CYTOTOXICITY AND GENOTOXICITY OF POLYETHYLENIMINE AND NICKEL CHLORIDE IN RED SEA BREAM（Pagrosomus major）FIN CELL LINE RSBF

A continuous marine fish cell line RSBF(i.e.Red Sea Bream Fin)was utilized to screen the cytotoxicity and genotoxicity of polyethylenimine(PEI)and nickel cholride(NiCl2)in this study on the deleterious effects of aquatic genotoxins on fish.At the 0.01 to 1μgml concentration tested,PEI had acute toxicity to the treated RSBF cells(IC50=1.12,0.92,0.88 and 0.64μg/ml PEI for time 0 h,24 h,48 h and 72 h after treatment,respectively)and markedly inhibited their proliferation in a dose-dependent manner.At the 0.001 to 5 μmol/L concentration tested,NiCl2 posed no acute toxicity but significantly stimulated their growth(107?214?of control).Random amplified polymorphic DNA(RAPD)technique was used to detect the genotoxic effects of PEI and NiCl2 by comparing the RAPD banding patterns of the control and treated cells.RAPD analysis indicated that at the concentrations tested,PEI was more genotoxic than NiCl2 to RSBF cells;that there was a slight dose-dependent response in the genotoxic effect of PEI bue not NiCl2;and that RAPD technique might provide a sensitive,non-specif-ic gentoxic endpoint.And the potent cytotoxicity and genotoxicity of PEI on fish cells showed that we should be cautious in utilizing it as gene vector in fish gene transfer and human gene therapy.     

The genotoxic effects of benzo[a]pyrene and methamidophos on black porgy evaluated by comet assay

In this study, two common pollutants （benzo[a]pyrene and methamidophos） in marine environment were tested by comet assay for their inducement of in vivo genotoxic effect to the blood cells of black porgy （ Acanthopagrus schlegeli）. The fish was exposed to 2 μg/L of benzo[a]pyrene （BaP） and methamidophos, and their mixture. The assay was performed on whole blood at 2 h, 5 h, 24 h and 96 h exposure intervals. A significant increase in DNA damage was observed in each treatment with the pollutants. Additive effect of BaP and methamidophos was also found in the experiment. However, the decrease ratios of DNA damage for 5 h and 96 h exposure interals compared with 2 h and 24 h exposure ones, respectively, were noticed. This phenomenon may be explained by the function of repairing process via enzyme cytochrome P450 in the animal. Evidence of the genotoxicity of organophosphorus pesticides （OPs） and polynuclear aromatic hydrocarbons （PAHs） on marine fish are discussed in this paper.     

CHARACTERIZATION OF LACTATE DEHYDROGENASE ISOZYME PATTERN AND MORPHOLOGY OF THREE MARINE FISH CELL LINES

Three continuous marine fish cell lines of FG (i. e., Hounder Gill) from flounder (Paralichthys olivaceus) gill, SPH (i. e. , Sea Perch Heart) from sea perch (Lateolabrax japonicus) heart and RSBF (i.e., Red Sea Bream Fin) from red sea bream (Pagrosomus major) fin, were characterized by lactate dehydrogenase (LDH) isozyme and morphological analysis. The LDH isozyme patterns of these three cell lines and their corresponding tissues of origin were investigated and compared. The results showed: (1) No difference was found in the LDH isozyme patterns of FG and flounder gill tissue. However, the LDH isozyme patterns of SPH and RSBF were significantly different from their corresponding tissues of origin; (2) LDH isozyme patterns of FG, SPH and RSBF were markedly different from each other and could serve as genetic markers for species identification and detection of cross contamination. Morphological change analysis of these three cell lines in comparison to their original tissues indicated that FG cells still appeared epithelioid without morphological transformation. However, morphological changes were found in SPH and RSBF compared to their original tissues. Therefore, the cellular morphology was still plastic in the relatively stable culture conditions, and it was possible that change of LDH patterns wasrelated to morphological changes of fish cells in vitro.     

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

The hybrid between olive flounder Paralichthys olivaceus and stone flounder Kareius bicoloratus was produced by artificial insemination of olive flounder eggs with stone flounder Sperm. Sinistral and dextral are two types of hybrid progeny after metamorphosis. Karyotypes of both hybrid flounders are the same as those of the two parental species. Of the 22 loci examined from 12 allozymes,12 confirmed hybridization of the paternal and matemal loci in hybrids and no difference was found in allozyme patterns of sinistral and dextral hybrid fishes. RAPD patterns of these specimens were also studied with 38 primers selected from 104 tested. Among them, the PCR products of 30 primers showed hybridization of the paternal and maternal bands. Genetic variation between hybrids and their parental stocks was analyzed by RAPD using 10 of the above 38 primers. The average heterozygosity and genetic distance were calculated. The results suggested that the filial generation could inherit a little more genetic materials from paternal fish than that from maternal fish.     

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

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

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. Supported by National Natural Science Foundation of China (No. 30571445), National High-Technology Research and Development Program (863 Program, No. 2006AA10A404), and Project from the Ministry of Science and Technology of China (No. 2006DKA30470-017)     

Effects of tributyltin （TBT） on enzyme activity and oxidative stress in hepatopancreas and hemolymph of small abalone, Haliotis diversicolor supertexta

We investigated the effect of tributyltin (TBT) exposure on the concentration of malondialdehyde (MDA) and the activity levels of the superoxide dismutase (SOD), catalase (CAT), and acid and alkaline phosphatase (ACP and AKP) enzymes in the small abalone, Haliotis diversicolor supertexta. We collected samples of the hepatopancreas and hemolymph 2, 6, 24, 48, 96, and 192 h after exposure to 0.35 μg (Sn)/L TBT. In the hepatopancreas, ACP activity was significantly higher in animals exposed to TBT 2, 24, and 96 h post-exposure compared with the control animals. AKP activity was also higher after 2 h, but SOD and CAT activity was unchanged. The concentration of MDA in the hemolymph was significantly higher than the control animals 2 and 6 h post-exposure. In the hemolymph of animals exposed to TBT, ACP activity was significantly lower than in the control animals 192 h post-exposure, whereas AKP activity was significantly lower 2 and 192 h post-exposure. Hemolymph SOD activity and levels of MDA were significantly lower than in the control animals 24 h after exposure but significantly higher after 96 h. Our results demonstrate that exposure to TBT cause rapid changes in ACP and AKP activity as well as altering the concentration of MDA in the hepatopancreas and hemolymph. SOD and CAT do not appear to be involved in the detoxification of TBT in the hepatopancreas of small abalone.     

蜂巢石斑鱼随机扩增多态性DNA的初步研究

采用RAPD技术分析了湛江沿海野生蜂巢石斑鱼的遗传多样性 ,从 6个系列 1 2 0个引物筛选出1 6个引物 ,对 1 0尾蜂巢石斑鱼进行RAPD分析 ,1 6个引物共检测到 1 5 4个位点 ,其中 ,多态位点比例 (P)为 6 5 5 8% ,平均个体间遗传相似系数 (S)为 0 7887,平均个体间遗传距离 (D)为 0 2 1 1 3,遗传多样性指数 (H)为 0 1 776。研究结果表明 ,湛江沿海的野生蜂巢石斑鱼的遗传多样性较高。     

Comparison of the <Emphasis Type="Italic">in vitro</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis> toxic effects of three sizes of zinc oxide (ZnO) particles using flounder gill (FG) cells and zebrafish embryos

Nano-sized zinc oxide (nZnO) particles are one kind of the most commonly used metal oxide nanoparticles (NPs). This study compared the cytotoxic and embryotoxic effects of three increasing sized ZnO particles (? 30 nm, 80-150 nm and 2 μm) in the flounder gill (FG) cells and zebrafish embryos, and analyzed the contribution of size, agglomeration and released Zn²⁺ to the toxic effects. All the tested ZnO particles were found to be highly toxic to both FG cells and zebrafish embryos. They induced growth inhibition, LDH release, morphological changes and apoptosis in FG cells in a concentration-, size- and time-dependent manner. Moreover, the release of LDH from the exposed FG cells into the medium occurred before the observable morphological changes happened. The ultrasonication treatment and addition of serum favored the dispersion of ZnO particles and alleviated the agglomeration, thus significantly increased the corresponding cytotoxicity. The released Zn²⁺ ions from ZnO particles into the extracellular medium only partially contributed to the cytotoxicity. All the three sizes of ZnO particles tested induced developmental malformations, decrease of hatching rates and lethality in zebrafish embryos, but size- and concentration- dependent toxic effects were not so obvious as in FG cells possibly due to the easy aggregation of ZnO particles in freshwater. In conclusion, both FG cells and zebrafish embryos are sensitive bioassay systems for safety assessment of ZnO particles and the environmental release of ZnO particles should be closely monitored as far as the safety of aquatic organisms is concerned.     

GENETIC DIFFERENTIATION WITHIN ERIOCHOIR SINENSIS (MILNE,EDWARDS) REVEALED BY ALLOZYME AND RAPD

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大口黑鲈仔幼鱼的饵料选择和生长的研究

全长5.8—6.4mm 具卵黄和油球的仔鱼开口摄食轮虫和无节幼体。稚鱼选择枝角类为主。幼鱼对桡足类的选择明显地高于枝角类,对轮虫和无节幼体则无选择性。早期幼鱼还摄食摇蚊幼虫,虾,草鱼苗和本种小鱼。26日龄幼鱼平均日增长1.18mm,日增重22.31mg。数理统计分析确定,全长与日龄呈直线关系。体重与日龄,全长对体重均呈幂函数关系。     

Genetic variation of natural and cultured stocks of Paralichthys olivaceus by allozyme and RAPD

Population genetics of the left-eyed flounder, Paralichthys olivaceus, including natural and cultured stocks distributed in the coastal waters near Qingdao of eastern maritime China, was analyzed in allozyme and RAPD. The results showed that among total 29 gene loci of 15 isozymes, 9 and 7 were po- lymorphic in natural and cultured stocks, respectively. The status of genetic diversity in P. olivaceus is low in terms of polymorphic loci in chi-square test and genetic departure index of Hardy-Weinberg equi- librium. More alleles in IDHP, CAT, GDH and Ldh-C allozymes were found in the fish, which could be used as markers in assortive breeding and distinguishing stock, population or species evolution. Total 88 and 86 RAPD bands ranging from 200 to 2 500 bp were recognized individually in average of 7.8–8.0 bands per primer. The genetic diversity in cultured stock is lower than that in natural ones showing an ob- viously decreasing genetic divergence. Therefore, effective countermeasures must be taken to protect ge- netic resources of marine cultured fishes. The 2 markers have their own pros and cons. Combining the 2 markers to investigate the genetic variation of populations is suggested. The results provide basic data of this flounder and they are useful for studying genetic improvement and genetic resources of the fish.     

EFFECT OF ARTIFICIAL REGULATIONS OF ARTEMIA n-3 HUFA CONTENT ON GROWTH AND SURVIVAL OF BLACK SEABREAM (SPARUS MACROCEPHLUS) LARVAE

ImODUCTIONStudiesonthenutritionmpuirementSoffishlarvaehavernadesomeprognaabroad,butwearenotawareofreportSonsuchstudiesinChinanow.ltwasdeter-Annedanhythatn-3HU-FA,espedally22f6(n-3)roHA)and2O5(n-3)(EPA)ises-sentialformarinefishlarvaegroWth.ThehighcontentofDHAinmarinefisheggSandthel0wcontent0fDHAinArtrmiashowthatArthetaasliwfndcandsaoothen-3HUFAreq~dmendfishlacae.bosdenistSdriitaln-3HUFAenriedArbotabyboingthemn-3HUFAcontainingbogtanulataldietS,etC.Watanabe,T.(l983)enri~livefe…     

ENCLOSURE EXPERIMENTS ON AND LACUSTRINE PRACTICE FOR ELIMINATING MICROCYSTIS BLOOM

ＩＮＴＲＯＤＵＣＴＩＯＮＭｉｃｒｏｃｙｓｔｉｓａｅｒｕｇｉｎｏｓａｗａｓｔｈｅｍａｉｎｂｌｏｏｍ ｆｏｒｍｉｎｇｓｐｅｃｉｅｓｉｎＤｏｎｇｈｕＬａｋｅ;ｏｃｃｕｒｒｅｄｏｎｌｙｓｐｏｒａｄｉｃａｌｌｙｉｎｔｈｅ 1 950ｓ,ｉｎｃｒｅａｓｅｄｒａｐｉｄｌｙｉｎｎｕｍｂｅｒｉｎｔｈｅ 1 960ｓ.Ｆｒｏｍｔｈｅｍｉｄｄｌｅｏｆｔｈｅ 1 970ｓｔｏｔｈｅｍｉｄｄｌｅｏｆｔｈｅ 1 980ｓ,ｔｈｉｃｋｌａｙｅｒｏｆｉｔｓｂｌｏｏｍｃｏｖｅｒｅｄｔｈｅｌａｋｅｓｕｒｆａｃｅｅｖｅｒｙｓｕｍｍｅｒ,ａｔｔｒａｃｔ ｉｎｇｆｏ…     

苯酚对奥尼罗非鱼非特异性免疫指标的影响

采用水体中常见污染物——苯酚对奥尼罗非鱼进行刺激,研究不同浓度苯酚对奥尼罗非鱼非特异性免疫功能的影响。奥尼罗非鱼分别暴露于0.005、0.025、0.125、0.625、3.125 mg/L等5个浓度的苯酚中8周,每两周采集抗凝血液和血清,用于检测非特异性免疫指标。结果表明:暴露于低浓度苯酚组(0.005 mg/L和0.025 mg/L)的奥尼罗非鱼NBT(氯化硝基四氮唑蓝)阳性细胞数等非特异性免疫指标相对于对照组无显著性差异(P>0.05),中浓度苯酚组(0.125 mg/L和0.625 mg/L)和高浓度苯酚组(3.125mg/L)对罗非鱼非特异性免疫指标的影响存在显著的时间效应和剂量效应(P<0.05),可减少NBT阳性细胞数、抑制超氧化物歧化酶活力和抗菌活力;促使溶菌酶活力和溶菌酶含量先升后降。表明中高浓度苯酚组对奥尼罗非鱼具有明显的免疫毒性。     

3-甲基胆蒽对斑马鱼组织病理效应的初步研究

分别用25、50、100、200和400μg/L的3-甲基胆蒽(3-MC)对斑马鱼进行水浴染毒,于第7天和第14天采集各染毒组和对照组活鱼的肝、鳃、心、肠和肾,进行病理组织学检查。结果发现,一定剂量的3-MC能导致肝细胞变性,胞浆内出现脂肪滴,严重时肝细胞坏死解体,界限模糊;心肌细胞萎缩坏死;肠粘膜上皮细胞纹状缘不整齐,有的脱落;鳃丝结构异常,鳃小片表面粗糙,上皮细胞不完整;肾小管上皮细胞肿胀,细胞界限不清,管腔内均质红染。供试斑马鱼的病理组织变化与3-MC的作用剂量和作用时间存在一定的关系。     

Effects of waterborne Fe(II) on juvenile turbot Scophthalmus maximus: analysis of respiratory rate, hematology and gill histology

The concentration of Fe(Ⅱ) is high in some groundwater supplies used in turbot culture,and the toxicity of waterborne Fe(Ⅱ) is unknown.We investigated the stress responses of juvenile turbot,Scophthalmus maximus,exposed to Fe(Ⅱ) of different concentrations (0.01,0.05,0.1,0.5,1,and 2 mg/L) for 1,7,14,and 28 d,under the same ambient conditions of other parameters.Changes in respiratory rate,hematological parameters,and gill structure were determined.The results show that waterborne Fe(Ⅱ) did not cause severe hematological perturbation to turbot.A low-medium Fe(Ⅱ) concentration (lower than 0.1 mg/L) could boost the respiratory rate,and caused no or very limited damage to fish.A high Fe(Ⅱ) concentration (0.1 mg/L or higher),however,caused gill damage,such as vacuoles in branchial lamellae,epithelial necrosis,and hypertrophy of epithelial cells,and even death after extended exposure time.Therefore,excess waterborne Fe(Ⅱ) and long-term exposure to Fe(Ⅱ) could be responsible for poor growth and high mortality of turbot in culture.The concentration of waterborne Fe(Ⅱ) in turbot culture should be kept below 0.1 mg/L.     

Effect of lead on cytoskeletal protein stability in crucian carp Carassius auratus

Inorganic lead (Pb) is one of the most common environmental pollutants. Much evidence indicates that Pb exposure could directly affect fish growth and development. In this study, we investigated the cytotoxic effects of Pb on cytoskeletal protein stability at both protein and mRNA level in crucian carp Carassius auratus. Pb(NO3)2 treatment in concentration of 100 μmol/L resulted in decreased expression of both α- and β-tubulin but γ-tubulin as assayed with SDS-PAGE, Western Blot, and ELISA. In vivo and in vitro analyses on protein expression of tubulins are consistent. The effect of Pb on mRNA expression varied among different tissues. Our results suggest that cytotoxicity of Pb at protein translation level is stronger than at mRNA expression level.