Copper (Cu) is a common contaminant in aquatic environments, which could cause physiological dysfunction in aquatic organisms. However, few studies have comprehensively examined the impact of copper toxicity in freshwater fish over the past decade. In this research, the oxidative stress, liver transcriptome, intestinal microbiota, and histopathology of common carp (C. carpio) in response to Cu exposure were studied, by exposing juvenile carp to 0.2 mg/ml Cu²⁺ for 30 days. The results revealed that Cu²⁺ could induce significant changes in malondialdehyde (MDA) content and antioxidant enzyme (superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx)) activity. The changes in antioxidant enzyme activities indicate that Cu can induce oxidative stress by generating reactive oxygen species (ROS) content. RNA-seq analysis of the liver identified 1069 differentially expressed genes (DEGs) after treatment with 2.0 mg/L Cu²⁺. Among the DEGs, 490 genes were upregulated and 579 genes were downregulated. GO functional enrichment analysis revealed that Cu could affect the fatty acid biosynthetic process, carnitine biosynthetic process, and activity of carboxylic acid transmembrane transporter. Meanwhile, the most significantly enriched KEGG pathway also included the lipid metabolism pathway. In addition, Cu²⁺ exposure increased bacterial richness and changed bacterial composition. At the phylum level, we found that the ratio of Bacteroidetes to Firmicutes was increased in the treatment carps, which can regulate intestinal epithelium function and reduce inflammation and immune responses. At the genus level, the abundances of 11 genera were significantly altered after exposure to Cu²⁺. The altered composition of the microbial community caused by Cu exposure may play a useful role in compensation of the intestinal lesions by Cu exposure. Furthermore, we found that Cu²⁺ exposure could cause histological alterations such as structural damage to the liver and intestines. The results of this research contribute to a better understanding of mechanisms related to Cu toxicity in fish.

Temperature variation structures the composition and diversity of gut microbiomes in ectothermic animals, key regulators of host physiology, with potential benefit to host or lead to converse results (i.e., negative). So, the significance of either effect may largely depend on the length of time exposed to extreme temperatures and how rapidly the gut microbiota can be altered by change in temperature. However, the temporal effects of temperature on gut microbiota have rarely been clarified. To understand this issue, we exposed two juvenile fishes (Cyprinus carpio and Micropterus salmoides), which both ranked among the 100 worst invasive alien species in the world, to increased environmental temperature and sampled of the gut microbiota at multiple time points after exposure so as to determine when differences in these communities become detectable. Further, how temperature affects the composition and function of microbiota was examined by comparing predicted metagenomic profiles of gut microbiota between treatment groups at the final time point of the experiment. The gut microbiota of C. carpio was more plastic than those of M. salmoides. Specifically, communities of C. carpio were greatly altered by increased temperature within 1 week, while communities of M. salmoides exhibit no significant changes. Further, we identified 10 predicted bacterial functional pathways in C. carpio that were temperature-dependent, while none functional pathways in M. salmoides was found to be temperature-dependent. Thus, the gut microbiota of C. carpio was more sensitive to temperature changes and their functional pathways were significantly changed after temperature treatment. These results showed the gut microbiota of the two invasive fishes differ in response to temperature change, which may indicate that they differ in colonization modes. Broadly, we have confirmed that the increased short-term fluctuations in temperatures are always expected to alter the gut microbiota of ectothermic vertebrates when facing global climate change.

Understanding how age and body size vary across elevations can provide insights into the evolution of life‐history traits in animals. In the present study, we compared the demographic (using skeletochronology) and morphological traits of the Tibetan toad (Bufo tibetanus) between two populations from different elevational habitats (2650 vs. 3930 m). We found that (1) the mean age and body size of females were significantly greater than those of males in both populations; (2) both sexes of toads from the higher elevation tended to be significantly older in age and larger in body size; (3) there was a significant positive relationship between age and body size within each sex of the toad at both elevations; and (4) growth rates varied between the two populations, with the higher rate observed in the lower‐elevation population. Our results suggested that factors other than age, such as elevation‐associated temperature, influence the observed differences in body size between the two populations. Future research at a broader range of elevations should focus on these factors and evaluate their influence on animal growth patterns.

The intestinal microbiota is vital for host immunity and metabolism, and its changes are associated with the development stage of hosts. However, little is known regarding how growth and development of anurans affect the diversity of their microbiota, which has a complex life cycle. The Tibetan toad (Bufo tibetanus) is a wild population in the high-altitude area of southwest China, which has special adaptability to the environment. Here, the microbial community of the Tibetan toad at six developmental stages (from the tadpole at Gosner stage 18 to the 8-year-old adult) was assessed using high-throughput 16S rRNA sequencing. The alpha diversity index analysis showed that the Chao, Ace, and Shannon indices were highest at Gosner stage 32 and decreased as development progressed, and their alpha diversity remained unchanged over time in adult stages. Beta diversity revealed that the gut microbiota structure differed significantly from Gosner stages 18 to 31, and it became similar to adult toads from Gosner stages 45 to 46 and in juvenile groups. At the phylum level, Firmicutes, Proteobacteria, and Actinobacteria were dominant phyla in tadpoles and adults. The relative abundance of Firmicutes and Proteobacteria in the adult group was significantly higher and lower than that of tadpoles, respectively. The linear discriminant analysis effect size (LEfSe) analysis identified seven phyla exhibiting significant differences during life stages: Verrucomicrobiota, Bacteroidota, and Proteobacteria (Gosner 18 to 31), Cyanobateria and Chloroflexi (Gosner 32 to 41), Actinobacteriota (Gosner 45 to 46), Desulfobacterota (juvenile group), and Firmicutes (adult group). A pathway enrichment analysis revealed that the metabolism and biosynthesis of secondary metabolites were significantly enriched across all developmental stages. This research unveiled variations in the intestinal microbiota composition during development in anurans. Factors such as developmental stage, habitat type and feeding habit jointly affected the gut microbial diversity and community composition in the Tibetan toad. The findings of this study can provide information for understanding the influence of historical developments on the intestinal microbiota and provide protection information for anurans.

Microcystin-LR (MC-LR), produced by Cyanobacteria, is being detected in many types of waters, posing a universal threat to aquatic animals. However, there have been few comprehensive endpoints assessed, including oxidative stress, transcriptome changes, intestinal microbiota, and histopathology, in anurans exposed to MC-LR. In this study, all these effects of MC-LR on Chinese brown frog (Rana chensinensis David, 1875) tadpoles were investigated by exposing the tadpoles to MC-LR at different concentrations (0, 0.1, 1.0, 5.0, and 10.0 μg/L) for 7 days. Our results revealed that treatment of tadpoles with the high MC-LR dosage (10.0 μg/L) induced a significant increase in malondialdehyde (MDA) content and decreases in superoxide dismutase (SOD) activity, glutathione peroxidase (GPx) activity, and total antioxidant capacity (TAC). RNA-seq analysis of the liver showed that the number of differentially expressed genes (DEGs) was 2361 under lower MC-LR stress (1.0 μg/L), while the number of DEGs increased to 3185 under higher MC-LR stress (10.0 μg/L). Gene Ontology analysis showed that several biological processes and molecular functions related to digestion were enriched in both MC-LR treated groups, such as digestion, serine-type endopeptidase activity, and serine-type peptidase activity. KEGG enrichment analysis also indicated that the digestion for pancreatic secretion, protein digestion and absorption, and fat digestion and absorption pathways were significantly enriched in the treatment groups. Additionally, the bacterial richness was elevated by MC-LR exposure. At the phylum level, treatment with MC-LR changed the relative abundances of Desulfobacterota, Fusobacteriota, and Actinobacteriota. At the genus level, MC-LR caused significant alterations in the abundances of 23 genera. Furthermore, examination of sections obtained from the livers and intestines of tadpoles in the treatment groups showed damaged histological structure. The knowledge from this study will have potential value for understanding the mechanisms related to MC-LR toxicity in anurans.