Genetic variation of white spot syndrome virus (WSSV) in Pacific white shrimp Litopenaeus vannamei (Boone 1931) culture of Thailand

White spot syndrome virus (WSSV) is highly pathogenic to penaeid shrimp and has caused significant economic losses in the shrimp farming industry in Thailand. Genotyping analysis was done in 124 WSSV isolates from cultured Pacific white shrimp Litopenaeus vannamei. These samples were obtained during 2007–2014 from eight provinces in Thailand. We investigated five variable loci in the virus genome: deletions in two variable regions, VR14/15 and VR23/24, and three variable number tandem repeats (VNTR) located in open reading frame (ORF) 75, 125 and 94. WSSV genotype was characterized as (X_14/15, X_23/24) (N₇₅‐N₁₂₅‐N₉₄) where X is the number of base pair deletion in the variable region and N is the number of repeat units (RUs) in a specific ORF. The deletion pattern in VR14/15 and VR23/24 regions characterized three WSSV genotypes. The most prevalent genotype was (5950_14/15, 10971_23/24), and it was found in all studied areas. At least 33 genotypes of WSSV were analysed based on 3 VNTR loci, indicating that the VNTRs of WSSV genome are highly variable. From 124 WSSV samples, two samples presented the characteristic of all five variable loci similar to WSSV collected during 2010 in Saudi Arabia (5950_14/15, 10971_23/24) (3₇₅‐6₁₂₅‐7₉₄). Many different WSSV genotypes shown in this study as compared to previously reported genotypes in Thailand suggests current status of disease epidemiology, as well as probable movements of WSSV between countries.     

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.     

White spot syndrome virus epizootic in cultured Pacific white shrimp Litopenaeus vannamei (Boone) in Taiwan

White spot syndrome virus (WSSV) has caused significant losses in shrimp farms worldwide. Between 2004 and 2006, Pacific white shrimp Litopenaeus vannamei (Boone) were collected from 220 farms in Taiwan to determine the prevalence and impact of WSSV infection on the shrimp farm industry. Polymerase chain reaction (PCR) analysis detected WSSV in shrimp from 26% of farms. Juvenile shrimp farms had the highest infection levels (38%; 19/50 farms) and brooder shrimp farms had the lowest (5%; one of 20 farms). The average extent of infection at each farm was as follows for WSSV‐positive farms: post‐larvae farms, 71%; juvenile farms, 61%; subadult farms, 62%; adult farms, 49%; and brooder farms, 40%. Characteristic white spots, hypertrophied nuclei and basophilic viral inclusion bodies were found in the epithelia of gills and tail fans, appendages, cephalothorax and hepatopancreas, and virions of WSSV were observed. Of shrimp that had WSSV lesions, 100% had lesions on the cephalothorax, 96% in gills and tail fans, 91% on appendages and 17% in the hepatopancreas. WSSV was also detected in copepoda and crustaceans from the shrimp farms. Sequence comparison using the pms146 gene fragment of WSSV showed that isolates from the farms had 99.7–100% nucleotide sequence identity with four strains in the GenBank database – China ( AF332093 ), Taiwan ( AF440570 and U50923 ) and Thailand ( AF369029 ). This is the first broad study of WSSV infection in L. vannamei in Taiwan.     

Cage culture of the Pacific white shrimp Litopenaeus vannamei (Boone, 1931) at different stocking densities in a shallow eutrophic lake

Postlarvae of Litopenaeus vannamei were acclimated and stocked in lake-based cages at the following stocking densities: 10, 20, 30 and 40 shrimp m⁻². Another set of shrimp was stocked in concrete tanks as reference samples at 30 shrimp m⁻². Significant differences were observed among stocking densities throughout the 95-day culture. The final weight at harvest decreased with increasing stocking density: mean weights of 23.3, 15.8, 13.0, 10.9 and 14.6 g for the 10, 20, 30, 40 shrimp m⁻² and reference tanks were observed respectively. There were no significant differences in survival throughout the culture period, ranging between 69% and 77%. Daily growth rates (range: 0.11–0.24 g day⁻¹) and specific growth rates (range: 3.54–4.34%) also differed significantly among stocking densities, both increasing with decreasing stocking density. The feed conversion ratio in the cages did not differ among the stocking densities, ranging from 1.53 to 1.65. The relationship between stocking density and mean individual weight at harvest followed the equation y =81.06 x ^−0.54 ( R ²=0.938) and that of stocking density and production (in g m⁻²) is y =58.01 x ^−0.46 ( R ²=0.834).     

Genetics of the resistance to hypoxia in postlarvae and juveniles of the Pacific white shrimp Penaeus (Litopenaeus) vannamei (Boone 1931)

The genetics of resistance to hypoxia was studied in shrimp using two approaches, heritability estimation and heterosis estimation. The heritability of resistance to hypoxia was evaluated in two populations of shrimp postlarvae families produced at different times, and heterosis was estimated through the reciprocal crosses of two genetically distinct shrimp populations at postlarvae and juvenile. Heritabilities of resistance to hypoxia in postlarvae were large and out of the range for the heritability (1.73±0.09, 1.35±0.14) regardless of population evaluated. When covariates (survival and mean total weight to postlarvae 15 days old) were introduced in the analyses to correct for effects during rearing of the larvae, the heritabilities did not change substantially for the first population, but were decreased for the second population depending on the covariate used for correction (1.15±0.10 and 1.08±0.11 for the covariates survival and total weight respectively). Those large heritabilities could be explained by the presence of confounded common environmental variance in the estimated genetic variance (maternal effects), or by dominance and epistatic effects being an important part of the estimated genetic variance. Heterosis analyses dissecting maternal effect indicated that both effects, maternal and dominance, are involved in the resistance to hypoxia at postlarvae, and that although maternal effects decreased at juvenile stage, the trait was still characterized by presenting a large heterosis value (62–65% for postlarvae and 36% for juveniles).     

Selectivity of particle size by the shrimp Litopenaeus vannamei (Boone, 1931) larvae

Because of the filter‐feeding behavior of shrimp larvae, it is important to define precisely the size of the particle ingested in the different stages until postlarval stage where raptorial habits are more evident than the filter‐feeding lifestyle. Selectivity assays were conducted by using Polystyrene DVB particles with diameter between 1 and 50 μm as food. A group of organisms from each stage were put into the particle suspension for 15 min to let the polystyrene particles be ingested. The particle distribution in the media and the content of the gut of the larvae were characterized with digital image processing analysis. The results were compared using Ivlev selectivity formula, which compares the frequency distribution of each size of the particle in the media and in the gut of larvae. The results of selectivity were adjusted with a third‐order polynomial regression to determine the optimum and preferred size of the food particles for each larval stage between Zoea I and Postlarva I. It is concluded that the different larval stages of Litopenaeus vannamei may be considered as a single group of larvae who ingest foods with size between 5.71 and 20.33 μm. The optimal size of the food ingested was 14.42 μm wide.     

Fish meal replacement with rice protein concentrate in a practical diet for the Pacific white shrimp, Litopenaeus vannamei Boone, 1931

Replacement of fish meal (FM) with rice protein concentrate (RPC) as a practical diet for the Pacific white shrimp, Litopenaeus vannamei, was evaluated. Five isonitrogenous (36.6% protein) diets, formulated by replacing 0, 25, 50, 75, and 100% of FM by RPC, were fed to shrimp (initial weight of 6.99 ± 0.08 g) five times daily to satiation for 60 days. Relatively high final weight (FW 17.64–18.25 g) and weight gain (WG 10.81–11.39 g) were obtained in treatments up to 50% of the plant protein inclusion. Above this inclusion level, FW (14.93–14.35 g) and WG (7.68–7.23 g) were reduced. Survival was high (≥95%) and similar for all diets. There were no significant differences (P > 0.05) in tail-muscle composition (moisture, protein, lipid, and ash) among different dietary treatments. Dispensable and indispensable amino acids of the tail muscle of shrimp fed with 25, 50, and 75% RPC were significantly higher than the FM (0%) and 100% RPC diets. A decreasing trend in apparent digestibility coefficient (excluding dry matter) for crude protein (90.52–52.41), ether extract (94.11–80.03), organic matter (87.25–50.16), and gross energy (89.41–55.24) was observed at higher RPC inclusion rates. The results suggest that RPC meal can be a potential candidate for FM replacement up to 50% of the protein in shrimp diets.     

Antioxidant effects of nanoencapsulated lipoic acid in tissues and on the immune condition in haemolymph of Pacific white shrimp Litopenaeus vannamei (Boone, 1931)

The antioxidant and oxidative damage responses and immune parameters in shrimp, Litopenaeus vannamei, reared in clear water (CW) and in a biofloc system (BFT) were evaluated. The animals were given feed supplemented with either nanocapsules containing the antioxidant lipoic acid (NCLA) or empty nanocapsules (NC). Antioxidant and oxidative stress analyses were evaluated by measuring glutathione S‐transferase activity (GST), reduced glutathione (GSH) concentration and lipid peroxidation (TBARS) in the gills, hepatopancreas and muscle. The immune parameters were then determined by the differential hemocyte count in the haemolymph. The final weight of the shrimp reared in both CW and BFT and treated with NCLA increased (p < .05). The shrimps reared in BFT showed increased GST activity in the gills and hepatopancreas after NCLA treatment (p < .05). Higher GSH levels were observed in the hepatopancreas with NCLA supplementation in CW and BFT (p < .05). Decreased levels of TBARS were observed in the gills and muscles of shrimp supplemented with NCLA and maintained in CW (p < .05). A decrease in the percentage of hyaline hemocytes and an increase in granular hemocytes were observed in shrimp treated with NCLA in CW and BFT (p < .05). The antioxidant treatment induced positive responses in terms of the zootechnical, biochemical and immunological parameters.     

Genetic differentiation in hatchery strains and wild white shrimp Penaeus (Litopenaeus) vannamei (Boone, 1931) from northwest Mexico

Ten enzymatic systems were analyzed to determine allozyme genetic differentiation among three hatchery strains (A, B and C) of white shrimp, Penaeus (Litopenaeus) vannamei, commonly used in shrimp farming in northwest Mexico. A wild population from northern Sinaloa was used as a reference. Fifteen loci were detected, nine of which were polymorphic (ACP-1*, ACP-2*, AKP-2*, EST-2*, EST-3*, EST-4*, EST-5*, LAP*, and LDH*). Polymorphism of A, B and C were 53, 53, and 40%. The mean observed heterozygosity per locus was 0.071, 0.093, and 0.050 without any significant difference among them or with respect to the wild population (0.056). In all samples observed heteroxygosity was smaller than expected. The mean number of alleles per locus was 1.8 in A and 1.87 in both B and C. Strain A was the only sample without rare alleles. Only EST-3* and LAP* of strain A were in Hardy-Weinberg equilibrium; the other loci were in disequilibrium in all samples. High inbreeding values and heterozygote deficiencies were detected in all samples. Distribution of allelic frequencies was heterogeneous among the samples (G-test), involving all polymorphic loci and suggesting a genetic differentiation. According to Fst, a moderate genetic differentiation (7.4%) was detected among the samples. Greater differences were between strains A and C. Based on genetic distance, the samples were grouped into two pairs, B–C and A-wild. Strain A is a young strain related to the wild sample, whereas strains B and C have a different geographic origin than the wild sample.     

低盐度条件下的凡纳滨对虾幼虾亮氨酸营养需求

为了探讨低盐度(0.6～0.8 g/L)条件下初始体质量为(0.38±0.004)g的凡纳滨对虾(Litopenaeus vannamei)幼虾的亮氨酸需求量,以鱼粉、花生麸和L-晶体氨基酸为蛋白源配制含粗蛋白400 g/kg的6种等氮饲料,标记为L7～L12组,各组亮氨酸水平分别为15.95、17.95、19.95、21.95、23.95和25.95 g/kg(饲料干物质).每组3个重复,每个重复30尾虾,进行56 d生长实验.结果表明,凡纳滨对虾幼虾的增重率随饲料亮氨酸水平的升高而升高,当亮氨酸水平达到23.95 g/kg(L11组)时,增重率达到最高值(1143.11±36.40)％,显著高于L7组(P＜0.05);亮氨酸水平继续提高,增重率变化不显著(P＞0.05).幼虾的蛋白质效率、全虾体蛋白沉积率和血淋巴总蛋白随着饲料亮氨酸水平的升高呈现升高趋势,最高值出现在L11组,并显著高于L7组(P＜0.05).各实验组饵料系数、血淋巴谷丙转氨酶、谷草转氨酶活性和尿素氮含量则呈现降低的趋势,最低值出现在L11组,且显著低于L7组(P＜0.05).以增重率为指标,根据折线模型可知,低盐度条件下凡纳滨对虾幼虾的亮氨酸最适需要量为24.80 g/kg饲料,即61.99 g/kg饲料蛋白.     

Quantitative dietary isoleucine requirement of juvenile Pacific white shrimp,Litopenaeus vannamei (Boone) reared in low-salinity water

This study was designed to determine the isoleucine requirement of juvenile Pacific white shrimp Litopenaeus vannamei (Boone) in low-salinity water (0.50–0.70 g L^?1). Six diets were formulated to contain 410 g kg^?1 crude protein with fish meal, peanut meal and pre-coated crystalline amino acids with different concentration of l-isoleucine (9.35, 11.85, 14.35, 16.85, 19.35, and 21.85 g kg^?1 dry diet, defined as diet I1, diet I2, diet I3, diet I4, diet I5 and diet I6, respectively.). Each diet was randomly assigned to triplicate treatments of 30 shrimps (0.43 ± 0.005 g), and the feed trial lasted for 8 weeks. The results indicated that the weight gain significantly increased with increasing isoleucine concentration up to 14.35 g kg^?1 (diet I3), whereas it was reduced in the treatments exceeding 16.85 g kg^?1 isoleucine (diet I4) significantly (P < 0.05). Moreover, the highest body protein deposition and protein efficiency ratio, and the lowest feed conversion ratio, haemolymph aspartate aminotransferase and alanine aminotransferase activities were also found at treatment I3–I4 (P < 0.05). And the haemolymph urea nitrogen concentration of shrimps-fed diet I6 was particularly higher than those fed with diets I1–I5 (P < 0.05). The results of polynomial regression based on weight gain, feed efficiency and body protein deposition indicated that the optimal dietary isoleucine requirement for L. vannamei reared in low-salinity water was 15.95 g kg^?1 isoleucine of dry diet, correspondingly 38.81 g kg^?1 of dietary protein.     

Quantitative dietary threonine requirement of juvenile Pacific white shrimp, Litopenaeus vannamei (Boone) reared in low-salinity water

An 8-week feeding trial was conducted to determine the threonine requirement of juvenile Pacific white shrimp Litopenaeus vannamei (Boone) in low-salinity water (0.50–1.50 g L⁻¹). Diets 1–6 were formulated to contain 360 g kg⁻¹ crude protein with fish meal, wheat gluten and pre-coated crystalline amino acids with six graded levels of l -threonine (9.9–19.0 g kg⁻¹ dry diet). Diet 7, which was served as a reference, contained only intact proteins (fish meal and wheat gluten). Each diet was randomly assigned to triplicate groups of 30 shrimps (0.48±0.01 g), each four times daily. Shrimps fed the reference diet had similar growth performance and feed utilization efficiency compared with shrimps fed the diets containing 13.3 g kg⁻¹ or higher threonine. Maximum specific growth rate (SGR) and protein efficiency ratio were obtained at 14.6 g kg⁻¹ dietary threonine, and increasing threonine beyond this level did not result in a better performance. Body compositions, triacyglycerol and total protein concentrations in haemolymph were significantly affected by the threonine level; however, the threonine contents in muscle, aspartate aminotransferase and alanine aminotransferase activities in haemolymph were not influenced by the dietary threonine levels. Broken-line regression analysis on SGR indicated that optimal dietary threonine requirement for L. vannamei was 13.6 g kg⁻¹ dry diet (37.8 g kg⁻¹ dietary protein).     

Nursery performance of Pacific white shrimp (Litopenaeus vannamei Boone, 1931) cultivated in a biofloc system: the effect of adding different carbon sources

Effect of different carbon sources on nursery performance of Pacific white shrimp (Litopenaeus vannamei) cultivated in biofloc system was investigated. Shrimp postlarvae (98.47 ± 8.6 mg) were fed for 32 days in tanks with water volume of 130 L and density of 1 individual L^?1. One control treatment and four biofloc treatments (BFT1, BFT2, BFT3 and BFT4) with adding different carbon sources including molasses, starch, wheat flour and mixture of them, respectively, were considered at equal weight ratios. According to the results, salinity, dissolved oxygen and pH were not significantly different among the biofloc treatments (P > 0.05). Maximum pH (8.27) and maximum dissolved oxygen (6.35 mg L^?1) were recorded in the control. Maximum (0.43 mg L^?1) and minimum (0.09 mg L^?1) ammonia were recorded in the control and BFT2, respectively (P < 0.05). Using simple carbohydrates (molasses and starch) lowered the ammonia concentration significantly. The highest increase in body weight (1640.43 ± 231.28 mg), growth rate, specific growth rate (8.97 ± 0.42% per day) and biomass (190.29 ± 26.83 mg) were found in BFT1 and the highest survival (90 ± 0.77%) was found in BFT4. The highest feed conversion (1.52 ± 0.23) and the lowest feed efficiency (66.81 ± 7.95) were observed in the control (P < 0.05). The proximate composition analysis revealed an increase in lipid and ash in biofloc treatments. Results indicated that using biofloc technology with zero‐water exchange system and adding carbon sources could help to recycle waste and improve the water quality. Moreover, the type of carbonaceous organic matter as a substrate for heterotrophic bacteria would be effective in degradation and metabolization of ammonia and nitrite.     

Stimulating effects of pond water on digestive enzyme activity in the Pacific white shrimp, Litopenaeus vannamei (Boone)

Crude enzyme extracts were obtained from the digestive glands of Pacific white shrimp, Litopenaeus vannamei (Boone), reared in oligotrophic well water and eutrophic shrimp pond water to compare digestive enzyme activity between the two groups. Specific activities of serine protease, collagenase, amylase, cellulase, lipase and acid phosphatase were significantly higher (P < 0.01) in pond water‐reared shrimp (PW shrimp) than in well water reared‐shrimp (WW shrimp). For most enzymes assayed, specific activity was more than two times higher in PW shrimp, and cellulase activity was over six times higher. In contrast, chitinase activity was significantly higher (P < 0.001) in WW shrimp. Higher specific activity of most digestive enzymes in PW shrimp was probably due to natural productivity in the pond water that served as a source of organic substrates, and this increased activity may contribute to the growth‐enhancing effect of shrimp pond water.     

Daily activity pattern of the marine shrimp Litopenaeus vannamei (Boone 1931) juveniles under laboratory conditions

Lack of information on the daily activity patterns of Litopenaeus vannamei (Boone 1931) might result in inadequate feed management strategies in shrimp farms. In order to provide information to improve feed management in shrimp farms, the daily activity of juvenile shrimp reared in glass aquaria was studied, using L. vannamei (7.57±1.01 g), equivalent to 33 shrimp m^?2. Thirty‐two shrimp were observed, half in light and half in dark phase, over 280 h in 15 min h^?1 windows. Feed was provided once a day, at 10% of tank biomass. Swimming, exploration of substrate, inactivity and cleaning were recorded through instantaneous focal sampling. During the dark phase, swimming was predominant and alternated with exploration. In the light phase, inactivity was predominant, and again alternated with exploration. Cleaning occurred evenly in both the light and the dark. Substrate exploration, which characterizes the search for food, occurred in both dark and light phases, with a most intense peak 7 h after light phase onset, indicating this as the optimal feed offer.     

Experimental white spot syndrome virus challenge of juvenile Litopenaeus vannamei (Boone) at different salinities

In recent years, the shrimp industry has turned to inland freshwater culture as one method to avoid problems such as the introduction of possible vectors of viral pathogens into seawater ponds. Our experiments evaluated susceptibility to white spot syndrome virus (WSSV) in Litopenaeus vannamei held under different salinity regimens. Juvenile L. vannamei that were conditioned at salinities of 35, 25, 15, 5 and 2 g L⁻¹ were challenged with WSSV. In order to assess the severity of white spot disease, histological analysis and nested polymerase chain reaction (PCR) tests were carried out on the challenged shrimp every 4 h after 48 h post challenge. The results indicated that significantly more severe infections resulted at 15‰ than at other salinities. Mortality could not be compared due to the sampling design and because severe WSSV infections occurred in all test groups such that few shrimp remained alive in each challenged group at the end of the test. Despite this, the results suggest that salinity may affect the course and outcome of WSSV infections.     

Effect of proteolytic enzyme addition to a practical feed on growth of the Pacific white shrimp, Litopenaeus vannamei (Boone)

Four commercially available proteolytic enzymes active over a wide pH range were assessed as shrimp feed additives. Feeds were formulated to contain 1% of the test enzymes by weight. A feed of the same composition with no enzyme addition served as a control. In vitro feed protein digestibility of the feeds was determined using a modified pepsin digestibility method. In vitro protein digestibility results indicated that commercially available proteases added to a shrimp feed formulation and then hot pelleted/dried did retain enzyme activity. Feeds exhibited low weight losses through leaching (≤ 10.9%) and low or no losses of protein content (≤ 2.7%), indicating that the test feeds were available in their entirety to the shrimp. An 8-week feeding trial using the enzyme-supplemented feeds was conducted to evaluate shrimp growth. Mean shrimp survival was high for all treatments (> 90%). One-way analysis of variance did not detect significant differences (P > 0.05) among feeds in terms of survival, weight gain, specific growth rates and feed–weight gain ratios. Although in vitro digestibility indicated the presence of active enzymes in the feed, the feeding trial revealed that shrimp growth was not enhanced by the inclusion of proteases in these feeds. This study provides sufficient proof to emphasize the importance of in vivo investigations before enzyme-supplemented feeds are used with shrimp.     

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.