| White spot syndrome virus(WSSV)is one of the most devastating pathogens of the cultured shrimp Litopenaeus vannamei,responsible for massive loss of its commercial products worldwide.WSSV was first recorded in Taiwan in 1992 and subsequently observed worldwide.Losses due to WSSV have been estimated at more US$8 billion since 2000.WSSV outbreaks occur continuously in China and exist widely in the wild.Macrobrachium nipponense is an important economic species that is farmed widely in China,with annual yields exceeding more than 270 thousand metric tonne.The most aquaculture area were Jiangsu,Anhui and Zhejiang Province.Macrobrachium nipponense is generally considered to be less prone to disease in culture.However.WSSV was firstly found in M.nipponense in Wuxi city,2015.Macrobrachium nipponense can take along with WSSV,but there is not out break due to WSSV.Macrobrachium nipponense can be a new host of WSSV or a new WSSV carrier,and it is a threat to the other crustaceans.The gills play a vital role in transporting respiratory gases and controlling the osmotic and ionic balances in aquatic organisms.Hepatopancrcas is the main immune organ in shrimp and the center for storage,metabolism,and detoxification.However,information on histological changes following infection of WSSV in M.nipponense is lacking.In this paper,we studied the pathogenicity of WSSV and WSSV induced histopathological changes in gill and hepatopancreas of M.nipponense.Using next generation sequencing and bioinformatics techniques for analyzing the transcriptome differences between the hepatopancreas of M.nipponense experimentally infection with WSSV and normal control prawns without previously annotated genomes as references.It will demonstrate some immune molecular mechanisms involved in WSSV infection,identify candidate immune-related genes against WSSV infection,and construct possible strategies to prevent the spread of WSSV in aquaculture of other crustaceans.1.Experimental inoculation of oriental river prawn M.nipponense infected with white spot syndrome virus(WSSV)We investigated the infectivity and pathogenicity of WSSV in adult M.nipponense using standardized conditions for Litopenaeus vannamei.The median lethal dose of WSSV in adult M nipponense was 103.84±0.06 copies g-1,which was about 1000-fold higher than in L.vannamei(100.59±0.22 copies g-1).WSSV was detected by two-step polymerase chain reaction in the gills,hepatopancreas,muscle,stomach,heart,gut,nerve,integument,pereopod,eyestalk,testis,and ovary of experimentally infected dead M.nipponense.2.Histopathology studies of oriental river prawn M.nipponense infected with white spot syndrome virus(WSSV)Lesions were observed histologically following WSSV injection(0,24,48,72 and 96 h),hepatopancreas showed histological lesions firstly(24 hpi),including increased numbers of F and R cells compared with 0 h hpi,as well as enlarged nuclei with marginalized chromatin,and often contained a distinct basophilic inclusion body.,pyknotic nuclei were more abundant than 0 hpi and the size of the B cells was decreased;then gill showed histological lesions subsequently(48 hpi),including hemocytic infiltration(HI)in the hemocoelic sinuses,fusion of lamellae(FL),and basophilic inclusion bodies.WSSV concentration of hepatopancreas at 0,24,48,72,96 hpi were 0,103.9,102.8,103.5,102 6 copies g-1.WSSV concentration of gills at 0,24,48,72,96 hpi were 0,0,103.0,103.5,102.7 copies g-1.WSSV concentration of muscle at each time point were 0 copies g-1 The clearance of WSSV were observed in hepatopancreas and gills came out together with the decrease in both WSSV concentration and number of basophilic inclusion bodies at 48 and 96 h post-inoculation,respectively.No histological lesions were detected in muscle from 0-96 h post-injection.The results show that the oriental river prawn M.nipponense can be infected by WSSV and the infections are self limiting over time,therefore,M.nipponensis may serve as a useful model for studying resistance to WSSV.3.Screeing of immune-related genes in oriental river prawn M.nipponenseWe applied the next generation sequencing and bioinformatics techniques for analyzing the transcriptome differences from the hepatopancreas of M.nipponense experimentally infection with WSSV among the survived,moribund and normal control prawns without previously annotated genomes as references.The Illumina Hiseq2500 TM sequencing platform was used to perform high-throughput sequencing.Then,the sequencing data were subjected to de novo assembly using the Trinity program.A total of 52,605,014,51,924,787 and 51,969,106 raw reads were produced in control,moribund and survived samples,respectively.After the removal of adapter sequence and low-quality reads,a total of 50,948,257,50,696,076,and 49.870,542 high quality clean reads that represent a total of 7,635,728,511(7.63 Gb),7,599,313,025(7.60 Gb),7,475,646,918(7.48 Gb)nucleotides were generated for the control,moribund and surivived samples,respectively.After the de novo assembly,in total 64,049 unigenes were obtained.In the comparison of moribund samples and control,a total of 1,960 genes were found to be aberrantly expressed,with 764 significantly up-regulated and 1,196 significantly down-regulated.In brief,among these 1,960 DEGs,1,329 genes were expressed in both control and moribund groups,including 443 significantly up-regulated genes and 886 significantly down-regulated genes.Moreover,321 genes were only expressed in the moribund samples after WSSV challenge.In the comparison of survived samples and control,a total of 4,311 genes were found to be aberrantly expressed,with 3,308 significantly up-regulated and 1,003 significantly down-regulated.In brief,among these 4,311 DEGs,2,531 genes were expressed in both control and survived samples,including 1,825 significantly up-regulated genes and 589 significantly down-regulated genes.Moreover,1,483 genes were only expressed in the survived samples.In the contrast of two comparison libraries,4,011 DEGs were only expressed in the comparison of survived and control library with 3,213 up-regulated genes and 798 down-regulated genes,and 1,660 DEGs were only expressed in the comparison of moribund and control library with 699 up-regulated genes and 991 down-regulated genes.The two comparison libraries were have 300 mutual DEGs with 95 up-regulated genes and 205 down-regulated genes.Numerous immune-related genes were obtained from two comparison libraries,the library of comparison between control and moribund samples and the library of comparison between control and survived samples.In total,88 immune-related genes were obtained.These immunes genes are groups into 13 functions,Which include antimicrobial(1 genes),Proteases(11 genes),protease inhibitors(2 genes),cell death(5 genes),cell adhesion(9 genes),heat shock proteins(2 genes),oxidative stress(10 genes),pathogen recognition immune receptors(4 genes),signaling pathway(Wnt signaling pathway-5 genes,PI3K/AKT/mTOR pathway-3 genes,MAPK signaling pathway-17 genes,JAK/STAT pathway-3 genes,Ubiquitin Proteasome Pathway-6 genes)and other immune genes(20 genes).4.The reponses of immune-related genes in oriental river prawn M.nipponense to WSSV challengeThe time-course mRNA expression changes of ten genes in hepatopancreas tissue after WSSV challenge were investigated by qRT-PCR.The ten genes were glutathione s-transferase(GST),copper/zinc superoxide dismutase 4(Cu/Zn SOD 4),heat shock protein 21(HSP21),interferon regulatory factor(IRF),extracellular signal-regulated kinases 1/2(ERK),MAP kinase-activated protein kinase 2(MKK2),MAP kinase-activated protein kinase 4(MKK4),c-Jun N-terminal kinase(JNK),inhibitor of apoptosis protein(IAP)and ferritin.The gene expression of the ten genes were all significantly changed after WSSV infection at 24 hpi in M.nipponense. |
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