Study On Physiological Response And Transcriptomie Analysis Of Exopalaemon Carinicauda To Hypoxia-reoxygenation Stress

Dissolved oxygen(DO)in water as a major source of oxygen for shrimp and a prerequisite for survival,playing an important role in the growth,development and metabolism of shrimp.Water temperature,water depth,air pressure and organic matter content and other factors can make the DO content below normal levels,causing shrimp stress and evasion reaction,inhibit the growth and development of shrimp,resulting in shrimp molt frequency reduced,slow growth,reduced immune function,and in severe cases,shrimp mortality,bringing huge economic losses to the shrimp aquaculture industry.The ridgetail white prawn,Exopalaemon carinicauda,is widely distributed along the coast of China,and has become a major shrimp species for marine pond culture because of its fast growth,good reproductive performance,delicious taste and high nutritional value.However,the oxygen demand of ridgetail white prawn is relatively high,especially in the hot summer season,which is prone to hypoxic mortality.At present,relatively few studies have been conducted on the effects of DO on ridgetail white prawn.In this paper,we studied the physiological response and transcriptomic changes of E.carinicauda in this process by simulating the gradual development of hypoxia in natural water by means of static chamber respiratory oxygen consumption,and initially revealed the hypoxic coping mechanism of E.carinicauda at the tissue,cellular and molecular levels.(1)Histology studies on E.carinicauda under hypoxia-reoxygenation stress.The changes of DO at 0,1,2,4,5 h of hypoxia and 1,4,8 h of reoxygenation were analyzed by simulating the gradual formation of hypoxia in natural water by means of static chamber respiratory oxygen consumption,and the histological characteristics of hepatopancreas,gills and muscles at 0,2,5 h of hypoxia and 1,8 h of reoxygenation were studied in E.carinicauda.The results show that over time,the dissolved oxygen concentration in the experimental group significantly decreased and was significantly lower than that in the control group(P<0.05).The dissolved oxygen concentration in the control group remained stable.After reoxygenation,the dissolved oxygen concentration in the experimental group quickly returned to the control level.With the prolongation of hypoxia,the hepatopancreas tissue of E.carinicauda showed vacuolation and the vacuolation phenomenon was increasing,the gill filaments in the gill tissue became thinner and longer,and the content of blood cells in the gill filaments increased significantly,the muscle fibers in the muscle tissue were loosely arranged,and the muscle fibers were gathered into bundles,after 8 h of reoxygenation,the morphological changes of the three tissues of E.carinicauda were not sufficient to recover to the level of the control group.(2)Effects of hypoxia and reoxygenation on respiratory metabolism enzyme and antioxidant enzyme activities in E.carinicauda.The changes of the main respiratory metabolism and antioxidant enzymes in the gill,hepatopancreas and muscle tissues of E.carinicauda at different time points of 0,1,2,4,5 h of hypoxia and 1,4,8 h of reoxygenation were studied.The results show that over time,cytochrome c oxidase(CCO)and succinate dehydrogenase(SDH)activities in the gill,hepatopancreas,and muscle tissue from the experimental group continuously decreased,lactic dehydrogenase(LDH)and fumaric reductase(FRD)activities continuously increased.In addition,the value of SDH/FRD showed a gradually decreasing trend.During reoxygenation,SDH,LDH,and FRD activities gradually returned to the levels of the control group in three organizations;CCO activity in muscle continuously increased but was significantly lower than that in the control group at 8 h(P<0.05).Furthermore,the value of SDH/FRD also showed a gradually increasing trend.Over time,the activities of superoxide dismutase(SOD),catalase(CAT),glutathione peroxidase(GP_X),and glutathione s-transferase(GST)in the gill,hepatopancreas,and muscle tissue showed a trend of first increasing and then decreasing,but the activities of peroxidase(POD)continuously decreased in the hepatopancreas,while fluctuating in both,the gill and the muscle tissue.During reoxygenation,the activities of SOD and CAT in the gill,hepatopancreas,and muscle tissue all showed a trend of first increasing and then decreasing,but showed no significant difference with the control group at 8 h(P>0.05).With the increase of recovery time,the activities of GP_X and GST in the three tissues returned to the level of the control group,but the activities of POD in gill and muscle tissue were significantly lower than those in the control group under reoxygenation at 8 h.The results show that with the continuous decrease in dissolved oxygen concentration,the aerobic metabolic level of E.carinicauda gradually decreased,and the anaerobic metabolic capacity gradually increased,while during reoxygenation,the aerobic metabolic capacity gradually recovered.Antioxidant enzymes such as SOD,CAT,POD,GP_X,and GST may play an important role in responding to oxidative damage during hypoxia and reoxygenation.(3)Transcriptome analysis of hub genes in the hepatopancreas of E.carinicauda in responses to hypoxia and reoxygenationTranscriptomic studies of hepatopancreas tissue in E.carinicauda after hypoxia and reoxygenation were performed to mine core genes in the hepatopancreas tissue response to hypoxia-reoxygenation.Transcriptome sequencing of hepatopancreas tissues of E.carinicauda at hypoxia 0,3,6 h and reoxygenation 1,8 h was performed,and a total of 93227 genes were obtained.In hepatopancreas tissues,4315 were identified differentially expressed genes(DEGs)responding to hypoxia and reoxygenation.Pathway enrichment analysis suggested that all DEGs were mainly enriched in ribosome biogenesis in eukaryotes,apoptosis,longevity regulating pathway,MAPK signaling pathway and protein processing in endoplasmic reticulum.In addition,20203 genes with the RPKM values≥1 were parsed into 20 modules via weighted gene co-expression network(WGCNA),3 of which were related with hypoxia and reoxygenation,then GO and KEGG enrichment analysis were performed for these 3 modules.Three hub genes were identified based on connectivity:RREB1(ras-responsive element-binding protein 1-like),UBE1(ubiquitin-activating enzyme E1)and an unknown gene.It is hypothesized that these three genes may play an important role in the response of hepatopancreas tissue to hypoxia and reoxygenation stress in E.carinicauda.(4)Trend analysis of differentially expressed genes in gill of E.carinicauda responses to hypoxia-reoxygenationTrend analysis study of differentially expressed genes in gill tissues of E.carinicauda were conducted by transcriptomic study of gill tissues of E.carinicauda after hypoxia and reoxygenation.Transcriptome sequencing of gill tissues of E.carinicauda at hypoxia 0,3,6 h and reoxygenation 1,8 h was performed.In gill tissues,4750 genes were identified as DEGs.All the DEGs were analyzed by trend analysis method,six gene expression trends were significantly enriched(P<0.01).The expression trend of genes that rose first,and fell later under hypoxia and rose again under reoxygenation were focused on.The KEGG pathway enrichment analysis showed that genes were significantly enriched(P<0.05)in Ribosome,Carbon metabolism,Oxidative phosphorylation,Biosynthesis of amino acids,Protein processing in endoplasmic reticulum,Glycolysis/Gluconeogenesis,Glutathione metabolism,and Protein export,which indicated that hypoxia-reoxygenation had a significant effect on protein synthesis and energy metabolism in the gill tissues of E.carinicauda.The results indicated that the gill tissues of E.carinicauda may synthesize protein and increase metabolic capacity to resist hypoxia in the early stage of hypoxia stress,but with the prolongation of hypoxia,both substance synthesis and energy metabolism were affected and decreased,after reoxygenation,with the prolongation of reoxygenation,the protein synthesis and energy metabolism were rose and recovered.In this paper,we investigated the effects of gradually changing hypoxia on the tissue,cellular and molecular levels of E.carinicauda from histoology,respiratory metabolism and antioxidant enzyme activity and transcriptomic studies of hepatopancreas and gill tissues,and initially revealed the physiological responses and molecular mechanisms of E.carinicauda in response to hypoxic stress.The results provide basic information for the prevention of hypoxic accidents in E.carinicauda at the immune level and for the development of new hypoxia-tolerant strains of E.carinicauda.