| Chinese mitten crab(Eirocheir sinensis)is an important aquatic economic animal in China.Dissolved oxygen(DO)has a severe influence on the growth and survival of juvenile E.Sinensis.High temperature,eutrophication and deterioration of water quality will result in a low DO environment in the breeding process of E.Sinensis.Low DO levels are detrimental to the health of E.Sinensis.So studying the adaptation mechanisms of juvenile E.sinensis in hypoxic environments is of great significance in elucidating hypoxia-tolerant traits and germplasm evaluation in crustaceans.Under hypoxia stress,E.sinensis initiates relevant molecular response mechanisms,in which the hypoxiainducible factor pathway(HIF)plays a key role in sensing oxygen and mediating the transcription of many genes to maintain oxygen homeostasis in cell.In this study,antioxidant parameters and glycolysis indexes related enzyme activities were measured in different tissues.Real-time quantitative PCR(q RT-PCR)was used to detect the expression patterns of HIF signaling pathway related genes in different tissues.To evaluate antioxidant status and glycolysis function of juvenile E.sinensis under hypoxia stress.At the same time,the effects of hypoxia stress on hepatopancreas metabolism of juvenile E.sinensis were analyzed by transcriptomics.The main research results are as follows:(1)Effects of hypoxia stress on antioxidant status of juvenile E.sinensisTwo groups were set up in the experiment,hypoxia group and reoxygenation group.In the hypoxia group,DO levels were controlled with nitrogen and air(1.0 ± 0.2 mg/L),and samples of hemolymph,gills,hepatopancreas and muscle tissues were collected at0,3,6,12 and 24 hours of stress,respectively.In the reoxygenation group,DO recovered to normal oxygen level(7.0 ± 0.2 mg/L)after 24 hours of hypoxia stress,and samples were collected at 1,3,6,12 and 24 hours of reoxygenation,respectively.The samples were used to determine antioxidant parameters,glycolysis indexes and gene expression.The results of antioxidant parameters showed that the activities of oxidative stress parameters(catalase(CAT),total superoxide dismutase(T-SOD),total antioxidant capacity(T-AOC)and malondialdehyde(MDA))in all tissues increased significantly under hypoxia stress and decreased gradually in the reoxygenation.The results showed that hypoxia stress activated the antioxidant defense system,and the antioxidant reaction was tissue specific.(2)Effects of hypoxia stress on glycolysis function of juvenile E.sinensisGlycolysis corresponding enzyme activity include hexokinase(HK),phosphofructokinase(PFK),pyruvate kinase(PK),pyruvic acid(PA),lactate dehydrogenase(LDH),lactic acid(LA),succinate dehydrogenase(SDH)and glycogen in hepatopancreas,hemolymph and gill.They all increased to different degrees under hypoxia stress and decreased gradually in the reoxygenation.Hypoxic stress activated the glycolytic pathway.The energy metabolic pathway changed accordingly from aerobic to anaerobic glycolysis.The gene expression data showed that genes related to hypoxia signaling pathways,including hypoxia-inducible factor-1α/β(HIF-1α/β),proline hydroxylase(PHD),factor inhibiting hypoxia-inducible factor(FIH)and glycolysis-related factors(HK and PK),were up-regulated in different tissues of juvenile E.sinensis after 24 h of hypoxia stress.The gene expression returned to control levels after reoxygenation.This indicates that the HIF signaling pathway and glycolytic metabolic were activated under hypoxia.(3)Transcriptome-based analysis of the effects of hypoxia stress on hepatopancreas metabolismThe hepatopancreas tissues of juvenile E.sinensis were sequenced in normoxia(7.0± 0.2 mg/L)and 6 hours after hypoxia(1.0 ± 0.2 mg/L)by transcriptome sequencing.A total of 2209 differential genes were identified,including 1024 up-regulated genes and1085 down-regulated genes.GO and KEGG enrichment analysis showed that the genes related to oxidative stress,glucose and lipid metabolism,carbohydrate metabolism and immune system were all significantly affected by hypoxia stress.Ten genes from the transcriptome data were randomly selected for q RT-PCR verification,and the results were consistent with the transcriptome data.There are five up-regulated genes including heat shock protein 70(HSP70),cytochrome P450 11A1(CYP11A1),pyruvate carboxylase(PC),inositol-trisphosphate 3-kinase(IT3K)and facilitated trehalose transporter Tret1(TRE).There are five down-regulated genes including glutathione Stransferase(GST),glyceraldehyde 3-phosphate dehydrogenase(GAPDH),hemocyanin(He),fatty acid-binding protein 1(FABP-1)and lipopolysaccharide and beta-1,3-glucan binding protein(GBP).Transcriptome data indicated that juvenile E.sinensis could maintain normal body functions through glucolipid metabolism and immune system under hypoxia stress,thus alleviating oxidative stress of juvenile E.sinensis. |
