Biochemical characterization of the cyclooxygenase enzyme in penaeid shrimp.

Cyclooxygenase (COX) is a two-step enzyme that converts arachidonic acid into prostaglandin H2, a labile intermediate used in the production of prostaglandin E2 (PGE2) and prostaglandin F2α (PGF2α). In vertebrates and corals, COX must be N-glycosylated on at least two asparagine residues in the N-(X)-S/T motif to be catalytically active. Although COX glycosylation requirement is well-characterized in many species, whether crustacean COXs require N-glycosylation for their enzymatic function have not been investigated. In this study, a 1,842-base pair cox gene was obtained from ovarian cDNA of the black tiger shrimp Penaeus monodon. Sequence analysis revealed that essential catalytic residues and putative catalytic domains of P. monodon COX (PmCOX) were well-conserved in relation to other vertebrate and crustacean COXs. Expression of PmCOX in 293T cells increased levels of secreted PGE2 and PGF2α up to 60- and 77-fold, respectively, compared to control cells. Incubation of purified PmCOX with endoglycosidase H, which cleaves oligosaccharides from N-linked glycoproteins, reduced the molecular mass of PmCOX. Similarly, addition of tunicamycin, which inhibits N-linked glycosylation, in PmCOX-expressing cells resulted in PmCOX protein with lower molecular mass than those obtained from untreated cells, suggesting that PmCOX was N-glycosylated. Three potential glycosylation sites of PmCOX were identified at N79, N170 and N424. Mutational analysis revealed that although all three residues were glycosylated, only mutations at N170 and N424 completely abolished catalytic function. Inhibition of COX activity by ibuprofen treatment also decreased the levels of PGE2 in shrimp haemolymph. This study not only establishes the presence of the COX enzyme in penaeid shrimp, but also reveals that N-glycosylation sites are highly conserved and required for COX function in crustaceans.

The effect of dietary supplementation of Astragalus membranaceus and Bupleurum chinense on the growth performance, immune-related enzyme activities and genes expression in white shrimp, Litopenaeus vannamei.

A 56-day feeding trial was conducted to investigate the effects of dietary supplementation of Astragalus membranaceus or/and Bupleurum chinense on the growth performance, immune enzymes, and related gene expression of Pacific white shrimp (Litopenaeus vanammei). Six experimental diets were formulated and supplemented with two levels (0.25% and 0.5%) of each herb and their combination. At the end of the trial, the specific growth rate and feed conversion ratio of shrimp were significantly (P < 0.05) improved by herbal diets. Besides, the activities of immune-related enzymes such as superoxide dismutase (SOD), alkaline phosphatase (AKP), and lysozyme in serum and hepatopancreas were significantly (P < 0.05) elevated in shrimp fed A. membranaceus or/and B. chinense. The high expression levels of immune deficiency (IMD), lysozyme, and Toll-like receptor mRNA directly or indirectly reflected the activation effect of innate immune in shrimp by dietary A. membranaceus or/and B. chinense. However, no significant difference (P > 0.05) among the herbal incorporated treatments was detected on the growth performance and immune response. In conclusion, the results suggest that A. membranaceus and B. chinense could be used as a beneficial feed additives and alternatives to antibiotics for white shrimp aquaculture.

Growth performance, immune response, and digestive enzyme activity in Pacific white shrimp, Penaeus vannamei Boone, 1931, fed dietary microbial lysozyme.

The present study investigates the effects of the dietary microbial lysozyme (ML) as an immunostimulant, on the growth performance, some immune parameters and digestive enzyme of Pacific white shrimp, Penaeus vannamei. Six hundred shrimps were obtained and randomly allocated into four groups as follows with three replicates. The shrimps were fed diets supplemented with 0 (control), 0.5, 1, and 2 g kg-1 ML for 4 months. The results indicated that dietary supplementation of ML significantly improved final weight, weight gain, average daily weight gain rate (ADG), feed conversion rate (FCR), and feed efficiency rate (FER) compared to the control (P ˂ 0.05). However, weight gain specific growth rate (SGR) and survival rate were not significantly affected by dietary ML (P ˃ 0.05). Dietary ML had a progressive effects on some immune parameters status including total antioxidant capacity (TAOC), superoxide dismutase (SOD), glutathione peroxidase (GPX), lysozyme (LYZ), alkaline phosphatase (ALP), phenoloxidase (PO) and acid phosphatase (ACP) activity as well as differential haemocyte count (DHC) and total haemocyte count (THC), in shrimps treated with the lysozyme than untreated shrimps (P ˂ 0.05). However, feeding with ML had no significant effect on plasma malondialdehyde (MDA) level (P ˃ 0.05). Furthermore, intestinal digestive enzymes (lipase, protease, and amylase) in shrimp fed with dietary ML were significantly (P ˂ 0.05) higher than those fed with non-supplemented control basal diet. Thus, the results indicate that oral administration of ML can be recommended for shrimp feed to improve immune response as well digestive enzymes activity modulation.

The role of delta-1-pyrroline-5-carboxylate dehydrogenase (P5CDh) in the Pacific white shrimp (Litopenaeus vannamei) during biotic and abiotic stress.

Proline (Pro) metabolism is intimately associated with stress adaptation. The catabolism of Pro includes two dehydrogenation reactions catalyzed by proline dehydrogenase (ProDH) and Δ1-pyrroline-5-carboxylate dehydrogenase (P5CDh). P5CDh is a mitochondrial matrix NAD+-dependent dehydrogenase that is critical in preventing P5C-Pro intensive cycling and avoiding ROS production from electron run-off. Little is known about the roles of P5CDh in invertebrates, however. We cloned the P5CDh sequence in the Pacific white shrimp, Litopenaeus vannamei, and found that LvP5CDh is expressed predominantly in pleopod, hepatopancreas and gill. Subcellular localization analysis revealed that LvP5CDh protein was mainly found in the cytoplasm. In addition, overexpressing LvP5CDh in cells reduced ROS formation and inhibited apoptosis induced by LC50 Cd2+. Shrimp were exposed to various stress factors including infection with Vibrio alginolyticus, (½ LC50 and LC50) Cd2+, acid (pH 5.6) and alkali stress (pH 9.3). Both biotic and abiotic stress resulted in increased LvP5CDh expression and Pro accumulation; V. alginolyticus infection, pH 9.3 and LC50 Cd2+ stress apparently stimulated the Glu pathway of Pro synthesis, while pH 5.6 and ½ LC50 Cd2+ stress promoted the Orn pathway of Pro synthesis. Silencing of Lvp53 in shrimp attenuated LvP5CDh expression during Cd2+ stress, but had no effect on LvP5CDh mRNA levels if no Cd2+ stress was imposed. Our study contributes to the functional characterization of LvP5CDh in biotic and abiotic stress and reveals it to protect against ROS generation, damage to the cell, including the mitochondria, and apoptosis. Thus, LvP5CDh plays a critical role in immune defense and antioxidant responses.

Effects of Dietary Clostridium butyricum on the Growth, Digestive Enzyme Activity, Antioxidant Capacity, and Resistance to Nitrite Stress of Penaeus monodon.

The present study investigated the effects of the dietary probiotic Clostridium butyricum (CB) on the growth, intestine digestive enzyme activity, antioxidant capacity and resistance to nitrite stress, and body composition of Penaeus monodon. For 56 days, shrimps were fed diets containing different levels of C. butyricum (1 × 109 CFU g-1), 0% (control), 0.5% (CB1), 1.0% (CB2), and 2.0% (CB3), as treatment groups, followed by an acute nitrite stress test for 48 h. The results indicated that dietary supplementation of C. butyricum increased the growth of shrimp in the CB2 and CB3 groups. The survival rate of shrimp increased after nitrite stress for 24 and 48 h. The intestine amylase and trypsin activities increased in all three C. butyricum groups, while the lipase activity was only affected in the CB3 group. The superoxide dismutase (SOD) activity as well as heat shock protein 70 (hsp70) and ferritin gene expression levels were increased in the intestines of shrimps cultured under normal conditions for 56 days, while the catalase (CAT) activity was not changed and glutathione peroxidase (GPx) activity was only increased in the CB2 and CB3 groups. After exposure to nitrite stress for 24 and 48 h, the intestine antioxidant enzyme (SOD, CAT, and GPx) activity and gene (hsp70 and ferritin) expression levels in the three C. butyricum groups were higher than those of the control. C. butyricum had no effects on the whole body composition of the shrimp. These results revealed that C. butyricum improved the growth as well as enhanced the intestine digestive enzyme and antioxidant activities of P. monodon against nitrite stress, and C. butyricum may be a good probiotic for shrimp aquaculture.

Dietary effects of succinic acid on the growth, digestive enzymes, immune response and resistance to ammonia stress of Litopenaeus vannamei.

Organic acids acts as an growth promoter and antimicrobial agent in aquaculture. The present study investigated the effects of a natural organic acid - succinic acid (SA) on the growth, digestive enzymes, immune response and resistance to ammonia stress of Litopenaeus vannamei. The shrimps were firstly fed with diets containing different levels of SA: 0% (Control), 0.25% (SA1), 0.50% (SA2), and 1.0% (SA3) (w/w) for 56 days, followed by an acute ammonia stress for 48 h. The results indicated that dietary of SA improved the growth of shrimp, and increased the survival rate of shrimp after ammonia stress for 48 h. The amylase, lipase and pepsin activity increased in hepatopancreas in three SA group, while trypsin activity was only increased in the SA1 and SA2 groups. At 56 d, T-NOS activity, proPO and HSP70 gene expression level increased in the three SA group, PO activity increased in the SA1 and SA2 groups, T-AOC content and Toll gene expression level increased in the SA2 and SA3 groups, Trx and SOD gene expression level increased in the SA2 group, while Imd, GS and GDH gene expression level was no changes. After exposure to ammonia stress for 48 h, immune biochemical parameters (T-AOC and PO) and genes (proPO, HSP70, Trx and GDH) expression level increased in the three SA group, T-NOS activity, Toll, Imd and GS gene expression level increased in the SA2 and SA3 groups, while SOD gene expression level increased in the SA1 and SA2 groups. These results indicated that SA improved growth, enhanced digestive and immune capacities of L. vannamei against ammonia stress, and may be a potential feed additive for shrimp. The optimal dietary supplementation dosage is 0.50% (w/w) in diet.

Effect of selenium on Penaeus monodon and Perna viridis: Enzyme activities and histopathological responses.

The study was carried out to evaluate enzyme activities and histopathological changes due to the effect of acute and chronic definitive toxicity of selenium (Se) on the post larvae (PL) of giant tiger shrimp (Penaeus monodon), and green mussel (Perna viridis). The 96-h Median Lethal concentration (LC50) for the PL of shrimp was 3.36 mg L-1 and the chronic value for the long-term survival endpoint in a 21-d exposure was 0.10 mg L-1. The green mussel 96-h LC50 was 28.41 mg L-1 and the chronic value for the long-term survival endpoint in a 30-d exposure was 3.06 mg L-1. Native polyacrylamide gel electrophoresis revealed altered diverse isoforms of esterase, superoxide dismutase and malate dehydrogenase activities in the PL of shrimp and green mussel exposed to sublethal concentration of Se. Cellular anomalies such as deformation and fusion of corneal cells, detachment of corneal cells from cornea facet and increased space between ommatidia were observed in the compound eye of PL of shrimp exposed to Se for 21-d. Shrinkage and clumping of mucous gland, degenerative changes in phenol gland, and ciliated epithelium were observed in the foot of green mussel exposed to Se for 30-d. This study shows that cellular anomalies in the compound eye of PL of P. monodon and foot tissues of P. viridis described would affect the vision of shrimp and byssus thread formation in green mussel.

Effects of Dietary Lactobacillus plantarum on Growth Performance, Digestive Enzymes and Gut Morphology of Litopenaeus vannamei.

A 15-day feeding trial was conducted to investigate the effect of dietary Lactobacillus plantarum on growth performance, digestive enzyme activities and gut morphology of juvenile Pacific white shrimp, Litopenaeus vannamei (initial body weight = 7.96 ± 0.59 g). Four microbound diets were formulated to contain fermentation supernatant (FS), live bacteria (LB), dead bacteria (DB), and cell-free extract (CE) of L. plantarum. Results indicated that final weight was significantly higher in FS, DB, and CE group in comparison to the control group (P < 0.05). The maximum weight gain rate (WGR) and specific growth rate (SGR) of the CE diet group were significantly higher than that of other groups (P < 0.05). The FCR of CE diet group was lower than that of the control, LB, DB, and FS diets groups (P < 0.05). The highest digestive enzyme activities (amylase, lipase, and pepsin activity) in the hepatopancreas and gut of shrimp were observed in the CE diet group. Histological study revealed that dietary CE diet could significantly increase the enterocytes height of shrimp. The administration of cell-free extract of L. plantarum could effectively improve the growth performance of L. vannamei via the improvement of digestive enzyme activities and the enterocytes height of shrimp. The results of this study will be essential to promote application of probiotics in shrimp aquaculture.

Identification and characterization of a laccase from Litopenaeus vannamei involved in anti-bacterial host defense.

Phenoloxidases (POs) are a family of enzymes including tyrosinases, catecholases and laccases, which play an important role in immune defences of various invertebrates. Whether or not laccase exists in shrimp and its function is still poorly understood. In this study, a laccase (LvLac) was cloned and identified from Litopenaeus vannamei for the first time. The full length of LvLac is 3406 bp, including a 2034 bp open reading frame (ORF) coding for a putative protein of 677 amino acids with a signal peptide of 33 aa. LvLac contains three Cu-oxidase domains with copper binding centers formed by 10 histidines, one cysteine and one methionine, respectively. Phylogenetic analysis revealed that LvLac was close to insects laccase 1 family. LvLac expression was most abundant in heart and the crude LvLac protein could catalyze the oxidation of hydroquinone. Real-time PCR showed that LvLac expression was responsive to Vibrio parahaemolyticus, Micrococcus lysodeikticus and white spot syndrome virus (WSSV) infection. Knockdown of LvLac enhanced the sensitivity of shrimps to V. parahaemolyticus and M. lysodeikticus challenge, suggesting that LvLac may play a positive role against bacterial pathogens.

Ontogeny of osmoregulation in the Pacific blue shrimp, Litopenaeus stylirostris (Decapoda, Penaeidae): Deciphering the role of the Na(+)/K(+)-ATPase.

The role of the main ion transporting enzyme Na+/K+-ATPase in osmoregulation processes was investigated in Litopenaeus stylirostris. The development and localization of the osmoregulation sites were studied during ontogenesis by immunodetection of Na(+)K(+)-ATPase using monoclonal antibodies and transmission electron microscopy (TEM). Osmoregulation sites were identified as the pleurae and branchiostegites in the zoeae and mysis stages. In the subsequent post-metamorphic stages the osmoregulatory function was mainly located in the epipodites and branchiostegites and osmotic regulation was later detected in the gills. The presence of ionocytes and microvilli in these tissues confirmed their role in ionic processes. The complete open reading frame of the mRNA coding for the α-subunit of Na+K+-ATPase was characterized in L. stylirostris. The resulting 3092-bp cDNA (LsNKA) encodes a putative 1011-amino-acid protein with a predicted molecular mass of 112.3kDa. The inferred amino acid sequence revealed that the putative protein possesses the main structural characteristics of the Na+K+-ATPase α-subunits. Quantitative RT-PCR analyses indicated that LsNKA transcripts did not significantly vary between the different developmental stages. The number of transcripts was about 2.5-fold higher in the epipodites and gills than in any other tissues tested in juveniles. A reverse genetic approach was finally implemented to study the role of LsNKA in vivo. Knockdown of LsNKA expression by gene-specific dsRNA injection led to an increase of shrimp mortality following an abrupt salinity change compared to control animals. These data strongly suggest that LsNKA plays an important role in osmoregulation when the shrimp are challenged by changing salinities.

Glucosamine: fructose-6-phosphate amidotransferase in the white shrimp Litopenaeus vannamei: characterization and regulation under alkaline and cadmium stress.

Heavy metal residues and chemical contaminators considered as relevant sources of aquatic environmental pollutants have a generally immunosuppressive effect on aquatic organisms, depressing metabolic activities and immune response. Glutamine: fructose-6-phosphate aminotransferase (GFAT, EC2.6.1.16) is the first, and rate-limiting, enzyme in the hexosamine biosynthetic pathway, and is involved in the regulation of chitin biosynthesis and glycosylation of proteins. We have isolated and characterized GFAT from the white shrimp Litopenaeus vannamei. Amino acid sequence similarity of the Lv-GFAT (L.vannamei-GFAT) was highest to GFATs isolated from insects and mammals (83 % similarity to that of Haemaphysalis longicornis). The open-reading frame of the Lv-GFAT codes for a protein of 41.6 kDa with a calculated isoelectric point of 5.03. RT-PCR assays showed that endogenous Lv-GFAT mRNA is most strongly expressed in the intestine. Further analysis of Lv-GFAT gene expression in hepatopancreas by quantitative real-time PCR demonstrated that Lv-GFAT transcript levels increased when the shrimp were exposed to alkaline pH (9.3) and cadmium stress, but the time when its mRNA expression level peaked differed under these stresses. We also first expressed the recombinant protein of GFAT from shrimps in Escherichia coli. Western blot analyses confirmed that the Lv-GFAT protein was strongly expressed in the hepatopancreas after exposure to the LC-Cd stress. These results suggest that Lv-GFAT expression is stimulated by alkaline pH and cadmium stress and that it may play important roles in resistance of shrimp to environmental stresses.

The nuclear encoded subunits gamma, delta and epsilon from the shrimp mitochondrial F1-ATP synthase, and their transcriptional response during hypoxia.

The mitochondrial FOF1 ATP synthase produces ATP in a reaction coupled to an electrochemical proton gradient generated by the electron transfer chain. The enzyme also hydrolyzes ATP according to the energy requirements of the organism. Shrimp need to overcome low oxygen concentrations in water and other energetic stressors, which in turn lead to mitochondrial responses. The aim of this study was to characterize the full-length cDNA sequences of three subunits that form the central stalk of the F1 catalytic domain of the ATP synthase of the white shrimp Litopenaeus vannamei and their deduced proteins. The effect of hypoxia on shrimp was also evaluated by measuring changes in the mRNA amounts of these subunits. The cDNA sequences of the nucleus-encoded ATPγ, ATPδ and ATPε subunits are 1382, 477 and 277 bp long, respectively. The three deduced amino acid sequences exhibited highly conserved regions when compared to homologous sequences, and specific substitutions found in shrimp subunits are discussed through an homology structural model of F1 ATP-synthase that included the five deduced proteins, which confirm their functional structures and specific characteristics from the cognate complex of ATP synthases. Genes expression was evaluated during hypoxia-reoxygenation, and resulted in a generalized down-regulation of the F1 subunits and no coordinated changes were detected among these five subunits. The reduced mRNA levels suggest a mitochondrial response to an oxidative stress event, similar to that observed at ischemia-reperfusion in mammals. This model analysis and responses to hypoxia-reoxygenation may help to better understand additional mitochondrial adaptive mechanisms.

Effects of dietary chlorogenic acid on growth performance, antioxidant capacity of white shrimp Litopenaeus vannamei under normal condition and combined stress of low-salinity and nitrite.

An eight-week feeding trial followed by an acute combined stress test of low-salinity and nitrite were performed to evaluate effects of chlorogenic acid (CGA) on growth performance and antioxidant capacity of white shrimp Litopenaeus vannamei. Shrimp were randomly allocated in 12 tanks (30 shrimp per tank) and triplicate tanks were fed with a control diet or diets containing different levels of CGA (100, 200 and 400 mg kg(-1) feed) as treatment groups. Growth performance including weight gain (WG), biomass gain (BG), feed conversion ratio (FCR), and feed intake were determined after feeding for 56 days. Antioxidant capacity were evaluated by determining the activity of total antioxidant status (TAS), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT) as well as the gene expression of GSH-Px and CAT in the hepatopancreas of shrimp at the end of feeding trial and again at the end of the combined stress test. The results indicated that supplemention of CGA had no significant effects on the growth performance and the activities of TAS, SOD, GSH-Px and CAT in hepatopancreas of shrimp cultured under normal conditions for 56 days. However, compared with the control group, CGA (200, 400 mg kg(-1) feed) significantly improved the resistance of L. vannamei against the combined stress of low-salinity and nitrite, as indicated by the significant (P < 0.05) higher survival, higher activities of TAS, GSH-Px and CAT, as well as higher transcript levels of GPx and CAT gene in shrimp treated with CGA in the combined tress test. Our findings suggested that CGA possessed dual-modulatory effects on antioxidant capacity of L. vannamei and could be a potential feed additive that can enhance shrimp resistance against environmental stresses. The recommended application dosage is 200 mg kg(-1) and further studies are needed to clarify the action model of CGA efficiency.

Identification of novel copper/zinc superoxide dismutase (Cu/ZnSOD) genes in kuruma shrimp Marsupenaeus japonicus.

Superoxide dismutases (SODs) protect cells from superoxides, but in invertebrates, they also have role in the innate immune system. In this study, the genes for five isoforms of copper/zinc superoxide dismutase (MjCu/ZnSOD) gene were identified and sequenced in kuruma shrimp, Marsupenaeus japonicus. The coding parts of the genes ranged from 516 to 585 bp in length and encoded from 172 to 194 amino acids. Structure, phylogenetic and BLAST analyses indicated that MjCu/ZnSOD isoform_3 and _5 belonged to extracellular Cu/ZnSOD (ecSOD) group while the other three isoforms belong to the intracellular Cu/ZnSOD family. In healthy shrimp, the highest expressions of isoform 2, 3 and 4 were in the gills, whereas the expression of isoform 5 was highest in hemocytes. Challenging the shrimp with WSSV and Vibrio penaeicida up-regulated the mRNA expressions of isoforms 3 and 5, suggesting that these isoforms have roles in the innate immune system of kuruma shrimp.

Effect of guava leaves on growth and the non-specific immune response of Penaeus monodon.

Guava (Psidium guajava L.) leaf extracts have antiviral and antibacterial activity against shrimp pathogens such as yellow-head virus (YHV), white spot syndrome virus (WSSV), and Vibrio harveyi, which make it a potential water disinfectant for use in shrimp culture. In this study, the safety of guava leaf supplementation in shrimp was evaluated by studying its influence on growth and the non-specific immune response of Penaeus monodon. Six diets containing different levels of guava leaves (0% [basal diet], 0.025% [G1], 0.05% [G2], 0.1% [G3], 0.2% [G4], and 0.4% [G5]) were fed to groups of shrimp (1.576 ± 0.011 g body weight) in triplicate for 56 days. Growth performance (final body weight, WG, PWG, SGR) of shrimp fed guava leaf diets was significantly higher (P < 0.05) than that of shrimp fed on the basal diet. The G1 diet resulted in the highest body weight gain (308.44%), followed by the G2 (295.45%), G3 (283.05%), G5 (281.29%), G4 (276.11%), and finally the basal diet (214.58%). Survival of shrimp in the G1 diet group was higher than that of shrimp in the control and the other experimental groups; however, no statistical differences (P > 0.05) were found. Dietary supplementation with guava leaf improved the activities of prophenoloxidase (PO) and nitric oxide synthase (NOS) in serum, and of superoxide dismutase (SOD), acid phosphatase (ACP), alkaline phosphatase (AKP), and lysozyme (LSZ) both in serum and hepatopancreas of shrimp. In the experimental groups, the activities of these enzymes followed a similar pattern of change; they increased initially at low levels of dietary supplementation and then decreased with increasing concentrations of dietary guava leaf. Serum PO and SOD activities in shrimp fed the G1 diet reached 7.50 U ml(-1) and 178.33 U ml(-1), respectively, with PO activity being significantly higher than in controls. In shrimp fed the G1 diet, SOD, ACP, and AKP activities in hepatopancreas were significantly higher than in the controls, reaching 57.32 U g(-1), 23.28 U g(-1), and 19.35 U g(-1) protein, respectively. The highest activities of serum ACP, AKP, LSZ, and of hepatopancreas LSZ, were observed in the G3 diet group. Total nitric oxide synthase (TNOS) activity was highest (64.80 U ml(-1)) in the G4 diet group, which was significantly higher than that observed in the control group. These results suggest that dietary guava leaf supplementation could enhance the growth performance and non-specific immune response of P. monodon. Therefore, guava leaf is considered safe for use as a water disinfectant in shrimp culture.

The second type of transglutaminase regulates immune and stress responses in white shrimp, Litopenaeus vannamei.

The total haemocyte count (THC), differential haemocyte count (DHC), phenoloxidase activity, respiratory bursts (release of superoxide anion), superoxide dismutase activity, and phagocytic activity and clearance efficiency to the pathogen Vibrio alginolyticus were measured when white shrimp, Litopenaeus vannamei, (7.5 ± 0.5 g) were individually injected with diethyl pyrocarbonate-water (DEPC-H2O) or different dsRNA at 3 days of injection. In addition, haemolymph glucose and lactate, and haemocytes crustacean hyperglycemic hormone (CHH), transglutaminase I (TGI), transglutaminase II (TGII) and clottable protein (CP) mRNA expression were determined for the shrimp that received DEPC-H2O and different dsRNA after 3 days, and then transferred to 22 and 28 °C from 28 °C. Results showed that respiratory burst, phagocytic activity and clearance efficiency significantly decreased, but hyaline cells significantly increased in the shrimp received LvTGII dsRNA after 3 days. In hypothermal stress studies, LvTGI and CHH were significantly up-regulated in LvTGII-depleted shrimp following exposure to 28 and 22 °C, and haemolymph glucose and lactate were significantly enhanced in LvTGII-depleted shrimp. The injection of LvTGII dsRNA also significantly increased the mortality of L. vannamei challenged with the pathogen V. alginolyticus. These results suggest that LvTGII is an important component on the immune resistance of shrimp, and is involved in the regulation of some immune parameters and carbohydrate metabolites, as well as has a complementary effect with LvTGI in immunological and physiological response of shrimp.

An evolutionarily ancient NO synthase (NOS) in shrimp.

Nitric oxide (NO) is a well known essential molecule that is involved in multiple functions such as neuron transduction, cardiac disease, immune responses, etc.; nitric oxide synthase (NOS) is a critical enzyme that catalyzes the synthesis of it. A very few crustacean NOS molecules were biochemically characterized so far. In the present study, we cloned and characterized a NOS cDNA from haemocytes of tiger shrimp (Penaeus monodon) (PmNOS). The full-length of PmNOS cDNA contained 3997 bp, including a 5'UTR of 249 bp, ORF of 3582 bp and a 3'UTR of 166 bp. The putative peptide was 1193 amino acid residues in length, with an estimated molecular weight of 134.7 kDa and pI 6.7. Structurally, PmNOS contained oxygenase and reductase domains at N-terminal and C-terminal, respectively, and connected with a calmodulin binding motif. The deduced amino acid sequence of PmNOS shared 98% identical to the Chinese shrimp (Fenneropenaeus chinensis) NOS. Phylogenetically, PmNOS clustered with invertebrate NOS, but not clustered with iNOS, eNOS or nNOS found in vertebrates. PmNOS mRNA was expressed in many tissues or organs including thoracic and ventral nerves, midgut, gill, eyestalk, haemocytes, subcuticular epithelium and heart, but not found in hepatopancreas, muscle and lymphoid organ. But there was no significant difference in PmNOS mRNA expression after stimulation with LPS either by different concentration or time course or against CpG-ODN 2006. The enzyme activities of rPmNOS or crude homogenates from different tissues were detected, and were shown its highest activity in thoracic and ventral nerves, moderate in midgut and haemocytes but the lowest activity were seen in muscle. The addition of NOS antibody against NADPH binding domain leads to less activity which suggested that NADPH was an essential cofactor for PmNOS catalytic activity. The calcium dependency of PmNOS was ascertained using calmodulin inhibitor, Trifluroperazine. To confirm the population of haemocyte which produce NOS, the florescence test was assayed, and it implicated that the production of NO was catalyzed by subset of granulocytic NOS. Since the MW range, inducible/noninducible transcript, calcium-dependent activity and tissue distribution, we suggest that PmNOS may recognize as an ancient NOS evolutionarily.

Identification and cloning of the second type transglutaminase from Litopenaeus vannamei, and its transcription following pathogen infection and in relation to the haemolymph coagulation.

Complementary (c)DNA encoding transglutaminaseII (TGII) messenger (m)RNA of white shrimp, Litopenaeus vannamei, was cloned from haemocytes by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) using oligonucleotide primers based on the TG sequence of the horseshoe crab, Tachypleus tridentatus (accession no.: BAA02134), tiger shrimp, Penaeus monodon (AAV49005; AAO33455), kuruma shrimp, Marsupenaeus japonicus (BAD36808) and Pacifastacus leniusculus (AAK69205) TG. The 2405-bp cDNA contained an open reading frame (ORF) of 2292 bp, a 31-bp 5'-untranslated region (UTR), and an 82-bp 3'-UTR containing a poly A tail. The molecular mass of the deduced amino acid (aa) sequence (764 aa) was 85.9 kDa with an estimated pI of 5.32. The L. vannamei TGII (abbreviated LvTGII) contains a typical TG-like homologue, two putative integrin binding motif (RGD and KGD), and five calcium-binding sites; three catalytic triad is present as in arthropod TG. Sequence comparison and phylogenetic analysis revealed that shrimp TG can be separated into two groups, STGI and STGII, and LvTGII is more closely related to STGII than to STGI. LvTGII mRNA was detected in all tested tissues of L. vannamei, and was highly expressed in haemocytes. The haemocytes of L. vannamei injected with Vibrio alginolyticus showed a significant increase of LvTGI and LvTGII mRNA expression at 6 h followed by a notable decrease at 24 h in LvTGI and a continually increase in LvTGII indicating a complementary effect, which implied that both LvTGs involved in the immune response of shrimp, and LvTGII was more important in the later defense response. The gene silencing of LvTGII in shrimp significantly decreased LvTGII expression and TG activity of haemocytes, and significantly increased clotting time of haemolymph, suggests that the cloned LvTGII is a clotting enzyme involved in haemolymph coagulation of L. vannamei. In conclusion, the cloned LvTGII is a clotting enzyme involved in coagulation of haemolymp and immune response of white shrimp, L. vannamei.

Dietary effect of Rubus coreanus ethanolic extract on immune gene expression in white leg shrimp, Penaeus vannamei.

The objective of this study was to evaluate the effect of dietary supplementation of a Rubus coreanus ethanolic extract on immunostimulatory response in white leg shrimp Penaeus vannamei. Shrimps with an average initial weight of 0.5 ± 0.04 g were collected and acclimatized for 10 days. Four experimental diets including a control diet, a probiotic diet and 0.25 and 0.5% of R. coreanus ethanolic extract (RcEE) diets were used to feed the shrimps. After 8 weeks of culture, shrimp fed with probiotic and 0.25% RcEE diet had showed significant enhancement in the growth while shrimp fed with 0.5% RcEE diet showed significantly increased expression of immune genes and antioxidant enzymes activities. One week of challenge experiments for all the four diets fed shrimps showed decreased cumulative mortality in the 0.5% RcEE diets fed shrimps, when compared with the probiotic and 0.25% RcEE diet fed shrimp groups. The results indicates that R. coreanus ethanolic extract could be used as a herbal immunostimulant for shrimps to increase its immunity and disease resistance against the bacterial pathogen, Vibrio alginolyticus.

Shrimp ATP synthase genes complement yeast null mutants for ATP hydrolysis but not synthesis activity.

The aim of this study was to examine the feasibility of employing a yeast functional complementation assay for shrimp genes by using the shrimp mitochondrial F(1)F(0)-ATP synthase enzyme complex as a model. Yeast mutants defective in this complex are typically respiratory-deficient and cannot grow on non-fermentable carbon sources such as glycerol, allowing easy verification of functional complementation by yeast growth on media with them as the only carbon source. We cloned the previous published sequence of ATP2 (coding for ATP synthase ß subunit) from the Pacific white shrimp Penaeus vannamei (Pv) and also successfully amplified a novel PvATP3 (coding for the ATP synthase γ subunit). Analysis of the putative amino acid sequence of PvATP3 revealed a significant homology with the ATP synthase γ subunit of crustaceans and insects. Complementation assays were performed using full-length ATP2 and ATP3 as well as a chimeric form of ATP2 containing a leader peptide sequence from yeast and a mature sequence from shrimp. However, the shrimp genes were unable to complement the growth of respective yeast mutants on glycerol medium, even though transcriptional expression of the shrimp genes from plasmid-borne constructs in the transformed yeast cells was confirmed by RT-PCR. Interestingly, both PvATP2 and PvATP3 suppressed the lethality of the yeast F(1) mutants after the elimination of mitochondrial DNA, which suggests the assembly of a functional F(1) complex necessary for the maintenance of membrane potential in the ρ(0) state. These data suggest an incompatibility of the shrimp/yeast chimeric F(1)-ATPase with the stalk and probably also the F(0) sectors of the ATP synthase, which is essential for coupled energy transduction and ATP synthesis.