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1.
Histological development of Japanese flounder Paralichthys olivaceus larval skin and ultrastructural difference of skin between reared normal and malpigmented Japanese flounder were studied with light microscopy (LM) and transmission electron microscopy (TEM). The results show that the skin develops slowly before the metamorphosis, while at the onset of metamorphosis, the skin develops quickly and becomes complete in structure till about 50d after being hatched. Ultrastructural observation on the normal and malpigmented skins shows that the iridophore and melanophore are adjacent to each other. Profile and structure of the two kinds of pigmcnt cells are more complete in the skin of normal ocular side than in the skin of pigmented blind side. The ultrastructure of typical chloride cell was observed in the skin of Japanese flounder larvae for the first time.  相似文献   

2.
The symmetrical body of flatfish larvae changes dramatically into an asymmetrical form after metamorphosis. The molecular mechanisms responsible for this change are poorly understood. As an initial step to clarify these mechanisms, we used representational difference analysis of cDNA for the identification of genes active during metamorphosis in the Japanese flounder, Paralichthys olicaceus. One of the up-regulated genes was identified as creatine kinase muscle type 1 (CK-M1). Sequence analysis of CK-M1 revealed that it spanned 1 708 bp and encoded a protein of 382 amino acids. The overall amino acid sequence of the CK-M1 was highly conserved with those of other organisms. CK-M1 was expressed in adult fish tissues, including skeletal muscle, intestine and gill. Whole mount in-situ hybridization showed that the enhanced expression of CK-M1 expanded from the head to the whole body of larvae as metamorphosis progressed. Quantitative analysis revealed stage-specific high expression of CK-M1 during metamorphosis. The expression level of CK-M1 increased initially and peaked at metamorphosis, decreased afterward, and finally returned to the pre-metamorphosis level. This stage-specific expression pattern suggested strongly that CK-M1 was related to metamorphosis in the Japanese flounder. Its specific role in metamorphosis requires further study.  相似文献   

3.
Flatfish or flounder moves one eye to change body proportion into vertebral asymmetry during metamorphosis, during which some become sinistral while others dextral. However, the mechanism behinds the eye-position has not been well understood. In this research, hybrids between Japanese flounder(♀) and stone flounder (♂) show mixed eye-location in both dextral type and sinistral type, and thus become good samples for studying the eye-migration. mRNAs from pro-metamorphosis sinistral and dextral hybrids larvae...  相似文献   

4.
Flatfish or flounder moves one eye to change body proportion into vertebral asymmetry during metamorphosis, during which some become sinistral while others dextral. However, the mechanism behinds the eye-position has not been well understood. In this research, hybrids between Japanese flounder(♀) and stone flounder (♂) show mixed eye-location in both dextral type and sinistral type, and thus become good samples for studying the eye-migration. mRNAs from pro-metamorphosis sinistral and dextral hybrids larvae were screened with classical differential display RT-PCR (DD-RT-PCR) and representational difference analysis of cDNA (cDNA-RDA); 30 and 47 putative fragments were isolated, respectively. The cDNA fragments of creatine kinase and trypsinogen 2 precursor genes isolated by cDNA-RDA exhibited eye-position expression patterns during metamorphosis. However, none of the fragments was proved to be related to flatfishes’ eye-position specifically. Therefore, further studies and more sensitive gene isolated methods are needed to solve the problems. Supported by National Natural Science Foundation of China (No. 30600455) and the National Basic Research Program of China (973 Program, No.2004CB117402)  相似文献   

5.
To reveal the key factor in self-healing from LCDV (lymphocystis disease virus)-infec Japanese flounder (Paralichthys olivaceus), serum proteins from self-healing and sick Japanese floun were separated by two-dimensional electrophoresis to screen differentially expressed proteins. Prot spots demonstrating changes greater than two-fold in the expression level were digested and furt identified in capillary liquid chromatography tandem mass spectrometry (LC-MS/MS). Two immuni relevant proteins were thus identified as transferrin and the complement component C3 of Japan flounder. These findings suggest that the two proteins may play important roles in the self-healing lymphocystis in Japanese flounder. This is an important theoretical foundation to promote self-healing LCDV-infected Japanese flounder by improving their innate immunity.  相似文献   

6.
We isolated a strain of lymphocystis disease virus (LCDV) from Japanese flounder (Paralichthys olivaceus) cultured in northern China. Based on published sequences of major capsid protein (MCP) gene of LCDV-cn (GenBank: AF126405), we designed two primer sets P1/P2 and P3/P4. We then used one-step or nested PCR and in-situ hybridization (ISH) to detect LCDV and identify the target tissues or cells in infected Japanese flounder. The PCR products were positive in purified viral supernatant, skin nodules, gut, gill, kidney, spleen, stomach, heart, and liver of Japanese flounder. We compared the DNA sequence with 14 MCP nucleotide sequences from GenBank, including Megalocytivirus (OFIV and RSIV), Iridovirus (CzIV and WIV), Ranavirus (TFV and FV3), and Lymphocystivirus (8 LCDV). Based on the alignment, we confirmed the PCR product was from Lymphocystivirus (GenBank accession number DQ279090 (LCDV-HD)). Using ISH, we noted the presence of LCDV in the skin nodules, gut, gill, spleen, stomach, and heart of spontaneously infected Japanese flounders. We successfully amplified LCDV fragments from Schlegel’s black rockfish (Sebastes schlegeli Higendorf), redwing sea robin (Lepidotrigla microptera Günther) and turbot (Scophthalmus maximus) using the one-step and nested PCR, suggesting the target genes can be widely detected in fish using this method.  相似文献   

7.
The hybrid between olive flounder Paralichthys olivaceus and stone flounder Kareius bicoloratus was produced by artificial insemination of olive flounder eggs with stone flounder Sperm. Sinistral and dextral are two types of hybrid progeny after metamorphosis. Karyotypes of both hybrid flounders are the same as those of the two parental species. Of the 22 loci examined from 12 allozymes,12 confirmed hybridization of the paternal and matemal loci in hybrids and no difference was found in allozyme patterns of sinistral and dextral hybrid fishes. RAPD patterns of these specimens were also studied with 38 primers selected from 104 tested. Among them, the PCR products of 30 primers showed hybridization of the paternal and maternal bands. Genetic variation between hybrids and their parental stocks was analyzed by RAPD using 10 of the above 38 primers. The average heterozygosity and genetic distance were calculated. The results suggested that the filial generation could inherit a little more genetic materials from paternal fish than that from maternal fish.  相似文献   

8.
Heat shock proteins 10/60(hsp10/60) are a family of conserved ubiquitously expressed heat shock proteins which are produced by cells in response to exposure to stressful conditions. Besides the chaperone and housekeeping functions, they are also known to be involved in immune response during bacterial infection. In this study, we identified and annotated 10 hsp10/60 genes through bioinformatic analysis in Japanese flounder(Paralichthys olivaceus). Among them one member of hsp10(hspe) family and nine members of hsp60(hspd) family were identified. Phylogenetic and selection pressure analysis showed that the hsp10/60 genes were evolutionarily constrained and their function was conserved. Besides, hsp10/60 genes were involved in different embryonic and larval stages and acted as the sentinel role in an unchallenged organism. In addition, we also observed the expression patterns of hsp10/60 genes after Edwardsiella tarda infection, for the first time in Japanese flounder. Eight out of 10 genes were differentially expressed after bacterial challenges, the significantly regulated expressions of flounder hsp10/60 genes after bacterial infections suggested their involvement in immune response in flounder. Our results provide valuable information for clarifying the evolutionary relationship, and early insights of the immune functions of hsp10/60 genes in Japanese flounder.  相似文献   

9.
A novel immune-related gene was expressed in Japanese flounder (Paralichthys olivaceus) injected with Vibrio anguillarum. The complete cDNA contained a 169 bp 5’UTR, a 336 bp open reading frame (ORF) encoding 111 amino acids and a 556bp 3’UTR. Six exons and five introns were identified in the PoIR2 gene. Blastp similarity comparison showed its encoding protein had 50% similarity to Danio rerio neuromedin S (NMS), but further alignment indicated they did not have NMS C-terminal conservational signature domain. So it was not defined as an NMS homologue. Protein structure analysis indicated it had a 26aa signal peptide and was a secretory pathway protein. RT-PCR demonstrated that the expression of PoIR2 was quickly induced and drastically increased in liver, kidney, spleen, gills, intestine, heart, and skeletal muscle after infected with V. anguillarum. These results indicated that the PoIR2 might play some important role in Japanese flounder immune response system. This gene was named PoIR2 (P.olivaceus immune-related gene 2, GenBank accession number: EU224372). The mature PoIR2 peptide was expressed in BL21(DE3) pLysS using pET-32a(+) vector and a great part of the recombinant mature peptide existed as soluble type.  相似文献   

10.
The hybrid between olive flounder Paralichthys olivaceus and stone flounder Kareius bicoloratus was produced by artificial insemination of olive flounder eggs with stone flounder sperm. Sinistral and dextral are two types of hybrid progeny after metamorphosis. Karyotypes of both hybrid flounders are the same as those of the two parental species. Of the 22 loci examined from 12 allozymes, 12 confirmed hybridization of the paternal and maternal loci in hybrids and no difference was found in allozyme patterns of sinistral and dextral hybrid fishes. RAPD patterns of these specimens were also studied with 38 primers selected from 104 tested. Among them, the PCR products of 30 primers showed hybridization of the paternal and maternal bands. Genetic variation between hybrids and their parental stocks was analyzed by RAPD using 10 of the above 38 primers. The average heterozygosity and genetic distance were calculated. The results suggested that the filial generation could inherit a little more genetic materials from paternal fish than that from maternal fish. Supported by National Natural Science Foundation of China (No. 30571445), National High-Technology Research and Development Program (863 Program, No. 2006AA10A404), and Project from the Ministry of Science and Technology of China (No. 2006DKA30470-017)  相似文献   

11.
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12.
Lymphocystis disease, caused by the lymphocystis disease virus (LCDV), is a significant worldwide problem in fish industry causing substantial economic losses. In this study, we aimed to develop the DNA vaccine against LCDV, using DNA vaccination technology. We evaluated plasmid pEGFP-N2-LCDV1.3 kb as a DNA vaccine candidate. The plasmid DNA was transiently expressed after liposome transfection into the eukaryotic COS 7 cell line. The distribution and expression of the DNA vaccine (pEGFP-N2-LCDV1.3kb) were also analyzed in tissues of the vaccinated Japanese flounder by PCR, RT-PCR and fluorescent microscopy. Results from PCR analysis indicated that the vaccine-containing plasmids were distributed in injected muscle, the muscle opposite the injection site, the hind intestine, gill, spleen, head, kidney and liver, 6 and 25 days after vaccination. The vaccine plasmids disappeared 100 d post-vaccination. Fluorescent microscopy revealed green fluorescence in the injected muscle, the muscle opposite the injection site, the hind intestine, gill, spleen, head, kidney and liver of fish 48 h post-vaccination, green fluorescence did not appear in the control treated tissue. Green fluorescence became weak at 60 days post-vaccination. RT-PCR analysis indicated that the mcp gene was expressed in all tested tissues of vaccinated fish 6–50 days post-vaccination. These results demonstrate that the antigen encoded by the DNA vaccine is distributed and expressed in all of the tissues analyzed in the vaccinated fish. The antigen would therefore potentially initiate a specific immune response. the plasmid DNA was injected into Japanese flounder (Paralichthys olivaceus) intramuscularly and antibodies against LCDV were evaluated. The results indicate that the plasmid encoded DNA vaccine could induce an immune response to LCDV and would therefore offer immune protection against LCD. Further studies are required for the development and application of this promising DNA vaccine.  相似文献   

13.
Japanese flounder is one of the most important commercial species in China; however, information on the genetic background of natural populations in China seas is scarce. The lack of genetic data has hampered fishery management and aquaculture development programs for this species. In the present study, we have analyzed the genetic diversity in natural populations of Japanese flounder sampled from the Yellow Sea (Qingdao population, QD) and East China Sea (Zhoushan population, ZS) using 10 polymorphic microsatellite loci and cytochrome c oxidase subunit I (COI) sequencing data. A total of 68 different alleles were observed over 10 microsatellite loci. The total number of alleles per locus ranged from 2 to 9, and the number of genotypes per locus ranged from 3 to 45. The observed heterozygosity and expected heterozygosity in QD were 0.733 and 0.779, respectively, and in ZS the heterozygosity values were 0.708 and 0.783, respectively. Significant departures from Hardy-Weinberg equilibrium were observed in 7 of the 10 microsatellite loci in each of the two populations. The COI sequencing analysis revealed 25 polymorphic sites and 15 haplotypes in the two populations. The haplotype diversity and nucleotide diversity in the QD population were 0.746±0.072 8 and 0.003 34±0.001 03 respectively, and in ZS population the genetic diversity values were 0.712±0.047 0 and 0.003 18±0.000 49, respectively. The microsatellite data (F_st =0.048 7, P<0.001) and mitochondrial DNA data (F_st =0.128, P<0.001) both revealed significant genetic differentiation between the two populations. The information on the genetic variation and differentiation in Japanese flounder obtained in this study could be used to set up suitable guidelines for the management and conservation of this species, as well as for managing artificial selection programs. In future studies, more geographically diverse stocks should be used to obtain a deeper understanding of the population structure of Japanese flounder in the China seas and adjacent regions.  相似文献   

14.
As a specific gene of fish,cytochrome P450c17-Ⅱ(CYP17-Ⅱ) gene plays a key role in the growth,development and reproduction level of fish.In this study,the single-stranded conformational polymorphism(SSCP) technique was used to characterize polymorphisms within the coding region of CYP17-Ⅱ gene in a population of 75 male Japanese flounder(Paralichthys olivaceus).Three single nucleotide polymorphisms(SNPs) were identified in CYP17-Ⅱ gene of Japanese flounder.They were c.G594A(p.G188R),c.G939A and c.G1502A(p.G490D).SNP1(c.G594A),located in exon 4 of CYP17-Ⅱ gene,was significantly associated with gonadosomatic index(GSI).Individuals with genotype GG of SNP1 had significantly lower GSI(P < 0.05) than those with geno-type AA or AG.SNP2(c.G939A) located at the CpG island of CYP17-Ⅱ gene.The mutation changed the methylation of exon 6.Indi-viduals with genotype AA of SNP2 had significantly lower serum testosterone(T) level and hepatosomatic index(HSI) compared to those with genotype GG.The results suggested that SNP2 could influence the reproductive endocrine of male Japanese flounder.How-ever,the SNP3(c.G1502A) located in exon 9 did not affect the four measured reproductive traits.This study showed that CYP17-Ⅱ gene could be a potentially useful candidate gene for the research of genetic breeding and physiological aspects of Japanese flounder.  相似文献   

15.
Follicle stimulating hormone β (FSHβ) of Japanese flounder (Paralichthys olivaceus) plays a key role in the regulation of gonadal development.This study aimed to investigate molecular genetic characteristics of the FSHβ gene and elucidate the effects of single nucleotide polymorphisms (SNPs) of FSHβ on reproductive traits in Japanese flounder.We used polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) and sequencing of the FSHβ gene in 60 individuals.We identified only an SNP (T/C) in the coding region of exon3 of FSHβ.The SNP (T/C) did not lead to amino acid changes at the position 340 bp of FSHβ gene.Statistical analysis showed that the SNP was significantly associated with testosterone (T) level and gonadosomatic index (GSI) (P0.05).Individuals with genotype TC of the SNP had significantly higher serum T levels and GSI (P0.05) than that of genotype CC.Therefore, FSHβ gene could be a useful molecular marker in selection for prominent reproductive trait in Japanese Flounder.  相似文献   

16.
Artificial gynogenesis is of great research value in fish genetics and breeding technology. However, existing studies did not explain the mechanism of some interesting phenomena. Severe developmental defects in gynogenetic haploids can lead to death during hatching. After diploidization of chromosomes, gynogenetic diploids may dispense from the remarkable malformation and restore the viability, although the development time is longer and the survival rate is lower compared with normal diploids. The aim of this study was to reveal key mechanism in haploid syndrome of Japanese flounder, a commercially important marine teleost in East Asia. We measured genome-scale gene expression of flounder haploid, gynogenetic diploid and normal diploid embryos using RNA-Seq, constructed a module-centric co-expression network based on weighted correlation network analysis(WGCNA) and analyzed the biological functions of correlated modules. Module gene content analysis revealed that the formation of gynogenetic haploids was closely related to the abnormality of plasma proteins, and the up-regulation of p53 signaling pathway might rescue gynogenetic embryos from haploid syndrome via regulating cell cycle arrest, apoptosis and DNA repair. Moreover, normal diploid has more robust nervous system. This work provides novel insights into molecular mechanisms in haploid syndrome and the rescue process by gynogenetic diploidization.  相似文献   

17.
The retinal structure and visual acuity in Japanese flounderParalichthys olivaceus at different stages of development were examined by light microscopy. The resolving power of the retina, the visual axis and the best visual field were estimated based on the distribution of cone cells in the retina. The visual system of the larvae appears poorly developed at hatching. The larvae with total length (TL) of less than 10 mm, have single cones only and the eyes were well pigmented. At 10–11 mm TL, most single cones fused to form double cones, with the single and double cones forming a mosaic pattern. From larvae to early juvenile the retina stretches, the cones increase in diameter and rods increase in number. Based on the highest density of the cones in the ventro-temporal region, the visual axis was orientated upforward. The resolving power of the retina in 40–530 mm TL Japanese flounder was found to range from 25.1 to 11.5 min. The results indicated continual improvements in the visual system of the growing fish towards higher resolving power, visual acuity and sensitivity. Project 39970578 supported by the NSFC, and also supported by the Ministry of Education Foundation for University Key Teachers.  相似文献   

18.
Histological study on the ontogeny of the lymphoid organs, kidney, thymus and spleen of Japanese flounder, Paralichthys olivaceus, from hatching to 40d was carried out. The pronephric kidney duct appeared early in hatching although the primordial haemopoietic stem cells wereobserved within a week after hatching. The spleen was first seen after 8d of hatching. The thymus appeared after 15d, situated near the pronephric kidney. Small lymphoid cells appeared during the later phase of the post-larval stage in the sequence of thymus, kidney and spleen. During the 40d of observations, there were no distinct inner or outer zones in thymus and no red or white pulp in spleen. These results suggest that the nonspecific defense immune system plays a very important role in the early larval stage of Japanese flounder.  相似文献   

19.
Histological study on the ontogeny of the lymphoid organs, kidney, thymus and spleen of Japanese flounder, Paralichthys olivaceus, from hatching to 40 d was carried out. The pronephric kidney duct appeared early in hatching although the primordial haemopoietic stem cells were observed within a week after hatching. The spleen was first seen after 8d of hatching. The thymus appeared after 15d, situated near the pronephric kidney. Small lymphoid cells appeared during the later phase of the post-larval stage in the sequence of thymus, kidney and spleen. During the 40d of observations, there were no distinct inner or outer zones in thymus and no red or white pulp in spleen. These results suggest that the nonspecific defense immune system plays a very important role in the early larval stage of Japanese flounder.  相似文献   

20.
Soybean stachyose (SBS) and phytic acid (PA) are anti-nutritional factors (ANF) which have deleterious effects on the growth and digestibility in fish. The present research studied the effects of dietary SBS and PA on the expression of three serine protease genes in the liver of Japanese flounder (Paralichthys olivaceus). These genes are trypsinogen 1 (poTRY), elastase 1 (poEL) and chymotrypsinogen 1 (poCTRY). Eight artificial diets with graded levels of supplemented ANFs were formulated to 4 levels of SBS ...  相似文献   

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