米氏凯伦藻与东海原甲藻共培养条件下的种群竞争

米氏凯伦藻(Karenia mikimotoi)与东海原甲藻(Prorocentrum donghaiense)为有害赤潮生物,两者经常形成复合型赤潮.该文设置东海原甲藻的起始密度为400 cells·mL-1,米氏凯伦藻分别为200 cells·mL-1、400 cells·mL-1和800 cells·mL-1,通过共培养实验,初步研究米氏凯伦藻和东海原甲藻的种间关系.结果表明:共培养条件下,受到东海原甲藻的影响,米氏凯伦藻的生长受到抑制.米氏凯伦藻不同的起始密度对东海原甲藻的生长有不同的影响,较低的起始密度(200 cells·mL-1、400cells·mL-1)促进东海原甲藻的生长,使其增长率提高,生长曲线达到拐点的时间提前;高的起始密度(800 cells·mL-1)对东海原甲藻的生长有一定的抑制作用,使其增长率降低,生长曲线达到拐点的时间推迟.     

不同营养条件对东海原甲藻和中肋骨条藻竞争参数的影响

以东海原甲藻和中肋骨条藻为研究对象,采用室内单种培养和混合培养,设置不同的氮、磷营养条件,研究了不同营养条件对两种微藻的生长状况和种间竞争参数的影响.结果表明:随着氮、磷浓度的增加,两种藻的最大生物量均呈增加趋势,混合培养中两种微藻的比生长率低于单独培养.在混合培养中,生长前期中肋骨条藻是优势种,随着培养时间的延长,东海原甲藻成为优势种,且优势种发生变化的时间与营养条件有关.混合培养中,东海原甲藻拐点出现时间在0.5 ～4.9 d,中肋骨条藻为0～2.6d,东海原甲藻拐点出现时间晚于中肋骨条藻.在各营养条件下,东海原甲藻对中肋骨条藻的竞争抑制参数β均高于中肋骨条藻对东海原甲藻的竞争抑制参数α,当N为128μmol·L-1、P为32 μmol·L-1时,东海原甲藻的竞争能力是中肋骨条藻的3.8倍,两者差异最为明显.     

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。     

米氏凯伦藻溶血毒素的溶血反应特征

探讨了温度、pH值、二价阳离子等对米氏凯伦藻(Karenia mikimotoi Hasen)溶血毒素溶血活性的影响,分析了米氏凯伦藻溶血毒素的溶血反应特征.结果表明,实验室培养米氏凯伦藻的溶血活性约为64.69±6.43 HU L-1,单个藻细胞的溶血活性为6.17±0.61×10-6 HU;在实验温度(0～37℃)下,溶血活性随温度的增加而增加;pH6.0时的溶血活性最高;Cu2+、Mg2+、Mn2+、Ca2+、Co2+、Zn2+和Hg2+等对米氏凯伦藻的溶血活性的影响不同.离子浓度为5 mmol/L时,Hg2+的抑制作用最强.高浓度Hg2+对红细胞的集合效应不但阻止了Hg2+进入血细胞诱导的溶血作用,而且阻止了毒素对细胞膜的破坏,但这种抑制作用可被EDTA消除.     

蒽和UV—B辐射对米氏凯伦藻生长的影响

为研究多环芳烃蒽（anthracene）和UV—B辐射对米氏凯伦藻（Kreniamikimotoi Hansen）的单独效应和联合毒性效应,采用实验生态学的方法,以米氏凯伦藻为实验材料,蒽质量浓度设为0．00、6．25、11．50、20．00、35．00、62．50μg／L,UV—B辐射剂量设为0．00、0．375、1．125、2．25、3．75、6．00J／m^2。实验结果表明：对米氏凯伦藻的生长,多环芳烃蒽具有抑制效应,小剂量的UV-B具有刺激作用,随着剂量的增加表现出抑制作用,蒽与UV—B的联合则表现出更强的抑制作用,二者表现为协同作用。蒽和UV—B对米氏凯伦藻的96h—EC。分别为15．35μg／L和2．843J／m^2,而蒽在UV—B辐射条件下的96h-EC50为7．376μg／L。     

营养盐限制条件下塔玛亚历山大藻对东海原甲藻的化感作用研究

为明确塔玛亚历山大藻(Alexandrium tamarense)对东海原甲藻(Prorocentrum donghaiense)生长的化感作用,研究了在N、P限制及正常营养盐条件下(又称富营养)塔玛亚历山大藻无细胞滤液对东海原甲藻生长的影响,并探讨了3种不同营养盐条件下两种藻共培养时的生长状况。结果表明,半连续培养时,营养盐限制下,塔玛亚历山大藻无细胞滤液对东海原甲藻的生长均有一定影响。N限制下,5 d后东海原甲藻藻密度显著低于未加滤液的对照组,藻密度为1.02×107 cells L-1,对照组为1.7×107 cells L-1;P限制下,东海原甲藻藻密度与对照组差异不显著,5 d后藻密度为1.44×107 cells L-1;富营养条件下,东海原甲藻藻密度与对照组无明显区别。共培养时,塔玛亚历山大藻对东海原甲藻生长的抑制作用更为显著,N、P限制下,4 d后东海原甲藻全部死亡,且聚集成团形成沉淀;富营养条件下,仍有少量东海原甲藻存活(藻密度3.3×104 cells L-1)。这表明,塔玛亚历山大藻对东海原甲藻的生长有一定的化感作用。营养盐限制可促进塔玛亚历山大藻化感物质的合成和释放,化感作用是塔玛亚历山大藻抑制东海原甲藻生长的原因之一。     

铁限制条件下东海原甲藻分泌铁载体

在铁限制条件下,进行东海原甲藻分泌铁载体的动态研究。对藻类在富铁与缺铁条件下生长状况、生长过程中分泌铁载体的情况以及海藻接种量对铁载体分泌的影响进行了连续观测,结果表明:东海原甲藻在缺铁条件下生长状况远不如在富铁条件下;随着藻类的生长,分泌铁载体不断增多,达指数生长期时,其分泌量也达到了最大值,之后藻类的生长和铁载体分泌都呈现下降趋势;高接种量东海原甲藻能分泌较多的铁载体,并在较短时间到达峰值。     

重金属Zn2+胁迫下米氏凯伦藻(Karenia mikimotoi)的生长生理响应研究

采用室内培养试验方法, 以米氏凯伦藻(Karenia mikimotoi)为生物材料, 设置不同浓度梯度的重金属Zn2+(0、1、5、10、15和20 mg•L-1), 主要测定藻细胞密度、光合色素、光合效率、抗氧化酶类、丙二醛(malondialdehyde, MDA)等相关指标, 探讨重金属Zn2+胁迫下米氏凯伦藻(Karenia mikimotoi)的生长和生理特征。结果发现, 在1和5 mg•L-1的Zn2+浓度下, 米氏凯伦藻细胞依然保持较好生长繁殖, 表明其具有一定的耐受性, 而随着重金属Zn2+浓度的提高, 细胞生长受到毒害抑制。光合色素含量呈现动态变化, 试验结束时(96 h)叶绿素a、叶绿素b和胡萝卜素含量有各自不同的变化趋势, 最大光能转化效率(Fv/Fm)表现出先升后降的趋势。Zn2+浓度为10 mg•L-1时, 超氧化物歧化酶(superoxide dismutase, SOD)活性显著高于对照; 过氧化氢酶(catalase, CAT)活性呈现出先上升后下降的趋势, 且5、10、15和20 mg•L-1 Zn2+浓度下米氏凯伦藻的过氧化氢酶(CAT)活性均显著高于对照; 10、15 mg•L-1 Zn2+浓度下米氏凯伦藻的总抗氧化能力(total antioxidant capacity, T-AOC)显著高于对照, 而20 mg•L-1 Zn2+浓度下的显著低于对照。丙二醛(MDA)呈现出随着Zn2+浓度提高而增加的趋势, 5、10、15和20 mg•L-1Zn2+浓度下米氏凯伦藻的丙二醛(MDA)均显著高于对照。结果可为了解重金属对海洋微藻的毒性作用提供数据参考。     

不同无机氮源对东海原甲藻生长的影响

在实验室条件下,研究了不同浓度、不同形态的氮(NaNO₃、NH₄Cl和NaNO₂)对东海原甲藻(Prorocentrum donghaiense)生长的影响。结果表明:在NH₄Cl浓度为5.20μmol·L^-1(N/P为8)时藻的比生长率最高,而N/P为32和100时,藻的生长明显受到抑制。在NaNO₃为氮源时,最适N/P为12(氮浓度为7.80μmol·L^-1)。而NaNO₂作氮源,N/P为16(10.40μmol·L^-1)时藻的比生长率最高,N/P为32和100时藻的生长也明显受到抑制。研究显示,东海原甲藻对无机氮NH₄Cl和NaNO₃和NaNO₂都可以利用,最适生长的N/P比范围在8～20之间,相对高的N/P(32、100)不利于东海原甲藻的生长。     

东海原甲藻对不同磷源的利用特征

采用单因子实验,以NaNO3作为唯一氮源,固定初始氮浓度为40μmol.L-1,设置六个初始磷浓度梯度:0.1μmol.L-1、0.5μmol.L-1、1.0μmol.L-1、2.0μmol.L-1、5.0μmol.L-1、10.0μmol.L-1,对比研究了东海原甲藻(Prorocentrum donghaiense)对4种不同磷源的利用特征及其这4种不同磷源对东海原甲藻的营养价值。结果表明:东海原甲藻既可以直接吸收利用无机磷——磷酸二氢钠(NaH2PO4),又可以不同程度地利用3种小分子有机磷:三磷酸腺苷二钠盐(ATP)、D-葡萄糖-6-磷酸钠(G-6-P)和甘油磷酸钠(G-P);东海原甲藻在以NaH2PO4、G-6-P和ATP分别作为磷源时的最大比生长率(μmax)值相差不大,3种磷源对最大生物量的贡献也基本相近;但是以G-P作为磷源时则明显有最低的μmax值,其对最大生物量的贡献也最低。东海原甲藻对这4种磷源的利用特征说明:长江口自然海水中与三种小分子有机磷结构相似的溶解有机磷在一定程度上也可以为东海原甲藻赤潮的爆发和维持提供磷源。     

三种甲藻对阳光紫外辐射响应机制的种间差异

<正>从发现南极臭氧空洞以来,光生物学的研究领域便出现了一个新的研究方向,即研究阳光紫外辐射(Ultraviolet radiation,UVR)对藻类的影响[1]。众多研究表明,阳光UVR对藻类具有明显的负面效应,如UVR抑制光合固碳和生长,并导致DNA和蛋白的损伤[2—4]。但也有少数研究发现,在一定条件下,阳光UVR具有正面效应,如紫外线A(UV-A)有利于DNA损伤后的修复[2],并     

Development and application of LSU rRNA probes for Karenia brevis in the Gulf of Mexico, USA

The brevetoxin producing dinoflagellate, Karenia brevis, is the target of several monitoring and research programs in the Gulf of Mexico, where it forms extensive and frequently long-lived annual blooms that can cause human intoxication and fish kills, as well as severe economic losses to coastal communities. Rapid, reliable methods for the detection and enumeration of K. brevis cells, as well as their discrimination from morphologically similar species, are valuable tools for managers and scientists alike. Our aim was to produce a species-specific molecular probe that would serve as a tool to facilitate the efficient and reliable detection of K. brevis in the Gulf of Mexico. We sequenced a fragment of the large-subunit ribosomal RNA gene (LSU rDNA) from five K. brevis cultures isolated from the Texas Gulf coast, the Florida Gulf coast, and the Atlantic coast of Florida, and detected no differences among these isolates. A consensus sequence was thus compiled and compared to a previously published sequence from Karenia mikimotoi, the closest known phylogenetic relative to K. brevis, for the purpose of identifying unique K. brevis signature sequences. Fluorescently-labeled (FITC) oligonucleotide probes targeting these regions of the K. brevis LSU rRNA were designed to include at least two base pair differences, as compared to K. mikimotoi. Among seven probes designed, one uniquely identified all K. brevis isolates to the exclusion of all other species tested (Kbprobe-7), including a Gulf of Mexico K. mikimotoi isolate (Sarasota, FL) and several additional Gymnodinium species, as well as other dinoflagellate, diatom, and raphidophyte taxa. Importantly, K. brevis cells in samples taken during a 2001 bloom, fixed with a mixture of modified saline ethanol and 10% formalin, and stored at 4 °C for 7 months were successfully labeled with Kbprobe-7. In addition, preliminary analysis of labeled cells by flow cytometry revealed that K. brevis could be distinguished from K. mikimotoi in solution, suggesting other potential applications of this probe.     

不同营养盐条件下赤潮高发区围隔生态系内多胺的变化

在东海赤潮爆发区域运用围隔生态系实验方法,研究了不同营养盐条件下围隔生态系内多胺浓度变化。结果表明:2010年选用东海原甲藻赤潮爆发处海水,东海原甲藻是各围隔生态系内主要优势种,没有种群演替现象发生。两种营养盐添加方式下各围隔内精胺浓度维持较高水平,都呈现先波折下降后波折上升的趋势,与东海原甲藻的生长变化正好相反;各围隔内腐胺浓度水平较高,变化起伏较大,其中有两个实验组腐胺整体变化趋势与东海原甲藻生长趋势类似;所有围隔内亚精胺浓度最低,波动较小。2011年取用中肋骨条藻赤潮爆发处海水,所有围隔生态系内优势种都发生了从中肋骨条藻到东海原甲藻的演替。各围隔生态系内腐胺浓度最高,在中肋骨条藻生长初期腐胺浓度下降,随着中肋骨条藻的生长有所上升,实验后期随着东海原甲藻的生长又整体呈现出下降趋势;各实验组精胺浓度较低,在中肋骨条藻消亡东海原甲藻出现的种群演替期间,都呈现出较大波动;各围隔内亚精胺浓度较低,在整个种群演替过程中没有明显的变化。围隔生态系中补充营养盐,通过对浮游植物生长的影响,间接影响围隔生态系内的多胺变化。     

Development of microsatellite markers in the marine phytoplankton Karenia mikimotoi (Dinophyceae)

The marine phytoplankton, Karenia mikimotoi, causes severe red tides which are associated with mass mortality of marine fish, and have expanded their distributions in the coastal waters of western Japan. To assess the dispersal mechanism, a population genetic study using highly polymorphic genetic markers is one of the crucial approaches. Here we developed 12 polymorphic microsatellite markers from K. mikimotoi. These loci provide a class of highly variable genetic markers, as the number of alleles ranged from 5 to 23, and the estimate of gene diversity was from 0.551 to 0.933 across the 12 microsatellites. We consider these loci potentially useful for detailing the genetic structure and gene flow among K. mikimotoi populations.     

环境因子对东海原甲藻生长及脲酶活性的影响

以我国东南沿海大规模赤潮原因种东海原甲藻为实验材料,研究了环境因子对其生长及脲酶活性的调控作用。结果表明,东海原甲藻的适宜生长温度为20—25℃,而25℃下脲酶活性最高。在光强2μmol m-2s-1条件下细胞密度显著下降(P0.05),但仍能维持较高的脲酶活性(9.405 fmol h-1个-1)。在盐度20—40范围内,东海原甲藻能够维持快速生长和较高的脲酶活性。氮源组成对东海原甲藻生长无显著影响,但对脲酶活性影响较大。具体而言,东海原甲藻脲酶活性与尿素浓度呈显著正相关关系,而无机氮源NH+4和NO-3对其脲酶活性具有显著的抑制作用,在氮缺乏条件下脲酶活性明显增强。东海原甲藻脲酶活性对环境温度、光照、盐度和营养的响应特征可能是在长期进化中形成的生态适应策略,使其在无机氮源不足时得以转而利用有机氮源,从而在资源竞争中占据有利地位。     

Development of an immunofluorescence technique for detecting <Emphasis Type="Italic">Prorocentrum donghaiense</Emphasis> Lu

Prorocentrum donghaiense Lu is a key harmful algal bloom (HAB) species which is widespread along the China Sea coast. In this study, P. donghaiense-specific antiserum was developed, and the detection method, based on immunofluorescence (IF), was optimized. The antiserum was raised using 0.5% paraformaldehyde-fixed whole cells (WC) as antigen. The titer and the specificity were examined using whole-cell IF. Results showed that ethanol was an effective decolorization reagent, and 80% ethanol was able to minimize autofluorescence of cells. Samples preserved by freezing at −20°C or −80°C remained above 85% detection efficiency after 1-month storage. The antiserum against WC had a high titer (1:10000), and exhibited high specificity at species level. The antiserum showed a weak cross reaction with the closely related species P. dentatum HK, P. dentatum CCMP1517 and P. minimum HK only at very low dilution (1:5). However, it did not cross-react with the species from the same genus or other phytoplankton species when the dilution reached or exceeded 1:100. Results from different P. donghaiense samples collected at different growth phases or grown under different nutrient conditions showed no significant difference in IF intensity. In addition, the antiserum exhibited high specific binding to P. donghaiense in both mixed phytoplankton samples and field samples. The results indicate that the technique is a potentially useful tool for the rapid identification of P. donghaiense and can facilitate the analysis of various natural samples.     

Morphological and genetic study of Prorocentrum donghaiense Lu from the East China Sea, and comparison with some related Prorocentrum species

In this paper, the detailed morphology of Prorocentrum donghaiense Lu from both field samples and cultures was examined, and a taxonomic comparison was made between P. donghaiense and some related Prorocentrum spp. using morphological and molecular data and other published information. There were distinct differences among these species in morphological characteristics that historically have been presented as conservative features. The discrepancies extended beyond that of individual variations within the same species due to environmental factors. Therefore, these morphological features may not be conservative but, rather, polymorphic depending on environmental conditions. Based on this analysis, we suggest that the high-biomass bloom-forming species in the East China Sea, previously reported as Prorocentrum dentatum Stein, is P. donghaiense Lu. The species reported from the East China Sea and Japanese and Korean waters appear to be the same species. Molecular data also suggest that P. dentatum (CCMP1517) and P. donghaiense are genetically identical. Therefore, the geographic distribution of P. donghaiense may be much wider than expected.     

Interactions between Prorocentrum donghaiense Lu and Scrippsiella trochoidea (Stein) Loeblich III under laboratory culture

Interactions between Prorocentrum donghaiense Lu and Scrippsiella trochoidea (Stein) Loeblich III, two species of causative bloom dinoflagellates in China, were investigated using bi-algal cultures under controlled laboratory conditions. The growth of P. donghaiense and S. trochoidea were significantly suppressed when the initial cell densities were set at 1.9 × 10⁴ cells mL⁻¹ or 1.9 × 10⁵ cells mL⁻¹ for P. donghaiense and 1.0 × 10⁴ cells mL⁻¹ for S. trochoidea when the initial size/density ratio was 1:1 or 10:1, respectively, but no out-competement was observed in either bi-algal culture by the end. The simultaneous assay on the culture filtrate showed that P. donghaiense filtrate prepared at a lower initial density (1.9 × 10⁴ cells mL⁻¹) stimulated the co-cultured S. trochoidea at a density of 1.0 × 10⁴ cells mL⁻¹, but filtrate at a higher density (1.9 × 10⁵ cells mL⁻¹) depressed its growth. Differently, the filtrate of S. trochoidea at a density of 1.0 × 10⁴ cells mL⁻¹ significantly suppressed the growth of P. donghaiense at a density of 1.9 × 10⁴ cells mL⁻¹, but had little stimulatory effect on P. donghaiense at a density of 1.9 × 10⁵ cells mL⁻¹compared to the control (P > 0.05). It is likely that these two species of microalgae interact 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. We then quantify their interactions in the bi-algal culture by using a mathematical model. The estimated parameters from the model showed that the inhibition exerted by S. trochoidea on P. donghaiense was about 43 and 24 times stronger than the inhibitory effect that P. donghaiense exerted on S. trochoidea when the initial size/density were 1:1 and 10:1, respectively. S. trochoidea seemed to have a survival strategy that was superior to P. donghaiense in the bi-algal culture under controlled laboratory conditions. We also observed a closely positive relationship between the initial cell density and its effect on the co-cultured microalga by measuring the fluorenscence: filtrate prepared from higher initial cell density had stronger interference on the co-cultured microalga. Moreover, pre-treated under different temperature conditions (30 °C, 60 °C and 100 °C) would significantly changed the effect of culture filtrate on the co-cultured microalga. Result inferred that P. donghaiense or S. trochoidea would release allelochemicals into the bi-algal culture medium and the allelochemicals might be a mixture with temperature-sensitive components in it.