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White spot syndrome virus (WSSV) is one of the most concerning pathogens in penaeid shrimp and can cause severe loss in shrimp aquaculture worldwide. Among the WSSV structural proteins, VP15, a DNA-binding protein located in the WSSV nucleocapsid, is an antiviral protein candidate to protect kuruma shrimp (Marsupenaeus japonicus) from WSSV infection. We identified that the truncated VP15, VP15

White spot syndrome virus (WSSV) is one of the most devastating pathogens in penaeid shrimp and can cause massive damage in shrimp aquaculture industries. Previously, the WSSV structural protein VP15 was identified as an antigenic reagent against WSSV infections. In this study, we truncated this protein into VP15(1−25), VP15(26−57), VP15(58−80), and VP15(1−25,58−80). The purified proteins from the E. coli expression system were assayed as potential protective agents in Kuruma shrimp (Marsupenaeus japonicus) using the prime-and-boost strategy. Among the four truncated constructs, VP15(26−57) provided a significant improvement in the shrimp survival rate after 20 days of viral infection. Subsequently, four peptides (KR11, SR11, SK10, and KK13) from VP15(26−57) were synthesized and applied in an in vivo assay. Our results showed that SR11 could significantly enhance the shrimp survival rate, as determined from the accumulated survival rate. Moreover, a multiligand binding protein with a role in the host immune response and a possible VP15-binding partner, MjgC1qR, from the host M. japonicus were employed to test its binding with the VP15 protein. GST pull-down assays revealed that MjgC1qR binds with VP15, VP15(26−57), and SR11. Taken together, we conclude that SR11 is a determinant antigenic peptide of VP15 conferring antiviral activity against WSSV.

Carp edema virus disease (CEVD), or koi sleepy disease, is caused by CEV. Here, we report the complete genome sequence of CEV strain FTI2020, isolated from koi carp. This sequence information has great potential for improving our understanding of the genetic characteristics of this piscine poxvirus.

The Gillespie algorithm, which is a stochastic numerical simulation of continuous-time Markovian processes, has been proposed for simulating epidemic dynamics. In the present study, using the Gillespie-based epidemic model, we focused on each single trajectory by the stochastic simulation to infer the probability of controlling an epidemic by non-pharmaceutical interventions (NPIs). The single trajectory analysis by the stochastic simulation suggested that a few infected people sometimes dissipated spontaneously without spreading of infection. The outbreak probability was affected by basic reproductive number but not by infectious duration and susceptible population size. A comparative analysis suggested that the mean trajectory by the stochastic simulation has equivalent dynamics to a conventional deterministic model in terms of epidemic forecasting. The probability of outbreak containment by NPIs was inferred by trajectories derived from 1000 Monte Carlo simulation trials using model parameters assuming COVID-19 epidemic. The model-based analysis indicated that complete containment of the disease could be achieved by short-duration NPIs if performed early after the import of infected individuals. Under the correctness of the model assumptions, analysis of each trajectory by Gillespie-based stochastic model would provide a unique and valuable output such as the probabilities of outbreak containment by NPIs.

We isolated a novel Aquareovirus (hirame aquareovirus: HAqRV) from Japanese flounder Paralichthys olivaceus suffering from reovirus-like infection. In electron microscopy, the spherical virion (75 nm in diameter) was observed with multi-layered capsid structure. The viral genome consisted of 11 segments and regions encoding 7 virion structural proteins and 5 non-structural proteins were predicted. The deduced amino acid sequences of those proteins were highly similar to those of the aquareoviruses. However, the similarity of complete genome sequence between the HAqRV and other aquareoviruses was less than 60%. Phylogenetic analyses based on the deduced amino acid sequences suggested that the HAqRV is not classified into the known species of Aquareovirus. Pathogenicity of HAqRV was clearly demonstrated in accordance with Koch's postulates by experimental infection using Japanese flounder. The results suggest that the HAqRV is a new Aquareovirus species which is highly virulent for the Japanese flounder at early life stages.

In shrimp aquaculture, overcrowded farming causes fluctuations in dissolved oxygen concentrations. Low-oxygen conditions (hypoxia) affect shrimp growth. Hypoxia-inducible factor (HIF) is a transcriptional factor in the basic helix-loop-helix/PAS family and is activated in response to hypoxic stress. However, little is known about HIF and other inhibitors of the HIF pathway in crustaceans. In this study, we cloned MjHIF-1α, an inhibitory factor, MjFIH-1 (factor inhibiting HIF-1α), and MjVHL (Von Hippel-Lindau tumor suppressor) from kuruma shrimp (Marsupenaeus japonicus). MjVHL is the first crustacean VHL ortholog to be cloned. MjHIF-1α, MjFIH-1, and MjVHL exhibit significant sequence similarity and share key functional domains with previously described vertebrate and invertebrate genes. As a result of gene expression analysis in various tissues, MjHIF-1α and MjVHL were more highly expressed in the intestine than in any other organ tissues. In hypoxia experiments, HIF-induced expression levels of MjHIF-1α in the hypoxic group increased significantly for 24 h after initiating hypoxia stimulation and expression of MjVHL decreased significantly for 6 h after hypoxia stimulation (P

Mass mortality of 0-year-old pearl oysters, Pinctada fucata (Gould), and anomalies in adults were observed in Japan’s major pearl farming areas in the summer of 2019 and 2020. Although adult oyster mortality was low, both adult and juvenile oysters underwent atrophy of the soft body, detachment of the mantle from nacre (the shiny inner surface of the valves), deposition of brownish material on the nacre, and loss of nacre luster. Infection trials were conducted to verify the involvement of pathogens in this phenomenon. Healthy adult pearl oysters were obtained from areas where this disease had not occurred to use as the recipients. The sources of infection were either affected adult oysters with atrophied soft bodies or batches of juveniles in which mortality had reached conspicuous levels. Transmission of the disease to the healthy oysters were tested either by cohabitation with affected oysters or by injections of the hemolymph of affected animals. The injection infection test examined the effects of filtration and chloroform exposure on the pathogen. Occurrence of the disease was confirmed by the appearance of brown deposits on the nacre and loss of nacre luster. The abnormalities of nacre were clearly reproduced in recipient shells in three out of four cohabitation trials with affected oysters. The disease was also reproduced in six out of six injection trails either with hemolymph filtered through 100 nm filter or with hemolymph treated with chloroform. In a serial passage with hemolymph injections, the disease was successfully transmitted through eight passages. These results suggest that the etiology of the disease is a non-enveloped virus with a diameter ≤100 nm.

To understand the regulation systems of appetite, bioactive peptides from the kuruma shrimp Marsupenaeus japonicus (Mj) were isolated and purified by reverse pharmacological assays using CHO cells expressing the Drosophila melanogaster G-protein-coupled receptors (GPCRs) CG5811 (a RYamide receptor) or CG14593 (a CCHamide-2 receptor). Four peptides having binding activity to GPCRs were obtained and named Mj RYamide-1, Mj RYamide-2, Mj RYamide-3, and Mj CCHamide. Genes encoding the prepropeptides of these peptides were identified using kuruma shrimp transcriptome databases. The Mj prepro-RYamide gene encodes a 130-amino acid polypeptide containing Mj RYamide-1, Mj RYamide-2, and Mj RYamide-3, whereas the Mj prepro-CCHamide gene encodes a 119-amino acid polypeptide containing a single Mj CCHamide peptide. The expression of these genes was confirmed in various neuronal organs including the brain and ventral nerve cord. In addition, prepro-RYamide gene expression is significantly reduced in the brain after starvation. RYamides may thus be associated with regulation of feeding or digestion. Changes in kayak (the c-fos ortholog in invertebrates) gene expression after administration of synthetic peptides were also investigated. Mj kayak expression levels are upregulated in hepatopancreas after treatment with Mj RYamide-3 or CCHamide. Thus, the peptides isolated in this study may have some regulatory effect on cellular metabolism in aquacultured invertebrates.

A new continuous cell line designated as SGA has been developed from the skin tissue of the freshwater mosquitofish Gambusia affinis. The cell line grew well in Leibovitz's L-15 medium supplemented with 15% fetal bovine serum at 28 °C. Immunophenotyping of the cell line showed the epithelial nature of the cells. Chromosome number analysis showed that SGA cells have a modal diploid chromosome number of 48. Replication of the two different strains of betanodavirus (RGNNV and SJNNV) in SGA cell line showed the maximum virus titre of 108.82 TCID50 mL−1 for RGNNV and 107.2 TCID50 mL−1 for SJNNV. The cytopathic effect in the cell line was observed at 3 days post-infection (dpi) and multiple vacuolations were observed at 7 dpi. Further, DsRed2 plasmid was efficiently delivered into SGA cell line and was found that these cells were transfectable and can be used for assessing gene promoter activity. In vivo challenge experiments using the RGNNV infected cell culture supernatant showed signs of the disease in healthy mosquitofish with mortality commencing from 15 dpi. The above results suggest that the cell line is permissive for propagating betanodavirus and also could be an essential tool for studying the molecular pathogenesis of betanodavirus infection.

The adjuvant effects of poly I:C and imiquimod during immunization with a recombinant protein, rVP28 derived from white spot syndrome virus (WSSV) was investigated in kuruma shrimp ( Marsupenaeus japonicus ). Shrimps were injected intramuscularly with different doses of rVP28, poly I:C and imiquimod, and combined rVP28 + poly I:C or imiquimod, and challenged with WSSV. Expression of innate immune-related genes was examined in the heart and lymphoid organ of combined rVP28 + poly I:C or imiquimod immunized shrimps at 1, 3 and 7 days after WSSV challenge. Shrimps which received rVP28 + poly I:C and rVP28 + imiquimod had significantly higher survivals of 52 and 58%, respectively compared to the rVP28 alone or PBS injected control groups ( P Statement of relevance Major contributors to economic losses in shrimp aquaculture are viral diseases, of which white spot syndrome virus (WSSV) is the most important one due to its rapid spread and economic impact. In this paper, we present an immunization method towards prevention of WSSV disease in kuruma shrimp using adjuvants such as poly I:C or imiquimod along with a sub-unit protein (rVP28).

In fish culture, cannibalism, which induces critical damage, is separated into two types: type I, which occurs in early larval stages when size heterogeneity is low; and type II, which starts in later larval and/or early juvenile stages following type I when size heterogeneity is high. In type II, the cannibal's maximum prey size is limited by its gape size relative to the prey fish body size. Ratios of prey to cannibal body size changes are associated with allometric factors and feeding behaviour with growth. In a previous study on grouper, the maximal prey size was estimated morphologically (i.e., the relationship between gape size and body height). This method is very simple and useful; however, it does not consider allometric factors and feeding behaviour changes; therefore, it probably causes a large estimation error. To overcome this, we present a new method for estimating maximal prey size to determine the size ranges for size grading of longtooth grouper, Epinephelus bruneus, juveniles using predation experiments. It is shown that grouper type II cannibalism is affected by two factors; in early juvenile stages by morphology and in later (TL over 50 mm) by prey size selectivity.

Aims: Lesions of DNA are removed by nucleotide excision repair (NER) process in the living systems. NER process-related host factors are believed to aid recovery steps during viral integration. Here, we report identification and characterization of a DNA repair molecule Rad23 from kuruma shrimp Marsupenaeus japonicus. Methods and Results: The full-length cDNA of M. japonicus Rad23 gene (MjRad23) has 1149 bp coding for a putative protein of 382 amino acids with a 5′ untranslated region (UTR) of 92 bp and 3′ UTR region of 1116 bp. Quantitative expression analysis revealed MjRad23 is constitutively expressed in all the organs of healthy shrimp, whereas with high level in muscle tissue. Although MjRad23 expression is observed in every haemolymph samplings to post-white spot syndrome virus infection, high expression is recorded at 2 h post infection (h.p.i.). MjRad23 consists of putative functional domains including one ubiquitin domain (UBQ), two ubiquitin-associated domains (UBA) and one heat-shock chaperonin-binding motif (STI1). Multiple alignment of MjRad23 with Rad23 of other species showed highly significant identity ranging from 37 to 53%; however, high homology is observed with Rad23 of Bombyx mori (BmRad23). UBQ domain region alignment revealed maximum of 66% homology with Rad23 of Apis melifera (AmRad23). MjRad23 clustered with invertebrate sector along with insect species in evolution analysis. Three-dimensional structural analyses demonstrated the highest identity between MjRad23 and human Rad23A (hHR23A). Conclusions: The present work revealed the presence of MjRad23 gene, which is essential in DNA repair process. Further studies are required to clarify the involvement of MjRad23 in NER process. Significance and Impact of the Study: This is the first report on identification and characterization of DNA repair protein in crustaceans, which will lead to further investigation to explore the molecular mechanisms behind the NER process.

A new myxosporean species, Myxobolus supamattayai n. sp., was isolated from wild mullet (Valamugil seheli) from the Andaman Sea, Thailand and described based on its morphology and molecular data. The myxosporean produced black plasmodia-like unique clinical sign on the skin with sporogonic stages and mature spores. Polysporous plasmodia, up to 2.5 mm in diameter, were found in epithelium tissue in the scale pocket. The spores measured 6.6 (6.2-7.0) μm in length, 6.5 (6.2-6.7) μm in width, smooth, and round board to ellipsoidal in valvular view. Spores were enclosed with intracapsular process which represents 5-7 and 11-12 in amount revealed in light microscopy and ultrastructure, respectively. The polar capsules were pyriform and of equal size, measuring 3.5 (3.4-3.6) μm in length and 2.0 (1.9-2.2) μm in width, with four to five turns of polar filament arranged perpendicularly to longitudinal axis of the polar capsule. In conclusion, this new species is entirely different from those previously described; however, this finding was assured by the partial sequence of SSU rRNA gene (1,666 bp) analysis that differed from all known species of Myxobolus Bütschli, 1882. The phylogenetic tree of the sequence data sets including those of freshwater and marine of Myxobolus spp. and the sister group (Henneguya spp.) was constructed to establish the relationship of this new species in Myxobolus clade and to explore its relations between their sister groups. Phylogenetic analysis indicated that a monophyletic group with Myxobolus spp. which infected mullet represents the newly formed species. These results suggested the presumably nearby evolution prospecting of Myxobolus species that were found in the same host.

Recent applications of RNA interference technology against the white spot syndrome virus (WSSV) were found to be promising and effective antiviral strategies. In the present study, a gene-specific full-length double-stranded RNA (dsRNA) was prepared for the WSSV-VP28 gene using the in vitro transcription method. Challenging experiments were performed by an intramuscular injection of VP28-dsRNA in Marsupenaeus japonicus, followed by WSSV infection after 24 h, expecting 95-100% mortality within in 9 days of duration. The results revealed that the protective efficiency of VP28-dsRNA against WSSV infection is more than 85%, 25 days post infection (dpi). Various assays including bioassay, reverse transcriptase polymerase chain reaction (RT-PCR), Western blotting, immunohistochemistry (IHC), histology and 4′,6-diamidino-2-phenylindole (DAPI) staining were performed to confirm the knockdown of the VP28 gene. No positive signals were observed in the gill and heart tissues of VP28-dsRNA/WSSV-infected shrimp 7 dpi by RT-PCR and Western blotting. No infection was observed in the VP28-dsRNA/WSSV-infected group through histology and IHC. DAPI staining of haemocytes revealed no signs of condensation and fragmentation in the VP28-dsRNA-administered group. The unrelated YHVGP116-dsRNA had no significant effect on the expression of the VP28 gene, confirming that the knockdown is specific to VP28-dsRNA.

Nitric oxide (NO) signaling is involved in many physiological processes in vertebrates and invertebrates. In crustaceans, nitric oxide synthase (NOS) plays a significant role in the regulation of the nervous system and in innate immunity. Here, we describe the entire cDNA sequence (4616 bp) of the kuruma shrimp Marsupenaeus japonicus NOS (Mj NOS) generated using the reverse transcriptase-polymerase chain reaction (RT-PCR) and 5′- and 3′- rapid amplification PCRs of cDNA ends from brain and gill mRNAs. The open reading frame of Mj NOS encoded a protein of 1187 amino acids with an estimated mass of 134 kDa, and had an 82.3% sequence homology with the NOS gene of the land crab Gecarcinus lateralis . Highly conserved amino acid sequences in heme and tetrahydrobiopterin were observed in the oxygenase domain. FMN, FAD and NADPH were found in the reductase domain. Mj NOS mRNA was constitutively expressed in the brain, gill, intestine, thoracic ganglion and testis of the kuruma shrimp. When Vibrio penaeicida was injected into the kuruma shrimp, Mj NOS was expressed in the brain, gill, heart, lymphoid organ, intestine and thoracic ganglion. Mj NOS expression in the gill reached its peak 12 h and decreased to its normal level 24 h after V. penaeicida injection.

Aim: In the present study, we have cloned a new family of anti-lipopolysaccharide factor (ALF) from haemocytes of kuruma shrimp Marsupenaeus japonicus (MjALF2) using RACE method. Methods and Results: Transcriptional analysis of MjALF2 gene in the organs of healthy shrimp revealed prominent expression in gills and muscle. In vitro LPS stimulation in the lymphoid organ cells resulted in significant increase in expression at 48, 8 and 12 h poststimulation, compared to the nonstimulated cells. In vivo injection of V. penaeicida does not show any high expression in time course assay. Phylogenetic analysis showed MjALF2 is placed in the group closer to P. monodon isoform 1 and 2 than to MjALF1. The full-length MjALF2 gene consists of 558 bp with a 363 -bp open reading frame, encoding 121 amino acids. The deduced peptide contains a putative signal peptide of 22 amino acids with molecular mass of about 13·8 kDa molecular mass. The deduced amino acid sequence of MjALF2 showed 83·3 and 56·7% identity with ALF sequences of P. monodon. Conclusions: The present work revealed the presence of MjALF2 gene, which is highly expressed in gills and muscle of healthy kuruma shrimp. Further studies are required to clarify the involvement of MjALF2 in immune responses for using as a therapeutic agent. Significance and Impact of the Study: Antimicrobial peptides are promising potential therapeutic agents for disease control in aquaculture. Understanding the relation of MjALF2 with innate immunity mechanism will lead to develop better health management strategies for long-term sustainability of the shrimp industry.

In this study, we have developed a DNA vaccine encoding viral envelope protein VP28 of penaeid rod-shaped DNA virus (PRDV, = WSSV). Protective efficacy of the DNA vaccine against PRDV was confirmed at 7 days post vaccination in kuruma shrimp Penaeus japonicus. The VP28 transcript derived from the vaccine was detected in various tissues at 1, 3 and 7 days post vaccination. Moreover, notable up-regulated expression of Rab7, penaeidin, lysozyme and crustin genes was observed upon DNA vaccination. These results suggest that the DNA vaccine significantly increased the protection and innate immune responses in kuruma shrimp against PRDV.

A ghrelin gene has been cloned and sequenced in common carp Cyprinus carpio. Ghrelin cDNA is composed of 461 bp [with a 36-bp 5′-untranslated region (UTR) and a 113-bp 3′-UTR], which translates into a protein of 103 amino acid residues. Carp ghrelin (preproghrelin) contained a predicted signal peptide of 26 amino acid residues, the ghrelin domain (Gly 27-Val 15) and C-terminal peptide (Gly 46-Phe 103). Homology analysis of the ghrelin domain of carp with that of other known ghrelin in vertebrates showed good similarity to teleost ghrelin (50–81.8%). Hydropathy analysis based on the deduced amino acid sequence of ghrelin domains in teleosts showed a similar profile. Carp ghrelin clustered with ghrelin of goldfish Carassius auratus and other teleosts, away from mammalian, reptilian, avian, amphibian and chondrichthian ghrelin, by phylogenetic analysis. Genomic organization of carp ghrelin gene was composed of four exons and three introns, which was the same as that of other teleosts and human ghrelin genes. The carp ghrelin gene was expressed in unstimulated tissues such as foregut, hindgut, spleen and brain. In spleen cells, expression of the ghrelin gene increased upon stimulation with lipopolysaccharide (LPS), phytohemagglutinin (PHA) or imiquimod. The identification of carp ghrelin gene and the analysis of the modulation of its expression in immune-activated conditions will allow a more complete analysis of the roles of ghrelin in teleosts.

A one-step, single tube, real-time loop-mediated isothermal amplification (real-time LAMP) assay was developed to detect a non-structural protein encoding gene of infectious hypodermal and haematopoietic necrosis virus (IHHNV). The real-time LAMP method for IHHNV is simple and rapid with specific amplification within 60 min at 63°C employing four primers. This method requires less time than the PCR method and is specific for IHHNV when analysed with PRDV (= WSSV), YHV and TSV DNA/cDNA, and healthy shrimp DNA. Sensitivity analysis revealed this method is capable of detecting as few as 102-103 copies/μL, suggesting it can be used as an alternative quantitative detection method for IHHNV in diagnostic laboratories.

This study aimed to analyze vancomycin-resistant enterococci (VRE) and their resistance genes, vanA and vanB, to examine their presence in sewage treatment systems. Water samples were collected from primary sedimentation tank inlet, aeration tank, final sedimentation tank overflow outlet, and disinfection tank. Enterococcal strains were determined their vancomycin susceptibility by the minimum inhibitory concentration (MIC) test. Vancomycin-resistance genes (vanA and vanB) were quantified by real-time PCR. The sewage treatment process indeed decreased the number of most enterococci contained in the entering sewage, with a removal rate of ≥ 5 log. The MIC test showed that two enterococcal strains resistant to a high concentration of vancomycin (>128 μg mL(-1)). However, most of the enterococcal strains exhibited sensitivity to vancomycin, indicating that VRE were virtually absent in the sewage treatment systems. On the other hand, vancomycin-resistance genes were detected in all the sewage samples, including those collected from the chlorination disinfection tank. The highest copy numbers of vanA (1.5 × 10(3) copies mL(-1)) and vanB (1.0 × 10(3) copies mL(-1)) were detected from the water sample of effluent water and chlorinated water, respectively. Therefore, antibiotic resistance genes remain in the sewage treatment plant and might discharged into water environments such as rivers and coastal areas.

Reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detecting the structural glycoprotein gene of yellow head virus (YHV). The RT-LAMP assay is a novel method of gene amplification that amplifies nucleic acid with high specificity, sensitivity and rapidity under isothermal conditions with a set of four specially designed primers that recognize six distinct sequences of the target. The whole procedure is very simple and rapid, and reaction time and temperatures were optimized for 60 min at 65 degrees C, respectively. Detection of gene amplification could be accomplished by agarose gel electrophoresis. The standardized RT-LAMP procedure was used to detect YHV in the heart and gill from infected shrimp. Thus, the RT-LAMP assay is extremely rapid, cost-effective, sensitive and specific and has potential usefulness for rapid diagnosis for YHV detection in shrimp.

The protective efficacy of oral delivery and intramuscular (i.m.) injection of a recombinant protein, rVP28 derived from white spot syndrome virus (WSSV) and synthesized using wheat germ cell-free technology was investigated in kuruma shrimp (Marsupenaeus japonicus). Shrimps were administered with the unpurified rVP28 and challenged with WSSV. Expression of innate immune-related genes was examined in intestine, heart, and lymphoid organ at 1, 3 and 7 days after oraladministration. Shrimps received rVP28 protein through i.m. injection or oral route and then challenged with WSSV displayed higher survival (90 and 85.7%, respectively) compared to the respective control groups. A significant up-regulation (P

In many physiological processes, including the innate immune system, free radicals such as nitric oxide (NO) and reactive oxygen species (ROS) play significant roles. In humans, 2 homologs of Dual oxidases (Duox) generate hydrogen peroxide (H(2)O(2)), which is a type of ROS. Here, we report the identification and characterization of a Duox from kuruma shrimp, Marsupenaeus japonicus. The full-length cDNA sequence of the M. japonicus Dual oxidase (MjDuox) gene contains 4695 bp and was generated using reverse transcriptase-polymerase chain reaction (RT-PCR) and random amplification of cDNA ends (RACE). The open reading frame of MjDuox encodes a protein of 1498 amino acids with an estimated mass of 173 kDa. In a homology analysis using amino acid sequences, MjDuox exhibited 69.3% sequence homology with the Duox of the red flour beetle, Tribolium castaneum. A transcriptional analysis revealed that the MjDuox mRNA is highly expressed in the gills of healthy kuruma shrimp. In the gills, MjDuox expression reached its peak 60 h after injection with WSSV and decreased to its normal level at 72 h. In gene knockdown experiments of free radical-generating enzymes, the survival rates decreased during the early stages of a white spot syndrome virus (WSSV) infection following the knockdown of the NADPH oxidase (MjNox) or MjDuox genes. In the present study, the identification, cloning and gene knockdown of the kuruma shrimp MjDuox are reported. Duoxes have been identified in vertebrates and some insects; however, few reports have investigated Duoxes in crustaceans. This study is the first to identify and clone a Dual oxidase from a crustacean species.

Signal transducer and activators of transcription (STAT) gene, suppressors of cytokine signaling (SOCS) has been isolated from kuruma shrimp, Marsupenaeus japonicus and characterized. The kuruma shrimp STAT (MjSTAT) cDNA was composed of 2901 bp consisting of 801 amino acid residues which includes a protein interaction domain, all alpha domain, DNA binding domain and SH2 domain. Homology analysis of MjSTAT showed 94.1% and 34.0% identities with Penaeus monodon STAT (PmSTAT) and Drosophila melanogaster STAT92E (DmSTAT), respectively. The kuruma shrimp SOCS (MjSOCS) cDNA was composed of 1675 bp consisting of 342 amino acid residues including a SH2 domain and C-terminal SOCS domain. Homology analysis of MjSOCS showed 52.6% and 21.3% identities with Chinese mitten crab (Eriocheir sinensis) SOCS2 and fruit fly (D. melanogaster) SOCS44A, respectively. The MjSTAT and MjSOCS genes are constitutively expressed in the muscle, stomach, brain and gill of kuruma shrimp. In lymphoid organ cells, an enhanced expression of both MjSTAT and MjSOCS genes are observed following stimulation with peptidoglycan and polycytidylic acid. These observations suggest that MjSTAT and MjSOCS might play a major role in the innate immune defense of kuruma shrimp. The discovery of JAK/STAT signaling pathway in shrimp will allow a complete and concrete understanding of shrimp cytokine signaling.

A one-step, single tube, real-time accelerated loop-mediated isothermal amplification (real-time LAMP) assay was developed separately to detect major shrimp viral diseases such as penaeid rod shaped DNA virus (PRDV), hypodermal and haematopoietic necrosis virus (IHHNV), Taura syndrome virus (TSV) and yellow head virus (YHV). Real-time LAMP method is more sensitive than other conventional PCR, RT-PCR and LAMP methods. The applicability of this assay was validated with plenty of viral samples collected from Japan and Thailand. Highly conserved regions of each viral genome developed separately were used to design the real-time LAMP primers. The real-time LAMP assay reported in this study is simple and rapid, where specific amplification is obtained for PRDV, IHHNV, TSV and YHV in 60 min under isothermal conditions at 63 o C employing six distinct sequences of the target gene. The quantification of viral load in the infected samples was determined from the standard curve based on their threshold time required for turbidity to occur in the reaction by precipitation of magnesium pyrophosphate. Sensitivity analysis revealed that all of these viruses can be detected up to 100 copies of template DNA, rendering it ten-fold more sensitive than conventional LAMP assay.

Vaccines (subunit and DNA) targeting major envelope proteins VP19 and/or VP28 of white spot syndrome virus (WSSV) in penaeid shrimp were developed and elicited good protection against white spot disease (WSD). However, the immune responses in shrimp after administration of these vaccines are not well understood. In this study, we developed a DNA vaccine encoding the VP28 envelope protein in kuruma shrimp (Marsupenaeus japonicus) and confirmed the potentiality of protection against WSSV infection. The efficacy of the DNA vaccine against WSSV infection was confirmed by WSSV artificial challenge at 7 days post vaccination in kuruma shrimp. However, the efficacy of the vaccine did not last 30 days post vaccination. The transcript of VP28 gene derived from expression vector in tissues of vaccinated shrimp was analyzed by RT-PCR. The transcript of VP28 gene was detected in various tissues including muscle, hemolymph, gill, intestine, stomach, heart, hepatopancreas and lymphoid organ tested at 1, 3 and 7 days post vaccination. Subsequently, the expression of innate immunerelated genes in intestine and lymphoid organ was analyzed at 1, 3 and 7 days post vaccination. The expression of innate immune-related genes such as Rab7, penaeidin, lysozyme, and crustin was up-regulated upon DNA vaccination. These results suggest that DNA vaccination induces significant protection against WSSV by stimulating innate immune responses in kuruma shrimp.

The scavenger receptor, Croquemort is a member of the CD36 superfamily comprising transmembrane proteins involved in the recognition of polyanionic ligands. Various researchers have proved that members of the CD36 superfamily are involved in immunity and developmental processes. In the present study, we report a cDNA encoding the kuruma shrimp, Marsupenaeus japonicus Croquemort scavenger receptor (MjSCRBQ) obtained from a cDNA library of lymphoid organ by RACE amplification. The full-length cDNA of 2098 bp consists an open reading frame of 1596 nucleotides that translates into a 532-amino acid putative protein, with a 5′ untranslated region of 323 bp and 3′ UTR of 153 bp. The MjSCRBQ is constitutively expressed in gills, heart, hemolymph, hepatopancreas, intestine, lymphoid organ, muscle, nerve, and stomach and at high levels in the brain. Expression analysis in lymphoid organs of shrimp infected with white spot syndrome virus (WSSV) revealed high levels of MjSCRBQ 72 and 120 h post-infection. The MjSCRBQ contains putative functional domains including transmembrane domains and a CD36 domain. Multiple alignments of MjSCRBQ amino acid sequences showed significant identity with Drosophila melanogaster SCRBQ (31%), Salmo salar SCRBQ (29%), Homo sapiens SCRBQ (28%) and Rattus norvegicus SCRBQ (30%). In a phylogenetic analysis, MjSCRBQ was identified in the invertebrate scavenger receptor cluster. This is the first report in crustaceans of the identification and characterization of a Croquemort scavenging receptor.

A tumor necrosis factor (TNF) gene has been isolated and characterized in kuruma shrimp, Marsupenaeus japonicus, providing the first conclusive evidence for the existence of the TNF ligand in shrimp. The kuruma shrimp TNF (MjTNF) cDNA was composed of 1868 bp with a 262 bp 5′-untranslated region (UTR) and a 220 bp 3′-UTR, which was translated into a protein of 462 amino acid residues that included a predicted transmembrane domain of 23 amino acid residues (Trp20–Val42) and the TNF family signature (Pro321–Leu448). Homology analysis of MjTNF showed 30.7% and 26.7% identities with fruit fly (Drosophila melanogaster) Eiger and human (Homo sapiens) ectodysplasin A, respectively. The MjTNF gene was constitutively expressed in unstimulated organs of shrimp such as the muscle, stomach, brain and gill. In lymphoid organ cells, an enhanced expression of the MjTNF gene was observed following stimulation with peptidoglycan and polycytidylic acid. A high expression level of MjTNF was observed in vivo 2 h and 4 h after stimulation with lipopolysaccharide and Vibrio penaeicida, respectively. These observations suggest that MjTNF plays a role in the innate immune defense in kuruma shrimp. The discovery of shrimp TNF will allow a more complete and concrete understanding of shrimp inflammatory responses.