| Macrobraehium rosenbergii is an important cultural freshwater prawns cultured in China due to its high growth performance,delicious taste,high economic benefit.Unfortunately,with expansion of the cultivation scale and density,epidemic infectious diseases occur frequently and a new slow-growth agent of M.rosenbergii has emerged in recent years,which have caused serious economic losses of M.rosenbergii industry.Since 2017,outbreaks of mass mortalities among cultured larva of M.rosenbergii occurred in different hatcheries of Gaoyou and Jiangdu counties of Jiangsu province,China.The epidemiological investigation showed that Enterobacter cloacae was the major pathogen which caused repeated infections in M.rosenbergii.In this study,the pathogenicity of E.cloacae and its induced immune response in M.rosenbergii were explored;The effects of E.cloacae infection on the growth of M.rosenbergii were also investigated;Furthermore,considering the importance of rpoS in environmental adaptation and virulence regulation in several other bacterial species,the functions of rpoS in the regulation of E.cloacae virulence and the response to diverse stresses were explored,which will provide references for revealing the repeated infection mechanism of E.cloacae in M rosenbergii.The main research results of this study are as follows:1.Outbreaks of mass mortalities occurred in different hatcheries of Gaoyou and Jiangdu counties from 2017 to 2019.The bacterial isolates from the diseased M.rosenbergii exhibited the same phenotypic traits and biochemical characteristics,and were identified as E.cloacae.In challenge test,the zoea and postlarva of M.rosenbergii infected with the representative E.cloacae XL3-1 developed similar pathological signs to the naturally diseased prawns,and the LD50 value of the strain XL3-1 to the zoea and postlarva were 3.2 × 106 CFU/mL and 3.5 × 106 CFU/mL,respectively.Histopathological analysis revealed that hepatopancreas and intestines of infected M.rosenbergii exhibited obvious inflammatory responses to E.cloacae infection.Detection of virulence-associated genes by PCR indicated that XL3-1 was positive for irp2,fhuA,sodB,sltA,flaD and OmpX.According to the antibiotic resistant assay,the isolate was sensitive to quinolones,while they were resistant to cephalosporins and penicillins.2.To investigate the immune response of M.rosenbergii against E.cloacae,we performed transcriptome analysis of the M.rosenbergii hepatopancreas with and without E.cloacae infection using RNA-seq.After assembly and annotation,29,731 high quality unigenes were obtained from RNA-seq data.Differential expression analysis revealed the existence of 2498 significantly differential expressed genes(DEGs)at 12 h post infection,with 1365 up-regulated and 1133 down-regulated genes.Among these DEGs,some well-known immune-related genes were up-regulated significantly,including C-type lectin 1,lectin 3,anti-lipopolysaccharide factor 2,Cu/Zn superoxide dismutase and heat shock protein 70.GO analysis showed some DEGs were clustered into immune related subcategories such as immune system process,response to stimulus,biological adhesion,and antioxidant activity.These DEGs were enriched into 216 KEGG pathways including a core set of immune correlated pathways notably in phagosome and lysosome.To further study the immune response of M.rosenbergii against E.cloacae,the expression level of six selected immune-related genes(ALF2,CLEC1,LEC3,hemocyaninl,HSP70 and SOD)based on the transcriptomic data were monitored at different point of time in hepatopancreas,hemolymph,gill and intestine by qRT-PCR.Results revealed the expression level of ALF2,LEC3,hemocyaninl,SOD up-regulated and reached the peak at 12 hpi in the hepatopancreas,the expression level of CLECI,HSP70 reached the peak at 24 hpi;The expression level of ALF2,HSP70 reached the peak at 6 hpi in the hemolymph,the expression level of hemocyaninl,SOD reached the peak at 12 hpi,the expression level of CLEC1,LEC3 reached the peak at 24 hpi;The expression level of hemocyaninl reached the peak at 6 hpi in the gill,the expression level of HSP70,SOD reached the peak at 12 hpi,the expression level of ALF2,CLEC1,LEC3 reached the peak at 24 hpi.The expression level of SOD reached the peak at 6 hpi in the intestine,the expression level of ALF2,CLEC1,HSP70 reached the peak at 12 hpi,the expression level of ALF2,CLEC1,LEC3 reached the peak at 24 hpi.These results showed that these immune-related genes participated in the immune response and can be considered as potential monitoring indicators of prawn's health status.3.M.has been severely affected by slowing growth in recent years.The epidemiological investigation showed E.cloacae has been detected with high prevalence in slow-growing prawns,while E.cloacae infection frequently occurred and caused mass mortalities in M.rosenbergii hatcheries.Therefore,the correlation between E.cloacae and slow-growing M.rosenbergii was explored in this study.The infectivity of slow-growing prawns to healthy M.rosenbergii was verified by co-culture of healthy and slow-growing M.rosenbergii.The result showed the slow-growing M.rosenbergii was infectious and caused slow growth of healthy M.rosenbergii.In addition,the growth indexes and the expression of growth-related genes of M.rosenbergii after challenging with E.cloacae were detected to investigate the effects of E.cloacae on the growth of M.rosenbergii.The results showed that the growth of M.rosenbergii was found to be significantly reduced at 30 and 60 days post infection,while the expressions of growth-related genes(CHIT3,Cat,JHEH,EcR,and RXR)were downregulated after E.cloacae infection,the expression of inhibiting growth related gene(EcR)was upregulated after E.cloacae infection.Similarly,the differential expression of these growth-related genes in M.rosenbergii after E.cloacae infection showed identical expression tendency in the slow-growing M.rosenbergii.The present results reveal a high prevalence of E.cloacae in slow-growing M.rosenbergii and suggest that this pathogen is the probable cause of the slow growth in M rosenbergii.4.Considering the continuous E.cloacae infection in M.rosenbergii and the importance of rpoS in environmental adaptation and virulence regulation in several other bacterial species,the role of rpoS in stress survival and virulence of E.cloacae was explored.In this study,RNAi technology was used to knockdown,poS in E.cloacae.The expression of rpoS in the rpoS-RNAi strain and the wild-type strain was detected by qRT-PCR.The results showed that the expression of rpoS in the rpoS-RNAi strain was reduced by 82.62%compared to the wild-type strain.The transcriptome analysis of the rpoS-RNAi and wild-type strains was used to explore the roles of rpoS in the stress response and virulence of regulation E.cloacae.The transcriptome analysis results revealed that there were a total of 488 differentially expressed genes,among which 30 genes were up-regulated and 458 genes were down-regulated.Among these DEGs,multiple genes related to environmental response,biofilm formation,bacterial type II secretory system,flagellin,fimbrillin,and chemotactic protein which associated with bacterial survival and virulence,were down-regulated significantly in the rpoS-RNAi strain.In addition,KEGG analysis showed that the differentially expressed genes of the rpoS-RNAi and wild-type strain were mainly enriched in two-component system,ABC transporters,flagellar assembly,bacterial chemotaxis and quorum sensing KEGG pathways,which associated with bacterial survival and virulence.These results indicated that rpoS was involved in stress response and virulence regulation of E.cloacae.5.To further determine the functional roles of rpoS in survival of E.cloacae,the expression levels of rpoS under environment stress were detected by qRT-PCR,and the effects of rpoS on bacterial growth and survival under stress conditions,the biofilm development were also investigated.The results showed that the expression levels of rpoS were upregulated after high salinity and starvation stress,indicating that rpoS might play important roles in survival;In comparison with the wild-type E.cloacae,RNAi-treated bacteria showed significantly impaired abilities of growth in diverse environments,survival in environmental stresses(starvation,salinity,low pH,and oxidative stress),biofilm production;And qRT-PCR analysis showed that the expression of rpoS could directly down-regulate the expression of several important stress resistance and biofilm formation relate genes.In addition,the effects of rpoS on E.cloacae virulence were also evaluated by motility test,adhesion test and virulence test.In comparison with wild-type E.cloacae,RNAi-treated bacteria showed significantly impaired abilities of movement,adhesion to cells,pathogenicity and colonization to M.rosenbergii;And qRT-PCR analysis showed that the expression of rpoS can directly down-regulate the expression of several important virulence-related genes,such as flagellin,fimbrillin,chemotactic protein,bacterial type II secretion system.In this study,the characterization of virulence properties of E.cloacae isolated from diseased M.rosenbergii was clarified;the effects of E.cloacae on growth of M.rosenbergii were investigated;the roles of rpoS in the survival and virulence regulation of E.cloacae were clarified.These results will provide theoretical support for prevention and control the diseases caused by E.cloacae in M.rosenbergii. |
PDF Full Text Request