Development of chitosan conjugated DNA vaccine against nodavirus in Macrobrachium rosenbergii (De Man, 1879)

The protective efficacy of a DNA construct containing extra small virus antisense (XSVAS) gene of nodavirus encapsulated with chitosan nanoparticles (NPs) was investigated in giant freshwater prawn Macrobrachium rosenbergii (De Man, 1879). The delivery was carried out using oral and immersion methods. A plasmid concentration of 100 ng μL^?1 when conjugated with chitosan NPs was found to be more effective in increasing the survivability of the infected prawn. The particle mean size, zeta potential and loading efficiency percentage were 297 nm, 27 mV and 85%, respectively. The ability of the chitosan to form a complex with the plasmid was studied by agarose gel electrophoresis. The NPs were characterized by atomic force microscopy (AFM). Persistence study showed the presence of the DNA construct up to 30th day post‐treatment. The oral treatment was found to be better than the immersion treatment for delivery of the chitosan‐conjugated DNA construct. This is probably the first report on the delivery of nanoconjugated DNA construct in M. rosenbergii, against nodavirus.     

Live transportation of Macrobrachium rosenbergii (De Man) in chilled sawdust

Three cooling rates of 1.26±0.09°C h^?1 within 8 h (slow, T₁), 2.52±0.18°C h^?1 within 4 h (moderate, T₂) and 5.04±0.36°C h^?1 within 2 h (fast, T₃) were tested to cold‐anaesthetize farm raised Macrobrachium rosenbergii (De Man) (45–52 g) in each case from 25°C down to 15±1°C in a refrigerated chilling tank, provided with aeration. The cold‐anaesthetized prawns subjected to each chilling rate were packed in an insulated cardboard box (triplicate) between two layers of moist and chilled (2–3°C) sawdust, and kept inside a chilled storage cabinet at 15±1°C, for set durations of 6, 9, 12, 15 and 18 h. Survival was determined by revitalizing the prawns in aerated water with an initial temperature of 20°C, which was raised to 29±1°C within 3 h. The experiment was repeated using berried females acclimated to brackishwater of 12 g L^?1 salinity and the percentage survival recorded after live storage for durations ranging from 6 to 24 h at intervals of 3 h. Statistically valid safe durations for obtaining 100% survival of the cold anaesthetized and live stored prawns were determined using probit analysis at the three chilling rates tested, and were found to be 7.39, 6.98 and 4.54 h in the case of adult prawns, and 7.87, 8.17 and 6.43 h for berried females for T₁, T₂ and T₃ respectively. For practical purposes, the durations that yielded 95% survival rates were computed to be 16.47, 12.14 and 8.35 h in the case of adult prawns and 18.49, 19.02 and 11.11 h for berried females for T₁, T₂, and T₃ respectively. The berried prawns revitalized after live storage were incubated in tanks and the zoea larvae reared up to postlarvae (PL‐5), and compared against a control. No significant difference was found in larval hatch fecundity, survival rate and the production of PL L^?1 between the treatment and control, indicating that the method of cold anaesthetization and live storage of berried prawns could be used for successful transportation of broodstock.     

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.     

Culture of Macrobrachium rosenbergii (De Man 1879) in experimental cages in a freshwater eutrophic lake at different stocking densities

Macrobrachium rosenbergii (de Man 1879) juveniles (0.4 g) were cultured in experimental cages (L × W × H: 2.5 × 1 × 1 m) in Laguna de Bay, the largest lake in the Philippines. The following stocking densities at four replicates each were used: 15, 30, 60 and 90 prawns m⁻² of cage bottom. The mean sizes at harvest after 5 months of culture ranged from 14.3 g for the highest stocking density to 26.3 g for the lowest. The mean size at harvest, daily growth rate and size class distribution were significantly influenced by stocking density, with those at the lowest stocking density showing significantly better growth and overall proportion of larger prawns. Heterogeneous individual growth (HIG) was fairly evident in all treatments. The percentage of blue‐clawed males was not influenced by treatment but the mean weight was significantly higher in the lower stocking densities. Both the percentage and mean weight of berried females were significantly higher in the lowest stocking density. Survival was the highest in the lower stocking densities (55.3%, 54.0%, 52.7% and 36.9% for 15, 30, 60 and 90 prawns m⁻² respectively). Feed conversion ratio (FCR) improved with decreasing stocking density, ranging from 2.1 to 3. As expected, yield per cropping increased with stocking density and ranged from 450 to 1089 g m⁻² yr⁻¹ of actual cage area. Production values obtained in the cage cultured M. rosenbergii were comparable to or even higher than those reported from pond culture, given that the stocking densities used in this study were generally higher than in ponds. The results show that the farming of M. rosenbergii in cages in lakes is a viable alternative to pond culture and has the potential of improve aquaculture production in lakeshore fish farming communities.     

Use of Cyclop-eeze as a substitute for Artemia nauplii in larval rearing of giant freshwater prawn Macrobrachium rosenbergii (De Man 1879)

Four feeding experiments, replacing 25% (T₁), 50% (T₂), 75% (T₃) and 100% (T₄), by dry weight, of the live feed Artemia nauplii for Cyclop‐eeze, a new larval feed that was claimed to contain the highest known levels of astaxanthin and omega‐3 polyunsaturated fatty acids, were compared against a control that was fed with Artemia and egg custard alone, to the larvae of giant freshwater prawn Macrobrachium rosenbergii (De Man 1879). Analysis of different production characteristics of the larvae revealed that the highest survival up to postlarvae (PL) stage was obtained for T₂ in which 50% of the Artemia nauplii were replaced by Cyclop‐eeze [freeze‐dried (FD) deep frozen (DF)], and the highest astaxanthin content of the larval tissue obtained in T₄ in which the larvae were fed 100% Cyclop‐eeze, although the survival rate was the lowest in this treatment. The costs of different treatments were also compared. The Artemia consumption million⁻¹ larvae was the highest in control (11490 g), followed by T₁ (8240 g), T₂ (4990 g), T₃ (3730 g) and T₄, which completely replaced Artemia from stage 5 onwards (1830 g). The highest consumption of Cyclop‐eeze million⁻¹ larvae was in T₄ (1670 and 10 880 g), followed by T₃ (850 and 5560 g), T₂ (410 and 2690 g) and T₁ (230 and 1490 g) of FD and DF, respectively. The astaxanthin contents of the late‐stage larvae fed under the four treatments were 24.90, 27.40, 28.60 and 35.60 μg g⁻¹ tissue for T₁, T₂, T₃ and T₄, respectively, while that of the control was 23.70 μg g⁻¹. The lowest cost of live feeds million⁻¹ PL was obtained for T₂ (US$ 428.60), followed by T₁ (US$ 490.46), control (US$ 529.07) and T₃ (US$ 583.26), while it was the highest for T₄ (US$ 890.93). The results indicated that Cyclop‐eeze could economically replace Artemia nauplii at 50% level that could significantly improve the survival and carotenoid composition of the larvae of M. rosenbergii.     

Effect of Bacillus subtilis on growth development and survival of larvae Macrobrachium rosenbergii (de Man)

A feeding experiment was conducted to investigate the effect of Bacillus subtilis bacterium, on larval growth and development rate of Macrobrachium rosenbergii (de Man) during February 28 to April 8, 2005 in University Putra Malaysia hatchery. Newly hatched larvae of M. rosenbergii were reared with two dietary treatments consisting of newly hatched Artemia salina nauplii with B. subtilis (10⁸ cells ml⁻¹), and newly hatched A. salina nauplii without B. subtilis carried out in triplicate in 60‐L aquarium (50 L⁻¹). After trial, the larvae that fed B. subtilis‐treated Artemia naupli were found to have higher survival and a faster rate of metamorphosis than larvae that were fed with nontreated Artemia naupli. There were significant differences between B. subtilis‐treated Artemia naupli and nontreated Artemia diet in larval growth and development rate of metamorphosis (P < 0.05). Larval survival after 40 days was significantly greater (P < 0.05) in the B. subtilis‐treated groups (55.3 ± 1.02) compared with the nontreated groups (36.2 ± 5.02%).     

Current status & prospects of farming the giant river prawn (Macrobrachium rosenbergii de Man 1879) in Thailand

Thailand was second only to P.R.China in annual production of giant river prawn. This article briefly describes the seed production of giant river prawn technology in Thailand and notes that broodstock management has paid scant attention to genetic issues. There are, however, ongoing selective breeding programmes on giant river prawn aimed to improve growth and disease resistance. In the conventional culture technology, postlarvae are stocked at a high density and prawns are harvested partially. The cropping period is long and the practice has led to disease outbreaks and deteriorated pond bottoms that result in frequent detection of antibiotic residue. The improved culture technology involves manual sexing of the prawns at 3 months and restocking only the male prawns into new ponds at low density. The technique gives higher yield and reduces incidence of disease and thus the need for prophylactics. The constraints to giant river prawn culture business in Thailand include low yield and a limited export market. The article describes three ways to expand and sustain the business of giant river prawn farming in the country: development of more genetically improved strains; culture of all‐male stock, and new culture techniques.     

Genetic diversity of cultured and wild populations of the giant freshwater prawn Macrobrachium rosenbergii (de Man, 1879) based on microsatellite analysis

Freshwater prawn (Macrobrachium rosenbergii) culture in the Western Hemisphere is primarily, if not entirely, derived from 36 individual prawns originally introduced to Hawaii from Malaysia in 1965 and 1966. Little information is available regarding genetic variation within and among cultured prawn stocks worldwide. The goal of the current study was to characterize genetic diversity in various prawn populations with emphasis on those cultured in North America. Five microsatellite loci were screened to estimate genetic diversity in two wild (Myanmar and India‐wild) and seven cultured (Hawaii‐1, Hawaii‐2, India‐cultured, Israel, Kentucky, Mississippi and Texas) populations. Average allelic richness ranged from 3.96 (Israel) to 20.45 (Myanmar). Average expected heterozygosity ranged from 0.580 (Israel) to 0.935 (Myanmar). Many of the cultured populations exhibited reduced genetic diversity when compared with the Myanmar and the India‐cultured populations. Significant deficiency in heterozygotes was detected in the India‐cultured, Mississippi and Kentucky populations (overall F_is estimated of 0.053, 0.067 and 0.108 respectively) reflecting moderate levels of inbreeding. Overall estimate of fixation index (F_st = 0.1569) revealed moderately high levels of differentiation among the populations. Outcome of this study provide a baseline assessment of genetic diversity in some available strains that will be useful for the development of breeding programmes.     

On the breeding behaviour and reproduction of the freshwater prawn,Macrobrachium rosenbergii (de Man 1879) (Decapoda‐Crustacea) under laboratory conditions

The giant freshwater prawn, Macrobrachium rosenbergii is one of the most common decapod species, and now getting more attention from the aquaculturists world wide due to its high market demand. It is commercially important because of its size as well as its eating flesh qualities. The breeding behaviour, reproduction and hatching of this species were observed for about 8 months during 2009. Juveniles (0.55 ± 0.177 g total weight and 2.7 ± 0.12 cm, total length) were reared in rounded fibre glass tanks (1.3 m, diameter). Pre‐mating moult occurs in prawns once the ovaries ripen in their carapace cavity so as to transform the prawn into berried stage. Recorded incubation period ranged from 18 to 24 days. The number of eggs ranged from 2050 to 150 500 and the fecundity ranged from 435.2 to 3849.1 eggs. Number of hatched larvae ranged from 1825 to 123 410 larvae for females of 4.71 to 39.1 g respectively. The number of eggs carried by female prawn was directly proportional to its body weight (no. of eggs = 3441.3 wt. of female – 32 292, r² = 0.819). (fecundity = 82.066 wt. of female ? 235.04, r² = 0.7779; fecundity = 317.86 length of female ? 2651, r² = 0.833). Hatching rate ranged from 65% to 91%, and there was a correlation between number of hatched larvae and size of female parent (no. of larvae = 10 369 length of female – 102 965, r² = 0.8159; no. of larvae = 2792.9 wt. of female – 26 268, r² = 0.829). This study can greatly help in the management strategies of prawn hatcheries and improve its hatching technology.     

Incidence of white muscle disease, a viral like disease associated with mortalities in hatchery-reared postlarvae of the giant freshwater prawn Macrobrachium rosenbergii (De Man) from the south-east coast of India

Incidence of post‐larval mortalities of 30–100% was reported from commercial freshwater prawn, Macrobrachium rosenbergii (De Man) hatcheries in Andhra Pradesh and Tamil Nadu (south‐eastern states of India) since 2001. Infected postlarvae (PL) exhibited clinical symptoms with lethargy, anorexia and whitening of abdominal muscles and the disease was identified as white muscle disease (WMD). The waterborne infection of WMD was induced in the laboratory by exposing uninfected and healthy M. rosenbergii PL to the filtered muscle homogenates of the naturally infected PL, resulting in mortality that reached 99% within 10 days post infection. Histopathological examination of the infected animals revealed highly necrotic musculature. Degenerated muscle areas showed aggregations of melanized nuclei, many of which looked like inclusion bodies. Bacteriological examination of affected PL showed the presence of Staphylococcus spp. as a predominant organism, while laboratory challenge of healthy PL with this bacterial isolate did not reproduce WMD.     

Immunomodulatory effect of Cynodon dactylon against white tail disease of giant freshwater prawn,Macrobrachium rosenbergii (de Man, 1879)

The giant freshwater prawn, Macrobrachium rosenbergii, is an economically important and extensively cultured crustacean worldwide. The viral pathogens, Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) are responsible for causing severe mortalities in the hatchery and nursery phases. This study investigates the protection of postlarvae of freshwater against white tail disease (WTD) using plant extract derived from Cyanodon dactylon and the modulation of the prawn non‐specific immunity. To determine the immunomodulatory effect of C. dactylon extract, the prawn was injected with plant extract and various immunological parameters were estimated. The immunological parameters such as proPO, SOD, THC and clotting time were found to be significantly higher in the plant extract‐injected prawn when compared with control groups. The results of real time PCR analysis revealed up regulation on the expression proPO, SOD and lysozyme genes in MrNV and XSV challenged prawn postlarvae treated with C. dactylon extract. Infectivity experiment showed high relative per cent survival in MrNV and XSV‐challenged prawn postlarvae treated with C. dactylon extract. These results strongly indicate that the administration of C. dactylon plant extract enhances immunity of the prawn. Based on the results, this study recommends that the immersion of postlarvae in C. dactylon plant extract is a potential prophylactic agent against WTD.     

Cold tolerance at three salinities in post-larval prawns,Macrobrachium rosenbergii (De Man)

Post-larval Macrobrachium rosenbergii acclimated to 27, 22, or 16°C and 5, 8, or 14 ‰ were temperature-shocked to 16°C or $\text{13°}\text{16°}\text{C}$ for 1 week. Survival was significantly better in animals acclimated to 22°C or below. Salinity had no significant effect on survival. Growth of post-larvae which had been maintained at 16°C or below for 3 weeks was followed for 1 month at 27°C; it was not significantly different from that of 27°C controls. Slower growth was observed in the 14‰ prawns. These data indicate that post-larvae stocked into cool ponds from warm hatcheries might benefit from pre-acclimation to lower temperatures. Exposure to cold does not adversely affect later growth.     

Bacterial microflora associated with farmed freshwater prawn Macrobrachium rosenbergii (de Man) and the aquaculture environment

The initial counts of bacteria associated with farmed giant freshwater prawn (Macrobrachium rosenbergii de Man) as well as with the water and sediment from two farms located at Kottayam district in Kerala (India) were determined. A total of 367 randomly selected isolates were characterized and identified. Prawn samples yielded mean microbiological counts of 4.92 log CFU g^?1 of shell with muscle and 7.78 log CFU g^?1 of intestine at 30°C. Bacterial numbers in the intestine of prawn were much higher than those in the pond water. Motile and non‐motile aerobic Gram‐negative bacteria together with Enterobacteriaceae accounted for 60–70% of the mesophiles isolated from water and prawn. However, the microorganisms isolated in significant numbers from shell with muscle and intestine of prawn were not recovered from the growing waters. Among Gram‐positive bacteria, Micrococcus, Bacillus, coryneforms, and Arthrobacter were found. Faecal coliforms and enterococci were found in significant numbers in M. rosenbergii. The rearing practices such as feeding and pond fertilization could have influenced the microflora in prawn. The commensal microflora of freshwater prawn included opportunistic pathogens such as Aeromonas spp. and Streptococcus spp. Storage of prawn at elevated temperatures can permit their survival and growth leading to quality loss. Care should be exercised during handling and processing to prevent contamination of edible meat.     

Liver oil of pharaoh cuttlefish Sepia pharaonis Ehrenberg, 1831 as a lipid source in the feed of giant freshwater prawn, Macrobrachium rosenbergii (De Man 1879)

The efficacy of pharaoh cuttlefish ( Sepia pharaonis Ehrenberg, 1831) liver lipid in the feed for juveniles of giant freshwater prawn Macrobrachium rosenbergii (De Man 1879) was tested by feeding five experimental diets prepared using clam meat, groundnut oil cake, wheat bran, tapioca powder, vitamin–mineral mixture and cellulose powder as the major ingredients. The test diets T₁, T₂, T₃, T₄ and T₅, containing five levels (1, 2, 3, 4 and 5%, respectively) of lipid, extracted from the liver of pharaoh cuttlefish, were fed to 30-day-old prawn juveniles for 60 days, in triplicates and compared against a control. Analysis of variance of the growth parameters showed that the juveniles fed with T₂, containing 2% cuttlefish liver lipid with a total lipid content of 9.85% and lipid–carbohydrate ratio of 1 : 3.8, showed significantly better growth ( P  < 0.05) with the highest weight gain of 118%, greatest food conversion and protein utilization efficiency, and the highest body protein content, although the survival rates remained unaffected among treatments. Growth of the juveniles, however, declined at lipid levels >2%. Addition of cuttlefish liver lipid led to an increase of ω-3 and ω-6 fatty acids in T₂, the proportion of ω-6 being higher than the former. The crude lipid content of the body of test animals also increased with the increase in dietary lipid from 1.51% in T₁ to 3.27% in T₅. The present study indicates that cuttlefish liver lipid can be successfully used as a substitute for conventional lipid sources in rearing juveniles of giant freshwater prawn, an eco-friendly practice in recycling cuttlefish waste – a discarded fishery by-product.     

罗氏沼虾缅甸野生群体和浙江养殖群体的遗传多样性比较

采用形态学方法、同工酶电泳技术和DNA分析方法对罗氏沼虾(Macrobrachium rosenbergii DeMan)缅甸野生群体(BNP种群)和浙江省养殖群体(ZCP种群)的遗传多样性进行比较分析。结果表明,两种群在外形、体色、生长等方面存在一定的差异;对罗氏沼虾11种酶的25个基因位点进行分析,得出BNP种群和ZCP种群的同工酶分析所得出的多态位点比例及平均杂合度分别为12％和:16％,0.0431和0.0501。根据同工酶的基因频率计算得两种群的遗传相似系数为0.9963,遗传距离为0.0037;22个经筛选的10bp随机引物共检测到139个RAPD位点,BNP种群的多态位点比例和遗传多态度(Ho)分别为33.81％和0.0940,高于ZCP种群的30.22％和0.0780,两个群体的平均遗传多态度(Hpop)为0.0860,90％以上的遗传变异是在群体内检测到的。根据RAPD扩增的基因频率得出两群体间的遗传距离为0.1206。无论是同工酶电泳结果还是RAPD分析结果,都表明罗氏沼虾缅甸野生群体的遗传多样性高于浙江养殖群体,说明人工养殖会导致罗氏沼虾遗传多样性的下降。结果还表明,罗氏沼虾群体遗传多样性水平欠丰富,因此需要对罗氏沼虾群体实行科学的管理。     

Experimental evaluation of the culture performance of three exotic and a ‘local’ Giant Freshwater Prawn (Macrobrachium rosenbergii,De Man 1879) culture strain in Fiji

Giant freshwater prawn (GFP; Macrobrachium rosenbergii) aquaculture has expanded rapidly since 1990. Most local culture industries, however, have developed in an unsystematic way. Fiji has a small culture industry producing the ‘Anuenue’ strain; however, performance of this strain has never been systematically evaluated. Recently, some Fijian farmers have reported declines in stock productivity. The current project evaluated the relative performance of three exotic strains with different genetic backgrounds from Malaysia, Indonesia and Vietnam, against the ‘local’ strain in Fiji in a 4 × 3 replicated pond trial experiment. A total of 5827 prawns were harvested after 143 days growout. Individual growth rate and relative survival of the Fiji strain were not statistically different from any of the introduced strains, but Vietnam strain was superior to that of the Malaysia strain. Genetic diversity showed significant differences in variability among strains, with the Malaysian strain displaying the lowest genetic diversity. Indonesia strain showed that females were reaching maturation earlier than other strains and were smaller in size. This study suggests that Malaysian and Indonesian strains would constitute a poor choice for Fiji, whereas the Vietnam strain consistently performed well on all criteria measured. High variation among replicate ponds within strains unfortunately confounded among‐strain variation.     

Identification of a putative cellulase gene in the giant freshwater prawn,Macrobrachium rosenbergii (De Man, 1879)

Nutrition plays an important role in the development of all organisms and in particular that of farmed aquatic species where costs associated with feed can often exceed 60% of total production costs. Crustacean species in addition, have the added metabolic requirement for regular moulting to allow normal growth and this requires large amounts of energy in the form of sugars (glucose). The current study explored the capacity of the giant freshwater prawn to produce endogenous cellulose‐degrading enzymes capable of extracting nutrients (simple sugars) from plant sources in formulated feeds used in the prawn aquaculture industry. We identified a putative cellulase cDNA fragment in the target organism of 1576 base pairs in length of non‐microbial origin that after protein modelling exhibited a TM‐score of 0.916 with a described cellulase reported from another crustacean species. The functional role of cellulase enzymes is to hydrolyse cellulose to glucose and the fragment identified in GFP was highly expressed in the hepatopancreas, the site of primary food digestion and absorption in crustaceans. Hepatopancreatic tissue from Macrobrachium rosenbergii also showed active digestion of cellulose to glucose following an endoglucanase assay. Cellulase gene(s) are present in the genomes of many invertebrate taxa and play an active role in the conversion of cellulose to available energy. Identification and characterization of endogenous cellulase gene(s) in giant freshwater prawn can assist development of the culture industry because the findings confirm that potentially greater levels of low‐cost plant‐material could be included in artificial formulated diets in the future without necessarily compromising individual growth performance. Ultimately, this development may contribute to more efficient, cost‐effective production systems for freshwater prawn culture stocks that meet the animal's basic nutritional requirements and that also support good individual growth rates.