Characterization,Expression Pattern Analysis,and Cellular Localization of Two Homologous MUC2 Genes in Japanese Flounder(Paralichthys olivaceus)

As a member of mucins family,MUC2 is an important component of mucus,which is characterized by tandem and irregular repeat sequences rich in threonine and serine.It is strongly expressed by goblet cells that are involved in innate immunity.Japanese flounder is an economically important marine flatfish.In this study,two homologous genes of MUC2 were identified,named MUC2-1 and MUC2-2.Depending on phylogenetic and structural analysis,MUC2-1 and MUC2-2 were clustered in two clades,respectively.Paralichthys olivaceus MUC2-1 showed a closer relationship with MUC2 in the higher vertebrates.Various healthy tissues were analyzed to determine the expression patterns,and both MUC2 genes showed high expression levels in the gills and intestines.Following Edwardsiella tarda challenge,P.olivaceus MUC2-1 and MUC2-2 both showed significant up-regulated expression in intestine and kidney.Moreover,MUC2-1 was significantly up-regulated in gill cell lines following PGN and polyI:C stimulation,and MUC2-2 was significantly up-regulated in gill cell lines following LPS stimulation.In addition,ISH results revealed that MUC2-1 and MUC2-2 showed the same tissue localization in intestine tissues,but displayed different localization in gill.The siRNA-mediated knockdown of MUC2-1 and MUC2-2 genes in the gill cell lines of Japanese flounder affected the expressions of galnt family genes and smad pathway members that are related to mucosal immunity.The results provided a valuable insight into understanding the functions of MUC2 on mucosal immune system in response to the invasion of bacterial pathogen.

牙鲆(Paralichthys olivaceus)抗缪勒氏管激素Ⅱ型受体基因(amhr2)表达特征分析及功能初探

抗缪勒氏管激素Ⅱ型受体(anti-Müllerian hormone receptorⅡ,Amhr2)是抗缪勒氏管激素(anti-Müllerian hormone,Amh)的特异受体。amhr2基因在鱼类性腺分化和发育中发挥重要作用,更决定了有的鱼种的性别,然而,相关功能研究较为有限。本研究旨在明晰amhr2在我国重要海水养殖鱼类—牙鲆(Paralichthys olivaceus)性腺分化和发育过程中的表达特征,并初步探究其功能。首先,克隆了牙鲆amhr2-CDS序列,共1536bp,编码511个氨基酸。系统发育分析显示牙鲆Amhr2近C端较为保守,与其他硬骨鱼类聚为一枝,并与上游sp1以及下游的prr13和PCBP2在基因组中共定位。蛋白结构分析显示,牙鲆Amhr2包含信号肽、跨膜结构域和保守的酪氨酸蛋白激酶结构域。进而,利用实时荧光定量PCR(q PCR)分析表明,牙鲆amhr2主要表达于性腺,且在精巢中的表达极显著高于卵巢(P<0.01),其在I-V期精巢中持续高表达,而在卵巢中仅在I期高表达,后显著下降(P<0.05)。性腺分化期基因的表达,是对雌核发育组(对照组,20±0.5℃,100%雌性)与雌核发育高温诱导组(HT组,28±0.5℃,100%雄性)鱼苗进行检测的,amhr2在对照组全长(totallength,TL)2cm的实验鱼性腺中表达最高,后逐渐下降;而HT组实验鱼性腺中的表达呈现先上升后下降的趋势,并在精巢开始分化的6cm TL时表达最高。利用Hela细胞进行亚细胞定位分析发现,Amhr2与其配体Amh均定位于细胞质。同时,通过原核表达重组牙鲆Amh,并用其孵育离体性腺组织,q PCR分析显示精巢中amhr2的表达没有显著变化,但卵巢中表达显著上升(P<0.05)。进一步通过双荧光素酶报告分析表明,Amh和Amhr2共转染能够显著抑制雌激素合成的关键芳香化酶基因cyp19a的表达(P<0.01)。综上所述,牙鲆amhr2主要表达于精巢,但在不同性腺发育时期表达不同,且其可能与Amh共同影响cyp19a的转录,对雄性表型形成的启动和性腺发育起作用。

Studies on the edwardsiellosis and characterization of pathogenic bacteria from diseased flounder (Paralichthys olivaceus L.) and turbot (Scophthalmus maximus L.)

Edwardsiellosis of flounder and turbot occurring in different mariculture farms during 2001～ 2004 was examined, including the conditions of disease occurrence, clinical signs and pathological changes. The results showed that all diseased fishes expressed bacterial septicaemia. A total of 148 strains were identified using a combination of traditional physiological and biochemical tests and partial 16S rRNA gene analysis. In addition, the mole fraction G ＋ C ratio of the DNA of representative strain of isolates and serum homology were detected, and pathogenicity tests of isolates were conducted by experimental infection. The results revealed that 148 strains were identified as E. tarda of genus Edwardsiella, all the isolates are of serologic similarity, and have strong pathogenicity to flounder and turbot.

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

The characteristics of a bacterium strain M3, isolated from cultured flounder Paralichthys olivaceus with remarkable external sign of skin ulcer during an epizootic outbreak, indicated that the bacterium belonged to the species Vibrio anguillarum . Challenge by I.M. (intramuscular injection), bath, and oral administration with M3 showed that it was highly pathogenic for Paralichthys olivacues . The LD 50 dose was 5.144×10 3 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.

Identification of candidate piRNAs in the gonads of Paralichthys olivaceus (Japanese flounder)

Piwi-interacting RNA （piRNA） plays an important role in the gonadal development and maintenance of Teleostei. In this study, piRNA libraries derived from the adult gonads of Japanese flounder （Paralichthys ofivaceus） were generated using next-generation sequencing technology. Using zebrafish piRNAs as a reference, 5 865 unique candidate piRNAs were identified; 289 candidate piRNA clusters （PRCs） were generated from the above piRNAs. Among the isolated candidate PRCs, a total of 38 ovary-specific, 45 ovary-bias, 24 testis-specific, and 131 testis-bias PRCs were found. The relative expression levels of seven PRCs were validated through quantitative reverse transcription-polymerase chain reaction. The results of this study will help facilitate exploration of the development and maintenance of the phenotypic sex mechanism in P. olivaceus.

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.

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.

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 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.

Molecular Characterization, Expression Profiles, and Immunostimulation Responses of TRAF6 and TAK1 in Japanese Flounder(Paralichthys olivaceus)

Tumor necrosis factor receptor-associated factor 6(TRAF6) and transforming growth factor-β-activated kinase 1(TAK1) are two important adaptor molecules in Toll-like receptor(TLR) signaling pathway. In this study, TRAF6(PoTRAF6) and TAK1(PoTAK1) were cloned and characterized in Japanese flounder(Paralichthys olivaceus). The full-length cDNA sequence of Po TRAF6 is 1953 bp, with an open reading frame(ORF) of 1713 bp encoding a putative protein of 570 amino acids. PoTRAF6 contains one really interesting new gene(RING) domain, two zinc fingers, one coiled-coil region, and one meprin and TRAF homology(MATH) domain, which shows a high similarity to TRAF6 s in other species. The full-length PoTAK1 cDNA sequence is 2086 bp, with an ORF of 1728 bp that encodes a putative protein of 575 amino acids. PoTAK1 contains a conserved serine/threonine protein kinase catalytic domain and a coiled-coil region. The promoter regions of PoTRAF6 and PoTAK1 were also analyzed to predict several potential transcription factor-binding sites. In addition, the expression patterns of these two genes were examined in developmental stages, different tissues, and challenged samples. PoTRAF6 and PoTAK1 were expressed during the whole developmental stages, and the highest expressions were in intestine and heart, respectively. In challenged embryonic cells with LPS, CpG ODN, and poly I:C, the expressions of PoTRAF6 and PoTAK1 were both up-regulated significantly. These results suggest that PoTRAF6 and Po TAK1 play crucial roles in immune responses and may be involved in the developmental process of Japanese flounder.

Cytogenetic characterization of olive flounder Paralichthys olivaceus: DNA content, karyotype, AgNORs and location of major ribosomal genes

A cytogenetic analysis of Paralichthys olivaceus was carried out using the flow cytometry method for DNA content, silver staining for the nucleolus organizer region （AgNORs） identification and one-color fluorescence in situ hybridization （FISH） for chromosomal mapping of major ribosomal genes. Nuclear DNA content was estimated by flow cytometry method using Gallus domesticus erythrocytes as the internal reference standard. The C-value of this species was （0.737±0.024） pg, and the DNA contents of each chromosome were estimated to be 16.51 Mb to 39.50 Mb after paired according to the average relative length. The FISH probe was made by PCR amplification of a DNA fragment containing internal transcribed spacers ITS1 between 18S and 5.8S ribosomal RNA gene, and labeled by PCR incorporation of bio-16-dUTP. FISH signals and AgNORs were both located on the secondary constrictions of chromosome 1. These results will provide a better understanding of the cytogenetic information of this species and would help for further research of the karyotype evolution in the order Pleuronectiformes.

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.

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 1±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.

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.

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, MyJ5 is the first gene to be expressed during the early stages of flounder development, followed by MyoD and myogenin. The expressions ofMyf5, 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.

Establishment and Characterization of Four Long-Term Cultures of Neural Stem/Progenitor Cell Lines from the Japanese Flounder Paralichthys olivaceus

Neurogenesis is an important progress wherein the neural stem cells(NSCs) differentiate into functional neurons under conductive conditions. Neurogenesis occurs continuously in different areas of the central neural system in adult teleosts compared with adult mammals. Therefore, NSC cell lines must be established to offer a valuable in vitro system for studies on neurogenesis and other related functions. In this study, four cell lines designated as PoB1, PoB2, PoBf and PoBh were established from the brain of the Japanese flounder Paralichthys olivaceus. The cell lines were sub-cultured over 150 times and still grew well in DMEM/F12 medium at 24℃. PoB1, PoB2, PoBf and PoBh were identified as neural stem/progenitor cell lines on the basis of the mRNA expression of nestin and/or aldh111 or slc6 a4 and the formation of neurospheres. The cells transfected with the pEGFP-C1 plasmid showed fluorescent signals with distinct reagent dependencies. The established cell lines from the brain of P. olivaceus offer a valuable system in vitro for the study of neurogenesis, fish neural regulation and endocrinology-related functions.

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 (0.00, 0.40, 0.80 and 1.50%), 4 levels of PA (0.00, 0.20, 0.40 and 0.80), respectively.Japanese flounder (initial weight 2.45 g ± 0.01 g) were fed with these diets for 10 weeks with three replications per treatment. At the end of 10 weeks, supplementation of 0.40% of dietary SBS or PA significantly increased the gene expression of poTRY and poCTRY (P<0.05). The same level of dietary SBS significantly decreased the gene expression of poEL. In comparison with the control group (ANF-free),dietary PA (0.2% and 0.8%) significantly decreased the gene expression of poTRY, poCTRY and poEL (P<0.05). However, excessive supplement of dietary SBS (1.5%) has no significant effects on these gene expressions (P>0.05). These results suggested that dietary SBS and dietary PA could directly affect the serine protease genes at the transcriptional level in Japanese flounder, and these genes'expression was more sensitive to dietary PA than to SBS under the current experimental conditions.

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 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.

Compensatory Growth of Juvenile Brown Flounder Paralichthys olivaceus Following Low Temperature Treatment for Different Periods

We investigated the effects of low temperature(8.5℃) on the growth and feeding rates and feed conversion efficiency of juvenile P.olivaceus with an average initial weight of 3.87 ± 0.06 g(mean ± SE).Fish were exposed to 8.5℃ for 0(control),1,2,3 and 4 weeks,and then to 20℃ for 10,9,8,7 and 6 weeks,respectively.Low temperature clearly led to growth depression.The weight of fish exposed to low temperature for 1 week was restored to that of control,while that of fish exposed to low temperature longer was significantly decreased(P < 0.05).During the entire low-temperature period,specific growth rate,feeding rate and feed conversion efficiency of the fish were significantly lower(P < 0.05) than those of control,while in the whole recovery period,specific growth and average feeding rate were markedly higher(P < 0.05) than those of control.At the end of experiment,only the feeding rate of the fish exposed to low temperature for 1 week was not significantly different from that of control(P > 0.05).Feeding rate and feed conversion efficiency were reduced at low temperature in juvenile P.olivaceus.The compensatory growth of juvenile P.olivaceus may therefore be attributed to the improvement of feeding rate.Our results suggested that growth depression occurs when juvenile P.olivaceus are exposed to low temperature for more than one week.

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）-infected Japanese flounder （Paralichthys olivaceus）, serum proteins from self-healing and sick Japanese flounder were separated by two-dimensional electrophoresis to screen differentially expressed proteins. Protein spots demonstrating changes greater than two-fold in the expression level were digested and further identified in capillary liquid chromatography tandem mass spectrometry （LC-MS/MS）. Two immunityrelevant proteins were thus identified as transferrin and the complement component C3 of Japanese flounder. These findings suggest that the two proteins may play important roles in the self-healing of lymphocystis in Japanese flounder. This is an important theoretical foundation to promote self-healing in LCDV-infected Japanese flounder by improving their innate immunity.

Characterization,tissue distribution,and expression of neuropeptide Yin 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.