Cloning and stage-specific expression of CK-M1 gene during metamorphosis of Japanese flounder, Paralichthys olivaceus

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.     

Induction of gyno-tetraploidy in Japanese flounder Paralichthys olivaceus

Journal of Oceanology and Limnology - Tetraploid fish are important for mass production of triploids in polyploid breeding. In this study, we reported a novel protocol for artificial induction of...     

Vertebral deformities in hatchery-reared and wild-caught juvenile Japanese flounder,Paralichthys olivaceus

The present study compared vertebral deformities of hatchery-reared and wild-caught juvenile Japanese flounder,Paralichthys olivaceus.A total of 362 hatchery-reared flounder(total length 122.5-155.8 mm) were collected from three commercial hatcheries located in Yantai,East China,and 89 wild fish(total length 124.7-161.3 mm) were caught off Yangma Island near Yantai City(37°27′N,121°36′E).All the fish were dissected,photographed,and images of the axial skeleton were examined for vertebral deformities.Compared with wild-caught flounder in which no deformed vertebrae were detected,48(13.3%) hatcheryreared fish had deformed vertebrae.The deformities were classified as compression,compression-ankylosis,and dislocation-ankylosis.The vertebral deformities were mainly localized between post-cranial vertebra 1and 3,with vertebrae number 1 as the most commonly deformed.The causative factors leading to vertebral deformities in reared Japanese flounder may be related to unfavorable temperature conditions,inflammation,damage,or rupture to the intervertebral ligaments under rearing conditions.Furthermore,no significant difference in the total number of vertebral bodies was observed between wild-caught(38.8±0.4) and hatchery-reared flounder(38.l±0.9)(P0.05).However,the number of vertebral bodies of hatchery-reared and wild-caught flounder ranged from 35 to 39 and from 38 to 39,respectively.     

Characterization,tissue distribution,and expression of neuropeptide Y in olive flounder Paralichthys olivaceus

Neuropeptide Y(NPY) is a 36-amino acid peptide of the neuropeptide Y family that plays key roles in the regulation of food intake. In this study,we focused on NPY m RNA expression changes around feeding time and during food deprivation in olive flounder. The olive flounder NPY m RNA levels were analyzed in different tissues and a high level of expression was detected in the brain. We also demonstrated a correlation between NPY expression levels in the brain and feeding schedule. NPY expression levels in olive flounder maintained on a daily scheduled feeding regimen increased shortly before feeding and decreased after the scheduled feeding time. Compared with the-1 h group before feeding,NPY expression in the 3 h group after feeding decreased significantly( P <0.05). Food deprivation led to an 81.7% decrease in NPY m RNA levels in the 24 h fasted group(P <0.05) and a 91.7% decrease in the 48 h fasted group(P <0.05). Therefore,our study demonstrates that NPY expression is associated with food intake in olive flounder. This result reveals the function of NPY in regulating food intake and its potential importance in olive flounder aquaculture.     

Development of retinal structure and visual acuity in Japanese flounder (Paralichthys olivaceus)

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.     

Proteomic aspects of infection by lymphocystis disease virus in Japanese flounder (Paralichthys olivaceus)

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.     

Effects of dietary soybean stachyose and phytic acid on gene expressions of serine proteases in Japanese flounder (Paralichthys olivaceus)

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 ...     

Histological change and heat shock protein 70 expression in different tissues of Japanese flounder Paralichthys olivaceus in response to elevated temperature

High temperature influences the homeostasis of fish.We investigated the effects of elevated temperature on tissues of Japanese flounder {Paralichthys olivaceus) by analyzing the histology and heat shock protein 70(hsp70) expression of fish reared in warm conditions.In this study,temperature was increased at l±0.5℃/day starting at 24±0.5℃,and was kept at that temperature for 5 days before the next rise.After raising temperature at the rate up to 32±0.5℃,tissue samples from midgut,spleen,stomach,liver,muscle,gill,heart,trunk kidney and brain were collected for histological analysis and mRNA assay.Almost all the tissues showed changes in morphological structure and hsp70 level at 32±0.5℃.Histological assessment of the tissues indicated that the gill had the most serious damage,including highly severe epithelial lifting and edema,curved tips and hyperemia at the ending of the lamellars,desquamation and necrosis.The next most severe damage was found in liver and kidney.The hsp70 levels in all the tissues first increased and then decreased.The gut,stomach,muscle,heart,and brain had the highest expressions in 6 h,whereas the spleen,liver,gill and kidney had the highest expressions in 2 h.Therefore,tissues with the most significant lesions(especially gill and liver) responded much earlier(2 h) in hsp70 expression than other tissues,and these tissues demonstrated the most marked histological disruption and elevated mRNA levels,making them ideal candidates for further studies on the thermal physiology of this species.     

Detection of lymphocystis disease virus in Japanese flounder Paralichthys olivaceus and other marine teleosts from northern China

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.     

Distribution and expression in vitro and in vivo of DNA vaccine against lymphocystis disease virus in Japanese flounder(Paralichthys olivaceus)

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 ana...     

Mutations in exons of the CYP17-II gene affect sex steroid concentration in male Japanese flounder (Paralichthys olivaceus)

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.     

Distribution and expression in vitro and in vivo of DNA vaccine against lymphocystis disease virus in Japanese flounder (Paralichthys olivaceus)

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.     

Screening of eye-position related genes with DD-RT-PCR and RDA in the hybrids between Japanese flounder Paralichthys olivaceus and stone flounder Kareius bicoloratus

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...     

Hybrids between olive flounder Paralichthys olivaceus and stone flounder Kareius bicoloratus: karyotype, allozyme and RAPD analyses

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.     

Tissue-specific bioaccumulation and oxidative stress responses in juvenile Japanese flounder (Paralichthys olivaceus) exposed to mercury

To understand mercury (Hg) toxicity in marine fish, we measured Hg accumulation in juvenile Japanese flounder (Paralichthys olivaceus) and assessed the effects on growth and antioxidant responses. After Hg exposure (control, 5, 40, and 160 μg/L Hg) for 28 d, fish growth was significantly reduced. The accumulation of Hg in fish was dose-dependent and tissue-specific, with the maximum accumulation in kidney and liver, followed by gills, bone, and muscle. Different antioxidants responded differently to Hg exposure to cope with the induction of lipid peroxidation (LPO), which was also tissue-specific and dosedependent. As Hg concentration increased, superoxide dismutase (SOD) and catalase (CAT) activities increased significantly, whereas glutathione S-transferase (GST) activity and glutathione (GSH) levels decreased significantly in the gills. SOD and glutathione peroxidase (GPx) activities and the GSH level increased significantly in the liver. SOD activity and GSH levels increased significantly, but CAT activity decreased significantly with an increase in Hg concentration in the kidney. LPO was induced significantly by elevated Hg in the gills and kidney but was least affected in the liver. Therefore, oxidative stress biomarkers in gills were more sensitive than those in the liver and kidney to Hg exposure. Thus, the gills have potential as bioindicators for evaluating Hg toxicity in juvenile flounder.     

Molecular identification,expression pattern,and in-vitro bioactivity analysis of insulin-like growth factor 2 in olive flounder Paralichthys olivaceus

Insulin-like growth factors (IGFs) are key regulators of development and growth. Here, we characterized the igf2 gene from olive flounder (Paralichthys olivaceus) and determined its temporal and spatial expression. We set up an in-vitro protein expression system in eukaryotic human embryonic kidney (HEK293T) cells and explored its effects on cell proliferation. The flounder igf2 cDNA contained a 648-bp open reading frame (ORF) encoding a protein of 215 amino acids (aa), which spanned the complete signal peptide (47 aa), mature peptide (70 aa), and E domain (98 aa). In adult flounder, igf2 mRNA was detected in all selected tissues. In early development, igf2 mRNA was detected throughout development from unfertilized eggs to hatching-stage embryos. In-situ hybridization analysis indicated that igf2 mRNA was specially expressed in the brain region, floor plate, hypochord, otic vesicle, and pectoral fin during embryogenesis. Western blotting analysis indicated that the soluble recombinant flounder IGF2 protein was successfully produced through eukaryotic expression in HEK293T cells. In addition, the recombinant IGF2 protein significantly promoted the proliferation of human cervical carcinoma (HeLa) and HEK293T cells. These results provide new information about the structural and functional conservation, expression patterns, and biological activity of the igf2 in teleosts.

     

Effects of dietary soy isoflavones on feed intake, growth performance and digestibility in juvenile Japanese flounder (Paralichthys olivaceus)

An 8-week feeding trial was conducted to investigate the effects of dietary soy isoflavones on feeding intake,growth performance,and digestion of juvenile Japanese flounder(Paralichthys olivaceus).Four isonitrogenous(49% crude protein) and isoenergetic(20.1 MJ kg-1) diets were formulated to contain four graded levels of soy isoflavones,namely,0,1,4 and 8 g soy isoflavones in 1 kg of diet.Each diet was randomly fed to triplicate tanks of fish(Initial average weight:2.58 g ± 0.01 g),and each tank was stocked with 35 fish.No significant difference was observed among diets with levels of 0,1 and 4 g kg-1 soy isoflavones in feed intake,weight gain,feed efficiency ratio(FER),proximate composition of fish whole body and apparent digestibility coefficients(ADC) of nutrients and energy(P>0.05).However,high dietary soy isoflavones level(8 g kg-1) significantly depressed weight gain,FER,whole-body crude lipid content of fish and ADC of nutrients(P<0.05).These results indicate that high level of dietary soy isoflavones(above 4 g kg-1) significantly depresses growth responses and FER of Japanese flounder.However,as the content of soy isoflavones in soybean meal is around 1 to 3 g kg-1,the adverse effects might be neglected when soybean products are used as a fish feed ingredient.     

A VIBRIO ANGUILIARUM STRAIN ASSOCIATED WITH SKIN ULCER ON CULTURED FLOUNDER, PARALICHTHYS OLIVACEUS

The characteristics of a bacterium strain M3, isolated from cultured flounderParalichthys olivaceus with remarkable external sign of skin ulcer during an epizootic outbreak, indicated that the bacterium belonged to the speciesVibrio anguillarum. Challenge by I.M. (intramuscular injection), bath, and oral administration with M3 showed that it was highly pathogenic forParalichthys olivacues. The LD₅₀ dose was 5.144×10³ CFU/ per fish infection by I.M. injection. Recovered inoculated bacteria from the surviving fish revealed that the asymptomatic carriers could be a latent contagious source. Study of the effect of bacterial culture CFS (cell-free-supernatant) showed that the exotoxins produced by M3 play an important role in its pathogenicity for flounder. The resistance of M3 to 36 out of 41 antibiotics indicated that the bacterial disease outbreak was mainly attributable to the frequent and excessive use of antimicrobial agents; and that vaccination would be an effective precaution against bacterial disease. Contribution No. 4107 from Institute of Oceanology, Chinese Academy of Science. Contribution No. 270 from Experimental marine Biology Laboratory, CAS. Funded by projects under the Major State Basic Research Development Program, Grant G1999012003.