Effects of dietary astaxanthin on growth,antioxidant capacity and gene expression in Pacific white shrimp Litopenaeus vannamei

Pacific white shrimp Litopenaeus vannamei (1050 individuals with initial weight of 1.01 ± 0.001 g) were fed either control diet or one of six dietary astaxanthin (AX) concentration (25, 50, 75, 100, 125 and 150 mg kg⁻¹) diets for 56 days in 35 tanks (30 shrimp per tank). After 56 days of culture, shrimp‐fed AX125 and AX150 diets had higher (P < 0.05) weight gain, specific growth rate, total antioxidant status and lower (P < 0.05) superoxide dismutase (SOD), catalase (CAT) than shrimp fed control diet. After low dissolved oxygen stress for 1 h, survival rate of shrimp fed AX75, AX100, AX125 and AX150 diets was higher (P < 0.05) than that of shrimp fed control diet. Hypoxia inducible factor‐1α (HIF‐1α), cytosolic manganese superoxide dismutase (cMnSOD) and CAT mRNA expression levels of shrimp fed seven diets were significantly down‐regulated under hypoxia than under normoxia, but their expression levels were higher under hypoxia in shrimp fed AX‐supplemented diets than in shrimp fed control diet. About 70‐kDa heat‐shock protein (Hsp70) mRNA expression level of shrimp fed seven diets was significantly up‐regulated under hypoxia than under normoxia, but its expression level was lower under hypoxia in shrimp fed AX‐supplemented diets than in shrimp fed control diet.     

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.     

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.     

七种不同结构糖源对凡纳滨对虾三大营养物质代谢的影响

本实验研究了不同结构糖源饲料对初始体质量为(0.14±0.01)g的凡纳滨对虾生长、体组成及相关代谢的影响。实验共设7个处理,基础饲料中分别添加7种结构不同的糖源:葡萄糖、果糖、葡萄糖+果糖(1∶1)、蔗糖、麦芽糖、玉米淀粉和糊精,添加水平为20%。每处理设3个平行,每个平行放50尾虾,实验期56 d。结果表明,实验饲料对凡纳滨对虾的特定生长率和蛋白质效率有显著影响,呈现多糖蔗糖其他,其中葡萄糖组最低;实验饲料对凡纳滨对虾的全虾水分、灰分、蛋白质无显著影响,但对脂肪、肝糖原、肌糖原的影响显著。不同结构的糖源饲料对糖代谢相关酶及产物产生显著影响,对脂肪和蛋白代谢酶及产物影响不显著。     

Influence of different dietary carbohydrate sources on the growth and intestinal microbiota of Litopenaeus vannamei at low salinity

Because of the increasing market potential of Pacific white shrimp (Litopenaeus vannamei) and limited availability of coastal areas for production, culture of L. vannamei at low salinity is a growing trend throughout the world. Dietary manipulation could improve the growth performance of L. vannamei at low salinity. This study reared L. vannamei with glucose, sucrose and corn starch as dietary carbohydrate sources, respectively, at a low salinity. The results indicated that the sucrose and glucose feeding groups showed better growth performance. An Illumina‐based sequencing method was used to examine the intestinal bacterial composition and the results indicated that Proteobacteria were the most prevalent members, but abundance of Actinobacteria decreased while Firmicutes increased in the corn starch‐fed group. Furthermore, bacteria related to complex carbohydrate degradation were in lower abundance, whereas the abundance of opportunistic pathogenic bacteria increased in corn starch‐fed group than the other two groups, suggesting that the diet imposes selective pressure on the intestinal microbiota. Complex carbohydrates were not the ideal energy sources for L. vannamei at low salinity because the host has higher energy demand in the stressful conditions while the complex carbohydrate degradation efficiency of the gut microbiota in L. vannamei is limited.     

Effect of Lead on Survival,Osmoregulation, and Histological Changes of the Gills of the White Shrimp,Litopenaeus vannamei,Boone, 1931

The purpose of this study was to evaluate the acute toxicity of lead for different life stages of shrimp, Litopenaeus vannamei, and to determine the effect of its sublethal concentrations on osmoregulatory capacity (OC) as well as the possible histological alteration in the gills of juvenile shrimp. The 24‐, 48‐, and 96‐h LC₅₀ values for lead to L. vannamei increased progressively with increasing life stage, from nauplii < zoeae < mysis < postlarvae < juvenile. After 15‐d exposure to 3.25, 6.50, and 9.75 mg Pb/L, the OC values of exposed shrimp were reduced by 39, 73, and 157%, respectively compared to control animals. Compared to controls, the lead concentrations in gill tissues increased significantly by 127,500, 137,500, and 141,000% in shrimps exposed to 3.25, 6.50, and 9.75 mg Pb/L, respectively. After lead exposure, hemocytic congestion in efferent vessels and multiple hyperplasia were observed in gill filaments, resulting in narrowed hemolymphatic lacuna. The histopathological effects increased with increasing Pb concentration.     

Effect of bioflocs on growth and immune activity of Pacific white shrimp,Litopenaeus vannamei postlarvae

The bioflocs technology (BFT) for shrimp production has been proposed as a sustainable practice capable of reducing environmental impacts and preventing pathogen introduction. The microbial community associated with BFT not only detoxifies nutrients, but also can improve feed utilization and animal growth. Biofloc system contains abundant number of bacteria of which cell wall consists of various components such as bacterial lipopolysaccharide, peptidoglycan and β‐1, 3‐glucans, and is known as stimulating nonspecific immune activity of shrimp. Bioflocs, therefore, are assumed to enhance shrimp immunity because they consume the bioflocs as additional food source. Although there are benefits for having an in situ microbial community in BFT systems, better understanding on these microorganisms, in particular molecular level, is needed. A fourteen‐day culture trial was conducted with postlarvae of Litopenaeus vannamei in the presence and absence of bioflocs. To determine mRNA expression levels of shrimp, we selected six genes (prophenoloxidase1, prophenoloxidase2, prophenoloxidase activation enzyme, serine proteinase1, masquerade‐like proteinase, and ras‐related nuclear protein) which are involved in a series of responses known as the prophenoloxidase (proPO) cascade, one of the major innate immune responses in crustaceans. Significant differences in shrimp survival and final body weights were found between the clear water and in the biofloc treatments. mRNA expression levels were significantly higher in the biofloc treatment than the clear water control. These results suggest that the presence of bioflocs in the culture medium gives positive effect on growth and immune‐related genes expression in L.vannamei postlarvae.     

Comparative study of the intracellular superoxide anion production in different penaeid species through the NBT‐reduction assay

The capacity of reactive oxygen intermediates production upon haemocyte stimulation is one of the most important immunoparameter utilized to assess the health status in cultivated shrimps. In the present study, we compared oxidative stress potential, by measuring the superoxide anion production in three penaeid shrimps: two wild Atlantic species, the pink shrimp Farfantepenaeus paulensis and the white shrimp Litopenaeus schmitti and one cultivated Pacific species, the white shrimp, Litopenaeus vannamei, through the nitro‐blue‐tetrazolium‐reduction assay. We also proposed an optimized experimental protocol for this assay, that produces rapid and consistent results with low levels of basal superoxide anion (O₂^?) production by unstimulated haemocytes and high levels of this oxygen radical after cell stimulation. Among the different cell elicitors used (zymosan, laminarin, lipopolysaccharide and phorbol myristate acetate), laminarin (β‐1,3‐glucans – 2 mg mL^?1) was the most potent cell activator for the haemocytes of all three penaeids and we recommend this immunostimulant to routinely evaluate shrimp respiratory burst. In general terms, the most elevated levels of O₂^? production, after cell stimulation with microbial components, were detected in L. schmitti. Interestingly, the stimulation profile of the haemocytes of L. vannamei was more similar to F. paulensis, than to L. schmitti, which is more phylogenetically related.     

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.     

Supplementation of sodium chloride in diets to improve the meat quality of Pacific white shrimp,Litopenaeus vannamei,reared in low‐salinity water

To investigate the effect of dietary sodium chloride (NaCl) on meat quality of white shrimp (Litopenaeus vannamei) reared in low‐salinity (2 g L^?1) water, shrimp were distributed into four groups (treatments T‐1, T‐2, T‐3 and control) with three replicates. All shrimps were completely randomised stocked into 12 tanks at an initial density of 40 shrimps per tank. Diets for the control, T‐1, T‐2 and T‐3 groups consisted of the basal diet supplemented with 0 g kg^?1, 10 g kg^?1, 20 g kg^?1 and 40 g kg^?1 of NaCl respectively. After 50 days, shrimps in T‐3 showed significantly better (P < 0.05) moisture, crude protein and ash than those of the control and T‐1. Higher muscle Na content was observed (P < 0.05) in T‐3 than that of the control. Significant increases (P < 0.05) in contents of inosinic acid, total free amino acid (TFAA) and essential free amino acid (EFAA) were also found in T‐3. Texture assays showed significant differences (P < 0.05) in hardness, adhesiveness and springiness between group T‐3 as compared with those of T‐1 and control. It indicated that dietary supplementation of NaCl appeared to be a promising practice to improve meat quality of white shrimp reared in low‐salinity waters.     

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.     

Physiological, metabolic and haematological responses in white shrimp Litopenaeus vannamei (Boone) juveniles fed diets supplemented with astaxanthin acclimated to low-salinity water

The effects of dietary astaxanthin supplemented at 0, 40, 80 or 150 mg astaxanthin kg⁻¹ on growth, survival, moult frequency, osmoregulatory capacity (OC) and selected metabolic and haematological variables in Litopenaeus vannamei acclimated to low‐salinity water (3 g L⁻¹) were evaluated. Supplemented astaxanthin at 80 mg kg⁻¹ improved growth, survival and moult frequency in shrimp. The lowest OC was also exhibited in shrimp fed with dietary astaxanthin at 80 mg kg⁻¹. Shrimp haemolymph concentrations of glucose, lactate, haemocyanin and total haemocyte count were all significantly enhanced by feeding the diet supplemented with 80 mg astaxanthin kg⁻¹ compared with shrimp fed with the other diets. On the basis of these results, dietary astaxanthin supplementation of 80 mg kg⁻¹ is recommended for juvenile L. vannamei cultured in low‐salinity water.     

Apparent energy,dry matter and amino acid digestibility of differently sourced soybean meal fed to Pacific white shrimp Litopenaeus vannamei

Due to the variations in nutrient quality of soybean meal (SBM) that is a result of differences in production location and processing specifications, a study was conducted to determine the fluctuations in apparent digestibility coefficients of differently sourced SBM fed to Pacific white shrimps (Litopenaeus vannamei). Twenty‐four SBM‐based diets were formulated by mixing a basal diet and test ingredients on a dry matter basis (70:30 ratio), while 1% chromic oxide was used as the inert marker. The digestibility trial was carried out in a semi‐closed recirculation system with six replicate groups per treatment (mean shrimp weight of 10.2 g). Significant differences were observed for apparent dry matter, energy and protein digestibility coefficients (p < .05 was considered significant) among 24 sources of SBM and digestibility values ranged from 45% to 90%, 56% to 93% and 87% to 98%, respectively. Based on multivariate analysis, acid detergent fibre, neutral detergent fibre, lignin, raffinose and trypsin inhibitor were screened as the key chemical characteristics in SBM that influenced digestibility of nutrients in Pacific white shrimps. Variations in growth performances of shrimp were in line with the variations in apparent digestibility coefficients of SBM verifying the importance of digestibility data in shrimp feed formulations.     

Dietary fulvic acid effects on survival and expression of immune‐related genes in Litopenaeus vannamei challenged with Vibrio parahaemolyticus

The effects of fulvic acid (FA) on survival and immune‐related gene expression were investigated in Litopenaeus vannamei challenged with Vibrio parahaemolyticus by immersion. Shrimp were fed with different dietary FA concentrations (1, 2, 4 and 6 g/kg feed) for 20 days (first bioassay) or 8 days (second bioassay, 2 g/kg feed of FA added every 2 days) and then challenged with V. parahaemolyticus. In a third bioassay, the expression of three immune‐related genes (translationally controlled tumour protein [TCTP], superoxide dismutase [SOD] and heat‐shock protein 70 [HSP70]) in haemocytes or hepatopancreas of experimental shrimp was measured by real‐time quantitative PCR at 0, 6, 12, 24, 48, 72 and 96 hr after FA (2 g/kg feed) administration. Fulvic acid increased survival at a concentration of 2 g/kg feed supplied every two days. Interestingly, TCTP gene expression was upregulated, whereas gene expression of SOD and HSP70 was downregulated. In conclusion, dietary fulvic acid improves survival in white shrimp challenged with V. parahaemolyticus and modulates the immune response. Therefore, FA merits further evaluation as prophylactic treatment in commercial shrimp farms.     

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.     

Selection of reference genes for the study of relative gene expression during development of Litopenaeus vannamei larvae

Pacific whiteleg shrimp (Litopenaeus vannamei) production is one of the most economically important aquaculture industries around the world. Gene expression studies during larval development are a key tool to elucidate biological aspects during metamorphosis and the effects of pathological conditions; the adequate quantification of gene expression can provide molecular markers and basic knowledge to optimize culture conditions, including the development of strategies for the prevention and control of diseases. In this context, the selection and validation of reference genes is a critical process to avoid bias due to inaccurate measurements and the consequent misinterpretation of biological processes. In this study, nine candidate reference genes (Clathrin, Cyt‐c, SubF0, EF1α, β‐Actin, GAPDH, TBP, AK and PK) were selected to test their expression stability during larval development of L. vannamei using geNorm, NormFinder, BestKeeper and the integrated tool RefFinder algorithms. Based on our analysis the most stable gene was SubF0, followed by GAPDH and EF1α. Relative expression of developmental genes (Twist, Mef‐2 and Ubx) was quantified using the three most stable reference genes (individually and combined), taking special attention to the expected expression and biological function of these genes. The expression of Mef‐2 was the most sensitive to the reference gene. However, the combination of the three most stable reference genes provided consistent results according to the expected expression patterns of developmental genes.     

Optimal dietary lipid requirement of postlarval white shrimp,Litopenaeus vannamei in relation to growth performance,stress tolerance and immune response

A 25‐day experiment was conducted to evaluate the optimal lipid level for postlarval Litopenaeus vannamei. Shrimp (1.7 mg) were fed five isonitrogenous diets containing grade levels of lipid (96.6, 114.3, 128.5, 136.5 and 154.5 g/kg diet, respectively). Each diet was assigned to four tanks (500 shrimp), and shrimp were fed six times a day. Weight gain was increased with the increasing dietary lipid levels, and the highest weight gain was observed in shrimp fed diet with 154.5 g/kg lipid (p < 0.05). On the contrary, the survival was lowest in shrimp fed the L15.45 and highest in shrimp fed the L11.43. Triglyceride in hepatopancreas was increased, and cholesterol was decreased with the increasing dietary lipid. Pyruvate kinase and AMPK mRNA expression were highest in shrimp fed the L12.85. Malondialdehyde in whole body was positively correlated with the dietary lipid levels. The mRNA expression of SOD and Caspase 3 was highest in shrimp fed the L12.85. After hypoxia stress, shrimp fed the L12.85 showed highest survival. The mRNA expression of superoxide dismutase and Akirin was highest in shrimp fed the L11.43 and L15.45, respectively. Based on the survival after 25‐day feeding trail and after the hypoxia stress, the optimal dietary lipid for postlarval L. vannamei should be 118–124 g/kg.