Non-toxic Microcystis Strains Research Articles

Assessment of different mcy genes for detecting the toxic to non-toxic Microcystis ratio in the field by multiplex qPCR

Harmful cyanobacterial blooms, especially Microcystis blooms, occur worldwide and draw widespread attention. The dynamics of microcystin-producing Microcystis and competition between microcystin-producing Microcystis and non-microcystin-producing Microcystis are key to predicting and treating Microcystis blooms. Multiplex qPCR is a useful tool to assess such issues. In this study, we developed multiplex qPCR methods with newly-designed probes and primers for the microcystin-synthesis related genes mcyA and mcyE. We used seven toxic Microcystis strains and four non-toxic Microcystis strains to compare the differences in the ratios of toxic and non-toxic Microcystis in mixed cultures, which were calculated using abundances of the genes mcyA, mcyB, mcyD, mcyE and phycocyanin (PC). We also compared traditional cell counting and multiplex qPCR. Hierarchical clustering and principal component analysis indicated that mcyD was the most suitable mcy gene for quantification in laboratory experiments. mcyB abundances were always higher; we suggest that the amount of toxic Microcystis measured using mcyB might overestimate the actual percentages.

Biometric and physiological responses of Egeria densa Planch. cultivated with toxic and non-toxic strains of Microcystis

Cyanobacterial blooms are becoming increasingly common in aquatic environments around the world, mainly due to eutrophication and climate change. Cyanotoxin-producing strains (e.g., microcystins (MC) producers) may be present in these blooms, affecting the growth of other aquatic organisms, such as aquatic macrophytes. In this study, we evaluated the morphometric and physiological responses of the aquatic macrophyte Egeria densa to the exposure to a toxic strain of Microcystis aeruginosa (MCs producer) and a non-toxic Microcystis panniformis (non-MC producer). The effects of Microcystis strains on E. densa growth and biomass were verified for five weeks (Experiment 1) and physiological responses were evaluated for 14days (Experiment 2). Prolonged exposure of E. densa to the MC producing strain reduced growth, accompanied by the inhibition of shoot and root emission. Both Microcystis strains caused a decrease in the content of photosynthetic pigments, like total chlorophyll and chlorophyll a and b, accompanied by an increase of carotenoids. At the beginning of the MC-producing strain exposure, E. densa showed an increase in the activity of the anti-oxidative enzymes superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX), accompanied by an increase in the levels of malondialdehyde, indicating lipid peroxidation. During the 14th day of exposure, the activity of antioxidant enzymes remained similar to the control, suggesting that E. densa has an efficient anti-oxidative system to control the reactive oxygen species produced in response to the stress caused by microcystins. However, when prolonged exposure occurred, possible damage to proteins may have affected the growth and development of E. densa. No changes were observed in the enzymatic activity of the plants exposed to the non-MC producing strain, suggesting that this cyanobacterial strain do not cause significant damage to the development of E. densa. These results are important for understanding the anti-oxidative defense mechanisms of aquatic macrophytes when coexisting with an MC producing strain.

PCR primers for selective detection of intra-species variations in the bloom-forming cyanobacterium, Microcystis

Members of the cyanobacterial genus Microcystis commonly form blooms in eutrophic freshwater systems, and some produce cyclic heptapeptide hepatotoxins called microcystins, thereby often causing serious water management problems. Microcystis species were unified into the single Microcystis aeruginosa classification based on 16S rRNA gene sequences and DNA–DNA re-association experiments; however, the morphological features of the organisms differ in different culturing conditions. Here, we describe a new real-time quantitative PCR (qPCR) method of determining Microcystis intradiversity using the SYBR Green I assay. We analyzed 71 Microcystis 16S-23S rDNA internal transcribed spacer region (16S-23S ITS) sequences, designed three group-specific PCR primers that successfully selected a morphologically M. wesenbergii-like non-toxic group (Group-3), and differentiated between M. viridis-like toxic group (Group-4) and M. aeruginosa-like Group-1 organisms including toxic and non-toxic Microcystis strains. The primers covered 76% of the Microcystis 16S-23S ITS regions from all over the world (six continents) included in GenBank. We constructed a mixed culture with representative Microcystis strains from each group, and estimated their cell densities by qPCR over 7 weeks. Group-1 and Group-3 grew exponentially for 4 weeks; however, the growth of Group-4 declined after 2 weeks, revealing different growth properties for the Microcystis groups in the mixed culture. Finally, we applied this method to natural Microcystis blooms at four freshwater sites, and found the dominance of Group-1 in three blooms and of Group-3 in one bloom, thereby showing the geographically uneven distribution of Microcystis genotypes. The developed qPCR technique targeting the 16S-23S ITS region is both rapid and simple and is useful for selective quantification of group variations among sympatric Microcystis genotypes, such as in mixed cultures and the natural environment.

The dynamics of toxic Microcystis strains and microcystin production in two hypertrofic South African reservoirs

The South African impoundments of Hartbeespoort and Roodeplaat experience excessive blooms of Microcystis species each year. Microcystins, produced primarily by strains of cyanobacteria belonging to the genera Microcystis, Anabaena and Planktothrix, are harmful cyanobacterial hepatotoxins. These bloom-forming cyanobacteria form toxic and non-toxic strains that co-occur and are visually indistinguishable, but can be identified and quantified molecularly. We described the relationships between microcystin production and the genotypic composition of the Microcystis community involved together with environmental conditions in both the Roodeplaat and Hartbeespoort reservoirs using quantitative real time PCR. DNA copy number of the Microcystis-specific 16S rRNA and toxin biosynthesis genes, mcyE and mcyB, were measured. Planktothrix spp. occurred in both reservoirs during autumn, but no toxin-producing species was present as measured with mcyE specific primers, whereas both toxic and non-toxic strains of Microcystis were recorded in both reservoirs, with Microcystis spp. dominating in the summer months. Water-surface temperature correlated strongly with microcystin concentration, mcyE and mcyB copy number. Microcystin production was associated by temperatures higher than 23°C. This suggests that should current environmental trends persist with surface water temperatures continuing to rise and more and more nutrients continued to be loaded into fresh water systems toxic Microcystis may outgrow non-toxic Microcystis and synthesise even more microcystins.

Feeding behavior of the invasive bivalve Limnoperna fortunei (Dunker, 1857) under exposure to toxic cyanobacteria Microcystis aeruginosa

The aim of this study was to test the effects of cyanobacteria toxicity on feeding behavior of the golden mussel Limnoperna fortunei. First, it was tested the hypothesis that L. fortunei preferentially graze on non-toxic phytoplankton and reject toxic cyanobacteria. Second, it was tested the hypothesis that toxic cyanobacteria negatively affect feeding and survival of L. fortunei. The present study is the first to evaluate the effects of toxic cyanobacteria on L. fortunei feeding and survival. In the short-term grazing, golden mussel filtration rates were evaluated in the presence of toxic and non-toxic strains of cyanobacteria Microcystis aeruginosa, and non-toxic phytoplankton Nitzschia palea. Highest filtration rates were registered when mussels fed on Nitzschia. Despite that, golden mussel expelled Nitzschia cells in large quantities and preferentially ingested Microcystis cells, both toxic and non-toxic strains. In the long-term grazing, mussels were exposed to toxic and non-toxic strains of Microcystis during 5 days. Filtration rates were not significantly different for toxic and non-toxic Microcystis throughout exposure period. The results have demonstrated cyanobacteria toxicity is not the main factor influencing L. fortunei feeding behavior. Survival of L. fortunei feeding on toxic cyanobacteria shows the potential of this invasive bivalve as a vector to the transference of cyanotoxins to higher trophic levels.

Effects of dissolved inorganic carbon on competition of the bloom-forming cyanobacterium Microcystis aeruginosa with the green alga Chlamydomonas microsphaera

The availability of dissolved inorganic carbon (DIC) may have a considerable impact on species competition in phytoplankton communities. The growth, photosynthetic characteristics and competition of three strains, the toxic Microcystis aeruginosa FACHB 912, nontoxic M. aeruginosa FACHB 469 and Chlamydomonas microsphaera FACHB 52, were investigated under two different DIC concentrations (0.365 and 7.658 mmol l−1 KHCO3). In monoculture, DIC concentration did not affect the specific growth rates of any of the three strains. However, when grown in mixed culture with C. microsphaera, both toxic and nontoxic Microcystis strains showed increased percentages under low DIC concentration but decreased percentages under high DIC concentration. After 12 days’ mixed culture, the percentage of M. aeruginosa FACHB 912 or FACHB 469 decreased by 9−22% in high DIC medium, but increased by 6–11% in low DIC medium. Low DIC concentration decreased the cell size, cellular chlorophyll fluorescence and photosynthetic capacity of C. microsphaera, but it had little effect on those of M. aeruginosa FACHB 912 and FACHB 469. The apparent photosynthetic affinities for DIC were significantly higher in the two M. aeruginosa strains than in C. microsphaera. From the higher DIC affinity, more efficient photosynthetic capacity and increased percentages in competition experiments under low DIC concentration, it can be concluded that the bloom-forming M. aeruginosa possesses a competitive advantage in DIC-limited conditions.

Effects of nitrogenous compounds and phosphorus on the growth of toxic and non-toxic strains of Microcystis during cyanobacterial blooms

Since the mid-twentieth century, both nutrient delivery rates and the frequency of harmful algal blooms (HABs) in coastal aquatic ecosystems have intensified. Recent studies have shown that nitrogen (N) or phosphorus (P) can limit primary production in freshwater systems, and Microcystis is able to utilize both inorganic and organic forms of N. The present study quantified the microcystin synthetase gene (mcyD) and the ribosomal RNA gene (16S) to assess how various nutri- ent sources affected the growth of toxic and non-toxic strains of Microcystis during natural blooms. During the present study, dense Microcystis blooms (>10 6 cell equivalents l -1 ) were observed within 2 contrasting ecosystems in the eastern USA: a tidal tributary and a eutrophic lake. In both systems, all Microcystis populations were stimulated by N more frequently than P during nutrient amendment experiments. The abundance of toxic strains of Microcystis was enhanced by nutrient enrichment more frequently (83% of experiments) than non-toxic strains (58% of experiments), suggesting that toxic strains may have a greater demand for both nutrients. Furthermore, abundances of toxic strains of Microcystis were enhanced by inorganic N more frequently (67% of experiments) than organic N (8% of experiments), while non-toxic strains were stimulated by organic N (50% of experiments) more frequently than inorganic N (25% of experiments). Inorganic P increased abundances of toxic strains of Microcystis more frequently than non-toxic strains (42 and 33% of experiments, respec- tively). Therefore, the dominance of toxic Microcystis may be influenced by both the concentration and species of nutrients, with higher concentrations of inorganic N and/or P likely promoting blooms dominated by toxic strains and potentially yielding higher microcystin concentrations.

Grazing by mesozooplankton and microzooplankton on toxic and non-toxic strains of Microcystis in the Transquaking River, a tributary of Chesapeake Bay

The objective of this study was to assess the ability of laboratory-reared and natural communities of zooplankton to graze on toxic and non-toxic strains of Microcystis during bloom events in the Transquaking River, a tributary of Chesapeake Bay. The dynamics of toxic and non-toxic strains were documented via quantification of the microcystin synthetase gene (mcyD) and ribosomal RNA gene, 16S. During the 2-year field study, Microcystis blooms were comprised of >106 cell equivalents L−1 and the percentage of toxic strains ranged from 1 to 100%. The natural microzooplankton community was able to graze on both strains of Microcystis in two-thirds of experiments, with a slight preference for the non-toxic strains in most experiments. Cultured mesozooplankters (Daphnia pulex and Hyalella azteca) grazed at least one strain (toxic or non-toxic) of Microcystis in only one-third of experiments conducted. In contrast, the wild mesozooplankton community grazed at least one strain of Microcystis in every experiment with electivity indices showing a preference towards toxic strains in most experiments. This finding demonstrates that natural mesozooplankton were better grazers of both toxic and non-toxic strains of Microcystis than their cultured counterparts. Overall, both microzooplankton and mesozooplankton were capable of grazing toxic and non-toxic strains of Microcystis with similar success and at similar rates. As such, the ability to synthesize microcystin does not seem to offer toxic Microcystis populations a significant defense against grazing by co-occurring zooplankton communities.

Genetic polymorphism in brazilian microcystis spp. (Cyanobacteria) toxic and non-toxic through RFLP-PCR of the cpcBA-IGS

The escalating occurrence of cyanobacterial toxic blooms demands a better understanding of genetic variability as an auxiliary expedient in species identification, collaborating with the monitoring of water destined to public supply. This study aimed at the unraveling of genetic polymorphism in the toxic and nontoxic strains of Microcystis (Cyanobacteria) species, isolated from diverse Brazilian localities through the RFLP-PCR technique applied to the c-phycocyanin encoding operon and its intergenic spacer (cpcBA-IGS). Eighteen strains belonging to M. aeruginosa, M. panniformis, M. protocystis and M. wesenbergii, plus two other unidentified strains, were analyzed by means of the morphological and molecular data. The molecular data constituted three groups with low similarity values unrelated to the geographical origin, toxicity or morphospecies. A high genetic variability among the studied populations was unveiled by the results. Brazilian populations of Microcystis spp. displayed high genetic diversity when compared to those from Australia, Japan, United States and Europe. This ample genetic diversity could be observed through the diverse eletrophoretic profiles obtained among the strains from a single species. The presence of toxic and non-toxic strains was observed in the same species, as M. aeruginosa.

The effects of temperature and nutrients on the growth and dynamics of toxic and non-toxic strains of Microcystis during cyanobacteria blooms

In temperate latitudes, toxic cyanobacteria blooms often occur in eutrophied ecosystems during warm months. Many common bloom-forming cyanobacteria have toxic and non-toxic strains which co-occur and are visually indistinguishable but can be quantified molecularly. Toxic Microcystis cells possess a suite of microcystin synthesis genes ( mcyA– mcyJ), while non-toxic strains do not. For this study, we assessed the temporal dynamics of toxic and non-toxic strains of Microcystis by quantifying the microcystin synthetase gene ( mcyD) and the small subunit ribosomal RNA gene, 16S (an indicator of total Microcystis), from samples collected from four lakes across the Northeast US over a two-year period. Nutrient concentrations and water quality were measured and experiments were conducted which examined the effects of elevated levels of temperatures (+4 °C), nitrogen, and phosphorus on the growth rates of toxic and non-toxic strains of Microcystis. During the study, toxic Microcystis cells comprised between 12% and 100% of the total Microcystis population in Lake Ronkonkoma, NY, and between 0.01% and 6% in three other systems. In all lakes, molecular quantification of toxic ( mcyD-possessing) Microcystis was a better predictor of in situ microcystin levels than total cyanobacteria, total Microcystis, chlorophyll a, or other factors, being significantly correlated with the toxin in every lake studied. Experimentally enhanced temperatures yielded significantly increased growth rates of toxic Microcystis in 83% of experiments conducted, but did so for non-toxic Microcystis in only 33% of experiments, suggesting that elevated temperatures yield more toxic Microcystis cells and/or cells with more mcyD copies per cell, with either scenario potentially yielding more toxic blooms. Furthermore, concurrent increases in temperature and P concentrations yielded the highest growth rates of toxic Microcystis cells in most experiments suggesting that future eutrophication and climatic warming may additively promote the growth of toxic, rather than non-toxic, populations of Microcystis, leading to blooms with higher microcystin content.

Microcystis genotype succession in relation to microcystin concentrations in freshwater lakes

AME Aquatic Microbial Ecology Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsSpecials AME 48:1-12 (2007) - doi:10.3354/ame048001 Microcystis genotype succession in relation to microcystin concentrations in freshwater lakes W. Edwin A. Kardinaal1,3,*, Ingmar Janse2,4,*, Miranda Kamst-van Agterveld2, Marion Meima2, Josje Snoek1, Luuc R. Mur1, Jef Huisman1, Gabriel Zwart1,2,5, Petra M. Visser1,** 1Aquatic Microbiology, Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, Nieuwe Achtergracht 127, 1018 WS, Amsterdam, The Netherlands 2Department of Microbial Wetland Ecology, Netherlands Institute of Ecology (NIOO-KNAW), Centre for Limnology, Rijksstraatweg 6, 3631 AC Nieuwersluis, The Netherlands 3Present address: DHV consultancy, PO Box 1132, 3800 BC Amersfoort, The Netherlands 4Present address: National Institute for Public Health and the Environment, PO Box 1, 3720 BA Bilthoven, The Netherlands 5Present address: Waterboard Peel en Maasvallei, PO Box 3390, 5902 RJ Venlo, The Netherlands *Both authors contributed equally to this work**Corresponding author. Email: petra.visser@science.uva.nl ABSTRACT: Potentially toxic Cyanobacteria, like Microcystis, form a serious threat in recreational waters and drinking-water reservoirs. We monitored the population dynamics of toxic and non-toxic Microcystis strains using rRNA of the internal transcribed spacer region in combination with DGGE to determine whether there is a seasonal succession of toxic and non-toxic Microcystis genotypes in freshwater lakes and, if so, whether this succession can explain seasonal dynamics of the toxin microcystin. We studied 3 lakes in The Netherlands, all dominated by Microcystis during summer. Coexistence of several genotypes was observed in all lakes. The seasonal succession in a deep, stratified lake started with a population consisting of several toxic genotypes at the onset of the bloom, which changed into a population dominated by non-toxic genotypes at the end of the bloom. In this lake, the genotype succession clearly accounted for the observed microcystin dynamics. In 2 unstratified lakes, we also observed a seasonal replacement of Microcystis genotypes; however, the relation between genotype succession and microcystin dynamics was less conspicuous, since toxic strains dominated throughout the bloom period. A seasonal succession of different Microcystis genotypes might often be a key mechanism determining microcystin concentrations in Microcystisdominated lakes. Therefore, factors driving the succession of toxic and non-toxic genotypes deserve further study. KEY WORDS: Harmful cyanobacteria · Microcystis · Microcystins · Genotypes · Succession · ITS · DGGE Full text in pdf format NextExport citation RSS - Facebook - Tweet - linkedIn Cited by Published in AME Vol. 48, No. 1. Online publication date: June 20, 2007 Print ISSN: 0948-3055; Online ISSN: 1616-1564 Copyright © 2007 Inter-Research.

Analysis of cyanobacteria from Lake Baikal and the Ust-Ilim Reservoir for the gene responsible for microcystin synthesis

Mass growth of cyanobacteria in various aquaticecosystems leads not only to technological and aes-thetic problems, but also to the contamination of waterwith substances toxic for humans and animals [1].Among cyanotoxins, mycrocystins are the best studied;in addition to their toxicity, microcystins have a carci-nogenic effect after a long-term action at low doses.Microcystins are produced by the members of fre-quently occurring cyanobaterial genera, such as Micro-cystis Kutzing, Anabaena, Oscillatoria, and Plankto-thrix Anagnostidis et Komarek [2]. Microcystins arecyclic peptides consisting of seven amino acids thathave the formula (D-Ala-L-X-D-MeAsp-L-Z-Adda-D-Glu-Mdha), where X and Z are variable L-amino acids,Adda is 3-amino-9-methoxy-2,6,8-trimethyl-10-phe-nyl-4,6-dicadinoic acid, D-MeAsp is 3-methylaspar-tamic acid, and Mdha is N-methyldehydroalanine [3].To date, about 60 variants of microcystins are known[4], which are synthesized outside ribosomes by meansof the thiotemplate mechanism [5, 6]. The microcystinsynthetase (mcy) gene cluster has a module structure,where each module is responsible for the synthesis ofspecific enzymes.Toxin production is often observed during waterbloom in eutrophic water bodies. In oligotrophic Alpinelakes, cases of animal poisoning with Microcystisaeruginosa toxins have been also reported [7]. Thus,the risk of toxic cyanobacterial growth is inherent inwaters differing in their trophic conditions. Both toxicand nontoxic strains of Microcystis, which are indistin-guishable in morphology, have been described [8]. InLake Vancy (Germany), microcystins appeared whenthe M. aeruginosa concentration reached 4 × 10

Influence of toxic and non-toxic phytoplankton on feeding and survival of Dreissena polymorpha (Pallas) larvae

Grazing and survival of larvae of the zebra mussel, Dreissena polymorpha, on a green alga and cyanobacteria were studied in laboratory experiments. Clearance rates of the larvae were determined for Chlamydomonas reinhardtii (green alga), two non-toxic and two toxic Microcystis aeruginosa strains (Cyanobacteria). Clearance rates of larvae on non-toxic Microcystis were significantly higher than on toxic Microcystis. The clearance rate on Chlamydomonas reinhardtii was in between the clearance rates on toxic and non-toxic Microcystis strains and not significantly different from them. Effects of toxicity of Microcystis on the survival of zebra mussel larvae was investigated in a short-term experiment. Survival of larvae fed toxic Microcystis was lower than that of larvae fed non-toxic Microcystis, but higher than that of starved larvae. This may imply that, for survival of zebra mussel larvae, it is better to have bad quality (toxic) food than no food.

Quantitative Detection of Toxic Strains of the Cyanobacterial Genus <i>Microcystis</i> by Competitive PCR

When module 1 of the microcystin synthetase gene was isolated from two strains, Microcystis aeruginosa NIES298 and M. viridis NIES102 with different types of module 3, the sequence identity was very high. Twenty toxic strains and 18 non-toxic strains of Microcystis were subjected to PCR amplification with primers (M1F3M1R3) designed from completely conserved regions of module 1. The specific PCR products were amplified from toxic strains and no amplified products were observed in the non-toxic strains. Prior to the development of the competitive PCR, template DNA was isolated from the Microcystis cultures using four isolation methods focused on removing polysaccharide. The potassium xanthogenate-sodium dodecyl sulfate procedure gave a high extraction yield and purity and was used for the preparation of template for competitive PCR. By competitive PCR with M1F3-M1R3, cultured cells of toxic Microcystis were quantitatively detected at 10–10 cells/mL. In environmental water samples from Lake Mikata and Lake Suigetsu, plots of the toxin concentration versus density of toxic cells measured by the competitive PCR showed a positive correlation (r 0.98), which suggested that competitive PCR with M1F3-M1R3 is useful for the quantitative detection of toxic Microcystis cells in complex environmental samples.

Effect of nitrogen and phosphorus on growth of toxic and nontoxic Microcystis strains and on intracellular microcystin concentrations.

The growth and intracellular microcystin concentration of two hepatotoxic and two nontoxic axenic Microcystis strains were measured in batch cultures with variable nitrogen (0.84-84 mg L(-1)) and phosphorus (0.05-5.5 mg L(-1)) concentrations. Growth was estimated by measuring dry weight, optical density, chlorophyll a, and cellular protein concentration. Microcystin concentrations in cells and in culture medium were measured by HPLC analysis. Both nontoxic strains needed less nutrients for their growth at low nutrient concentrations. With high nutrient concentrations the toxic strains grew better than the nontoxic strains. Growth and intracellular microcystin concentration did not correlate in the hepatotoxic strains. Multivariate regression analysis together with mathematical modeling revealed a significant interactive effect of nitrogen and phosphorus, which partly explains the controversial results obtained in previous studies. In this study we have shown that variation of nitrogen and phosphorus concentrations influence the growth and the microcystin production of Microcystis strains and that the strains differ in their response to nutrients. High levels of nitrogen and phosphorus in freshwaters may favor the growth of toxic Microcystis strains over nontoxic ones.