塔玛亚历山大藻对氮和磷的吸收及其生长特性

参照塔玛亚历山大藻(Alexandrium tamarense)赤潮爆发时的物理条件,以f／2加富的人工海水为培养基,设定了不同的氮、磷水平,研究了在室内批量培养条件下,塔玛亚历山大藻对无机氮、磷的吸收和无机氮、磷对塔玛亚历山大藻细胞生长的影响．结果表明,3种氮浓度条件下,塔玛亚历山大藻的比生长速率几乎没有差异,但低氮(0．0882mmol·L-1)条件下,藻细胞的生物量最低;中氮(0．882mmol·L-1)条件下,藻细胞具有最大的生物量,分别比高氮(2．646mmol·L-1)和低氮下增加44．7％和53．6％．随着培养基中磷浓度的升高,藻细胞生物量也升高,在高磷(0．108mmol·L-1)条件下达到最大值17200cell·ml-1,但在中磷(0．036mmol.L-1)条件下藻细胞具有最大的比生长速率．藻细胞对氮、磷的吸收速率与生长状态有密切关系,氮、磷限制条件下生长的藻细胞对氮、磷有快速的吸收．研究显示,低的N／P比有利于塔玛亚历山大藻的生长分裂,对数生长后期适当补氮则有利于其生物量的积累．     

N、P营养盐对塔玛亚历山大藻(Alexandrium tamarense)生长的影响

模拟自然海水营养盐浓度状况,在N、P浓度分别为10-500μg L-1 N和0.74-74μg L-1 P时,研究N、P双因子限制(N、P浓度同时降低,N:P固定为15:1)及单因子限制(保持N或P为最高浓度,只降低一种营养盐浓度)对有毒赤潮藻塔玛亚历山大藻(Alexandrium tamarense)生长的影响。结果表明,塔玛亚历山大藻细胞能较快进入对数生长期,但N、P双因子限制能明显影响其生长,在N、P浓度分别低于100μg L-1 N和15μg L-1 P时,细胞密度无明显增长;而N或P分别受限时,生长态势明显优于N、P同时受到限制的试验组,而且N、P单因子中度限制对生长影响较小。结果说明塔玛亚历山大藻对单因子营养元素限制较强的适应能力,可使其在常常出现单营养因子限制的自然水体中维持一定生长速率和细胞密度,并有助于滤食该藻的贝类体内麻痹性贝类毒素的积累。     

Species boundaries and global biogeography of the Alexandrium tamarense complex (Dinophyceae)1

Alexandrium catenella (Whedon et Kof.) Balech, A. tamarense (M. Lebour) Balech, and A. fundyense Balech comprise the A. tamarense complex, dinoflagellates responsible for paralytic shellfish poisoning worldwide. The relationships among these morphologically defined species are poorly understood, as are the reasons for increases in range and bloom occurrence observed over several decades. This study combines existing data with new ribosomal DNA sequences from strains originating from the six temperate continents to reconstruct the biogeography of the complex and explore the origins of new populations. The morphospecies are examined under the criteria of phylogenetic, biological, and morphological species concepts and do not to satisfy the requirements of any definition. It is recommended that use of the morphospecies appellations within this complex be discontinued as they imply erroneous relationships among morphological variants. Instead, five groups (probably cryptic species) are identified within the complex that are supported on the basis of large genetic distances, 100% bootstrap values, toxicity, and mating compatibility. Every isolate of three of the groups that has been tested is nontoxic, whereas every isolate of the remaining two groups is toxic. These phylogenetic groups were previously identified within the A. tamarense complex and given geographic designations that reflected the origins of known isolates. For at least two groups, the geographically based names are not indicative of the range occupied by members of each group. Therefore, we recommend a simple group‐numbering scheme for use until the taxonomy of this group is reevaluated and new species are proposed.     

塔玛亚历山大藻的生长研究

在室内条件下研究了温度、N和P、维生素、抗生素对有毒赤潮甲藻塔玛亚历山大藻(香港株Ⅱ)生长的影响。结果表明,塔玛亚历山大藻的最适生长温度为21—25℃,最适N、P浓度分别为882—1765μmol/L和18—72μmol/L。复合维生素B1、B6、B12的加入有利于塔玛亚历山大藻的生长,而50U/mL以上的抗生素(氨苄青霉素液体)则对其有明显的抑制作用。     

Growth and phosphate uptake kinetics of the toxic dinoflagellate Alexandrium tamarense from Hiroshima Bay in the Seto Inland Sea, Japan

Shellfish poisoning by the toxic dinoflagellate Alexandrium tamarense (Lebour) Balech occurred for the first time in Hiroshima Bay, Japan, in 1992. Oyster culture in the bay produces as much as 60% of the total production in Japan, and it suffered severe damage. In the present study, we experimentally investigated the growth rate and phosphate uptake kinetics of A. tamarense, Hiroshima Bay strain. A short-term phosphate uptake experiment revealed that the maximum uptake rate was 1.4 pmol P cell^-1 per h and the half-saturation constant was 2.6 umol L^-1. In semicontin-uous culture, the maximum specific growth rate and the minimum phosphorus cell quota were 0.54 day^-1 and 0.56 pmol P cell^-1, respectively. These uptake rates suggest that A. tamarense is a poor phosphorus competitor compared with other species. However, the large phosphorus storage capacity (Q_pmax/q_o= 36), the surge phosphorus uptake ability (V_s/V_i= 4.1) and the low growth rate would be advantageous for surviving brief periods of phosphorus limitation which frequently occur in Hiroshima Bay.     

Development of microsatellite markers in the toxic dinoflagellate Alexandrium tamarense (Dinophyceae)

Outbreaks of paralytic shellfish poisoning caused by the toxic dinoflagellate Alexandrium tamarense (Dinophyceae) are currently a serious problem from an economic and food hygiene point of view throughout the world. We isolated 13 polymorphic microsatellite loci from this species. These loci provided microsatellite markers with high polymorphism ranging from four to 15 alleles per locus and gene diversity between 0.632 and 0.974. The markers are available for more detailed investigations of genetic structure and gene flow of A. tamarense populations.     

长江口塔玛亚历山大藻孢囊的形成、发展及其与赤潮动力学的关系(英文)

The toxic dinoflagellate—Alexandrium tamarense (Lebour) Balech, formed resting cysts in f/2 media with low nitrate concentrations. Among the concentrations tested, f/20 NO3^- was the most effective to induction with an encystment percentage of 2.0 in batch culture. About 73.2% and 17.6% of cysts were produced on 8 and 9 d after transferring. Newly formed cysts developed accumulation body 3d later and kept forming mucilaginous substance, which might help their dispersal and survival. The mandatory dormancy period of resting cysts was 15 and 10d when stored at 15 and 20℃ respectively. The cysts could germinate without temperature change, with germination of 75.6% 20d after formation at 20℃. The Alexandrium cyst density in the surface sediment of DG-26 station reached above 25 cysts/g in May and November of 2002, and dropped to 4.5 and 0.9 cysts/g in August of 2002 and February of 2003, suggesting that Alexandrium cysts might have germinated in spring and autumn 2002. Cysts produced during the bloom returned to water column soon, whatever the season and water temperature were. The cyst density in the surface sediment at DG-26 station in May, 2003 was only 3.3 cysts/g and the cysts were newly formed. In the Yangtse River estuary, the inoculum size was not a major factor to determine the outbreak of A.tamarense bloom.     

长江口塔玛亚历山大藻孢囊的形成、发展及其与赤潮动力学的关系

有毒甲藻棗塔玛亚历山大藻(Alexandrium tamarense(Leboru)Balech)在低氮的F\2培养液中会形成休眠孢囊.在试验的递度中,f\20NO3-诱导效率最高,一次性培养中孢囊形成率达到2%.大约73.2%和17.6%的孢囊在接种后的第八天和第九天形成.新形成的孢囊3d后红色体开始出现,并持续地分泌粘性物质,这可能有助于孢囊的扩散和生存.孢囊在15和20℃保存下的休眠期分别为15和10d.孢囊需要温度的改变就能萌发,在20℃条件下孢囊密度分别降到了4.5和0.9个\g,说明2002年亚历山大大藻孢囊在春季和各有一次萌发.赤潮发生过程中产生的孢囊会很快通过萌发回到水体中,无论季节和水温如何.2003年5月DG-26站位表层沉积物中亚历山大藻孢囊密度只有3.3个\g,但这些孢囊均是新形成的.在长江口,种群初始的大小不是决定塔玛亚历山大藻赤潮发生的关键因素.     

Geographic differences in paralytic shellfish poisoning toxin profiles among Japanese populations of Alexandrium tamarense and A. catenella (Dinophyceae)

To reconsider whether toxin profile could be used as a marker for populations from different geographical areas, clonal isolates of the toxic dinoflagellates Alexandrium tamarense (Lebour) Balech and Alexandrium catenella (Whedon et Kofoid) Balech from Ofunato Bay (Iwate Prefecture), Atsumi Bay (Aichi Prefecture), Tanabe Bay (Wakayama Prefecture), Harima‐Nada (Kagawa Prefecture), Uranouchi Bay (Kochi Prefecture), Hiroshima Bay (Hiroshima Prefecture) and Yamakawa Bay (Kagoshima Prefecture), which were identified on the basis of morphotaxonomy, immunological and molecular biological techniques, were subjected to analysis of paralytic shellfish poisoning toxins by high performance liquid chromatography‐fluorometric method. All the isolates except A. tamarense OF152 from Ofunato Bay contained mainly N‐sulfocarbamoyl toxins (C1 +2) with various amounts of derivatives, and a typical north‐to‐south trend of decreasing toxicity was observed. In both A. tamarense and A. catenella, toxin profiles were rather constant within a geographical area and divergent among different geographical areas. The toxin profiles of A. tamarense from Harima‐Nada were well conserved among different bloom years. Toxin profile showed that isolates of A. tamarense from Ofunato Bay, A. tamarense from Harima‐Nada isolated in 1988 and A. catenella from Uranouchi Bay were heterogeneous. However, only two or three groups of isolates with different toxin profiles were observed in a geographical region, suggesting that several representative isolates express the genotype in a given region. These observations confirmed that toxin composition could be used as a marker to discriminate different geographical populations of these species.     

Comparative study of the life cycles of Alexandrium tamutum and Alexandrium minutum (Gonyaulacales,Dinophyceae) in culture1

The microalgal genus Alexandrium includes species known to produce paralytic shellfish poisoning (PSP). Due to the importance of discriminating between HAB‐forming species, we compared the undescribed life‐cycle pattern of Alexandrium tamutum Montresor, Beran et U. John and of its toxic relative Alexandrium minutum Halim. Sexual stages, asexual and sexual division, mating type, and nuclear morphology were studied in both species. Sexual cysts are known to be morphologically identical. However, the relative size of the U‐shaped nucleus may be used to differentiate between the cysts of these species since DNA packaging in the resting cysts was lower in A. tamutum than in A. minutum, species in which the planozygote nucleus was reduced to half its volume prior to encystment. The dormancy period of the cysts was <20 d for A. tamutum, but longer than 1 month for A. minutum. In both species, cyst appearance needed to be explained by the existence of more than two sexual types (+/–), which indicates a complex heterothallic mating type. However, planozygotes of both species may divide instead of encysting. This characteristic was used for nutritional and heritage studies. Isolated planozygotes of both species encysted in larger percentages in medium deficient in both nitrates and phosphates (L/15) than in medium without phosphates added (L‐P), a medium in which most planozygotes neither divide nor encyst. Parental strains of A. minutum with and without the ventral pore formed planozygotes and, later, offspring with the ventral pore, although apparently smaller than usual. A synchronization–flow cytometry method for discriminating diploids formed by sexual fusion (planozygotes) from cells with 2C DNA content resulting from self‐duplication of DNA (dividing cells) was described. The results indicated that the maximum percentage of A. minutum planozygotes (20%) was achieved only 3 to 5 d after crossing the parental strains, and that light might not be needed for the sexual fusion and formation of planozygotes.     

塔玛亚历山大藻和东海原甲藻对褶皱臂尾轮虫种群数量的影响

通过对2002年5月东海赤潮原因种一东海原甲藻和亚历山大藻的单一藻种和两种藻混合情况下对褶皱臂尾轮虫种群数量影响的实验研究,发现塔玛亚历山大藻(ATHK)对轮虫有致死作用,其48hLC50为1300cell·ml^-1．藻的各组分毒性比较研究表明,只有藻液和藻细胞具有这种毒害作用,藻在早期生长阶段的毒害作用较强,毒性大小与藻细胞活性相关．东海原甲藻在高密度(4×10^4、5×10^4、10×10^4cell·ml^-1)时对轮虫种群数量在第5d时开始有影响;东海原甲藻在低密度(1×10^4、2×10^4、3×10^4cell·ml^-1)时,轮虫能够以其为食并进行生长繁殖．两种藻混合情况下,东海原甲藻能够减轻塔玛亚历山大藻对轮虫的毒害作用．实验结果表明,此次赤潮对东海的微型浮游动物种群能够产生一定的影响．     

溶藻细菌BS03(Microbulbifer sp.)对塔玛亚历山大藻生长及抗氧化系统的影响

【目的】研究溶藻细菌BS03(Microbulbifer sp.)胁迫下塔玛亚历山大藻细胞光合作用、抗氧化酶系统和半胱氨酸蛋白酶3(Caspase-3)变化,探讨溶藻细菌BS03对塔玛亚历山大藻的溶藻机制。【方法】通过0.5%、1.0%、1.5%、2.0%不同终浓度BS03上清液处理藻细胞后12、24、36、48h取样,测定溶藻过程藻细胞光合色素、叶绿素荧光效率、抗氧化酶系统、Caspase酶活性变化。【结果】(1)BS03上清液处理藻细胞后,藻细胞叶绿素a含量和叶绿素荧光Fv/Fm比值随BS03上清液处理时间延长和浓度的增加呈逐渐下降趋势;低浓度处理组藻细胞类胡萝卜素含量上升到一峰值,高于对照组后逐渐回落,而高浓度处理组类胡萝素含量呈下降趋势,低于对照组;(2)藻细胞抗氧化酶保护系统(SOD和CAT)活性随着BS03上清液处理浓度增加而升高,但随着处理时间的延长呈现先上升后下降趋势。藻细胞膜脂过氧化产物MDA积累量随着BS03上清液处理时间延长和处理浓度的增加而显著提高;(3)处理组藻细胞Caspase-3活性显著高于对照组,呈现出类似程序性死亡特征。【结论】BS03的抑藻机理可能是通过抑制藻细胞光合作用,降低抗氧化酶活性、加大膜脂过氧化起到对塔玛亚历山大藻的溶解作用,并呈现出类程序性死亡特征。     

Purification and characterization of a novel high molecular weight exotoxin produced by red tide phytoplankton,Alexandrium tamarense

Our recent studies have demonstrated that the aqueous extract prepared from Alexandrium tamarense, a harmful red tide phytoplankton, showed cytotoxicity on Vero cells. In this study, the toxic substance was purified from the culture supernatant of A. tamarense. Based on the gel‐filtration profile, the molecular mass of a purified toxin was estimated to be about 1,000 kDa. On sodium dodecylsulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) analysis, a main band with molecular mass of 1,000 kDa was detected with periodic acid‐Schiff (PAS) staining, but no protein bands were detected by Coomassie brilliant blue (CBB) protein staining. Sugar composition analysis of the toxin suggested that the toxin contains galactose, fucose, mannose, N‐acetylglucosamine, xylose, and other minor saccharides, whereas no significant levels of amino acids were detected by amino acid analysis. These results suggest that the toxin is a polysaccharide‐based compound. The toxin showed cytotoxic effects on various cell lines in a concentration‐dependent manner. Among the cell lines tested, U937 cells were the most susceptible to the toxin. In U937 cells treated with the toxin, a typical apoptotic nuclear morphological change and DNA fragmentation were observed. This is the first report demonstrating that a polysaccharide‐based toxin isolated from red tide phytoplankton can induce apoptotic cell death. © 2008 Wiley Periodicals, Inc. J Biochem Mol Toxicol 22:405–415, 2008; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20253     

The application of a molecular clock based on molecular sequences and the fossil record to explain biogeographic distributions within the Alexandrium tamarense "species complex" (Dinophyceae)

The cosmopolitan dinoflagellate genus Alexandrium, and especially the A. tamarense species complex, contain both toxic and nontoxic strains. An understanding of their evolution and paleogeography is a necessary precursor to unraveling the development and spread of toxic forms. The inclusion of more strains into the existing phylogenetic trees of the Alexandrium tamarense species complex from large subunit rDNA sequences has confirmed that geographic distribution is consistent with the molecular clades but not with the three morphologically defined species that constitute the complex. In addition, a new clade has been discovered, representing Mediterranean nontoxic strains. The dinoflagellates fossil record was used to calibrate a molecular clock: key dates used in this calibration are the origins of the Peridiniales (estimated at 190 MYA), Gonyaulacaceae (180 MYA), and Ceratiaceae (145 MYA). Based on the data set analyzed, the origin of the genus Alexandrium was estimated to be around late Cretaceous (77 MYA), with its earliest possible origination in the mid Cretaceous (119 MYA). The A. tamarense species complex potentially diverged around the early Neogene (23 MYA), with a possible first appearance in the late Paleogene (45 MYA). A paleobiogeographic scenario for Alexandrium is based on (1) the calculated possible ages of origination for the genus and its constituent groups; (2) paleogeographic events determined by plate movements, changing ocean configurations and currents, as well as climatic fluctuations; and (3) the present geographic distribution of the various clades of the Alexandrium tamarense species complex.     

有害赤潮藻种塔玛亚历山大藻的研究进展

塔玛亚历山大藻(Alexandrium tamarense)是一种全球分布的有毒涡鞭毛藻,在中国的渤海、东海和南海海域均有分布。塔玛亚历山大藻是一种主要的有毒赤潮原因种,而且它能产生可经食物链传递后在贝类、鱼类等生物体内大量蓄积的麻痹性贝毒素,对水域环境和人类健康都具有极大的危害。因此,针对塔玛亚历山大藻的产生、发展以及毒素的特征等开展了广泛而深入的研究。本文主要从塔玛亚历山大藻的生长环境、分子鉴定、藻际竞争关系以及治理等方面进行了综述,为研究塔玛亚历山大藻的利弊和监控提供参考。     

ENVIRONMENTAL FACTORS CONTROLLING ALEXANDRIUM TAMARENSE (DINOPHYCEAE) GROWTH RATE DURING A RED TIDE EVENT IN THE ST. LAWRENCE ESTUARY (CANADA)1

The dinoflagellate Alexandrium tamarense (Lebour) Balech 1985 is responsible for recurrent outbreaks of paralytic shellfish poisoning in the St. Lawrence Estuary. In July 1998, an A. tamarense red tide developed in the estuary with maximum cell concentrations reaching 2.3 × 10⁶ cells·L^?1 in brackish surface waters. To estimate the growth rate of these cells, surface water samples from different locations and days during the bloom were incubated for 5 to 9 days under in situ temperature and light conditions. Growth rates varied both spatially and temporally between 0 and 0.55 day^?1, reaching the maximum growth rate reported for this species in culture. High growth rates were measured even during the peak of the red tide, suggesting that the extremely high cell concentrations observed did not solely result from aggregation or physical concentration but also involved active cellular growth. Alexandrium tamarense cells were found over a large range of salinity (20.8–29.5 psu), but high densities and significant growth were only measured when salinity was lower than 24.5 psu. Under these conditions, the number of divisions achieved by A. tamarense was proportional to the amount of nitrate available at the beginning of the incubations, whereas variations in growth rate were apparently controlled by the availability of phosphate. We hypothesize that the ability of A. tamarense to perform vertical migrations and acquire nitrate at night pushes this species toward phosphate limitation in the St. Lawrence Estuary.     

塔玛亚历山大藻对黑褐新糠虾存活、生长以及种群繁殖的影响

通过塔玛亚历山大藻 ( Alexandrium tamarense)对黑褐新糠虾 ( N eomysis awatschensis)的急性和慢性毒性作用研究 ,发现塔玛亚历山大藻对黑褐新糠虾的存活、生殖、生长等有不利影响 ,影响程度随塔玛亚历山大藻藻细胞密度的增加而增加。在 96 h急性毒性实验中 ,塔玛亚历山大藻对黑褐新糠虾的半致死密度为 70 0 0 cells/ml,去藻过滤液中糠虾的死亡率为 2 5 %。在 6 2 d的慢性毒性实验中 ,密度为 90 0 cells/ml的塔玛亚历山大藻对黑褐新糠虾的繁殖有严重影响 ,在此影响下的实验组亲虾产幼虾总数只有 2 7尾 ,仅为对照组产幼虾数目的 1 6 .4 % ;其总产幼虾天数、日最高产幼数分别只有对照的 32 %、4 1 % ,其初次产虾日期也推迟了 3d,并出现了 3次生殖中断。塔玛亚历山大藻对黑褐新糠虾亲虾的存活、生长也有一定的影响 ,处在密度为 90 0 cells/ml塔玛亚历山大藻中的黑褐新糠虾亲虾的存活率只有对照的 6 3% ,糠虾亲虾的体长和体重分别为对照组亲虾的 95 .6 %和 81 .9% ,但差异尚不显著 ( P>0 .0 5 )     

Morphology, phylogenetic position, and ecophysiology of Alexandrium ostenfeldii (Dinophyceae) from the Bohai Sea, China

Alexandrium ostenfeldii is a potentially toxic dinoflagellate that often occurs in coastal areas at high latitudes.Here we report the presence of A.ostenfeldii in the Bohai Sea,China,for the first time.The vegetative cells ofA.ostenfeldii are characterized by a narrow first apical plate and a large ventral pore located on the anterior right side.Partial large subunit sequence comparison revealed that the Chinese strain differs from the Finnish strains at only three positions,and from A.peruvianum of Spain at five positions.Maximum parsimony analysis revealed that A.ostenfeldii from China and Finland and A.peruvianum from Spain grouped together.They were the nearest sister group to a clade with A.ostenfeldii from New Zealand,Europe,and North America.In culture,growth did not occur at temperatures below 9 ℃ and occurred at salinities between 7 and 27 psu.It took 10-20 days for newly formed cysts to mature at 20 ℃.Lower temperature delayed germination,but the germination rate exceeded 90％ at temperatures from 12 to 24 ℃.No germination occurred below 9 ℃ after 1 month of incubation.The Chinese strain ofA.ostenfeldii produced neither spirolides nor paralytic shellfish poisoning toxins.     

Morphology,phylogenetic position and ecophysiology of Alexandrium ostenfeldii (Dinophyceae) from the Bohai Sea,China

Alexandrium ostenfeldii is a potentially toxic dinoflagellate that often occurs in coastal areas at high latitudes. Here we report the presence of A. ostenfeldii in the Bohai Sea, China, for the first time. The vegetative cells of A. ostenfeldii are characterized by a narrow first apical plate and a large ventral pore located on the anterior right side. Partial large subunit sequence comparison revealed that the Chinese strain differs from the Finnish strains at only three positions, and from A. peruvianum of Spain at five positions. Maximum parsimony analysis revealed that A. ostenfeldii from China and Finland and A. peruvianum from Spain grouped together. They were the nearest sister group to a clade with A. ostenfeldii from New Zealand, Europe, and North America. In culture, growth did not occur at temperatures below 9 °C and occurred at salinities between 7 and 27 psu. It took 10–20 days for newly formed cysts to mature at 20 °C. Lower temperature delayed germination, but the germination rate exceeded 90% at temperatures from 12 to 24 °C. No germination occurred below 9 °C after 1 month of incubation. The Chinese strain of A. ostenfeldii produced neither spirolides nor paralytic shellfish poisoning toxins.