| The Chinese mitten crab（Eriocheir sinensis）,commonly known as river crab,belongs to the genus Eriocheir sinensis,a genus of crustaceans,family Archaeidae,and is a special aquaculture animal favored by the market.Vibrio parahaemoilticus is a Gram-negative bacterium widely found in seawater and estuarine environments,and is a highly pathogenic conditional pathogen.The main clinical symptom of Vibrio parahaemoilticus infection in crab is hepatopancreas necrosis.To date,the immune response mechanism of E.sinensis against Vibrio parahaemolyticus infection is not known.In this experiment,we compared the differentially expressed genes in the susceptible,resistant and control groups after Vibrio parahaemolyticus infection,screened the significantly different genes for quantitative analysis,and investigated the role of the differentially expressed genes in the process of Vibrio resistance in river crabs,to provide basic research to reveal the immune defense mechanism of E.sinensis infection.The data,the specific findings of the study are as follows.The pre-test yielded a semi-lethal concentration of 1.78×10~6 CFU/m L for Vibrio parahaemolyticus infection of E.sinensis for 24 h.The hepatopancreas pathology results showed that the color of the hepatopancreas of the diseased crab changed from bright yellow to white,and the hepatocytes were crumbled and necrotic at 72 h after infection,indicating that the causative bacteria had severely damaged the hepatopancreas tissue of the diseased crab.Individuals that were near death or lost the ability to move on their own within 6 hours after attack were defined as the susceptible group（SC）;those in good condition 24 hours after attack were defined as the disease-resistant group（AI）;those injected with PBS were used as the control group（CG）.Three replicates of each group were performed,and the samples of the three groups were analyzed by comparative transcriptome high-throughput sequencing.The results showed that a total of 78,409 Unigenes were assembled,which were annotated in the GO database to functional classifications including signaling,immune system processes and antioxidant activity;in the KOG database were mainly annotated to signal transduction mechanisms,accounting for 11.83%of the total annotated genes;in the KEGG database were mainly mapped to functional classifications including cell growth and death,transport and catabolism,the signal transduction,and immune system-related signaling pathways.The GO and KEGG databases were enriched for differential genes.The differential genes in the GO database were mainly enriched in carbohydrate metabolic processes,transmembrane transport and oxidoreductase activity;the differential genes in the KEGG analysis were mainly enriched in ribosomal pathways,protein processing in the endoplasmic reticulum and lysosomal pathways.A total of 342 immune-related differentially expressed genes were screened,mainly enriched to 22 signaling pathways.It is hypothesized that these differentially expressed genes are involved in the body’s immune response and defense process against bacterial invasion,mainly through these cellular signaling pathways.Eriocheir sinensis showed a highly significant increase（P（27）0.01）in glutathione（ALT）andglutathione（AST）activities in the hepatopancreas after infection with Vibrio parahaemolyticus.The activities of superoxide dismutase（SOD）,total antioxidant capacity（T-AOC）,phenol oxidase（PO）,alkaline phosphatase（AKP）and acid phosphatase（ACP）enzymes showed a trend of increasing and then decreasing,and the difference between the test group and the control group was significant（P（27）0.05）;the activity of lysozyme（LZM）increased with the duration of infection and showed a continuous decrease with the increase of infection time.The above results indicated that Vibrio parahaemolyticus infection stimulated oxidative stress and the immune defense system of the crab,resulting in a significant increase in hepatic function,antioxidant and immune-related enzyme activities in the hepatopancreas.The changes in the expression of immune-related genes in the hepatopancreas tissue of E.sinensis were compared and analyzed by fluorescence quantitative PCR at various time periods.The results showed that the expression of superoxide dismutase（SOD）and hemocyanin（HC）genes in the hepatopancreas of E.sinensis showed a pattern of increasing and then decreasing;the highest expression was reached at 12 h after infection,with highly significant difference（P（27）0.01）,and then gradually decreased and returned to normal level;the expression of serine protease of the clamped structural domain（CDSP）and The expression of CDSP and integrin genes also showed a decreasing pattern,and the expression in the test group was highly significantly increased at 6 hours after infection（P（27）0.01）,and then began to decrease;while the expression of lysozyme（LZM）gene showed an up-regulation at 6 hours after infection,and then continued to decrease,and the difference was significant compared with the control group（P（27）0.05）.This indicates that the expression of superoxide dismutase（SOD）,hemocyanin（HC）gene,lysozyme（LZM）,clamping domain serine protease（CDSP）and integrin gene in the hepatopancreas of E.sinensis was up-regulated at 6 h post-infection,which is related to the resistance of the crab to Vibrio infection and can be an important indicator to evaluate the resistance of E.sinensis to Vibrio parahaemolyticus. |
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