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.     

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.     

Histopathological changes in giant freshwater prawn Macrobrachium rosenbergii (de Man 1879) fed with probiotic Bacillus licheniformis upon challenge with Vibrio alginolyticus

The effect of dietary supplementation of probiotic bacterium Bacillus licheniformis on the histopathological changes in Macrobrachium rosenbergii juveniles (4.0 ± 0.02 g) challenged with known pathogenic strain of Vibrio alginolyticus are reported. Two isocaloric basal diets supplemented with probiotic bacteria B. licheniformis (1.0 × 10⁹ cfu/g feed) and other without probiotic supplementation were fed to the M. rosenbergii juveniles for 45 days. The histological observations revealed no significant changes in the hepatopancreas and gut tissues of both the experimental and the control groups which indicate that the present bacterium is a safe candidate probiont for the host. Prawns were challenged with V. alginolyticus after 45 days of feeding with probiotic diet. The histopathological studies of the hepatopancreas revealed that M. rosenbergii fed with probiotic‐supplemented diet showed less changes as compared to the prawns fed with control diet on second and fourth day of post‐experimental challenge with V. alginolyticus. Histopathological observations revealed that the gills of the prawns fed with control diet were severely affected in comparison to the prawns fed with probiotic‐supplemented diet after challenging with V. alginolyticus. Results from this study revealed the improved protection by dietary incorporation of B. licheniformis in reducing the histopathological manifestations due to V. alginolyticus infection in freshwater prawn.     

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.     

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.     

Immunomodulatory effect of recombinant RNA‐dependent RNA polymerase protein of Macrobrachium rosenbergii nodavirus in giant freshwater prawn M. rosenbergii

The present study evaluated the role of recombinant RNA‐dependent RNA polymerase (RdRp) protein of Macrobrachium rosenbergii nodavirus (MrNV) in modulating the immune response and in reducing MrNV load in infected prawn. In the first experiment, prawns (25–30 g) were injected with recombinant RdRp protein (RP) at a concentration of 0, 1.0 and 10 μg, and immune parameters and expression of some immune‐related genes were measured up to 14 days post injection (p.i.). In the second experiment, early juveniles were injected with a similar dose of RdRp and animals were challenged by immersion with MrNV. The infection status was detected in muscles by nested RT‐PCR up to 21 days post challenge. Prawn injected with higher concentration of RP showed significantly higher total haemocyte count at different period post injection. Significant up‐regulation of immune‐related genes was observed within 24 h in prawn treated with lower dose of RP and after 7 days p.i. at higher level of RP injection compared with adult control. Most of the tested samples (63%) were found to be RT‐PCR positive for MrNV at 48 h of post‐immersion challenge. After 14 days, MrNV was detected only in control prawn, while both RP‐injected groups were MrNV negative. This study elucidated the potential viral load reduction role played by RdRP in MrNV‐infected prawn.     

Growth and immune response of giant freshwater prawn Macrobrachium rosenbergii (De Man) postlarvae fed diets containing Chlorella vulgaris (Beijerinck)

A 50‐day growth trial was conducted to evaluate the efficacy of Chlorella vulgaris (Beijerinck) as an ingredient in the diets of giant freshwater prawn Macrobrachium rosenbergii (De Man) postlarvae (PL30). Immune response (total haemocyte count and prophenoloxidase activity) was also assessed by subjecting postlarvae to a challenge test against Aeromonas hydrophila (Chester) for 14 days. Iso‐nitrogenous and iso‐lipidic test diets were prepared using a fishmeal‐based‐positive control diet (D0) and four basal diets with inclusion levels of 2% (D2), 4% (D4), 6% (D6) and 8% (D8) C. vulgaris. Postlarvae of M. rosenbergii were randomly stocked (mean initial body weight of 0.19 ± 0.02 g) in 30‐L tanks in three replicates per dietary treatment for evaluation of growth performance. Another set of postlarvae (mean initial body weight of 1.25 ± 0.02 g) was randomly distributed in 95‐L tanks in three replicates per dietary treatment for the assessment of immune response. Results showed that specific growth rate was significantly higher (P < 0.05) in postlarvae fed D4 and D6. Variations in values for carcass protein, lipid, moisture and ash were also evident. Postlarvae fed diets with Chlorella showed increased prophenol oxidase activity and total haemocyte counts. Moreover, survival rate after challenge with A. hydrophila was significantly increased (P < 0.05). Inclusion of C. vulgaris in diets enhanced immune response and resistance of M. rosenbergii postlarvae against A. hydrophila infection.     

Evaluation of Lactobacillus plantarum as a water additive on host associated microflora,growth, feed efficiency and immune response of giant freshwater prawn,Macrobrachium rosenbergii (de Man, 1879)

A 90‐day experiment was conducted by rearing 1020 prawn juveniles (0.54 ± 0.03 g) in water supplemented with three different concentrations of probiotic bacteria viz. T1 (10⁷ cfu L^?1), T2 (10⁸ cfu L^?1), T3 (10⁹ cfu L^?1) and the control (C) (unsupplemented water), to evaluate probiotic effect of Lactobacillus plantarum. In the present study, the growth parameters (WG%, SGR) and feed utilization parameters (FCR, PER) significantly improved (P < 0.05) in T3. The growth and feed utilization parameters though improved marginally in T1 and T2, the difference was not significant (P > 0.05) compared to the control. The gastro‐intestinal Lactobacillus sp. count increased significantly (P < 0.05) in all the treatment groups, whereas the decrease in harmful bacteria was significant (P < 0.05) in T3 compared to the control. Similarly, the Lactobacillus sp. count in culture water increased significantly (P < 0.05) in all the experimental groups, whereas the decrease in harmful bacteria was significant (P < 0.05) in T2 and T3. The immune parameters (THC, PO and RB activity) and clearance efficiency significantly improved (P < 0.05) in T3 with concurrent decrease (P < 0.05) in cumulative mortality against Aeromonas hydrophila challenge. However, water quality did not improved (P > 0.05) in any of the treatment groups. The results indicate that Lactobacillus plantarum at a minimum concentration of 10⁹ cfu L^?1 could be used as water additive to confer its probiotic effect in prawn, Macrobrachium rosenbergii. Moreover, future studies with higher probiotic concentrations should be conducted for its efficient commercial scale field application.     

Heritability for growth traits in giant freshwater prawn,Macrobrachium rosenbergii (de Mann 1879) based on best linear unbiased prediction methodology

In this study, heritability was estimated for growth‐related traits of giant freshwater prawn (Macrobrachium rosenbergii) before and after morphological sexual differentiation. Estimation was made on data from 16 full‐sib and eight half‐sib families. The variance estimation was performed using a univariate mixed linear animal model and variance components were analysed following an animal model using restricted maximum likelihood procedure using average information algorithm. Heritability estimates (h²) varied considerably with ages. At 2 months old, h²for carapace length (CL; 0.35±0.15) and body weight (BW; 0.26±0.13) were higher than those estimated at 5 months old, based on mixed sex data. However, when data were sorted by sex, h² calculated from data of females were higher than those of males for CL (0.26±0.16 vs. 0.10±0.06), BW (0.28±0.17 vs. 0.12±0.08), body length (0.40±0.17 vs. 0.11±0.07), total length (0.47±0.18 vs. 0.11±0.07) and claw length (0.29±0.16 vs. 0.03±0.04). The same trend was observed for traits at 6 months old in both bulk and individual rearing.     

Current status and prospects of farming the giant river prawn Macrobrachium rosenbergii (De Man) and the monsoon river prawn Macrobrachium malcolmsonii (H.M. Edwards) in India

Freshwater prawn production in India that includes farming and wild capture of the giant freshwater prawn, Macrobrachium rosenbergii and the monsoon river prawn, M. malcolmsonii has increased steadily since 1999 reaching a peak output of 42 780 t in 2005, but then declined to 6568 t in 2009–2010. Stunted growth and diseases in ponds because of poor seed quality and the broodstock which had been inbred over several generations; pond water quality issues; and increased cost of production on account of feed, labour and the mandatory certification requirements are suggested to be some of the factors leading to the production declines. While majority of the output occurs in Andhra Pradesh, single crop paddy–prawn production systems in the low‐lying fields of Kerala have helped gradual transformation to a sustainable, organic mode of farming of both rice and prawns, suitable for other states of India. Although the trends by June 2011 indicate that the sector is set to a revival, future prospects of freshwater prawn farming in India will also depend on the expansion of whiteleg shrimp Litopenaeus vannamei that was introduced recently in India and provided a more profitable opportunity for farming.     

Production of recombinant capsid protein of Macrobrachium rosenbergii nodavirus (r‐MCP43) of giant freshwater prawn,M. rosenbergii (de Man) for immunological diagnostic methods

White tail disease (WTD) caused by Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) is a serious problem in prawn hatcheries. The gene for capsid protein of MrNV (MCP43) was cloned into pRSET B expression vector. The MCP43 protein was expressed as a protein with a 6‐histidine tag in Escherichia coli GJ1158 with NaCl induction. This recombinant protein, which was used to raise the antiserum in rabbits, recognized capsid protein in different WTD‐infected post‐larvae and adult prawn. Various immunological methods such as Western blot, dot blot and ELISA techniques were employed to detect MrNV in infected samples using the antiserum raised against recombinant MCP43 of MrNV. The dot blot assay using anti‐rMCP43 was found to be capable of detecting MrNV in WTD‐infected post‐larvae as early as at 24 h post‐infection. The antiserum raised against r‐MCP43 could detect the MrNV in the infected samples at the level of 100 pg of total protein. The capsid protein of MrNV estimated by ELISA using anti‐rMCP43 and pure r‐MCP43 as a standard was found to increase gradually during the course of infection from 24 h p.i. to moribund stage. The results of immunological diagnostic methods employed in this study were compared with that of RT‐PCR to test the efficiency of antiserum raised against r‐MCP43 for the detection of MrNV. The Western blot, dot blot and ELISA detected all MrNV‐positive coded samples as detected by RT‐PCR.     

Interactions of different sodium and potassium concentrations on Macrobrachium rosenbergii (de Man) offspring quality parameters

The present study evaluated various sodium and potassium concentrations in hatchery water to determine which proportions would be optimal for Macrobrachium rosenbergii larviculture. Using a closed RAS system (60‐L), experiments were conducted in two stages. In the first stage, larval quality parameters were compared among triplicate treatments of sodium (2000, 3000, 4000 and 5000 mg L^?1) and potassium (100, 150, 200 and 250 mg L^?1). During the second stage, these same parameters were compared from interactions of the two best concentrations determined in the first stage. Initial larval density was fixed at 100 larvae L^?1 and larval quality parameters such as larval stage index (LSI), larval condition index (LCI), larvae dry weight, survival (%), LC₅₀‐24 h for formalin stress and time of the first postlarvae (PL) appearance were measured. Results showed that during the early larval period time LSI, LCI and survival parameters were affected only by potassium and the interaction with sodium was not significant. At a later period of the larval development, interactions between both sodium and potassium were measurable for LSI (P < 0.05) while the interactions on LCI and survival were not significant. Measurable differences among the combined treatments 4000 mg L^?1 sodium and 150 mg L^?1 potassium resulted in the best performance for M. rosenbergii larviculture. This concentration also provided the highest final survival to PL metamorphosis (40.6 ± 2.5%) which was at least 10% higher than the other treatments.     

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.     

Resistance to vibriosis,production performance and tolerance to stress of the selected giant freshwater prawn (Macrobrachium rosenbergii)

The purpose of this study was to evaluate the resistance to vibriosis, growth, survival and tolerance to stress of the selected prawn, second generation, compared to a non‐selected control. The first generation of selected giant freshwater prawn, which has 10.4% higher of resistance, was used to attain disease resistant freshwater prawn (Macrobrachium rosenbergii) generation through challenge test‐based selection. Resistance test was conducted by infecting the prawn (mean body weight of 10.29 ± 1.40 g) with pathogenic Vibrio harveyi (5 × 10⁵ cfu prawn^?1). The growth and survival of the prawn were evaluated by rearing the two populations of prawn in both nursery and grow‐out phases. Stress tolerance test was done by evaluating the viability of postlarvae exposed to environmental stressors, i.e. temperature, salinity, NH₃ and formaldehide. Post‐challenge survival of the selected prawn (55.0 ± 5.0%) was about 46% higher than that of the control (37.5 ± 7.5%). The survival of the selected prawn in nursery culture (77.16 ± 0.841%) was significantly higher (p < .05) than that of the control (51.31 ± 2.938%), while the survival in grow‐out culture was similar (p > .05). The growth of selected prawn (4.99 ± 0.03% day^?1) was significantly higher than that of the control (4.81 ± 0.05% day^?1). There was no difference between treatments on the tolerance level against the tested environmental stressor. Overall data suggested that the selected prawn showed better performance in growth and resistance against vibriosis.     

Effects of various sodium adsorption ratio (SAR) mediums on larval performance of the freshwater prawn Macrobrachium rosenbergii (de Man)

The sodium adsorption ratio (SAR) is used to describe the ratio of sodium to calcium and magnesium in water that is very important in both aquaculture and agriculture. SAR treatments including of 20, 30, 40, 50 and 60 were adjusted in 10‐L containers. Initial larval density was fixed at 100 larvae L^?1 and during the culture period, various larval quality parameters were evaluated. Our findings illustrated that at initial stages of larval development, different SAR treatments affected the larval condition index (LCI), but no influences on larval stage index (LSI) were apparent. Importantly, this process at the final larval stages became reversed. In addition, survival at SAR 30 was 12.4% greater than among any other treatments. The highest larval resistance to stress (191 ± 5 ppm), as measured by the 24‐h LC₅₀ formalin test also was observed for SAR 30, at the 11th larval stage. Analysis indicated that calcium absorption during larval developmental stages is affected by SAR levels in the environment and also showed that the optimal SAR treatment was 30 for Macrobrachium rosenbergii larviculture in inland hatchery brackish water. According to our findings and desired SAR for the environment and agricultural soil (<15), effluent of M. rosenbergii inland hatcheries must be treated at least two times by dilution and adding calcium and magnesium salts.     

Evidence of rapid transfer and bioaccumulation of Microcystin‐LR poses potential risk to freshwater prawn Macrobrachium rosenbergii (de Man)

Microcystins accumulate in aquatic organisms and can be transferred to higher trophic levels, eventually affecting vector animals and consumers. We examined three levels of an aquatic food chain (Microcystis aeruginosa, Daphnia magna and Macrobrachium rosenbergii) to identify the transfer efficiency and risk of microcystin on prawns. Samples were analysed using ultra performance liquid chromatography‐mass spectrometry (MS)/MS and microcystin‐LR (MC‐LR) distributions in prawn tissues were studied. The results showed that prawns accumulate MC‐LR both directly from M. aeruginosa and indirectly through D. magna which was pre‐exposed to M. aeruginosa. MC‐LR was detected in the gills, digestive tracts and hepatopancreas of the prawns 2 h after exposure. MC‐LR accumulated in prawns to 0.49 ± 0.04 μg g^?1 dry weight in hepatopancreas within 24 h, while it was not detected in muscle samples, and rarely appeared in blood samples in such a short period. Although MC‐LR was not detected in muscle, the head including hepatopancreas of the prawns accumulated troublesome amounts of MC‐LR. These results demonstrate that microcystis blooms in prawn farming potentially pose a risk to human consumers, although prawns may be exposed to the bloom for a very short time, hence regular monitoring of blue green algae population is recommended.