| The plateau brown frog(Rana kukunoris)is endemic to the Qinghai-Tibet Plateau and is distributed between the altitude range of 2000～4000 m above sea level.Environmental factors such as low oxygen,low temperature and strong ultraviolet radiation at high altitude brings great pressure on the survival of animals.In the longterm evolutionary process,highland animals have developed unique adaptive characteristics at the morphological,behavioral,physiological and molecular levels in order to adapt to the high altitude environment,both in phenotypic and genetic adaptations.Because of its late establishment as an independent species,there is a lack of research on various aspects of Rana kukunoris.In this paper,we investigated the adaptation characteristics of Rana kukunoris to high altitude environment by transcriptome,metabolome,microbiome and histomorphology.We compared the differential expressed genes and metabolites in high altitude population of Rana kukunoris with those in low altitude population.We also screened key genes and metabolic pathways in response to altitude stress by multi-omics association analysis.Microflora associated with high altitude acclimation in Rana kukunoris were screened by comparison of symbiotic microorganism.Combined with the results of morphological comparison and metabolism-related enzyme activity comparison,the altitude-related metabolic adaptation patterns of Rana kukunoris was analyzed.These results provided a theoretical basis for revealing the mechanism of Rana kukunoris response to high altitude stress.The main findings are as follows.(1)Transcriptomic analysis of Rana kukunoris response to high altitude stressThe transcriptomic data of high and low altitude populations of Rana kukunoris were compared to screen out differentially expressed genes,were compared to screen out differentially expressed genes,and 3159 up-regulated genes and 1716 downregulated genes were found in different tissues of adults;3905 up-regulated genes and 2101 down-regulated genes were found in juveniles.This indicates that juveniles are subjected to stronger selection pressure by high altitude stress.This indicates that juveniles are subjected to stronger selection pressure by high altitude stress.These genes were mainly classified in GO terms of cellular processes,metabolic processes and bioregulatory processes.Larval beta globin,Tcl-like transposon,PTBP1 and NR4A1 genes were found to be up-regulated in all four tissues in adults,while C1R and CPA3 were found to be down-regulated in all four tissues in juveniles.In adults,aldosterone synthesis and secretion showed up-regulated in heart,muscle and skin tissues;arginine and proline metabolism was up-regulated in all four tissues;myocardial contraction pathway and adrenergic transmission pathway were upregulated in heart;oxidative phosphorylation,glycolysis and gluconeogenesis,and fatty acid degradation pathways were up-regulated in liver;and circadian pathway was upregulated in skin.In juvenile liver,digestion and absorption related pathways were up-regulated;in skin,circadian pathways were also up-regulated;in heart and liver,immune related pathways were down-regulated;in all four tissues,lysosomal pathways were down-regulated;and in skin,prolyl hydroxylase genes in the hypoxiainducible factor pathway were down-regulated.(2)Metabolomic analysis of Rana kukunoris response to high altitude stressThe metabolites in plasma were identified by LC-MS,and 760 and 394 metabolites were obtained in positive and negative ion mode,respectively.The up-regulated pathways in high-altitude adults were glutamate and glutamine metabolism,arginine and proline metabolism,steroid hormone biosynthesis,pentose phosphate pathway,arachidonic acid metabolism and glycerophospholipid metabolism;the down-regulated pathways were mainly fatty acid biosynthesis,pantothenic acid and coenzyme A biosynthesis,base metabolism and branched-chain amino acid metabolism.The upregulation pathways in juvenile were pentose phosphate pathway,arginine,proline and glutamate metabolism,glycerophospholipid metabolism,tricarboxylic acid cycle,pantothenic acid and coenzyme A biosynthesis and branched-chain amino acid metabolism;the down-regulation pathways were fatty acid biosynthesis,arachidonic acid metabolism and steroid biosynthesis pathways.(3)Comparison of metabolism related enzyme activities in Rana kukunoris from different altitudesIn this paper,the activities in liver and muscle tissues of Rana kukunoris from different altitudes were compared.The enzymes examined included lactate dehydrogenase and pyruvate kinase associated with anaerobic metabolism,citrate synthase and malate dehydrogenase associated with aerobic metabolism,cytochrome C oxidase associated with the electron transport chain and glutamate dehydrogenase,one of the amino acid metabolizing enzymes.It was found that all the enzyme activities tested showed that the enzyme activities of high altitude population was higher than that of low altitude population,and the enzyme activities of juveniles were higher than that of adults.(4)Comparison of symbiotic microorganisms of Rana kukunoris from different altitudesIn order to compare the differences in symbiotic microorganisms between high and low altitude populations,microbial samples were collected from the gut,body surface and habitat environments of Rana kukunoris.A total of 5,452,216 AS Vs were identified,and annotated to 2 Kingdoms,63 Phyla,161 Classes,381 Orders,630 Families,1,394 Genera and 997 Species.The α diversity of the environmental microorganisms was found to be higher than that of the skin microorganisms,and the skin microorganisms was higher than that of the intestinal microorganisms,while the intestinal microorganism of the high altitude population was higher than that of the low altitude population.Adonis analysis showed significant differences in β diversity between groups.Both high and low altitude intestinal microorganisms were dominated by Firmicutes,Bacteroidota,Desulfobacterota,Proteobacteria,Verrucomicrobiota,and Actinobacteriota at the Phylum level.Using LEfSe analysis,we identified that the significantly enriched intestinal microorganisms of high altitude population including Lachnospiraceae,Akkermansiaceae,Eubacterium coprostanoligenes,and Oscillospiraceae at the Family level,and the significantly enriched intestinal microorganisms of low altitude population including Desulfovibrionaceae and Mycoplasmataceae at the Family level.(5)Morphological comparison of Rana kukunoris at different altitudesComparison of morphological data revealed that the body size of high and low altitude populations of Rana kukunoris did not follow Bergman’s law,but followed the hypothesis of metabolic cold acclimation at high altitude.The pattern of variation in sexual dimorphism was opposite to Rensch’s law,while forelimb length followed Allen’s law.These results suggest that Rana kukunoris have a particular morphological adaptation to high altitude.(6)Association analysis of transcriptomic and metabolomicNeither transcriptomic nor metabolomic results showed a general high expression of genes or upregulation of metabolites associated with the HIF(Hypoxia-inducible factor)pathway,suggesting that hypoxia may not be a major selection pressure on Rana kukunoris from high altitude population.Both transcriptomic and metabolomic results showed upregulation of the arginine and proline metabolic pathways,glutamate and glutamine metabolic pathways,and elevated glutamate dehydrogenase activity,which catalyzes the conversion between glutamate and α-ketoglutarate,suggesting that glutamate and glutamine may play an important role in high altitude adaptation in Rana kukunoris.Aldosterone was upregulated in multiple tissues of Rana kukunoris from high altitude population,suggesting an active in regulation of Na+ and H2O in high-altitude adaptation.The pentose phosphate pathway is widely upregulated in high altitude populations and is capable of producing large amounts of NADPH to provide reducing power to maintain the homeostasis of the organism and against to oxidative damage.The above results preliminarily revealed the molecular regulatory mechanisms of Rana kukunoris response to high altitude stress,and screened and identified some candidate genes for altitude response,which provide a theoretical basis for further revealing the high altitude adaptation mechanisms of Rana kukunoris and other Tibetan Plateau amphibians,as well as population conservation and exploitation in the future. |
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