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 oliv...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.展开更多
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 sho...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.展开更多
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 t...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.展开更多
Appropriate pathogenic bacteria were isolated from diseased (or dead) flounder (Paralichthys olivaceus L) occurring in a mariculture farms of Qinhuangdao, which all diseased fishes expressed bacterial septicaemia. The...Appropriate pathogenic bacteria were isolated from diseased (or dead) flounder (Paralichthys olivaceus L) occurring in a mariculture farms of Qinhuangdao, which all diseased fishes expressed bacterial septicaemia. The phenotypic information of the 12 pure cultures was studied extensively, including morphological characteristics, colony characteristics in different media, physiological and biochemi- cal characteristics, and the mole fraction G+C ratio of the DNA for representative strain. The results show that the isolates belong to a new species of Vibrio, and are designated as Vibrio qinhuangdaora sp.nov. by the area from which the diseased flounders are collected (Qinhuangdao) based on its biological properties following Rules of International Code of Nomenclature of Bacteria. At the same time, studies on the serum homology of isolates, and pathogenicity of isolates were conducted by experimental infection. The results show that all the isolates are of serologic similarity, and the isolates have strong pathogenicity to flounder. The studies can extensively reflect the main biological properties of this new species of Vibrio.展开更多
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-dimensio...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.展开更多
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 durin...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.展开更多
Some germ cell marker genes, such as vasa, nanos, and dead end( dnd), have been identified in fish. Recently, lymphocyte antigen 75(Ly75/CD205) has been identified as a mitotic germ cell-specific cell-surface marker i...Some germ cell marker genes, such as vasa, nanos, and dead end( dnd), have been identified in fish. Recently, lymphocyte antigen 75(Ly75/CD205) has been identified as a mitotic germ cell-specific cell-surface marker in several fish species. In this study, the Japanese flounder Paralichthys olivaceus ly75 homolog( ly75) was cloned and its expression pattern in gonads was analyzed. The full-length c DNA of ly75 was 7 346 bp, with an open reading frame(ORF) of 5 229 bp. The ORF encoded a protein containing 1 742 amino acids with a predicted molecular mass of 196.89 k Da. In adult tissues, ly75 transcripts were detected in all analyzed tissues but abundantly in the testis. In in-situ hybridization analyses, ly75 mRNA was predominantly localized in oocytes in the ovary and spermatogonia in the testis, but ly75 mRNA was not detected in oogonia, spermatocytes, spermatids, or spermatozoa. These results indicated that ly75 could be a potential germ cell-specific marker in P. olivaceus, as in other fishes.展开更多
In vitro acute cytotoxicity of neonicotinoid insecticide imidacloprid (IMI) to the gill cell line of flounder (FG) that collected in the gill of Paralichthys olivaceus, was examined by 3 widely used endpoint bioassays...In vitro acute cytotoxicity of neonicotinoid insecticide imidacloprid (IMI) to the gill cell line of flounder (FG) that collected in the gill of Paralichthys olivaceus, was examined by 3 widely used endpoint bioassays: NR (neutral red), MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) and TCP (total cell protein). The result shows that the IMI increased at concentrations ≥0.5 μg/ml. The IC50 value of NR, MTT, and TCP was 41.86, 38.46, and 39.08 μg/ml, respectively. The ultrastructural observation revealed that the mitochondria of the cells exposed to 60 μg/ml IMI for 48 h were severely damaged, swollen or disrupted, while their nuclei and rough endoplasmic reticulum (RER) remained normal. This would suggest that the mitochondria are probably the primary target of IMI.展开更多
In aquaculture,selective breeding for survival till harvest have become an alternative strategy for improving disease resistance and production.However,knowledge of genetic parameters of harvest survival,e.g.,heritabi...In aquaculture,selective breeding for survival till harvest have become an alternative strategy for improving disease resistance and production.However,knowledge of genetic parameters of harvest survival,e.g.,heritability and genetic correlations between survival and growth rate traits,is still scarce.The aims of this study were to estimate genetic parameters for harvest survival and growth rate traits under commercial farming conditions in olive flounder(Paralichthys olivaceus).Harvest survival was defined as a binary trait;growth traits were measured as average daily gain(ADG),specific growth rate(SGR),daily growth coefficient(DGC)and body weight(BW).Data from a population of 241 full-sib families(involving 39,904 individuals,four generations)were used.Heritabilities of survival were low but significant,which were 0.15±0.04 and 0.22±0.01 on observed and underlying scale,respectively.Heritability estimates for ADG,SGR and DGC were medium to high,which were 0.33±0.06,0.83±0.07,0.58±0.07,respectively.While the heritability of BW is of low magnitude(0.17±0.08).The genetic correlations between harvest survival and three growth rate traits(i.e.,ADG,SGR and DGC)were very strong(ranging from 0.66 to 0.79),which is an exciting result.However,the genetic correlation between harvest survival and BW was much lower(0.17±0.08).These results suggest that selection for harvest survival would consequentially result in concomitant increase of growth rate,and vice versa.Our findings revealed novel insights into the genetic improvement of growth rate and harvest survival through genetic selection in olive flounder.展开更多
The aim of this study was to evaluate genetic parameters of growth traits for improving selective breeding in olive flounder(Paralichthys olivaceus).A total of 82 full-sib families from 2 year-classes with complete pe...The aim of this study was to evaluate genetic parameters of growth traits for improving selective breeding in olive flounder(Paralichthys olivaceus).A total of 82 full-sib families from 2 year-classes with complete pedigree information were used to estimate heritabilities and genetic/phenotypic correlations for 4 growth performance traits(body weight BW,total length TL,body height BH and condition factor K)based on uni-trait and multi-trait linear mixed models,respectively.Data were analyzed by the restricted maximum likelihood(REML)method.The heritabilities of BW,TL and BH were 0.81,0.70 and 0.61,which are relatively high,however,heritability for K is low(0.11).For all growth traits,the common full-sib effects(c^(2))were at a low to moderate level(0.14–0.20);the maternal effects(m^(2))of the traits(except K)were also detected(0.15–0.19).The phenotypic and genetic correlations among BW,TL and BH were highly positive(0.70–0.91),however,the correlations between K and the other 3 traits was low or negligible(−0.22–0.26).According to our results,these traits(except K)could be selected and enhanced simultaneously.In addition,direct selection based on TL or BH,which is an easily measurable trait,will be more favorable than selection on BW.Moreover,our results indicated that growth parameters estimated in the juvenile stage could be used as a predictor of growth performance at later growth stages.展开更多
基金Supported by the National High Technology Research and Development Program of China(863 Program)(Nos.2012AA10A408,2012AA10A402)the National Natural Science Foundation of China(No.41276171)the National Flatfish Industry System Construction Program(No.nycytx-50-G03)
文摘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.
文摘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.
文摘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.
基金supported by the National“863”Project of China under contract No.2001 AA628020the National Natural Science Foundation of China un-der contract No.30271016+1 种基金Projects under the Major State Basic Research Development Program of China under contract No.G1999012002Science Tech-nology Department Project of Hebei Province under contract No.302431.
文摘Appropriate pathogenic bacteria were isolated from diseased (or dead) flounder (Paralichthys olivaceus L) occurring in a mariculture farms of Qinhuangdao, which all diseased fishes expressed bacterial septicaemia. The phenotypic information of the 12 pure cultures was studied extensively, including morphological characteristics, colony characteristics in different media, physiological and biochemi- cal characteristics, and the mole fraction G+C ratio of the DNA for representative strain. The results show that the isolates belong to a new species of Vibrio, and are designated as Vibrio qinhuangdaora sp.nov. by the area from which the diseased flounders are collected (Qinhuangdao) based on its biological properties following Rules of International Code of Nomenclature of Bacteria. At the same time, studies on the serum homology of isolates, and pathogenicity of isolates were conducted by experimental infection. The results show that all the isolates are of serologic similarity, and the isolates have strong pathogenicity to flounder. The studies can extensively reflect the main biological properties of this new species of Vibrio.
基金Supported by the National High Technology Research and Development Program of China (863 program) (No. 2006AA100309)
文摘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.
基金Supported by the National High Technology Research and Development Program of China(863 Program)(Nos.2012AA10A408,2012AA092203)the National Natural Science Foundation of China(No.31128017)the National Key Basic Program of Science and Technology-Platforms of Aquaculture Stock Resources(No.2006DKA30470017)
文摘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.
基金Supported by the National Natural Science Foundation of China(Nos.31372514,31472264,31572602)the China Agriculture Research System(No.CARS-47)+1 种基金the Scientific and Technological Innovation Project Financially Supported by Qingdao National Laboratory for Marine Science and Technology(Nos.2015ASKJ02,2015ASKJ02-03-03)the Youth Innovation Promotion Association CAS and Chinese Academy of Science and Technology Service Network Planning(No.KFJ-EW-STS-060)
文摘Some germ cell marker genes, such as vasa, nanos, and dead end( dnd), have been identified in fish. Recently, lymphocyte antigen 75(Ly75/CD205) has been identified as a mitotic germ cell-specific cell-surface marker in several fish species. In this study, the Japanese flounder Paralichthys olivaceus ly75 homolog( ly75) was cloned and its expression pattern in gonads was analyzed. The full-length c DNA of ly75 was 7 346 bp, with an open reading frame(ORF) of 5 229 bp. The ORF encoded a protein containing 1 742 amino acids with a predicted molecular mass of 196.89 k Da. In adult tissues, ly75 transcripts were detected in all analyzed tissues but abundantly in the testis. In in-situ hybridization analyses, ly75 mRNA was predominantly localized in oocytes in the ovary and spermatogonia in the testis, but ly75 mRNA was not detected in oogonia, spermatocytes, spermatids, or spermatozoa. These results indicated that ly75 could be a potential germ cell-specific marker in P. olivaceus, as in other fishes.
基金the Ministry of Science and Technology (MOST) of China (No. 2001AA649040)the Scientific-Research Fund of Qingdao University of Science and Technology (No.0022168)
文摘In vitro acute cytotoxicity of neonicotinoid insecticide imidacloprid (IMI) to the gill cell line of flounder (FG) that collected in the gill of Paralichthys olivaceus, was examined by 3 widely used endpoint bioassays: NR (neutral red), MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) and TCP (total cell protein). The result shows that the IMI increased at concentrations ≥0.5 μg/ml. The IC50 value of NR, MTT, and TCP was 41.86, 38.46, and 39.08 μg/ml, respectively. The ultrastructural observation revealed that the mitochondria of the cells exposed to 60 μg/ml IMI for 48 h were severely damaged, swollen or disrupted, while their nuclei and rough endoplasmic reticulum (RER) remained normal. This would suggest that the mitochondria are probably the primary target of IMI.
基金supported by Central Public-interest Scientific Institution Basal Research Fund CAFS(2016HY-ZD0201).
文摘In aquaculture,selective breeding for survival till harvest have become an alternative strategy for improving disease resistance and production.However,knowledge of genetic parameters of harvest survival,e.g.,heritability and genetic correlations between survival and growth rate traits,is still scarce.The aims of this study were to estimate genetic parameters for harvest survival and growth rate traits under commercial farming conditions in olive flounder(Paralichthys olivaceus).Harvest survival was defined as a binary trait;growth traits were measured as average daily gain(ADG),specific growth rate(SGR),daily growth coefficient(DGC)and body weight(BW).Data from a population of 241 full-sib families(involving 39,904 individuals,four generations)were used.Heritabilities of survival were low but significant,which were 0.15±0.04 and 0.22±0.01 on observed and underlying scale,respectively.Heritability estimates for ADG,SGR and DGC were medium to high,which were 0.33±0.06,0.83±0.07,0.58±0.07,respectively.While the heritability of BW is of low magnitude(0.17±0.08).The genetic correlations between harvest survival and three growth rate traits(i.e.,ADG,SGR and DGC)were very strong(ranging from 0.66 to 0.79),which is an exciting result.However,the genetic correlation between harvest survival and BW was much lower(0.17±0.08).These results suggest that selection for harvest survival would consequentially result in concomitant increase of growth rate,and vice versa.Our findings revealed novel insights into the genetic improvement of growth rate and harvest survival through genetic selection in olive flounder.
基金This work was supported by Central Public-interest Scientific Institution Basal Research Fund,CAFS,China(NO.2016HY-ZD0201)Taishan Scholar Project Fund of Shandong of China.
文摘The aim of this study was to evaluate genetic parameters of growth traits for improving selective breeding in olive flounder(Paralichthys olivaceus).A total of 82 full-sib families from 2 year-classes with complete pedigree information were used to estimate heritabilities and genetic/phenotypic correlations for 4 growth performance traits(body weight BW,total length TL,body height BH and condition factor K)based on uni-trait and multi-trait linear mixed models,respectively.Data were analyzed by the restricted maximum likelihood(REML)method.The heritabilities of BW,TL and BH were 0.81,0.70 and 0.61,which are relatively high,however,heritability for K is low(0.11).For all growth traits,the common full-sib effects(c^(2))were at a low to moderate level(0.14–0.20);the maternal effects(m^(2))of the traits(except K)were also detected(0.15–0.19).The phenotypic and genetic correlations among BW,TL and BH were highly positive(0.70–0.91),however,the correlations between K and the other 3 traits was low or negligible(−0.22–0.26).According to our results,these traits(except K)could be selected and enhanced simultaneously.In addition,direct selection based on TL or BH,which is an easily measurable trait,will be more favorable than selection on BW.Moreover,our results indicated that growth parameters estimated in the juvenile stage could be used as a predictor of growth performance at later growth stages.
