| Sander lucioperca is one of the most attractive freshwater fish in the aquaculture industry,which has outstanding properties like quick growth,robust disease resistance,and high nutritional content.Yet,the Sander lucioperca is a ferocious,aggressive fish with a quick temper who is susceptible to a variety of stresses in intensive raising.Dissolved oxygen is the most crucial component of water quality,and hypoxia frequently occurs in aquatic situations.Research have demonstrated that hypoxia has an impact on fish development,reproduction,metabolism,immunological function,and other vital life processes,directly threatening fish survival.Nonetheless,research on the mechanisms of hypoxia adaptation in Sander lucioperca is still in its early stages.Therefore,in order to explore the physiological response of Sander lucioperca to hypoxia and its molecular regulation mechanism,this study is the subject of the Sander lucioperca,firstly,we conducted hypoxia stress and normoxia recovery trial;then by microscopic observation of magnesium,liver’s structural changes,detection of antioxidant indicators and changes in blood composition.Subsequently,RNA-sequencing(RNA-seq)was used to dissect the main differentially expressed genes involved in the regulation of hypoxia adaptation in gill tissues and the signaling pathways in which they are located.Further,the screened oxygen-sensing genes hif-1α and phd2,which have important transcriptional regulatory roles in the HIF-1 signaling pathway,were cloned with a full-length c DNA sequence.And their tissue expression patterns and response patterns to hypoxia and reoxygenation in gill and liver tissues were revealed at the transcriptional regulatory level.The results clarified the physiological effects of hypoxic stress on Sander lucioperca,thus clarifying the physiological and biochemical responses of Sander lucioperca to hypoxia and reoxygenation as well as the basis of molecular responses.The main findings are as follows.(1)Physiological response of Sander lucioperca under hypoxic stress.To investigate the effects of hypoxia and reoxygenation on the physiological and biochemical indexes of Sander lucioperca,this study was conducted to examine the changes of antioxidant indexes as well as blood composition by microscopic observation of gill and liver tissue sections made by HE staining method in normoxia,hypoxia for 9h,reoxygenation for 1 h and reoxygenation for 12 h,respectively.The results showed that hypoxic stress led to "S"-shaped curling,terminal enlargement and adhesion of gill lamellae in the gill tissue of Sander lucioperca,and the flattened epithelial cells were enlarged,vacuolated and gradually ruptured,which were shed in the interstices of gill lamellae,causing serious damage to gill tissue,and reoxygenation for 12 h failed to alleviate these oxidative damages.Hypoxic stress caused the hepatocytes in the liver tissue to be loosely arranged and scattered,with lesions such as cell vacuolation,nuclear consolidation and nuclear fragmentation,etc.The damaged area recovered after reoxygenation,but still accompanied by vacuolation of hepatocytes;indicating that hypoxic stress caused serious tissue damage to the gills and liver of Sander lucioperca.Among the antioxidant indexes,superoxide dismutase(SOD)and catalase(CAT)activity in gills increased significantly(P<0.05)in the first 3 h of hypoxic stress,and synergized with glutathione peroxidase(GSH-PX)to scavenge reactive oxygen species(ROS)after3 h,and all returned to normal levels after 12 h of reoxygenation.Lactate dehydrogenase(LDH)showed a decreasing-increasing trend during hypoxia,with the weakest activity at6 h and the strongest activity at 9 h.Total antioxidant capacity(T-AOC)decreased significantly under hypoxic stress,and malondialdehyde(MDA)content increased significantly during stress and was still significantly higher than that of the control group after 12 h of reoxygenation(P<0.05).During hypoxic stress,SOD,CAT,GSH-PX and TAOC activity in the liver increased significantly and remained significantly higher than normal after 12 h of reoxygenation(P<0.05);MDA content accumulated gradually and was highest at 9 h of hypoxia;indicating that the gills and liver of Sander lucioperca resist oxidative stress by activating antioxidant enzymes and enhancing anaerobic metabolism.The number of erythrocytes,leukocytes and hemoglobin in the blood showed a decreasing-increasing trend after the hypoxic emergency,gradually increased at 3 h of hypoxia,and basically returned to the normal state after reoxygenation,indicating that Sander lucioperca adapted to the hypoxic environment by improving the oxygen-carrying capacity and immunity of the blood.This study showed that the gills and liver of Sander lucioperca responded to hypoxic stress by enhancing antioxidant capacity,immunity,blood oxygen-carrying capacity and anaerobic glycolysis,while hypoxic stress also induced irreversible tissue damage in the gills and liver.(2)Analysis of differentially expressed genes in gill tissues of Sander lucioperca.under hypoxic stressTo investigate the molecular regulation mechanism of Sander lucioperca gill in response to hypoxia and reoxygenation,this study analyzed the response patterns of gill under normoxia,hypoxia 9 h,reoxygenation 1 h and reoxygenation 12 h conditions at the transcriptional level using RNA-seq technique.The results revealed that a total of 2925 differentially expressed genes(DEGs)were screened and identified,and the expression trends of 12 DEGs verified by quantitative real-time PCR were fully consistent with them.GO annotation and KEGG enrichment analysis showed that DEGs were mainly enriched in phagosome,HIF-1 signaling pathway,biosynthesis of amino acids and ABC transporter pathways during hypoxic stress.DEGs are significantly enriched in cell cycles,DNA replication and cytokine-cytokine receptor interaction pathways after recovery from normoxia.The egln family genes were widely expressed in the HIF-1 signaling pathway;suggesting that hypoxic stress may induce an innate immune response in Sander lucioperca gills,activating HIF-1,phagosome,and cell proliferation pathways to scavenge damaged proteins and enhance gill immune defense.(3)Response of the PHD2—HIF1 signaling pathway in Sander lucioperca.under hypoxic stressBased on transcriptome sequencing analysis,the PHD2—HIF1 signaling pathway has an important regulatory role in Sander lucioperca gill in response to hypoxia,and hypoxia-inducible factor 1 a(hif-1α)and proline hydroxylase 2(phd2)are the most critical transcriptional regulators.Therefore,in this study,the c DNA full-length sequences of hif-1α and phd2 genes were obtained by cloning using RACE technique,and bioinformatics analysis was performed.q RT-PCR was used to detect the tissue distribution of hif-1α and phd2 as well as the transcriptional regulation patterns at different times of hypoxia and reoxygenation.The results showed that the c DNA of hif-1α was 3535 bp,encoding 749 amino acids;the 5′-UTR(Untranslated region)and 3′-UTR were 226 bp and 1059 bp,respectively.The full-length c DNA of phd2 is 1913 bp,containing 255 bp 5′-UTR and 578 bp 3′-UTR,encoding a total of 359 amino acids.They all have characteristic structural domains of their respective gene families with a high level of conservation.The amino acid homology comparison and phylogenetic evolutionary analysis showed that the amino acid sequences encoded by hif-1α and phd2 had the highest homology with Perca fluviatilis,and were the closest relatives to Perca fluviatilis,Perca flavescens,and Siniperca chuatsi.The q RT-PCR showed that hif-1α and phd2 were the most highly expressed in the brain and gills and the least in the skin and muscle of Sander lucioperca.The expression levels of hif-1α and phd2 in gill tissues were significantly increased(P<0.05)before 6 h of hypoxia,and then both gradually decreased,and only phd2 returned to the initial level by 12 h of reoxygenation.There was no significant change in hif-1α and phd2 in the liver before 3 h of hypoxia,which rose to the highest expression level by 9 h of hypoxia(P<0.05),and both showed a decreasingincreasing trend after reoxygenation.hif-1α returned to normal expression level after 12 h.Sander lucioperca utilizes feedback regulation of hif-1α and phd2 in response to hypoxic stress to enhance organismal adaptation to changes in dissolved oxygen.In conclusion,this study provides a reference for understanding the physiological response of fish to hypoxic reoxygenation and its molecular regulatory mechanism,and also provides theoretical support for breeding new species of Sander lucioperca tolerant to hypoxia as well as culture management. |
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