Roles of Gonadotropin Receptors in Sexual Development of Medaka.

The gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), are secreted from the pituitary and bind to the FSH receptor (FSHR) and LH receptor (LHR) to regulate gonadal development in vertebrates. Previously, using fshr-knockout (KO) medaka (Oryzias latipes), we demonstrated that FSH regulates ovarian development by elevating estrogen levels. However, the lhr-KO phenotype in medaka is poorly characterized. Here, we generated lhr-KO medaka using the transcription activator-like effector nuclease (TALEN) technique. We analyzed its phenotype and that of fshr-KO, lhr;fshr double-heterozygotes (double-hetero), and double-KO fish. All genetically male medaka displayed normal testes and were fertile, whereas fshr-KO and double-KO genetically female fish displayed small ovaries containing many early pre-vitellogenic oocytes and were infertile. Although lhr-KO genetically female fish had normal ovaries with full-grown oocytes, ovulation did not occur. Levels of 17α,20ß-dihydroxy-4-pregnen-3-one, which is required for meiotic maturation of oocytes and sperm maturation in teleost fish, were significantly decreased in all KO female medaka ovaries except for double-heteros. Further, 17ß-estradiol levels in fshr-KO and double-KO ovaries were significantly lower than those in double-heteros. These findings indicate that LH is necessary for oocyte maturation and FSH is necessary for follicle development, but that neither are essential for spermatogenesis in medaka.

Development of a Novel Enhanced Biosensor System for Real-Time Monitoring of Fish Stress Using a Self-Assembled Monolayer.

Wireless biosensor systems were developed in our lab for monitoring blood glucose concentrations in fish as an indicator of fish stress. However, uniform immobilization of the enzyme on the surface of the electrode is difficult, so the sensor response is typically reduced at a range of high glucose concentrations during the stress monitoring. In this study, we attempted to enhance sensor response by using a self-assembled monolayer-immobilized enzyme. Glucose oxidase was immobilized on a working electrode modified with a self-assembled monolayer. The proposed biosensor showed a good correlation between the output current and the glucose concentration range of 10⁻3500 mg dL-1 under an optimized working condition. The dynamic measurement range of this newly developed sensor is significantly improved, especially over a high concentration range, which helps the sensor to achieve better performance in dramatic changes in the stress response of fish. In addition, we used biological samples from test fish and obtained a good correlation coefficient between the sensor output current and the glucose concentration using a conventional method. The proposed wireless biosensor system was also applied to monitor fish stress responses in real time through different stressors and to obtain some precise data that reflect real fish stress responses.

Novel method for mass producing genetically sterile fish from surrogate broodstock via spermatogonial transplantation†.

A stable system for producing sterile domesticated fish is required to prevent genetic contamination to native populations caused by aquaculture escapees. The objective of this study was to develop a system to mass produce stock for aquaculture that is genetically sterile by surrogate broodstock via spermatogonial transplantation (SGTP). We previously discovered that female medaka carrying mutations on the follicle-stimulating hormone receptor (fshr) gene become sterile. In this study, we demonstrated that sterile hybrid recipient females that received spermatogonia isolated from sex-reversed XX males (fshr (-/-)) recovered their fertility and produced only donor-derived fshr (-) X eggs. Natural mating between these females and fshr (-/-) sex-reversed XX males successfully produced large numbers of sterile fshr (-/-) female offspring. In conclusion, we established a new strategy for efficient mass production of sterile fish. This system can be applied to any aquaculture species for which SGTP and methods for producing sterile recipients can be established.

Comprehensive validation of T- and B-cell deficiency in rag1-null zebrafish: Implication for the robust innate defense mechanisms of teleosts.

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.

New approach for monitoring fish stress: A novel enzyme-functionalized label-free immunosensor system for detecting cortisol levels in fish.

Fishes display a wide variation in their physiological responses to stress, which is clearly evident in the plasma corticosteroid changes, chiefly cortisol levels in fish. As a well-known indicator of fish stress, a simple and rapid method for detecting cortisol changes especially sudden increases is desired. In this study, we describe an enzyme-functionalized label-free immunosensor system for detecting fish cortisol levels. Detection of cortisol using amperometry was achieved by immobilizing both anti-cortisol antibody (selective detection of cortisol) and glucose oxidase (signal amplification and non-toxic measurement) on an Au electrode surface with a self-assembled monolayer. This system is based on the maximum glucose oxidation output current change induced by the generation of a non-conductive antigen-antibody complex, which depends on the levels of cortisol in the sample. The immunosensor responded to cortisol levels with a linear decrease in the current in the range of 1.25-200ngml-1 (R=0.964). Since the dynamic range of the sensor can cover the normal range of plasma cortisol in fish, the samples obtained from the fish did not need to be diluted. Further, electrochemical measurement of one sample required only ~30min. The sensor system was applied to determine the cortisol levels in plasma sampled from Nile tilapia (Oreochromis niloticus), which were then compared with levels of the same samples determined using the conventional method (ELISA). Values determined using both methods were well correlated. These findings suggest that the proposed label-free immunosensor could be useful for rapid and convenient analysis of cortisol levels in fish without sample dilution. We also believe that the proposed system could be integrated in a miniaturized potentiostat for point-of-care cortisol detection and useful as a portable diagnostic in fish farms in the future.

TALENs-mediated gene disruption of myostatin produces a larger phenotype of medaka with an apparently compromised immune system.

Although myostatin, a suppressor of skeletal muscle development and growth, has been well studied in mammals, its function in fish remains unclear. In this study, we used a popular genome editing tool with high efficiency and target specificity (TALENs; transcription activator-like effector nucleases) to mutate the genome sequence of myostatin (MSTN) in medaka (Oryzias latipes). After the TALEN pair targeting OlMyostatin was injected into fertilized medaka eggs, mutant G0 fish carrying different TALENs-induced frameshifts in the OlMSTN coding sequence were mated together in order to transmit the mutant sequences to the F1 generation. Two F1 mutants with frameshifted myostatin alleles were then mated to produce the F2 generation, and these F2 OlMSTN null (MSTN(-/-)) medaka were evaluated for growth performance. The F2 fish showed significantly increased body length and weight compared to the wild type fish at the juvenile and post-juvenile stages. At the post-juvenile stage, the average body weight of the MSTN(-/-) medaka was ∼25% greater than the wild type. However, we also found that when the F3 generation were challenged with red spotted grouper nervous necrosis virus (RGNNV), the expression levels of the interferon-stimulated genes were lower than in the wild type, and the virus copy number was maintained at a high level. We therefore conclude that although the MSTN(-/-) medaka had a larger phenotype, their immune system appeared to be at least partially suppressed or undeveloped.

Leptin receptor-deficient (knockout) medaka, Oryzias latipes, show chronical up-regulated levels of orexigenic neuropeptides, elevated food intake and stage specific effects on growth and fat allocation.

The first studies that identified leptin and its receptor (LepR) in mammals were based on mutant animals that displayed dramatic changes in body-weight and regulation of energy homeostasis. Subsequent studies have shown that a deficiency of leptin or LepR in homoeothermic mammals results in hyperphagia, obesity, infertility and a number of other abnormalities. The physiological roles of leptin-mediated signaling in ectothermic teleosts are still being explored. Here, we produced medaka with homozygous LepR gene mutation using the targeting induced local lesions in a genome method. This knockout mutant had a point mutation of cysteine for stop codon at the 357th amino acid just before the leptin-binding domain. The evidence for loss of function of leptin-mediated signaling in the mutant is based on a lack of response to feeding in the expression of key appetite-related neuropeptides in the diencephalon. The mutant lepr−/− medaka expressed constant up-regulated levels of mRNA for the orexigenic neuropeptide Ya and agouti-related protein and a suppressed level of anorexigenic proopiomelanocortin 1 in the diencephalon independent of feeding, which suggests that the mutant did not possess functional LepR. Phenotypes of the LepR-mutant medaka were analyzed in order to understand the effects on food intake, growth, and fat accumulation in the tissues. The food intake of the mutant medaka was higher in post-juveniles and adult stages than that of wild-type (WT) fish. The hyperphagia led to a high growth rate at the post-juvenile stage, but did not to significant alterations in final adult body size. There was no additional deposition of fat in the liver and muscle in the post-juvenile and adult mutants, or in the blood plasma in the adult mutant. However, adult LepR mutants possessed large deposits of visceral fat, unlike in the WT fish, in which there were none. Our analysis confirms that LepR in medaka exert a powerful influence on the control on food intake. Further analyses using the mutant will contribute to a better understanding of the role of leptin in fish. This is the first study to produce fish with leptin receptor deficiency.

Homologue gene of bile acid transporters ntcp, asbt, and ost-alpha in rainbow trout Oncorhynchus mykiss: tissue expression, effect of fasting, and response to bile acid administration.

Bile acid transporters belonging to the SLC10A protein family, Na+ taurocholate cotransporting polypeptide (NTCP or SLC10A1), apical sodium-dependent bile salt transporter (ASBT or SLC10A2), and organic solute transporter alpha (Ost-alpha) have been known to play critical roles in the enterohepatic circulation of bile acids in mammals. In this study, ntcp, asbt, and ost-alpha-1/-2 cDNA were cloned, their tissue distributions were characterized, and the effects of fasting and bile acid administration on their expression were examined in rainbow trout Oncorhynchus mykiss. The structural characteristics of Ntcp, Asbt, and Ost-alpha were well conserved in trout, and three-dimensional structure analysis showed that Ntcp and Asbt were similar to each other. Tissue distribution analysis revealed that trout asbt was primarily expressed in the hindgut, while ntcp expression occurred in the brain, and ost-alpha-1/-2 was mainly expressed in the liver or ovary. Although asbt and ost-alpha-1 mRNA levels in the gut increased in response to fasting for 4 days, ost-alpha-1 expression in the liver decreased. Similarly, bile acid administration increased asbt and ost-alpha-1 expression levels in the gut, while those of ntcp and ost-alpha-2 in the liver decreased. These results suggested that the genes asbt, ntcp, and ost-alpha are involved in bile acid transport in rainbow trout.

Design, evaluation, and screening methods for efficient targeted mutagenesis with transcription activator-like effector nucleases in medaka.

Genome editing using engineered nucleases such as transcription activator-like effector nucleases (TALENs) has become a powerful technology for reverse genetics. In this study, we have described efficient detection methods for TALEN-induced mutations at endogenous loci and presented guidelines of TALEN design for efficient targeted mutagenesis in medaka, Oryzias latipes. We performed a heteroduplex mobility assay (HMA) using an automated microchip electrophoresis system, which is a simple and high-throughput method for evaluation of in vivo activity of TALENs and for genotyping mutant fish of F1 or later generations. We found that a specific pattern of mutations is dominant for TALENs harboring several base pairs of homologous sequences in target sequence. Furthermore, we found that a 5' T, upstream of each TALEN-binding sequence, is not essential for genomic DNA cleavage. Our findings provide information that expands the potential of TALENs and other engineered nucleases as tools for targeted genome editing in a wide range of organisms, including medaka.

New approach for fish breeding by chemical mutagenesis: establishment of TILLING method in fugu (Takifugu rubripes) with ENU mutagenesis.

BACKGROUND: In fish breeding, it is essential to discover and generate fish exhibiting an effective phenotype for the aquaculture industry, but screening for natural mutants by only depending on natural spontaneous mutations is limited. Presently, reverse genetics has become an important tool to generate mutants, which exhibit the phenotype caused by inactivation of a gene. TILLING (Targeting Induced Local Lesions IN Genomes) is a reverse genetics strategy that combines random chemical mutagenesis with high-throughput discovery technologies for screening the induced mutations in target genes. Although the chemical mutagenesis has been used widely in a variety of model species and also genetic breeding of microorganisms and crops, the application of the mutagenesis in fish breeding has been only rarely reported. RESULTS: In this study, we developed the TILLING method in fugu with ENU mutagenesis and high-resolution melting (HRM) analysis to detect base pair changes in target sequences. Fugu males were treated 3 times at weekly intervals with various ENU concentrations, and then the collected sperm after the treatment was used to fertilize normal female for generating the mutagenized population (F1). The fertilization and the hatching ratios were similar to those of the control and did not reveal a dose dependency of ENU. Genomic DNA from the harvested F1 offspring was used for the HRM analysis. To obtain a fish exhibiting a useful phenotype (e.g. high meat production and rapid growth), fugu myostatin (Mstn) gene was examined as a target gene, because it has been clarified that the mstn deficient medaka exhibited double-muscle phenotype in common with MSTN knockout mice and bovine MSTN mutant. As a result, ten types of ENU-induced mutations were identified including a nonsense mutation in the investigated region with HRM analysis. In addition, the average mutation frequency in fugu Mstn gene was 1 mutant per 297 kb, which is similar to values calculated for zebrafish and medaka TILLING libraries. CONCLUSIONS: These results demonstrate that the TILLING method in fugu was established. We anticipate that this TILLING approach can be used to generate a wide range of mutant alleles, and be applicable to many farmed fish that can be chemically mutagenized.

Postprandial response and tissue distribution of the bile acid synthesis-related genes, cyp7a1, cyp8b1 and shp, in rainbow trout Oncorhynchus mykiss.

In mammals, cholesterol 7α-hydroxylase (CYP7A1) and sterol 12α-hydroxylase (CYP8B1) are rate-limiting enzymes in bile acid synthesis. In addition, a small heterodimer partner (SHP) is also known to inhibit bile acid synthesis via the suppression of CYP7A1 and CYP8B1 expression. However, little information is currently available regarding primary structure of the genes involved in bile acid synthesis in fish. We therefore cloned cyp7a1, cyp8b1 and shp genes from rainbow trout and obtained cDNAs encoding two isoforms each of Cyp7a1 (-1 and -2), Cyp8b1 (-1 and -2) and Shp (-1 and -2). Both cyp7a1-1 and -2 encoded proteins of 512 amino acids. Trout cyp7a1-1 was expressed not only primarily in the kidney, pyloric caecum and mid-gut, but also weakly in the liver, eye, gill and ovary. cyp7a1-2 was highly expressed in the liver, pyloric caecum and mid-gut. cyp8b1-1 and -2, which encoded proteins of 512 and 509 amino acids, respectively, were principally expressed in the liver. Both shp-1 and -2, which encoded proteins of 288 and 290 amino acids, respectively, were strongly expressed in the liver, but shp-2 was also highly expressed in the gallbladder and digestive tract. The temporal changes in the expression of cyp7a1-1/-2, cyp8b1-1/-2 and shp-1/-2 in the liver were assessed after consumption of a single meal. Expression of cyp7a1-1/-2 and cyp8b1-1/-2 increased within 3h post feeding (hpf) when the stomach was still approximately 84% full and the gallbladder was almost completely empty. Although the expression of shp-1 did not change after feeding, the expression pattern of shp-2 was inversely related to the expression patterns of cyp7a1-1/-2 and cyp8b1-1/-2. Specifically, shp-2 expression decreased until 3 hpf before returning to initial levels at 24 hpf. These findings suggest that Cyp7a1s/8b1s and Shp-2 function antagonistically in bile acid synthesis in rainbow trout.

Prevalence of red sea bream iridovirus among organs of Japanese amberjack (Seriola quinqueradiata) exposed to cultured red sea bream iridovirus.

Red sea bream iridovirus (RSIV) is a representative of the genus Megalocytivirus which causes severe disease to aquaculture fish, mainly in Japan and South-east Asia. However, information to assess the viral kinetics of RSIV in fish is limited since reports on experimental infection by the immersion route, which is the natural infection route, are scarce. In this study, a method to evaluate the titre of RSIV was first developed. Experimental infections were continuously performed using RSIV cell culture as the inoculum to juvenile Japanese amberjack (Seriola quinqueradiata) (initial body weight 12.2 g) by immersion at three different concentrations. In addition, to investigate the prevalence of the virus among the organs of experimentally infected fish, viral DNA was measured at selected times by the real-time PCR method following viral inoculation by immersion. The developed titration method showed a 10(2) increase in sensitivity compared with the conventional method. We demonstrated that grunt fin cells can be used for continuous passage of RSIV. In the experimental infection, fish which were intraperitoneally injected with the RSIV cell culture or immersed with RSIV cell culture at 10(-2) and 10(-3) dilutions showed cumulative mortalities of 100 %. The results of measurements of the viral DNA of several organs from infected fish strongly suggest that the spleen is the target organ of RSIV in Japanese amberjack. Since the viral genome was detected from all the tested organs of two of five surviving fish which appeared to completely recover from the disease, it is suggested that these fish may become carriers.

Adaptive immune response to Edwardsiella tarda infection in ginbuna crucian carp, Carassius auratus langsdorfii.

Edwardsiella tarda is an intracellular pathogen that causes edwardsiellosis in fish. Although cell-mediated immunity and innate immunity play a major role in protection against intracellular bacterial infection in mammals, their importance in protecting fish against E. tarda infection remain unclear. In this study, we examined cell-mediated and humoral immune responses in ginbuna crucian carp (Carassius auratus langsdorfii) after E. tarda infection. Innate immunity was observed to be the principal immune system for eliminating the majority of E. tarda, while a proportion of the bacteria might be resistant to its bactericidal activity. Bacterial clearance in kidney and spleen was also observed following higher cytotoxic activities of cytotoxic T lymphocytes (CTLs) and increased numbers of CD8α(+) cells, suggesting that CTLs might contribute to the elimination of E. tarda-infected cells with specific cytotoxicity. On the other hand, E. tarda-specific antibody titers did not increase until after bacterial clearance, indicating that induction of humoral immunity would be too late to provide protection against infection. Overall, these data suggest that both cell-mediated immunity and innate immunity may play important roles in the protection against intracellular bacterial infection, as they do in mammals. Our study would also contribute toward the understanding of immune responses that provide protection against other intracellular pathogens.

Myostatin-deficient medaka exhibit a double-muscling phenotype with hyperplasia and hypertrophy, which occur sequentially during post-hatch development.

Myostatin (MSTN) functions as a negative regulator of skeletal muscle mass. In mammals, MSTN-deficient animals result in an increase of skeletal muscle mass with both hyperplasia and hypertrophy. A MSTN gene is highly conserved within the fish species, allowing speculation that MSTN-deficient fish could exhibit a double-muscled phenotype. Some strategies for blocking or knocking down MSTN in adult fish have been already performed; however, these fish show either only hyperplastic or hypertrophic growth in muscle fiber. Therefore, the role of MSTN in fish myogenesis during post-hatch growth remains unclear. To address this question, we have made MSTN-deficient medaka (mstnC315Y) by using the targeting induced local lesions in a genome method. mstnC315Y can reproduce and have the same survival period as WT medaka. Growth rates of WT and mstnC315Y were measured at juvenile (1-2wk post-hatching), post-juvenile (3-7wk post-hatching) and adult (8-16wk post-hatching) stages. In addition, effects of MSTN on skeletal muscle differentiation were investigated at histological and molecular levels at each developmental stage. As a result, mstnC315Y show a significant increase in body weight from the post-juvenile to adult stage. Hyper-morphogenesis of skeletal muscle in mstnC315Y was accomplished due to hyperplastic growth from post-juvenile to early adult stage, followed by hypertrophic growth in the adult stage. Myf-5 and MyoD were up-regulated in mstnC315Y at the hyperplastic growth phase, while myogenin was highly expressed in mstnC315Y at the hypertrophic growth phase. These indicated that MSTN in medaka plays a dual role for muscle fiber development. In conclusion, MSTN in medaka regulates the number and size of muscle fiber in a temporally-controlled manner during posthatch growth.

Expression profiles of cytokines released in intestinal epithelial cells of the rainbow trout, Oncorhynchus mykiss, in response to bacterial infection.

To determine whether fish intestinal epithelial cells (IECs) contribute to mucosal immunity, we established a method for isolating IECs from the rainbow trout Oncorhynchus mykiss and examined cytokine production in these cells. Components of the intestinal epithelium were released by incubation of intestinal pieces with 1mM dithiothreitol (DTT)/ethylenediamine tetraacetic acid (EDTA). The IEC-rich fraction (purity >90%; survival rate approximately 95%) was obtained by centrifugation on a 35%/40% Percoll gradient, followed by magnetic cell sorting using an anti-trout IgM antiserum. The gene expression profiles of 14 cytokines in trout IECs were investigated after culturing the cells for 6h with or without the pathogenic bacterium Aeromonas salmonicida. Trout IECs could produce several cytokines, of which IL-1beta and TNFalpha2 were upregulated when the cells were stimulated with live A. salmonicida. Immunohistochemical analyses with the anti-trout TNF antibody confirmed that the TNF protein was present in the IECs of trout that were intra-anally challenged with live A. salmonicida. These results show that trout IECs are an important trigger of the intestinal immune system. Further, formalin-killed A. salmonicida, conditioned medium of this bacterium, or live nonpathogenic Escherichia coli could not upregulate the expression of these cytokines. These results indicate that the production of inflammatory cytokines by IECs is caused by the adhesion of A. salmonicida, but is not due to only simple ligand-receptor interactions between the surface molecules of IECs and the bacterium or in response to bacterial secretions.

Macrophage migration inhibitory factor (MIF) is essential for development of zebrafish, Danio rerio.

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.

Genomic organization and expression of CD8alpha and CD8beta genes in fugu Takifugu rubripes.

We have cloned cDNAs encoding the alpha and beta chains of CD8 from the tiger pufferfish (fugu), Takifugu rubripes. The cDNA sequences encode a putative leader peptide, extracellular immunoglobulin variable region-like domain, stalk region, transmembrane region, and cytoplasmic tail. A protein tyrosine kinase p56lck binding motif was not found in the putative fugu CD8alpha cytoplasmic tail. O-linked glycosylation sites were found in the stalk of both CD8 chains, suggesting possible stalk formation. Phylogenetic analysis showed that fugu CD8alpha and CD8beta chains cluster with other vertebrate CD8alpha and CD8beta chains, respectively. The fugu CD8 genes comprise six exons separated by five introns. The genes are tandemly aligned 3.6 kb apart and are in the same transcription orientation. Quantitative RT-PCR analysis demonstrated that fugu CD8 is expressed predominantly in lymphoid tissues. In situ hybridization showed that fugu CD8 genes are expressed in thymocytes and lymphocytes within lymphoid organs. Molecular characterization of CD8 in fish provides the basis for development of specific antibodies to identify T-cell subsets, as well as potentially important insights into the evolution of CD8 and the adaptive immunity.

Polymorphism of two very similar MHC class Ib loci in rainbow trout (Oncorhynchus mykiss).

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.

Expression profiles of TCRbeta and CD8alpha mRNA correlate with virus-specific cell-mediated cytotoxic activity in ginbuna crucian carp.

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.

Two interleukin (IL)-15 homologues in fish from two distinct origins.

Here, we report two distinct genes in teleosts that are homologous to interleukin (IL)-15. The two genes, isolated from fugu (Takifugu rubripes), resemble to mammalian IL-15 but differ from IL-2 and IL-21 in their amino acid sequences, the possessing of an extraordinary long signal peptide and more widespread tissue localization. In addition, multiple out-of-frame AUG codons, the negative translational regulators of mammalian IL-15 genes were also detected in the 5'-UTR of the two genes. Fugu IL-15 homologues also contain four conserved cysteines allowing the formation of two disulfide bridges along with four predicted alpha-helices. Genomic analysis showed that one of the fugu IL-15 homologues possessed six coding exons and exhibited a similar exon-intron organization and synteny structure to that of mammalian and chicken IL-15 genes. Conversely, the other fugu IL-15 homologue possesses four exons and exhibits a different synteny structure with that of IL-15, suggesting that the two genes were derived from two different origins. Moreover, the two genes also differ from each other in tissue localizations and in their expression in response to mitogens. The existence of these two IL-15 homologues in telesots was further supported by their characterization in zebrafish Danio rerio, and the green-spotted pufferfish Tetraodon nigroviridis. The discovery of two distinct IL-15 homologues in fish will assist investigations into the evolution of these genes and their relative contribution to the fish immune system.