Immunostimulatory effect of mangrove‐derived marine yeasts in Penaeus monodon

This study attempted to assess the efficacy of mangrove‐derived marine yeasts as a source of immuno‐stimulant in the tiger shrimp, Penaeus monodon. The shrimp was fed on the diets in supplementation with yeast biomass at 10% for a period of 15 days and then challenged with the pathogenic Vibrio cholerae. The animals were assessed for immune responses in terms of total haemocyte counts, phenol oxidase and endobiotics. Among the yeasts tested, Rhodotorula minuta was found to have high immunostimulatory effect in the shrimps especially after challenge with the pathogenic vibrio.     

Estimates of heritability and genetic correlations for growth‐related traits in the tiger prawn Penaeus monodon

Heritabilities and genetic and phenotypic correlations were estimated for body weight (BW), body length (BL), carapace length (CL), carapace width (CW) and carapace height (CH) of the Penaeus monodon from measurements on progeny at 6 months of age resulting from a nested mating design. Estimation was made on data from 41 full‐sib and 10 half‐sib families. The estimated heritabilities for these traits were 0.13–0.24, and the significantly phenotypic and genetic correlations were 0.81–0.94 and 0.88–1.00, respectively. The present results indicate that genetic improvement through selective breeding conducted on P. monodon might be feasible and would be an efficient method to obtain a positive response to selection.     

Comparison of L‐lysine·HCl and L‐lysine sulphate in the feed of Penaeus monodon and re‐evaluation of dietary lysine requirement for P. monodon

Two trials were conducted to compare L‐lysine HCl and L‐lysine sulphate regarding its availability to Penaeus monodon, and further evaluate the optimum dietary lysine requirement. In experiment 1, five experimental diets were formulated (D1, D2, D3, D4 and D5), a basal diet (D1), aimed at a low‐lysine concentration (2.22% dry matter), with lysine concentration of the other four diets increasing in two 0.25% L‐lysine intervals from either L‐lysine HCl (D2 and D3) or L‐lysine sulphate (D4 and D5). Each diet was fed at a restricted rate to three groups of 40 shrimp for 74 days. The highest values of growth performance (weight gain, WG; specific growth rate, SGR) and survival were observed with shrimp fed the L‐lysine HCl diet. Feed efficiency (FE) of shrimp fed D2 was significantly higher than that of shrimp fed D1 and D5 (P < 0.05), but without significant difference with shrimp fed D3 and D4 (P > 0.05). In experiment 2, six diets (d1, d2, d3, d4, d5 and d6) were formulated with six graded levels of lysine (2.21%, 2.41%, 2.59%, 2.87%, 3.11% and 3.29% of diet). Each diet was randomly assigned to triplicate groups of 40 shrimp for 74 days. WG, SGR and survival increased increasing levels of lysine up to 2.41% of diet and reached an apparent plateau. Broken‐line model analysis on WG and SGR indicated that the optimum dietary lysine level for optimal growth of shrimp was 2.37% of diet, corresponding to 5.78% of dietary protein. In conclusion, results of this trial suggest that L‐lysine HCl is superior to L‐lysine sulphate when fed to Penaeus monodon and optimal growth can be obtained at lysine levels corresponding to 2.37% of diet, or 5.78% of dietary protein in this specie.     

Enhancement of innate immune system,survival and yield in Penaeus monodon reared in ponds using Streptococcus phocae PI80

The objective of this study was to evaluate the effect of dietary and water supplementation of probiotic Streptococcus phocae PI80 on growth, immune response and feed utilization of tiger shrimp Penaeus monodon in earthen ponds. The probiotic bacterium S. phocae PI80 was cultured in large fermenter (50 L) by adding additional carbon source in the form of molasses and glucose along with yeast extract as nitrogen source to enrich S. phocae PI80 biomass. This enriched S. phocae PI80 was administered to shrimp in feed (6.5 × 10¹³ bacterial cells mL^?1) as well as in pond water (5 L/pond). Shrimp growth performance was significantly improved (P < 0.05) in 120 days culture when the average body weight of treated molasses + yeast extract (MY) (28.41 ± 0.874 g), glucose + yeast extract (GY) (27.013 ± 0.698 g) was significantly higher than control (23.63 ± 0.684 g). Food conversion ratio FCR was also found to be reduced significantly in ponds treated with probiotics when compared with control pond (1.89 ± 0.09). Vibrio and luminescent bacteria were found to be lower in the treatment receiving MY group, and we hypothesize that this may lead to greater shrimp survival. Furthermore, fermentation product of S. phocae PI80 added to pond water and feed additives enhanced the shrimp immune system. The results indicated that total haemocytes count (THC), phenoloxidase (PO) activity, NBT reductase assay and phagocytic activity significantly increased in shrimps treated with S. phocae PI80. Our study demonstrates that administration of S. phocae PI80 in the water and feed at 6.5 × 10¹³ colony‐forming units (CFU) mL^?1 bacterial cells induce immune modulation and enhances the immune ability of P. monodon in pond‐reared shrimp and increased the shrimp production.     

Effect of vitamin E on spermatophore regeneration and quality of pond‐reared,black tiger shrimp (Penaeus monodon)

The objective of the present work was to evaluate the effects of different levels of vitamin E in feed on the spermatophore regeneration and quality of male Penaeus monodon. The experiment was carried out with the four following treatments: the basal diet no added vitamin E, the diet added 200, 600 and 1,000 mg/kg respectively. Spermatophore regeneration and quality were evaluated by spermatophore weight, sperm count and spermatophore absence rates, which male P. monodon were extruded spermatophore for feeding 20 and 40 days. In the experiment, the weight of the twice regenerated spermatophore of the males added to the vitamin E group was higher than that of the untreated control group. The weight of the first regenerated spermatophore with the addition of 1,000 mg/kg group was the highest and significantly higher than the control group (p < .05), but there was no significant difference among the three groups with different levels of vitamin E. The weight of the second regenerated spermatophore with the addition of 600 mg/kg group was the highest, followed by 1,000 mg/kg group, both of which were higher than the control group and the addition of 200 mg/kg group. Within the same group, the regeneration spermatophore weight showed overall upward trend as the feeding time, twice regenerate experiment spermatophore weight with added to the vitamin E groups were significantly higher than the initial value (p < .05), but three spermatophore weight of male shrimp at the control group had no significant difference. The sperm quantity and the percentage of normal sperm of the twice regenerated spermatophore of the males with added to the vitamin E group was higher than that of the untreated control group, and those of the addition of 200 mg/kg group was significantly higher than that of control group (p < .05). The total number of sperm and the percentage of living sperm of male shrimp in the experimental group decreased with the increase of vitamin E in the feed. Within the same group, the total number of sperm and the percentage of living sperm of male shrimp with added to the vitamin E groups showed overall upward trend as the feeding time and were significantly higher than the initial value (p < .05), but the control group was slightly down and had no significant difference. Comprehensive sperm weight, sperm quantity and living sperm percentage of three indicators, that adding 200 mg/kg of vitamin E in feed could effectively promote the spermatophore regeneration in the male P. monodon and improve the sperm quantity. The experimental results provide a scientific basis for the breeding of P. monodon.     

In vitro study of a putative role of gonad‐inhibiting hormone in oocyte growth stimulation in Penaeus monodon

Ovarian development in crustacean is controlled by several factors, among which a neuropeptide gonad‐inhibiting hormone (GIH) is known to inhibit vitellogenin (Vg) synthesis in the ovary. It has been postulated that GIH may control Vg synthesis by inhibiting the release of gonad‐stimulating factor (GSF) from brain and thoracic ganglia. To prove this hypothesis, this study was primarily aimed to investigate the influence of GIH on the release of GSF from thoracic ganglia of Penaeus monodon. Our result showed that GIH did not suppress the release of putative GSF from thoracic ganglia by calcium ionophore A23187 as the induction of oocyte growth in the ovary explants that were cocultured with thoracic ganglia in the presence of A23187 was not affected by the addition of recombinant GIH protein. In addition and interestingly, when the ovary explants were incubated with the recombinant GIH alone, the oocyte growth was increased at the rate comparable to that induced by A23187 in the presence of thoracic ganglia. Hence, our in vitro study demonstrated that the stimulation of GSF released from thoracic ganglia is independent of GIH, and that the GIH has a dual function in oocyte growth stimulation and inhibition of Vg synthesis in the early stage of ovarian development. This expands our knowledge on the regulation of ovarian development in shrimp by GIH. Further in vivo studies in this novel aspect of GIH function will be useful for the improvement of shrimp ovarian maturation in the future.     

Identification of RAPD‐SCAR marker linked to white spot syndrome virus resistance in populations of giant black tiger shrimp,Penaeus monodon Fabricius

White spot disease (WSD) caused by white spot syndrome virus (WSSV) creates severe epizootics in shrimp aquaculture industry worldwide. Despite several efforts, no such permanent remedy was yet developed. Selective breeding using DNA markers would be a cost‐effective strategy for long‐term solution of this problem. In the present investigation, out of 30 random primers, only one primer produced a statistically significant (P < 0.01) randomly amplified polymorphic DNA (RAPD) marker of 502 bp, which provided a good discrimination between disease resistant and disease susceptible populations of Penaeus monodon from three geographical locations along the East coast of India. Because RAPD markers are dominant, a sequence characterized amplified region (SCAR) marker was developed by cloning and sequencing of 502 bp RAPD fragment, which generates a single 457 bp DNA fragment after PCR amplification only in the disease resistant shrimps. Challenge experiment was also conducted to validate this 457 bp SCAR marker, and the results suggested that the WSSV loads were 2.25 × 10³ fold higher in disease susceptible than that in disease resistant shrimps using real‐time PCR. Therefore, this 457 bp DNA SCAR marker will be very valuable towards the development of disease‐free shrimp aquaculture industry.     

Artificial substratum consisting of poly‐β‐hydroxybutyrate‐based biodegradable plastic improved the survival and overall performance of postlarval tiger shrimp Penaeus monodon

The use of artificial substratum consisting of poly‐β‐hydroxybutyrate (PHB)‐based biodegradable plastic for penaeid shrimp culture was investigated in the present study. The survival of postlarval tiger shrimp Penaeus monodon (30 ± 5 mg) provided with PHB substratum made out of PHB type DP9002 (Metabolix GmbH, Köln, Germany) was 88.7 ± 3.4% and this was significantly higher as compared to postlarvae provided conventional substratum consisting of polyvinylchloride (PVC) pipes (67.3 ± 6.5%). However, no significant weight improvement was observed for the postlarval tiger shrimp indicating that PHB could not be used as growth promoter. Nevertheless, a trend of improved robustness against adverse environmental conditions (lethal ammonium chloride concentration) and increased resistance to pathogenic Vibrio was observed in postlarval tiger shrimp provided with PHB substratum as compared to postlarvae provided with PVC substratum. Results indicate higher preference by postlarvae on PHB substratum over PVC substratum. Overall, this study indicates the potential of artificial substratum consisting of PHB‐based biodegradable plastic as replacement for conventional substratum consisting of PVC pipes in enhancing the survival of postlarval tiger shrimp and improving its performance against adverse environmental conditions and disease resistance.     

Evaluation of growth,feed utilization efficiency and immune parameters in tiger shrimp (Penaeus monodon) fed diets supplemented with or diet fermented with gut bacterium Bacillus sp. DDKRC1. isolated from gut of Asian seabass (Lates calcarifer)

An experiment was conducted to assess the effect of supplementation of cellulolytic gut bacterium with plant protein‐based diet on growth, digestibility and digestive enzyme status of tiger shrimp, Penaeus monodon. Bacillus sp. DDKRC1. (JN641289), a potential cellulolytic bacterium, isolated from the gut of Asian seabass was used as feed supplement in this experiment. The shrimps of group I, II and III were fed with control diet (without bacterial supplement, D1), diet supplemented with live Bacillus sp. DDKRC1. at 2.94 × 10⁷ cfu per 100 g feed (D2) and diet fermented (48 hr) with same bacteria (D3) respectively. At the end of the 42 days experiment, shrimps of group II showed significantly better (p < .01) growth and protein efficiency ratio (PER) and lower (p < .01) feed conversion ratio (FCR) as compared to group I and III. Dry matter, cellulose, hemicellulose and lipid digestibilities were significantly (p < .01) higher in group II than other two groups. Cellulase, amylase and protease activities in the gastrointestinal (GI) tract were significantly (p < .01) higher in group II as compared to other two groups. Total heterotrophic count, amylolytic, cellulolytic and proteolytic bacterial counts in gut were significantly (p < .01) higher in group II as compared to other two groups. Haematological analysis indicated better (p < .05) immune response in groups II and III than control group. Diets D2 and D3 showed better effect on growth, digestibility, FCR, survival and immune response of P. monodon as compared to D1.     

Cloning,characterization, expression analysis and RNAi of Retinoblastoma‐like gene from black tiger shrimp (Penaeus monodon)

Retinoblastoma (Rb) is a multifunctional regulator involved in several key cellular processes, such as cell cycle control, cell differentiation, tumorigenesis and senescence. In this study, an Rb‐like gene, PmRBL, was cloned from black tiger shrimp (Penaeus monodon). The full‐length cDNA sequence of PmRBL is 4,069 bp with an open reading frame of 3,243 bp, which encodes 1,080 amino acids. Quantitative real‐time PCR (qRT‐PCR) analysis indicated that PmRBL was highly expressed in the gills, hepatopancreas and ovaries of P. monodon. The highest PmRBL expression levels were observed in stage III of the ovarian development in P. monodon. RNA interference experiments were conducted to examine the expression profiles of PmRBL, PmCDK2 and PmCyclin E. The knockdown of PmRBL in the ovary and hepatopancreas by dsRNA‐RBL was successful. After dsRNA‐RBL was injected into the shrimp, the relative expression levels of PmCDK2 and PmCyclin E were upregulated at 12–72 hr in the ovaries and hepatopancreas. The localization and level of PmRBL expression in the ovary and hepatopancreas were investigated through in situ hybridization, which revealed consistent results with those of qRT‐PCR. Therefore, PmRBL, PmCDK2 and PmCyclin E may be involved in vitellogenin synthesis and ovarian maturation in P. monodon.     

Analysis of viral load between different tissues and rate of progression of white spot syndrome virus (WSSV) in Penaeus monodon

At present the most common and most devastating disease of shrimp is caused by the white spot syndrome virus (WSSV), which has spread throughout the world mainly by different species of crustaceans carrying the virus. After experimental injection of Penaeus monodon with a known copy number of WSSV in the abdominal muscle, the rate of viral progression in different tissues at 12, 24, 36 and 48 hpi (hours post infection) was assessed using quantitative real‐time PCR. At 12 hpi the viral load was highest in haemocytes followed by pleopod, muscle and gills whereas at 48 hpi, the gills, the main target of WSSV, showed the highest viral load followed by pleopod, muscle and haemocytes. Viral copy number in the haemocytes was the lowest beyond 12 hpi indicating a remarkable reduction in the rate of viral replication in haemocytes compared with other tissues. The viral load in haemocytes, though increased again beyond 36 hpi, never surpassed the load in the other tissues. The real‐time PCR assay with its high sensitivity and wide dynamic range make it ideal for detecting low‐level WSSV infections that can occur in apparently healthy P. monodon.     

Apparent digestibility coefficient of poultry by‐product meal (PBM) in diets of Penaeus monodon (Fabricius) and Litopenaeus vannamei (Boone), and replacement of fishmeal with PBM in diets of P. monodon

Nutrient apparent digestibility coefficients (ADCs) of pet food grade poultry by‐product meal (PBM) were determined for black tiger prawn, Penaeus monodon and Pacific white shrimp, Litopenaeus vannamei by the indirect method (reference diet and test diet at 7:3 ratio). Subsequently, an 8‐week growth trial was conducted to evaluate the effects of substitution of fishmeal (FM) with PBM in diets of P. monodon (initial weight = 0.21 ± 0.01 g). In the growth trial, six isonitrogenous and isoenergetic diets PBM0, PBM25, PBM50, PBM75, PBM100 and PBMA100, containing a gradient of PBM 0, 88.7, 177.4, 266, 354.7 and 354 g kg^?1 to replace 0, 92.5, 185, 277.5, 370 and 370 g kg^?1 FM were fed to four replicate groups respectively. The diet PBMA100 was supplemented with DL‐Met to be similar to PBM0. The results showed that both P. monodon and L. vannamei had relatively high ADC of crude protein (77.6% and 84.2% respectively) and gross energy (72.8% and 84.0% respectively) for PBM. Litopenaeus vannamei showed significantly higher digestion ability for PBM than P. monodon (P < 0.05). In growth trial, no significant difference in growth performance was observed among shrimp fed the experimental diets. DL‐Met supplementation did not improve the growth of P. monodon. PBM is a suitable protein ingredient for P. monodon feeds and can be used up to 354.7 g kg^?1 to totally replaced FM.     

Development of a monoclonal antibody‐based flow‐through immunoassay (FTA) for detection of white spot syndrome virus (WSSV) in black tiger shrimp Penaeus monodon

A flow‐through immunoassay (FTA), an improved version of immunodot, was developed using a nitrocellulose membrane baked onto adsorbent pads enclosed in a plastic cassette to detect white spot syndrome virus (WSSV) in shrimp. Sharp purple dots developed with WSSV against the white background of the nitrocellulose membrane. The detection limits of WSSV by the FTA and immunodot were 0.312 and 1.2 μg mL^?1 crude WSSV protein, respectively. The FTA could be completed in 8–10 min compared with 90 min for immunodot. The FTA was 100 times more sensitive than 1‐step polymerase chain reaction (PCR) and in between that of the 1‐ and 2‐step PCR protocol recommended by the Office of International Epizootics (OIE). In experimental, orally infected shrimp post‐larvae, WSSV was first detected 14, 16 and 18 h post‐infection (hpi) by FTA, immunodot and one‐step PCR, respectively. The FTA detected WSSV 2 and 4 h earlier than immunodot and one‐step PCR, respectively. The FTA was more sensitive (25/27) than one‐step PCR (23/27) and immunodot (23/27) for the detection of WSSV from white spot disease outbreak ponds. The reagent components of the FTA were stable giving expected results for 6 m at 4–8 °C. The FTA is available as a rapid test kit called ‘RapiDot’ for the early detection of WSSV under field conditions.     

Evidence for the presence of white spot syndrome virus (WSSV) and monodon baculovirus (MBV) in wild Penaeus monodon (Fabricius) broodstock,in the southeast coast of India

A survey on the presence of the viruses of two economically significant diseases, white spot syndrome virus (WSSV) and monodon baculovirus (MBV) in wild‐collected Penaeus monodon broodstock, was conducted during different seasons of the year in two major coastal areas of southeast India. The broodstock were collected along the coast of Tamil Nadu and Andhra Pradesh during summer, premonsoon, monsoon and post‐monsoon seasons for three consecutive years. A total of 7905 samples were collected and subjected to MBV screening, and 6709 samples that were screened as MBV negative were diagnosed for WSSV. MBV was detected using rapid malachite green staining and WSSV by nested polymerase chain reaction. Prevalence data of the viruses were analysed using the EpiCalc 2000 program at 95% confidence interval. Samples collected from the Andhra Pradesh coast displayed a slightly higher prevalence of WSSV and MBV infection than those collected from Tamil Nadu, although this difference was not statistically significant (P > 005). In addition, it was found that the prevalence of both WSSV and MBV infections fluctuated according to season. Data on prevalence of these viruses in broodstock would be useful to develop strategies for shrimp health management along the southeast coast of India.     

Ovarian development and nutritional quality changes of the wild‐caught Portunus trituberculatus during the artificial fattening period

A female swimming crab (Portunus trituberculatus) with fully developed ovaries is considered one of the most nutritious types of seafood. The fishing yield of P. trituberculatus in China has always been high; however, its economic value has been low and unreliable due to poor ovarian development and low nutritional value. Artificial fattening is an effective way to improve the value of wild‐caught crabs, and this study focused on analysing the ovarian development and nutritional quality of the female P. trituberculatus during an artificial fattening period. Therefore, this study provides scientific evidence on the effects of artificial fattening on female P. trituberculatus and can guide the development of nutritional quality regulations for wild‐caught female crabs during the fattening process. The results showed that the ovaries of the wild female P. trituberculatus developed gradually. The ovarian morphology became bulkier, while the gonadosomatic index (GSI) and total edible yield (TEY) were significantly improved after artificial fattening. Significant changes in the nutritional components of the edible tissues were observed, and the moisture content in the ovaries and hepatopancreas decreased continually, while both the protein and fat content in the ovaries significantly increased. Moreover, the fat content in the hepatopancreas significantly increased. The saturated fatty acids(ΣSFA) in the ovaries and hepatopancreas had an increasing trend after fattening, while ΣPUFA and Σn‐3PUFA first decreased and then increased. In contrast, the ratio of Σn‐3PUFA to Σn‐6PUFA decreased steadily. However, the fatty acid composition of the muscles did not change significantly. The levels of all the amino acids increased continuously during the fattening process, and EAA increased significantly in the early stages of the fattening period. Consequently, all amino acids became nonlimiting amino acids after fattening. Furthermore, the ratio of EAA/NEAA in the ovaries increased continually until the end of the fattening period. On the other hand, the fatness and TEY increased significantly after artificial fattening. In addition, the nutritional value significantly improved with the exception of the high‐quality fatty acids in the ovaries and hepatopancreas, which were slightly lower in both.     

Effects of biofloc under different carbon sources and protein levels on water quality,growth performance and immune responses in black tiger shrimp Penaeus monodon (Fabricius, 1978)

A 75‐day 2 × 3 factorial experiment was conducted to evaluate the effect of two levels of dietary protein (32 and 40%) and two different carbon sources (rice flour–R and molasses‐M), and without carbohydrate (control–C) in black tiger shrimp Penaeus monodon juveniles (3.37 ± 0.04 g) at 100 nos. m^?3 in 100 L fibre reinforced plastic tanks. Biofloc volume and total suspended solid were higher in molasses added groups (32 + M and 40 + M) followed by rice flour (32 + R and 40 + R) and controls (32C and 40C). Molasses and rice flour addition significantly reduced (P < 0.01) the total ammonia–N compared to controls. The highest Vibrio, Bacillus and Lactobacillus counts were recorded in 40 + M, 32 + M and 32 + R respectively. Among the treatments, significantly higher (P < 0.01) final body weight was recorded in 40 + R (8.5 ± 0.3 g), 40 + M (7.8 ± 0.3 g) and 32 + R (7.5 ± 0.4 g) compared to control groups, 32C (6.1 ± 0.3 g), 40C (6.4 ± 0.3 g) and molasses added group, 32 + M (5.7 ± 0.4 g). Rice flour supplementation significantly increased (P < 0.01) the total haemocyte count (×10⁶ cells mL^?1) in 32 + R (45.7 ± 3.7) and 40 + R (44.3 ± 3.1) compared to controls, 32C (27.3 ± 3.4) and 40C (25.8 ± 0.9). Similarly, higher superoxide dismutase, catalase, serum protein and glucose were recorded in the rice flour added groups, 40 + R followed by 32 + R. Among the treatments, the highest level of prophenoloxidase (OD 490 nm, 0.3 ± 0.0) and survival after challenge with Vibrio harveyi (55.6%) was observed in 32 + R. The study elucidates that rice flour addition produces optimum level of biofloc with better growth and immune responses compared to molasses and control. Furthermore, rice flour addition at 32% protein level could replace 40% protein feed.     

Ingestion of food pellets containing Escherichia coli overexpressing the heat‐shock protein DnaK protects Penaeus vannamei (Boone) against Vibrio harveyi (Baumann) infection

Feeding aquatic animals with bacterial encapsulated heat‐shock proteins (Hsps) is potentially a new method to combat vibriosis, an important disease affecting aquatic animals used in aquaculture. Food pellets comprised of shrimp and containing Escherichia coli overexpressing either DnaK‐DnaJ‐GrpE, the prokaryotic equivalents of Hsp70‐Hsp40‐Hsp20, or only DnaK were fed to juveniles of the white leg shrimp Penaeus vannamei, and protection against pathogenic Vibrio harveyi was determined. Maintaining pellets at different temperatures for varying lengths of time reduced the number of live adhering E. coli, as did contact with sea water, demonstrating that storage and immersion adversely affected bacterial survival and attachment to pellets. Feeding P. vannamei with E. coli did not compromise their survival, indicating that the bacteria were not pathogenic to shrimp. Feeding P. vannamei with pellets containing bacteria overproducing DnaK (approximately 60 cells g^?1 pellets) boosted P. vannamei survival twofold against V. harveyi, suggesting that DnaK plays a role in Vibrio tolerance. Pellets containing DnaK were effective in providing protection to P. vannamei for up to 2 weeks before loss of viability and that DnaK encapsulated by these bacteria enhanced shrimp resistance against Vibrio infection.