Bile acids (BAs) are cholesterol derivatives synthesized by the liver, exhibit variation between different species. Researchers have long appreciated that microbiota play the roles in the biotransformation of BAs. However, relatively few studies have been reported on microbial-mediated production and transformation of BAs in amphibians. Our focus here is principally on difference of intestinal microbial diversity and BAs profiles between two common amphibians, Bufo gargarizans (B. gargarizans) and Rana chensinensis (R. chensinensis) tadpoles, through intestinal targeted BAs metabolomics and fecal metagenomic sequencing. The results demonstrated that B. gargarizans possessed higher levels of total BAs and higher ratio of unconjugated / conjugated BAs. In addition, the relative abundance of microbiota with bile salt hydrolase (BSH) activity in B. gargarizans was significantly higher than that of R. chensinensis, which may facilitate the conversion of conjugated to unconjugated BAs. Meanwhile the higher prevalence of bile-acid-induced (BAI) gene encoding microbiota in R. chensinensis may promote the synthesis of deoxycholic acid (DCA). Furthermore, discrepancies in virulence factors (VFs) and energy metabolism were observed between the two species, which may be linked to differences in the microbiota. This study revealed substantial differences in intestinal microbes and BAs across amphibian species, emphasizing the significant impact of intestinal microbes on BAs metabolism.

Amphibians are extremely sensitive to environmental changes, and their gut microbiome may have different responses to environmental changes. Here, metagenomic sequencing was used to investigate the intestinal microbiota of the Asiatic toad (Bufo gargarizans) from three different habitats (city areas, transition areas, and wild areas) of Sichuan Province, China. The results showed that Proteobacteria, Firmicutes, and Fusobacteria were the main bacteria in the gut of B. gargarizans. There were significant differences in the composition and function of the gut microbiome among the samples from the three different habitats. Enterobacteriaceae showed significant changes in the three habitats and occupied a high relative abundance in the city areas, especially for Citrobacter. Especially, antibiotic resistance genes (ARGs) and virulence factors (VFs) were significantly increased in city areas. We performed de novo assembly of the metagenome-assembled genomes (MAGs). In total, 322 nonredundant MAGs were reconstructed, 304 of which might be potential novel genomes. Among the 13 species-level genome bins (SGBs) belonging to Enterobacteriaceae, the one belonging to Citrobacter portucalensis annotated the most types of ARGs and VFs. Phylogenetic and functional analyses of the assembled C. portucalensis MAG and public genome data were carried out, suggesting that it may play a potential role in intestinal diseases in amphibians. Our study revealed the differences in the gut microbiome of B. gargarizans across different habitats and suggests that amphibian intestinal microbiota could serve as environmental indicators to reflect environmental changes and human activities. The reconstructed MAGs expanded our understanding of the gut microbiota in amphibians, which may serve as a substantial reservoir for microbiome resources.

Lead (Pb), a prevalent heavy metal contaminant in aquatic environments, has complex effects on the gut microbiome function of aquatic animals. In this study, metagenomic analysis of Bufo gargarizans tadpoles was carried out following Pb exposure. Moreover, histological analysis was performed on the intestines. The results showed that Pb exposure induced histological damage to the intestinal epithelium. Significant differences in microbial abundance and function were detected in the 200 μg/L Pb group compared to the control group. Specifically, an increase in Bosea and Klebsiella was noted at 200 μg/L Pb, which potentially could induce inflammation in tadpoles. Notably, the decrease in the abundance of glycoside hydrolases subsequent to exposure to 200 μg/L Pb is likely to attenuate carbohydrate metabolism. Furthermore, increased fluoroquinolone-related antibiotic resistance genes (ARGs), phenolic-related ARGs, and iron uptake systems following 200 μg/L Pb exposure might heighten the disease risk for tadpoles. These discoveries augment our comprehension of the influences of Pb on the intestinal well-being of amphibians and offer valuable insights for further assessment of the ecological risks that Pb poses to amphibians.

Environmental heavy metal contamination, combined with inappropriate use of fungicides, has led to the co-existence of lead (Pb) and iprodione (IPR), presenting signification risks to ecosystems and human health. The toxic effects resulting from concurrent exposure to Pb and IPR, however, remain poorly understood. In the study, we conducted a comprehensive 60-day subchronic study to investigate the toxic effects on the liver and gut in parental male zebrafish through employing multi-omics analyses. We also explored the potential transgenerational toxicity to unexposed offspring embryos. The results demonstrated that exposure to both Pb and IPR exacerbated intestinal pathological damage, decreased the expression of intestinal tight junction molecules, and activated the expression of intestinal inflammatory molecules in the gut. Metabolic and microbial analyses, utilizing 16S rRNA sequencing and non-targeted metabolic profiling, revealed alterations in the intestinal flora structure and disruptions in metabolite synthesis. Notably, we observed a significant negative correlation between the abundance of the Lactobacillus genus and uracil synthesis. Furthermore, liver RNA-seq analysis identified a marked enrichment of the P53 signaling pathway, confirmed by the activation of P53-mediated apoptotic markers, which was consistent with the observed increase in inflammatory infiltration and pathological damage within the liver. Importantly, P53-mediated apoptosis and inflammatory responses were activated in offspring embryos, suggesting that long-term parental exposure to Pb and IPR may induce transgenerational toxicity, potentially impacting offspring health. Despite the identification of these molecular changes, the phenotypic effects remain to be elucidated. Future studies are necessary to evaluate the potential phenotypic changes in offspring to fully understand the long-term effects of Pb and IPR exposure. Overall, these findings enhance the understanding of the molecular mechanisms underlying the toxic effects of Pb and IPR and emphasize the importance of a comprehensive risk assessment of environmental pollutants.

The gut microbiota is the largest and most complex microecosystem in animals. It is influenced by the host's dietary habits and living environment, and its composition and diversity play irreplaceable roles in animal nutrient metabolism, immunity, and adaptation to the environment. Although the gut microbiota of red deer has been studied, the composition and function of the gut microbiota in Gansu red deer (Cervus elaphus kansuensis), an endemic subspecies of red deer in China, has not been reported. In this study, the composition and diversity of the gut microbiome and fecal metabolomics of C. elaphus kansuensis were identified and compared for the first time by using 16S rDNA sequencing, metagenomic sequencing, and LC-MS/MS. There were significant differences in gut microbiota structure and diversity between wild and farmed C. elaphus kansuensis. The 16S rDNA sequencing results showed that the genus UCRD-005 was dominant in both captive red deer (CRD) and wild red deer (WRD). Metagenomic sequencing showed similar results to those of 16S rDNA sequencing for gut microbiota in CRD and WRD at the phylum and genus levels. 16S rDNA and metagenomics sequencing data suggested that Bacteroides and Bacillus might serve as marker genera for CRD and WRD, respectively. Fecal metabolomics results showed that 520 metabolites with significant differences were detected between CRD and WRD and most differential metabolites were involved in lipid metabolism. The results suggested that large differences in gut microbiota composition and fecal metabolites between CRD and WRD, indicating that different dietary habits and living environments over time have led to the development of stable gut microbiome characteristics for CRD and WRD to meet their respective survival and reproduction needs. KEY POINTS: • Environment and food affected the gut microbiota and fecal metabolites in red deer • Genera Bacteroides and Bacillus may play important roles in CRD and WRD, respectively • Flavonoids and ascorbic acid in fecal metabolites may influence health of red deer.

Lead (Pb) is a ubiquitously detected heavy metal pollutant in aquatic ecosystems. Previous studies focused mainly on the response of gut microbiota to Pb stress, with less emphasis on gene expression in intestine, thereby limiting the information about impacts of Pb on intestinal homeostasis in amphibians. Here, microbial community and transcriptional response of intestines in Rana zhenhaiensis tadpoles to Pb exposure were evaluated. Our results showed that 10 μg/L Pb significantly decreased bacterial diversity compared to the controls by the Simpson index. Additionally, 1000 μg/L Pb exposure resulted in a significant reduction in the abundance of Fusobacteriota phylum and Cetobacterium genus but a significant expansion in Hafnia-Obesumbacterium genus. Moreover, transcriptome analysis revealed that about 90 % of the DEGs (8458 out of 9450 DEGs) were down-regulated in 1000 μg/L Pb group, mainly including genes annotated with biological functions in fatty acid degradation, and oxidative phosphorylation, while up-regulated DEGs involved in metabolism of xenobiotics by cytochrome P450. The expression of Gsto1, Gsta5, Gstt4, and Nadph showed strong correlation with the abundance of genera Serratia, Lactococcus, and Hafnia-Obesumbacterium. The findings of this study provide important insights into understanding the influence of Pb on intestinal homeostasis in amphibians.

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 nitrate‑nitrogen (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.

Zinc (Zn) contaminants in the aquatic environment have an intricate impact on amphibians. Amphibian gut and skin microbiota are participated in regulating their normal physiological functions. Here, we investigated the effects of Zn on the gut and skin tissues and microbiota of Bufo gargarizans and Rana chensinensis tadpoles using histological methods and 16S rRNA sequencing technology. Our results showed a decrease in the height of enterocytes and skin epithelial cells after Zn treatment. Furthermore, Zn exposure elicited alterations in the composition and structure of the gut and skin microbiota at the phylum and genus levels in Bufo gargarizans and Rana chensinensis tadpoles. The feature predictions revealed an elevation in the abundance of potentially pathogenic bacteria and stress-tolerant bacteria in the gut and skin of both tadpoles after zinc exposure. We also speculated that microbiota from various species and organs exhibit varying degrees of sensitivity to zinc based on the functional predictions results. In the context of increasing environmental pollution and the global amphibians decline, our research enriches the current understanding of effects of zinc on amphibian microbiota and provides new framework for artificial breeding and amphibian conservation.

Ciprofloxacin (CIP) and levofloxacin (LEV) are broad-spectrum antibiotics with potent antibacterial activity. Although many studies have shown that antibiotics can lead to gut microbiota disruption, the effects of CIP and LEV on gut microbial colonization at the embryonic stage remain poorly characterized. Here, we evaluated the response of Bufo gargarizans embryos in terms of gut microbiota colonization, growth and developmental stages to CIP and LEV exposure. Embryos treated with 100 μg/L CIP and LEV exhibited significantly reduced diversity and richness of the gut microbiota, as well as altered community structure. Both CIP and LEV treatments resulted in an increase in the pathogenic bacteria Bosea and Aeromonas, and they appeared to be more resistant to CIP than LEV. Additionally, CIP exposure caused reduced total length and delayed the development in B. gargarizans embryos, while LEV increased the total length and promoted embryonic development. The present study revealed the adverse effects of CIP and LEV exposure on host gut microbiota, growth and development during the embryonic stage, and contributed new perspectives to the evaluation of early aquatic ecological risk under CIP and LEV exposure.

Cu and Pb are ubiquitous environmental contaminants, but there is limited information on their potential impacts on gut microbiota profile in anuran amphibians at different developmental stages during metamorphosis. In this study, Bufo gargarizans tadpoles were chronically exposed to Cu alone or Cu combined with Pb from Gs26 throughout metamorphosis. Morphology of tadpoles, histological characteristic and bacterial community of intestines were evaluated at three developmental stages: Gs33, Gs36, and Gs42. Results showed that Cu and Cu + Pb exposure caused various degrees of morphological and histological changes in guts at tested three stages. In addition, bacterial richness and diversity in tadpoles especially at Gs33 and Gs42 were disturbed by Cu and Cu + Pb. Beta diversity demonstrated that the bacterial community structures were influenced by both heavy metals exposure and developmental stages. Alterations in taxonomic composition were characterized by increased abundance of Proteobacteria and Firmicutes, reduction of Fusobacteriota, as well as decreased Cetobacterium and increased C39 at all three stages. Overall, response of gut bacterial diversity and composition to Cu stress depends on the developmental stage, while the altered patterns of bacterial community at Cu stress could be modified further by the presence of Pb. Moreover, predicted metabolic disorders were associated with shifts in bacterial community, but needs integrated information from metagenomic and metatranscriptomic analyses. These results contribute to the growing body of research about potential ecotoxicological effects of heavy metals on amphibian gut microbiota during metamorphosis.

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.

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.