Effect of dietary ascorbic acid on growth and non-specific immune responses of tiger puffer, Takifugu rubripes

We report nutritional physiology and non-specific immune responses of ascorbic acid (AA) in puffer fish for the first time. This study aimed to examine the essentiality and requirements of AA in diets for the tiger puffer, Takifugu rubripes based on growth performance, liver AA and bone collagen concentration, and non-specific immune responses. Five casein-gelatin based semi-purified diets were formulated to contain five graded levels of l-ascorbyl-2-monophosphate at 0, 40, 80, 160 and 700mg/kg (designated as AMP0, AMP40, AMP80, AMP160 and AMP700, respectively) and fed to triplicate groups of fish. After 10weeks of feeding trial, growth performances of fish (initial body weight, 35g) fed the AMP0 were significantly lower compared to that of fish fed diets supplemented with AMP. The fish fed the AMP0 diet also exhibited significantly lower hematocrit, condition factor and hepatosomatic index compared to the fish fed diets supplemented with AMP. Phagocytic activity (NBT assay) was significantly lower in fish fed the AMP0 diet than in fish fed the AMP containing diets. Plasma lysozyme activity of fish fed the AMP80 and AMP160 was significantly higher than that of fish fed the AMP0. Dietary supplementation of AMP significantly increased the liver superoxide dismutase in the fish. Myeloperoxidase activity of fish fed the AMP0 was significantly lower compared to that of fish fed the AMP containing diets. Bone collagen level tended to increase numerically and total AA concentration in liver of fish was significantly increased in a dose dependent manner by the supplementation of AMP. Therefore, tiger puffer requires exogenous ascorbic acid and the optimum dietary level could be 29mg AA/kg diet for normal growth and physiology. Dietary AA concentration over 82mg/kg could be required to enhance non-specific immune responses of the fish. However, it does not seem that the fish needs an overdose of dietary AA (>160mg/kg) for better non-specific immune responses.     

Developmental study of the shortened spinal cord in the adult tiger puffer fish, Takifugu rubripes (Teleostei)

ABSTRACT The spinal cords of vertebrates are generally divided into the cord proper and the minute filum terminale. While the spinal cord extends the entire length of the vertebral canal in the adult tiger puffer, Takifugu rubripes, the cord proper is greatly reduced in length and almost all of the canal is occupied by the filum terminale, which is tape-like rather than thread-like. The dorsal and ventral roots of the spinal nerves extend, respectively, above and below the filum terminale; as a whole, these form a massive cauda equina. Supramedullary cells are found in the rostral half of the medulla oblongata caudal to the cerebellum. In 4-mm long tiger puffers, the spinal cord is cylindrical and supramedullary cells are found in the rostral half of the cord. In 7-mm puffers, the longitudinally arranged ventral roots appear ventrally in the middle portion of the spinal cord. In 15-mm puffers, the dorsal and ventral roots run longitudinally along the spinal cord and have noticeably increased in number. Supramedullary cells are located in the rostral 15% of the cord. In 21-mm puffers, the spinal cord in large part becomes dorsoventrally flattened. In 30-mm puffers, the spinal cord becomes much flatter, and supramedullary cells now are located mainly in the medulla oblongata. These observations indicate that formation of the shortened spinal cord proper is due to at least two developmental processes. First, the elongation of the spinal cord proper is remarkably less than that of the vertebral canal. Second, the bulk of the spinal cord proper is translocated to the cranial cavity, where it is transformed into part of the medulla oblongata.     

Monoclonal antibodies recognising serum immunoglobulins and surface immunoglobulin-positive cells of puffer fish, torafugu (Takifugu rubripes)

Immunoglobulin of the torafugu, Takifugu rubripes, was purified by a combination of precipitation by low ionic strength dialysis and gel filtration. The Ig was used to immunise mice for the production of monoclonal antibody (MAb). Supernatants of hybridoma cultures were screened by enzyme-linked immunosorbent assay using purified-torafugu Ig-coated plates, and two stable hybridomas producing MAbs against torafugu Ig were obtained. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis under reducing conditions and Western blotting indicated that one MAb (16F3) was specific for the deglycosylated heavy chain of torafugu, and the other MAb (4H5) did not bind to the reduced Ig, suggesting that 4H5 recognised the higher-order structure of Ig. Under non-reduced conditions, both MAbs recognised mainly a 750 kDa band and also minor bands of 672, 410 and 205 kDa. MAb 16F3- and 4H5-primed magnetic beads (Dynabeads) adsorbed 84.9+/-3.3% and 63.6+/-4.4% of the torafugu Ig, respectively. The Ig adsorbed by MAb 16F3-primed Dynabeads was reactive to 4H5 on immunoblotting, and vice versa, indicating that the epitopes for both MAbs are held on the same Ig molecule. Both of these MAbs cross-reacted extensively with the Ig of other Takifugu species, but not with other genus. The MAbs were used to identify surface Ig-positive lymphocytes in the spleen, pronephros, peripheral blood and thymocytes of torafugu by flow cytometry. Flow cytometric analysis of the cells in the lymphocyte-enriched fraction revealed that 50.2+/-6.9% in the PBL, 11.8+/-1.7% in the mesonephros, 13.3+/-2.1% in the pronephros, 42.5+/-4.3% in the spleen and 3.2+/-0.6% in thymus were reactive to 4H5 or 16F3.     

Molecular cloning and expression of gelatinases (MMP-2 and MMP-9) in the pufferfish Takifugu rubripes

To determine the metabolism location of the extra-cellular matrix proteins in fugu (Takifugu rubripes), we cloned the cDNAs of the fugu gelatinases, matrix metalloproteinase-2 (MMP-2) and MMP-9, and examined their expressions in various adult tissues using a quantitative real-time PCR. The expression profiles of fugu gelatinases were different among tissues. FgMMP-9 mRNA was abundant in tissues that contain blood cells abundantly where fgMMP-2 mRNA was little expressed. We also examined the expression of these genes in fugu embryos during development using a whole mount in situ hybridization. Fugu MMP-2 mRNA was expressed in the pharyngeal area and mesenchyme in embryos at 80 hours post fertilization (hpf). While fugu MMP-9 mRNA was expressed in the vent at 140 hpf and the caudal end of the fin fold at 172 hpf. Although fugu MMP-2 mRNA was expressed in the pectoral fin bud at 120 hpf, fugu MMP-9 mRNA did not appear in this tissue until 10 days post fertilization (dpf). These data show expression profiles differ between the fugu gelatinases and suggest expressions of these genes are controlled at the matrix protein degradation site in fugu embryos during development.     

Experimental infection of several fish species with the causative agent of Kuchijirosho (snout ulcer disease) derived from the tiger puffer Takifugu rubripes.

Kuchijirosho (snout ulcer disease) is a fatal epidemic disease which affects the tiger puffer, Takifugu rubripes, a commercial fish species in Japan and Korea. To assess the possibility that non-tiger puffer fish can serve as reservoirs of infection, 5 fish species were challenged by infection with the extracts of Kuchijirosho-affected brains from cultured tiger puffer: grass puffer T. niphobles, fine-patterned puffer T. poecilonotus, panther puffer T. pardalis, red sea bream Pagrus major, and black rockfish Sebastes schlegeli. When slightly irritated, all these species, especially the puffer fish, exhibited typical signs of Kuchijirosho, i.e., erratic swimming, biting together and bellying out (swelling of belly), as generally observed in tiger puffers affected by Kuchijirosho. Although the mortalities of the 2 non-puffer species were lower, injection of the extracts prepared from the brains of both inoculated fish into tiger puffer resulted in death, indicating that the inoculated fish used in this experiment have the potential to be infected with the Kuchijirosho agent. Condensations of nuclei or chromatin in the large nerve cells, which is a major characteristic of Kuchijirosho, were histopathologically observed to some extent in the brains of all kinds of puffer fish species infected. These findings suggest that the virus can spread horizontally among wild and cultured puffers and even among fishes belonging to different orders.     

Neuropeptide Y (NPY) and depression: from animal studies to the human condition

Neuropeptide Y (NPY) is widely distributed throughout the central nervous system (CNS) and is one of the most conserved peptides in evolution, suggesting an important role in the regulation of basic physiological functions. In addition, both pre-clinical and clinical evidence have suggested that NPY, together with its receptors, may have a direct implication in several psychiatric disorders, including depression and related illnesses. NPY-like immunoreactivity and NPY receptors are expressed throughout the brain, with varying concentrations being found throughout the limbic system. Such brain structures have been repeatedly implicated in the modulation of emotional processing, as well as in the pathogenesis of depressive disorders. This review will concentrate on the distribution of NPY, its receptors, and the putative role played by this peptide in depressive illness based on both pre-clinical and clinical evidence.     

A trans-species missense SNP in Amhr2 is associated with sex determination in the tiger pufferfish, Takifugu rubripes (fugu)

Heterogametic sex chromosomes have evolved independently in various lineages of vertebrates. Such sex chromosome pairs often contain nonrecombining regions, with one of the chromosomes harboring a master sex-determining (SD) gene. It is hypothesized that these sex chromosomes evolved from a pair of autosomes that diverged after acquiring the SD gene. By linkage and association mapping of the SD locus in fugu (Takifugu rubripes), we show that a SNP (C/G) in the anti-Müllerian hormone receptor type II (Amhr2) gene is the only polymorphism associated with phenotypic sex. This SNP changes an amino acid (His/Asp384) in the kinase domain. While females are homozygous (His/His384), males are heterozygous. Sex in fugu is most likely determined by a combination of the two alleles of Amhr2. Consistent with this model, the medaka hotei mutant carrying a substitution in the kinase domain of Amhr2 causes a female phenotype. The association of the Amhr2 SNP with phenotypic sex is conserved in two other species of Takifugu but not in Tetraodon. The fugu SD locus shows no sign of recombination suppression between X and Y chromosomes. Thus, fugu sex chromosomes represent an unusual example of proto-sex chromosomes. Such undifferentiated X-Y chromosomes may be more common in vertebrates than previously thought.     

Characterization and tissue distribution of multiple agouti-family genes in pufferfish, Takifugu rubripes

Four types of agouti-family genes (AGRP1, AGRP2, ASIP1 and ASIP2) were obtained from torafugu, Takifugu rubripes. Their characterization and structure were analyzed to elucidate the relationship among the torafugu agouti-family genes. Both AGRP1 and AGRP2 showed genomic synteny with the human AGRP gene. Phylogenetic tree analysis showed that AGRP1 formed a cluster with human AGRP. We inferred that torafugu AGRP1 and AGRP2 are orthologs of human AGRP and that they are paralogous genes derived from genome duplication occurred in the teleost phylogeny. Torafugu ASIP1 showed genomic synteny with the human ASIP, but ASIP2 did not. The ASIP1 expression level was about five times higher in the white ventral skin than in the black dorsal skin. Therefore, we concluded that torafugu ASIP1 is an ortholog of human ASIP, nevertheless, we are unable to determine if torafugu ASIP2 is a paralog of ASIP1 or not.     

Neuropeptide Y receptor(s) mediating feeding in the rat: characterization with antagonists

The present study evaluated the effect of the neuropeptide Y (NPY) Y1 receptor antagonists BIBO 3304 and SR 120562A and of the Y5 receptor antagonists JCF 104, JCF 109, and CGP 71683A on feeding induced either by NPY or food deprivation. In a preliminary experiment, NPY was injected into the third cerebroventricle (3V) at doses of 0.07, 0.15, 0.3, or 0.6 nmol/rat. The dose of 0.3 nmol/rat, which produced a cumulative 2-h food intake of 11.2 +/- 1.9 g/kg body weight, was chosen for the following experiments. The antagonists were injected in the 3V 1 min before NPY. The Y1 receptor antagonist BIBO 3304 significantly inhibited NPY-induced feeding at doses of 1 or 10 nmol/rat. The Y1 receptor antagonist SR 120562A, at the dose of 10 but not of 1 nmol/rat, significantly reduced the hyperphagic effect of NPY, 0.3 nmol/rat. The Y5 receptor antagonists JCF 104 and JCF 109 (1 or 10 nmol/rat) and CGP 71683A (10 or 100 nmol/rat) did not significantly modify the effect of NPY, 0.3 nmol/rat. However, JCF 104 (10 nmol/rat) and CGP 71683A (100 nmol/rat), but not JCF 109 (10 nmol/rat), significantly reduced food intake during the interval from 2 to 4 h after injection of a higher dose, 0.6 nmol/rat, of NPY. Feeding induced by 16 h of food deprivation was significantly reduced by the Y1 receptor antagonist BIBO 3304 (10 nmol/rat), but it was not significantly modified by the same dose of SR 120562A or JCF 104. These findings support the idea that the hyperphagic effect of NPY is mainly mediated by Y1 receptors. The results obtained with JCF 104 and CGP 71683A suggest that Y5 receptors may have a modulatory role in the maintenance of feeding induced by rather high doses of NPY after the main initial feeding response.     

Neuropeptide Y (NPY) functional group mimetics: design, synthesis, and characterization as NPY receptor antagonists

N,N′-bis-[2-N-(O-2,6-dichlorobenzyl-L-tyrosyl)aminoethylguanyl]cystamine 3 and N,N′-bis-[2-N-(O-2,6-dichlorobenzyl-L-tyrosyl)aminoethyl]-1,6-hexanediguanidine 4 have been designed as neuropeptide Y (NPY) functional group mimetics. Both 3 and 4 displace N-[propionyl-³H]-NPY from rat brain binding sites, and are NPY receptor antagonists in rat femoral artery ring segments.     

Cloning, expression, and characterization of TNFSF14 (LIGHT) gene in mefugu, Takifugu obscurus

Tumor necrosis factor receptor-associated factor 6 (TRAF6), which plays an important role in inflammation and immune response, is an essential adaptor protein for the NF-κB (nuclear factor κB) signaling pathway. Recent studies have shown that TRAF6 played an important role in tumorigenesis and invasion by suppressing NF-κB activation. However, up to now, the biologic role of TRAF6 in glioma has still remained unknown. To address the expression of TRAF6 in glioma cells, four glioma cell lines (U251, U-87MG, LN-18, and U373) and a non-cancerous human glial cell line SVG p12 were used to explore the protein expression of TRAF6 by Western blot. Our results indicated that TRAF6 expression was upregulated in human glioma cell lines, especially in metastatic cell lines. To investigate the role of TRAF6 in cell proliferation, apoptosis, invasion, and migration of glioma, we generated human glioma U-87MG cell lines in which TRAF6 was either overexpressed or depleted. Subsequently, the effects of TRAF6 on cell viability, cell cycle distribution, apoptosis, invasion, and migration in U-87MG cells were determined with 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl tetrazolium bromide (MTT) assay, flow cytometry analysis, transwell invasion assay, and wound-healing assay. The results showed that knockdown of TRAF6 could decrease cell viability, suppress cell proliferation, invasion and migration, and promote cell apoptosis, whereas overexpression of TRAF6 displayed the opposite effects. In addition, the effects of TRAF6 on the expression of phosphor-NF-κB (p-p65), cyclin D1, caspase 3, and MMP-9 were also probed. Knockdown of TRAF6 could lower the expression of p-p65, cyclin D1, and MMP-9, and raise the expression of caspase 3. All these results suggested that TRAF6 might be involved in the potentiation of growth, proliferation, invasion, and migration of U-87MG cell, as well as inhibition of apoptosis of U-87MG cell by abrogating activation of NF-κB.     

cDNA cloning and mRNA expression of neuropeptide Y in orange spotted grouper, Epinephelus coioides

A full-length cDNA encoding the neuropeptide Y (NPY) was cloned from the hypothalamus of orange spotted grouper (Epinephelus coioides) by rapid amplification of cDNA ends approaches. The NPY cDNA sequence is 688 bp long and has an open reading frame of 300 bp encoding prepro-NPY with 99 amino acids. The deduced amino acid sequences contain a 28-amino-acids signal peptide followed by a 36-amino-acids mature NPY peptide. mRNA expression of NPY was determined using semi-quantitative RT-PCR followed by Southern blot analysis. NPY mRNA was expressed in olfactory bulb, telencephalon, pituitary, hypothalamus, optic tectum-thalamus, medulla oblongata, cerebellum and spinal cord. Low levels of NPY mRNA expression were found in retina, ovary and stomach, while much lower levels of expression were detected in liver, heart, gill, skin, anterior intestine, thymus and blood. No NPY mRNA expression was observed in unfertilized eggs, newly fertilized eggs, 16-cells stage and morula stage of the embryo and lower levels of expression were detected in the blastula, gastrula and neurula stages. It was highly expressed from lens formation stage to 52-day-old larval stage. NPY might be involved in the late embryonic and larval development of the orange spotted grouper.     

Molecular cloning and expression of pyruvate kinase from globefish (Fugu rubripes) skeletal muscle

A cDNA clone encoding pyruvate kinase (PK) was isolated from a skeletal muscle cDNA library of globefish (Fugu rubripes), which is a kind of lower vertebrate. The full-length cDNA of globefish skeletal muscle pyruvate kinase (FM-PK) is approximately 2 kb and encodes a protein comprising 530 amino acids. The FM-PK gene is spanning approximately 4.8 kb and consists of 11 exons. FM-PK mRNA was detected in muscle and heart using Northern blots. The recombinant FM-PK (rFM-PK) was expressed in a baculovirus-insect cell system and purified using ion-exchange chromatography. The purified rFM-PK was shown to exist a 230 kDa homotetramer composed of 57 kDa subunits. Gel filtration showed 230000 as the tetramer of the subunit. The apparent K(m) (or S(0.5)) and the Hill coefficient for phosphoenolpyruvate (PEP) and ADP are 0.14 mM, 1.3 and 0.30 mM 0.98 at pH 7.4, respectively, when the enzyme is saturated with the second substrate. The rFM-PK is strongly activated by fructose-1,6-bisphosphate, the apparent K(m) for PEP changes to 0.059 mM and the Hill coefficient to 1.1. ATP, which is the product of the enzyme reaction, inhibits activity. This is the first report to show the full-length cDNA and amino acid sequence of PK for a species of fish.     

Evidence for up-estuary transport of puffer Takifugu larvae (Tetraodontidae) in Ariake Bay, Japan

The present study determined whether puffer Takifugu rubripes and T. xanthopterus larvae use selective tidal stream transport (STST) for migration into the nursery area. The influence of the tidal cycle on the vertical distribution of Thkifugu larvae was investigated during a 24 h sampling period at one location off Shimabara Peninsula in Ariake Bay. Samples were collected in three depth layers, from near the sea floor to near the surface (5, 20 and 30 m depth). The change in vertical distribution in relation to tidal phase was not observed. This data did not support STST hypothesis. Diel vertical migration was observed irrespective of tidal phase, where larvae migrated to the middle layer during the night, and sank to the bottom layer during the day, however, larvae hardly emerged into the surface layer during the study period. In Ariake Bay, the residual current leads to a layered vertically stratified structure, in which surface water flows towards the mouth and the middle-bottom water flows toward inner part of the Bay. It is suggested that Takifugu larvae use not STST but residual currents for transport into the nursery ground, namely, undergoing nocturnal diel vertical migration in the water column between the middle layer and the bottom layer where the net flow is northward.