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.     

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。     

罗氏沼虾胚胎附着机制的初步研究

甲壳动物十足目的大多数种类,其受精卵自产出后就附着于亲体腹部,并完成胚胎发育过程直至孵化,这对于胚胎的发育、保护和幼体散布有重要的生物学意义。胚胎的附着机制较为复杂,与刚毛、卵柄、卵索、卵膜和黏液腺有关:卵柄和卵索的形成决定了胚胎发育的顺利进行;卵膜的变化影响着胚胎的附着;黏液腺在胚胎附着过程中形成卵柄和外层卵膜,胚胎是依靠卵柄或卵索附着于腹肢的刚毛上,进而完成胚胎发育过程。本研究采用常规组织学和扫描电镜的方法研究了罗氏沼虾(Macrobrachium rosenbergii)胚胎及其附着结构。结果表明,胚胎附着与母体腹肢的结构、黏液腺分泌的黏液相关,也与胚胎表面结构、次级卵膜的形态和结构密切相关。原肢底节和内肢刚毛密而长,基部有细小的分支,有利于胚胎的附着,外肢刚毛羽状则有利于胚胎的托附和保护。抱卵雌虾腹肢的原肢和内肢上布满腹肢黏液腺分泌的黏液;携卵刚毛中空、壁厚,刚毛外壁由微绒毛状结构组成,扩大了携卵刚毛的表面积,以利胚胎附着。胚胎外表面由初级卵膜和次级卵膜组成,次级卵膜主要由卵巢黏液腺分泌的黏液参与形成。排卵时,受精卵依靠坚韧的次级卵膜相互粘连,同时,部分受精卵在刚毛上滑动、旋转,并借助次级卵膜逐渐形成了卵柄或卵索固着在腹肢刚毛上。从受精卵到胚胎孵化,胚胎之间最主要的连接方式是依靠次级卵膜的连接。胚胎的表面有许多褶皱和黏液,利于胚胎的附着以及胚胎对溶氧和小分子物质等的通透。以上结构与附着相适应,保证了胚胎发育的顺利进行和幼体的正常孵化。本研究旨为进一步认识甲壳动物胚胎附着机制、胚胎发育和人工育苗的研究提供参考。[中国水产科学,2007,14(1):67-73]     

Acute Toxicity of Copper to Juvenile Freshwater Prawns,Macrobrachium rosenbergii

Abstract

Copper sulfate is an algicide that is commonly used for phytoplankton and filamentous algae control and has been used as a therapeutant in aquaculture. The objectives of this study were to determine the acute toxicity of copper sulfate and the safe level for use in freshwater prawn, Macrobrachium rosenbergii, production ponds in a high calcium and alkalinity environment. Six concentrations of copper sulfate (0, 0.2, 0.4, 0.6, 0.8, 1.0 mg/L) were tested in 8-L glass aquaria for 48 hours with three replicate aquaria per treatment. Concentrations of calcium hardness and alkalinity were set at 100 mg/L using calcium chloride and sodium bicarbonate, respectively. After 48 hours, survival of the control treatment (0% CuSO₄) averaged 97%, which was significantly higher (P< 0.05) than that of all other treatments. The survival in the 0.2 mg/L and 0.4 mg/L (70% and 73%, respectively) concentrations of CuSO₄ were significantly greater (P< 0.05) than higher dose treatments; but were not significantly different from each other (P> 0.05). Treatments containing 0.6, 0.8, and 1.0-mg/L copper sulfate demonstrated a dramatic decrease in prawn survival, which averaged 30, 7, and 0%, respectively. Regression analysis of the data predicted 48-hour LC₅₀ for copper sulfate tobe0.46 mg/L. Since recommended application rates for use of copper sulfate as an algicide are 1.0 mg/L or more for water with alkalinities of 100 mg/L, copper sulfate treatments are not recommended for prawn production ponds.     

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。     

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"白体病"肌肉组织的超微病理变化

在江苏扬州的罗氏沼虾(Mocrobrochium rosenbergii)养殖区,选取具“白体病”典型症状的活体仔虾和幼虾,对其腹部肌肉的病变部位进行显微和超微结构观察,未见病原生物;光镜下未见明显病理变化;其超微病理变化主要在病程中后期可见,病变部位肌丝断解、形成肌溶灶;线粒体变性、并呈空泡化;特别是肌原纤维间脂滴明显增多,呈串珠状排列。分析该病可能是一种线粒体肌病,属脂质沉积性肌病。     

应用动物模型对罗氏沼虾育种估计的差别分析

为准确评估罗氏沼虾的育种值,利用罗氏沼虾106个家系中2628尾个体的体重资料,分别考虑日龄、性别和雌虾出池时是否抱卵等因素,设计了4种单性状动物模型,应用BLUP法估计个体的体重育种值。4种模型所估计的体重遗传力差别较大,遗传力范围在0.02～0.13之间。分析表明,家系混养前日龄与家系出池体重测定值存在极显著的相关(r=0.434,P<0.01),说明家系混养前日龄对混养后家系个体的体重有显著影响,提示在动物模型设计时,应该考虑家系混养前日龄作协变量;在考虑性别效应、雌虾出池时的抱卵效应,并以家系混养前日龄为协变量,获得的育种值估计较其他模型更为准确。实验还对育种值选择与表型值选择的效率进行了比较。结果表明,依据育种值选择与依据表型值选择的结果存在着较大差异,两种选择方法选取的前53个家系育种值的平均值分别为0.70和0.58g,前者比后者提高20.7%,提示依据育种值的选择效率要高于表型值选择效率。     

Ovarian histology of stunted pond-reared Macrobrachium rosenbergii females

The present study describes the ovarian histology of stunted freshwater prawn Macrobrachium rosenbergii. The ovarian maturation of stunted animals was examined and compared with similar‐aged normal females. Ten animals of the stunted group and each maturation stage of the normal group were sampled from the same pond and had their ovaries removed for histological analysis. Body weight, body length, ovarian weight and gonadosomatic index (GSI) were recorded for each female. The diameters of the different oocyte types were compared among groups through histological assessments. The ovarian histology of stunted M. rosenbergii females indicated that although the somatic growth is severely affected (7.6 g), some energy has been placed on the vitellogensis. Stunted females showed the simultaneous occurrence of previtellogenic, vitellogenic and mature oocytes in their ovarian tissue, but overall oocyte diameter and GSI (1.02%) were significantly affected when compared with normal females.     

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.     

外源Fe3+诱导罗氏沼虾铁蛋白mRNA的表达

探讨铁蛋白ferritin基因在罗氏沼虾中的组织表达分布及外源Fe3+对ferritin mRNA表达的影响。通过RT-PCR表明,ferritin mRNA在罗氏沼虾精巢、卵巢、肌肉、心脏、中肠腺和血淋巴中均有表达;荧光定量PCR结果证实了ferritin mRNA在中肠腺中表达量最高,其次是精巢。对罗氏沼虾进行FeCl3溶液肌肉注射后,荧光定量PCR结果表明,肌肉中ferritin mRNA表达量在注射后3 h增加了61倍,高水平的表达一直维持到8 h,随后在12 h表达量急剧地下降;卵巢中ferritin mRNA表达量在3 h有近4倍显著增加,在注射后48 h内一直维持在较高水平;心脏ferritin mRNA表达量在24 h后增加了8倍,48 h后表达量开始下降;在血淋巴、中肠腺和精巢注射前后均未发现有显著性差异(P>0.05)。以上实验结果表明,外源Fe3+可以诱导罗氏沼虾铁蛋白mRNA的表达。     

Molecular isolation and characterization of a haemocyanin of Macrobrachium rosenbergii reveal its antibacterial activities

Haemocyanin is a multi‐subunit protein complex found in the haemolymph and is involved in the immune system of crustaceans. In this study, a haemocyanin gene of Macrobrachium rosenbergii, designated MrHc, was successfully isolated. The MrHc gene contained an open reading frame (ORF) of 1,992 nucleotides, encoding a protein of 663 amino acid residues with a molecular mass of 76.5 kDa. The deduced amino acid sequence contained distinct structural motifs of the haemocyanin superfamily, including an all‐alpha domain, a copper‐containing domain and an immunoglobulin‐like domain. Based on the phylogenetic analysis, the MrHC protein demonstrated a close relationship with the haemocyanins of Palaemon carinicauda and Macrobrachium nipponense. The MrHc gene was expressed in various shrimp tissues, including the hepatopancreas, gill, haemocytes, stomach and muscle. After Macrobrachium rosenbergii nodavirus (MrNV) challenge tests, the MrHc gene was up‐regulated 237‐fold at day 2. A recombinant protein of the MrHc immunoglobulin‐like domain exhibited antibacterial activity against Vibrio vulnificus, V. parahaemolyticus, Aeromonas caviae, A. veronii, A. hydrophila and Bacillus cereus. This study suggested that MrHc may play important roles in the shrimp innate immune response to MrNV infection and bacterial infection.     

罗氏沼虾胚胎发育期主要消化酶和同工酶特性

采用生物化学方法分别测定了罗氏沼虾胚胎发育期5种消化酶:胃蛋白酶、类胰蛋白酶、淀粉酶、纤维素酶和脂肪酶的比活力,测定了6种同工酶:酯酶(EST)、苹果酸脱氢酶(MDH)、谷氨酸脱氢酶(GDH)、碱性磷酸酶(ALP)、乳酸脱氢酶(LDH)和淀粉酶(AMY)在成熟卵细胞和胚胎发育时期的表达特征。结果显示:5种消化酶的活力表现出两种变化趋势,即胃蛋白酶、类胰蛋白酶、淀粉酶和纤维素酶的酶活力呈现出“高—低—高”的变化趋势,而脂肪酶活力则呈现出下降的变化趋势。6种同工酶酶谱则随胚胎发育渐趋复杂,酶活性随之增强。研究表明,所测定的消化酶以及同工酶在罗氏沼虾受精卵期均已表现出酶活性,此阶段为卵源性的酶;原肠期是胚胎发育中一个较为重要的时期,胚胎内酶基因表达能力开始逐渐增强,开始合成一些酶类,为胚胎发育后期利用卵黄物质提供了保证。消化酶以及同工酶对卵黄物质的利用和胚胎发育的物质代谢起重要作用。     

Characterization and pathogenicity studies of Vibrio parahaemolyticus isolated from diseased freshwater prawn, Macrobrachium rosenbergii (de Man)

Vibrio parahaemolyticus, an important pathogen of freshwater prawn, Macrobrachium rosenbergii causes high mortality. Four strains of V. parahaemolyticus were isolated from the gill, haepatopancreas, hemolymph and carapace of diseased prawn. The species were characterized based on biochemical and serological tests. On performing an antibiotic assay, these strains were found to be highly susceptible to cephalaxin, tetracycline and erythomycin. During the in vitro pathogenicity test, all the strains were found to be positive to the Congo red binding assay and to be haemolytic in nature, whereas an in vivo pathogenicity test revealed that 2 × 10⁹ CFUs mL^?1of bacteria induced disease symptoms such as black colouration on the carapace, red discolouration of the exoskeleton and loss of appendages within 6 days and 80% mortality. In histopathological studies, a prominent necrosis was seen in gill lamellae, and branchial arches were thickened at places due to hyperplasia and haemocytic infiltration. Hepatopancreatic tissue showed dilation of tubules, vacuolation of hepatocytes and marked necrosis in acinar cells. Development of an immunological technique for the detection and screening of V. parahaemolyticus infection and its treatment is highly important in freshwater prawn aquaculture.     

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.