Muscle growth was investigated in Harpagifer species with sub-Antarctic (Harpagifer bispinis, environmental temperature (ET) 4-11°C) and Antarctic (Harpagifer antarcticus, ET -1.5 to +1°C) distributions. The trunk musculature of 1 d-old larval H. antarcticus, 0.84-0.93cm standard length (SL), contained 1025±76 fast muscle fibres per cross-section (Mean±SE, n=6). Zones of muscle fibre formation were restricted to the dorsal and ventral cones of the myotomes. The maximum diameter of fast muscle fibres increased linearly with standard length and was 270 μm at 10.1cm SL. The density of myogenic progenitor cells, determined by staining sections for c-met and myogenic regulatory factors (MyoD, myf-5, myf-6, myogenin), decreased with increasing body length. The number and size distribution of fast muscle fibres with respect to standard length was indistinguishable in H. bispinis and H. antarcticus, although the number of c-met^+ve cells and the maximum body size was higher in the Antarctic species. Comparative studies indicate that a large maximum fibre diameter is a general characteristic of the Notothenioideii. This may reflect the radiation from a small bodied ancestor with a relatively low fibre number, although adaptive explanations related to metabolic rate reduction cannot be excluded.

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To elucidate the molecular mechanism and in vivo regulation of heat shock response during development, the heat-inducible HSP 70 cDNA and its transactivating factor HSF cDNA were isolated from zebrafish embryos. The amino acid sequence of zebrafish HSP 70 closely related to that of other vertebrate heat inducible HSP 70 with 75-86% identities. The DNA binding domain, the hydrophobic repeat HR-A/B and the carboxyl-terminal heptad repeat HR-C of the HSF are well conserved in the zebrafish HSF. The spatial and developmental expression patterns of the HSF gene in embryos were suggested to determine the heat-inducible expression of heat shock genes during zebrafish development.

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Creatine kinase plays a key role in the energy metabolism of cells that have fluctuating energy requirements. In vertebrates, the creatine kinase isoenzyme family consists two cytosolic and two mitochondrial forms. Only one M-CK isoform was known until we identified another three carp M-CK isoenzymes. Herein, we examine the effect of temperature changes on the temporal and spatial expressions of these three M-CK isoenzymes. Enzyme-linked immunosorbent assay experimental results indicate that these three M-CK isoforms are expressed at each temperature examined. Consistent with this result, immuno- histochemical staining also showed that these M-CK isoenzymes are co-localized in the same cellular compartment. In addition, the formation of both homo- and heterogeneous complexes among M 1-, M 2, and M3-CK was darified by coimmunoprecipitation and in vitro transcription and translation coupled GST-pull-down assay. We also generated M-CK homo- and heterodimer models to demonstrate the interactions of these monomers. Therefore, we propose that M-CK homo- or heterodimer formation undergoes an exchange according to acclimation to mircoenvironmental temperatures and relative pH values. We think that this MM-CK homo- and heterodimer exchange plays an important role as a temporal energy buffer with a spatial buffering function.

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The effect of cold shock and increased levels of circulating cortisol on heat shock proteins (Hsp) levels in rainbow trout (Oncorhynchus mykiss) were examined. Cold shock: When the cultured hepatocytes were exposed to cold (at 4°C for 2h), Hsp 70 level in hepatocytes were similar to that in control. However, Hsp 30 was markedly induced by cold shock. Cortisol: The high concentration of circulating cortisol (pharmacological levels) was found to reduce the Hsp 70 levels in liver and gill of cortisol implanted fish (50 μg cortisol/g body weight) exposed to heat shock (at 22°C for 2h) compared to the sham. These results suggest that the expressions of Hsp 30 and 70 in cell may be affected by cold stress response and circulating cortisol levels in fish, respectively. Furthermore, cellular stress response, such as Hsp expression, might be related with neuroendocrine/endocrine system in fish.

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Marine organisms including fish (vertebrate) and shellfish (invertebrate) adapt to changes in the osmolality at the cellular level. Fish can maintain the osmolality of body fluid within a certain range due to the highly developed endocrinological system, while shellfish cannot regulate the osmolality of body fluid due to the lack of it. This suggests that shellfish cells are more resistant against osmotic change than fish cells. To compare the osmo-responsive system of fish and shellfish at cellular level, we focussed on the amino acid transporters and examined the expression patterns of their mRNAs following the osmotic change. When examined the expression patterns of fish taurine transporter mRNA following the osmotic change by using EPC cells, cultured carp epidermal cells, specific induction by hyper-osmotic stress was observed. Hyper-osmolality induced expression was also observed in tilapia tissues when tilapia acclimatized to fresh water was immediately exposed to 70% seawater. In contrast to fish, mRNA of oyster amino acid transporter was induced both by hyper- and hypo-osmotic stresses. Unexpected induction of oyster transporter mRNA by hypo-osmotic stress suggests a difference in the promoter function of the transporter genes between fish and shellfish.

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Genetic responses of the aquaculturable seaweed Porphyra yezoensis to pine needle ash have been compared using differential display technique. The tissue viability was assessed to evaluate the stress level with triphenyltetrazolium chloride. Total RNA, from tissues challenged in seawater containing ash, was reverse transcribed and amplified by PCR with arbitrary primers. The genetic fragments responded by the stress were selectively isolated from agarose gel and sequenced with DNA auto sequencer. According to sequence analysis by the NCBI BLASTX program, ash-suppressed genes (1065 bp, 347 bp) were putative glutaredoxin-like protein and hypothetical protein. Pine needle ashinducible genes (939 bp, 556 bp) were recognized as hypothetical protein and phosphoenolpyruvate-protein phosphotransferase, respectively.

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The thermal stress responses of a sterile mutant of Ulva pertusa were investigated at 20 C and 30 C. The amounts of the photosynthetic pigments and the major free amino acids increased 1.4-2.4 and 1.9-10.5 fold, respectively, in response to thermal stress. Total carbon and nitrogen contents also increased in the 30 C-cultivated alga. The changes in chemical components due to thermal stress account for the morphological changes such as darkening of chloroplasts, thickened cell walls, and enlarged cytoplasm in the 30 C-cultivated alga. Isozyme assay showed that both temperature-treated algae possessed NAD⁺- and NADP⁺-specific glutamate dehydrogenase (NAD/NADP-GDH) and that the 30 C-cultivated alga had an additional NADP⁺ -specific GDH (NADP-GDH). Two cDNA clones encoding GDH-L and GDH-S were isolated from the 30 C-cultivated alga. mRNA encoding GDH-L was constitutively expressed in the algae, whereas expression of that encoding GDH-S was induced by thermal stress. These results suggest that NADP-GDH compensates for the drop in NAD/NADP-GDH activity under thermal stress conditions and that NAD/NADP-GDH and NADP-GDH in the alga correspond to GDH-L and GDH-S, respectively.

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One explanation for the predominance of diatoms in marine ecosystem is their high adaptability to different environmental conditions. However, little is known about the physiological mechanisms concerned at the molecular level. Recent works on heat stress responses of various diatom species have indicated that diatoms markedly change their gene expression profiles and produce various molecules to respond to heat stress. Recently, we have isolated a novel heat-stress-responsive gene, Hl-5, from the marine diatom Chaetoceros compressum. Two types of transcripts were produced from the single Hl-5 gene by heat stress-dependent RNA splicing, encoding structurally different proteins, the functions of which are apparently different. This gene may be a promising molecule to give us a new insight for better understanding of heat stress responses in diatoms.

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Fish into which foreign DNA (transgene) have been artificially introduced and integrated in their genomes are called transgenic fish. Since the mid 1980 s, introduction of desired foreign DNA into unfertilized or newly fertilized eggs by microinjection or electroporation has produced many species of transgenic fish. More recently, infecting newly fertilized eggs or the immature gonads of male fish with replication-defective pantropic retroviral vectors carrying the desired foreign DNA has also produced transgenic finfish. These transgenic fish could serve as excellent experimental models for basic scientific investigations as well as biotechnological applications. In this paper, we will review the current status of the transgenic fish technology and provide data from our own research to show the feasibility of producing disease resistant fish strains for aquaculture via manipulation of antimicrobial peptide genes.

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The transgenic technology has been widely used in biotechnology, from generation of genetically modified (GM) foods to production of pharmaceutical proteins. Although fast-growing transgenic fish using exogeneous growth hormone gene have been successfully produced, the marketing of such GM food fish remains to face an uphill battle. In the present study, we attempted to use the transgenic technology to develop novel varieties of ornamental fish. So far, we have succeeded in generation of several colourful transgenic zebrafish by transferring a jellyfish green fluorescent protein (gfp) gene and an anemone red fluorescent protein (rfp) gene. These transgenic zebrafish displayed green, red, yellow or orange fluorescent colors under both daylight and uv light. Thus, our work demonstrated the feasibility to generate novel varieties of ornamental fish by the transgenic techniques. Currently we are conducting mating behaviour experiments using these transgenic zebrafish. We are also aiming at development of biomonitoring transgenic fish by using some inducible gene promoters that can respond certain environmental pollutants.

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To develop a cell-mediated gene transfer technique in fish, a cell line that can differentiate into the germ cell lineage is needed. In this study, we developed techniques to identify, visualize, and isolate live primordial gems cells (PGCs) for in vitro culture and later for gene transfer work. In many animals, the vasa gene products have been found in the germ cell lineage, and we have previously identified and characterized its homologue (RtVLG) in rainbow trout. Whole mount in situ hybridization of trout embryos at various developmental stages revealed that transcripts were localized to PGCs, suggesting that RtVLG transcription can be used as a marker for PGCs. To identify PGCs in vivo, we constructed a plasmid containing the green fluorescent protein (GFP) gene driven by the RtVLG regulatory regions (pvasa-GFP), microinjected it into fertilized eggs, and raised mature fish. The F1 generation of these transgenic trout showed green fluorescence only in PGCs in all developmental stages. This expression pattern was consistent with that of the endogenous RtVLG gene. To obtain PGCs for in vitro culture, the genital ridges were excised from transgenic embryos, dissociated by trypsin, and a flow cytometer was used to sort them into GFP-positive and GFP-negative fractions. RT-PCR analysis revealed that RtVLG was expressed only in GFP-positive cells. These results confirm that the isolated GFP-positive cells are PGCs. We are presently attempting to establish cell lines for further work.

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Growth hormone (GH) gene expression has been examined in control and transgenic coho salmon containing a transgene comprised of the sockeye salmon GH 1 gene under the control of the MT-B promoter from the same species. This transgene dramatically enhances the growth of salmonids, and raises serum GH levels some forty-fold. Transcript levels from this transgene were detected by RT-PCR in all tissues examined, and an obvious increase of the transgene expression was obtained in some developing stage. GH mRNA levels were also examined in the pituitary gland, and were found to be significantly lower (p<0.01) in transgenic compared to nontransgenic salmon of the same size. Pituitary glands of transgenic were also smaller than control of the same size. The transgenic fish changed a surface area in the anterior intestine, and the intestine of GH transgenic salmon had more folds and the path tracing the inner circumference around the intestinal folds of a cross section was about 2.7 times longer than in control salmon. Further, the transgenic salmon increased ability to compete for food, and they consumed 2.9 times more pellets that the non-transgenic controls.

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A marine red alga Porphyra has recently received great interest not only as a valuable marine crop but also as a model plant for fundamental and applied studies in marine sciences. Porphyra has a unique dimorphic life cycle which consists of a leafy gametophytic generation and a filamentous sporophytic one. It completes its life cycle in laboratory culture within a few months and has a small number of chromosomes (2-7) in the haploid phase. The haploid genome sizes in the genus Porphyra (2.7-5.3×10⁸ bp) are in the same order of magnitude as those of Arabidopsis thaliana. Genetic analyses, including both classical and modern molecular studies are in progress. Now, we are performing the following studies in order to make Porphyra a sophisticated model organism: cryo-preservation, establishment of mutant cell lines, development of host-vector system for transformation, Porphyra genome project which includes genetic map, EST, DNA macro/micro array, etc. We will introduce arrangements for an infrastructure of advanced research on Porphyra yezoensis which is a key to opening the door towards a new marine botany world.

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The majority of species and strains reared globally for aquaculture are relatively unimproved for commercially important traits. The potential for genetic improvement in fish species compared with domestic livestock, is very high. Therefore, we are integrating molecular genetic technologies into aquaculture to help solve some of the major genetic problems. Our long-term goal is to use genetic markers to increase the efficiency of artificial selection in fish stock improvement. To do this, marker-assisted selection (MAS) has been proposed. MAS can be carried out with an understanding of the linkage relationships between quantitative trait loci (QTL) and markers. To identify QTL controlling traits of economic importance, a genetic linkage map is required, with variable markers distributed throughout the genome. We have constructed a genetic linkage map for rainbow trout using 192 microsatellite, 3 RAPD, 5 ESMP, and 7 allozyme markers in three backcross families. As a first step towards MAS, some QTLs associated with economically important traits have been identified using this linkage map. The genetic linkage map based on microsatellites could be useful for QTL analysis in aquaculture.

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A cDNA library was constructed from hemocytes of the black tiger shrimp Penaeus monodon that were infected with the luminous bacteria, Vibrio harveyi, to survey the immune-related genes which are induced and expressed in hemocytes. Four hundred and nine cDNA clones were sequenced and subjected to homology searches in the DNA databases. One hundred and eighty one clones (44%) showed significant homology to known genes but the remaining sequence (56%) did not match to any sequence in the GenBank. Forty one clones (10%) were identified as putative immunerelated genes and represented 14 different immune genes. Antimicrobial peptides were the most common group of immunerelated ESTs found in infected hemocytes. The ESTs coding for putative anti-lipopolysaccharide factor (16 clones) and 11.5 kDa antibacterial protein (8 clones) were predominate among the immune genes indicating the high abundance of these transcripts in infected hemocytes. High expression of these antimicrobial proteins suggests that they play a major role in the host respose to V. harveyi infection.

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Species-specific markers of three commercially cultured oysters (Crassostrea belcheri, C. iredalei and Saccostrea cucullata) in Thailand were identified by RAPD-PCR and PCR-RFLP analyses. Two hundred and fifty-four RAPD fragments were generated across overall samples in this study. High levels of genetic polymorphism were observed in Thai oysters. Ten, five and two species-diagnostic RAPD markers were found in C. belcheri, C. iredalei and S. cucullata, respectively. In addition, restriction analysis of the amplified COI gene segment with Acs I, Dde I and Mbo I provided 6, 13 and 22 restriction patterns, respectively. Species-specific RFLP patterns were also found in those oysters. A molecular taxonomic key based on RAPD and RFLP markers was constructed and successfully used for identification of the species origins of C. belched, C. iredalei and S. cucullata in Thailand

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Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of 16S ribosomal (r) DNA was used to identify species-specific markers of three abalone species; Haliotis asinina, H. ovina and H. varia in Thailand. A total of 10 composite haplotypes were found across geographically different samples of these abalone. Species-specific composite haplotypes of each abalone were found. Intraspecific genetic differentiation was clearly observed in H. ovina but not in H. asinina and H. varia. The 16 S rDNA of an individual representing major composite haplotypes AAAA, ABBB, ARAB, BABG and BABC were cloned and sequenced. Comparisons of 16 S rDNA sequences suggest the possibility of developing a species-specific PCR for each abalone species.

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An expressed sequence tag analysis (EST) was conducted for discovery of immune- and defense-related genes of Japanese flounder, Paralychthys olivaceus and kuruma shrimp, Penaeus japonicus. Eight cDNA libraries were constructed from liver, spleen, skin, kidney, and leukocytes of Japanese flounder. We also constructed two cDNA libraries from healthy and white spot syndrome virus (WSSV)-infected kuruma shrimp hemocytes. We sequenced more than 2, 800 clones and 1, 000 clones of Japanese flounder cDNA libraries and kuruma shrimp cDNA libraries, respectively. We found many putative biodefence and immune response related genes from Japanese flounder, such as cytokines (interleukin 1 β, tumor necrosis factor, chemokines, etc), cytokine receptors (TNFR 1 and 2, IL 1 R, IL 6 R, chemokine receptors, toll like receptors, etc), cell surface molecules (T cell receptors, IgM, IgD, CD 3, CD 8, etc), transcription factors (IRFs, CEBP, Stat 3, etc), complement component (C 3, C 8, C 9), antimicrobial proteins (lysozymes, transferrin, Mx), proteases and protease inhibitors. There were several kuruma shrimp ESTs for immune- and defense-related genes including the prophenoloxidase system, antimicrobial peptides and an antimicrobial protein, membrane-associated proteins, and soluble proteins. The immune- and defense-related cDNAs that are discovered by EST analysis could be useful tools for studying the immune- and defense-response in fish and shrimp.

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The siderophore anguibactin is an important component of the pJM 1 plasmidmediated iron uptake system that is essential for virulence in this pathogenic vibrio. Genetic and physiological analysis has led us to the identification and cloning of genes encoded on the pJM 1 plasmid that play an essential role in anguibactin biosynthesis. DNA sequence and protein analysis revealed that these genes encode polypeptides which possess domains found in nonribosomal peptide synthetases (NRPSs), originally identified as components of the biosynthetic machinery for the synthesis of antibiotics in Gram-positive bacteria. These proteins have been named AngB, AngM, AngN, and AngR and possess modules that could be involved in one or more of the following reactions during the biosynthesis of anguibactin: peptidyl carrier protein (PCP), involved in thioester formation; condensation (C), intervening in peptide bond formation, cyclization (Cy), involved in both condensation and heterocycle formation and adenylation (A), which is responsible for substrate activation. AngB is an isochorismate lyase that also operates as an aryl carrier (ArCP) protein during siderophore assembly. The combination of our in vivo genetic and in vitro biochemical approaches has resulted in the elucidation of the mechanism of anguibactin biosynthesis and its contribution to the virulence repertoire of this fish pathogen.

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Genome analysis was conducted to detect the virulence genes in Pasteurella piscicida. To date we have analyzed 366 clones from plasmid genomic library and 507 clones from cosmid genomic library, from which we determined a total of 932, 722 by nucleotide sequences. As a result of comparison of these clones to the GenBank database, five clones from plasmid library and 11 clones from cosmid library have homology with deduced bacterial virulence genes. In the plasmid library, these are: DsbA, LspA2, nucleotide sugar epimerase, OpsX and PIdA . In the cosmid library, these are: delta endotoxi, hemolysin, heme receptor, hyahronidase, immature fimbrillin, lipidA dissacharide synthase, lipidA 4'-kinase, neurotoxin, PIdA, TonB and Wzz. We consider that these results are useful information to identify virulence genes in P. piscicida, that will now allow further analysis of this bacterium's pathogenicity.

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The toxR gene was cloned from Vibrio anguillarum isolated from diseased Ayu (Plecoglossus altivelis). Analysis of the toxR sequence suggested that the V. anguillarum ToxR is likely to encode the functions shared by ToxRs of vibrios so far reported. There was no significant difference in the ability to produce a protease (s), a hemolysin (s), or in pathogenicity to ayu (intramuscular injection and immersion methods) between PT-24 Δ toxR, an isogenic toxR mutant of PT-24, and PT-24 (an ayu isolate). The expression of two major outer membrane proteins was different between the two strains. The results clearly indicate that ToxR is not involved in regulation of virulence in V. anguillarum and suggest that the ancestral role of ToxR in vibrios is not virulence regulation but regulation of outer membrane expression, in response to environmental change. A DNA probe method and a PCR method targeting the toxR gene were developed for identification of V. anguillarum, and they were shown to be very specific.

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The complete genome (112.4 kb) of red sea bream iridovirus (RSIV) (strain Ehime-1) was sequenced. RSIV causes the red sea bream iridoviral disease (RSIVD), an important disease affecting cultured marine fish species. A circular physical map of the RSIV genome supports the fact that RSIV is member of family Iridoviridae. Size and G+C content of the RSIV genome is compared to those of other iridoviruses, from which it is determined that RSIV was similar to those of frog virus 3 (FV3), the vertebrate iridovirus and has similarity to the genus Ranavirus. However, phylogenetic analysis of MCP genes reveals that RSIV should be classified as new genus of the family Iridoviridae. In addition two genes were possibly identified which encode prospective protective antigens thatwould have potential for the production of a recombinant vaccine.

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Studies about the ways in which hemocytes are produced and differentiate in crustaceans are important for basic understanding of the immune defence in these animals.We have also for the first time from any invertebrate made a cell culture from hematopoietic stem cells of freshwater crayfish as well as from tiger shrimp, which we can manipulate to differentiate in vitro. The hemocyte precursors do not express prophenoloxidase, but this transcript can be induced in vitro by addition of a factor. We have also confirmed that hematopoietic cells from the tiger shrimp differentiate in vitro as a response to a similar factor.

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The Cypridina luciferin analogue (CLA), a highly sensitive chemiluminescence (CL) probe, was used to detect superoxide (O₂) in kuruma prawn hemocytes. The CLA-dependent CL of the shrimp hemocyte after phorbol 12-myristate 13-acetate (PMA) stimulation was inhibited by superoxide dismutase. Nitric oxide (NO) production from the shrimp hemocytes was detected by fluorometric measurement after co-stimulation with lipopolysaccharides (LPS) and recombinant human interferon γ in vitro. The supernatant of a hemocyte culture after stimulation showed an increase in the fluorescence intensity, and this intensity decreased with the addition of N^G-monomethyl-L-arginine (L-NMMA). These results show that shrimp hemocytes can produce O₂ and NO and these free radicals play an important role in the shrimp defense network.

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In this study, we showed the lack of defense responses and apoptotic death in the Pacific oyster hemocytes when the hemocytes were in vitro exposed to three species of bacteria. The oyster hemocytes exerted a high phagocytic ability against the live cells of both Escherichia coli (45.9%) and Planococcus citreus (33.7%), whereas the hemocytes produced only a slight phagocytosis of live Vibrio tubiashii cells (0.4%). Moreover, the oyster hemocytes were attracted to live E. coli but not to live V tubiashii cells and their culture filtrate. Migration to both live V. tubiashii cells and their culture filtrate was significantly lower than that to the control (showing random motility, p<0.05, Dunnett's test). In addition to the above results, the oyster hemocytes exposed to live V. tubiashii showed a high mortality at 71.6% after 6-hr incubation. On the other hand, the oyster hemocytes underwent apoptotic cell death due to the phagocytosis of live P. citreus cells. Apoptosis was also induced in the hemocytes ingesting pasteurized P. citreus cells, whereas the hemocytes phagocytizing autoclave-killed P. citreus did not. The apoptosis in the oyster hemocytes was markedly blocked by treatment with antioxidants, especially catalase and N-acetyl-L-cysteine.

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Megabalanus rosa contained the C-type lectins as the major constituents of hemolymph proteins. BRA-1 (330 kDa) and BRA-2 (140 kDa) were composed of identical subunits (173 amino acids), and BRA-3 (64 kDa) of four same subunits (138 amino acids). These lectins were present in various tissues other than the hemolymph. However, only BRA-2 was detected in the organic matrix of shell by western blotting. Both BRA-2 and BRA-3 showed high association constants to calcium ions and inhibitory activity to CaCO₃ crystallization. The immobilized BRA-2 was found to promote the CaCO₃ crystallization. M. rosa lectins also showed opsonic activity when checked with murine macrophage. The gene structures of BRA-2 and BRA-3 were different from each other. These results suggest the participation in defense as well as biomineralization and the allotted biological role of M. rosa humoral lectins.

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Eosinophilic granular cells (EGCs) of fish are the granulocytes existing in the tissue. Their morphological characteristics have led to suggestion that fish EGCs are analogous to mammalian mast cells. It has been postulated that EGCs are involved in defense mechanisms, with EGCs degranulating after injection of non-self materials. The aim of this study is to determine the role of tilapia EGCs in inflammatory responses.
Neutrophil migration was observed after the degranulation of tilapia EGCs. Substance P (neurotransmitter) or anaphylatoxins (C 3 a and C 5 a) induced the EGCs degranulation. In the in vitro experiments, supematants of the degranulated EGCs induced neutrophil migration, indicating neutrophil migration-stimulating factor (s) were released after the degranulation of tilapia EGCs.
Injection with tilapia EGC lysates enhanced the vascular permeability, and the lysates induced the Ca²⁺ uptake by cultured tilapia vascular endothelial cells. These results indicate that tilapia EGC products directly activate the endothelial cells and increase the vascular permeability. The EGC products also induced expression of the neutrophil adhesion molecules by cultured vascular endothelial cells, because neutrophil adhesion to the endothelial cells increased after stimulating the endothelial cells with the EGC lysates.
These functions and properties of tilapia EGCs are resembled to those of mammalian mast cells, and EGCs of tilapia appear to be homologous to mammalian mast cells.

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In all teleost fish, the immune-endocrine interaction is one of the most important communication network to ensure physiological homeostasis during the spawning season. In this review, we discuss on the effects of endocrine secretions on the immune system of different fish species with the special reference to reproduction. High levels of sex steroids in mature goldfish related with high IgM levels. High water temperature also enhanced the immunity. In rainbow trout, which is susceptible to disease during the spawning season, plasma IgM levels and the number of IgM producing cells decrease with the increase of sex steroids, i.e., testosterone, estradiol-17β and 11-ketotestosterone. In addition, administration of sex steroids as well as cortisol caused immunosuppression. These steroids also inhibited in vitro IgM production of lymphocytes with the reduction of mRNA transcription of both membrane and secretory form of IgM. Such immunosuppressive functions of sex steroids were not recognized in cyprinid fish, which can keep their health strong during the spawning season, although cortisol acts as an immune suppressor. Higher sensitivity against steroids in leukocytes of salmonid fish may cause the high disease susceptibility of rainbow trout during the spawining season.

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The complement system is a major humoral factor for tagging and killing target microorganisms. A striking feature of bony fish complement is the diversity of several complement components. The present review is focused on the diversity of C 3, C 4, and C 5, which belong to the α₂-macroglobulin (α₂M) family. Except for C 5, they contain an internal thioester bond, which mediates covalent binding of the proteins to target cells and molecules. In contrast to mammals, carp has multiple C 3 isofcxms, including novel isoforms that lack the catalytic histidine residue that primarily determines the reactivity of the thioester to amino- and hydroxyl-groups and thereby influences the binding specificity of the components. Protein analyses revealed that the C 3 isoforms differ in the hemolytic activity and the binding specificity. Carp also have two diverged C 4 isotypes, designated C4A and C4B. C4B has the catalytic histidine and C4A does not, like human C4B and C4A isotypes respectively, predicting their difference in the binding specificity. C 5 is also present in two distinct forms in carp. One of them contains a potentially deleterious amino acid substituion. These diversity in the complement components may represent a unique strategy to enhance innate immunity of bony fish.

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Cytotoxic T cells taken from an individual recovering from a viral infection are capable of killing only virally infected cells which share an MHC haplotype with the host (MHC restriction). Very recently extensive allelic polymorphism of MHC class I genes has been demonstrated in both elasmobranchs and teleosts. Accordingly, we should use effector and target cells with the same MHC haplotype to study the MHC-restricted, antigen-specific cytotoxic T cell response in fish. In this symposium our recent studies on polymorphism and function of shark and rainbow trout MHC I genes were first described, followed by MHC-restricted cell-mediated cytotoxicity against virus-infected cell lines using homozygous clonal rainbow trout (C 25 clone) sensitized by DNA immunization with IHNV-G or by infection with IHNV and trout cell line (RTG-2) which shares the same MHC I allele with the trout.

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We briefly review our recent studies on in vitro and in vivo virus-specific cell-mediated cytotoxicity in clonal ginbuna crucian carp. We have demonstrated specific cell-mediated cyototxicity against virus-infected cells, employing clonal ginbuna crucian carp (S3n clone) and the syngeneic cell lines (CFS) established from the S3n clone. Moreover, we have demonstrated an in vivo role of specific cytotoxic cells against viral infection, using CHNV which has a virulence to ginbuna. We found that 1) there is an inverse relationship between cytotoxic activity and viral load and 2) adoptive transfer of immune leukocytes prevented viral infection. Viral titers of tissues from infected fish were remarkably reduced 8 days after infections when specific cytotoxic activity reached a peak. This result suggested that specific cytotoxic cells were responsible for the early control of CHNV replication. On the other hand, CHNV-specific antibody increased after virus was eliminated by cytotoxic activities. These results suggested that specific cytotoxic cells of fish play an important role in specific immunity to viruses.

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A DNA vaccine against infectious hematopoietic necrosis virus (IHNV) was the first DNA vaccine developed for use in salmonid fish. Protective immunogenicity was only observed with the glycoprotein (G) gene in rainbow trout Oncorhynchus mykiss using the DNA vaccine technology. A similar significant level of protection was also observed in Atlantic salmon Salmo salar vaccinated with a naked plasmid encoding the G protein and assessed for protection by immersion and cohabitation IHNV challenges. In both studies vaccinated fish seroconverted and this sera was shown to be protective after passive transfer and subsequent challenge with IHNV. Vaccine doses as low as 1-10 ng provided significant protection to rainbow trout fry (mean weight, 0.8-1.8g) and an optimal dose of 0.1 μg was selected. Rainbow trout immunized with this dose were shown to significantly protected against a broad range of viral strains from different geographic locations and host species. Although intramuscular injection is the principle mode of vaccination, gene gun immunization induced protective immunity in fry, while intraperitoneal injection provided partial protection. This vaccine was also shown to provide significant protection as soon as 4 days after intramuscular vaccination and provided a significant level of crossprotection against another fish rhabdovirus, viral hemorrhagic septicemia virus, for a transient period of time.

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The potential for genetic immunization, following DNA particle bombardment for juvenile (4.7cm ±0.3) Japanese flounder Paralichthys olivaceus was examined. Histopathology, at 3 hours post bombardment showed considerable damage to fish epithelial and dermal tissues when bombarded at pressures greater than 200 psi, with many DNA-coated gold particles found to be present. Histopathology, examined at 1, 7, 14 and 28 days post-bombardment showed little pathology at 150 psi with few DNA-coated particles, whereas at 300 psi there was significant pathology observed with many DNA-coated particles seen in conjunction with the cytoplasm of inflammatory cells. Between days 14 and 28 post-bombardment complete epithelial coverage was observed with tissue damage restricted to the dermal layer. CAT assay, following administration of plasmid DNA constructs at 250 psi, showed long term and stable expression of the CAT protein from day 1 to day 60. The transcription activity of two promoters, pCMV-CAT and pSV2-CAT showed marginally greater activity in the former. We conclude that the potential for DNA vaccination of juvenile flounder to be a viable option.

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Immunostimulants in aquaculture has created lot of interest as a suitable prophylaxis agent to control fish diseases. The efficacies of immunostimulants such as glucan and lactoferrin have been well studied in fish. Recently, new immunostimulants, like the nucleotides and CpG motif of DNA have been reported. Usually, immunostimulants can increase the activities of phagocytic cells such as phagocytosis, NBT and chemotaxis. Several immunostimulants also stimulate the natural killer cells, complement, lysozyme and antibody responses of fish. Immunostimulants activate several cytokines such as interleukin 1β and CC chemokines. The activation of these immunological functions is associated with increased protection against infectious diseases. For an effective use of immunostimulants, the timing, dosage, mode of administration and the physiological condition of fish need to be taken into consideration.

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An outer membrane protein (OMP) of the fish pathogenic bacterium Edwardsielia tarda was investigated on its antigenic property. Bacterial agglutination reaction and SDS-PAGE /westem blotting analysis using selected rabbit antisera demonstrated that a 37 kDa OMP was a common antigen among 17 E. tarda strains. Immunoelectron microscopy demonstrated that the 37 kDa OMP located on/in the outer membrane of the bacterium. Immunization of Japanese eel with the 37 kDa OMP prepared from two different serotype strains showed protection against intraperitoneal injection with one strain. Immunization of Japanese flounder with the 37 kDa OMP prepared from one strain also showed protection against injection with two different serotype strains. The results indicate that the 37 kDa OMP is a cell surface protective antigen commonly effective against different serotypes of E. tarda.

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To understand adaptive immune system in fish, we identified and characterized three kinds of T-cell antigen receptors (TCR α, β, and δ) and one of cytotoxic T cell co-receptor (CD8α) from Japanese flounder (Paralichthys olivaceus) leukocytes cDNA library. From the study of genomic DNA organization the constant genes for TCR α, β, and δ using BAC genomic DNA clones, it was revealed clearly that the TCR α constant gene and TCR δ constant gene are linked at the same locus; similar with that of human and mouse. The expression patterns of TCR α, β, and δ, and CD8α were tested by Northern blot hybridization or RT-PCR and in situ hybridization (ISH). The results of Northern blotting and RT-PCR showed that the lymphoid organs (kidney, spleen and leukocyte cells) and also gill and intestine which have direct contact with pathogens, highly express mRNA of TCR α, β, and δ, CD8α chains. The ISH with anti-sense RNA probes of these cDNA molecules allowed to confirm their respective expression and distribution patterns in some lymphoid organs (peripheral blood and head kidney).

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Marine hyperthermophilic microorganisms are valuable resources for thermostable enzymes which are well suited for biotechnological applications. A novel DNA endonuclease I-Ape I was found from the hyperthermophilic archaeon Aeropyrum pernix and characterized. Of particular interest is that I-Ape I is encoded in the optional intron Iα within the 16 S rRNA gene. Probably, I-Ape I confer mobility to the host intron Iα, like group I intron homing endonucleases from eukaryotes and bacteriophages. Also, this rarecutting enzyme is a useful tool for genome mapping.

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We identified a Na⁺- and Cl^--dependent high affinity taurine transporter as a hyperosmotic stress-inducible gene in a cultured common carp cell (EPC). Since the taurine transporter concentrates taurine from extracellular fluid to cell, upregulation of taurine transporter expression promotes the accumulation of taurine during hyperosmotic stress, suggesting the role of taurine as a compatible organic osmolyte. In tissues of tilapia (Oreochromis mossambicus), taurine transporter mRNA was also increased during high-salinity adaptation. Although increase in taurine transporter mRNA was detected in all the tissues examined, total tissue taurine content did not always increased during high-salinity adaptation. Immunohistochemistry indicated the localization of taurine transporter protein in basolateral membrane of corium and epidermal cells of pectoral fin. These results suggest that polarized transport and subsequent changes in distribution of taurine in a tissue are critical for high-salinity adaptation of an individual fish.

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Common carp had extraordinarily high Zn concentration of 300-500 μg/(g fresh tissue) in its digestive tract tissue. The high Zn concentration was not caused by the feeding habits of common carp, but because the fish had a much higher Zn absorption ability. The accumulation of Zn in the digestive tract tissue of the fish was saturable and reversible. Subcellular fractionation of the digestive tract tissue showed that 60-88% of the total cellular Zn was in nuclei/cell debris fraction, and only 12-40% was in all other fractions including cytosol, mitochondria and microsome fractions. Experiments indicated that in the nuclei/cell debris fraction, the Zn was most probably bound with thiol groups. The in vitro binding of ⁶⁵Zn to the nuclei/cell debris fraction was characterized according to the models used in receptor-binding assays method. Linearity of the Scatchard plot (r=0.9988) was strongly suggestive of the presence of a single high-affinity binding site. It is concluded that the main Zn binding substance in the nuclei/cell debris fraction of digestive tract tissue of common carp is a Zn specific binding protein. It is proposed that most of the Zn binding protein is located on the basolateral plasma membranes of the epithelial cells and surrounding muscle cells which are attached to collagen type IV of basal laminae.

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Fish species have recently become powerful model systems for analysis of vertebrate development and human disease. High throughput gene and expressed sequence tag (EST) mapping projects in fish are now facilitating our understanding of the relationship between vertebrates genomes and will help identify roles for human genes from fish mutations. Mutant screens in zebrafish are performed routinely, resulting in sizable collections of mutations causing a variety of developmental and physiological defects including digestive organ and lipid processing. Fish gene maps showed duplicated chromosome segments and blocks of numerous conserved syntenies between fish and human. The function of putatively orthologous and paralogous genes can, nonetheless, be different. In the context of the current revolution linking genes and pathways to integrative physiology, numerous fields, including fish physiology, are being redefined. Fish species are useful animal models for studying lipid metabolism and transport. The task of linking genes to function improve our knowledge of the molecular basis of lipid and lipoprotein metabolism in fish, and support the existence of related lipid processing mechanisms in mammals and teleosts.

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Forty species of marine mammals have been reported from Chinese waters. Studies on systematics and taxonomy, morphology, and conservation biology of Chinese marine mammals are briefly reviewed. Brief accounts of systematic and taxonomic studies on Lipotes, Neophocaena, Tursiops, Sousa and Delphinus are included. Studies of gene sequences of Lipotes and other river dolphins in recent years support ranking Lipotidae as a family. The studies of structure and function were mainly on Lipotes and Neophocaena. The researches on conservation biology of Lipotes, Neophocaena, Sousa and other marine mammals in the past decades have provided scientific bases for conservation actions. Natural reserves for Lipotes and Neophocaena, Sousa, Dugong and Phoca largha were established.

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Lipoprotein lipase (LPL) is a key enzyme of lipid metabolism. The elucidation of the structure, function and regulation of LPL is important for understanding the lipid metabolism in fish. Recently, we have cloned the LPL gene of red sea bream (Pagrus major) and characterized it by cDNA and genomic structure analysis. Subsequently, using the cloned sequence, gene expression has been investigated. We review in this report the features of red sea bream LPL gene. Red sea bream LPL gene spans 6.3 kb of the genome and is organized into ten exons and nine introns. The deduced amino acid sequences showed high degree of similarities to the LPLs of other animals. In a 1.1 kb 5' flanking region, the homologous sequences for the response elements of Insulin, glucocorticoid and thyroid hormone were detected. These results suggest the hormonal regulation of the LPL gene expression. Red sea bream LPL gene was expressed in various tissues including adipose tissue, heart, liver (hepatopancreas) and muscle. A fourteen-week feeding experiment was conducted to investigate the effects of feeding condition and dietary lipid level. The results suggest that LPL gene expression in visceral adipose tissue and liver is regulated in tissue-specific manner. The expression level in adipose tissue was down-regulated during starvation whereas it was up-regulated in liver. The effects of dietary lipid level on the gene expression were not observed. These results will facilitate further study on the function and regulation of the LPL in fish.

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Vitellogenesis in oviparous species corresponds to accumulation of nutrients in oocytes and their storage as yolk used by embryos and larvae during their development. In fish the main yolk component is vitellogenin (VTG) a glycophospholipoprotein synthesized by the liver under estrogenic induction and specifically incorporated into oocytes by receptor-mediated endocytosis. We have cloned the 91 kDa VTG receptor in rainbow trout. Its transcription occurs very early in young previtellogenic ovarian follicles when the oocyte sets up the necessary molecules and tools for its subsequent endocytotic activity. Lipid droplets are the other important yolk moiety. They could come from the sequestration of VLDL in outer somatic oocyte envelopes by specific receptors with O-linked sugar domain. These two ovarian receptors are members of the LDLR superfamily. They exhibit strong homology with oviparous ovarian follicle receptors. A review of oviparous species LRPs ensuring a pivotal event in the reproductive function is given, with special emphasis to chicken yolk receptors.

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The plasmalogen content and composition in the low density lipoprotein (LDL) and high density lipoprotein (HDL) of the fish were analyzed. The composition of the plasmalogen in the carp plasma LDL phospholipids was 0.94% and 0.23% in the HDL; the LDL phospholipids in the rainbow trout were 0.44% and the HDL was 0.18%. Aldehydes from the plasmalogen were derivatized with dansylhydrazides and separated by high performance liquid chromatography (HPLC), their presence was detected using a fluorescence detector. Hexadecanal (C 16:0), octadecanal (C 18:0) and octadecenal (C 18:1) were determined to be the major components in the carp and rainbow trout. The plasmalogen content in the plasma lipoprotein phospholipid was much lower than that in the brain, heart and ordinary muscle. Although the alkyl dihydroxyacetone phosphate (DHAP) synthetic ability was recognized in these tissues, the alkyl DHAP was also synthesized in the liver. It was indicated that lipoprotein included in plasmalogen was synthesized in the liver.

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As a series of studies to clarify the relationship between serum lipoprotein profile and nutritional physiological metabolism of fat in the fish body, the difference of serum lipoprotein profile between cultured and wild red sea bream Pagrus major was examined by disc electrophoresis and density gradient ultracentrifugation. The low density lipoprotein (LDL) level was higher in cultured red sea bream than in wild red sea bream. In the high density lipoprotein (HDL), though the HDL₃ level was similar between both groups, the HDL₂ level was remarkably lower in cultured fish than in wild fish. Both the levels of thickness coefficient and fat in internals within the limits of macroscopic observation were significantly higher in cultured fish than in wild fish, and the levels of triglyceride and cholesterol contents in serum also exhibited a higher tendency in cultured fish. From these findings, it was suggested that in cultured fish, the nutritional physiological condition of fat in the fish body might be unbalanced toward fat accumulation. Furthermore, although the function of HDL₂ in relation to fat carrier in serum of fish could not be clarified, it was considered that the HDL₂ might be not specially concerned with fat accumulation.

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A cDNA encoding a rainbow trout homologue of mammalian heart-type FABP was first isolated. Its sequence information was obtained from nucleotides (nt)-53 to +597, excluding the poly (A) tail, and an open reading frame of 399 nt was shown coding for 133 amino acids. A polyadenylation consensus signal was found 24 nt upstream from poly (A) tail. The coding sequence was interrupted by three introns of 2, 218, 891 (887) and 137 nt. Mammalian heart-type FABP has been known to be distributed in various tissues, except for liver, while reverse transcription-polymerase chain reaction showed high expression of heart-type FABP in liver, heart, muscle, intestine and brain of rainbow trout. The expression of heart-type FABP in fish liver seemed to be associated with lipid accumulation.

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Eel hepatocytes actively syntesize and secrete a lipoprotein. It consists of about 60% triacylglyceroi and 10% protein, and main apolipoproteins are apoB and apoA. Its average particle size is 750 nm. These characteristics show it is a chylomicron llike lipoprotein. Thyroxin (T 4) at 10^-8M activates the lipid synthesis in the hepatocytes and increases hepatocellular lipid. The synthesis of the chyloimicron Ilike lipoprotein is also stimulated by T 4, but T 4 does not affect the synthesis of other secreted proteins. T 4 specifically stimulates the synthesis of the lipoprotein. Though docosahexaenoic acid (DHA) stimulates lipid synthesis in the hepatocytes, DHA inhibits the synthesis and secretion of the lipoprotein. However, DHA does not in hibit the synthesis of other secreted proteins. DHA specifically in hibits the synthesis and secretion of the lipoprotein, but eicosapentaenoic acid does not in hibit HDL specifically binds HDL -binding protein (HBP) of the plasma membrane. The binding of HDL to the hepatocytes is saturated at concentrations over 100 μg HDL/ml and Kd value is 20 μg HDL/ml. HDL and apoAl are ligands of HBP. We suppose that HDL and free apoAl in eel serum take part in lipid transport through the mediation of the HBP.

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