| The Eriocheir sinensis（E.sinensis）is an important species of special freshwater farmed crab in China,with a total national production of approximately 770,000 tonnes in 2020.Currently,bacterial diseases are one of the important factors limiting the development of E.sinensis farming.Klebsiella pneumoniae（K.pneumoniae）is a common pathogenic bacterium capable of infecting farmed animals such as fish,shrimps and crabs,causing significant economic losses to the aquaculture industry.Therefore,studying the infectivity and pathogenicity of K.pneumoniae on E.sinensis and revealing its pathogenic mechanism can help the healthy culture and bacterial disease control of E.sinensis.The main studies are as follows.（1）A strain of K.pneumoniae type K57 was isolated and identified from the hepatopancreas of E.sinensis with an LD50 of 2.11 x 107 CFU/m L for 24 h.The infection of K.pneumoniae caused tissue damage to the hepatopancreas of E.sinensis,and the activity of some antioxidant-related enzymes,such as SOD,CAT and TAOC,in the hepatopancreas was significantly increased(The activity of ACP and AKP enzymes was significantly reduced at 6,12 and 24 h（P < 0.05）.Drug sensitivity tests showed that cefixime,ofloxacin,nalidixic acid,ciprofloxacin,chloramphenicol and tetracycline were strongly inhibitory to K.pneumoniae K57;while amoxicillin,rifampicin and amikacillin had lower inhibitory effects.Group definition: According to the different clinical manifestations 24 h after infection with K.pneumoniae,individuals on the verge of death were defined as the susceptible group（SG）,and asymptomatic individuals were defined as the anti-susceptible group（AI）,and PBS injection as the control group（CG）.（2）The transcriptome sequencing results showed that 10,940 differentially expressed genes（DEGs）were identified between the SG and CG groups,of which 5,449 were upregulated and 5,491 were down-regulated（P < 0.05）.8,495 DEGs were identified between the SG and AI groups,of which 4,377 and 4,118 genes were up-regulated and down-regulated respectively（P < 0.05）.(3,379 DEGs were identified between the AI and CG groups,including 1,667 up-regulated and 1,712 down-regulated genes（P < 0.05）.Twenty DEGs were randomly validated using qRT-PCR and the results matched the RNA-seq trend,demonstrating the high accuracy of the RNA-Seq results.GO functional annotation showed that the upregulated DEGs in the SG and CG groups were significantly annotated in the GTPase activity class,kinase activity,cell adhesion class,signal transduction and other functions,while the down-regulated DEGs were significantly annotated in the oxidative The KEGG pathway enrichment analysis showed that the DEGs upregulated in the SG and CG groups were significantly enriched in the immune system.DEGs were significantly enriched in pathways related to immune response and apoptosis-like functions,while those down-regulated were significantly enriched in signalling pathways related to digestion and absorption,metabolism and biosynthesis（P < 0.05）.The down-regulated DEGs were significantly enriched in functions related to metabolism,digestion and absorption,and biosynthesis（P < 0.05）.Five immunerelated DEGs,SOD,CTL,Tollip,Dorsal and CaM,were screened for expression in different time sequences based on GO functional annotation,KEGG pathway enrichment analysis and immune-related processes,and 18 immune-related DEGs were screened for comparison between groups.The results showed that SOD expression was significantly up-regulated at 3 h and 6 h（P < 0.05）.CTL expression was significantly up-regulated from 3 h（P < 0.05）,significantly down-regulated at 12 h and 24 h（P < 0.05）and returned to the control level after48 h.The expression of Tollip,Dorsal and CaM was significantly up-regulated at 3 h postinfection（P < 0.05）and at 6 Some of the transducers and effectors in the Toll pathway,IMD pathway and MAPK pathway may be involved in the immune response to K.pneumoniae infection in E.sinensis.（3）The effect of K.pneumoniae infection on the intestinal flora of E.sinensis was analyzed based on 16 S r RNA gene amplicon sequencing technology.We found that the abundance of Alphaproteobacteria in the SG and AI groups was significantly lower than that in the CG group（P < 0.05）,but the abundance of Enterobacterales and Enterobacteriaceae in the SG and AI groups was significantly lower than that in the CG group（P < 0.05）.(The abundance of Enterobacterales,Enterobacteriaceae,Prolixibacteraceae and Roseimarinus in the SG and AI groups was significantly higher than that in the CG group（P < 0.05）.The abundance of Firmicutes,Erysipelotrichales and Erysipelotrichaceae was significantly lower in the SG group than in the AI group（P < 0.05）,and the abundance of Acinetobacter was significantly higher in the SG group than in the AI group（P < 0.05）.Based on the differences in intestinal flora abundance between groups and PCA analysis,it was concluded that acute infection with K.pneumoniae affected the composition and structure of the intestinal microbial community of E.sinensis,increasing the abundance of harmful intestinal bacteria and a decrease in the abundance of beneficial bacteria.In summary,this study initially revealed the changes in histopathological and enzymatic activity indicators of the hepatopancreas of E.sinensis infection over time,further investigated the differentially expressed genes screened,analysed their main functional annotations and enriched signalling pathways,and combined with relevant immune signalling pathways for immune differentially expressed genes,and revealed that acute infection with K.pneumoniae The relationship between pathogenic bacteria and intestinal flora species and numbers were investigated.These new findings provide basic data for the healthy breeding and prevention of bacterial diseases in E.sinensis. |
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