Protection of Penaeus monodon against white spot syndrome virus using a WSSV subunit vaccine

Although invertebrates lack a true adaptive immune response, the potential to vaccinate Penaeus monodon shrimp against white spot syndrome virus (WSSV) using the WSSV envelope proteins VP19 and VP28 was evaluated. Both structural WSSV proteins were N-terminally fused to the maltose binding protein (MBP) and purified after expression in bacteria. Shrimp were vaccinated by intramuscular injection of the purified WSSV proteins and challenged 2 and 25 days after vaccination to assess the onset and duration of protection. As controls, purified MBP- and mock-vaccinated shrimp were included. VP19-vaccinated shrimp showed a significantly better survival (p<0.05) as compared to the MBP-vaccinated control shrimp with a relative percent survival (RPS) of 33% and 57% at 2 and 25 days after vaccination, respectively. Also, the groups vaccinated with VP28 and a mixture of VP19 and VP28 showed a significantly better survival when challenged two days after vaccination (RPS of 44% and 33%, respectively), but not after 25 days. These results show that protection can be generated in shrimp against WSSV using its structural proteins as a subunit vaccine. This suggests that the shrimp immune system is able to specifically recognize and react to proteins. This study further shows that vaccination of shrimp may be possible despite the absence of a true adaptive immune system, opening the way to new strategies to control viral diseases in shrimp and other crustaceans.     

Hepatopancreatic nuclease of black tiger shrimp Penaeus monodon unlikely to be involved in viral triggered apoptosis

Nucleases are phosphodiesterases that hydrolyze DNA and/or RNA. In a search for shrimp nucleases involved in apoptosis, we discovered a nuclease from hepatopancreatic cDNA of the black tiger shrimp Penaeus monodon. The full-length nuclease gene was amplified and revealed to contain 1668bp corresponding to 381 deduced amino acid residues in the mature enzyme. Sequence analysis indicated 83% nucleic acid identity and 89% amino acid identity to a nuclease from the Kuruma shrimp Penaeus japonicus (also called Marsupenaeus japonicus). Comparative analysis of sequences, conserved motifs and phylogenetic trees indicated that P. monodon nuclease (PMN) belonged to the family of DNA/RNA non-specific endonucleases (DRNSN). RT-PCR analysis using primers specific for PMN mRNA with seven different shrimp tissues revealed that expression in normal shrimp was restricted to the hepatopancreas. Semiquantitative RT-PCR analysis of PMN using hepatopancreatic mRNA from normal shrimp and from shrimp challenged with white spot syndrome virus (WSSV) indicated significant up-regulation of PMN in the hepatopancreas (P<0.05) at the early stage of viral infection but a return to baseline levels as gross signs of disease developed. At the same time, expression was always confined to the hepatopancreas and never seen in other tissues, including those reported to be prime targets for WSSV and subject to increased levels of apoptosis after infection. The results suggested that PMN is probably a digestive enzyme that is unlikely to be involved in hallmark DNA digestion associated with apoptosis.     

YHV-protease dsRNA inhibits YHV replication in Penaeus monodon and prevents mortality

Yellow head virus infects cultured shrimps and causes severe mortality resulting in a great economic loss. Haemolymph injection of dsRNA(pro) corresponding to the protease motif of YHV genome resulted in a complete inhibition of YHV replication. The effect of dsRNA lasted for at least 5 days. Injecting sequence-unrelated dsRNA(gfp) or dsRNA(TSV-pol) also resulted in an inhibition of YHV replication but at a comparatively much less extent. Shrimp mortality was monitored for 10 days when more than 90% shrimps receiving no dsRNA died within 8 dpi. However, those receiving dsRNA(pro) showed no mortality. A partial mortality was observed among the shrimps receiving dsRNA(gfp) or dsRNA(TSV-pol). Thus, Penaeus monodon possesses the sequence-specific protection to YHV infection, most likely through the RNAi pathway, in addition to sequence-independent protection. It gives a new notion that dsRNA induction of antiviral immunity in shrimp goes through two pathways, sequence-independent and sequence-dependent.     

In vitro toxicity of nitrite on haemocytes of the tiger shrimp, Penaeus monodon, using flow cytometric analysis

This study investigated the in vitro effects of nitrite on reactive oxygen species (ROS) production, NO production, esterase activity and cell apoptosis of Penaeus monodon haemocytes. Haemocytes were in vitro exposed to different dose of nitrite (0, 0.1, 0.5, 1, 5 and 10 μM). Cellular responses of nitrite-treated haemocytes were determined by flow cytometry. The results revealed that haemocytes treated by nitrite in vitro showed conspicuous time- and dose-dependent decreases in ROS and NO production as well as esterase activity. Additionally, 0.1 and 0.5 μM nitrite did not affect the apoptotic cell ratio during the 3h experimental time, while significant increases in apoptotic cells were observed after haemocyte exposure to nitrite at 1 μM for 3h, and at 5 or 10 μM for 1h. These results indicated that nitrite suppresses cellular functions, including production of ROS and NO, and activity of esterase. Cell apoptosis of haemocytes would be induced by extracellular nitrite as doses exceed 1 μM.     

A novel medium for the development of in vitro cell culture system from Penaeus monodon

Lack of a valid shrimp cell line has been hampering the progress of research on shrimp viruses. One of the reasons identified was the absence of an appropriate medium which would satisfy the requirements of the cells in vitro. We report the first attempt to formulate an exclusive shrimp cell culture medium (SCCM) based on the haemolymph components of Penaeus monodon prepared in isosmotic seawater having 27 ‰ salinity. The SCCM is composed of 22 amino acids, 4 sugars, 6 vitamins, cholesterol, FBS, phenol red, three antibiotics, potassium dihydrogen phosphate and di-sodium hydrogen phosphate at pH 6.8–7.2. Osmolality was adjusted to 720 ± 10 mOsm kg⁻¹ and temperature of incubation was 25 ºC. The most appropriate composition was finally selected based on the extent of attachment of cells and their proliferation by visual observation. Metabolic activity of cultured cells was measured by MTT assay and compared with that in L-15 (2×), modified L-15 and Grace’s insect medium, and found better performance in SCCM especially for lymphoid cells with 107 % increase in activity and 85 ± 9 days of longevity. The cells from ovary and lymphoid organs were passaged twice using the newly designed shrimp cell dissociation “cocktail”.     

对虾白斑综合症病毒囊膜蛋白VP19的融合表达及其抗病毒感染作用

根据GenBank上WSSV囊膜蛋白基因vp19的序列,设计并合成引物,PCR扩增得到vp19基因并克隆到pGEM‐T载体中,经过BamHⅠ/HindⅢ酶切、连接并将vp19插入到pET32b表达载体中。用重组质粒pET32b-vp19转化大肠杆菌Origam(iDE3)pLysS,在IPTG诱导下,融合蛋白Trx-VP19以可溶性的形式得到表达,经SDS-PAGE和Western-blot检测显示其分子量与预期的大小相符合。目的蛋白经Ni2 柱纯化并定量后分别直接注射鳌虾和包被饲料投喂鳌虾。实验结果表明注射Trx-VP19可以提高鳌虾个体抗WSSV感染力的作用。     

Up-regulation of ribophorin I after yellow head virus (YHV) challenge in black tiger shrimp Penaeus monodon

This work constitutes the second report from a continuing investigation of shrimp genes that may be involved in apoptosis associated death resulting from yellow head virus (YHV) infection. Here, we describe from the black tiger shrimp Penaeus monodon, a ribophorin I-like gene that is probably a subunit of the oligosaccharyltransferase complex (OST), a key enzyme in N-linked glycosylation that occurs in the endoplasmic reticulum. The OST complex also contains DAD1 (defender against apoptotic death 1) that has been reported to control apoptosis and that we have previously reported from P. monodon. The full length ribophorin I of P. monodon comprised 2157 bp with the ORF of 1806 bp corresponding to 601 deduced amino acids and three putative N-linked glycosylation sites. Analysis revealed hydrophobic properties implying that it could be a membrane protein. Tissue distribution analysis using real-time RT-PCR with SYBR Green revealed that ribophorin I was endogenously expressed in all examined tissues of normal shrimp. However, unlike DAD1 that was down-regulated after YHV challenge, ribophorin I expression was up-regulated and remained high until the moribund stage.     

In vivo therapeutic potentiality of red seaweed,Asparagopsis (Bonnemaisoniales,Rhodophyta) in the treatment of Vibriosis in Penaeus monodon Fabricius

The crude extract of the red seaweed, Asparagopsis sp. was evaluated for in vivo antibacterial activity against the shrimp vibrio pathogens. The algal extract was rationalized with commercial shrimp feed and orally administered for different duration of time followed by the artificial bacterial challenge experiment. In dose titration experiments, the oral administration of Asparagopsis sp. at a dosage of 850 mg kg^–1 of biomass was highly efficacious in the treatment of natural infestations of Vibriosis in Penaeus monodon. The results of the confirmatory dose experiment revealed that the prophylactic treatment with moderate dose of 850 mg kg^–1 of biomass day^–1 for four weeks followed by 14 days of post infection therapy was highly effective in controlling Vibrio infection in shrimps. Moreover, results of the percent survival index and microbiological analysis clearly show that Asparagopsis extract incorporated medicated feed had broad therapeutic potential for managing shrimp Vibriosis. In addition, in vivo trials and results obtained in this work are based on the crude organic extract sourced from an unidentified Asparagopsis cryptic lineage, therefore further molecular analysis to identify the species will be required.     

Receptor for Activated C Kinase-1 protein from Penaeus monodon (Pm-RACK1) participates in the shrimp antioxidant response

Cellular oxidative stress responses are caused in many ways, but especially by disease and environmental stress. After the initial burst of reactive oxygen species (ROS), the effective elimination of ROS is crucial for the survival of organisms and is mediated by antioxidant defense mechanisms. In this paper, we investigate the possible antioxidant function of Penaeus monodon Receptor for Activated C Kinase-1 (Pm-RACK1). When Pm-RACK1 was over-expressed in Escherichia coli cells or Spodoptera frugiperda (Sf9) insect cells exposed to H₂O₂, it significantly protected the cells from oxidative damage induced by H₂O₂. When recombinant Pm-RACK1 protein was expressed as a histidine fusion protein in E. coli and purified with a Ni²⁺-column it possessed antioxidant functions that protected DNA from metal-catalyzed oxidation. Shrimp (Penaeus vannamei) held at an alkaline pH had a much higher hepatopancreatic expression of Pm-RACK1 than in those held at pH 7.4. The exposure of shrimp to alkaline pH is also known to increase ROS production. These results provide strong evidence that Pm-RACK1 can participate in the shrimp antioxidant response induced by the formation of ROS.     

Multifactorial interaction of growth factors on Penaeus monodon lymphoid cells and the impact of IGFs in DNA synthesis and metabolic activity in vitro

Development of continuous cell lines from shrimp is essential to investigate viral pathogens. Unfortunately, there is no valid cell line developed from crustaceans in general and shrimps in particular to address this issue. Lack of information on the requirements of cells in vitro limits the success of developing a cell line, where the microenvironment of a cell culture, provided by the growth medium, is of prime importance. Screening and optimization of growth medium components based on statistical experimental designs have been widely used for improving the efficacy of cell culture media. Accordingly, we applied Plackett–Burman design and response surface methodology to study multifactorial interactions between the growth factors in shrimp cell culture medium and to identify the most important ones for growth of lymphoid cell culture from Penaeus monodon. The statistical screening and optimization indicated that insulin like growth factor-I (IGF-I) and insulin like growth factor-II (IGF-II) at concentrations of 100 and 150 ng ml⁻¹, respectively, could significantly influence the metabolic activity and DNA synthesis of the lymphoid cells. An increase of 53 % metabolic activity and 24.8 % DNA synthesis could be obtained, which suggested that IGF-I and IGF-II had critical roles in metabolic activity and DNA synthesis of shrimp lymphoid cells.

Electronic supplementary material

The online version of this article (doi:10.1007/s10616-014-9697-0) contains supplementary material, which is available to authorized users.     

Identification of a putative egg-laying hormone in neural and ovarian tissues of the black tiger shrimp, Penaeus monodon, using immunocytochemistry

The existence of an egg-laying hormone (ELH) was identified for the first time in the black tiger shrimp, Penaeus monodon, by means of immunoenzyme and immunofluorescence techniques. This was achieved using a polyclonal antibody produced against expressed recombinant ELH of the female Australian blacklip abalone, Haliotis rubra. The shrimp ELH reactive material was found to be localised within female neurosecretory tissues and the secretory tissue of the antennal gland, but was not identified in the X-organ sinus gland within the eyestalk. It was also present in the ovary, where the amount of ELH present was observed to be greatest in the period prior to spawning. These findings implied that the induction of P. monodon spawning might be involved with humoral regulation relating to ELH expression.     

Gene silencing studies in the gymnosperm species Pinus radiata

A biolistic transformation procedure was used to transform embryogenic Pinus radiata tissue with constructs containing the Zea mays UBI1 (ubiquitin)-promoter followed by the P. radiata CAD (cinnamyl alcohol dehydrogenase) cDNA in sense or anti-sense orientation or in the form of an inverted-repeat. The effect of the different constructs on silencing the endogenous CAD gene was monitored in embryogenic tissue and somatic seedlings of 28 P. radiata transclones. Quantitative CAD measurements demonstrated that the construct containing an inverted-repeat of the CAD cDNA was most efficient in triggering gene silencing in P. radiata. Northern hybridization experiments with silenced transclones revealed that reduced CAD activities were the result of reduced steady state levels of the targeted CAD mRNA. Monitoring of the activity of the UBI1-promoter in the P. radiata transclones and heat-shock experiments with transgenic somatic P. radiata seedlings indicated that gene silencing is positively correlated with the expression level of the transgene. The obtained data are also consistent with a role for the expression level of the endogenous CAD gene in gene silencing.