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.     

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.     

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.     

Nitrogen Forms Influence Microcystin Concentration and Composition via Changes in Cyanobacterial Community Structure

The eutrophication of freshwaters is a global health concern as lakes with excess nutrients are often subject to toxic cyanobacterial blooms. Although phosphorus is considered the main element regulating cyanobacterial biomass, nitrogen (N) concentration and more specifically the availability of different N forms may influence the overall toxicity of blooms. In this study of three eutrophic lakes prone to cyanobacterial blooms, we examined the effects of nitrogen species and concentrations and other environmental factors in influencing cyanobacterial community structure, microcystin (MC) concentrations and MC congener composition. The identification of specific MC congeners was of particular interest as they vary widely in toxicity. Different nitrogen forms appeared to influence cyanobacterial community structure leading to corresponding effects on MC concentrations and composition. Total MC concentrations across the lakes were largely explained by a combination of abiotic factors: dissolved organic nitrogen, water temperature and ammonium, but Microcystis spp. biomass was overall the best predictor of MC concentrations. Environmental factors did not appear to affect MC congener composition directly but there were significant associations between specific MC congeners and particular species. Based on redundancy analyses (RDA), the relative biomass of Microcystis aeruginosa was associated with MC-RR, M. wesenbergii with MC-LA and Aphanizomenon flos-aquae with MC-YR. The latter two species are not generally considered capable of MC production. Total nitrogen, water temperature, ammonium and dissolved organic nitrogen influenced the cyanobacterial community structure, which in turn resulted in differences in the dominant MC congener and the overall toxicity.     

Evaluating putative ecological drivers of microcystin spatiotemporal dynamics using metabarcoding and environmental data

Microcystin is a cyanobacterial hepatotoxin of global concern. Understanding the environmental factors that cause high concentrations of microcystin is crucial to the development of lake management strategies that minimize harmful exposures. While the literature is replete with studies linking cyanobacterial production of microcystin to changes in various nutrients, abiotic stressors, grazers, and competitors, no single biotic or abiotic factor has been shown to be reliably predictive of microcystin concentrations in complex ecosystems. We performed random forest regression analyses with 16S and 18S rRNA gene sequencing data and environmental data to determine which putative ecological drivers best explained spatiotemporal variation in total microcystin and several individual congeners in a eutrophic freshwater reservoir. Model performance was best for predicting concentrations of the congener MC-LR, with ca. 88% of spatiotemporal variance explained. Most of the variance was associated with changes in the relative abundance of the cyanobacterial genus Microcystis. Follow-up RF regression analyses revealed that factors that were the most important in predicting MC-LR were also the most important in predicting Microcystis population dynamics. We discuss how these results relate to prevailing ecological hypotheses regarding the function of microcystin.     

Spatial and temporal diversity of microcystins and microcystin-producing genotypes in Oneida Lake, NY

Oneida Lake is a shallow, eutrophic lake with a well-established cyanobacterial population with reported toxic blooms containing hepatotoxic microcystins (MC). Peak bloom events from the summers of 2002 and 2003 were analyzed to determine the principal cyanobacterial genera containing microcystin synthetase (mcy) genes. Sequence analysis of a partial mcyA amplicon targeting Microcystis, Anabaena and Planktothrix sp. indicated that Microcystis sp. was the dominant mcy genotype. This Microcystis clade was split into two distinct sub-clades. Bloom events contained members of both sub-clades with the higher MC concentrations found when both sub-clades were present in near equal proportions. The proportion of Microcystis containing the mcyD gene ranged from 0 to 37% of the total Microcystis population as determined by quantitative PCR (qPCR). The total concentration of Microcystis containing mcyD genes was linearly related to the concentration of MCs (r² = 0.63). The relationship between mcy genotype and physiochemical variables was examined to determine the factor(s) controlling the periodicity in MC production in Oneida Lake. Multivariate statistical analyses, used to correlate the continuous-response variables, revealed a strong relationship between chlorophyll a, MCs and total Microcystis.     

The Role of Nitrogen Fixation in Cyanobacterial Bloom Toxicity in a Temperate,Eutrophic Lake

Toxic cyanobacterial blooms threaten freshwaters worldwide but have proven difficult to predict because the mechanisms of bloom formation and toxin production are unknown, especially on weekly time scales. Water quality management continues to focus on aggregated metrics, such as chlorophyll and total nutrients, which may not be sufficient to explain complex community changes and functions such as toxin production. For example, nitrogen (N) speciation and cycling play an important role, on daily time scales, in shaping cyanobacterial communities because declining N has been shown to select for N fixers. In addition, subsequent N pulses from N₂ fixation may stimulate and sustain toxic cyanobacterial growth. Herein, we describe how rapid early summer declines in N followed by bursts of N fixation have shaped cyanobacterial communities in a eutrophic lake (Lake Mendota, Wisconsin, USA), possibly driving toxic Microcystis blooms throughout the growing season. On weekly time scales in 2010 and 2011, we monitored the cyanobacterial community in a eutrophic lake using the phycocyanin intergenic spacer (PC-IGS) region to determine population dynamics. In parallel, we measured microcystin concentrations, N₂ fixation rates, and potential environmental drivers that contribute to structuring the community. In both years, cyanobacterial community change was strongly correlated with dissolved inorganic nitrogen (DIN) concentrations, and Aphanizomenon and Microcystis alternated dominance throughout the pre-toxic, toxic, and post-toxic phases of the lake. Microcystin concentrations increased a few days after the first significant N₂ fixation rates were observed. Then, following large early summer N₂ fixation events, Microcystis increased and became most abundant. Maximum microcystin concentrations coincided with Microcystis dominance. In both years, DIN concentrations dropped again in late summer, and N₂ fixation rates and Aphanizomenon abundance increased before the lake mixed in the fall. Estimated N inputs from N₂ fixation were large enough to supplement, or even support, the toxic Microcystis blooms.     

Identifying the Source of Unknown Microcystin Genes and Predicting Microcystin Variants by Comparing Genes within Uncultured Cyanobacterial Cells

While multiple phylogenetic markers have been used in the culture-independent study of microcystin-producing cyanobacteria, in only a few instances have multiple markers been studied within individual cells, and in all cases these studies have been conducted with cultured isolates. Here, we isolate and evaluate large DNA fragments (>6 kb) encompassing two genes involved in microcystin biosynthesis (mcyA2 and mcyB1) and use them to identify the source of gene fragments found in water samples. Further investigation of these gene loci from individual cyanobacterial cells allowed for improved analysis of the genetic diversity within microcystin producers as well as a method to predict microcystin variants for individuals. These efforts have also identified the source of the novel mcyA genotype previously termed Microcystis-like that is pervasive in the Laurentian Great Lakes and they predict the microcystin variant(s) that it produces.Microcystin-producing cyanobacteria are common nuisance organisms in harmful algal blooms in freshwaters around the world (4). This genetically diverse group (based on 16S rRNA, mcyA, mcyD, and mcyE gene sequences [6, 10, 15, 16, 22]) ranges in morphology from unicellular and colonial cocci to large filamentous strands. Many species can produce a variety of secondary metabolites that can act as hepatatoxins upon ingestion by animals (e.g., variants of microcystin) (4, 33). Microcystin production reduces the water quality in reservoirs used by human populations and fishery resources, and production of these toxins by this group of cyanobacteria makes them important organisms for continued observation and study (4, 33, 36). Much effort has been expended over the past 15 years to characterize the genomic and structural components of the microcystin (mcy) synthetase operon responsible for the production of microcystins. Several complete DNA sequences of the mcy synthetase operon are currently available in GenBank (3, 11, 29, 31).Although the mechanisms of microcystin production are now better understood, recent analyses of mcyA gene fragments from Lakes Erie and Ontario indicated a microcystin toxin producer of unknown phylogeny (7, 28). This discrepancy suggested a need for improved molecular characterization of naturally occurring microcystin producers, which spurred our research to identify the source of several unusual mcyA fragments from the cyanobacterial community (7, 28). It was apparent from initial sequence data that these mcyA gene fragments, termed Microcystis-like, were highly similar to those from Microcystis spp. (colonial or unicellular cocci). However, they contained a 6-nucleotide insert consistent with mcyA genes from filamentous cyanobacteria (e.g., Anabaena, Nostoc, and Planktothrix) (28). These preliminary findings suggested that these unusual mcyA fragments either came from (i) a novel species or strain, (ii) an ancestral Microcystis, (iii) the highly unlikely hybridization of colonial cocci and filamentous cyanobacteria, or (iv) a chimera of cocci and filamentous PCR products. To identify the source of these mcyA gene fragments from uncultured cyanobacteria, we used culture-independent methods to amplify and isolate long regions of the mcy synthetase operon for the simultaneous analysis of two genes, mcyA and mcyB, in one individual from a population. This approach ensures that both genes are contained on the same DNA molecule, thus allowing for more continuous sequence information to use in comparative phylogenetic analyses than previously described. We also envisioned that this mcy gene combination would provide an improved diagnostic tool for determining the genetic potential of naturally occurring cyanobacteria to produce specific microcystin variants by comparing the phylogenetic marker in mcyA to the predictor of amino acid incorporation (via an adenylation domain) in mcyB1.     

Seasonal Variation and Indirect Monitoring of Microcystin Concentrations in Daechung Reservoir, Korea

Physicochemical and biological water quality, including the microcystin concentration, was investigated from spring to autumn 1999 in the Daechung Reservoir, Korea. The dominant genus in the cyanobacterial blooming season was Microcystis. The microcystin concentration in particulate form increased dramatically from August up to a level of 200 ng liter⁻¹ in early October and thereafter tended to decrease. The microcystin concentration in dissolved form was about 28% of that of the particulate form. The microcystins detected using a protein phosphatase (PP) inhibition assay were highly correlated with those microcystins detected by a high-performance liquid chromatograph (r = 0.973; P < 0.01). Therefore, the effectiveness of a PP inhibition assay for microcystin detection in a high number of water samples was confirmed as easy, quick, and convenient. The microcystin concentration was highly correlated with the phytoplankton number (r = 0.650; P < 0.01) and chlorophyll-a concentration (r = 0.591; P < 0.01). When the microcystin concentration exceeded about 100 ng liter⁻¹, the ratio of particulate to dissolved total nitrogen (TN) or total phosphorus (TP) converged at a value of 0.6. Furthermore, the microcystin concentration was lower than 50 ng liter⁻¹ at a particulate N/P ratio below 8, whereas the microcystin concentration varied quite substantially from 50 to 240 ng liter⁻¹ at a particulate N/P ratio of >8. Therefore, it seems that the microcystin concentration in water can be estimated and indirectly monitored by analyzing the following: the phytoplankton number and chlorophyll-a concentration, the ratio of the particulate and the dissolved forms of N and P, and the particulate N/P ratio when the dominant genus is toxigenic Microcystis.     

Role of Microcystins in Poisoning and Food Ingestion Inhibition of Daphnia galeata Caused by the Cyanobacterium Microcystis aeruginosa

The effects of microcystins on Daphnia galeata, a typical filter-feeding grazer in eutrophic lakes, were investigated. To do this, the microcystin-producing wild-type strain Microcystis aeruginosa PCC7806 was compared with a mcy⁻ PCC7806 mutant, which could not synthesize any variant of microcystin due to mutation of a microcystin synthetase gene. The wild-type strain was found to be poisonous to D. galeata, whereas the mcy⁻ mutant did not have any lethal effect on the animals. Both variants of PCC7806 were able to reduce the Daphnia ingestion rate. Our results suggest that microcystins are the most likely cause of the daphnid poisoning observed when wild-type strain PCC7806 is fed to the animals, but these toxins are not responsible for inhibition of the ingestion process.     

Microcystins derived from lysing Microcystis cells do not cause negative effects on crustacean zooplankton in Lake Taihu, China

Microcystins (MCs) have a toxic effect on crustacean zooplankton in the laboratory, but there is little or no unequivocal evidence in the literature of their lethal effects on crustacean zooplankton in the field. We used the natural microcystins extracted from Microcystis spp. to test if they could cause any negative effects on crustacean zooplankton. We conducted three experiments in enclosures with water from Lake Taihu, China, and microcystins derived by extraction from Microcystis spp. collected from the lake when the species was in bloom conditions. Initial concentrations of extracellular microcystins (EMCs = MC-RR + MC-LR + MC-YR) ranged from 9.7 to 44.9 μg/L in treatments with microcystin addition. Microcystin concentrations sharply decreased on second day in all the three experiments. EMCs at the end of the experiments varied from only 2.7 to 14.2 % of the levels at the start of the experiments. The dominant species of crustacean zooplankton in the lake were Bosmina longirotris, Ceriodaphnia cornuta, Mesocyclops spp., Limnoithona sinensis, Sinocalanus dorrii and Schmackeria inopinus. ANOVA analysis showed that the density and biomass of cladoceran and copepod did not significantly differ between treatments with microcystin addition and controls. Our results indicate that microcystins derived from lysing Microcystis do not cause any negative effects on crustacean zooplankton.     

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.     

Depth profiles of cyanobacterial hepatotoxins (microcystins) in three Turkish freshwater lakes

The Turkish freshwater lakes, Sapanca, Iznik and Taskisi (Calticak) have been enriched with nutrients from agriculture and domestic sources for many years. A major bloom of cyanobacteria (blue-green algae) in Lake Sapanca was recorded in May 1997, closely followed by a fish kill. Investigations were subsequently made on the cyanobacteria and water quality of the lakes, including analysis for cyanobacterial hepatotoxins (microcystins) in the filtered particulate fraction. Samples, taken from the beginning of May to end of August 1998, were analysed for microcystins by high–performance liquid chromatography with photodiode array detection (HPLC-PDA), protein phosphatase inhibition assay (PPIA) and an enzyme-linked immunosorbent assay (ELISA). No microcystins were detected in the water column in Lake Sapanca above 10 m, but toxins were found in filtered cyanobacterial samples from 20 m depth at a concentration of 3.65 μg l^?1 microcystin–LR equivalents. Ninety percent of the microcystin pool detected in L. Sapanca was found between depths of 15 and 25 m. The principal microcystin detected by HPLC-PDA was similar to microcystin–RR. Two unidentified microcystin variants were found in Lake Taskisi surface samples at a concentration of 2.43 μg l^?1 microcystin–LR equivalents in the filtered cyanobacterial cell fraction. Although 10 water samples (10 × 5 l) were taken from Lake Iznik (surface to 20 m, 5 m intervals), no microcystins were detected by HPLC-PDA (limit of detection 10 ng). The depth at which microcystins were detected in L. Sapanca coincided with the draw-off depth for the drinking water supply for the city of Sakarya     

Stability of toxigenic Microcystis blooms

This is the first detailed study on the occurrence of cyanobacterial toxins in India, where we studied five eutrophic, temple ponds in the vicinity of Varanasi city, Uttar Pradesh, which continuously supported blooms of Microcystis sp. for several years. Bloom material from all five ponds was sampled bi-monthly from September 2003 to August 2004. Analysis of extracts by high-performance liquid chromatography (HPLC) indicated that microcystin-RR (MC-RR) was present all year round at high concentrations (311–1540 μg/g, dry weight), posing a significant health hazard especially since all five ponds are widely used for bathing, washing, cattle drinking supply, irrigation and recreation. In addition, there was unusually low temporal variation in concentration of MC-RR in each pond, <20% variation in four out of five ponds throughout the year.Characterization of microcystin composition of several bloom samples from this study by HPLC–PDA/MS confirmed that additional microcystins were present in many of the samples. The rarely reported, MC-AR was frequently detected in bloom samples from three of the ponds (Adityanagar, Durgakund and Sankuldhara), where it typically represented 20% of the microcystin pool. MC-WR was frequently found in samples from Adityanagar and Sankuldhara, representing 5–10% of the microcystin pool. MC-LR, along with the previously unreported MC-AHar, each represented approximately 5% of the microcystin pool when present. Bloom samples from each pond had a characteristic microcystin profile, when sampled from 2003 to 2006, suggesting persistent species/strain domination.The perennial and consistent nature of the toxic Microcystis blooms in these ponds is highly unusual, in contrast to the commonly encountered temporal and spatial variation of toxigenic and non-toxigenic species. Laboratory isolates from several ponds were shown to produce microcystins, showing similar microcystin composition to the original bloom material.     

Controlling Harmful Cyanobacteria: Taxa-Specific Responses of Cyanobacteria to Grazing by Large-Bodied Daphnia in a Biomanipulation Scenario

Lake restoration practices based on reducing fish predation and promoting the dominance of large-bodied Daphnia grazers (i.e., biomanipulation) have been the focus of much debate due to inconsistent success in suppressing harmful cyanobacterial blooms. While most studies have explored effects of large-bodied Daphnia on cyanobacterial growth at the community level and/or on few dominant species, predictions of such restoration practices demand further understanding on taxa-specific responses in diverse cyanobacterial communities. In order to address these questions, we conducted three grazing experiments during summer in a eutrophic lake where the natural phytoplankton community was exposed to an increasing gradient in biomass of the large-bodied Daphnia magna. This allowed evaluating taxa-specific responses of cyanobacteria to Daphnia grazing throughout the growing season in a desired biomanipulation scenario with limited fish predation. Total cyanobacterial and phytoplankton biomasses responded negatively to Daphnia grazing both in early and late summer, regardless of different cyanobacterial densities. Large-bodied Daphnia were capable of suppressing the abundance of Aphanizomenon, Dolichospermum, Microcystis and Planktothrix bloom-forming cyanobacteria. However, the growth of the filamentous Dolichospermum crassum was positively affected by grazing during a period when this cyanobacterium dominated the community. The eutrophic lake was subjected to biomanipulation since 2005 and nineteen years of lake monitoring data (1996–2014) revealed that reducing fish predation increased the mean abundance (50%) and body-size (20%) of Daphnia, as well as suppressed the total amount of nutrients and the growth of the dominant cyanobacterial taxa, Microcystis and Planktothrix. Altogether our results suggest that lake restoration practices solely based on grazer control by large-bodied Daphnia can be effective, but may not be sufficient to control the overgrowth of all cyanobacterial diversity. Although controlling harmful cyanobacterial blooms should preferably include other measures, such as nutrient reductions, our experimental assessment of taxa-specific cyanobacterial responses to large-bodied Daphnia and long-term monitoring data highlights the potential of such biomanipulations to enhance the ecological and societal value of eutrophic water bodies.     

Metagenomic Identification of Bacterioplankton Taxa and Pathways Involved in Microcystin Degradation in Lake Erie

Cyanobacterial harmful blooms (CyanoHABs) that produce microcystins are appearing in an increasing number of freshwater ecosystems worldwide, damaging quality of water for use by human and aquatic life. Heterotrophic bacteria assemblages are thought to be important in transforming and detoxifying microcystins in natural environments. However, little is known about their taxonomic composition or pathways involved in the process. To address this knowledge gap, we compared the metagenomes of Lake Erie free-living bacterioplankton assemblages in laboratory microcosms amended with microcystins relative to unamended controls. A diverse array of bacterial phyla were responsive to elevated supply of microcystins, including Acidobacteria, Actinobacteria, Bacteroidetes, Planctomycetes, Proteobacteria of the alpha, beta, gamma, delta and epsilon subdivisions and Verrucomicrobia. At more detailed taxonomic levels, Methylophilales (mainly in genus Methylotenera) and Burkholderiales (mainly in genera Bordetella, Burkholderia, Cupriavidus, Polaromonas, Ralstonia, Polynucleobacter and Variovorax) of Betaproteobacteria were suggested to be more important in microcystin degradation than Sphingomonadales of Alphaproteobacteria. The latter taxa were previously thought to be major microcystin degraders. Homologs to known microcystin-degrading genes (mlr) were not overrepresented in microcystin-amended metagenomes, indicating that Lake Erie bacterioplankton might employ alternative genes and/or pathways in microcystin degradation. Genes for xenobiotic metabolism were overrepresented in microcystin-amended microcosms, suggesting they are important in bacterial degradation of microcystin, a phenomenon that has been identified previously only in eukaryotic systems.     

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

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

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.