东湖蓝藻水华毒性的研究 Ⅱ.季节变化及微囊藻的毒性

1984年至1986年间武汉东湖(包括湖边池塘)的水华有7种,即铜绿微囊藻、大型铜绿微囊藻、边缘微囊藻、水华鱼腥藻、卷曲鱼腥藻、美丽颤藻和束丝藻。经生物测定,除束丝藻未测、卷曲鱼腥藻和美丽颤藻未测出毒性外,其余4种皆为有毒水华。东湖的水华随着季节变化而有不同类型的更迭,其出现格局为:卷曲鱼腥藻、微囊藻、颤藻、微囊藻。微囊藻水华的毒性在不同季节也有较大差异,毒性最低在8—9月份,最高在12月份(LD_(50)=24mg/kg鼠重),随着温度的降低而提高。讨论了某些环境参数与微囊藻水华形成及其毒性变化的关系。此外,还用脑内注射和腹腔注射方法,研究了微囊藻毒素的毒性表现。     

Taxonomic notes on water bloom forming Microcystis species (Cyanophyta) from China—An example from samples of the Dianchi Lake

Ten common species of Microcystis, based on the examination of water samples from the Dianchi Lake, Yunnan, China, were morphologically described, and their taxonomy was also discussed. They are Microcystis aeruginosa, M. botrys, M. firma, M. flos-aquae, M. ichthyoblabe, M. novacekii, M. pseudofilamentosa, M. smithii, M. viridis and M. wesenbergii. Taxonomic status of other Microcystis species reported in China was also evaluated. Key words Cyanophyta, Microcystis, morphology, taxonomy, China.     

中国淡水微囊藻属常见种类的分类学讨论——以滇池为例

根据中国云南滇池藻类样品的观察结果,对中国分布的淡水微囊藻属Microcystis10个常见种的形态特征进行了描述,同时对它们的分类学进行了讨论,并整理出分类检索表。这10种微囊藻是铜绿微囊藻M.aeruginosa、放射微囊藻M.botrys、坚实微囊藻M.firma、水华微囊藻M.flos-aquae、鱼害微囊藻M.ichthyoblabe、挪氏微囊藻M.novacekii、假丝微囊藻M.pseudofilamentosa、史密斯微囊藻M.smithii、绿色微囊藻M.viridis、惠氏微囊藻M.wesenbergii。最后还讨论了中国报道的其它微囊藻种类的分类学状况。     

Phylogenetic Inference of Colony Isolates Comprising Seasonal Microcystis Blooms in Lake Taihu, China

Blooms of the toxin-producing cyanobacterium, Microcystis spp., are an increasingly prevalent water quality problem and health hazard worldwide. China's third largest lake, Lake Taihu, has been experiencing progressively more severe Microcystis blooms over the past three decades. In 2009 and 2010, individual Microcystis colonies, consisting of four different morphospecies, were isolated and genotyped using a whole-cell multiplex PCR assay. The 16S-23S rDNA-ITS sequences were aligned based on Bayesian inference and indicated that one morphospecies was genetically unique (Microcystis wesenbergii) and three were indistinguishable (Microcystis aeruginosa, Microcystis flos-aquae, and Microcystis ichthyoblabe). Microcystin (mcyB) genes were detected intermittently in two of the morphospecies while the other two morphospecies lacked the mcyB gene in all samples. Water temperature was found to influence bloom formation and morphotype prevalence, and chlorophyll a and temperature were positively and significantly correlated with microcystin concentration. Cooler water temperatures promoted toxigenic strains of Microcystis. Wind appeared to influence the distribution of morphotypes across the lake, with M. aeruginosa and M. ichthyoblabe being more susceptible to wind stress than M. wesenbergii and M. flos-aquae. The results of this study indicated that the blooms were composed of a variety of Microcystis morphospecies, with more genotypes observed than can be attributed to individual morphotypes. We conclude that morphology is not a reliable indicator of toxigenicity in Lake Taihu, and caution should be exercised when the M. aeruginosa morphotype is present because it is capable of producing MC-LR, the most toxic microcystin isoform.     

微囊藻群体细胞数量估算的一种简单方法

采用加酸水解和人工计数的方法,对巢湖铜绿微囊藻、放射微囊藻、惠氏微囊藻、坚实微囊藻、绿色微囊藻、挪氏微囊藻、水华微囊藻、鱼害微囊藻所含细胞数进行了估算.通过统计分析,建立了微囊藻群体最大投影面积与所含细胞数的回归方程模型,通过这些模型可以估算群体微囊藻所含的细胞数.     

PROPERTIES OF MICROCYSTIS AERUGINOSA AND M. FLOS-AQUAE (CYANOPHYTA) IN CULTURE: TAXONOMIC IMPLICATIONS1

Cultures were cloned from a sample containing Microcystis aeruginosa, M. flos-aquae and a few morphological intermediates. The M. aeruginosa cultures remained distinct from the M. flos-aquae cultures in (a) cell size, (b) cell aggregation pattern, (c) width of the mucilage surrounding the multicellular colonies, (d) sharpness of the mucilage boundary, (e) efect of 0.1–1.0 μM calcium chloride on the disaggregation of multicellular colonies, (f) frequency of mucilage mutants and (g) colony morphology on agar media. No M. flos-aquae culture produced morphs resembling M. aeruginosa, inconsistent with proposals that M. flos-aquae is a developmental stage or environmentally-induced variant of M. aeruginosa. After longterm cultivation, but not soon after origanal isolation, several M. aeruginosa cultures contained mutants with diminished mucilage production and an altered colony shape.     

NaNO3胁迫对两种微囊藻VOCs释放的影响

为了揭示硝酸盐过量积累对藻类产生胁迫后对藻类挥发性有机化合物(VOCs)释放以及水体气味的影响, 研究以形成蓝藻水华的主要种类铜绿微囊藻和水华微囊藻为材料, 在NaNO3胁迫下, 对其细胞生长和释放的VOCs进行测定分析。结果表明, NaNO3胁迫24h后, 铜绿微囊藻和水华微囊藻细胞生长均受到明显影响, 与对照相比细胞密度分别降低了29.6%和43.0%。在正常条件下, 铜绿微囊藻和水华微囊藻分别释放出26和27种化合物, 其主要类型是硫化物、烃类、萜烯类、苯类、醛类和酯类化合物。在NaNO3胁迫下, VOCs含量均明显增加, 其中铜绿微囊藻释放的此6类VOCs含量分别增加了60.5%、14.3%、136.6%、92.1%、730.0%和120.7%, 水华微囊分别增加了172.7%、162.5%、154.0%、55.9%、51.2%和109.4%。此外, 铜绿微囊藻VOCs中出现4种新成分, 水华微囊藻VOCs中出现1种新成分。由此可见, 硝酸盐过量积累对藻细胞产生胁迫后会诱导其释放出大量VOCs, 从而增加水体气味、破坏水质。       

STRONG PROBABILITY OF LETHAL TOXICITY IN THE BLUE-GREEN ALGA MICROCYSTIS VIRIDIS LEMMERMANN1

Lethal toxicity (intraperitoneal, mouse) was examined in relation to Species composition of samples containing bloom-forming Microcystis populations from natural waters and correlated with toxicity of laboratory strains of four Microcystis formas and species. Toxicity was not always associated with the presence of M. aeruginosa f . aeruginosa Elenkin. A sample with almost all cells of M. aeruginosa f . aeruginosa showed no toxicity, However samples comprised of a high percentage of M. viridis Lemmermann often showed lethal toxicity. Toxicity tests were done on culture strains M. aeruginosa f aeruginosa, M. aeruginosa f flos-aquae Elenkin , M. viridis and M. wesenbergii Kamárek. All five cultured strains of M. viridis were found to be toxic, while only one out of nine strains of M. aeruginosa f . aeruginosa was toxic. Six strains of M. wesenbergii showed no toxicity, It is recommended that attention should be paid to the occurrences and possibility of toxic bloom of M. viridis from the standpoint of water management and public health .     

Potassium Salts Inhibit Growth of the Cyanobacteria Microcystis spp. in Pond Water and Defined Media: Implications for Control of Microcystin-Producing Aquatic Blooms

Ten metals were assayed in 21 Indian ponds which comprised three groups: (i) eutrophic alkaline ponds containing <2.5 mM potassium and thick growths of Microcystis aeruginosa or Microcystis flos-aquae during most of the year, (ii) equally eutrophic alkaline ponds containing >2.8 mM potassium and no detectable Microcystis growth, and (iii) oligo- or mesotrophic ponds with various potassium and hydrogen ion concentrations and no persistent Microcystis blooms. The effects of potassium on Microcystis growth were examined in filter-sterilized pond water and in defined culture media. A 50% reduction in the 10-day yield of cultured M. aeruginosa was observed in DP medium and pond water supplemented with 1 and 3 mM KCl, respectively. In contrast, the addition of 2 to 30 mM NaCl did not suppress the growth of M. aeruginosa in either DP medium or pond water. Both 5 mM KCl and 20 mM KHCO(inf3) in J medium strongly inhibited the growth of M. flos-aquae C3-9, whereas 5 to 30 mM NaCl had no effect and 20 mM NaHCO(inf3) was stimulatory. For pond water cultured with a mixture of M. aeruginosa and the duckweed Wolffia arrhiza, M. aeruginosa dominated in unsupplemented water and W. arrhiza dominated in water supplemented with 4.8 mM KCl. Implications for the ecology and control of Microcystis blooms are discussed.     

滇池中溶藻细菌的分离鉴定及其溶藻效应

【背景】藻类水华或赤潮在世界范围内频发,带来各种危害,亟需找到有效途径控制水华或赤潮。溶藻细菌具有杀死藻类控制藻类生物量的能力,可以作为防治水华和赤潮的有效工具。【目的】分离并鉴定滇池中的铜绿微囊藻(Microcystisaeruginosa)及其溶藻细菌,对溶藻菌作用于铜绿微囊藻的溶藻效应进行研究,初步了解其溶藻特性与溶藻机制。【方法】采用LB平板稀释涂布,再经多次划线分离纯化细菌,测定16SrRNA基因序列以鉴定细菌种类;采用毛细管分离的方法分离铜绿微囊藻,并测定其cpcBA基因序列以鉴定蓝藻种类;采用热乙醇法提取叶绿素a,从而计算溶藻效率;基于过氧化氢酶(CAT)、还原型谷胱甘肽(GSH)和丙二醛(MDA)探究藻细胞在溶藻菌处理下的抗氧化系统响应。【结果】共分离获得11株微囊藻和17株针对铜绿微囊藻的高效溶藻菌。选取其中一株生长速度最快的铜绿微囊藻DCM4和一株溶藻效果最好的溶藻菌Sp37 (Bacillus siamensis)进行后续研究。Sp37对DCM4的4 d溶藻率达到92.4%±1.5%,且对微囊藻属的水华微囊藻(M. flos-aquae)和惠氏微囊藻(M.wesenbergii)均有溶藻效果,而对绿藻没有溶藻效果。Sp37的原菌液和无菌滤液对DCM4的4d溶藻率分别为86.8%±4.3%和81.1%±2.2%,两者没有显著差异(P0.05)。Sp37菌体对DCM4的溶藻率为25.4%±7.3%。Sp37无菌滤液经不同温度和pH处理之后的溶藻率与未经处理的无菌滤液的溶藻率无明显差异。Sp37无菌滤液处理藻细胞会使藻细胞的CAT、GSH和MDA含量发生变化。【结论】菌株Sp37对铜绿微囊藻DCM4具有高效的溶藻作用,而且对微囊藻属具有一定的溶藻特异性。Sp37是通过分泌胞外物质间接溶藻,且溶藻物质具有热稳定性和酸碱稳定性。Sp37无菌滤液处理藻细胞会触发藻细胞抗氧化系统,并且会损伤藻细胞膜。Sp37无菌滤液很可能是通过对藻细胞造成氧化胁迫,最终导致藻细胞死亡的。     

以浮游植物评价达赉湖水质污染及营养水平

1987—1988年在30个样点,四季采样,对达赉湖的浮游植物进行了种类组成,生物量、种群数量、优势种、污染指示种、硅藻指数、综合指数等群落生态学的初步研究,应用后五项参数对达赉湖水质污染及营养水平进行了评价。达赉湖浮游植物年均值达54.7×106个/L(细胞数,或个体数为2.3×106个/L),硅藻指数为149.3,综合指数为5.6。群落组成中污染指示种占65%,春季以绿藻的十字藻、卵囊藻为优势种,其它3个季节均以蓝藻中的微囊藻、鱼腥藻、腔球藻占优势,表明达赉湖已受到中等程度污染,属于蓝、绿藻型富营养湖。       

四种浮游生物的碎屑形成过程

本文就不同学者的观点,讨论了有机碎屑的具体定义问题。从有机碎屑产生的具体途径来看,细菌侵入已死亡的有机体这一时刻,就是碎屑形成的开始。根据Golterman的藻类矿化概念,以细菌侵入藻细胞这一时相作为有机碎屑的开始期;作者将隆线溞,水华束丝藻、螺旋鱼腥藻和铜绿微囊藻的碎屑开始期分别定为:心跳停止和细菌入侵(枝角类)、藻丝体断裂期(水华束丝藻及螺旋鱼腥藻)、灰蓝色细胞质显现期(铜绿微囊藻)。碎屑开始期及其后的各种形态均属碎屑范畴。       

三种水华蓝藻对不同磷浓度生理响应的比较研究

本实验研究了铜绿微囊藻(Microcystis aeruginosa FACHB469)、水华鱼腥藻(Anabaena flos-aquae FACHB245)和浮游颤藻(Oscillatoria planctonicaFACHB708)对磷浓度变化的生理响应。结果表明,在缺磷条件下,A. flos-aquae对低磷环境的适应能力较强,O. planctonica其次,M. aeruginosa最差;在磷充足条件,微囊藻对磷过量吸收的能力明显高于其他两种蓝藻。三种蓝藻胞外碱性磷酸酶活性(APA)与培养基中磷浓度呈负相关性,其产生碱性磷酸酶(AP)的能力由高至低为:A. flos-aquae>O. planctonica>M. aeruginosa。磷缺陷时A. flos-aquae产生的胞外APA约是M. aeruginosa的10倍,是O. planctonica的5倍。     

Laboratory evaluation of six algal species for larval nutritional suitability of the pestiferous midge Glyptotendipes paripes (Diptera: Chironomidae)

Glyptotendipes paripes Edwards midge larval growth, development, survival, emerging adult size, and food digestibility when provided with six species of algae as food were studied in the laboratory. For the study, eggs from G. paripes adults maintained in the laboratory were reared to the adult stage at 30 degrees C for 60 d on pure culture of each algal species at densities of 0.4, 0.1, and 0.02 mg of algae (fresh weight) per milliliter, as a sole food source. All larvae reared on Microcystis sp., Botryoccocus braunii, and Scenedesmus quadricauda died before completing development. The only larvae to complete development to adult were those reared on 0.4 mg/ml Lyngbia cf. aeruginosa (44.0 d), Anabaena flos-aquae (29.7 d), and Chlorella keslerii (44.8 d). No significant differences in body size of the adults achieving complete development on the three algal species were found. Algal digestion, measured by comparing amounts of live and dead algal cells in remains of cultures used for feeding and in larval excrement, revealed that >95% of all L. cf. aeruginosa, A. flos-aquae, and Microcystis sp. cells were digested; for C. keslerii, 13% of cells were digested, whereas little or no digestion of B. braunii and S. quadricauda was observed. To evaluate the effects of algal species on larval growth, laboratory-reared (on artificial food) late third/early fourth instars of G. paripes were fed individual algal species, and 10 d later, body mass changes were recorded and compared with nonfed larvae. Body mass of larvae reared on L. cf. aeruginosa and A. flos-aquae significantly increased, whereas those provided Microcystis sp. and the nonfed larvae showed significant body mass reductions. Overall, B. braunii and S. quadricauda were not suitable as larval food, probably due to their low digestibility, and Microcystis sp. because of its toxicity. This study identifies some algae that do and others that do not support G. paripes larval growth. The information is useful in understanding the feeding habits of G. paripes larvae as some of these algae occur in the larval environments of this pestiferous midge.     

Temporal and spatial distribution of planktic cyanobacteria in Lake Kastoria, Greece, a shallow, urban lake

The temporal and spatial distribution of planktic cyanobacteria and some environmental parameters were studied in the shallow, urban Lake Kastoria, Greece from June 1996 to June 1997. Water temperature varied from 6–27 °C, pH from 7.5–8.9 and dissolved O₂ concentration from 0.7–12 mg m^-3 10^-3. The mean annual Chl a concentration was 83 mg Chl a m^-3 indicative of the eutrophic-hypertrophic state of the lake. Cyanobacterial biomass ranged from 11–238 g FW m^-3, constituting about 90% of the total phytoplankton biomass throughout the year. Cyanobacterial biomass was non-uniformly distributed both vertically and horizontally from August to November 1996 and resulted mainly from the distribution of Microcystis. Seven cyanobacterial taxa were reported for the first time in Lake Kastoria. Six taxa were dominant: Microcystis aeruginosa, M. flos-aquae, M. novacekii. Limnothrix redekei, Anabaena sp. and Cylindrospermopsis raciborskii. The dominant cyanobacterial taxa can be grouped on the basis of their distribution patterns (1) Microcystis species: maximum biomass occurring at pH > 8, temperature 12–17 °C, depth < 0.2 m; (2) Anabaena sp. and Cylindrospermopsis raciborskii : maximum biomass at temperatures 23–26 °C; (3) Limnothrix redekei : maximum biomass at temperatures 6–27 °C. Usually, non-uniform, vertical distributions of cyanobacterial biomass were associated with the formation of temperature, pH and O₂ gradients. L. redekei was considered to be a key lake organism since it contributed up to 59% of the cyanobacterial biomass. Interestingly, three of the dominant cyanobacteria Microcystis aeruginosa, Anabaena sp. and Cylindrospermopsis raciborskii belong to genera that include toxin-producing species.     

Toxicity of Microcystis species isolated from natural blooms and purification of the toxin.

Microcystis strains (2 toxic and 18 nontoxic to mice) were isolated from toxic waterblooms that had been collected from Lake Kasumigaura, Ibaraki Prefecture, Japan, in August 1985. Thirteen of the strains (2 toxic and 11 nontoxic) were Microcystis aeruginosa, 2 (nontoxic) were Microcystis wesenbergii, and the other 5 were difficult to identify. Six (1 toxic and 4 nontoxic M. aeruginosa and 1 M. wesenbergii) of these 20 strains were established as axenic cultures. A toxic and axenic strain of M. aeruginosa, K-139, was used to study the relationship between growth conditions and toxicity. Cells in early-to-mid-log phase showed the highest toxicity (50% lethal dose, 7.5 mg of cells per kg of mouse), and maximum toxicity was not affected by growth temperatures between 22 and 30 degrees C. Purification and characterization of the toxins from K-139 cells were also conducted, and at least two toxins were detected. One of the toxins (molecular mass, 980 daltons) has not been reported previously. The main target of the toxin in mice was the liver. Marked congestion and necrosis in the parenchymal cells around the central veins of the liver were observed microscopically in specimens that had been prepared from the mice with acute toxicity after injection with the toxin.     

The amino acid sequence of low-potential cytochrome c550 from the cyanobacterium Microcystis aeruginosa

The low-potential cytochrome c550 has been purified from the cyanobacterium Microcystis aeruginosa and its amino acid sequence has been determined. The protein contains 135 amino acid residues with the Cys-X-X-Cys-His heme binding site at residues 37 to 41. The sequence from residue 28 to 45 shows similarity to cytochrome c553 residues 1 to 18 when the heme binding sites are aligned. Another region of similarity is in the carboxyl-terminal regions of these two proteins. The two aligning regions of cytochrome c553 correspond to helical segments in other related cytochromes. A partial sequence of cytochrome c550 from Aphanizomenon flos-aquae was obtained and showed a 48% identity to the sequence of the M. aeruginosa cytochrome. The single methionine residue in cytochrome c550 of M. aeruginosa occurs at position 119 but there is no methionine in this region in the A. flos-aquae cytochrome, indicating that methionine is not the sixth ligand to the heme iron atom. Histidine 92 is a possible sixth ligand in M. aeruginosa cytochrome c550. The far-uv circular dichroism spectrum indicates that this protein is approximately 17% alpha helix, 42% beta-pleated sheet, and 41% random coil.     

大型海藻内共生真菌次生代谢产物中抑制水华蓝藻物质的分离与活性

为获得能明显抑制水华蓝藻的化感物质,本研究从28株大型海藻内共生真菌中筛选出了4株菌株,其粗提物能显著抑制水华鱼腥藻（Anabaenaflos-aquae）和铜绿微囊藻（Microcystis aeruginosa）的生长。对这4株真菌扩大培养后,检测了从其次生代谢产物中分离出的32种单体化合物的抑藻活性。 其中, 化合物sterigmatocystin、（2’E,4’E,6’E）-6-（1’-carboxyocta-2’,4’,6’-triene）-11,12-epoxy-9,11-dihydroxydrim-7-ene、 6,8-di-O-methylnidurufin和aversin对水华鱼腥藻的抑制率显著大于50%（P 〈 0.05）, 24 h半数有效浓度（EC50,24h）分别为6.865、4.302、1.824、2.319 μg/mL;sterigmatocystin、indol-3-carboxaldehyde和（2’E,4’E,6’E）-6-（1’-carboxyocta-2’,4’,6’-triene）-11,12-epoxy-9,11-dihydroxydrim-7-ene对铜绿微囊藻的抑制率显著大于50%（P 〈 0.05）, EC50,24h 分别为4.167、5.374、8.386 μg/mL。     

Occurrence of mycosporine-like amino acids (MAAs) in the bloom-forming cyanobacterium Microcystis aeruginosa

Here we report the finding of two mycosporine-like amino acids(shinorine and Porphyra-334) in both a culture of the cyanobacteriumMicrocystis aeruginosa isolated from Lake Taihu (China) anda natural phytoplankton sample collected from this lake whichincluded Microcystis spp. Our results are the first to clearlydocument the occurrence of these UV-sunscreen compounds in afreshwater bloom-forming cyanobacterium.     

PREPARATION OF METABOLICALLY ACTIVE PROTOPLASTS OF BLUE-GREEN ALGAE1

Protoplasts that remained metabolically active for several days were obtained by treating cells of Microcystis aeruginosa, Anacystis nidulans, and Anabaena flos-aquae with lysozyme. Photosynthetic and respiratory rates were near those of control cells. Lysozymeresistant cells were isolated which may be genetic mutants.