Chemical composition of a hot water crude extract (HWCE) from Ulva intestinalis and its potential effects on growth performance, immune responses, and resistance to white spot syndrome virus and yellowhead virus in Pacific white shrimp (Litopenaeus vannamei).

In the present study, a hot water crude extract from Ulva intestinalis (Ui-HWCE) was used as a dietary supplement, and the effects on growth, immune responses, and resistance against white spot syndrome virus (WSSV) and yellowhead virus (YHV) infection in Pacific white shrimp (Litopenaeus vannamei) were investigated. Chemical analyses of Ui-HWCE revealed 13.75 ± 0.41% sulfate, 37.86 ± 5.96% uronic acid, and 46.63 ± 5.16% carbohydrate contents. The monosaccharide content of Ui-HWCE contained glucose (6.81 ± 0.94%), xylose (4.15 ± 0.11%), and rhamnose (25.84 ± 0.80%). Functional group analysis of Ui-HWCE by Fourier transform infrared (FTIR) spectroscopy revealed a typical infrared spectrum of ulvan similar to the infrared spectrum of commercially purified ulvan from Ulva armoricana (77.86 ± 2.19% similarity). Ui-HWCE was added to shrimp diets via top-dressing at 0, 1, 5, and 10 g/kg diet. After 28 days, Ui-HWCE supplementation at 5 g/kg diet efficiently improved shrimp growth performance, as indicated by weight gain, average daily growth, specific growth rates, and villus height determined by observing gut morphology. Additionally, Ui-HWCE feed supplementation at 5 g/kg diet significantly increased immune responses against a pathogenic bacterium (Vibrio parahaemolyticus AHPND stain), including phagocytic activity and clearance efficiency. Furthermore, Ui-HWCE feed supplementation upregulated the expression of several immune-related genes in the hemocytes and gills. Ui-HWCE supplementation at 1 and 5 g/kg resulted in effective anti-YHV but not anti-WSSV activity, which significantly decreased the mortality rate and YHV burden in surviving shrimp. It was concluded that Ui-HWCE supplied at 5 g/kg diet exhibits growth-promoting, immune-stimulatory, and antiviral activity that could protect L. vannamei against YHV infection.

Dietary Bacillus amyloliquefaciens enhance survival of white spot syndrome virus infected crayfish.

Bacillus amyloliquefaciens, which is closely related to Bacillus subtilis, produces a series of metabolites that can inhibit the growth of fungi and bacteria. Here, we investigated the effect of B. amyloliquefaciens used as a probiotic on the innate immunity of the crayfish Procambarus clarkii when challenged with white spot syndrome virus (WSSV). Dietary B. amyloliquefaciens supplement significantly reduced the mortality of WSSV-challenged crayfish and reduced copy numbers of WSSV. The quantitative reverse transcription-polymerase chain reaction results showed that B. amyloliquefaciens supplement increased the expression of several immune-related genes, including Toll-like receptor, NF-κB and C-type-lectin. Further analysis showed that B. amyloliquefaciens supplement also had an effect on three immune parameters, including total hemocyte count, phenoloxidase activity and superoxide dismutase activity. In both infected and uninfected crayfish, B. amyloliquefaciens supplement significantly decreased hemocyte apoptosis. Our results showed that B. amyloliquefaciens can regulate innate immunity of crayfish and reduce the mortality following WSSV challenge. This study provides a novel insight into the potential for therapeutic or prophylactic intervention with B. amyloliquefaciens to regulate crayfish immunity and protect against WSSV infection, and also provides a theoretical basis for the use of probiotics as aquatic feed additives.

Dietary Hizikia fusiforme enhance survival of white spot syndrome virus infected crayfish Procambarus clarkii.

The sea vegetable Hizikia fusiforme is not only a good source of dietary fiber but also enhances immunity. In this study, we investigated the effects of H. fusiforme on innate immunity in invertebrates, using white spot syndrome virus (WSSV) challenge in the crayfish, Procambarus clarkii. Supplementation with H. fusiforme significantly reduced mortality caused by WSSV infection and also reduced copy numbers of the WSSV protein VP28. Quantitative reverse transcription-polymerase chain reaction showed that supplementation of feed with H. fusiforme increased the expression of immune-related genes, including NF-κB and crustin 1. Further analysis showed that supplementation with H. fusiforme also affected three immune parameters, total hemocyte count, and phenoloxidase and superoxide dismutase activity. H. fusiforme treatment significantly increased hemocyte apoptosis rates in both WSSV-infected and uninfected crayfish. H. fusiforme thus regulates the innate immunity of crayfish, and both delays and reduces mortality after WSSV challenge. Our study demonstrates the potential for the commercial use of H. fusiforme, either therapeutically or prophylactically, to regulate the innate immunity and protect crayfish against WSSV infection.

Hesperetin protects crayfish Procambarus clarkii against white spot syndrome virus infection.

Hesperetin is a natural flavanone compound, which mainly exists in lemons and oranges, and has potential antiviral and anticancer activities. In this study, hesperetin was used in a crayfish pathogen challenge to discover its effects on the innate immune system of invertebrates. The crayfish Procambarus clarkii was used as an experimental model and challenged with white spot syndrome virus (WSSV). Pathogen challenge experiments showed that hesperetin treatment significantly reduced the mortality caused by WSSV infection, while the VP28 copies of WSSV were also reduced. Quantitative reverse transcriptase polymerase chain reaction revealed that hesperetin increased the expression of several innate immune-related genes, including NF-kappaB and C-type lectin. Further analysis showed that hesperetin treatment plays a positive effects on three immune parameters like total hemocyte count, phenoloxidase and superoxide dismutase activity. Nevertheless, whether or not infected with WSSV, hesperetin treatment would significantly increase the hemocyte apoptosis rates in crayfish. These results indicated that hesperetin could regulate the innate immunity of crayfish, and delaying and reducing the mortality after WSSV challenge. Therefore, the present study provided novel insights into the potential therapeutic or preventive functions associated with hesperetin to regulate crayfish immunity and protect crayfish against WSSV infection, provide certain theoretical basis for production practice.

Effect of Argemone mexicana active principles on inhibiting viral multiplication and stimulating immune system in Pacific white leg shrimp Litopenaeus vannamei against white spot syndrome virus.

Argemone mexicana called as Mexican prickly poppy is a species of poppy found in Mexico and now widely naturalized in many parts of the world with broad range of bioactivities including anthelmintic, cures lepsory, skin-diseases, inflammations and bilious fevers. Plant parts of A. mexicana were serially extracted with hexane, ethyl acetate, methanol and performed antiviral and immunostimulant screening against WSSV and Vibrio harveyi respectively. The control groups succumbed to death 100% within three days, whereas the mortality was significantly (P < 0.5) reduced to 17.43 and 7.11 in the ethyl acetate extracts of stem and root treated shrimp group respectively. The same trend was reflected in the immunostimulant screening also. Different diets were prepared by the concentrations of 100 (AD-1), 200 (AD-2), 300 (AD-3) and 400 (AD-4) mg kg-1 using A. mexicana stem and root ethyl acetate extracts and fed to Pacific white leg shrimp Litopenaeus vannamei weighed about 9.0 ±â€¯0.5 g for 30 days. The control groups fed with the normal diets devoid of A. mexicana extracts. The antiviral screening results revealed that, the ethyl acetate extract of the stem and root were effectively suppressed the WSSV and it reflected in the lowest cumulative mortality of treated shrimps. After termination of feeding trials, group of shrimps from control and each experimental group were challenged with virulent WSSV by intramuscular (IM) injection and studied cumulative mortality, molecular diagnosis by quantitative real time PCR (qRT-PCR), biochemical, haematological and immunological parameters. Control group succumbed to 100% death within four days, whereas the survival was significantly (P < 0.001) increased to 30, 45, 75 and 79% in AD1, AD-2, AD-4 and AD-5 diets fed shrimp groups respectively. qRT PCR results with positive correlation analysis revealed that, the WSSV copies were gradually decreased when increasing the A. mexicana extracts in the diets. The highest concentrations (300 and 400 mg g-1) of A. mexicana extracts in the diets helped to reduce the protein level significantly (P < 0.05) after WSSV challenge. The diets AD-3 and AD-4 also helped to decrease the coagulation time of maximum 64-67% from control groups and maintained the normal level of total haemocyte, oxyhaemocyanin level after WSSV challenge. The proPO level was significantly increased (Column: F = 35.93; P ≤ 0.001 and Row: F = 37.14; P ≤ 0.001) in the AD1-AD-4 diet fed groups from the control diet fed groups. The lowest intra-agar lysozyme activity of 1.63 mm found in control diet fed group and the activity were significantly (P < 0.05) increased to 4.86, 7.89, 9.12 and 10.45 mm of zone of inhibition respectively in AD1 to AD4 diet fed groups.

A CqFerritin protein inhibits white spot syndrome virus infection via regulating iron ions in red claw crayfish Cherax quadricarinatus.

It is well known that iron is an essential element for all living organism. The intracellular iron availability is also important for the host's innate immune response to various pathogens, in which the iron homeostasis can be regulated by ferritin due to its iron storage property. In this study, a full-length cDNA sequence of ferritin (named as CqFerritin) was identified with 1410 bp from red claw crayfish Cherax quadricarinatus, which contained an open reading frame of 513 bp, encoding 170 amino acids with a conserved ferritin domain. Tissue distribution analysis demonstrated that CqFerritin was widely expressed in various tissues with high presence in haemocyte, haematopoietic tissue (Hpt) and heart, while lowest expression in hepatopancreas. In addition, loss-of-function of CqFerritin by gene silencing resulted in significantly higher expression of an envelope protein VP28 of white spot syndrome virus (WSSV) in red claw crayfish Hpt cell cultures, indicating the potential antiviral response of CqFerritin. To further explore the effect on WSSV replication by CqFerritin, recombinant CqFerritin protein (rCqFerritin) was transfected into Hpt cells followed by WSSV infection. Importantly, the replication of WSSV was obviously decreased in Hpt cells if transfected with rCqFerritin protein, suggesting that CqFerritin had clearly negative effect on WSSV infection. Furthermore, intracellular accumulation of iron ions was found to promote the WSSV replication in a dose-dependent manner, illustrating that the iron level regulated by CqFerritin was likely to be vital for WSSV infection in red claw crayfish. Taken together, these data suggest that CqFerritin plays an important role in immune defense against WSSV infection in a crustacean C. quadricarinatus.

Role of Litopenaeus vannamei Yin Yang 1 in the Regulation of the White Spot Syndrome Virus Immediate Early Gene ie1.

Yin Yang 1 (YY1) is a multifunctional zinc finger transcription factor that regulates many key cellular processes. In this study, we report the cloning of YY1 from Litopenaeus vannamei shrimp (LvYY1). This study shows that LvYY1 is ubiquitously expressed in shrimp tissues, and knockdown of LvYY1 expression by double-stranded RNA (dsRNA) injection in white spot syndrome virus (WSSV)-infected shrimp reduced both mRNA levels of the WSSV immediate early gene ie1 as well as overall copy numbers of the WSSV genome. The cumulative mortality rate of infected shrimp also declined with LvYY1 dsRNA injection. Using an insect cell model, we observed that LvYY1 activates ie1 expression, and a mutation introduced into the ie1 promoter subsequently repressed this capability. Moreover, reporter assay results suggested that LvYY1 is involved in basal transcriptional regulation via an interaction with L. vannamei TATA-binding protein (LvTBP). Electrophoretic mobility shift assay (EMSA) results further indicated that LvYY1 binds to a YY1-binding site in the region between positions -119 and -126 in the ie1 promoter. Chromatin immunoprecipitation analysis also confirmed that LvYY1 binds to the ie1 promoter in WSSV-infected shrimp. Taken together, these results indicate that WSSV uses host LvYY1 to enhance ie1 expression via a YY1-binding site and the TATA box in the ie1 promoter, thereby facilitating lytic activation and viral replication.IMPORTANCE WSSV has long been a scourge of the shrimp industry and remains a serious global threat. Thus, there is a pressing need to understand how the interactions between WSSV and its host drive infection, lytic development, pathogenesis, and mortality. Our successful cloning of L. vannamei YY1 (LvYY1) led to the elucidation of a critical virus-host interaction between LvYY1 and the WSSV immediate early gene ie1 We observed that LvYY1 regulates ie1 expression via a consensus YY1-binding site and TATA box. LvYY1 was also found to interact with L. vannamei TATA-binding protein (LvTBP), which may have an effect on basal transcription. Knockdown of LvYY1 expression inhibited ie1 transcription and subsequently reduced viral DNA replication and decreased cumulative mortality rates of WSSV-infected shrimp. These findings are expected to contribute to future studies involving WSSV-host interactions.

Dietary chitosan nanoparticles protect crayfish Procambarus clarkii against white spot syndrome virus (WSSV) infection.

Chitosan nanoparticles have exhibited potential antibacterial activity or anticancer activity as their unique character. In this study, we investigated the effect of chitosan nanoparticles protect crayfish Procambarus clarkii against WSSV. Chitosan (from crab shell) nanoparticles were prepared by ultrafine milling. The physicochemical properties of the nanoparticles were determined by particle size measure, zeta potential analysis and scanning electron microscope observation. The total hemocyte count (THC), phenoloxidase (PO) and superoxide dismutase (SOD) activity were measured at days 1, 4, 9 and 12, and the survival rate was also recorded after WSSV challenge. The results showed that chitosan nanoparticles could enhance the survival rate of WSSV-challenged crayfish. And crayfish fed diets supplemented with 10 mg/g chitosan nanoparticles (65% mortality) showed a significantly higher survival rate when compared to the control group (100% mortality). The analysis of immunological parameters revealed that 10 mg/g chitosan nanoparticles showed significantly higher level of prophenoloxidase (proPO), superoxide dismutase (SOD) and total hemocyte count (THC) when compared to the control group. It was found that chitosan nanoparticles could inhibit WSSV replication in crayfish. Our results demonstrated that dietary chitosan nanoparticles effectively improve innate immunity and survival of P. clarkii challenged with WSSV.

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.

Sulfated galactans isolated from the red seaweed Gracilaria fisheri target the envelope proteins of white spot syndrome virus and protect against viral infection in shrimp haemocytes.

The present study was aimed at evaluating an underlying mechanism of the antiviral activity of the sulfated galactans (SG) isolated from the red seaweed Gracilaria fisheri against white spot syndrome virus (WSSV) infection in haemocytes of the black tiger shrimp Penaeus monodon. Primary culture of haemocytes from Penaeus monodon was performed and inoculated with WSSV, after which the cytopathic effect (CPE), cell viability and viral load were determined. Haemocytes treated with WSSV-SG pre-mix showed decreased CPE, viral load and cell mortality from the viral infection. Solid-phase virus-binding assays revealed that SG bound to WSSV in a dose-related manner. Far Western blotting analysis indicated that SG bound to VP 26 and VP 28 proteins of WSSV. In contrast to the native SG, desulfated SG did not reduce CPE and cell mortality, and showed low binding activity with WSSV. The current study suggests that SG from Gracilaria fisheri elicits its anti-WSSV activity by binding to viral proteins that are important for the process of viral attachment to the host cells. It is anticipated that the sulfate groups of SG are important for viral binding.

Immune responses of prophenoloxidase and cytosolic manganese superoxide dismutase in the freshwater crayfish Cherax quadricarinatus against a virus and bacterium.

Prophenoloxidase (proPO) and cytosolic manganese superoxide dismutase (cytMnSOD) play crucial roles in crustacean innate immunity. In the present study, both of the above genes were cloned from hemocytes of the red claw crayfish Cherax quadricarinatus. A phylogenetic analysis of the amino acid sequences showed that C. quadricarinatus proPO and cytMnSOD were more closely related to the proPO and cytMnSOD of other crayfish than to those of penaeids, crabs, lobsters, or freshwater prawns. A tissue distribution analysis revealed that proPO was primarily expressed in hemocytes, gills, and the heart, while cytMnSOD was detected in all tissues examined. All of the crayfish artificially infected with white spot syndrome virus (WSSV) died within 4 days. According to a non-lethal dose, there was no mortality in crayfish when infected deliberately with Aeromonas hydrophila. Total hemocyte counts (THCs) had significantly decreased in crayfish at 48 and 72 h after infection with WSSV compared to the control group. In contrast, THCs of crayfish after A. hydrophila challenge had recovered by 48 and 72 h from a lower level at 24 h. There were similar responses in enzyme activities toward WSSV and A. hydrophila infection. Phenoloxidase (PO) and superoxide dismutase (SOD) activities per hemocyte significantly increased from 48 to 72 h compared to the control group. After WSSV challenge, expressions of proPO and cytMnSOD transcripts in hemocytes significantly decreased at 12h, then had respectively recovered and increased at 24 h. At 48-72 h, transcript levels were finally downregulated. No significant differences in the expression profiles of proPO and cytMnSOD were observed between the A. hydrophila-infected and control groups, besides the significant upregulation at 24h post-infection. These results implicate proPO and cytMnSOD in the immune response, and they presented similar expression patterns, although different defense mechanisms may exist for crayfish induced by WSSV and A. hydrophila.

Susceptibility of juvenile European lobster Homarus gammarus to shrimp products infected with high and low doses of white spot syndrome virus.

White spot syndrome virus (WSSV) is the most important pathogen known to affect the sustainability and growth of the global penaeid shrimp farming industry. Although most commonly associated with penaeid shrimp farmed in warm waters, WSSV is also able to infect, cause disease in and kill a wide range of other decapod crustaceans, including lobsters, from temperate regions. In 2005, the European Union imported US$500 million worth of raw frozen or cooked frozen commodity products, much of which originated in regions positive for white spot disease (WSD). The presence of WSSV within the UK food market was verified by means of nested PCR performed on samples collected from a small-scale survey of supermarket commodity shrimp. Passage trials using inoculum derived from commodity shrimp from supermarkets and delivered by injection to specific pathogen-free Pacific white shrimp Litopenaeus vannamei led to rapid mortality and pathognomonic signs of WSD in the shrimp, demonstrating that WSSV present within commodity shrimp was viable. We exposed a representative European decapod crustacean, the European lobster Homarus gammarus, to a single feeding of WSSV-positive, supermarket-derived commodity shrimp, and to positive control material (L. vannamei infected with a high dose of WSSV). These trials demonstrated that lobsters fed positive control (high dose) frozen raw products succumbed to WSD and displayed pathognomonic signs associated with the disease as determined by means of histology and transmission electron microscopy. Lobsters fed WSSV-positive, supermarket-derived commodity shrimp (low dose) did not succumb to WSD (no mortality or pathognomonic signs of WSD) but demonstrated a low level or latent infection via PCR. This study confirms susceptibility of H. gammarus to WSSV via single feedings of previously frozen raw shrimp products obtained directly from supermarkets.

Protection of Penaeus monodon against white spot syndrome virus by inactivated vaccine with herbal immunostimulants.

To improve the immune response in tiger shrimp Penaeus monodon against WSSV infection, juveniles (350 ± 10 mg) were vaccinated with formalin-inactivated WSSV and fed with herbal immunostimulants. The methanolic extracts of herbal immunostimulants such as Acalypha indica, Cynodon dactylon, Picrorrhiza kurrooa, Withania somnifera and Zingiber officinalis were incorporated in formulated diets at different concentrations; 250 (ED(1)), 500 (ED(2)), 1000 (ED(3)) and 2000 (ED(4)) mg kg(-1) of feed and fed for 60 days after vaccination. After 30 and 60 days intervals of feeding, the shrimps were challenged with WSSV, which were isolated and propagated from the infected crustaceans. The shrimps fed with control diets (C(1)) succumbed to death within 5 days after WSSV challenge, when no vaccination and immunostimulations were given. The other control groups (C(2) and C(3)) had slight improvements in all parameters including survival. The percentage survival was significantly (P < 0.05) increased to 30, 50 and 60% in the ED(2), ED(3) and ED(4) diets respectively after 60 days challenging. The better haematological, biochemical and immunological parameters were also found in the herbal extracts supplemented diets fed vaccinated shrimps. The present study revealed that the combined effect of immunostimulation and vaccination helped to boost the immune system against WSSV infection and hence this application can be adopted for shrimp culture.

Studies on the immunomodulatory effect of polysaccharide gel extracted from Durio zibethinus in Penaeus monodon shrimp against Vibrio harveyi and WSSV.

Oral administration of polysaccharide gel (PG) in shrimp diets revealed immunostimulating potential and disease resistance in Penaeus monodon (black tiger shrimp). PG from the fruit-rind of Durio zibethinus has been characterized to be a pectic polysaccharide with immunomodulating and antibacterial activities. PG inhibited growth of the shrimp bacterial pathogen, Vibrio harveyi 1526, by agar diffusion and broth microdilution tests. Clear inhibition zones on agar plates were observed at the lowest PG concentration of 3.1 mg/ml, where minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values for PG were 6.3 and 12.5 mg/ml, respectively. Each group of juvenile shrimps, initial mean body weight 0.29 +/- 0.04 g, was housed in a closed-recirculating treated water system and was fed with PG-supplemented diets containing 1, 2 and 3% PG or shrimp basal diet in the control group for 8 and 12 weeks. PG-supplemented diets did not contribute to the overall growth of black tiger shrimp. The immune response was evaluated by analysis of prophenoloxidase activity and total hemocyte count in the shrimp fed PG-supplemented diets for 12 weeks. Prophenoloxidase activity in shrimp fed the 1, 2 and 3% PG-supplemented diet and total hemocyte count in shrimp fed the 1 and 2% PG-supplemented diet were higher (P < 0.05) than those of the control group. The percent survival was higher in groups fed the 1-3% PG-supplemented diets in challenge tests with either white spot syndrome virus (WSSV) or the bacterium V. harveyi 1526 than that of the control group. Relative percent survival (RPS) values in groups fed the 2% PG-supplemented diet showed the highest RPS value for disease resistance of 100% (at Day 6) and 36% (at Day 4) in treated shrimp against viral and bacterial infection, respectively. Mortality of PG-supplemented diets in treated shrimps against WSSV infection was also found to be much lower (P < 0.05) than that of the control group.

Comparative analysis of differentially expressed genes in normal and white spot syndrome virus infected Penaeus monodon.

BACKGROUND: White spot syndrome (WSS) is a viral disease that affects most of the commercially important shrimps and causes serious economic losses to the shrimp farming industry worldwide. However, little information is available in terms of the molecular mechanisms of the host-virus interaction. In this study, we used an expressed sequence tag (EST) approach to observe global gene expression changes in white spot syndrome virus (WSSV)-infected postlarvae of Penaeus monodon. RESULTS: Sequencing of the complementary DNA clones of two libraries constructed from normal and WSSV-infected postlarvae produced a total of 15,981 high-quality ESTs. Of these ESTs, 46% were successfully matched against annotated genes in National Center of Biotechnology Information (NCBI) non-redundant (nr) database and 44% were functionally classified using the Gene Ontology (GO) scheme. Comparative EST analyses suggested that, in postlarval shrimp, WSSV infection strongly modulates the gene expression patterns in several organs or tissues, including the hepatopancreas, muscle, eyestalk and cuticle. Our data suggest that several basic cellular metabolic processes are likely to be affected, including oxidative phosphorylation, protein synthesis, the glycolytic pathway, and calcium ion balance. A group of immune-related chitin-binding protein genes is also likely to be strongly up regulated after WSSV infection. A database containing all the sequence data and analysis results is accessible at http://xbio.lifescience.ntu.edu.tw/pm/. CONCLUSION: This study suggests that WSSV infection modulates expression of various kinds of genes. The predicted gene expression pattern changes not only reflect the possible responses of shrimp to the virus infection but also suggest how WSSV subverts cellular functions for virus multiplication. In addition, the ESTs reported in this study provide a rich source for identification of novel genes in shrimp.

The cytosolic manganese superoxide dismutase from the shrimp Litopenaeus vannamei: molecular cloning and expression.

Manganese containing superoxide dismutase (SOD) is normally a nuclear-encoded mitochondrial enzyme in eukaryotic organisms; however, a cytoplasmic manganese SOD (cMnSOD) was found in crustaceans that use hemocyanin as oxygen carrier. The complete cDNA and deduced amino acid sequence of a cMnSOD from Litopenaeus vannamei were determined. The coding sequence predicts a 287 residues protein with a unique 61 amino acids extension at the N-terminus and lacking a mitochondrial-targeting sequence. Phylogenetic analysis clusters cMnSODs and mitochondrial MnSODs in two separate groups. cMnSOD transcripts were detected in hemocytes, heart, hepatopancreas, intestine, nervous system, muscle, pleopods and gills. Since hemocytes are key defense cells and their reactions produce superoxide radicals, the infection by white spot syndrome virus on the cMnSOD transcript levels were investigated and found to increase transiently 1h post-infection and then decrease as the viral infection progressed to levels significantly lower than uninfected controls by 12h post-infection.