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.     

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.     

Establishment and characterization of a testicular Sertoli cell line from olive flounder Paralichthys olivaceus

The culture of Sertoli cells has become an indispensable resource in studying spermatogenesis.A new Sertoli cell line(POSC) that consisted predominantly of fibroblast-like cells was derived from the testis of the olive flounder Paralichthys olivaceus and sub-cultured for 48 passages.Analysis of the mtDNA COI gene partial sequence confirmed that the cell line was from P.olivaceus.Cells were optimally maintained at 25℃ in DMEM/F12 medium supplemented with fetal bovine serum,basic fibroblast growth factor,and epidermal growth factor.The growth curve of POSC showed a typical "S" shape.Chromosome analysis revealed that the cell line possessed the normal P.olivaceus diploid karyotype of 2n=48t.POSC expressed dmrt1 but not vasa,which was detected using RT-PCR and sequencing.Immunocytochemistry revealed that the cells exhibited the testicular Sertoli cell marker FasL.Therefore,POSC appeared to consist of testicular Sertoli cells.Bright fluorescent signals were observed after the cells were transfected with pEGFP-N3 plasmid,with the transfection efficiency reaching 10%.This research not only offers an ideal model for further gene expression and regulation studies on P.olivaceus,but also serves as valuable material in studying fish spermatogenesis,Sertoli cell-germ cell interactions,and the mechanism of growth and development of testis.     

Analysis of IL-2-1ike factor in lymphocyte culture supernatant of olive flounder, Paralichthys oliveaceus

To study immune mechanism of fish lymphocyte we performed a proliferation assay and ELISA using monoclonal antibody against human IL-2. The result showed that an interleukin-2 (IL-2)-like factor was detected in the supernatant of plant haemoglutinin (PHA)-stimulated lymphocyte culture from peripheral blood, spleen and head kidney of olive flounder,Paralichthys olivaceus. The quantities of IL-2-like factor in the supernatant from different lymphoid tissues were quite different. The IL-2 like factor in the supernatant from cultured head kidney lymphocytes was much higher than those of peripheral blood lymphocytes and spleen lymphocytes (P<0.01). The IL-2 activity was found in either mouse thymocyte proliferation assay or flounder head kidney lymphocyte proliferation assay and shown to have obvious enhancing effect on proliferation of the above two types of cell. The recombinant human IL-2, (rhIL-2) was able to stimulate flounder thymocyte proliferation and used to detect the IL-2 receptor (IL-2R) on the surface of flounder lymphocyte. The cross-reaction between the lymphocytes of flounder peripheral blood and CD25(IL-2R) was detected with flow cytometry and shown that the percentage of CD25-positive cell in peripheral blood was 7.74±0.67%. This work was supported by National “973” Project G1999012003, G19999012006.     

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.     

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.     

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.     

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.     

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.     

Effects of florfenicol exposure on growth,development and antioxidant capacity of flounder Paralichthys olivaceus larvae at different developmental stages

Antibiotics are widespread in various environmental media,and may pose a potential threat to aquatic ecosystems and non-target aquatic organisms.Florfenicol(FLO) is one of the most commonly used antibiotics in aquaculture,and extensively used to substitute chloramphenicol with its strong sterilization and low adverse ef fect.In this study,flounder Paralichthys olivaceus,an important economic fish species in seawater was used as an experimental subject.Five exposure concentrations of FLO(including environmentrelated concentrations) were set at 0,0.01,0.1,1,and 10 mg/L.Ef fects of FLO exposure for 168 h on growth and development,motor behavior,antioxidant enzyme activity,malondialdehyde(MDA) content,and thyroid hormone level of P.olivaceus larvae were studied in pre-larvae(1 dpf) and post-larvae(20 dpf).The results show that the short-term FLO exposure could promote the larvae growth to some degrees,but inhibit them as the exposure time prolonged.For pre-larvae,FLO at 0.01 mg/L could stimulate the motor nerve system and increase the swimming ability,but inhibited it at 1 mg/L.With the increasing dosage of FLO,the superoxide dismutase(SOD) and MDA contents were elevated,reaching the maximum in the 1 mg/L FLO group.The pre-larvae were more sensitive than the post-larvae to FLO in the environment,and the growth and immune resistance could be damaged with long exposure.Post-larvae were more tolerant to external pollutants,FLO at 1 mg/L could promote the motor behavior and reduce SOD and MDA contents.Therefore,FLO can be used as an antibiotic at a proper concentration but as a drug to prevent disease in a long-term way.     

Quantitative comparison of the expression of myogenic regulatory factors in flounder (Paralichthys olivaceus) embryos and adult tissues

MyoD, Myf5, and myogenin are myogenic regulatory factors that play important roles during myogenesis. It is thought that MyoD and Myf5 are required for myogenic determination, while myogenin is important for terminal differentiation and lineage maintenance. To better understand the function of myogenic regulatory factors in muscle development of flounder, an important economic fish in Asia, real-time quantitative RT-PCR was used to characterize the expression patterns of MyoD, Myf5, and myogenin at early stages of embryo development, and in different tissues of the adult flounder. The results show that, Myf5 is the first gene to be expressed during the early stages of flounder development, followed by MyoD and myogenin. The expressions of Myf5, yoD, and myogenin at the early stages have a common characteristic: expression gradually increased to a peak level, and then gradually decreased to an extremely low level. In the adult flounder, the expression of the three genes in muscle is much higher than that in other tissues, indicating that they are important for muscle growth and maintenance of grown fish. During embryonic stages, the expression level of MyoD might serve an important role in the balance between muscle cell differentiation and proliferation. When the MyoD expression is over 30% of its highest level, the muscle cells enter the differentiation stage.     

Quantitative comparison of the expression of myogenic regulatory factors in flounder (Paralichthys olivaceus)embryos and adult tissues

MyoD,Myf5,and myogenin are myogenic regulatory factors that play important roles during myogenesis.It is thought that MyoD and Myf5 are required for myogenic determination,while myogenin is important for terminal differentiation and lineage maintenance.To better understand the function of myogenic regulatory factors in muscle development of flounder,an important economic fish in Asia,real-time quantitative RT-PCR was used to characterize the expression patterns of MyoD,Myf5, and myogenin at early stages of embryo development,and in different tissues of the adult flounder.The results show that,Myf5 is the first gene to be expressed during the early stages of flounder development, followed by MyoD and myogenin.The expressions of Myf5,MyoD,and myogenin at the early stages have a common characteristic:expression gradually increased to a peak level,and then gradually decreased to an extremely low level.In the adult flounder,the expression of the three genes in muscle is much higher than that in other tissues,indicating that they are important for muscle growth and maintenance of grown fish.During embryonic stages,the expression level of MyoD might serve an important role in the balance between muscle cell differentiation and proliferation.When the MyoD expression is over 30%of its highest level,the muscle cells enter the differentiation stage.     

Quantitative trait loci detection of E dwardsiella tarda resistance in Japanese flounder Paralichthys olivaceus using bulked segregant analysis

In recent years, Edwardsiella tarda has become one of the most deadly pathogens of Japanese fl ounder( Paralichthys olivaceus), causing serious annual losses in commercial production. In contrast to the rapid advances in the aquaculture of P. o livaceus, the study of E. tarda resistance-related markers has lagged behind, hindering the development of a disease-resistant strain. Thus, a marker-trait association analysis was initiated, combining bulked segregant analysis(BSA) and quantitative trait loci(QTL) mapping. Based on 180 microsatellite loci across all chromosomes, 106 individuals from the F1333(♀: F0768 ×♂: F0915)(Nomenclature rule: F+year+family number) were used to detect simple sequence repeats(SSRs) and QTLs associated with E. tarda resistance. After a genomic scan, three markers(Scaffold 404-21589, Scaffold 404-21594 and Scaffold 270-13812) from the same linkage group(LG)-1 exhibited a signifi cant difference between DNA, pooled/bulked from the resistant and susceptible groups( P 0.001). Therefore, 106 individuals were genotyped using all the SSR markers in LG1 by single marker analysis. Two different analytical models were then employed to detect SSR markers with different levels of signifi cance in LG1, where 17 and 18 SSR markers were identifi ed, respectively. Each model found three resistance-related QTLs by composite interval mapping(CIM). These six QTLs, designated q E1–6, explained 16.0%–89.5% of the phenotypic variance. Two of the QTLs, q E-2 and q E-4, were located at the 66.7 c M region, which was considered a major candidate region for E. tarda resistance. This study will provide valuable data for further investigations of E. tarda resistance genes and facilitate the selective breeding of disease-resistant Japanese fl ounder in the future.     

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.     

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...     

UP-REGULATION OF HEPATIC RECEPTOR FOR GROWTH HORMONE IN THE FLOUNDER （PARALICHTHYS OLIVACEUS） AFTER ORAL ADMINISTRATION WITH EXOGENOUS GH

The iodination efficiency of salmon GH (sGH) was 38.82%,using a modification of the chloramine-T method. The specific activity of the ^125I-sGH was about 40 μCi/μg protein. The results of binding assay showed a single class of high affinity and low-capacity binding site in flounder liver. Long-term administration with exogenous GH can induce the up-regulation of hepatic GH receptor in total binding capacity though there was no significant difference of association constant among any groups. Con-sidering that there was no significant difference in capacity of free binding sites of livers from control and experimental fish, this result also indicated that the liver from experimental fish, compared to that from control fish, had more occupied binding sites.