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.     

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.     

Identification and genetic characterization of yeasts isolated from freshwater prawns, Macrobrachium rosenbergii de Man, in Taiwan

Since 1987, farmers in southern Taiwan have reported significant disease-related mortalities in freshwater prawns. Most mortalities have occurred during the winter, and usually almost all the adult prawns die within 4-5 days after clinical signs appear. Histopathological studies show that cells in hepatopancreatic ducts and tubules are vacuolized and degenerated. Large numbers of membrane-bound yeast aggregates are observed in the affected tissues. Two hundred and seventeen yeast isolates were obtained from diseased prawns, pond water and sediments from six areas. DNA restriction fragment length polymorphism (RFLP) was used to analyse the yeast genomes and to categorize them into five groups. Conventional biochemical and metabolic methods were then used to identify the yeasts of each group. The results showed that two strains of Candida sake (I and II), Pichia anomala, Endomyces fibuliger and Candida famata were present. In addition, the RAPD (random amplified polymorphic DNA) method was used to determine their genomic similarities. Two strains (I and II) of C. sake were the most similar (72%). C. sake I appears to be the primary causative agent of disease, based on frequencies of occurrence of the yeasts found in the diseased prawns.     

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.     

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.     

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.     

Preparation of monoclonal antibody against Macrobrachium rosenbergii Nodavirus and application of TAS-ELISA for virus diagnosis in post-larvae hatcheries in east China during 2000-2004

“Whitish muscle disease” of Macrobrachium rosenbergii, also called “whitish disease” or “white tail disease”, is a new serious epizootic disease that has occurred in recent years in giant freshwater prawn culture regions, mainly in southern China. This disease occurred in post-larvae 3-5 days to 3 weeks after desalting. Clinical signs include the development of white spot in muscles or milky muscles throughout the body, causing serious loss in few days, with a mortality rate of 40-90%. A 26-27 nm icosahedral non-enveloped virus, identified as M. rosenbergii Nodavirus (MrNV), was confirmed as the aetiological agent. Twelve hybridomas strongly secreting monoclonal antibodies (Mabs) against MrNV were shown to be specific for MrNV and reacted with MrNV 42 kDa coat protein by Western blot. A triple antibody enzyme-linked immunosorbent assay (TAS-ELISA) was developed and shown to be a useful diagnostic tool for MrNV infection.     

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.     

Production Comparison of Three Genetic Strains of Freshwater Prawn,Macrobrachium rosenbergii,Raised Under Two Pond Management Technologies

Three genetic strains (Texas [cultured], Hawaii [cultured], and Myanmar [wild]) of freshwater prawn, Macrobrachium rosenbergii, were characterized and compared under two pond grow‐out management technologies using a 3 × 2 factorial design. Juvenile prawns (45 d nursed juveniles) from each strain were stocked at individual average weights of 0.4 ± 0.3 g (Texas), 0.3 ± 0.2 g (Hawaii), and 0.3 ± 0.2 g (Myanmar). The low input management technology prawns were stocked at 24,700 /ha with no added substrate. The high input management technology prawns were stocked at 74,100 /ha with the addition of artificial substrate. Each of the six treatment combinations were replicated in three, 0.04 ha earthen ponds (total of 18 ponds). Prawns were fed a sinking pellet (32% protein) once daily at a standardized rate. After 112 d, prawns were harvested, bulk weighed, and counted. Survival of Texas strain (95%) was significantly higher (P ≤ 0.05) than Myanmar strain (77–80%) under both management technologies with survival of Hawaii strain (86–91%) intermediate and not significantly different (P > 0.05) from other strains. Under both management technologies, average weight, total production, and marketable percentage (>20 g) was significantly better (P ≤ 0.05) in Texas and Hawaii strains in comparison to the Myanmar strain. These data appear to indicate that the cultured strains evaluated in this study demonstrate positive impacts of domestication and do not indicate inbreeding depression.     

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.     

Reproductive Performance of Macrobrachium rosenbergii Females in Captivity

Abstract.— The breeding frequency, fecundity, egg hatchability, larval output and viability of pond-reared, mature Macrobrachium rosenbergii females were individually followed up during 180 d. Sexually mature prawns were maintained under controlled laboratory conditions (28.7 C and 12-h light/d) in a 190-L freshwater recirculation system and fed a formulated diet. Ovarian development, moulting and spawning events were checked daily. At least six consecutive moults were recorded for each of the 18 females. The duration of the intermoult period averaged 27.5 d and was not affected by ovarian development or spawning, but intermoult periods followed by spawning had significantly lower growth rates. From a total of 126 moulting events recorded, egg laying successfully followed 76 (60.3%) of them. The number of eggs per spawn (NES) varied from 26,587 to 74,775 for females weighing 20.0 to 55.8 g. The relationship between NES and female size (W; in g) was found to be NES = 484 + 1454W ( r ²= 0.74). Results suggest that the number of viable larvae produced per egg clutch may be increased by in vitro incubation, as the losses of eggs, which usually occur under in vivo incubation, are prevented. The present study illustrates that under adequate and stable rearing conditions, M. rosenbergii females are able to spawn up to five times during 180 d, in comparison to three to five times per year as reported for wild prawn populations.     

Fecundity changes in Macrobrachium rosenbergii (de Man) during egg incubation

Abstract. For a study of the fecundity of Macrobrachium rosenbergii , a total of 117 berried females were sampled from ponds. Fecundity was found to vary from 1216 to 89747 eggs for prawns measuring 9.0cm to 15.8cm total length and weighing 6.22 to 45.80g. The relationship between fecundity (EN) and total length (TL) was found to be EN = 0.001876 TL^6.3617.
The average egg number/g total body weight for prawns having orange, yellow and grey eggs was 1132.7 ± 484.1,766.4 ± 524.3 and 745.5 ± 487.2, respectively, a decrease of 32.3% when orange eggs became yellow and 34.3% when they turned grey. This decrease was probably due to unfertilized eggs dropping off and some eggs being eaten by the brooders during the incubation period.
Egg numbers/g egg weight were found to be 11360.1 ± 1642.1,10715.7 ± 2725.3 and 8634.0 ± 1892.3 for orange, yellow and grey eggs, respectively. When compared to orange eggs, yellow eggs were slightly heavier (6%), but grey eggs were significantly heavier by 31.6%. The increase in the weight of the grey eggs can be explained by the elongation of fully formed organs and appendages by about 17 days post fertilization.     

Amino Acid Composition of Developing Larval Freshwater Prawn Macrobrachium rosenbergii

Abstract.— Amino acid profiles of freshwater prawn Macrobrachium rosenbergii were determined during early larval stages (I-IX) to provide some baseline biochemical information of the growing larvae. The larvae obtained from several females were stocked into three 250-L tanks at a density of 30 larvae/L. The feeding regime consisted of newly hatched Artemia nauplii and egg custard containing 5% cod liver oil. For each developmental stage, larval samples from each experimental tank were pooled, freeze dried and after acid hydrolysis, the quantification of amino acids was done by a reverse phase high performance liquid chromatography ( N = 2). The highest represented of the total amino acids were for glutamic acid and phenylalanine (with cystine) with ranges from 13.4–16.6 and 9.7–11.5%, respectively, whereas tryptophan (1.4–1.6%). methionine (1.4–2.7%) and histidine (2.9–4.2%) were relatively lower. The levels of the following essential amino acids did not significantly change during larval development: arginine, leucine, phenylalanine (plus cystine), threonine, tryptophan and valine. Despite statistically significant changes that were observed in levels of some amino acids, amino acid composition appeared to be relatively unchanged during the larval growth. The results may suggest that the amino acid requirements of the freshwater prawn is relatively constant during larval life and can be satistied by a suitable protein source that resembles the larval amino acid profile.     

罗氏沼虾不同品系的养殖对比研究

对罗氏沼虾(Macrobraciumrosenbergii)四个选育系——新引进系、原引进系、正交系和反交系,进行了养殖对比研究。研究结果表明:新引进系的生产性能最优,试验池单位养殖产量比反交系增产3·40%,比正交系增产8·35%,比原引进系增产12·16%;试验塘单位养殖产量比反交系增产21·24%,比正交系增产27·60%,比原引进系增产11·12%;杂交系的杂种优势不明显。