Due to the discharge of industrialwastewater,urban domestic sewage,and intensive marine aquaculture tailwater,nitrate(NO_(3)^(−))pollution has emerged as a significant issue in offshore waters.Nitrate pollution affect...Due to the discharge of industrialwastewater,urban domestic sewage,and intensive marine aquaculture tailwater,nitrate(NO_(3)^(−))pollution has emerged as a significant issue in offshore waters.Nitrate pollution affects aquatic life and may interact with other pollutants,leading to comprehensive toxicity.Cadmium(Cd^(2+))is the most widespread metal contaminant,adversely affecting aquatic life in the coastal waters of China.Despite this,few studies have focused on the synergistic toxicity of NO_(3)^(−)and Cd^(2+)in marine organisms.This study conducted a 30-day exposure experiment on marine Japanese flounder(Paralichthys olivaceus)to explore the synergistic toxicity of NO_(3)^(−)and Cd^(2+).Our results demonstrated that the exposure to Cd^(2+)alone induced slight histopathological changes in the liver.However,malformations such as hepatic vacuolar degeneration and sinusoid dilatationwere exacerbated under co-exposure.Moreover,co-exposure induced the downregulation of antioxidants and the upregulation of the product malonaldehyde(MDA)from lipid peroxidation,indicating potent oxidative stress in the liver.The increased mRNA expression of IL-8,TNF-α,and IL-1β,along with the decreased expression level of TGF-β,indicated a synergistic inflammatory response in the organisms.Furthermore,the co-exposure led to an abnormal expression of P53,caspase-3,caspase-9,Bcl-2,and Bax,and disturbed the apoptosis in the liver through TUNEL staining analysis.Overall,our results imply that co-exposure synergistically affects inflammation,redox status,and apoptosis in flounders.Therefore,the findings from this study provide valuable perspectives on the ecological risk assessment of marine teleosts co-exposure to NO_(3)^(−)and Cd^(2+).展开更多
Circular RNAs(circRNAs)are a type of endogenous competitive RNA that can perform various functions and regulate a number of biological activities in organisms.Many studies have shown that circRNAs can modulate skeleta...Circular RNAs(circRNAs)are a type of endogenous competitive RNA that can perform various functions and regulate a number of biological activities in organisms.Many studies have shown that circRNAs can modulate skeletal muscle development in animals.However,the effects of circRNAs on the skeletal muscle development of Japanese flounder and its regulatory mechanisms remain unclear.We screened circRNA_2111 from previously obtained transcriptomic data related to Japanese flounder skeletal mus-cle development.Derived from the tefb gene,it has been designated as circtefb.Circtefb was formed by the reverse splicing of two exons of tefb.Circtefb was more stable than its corresponding linear RNA.There was a significant difference in the expression of circtefb at different stages P1(90 d),P2(12 months)and P3(24 months)of Japanese flounder(p<0.05).Over-expression of circtefb in Japanese flounder myocytes resulted in a significant upregulation of proliferation marker genes ccnd1,ccnd2a and pcna(p<0.05),suggesting that it promoted proliferation of Japanese flounder myocytes.The expressions of ccnd1,ccnd2a,and pcna were signifi-cantly decreased after the transfection of pol-miR-138 into Japanese flounder myocytes(p<0.05).In addition,circtefb can suppress pol-miR-138 function by binding to it,which was detected by dual-Luciferase reporter assay and co-transfection of circtefb and pol-miR-138,thereby promoting the proliferation of Japanese flounder myocytes.The results indicated the function of circRNAs in Japanese flounder muscle development,,which is also helpful to understand the growth and development of other fish species.展开更多
Mucin family members play an indispensable role in immunity against pathogen invasion.Currently,there are very few studies on members of the mucin family in Japanese flounder Paralichthys olivaceus.In this regard,we c...Mucin family members play an indispensable role in immunity against pathogen invasion.Currently,there are very few studies on members of the mucin family in Japanese flounder Paralichthys olivaceus.In this regard,we characterized the mucin members in Japanese flounder as well as their involvement in response to pathogen infection.In our results,a total of 9 mucin genes were identified based on the whole genome database of flounder.Among them,MUC2-1,MUC2-2,MUC5AC-1,MUC5AC-2 and MUC5B are secreted mucins,while MUC3A,MUC3B,MUC13 and MUC15 are membrane binding mucins.The collinearity results showed that the adjacent MUC genes of flounder had collinearity relationship with many other teleosts.Phylogenetic tree results showed that the mucin gene of flounder was divided into several subfamilies.In addition,the expression patterns of flounder mucin family members were examined in 11 healthy tissues.The expression changes of mucin gene were also detected in six immune tissues(gill,intestine,skin,liver,kidney,spleen)after pathogen infection,revealing their role in disease resistance.Collinear analysis indicates the adjacent mucin genes.According to the protein interaction network(PPI)results,the mucin genes interact with the galnt genes.The results provide a solid foundation for further research on the function of mucins in mucosal immunity of flounder.展开更多
The survival rate, feeding, growth of juvenile Starry flounder were studied at different salinities. The results showed that when the fish was directly translated from salinity of 32 to 0 and more than 0, the survival...The survival rate, feeding, growth of juvenile Starry flounder were studied at different salinities. The results showed that when the fish was directly translated from salinity of 32 to 0 and more than 0, the survival rate was 100% in 96 hours. If the fish was acclimatized, it was still 100% in 42 days. The fish at the salinity of 0 - 16 grew faster than others; feeding rate varied as salinity changed, and the highest one was at salinity of 32 and the lowest one was at the salinity of 16. As to feed conversion efficiency, the highest one was at the salinity of 16, and the lowest one was at the salinity of 24. There were no significant differences among the treatments （P＆gt;0.05）. It was found that low salinity benefited for recovering albinism.展开更多
The toxic mechanism of herbicide butachlor to induce extremely high lethality in marine flatfish flounder, Paralichthys Olivaceus, was analyzed by histopathological examination, antioxidant enzymes activities and ATP ...The toxic mechanism of herbicide butachlor to induce extremely high lethality in marine flatfish flounder, Paralichthys Olivaceus, was analyzed by histopathological examination, antioxidant enzymes activities and ATP content assay. Histopathological examination of gill, liver and kidney of exposed fishes showed that gill was a target organ of butachlor. The butachlor seriously impaired the respiration of gills by a series of lesions such as edema, lifting and detachment of lamellar epithelium, breakdown of pillar cells, and blood congestion. The dysfunction of gill respiration caused suffocation to the exposed flounder with extremely high acute lethality. Antioxidant enzyme activity assay of the in vitro cultured flounder gill （FG） ceils exposed to butachlor indicated that butachlor markedly inhibited the antioxidant enzyme activities of Superoxide dismutase （SOD）, catalase （CAT） and glutathione peroxidase （GPX）. Furthermore, along with the decline of antioxidant enzyme activities, ATP content in the exposed FG cells decreased, too. This infers that the oxidative stress induced by butachlor can inhibit the production of cellular ATP. Similar decrease of ATP content was also observed in the exposed flounder gill tissues. Taken together, as in FG cells, butachlor possibly induced a short supply of ATP in pillar cells by inhibiting the antioxidant enzyme activities and then affecting the contractibility of the pillar cells, which in turn resulted in the blood congestion and suffocation of exposed flounder.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
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(...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.展开更多
[Objective] The paper aimed to clone the full length gene of Toll-like recep- tors （TLRs） in Japanese flounder （Paralichthys olivaceus）, and analyze their structural features and expression regularity. [Method] Th...[Objective] The paper aimed to clone the full length gene of Toll-like recep- tors （TLRs） in Japanese flounder （Paralichthys olivaceus）, and analyze their structural features and expression regularity. [Method] The full length cDNA sequence of Toll like receptor 1（TLR1） gene was identified from Japanese flounder head kidney by ho- mologous cloning and rapid amplification cDNA ends （RACE）. The bioinformatics and expression model of this gene was analyzed. [Result] The TLR1 cDNA was 2 947 bp, a 2 418 bp open reading frame （ORF）, encoding 805 amino acid （aa） residues, including signal peptide, six leucine-rich repeat（LRR） motifs, two transmembrane zones and one TolI/IL 1 receptor （TIR） domain. The molecular weight of the deduced protein was 91.15 KDa, and the isoelectric point was 6.49. The amino acid sequence of Japanese flounder TLR1 possessed 69%-35% identity with the TLRls of other verte- brates, further analysis showed that the TIR domain of Japanese flounder TLR1 shared 84%-62% identities with TIR domains in other vertebrates. Japanese flounder TLR1 protein firstly clustered with TLRls in Epinephelus coioides in the phylogenetic analysis. The transcription of Japanese flounder TLR1 was examined by real-time quantitative PCR, and its mRNA was mainly detected in liver, heart and spleen. [Conclusion] The results lay a foundation for further studying the functions of TLR1 and developing immune potentiator in Japanese flounder.展开更多
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
Artificial gynogenesis is of great research value in fish genetics and breeding technology. However, existing studies did not explain the mechanism of some interesting phenomena. Severe developmental defects in gynoge...Artificial gynogenesis is of great research value in fish genetics and breeding technology. However, existing studies did not explain the mechanism of some interesting phenomena. Severe developmental defects in gynogenetic haploids can lead to death during hatching. After diploidization of chromosomes, gynogenetic diploids may dispense from the remarkable malformation and restore the viability, although the development time is longer and the survival rate is lower compared with normal diploids. The aim of this study was to reveal key mechanism in haploid syndrome of Japanese flounder, a commercially important marine teleost in East Asia. We measured genome-scale gene expression of flounder haploid, gynogenetic diploid and normal diploid embryos using RNA-Seq, constructed a module-centric co-expression network based on weighted correlation network analysis(WGCNA) and analyzed the biological functions of correlated modules. Module gene content analysis revealed that the formation of gynogenetic haploids was closely related to the abnormality of plasma proteins, and the up-regulation of p53 signaling pathway might rescue gynogenetic embryos from haploid syndrome via regulating cell cycle arrest, apoptosis and DNA repair. Moreover, normal diploid has more robust nervous system. This work provides novel insights into molecular mechanisms in haploid syndrome and the rescue process by gynogenetic diploidization.展开更多
A novel continuous ovary cell line from barfin flounder(Verasper moseri)(BFO cell line) was established with its primitive application in transgenic expression demonstrated in this study. Primarily cultured cells grew...A novel continuous ovary cell line from barfin flounder(Verasper moseri)(BFO cell line) was established with its primitive application in transgenic expression demonstrated in this study. Primarily cultured cells grew well at 22℃ in Dulbecco's modified Eagle medium/F12 medium(DMEM/F12, 1:1; p H 7.2) supplemented with 20% fetal bovine serum(FBS), carboxymethyl chitooligosaccharide, basic fibroblast growth factor(b FGF) and insulin-like growth factor-I(IGF-I). The primary BFO cells in fibroblastic morphology proliferated into a confluent monolayer about 2 weeks later, and were able to be subcultured. Impacts of medium and temperature on the growth of the cells were examined. The optimum growth was found in DMEM/F12 with 20% FBS and at 22℃. The BFO cells can be continuously subcultured to Passage 120 steadily with a population doubling time of 32.7 h at Passage 60. Chromosome analysis revealed that 72% of BFO cells at Passage 60 maintained the normal diploid chromosome number(46) with a normal karyotype of 2st+44t. The results of gene transformation indicated that green fluorescence protein(GFP) positively expressed in these cells after being transformed with pc DNA3.1-GFP. Therefore, a continuous and transformable BFO cell line was successfully established, which may serve as a useful tool for cytotechnological manipulation and transgenic modification of this fish.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
Follicle stimulating hormone β (FSHβ) of Japanese flounder (Paralichthys olivaceus) plays a key role in the regulation of gonadal development.This study aimed to investigate molecular genetic characteristics of the ...Follicle stimulating hormone β (FSHβ) of Japanese flounder (Paralichthys olivaceus) plays a key role in the regulation of gonadal development.This study aimed to investigate molecular genetic characteristics of the FSHβ gene and elucidate the effects of single nucleotide polymorphisms (SNPs) of FSHβ on reproductive traits in Japanese flounder.We used polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) and sequencing of the FSHβ gene in 60 individuals.We identified only an SNP (T/C) in the coding region of exon3 of FSHβ.The SNP (T/C) did not lead to amino acid changes at the position 340 bp of FSHβ gene.Statistical analysis showed that the SNP was significantly associated with testosterone (T) level and gonadosomatic index (GSI) (P<0.05).Individuals with genotype TC of the SNP had significantly higher serum T levels and GSI (P<0.05) than that of genotype CC.Therefore, FSHβ gene could be a useful molecular marker in selection for prominent reproductive trait in Japanese Flounder.展开更多
Zooplankton constitutes a major part of the diet for fish larvae in the marine food web, and it is generally believed that copepods can meet the nutritional requirements of fish larvae. In this study, calanoid copepod...Zooplankton constitutes a major part of the diet for fish larvae in the marine food web, and it is generally believed that copepods can meet the nutritional requirements of fish larvae. In this study, calanoid copepod Schmackeria poplesia, rotifer Brachionus plicatilis and anostraca crustacean Artemia sp. were analyzed for fatty acid contents, and were used as live food for cul- turing larval Japanese flounder, Paralichthys olivaceus. The total content of three types of HUFAs （DHA, EPA and ARA） in S. po- plesia was significantly higher than that in the other two live foods （P〈O.O1）. Three live organisms were used for raising larvae and juveniles of Paralichthys olivaceus respectively for 15 and 10 d. Then the growth, survival and fatty acid composition of the larvae and juveniles were investigated. The results showed that the larvae and juveniles fed with copepods （S. poplesia） had significantly higher growth rate than those fed with the other two organisms （P〈0.01）. The survival of the flounder larvae fed with copepods was significantly higher than that of the others （P〈0.01）, and the survival of the juvenile fish fed with copepods was higher than that fed with Artemia （P〈0.05）. The contents of three types of HUFAs （DHA, EPA and ARA） and the ratio of DHA/EPA in larval and juve- nile flounder P. olivaceus were analyzed. The results showed that the contents of DHA, EPA and ARA in the larvae and juveniles fed with S. poplesia were higher than those fed with a mixed diet orArtemia only, and the ratio of EPA/ARA in larvae and juveniles of P. olivaceus fed with S. poplesia was lower than that in the case of feeding with a mixed diet or Artemia only. The present data showed that copepod is the best choice for feeding the larvae and juveniles of fish considering its effects on the survival, growth and nutrition composition of the fish.展开更多
The expressed sequence tags（ESTs）of Japanese flounder,Paralichthys olivaeeus,were selected from GenBank to identify simple sequence repeats（SSRs）or microsatellites.A bioinformatic analysis of 11111 ESTs identified...The expressed sequence tags（ESTs）of Japanese flounder,Paralichthys olivaeeus,were selected from GenBank to identify simple sequence repeats（SSRs）or microsatellites.A bioinformatic analysis of 11111 ESTs identified 751 SSR-containing ESTs,including 440 dinucleotide,254 trinucleotide,53 tetranueleotide,95 pentanucleotide and 40 hexanucleotide microsatellites respectively.The CA/TG and GA/TC repeats were the most abundant microsatellites.AT-rich types were predominant among trinucleotide and tetranucleotide microsatellites.PCR primers were designed to amplify 10 identified microsatellites loci.The PCR results from eight pairs of primers showed polymorphisms in wild populations.In 30 wild individuals,the mean observed and expected heterozygosities of these 8 polymorphic SSRs were 0.71 and 0.83 respectively and the average PIC value was 0.8.These microsatellite markers should prove to be a useful addition to the microsatellite markers that are now available for this species.展开更多
Tissue expression and stock variation of isozymes of stone flounder （Kareius bicoloratus） were analyzed with horizontal starch gel electrophoresis. For the fourteen enzymes assayed, 31 loci were recorded. The result...Tissue expression and stock variation of isozymes of stone flounder （Kareius bicoloratus） were analyzed with horizontal starch gel electrophoresis. For the fourteen enzymes assayed, 31 loci were recorded. The results indicated that all the isozymes examined were obviously tissue-specific. The expressions of SOD＊, GDH＊, G3PDH-2＊ and ADH-2＊ were detected only in liver, SDH-1＊, MDH-1＊ and ADH-1＊ only in muscle, and LDH-B＊ and LDH-C＊ only in eyes. In comparison, MDH-2＊, GPI-3＊ and SDH-2＊ were detected in all tissues examined. Other loci examined were detected in a variety of tissues. Muscle and liver were selected to detect the isozyme variation of the two geographic stocks of Qingdao and Weihai, Shandong Province, China. The percentages of polymorphic loci （P0.99） were 29.17% and 25.00%, the observed heterozygosities （H0） were 0.028 ±0.014 and 0.040 ± 0.019, and the expected heterozygosities （He） were 0.039±0.017 and 0.052±0.022 in Qingdao and Wethai stock, respectively. The coefficient of gene differentiation （Fst） and genetic distance （D） between the two stocks was 0.012 and 0.0011, respectively, indicating that the genetic differentiation is low between them. Compared with other species of Pleuronectiformes, both the percentage of polymorphic loci and the mean heterozygosity ofK. Bicoloratus were at a middle level.展开更多
The FG 9307 cell line derived from the gill of flounder Paralichthys olivaceus was used in the present study to determine the acute cytotoxic effects of the organophosphorous pesticide, profenofos. It was found that t...The FG 9307 cell line derived from the gill of flounder Paralichthys olivaceus was used in the present study to determine the acute cytotoxic effects of the organophosphorous pesticide, profenofos. It was found that the cell growth rate was markedly reduced by profenofos at the concentrations of 2.5, 5 and 7.5 mg/L tested; and that the fine structures of the cells were also altered by profenofos, as evidenced by dilation of nuclear membranes and mitochondria cristae, and presence of enlarged lysosomes with engulfed organelles and numerous vacuoles in the cytoplasm. Probably, mitochondria, the cell energy generating sites, are the most prominent sites of profenofos cytotoxity in the cells. This seems to be the first report of the use of marine fish cell line for evaluation of the acute in vitro cytotoxicity of organophosphorus pesticide.展开更多
The in vitro acute cytotoxicity of the neonicotinoid insecticide acetamiprid to FG cell line, derived from the gill of flounder Paralichthys olizaceus, was tested by the three widely used endpoint bioassays-neutral r...The in vitro acute cytotoxicity of the neonicotinoid insecticide acetamiprid to FG cell line, derived from the gill of flounder Paralichthys olizaceus, was tested by the three widely used endpoint bioassays-neutral red （NR） assay, tetrazolium （MTT） assay and cell protein assay. It was found that acetamiprid was increasingly toxic to FG cells at concentrations of 1 μg/cm^3 or above, and the inhibitory concentration 50% values for NR, MTF, and cell protein assays were 38.38, 36.27 and 32.03 μg/cm^3, respectively. This appeared to be the first report on the in vitro cytotoxicity of acetamiprid to non-mammalian vertebrate cells. Ultrastructural examination revealed that for the cells exposed to 60 μg/cm^3 acetamiprid for 48 h, their mitochondria were severely damaged with the cristae swelled up or disrupted, while their nuclei and rough endoplasmic reticlum （RER） appeared to be still normal. This suggests that mitochondria are possibly the primary target of acetamiprid.展开更多
Flounder gill （FG） cells were used to isolate lymphocystis disease virus （LCDV） and two monoclonal antibodies （Mabs） （1A8 and 3G3） against LCDV were used to trace LCDV infection to FG cells. FG monolayer cells...Flounder gill （FG） cells were used to isolate lymphocystis disease virus （LCDV） and two monoclonal antibodies （Mabs） （1A8 and 3G3） against LCDV were used to trace LCDV infection to FG cells. FG monolayer cells was inoculated with LCDV supernatant, obtained from lymphocystis cells of diseased flounder, Paralichthys olivaceus. LCDV infection was detected with Mabs employing immunocytochemical assay （ICA） and indirect immunofluorescence assay test （IIFAT） technique. Detected by IIFAT, they were specifie for LCDV. The results of experimental infection illustrated that FG cells was sensitive to LCDV, and showed virus-infection positive detected by ICA. Cytopathic effect （CPE） occurred 1-2 days post inoculation （PI）, and half tissue culture infection dosage （TCID50） of vires supematant was 2^2.57 per 40μl. Tracing by IIFAT showed that LCDV positive signal first appeared at the cell membrane immediately PI, and then in cytoplasm at 24h PI, it reached the strongest positive at 48-72 h PI, and began to decrease at 96h PI.展开更多
In this study, we determined the cytotoxicity and genotoxicity of carbamate insecticide carbaryl to flounder gill （FG） cells and its teratogenicity to zebrafish embryos. The cytotoxicity of carbaryl to FG cells was ...In this study, we determined the cytotoxicity and genotoxicity of carbamate insecticide carbaryl to flounder gill （FG） cells and its teratogenicity to zebrafish embryos. The cytotoxicity of carbaryl to FG cells was determined with methods including MTT and neutral red uptaking （NRU）, lactate dehydrogenase （LDH） releasing and Hoechst 33342 and propidiurn idodide （PI） double staining. Moderate cytotoxicity in a concentration-dependent manner was observed. The 24h-IC50 value of 53.48 ± 1.21, 59.13 ± 1.19 and 46.21 ± 1.24 mgL 1 carbaryl was obtained through MTT, NRU and LDH assays, respectively. Double fluorescence staining dem- onstrated that carbaryl induced the death of FG cells mainly through necrosis. There was no significant genotoxicity found in the FG cells exposed to the highest testing concentration of carbaryl （20mgL-1, P〉0.05） as was demonstrated by Comet assay. Zebrafish embryos exposed to carbaryl at concentrations 〉10 mg L-1 displayed moderate toxic effects on the survival, spontaneous movement, hatching, heart rates of the embryos and their development, which were evidenced by yolk and pericardial sac edemas, body length reduction and tail flexure in time- and concentration-dependent manners at specific stages. The 24h-, 48h- and 96h-LC50 values of carbaryl to zebra fish embryos were 41.80 ± 1.10, 17.80 ± 1.04 and 14.46 ± 1.05 mg L-1, respectively. These results suggested that car- baryl is moderately toxic to FG ceils cultured in vitro and zebrafish embryos, and the FG cells were similar to zebrafish embryos in their sensitivity to carbaryl as 24h-IC50 and LC50 indicated.展开更多
基金supported by the National Natural Science Foundation of China(No.32202963)the Natural Science Foundation of Jiangsu Province(No.BK20220681)+3 种基金the Doctoral Program of Entrepreneurship and Innovation in Jiangsu Province(No.JSSCBS20221625)the Scientific Research Foundation Program of Jiangsu Ocean University(No.KQ22009)the Undergraduate Innovation&Entrepreneurship Training Program of Jiangsu Province,China(No.SY202411641631001)the Postgraduate Research&Practice Innovation Program of Jiangsu Province(No.KYCX2023-112).
文摘Due to the discharge of industrialwastewater,urban domestic sewage,and intensive marine aquaculture tailwater,nitrate(NO_(3)^(−))pollution has emerged as a significant issue in offshore waters.Nitrate pollution affects aquatic life and may interact with other pollutants,leading to comprehensive toxicity.Cadmium(Cd^(2+))is the most widespread metal contaminant,adversely affecting aquatic life in the coastal waters of China.Despite this,few studies have focused on the synergistic toxicity of NO_(3)^(−)and Cd^(2+)in marine organisms.This study conducted a 30-day exposure experiment on marine Japanese flounder(Paralichthys olivaceus)to explore the synergistic toxicity of NO_(3)^(−)and Cd^(2+).Our results demonstrated that the exposure to Cd^(2+)alone induced slight histopathological changes in the liver.However,malformations such as hepatic vacuolar degeneration and sinusoid dilatationwere exacerbated under co-exposure.Moreover,co-exposure induced the downregulation of antioxidants and the upregulation of the product malonaldehyde(MDA)from lipid peroxidation,indicating potent oxidative stress in the liver.The increased mRNA expression of IL-8,TNF-α,and IL-1β,along with the decreased expression level of TGF-β,indicated a synergistic inflammatory response in the organisms.Furthermore,the co-exposure led to an abnormal expression of P53,caspase-3,caspase-9,Bcl-2,and Bax,and disturbed the apoptosis in the liver through TUNEL staining analysis.Overall,our results imply that co-exposure synergistically affects inflammation,redox status,and apoptosis in flounders.Therefore,the findings from this study provide valuable perspectives on the ecological risk assessment of marine teleosts co-exposure to NO_(3)^(−)and Cd^(2+).
基金supported by the National Key R&D Program of China(No.2022YFD2400401)the Na-tional Natural Science Foundation of China(No.31672642).
文摘Circular RNAs(circRNAs)are a type of endogenous competitive RNA that can perform various functions and regulate a number of biological activities in organisms.Many studies have shown that circRNAs can modulate skeletal muscle development in animals.However,the effects of circRNAs on the skeletal muscle development of Japanese flounder and its regulatory mechanisms remain unclear.We screened circRNA_2111 from previously obtained transcriptomic data related to Japanese flounder skeletal mus-cle development.Derived from the tefb gene,it has been designated as circtefb.Circtefb was formed by the reverse splicing of two exons of tefb.Circtefb was more stable than its corresponding linear RNA.There was a significant difference in the expression of circtefb at different stages P1(90 d),P2(12 months)and P3(24 months)of Japanese flounder(p<0.05).Over-expression of circtefb in Japanese flounder myocytes resulted in a significant upregulation of proliferation marker genes ccnd1,ccnd2a and pcna(p<0.05),suggesting that it promoted proliferation of Japanese flounder myocytes.The expressions of ccnd1,ccnd2a,and pcna were signifi-cantly decreased after the transfection of pol-miR-138 into Japanese flounder myocytes(p<0.05).In addition,circtefb can suppress pol-miR-138 function by binding to it,which was detected by dual-Luciferase reporter assay and co-transfection of circtefb and pol-miR-138,thereby promoting the proliferation of Japanese flounder myocytes.The results indicated the function of circRNAs in Japanese flounder muscle development,,which is also helpful to understand the growth and development of other fish species.
基金supported by Young Experts of Taishan Scholars(No.tsqn 201909130)the Science and Technology Support Plan for Youth Innovation of Colleges and Universities in Shandong Province(No.2019KJF003)+1 种基金the Shandong Technical System of Fish Industry(No.SDAIT12-03),the Natural Science Foundation of Shandong Province(No.ZR2022QC037)the High-Level Talents Research Fund of Qingdao Agricultural University(No.663/1120033)。
文摘Mucin family members play an indispensable role in immunity against pathogen invasion.Currently,there are very few studies on members of the mucin family in Japanese flounder Paralichthys olivaceus.In this regard,we characterized the mucin members in Japanese flounder as well as their involvement in response to pathogen infection.In our results,a total of 9 mucin genes were identified based on the whole genome database of flounder.Among them,MUC2-1,MUC2-2,MUC5AC-1,MUC5AC-2 and MUC5B are secreted mucins,while MUC3A,MUC3B,MUC13 and MUC15 are membrane binding mucins.The collinearity results showed that the adjacent MUC genes of flounder had collinearity relationship with many other teleosts.Phylogenetic tree results showed that the mucin gene of flounder was divided into several subfamilies.In addition,the expression patterns of flounder mucin family members were examined in 11 healthy tissues.The expression changes of mucin gene were also detected in six immune tissues(gill,intestine,skin,liver,kidney,spleen)after pathogen infection,revealing their role in disease resistance.Collinear analysis indicates the adjacent mucin genes.According to the protein interaction network(PPI)results,the mucin genes interact with the galnt genes.The results provide a solid foundation for further research on the function of mucins in mucosal immunity of flounder.
基金supported by the Key Projects of Science and Technology Department of Liaoning province "The Research and Demonstration of the Efficient and Safe Feeding Technology of Seafood" (No. 2008203002)
文摘The survival rate, feeding, growth of juvenile Starry flounder were studied at different salinities. The results showed that when the fish was directly translated from salinity of 32 to 0 and more than 0, the survival rate was 100% in 96 hours. If the fish was acclimatized, it was still 100% in 42 days. The fish at the salinity of 0 - 16 grew faster than others; feeding rate varied as salinity changed, and the highest one was at salinity of 32 and the lowest one was at the salinity of 16. As to feed conversion efficiency, the highest one was at the salinity of 16, and the lowest one was at the salinity of 24. There were no significant differences among the treatments （P＆gt;0.05）. It was found that low salinity benefited for recovering albinism.
基金supported by the National Basic Research Program of China (Grant No 2006CB101805)Undergraduate Innovative Experiment Program of China (Grant Number 091042328)
文摘The toxic mechanism of herbicide butachlor to induce extremely high lethality in marine flatfish flounder, Paralichthys Olivaceus, was analyzed by histopathological examination, antioxidant enzymes activities and ATP content assay. Histopathological examination of gill, liver and kidney of exposed fishes showed that gill was a target organ of butachlor. The butachlor seriously impaired the respiration of gills by a series of lesions such as edema, lifting and detachment of lamellar epithelium, breakdown of pillar cells, and blood congestion. The dysfunction of gill respiration caused suffocation to the exposed flounder with extremely high acute lethality. Antioxidant enzyme activity assay of the in vitro cultured flounder gill （FG） ceils exposed to butachlor indicated that butachlor markedly inhibited the antioxidant enzyme activities of Superoxide dismutase （SOD）, catalase （CAT） and glutathione peroxidase （GPX）. Furthermore, along with the decline of antioxidant enzyme activities, ATP content in the exposed FG cells decreased, too. This infers that the oxidative stress induced by butachlor can inhibit the production of cellular ATP. Similar decrease of ATP content was also observed in the exposed flounder gill tissues. Taken together, as in FG cells, butachlor possibly induced a short supply of ATP in pillar cells by inhibiting the antioxidant enzyme activities and then affecting the contractibility of the pillar cells, which in turn resulted in the blood congestion and suffocation of exposed flounder.
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 Natural Science Foundation of China (No. 30371120)Modern Agro-industry Technology Research System (No. nycytx50-G07)
文摘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.
基金Supported by Natural Science Foundation of Tianjin City(10JCYBJC09100)Doctoral Fund of Tianjin Normal University(52XB1004)Open Research Fund for Municipal Key Laboratory of Tianjin Normal University~~
文摘[Objective] The paper aimed to clone the full length gene of Toll-like recep- tors （TLRs） in Japanese flounder （Paralichthys olivaceus）, and analyze their structural features and expression regularity. [Method] The full length cDNA sequence of Toll like receptor 1（TLR1） gene was identified from Japanese flounder head kidney by ho- mologous cloning and rapid amplification cDNA ends （RACE）. The bioinformatics and expression model of this gene was analyzed. [Result] The TLR1 cDNA was 2 947 bp, a 2 418 bp open reading frame （ORF）, encoding 805 amino acid （aa） residues, including signal peptide, six leucine-rich repeat（LRR） motifs, two transmembrane zones and one TolI/IL 1 receptor （TIR） domain. The molecular weight of the deduced protein was 91.15 KDa, and the isoelectric point was 6.49. The amino acid sequence of Japanese flounder TLR1 possessed 69%-35% identity with the TLRls of other verte- brates, further analysis showed that the TIR domain of Japanese flounder TLR1 shared 84%-62% identities with TIR domains in other vertebrates. Japanese flounder TLR1 protein firstly clustered with TLRls in Epinephelus coioides in the phylogenetic analysis. The transcription of Japanese flounder TLR1 was examined by real-time quantitative PCR, and its mRNA was mainly detected in liver, heart and spleen. [Conclusion] The results lay a foundation for further studying the functions of TLR1 and developing immune potentiator in Japanese flounder.
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.
基金supported by the Scientific and Technological Innovation Project of Qingdao National Laboratory for Marine Science and Technology (No. 2015A SKJ02)the National Natural Science Foundation of China (No. 31540063)
文摘Artificial gynogenesis is of great research value in fish genetics and breeding technology. However, existing studies did not explain the mechanism of some interesting phenomena. Severe developmental defects in gynogenetic haploids can lead to death during hatching. After diploidization of chromosomes, gynogenetic diploids may dispense from the remarkable malformation and restore the viability, although the development time is longer and the survival rate is lower compared with normal diploids. The aim of this study was to reveal key mechanism in haploid syndrome of Japanese flounder, a commercially important marine teleost in East Asia. We measured genome-scale gene expression of flounder haploid, gynogenetic diploid and normal diploid embryos using RNA-Seq, constructed a module-centric co-expression network based on weighted correlation network analysis(WGCNA) and analyzed the biological functions of correlated modules. Module gene content analysis revealed that the formation of gynogenetic haploids was closely related to the abnormality of plasma proteins, and the up-regulation of p53 signaling pathway might rescue gynogenetic embryos from haploid syndrome via regulating cell cycle arrest, apoptosis and DNA repair. Moreover, normal diploid has more robust nervous system. This work provides novel insights into molecular mechanisms in haploid syndrome and the rescue process by gynogenetic diploidization.
基金supported by grants from the National 863 High Technology Research Foundation of China(2006AA10A401)the National Natural Science Foundation of China(31001100)
文摘A novel continuous ovary cell line from barfin flounder(Verasper moseri)(BFO cell line) was established with its primitive application in transgenic expression demonstrated in this study. Primarily cultured cells grew well at 22℃ in Dulbecco's modified Eagle medium/F12 medium(DMEM/F12, 1:1; p H 7.2) supplemented with 20% fetal bovine serum(FBS), carboxymethyl chitooligosaccharide, basic fibroblast growth factor(b FGF) and insulin-like growth factor-I(IGF-I). The primary BFO cells in fibroblastic morphology proliferated into a confluent monolayer about 2 weeks later, and were able to be subcultured. Impacts of medium and temperature on the growth of the cells were examined. The optimum growth was found in DMEM/F12 with 20% FBS and at 22℃. The BFO cells can be continuously subcultured to Passage 120 steadily with a population doubling time of 32.7 h at Passage 60. Chromosome analysis revealed that 72% of BFO cells at Passage 60 maintained the normal diploid chromosome number(46) with a normal karyotype of 2st+44t. The results of gene transformation indicated that green fluorescence protein(GFP) positively expressed in these cells after being transformed with pc DNA3.1-GFP. Therefore, a continuous and transformable BFO cell line was successfully established, which may serve as a useful tool for cytotechnological manipulation and transgenic modification of this fish.
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.
基金supported by the open-fund of Key Laboratory of Fisheries Genetic Resources and Aquaculture,Chinese Academy of Fisheries Sciences of China (2008B1207)New Teacher Special Fund of Doctor of Ministry of Education of China (20090132120006)
文摘Follicle stimulating hormone β (FSHβ) of Japanese flounder (Paralichthys olivaceus) plays a key role in the regulation of gonadal development.This study aimed to investigate molecular genetic characteristics of the FSHβ gene and elucidate the effects of single nucleotide polymorphisms (SNPs) of FSHβ on reproductive traits in Japanese flounder.We used polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) and sequencing of the FSHβ gene in 60 individuals.We identified only an SNP (T/C) in the coding region of exon3 of FSHβ.The SNP (T/C) did not lead to amino acid changes at the position 340 bp of FSHβ gene.Statistical analysis showed that the SNP was significantly associated with testosterone (T) level and gonadosomatic index (GSI) (P<0.05).Individuals with genotype TC of the SNP had significantly higher serum T levels and GSI (P<0.05) than that of genotype CC.Therefore, FSHβ gene could be a useful molecular marker in selection for prominent reproductive trait in Japanese Flounder.
基金supported by the National High-Tech Research and Development Program of China (863 Program, Nos. 2002AA629100 and 2004AA626100
文摘Zooplankton constitutes a major part of the diet for fish larvae in the marine food web, and it is generally believed that copepods can meet the nutritional requirements of fish larvae. In this study, calanoid copepod Schmackeria poplesia, rotifer Brachionus plicatilis and anostraca crustacean Artemia sp. were analyzed for fatty acid contents, and were used as live food for cul- turing larval Japanese flounder, Paralichthys olivaceus. The total content of three types of HUFAs （DHA, EPA and ARA） in S. po- plesia was significantly higher than that in the other two live foods （P〈O.O1）. Three live organisms were used for raising larvae and juveniles of Paralichthys olivaceus respectively for 15 and 10 d. Then the growth, survival and fatty acid composition of the larvae and juveniles were investigated. The results showed that the larvae and juveniles fed with copepods （S. poplesia） had significantly higher growth rate than those fed with the other two organisms （P〈0.01）. The survival of the flounder larvae fed with copepods was significantly higher than that of the others （P〈0.01）, and the survival of the juvenile fish fed with copepods was higher than that fed with Artemia （P〈0.05）. The contents of three types of HUFAs （DHA, EPA and ARA） and the ratio of DHA/EPA in larval and juve- nile flounder P. olivaceus were analyzed. The results showed that the contents of DHA, EPA and ARA in the larvae and juveniles fed with S. poplesia were higher than those fed with a mixed diet orArtemia only, and the ratio of EPA/ARA in larvae and juveniles of P. olivaceus fed with S. poplesia was lower than that in the case of feeding with a mixed diet or Artemia only. The present data showed that copepod is the best choice for feeding the larvae and juveniles of fish considering its effects on the survival, growth and nutrition composition of the fish.
基金supported by the National High Technology Research and Development Program of China (Grant No.2006AA10A404)the National Natural Science Foundation of China (Grant No.30671624)
文摘The expressed sequence tags（ESTs）of Japanese flounder,Paralichthys olivaeeus,were selected from GenBank to identify simple sequence repeats（SSRs）or microsatellites.A bioinformatic analysis of 11111 ESTs identified 751 SSR-containing ESTs,including 440 dinucleotide,254 trinucleotide,53 tetranueleotide,95 pentanucleotide and 40 hexanucleotide microsatellites respectively.The CA/TG and GA/TC repeats were the most abundant microsatellites.AT-rich types were predominant among trinucleotide and tetranucleotide microsatellites.PCR primers were designed to amplify 10 identified microsatellites loci.The PCR results from eight pairs of primers showed polymorphisms in wild populations.In 30 wild individuals,the mean observed and expected heterozygosities of these 8 polymorphic SSRs were 0.71 and 0.83 respectively and the average PIC value was 0.8.These microsatellite markers should prove to be a useful addition to the microsatellite markers that are now available for this species.
文摘Tissue expression and stock variation of isozymes of stone flounder （Kareius bicoloratus） were analyzed with horizontal starch gel electrophoresis. For the fourteen enzymes assayed, 31 loci were recorded. The results indicated that all the isozymes examined were obviously tissue-specific. The expressions of SOD＊, GDH＊, G3PDH-2＊ and ADH-2＊ were detected only in liver, SDH-1＊, MDH-1＊ and ADH-1＊ only in muscle, and LDH-B＊ and LDH-C＊ only in eyes. In comparison, MDH-2＊, GPI-3＊ and SDH-2＊ were detected in all tissues examined. Other loci examined were detected in a variety of tissues. Muscle and liver were selected to detect the isozyme variation of the two geographic stocks of Qingdao and Weihai, Shandong Province, China. The percentages of polymorphic loci （P0.99） were 29.17% and 25.00%, the observed heterozygosities （H0） were 0.028 ±0.014 and 0.040 ± 0.019, and the expected heterozygosities （He） were 0.039±0.017 and 0.052±0.022 in Qingdao and Wethai stock, respectively. The coefficient of gene differentiation （Fst） and genetic distance （D） between the two stocks was 0.012 and 0.0011, respectively, indicating that the genetic differentiation is low between them. Compared with other species of Pleuronectiformes, both the percentage of polymorphic loci and the mean heterozygosity ofK. Bicoloratus were at a middle level.
文摘The FG 9307 cell line derived from the gill of flounder Paralichthys olivaceus was used in the present study to determine the acute cytotoxic effects of the organophosphorous pesticide, profenofos. It was found that the cell growth rate was markedly reduced by profenofos at the concentrations of 2.5, 5 and 7.5 mg/L tested; and that the fine structures of the cells were also altered by profenofos, as evidenced by dilation of nuclear membranes and mitochondria cristae, and presence of enlarged lysosomes with engulfed organelles and numerous vacuoles in the cytoplasm. Probably, mitochondria, the cell energy generating sites, are the most prominent sites of profenofos cytotoxity in the cells. This seems to be the first report of the use of marine fish cell line for evaluation of the acute in vitro cytotoxicity of organophosphorus pesticide.
文摘The in vitro acute cytotoxicity of the neonicotinoid insecticide acetamiprid to FG cell line, derived from the gill of flounder Paralichthys olizaceus, was tested by the three widely used endpoint bioassays-neutral red （NR） assay, tetrazolium （MTT） assay and cell protein assay. It was found that acetamiprid was increasingly toxic to FG cells at concentrations of 1 μg/cm^3 or above, and the inhibitory concentration 50% values for NR, MTF, and cell protein assays were 38.38, 36.27 and 32.03 μg/cm^3, respectively. This appeared to be the first report on the in vitro cytotoxicity of acetamiprid to non-mammalian vertebrate cells. Ultrastructural examination revealed that for the cells exposed to 60 μg/cm^3 acetamiprid for 48 h, their mitochondria were severely damaged with the cristae swelled up or disrupted, while their nuclei and rough endoplasmic reticlum （RER） appeared to be still normal. This suggests that mitochondria are possibly the primary target of acetamiprid.
文摘Flounder gill （FG） cells were used to isolate lymphocystis disease virus （LCDV） and two monoclonal antibodies （Mabs） （1A8 and 3G3） against LCDV were used to trace LCDV infection to FG cells. FG monolayer cells was inoculated with LCDV supernatant, obtained from lymphocystis cells of diseased flounder, Paralichthys olivaceus. LCDV infection was detected with Mabs employing immunocytochemical assay （ICA） and indirect immunofluorescence assay test （IIFAT） technique. Detected by IIFAT, they were specifie for LCDV. The results of experimental infection illustrated that FG cells was sensitive to LCDV, and showed virus-infection positive detected by ICA. Cytopathic effect （CPE） occurred 1-2 days post inoculation （PI）, and half tissue culture infection dosage （TCID50） of vires supematant was 2^2.57 per 40μl. Tracing by IIFAT showed that LCDV positive signal first appeared at the cell membrane immediately PI, and then in cytoplasm at 24h PI, it reached the strongest positive at 48-72 h PI, and began to decrease at 96h PI.
基金supported by the grants from National High-tech R&D Program of China (863 Program, Grant No. 2012AA 10A402)National Natural Science Foundation of China (Grant Nos. 31172391 and 31472274)+1 种基金Key Laboratory Open Foundation of Marine Bioactive Substances and Modern Analytical Technology, State Oceanic Administration (No. MBSMAT-2011-01)Fundamental Research Funds for the Central Universities of China (Grant No. 201122005)
文摘In this study, we determined the cytotoxicity and genotoxicity of carbamate insecticide carbaryl to flounder gill （FG） cells and its teratogenicity to zebrafish embryos. The cytotoxicity of carbaryl to FG cells was determined with methods including MTT and neutral red uptaking （NRU）, lactate dehydrogenase （LDH） releasing and Hoechst 33342 and propidiurn idodide （PI） double staining. Moderate cytotoxicity in a concentration-dependent manner was observed. The 24h-IC50 value of 53.48 ± 1.21, 59.13 ± 1.19 and 46.21 ± 1.24 mgL 1 carbaryl was obtained through MTT, NRU and LDH assays, respectively. Double fluorescence staining dem- onstrated that carbaryl induced the death of FG cells mainly through necrosis. There was no significant genotoxicity found in the FG cells exposed to the highest testing concentration of carbaryl （20mgL-1, P〉0.05） as was demonstrated by Comet assay. Zebrafish embryos exposed to carbaryl at concentrations 〉10 mg L-1 displayed moderate toxic effects on the survival, spontaneous movement, hatching, heart rates of the embryos and their development, which were evidenced by yolk and pericardial sac edemas, body length reduction and tail flexure in time- and concentration-dependent manners at specific stages. The 24h-, 48h- and 96h-LC50 values of carbaryl to zebra fish embryos were 41.80 ± 1.10, 17.80 ± 1.04 and 14.46 ± 1.05 mg L-1, respectively. These results suggested that car- baryl is moderately toxic to FG ceils cultured in vitro and zebrafish embryos, and the FG cells were similar to zebrafish embryos in their sensitivity to carbaryl as 24h-IC50 and LC50 indicated.
