A simple filter paper-based method for transporting and storing Enterocytozoon hepatopenaei DNA from infected Litopenaeus vannamei tissues.

The microsporidian parasite Enterocytozoon hepatopenaei (EHP) causes a significant negative impact in shrimp aquaculture. A diagnostic procedure for detecting EHP in shrimp was developed, but transportation of the infected shrimp samples from the farm / hatcheries to the laboratory is burdensome and preservation of the tissues is problematic. Here, we developed a simple method of transporting nucleic acid without preservatives using the Flinders Technology Associates filter card (FTA matrix card; Whatman). DNA can be stored and extracted without the need for centrifugation and hazardous chemicals. EHP infected shrimp homogenate was spotted on a FTA matrix card and stored at room temperature. Storage stability was confirmed by analysis at different time points and we efficiently recovered DNA up to 6 months post spotting. The recovery efficiency of FTA-DNA was compared with the existing DNA extraction methods DNeasy® Blood & Tissue kit method and Guanidine hydrochloride method. The efficiency of extraction and sensitivity of the DNA in the FTA card confirmed that recovery of EHP-DNA from the FTA matrix was superior to with other methods.

Molecular characterization and gene expression analysis of hypoxia-inducible factor and its inhibitory factors in kuruma shrimp Marsupenaeus japonicus.

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 < 0.05).

Characterization of xanthine dehydrogenase and aldehyde oxidase of Marsupenaeus japonicus and their response to microbial pathogen.

Reactive oxygen species (ROS) play key roles in many physiological processes. In particular, the sterilization mechanism of bacteria using ROS in macrophages is a very important function for biological defense. Xanthine dehydrogenase (XDH) and aldehyde oxidase (AOX), members of the molybdo-flavoenzyme subfamily, are known to generate ROS. Although these enzymes occur in many vertebrates, some insects, and plants, little research has been conducted on XDHs and AOXs in crustaceans. Here, we cloned the entire cDNA sequences of XDH (MjXDH: 4328 bp) and AOX (MjAOX: 4425 bp) from Marsupenaeus japonicus (kuruma shrimp) using reverse transcriptase-polymerase chain reaction (RT-PCR) and random amplification of cDNA ends (RACE). Quantitative real-time RT-PCR transcriptional analysis revealed that MjXDH mRNA is highly expressed in heart and stomach tissues, whereas MjAOX mRNA is highly expressed in the lymphoid organ and intestinal tissues. Furthermore, expression of MjAOX was determined to be up-regulated in the lymphoid organ in response to Vibrio penaeicida at 48 and 72 h after injection; in contrast, hydrogen peroxide (H2O2) concentrations increased significantly at 6, 12, 48, and 72 h after injection with white spot syndrome virus (WSSV) and at 72 h after injection with V. penaeicida. To the best of our knowledge, this study is the first to have identified and cloned XDH and AOX from a crustacean species.

Enhancement of immune response and resistance against Vibrio parahaemolyticus in kuruma shrimp (Marsupenaeus japonicus) by dietary supplementation of ß-1,4-mannobiose.

Supplementation of prebiotic carbohydrates can act as a potent immunomodulator and have the efficacy to induce immune-related genes which are involved in host defense. Pure ß-1,4-mannobiose (MNB) showed activation of prophenoloxidase system of shrimp hemocytes in vitro. The resistance of kuruma shrimp Marsupenaeus japonicus against Vibrio parahaemolyticus was examined after the shrimp were fed with 0 (control), 0.02, 0.2, and 2% MNB supplemented diets. The results showed significantly higher survival rates in MNB supplemented shrimp than those of the control one from 2 to 12 days post challenge. In another experiment, the hemocyte count, ROS production, phagocytic, phenoloxidase and bactericidal activities, and expression of immune-related genes were investigated in the control and MNB supplemented groups at day 1, 4, 6, 8 and 11 of the feeding. These immune parameters were significantly enhanced in MNB supplemented groups. Furthermore, the gene expression analysis showed that transcripts of lysozyme, crustin, penaeidin and TNF were significantly up-regulated in hemolymph, lymphoid organs and intestines of MNB treated shrimp. Overall, the results provided evidence that MNB supplementation could improve the immune response and increase shrimp resistance against V. parahaemolyticus infection.

CpG ODN mimicking CpG rich region of myxosporean Myxobolus supamattayai stimulates innate immunity in Asian sea bass (Lates calcarifer) and defense against Streptococcus iniae.

Oligodeoxynucleotides (ODNs) containing unmethylated cytosine-phosphate-guanine CpG dinucleotides within specific sequence contexts (CpG motifs) have been reported as pathogen-associated molecular patterns (PAMPs). Its immunostimulatory effects have been demonstrated in diverse vertebrate models. CpG ODN is typically found in bacterial or viral genome and recognized by a non-self recognition receptor Toll-like receptor9 (TLR9). Here, a new CpG ODN 1013 which mimics sequence of SSU rDNA of early eukaryotic organism myxosporidia, Myxobolus supamattayai, was employed to stimulate the immune responses of Asian sea bass Lates calcarifer. Its immunostimulant potentiality was comparatively compared with that of CpG ODN 1668, a widely used as functional immunostimulant. Both unmethylated CpG ODNs with some modified phosphorothioated positions were intraperitoneally injection (5 µg/fish). Hematological examination, immunological assays and immune-related genes expression were evaluated 12 h, 1, 3 and 5 d after post CpG ODN challenge. The immunosimulatory effect of these CpG ODNs on fish immunity to protect the bacterial pathogen Streptococcus iniae was also determined. The results demonstrated that these two CpG ODNs could induce immune responses in Asian sea bass including the significant (P < 0.05) increase level of WBC, peroxidase activity and oxidative radicals in head kidney (HK) leukocyte, serum innate immune parameters and up-regulation of four immune responsive genes compared with the control group. Most of immune responses induced by ODN 1668 were strong within 1 d but lesser extended while ODN 1013 prolonged the stimulatory effects during the whole experimental period. After challenge with S. iniae, the survival proportion in ODN 1013-treated fish was apparently higher than that treated with ODN 1668 and PBS, respectively. The results together suggested that CpG ODN 1013 enhanced innate immune responses, including humoral and cellular responses, through TLR9 mediated signaling pathway which is mainly contribute to the protective immunity in Asian sea bass against S. iniae infection. These findings can lead to a new approach in immunostimulant development by using the novel CpG ODN originating from the parasite M. supamattayai, besides those from bacterial and viral genomes, for disease control in fish host.

Deciphering of the Dual oxidase (Nox family) gene from kuruma shrimp, Marsupenaeus japonicus: full-length cDNA cloning and characterization.

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.

A new host record of Sphaerospora epinepheli (Myxosporea: Bivalvulida) occurring on orange-spotted grouper Epinephelus coioides from Thailand: epidemiology, histopathology and phylogenetic position.

In 1991, the first record of Sphaerospora epinepheli was described as a kidney parasite of wild and cultured malabar grouper, Epinephelus malabaricus, along coastlines of Thailand, the Gulf of Thailand and the Andaman Sea. However, the present study detected high infection of this parasite in kidney renal tubes of orange spotted grouper, Epinephelus coioides, collected from Andaman Sea. The highest infection rate of 36.82% was observed during the rainy season in 2009 in Phang-Nga Bay, in the north of Andaman Sea, which is an important grouper production site in Thailand. The biological and histopathological data of the parasite in this new host record are presented. Species classification is described based on morphological data of mature spore and molecular analysis of myxosporean 18S rDNA phylogeny including that of S. epinepheli which infected E. malabaricus. The genetic position of this parasite found in two host species was also studied. The phylogenetic tree analysis of small-subunit rDNA sequences of S. epinepheli from both infected hosts was constructed using two algorithms, maximum likelihood (ML) and Bayesian inference (BI). They were placed in the clustered basal sphaerosporid clade that contain four long SSU rDNA sphaerosporid species including Sphaerospora truttae, Sphaerospora elegans, Sphaerospora ranae, Sphaerospora fugu and Bipteria formosa with strong bootstrap supports. Histopathologically, renal intratubular myxosporean spores were associated with tubulonephosis, tubular necrosis, chronic interstitial nephritis and mimic membranoproliferative glomerulonephritis. This myxosporean parasite appears to be a significant pathogen on the basis of pathological changes in the renal tubules and is highly distributed in orange-spotted grouper.

Molecular cloning and characterization of the NADPH oxidase from the kuruma shrimp, Marsupenaeus japonicus: early gene up-regulation after Vibrio penaeicida and poly(I:C) stimulations in vitro.

Free radicals such as nitric oxide (NO) and reactive oxygen species (ROS) are involved in many physiological processes. In humans, there are 5 homologs of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (Noxes) that generate superoxide (O(2)(-)), which can dismute to produce ROS, and play significant roles in innate immunity and cell proliferation. Though Noxes have been identified in vertebrates (humans and fishes) and some insects, there are very few reports investigating Noxes in crustaceans. In the present study, we describe the entire cDNA sequence (4216 bp) of Marsupenaeus japonicus (kuruma shrimp) Nox (MjNox) generated using reverse transcriptase-polymerase chain reaction (RT-PCR) and random amplification of cDNA ends (RACE). The open reading frame of MjNox encodes a protein of 1280 amino acids with an estimated mass of 146 kDa that has 46.8% sequence homology with the Nox gene of the fruit fly, Drosophila melanogaster. Highly conserved amino acid sequences were observed in the NADPH binding domain. Transcriptional analysis revealed that MjNox mRNA is highly expressed in the lymphoid organ, hepatopancreas and hemocytes of the healthy kuruma shrimp. In the hemocytes, MjNox expression reached its peak 4 h after stimulation with either Vibrio penaeicida or poly(I:C) and decreased to its normal level after 12 h.This study is the first to identify and clone a Nox family member (MjNox) from a crustacean species.

Expression of the serum opacity factor gene and the variation in its upstream region in Streptococcus dysgalactiae isolates from fish.

Group C Streptococcus dysgalactiae (GCSD) is a pathogen of farmed fish. Almost all GCSD isolates from Asian countries, including Japan, Taiwan, Malaysia, and China, have a serum opacity factor (SOF-FD). Although the SOF-FD sequences in different GCSD isolates are identical, different opacification activities are observed. Three types of variations were observed in the upstream sequence of the sof-FD gene in GCSD isolates with different SOF-FD activities. Type 1 was characterized by insertion of an IS981-like element into the upstream region of the sof-FD gene. In Type 2, an IS981-like element was inserted into the upstream region in a direction opposite to that in Type 1. In Type 3, no IS element was inserted. Type 1 was predominant among Japanese isolates (129 of 133). Isolates from other Asian countries were generally Type 3 (13 of 16). Except for 1 strain, Type 1 strains exhibited opacification activities with optical densities (ODs)>0.6, while Type 2 and Type 3 strains have low opacification activities (ODs >0.2). Only Type 1 strains have putative -10 and -35 promoter regions upstream of the sof-FD gene, and the expression level of the sof-FD gene was higher in Type 1 strains than in Type 2 and Type 3 strains.

Cloning and expression of serum opacity factor in fish pathogenic Streptococcus dysgalactiae and its application to discriminate between fish and mammalian isolates.

Lancefield group C Streptococcus dysgalactiae (GCSD) is known as a causative agent of bovine mastitis and cardiopulmonary diseases in humans. Recently, GCSD has been isolated from diseased fish in Japan. Almost all culture supernatants and sodium dodecyl sulfate extracts obtained from GCSD isolated from farmed fish possessed serum opacity activity. Serum opacity factor (SOF) is a bifunctional cell-associated protein that causes serum opacification. In this study, a gene coding SOF, which was named sof-FD, was identified from GCSD isolated from fish. The amino acid sequence of sof-FD showed 40.1-46.5% identity to those of other SOFs from mammalian strains of S. dysgalactiae and Streptococcus pyogenes. Repetitive fibronectin binding domains were also observed in sof-FD, the structures of which were similar to those of other SOFs, as previously reported. The amino acid sequence of SOF was identical among fish isolates. A primer set targeting the sof-FD gene was designed and applied to a PCR assay for discriminating fish isolates from mammalian isolates.

Class B scavenger receptor, Croquemort from kuruma shrimp Marsupenaeus japonicus: Molecular cloning and characterization.

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.

The concentration of white spot disease virus for its detection in sea water using a combined ferric colloid adsorption- and foam separation-based method.

The white spot disease virus (WSDV), which is most virulent in shrimp, is a cause of serious damage in the shrimp production industry. However, it is difficult to track the infection route and behaviour of WSDV in shrimp farms because it is present at extremely low concentrations in culture sea water. In this study, the concentration of WSDV in sea water foam was examined using dispersed bubbles and milk casein as a surface-active protein. WSDV concentrations were assessed using real-time PCR. When ferric colloid adsorption was performed prior to foam separation, WSDV was effectively removed from sea water and concentrated in the generated foam within 5 min. The removal efficiency was greater than 90% at the optimum iron and casein concentrations of 1mg Fe/l and 1mg/l, respectively. Furthermore, to analyse the dissolution of the collected ferric colloid, the WSDV concentration in the colloid-dissolved solution was set to be 200-fold higher than that found in raw water. This represents a novel method of concentrating WSDV for its detection in sea water using a combination of ferric colloid adsorption and foam separation that is easy to perform, rapid and efficient.

Myxobolus supamattayai n. sp. (Myxosporea: Myxobolidae) from Thailand parasitizing the scale pellicle of wild mullet (Valamugil seheli).

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.

Molecular cloning and characterization of the nitric oxide synthase gene from kuruma shrimp, Marsupenaeus japonicus.

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.

A novel gene of tumor necrosis factor ligand superfamily from kuruma shrimp, Marsupenaeus japonicus.

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.

Real-time reverse transcription loop-mediated isothermal amplification for rapid detection of yellow head virus in shrimp.

A real-time reverse transcription loop-mediated isothermal amplification (real-time RT-LAMP) method was applied for detecting the replicase polyprotein-encoding gene of yellow head virus (YHV) in shrimp, Penaeus monodon. It is a novel, gene-specific assay that amplifies nucleic acid with high specificity, sensitivity and rapidity under isothermal conditions using a set of six specially designed primers that recognize eight distinct sequences of the target gene. This method works with even low copies of DNA and is based on magnesium pyrophosphate turbidity detection by an inexpensive photometer for quantitative analysis. A user-friendly protocol was developed with optimal conditions standardized at 63 degrees C for 60 min. With this protocol, the assay sensitivity was 10 times higher than the widely used YHV nested RT-PCR system. Cross-reactivity analysis using other shrimp virus DNA/cDNA and YHV-negative shrimp demonstrated high specificity of the assay. The real-time RT-LAMP method was performed also for an internal control gene, EF-1alpha, to compare with the expressions of the YHV gene in different organs of infected shrimp, and the resulting standard curves showed high correlation coefficient values. These results suggest that this assay is applicable widely as a new quantitative detection method in the pursuit of YHV-free shrimp culture.

Establishment of loop-mediated isothermal amplification method (LAMP) for the detection of Vibrio nigripulchritudo in shrimp.

LAMP is a novel method that amplifies DNA with high specificity and rapidity under isothermal conditions. In this study, using the LAMP method, a diagnostic protocol was developed for the detection of Vibrio nigripulchritudo in shrimps. Vibrio nigripulchritudo is associated with distinct shrimp diseases (vibriosis) and is considered one of the threatening pathogens in shrimp industry. After initial cloning and sequencing of the intergenic spacer region (ITS) between 16S and 23S rRNA genes of V. nigripulchritudo, a set of four primers - two inner and two outer - were designed for use in the LAMP reaction. Reaction time and temperature were optimized for 60 min at 63 degrees C, respectively. The detection limit of V. nigripulchritudo by LAMP was 10(2) CFU mL(-1) but PCR could detect up to 10(3) CFU mL(-1). The LAMP method could detect the presence of V. nigripulchritudo from heart, lymphoid organ, and muscle of experimentally infected shrimps with V. nigripulchritudo. This study established a highly sensitive and a rapid diagnostic procedure for detection of V. nigripulchritudo in shrimps. The method developed in this study could be very useful for routine shrimp disease diagnostics.

Identification of cDNA encoding Toll receptor, MjToll gene from kuruma shrimp, Marsupenaeus japonicus.

Toll receptors are cell-surface receptors acting as pattern recognition receptors (PRRs) that are involved in the signaling pathway for innate immunity activation and are genetically conserved from insects to mammals. Tolls from penaeid shrimp are found in white leg shrimp Litopenaeus vannamei (lToll) and black tiger shrimp Penaeus monodon (PmToll). However, the molecular ligand-recognition patterns and identification of these penaeid Toll classes remain unknown. Here, we report cDNA cloning of a new type of Toll receptor gene (MjToll) from kuruma shrimp, Marsupenaeus japonicus, and the modulation of expression by immunostimulation. The full length cDNA of MjToll gene has 3095 nucleotides coding for a putative protein of 1009 amino acids. The MjToll gene is constitutively expressed in the gill, gut, lymphoid organ, heart, hematopoietic organ, hemocyte, ventral abdominal nerve cord, eyestalk neural ganglia and brain tissues. The MjToll gene expression was significantly increased (76-fold) as compared to a control in lymphoid organ stimulated with peptidoglycan at 12h, in vitro. lToll gene showed high similarity to PmToll gene with 96.9% identity; however, MjToll gene exhibited a percentage identity of 59% with that of penaeid Toll homologues. Therefore, this suggests that the identified MjToll gene belongs to the other class of Toll receptors in shrimp.

Detection of yellow head virus in shrimp by loop-mediated isothermal amplification (LAMP).

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.

Replication of white spot syndrome virus in ovarian primary cultures from the kuruma shrimp, Marsupenaeus japonicus.

Propagation of white spot syndrome virus (WSSV) was investigated in primary ovarian cultures from the kuruma shrimp Marsupenaeus japonicus. A WSSV strain, purified by sucrose density gradient centrifugation, was inoculated into 10-day-old primary ovarian cultures. WSSV infection induced marked cytopathic effect (CPE) on primary ovarian cells. Initially, virus-infected cells began to shrink 72 h post-inoculation, followed by the rounding of most cells which detached finally from flask surface. Electron microscopic observations clearly showed that the replication of WSSV occurred in nuclei of ovarian cells. Immunoblot analysis with antibodies against the WSSV envelope protein VP28 provided the evidence that the levels of WSSV antigens in culture supernatant gradually increased during the period between 24 and 120 h after virus inoculation. The results suggest that the use of primary ovarian cultures of the kuruma shrimp will facilitate characterization of the WSSV infection.