Identification,Characterization, and Mapping of a Novel SNP Associated with Body Color Transparency in Juvenile Red Sea Bream (<Emphasis Type="Italic">Pagrus major</Emphasis>)

We previously reported a body color deformity in juvenile red sea bream, which shows transparency in the juvenile stage because of delayed chromatophore development compared with normal individuals, and this finding suggested a genetic cause based on parentage assessments. To conduct marker-assisted selection to eliminate broodstock inheriting the causative gene, developing DNA markers associated with the phenotype was needed. We first conducted SNP mining based on AFLP analysis using bulked-DNA from normal and transparent individuals. One SNP was identified from a transparent-specific AFLP fragment, which significantly associated with transparent individuals. Two alleles (A/G) were observed in this locus, and the genotype G/G was dominantly observed in the transparent groups (97.1%) collected from several production lots produced from different broodstock populations. A few normal individuals inherited the G/G genotype (5.0%), but the A/A and A/G genotypes were dominantly observed in the normal groups. The homologs region of the SNP was searched using a medaka genome database, and intron 12 of the Nell2a gene (located on chromosome 6 of the medaka genome) was highly matched. We also mapped the red sea bream Nell2a gene on the previously developed linkage maps, and this gene was mapped on a male linkage group, LG4-M. The newly found SNP was useful in eliminating broodstock possessing the causative gene of the body color transparency observed in juvenile stage of red sea bream.     

Comparison of UV-B tolerance between wild and hatchery-reared juveniles of red sea bream (Pagrus major) and black sea bream (Acanthopagrus schlegeli)

The amount of ultraviolet (UV)-B radiation reaching the sea surface has increased due to ozone depletion. Several laboratory studies have highlighted the negative impacts of UV radiation on fish using hatchery-reared specimens. However, potential differences in UV tolerance between wild and hatchery-reared fish have been given little consideration. Wild and reared juveniles of red sea bream and black sea bream were exposed to one of four different UV-B radiation levels (1.8; 1.1; 0.4; 0?W/m²) for 4?h. Survival rate was measured every 2?h for a period of 24?h (red sea bream) or 48?h (black sea bream) following exposure. Wild and reared juvenile red sea bream were characterized by similar survival rate, with survival declining to almost 0?% 24?h after exposure at the 1.1 and 1.8?W/m² levels. In black sea bream, wild individuals showed significantly higher survival than reared fish in levels 1.1 and 1.8?W/m². Melanophore density was also measured since melanin absorbs UV radiation. Wild black sea bream showed higher melanophore density compared to reared individuals, while no such difference was observed in red sea bream. We conclude that wild black sea bream juveniles acquire higher UV tolerance partly by increasing melanophore density through exposure to UV radiation. Our results indicate that the predicted impacts of UV radiation on fish populations solely based on experimentation with hatchery-reared specimens may be overestimated for some species.     

Inference of Potential Genetic Risks Associated with Large-Scale Releases of Red Sea Bream in Kanagawa Prefecture, Japan Based on Nuclear and Mitochondrial DNA Analysis

Since 1978, millions of hatchery-reared red sea bream (Pagrus major) juveniles have been released in Sagami Bay and Tokyo Bay in Kanagawa Prefecture, Japan. The stock enhancement program has contributed to total catch; however, no information regarding the genetic interactions with wild counterparts is available. Here, we combined 15 microsatellite loci and mitochondrial D-loop sequencing to characterize the genetic resources of red sea bream in Sagami Bay and Tokyo Bay and to elucidate the potential harmful genetic effects associated with fish releases. Both types of markers evidenced higher levels of genetic diversity in wild samples (SB and TB) compared with offspring before stocking (H07 and H08) as well as a hatchery-released sample recaptured in Sagami Bay (HR). Microsatellite F _ST estimates and Bayesian clustering analysis found significant genetic differences among samples (F _ST?=?0.013–0.054), except for the two wild samples (F _ST?=?0.002) and HR vs. H07 (F _ST?=?0.007). On the other hand, mitochondrial-based Ф _ST suggested haplotypic similarity between SB, H07, and HR. The low effective number of females contributing to the offspring over multiple generations may be responsible for the lack of haplotypic differentiation. Moreover, the putative hatchery origin to three fish (8 %) without deformity in the inter-nostril epidermis was inferred for the first time. Our results showed the usefulness of combining nuclear and mitochondrial markers to elucidate genetic interactions between hatchery-released and wild red sea bream and warned about potential harmful genetic effects should interbreeding takes place.     

Effects of Large-Scale Releases on the Genetic Structure of Red Sea Bream (Pagrus major,Temminck et Schlegel) Populations in Japan

Large-scale hatchery releases are carried out for many marine fish species worldwide; nevertheless, the long-term effects of this practice on the genetic structure of natural populations remains unclear. The lack of knowledge is especially evident when independent stock enhancement programs are conducted simultaneously on the same species at different geographical locations, as occurs with red sea bream (Pagrus major, Temminck et Schlegel) in Japan. In this study, we examined the putative effects of intensive offspring releases on the genetic structure of red sea bream populations along the Japanese archipelago by genotyping 848 fish at fifteen microsatellite loci. Our results suggests weak but consistent patterns of genetic divergence (F_ST = 0.002, p < 0.001). Red sea bream in Japan appeared spatially structured with several patches of distinct allelic composition, which corresponded to areas receiving an important influx of fish of hatchery origin, either released intentionally or from unintentional escapees from aquaculture operations. In addition to impacts upon local populations inhabiting semi-enclosed embayments, large-scale releases (either intentionally or from unintentional escapes) appeared also to have perturbed genetic structure in open areas. Hence, results of the present study suggest that independent large-scale marine stock enhancement programs conducted simultaneously on one species at different geographical locations may compromise native genetic structure and lead to patchy patterns in population genetic structure.     

Effects of exogenous cortisol and seawater adaptation on thyroid hormone receptors in the smolt stage of the sockeye salmon,Oncorhynchus nerka

The objective of this investigation was to quantify how thyroid hormone receptors of the sockeye salmon, Oncorhynchus nerka, respond to salinity changes from freshwater (FW) to seawater (SW) conditions. Thyroid hormone receptors (TRs) mRNA and protein expressions levels significantly increased when the fish were transferred from FW to SW, and the plasma T₃ and T₄ levels were significantly highest at 50 % SW and then maintained as control. Moreover, these parameters were significantly lower in the cortisol-injected groups than in the control. Hence, TRs, T₃, and T₄ may play a role in SW adaptation, when the fish migrate from FW to SW environments. We showed a negative correlation between cortisol and thyroid hormone levels, and a significant increase in plasma K⁺ levels in the kidney when the fish were transferred to SW, with levels being significantly lower in the cortisol-injected group. Hence, cortisol appears to be a stress hormone, and the plasma Na⁺ and Cl^- levels significantly increased when the fish were transferred to SW, with levels being significantly lower in the cortisol-injected group. These results indicate that cortisol modulates ion transportation in the plasma.     

Red sea bream iridoviral disease

The first outbreak of red sea bream iridoviral disease caused by red sea bream iridovirus (RSIV) was recorded in cultured red sea bream Pagrus major in Shikoku Island, Japan in 1990. Since 1991, the disease has caused mass mortalities of cultured marine fishes not only red sea bream but also many other species. The affected fish were lethargic and exhibited severe anemia, petechiae of the gills, and enlargement of the spleen. The causative agent was a large, icosahedral, cytoplasmic DNA virus classified as a member of the family Iridoviridae and was designated as red sea bream iridovirus (RSIV). The genome of RSIV is liner dsDNA and considered to be circularly permitted and terminally redundant like other iridoviruses. The length of physical map of RSIV genome is 112,415bp. An indirect immunofluorescence test with a monoclonal antibody and PCR are commonly used for the rapid diagnosis of RSIV infected fish in the field. For the control of this disease, a formalin-killed vaccine against red sea bream iridoviral disease was developed and now commercially available.     

Thyroid hormone divergence between two closely related but ecologically diverse cyprinid fish species (Cyprinidae)

Thyroid hormones (THs) are involved in regulation of many life history traits of fishes. Development, growth, reproduction and shaping the adult phenotypes were found to be regulated by THs. Regulatory evolution is one of the main factors driving morpho-ecological diversification and speciation. We tested a hypothesis whether closely related but ecologically diverged species may have a diverse level of THs. We compared seasonal levels of plasma concentrations of triiodothyronine (T₃) in two cyprinid species, benthophagous bream Abramis brama and zooplanktivorous blue bream Ballerus ballerus. Significant differences in T₃ concentrations were detected. B. ballerus had substantially reduced T₃ levels as compared to most other species of fish. Two hypotheses explaining the thyroid divergence are discussed. Despite differences in absolute T₃ concentrations, both species manifest similar seasonal cycling with increased plasma T₃ levels in warmer seasons and decreased T₃ in colder seasons. Several peaks of plasma T₃ level were registered: at pre-spawning periods, at mid-summer and in the early autumn. The pre-spawning peak noted in cyprinids was a novel observation. Significant positive correlations were detected between water temperature and plasma T₃ concentrations, as well as between the duration of daylight and T₃ plasma concentrations in both species.     

Juvenile bisexuality in the red sea bream,Pagrus major

The histology of the gonad of the red sea bream,Pagrus major, was examined in order to study the early gonadal development, sexual maturation and sex ratio in a natural population. A total of 1,117 fish between the ages of 4 months and 8 years were examined. Gonads of 4-month-old fish were either sexually undifferentiated with a central cavity, or ovarian in form. Gonads of 12- and 18-month-old fish were ovaries or bisexual gonads, while those of 2-year-old fish were ovaries, bisexual gonads or testes. Fish aged between 3 and 8 years had ovaries or testes, except for a few bisexual gonads found in 3- and 4-year-old fish. The chronological appearance of females, hermaphrodites and males in that order, and histological evidence, suggested that the testis originates from the ovary via a bisexual gonad in the juvenile stage. The sex ratio of females to males at the age of 2 years and over was about 1:1, suggesting that hermaphroditic red sea bream appear in about 50% of the juvenile population. The sexual pattern in this species, therefore, is concluded to be gonochorism with a bisexual juvenile stage.     

Organization of the lipoprotein lipase gene of red sea bream Pagrus major

Lipoprotein lipase (LPL) is a key enzyme of lipid deposition and metabolism. To investigate the mechanism of lipid deposition in fish, as a first step, we have characterized the LPL gene of a marine teleost red sea bream Pagrus major by cDNA and genomic structure analysis. The red sea bream LPL gene encodes 511 amino acids and spans approximately 6.3 kb of the genome. The coding region is organized into ten exons and nine introns. In comparison with the LPL of other animals, the deduced amino acid sequence shows a high degree of similarity with a conservation of functional domains, e.g. catalytic triad, N-glycosylation sites, lipid and heparin binding regions. The 1.1 kb of 5′ flanking region contains two CCAAT, sequences homologous to Oct-I site and response elements for hormones including glucocorticoid, insulin and thyroid hormone. The results of the present study will facilitate further study of the function and regulation of the LPL in non-mammalian vertebrates.     

Effectiveness of a vaccine against red sea bream iridoviral disease in a field trial test.

Since 1990, red sea bream iridovirus (RSIV) has caused high mortalities in the summertime in cultured red sea bream Pagrus major in southwest Japan. To establish control measures for red sea bream iridoviral disease (RSIVD), the effectiveness of a formalin-killed viral vaccine was evaluated in a field trial. Two groups each consisting of 1000 juvenile red sea bream were either intraperitoneally inoculated with vaccine (vaccinated group) or were not vaccinated (non-vaccinated group). After vaccination, the fish were held for 1 wk, then transferred to a marine net pen and observed for 12 wk. The cumulative mortalities caused by RSIVD in the vaccinated group or control group were 19.2 and 68.5%, respectively. Additionally, the presence of virus antigen in the spleen was investigated and body weight was measured 6 and 12 wk post vaccination. In the vaccinated group, viral antigen was not detected. The increase in body weight of vaccinated fish was significantly (p < 0.05) greater than that of control fish. These results suggest that the vaccine against RSIVD was effective in 1 field trial.     

Ontogeny of gene expression of group IB phospholipase A₂ isoforms in the red sea bream, Pagrus (Chrysophrys) major

The red sea bream (Pagrus major) was previously found to express mRNAs for two group IB phospholipase A₂ (PLA₂) isoforms, DE-1 and DE-2, in the digestive organs, including the hepatopancreas, pyloric caeca, and intestine. To characterize the ontogeny of the digestive function of these PLA₂s, the present study investigated the localization and expression of DE-1 and DE-2 PLA₂ genes in red sea bream larvae/juveniles and immature adults, by in situ hybridization. In the adults, DE-1 PLA₂ mRNA was expressed in pancreatic acinar cells. By contrast, DE-2 PLA₂ mRNA was detected not only in digestive tissues, such as pancreatic acinar cells, gastric glands of the stomach, epithelial cells of the pyloric caeca, and intestinal epithelial cells, but also in non-digestive ones, including cardiac and lateral muscle fibers and the cytoplasm of the oocytes. In the larvae, both DE-1 and DE-2 PLA₂ mRNAs first appeared in pancreatic tissues at 3 days post-hatching (dph) and in intestinal tissue at 1 dph, and expression levels for both gradually increased after this point. In the juvenile stage at 32 dph, DE-1 PLA₂ mRNA was highly expressed in pancreatic tissue, and DE-2 PLA₂ mRNA was detected in almost all digestive tissues, including pancreatic tissue, gastric glands, pyloric caeca, and intestine, including the myomere of the lateral muscles. In conclusion, both DE-1 and DE-2 PLA₂ mRNAs are already expressed in the digestive organs of red sea bream larvae before first feeding, and larvae will synthesize both DE-1 and DE-2 PLA₂ proteins.     

Absence of A Thyroid Phenotype in Sortilin-Deficient Mice

Objective: The Vps10p family member sortilin is expressed in thyroid epithelial cells where it contributes to recycling of the thyroid hormone precursor thyroglobulin (Tg), a process that is thought to render hormone release more effective. Here we investigated the functional impact of sortilin in the thyroid gland using sortilin-deficient mice.Methods: We measured free T₄, thyroid-stimulating hormone (TSH) and Tg serum levels and studied thyroid morphology in 14 sortilin-deficient (Sort1)^-/-and 12 wildtype (WT) mice.Results: Serum free T₄ levels did not differ between Sort1^-/-and WT females but were significantly lower in Sort1^-/-males compared with WT (P = .0424). Neither serum TSH nor Tg levels differed between Sort1^-/-and WT mice, regardless of sex. On the same line, no thyroid histology differences were observed.Conclusion: Our findings seem to exclude a role of sortilin in thyroid hormone secretion, although it is possible that the absence of sortilin may result in a thyroid phenotype if combined with other molecular defects of thyroid hormone synthesis and secretion or under iodine deficiency.Abbreviations: T₄ = thyroxine Sort1 = Sortilin 1 Tg = thyroglobulin TSH = thyroid-stimulating hormone WT = wild type     

Antioxidant responses and lipid peroxidation in gills and erythrocytes of fish (Rhabdosarga sarba) upon exposure to Chattonella marina and hydrogen peroxide: Implications on the cause of fish kills

Chattonella marina, a red tide or harmful algal bloom species, has caused mass fish kills and serious economic loss worldwide, and yet its toxic actions remain highly controversial. Previous studies have shown that this species is able to produce reactive oxygen species (ROS), and therefore postulated that ROS are the causative agents of fish kills. The present study investigates antioxidant responses and lipid peroxidation in gills and erythrocytes of fish (Rhabdosarga sarba) upon exposure to C. marina, compared with responses exposed to equivalent and higher levels of ROS exposure. Even though C. marina can produce a high level of ROS, gills and erythrocytes of sea bream exposed to C. marina for 1 to 6 h showed neither significant induction of antioxidant enzymes nor lipid peroxidation. Antioxidant responses and oxidative damage did not occur as fish mortality began to occur, yet could be induced upon exposure to artificially supplied ROS levels an order of magnitude higher. The result of this study implies that ROS produced by C. marina is not the principal cause of fish kills.     

Morphometric comparison and differentiation between artificially-released and wild red sea bream (Pagrus major)

The objective of this study is to develop a method of differentiation of hatchery-reared and artificially-released red sea bream (Pagrus major) from wild fish, based upon their morphometric differences. Morphometric measurements were done on fork length and 14 other characters. Among these charac)ters, significant differences between hatchery-reared and wild red sea bream were observed in body height, height at eye, eye diameter and upper jaw length. Discriminant functions were effective in differentiating artificial fish from wild fish.     

Characterization of Pseudomonas spp. Associated with Spoilage of Gilt-Head Sea Bream Stored under Various Conditions

The population dynamics of pseudomonads in gilt-head sea bream Mediterranean fish (Sparus aurata) stored under different conditions were studied. Phenotypic analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins were performed to identify a total of 106 Pseudomonas strains isolated from S. aurata stored under different temperatures (at 0, 10, and 20°C) and packaging conditions (air and a modified atmosphere of 40% CO₂-30% N₂-30% O₂). Pseudomonas lundensis was the predominant species, followed by Pseudomonas fluorescens, while Pseudomonas fragi and Pseudomonas putida were detected less frequently. Fluorescent Pseudomonas strains dominated under air conditions, while proteolytic and less lipolytic strains dominated under modified-atmosphere packaging. Different storage conditions appear to govern the selection of pseudomonads in gilt-head sea bream fish.     

Pathogenicity of Vibrio alginolyticus for Cultured Gilt-Head Sea Bream (Sparus aurata L.)

The in vivo and in vitro pathogenic activities of whole cells and extracellular products of Vibrio alginolyticus for cultured gilt-head sea bream were evaluated. The 50% lethal doses ranged from 5.4 × 10⁴ to 1.0 × 10⁶ CFU/g of body weight. The strains examined had the ability to adhere to skin, gill, and intestinal mucus of sea bream and to cultured cells of a chinook salmon embryo cell line. In addition, the in vitro ability of V. alginolyticus to adhere to mucus and skin cells of sea bream was demonstrated by scanning electron microscopy. The biological activities of extracellular products of V. alginolyticus were hydrolytic activities; the products were able to degrade sea bream mucus. V. alginolyticus was cytotoxic for fish cell lines and lethal for sea bream. Moreover, the extracellular products could degrade sea bream tissues. However, experiments performed with the bath immersion inoculation technique demonstrated that V. alginolyticus should be considered a pathogen for sea bream only when the mucus layer is removed and the skin is damaged.     

Thyroid hormone receptor inhibits hepatoma cell migration through transcriptional activation of Dickkopf 4

Triiodothyronine (T₃) is a potent form of thyroid hormone mediates several physiological processes including cellular growth, development, and differentiation via binding to the nuclear thyroid hormone receptor (TR). Recent studies have demonstrated critical roles of T₃/TR in tumor progression. Moreover, long-term hypothyroidism appears to be associated with the incidence of human hepatocellular carcinoma (HCC), independent of other major HCC risk factors. Dickkopf (DKK) 4, a secreted protein that antagonizes the canonical Wnt signaling pathway, is induced by T₃ at both mRNA and protein levels in HCC cell lines. However, the mechanism underlying T₃-mediated regulation of DKK4 remains unknown. In the present study, the 5′ promoter region of DKK4 was serially deleted, and the reporter assay performed to localize the T₃ response element (TRE). Consequently, we identified an atypical direct repeat TRE between nucleotides −1645 and −1629 conferring T₃ responsiveness to the DKK4 gene. This region was further validated using chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA). Stable DKK4 overexpression in SK-Hep-1 cells suppressed cell invasion and metastatic potential, both in vivo andin vitro, via reduction of matrix metalloproteinase-2 (MMP-2) expression. Our findings collectively suggest that DKK4 upregulated by T₃/TR antagonizes the Wnt signal pathway to suppress tumor cell progression, thus providing new insights into the molecular mechanism underlying thyroid hormone activity in HCC.     

Memory retention capacity using two different training methods,appetitive and aversive learning,in juvenile red sea bream Chrysophrys major

Memory retention based on appetitive and aversive learning was studied in juvenile red sea bream Chrysophrys major. The fish were individually trained via appetitive and aversive learning. In general, they retained appetitive memories for 30 days, but not for 60 days. Conversely, aversive memory endured for 1 day, but not for 3 days or longer. Analyses at the individual level revealed that some fish retained appetitive memories for 60 days, whereas others lost it within 3 days; this suggests considerable variability in memory retention capacity among individual fish. The memory duration for aversive learning was remarkably short, which should be considered when releasing trained fish into the wild for stock enhancement. Furthermore, the high inter-individual variability suggests that evaluating memory retention capacity through group experiments might lead to overestimation of fishes’ ability.     

Increased Oxidative Metabolism and Neurotransmitter Cycling in the Brain of Mice Lacking the Thyroid Hormone Transporter Slc16a2 (Mct8)

Mutations of the monocarboxylate transporter 8 (MCT8) cause a severe X-linked intellectual deficit and neurological impairment. MCT8 is a specific thyroid hormone (T₄ and T₃) transporter and the patients also present unusual abnormalities in the serum profile of thyroid hormone concentrations due to altered secretion and metabolism of T₄ and T₃. Given the role of thyroid hormones in brain development, it is thought that the neurological impairment is due to restricted transport of thyroid hormones to the target neurons. In this work we have investigated cerebral metabolism in mice with Mct8 deficiency. Adult male mice were infused for 30 minutes with (1-¹³C) glucose and brain extracts prepared and analyzed by ¹³C nuclear magnetic resonance spectroscopy. Genetic inactivation of Mct8 resulted in increased oxidative metabolism as reflected by increased glutamate C4 enrichment, and of glutamatergic and GABAergic neurotransmissions as observed by the increases in glutamine C4 and GABA C2 enrichments, respectively. These changes were distinct to those produced by hypothyroidism or hyperthyroidism. Similar increments in glutamate C4 enrichment and GABAergic neurotransmission were observed in the combined inactivation of Mct8 and D2, indicating that the increased neurotransmission and metabolic activity were not due to increased production of cerebral T₃ by the D2-encoded type 2 deiodinase. In conclusion, Mct8 deficiency has important metabolic consequences in the brain that could not be correlated with deficiency or excess of thyroid hormone supply to the brain during adulthood.