Extra small virus-like particles (XSV) and nodavirus associated with whitish muscle disease in the giant freshwater prawn, Macrobrachium rosenbergii

A disease of Macrobrachium rosenbergii, the giant freshwater prawn, farmed in China was recently recorded in Zhejiang, Jiangsu, Shanghai, Guangxi and Guangdong provinces. The clinical sign of the disease, which develops in post-larvae (PL), is a whitish appearance of the muscles, particularly noticeable in the abdomen. Mortalities may reach 100% in some hatcheries. Investigations by transmission electron microscopy after negative staining of diseased PL homogenates showed the presence of two types of viral particles: one, unenveloped, icosahedral in shape, 26-27 nm in diameter, the second, much smaller, about 14-16 nm in diameter, designated extra small virus particle (XSV). The large virus has a genome with two pieces of ssRNA (RNA-1 and RNA-2), of 3 and 1.2 kb, respectively. Hybridization tests confirmed that this large virus is closely related to M. rosenbergii nodavirus (MrNV) which was isolated from diseased prawns in a hatchery in the French West Indies. Its very small size and hypothesized biochemical and biological characteristics suggest XSV is a new type of crustacean virus. As XSV has always been found associated with the larger virus (nodavirus) and is located in muscle and connective cells of diseased animals, it could be an autonomous virus, a helper-type virus or a satellite-like virus.     

Partial susceptibility of the SSN-1 fish cell line to a crustacean virus: a defective replication study

This study evaluated the possible use of the fish SSN-1 cell line to investigate the development of Macrobrachium rosenbergii nodavirus (MrNV). Cells were incubated with viral particles and cytopathic effects were observed. De novo synthesis of viral capsid proteins was shown by immuno-fluorescence labelling and a sandwich ELISA test. Viral genomic replication was demonstrated by RT-PCR using primers specific to RNA-1 as well as by quantitative RT-PCR (RT-qPCR). Using electron microscopy, only a few empty particles were observed and attempts to isolate complete infectious particles or to re-infect healthy cells (second passage) were unsuccessful. As complete viral particles were rarely observed, it appeared that defaults in MrNV virogenesis might arise resulting in the formation of scarce and non-infectious particles. SSN-1 cells were found to be partially permissive to MrNV infection that induced cell lysis, but key elements for viral infection were lacking such as regulatory factors for gene replication or post-translational modifications.     

Rapid detection of Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV), the pathogenic agents of white tail disease of Macrobrachium rosenbergii (De Man), by loop-mediated isothermal amplification

A loop-mediated isothermal amplification (LAMP) procedure is described for rapid diagnosis of white tail disease, a viral disease caused by Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV), in the giant freshwater prawn, Macrobrachium rosenbergii. This method was more sensitive than conventional RT-PCR for detecting the two viruses. A set of four primers, two outer and two inner, were designed for MrNV detection. An additional pair of loop primers was also used in an accelerated LAMP reaction for detection of XSV. Time and temperature conditions were optimized for detection of the two viruses. The LAMP reaction is highly suited for disease diagnosis in developing countries as amplification of DNA can be detected without the use of agarose gel electrophoresis, by the production of whitish precipitate of magnesium pyrophosphate as a by-product.     

A sandwich enzyme linked immunosorbent assay (S-ELISA) for detection of MrNV in the giant freshwater prawn,Macrobrachium rosenbergii (de Man)

A sandwich enzyme-linked immunosorbent assay (S-ELISA) was developed to improve diagnosis of white tail disease of the giant freshwater prawn, Macrobrachium rosenbergii, caused by the nodavirus, MrNV. Polyclonal antibodies were produced by immunization of Balb/C mice using a purified suspension of the virus and IgG anti-MrNV were purified from ascitic fluid. A sandwich method was successfully developed, coating first with unlabelled antibody and detecting trapped antigens with a second biotinylated antibody. Reaction was demonstrated using an avidin-peroxidase conjugate. Tissue extracts from M. rosenbergii infected with MrNV or purified viral extracts (control) were successfully identified in an individual ELISA, thus confirming the validity of the method. This S-ELISA should be the technique of choice for epidemiological studies of this disease and is a rapid and inexpensive assay with high specificity and sensitivity.     

White tail disease of the giant freshwater prawn, Macrobrachium rosenbergii: separation of the associated virions and characterization of MrNV as a new type of nodavirus

White tail disease of the farmed freshwater prawn, Macrobrachium rosenbergii, is the cause of mortalities in the French West Indies, China and India. Two different sized particles, both developing in the cytoplasm of target cells, are found associated with diseased animals. These two viruses were separated, purified and subsequently characterized. The larger one, called MrNV, is icosahedral in shape and 27 nm in diameter. Its genome is composed of two fragments of linear single-stranded RNA (ss-RNA), of 2.9 and 1.3 kb, respectively and its capsids exhibited a single polypeptide of 43 kDa. These characteristics and the partial sequence of a cloned fragment of RNA-1 suggest this agent is a member of the family Nodaviridae, but with differences from both the genera Alphanodavirus and Betanodavirus. The smaller virus, named XSV, is icosahedral in shape, 15 nm in diameter, possesses a linear ss-RNA genome of about 0.9 kb, and its capsid exhibits two polypeptides of 16 and 17 kDa, respectively. The relationships between these two viruses remain unknown.     

Non‐permissive C6/36 cell culture for the Australian isolate of Macrobrachium rosenbergii nodavirus

Macrobrachium rosenbergii nodavirus (MrNV) that causes white tail disease (WTD) is an emerging disease that contributes to serious production losses in Macrobrachium hatcheries worldwide. Mosquito cell lines (C6/36) have been reported to support the growth of MrNV and used to observe the cytopathic effects (CPE) in infected cells. This study determined the susceptibility of C6/36 mosquito cells to the Australian isolate of MrNV in order to use fewer animals in further investigations. Different staining methods were used to observe MrNV viral activity in C6/36 cells. Typical cytopathic effects such as vacuolation and viral inclusion bodies were observed in infected C6/36 cells with H&E and Giemsa staining. With acridine orange, it was easier to detect presumptive MrNV messenger ribonucleic acid in the infected cells. Using neutral red staining to measure mitochondrial activity showed light absorption of infected cells maximized at day 4 (O.D. = 0.6) but was significantly lower (chi‐square = 41.265, df = 1, P < 0.05) than control groups (O.D. = 2) which maximized at day 12. Using trypan blue staining to count the number of cells with disrupted cell membranes, the maximum number of presumptively dead cells at day 8 (4 × 10⁵ cells) in infected treatments was higher than the control treatment at day 10 (1.8 × 10⁵ cells). However, TaqMan real‐time PCR did not confirm the replication of MrNV in the cells over 14 days. The mean viral copies and mean cycle times of positive samples were stable at 2.07 × 10⁴ and 24.12, respectively. Limited evidence of viral replication was observed during four serial passages. This study determined the mortality of the C6/36 cell line to the Australian isolate of MrNV but suggests limited patent replication was occurring. Trying different cell lines or adapting the virus to the C6/36 cells may be necessary to successfully replicate Australian MrNV in cell lines.     

罗氐沼虾3个Dmrt基因的序列分析

Dmrt基因家族是一个与性别决定相关的基因家族。迄今，已在鱼类、爬行类、鸟类、哺乳类等高等动物中检测到了Dmrt基因的存在。为了进一步探讨该家族在系统进化中的保守性，本研究采用简并PCR技术，扩增了罗氏沼虾(Macrobrachium rosenbergii)Dmrt基因的DM结构域。经序列分析，获得了Dmrt基因家族的3个成员，其编码序列分别与人DMRT2、DMRT3、DMRT14基因DM结构域编码序列的相似性分别为93％、80％和95％，根据罗氏沼虾的拉丁名分别命名为MrDmrt2、MrDmrt3、MrDmrt4。与其他动物相关的Dmrt基因进行聚类分析，结果表明，不同进化地位动物的Dmrt基因DM域编码序列存在高度的同源性，显示Dmrt基因在系统进化上高度保守，序列上的相似性可能暗示着它们在功能上的保守性。     

罗氏沼虾18SrRNA基因生物素标记探针的制备及应用

探针(probe)是带有标记的特定DNA(RNA)片段,是核酸杂交鉴定特定基因以及研究特定基因组织表达的重要工具[1]。PCR标记法是近年来发展起来的酶促标记基因探针的方法之一[2]。常用的标记物有放射性物质(如γ 32P)和非放射性物质(地高辛,生物素,荧光素等)[2]。在非放射性的标记物质中,生物素由于具有较高的化学稳定性和使用安全等优点而受到瞩目[3]。在研究特定基因组织表达的过程中,由于仪器精确度限制和操作误差等因素,使用于杂交的各样品上样量难以达到绝对一致。如没有内参照杂交信号间的可比性就会降低而影响实验结果的可靠性。脊…     

患铁虾综合征罗氏沼虾眼柄转录组研究

为探索罗氏沼虾(Macrobrachium rosenbergii)铁虾综合征(IPS)的分子机制,采用高通量测序平台(Illumina Hiseq-2500)分别对患IPS罗氏沼虾(IPS虾)和正常罗氏沼虾开展转录组测序,进行生物信息学分析。结果显示,高通量测序共获得56.42 G高质量数据,拼接后得到221 901条单基因序列(unigene),长度范围为201~30 985 bp,平均长度为1572 bp,N50长度为2867 bp,N90长度为646 bp。将单基因序列分别在Nr、Nt、Swissprot、KEGG、KOG、GO、PFAM数据库进行序列比对及功能注释,103 570条得到注释,其中,GO数据库注释到的单基因序列最多。差异表达分析显示,2003个基因在IPS虾眼柄中差异表达,包括1209个上调基因和794个下调基因,516个基因被注释到242条KEGG通路中,翻译、信号转导和免疫系统富集的差异基因数目最多。催乳素、雌激素、胰岛素、促性腺激素释放激素、胰高血糖素、催产素、谷氨酸能突触、血清素能突触等与生殖调控相关激素的代谢过程在IPS虾与正常虾眼柄之间存在差异。此外,一些已被证明在免疫反应中起重要作用的基因在IPS虾眼柄中显著上调,如血管内皮生长因子受体1、丝氨酸蛋白酶抑制剂6、C型凝集素、芳基硫酸酯酶B、酚氧化酶原激活酶2a、组织蛋白酶B、组织蛋白酶L、甲壳类抗菌肽4等。同时,注释到溶酶体、吞噬体、抗原处理与呈递、细胞凋亡、内吞作用等多条与免疫相关的途径,支持近期研究得出的罗氏沼虾IPS与病原感染相关的结论。本研究为解析罗氏沼虾IPS的成因和分子机制提供了数据支撑。     

罗氏沼虾冷冻调理品的开发利用

对罗氏沼虾（Ｍａｃｒｏｂｒａｃｈｉｕｍｒｏｓｅｎｂｅｒｇｉ）冷冻调理品的加工方法和保鲜技术进行了研究。试验结果表明,加工后的罗氏沼虾,其保藏性能增强,加添加剂后罗氏沼虾的鲜度下降明显减慢。     

A test of two methods for marking larvae and postlarvae of the giant freshwater prawn, Macrobrachium rosenbergii

This paper describes the successful use of two tagging systems, both produced by Northwest Marine Technology Inc., on larval and postlarval giant freshwater prawns, Macrobrachium rosenbergii. Visible implant (VI) elastomer tags (a coloured liquid that solidifies under the epidermis) were used on stage XI larval prawns (mean weight 0.01 g) and postlarval prawns (mean weight 0.07 g). VI alphanumeric tags (small biocompatible plastic labels also inserted under the epidermis) were tested on postlarval prawns (from a weight of 0.5 g). Tags were inserted using clove oil as anaesthetic, and survival, mortality and growth rates of tagged animals were compared with those of controls that were handled but not anaesthetized or marked. Twenty per cent of the larval prawns (the smallest of the group) died just after tagging, but thereafter the remaining prawns survived well, as did all the tagged postlarval prawns. Visibility of the VI elastomer tags in larval prawns deteriorated with time, though 79% of marks were still visible to the naked eye 70 days after tagging. VI elastomer tags in the postlarval group remained clearly visible for up to 100 days. Visibility of the VI alphanumeric tags fell shortly after tagging, but remained adequate thereafter. Moult rates in control and tagged animals were the same in larvae with VI elastomer tags and postlarvae with VI alphanumeric tags, but the moult rate in the postlarval prawns given elastomer tags was slower than in controls. Rates of growth were similar in tagged (elastomer and alphanumeric) and control postlarval prawns, once the size‐dependent mortality of tagged larval prawns was taken into account. We conclude that VI elastomer tags could be used to mark small numbers of individual larval and immediately postlarval prawns for periods of several months, and that VI alphanumeric tags could be used to mark an unlimited number of individuals from a size of approximately 0.5 g.     

罗氏沼虾不同养殖条件下的生长和存活率相关分析

为探讨不同养殖条件下罗氏沼虾重要经济性状的相关性,以106个罗氏沼虾家系为材料,将每个家系随机分成两组,分别放入两个大小不同的池塘中,以不同的养殖密度养殖,两个月后测定每个家系的头胸甲长、腹长、体长、体重及存活率,并以家系为单位进行同一性状在两种不同养殖环境条件下的相关分析。结果表明,罗氏沼虾头胸甲长、腹长和体长在两种不同养殖环境条件下的对应性状间均呈不显著正相关(P>0.05),相关系数依次为r=0.109、r=0.15和r=0.143;体重呈显著正相关(r=0.223,P<0.05);存活率呈极显著正相关(r=0.862,P<0.01)。头胸甲长、腹长和体长的变异系数范围在10.85～18.09之间;两个土池中体重的变异系数分别为36.80和43.47,说明罗氏沼虾体重性状具有较大的遗传选择潜力。研究结果表明,罗氏沼虾在不同养殖条件下,以体重、存活率为选育指标,尽管选择趋势不变,但选种效果有差别。     

罗氏沼虾大规模家系构建与培育技术研究

为建立罗氏沼虾育种项目,2006年利用罗氏沼虾3个群体,通过巢式交配设计,实现了大规模构建罗氏沼虾父系半同胞家系。实验中,每箱放置亲虾1雄5雌,按交配设计共布置100个交尾网箱;通过对培育条件及幼体数量的标准化,使每个家系在幼体培育的各个阶段的条件尽量保持一致,减少由于条件不一致造成的家系间的环境偏差。结果显示,500尾雌虾抱卵虾为271尾,亲虾抱卵率平均为54.2%,且每个网箱出现两尾以上抱卵虾的网箱数为92个,占全部网箱数的92%;通过标准化培育,最终建立了123个家系,其中含有父系半同胞家系37个。本试验结果为建立罗氏沼虾全同胞和半同胞家系提供了依据。     

不同来源罗氏沼虾幼体的培育对比试验

2010年2月,对采自江苏吴江、广西三塘和广西南宁国家级良种场共3种不同来源的罗氏沼虾（Macrobra-chium rosenbergii）进行从幼体阶段至变态苗阶段的培育试验;干量容积法推算产苗量,计算育苗成活率。试验结果表明,3种不同来源幼体的育苗成活率分别为73.6%、65.2%和90.4%,成活率差异明显;其中,国家级广西南宁罗氏沼虾良种场的育苗成活率最高。在育苗生产中,必须注重罗氏沼虾亲本的种质复壮与培育,才能保证幼虾的质量,有利于提高其养殖成活率。     

罗氏沼虾引种复壮技术的研究

长期以来,国内进行罗氏沼虾人工繁殖一直以池塘饲养的成虾作亲本,由此造成该虾种质退化,养殖经济效益下降.为提高其种质和养殖经济效益,于1996年和1997年从马来西亚引进野生原种繁育虾苗,进行复壮技术的研究.研究结果表明,野生种子代和杂交种子代的体长、体重明显高出本地的驯养种.种群间具有标准差、变异系数、第二步足与体长的比值相对较小、额齿数相对较多的特点.通常野生原种体形细长、体色淡黄透明、摄食旺盛、抗病力强、性成熟个体较大.野生种子代和杂交种子代幼体活力强,摄食旺盛,变态期延长,但人工育苗的难度大.研究结果显示,采用繁育难度相对较低的雌(本地驯养种)×雄(野生种)配组方式繁育杂交苗,难度较小,效果好.