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.     

Molecular genetic identification and seasonal succession of toxigenic cyanobacteria in phytoplankton of the Rybinsk Reservoir (Russia)

Data on the hepato- and neurotoxigenic cyanobacteria in phytoplankton of the Rybinsk Reservoir have been obtained for the first time. Different methods for revealing toxigenic cyanobacteria (light microscopy, PCR analysis, and enzyme-linked immunosorbent assay) demonstrate the same results. Hepatotoxins microcystins and for the first time neurotoxins saxitoxins were detected in the reservoir, whereas cylindrospermopsin and anatoxin-a were not revealed. The presence of mcyE and stxA genes responsible for microcystin and saxitoxin biosynthesis in total phytoplanktonic DNA is demonstrated. The following three genera of cyanobacteria containing mcyE gene are identified: Microcystis (M. aeruginosa, M. viridis), Planktothrix (P. agardhii), and Dolichospermum (Anabaena). It is hypothesized that saxitoxin-producing cyanobacteria Dolichospermum (Anabaena) inhabit the Rybinsk Reservoir.     

Development of a Universal Microarray Based on the Ligation Detection Reaction and 16S rRNA Gene Polymorphism To Target Diversity of Cyanobacteria

The cyanobacteria are photosynthetic prokaryotes of significant ecological and biotechnological interest, since they strongly contribute to primary production and are a rich source of bioactive compounds. In eutrophic fresh and brackish waters, their mass occurrences (water blooms) are often toxic and constitute a high potential risk for human health. Therefore, rapid and reliable identification of cyanobacterial species in complex environmental samples is important. Here we describe the development and validation of a microarray for the identification of cyanobacteria in aquatic environments. Our approach is based on the use of a ligation detection reaction coupled to a universal array. Probes were designed for detecting 19 cyanobacterial groups including Anabaena/Aphanizomenon, Calothrix, Cylindrospermopsis, Cylindrospermum, Gloeothece, halotolerants, Leptolyngbya, Palau Lyngbya, Microcystis, Nodularia, Nostoc, Planktothrix, Antarctic Phormidium, Prochlorococcus, Spirulina, Synechococcus, Synechocystis, Trichodesmium, and Woronichinia. These groups were identified based on an alignment of over 300 cyanobacterial 16S rRNA sequences. For validation of the microarrays, 95 samples (24 axenic strains from culture collections, 27 isolated strains, and 44 cloned fragments recovered from environmental samples) were tested. The results demonstrated a high discriminative power and sensitivity to 1 fmol of the PCR-amplified 16S rRNA gene. Accurate identification of target strains was also achieved with unbalanced mixes of PCR amplicons from different cyanobacteria and an environmental sample. Our universal array method shows great potential for rapid and reliable identification of cyanobacteria. It can be easily adapted to future development and could thus be applied both in research and environmental monitoring.     

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.     

PCR-based identification of microcystin-producing genotypes of different cyanobacterial genera

Microcystins are harmful hepatotoxins produced by many, but not all strains of the cyanobacterial genera Anabaena, Microcystis, Anabaena, Planktothrix, and Nostoc. Waterbodies have to be monitored for the mass development of toxic cyanobacteria; however, because of the close genetic relationship of microcystin-producing and non-producing strains within a genus, identification of microcystin-producers by morphological criteria is not possible. The genomes of microcystin-producing cells contain mcy genes coding for the microcystin synthetase complex. Based on the sequence information of mcy genes from Microcystis and Planktothrix, a primer pair for PCR amplification of a mcyA gene fragment was designed. PCR with this primer pair is a powerful means to identify microcystin-producing strains of the genera Anabaena, Microcystis, and Planktothrix. Moreover, subsequent RFLP analysis of the PCR products generated genus-specific fragments and allowed the genus of the toxin producer to be identified. The assay can be used with DNA from field samples.Abbreviations RFLP Restriction fragment length polymorphism - MALDI-TOF Matrix-assisted laser desorption/ionization-time of flight spectrometry - HPLC High performance liquid chromatography     

Phylogenies of Microcystin-Producing Cyanobacteria in the Lower Laurentian Great Lakes Suggest Extensive Genetic Connectivity

Lake St. Clair is the smallest lake in the Laurentian Great Lakes system. MODIS satellite imagery suggests that high algal biomass events have occurred annually along the southern shore during late summer. In this study, we evaluated these events and tested the hypothesis that summer bloom material derived from Lake St. Clair may enter Lake Erie via the Detroit River and represent an overlooked source of potentially toxic Microcystis biomass to the western basin of Lake Erie. We conducted a seasonally and spatially resolved study carried out in the summer of 2013. Our goals were to: 1) track the development of the 2013 summer south-east shore bloom 2) conduct a spatial survey to characterize the extent of toxicity, taxonomic diversity of the total phytoplankton population and the phylogenetic diversity of potential MC-producing cyanobacteria (Microcystis, Planktothrix and Anabaena) during a high biomass event, and 3) compare the strains of potential MC-producers in Lake St. Clair with strains from Lake Erie and Lake Ontario. Our results demonstrated a clear predominance of cyanobacteria during a late August bloom event, primarily dominated by Microcystis, which we traced along the Lake St. Clair coastline downstream to the Detroit River''s outflow at Lake Erie. Microcystin levels exceeded the Province of Ontario Drinking Water Quality Standard (1.5 µg L⁻¹) for safe drinking water at most sites, reaching up to five times this level in some areas. Microcystis was the predominant microcystin producer, and all toxic Microcystis strains found in Lake St. Clair were genetically similar to toxic Microcystis strains found in lakes Erie and Ontario. These findings suggest extensive genetic connectivity among the three systems.     

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.     

Grazing on colonial and filamentous, toxic and non-toxic cyanobacteria by the zebra mussel Dreissena polymorpha

Colony forming and toxic cyanobacteria form a problem in surfacewaters of shallow lakes, both for recreation and wildlife. Zebramussels, Dreissena polymorpha, have been employed to help torestore shallow lakes in the Netherlands, dominated by cyanobacteria,to their former clear state. Zebra mussels have been presentin these lakes since they were created in the 19th century bythe excavation of peat and are usually not considered to bean invasive species. Most grazing experiments using Dreissenahave been performed with uni-cellular phytoplankton laboratorystrains and information on grazing of larger phytoplankton taxahardly exists. To gain more insight in to whether D. polymorphais indeed able to decrease cyanobacteria in the phytoplankton,we therefore performed grazing experiments with zebra musselsand two species of cyanobacteria, that greatly differ in shape:colony forming strains of Microcystis aeruginosa and the filamentousspecies Planktothrix agardhii. For both species a toxic anda non-toxic strain was selected. We found that zebra musselscleared toxic Planktothrix at a higher rate than non-toxic Planktothrix,toxic or non-toxic Microcystis. Clearance rates between theother strains were not significantly different. Both phytoplanktonspecies, regardless of toxicity, size and shape, were foundin equal amounts (based on chlorophyll concentrations) in theexcreted products of the mussels (pseudofaeces). The resultsshow that zebra mussels are capable of removing colonial andfilamentous cyanobacteria from the water, regardless of whetherthe cyanobacteria are toxic or not. This implies that the musselsmay be used as a biofilter for the removal of harmful cyanobacterialblooms in shallow (Dutch) lakes where the mussels are alreadypresent and not a nuisance. Providing more suitable substratefor zebra mussel attachment may lead to appropriate mussel densitiescapable of filtering large quantities of cyanobacteria.     

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.     

Comparison of Quantitative PCR and Droplet Digital PCR Multiplex Assays for Two Genera of Bloom-Forming Cyanobacteria,Cylindrospermopsis and Microcystis

The increasing occurrence of harmful cyanobacterial blooms, often linked to deteriorated water quality and adverse public health effects, has become a worldwide concern in recent decades. The use of molecular techniques such as real-time quantitative PCR (qPCR) has become increasingly popular in the detection and monitoring of harmful cyanobacterial species. Multiplex qPCR assays that quantify several toxigenic cyanobacterial species have been established previously; however, there is no molecular assay that detects several bloom-forming species simultaneously. Microcystis and Cylindrospermopsis are the two most commonly found genera and are known to be able to produce microcystin and cylindrospermopsin hepatotoxins. In this study, we designed primers and probes which enable quantification of these genera based on the RNA polymerase C1 gene for Cylindrospermopsis species and the c-phycocyanin beta subunit-like gene for Microcystis species. Duplex assays were developed for two molecular techniques—qPCR and droplet digital PCR (ddPCR). After optimization, both qPCR and ddPCR assays have high linearity and quantitative correlations for standards. Comparisons of the two techniques showed that qPCR has higher sensitivity, a wider linear dynamic range, and shorter analysis time and that it was more cost-effective, making it a suitable method for initial screening. However, the ddPCR approach has lower variability and was able to handle the PCR inhibition and competitive effects found in duplex assays, thus providing more precise and accurate analysis for bloom samples.     

Multiplex PCR for the detection of toxigenic cyanobacteria in dietary supplements produced for human consumption

The production of food supplements containing cyanobacteria is a growing worldwide industry. While there have been several reports of health benefits that can be gained from the consumption of these supplements, there have also been a growing number of studies showing the presence of toxins some of which (for example microcystins) are known to affect human health. In this paper, we report a multiplex polymerase chain reaction (PCR) technique that can be used to identify microcystin contamination in dietary supplements produced for human consumption. This method involves a PCR reaction containing three primer pairs, the first of which is used to amplify a 220-bp fragment of 16s rDNA specific to Microcystis, the most common microcystin-producing cyanobacterium. The second primer pair is used to amplify a 300-bp fragment of the mcyA gene, linked to microcystin biosynthesis in Anabaena, Microcystis, and Planktothrix. A third primer pair, used as a positive control, results in the amplification of a 650-bp fragment from the phycocyanin operon common to all cyanobacteria. This technique was found to be useful for detecting the presence of toxigenic Microcystis in all dietary supplements produced from the nontoxic cyanobacterium Aphanizomenon flos-aquae.     

Molecular techniques for the early warning of toxic cyanobacteria blooms in freshwater lakes and rivers

The aim of this work was to test the efficacy of molecular techniques for detecting toxigenic cyanobacteria in environmental water samples collected from freshwater lakes, rivers and reservoirs in Portugal. Of 26 environmental samples tested, 21 were found to contain Microcystis using a genus-specific polymerase chain reaction (PCR). Another primer pair was applied to the same DNA template to test for the presence of microcystin synthetase genes. This primer pair resulted in the formation of a PCR product in 15 of the samples containing Microcystis and one sample that did not give a positive result in the Microcystis genus-specific PCR. A restriction assay using the enzyme EcoRV was then applied to show that in most cases, the gene fragment was from toxigenic strains of Microcystis and, in one above-mentioned case, from a microcystin-producing strain of Planktothrix. All environmental samples were examined microscopically to confirm the presence of cyanobacteria species. Samples were also tested for the presence of microcystins using the ELISA plate assay. There was good agreement between the results obtained with molecular techniques and those obtained from microscopy and chemical methods. The PCR techniques applied in this paper were found to be useful, particularly when the concentration of the target organism was very low compared with other organisms. This technique can be used to detect inocula for cyanobacterial populations and therefore provide a useful tool for assessing under which conditions particular species can grow into bloom populations.     

Molecular (PCR-DGGE) versus morphological approach: analysis of taxonomic composition of potentially toxic cyanobacteria in freshwater lakes

Background

The microscopic Utermöhl method is commonly used for the recognition of the presence and taxonomic composition of potentially toxic cyanobacteria and is especially useful for monitoring reservoirs used as drinking water, recreation and fishery resources. However, this method is time-consuming and does not allow potentially toxic and nontoxic cyanobacterial strains to be distinguished. We have developed a method based on denaturing gradient gel electrophoresis (DGGE) of the marker gene ITS and the mcy-gene cluster, and DNA sequencing. We have attempted to calibrate the DGGE-method with a microscopic procedure, using water samples taken in 2011 from four lakes of the Great Mazurian Lakes system.

Results

Results showed that the classic microscopic method was much more precise and allowed the classification of the majority of cyanobacterial taxa to the species or genus. Using the molecular approach, most of the sequences could only be assigned to a genus or family. The results of DGGE and microscopic analyses overlapped in the detection of the filamentous cyanobacteria. For coccoid cyanobacteria, we only found two taxa using the molecular method, which represented 17% of the total taxa identified using microscopic observations. The DGGE method allowed the identification of two genera of cyanobacteria (Planktothrix and Microcystis) in the studied samples, which have the potential ability to produce toxins from the microcystins group.

Conclusions

The results confirmed that the molecular approach is useful for the rapid detection and taxonomic distinction of potentially toxic cyanobacteria in lake-water samples, also in very diverse cyanobacterial communities. Such rapid detection is unattainable by other methods. However, with still limited nucleotide sequences deposited in the public databases, this method is currently not sufficient to evaluate the entire taxonomic composition of cyanobacteria in lakes.     

Diversity of Microcystin-Producing Cyanobacteria in Spatially Isolated Regions of Lake Erie

The diversity of microcystin-producing cyanobacteria in the western basin of Lake Erie was studied using sequence analysis of mcyA gene fragments. Distinct populations of potentially toxic Microcystis and Planktothrix were found in spatially isolated locations. This study highlights previously undocumented diversity of potentially toxic cyanobacteria.     

The apoptosis-inducing activity towards leukemia and lymphoma cells in a cyanobacterial culture collection is not associated with mouse bioassay toxicity

Cyanobacteria (83 strains and seven natural populations) were screened for content of apoptosis (cell death)-inducing activity towards neoplastic cells of the immune (jurkat acute T-cell lymphoma) and hematopoetic (acute myelogenic leukemia) lineage. Apoptogenic activity was frequent, even in strains cultured for decades, and was unrelated to whether the cyanobacteria had been collected from polar, temperate, or tropic environments. The activity was more abundant in the genera Anabaena and Microcystis compared to Nostoc, Phormidium, Planktothrix, and Pseudanabaena. Whereas the T-cell lymphoma apoptogens were frequent in organic extracts, the cell death-inducing activity towards leukemia cells resided mainly in aqueous extracts. The cyanobacteria were from a culture collection established for public health purposes to detect toxic cyanobacterial blooms, and 54 of them were tested for toxicity by the mouse bioassay. We found no correlation between the apoptogenic activity in the cyanobacterial isolates with their content of microcystin, nor with their ability to elicit a positive standard mouse bioassay. Several strains produced more than one apoptogen, differing in biophysical or biological activity. In fact, two strains contained microcystin in addition to one apoptogen specific for the AML cells, and one apoptogen specific for the T-cell lymphoma. This study shows the potential of cyanobacterial culture collections as libraries for bioactive compounds, since strains kept in cultures for decades produced apoptogens unrelated to the mouse bioassay detectable bloom-associated toxins.     

Rapid quantification of mcyB copy numbers on dry chemistry PCR chips and predictability of microcystin concentrations in freshwater environments

Microcystin-producing cyanobacteria cause serious water quality problems worldwide, which has led to growing pressure for more intensive monitoring. Molecular biology methods that are based on identification and enumeration of biosynthetic genes, such as quantitative PCR, show promise in this respect. To be practical in a wide range of settings, these methods need to be usable also by laboratory personnel who do not have previous experience in PCR setup. Here we present a real-time quantitative mcyB dry chemistry PCR assay capable of identifying the three globally most common microcystin-producing cyanobacterial genera, Anabaena, Microcystis and Planktothrix. It minimizes the amount of liquid handling and avoids direct contact with the PCR reagents at the time of analysis. Large quantities of virtually identical chips can be manufactured, improving the comparability of results. Using the dry chemistry PCR chips, freshwater environmental samples from Finnish and Estonian lakes, rivers and reservoirs were analyzed for mcyB. The chip format was found to be highly suitable for water sample analysis due to its ease-of-use, good sensitivity and amplification efficiency. Significant positive correlation (Spearman's rank correlation, ρ > 0.66, P < 0.001) was observed between combined mcyB copy numbers from Microcystis, Anabaena, Planktothrix and total microcystin concentrations, regardless of the method used to measure the toxins (ELISA or LC–MS). Positive correlations were observed also for single lakes.     

Toxic cyanobacteria (blue-green algae) in Finnish fresh and coastal waters

A survey of the occurrence of toxic blooms of cyanobacteria in Finnish fresh and coastal waters was made during 1985 and 1986. Toxicity of the freeze-dried water bloom samples was tested by mouse-bioassay (i.p.). Forty-four per cent (83/188) of the bloom samples were found to be lethally toxic. Hepatotoxic blooms (54) were almost twice as common as neurotoxic ones (29). Anabaena was the most frequently found genus in toxic and non-toxic blooms and it was present in all neurotoxic samples. Statistical associations were found between hepatotoxicity and incidence of Microcystis aeruginosa, M. viridis, M. wesenbergii, Anabaena flos-aquae and Anabaena spiroides. Neurotoxicity was statistically associated with Anabaena lemmermannii, Anabaena flos-aquae and Gomphosphaeria naegeliana. Isolation of strains of cyanobacteria confirmed the occurrence of hepatotoxic and neurotoxic strains of Anabaena, as well as hepatotoxic strains of Microcystis and Oscillatoria species.Toxic blooms caused cattle poisonings at three different lakes during the study period. Toxic blooms also occurred in drinking water sources. Our study shows that toxic cyanobacteria are more common in Finnish lakes than would be expected on the basis of animal poisonings. The results of this study show the existence of toxic cyanobacteria in Finnish water supplies and the need for their continued study as agents of water based disease.     

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.     

Recombination and selectional forces in cyanopeptolin NRPS operons from highly similar,but geographically remote <Emphasis Type="Italic">Planktothrix</Emphasis> strains

Background  

Cyanopeptolins are nonribosomally produced heptapetides showing a highly variable composition. The cyanopeptolin synthetase operon has previously been investigated in three strains from the genera Microcystis, Planktothrix and Anabaena. Cyanopeptolins are displaying protease inhibitor activity, but the biological function(s) is (are) unknown. Cyanopeptolin gene cluster variability and biological functions of the peptide variants are likely to be interconnected.     

Estimating toxic cyanobacteria in a Brazilian reservoir by quantitative real-time PCR, based on the microcystin synthetase D gene

The production of microcystin toxins by cyanobacteria is an intrapopulation feature and the toxic and nontoxic genotypes can be separated only through molecular analyses targeting the mcy markers. Quantitative real-time PCR (qPCR) is a procedure that has been established, not only to detect but to specifically quantify these genotypes. In the present work, primers were designed for the mcyD region to estimate the number of cyanobacteria that are potential microcystin producers. Laboratory tests to verify the efficiency and the specificity of the primers were performed. The methodology was first established for single strain cultures and thereafter was applied in environmental water samples, from a reservoir located in the Brazilian savannah (“cerrado”). The results were very satisfactory, demonstrating the high efficiency and the specificity of the primers used, and their ability to detect different cyanobacteria genera. Of particular interest were the results showing a high proportion of toxic strains (as high as 100 %) in the environmental samples, as previously reported in another tropical system. Furthermore, the occurrence of a smaller fraction of toxic strains at high cyanobacteria densities, and of more toxic populations when fewer cyanobacteria were present, deserves further investigation. Although records of cyanobacteria blooms are very common in the tropics and suggest an increasing incidence of toxic populations, the present research is one of the few applying qPCR in a tropical environment. The results obtained here, by a technique that allows a more precise quantification and in situ follow-up of changes in toxicity, will make possible new observations of seasonal and spatial dynamics in these environments.