, prior to the completion of metamorphosis. There was variability in infection outcome of exposed individuals, with a subgroup of animals (n = 5/29) apparently clearing their infection while the majority (n = 21/29) became moribund with high infection burdens. This study demonstrates the role that metamorphic restructuring plays in shaping Bd infection dynamics and raises the concern that substantial Bd-associated mortality could be overlooked in the field due to the often cryptic nature of these latter metamorphic stages. We recommend future studies that directly examine the host immune response to Bd infection throughout metamorphosis, incorporating histological and molecular methods to elucidate the mechanisms responsible for the observed trends.

Do immune system changes at metamorphosis predict vulnerability to chytridiomycosis? An update.

Amphibians are among the vertebrate groups suffering great losses of biodiversity due to a variety of causes including diseases, such as chytridiomycosis (caused by the fungal pathogens Batrachochytrium dendrobatidis and B. salamandrivorans). The amphibian metamorphic period has been identified as being particularly vulnerable to chytridiomycosis, with dramatic physiological and immunological reorganisation likely contributing to this vulnerability. Here, we overview the processes behind these changes at metamorphosis and then perform a systematic literature review to capture the breadth of empirical research performed over the last two decades on the metamorphic immune response. We found that few studies focused specifically on the immune response during the peri-metamorphic stages of amphibian development and fewer still on the implications of their findings with respect to chytridiomycosis. We recommend future studies consider components of the immune system that are currently under-represented in the literature on amphibian metamorphosis, particularly pathogen recognition pathways. Although logistically challenging, we suggest varying the timing of exposure to Bd across metamorphosis to examine the relative importance of pathogen evasion, suppression or dysregulation of the immune system. We also suggest elucidating the underlying mechanisms of the increased susceptibility to chytridiomycosis at metamorphosis and the associated implications for population persistence. For species that overlap a distribution where Bd/Bsal are now endemic, we recommend a greater focus on management strategies that consider the important peri-metamorphic period.

Synchrotron-Based Imaging Reveals the Fate of Selenium in Striped Marsh Frog Tadpoles.

Synchrotron-based X-ray fluorescence microscopy (XFM) coupled with X-ray absorption near-edge structure (XANES) imaging was used to study selenium (Se) biodistribution and speciation in Limnodynastes peronii tadpoles. Tadpoles were exposed to dissolved Se (30 µg/L) as selenite (SeIV) or selenate (SeVI) for 7 days followed by 3 days of depuration. High-resolution elemental maps revealed that Se partitioned primarily in the eyes (specifically the eye lens, iris, and retinal pigmented epithelium), digestive and excretory organs of SeIV-exposed tadpoles. Speciation analysis confirmed that the majority of accumulated Se was converted to organo-Se. Multielement analyses provided new information on Se colocalization and its impact on trace element homeostasis. New insights into the fate of Se on a whole organism scale contribute to our understanding of the mechanisms and risks associated with Se pollution.

Immunological Aspects of Chytridiomycosis.

Amphibians are currently the most threatened vertebrate class, with the disease chytridiomycosis being a major contributor to their global declines. Chytridiomycosis is a frequently fatal skin disease caused by the fungal pathogens Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal). The severity and extent of the impact of the infection caused by these pathogens across modern Amphibia are unprecedented in the history of vertebrate infectious diseases. The immune system of amphibians is thought to be largely similar to that of other jawed vertebrates, such as mammals. However, amphibian hosts are both ectothermic and water-dependent, which are characteristics favouring fungal proliferation. Although amphibians possess robust constitutive host defences, Bd/Bsal replicate within host cells once these defences have been breached. Intracellular fungal localisation may contribute to evasion of the induced innate immune response. Increasing evidence suggests that once the innate defences are surpassed, fungal virulence factors suppress the targeted adaptive immune responses whilst promoting an ineffectual inflammatory cascade, resulting in immunopathology and systemic metabolic disruption. Thus, although infections are contained within the integument, crucial homeostatic processes become compromised, leading to mortality. In this paper, we present an integrated synthesis of amphibian post-metamorphic immunological responses and the corresponding outcomes of infection with Bd, focusing on recent developments within the field and highlighting future directions.

Physiological stress response of the scleractinian coral Stylophora pistillata exposed to polyethylene microplastics.

We investigated physiological responses including calcification, photosynthesis and alterations to polar metabolites, in the scleractinian coral Stylophora pistillata exposed to different concentrations of polyethylene microplastics. Results showed that at high plastic concentrations (50 particles/mL nominal concentration) the photosynthetic efficiency of photosystem II in the coral symbiont was affected after 4 weeks of exposure. Both moderate and high (5 and 50 particles/mL nominal) concentrations of microplastics caused subtle but significant alterations to metabolite profiles of coral, as determined by Nuclear Magnetic Resonance (NMR) spectroscopy. Specifically, exposed corals were found to have increased levels of phosphorylated sugars and pyrimidine nucleobases that make up nucleotides, scyllo-inositol and a region containing overlapping proline and glutamate signals, compared to control animals. Together with the photo-physiological stress response observed and previously published literature, these findings support the hypothesis that microplastics disrupt host-symbiont signaling and that corals respond to this interference by increasing signaling and chemical support to the symbiotic zooxanthellae algae. These findings are also consistent with increased mucus production in corals exposed to microplastics described in previous studies. Considering the importance of coral reefs to marine ecosystems and their sensitivity to anthropogenic stressors, more research is needed to elucidate coral response mechanisms to microplastics under realistic exposure conditions.

Compensatory indirect effects of an herbicide on wetland communities.

The direct effects of large-scale disturbances are readily studied because their effects are often apparent and result in large changes to ecosystems. Direct effects can cascade through the ecosystem, leading to indirect effects that are often subtle and difficult to detect. Managing anthropogenic disturbances, such as chemical contamination, requires an understanding of both direct and indirect effects to predict, measure, and characterize the impact. Using a replicated whole-ecosystem experiment and path analyses (assesses the effects of a set of variables on a specified outcome, similar to multiple regression), we examined the direct and indirect effects of a glyphosate-based herbicide and nutrient enrichment on wetland communities. The latter did not impact any measured endpoints. The strongest drivers of macrophyte, benthic invertebrate, and amphibian assemblages were the ephemerality and the size of wetlands, factors which were not altered by herbicide applications. The herbicide had a direct negative effect on macrophyte cover, amphibian larval abundance, and the proportion of predatory benthic invertebrates. However, both amphibians and invertebrates were positively affected by the reduction in the macrophyte cover caused by the herbicide applications. The opposing directions of the direct and indirect effects lead to no net change in either group. The compensatory dynamics observed herein highlight the need for a better understanding of indirect effects pathways to determine whether common anthropogenic disturbances alter the ecological communities in small wetland ecosystems.

Contaminant-induced behavioural changes in amphibians: A meta-analysis.

Environmental contamination contributes to the threatened status of many amphibian populations. Many contaminants alter behaviour at concentrations commonly experienced in the environment, with negative consequences for individual fitness, populations and communities. A comprehensive, quantitative evaluation of the behavioural sensitivity of amphibians is warranted to better understand the population-level and resultant ecological impacts of contaminants. We conducted a systematic review and meta-analysis evaluating behavioural changes following exposure to contaminants. Most studies were conducted in North America and Europe on larval stages, and 64% of the 116 studies focussed on the effects of insecticides. We found that a suite of contaminants influence a wide range of behaviours in amphibians, with insecticides typically invoking the strongest responses. In particular, insecticides increased rates of abnormal swimming, and reduced escape responses to simulated predator attacks. Our analysis identified five key needs for future research, in particular the need: (1) for researchers to provide more details of experimental protocols and results (2) to develop a strong research base for future quantitative reviews, (3) to broaden the suite of contaminants tested, (4) to better study and thus understand the effects of multiple stressors, and (5) to establish the ecological importance of behavioural alterations. Behavioural endpoints provide useful sub-lethal indicators of how contaminants influence amphibians, and coupled with standard ecotoxicological endpoints, can provide valuable information for population models assessing the broader ecological consequences of environmental contamination.

NMR-based lipidomics of fish from a metal(loid) contaminated wetland show differences consistent with effects on cellular membranes and energy storage.

Metals and metalloids are priority contaminants due to their non-degradable and bioaccumulative nature, and their ability to regulate and perturb diverse physiological processes in various species. Metal(loid)s are known to cause oxidative stress through production of reactive oxygen species (ROS), thus related endpoints like lipid peroxidation (LPO) have received considerable attention as biomarkers of exposure. However, the implications of metal(loid) toxicity including LPO on actual lipid profiles of species inhabiting contaminated systems are poorly understood. Here we applied Nuclear Magnetic Resonance (NMR) spectroscopy for untargeted lipidomics of mosquitofish (Gambusia holbrooki) collected from reference and metal(loid)-contaminated wetlands. We measured a range of trace elements in water and fish using inductively coupled plasma - mass spectrometry (ICP-MS), and interpreted site differences in the lipid profiles of mosquitofish in the context of known physiological responses to sub-lethal metal(loid) exposure. Results indicate deregulation of cellular membrane lipids (i.e., glycerophospholipids, cholesterol and sphingolipids) and increased energy storage molecules (i.e., triacylglycerols and fatty acids) in fish from the contaminated wetland. These responses are consistent with the recognised induction of oxidative stress pathways in organisms exposed to metal(loid)s and could also be symptomatic of mitochondrial dysfunction and endocrine disruption. It is difficult to attribute metal(loid)s as the sole factor causing differences between wetlands, and a more controlled experimental approach is therefore warranted to further explore mechanistic pathways. Nevertheless, our study highlights the benefits of untargeted 1H NMR-based lipidomics as a relatively fast and simple approach for field-scale assessment and monitoring of organisms inhabiting metal(loid) contaminated environments.

Untargeted NMR-based metabolomics for field-scale monitoring: Temporal reproducibility and biomarker discovery in mosquitofish (Gambusia holbrooki) from a metal(loid)-contaminated wetland.

There is considerable interest in applying omics techniques, which have proven extremely valuable for laboratory-based toxicology studies, towards field-scale ecotoxicology and environmental monitoring. Concerns that confounding factors in natural ecosystems may exacerbate variability in omics datasets must be addressed to validate the transition from laboratory to field. This study explores how temporal variability related to seasonal and climatic trends influence qualitative and quantitative metabolomics outcomes, in fish from reference and metal(loid)-polluted wetlands in Australia. Female mosquitofish (Gambusia holbrooki) were sampled on two separate occasions, from a rehabilitated tailings wetland at the site of historic antimony (Sb) processing and a reference wetland with comparable water quality. The first sampling coincided with greater monthly rainfall and colder water temperature, whereas the second sampling was drier and water was warmer. Despite temporal changes and associated differences in metal(loid) concentrations, site differences in metabolite profiles were qualitatively very similar between sampling events. However, quantitative differences were observed, with a greater number of significantly altered metabolites identified during the second sampling event, which coincided with greater metal(loid) concentrations in both water and fish. The majority of identified metabolites were elevated in fish from the contaminated wetland, but with notable decreases in several metabolites that are known to play a role in various aspects of metal(loid) binding, detoxification and excretion. Specifically, decreased aspartate, histidine, myo-inositol, taurine and choline were observed in fish from the contaminated wetland, and may therefore represent a metabolite suite that is broadly indicative of metal toxicity. Quantitative differences between sampling events are suggestive of a dose-response relationship observable at the cellular level which, if harnessed, may be useful for assigning levels of concern based on the degree of change in a multi-parameter set of metabolite biomarkers.

Application of nuclear techniques to environmental plastics research.

Plastic pollution is ubiquitous in aquatic environments and its potential impacts to wildlife and humans present a growing global concern. Despite recent efforts in understanding environmental impacts associated with plastic pollution, considerable uncertainties still exist regarding the true risks of nano- and micro-sized plastics (<5 mm). The challenges faced in this field largely relate to the methodological and analytical limitations associated with studying plastic debris at low (environmentally relevant) concentrations. The present paper highlights how radiotracing techniques that are commonly applied to trace the fate and behaviour of chemicals and particles in various systems, can contribute towards addressing several important and outstanding questions in environmental plastic pollution research. Specifically, we discuss the use of radiolabeled microplastics and/or chemicals for 1) determining sorption/desorption kinetics of a range of contaminants to different types of plastics under varying conditions, 2) understanding the influence of microplastics on contaminant and nutrient bioaccumulation in aquatic organisms, and 3) assessing biokinetics, biodistribution, trophic transfer and potential biological impacts of microplastic at realistic concentrations. Radiotracer techniques are uniquely suited for this research because of their sensitivity, accuracy and capacity to measure relevant parameters over time. Obtaining precise and timely information on the fate of plastic particles and co-contaminants in wildlife has widespread applications towards effective monitoring programmes and environmental management strategies.

Uptake and tissue distributions of cadmium, selenium and zinc in striped marsh frog tadpoles exposed during early post-embryonic development.

Metals and metalloids released through anthropogenic activities can accumulate in aquatic organisms, resulting in adverse effects in sensitive species. We investigated the influence of feeding regime and exposure complexity (i.e., mixture) on bioaccumulation kinetics and body distribution of common metal(loid) pollutants in Limnodynastes peronii during early post-embryonic development. Tadpoles were exposed to radiolabelled 109Cd, 75Se and 65Zn alone and in a mixture for 4 days, followed by 3 days depuration in clean water. One group was fed directly in exposure aquaria, whereas a second group was transferred to clean water for feeding, to investigate the potential influence of sorption to food on uptake. Bioconcentration factor and retention was observed to be greatest for Se. Results demonstrate that tadpoles accumulated and retained half the amount of Cd when exposed in mixture, suggesting that Se and/or Zn may have antagonistic effects against Cd uptake. Additionally, tadpoles fed directly in exposure water accumulated 2-3-times more Cd and Zn compared to tadpoles fed in clean water, indicating that the presence of food particles is an important factor that may influence uptake. Interestingly, this had a negligible impact on Se uptake. The study reveals how exposure conditions can influence the bioaccumulation of metal(loid)s, highlighting experimental factors as important considerations for both controlled toxicity experiments and for understanding exposure risks for amphibian populations.

Bioaccumulation and Biodistribution of Selenium in Metamorphosing Tadpoles.

Selenium is an important macronutrient with a very narrow margin between essentiality and toxicity. Amphibians are hypothesized to be particularly sensitive due to the potential for metamorphosis-driven mobilization, which could transfer or concentrate contaminant burdens within specific organs. We explored the potential role of tissue degeneration and remodeling during anuran metamorphosis as a mechanism for altering tissue-specific Se burdens. Limnodynastes peronii tadpoles were exposed to dissolved 75Se (as selenite) for 7 days and depurated until completion of metamorphosis. Bioaccumulation and retention kinetics were assessed in whole tadpoles and excised tissues using gamma spectroscopy, and temporal changes in biodistribution were assessed using autoradiography. Tadpoles retained Se throughout metamorphosis, and partitioned the element predominantly within digestive and excretory tissues, including livers > mesonephros > guts > gallbladder. Importantly, our results demonstrate that Se biodistribution varies significantly throughout development. This is indicative of tissue transference, and particularly in tissues developing de novo after depuration. To the best of our knowledge, this is the first study demonstrating Se transference during metamorphic tissue remodelling. Further research is warranted to explore the fate and metabolism of Se (and other metal and metalloids) during anuran development and the implications of transference for influencing toxicity.

Altered bioenergetics and developmental effects in striped marsh frog (Limnodynastes peronii) tadpoles exposed to UV treated sewage.

Effectively treating domestic wastewater so that it can be safely discharged or reused is critical for maintaining the integrity of freshwater resources, and for protecting the health of animals that rely on these systems. Amphibians are currently facing widespread population declines, so there is a particularly urgent need to investigate exposure scenarios that might result in weakened amphibian populations. Domestic sewage has received little attention as a possible factor that could influence the survival, growth and development, or general health of amphibians. However, wastewater reuse for crop irrigation and other purposes is increasing and holding ponds and constructed wetlands exist at many wastewater treatment facilities, introducing conceivable pathways that could result in the exposure of amphibians to treated wastewater. We exposed developing striped marsh frog (Limnodynastes peronii) tadpoles, to control water and 12.5, 25, 50 and 100% UV treated domestic sewage, and quantified effects on growth and development, hepatic energy reserves, and enzymatic pathways associated with detoxification and oxidative stress. Growth and development were accelerated and relative liver size was increased in exposed animals. The exposure resulted in an apparently hormetic increase in hepatic triglycerides and dose-dependent reduction in glycogen stores, as well as increased lipase and NADPH activity, indicating a general disruption to energy metabolism and/or mobilization. Contrary to expectations based on published studies with fish, we found no evidence of lipid peroxidation or induction of the detoxification enzyme Superoxide Dismutase (SOD), however, this may reflect the use of UV treatment as opposed to chlorination for disinfection. Chemical analysis and risk-based prioritization consistently identified fluoxetine, triclosan and diazinon as high-risk contaminants in the wastewater, with nonylphenol and mestranol flagged as risks during one early collection. Research is needed to explore the potential for these specific contaminants to elicit the responses identified in the present study, and to perform similar assessments using wastewater from other locations with different treatment options.

Individual and mixture toxicity of pharmaceuticals naproxen, carbamazepine, and sulfamethoxazole to Australian striped marsh frog tadpoles (Limnodynastes peronii).

Nonsteroidal human pharmaceuticals are prevalent in domestic wastewater and may find their way into the environment at low concentrations. Since most pharmaceuticals are designed to be biologically active at low concentrations, there is a risk that these compounds may affect aquatic wildlife. Of particular concern is the occurrence of pharmaceutical mixtures, which may lead to increased adverse effects compared to individual compounds. Interactive effects were previously demonstrated for amphibians exposed to pesticide mixtures, but no such studies investigating responses of amphibians to pharmaceutical mixtures are apparently available. Results demonstrated increased toxicity (loss of tactile response) of striped marsh frog (Limnodynastes peronii) tadpoles exposed to a mixture of naproxen, carbamazepine, and sulfamethoxazole, compared to exposures to the individual compounds. Significant time × treatment interactions were observed for tadpole development following chronic exposures to 10 or 100 µg/L of each compound and the mixture; however, responses were weak and main treatment effects were not significant. Despite minor effects at low exposure concentrations, results demonstrated a potential for mixtures of nonsteroidal pharmaceuticals commonly occurring in wastewater to influence amphibian development. With the vast numbers of pharmaceuticals that exist and are found in the environment, this work highlights a need for further research into mixtures of pharmaceutically active wastewater contaminants. Further, since pharmaceuticals exert extremely varied biological actions, it is suggested that future investigations would benefit from inclusion of endpoints that are indicative of physiological or metabolic performance, as well as assessment of sensitive behavioral responses.

Effects of naphthenic acid exposure on development and liver metabolic processes in anuran tadpoles.

Naphthenic acids (NA) are used in a variety of commercial and industrial applications, and are primary toxic components of oil sands wastewater. We investigated developmental and metabolic responses of tadpoles exposed to sub-lethal concentrations of a commercial NA blend throughout development. We exposed Lithobates pipiens tadpoles to 1 and 2 mg/L NA for 75 days and monitored growth and development, condition factor, gonad and liver sizes, and levels of liver glucose, glycogen, lipids and cholesterol following exposure. NA decreased growth and development, significantly reduced glycogen stores and increased triglycerides, indicating disruption to processes associated with energy metabolism and hepatic glycolysis. Effects on liver function may explain reduced growth and delayed development observed in this and previous studies. Our data highlight the need for greater understanding of the mechanisms leading to hepatotoxicity in NA-exposed organisms, and indicate that strict guidelines may be needed for the release of NA into aquatic environments.