Effects Of Acute Vibrio Alginolyticus Infection On The Immune Defense And Transcriptome Of The Eriocheir Sinensis

The Chinese mitten crab（Eriocheir sinensis）,commonly known as the hairy crab and river crab,is one of the most important species of shrimp and crab farming in China,with a delicious taste,rich in nutrition and of great economic value.Eriocheir sinensis has a migratory habit,usually growing and developing in freshwater,and migrating to seawater or estuarine areas to reproduce and reproduce when the gonads are mature.Vibrio alginolyticus is a group of partly anaerobic Gram-negative,short-form bacilli that do not have budding spores or pods.It is widely distributed in aquaculture waters,estuaries,rivers and brackish lakes,and is highly pathogenic and infectious to both aquatic organisms and humans.In this experiment,the immune defence mechanism of E.sinensis against Vibrio alginolyticus infection was investigated in terms of clinical symptoms,histopathological changes,enzyme activity changes,immune-related gene expression and transcriptomics,and transcriptome changes and differentially expressed genes were analysed to investigate the immune defence mechanism of E.sinensis against bacterial infection and to provide a basis for healthy breeding of E.sinensis.The main methods and results are as follows（1）A model for acute infection with Vibrio alginolyticus in Eriocheir sinensis was established by laboratory attack tests.The 24-h LD₅₀was calculated to be 1.93×10~6CFU/m L according to Kou’s modified formula.（2）Eriocheir sinensis was infected with Vibrio alginolyticus and showed clinical signs such as reduced vigour,reduced locomotion,no avoidance behaviour after stimulation,and failure to turn over quickly after artificial inversion.The hepatopancreas was found to be pale in colour and some of the gill filaments were blackened after autopsy.Histopathological analysis showed that compared to the control group,the hepatopancreas showed obvious lesions 12 h after bacterial infection;swelling of the tips of the gill filaments,irregular arrangement of the gill filaments and atrophy of the epithelial cells.Vibrio alginolyticus infection caused damage to the hepatopancreas and gill tissue of E.sinensis,with the most serious organ damage occurring between 12 and 24 h after infection.（3）The results of the enzyme activity assay showed that the lysozyme（LZM）activity in the hepatopancreas tissue of Eriocheir sinensis infected with Vibrio alginolyticus gradually increased from 6 to 48 h and gradually decreased from 48 to 72 h,all of which were significantly higher than that of the control group（P<0.01）.The activity of superoxide dismutase（SOD）and phenol oxidase（PO）gradually increased from 0 to 6 h and was significantly higher than that of the control group（P<0.01）,gradually decreased from 6 to 12h,increased again from 12 to 24 h,gradually decreased again from 24 to 72 h,and returned to a level close to that of the control group at 72 h.The overall trend of glutathione reductase（GR）decreased and then increased,and alkaline phosphatase（AKP）decreased and then increased,and then decreased and then continued to increase after being infected.The above results demonstrated that Vibrio alginolyticus infection activated the immune-related enzyme activity in the hepatopancreas tissue of E.sinensis,causing changes in the activity of immune-related enzymes such as lysozyme.（4）Near death susceptible individuals within 12 h of infection were defined as susceptible group（SIS）,disease resistant surviving individuals within 24 h were defined as disease resistant group（ASX）,and the PBS injection group within 24 h was used as control group（CG）.Nine crabs were randomly collected from each group,RNA was extracted from hepatopancreatic tissue,and after quantification,three random equivalents were evenly mixed into one sample for high-throughput transcriptome sequencing.The obtained Unigenes were annotated in seven databases,NR,NT,KO,Swissprot Pfam,GO and KOG.2.98%of Unigenes were annotated in all databases,45.25%of Unigenes were annotated in at least one database and 54.75%of Unigenes were not annotated in all databases.GO analysis showed that 21,633 Unigenes were clustered by molecular function,biological process and cellular component;KOG analysis,a total of 6,113 Unigenes were grouped into 26 functional categories;7,870 Unigenes were mapped to the KEGG pathway database.（5）A total of 10,547 differentially expressed genes were identified（P<0.05）.Among them,6,232 differentially expressed genes（P<0.05）were identified between the susceptible group（SIS）and the control group（CG）,including 3,170 up-regulated genes such as:anti-lipopolysaccharide factor 2,phosphoenolpyruvate carboxykinase,and 3,062down-regulated genes such as:alkaline phosphatase,chitinase;4,144 differentially expressed genes（P<0.05）were identified between the disease-resistant group（ASX）and the control group（CG）（P<0.05）,including 1,670 up-regulated genes,e.g.ADP-nucleotidylation factor,clip structural domain serine protease,and 2,474 down-regulated genes,e.g.glutathione S-transferase Mu 1,carboxypeptidase B.6,104 differentially expressed genes were identified between the susceptible group（SIS）and the resistant group（ASX）（P<0.05）.These included3,276 up-regulated genes such as B-lymphocytoma-2,NF-kappa B inhibitor alpha and 2,828down-regulated genes such as phosphoenolpyruvate carboxykinase,calcineurin.KEGG enrichment of differential genes revealed that the main enriched pathways were:MAPK signaling pathway,lysosomes,metabolism of glutathione,PPAR signaling pathway,etc.（6）Six genes,including Arf,Laccases2 and Bcl2,were screened out from the identified differential genes and further quantified by RT-q PCR for fluorescence analysis of m RNA expression at 6 h,12 h,24 h,48 h and 72 h after infection.The results showed that the expression of the above genes changed significantly after Vibrio alginolyticus infection,suggesting that these genes may play an important role in the regulation of immunity in E.sinensis.In conclusion,Vibrio alginolyticus infects E.sinensis,causing tissue damage to the hepatopancreas and gills,resulting in changes in the activity of immune-related enzymes such as lysozyme and improving the antioxidant capacity of the organism and immune defence;at the same time,it leads to differential expression of immune-related genes in the transcriptome of hepatopancreas tissue of E.sinensis with different clinical symptoms,thereby improving the body’s immune performance.