产麻痹性贝毒塔玛亚历山大藻共培养菌群的物种多样性研究

产毒赤潮藻与其共生菌的互作关系是揭示麻痹性贝毒(PSP)产生机制的关键,而其共培养菌群的分离及其多样性解析是开展此研究的必要前提。塔玛亚历山大藻(Alexandrium tamarense)是典型产PSP赤潮原因甲藻,为揭示其共培养菌群的物种多样性信息,本文通过免培养海洋微生物高通量测序技术,解析了东海塔玛亚历山大藻(at3#)共培养菌群的种类、丰度及多样性信息。结果表明,其共培养菌群共有87个种与已有数据库匹配,此外尚有5%的未知种。其包含57个属,其中优势菌属5个,分别为Rhodobacteraceae、Marinobacter、Methylophaga、Nitratireductor及Phycisphaera。研究表明东海产PSP塔玛亚历山大藻(at3#)含有物种丰富的共培养菌群,且具有发掘海洋特殊生境微生物新种属的良好潜力。     

塔玛亚历山大藻产麻痹性贝类毒素能力的研究

为探究塔玛亚历山大藻(Alexandrium tamarense)产麻痹性贝类毒素(paralytic shellfish poisoning,PSP)的能力,通过设置不同空间效应、盐度和营养素浓度对塔玛亚历山大藻进行培养,分析不同培养条件下塔玛亚历山大藻产麻痹性贝类毒素的能力.结果表明:1)塔玛亚历山大藻中至少含有1...     

不同温度对微小亚历山大藻生长和藻毒力的影响

<正>水体中的藻类是麻痹性贝毒的主要来源,现已知海洋中有13种单细胞的甲藻类可产生麻痹性贝毒。目前在我国海区已经发现的产毒甲藻主要有3种:塔玛亚历山大藻(Akexandrium tamarense)、微小亚历山大藻(Alezandrium minudum)、链状亚历山大藻(Alexandrium acatenella)。微小亚历山大藻在我国台湾沿海较常见。因此,有必要针对我国沿海的微小     

3种滤食性贝类对塔玛亚历山大藻的摄食研究

20 0 3年 4月在实验室内采用静水法 ,研究了长牡蛎、紫贻贝、栉孔扇贝对塔玛亚历山大藻的清滤率 (CR)和摄食率 (IR) ,实验时间持续 3d ,水温 17℃。实验中每 1种贝都分别投喂 :(1)叉鞭金藻 (对照藻 ) ,(2 )叉鞭金藻 +塔玛亚历山大藻去藻过滤液 1.5L ,(3)叉鞭金藻 +塔玛亚历山大藻细胞。结果表明 ,塔玛亚历山大藻过滤液 ,对 3种贝的清滤率的无明显影响 (P >0 .0 5 ) ;3种贝对塔玛亚历山大藻和叉鞭金藻混合液的清滤率与对照组相比明显降低 (P <0 .0 1) ;而对塔玛亚历山大藻的摄食率和保留率 3d间无显著变化 ;实验期间 ,随时间的变化 ,牡蛎对混合液的清滤率变化最大 ,贻贝次之 ,扇贝则无明显著变化 (P >0 .0 5 ) ;如果以摄食率作为指标 ,贻贝在控制赤潮方面的潜力较大 ,值得今后进一步研究。     

环境因子对微小亚历山大藻Amtk-9生长与产毒的综合影响

微小亚历山大藻是产生麻痹性贝毒的主要生物之一。产毒藻株在不同培养条件下所产生的毒素总量有差异,因此研究环境因子对微小亚历山大藻生长与产毒的综合影响,找到微小亚历山大藻生长和产毒的适宜条件,对麻痹性贝毒在赤潮中是否造成贝类蓄毒有十分重要意义。本文设计了一个四因素三水平的正交实验,研究温度、盐度、营养盐、光照强度等环境因子对微小亚历山大藻Amtk-9生长与产毒的综合影响。结果表明:在实验范围内,微小亚历山大藻Amtk-9生长最佳条件为温度25℃,盐度30,培养基中氮含量为基础f/2培养液配方中氮磷含量的2倍,光照强度10000 lx。最佳产毒条件除了氮磷含量为基础f/2培养液配方中氮磷含量的3倍外,其它条件与生长的最佳条件相同。盐度、营养盐、光照这三个因素的不同水平对微小亚历山大藻Amtk-9指数生长期细胞比增长率、指数生长末期单位细胞含毒量均有极显著影响(F>F0.01)。温度对微小亚历山大藻Amtk-9的生长和产毒则无显著影响(F相似文献   

塔玛亚历山大藻对中国明对虾肝胰腺和鳃SOD、GST和MDA的影响

本文选择在我国分离得到的一株能典型产生麻痹性贝毒(PSP,Paralytic Shellfish Poison)的有毒赤潮甲藻塔玛亚历山大藻(Alexandrium tamarense,ATHK株),研究其是否能通过引发中国明对虾的脂质过氧化作用, 使机体产生过多活性氧自由基, 导致氧化损伤而发挥其毒性作用。塔玛亚历山大藻粗提液经肌肉注射方式染毒中国明对虾,于染毒后1、3、6、12、24和48 h 测定肝胰腺和鳃SOD,GST活力和MDA含量的影响。结果显示,染毒后 1-6 h, 对虾肝胰腺和鳃组织SOD,GST酶活力均增加,12和48h鳃组织的上述指标受到抑制。对虾肝胰腺MDA含量除1h外未见明显改变,鳃中MDA含量随时间增加呈升高趋势。研究表明：塔玛亚历山大藻粗提液在中国明对虾体内代谢过程中能诱导对虾产生活性氧自由基,塔玛亚历山大藻粗提液能诱导MDA含量增加,降低SOD和GST活力,能够引发鳃的脂质过氧化,对鳃造成氧化损伤。     

细胞凝集素探针对典型赤潮生物的定量检测

采用14种异硫氰酸荧光素（FITC）标记的细胞凝集素探针对23株典型的有害赤潮生物进行了检测,结合流式细胞仪、荧光显微镜、荧光分光光度计对探针标记区分和识别目标藻的特异性和有效性进行定性和定量测定。结果表明,DBA可以把裸甲藻（Gymnodinium）GIXM01株与其他裸甲藻区分开：SBA、WGA和PNA探针能定性或定量地区分形态相似的裸甲藻GMDH01和TPXM01。PHA-E和SJA能分别将塔玛亚历山大藻（Alexandrium tamarense）ATDH04、ATMJ02株系与同种其他的藻株区分开：PNA也能从同种其他株系中识别塔玛亚历山大藻ATDH01,ATDH02,ATDH03株系：UEAⅠ能从同种藻株中识别塔玛亚历山大藻ATCI01．JN、ATCI01藻株;RCA120可以区分产毒水平不同的亚历山大藻属（Alexandrium）的ASPGX01和ASPGX02。DBA、SBA、PNA和LCA探针能定性和定量区分微小原甲藻（Prorocentrum minimum）PMDH01和PMXM01;而PNA探针仅能定量区分原甲藻属（Prorocentrum）PDDH01与PMXM01。用WGA探针结合流式细胞仪、荧光显微镜、荧光分光光度等检测手段可以把探针标记的目标裸甲藻GSPXM01和TPXM01从米氏凯伦藻（Karenia mikimotoi）、东海原甲藻（P.donghaiense）和微小原甲藻（P.minimum）等组成的混合样品中区分开。[中国水产科学,2008,15（2）：279-292]     

4种海洋微藻酸性磷酸酶的分离纯化及其性质的研究

提取、纯化了4种海洋徽藻的酸性磷酸酶,确定了酸性磷酸酶的条件并研究其性质.结果显示,在37℃条件下,塔玛亚历山大藻酸性磷酸酶回收得率低,但酶活最高;确定各徽藻酶促反应初速度均恒定的时间为15 min;三角褐指藻、塔胞藻、塔玛亚历山大藻、海链藻的酸性磷酸酶分别在酶浓度为0.96、0.29、0.25、14.9 mg/mL酶促反应速度达最高;三角褐指藻、塔胞藻、海链藻的酸性磷酸酶最适pH值为3,而塔玛亚历山大藻为4;37℃下各藻产生的酸性磷酸酶米氏常数为:塔玛亚历山大藻＜塔胞藻＜三角褐指藻＜海链藻.试验证实,塔玛亚历山大藻在磷源限制条件下,较其他3种海洋徽藻更易于利用有机溶解磷;塔玛亚历山大藻在利用海洋微藻生产酸性磷酸酶方面极具应用前景.     

营养环境对微小亚历山大藻C4生长和产毒的影响

为考察营养成分以及培养基础液再利用对一株微小亚历山大藻的生长和产毒的影响,采用单因素实验分别研究了氮(NaNO_3)、磷(NaH_2PO_4)、微量元素(FeCl_3、Na2EDTA、CuSO_4、Na_2MoO_4、ZnSO_4、CoCl_2、MnCl_2)、维生素(Vitamin B12、Vitamin H、Vitamin B1)、碳(NaHCO_3)的不同含量以及海水(培养基础液)利用方式(不循环与循环利用)对微小亚历山大藻C4生长、毒素含量(μmol/L)与毒素组成的影响。结果显示,氮、磷浓度对C4藻的毒素总含量(μmol/L)有显著影响,其他因素影响均不显著;氮、磷、碳及微量元素的浓度对GTX1/4(GTX1+GTX4)在总毒素(GTX1+GTX2+GTX3+GTX4)中所占比例均影响显著,而维生素浓度和海水循环利用对GTX1/4所占比例影响均不明显。氮浓度在0~883μmol/L范围内,毒素含量与氮浓度呈正相关关系,氮浓度进一步增加,毒素含量没有明显变化;磷浓度在0~145.2μmol/L范围内增加,毒素含量先增后降,最终保持稳定的趋势。最佳的产毒条件为氮883μmol/L,磷18.15μmol/L,微量元素为f/2海水培养基中微量元素的0.5倍,碳不添加。     

不同氮磷比对海洋赤潮藻碳、氮稳定同位素组成的影响

为查明营养盐对不同赤潮藻不同生长阶段的影响,通过测定不同营养条件下培养的7种赤潮藻δ13 C和δ15 N值,比较不同生长阶段、不同藻种之间稳定同位素组成的差别。结果显示,7种赤潮藻δ13 C和δ15 N值存在一定的差异,海洋原甲藻具有较高的δ13 C值;海洋球石藻δ13 C值相对较小;新月菱形藻、威氏海链藻和塔玛亚历山大藻具有相似的δ13 C值。强壮前沟藻、海洋原甲藻和塔玛亚历山大藻δ15 N值要高于其他赤潮藻,威氏海链藻、海洋球石藻、中肋骨条藻和新月菱形藻具有相似的δ15 N值。塔玛亚历山大藻在NP、3N、1/3P和3P组别指数期δ13 C值要低于稳定期,其他6种赤潮藻指数期δ13 C值要高于稳定期。几种赤潮藻δ13 C值与NO3-和PO3-4浓度普遍呈显著正相关;δ15 N值与NO3-浓度普遍呈显著负相关,与PO3-4浓度普遍呈显著正相关。     

Utilization of dissolved organic phosphorus by the two toxic dinoflagellates, Alexandrium tamarense and Gymnodinium catenatum (Dinophyceae)

ABSTRACT: The utilization of dissolved organic phosphorus (DOP) by the two toxic dinoflagellates Alexandrium tamarense (Lebour) Balech and Gymnodinium catenatum Grahamm which were isolated from Hiroshima Bay, Japan, was studied. Alexandrium tamarense grew poorly on fructose-6-phophate, glucose-1-phosphate, glycerophosphate, and ribose-5-phosphate with a phosphomonoester bond, although it grew well on the nucleotides adenosine-5-diphosphate (ADP) and adenosine triphosphate (ATP), as well as on dissolved inorganic phosphorus (DIP; as metaphosphate, pyrophosphate, tripolyposphate and orthophosphate). The results imply that A. tamarense was able to utilize DOP and DIP from ambient water using nucleotidase, pyrophosphatase and poly-phosphatase, which hydrolyze phosphodiesters. In contrast, G. catenatum was able to utilize DOP compounds of various molecular weights and structures as well as DIP. In time-course experiments, alkaline phosphatase activity (APA) was induced at orthophosphate concentrations of 0.43 mmol/L and 3.3 mmol/L for A. tamarense and G. catenatum , respectively, and APA increased with orthophosphate depletion. The experiments also demonstrated that APA was maximum at the optimum temperatures for the growth of A. tamarense and G. catenatum ; that is, 15°C and 25°C, respectively. These results suggest that the DIP-depleted conditions in Hiroshima Bay might have led to the outbreaks of noxious dinoflagellates in recent years.     

塔玛亚历山大藻毒素及其对中国对虾的急性毒性

利用美国分析化学家协会(Association of Official Analytical Chemists, AOAC)所推荐的小鼠生物法(mouse bioassay, MBA), 测定了塔玛亚历山大藻(Alexandrium tamarense)(ATHK藻株)毒素粗提液的麻痹性贝毒(paralytic shellfish poisoning, PSP)毒性;采用浸浴方式, 研究了该藻株对中国对虾的急性毒性; 采用HE染色方法, 分别对有毒藻浸浴处理和毒素粗提液注射处理后96 h中国对虾(Fenneropenaeus chinensis)的鳃和肝胰腺石蜡切片进行观察。结果表明, 该藻株麻痹性贝毒毒性为3.9×10^–5 MU/cell (相当于7.3 pg STX Equal/cell), 在同种藻株中属低毒藻株; 在96 h急性毒性实验中, 塔玛亚历山大藻对中国对虾的半致死浓度(LC₅₀)为1.0×10⁴ cells/mL, 安全浓度(SC)为1.0×10³ cells/mL; 石蜡切片观察发现, 塔玛亚历山大藻与毒素粗提液分别引起了鳃和肝胰腺组织出现细胞肿胀、空泡化等病理变化。以上研究结果提示, 塔玛亚历山大藻对中国对虾有急性致死作用。为了保证中国对虾的健康养殖, 养殖水体中的塔玛亚历山大藻浓度至少应该控制在1.0×10³ cells/mL以下; 鳃的病变直接导致对虾窒息死亡可能是塔玛亚历山大藻暴露后对虾急性致死作用的最重要原因;塔玛亚历山大藻所产的PSP毒素可引起虾体代谢和解毒的主要器官—肝胰腺的病变。     

Development of a quantification assay for the cysts of the toxic dinoflagellate <Emphasis Type="Italic">Alexandrium tamarense</Emphasis> using real-time polymerase chain reaction

ABSTRACT:   The cysts of toxic dinoflagellate Alexandrium tamarense are the seed population for the bloom responsible for paralytic shellfish poisoning (PSP). However, it is impossible to identify the Alexandrium spp. cyst on the basis of morphological features. In this study, we prepared A. tamarense cysts by sexual conjugation in laboratory conditions and developed an efficient DNA extraction method for polymerase chain reaction (PCR) assay. Using the A. tamarense cysts, we established the identification and quantification method showing the species specificity and the high sensistivity for A. tamarense cysts using real-time PCR. This assay was also able to detect and quantify the A. tamarense cysts accurately when mixed with excess cysts of A. catenella (Whedon and Kofoid) Balech prepared by conjugation experiment.     

塔玛亚历山大藻对海湾扇贝胚胎发育过程的影响

研究了塔玛亚历山大藻（Alexandrium tamarense）ATHK株对海湾扇贝受精卵孵化的影响,并探讨了可能的影响机制。将海湾扇贝受精卵暴露于不同密度的塔玛亚历山大藻ATHK中,观察其对胚胎孵化各阶段的影响,并利用光镜和透射电镜对实验组和对照组胚胎的外部形态和内部结构进行连续观察。结果表明：该藻对海湾扇贝受精卵孵化有显著的抑制作用,其IC50约为800 mL-1;2000 mL-1的ATHK延缓或破坏了胚胎的正常发育,胚胎内部出现大量溶酶体,原肠胚腔不能形成,且部分细胞出现胞质肿胀、线粒体自溶的现象,细胞不能进行正常的功能性分化;观察发现实验组中有些担轮幼虫的外形畸变,鞭毛发育迟缓;至24 h（D型幼虫）时,ATHK组中的D型幼虫数量远远低于对照组（P<0.05）,且死亡率明显高于对照组。观察还发现ATHK藻细胞和胚胎碰撞接触的现象,这种碰撞接触可能对有害物质的释放和转移有一定的刺激作用。     

Paralytic shellfish poisoning toxin analysis of the genus <Emphasis Type="Italic">Alexandrium</Emphasis> (Dinophyceae) occurring in Korean coastal waters

ABSTRACT:   Paralytic shellfish poisoning (PSP) toxins produced by Alexandrium isolates from Korea were analyzed by high-pressure liquid chromography. Species designation of the regional isolates was determined by morphological criteria and ribotyping inferred from sequences of the 28S rDNA D1-D2 region. Toxin analysis performed at the exponential growth phase, revealed that the two strains of A. fraterculus were non-toxic, while the strains of A. tamarense and A. catenella were toxic. Toxic isolates DPC7 and DPC8 of A. catenella produced GTX1, 2, 3, 4, 5, dcGTX2, 3, C1, 2, neoSTX and STX with trace or non-detectable levels of C3 and C4, while isolates UL7, KDW981, SJW97043, SJW97046, KJC97111 and KJC97112 of A. tamarense produced GTX1, 2, 3, 4, dcGTX3, C1, 2, neoSTX with trace or non-detectable levels of C3, 4, dcSTX and STX, and no GTX5 and dcGTX2. The major toxins produced by A. catenella were C1 +2, and those of A. tamarense were C1 +2 and GTX4 in most of the isolates. A. tamarense strains other than SJW97046 produced a relatively high proportion of carbamate toxins, reflecting the high toxicity scores of shellfish intoxication in sampled coastal areas. Two representative toxic isolates, A. tamarense SJW97043 and A. catenella DPC7, were cultured for 30 days in batch mode and subjected to toxin analysis at 5-day intervals. Comparison of toxin productivity in terms of total toxin content, toxin components, and their variations with culture age revealed marked differences between the two strains.     

Distribution and abundance of resting cysts of the toxic Alexandrium spp. (Dinophyceae) in sediments of the western Seto Inland Sea, Japan

Sediment samples were collected from 135 stations in the western part of the Seto Inland Sea (Iyo Nada, Suo Nada, Beppu Bay, Tokuyama Bay, Hiroshima Bay, Aki Nada, Hiuchi Nada and Bingo Nada) to determine the horizontal distribution and abundance of resting cysts of Alexandrium spp. ( A. tamarense  +  A. catenella ). Enumeration of the cysts was performed using the primuline-staining direct count method. Cysts of Alexandrium spp. were rarely found in Iyo Nada, Suo Nada and Beppu Bay, but were widely distributed in Tokuyama Bay, Hiroshima Bay, Aki Nada, Hiuchi Nada and Bingo Nada. Cyst concentrations ranged from not detected (ND) to 14, ND to 17, ND to 4, 93 to 8137, 8 to 4454, ND to 6, ND to 18 and 4–29 cysts/cm³ wet sediment in Iyo Nada, Suo Nada, Beppu Bay, Tokuyama Bay, Hiroshima Bay, Aki Nada, Hiuchi Nada and Bingo Nada, respectively. The majority of cysts occurred in Tokuyama Bay and Hiroshima Bay, where higher densities were observed in the inner bay and along the coastal margin. Relatively higher cyst concentrations were observed at stations with a higher mud content. The abundance of Alexandrium spp. cysts in western Seto Inland Sea is lower than in the eastern Seto Inland Sea, except for Tokuyama Bay and Hiroshima Bay. However, because sporadic blooms of Alexandrium have been observed, continuing monitoring is necessary to prevent paralytic shellfish poisoning outbreaks in this area.     

淡水环境下3种红树植物对氮磷的去除效应应

2010年7 ~ 12月,通过水培盆栽试验研究了桐花（Aegiceras corniculatum）、海莲（Bruguiera sexangula）和无瓣海桑（Sonneratia caseolaris）对生活污水的净化效果,设置了3个浓度梯度,即N1P1（TN 为3.96 mg• L-1,TP为 0.47 mg• L-1）,N2P2（TN为 19.8 mg•L-1,TP 为2.35 mg•L-1）和N3P3（TN为 39.6 mg•L-1,TP为 4.7 mg•L-1）。研究结果表明：随着水培时间的增加,水体盐度、总氮和总磷含量显著下降（P <0.001）。桐花、海莲和无瓣海桑对生活污水总氮去除率达74.5% ~ 97.4%,总磷去除率达72.3% ~ 95.7%。栽培有红树植物的系统对氮磷的去除率显著高于无植物系统,分别为无植物的1.28倍 ~ 3.2倍和1.24倍 ~ 2.71倍。红树植物氮磷质量分数分别为6.81 mg•g-1±0.85 mg•g-1 和 1.41 mg•g-1±0.46 mg•g-1。当污水浓度低时（N1P1）,红树植物主要是通过累积作用去除水体氮磷,占总去除效应的42.2% ~ 63.3%和46.1% ~ 85.8%。当污水浓度高时（N3P3）,红树植物氮磷累积量仅占4.5% ~ 10.4%和6.7% ~ 14.9%。