Physiological And Biochemical Characteristics And Underlying Molecular Mechanisms Of Hibernation In Nanorana Parkeri

The Xizang plateau frog Nanorana parkeri,an extant highest-distribution amphibian in the world,lives on the southern Tibetan Plateau.It inhabits from 2850 to 5100 m above sea level.The Tibetan Plateau,known as the ?roof of the world?,is characterized by low temperatures and hypoxia.In the long-term evolution,high-altitude animals exhibit a series of adaptive characteristics including morphological structure,behavior,physiological and molecular level.Hibernation,as a common winter survival strategy,allows organisms to cope with prolonged cold and starvation,which plays a key role in the high-altitude adaptation of animals.Heretofore,no systematic study on the hibernation of amphibians living on the Tibetan Plateau has been found.In the present study,summer-active and hibernating N.parkeri were used to investigate the seasonal differences in the morphology,metabolic rate,heart rate,mitochondrial respiration rate,metabolites,activities and relative mRNA expression of metabolic enzymes,oxidative stress,oxidative damage and antioxidant capacity with physiological,biochemical and molecular biology methods.The transcriptional and metabolic regulation mechanisms of hibernation were explored by transcriptomics and metabonomics technique.Moreover,the freeze tolerance and underlying molecular mechanisms were also studied using metabonomics.Compared to summer-active frogs,the body mass,body mass index(BMI)and hepatosomatic index(HIS)was significantly increased in hibernating N.parkeri.The resting metabolic rate of N.parkeri decreased by 76% and 66% at 8 °C and 20 °C during hibernation,respectively.The mitochondria respiration rates of the liver in hibernating frogs decreased by 76% in the state III at 8 °C,65% in the state III at 20 °C,44% in the state IV at 8 °C,and 56% in the state IV at 20 °C,respectively.Hibernation resulted in a 46% decrease in the state III respiration rate of muscle mitochondria at 20 °C,but the state IV respiration rate showed no seasonal difference.The activity of CCO in the liver and muscle showed a remarkable reduction in hibernating frogs,and the mRNA level of COX1,COX2,and COX3 also declined sharply.These results suggested that hibernation was characterized by metabolic depression in N.parkeri at the level of whole-animal and mitochondria,which is essential for energy conservation and life maintenance.However,lactate dehydrogenase(LDH)activity in the liver and muscle tissue increased significantly,accompanied by the augment of the mRNA of LDH subunit A,indicating that the effect of low temperature on anaerobic metabolism was presumably compensated by the up-regulation of LDH during hibernation.Moreover,a high level of glycogen in liver and muscle can provide large energy reserves for the successful overwintering of N.parkeri.We compared the indices of oxidative stress,oxidative damage,and the activities of antioxidant enzymes in liver,brain,heart,and muscle of summer-active and hibernating N.parkeri.Obtained results showed that the level of total glutathione and reduced glutathione in liver,heart,and muscle significantly decreased in hibernating N.parkeri,while the ratio of GSSG/GSH markedly increased in all tested tissues except muscle.Compared to summer-active frogs,significant increases in TBARS were observed in above-mentioned tissues during hibernation,and the lipid peroxides level also clearly elevated in these tissues except the liver.In the liver and brain,the level of carbonyl proteins was significantly higher in winter than in summer.The activity of antioxidant enzymes reduced in the liver of hibernating N.parkeri,and the total antioxidant capacity in above-mentioned tissues also decreased significantly.These results suggested that hibernation induced oxidative stress with suppression of antioxidant defense in N.parkeri,which was supported by oxidative damage to lipids and proteins.Comparative transcriptome analysis of liver and muscle between summer-active and hibernating N.parkeri showed a total of 4387 differentially expressed genes in the liver.Of these,the expression of 1992 genes(45%)were significantly down-regulated,and the expression of 2395 genes(55%)were significantly up-regulated during hibernation.Of 2434 differentially expressed genes in the muscle,1425 genes(59%)were down-regulated,and 1009 genes(41%)were up-regulated during hibernation.The GO enrichment analysis revealed that up-regulated genes(NOX1,TNFRSF9,CD28,BTK,IRF5,IRF8)in the liver were significantly enriched in the immune response-related processes(GO:0001817,GO:0002764,GO:0002521).This may be a compensatory adjustment of the immune system for the effect of winter hypothermia,which was helpful to maintain normal immune defense function and was beneficial to the successful overwintering of N.parkeri.Genes related to muscle contraction function were significantly up-regulated in the muscle during hibernation,such as LOC108794414,MYLIP,TNNT1,and TNNT2.We believed that these up-regulated genes would help to prevent muscle from disuse atrophy,and would benefit activities(such as foraging and reproduction)after awakening from hibernation.The down-regulated genes in the liver and muscle were significantly enriched in the energy metabolism processes(GO:0006631,GO:1901605).Moreover,KEGG enrichment results showed that the genes participated in the carbon metabolism(npr01200),fatty acid metabolism(npr01212),glycine,serine and threonine metabolism(npr00260),glycerophospholipid metabolism(npr00564)were down-regulated in the liver.This indicated that the metabolism was depressed in hibernating N.parkeri at the level of gene expression.The down-regulation of COX1,COX2,and COX3 was consistent with the results of enzyme activity and real-time quantitative PCR experiments.The metabolism-related genes were markedly down-regulated,which contributed to reducing energy consumption during hibernation.Additionally,the genes encoding antioxidant enzymes in the liver were significantly down-regulated,such as SOD2,GSTZ1,GSTCD,which was consistent with the results of enzyme activity,suggesting that the antioxidant capacity was decreased during hibernation.The differences of metabolites between summer-active and hibernating N.parkeri were analyzed in the liver and muscle using GC-MS metabonomics.A total of 35 differential metabolites were identified in the liver,including 13 up-regulated metabolites and 22 down-regulated metabolites in hibernating frogs.A total of 22 differential metabolites were identified in the muscle,including 9 up-regulated metabolites and 13 down-regulated metabolites in hibernating frogs.Metabolites participated in glycerol phospholipid metabolism(npr00564)were up-regulated in liver and muscle,while metabolites in the amino acid synthesis and metabolism pathways were down-regulated significantly during hibernation.These pathways included phenylalanine,tyrosine and tryptophan biosynthesis(npr00400),valine,leucine and isoleucine biosynthesis(npr00290),phenylalanine metabolism(npr00360),and glycine,serine and threonine metabolism(npr00260).These results were coincident with the fasting state and the inhibited protein degradation during hibernation.Moreover,lactate was significantly accumulated in the liver,suggesting that the anaerobic metabolic capacity was increased during hibernation.In fact,lactate can also act as a cryoprotectant in hibernating N.parkeri.The concentration of maltose,fructose,and inositol was obviously elevated,which may be related to their cryoprotective and osmoregulatory functions during hibernation.The freezing experiments revealed that the hibernating N.parkeri shows the modest freeze tolerance capacity relative to other published freeze-tolerant species.Freezing exposure induced partial dehydration in the muscle,which served to reduce the amount of ice in tissues.We analyzed the metabolomics of liver and muscle in N.parkeri before and after freezing exposure.A total of 33 differential metabolites were identified in the liver,including 29 up-regulated metabolites and 4 down-regulated metabolites with freezing exposure.A total of 36 differential metabolites were identified in the muscle,including 25 up-regulated metabolites and 11 down-regulated metabolites with freezing exposure.Freezing significantly affected alanine,aspartic acid and glutamic acid metabolism(npr00250),arginine and proline metabolism(npr00330),D-glutamine and D-glutamate metabolism(npr00471).After freezing exposure,the contents of ornithine,melezitose,and maltotriose accumulated significantly.They may be involved in the cryoprotection process.The augment of unsaturated fatty acid would maintain the membrane fluidity at the low temperature and contribute to the freeze tolerance of N.parkeri.Additionally,the content of 8-hydroxy-2-deoxyguanine and hypoxanthine showed a remarkable elevation,indicating that freezing induced oxidative stress in hibernating N.parkeri.In summary,our present study represented an elaborate investigation in the physiological and biochemical characteristics and underlying molecular mechanisms of hibernation in N.parkeri.These findings could aid in elucidating the mechanism underlying metabolic depression associated with hibernation,filling the gap of hibernation research of amphibians living on the Tibetan Plateau,further enriching and improving the theory of hibernation biology research,and contribute to the conservation of this species and the ecological environment of Tibetan Plateau.In addition,our investigation on the freeze tolerance and underlying molecular mechanisms would lay a foundation for cryobiological studies of amphibians living on the Tibetan Plateau,and provide a reference for the research and development of cryo-medicine.