鼠尾藻对赤潮异弯藻和中肋骨条藻的抑制作用

通过共培养的方法,研究了鼠尾藻（Surgassum thunbergii）培养水的过滤液、新鲜组织、干粉末、水溶性抽提液对赤潮异弯藻（Heterosigma akashiwo）和中肋骨条藻（Skeletonema costatum）生长的抑制效应,建立了分隔共培养系统并证明了抑制物质的存在,排除了细胞直接接触抑制的可能性.结果表明,在共培养实验中,鼠尾藻新鲜组织、干粉末及水溶性抽提液对赤潮异弯藻和中肋骨条藻的生长具有显著的抑制作用,且在较高浓度下对两种赤潮微藻的生长具有致死效应.在一次性及半连续培养方式下,鼠尾藻培养水过滤液对赤潮异弯藻的生长无抑制效应,而对中肋骨条藻的生长具有显著的抑制效应.     

一次陆源降雨污水引起血红哈卡藻赤潮的成因

以2007年烟台四十里湾海域血红哈卡藻(Akashiwo sanguinea Hirasaka)赤潮为对象,研究赤潮消长与水环境因子的关系。研究发现大量陆源降雨污水输入后,海水盐度急剧下降、营养盐大幅增加,特别是活性磷酸盐浓度明显增加,促进了血红哈卡藻的生长繁殖并最终形成赤潮。赤潮发生前海区第一优势种为尖刺拟菱形藻(Pseudo-nitzschia pungens Halse),优势度0.47(0.42—0.52),多样性指数2.63(2.43—2.89);赤潮发生时血红哈卡藻密度范围1.05×105—4.10×106个/L,优势度0.92(0.83—0.99),多样性指数0.27(0.15—0.64);赤潮消退后中肋骨条藻(Skeletonema costatum Cleve)为第一优势种,优势度0.49(0.43—0.55),多样性指数2.46(2.19—2.84)。赤潮的发生、发展、消亡与化学需氧量(COD)、无机氮(DIN)、活性磷酸盐(DIP)、富营养化指数(E)呈显著正相关(P<0.05),与盐度呈显著负相关(P<0.01)。赤潮前、后该海域为贫营养、P限制、叶绿素a含量中等,赤潮期间该海域为富营养、P限制、叶绿素a含量高。通过影响力评定,活性磷酸盐、COD、盐度是此次赤潮发生的主要诱发因子,当活性磷酸盐含量低于0.3μmol/L时,硅藻逐渐取代甲藻,此次赤潮消散。     

赤潮藻中肋骨条藻的光合作用对海水pH和N变化的响应

为探讨赤潮发生时中肋骨条藻 (Skeletonemacoatatum)的光合作用生理变化 ,研究了不同无机氮 (N)水平上 ,海水pH值升高对其胞外碳酸酐酶 (CA)和光合生理特性的影响。海水pH从 8.2升至 8.7时 ,中肋骨条藻胞外CA被诱导 ,细胞对无机碳的亲和力 (1/Km)提高 ;在pH8 7时 ,高N条件下的胞外CA活性是低N条件下的 3倍 ,1/Km 值也提高了 80 %。单位叶绿素a的最大净光合能力 (Pam)在不同pH和N水平上没有显著差异 ;但单位细胞的最大净光合能力 (Pcm)提高了 10 0 %。这些结果表明 ,赤潮发生时 ,中肋骨条藻通过启动无机碳浓缩机制 (CCM) ,提高细胞对无机碳利用效率 ,使其在低CO2 (高pH)环境下维持光合机构正常运行 ;充足的N源有利于提高CCM的效率 ,从而提高CO2 环境下的光合固碳能力。     

赤潮水体中胶体物质对赤潮异弯藻(Heterosigma akashiwo)和中肋骨条藻(Skeletonema costatum)生长的影响

2004年2月9～10日在胶州湾发生的柔弱几内亚藻(Guinardia delicatula)赤潮期间分别在3个典型赤潮站位采集表层水样,利用过滤(0.7μm)和切向超滤(1000 Daltons)分离赤潮暴发水体中的胶体物质,分析了不同滤液及截留液的有机碳含量,并将赤潮异弯藻 (Heterosigma akashiwo)和中肋骨条藻(Skeletonema costatum)在<1000 Daltons的超滤液及具有不同含量胶体物质(1000 Daltons～0.7μm)的截留液中进行培养.结果发现在发生柔弱几内亚藻赤潮期间水体中的溶解有机碳浓度会增加,特别是胶体有机碳浓度会成倍增长;在不同胶体含量海水中培养的赤潮异弯藻的最大生长率μmax和最大生物量Bf与对照组相比会随着胶体含量的增加而显著增大,而中肋骨条藻的最大生长率μmax和最大生物量Bf与对照组相比会随着胶体含量增加而显著降低,表明柔弱几内亚藻赤潮水体中的胶体物质对赤潮异弯藻的生长起促进作用,而对中肋骨条藻的生长起抑制作用.这说明柔弱几内亚藻在增殖过程中所产生的胶体物质具有他感作用,会影响其它微藻的生长,从而可能对柔弱几内亚藻赤潮的形成起重要作用.     

广州珠江口海域中肋骨条藻的周年变化及其与环境因子的关系

2004年3月至2005年3月对广州海域(22°33′～22°41′N,113°37′～113°42′E)进行了调查研究,分析结果表明,广州海域中肋骨条藻数量的空间平面分布不均匀,分布格局呈内河口向外河口递增的趋势;中肋骨条藻数量的季节波动模式为单峰型;中肋骨条藻的密度与活性磷酸盐含量存在显著的负相关关系,与硅酸盐含量存在显著的正相关关系;与温度、盐度、亚硝酸盐、铵盐、透明度、硝酸盐、COD及pH的相关性不显著,但与DO存在一定的正相关性,但相关性均不显著;活性磷酸盐和硅酸盐是广州海域中肋骨条藻的主要影响因素。     

敌百虫对中肋骨条藻生长的影响

研究了有机磷农药敌百虫对中肋骨条藻Skeletonema costatum生长的影响。相对于水生甲壳类和鱼类,敌百虫对中肋骨条藻毒性较低,72 h半抑制浓度(EC₅₀)为84.8 mg/L。在31 d的亚慢性暴露下,低浓度组(≤10 mg/L)藻细胞生长趋势与对照组相似;50 mg/L的敌百虫在暴露初期对骨条藻的生长具有明显抑制作用,但在暴露15 d后,该浓度组藻细胞密度仍保持增长,最大细胞密度为2.77×10⁷ cells/mL,是对照组的137.7%。100 mg/L则能完全抑制藻细胞的生长。本实验结果说明敌百虫等有机磷农药的广泛使用可能会刺激浮游植物的生长,从而增加引发赤潮的风险。     

尿素对中肋骨条藻与米氏凯伦藻生长的影响

采用实验室一次性培养,研究了尿素对我国东海赤潮优势藻中肋骨条藻(Skeletonema costatum)和米氏凯伦藻(Karenia mikimotoi)生长的影响。结果表明,中肋骨条藻和米氏凯伦藻均能在不同比例尿素的条件下较好地生长。随着培养液中尿素比例的增大,中肋骨条藻细胞生长速率(0.91—0.82/d)逐渐减小,平台期最大生物量(2.0×10~5—1.2×10~5个/m L)也逐渐减小,而米氏凯伦藻细胞的生长速率(0.36—0.51/d)逐渐增大,最大生物量基本不变(约1.1×10~4个/m L)。在平台期中肋骨条藻培养液中氮盐浓度最低下降到2.5μmol/L左右维持不变,而米氏凯伦藻氮盐浓度最低下降到1.0μmol/L左右。在指数生长期,随着细胞的生长溶解有机氮(DON,Dissolved Organic Nitrogen)含量迅速增加,中肋骨条藻介质中DON的浓度达到最大值(5—6μmol/L),然后浓度基本不变。米氏凯伦藻介质中DON在指数生长阶段达到最大值(2—3μmol/L)后开始下降。中肋骨条藻单细胞颗粒氮的含量(约为10~(-6)μmol,平台期约为10~(-7)μmol)要远远小于米氏凯伦藻(指数期约为10~(-4)μmol,平台期约为10~(-6)μmol)。研究表明,两种藻对尿素的吸收利用存在明显差异,在较低的溶解无机氮和较高的溶解有机氮环境中,甲藻有更好的适应性,该研究对于解释我国长江口春季硅藻和甲藻赤潮的演替有借鉴的意义。     

增殖细胞核抗原基因表达量与中肋骨条藻生长的关系

对中国近海常见的赤潮藻-中肋骨条藻（Skeletonema costatum）生长相关的分子标记进行了研究,通过RT-PCR和RACE技术获得了其PCNA基因的部分序列,首次克隆测序得到了714bp的中肋骨条藻细胞色素b（Cytochrome b,Cyt b）基因序列。根据得到的PCNA与Cyt b基因序列,分别建立了检测中肋骨条藻PCNA与Cyt b基因的实时荧光定量PCR的标准曲线,并通过对已知浓度样品的检测证明了所建立标准曲线的准确性。以上述建立的方法为基础,研究了中肋骨条藻生长率与增殖细胞核抗原基因表达量之间的关系。结果发现,平均单细胞中PCNA表达量在培养的不同阶段变化很大,且变化趋势与生长率表现出良好的一致性,说明PCNA表达量与细胞分裂密切相关,体现了其作为细胞增殖指标的潜能;而Cyt b基因表达稳定,表达量变化很小,说明Cyt b基因是一个潜在的良好的内参基因。基于此,提出以PCNA为目的基因、Cyt b为内参基因,将PCNA基因的相对表达量（REQ）作为中肋骨条藻生长率的一个指标,并建立了实验室条件下PCNA相对表达量与中肋骨条藻生长率之间的关系式。     

粤东柘林湾中肋骨条藻(Skeletonema costatum)种群生态学

于2000年5月～2004年12月对粤东大规模养殖区柘林湾的赤潮藻中肋骨条藻（Skeletonema costatum）种群的时空分布进行了长达5a的调查。结果表明,中肋骨条藻种群密度的周年变动模式基本为双峰型,平面分布没有显著的空间差异。调查期间,中肋骨条藻种群密度的站位实测值为0～1．4×10^7cells／dm^3,总均值为3．3×10^5cells／dm^3,占浮游植物总细胞数的67．1％,为调查海区第1优势种。在总共1045份样品中,有中肋骨条藻出现的样品数为1020份。其中,种群密度大于10^6cells／dm^3的样品有65份,大于10^7cells／dm。则有4份。以大于10^6cells／dm^3为中肋骨条藻的赤潮密度标准,在调查期间至少于2000年、2003年发生4次赤潮。运用灰关联理论对中肋骨条藻种群密度与13个环境因子的关系进行排序分析发现,水温、pH值和浮游动物是影响柘林湾中肋骨条藻种群时空分布的关键因子。水温还与中肋骨条藻种群密度的对数值具极显著意义的线性关系,而达到赤潮密度的样品均落在24．5～32．0℃区间,即每年的5～9月份高温季节。由于柘林湾浮游动物的年高峰期也出现在高温季节,说明浮游动物摄食压力的存在可能是柘林湾中肋骨条藻赤潮发生的重要抑制因子。2004年调查海区中肋骨条藻种群密度和在浮游植物群落中的优势度骤然降低,可能与水体营养盐结构和Fe含量的变化有关。因此,长期调查与监测对于研究海湾生态学和赤潮发生机制是极为重要的。     

氮和磷浓度对中肋骨条藻和锥状斯氏藻种间竞争的影响

采用室内单养和混养方法, 设置不同的氮、磷营养条件, 研究了氮、磷对中肋骨条藻(Skeletonema costatum)和锥状斯氏藻(Scrippsiella trochoidea)种间竞争的影响。结果表明: 混养时各氮和磷浓度下均呈现培养初期中肋骨条藻为优势种、培养后期锥状斯氏藻为优势种的变化趋势, 但随着氮、磷浓度的升高, 中肋骨条藻作为优势种的时间延长; 与单养时相比, 混养中两种微藻的最大密度受到不同程度的抑制, 表现出氮、磷浓度越高, 受抑制的程度越大的特征, 且与锥状斯氏藻相比, 中肋骨条藻的最大密度受到抑制的程度更大。混养时两种微藻均是在氮、磷浓度最高时, 抑制起始点出现时间最长, 随着氮、磷浓度的降低, 抑制起始点出现时间缩短; 各氮、磷浓度条件下, 锥状斯氏藻对中肋骨条藻的竞争抑制参数明显高于中肋骨条藻对锥状斯氏藻的竞争抑制参数, 当氮浓度为512 μmol·L^-1、磷浓度为2 μmol·L ^-1时, 竞争结果是锥状斯氏藻获胜; 其余氮、磷浓度条件下为两种微藻不稳定共存。     

Bacterial community dynamics during a bloom caused by Akashiwo sanguinea in the Xiamen sea area,China

Phytoplankton blooms are a worldwide ecological problem and one of the major algae that cause phytoplankton blooms is Akashiwo sanguinea. Though much research has addressed the abiotic causes (e.g. growth condition) of A. sanguinea blooms, few studies have examined the dynamics of microbial communities associated with these blooms. In this study, polymerase chain reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE) analysis of 16S rDNA genes was used to document changes in the phylogenetic diversity of microbial communities associated with an A. sanguinea bloom that occurred in the Xiamen sea in May 2010. Surface sea water was sampled once a day within five consecutive days at four sites, and the microbial community composition was determined using DGGE. Sea water concentrations of chlorophyll a, nitrate and phosphate were also measured. The results indicated that the A. sanguinea bloom was probably stimulated by low salinity (26–30‰) and ended probably because inorganic nutrients were consumed and resulted in a N/P ratio unfavorable for this alga. Gammaproteobacteria populations increased significantly during bloom declines and then decreased post-bloom. Divergences in the microbial community composition during different bloom periods were the result of changes in Candidatus, Pelagibacter, Alteromonas, Rhodobacteraceae, Vibrio and Pseudoalteromonas populations. Sediminimonas qiaohouensis was the first bacterium shown to be significantly negatively correlated with A. sanguinea concentration. This study indicated that bacteria may play an important role in A. sanguinea–bloom regulation and provides a deeper insight into bacterial community succession during and after an A. sanguinea–bloom.     

Development of microbial community during Skeletonema costatum detritus degradation

Microbial degradation of algal detritus was studied experimentally usingthe diatom Skeletonema costatum prekilled culture as a substrate for themarine microbial community. The qualitative and quantitative changes in themicrobial community and the algal detritus structure were followed during 11days of incubation at the water temperature of 20°C. Most of thebacterial parameters (epifluorescence microscopy counts of free-living andattached cells, mean cell volume, biomass and productivity) were the highestafter 24 h from the algal detritus addition to the microbial community. Thebacterial peak (3.6×10⁶ cells ml^™1,biomass 3.1 μgC ml^™1, net production 66×4 ngC ml^™1h^™1) was followed by a precipitous increaseof homogenous nanoflagellate population with a maximum number of2.6×10⁵ cells ml^™1, which in turn declinedquickly after ciliates appeared in the community. The bacterial production,initiated by the supplement of algal detritus, was totally ingested bymicrozoans within 3 days. Changes of the structure of marine bacterialassemblage and relative increase of the amount of attached bacteria duringthe period of massive development of nanoflagellates emphasized theimportance of small flagellates predation on the free-living average size(cell volume 0.1–0.2 μm³) pelagic bacteria. After11 days of incubation on algal detritus the initial bacterial assemblage wasreplaced by the mixed succession of bacteria, flagellates and ciliates at aproportion of 1000 : 1.5 : 0.2. The stabilization of microbialcommunity and changes in algal detritus structure allow to expect thatpelagic microbial utilization of Skeletonema costatum bloom(53×10⁶ cells l^™1), could be finishedwithin a period of 8–11 days at the summer water temperature. This revised version was published online in July 2006 with corrections to the Cover Date.     

Dynamics of bacterial community composition and activity during a mesocosm diatom bloom

Bacterial community composition, enzymatic activities, and carbon dynamics were examined during diatom blooms in four 200-liter laboratory seawater mesocosms. The objective was to determine whether the dramatic shifts in growth rates and ectoenzyme activities, which are commonly observed during the course of phytoplankton blooms and their subsequent demise, could result from shifts in bacterial community composition. Nutrient enrichment of metazoan-free seawater resulted in diatom blooms dominated by a Thalassiosira sp., which peaked 9 days after enrichment ( approximately 24 microg of chlorophyll a liter(-1)). At this time bacterial abundance abruptly decreased from 2.8 x 10(6) to 0.75 x 10(6) ml(-1), and an analysis of bacterial community composition, by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene fragments, revealed the disappearance of three dominant phylotypes. Increased viral and flagellate abundances suggested that both lysis and grazing could have played a role in the observed phylotype-specific mortality. Subsequently, new phylotypes appeared and bacterial production, abundance, and enzyme activities shifted from being predominantly associated with the <1.0-microm size fraction towards the >1.0-microm size fraction, indicating a pronounced microbial colonization of particles. Sequencing of DGGE bands suggested that the observed rapid and extensive colonization of particulate matter was mainly by specialized alpha-Proteobacteria- and Cytophagales-related phylotypes. These particle-associated bacteria had high growth rates as well as high cell-specific aminopeptidase, beta-glucosidase, and lipase activities. Rate measurements as well as bacterial population dynamics were almost identical among the mesocosms indicating that the observed bacterial community dynamics were systematic and repeatable responses to the manipulated conditions.     

Dynamics in bacterial surface properties of a natural bacterial community in the coastal North Sea during a spring phytoplankton bloom

The hydrophilic and hydrophobic properties of single cells of natural bacterioplankton communities were determined using a recently developed staining method combined with confocal laser scanning microscopy and advanced image analysis. On an average, about 50% of the bacterial cell area was covered by hydrophobic and only 16% by hydrophilic properties, while about 72% was covered by the genome. However, the size of these properties was independent of the bacterial cell size. Bacterial hydrophobicity was positively correlated with ambient NH(4)(+) concentrations and negatively correlated with overall bacterial abundance. The expression of hydrophilicity was more dynamic. Over the spring phytoplankton bloom, the bacterioplankton ratio(phil/phob) repeatedly reached highest values shortly before peaks in bacterioplankton abundance were observed, indicating a direct and fast response of bacterial surface properties, especially hydrophilicity, to changing environmental conditions. Compared to bacterial strains, recently studied with the same method, cells of marine bacterioplankton communities are much smaller and less frequently covered by hydrophobic or hydrophilic properties. While the percentage area covered by the genome is essentially the same, the percentage area covered by hydrophobic or hydrophilic properties is much smaller.     

Cytometric quantification of nitrate reductase by immunolabeling in the marine diatom Skeletonema costatum

BACKGROUND: The uptake of nitrate by phytoplankton is a central issue in biological oceanography due to its importance to primary production and vertical flux of biogenic carbon. Nitrate reductase catalyzes the first step of nitrate assimilation, the reduction of NO(3) to NO(2). A cytometric protocol to detect and quantify relative changes in nitrate reductase (NR) protein content of the marine centric diatom Skeletonema costatum is presented. METHODS: Immunolabeling of NR protein was achieved with polyclonal antibodies raised against S.costatum NR. Antisera specific to a NR protein subunit and to a NR polypeptide sequence were compared, and cytometric results of NR protein abundance were related to Western analyses. Changes in cellular NR abundance and activity were followed during an upwelling simulation experiment in which S. costatum was exposed to a shift from ammonia to nitrate as major nitrogen source. RESULTS: NR protein could be detected in NO(3)-grown cells and at extremely low levels hardly discernible by Western Blot densiometry in NH(4)-grown cells. The protocol allowed observation of early stages of NR induction during an upwelling simulation. NR abundance increased after the nutrient shift to reach a new physiological "steady-state" 96 hrs later. NR activity exhibited diel variation with maxima at mid-day. NR abundance as estimated by both flow cytometry and Western analysis exhibited a hyperbolic relationship to NR activity. This pattern suggests post-translational activation of NR protein. CONCLUSIONS: The presented protocol allows the differentiation of NH(4)- versus NO(3)-grown algae as well as the monitoring of early stages in the induction of nitrate assimilatory capacities.     

Alkaline phosphatase activity of Skeletonema costatum populations in the Trondheimsfjord

During May and June of 1975 and 1976, unialgal blooms of themarine diatom Skeletonema costatum in the Trondheimsfjord exhibitedsignificant alkaline phosphatase activity, as measured by afluorometric method. The fairly high activities of some populationscorrelated well with clear indications of P-limitation obtainedby measurement of N/P, ß-l,3-glucan/P and protein/ß-l,3-glucanratios. Very few significant activities were measured in populationswith a cellular N/P ratio below 14, which agrees well with anN to P ]balance point’ of 11-12 for S. costatum. The activitywas detected in brackish water with S <30 and was foundto be closely related with the cellular phosphorus content,low content leading to high phosphatase activity. The ratioof phosphatase activity to cellular phosphorus is proposed asa sensitive parameter for phosphorus-limited growth. No enzymeactivity could be detected in coastal waters outside the fjord.     

Copper binding ability of the extracellular organic matter released by Skeletonema costatum

Abstract

The copper complexing ability of the exudates produced during the exponential growth phase by Skeletonema costatum has been investigated by a ligand-competition technique involving copper sorption onto C-18 Sep-Pak cartridges. Two ligands with different affinity for copper were required for the best fit of the copper complexation data in seawater with and without exudates: a strong ligand with a log K close to 13 and a weaker ligand with a log K close to 9. The culture increased both L₁ and L₂ ligand concentrations, already present in seawater, by a factor close to 4 after the first 72 hours of growth. The presence of class 1 stronger ligands in copper binding organics produced by the diatom is discussed in relation to natural copper speciation in the sea.     

Nitrate Utilization by the Diatom Skeletonema costatum: II. Regulation of Nitrate Uptake

Nitrate utilization has been characterized in nitrogen-deficient cells of the marine diatom Skeletonema costatum. In order to separate nitrate uptake from nitrate reduction, nitrate reductase activity was suppressed with tungstate. Neither nitrite nor the presence of amino acids in the external medium or darkness affects nitrate uptake kinetics. Ammonium strongly inhibits carrier-mediated nitrate uptake, without affecting diffusion transfer. A model is proposed for the uptake and assimilation of nitrate in S. costatum and their regulation by ammonium ions.     

Nitrate Utilization by the Diatom Skeletonema costatum: I. Kinetics of Nitrate Uptake

Nitrate uptake has been studied in nitrogen-deficient cells of the marine diatom Skeletonema costatum. When these cells are incubated in the presence of nitrate, this ion is quickly taken up from the medium, and nitrite is excreted by the cells. Nitrite is excreted following classical saturation kinetics, its rate being independent of nitrate concentration in the incubation medium for nitrate concentration values higher than 3 micromolar. Nitrate uptake shows mixed-transfer kinetics, which can be attributed to the simultaneous contributions of mediated and diffusion transfer. Cycloheximide and p-hydroxymercuribenzoate inhibit the carrier-mediated contribution to nitrate uptake, without affecting the diffusion component. When cells are preincubated with nitrate, the net nitrogen uptake is increased.     

Isolation and characterization of glutamine synthetase from the marine diatom Skeletonema costatum.

Two peaks of glutamine synthetase (GS) activity were resolved by anion-exchange chromatography from the marine diatom Skeletonema costatum Grev. The second peak of activity accounted for greater than 93% of total enzyme activity, and this isoform was purified over 200-fold. Results from denaturing gel electrophoresis and gel-filtration chromatography suggest that six 70-kD subunits constitute the 400-kD native enzyme. The structure of the diatom GS, therefore, appears more similar to that of a type found in bacteria than to the type common among other eukaryotes. Apparent Michaelis constant values were 0.7 mM for NH4(+), 5.7 mM for glutamic acid, and 0.5 mM for ATP. Enzyme activity was inhibited by serine, alanine, glycine, phosphinothricin, and methionine sulfoximine. Polyclonal antiserum raised against the purified enzyme localized a single polypeptide on western blots of S. costatum cell lysates and recognized the denatured, native enzyme. Western analysis of the two peak fractions derived from anion-exchange chromatography demonstrated that the 70-kD protein was present only in the later eluting peak of enzyme activity. This form of GS does not appear to be unique to S. costatum, since the antiserum recognized a similar-sized protein in cell lysates of other chromophytic algae.