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.     

Evaluating the effects of dietary resistant starch on growth,digestion, metabolism,immunity and resistance to ammonia stress in Litopenaeus vannamei

Resistant starch (RS) is considered to be a good alternative to antibiotics. A 56‐day feeding trial was followed by induction of acute ammonia stress to evaluate the effects of dietary RS on growth, digestion, metabolism, immunity and resistance to ammonia stress in Litopenaeus vannamei. The four diets used differed only in RS content: 0 g/kg (Control), 10 g/kg (RS1), 30 g/kg (RS2) and 50 g/kg (RS3). The results showed that dietary RS improved the growth and the survival rates of shrimp exposed to ammonia stress. At 56 days, the activity of digestive and metabolic enzymes (amylase, lipase, pepsin, hexokinase and pyruvate kinase) was increased in the three RS groups, while trypsin activity only increased in the RS1 and RS2 groups; immune‐related parameters (the total antioxidant capacity; the activity of superoxide dismutase, total nitric oxide synthase and phenoloxidase; and the expression of heat‐shock protein 70, thioredoxin, prophenoloxidase, lysozyme and toll gene) were increased in the three RS groups. After exposure to ammonia stress, all the selected immune parameters of the three RS groups were higher than those of the control group at 24 hr. Therefore, dietary RS improved the growth, digestion, metabolism, immunity and resistance to ammonia stress in L. vannamei.     

Effect of polysaccharides extracts of algae Ulva rigida on growth,antioxidant, immune response and resistance of shrimp,Litopenaeus vannamei against Photobacterium damselae

This study was carried out to investigate the effect of water‐soluble polysaccharides extract of algae Ulva rigida (WPU) as dietary supplement on growth performance, antioxidant enzyme activity, lysozyme and phenoloxidase activity, and resistance of shrimp (Litopenaeus vannamei) subjected to bacterial infection with Photobacterium damselae. Three replicate groups of shrimp (1.0 g) were fed four diets containing four levels, 0 or control, 0.5, 1 and 1.5 g/kg of WPU for 8 weeks over the growth trial. Thereafter, 30 shrimps from each dietary treatment were infected with bacteria P. damselae to evaluate disease resistance of infected shrimp. The results of this study showed that WPU was effective as a growth promoter for L. vannamei. The best growth rate was observed in shrimp fed 1.5 g/kg of WPU diet. Regarding antioxidant defences, the diets supplemented with three levels of WPU stimulated glutathione peroxidase and catalase activates in experimental shrimps. MDA content of L. vannamei‐fed diet containing WPU 1.5 and WPU 1.0 was lower than WPU 0 and WPU 0. 5 diets. Also, lysozyme and phenoloxidase activities of shrimp receiving WPU at 1.0 and 1.5 level were significantly higher than those fed WPU 0 and WPU 0.5 diets. In addition, using WPU extract in all diets decreased mortality in L. vannamei in a dose‐dependent manner after challenge with P. damselae. These results suggest that incorporation of water‐soluble polysaccharides from green algae U. rigida at 1.5 g/kg doses improves growth and antioxidant activity and enhances the immune responses in shrimp L. vannamei.     

Growth,body composition,ammonia tolerance and hepatopancreas histology of white shrimp Litopenaeus vannamei fed diets containing different carbohydrate sources at low salinity

Juvenile Litopenaeus vannamei farmed at 3.0 psu were fed five diets containing glucose, sucrose, wheat starch, corn starch or potato starch as the carbohydrate (CBH) source. Shrimp were fed for 50 days to explore the effect of dietary CBH source on growth, body composition and ammonia tolerance. The specific growth rate of body length of shrimp fed glucose was the highest and significantly higher than those fed potato starch. The survival rate of shrimp fed glucose was 89.44%, and it was the highest and significantly higher than those fed wheat starch. Whole shrimp body crude protein and lipid of the corn starch group were 140.2 g kg^?1 and 10.1 g kg^?1 respectively. And they were significantly higher than those fed wheat starch. Shrimp fed potato starch had higher hepatopancreas and muscle glycogen. Shrimp fed sucrose had higher glucose‐6‐phosphate dehydrogenase and lower pyruvate kinase activities (P < 0.05). Besides, shrimp fed starch produced more B cells in hepatopancreas tubules than those fed glucose or sucrose. Shrimp fed different sources of CBH differed in the number of R cells. After 96‐h of ammonia nitrogen challenge, the survival rate of the treatments from high to low in turn was glucose, wheat starch, corn starch, sucrose and potato starch, and no significant differences were observed among all treatments. Based on shrimp growth and the economic problems of practical production, we recommend wheat starch as CBH source in practical diets for L. vannamei farmed at low salinities.     

Effect of dietary lipid level on growth,lipid metabolism and health status of the Pacific white shrimp Litopenaeus vannamei at two salinities

Three isonitrogenous diets containing 60 g kg^–1, 90 g kg^–1 or 120 g kg^–1 lipid were formulated and fed to the Litopenaeus vannamei (2.00 ± 0.08 g) under two salinities (25 or 3 psu) in triplicate for 8 weeks. Shrimp fed 90 g kg^–1 lipid had higher weight gain and specific growth rate than shrimp fed the other two diets regardless of salinity, and the hepatosomatic index increased with increasing dietary lipid at both salinities. The shrimp at 3 psu had significantly lower survival and ash content, higher condition factor, weight gain and specific growth rate than the shrimp at 25 psu. Increasing dietary lipid level induced the accumulation of serum MDA regardless of salinity, and at 3 psu, it reduced the serum GOT and GPT activities and the mRNA expression of TNF‐α in intestine and gill of L. vannamei. The hepatopancreatic triacylglycerol lipase (TGL) and CPT‐1 mRNA expression showed the highest value in shrimp fed 90 g kg^–1 lipid diet at 3 psu. This study indicates that 120 g kg^–1 dietary lipid may negatively affect the growth and induce oxidative damage in shrimp, but can improve immune defence at low salinity; 60 g kg^–1 dietary lipid cannot afford the growth and either has no positive impact on the immunology for L. vannamei at 3 psu.     

Growth parameters of whiteleg shrimp Litopenaeus vannamei and red seaweed Gracilaria corticata in integrated culturing method under zero water exchange system

Growth parameters of whiteleg shrimp Litopenaeus vannamei and red seaweed Gracilaria corticata were measured using integrated culturing method under zero‐water exchange system in a 45‐day period. A 2 × 3 factorial design was used with two levels of shrimp stocking densities and three levels of seaweed weight densities. G. corticata was cultured on a net tied to a round polyethylene frame. Culture tanks were filled with 750‐L filtered seawater. A 40‐W compact fluorescent lamp was hung over each tank to provide adequate and sufficient light for seaweed growth. Growth parameters of shrimp and seaweed such as specific growth rate (SGR), weight gained (WG) and average daily growth (ADG) were computed based on the initial and final weight of shrimp and seaweed. The maximum and minimum SGR of L. vannamei (1.97 and 1.69%/day) were observed in treatment S₁A₃ (25 shrimp/m² and 400 g seaweed/m²) and S₂A₁ (50 shrimp/m² without seaweed) respectively. The best survival rate (94.67 ± 1.33%), WG (129.9 ± 2.9%) and feed conversion ratio (1.67 ± 0.04) were also observed in treatment S₁A₃. The SGR of G.corticata in the treatment S₁A₃ (1.97 ± 0.00%/day) was significantly higher, compared to others. Strong positive correlations were obtained between the density of G. corticata and the growth parameters of L. vannamei. The red seaweed G. corticata could boost the growth parameters, survival rate and total production of L. vannamei under zero‐water exchange system.     

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.     

Effects of dietary supplementation with freeze‐dried powder of Ampithoe sp. on the growth performance,energy metabolism,and ammonia‐nitrogen tolerance of the Pacific white shrimp,Litopenaeus vannamei

This study evaluated the influence of dietary supplementation with freeze‐dried powder of Ampithoe sp. (FDPA) on the growth, energy metabolism, and resistance to ammonia‐nitrogen stress in Litopenaeus vannamei. There were four treatment groups: a 0% group (no FDPA addition), a 33% group, a 66% group (33% and 66% of the shrimp diet, respectively, replaced with FDPA), and a 100% group (only FDPA). The results of this study suggested a positive effect of FDPA supplementation on shrimp survival: the supplemented groups had significantly higher survival than the 0% group (p < 0.05). The body length, body weight, and specific growth rate (SGR) of the 33% group were higher than those of the other groups and were significantly higher than that of the 100% group (p < 0.05). FDPA feeding had a negative effect on carbohydrate metabolism pathways and energy consumption due to decreases in pyruvate kinase (PK), phosphofructokinase (PFK), succinate dehydrogenase (SDH), lactic dehydrogenase (LDH), and respiratory electron transport system (ETS) activity in shrimp fed FDPA during the culture period. The shrimp in the 33% group exhibited good resistance to ammonia‐nitrogen stress. Additionally, the glycolysis pathway and energy consumption of shrimp in the 33% group were enhanced during the ammonia‐nitrogen stress period. Consequently, it was inferred that FDPA supplementation could improve the resistance of shrimp to ammonia‐nitrogen stress (in the 33% group), which might be related to the effects of the supplement on energy metabolism pathways, particularly in terms of enhancing glycolysis to provide sufficient energy for the stress response.     

Effects of crude extracellular protein and crude intracellular polysaccharides of Vibrio alginolyticus on the growth,energy metabolism regulation and WSSV resistance of Litopenaeus vannamei

This study aimed to investigate the effects of adding crude extracts of the extracellular protein (Ex‐Pro) and intracellular polysaccharides (In‐Poly) of Vibrio alginolyticus to diets, at a dosage of 10 g/kg, on the growth, energy metabolism and WSSV (White Spot Syndrome Virus) resistance of Litopenaeus vannamei (initial weight, 0.88 ± 0.04 g/shrimp). Growth and survival rate were not significantly affected by the crude extracts (p > 0.05). Shrimp fed Ex‐Pro crude extracts showed higher succinate dehydrogenase (SDH) activity in the hepatopancreas and muscle but lower lactate dehydrogenase (LDH) activity in the muscle (p < 0.05). The activities of hexokinase (HK), pyruvate kinase (PK), LDH and SDH in the hepatopancreas and the activity of SDH in the muscle were significantly increased by feeding In‐Poly crude extracts (p < 0.05). In contrast, the content of fatty acid synthase (FAS) in the muscle was significantly reduced by the crude extracts (p < 0.05). The contents of glucose and triglyceride and the activity of the electron transport system in the hepatopancreas were significantly increased by the crude extracts (p < 0.05), and the WSSV resistance of the shrimp was increased. These results indicated that the Ex‐Pro and In‐Poly crude extracts of V. alginolyticus could affect energy metabolism, and there was a correlation between WSSV resistance and energy metabolism in L. vannamei.     

Advances in the study of activity additivity of supplemented proteases to improve digestion of feed protein by Penaeus vannamei

In vitro assays used porcine or bovine trypsin as models of exogenous enzymes to determine functioning in the presence of enzymatic extracts from the digestive gland of whiteleg shrimp Penaeus vannamei. Using electrophoresis and zymograms, when enzymes from the shrimp were mixed in the absence of protein substrate, they hydrolysed the trypsin from bovine or porcine origin. Porcine or bovine trypsin, when mixed with shrimp enzymes in pH‐stat assays in the presence of shrimp commercial feed, fish meal, or casein, there was added activity to hydrolyse the protein substrate. Hydrolysis of protein substrate was twofold to threefold stronger if exogenous enzymes were added. Results suggest that porcine or bovine trypsin could be used as feed supplements for whiteleg shrimp P. vannamei to enhance hydrolysis of proteins in feeds, because the commercial enzymes contributed to the hydrolysis of the protein in the three substrates in the presence of shrimp enzymes.     

Effects of dietary phytase supplementation on growth performance and apparent digestibility coefficients of Pacific White Shrimp Litopenaeus vannamei

A growth trial and a digestibility trial were conducted to evaluate the efficacy of phytase supplemented to practical shrimp feeds. The 5 weeks growth trial evaluated the effects of phytase supplementation in replete phosphorus (P) diets on the performances and compositions of juvenile Pacific white shrimp, Litopenaeus vannamei. No significant differences were observed in final biomass, final mean weight, weight gain, feed conversion ratio (FCR), protein retention and survival across all the treatments. Shrimp reared on the P deficient diet had significantly higher P retention and lower whole body P levels as compared to shrimp fed the other diets. Copper content in the whole shrimp body was significantly increased in the treatment supplemented with 1,000 IU kg^?1 feed phytase. The digestibility trial was conducted to study the combined effects of phytase supplementation levels and diet type (plant‐based versus fishmeal‐based) on apparent digestibility coefficients of Pacific white shrimp, Litopenaeus vannamei. There were no effects of diet type so the data was combined. Phytase incorporation at both 500 and 2,000 IU kg^?1 significantly improved protein digestibility, whereas P digestibility was enhanced when 2,000 IU kg^?1 phytase was supplemented to the diet. Apparent digestibility coefficients of alanine, arginine, aspartic acid, cysteine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, ornithine, phenylalanine, proline, serine, threonine, tyrosine and valine were significantly increased when fed diets contained 500 and 2,000 IU kg^?1 phytase supplementation. Results of this work demonstrate that under the conditions of the study growth was not enhanced by phytase supplementation in P replete diets. However, nutrient retention for Cu and digestibility of P, protein and a number of amino acids were enhanced.     

Effects of Vibrio harveyi on plasma clotting protein (coagulogen) of white shrimp Litopenaeus vannamei

Blood clotting exhibits various important functions, including the prevention of body fluid loss and invasion of pathogens in shrimp. The effects of pathogenic Vibrio harveyi on plasma of white shrimp (Litopenaeus vannamei) in vitro and in vivo were investigated in this study. The clotting protein (coagulogen) in plasma of white shrimp pre‐incubated with extracellular products (ECP) of V. harveyi was found apparently decreased and fast‐migrated in crossed immunoelectrophoresis (CIE) gels. In addition, the coagulogen had been degraded to many low molecular‐weight protein bands in plasma pre‐incubated with ECP on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) gels. When pre‐challenged with bacterial cells and ECP of V. harveyi, the white shrimp began to die at about 30 and 16 h respectively. Moreover, plasma coagulogen was decreased more obvious in shrimp challenged with ECP than that with bacterial cells as visualized in CIE gels, and total plasma protein in both group of shrimp were all decreased. Haemolymph withdrawn from moribund shrimp pre‐challenged with V. harveyi or its ECP was observed unclottable. However, the addition of clotting factors (transglutaminase and/or Ca²⁺) to these unclottable plasma could apparently promote their re‐clotting ability as jelly‐like solid observed in microtubes. The recovery of clotting ability of plasma from moribund shrimp was due to the reformation of coagulogen (200 kDa) after adding the two clotting factors as shown on CIE and SDS‐PAGE gels. The present results suggest that the infection of V. harveyi in white shrimp may not only degrade coagulogen but also influence the presence of transglutaminase and Ca²⁺ ion.     

Anti‐PirA‐like toxin immunoglobulin (IgY) in feeds passively immunizes shrimp against acute hepatopancreatic necrosis disease

Acute hepatopancreatic necrosis disease (AHPND), caused by a toxin‐producing Vibrio parahaemolyticus strain, has become a serious threat to shrimp aquaculture. The need to regulate antibiotic use prompted the development of alternative ways to treat infections in aquaculture including the use of chicken egg yolk immunoglobulin (IgY) for passive immunization. This study evaluated the protective effect of IgY against AHPND infection in Litopenaeus vannamei (Boone). IgY was isolated from eggs laid by hens immunized with recombinant PirA‐like (rPirA) and PirB‐like (rPirB) toxins. Whole‐egg powders having IgY specific to rPirA (anti‐PirA‐IgY) and rPirB (anti‐PirB‐IgY) and IgY from non‐immunized hen (control‐IgY) were mixed with basal diets at 20% concentrations and used to prefeed shrimp 3 days before the bacterial challenge test. Survival rates of the challenged shrimp fed the anti‐PirA‐IgY, anti‐PirB‐IgY and control‐IgY diets were 86%, 14% and 0%, respectively. Only the feed containing anti‐PirA‐IgY protected shrimp against AHPND. Increasing the concentration of rPirA antigen to immunize hens and lowering the amount of egg powder in feeds to 10% consistently showed higher survival rates in shrimp fed with anti‐PirA‐IgY (87%) compared with the control (12%). These results confirm that addition of anti‐PirA‐IgY in feeds could be an effective prophylactic method against AHPND infection in shrimp.     

Effect of recombinant crustacean hyperglycemic hormones rCHH‐B1 and rCHH‐B2 on lipid metabolism in the Pacific white shrimp Litopenaeus vannamei

The crustacean hyperglycemic hormones (CHH) are pleiotropic neuropeptides controlling diverse processes in the life cycle of crustaceans. In the Pacific white shrimp Litopenaeus vannamei, CHH‐B1 and CHH‐B2 are generated by alternative splicing of the same gene, and the recombinant versions of these peptides have shown differential effects on glucose metabolism and osmoregulation in this species. In this work, we explored the role of both recombinant variants, expressed in yeast Pichia pastoris, on lipid metabolism. The results showed that rCHH‐B1 and rCHH‐B2 injections into eyestalk‐ablated shrimp significantly increased the phospholipid and triglyceride levels in haemolymph and restored free fatty acid levels, suggesting that these CHH variants mobilize lipid reserves in shrimp. The effects on lipids were concomitant to its hyperglycemic activity, suggesting that both variants, in addition to their hyperglycemic activity, have hyperlipidemic functions.     

Response of facilitative glucose transporter 1 to salinity stress and dietary carbohydrate nutrition in white shrimp Litopenaeus vannamei

Facilitative glucose transporter 1 (GLUT1) is a transporter protein for glucose transport via the plasma membrane of the cells to provide energy through carbohydrate metabolism. GLUT1 cDNA from Litopenaeus vannamei was obtained and analysed in this study. Full‐length GLUT1 cDNA is 2062 bp long and contained a 1506‐bp ORF encoding a 502 amino acid protein, a 270‐bp 5′UTR and a 284‐bp 3′UTR. When shrimp were under acute low salinity stress, the expression in hepatopancreas, muscle, gill and eyestalk was all up‐regulated at 12 h (P < 0.05) and 96 h (P < 0.05), while the expression in the four tissues was all down‐regulated at 6 h (P < 0.05) and 48 h (P < 0.05) . The expression in the muscle of shrimp at water salinity of 3 was lower than that at water salinity of 30 independent of dietary carbohydrate levels, while expression in hepatopancreas, gill and eyestalk was up‐regulated at 200 and 300 g kg^?1 carbohydrate levels. The expression in all tissues fed glucose was up‐regulated when compared to the expression in shrimp held at a water salinity of 30. This study suggests that GLUT1 is a conserved protein in L. vannamei, and changes in expression due to environmental salinity and dietary carbohydrate level and source.