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题名: 滇池与洱海的鱼腥藻及其生态生理与毒理学研究
作者: 潘晓洁
答辩日期: 2008-06-18
导师: 刘永定
授予单位: 中国科学院水生生物研究所
授予地点: 水生生物研究所
学位: 博士
关键词: 水华 ; 鱼腥藻 ; 浮游植物种群演替 ; 典型对应分析 ; 系统进化 ; 生长生理特性 ; 毒理
其他题名: Studies on the Species Composition, Ecophysiology and Toxicology of Anabaena spp. in Lakes Dianchi and Erhai
摘要: 鱼腥藻(Anabaena)中有多个物种是淡水水华蓝藻常见的优势种,其广泛分布在我国湖泊、河流、水库、池塘中,且鱼腥藻的某些种类可产生毒素、释放异味物质,对环境和人畜健康造成很大危害,但国内有关鱼腥藻水华及水体鱼腥藻的研究报道并不是很多。鱼腥藻属是滇池和洱海蓝藻水华中常见的组成种类,但目前还没有对其系统分析的研究报道,故无法正确评价两湖泊鱼腥藻的分布现状及危害。本研究以滇池和洱海两高原湖泊为背景,对其中的鱼腥藻进行了分离和鉴定,系统研究了两高原湖泊鱼腥藻的生态生理及毒理特征,结果如下: 1)调查了滇池2002和2003年浮游植物和理化指标,分析了滇池鱼腥藻分布现状,探讨了影响滇池各浮游植物种群生长的环境因子。研究结果表明滇池水体富营养化严重,大多数月份微囊藻占浮游植物总生物量的90%以上,为水体绝对优势种;冬、春季节鱼腥藻生物量有所增加,数量可与微囊藻相当,约占浮游植物总量的30%左右;束丝藻和鱼腥藻与水体透明度显著相关,冬春季节水体透明度较高,束丝藻和鱼腥藻生物量达到峰值,而此时相对其他微囊藻水华暴发月份水体叶绿素含量稍低。 2)调查了洱海2006年6-10月蓝藻水华大量暴发季节各理化因子及浮游植物生物量的时空分布,定性定量分析了浮游植物种群,进一步分析了洱海藻类水华暴发季节水质营养水平及浮游植物多样性,并探讨了洱海浮游植物种群演替的环境机制。研究结果表明,洱海蓝藻水华大量暴发的6-10月份,蓝藻数量高达107 cells/L,水华种类为鱼腥藻(Anabaena)和微囊藻(Microcystis)两属的一些种。两属的种类交替大量生长形成水华,洱海浮游植物种群存在明显的演替。洱海水质近些年有所下降,分析6-10月洱海营养水平,其中8月营养水平最高,此时浮游植物生物量最大(以叶绿素 a表示)。典型对应(CCA)分析了鱼腥藻和微囊藻生物量同各理化因子的关系,结果显示总磷和温度可能是驱动鱼腥藻水华和微囊藻水华转换的关键环境因子。 3) 进一步分析了洱海浮游植物和水质的周年变化。综合营养状态指数分析显示处于中营养状态的洱海,已处于十分敏感的富营养化转型的关键期;不同月份营养状态略有不同,表现为夏秋季节高,冬春季节低的特点。就单个营养指标而言,总氮是洱海主要的超标污染物。洱海浮游植物群落具有明显的季节演替规律:1~3月份浮游植物群落以硅藻为优势种,5~6月份以束丝藻为主要优势种,7~8月份以鱼腥藻为主要优势种, 9~11月份以微囊藻为主要水华优势种。统计分析表明总氮、总磷、温度是影响洱海藻类大量生长的主要环境因子,其中总磷和温度在周年浮游植物种群演变中起着关键作用。 4)在滇池和洱海鱼腥藻水华发生时,分离、纯化主要鱼腥藻藻株,选取代表性的七株鱼腥藻依据形态特征并结合16S rRNA基因序列分析进行了鉴定。依据孢子的形态和藻丝的宽度可将这七个藻株分成三组;观察16S rRNA基因序列形成的邻接树,这七株鱼腥藻分布在三个不同的亚群里,并且系统进化分析结果与形态鉴定一致。藻株EH-2、EH-3和EH-4在同一组内,系统进化关系与Anabaena circinalis 及 Anabaena crassa株系最接近。藻株DC-1、DC-2 和 EH-1组成另一株系,依据形态学特征和16S rRNA基因序列分析,可鉴定为Anabaena flos-aquae。藻株DC-3 与其他六株藻株不同,为Anabaena cylindrica。 5)通过研究生长状况、色素组成及光合效率,分析比较了所分离获得的鱼腥藻不同藻株间在相同实验室培养条件下的生长差异。研究结果表明,培养期间各藻株生长状态良好,均保持了较高的光合效率,光合色素含量与生长状况明显相关,而生长状况与藻类种类有关;在实验室培养条件下,各藻株的生长状况与在自然水体中明显不同,这可能与培养条件的差异以及藻类本身的生物学特性有关;结果证明,在光合色素含量相对较低时,为保证藻株正常生长,光系统Ⅱ(PSⅡ)的最大光量子产量(Yield)、最大相对电子传递速率(rETRmax)和P-I曲线初始斜率(Alpha)相对增大,补偿光合作用。 6)对洱海卷曲鱼腥藻生长生理特性进行了初步研究,以期为探讨洱海鱼腥藻水华发生的环境影响因素提供基础的参考资料。实验结果表明,氮含量1.5 mmol/L、磷含量12 µmol/L、光照强度30 µE/ m﹒s、pH值8-10及温度25℃时,卷曲鱼腥藻生长状况最好,生物量及相对生长速率较高。不同氮、磷浓度下的氮磷代谢活性表明:氮浓度在0-0.36mmol/L时,硝酸还原酶活性随着氮浓度的增加而增强;氮浓度在0.36-6 mmol/L时,酶活性由其生长状况决定,生长越好,酶活性越高。碱性磷酸酶受磷影响较大,随着磷浓度的增加其活性逐渐减弱,磷充足时,氮对其活性并无显著影响。此外,洱海卷曲鱼腥藻可在低的氮、磷浓度下生长,这与其氮磷代谢活性的调节作用有关。 7)制备分离藻株细胞注射液和细胞抽提液,腹腔注射小白鼠,观察小白鼠中毒症状,并在其死亡时间或24小时后解剖分离脏器,观察组织病理变化,探讨不同藻株的毒性及差异,分析细胞壁脂多糖毒素对细胞内毒素的毒性作用。结果表明,七株藻株皆为有毒藻株,但毒性大小不同;细胞壁脂多糖类毒素可增强细胞内毒素的作用。 8)通过超微结构观察、组织切片病理检查和血清生化指标分析,进一步研究了滇池水华鱼腥藻对小白鼠的毒性影响。研究结果表明滇池水华鱼腥藻粗毒素提取液可引起小鼠肝、肾、肺、脾、肠、心等组织超微结构的明显改变;组织切片病理检查发现,小鼠肝、肾和肺等组织严重损伤,出现出血、空泡、细胞坏死等损害,但这些损害在不同组织中程度不同;血清肝功能指标、肾功能指标以及反映机体免疫功能的指标发生明显变化。通过组织病理学观察和生物化学分析进一步证实滇池水华鱼腥藻是有毒的,对哺乳动物存在潜在的危害,滇池鱼腥藻水华的发生和危害需得到重视。
英文摘要: Cyanobacterial blooms are a major worldwide environmental problem. Among the bloom-forming cyanobacteria, Anabaena is one of the common planktonic genera in freshwater bodies. Like many other cyanobacteria, a number of Anabaena species can produce toxins or release odors into drinking and recreational waters. Thus, the surface blooms of Anabaena pose potential risks to aquatic environments and human health. In China, Anabaena is one of the most prevalent planktonic cyanobacterial genera, and it is frequently found in lakes Dianchi and Erhai. But few researches on blooming Anabaena and its toxicity in China has been reported. In the present study, the species composition, ecophysiology and toxicology of Anabaena spp. in lakes Dianchi and Erhai were investigated. The main objective of this study was to elucidate the distribution of Anabaena species and the mechanism of phytoplankton succession, to detect the toxin and to evaluate the toxic effects of Anabaena on mammal. The main results are summarized as follows: 1) Composition of phytoplankton and distribution of physicochemical factors in Lake Dianchi in years 2002 and 2003 were investigated to present the existence of Anabaena and analyze the correlation of phytoplankton species with the environmental factors. Results showed that Lake Dianchi has been in a hypereutrophic status; Microcystis is the dominant species in most months of the year, and its population dominance held more than 90%; Anabaena occurred heavily in winter and spring, and its population dominance can account for about 30%; SD was higher and Chla was lower during the occurrence of Anabaena or Aphanizomenon bloom in the cooling season. 2) Field studies were conducted in Lake Erhai from June to October of the year 2006. The investigation on phytoplankton composition showed that the cyanobacterial blooms occurred in the whole investigated period, phytoplankton abundance reached to 107 cells/L and more, and dominant species of cyanobacterial blooms displayed an obvious succession from Anabaena to Microcystis. Analysis of water quality showed that Lake Erhai remained at a mesotrophic status presently, but it was experiencing a transition from the mesotrophic to eutrophic condition; the trophic level was higher in August with lower phytoplankton diversity, and the biomass of phytoplankton (Chl a) was the most in this month. In addition, it was suggested that TP and T were the important factors to drive the succession between Anabaena bloom and Microcystis bloom by using CCA analysis. 3) Annual variations of phytoplankton composition and water quality in Lake Erhai were investigated. Results showed that the trophic status of Lake Erhai was probably transferring from mesotrophic to eutrophic, and the trophic levels were higher in the summer and autumn than in the winter and spring. TN in higher level was the primary pollution. Obvious process of phytoplankton succession was, with the dominant species one after the other as the followings: Diatom from January to March; Aphanizomenon in May and June; Anabaena in July and August; Microcystis from September to November. Correlative analysis showed that TN, TP and T were the important factors in changing the phytoplankton abundance, and TP and T were more important for phytoplankton succession. 4) Morphological characteristics and phylogenetic relationships of seven Anabaena strains isolated and purified from the two plateau lakes Dianchi and Erhai were investigated. Phylogenetic relationships were determined with the 16S rRNA gene tree which was calculated and constructed by using the neighbour joining algorithm. The seven strains were reasonably divided into three groups with the morphology of akinetes and the width of trichomes. The 16S rRNA gene tree also resulted in three groups of the seven strains, and the phylogenetic relationships of the studied strains were in agreement with the taxonomic classification. Strains EH-2, EH-3 and 4 were classified as Anabaena circinalis or Anabaena crassa. Strains DC-1, DC-2 and EH-1 were identified as Anabaena flos-aquae. Strain DC-3 was different from the other test strains and determined as Anabaena cylindrica. 5) In the study, all strains tested grew well, but the growth was different among strains. The obvious correction was found between growth and photosynthetic pigments. In addition, Yield, rETRmax and Alpha were all high to hold effective photosynthesis. This study provided some basic information and indicated significant ecological and physiological meanings for further researches. 6) In this study, the dominant species from the bloom of Lake Erhai, Anabaena circinalis, was isolated and purified. The growth and physiological characteristics of an Anabaena circinalis strain under different environmental conditions were studied to evaluate the influence of environmental factors on Anabaena bloom formation. Results showed that the Anabaena circinalis strain had best growth at temperature of 25℃, light intensity of 30 μE/ m﹒s, pH of 8.0-10.0, nitrogen 1.5mmol/L and phosphorus 12 µmol/L. The activities of nitrate reductase (NR) and alkaline phophatase (APA), which indicate metabolism of nitrogen and phosphorus, were measured with different concentrations of nitrogen and phosphorus in the medium. NR activity of the Anabaena circinalis strain increased with the raising concentration of nitrogen when nitrogen supply level was low (0 mmol/L-0.36 mmol/L). The NR activity was related to the growth under rich nitrogen supply (0.36 mmol/L-6 mmol/L), i.e. the better, the strain’s growth and the higher, the NR activity. APA activity of the Anabaena circinalis strain largely depended on the phosphorus concentration. APA activity was lower with more phosphorus supply. And Nitrogen did not play an important role in the activity change of APA when phosphorus supply was rich. In addition, it was observed that the Anabaena circinalis strain can grow with low concentration of nitrogen or phosphorus. This may be related to special regulatory mechanism of nitrogen fixing and phosphorus utilizing of Anabaena. 7) The toxicological effects induced by non-centrifuged extracts and centrifuged extracts from the cultures of the isolated strains were assessed with mouse by intraperitoneal (i.p.) injection. Results showed that the seven strains isolated from lakes Dianchi and Erhai all presented toxic effects on mouse, and LPS was proved to increase the toxic effects on mouse for cellular toxins. 8) In the present study, the mouse toxicity of an Anabaena flos-aquae strain isolated from Lake Dianchi was further investigated. The lesions of ultrastructur in liver, kidney, lung, spleen, intestines and myocardiocyte were characterized. Significant alterations were found in the serum biochemical parameters indicating changes in the hepatic functions, renal functions, and immune functions. Histopathological observations with haematoxilin and eosin (HE) stain also showed that severe lesions were in the livers, kidneys and lungs of the mice. The alterations of biochemical parameters and the severities of histological lesions were in a dose-dependent manner. Based on the biochemical and histological studies, and the above mentioned biological toxicity testing, this research firstly showed the presence of toxin producing Anabaena species in Lake Dianchi and the toxic effects of its crude extracts on mammals.
语种: 中文
内容类型: 学位论文
URI标识: http://ir.ihb.ac.cn/handle/342005/12300
Appears in Collections:中科院水生所知识产出(2009年前)_学位论文

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滇池与洱海的鱼腥藻及其生态生理与毒理学研究.潘晓洁[d].中国科学院水生生物研究所,2008.20-25
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