Background: Metamorphic climax is the crucial stage of amphibian metamorphosis responsible for the morphological and functional changes necessary for transition to a terrestrial habitat. This developmental period is sensitive to environmental changes and pollution. Understanding its metabolic basis and requirements is significant for ecological and toxicological research. Rana omeimontis tadpoles are a useful model for investigating this stage as their liver is involved in both metabolic regulation and fat storage. Results: We used a combined approach of transcriptomics and metabolomics to study the metabolic reorganization during natural and T3-driven metamorphic climax in the liver and tail of Rana omeimontis tadpoles. The metabolic flux from the apoptotic tail replaced hepatic fat storage as metabolic fuel, resulting in increased hepatic amino acid and fat levels. In the liver, amino acid catabolism (transamination and urea cycle) was upregulated along with energy metabolism (TCA cycle and oxidative phosphorylation), while the carbohydrate and lipid catabolism (glycolysis, pentose phosphate pathway (PPP), and β-oxidation) decreased. The hepatic glycogen phosphorylation and gluconeogenesis were upregulated, and the carbohydrate flux was used for synthesis of glycan units (e.g., UDP-glucuronate). In the tail, glycolysis, β-oxidation, and transamination were all downregulated, accompanied by synchronous downregulation of energy production and consumption. Glycogenolysis was maintained in the tail, and the carbohydrate flux likely flowed into both PPP and the synthesis of glycan units (e.g., UDP-glucuronate and UDP-glucosamine). Fatty acid elongation and desaturation, as well as the synthesis of bioactive lipid (e.g., prostaglandins) were encouraged in the tail during metamorphic climax. Protein synthesis was downregulated in both the liver and tail. The significance of these metabolic adjustments and their potential regulation mechanism are discussed. Conclusion: The energic strategy and anabolic requirements during metamorphic climax were revealed at the molecular level. Amino acid made an increased contribution to energy metabolism during metamorphic climax. Carbohydrate anabolism was essential for the body construction of the froglets. The tail was critical in anabolism including synthesizing bioactive metabolites. These findings increase our understanding of amphibian metamorphosis and provide background information for ecological, evolutionary, conservation, and developmental studies of amphibians.

Background: Captivity and artificial food provision are common conservation strategies for the endangered golden snub-nosed monkey (Rhinopithecus roxellana). Anthropogenic activities have been reported to impact the fitness of R. roxellana by altering their gut microbiota, a crucial indicator of animal health. Nevertheless, the degree of divergence in gut microbiota between different anthropogenically-disturbed (AD) R. roxellana and their counterparts in the wild has yet to be elucidated. Here, we conducted a comparative analysis of the gut microbiota across nine populations of R. roxellana spanning China, which included seven captive populations, one wild population, and another wild population subject to artificial food provision. Results: Both captivity and food provision significantly altered the gut microbiota. AD populations exhibited common variations, such as increased Bacteroidetes and decreased Firmicutes (e.g., Ruminococcus), Actinobacteria (e.g., Parvibacter), Verrucomicrobia (e.g., Akkermansia), and Tenericutes. Additionally, a reduced Firmicutes/Bacteroidetes ratiosuggested diminished capacity for complex carbohydrate degradation in captive individuals. The results of microbial functional prediction suggested that AD populations displayed heightened microbial genes linked to vitamin and amino acid metabolism, alongside decreased genes associated antibiotics biosynthesis (e.g., penicillin, cephalosporin, macrolides, and clavulanic acid) and secondary metabolite degradation (e.g., naphthalene and atrazine). These microbial alterations implied potential disparities in the health status between AD and wild individuals. AD populations exhibited varying degrees of microbial changes compared to the wild group, implying that the extent of these variations might serve as a metric for assessing the health status of AD populations. Furthermore, utilizing the individual information of captive individuals, we identified associations between variations in the gut microbiota of R. roxellana and host age, as well as pedigree. Older individuals exhibited higher microbial diversity, while a closer genetic relatedness reflected a more similar gut microbiota. Conclusions: Our aim was to assess how anthropogenic activities and host factors influence the gut microbiota of R. roxellana. Anthropogenic activities led to consistent changes in gut microbial diversity and function, while host age and genetic relatedness contributed to interindividual variations in the gut microbiota. These findings may contribute to the establishment of health assessment standards and the optimization of breeding conditions for captive R. roxellana populations.

Declining body size is a universal ecological response to globalwarming in ectotherms. Ectotherms growfaster but mature at a smaller size at higher temperatures. This phenomenon is known as the temperature-size rule (TSR). However, we know little about the details of the relationship between temperature and size. Here, this issue was studied in the Chinese giant salamander (Andrias davidianus), one of the largest extant amphibians and a flagship species of conservation in China. Warm-acclimated A. davidianus larvae (25 degrees C) had accelerated development but little superiority in body growth when compared to their 15 degrees C counterparts when fed with red worm. This predicts a drastic decrease in adult body size with warming. However, a fish diet (more abundant lipid and protein) improved the growth performance at 25 degrees C. The underlying mechanism was studied. Warm-acclimated larvae had enlarged livers but shortened tails (fat depot). Their livers suffered fromenergy deficiencies and decreased protein levels, even when protein synthesis and energy metabolismwere transcriptionally upregulated. This could be a direct explanation for their poor growth performance. Further analyses revealed a metabolic disorder resembling mammal glycogen storage disease in warm-acclimated larvae, indicating deficiency in glycogen catabolism. This speculation is consistent with their increased lipid and amino acid catabolism and explained the poor energy conditions of the warm-acclimated larvae. Additionally, a deficiency in glycogen metabolism explains the different efficiency of worm and fish diets in supporting the growth of warm-acclimated larvae, even when both diets were provided sufficiently. In conclusion, our results suggest that the relationship between temperature and body size can be flexible, which is a significant finding in terms of the TSR. The underlying metabolic and nutrient mechanisms were revealed. This knowledge can help deepen our understanding of the consequences of warming and can contribute to the conservation of A. davidianus.

Vertebrates use different pigmentation strategies to adapt to various environments. A large amount of research has been done on disclosing the mechanisms of pigmentation strategies in vertebrates either under light, or, living in constant darkness. However, less attention has been paid to non-obligate, darkness dwellers. Red-spotted toothed toads Oreolalax rhodostigmatus (Megophryidae; Anura) from the karst mountainous region of southwestern China are non-obligate cave dwellers. Most tadpoles of the species possess transparent skin as they inhabit the dark karst caves. But remarkably, the transparent tadpoles can darken just within 15 h once exposed to light. Obviously, it is very significant to reveal molecular mechanisms of the unexpected rapid-darkening phenomenon. We compared the transcriptomes of O. rhodostigmatus tadpoles with different durations of light exposure to investigate the cellular processes and potential regulation signals for their light-induced rapid darkening. Genes involved in melanogenesis (i.e. TYR, TYRP1 and DCT) and melanocyte proliferation, as well as their transcriptional factor (MITF), showed light-induced transcription, suggesting a dominating role of morphological color change (MCC) in this process. Transcription of genes related to growth factor, MAPK and PI3K-Akt pathways increased with time of light exposure, suggesting that light could induce significant growth signal, which might facilitate the rapid skin darkening. Most importantly, an in-frame deletion of four residues was identified in O. rhodostigmatus melanocortin-1 receptor (MC1R), a critical receptor in MCC. This deletion results in a more negatively charged ligand pocket with three stereo-tandem aspartate residues. Such structural changes likely decrease the constitutive activity of MC1R, but increase its ligands-dependent activity, thus coordinating pigment regression and rapid melanogenesis in the dark and light, respectively. Our study suggested that rapid MCC was responsible for the light-induced rapid darkening of O. rhodostigmatus tadpoles. Genetic mutations of MC1R in them could explain how these non-obligate cave dwellers coordinate pigment regression and robust melanogenesis in darkness and light, respectively. To our knowledge, this is the first study that reports the association between pigmentation phenotype adaptation and MC1R mutations in amphibians and/or in non-obligate cave dwellers.