Response characteristic and potential molecular mechanism of tail resorption in Bufo gargarizans after exposure to lead and copper, alone or combined.

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.

Intestinal response of Rana chensinensis larvae exposed to Cr and Pb, alone and in combination.

Although numerous investigations on the adverse impact of Cr and Pb have been performed, studies on intestinal homeostasis in amphibians are limited. Here, single and combined effects of Cr (104 µg/L) and Pb (50 µg/L) on morphological and histological features, bacterial community, digestive enzymes activities, as well as transcriptomic profile of intestines in Rana chensinensis tadpoles were assessed. Significant decrease in the relative intestine length (intestine length/snout-to-vent length, IL/SVL) was observed after exposure to Pb and Cr/Pb mixture. Intestinal histology and digestive enzymes activities were altered in metal treatment groups. In addition, treatment groups showed significantly increased bacterial richness and diversity. Tadpoles in treatment groups were observed to have differential gut bacterial composition from controls, especially for the abundance of phylum Proteobacteria, Firmicutes, Verrucomicrobia, Actinobacteria, and Fusobacteria as well as genus Citrobacter, Anaerotruncus, Akkermansia, and Alpinimonas. Moreover, transcriptomic analysis showed that the transcript expression profiles of GPx and SOD isoforms responded differently to Cr and/or Pb exposure. Besides, transcriptional activation of pro-apoptotic and glycolysis-related genes, such as Bax, Apaf 1, Caspase 3, PK, PGK, TPI, and GPI were detected in all treatment groups but downregulation of Bcl2 in Pb and Cr/Pb mixture groups. Collectively, these results suggested that Cr and Pb exposure at environmental relevant concentration, alone and in combination, could disrupt intestinal homeostasis of R. chensinensis tadpoles.

Computational Investigation of the Bisphenolic Drug Metabolism by Cytochrome P450: What Factors Favor Intramolecular Phenol Coupling.

Intramolecular phenol coupling reactions of alkaloids can lead to active metabolites catalyzed by the mammalian cytochrome P450 enzyme (P450); however, the mechanistic knowledge of such an "unusual" process is lacking. This work performs density functional theory computations to reveal the P450-mediated metabolic pathway leading from R-reticuline to the morphine precursor salutaridine by exploring possible intramolecular phenol coupling mechanisms involving diradical coupling, radical addition, and electron transfer. The computed results show that the outer-sphere electron transfer with a high barrier (>20.0 kcal/mol) is unlikely to happen. However, for inter-sphere intramolecular phenol coupling, it reveals that intramolecular phenol coupling of R-reticuline proceeds via the diradical mechanism consecutively by compound I and protonated compound II of P450 rather than the radical addition mechanism. The existence of a much higher radical rebound barrier than that of H-abstraction in the quartet high-spin state can endow the R-reticuline phenoxy radical with a sufficient lifetime to enable intramolecular phenol coupling, while the H-abstraction/radical rebound mode with a negligible rebound barrier leading to phenol hydroxylation can only happen in the doublet low-spin state. Therefore, the ratio [coupling]/[hydroxylation] can be approximately reflected by the relative yield of the high-spin and low-spin H-abstraction by P450, which thus can provide a theoretical ratio of 16:1 for R-reticuline, which is in accordance with previous experimental results. Especially, the high rebound barrier of the phenoxy radical derived from the weak electron-donating ability of the phenoxy radical is revealed as an intrinsic nature. Therefore, the revealed intramolecular phenol coupling mechanism can be potentially extended to several other bisphenolic drugs to infer groups of unexpected metabolites in organisms.

Using Physical Organic Chemistry Knowledge to Predict Unusual Metabolites of Synthetic Phenolic Antioxidants by Cytochrome P450.

Biotransformation, especially by human CYP450 enzymes, plays a crucial role in regulating the toxicity of organic compounds in organisms, but is poorly understood for most emerging pollutants, as their numerous "unusual" biotransformation reactions cannot retrieve examples from the textbooks. Therefore, in order to predict the unknown metabolites with altering toxicological profiles, there is a realistic need to develop efficient methods to reveal the "unusual" metabolic mechanism of emerging pollutants. Combining experimental work with computational predictions has been widely accepted as an effective approach in studying complex metabolic reactions; however, the full quantum chemical computations may not be easily accessible for most environmentalists. Alternatively, this work practiced using the concepts from physical organic chemistry for studying the interrelationships between structure and reactivity of organic molecules, to reveal the "unusual" metabolic mechanism of synthetic phenolic antioxidants catalyzed by CYP450, for which the simple pencil-and-paper and property-computation methods based on physical organic chemistry were performed. The phenol-coupling product of butylated hydroxyanisole (BHA) (based on spin aromatic delocalization) and ipso-addition quinol metabolite of butylated hydroxytoluene (BHT) (based on hyperconjugative effect) were predicted as two "unusual" metabolites, which were further confirmed by our in vitro analysis. We hope this easily handled approach will promote environmentalists to attach importance to physical organic chemistry, with an eye to being able to use the knowledge gained to efficiently predict the fates of substantial unknown synthesized organic compounds in the future.

Precision Biotransformation of Emerging Pollutants by Human Cytochrome P450 Using Computational-Experimental Synergy: A Case Study of Tris(1,3-dichloro-2-propyl) Phosphate.

Precision biotransformation is an envisioned strategy offering detailed insights into biotransformation pathways in real environmental settings using experimentally guided high-accuracy quantum chemistry. Emerging pollutants, whose metabolites are easily overlooked but may cause idiosyncratic toxicity, are important targets of such a strategy. We demonstrate here that complex metabolic reactions of tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) catalyzed by human CYP450 enzymes can be mapped via a three-step synergy strategy: (i) screening the possible metabolites via high-throughout (moderate-accuracy) computations; (ii) analyzing the proposed metabolites in vitro by human liver microsomes and recombinant human CYP450 enzymes; and (iii) rationalizing the experimental data via precise mechanisms using high-level targeted computations. Through the bilateral dialogues from qualitative to semi-quantitative to quantitative levels, we show how TDCIPP metabolism especially by CYP3A4 generates bis(1,3-dichloro-2-propyl) phosphate (BDCIPP) as an O-dealkylation metabolite and bis(1,3-dichloro-2-propyl) 3-chloro-1-hydroxy-2-propyl phosphate (alcoholß-dehalogen) as a dehalogenation/reduction metabolite via the initial rate-determining H-abstraction from αC- and ßC-positions. The relative yield ratio [dehalogenation/reduction]/[O-dealkylation] is derived from the relative barriers of H-abstraction at the ßC- and αC-positions by CYP3A4, estimated as 0.002 to 0.23, viz., an in vitro measured ratio of 0.04. Importantly, alcoholß-dehalogen formation points to a new mechanism involving successive oxidation and reduction functions of CYP450, with its precursor aldehydeß-dehalogen being a key intermediate detected by trapping assays and rationalized by computations. We conclude that the proposed three-step synergy strategy may meet the increasing challenge of elucidating biotransformation mechanisms of substantial synthesized organic compounds in the future.

Copper-induced sublethal effects in Bufo gargarizans tadpoles: growth, intestinal histology and microbial alternations.

Copper (Cu) is one of the environmental contaminations which can pose significant risks for organisms. The current study explores the effects of Cu exposure on the growth, intestinal histology and microbial ecology in Bufo gargarizans. The results revealed that 0.5-1 µM Cu exposure induced growth retardation (including reduction of total body length and wet weight) and intestinal histological injury (including disordered enterocyte, changes in the villi and vacuoles) of tadpoles. Also, high-throughput sequencing analysis showed that Cu exposure caused changes in richness, diversity and structure of intestinal microbiota. Moreover, the composition of intestinal microbiota was altered in tadpoles exposed to different concentrations of Cu. At the phylum level, we observed the abundance of proteobacteria was increased, while the abundance of fusobacteria was decreased in the intestinal microbiota of tadpoles exposed to 1 µM Cu. At the genus level, a reduced abundance of kluyvera and aeromonas was observed in the intestinal microbiota of tadpoles under the exposure of 0-0.5 µM Cu. Finally, functional predictions revealed that tadpoles exposed to copper may be at a higher risk of developing metabolic disorders or diseases. Above all, our results will develop a comprehensive view of the Cu exposure in amphibians and will yield a new consideration for sublethal effects of Cu on aquatic organisms.

Biotransformation Mechanism of Pesticides by Cytochrome P450: A DFT Study on Dieldrin.

Pesticide biotransformation, especially by cytochrome P450 enzymes (CYPs), may produce metabolites with substantially altered toxicological and physicochemical profiles, which has drawn great attention as a basis for environmental risk assessment. CYPs are active in the metabolism of various reactions of pesticides, and there are potentially different short-lived oxidant species in CYPs (Compound I vs Compound 0), which make elucidating their biotransformation mechanism challenging. To facilitate this task, we performed density functional theory (DFT) calculations to explore the puzzling bifurcation pathways of dieldrin by CYPs. The results show that the two-oxidant mechanism does not work, while the bifurcation pathways are within the mechanistic framework of a two-state reactivity of Compound I. Specifically, 9-hydroxy-dieldrin as a hydroxylation product is formed via H-abstraction and essentially barrierless C-9 alkyl radical rebound in the doublet state; while 3-ketone-dieldrin as a dechlorination product is formed via H-abstraction, C-9 alkyl radical cyclization, and C-3 cyclized radical rebound in the quartet state followed by HCl elimination, originating from a significant barrier for C-9 alkyl radical rebound in the quartet state to provide this radical sufficient lifetime for cyclization. Thus, the ratio [dechlorination]/[hydroxylation] can be estimated as 1:35, consistent with the experimental findings. We envision that application of computational chemistry has a great potential in revealing the complex biotransformation mechanisms of pesticides.

Computational Biotransformation Profile of Emerging Phenolic Pollutants by Cytochromes P450: Phenol-Coupling Mechanism.

Phenols are ubiquitous environmental pollutants, whose biotransformation involving phenol coupling catalyzed by cytochromes P450 may produce more lipophilic and toxic metabolites. Density functional theory (DFT) computations were performed to explore the debated phenol-coupling mechanisms, taking triclosan as a model substrate. We find that a diradical pathway facilitated by compound I and protonated compound II of P450 is favored vs alternative radical addition or electron-transfer mechanisms. The identified diradical coupling resembles a "two-state reactivity" from compound I characterized by significantly high rebound barriers of the phenoxy radicals, which can be formulated into three equations for calculating the ratio [coupling]/[hydroxylation]. A higher barrier for rebound than for H-abstraction in high-spin triclosan can facilitate the phenoxy radical dissociation and thus enable phenol coupling, while H-abstraction/radical rebound causing phenol hydroxylation via minor rebound barriers mostly occurs via the low-spin state. Therefore, oxidation of triclosan by P450 fits the first equation with a ratio [coupling]/[hydroxylation] of 1:4, consistent with experimental data indicating different extents of triclosan coupling (6-40%). The high rebound barrier of phenoxy radicals, as a key for the mechanistic identification of phenol coupling vs hydroxylation, originates from their weak electron donor ability due to spin aromatic delocalization. We envision that the revealed mechanism can be extended to the cross-coupling reactions between different phenolic pollutants, and the coupling reactions of several other aromatic pollutants, to infer unknown metabolites.

Changes in intestinal microbiota of Bufo gargarizans and its association with body weight during metamorphosis.

The assembly of intestinal microbial communities can play major roles in animal development. We hypothesized that intestinal microbial communities could mirror the developmental programs of amphibian metamorphosis. Here, we surveyed the morphological parameters of the body and intestine of Bufo gargarizans at varying developmental stages and inventoried the intestinal microbial communities of B. gargarizans at four key developmental stages via 16S rDNA gene sequencing. Firstly, our survey showed that during metamorphosis, body weight and intestinal weight were reduced by 56.8 and 91.8%, respectively. Secondly, the gut bacterial diversity of B. gargarizans decreased with metamorphosis and the composition of the tadpoles' intestinal microbiota varied across metamorphosis. Compared to aquatic larvae, terrestrial juveniles showed major shifts in microbial composition, including reduction in Proteobacteria and Actinobacteria, increases in Bacteroidetes and Fusobacteria, and the appearance of Verrucomicrobia. Firmicutes in four developmental stages showed similar abundance at the phylum level, but in each stage was driven by distinct genera. Enterobacter, Aeromonas, Mucinivorans and Bacteroides also changed in abundance and were found to be significantly correlated with loss of body or intestinal tissue during metamorphosis. These results indicate a shift in intestinal microbial community composition throughout amphibian metamorphosis.

Metabolic Mechanism of Aryl Phosphorus Flame Retardants by Cytochromes P450: A Combined Experimental and Computational Study on Triphenyl Phosphate.

Understanding metabolic mechanisms is critical and remains a difficult task in the risk assessment of emerging pollutants. Triphenyl phosphate (TPHP), a widely used aryl phosphorus flame retardant (aryl-PFR), has been frequently detected in the environment, and its major metabolite was considered as diphenyl phosphate (DPHP). However, knowledge of the mechanism for TPHP leading to DPHP and other metabolites is lacking. Our in vitro study shows that TPHP is metabolized into its diester metabolite DPHP and mono- and dihydroxylated metabolites by cytochromes P450 (CYP) in human liver microsomes, while CYP1A2 and CYP2E1 isoforms are mainly involved in such processes. Molecular docking gives the conformation for TPHP binding with the active species Compound I (an iron IV-oxo heme cation radical) in specific CYP isoforms, showing that the aromatic ring of TPHP is likely to undergo metabolism. Quantum chemical calculations have shown that the dominant reaction channel is the O-addition of Compound I onto the aromatic ring of TPHP, followed by a hydrogen-shuttle mechanism leading to ortho-hydroxy-TPHP as the main monohydroxylated metabolite; the subsequent H-abstraction-OH-rebound reaction acting on ortho-hydroxy-TPHP yields the meta- and ipso-position quinol intermediates, while the former of which can be metabolized into dihydroxy-TPHP by fast protonation, and the latter species needs to go through type-I ipso-substitution and fast protonation to be evolved into DPHP. We envision that the identified mechanisms may give inspiration for studying the metabolism of several other aryl-PFRs by CYP.

Endocrine disruption, oxidative stress and lipometabolic disturbance of Bufo gargarizans embryos exposed to hexavalent chromium.

The aim of the current study was to determine the potential developmental and metabolic abnormalities caused by Cr (VI) exposure on Bufo gargarizans (B. gargarizans) embryos. B. gargarizans embryos were treated with different concentrations of Cr (VI) (13, 52, 104, 208, and 416 µg Cr6+ L-1) for 6 days. Morphological abnormalities, total length, weight and developmental stage were monitored. Malformations of embryos were also examined using scanning electron microscopy (SEM). In addition, the transcript levels of several genes associated with lipid metabolism, oxidative stress, and thyroid hormones signaling pathways were also determined. Our results showed a time-dependent inhibitory effect of Cr (VI) on the growth and development of B. gargarizans embryos. On day 4, total length, weight, and developmental stage were significantly lower at 416 µg Cr6+ L-1 relative to control embryos. On day 6, significant reductions in total length, weight, and developmental stage were observed at 104, 208, and 416 µg Cr6+ L-1. Malformed embryos were found in all Cr (VI) treatments, which were characterized by axial flexures, yolk sac edema and rupture, surface tissue hyperplasia, stunted growth, wavy fin and fin flexure. RT-qPCR results showed that exposure to Cr (VI) down-regulated TRß and Dio2 mRNA expression and up-regulated Dio3 mRNA level at 416 µg Cr6+ L-1. The transcript levels of SOD and GPx were upregulated at 52, 208, and 416 µg Cr6+ L-1, while the transcript level of HSP90 was downregulated at 52, 208, and 416 µg Cr6+ L-1. Also, mRNA expression of lipid synthesis-related genes (FAE and ACC) were significantly downregulated in embryos treated with 208 and 416 µg Cr6+ L-1, but mRNA expression of fatty acid ß-oxidation-related genes (ACOX, CPT, and SCP) was significantly upregulated at 416 µg Cr6+ L-1. Therefore, our results suggested that Cr (VI) could disrupt thyroid endocrine pathways and lipid synthesis, leading to the inhibition of growth and development in B. gargarizans embryos. Furthermore, the decreased ability of scavenging ROS induced by Cr (VI) might be responsible for the teratogenic effects of Cr (VI).

Chronic Effects of Fluoride Exposure on Growth, Metamorphosis, and Skeleton Development in Bufo gargarizans Larvae.

Bufo gargarizans tadpoles were chronically exposed to waterborne fluoride at measured concentrations ranging from 0.4 to 61.2 mg F-/L for 70 days from Gosner stage 26 to completion of metamorphosis. The chronic exposure caused a concentration-dependent mortality in all tested fluoride concentrations. Total length, snout-to-vent length (SVL), body mass, and developmental stage of tadpoles were significantly inhibited at 42.6 mg F-/L. In addition, significant metamorphic delay and increase in size at completion of metamorphosis occurred after exposure to 19.8 mg F-/L. Moreover, 19.8 mg F-/L suppressed the bone mineralization of larvae at completion of metamorphosis. However, the bone mineralization could be enhanced by 4.1 mg F-/L. In conclusion, our results suggested that the presence of high concentrations of fluoride could increase mortality risk, delay metamorphosis, and suppress skeletal ossification in B. gargarizans larvae.

Effects of fluoride on development and growth of Rana chensinensis embryos and larvae.

The present study examined the adverse effects of fluoride exposure on embryos and larvae of Rana chensinensis. Survival, morphological abnormalities, growth and development, time to metamorphosis and size at metamorphic climax of R. chensinensis were examined. Our results showed that embryos malformation occurred in all fluoride treatments. Morphological abnormalities of embryos are characterized by axial flexures, the extrusion of fin axis, edema, and ruffled dorsal and ventral fin. Additionally, 4.1mg F(-)/L and above could significantly inhibit embryos growth and development. On day 15, total length and weight of tadpole were significantly lower in 19.6 and 42.4 mg F(-)/L treatments compared to control. However, significant reductions in total length and weight were observed only at 42.4 mg F(-)/L on day 30. Moreover, significant metamorphic delay and decrease in the size at metamorphic climax were found in larvae exposed to 42.4 mg F(-)/L. Taken together, embryos of R. chensinensis are more vulnerable to fluoride exposure than their tadpoles. Our results suggested that the presence of high concentrations fluoride might increase mortality risk and a reduction in juvenile recruitment in the field by increasing embryos malformation, delaying metamorphosis and decreasing size at metamorphosis.

Chronic effects of triclosan on embryonic development of Chinese toad, Bufo gargarizans.

Triclosan (TCS) is commonly used worldwide in a range of personal care and sanitizing products. The aim of this study was to evaluate potential effects of TCS exposure on embryonic development of Bufo gargarizans, an endemic frog species in China. Standard Gosner stage 3 B. gargarizans embryos were exposed to 10 ~ 150 µg/L TCS during embryogenesis. Survival, total length, weight, developmental stage, duration of different embryo stages, malformation, and type II and III deiodinase (D2 and D3) expression were measured. Inhibitory effects on embryo developmental stage, total length and weight were found at 30 ~ 150 µg/L TCS. Moreover, the duration of embryonic development was increased at gastrula, neural, circulation, and operculum development stage in TCS-treated groups. In addition, TCS exposure induced morphological malformations in B. gargarizans embryos, which are characterized by hyperplasia, abdominal edema, and axial flexures. Furthermore, our results showed that the expression of D2 in embryos was probably down-regulated at 60 and 150 µg/L TCS, but its spatial expression patterns was not affected by TCS. In summary, our study suggested that TCS exposure not only resulted in delayed growth and development but also caused teratogenic effects in B. gargarizans embryos, and the developmental effects of TCS at high concentrations may be associated with disruption of THs homeostasis. Although further studies are necessary, the present findings could provide a basis for understanding on harmful effects and the potential mechanisms of TCS in amphibian embryos.

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

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

[Preparation of Chitosan-modified Biochar and Its Adsorption Mechanism for Cd2+ in Aqueous Solution].

Chitosan-modified biochar （CBC） was prepared as a low-cost and highly efficient adsorbent for Cd2+ in aqueous solutions. Batch adsorption experiments were conducted to evaluate the adsorption performance. Characterization experiments with SEM-EDS， FTIR， and XPS were used to analyze the surface microstructure and chemical composition of the adsorbent. The results showed that the adsorption performance of CBC was remarkably improved by the introduction of surface functional groups （-OH， -C=O， and -NH2）. The pseudo-second-order kinetic model and Langmuir model were better for describing the kinetics and isotherms for Cd2+ adsorption onto CBC， indicating that the adsorption rate was determined by the active sites and controlled by monolayer chemisorption. The adsorption process was endothermic spontaneous， and the key mechanisms involved complexation， precipitation， cation exchange， and cation-π bonds. After five instances of adsorption-desorption cycles， the adsorption capacity of CBC for Cd2+ still remained above 80% of the initial adsorption capacity， indicating that CBC had a favorable recyclability. The current work embodies the concept of green chemistry， and the prepared chitosan-modified biochar was a promising adsorbent for the removal of Cd2+ in wastewater and soil.

Effects of fluoride on metamorphosis, thyroid and skeletal development in Bufo gargarizans tadpoles.

This study examined the effects of chronic fluoride exposure on metamorphosis, thyroid and skeletal development in tadpoles of Chinese Toad, Bufo gargarizans. The tadpoles were exposed to fluoride concentrations either at 0, 1, 5, 10, or at 50 mg L(-1) from Gosner stage 26 to Gosner stage 42. Body weight, total length and percentage of tadpoles reaching metamorphosis climax were recorded, and thyroid histological examinations were employed. In addition, mRNA expression of both deiodinase type 2 (D2) and deiodinase type 3 (D3) was analyzed by using RT-PCR and skeletal systems were investigated by using double-staining methodology at stage 42. Results showed that total length and body weight were unaffected by fluoride exposure at all concentrations while metamorphosis was strongly inhibited only by 50 mg L(-1) fluoride. Histomorphological measurements showed the percentage of colloid depletion in thyroid gland increased significantly, while the average diameter of follicles was significantly shorter at 50 mg L(-1) concentration. In addition, fluoride at 5 mg L(-1) can stimulate bone mineralization, while fluoride at 50 mg L(-1) can retard deposition of calcium. In conclusion, our study suggests that 50 mg L(-1) fluoride could damage follicular cells in thyroid gland and induce a sharp reduction in thyroid hormone probably through the up-regulation of D3 mRNA expression, and these influences on thyroid system may delay metamorphosis as well as ossification in bone tissue by inhibiting calcium deposition.

Comparison of the characteristics of gut microbiota response to lead in Bufo gargarizans tadpole at different developmental stages.

In amphibians, lead (Pb) exposure could alter the composition and structure of gut microbiota, but changes involving microbiota of several successive phases following Pb exposure have been less studied. In the present study, we compared the effects of Pb exposure on morphological parameters and gut microbiota of Bufo gargarizans at Gosner stage (Gs) 33, Gs36, and Gs42. Our results showed that total length (TL), snout-vent length (SVL), and body wet weight (TW) of B. gargarizans at Gs33, as well as TL and SVL at Gs42, were significantly increased after Pb exposure. In addition, high-throughput sequencing analysis indicated that gut microbiota has distinct responses to Pb exposure at different developmental stages. The diversity of gut microbiota was significantly reduced under Pb exposure at Gs33, while it was significantly increased at Gs42. In terms of community composition, Spirochaetota, Armatimonadota, and Patescibacteria appeared in the control groups at Gs42, but not after Pb treatment. Furthermore, functional prediction indicated that the relative abundance of metabolism pathway was significantly decreased at Gs33 and Gs36, and significantly increased at Gs42. Our results fill an important knowledge gap and provide comparative information on the gut microbiota of tadpoles at different developmental stages following Pb exposure.

Copper-mediated shifts in transcriptomic responses of intestines in Bufo gargarizans tadpoles to lead stress.

The differential transcriptomic responses of intestines in Bufo gargarizans tadpoles to Pb alone or in the presence of Cu were evaluated. Tadpoles were exposed to 30 µg/L Pb individually and in combination with Cu at 16 or 64 µg/L from Gosner stage (Gs) 26 to Gs 38. After de novo assembly, 105,107 unigenes were generated. Compared to the control group, 7387, 6937, and 11139 differentially expressed genes (DEGs) were identified in the treatment of Pb + Cu0, Pb + Cu16, and Pb + Cu64, respectively. In addition, functional annotation and enrichment analysis of DEGs revealed substantial transcriptional reprogramming of diverse molecular and biological pathways were induced in all heavy metal treatments. The relative expression levels of genes associated with intestinal epithelial barrier and bile acids (BAs) metabolism, such as mucin2, claudin5, ZO-1, Asbt, and Ost-ß, were validated by qPCR. This study demonstrated that Pb exposure induced transcriptional responses in tadpoles, and the responses could be modulated by Cu.

Effects of temperature on intestinal microbiota and lipid metabolism in Rana chensinensis tadpoles.

Climate change such as global warming is considered a major threat to amphibians. The guts of amphibians are home to trillions of microbes, which are key regulators of gastrointestinal digestion and play a crucial role in lipid metabolites. The aim of this study was to evaluate the effect of temperature change on intestinal microbiota and lipid metabolism in Rana chensinensis tadpoles. Morphological and intestinal microbiota data of R. chensinensis larvae exposed to different temperatures (15 °C, 21 °C, and 26 °C) were measured. The results show that the warm temperature causes histological damage to the intestinal epithelium. In addition, temperature treatments alter the diversity and composition of gut microbes in R. chensinensis tadpoles. At the phylum level of intestinal microbial community, Campilobacterota was detected only in the warm group. At the genera level, unclassified_f__Enterobacteriaceae was markedly declined in the warm group but was notably enriched in the cold group. For lipid metabolism-related genes, the expression levels of GPR109A, HDAC1, and APOA-I decreased significantly in both warm and cold treatment groups, while the expression levels of CLPS and LIPASE increased significantly. Collectively, these observations demonstrated that warm and cold temperatures may reduce the immune capacity of tadpoles by changing the composition of intestinal microorganisms and the expression of genes related to lipid metabolism, affecting the survival of tadpoles.