Winter break? The effect of overwintering on immune gene expression in wood frogs.

Among terrestrial ectotherms, hibernation is a common response to extreme cold temperatures and is associated with reduced physiological rates, including immunity. When winter wanes and temperatures increase, so too do vital rates of both ectothermic hosts and their parasites. Due to metabolic scaling, if parasite activity springs back faster than host immune functions then cold seasons and transitions between cold and warm seasons may represent periods of vulnerability for ectothermic hosts. Understanding host regulation of physiological rates at seasonal junctions is a first step toward identifying thermal mismatches between hosts and parasites. Here we show that immune gene expression is responsive to transitions into and out of the cold season in a winter-adapted amphibian, the wood frog (Lithobates sylvaticus), and that frogs experienced parasitism by at least two nematode species throughout the entirety of the cold season. In both splenic and skin tissues, we observed a decrease in immune gene expression going from fall to winter, observed no changes between winter and emergence from hibernation, and observed increases in immune gene expression after hibernation ended. At all timepoints, differentially expressed genes from spleens were more highly enriched for immune system processes than those from ventral skin, especially with respect to terms related to adaptive immune processes. Infection with nematode lungworms was also associated with upregulation of immune processes in the spleen. We suggest that rather than being a period of stagnation, during which physiological processes and infection potential cease, the cold season is immunologically dynamic, requiring coordinated regulation of many biological processes, and that the reemergence period may be an important time during which hosts invest in preparatory immunity.

Integrated biomarker-based ecological risks assessment of tadpole responses to tris(2-chloroethyl) phosphate, tris(1-chloro-2-propyl) phosphate, and their combined environmental exposure.

Tris(2-chloroethyl) phosphate (TCEP) and tris(1-chloro-2-propyl) phosphate (TCPP) are common chlorinated organophosphorus flame retardants (OPFRs) used in industry. They have been frequently detected together in aquatic environments and associated with various hazardous effects. However, the ecological risks of prolonged exposure to these OPFRs at environmentally relevant concentrations in non-model aquatic organisms remain unexplored. This study investigated the effects of long-term exposure (up to 25 days) to TCEP and TCPP on metamorphosis, hepatic antioxidants, and endocrine function in Polypedates megacephalus tadpoles. Exposure concentrations were set at 3, 30, and 90 µg/L for each substance, conducted independently and in equal-concentration combinations, with a control group included for comparison. The integrated biomarker response (IBR) method developed an optimal linear model for predicting the overall ecological risks of TCEP and TCPP to tadpoles in potential distribution areas of Polypedates species. Results showed that: (1) Exposure to environmentally relevant concentrations of TCEP and TCPP elicited variable adverse effects on tadpole metamorphosis time, hepatic antioxidant enzyme activity and related gene expression, and endocrine-related gene expression, with their combined exposure exacerbating these effects. (2) The IBR value of TCEP was consistently greater than that of TCPP at each concentration, with an additive effect observed under their combined exposure. (3) The ecological risk of tadpoles exposed to the combined presence of TCEP and TCPP was highest in China's Taihu Lake and Vietnam's Hanoi than in other distribution locations. In summary, prolonged exposure to environmentally relevant concentrations of TCEP and TCPP presents potential ecological risks to amphibian tadpoles, offering insights for the development of policies and strategies to control TCEP and TCPP pollution in aquatic ecosystems. Furthermore, the methodology employed in establishing the IBR prediction model provides a methodological framework for assessing the overall ecological risks of multiple OPFRs.

Longer days, larger grays: carryover effects of photoperiod and temperature in gray treefrogs, Hyla versicolor.

Environmental conditions like temperature and photoperiod can strongly shape organisms' growth and development. For many ectotherms with complex life cycles, global change will cause their offspring to experience warmer conditions and earlier-season photoperiods, two variables that can induce conflicting responses. We experimentally manipulated photoperiod and temperature during gray treefrog (Hyla versicolor) larval development to examine effects at metamorphosis and during short (10-day) and long (56-day) periods post-metamorphosis. Both early- and late-season photoperiods (April and August) decreased age and size at metamorphosis relative to the average-season (June) photoperiod, while warmer temperatures decreased age but increased size at metamorphosis. Warmer larval temperatures reduced short-term juvenile growth but had no long-term effect. Conversely, photoperiod had no short-term carryover effect, but juveniles from early- and late-season larval photoperiods had lower long-term growth rates than juveniles from the average-season photoperiod. Similar responses to early- and late-season photoperiods may be due to reduced total daylight compared with average-season photoperiods. However, juveniles from late-season photoperiods selected cooler temperatures than early-season juveniles, suggesting that not all effects of photoperiod were due to total light exposure. Our results indicate that despite both temperature and photoperiod affecting metamorphosis, the long-term effects of photoperiod may be much stronger than those of temperature.

Voriconazole successfully treats chytridiomycosis in frogs.

Chytridiomycosis is a devastating disease and is a key cause of amphibian population declines around the world. Despite active research on this amphibian disease system for over 2 decades, we still do not have treatment methods that are safe and that can be broadly used across species. Here, we show evidence that voriconazole is a successful method of treatment for 1 species of amphibian in captivity and that this treatment could offer benefits over other treatment options like heat or itraconazole, which are not able to be used for all species and life stages. We conducted 2 treatments of chytridiomycosis using voriconazole. The treatment was effective and resulted in 100% pathogen clearance, and mortality ceased. Additionally, treating frogs with voriconazole requires less handling than treatment methods like itraconazole and requires no specialized equipment, like heat treatment. We highlight that clinical treatment trials should be conducted to identify an optimum dosage and treatment time and that trials should test whether this treatment is safe and effective for tadpoles and other species.

Combined effect and mechanism of microplastic with different particle sizes and levofloxacin on developing Rana nigromaculata: Insights from thyroid axis regulation and immune system.

Microplastics (MPs) usually appear in the aquatic environment as complex pollutants with other environmental pollutants, such as levofloxacin (LVFX). After 45-day exposure to LVFX and MPs with different particle sizes at environmental levels, we measured the weight, snout-to-vent length (SVL), and development stages of Rana nigromaculata. Furthermore, we analyzed proteins and genes related to immune system and thyroid axis regulation, intestinal histological, and bioaccumulation of LVFX and MPs in the intestine and brain to further explore the toxic mechanism of co-exposure. We found MPs exacerbated the effect of LVFX on growth and development, and the order of inhibitory effects is as follows: LVFX-MP3>LVFX-MP1>LVFX-MP2. 0.1 and 1 µm MP could penetrate the blood-brain barrier, interact with LVFX in the brain, and affect growth and development by regulating thyroid axis. Besides, LVFX with MPs caused severer interference on thyroid axis compared with LVFX alone. However, 10 µm MP was prone to accumulating in the intestine, causing severe histopathological changes, interfering with the intestinal immune system and influencing growth and development through immune enzyme activity. Thus, we concluded that MPs could regulate the thyroid axis by interfering with the intestinal immune system.

Neural and sensory basis of homing behavior in the invasive cane toad, Rhinella marina.

The behavioral, sensory, and neural bases of vertebrate navigation are primarily described in mammals and birds. However, we know much less about navigational abilities and mechanisms of vertebrates that move on smaller scales, such as amphibians. To address this knowledge gap, we conducted an extensive field study on navigation in the cane toad, Rhinella marina. First, we performed a translocation experiment to describe how invasive toads in Hawai'i navigate home following displacements of up to one kilometer. Next, we tested the effect of olfactory and magnetosensory manipulations on homing, as these senses are most commonly associated with amphibian navigation. We found that neither ablation alone prevents homing, suggesting that toad navigation is multimodal. Finally, we tested the hypothesis that the medial pallium, the amphibian homolog to the hippocampus, is involved in homing. By comparing neural activity across homing and non-homing toads, we found evidence supporting the involvement of the medial pallium, lateral pallium, and septum in navigation, suggesting a conservation of neural structures supporting navigation across vertebrates. Our study lays the foundation to understand the behavioral, sensory, and neural bases of navigation in amphibians and to further characterize the evolution of behavior and neural structures in vertebrates.

Sexy fingers: Pheromones in the glands of male dendrobatid frogs.

Many animals exchange chemicals during courtship and mating. In some amphibians, sexual chemical communication is mediated by pheromones produced in male breeding glands that are transferred to the female's nostrils during mating. This has been mostly studied in salamanders, despite frogs having similar glands and courtship behaviours suggestive of chemical communication. In Neotropical poison frogs (Dendrobatidae and Aromobatidae), males of many species develop breeding glands in their fingers, causing certain fingers to visibly swell. Many also engage in cephalic amplexus, whereby the male's swollen fingers are placed in close contact with the female's nares during courtship. Here, we investigate the possible roles of swollen fingers in pheromone production using whole-transcriptome sequencing (RNAseq). We examined differential gene expression in the swollen versus non-swollen fingers and toes of two dendrobatid species, Leucostethus brachistriatus and Epipedobates anthonyi, both of which have specialised mucous glands in finger IV, the latter of which has cephalic amplexus. The overwhelming pattern of gene expression in both species was strong upregulation of sodefrin precursor-like factors (SPFs) in swollen fingers, a well-known pheromone system in salamanders. The differentially expressed SPF transcripts in each species were very high (>40), suggesting a high abundance of putative protein pheromones in both species. Overall, the high expression of SPFs in the swollen fingers in both species, combined with cephalic amplexus, supports the hypothesis that these traits, widespread across members of the subfamilies Colostethinae and Hyloxalinae (ca. 141 species), are involved in chemical signalling during courtship.

Muchos animales intercambian sustancias químicas durante el cortejo y el apareamiento. En algunos anfibios, la comunicación química sexual está mediada por feromonas producidas en las glándulas reproductoras de los machos que se transfieren a las hembras durante el apareamiento. Esto se ha estudiado sobre todo en salamandras, a pesar de que las ranas tienen glándulas similares y comportamientos de cortejo que sugieren una comunicación química. En las ranas venenosas neotropicales (Dendrobatidae y Aromobatidae), los machos de muchas especies desarrollan glándulas en los dedos, lo que hace que algunos dedos se vean hinchados. Asimismo, varias especies presentan amplexo cefálico, comportamiento de cortejo en el cual los dedos hinchados entran en estrecho contacto con las narinas y boca de la hembra. En este estudio investigamos las posibles funciones de los dedos hinchados en la producción de feromonas mediante la secuenciación del transcriptoma completo (RNAseq). Examinamos la expresión génica diferencial en los dedos hinchados y no hinchados de dos especies de dendrobátidos, Leucostethus brachistriatus y Epipedobates anthonyi, ambos con glándulas mucosas especializadas en el dedo IV, y esta última especie, con amplexo cefálico. El patrón abrumador de expresión génica en ambas especies fue la alta expression de Sodefrin Precursor­Like Factor (SPF) en los dedos hinchados, un sistema de feromonas ampliamente conocido en las salamandras. El número de transcritos SPF expresados diferencialmente en cada especie fue muy elevado (>40), lo que sugiere una gran abundancia de feromonas proteicas putativas en ambas especies. En general, la elevada expresión de SPF en los dedos hinchados en ambas especies, combinada con el amplexo cefálico, apoya la hipótesis de que estos rasgos, muy extendidos entre los miembros de las subfamilias Colostethinae e Hyloxalinae (aprox 141 especies), están implicados en la señalización química durante el cortejo.

Selected Wildlife Trematodes.

The trematodes are a species-rich group of parasites, with some estimates suggesting that there are more than 24,000 species. However, the complexities associated with their taxonomic status and nomenclature can hinder explorations of the biology of wildlife trematodes, including fundamental aspects such as host use, life cycle variation, pathology, and disease. In this chapter, we review work on selected trematodes of amphibians, birds, mammals, and their snail intermediate hosts, with the goal of providing a tool kit on how to study trematodes of wildlife. We provide a brief introduction to each group of wildlife trematodes, followed by some examples of the challenges each group of trematodes has relative to the goal of their identification and understanding of the biology and interactions these organisms have with their wildlife hosts.

Dynamics of Amphibian Pathogen Detection Using Extended Museum Specimens.

Natural history collections have long served as the foundation for understanding our planet's biodiversity, yet they remain a largely untapped resource for wildlife disease studies. Extended specimens include multiple data types and specimen preparations that capture the phenotype and genotype of an organism and its symbionts-but preserved tissues may not always be optimized for downstream detection of various pathogens. Frogs are infected by an array of pathogens including Batrachochytrium dendrobatidis (Bd), Ranavirus (Rv), and Amphibian Perkinsea (Pr), which provides the opportunity to study differences in detection dynamics across tissue types. We used quantitative PCR protocols to screen two tissue types commonly deposited in museum collections, toe clips and liver, from two closely related host species, Rana catesbeiana and Rana clamitans. We compared Bd, Rv, and Pr infection prevalence and intensity between species and tissue types and found no significant difference in prevalence between species, but Bd intensity was higher in R. clamitans than R. catesbeiana. Toe tissue exhibited significantly higher Bd infection loads and was more useful than liver for detecting Bd infections. In contrast, Rv was detected from more liver than toe tissues, but the difference was not statistically significant. Our results support the use of extended specimen collections in amphibian disease studies and demonstrate that broader tissue sampling at the time of specimen preparation can maximize their utility for downstream multipathogen detection.

Baseline and stress-induced changes in plasma bacterial killing ability against gram-negative bacteria are partially mediated by the complement system in Rhinella diptycha toads.

The plasma bacterial killing ability (BKA) is modulated by the stress response in vertebrates, including amphibians. The complement system is an effector mechanism comprised of a set of proteins present in the plasma that once activated can promote bacterial lysis. Herein, we investigated whether changes in plasma BKA as a result of the acute stress response and an immune challenge are mediated by the complement system in Rhinella diptycha toads. Additionally, we investigated whether the observed changes in plasma BKA are associated with changes in plasma corticosterone levels (CORT). We subjected adult male toads to a restraint or an immune challenge (with three concentrations of Aeromonas hydrophila heat inactivated), and then evaluated the plasma BKA against A. hydrophila, in vitro. We determined the complement system activity on plasma BKA, by treating the plasma (baseline, 1 h and 24 h post-restraint, and after the immune challenge) with ethylenediaminetetraacetic acid, heat, or protease. Our results showed increased CORT 1 h and 24 h after restraint and decreased plasma BKA 24 h post-restraint. The inhibitors of the complement system decreased the plasma BKA compared with untreated plasma at all times (baseline, 1 h, and 24 h after restraint), demonstrating that the plasma BKA activity is partially mediated by the complement system. The immune challenge increased CORT, with the highest values being observed in the highest bacterial concentration, compared with control. The plasma BKA was not affected by the immune challenge but was demonstrated to be partially mediated by the complement system. Our results demonstrated that restraint and the immune challenge activated the hypothalamus-pituitary-interrenal axis, by increasing plasma CORT levels in R. diptycha. Also, our results demonstrated the complement system is participative in the plasma BKA for baseline and post-stress situations in these toads.

Exploring the effects of environmentally relevant concentrations of tris(2-chloroethyl) phosphate on tadpole health: A comprehensive analysis of intestinal microbiota and hepatic transcriptome.

Tris(2-chloroethyl) phosphate (TCEP), a chlorinated organophosphate ester, is commonly found in aquatic environments. Due to its various toxic effects, it may pose a risk to the health of aquatic organisms. However, the potential impacts of TCEP exposure on the intestinal microbiota and hepatic function in amphibians have not been reported. This study investigated the impact of long-term exposure to environmentally relevant concentrations of TCEP (0, 3, and 90 µg/L) on the intestinal microbiota and hepatic transcriptome of Polypedates megacephalus tadpoles. The results showed that the body size of the tadpoles decreased significantly with an increase in TCEP concentration. Additionally, TCEP exposure affected the diversity and composition of the intestinal microbiota in tadpoles, leading to significant changes in the relative abundance of certain bacterial groups (the genera Aeromonas decreased and Citrobacter increased) and potentially promoting a more even distribution of microbial species, as indicated by a significant increase in the Simpson index. Moreover, the impact of TCEP on hepatic gene expression profiles in tadpoles was significant, with the majority of differentially expressed genes (DEGs) (709 out of 906 total DEGs in 3 µg/L of TCEP versus control, and 344 out of 387 DEGs in 90 µg/L of TCEP versus control) being significantly down-regulated, which were primarily related to immune response and immune system process. Notably, exposure to TCEP significantly reduced the relative abundance of the genera Aeromonas and Cetobacterium in the tadpole intestine. This reduction was positively correlated with the down-regulated expression of immune-related genes in the liver of corresponding tadpoles. In summary, these findings provide empirical evidence of the potential health risks to tadpoles exposed to TCEP at environmentally relevant concentrations.

Amphibian mast cells serve as barriers to chytrid fungus infections.

Global amphibian declines are compounded by deadly disease outbreaks caused by the chytrid fungus, Batrachochytrium dendrobatidis (Bd). Much has been learned about the roles of amphibian skin-produced antimicrobial components and microbiomes in controlling Bd, yet almost nothing is known about the roles of skin-resident immune cells in anti-Bd defenses. Mammalian mast cells reside within and serve as key immune sentinels in barrier tissues like skin. Accordingly, we investigated the roles of Xenopus laevis frog mast cells during Bd infections. Our findings indicate that enrichment of X. laevis skin mast cells confers anti-Bd protection and ameliorates the inflammation-associated skin damage caused by Bd infection. This includes a significant reduction in infiltration of Bd-infected skin by neutrophils, promoting mucin content within cutaneous mucus glands, and preventing Bd-mediated changes to skin microbiomes. Mammalian mast cells are known for their production of the pleiotropic interleukin-4 (IL4) cytokine and our findings suggest that the X. laevis IL4 plays a key role in manifesting the effects seen following cutaneous mast cell enrichment. Together, this work underscores the importance of amphibian skin-resident immune cells in anti-Bd defenses and illuminates a novel avenue for investigating amphibian host-chytrid pathogen interactions.

Host Species and Environment Shape the Skin Microbiota of Mexican Axolotls.

Skin microbiomes in amphibians are complex systems that can be influenced by biotic and abiotic factors. In this study, we examined the effect of host species and environmental conditions on the skin bacterial and fungal microbiota of four obligate paedomorphic salamander species, commonly known as axolotls (Ambystoma andersoni, A. dumerilii, A. mexicanum, and A. taylori), all of them endemic to the Trans-Mexican Volcanic Belt. We found that despite their permanent aquatic lifestyle, these species present a host-specific skin microbiota that is distinct from aquatic communities. We identified skin-associated taxa that were unique to each host species and that differentiated axolotl species based on alpha and beta diversity metrics. Moreover, we identified a set of microbial taxa that were shared across hosts with high relative abundances across skin samples. Specifically, bacterial communities were dominated by Burkholderiales and Pseudomonadales bacterial orders and Capnodiales and Pleosporales fungal orders. Host species and environmental variables collectively explained more microbial composition variation in bacteria (R2 = 0.46) in comparison to fungi (R2 = 0.2). Our results contribute to a better understanding of the factors shaping the diversity and composition of skin microbial communities in Ambystoma. Additional studies are needed to disentangle the effects of specific host associated and environmental factors that could influence the skin microbiome of these endangered species.

Effects of amphibian genetic diversity on ecological communities.

The amount of genetic diversity within a population can affect ecological processes at population, community, and ecosystem levels. However, the magnitude, consistency, and scope of these effects are largely unknown. To investigate these issues, we conducted two experiments manipulating the amount of genetic diversity and environmental factors in larval amphibians. The first experiment manipulated wood frog genetic diversity, the presence or absence of caged predators, and competition from leopard frogs to test whether these factors affected survival, growth, and morphology of wood frogs and leopard frogs. The second experiment manipulated wood frog genetic diversity, the presence or absence of uncaged predators, and resource abundance to test whether these factors affected wood frog traits (survival, morphology, growth, development, and behavior) and other components of the ecological community (zooplankton abundance, phytoplankton, periphyton, and bacterial community structure). Genetic diversity did not affect wood frog survival, growth, and development in either experiment. However, genetic diversity did affect the mean morphology of wood frog tadpoles in the first experiment and the abundance and distribution of zooplankton in the second experiment. It did not affect phytoplankton abundance, periphyton abundance, or bacterial community structure. While effect sizes (Cohen's d) of genetic diversity were approximately half those of environment treatments, the greatest effect sizes were for interaction effects between genetic diversity and environment. Our results indicate that genetic diversity can have a large effect on ecological processes, but the direction of those effects is highly dependent upon environmental conditions, and not easily predicted from simple measures of traits.

Archival mitogenomes identify invasion by the Batrachochytrium dendrobatidis CAPE lineage caused an African amphibian extinction in the wild.

Outbreaks of emerging infectious diseases are influenced by local biotic and abiotic factors, with host declines occurring when conditions favour the pathogen. Deterioration in the population of the micro-endemic Tanzanian Kihansi spray toad (Nectophrynoides asperginis) occurred after the construction of a hydropower dam, implicating habitat modification in this species decline. Population recovery followed habitat augmentation; however, a subsequent outbreak of chytridiomycosis caused by Batrachochytrium dendrobatidis (Bd) led to the spray toad's extinction in the wild. We show using spatiotemporal surveillance and mitogenome assembly of Bd from archived toad mortalities that the outbreak was caused by invasion of the BdCAPE lineage and not the panzootic lineage BdGPL. Molecular dating reveals an emergence of BdCAPE across southern Africa overlapping with the timing of the spray toad's extinction. That our post-outbreak surveillance of co-occurring amphibian species in the Udzungwa Mountains shows widespread infection by BdCAPE yet no signs of ill-health or decline suggests these other species can tolerate Bd when environments are stable. We conclude that, despite transient success in mitigating the impact caused by dams' construction, invasion by BdCAPE caused the ultimate die-off that led to the extinction of the Kihansi spray toad.

Behavioral responses during sickness in amphibians and reptiles: Concepts, experimental design, and implications for field studies.

In ectothermic vertebrates, behavioral fever, where an individual actively seeks warmer areas, seems to be a primary response to pathogens. This is considered a broad and evolutionarily conserved response among vertebrates. Recent population declines in amphibians are associated with an increase of infectious disease driven largely by climate change, habitat degradation, and pollution. Immediate action through research is required to better understand and inform conservation efforts. The literature available, does not provide unifying concepts that can guide adequate experimental protocols and interpretation of data, especially when studying animals in the field. The aim of this review is to promote common understanding of terminology and facilitating improved comprehension and application of key concepts about the occurrence of both sickness behavior or behavioral fever in ectothermic vertebrates. We start with a conceptual synthesis of sickness behavior and behavioral fever, with examples in different taxa. Through this discussion we present possible paths to standardize terminology, starting from original use in endothermic tetrapods which was expanded to ectothermic vertebrates, particularly amphibians and reptiles. This conceptual expansion from humans (endothermic vertebrates) and then to ectothermic counterparts, gravitates around the concept of 'normality'. Thus, following this discussion, we highlight caveats with experimental protocols and state the need of a reference value considered normal (RVCN), which is different from experimental control and make recommendations regarding experimental procedures and stress the value of detailed documentation of behavioral responses. We also propose some future directions that could enhance interaction among disciplines, emphasizing relationships at different levels of biological organization. This is crucial given the increasing convergence of fields such as thermal physiology, immunology, and animal behavior due to emerging diseases and other global crises impacting biodiversity.

The genomic evolution of visual opsin genes in amphibians.

Among tetrapod (terrestrial) vertebrates, amphibians remain more closely tied to an amphibious lifestyle than amniotes, and their visual opsin genes may be adapted to this lifestyle. Previous studies have discussed physiological, morphological, and molecular changes in the evolution of amphibian vision. We predicted the locations of the visual opsin genes, their neighboring genes, and the tuning sites of the visual opsins, in 39 amphibian genomes. We found that all of the examined genomes lacked the Rh2 gene. The caecilian genomes have further lost the SWS1 and SWS2 genes; only the Rh1 and LWS genes were retained. The loss of the SWS1 and SWS2 genes in caecilians may be correlated with their cryptic lifestyles. The opsin gene syntenies were predicted to be highly similar to those of other bony vertebrates. Moreover, dual syntenies were identified in allotetraploid Xenopus laevis and X. borealis. Tuning site analysis showed that only some Caudata species might have UV vision. In addition, the S164A that occurred several times in LWS evolution might either functionally compensate for the Rh2 gene loss or fine-tuning visual adaptation. Our study provides the first genomic evidence for a caecilian LWS gene and a genomic viewpoint of visual opsin genes by reviewing the gains and losses of visual opsin genes, the rearrangement of syntenies, and the alteration of spectral tuning in the course of amphibians' evolution.

Impacts of Exposure to Ultraviolet Radiation and an Agricultural Pollutant on Morphology and Behavior of Tadpoles (Limnodynastes tasmaniensis).

Amphibians are the most threatened vertebrate class globally. Multiple factors have been implicated in their global decline, and it has been hypothesized that interactions between stressors may be a major cause. Increased ultraviolet (UV) radiation, as a result of ozone depletion, has been identified as one such stressor. Exposure to UV radiation has been shown to have detrimental effects on amphibians and can exacerbate the effects of other stressors, such as chemical pollutants. Chemical pollution has likewise been recognized as a major factor contributing to amphibian declines, particularly, endocrine-disrupting chemicals. In this regard, 17ß-trenbolone is a potent anabolic steroid used in the agricultural industry to increase muscle mass in cattle and has been repeatedly detected in the environment where amphibians live and breed. At high concentrations, 17ß-trenbolone has been shown to impact amphibian survival and gonadal development. In the present study, we investigated the effects of environmentally realistic UV radiation and 17ß-trenbolone exposure, both in isolation and in combination, on the morphology and behavior of tadpoles (Limnodynastes tasmaniensis). We found that neither stressor in isolation affected tadpoles, nor did we find any interactive effects. The results from our 17ß-trenbolone treatment are consistent with recent research suggesting that, at environmentally realistic concentrations, tadpoles may be less vulnerable to this pollutant compared to other vertebrate classes. The absence of UV radiation-induced effects found in the present study could be due to species-specific variation in susceptibility, as well as the dosage utilized. We suggest that future research should incorporate long-term studies with multiple stressors to accurately identify the threats to, and subsequent consequences for, amphibians under natural conditions. Environ Toxicol Chem 2024;43:1615-1626. © 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Toxicity comparison and risk assessment of two chlorinated organophosphate flame retardants (TCEP and TCPP) on Polypedates megacephalus tadpoles.

Tris(2-chloroethyl) phosphate (TCEP) and tris(1­chloro-2-propyl) phosphate (TCPP) are widely used as chlorinated organophosphate flame retardants (OPFRs) due to their fire-resistance capabilities. However, their extensive use has led to their permeation and pollution in aquatic environments. Using amphibians, which are non-model organisms, to test the toxic effects of OPFRs is relatively uncommon. This study examined the acute and chronic toxicity differences between TCEP and TCPP on Polypedates megacephalus tadpoles and evaluated the potential ecological risks to tadpoles in different aquatic environments using the risk quotient (RQ). In acute toxicity assay, the tadpole survival rates decreased with increased exposure time and concentrations, with TCEP exhibiting higher LC50 values than TCPP, at 305.5 mg/L and 70 mg/L, respectively. In the chronic assay, prolonged exposure to 300 µg/L of both substances resulted in similar adverse effects on tadpole growth, metamorphosis, and hepatic antioxidant function. Based on RQ values, most aquatic environments did not pose an ecological risk to tadpoles. However, the analysis showed that wastewater presented higher risks than rivers and drinking water, and TCPP posed a higher potential risk than TCEP in all examined aquatic environments. These findings provide empirical evidence to comprehend the toxicological effects of OPFRs on aquatic organisms and to assess the safety of aquatic environments.

Gut microbiota-bile acid crosstalk contributes to intestinal damage after nitrate exposure in Bufo gargarizans tadpoles.

Bile acids (BAs) are amphipathic steroid acids whose production and diversity depend on both host and microbial metabolism. Nitrate (NO3-) is a widespread pollutant in aquatic ecosystems, which can cause rapid changes in microbial community structure and function. However, the effect of gut microbiota reshaped by nitrate­nitrogen (NO3-N) on BAs profiles remains unclarified. To test this, intestinal targeted BAs metabolomics and fecal metagenomic sequencing were performed on Bufo gargarizans tadpoles treated with different concentrations of NO3-N. NO3-N exposure induced a reduction in the abundance of microbiota with bile acid-inducible enzymes (BAIs) and/or hydroxysteroid dehydrogenases (HSDHs), thus inhibiting the conversion of primary BAs to secondary BAs. Inhibition of BAs biotransformation decreased protective hydrophilic BAs (UDCA) and increased toxic hydrophobic BAs (CA and CDCA), which may contribute to intestinal histopathological damage. Moreover, we found that NO3-N treatment increased microbial virulence factors and decreased Glycoside hydrolases, further highlighting the deleterious risk of NO3-N. Overall, this study shed light on the complex interactions of NO3-N, gut microbiota, and BAs, and emphasized the hazardous effects of NO3-N pollution on the health of amphibians.