An immersion of Gracilaria tenuistipitata extract improves the immunity and survival of white shrimp Litopenaeus vannamei challenged with white spot syndrome virus

The innate immunity and resistance against white spot syndrome virus (WSSV) in white shrimp Litopenaeus vannamei which received the Gracilaria tenuistipitata extract were examined. Shrimp immersed in seawater containing the extract at 0 (control), 400 and 600 mg L(-1) for 3 h were challenged with WSSV at 2 × 10(4) copies shrimp(-1). Shrimp not exposed to the extract and not received WSSV challenge served as unchallenged control. The survival rate of shrimp immersed in 400 mg L(-1) or 600 mg L(-1) extract was significantly higher than that of challenged control shrimp over 24-120 h. The haemocyte count, phenoloxidase activity, respiratory burst, superoxide dismutase activity, and lysozyme activity of shrimp immersed in 600 mg L(-1) extract were significantly higher than those of unchallenged control shrimp at 6, 6, 6, 6, and 6-24 h post-challenge. In another experiment, shrimp which had received 3 h immersion of 0, 400, 600 mg L(-1) extract were challenged with WSSV. The shrimp were then received a booster (3 h immersion in the same dose of the extract), and the immune parameters were examined at 12-120 h post-challenge. The immune parameters of shrimp immersed in 600 mg L(-1) extract, and then received a booster at 9, 21, and 45 h were significantly higher than those of unchallenged control shrimp at 12-48 h post-challenge. In conclusion, shrimp which had received the extract exhibited protection against WSSV as evidenced by the higher survival rate and higher values of immune parameters. Shrimp which had received the extract and infected by WSSV showed improved immunity when they received a booster at 9, 21, and 45 h post-WSSV challenge. The extract treatment caused less decrease in PO activity, and showed better performance of lysozyme activity and antioxidant response in WSSV-infected shrimp.     

Influence of selected Indian immunostimulant herbs against white spot syndrome virus (WSSV) infection in black tiger shrimp, Penaeus monodon with reference to haematological, biochemical and immunological changes

Immunostimulants are the substances, which enhance the non-specific defence mechanism and provide resistance against the invading pathogenic micro-organism. In order to increase the immunity of shrimps against the WSSV, the methanolic extracts of five different herbal medicinal plants like Cyanodon dactylon, Aegle marmelos, Tinospora cordifolia, Picrorhiza kurooa and Eclipta alba were selected and mixed thoroughly in equal proportion. The mixed extract was supplemented with various concentrations viz. 100 (A), 200 (B), 400 (C), and 800 (D) mgkg(-1) through artificial diets individually. The prepared diets (A-D) were fed individually to WSSV free healthy shrimp Penaeus monodon with an average weight of 8.0+/-0.5g for 25 days. Control diet (E), devoid of herbal extract was also fed to shrimps simultaneously. After 25 days of feeding experiment, the shrimps were challenged with WSSV, which were isolated and propagated from the infected crustaceans. The shrimps succumbed to death within 7 days when fed on no herbal immunostimulant diet (E). Among the different concentrations of herbal immunostimulant supplemented diets, the shrimps fed on diet D (800mgkg(-1)) significantly (P<0.0001) had more survival (74%) and reduction in the viral load. Also the better performance of haematological, biochemical and immunological parameters was found in the immunostimulant incorporated diets fed shrimps. The present work revealed that the application of herbal immunostimulants will be effective against shrimp viral pathogenesis and they can be recommended for shrimp culture.     

Dietary administration of a Gracilaria tenuistipitata extract enhances the immune response and resistance against Vibrio alginolyticus and white spot syndrome virus in the white shrimp Litopenaeus vannamei

The haemogram, phenoloxidase (PO) activity, respiratory bursts (RBs), superoxide dismutase (SOD) activity, glutathione peroxidase (GPx) activity, lysozyme activity, and the mitotic index of haematopoietic tissue (HPT) were examined after the white shrimp Litopenaeus vannamei had been fed diets containing the hot-water extract of Gracilaria tenuistipitata at 0 (control), 0.5, 1.0, and 2.0 g kg(-1) for 7-35 days. Results indicated that these parameters directly increased with the amount of extract and time, but slightly decreased after 35 days. RBs, SOD activity, and GPx activity reached the highest levels after 14 days, whereas PO and lysozyme activities reached the highest levels after 28 days. In a separate experiment, white shrimp L. vannamei, which had been fed diets containing the extract for 14 days, were challenged with Vibrio alginolyticus at 2 × 10(6) cfu shrimp(-1) and white spot syndrome virus (WSSV) at 1 × 10(3) copies shrimp(-1), and then placed in seawater. The survival rate of shrimp fed the extract-containing diets was significantly higher than that of shrimp fed the control diet at 72-144 h post-challenge. We concluded that dietary administration of the G. tenuistipitata extract at ≤1.0 g kg(-1) could enhance the innate immunity within 14 days as evidenced by the increases in immune parameters and mitotic index of HPT in shrimp and their enhanced resistance against V. alginolyticus and WSSV infections. Shrimp fed the extract-containing diets showed a higher and continuous increase in the humoral response indicating its persistent role in innate immunity.     

White shrimp Litopenaeus vannamei immersed in seawater containing Sargassum hemiphyllum var. chinense powder and its extract showed increased immunity and resistance against Vibrio alginolyticus and white spot syndrome virus

This study was to examine the immune response of white shrimp Litopenaeus vannamei and its resistance against Vibrio alginolyticus and WSSV when shrimp received the Sargassum hemiphyllum var. chinense powder and its hot-water extract. Both powder and extract showed activation of prophenoloxidase and generation of superoxide anion in the shrimp in vitro. The haemocyte count, phenoloxidase (PO) activity, respiratory burst, and lysozyme activity were examined after the shrimp were immersed in seawater containing S. hemiphyllum var. chinense powder or its extract at 0, 100, 300, and 500 mg L?1 for 1, 3, and 5 h. These immune parameters of shrimp immersed in 300 and 500 mg L?1 powder, and 100 and 300 mg L?1 extract were significantly higher than those of control shrimp after 3 h, but slightly decreased after 5 h. In another experiment, shrimp immersed in seawater containing the powder or the extract at 0, 100, 300, and 500 mg L?1 after 3 h were challenged with V. alginolyticus at 8 × 10? colony-forming unit (cfu) shrimp?1, or challenged with WSSV at 1 × 10? copies shrimp?1, and then placed in seawater. Survival rate of shrimp immersed in 500 mg L?1 powder was significantly higher than that of control shrimp after 24-120 h in the V. alginolyticus-challenge test, and after 72 h in the WSSV-challenge test, respectively. Survival rate of shrimp immersed in 300 mg L?1 extract was significantly higher than that of control shrimp after 72-120 h in both V. alginolyticus-challenge and WSSV-challenge tests. It was concluded that the shrimp immersed in seawater containing the powder at 500 mg L?1, and the extract at 300 mg L?1 had increased immunity and resistance against V. alginolyticus infection, and the shrimp that received extract at 300 mg L?1 showed resistance against WSSV infection.     

Effects of immunostimulants targeting Ran GTPase on phagocytosis against virus infection in shrimp

The global shrimp aquaculture has been consistently beset by diseases that cause severe losses in production. To fight various harmful pathogens, the enhanced shrimp immunity by immunostimulants would play key roles against the invading pathogens. In aquaculture, however, the target proteins/genes which can be used for the screening of immunostimulants are very limited. Based on our previous study, in the present study, the shrimp Ran protein, which was required in shrimp antiviral phagocytosis, was used as the target protein to screen for immunostimulants. The GTPase activity assays showed that the IL-4 and lysophosphatidylcholine molecules could enhance the activity of Ran protein, suggesting that the two molecules might function in phagocytosis. When the IL-4 and lysophosphatidylcholine were respectively injected into shrimp, the results indicated that the two molecules enhanced the hemocytic phagocytosis against white spot syndrome virus (WSSV), suggesting that they improved the activity of phagocytosis through the activation of the Ran protein. It was evidenced that the enhancement of phagocytosis activity effectively inhibited the WSSV infection in shrimp, which further led to the decrease of mortalities of WSSV-infected shrimp. Therefore, our study presented a novel strategy for the screening of immunostimulants by using the key proteins in immune responses of aquatic organisms as the target proteins, which would be very helpful for the development of efficient approaches to prevent the aquatic organisms from pathogen infections.     

Comparison of antiviral efficiency of immune responses in shrimp

The antiviral effectiveness of three major immune responses including phagocytosis, apoptosis and proPO system in the shrimp Marsupenaeus japonicus was characterized. It was found that the suppression of apoptosis and phagocytosis in vivo by their corresponding inhibitors resulted in the increase of white spot syndrome virus (WSSV) copies and shrimp mortality, whereas the inhibition of phenol oxidase generated the least influence on WSSV infection and shrimp mortality. These results suggest that apoptosis and phagocytosis were the essential immune responses to protect shrimp from virus infection, while the phenol-oxidase-dependent proPO system plays a comparatively minor role in antiviral defense of shrimp.     

Dietary supplementation with Cynodon dactylon (L.) enhances innate immunity and disease resistance of Indian major carp, Catla catla (Ham.)

Indian major carp (Catla catla) was subjected to study the immunostimulatory effects when the grass Cynodon dactylon(L) ethanolic extract administrated as feed supplement. C. catla was fed with 0% (Control), 0.05% (group I), 0.5% (group II) and 5% (group III) extract provided for 60 days. Blood samples were collected at every 10 days of interval up to 60 days for analyzing the non-specific humoral (lysozyme activity, antiprotease activity and haemolytic complement) and cellular (production of reactive oxygen and nitrogen species, myeloperoxidase activity) immune response study. The results indicate that C. dactylon ethanolic extract administered as feed supplement significantly (P < 0.05) enhanced most of the non-specific immune parameters tested. Among the experimental diet groups, significantly increased response of non-specific immunity was seen in group III (5%). Disease resistant analysis against Aeromonas hydrophila was performed in control group and plant extract treated fish for 7, 14, 21 and 28 days. Relative percent survival rate (RPS) was observed in treated samples, which is directly proportional to concentration of the extract. Additionally, electron microscopic studies and gelatin zymography for Matrix Metalo Proteinase (MMPs) were examined in spleen at 7th and 28th days of feeding. Administration of C. dactylon mixed diet delayed the lymphocyte destruction with positive ultrastructural changes. An induced stress (A. hydrophila infection) was observed by using MMPs expression, which was reduced in the experimental diet groups than the control. All these experimental results prove that C. dactylon ethanolic extract enhances the immunity of Catla fish.     

对虾抗病毒免疫研究现状

白斑综合征病毒（white spot syndrome virus,wssv）,是对虾养殖中主要的致病病毒,在全球范围内引起急性感染和致死反应,带来巨大的经济损失。之前的研究主要集中在对虾的先天免疫反应上,在抗病毒反应方面的尚所知有限。总结了近年来对虾抗病毒免疫研究取得的主要成果,概括了病毒侵染对虾后分子水平上的改变,旨在为更加有效的预防和治疗白斑综合症提供参考。     

Enzyme E2 from Chinese white shrimp inhibits replication of white spot syndrome virus and ubiquitinates its RING domain proteins

Recent studies have shown that the ubiquitin (Ub) proteasome pathway (UPP) is closely related to immune defense. We have identified a ubiquitin-conjugating enzyme, E2, from the Chinese white shrimp, Fenneropenaeus chinensis (FcUbc). Injection of recombinant FcUbc protein (rFcUbc) reduced the mortality of shrimp infected with white spot syndrome virus (WSSV) and inhibited replication of WSSV. rFcUbc, but not a mutant FcUbc (mFcUbc), bound to WSSV RING domains (WRDs) from four potential E3 ligase proteins of WSSV in vitro. Importantly, rFcUbc could ubiquitinate the RING domains (named WRD2 and WRD3) of WSSV277 and WSSV304 proteins in vitro and the two proteins in WSSV-infected Drosophila melanogaster Schneider 2 (S2) cells. Furthermore, overexpression of FcUbc increased ubiquitination of WSSV277 and WSSV304 during WSSV infection. In summary, our study demonstrates that FcUbc from Chinese white shrimp inhibited WSSV replication and could ubiquitinate WSSV RING domain-containing proteins. This is the first report about antiviral function of Ubc E2 in shrimp.     

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.     

Contribution of the Argonaute-1 Isoforms to Invertebrate Antiviral Defense

Argonaute (Ago) protein, the central component of the RNA interference (RNAi) pathway, plays important roles in host innate antiviral immunity. Most organisms harbor a large number of different Ago proteins and isoforms; however, the roles of Ago isoforms in immune defense against pathogens remain unclear. In the present study, three Argonaute-1 (Ago1) isoforms, termed Ago1A, Ago1B, and Ago1C, were found in Marsupenaeus japonicus shrimp. Quantitative real-time PCR (polymerase chain reaction) revealed that isoforms Ago1A and Ago1B containing an insertion sequence in the PIWI domain, were significantly up-regulated in lymphoid organ and hemolymph, and also upon white spot syndrome virus (WSSV) challenge, indicating the involvement of Ago1A and Ago1B in antiviral immunity. The results showed that silencing of Ago1A with a sequence-specific siRNA led to a significant increase of WSSV loads. It was revealed that knockdown of Ago1B mRNA by 37–70% resulted in higher virus loads in shrimp. However, upon silencing Ago1B by more than 85%, a two-fold increase in Ago1A mRNA was observed but viral load was the same as untreated controls challenged with WSSV, suggesting that the simultaneous up-regulation of Ago1A might compensate for the loss of Ago1B. These data indicated that Ago1A played more important roles in the antiviral immune response than Ago1B. The simultaneous inhibition of Ago1A and Ago1B resulted in a greater increase in viral loads than Ago1A or Ago1B alone, indicating that Ago1A and Ago1B isoforms were involved in shrimp antiviral immunity. It was revealed that Ago1C had no effect on virus infection. Therefore, the current study presented the first report on the contribution of Ago isoforms in the invertebrate defense against virus infection.     

Enhancement of Shrimp Antiviral Immune Response Through Caspase-Dependent Apoptosis by Small Molecules

Epidemic diseases cost large amount of economic loss in the shrimp aquaculture. To control the epidemic diseases, it is a very efficient approach to enhance the shrimp immunity by immunostimulants. In aquaculture, however, the applications of the available immunostimulants are very limited due to the lack of information about the roles of these immunostimulants in animal immunity. In the present study, a caspase protein (PjCaspase), required in shrimp antiviral apoptosis, was used as the target protein to screen for small molecules which would enhance the shrimp immunity. Based on screening using the EGFP-PjCaspase fusion protein in insect cells, four small molecules could enhance the activity of PjCaspase protein. Among them, IL-2 and evodiamine were further evidenced to enhance the apoptotic activity of shrimp hemocytes in vivo, suggesting that the small molecules improved the activity of apoptosis through the activation of the PjCaspase protein. The results indicated that the enhancement of apoptotic activity effectively inhibited the white spot syndrome virus (WSSV) infection in shrimp, which further led to the decrease of mortalities of WSSV-infected shrimp. Therefore, our study, for the first time, presented that the strategy using the key proteins in immune responses of aquatic organisms as the target proteins was a very efficient approach for the screening of immunostimulants to prevent the aquatic organisms from pathogen infections.     

Vp28 of shrimp white spot syndrome virus is involved in the attachment and penetration into shrimp cells

White spot disease (WSD) is caused by the white spot syndrome virus (WSSV), which results in devastating losses to the shrimp farming industry around the world. However, the mechanism of virus entry and spread into the shrimp cells is unknown. A binding assay in vitro demonstrated VP28-EGFP (envelope protein VP28 fused with enhanced green fluorescence protein) binding to shrimp cells. This provides direct evidence that VP28-EGFP can bind to shrimp cells at pH 6.0 within 0.5 h. However, the protein was observed to enter the cytoplasm 3 h post-adsorption. Meanwhile, the plaque inhibition test showed that the polyclonal antibody against VP28 (a major envelope protein of WSSV) could neutralize the WSSV and block an infection with the virus. The result of competition ELISA further confirmed that the envelope protein VP28 could compete with WSSV to bind to shrimp cells. Overall, VP28 of the WSSV can bind to shrimp cells as an attachment protein, and can help the virus enter the cytoplasm.     

Contribution of the caspase gene sequence diversification to the specifically antiviral defense in invertebrate

Vertebrates achieve adaptive immunity of all sorts against pathogens through the diversification of antibodies. However the mechanism of invertebrates' innate immune defense against various pathogens remains largely unknown. Our study used shrimp and white spot syndrome virus (WSSV) to show that PjCaspase, a caspase gene of shrimp that is crucial in apoptosis, possessed gene sequence diversity. At present, the role of gene sequence diversity in immunity has not been characterized. To address this issue, we compared the PjCaspase gene sequence diversities from WSSV-free and WSSV-resistant shrimp. The sequence analysis indicated that the PjCaspase gene from the WSSV-resistant shrimp contained a special fragment, designated as fragment 3 (221-229 aa). Down-regulation or overexpression of the PjCaspase gene containing fragment 3 led to significant inhibition or enhancement of virus-induced apoptosis, but had no effect on bacterium challenge. We found evidence that the silencing or overexpression of this gene led to a 7-fold increase or 11-fold decrease of WSSV copies, respectively. Our results suggested that the PjCaspase gene containing fragment 3 provided the molecular basis for the antiviral defense of shrimp. This study represented the first report of the role of gene sequence diversity in the immunity of an invertebrate against virus infection. Invertebrates may employ this gene sequence diversity as a system to avoid pathogen interference with their immune response.     

The role of white spot syndrome virus (WSSV) VP466 protein in shrimp antiviral phagocytosis

Widespread evidence indicates that the structural proteins of virus play very important roles in virus-host interactions. However, the effect of viral proteins on host immunity has not been addressed. Our previous studies revealed that the host shrimp Rab6 (termed as PjRab previously), tropomyosin, β-actin and the white spot syndrome virus (WSSV) envelope protein VP466 formed a complex. In this study, the VP466 protein was shown to be able to bind host Rab6 protein and increase its GTPase activity in vivo and vitro. Thus, VP466 could function as a GTPase-activating protein (GAP) of Rab6. In the VP466-Rab-actin pathway, the increase of the Rab6 activity induced rearrangements of the actin cytoskeleton, resulting in the formation of actin stress fibers which promoted the phagocytosis against virus. Therefore our findings revealed that a viral protein could be employed by host to initiate the host immunity, representing a novel molecular mechanism in the virus-host interaction. Our study would help to better understand the molecular events in immune response against virus infection in invertebrates.     

VP37 of white spot syndrome virus interact with shrimp cells

Aims:  To investigate VP37 [WSV 254 of White spot syndrome virus (WSSV) genome] interacting with shrimp cells and protecting shrimp against WSSV infection.
Methods and Results:  VP37 was expressed in Escherichia coli and was confirmed by Western blotting. Virus overlay protein binding assay (VOPBA) technique was used to analyse the rVP37 interaction with shrimp and the results showed that rVP37 interacted with shrimp cell membrane. Binding assay of recombinant VP37 with shrimp cell membrane by ELISA confirmed that purified rVP37 had a high-binding activity with shrimp cell membrane. Binding of rVP37 to shrimp cell membrane was a dose-dependent. Competition ELISA result showed that the envelope protein VP37 could compete with WSSV to bind to shrimp cells. In vivo inhibition experiment showed that rVP37 provided 40% protection. Inhibition of virus infection by rVP37 in primary cell culture revealed that rVP37 counterparted virus infection within the experiment period.
Conclusions:  VP37 has been successfully expressed in E . coli . VP37 interacted with shrimp cells.
Significance and Impact of the Study:  The results suggest that rVP37 has a potential application in prevention of virus infection.     

Feeding hermit crabs to shrimp broodstock increases their risk of WSSV infection

White spot syndrome virus (WSSV) is a serious shrimp pathogen that has spread globally to all major shrimp farming areas, causing enormous economic losses. Here we investigate the role of hermit crabs in transmitting WSSV to Penaeus monodon brooders used in hatcheries in Vietnam. WSSV-free brooders became PCR-positive for WSSV within 2 to 14 d, and the source of infection was traced to hermit crabs being used as live feed. Challenging hermit crabs with WSSV confirmed their susceptibility to infection, but they remained tolerant to disease even at virus loads equivalent to those causing acute disease in shrimp. As PCR screening also suggests that WSSV infection occurs commonly in hermit crab populations in both Vietnam and Taiwan, their use as live feed for shrimp brooders is not recommended.