节旋藻和螺旋藻对7种抗生素敏感性的比较研究

对两种丝状蓝藻(钝顶节旋藻和盐泽螺旋藻)在基因工程中常用作选择试剂的7种抗生素——氯霉素、氨苄青霉素、红霉素、链霉素、卡那霉素、庆大霉素和新霉素的敏感性作比较实验.结果表明,两种蓝藻对抗生素的敏感性既有共同的特点,也有明显的差异.它们对红霉素、氯霉素和链霉素最敏感,致死浓度分别为0.1,0.5和5μg/cm3.两种蓝藻对氨苄青霉素比较敏感,1μg/cm3的氨苄青霉素即可抑制Arthrospira.341和Spirulina.351的生长,但6d后生长恢复.Arthrospira.341和Spirulina.351对卡那霉素、庆大霉素和新霉素均有抗性,而且存在很大差异:300μg/cm3的卡那霉素对Arthrospira.341的生长仍然没有影响,但对于Spirulina.351,50μg/cm3的卡那霉素即对其生长有明显抑制作用;200μg/cm3的卡那霉素即可将其全部致死.200μg/cm3的庆大霉素和300μg/cm3的新霉素不能抑制Arthrospira.341和Spirulina.351的生长,但在这两种抗生素环境中两种藻的生长状态有很大差异.并验证了氯霉素、红霉素和链霉素是节旋藻和螺旋藻基因转化过程中的有效的抗性选择剂,也从对抗生素敏感性方面表明节旋藻和螺旋藻两个属的遗传差异.     

东海藻华高发区塔玛亚历山大藻(Alexandrium tamarense)的形态、分子及分布特征

本文对分离自我国东海藻华高发区的亚历山大藻进行了详细的形态特征描述和分子系统进化分析,通过运用光学显微镜、荧光显微镜、扫描电镜和分子生物学方法,确定该藻株为塔玛亚历山大藻Alexarium tamarense(Lebour)Balech,并对其ITS序列进行了系统进化分析。该藻体长21—35μm,体宽20—33μm,ITS序列长度为592bp,系统发育树中与中国东海、南海两株A.tamarense以及一株代表"亚洲温带"基因型的A.catenella聚合在一起,支持率为100%,形态特征与A.tamarense基本一致,核糖体基因型则为塔玛复合种"亚洲温带"基因型。通过现场观测,研究浙南至闽北东海藻华高发区的塔玛亚历山大藻分布变化特点,在东海原甲藻藻华消散期,塔玛亚历山大藻逆势而上,其细胞密度达到1.7×104cell/L,并逐渐向沿岸靠近,这与季风转向,台湾暖流入侵及水体层化密切相关。     

环境因子对塔玛亚历山大藻生长的综合影响

利用多因子实验设计,研究了主要环境因子温度、盐度和光照强度对麻痹性贝毒产毒藻——塔玛亚历山大藻(Alexandrium tamarense)生长的影响.经三维ANOVA统计分析(Tukey test)结果表明,在实验条件范围内(温度12,19,25,32℃;盐度10,18,25,30,35和光照强度0.02×1016,0.08×1016,0.3×1016,1.6×1016s-1/cm2),温度、光照强度和盐度对藻生长率有显著的影响(P<0.001).光照强度和温度、盐度和温度、光照强度和盐度以及这3个因子之间存在显著的相互作用(P<0.001),在本实验中该藻生长的最适条件是19℃,1.6×1016s-1/cm2,盐度30.根据塔玛亚历山大藻的生长特性,我们探讨了这些环境因子在塔玛亚历山大藻赤潮形成机制中的作用.     

东海原甲藻与塔玛亚历山大藻种群增长过程与种间竞争研究

针对我国东海2种重要赤潮肇事生物东海原甲藻(Prorocentrum donghaiense)和塔玛亚历山大藻(Alexan-drium tamarense)进行了种群增长过程和种间竞争的实验研究。结果表明,单种培养时,不同氮磷比对东海原甲藻和亚历山大藻种群增长过程没有明显的影响,但是会影响其最终的细胞密度;在混合培养时,营养盐组成比例不同对东海原甲藻和塔玛亚历山大藻的种间竞争过程有影响,但不会影响其最后竞争结果。竞争物种的细胞起始密度对种间竞争结果有明显的影响,P.donghaiense和A.tamarense的接种细胞密度比为2 000∶100和2 000∶200时,东海原甲藻占优势;当接种细胞密度比为4 000∶400时,塔玛亚历山大藻占优势。     

四种/株亚历山大藻(Alexandrium)毒性的比较研究

通过对塔玛亚历山大藻(ATHK和AT-6藻株)、链状亚历山大藻(ACDH藻株)和微小亚历山大藻(AM-1藻株)的PSP毒素含量和组成以及它们分别对轮虫、黑褐新糠虾和鲈鱼存活影响的比较研究发现:塔玛亚历山大藻AT-6藻株不产生麻痹性贝毒毒素PSP,而塔玛亚历山大藻ATHK、链状亚历山大藻ACDH和微小亚历山大藻AM-1均产生PSP毒素,其总含量分别为19.74、5.395、5.57fmol/cell(2.60、0.36、1.61pgSTXeq/cell)。塔玛亚历山大藻AT-6对轮虫、黑褐新糠虾和鲈鱼这三种生物均没有不利影响;对于轮虫,塔玛亚历山大藻ATHK和链状亚历山大藻ACDH的96hLC50分别为:200和1200cells/ml,而微小亚历山大藻AM-1无不利影响;对于糠虾,塔玛亚历山大藻ATHK、链状亚历山大藻ACDH和微小亚历山大藻AM-1的96hLC50分别为7000、11000、16000cells/ml;对于鲈鱼,这三株藻的96hLC50分别为3700、4000、20000cells/ml。四株亚历山大藻对三种生物的毒性大小与其PSP毒素含量和组成无直接的相关关系,其毒性作用可能来自其他未知毒性物质。塔玛亚历山大藻ATHK不同组分分别对三种生物的毒性比较研究表明:藻细胞重悬液和藻液对三种生物的不利影响最为显著,去藻过滤液和细胞碎片对轮虫和鲈鱼没有明显影响,但糠虾的存活率有所下降;细胞内容物对轮虫和糠虾也无影响,但对鲈鱼的存活有一定影响。以上研究表明:不同亚历山大藻的毒性大小存在差别,其对不同生物的危害机制也不相同,除PSP毒素外,亚历山大藻可能还存在其他的毒性物质。     

雨生红球藻(Haematococcus pluvialis)基因工程选择试剂的筛选

采用细胞计数法，研究了重要经济微藻——雨生红球藻对10种基因工程常用选择试剂的敏感性。结果表明，雨生红球藻对除草剂草丁膦和抗生素Zeocin比较敏感，50μg／ml的草丁膦和25μg／ml的Zeocin可明显地抑制雨生红球藻生长，尤其是50μg／ml的草丁膦能导致细胞两周内全部死亡；高浓度的卡那霉素和硫酸链霉素也可抑制雨生红球藻的生长；雨生红球藻对包括氯霉素和G418在内的其他6种试剂不敏感，即使处理浓度高达1000μg／ml。本文结果提示草丁膦可能是雨生红球藻基因工程适宜的选择试剂，其相应的抗性基因bar可能成为雨生红球藻遗传转化的选择标记基因。     

塔玛亚历山大藻和赤潮异弯藻对黑褐新糠虾和卤虫的急性毒性作用

以黑褐新糠虾和卤虫为实验材料,研究了两种典型有毒有害赤潮藻,塔玛亚历山大藻和赤潮异弯藻的毒性.结果表明塔玛亚历山大藻和赤潮异弯藻都能影响糠虾的存活,在9 h内塔玛亚历山大藻和赤湖异弯藻对糠虾的半致死浓度分别约为7000和450 000个/cm3;在24 h内塔玛亚历山大藻和赤潮异弯藻对卤虫存活没有显著影响,而赤潮异弯藻能对卤虫的活力产生强烈的抑制作用,0.1,1.5,5和24 h的半抑制浓度分别为15 000,7500,4500,3000个/cm3,但在赤潮异弯藻较低浓度为3000个/cm3时,经过48 h卤虫的运动能力能够恢复到正常水平.结果表明,这两种有害藻对同一种生物的毒性大小存在差异,而这两种甲壳类生物对同一种有害藻的反应也不相同.     

生物胺对赤潮藻生长的影响作用初探

选取东海赤潮高发区常见的两种甲藻[东海原甲藻(Prorocentrum donghaiense)、塔玛亚历山大藻(Alexandrium tamarense)]和两种硅藻[中肋骨条藻(Skeletonema costatum)、海链藻(Thalassiosirasp.)],以2-苯基乙胺、腐胺、亚精胺、精胺四种赤潮水体中常见的生物胺为因素,设置0、5、25、100nmol/L四个浓度水平,进行L16(45)正交添加培养实验。根据Logistic生长模型进行曲线拟合,分析得到的生长参数。结果显示,不同的生物胺对各赤潮藻生长影响的大小、趋势均存在差异。其中,2-苯基乙胺对四种赤潮藻的生长影响最显著。高浓度的2-苯基乙胺对中肋骨条藻的生长具有明显的抑制作用,对塔玛亚历山大藻的生长具有明显的促进作用。多胺物质(腐胺、亚精胺和精胺)对甲藻(东海原甲藻和塔玛亚历山大藻)生长的促进作用大于硅藻(海链藻和中肋骨条藻)。多胺中的亚精胺对塔玛亚历山大藻和海链藻的生长影响最大,精胺对东海原甲藻和中肋骨条藻的生长影响最大。多胺可能是2010年东海赤潮由中肋骨条藻向东海原甲藻演替的一个诱导因素,其中精胺可能发挥的作用较大。     

厦门湾沉积物中有害有毒甲藻休眠孢囊及塔玛亚历山大藻的潜在危害

本工作通过分析厦门湾表层沉积物中的甲藻休眠孢囊，发现了1种有毒塔玛亚历山大藻(Alexandrium tamarense)休眠孢囊，及两种有害甲藻休眠孢囊：锥状斯氏藻(Scrippsiella trochoidea)、具刺膝沟藻(Gonyaulax spinifera)．对塔玛亚历山大藻进行了培养。并初步研究了该藻的毒性及贝类对其毒素的积累．研究结果表明，厦门海域沉积物中的塔玛亚历山犬藻是有毒的．它的较适宜生长环境条件是：水温20～25℃．盐度为25～30，光照度为3000～50001x．塔玛亚历山大藻在每年的4月26日至6月10日、10月11日至11月20日可能会形成有害藻华，危害厦门海域的海洋生物并可能威胁到人类的健康和生命安全．     

硝基苯和间苯二酚对3 种海洋微藻的毒理效应

通过研究硝基苯和间苯二酚对海洋微藻的毒理效应,旨在提示化工废水污染对水环境破坏的严重性,揭示海洋微藻类净化废水的潜在应用。本试验用塔玛亚历山大藻(Alexandrium tamarense)、海链藻(Thalassiosira weissflogii)和绿色巴夫藻(Pavlova viridis)为供试藻种,以微藻生长量及叶绿素 a 的变化为参数,研究不同质量浓度的硝基苯和间苯二酚对3种海洋微藻的毒理效应。结果表明：硝基苯对塔玛藻和巴夫藻的生长具有一定的抑制作用,质量浓度为12mg/L时抑制率分别为22.9%和20.04%,但对海链藻的生长在一定程度上有促进作用。间苯二酚对海链藻和巴夫藻的生长具有抑制作用,质量浓度为12mg/L 时其抑制率分别为26.11%和23.6%,对塔玛亚历山大藻的生长有一定的促进作用。对塔玛亚历山大藻和巴夫藻而言,在硝基苯质量浓度为3mg/L 时,叶绿素 a 含量均达到最低,其叶绿素 a含量分别为对照组的68.6%和50.9%,对于海链藻,硝基苯对其叶绿素a的影响波动不大。间苯二酚对3种海洋微藻叶绿素 a 含量的影响为绿色巴夫藻>海链藻>塔玛亚历山大藻,其叶绿素 a 含量分别是对照组的1.1%,24.9%,88%。     

Differences in abundance and distribution of Alexandrium cysts in Sendai Bay,northern Japan,before and after the tsunami caused by the Great East Japan Earthquake

The tsunami caused by the Great East Japan Earthquake on 11 March 2011 greatly influenced the coastal benthic environment on the northern Pacific coast of Japan. We used the direct count method to investigate changes in the abundance and distribution of Alexandrium (Alexandrium tamarense and Alexandrium catenella) cysts before and after the tsunami in Sendai Bay. Densities of Alexandrium cysts in sediments collected in summer 2011 ranged from 0 to 8,190 cysts cm^?3. In the western part of the bay, the density increased greatly after the tsunami, the highest density being approximately 10 times the density recorded in 2005. Molecular identification of single cysts with multiplex polymerase chain reaction (PCR) showed that Alexandrium tamarense dominated the cyst population in the southwestern part of the bay in 2011. Furthermore, accumulation of cysts on the surface sediment after disturbance of the sediment was confirmed by a laboratory experiment. The main factor causing the drastic changes in abundance and distribution of Alexandrium cysts after the earthquake was considered to be vertical and horizontal redistribution of the cysts in sediments after the tsunami.     

On an extremely dense bloom of the dinoflagellate Alexandrium tamarense in lagoons of the PO river delta: Impact on the environment

An extremely dense bloom of the potentially toxic dinoflagellate Alexandrium tamarense was observed in the lagoons of Cà Pisani (Veneto, Italy) in summer 1993. The lagoons were experiencing a significant eutrophication impact, receiving waste waters from intensive fish culture plants. During their bloom dinoflagellates in the lagoons reached densities of 2 to 4 × 10⁶ cells·dm⁻³ and a biomass of over 100 g·m⁻³. The bloom produced drastic ecological changes in the lagoons. It caused nocturnal anoxia, mortality of macrophytes and the build-up of labile organic matter in the water column. Grazing by the tintinnid Favella sp. contributed to the termination of the bloom of the flagellates. The results show that coastal aqua culture probably stimulates dinoflagellate blooms in shallow brackish lagoons.     

Molecular analysis of complete ssu to lsu rdna sequence in the harmful dinoflagellatealexandrium tamarense (korean isolate, HY970328M)

New PCR primers (N=18) were designed for the isolation of complete SSU to LSU rDNA sequences from the dinoflagellateAlexandrium tamarense. Standard PCR, employing each primer set selected for amplifications of less than 1.5 kb, successfully amplified the expected rDNA regions of A. tamarense (Korean isolate, HY970328M). Complete SSU, LSU rDNAs and ITS sequences, including 5.8S rDNA, were recorded at 1,800 bp, 520 bp and 3,393 bp, respectively. The LSU rDNA sequence was the first report inAlexandrium genus. No intron was found in the LSU rRNA coding region. Twelve D-domains within the LSU rDNA were put together into 1,879 bp (44.4% G+C), and cores into 1514 bp (42.8% G+C). The core sequence was significantly different (0.0867 of genetic distance, 91% sequence similarity) in comparison withProrocentrum micans (GenBank access. no. X16108). The D2 region was the longest in length (300 bp) and highly variable among the 12 D-domains. In a phylogenetic analysis using complete LSU rDNA sequences of a variety of phytoplankton,A tamarense was clearly separated with high resolution against other species. The result suggests that the sequence may resolve the taxonomic ambiguities ofAlexandrium genus, particularly of the tamarensis complex.     

Toxic effect of dinoflagellate Alexandrium species on Cladocera Moina mongolica Daday

The main objective of this study was to investigate toxic effects of the dinoflagellate Alexandrium species,on cladocera Moina mongolica in the laboratory.Ten strains of Alexandrium species,including Alexandrium tamarense(AT-6,ATHK,ATCI02,ATCI03 and AT5-1),A.catenella(ACDH),A.affine(AC-1 and AS-1),A.lusitanicum and A.minutum were tested.The results showed that A.tamarense(ATHK,ATCI02 and AT5-1) and A.affine(AC-1 and AS-1),but not A.tamarense(AT-6 and ATCI02),A.catenella(ACDH),A.lusitanicum and A.minutum had significantly negative effects on the survival of M.mongolica;when exposed to these ten strains of Alexandrium species in densities of 3 000 cells/cm 3 for 7 d,respecyively.M.mongolica could feed on A.tamarense(AT-6,ATHK,ATCI02,ATCI03 and AT5-1),A.catenella,A.lusitanicum and A.minutum,but exhibited little or no grazing on A.affine(AC-1 and AS-1),based on the changes in gut pigment after exposure to alga for 1 and 12 h.A.affine AC-1,which had the strongest toxicity on the survival of M.mongolica,was chosen to further study the negative effects on M.mongolica.The results showed that the effect of A.affine AC-1 on the survival of M.mongolica was density-dependent,and its lethal effects on one-day,two-day and three-day old M.mongolica were exacerbated with increasing age;The whole algal culture,re-suspended algal cells,cell fragments and cell contents all had adverse impacts on the survival of M.mongolica.Moreover,both juvenile numbers and life-span time of M.mongolica were decreased significantly when they were cultured in A.affine AC-1 of 10 and 50 cells/cm 3 ,combined with 3 × 10 6 cells/cm 3 of Chlorella spp.,respectively.As a non-PSP producer,A.affine may produce other toxins,responsible for the strong negative effects on M.mongolica.     

日本真海带对硒的积累和生物转化

在海水中添加亚硒酸盐(Na₂SeO₃)及不同浓度的氮磷营养盐(NaNO₃+NaHO₂PO₄),研究海带对硒的积累,通过分离测定海带富硒前后各生化组分的含硒量来探讨硒的生物转化问题.结果表明天然海带含硒量(鲜重)为0.451~0.596μg/g,海带富硒最佳的Na2SeO₃浓度为200mg/dm3,培养56h后的富硒倍率约为50.在天然海带中,硒主要以有机态存在,有机硒占总硒含量的86.22%;在含有200mg/dm3NaO₂SeO₃的海水中培养56h后测得海带的总硒为24.481μg/g,其中有机硒含量为16.703μg/g,无机硒为4.714μg/g;在添加氮磷营养盐的含硒海水中,海带的富硒能力得到进一步的提高,在适宜的氮磷条件下(150mg/dm3NaNO₃和25mg/dm3NaHO₂PO₄)总硒含量达到33.649μg/g,而无机硒含量基本不变,为4.497μg/g,因此提高部分转化为有机硒,有机硒含量达到24.678μg/g.这说明海带具有通过自身代谢将无机硒转化为有机硒的较强的能力.     

赤潮多发区塔玛亚历山大藻的麻痹性贝毒预警值研究

以厦门西海域养殖的翡翠贻贝为实验对象,在实验室内对塔玛亚历山大藻(Alexandrium tamarense(Lebour)Balech)采用单种培养技术,运用“麻痹性贝毒小白鼠生物检测法”进行毒性实验,研究了塔玛亚历山大藻对翡翠贻贝的麻痹性贝毒的毒力,藻密度为1×105个/dm3时,白鼠未死亡;藻密度为1×106个/dm3时,白鼠发生死亡.厦门西海域近年来暴发的赤潮一般起始于局部海区无害硅藻类浮游植物,在生态环境恶化下,硅藻类赤潮在生存竞争中消退,最终暴发有害赤潮.以试验结果为依据,根据厦门海域实际情况,参考各国贝毒临界值行动标准,提出适合南方赤潮多发区塔玛亚历山大藻的麻痹性贝毒藻密度预警值为1×105个/dm3.     

紫外光对有毒甲藻塔玛亚历山大藻的生态学效应

对1991年分离自南海大鹏湾海域的塔玛亚历山大藻单克隆培养林在不同光照条件、辐射强度（9.5，5．9W／m2）（辐射剂量为：95，190，380,760,1520J/m2）和营养盐（N，P）浓度下，进行紫外辐射敏感性的研究。结果表明，紫外辐射可以导致细胞变形、增大和死亡。不论在什么条件下，低剂量的紫外辐射（95J／m2）就能使该藻的存活率和生长率大幅下降。此后，随着辐射剂量的增加，虽然存活率和生长率继续下降，但下降幅度渐缓。充足的营养盐和辐射处理后的连续光照对藻群的恢复有益，但在缺氮和缺磷条件下培养的藻群，可能由于生理活性减弱而导致对紫外辐射敏感性的迟钝。辐射强度对该藻的紫外辐射敏感性的影响十分显著，而且与辐射剂量具有明显的双重效应。     

Flushing rate and salinity may control the blooms of the toxic dinoflagellate Alexandrium tamarense in a river/estuary in Osaka Bay, Japan

The first observation of a red tide of the toxic dinoflagellate Alexandrium tamarense in the Yodo River estuary, Osaka Bay, Japan was in the spring of 2007. To clarify which environmental factors controlled the abundance of A. tamarense, field surveys were conducted in 2008 and 2009. In 2008, the increase of A. tamarense occurred from early to mid-March and temperatures ranged from 9 to 13 °C. In contrast, in 2009, the increase was 1 month later, from early to mid-April and therefore temperatures were higher. In both years, A. tamarense was most abundant when salinities were relatively high (15–25), river discharge was low, and the water column was stable. There were no cells during periods of low salinity (<10), high discharge. In 2008, possibly silicate and phosphate were limiting during the simultaneous diatom and A. tamarense bloom in early March with silicate restricting the further growth of diatoms. In this estuarine basin, freshwater discharge from the weir increased the flow rate at stations downstream of the weir and deterred bloom formation by A. tamarense. Conversely, the lack of discharge increased the salinity and stability of the water column and decreased in the flow rate in the river/estuary. This process promoted the initiation of the bloom of A. tamarense and the subsequent accumulation of paralytic shellfish toxin in the brackish water clam Corbicula japonica. It is recommended that a constant discharge from the weir is maintained in order to prevent blooms of A. tamarense and subsequent paralytic shellfish poisoning (PSP) incidents in this area.