Shrimp Hemolymph Research Articles

Molecular Detection of Taura Syndrome Virus Infections in White Shrimp (Litopenaeus vannamei) and Giant Prawn (Macrobrachium rosenbergii)

Abstract Giant prawn (Macrobrachium rosenbergii) and vaname shrimp (Litopenaeus vannamei) are types of shrimp that became excellent commodities in the fisheries sector. However, one of the obstacles in the vaname shrimp aquaculture is a disease caused by the infection of Taura Syndrome Virus (TSV). One of the consequence of raising the vaname shrimp in Indonesia is the possibility of spreading TSV infection in another shrimp species. TSV infection in giant prawns in Indonesia has not been reported. The aims of this study were: 1. To determine the resistance of giant prawns toward TSV infection and 2. To detect molecularly using RT-PCR technique the presence of viruses TSV on vaname shrimp or giant prawns infected with 3 different doses (0.05 ml; 0.10 ml and 0.15 ml) of TSV inoculum using a pair of specific primers for TSV 9992F (5'-AAG CTT GCG TAG ACA GCC-3') and 9195R (5'-TCA AGA ATG GCT TCC TGG-3'). The research results showed that vaname shrimp mortality infected by 0.05 ml; 0.10 ml and 0.15 ml TSV inoculums were 14.28%, 42.86%, and 57.14%, respectively. Whereas the giant prawns mortality that were infected using the same dose of TSV inoculums were 0%, 8.33%, and 8.33%, respectively. Positive result was detected molecularly only from haemolymph of vaname shrimp infected using 0.15 ml of TSV inoculum. On the other hand, positive results were detected in pleopod and the gill of vaname shrimp infected using 0.05 ml; 0.10 ml or 0.15 ml of TSV inoculums. In giant prawns, infection using 3 different doses of TSV inoculums causes negative result molecularly. Based on all of the facts, it can be concluded that, giant prawns has the higher resistance to TSV infection than that of vaname shrimp.

Immune responses of Litopenaeus vannamei to thermal stress: a comparative study of shrimp in freshwater and seawater conditions

The effects of hypothermal (22–16 °C) and hyperthermal (22–28 °C) stress on the immune system responses of the shrimp Litopenaeus vannamei cultured in either freshwater or seawater were measured and compared. The following immune system indicators were measured for comparison: total hemocyte count (THC), activity of phenoloxidase (PO), nitric oxide synthase (NOS), superoxidase (SOD), and malondialdehyde (MDA) content. Thermal stress significantly decreased THC in both freshwater and seawater shrimp within 6–12 h (P < 0.05). After hypothermal stress, all shrimp had a significantly lower THC level than their prechallenge levels (P < 0.05). Under both types of thermal stress, the activity of PO, NOS, and SOD first increased and then decreased. After 48 h of thermal stress, shrimp PO and NOS activity decreased in both freshwater and seawater. After 48 h of thermal stress, the reduction in the SOD activity in the hemolymph of freshwater shrimp was greater than that in seawater shrimp. During exposure to stress, the MDA content in freshwater shrimp was significantly higher than in seawater shrimp, which demonstrated that lipids in freshwater shrimp were more susceptible to peroxidation than those in seawater shrimp, particularly at low temperatures. Large temperature fluctuations, particularly sudden cooling, should be avoided when rearing L. vannamei because of high rates of lipid peroxidation and decreased immunity. These effects are more marked in freshwater than in seawater.

Molecular characterization, immune responsive expression and functional analysis of QM, a putative tumor suppressor gene from the Pacific white shrimp, Litopenaeus vannamei

The QM, firstly identified as a putative tumor suppressor gene from human, has been confirmed to possess varieties of functions in a range of organisms. In the present study, the cDNA that encodes a 220-amino-acid QM protein with calculated molecular mass of 25.5 kDa and isoelectric point of 10.07 was characterized from the Pacific white shrimp Litopenaeus vannamei. Analysis of the deduced amino acid sequence of LvQM revealed that it contained a series of conserved functional motifs. Quantitative real-time PCR (qRT-PCR) results showed that the transcript of LvQM was extensively distributed in the tissues under investigation and most highly expressed in gill. After challenged with Vibrio anguillarum, the LvQM transcripts were significantly increased (P < 0.05) both in hepatopancreas and hemocytes in the early experimental phase. When LvQM was knocked down by RNA interference (RNAi), the transcript of prophenoloxidase (proPO) and the phenoloxidase activity (PO) in shrimp hemolymph were dramatically decreased, while the mortality was significantly increased. Furthermore, the recombinant LvQM protein (rLvQM) was successfully expressed in Escherichia coli BL21 (DE3)-pLysS. Injecting the purified rLvQM mixed with V. anguillarum markedly increased the clearance rate of bacteria and PO activity in the shrimp hemolymph. Hence, we conclude that LvQM was involved in the host defense of L. vannamei, probably as a positive regulator to phenoloxidase activity.

Phenotyping of vibrios isolated from marine shrimp hemolymph

The phenotypic characterization of 100 Vibrio strains isolated from the hemolymph of cultured Pacific white shrimp, Litopenaeus vannamei, was performed. This process allowed the identification of 10 species: V. navarrensis (n = 53), V. brasiliensis (n = 15), V. parahaemolyticus (n = 10), V. xuii (n = 8), V. cholerae (n = 5), V. coralliilyticus (n = 4), V. neptunis (n = 2), V. alginolyticus (n = 1), V. diazotrophicus (n = 1), and V. vulnificus B3 (n = 1). These results suggest that penaeid hemolymph may be colonized by vibrios with no imposition or necessity of pathological compromising.

N-Linked glycosylation is essential for the yellow head virus replication cycle

Yellow head virus (YHV) particles contain a nucleocapsid protein (p20) and two envelope glycoproteins (gp116 and gp64). The glycans attached to the two glycoproteins are N-linked and are complex and high mannose types, respectively. Here, we show that treatment with the N-linked glycosylation inhibitor tunicamycin in YHV-infected black tiger shrimp (Penaeus monodon) resulted in less severe yellow head disease and reduced mortality when compared with untreated control shrimp. Quantitative real-time reverse transcription PCR analysis also revealed lower YHV copy numbers in the haemolymph of treated than control shrimp. This was concurrent with less intense immuno-reactions in tissues of treated versus untreated shrimp using mAbs against all three YHV structural proteins. In addition, transmission electron microscopy of lymphoid organ tissue of the treated and untreated shrimp [eight collected at 36 h and eight at 48 h post-infection (p.i.)] revealed only unenveloped nucleocapsids in all but one of the treated shrimp (collected at 48 h p.i.). By contrast, all the untreated shrimp showed a mixture of many unenveloped and enveloped virions. These results were supported by purification of YHV from the cell-free haemolymph of treated and untreated shrimp followed by YHV structural protein analysis by SDS-PAGE. It revealed three expected structural protein bands (116, 64 and 20 kDa) from the untreated shrimp but no structural protein bands from the tunicamycin-treated shrimp (confirmed by Western blot analysis). Overall, the results indicated that blocking glycosylation with tunicamycin inhibited the formation of mature YHV virions and their subsequent release into shrimp haemolymph, reducing the severity of disease.

Purification and identification of a clotting protein from the hemolymph of Chinese shrimp (Fenneropenaeus chinensis)

The clotting protein (CP) plays important and diverse roles in crustaceans, such as coagulation and lipid transportation. A clotting protein was purified from the hemolymph of Chinese shrimp Fenneropenaeus chinensis (named as Fc-CP) with Q sepharose HP anion-exchange chromatography and phenyl sepharose HP hydrophobic interaction chromatography. Fc-CP was able to form stable clots in vitro in the presence of hemocyte lysate and Ca2+, suggesting that the clotting reaction is catalyzed by a Ca2+-dependent transglutaminase in shrimp hemocytes. The molecular mass of Fc-CP was 380 kDa under non-reducing conditions and 190 kDa under reducing conditions as was determined with SDS-PAGE. CP exists as disulfide-linked homodimers and oligomers. The N-terminal amino acid sequence of Fc-CP was identical to that of shrimps including Penaeus monodon, Farfantepenaeus paulensis and Litopenaeus vannamei; and similar to that of other decapods. The purified Fc-CP was digested with trypsin and verified on an ABI 4700 matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF/TOF) mass spectrometry. Our results will aid to better understanding the coagulation mechanism of shrimp hemolymph.

Peptidomic analysis of Chinese shrimp (Fenneropenaeus chinensis) hemolymph by magnetic bead-based MALDI-TOF MS

Peptides in shrimp hemolymph play an important role in the innate immune response. Analysis of hemolymph will help to detect and identify potential novel biomarkers of microbial infection. We used magnetic bead-based purification (ClinProt system) and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) to characterize shrimp hemolymph peptides. Shrimp serum and plasma were used as the source of samples for comparative analysis, and it was found that serum was more suitable for shrimp hemolymph peptidomic analysis. To screen potential specific biomarkers in serum of immune-challenged shrimps, we applied magnetic bead-based MALDI-TOF MS to serum samples from 10 immune-challenged and 10 healthy shrimps. The spectra were analyzed using FlexAnalysis 3.0 and ClinProTools 2.1 software. Thirteen peptide peaks significantly different between the two groups were selected as candidate biomarkers of lipopolysaccharide (LPS)-infection. The diagnostic model established by genetic algorithm using five of these peaks was able to discriminate LPS-challenged shrimps from healthy control shrimps with a sensitivity of 90% and a specificity of 100%. Our approach in MALDITOF MS-based peptidomics is a powerful tool for screening bioactive peptides or biomarkers derived from hemolymph, and will help to enable a better understanding of the innate immune response of shrimps.

Activation of an immune response in Litopenaeus vannamei by oral immunization with phagocytosis activating protein (PAP) DNA

The phagocytosis activating protein (PAP) gene has been reported to stimulate the phagocytic activity of shrimp hemocytes and to protect shrimp from several pathogens. In this study oral administration of the chitosan-PAP gene to shrimp was investigated for its ability to induce immunity. The PAP gene was cooperated into a phMGFP plasmid, named PAP-phMGFP. Chitosan-PAP-phMGFP nanoparticles were formed by mixing a low molecular weight chitosan (50 kDa) and a high molecular weight chitosan (150 kDa) with various ratios of PAP-phMGFP. The optimal ratio of chitosan PAP-phMGFP nanoparticles was first determined by transfection into Chinese Hamster Ovary (CHO) cells before being used for oral immunization in shrimp. The chitosan-PAP-phMGFP nanoparticles at a ratio of 2:1 with the low molecular weight chitosan were optimum for transfecting the CHO cells. The shrimp were then fed with 25, 50, 100 and 150 μg/shrimp/day of chitosan-PAP-phMGFP (2:1) nanoparticles then challenged by the white spot syndrome virus (WSSV). Shrimp fed with 50 μg of chitosan-PAP-phMGFP nanoparticles per day for 7 consecutive days, produced the highest relative percent survival (RPS) (94.45 ± 9.86%). The presence of PAP-phMGFP was detected in every shrimp tissue including the hemolymph, lymphoid organ, heart, hepatopancreas, intestine and muscle. The folds increase of the PAP gene expression increased significantly together with an increase of the phagocytic activity in the immunized shrimp. The stability of the PAP-phMGFP in the immunized shrimp hemolymph was detected by determination of the expression of the GFP at various days after immunization ceased. GFP expression was detected until the 15th day but not at the 30th day after immunization ceased. A quantitative analysis of the WSSV copies in shrimp heart tissue was significantly reduced in the immunized shrimp. In addition, chitosan-PAP-phMGFP nanoparticles protected shrimp against WSSV, Yellow head virus (YHV) and Vibrio harveyi with RPS values of 83.34 ± 7.86%, 55.56 ± 15.72% and 53.91 ± 5.52%, respectively. This study therefore confirms the role of the PAP gene in shrimp immunity and may lead to the development of a way to prevent microbial diseases of shrimp at an industrial level by appropriate feeding of a chitosan/DNA complex.

Identification of haematological markers in shrimp health assessment from the immune profile of Fenneropenaeus indicus on β-1,3-glucan administration and white spot syndrome virus challenge

Haematological alterations in Fenneropenaeus indicus fed marine yeast glucan-incorporated diet and challenged with white spot syndrome virus were analysed. Adult F. indicus 16.45 ± 2.12 g (mean ± SD) were reared in 25 ‰ sea water and allowed to acclimate for a period of 7 days. Diet was prepared incorporating glucan (0.2 %) extracted from marine yeast, Debaryomyces hansenii S169. The shrimps were reared on experimental diet for 28 days and then challenged with white spot syndrome virus through oral administration. Random sampling was done (n = 6 shrimps) on 1st, 15th and 28th day of the experiment and on post-challenge day 2 (PCD 2) and PCD 7 for haematological analysis. The immune parameters viz. plasma protein, total haemocyte count, phenoloxidase activity, superoxide anion production, alkaline phosphatase and acid phosphatase activities were estimated in the haemolymph. Superoxide dismutase, catalase, glutathione peroxidase, hydroperoxide, conjugated dienes and malondialdehyde concentrations in the haemolymph of shrimps were also analysed. Analysis of variance showed that there are significant differences (p < 0.05) in the immune and antioxidant parameters in different treatment groups of F. indicus. The shrimps fed glucan-incorporated diet showed higher survival rate with comparatively lower accumulation of lipid peroxidation products. Correlation coefficients showed that all haematological variables except hydroperoxides and conjugated dienes exhibited positive correlation with the survival rate. The immune variables and antioxidant parameters exhibited a greater degree of correlation with each other. When multiple regression of survival rate on all immune parameters was considered, the amount of variability explained was 93 % (R Square = 0.932). When significant regression coefficients among the immune parameters were taken into account, it was found that total haemocyte count (p < 0.001), phenoloxidase activity (p < 0.05), alkaline phosphatase activity (p < 0.05) and plasma protein concentration (p < 0.05) together are explaining 84 % (R 2 = 0.842) of the variability, indicating that these four parameters are mainly supporting the survival. The amount of variability explained by the antioxidant parameters was 94 % (R 2 = 0.938). When significant regression coefficients among the antioxidant parameters were taken into account, it was found that superoxide dismutase activity (p < 0.01) and catalase activity (p < 0.05) together are explaining 87.4 % (R 2 = 0.874) of the variability, indicating that these two antioxidants act as the major immune effectors supporting the survival in shrimps.

绿原酸对凡纳滨对虾抗氧化系统及抗低盐度胁迫的影响

PDF HTML阅读 XML下载 导出引用 引用提醒 绿原酸对凡纳滨对虾抗氧化系统及抗低盐度胁迫的影响 DOI: 10.5846/stxb201304160720 作者: 作者单位: 农业部水产品加工重点实验室 中国水产科学研究院 南海水产研究所,建明工业珠海有限公司,农业部海洋渔业可持续发展重点实验室 中国水产科学研究院 黄海水产研究所;农业部海洋渔业可持续发展重点实验室 中国水产科学研究院 黄海水产研究所,农业部水产品加工重点实验室 中国水产科学研究院 南海水产研究所,农业部水产品加工重点实验室 中国水产科学研究院 南海水产研究所,农业部水产品加工重点实验室 中国水产科学研究院 南海水产研究所,农业部水产品加工重点实验室 中国水产科学研究院 南海水产研究所 作者简介: 通讯作者: 中图分类号: 基金项目: 中央级公益性科研院所基本科研业务费专项资金(2011TS05,2013TS03);农业部水产品加工重点实验室开放基金课题(nybjg201205) Effects of dietary chlorogenic acid supplementation on antioxidant system and anti-low salinity of Litopenaeus vannamei Author: Affiliation: Key Laboratory of Aquatic Product Processing,Kemin Industries Zhuhai Co,Ltd,Key Laboratory for Sustainable Development of Marine Fisheries,Key Laboratory of Aquatic Product Processing,Key Laboratory of Aquatic Product Processing,Key Laboratory of Aquatic Product Processing,Key Laboratory of Aquatic Product Processing Fund Project: 摘要 | 图/表 | 访问统计 | 参考文献 | 相似文献 | 引证文献 | 资源附件 | 文章评论 摘要:对绿原酸调节凡纳滨对虾(Litopenaeus vannamei)血淋巴抗氧化系统功能及抗低盐度胁迫的效果进行了评价。360尾凡纳滨对虾随机分为4组,分别投喂含有0、100、200和400 mg/kg绿原酸的饲料28 d,随后将对虾从盐度为32的天然海水直接转入至盐度为10的水中胁迫72 h。结果表明,在正常养殖条件下,绿原酸对凡纳滨对虾的成活率、血淋巴总抗氧化能力(Total antioxidative capacity, T-AOC)、超氧化物歧化酶(Superoxide dismutase, SOD)及过氧化氢酶(Catalase, CAT)活力均无明显影响,然而投喂含有绿原酸的饲料14 d,对虾血淋巴谷胱甘肽过氧化物酶(Glutathione peroxidase, GPx)活性和血淋巴细胞GPx和CAT基因表达水平均显著高于对照组(P<0.05);低盐度胁迫24 h,绿原酸组凡纳滨对虾的存活率较对照组提高10%,但各组之间无显著性差异(P>0.05);低盐度胁迫24 h,各组凡纳滨对虾血淋巴T-AOC、SOD和GPx活性与胁迫前相比均显著增加,说明低盐度胁迫条件下机体产生了抗氧化胁迫反应,同时绿原酸组对虾血淋巴GPx、CAT活性均显著高于对照组(P<0.05);低盐度胁迫72 h,绿原酸组对虾血淋巴T-AOC、GPx和CAT活性和血淋巴细胞GPx和CAT基因表达水平均明显高于对照组。上述结果表明绿原酸可有效调节凡纳滨对虾的抗氧化系统功能,增强对虾对于低盐度胁迫下的适应能力。 Abstract:Intensively-reared white shrimp Litopenaeus vannamei are subjected to various environmental stresses that can have severe effects on their physiology. For example, low salinity stress decreases the activity of antioxidant enzymes. How to reduce the adverse effects of multiple environmental stresses on shrimp has become a new challenge in aquaculture. Chlorogenic acid has been documented to effectively scavenge free radicals and improve antioxidant defenses in humans and animals. The objectives of this study were to evaluate the effects of chlorogenic acid on the antioxidant system of L. vannamei, and determine whether it protects the shrimp against low salinity stress. A total of 360 shrimps with an average initial body weight of about 6.7 g were divided randomly into four replicated (×3) treatments, and reared indoors in 500-L glass-fiber tanks containing 300 L of natural seawater. The shrimp were fed one of four diets, a control diet without chlorogenic acid, and three diets containing different levels (100, 200, 400 mg/kg diet) of chlorogenic acid. Shrimps in each treatment were hand-fed one of the four diets to apparent satiation three times daily; uneaten food and feces were removed by siphoning. Prior to the beginning of the feeding trial, all shrimps were acclimatized for one week. Each tank was continuously oxygenated using an air pump, and 30% of the seawater was renewed daily. During the experimental period, water temperature, salinity, pH and other water quality parameters were kept stable. After being fed for 28 days, all shrimps were subjected to acute low salinity stress in which salinity was decreased rapidly from 32 to 10 for 72 h. The results indicated that at normal salinity (32), there were no significant differences in the survival, the total antioxidant status (T-AOC), superoxide dismutase (SOD) and catalase (CAT) activities of the hemolymph of white shrimps that were fed diets with or without chlorogenic acid. However, the glutathione peroxidase (GPx) activity and gene expression of GPx and CAT of shrimp hemolymph increased significantly (P<0.05) when shrimps were, for 14 days, fed diets containing different concentrations of chlorogenic acid. After exposure to low-salinity seawater for 24 h, survival of shrimps fed diets supplemented with chlorogenic acid increased by 10% compared with the control treatment, although the difference between the control and treatments was not significant (P>0.05). Activities of T-AOC, SOD and GPx of shrimp hemolymph in all treatments increased significantly after 24 h of low salinity stress, which suggests that low salinity induced an antioxidant stress response in the shrimps. Moreover, GPx and CAT activities of hemolymph in shrimp fed diets supplemented with chlorogenic acid were significantly higher than those fed the control diet after 24 h of low salinity stress (P<0.05). Over a period of 72 h of exposure to low salinity, activities of T-AOC, GPx and CAT, along with gene expression of GPx and CAT of hemolymph in white shrimp fed dietary chlorogenic acid, were significantly higher than those measured in the control animals. In conclusion, the findings of this study suggest that chlorogenic acid can serve as an effective antioxidant to regulate the antioxidant defense system of white shrimp and improve resistance against low salinity stress. 参考文献 相似文献 引证文献

Antioxidant enzyme activity in pacific whiteleg shrimp (Litopenaeus vannamei) in response to infection with white spot syndrome virus

White spot syndrome virus is a virulent pathogen that affects most crustaceans. Reactive oxygen species are produced by the immune system that defends the host against foreign microorganisms. This study examined the antioxidant enzymes and oxidative damage in tissues (hemolymph, digestive gland, gills, and muscle) of Pacific whiteleg shrimp (Litopenaeus vannamei) infected with the disease by intramuscular injection of the virus. Viral loads were confirmed by real-time PCR. The levels of oxidative damage (lipid peroxidation and protein carbonyl), the activities of antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, and glutathione S-transferase) were quantified in uninfected and infected tissues. Lipid peroxidation and protein carbonyl levels were higher in infected specimens compared to uninfected controls. A significant reduction in antioxidant enzyme activities was found at 48h post-infection in all tissues analyzed. These results suggest that oxidative stress and tissue damage via inactivation of antioxidant enzymes in infected shrimps result in system failure and sudden death.

Effect of Seawater on the Activity of Antibiotics Against Vibrios Isolated from the Hemolymph of Cultured Pacific White Shrimp

The purpose of this study was to evaluate the effect of seawater (SW) on the activity of antibiotics belonging to 10 families (aminoglycosides, aminopenicillins, carbapenems, β‐lactams, chloramphenicols, monobactams, nitrofurans, quinolones, sulfonamides, and tetracyclines) against Vibrio strains isolated from hemolymph of Pacific white shrimp, Litopenaeus vannamei, farmed in Northeastern Brazil and standard strain Vibrio cholera ATCC 19582. Susceptibility of the strains to antibiotics was determined by disk diffusion method and the minimum inhibitory concentration was determined by macrodilution method. The media Mueller–Hinton agar and broth used in the above methods were diluted in distilled water (control, 1% NaCl, pH 7.5) and SW (2.5% NaCl, pH 7.5). The antibiotics most affected by dilution in SW were tetracycline, penicillin, cephalothin, aztreonam, ampicillin, and imipenem, as indicated by a considerable increase in the number of strains classified as intermediate or resistance. Thus, in this study, the efficiency of these antibiotics on Vibrio strains was found to be reduced by contact with SW.

Use of molasses as a carbon source during the nursery rearing ofFarfantepenaeus brasiliensis(Latreille, 1817) in a Biofloc technology system

The present work evaluated the use of molasses as a carbon source during the nursery rearing of Farfantepenaeus brasiliensis in a Biofloc technology system. During a 30 days trial, three replicate tanks were randomly assigned to the following treatments: 1.) molasses (with molasses addition) and 2.) control (without molasses addition). Bacteriological analysis was used to quantify the abundance of presumptive Vibrio spp. between control and molasses treatment. The concentration of this microorganism was lower in molasses compared with control. For the immunological analysis, shrimp haemolymph was collected to determine the total haemocyte count and the total protein concentration. The immunological results were not different between treatments. The performance results of shrimp reared with molasses addition showed that the survival rate (88.87 ± 6.36), the mean final weight (1.22 ± 0.38) and the specific growth rate (0.0309 ± 1.06) were significantly higher compared with control (80.5 ± 2.42; 1.03 ± 0.13; 0.0256 ± 0.97 respectively). Moreover, the addition of molasses contributed to the maintenance of water quality and lower concentration of presumptive Vibrio spp. The control presented an unstable variation of Vibrio spp. reaching values of 80 × 102 CFU/ml, while the highest result of molasses was 20 × 102 CFU/ml, confirming the beneficial effects of molasses addition.

Decreased level of crustacean hyperglycemic hormone (CHH) in black tiger shrimp Penaeus monodon suffering from Monodon Slow-Growth Syndrome (MSGS)

Monodon Slow-Growth Syndrome (MSGS), a pathological condition in the black tiger shrimp Penaeus monodon, is associated with Laem–Singh virus (LSNV) infection. Infected shrimp grow slowly when the virus invades the part of the retina called the zona fasciculata. Since molt-inhibiting hormone (MIH) and crustacean hyperglycemic hormone (CHH) are synthesized in the optic lobe and are related to molting activity and growth of the shrimp, the purpose of this study is therefore to determine the levels of these hormones and related parameters in MSGS shrimp. P. monodon juveniles were sampled from normal and MSGS ponds and were divided into small-negative, large-negative, small-positive and large-positive groups, depending on the size of the shrimp and whether they were LSNV-negative or LSNV-positive. Individual shrimp were measured for duration of each molt stage and molt interval. Levels of MIH1 and CHH1 transcripts were determined from the optic lobe, using real-time reverse transcriptase–polymerase chain reaction. The results revealed that the small-positive and the small-negative shrimp did not differ in durations of molt stages or molt intervals, as well as on the levels of MIH1 transcript in the optic lobe, MIH1 peptide in the optic lobe and hemolymph, or ecdysteroids in the hemolymph. Differences in molting activities and related transcript and hormones were observed between the small- and large-sized shrimp, but not in the status of LSNV infection. While levels of CHH1 transcript in the optic lobe of both small-negative and small-positive shrimp did not differ, levels of CHH1 peptide, as well glucose, in the hemolymph of the small-positive shrimp were significantly lower than those of the small-negative group. Levels of glycogen in the hepatopancreas of the small-positive shrimp were also significantly higher than that of the small-negative ones. The results suggest that growth retardation in MSGS shrimp is related to the suppression of the release of CHH1 peptide by LSNV invasion in the zona fasciculata, consequently causing decreased hepatopancreatic glycogenolysis and persistent hypoglycemia, resulting in growth stunting.

Effects of water temperature and Na+:K+ ratio on physiological and production parameters of Litopenaeus vannamei reared in low salinity water

Two experiments were conducted to examine the simultaneous effects of water temperature and Na+:K+ ratio on growth, survival, and hemolymph osmolality of Litopenaeus vannamei reared in 4‰ salinity well-water. In one study, a factorial experiment was conducted with levels of water temperature of 26 and 30°C, and Na+:K+ ratios of 40:1, 80:1, and 120:1. Growth of shrimp was significantly higher at the Na+:K+ ratio of 40:1 than at 120:1, but not higher than at 80:1. Also, growth of shrimp was statistically greater at 30°C than at 26°C. Survival rate of shrimp was neither affected by Na+:K+ ratio nor by temperature. In another factorial experiment, a lower range of experimental temperatures (20 and 24°C) and Na+:K+ ratios of 40:1 and 120:1 were tested. Both growth and survival of L. vannamei reared at the Na+:K+ ratio of 40:1 were significantly higher than at the ratio of 120:1. In turn, survival rate of shrimp, but not growth, was significantly higher at 24°C than at 20°C. No differences among treatments were observed for shrimp hemolymph osmolality measurements in both experiments. Results of the present study agree with previous reports of negative effects of high aqueous Na+:K+ ratio (or K+ deficient levels) on shrimp performance. At the same time, they represent the first report of a strong sensitivity of L. vannamei survival to the aqueous Na+:K+ ratio at low temperature.

Real time PCR quantification of WSSV infection in specific pathogen free (SPF) Litopenaeus vannamei (Boone, 1931) exposed to antiviral nucleotide

ObjectiveTo investigate the level of WSSV transmission from the infected tiger prawn Penaeus monodon (P. monodon) to specific pathogen free Litopenaeus vannamei (L. vannamei) in laboratory captivity condition in relation to PCR detection, histopathological observation and viral genome sequence. MethodsViral DNA was isolated from purified virions by treatment with proteinase K (0.2 mg/mL) and Sarkosyl (1%). The purity and concentration of the DNA were determined by agarose gel electrophoresis. Moribund and dead shrimp were removed and processed for indirect immunofluorescence (IIF) analysis. Histological observation of infected L. vannamei shrimps were revealed by the degenerated cells which were characterized by intranuclear inclusions in the tissues of WSSV infected mid-gut gland, lymphoid organ, gill lamellae and gut epithelium. Total DNA was extracted, from shrimp hemolymph and tissues, with a High Puree PCR template preparation kit. WSSV-DNA was detected using a commercial 2-step PCR detection kit. ResultsThe present study compares the real-time PCR results with SYBR Green I concentration ranging from 0.2 to 0.7×. The positive standard was used in the range of 102, 104 106, 108 and 1010 copies/ng of DNA in general. The PCR analysis showed the appearance of a prominent band from the PCR amplified product of WSSV-DNA at internal control band of 848 bp. Moderate and severe levels were observed as 650 bp and 910 bp (200 & 2 000 copies) in various transmission routes. The WSSV content in moribund shrimp of all the experimental species (L. vannamei) approximately ranged in nucleotide application by quantification method from 0.000 001 WSSV copies/μg of total DNA. In whole moribund infection animal, approximately 0.02 WSSV copies/μg of DNA was detected in nucleotide applied animal. ConclusionThese results indicate that wild brood stock and native culture shrimp P. monodon may be infected with WSSV and can get transferred into the SPF L. vannamei farming environment. Based on the studies, we made in captivity condition in different WSSV transmission route in dissimilar infection range with the use of nucleotide for antiviral drugs. There is an urgent need to address and develop antiviral drugs and molecular based viral genome technique for control measures to salt away the aquaculture environments.

Antimicrobial effect of Calotropis procera active principles against aquatic microbial pathogens isolated from shrimp and fishes

ObjectiveTo study the influence of Calotropis procera (C. procera) active principles against aquatic microbial pathogens isolated from shrimp and fishes. MethodsC. procera leaf powder was serially extracted with hexane, ethyl acetate and methanol and screened by antibacterial, antifungal and antiviral activity against aquatic pathogens which isolated from shrimp/fish. After initial screening, the active extract was purified through column chromatography and again screened. Finally the active fractions were characterized by phytochemical analysis and GC-MS analysis. ResultsIn vitro antibacterial, antifungal and antiviral screening revealed that, the ethyl acetate extracts were effectively suppressed the bacterial pathogens Pseudomonas aeruginosa (P. aeruginosa), Vibrio harveyi (V. harveyi) and Aeromons hydrophila (A. hydrophila) of more than 20 mm zone of inhibition; the fungi Fusarium sp and the killer virus WSSV. The ethyl acetate extracts of C. procera incubated WSSV was failed to multiply its progeny in the in vivo system of shrimp P. monodon. The shrimp had 80% survival after WSSV challenge from the control group significantly (P<0.001) and also PCR detection confirmed that no WSSV transcription found in shrimp haemolymph. After purified the ethyl acetate extracts again antimicrobial screening performed and it concluded that the fraction namely F-II was effectively suppressed the bacterial growth and WSSV due to its enriched active principles such as cardiac glycosides, Phenols, alkaloids, Tannin and quinines. Surprisingly this fraction, F-II was effectively controlled the WSSV at 90% level at a highest significant level (P<0.001). Finally the structural characterization by GC-MS analysis revealed that, the F-II fraction contained Phenols including several other compounds such as 2,4-bis(1,1-dimethylethyl)-, Methyl tetradecanoate, Bicyclo[3.1.1] heptane, 2,6,6-trimethyl-, (1α,2β,5α)-and Hexadecanoic acidetc. ConclusionsThe present study revealed that there is a possibility for developing new eco-friendly antibacterial and antiviral drugs from C. procera against aquatic important pathogens.

Pharmacological level of copper induces the immune and antioxidant mechanisms of Fenneropenaeus indicus conferring better protection against white spot syndrome virus infection

Effect of ambient copper on the immune, antioxidant and lipid peroxidation parameters of Fenneropenaeus indicus and its susceptibility to white spot syndrome virus under heavy metal (Cu2+) exposure has been studied. Adult shrimps were acclimated to 25‰ salinity for a period of 7 days, and after 12 h of starvation, shrimps were dosed with 0.075, 0.150, 0.225 and 0.30 ppm Cu2+ by adding appropriate quantities of copper sulphate solution. After 14 days of metal exposure, the shrimps were challenged with white spot syndrome virus through oral administration and the immune and antioxidant parameters were analysed. Analysis of variance showed that there were significant differences (P < 0.05) in the immune and antioxidant parameters and lipid peroxidation product in different treatment groups of F. indicus compared to untreated group. The immune parameters and antioxidant enzymes have been significantly lowered (P < 0.05) in the haemolymph of shrimps dosed with higher concentrations of copper sulphate leading to lipid peroxidation and accumulation of malondialdehyde. However, there was less oxidative stress in shrimps exposed to 0.075 ppm Cu2+. The present study showed that high levels of Cu2+ enhanced the mortality of F. indicus with concomitant reduction in immune and antioxidant parameters. A concentration of 0.075 ppm Cu2+ in the rearing water was found to have beneficial effect in shrimps in terms of immunostimulation and higher survival against WSSV infection. This can be adopted as a pharmacological approach towards shrimp health management in culture systems.

Production and characterization of monoclonal antibodies against the haemocyanin of the Chinese shrimp, Fenneropenaeus chinensis (Osbeck, 1765)

Two protein bands with molecular masses of 73 kDa and 75 kDa were purified from haemolymph of the Chinese shrimp, Fenneropenaeus chinensis (Osbeck, 1765), by affinity chromatography coupled with purified white spot syndrome virus, which were identified as haemocyanin subunits by mass spectrometry. Fourteen hybridomas secreting monoclonal antibodies against Chinese shrimp haemocyanin were obtained by immunization of Balb/C mice with purified haemocyanin preparation, which were selected on the basis of the indirect enzyme-linked immunosorbent assay (ELISA). Three of these, designated as 2H3, 4E5, and 1G8 were cloned by limiting dilution and characterized with western blotting. Under reducing conditions in Western blotting, all three MAbs could specifically react with 73 and 75 kDa subunits of haemocyanin of F. chinensis. MAb 2H3 could also react with the native haemocyanin in haemolymph by immunoprecipitation, and cross-react with the haemocyanins of Marsupenaeus japonicus (Bate, 1888), Litopenaeus vannamei (Boone, 1931) and Procambarus clarkii (Girard, 1852).

A novel lectin domain-containing protein (LvCTLD) associated with response of the whiteleg shrimp Penaeus (Litopenaeus) vannamei to yellow head virus (YHV)

When using mRNA from gills of normal whiteleg shrimp Penaeus (Litopenaeus) vannamei as the tester and mRNA from yellow head virus (YHV)-infected shrimp as the driver, subtractive suppression hybridization (SSH) revealed that a novel EST clone of 198bp with a putative C-type lectin-like domain (CTLD) was downregulated in YHV-infected shrimp. The clone nucleotide sequence had 99% identity with one contig MGID1052359 (1,380bp) reported in an EST database of P. vannamei, and the presence of this target in normal shrimp was confirmed by RT-PCR using primers designed from the MGID1052359 sequence. Analysis of the primary structure of the deduced amino acid (a.a.) sequence of the contig revealed a short portion (40 a.a. residues) at its N-terminus with high similarity to a low density lipoprotein receptor (LDLR) class A domain and another 152 a.a. residues at its C-terminus with high similarity to a C-type lectin domain. Thus, the clone was named LvCTLD and three recombinant proteins (LvCTLD, the LDLR domain and the CTLD domain) were synthesized in a bacterial system based on its sequence. An in vitro encapsulation assay revealed that Sepharose 4B beads coated with rLvCTLD were encapsulated by shrimp hemocytes and that melanization followed by 24h post-encapsulation. The encapsulation activity of rLvCTLD was inhibited by 100mM galactose, but not mannose or EDTA. In vivo injection of rLvCTLD or rLvCTLD plus YHV resulted in a significant elevation of PO activity in the hemolymph of the challenged shrimp when compared to shrimp injected with buffer, suggesting that rLvCTLD could activate the proPO system. An ELISA test revealed that rLvCTLD could bind to YHV particles in the presence of shrimp hemolymph. Phylogenetic analysis suggested that the LvCTLD sequence was more closely related to an antiviral gene found in Penaeus monodon (PmAV) than to other reported shrimp lectins. Taken together, we conclude that a novel shrimp LvCTLD is a host recognition molecule involved in the shrimp defense mechanism against YHV via recruitment of hemocytes, probably at the site of viral infection, and via activation of the proPO system.