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The structures of fish serum immunoglobulin differ among different fish species. In this study, we accidently isolated a rabbit immunoglobulin (Ig) light chain bound to serum immunoglobulin from different marine fish species using phage display. Fish Ig was separated using a protein A column.The phage library was generated from variable regions of rabbit spleen B cells immunized with bluefin tuna Thunnus orientalis Ig. Fish Ig–specific phages were enriched using two rounds of bio-panning with yellowtail Seriola quinqueradiata serum Ig, followed by two rounds of bio-panning with red seabream Pagrus major serum Ig.The enriched phages demonstrated an increase in binding specificity to the tuna, yellowtail, and red seabream Igs compared to the phages listed in the unpanned library.A recombinant protein of a single clonal phage, which encodes the rabbit Ig light chain, was produced, and the binding specificities to fish Igs were analyzed using enzyme-linked immunosorbent assay (ELISA) and western blotting.The recombinant protein exhibited binding properties to fish Igs in the ELISA. However, the recombinant protein that bound to serum protein(s), but not IgM, was detected via western blotting.The recombinant protein may provide a novel information on the common structural feature in the fish immunoglobulins., 公開日: 2023-07-21

Red sea bream iridovirus (RSIV) is the pathogen that causes red sea bream iridoviral disease. It causes a huge loss to the Japanese aquaculture industry. In 2021, outbreaks of red sea bream iridovirus occurred in South Japan. This study analysed nine whole-genome sequences of RSIV isolated in Oita and Ehime Prefectures in 2021 using a short-read next-generation sequencer. Nine isolates had highly uniform sequences, and there was no variant depending on locations or host species. Phylogenetic analyses with other reported megalocytivirus isolates showed that RSIV isolated in 2021 was genetically different from RSIV previously isolated in Oita and Ehime Prefectures in 2017-2019. These results suggest that RSIV isolated in Oita and Ehime Prefectures in 2021 might spread from a common ancestor different from the recent one. Additionally, it was found that RSIV isolated in 2021 had sequence mutations on protein-coding sequences that may be involved in viral pathogenicity and infectivity.

Virus like particles (VLPs) are non-infectious nanoparticles containing repetitive, high density viral epitopes on the surface and can prevent viral infections in aquatic animals. Here, we evaluated the immuno-stimulation effect of infectious hypodermal and hematopoietic necrosis virus like particle (IHHNV-VLP) using a next generation sequencing in Fenneropenaeus merguiensis to identify the important immune-related genes that may prevent viral infection. The in situ target of IHHNV was predominantly found in gill tissue following IHHNV-VLP administration in juvenile shrimp. Comparative transcriptome analysis in the injected gills showed that there were 326 unigenes expressed differently than the mock-injected samples. One of the most differential genes between the two animal groups was the antioxidative gene, peroxiredoxin (FmPrx), that was up-regulated after 6 h post-VLP injection. Phylogenetic tree analysis showed that this gene could be found among many shrimp species and was closely clustered among Prx families. The expression of FmPrx was also detected in all tissues examined, thus suggesting the multi-functional roles of this gene in many tissues. Administration of IHHNV-VLP in vivo led to a significant increase in peroxidase activity in gill tissue-approximately two-fold versus control animals; the WSSV copy number was significantly reduced. These data suggest that IHHNV-VLP exerts an immune-stimulating effect by enhancing the level of immune-related genes including FmPrx and its corresponding peroxidase activity, which are a well-known part of the shrimp innate immune system.

Red sea bream iridovirus (RSIV) belonging to the genus Megalocytivirus of the family Iridoviridae is the cause of serious mass mortality of cultured marine fishes. RSIV-type megalocytiviruses show extremely high nucleotide sequence identities. Thus, epidemiological studies on this virus are limited. This study developed two primer sets amplifying the regions possessing single nucleotide polymorphism (SNP) to determine the relationships and divergence of RSIV-type megalocytiviruses isolated from cultured marine fishes in Japan. The two regions were designed according to the genome sequences of the representative RSIV genotype II of megalocytivirus members in GenBank. The SNP 1 and 2 regions have sequences homologous to hypothetical protein ORF 24 and ORF 31, respectively, of RSIV (accession no. AP017456.1). By sequencing the regions, 53 polymorphic sites were identified. The phylogenetic analysis of 25 RSIV-type megalocytivirus isolates, classified into RSIV cluster, was clustered into eight haplotypes (seven haplotypes from Oita, two haplotypes from Ehime, and one haplotype shared between Oita and Ehime). These findings suggested that SNP in the RSIV genome is a powerful application for the detection and identification of RSIV-type megalocytiviruses.

Here, we investigated the effect of dietary cysteic acid on the growth performance, sulfur amino acid content, and gene expression levels of taurine-synthesizing enzymes, growth hormone (GH), and insulin-like growth factor 1 (IGF-1) in Japanese flounder Paralichthys olivaceus. Juvenile flounder (0.9 g) were fed one of four diets for 30 days: with 0.25, 0.5, or 1.0% cysteic acid (C0.25, C0.5, C1.0) supplementation and without supplementation (control). Fish in the C0.25 and C0.5 groups showed significantly better growth than those in the control group (P 0.05), the expression level of IGF-1 in C1.0 fish was significantly higher than that in controls (P

Gene expression profiles during the transition from fasting to refeeding were investigated in the gut and liver of Masu salmon Oncorhynchus masou masou. Fish were starved for 3 days and then fed at 3% of body weight. The gut and liver were resected before and after feeding, and the tissues were used for transcriptome analyses. Twenty-nine and 45 genes were more than eightfold differently expressed in the gut and liver, respectively. Genes involved in fatty acid and carbohydrate metabolism were differentially expressed in the liver tissues. Genes involved in protein folding, such as GRP78 and endoplasmin, were significantly upregulated in both organs at 6, 12, and 24 h after feeding. Furthermore, glycogen synthase kinase (GSK) 3-binding protein, a negative regulator of GSK with pivotal roles in stress response, was downregulated in both organs 6 h after feeding. These results suggest that the change in nutritional status from fasting to refeeding is accompanied by cellular stress.

Acute hepatopancreatic necrosis disease (AHPND), caused by a toxin-producing Vibrio parahaemolyticus strain, has become a serious threat to shrimp aquaculture. The need to regulate antibiotic use prompted the development of alternative ways to treat infections in aquaculture including the use of chicken egg yolk immunoglobulin (IgY) for passive immunization. This study evaluated the protective effect of IgY against AHPND infection in Litopenaeus vannamei (Boone). IgY was isolated from eggs laid by hens immunized with recombinant PirA-like (rPirA) and PirB-like (rPirB) toxins. Whole-egg powders having IgY specific to rPirA (anti-PirA-IgY) and rPirB (anti-PirB-IgY) and IgY from non-immunized hen (control-IgY) were mixed with basal diets at 20% concentrations and used to prefeed shrimp 3 days before the bacterial challenge test. Survival rates of the challenged shrimp fed the anti-PirA-IgY, anti-PirB-IgY and control-IgY diets were 86%, 14% and 0%, respectively. Only the feed containing anti-PirA-IgY protected shrimp against AHPND. Increasing the concentration of rPirA antigen to immunize hens and lowering the amount of egg powder in feeds to 10% consistently showed higher survival rates in shrimp fed with anti-PirA-IgY (87%) compared with the control (12%). These results confirm that addition of anti-PirA-IgY in feeds could be an effective prophylactic method against AHPND infection in shrimp.

Crustacean hemocytes, which have usually been classified morphologically based on methods using Giemsa or May-Giemsa stains, have recently been categorized using monoclonal antibodies or marker genes. However, these latter techniques are not yet widely used, and different classification methods are used for hemocytes among laboratories. Therefore, we aimed to develop a molecular classification method that can be widely used by researchers. The method we have developed uses lectins and magnetic-activated cell sorting (MACS) to isolate sub-populations of hemocytes. Two lectins, wheat germ agglutinin (WGA) and tomato lectin (Lycopersicon esculentum lectin; LEL), characteristically bind to hemocytes, which allows them to be classified into three sub-populations. Furthermore, different sub-populations of hemocyte can be isolated by using LEL and MACS. These sub-populations were characterized as non-granular and granular hemocytes, and the accumulation patterns of the gene transcripts were consistent with the results of a functional analysis reported previously. The lectin-based hemocyte isolation method developed in this study has good reproducibility.

Fc receptors (FcRs) are specific to the Fc portion of immunoglobulin (Ig) molecules. Here, four Fc receptor-like proteins, JF-FcR-like protein 1-4, were identified in Japanese flounder. Their open reading frames encoded 358, 255, 519 and 441 amino acid residues, respectively. JF-FcR-like protein mRNAs were mainly detected in kidney and spleen of healthy fish. Injection of formalin-killed cells (FKCs) of Edwardsiella tarda significantly increased the spleen mRNA levels of JF-FcR-like protein 1 but not the other JF-FcR-like proteins. Injection of FKC of Streptococcus iniae did not significantly affect any of the JF-FcR-like protein mRNAs. These findings suggest that the FcR-like proteins have different involvements in pathogen responses.

The white spot syndrome virus (WSSV) has been considered a serious threat to shrimp aquaculture. Besides, the activation of cell metabolism as an immune reaction to the virus is now recognized as a piece of the pivotal puzzle of the antiviral responses. Hence, this study explores the relationship between metabolic gene expression and antiviral responses in shrimp using transcriptome analysis. The RNA-seq libraries of Fenneropenaeus merguensis hemocytes after WSSV challenge at early (6 hpi) and late (24 hpi) stages of infection were analyzed to identify differentially expressed genes (DEGs) that the WSSV subverted the expression. One-hundred-thirty-three DEGs that were expressed in response to WSSV infection at both stages were identified. Based on the GO annotation, they were related to innate immunity and metabolic pathway. The expression correlation between "full term" (NGS) and qRT-PCR of 16 representative DEGs is shown. Noticeably, the expression profiles of all the selected metabolic genes involved in glucose metabolism, lipid metabolism, amino acid metabolism, and nucleotide metabolism showed a specific correlation between NGS and qRT-PCR upon WSSV infection. Of these, we further characterized the function related to the WSSV response of glutamine: fructose-6-phosphate aminotransferase (FmGFAT), the rate-limiting enzyme of the hexosamine biosynthesis pathway, which was found to be up-regulated at the late stage of WSSV infection. Suppression of FmGFAT by RNA interference resulted in postponing the death of WSSV-infected shrimp and reduction of viral copy number. These results suggested that the FmGFAT is linked between metabolic change and WSSV responses in shrimp, where the virus-induced metabolic rewiring hijack biological compounds and/or energy sources to benefit the viral replication process.

We evaluated the effects of Freund's adjuvants (FCA/FIA) on protection and immune response of Japanese flounder Paralichthys olivaceus immunized by the formalin-killed cell (FKC) of Edwardsiella tarda. Combination of FKC and FCA/FIA did not confer protection against the challenge, while they significantly induced higher antibody titers than that of FKC only. The suppression of FKC-dependent induction of interferon γ (IFNγ) mRNA levels by FCA/FIA was observed by gene expression profiling. Similarly, interleukin (IL)-12 p35 mRNA levels were not detected after FKC+FCA or +FIA. These results suggest that the mineral oil in Freund's adjuvants might suppress the signaling pathway(s) that induce IFNγ and IL-12 gene expression.

Although microorganisms in pond water and shrimp gut have been thought to be important factors in shrimp health, knowledge of the microbiota surrounding the shrimp is limited. In this study, we analyzed bacterial and eukaryotic microbiota in the pond water of whiteleg shrimp Litopenaeus vannamei, and bacterial compositions in the stomach and midgut of shrimp that are reared in the ponds.Microorganisms were collected at three sites in each of two ponds using filter membranes for 7 consecutive days.Amplicons of 16S rDNA and 18S rDNA were generated. Shrimp were also collected from the ponds on the same days and 16S rDNA was amplified from their stomachs and midguts and analyzed.Results showed that the bacterial communities in the surface water were distributed uniformly and were relatively stable, and the differences between the ponds were small. However, a principal coordinate analysis indicated significant differences in the composition of bacteria in the water of each pond and the microbiota shifted continuously during the sampling period.The eukaryotic plankton in the water changed dynamically and consecutively including the alternations of the dominant families in 1 to 2 days.The bacterial compositions of the digestive tracts, especially the stomach, clearly differed between the two ponds.Also, a bacterium, classified as Candidatus Bacilloplasma in class Mollicutes which was found in the stomachs might be a novel lineage of Mollicutes.Analysis of the combination of bacterial and eukaryotic microbiota, and not only the intestine (midgut) but also the stomach microbiota would be useful to understand microbiome in shrimp aquaculture., 公開日: 2024-03-20

Polymorphic simple sequence repeat (SSR) markers of Ecklonia radicosa were developed using Illumina MiSeq next-generation sequencing (NGS) analysis. In total, 3112 SSRs (di- to hexanucleotide motifs repeated more than six times) were identified from 61.5-Mb assembled genomic DNA using MISA perl script. Among the SSRs, di- (853 loci, 27.4%) and trinucleotides (1813 loci, 58.3%) were dominant, while tetra- (172 loci, 5.5%), penta- (175 loci, 5.6%), and hexanucleotides (99 loci, 3.2%) were not common. After specific amplification and polymorphic tests of 75 selected loci (tri- to hexanucleotide motifs repeated more than eight times), 11 SSR markers (Eradic01–11) were successfully developed. The range of the number of alleles, observed heterozygosity, and expected heterozygosity in the 11 markers was 3–13, 0.200–0.683, and 0.258–0.864, respectively. Polymorphic information content (PIC) analysis indicated that eight markers (Eradic01, 02, 04, 05, 06, 08, 09, and 11) were highly informative (PIC > 0.5) and the other three were reasonably informative (0.5

A viral responsive protein (MjVRP) was characterized from Marsupenaeus japonicus haemocytes. In amino acid homology and phylogenetic tree analyses, MjVRP clustered in the same group with the viral responsive protein of Penaeus monodon (PmVRP15), showing 34% identity. MjVRP transcripts were mainly expressed in haemocytes and the lymphoid organ. Western blotting likewise showed that MjVRP was strongly expressed in haemocytes and the lymphoid organ. Immunostaining detected MjVRP within the cytosol next to the perinuclear region in some haemocytes. Experimental challenge with white spot syndrome virus (WSSV) significantly up-regulated the mRNA level of MjVRP in the M. japonicus haemocytes at 6 and 48 h. Flow cytometry and indirect immunofluorescence assays revealed that the ratio of MjVRP+ haemocytes significantly increased 24 and 48 h post-WSSV infection. These results suggest that MjVRP+ haemocytes have a supporting role in the pathogenesis of WSSV.

Two novel type I interferon genes (JfIFN3 and JfIFN4) have been identified in Japanese flounder Paralichthys olivaceus. Open reading frames of JfIFN3 and JfIFN4 were 555bp and 528bp, encoding 184aa and 175aa, respectively. The genomic structures of JfIFN3 and JfIFN4 are composed of 5 exons and 4 introns. JfIFN4 has 2 conserved cysteine residues, while JfIFN3 has 4. JfIFN3 and JfIFN4 showed the highest amino acid sequence identities to turbot IFN1 (74%) and IFN2 (62%), respectively. Interestingly, JfIFN3 and JfIFN4 were clustered in distinct branches with JfIFN1 and JfIFN2, which have reported so far. The mRNA levels of JfIFN4 were apparently increased in the kidney and spleen at 3 h after ployI:C injection, while JfIFN1-3 were not detected by RT-PCR.

Neobenedenia melleni is a monogenean parasite that causes significant mortality and economic losses in fish aquaculture. Changes in the antigenic composition of this parasite occur during its developmental stages. In this study, we evaluated humoral parameters in serum and transcriptional immune responses of yellowtail naturally infected with N. melleni. In addition, in vitro assays were performed to study the stimulatory effects of antigens from larvae and adults on spleen leucocytes from non-infected fish at 6 and 24 h post-stimulation. The results showed enhanced total protein, myeloperoxidase and antiprotease activities in N. melleni-infected fish compared with non-infected ones. The induction of Toll-like receptors (TLRs) and pro-inflammatory cytokines in spleen leucocytes during natural infection with N. melleni suggests that these immune-related genes play an important role in the initiation of the immune defence mechanism for controlling parasite infection. Interestingly, the magnitude of in vitro responses of spleen leucocytes was dependent on the parasitic stage. An important stimulation of gene expression by adult antigens on spleen leucocytes was observed. Differential expression patterns of TLRs and target cytokines in yellowtail leucocytes in both in vivo and in vitro studies suggest that the quality of yellowtail immune response is conditioned by N. melleni development.

Suppressor of cytokine signaling (SOCS) family members are key regulators of the immune system, particularly cytokine action, and have now been discovered in a number of fish species. Here we identified eight SOCS proteins (CISH, SOCS1a, SOCS1b, SOCS3a, SOCS3b, SOCS5, SOCS6 and SOCS9) in the Japanese flounder and analyzed their mRNA expressions after injection of poly (I:C) and formalin-killed cells (FKC) of Edwardsiella tarda. The expressions of all eight SOCS genes were detected in all the tissues examined. Stimulation of Japanese flounder reared at 15 or 25 °C with poly (I:C) affected the gene expressions of CISH, SOCS1a, SOCS1b and SOCS3a. All SOCS genes mRNA levels were significantly changed after FKC injection. Significant up-regulation of SOCS1a, SOCS1b, SOCS3a and SOCS3b genes was detected at 3, 12 and 24 hpi. SOCS5 and SOCS6 genes were significantly down-regulated at 3 hpi. SOCS9 gene was significantly up-regulated at 12 hpi. These results suggest that all eight of the SOCS genes are involved in immune responses, and that the CISH, SOCS1 and SOCS3 genes have functions distinct from those of the other SOCS members.

In invertebrates, hemocytes play an important role in immune responses. Recently, a novel form of innate immune mechanism called extracellular traps (ETs) was identified in shrimps, where DNA and antimicrobial peptides form complex structure to entrap the invading microbes. In this study, we detected the formation of ETs from hemocytes of kuruma shrimp in response to various stimulations, including phorbol myristate acetate (PMA), lipopolysaccharide (LPS), peptidoglycan (PGN) and Escherichia coli. E. coli cells were also found to be trapped by ET fibers. Fluorescence imaging revealed that c-type lysozyme proteins were released around the ET complex after E. coli stimulation, suggesting the presence of a coupled antimicrobial immune response involving ET formation and AMP release.

Continuous shrimp cell cultures offer opportunities for studying shrimp pathogens at the cellular and molecular level and developing diagnostic tools. However, no continuous shrimp cell lines have yet been successfully established. This might be due to the lack of information on the molecular mechanisms that control shrimp cell proliferation and cell arrest under culture conditions. In this study, differentially expressed genes (DEGs) in shrimp primary culture cells were determined by comparison with normal tissue (in vivo). This study aimed to comprehensively identify key regulator genes that control cell proliferation and cell cycle arrest in culture conditions. The primary shrimp cells were derived from testicular tissue and cultured in vitro. RNA sequencing (RNA-Seq) was performed to investigate change in gene expression level across the entire transcriptome between shrimp primary cells and normal tissue (in vivo). RNA-seq results revealed over 100 genes with distinct gene expression patterns between primary cells and normal tissue. The DEG results and functional gene analysis showed a clear difference in gene expression patterns of cell cycle-related genes between primary cells and normal tissue. LRWD1, TMEM127, CDCA3, PPP2R1A, and GOLGA2, which are cell cycle-related genes, were downregulated in shrimp primary cells. These genes are required for G1/S phase transition, entry into mitosis, and cell proliferation control. However, a gene that induces cell cycle arrest, ARAF, was upregulated in culture conditions. The results demonstrated that these genes might play essential roles in cell proliferation and cell arrest under culture conditions. These candidate genes may be alternative potential targets for genetic manipulation to maintain cell proliferation and establishment of a shrimp continuous cell line.

Arginine is a versatile amino acid that plays important roles in various physiological functions. However, supplementation with arginine can also increase urea excretion of the fish. Thus, given than ornithine and citrulline can potentially be used to increase arginine availability or improve fish immunity, this study aimed to compare the effects of dietary supplementation of arginine, ornithine, and citrulline on the survival of rainbow trout Oncorhynchus mykiss against Vibrio anguillarum. Dietary treatment consisted of a control diet (CTRL) and three treatment diets comprising CTRL supplemented with 2% L-arginine (+ARG), L-ornithine (+ORN), or L-citrulline (+CIT). Protein and lipid content of the diets were confirmed at 48.9 and 18.9%, 53.1 and 18.7%, 51.7 and 18.1% and 52.9 and 18.9% for the CTRL, +ARG, +ORN & + CIT diets respectively. Postprandial plasma amino acid levels were measured at 6, 15, and 30 h after feeding. A feeding trial was conducted for 30 days, and at the end of the trial, the rainbow trout were challenged with V. anguillarum. At 24 h post-injection, plasma amino acid and renal gene expression levels were examined. Fish exhibited better weight gain in +ARG treatment compared to +CIT treatment. Similar postprandial levels of plasma arginine were observed in +ARG and + CIT, and the levels in both treatments were significantly higher than those in CTRL. A survival analysis revealed that fish in +CIT (69.2%) were more resistant than those in CTRL (25.0%). There was significantly higher inducible nitric oxide synthase (iNOS) expression in the kidneys of fish in +CIT than those in CTRL. A significant difference in interleukin-1-beta expression was found between +ORN and CTRL treatments, while there was no significant difference in arginase II expression among the treatments. These result shows that citrulline supplementation can be used to increase arginine availability and better at enhancing the immune performance of rainbow trout compared to arginine supplementation when challenged with V. anguillarum.

White tail disease caused by Macrobrachium rosenbergii nodavirus (MrNV) infection takes place only in nauplii, not adults, of M. rosenbergii prawn. Hemocyte homeostasis and immune-related functions derived from the hematopoietic tissue (Hpt) in adult prawn are presumed to play roles in resisting viral infection. To elucidate the role of the Hpt cell response to MrNV, a comparative transcriptome analysis was performed with MrNV-infected prawn at various time intervals. The results showed that there were 462 unigenes that were differentially expressed between mock and infected samples. BlastX sequence analysis revealed that two proteins, crustacean hematopoietic factor (CHF) and cell growth-regulating zinc finger protein (Lyar), are involved in hemocyte hematopoiesis and are up-regulated during MrNV infection. In fact, genes involved in cell growth regulation and immunity were highly expressed at 6 h and decreased within 24 h post-infection. Localization studies in the Hpt tissue revealed the presence of anti-lipopolysaccharide factor (ALF) and CHF mRNAs in Hpt cells. Considering these findings, we concluded that resistance to MrNV infection in adult prawn is due to an increase in humoral immune factors and the acceleration of hemocyte homeostasis by the dual roles of the Hpt organ in M. rosenbergii.

Acute hepatopancreatic necrosis disease (AHPND) is a shrimp farming disease, caused by the pathogenic Vibrio parahaemolyticus carrying a plasmid encoding Vp_PirAB-like toxins. Formalin-killed cells of V. parahaemolyticus AHPND-causing strain D6 (FKC-VpD6) were used to select Vp_PirAB-like toxin-resistant Litopenaeus vannamei by oral administration. Stomach and hepatopancreas tissues of shrimps that survived for one week were subjected to RNA sequencing. Differentially expressed genes (DEGs) between surviving shrimp, AHPND-infected shrimp, and normal shrimp were identified. The expressions of 10 DEGs were validated by qPCR. Only one gene (a gene homologous to L. vannamei anti-lipopolysaccharide factor AV-R isoform (LvALF AV-R)) was expressed significantly more strongly in the hepatopancreas of surviving shrimp than in the other groups. Significantly higher expression of LvALF AV-R was also observed in shrimp that survived two other trials of FKC-VpD6 selection. Recombinant ALF AV-R bound to LPS, PGN, Gram-negative bacteria, and some Gram-positive bacteria in ELISAs. ALF AV-R recombinant protein did not interact with native Vp_PirAB-like toxin in an ELISA or a Far-Western blot. For L. vannamei orally fed ALF AV-R protein for 3 days, the survival rate following challenge with VpD6-immersion was not significantly different from that of shrimp fed two control diets. These results suggest that LvALF AV-R expression was induced in the hepatopancreas of shrimp in response to the presence of Vp_PirAB-like toxin, although other factors might also be involved in the resistance mechanism.

Cysteine sulfinic acid decarboxylase catalyzes reaction of decarboxylation of cysteine sulfinic acid into hypotaurine. This step is considered as a rate limiting step of taurine biosynthesis in animals. Because of lower CD activity, marine fish has limited ability of de novo taurine synthesis and requires taurine in food. However, if we can develop a method to control CSD activity in marine fish, supplement of taurine to diet of marine fish is not necessary. In order to develop this technique, we have to isolate CSD from marine fish. This study, thereby, is conducted to isolate CSD from red sea bream Pagrus major and yellowtail Seriola quinqueradiata. We also analyze the expression of CSD in red sea bream, yellowtail, Japanese seabass Lateolabrax japonicus, and barfin flounder Verasper moseri. Total RNA was extracted from liver of red sea bream and yellowtail. Primers are designed against sequence of highly conserved region of reported CSD in other animals for RT-PCR. 776 bp and 725 bp partial CSD sequences were successfully cloned from red sea bream and yellowtail. By 5′ and 3′ RACE methods for cloned partial CSD sequences, total length of CSD (1882 bp and 1821 bp) from red sea bream and yellowtail was determined. Classification of deduced amino acid sequence revealed that red sea bream and yellowtail CSD showed 88.8% homology similar to Nile tilapia, platyfish, etc. Domain analysis shows that pyridoxine binding region was conserved in CSD from both species. Expression analysis of CSD from red sea bream, Japanese seabass, barfin flounder and yellowtail indicated that CSD is expressed in a variety of tissues but commonly in the liver and pyloric ceca of all species examined. Additionally, strong CSD expression was observed in the heart in all species examined except for Japanese seabass.

Gene expression profiling of poly (I:C)-treated Japanese flounder, Paralichthys olivaceus, under different temperatures was investigated using microarray analysis. The response was analyzed in spleen tissue at 3 and 24 h post injection (hpi) at 15 °C and 25 °C. A large number of genes in fish treated with poly (I:C) at 25 °C were expressed at 3 hpi, whereas the expression profiles at 24 hpi appeared to be similar to those of the controls. Cluster analysis of the different expression profiles showed three distinct groups of up-regulated genes in fish reared at 15 °C. These were early (3 hpi), early-to-late (3 and 24 hpi), and late (24 hpi) up-regulated genes. These genes included type I IFN-related genes and inflammatory genes. Among the up-regulated genes, most of the type I IFN-related genes played early-to-late- and late-responding genes at 15 °C but early-responding genes at 25 °C. Thus, several up-regulated genes in these groups from the microarray result were further verified by qPCR. These results indicate that the type I IFN gene expressions of P. olivaceus treated with poly (I:C) can be regulated in a temperature-dependent manner.

Partial sequences of cytokine genes IFNγ, IL-1β, IL-8, IL-10, and TNFα were identified from next-generation sequencing data for three amberjack species: Seriola quinqueradiata, S. lalandi and S. dumerili. The cytokine genes had more than 90 % nucleotide identity among the three species. Quantitative PCR (qPCR) primers were designed from the consensus sequences to detect cytokine genes. The specificities of consensus qPCR primers were investigated in the head kidney of three amberjack species treated with formalin-killed cells (FKC) of Vibrio anguillarum and polyinosinic-polycytidylic acid [poly(I:C)] at 3 h post injection (hpi) using qPCR. The primers detected up-regulation of certain cytokine genes after FKC of V. anguillarum and poly(I:C) stimulation in all species examined. Consequently, the primers are suitable for studying and detecting cytokine genes after vaccination and pathogen infection in these three amberjack species.

Penaeidins are a unique family of antimicrobial peptides specific to penaeid shrimp and have been reported mainly function as anti-bacterial and anti-fungal. In order to investigate whether penaeidins could also respond to virus or not, we examined the effect of WSSV on MjPen-II (penaeidin in kuruma shrimp, Marsupenaeus japonicus) expression. In the control group, MjPen-II transcript level can be detected in almost all test tissues but was expressed most strongly in hemocytes. After WSSV infection, MjPen-II transcript level was significantly downregulated in hemocytes. Moreover, the proportion of MjPen-II+ hemocytes was not significantly different between non-infected and WSSV-infected shrimp, but the number of MjPen-II+ highly expressing hemocytes decreased after infection. In addition, MjPen-II was observed in the cytoplasm of granule-containing hemocytes. These results suggest that WSSV suppresses MjPen-II expression in hemocytes.

Shrimp, as invertebrates, have an open vasculature that allows circulating hemocytes to infiltrate the tissues, where they are referred to as sessile hemocytes. Sessile hemocytes are known to express immune-related genes, but it is not known whether their functions differ from those of circulating hemocytes. To answer this question, we enriched them from suspensions of different tissues using discontinuous density gradient centrifugation and analyzed their transcripts by RNA-seq. The results suggest that circulating hemocytes and sessile hemocytes of the gills are in a state that could react quickly to pathogens, immune-related genes expression of sessile hemocytes differ from circulating hemocytes, and the gills, heart and lymphoid organs have cells that express immune-related genes that are different from hemocytes.

cDNA of a newly recognized white spot syndrome virus (WSSV)-induced gene (MjVIG1) was characterized from Marsupenaeus japonicus hemocytes; this gene encodes a protein that lack similarity to any known characterized protein. To identify this novel gene, we mainly conducted transcript level analysis, immunostaining and flow cytometry after WSSV infection. MjV1G1 transcript levels were also measured after Yellow head virus (YHV) and Vibrio parahaemolyticus infection tests. In non-infected and WSSV-infected shrimp, MjVIG1 was observed in granule-containing hemocytes. In addition, the MjVIG1 transcript level and ratio of MjVIG1-positive hemocytes both significantly increased, and number of MjVIG1-positive hemocytes slightly increased after WSSV infection. In contrast, MjVIG1 transcript level did not change after YHV and V. parahaemolyticus infection. These results indicated that MjVIG1 might be a WSSV-specific induced gene in M. japonicus hemocytes.

Influence of temperature on the susceptibility of fish against virus infection has been studied for a decade. Recent reports have been shown the effects of rearing temperatures on the fish immune system against virus infection. However, the roles of temperature in regulation of type I interferon (IFN) system has not yet been investigated. Thus, the effects of temperature on type I IFN response were investigated in this study using poly (I:C) injection in sevenband grouper and Mx gene was used as a marker for type I IFN expression. Quantitative real-time PCR (qPCR) result showed that Mx expression profiles were moderately different between temperatures. The highly up-regulated Mx transcripts at 3 h post injection (hpi) were observed in high temperatures (25 °C and 30 °C) but not in low temperatures (15 °C and 20 °C). Meanwhile, low temperatures (15 °C and 20 °C) could detect the highly up-regulated Mx transcripts at 24 hpi. Expression of Mx transcripts was also observed at 72 hpi at 15 °C. Poly (I:C)-injected fish were challenged with RGNNV after 72 and 168 hpi. At 72 hpi, 100% of fish survived at all temperatures, whereas 95% survival rate was observed at 168 hpi at 25 °C during 14 days of observation. To further verify the duration period of an antiviral state at different temperatures, qPCR and endpoint dilution assay were used to quantify the number of virus in fish challenged with RGNNV. The reduction of viral copy numbers and viral titers could be observed at 72 and 168 hpi. However, high viral copy numbers and viral titers could be detected at 168 hpi at 30 °C. These results demonstrate that temperatures influenced on the Mx expression profiles and the duration period of an antiviral state efficiently interfered with virus replication at different temperatures.

Cyprinid herpesvirus 3 (CyHV-3) infection in common carp Cyprinus carpio L. and its ornamental koi varieties can induce the severe systemic disease known as koi herpesvirus disease. This disease is characterised by a rapid replication and spreading of the virus through multiple organs and results in a fast onset of mortality (starting on Day 6 post infection) in up to 100% of infected fish. During the first phase of viral infections, type I interferons (IFNs) have gen- erally been proven to be essential in inducing an innate immune response; however, very little is known about the type I IFN response to herpesviruses in fish. The aim of this work was to study the type I IFN responses during CyHV-3 infection in 2 genetically divergent lines of common carp which presented differing survival rates. Our results show that CyHV-3 induced a systemic type I IFN response in carp, and the magnitude of type I IFN expression is correlated with the virus load found in skin and head kidney. In this in vivo experimental setup, the level of type I IFN response cannot be linked with higher survival of carp during CyHV-3 infection.

Shrimp, like other invertebrates, relies solely on its innate immune system, to combat invading pathogens. The invertebrate immune system has ancient origins that involve cellular and humoral responses. The clotting system of the humoral immune response is the first line of defense against pathogens and also serves to prevent blood loss during injury and wound healing. Tranglutaminase and clotting protein are molecules involved in the blood clotting system of crayfish and shrimp. Studies have shown that the shrimp clotting system is linked with the activation of antimicrobial peptides, similar to that of the horseshoe crab. Unlike the horseshoe crab and crayfish blood coagulation which are well studied systems, blood clotting in shrimp remains poorly understood. Here we review the shrimp clotting system and its involvement in innate immunity.

DNA vaccination is one method to protect farmed fish from viral and bacterial diseases. Chimeric antigens encoded by DNA vaccines have been shown to increase the resistance to viral diseases. Here, we sequenced the gene encoding lysosome-associated membrane protein-1 from Japanese flounder, Paralichthys olivaceus, (JfLAMP-1) and assessed its use in a chimeric DNA vaccine fused with the major capsule protein (MCP) from red seabream iridovirus (RSIV). JfLAMP-1 cDNA has a length of 1248 bp encoding 415 aa, which contains transmembrane and cytoplasmic domains. JfLAMP-1 is constitutively expressed in several tissues and its expression in spleen was upregulated following injection of formalin-killed cells (FKC) of Edwardsiella tarda. Immunofluorescence analysis showed that JfLAMP-1 is distributed in the small and large granules in the cytoplasm and groups close to the nucleus. The DNA encoding the luminal domain of JfLAMP-1 was replaced with the gene for the RSIV MCP, and the construct was cloned in an expression vector (pCIneo). Fish vaccinated with pCLAMP-MCP had significantly higher antibody levels than fish vaccinated with pCIneo vector harboring the MCP gene (p < 0.05) at day 30 post-vaccination.

Phage display libraries are used to screen for nucleotide sequences that encode immunoglobulin variable (V) regions that are specific for a target antigen. We previously constructed an immunoglobulin new antigen receptor (IgNAR) phage display library. Here we used this library to obtain an IgNAR V region that is specific for viral hemorrhagic septicemia virus (VHSV). A phage clone (clone 653) was found to be specific for VHSV by the biopanning method. The V region of clone 653 was used to construct a 6 × His tagged recombinant IgNAR-653 V protein (rIgNAR-653) using the Escherichia coli pET system. The rIgNAR-653 protein bound specifically to VHSV, confirming its activity.

Gene in the interleukin 1 family plays a central role in the regulation of immune and inflammatory responses. Here, we describe two novel IL-1-like gene in Japanese flounder Paralichthys olivaceus (jfIL1-L1 and jfIL1-L2). jfIL1β-L1 was homologous to Nile Tilapia IL-1β-like gene and Arctic char IL-1, and jfIL1β-L2 showed homology to hypothetical protein LOC100699119 of Nile Tilapia and rainbow trout Oncorhynchus mykiss IL-1 receptor agonist (RA). The deduced amino acids sequences of these IL-1β-like genes showed very low identities to the Japanese flounder IL-1 (jfIL-1). Phylogenetic analysis confirmed that jfIL1β-L1 and -L2 were distinct from jfIL-1. The gene encoding the predicted ORF of jfIL1β-L1 and -L2 is divided into 6 exons and 7 exons, respectively. Transcripts of jfIL1β-L1 were detected in gills, intestine, kidney and spleen, and those of jfIL1β-L2 were detected in gills, intestine and spleen. The mRNA levels of jfIL1β-L1 and -L2 were not effect or slightly decreased by treatment with LPS and the formalin-killed cells of Edwardsiella tarda whilst mRNA levels of jfIL1β were significantly increased in the kidney and spleen at 6 h by these treatments.