Nobuaki Okamoto

Quantitative trait loci (QTLs) associated with resistance/susceptibility to infectious pancreatic necrosis virus (IPNV) in rainbow trout (Oncorhynchus mykiss)

Infectious pancreatic necrosis (IPN) is a well-known acute viral disease of salmonid species. We have identified quantitative trait loci (QTLs) associated with resistance to this disease in rainbow trout. We searched for linkage among 51 microsatellite markers used to construct a framework linkage map in backcross families of rainbow trout (Oncorhynchus mykiss), produced by crossing IPN-resistant (YN-RT201) and -susceptible (YK-RT101) strains. Two putative QTLs affecting disease resistance were detected on chromosomes A (IPN R S-1) and C (IPN R/S-2), respectively, suggesting that this is a polygenic trait in rainbow trout. These markers have great potential for use in marker-assisted selection (MAS) for IPN resistance and provide the basis for cloning of IPN resistance genes. Clarification of the genetic bases of complex traits has broad implications for fundamental research, but will also be of practical benefit to fish breeding.

A Linkage Map for Brown Trout (Salmo trutta): Chromosome Homeologies and Comparative Genome Organization With Other Salmonid Fish

We report on the construction of a linkage map for brown trout (Salmo trutta) and its comparison with those of other tetraploid-derivative fish in the family Salmonidae, including Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss), and Arctic char (Salvelinus alpinus). Overall, we identified 37 linkage groups (2n = 80) from the analysis of 288 microsatellite polymorphisms, 13 allozyme markers, and phenotypic sex in four backcross families. Additionally, we used gene–centromere analysis to approximate the position of the centromere for 20 linkage groups and thus relate linkage arrangements to the physical morphology of chromosomes. Sex-specific maps derived from multiple parents were estimated to cover 346.4 and 912.5 cM of the male and female genomes, respectively. As previously observed in other salmonids, recombination rates showed large sex differences (average female-to-male ratio was 6.4), with male crossovers generally localized toward the distal end of linkage groups. Putative homeologous regions inherited from the salmonid tetraploid ancestor were identified for 10 pairs of linkage groups, including five chromosomes showing evidence of residual tetrasomy (pseudolinkage). Map alignments with orthologous regions in Atlantic salmon, rainbow trout, and Arctic char also revealed extensive conservation of syntenic blocks across species, which was generally consistent with chromosome divergence through Robertsonian translocations.

Role of p300, a transcriptional coactivator, in signalling of TGF-beta.

Smad proteins are novel transcriptional regulators mediating the signalling of the transforming growth factor‐β (TGF‐β) superfamily. Coactivators such as p300/CBP promote transactivation by various transcription factors through a direct interaction with them. Adenoviral oncoprotein E1A, which binds p300, was shown to inhibit the signalling of TGF‐β. These findings raise the possibility that p300 may be involved in TGF‐β signalling.

A genetic linkage map of the Japanese flounder, Paralichthys olivaceus

Abstract We report the first genetic linkage map of the Japanese flounder ( Paralichthys olivaceus ) constructed with 111 microsatellite markers and 352 AFLP fragments. The parental male linkage map consisted of 25 linkage groups while the female map consisted of 27 groups, with an average resolution of 8 and 6.6 cM, respectively. We have identified linkage among 96% of the markers and the total map length was estimated to be around 1000–1200 cM. This study reports the first low-density linkage map for the Japanese flounder and describes differences in sex recombination. Recombination rates were higher in male flounder compared to the female (7.4:1), a rare condition among vertebrates. This map is a starting point for the mapping of single loci and quantitative traits in flatfish species.

Linkage analysis of quantitative trait loci associated with spawning time in rainbow trout (Oncorhynchus mykiss)

We have used 54 microsatellite markers to search for quantitative trait loci (QTL) associated with spawning time in rainbow trout (Oncorhynchus mykiss). Linkage tests have been conducted in a backcross family produced by crossing spring and fall spawning stains in Ontario, Canada. We have mapped thirteen QTL markers for spawning time representing seven linkage groups. Eight of these QTL markers from five linkage groups show repeatable effects in two sampling years. These results suggest this trait is highly polygenic in rainbow trout.

Cytotoxic T cell function in fish

Fish possess immunoglobulins, major histocompatibility complex (MHC), T-cell receptors, and lymphocyte populations analogous to B and T cells and can evoke specific immune responses against a variety of antigens. However, T-cell subsets have yet to be demonstrated and the information on cell-mediated immunity is limited. Here we briefly review our recent studies on specific cell-mediated immunity, particularly on cytotoxic T-cell function employing isogeneic fish and cell lines. Analyses of the graft-versus host reaction (GVHR) and cell-mediated cytotoxicity (CMC) against allogeneic erythrocytes or cell lines show alloantigen-specific cytotoxicity in clonal ginbuna crucian carp. We also describe specific cytotoxicity against virus-infected cells using clonal ginbuna and a syngeneic cell line. Lastly, we report MHC-restriction in CMC against virus-infected cells using homozygous clonal rainbow trout and trout cell line which share the same MHC class I allele. These studies on CMC strongly suggest the presence of antigen specific cytotoxic T cells in teleosts and functional similarities between the immune systems of fish and higher vertebrates. Experimental model systems established in these studies can be applied to the investigation of protective antigens to induce cell-mediated immunity for the development of fish vaccines.

Nonspecific immune response of rainbow trout (Oncorhynchus mykiss Walbaum) in relation to different status of vitamin E and highly unsaturated fatty acids

This study was designed to examine the effects of dietary vitamin E (VE) on modulation of immune responses when supplied with two levels of n-3 highly unsaturated fatty acids (n-3 HUFA) in rainbow trout, Oncorhynchus mykiss. Six semipurified diets were prepared containing three levels of dietary VE (0, 100 or 1000 mg alpha-tocopheryl acetate kg(-1) diet) and n-3 HUFA either at 20 or 48% of dietary lipid provided from fish oil or docosahexaenoic acid (DHA) concentrated fish oil respectively. The diets were fed to rainbow trout (100 g initial mean weight) for 15 weeks. The VE, vitamin C (VC) content in plasma and tissues and the nonspecific immune responses, both humoral (alternative complement activity, total immunoglobulin) and cellular (phagocytosis, nonspecific cytotoxicity) were examined. VE contents in the kidney reflected the dietary input but were lower in fish fed 48% n-3 HUFA diets, and could have impaired some of immune responses compared to fish fed 20% n-3 HUFA. VC contents in kidney followed the same pattern as VE. Both humoral and cellular immune functions deteriorated in fish fed VE deficient diets whereas improvement in most of the parameters corresponded to its supplementation. However, the higher dose of dietary VE did not substantially enhance the responses assayed compared to the 100 mg dose. Besides clearly indicating the role of VE in maintaining the immune functions in fish in relation to dietary n-3 HUFA, this study has revealed that optimum health benefits could be achieved when VE is maintained slightly above the levels generally recommended for normal growth.

A second generation genetic linkage map of Japanese flounder ( Paralichthys olivaceus )

BackgroundJapanese flounder (Paralichthys olivaceus) is one of the most economically important marine species in Northeast Asia. Information on genetic markers associated with quantitative trait loci (QTL) can be used in breeding programs to identify and select individuals carrying desired traits. Commercial production of Japanese flounder could be increased by developing disease-resistant fish and improving commercially important traits. Previous maps have been constructed with AFLP markers and a limited number of microsatellite markers. In this study, improved genetic linkage maps are presented. In contrast with previous studies, these maps were built mainly with a large number of codominant markers so they can potentially be used to analyze different families and populations.ResultsSex-specific genetic linkage maps were constructed for the Japanese flounder including a total of 1,375 markers [1,268 microsatellites, 105 single nucleotide polymorphisms (SNPs) and two genes]; 1,167 markers are linked to the male map and 1,067 markers are linked to the female map. The lengths of the male and female maps are 1,147.7 cM and 833.8 cM, respectively. Based on estimations of map lengths, the female and male maps covered 79 and 82% of the genome, respectively. Recombination ratio in the new maps revealed F:M of 1:0.7. All linkage groups in the maps presented large differences in the location of sex-specific recombination hot-spots.ConclusionsThe improved genetic linkage maps are very useful for QTL analyses and marker-assisted selection (MAS) breeding programs for economically important traits in Japanese flounder. In addition, SNP flanking sequences were blasted against Tetraodon nigroviridis (puffer fish) and Danio rerio (zebrafish), and synteny analysis has been carried out. The ability to detect synteny among species or genera based on homology analysis of SNP flanking sequences may provide opportunities to complement initial QTL experiments with candidate gene approaches from homologous chromosomal locations identified in related model organisms.

Specific cell-mediated cytotoxicity against a virus-infected syngeneic cell line in isogeneic ginbuna crucian carp.

In the present study, we attempted to determine whether virus-specific cell-mediated cytotoxicity occurs in fish. Experiments were conducted with clonal ginbuna crucian carp (Carassius auratus langsdorfii) (S3n strain), and the syngeneic cell line (CFS). Two infectious viruses were used: infectious pancreatic necrosis virus (IPNV) and eel virus from America (EVA). Peripheral blood leukocytes, which consisted almost entirely (95%) of lymphocytes and thrombocytes, from S3n ginbuna immunized with virus-infected CFS cells lysed the virus-infected CFS cells (immunogen) more completely than CFS cells infected with a different virus (non-immunogen). This specific cell-mediated cytotoxicity of ginbuna against virus-infected CFS cells was efficiently induced as a result of in vivo secondary immunization. A significant specific cytotoxic activity peaked during 7-10 days after the secondary immunization. In addition, effector cells immunized with IPNV-infected syngeneic cells did not lyse IPNV-infected xenogeneic cells. These results support the hypothesis that fish exhibit specific cytotoxicity against virus-infected cells, resembling the specific cytotoxicites of higher vertebrates.

Neutrophilic granulocytes in carp, Cyprinus carpio, possess a spontaneous cytotoxic activity

This study demonstrates for the first time that carp (Cyprinus carpio) neutrophilic granulocytes from the head kidney possess potent spontaneous cytotoxic activity against several human tumor cell lines. Carp head kidney cells isolated at a density of 1.09 g/mL contained more than 90% neutrophilic granulocytes. These cells were round and approximately 10 millimicrons in diameter with reniform or polymorphic nuclei and slightly eosinophilic cytoplasm when stained with Giemsa. Electron microscopy revealed that the cytoplasm contained numerous oval granules, some of which contained a dense rod-shaped core. The neutrophilic granulocytes readily formed conjugates with the human target cells and rapidly killed them. The neutrophilic granulocytes killed human derived target cells better than murine derived target cells. Inhibition of cytotoxicity by catalase suggested that the production of H2O2 is involved as a mediator in the cytotoxic reaction. The size and granularity of the carp effector cells indicate that they are different from the small agranular nonspecific cytotoxic cells (NCC) described in the channel catfish.