Effect of starvation and refeeding on the hepatopancreas of whiteleg shrimp Penaeus vannamei (Boone) using computer‐assisted image analysis

Under normal farming conditions, shrimp can experience starvation periods attributable to disease outbreaks or adverse environmental conditions. Starvation leads to significant morphological changes in the hepatopancreas (HP), being the main organ for absorption and storage of nutrients. In the literature, limited research has described the effect on the HP of periods of starvation followed by refeeding and none in whiteleg shrimp (Penaeus vannamei) using computer‐assisted image analysis (CAIA). This study describes the effect of starvation and starvation followed by refeeding on the HP of whiteleg shrimp using CAIA. Visiopharm^® software was used to quantify the following morphological parameters, measured as ratio to the total tissue area (TLA): total lumen area (TLA:TTA), haemocytic infiltration area in the intertubular spaces (HIA:TTA), B‐cell vacuole area (VBA:TTA), lipid droplet area within R cells (LDA:TTA) and F‐cell area (FCA:TTA). Significant changes were measured for HIA:TTA and LDA:TTA during starvation (increase in HIA:TTA associated with decrease in LDA:TTA) and starvation followed by refeeding (decrease in HIA:TTA associated with increase in LDA:TTA). In the future, HIA:TTA and LDA:TTA have the potential to be used in a pre‐emptive manner to monitor the health of the HP, facilitate early diagnosis of diseases and study the pathophysiology of the organ.     

Optimization of fixation methods for image analysis of the hepatopancreas in whiteleg shrimp,Penaeus vannamei (Boone)

Pathology in penaeid shrimps relies on histology, which is subjective, time‐consuming and difficult to grade in a reproducible manner. Automated image analysis is faster, objective and suitable for routine screening; however, it requires standardized protocols. The first critical step is proper fixation of the target tissue. Bell & Lightner's (A Handbook of Normal Penaeid Shrimp Histology, 1988, The World Aquaculture Society, Baton Rouge) fixation protocol, widely used for routine histology of paraffin sections, is not optimized for image analysis, and no protocol for frozen sections is described in the available literature. Therefore, the aim of this study was to optimize fixation of the hepatopancreas (HP) from whiteleg shrimp (Penaeus vannamei) for both paraffin and frozen sections using a semiquantitative scoring system. For paraffin sections, four injection volumes and three injection methods were compared, for frozen sections, four freezing methods and four fixation methods. For paraffin sections, optimal fixation was achieved by increasing threefold the fixative volume recommended by Bell and Lightner, from 10% to 30% of the shrimp body weight, combined with single injection into the HP. Optimal fixation for frozen sections was achieved by freezing the cephalothorax with liquid nitrogen, followed by fixation of the section with 60% isopropanol. These optimized methods enable the future use of image analysis and improve classical histology.     

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.     

Induction of spawning in Pacific white shrimp Litopenaeus vannamei (Boone, 1931) by injection of its molt inhibiting hormone isoform II produced in E. coli

The optimization of reproductive parameters in shrimp farming continues to be a challenge for most producing countries. Although the crustacean neuropeptides have been studied extensively in the last two decades, the functions of most of these neuropeptides remained putative. Among them, molt‐inhibiting hormone isoform II (MIH II) has shown an important role in vitellogenesis. In this study, the cDNA encoding mature MIH II peptide was isolated by RT‐PCR from the L. vannamei eyestalk. The cDNA was cloned into pET28a bacterial expression vector. Recombinant MIH II was obtained in the form of insoluble inclusion bodies and purified to ~88% purity. Two doses of rMIH II and a negative control group were assayed in vivo. The stages of ovarian maturation and spawning were recorded during 72 hr post‐injection. The results showed that ovarian maturation occurred approximately in 9% and 33% of females injected with rMIH II at the doses of 300 and 600 ng/gbw respectively. Neither maturation nor spawning was detected in the negative control group. Females injected with 600 ng/gbw, which showed vitellogenic stages III and IV, spawned. These preliminary results argue that the hormone rMIH II could be a promising candidate to induce spawning in L. vannamei shrimp.     

Aspergillus niger fermented plant protein mix as a potential substitute for fishmeal in the diet of Penaeus vannamei (Boone, 1931)

A 45‐day feeding trial was conducted to evaluate the utilization of fermented plant protein mix (FPPM) as a fishmeal substitute in the diet of Penaeus vannamei. Six isonitrogenous and isolipidic diets were formulated by substituting fishmeal at 0% (control), 50%, 60%, 70%, 80% and 90% (FR‐0, FR‐50, FR‐60, FR‐70, FR‐80 and FR‐90, respectively) using FPPM. Shrimps fed with diets FR‐50, FR‐60 and FR‐70 had no significant difference in weight gain (173.22%–178.07%) with the control group (179.50%). However, the broken‐line regression indicated that 70.8% fishmeal could be substituted by FPPM. The trend of digestibility was reflected in growth performance. Survival had no significant difference among the treatments. Fishmeal substitution had significant (p < .05) variations in protease and amylase activity, but not in lipase. Except lipid, other body composition was not affected due to dietary change. A decrease (p < .05) in haemolymph total protein, cholesterol and triglycerides was observed to increase in fishmeal substitution. A higher fishmeal substitution (FR‐90) had significantly (p < .05) lower total haemocyte count and the reverse was true for phenoloxidase. Results conclude that a combination of fermented ingredients would be more effective rather using alone in shrimp feed, with a great potential in reducing the pressure on fishmeal.     

Studies on the occurrence of infectious myonecrosis virus in pond‐reared Litopenaeus vannamei (Boone, 1931) in India

Whiteleg shrimp, Litopenaeus vannamei, with clinical sign of muscle opaqueness with reddish colour at the distal abdominal segments were observed in farms located in West Bengal State, India. The mortality of shrimp in all disease outbreak ponds ranged from 20% to 50%, and mortality increased gradually. The RT‐PCR assay of these samples using primer sets specific to infectious myonecrosis virus (IMNV) revealed its presence in the disease outbreak ponds. The IMNV infection was reproduced in healthy shrimp by intramuscular injection to satisfy River's postulates. The virus caused mortality in intramuscularly challenged shrimp, but failed to cause mortality by oral route. Tissue distribution of IMNV in infected shrimp by RT‐PCR assay revealed the presence of this virus in haemolymph, gill, hepatopancreas and muscle. This study confirms that the disease outbreak which occurred in the shrimp farms located at Purba Medinipur District, West Bengal, India, was due to IMNV.     

Artificial substrates in zero‐water‐exchange culture system regulate the rearing performance of Pacific white shrimp Litopenaeus vannamei (Boone, 1931) under the winter indoor condition

Effects of artificial substrates in zero‐water‐exchange culture system on the rearing performance of Litopenaeus vannamei under winter indoor condition were investigated in this study. Growth, survival, feed conversion rate (FCR), production rate of L. vannamei and water quality were compared between artificial substrate‐treated group (AST) and control group (without artificial substrates presented in the rearing environment). Artificial substrates can significantly improve the water quality, the ammonia and nitrite‐N concentrations in the AST group were significantly lower than in the control group (P < 0.05), and the total heterotrophic bacteria and Vibrio spp. were also significantly lower in the AST group (P < 0.05). The survival, growth and production rate of L. vannamei in the AST group were significantly higher than in the control group (P < 0.05). Significantly lower FCR was observed in the AST group (P < 0.05). Results from this study indicate that the utilization of artificial substrates in the indoor shrimp culture system could effectively control the water quality, improve the survival and growth of shrimp and significantly reduce the FCR. This study provides a guideline for employing artificial substrates in rearing of shrimp in the zero‐water‐exchange culture system under lower temperature, which could be applicable to other similar species.     

The induction of Hsp70 synthesis by non‐lethal heat shock confers thermotolerance and resistance to lethal ammonia stress in the common carp,Cyprinus carpio (Linn)

Exposure to heat‐shock protein (Hsp) stimulating factors induces Hsp accumulation and confers tolerance to lethal ammonia stress on the common carp Cyprinus carpio. This study investigated whether a non‐lethal heat shock bestowed similar protective effects against ammonia and induced thermotolerance, both thought to be rendered by increased amounts of Hsps. The 30‐min lethal temperature (30 min LHT) and 1‐h lethal ammonia concentration (1 h LCT) for this species occurred at 41°C and 14.2 mg/L NH₃ respectively. Heating juvenile carp (5 cm) from 28°C to 32, 34 and 38°C, with a subsequent 8‐h recovery period augmented tolerance to lethal heat and ammonia perturbation by two to threefold as compared with animals held at 28°C. Protection occurred in conjunction with Hsp70 accumulation in gills, substantiating the role of this Hsp in enhancing the stress tolerance of common carp.     

The effects of dietary lipid levels on performance and heat‐shock protein response of juvenile white seabass,Atractoscion nobilis

A feeding trial was conducted to study the effect of dietary lipid on growth performance and heat‐shock protein (HSP70 and HSP60) response of white seabass (WSB), Atractoscion nobilis. Five diets were formulated to contain 440 g kg^?1 protein from 300 g kg^?1 fish meal, 240 g kg^?1 soybean meal and 100 g kg^?1 soy protein concentrate with different levels of lipid: 100, 120, 140, 160 or 180 g kg^?1. At the end of the trial, heat shock response based on HSP70 and HSP60 was measured in liver and white muscle from fish at ambient temperature and temperature shock conditions. Final weight and percent gain were significantly higher for fish fed the 100 g kg^?1 lipid diet than for fish fed the rest of the diets (P ≤ 0.05). Feed conversion ratio was lowest for fish fed the 100 g kg^?1 lipid diet. The HSP70 and HSP60 responses were positively correlated to dietary lipid levels following temperature shock. At ambient temperature, HSP60 and HSP70 responses in muscle and HSP60 response in liver increased with dietary lipid level. Temperature shock significantly increased the HSP response of fish in all treatments. Results of this study demonstrated that a moderate (110–120 g kg^?1) level of dietary lipids would be recommended for production diets but a higher dietary lipid level may be required for optimal stress tolerance.     

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.     

Enhanced immune responses and effectiveness of refined outer membrane protein vaccines against Vibrio harveyi in orange‐spotted grouper (Epinephelus coioides)

Vibriosis is a severe infection occurring in many commercially important marine fish species. In this study, vaccines containing Vibrio harveyi recombinant outer membrane protein K (rOmpK), outer membrane protein U (rOmpU) and rOmpK‐OmpU fusion protein in addition to the metabolizable Montanide^TM ISA 763 A VG adjuvant were developed and evaluated in the orange‐spotted grouper. The results indicate that recombinant V. harveyi protein‐based vaccines resulted in a remarkably higher expression of IL‐1β and IL‐8 at 24 hr, and greater antibody production, as early as 2 weeks postimmunization. Notably, enhanced immune responses and significant protective efficacy against V. harveyi infections were observed in the fusion protein vaccine‐injected fishes with relative per cent survival value of 81.8%. Additionally, the rOmpK‐OmpU antisera presented a high bactericidal effect on not only V. harveyi, but also Vibrio parahaermolyticus and Vibrio alginolyticus. Our results demonstrated that the fusion protein rOmpK‐OmpU was an effective vaccine candidate that exhibited potentially great versatility for controlling vibrio infections.     

A chimeric recombinant crustacean hyperglycemic hormone from Litopenaeus schmitti (Burkenroad) obtained as C‐terminus fusion protein boost hemolymph glucose concentration in Litopenaeus vannamei (Boone)

To date, no hormonal treatments are available for control of shrimp reproduction and only eyestalk ablation is of practical use. The crustacean hyperglycemic hormone (CHH) is the most abundant neuropeptide of the eyestalk CHH family. It plays an important role in the regulation of hemolymph glucose levels, as its principal function, but it is also implicated in additional physiological processes such as moulting and reproduction. In the present study, the cDNA encoding Litopenaeus schmitti (Burkenroad) mature CHH was cloned into the Escherichia coli pTYB2 expression vector. Using this strategy we have obtained, for the first time, the recombinant CHH from L. schmitti with its C‐terminus fused to an intein tag. The expected fused protein of about 63 KDa was expressed in E. coli forming inclusion bodies. It was purified in a soluble form by electroelution following molecular size fractionation in sodium dodecil sulphate polyacrylamide gel electrophoresis. The ability of the chimeric CHH protein to elevate glucose levels in the hemolymph of the eyestalk‐ablated Litopenaeus vannamei (Boone) shrimps indicates that its biological activity as hyperglycemic protein is preserved. The results provide an alternative tool to obtain soluble recombinant proteins from the CHH family of neuropeptides to get a better understanding of shrimp endocrinology.     

Impact of a reduced water salinity on the composition of Vibrio spp. in recirculating aquaculture systems for Pacific white shrimp (Litopenaeus vannamei) and its possible risks for shrimp health and food safety

Tropical shrimp, like Litopenaeus vannamei, in land‐based recirculating aquaculture systems (RAS) are often kept at low water salinities to reduce costs for artificial sea salt and the amount of salty wastewater. Although these shrimp are tolerant against low salinities, innate immunity suppression and changes in the microbial composition in the water can occur. As especially Vibrio spp. are relevant for shrimp health, alterations in the species composition of the Vibrio community were analysed in water from six RAS, run at 15‰ or 30‰. Additionally, pathogenicity factors including pirA/B, VPI, toxR, toxS, vhh, vfh, tdh, trh, flagellin genes and T6SS1/2 of V. parahaemolyticus were analysed. The Vibrio composition differed significantly depending on water salinity. In RAS at 15‰, higher numbers of the potentially pathogenic species V. parahaemolyticus, V. owensii and V. campbellii were detected, and especially in V. parahaemolyticus, various pathogenicity factors were present. A reduced salinity may therefore pose a higher risk of disease outbreaks in shrimp RAS. Because some of the detected pathogenicity factors are relevant for human health, this might also affect food safety. In order to produce healthy shrimp as a safe food for human consumption, maintaining high water salinities seems to be recommendable.     

Growth performance,haematological parameters,antioxidant status and salinity stress tolerance of juvenile Pacific white shrimp (Litopenaeus vannamei) fed different levels of dietary myo‐inositol

A 10‐week growth trial was run to evaluate effects of myo‐inositol (MI) on growth performance, haematological parameters, antioxidative capacity and salinity stress tolerance of Litopenaeus vannamei. Six practical diets supplemented with graded levels of MI (designated as MI0, MI600, MI1200, MI2400, MI 3600 and MI4800 for 448.8, 974.2, 1568.0, 2810.6, 3835.5 and 4893.6 mg/kg diet, respectively) were fed to six replicate groups of L. vannamei (mean initial body weight 0.63 ± 0.00 g). The results showed that significant increment of growth performance was observed in shrimp fed MI600 diet than those fed MI1200 diet. Lipid concentration in whole body of the shrimp fed MI600 diet was significantly increased. Shrimp fed MI0 diet had lower total protein (TP) as compared to shrimp fed the MI‐supplemented diets (except MI4800 diet). In general, lower activities of antioxidant enzymes and higher malondialdehyde (MDA) content in haemolymph and hepatopancreas were recorded in shrimp fed MI0 diet, compared to those fed the MI‐supplemented diets. Reduced survival after 7‐h salinity stress was present in shrimp fed MI0 diet as compared to those fed MI4800 diet. Dietary MI requirement for glutathione peroxidase activity of L. vannamei was 2705 mg/kg diet.     

Hypoxia‐induced changes in survival,immune response and antioxidant status of the Pacific white shrimp (Litopenaeus vannamei) fed with graded levels of dietary myo‐inositol

A 3‐hr experiment was conducted to investigate the effects of dietary myo‐inositol (MI) supplementation on survival, immune response and antioxidant abilities in Litopenaeus vannamei under acute hypoxia stress. Six practical diets were formulated with supplementation of graded levels (control group 0, 0.1, 0.2, 0.4, 0.8 and 1.6 g/kg dry diet) of MI and were randomly assigned to triplicate groups of L. vannamei (mean weight 0.40 ± 0.00 g) for 8 weeks. Ten healthy shrimp (final mean weight approximately 11–14 g) randomly selected from each tank were exposed to hypoxia stress after feeding trial. After 3‐hr acute hypoxia stress, survival of shrimp fed MI‐supplemented diets (except 0.1 and 0.4 g/kg diets) was significantly increased compared with the control group. Shrimp fed control diet had lower activities of alkaline phosphatase (AKP), acid phosphatase (ACP), total antioxidant capacity (T‐AOC) and glutathione peroxidase (GPX), and higher malondialdehyde (MDA) and protein carbonyl (PC) contents in hepatopancreas than those fed the MI‐supplemented diets. In addition, mRNA expression levels of heat shock protein 70 (Hsp70), catalase (CAT) and penaeidin were significantly differentially regulated in hepatopancreas. In summary, dietary MI supplementation may have a positive effect on improving resistance to acute hypoxia stress of L. vannamei.     

Effect of acclimation temperature on thermoregulatory behaviour,thermal tolerance and respiratory metabolism of Lutjanus guttatus and the response of heat shock protein 70 (Hsp70) and lactate dehydrogenase (Ldh‐a) genes

This study evaluated the effect of different environmental temperatures in the physiology of Lutjanus guttatus juveniles by analysing their thermoregulatory behaviour, thermal tolerance, oxygen consumption rates and thermal metabolic scope. Jointly, the effect of acclimation and critical temperatures on heat shock protein 70 (Hsp70) and lactate dehydrogenase (Ldh‐a) gene expressions were also analysed using acclimation temperatures of 20, 23, 26, 29 and 32°C. The results showed that the final preferred temperature in juvenile snapper was 26°C with a thermal window of 336.5°C², which was related to an optimal temperature for their physiology determined by the routine metabolic rate and thermal metabolic scope. At temperatures from 20 to 26°C, the routine metabolic rate and Hsp70 and Ldh‐a genes had the lowest values related to a basal expression level. At acclimation temperatures from 29 to 32°C and after critical thermal maximum (CT_max) limit, the relative expression of Hsp70 and Ldh‐a genes increased significantly, but the main response at CT_max was the upregulation of Hsp70 gene.     

Rainbow trout Oncorhynchus mykiss (Walbaum) type IV ice‐structuring protein LS‐12 in the acute‐phase response to Flavobacterium psychrophilum infection

A previous proteomic study examining the plasma acute‐phase response of rainbow trout to sterile inflammation highlighted an unidentified 9.5‐kDa spot using 2D‐PAGE, which was dramatically increased. The 15 amino acid sequence obtained from this protein spot allowed rapid amplification of cDNA ends PCR to generate a 443‐bp nucleotide sequence that was 98.6% similar to type‐4 ice‐structuring protein LS‐12 from Atlantic salmon Salmo salar Linnaeus. Quantitative reverse translation PCR and an ELISA were used to measure gene expression and plasma concentrations of LS‐12 following experimental intraperitoneal injection of rainbow trout with either 10⁶ or 10⁸ colony‐forming units (CFU) of Flavobacterium psychrophilum. There was no significant change in the plasma concentration of LS‐12 up to 15 days post‐infection in any group. Hepatic LS‐12 gene expression was significantly reduced at 3 and 6 days (p < 0.001) post‐infection in fish injected with 10⁸ CFU of F. psychrophilum relative to control fish, while branchial or head kidney expression was unchanged. Infected fish had significantly increased hepatic gene expression of serum amyloid A, confirming an acute‐phase response. Under the conditions used, LS‐12 is not a positive acute‐phase protein in rainbow trout.     

Effects of continuous and alternate administration of β‐glucan and mannan‐oligosaccharide on the growth,immunity and resistance against Vibrio splendidus of sea cucumber Apostichopus japonicus (Selenka)

A 4‐week growth trial was conducted to compare the effects of different feeding strategies of dietary immunostimulants on the growth, immunity and resistance against Vibrio splendidus of sea cucumbers Apostichopus japonicus (Selenka). Six feeding strategies were set, including feeding immunostimulants‐free diet continuously (control), feeding dietary β‐glucan (1.25 g kg^?1 diet) continuously, feeding dietary mannan‐oligosaccharides (MOS; 2.00 g kg^?1 diet) continuously, feeding β‐glucan 2 days followed by MOS 5 days alternately, feeding β‐glucan 5 days followed by MOS 2 days alternately and feeding β‐glucan 7 days followed by MOS 7 days alternately. The sea cucumbers fed immunostimulants showed higher specific growth rate (SGR) and lower cumulative mortality than control (P < 0.05). When sea cucumbers were fed with β‐glucan continuously, total coelomocytes counts and superoxide anion were significantly higher than control on the 4th day (P < 0.05). However, these two immune parameters were not significantly higher than those in control after the 18th day (P > 0.05). While sea cucumbers continuously fed MOS, these two immune parameters were not significantly higher than control until the 15th day. All immune parameters of the sea cucumbers fed with β‐glucan and MOS alternately were significantly higher than those in control during the experiment (P < 0.05). The sea cucumbers fed with β‐glucan 7 days followed MOS 7 days alternately showed the highest SGR and second lowest cumulative mortality. It was suggested that this feeding strategy is most suitable for sea cucumbers.