海洋卡盾藻与中肋骨条藻和锥状斯氏藻种间竞争研究

研究了不同起始细胞密度与硅酸盐浓度对海洋卡盾藻(Cm)与中肋骨条藻(Sc)及锥状斯氏藻(St)之间种间竞争的影响,利用竞争抑制参数对相互间的竞争关系进行了分析,并引入体积竞争抑制系数的概念。结果表明:海洋卡盾藻的环境容量不受起始细胞密度(100—3600cells/mL)的影响,但随着起始接种密度的增加,达到最大细胞密度和进入稳定生长期的时间均提前。在硅缺乏的条件下,中肋骨条藻仍能保持一定生长,并能在与海洋卡盾藻之间的种间竞争中保持数量优势,但硅酸盐浓度的增加有利于其种间竞争。初始细胞密度和接种比例对3种赤潮藻类之间的种间竞争影响明显,总体来说海洋卡盾藻在竞争中处于劣势。当海洋卡盾藻细胞密度远远高于中肋骨条藻时(Cm:Sc=6:1),海洋卡盾藻才能在与中肋骨条藻的竞争中取胜;而在与海洋卡盾藻共培养条件下,锥状斯氏藻具有一定竞争优势,其对海洋卡盾藻的体积抑制系数(α’)是后者(β’)的27—100倍。但在所有处理中均没有绝对优胜者,竞争结果都会出现不稳定的平衡状态。       

温度、盐度、光照对海洋卡盾藻生长和产毒的影响

在正交试验条件下,分析了盐度、温度、光照强度对海洋卡盾藻生长和产毒的影响.结果表明：在盐度22、33、45和温度20 ℃、25 ℃、30 ℃以及光照强度2000、3000、4500 lx条件下,三因素对海洋卡盾藻生长的影响均不显著,盐度是影响海洋卡盾藻产毒的主要因子;盐度45、温度30 ℃、光照2000 lx下海洋卡盾藻的比生长速率最大,盐度22、温度20 ℃、光照4500 lx下海洋卡盾藻的产毒能力最强;低盐条件不利于海洋卡盾藻的生长,但有利于溶血毒素的合成;当海洋卡盾藻生长受到限制时,其溶血毒素合成增多.     

大亚湾春季卡盾藻种群动态及其赤潮成因分析

研究了2000年春季大亚湾古老卡盾藻/海洋卡盾藻(Chattonella antique/marina)种群动态及其与环境因子的关系。卡盾藻是一种沿岸分布的有害赤潮针孢藻,2000年春在大亚湾海域高密度高频率出现,最高细胞密度为129cells/mL。大亚湾合适的水温、盐度及丰富的营养盐含量为卡盾藻的生长提供了良好的环境条件,而较低的Si∶N及持续的阴雨天气抑制了小型硅藻的生长,使卡盾藻在浮游植物种群竞争中占据一定地位。孢囊萌发被认为是水体中卡盾藻营养细胞及其赤潮发生的种源,但孢囊需经过4个月以上、11℃以下低温处理后方可成熟和萌发,大亚湾冬季水温,不能满足卡盾藻孢囊休眠成熟的需要。作者推测,2000年春大亚湾高密度卡盾藻营养细胞的种源可能来源于由船舶压舱水所带来的成熟孢囊。       

香港及珠江口海域有害赤潮发生机制初步探讨

回顾了香港及珠江口海域赤潮发生和藻毒素污染的历史及研究状况,结合3种代表赤潮藻种最适光温盐生长实验,4种不同形态氮营养盐对几种常见赤潮藻种生长影响实验,以及不同营养盐,温度,盐度条件下赤潮藻群落变化过程实验,初步探讨了物理,化学,生物等环境因子在香港和珠江口海域赤潮发生中的作用机制。     

N、P营养盐对海洋卡盾藻（Chattonella marina）生长的影响

在实验室条件下,设置不同的N、P浓度,研究N、P双因子限制(N:5-500ug·L-1,P:0.74-74ug·L-1,N:P=15)及单因子限制(N:500ug·L-1,P:0.74-741ug·L-1和P:74ug·L-1,N:5-500ug·L-1)对海洋卡盾藻生长的影响.结果表明:在高N、P浓度(N:500ug·L-1,P:74ug·L-1)条件下海洋卡盾藻具有相对较高的比生长率(O.788/d),稳定期持续时间较长,最大细胞密度可达8850 cells/ml.N、P限制能明显抑制海洋卡盾藻的生长,而N限制对海洋卡盾藻生长影响更大,N限制组的比生长率和稳定期细胞密度均明显低于P限制组和N、P双因子限制组.结果说明海洋卡盾藻生长对N变化更为敏感,但随着海洋污染的加剧,海水中N含量持续上升,卡盾藻可迅速爆发性增长并引发赤潮,这也许是近年来我国沿海卡盾藻赤潮频繁发生的重要原因之一.     

小定鞭藻毒素的分离与鉴定

从大量死鱼的鱼池中收集分离出小定鞭藻Ｐｒｙｍｎｅｓｉｕｍｐｏｒｖｕｍ的毒株,并在实验室成功地进行了单种培养,当温度２３℃,光照６００－８００ｌｘ盐度约１２—１６‰左右时,该藻在海水及人工海水培养基中均生长良好,在对数生长末期到平衡期溶血毒素活性最高。从藻细胞及浓缩的培养液中提取出二种毒素：溶血毒素（Ｈａｅｍｏｌｙｔｉｃｔｏｘｉｎｓ）和鱼毒素（Ｉｃｈｔｈｙｏｔｏｘｉｎｓ）。用新鲜牛血球测定了溶血毒素活性;用孔雀鱼测定了鱼毒素活性。用部分纯化的溶血毒素经元素分析、红外光谱、核磁共振及ＦＡＢ质谱测定,结果显示该藻溶血毒素可能是一个糖脂。     

实验室培养球形棕囊藻溶血毒素的提取、分离及其生成特征

研究从实验室培养的球形棕囊藻(Phaeocystis globosa)提取溶血毒素的条件,探讨不同生长期球形棕囊藻溶血毒素的生成特征,用薄层色谱法对溶血成份进行了初步分析。结果表明,球形棕囊藻细胞壁的超声波破碎最适条件为：功率600W,4℃下处理30min。对数生长期、平稳期和衰亡期藻细胞适宜处理量分别为每次3000、2000、1000ml;在球形棕囊藻生长的平稳期和衰亡期具有明显的溶血活性,对数生长期溶血活性很低,甚至检测不到。球形棕囊藻溶血毒素至少含有4种糖脂类化合物。     

扇贝毒素pectenotoxins(PTXs)研究进展

扇贝毒素(pectenotoxins,PTXs)是一类聚醚大环内酯结构的脂溶性海洋生物毒素,是由海洋甲藻中的鳍藻属Dinophysisspp.的几个种产生的,1984年首次从日本的养殖扇贝Patinopecten yessoensis中发现鉴定,具有很高的小鼠腹腔注射致死毒性。近年发现的地理区域不断扩大,我国尚属空白。就这一毒素的结构、来源生物、毒性、携带生物、地理分布、降解代谢及风险评估等研究现状作一系统综述,并分析展望了今后我国藻毒素研究的重点方向。     

赤潮藻毒素危害及其检测方法研究进展

近年来,世界各地包括我国部分沿海地区赤潮发生过度频繁,许多赤潮藻毒素不但使其他海洋生物中毒,对人类健康也有潜在的危害,因此对赤潮藻毒素的研究已经成为海洋环境科学研究的重要内容。我们介绍了赤潮藻毒素的危害,并详细综述了赤潮藻毒素检测技术研究进展。     

海洋微藻溶血毒素研究进展

对海洋微藻溶血毒素的类型、理化性质、生物合成和毒性作用进行了综述,分析了存在的问题和应用前景的展望。     

Morphology and germination characteristics of the cysts of Chattonella ovata (Raphidophyceae), a novel red tide flagellate in the Seto Inland Sea, Japan

To elucidate the mechanism of bloom outbreaks of Chattonella ovata (Raphidophyceae), we investigated the cysts of C. ovata and succeeded in finding them from the bottom sediments of Hiroshima Bay. The morphology of the cysts was mostly hemispherical in shape, with a diameter of ca. 30 μm and height of ca. 20 μm. The cysts were usually adhering to solid materials, such as diatom frustules, yellow-greenish in color and had several dark brown grains. The cyst wall was smooth and had no ornamentation. Because the morphological characteristic of the cysts was in general agreement with those of Chattonella antiqua and Chattonella marina, it was difficult to differentiate the cysts of these three species. Germination of the cysts of C. ovata was observed at temperatures from 17.5 to 30 °C, but not at 15 °C or below. The number of the germinated cysts increased with increasing temperature and the optimum temperature for germination was 30 °C. Although cysts of C. antiqua and C. marina germinated at temperatures from 15 to 30 °C, optimum temperature of germination was 22.5 °C. The lower limit and optimum temperatures for germination of C. ovata cysts was higher than for C. antiqua and C. marina. The role of cysts in the population dynamics of C. ovata is discussed.     

Methods for Quantitative Detection of Antibody-induced Complement Activation on Red Blood Cells

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver (extravascular hemolysis) or leading to intravascular lysis of RBCs. Alloantibodies (e.g. ABO) or autoantibodies to RBC antigens (as seen in autoimmune hemolytic anemia, AIHA) leading to complement activation are potentially harmful and can be - especially when leading to intravascular lysis - fatal¹. Currently, complement activation due to (auto)-antibodies on RBCs is assessed in vitro by using the Coombs test reflecting complement deposition on RBC or by a nonquantitative hemolytic assay reflecting RBC lysis^1-4. However, to assess the efficacy of complement inhibitors, it is mandatory to have quantitative techniques. Here we describe two such techniques. First, an assay to detect C3 and C4 deposition on red blood cells that is induced by antibodies in patient serum is presented. For this, FACS analysis is used with fluorescently labeled anti-C3 or anti-C4 antibodies. Next, a quantitative hemolytic assay is described. In this assay, complement-mediated hemolysis induced by patient serum is measured making use of spectrophotometric detection of the released hemoglobin. Both of these assays are very reproducible and quantitative, facilitating studies of antibody-induced complement activation.     

大叶藻对氮磷营养盐的吸收动力学特征

营养盐是影响海草生长的关键因子, 目前有关海草不同组织对不同形式氮和磷的吸收特征尚不明确。该研究通过利用海草地上和地下组织分隔培养装置, 设置不同的氨态氮、硝态氮和磷酸盐浓度, 探究了大叶藻(Zostera marina)植株及其地上和地下组织对氮磷营养盐的吸收动力学特征。结果显示: (1)大叶藻对氮磷的吸收符合饱和吸收动力学特征, 吸收速率和水体氮磷浓度可用米式方程描述; (2)大叶藻植株对NH₄⁺-N的最大吸收速率(V_max, 52 μmol·g^-1·h^-1)显著高于其对NO₃^--N的V_max (39 μmol·g^-1·h^-1); (3)大叶藻地上组织和地下组织均可吸收氮磷, 但地上组织对氨态氮、硝态氮、磷酸盐的V_max分别为43.1、30.5和15.6 μmol·g^-1·h^-1, 为地下组织的2.6、1.2和6倍。结果表明, 大叶藻对氨态氮的吸收能力高于硝态氮, 且对氮磷的吸收可能主要依赖地上组织(叶片)。结果为查明大叶藻对氮磷的吸收利用机制及评估大叶藻的海洋生态效应提供了理论依据。     

Uptake kinetics of nitrogen and phosphorus by Zostera marina

Aims Nutrient availability is an external factor that affect the growth of seagrasses. However, the demand for and absorption of different forms of nitrogen and phosphorus by different tissues of seagrasses are unclear. In this study, the uptake of nitrogen and phosphorus by Zostera marina was studied to determine the nutrient uptake kinetics. The objectives of this research are to: 1) investigate the absorption characteristics of ammonia nitrogen, nitrate nitrogen and phosphorus in Z. marina; 2) evaluate the differences in absorption between the different forms of nitrogen; and 3) analyse the differences in absorption between the different tissues of Z. marina.Methods Equipment was used to separate the aboveground and belowground tissues of Z. marina. Six concentration levels of ammonia nitrogen, nitrate nitrogen and phosphorus were established to experimentally test the uptake kinetics of nutrients by Z. marina. The nutrient concentrations in different parts of seawater column were measured to determine the nutrient changes and calculate the kinetic characteristics of nutrient uptake.Important findings 1) The absorption of ammonia nitrogen, nitrate nitrogen and phosphorus by Z. marina was consistent with the characteristics of saturated absorption kinetics. The relationship between the absorption rate and the nutrient concentrations in water could be described by the Michaelis-Menten equation. 2) The maximum absorption rate (V_max) of ammonia nitrogen by Z. marina (52 μmol·g^-1·h^-1) was significantly higher than that of nitrate nitrogen (39 μmol·g^-1·h^-1). 3) Both aboveground and belowground tissues of Z. marina could absorb nutrient, but the V_max of leaves (aboveground tissues) for ammonia nitrogen, nitrate nitrogen and phosphorus absorption were 43.1, 30.5 and 15.6 μmol·g^-1·h^-1, respectively, which were 2.6-fold, 1.2-fold and 6-fold higher than the corresponding V_max of belowground tissues. The results show that the absorption capacity of Z. marina for ammonia nitrogen is higher than that for nitrate nitrogen, and the absorption of nitrogen and phosphorus may depend primarily on the aboveground tissues (leaves). The results provide a theoretical basis for the study of the mechanisms of nitrogen and phosphorus uptake and utilization by Z. marina and the evaluation of marine ecological impacts.     

Comparison of hemolytic activities among strains of Heterocapsa circularisquama isolated in various localities in Japan

Heterocapsa circularisquama (Dinophyceae), a red tide dinoflagellate, is toxic to bivalve molluscs such as the pearl oyster (Pinctada fucata), but no harmful effects of this alga on fish have been observed so far. We found that 7 strains of H. circularisquama showed hemolytic activities toward rabbit erythrocytes in a cell-density dependent manner, but to quite different extents. The strains which are known to be highly toxic to bivalves tend to show stronger hemolytic activities and vice versa, suggesting that the hemolytic activity is parallel with the shellfish toxicity of H. circularisquama. Since the hemolytic assay is simple, semiquantitative, and reproducible, this assay is useful not only to search for certain toxins responsible for the shellfish toxicity of H. circularisquama but also to estimate the potential toxicity of a newly appeared strain of H. circularisquama.     

A RE-EVALUATION OF CORALLINE RED ALGAL TAXONOMY USING ULTRA-STRUCTURAL INFORMATION

The marine dinoflagellate, Gymnodinium breve (Davis), produces several neurotoxins that cause neurotoxic shellfish poisoning (nsp), massive fish kills and respiratory irritation in marine mammals and humans. The common method for discerning toxic levels of G. breve for public health advisories is enumeration of live cells in a given water mass. In this study, laboratory cultures, as well as natural blooms, were added to a stirred ultra-filtration cell concentrator to separate viable cells containing intra-cell toxins from ambient water containing extra-cell toxins. Methods were validated using various mixtures of lysed and whole G. breve laboratory culture. Extractions and recovery of brevetoxins were done using a C-18 bonded-phase glass fiber extraction disc eluted with methanol. Total PbTx toxin concentrations were quantified by HPLC/UV using a C-18 column and an 85:15 methanol:water (1 ml min⁻¹) isocratic elution at 215 nm. This method of separation and extraction was subsequently applied to water samples collected during natural blooms along two different areas of the Florida Gulf coast. The results indicated that early stages of G. breve blooms contained primarily intra-cell toxins with extra-cell toxins increasing as the bloom progressed, even though very few viable G. breve cells were present. This suggests that enumeration of cells alone may be insufficient and additional toxin quantitation is necessary.     

A new simple screening method for the detection of cytotoxic substances produced by harmful red tide phytoplankton

A highly sensitive, but simple and quantitative, cytotoxic assay method for the detection of toxic substances produced by red tide phytoplankton was developed by utilizing Vero cells which were the most resistant to seawater among the six cell lines tested. Heterocapsa circularisquama, which is known to be highly toxic to shellfish, showed cytotoxicity to Vero cells in a cell-density dependent manner when Vero cells were directly exposed to the cell suspension of H. circularisquama in seawater-based plankton culture medium, whereas Heterocapsa triquetra, which is morphologically similar to H. circularisquama but non-toxic to shellfish, showed no cytotoxic effect. Since the potent cytotoxicity was also detected in the cell-free culture supernatant of H. circularisquama, it was suggested that a certain cytotoxic substance is extracellularly secreted by H. circularisquama. Furthermore, by this direct exposure method, we found that Alexandrium fraterculus, Alexandrium tamiyavanichii, Alexandrium tamarense, and Alexandrium affine but not Alexandrium taylorii and Alexandrium catenella cause toxic effect on Vero cells with different extent depending on species. By gel-filtration and subsequent two cytotoxicity assays using Vero and mouse neuroblastoma cell line (Neuro-2a), we found that high molecular weight cytotoxic substance distinct from paralytic shellfish poisoning toxins is present in the aqueous extract of A. tamarense. These results suggest that our 96-well microplate cytotoxicity assay using Vero cells is useful not only as a primary screening assay for the detection of potential toxic activity of harmful phytoplankton but also as a quantitative routine toxicity assay for following the active substances during the extraction and purification processes.     

Toxicity of Karlodinium micrum (Dinophyceae) associated with a fish kill in a South Carolina brackish retention pond

A dinoflagellate bloom was found associated with a fish kill event in a South Carolina brackish water retention pond. A multi-analytical approach was used to confirm the identity of the bloom dinoflagellate and evaluate its potential toxicity. Karlodinium micrum was confirmed through light microscopy, pigment profile comparisons, species-specific PCR, and gene sequence data. Necropsy findings on several fish were suggestive of an acute kill event. Toxicity of filtrate from bloom samples was tested by a hemolytic assay using rainbow trout (Oncorhynchus mykis) erythrocytes and an ichthyotoxicity assay using larval zebrafish (Danio rerio). Hemolytic activity was measurably high (>80% hemolysis) in both whole filtrate and fractionated filtrate (from the 80% MeOH C₁₈ column elution). This fraction also demonstrated high ichthyotoxic activity as exposed fish experienced rapid death. These results implicate toxic K. micrum as a causative factor in fish death in a non-aquaculture brackish pond associated with a housing development, and extend recent findings linking this species to fish kills in aquaculture ponds.     

Identification of a new RTX-like gene cluster in Vibrio cholerae

A gene cluster containing two genes in tandem has been identified in Vibrio cholerae ElTor N16961. Each has more than one cadherin domain and is homologous to the RTX toxin family and was common in various V. cholerae strains. Insertional mutagenesis demonstrated that each gene has a role in Hep-2 cell rounding, hemolytic activity towards human and sheep RBCs and biofilm formation. The mutants showed reduced adherence to intestinal epithelial cells as well as reduction of in vivo colonization in suckling mice. These two genes thus code for RTX-like toxins in V. cholerae and are associated with the pathogenecity of this organism.