大菱鲆趋化因子CCL34基因的鉴定及其响应细菌感染的表达特征

为了研究大菱鲆(Scophthalmus maximus)中趋化因子(Chenokine)的特征及其在大菱鲆免疫过程中发挥的作用,设计了本文中的实验。本实验从大菱鲆基因组和转录组数据库中鉴定了一个硬骨鱼特有的趋化因子CC亚家族成员—CCL34,并选取大菱鲆的8个健康组织以及2种细菌感染后的肠道和皮肤组织,采用实时荧光定量PCR(qRT-PCR)对其表达特征进行研究。序列分析结果显示该趋化因子全长mRNA包含1个327 bp的5’非编码区(UTR),1个246 bp的3’非编码区,和1个编码103个氨基酸残基长度为312 bp的开放阅读框(ORF)。此CCL34基因含有4个外显子和3个内含子。通过系统发育分析、共线性分析,将该大菱鲆趋化因子命名为CCL34。实时荧光定量PCR表明CCL34在大菱鲆健康组织中普遍表达,尤其在肾脏、肝脏、皮肤中有高水平表达。鳗弧菌(Vibrio anguillarum)和无乳链球菌(Streptococcus agalactiae)感染后,肠道组织CCL34基因表达量较对照组显著性上调(p<0.05),这表明CCL34可能在大菱鲆的肠道粘膜免疫中起重要作用。本研究为趋化因子家族功能的研究奠定了基础,为增强鱼类免疫力和抗病能力提供了理论依据。     

Changes of melatonin and its receptors in synchronizing turbot (Scophthalmus maximus) seasonal reproduction and maturation rhythm

In most fish, reproduction is seasonal or periodic under the suitable conditions. In turbot(Scophthalmus maximus) farms, one of the most economically important marine flatfish species, changes in daylength could cause changes in the spawning time. In this study, to characterize the regulation of reproductive physiology following light signals, three melatonin receptors(Mtnr) investigated in turbot were named sm Mtnr1, sm Mtnr2,and sm Mtnr1 c. Distinct expression profiles demonstrated that Mtnr m...     

牛蒡寡糖对大菱鲆生长和免疫机能的影响

将自主研发的牛蒡寡糖作为添加剂添加到大菱鲆的基础饲料中,探讨其对大菱鲆(Scophthalmus maxi-mus)的生长和免疫机能的影响。经过60 d不同剂量(1.0%,2.0%,4.0%,6.0%)的饲喂试验,分别从每组随机抽取鱼体,测定其生长指标和血清中ACP,AKP,LSZ,SOD和PO等多种免疫相关酶的活力以及血液中白细胞的吞噬活性。结果表明,牛蒡寡糖作为一种非特异性免疫调节剂对大菱鲆表现出优良的促生长作用,还能显著提高大菱鲆的免疫相关酶活力和白细胞的吞噬活性,从而提高大菱鲆的非特异性免疫力,其适宜添加量为4.0%。牛蒡寡糖可以开发成为水产动物的促生长剂和免疫增强剂。     

水位深度对工厂化流水养殖大菱鲆生长的影响

为探讨水深对工厂化流水养殖水环境的影响,本实验将9 000尾初始体质量为141.62±24.47 g的大菱鲆(Scophthalmus maximus)按照低水深(20 cm)、中水深(40 cm)、高水深(60 cm)条件分为3个不同养殖水深组,实验周期为80 d。实验期间,跟踪检测长期和特定时期(投喂后8 h内)不同养殖水深水体中总氨氮(TAN)、亚硝酸盐(NO₂^--N)、固体悬浮物(SS)、化学需氧量(COD)等参数,并在实验结束时对大菱鲆成活率、体质量、饲料系数水平进行测量。研究表明,水池内水流速度与水深呈负相关,但各组间无显著性差异。高水深组的固体悬浮物含量显著(P<0.05)低于其他两组,低水深组的化学需氧量显著(P<0.05)低于其他两组,各水深组中氨氮、亚硝酸盐都在大菱鲆幼鱼安全浓度范围内,且无显著性差异。在投喂后,固体悬浮物含量在各水深组中呈先升高后降低趋势,其中低水深组波动最大。氨氮含量在投喂后3 h开始上升,其中低水深组涨幅最大。各水深组中化学需氧量随着投喂时间延长而逐渐积累,而亚硝酸盐含量基本保持不变。实验结束,低水深组中大菱鲆增质量率、特定生长率、体质量变异系数均显著(P<0.05)高于高水深组,而存活率、肥满度、饲料系数在各组之间没有显著差异。研究结果显示,增加水深有利于提高养殖水环境水质恶化的缓冲能力。在保证养殖系统水质指标安全的前提下,低水深在大菱鲆工厂化流水养殖中是一个可行的方案。     

大菱鲆免疫球蛋白轻链IgL全长cDNA的分离、鉴定及表达分析

实验采用末端快速扩增(RACE)技术从大菱鲆脾脏cDNA文库中筛选得到了免疫球蛋白轻链IgL的全长cD-NA片段。该序列包含47 bp的5′末端非编码区(5′UTR),738 bp的开放阅读框(ORF)和202 bp 3′UTR,整个开放阅读框编码246个氨基酸。系统发生分析表明大菱鲆IgL基因与五条鰤的IgL基因起源关系最近。RT-PCR分析表明,大菱鲆IgL基因只在正常脾脏、肾脏和头肾组织中表达;IgL在大菱鲆胚胎发育细胞期就已开始表达,在大菱鲆胚胎尾芽期和体节期表达持续增强;在鳗弧菌感染大菱鲆脾脏和头肾早期均检测到IgL基因强烈表达,后期表达逐渐减弱;大菱鲆胚胎细胞(TEC)在用鳗弧菌感染48h后,IgL开始表达。这些结果均表明IgL基因在大菱鲆免疫应答中起着重要作用。     

养殖水体中氨氮对大菱鲆的急性毒性效应研究

为了评估大菱鲆(Scophthalmus maximus)耐氨氮性状的遗传参数和为循环水养殖大菱鲆提供科学指导,作者研究了养殖海水中氨氮对4月龄和8月龄大菱鲆的急性毒性效应,同时对比了氨氮对白化和正常个体、雌性和雄性个体的急性毒性效应差别。结果表明:非离子氨对4月龄大菱鲆的24、48、72、96 h LC50分别为2.19、1.94、1.80、1.72 mg/L,对8月龄大菱鲆的24、48、72、96 h LC50分别为3.64、3.02、2.93、2.86 mg/L,非离子氨对4月龄和8月龄大菱鲆的安全质量浓度分别为0.17和0.29 mg/L;氨氮对白化和正常大菱鲆以及雌性和雄性大菱鲆的急性毒性效应均没有显著性差异,研究结果为进一步选育大菱鲆的耐氨氮品系提供科学依据。     

Morphological and histological changes in the brains of turbot (Scophthalmus maximus) with gonadal development

The brain plays a critical role in controlling reproduction through the hypothalamus-pituitary-gonadal (HPG) axis in vertebrates. Turbot (Scophthalmus maximus) has become an economically important marine fish in Europe and North China. Previous research investigating turbot reproduction has focused on the role of the HPG axis in regulating egg and sperm production. However, the morphology and histology of the organs in the HPG axis have not been studied. In this study, we investigated the morphology and histology of brains in female and male turbot at different stages of gonadal development. The results showed that the brains of both female and male turbot were composed of seven parts that are typical of advanced teleosts: the telencephalon, diencephalon, cerebellum, hypothalamus, pituitary gland, myelencephalon, and olfactory bulbs. The telencephalon was well-developed and contained five distinct lobes, with the contiguous diencephalon at the caudal portion. The torus longitudinales and rostral torus semicircularis of the mesencephalon flattened along the dorsal surface, and the rostral corpus cerebellum was located in the dorsal portion. The actual total brain volume in mature males was significantly greater (p<0.05) than that of females with gonadal development. Notably, the pituitary volume in male turbot significantly increased (p<0.05) from immature to mature stage, but this difference did not occur in females. The data together illustrate a distinct sex difference in the turbot brain during gonadal development, providing insight into their HPG axes.     

大菱鲆家系选育二代7种免疫因子的分析

为选育高成活率的大菱鲆(Scophthalmus maximus)抗鳗弧菌(Vibrio anguillarum)群体,对30个大菱鲆选育二代家系(F2)和1个普通养殖群体进行鳗弧菌攻毒实验(周期为14 d),统计分析死亡率。选取死亡率不同的6个选育家系和普通养殖群体构成7个实验组,采用半定量-聚合酶链反应(semi-quantitative polymerase Chain reaction,RT-PCR)技术对它们肝脏、脾脏、头肾的相关免疫因子——溶菌酶(Lysozyme)、抗菌肽(Hepcidin)、热激蛋白70(HSP70)、热激蛋白90(HSP90)、免疫球蛋白(Ig M)、C-型凝集素(C-type lectin)、Lily-型凝集素(Lily-type lectin)的表达量开展研究,并应用SPSS 16.0软件对攻毒前后各免疫因子的表达量与攻毒后存活率之间的相关性进行分析。研究结果表明:攻毒前后选育家系幼鱼肝脏、脾脏、头肾中7个免疫因子的表达量普遍高于普通养殖幼鱼,且在7个实验组中,攻毒后的表达量相较攻毒前均呈现下降趋势;攻毒前,肝脏中lysozyme、Ig M的表达量与存活率为极显著性正相关(P0.01),HSP70、HSP90、C-type lectin、Lily-type lectin的表达量与存活率为显著性正相关(P0.05);脾脏中,Hepcidin、HSP70和HSP90的表达量与存活率为极显著性正相关(P0.01);头肾中,HSP70的表达量与存活率为显著正相关(P0.05)。综上,可以推断选育家系相对于普通养殖群体大菱鲆抗鳗弧菌性能更强,且7种免疫因子在鳗弧菌感染鱼体的过程中发挥重要的抗感染作用;从F2中获得一个抗鳗弧菌性能较强的家系(23号),可用于今后大菱鲆抗鳗弧菌品系的选育指导工作,为大菱鲆高成活率群体的选育提供参考资料和理论依据。     

大菱鲆亲子鉴定的微卫星多重PCR技术建立及应用

用8个微卫星标记组合建立了2个微卫星多重PCR体系,对大菱鲆7个人工选育家系进行了系谱认证、亲子鉴定和遗传多样性研究。2个多重PCR体系中8个微卫星位点共检测到等位基因49个,每个位点等位基因数为3~8个。根据已知亲本及子代基因型,成功推断出了3个缺失亲本的基因型。在双亲未知的情况下2个多重PCR的8个微卫星位点累积排除概率是96.58%,已知1个亲本时累积排除率为99.71%,亲子鉴定准确率为96.42%。采用70个个体进行双盲验证,利用UPGMA法对7个家系的70个体进行了聚类分析,同一家系95.71%的个体聚类分析结果与系谱来源一致。Cervus 2.0软件亲子鉴定结果表明亲子鉴定准确率为92.86%。2个多重PCR体系的建立为大菱鲆不同家系混养后的亲子鉴定、系谱分析和分子辅助家系管理提供了技术手段。     

Accumulation and effects of benzo(a)pyrene on cytochrome P450 1A in waterborne exposed and intraperitoneal injected juvenile turbot (Scophthalmus maximus)

Juvenile turbot (Scophthalmus maximus) were exposed to benzo(a)pyrene (BaP) for 14 d using a glass bead generator flow-through system. Exposure was followed by a recovery period of 16 d. The highest BaP concentration in the edible portion of the fish, 16.5 ± 4.3 μg BaP/kg, was observed on the first day. Then concentrations dropped following first-order kinetics. BaP was below detection level at the end of the experiment. A statistically significant increase in bile fluorescence was observed from day 9 until the end of the experiment, suggesting the elimination of BaP metabolites by this route. No significant differences between control and exposed fish in EROD activity and CYP1A concentration, measured by immunodetection method, were observed. Intraperitoneal injection of 2.5 mg BaP/kg in juvenile turbot induced EROD activity. Under waterborne experimental conditions, bile fluorescence was observed to be a more sensitive biomarker of BaP exposure than EROD activity and CYP1A measurement.     

不同脂肪水平下添加胆汁酸对大菱鲆(Scophthalmus maximus)幼鱼生长、体组成和脂肪代谢的影响

在10%和18%两个脂肪水平下,分别添加0%、1.5%的胆汁酸,配制成四种等氮饲料,投喂初始平均体重为(45.78±0.11)g的大菱鲆(Scophthalmus maximus)幼鱼8周,研究两个脂肪水平下添加胆汁酸对大菱鲆幼鱼生长、体组成和脂肪代谢的影响。结果表明:饲料脂肪水平升高或添加胆汁酸均能显著提高大菱鲆幼鱼增重率、特定生长率、蛋白质效率,降低饵料系数(P0.05);相同脂肪水平下,添加胆汁酸显著提高了实验鱼的生长,降低了肝体比(P0.05)。随着饲料脂肪水平的升高,组织中粗脂肪含量呈上升的趋势;而添加胆汁酸降低了组织中脂肪的蓄积,但全鱼与肌肉中水分、粗蛋白含量主要受饲料中胆汁酸水平的影响(P0.05)。胆汁酸能显著降低大菱鲆幼鱼肌肉PUFA含量(P0.05),增加SFA、MUFA含量。饲料添加胆汁酸能降低实验鱼血清甘油三酯、总胆固醇、高密度脂蛋白胆固醇、低密度脂蛋白胆固醇含量及谷草转氨酶、谷丙转氨酶活性(P0.05),但饲料脂肪水平对血液生化指标的影响呈相反趋势。饲料脂肪水平的升高或添加胆汁酸均能提高实验鱼肝脏脂蛋白脂酶、肝脂酶、总脂酶和肠道脂肪酶活性(P0.05),且胆汁酸和脂肪二者之间的交互作用对脂肪代谢有显著影响(P0.05)。综上所述,在2个脂肪水平下添加胆汁酸均能促进大菱鲆幼鱼的生长,提高饲料利用状况,促进脂肪消化吸收,降低组织中粗脂肪含量。     

Enzymatic activities in spermatozoa and butyltin concentrations in Baltic turbot (Scophthalmus maximus)

Spermatozoal enzymes of fish (NAD+- and NADP-dependent dehydrogenases and creatine kinase (CK)) were previously determined to be sensitive to tributyltin (TBT) in laboratory experiments and were thus indicated for use as biomarkers for TBT exposure. However, the potential ability of spermatozoal enzymes as biomarkers of TBT exposure has never been recapitulated in a field study. For this purpose, the kinetic activities of spermatozoal enzymes of the natural turbot Scophthalmus maximus population from the Gulf of Gdańsk (GDA) and the Pomeranian Bay (POM) in the southern Baltic Sea were measured. Gas chromatography/high-resolution mass spectrometry was used to determine the concentrations of TBT and its breakdown products, dibutyltin (DBT) and monobutyltin (MBT), in the muscle, liver and testes of the male turbot. Males from GDA had significantly higher enzymatic activities and butyltin (BT) content in tissues than those from POM. A general linear model (GLM) showed that lactate dehydrogenase (LDH), malate dehydrogenase (MDH), glucose-6-phosphate dehydrogenase (G6PDH) and CK activities increased significantly with BT concentration in the testes and liver. We indicate the potential effects of TBT pollution on the spermatozoal enzymes of Baltic turbot.     

Correlative changes in metabolism and DNA damage in turbot (Scophthalmus maximus) exposed to benzo[a]pyrene

Juvenile turbot (Scophthalmus maximus) were injected intraperitoneally with either corn oil or 5 mg/kg benzo[a]pyrene (BaP) dissolved in corn oil and sampled I and 3 days after injection. After 1 day, no elevation of 7-ethoxyresorufin O-deethylase (EROD) activity was observed, however bile metabolites (BaP-7,8 dihydrodiol representing 70% of the total metabolites) and a single hepatic DNA adduct spot (0.47 adducts/10(8) nucleotides) identified by 32P-postlabelling were formed. No BaP metabolites or DNA adducts were observed in either control or carrier control fish. Fish sampled after 3 days reported 5-fold higher (P < 0.05) levels of EROD activity, a shift in the bile metabolite profile towards BaP phenol formation (1OH and 30H BaP comprising up to 60% of total metabolites detected) and the formation of two adduct spots (0.86 and 0.71 adducts/10(8) nucleotides). These results show that BaP can be metabolised and form hydrophobic DNA adducts in turbot without EROD elevation. Following EROD elevation, a shift in the profile of both BaP metabolites and BaP metabolite-DNA interactions occurs indicative of other oxidative processes.     

大菱鲆精子运动特征与精液pH的相关性研究

为探究大菱鲆(Scophthalmus maximus)精液质量与pH的关系及甘油对大菱鲆精子激活的影响,本研究通过计算机辅助精子分析系统分析检测了威海和烟台4家大菱鲆育苗场(A、B、C、D)的精子质量。研究发现,不同育苗场的大菱鲆精子活力存在显著的差异,A2A1DCB,精子预测寿命分别为11.7、7.1、6.8、4.6、3.3 min,所取精子的质量的存在显著差异。研究结果发现,大菱鲆精子运动特性与精子活化率相关性显著,其中精子活化率与平均曲线运动速度、平均直线运动速度、平均路径速度、精子头侧摆幅度、精子平均鞭打频率呈显著正相关(P0.05),与运动直线性、运动摆动性、运动前向性等参数无显著相关性;大菱鲆精子运动特性受pH影响显著,精液pH与平均曲线运动速度、平均直线运动速度、平均路径速度、精子头侧摆幅度、精子平均鞭打频率呈显著正相关(P0.05),而与运动直线性、运动摆动性、运动前向性等参数无显著相关性。同时还发现,精子活化率高、寿命长的大菱鲆精液pH偏弱碱性(pH=8.0),即大菱鲆精子适宜在弱碱性环境中活动。除此之外,向激活海水中加入梯度甘油(0.1、1、10、100、500、1 000 mmol/L),发现可以显著提高大菱鲆精子寿命,其中甘油浓度为100 mmol/L时,精子寿命延长至15.17 min±0.29 min,推测甘油在精子激活后可能被精子吸收并氧化分解提供能量。     

大菱鲆对迟缓爱德华氏菌的免疫应答

用制备的迟缓爱德华氏菌灭活全菌和主要外膜蛋白(OMP)对大菱鲆进行腹腔注射及肛门灌注免疫,以Ig+细胞数量及血清抗体效价为指标,评价和比较大菱鲆对不同抗原的免疫应答水平.结果显示,注射2种抗原后,外周血中Ig+细胞数量及血清抗体效价均呈明显增加趋势,注射OMP大菱鲆的外周血Ig+细胞数量及血清抗体效价不显著低于注射灭活全菌大菱鲆(外周血Ig+细胞:P=0.390;抗体效价:P=0.300);肛门灌注2种抗原后,大菱鲆外周血和后肠中Ig+细胞数量及血清凝集效价亦均呈增加趋势,灌注OMP大菱鲆后肠、外周血中Ig+细胞数量和血清抗体效价均不显著高于灌注灭活全菌大菱鲆(外周血Ig+细胞:P=0.056;后肠Ig+细胞:P=0.163;抗体效价:P=0.195).结果表明,注射和肛门灌注迟缓爱德华氏菌灭活全菌和OMP均能够诱导大菱鲆产生针对迟缓爱德华氏菌的特异性免疫应答.本文结果对鱼类疫苗的研制具有参考价值.     

Annual and spatial variation in the abundance length and condition of juvenile turbot (Psetta maxima L.) on nursery grounds on the west coast of Ireland: 2000–2007

Turbot (Psetta maxima Linnaeus) is a high value commercially exploited marine flatfish which occurs in European waters, from the Northeast Atlantic to the Arctic Circle, the Baltic and Mediterranean Sea. In Ireland, turbot are the most valuable commercial non-quota species. Very little is known about their population dynamics in the wild, in particular during the sandy beach nursery phase of the life history. In 2000, a survey was established to assess flatfish species on nursery grounds on the west coast of Ireland. Eleven sandy beaches were assessed for 0+ turbot by beach seining, over an eight year period (2000–2007) during the months of August and September. The objective of the study was to estimate juvenile turbot abundance and size structure to determine if any spatial and annual trends existed. Large scale variability in the recruitment of fish to nursery grounds may be indicative of fluctuations in the adult stock. Turbot were found to recruit to five beaches consistently over the eight year period. Temporal and spatial variability in the relative abundance and length of turbot was discerned, with no apparent overall trend. However, certain nursery grounds were shown in most of the years examined to support higher abundances of turbot in comparison to other areas over the eight year period. Turbot abundances on nursery grounds were significantly correlated with mean spring sea temperatures during the pelagic stage. The condition of turbot did not significantly differ on an annual or spatial scale. Mean densities of 0+ turbot along the Irish coast were found to be similar and at times higher than other areas in Europe, ranging from 0.1 (± 0.3) individuals 1000 m^− 2 to 18.5 (± 6.9) individuals 1000 m^− 2. Mean turbot total length on beaches ranged from 3.8 cm (± 0.6) to 6.6 cm (± 4.3). The observed spatial and temporal variability in abundance and length highlights the need for long-term studies when assessing juvenile flatfish populations. Results from the present study have provided much needed baseline data on wild juvenile turbot populations which is severely lacking for this species both on an Irish and on a European scale.     

Hepatic biomarkers of sediment-associated pollution in juvenile turbot, Scophthalmus maximus L

Hatchery-reared turbot (Scophthalmus maximus L.) were exposed for 3 weeks, under laboratory conditions, to sediment collected from polluted sites in Cork Harbour and a reference site at Ballymacoda, Co. Cork, Ireland. The potential of surficial sediment for inducing hepatic biomarkers was assessed at two levels of biological organisation: expression of cytochrome P450 [Western blotting analysis and 7-ethoxy-resorufin O-dealkylase (EROD), 7-benzoxy resorufin O-dealkylase (BROD), 7-methoxy resorufin O-dealkylase (MROD), 7-pentoxy-resorufin O-dealkylase (PROD) activities] and DNA integrity (Comet assay). Positive controls were generated, either by exposing turbot to cadmium chloride-spiked seawater (Comet assay) or to beta-naphthaflavone by intraperitoneal injection (cytochrome P450 induction). The induction of cytochrome P450 activity (EROD, MROD and PROD) in animals following a 7-day exposure to contaminated sediments was significantly higher than those exposed to reference site sediment and remained elevated thereafter; BROD was not induced. DNA single-strand breaks were also significantly higher following exposure to contaminated sediments throughout the experiment. Although no direct correlation between induction of alkoxyresorufin O-dealkylase activities and a particular chemical class was established, the induction of MROD and PROD activities in fish exposed to sediments containing complex contaminant mixtures, appeared to be more sensitive than conventional EROD activity assays. We conclude from the present laboratory study that S. maximus is a suitable sentinel species for the assessment of moderately contaminated sediments and therefore allows for the further development of this model for future, ecologically relevant, field studies.     

养殖密度对陆基工厂化循环水养殖系统中大菱鲆(Scophthalmus maximus)幼鱼生长和胁迫反应的影响研究

Stocking density is widely recognized as a critical factor in aquaculture and a potential source of long-term stress.The influence of stocking density on growth and stress response of juvenile turbot(Scophthalmus maximus, ~3–75g, initial to final weight) was examined in fish held under low(LD, ~0.21–5.31 kg/m~2, initial to final density),medium(MD, ~0.42–10.81 kg/m~2) and high stocking density(HD, ~0.63–14.27 kg/m~2) for 120 days in a recirculating aquaculture system(RAS). In this trial, the growth curve for weight of juvenile turbot in RAS, all fitted by the Schnute model. No significant difference was found in growth performance among the three densities until at the final sampling(Day 120). The final weight and body weight increase(BWI) in the HD group were significantly lower than in other groups(P0.05, weight:(75.83±2.49) g,(75.39±2.08) g,(65.72±2.86) g and BWI:(2 436.12±28.10)%,(2 421.29±4.64)%,(2 097.88±20.99)% in LD, MD and HD groups, respectively). Similarly, the specific growth rate(SGR), feed conversion ratio(FCR) and coefficient of variation for weight(CV_w) were adversely affected by high stocking density(P0.05). However, there was no difference in survival and Fulton's condition factor(K) of turbot among the different groups. Physiological analyses demonstrated a clear increase in the plasma cortisol level and an obvious decrease in growth hormone(GH) concentration in the HD group on Day120(P0.05). There was no significant effect of stocking density on plasma glucose, Cl– and protein levels. All these findings would provide a reference for selecting the optimal stocking density of juvenile turbot in RAS.     

Rhodamine exclusion activity in primary cultured turbot (Scophthalmus maximus) hepatocytes

Cellular detoxification by direct processes has been investigated in fish by studying the ability of hepatocytes prepared from juvenile aquarium-reared turbot (Scophthalmus maximus) to actively exclude the fluorescent dye rhodamine B (RB). Cell viability was studied by measurements of non-specific esterase activity using fluorescein diacetate. This revealed that turbot hepatocytes can be cultured for a few days with a viability decreasing to 38% after 24 h. The 24-h cultured cells have been used to study the rhodamine B exclusion activity using confocal laser microscopy. Hepatocytes accumulated the dye in a competitive manner with verapamil, thus suggesting that they express a transport system similar to the P-glycoprotein-mediated multixenobiotic resistance process. Incubation of cells with 1 microM RB and 20 microM verapamil led to a 26% increase of cellular fluorescence as compared to the accumulation in absence of competitor. Rhodamine B accumulated in the whole cytoplasm, with more concentrated areas that might correspond to the lysosomal compartment and the cell membrane.