Determination of oligopeptide diversity within a natural population of Microcystis spp. (cyanobacteria) by typing single colonies by matrix-assisted laser desorption ionization-time of flight mass spectrometry.

Besides the most prominent peptide toxin, microcystin, the cyanobacteria Microcystis spp. have been shown to produce a large variety of other bioactive oligopeptides. We investigated for the first time the oligopeptide diversity within a natural Microcystis population by analyzing single colonies directly with matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The results demonstrate a high diversity of known cyanobacterial peptides such as microcystins, anabaenopeptins, microginins, aeruginosins, and cyanopeptolins, but also many unknown substances in the Microcystis colonies. Oligopeptide patterns were mostly related to specific Microcystis taxa. Microcystis aeruginosa (Kütz.) Kütz. colonies contained mainly microcystins, occasionally accompanied by aeruginosins. In contrast, microcystins were not detected in Microcystis ichthyoblabe Kütz.; instead, colonies of this species contained anabaenopeptins and/or microginins or unknown peptides. Within a third group, Microcystis wesenbergii (Kom.) Kom. in Kondr., chiefly a cyanopeptolin and an unknown peptide were found. Similar patterns, however, were also found in colonies which could not be identified to species level. The significance of oligopeptides as a chemotaxonomic tool within the genus Microcystis is discussed. It could be demonstrated that the typing of single colonies by MALDI-TOF MS may be a valuable tool for ecological studies of the genus Microcystis as well as in early warning of toxic cyanobacterial blooms.     

Peptide diversity in strains of the cyanobacterium <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis> isolated from Portuguese water supplies

Strains of the cyanobacterium Microcystis aeruginosa were isolated into pure culture from a variety of lakes, rivers, and reservoirs in Portugal. Samples were tested with matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry (MALDI-TOF MS) to investigate the presence of various peptide groups including aeruginosins, microginins, anabaenopeptins, cyanopeptilins, microcystins, and microviridins and other peptide-like compounds. Binary data, based on the presence and absence of different peptide groups, were analyzed by phylogenetic inference. DNA was also extracted from the samples and tested using a range of primers. Those strains that gave positive results for a Microcystis-specific primer pair were further analyzed for the presence of genes linked to the biosynthesis of microginin and microcystin. The results showed that a wide range of microcystin forms were produced by the strains among which MC-LR, -FR, -RR, -WR, and -YR were the most common. The peptide profiles obtained from the MALDI analysis were assessed using cluster analysis which resulted in the formation of distinct groups or chemotypes.     

LOCALIZATION OF MICROCYSTIN SYNTHETASE GENES IN COLONIES OF THE CYANOBACTERIUM MICROCYSTIS USING FLUORESCENCE IN SITU HYBRIDIZATION1

To better understand the production of microcystins (MCs) in Microcystis colonies, fluorescence in situ hybridization (FISH) methods were developed to detect DNA involved in the synthesis of these cyanobacterial hepatotoxins. Using colonies of Microcystis aeruginosa (Kütz.) Kütz. isolated from environmental blooms of cyanobacteria and from a colony‐forming, MC‐producing laboratory strain of Microcystis, amplified PCR products were observed, coincident with positive controls. The total MC content of individual colonies of Microcystis, determined by ELISA, showed a positive correlation with colony cross‐sectional area. FISH analysis of Microcystis colonies gave high fluorescence in comparison to negative controls, indicating the presence of MC synthetase DNA (mcyA) in situ. FISH analysis for MC synthetase genes has the potential to be developed into an effective early warning tool for drinking and recreational water management.     

Effects of nutrient and light availability on production of bioactive anabaenopeptins and microviridin by the cyanobacterium <Emphasis Type="Italic">Planktothrix agardhii</Emphasis>

Cyanobacteria produce a large number of bioactive oligopeptides with yet unknown functions. Here the effect of environmental factors on the production of two anabaenopeptins and a microviridin by Planktothrix agardhii was investigated. The results were compared with previous findings on the production of a third family of oligopeptides, the highly toxic microcystins, to test the hypothesis that environmental factors affect the production of different types of cyanobacterial bioactive oligopeptides in a similar manner. Despite marked changes in culture conditions, variations in the amount of cell-bound anabaenopeptins and microviridin I per biomass unit did not exceed a factor of 5. High amounts of cell-bound anabaenopeptins and microviridin I per Planktothrix biovolume were associated with a high availability of nitrogen and phosphorus. The production of anabaenopeptins and microviridin continued as long as the Planktothrix cultures grew. In all cases the oligopeptide net production rate was linearly correlated to the growth rate of Planktothrix, identifying the growth activity as a major regulator of anabaenopeptin and microviridin production. The concentration of anabaenopeptins and microviridins in nature may therefore be estimated from the biomass concentration of their producers. These findings are similar to those previously reported for microcystins and thus support the idea that different types of bioactive cyanobacterial oligopeptides may share common regulation patterns. This may be seen as a hint to a mutual function of cyanobacterial oligopeptides, although further studies are needed to draw final conclusions on this issue. Handling editor: J. Padisak     

DISTRIBUTION OF OLIGOPEPTIDE CHEMOTYPES OF THE CYANOBACTERIUM PLANKTOTHRIX AND THEIR PERSISTENCE IN SELECTED LAKES IN FENNOSCANDIA1

Eighty‐seven Planktothrix (Anagnostidis and Komàrek 1988) strains isolated from 13 lakes in Scandinavia and Finland between 1964 and 2007 were screened for oligopeptides. Forty‐six individual compounds were detected in total, belonging to the structural classes anabaenopeptins (six variants), aeruginosins (six variants), cyanopeptolins (21 variants), microcystins (five variants), microginins (two variants), and microviridins (two variants). Oscillatorin was also found. Three additional compounds could not be assigned to known oligopeptide classes. Thirty oligopeptides have not been described in previous studies. Of these new compounds, five were aeruginosins and 20 cyanopeptolins. The number of oligopeptides per strain ranged from one to 13. No oligopeptide‐free strains were found, suggesting that oligopeptide production is vital for Planktothrix. On the basis of their oligopeptide patterns, the Planktothrix strains of the present study were assigned to 17 chemotypes. Three major chemotypes occurred in up to six lakes. One chemotype occurred in lakes around the city of Oslo (Norway), on the Finnish island Fasta Åland, which is situated in the Baltic Sea, and on the Finnish mainland. This wide distribution suggests that chemotypes can be subjects of recurrent dispersal and/or strong directional selection. Lake size, maximum depth, and nutrient availability appeared to be of minor importance for the ability of some chemotypes to colonize a water body successfully as long as the general requirements of Planktothrix were met. Four chemotypes were reisolated from the Oslo lake district over a period of 33–40 years, suggesting that they have been members of local Planktothrix populations for decades.     

Detection and identification of oligopeptides in Microcystis (cyanobacteria) colonies: toward an understanding of metabolic diversity

Cyanobacteria and particularly Microcystis sp. (Chroococcales) are known to produce a multitude of peptide metabolites. Here we report on the mass spectral analysis of cyanobacterial peptides in individual colonies of Microcystis sp. collected in a drinking water reservoir. A total number of more than 90 cyanopeptides could be detected, 61 of which could be identified either as known peptides or new structural variants of known peptide classes. For 18 new peptides flat structures are proposed. New congeners differed from known ones mainly in chlorination (aeruginosins), methylation (microginins), or amino acid sequences (cyanopeptolins). The high number of peptides and especially the new peptides underline the capability of Microcystis strains as producers of a high diversity of potentially bioactive compounds.     

Seasonal variations of Microcystis populations in sediments of Lake Biwa, Japan

Seasonal variations of colony numbers of Microcystis aeruginosa(Kütz.) Kütz. and M. wesenbergii(Komárek) Komárek in N. V. Kondrat. in sediments of Lake Biwa were investigated over a period of 1 year. At two stations located in the shallow South Basin of Lake Biwa (ca. 4 m water depth), the colony number of Microcystisfluctuated seasonally. The number had a tendency to gradually decrease from winter to early summer, while it increased through mid-summer and autumn. Since the Microcystispopulation in sediment was rather small, intensive growth and accumulation in the water column should be important for the formation of Microcystisblooms in Lake Biwa. Microcystiscolonies in the sediment samples after June were observed to be floating in a counting chamber under a microscope. The observation suggests that the recruitment of Microcystis colonies into the water column mostly occurs in early summer. The number of Microcystiscolonies in the deep North Basin of Lake Biwa (70 – 90 m water depth) was larger than in the South Basin. Because the seasonal variation of colony numbers was not observed in the North Basin, and Microcystiscells do not have gas vesicles, these colonies will not return into the water column. The colonies isolated from the sediment of the North Basin were able to grow in cultured conditions, in the same way as those from the sediment of the South Basin. Therefore, Microcystiscolonies may survive for a long time under stable conditions of low temperature (ca. 8 °C) and darkness, in the sediment of the deep North Basin, accumulating gradually each year.     

Quantitative Real-Time PCR for Determination of Microcystin Synthetase E Copy Numbers for Microcystis and Anabaena in Lakes

Cyanobacterial mass occurrences in freshwater lakes are generally formed by Anabaena, Microcystis, and Planktothrix, which may produce cyclic heptapeptide hepatotoxins, microcystins. Thus far, identification of the most potent microcystin producer in a lake has not been possible due to a lack of quantitative methods. The aim of this study was to identify the microcystin-producing genera and to determine the copy numbers of microcystin synthetase gene E (mcyE) in Lake Tuusulanjärvi and Lake Hiidenvesi in Finland by quantitative real-time PCR. The microcystin concentrations and cyanobacterial cell densities of these lakes were also determined. The microcystin concentrations correlated positively with the sum of Microcystis and Anabaena mcyE copy numbers from both Lake Tuusulanjärvi and Lake Hiidenvesi, indicating that mcyE gene copy numbers can be used as surrogates for hepatotoxic Microcystis and Anabaena. The main microcystin producer in Lake Tuusulanjärvi was Microcystis spp., since average Microcystis mcyE copy numbers were >30 times more abundant than those of Anabaena. Lake Hiidenvesi seemed to contain both nontoxic and toxic Anabaena as well as toxic Microcystis strains. Identifying the most potent microcystin producer in a lake could be valuable for designing lake restoration strategies, among other uses.     

Modulation and adaptation of carbonic anhydrase activity in Microcystis spp. under different environmental factors

The carbonic anhydrase (CA) activities were determined in three cyanobacterial species, namely Microcystis aeruginosa Kütz., Microcystis viridis (A.Br.) Lemm, and Microcystis wesenbergii (Kom.) Kom, which were dominant in a lake (Dianchi Lake) subject to major blooms. In more detailed experiments on M. aeruginosa, the effects of inorganic carbon, pH, temperature, nitrogen/phosphorus ratio, glucose, and light intensity on CA activity were also investigated. Because of the relatively alkaline pH value of the culture media for the optimum growth of algal cells, bicarbonate ions were the main form of exogenous inorganic carbon. The results showed that the CA activity of M. aeruginosa was influenced dramatically by the concentration of bicarbonate. Consequently, it was suggested that bicarbonate ions were the main form of exogenous inorganic carbon that M. aeruginosa could utilize. Cultures grown in the dark exhibited CA activity six times higher than that of cells cultured mixotrophically with the addition of glucose. Features of eutrophic water bodies promoted an increase in CA activity, and the resulting higher CA activity would accelerate the utilization of inorganic carbon and favor the growth and blooming of Microcystis spp. in eutrophic lakes. Although the experiments were carried out under controlled experimental conditions, they could provide some basic data that would prove useful for the control of cyanobacterial blooms in nature.     

Inhibition of seagrass photosynthesis by ultraviolet-B radiation

Effects of ultraviolet-B radiation on the photosynthesis of seagrasses (Halophila engelmanni Aschers, Halodule wrightii Aschers, and Syringodium filiforme Kütz) were examined. The intrinsic tolerance of each seagrass to ultraviolet-B, the presence and effectiveness of photorepair mechanisms to ultraviolet-B-induced photosynthetic inhibition, and the role of epiphytic growth as a shield from ultraviolet-B were investigated.     

Variation between strains of the cyanobacterium Microcystis aeruginosa isolated from a Portuguese river

AIMS: The aim of this study was to investigate toxicological differences between strains of the cyanobacterium Microcystis aeruginosa isolated from a potable water supply in the north of Portugal over a 2-month period. METHODS AND RESULTS: Twenty-six strains of M. aeruginosa were isolated, grown in pure culture, and tested using a range of techniques including matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), ELISA and a PCR procedure targeting the genes implicated in the production of toxic microcystins. There was considerable variation with respect to the amounts of microcystin produced by each of the strains as measured by ELISA, with values ranging from 0.02 to 0.53% dry weight. The results of the MALDI-TOF MS analysis demonstrated the presence of several chemically distinct forms of microcystin as well as aeruginosins, anabaenopeptins and several other unidentified peptide-like compounds. CONCLUSIONS: The growth of individual strains that comprise bloom populations, with unique 'chemotypes' can potentially be an important factor affecting the toxicity of bloom populations. Molecular probes, targeting the genes responsible for microcystin production were shown to be useful for distinguishing between toxic and nontoxic strains and showed good agreement with the results obtained from the other analyses. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study show that the analysis of cyanobacterial bloom populations at the subspecies (strain) level can potentially provide important information regarding the toxin-producing potential of a cyanobacterial bloom and could be used as an 'early warning' for toxic bloom development.     

THE ACCUMULATION OF ELECTROLYTES : XI. ACCUMULATION OF NITRATE BY VALONIA AND HALICYSTIS

The nitrate concentration in the sap of Valonia macrophysa, Kütz., is at least 2000 times that of the sea water, and in Halicystis Osterhoutii, Blinks and Blinks, at least 500 times that of the sea water.     

Temporal evolution and vertical stratification of Microcystis toxic potential during a first bloom event

In order to study the setup of a Microcystis bloom and the evolution of its toxic potential, we studied the temporal and vertical variations in Microcystis aeruginosa abundance, microcystins (MC) concentrations (intracellular and extracellular), and the relative proportion of potentially microcystin-producing cells (MC-producing cells) in relation to physicochemical parameters in the recently setup Moroccan reservoir “Yaacoub Al Mansour.” The Microcystis bloom appeared relatively late in the season and was associated with a low proportion of MC-producing cells in the water surface layer, probably related to non-limiting nutrient concentrations. Interestingly, the setup of the bloom leads to a vertical gradient, showing a decrease in Microcystis cell abundance inversely coupled with an increase in the proportion of MC-producing cells. Thus, this can be the result of the growth where non-MC-producing cells remain in the lighted water layer easier than MC-producing ones. Nevertheless, parameters other than light intensity may influence the toxic potential of bloom as no vertical pattern was observed concerning microcystins cellular quotas. The high microcystins concentrations measured in the deep water layer have also proved the importance of considering the deep part of aquatic ecosystem in the management of health risks associated with cyanobacterial proliferations.     

Photosynthetic response of seagrasses to ultraviolet-a radiation and the influence of visible light intensity

Inhibition of photosynthesis by ultraviolet-A radiation (UV-A, 315-380 nanometers) was examined in three marine angiosperms: Halophila engelmannii Aschers, Halodule wrightii Aschers, and Syringodium filiforme Kütz. Sensitivity to UV-A and photosensitization to UV-A by photosynthetically active radiation (PAR, 380-700 nanometers) were characterized.     

Biodegradation of Microcystins during Gravity-Driven Membrane (GDM) Ultrafiltration

Gravity-driven membrane (GDM) ultrafiltration systems require little maintenance: they operate without electricity at ultra-low pressure in dead-end mode and without control of the biofilm formation. These systems are already in use for water purification in some regions of the world where adequate treatment and distribution of drinking water is not readily available. However, many water bodies worldwide exhibit harmful blooms of cyanobacteria that severely lower the water quality due to the production of toxic microcystins (MCs). We studied the performance of a GDM system during an artificial Microcystis aeruginosa bloom in lake water and its simulated collapse (i.e., the massive release of microcystins) over a period of 21 days. Presence of live or destroyed cyanobacterial cells in the feed water decreased the permeate flux in the Microcystis treatments considerably. At the same time, the microbial biofilms on the filter membranes could successfully reduce the amount of microcystins in the filtrate below the critical threshold concentration of 1 µg L⁻¹ MC for human consumption in three out of four replicates after 15 days. We found pronounced differences in the composition of bacterial communities of the biofilms on the filter membranes. Bacterial genera that could be related to microcystin degradation substantially enriched in the biofilms amended with microcystin-containing cyanobacteria. In addition to bacteria previously characterized as microcystin degraders, members of other bacterial clades potentially involved in MC degradation could be identified.     

Microcystin mcyA and mcyE Gene Abundances Are Not Appropriate Indicators of Microcystin Concentrations in Lakes

Cyanobacterial harmful algal blooms (cyanoHABs) are a primary source of water quality degradation in eutrophic lakes. The occurrence of cyanoHABs is ubiquitous and expected to increase with current climate and land use change scenarios. However, it is currently unknown what environmental parameters are important for indicating the presence of cyanoHAB toxins making them difficult to predict or even monitor on time-scales relevant to protecting public health. Using qPCR, we aimed to quantify genes within the microcystin operon (mcy) to determine which cyanobacterial taxa, and what percentage of the total cyanobacterial community, were responsible for microcystin production in four eutrophic lakes. We targeted Microcystis-16S, mcyA, and Microcystis, Planktothrix, and Anabaena-specific mcyE genes. We also measured microcystins and several biological, chemical, and physical parameters—such as temperature, lake stability, nutrients, pigments and cyanobacterial community composition (CCC)—to search for possible correlations to gene copy abundance and MC production. All four lakes contained Microcystis-mcyE genes and high percentages of toxic Microcystis, suggesting Microcystis was the dominant microcystin producer. However, all genes were highly variable temporally, and in few cases, correlated with increased temperature and nutrients as the summer progressed. Interestingly, toxin gene abundances (and biomass indicators) were anti-correlated with microcystin in all lakes except the largest lake, Lake Mendota. Similarly, gene abundance and microcystins differentially correlated to CCC in all lakes. Thus, we conclude that the presence of microcystin genes are not a useful tool for eliciting an ecological role for toxins in the environment, nor are microcystin genes (e.g. DNA) a good indicator of toxins in the environment.     

Changes in the bacterial community and extracellular compounds associated with the disaggregation of Microcystis colonies

Microcystis is a well-studied type of bloom-forming genus cyanobacteria that occurs as colonies in lakes. However, whenever Microcystis colonies are transferred to the laboratory, they always disaggregate into a unicellular form. The mechanism underlying this disaggregation of Microcystis colonies remains uncharacterized. Here, we report on the changes in morphology and the changes in the composition of the associated bacterial community of Microcystis wesenbergii colonies. Denaturing gradient gel electrophoresis analysis (DGGE) showed that the diversity of the associated bacterial community decreased during the disaggregation of Microcystis colonies. Two γ-Proteobacteria and one Bacteroidetes species from the mucilage of Microcystis colonies were not detected following colony disaggregation, suggesting that these species may influence Microcystis colony morphology. Solid phase microextraction and gas chromatography–mass spectrometry (SPME GC/MS) analysis revealed that seven of the forty-one extracellular compounds detected were exclusively present in the media of the Microcystis colony extracts; these compounds may be secreted by bacteria and may be a beneficial role in Microcystis colony maintenance. The results of this study indicate that changes in the composition of the bacterial community associated with Microcystis colonies are likely responsible for the disaggregation of these colonies in the laboratory.     

Application of Bayesian network including Microcystis morphospecies for microcystin risk assessment in three cyanobacterial bloom-plagued lakes,China

Microcystis spp., which occur as colonies of different sizes under natural conditions, have expanded in temperate and tropical freshwater ecosystems and caused seriously environmental and ecological problems. In the current study, a Bayesian network (BN) framework was developed to access the probability of microcystins (MCs) risk in large shallow eutrophic lakes in China, namely, Taihu Lake, Chaohu Lake, and Dianchi Lake. By means of a knowledge-supported way, physicochemical factors, Microcystis morphospecies, and MCs were integrated into different network structures. The sensitive analysis illustrated that Microcystis aeruginosa biomass was overall the best predictor of MCs risk, and its high biomass relied on the combined condition that water temperature exceeded 24 °C and total phosphorus was above 0.2 mg/L. Simulated scenarios suggested that the probability of hazardous MCs (≥1.0 μg/L) was higher under interactive effect of temperature increase and nutrients (nitrogen and phosphorus) imbalance than that of warming alone. Likewise, data-driven model development using a naïve Bayes classifier and equal frequency discretization resulted in a substantial technical performance (CCI = 0.83, K = 0.60), but the performance significantly decreased when model excluded species-specific biomasses from input variables (CCI = 0.76, K = 0.40). The BN framework provided a useful screening tool to evaluate cyanotoxin in three studied lakes in China, and it can also be used in other lakes suffering from cyanobacterial blooms dominated by Microcystis.     

First report of cyanobacterial diversity and microcystins in a Microcystis strain from Sidi Boughaba,a Moroccan coastal lagoon

The cyanobacterial diversity of Sidi Boughaba, a Moroccan coastal lagoon and Ramsar site, was evaluated and its potentially toxic species were isolated and characterised. This study was the first time that cyanobacterial diversity and cyanotoxin production have been characterised in a Moroccan coastal lagoon. Samples collected in June 2004, July 2008 and August 2013 contained 41 species. Three strains of cyanobacteria were isolated, cultured and assessed for their potentially toxic levels of microcystins. Only Microcystis flos-aquae exhibited the presence of microcystins, at a concentration of 823.41 µg g?1 as MC-LR equivalents. MC-RR and MC-WR were also detected. The presence of toxic Microcystis and other potentially toxic strains, especially in periods of bloom proliferations, could pose environmental and health hazards. Cyanotoxin monitoring of this lagoon is highly recommended.     

Grazing on toxic cyanobacterial blooms by tadpoles of edible frog Rana grylio

Tadpoles of Rana grylio were raised as edible frogs in fishponds of Guanqiao in Wuhan City, Hubei, China, during cyanobacterial blooms from June to October. The dominant cyanobacterial species was Microcystis, which was found to be lethally toxic by intraperitoneal (i.p.) mouse bioassay. Little is known about the effect of tadpoles on toxic cyanobacterial blooms. To evaluate the potential of the tadpoles to graze on cyanobacterial blooms, the tadpoles were fed on Microcystis collected from the field in the laboratory. The Microcystis cells decreased from 1.19 × 10⁷ cells mL^?1 to 3.23 × 10⁶ cells mL^?1, with a sharp reduction of 73% of the initial Microcystis population observed in the first 24 h after introduction of the tadpoles. The ponds containing tadpoles had a markedly lower density of Microcystis than those lacking tadpoles. Tadpoles exposed to either cultured Microcystis aeruginosa (NIES–90, 2.768 µg microcystins mg^–1 dw^–1) cells or lysed M. aeruginosa cells grew well, however, indicating that they were unaffected by Microcystis toxins. We found a significant increase in tadpole body weight after feeding on either field Microcystis or cultured M. aeruginosa. The mean increase in individual body weight was 20 mg day^?1 when fed on Microcystis from the pond, and 7 mg day^?1 when fed on M. aeruginosa from culture. Our study strongly suggested that there is a direct trophic relationship between R. grylio tadpoles and toxic Microcystis blooms and they possess the potential to graze on toxic Microcystis. The results imply that R. grylio tadpoles may play an important ecological role in reducing toxic cyanobacterial blooms caused by Microcystis.