Shrimp Tissues Research Articles

Fatty acid synthase plays a positive role in shrimp immune responses against Vibrio parahaemolyticus infection

Fatty acid synthase (FAS) is an important enzyme that catalyzes the synthesis of fatty acids. In this study, the role of the FAS gene from pacific white shrimp Litopenaeus vannamei (LvFAS) in immune responses against Vibrio parahaemolyticus infection was studied. The expression of LvFAS could be up-regulated upon infection of V. parahaemolyticus and stimulation of lipopolysaccharide and poly (I:C). The promoter of LvFAS was predicted to harbor a NF-κB binding site and dual-luciferase reporter assays demonstrated that the NF-κB family proteins Relish, sRelish and Dorsal could activate the transcription of LvFAS. After knockdown of LvFAS expression using RNAi strategy, both the mortality of V. parahaemolyticus infected shrimps and the bacterial load in shrimp tissues were significantly increased. Meanwhile, the expression of many immune-responsive genes, such as antimicrobial peptides, C-type lectins (CTLs), lysozyme and hemolin, was down-regulated. These suggested that LvFAS could play a positive role in anti-V. parahaemolyticus responses in shrimp. To our knowledge, this is the first study that investigates the role of FAS in antibacterial immunity in animals, which may indicate the relationship between the anabolism of fatty acids and immune responses in crustaceans.

A novel C-type lectin in the black tiger shrimp Penaeus monodon functions as a pattern recognition receptor by binding and causing bacterial agglutination

C-type lectins are pattern recognition proteins that play important roles in innate immunity in invertebrates by mediating the recognition of pathogens. In this study, a novel C-type lectin gene, PmCLec, was cloned and characterized from the black tiger shrimp Penaeus monodon. The open reading frame of PmCLec is 657 bp in length. It encodes a predicted protein of 218 amino acids with a calculated molecular mass and an isoelectric point of 24086 Da and 4.67, respectively. Sequence analysis of PmCLec showed similarity to members of the C-type lectin gene superfamily. The deduced protein contains a single carbohydrate recognition domain (CRD) and four conserved cysteine residues (Cys58, Cys126, Cys141, Cys149) that are involved in the formation of disulfide bridges. PmCLec transcripts are expressed in various tiger shrimp tissues, with the highest expression in the lymphoid organ. RNAi-mediated silencing of PmCLec resulted in higher cumulative mortality of knockdown shrimp after Vibrio harveyi infection compared to the control groups. Recombinant PmCLec was successfully expressed in the E. coli system. In the presence of Ca2+, purified rPmCLec protein binds and agglutinates Gram-positive bacteria (Staphylococcus aureus, S. hemolyticus), but only slightly binds and agglutinates E. coli and could not bind to the Gram-negative bacteria Bacillus megaterium and Vibrio harveyi. These results suggest that PmCLec functions as a pattern recognition receptor that is implicated in shrimp innate immunity.

Effective cleanup for the determination of six quinolone residues in shrimp before HPLC with diode array detection in compliance with the European Union Decision 2002/657/EC.

A high-performance liquid chromatographic method was developed for the determination of six quinolone residues (ciprofloxacin, enrofloxacin, sarafloxacin, oxolinic acid, nalidixic acid, and flumequine) in shrimp tissue samples. Separation was carried out by a LiChrospher® 100 RP-8e column, running at a 22 min gradient elution program, and the mobile phase consisted of citric acid (0.4 mol/L), acetonitrile and methanol. Detection was achieved by a diode array detector, monitoring at 255 and 275 nm. Sample preparation included initial extraction with citric acid solution and further clean-up by solid-phase extraction, employing Lichrolut RP-18 cartridges. Validation was performed according to the European Union Decision 2002/657/EC. The detection capability was 127.2 μg/kg for ciprofloxacin, 115.2 μg/kg for enrofloxacin, 126.2 μg/kg for sarafloxacin, 113.1 μg/kg for oxolinic acid, 125.2 μg/kg for nalidixic acid, and 239.0 μg/kg for flumequine. Recoveries ranged between 83.0 and 121.6%. The Youden test was applied to study the method ruggedness.

Ovarian Transcriptome Analysis of Vitellogenic and Non-Vitellogenic Female Banana Shrimp (Fenneropenaeus merguiensis).

The banana shrimp (Fenneropenaeus merguiensis) is one of the most commercially important penaeid species in the world. Its numbers are declining in the wild, leading to a loss of broodstock for farmers of the shrimp and a need for more successful breeding programs. However, the molecular mechanism of the genes involved in this shrimp’s ovarian maturation is still unclear. Consequently, we compared transcriptomic profiles of ovarian tissue from females in both the vitellogenic stage and the non-vitellogenic stage. Using RNA-Seq technology to prepare the transcriptome libraries, a total of 12,187,412 and 11,694,326 sequencing reads were acquired from the non-vitellogenic and vitellogenic stages respectively. The analysis of the differentially expressed genes identified 1,025 which were significantly differentially expressed between the two stages, of which 694 were up-regulated and 331 down-regulated. Four genes putatively involved in the ovarian maturation pathway were chosen for validation by quantitative real-time PCR (RT-qPCR). The data from this study provided information about gene expression in ovarian tissue of the banana shrimp which could be useful for a better understanding of the regulation of this species’ reproductive cycle.

White Spot Syndrome Virus (WSSV) and Necrotizing Hepatopancreatitis (NHP) detection in wild shrimp of the San Andrés Lagoon, Mexico

The presence of diseases caused by virus and bacteria pose a threat to the capture and commercialization of shrimp, and may cause significant economic damage. Nocturnal monthly sampling were conducted to detect the presence of IHHNV, WSSV, and NHP in San Andres Lagoon in Tamaulipas, Mexico, an important coastal ecosystem due to its shrimp fishery and the existence of shrimp farms in the area. Polymerase chain reaction (PCR) analysis in the shrimp tissue did not detect the presence of IHHNV, however, WSSV was detected, as well as NHP during July and August, when low salinities and high temperatures were recorded.

Immunophysiological Response of Pacific White Shrimp Exposed to a Probiotic Mixture of Proteobacteria and Firmicutes in Farm Conditions

AbstractThe effect of a probiotic mixture (Proteobacteria–Firmicutes) on the immune and physiological responses of Pacific white shrimp Litopenaeus vannamei reared at commercial scale was evaluated. Shrimp were cultured into two different systems: a traditional system and a probiotic‐based system using the probiotic mixture every day of culture. Hemolymph parameters and the expression of immune‐related genes were monitored during the culture cycle. Shrimp reared in the probiotic‐based system registered an upregulation of clotting protein (CP), prophenoloxidase (proPO), alpha‐2‐macrogolbulin (A2M), superoxide dismutase (SOD), and heat shock protein (hsp) 60 genes in hemocytes compared with those reared in the traditional system. Also, high transcription levels of CP, proPO, transglutaminase, beta glucan binding protein, SOD, hsp60, and hsp90 genes were detected in the hepatopancreatic tissue of shrimp reared with probiotics. While no significant differences were detected for hemolymphatic concentrations of glucose, lactate, and protein, lower concentrations of cholesterol and acylglycerides were detected in shrimp cultured with probiotics; in addition, we detected a higher activity of proPO and a lower activity of aminotransferases, and recorded better survival in these shrimp. Results suggest that the probiotic mixture we used had an immunostimulatory effect on the shrimp; the upregulation of all the genes studied suggests the activation of the complex immune network. From a physiological perspective, the results also indicate that probiotics can be effective at the commercial shrimp farm scale.

Trace metal distribution in white shrimp (Litopenaeus schmitti) tissues from a Brazilian coastal area

Biomonitoring in situ was performed in order to assess Sepetiba Bay’s Litopenaeus schmitti efficiency as environmental contamination biomonitor, an area historically contaminated by metals and where this shrimp is the second most abundant species. Trawlings were conducted during rainy and dry seasons close to the main pollution sources to evaluate Cd, Cr, Cu, Mn, Pb, and Zn concentrations in muscle, exoskeleton, and whole body. After specimen classification, biometrics measurements, sex and age determination, samples were dried (50 °C) and grinded before acid extraction of metals, followed by ICP OES quantification. Concentrations were higher in whole shrimp samples, followed by exoskeleton and subsequently muscle samples. The descending concentration order was Zn > Cu > Mn > Cr > Cd > Pb. Comparing previous studies in the area, Pb and Zn concentrations have decreased and Cu has increased. Males and females showed no significant differences. Adults showed higher concentrations of Cr and Zn than juveniles, but bioconcentration factor did not exceed 10−2, except for Cu (10−1). Nevertheless, it did not mean intake absence, once concentrations of Cr and Cu reached values above and close, respectively, to the consumption law.

Involvement of a LysM and putative peptidoglycan-binding domain-containing protein in the antibacterial immune response of kuruma shrimp Marsupenaeus japonicus.

Lysin motif (LysM) is a peptidoglycan and chitin-binding motif with multiple functions in bacteria, plants, and animals. In this study, a novel LysM and putative peptidoglycan-binding domain-containing protein was cloned from kuruma shrimp (Marsupenaeus japonicus) and named as MjLPBP. The cDNA of MjLPBP contained 1010 nucleotides with an open reading frame of 834 nucleotides encoding a protein of 277 amino acid residues. The deduced protein contained a Lysin motif and a transmembrane region, with a calculated molecular mass of 31.54kDa and isoelectric point of 8.61. MjLPBP was ubiquitously distributed in different tissues of shrimp at the mRNA level. Time course expression assay showed that MjLPBP was upregulated in hemocytes of shrimp challenged with Vibrio anguillarum or Staphylococcus aureus. MjLPBP was also upregulated in hepatopancreas after white spot syndrome virus and bacteria challenge. The recombinant protein of MjLPBP could bind to some Gram-positive and Gram-negative bacteria and yeast. Further study found that rMjLPBP bound to bacterial cell wall components, including peptidoglycans, lipoteichoic acid, lipopolysaccharide, and chitin. The induction of several antimicrobial peptide genes and phagocytosis-related gene, such as anti-lipopolysaccharide factors and myosin, was depressed after knockdown of MjLPBP. MjLPBP could facilitate V.anguillarum clearance invivo. All the results indicated that MjLPBP might play an important role in the innate immunity of shrimp.

Contribution of bioflocs to the culture of Litopenaeus vannamei post-larvae determined using stable isotopes

The aim of this study was to use preselected quality indicators for Litopenaeus vannamei post-larvae and the stable isotopes technique with δ13C and δ15N to determine the influence of bioflocs in shrimp feeding during the nursery phase, between PL1 and PL30. A control treatment (CT) with water renewal was compared to a biofloc treatment (BT) that received organic carbon fertilizations. Different types of commercial feed (Stresspak and Flake-INVE™, PL40-GUABI™), microalgae (Chaetoceros muelleri) and Artemia sp. nauplii were used as food sources. The physical and chemical parameters of water and ammonia were monitored daily, and nitrite, nitrate and alkalinity were measured weekly. Suspended solids and the microorganisms of bioflocs were characterized. At the end of the experiment, fifteen shrimps of each replicate were collected to evaluate nine larvae quality indicators. Isotopic values of 13C and 15N of food sources and shrimp tissues were collected on days 10, 20 and 30, and a Bayesian model of isotopic mixture measured the contributions of these sources to the shrimp biomass. Salinity, alkalinity and nitrite differed significantly between the treatments but were appropriate for post-larvae production. The larvae quality conditions in the CT treatment were ranked as “good,” whereas the BT conditions were considered “excellent” according to the analysis of larval quality parameters. The stable isotopes analysis showed that the PL40 GUABI™ feed was the most important food source throughout the experimental period in the CT. In the BT (10th and 20th days), the bioflocs and commercial feeds (Flake-INVE™, PL40-GUABI™) did not present significant variations in their contribution ratios; however, on the 30th day, the contribution of the PL40-GUABI™ feed was higher when compared to bioflocs. In general, the commercial feed played a more important role as a food source for Litopenaeus vannamei larvae during the nursery phase, although the consumption of bioflocs led to a better larvae quality.

Some Limiting Factors in Superintensive Production of Juvenile Pacific White Shrimp, Litopenaeus vannamei, in No‐water‐exchange, Biofloc‐dominated Systems

AbstractSuperintensive shrimp culture in zero‐exchange, biofloc‐dominated production systems is more biosecure and sustainable than traditional shrimp farming practices. However, successful application of this technology depends upon optimizing dietary formulations, controlling Vibrio outbreaks, and managing accumulative changes in water quality and composition. A 49‐d study investigated the effect of two commercial feeds of differing protein content and an indoor limited‐exchange, biofloc‐dominated culture environment on Litopenaeus vannamei performance and tissue composition, water quality and ionic composition, and Vibrio dynamics. Juveniles (5.3 g) were stocked at 457/m3 into four 40 m3 shallow raceways containing biofloc‐dominated water and fed one of two commercial feeds with differing protein content, 35 or 40%. Shrimp performance, Vibrio populations, and changes in shrimp and culture water composition were monitored. There were no significant differences (P > 0.05) in shrimp performance (survival, weight, growth, specific growth rate, total biomass, yield, feed conversion ratio, and protein efficiency ratio) or proximate composition between feed types. The 40% protein feed resulted in higher culture water nitrate and phosphate concentrations, alkalinity consumption and bicarbonate use, and higher phytoplankton density. The presence of Vibrio, specifically Vibrio parahaemolyticus, reduced shrimp survival. This survival decrease corresponded with increased culture water Vibrio concentrations. Culture water K+ and Mg2+ increased significantly (P < 0.05), and Sr2+, Br−, and Cl− decreased significantly (P < 0.05) over time. While Cu2+ and Zn2+ did increase in shrimp tissue, no heavy metals accumulated to problematic levels in culture water or shrimp tissue. These results demonstrate the importance of monitoring Vibrio populations and ionic composition in limited‐exchange shrimp culture systems.

Efficacy of inorganic and chelated trace minerals (Cu, Zn and Mn) premix sources in Pacific white shrimp, Litopenaeus vannamei (Boone) fed plant protein based diets

The present experiment was conducted to evaluate the efficacy of trace minerals (Cu, Zn and Mn) premixes from inorganic and chelated (chelated to 2-hydroxy-4-methylthiobutanoic acid or hydroxy analog of methionine; Mintrex™) sources, in juvenile Pacific white shrimp, (Litopenaeus vannamei) fed plant protein based practical diets. Eight experimental diets comprising a trace minerals (Cu, Zn and Mn) deficient Basal control, and diets supplemented with the trace mineral premixes at four different levels of 2.5 (M2.5), 5 (M5), 7.5 (M7.5) and 8.5g/kg (M8.5) from chelated source and at three different levels of 5 (I5), 8.5 (I8.5) and 20g/kg (I20) from inorganic source were formulated. Eleven numbers of juvenile shrimp averaging 0.6±0.01g (mean±SD) were fed one of the eight experimental diets in triplicate groups for 8weeks. At the end of the feeding trial, shrimp fed M2.5 and I8.5 diets exhibited the similar final weight (FW) and weight gain (WG) (P<0.05). Performance for Cu and Zn content in the hepatopancreas tissues and whole body showed equal efficiency of M5 compared to I8.5 diet (P<0.05). Whereas, nonspecific enzyme, CuZn super oxide dismutase (CuZn SOD) from the serum and hepatopancreas tissue was recorded to be peaked for the group of shrimp fed M5 diet. Moreover, plasma protein and glucose levels were recorded to be similar between the groups of shrimp fed M2.5 and I20 diets (P<0.05). Therefore, the present experiment demonstrated, a higher efficiency of chelated over inorganic source of trace mineral premix in Pacific white shrimp, (L. vannamei) fed plant protein based diets. Statement of relevanceThe significance of trace mineral bioavailability has become more important as the composition of majority of commercial fish feed has been changing and there is an increased use of dietary plant protein. As a result, the bioavailability of trace minerals is being adversely affected by the presence of antagonistic factor such as phytic acid in plant protein. Even though, our knowledge in fish nutrition has advanced significantly, the information on trace minerals requirement is still limited and fragmentary. Inorganic form (sulfate/nitrate) of trace mineral has traditionally been used in aquafeed formulation. However, the limited bioavailability of inorganic source of trace mineral due to its higher affinity to antinutrients has hastened the search for alternative form of inorganic trace minerals. Overall performance observed in the present experiments vouched the potential benefit of using chelated trace mineral, Cu, Zn & Mn premix in marine shrimp, Pacific white shrimp to promote the optimum growth, trace minerals saturation in shrimp body and tissue as well as to ensure the optimum enzyme activity and health of cultured shrimp. The present experiment opens a new avenue to compare the efficacy of inorganic and chelated source of trace minerals in other commercially important marine and fresh water fish species.Whereas, the sustainability issue has put a new dimension in aquafeed formulation with a wide array of new ingredients and additives, on the other hand, the importance of basic nutrient such as trace minerals is still in sideline. Substantial investment and integrated scientific efforts are warranted to bridge the knowledge gap and further improve our understandings on the significance of dietary trace mineral in fish nutrition and health at the least cost to environment.

Acidic Electrolyzed Water as a Novel Transmitting Medium for High Hydrostatic Pressure Reduction of Bacterial Loads on Shelled Fresh Shrimp.

Acidic electrolyzed water (AEW), a novel non-thermal sterilization technology, is widely used in the food industry. In this study, we firstly investigated the effect of AEW as a new pressure transmitting medium for high hydrostatic pressure (AEW-HHP) processing on microorganisms inactivation on shelled fresh shrimp. The optimal conditions of AEW-HHP for Vibrio parahaemolyticus inactivation on sterile shelled fresh shrimp were obtained using response surface methodology: NaCl concentration to electrolysis 1.5 g/L, treatment pressure 400 MPa, treatment time 10 min. Under the optimal conditions mentioned above, AEW dramatically enhanced the efficiency of HHP for inactivating V. parahaemolyticus and Listeria monocytogenes on artificially contaminated shelled fresh shrimp, and the log reductions were up to 6.08 and 5.71 log10 CFU/g respectively, while the common HHP could only inactivate the two pathogens up to 4.74 and 4.31 log10 CFU/g respectively. Meanwhile, scanning electron microscopy (SEM) showed the same phenomenon. For the naturally contaminated shelled fresh shrimp, AEW-HHP could also significantly reduce the micro flora when examined using plate count and PCR-DGGE. There were also no significant changes, histologically, in the muscle tissues of shrimps undergoing the AEW-HHP treatment. In summary, using AEW as a new transmitting medium for HHP processing is an innovative non thermal technology for improving the food safety of shrimp and other aquatic products.

Enhancement and confirmation of white spot syndrome virus detection using monoclonal antibody specific to VP26

A portion of the VP26 gene (VP26F109) encoding a structural protein of white spot syndrome virus was expressed, purified by SDS-PAGE and used for immunization of Swiss mice for monoclonal antibody (MAb) production. Three groups of MAbs specific to different epitopes on VP26 were selected; these MAbs can be used to detect natural WSSV infection in Penaeus vannamei using dot blotting, Western blotting or immunohistochemistry without cross-reaction with other shrimp tissues or other common shrimp viruses. The detection sensitivity of the MAbs was ranged 7–14 fmole per spot of the rVP26F109 as determined using dot blotting. A combination of three MAbs specific to VP26 with MAbs specific to VP28, VP19 and ICP11 increased the detection sensitivity of WSSV during early infection. Therefore, the MAbs specific to VP26 could be used to confirm and to enhance the detection sensitivity for WSSV infection in shrimp with various types of antibody-based assays.

In situ DIG-labeling, loop-mediated DNA Amplification (ISDL) for highly sensitive detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV)

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is also known as Penaeus stylirostris densovirus (PstDNV). Since it is listed as a reportable crustacean disease by the World Organization for Animal Health (OIE), many countries require testing for IHHNV in shrimp stocks imported for aquaculture, necessitating rapid and highly sensitive detection methods to guarantee and maintain absence of IHHNV in traded stocks. Here we describe a new, highly sensitive method for detection of IHHNV in shrimp tissues. It involves in situ loop-mediated DNA amplification (IS-LAMP) employing digoxigenin-11-dUTP (DIG-dUTP) as part of the deoxynucleotide triphosphate (dNTP) mix. This resulted in DIG-labeling of the DNA amplicons (i.e., in situ, DIG-labeling LAMP or ISDL) allowing direct in situ, immuno-histochemical detection of IHHNV using an anti-DIG antibody conjugated with alkaline phosphatase, and eliminating the lengthy hybridization step of conventional ISH. The target region for the method is at the 3'end of the IHHNV genome that has been reported least likely to give rise to endogenous viral elements (EVE) in the shrimp genome. When compared to the results for IHHN detection by conventional in situ hybridization (ISH) using a DIG-labeled oligonucleotide probe, signals from the ISDL method were stronger and also gave positive results with some tissues unreactive by the conventional ISH method. Additional comparison of the two methods revealed that the proteinase-K digestion step needed for normal ISH was unnecessary for ISDL. This further shortened the assay time and improved the tissue integrity necessary to clearly indicate the location of positive hybridization signals. The ISDL method gave no cross- reactions with tissues of shrimp infected with other common pathogens, and the total time needed to carry out ISDL was one-third of that needed for conventional ISH. Statement of relevanceThis work describes an in situ, DIG-labeling LAMP (ISDL) method, as a new, highly sensitive method for detection of IHHNV in shrimp tissues. The developed method can be used as a confirmatory test carried out with tissue sections to confirm the results from molecular assays, especially when immune-histochemical methods are not available to do so. ISDL is especially important for molecular detection from a new or unexpected location and for molecular detection in newly suspected carriers to determine whether they are actually infected or just mechanical carriers.

Assessment of heavy metals contamination and human health risk in shrimp collected from different farms and rivers at Khulna-Satkhira region, Bangladesh.

This study is aimed to assess the heavy metals contamination and health risk in Shrimp (Macrobrachium rosenbergii and Penaeus monodon) collected from Khulna-Satkhira region in Bangladesh. The results showed that the Pb concentrations (0.52–1.16 mg/kg) in all shrimp samples of farms were higher than the recommended limit. The Cd levels (0.05–0.13 mg/kg) in all samples and Cr levels in all farms except tissue content at Satkhira farm were higher than the permissible limits. The individual concentration of Pb, Cd, and Cr between shrimp tissue and shell in all rivers and farms were not statistically significant (P > 0.05). Target hazard quotient (THQ) and hazard index (HI) were estimated to assess the non-carcinogenic health risks. Shrimp samples from all locations under the current study were found to be safe for consumption, the possibility of health risk associated with non-carcinogenic effect is very low for continuous consumption for 30 years.

A Sensitive and Validated Method for Determination of T-2 and HT-2 Toxin Residues in Shrimp Tissues by LC-MS/MS

Accurate analyses of T-2 and HT-2 toxin in aquatic organisms including shrimp are important as these two toxins are increasingly detected in aquatic cereal-based feed. Therefore, the potential for these toxins to enter the human food chain from contaminated fish and shrimp products is very real. A rapid, sensitive, and validated method for simultaneous determination of T-2 and HT-2 toxins was developed using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method following extraction of the two toxins from shrimp tissues with ethyl acetate. This method is simple in that additional solid-phase extraction is not required to isolate and purify the toxins. LC was performed on an analytical Hypersil GOLD column. The mobile phase consisted of methanol and 5 mM ammonium acetate containing 0.1 % formic acid. The MS/MS ion transitions for both the T-2 toxin (484.20 → 214.87) and HT-2 toxin (442.20 → 214.96) were monitored. And the most intense transition ion product (m/z) of T-2 and HT-2 used for quantification on the SRM mode of a mass spectrometer was 304.95 and 262.91, respectively. The results linearly correlated with coefficients >0.9990. The limits of quantification ranged from 0.02 to 0.51 ng·g−1 and from 0.17 to 4.48 ng·g−1 for T-2 and HT-2, respectively, depending on the shrimp tissue type. The overall extraction recovery for both toxins ranged between 84 and 111 % with RSD values less than 15.0 %, indicative of good replication. Furthermore, the recovery and precision levels were within the predefined limits (≤15 %) at all concentrations. The application of this method to study the accumulation of T-2 toxin in shrimp showed that it can be successfully used to monitor even very low tissue toxin concentrations. Research is in progress to extend this method for the measurement of T-2 and HT-2 in aquatic foods that enter the human food chain.

Molecular Characterization of Differentially Expressed ADP Ribosylation Factor from WSSV Infected Black Tiger Shrimp Penaeus monodon

Suppression subtractive hybridization (SSH) cDNA library was constructed using hepatopancreas of the WSSV infected black tiger shrimp, Penaeus monodon. The differentially expressed ESTs of SSH cDNA library represented different functional categories belonging to energy and metabolism, osmoregulation, cytoskeletal and structural proteins, membrane traffic and organelle structure, immune genes, protein folding, transport and synthesis, ovarian and oocyte development, nucleic acid processing, cell cycle and heat shock protein. The ADP ribosylation factor (Arf) was one of the differentially expressed gene which was identified from the SSH cDNA library. The full length cDNA of P. monodon Arf (pmArf) was cloned by 3′- and 5′-rapid amplification of cDNA ends (3′/5′ RACE). The complete nucleotide sequence of pmArf encoded protein sequence of 180 amino acids revealed the characteristic conserved features of the Arf family proteins represented by the P-loop, the switch regions and interswitch region. The sequence homology indicated that the pmArf belonged to class II of Arf family. The gene expression analysis of pmArf and other immune related genes (penaeidin, anti-lipopolysaccharide factor and QM) in the gills, hepatopancreas and gut tissues of the WSSV infected shrimp was carried out by Real-Time PCR. The pmArf gene was up-regulated with highest gene expression levels at 24 h (1.33-fold) in the gill tissues and at 48 h in the gut tissues (1.44-fold) and hepatopancreas (1.46-fold) of WSSV infected shrimp, indicating its potential role in immune response against WSSV infection.

Dietary astaxanthin levels affect colour, growth, carotenoid digestibility and the accumulation of specific carotenoid esters in the Giant Tiger Shrimp,Penaeus monodon

The carotenoid astaxanthin (Axn) plays a vital role in shrimp pigmentation, with direct influence on product quality, and forms a significant cost component of shrimp aquaculture feeds. However, the effects of dietary Axn on other measures of shrimp physiological performance are varied, and the efficiency of carotenoid uptake from the diet and deposition in shrimp tissues is poorly defined. This study fed juvenile shrimp (Penaeus monodon) diets that contained 0, 25, 50 or 100 mg kg−1 Axn for 6 weeks. Shrimp fed carotenoid-free diets had significantly reduced colour and growth than those fed carotenoids, but survival was unaffected. Carotenoid digestibility improved as dietary carotenoid levels increased, and was 98.5% in shrimp fed 100 mg kg−1 Axn. After 6 weeks, whole body carotenoid levels were significantly depleted in 0 or 25 mg kg−1 fed shrimp, compared with those fed 50 or 100 mg kg−1 or compared with initial shrimp. This study also showed that Axn monoesters were enriched with saturated fatty acids, whereas Axn diesters were enriched with monounsaturated and polyunsaturated fatty acids. Combined, these studies demonstrate that a total dietary carotenoid intake of between 25 and 50 mg kg−1 Axn is required for normal shrimp growth and health in P. monodon. Evidence suggests that there is a functional role for the accumulation of carotenoids and the formation of specific Axn fatty acid esters, and these may be linked to the metabolism, storage, mobilization or deposition of Axn within various tissues.

A homologue gene of β-catenin participates in the development of shrimps and immune response to bacteria and viruses.

β-Catenin is a multifunctional protein that is involved in many physiological processes, including development, cell proliferation, cell migration, and apoptosis. However, the function of β-Catenin in crustacean is unknown. In this study, the first shrimp homologous gene of β-catenin in Marsupenaeus japonicus (i.e., Mjβ-catenin) was identified and characterized. The full-length of the complementary DNA of Mjβ-catenin is 3130 bp, including a 2463 bp open reading frame that encodes 821 amino acid. Multiple alignment of β-Catenin proteins suggested that the Armadillo/β-Catenin-like repeat domains were conserved. Phylogenetic analysis showed that β-Catenin from shrimp was clustered into one group with invertebrate β-Catenin. The transcription of β-catenin in various development stages of shrimp was detected and persistently increased as the shrimp matured. In adult shrimp, β-catenin was widely distributed in detected tissues and has the relatively high expression level in gills, hemocytes, testes, and ovaries. The transcripts of β-catenin in tissues of adult shrimp were significantly up-regulated at various time points after infecting with Staphylococcus aureus, Vibrio anguillarum, and white-spot syndrome virus. Furthermore, knockdown of β-catenin resulted in impaired bacterial clearance ability and increased virus copy in shrimp in vivo. Therefore, β-Catenin in shrimp participates in the development and immune response of shrimps.

A shrimp pacifastin light chain-like inhibitor: Molecular identification and role in the control of the prophenoloxidase system

Pacifastin is a recently classified family of serine proteinase inhibitors that play essential roles in various biological processes, including in the regulation of the melanization cascade. Here, a novel pacifastin-related gene, termed PmPacifastin-like, was identified from a reverse suppression subtractive hybridization (SSH) cDNA library created from hemocytes of the prophenoloxidase PmproPO1/2 co-silenced black tiger shrimp Penaeus monodon. The full-length sequences of PmPacifastin-like and its homologue LvPacifastin-like from the Pacific white shrimp Litopenaeus vannamei were determined. Sequence analysis revealed that both sequences contained thirteen conserved pacifastin light chain domains (PLDs), followed by two putative kunitz domains. Expression analysis demonstrated that the PmPacifastin-like transcript was expressed in all tested shrimp tissues and larval developmental stages, and its expression responded to Vibrio harveyi challenge. To gain insight into the functional roles of PmPacifastin-like protein, the in vivo RNA interference experiment was employed; the results showed that PmPacifastin-like depletion strongly increased PO activity. Interestingly, suppression of PmPacifastin-like also down-regulated the expression of the proPO-activating enzyme PmPPAE2 transcript; the PmPacifastin-like transcript was down-regulated after the PmproPO1/2 transcripts were silenced. Taken together, these results suggest that PmPacifastin-like is important in the shrimp proPO system and may play an essential role in shrimp immune defense against bacterial infection. These results also expand the knowledge of how pacifastin-related protein participates in the negative regulation of the proPO system in shrimp.