Toxic and non-toxic strains of Microcystis aeruginosa induce temperature dependent allelopathy toward growth and photosynthesis of Chlorella vulgaris

Global warming was believed to accelerate the expansion of cyanobacterial blooms. However, the impact of changes due to the allelopathic effects of cyanobacterial blooms with or without algal toxin production on the ecophysiology of its coexisting phytoplankton species arising from global warming were unknown until recently. In this study, the allelopathic effects of toxic and non-toxic Microcystis aeruginosa strains on the growth of green alga Chlorella vulgaris and photosynthesis of the co-cultivations of C. vulgaris and toxic M. aeruginosa FACHB-905 or non-toxic M. aeruginosa FACHB-469 were investigated at different temperatures. The growth of C. vulgaris, co-cultured with the toxic or non-toxic M. aeruginosa strains, was promoted at 20 °C but inhibited at temperatures ≥25 °C. The inhibitory effects of the toxic and non-toxic M. aeruginosa strains on of the co-cultivations (C. vulgaris and non-toxic M. aeruginosa FACHB-469 or toxic M. aeruginosa FACHB-905) also linearly increased with elevated temperatures. Furthermore, toxic M. aeruginosa FACHB-905 induced more inhibition toward growth of C. vulgaris or P_max and R_d of the mixtures than non-toxic M. aeruginosa FACHB-469. C. vulgaris dominated over non-toxic M. aeruginosa FACHB-469 but toxic M. aeruginosa FACHB-905 overcame C. vulgaris when they were co-cultured in mesocosms in water temperatures from 20 to 25 °C. The results indicate that allelopathic effects of M. aeruginosa strains on C. vulgaris are both temperature- and species-dependent: it was stimulative for C. vulgaris at low temperatures such as 20 °C, but inhibitory at high temperatures (≥25 °C); the toxic strain was determined to be more harmful to C. vulgaris than the non-toxic one. This suggests that global warming may aggravate the ecological risk of cyanobacteria blooms, especially those with toxic species as the main contributors.     

7-Deoxy-desulfo-cylindrospermopsin and 7-deoxy-desulfo-12-acetylcylindrospermopsin: Two new cylindrospermopsin analogs isolated from a Thai strain of Cylindrospermopsis raciborskii

As environmental changes such as eutrophication lead to increased size and frequency of cyanobacterial blooms, research into the toxins produced by these blooms becomes increasingly important. One of the common toxins produced by cyanobacterial blooms is cylindrospermopsin (1), a potent inhibitor of protein synthesis. To date, only two additional analogs of cylindrospermopsin have been isolated, namely 7-epicylindrospermopsin (2) and 7-deoxy-cylindrospermopsin (3). This report details the isolation and structure determination of an additional two new analogs, 7-deoxy-desulfo-cylindrospermopsin (4) and 7-deoxy-desulfo-12-acetylcylindrospermopsin (5). These are the first new analogs of cylindrospermopsin to be reported in over a decade. Based on their structural features, it is likely that these new analogs also possess the harmful biological activities displayed by the rest of the cylindrospermopsin family.     

Intraspecific variability in response to phosphorus depleted conditions in the cyanobacteria Microcystis aeruginosa and Raphidiopsis raciborskii

Phosphorus loading plays an important role in the occurrence of cyanobacterial blooms and understanding how this nutrient affects the physiology of cyanobacteria is imperative to manage these phenomena. Microcystis aeruginosa and Raphidiopsis raciborskii are cyanobacterial species that form potentially toxic blooms in freshwater ecosystems worldwide. Blooms comprise numerous strains with high trait variability, which can contribute to the widespread distribution of these species. Here, we explored the intraspecific variability in response to phosphorus depleted conditions (P-) testing five strains of each species. Strains could be differentiated by cell volume or genetic profiles except for those of the same species, sampling location and date, though these presented differences in their response to (P-). Although differently affected by (P-) over 10 days, all strains were able to grow and maintain photosynthetic activity. For most M. aeruginosa and R. raciborskii strains growth rates were not significantly different comparing (P+) and (P-) conditions. After ten days in (P-), only one M. aeruginosa strain and two R. raciborskii strains showed reduction in biovolume yield as compared to (P+) but in most strains chlorophyll-a concentrations were lower in (P-) than in (P+). Reduced photosystem II efficiency was found for only one R. raciborskii strain while all M. aeruginosa strains were affected. Only two M. aeruginosa and one R. raciborskii strain increased alkaline phosphatase activity under (P-) as compared to (P+). Variation in P-uptake was also observed but comparison among strains yielded homogeneous groups comprised of representatives of both species. Comparing the response of each species as a whole, the (P-) condition affected growth rate, biovolume yield and chlorophyll yield. However, these parameters revealed variation among strains of the same species to the extent that differences between M. aeruginosa and R. raciborskii were not significant. Taken together, these results do not support the idea that R. raciborskii, as a species, can withstand phosphorus limitation better than M. aeruginosa and also point that the level of intraspecific variation may preclude generalizations based on studies that use only one or few strains.     

Isolation and characterization of a novel Lambda-like phage infecting the bloom-forming cyanobacteria Cylindrospermopsis raciborskii

Cylindrospermopsis raciborskii is a central bloom-forming cyanobacteria. However, despite its ecological significance, little is known of its interactions with the phages that infect it. Currently, only a single sequenced genome of a Cylindrospermopsis-infecting phage is publicly available. Here we describe the isolation and characterization of Cr-LKS3, a second phage infecting Cylindrospermopsis. Cr-LKS3 is a siphovirus with a higher genome similarity to prophages within heterotrophic bacteria genomes than to any other cyanophage/cyano-prophage, suggesting that it represents a novel cyanophage group. The function, order and orientation of the 72 genes in the Cr-LKS3 genome are highly similar to those of Escherichia virus Lambda (hereafter Lambda), despite the very low sequence similarity between these phages, showing high evolutionary convergence despite the substantial difference in host characteristics. Similarly to Lambda, the genome of Cr-LKS3 contains various genes that are known to be central to lysogeny, suggesting it can enter a lysogenic cycle. Cr-LKS3 has a unique ability to infect a host with a dramatically different GC content, without carrying any tRNA genes to compensate for this difference. This ability, together with its potential lysogenic lifestyle shed light on the complex interactions between C. raciborskii and its phages.     

Membrane effects of toxins isolated from a cyanobacterium, Cylindrospermopsis raciborskii, on identified molluscan neurones

The effect of anatoxin (ANTX), the crude extract (AlgTX) and purified fraction (F1) isolated from cyanobacterium C. raciborskii was studied on the neurones of two snail species. ANTX and AlgTX exerted excitatory, inhibitory and biphasic effects on the spontaneous activity of identified neurones. Both ANTX and AlgTX elicited an inward current, which could be decreased by curare or amiloride. On the contrary, F1 had no direct effect on the spontaneous activity; it was not able to induce conductance changes of the neuronal membrane, but it did antagonise the acetylcholine (ACh)-induced inward current. We concluded that ANTX affects the neuronal membrane of neurones acting on ACh receptors. The AlgTX had similar effects, and therefore the extract of C. raciborskii may contain an ANTX-like component. The purified fraction prolonged and decreased the ACh-elicited response, but had no direct membrane effect. We suggest, therefore, that both AlgTX and the purified fraction F1 interact with the ACh receptor, but they have different binding sites on the neuronal ACh receptor-ion channel complex. The possible neurotoxic effects of the C. raciborskii extract and F1 are demonstrated for the first time; the molecular mechanism of their action, however, remains to be elucidated.     

不同氮、磷浓度对铜绿微囊藻生长、光合及产毒的影响

对一株从野外分离得到的铜绿微囊藻产毒株进行分批培养,在不同的氮磷条件下研究其生长、光合荧光及毒素含量的变化。结果表明:正磷酸盐浓度不变时,铵氮浓度的改变对铜绿微囊藻的生长有明显影响。叶绿素a(Chl.a)含量在铵氮浓度为1.83-18.3mg/L时明显较大;微囊藻毒素(包括MC-LR和MC-RR)的含量在铵氮浓度为1.83mg/L时达到最大;当铵氮浓度为0-1.83mg/L时,随着铵氮浓度升高,可变荧光FV和MC的产量均增大,同时MC异构体的种类增多;铵氮浓度过大对M.aeruginosa的生长、生理和产毒均有抑制作用。在另一组实验中,即铵氮浓度不变而正磷酸盐浓度增大时,Chl.a含量呈总体下降的趋势,并且与FV/Fm呈显著正相关关系(P<0.01,r=0.97),MC(MC-LR和MC-RR)的含量在正磷酸盐浓度小于0.56mg/L时明显升高,MC-LR与FV/Fm呈显著正相关关系(P<0.01,r=0.967)。       

陆生植物化感抑制铜绿微囊藻作用效应及机制研究进展

抑制藻类繁殖,控制水华爆发,是对富营养化水体完成生态修复过程中面临的首要挑战,如何高效安全的抑藻是亟待解决的问题.利用植物化感作用控制富营养化水体中有害藻类大量繁殖是有效的手段之一,但是利用陆生植物化感物质进行抑藻的研究较少.本文从草本植物(菊科/罂粟科、百合科、禾本科)、木本植物以及中草药类植物三大类详细介绍了陆生植物对水华爆发中的主要藻类铜绿微囊藻的抑制效应,并且针对化感物质的分离鉴定和物质基础、作用机制等进行阐述,最后对利用陆生植物化感物质开发抑藻剂的研究思路和注意问题提出了建议.     

铜绿微囊藻(Microcystis aeruginosa)对惠氏微囊藻(Microcystis wesenbergii)的化感作用

通过混合培养和添加过滤液两种方式观察铜绿微囊藻和惠氏微囊藻的生长曲线,探讨两种微囊藻之间的化感作用。结果表明:在混合培养条件下,两者能够形成相互抑制作用;当两者起始藻密度高于0.5×106cells.mL-1、混合比为1:1时,惠氏微囊藻的生长因化感作用而受到显著抑制(P<0.05),同时惠氏微囊藻也会对铜绿微囊藻产生一定的胁迫作用;处于对数生长期的铜绿微囊藻过滤液能抑制惠氏微囊藻的生长,且惠氏微囊藻起始藻密度低于0.5×106cells.mL-1,连续滴加该过滤液后,其生长受到极显著抑制(P<0.01)。     

Simultaneous cultivation of Spirulina platensis and the toxigenic cyanobacteria Microcystis aeruginosa

Mangueira Lagoon, located in the extreme south of Brazil, has water with physicochemical characteristics such as alkaline pH and carbonate levels propitious for the growth of the cyanobacterium Spirulina platensis. Previously published studies have shown that Mangueira Lagoon water supplemented with small quantities of carbon and nitrogen is suitable for S. platensis cultivation and can significantly reduce production costs. We studied mixed cultures of Spirulina platensis and the toxic cyanobacterium Microcystis aeruginosa using a 2(3) factorial design in which the three factors were the initial biomass concentration of S. platensis and M. aeruginosa and the type of culture medium (100% Zarrouk's medium or 80% Mangueira Lagoon water plus 20% Zarrouk's medium). The highest S. platensis maximum specific growth rate (mu(max)) occurred in the culture with the highest M. aeruginosa biomass concentration and when undiluted culture medium was used (micro(max) = 0.283 d(-1)). The highest M. aeruginosa specific death rate (k) was obtained in the presence of S. platensis (k = 0.555 d(-1)) and was independent of the initial M. aeruginosa biomass concentration and culture medium, demonstrating that S. platensis cultures are not susceptible to contamination by M. aeruginosa. The culture medium had no significant influence (p > 0.05) on S. platensis micro(max) values, indicating that production costs could be reduced by using a medium consisting of 80% Mangueira Lagoon water plus 20% Zarrouk's medium.     

Allelopathic effects of Microcystis aeruginosa on green algae and a diatom: Evidence from exudates addition and co-culturing

Allelopathic interactions among phytoplankton species are regarded as one of the important factors contributing to phytoplankton species competition and succession. The role and extent of allelopathic effects of blooming freshwater cyanobacteria on other phytoplankton species in eutrophied waters, however, are still unknown. We examined the allelopathic effect of Microcystis aeruginosa on two common green algae (Scenedesmus quadricauda, Chlorella pyrenoidosa) and a diatom (Cyclotella meneghiniana) by adding exudates from different growth phases and in co-culture tests. Exudates of M. aeruginosa from the exponential growth phase and the stationary phase significantly inhibited the growth of S. quadricauda, C. pyrenoidosa and C. meneghiniana, whereas those from the decline phase increased their growth. The presence of M. aeruginosa extremely inhibited the growth of all tested species in co-cultures within 24 h. Our results indicate that under the tested environmental conditions (25 °C, light 80 μmol quanta m⁻² s⁻¹, manual shaking twice a day), allelopathic effects of M. aeruginosa on other phytoplankton species can significantly contribute to their competitive success.     

Identification of teratogenic polymethoxy-1-alkenes from Cylindrospermopsis raciborskii,and taxonomically diverse freshwater cyanobacteria and green algae

Cylindrospermopsis raciborskii is among the most commonly recognized toxigenic cyanobacteria associated with harmful algal blooms (HAB) in freshwater systems, and specifically associated with multiple water-soluble toxins. Lipophilic metabolites from C. raciborskii, however, were previously shown to exert teratogenicity (i.e. inhibition of vertebrate development) in the zebrafish (Danio rerio) embryo model, specifically suggesting the presence of additional bioactive compounds unrelated to the currently known toxins. In the present study, a series of known teratogenic polymethoxy-1-alkenes (PMA) were identified, purified and chemically characterized from an otherwise well-characterized strain of toxigenic C. raciborskii. Although PMA have been previously identified in other cyanobacteria, this is the first time they have been identified from this recognized HAB species. Following their identification from C. raciborskii, the taxonomic distribution of the PMA was additionally investigated by chemical screening of a freshwater algal (i.e. cyanobacteria, green algal) culture collection. Screening suggests that these compounds are distributed among phylogenetically diverse taxa. Furthermore, parallel screening of the algal culture collection, using the zebrafish embryo model of teratogenicity, the presence of PMA was found to closely correlate with developmental toxicity of these diverse algal isolates. Taken together, the data suggest PMA contribute to the toxicity of C. raciborskii, as well as apparently several other taxonomically disparate cyanobacterial and green algal genera, and may, accordingly, contribute to the toxicity of diverse freshwater HAB.     

Optimization of 2D-PAGE protocols for proteomic analysis of two nonaxenic toxin-producing freshwater cyanobacteria: Cylindrospermopsis raciborskii and Raphidiopsis sp.

Aims:  To optimize a protocol for the extraction and an in-depth analysis of the soluble protein fraction of two nonaxenic toxin-producing cyanobacteria Cylindrospermopsis raciborskii (hepatotoxin-producing), and Raphidiopsis sp. (neurotoxin-producing), using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE).
Methods and Results:  The soluble protein fractions from strains of C. raciborskii and Raphidiosis sp. with different toxicity phenotypes were analysed by 2D-PAGE. Specific protocols were optimized specifically for each strain. Between 500 and 700 sharp protein spots were distinguished in a single 4–7 pH range 2D-PAGE for each cyanobacterium. Comparison of the protein maps of C. raciborskii CS-505 (a cylindrospermopsin-producing strain) and Raphidiopsis sp. D9 (saxitoxin-producing strain) against the nontoxic C. raciborskii strain CS-509 revealed many unique proteins in each protein map. We confirmed that the resolved proteins were cyanobacterial by identifying three randomly chosen protein spots from a Raphidiopsis sp. strain D9 2D-PAGE, using high-performance liquid chromatography (HPLC) tandem mass spectrometry (MS).
Conclusions:  The 2D-PAGE conditions presented here provide a robust protocol for proteomic studies in two CYN- and STX-producing model organisms, C. raciborskii and Raphidiopsis sp.
Significance and Impact of the Study:  We present the first protocols for proteomic analyses of Cylindrospermopsis raciborskii and Raphidiopsis sp.     

Genetic characterization of Cylindrospermopsis raciborskii (cyanobacteria) isolates from diverse geographic origins based on nifH and cpcBA-IGS nucleotide sequence analysis

Isolates of the toxic, N(2)-fixing species Cylindrospermopsis raciborskii from various geographic locations were analyzed with respect to their genetic diversity based on the nifH and cpcBA-IGS genes. Gene sequences clustered according to their geographic origin, with the nifH sequences separating into European, Australian, and American groups and the cpcBA-IGS sequences separating into American and European or Australian groups. PCR primers for both genes were designed to exclusively amplify DNA from Cylindrospermopsis species, and an additional primer set for cpcBA-IGS was designed to specifically amplify the American C. raciborskii strains.     

A comparison of toxins isolated from the cyanobacteria Oscillatoria agardhii and Microcystis aeruginosa

1. A toxin isolated from a strain of Oscillatoria agardhii var. was compared to a peptide toxin isolated from Microcystis aeruginosa. 2. The Oscillatoria toxin possessed similar hepatotoxic properties on mice as the Microcystis toxin but had a higher LD50 than the latter; 320 micrograms/kg compared to 43 micrograms/kg (i.p. mouse), respectively. 3. Ultra-violet and infra-red spectra showed that the Oscillatoria toxin is a peptide which is not identical to the Microcystis toxin. 4. The spectra also indicated some structural similarities in these toxins.     

Allelopathic effect of Microcystis aeruginosa on Microcystis wesenbergii: microcystin-LR as a potential allelochemical

Microcystis aeruginosa and Microcystis wesenbergii are two cyanobacteria commonly found in eutrophic shallow lakes. Previous studies reported that microcystin-producing M. aeruginosa could have an increased competitive potential on other algae and aquatic plants, and microcystin-LR (MC-LR) was regarded as an allelochemical. Based on this hypothesis, the allelopathic interaction between these two cyanobacteria was studied for the first time under laboratory conditions, and potential allelochemicals were screened. Cyanobacteria biomass and microcystin-LR (MC-LR) concentration were monitored under different culture conditions. The potential allelochemicals from M. aeruginosa were investigated by extract fractionation and GC(LC)/MS analysis. The growth of M. wesenbergii was inhibited by the addition of cell-free filtrates of M. aeruginosa whereas M. aeruginosa was promoted by the addition of cell-free filtrates of M. wesenbergii. The higher polarity the extract of M. aeruginosa is, the stronger the inhibition effect of the extract on M. wesenbergii will be. According to our results, M. aeruginosa has a significant allelopathic inhibition effect on M. wesenbergii. Allelopathic compounds from M. aeruginosa have synergistic effects on inhibition of M. wesenbergii. Besides microcystin, there may be other allelopathic compounds in M. aeruginosa.     

Effects of Interspecific Interactions between Microcystis aeruginosa and Chlorella pyrenoidosa on Their Growth and Physiology

Interactions between Microcystis aeruginosa and Chlorella pyrenoidosa were analyzed by flow cytometry and by phytoplankton pulse‐amplitude‐modulated fluorimetry (Phyto‐PAM) in joint cultures as well as in cultures separated by dialysis membranes. Results showed that the growth of C. pyrenoidosa was greater than that of M. aeruginosa, and that the growth of M. aeruginosa but not the growth of C. pyrenoidosa was significantly inhibited by the interactions between M. aeruginosa and C. pyrenoidosa. Culture filtrates of these two algae showed no apparent effects on the growth of the competing species. For M. aeruginosa, decreases in esterase activity, chlorophyll a fluorescence, and maximum quantum yield were observed in joint cultures, indicating that the metabolic activity and photosynthetic capacity of M. aeruginosa were suppressed. Light limitation from the shading effect of C. pyrenoidosa may be the main reason for such inhibition. For C. pyrenoidosa, esterase activity was suppressed in membrane‐separated and joint cultures, suggesting that C. pyrenoidosa was probably affected by allelopathic substances secreted by M. aeruginosa. However, no significant difference was observed in the chlorophyll a fluorescence and maximum quantum yield of C. pyrenoidosa in the two cultures. In addition, interspecific interactions induced a reduction in size in both M. aeruginosa and C. pyrenoidosa, which may contribute to the development of C. pyrenoidosa dominance in the present study. (© 2007 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Growth and temperature‐related phenotypic plasticity in the cyanobacterium Cylindrospermopsis raciborskii

Cylindrospermopsis raciborskii is an invasive and potentially toxic cyanobacterium, which has recently spread worldwide, mainly because of its tolerance to a wide range of climatic conditions. C. raciborskii is able to change several traits in response to environmental changes and its morphology is also affected by these changes (especially in nutrients). We also expected temperature to affect the morphology of this cyanobacterium. We examined the growth and morphology of C. raciborskii at different temperatures and compared laboratory results to the morphology of this cyanobacterium in situ. As expected, growth rates increased with temperature. In addition, a high carrying capacity at 32°C suggests that this cyanobacterium is able to form more dense blooms at high temperatures. Fragile trichomes and low growth rates were observed at 12°C. An increase in the growth rate related to temperature resulted in a decrease in trichome length, with shorter trichomes at 32°C. The same pattern was observed in wild populations of C. raciborskii in a tropical reservoir, where shorter trichomes were observed in warmer months, when biomass was highest. This species' high ability to adapt to different environmental conditions throughout the year (i.e., nutrients, temperature) may have provided it with an additional advantage to increase its perennial blooms, mainly in tropical regions.     

蒜头果他感作用的初步研究

蒜头果具有自毒现象 ,为研究其他感作用 ,用蒜头果新鲜果皮与蒸馏水按 1∶1比例进行浸提 ,以蒸馏水 (A1 ,对照 )、稀释 4倍液 (A2 )、稀释 2倍液 (A3 )和母液 (A4)对萝卜、马尾松、重阳木种子进行随机区组发芽试验 ,3个重复。结果表明 :蒜头果果皮水浸提液的他感作用极其显著 ,但对不同植物的影响不同 ,不同浓度浸提液对萝卜全部表现为抑制作用 ,对其它植物既有抑制作用也有促进作用。浸提液明显抑制所供试植物幼苗生长 ,抑制强度顺序是A4>A3 >A2 。不同植物的敏感程度是萝卜 >重阳木 >马尾松