Expression and activity of a multixenobiotic resistance system in the Pacific oyster Crassostrea gigas

The multidrug resistance (MDR) mechanism corresponds to a defence system relying on the expression of high molecular membrane proteins that can actively lower the intracellular concentration of a wide variety of toxins, thus maintaining them below their toxic level. Using RT-PCR, expression levels of a gene belonging to the class I of mammalian mdr genes, has been assessed in different developmental stages of the oyster Crassostrea gigas. While no expression was found in the oocyte or the trocophore stage, a rise of mRNA content was observed from the veliger stage to the juvenile stage, thus indicating the induction of the system as the animal is developing in the environment. The incubation of gill fragments in the dye rhodamine B and subsequent measurements of intracellular fluorescence using a microplate reader indicates that the system can effectively decrease the accumulation of the test compound in a competitive manner with known inhibitors or environmental contaminants as observed in vertebrate cells. The oyster MXR system is thus becoming active in adult oyster and could be of importance in environmentally contaminated areas.     

Metal speciation and toxicity of Tamar Estuary water to larvae of the Pacific oyster, Crassostrea gigas

As part of the PREDICT Tamar Workshop, the toxicity of estuarine waters in the Tamar Estuary (southwest England) was assessed by integration of metal speciation determination with bioassays. High temporal resolution metal speciation analysis was undertaken in situ by deployment of a Voltammetric In situ Profiling (VIP) system. The VIP detects Cd (cadmium), Pb (lead) and Cu (copper) species smaller than 4?nm in size and this fraction is termed 'dynamic' and considered biologically available. Cadmium was mainly present in the dynamic form and constituted between 56% and 100% of the total dissolved concentration, which was determined subsequently in the laboratory in filtered discrete samples. In contrast, the dynamic Pb and Cu fractions were less important, with a much larger proportion of these metals associated with organic ligands and/or colloids (45-90% Pb and 46-85% Cu), which probably reduced the toxicological impact of these elements in this system. Static toxicity tests, based on the response of Crassostrea gigas larva exposed to discrete water samples showed a high level of toxicity (up to 100% abnormal development) at two stations in the Tamar, particularly during periods of the tidal cycle when the influence of more pristine coastal water was at its lowest. Competitive ligand-exchange Cu titrations showed that natural organic ligands reduced the free cupric ion concentration to levels that were unlikely to have been the sole cause of the observed toxicity. Nonetheless, it is probable that the combined effect of the metals determined in this work contributed significantly to the bioassay response.     

福建太平洋牡蛎种群微卫星DNA分析

本文采用Ucdcg153、157、202微卫星引物对福建两个太平洋牡蛎(学名为长牡蛎Crassostrea gigas)养殖种群进行扩增分析和序列测定.与采自漳浦旧镇的近江牡蛎(C. ariakensis)比较,前者对以上三种引物全部呈阳性反应,后者只对Ucdcg157显阳性.与Genebank提供的相关序列比较,福建太平洋牡蛎与Genebank样品应属同源,其中漳浦霞美和厦门白礁的样品则可能代表同一种群的两个衍生品系.本项调查结果同时显示,近亲繁殖的育苗方式已经导致福建太平洋牡蛎种群裂化;任其发展不利于维护良种优势.本次采集的样品中未发现显示葡萄牙牡蛎、熊本牡蛎和美洲牡蛎微卫星特征的个体.     

长牡蛎贝壳损伤后的再固着能力初探

牡蛎是重要的养殖贝类,营固着生活,能聚集形成牡蛎礁,在海洋生态系统中发挥着重要作用。本研究以成体长牡蛎为研究对象,将牡蛎从附着基上剥离下来,去掉其左壳腹缘端的表面鳞片并打磨平整,露出棱柱层,将其紧密粘合于水泥砖、橡胶片、磨砂玻璃三种固着基表面,背缘端用水泥固定,观察并记录牡蛎贝壳的修复及再固着情况。结果发现：左壳损伤后,在贝壳边缘会生长出新的透明壳膜,此时壳膜虽然会紧靠固着基表面生长,但却不能再次固着;将牡蛎从固着基上剥离下来,在三种固着基表面均未发现有“附着斑”形成。因此,为了进一步研究左壳损伤后贝壳修复的分子机制以及不能再次固着的原因,对长牡蛎左壳损伤后的外套膜转录组进行了分析,发现在损伤后的第1 d、第5 d、第9 d、第13 d、第17 d和第21 d,左侧外套膜中差异上调表达的基因显著富集在TGF-β等信号通路,而下调表达的基因显著富集在对温度以及对非生物刺激反应相关通路。通过基因表达数据,还发现壳损伤后Cgfmp、CgTyr、EGF-P1等与黏附相关的基因呈下调表达,这可能是导致成体长牡蛎无法再次固着的原因。长牡蛎贝壳损伤后再固着能力的研究,为贝类生物矿化、附着等机制的研究...     

双酚A暴露对不同黑色素含量长牡蛎的影响研究

长牡蛎的壳色有很多种,其中壳色较深个体(黑壳)比较浅个体(白壳)的外套膜中含有更多的黑色素。本研究采用高效液相色谱-质谱法(HPLC-MS/MS)测定天然海水中白壳与黑壳长牡蛎外套膜双酚A (BPA)含量,发现黑壳长牡蛎外套膜中的BPA含量较白壳长牡蛎低(P0.05)。由于黑色素合成过程中的关键——酶酪氨酸酶是一种酚氧化酶,可以通过对氧化酚类或芳胺类等多种底物的氧化起催化作用,推测其在BPA等酚类毒物的降解中也发挥重要作用。为了进一步研究不同黑色素含量牡蛎对BPA降解能力和对BPA损伤抵抗能力的差异,本实验进行了浓度为1 mg/L,1.5 mg/L和2 mg/L的BPA亚慢性暴露实验:通过对暴露后长牡蛎外套膜组织中BPA含量分析发现,白壳与黑壳长牡蛎外套膜中BPA含量均随着暴露浓度的升高而升高,且3种浓度BPA亚慢性暴露后,黑壳长牡蛎外套膜BPA含量均低于白壳长牡蛎,但是差异不显著(P0.05)。采用3种浓度BPA亚慢性暴露后,黑壳长牡蛎外套膜组织中活性氧(ROS)荧光值均显著低于白壳长牡蛎(P0.01)。通过对外套膜组织学观察发现,2 mg/L的高浓度BPA暴露使白壳长牡蛎外套膜组织发生了明显的病理变化,黑壳长牡蛎外套膜未发生明显的病理变化。可见,黑壳长牡蛎对BPA暴露可能具有更强的抵抗能力。     

Multi-technique approach to characterise the effects of cryopreservation on larval development of the Pacific oyster (Crassostrea gigas)

Cryopreservation experiments were conducted on D-stage larvae of the Pacific oyster (Crassostrea gigas) to investigate the effects of two cryoprotectant solutions and three cooling rates on larval development from 1 to 22 days post-fertilisation. Cryoprotectant solutions were made up to final concentrations (after 1:1 dilution with larvae) of 10% ethylene glycol, 1% polyvinylpyrrolidone and either 0.2 or 0.4 M trehalose. Three cooling rates (0.5, 1 and 2 °C min^?1 between ?10 and ?35 °C post-holding) were tested in an orthogonal design with the two cryoprotectants. Results indicate that control larvae out-performed all cryopreservation treatments for survival, feeding consumption and shell length parameters. However, larvae exposed to 0.4 M trehalose did considerably better than those exposed to 0.2 M trehalose, regardless of cooling rate conditions. Scanning electron and light microscopy observations were used to assess larval morphology and organogenesis, indicating that treatments with surviving larvae were morphologically and developmentally similar to control larvae.     

长牡蛎新型模式识别受体CgLRRC69的克隆和功能鉴定

在双壳类软体动物中, 血淋巴细胞介导的吞噬作用是清除入侵微生物的主要方式。本文在长牡蛎中鉴定了一个包含富含亮氨酸重复序列(Leucine-rich repeat, LRR)结构域的新型基因, 命名为CgLRRC69。对该基因的组织分布分析表明, CgLRRC69 mRNA在血淋巴细胞、鳃、肌肉、外套膜、心脏、消化腺和性腺中广泛表达。副溶血弧菌感染可以显著地刺激CgLRRC69在血淋巴细胞中表达, 并且在感染后6h达到峰值。同时, 酶联免疫吸附实验发现CgLRRC69可以特异性结合脂多糖(lipopolysaccharide, LPS), 表明它可能在免疫防御中有功能。吞噬实验结果显示, CgLRRC69重组蛋白可以显著地提高血淋巴细胞的吞噬能力; RNAi干扰CgLRRC69在牡蛎体内的表达, 显著降低了血淋巴细胞对细菌的清除能力。因此, 这些结果揭示了CgLRRC69作为一种新型模式识别受体, 可以特异性识别革兰氏阴性菌的主要成分LPS, 通过调理作用有效地清除细菌。     

长牡蛎繁殖周期、生化成分的季节变化与环境因子的关系

为了阐明长牡蛎的繁殖策略,自2004年3月—2005年2月对乳山湾养殖海区的环境因子(水温、盐度、叶绿素a含量)、长牡蛎繁殖周期及其生化成分(糖原、脂肪、蛋白质、RNA/DNA比值)的季节变化进行研究。结果显示,水温在一年中呈现显著变化,夏季水温最高,最高值为29.5℃(8月),冬季水温最低,最低值为1.8℃(1月);盐度在夏季雨水较多的时候略有降低;叶绿素a含量在夏末秋初(8月和9月)和春季(4月)有2个峰值,冬季最低。长牡蛎的繁殖周期分为冬季的休止期和从春季到夏季的繁殖期,在长牡蛎的繁殖期,条件指数逐渐下降到最低值,表明繁殖期长牡蛎生长变慢。在长牡蛎的配子发生过程中软体部蛋白质和脂肪含量略有上升,而糖原含量显著下降,表明长牡蛎配子的发生需要储存的糖原提供能量;RNA/DNA比值在配子发生过程中逐渐升高,显示RNA/DNA比值可以作为一个指标来指示长牡蛎的性成熟。配子发生过程中,长牡蛎的灰分含量逐渐升高,表明长牡蛎通过分解自身的贮能物质提供能量,使体内有机物含量减少,从而导致灰分含量上升。实验结果表明长牡蛎的配子发生类型为保守种。     

长牡蛎Wnt5b基因的鉴定及在黑色素生成中的作用

Wnt基因家族编码一类保守的分泌型糖蛋白,广泛参与细胞分化与增殖、细胞运动、凋亡、免疫调控以及色素生成等生物学过程。本研究鉴定了一个长牡蛎Wnt家族基因——CgWnt5b,比较了黑、白壳色长牡蛎中CgWnt5b转录水平的表达情况,并对其在调控黑色素生成方面的功能进行初步研究。研究结果显示,CgWnt5b编码序列全长1116 bp,编码371个氨基酸。生物信息学分析预测CgWnt5b蛋白相对分子量为42 KDa,等电点为9.28。对CgWnt5b与哺乳动物Wnt基因家族19个成员进行系统发育分析,确定了CgWnt5b为哺乳动物Wnt5a和Wnt5b的同源基因。荧光实时定量PCR检测长牡蛎外套膜、鳃、消化腺、闭壳肌、性腺、唇瓣以及血淋巴组织中CgWnt5b的mRNA分布,结果显示,CgWnt5b在外套膜、闭壳肌和唇瓣组织中的相对表达水平较高,在消化腺、鳃、血淋巴和性腺组织中相对表达量较低。黑壳长牡蛎中CgWnt5b mRNA表达量显著低于白壳长牡蛎,且siRNA敲降CgWnt5b的表达后,多个黑色素生成相关酪氨酸酶基因的表达显著上调。综上,本研究在长牡蛎中鉴定了一个Wnt家族成员CgWnt5b,可能通过抑制黑色素生成关键基因TYR的表达抑制长牡蛎中黑色素的生成。     

近江牡蛎与太平洋牡蛎的同工酶鉴别

福建省罗源湾于上世纪80年代初先后引进来自日本、台湾和澳大利亚的太平洋牡蛎．该品种引进后不久就开始出现养殖品质退化的现象；对此，有人怀疑是与本地近江牡蛎杂交所致。本文通过同工酶分析，确认罗源湾太平洋牡蛎被测样品中不舍与近江牡蛎杂交的个体．苹果酸脱氢酶电泳可作为鉴定相关种群的技术手段．但是，本项调查结果也显示罗源湾太平洋牡蛎很可能不是纯系品种，培育纯化品系、加强种质管理对振兴福建牡蛎产业具有重要意义。     

太平洋牡蛎精液的超低温保存

利用解剖法采集太平洋牡蛎精液 ,用自然海水做基础溶液 ,配制不同浓度的 DMSO(二甲亚砜 )作为超低温保护剂 ,在液氮 (L N2 )面上不同高度进行预处理 ,投入 LN2 内保存 ,升温解冻后 ,检测精子成活率。结果表明 ,DMSO处理浓度、预处理温度及时间和升温方式对超低温保存成活率均具有显著影响。其中 ,用 10 % DMSO在 LN2 面上 17cm预处理 6 min,采用 16～ 17℃流水不完全解冻 ,效果最好 ,超低温保存精子成活率可达 70 %。与新鲜精子相比 ,在 L N2 内保存一天的精子 ,授精能力没有明显变化。     

Trophic transfer of benzo[a]pyrene metabolites between benthic marine organisms

This study was undertaken to determine the potential for trophic transfer of polycyclic aromatic hydrocarbon (PAH) metabolites from infaunal organisms to bottom-feeding fish. Winter flounder, Pseudopleuronectes americanus, were given single oral doses of ground polychaetes (Nereis virens), either treated with pure [¹⁴C]henzo[a]pyrene (BaP) or containing a mixture of naturally produced radiolabeled BaP and BaP metabolites. Fish were sacrificed 24 h after feeding and total accumulated radioactivity and metabolite class profiles determined in major organs. Metabolites produced by worms were absorbed by flounder, although as a percentage of dose given they were less available than parent BaP. Comparison of metabolite profiles in the worm diet and in target organs in the fish indicated that metabolites accumulated through the diet can be further modified by the prey organism and can lead to the formation of bound residues. These results demonstrate that PAH metabolites in the diet are available for accumulation. Furthermore, metabolites absorbed appear to be susceptible to metabolic alteration by consumer organisms.     

太平洋牡蛎人工培育群体的基因型及杂合度研究

实验运用淀粉凝胶电泳的方法检测了１龄养殖太平洋牡蛎Ｃｒａｓｏｓｔｒｅａｇｉｇａｓ５个基因座位上的基因型和杂合度。根据孟德尔自由分离规律,从子代的基因型推断出亲代可能的基因。杂合度的分析表明,人工培育的１龄太平洋牡蛎在Ａａｔ２、Ａｈ１、Ａｈ２、Ｉｄｈ１和Ｌａｐ５个多态位点上的杂合度符合Ｈ－Ｗ平衡规律,未发现明显的杂合子缺失现象。     

太平洋牡蛎精子形成的研究

透射电镜下研究了太平洋牡蛎 (Crassostrea gigas Thunberg)的精子形成过程。精子细胞中含有前顶体颗粒、线粒体、高尔基体、中心粒等多种细胞结构 ,线粒体、前顶体颗粒的数量较多。在精子形成过程中 ,前顶体颗粒逐渐汇集、愈合成顶体泡 ,顶体泡覆盖在细胞核的一端逐渐发育为顶体 ;线粒体则向顶体相反的方向移动 ,最后移到核后端形成 4个较大的线粒体球 ;中心粒移到核后端由远端中心粒形成轴丝 ;细胞核发生致密 ,形态发生变化 ,最后形成杯状的精子核 ;多余的细胞质被抛弃。     

太平洋牡蛎多倍体研究

本文综述了太平洋牡蛎多倍体产生的途径、诱导方法、诱导结果、诱导机制、倍性鉴别方法、繁殖、生长、生理指标、抗逆性和口味等。提出。６－ＤＭＡＰ诱导牡蛎三倍体的技术前景广阔;四倍体与二倍体杂交的方法是产生三倍体的最好途径;四倍体是生物方法产生三倍体的中间材料,可以存活,应加大力度地研究和开发;三倍体的性腺能够发育,并可产生具有繁殖力的配子;在良好的环境里,四倍体的生长和抗逆性较二倍体优势;在繁殖季节,三倍体的生化组成等指标比二倍体高,口味较好。     

Culture environment and hatchery of origin influence growth,condition and feeding organ morphology in the Pacific oyster Crassostrea gigas in South Africa

South Africa lacks a commercial oyster hatchery. To inform the sourcing of seed for future hatchery establishments, we compared half-sib Pacific oyster Crassostrea gigas cohorts from hatcheries in Namibia and Chile. We measured oyster growth, mortality, condition and feeding organ morphology in Algoa Bay (AB, Eastern Cape) and Saldanha Bay (SB, Western Cape), South Africa, from July 2011 to June 2012. Within SB, 14.3% of mean daily sea temperatures exceeded this species’ thermal optimum of 19 °C, compared to 50.5% in AB. Food abundance (mean daily chlorophyll a concentration) in SB (7.8 mg m^–3) was double that in AB (3.9 mg m^–3) where, presumably to increase particle clearance rates in a relatively phytoplankton-poor environment, oysters had larger gill:palp surface area ratios. Plankton fatty acid profiles (indicators of food quality) differed between locations. In AB, instantaneous growth rates differed between cohorts, and trends varied seasonally. Within both locations, condition index was usually higher in Chilean oysters, whereas shell density was higher in Namibian oysters. In AB only, Chilean seed suffered substantially higher summer mortalities than Namibian seed, suggesting that the latter are more suited to temperatures in AB. AB should also be assessed for culture of the indigenous oyster species that occur there.     

Seasonal variation in the reproductive activity and biochemical composition of the Pacific oyster (Crassostrea gigas) from the Marlborough Sounds,New Zealand

Reproductive cycles of the Pacific oyster Crassostrea gigas (Thunberg) from the Marlborough Sounds, New Zealand, were followed between June 1998 and January 2000. Histological examination of the gonad confirms an annual cycle with a winter inactive period followed by rapid gonad development and a single short spawning period. The population gonad index correlated with seawater temperature and changes in tissue dry weight, condition index, and biochemical components. In winter, few individuals with early gametogenic stages were present and rapid development of primary oocytes (diam. 11 μm) occurred during spring (September‐November). The developmental rate and the diameter of mature oocytes (37 μm) was similar for the 1998 and 1999 seasons. For a standard 110‐mm‐length oyster, maximal tissue body weight and condition index were recorded in December. Rapid weight loss in January was length dependent and was attributed to spawning. Temperature was the environmental variable which best correlated with the timing of gametogenesis. Food availability (phytoplankton biomass) may have been responsible for inter‐annual variations. The biochemical composition (% glycogen, lipid, protein) of separated gonad and somatic tissues were variable seasonally and annually. Gametogenesis (oocyte diameter) was associated with increased gonad protein and glycogen and a decrease in lipid concentrations. These changes are similar to those in Pacific oyster populations from other parts of the world.