Effects Of Hypoxic Stress On Mitten Crab Hemolymphocytes Based On Multi-omics Analysis

The Chinese mitten crab,Eriocheir sinensis,is a traditional Chinese aquatic product due to its high nutritional value and economic value.However,the aquatic environment where crabs live in is often caused hypoxia by natural factors(e.g.,temperature,geographic location and water flow)and human factors(e.g.,nutritent enrichment,pollutants and high-density breeding),leading to slow growth and weaken immunity.E.sinensis tremor disease is a harmful aquatic disease which the pathogen is Spiroplasma eriocheiris.The infected crabs are often accompanied by symptoms such as sudden loss of food intake and intermittent tremor of appendages.The disease is highly contagious and has a high mortality rate,which can easily cause a lot of economic losses for farmers.In this study,E.sinensis was used as the research object.Firstly,comparative transcriptomics and proteomics were used to analyze under hypoxia stress.Then,Es Integrin1 and Es Integrin2 were selected and their functions were preliminarily explored.Finally,the mortality and changes of related physiological and biochemical indexes after S.eriocheiris infection were investigated under hypoxia stress.These data are expected to provide some theoretical basis for the related molecular regulatory mechanisms of crabs in response to hypoxia stress and S.eriocheiris infection.The paper mainly includes the following 4 parts:1.Transcriptome analysis of E.sinensis hemocytes in response to hypoxia.The transcriptomic profiles of E.sinensis hemocytes after 24 h of hypoxia and normoxia were obtained using Nova Seq 6000.128,614 unigenes were aquired with an average length of 2,634 bp through sequence splicing,annotation and screening analysis.Meanwhile,a total of 2,037 differentially expressed genes were identified,including 1,068 up-regulated and 969 down-regulated genes.GO analysis shows that metabolic processes such as glycolytic process,ADP metabolic process and ribonucleoside diphosphate metabolic process play an important role in hypoxia.Some immune-related pathways(ECM-receptor interaction,gap junction,autophagy and so on)and metabolic pathways(cysteine and methionine metabolism,glycolysis,ether lipid metabolism and so on)were identified via KEGG pathway analysis.Finally,8selected genes from these pathways were verified by RT-q PCR analysis.The results showed 5 up-regulated genes(integrin beta 1,laminin subunit alpha-like,phosphoinositide-3 kinase,phosphoglycerate mutase 2 and,relish)and 3 downregulated genes(90 k Da heat shock protein,aspartate aminotransferase and,tubulin alpha)which is correspond with the transcriptome analysis.2.Proteomics analysis of E.sinensis hemocytes in response to hypoxia.A total of 19,432 peptides and 3,005 proteins were identified by the proteomic analysis of the hemocytes of E.sinensis after 24 h of hypoxia using i TRAQ technique.Compared with normoxia group,there were 664 significantly different proteins in hemocytes under hypoxia stress,including 567 up-regulated proteins and 97 downregulated proteins.KEGG pathway enrichment analysis showed that ECM-receptor interaction,PI3K-Akt signaling pathway,focal adhesion,and apoptosis were enriched.The activities of antioxidant enzymes(PO,CAT,SOD,MDA)in the hemocytes of E.sinensis under hypoxia stress for 24 h were detected.The results showed that the enzymes activities in the hypoxia group were significantly higher than the normoxia group,indicating that the hypoxia stress could regulate the activity of antioxidant enzymes to protect the cells from damage.3.Functional study of Es Integrin1 and Es Integrin2.Two hypoxia-related integrin genes,Es Integrin1 and Es Integrin2,were screened by transcriptomics.The full length of Es Integrin1 is 2,790 bp,the open reading frame(ORF)encodes 782 amino acids.The full length of Es Integrin2 is 3,112 bp,and the ORF encodes 972 amino acids.Both protein sequences contain typical integrin domains,such as integrin beta subunits(INB),epidermal growth factor-like domain(EGF).The results of RT-q PCR showed that these two genes were highly transcribed in hemocytes of E.sinensis;the transcription of Es Integrin1 was significantly up-regulated after S.eriocheiris infection,but Es Integrin2 was significantly down-regulated,indicating that Es Integrin1 and Es Integrin2 may involved in the resistance process of S.eriocheiris infection,and play their own immune functions.4.Changes of physiological and biochemical indexes of E.sinensis infected with S.eriocheiris under hypoxia stress.The mortality of E.sinensis and the copy number of S.eriocheiris in hemocytes in hypoxia group was significantly higher than that in normoxia group.The total cell number of E.sinensis did not change significantly under different dissolved oxygen.The necrosis rate and apoptosis rate of hemocytes of crabs after S.eriocheiris infection under hypoxia were significantly higher than those in normoxic group.Compared with the control group,the hepatopancreas and gill tissue of crabs were analyzed.Multiple vesicles appeared in the hepatopancreas tissue and the gill tissue structure was diffuse after long-term hypoxia stress,with structural damage of different degrees in both tissues.These results indicate that hypoxia stress has a synergistic effect on S.eriocheiris infection and has harmful effects on the physiology and biochemistry of E.sinensis.