Changes in the intestinal bacterial community during the growth of white shrimp,Litopenaeus vannamei

In this study, we documented the changes in the intestinal bacterial community at four stages in Litopenaeus vannamei: 14 days postlarvae (L14) and 1‐, 2‐ and 3‐month old juveniles (J1, J2, J3), using 454 pyrosequencing techniques. The intestinal bacterial community was dominated by three bacterial phyla, Proteobacteria, Bacteroidetes and Actinobacteria at all stages. However, the relative abundance and bacterial lineages varied at the family level. The intestinal bacterial community of L14 and J1 was similar, with dominant members belonging to the Comamonadaceae of Betaproteobacteria. Conversely, bacterial members affiliated to Flavobacteriaceae of Bacteroidetes were dominant in J2 and Vibrionaceae of Gammaproteobacteria was dominant in J3. The abundance of Microbacteriaceae of Actinobacteria also fluctuated during the four stages. Bacterial members of Flavobacteriaceae and Rhodobacteraceae (Alphaproteobacteria) were present through all growth stages, and likely form the intestinal core microbiome of L. vannamei. However, they varied at the operational taxonomic unit (OTU) level through the growth stages. The intestinal bacterial community of pond‐rearing shrimp included the three main bacterial phyla identified above, and an additional group, Mycoplasmataceae of Mollicutes. Our results demonstrate that the intestinal bacterial community of L. vannamei was highly dynamic during the growth stages. Bacterial members belonging to Commamonadaceae dominated in the earlier growth stage of shrimp, possibly influenced by feeding with Artemia nauplii, but there was a shift to Flavobacteriaceae in the mid and Vibrionaceae in the late growth stages.     

In vivo evaluation of the effects of commercial Bacillus probiotics on survival and development of Litopenaeus vannamei larvae during the early hatchery period

Three in vivo experiments were conducted to measure the effectiveness of commercial Bacillus probiotics on survival and development of Litopenaeus vannamei larvae from nauplii 3–4 (N_3–4) to zoea 3 (Z₃) stages. Experiment I: Nine commercial Bacillus probiotics were individually added to larvae twice at N₆ and Z₁ at levels of 2, 20, and 100 mg L^?1. Only six of nine products at 20 mg L^?1 exhibited higher or significantly higher larval survival (P < 0.05) than the control. Experiment II: Two superior products from the first experiment were administered to larvae with six dose frequencies at 20 mg L^?1. For both products, three doses, once at each of N_3–4, N₆ and Z₁ stages, yielded the best larval survival and development rates (P < 0.05), and these were confirmed by enhanced activities of tryptase and amylase. Experiment III: The isolates of these two products, identified as Bacillus subtilis and Bacillus licheniformis, were delivered to larvae singly at concentrations of 1.0 × 10⁸ and 1.0 × 10⁹ CFU L^?1, or at the same concentrations by mixing the two equally. At 1.0 × 10⁹ CFU L^?1, B. subtilis exerted more beneficial effects on larvae than B. licheniformis or the mixture. Therefore, the optimal dosage and dose frequency of commercial Bacillus products should be evaluated prior to large‐scale application in shrimp hatcheries to avoid futility or even adverse effects, as spore counts and non‐bacterial ingredients are key factors influencing the efficacy of Bacillus probiotics.     

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.     

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.     

Molecular and functional characterization of Raptor in mTOR pathway from Litopenaeus vannamei

Raptor, a member of the target of rapamycin complex 1 (TORC1), participates in the formation of complex proteins related to the mechanistic target of rapamycin (mTOR) signalling. In this study, a 5,020 bp cDNA of Raptor with an open reading frame (ORF) of 3,804 bp encoding for 1,267 amino acids was cloned from Litopenaeus vannamei. The protein contains three conserved domains: Raptor N, HEAT and WD40 domains. The expression of Raptor gene was detected by qRT‐PCR in different tissues of L. vannamei, including hepatopancreas, intestinal, stomach, eyestalk, gill and muscle. The mRNA expression profiles of Raptor in muscle were also analysed under suppression or stimulation of mTOR signalling pathway. The level of Raptor mRNA significantly increased either at 0.5–6 hr after an injection of rapamycin (RAPA) or after 3 days starvation. Leucine or arginine alleviated the up‐regulation of Raptor gene expression caused by RAPA or starvation. The Raptor gene was successfully suppressed using RNA interference (RNAi) technology, and the gene expression and the protein phosphorylation level of 4EBP1 and S6K were significantly decreased. The results of the study suggested that the expression of Raptor was sensitive to the immunology status of L. vannamei and participated in nutritional metabolism.     

Evaluation of frozen Umbrella‐stage Artemia as first animal live food for Litopenaeus vannamei (Boone) larvae

An alternative larval shrimp feeding regime, in which umbrella‐stage Artemia were constituting the first zooplankton source was evaluated in the culture of Litopenaeus vannamei. In a preliminary experiment, umbrella‐stage Artemia were fed to larvae from zoea 2 (Z2) to mysis 2 (M2) stages to identify the larval stage at which raptorial feeding starts and to determine daily feeding rates. The following experiment evaluated the performance of two feeding regimen that differed during the late zoea/early mysis stages: a control treatment with frozen Artemia nauplii (FAN), and a treatment with frozen umbrella‐stage Artemia (FUA). The ingestion rate of umbrella‐stage Artemia increased from nine umbrella per larvae day^?1 at Z2 stage to 21 umbrella per larvae day^?1 at M2. A steep increase in ingestion and dry weight from Z3 to M2 suggests a shift to a raptorial feeding mode at the M1 stage. Treatment FUA exhibited a significantly higher larval stage index (P < 0.05) during the period that zoea larvae metamorphosed to the mysis stage, and a higher final biomass, compared with treatment FAN. Based on these results and on practical considerations, a feeding regime starting with umbrella‐stage Artemia from Z2 sub‐stage can be recommended for L. vannamei larvae rearing.     

Effects of dietary marine microalgae,Tetraselmis suecica,on production,gene expression,protein markers and bacterial count of Pacific white shrimp Litopenaeus vannamei

The marine flagellated Chlorophyta Tetraselmis suecica is among the most important live food species in marine aquaculture. In the present study, the effects of dietary supplementation of dried marine microalgae, Tetraselmis suecica, on growth performance; feed utilization; chemical composition; gene expression of superoxide dismutase (SOD), glutathione peroxidase (GPx) and insulin‐like growth factor 2 (IGF‐II) gene of Pacific white shrimp, Litopenaeus vannamei; muscle protein polymorphism; and microbial count were assessed and evaluated. Three hundred and sixty L. vannamei (postlarvae) Pls (0.124 ± 0.002 g) were randomly stocked into 40‐L glass aquaria (30 shrimp/aquarium) and fed three times daily four tested diets: a basal diet (control), diet incorporated with 2.5 g kg^?1 dried T. suecica (T1), 5 g kg^?1 dried T. suecica (T2) and 7.5 g kg^?1 dried T. suecica (T3) in triplicates, for 90 days. At the end of the trial, the survival rate (SR) of L. vannamei fed diets supplemented with different levels of T. suecica was significantly (p < .05) higher than the control diet. The highest weight gain and specific growth rate and the best feed conversion ratio were recorded on L. vannamei fed a diet supplemented with a 7.5 g/kg dried T. suecica. The highest protein, lipid and ash contents were obtained in L. vannamei fed the diet containing 7.5 g/kg T. suecica, when compared with the remaining tested diets. The gene expression of antioxidant genes SOD and GPx was the lowest in the T3 group in comparison with the control group. Meanwhile, expression level of IGF‐II was higher in the T2 group. The total heterotrophic bacterial count was significantly (p < .05) increased with the cumulative T. suecica level, while no significant (p > .05) differences were found in the total Vibrio count among treatments. Overall, the present results have shown that the diet supplemented with the highest inclusion level of dried T. suecica resulted in improved growth and nutrient utilization.     

Experimental horizontal transmission of Enterocytozoon hepatopenaei in post‐larvae of whiteleg shrimp,Litopenaeus vannamei

Enterocytozoon hepatopenaei (EHP) is a microsporidian parasite that causes hepatopancreatic microsporidiosis (HPM) in penaeid shrimp. HPM was observed in several countries, including Thailand and India; it has become a prominent pathogen in shrimp culture. Based on observations on EHP infection in the wild, the route of transmission has been hypothesized. Identification of artificial EHP infection procedures can facilitate our understanding of EHP transmission. Experimental transmission of EHP was attempted using the immersion and oral infections of infection. In the immersion mode, post‐larvae (PL) were exposed to an EHP tissue homogenate (0.2%) by immersion for 48 hr. Experimental samples were collected at various time points, and infection was confirmed using polymerase chain reaction, haematoxylin and eosin staining, transmission electron microscopy and modified trichrome staining. All test results revealed successful EHP transmission. Similar results were obtained through oral infection (oral infection). Innate immune gene expression patterns during infection were analysed; prophenoloxidase, crustin and superoxide dismutase were upregulated at 6, 6 and 48 hr post‐challenge, respectively. Experimental infection procedures facilitate the development of diagnostic and prevention strategies. This is the first study demonstrating the experimental transmission of EHP in shrimp PL.     

Evaluation of Schizochytrium meal in microdiets of Pacific white shrimp (Litopenaeus vannamei) larvae

A feeding trial was conducted to assess the effects of dietary Schizochytrium meal supplementation on survival, growth performance, activities of digestive enzymes and fatty acid composition in Pacific white shrimp (Litopenaeus vannamei) larvae (initial body weight 4.21 ± 0.10 mg). Four isonitrogenous and isolipidic diets were formulated to contain graded levels of Schizochytrium meal: 0% (S0, the control diet), 2% (S2), 4% (S4) and 6% dry matter (S6). Results showed that there was no significant difference in survival of shrimps among dietary treatments (P > 0.05). Shrimps fed diets with 2% and 4% microalgae meal had significantly higher specific growth rate (SGR) than that of shrimps fed diets with 0% and 6% microalgae meal, and no significant differences were observed between shrimps fed diets with 2% and 4% microalgae meal (P > 0.05). Activity of trypsin in the pancreatic and intestinal segments, and activity of amylase in the pancreatic segments were not significantly affected by dietary microalgae meal levels (P > 0.05). Specific activities of both alkaline phosphatase and leucine‐aminopeptidase in intestine and purified brush border membrane of intestine were significantly higher in shrimps fed diet with 2% microalgae meal (P < 0.05). There were no significant differences in C18:2n‐6, n‐3 fatty acids, n‐6fatty acids, PUFA and n‐3/n‐6 in muscle samples among dietary treatments. C16:1n‐7, C18:1n‐9, MUFA, C18:3n‐3 and C20:5n‐3 decreased, however, C20:4n‐6 increased in the muscle as dietary microalgae meal level increased. In conclusion, 4% Schizochytrium meal in microdiets of shrimps can improve growth performance and may be a valuable additive in the microdiets of shrimps.     

Dietary niacin levels in practical diets for Litopenaeus vannamei to support maximum growth

This study investigated the effects of dietary niacin on growth performance, feed utilization and non‐specific immune response in juvenile Pacific white shrimp. Six isonitrogenous and isolipidic practical diets were formulated with graded niacin levels of 10.9, 65.8, 121.2, 203.4, 387.5 and 769.3 mg kg^?1 of dry diet, respectively. Results indicated that per cent weight gain (WG), specific growth rate (SGR), feed efficiency (FE), protein efficiency ratio (PER) and protein productive value (PPV) were significantly influenced by the dietary niacin levels. The maximum WG and SGR occurred at 121.2 mg kg^?1 niacin diet. However, survival and proximate composition of whole body were not significantly affected by the dietary niacin levels. Dietary niacin levels had no significant effects on the total protein, glucose, triacylglycerol and cholesterol contents in the haemolymph. The activity of catalase and lysozyme in the haemolymph was significantly affected by dietary niacin levels. Based on a two‐slope regression analysis of SGR against dietary niacin level, the dietary niacin requirement of juvenile Pacific white shrimp was 109.6 mg kg^?1.     

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.     

Isolation and identification of Vibrio campbellii as a bacterial pathogen for luminous vibriosis of Litopenaeus vannamei

Outbreak of luminescent disease was reported from Litopenaeus vannamei shrimp farms in Zhangpu County, Southern China during May–July 2011. The clinical signs included fluorescent, less food consumption and high mortality. Bacteria were isolated from the infected shrimps. The pathogen, a luminescent bacterium named VH1 was identified as Vibrio campbellii based on MLSA analysis (16S rDNA, rpoD and toxR). The haemolysin (hly) gene specific in V. campbellii was detected in strain VH1. Pathogenicity test using immersion infection confirmed that strain VH1 was virulent to L. vannamei postlarvae and juveniles, and the LC₅₀ value was 1.55 × 10⁶ CFU mL^?1 and 1.7 × 10⁶ CFU mL^?1 respectively.     

Successful rearing of whiteleg shrimp Litopenaeus vannamei larvae fed a desiccation‐tolerant nematode to replace Artemia

The nematode Panagrolaimus sp. was tested as live feed to replace Artemia nauplii during first larval stages of whiteleg shrimp Litopenaeus vannamei. In Trial 1, shrimp larvae were fed one of four diets from Zoea 2 to Postlarva 1 (PL1): (A) Artemia nauplii, control treatment; (NC) nematodes enriched in docosahexaenoic acid (DHA) provided by the dinoflagellate Crypthecodinium cohnii; (N) non‐enriched nematodes; and (Algae) a mixture of microalgae supplemented in C. cohnii cells. In Trial 2, shrimp were fed (A), (NC) and a different treatment (NS) with nematodes enriched in polyunsaturated fatty acids (PUFAs) provided by the commercial product S.presso^®, until Postlarva 6 (PL6). Mysis 1 larvae fed nematodes of the three dietary treatments were 300 μm longer (3.2 ± 0.3 mm) than control larvae. At PL1, control shrimp were 300 μm longer (4.5 ± 0.3 mm) than those fed DHA‐enriched or PUFAs‐enriched nematodes. No differences were observed in length and survival at PL6 between control larvae and those fed DHA‐enriched nematodes (5.1 ± 0.5 mm; 33.1%–44.4%). Shrimp fed microalgae showed a delay in development at PL1. This work is the first demonstration of Panagrolaimus sp. suitability as a complete substitute for Artemia in rearing shrimp from Zoea 2 to PL6.     

Minimum water exchange spares the requirement for dietary methionine for juvenile Litopenaeus vannamei reared under intensive outdoor conditions

We examined if minimum water exchange could spare dietary methionine (Met) required for maximum growth performance of juvenile Litopenaeus vannamei reared in an intensive outdoor system. Shrimp of 1.98 ± 0.13 g were stocked at 70 animals/m² and reared for 72 days in 50 tanks of 1 m³ under flow‐through (14.4% a day) and static (1.4%–2.9% a day) green‐water conditions at 32.0 ± 3.7 g/L salinity. Five diets with a minimum inclusion of fishmeal supplemented with a dipeptide, dl ‐methionyl‐dl ‐methionine, were formulated to contain increasing levels of Met, 4.8, 6.2, 7.2, 8.1 or 9.4 g/kg (on a dry matter basis). Each of the five diets were fed four times daily to five replicate groups. Dietary Met and water exchange significantly influenced shrimp survival, gained yield, apparent feed intake, food conversion ratio and final body weight (p < .05). Raising shrimp under limited water exchange, i.e., static versus flow‐through spared the dependence on higher levels of dietary Met to maximize shrimp body weight, from 9.4 g/kg to 8.0 g/kg (14.0 and 12.6 g/kg Met+Cys respectively). In an intensive rearing system, a reduction in water exchange is desirable as it leads to a lower need for supplemental dietary Met.     

Effect of different total suspended solids levels on a Litopenaeus vannamei (Boone, 1931) BFT culture system during biofloc formation

In a Biofloc Technology System (BFT), there is constant biofloc formation and suspended solids accumulation, leading to effects on water quality parameters that may affect the growth performance of cultured shrimp. This study aimed to analyse during biofloc formation the effect of different total suspended solids (TSS) levels on water quality and the growth performance of Litopenaeus vannamei shrimp in a BFT system. A 42‐day trial was conducted with treatments of three ranges of TSS: 100–300 mg L^?1 as low (TL), 300–600 as medium (TM) and 600–1000 as high (TH). The initial concentrations of 100 (TL), 300 (TM) and 600 mg L^?1 (TH) were achieved by fertilization before starting the experiment. Litopenaeus  vannamei juveniles with an average weight of 4.54 ± 1.19 g were stocked at a density of 372 shrimp m^?3. Physical and chemical water parameters and shrimp growth performance were analysed. After 6 weeks, TSS mean concentrations were 306.37, 532.43 and 745.2 mg L^?1 for, respectively, TL, TM and TH treatments. Significant differences (P < 0.05) were observed in TSS, settleable solids, pH, alkalinity and nitrite, especially between the TL and TH treatments. Similarly, differences (P < 0.05) were observed in the growth performance parameters, specifically final weight, survival, feed conversion and productivity. The water quality parameters at lower range of total suspended solids concentration (TL) treatment resulted in a better performance of L. vannamei in the BFT system. The maintenance at range of 100–300 mg L^?1 TSS is thus important to the success of shrimp culture.     

Apparent digestibility of animal,plant and microbial ingredients for Pacific white shrimp Litopenaeus vannamei

A digestibility trial was conducted to determine apparent digestibility coefficients of dry matter, protein, energy and amino acids of animal, plant and microbial ingredients for Pacific white shrimp, Litopenaeus vannamei. The tested ingredients included traditional soybean meal (TSBM), PepsoyGen soybean meal (PSBM), NutriVance soybean meal (NSBM), fish meal (FM), poultry meal (PM), squid hydrolysis (SQH), scallop hydrolysis (SCH), flash dried yeast (FDY), two batches of Ulva meal (UMF and UMS) and bacteria biomass (BB). A basal diet was formulated and produced along with the experimental diets which included 300 g/kg of each ingredient and 700 g/kg of the basal diet. Juvenile shrimp (initial mean weight: 12 g, six shrimp/tank, n = 3) were stocked in a recirculation system. Apparent dry matter, protein and energy digestibility coefficients ranged from ?40.11% to 78.51%, 15.17% to 97.03% and 13.33% to 82.56% among different protein sources, respectively. In general, protein and energy digestibilities in soy sources (77.6% to 97.03% and 62.77% to 82.56%, respectively) are higher than the tested animal protein (51.39% to 71.41% and 45.29% to 69.77%, respectively) and single‐cell protein sources (15.17% to 53.47% and 13.33% to 40.39%, respectively). Among the three soybean sources, TSBM showed highest protein and energy digestibility. Apparent individual amino acid digestibility coefficients were also variable among different types of ingredients, and there was a reasonable correspondence to protein digestibility. The most digestible feed ingredients for Pacific white shrimp in this study were conventional soybean meal (SBM) and NutriVance soybean meal (NSBM), which indicated that these ingredients are good protein and amino acid sources for Pacific white shrimp. Resultant digestibility data may provide useful information to commercial shrimp feed industry.