Developmental temperature modulates microplastics impact on amphibian life history without affecting ontogenetic microplastic transfer.

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.

Cadmium bioaccumulation dynamics during amphibian development and metamorphosis.

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.

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.

Hydroxylated polychlorinated biphenyls may affect the thyroid hormone-induced brain development during metamorphosis of Xenopus laevis by disturbing the expression of matrix metalloproteinases.

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 T3 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.

Transcriptomic profiling of Rana [Lithobates] catesbeiana back skin during natural and thyroid hormone-induced metamorphosis under different temperature regimes with particular emphasis on innate immune system components.

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.

How the Xenopus eleutheroembryonic thyroid assay compares to the amphibian metamorphosis assay for detecting thyroid active chemicals.

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.

Comparing the effects and potencies of perchlorate and nitrate on amphibian metamorphosis using a modified amphibian metamorphosis assay (AMA).

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 (ClO4 -) or nitrate (NO3 -) for 32 days. Larvae were exposed to 0.0 (control), 5, 25, 125, 625, and 3125 µg/L ClO4 -, or 0 (control), 23, 71, 217, 660, and 2000 mg/L NO3 -. The primary endpoints were survival, hind limb length (HLL), forelimb emergence and development, developmental stage (including time to NF stage 62 [MT62]), thyroid histopathology, wet weight, and snout-vent length (SVL). Developmental delay as evidenced by altered stage distribution and increased MT62, 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 ClO4 -. The no observed effect concentration (NOEC) for developmental endpoints was 25.0 µg/L ClO4 - and the NOEC for growth endpoints was 3125 µg/L ClO4 -. Exposure to nitrate did not adversely affect MT62, but a decreasing trend in stage distribution and median developmental stage at ≥217 mg/L NO3 - was observed. No histopathologic effects associated with nitrate exposure were observed. An increasing trend in SVL-normalized HLL was observed at 2000 mg/L NO3 -. Nitrate did not alter larval growth. The NOEC for developmental endpoints was 71 mg/L NO3 -, and 2000 mg/L NO3 - 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.

The role of phenotypic plasticity and corticosterone in coping with pond drying conditions in yellow-bellied toad (Bombina variegata, Linnaeus 1758) tadpoles.

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.

Long-term, landscape-level assessment of aquatic pesticide exposure to identify amphibian ontological traits affecting vulnerability.

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.

Evaluating potential developmental toxicity of perfluoroalkyl and polyfluoroalkyl substances in Xenopus laevis embryos and larvae.

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.

The glutathione biosynthesis is involved in metamorphosis, antioxidant function, and insecticide resistance in Tribolium castaneum.

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.

Effects of perchlorate and exogenous T4 exposures on development, metamorphosis and endochondral ossification in Bufo gargarizans larvae.

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.

Drosophila E93 promotes adult development and suppresses larval responses to ecdysone during metamorphosis.

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 â€‹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.

Tissue and salinity specific Na+/Cl- cotransporter (NCC) orthologues involved in the adaptive osmoregulation of sea lamprey (Petromyzon marinus).

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 (Isc) in the proximal and distal intestine, respectively. Luminal application of bumetanide (an NKCC inhibitor) did not affect intestinal Isc. 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.

Early-life exposure to Ivermectin alters long-term growth and disease susceptibility.

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.

Larval metamorphosis is inhibited by methimazole and propylthiouracil that reveals possible hormonal action in the mussel Mytilus coruscus.

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.

Biochemical and developmental effects of thyroid and anti-thyroid drugs on different early life stages of Xenopus laevis.

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.

The plant-derived triterpenoid, cucurbitacin B, but not cucurbitacin E, inhibits the developmental transition associated with ecdysone biosynthesis in Drosophila melanogaster.

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.

Adenosine Triggers Larval Settlement and Metamorphosis in the Mussel Mytilopsis sallei through the ADK-AMPK-FoxO Pathway.

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.

Effect of ethylenethiourea on metamorphosis and ovary development: A comparative study of three larval frogs.

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.