| The oriental river prawn,Macrobrachium nipponensis is one of the most important freshwater economic shrimps in China.This freshwater shrimp has become a leading breed in aquaculture industry,because of its advantages of high fecundity,strong adapt-ability,wide appetite,tender meat,delicious taste,rich nutrition,which can be listed all the year round.Also,it has become one of the important ways to increase agricultural efficiency and farmers’ income.However,with the expansion of the scale of cultivation,the gradual deterioration of water ecological environment,various diseases are con-stantly emerging,especially bacterial diseases,which cause great harm for the healthy and sustainable development of M.nipponensis.In September 2017,an outbreak of mass mortality break out at the river prawn farms in Yangzhou city,Jiangsu province,P.R.China,the body surface and hepatopancreas of the diseased shrimps are red,the vitality is weakened,the appetite is decreased,the response to external stimulation is slow,and then a large number of deaths are caused.At present,the cause of the outbreak of this disease has not been reported.In this study,we identified the pathogen XL1 which caused the disease of M nipponensis,explored the pathogenicity of the stain XL1 and the host immune response,and screened a Bacillus CPA 1-1 which has good antibacterial effect on the XL1,to establish a theoretical basis for the prevention and treatment of diseases.1.In this study,the dominant strain XL1 was isolated from hepatopancreas of dis-eased M.nipponensis,and its taxonomic position,pathogenicity,carrying of virulence related genes,secretion of extracellular enzymes and pathological changes in tissues of shrimp after infection were studied.Non-O1 Vibrio cholerae was identified by bio-chemical characteristics and 16S rRNA and gyrB homologous analysis.The infection test showed that the strain XL1 was pathogenic to M.nipponensis,and the half lethal dose(LD50)were 4.09×104 CFU/mL.Detection of virulence-associated genes by PCR indicated that strain XL1 was positive for Mp,HlyA,RtxA,OmpU,Ace,Zot and T6SS.Furthermore,the results of extracellular enzyme analysis revealed that the strain can produce lecithinase,amylase,gelatinase and hemolysin.Histopathological analysis re-vealed that the hepatic tubule lumen and the gap between the hepatic tubules became larger,and the brush border disappeared in the hepatopancreas.2.To study the immune response against non-O1 V.cholerae,in this study,we performed transcriptome analysis of M.nipponense at 6 h and 12 h post-infection(hpi),and differentially expressed genes(DEGs)(P<0.05)were revealed by comparison of gene expression levels between 6 and 12 hpi.The results showed that a total of 189 DEGs including 104 up-regulated genes and 85 down-regulated genes at 6 hpi,56 DEGs at 12 hpi were identified,which was much lower than that in the comparison of at 6 hpi and the up-regulated and down-regulated genes were 39 and 17,respectively.Rap1 signaling pathway,MAPK signaling pathway,ECM-receptor interaction,Endo-cytosis,Hippo signaling pathway,Ubiquitin mediated proteolysis and Phagosome were positively modified after non-O1 V.cholerae challenge,and most the immune-related pathways were activated at 6 hpi.We selected 6 DEGs and validated their expression level by RT-qPCR.The data generated in this study may provide a valuable resource for further immune response research and offer improved strategies against non-O1 V.cholerae infection in shrimp.In order to further study the effect of non-O1 V.cholerae on the immunity of M.nipponensis,Quantitive real-time PCR(qRT-PCR)was undertaken to measure mRNA expression levels for thirteen immune related genes in M.nipponense after non-O1 V.cholerae infection at different time.The transcriptional analysis of these immune re-lated genes demonstrated that the expression levels of dorsal,relish,p38,crustin1,crustin2,crustin3,hemocyanin,i-lysozyme,anti-lipopolysaccharide factors 1,anti-lip-opolysaccharide factors 2,prophenoloxidase were significantly up-regulated in hemo-lymph of M nipponense post-infection.These results revealed varying expression pro-files and clear transcriptional activation of these immune related genes in hemolymph,which will contribute to better understand the pathogenesis and host defensive system in non-O1 V.cholerae invasion.3.To study the probiotic mechanism of bacillus against M nipponense,a strain of CPA 1-1 with obvious antagonistic activity was screened from healthy M.nipponense by double-layer plate method.Strain CPA1-1 was identified as Bacillus velezensis through morphological,physiological and biochemical charact eristics in combination with 16S rRNA and gyrB sequence analysis.The growt h of characteristics,inhibition effect and the carrying of antibiotic-related genes of the strain were also studied.The results showed that the minimum inhibito ry concentration of CPA1-1 fermentation broth against non-O1 V.cholerae was 2.7×105 CFU/mL.B.velezensis CPA1-1 contains genes such as srfAA,ituA,fen A,dhbA,bmyA,beaS and dfnA encoding the biosynthesis of various peptidogly cans,polyketose compounds and antimicrobial proteins.Also,the different conc entrations of B.velezensis fermentation broth on immune-related genes and enz ymes in hepatopancreatine and haemolymph of M.nipponense were studied.Th e stain CPA 1-1 can stimulate the expression level of immune related genes and the activity of immune enzyme and reduce the content of ammonia nitrogen a nd nitrite nitrogen in aquaculture water.Alao,the results of challenge test sho wed that the survival rate and the disease resistant ability of M.nipponense co uld be improved.This study provides a theoretical basis for the rational use of bacillus in M nipponense culture. |
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