Total Microcystin Research Articles

Multi-scale strategies for the monitoring of freshwater cyanobacteria: Reducing the sources of uncertainty

Cyanobacterial blooms are a frequent phenomenon in eutrophic freshwaters worldwide and are considered potential hazards to ecosystems and human health. Monitoring strategies based on conventional sampling often fail to cover the marked spatial and temporal variations in cyanobacterial distribution and fluctuating toxin concentrations inherent to cyanobacterial blooms. To deal with these problems, we employed a multi-scale approach for the study of a massive Microcystis bloom in Tajo River (Spain) utilizing 1) remote sensing techniques, 2) conventional water sampling and 3) analysis of chemotypical subpopulations. Tajo River at the study area is influenced by high temperatures waters diverted upstream from a nuclear power plant, the presence of a dam downstream and a high nutrient load, which provide optimal conditions for massive cyanobacterial proliferation. MERIS imagery revealed high Chl-a concentrations that rarely fell below 20 μg L−1 and moderate spatiotemporal variations throughout the study period (March–November 2009). Although the phytoplanktonic community was generally dominated by Microcystis, sampling points highly differed in cyanobacterial abundance and community composition. Microcystin (MC) concentrations were highly heterogeneous, varying up to 3 orders of magnitude among sampling points, exceeding in some cases WHO guideline values for drinking and also for recreational waters. The analysis of single colonies by MALDI-TOF MS revealed differences in the proportion of MC-producing colonies among points. The proportion of toxic colonies showed a highly significant linear correlation with total MC: biovolume ratio (r2 = 0.9; p < 0.001), evidencing that the variability in toxin concentrations can be efficiently addressed by simple analysis of subpopulations. We propose implementing a multi-scale monitoring strategy that allows covering the spatiotemporal heterogeneities in both cyanobacterial distribution (remote sensing) and MC concentrations (subpopulation analysis) and thereby reduce the main sources of uncertainty in the assessment of the risks associated to bloom events.

Pseudomonas aeruginosa UCBPP-PA14 a useful bacterium capable of lysing Microcystis aeruginosa cells and degrading microcystins

To identify a useful bacterium capable of controlling both Microcystis aeruginosa and microcystins (MCs), 30 strains of Pseudomonas were screened. Two of them (Pseudomonas aeruginosa UCBPP-PA14 and Pseudomonas putida KCCM 10464) could cause significant lysis of M. aeruginosa. PA14 exhibited higher degradation activity against microcystins than KCCM 10464, and hence, it was selected as the bacterium for further analysis. Following its introduction into M. aeruginosa culture (105 cells mL-1) at densities of 107, 105, and 103 PA14 cells mL-1, higher initial inoculations of PA14 removed correspondingly more M. aeruginosa cells (100%, 100%, and 92% at 15, 30, and 10 days, respectively) and degraded microcystin (extracellular MCs: 83.7%, 77.7%, and 51.6% at 30 days; total MCs: 91.0%, 86.9%, and 61.6% at 30 days, respectively). However, the activity of PA14 diminished when its density decreased to less than 106 cells mL-1. At three initial algal densities (106, 105, and 103 cells mL-1), PA14 at a density of 105 cells mL-1 easily and quickly removed algal cells (100%, 100%, and 97.3% at 8, 16, and 30 days, respectively). Host range assays showed that at lower initial PA14 inoculation (105 cells mL-1), the algicidal activity of PA14 was effective species-specifically on M. aeruginosa, while at higher initial inoculation (107 cells mL-1), a wider algicidal range regardless of the general taxonomical relationships was observed. These results indicate that inoculation with 105 Pseudomonas aeruginosa PA14 cells mL-1 into developing natural algal blooms can remove both M. aeruginosa and MCs without causing problems for other algae species.

Supported liquid membrane-liquid chromatography–mass spectrometry analysis of cyanobacterial toxins in fresh water systems

Harmful algal blooms (HABs) are increasingly becoming of great concern to water resources worldwide due to indiscriminate waste disposal habits resulting in water pollution and eutrophication. When cyanobacterial cells lyse (burst) they release toxins called microcystins (MCs) that are well known for their hepatotoxicity (causing liver damage) and have been found in eutrophic lakes, rivers, wastewater ponds and other water reservoirs. Prolonged exposure to low concentrated MCs are equally of health importance as they are known to be bioaccumulative and even at such low concentration do exhibit toxic effects to aquatic animals, wildlife and human liver cells. The application of common treatment processes for drinking water sourced from HABs infested reservoirs have the potential to cause algal cell lyses releasing low to higher amounts of MCs in finished water. Trace microcystins in water/tissue can be analyzed and quantified using Liquid chromatography–electrospray ionization-mass spectrometry (LC–ESI-MS) following solid-phase extraction (SPE) sample clean-up procedures. However, extracting MCs from algal samples which are rich in chlorophyll pigments and other organic matrices the SPE method suffers a number of drawbacks, including cartridge clogging, long procedural steps and use of larger volumes of extraction solvents. We applied a supported liquid membrane (SLM) based technique as an alternative sample clean-up method for LC–ESI-MS analysis of MCs from both water and algal cells. Four (4) MC variants (MC-RR, -YR, -LR and -WR) from lyophilized cells of Microcystis aeruginosa and water collected from a wastewater pond were identified) and quantified using LC–ESI-MS following a SLM extraction and liquid partitioning step, however, MC-WR was not detected from water extracts. Within 45min of SLM extraction all studied MCs were extracted and pre-concentrated in approximately 15μL of an acceptor phase at an optimal pH 2.02 of the donor phase (sample). The highest total quantifiable intracellular and extracellular MCs were 37.039±0.087μg/g DW and 5.123±0.018μg/L, respectively. The concentrations of MC-RR were the highest from all samples studied recording maximum values of 21.579±0.066μg/g DW and 3.199±0.012μg/L for intracellular and extracellular quantities, respectively.

Increasing Microcystis cell density enhances microcystin synthesis: a mesocosm study

An experimental protocol using mesocosms was established to study the effect of Microcystis sp. cell abundance on microcystin production. The mesocosms (55 L) were set up in a shallow eutrophic lake and received either no (control), low (to simulate a moderate surface accumulation), or high (to simulate a dense surface scum) concentrations of Microcystis sp. cells collected from the lake water adjacent to the mesocosms. In the low- and high-cell addition mesocosms (2 replicates of each), the initial addition of Microcystis sp. cells doubled the starting cell abundance from 500 000 to 1 000 000 cells mL−1, but there was no detectable effect on microcystin quotas. Two further cell additions were made to the high-cell addition mesocosms after 60 and 120 min, increasing densities to 2 900 000 and 7 000 000 cells mL−1, respectively. Both additions resulted in marked increases in microcystin quotas from 0.1 pg cell−1 to 0.60 and 1.38 pg cell−1, respectively, over the 240 min period. Extracellular microcystins accounted for <12% of the total microcystin load throughout the whole experiment. The results of this study indicate a relationship between Microcystis cell abundance and/or mutually correlated environmental parameters and microcystin synthesis.

Adaptation of freshwater mussels to cyanobacterial toxins: Response of the biotransformation and antioxidant enzymes

Freshwater mussels such as the invasive Dreissena polymorpha and the indigenous Unio tumidus nourish by high filtration rates and may accumulate cyanobacteria and their toxins during cyanobacterial blooms. Physiological adaptations to cyanotoxins enable organisms to endure cyanobacterial blooms but may differ between species. Biotransformation and excretion capacities for cyanobacteria and anthropogenic pollutants have been demonstrated for Dreissena polymorpha but less for unionid species. This study compares the activities of biotransformation (glutathione S-transferase, GST) and antioxidant enzymes (superoxide dismutase, SOD and catalase, CAT) in Dreissena polymorpha to Unio tumidus in response to cyanotoxin exposure (10μgL−1 and 50μgL−1 microcystin-LR, respectively, total microcystin from a cyanobacterial crude extract) for 24h and 7d exposure duration. Enzyme activities in Dreissena polymorpha were measured in the whole mussel tissue, digestive gland and in gills and in Unio tumidus in the digestive gland, gills, mantle, foot as well as in the remaining tissue. The sGST was elevated for the entire exposure period in the whole mussel tissue of Dreissena polymorpha but despite higher basal activities in digestive gland and gills of Unio tumidus, it was rather inhibited or unaltered in most of their tissues. Elevated SOD activity indicated oxidative stress response in Dreissena polymorpha, but not in Unio tumidus. The CAT activity was barely affected in both species, rather inhibited in Unio tumidus, despite again higher basal activities in digestive gland and remaining tissue. Compared to the indigenous Unio tumidus, the investigated biotransformation and oxidative stress combating enzymes respond stronger in the invasive Dreissena polymorpha.

Toxic cyanobacterial breakthrough and accumulation in a drinking water plant: A monitoring and treatment challenge

The detection of cyanobacteria and their associated toxins has intensified in recent years in both drinking water sources and the raw water of drinking water treatment plants (DWTPs). The objectives of this study were to: 1) estimate the breakthrough and accumulation of toxic cyanobacteria in water, scums and sludge inside a DWTP, and 2) to determine whether chlorination can be an efficient barrier to the prevention of cyanotoxin breakthrough in drinking water. In a full scale DWTP, the fate of cyanobacteria and their associated toxins was studied after the addition of coagulant and powdered activated carbon, post clarification, within the clarifier sludge bed, after filtration and final chlorination. Elevated cyanobacterial cell numbers (4.7×106cells/mL) and total microcystins concentrations (up to 10mg/L) accumulated in the clarifiers of the treatment plant. Breakthrough of cells and toxins in filtered water was observed. Also, a total microcystins concentration of 2.47μg/L was measured in chlorinated drinking water. Cyanobacterial cells and toxins from environmental bloom samples were more resistant to chlorination than results obtained using laboratory cultured cells and dissolved standard toxins.

Influences of two antibiotic contaminants on the production, release and toxicity of microcystins

The influences of spiramycin and amoxicillin on the algal growth, production and release of target microcystins (MCs), MC-LR, MC-RR and MC-YR, in Microcystis aeruginosa were investigated through the seven-day exposure test. Spiramycin were more toxic to M. aeruginosa than amoxicillin according to their 50 percent effective concentrations (EC 50) in algal growth, which were 1.15 and 8.03 μg/l, respectively. At environmentally relevant concentrations of 100 ng/l–1 μg/l, spiramycin reduced the total MC content per algal cell and inhibited the algal growth, while exposure to amoxicillin led to increases in the total MC content per algal cell and the percentage of extracellular MCs, without affecting the algal growth. Toxicity of MCs in combination with each antibiotic was assessed in the luminescent bacteria test using the toxic unit (TU) approach. The 50 percent effective concentrations for the mixtures (EC 50 mix ) were 0.56 TU and 0.48 TU for MCs in combination with spiramycin and amoxicillin, respectively, indicating a synergistic interaction between MCs and each antibiotic (EC 50 mix <1 TU). After seven-day exposure to 100 ng/l–1 μg/l of antibiotics, spiramycin-treated algal media and amoxicillin-treated algal media showed significantly lower ( p<0.05) and higher ( p<0.05) inhibition on the luminescence of Photobacterium phosphoreum, respectively, compared with the untreated algal medium. These results indicated that the toxicity of MCs were alleviated by spiramycin and enhanced by amoxicillin, and the latter effect would increase threats to the aquatic environment.

Optimal strategies for determination of free/extractable and total microcystins in lake sediment

The optimization of analytical procedures for the quantification of free and total microcystins (MCs) in natural sediments was systematically examined based on solvent extraction and Lemieux oxidation. In this optimized analytical procedure, a sequential solvent extraction using 50% (v/v) methanol and EDTA-sodium pyrophosphate was selected as the optimal extraction solvent for free MCs analysis, after which the purified extracts and sediment residuals were applied to the optimized Lemieux oxidation for determination of total MCs in lake sediments. The optimized procedures were shown to be efficient and reliable for the routine analysis of both free and total MCs in lake sediment samples, as indicated by the minimal adverse impact of sediment organic matter on the recovery of free MCs and yield of MMPB (2-methyl-3-methoxy-4-phenylbutyric acid). Finally, the developed procedures were applied to field sediment samples collected from Lake Dianchi during a bloom season and seven of thirty samples showed positive results.

Initial studies on the occurrence of cyanobacteria and microcystins in Irish lakes

We report the results of a synoptic survey at 14 sites across the north of Ireland undertaken to determine the occurrence of cyanobacteria and their constituent microcystin cyanotoxins. Seven microcystin toxins were tested for, and five of which were found, with MC-LR, MC-RR, and MC-YR being the most prevalent. Gomphosphaeria spp and Microcystis aeruginosa were the most dominant cyanobacterial species encountered. Together with Aphanizomenon flos-aquae, these were the cyanobacteria associated with the highest microcystin concentrations. The occurrence of several microcystin toxins indicates that there may potentially be more than one cyanobacteria species producing microcystins at many sites. Total microcystin concentrations varied over three orders of magnitude dividing the sites into two groups of high (>1000 ngMC/μgChla, six sites) or low toxicity (<200 ngMC/μgChla, eight sites).

The effect of sampling scales on the interpretation of environmental drivers of the cyanotoxin microcystin

Microcystins (MC), a class of cyanobacterial toxins, were measured in one shallow moderately eutrophic lake over 3 years and compared with results from multi-lake regional studies taken from the literature to understand how the choice of sampling regime affects observed correlations between microcystins and environmental conditions. Our results show that correlations between MC seston content and environmental factors are quite dependent upon the temporal sampling scale. The within-year variation in total MC seston content was high, almost an order of magnitude higher than that of the abiotic variables measured and that of algal biomass (Chl-a) but similar to the variation in the density of cladoceran zooplankton. At the temporal scale of seasons, no significant correlations could be detected between MC seston content and environmental variables, but at the monthly scale, pH and temperature were positively correlated with MC seston content. At the weekly scale, reactive phosphate was weakly correlated with MC seston content (with a lag of 12 d) as detected through time series analysis. Furthermore, the factors previously associated with microcystins at a larger spatial scale in among-lake studies (namely total phosphorus, total nitrogen, and light attenuation) did not explain the temporal MC seston content variation within one lake at any sampling scale.

Effects of inorganic arsenic on growth and microcystin production of a Microcystis strain isolated from an algal bloom in Dianchi Lake, China

Our previous data have shown that inorganic arsenic concentrations were high in Dianchi Lake, China, where Microcystis blooms often occur. To explore the relationship between arsenic and the growth of Microcystis, the effects of arsenite [As(III)] and arsenate [As(V)] on the growth and toxin production of M. aeruginosa strain FACHB 905 were tested. Results showed that M. aeruginosa FACHB 905 was tolerant to inorganic arsenic and its growth was not inhibited when the concentration of As(III) was below 10(-5) mol L(-1) or that of As(V) below 10(-3) mol L(-1). Total microcystin production was stimulated in the presence of 10(-7) mol L(-1) As(III) and the response of this M. aeruginosa strain to As(III) seemed to be a typical inverted U-shaped hormesis. The content increase of microcystin-LR per cell indicated that the toxicity was enhanced as M. aeruginosa FACHB 905 was exposed to As(V). Considering the relatively high concentration of inorganic arsenic in Dianchi Lake (139 mu g L(-1) in epilimnetic water), the origin of the M. aeruginosa strain, inorganic arsenic favors survival of M. aeruginosa FACHB 905 and may stimulate its microcystin production and cellular toxicity.

The influence of environmental conditions and hydrologic connectivity on cyanobacteria assemblages in two drowned river mouth lakes

We evaluated the temporal and spatial variability of cyanotoxins, water chemistry, and cyanobacteria communities in two lakes of different trophic status. Bear Lake is a hypereutrophic system that flows into mesotrophic Muskegon Lake. Total microcystins (MC) in Bear Lake (mean, 1.66 μg/L) were composed of multiple structural analogs: 43% MC-LR, 50% MC-RR, and 7% MC-YR. Total microcystins in Muskegon Lake (mean, 0.52 μg/L) consisted of MC-LR (76%), MC-RR (14%), MC-YR (6%), and MC-LA (3%). The lakes were dominated by the cyanobacteria Microcystis spp., which accounted for 75% of phytoplankton biovolume in Bear Lake and > 90% in Muskegon Lake. Total microcystin concentration was positively correlated with cyanobacteria biovolume and turbidity (Muskegon Lake) and total phosphorus (Bear Lake), while negatively correlated with ammonia (Bear Lake) and nitrate (both lakes). The relationships between microcystins and environmental factors differed between lakes, despite hydrologic connectivity, suggesting that local conditions have a greater influence on toxin production than regional effects. Cylindrospermopsis raciborskii was found in both systems; however, the assemblage does not appear to be capable of producing cylindrospermopsin due to the absence of the PKS gene. Although the Bear Lake discharge appears to be the source of C. raciborskii, the physical/chemical properties of Muskegon Lake (lower turbidity and temperature, higher nitrate) may constrain the growth of this invasive species. Thus, local conditions in each lake are important in determining which species are capable of maintaining a viable population.

Seasonal development of cyanobacteria and microcystin production in Ugandan freshwater lakes

AbstractThis study investigated the seasonal development of phytoplankton and potential microcystin (MC)‐producing cyanobacteria and MC concentrations in freshwater lakes in Uganda. During 1 year (May 2007–April 2008), monthly measurements were made of chemical and physical characteristics, phytoplankton composition and MC concentrations in a hypertrophic crater lake (Lake Saka), in shallow eutrophic lakes (Lakes Mburo, George and Edward) and in Lake Victoria (Murchison Bay, Napoleon Gulf). Throughout the study period, cyanobacteria (composed of the genera Anabaena, Aphanocapsa, Chroococcus, Cylindrospermopsis, Microcystis, Planktolyngbya and Planktothrix) dominated, always contributing &gt;50% to the total phytoplankton biovolume. All samples from all sampling sites were found to contain MC. Samples from Lake Saka had the maximum MC concentration (10 μg L−1) in July 2007. The minimum concentration (0.02 μg L−1) was recorded in Lake George for the months of May and June 2007 and January and April, 2008. Intermediate MC concentrations (0.1–2.5 μg MC‐LR eq. L−1) were observed at all the sampling sites in the other three lakes. Highly significant positive linear relationships between the total MC concentration and Microcystis cell numbers were observed for all sampling sites. Relating the total MC concentrations to Microcystis cells revealed a &gt;100‐fold variation in the average MC contents per cell between lakes. While Microcystis from Lake George exhibited the lowest MC cell quotas (0.03–1.24 fg cell−1), Microcystis from Lake Saka consistently exhibited maximum MC cell contents (14–144 fg cell−1). It was concluded that MC production was because of the occurrence of Microcystis at all sampling sites. The populations differed consistently between sites and independent of the season in their average MC content per cell.

Accumulation of Microcystins in Nile Tilapia, Oreochromis niloticus L., and Effects of a Complex Cyanobacterial Bloom on the Dietetic Quality of Muscles

The aim of this study was to investigate the kinetics of accumulation and elimination of microcystins in the tissues of Nile tilapia (Oreochromis niloticus) and to evaluate the effect of cyanobacterial exposure on fish muscle quality (levels of total fat and ash, protein, dry matter and the composition of saturated, monounsaturated and polyunsaturated fatty acids). Fish were exposed for 28 days to a natural cyanobacterial bloom with total microcystin concentration around 1,200 μg g⁻¹ biomass dry weight. The hepatopancreas accumulated microcystins up to 350 ng g⁻¹ fresh weight, but concentrations in muscle were generally below the detection limit (2 ng g⁻¹ fresh weight). Following the exposure, fish were moved to the clean water, but only minor microcystin removal from the hepatopancreas was observed during a 4 week depuration period. Exposures of tilapia to the complex cyanobacterial bloom had only minor and temporary impacts on the investigated parameters of dietetic quality.

Switching toxin production on and off: intermittent microcystin synthesis in a Microcystis bloom

Toxic cyanobacterial blooms are increasing in prevalence. Microcystins are the most commonly produced cyanotoxin. Despite extensive research the variables regulating microcystin production remain unclear. Using a RT-QPCR assay that allowed the precise measurement of mcyE transcriptional gene expression and an ELISA that enabled small changes in total microcystin concentrations to be monitored, we demonstrate for the first time that microcystin production is not always constitutive and that significant up- and downregulation in microcystin synthesis can occur on time scales of 2-6 h. Samples were collected over 3 days from a small eutrophic lake during a dense microcystin-producing Microcystis bloom. McyE gene transcripts were detected in only four out of 14 samples. Vicissitudes in both microcystin quotas and extracellular microcystin levels corresponded with changes in mcyE expression. During the period of exalted microcystin synthesis Microcystis sp. cell concentrations increased from 70 000 cells ml(-1) to 4 000 000 cells ml(-1) . These data provide compelling evidence that changes in Microcystis cell concentrations influence microcystin production.

Cyanotoxin production in seven Ethiopian Rift Valley lakes

We hypothesized that unusual deaths and illnesses in wild and domestic animals in lake areas of the Rift Valley south of Addis Ababa were caused by toxic cyanobacteria. In the first cyanotoxic analyses conducted in samples from Ethiopia, we found lakes Chamo, Abaya, Awassa, Chitu, Langano, Ziway, and Koka all had concentrations of microcystins (MC) ranging from trace to hazardous, whereas only traces less than limits of detection (LOD) of cylindrospermopsin (CYN) were found. In the December 2006 dry season we sampled the lakes for analyses of MC, CYN, species structures, and calculations of cyanobacteria biomass. We used the Utermöhl technique to analyse cyanobacterial biomass and monitored MC toxins using HPLC-DAD, LC-ESI-MS-MRM, and ELISA-test and CYN with HPLC-DAD and ELISA. The various toxicity tests coincided well. In 4 of the lakes (Chamo, Langano, Ziway, and Koka), the inter-lake range of total MC concentration was 1.3–48 μg L−1; in 3 (Abaya, Awassa, and Chitu), we found only traces of MC. Microcystis aeruginosawas the dominant species, with Microcystis panniformis, Anabaena spiroides, and Cylindrospermopsisspp. as subdominants. The MC concentration, especially in Lake Koka, exceeded levels for serious health hazards for humans, cattle, and wildlife.

Cyanotoxin Mixtures and Taste-and-Odor Compounds in Cyanobacterial Blooms from the Midwestern United States

The mixtures of toxins and taste-and-odor compounds present during cyanobacterial blooms are not well characterized and of particular concern when evaluating potential human health risks. Cyanobacterial blooms were sampled in twenty-three Midwestern United States lakes and analyzed for community composition, thirteen cyanotoxins by liquid chromatography/mass spectrometry and immunoassay, and two taste-and-odor compounds by gas chromatography/mass spectrometry. Aphanizomenon, Cylindrospermopsis and/or Microcystis were dominant in most (96%) blooms, but community composition was not strongly correlated with toxin and taste-and-odor occurrence. Microcystins occurred in all blooms. Total microcystin concentrations measured by liquid chromatography/mass spectrometry and immunoassay were linearly related (r(s) = 0.76, p < 0.01) and LC/MS/MS concentrations were lower than or similar to ELISA in most (85%) samples. Geosmin (87%), 2-methylisoborneol (39%), anatoxin-a (30%), saxitoxins (17%), cylindrospermopsins (9%), and nodularin-R (9%) also were present in these blooms. Multiple classes of cyanotoxins occurred in 48% of blooms and 95% had multiple microcystin variants. Toxins and taste-and-odor compounds frequently co-occurred (91% of blooms), indicating odor may serve as a warning that cyanotoxins likely are present. However, toxins occurred more frequently than taste-and-odor compounds, so odor alone does not provide sufficient warning to ensure human-health protection.

Real-Time PCR법과 LC/MS법을 이용한 수계중의 마이크로시스틴 검출방법 비교연구

We performed a comparative analysis using a Real-time PCR (Polymerase Chain Reaction) and LC/MS (Liquid-Chromatograph/Mass Spectrometer) method in order to detect microcystin in environmental sources. Among the three different primer sets tested for microcystin using three positive strains of Microcystis aeruginosa by Real-time PCR assay, only TOX2P/TOX2M primer pairs were able to detect Microcystis aeruginosa. According to the results of a survey carried out from June 2009 to September 2009, 11 out of 11 (100%) raw water samples were were found to have microcystin when the Real-Time PCR and LC/MS method was used, with total microcystin concentration ranging from 5.98~219.0 ㎍/l. A microcystin removal treatment process was used to ensure entire removal, by passing it through a BAC filtration step. It was concluded that real-time PCR assay can be used to estimate micrucystin detection more rapidly and easily than the LC/MS method.

Detection of Microcystin-Producing Cyanobacteria in Missisquoi Bay, Quebec, Canada, Using Quantitative PCR

Toxic cyanobacterial blooms, as well as their increasing global occurrence, pose a serious threat to public health, domestic animals, and livestock. In Missisquoi Bay, Lake Champlain, public health advisories have been issued from 2001 to 2009, and local microcystin concentrations found in the lake water regularly exceeded the Canadian drinking water guideline of 1.5 microg liter(-1). A quantitative PCR (Q-PCR) approach was developed for the detection of blooms formed by microcystin-producing cyanobacteria. Primers were designed for the beta-ketoacyl synthase (mcyD(KS)) and the first dehydratase domain (mcyD(DH)) of the mcyD gene, involved in microcystin synthesis. The Q-PCR method was used to track the toxigenic cyanobacteria in Missisquoi Bay during the summers of 2006 and 2007. Two toxic bloom events were detected in 2006: more than 6.5 x 10(4) copies of the mcyD(KS) gene ml(-1) were detected in August, and an average of 4.0 x 10(4) copies ml(-1) were detected in September, when microcystin concentrations were more than 4 microg liter(-1) and approximately 2 microg liter(-1), respectively. Gene copy numbers and total microcystin concentrations (determined by enzyme-linked immunosorbent assay [ELISA]) were highly correlated in the littoral (r = 0.93, P < 0.001) and the pelagic station (r = 0.87, P < 0.001) in 2006. In contrast to the situation in 2006, a cyanobacterial bloom occurred only in late summer-early fall of 2007, reaching only 3 x 10(2) mcyD(KS) copies ml(-1), while the microcystin concentration was barely detectable. The Q-PCR method allowed the detection of microcystin-producing cyanobacteria when toxins and toxigenic cyanobacterial abundance were still below the limit of detection by high-pressure liquid chromatography (HPLC) and microscopy. Toxin gene copy numbers grew exponentially at a steady rate over a period of 7 weeks. Onshore winds selected for cells with a higher cell quota of microcystin. This technique could be an effective approach for the routine monitoring of the most at-risk water bodies.

Spatiotemporal Variations in Microcystin Concentrations and in the Proportions of Microcystin-Producing Cells in Several Microcystis aeruginosa Populations

With the aim of explaining the variations in microcystin (MC) concentrations during cyanobacterial blooms, we studied several Microcystis aeruginosa populations blooming in different freshwater ecosystems located in the same geographical area. As assessed by real-time PCR, it appeared that the potentially MC-producing cells (mcyB(+)) were predominant (70 to 100%) in all of these M. aeruginosa populations, with the exception of one population in which non-MC-producing cells always dominated. Apart from the population in the Grangent Reservoir, we found that the proportions of potentially MC-producing and non-MC-producing cells varied little over time, which was consistent with the fact that according to a previous study of the same populations, the intergenic transcribed spacer (ITS) genotype composition did not change (38). In the Grangent Reservoir, the MC-RR variant was the dominant microcystin variant throughout the bloom season, despite changes in the ITS composition and in the proportions of mcyB(+) cells. Finally, the variations in total MC concentrations (0.3 to 15 microg liter(-1)) and in the MC cellular quotas (0.01 to 3.4 pg cell(-1)) were high both between and within sites, and no correlation was found between the MC concentrations and the proportion of mcyB(+) cells. All of these findings demonstrate that very different results can be found for the proportions of potentially MC-producing and non-MC-producing cells and MC concentrations, even in M. aeruginosa populations living in more or less connected ecosystems, demonstrating the importance of the effect of very local environmental conditions on these parameters and also the difficulty of predicting the potential toxicity of Microcystis blooms.