Preliminary Study On Transmission Of Shrimp Viruses Between Coastal Ponds-offshore Sea And Genetic Evolution Of The Virus

Since the 1980 s,with the rapid development of the shrimp aquaculture industry in many countries,shrimp diseases have been caused more and more attention.Shrimp diseases are mainly divided into infective and non-infectious diseases.The main pathogens of infectious diseases include viruses,fungi,bacteria and parasites,amnong which the viral infectious diseases are the most serious.Up to now,more than 20 kinds of viral shrimp diseases have been reported.The major shrimp viral diseases that threaten the world shrimp aquaculture include,White spot syndrome virus disease(WSD),Disease of baculovirus penaei(BP),Infectious hypodermal and hematopoietic necrosis(IHHN),Yellow head disease(YHD),Taura syndrome virus disease(TSD),Infectious muscular necrosis(infectious)myonecrosis(IMN),Macrobrachium rosenbergii whitish muscle disease(WTD),Monodon baculovirus disease(MBV)and Viral covert mortality disease(VCMD).The prevalence of above-mentioned shrimp diseases has resulted in huge economic losses ofi the aquaculture industry.In the past decades,research mainly focused on the etiology and pathogenesis of the aquaculture diseases,immune and defense mechanism of the hosts.There are few reports involving the origination,transmission,evoluation and impact to the wild populations in offshore sea of the pathogens.The lack of such knowledge caused that it is hard to reveal the origin and fate of the shrimp pathogenes,it is impossible to effectively evaluate the potential impact of the pathogens to the wild population and offshore ecological environment,it is difficult to explore the decisive factors triggering the epidemic diseases and support the work on the early warning and prevention of new emerging diseases.Based on this,the present study investigated the epidemiology of CMNV,the infectious agentof VCMD,in aquaculture shrimp in coastal provinces in 2013~2015.Full-scale survey on host ranges,histological characteristics of infection,range and rate of prevelence of CMNV were also condected in wild shrimp in seas including the Bohai Sea,the Yellow Sear and the East China Sea.The prevalence rate and evolution of CMNV both from coastal ponds and wild seas were compared.Meanwhile,prevalence of the other two major shrimp diseases,WSD and IHHN,in the Bohai Sea were also investigated.Firstly,the prevalence and distribution of CMNV in aquaculture ponds in coastal provinces was surveyed based on the analysis of reverse transcription loop-mediated isothermal amplification(RT-LAMP),reverse transcription nested PCR(RT-nPCR),phylogenetic tree,histopathology,RNA in situ hybridization(ISH)and transmission electron microscopy(TEM).RT-LAMP results showed that CMNV positive samples appeared in the cultured crustaceans including Litopenaeus vannamei,Fenneropenaeus chinensis,Marsupenaeus japonicus,Penaeus monodon,and Macrobrachium rosenbergii,and mostly distributed the coastal provinces in China.The prevalence rates of CMNV among the collected samples in 2013,2014 and 2015 were 45.93%(130/283),27.91%(84/301)and 20.85%(54/259),respectively.Histopathological analysis showed that the whitish muscle lesions of L.vannamei,M.rosenbergii,M.japonicas,and P.monodon showed muscle fragmentation tending towards coagulative,muscular lysis,and myonecrosis.Vacuolation in the cytoplasm of hepatopancreocytes and the eosinophilic inclusions at different developmental stages were observed within the tubular epithelium of hepatopancreas.CMNV infection in M.japonicas and P.monodon was verified by ISH.The presence of CMNV-like particles was confirmed by TEM analysis in the CMNV positive samples diagnosed by RT-LAMP.The high prevalence and wide epidemic distribution of CMNV in this investigation revealed that it was necessary to pay close attention to the high risk of CMNV transmission in farmed crustaceans.Secondly,to evaluate the possibility of CMNV spread in wild marine,the dominant cold water species Crangon affinis,and the dominant warm water species Metapenaeopsis dalei in the Yellow Sea and East China Sea,were sampled and inspected by using the methods of RT-LAMP,RT-nPCR,histopathology,ISH and TEM.The results of RT-LAMP tests showed that 26.8%(30/112)individuals of C.affinis collected from 40 sampling sites were CMNV positive;35.94%(23/64)individuals of M.dalei collected from 27 sampling sites were CMNV positive.The CMNV positive rates of the sampling sites for C.affinis and M.dalei were 52.5%(21/40)and 51.85%(14/27),respectively.RT-nPCR and sequence alignment based on partial RNA-dependent RNA polymerase(RdRp)gene of CMNV indicated that the RdRp genes of CMNV isolates from C.affinis and M.dalei possessed 99% homology with that from the original CMNV isolate.Histopathological inspection revealed the obvious damages of retinular cell,ventral nerve cord,midgut epithelium,and nauplii of C.affinis with CMNV infection,as well as the extensive muscle necrosis in C.affinis and M.dalei adults with CMNV infection.The hybridization signal of CMNV probes in ISH verified the CMNV infection in the retinular cells,ventral nerve cord and midgut epithelium of C.affinis,as well as in the muscle tissues both of C.affinis and M.dalei adult individuals,and C.affinis nauplii.TEM analysis was further confirmed the presence of inclusion bodies and CMNV particles in the cytoplasm of retinular cells of eyestalk from the CMNV positive C.affinis and M.dalei samples diagnosed by ISH.The natural infection and high prevalence of CMNV in C.affinis and M.dalei revealed by this study indicated that CMNV had spreaded in wild marine and was threatening the dominant species of marine crustaceans in the Yellow Sea and East China Sea.Meanwhile,the results reminded that ecological risk of CMNV spread and epidemic in wild marine was unnegligible.Thirdly,in an attempt to investigate the prevalence of CMNV in the marine in different years and uncover the phylogenetic relationship between the CMNV isolates from terrestrial aquaculture ponds and the CMNV isolates from wild species in the marine,suveys were conducted in the Bohai Sea,Yellow Sea and East China Sea in 2016 and 2017.Wild shrimp amples of 27 different species were collected and analyzed by methods of RT-LAMP,RT-nPCR,histopathology,ISH and TEM.RT-LAMP assays showed that 18 different species of the wild shrimp collected from different sea areas were CMNV positive.The CMNV positive rate of samples in the Bohai Sea area rose from 29.04%(61/210)in 2016 to 30.93% in 2017.The CMNV positive rate of samples in the Bohai Sea,Yellow Sea and East China Sea rose from 9.80% in 2016 to 29.10% in 2017.Sequencing of the RdRp target gene fragment produced by RT-nPCR and multiple sequence alignments indicated that the CMNV target genes from the wild marine showed a certain degree variation compared with those from terrestrial culture ponds.The phylogenetic tree showed that the sequences of CMNV RdRp genes from C.affinis inYellow Sea shared higher homology with that from aquacultured shrimp in Shandong and Jiangshu provinces,and shared lower homology with that from cultured shrimp in Guangxi and Hainan provinces.Histopathology analysis and RNA ISH was performed on the CMNV positive samples determined by RT-LAMP.The Histopathological results showed that severe vacuolation could be observed in the hepatopancreas tubules of Plesionika izumiae,Palaemon gravieri,and C.affinis.ISH results indicated that significant positive hybridization signals appeared in the necrotic hepatopancreas tubules of P.izumiae,P.gravieri,and C.affinis,in the ventral nerve cord of P.gravieri and in the coagulative necrotic muscles of M.dalei.TEM analysis revealted the presence of a large number of viral inclusion bodies and CMNV-like particles in the tissues of C.affinis,M.dalei,P.gravieri,Solenocera crassicornis,P.japonicus,and Parapenaeopsis hardwickii.These results suggested that the prevalence of CMNV in widely ranges in the wild ocean and its ecological risk could not be ignored.The prevalence of WSSV and IHHNV in wild shrimp collected from the Bohai Sea in 2015 was investigated using the RT-LAMP method.The results showed that WSSV positive samples were identified in the 9 wild shrimp's species,including P.gravieri,C.affinis,Exopalaemon carinicauda,Alpheus japonicus,Alpheus distinguendus,Trachypenaeus curvirostris,Latreutes planirostris,Acetes chinensis,and Leptochela gracilis.47.79%(130/272)individuals collected from 39 sampling sites were WSSV positive.The WSSV positive rate of sampling sites was 97.44%b(38/39).The IHHNV positive rate of in the 272 sampleswas 5.88%(26/272),the IHHNV positive rate of sampling sites was 43.59%(17/39).IHHNV positive samples were found in 4 wild shrimp species including P.gravieri,T.curvirostris,L.planirostris and A.chinensis.The abovemented results indicated that WSSV and IHHNV did exist in the wild shrimp in the Bohai Sea,which supplied an additional evidence of presence of the aquaculture shrimp pathogens in wild sea.The results of CMNV epidemiological investigation in aquacultured shrimps in coastal province in 2013~2015 demonstrated the high prevalence rate,wide prevalence in the main shrimp farming areas in China.The results of aforementioned survey on CMNV prevalence in wild shrimp species in wild sea indicated that CMNV could infected many wild shrimp species in widely wild marine area including the Bohai Sea,Yellow Sea and East China Sea,and that CMNV prevalence rates increased from 2016 to 2017 in the three marines investigated.The newly research results of our laboratory showed that the prevalence rate of the original CMNV isolate was decreasing and the prevalence rate of CMNV variant was increasing.With a view to the characteristics of geographic isolation of different CMNV isolates whether which was from terrestrial aquaculture ponds or from wild marine,the close correlation between high conservation of CMNV RdRp genes and adjacency in geographical locations of CMNV isolates,and vice versa,indicated that interchange of CMNV happened between the land-based aquaculture ponds and the offshore waters(even the open marine)along with interchange of seawater and organisms in aquaculture activity.The present study indicated that CMNV,as well as WSSV and IHHNV,had spreaded in wild marine and was threatening the dominant species of marine crustaceans in the Yellow Sea and East China Sea,which supplied the detailed and solid evidences to prove transmission of shrimp viruses between coastal ponds-offshore sea.Meanwhile,the results reminded that ecological risk of aquaculture animal viruses spread and epidemic in wild marine was unnegligible.