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 Haemolymph withdrawn from moribund shrimp pre‐challenged with V. harveyi or its ECP was observed unclottable.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.     

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.     

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.     

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.     

Evaluation of a novel bacterial biomass as a substitution for soybean meal in plant‐based practical diets for Pacific white shrimp Litopenaeus vannamei

Three growth trials and a digestibility trial were designed to evaluate the efficacy of a novel bacterial biomass (BB) in commercial‐type feed formulation for Pacific white shrimp, Litopenaeus vannamei. In trial 1, the basal diet was supplemented with 0, 60 and 120 g/kg BB to replace soybean meal (SBM). Significant improvement was observed in the survival when BB was incorporated in the diets. However, shrimp fed diets containing 120 g/kg BB exhibited significantly lower weight gain (WG) and higher feed conversion ratio (FCR). To confirm the results from trial 1 and explore the effects of BB supplementation at low levels, the basal diet was incorporated with 0, 10, 20, 40, 60 and 120 g/kg BB to replace SBM in trial 2. Significant reductions in WG, FCR, lipid content of whole body, protein retention efficiency and most amino acids retention efficiency were detected in shrimp fed with diet containing 120 g/kg BB. Trial 3 was designed to elucidate whether the digestible protein is the cause of reduced growth. No improvements in terms of growth performance and FCR were detected in the treatments balanced for digestible protein. Apparent digestibility coefficients of energy, protein and amino acid (AA) for BB were determined using chromic oxide as an inert marker and the 70:30 replacement technique. The energy, protein and individual amino acid digestibility coefficients of BB were significantly lower than those of fish meal (FM) and SBM that were given at the same time. Results of this study indicated that BB can be utilized up to 40 g/kg in shrimp feed without causing a decrease in growth. However, supplementations (≥60 g/kg) of BB can result in negative effects on growth response, FCR and protein as well as amino acids retention efficiency. At the lower levels of inclusion, shrimp performance was improved when BB was supplemented on a digestibility basis; however, at the higher level of inclusion, there was no improvement, indicating there may be other nutrients limiting. Based on enhanced survival in the treatment with BB supplementation in trial 1, further research regarding the immune effects of BB in practical shrimp feed will be necessary.     

一株凡纳滨对虾病原菌的分离、鉴定及其致病力分析

从患病凡纳滨对虾肝胰腺中分离出菌株20100612001,经人工感染实验证实,该分离菌株对凡纳滨对虾的半数致死量为1.44×106CFU/ml。形态学观察和革兰氏染色表明,该菌株为革兰氏阴性,无芽孢,有一根端极鞭毛;呈球杆状、球状、棒状或梨状且有两极浓染现象;在2216E培养基上为透明或半透明的圆形菌落,而在TCBS选择性培养基上为绿色或蓝绿色菌落。经Biolog碳源利用反应、脂肪酸气相色谱分析得出,该菌株与副溶血弧菌、需钠弧菌等的生理生化特性最相似;16SrD-NA序列测定表明,该菌株与弧菌属中几株病原菌的同源性均达到98.9%以上。在分子进化树中该菌株与副溶血弧菌Vibrio parahaemolyticus的进化地位最接近。综合上述实验结果分析得出,该细菌分离物为副溶血弧菌。     

Improvement of growth performance,water quality and disease resistance against Vibrio harveyi of postlarval whiteleg shrimp (Litopenaeus vannamei) by administration of mixed microencapsulated Bacillus probiotics

The objective of this study was to investigate the effects of mixed Bacillus on growth, water quality and disease resistance against Vibrio harveyi in whiteleg shrimp (Litopenaeus vannamei). Postlarval shrimp (PL₃₀) were fed with (a) a basal diet (the control), (b) a diet containing mixed freeze‐dried Bacillus probiotics (FB) and (c) addition of mixed microencapsulated Bacillus probiotics (MB) in culture water. Addition of FB and MB probiotics improved (p < .05) growth, feed efficiency, survival and culture water quality (ammonia and nitrite) compared to the control group although there was no difference (p > .05) between the two treated groups. Bacillus numbers in gastrointestinal tracts and culture water of FB‐ and MB‐administrated shrimp were higher (p < .05) than in the control. After a 30‐day culture, shrimp were infected with V. harveyi and monitored for 10 days. A significant reduction (p < .05) in cumulative mortality was observed in FB‐ and MB‐supplemented shrimp (43.24% and 45.05%, respectively), compared to the control (63.06%). This finding demonstrated that administration of microencapsulated probiotics was as effective as freeze‐dried probiotics for improving growth, feed efficiency, survival, Bacillus in gastrointestinal tracts, water quality (ammonia and nitrite) and conferring disease resistance to V. harveyi.     

Effects of Lactobacillus pentosus on the growth performance,digestive enzyme and disease resistance of white shrimp,Litopenaeus vannamei (Boone, 1931)

The objective of this study was to evaluate the probiotic properties of lactic acid bacteria (LAB) strains isolated from digestive tract of white shrimp Litopenaeus vannamei. Eighteen LAB colonies were isolated and one bacterium was found capable of producing three extracellular enzymes (protease, cellulose and lipase) simultaneously and exhibited antagonistic activity against shrimp pathogens (Vibrio vulnificus, V. rotiferianus and V. campbellii). The putative probiotic strain AS13 was identified as Lactobacillus pentosus based on 16S rRNA sequencing. The L. vannamei were fed diet containing 0 (control), 10⁶, 10⁷ and 10⁸ CFU g^?1 bacterial cells of AS13 for 28 days. The results showed that supplementation of L. pentosus significantly improved the growth performance and feed utilization in the treated groups over the control. Similarly, digestive enzyme activities were elevated in the intestines of treated groups. Moreover, feeding of supplemented diets containing AS13 significantly reduced the mortality rate caused by pathogenic Vibrio species (V. vulnificus, V. rotiferianus and V. campbellii). Our results indicated L. pentosus AS13 addition at 10⁷ CFU g^?1 can effectively enhance the growth performance, feed utilization, digestive enzymes and disease resistance of L. vannamei in the laboratory condition.     

Effects of low salinity exposure on immunological,physiological and growth performance in Litopenaeus vannamei

Growth, immunological and physiological parameters of white shrimp Litopenaeus vannamei reared at different salinity levels (1, 10, 15, 25 and 35 g/L) at stocking density of 214 shrimp/m³ were examined at 1, 30 and 63 days. Results showed that the total haemocyte count (THC) of shrimp decreased with time at all salinity levels, indicating a potential reduction in the resistance of shrimp against pathogens, since a low value of THC indicates a perturbation of the immune system. Glucose and protein values observed in the haemolymph throughout the study indicate that shrimp adapted well to low salinities (1, 10 and 15 g/L). Although of those shrimp reared at 10 g/L only 83.3% survived, at this salinity, shrimp depicted a higher glucose concentration in haemolymph at the beginning and end of the study.     

Effects of dietary herbal formulae combined by Astragalus polysaccharides,chlorogenic acid and allicin in different combinations and proportions on growth performance,non‐specific immunity,antioxidant status,vibriosis resistance and damage indexes of Litopenaeus vannamei

In this study, a feeding trial followed by a challenge test was performed to evaluate effects of six herbal formulae which were different combinations of Astragalus polysaccharides (APS), chlorogenic acid (CGA) and allicin on growth performance, non‐specific immune response, antioxidant capacity, disease resistance and biomolecule damage of white shrimp Litopenaeus vannamei. Shrimp were fed seven diets, control diet (basal diet with no herbal formulae) and six herbal formula diets (G1–G6, basal diet supplemented with 0.1% APS + 0.05% allicin, 0.1% APS + 0.1% allicin, 0.1% CGA + 0.05% allicin, 0.1% CGA + 0. 1% allicin, 0.1% APS + 0.1% CGA and 0.1% APS + 0.1% CGA + 0.05% allicin respectively), for 21 days. After that, shrimp were challenged with Vibrio harveyi and then the cumulative mortality of shrimp was recorded for 7 days post challenge. The results showed that there were no significant differences in growth performance among all groups, while the non‐specific immune responses and antioxidant indexes were significantly improved (p < .05) in shrimp fed herbal formula diets when compared to the control. Meanwhile, the lowest cumulative mortality was observed in shrimp fed herbal formula diets with 0.1% APS, 0.1% CGA and 0.05% allicin supplementation after V. harveyi challenge. Additionally, herbal formulae could not cause biomolecule damage to the hepatopancreas of shrimp. In conclusion, these results indicated that synergistic effect of APS, CGA and allicin helped to boost immunity, antioxidant capacity and disease resistance of shrimp without biomolecule damage.     

The evaluation of practical diets on a basis of digestible crude protein,lysine and methionine for Litopenaeus vannamei

A feeding trial was conducted to evaluate the efficacy of replacing fish meal (FM) with blood meal (BM), poultry by‐product meal (PBM), meat and bone meal (MBM) and shrimp head meal (SHM), rapeseed meal (RM) and peanut meal (PM) on a digestible basis of crude protein and lysine and methionine in five practical diets for the Pacific white shrimp at the FM levels of 300, 250, 200, 150 and 100 g kg^?1 under laboratory conditions. Each of the five experimental diets was hand‐fed to four replicate tanks of shrimp with an average weight of 0.33 ± 0.03 g to satiation at each meal. The shrimp were fed three times a day over a six‐week period. The per cent weight gain of initial body weight (WG%) was significantly lower in shrimp fed 100 g kg^?1 FM diet, but the value for hepatosomatic index (HSI) and the level of blood urea nitrogen (BUN) tended to be higher in shrimp fed 100 g kg^?1 FM diet than those in shrimp fed other diets. The lowest value for feeding rate (FR) occurred for shrimp fed the basal diet and was significantly lower than that in shrimp fed the FM diets at 100–150 g kg^?1. Shrimp fed diets containing 200 g kg^?1 or lower FM had significantly lower feed utilization than those fed the 250 g kg^?1 FM diet and the basal diet. The protein efficiency ratio (PER) in the shrimp fed the basal diet was significantly higher than in the other FM diets. Decreasing the FM replacement level significantly reduced nutrient digestibility except in the cases of ash and gross energy, but it did not affect the survival, condition factor (CF), body composition, digestive enzyme activity or plasma transaminase activity. The results of the study indicate that feeding a diet formulated on a digestible basis and involving FM replacement with other protein sources at a greater replacement proportion will not produce a level of shrimp growth equal to that achieved by feeding the basal diet.     

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.     

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.     

Bacterial population in intestines of white shrimp,Litopenaeus vannamei fed a synbiotic containing Lactobacillus plantarum and galactooligosaccharide

The aims of this study were to unravel the intestinal microbiota of Litopenaeus vannamei after being fed a diet without (control) or with the synbiotic (SYN) for 60 days using next‐generation sequencing technology to see if changes in the intestinal microbiota were involved in the improved growth performance and health status of the shrimp. Next‐generation sequencing data showed that six phyla, 11 classes, 19 orders, 30 families, 58 genera and 73 species with taxonomic names assigned were detected. The majority of the operational taxonomic units (OTUs) was shared between the SYN and control shrimp and comprised 37 OTUs. However, intestinal biodiversity analyses revealed that SYN‐fed shrimp had a higher species richness, evenness and Shannon–Weaver index than did shrimp fed the control diet, but without reaching statistical significance. Interestingly, shrimp fed the SYN diet exhibited improved colonization of Lactobacillus plantarum and reduced prevalences of Vibrio harveyi and Photobacterium damselae in the intestines. These findings indicate that the SYN was able to modulate the intestinal bacterial community of shrimp and could be used to control vibriosis in shrimp.     

Proteome modifications of Pacific white shrimp (Litopenaeus vannamei) muscle under biofloc system

The objective of the study was to examine the effects of biofloc technology on the muscle proteome of Litopenaeus vannamei. Two biofloc treatments and one control were compared: biofloc‐based tanks under zero‐water exchange fed with 150 g/kg crude protein (BF15), or with 250 g/kg crude protein (BF25) diets, and clear water tanks with 50% of daily water exchange stocked with shrimp fed with similar amount of a 250 g/kg crude protein diet, referred to as control. The shrimp (5.28 ± 0.42 g) were divided into the 300‐L fibreglass tanks (water volume of 200 L) at a density of 35 shrimp per tank and were cultured for 35 days. The biofloc groups displayed better growth and survival compared to the control. The muscle tissue from the control and BF25 groups was subjected to proteomic analysis. Lactate dehydrogenase, enolase, arginine kinase, mitochondrial ATP synthase subunit alpha, mitochondrial ATPase inhibitor factor 1 precursor, serpin 3 and myeloid differentiation factor 88 had an increased abundance in the BF25 group, while myosin heavy chain type 1 and myosin heavy chain type 2 showed a decreased abundance. The results indicate that biofloc technology could alter the expression of proteins involved in structure, metabolism and immune status of cultured shrimp.     

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.