Adaptative strategies of the frog,Nanorana parkeri,to extreme environments at high altitude are linked with the evolution of their own genetic mechanisms and phenotypic traits.However,to date,the roles of symbiotic microbiomes in host adaptation to environmental extremes remain enigmatic.In the present study,the 16S rRNA gene amplicon coupled with metagenomic sequencing was used to explore composition as well as potential functions of microbiomes in the gut of N.parkeri collected at both low (3400 m above sea level (a.s.l.)) and high (4600 m a.s.l.) altitudes on the Qinghai-Xizang Plateau.We found that the phylum Firmicutes and genera,such as unclassified_Peptostreptococcaceae,unclassified_Lachnospiraceae,Breznakia,and unclassified_Ruminococcaceae,dominated the core gut microbiomes at both altitudes.High-altitude frogs have a lower alpha diversity of gut microbiome than low-altitude individuals.Moreover,two potentially butyrate-producing bacterial genera,Anaerovorax and Pygmaiobacter,exhibited higher relative abundances in high-altitude individuals versus low-altitude frogs.Notably,at high altitude,families such as antibacterial GH90 and GT103,associated with inflammation attenuation,showed a significantly high relative abundance,whereas GT48,contributing to the synthesis of fungal cell walls,exhibited a significant decrease in the relative abundance.The current study provides novel insights into the role of gut microbiomes in the adaptation of amphibians to high-altitude environments.

BACKGROUND:In response to seasonal cold and food shortage, the Xizang plateau frogs, Nanorana parkeri (Anura: Dicroglossidae), enter a reversible hypometabolic state where heart rate and oxygen consumption in skeletal muscle are strongly suppressed. However, the effect of winter hibernation on gene expression and metabolic profiling in these two tissues remains unknown. In the present study, we conducted transcriptomic and metabolomic analyses of heart and skeletal muscle from summer- and winter-collected N. parkeri to explore mechanisms involved in seasonal hibernation.RESULTS:We identified 2407 differentially expressed genes (DEGs) in heart and 2938 DEGs in skeletal muscle. Enrichment analysis showed that shared DEGs in both tissues were enriched mainly in translation and metabolic processes. Of these, the expression of genes functionally categorized as "response to stress", "defense mechanisms", or "muscle contraction" were particularly associated with hibernation. Metabolomic analysis identified 24 and 22 differentially expressed metabolites (DEMs) in myocardium and skeletal muscle, respectively. In particular, pathway analysis showed that DEMs in myocardium were involved in the pentose phosphate pathway, glycerolipid metabolism, pyruvate metabolism, citrate cycle (TCA cycle), and glycolysis/gluconeogenesis. By contrast, DEMs in skeletal muscle were mainly involved in amino acid metabolism.CONCLUSIONS:In summary, natural adaptations of myocardium and skeletal muscle in hibernating N. parkeri involved transcriptional alterations in translation, stress response, protective mechanisms, and muscle contraction processes as well as metabolic remodeling. This study provides new insights into the transcriptional and metabolic adjustments that aid winter survival of high-altitude frogs N. parkeri.

Organisms have evolved a range of phenotypic and genetic adaptations to live in different environments along an altitudinal gradient. Herein, we studied the widely distributed Chinese toad, Bufo gargarizans, as a model and used an integrated phenotype-genotype approach to assess adaptations to different altitudinal environments. Comparison of populations from four altitudes (50 m, 1200 m, 2300 m, and 3400 m) showed more effective defenses among high-altitude toads. These included thickened epidermis, more epidermal capillaries and granular glands, greater gland size in skin, and higher antioxidant enzyme activities in plasma. High-altitude toads also showed increased erythrocytes and hematocrit and elevated hemoglobin concentration, potentially improving oxygen delivery. Elevated altitude led to a metabolic shift from aerobic to anaerobic metabolism, and high-altitude populations favored carbohydrates over fatty acids to fuel for energy metabolism. Differentially expressed genes were associated with adaptive phenotypic changes. For instance, expression of genes associated with fatty acid metabolism showed greater suppression at high altitude (3400 m), consistent with decreased flux of β-hydroxybutyric acid and lower free fatty acids levels. Moreover, down-regulation of genes involved in carbon metabolism processes at high altitude (3400 m) were coincident with reduced TCA cycle flux. These results suggest that high-altitude toads adopt a metabolic suppression strategy for survival under harsh environmental conditions. Moreover, the hypoxia-inducible factor signaling cascade was activated at high altitude. Collectively, these results advance our comprehension of adaptation to high-altitude environments by revealing physiological and genetic mechanisms at work in Chinese toads living along altitudinal gradients.

Changes in protein abundance and reversible protein phosphorylation (RPP) play important roles in regulating hypometabolism but have never been documented in overwintering frogs at high altitudes. To test the hypothesis that protein abundance and phosphorylation change in response to winter hibernation, we conducted a comprehensive and quantitative proteomic and phosphoproteomic analysis of the liver of the Xizang plateau frog, Nanorana parkeri, living on the Qinghai-Xizang (Tibet) Plateau (QTP). In total, 5 170 proteins and 5 695 phosphorylation sites in 1 938 proteins were quantified. Based on proteomic analysis, 674 differentially expressed proteins (438 up-regulated, 236 down-regulated) were screened in hibernating N. parkeri versus summer individuals. Functional enrichment analysis revealed that higher expressed proteins in winter were significantly enriched in immune-related signaling pathways, whereas lower expressed proteins were mainly involved in metabolic processes. A total of 4 251 modified sites (4 147 up-regulated, 104 down-regulated) belonging to 1 638 phosphoproteins (1 555 up-regulated, 83 down-regulated) were significantly changed in the liver. During hibernation, RPP regulated a diverse array of proteins involved in multiple functions, including metabolic enzymatic activity, ion transport, protein turnover, signal transduction, and alternative splicing. These changes contribute to enhancing protection, suppressing energy-consuming processes, and inducing metabolic depression. Moreover, the activities of phosphofructokinase, glutamate dehydrogenase, and ATPase were all significantly lower in winter compared to summer. In conclusion, our results support the hypothesis and demonstrate the importance of RPP as a regulatory mechanism when animals transition into a hypometabolic state.

Extreme environmental conditions at high altitude, such as hypobaric hypoxia, low temperature, and strong UV radiation, pose a great challenge to the survival of animals. Although the mechanisms of adaptation to high-altitude environments have attracted much attention for native plateau species, the underlying metabolic regulation remains unclear. Here, we used a multi-platform metabolomic analysis to compare metabolic profiles of liver between high- and low-altitude populations of toad-headed lizards, Phrynocephalus vlangalii, from the Qinghai–Tibet Plateau. A total of 191 differential metabolites were identified, consisting of 108 up-regulated and 83 down-regulated metabolites in high-altitude lizards as compared with values for low-altitude lizards. Pathway analysis revealed that the significantly different metabolites were associated with carbohydrate metabolism, amino acid metabolism, purine metabolism, and glycerolipid metabolism. Most intermediary metabolites of glycolysis and the tricarboxylic acid cycle were not significantly altered between the two altitudes, but most free fatty acids as well as β-hydroxybutyric acid were significantly lower in the high-altitude population. This may suggest that high-altitude lizards rely more on carbohydrates as their main energy fuel rather than lipids. Higher levels of phospholipids occurred in the liver of high-altitude populations, suggesting that membrane lipids may undergo adaptive remodeling in response to low-temperature stress at high altitude. In summary, this study demonstrates that metabolic profiles differ substantially between high- and low-altitude lizard populations, and that these differential metabolites and metabolic pathways can provide new insights to reveal mechanisms of adaptation to extreme environments at high altitude.

Nanorana parkeri (Anura, Dicroglossidae) is a unique frog living at high altitude on the Tibetan plateau where they must endure a long winter dormancy at low temperatures without feeding. Here, we presented a comprehensive transcriptomic and metabolomic analysis of liver tissue from summer-active versus overwintering N. parkeri, providing the first broad analysis of altered energy metabolism and gene expression in this frog species. We discovered that significantly up-regulated genes (2,397) in overwintering frogs mainly participated in signal transduction and immune responses, phagosome, endocytosis, lysosome, and autophagy, whereas 2,169 down-regulated genes were mainly involved in metabolic processes, such as oxidation-reduction process, amino acid metabolic process, fatty acid metabolic process, and TCA cycle. Moreover, 35 metabolites were shown to be differentially expressed, including 22 down-regulated and 13 up-regulated in winter. These included particularly notable reductions in the concentrations of most amino acids. These differentially expressed metabolites were mainly involved in amino acid biosynthesis and metabolism. To sum up, these findings suggest that gene expression and metabolic processes show adaptive regulation in overwintering N. parkeri, that contributes to maintaining homeostasis and enhancing protection in the hypometabolic state. This study has greatly expanded our understanding of the winter survival mechanisms in amphibians.

The Xizang plateau frog, N. parkeri (Anura: Dicroglossidae), is endemic to the Tibetan Plateau, ranging from 2,850 to 5,100 m above sea level. The present study explores physiological and biochemical adaptations to high altitude in this species with a particular emphasis on parameters of hematology, oxidative stress, and antioxidant defense in adult and juvenile N. parkeri collected from high (4,600 m a.s.l) and low (3,400 m a.s.l) altitudes. Hematological results showed that hemoglobin concentration ([Hb]), hematocrit (Hct), and red blood cell (RBC) counts were significantly higher in high-altitude N. parkeri. High-altitude juveniles had lower RBC sizes than low-altitude juveniles. Higher levels of GSH and GSSG were indicated only in juveniles from high altitude, not in adults. High-altitude individuals also showed lower oxidative damage, assessed as malondialdehyde (MDA) and carbonyl groups (CG) in the liver. High-altitude adults also showed higher activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), and glutathione-S-transferase (GST) as well as total antioxidant capacity (T-AOC) in the liver as compared to low-altitude adults. Moreover, higher GPX activity and T-AOC were observed in the heart and brain of high-altitude adults. Liver CAT, GPX, and T-AOC showed significant increases in high-altitude juveniles. Vitamin C content was also higher in the heart of high-altitude frogs compared to low-altitude individuals. In summary, the high-altitude population of N. parkeri showed more robust hematological parameters, less oxidative damage, and stronger antioxidant defenses than the low-altitude population, all contributing to increased protection for survival in high-altitude environments.

Climate warming is intensifying on the Tibetan Plateau and poses a serious threat to amphibians that live there. Although hibernation physiology and ecology of Nanorana parkeri, a frog species native to the Tibetan plateau, has been well studied, little information is available about the physiological and biochemical responses to acute rising temperature. Here, we conducted an acute warming experiment comparing hibernating N. parkeri (acclimated at 4 °C) and frogs acutely warmed to 10 °C for 12 h, comparing indicators of oxidative stress and antioxidant defense between the two groups. Acute warming led to a significant increase in the content of oxidized glutathione and the ratio of oxidized:reduced glutathione in liver and muscle, indicating that rapid warming causing oxidative stress could be a negative factor for frogs inhabiting the Tibetan plateau. Malondialdehyde content increased by 57% only in muscle but decreased significantly in three tissues. Protein carbonyl groups rose by 15% in brain, 28% in liver, and 21% in muscle after heat exposure but vitamin C content in heart decreased by 44%. Acute heat exposure also induced tissue-specific upregulation of antioxidant enzyme activities. Catalase activity increased by 27% in heat-exposed frogs, as compared to controls, and glutathione peroxidase activity rose by 12% in brain, 30% in liver, and 12% in muscle. Glutathione-S-transferase activity was also enhanced in heart and muscle of heat-exposed frogs. Acute warming also resulted in a rise in total antioxidant capacity in all tissues examined except kidney, relative to controls. In summary, our findings show that acute heat exposure to hibernating N. parkeri causes a tissue-specific increase in oxidative damage and antioxidant defenses, with skeletal muscle being the most affected tissue. These results reveal the physiological responses to acute temperature change in overwintering N. parkeri.

Many animals lower their metabolic rate in response to low temperatures and scarcity of food in the winter in phenomena called hibernation or overwintering. Living at high altitude on the Tibetan Plateau where winters are very cold, the frog Nanorana parkeri , survives in one of the most hostile environments on Earth but, to date, relatively little is known about the biochemical and physiological adjustments for overwintering by this species. The present study profiled changes in plasma metabolites of N. parkeri between winter and summer using UHPLC-QE-MS non-target metabolomics in order to explore metabolic adaptations that support winter survival. The analysis showed that, in total, 11 metabolites accumulated and 95 were reduced in overwintering frogs compared with summer-active animals. Metabolites that increased included some that may have antioxidant functions (canthaxanthin, galactinol), act as a metabolic inhibitor (mono-ethylhexylphthalate), or accumulate as a product of anaerobic metabolism (lactate). Most other metabolites in plasma showed reduced levels in winter and were generally involved in energy metabolism including 11 amino acids (proline, isoleucine, leucine, valine, phenylalanine, tyrosine, arginine, tryptophan, methionine, threonine and histidine) and 4 carbohydrates (glucose, citrate, succinate, and malate). Pathway analysis indicated that aminoacyl-tRNA biosynthesis, phenylalanine, tyrosine and tryptophan biosynthesis, and nitrogen metabolism were potentially the most prominently altered pathways in overwintering frogs. Changes to these pathways are likely due to fasting and global metabolic depression in overwintering frogs. Concentrations of glucose and urea, commonly used as cryoprotectants by amphibians that winter on land, were significantly reduced during underwater hibernation in N. parkeri . In conclusion, winter survival of the high-altitude frog, N. parkeri was accompanied by substantial changes in metabolomic profiles and this study provides valuable information towards understanding the special adaptive mechanisms of N. parkeri to winter stresses.

The Xizang plateau frog Nanorana parkeri can tolerate brief and partial freezing of their body. To determine the significant role of antioxidant defense and non-specific immune defense in freezing survival of this species, we assayed parameters of oxidative damage, antioxidant defense and non-specific immune enzymes during freezing exposure (-2 degrees C for 12 h) in five organs (heart, brain, liver, kidney, and skeletal muscle). The results showed that freezing led to a significant rise in the content of malondialdehyde (MDA) and carbonyl groups (CG) in brain, liver and kidney tissues. The activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) increased significantly in brain and liver tissues with an augmentation of total antioxidant capacity (TAOC). Apparent increments in muscle SOD activity and liver GST activity were also observed during freezing exposure. Vitamin C content significantly decreased in liver and kidney but a significant increase occurred in brain. Activities of non-specific immune enzymes, acid phosphatase (ACP) and alkaline phosphatase (AKP), were also assessed. ACP activity was significantly reduced in all five tissues tested whereas AKP activity decreased significantly in four tissues but rose in brain. In summary, freezing is accompanied by oxidative stress in the highaltitude frog, N. parkeri, as documented by increases in the content of MDA and CG in tissues. Freezing exposure also induced tissue-specific changes in the antioxidant defenses showing that activation of antioxidant systems is a part of the survival strategy of this in a high-altitude frog during freezing. Such up-regulation of antioxidant enzymes suggests a particularly important role for them in the liver and brain, serving as an anticipatory mechanism to deal with the ROS challenge during freeze/thaw episodes. Our findings contribute to extending the current understanding of the mechanisms of freeze tolerance in high-altitude frogs.

Cold hardiness is a key determinant of the distribution and abundance of ectothermic animals, and thermal acclimation can strongly influence stress tolerance phenotypes. However, the effect of cold acclimation on oxidative stress and antioxidant defenses is still not well understood. Here, we investigated the effects of long-term cold exposure (30 days at 4 °C in darkness versus 30 days at 20 °C in natural light) on the redox state and antioxidant defenses of the high-altitude frog, Nanorana pleskei, indigenous to the Tibetan plateau. We found that cold acclimation, under conditions mimicking winter, led to a significant increase in the ratio of oxidized glutathione (GSSG) to its reduced form (GSH) in liver and skeletal muscle tissues, suggesting that cold exposure induced oxidative stress in this species. Furthermore, malondialdehyde (MDA) contents were significantly augmented in heart, liver and muscle, indicating cold-related oxidative damage in these tissues. In the brain, GST activity, total antioxidant capacity (T-AOC), and vitamin C content showed a significant reduction after cold acclimation. In liver, an apparent decrease was also observed in the activities of SOD and GST, as well as T-AOC, whereas CAT and GPX activities showed a prominent increase in cold-acclimated groups. In kidney, there was a significant decrease in most antioxidant enzyme activities except for SOD and GST activity. In skeletal muscle, the activity of SOD, CAT, GR as well as T-AOC significantly decreased but GPX activity showed a significant increase in cold-acclimated frogs. These findings indicate that, in general, cold acclimation induces a suppression of the antioxidant defense system. Overall, our present study systematically describes the responses of antioxidant defenses to long-term cold acclimation and these findings contribute to extending the current understanding of the mechanisms of cold tolerance in high-altitude frogs.

Author affiliations: 9 Nanjing Normal University, College of life Sciences, Nanjing 210046, China. 10 PubBio-Tech Services Corporation, Wuhan 430070, China. 11 Jiangsu Academy of Forestry, Nanjing, China. 12 Shanghai Majorbio Bio-pharm Biotechnology Co. Ltd., Shanghai, China. 13 DNA Stories Bioinformatics Center, Chengdu, 610021, China. 14 Jiangsu Dafeng Milu National Nature Reserve, Dafeng, China. 15 University of Nebraska at Omaha, Omaha, USA. 16 17 *X.Z., C.D., J.D., Y.T. and X.Z. contributed equally to this work. 18 †To whom correspondence should be addressed. E-mail: zhulf@ioz.ac.cn 19

The milu (Père David’s deer, Elaphurus davidianus ) has become a classic example of how highly endangered animal species can be rescued. However, the mechanisms that underpinned this population recovery remain largely unknown. As part of this study, we sequenced and analyzed whole genomes from multiple captive individuals. Following this analysis, we observed that the milu experienced a prolonged population decline over the last 200,000 years, which led to an elongated history of inbreeding. This protracted inbreeding history facilitated the purging of deleterious recessive alleles, thereby ameliorating associated threats to population viability. Because of this phenomenon, milu are now believed to be less susceptible to future inbreeding depression occurrences. SNP distribution patterns confirmed inbreeding history and also indicated sign of increased and increasing diversity in the recovered milu population. A selective sweep analysis identified two outlier genes ( CTSR2 and GSG1 ) that were related to male fertility. Furthermore, we observed strong signatures of selection pertaining to the host immune system, including six genes ( SERPINE1, PDIA3, CD302, IGLL1, VPREB3 , and CD53 antigen ), which are likely to strengthen resistance to pathogens. We also identified several adaptive features including the over-representation of gene families encoding for olfactory receptor activity, a high selection pressure pertaining to DNA repair and host immunity, and tolerance to high-salt swamp diets. Moreover, glycan biosynthesis, lipid metabolism, and cofactor and vitamin metabolism were all significantly enriched in the gut microbiomes of milu. We speculate that these characteristics play an important role in milu energy metabolism, immunity, development, and health. In conclusion, our findings provide a unique insight into animal population recovery strategies.