Nitrate and phosphate supplementation to increase toxin production by the marine dinoflagellate Alexandrium tamarense

Alexandrium tamarense toxins have great value in biotechnology research as well as important in connection with shellfish poisoning. The influence of nitrate or nitrate and phosphate supplementation on cell biomass and toxin content were investigated in batch cultures. When cultures at low nitrate (88.2 mu M NaNO3) Were supplemented with 793.8 mu M NaNO3 at day 10 the cell density and cellular toxin contents were increased by 6-29% and 20-76%, respectively, compared with controls, and maximal values were 43,600 cells/ml (day 38) and 0.91 pg/cell (day 31). Supplementation with nitrate at day 14 or with nitrate and phosphate at day 10/14 to the cultures did not increase the cell density compared with the non-supplemented middle nitrate or high phosphate (108 mu M NaH2PO4) cultures, respectively, but increased the cellular toxin contents by an average of 52%. The results showed that supplementation with nitrate or with nitrate and phosphate at different growth phases of the cultures increased toxin yield by an average of 46%. Supplementation with nitrate at selected times to maintain continuous low level of nitrate might contribute to the effective increase of toxin yield of A. tamarense. (c) 2005 Elsevier Ltd. All rights reserved.

Improvement in growth and toxin production of Alexandrium tamarense by two-step culture method

The growth and toxin content of the dinoflagellate Alexandrium tamarense ATHK was markedly affected by culture methods. In early growth phase at lower cell density static or mild agitation methods were beneficial to growth, but continuous agitation or aeration, to some extent, had an adverse effect on cell growth. Static culture in 2 L Erlenmeyer flasks had the highest growth rate (0.38 d(-1)) but smaller cell size compared with other culture conditions. Cells grown under aerated conditions possessed low nitrogen and phosphorus cell yields, namely high N and P cell-quota. At day 18, cells grown in continuous agitated and 1 h aerated culture entered the late stationary phase and their cellular toxin contents were higher (0.67 and 0.54 pg cell(-1)) compared with cells grown by other culture methods (0.27-0.49 pg cell(-1)). The highest cell density and cellular toxin content were 17190 cells mL(-1) and 1.26 pg cell(-1) respectively in an airlift photobioreactor with two-step culture. The results indicate that A. tamarense could be grown successfully in airlift photobioreactor by a two-step culture method, which involved cultivating the cells statically for 4 days and then aerating the medium. This provides an efficient way to enhance cell and toxin yield of A. tamarense.

Positive effects of continuous low nitrate levels on growth and photosynthesis of Alexandrium tamarense (Gonyaulacales, Dinophyceae)

The growth and photosynthesis of Alexandrium tamarense (Lebour) Balech in different nutrient conditions were investigated. Low nitrate level (0.0882 mmol/L) resulted in the highest average growth rate from day 0 to day 10 (4.58 x 10(2) cells mL(-1) d(-1)), but the lowest cell yield (5420 cells mL(-1)) in three nitrate level cultures. High nitrate-grown cells showed lower levels of chlorophyll a-specific and cell-specific light-saturated photosynthetic rate (P-m(chl a) and P-m(cell)), dark respiration rate (R-d(chl a) and R-d(cell)) and chlorophyll a-specific apparent photosynthetic efficiency (alpha(chl a)) than was seen for low nitrate-grown cells; whereas the cells became light saturated at higher irradiance at low nitrate condition. When cultures at low nitrate were supplemented with nitrate at 0.7938 mmol/L in late exponential growth phase, or with nitrate at 0.7938 mmol/L and phosphate at 0.072 mmol/L in stationary growth phase, the cell yield was drastically enhanced, a 7-9 times increase compared with non-supplemented control culture, achieving 43 540 cells mL(-1) and 52 300 cells mL(-1), respectively; however, supplementation with nitrate in the stationary growth phase or with nitrate and phosphate in the late exponential growth phase increased the cell yield by no more than 2 times. The results suggested that continuous low level of nitrate with sufficient supply of phosphate may facilitate the growth of A. tamarense.

塔玛亚历山大藻(Alexandrium tamarense)对鲈鱼(Lateolabrax japonicus) 的危害机制研究

本论文选择在我国分离得到的一株有毒赤潮甲藻-塔玛亚历山大藻（Alexandrium tamarense，ATHK株），研究了其对一种我国沿海常见和典型养殖鱼类鲈鱼（Lateolabrax japonicus）的危害机制。首先研究了塔玛亚历山大藻（ATHK）对鲈鱼鳃结构的影响及其溶血毒性； 然后采用腹腔注射的方法，研究了高剂量塔玛亚历山大藻毒素（ATHK毒素：约为1.6×105 细胞，相应PSP为0.886µg STX Equal，相当于每克湿重的鲈鱼PSP注射量为0.0118µg STX Equal）和低剂量塔玛亚历山大藻毒素（ATHK毒素： 约为0.16×105 细胞，相应PSP为0.0886µg STX Equal，相当于每克湿重的鲈鱼PSP注射量为0.00118µg STX Equal）在鲈鱼体内代谢过程中对鲈鱼肝脏、肾脏和鳃组织的超微结构、Na+K+-ATPase活性、肝脏功能和肾脏功能的影响、以及对抗氧化系统酶活性和异生物质代谢酶的影响，以期从不同方面了解塔玛亚历山大藻及其所产水溶性毒素（ATHK毒素）对鲈鱼的毒害效应及机制，为有毒赤潮的有效管理提供一定的科学依据。 塔玛亚历山大藻（ATHK）对鲈鱼鳃组织影响的实验结果表明，该藻使鲈鱼鳃组织出现水肿现象，细胞间隙变大；粘液细胞颗粒不规则，颜色加深，颗粒发生凝集，有板结状；氯细胞线粒体内部基质凝集。不产PSP的一种亚历山大藻（AT-6）也使鲈鱼鳃出现水肿，且使细胞出现一定的固缩现象。显微镜观察发现鲈鱼鳃丝间存在有这两种亚历山大藻细胞。由此推测塔玛亚历山大藻ATHK和AT-6的表面结构可能具有能导致鲈鱼鳃组织水肿的机械作用。对人血细胞溶血实验结果表明塔玛亚历山大藻（ATHK）具有较强的溶血毒性，大小与藻的生长阶段和细胞密度都有一定的关系：指数期的溶血毒性最大，随细胞数目的增多，活性逐渐加大；藻细胞、细胞碎片、细胞内容物都有一定的溶血毒性，其中细胞碎片的活性最大。通过11种（株）产PSP的亚历山大藻、不产PSP的亚历山大藻以及标准PSP的实验结果表明这种溶血毒性是由藻细胞的其它非PSP物质造成的，且这种溶血毒性在产PSP的亚历山大藻中具有一定的普遍性。 塔玛亚历山大藻（ATHK）对鲈鱼组织超微结构实验结果表明：ATHK毒素（约为1.6×105 细胞，相应PSP为0.886µg STX Equal，相当于每克湿重的鲈鱼PSP注射量为0.0118µg STX Equal）能导致鲈鱼组织细胞超微结构发生剧烈的变化，主要表现在：肝细胞细胞膜有肿胀现象，部分膜边缘溶解；细胞质糖原颗粒化；核糖体脱落，仅见滑面内质网；细胞质和线粒体内都出现空泡，且线粒体的嵴状结构也发生变化；核膜溶解比较严重，核质外溢，且异染色质边际化。前肾细胞超微结构的变化主要是淋巴细胞核质出现空泡，核膜有溶解迹象；Ⅰ型粒细胞颗粒膨大，伪足增多且变长；Ⅱ型颗粒细胞颗粒增多，内部出现空腔，细胞膜和核膜溶解，胞质、细胞器和核质外溢。鳃组织中氯细胞的核膜局部溶解，核仁弥散，线粒体膜溶解，微细小管膨大；粘液颗粒膜溶解，内部结构遭受破坏；扁平细胞核膜及线粒体膜几乎全部溶解。因此，我们的结果表明，ATHK毒素能作用于鲈鱼细胞的内膜和外膜系统，使膜发生溶解、脱落等变化；比较注射同样剂量大小ATHK毒素120h和240h时鲈鱼组织超微结构发现，细胞超微结构在一定程度上能够恢复。 ATHK毒素对Na+K+-ATPase活性、肝脏功能以及肾脏功能的影响结果表明，0.16×105―1.6×105细胞范围内的ATHK毒素可以显著影响肝脏和鳃组织中的Na+K+-ATPase，使这两种组织中的Na+K+-ATPase活性出现不同程度的下降； 而且还能够显著抑制肝脏中谷丙转氨酶的活性，最大抑制率为95%。但此范围内的ATHK毒素不能显著影响肾脏中的Na+K+-ATPase活性以及尿素氮含量。因此，ATHK毒素对Na+K+-ATPase活性的抑制则会导致细胞能量的缺失，使细胞进一步发生其它变化，而ATHK毒素对肝脏功能完整性的影响则可能会抑制对蛋白质的分解代谢。 ATHK毒素对鲈鱼肝脏、肾脏和鳃组织中的、超氧化物歧化酶（SOD）、过氧化氢酶（CAT）、谷胱肝肽过氧化物酶（GSH-Px）以及谷胱肝肽转硫酶（GST）活性变化影响的结果表明：高剂量（约为1.6×105 细胞，相应PSP为0.886µg STX Equal，相当于每克湿重的鲈鱼PSP注射量为0.0118µg STX Equal）ATHK毒素能显著诱导鲈鱼肝脏和鳃组织中SOD、GSH-Px以及GST酶活性，最大变化范围为正常状态下的3-4倍，对肝脏中CAT酶活性具有一定的抑制作用，对鳃中的CAT抑制效应则不显著；但此剂量的ATHK毒素仅对肾脏鳃中的GSH-Px活性有一定的诱导作用，对SOD、CAT以及GST的活性没有显著影响。低剂量（约为0.16×105 细胞，相应PSP为0.0886µg STX Equal，相当于每克湿重的鲈鱼PSP注射量为0.00118µg STX Equal）ATHK毒素也能诱导鲈鱼肝脏和鳃组织中SOD、GSH-Px以及GST酶活性，其在第一个24h内的诱导效果与临界致死毒素剂量相似； 且连续注射低剂量ATHK毒素则对肝脏和鳃中这三种酶活性具有累加的诱导作用，使这三种酶活性的变化范围为正常的5倍；低剂量ATHK毒素对肝脏中CAT酶活性也具有抑制作用，但对鳃中CAT酶活性的抑制作用并不显著。 同样，低剂量ATHK毒素除对肾脏中GSH-Px活性具有一定诱导效应外，对SOD、CAT以及GST都没有显著影响。 SOD、GSH-Px以及GST酶活性的显著升高表明ATHK毒素在鲈鱼体内代谢过程中能诱导鲈鱼产生活性氧自由基，且GST活性的升高则说明作为细胞色素P450依赖的异生物质代谢酶，GST在ATHK毒素代谢过程中可能可以加速ATHK毒素的代谢。推测鲈鱼可以通过这三种酶降低ATHK毒素以及次生毒物活性氧自由基对鲈鱼的危害。

塔玛亚历山大藻（Alexandrium tamarense）对双壳的影响研究

本文选取麻痹性贝毒产毒藻塔玛亚历山大（Alexandrium tamarense）以及双壳类为研究对象，进行了塔玛亚历山大藻对双壳类生命活动的影响，麻痹性贝毒在紫贻贝体内累积、转化与排出动力学过程的初步研究。实验证实，塔玛亚历山大藻能对栉孔扇贝受精卵至早期D型幼虫的整个发育阶段产生不利影响，抑制受精卵的孵化。经多方验证，我们推测这一抑制作用主要是由塔玛亚历山大藻藻细胞表面物质引起的。通过塔玛亚历大藻对栉孔扇贝和墨西哥湾贝早期发育的影响研究发现：产毒藻对其受精卵、早期D形幼虫、眼点幼虫、仔贝都有明显的影响。（1）两株塔玛亚历山大藻（ATHK、ATCI02）都能抑制栉孔扇贝受精卵的孵化，EC50分别是（1010、1580cells/mL）。指数生长期的藻作用最强，并发现毒性作用可能来源于细胞表面一种不同于PSP的毒性物质。（2）ATHK对D形幼虫有致死作用，死亡率随作用时间的延长和藻密度的增加而增加。栉孔扇贝的早期D形幼虫暴露于细胞密度为10，000/cells/mL的ATHK中14天，死亡就率达100％；在实验密度10，000cells/mL的48小时急性致毒实验中，墨西哥湾扇贝的早期D形幼虫的游泳能力受到了一定程度的抑制。（3）实验条件下未发现ATHK对墨西哥湾扇贝眼点幼虫的变态、存活产生明显影响，但变态后稚贝的个体大小与对照组相比有明显差异，表明有毒藻对变态过程幼体的生长有影响。（4）在1小时、5小时的急性致毒实验中ATHK对墨西哥湾扇贝仔贝（壳高：5mm）的爬升能力产生了明显的抑制（1hEC50 ＝ 1，000cells/mL）。作用5小时后仔贝的附着率与对照组相比显著降低。两次48小时急性实验的结果都显示高密度的塔玛亚历山大藻（ATHK）能抑制紫贻贝成体的滤水率，平均EC50为6000cells/mL。以（产麻痹性贝毒）的塔玛亚历山大藻（ATHK）投喂紫贻贝（Mytilus edulis），研究了麻痹性贝毒在紫贻贝体内的累积、转化与排出规律。结果表明，在八天的累积实验阶段，紫贻贝消化腺和肌肉组织中的毒素含量均随实验时间的延长而逐渐增加；累积实验结束时，平均每只贝体内的毒素量为13.40nmol，累积率为12.45％（以每只贝的总染毒量为107.67nmol计），毒性水平为12.24μgSTXeq／100g，还未达到国际上公认的贝类食用卫生标准（80μgSTXeq／100g）；贻贝消化腺的累积能力远远高于肌肉组织，累积实验结束时，消化腺中的毒素含量为13.07nmol，累积率为12.14％，而肌肉组织中的毒素量只有0.33nmol，累积率只有0.31％。消化腺中累积的毒素占贝体内毒素总量的97.5％。在八天的排出实验阶段贝体内的毒素总量有下降的趋势，只有进一步延长自净的时间，才能得到更明了的排出规律。

塔玛亚历山大藻（Alexandrium tamarense）产毒生理学研究

在实验室内建立了Oshima 提出的针对麻痹性贝毒毒素的高效液相色谱分析方法,并根据实验室条件对分析过程进行了完善.同时对另一重要的麻痹性贝毒分析方法-Thielert分析方法进行了改进，在保持原方法对neoSTX, dcSTX 和STX等毒素较高分辩率和检出限的基础上， 提高了原方法中GTX1 和GTX4，GTX3和GTX2的分辩率，使之可以分析已知的大多数麻痹性贝毒毒素.应用建立的高效液相色谱技术对从中国南海海域分离的两株塔玛亚历山大藻ATHK 和ATCI03的毒素含量及组成进行了分析，结果表明两株塔玛亚历山大藻均能产生毒素，但在毒素组成和含量方面存在明显差别。批次培养结果表明每细胞毒素含量在对数生长期初期最高，之后逐渐降低。以两株有毒藻中毒素含量较高的ATHK 作为模型，研究了光照强度、盐度以及营养盐（N，P）对有毒藻生长和毒素产生的影响。结果表明在实验光照强度内（2100lux-4700lux），塔玛亚历山大藻的生长速率随光照强度的增加而增加，对相同细胞密度的藻采样分析结果表明毒素含量在场300Lux最大；而在同一天取样分析的结果表明毒草素含量随光照强度的降低而增加。在实验盐度下相差不大。而毒素含量在职4S相对偏低。一次培养结果表明，在氮、磷营养盐充足的条件下，毒素今是在对数生长初期达到最高，之后逐渐降低，起始时较低的氮浓度对对数生长初期毒素含量影响不大，但对数生长期中后期的细胞内毒素含量明显降低。而起始时较低的磷浓度则使对数期中后期的细胞内毒素含量保持稳定或逐渐增加。培养液中氮、磷的供应情况直接决定细胞骨毒素含量的变化；而培养液起始磷比在决定细胞毒素含量弯化方面也有一定的作用。半连续培养结果表明氮限制恒化培养使每细胞毒素含量降低，而磷限制恒化培养使每细胞毒素含量升高，营养充足的恒浊培养得到的藻细胞毒素今是介于二者之间。通过分析氮、磷限制下半连续恒化培养的塔玛亚历山大藻细胞内毒素含量、精氨酸水平、谷氨酰胺/谷氨酸比值（Gln/Glu）等，研究了营养盐对毒素产生的影响机制。结果表明，细胞内毒素含量与精氨酸水平以及Gln/Glu比值存在明显相关关系。在此基础上提出了氮、磷对毒素产生可能的影响机制。即外界环境中的氮通过影响细胞内精氨酸水平而影响毒素含量；磷则是通过改变细胞的氮代谢途径而影响毒素的合成。磷的缺乏可能导致细胞正常的代谢停滞，为缓解细胞内氮过剩造成的毒性效应，作为氨基酸合成重要中间产物的谷氨酰氨转向合成精氨酸，造成精氨酸累积，累积的精氨酸双进一步生成麻痹性贝毒毒素。缺磷条件下毒素含量的增加很可能是对磷缺乏导致的细胞内氨积累的一种缓解措施.

Effects of the dinoflagellate Alexandrium tamarense on early development of the scallop Argopecten irradians concentricus

The effects of Alexandrium tamarense (strain ATHK) on early development of the bay scallop Argopecten irradians concentricus were studied under laboratory conditions. The algal culture was verified by HPLC to produce paralytic shellfish poisoning (PSP) at a level of 37.48 fmol/cell. Survival of the scallop larvae was not affected when they were grown with A. tamarense at concentrations of 500-10,000 cells/ml for 48 h. However, the activity of D-shape larvae was inhibited after 48-h exposure to A. tamarense at the algal cell density of 10,000 cells/ml. Scallop growth was inhibited significantly by A. tantarense during a 14-day exposure starting at the eye-spot larval stage. The size of juvenile scallops in the group of 10,000 cells/ml was only about 32% of that of the controls, although no obvious effect of A. tamarense was found on the rate of larval metamorphosis. All juvenile scallops survived in algal concentrations of 600-2400 cells/ml, however, attachment rates were significantly lower than control values after a 5-h exposure to A. tamarense at concentrations >600 cells/ml, while they were not obviously reduced after only 1 h of exposure. At concentrations >600 cells/ml, the climbing ability of juveniles was clearly reduced by exposure to A. tamarense after only 1 h. The climbing rate and height were only 55% and 45%, respectively, of those of the controls, when exposed to A. tantarense at a concentration of 600 cells/ml. The results indicated that A. tamarense blooms may have detrimental impacts on shellfish at early life stages, therefore, special attention should be paid to the toxic algal blooms in shellfish breeding area. (C) 2003 Elsevier Science B.V. All rights reserved.

Study on impact of dinoflagellate Alexandrium tamarense on life activities of marine bivalves

The effects of a PSP producing dinoflagellate Alexandrium tamarense on marine bivalves at their several important life,stages: egg, D - shape larva, eyespot larva, juvenile and adult, were studied! The results show that the hitching survival, activity, filtration and! growth were adversely affected by the alga and the impact was significantly increased with the increase of algal density. The inhibitory effect on egg hatching was most significant, which the hatching rate was only 30% of the control when exposed to the alga at 100 cell/cm(3) after 36 h. Further experiments show that the algal culture, re-suspended cells and cell fragments had the inhibitory effect, while no such effect was from the cell-free medium, cell contents and standard STX. The results indicate that the alga could produce unknown toxins, rather than PSP, associated with the cell surface.

Inhibition of egg hatching success and larvae survival of the scallop, Chlamys farreri, associated with exposure to cells and cell fragments of the dinoflagellate Alexandrium tamarense

We report an apparently novel toxic effect of the dinoflagellate Alexandrium tamarense, manifested by inhibition of the egg hatching success of the scallop, Chlamys farreri. The hatching rate of C. farreri approached only 30% of controls when its fertilised eggs were exposed for 36 h to A. tamarense cells or cellular fragments at a concentration of 100 cells/ml, and the hatching rate was just 5% after exposure to A. tamarense of 500 cells/ml. Similar exposures of the fertilised scallop eggs to two other algal species, the diatom Phaeodactylum tricornutum and the raphidophyte Heterosigma carterae, resulted in no such toxicity or inhibitory effects.. Likewise, exposure of eggs to standard STX toxin. as well as to A. tamarense cell contents (supernant of re-suspended algal cells following ultrasonication and centrifugation), did not elicit this inhibitory response. However, exposure of the scallop eggs to cell cultures, intact algal cells, or cell fragments of A. tamarense produced marked toxicity. The alga also influenced larvae at early D-shape stage of scallop. The survival rates began to decrease significantly after exposed for 6 days at concentration of 3000 cells/ml and above: no larvae could survive after 14-day exposure to A. tamarense at 10,000 cells/ml or 20-day at 5000 cells/ml. The results indicated the production of novel substances from A. tamarense which can cause adverse effects on egg hatching and survival of the scallop larvae, The experiment also found that the developmental stages before blastula was the developmental period most sensitive to the A. tamarense toxin(s) and the alga at early exponential stage had the strongest effect on egg hatching comparing with other growth phases. The adverse effect of A. tamarense on early development of scallops may cause decline of shellfish population and may have further impact on marine ecosystem. (C) 2001 Elsevier Science Ltd. All rights reserved.

Interactions between the bloom-forming dinoflagellates Prorocentrum donghaiense and Alexandrium tamarense in laboratory cultures

Interactions between Prorocentrum donghaiense and Alexandrium tamarens, two bloom-forming dinoflagellates, were investigated using bi-algal cultures. All R donghaiense died, but A. tamarense was hardly affected by the end of the experiment when the initial cell density was set at 1.0 X 10(4) cells mL(-1) for P. donghaiense and 0.28 x 10(4) cells mL(-1) for A. tamarense. However, significant growth suppression occurred in either species when the initial cell density of P donghaiense increased to I. 0 X 105 Cells mL(-1) in the bi-algal culture, but no out-competement was observed. The simultaneous assay on the culture filtrates showed that P donghaiense filtrate prepared at a lower initial density (1.0 X 10(4) cells mL(-1)) stimulated growth of the co-cultured A. tanzarense (0.28 x 10(4) cells mL(-1)), but filtrate at a higher initial density (1.0 x 10(5) cells mL(-1)) depressed its growth. The filtrate of A. tamarense at a density of 0.28 x 10(4) cells mL(-1) killed all R donghaiense at a lower density (1.0 x 10(4) cells mL(-1)), but only exhibited an inhibitory effect on it at a higher density (1.0 x 10(5) cells mL(-1)). It is likely that these two species of microalgae interfere with each other mainly by releasing allelochemical substance(s) into the culture medium, and a direct cell-to-cell contact was not necessary for their mutual interaction. The allelopathic test further proved that A. tamarense could affect the growth of co-cultured P. donghaiense by producing allelochemical(s); moreover, A. tamarense culture filtrate at the stationary growth phase (SP) had a strongly inhibitory effect on P donghaiense compared to that at the exponential phase (EP). Results also demonstrated a dose-dependent relationship between the microalgal initial cell density and the degree of the allelopathic effect. The growth of R donghaiense and A. tamarense in the bi-algal cultures was simulated using a mathematical model to quantify the interaction. The estimated parameters from the model showed that the inhibition exerted by A. tamarense on P. donghaiense was about 17 and 8 times stronger than the inhibition P. donghaiense exerted on A. tamarense, when the initial cell density was set at 1.0 X 10(4) and 1.0 X 10(5) cells mL(-1) for P donghaiense, respectively. and 0.28 x 10(4) cells mL(-1) for A. tamarense in the bi-algal cultures. A. tamarense seems to have a survival strategy that is superior to that of P. donghaiense in bi-algal cultures under controlled laboratory conditions. (c) 2006 Elsevier B.V. All rights reserved.

Biological activity of a red-tide alga - A-tamarense under co-cultured condition with bacteria

The relationship between Alexandrium tamarense (Lebour) Balech, one of red-tide alga, and two strains of marine bacteria, Bacillius megaterium(S-7) and B. halmapulus(S-10) isolated from Xiamen Western Sea, was investigated by evaluating the growth state of A. tamarense and the variation of P-glucosidase activity in co-culture system. The results showed the growth and multiplication of the alga were related with the concentration, genus speciality of the bacteria, and growth stage of the alga itself. The growth of A. tamarense was obviously inhibited by S7 and S, at high concentration. Either inhibition or promotion contributed much more clearly in earlier than in later stage of the growth of the alga. Furthermore, there was a roughly similar variation trend of the activity of extra-cellular enzyme, beta-glucosidase, in the water of the separately co-cultured bacteria S-7 and S-10 with the alga. The beta-glucosidase activity (beta-GlcA) rapidly increased during the later algal growth accompanying the increase of the lysis of the alga cells. The obvious inhibition of A. tamarense by marine bacteria at high concentration and evident increase of beta-GlcA in co-colture system would help us in better understanding the relationship between red-tide alga and bacteria, and also enlightened us the possible use of bacteria in the bio-control of red-tide.

Experimental study on the impact of dinoflagellate Alexandrium species on populations of the rotifer Brachionus plicatilis

To investigate harmful effects of the dinoflagellate Alexandrium species on microzooplankton, the rotifer Brachionus plicatilis was chosen as an assay species, and tested with 10 strains of Alexandrium including one known non-PSP-producer (Alexandrium tamarense, AT-6). HPLC analysis confirmed the PSP-content of the various strains: Alexandrium lusitanicum, Alexandrium minutum and Alexandrium tamarense (ATHK, AT5-1, AT5-3, ATC102, ATC103) used in the experiment were PSP-producers. No PSP toxins were detected in the strains Alexandrium sp1, Alexandrium sp2. Exposing rotifer populations to the densities of 2000 cells ml(-1) of each of these 10 Alexandrium strains revealed that the (non-PSP) A. tarnarense (AT-6) and two other PSP-producing algae: A. lusitanicum, A. minutum, did not appear to adversely impact rotifer populations. Rotifers exposed to these three strains were able to maintain their population numbers, and in some cases, increase them. Although some increases in rotifer population growth following exposures to these three algal species were noted, the rate was less than for the non-exposed control rotifer groups. In contrast, the remaining seven algal strains (A. tamarense ATHK, AT5-1, AT5-3, ATC102, ATC103; also Alexandrium sp1 and Alexandrium sp2) all have adverse effects on the rotifers. Dosing rotifers with respective algal cell densities of 2000 cells ml-1 each, for Alexandrium spl, Alexandrium sp2, and A. tamarense strains ATHK and ATC103 showed mean lethal time (LT50) on rotifer populations of 21, 28, 29, and 36h, respectively. The remaining three species (A. tamarense strains AT5-1, AT5-3, ATC102) caused respective mean rotifer LT50S of 56, 56, and 71 h, compared to 160 h for the unexposed "starved control" rotifers. Experiments to determine ingestion rates for the rotifers, based on changes in their Chlorophyll a content, showed that the rotifers could feed on A. lusitanicum, A. minutum and A. tamarense strain AT-6, but could graze to little or no extent upon algal cells of the other seven strains. The effects on rotifers exposed to different cell densities, fractions, and growth phases of A. tamarense algal culture were respectively compared. It was found that only the whole algal cells had lethal effects, with strongest impact being shown by the early exponential growth phase of A. tamarense. The results indicate that some toxic mechanism(s), other than PSP and present in whole algal cells, might be responsible for the adverse effects on the exposed rotifers. (C) 2004 Elsevier B.V. All rights reserved.

Allelopathic interactions between the macroalga Ulva pertusa and eight microalgal species

HETEROSIGMA-AKASHIWO RAPHIDOPHYCEAE; CENTRAL VENICE LAGOON; ALEXANDRIUM-TAMARENSE; RED-TIDE; COASTAL LAGOONS; PHYTOPLANKTON; GROWTH; BAY; DINOFLAGELLATE; COMPETITION

粘土矿物及无机絮凝剂对有害赤潮的治理研究

在害赤潮作为一种全球性的海洋灾害，给海洋环境、水产养殖业和人类健康造成巨大危害，研究赤潮的发生规律，探索新的、经济有效的赤潮的防治方法具有重要的科学意义和应用前景。本文针对中国沿海常见赤潮生物种，研究了粘土矿物双赤潮生物基本生理指标的影响，进一步探讨了粘土矿物的控制赤潮机制；并针对粘土矿物去除效率不高的特点，制备出具有较高絮凝胶效率的改性粘土；此外，本文还首次将无机高分子絮凝效率的改性粘土；此外，本文还首次将无机高分子絮凝剂MMH（混合金属氢氧化物正电胶体）和PSMS（聚硅酸金属盐）用于赤潮治理研究中，并探索了上述赤潮防治剂的环境生态效应，主要成果与内容有：1、粘土矿物的筛选 采集国内外近30种粘土矿物，分别考察了它们对中国沿海常见赤潮生物种赤潮异弯藻（Heterosigma akashiwo）和塔玛亚历山大藻（Alexandrium tamarense）的去除效果。结果表明，不同粘土矿物对赤潮生物的絮凝能力有显著差别，实验筛选出去除效果较好的几种粘土，分别为来自美国的Chemically treated kaolin、WHOI土、江苏吴县高岭土及苏州特号土等。通过絮凝实验得到上述几种粘土矿物对两种赤潮生物的絮凝曲线，发现不同粘土矿物对赤潮生物的最大去除率及达到最大去除率所需的粘土浓度有较大差别，同一粘土矿物对不同赤潮生物的去除效果也不相同。塔玛亚历山大藻较赤潮异变藻更易被粘土矿物去除。2、粘土矿物对赤潮生物的生理效应 研究了高岭土对赤潮异弯藻和塔玛来历山大藻生长繁殖、叶绿素a含量、光合作用以及呼吸作用的影响。结果表明，高岭土对赤潮生物生长、叶绿素a含量及光合作用均有抑制作用，表现为随粘土浓度的增加及作用时间的延长，抑制作用增强，并认为这可能与粘土的加入导致光照效应降低及藻类生长的营养环境改变有关。实验同时发现高岭土对不同赤潮生物的抑制程度不同，塔玛亚历山大藻上述各项生理指标比赤潮异弯藻更易受粘土影响。3、无机絮凝剂的制备及絮凝作用研究 主要考察了无机高分子絮凝剂MMH及PSMS（聚硅酸金属盐），包括聚硅酸氯化铝（PSAC）和聚硅酸硫酸铝（PSAS）的合成及对赤潮生物的絮凝作用。在MMH的合成过程中，考察了镁铝摩尔比及制备温度对MMH絮凝赤潮异弯藻的影响，认为镁铝摩尔比为2的条件下制备的MMH具有较好的絮凝和沉降性能，其絮凝赤潮异弯藻的最佳用量为1.5mmolAl/L。在PSAS的合成中采用三因子三水平的正交试验设计，得到PSAS的最佳制备条件为：SiO_2浓度 = 2%， Al~(3+)/SiO_2 = 1, pH = 4。絮凝实验结果表明，PSAS对赤潮生物的絮凝效果优于传统絮凝剂AS（硫酸铝），同时PSAS对不同赤潮生物的絮凝效果也不同，认为这主要与不同赤潮生物的形态结构、运动性及胞外分泌物有关。4、粘土矿物表面改性及机理研究 为提高粘土矿物去除赤潮生物的效率，分别用插入法和表面吸附法制备改性粘土。实验得出插入法的最佳制备条件为：制备温度70 ℃，镁离子浓度2mol/L；表面吸附法中粘土和MMH（混合金属氢氧化物胶体）的适宜配比为：粘土/MMH=10。改性后粘土去除赤潮异弯藻的效率较改性前提高2-3倍。相关分析表明，改性粘土去除赤潮生物的效率与改性后表面电位呈正相关，两种情况下R~2均大于0.8，说明表面电位是影响粘土矿物去除赤潮生物的一个重要因素。5、赤潮治理的现场模拟实验及环境生态效应 在1999年夏季胶州湾中肋骨条藻（Skeletonema costatum）赤潮的发生现场进行了改性粘土及PSAS对藻类去除率、悬浮物、DO、COD、pH等指标的影响研究。结果表明，改性粘土及PSAS对现场中肋骨条藻有较高的去除率，1g/L的改性高岭土和改性蒙脱土对中肋骨条藻的去除率分别可达77%和93%，1ml/L的PSAS去除率达90%以上。DO的含量轻微下降，不致影响其他生物的生长，COD下降幅度较大，达50-80%。6、赤潮防治剂对中国对虾的毒性研究 进行了粘土、次氯酸钙、氯化铝和氯化铁对中国对虾的毒性试验，结果表明，1g/L的高岭土对虾基本无毒性；次氯酸钙在低浓度下对对虾无毒，在高浓度下对对虾有急性毒性效应，安全使用浓度为0.5ml/L游离氯；氯化铝和氯化铁在对虾体内有一定的积累，其长期的毒性效应有待于进一步的分析。上述几种试剂均可用于粘土矿物的改性处理中，是比较安全的赤潮防治剂。

麻痹性贝毒毒素的制备和活性研究

麻痹性贝毒（Paralytic Shellfish Poisons, PSP）毒素主要是由海洋甲藻产生的一类活性物质，在赤潮毒素监测、神经与分子生物学基础研究以及新型药物开发方面都有潜在的应用价值。然而，纯化毒素的缺乏在很大程度上限制了相关工作的开展。本文通过大量培养塔玛亚历山大藻（Alexandrium tamarense）来提取PSP毒素，完善了毒素的分离纯化过程，并对纯化毒素的活性进行了研究和探讨。在实验室进行了塔玛亚历山大藻的规模培养，对其生长和培养条件进行了优化，探讨了有毒藻大量培养的采收方式，并获取了一批用于纯化毒素的有毒藻细胞。将藻细胞破碎后离心分离，提取出含PSP的粗毒素，再用大孔径凝胶过滤去除粗毒素中的大部分杂质，然后用小孔径凝胶和离子交换树脂将不同毒素逐级分离、纯化。结果表明，大孔径凝胶能有效地去除粗毒素中的杂质，小孔径凝胶过滤能将膝沟藻毒素（GTX）和C毒素分开，应用离子交换树脂能将GTX2,3和GTX1,4分开，最终得到GTX2,3和GTX1,4的纯化产品。分离纯化过程始终以小鼠法和荧光法进行毒素在线监测。以提取的毒素对小鼠进行毒性测试，小鼠呈现典型的PSP毒素中毒死亡症状；电生理分析的结果表明PSP毒素能够有效地阻断小鼠运动神经末梢钠电流，并可逆地阻断NG108-15全细胞的钠电流，与PSP毒素的作用机理一致；高效液相色谱分析结果表明，纯化毒素与标准毒素的保留时间完全一致；经质谱分析，纯化毒素与标准毒素具有相同的分子离子峰和碎片离子峰。应用运动模型研究了纯化毒素的药理活性。分别采用家兔椎管麻醉法、小白鼠热板法、小白鼠攀网法研究了GTX2,3对家兔的局麻作用、对小鼠的镇痛和肌松作用。结果表明，GTX2,3按剂量0.4μg STX equivalent/kg给予家兔椎管注射时，具有显著的局麻作用。GTX2,3按剂量5.99μg STX equivalent/kg给予小鼠肌肉注射时，具有显著的镇痛作用。GTX2,3按剂量6.49μg STX equivalent/kg给予小鼠皮下注射时，具有显著的肌松作用。