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The neuropeptide prothoracicotropic hormone (PTTH) plays a key role in regulating ecdysone synthesis and promoting insect metamorphosis. Pyriproxyfen is a juvenile hormone analogue. We previously reported that pyriproxyfen disrupts ecdysone secretion and inhibits larval-pupal metamorphosis in silkworms. However, the specific molecular mechanisms by which pyriproxyfen interferes with ecdysone signaling remain to be elucidated. Herein, the RNA-seq analysis on the ecdysone-secretion organ prothoracic gland (PG) was conducted following pyriproxyfen exposure. A total of 3774 differentially expressed genes (DEGs) were identified, with 1667 up-regulated and 2107 down-regulated. KEGG analysis showed that DEGs were enriched in the MAPK signaling pathway, a conserved pathway activated by PTTH binding to Torso, which regulates the ecdysone synthesis. qRT-PCR results indicated a significant up-regulation in PTTH transcription level, while the transcription levels of torso and downstream MAPK pathway genes, Ras2, Raf and ERK, were down-regulated 24 h post-pyriproxyfen treatment. Consistent with these transcriptional changes, PTTH titers in the brain also increased following pyriproxyfen treatment. These results suggest that pyriproxyfen induces abnormal metamorphosis in silkworms by impairing PTTH-Torso signaling. This study enhances our understanding of the molecular mechanisms of pyriproxyfen-induced larval-pupal abnormal metamorphosis in silkworms, and also provides insights for developing detoxification strategies for juvenile hormone analog pesticides to non-target organisms., Competing Interests: Declaration of competing interest The authors that there are no conflicts of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Hepatic enzyme induction, an inherent defense system against xenobiotics, is known to simultaneously affect endocrine system functions in mammals under specific conditions, particularly thyroid hormone (TH) regulation. While this phenomenon has been studied extensively, the pathway leading to this indirect thyroid effect in mammals has unclear applicability to amphibians, despite the importance of amphibian species in assessing thyroid-disruptive chemicals. Here, we investigated the effects of three well-known mammalian enzyme inducers-β-naphthoflavone (BNF), pregnenolone carbonitrile (PCN), and sodium phenobarbital (NaPB)-on the gene expression of phase-I and phase-II metabolizing enzymes in Xenopus laevis tadpoles. Waterborne exposure to BNF and PCN significantly induced the expression of both phase-I (cytochrome P450, CYP) and phase-II enzymes (UDP-glucuronosyltransferase, UGT and sulfotransferase, SULT), but in different patterns, while NaPB exposure induced CYP2B expression without affecting phase-II enzymes in tadpoles, in contrast to mammals. Furthermore, an ex vivo hepatic enzyme activity assay confirmed that BNF treatment significantly increased phase-II metabolic activity (glucuronidation and sulfation) toward TH. These results suggest the potential for certain mammalian enzyme inducers to influence TH clearance in X. laevis tadpoles. Our findings provide insights into the profiles of xenosensing activity and enzyme induction in amphibians, which can facilitate a better understanding of the mechanisms of indirect effects on the thyroid system via hepatic enzyme induction in nonmammalian species., (© 2024 John Wiley & Sons Ltd.)

To characterize the potential for exposure of amphibian terrestrial life stages to plant protection products (PPP), we studied the occurrence and habitat use of adult and metamorph common frogs (Rana temporaria) and common toads (Bufo bufo) in an agricultural landscape in Germany. The four selected study sites were breeding ponds with approximately 80% agricultural land within a 1-km radius. Adults were monitored by radio tracking for two years, and metamorph numbers were assessed for one summer using pitfall traps alongside drift fences. The results demonstrate that adults were rarely present in arable fields at any of the sites (overall 0.5% and 4% of total observations for frogs and toads, respectively). Metamorph captures in arable fields were more variable, ranging from 1.2% to 38.8% (frogs) and from 0.0% to 26.1% (toads) across study sites. Unsurprisingly, most captures in arable fields for both toad and frog metamorphs occurred at the site where the pond was completely surrounded by arable fields. Overall, the presence of adult amphibians in arable fields was limited and, for the metamorphs, occurred primarily when crops were denser and PPP spray interception higher. Diurnal hiding behavior was observed with the highest activity recorded at night, further reducing the risk of dermal exposure from direct PPP overspraying. In addition, it appeared that alternative habitats, such as woody structures or water bodies in the broader surrounding area, were preferred by the animals over the arable areas. The use of buffer zones around water bodies in agricultural areas would be an effective risk mitigation measure to protect terrestrial adults and metamorphs residing there and would reduce spray drift entry into water bodies during PPP application. It is hoped that these results will contribute to the discussion of risk assessment and mitigation options for amphibians. Integr Environ Assess Manag 2024;20:2218-2230. © 2024 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC)., (© 2024 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).)

Objective: Our research aims to investigate the specific mechanisms by which copper inhibits the asexual proliferation of Aurelia coerulea polyps., Methods: Aurelia coerulea polyps were exposed to various CuSO 4 concentrations to study metamorphosis and budding proliferation. Oxidative stress markers (ROS, MDA, CAT, H 2 O 2 , T-AOC, SOD) were measured in polyps and early strobilae. Transcriptomic analysis were used to compare differences in gene expression and enrichment pathways between untreated and copper-exposed polyps. Additionally, RT-qPCR was used to analyze the expression of key molecules. Antioxidant L-Ascorbic acid was applied to determine the role of oxidative stress in asexual reproduction of Aurelia coerulea polyps when exposed to copper., Results: Copper inhibited strobilization and budding of Aurelia coerulea polyps in a dose-dependent manner, in which oxidative stress was involved. Transcriptomic data suggested that the DNA replication pathway was significantly enriched in early strobilae compared to polyps. However, copper treatment repealed the difference of DNA replication pathway between early strobilae compared and polyps. Transcriptomic data suggested that alanine, aspartate, and glutamate metabolism pathways were enriched in untreated budding polyps compared to copper-exposed polyps. After applying the antioxidant L-Ascorbic acid to copper-exposed polyps, various oxidative indicators changed to different extents, with increases in ROS, MDA, CAT, H 2 O 2 , and SOD and a decrease in T-AOC. Further more, the time required for polyps to develop into early strobila was shortened, indicating that the delay in metamorphosis caused by copper exposure was effectively alleviated. And the budding rate increased, indicating that the inhibition of budding proliferation caused by copper exposure was effectively alleviated. The expression of key genes were consist with the transcriptomic sequencing results., Conclusion: Copper exposure causes oxidative stress resulting in the inhibition of asexual reproduction in Aurelia coerulea polyps, including metamorphosis and budding., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Tail resorption during amphibian metamorphosis is one of the most dramatic processes that is obligatorily dependent on thyroid hormone (TH). Heavy metals could result in thyroid gland damages and disturb TH homeostasis. Lead (Pb) and copper (Cu) often co-exist in natural aquatic ecosystems. However, there is still little information on how tail resorption responds to alone or combined exposure to Pb and Cu. Our study investigated the effects of Pb and Cu alone or combined exposure on the morphological parameters of the tail, histological changes of thyroid gland and tail, and gene expression programs involved in cell death of the tail in Bufo gargarizans tadpoles at the climax of metamorphosis. Results demonstrated that Pb, Cu and Pb-Cu mixture exposure resulted in a significantly longer tail compared with control. Damages to notochord, muscle, skin and spinal cord of the tail were found in Pb and Cu exposure groups. The colloid area, the height of follicular cells and number of phagocytic vesicles of thyroid gland in Pb-Cu mixture exposure groups were significantly reduced. In addition, the expression levels of TH, apoptosis, autophagy, degradation of cellular components and oxidative stress-related genes in the tail were significantly altered following Pb and Cu exposure. The present work revealed the relationship between environmental pollutants and tail resorption, providing scientific basis for amphibian protection., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The chronic toxicity of short chain perfluoroalkyl sulfonic acids (PFSAs), such as perfluorobutanesulfonic acid (PFBS) and perfluorohexanesulfonic acid (PFHxS), are relatively understudied despite the increasing detection of these compounds in the environment. We investigated the chronic toxicity and bioconcentration of PFBS and PFHxS using northern leopard frog (Rana [Lithobates] pipiens) tadpoles. We exposed Gosner stage (GS) 25 tadpoles to either PFBS or PFHxS at nominal concentrations of 0.1, 1, 10, 100, and 1000 μg/L until metamorphosis (GS42). We then assessed tadpole growth, development, stress, and immune metrics, and measured fatty acid (FA) composition and PFSA concentrations in liver and whole-body tissues. Tadpole growth and development measures were relatively unaffected by PFSA exposure. However, tadpoles exposed to 1000 μg/L PFBS or PFHxS had significantly increased hepatosomatic indexes (HSI) relative to controls. Further, tadpoles from the 1000 μg/L PFHxS treatment had altered FA profiles relative to controls, with increased total FAs, saturated FAs, monounsaturated FAs, and omega-6 polyunsaturated FAs. In addition, tadpoles from the 1000 μg/L PFHxS treatment had a higher probability of waterborne corticosterone detection. These results suggest that PFBS and PFHxS influence the hepatic health of tadpoles, and that PFHxS may alter lipid metabolism in tadpoles. We also observed a higher probability of tadpoles being phenotypically female after exposure to an environmentally relevant concentration (0.1 μg/L) of PFHxS, suggesting that PFHxS may exert endocrine disrupting effects on tadpoles during early development. The measured bioconcentration factors (BCFs) for both compounds were ≤10 L kg -1 wet weight, suggesting low bioconcentration potential for PFBS and PFHxS in tadpoles. Many of the significant effects observed in this study occurred at concentrations several orders of magnitude above those measured in the environment; however, our work shows effects of PFSAs exposure on amphibians and provides essential information for ecological risk assessments of these compounds., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Crown Copyright © 2024. Published by Elsevier Ltd. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

This study examines how temperature influences the response of Japanese tree frogs (Dryophytes japonicus) to microplastic (MP) pollution, assessing whether temperature can regulate the harmful effects of MPs on their life history and the dispersal of MPs across habitats. This analysis aims to understand the ecological and physiological ramifications of MP pollution. Our results demonstrated an ontogenetic transfer of MP particles across amphibian metamorphosis, possibly allowing and facilitating the translocation of MPs across ecosystems. Temperature did not significantly affect the translocation of aquatic MPs to land. However, high temperatures significantly reduced mortality and hindlimb deformities caused by MPs, thereby mitigating their harmful impact on amphibian life histories. Importantly, our study found that MPs cause hindlimb deformities during amphibian metamorphosis, potentially linked to oxidative stress. Additionally, MP exposure and ingestion induced a plastic response in the morphology of the digestive tract and changes in the fecal microbiome, which were evident at high temperatures but not at low temperatures. The effects of MPs persisted even after the frogs transitioned to the terrestrial stage, suggesting that MPs may have complex, long-term impacts on amphibian population sustainability. Our results enhance the understanding of the intricate environmental challenges posed by MPs and underscore the significant role of temperature in ectotherms regarding ontogenetic impacts and pollutant interactions., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

According to the World Health Organisation and European Commission definitions, substances shall be considered as having endocrine disrupting properties if they show adverse effects, have endocrine activity and the adverse effects are a consequence of the endocrine activity (using a weight-of-evidence approach based on biological plausibility), unless the adverse effects are not relevant to humans or non-target organisms at the (sub)population level. To date, there is no decision logic on how to establish endocrine disruption via the thyroid modality in non-mammalian vertebrates. This paper describes an evidence-based decision logic compliant with the integrated approach to testing and assessment (IATA) concept, to identify thyroid-mediated effect patterns in aquatic vertebrates using amphibians as relevant models for thyroid disruption assessment. The decision logic includes existing test guidelines and methods and proposes detailed considerations on how to select relevant assays and interpret the findings. If the mammalian dataset used as the starting point indicates no thyroid concern, the Xenopus Eleutheroembryonic Thyroid Assay allows checking out thyroid-mediated activity in non-mammalian vertebrates, whereas the Amphibian Metamorphosis Assay or its extended, fixed termination stage variant inform on both thyroid-mediated activity and potentially population-relevant adversity. In evaluating findings, the response patterns of all assay endpoints are considered, including the direction of changes. Thyroid-mediated effect patterns identified at the individual level in the amphibian tests are followed by mode-of-action and population relevance assessments. Finally, all data are considered in an overarching weight-of-evidence evaluation. The logic has been designed generically and can be adapted, e.g. to accommodate fish tests once available for thyroid disruption assessments. It also ensures that all scientifically relevant information is considered, and that animal testing is minimised. The proposed decision logic can be included in regulatory assessments to facilitate the conclusion on whether substances meet the endocrine disruptor definition for the thyroid modality in non-mammalian vertebrates., Competing Interests: Declaration of competing interest Administrative support, article publishing charges and the fees for writing assistance were provided by CropLife Europe. The employment affiliation of the authors is listed on the cover page. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Amphibians worldwide are threatened by habitat loss, some of which is driven by a changing climate, as well as exposure to pesticides, among other causes. The timing and duration of the larval development phase vary between species, thereby influencing the relative impacts of stochastic hydroregime conditions as well as potential aquatic pesticide exposure. We describe the stages of breeding through metamorphosis for eight amphibian species, based on optimal hydroregime conditions, and use a model of pesticide fate and exposure representative of central Florida citrus groves to simulate hydrodynamics based on observed weather data over a 54-year period. Using the Pesticide in Water Calculator and Plant Assessment Tool, we estimated daily wetland depth and pyraclostrobin exposure, with label-recommended application quantities. Species' timing and duration of larval development determined the number of years of suitable hydroregime for breeding and the likelihood of exposure to peak aquatic concentrations of pyraclostrobin. Although the timing of pesticide application determined the number of surviving larvae, density-dependent constraints of wetland hydroregime also affected larval survival across species and seasons. Further defining categorical amphibian life history types and habitat requirements supports the development of screening-level assessments by incorporating environmental stochasticity at the appropriate temporal resolution. Subsequent refinement of these screening-level risk assessment strategies to include spatially explicit landscape data along with terrestrial exposure estimates would offer additional insights into species vulnerability to pesticide exposure throughout the life cycle. Computational simulation of ecologically relevant exposure scenarios, such as these, offers a more realistic interpretation of differential agrichemical risk among species based on their phenology and habits and provides a more situation-specific risk assessment perspective for threatened species. Integr Environ Assess Manag 2024;20:1667-1676. Published 2024. This article is a U.S. Government work and is in the public domain in the USA., (Published 2024. This article is a U.S. Government work and is in the public domain in the USA.)

Anthranilic diamides (AD) are a modern class of insecticides used as alternatives to pyrethroids and neonicotinoids, particularly against lepidopteran pests. Despite their widespread use and presence in surface waters, little is known regarding their effects on amphibians. The aim of this study was to examine the effects of environmentally-relevant concentrations of AD insecticides chlorantraniliprole (CHLO) and cyantraniliprole (CYAN) on metamorphosis of the toad Rhinella arenarum . Tadpoles were exposed to CHLO or CYAN at concentrations ranging from 5 and 5000 µg/L from stage 27 until metamorphosis completion. Both insecticides produced a non-monotonic acceleration of the time required for individuals to progress through development and a decrease in the proportion of individuals completing metamorphosis, although a delay in metamorphosis was also observed at 5 µg/L of CHLO. Snout-vent length and body weight of metamorphosed toads were not markedly affected by either insecticide. CHLO was more toxic than CYAN, with a lowest observed effect concentration (LOEC) for CHLO on time to metamorphosis defined as 5 µg/L compared to 5000 µg/L for CYAN. The LOEC for reduced metamorphic success defined as 50 µg/L for CHLO compared to 500 µg/L for CYAN. As most effects occurred after stage 39, when metamorphosis depends upon thyroid hormones, it is conceivable that that AD insecticides act as endocrine disruptors. These findings suggest that contamination of surface waters with CHLO and CYAN may disrupt amphibian development in the wild and warrant further research to investigate the possibility of endocrine-disruption by ADs.

Cadmium pollution poses a significant threat to aquatic ecosystems due to its propensity to bioaccumulate and cause toxicity. This study assessed the complex dynamics of cadmium uptake, accumulation and distribution across anuran development to provide new insights into the fate of cadmium burdens during metamorphosis and compare the susceptibility of different life stages to cadmium accumulation. Tadpoles of various developmental stages were exposed to dissolved 109-cadmium and depurated in clean water in a series of experiments. Temporal changes in whole-body and tissue concentrations were analysed using gamma spectroscopy, and anatomical distributions were visualised using autoradiography. Results showed that animals exposed at the onset of metamorphic climax (forelimb emergence) retained significantly less cadmium than animals exposed through larval stages. After exposure, cadmium partitioned predominantly in the skin, gills and remains of metamorphs, whereas larvae accumulated cadmium predominately through their gut. This shows a shift in the primary route of uptake at the onset of climax, which relates to the structural and functional changes of uptake sites through metamorphosis. During climax, some cadmium was redistributed in tissues developing de novo, such as the forelimbs, and concentrated in the regressing tail. Our findings highlight the need for stage-specific considerations in assessing exposure risks., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Chantal Lanctot reports equipment, supplies and travel were provided by Australian Nuclear Science and Technology Organisation. Chantal Lanctot reports financial support was provided by Australian Research Council. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Among human actions threatening biodiversity, the release of anthropogenic chemical pollutants which have become ubiquitous in the environment, is a major concern. Chemical pollution can induce damage to macromolecules by causing the overproduction of reactive oxygen species, affecting the redox balance of animals. In species undergoing metamorphosis (i.e. the vast majority of the extant animal species), antioxidant responses to chemical pollution may differ between pre- and post-metamorphic stages. Here, we meta-analysed ( N  = 104 studies, k =  2283 estimates) the impact of chemical pollution on redox balance across the three major amphibian life stages (embryo, tadpole, adult). Before metamorphosis, embryos did not experience any redox change while tadpoles activate their antioxidant pathways and do not show increased oxidative damage from pollutants. Tadpoles may have evolved stronger defences against pollutants to reach post-metamorphic life stages. In contrast, post-metamorphic individuals show only weak antioxidant responses and marked oxidative damage in lipids. The type of pollutant (i.e. organic versus inorganic) has contrasting effects across amphibian life stages. Our findings show a divergent evolution of the redox balance in response to pollutants across life transitions of metamorphosing amphibians, most probably a consequence of differences in the ecological and developmental processes of each life stage.

Amphibian larvae inhabiting temporary ponds often exhibit the capacity to accelerate development and undergo metamorphosis in challenging conditions like desiccation. However, not all species exhibit this ability, the yellow-bellied toad (Bombina variegata) is one such example. The underlying mechanisms behind the inability to accelerate development under desiccation remain largely unexplored. The hypothalamic-pituitary-interrenal (HPI) axis and corticosterone (CORT), which act synergistically with thyroid hormone, are thought to facilitate metamorphosis in response to desiccation stress. In this study, we aimed to investigate whether modification in the HPI axis, particularly CORT levels, contributes to the absence of adaptive plasticity in B. variegata under desiccation stress. The study design included four treatments: high water level, high water level with exogenous CORT, low water level, and low water level with metyrapone (a CORT synthesis inhibitor). The main objective was to evaluate the effects of these treatments on whole-body corticosterone levels, life history, morphological traits, and oxidative stress parameters during the prometamorphic and metamorphic climax developmental stages. While low water level had no effect on total corticosterone levels, larval period, body condition index, and metamorphic body shape, it negatively affected metamorph size, mass, and growth rate. Our findings suggest that constant exposure to desiccation stress over generations may have led to modifications in the HPI axis activity in B. variegata, resulting in adaptation to changes in water level, evident through the absence of stress response. Consequently, CORT may not be a relevant stress indicator in desiccation conditions for this species., (© 2024 The Authors. Journal of Experimental Zoology Part A: Ecological and Integrative Physiology published by Wiley Periodicals LLC.)

A modified amphibian metamorphosis assay was performed in which Nieuwkoop and Faber (NF) stage 47 Xenopus laevis larvae were exposed to different concentrations of either perchlorate (ClO 4 - ) or nitrate (NO 3 - ) for 32 days. Larvae were exposed to 0.0 (control), 5, 25, 125, 625, and 3125 μg/L ClO 4 - , or 0 (control), 23, 71, 217, 660, and 2000 mg/L NO 3 - . The primary endpoints were survival, hind limb length (HLL), forelimb emergence and development, developmental stage (including time to NF stage 62 [MT 62 ]), thyroid histopathology, wet weight, and snout-vent length (SVL). Developmental delay as evidenced by altered stage distribution and increased MT 62 , a higher degree of thyroid follicular cell hypertrophy, and an increase in the prevalence of follicular cell hyperplasia was observed at concentrations ≥125 μg/L ClO 4 - . The no observed effect concentration (NOEC) for developmental endpoints was 25.0 μg/L ClO 4 - and the NOEC for growth endpoints was 3125 μg/L ClO 4 - . Exposure to nitrate did not adversely affect MT 62 , but a decreasing trend in stage distribution and median developmental stage at ≥217 mg/L NO 3 - was observed. No histopathologic effects associated with nitrate exposure were observed. An increasing trend in SVL-normalized HLL was observed at 2000 mg/L NO 3 - . Nitrate did not alter larval growth. The NOEC for developmental endpoints was 71 mg/L NO 3 - , and 2000 mg/L NO 3 - for growth endpoints. The present study provided additional evidence that the effects and potency of nitrate and perchlorate on metamorphosis and growth in X. laevis are considerably different., (© 2024 John Wiley & Sons Ltd.)

As part of the US Environmental Protection Agency's perfluoroalkyl and polyfluoroalkyl substances (PFAS) Action Plan, the agency is committed to increasing our understanding of the potential ecological effects of PFAS. The objective of these studies was to examine the developmental toxicity of PFAS using the laboratory model amphibian species Xenopus laevis. We had two primary aims: (1) to understand the developmental toxicity of a structurally diverse set of PFAS compounds in developing embryos and (2) to characterize the potential impacts of perfluorooctanesulfonic acid (PFOS), perfluorohexanesulfonic acid (PFHxS), perfluorooctanoic acid (PFOA), and hexafluoropropylene oxide-dimer acid (HFPO-DA a.k.a. GenX), on growth and thyroid hormone-controlled metamorphosis. We employed a combination of static renewal and flow-through exposure designs. Embryos were exposed to 17 structurally diverse PFAS starting at the midblastula stage through the completion of organogenesis (96 h). To investigate impacts on PFOS, PFOA, PFHxS, and HFPO-DA on development and metamorphosis, larvae were exposed from premetamorphosis (Nieuwkoop Faber stage 51 or 54) through pro metamorphosis. Of the PFAS tested in embryos, only 1H,1H,10H,10H-perfluorodecane-1,10-diol (FC10-diol) and perfluorohexanesulfonamide (FHxSA) exposure resulted in clear concentration-dependent developmental toxicity. For both of these PFAS, a significant increase in mortality was observed at 2.5 and 5 mg/L. For FC10-diol, 100% of the surviving embryos were malformed at 1.25 and 2.5 mg/L, while for FHxSA, a significant increase in malformations (100%) was observed at 2.5 and 5 mg/L. Developmental stage achieved was the most sensitive endpoint with significant effects observed at 1.25 and 0.625 mg/L for FC10-diol and FHxSA, respectively. In larval studies, we observed impacts on growth following exposure to PFHxS and PFOS at concentrations of 100 and 2.5 mg/L, respectively, while no impacts were observed in larvae when exposed to PFOA and HFPO-DA at concentration of 100 mg/L. Further, we did not observe impacts on thyroid endpoints in exposed larvae. These experiments have broadened our understanding of the impact of PFAS on anuran development., (Published 2024. This article is a U.S. Government work and is in the public domain in the USA.)

Tris(2-chloroethyl) phosphate (TCEP) and tris(1‑chloro-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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Reduced glutathione (GSH) synthesis is vital for redox homeostasis, cell-cycle regulation and apoptosis, and immune function. The glutamate-cysteine ligase catalytic subunit (Gclc) is the first and rate-limiting enzyme in GSH synthesis, suggesting the potential use of Gclc as a pesticide target. However, the functional characterization of Gclc, especially its contribution in metamorphosis, antioxidant status and insecticide resistance, is unclear in Tribolium castaneum., Results: In this study, we identified and cloned Gclc from T. castaneum (TcGclc) and found that its expression began to increase significantly from the late larvae (LL) stage (3.491 ± 0.490-fold). Furthermore, RNA interference-mediated knockdown of TcGclc resulted in three types of aberration (100% total aberration rate) caused by the downregulation of genes related to the 20-hydroxyecdysone (20E) pathway. This deficiency was partially rescued by exogenous 20E treatment (53.1% ± 3.2%), but not by antioxidant. Moreover, in the TcGclc knockdown group, GSH content was decreased to 62.3%, and total antioxidant capacity, glutathione peroxidase and total superoxide dismutase activities were reduced by 14.6%, 83.6%, and 82.3%, respectively. In addition, treatment with different insecticides upregulated expression of TcGclc significantly compared with a control group during the late larval stage (P < 0.01)., Conclusion: Our results indicate that TcGclc has an extensive role in metamorphosis, antioxidant function and insecticide resistance in T. castaneum, thereby expanding our understanding of GSH functions and providing a scientific basis for pest control. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

As amphibians undergo thyroid hormone (TH)-dependent metamorphosis from an aquatic tadpole to the terrestrial frog, their innate immune system must adapt to the new environment. Skin is a primary line of defense, yet this organ undergoes extensive remodelling during metamorphosis and how it responds to TH is poorly understood. Temperature modulation, which regulates metamorphic timing, is a unique way to uncover early TH-induced transcriptomic events. Metamorphosis of premetamorphic tadpoles is induced by exogenous TH administration at 24 °C but is paused at 5 °C. However, at 5 °C a "molecular memory" of TH exposure is retained that results in an accelerated metamorphosis upon shifting to 24 °C. We used RNA-sequencing to identify changes in Rana (Lithobates) catesbeiana back skin gene expression during natural and TH-induced metamorphosis. During natural metamorphosis, significant differential expression (DE) was observed in >6500 transcripts including classic TH-responsive transcripts (thrb and thibz), heat shock proteins, and innate immune system components: keratins, mucins, and antimicrobial peptides (AMPs). Premetamorphic tadpoles maintained at 5 °C showed 83 DE transcripts within 48 h after TH administration, including thibz which has previously been identified as a molecular memory component in other tissues. Over 3600 DE transcripts were detected in TH-treated tadpoles at 24 °C or when tadpoles held at 5 °C were shifted to 24 °C. Gene ontology (GO) terms related to transcription, RNA metabolic processes, and translation were enriched in both datasets and immune related GO terms were observed in the temperature-modulated experiment. Our findings have implications on survival as climate change affects amphibia worldwide., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Thyroid hormones are primarily responsible for the brain development in perinatal mammals. However, this process can be inhibited by external factors such as environmental chemicals. Perinatal mammals are viviparous, which makes direct fetal examination difficult., Methods: We used metamorphic amphibians, which exhibit many similarities to perinatal mammals, as an experimental system. Therefore, using metamorphic amphibians, we characterized the gene expression of matrix metalloproteinases, which play an important role in brain development., Results: The expression of many matrix metalloproteinases (mmps) was characteristically induced during metamorphosis. We also found that the expression of many mmps was induced by T 3 and markedly inhibited by hydroxylated polychlorinated biphenyls (PCBs)., Conclusion: Overall, our findings suggest that hydroxylated PCBs disrupt normal brain development by disturbing the gene expression of mmps., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

The Xenopus Eleutheroembryonic Thyroid Assay (XETA) was recently published as an OECD Test Guideline for detecting chemicals acting on the thyroid axis. However, the OECD validation did not cover all mechanisms that can potentially be detected by the XETA. This study was therefore initiated to investigate and consolidate the applicability domain of the XETA regarding the following mechanisms: thyroid hormone receptor (THR) agonism, sodium-iodide symporter (NIS) inhibition, thyroperoxidase (TPO) inhibition, deiodinase (DIO) inhibition, glucocorticoid receptor (GR) agonism, and uridine 5'-diphospho-glucuronosyltransferase (UDPGT) induction. In total, 22 chemicals identified as thyroid-active or -inactive in Amphibian Metamorphosis Assays (AMAs) were tested using the XETA OECD Test Guideline. The comparison showed that both assays are highly concordant in identifying chemicals with mechanisms of action related to THR agonism, DIO inhibition, and GR agonism. They also consistently identified the UDPGT inducers as thyroid inactive. NIS inhibition, investigated using sodium perchlorate, was not detected in the XETA. TPO inhibition requires further mechanistic investigations as the reference chemicals tested resulted in opposing response directions in the XETA and AMA. This study contributes refining the applicability domain of the XETA, thereby helping to clarify the conditions where it can be used as an ethical alternative to the AMA., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: David Du Pasquier, Benoit Salinier, Gregory Lemkine, Barbara Robin-Duchesne reports financial support was provided by CropLife Europe. Laurent Lagadic reports a relationship with Bayer CropScience AG that includes: employment. This work was supported by CropLife Europe as a Company Investment Project and research and development funds from Corteva Agriscience USA. The employment affiliation of the authors is listed on the cover page. BRD, BS, DDP and GL are affiliated to Laboratoire WatchFrog, developer of the Xenopus Eleutheroembryonic Thyroid Assay (XETA) and coordinator of the validation of OECD TG 248. AJ, JL, JRW, KKC, LL, LW and OK are affiliated to companies that manufacture agrochemicals, some of which having been used in the present study. There are no other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Several endocrine-disrupting chemicals (EDCs) have been proven to interfere with the physiological function of thyroid hormone (TH), which affected growth and development. However, few studies have investigated the effects of EDCs on TH axis with consequence for skeletal development in amphibians. This study thus examined the potential role of perchlorate and T4 in growth, development and endochondral ossification during metamorphosis of Bufo gargarizans. Our studies showed that NaClO₄ treatment caused weight gain and delayed the developmental stage in B. gargarizans tadpoles, while T4 decreased body size and survival rate, accelerated metamorphic duration and increased the risk of early death. Histological sections suggested that NaClO₄ and T4 treatments caused damages to thyroid tissue, such as decreased thyroid gland size, follicle size, colloid area, the height of follicular epithelial cells and the number of follicles. In addition, the double skeletal staining and RT-qPCR showed that NaClO₄ and T4 treatments inhibited the endochondral ossification by regulating TH synthesis (TRs, Dios) and endochondral ossification-related genes (MMPs, Runxs, VEGFs and VEGFRs) expression levels, which might affect terrestrial locomotion and terrestrial life. Altogether, these thyroid injury and gene expression changes as caused by NaClO₄ and T4 may have an influence on development and endochondral ossification during the metamorphosis of amphibians., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Pulses of the steroid hormone ecdysone act through transcriptional cascades to direct the major developmental transitions during the Drosophila life cycle. These include the prepupal ecdysone pulse, which occurs 10 ​hours after pupariation and triggers the onset of adult morphogenesis and larval tissue destruction. E93 encodes a transcription factor that is specifically induced by the prepupal pulse of ecdysone, supporting a model proposed by earlier work that it specifies the onset of adult development. Although a number of studies have addressed these functions for E93, little is known about its roles in the salivary gland where the E93 locus was originally identified. Here we show that E93 is required for development through late pupal stages, with mutants displaying defects in adult differentiation and no detectable effect on the destruction of larval salivary glands. RNA-seq analysis demonstrates that E93 regulates genes involved in development and morphogenesis in the salivary glands, but has little effect on cell death gene expression. We also show that E93 is required to direct the proper timing of ecdysone-regulated gene expression in salivary glands, and that it suppresses earlier transcriptional programs that occur during larval and prepupal stages. These studies support the model that the stage-specific induction of E93 in late prepupae provides a critical signal that defines the end of larval development and the onset of adult differentiation., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Two orthologues of the gene encoding the Na + -Cl - cotransporter (NCC), termed ncca and nccb, were found in the sea lamprey genome. No gene encoding the Na + -K + -2Cl - cotransporter 2 (nkcc2) was identified. In a phylogenetic comparison among other vertebrate NCC and NKCC sequences, the sea lamprey NCCs occupied basal positions within the NCC clades. In freshwater, ncca mRNA was found only in the gill and nccb only in the intestine, whereas both were found in the kidney. Intestinal nccb mRNA levels increased during late metamorphosis coincident with salinity tolerance. Acclimation to seawater increased nccb mRNA levels in the intestine and kidney. Electrophysiological analysis of intestinal tissue ex vivo showed this tissue was anion absorptive. After seawater acclimation, the proximal intestine became less anion absorptive, whereas the distal intestine remained unchanged. Luminal application of indapamide (an NCC inhibitor) resulted in 73% and 30% inhibition of short-circuit current (I sc ) in the proximal and distal intestine, respectively. Luminal application of bumetanide (an NKCC inhibitor) did not affect intestinal I sc . Indapamide also inhibited intestinal water absorption. Our results indicate that NCCb is likely the key ion cotransport protein for ion uptake by the lamprey intestine that facilitates water absorption in seawater. As such, the preparatory increases in intestinal nccb mRNA levels during metamorphosis of sea lamprey are likely critical to development of whole animal salinity tolerance., (© 2021. The Author(s).)

Ivermectin is a broad-spectrum antiparasitic medicine, which is often used as a treatment for parasites or as a prophylaxis. While studies have looked at the long-term effects of Ivermectin on helminths, studies have not considered the long-term impacts of this treatment on host health or disease susceptibility. Here, we tracked the effects of early life Ivermectin treatment in Cuban tree frogs (Osteopilus septentrionalis) on growth rates, mortality, metabolically expensive organ size, and susceptibility to Batrachochytrium dendrobatidis (Bd) infection. One year after exposure, there was no effect of Ivermectin exposure on frog mass (X21 = 0.904, p = 0.34), but when tracked through the exponential growth phase (~2.5 years) the Ivermectin exposed individuals had lower growth rates and were ultimately smaller (X21 = 7.78, p = 0.005; X21 = 5.36, p = 0.02, respectively). These results indicate that early life exposure is likely to have unintended impacts on organismal growth and potentially reproductive fitness. Additionally, we exposed frogs to Bd, a pathogenic fungus that has decimated amphibian populations globally, and found early life exposure to Ivermectin decreased disease susceptibility (disease load: X21 = 17.57, p = 0.0002) and prevalence (control: 55%; Ivermectin: 22%) over 2 years after exposure. More research is needed to understand the underlying mechanism behind this phenomenon. Given that Ivermectin exposure altered disease susceptibility, proper controls should be implemented when utilizing this drug as an antiparasitic treatment in research studies., Competing Interests: The authors have declared that no competing interests exist.

The effects of two drugs containing the synthetic thyroid hormone levothyroxine (LEV) and an anti-thyroid drug containing propylthiouracil (PTU) on the three early life stages of Xenopus laevis were evaluated with the Frog Embryo Teratogenesis Assay-Xenopus, Tadpole Toxicity Test, and Amphibian Metamorphosis Assay using biochemical and morphological markers. Tested drugs caused more effective growth retardation in stage 8 embryos than stage 46 tadpoles. Significant inhibition of biomarker enzymes has been identified in stage 46 tadpoles for both drugs. AMA test results showed that LEV-I caused progression in the developmental stage and an increase in thyroxine level in 7 days exposure and growth retardation in 21 days exposure in stage 51 tadpoles. On the other hand, increases in lactate dehydrogenase activity for both drugs in the AMA test may be due to impacted energy metabolism during sub-chronic exposure. These results also show that the sensitivity and responses of Xenopus laevis at different early developmental stages may be different when exposed to drugs., (Copyright © 2021 Elsevier B.V. All rights reserved.)

In insects, some sterols are essential not only for cell membrane homeostasis, but for biosynthesis of the steroid hormone ecdysone. Dietary sterols are required for insect development because insects cannot synthesize sterols de novo. Therefore, sterol-like compounds that can compete with essential sterols are good candidates for insect growth regulators. In this study, we investigated the effects of the plant-derived triterpenoids, cucurbitacin B and E (CucB and CucE) on the development of the fruit fly, Drosophila melanogaster. To reduce the effects of supply with an excess of sterols contained in food, we reared D. melanogaster larvae on low sterol food (LSF) with or without cucurbitacins. Most larvae raised on LSF without supplementation or with CucE died at the second or third larval instar (L2 or L3) stages, whereas CucB-administered larvae mostly died without molting. The developmental arrest caused by CucB was partially rescued by ecdysone supplementation. Furthermore, we examined the effects of CucB on larval-prepupal transition by transferring larvae from LSF supplemented with cholesterol to that with CucB just after the L2/L3 molt. L3 larvae raised on LSF with CucB failed to pupariate, with a remarkable developmental delay. Ecdysone supplementation rescued the developmental delay but did not rescue the pupariation defect. Furthermore, we cultured the steroidogenic organ, the prothoracic gland (PG) of the silkworm Bombyx mori, with or without cucurbitacin. Ecdysone production in the PG was reduced by incubation with CucB, but not with CucE. These results suggest that CucB acts not only as an antagonist of the ecdysone receptor as previously reported, but also acts as an inhibitor of ecdysone biosynthesis., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Larval metamorphosis in bivalves is a key event for the larva-to-juvenile transformation. Previously we have identified a thyroid hormone receptor (TR) gene that is crucial for larvae to acquire "competence" for the metamorphic transition in the mussel Mytilus courscus (Mc). The mechanisms of thyroid signaling in bivalves are still largely unknown. In the present study, we molecularly characterized the full-length of two iodothyronine deiodinase genes (McDx and McDy). Phylogenetic analysis revealed that deiodinases of molluscs (McDy, CgDx and CgDy) and vertebrates (D2 and D3) shared a node representing an immediate common ancestor, which resembled vertebrates D1 and might suggest that McDy acquired specialized function from vertebrates D1. Anti-thyroid compounds, methimazole (MMI) and propylthiouracil (PTU), were used to investigate their effects on larval metamorphosis and juvenile development in M. coruscus. Both MMI and PTU significantly reduced larval metamorphosis in response to the metamorphosis inducer epinephrine. MMI led to shell growth retardation in a concentration-dependent manner in juveniles of M. coruscus after 4 weeks of exposure, whereas PTU had no effect on juvenile growth. It is hypothesized that exposure to MMI and PTU reduced the ability of pediveliger larvae for the metamorphic transition to respond to the inducer. The effect of MMI and PTU on larval metamorphosis and development is most likely through a hormonal signal in the mussel M. coruscus, with the implications for exploring the origins and evolution of metamorphosis., (© 2021. The Author(s).)

Settlement and metamorphosis of planktonic larvae into benthic adults are critical components of a diverse range of marine invertebrate-mediated processes such as the formation of mussel beds and coral reefs, the recruitment of marine shellfisheries, and the initiation of macrobiofouling. Although larval settlement and metamorphosis induced by natural chemical cues is widespread among marine invertebrates, the mechanisms of action remain poorly understood. Here, we identified that the molecular target of adenosine (an inducer of larval settlement and metamorphosis from conspecific adults in the invasive biofouling mussel Mytilopsis sallei ) is adenosine kinase (ADK). The results of transcriptomic analyses, pharmacological assays, temporal and spatial gene expression analyses, and siRNA interference, suggest that ATP-dependent phosphorylation of adenosine catalyzed by ADK activates the downstream AMPK-FoxO signaling pathway, inducing larval settlement and metamorphosis in M. sallei . This study not only reveals the role of the ADK-AMPK-FoxO pathway in larval settlement and metamorphosis of marine invertebrates but it also deepens our understanding of the functions and evolution of adenosine signaling, a process that is widespread in biology and important in medicine.

Amphibian endocrine systems interact with each other during normal development. Interference with one of the endocrine systems may influence others. We studied the effect of a thyroid inhibitor (ethylenethiourea [ETU]) on metamorphosis and ovary development of three species, Sphaerotheca pashchima, Indosylvirana caesari, and Euphlyctis cyanophlyctis with different larval durations. We treated the tadpoles of these species with 50, 100, and 200 mg/L concentrations of ETU and studied their larval duration, size at metamorphosis, and ovary development. The results revealed that ETU affects metamorphosis, depending on the species and concentration. ETU delayed metamorphosis of E. cyanophlyctis tadpoles and did not affect metamorphosis in S. pashchima tadpoles. Lower concentrations of ETU stimulated metamorphosis in I. caesari tadpoles while high concentration delayed metamorphosis. In the tadpoles (E. cyanophlyctis) treated with higher concentrations of ETU, ovary development was advanced with an increased size of the diplotene oocytes. Oocyte size was smaller in the tadpoles (of I. caesari) treated with lower concentrations of ETU. These results demonstrated that the tadpoles of these species show different responses to the thyroid inhibitor, possibly due to the differences in the larval duration and sensitivity. Inhibition or acceleration of metamorphosis did not interfere in the ovary development of E. cyanophlyctis and I. caesari. These results will be useful in understanding the impact of endocrine disruptors on the interaction between thyroid and sex steroid hormones., (© 2021 Wiley Periodicals LLC.)

In holometabolous insects, many tissues and organs such as the fat body and midgut undergo a remodeling process during metamorphosis. Larval fat body cells are eliminated by programmed cell death (PCD), while tissue cells that adapt to adult life are formed by stem cells. In this study, we analyzed the features of the remodeling period of Galleria mellonella fat body in terms of PCD types, apoptotic and autophagic cell death characteristics. Besides, the effects of juvenile hormone (JH) on these processes were evaluated under the modified hormonal conditions via applications of JH analog, fenoxycarb. Several hallmarks of apoptotic and autophagic cell death were analyzed by morphological, biochemical, and molecular methods. The results of the present study have ascertained that the degeneration process of larval cells occurs via autophagic cell death accompanied by caspase-3 activity during the pupal period and it is regulated by 20-hydroxyecdysone (20HE) mediated by ecdysone receptor B1 (EcR-B1). Increased activity of the acid phosphatase and upregulation of ATG6 and ATG8 in parallel with the formation of autophagosomes in the fat body of Galleria during the pupal period strongly indicated that autophagy was the key player in the remodeling processes., (© 2021 Wiley Periodicals LLC.)

Diisononyl phthalate (DINP) as one of the most commonly used phthalates, has been found in various environmental samples and is considered to have potential risks to ecosystem. Till now, DINP has no clear effect consensus on insects from development to behavior and even mechanisms. Here, Drosophila melanogaster was selected as model organisms and the toxic effects of DINP (0.1%, 0.2%, 0.5% and 1.0%) (v/v) on its metamorphosis, crawling behavior, intestinal cells and cellular redox balance were investigated. During metamorphosis process, lower hatching rate, longer development time, lighter body weight and malformation were observed at high concentration groups. The crawling ability of larvae was severely inhibited by DINP and the movement distance was drastically reduced. DINP could cause severe damage to the larval intestinal cells in the dose-dependent and time-dependent manners. DINP was found to induce redox imbalance with activities of two important antioxidant enzymes (catalase (CAT) and superoxide dismutase (SOD)) increasing, and reactive oxygen species (ROS) level fluctuation in larvae. Our findings provide theoretical basis and data support for scientific management of DINP to reduce ecological risk., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Animal steroid hormones initiate signaling by passive diffusion into cells and binding to their nuclear receptors to regulate gene expression. Animal steroid hormones can initiate signaling via G protein-coupled receptors (GPCRs); however, the underlying mechanisms are unclear. Here, we show that a newly discovered ecdysone-responsive GPCR, ErGPCR-3, transmits the steroid hormone 20-hydroxyecdysone (20E) signal by binding 20E and promoting its entry into cells in the lepidopteran insect Helicoverpa armigera Knockdown of ErGPCR-3 in larvae caused delayed and abnormal pupation, inhibited remodeling of the larval midgut and fat body, and repressed 20E-induced gene expression. Also, 20E induced both the interaction of ErGPCR-3 with G proteins and rapid intracellular increase in calcium, cAMP and protein phosphorylation. ErGPCR-3 was endocytosed by GPCR kinase 2-mediated phosphorylation, and interacted with β-arrestin-1 and clathrin, to terminate 20E signaling under 20E induction. We found that 20E bound to ErGPCR-3 and induced the ErGPCR-3 homodimer to form a homotetramer, which increased 20E entry into cells. Our study revealed that homotetrameric ErGPCR-3 functions as a cell membrane receptor and increases 20E diffusion into cells to transmit the 20E signal and promote metamorphosis., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Background and Objective: The worldwide fly species, Muscina stabulans (Diptera: Muscidae) is known as 'false stable fly'. It has veterinary, forensic and medical importance. The present study aimed to examine the toxicity of novaluron (chitin synthesis inhibitor) via its effect on the growth and reproductive potential of M. stabulans., Materials and Methods: The early last (3rd) instar larvae and prepupae have been treated with novaluron using five doses: 5.0, 1.0, 0.5, 0.1 and 0.01 μg/larva. Student's t-test analysis has been used for data processing as well as refined by Bessel correction for significant differences among means., Results: Current study revealed that, after the treatment different mortalities of larva, pupa and adult have been estimated. LD50 values of novaluron were 0.018 and 0.057 μg/insect, respectively. Furthermore, the larval period was insignificantly shortened while the pupal duration has been significantly extended and the developmental rate has been slightly enhanced. On the other hand, the adult longevity of females was considerably reduced and the adult emergence was considerably blocked, regardless the dose. However, only after the treatment, some deformed pupae were observed and some adult deformities were observed. Both fecundity and fertility were drastically reduced and sterilizing activity on novaluron increased in a dose-dependent course, regardless the time of treatment., Conclusion: Novaluron can be used as an effective IGR in the integrated control program for this medically and veterinary serious fly.

To regulate the presence of contaminants in Brazilian water, the Brazilian Environmental Council (CONAMA) promulgates regulations regarding the concentrations of given compounds that are supposed to be safe for aquatic life. Considering these regulations, this study tested the effects of considered safe levels of lithium (2.5 mgL -1 ) and selenium (0.01 mgL -1 ), isolated and mixed, on the American bullfrog (Lithobates catesbeianus) tadpoles. The evaluation was done through the use of biomarkers of larval development as total wet weight (TWW), snout-vent-length (SVL), hind-limb-length (HLL), activity level (AL), histologic evaluation of the thyroid gland and the mortality rate. The tadpoles were allocated into four groups (n = 20 each): a control group (CT); a group exposed to lithium (LI), a group exposed to selenium (SE), and a group exposed to both lithium and selenium (SELI). The whole assay was carried out over 21 days, with two rounds of data collection (on 7th and 21st day) to evaluate the responses over time. A statistical reduction in the AL was observed in the tadpoles from the LI and SELI groups after 7 days of exposure, the same pattern was observed after 21 days. Histological analyses of the thyroid gland showed signs of up-regulation (i.e. statistic reduction in number and area of the follicles, as well a significant reduction in the area of the gland) in all exposed groups, which represents an endocrine response as an adaptative strategy to deal with polluted aquatic environment. The stress triggered by the polluted medium is discussed., (Copyright © 2020. Published by Elsevier Inc.)

Sodium perchlorate (NaClO 4 ) and exogenous L-thyroxine (T4), two kinds of endocrine-disrupting chemicals (EDCs), mainly affect the circulating thyroid hormones, which regulate the initiation and rate of metamorphosis in amphibian. The aim of this study is to evaluate the potential role of EDCs in regulating the development of tadpoles and leptin signaling pathway of liver during the metamorphosis of Bufo gargarizans. There was completely opposite result of average development stage of tadpoles and morphological parameters between the NaClO 4 and T4 exposure groups. Histological analysis revealed that NaClO 4 and T4 exposure both caused liver injury, such as the decreased size of hepatocytes, atrophy of nucleus, increased melanomacrophage centres and disappearance of hepatocyte membranes. In addition, the results of RT-qPCR revealed that NaClO 4 treatment significantly inhibited the transcript levels of genes related to thyroid hormone (D2, TRα and TRβ) and leptin signaling pathway (LepR, JAK1, JAK2, and TYK2), while there was an increase of mRNA expression of these genes in the liver of tadpoles administrated with T4 compared with control. This work lays an important foundation for assessing the risk of EDCs in relation to amphibian development during metamorphosis., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Senegalese sole is an economically important flatfish species in aquaculture and an attractive model to decipher the molecular mechanisms governing the severe transformations occurring during metamorphosis, where retinoic acid seems to play a key role in tissue remodeling. In this study, a robust sole transcriptome was envisaged by reducing the number of assembled libraries (27 out of 111 available), fine-tuning a new automated and reproducible set of workflows for de novo assembling based on several assemblers, and removing low confidence transcripts after mapping onto a sole female genome draft. From a total of 96 resulting assemblies, two "raw" transcriptomes, one containing only Illumina reads and another with Illumina and GS-FLX reads, were selected to provide SOLSEv5.0, the most informative transcriptome with low redundancy and devoid of most single-exon transcripts. It included both Illumina and GS-FLX reads and consisted of 51,348 transcripts of which 22,684 code for 17,429 different proteins described in databases, where 9527 were predicted as complete proteins. SOLSEv5.0 was used as reference for the study of retinoic acid (RA) signalling in sole larvae using drug treatments (DEAB, a RA synthesis blocker, and TTNPB, a RA-receptor agonist) for 24 and 48 h. Differential expression and functional interpretation were facilitated by an updated version of DEGenes Hunter. Acute exposure of both drugs triggered an intense, specific and transient response at 24 h but with hardly observable differences after 48 h at least in the DEAB treatments. Activation of RA signalling by TTNPB specifically increased the expression of genes in pathways related to RA degradation, retinol storage, carotenoid metabolism, homeostatic response and visual cycle, and also modified the expression of transcripts related to morphogenesis and collagen fibril organisation. In contrast, DEAB mainly decreased genes related to retinal production, impairing phototransduction signalling in the retina. A total of 755 transcripts mainly related to lipid metabolism, lipid transport and lipid homeostasis were altered in response to both treatments, indicating non-specific drug responses associated with intestinal absorption. These results indicate that a new assembling and transcript sieving were both necessary to provide a reliable transcriptome to identify the many aspects of RA action during sole development that are of relevance for sole aquaculture.

Background: Nitric oxide (NO) is presumed to be a regulator of metamorphosis in many invertebrate species, and although NO pathways have been comparatively well-investigated in gastropods, annelids and crustaceans, there has been very limited research on the effects of NO on metamorphosis in bivalve shellfish., Results: In this paper, we investigate the effects of NO pathway inhibitors and NO donors on metamorphosis induction in larvae of the Pacific oyster, Crassostrea gigas. The nitric oxides synthase (NOS) inhibitors s-methylisothiourea hemisulfate salt (SMIS), aminoguanidine hemisulfate salt (AGH) and 7-nitroindazole (7-NI) induced metamorphosis at 75, 76 and 83% respectively, and operating in a concentration-dependent manner. Additional induction of up to 54% resulted from exposures to 1H-[1,2,4]Oxadiazole[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylyl cyclase, with which NO interacts to catalyse the synthesis of cyclic guanosine monophosphate (cGMP). Conversely, high concentrations of the NO donor sodium nitroprusside dihydrate in combination with metamorphosis inducers epinephrine, MK-801 or SMIS, significantly decreased metamorphosis, although a potential harmful effect of excessive NO unrelated to metamorphosis pathway cannot be excluded. Expression of CgNOS also decreased in larvae after metamorphosis regardless of the inducers used, but intensified again post-metamorphosis in spat. Fluorescent detection of NO in competent larvae with DAF-FM diacetate and localisation of the oyster nitric oxide synthase CgNOS expression by in-situ hybridisation showed that NO occurs primarily in two key larval structures, the velum and foot. cGMP was also detected in the foot using immunofluorescent assays, and is potentially involved in the foot's smooth muscle relaxation., Conclusion: Together, these results suggest that the NO pathway acts as a negative regulator of metamorphosis in Pacific oyster larvae, and that NO reduction induces metamorphosis by inhibiting swimming or crawling behaviour, in conjunction with a cascade of additional neuroendocrine downstream responses.

Thyroid hormone (T3) plays pivotal roles in vertebrate development, acting via nuclear T3 receptors (TRs) that regulate gene transcription by promoting post-translational modifications to histones. Methylation of cytosine residues in deoxyribonucleic acid (DNA) also modulates gene transcription, and our recent finding of predominant DNA demethylation in the brain of Xenopus tadpoles at metamorphosis, a T3-dependent developmental process, caused us to hypothesize that T3 induces these changes in vivo. Treatment of premetamorphic tadpoles with T3 for 24 or 48 hours increased immunoreactivity in several brain regions for the DNA demethylation intermediates 5-hydroxymethylcytosine (5-hmC) and 5-carboxylcytosine, and the methylcytosine dioxygenase ten-eleven translocation 3 (TET3). Thyroid hormone treatment induced locus-specific DNA demethylation in proximity to known T3 response elements within the DNA methyltransferase 3a and Krüppel-like factor 9 genes, analyzed by 5-hmC immunoprecipitation and methylation sensitive restriction enzyme digest. Chromatin-immunoprecipitation (ChIP) assay showed that T3 induced TET3 recruitment to these loci. Furthermore, the messenger ribonucleic acid for several genes encoding DNA demethylation enzymes were induced by T3 in a time-dependent manner in tadpole brain. A TR ChIP-sequencing experiment identified putative TR binding sites at several of these genes, and we provide multiple lines of evidence to support that tet2 contains a bona fide T3 response element. Our findings show that T3 can promote DNA demethylation in developing tadpole brain, in part by promoting TET3 recruitment to discrete genomic regions, and by inducing genes that encode DNA demethylation enzymes., (© Endocrine Society 2020. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The cranial ossification sequence in Pleurodeles waltl is widely used in phylogenetic analyses of amphibian origin and evolution. However, the patterns published to date are far from completely resolved and contain certain discrepancies. Based on a large sample of P. waltl specimens ranging from early post-hatching larvae to post-metamorphic newts, we determined the most common cranial ossification sequence and revealed its intraspecific variations. Since thyroid hormones (THs) are involved in the mediation of skull development in salamanders, we studied the role of THs in the cranial development of P. waltl. The normal sequence and timing of bone appearance were compared with those in larvae reared under conditions of high (in 1 and 2 ng mL -1 triiodothyronine) and low [in 0.02% thiourea (TU), which inhibits thyroid gland activity] TH levels. Metamorphosis was greatly accelerated in the TH-treated larvae and was arrested in the TU-treated larvae, which retained the larval pattern of the palate and rudimentary external gills even after 2 years of the experiment. Early-appearing bones (the coronoid, vomer, palatine, dentary, squamosal, premaxilla, parasphenoid, pterygoid, prearticular, vomer, frontal, parietal, exoccipital, in this order) arise at the same stages and ages, and follow the same ossification sequence under different TH levels. The timing of the appearance of bones normally arising in the late larval and metamorphic periods (the quadratojugal, orbitosphenoid, prootic, maxilla, nasal, os thyroideum, prefrontal, quadrate, in this order) changes depending on the TH level. The maxilla and nasal display the most pronounced reaction to changes in the TH level: they appear precociously in TH-treated animals, while their appearance is postponed and they remain rudimentary in TU-treated animals. Because of different responses to THs, the order in which late-arising bones appear changes depending on the TH level. Although bones appearing early in larval ontogeny (e.g. the premaxilla, vomer, squamosal, palatine) display no TH-induced reaction when they start to develop, their further differentiation shows dependence on THs, and these bones become TH-inducible closer to metamorphosis. These findings indicate that TH involvement in the mediation of cranial development changes from minimal (if at all) in its early stages to maximal during metamorphosis. It is likely that the appearance of bones early in development is mediated by factors other than THs. Their further development is accompanied by changes in the mechanisms mediating their morphological differentiation. That is, likely non-hormonal mediation becomes replaced or/and complemented by hormonal mediation. The constituent parts of the same bone may exhibit differences in their reactions to changes in TH levels. Although in normal development, the overall cranial ossification sequence is constant, there was variation in the order in which late-appearing bones was recorded. These observations suggest that this variation results from individual variability in the internal TH level. Comparison with other salamanders suggests that (a) the pattern of TH mediation described in P. waltl is common for cranial development of metamorphosing urodeles and (b) the same bone may differ in its TH dependence in different salamanders, e.g. there are interspecific variations in the degree of TH dependence of individual cranial bones., (© 2020 Anatomical Society.)

Concern over global amphibian declines and possible links to agrochemical use has led to research on the endocrine disrupting actions of agrochemicals, such as fertilizers, fungicides, insecticides, acaricides, herbicides, metals, and mixtures. Amphibians, like other species, have to partition resources for body maintenance, growth, and reproduction. Recent studies suggest that metabolic impairments induced by endocrine disrupting chemicals, and more particularly agrichemicals, may disrupt physiological constraints associated with these limited resources and could cause deleterious effects on growth and reproduction. Metabolic disruption has hardly been considered for amphibian species following agrichemical exposure. As for metamorphosis, the key thyroid hormone-dependent developmental phase for amphibians, it can either be advanced or delayed by agrichemicals with consequences for juvenile and adult health and survival. While numerous agrichemicals affect anuran sexual development, including sex reversal and intersex in several species, little is known about the mechanisms involved in dysregulation of the sex differentiation processes. Adult anurans display stereotypical male mating calls and female phonotaxis responses leading to successful amplexus and spawning. These are hormone-dependent behaviours at the foundation of reproductive success. Therefore, male vocalizations are highly ecologically-relevant and may be a non-invasive low-cost method for the assessment of endocrine disruption at the population level. While it is clear that agrochemicals disrupt multiple endocrine systems in frogs, very little has been uncovered regarding the molecular and cellular mechanisms at the basis of these actions. This is surprising, given the importance of the frog models to our deep understanding of developmental biology and thyroid hormone action to understand human health. Several agrochemicals were found to have multiple endocrine effects at once (e.g., targeting both the thyroid and gonadal axes); therefore, the assessment of agrochemicals that alter cross-talk between hormonal systems must be further addressed. Given the diversity of life-history traits in Anura, Caudata, and the Gymnophiona, it is essential that studies on endocrine disruption expand to include the lesser known taxa. Research under ecologically-relevant conditions will also be paramount. Closer collaboration between molecular and cellular endocrinologists and ecotoxicologists and ecologists is thus recommended., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Larval metamorphosis and recruitment represent critical life-history transitions for most teleost fishes. While the detrimental effects of anthropogenic stressors on the behavior and survival of recruiting fishes are well-documented, the physiological mechanisms that underpin these patterns remain unclear. Here, we use pharmacological treatments to highlight the role that thyroid hormones (TH) play in sensory development and determining anti-predator responses in metamorphosing convict surgeonfish, Acanthurus triostegus. We then show that high doses of a physical stressor (increased temperature of +3 °C) and a chemical stressor (the pesticide chlorpyrifos at 30 µg L -1 ) induced similar defects by decreasing fish TH levels and affecting their sensory development. Stressor-exposed fish experienced higher predation; however, their ability to avoid predation improved when they received supplemental TH. Our results highlight that two different anthropogenic stressors can affect critical developmental and ecological transitions via the same physiological pathway. This finding provides a unifying mechanism to explain past results and underlines the profound threat anthropogenic stressors pose to fish communities.

Chemical safety evaluation is in the midst of a transition from traditional whole-animal toxicity testing to molecular pathway-based in vitro assays and in silico modeling. However, to facilitate the shift in reliance on apical effects for risk assessment to predictive surrogate metrics having characterized linkages to chemical mechanisms of action, targeted in vivo testing is necessary to establish these predictive relationships. In this study, we demonstrate a means to predict thyroid-related metamorphic success in the model amphibian Xenopus laevis using relevant biochemical measurements during early prometamorphosis. The adverse outcome pathway for thyroperoxidase inhibition leading to altered amphibian metamorphosis was used to inform a pathway-based in vivo study design that generated response-response relationships. These causal relationships were used to develop Bayesian probabilistic network models that mathematically determine conditional dependencies between biochemical nodes and support the predictive capability of the biochemical profiles. Plasma thyroxine concentrations were the most predictive of metamorphic success with improved predictivity when thyroid gland sodium-iodide symporter gene expression levels (a compensatory response) were used in conjunction with plasma thyroxine as an additional regressor. Although thyroid-mediated amphibian metamorphosis has been studied for decades, this is the first time a predictive relationship has been characterized between plasma thyroxine and metamorphic success. Linking these types of biochemical surrogate metrics to apical outcomes is vital to facilitate the transition to the new paradigm of chemical safety assessments., (Published by Oxford University Press on behalf of the Society of Toxicology 2020. This work is written by US Government employees and is in the public domain in the US.)

Cadmium (Cd) is carcinogenic to human and it also has adverse effects on aquatic life such as amphibian larvae. However, its influences on amphibian gut morphology and development as well as intestinal microbiota are still hardly understood. In this study, we examined the effects of chronic cadmium exposure on the gut of tadpoles at Gosner stage 42 of metamorphic climax by using Bufo gargarizans as a model species. Tadpoles were exposed to cadmium concentrations at 0, 5, 100 and 200 μg L -1 from Gosner stage 26-42. The results showed that high cadmium (100 and 200 μg L -1 ) exposure caused significant decrease of body length and weight but significant increase of intestinal length and weight. Moreover, severe histopathological damages were induced by high Cd exposure. In addition, microbial communities in the gut of tadpoles in high cadmium exposure groups were remarkably different from those in control group. Unexpectedly, species diversity and richness were higher in the intestinal microbiota of 200 μg L -1 cadmium exposure group. Furthermore, the abundance of prevalent phyla, families and genera of intestinal microbiota were changed by cadmium exposure. Meanwhile, cadmium exposure perturbed gut renewal functions and the relative mRNA expression of genes involved in canonical and non-canonical Wnt signaling pathway was seriously affected by high cadmium exposure. We concluded that cadmium could be harmful to tadpole health by inducing intestinal histopathological damages, gut remodeling inhibition and intestinal microbiota alterations., Competing Interests: Declaration of competing interest The authors declared no competing interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The adverse effects of triphenyltin (TPT) on aquatic systems have attracted much attention because TPT is widely used and prevalent in aquatic environments. Here, zebrafish embryos/larvae were exposed to TPT (0, 0.039, 0.39, and 3.9 nM; 0, 15, 150 and 1500 ng/L) for 7 or 14 days to determine its toxic effects on the hypothalamic-pituitary-thyroid (HPT) axis. The results showed that whole-body total T4 and T3 levels were significantly decreased, which was accompanied by the significant upregulation of the expression of the dio1, dio2 and ugt1ab genes after exposure to TPT for 7 and 14 days. Genes related to thyroid hormone synthesis (crh, tshβ, nis, tpo and tg) were upregulated at both 7 and 14 days after TPT exposure. This might have been due to the positive feedback regulation of the HPT axis, which is caused by a decrease in thyroid hormone in the whole body in zebrafish. In addition, the survival rates and body lengths were reduced after treatment with TPT for 7 and 14 days. This indicated that TPT caused adverse effect on the development of zebrafish embryos/larvae. In summary, the results suggested that TPT caused thyroid disruption and developmental toxicity in zebrafish larvae., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The effects of microplastic on mortality and sublethal responses on larval development of meroplankton are still largely unknown. Present study investigated the effects of four sizes of virgin spherical polystyrene microplastics (diameter 1.7, 6.8, 10.4, 19.0 μm) on naupliar (stage II-VI) and cypris larvae of barnacle Amphibalanus amphitrite at environmentally relevant concentrations (1, 10, 100, 1000 beads mL -1 ). Essential life-history traits, including mortality, development time and rates of growth, settling, and metamorphosis were measured throughout the entire larval development. Feeding experiments were conducted to evaluate if microplastics decreased naupliar feeding due to physical impacts or selective feeding of nauplii. The results showed that A. amphitrite stage II nauplii were able to ingest and efficiently egest all sizes of microplastics. All the life-history endpoints measured were not significantly affected by all sizes of microplastics at any concentration tested. Presence of all sizes of microplastics did not cause physical interference on naupliar feeding and all stages of nauplius larvae (stage III-VI) did not selectively feed on microplastics. However, the feeding ability of stage III nauplius appeared to be affected by 1.7 μm at 1000 beads mL -1 which was possibly due to individual variations rather than microplastics' impacts. Overall, the full larval development of barnacle A. amphitrite was not affected by microplastics at environmentally relevant concentrations under laboratory condition., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Neonicotinoid insecticides are associated with a decline in the diversity and distribution of bees and wasps (Hymenoptera: Aculeata). The effects of neonicotinoids on the metamorphosis of aculeates have never been addressed in detail; however, recent evidence suggests that neonicotinoids induce wing abnormalities. We hypothesized that the metamorphosis success of bees and wasps differs in response to contact exposure to field-realistic concentrations of neonicotinoid insecticides or in response to combined exposure to neonicotinoid insecticides and benzimidazole fungicides. We treated prepupae of the model crabronid wasp Pemphredon fabricii with field-realistic concentrations of four neonicotinoids, acetamiprid, imidacloprid, thiacloprid and thiamethoxam, and/or with the benzimidazole fungicide thiabendazole. Treatment with acetamiprid or imidacloprid decreased the pupation rates to only 39% and 32%, respectively. Treatment with thiacloprid or thiamethoxam did not affect the pupation rate when applied alone, but the subsequent treatment of thiacloprid- or thiamethoxam-treated prepupae with thiabendazole led to significant decreases in pupation rates. A high concentration of acetamiprid, which severely affected the pupation rates, had moderate effects on metamorphosis into adults, resulting in 53% metamorphosis success (as opposed to 95% metamorphosis success in the water-treated group). However, imidacloprid or thiamethoxam treatment resulted in only 5%-10% metamorphosis success into adults. Overall survival decreased in response to treatment with any of the neonicotinoids or benzimidazoles or their combinations, with extremely low survival (<2%) following combined treatment with imidacloprid and thiabendazole or thiamethoxam and thiabendazole. In conclusion, neonicotinoids alter insect metamorphosis success, which can be further potentiated by their combination with other agrochemicals, such as benzimidazoles.

Anthropogenic activities promote changes in community structure and decrease the species abundance of amphibians. The aim of this study was to assess potential alterations in the antioxidant system and cholinesterase activity, histopathological and oxidative damage in Lithobates catesbeianus tadpoles exposed to water from the Cascavel River, in Southern Brazil. Water samples (140 L each) were collected from the headwater, urban and rural areas of the river. Tadpoles were acclimated for seven days. After acclimatization tadpoles were reared in water from the river, except for the control aquarium. After seven days, a portion of the liver was removed and prepared for cholinesterase (ChE), superoxide dismutase (SOD), catalase (CAT) and lipid peroxidation (LPO) analysis; another part of the tissue was prepared for histological examination. An elevation of CAT activity was observed for water from both urban and rural environments. A decrease in LPO reaction was detected, mainly among the tadpoles exposed to water from the rural area. These alternations can cause delay the metamorphosis and lead to metabolic dysfunction, interfering in survival capacity and diminishing, not only individual fitness, but that of the whole population.

Synthetic compounds that mimic the action of juvenile hormones (JHs) are founding members of a class of insecticides called insect growth regulators (IGRs). Like JHs, these juvenoids block metamorphosis of insect larvae to reproductive adults. Many biologically active juvenoids deviate in their chemical structure considerably from the sesquiterpenoid JHs, raising questions about the mode of action of such JH mimics. Despite the early deployment of juvenoid IGRs in the mid-1970s, their molecular effect could not be understood until recent discoveries of JH signaling through an intracellular JH receptor, namely the ligand-binding transcription factor Methoprene-tolerant (Met). Here, we briefly overview evidence defining three widely employed and chemically distinct juvenoid IGRs (methoprene, pyriproxyfen, and fenoxycarb), as agonist ligands of the JH receptor. We stress that knowledge of the target molecule is critical for using these compounds both as insecticides and as research tools., (© 2019 Wiley Periodicals, Inc.)

The micronucleus test has been applied for more than three decades in tadpoles, generating an early warning of environmental quality. In this study, we reviewed 48 articles on the micronucleus test in tadpoles, published between 1987 and 2018. The findings reveal that pesticides have been the main topic discussed in the induction of micronucleus and other nuclear abnormalities in anuran larvae to the detriment of the widespread use of compounds used in agriculture. In addition to pesticides, a number of other xenobiotic agents have been targeted for genotoxic damage, such as heavy metals, radiation and wastewater. An appeal is reported to environmental contaminants, which when released naturally into the environment or because of human activities may contaminate aquatic habitats, threatening populations of tadpoles that depend on these environments for their survival. Larvae can bioaccumulate these contaminants that cause progressive impacts, ranging from DNA damage to metamorphosis delays, as well as malformations. We found that Argentina is the main driving force for the application of this test in anuran larvae along with Brazil. Different erythrocyte malformations have been reported for the erythrocyte nuclear abnormalities test, binucleated cells, nuclear buds, notched, lobed, reniform, nuclear bebbled, anucleated, picnotic and apoptotic cells are the most cited. In summary, the presence of chemical or physical agents, along with other disturbances of the habitat, can have a significant impact on the life history of the species, contributing to the decline of anuran populations., (Copyright © 2019 Elsevier Ltd. All rights reserved.)