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In this study, we investigated the influence of diurnal sampling bias on the community structure of plankton by comparing the biodiversity among seawater samples (n=9) obtained every 3h for 24h by using massively parallel sequencing (MPS)-based plankton monitoring at a fixed point conducted at Himedo seaport in Yatsushiro Sea, Japan. The number of raw operational taxonomy units (OTUs) and OTUs after re-sampling was 507-658 (558 ± 104, mean ± standard deviation) and 448-544 (467 ± 81), respectively, indicating high plankton biodiversity at the sampling location. The relative abundance of the top 20 OTUs in the samples from Himedo seaport was 48.8-67.7% (58.0 ± 5.8%), and the highest-ranked OTU was Pseudo-nitzschia species (Bacillariophyta) with a relative abundance of 17.3-39.2%, followed by Oithona sp. 1 and Oithona sp. 2 (Arthropoda). During seawater sampling, the semidiurnal tidal current having an amplitude of 0.3ms(-1) was dominant, and the westward residual current driven by the northeasterly wind was continuously observed during the 24-h monitoring. Therefore, the relative abundance of plankton species apparently fluctuated among the samples, but no significant difference was noted according to G-test (p0.05). Significant differences were observed between the samples obtained from a different locality (Kusuura in Yatsushiro Sea) and at different dates, suggesting that the influence of diurnal sampling bias on plankton diversity, determined using the MPS-based survey, was not significant and acceptable.

Next-generation sequencing technologies have allowed the rapid determination of the complete genomes of many organisms. Although shotgun sequences from large genome organisms are still difficult to reconstruct perfect contigs each of which represents a full chromosome, those from small genomes have been assembled successfully into a very small number of contigs. In this study, we show that shotgun reads from phage genomes can be reconstructed into a single contig by controlling the number of read sequences used in de novo assembly. We have developed a pipeline to assemble small viral genomes with good reliability using a resampling method from shotgun data. This pipeline, named V-GAP (Viral Genome Assembly Pipeline), will contribute to the rapid genome typing of viruses, which are highly divergent, and thus will meet the increasing need for viral genome comparisons in metagenomic studies.

Pathogen dynamics in environmental water are considered to be highly associated with the prevalence of fish disease. To examine the dynamics of red sea bream iridovirus (RSIV), which has caused serious damage in marine aquacultures in Japan, we developed a highly sensitive and quantitative method by combining the viral concentration and real-time PCR to detect the virus in seawater. The iron flocculation method employed here successfully concentrated 500 mL of artificial seawater spiked with RSIV in a 0.1-mL DNA solution at a 5000 concentration magnification, and the recovery rate of the RSIV genome by real-time PCR was higher than 80%. The detection limit of this method was approximately 8.0 × 10 1 RSIV copies L − 1 of sample water, which is a more than 1000 times improvement compared to the conventional DNA extraction method without concentration of water sample. Using this method, we detected the RSIV genome from the rearing water for experimentally infected Japanese amberjack Seriola quinqueradiata at ranges from 10 2.1 to 10 5.5 copies L − 1 . The RSIV copy number in the rearing water was increased more than 5 days before mortality started and it might become an indicator of disease prediction. These results indicate that the iron flocculation method coupled with PCR detection is useful for monitoring RSIV dynamics in the aquaculture setting. Statement of relevance Monitoring of RSIV in environmental water.

Edwardsiellosis, which is caused by Edwardsiella tarda, a Gram-negative bacterium, is one of the most serious infectious diseases in both marine and freshwater fish farms worldwide. Previously, we reported the complete genome sequences of three E. tarda-lytic bacteriophages (two podoviruses and a myovirus), which were isolated from fish tissues and fish-rearing seawater. Further genomic information regarding E. tarda phages is important for understanding phage-host interactions as well as for applications of the phages for the control of disease. Here, we report the complete genome sequence of a novel E. tarda phage (GF-2) of myovirus morphology (family Myoviridae), isolated from tissue homogenates of a cultured Japanese flounder (Paralichthys olivaceus) that succumbed to edwardsiellosis in Japan. The size of the entire genome was 43,129 bp, with a GC content of 51.3 % and containing 82 open reading frames (ORFs). The GF-2 genome possesses lysogeny-related genes that have not been found in the reported Edwardsiella phage genomes. Comparative genomics of Edwardsiella myophages suggest that the C-terminal domains of the tail fiber proteins have relevance to their host specificity. Thus, GF-2 genome information provides a novel resource for our understanding of the molecular mechanisms involved in their host specificity and for detection of E. tarda in aquaculture environments.

Herpesviral haematopoietic necrosis has caused great economic damage to goldfish Carassius auratus aquaculture in Japan. The existence of cyprinid herpesvirus 2 (CyHV-2), the causative agent, has also been reported from several other countries. To prevent spread to other areas, basic virological information such as viral kinetics in infected fish is essential. Experimental infection trials using reliably prepared CyHV-2 for defining viral kinetics are difficult to carry out because successful and sustainable propagation of this virus in cell culture has previously been limited. Here we describe a method for sustainable propagation of CyHV-2 in cell culture, and the results of fish infection experiments using the propagated virus. We found that goldfish fin (GFF) cells and standard Ryukin Takafumi (SRTF) cells established from goldfish fin can be used for continuous propagation of CyHV-2. Experimental infections using 2 varieties of goldfish, Ryukin and Edonishiki, were performed with the virus passaged 7 times in GFF cells. In transmission experiments with water temperature at 20°C, cumulative mortality was 30% in Ryukin infected by immersion, and 90 and 100% in Edonishiki and Ryukin intraperitoneally injected with the virus, respectively. In an experiment carried out at 25°C, 90% of Edonishiki challenged by immersion died. PCR detection of viral DNA from the organs of infected fish showed that systemic infection occurs and also that the kidney is a main viral multiplication site. Moreover, CyHV-2 was successfully re-isolated in GFF cells from the dead fish.

Interferon gamma (IFNγ), a Th1-derived cytokine is one of the key molecules inducing cell-mediated immune responses in mammals. The expression of 2 distinct IFNγ (ifng1 and ifng2) and IFNγrel (ifngrel) genes was examined in kidney leukocytes from clonal ginbuna crucian carp Carassius auratus langsdorfii. The expression of IFNγ and IFNγrel genes was induced in kidney leukocytes by stimulation with lipopolysaccharide or phytohemagglutinin in a dose-dependent manner. The expression levels of IFNγ and IFNγrel genes in kidney leukocytes from allo-sensitized fish cocultured with allogeneic cells were higher than those from fish cocultured with isogeneic cells. The highest expression of ifng1 was observed at day 1, whereas that of ifng2 and ifngrel was detected at day 2 after cocultivation with allogeneic cells. However, the expression pattern of ifng2 and ifngrel in kidney leukocytes from allo-sensitized fish by scale-grafting was similar to those from non-sensitized fish. These results indicate that ifng1 is a major isoform of IFNγ related to antigen-specific cell-mediated immunity (CMI), and will be a useful marker to evaluate induction of CMI in ginbuna crucian carp. In addition, IFNγ would be a crucial type-II interferon compared to IFNγrel, relevant to cell-mediated immunity in fish as in mammals.

The presence of helper and cytotoxic T cells in fish has been suggested, although T cell subsets have yet to be identified at the cellular level. In order to investigate the functions of CD4 and CD8α positive T cells we attempted to produce and characterize monoclonal antibodies (mAbs) against teleost CD4 and CD8α. Here we report the successful production of mAbs against CD4 and CD8α in clonal ginbuna crucian carp Carassius auratus langsdorfii and the function of CD4 positive T cells. In this study we demonstrate the presence of teleost CD4- and CD8α-positive T cell subsets with morphology, tissue distribution and gene expression similar to those of mammalian CD4- and CD8-positive T lymphocytes. Using mAbs we found that CD4/CD8 double positive T cells are only present in the thymus, suggesting that it is the site of T cell development. We further demonstrated in vitro proliferation of CD4 positive T cells by allogeneic combination of mixed leukocyte culture and antigen-specific proliferation of CD4 positive T cells after in vitro sensitization with OVA. In our previous study we showed that CD8α-positive lymphocytes are the primary cell type showing specific cytotoxicity against allogeneic targets. Collectively, these findings suggest that CD4 and CD8α positive T cells in ginbuna are equivalent to helper and cytotoxic T lymphocytes (CTL) in mammals, respectively. This is the first report to show the characteristics and functions of CD4 positive T cells in fish and these findings shed light into the evolutionary origins and primordial functions of helper T cells.

The clonal triploid ginbuna crucian carp Carassius auratus langsdorfii, a naturally occurring gynogenetic fish, is a useful model for studying T-cell-mediated immunity. CD4, a T-cell receptor (TCR) coreceptor, is a membrane-bound glycoprotein found on helper T-cells, and assists in the binding of major histocompatibility complexes. In the present study, full-length cDNAs encoding the CD4 molecule from the S3n strain of ginbuna crucian carp were cloned and characterized. 5'-rapid amplification of cDNA ends (RACE) and 3'-RACE yielded two distinct cDNA clones of CD4 homolog from the ginbuna, and these sequences share 95% identity at the amino acid level. These ginbuna CD4 molecules consisted of a signal peptide, immunoglobulin superfamily (IgSf) like domains, a transmembrane domain, and a cytoplasmic domain similar to other known CD4. A tyrosine protein kinase p56(lck) binding motif is conserved in the cytoplasmic tail of ginbuna CD4. Phylogenetic tree analysis indicated that ginbuna CD4 sequences are closely related to CD4L-1 from other fish species. Expression of ginbuna CD4 mRNA was detected in the gill, thymus, head kidney, trunk kidney and peripheral blood leukocytes, indicating that its expression pattern is similar to that of ginbuna TCR beta mRNA. The results suggest that ginbuna CD4 sequences are useful as molecular probes for helper T-cells.

Macrophage migration inhibitory factor (MIF) was discovered as the first cytokine that inhibited the random migration of macrophages. Recently, MIF has been reported to be involved in embryonic development in higher vertebrates. In fish, however, nothing is known about the function of MIF at early life stages, although immunological functions of MIF have been reported in adult fish. To elucidate the function of MIF during embryonic development in fish, we examined expression patterns and function of the zebrafish MIF gene using antisense morpholino-mediated knockdown (morpholino oligonucleotide-MO). In whole-mount in situ hybridization analysis, zebrafish MIF mRNA was detected in developing eyes, tectum, branchial arches, pectoral fin buds, liver and gut. The onset of MIF mRNA expression coincided with the beginning of tissue differentiation during embryogenesis. MIF-MO-injected embryos (morphants) displayed malformed eyes, abnormal swelling in the tectum and fourth ventricle region, and undeveloped jaw cartilage and pectoral fins. An increased number of apoptotic cells in the eye and neural tissues were observed in MIF morphants by histological analysis and acridine orange staining. Moreover, proliferating cell nuclear antigen (PCNA)-positive cells were reduced in morphant eyes. These results suggest that MIF is essential for normal embryonic development even at the level of teleosts and that it functions as a growth factor for the proliferation and differentiation of embryonic tissues.

In the adaptive immune system of mammals, naive helper T (Th) cells differentiate into Th1 or Th2 cells. The T-box expressed in T cells (T-bet) is a member of a family of T-box transcription factors that regulates the expression of IFN-gamma and plays a crucial role in Th1 cell differentiation and cell-mediated immunity. We cloned and sequenced T-bet cDNA for the first time from non-mammalian species, ginbuna crucian carp. Ginbuna T-bet was composed of 608 predicted amino acids and showed 41.5% identity with human T-bet (Tbx21), and human and ginbuna T-bet share 77.3% identity in their T-box regions. Comparative genomic analysis showed conserved synteny in these regions between zebrafish, fugu, medaka and human T-bet. Phylogenetic analysis indicated that ginbuna T-bet is closely related to that of mouse and human. In unstimulated fish, ginbuna T-bet mRNA was strongly expressed in peripheral blood leukocytes (PBL), head kidney (HK) and spleen. RT-PCR analysis in kidney cells sorted by FACS revealed that T-bet was strongly expressed in surface-IgM-negative lymphocytes in comparison to IgM-positive lymphocytes. These results suggest that ginbuna T-bet is involved in the immune system, especially in T-cell function, and is an important tool to analyze teleost cell-mediated immunity.

GATA3, a transcriptional activator, plays a critical role in the development of T-cells and differentiation to T helper type 2 cells. To date, no information is available on the role of GATA3 in the teleost immune system. We identified full-length cDNA and alternatively spliced variants of ginbuna crucian carp GATA3 (gbGATA3). The gbGATA3 gene is transcribed into multiple splice variants lacking either one or both zinc finger domains, although the sequences of both domains are fully conserved between ginbuna and other vertebrates. We found that alternative splice site and stop codon in gbGATA3 intron 3, located between exons that separately encode the two zinc finger domains, are conserved among teleosts, suggesting that teleost GATA3 gene can be translated into multiple isoforms. RT-PCR analysis revealed that the gbGATA3 is strongly expressed in the brain, thymus and gill of unstimulated fish. Moreover, gbGATA3 expression was detected in surface-IgM-negative lymphocytes among kidney cells sorted by FACS. Real-time PCR demonstrated that expression levels of full-length gbGATA3 and the splice variants differed with tissue type, but full length was always the predominantly expressed form. These results suggest that gbGATA3, including its splice variants, is involved in teleost T-cell function.

To identify viral proteins that induce cell-mediated cytotoxicity (CMC) against viral hemorrhagic septicemia virus (VHSV)-infected cells, rainbow trout were immunized with DNA vectors encoding the glycoprotein G or the nucleocapsid protein N of VHSV. The G protein was a more potent trigger of cytotoxic cells than the N protein. Peripheral blood leukocytes (PBL) isolated from trout immunized against the G protein killed both VHSV-infected MHC class I matched (RTG-2) and VHSV-infected xenogeneic (EPC) target cells, suggesting the involvement of both cytotoxic T lymphocytes (CTL) and NK cells, respectively. In contrast, PBL from trout that were immunized against the N protein only killed VHSV-infected RTG-2 cells, indicating that this protein only elicits a CTL response. Further, a significant killing capacity of these PBL was only observed during summer months. PBL from fish that were immunized against the VHSV G protein significantly killed VHSV-infected but not infectious hematopoietic necrosis virus (IHNV)-infected targets indicating antigen specificity. Thus, this is the first report on cytotoxic immune responses after DNA vaccination in fish. Furthermore, cells isolated from the inflamed site of DNA injection were stained and transferred to isogeneic DNA-vaccinated recipients. Most of the stained donor leukocytes accumulated at the recipients' DNA injection site showing, for the first time, leukocyte homing in fish. Transferred donor leukocytes mainly migrated to the homologous vaccine injection site rather than to injection sites of heterologous vaccines, suggesting the antigen specificity of homing. By demonstrating CMC responses to distinct viral proteins and homing in rainbow trout, these results substantially contribute to the understanding of the teleost immune system.

Lck is a member of the Src-related protein tyrosine kinase family. In mammals, it is predominantly expressed in T cells, and plays crucial role in T-cell maturation and activation. In this report, the cloning, characterization, and expression analysis of lck genes from the S3n strain of ginbuna crucian carp Carassius auratus langsdorfii is described. Three distinct lck homologs, designated as gblck-1, gblck-2, and gblck-3 were isolated from ginbuna thymus. The deduced amino acid sequences of gblck-1 and gblck-2 cDNAs exhibit conserved lck essential motif and overall structures, but gblck-3 lacks an essential region related to regulation of Src kinase activity. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis demonstrated that gblck-1 was expressed in peripheral blood leukocytes (PBLs), lymphoid organs, and mucosal tissues, as has been reported for T-cell marker genes, i.e. CD3, TCR, and lck in other fish species. In contrast, the expression of gblck-2 and gblck-3 were observed only in PBLs and lymphoid organs. In the sorted lymphocytes from kidney leukocytes using antiginbuna IgM monoclonal antibody, only slg−lymphocytes expressed lck genes as well as the TCR\ gene. Collectively, these results suggest that lck is a useful marker for the identification of T cells.

The complement system plays key roles in innate and adaptive immunity through mediating phagocytosis, chemotaxis and cell lysis. Complement component C3 is a central component in the complement cascade and belongs to the acute-phase proteins whose synthesis increase immediately upon inflammatory stimuli. The liver is the main producer of C3 and it is a well-known fact that the mammalian monocyte-macrophage lineage is a major contributor to extrahepatic C3. Immunomodulators, such as LPS and beta-glucan, can stimulate complement, lysozyme, natural killer cells and antibody responses in fish, thus enhancing the resistance to bacterial pathogens and parasitic infections. The aim of this study was to assess the effects of LPS and beta-glucan on the expression of interleukins (IL-1beta1, IL-1beta2 and IL-6) and the modulated expression of C3 subtypes (C3-1, C3-3 and C3-4) in the rainbow trout (Oncorhynchus mykiss) using real-time RT-PCR. From in vitro studies, we demonstrated that head kidney macrophages from rainbow trout and Atlantic salmon showed no basal transcription of C3. After immunostimulation, the cells responded by increased levels of ILs, but transcription of C3 was not induced. In contrast to the in vitro findings, the rainbow trout complement C3 subtypes were differentially regulated 48 h after in vivo stimulation with LPS and beta-glucan. These results support the previous findings of absence of C3 in macrophages of the spotted wolffish (Anarhichas minor) and is the first functional study showing differential regulation of the C3 subtypes in any vertebrate.

Mammalian cytotoxic T cells as part of the adaptive immune system recognize virus-infected target cells by binding of their T-cell receptors (TCR) to classical MHC class I molecules loaded with viral peptides. Our previous studies have shown that the allele of the single dominant polymorphic classical MHC class I locus Onmy-UBA is identical in the rainbow trout clone C25 and in the permanent rainbow trout cell line RTG-2. This enabled us to develop an assay to measure antiviral cytotoxicity in rainbow trout using a system of MHC class I-matched effector and target cells. Peripheral blood leucocytes (PBL) isolated from low dose viral haemorrhagic septicaemia virus (VHSV)-infected rainbow trout killed MHC class I-matched and later also xenogeneic MHC class I-mismatched VHSV-infected cells. When compared to PBL from uninfected control fish PBL from infected fish showed a higher transcriptional level of the CD8alpha gene which is a typical marker for mammalian cytotoxic T cells. Concurrently, the expression of the natural killer cell enhancement factor (NKEF)-like gene was enhanced as measured by real-time RT - PCR. Taken together, these results suggest that both innate and adaptive cell-mediated immune responses represented by NK and cytotoxic T cells, respectively, are triggered after VHSV infection. PBL that were able to kill VHSV-infected MHC class I-mismatched xenogeneic cells were generated later during infection than PBL capable of lysing VHSV-infected MHC class I-matched targets. This is contradictory to the generally accepted rule that innate immune mechanisms represent the first line of defence after viral infections.

Most of the previously studied teleost MHC class I molecules can be classified into two broad lineages: "U" and "Z/ZE." However, database reports on genes in cyprinid and salmonid fishes show that there is a third major lineage, which lacks detailed analysis so far. We designated this lineage "L" because of an intriguing linkage characteristic. Namely, one zebrafish L locus is closely linked with MHC class II loci, despite the extensively documented nonlinkage of teleost class I with class II. The L lineage consists of highly variable, nonclassical MHC class I genes, and has no apparent orthologues outside teleost fishes. Characteristics that distinguish the L lineage from most other MHC class I are (1) absence of two otherwise highly conserved tryptophan residues W51 and W60 in the alpha1 domain, (2) a low GC content of the alpha1 and alpha2 exons, and (3) an HINLTL motif including a possible glycosylation site in the alpha3 domain. In rainbow trout (Oncorhynchus mykiss) we analyzed several intact L genes in detail, including their genomic organization and transcription pattern. The gene Onmy-LAA is quite different from the genes Onmy-LBA, Onmy-LCA, Onmy-LDA, and Onmy-LEA, while the latter four are similar and categorized as "Onmy-LBA-like." Whereas the Onmy-LAA gene is organized like a canonical MHC class I gene, the Onmy-LBA-like genes are processed and lack all introns except intron 1. Onmy-LAA is predominantly expressed in the intestine, while the Onmy-LBA-like transcripts display a rather homogeneous tissue distribution. To our knowledge, this is the first description of an MHC class I lineage with multiple copies of processed genes, which are intact and transcribed. The present study significantly improves the knowledge of MHC class I variation in teleosts.

Previous typing of microsatellite markers in a BC1F1 backcross rainbow trout Oncorhynchus mykiss revealed two quantitative trait loci (QTL) with a significant impact on suscep- tibility to infectious pancreatic necrosis. In the present study, additional genetic markers were mapped using the same strain in order to increase QTL resolution. A total of 199 individuals were screened at 226 marker loci (106 microsatellites, 116 amplified fragment length polymorphism markers, classical major histocompatibility complex molecules (MHC class I, nonclassical MHC class I, MHC class II) and tyrosinase) and the QTL were determined by single-point and interval mappings. Seven QTL were distinguished in the linkage groups RT-11, 12, 17, 23, 26, 29 and 31, in addition to the two already known in RT-3 and 22. The major QTL were those in RT-3, 12 and 22; whereas the classical MHC class I locus Onmy-UBA and the MHC class II locus Onmy-DAB were not associated with any of the QTL, a nonclassical MHC class I region with Onmy-UCA, Onmy-UDA, and Onmy-UEA mapped to the major QTL on RT-3.

Bluefin tunas are one of the most important fishery resources worldwide. Because of high market values, bluefin tuna farming has been rapidly growing during recent years. At present, the most common form of the tuna farming is based on the stocking of wild-caught fish. Therefore, concerns have been raised about the negative impact of the tuna farming on wild stocks. Recently, the Pacific bluefin tuna (PBT), Thunnus orientalis, has succeeded in completing the reproduction cycle under aquaculture conditions, but production bottlenecks remain to be solved because of very little biological information on bluefin tunas. Functional genomics approaches promise to rapidly increase our knowledge on biological processes in the bluefin tuna. Here, we describe the development of the first 44K PBT oligonucleotide microarray (oligo-array), based on whole-genome shotgun (WGS) sequencing and large-scale expressed sequence tags (ESTs) data. In addition, we also introduce an initial 44K PBT oligo-array experiment using in vitro grown peripheral blood leukocytes (PBLs) stimulated with immunostimulants such as lipopolysaccharide (LPS: a cell wall component of Gram-negative bacteria) or polyinosinic:polycytidylic acid (poly I:C: a synthetic mimic of viral infection). This pilot 44K PBT oligo-array analysis successfully addressed distinct immune processes between LPS- and poly I:C- stimulated PBLs. Thus, we expect that this oligo-array will provide an excellent opportunity to analyze global gene expression profiles for a better understanding of diseases and stress, as well as for reproduction, development and influence of nutrition on tuna aquaculture production.

Our previous studies have demonstrated that virus-specific cell-mediated cytotoxicity of sensitized leukocytes can be induced using clonal ginbuna crucian carp and their syngeneic cell lines. In the present study, we attempt to determine if virus-specific cytotoxic cell populations of fish express CD8alpha and TCRbeta genes. Leukocytes from ginbuna crucian carp were separated into four fractions by immunomagnetic separation and density gradient centrifugation: Fraction A, leukocytes with a density of 1.08 g/ml (primarily lymphocytes); Fraction B, sIg-negative leukocytes with density of 1.08 g/ml; Fraction C, sIg-positive cells (primarily B-lymphocytes); Fraction D, leukocytes with a density of 1.08-1.09 g/ml (primarily neutrophils). Leukocytes in all fractions from uninfected fish do not exhibit cytotoxic activity against virus-infected syngeneic cells and weakly express CD8alpha and TCRbeta mRNAs. In contrast, leukocytes in fractions A and B from virus-infected fish exhibit a high level of cytotoxic activity and strongly express CD8alpha and TCRbeta mRNAs. In addition, mRNA expressions of CD8alpha and TCRbeta in effector cells are upregulated by cocultivation with virus-infected target cells but not uninfected ones. The present study suggests that fish possess virus-specific cytotoxic cells with phenotype and gene expression pattern similar to those of CTLs in mammals.

As part of an ongoing elucidation of rainbow trout major histocompatibility complex (MHC) class I, the polymorphism of two MHC class Ib loci was analyzed. These loci, Onmy-UCA and Onmy-UDA, are situated head-to-tail and share more than 89% nucleotide identity in their open reading frames. They share 80% identity with some trout Ia alleles. The deduced amino acid sequences suggest that the UCA and UDA molecules are transported to endosomal compartments and may bind peptides in their binding groove. Our survey revealed seven UCA and eight UDA alleles. Similarity indices overlap when comparing within and between UCA and UDA alleles and some cross-locus motif variation is observed. In most trout both UCA and UDA transcripts were found. However, there probably is functional redundancy, because some trout lacked transcription of one of the two loci. Furthermore, for some UCA and UDA alleles, splicing deficiencies, early stop codons, and upstream start codons were found, which may interfere with efficient protein expression. The present study is the first extensive report on MHC class Ib polymorphism assigned to locus in ectotherm species.

Like mammalian leucocytes, white blood cells of fish are able to kill altered (e.g. virus-infected) and foreign (allogeneic or xenogeneic) cells. The existence of natural killer (NK)-like and specific cytotoxic cells in fish was first shown using allogeneic and xenogeneic effector/target cell systems. In addition to in vivo and ex vivo studies, very important contributions were made by in vitro analysis using a number of different long-term cytotoxic cell lines established from channel catfish. In mammals, specific cell-mediated cytotoxicity (CMC) as part of the adaptive immune response requires a number of key molecules expressed on effector leucocytes and target cells. CD8+ T lymphocytes kill infected cells only, if their antigen receptor (TCR) matches the MHC class I with bound peptide of the target cell. Expression patterns of the fish gene homologues for TCR, CD8 and MHC class I, as well as related genes, are in agreement with similar function. Convenient systems for the analysis of specific CMC have only recently become available for fish with the combination of clonal fish with syngeneic or allogeneic but MHC class I matching cell lines. It was demonstrated that both, NK- and cytotoxic T (Tc) cells are involved in the killing of virus infected MHC class I matching and mismatching target cells. Analysis of these lymphocyte subsets is only starting for fish. There is also evidence that the different viral proteins trigger different subsets of killer cells. This review further discusses findings on fish CMC with regard to temperature/seasons and ontogeny.

Although polymorphism in major histocompatibility complex (MHC) genes has been thought to confer populations with protection against widespread decimation by pathogens, this hypothesis cannot explain the type of large allelic diversity in classical MHC class I (Ia) in rainbow trout. Based on expression of Onmy-UBA (MHC class Ia) in trout neurons, we hypothesized that polymorphism in trout class Ia may contribute to polymorphism in behavioral traits. The present study examined whether polymorphism in Onmy-UBA was associated with behavioral variation in Donaldson rainbow trout (Oncorhynchus mykiss) using experiments on food competition, lure-catch, fright recovery, diel locomotor activity and activity characterized as dominance or aggression. These behavioral traits were investigated in fish having Onmy-UBA*401/*401 or *4901/*4901 homozygous, or Onmy-UBA*401/*4901 heterozygous genotypes (referred to as BB, FF and BF, respectively). The BB fish exhibited boldness, aggression, faster growth and crepuscular activity, while the FF fish showed little boldness, smaller body size, and diurnal activity with no aggressive behavior. The BF fish displayed traits intermediary to those of the BB and FF fish. These results are consistent with polymorphism in a single MHC class Ia locus driving variation in neural circuits, thereby creating behavioral variation in the trout. This is the first study in any animal to show a potential correlation between polymorphism in MHC class Ia genes with polymorphism of behavioral traits such as aggression.

We cloned and sequenced full-length cDNAs for two types of CD8alpha from the S3n strain of ginbuna crucian carp (Carassius auratus langsdorfii) and quantified the expression of CD8alpha genes after sensitization by scale grafting, employing a model system of clonal triploid ginbuna and tetraploid ginbuna-goldfish hybrids. RT-PCR yielded four different fragments of CD8alpha homologue from the S3n strain of ginbuna and these sequences were classified into two groups. The two types of ginbuna CD8alpha (gbCD8alpha) were also found in other strains of triploid ginbuna and goldfish, which are a subspecies of ginbuna. The gbCD8alpha chains consisted of a signal peptide, Ig superfamily (IgSf) V-like domain, hinge, transmembrane domain, and cytoplasmic domain similar to other known CD8alpha. Phylogenetic analysis indicated that both types of gbCD8alpha are closely related to CD8alpha from other vertebrates. Expression of both types of gbCD8alpha mRNA was detected in the gill, thymus, head kidney, posterior kidney, spleen, intestine and peripheral blood leucocytes. In addition, quantitative real-time PCR analysis demonstrated that copy numbers of both gbCD8alpha gene products in kidney cells increased significantly following grafting with allogeneic but not isogeneic scales, and that regulation of expression correlated with that of TCRbeta. Expression of both gbCD8alpha genes after second scale allografting was elevated compared to that after the first set of grafting. These results suggest that expression analysis of these two gbCD8alpha sequences provides a useful tool to address the involvement of cytotoxic T-lymphocytes during the cell-meditated immune response in fish.

The superoxide-producing NADPH oxidase complex of phagocytes plays a crucial role in host defenses against microbial infection. NADPH oxidase consists of a membrane heterodimeric protein, composed of gp91phox and p22phox, and cytosolic proteins, p40phox, p47phox and p67phox. In the present study, cDNAs of all the components of NADPH oxidase were cloned from peripheral white blood cells of the Japanese pufferfish utilizing the reverse transcription-polymerase chain reaction. The sequences of these cDNAs showed that the pufferfish gp91phox, p22phox, p40phox, p47phox and p67phox clones contained open reading frames encoding 565, 186, 348, 423 and 495 amino acids, respectively. Comparison of the deduced amino acid sequences showed that the pufferfish gp91phox, p22phox, p40phox, p47phox and p67phox sequences shared 68.0, 61.8, 53.8, 54.7 and 41.9% identity with those of human components, respectively. gp91phox has three potential N-linked glycosylation sites. gp91phox and p22phox have six and three hydrophobic regions, respectively, that are predicted to be transmembrane regions. p47phox and p67phox have two potential Src homology 3 domains and p40phox has one. The functional domains are highly conserved in many animals, though the sequence of the components of the pufferfish showed low homology with that of mammals. The Fugu NADPH oxidase genes were expressed in various tissues of unstimulated fish. The level of gp91phox, p47phox and p67phox expression were high only in the blood and kidney, while p22phox and p40phox were constitutively expressed in a wide range of tissues. These results suggest that Japanese pufferfish NADPH oxidase components possess functional activities similar to those of human.

Despite accumulating sequence data, information on the function of major histocompatibility complex (MHC) genes in fish is scarce. In contrast to the genome organization in higher vertebrates, the polymorphic MHC class I and II genes are not linked in the teleost genome. A previous study found an MHC class II linkage group to be a major determinant in the rejection of allogeneic scales by a teleost species (Cardwell et al. 2001). The present study investigated whether the teleost MHC class I linkage group can be involved in allograft rejection. Erythrocytes were chosen as grafts since they express MHC class I, but do not express class II. Rainbow trout erythrocytes expressing different MHC class I alleles were differentially stained, mixed and injected into recipients that were of the same sibling group as the donors. The MHC class I linkage group was the major determinant for in vivo graft rejection.

We have isolated and characterized cDNAs and genes for pufferfish, Fugu rubripes, (Fugu) orthologues of mammalian interleukin (IL)-12 subunits (IL-12 p35 and IL-12 p40). The deduced amino acid sequences of the Fugu IL-12 subunits showed homology with mammalian IL-12 subunits (p35: 50.4-58.0% similarity; p40: 51.2-55.4% similarity). Phylogenetic analysis confirmed that Fugu IL-12 p35 and p40 genes cluster with their mammalian counterpart lineages. The genomic organization of each of the Fugu IL-12 subunit genes is similar to that of the corresponding mouse IL-12 subunit genes, although the Fugu genes are very compact due to small intron size. Comparative genomic analysis showed conserved syntenies within the IL-12 p35 and p40 regions between Fugu and human, indicating that the Fugu IL-12 p35 and p40 genes are orthologues for mammalian IL-12 p35 and p40 encoding genes, respectively. Expression of IL-12 p35 mRNA was observed in lymphoid tissues and several non-lymphoid tissues, while expression of IL-12 p40 mRNA was constitutive and nearly ubiquitous. In the spleen and head kidney, expression of IL-12 p35 was induced by polyriboinosinic polyribocytidylic acid [poly(I:C)] and not by lipopolysaccharide (LPS), while expression of IL-12 p40 was constitutive and unresponsive to both poly(I:C) and LPS. These results indicate that IL-12 levels are regulated by production of IL-12 p35 mRNA and suggest that IL-12 in fish may be involved in antiviral defense. This is the first report of the identification and characterization of IL-12 subunit cDNAs and genes in a non-mammalian vertebrate.

A putative G-protein coupled receptor (GPCR) gene belonging to the rhodopsin-like family was detected in rainbow trout and designated LPSenhR-1. Only moderate homology (

Bacterial coldwater disease is considered to be one of the most serious diseases in ayu Plecoglossus altivelis in Japan. We evaluated the efficacy of three highly water-soluble adjuvants (Montanidae IMS-1311, IMS-1312 and IMS-2212) combined with formalin-killed bacterin of Flavobacterium psychrophilum (FKB) in ayu, in comparison with a minimally water-soluble adjuvant (Montanidae ISA-763A). The retention period and toxicity of these adjuvants in ayu were also examined. Experimental challenge 4 wk after vaccination showed that FKB adjuvanted with IMS-1311 and IMS-1312 had higher potency than FKB alone and significantly (p

In rainbow trout there is only one dominant classical MHC class I locus, Onmy-UBA , for which four very different allelic lineages have been described. The purpose of the present study was to determine if Onmy-UBA polymorphism could be used for strain characterisation. This was performed by lineage-specific PCR investigation of 30 fish, each of the Nikko and Donaldson strains, and by sequence analysis of 25 of the amplified DNA fragments. Two new MHC class I lineages were detected in addition to the four previously described lineages, thus six distinct lineages were observed within the fish examined (Sal-MHCIa*A–F). The distribution of lineages appeared to be strain-specific. For example, the lineage Sal-MHCIa*A was very common in the Nikko strain but could not be detected in the Donaldson strain. Analysis of MHC class I variation may help to elucidate relationships between strains and the roles of MHC alleles in disease resistance.

Ubiquitin is a small protein involved in intracellular proteolysis. It is highly conserved throughout eukaryotic phyla and has been detected in such diverse species as yeast, barley, Drosophila and man. A previous study showed that chromatin of rainbow trout testis contains free ubiquitin with a sequence similar to that of other phyla. In the present study, which focused on rainbow trout but included eleven other species, it is shown that fish ubiquitin genetic organisation and expression are similar to those of other phylogenetic groups through the following set of observations: (a) Multiple loci were detected, (b) These loci encode repeats of ubiquitin, (c) Although the DNA sequences are not conserved, the encoded amino acid sequences are fully conserved, (d) The expression of ubiquitin was influenced by cell culture conditions and viral infection.

The classical MHC class I genes have been known to be highly polymorphic in various vertebrates. To date, putative allelic sequences of the classical MHC class I genes in teleost fish have been reported in several studies. However, the establishment of their allelic status has been hampered in most cases by the lack of appropriate genomic information. In the present study, using heterozygous and homozygous fish, we obtained classical-type MHC class I sequences of rainbow trout (Oncorhynchus mykiss) and investigated their allelic relationship by gene amplification and Southern and Northern hybridization analyses. The results indicated that all MHC class I sequences we obtained were derived from a single locus. Based on this, a unique polymorphic nature of the MHC class I locus of rainbow trout has been revealed. The mosaic combination of highly divergent ancient sequences in the peptide-binding domains is notable, and the variable nature around the boundary between the α3 and transmembrane domains is unprecedented.

rag1−/− zebrafish have been employed in immunological research as a useful immunodeficient vertebrate model, but with only fragmentary evidence for the lack of functional adaptive immunity. rag1-null zebrafish exhibit differences from their human and murine counterparts in that they can be maintained without any specific pathogen-free conditions. To define the immunodeficient status of rag1−/− zebrafish, we obtained further functional evidence on T- and B-cell deficiency in the fish at the protein, cellular, and organism levels. Our developed microscale assays provided evidence that rag1−/− fish do not possess serum IgM protein, that they do not achieve specific protection even after vaccination, and that they cannot induce antigen-specific CTL activity. The mortality rate in non-vaccinated fish suggests that rag1−/− fish possess innate protection equivalent to that of rag1+/− fish. Furthermore, poly(I:C)-induced immune responses revealed that the organ that controls anti-viral immunity is shifted from the spleen to the hepatopancreas due to the absence of T- and B-cell function, implying that immune homeostasis may change to an underside mode in rag-null fish. These findings suggest that the teleost relies heavily on innate immunity. Thus, this model could better highlight innate immunity in animals that lack adaptive immunity than mouse models.

We present the complete genome sequence for a novel Edwardsiella ictaluri -specific bacteriophage, PEi21, isolated from river water in Japan. An initial comparative genome analysis revealed that the phage was closely related to the previously reported Edwardsiella tarda phage MSW-3 isolated from a red sea bream farm in Japan.

We investigated the potency of two oil adjuvants, Montanidae ISA 763A (763A) and a squalene emulsion (Squalene), to enhance the response of ayu Plecoglossus altivelis to a formalin-killed bacterin (FKB) made from the coldwater disease etiologic agent, Flavobacterium psychrophilum. Ayu were challenged 4 wk after vaccination by an intramuscular injection with live pathogenic F. psychrophilum. Mortalities of fish injected with FKB of F. psychrophilum combined with either adjuvant showed that the adjuvanted vaccines had significantly higher (Pl0.05) potencies than the FKB vaccine alone or sterile distilled water. Moreover, both adjuvanted vaccines produced significantly higher antibody titers than the FKB vaccine without adjuvant. From these investigations it is concluded that the use of Squalene and 763A adjuvants provides enhanced protection against coldwater disease in ayu compared to the use of FKB alone.

To elucidate the process of the uptake of suspended particles into the skin, fins and gills of rainbow troutOncorhynchus mykiss, 100 fingerlings were immersed in a suspension of 1μm diameter fluorescent microspheres and rinsed with water. The fate of microspheres adhering to body surface was traced for elapsed times ranging from 1 min to 24 h. Although most microspheres were removed in a 2 h-rinse with water, a few were observed by light microscopy to be located in both epidermal and dermal tissues which had microscopic injuries. As observed by electron microscopy, microspheres were taken up by migrating epithelial cells, or embedded in the dermis covered with these cells. Some of those microspheres embedded in dermis were endocytosed by macrophages. Such features were confirmed in artificial wound experiments. We conclude that microscopic injuries to trout could be an important route for the uptake of antigen administered by immersion, 2 to 24 h after fish suffer from injuries.

A model system of clonal triploid ginbuna and tetraploid ginbuna-goldfish hybrids was employed to demonstrate the presence of graft-versus-host reaction (GVHR) in a teleost fish. Tetraploid scale grafts on triploid clone members evoked an acute rejection, whereas the reverse transplants were accepted. When sensitized triploid cells were injected into tetraploid recipients, a typical GVHR was induced, leading to death of the recipients within one month. The onset of illness appeared about one week after cell injection as a loss of appetite and constipation, followed by a scale protrusion, severe haemorrhage, local destruction of the ventral skin and prominent splenomegaly. GVHR was most effectively induced by head-kidney cells and peripheral blood leukocytes (PBL), followed by spleen and thymus cells. Donors had to be sensitized at least twice by scale grafting to induce the reaction. A considerable number of recipients injected with cells from donors which had been sensitized by allogenetically different tetraploids died, suggesting a limited polymorphism or heavy cross-reactions between the alleles of the histocompatibility antigens. Ploidy analyses revealed that donor cells greatly increased in the host liver and spleen, constituting approximately 30% of total cells after 2 3 weeks. Most of these features of acute GVHR observed in this fish system are quite similar to those found in mammals and birds. thereby suggesting the presence of allo-reactive cytotoxic T lymphocytes in teleosts.

In order to clarify the effect of a prolonged exposure period on vaccine effectiveness, juvenile rainbow trout were immersed in 100, 1, 000 and 10, 000 times dilutions of a commercial vibrio bacterin for 3 min (brief immersion, BI) and for 24 h (prolonged immersion, PI). Two weeks and 2, 4 and 6 mo after the vaccination, fish were challenged by waterborne exposure to Vibrio ordalii. Prolonged exposure to dilute bacterin resulted in better protection than the standard BI method. Two weeks after vaccination, the cumulative mortality was 0% in the group vaccinated by PI with the 100, 1, 000 and 10, 000 times diluted vaccine. In contrast, mortality was 58 and 77% in the groups vaccinated by BI with the 100 and 10, 000 times diluted vaccine respectively, and was 100% in the unimmunized control group. Furthermore, 6 mo after vaccination, the mortality was 20% (100 times dilution) to 77% (10, 000 times dilution) in the PI group, while it was 64% (100 times dilution) to 93% (10, 000 times dilution) in the BI group. Mortality in the corresponding control group was 97%. These results suggest that prolonged exposure period improves vaccine effectiveness.

This study examined the uptake of BSA-conjugated 1 μ m fluorescent latex microspheres from bath suspensions by 1·5–3·0 g rainbow trout, Oncorhynchus mykiss (Walbaum). Uptake was quantified using a novel method based on dissolving fish tissues (excluding the gastrointestinal tract) in an alkaline solution and capturing microspheres on a filter for microscopic counting. In suspensions of 1·0×10 7 particles ml −1 , the rate of uptake was approximately linear over 48 h of continuous immersion, with a 24 h mean±SE of 98 000±12 000 microspheres fish −1 . Uptake was logarithmically proportional to particle concentration in suspensions of 1·0×10 5 , 1·0×10 6 and 1·0×10 7 particles ml −1 . Prolonged immersion in a dilute suspension resulted in greater uptake than brief immersion in concentrated suspensions. Quantification of microspheres in separate tissues indicated that both skin and gill participate in uptake, with the skin playing a major role. Particles remained in skin and gill tissues for at least 24 days following exposure, while a minority were transported to the spleen and kidney. This observation supports an important role for local immunity in the protective responses generated during immersion immunisation. Histologically, microspheres were observed primarily within epithelial cells and underlying phagocytes of both skin and gill. Collectively these findings describe a new method for quantifying antigen uptake, which was used to demonstrate the effectiveness of prolonged exposure to dilute antigen suspensions and the importance of the skin as a site of antigen uptake during immersion immunisation.

Cell-mediated cytotoxicity of clonal ginbuna crucian carp leukocytes against allogeneic erythrocytes is described using a sensitive non-radioactive in vitro assay. Hemoglobin released from target erythrocytes after cell-mediated erythrolysis was detected by tetramethylbenzidine (TMB). TMB assay showed clear correlation with a 51Cr-release assay and even exhibited higher cytotoxicity. The use of erythrocytes as target cells has several advantages over a conventional 51Cr-release assay. Erythrocytes do not have cytotoxic activity, are relatively homogeneous, are available in large numbers and erythrocyte donors need not be killed. Leukocytes from fish sensitized by erythrocyte injection or scale grafting efficiently lysed allogeneic erythrocytes, but did not kill isogeneic or autologous erythrocytes. In contrast, leukocytes from unsensitized fish did not lyse allogeneic erythrocytes and repeated sensitizations by allogeneic grafts were necessary to induce cytotoxic cells. Effector cells isolated from peripheral blood showed a higher cytotoxic effect toward allogeneic target cells than effector cells isolated from kidney. These studies support the hypothesis that fish are capable of a genetically restricted specific cell-mediated cytotoxicity.

Immersion in bath suspensions for extensive time periods has been reported to enhance the uptake of particulate antigen by rainbow trout, Oncorhynchus mykiss. In order to determine if prolonged immersion also enhances soluble antigen uptake, juvenile rainbow trout, weighing 15 g, were immersed in BSA solutions at 15°C using exposure periods ranging from 3 minutes to 48 hours. BSA uptake, measured as plasma concentration, was significantly dependent upon the exposure period (p < 0.001), demonstrating that prolonged immersion enhances the uptake of soluble antigen as well as particulate antigen. Furthermore, we found that BSA uptake was significantly dependent upon the BSA concentration in the bath solution during prolonged immersion. The plasma BSA concentration Cp (μg/ml) after immersion was related to time and concentration by the equation Cp (Cb, t) = 3.47·10-3·Cb·√t, where t is the exposure period in hours and Cb is the BSA concentration of the bath solution (μg/ml). The correlation coefficient of this formula is 0.99. Thus, the optimal combination of exposure period and antigen concentration in the immersion solution can be selected.

We report the isolation and extensive analysis of highly polymorphic MHC class I genes from sharks (Triakis scyllia), which belong to the most primitive vertebrate group with jaws, the cartilaginous fish. Predicted complete peptide-binding domains showed retention of the critical amino acid residues that would interact with antigenic peptide termini and revealed extensive allelic polymorphisms comparable to those of classic human MHC class I molecules. Mosaic structures were apparent in these domains, suggesting recombinational mechanisms to create allelic diversity. The present study demonstrates the establishment of the basic strategy for antigen-presentation employed by MHC class I molecules and documents complete divergence of two polymorphic MHC classes at a phylogenetically primitive stage of vertebrate evolution.