Disruption of the Thyroid System by the Thyroid-Disrupting Compound Aroclor 1254 in Juvenile Japanese Flounder (Paralichthys olivaceus)

Polychlorinated biphenyls (PCBs) are a group of persistent organochlorine compounds that have the potential to disrupt the homeostasis of thyroid hormones (THs) in fish, particularly juveniles. In this study, thyroid histology, plasma TH levels, and iodothyronine deiodinase (IDs, including ID₁, ID₂, and ID₃) gene expression patterns were examined in juvenile Japanese flounder (Paralichthys olivaceus) following 25- and 50- day waterborne exposure to environmentally relevant concentrations of a commercial PCB mixture, Aroclor 1254 (10, 100, and 1000 ng/L) with two-thirds of the test solutions renewed daily. The results showed that exposure to Aroclor 1254 for 50 d increased follicular cell height, colloid depletion, and hyperplasia. In particular, hypothyroidism, which was induced by the administration of 1000 ng/L Aroclor 1254, significantly decreased plasma TT₄, TT₃, and FT₃ levels. Profiles of the changes in mRNA expression levels of IDs were observed in the liver and kidney after 25 and 50 d PCB exposure, which might be associated with a reduction in plasma THs levels. The expression level of ID₂ mRNA in the liver exhibited a dose-dependent increase, indicating that this ID isotype might serve as sensitive and stable indicator for thyroid-disrupting chemical (TDC) exposure. Overall, our study confirmed that environmentally relevant concentrations of Aroclor 1254 cause significant thyroid disruption, with juvenile Japanese flounder being suitable candidates for use in TDC studies.     

Geographic Variations in Dorsal and Anal Ray Counts of Juvenile Japanese Flounder, Paralichthys olivaceus, in the Japan Sea

Meristic counts of early and late wild settled juveniles of Japanese flounder, Paralichthys olivaceus (Paralichthyidae), in Wakasa Bay, Japan, were compared, with specimens collected from 26 sites spread along ca. 1700km of coastline facing the Japan Sea. Mean dorsal and anal ray counts of the early settled group at Kyoto were significantly larger than those of the late settled group. A discontinuous geographic boundary in mean dorsal and anal ray counts was found around Wakasa Bay and the Noto Peninsula with both means significantly larger in the southern group than in the northern group. Mean ray counts of the early and late settlers at Kyoto were similar to those of the southern and northern groups, respectively. The effect of water temperature on meristic characters was tested on two groups of laboratory-reared juveniles. Mean dorsal and anal ray counts were larger in juveniles reared at higher water temperatures. The early and late settler groups were estimated to hatch in late February and late April, respectively, representing negative response of meristic characters to water temperatures if these two groups hatched out in Wakasa Bay. Differences in the dorsal and anal ray counts between the early and late settled groups at Kyoto can, therefore be attributed to genotypic, not phenotypic variation. This suggests the possibility of transport of larvae of the early group from areas further south to Wakasa Bay by the warm Tsushima Current, a branch of the Kuroshio, which flows northeastward along the coast of the Japan Sea, particularly when the temperature gradation between the south and north areas during these seasons is considered.     

Inheritance Characteristics of Microsatellite DNA Loci in Experimental Families of Japanese Flounder Paralichthys olivaceus

Allele segregating patterns of microsatellite DNA loci in 5 experimental families of Japanese flounder Paralichthys olivaceus and genotype frequencies for deviation from Hardy-Weinberg expectations (HWE) in natural P. olivaceus populations were studied to assess inheritability. Of the 12 microsatellite loci examined, 1 locus had a possibility of scoring errors of heterozygous individuals caused by unreproducible polymerase chain reaction amplifications of a particular allele. At the remaining 11 loci, almost all of alleles were segregated according to Mendelian transmission, and observed genotype frequencies in natural populations were consistent with HWE. The results demonstrated here would provide useful information supporting the suitability of these microsatellite loci as inheritable P. olivaceus genetic markers. Received September 25, 2000; accepted March 1, 2001     

Molecular Characterization and Expression Analysis of ATP-Gated P2X7 Receptor Involved in Japanese Flounder (Paralichthys olivaceus) Innate Immune Response

ATP-gated P2X7 receptor (P2RX7) channel is a key component for purinergic signaling and plays important roles in the innate immune response in mammals. However, the expression, molecular properties and immune significances of P2RX7 in lower vertebrates are still very limited. Here we identified and characterized a novel bony fish P2RX7 homologue cDNA, termed poP2RX7, in Japanese flounder (Paralichthys olivaceus). PoP2RX7 protein shares about 60–88% sequence similarity and 45–78% sequence identity with known vertebrate P2RX7 proteins. Phylogenetic analysis placed poP2RX7 and other P2RX7 proteins within their own cluster apart from other P2RX members. While the functional poP2RX7 channel shares structural features in common with known P2RX7 homologs, electrophysiological studies revealed that BzATP, the more potent agonist for known mammalian and fish P2RX7s, shows similar potency to ATP in poP2RX7 activation. poP2RX7 mRNA constitutively expressed in all examined tissues from unstimulated healthy Japanese flounder with dominant expression in hepatopancreas and the lowest expression in head kidney, trunk kidney, spleen and gill. poP2RX7 mRNA expression, however, was significantly induced in Japanese flounder head kidney primary cells by Poly(I:C) and bacterial endotoxin LPS stimulations. In vivo experiments further revealed that poP2RX7 gene expression was substantially up-regulated by immune challenge with infectious bacteria Edwardsiella tarda and Vibrio anguillarum. Moreover, activation of poP2RX7 results in an increased gene expression of multifunctional cytokines IL-1β and IL-6 in the head kidney primary cells. Collectively, we identified and characterized a novel fish P2RX7 homolog which is engaged in Japanese flounder innate immune response probably through modulation of pro-inflammatory cytokines expression.     

微卫星评价牙鲆雌核发育二倍体纯合性

采用8个微卫星座位分别对牙鲆减数雌核发育二倍体家系和卵裂雌核发育二倍体家系的纯合性进行检验。卵裂雌核发育二倍体在所有检测座位全部纯合。减数雌核发育二倍体在部分座位发生纯合,但未发现在所有座位全部纯合的个体,在Poli9TUF、Poli9-8TUF、Poli11TUF、Poli13TUF、Poli23TUF、Poli30TUF、Poli123TUF和Poli130TUF座位,杂合子比例分别为1·0000、1·0000、0·1944、0·9459、0·8611、1·0000、0·7778和0·8000,平均杂合子比例为0·8224。由此表明,牙鲆除了Poli11TUF外,在其余7个座位均具有很高的重组率。研究结果显示,牙鲆卵裂雌核发育二倍体一代即可形成纯合子;而减数雌核发育二倍体由于具有较高的重组率,使其与母本的遗传同质性较高。     

Microarray analyses of gene expression in Japanese flounder Paralichthys olivaceus leucocytes during monogenean parasite Neoheterobothrium hirame infection

In this study, the gene expression patterns of peripheral blood leucocytes (PBL) from Japanese flounder Paralichthys olivaceus were analyzed during the course of monogenean parasite Neoheterobothrium hirame infection in order to select candidates for molecular biomarkers of infection. cDNA microarray analysis was performed to compare the gene expression patterns of PBL between infected and non-infected fishes. Among the 797 genes analyzed, 45 genes (5.6%) changed their expression levels. These genes included specific and non-specific immune-related genes (matrix metalloproteinase[MMP]-9, MMP-13, leukotriene B4 receptor, CD20 receptor, MHC [major histocompatibility complex] Class I, MHC Class II beta-chain, immunoglobulin light chain and immunoglobulin heavy chain). Significant up- and down-regulation of some unknown genes was also observed. Several candidates for infection-marker genes were selected for further study. These genes included MMP-9, MMP-13, leukotriene b4 receptor, CD20 receptor, immunoglobulin heavy chain, immunoglobulin light chain and unknown genes coded as B613, E25, LB3(8), WE2(3), WE8-18R and WF12-18R.     

A Genome Scan for Quantitative Trait Loci Associated with Vibrio anguillarum Infection Resistance in Japanese Flounder (Paralichthys olivaceus) by Bulked Segregant Analysis

A recent genetic linkage map was employed to detect quantitative trait loci (QTLs) associated with Vibrio anguillarum resistance in Japanese flounder. An F1 family established and challenged with V. anguillarum in 2009 was used for QTL mapping. Of the 221 simple sequence repeat (SSR) markers used to detect polymorphisms in the parents of F1, 170 were confirmed to be polymorphic. The average distance between the markers was 10.6 cM. Equal amounts of genomic DNA from 15 fry that died early and from 15 survivors were pooled separately to constitute susceptible bulk and resistance bulk DNA. Bulked segregant analysis and QTL mapping were combined to detect candidate SSR markers and regions associated with the disease. A genome scan identified four polymorphic SSR markers, two of which were significantly different between susceptible and resistance bulk (P?=?0.008). These two markers were located in linkage group (LG) 7; therefore, all the SSR markers in LG7 were genotyped in all the challenged fry by single marker analysis. Using two different models, 11–17 SSR markers were detected with different levels of significance. To confirm the associations of these markers with the disease, composite interval mapping was employed to genotype all the challenged individuals. One and three QTLs, which explained more than 60 % of the phenotypic variance, were detected by the two models. Two of the QTLs were located at 48.6 cM. The common QTL may therefore be a major candidate region for disease resistance against V. anguillarum infection.     

Developmental Sequence of Chloride Cells in the Body Skin and Gills of Japanese Flounder (Paralichthys olivaceus) Larvae

The developmental sequence of chloride cells was examined in both the body skin and gills of Japanese flounder (Paralichthys olivaceus) larvae by whole-mount immunocytochemistry using an antiserum specific for Na(+),K(+)-ATPase. In premetamorphic larvae at 0 and 4 days after hatching (days 0 and 4), immunoreactive chloride cells were distributed only in the yolk-sac membrane and body skin. Premetamorphic larvae at days 8-18 possessed both cutaneous and branchial chloride cells. Large chloride cells in the skin of premetamorphic larvae often formed multicellular complexes, suggestive of their ion-secreting function. Cutaneous chloride cells decreased in size and density at the beginning of metamorphosis (days 21 and 24), and disappeared at the metamorphic climax (days 28 and 33). In contrast, branchial chloride cells first appeared at day 8, and increased during metamorphosis. These results indicate that the site for ion secretion in seawater may shift from cutaneous to branchial chloride cells during metamorphosis. The appearance of branchial chloride cells before the differentiation of gill lamellae suggests that the primary function of the gills during the early development is ion regulation rather than gas exchanges.     

Sequences of 596 cDNA Clones (565,977 bp) of Japanese Flounder (Paralichthys olivaceus) Leukocytes Infected with Hirame Rhabdovirus

We have partially sequenced 785 sequences of 596 independent complementary DNA clones isolated from a cDNA library of Japanese flounder leukocytes infected with hirame rhabdovirus. These sequences consist of a total of 565,977 base pairs. The average size of the sequenced lengths was 721 bp. Of 596 clones, 386 (64.8%) were identified as previously reported genes by the BLASTN and BLASTX programs. About 30% of the identified clones could be recognized by only the BLASTX program. A total of 251 distinct genes were identified, and 181 of these genes are the first such genes reported from the teleostei. Approximately 27% of the identified Japanese flounder genes appear to be associated with cell division, cell structure or motility, and basic energy metabolism, 29% with gene or protein expression, and 17% with cell signaling, cell communication, and cell or organism defense. The most frequently identified expressed sequence tags of leukocytes of Japanese flounder were gelatinase b and ribosomal protein L23, which both had 1.34% prevalence. Received February 12, 1999; accepted April 13, 1999     

Changes of the Serum Cytokine Contents in Broilers Fed on Diets Supplemented with Nickel Chloride

Cytokines are immunoregulatory proteins which play an important role in the immune system. The purpose of this study was to examine the serum cytokine contents including interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-10 (IL-10), interferon gamma (IFN-γ), and tumor necrosis factor-alpha (TNF-α) induced by dietary nickel chloride in broilers by enzyme-linked immunospecific assay. A total of 240 one-day-old avian broilers were divided into four groups and fed on a corn–soybean basal diet as control diet or the same basal diet supplemented with 300, 600, and 900 mg/kg of nickel chloride. During the experimental period of 42 days, the results showed that the serum IL-2, IL-4, IL-6, IL-10, IFN-γ and TNF-α contents were lower (p?<?0.05 or p?<?0.01) in the 300, 600, 900 mg/kg groups than those in the control group. It was concluded that dietary nickel chloride in the range of 300 to 900 mg/kg could reduce the serum cytokine contents, which could finally impact the immune function in broilers.     

Construction of High-Density Genetic Linkage Maps and Mapping of Growth-Related Quantitative Trail Loci in the Japanese Flounder (Paralichthys olivaceus)

High-density genetic linkage maps were constructed for the Japanese flounder (Paralichthys olivaceus). A total of 1624 microsatellite markers were polymorphic in the reference family. Linkage analysis using JoinMap 4.0 resulted in the mapping of 1487 markers to 24 linkage groups, a result which was consistent with the 24 chromosomes seen in chromosome spreads. The female map was composed of 1257 markers, covering a total of 1663.8 cM with an average interval 1.35 cM between markers. The male map consisted of 1224 markers, spanning 1726.5 cM, with an average interval of 1.44 cM. The genome length in the Japanese flounder was estimated to be 1730.3 cM for the females and 1798.0 cM for the males, a coverage of 96.2% for the female and 96.0% for the male map. The mean recombination at common intervals throughout the genome revealed a slight difference between sexes, i.e. 1.07 times higher in the male than female. High-density genetic linkage maps are very useful for marker-assisted selection (MAS) programs for economically valuable traits in this species and for further evolutionary studies in flatfish and vertebrate species. Furthermore, four quantiative trait loci (QTL) associated with growth traits were mapped on the genetic map. One QTL was identified for body weight on LG 14 f, which explained 14.85% of the total variation of the body weight. Three QTL were identified for body width on LG14f and LG14m, accounting for 16.75%, 13.62% and 13.65% of the total variation in body width, respectively. The additive effects were evident as negative values. There were four QTL for growth traits clustered on LG14, which should prove to be very useful for improving growth traits using molecular MAS.     

Gene expression of leucocytes in vaccinated Japanese flounder (Paralichthys olivaceus) during the course of experimental infection with Edwardsiella tarda

In this paper, we focused on the detection of differentially expressed genes in peripheral blood leucocytes (PBL) during the course of Edwardsiella tarda infection in vaccinated and non-vaccinated Japanese flounder (Paralichthys olivaceus). cDNA microarray analysis was performed to compare the gene expression patterns of the PBL between the vaccinated and non-vaccinated fish in response to E. tarda inoculation. Fish were vaccinated twice, at a two-week interval and experimentally challenged with E. tarda two weeks after the second vaccination. Among the 1187 analyzed genes, 42 genes were up-regulated during the course of infection either in vaccinated or non-vaccinated fish. These genes included immune-related genes, such as MMP-9, MMP-13, CXC chemokine, CD20 receptor and hepcidin. Some immune-related genes were down-regulated after the E. tarda challenge, i.e. interferon inducible Mx protein, MHC class II-associated invariant chain, MHC class II alpha and MHC class II beta encoding genes, immunoglobulin light chain precursor, immunoglobulin light chain and IgM. These responses are thought to be a common reaction of Japanese flounder PBL in the course of edwardsiellosis, irrespective of immunized condition. Ten genes were significantly up-regulated only in vaccinated fish, and 11 genes were significantly up-regulated only in non-vaccinated fish. These genes may have a correlation with the efficacy of vaccination, although we have no evidence to link the different gene expression patterns and the efficacy of vaccination at present.     

Infection with Edwardsiella tarda causes hypertrophy of liver cells in the Japanese flounder Paralichthys olivaceus

To study the direct cause of liver enlargement in the Japanese flounder Paralichthys olivaceus infected with Edwardsiella tarda, the fish were challenged with E. tarda and reared without feeding. The liver of fish exposed to the bacteria was markedly enlarged compared to that of the controls while no severe histopathological change appeared in the organ during the experiments. No notable difference was observed in the crude fat, glycogen, and water content of the liver between challenged and control fish. The size of liver cells and nuclei of the challenged fish was apparently larger than that of the controls. Analysis of crude DNA in the liver suggested that the number of liver cells of starved control fish significantly decreased during the experiment while that of the challenged fish was maintained at a level of the initial control. RNA/DNA ratio of the liver of challenged fish clearly increased while it decreased in the control fish during the experiment. These observations suggest that liver enlargement of flounder infected with E. tarda, at least in the early stage of infection, is not a result of any readily observable histopathological changes and that E. tarda infection causes hypertrophy of the cells, as well as preventing decrease in liver cell number.     

Changes of IgA+ Cells and Cytokines in the Cecal Tonsil of Broilers Fed on Diets Supplemented with Vanadium

The cecal tonsil of broiler is known as a secondary lymphoid tissue, which is involved in antigen-specific humoral immune responses. The purpose of this study was to investigate the effects of dietary vanadium on the tissue distribution and quantity of immunoglobulin A-positive (IgA(+)) cell in the cecal tonsil by immunohistochemistry. Simultaneously, the changes in interleukin-6 (IL-6), interleukin-10 (IL-10), interferon gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) contents in the cecal tonsil were also quantified by enzyme-linked immunosorbent assay (ELISA). A total of 420 one-day-old avian broilers were divided into six groups and fed on a corn-soybean basal diet (control diet) or the same diet supplemented respectively with 5, 15, 30, 45, and 60 mg/kg of vanadium in the form of ammonium metavanadate for 42 days. The results showed that the population of the IgA(+) cells in the cecal tonsil were significantly lower (p < 0.05 or p < 0.01) in the 45 and 60 mg/kg groups than that in the control group. Meanwhile, IL-10, IFN-γ and TNF-α contents in the cecal tonsil were significantly decreased (p < 0.05 or p < 0.01) in the 30, 45 and 60 mg/kg groups in comparison with those of the control group. However, IL-6 content in the cecal tonsil was only decreased (p < 0.05 or p < 0.01) in 60 mg/kg at 14 and 28 days of age. In conclusion, dietary vanadium in excess of 30 mg/kg reduced the numbers of the IgA(+) cells and changed the contents of the abovementioned cytokines in the cecal tonsil, which may finally impact the function of local mucosal humoral immunity in broilers.