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.     

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.     

广东省水库微囊藻的产毒特征和ITS序列的遗传多样性分析

为了解广东省水库微囊藻的产毒特征和ITS 序列的遗传多样性,从广东省供水水库中分离得到28 株微囊藻(Microcystisspp.),对它们的产毒特征和15 株微囊藻的ITS 序列进行了分析.高效液相色谱(HPLC)和微囊藻毒素合成酶基因mcyE 的检测结果表明,广东省水库中的微囊藻以产毒藻株占优势,微囊藻毒素的主要类型为MC-RR.广东省15 株藻株的ITS 序列相似性大于93.2%,在用相邻法(NJ)构建的系统树上,不同形态的种和不同地理区域的藻株没有区分开,产毒和非产毒藻株没有形成独立分支.这说明微囊藻ITS 序列的遗传多样性较低,ITS 序列和mcyE 存在没有相关性,表型不能够反映藻株的进化关系.因此,有必要将藻类传统分类方法与分子方法结合起来对蓝藻进行重新分类.     

Development and application of a multiplex qPCR technique to detect multiple microcystin-producing cyanobacterial genera in a Canadian freshwater lake

The emergence and persistence of complex blooms comprising multiple toxigenic cyanobacteria genera pose significant challenges for water quality management worldwide. The co-occurrence of morphologically indistinguishable toxic and non-toxic strains makes monitoring and control of these noxious organisms particularly challenging. Conventional monitoring approaches are not only incapable of discriminating toxic from non-toxic strains but also have proven to be less sensitive and specific. In this study, a multiplex quantitative real-time polymerase chain reaction (qPCR) approach was developed and tested for its sensitivity and specificity at detecting, differentiating and estimating potentially toxic Anabaena, Microcystis and Planktothrix genotype compositions in environmental samples. The oligonucleotide primers and probes utilized were designed to target portions of the microcystin synthetase (mcy) E gene that encode synthesis of the unique 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid (ADDA) moiety of microcystins in the three target genera. Laboratory evaluation showed the developed assay to be highly sensitive and specific at detecting and quantifying targeted genera. Indeed, the assay standards for the Anabaena, Microcystis and Planktothrix reactions attained efficiencies above 90 %, with coefficients of determination consistently above 0.99. Analysis of water samples from Missisquoi Bay, Quebec, Canada, resulted in successful detection and quantification of target toxigenic cyanobacteria even when cell numbers were below the detection limit for the conventional microscopy methods. Furthermore, toxigenic Microcystis spp. were found to be the main putative microcystin-producing cyanobacteria in the study lake. The qPCR technique developed in this study therefore offers simultaneous detection, differentiation and quantification of multiple toxigenic cyanobacteria that otherwise cannot be accomplished by current monitoring approaches.     

Detection of potential microcystin-producing cyanobacteria in Brazilian reservoirs with a mcyB molecular marker

One of the most serious problems related to water eutrophication is the occurrence of increasingly frequent blooms of toxic cyanobacteria in freshwater ecosystems. Microcystin (MCYST) molecular markers may be used for the detection of toxic cyanobacteria, both cultivated strains and environmental samples, independently of their taxonomic category and production of the toxin at the moment of analysis. Sixty Microcystis spp. strains from 15 water reservoirs of south, southeastern and northeastern Brazil were analyzed by polymerase chain reaction (PCR) with oligonucleotide primers for mcyB gene of the operon that encodes a microcystin synthetase. It was found out that the presence of a unique amplified product of approximately 780 bp in 18 strains, indicated the presence of the microcystin-producing genotype. There was correspondence between the presence of the mcyB gene and microcystin determined by ELISA. Eight reservoirs contained toxic strains, two of these reservoirs being used mainly for public water supply. The coexistence of a mixture of toxic and non-toxic genotypes in populations of several reservoirs was found. Thus, it is evident that Microcystis populations present in blooms compose a mosaic, with genetically different individuals within the same population, each one, possibly, with its own tolerance to environmental factors and with distinct toxicity potential.     

Estimating microcystin levels at recreational sites in western Lake Erie and Ohio

Cyanobacterial harmful algal blooms (cyanoHABs) and associated toxins, such as microcystin, are a major global water-quality issue. Water-resource managers need tools to quickly predict when and where toxin-producing cyanoHABs will occur. This could be done by using site-specific models that estimate the potential for elevated toxin concentrations that cause public health concerns. With this study, samples were collected at three Ohio lakes to identify environmental and water-quality factors to develop linear-regression models to estimate microcystin levels. Measures of the algal community (phycocyanin, cyanobacterial biovolume, and cyanobacterial gene concentrations) and pH were most strongly correlated with microcystin concentrations. Cyanobacterial genes were quantified for general cyanobacteria, general Microcystis and Dolichospermum, and for microcystin synthetase (mcyE) for Microcystis, Dolichospermum, and Planktothrix. For phycocyanin, the relations were different between sites and were different between hand-held measurements on-site and nearby continuous monitor measurements for the same site. Continuous measurements of parameters such as phycocyanin, pH, and temperature over multiple days showed the highest correlations to microcystin concentrations. The development of models with high R² values (0.81–0.90), sensitivities (92%), and specificities (100%) for estimating microcystin concentrations above or below the Ohio Recreational Public Health Advisory level of 6 μg L⁻¹ was demonstrated for one site; these statistics may change as more data are collected in subsequent years. This study showed that models could be developed for estimates of exceeding a microcystin threshold concentration at a recreational freshwater lake site, with potential to expand their use to provide relevant public health information to water resource managers and the public for both recreational and drinking waters.     

Detection of Microcystin-Producing Cyanobacteria in Finnish Lakes with Genus-Specific Microcystin Synthetase Gene E (mcyE) PCR and Associations with Environmental Factors

We studied the frequency and composition of potential microcystin (MC) producers in 70 Finnish lakes with general and genus-specific microcystin synthetase gene E (mcyE) PCR. Potential MC-producing Microcystis, Planktothrixand Anabaena spp. existed in 70%, 63%, and 37% of the lake samples, respectively. Approximately two-thirds of the lake samples contained one or two potential MC producers, while all three genera existed in 24% of the samples. In oligotrophic lakes, the occurrence of only one MC producer was most common. The combination of Microcystis and Planktothrix was slightly more prevalent than others in mesotrophic lakes, and the cooccurrence of all three MC producers was most widespread in both eutrophic and hypertrophic lakes. The proportion of the three-producer lakes increased with the trophic status of the lakes. In correlation analysis, the presence of multiple MC-producing genera was associated with higher cyanobacterial and phytoplankton biomass, pH, chlorophyll a, total nitrogen, and MC concentrations. Total nitrogen, pH, and the surface area of the lake predicted the occurrence probability of mcyE genes, whereas total phosphorus alone accounted for MC concentrations in the samples by logistic and linear regression analyses. In conclusion, the results suggested that eutrophication increased the cooccurrence of potentially MC-producing cyanobacterial genera, raising the risk of toxic-bloom formation.     

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.     

The rise of potentially toxin producing cyanobacteria in Lake Naivasha,Great African Rift Valley,Kenya

Lake Naivasha, an important inland water ecosystem and a crucial freshwater resource in the Great African Rift Valley, has displayed clear signals of degradation in recent decades. We studied the phytoplankton composition and biomass levels in the period 2001–2013 and noted a progressive increase in the occurrence of potentially toxic cyanobacteria. Analyses for the presence of cyanotoxins such as microcystins (MC), cylindrospermopsin (CYN) and anatoxin-a (ATX-a) were carried out on samples collected in 2008–2013. Among the cyanotoxins tested, low concentrations of MC were detected in the lake. This is the first record of the occurrence of MC in Lake Naivasha. For the first time, molecular phylogenetic investigations of field clones of cyanobacteria from Lake Naivasha were carried out to establish the taxa of the dominant species. Amplification of the aminotrasferase (AMT) domain responsible for cyanotoxin production confirmed the presence of the mcyE gene belonging to the microcystin synthesis gene cluster in field samples containing Microcystis and Planktothrix species. These findings suggest that toxin producing cyanobacteria could become a threat to users of this over-exploited tropical lake in the near future.     

Ecological Dynamics of Toxic Microcystis spp. and Microcystin-Degrading Bacteria in Dianchi Lake,China

Toxic cyanobacterial blooms directly threaten both human safety and the ecosystem of surface waters. The widespread occurrence of these organisms, coupled with the tumor-promoting properties of the microcystin toxins that they produce, demands action to mitigate their potential impacts and, thus, a robust understanding of their ecological dynamics. In the present work, the abundance of toxic Microcystis spp. and microcystin (MC)-degrading bacteria in Dianchi Lake, located in Yunnan Province, China, was studied using quantitative PCR. Samples were taken at monthly intervals from June 2010 to December 2011 at three sampling stations within this freshwater lake. Results revealed that variation in the abundance of both total Microcystis spp. and toxic Microcystis spp. exhibited similar trends during the period of the algal bloom, including the reinvasion, pelagic growth, sedimentation, and overwintering periods, and that the proportion of toxic Microcystis was highest during the bloom and lowest in winter. Importantly, we observed that peaks in mlrA gene copy numbers of MC-degrading bacteria occurred in the months following observed peaks in MC concentrations. To understand this phenomenon, we added MCs to the MC-degrading bacteria (designated strains HW and SW in this study) and found that MCs significantly enhanced mlrA gene copy numbers over the number for the control by a factor of 5.2 for the microcystin-RR treatment and a factor of 3.7 for the microcystin-LR treatment. These results indicate that toxic Microcystis and MC-degrading bacteria exert both direct and indirect effects on each other and that MC-degrading bacteria also mediate a shift from toxic to nontoxic populations of Microcystis.     

The diversity and distribution of toxigenic Microcystis spp. in present day and archived pelagic and sediment samples from Lake Erie

The reoccurrence of significant cyanobacterial blooms in Lake Erie during the last 13 years has raised questions concerning the long-term persistence of microcystin-producing cyanobacteria and the presence of natural sediment reservoirs for potentially toxic cyanobacteria in this large lake system. To address these questions, we analyzed phytoplankton and sediment samples which were collected and preserved in the 1970s as well as samples collected in 2004 from locations within Lake Erie. The identification of microcystin-producing cyanobacteria in Lake Erie was examined via PCR amplification of the mcyA gene fragment. Based on the high % sequence similarity, the mcyA sequences from all 1970s phytoplankton and sediment samples were determined to belong to Microcystis spp., in spite of reports suggesting that Lake Erie was dominated by filamentous cyanobacteria in the 1970s. In sediment samples from 2004, signature genes for Microcystis were distributed and preserved not only in the surface sediments but also up to 10–12 cm in depth. Based on cell quantities determined by a quantitative polymerase chain reaction (qPCR) method, 0.18% of eubacteria in the sediments were Microcystis cells, of which 4.8% were potential microcystin producers. In combination with experiments showing that Microcystis cells can be cultured from Lake Erie surface sediments, this paper demonstrates the potential for these sediments to act as a reservoir for pelagic Microcystis populations and that the composition of the population of microcystin-producing cyanobacteria in Lake Erie has not changed remarkably since the 1970s.     

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.     

Transposons Inactivate Biosynthesis of the Nonribosomal Peptide Microcystin in Naturally Occurring Planktothrix spp.

The filamentous cyanobacteria Planktothrix spp. occur in the temperate region of the Northern hemisphere. The red-pigmented Planktothrix rubescens bacteria occur in deep, physically stratified, and less eutrophic lakes. Planktothrix is a known producer of the toxic heptapeptide microcystin (MC), which is produced nonribosomally by a large enzyme complex consisting of peptide synthetases and polyketide synthases encoded by a total of nine genes (mcy genes). Planktothrix spp. differ in their cellular MC contents as well as the production of MC variants; however, the mechanisms favoring this diversity are not understood. Recently, the occurrence of Planktothrix strains containing all mcy genes but lacking MC has been reported. In this study, 29 such strains were analyzed to find out if mutations of the mcy genes lead to the inability to synthesize MC. Two deletions, spanning 400 bp (in mcyB; one strain) and 1,869 bp (in mcyHA; three strains), and three insertions (IS), spanning 1,429 bp (in mcyD; eight strains), 1,433 bp (in mcyEG; one strain), and 1,433 bp (in mcyA; one strain), were identified. Though found in different genes and different isolates and transcribed in opposite directions, IS were found to be identical and contained conserved domains assigned to transposable elements. Using mutation-specific primers, two insertions (in mcyD and mcyA) and one deletion (in mcyHA) were found regularly in populations of P. rubescens in different lakes. The results demonstrate for the first time that different mutations resulting in inactivation of MC synthesis do occur frequently and make up a stable proportion of the mcy gene pool in Planktothrix populations over several years.     

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.     

Rising temperatures may increase growth rates and microcystin production in tropical Microcystis species

Rising temperatures (1.4–6 °C) due to climate change have been predicted to increase cyanobacterial bloom occurrences in temperate water bodies; however, the impacts of warming on tropical cyanobacterial blooms are unknown. We examined the effects of four different temperatures on the growth rates and microcystin (MC) production of five tropical Microcystis isolates (M. ichthyoblabe (two strains), M. viridis, M. flos-aquae, and M. aeruginosa). The temperature treatments are based on current temperature range in Singapore's reservoirs (27 °C and 30 °C), as well as projected mean (33 °C) and maximum temperatures (36 °C) based on tropical climate change estimates of +6 °C in air temperature. Increasing temperatures did not significantly affect the maximum growth rates of most Microcystis strains. Higher growth rates were only observed in one M. ichthyoblabe strain at 33 °C and M. flos-aquae at 30 °C where both were isolated from the same reservoir. MC-RR and MC-LR were produced in varying amounts by all four species of Microcystis. Raised temperatures of 33 °C were found to boost total MC cell quota for three Microcystis strains although further increase to 36 °C led to a sharp decrease in total MC cell quota for all five Microcystis strains. Increasing temperature also led to higher MC-LR:MC-RR cell quota ratios in M. ichthyoblabe. Our study suggests that higher mean water temperatures resulting from climate change will generally not influence growth rates of Microcystis spp. in Singapore except for increases in M. ichthyoblabe strains. However, toxin cell quota may increase under moderate warming scenarios depending on the species.     

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.     

Estimating spatial variation in the abundance of potential microcystin-producing Microcystis spp. using real-time PCR during summer bloom in Lake Taihu

Lake Taihu, which is the third largest freshwater lakes in China, is a hypertrophic shallow lake in eastern China that has experienced lake-wide cyanobacterial blooms annually during the last few decades. In this study, quantitative real-time PCR assays targeting on phycocyanin intergenic spacer (PC-IGS) and a microcystin synthetase gene mcyD were established, respectively. Water samples collected from eight sampling sites (including Zhushan Bay (N5), Meiliang Bay (N2), Gonghu Bay (N4), West lake areas (W2 and W4), south-middle lake areas (S2, S4 and S5)) in August of 2009 and 2010 were analyzed using real time PCR for the distribution and abundance of toxic and total Microcystis populations. The results showed that Microcystis exists as a mixed population of potential toxic and non-toxic genotypes, and there was significant spatial changes in the abundance of potential toxic Microcystis on the basis of quantification by quantitative real-time PCR analysis: the abundance of toxic Microcystis population in 2009 and 2010 varied from 4.08 × 10⁴ to 8.28 × 10⁶ copies mL^?1, from 4.45 × 10⁵ to 5.22 × 10⁷ copies mL^?1, respectively. Meanwhile the ratio of the mcyD subpopulation to the total Microcystis varied considerably, from 5.7% to 41.1% in 2009 and from 10.3% to 65.8% in 2010 in all sampling sites, and the value is high in Zhushan Bay and Meiliang Bay with the high level of eutrophication. Correlation analysis showed the abundance of toxic and total Microcystis being strongly related (P < 0.01). However, there is different effects of environmental factors on the abundance of toxic and non-toxic Microcystis populations. The abundance of toxic and total Microcystis populations were positively correlated with chlorophyll-a (Chl-a) concentration (P < 0.01) suggesting that Microcystis is dominated genera of cyanobacterial bloom in Lake Taihu. It was also found that the abundance of toxic Microcystis and the proportion of toxic subpopulation to the total Microcystis were positively correlated with total phosphorus and orthophosphate concentrations (P < 0.01), whereas there was no significant correlation with total nitrogen and nitrate concentration (P > 0.05). All data suggest that phosphorus concentration is a critical factor for determining the abundance of toxic Microcystis population.     

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 involvement of α-proteobacteria Phenylobacterium in maintaining the dominance of toxic Microcystis blooms in Lake Taihu,China

Lake Taihu in China has suffered serious harmful cyanobacterial blooms for decades. The algal blooms threaten the ecological sustainability, drinking water safety, and human health. Although the roles of abiotic factors (such as water temperature and nutrient loading) in promoting Microcystis blooms have been well studied, the importance of biotic factors (e.g. bacterial community) in promoting and meditating Microcystis blooms remains unclear. In this study, we investigated the ecological dynamics of bacterial community, the ratio of toxic Microcystis, as well as microcystin in Lake Taihu. High-throughput 16S rRNA sequencing and principal component analysis (PCA) revealed that the bacteria community compositions (BCCs) clustered into three groups, the partitioning of which corresponded to that of groups according to the toxic profiles (the ratio of toxic Microcystis to total Microcystis, and the microcystin concentrations) of the samples. Further Spearman's correlation network showed that the α-proteobacteria Phenylobacterium strongly positively correlated with the toxic profiles. Subsequent laboratory chemostats experiments demonstrated that three Phenylobacterium strains promoted the dominance of the toxic Microcystis aeruginosa PCC7806 when co-culturing with the non-toxic PCC7806 mcyB⁻ mutant. Taken together, our data suggested that the α-proteobacteria Phenylobacterium may play a vital role in the maintenance of toxic Microcystis dominance in Lake Taihu.     

The toxicity and enzyme activity of a chlorine and sulfate containing aeruginosin isolated from a non-microcystin-producing Planktothrix strain

The toxicity of six different Planktothrix strains was examined in acute toxicity assays with the crustacean Thamnocephalus platyurus. The presence of toxicity in two strains could be explained by the occurrence of microcystins. The other four Planktothrix strains were not able to produce microcystins due to different mutations in the microcystin synthetase (mcy) gene cluster. In these strains, toxicity was attributed to the presence of chlorine and sulfate containing compounds. The main representative, called aeruginosin 828A, of such a compound in the Planktothrix strain 91/1 was isolated, and structure elucidation by 2D NMR and MS methods revealed the presence of phenyllactic acid (Pla), chloroleucine (Cleu), 2-carboxy-6-(4′-sulfo-xylosyl)-octahydroindole (Choi), and 3-aminoethyl-1-N-amidino-Δ-3-pyrroline (Aeap) residues. Aeruginosin 828A was found to be toxic for T. platyurus with a LC₅₀ value of 22.4 μM, which is only slightly higher than the toxicity found for microcystins. Additionally, very potent inhibition values for thrombin (IC₅₀ = 21.8 nM) and for trypsin (IC₅₀ = 112 nM) have been determined for aeruginosin 828A. These data support the hypothesis that aeruginosins containing chlorine and sulfate groups, which were found in microcystin-deficient Planktothrix strains, can be considered as another class of toxins.