Microcystin-producing Microcystis Research Articles

Dynamic characteristics of total and microcystin-producing Microcystis in a large deep reservoir

Microcystis, one of the common cyanobacteria, often causes blooms in reservoirs, which has seriously threatened the safety of drinking water worldwide. To identify the growth characteristic of total and microcystin-producing Microcystis in large deep reservoirs, we used Quantitative PCR (qPCR) to measure the cell density of total and microcystin-producing Microcystis and monitored water quality in the water samples collected in Dongzhang Reservoir once a month. Microcystis blooms occurred in Dongzhang Reservoir in April 2017, which was composed of microcystin-producing and non-microcystin-producing Microcystis. Water temperature, dissolved oxygen, pH, and chlorophyll-a showed significant vertical stratification during Microcystis blooms. Total and microcystin-producing Microcystis grew rapidly under the high concentration of total phosphorus and rising water temperatures. Nitrate-nitrogen had a significant linear correlation with the abundance of microcystin-producing Microcystis. Our results indicated that nutrients and water temperature could be key triggers of Microcystis blooms and nitrate-nitrogen potentially regulates the competition between microcystin-producing and non-microcystin-producing Microcystis. This study improves our understanding of the characteristics of Microcystis blooms and the competition between microcystin-producing and non-microcystin-producing Microcystis in large deep reservoirs.

Survival and Accumulation of Microcystins in a Native Amphipod Shredder, Gammarus Pulex under Environmentally Relevant Cyanotoxin Exposure

Microcystin release in freshwaters is a major public health and ecological threat, affecting survival and trophic interactions across food web components. Although bioaccumulation has been suggested to represent an important pathway for microcystin uptake and trophic transfer across different food web compartments, it is rarely known how microcystin exposure may affect survival and accumulation among lower trophic levels under environmentally realistic scenario. Therefore, in this study, survival and microcystin accumulations in the UK native amphipod species, Gammarus pulex were monitored during a 96-hour static acute toxicity experiment. Animals were exposed to a range of environmentally relevant microcystin concentrations (0.01–10μg/L) across two treatment types, purified MC-LR and crude extract of microcystin-producing Microcystis aeruginosa. Survival of G. pulex individuals across the two treatments showed no evidence of significant difference from the control. Microcystin concentrations observed in G. pulex exposed to the purified MC-LR and crude extract ranged from (12.8 – 27.5) ng/g of MC-LR and (5.6-12.8) ng/g of MC-LR respectively. Microcystin concentrations measured in animals showed strong positive correlations with toxin concentrations in the experimental media across the two treatments, suggesting G. pulex can bioaccumulate microcystins, therefore may represent a potential vector for microcystin exposure to higher food web consumers. These results highlight the fact that exposure of benthic food web components to environmental microcystin concentrations may constitute an important route of toxin transfer within freshwater food webs.

Hepatotoxic shellfish poisoning: Accumulation of microcystins in Eastern oysters (Crassostrea virginica) and Asian clams (Corbicula fluminea) exposed to wild and cultured populations of the harmful cyanobacteria, Microcystis

The Asian clam (Corbicula fluminea) and eastern oyster (Crassostrea virginica) are important resource bivalves found in and downstream of waterways afflicted with cyanobacterial harmful algae blooms (CHABs), respectively. This study examined the potential for C. fluminea and C. virginica to become vectors of the hepatotoxin, microcystin, from the CHAB Microcystis. Laboratory experiments were performed to quantify clearance rates, particle selection, and accumulation of the hepatotoxin, microcystin, using a microcystin-producing Microcystis culture isolated from Lake Erie (strain LE-3) and field experiments were performed with water from Microcystis blooms in Lake Agawam, NY, USA. Clearance rates of Microcystis were faster (p<0.05) than those of Raphidocelis for C. fluminea, while C. virginica cleared Microcystis and Tisochrysis at similar rates. For both bivalves, clearance rates of bloom water were slower than cultures and clams displayed significantly greater electivity for green algae compared to wild populations of cyanobacteria in field experiments while oysters did not. In experiments with cultured Microcystis comprised of single and double cells, both bivalves accumulated >3 µg microcystins g − 1 (wet weight) in 24 – 72 h, several orders of magnitude beyond California guidance value (10 ng g − 1) but accumulated only up to 2 ng microcystins g − 1 when fed bloom water dominated by large Microcystis colonies for four days. For Asian clams, clearance rates and tissue microcystin content decreased when exposed to toxic Microcystis for 3 – 4 days. In contrast, eastern oysters did not depurate microcystin over 3 - 4-day exposures and accumulated an order of magnitude more microcystin than clams. This contrast suggests Asian clams are likely to accumulate minor amounts of microcystin by reducing clearance rates during blooms of Microcystis, selectively feeding on green algae, and depurating microcystin whereas oysters are more likely to accumulate microcystins and thus are more likely to be a vector for hepatotoxic shellfish poisoning in estuaries downstream of Microcystis blooms.

Depth-Dependent Spatiotemporal Dynamics of Overwintering Pelagic Microcystis in a Temperate Water Body.

Cyanobacteria in the genus Microcystis are dominant components of many harmful algal blooms worldwide. Their pelagic–benthic life cycle helps them survive periods of adverse conditions and contributes greatly to their ecological success. Many studies on Microcystis overwintering have focused on benthic colonies and suggest that sediment serves as the major inoculum for subsequent summer blooms. However, the contemporaneous overwintering pelagic population may be important as well but is understudied. In this study, we investigated near-surface and near-bottom pelagic population dynamics of both microcystin-producing Microcystis and total Microcystis over six weeks in winter at Dog Lake (South Frontenac, ON, Canada). We quantified relative Microcystis concentrations using real-time PCR. Our results showed that the spatiotemporal distribution of overwintering pelagic Microcystis was depth dependent. The abundance of near-bottom pelagic Microcystis declined with increased depth with no influence of depth on near-surface Microcystis abundance. In the shallow region of the lake (<10 m), most pelagic Microcystis was found near the lake bottom (>90%). However, the proportion of near-surface Microcystis rose sharply to over 60% as the depth increased to approximately 18 m. The depth-dependent distribution pattern was found to be similar in both microcystin-producing Microcystis and total Microcystis. Our results suggest the top of the water column may be a more significant contributor of Microcystis recruitment inoculum than previously thought and merits more attention in early CHAB characterization and remediation.

Insight Into the Molecular Mechanisms for Microcystin Biodegradation in Lake Erie and Lake Taihu.

Microcystins are potent hepatotoxins that are frequently detected in fresh water lakes plagued by toxic cyanobacteria. Microbial biodegradation has been referred to as the most important avenue for removal of microcystin from aquatic environments. The biochemical pathway most commonly associated with the degradation of microcystin is encoded by the mlrABCD (mlr) cassette. The ecological significance of this pathway remains unclear as no studies have examined the expression of these genes in natural environments. Six metatranscriptomes were generated from microcystin-producing Microcystis blooms and analyzed to assess the activity of this pathway in environmental samples. Seventy-eight samples were collected from Lake Erie, United States/Canada and Lake Tai (Taihu), China, and screened for the presence of mlr gene transcripts. Read mapping to the mlr cassette indicated transcripts for these genes were absent, with only 77 of the collective 3.7 billion reads mapping to any part of the mlr cassette. Analysis of the assembled metatranscriptomes supported this, with only distantly related sequences identified as mlrABC-like. These observations were made despite the presence of microcystin and over 500,000 reads mapping to the mcy cassette for microcystin production. Glutathione S-transferases and alkaline proteases have been previously hypothesized to be alternative pathways for microcystin biodegradation, and expression of these genes was detected across space and time in both lakes. While the activity of these alternative pathways needs to be experimentally confirmed, they may be individually or collectively more important than mlr genes in the natural environment. Importantly, the lack of mlr expression could indicate microcystin biodegradation was not occurring in the analyzed samples. This study raises interesting questions about the ubiquity, specificity and locality of microcystin biodegradation, and highlights the need for the characterization of relevant mechanisms in natural communities to understand the fate of microcystin in the environment and risk to public health.

Annual variations in microcystin occurrence in Upper Klamath Lake, Oregon, based on high-throughput DNA sequencing, qPCR, and environmental parameters

Eldridge SLC, Wood TM. 2019. Annual variation in microcystin occurrence in Upper Klamath Lake, Oregon, based on high-throughput DNA sequencing, qPCR, and environmental parameters. Lake Reserv Manage. 36:31–44.Cyanobacteria-dominated blooms in Upper Klamath Lake, Oregon, create poor water quality and produce microcystins that may be detrimental to local wildlife and human health. Genetic tools, including high-throughput DNA sequencing and quantitative polymerase chain reaction (qPCR), have been shown to improve the identification and quantification of key groups associated with these blooms over more traditional techniques. We examined the seasonal and interannual variations in nutrient (nitrogen and phosphorus) concentrations between 2013 and 2014 to describe the relations between these factors and the growth dynamics of Aphanizomenon and toxigenic Microcystis as described by DNA sequencing and qPCR. Although total nutrients and chlorophyll a concentrations were similar between years, qPCR results showed the cyanobacterial populations to be 40 times larger in 2014 and indicated a large shift from an Aphanizomenon-dominant, low microcystin-level regime in 2013 to one dominated later in the season by microcystin-producing Microcystis in 2014. In both years, the transition from Aphanizomenon to Microcystis was coincident with a late-season increase in nitrite-plus-nitrate concentrations and in dissolved inorganic nitrogen to dissolved inorganic phosphorus (DIN:DIP) ratios. However, these increases did not explain the large interannual differences in total cyanobacteria abundance. Rather, we hypothesized that year-to-year differences in bioavailable phosphorus, which also manifested as lower total nitrogen to total phosphorus (TN:TP) ratios, were responsible.

DETECTION OF POTENTIALLY TOXIGENIC MICROCYSTIS STRAINS FROM DAU TIENG RESERVOIR

Microcystis is a bloom-forming, common cyanobacterium in Dau Tieng reservoir used for public water supply. To assess the presence of potentially microcystin-producing Microcystis, molecular techniques were conducted and acute toxicity bioassays were performed with the microcrustacean Daphnia magna exposed to cyanobacterial crude extracts. Potentially toxigenic of isolated strains was characterized by amplifying mcyD genes and identification of Microcystis was confirmed by 16S rRNA amplification. Microcystins (MCs) concentration in bloom samples and cultured strains were quantified by High Performance Liquid Chromatography (HPLC). Results showed that there were 9/15 strains showed positive with the mcyD marker indicating that they are toxic strains. Three MCs variants including MC-RR, -LR and -YR were found in all extracts of toxic strains with the highest concentration of 1,218 μg/g dry weight (DW). The acute toxicity bioassays revealed that both toxic and non-toxic crude extracts elicited significant lethal effects on the tested animal with LC50 values ranged from 189-411 mg DW/L. The toxic effects of isolated strains were independent from the MCs concentration in some strains suggesting the presence of other metabolites contributed to the biological effects. In conclusion, microcystin-producing Microcystis from the Dau Tieng reservoir warn about possible toxic effects for aquatic biota and human health.

Transcriptomic Analysis Reveals the Pathways Associated with Resisting and Degrading Microcystin in Ochromonas.

Toxic Microcystis bloom is a tough environment problem worldwide. Microcystin is highly toxic and is an easily accumulated secondary metabolite of toxic Microcystis that threatens water safety. Biodegradation of microcystin by protozoan grazing is a promising and efficient biological method, but the mechanism in this process is still unclear. The present study aimed to identify potential pathways involved in resisting and degrading microcystin in flagellates through transcriptomic analyses. A total of 999 unigenes were significantly differentially expressed between treatments with flagellates Ochromonas fed on microcystin-producing Microcystis and microcystin-free Microcystis. These dysregulated genes were strongly associated with translation, carbohydrate metabolism, phagosome, and energy metabolism. Upregulated genes encoding peroxiredoxin, serine/threonine-protein phosphatase, glutathione S-transferase (GST), HSP70, and O-GlcNAc transferase were involved in resisting microcystin. In addition, genes encoding cathepsin and GST and genes related to inducing reactive oxygen species (ROS) were all upregulated, which highly probably linked with degrading microcystin in flagellates. The results of this study provided a better understanding of transcriptomic responses of flagellates to toxic Microcystis as well as highlighted a potential mechanism of biodegrading microcystin by flagellate Ochromonas, which served as a strong theoretical support for control of toxic microalgae by protozoans.

The microbiome and antibiotic resistance in integrated fishfarm water: Implications of environmental public health

Aquaculture, the production of farm-raised fish, is a major industry that employs and feeds millions of individuals across the globe, but which may also be a nexus of emerging public health threats. This study examined potential health risks associated with integrated aquaculture operations by with One Health approach using a suite of tools to study water contamination sources, pathogens, antibiotic resistant bacteria, and bacterial community in the water from fishponds. Water samples from 27 fishponds across 9 villages were collected in Jiangmen City, China. Microbial source tracking, pathogens (including Salmonella and Arcobacter), toxin-producing Microcystis, and antibiotic resistant bacteria (ARB) resistant to tetracycline, sulfonamide, and carbapenem were quantified with qPCR. Bacterial community was determined with next-generation sequencing. All ponds exceeded E. coli single-day maximum criteria of US, and 67% ponds exceeded World Health Organization (WHO) waste-fed aquaculture guidelines for protecting consumers and pond workers, representing a high degree of fecal contamination and potential pathogen risks in these ponds. The majority of the ponds were positive for human- (84%) and pig- (41%) associated fecal contamination. Salmonella and microcystin-producing Microcystis were detected in 37% and 15% of the ponds, respectively, while Arcobacter was not detected in any ponds. ARB were highly prevalent. Among the measured factors, canonical correspondence analysis and network analysis demonstrated that secchi depth, temperature and conductivity were the major environmental elements impacting the bacterial community structure, while Proteobacteria and Actinobacteria were the major biological factor. This study demonstrated the presence of intersecting health risk factors in aquaculture facilities and can lay the foundation for addressing these risks in aquaculture management in rural China, with potential applicability in other developing regions dependent on aquaculture.

First report of microcystin-producing Microcystis (Chroococales, Cyanobacteria) in a central highland Vietnam lake

First report of microcystin-producing Microcystis (Chroococales, Cyanobacteria) in a central highland Vietnam lake

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

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

Fluorescence insitu hybridization of Microcystis strains producing microcystin using specific mRNA probes.

Cyanobacteria producing toxic compounds (cyanotoxins) are present in the environment and in water bodies. Their presence poses a threat on human and animal health. It is thus important to detect, identify and enumerate these toxic Cyanobacteria. Using tyramide signal amplification fluorescence insitu hybridization (TSA-FISH) and specific probes, with confocal laser scanning microscopy, we have specifically detected Microcystis strains producing microcystin toxins. The data presented here might be applied to the monitoring of water bodies at early stages and all along the formation of Microcystis blooms.

Fine‐scale cryogenic sampling of planktonic microbial communities: Application to toxic cyanobacterial blooms

A lack of fine-scale methods for sampling planktonic microbial populations hinders advancement in understanding the responses of these communities to environmental conditions. Current methods provide resolution at scales of centimeters to meters, but not at the millimeter-scale required to understand highly stratified communities. To address this we developed two cryogenic sampling tools to collect spatially-precise samples from aquatic environments while simultaneously preserving the microbial communities. The application of these samplers was examined over a 5.5 h period using a cyanobacterial scum (Microcystis) formed in experimental mesocosms. A cryogenic “surface snatcher” collected a discrete layer (ca. 1 mm) of surface water. Compared to conventional surface sampling methods, the surface snatcher samples contained up to 22-times more microcystin, indicating that less underlying water was incorporated into the sample. A cryogenic “cold finger” sampler was used to collect vertical profiles of the upper 40 mm of the water column. This profiler provided new insights into the fine-scale structure of Microcystis scums, demonstrating that more microcystin-producing Microcystis was contained in the surface 5 mm than the 35 mm below. The results also showed that upregulation of microcystin production was highly localized in the top 2.5 mm of the Microcystis scum. Our results demonstrate that extreme changes in cyanobacterial communities can occur over small distances, and indicate that sampling resolution is of great importance for improving knowledge on cyanobacterial blooms and toxin production. While this study focused on microcystin-producing Microcystis, the cryogenic sampling tools described here could be applied to any planktonic microbial community.

Dominance and succession of Microcystis genotypes and morphotypes in Lake Taihu, a large and shallow freshwater lake in China

Lake Taihu, which is the third largest freshwater lake in China, has experienced extensive cyanobacterial (Microcystis spp.) blooms over the past two decades. However, the distribution, dynamics and succession of the blooms have not been fully studied. To better understand the basic characteristics of Microcystis blooms in Lake Taihu, samples were collected from December 2008 to December 2009. The distribution and dynamics of different Microcystis morphotypes were characterized. Microcystis genotypes were analyzed also by sequencing the clone library of the internal transcribed spacer of the rRNA operon (ITS). The abundance of total Microcystis and the proportion of microcystin-producing subpopulation were estimated by using a quantitative PCR assay. Marked succession in both morphotypes and genotypes of the Microcystis population occurred during the course of the Microcystis bloom. The 2337 ITS sequences were obtained and were revealed to contain 618 Microcystis genotypes, which was the highest Microcystis genetic diversity reported in Lake Taihu. The T1 genotype, which was characterized by strains of Microcystis flos-aquae, was the most dominant genotype during winter and spring around the entire lake and likely acted as the main inoculum for forming blooms the following year. Water temperature periodically affected the succession of both Microcystis genotypes and morphotypes, whereas the micro-environment influenced the spatial distribution of Microcystis genotypes and morphotypes. High ratios of mcyD containing Microcystis subpopulations were detected during the onset and later bloom phases. A redundancy analysis (RDA) indicated that the water temperature and PO4-P were the major factors controlling both the total Microcystis abundance and the proportion of microcystin-producing Microcystis in hyper-eutrophic waters.

Stress-responsive expression of a glutathione S-transferase (delta) gene in waterflea Daphnia magna challenged by microcystin-producing and microcystin-free Microcystis aeruginosa

Harmful cyanobacterial blooms resulting from eutrophication and global warming have emerged as a worldwide environmental concern. Some zooplankton populations, including Daphnia, have been shown to adapt locally to microcystin-producing Microcystis. Previous in vitro experiments indicate that glutathione-S-transferase (GST) may act as the first step of detoxification in Daphnia by conjugating microcystins (MCs) with glutathione. The GST family is categorized into many classes, and different classes present distinct responses to MC detoxification. To date, however, the molecular mechanism of single class GST participation in buffering the toxic effects of MCs in Daphnia remains poorly known. In this study, a full-length delta-GST cDNA of Daphnia magna (Dm-dGST) was isolated and characterized through bioinformatics. Differential gene expression studies revealed that short-term exposure to microcystin-producing (MP) Microcystis aeruginosa increased Dm-dGST transcript levels. By contrast, long-term exposure to MP or microcystin-free (MF) M. aeruginosa decreased Dm-dGST transcript levels. Together with changes in three other antioxidation biomarkers (catalase, CuZn- and Mn-superoxide dismutase), it is concluded that Dm-dGST can potentially biotransform MCs to reduce their toxicity. The present study highlights the importance of Dm-dGST in response to MC toxicity and may thus facilitate future research on the molecular mechanisms of MC tolerance in zooplankton under an increasing eutrophic world.

The trophic state of lake water regulates spatial-temporal variations of bloom-forming Microcystis

Although biomass variations in Microcystis and microcystin have been widely reported, few studies have addressed whether different trophic states of natural lake water affect the spatial-temporal variations in abundances of microcystin-producing Microcystis in a given bloom. In this study, we used a harmful algal bloom in Chaohu Lake, eastern China, as an example to investigate the mutual relationship between different nutrient states and environmental factors, and the impact on Microcystis. Overall, cyanobacteria and Microcystis were more abundant in the middle and western parts of the lake under high nutrients levels, while in the eastern part, nutrient concentrations were low enough to limit biomass, and their fluctuations affected the contents of toxic Microcystis. Moreover, microcystin concentration was correlated positively to nutrient levels and Microcystis biomass during bloom developing in 2013 from June to August. Temporally, the cellular content of total microcystin was lowest when the bloom peaked in intensity. Our results suggest that lake eutrophication not only results in cyanobacterial blooms, but may also increase the proportion of toxic Microcystis species and their cell-bound MCs contents (i.e. microcystin cell quotas) under mild eutrophication. The present investigation provided molecular evidence for the selection of MC-producing and non-MC-producing genotypes. The current study provides new evidence advocating the monitoring of partitions of large lakes when studying cyanobacteria and toxin-contaminated freshwaters, which will be beneficial for both water agencies and water researchers.

Analysis of phytoplankton in Tsimlyansk Reservoir (Russia) for the presence of cyanobacterial hepato- and neurotoxins

Although the water bodies of southern Russia experience the most extreme effects of cyanobacterial blooms, molecular genetic data on the composition of toxigenic cyanobacteria in this region have been absent. Screening for the genes responsible for the synthesis of hepatotoxins (microcystins and cylindrospermopsin) and neurotoxins (anatoxin-a and saxitoxins) in cyanobacteria from the Tsimlyansk reservoir on the Don River was carried out. The presence of microcystin-producing Microcystis and Planktothrix populations, as well as of cyanobacteria capable of synthesis of a neurotoxin anatoxin-a was revealed by polymerase chain reaction (PCR). A hypothesis of the presence of anatoxin-a-producing Planktothrix rubescens population in the phytoplankton of the Tsimlyansk reservoir is proposed. The obtained PCR data were confirmed by the results of enzyme-linked immunosorbent assay (ELISA) and liquid chromatography/mass-spectrometry (LC/MS). Anatoxin-a and five microcystin variants were identified in the phytoplankton biomass.

Sensitive and rapid detection of microcystin synthetase E Gene (mcyE) by loop-mediated isothermal amplification: A new assay for detecting the potential microcystin-producing Microcystis in the aquatic ecosystem

In order to develop a new molecular technique that has the potential to assist with monitoring and management of water bodies for potential microcystin producing cyanobacterial species that occur in mixed populations in many regions of the world, we designed a new loop-mediated isothermal amplification (LAMP) assay based on microcystin biosynthesis genes. Four sets of primers were designed to recognize six distinct sequences on target the mcyE gene that encodes a protein (McyE) being responsible to catalyze the addition of d-glutamate to Adda. One set (MCYE2) was selected as the most appropriate set of primers for its rapid detection. The specificity and sensitivity of the primers in the LAMP reactions for mcyE detection were determined. Two methods, namely, monitoring of turbidity and addition of calcein to the reaction tube, were used to determine negative and positive results. The results showed that target DNA was amplified and visualized by the two detection methods within 40min at an isothermal temperature of 61°C. For the sensitivity of LAMP, the detection limit was 8.5pg/μl (approximately 17pg) DNA. The eleven microcystin producing and four non-toxic cyanobacterial strains were selected for testing of specificity. The results of the amplification were positive with all microcystin-producing strains tested and not with four non-toxic strains, which showed that the primers had good levels of specificity. For testing the application of LAMP assay in the aquatic ecosystem, seven environmental samples from ponds and lakes in Ningbo City were also analyzed using the LAMP targeting the mcyE gene as well as an ELISA assay. Compared with these results of ELISA assay, LAMP assay is satisfied. All of these validated LAMP method being fast, simple and low in cost is a potentially valuable means for potential toxic of cyanobacterial blooms detection, especially for routine monitoring purposes in future.

Diversity of cyanobacteria and cyanotoxins in Hartbeespoort Dam, South Africa

The South African Hartbeespoort Dam is known for the occurrence of heavy Microcystis blooms. Although a few other cyanobacterial genera have been described, no detailed study on those cyanobacteria and their potential toxin production has been conducted. The diversity of cyanobacterial species and toxins is most probably underestimated. To ascertain the cyanobacterial composition and presence of cyanobacterial toxins in Hartbeespoort Dam, water samples were collected in April 2011. In a polyphasic approach, 27 isolated cyanobacterial strains were classified morphologically and phylogenetically and tested for microcystins (MCs), cylindrospermopsin (CYN), saxitoxins (STXs) and anatoxin-a (ATX) by liquid chromatography–tandem mass spectrometry (LC–MS/MS) and screened for toxin-encoding gene fragments. The isolated strains were identified as Sphaerospermopsis reniformis, Sphaerospermopsis aphanizomenoides, Cylindrospermopsis curvispora, Raphidiopsis curvata, Raphidiopsis mediterrranea and Microcystis aeruginosa. Only one of the Microcystis strains (AB2011/53) produced microcystins (35 variants). Forty-one microcystin variants were detected in the environmental sample from Hartbeespoort Dam, suggesting the existence of other microcystin producing strains in Hartbeespoort Dam. All investigated strains tested negative for CYN, STXs and ATX and their encoding genes. The mcyE gene of the microcystin gene cluster was found in the microcystin-producing Microcystis strain AB2011/53 and in eight non-microcystin-producing Microcystis strains, indicating that mcyE is not a good surrogate for microcystin production in environmental samples.

Sedimentation Patterns of Toxin-Producing Microcystis Morphospecies in Freshwater Reservoirs

Understanding the annual cycle of Microcystis is essential for managing the blooms of this toxic cyanobacterium. The current work investigated the sedimentation of microcystin-producing Microcystis spp. in three reservoirs from Central Spain during the summer and autumn of 2006 and 2007. We confirmed remarkable settling fluxes during and after blooms ranging 106–109 cells m−2 d−1, which might represent 0.1%–7.6% of the organic matter settled. A comprehensive analysis of the Valmayor reservoir showed average Microcystis settling rates (0.04 d−1) and velocities (0.7 m d−1) that resembled toxin settling in the same reservoir and were above most reported elsewhere. M. aeruginosa settling rate was significantly higher than that of M. novacekii and M. flos-aquae. Despite the fact that colony sizes did not differ significantly in their average settling rates, we observed extremely high and low rates in large colonies (>5000 cells) and a greater influence of a drop in temperature on small colonies (<1000 cells). We found a 4–14 fold decrease in microcystin cell quota in settling Microcystis of the Cogotas and Valmayor reservoirs compared with pelagic populations, and the hypothetical causes of this are discussed. Our study provides novel data on Microcystis settling patterns in Mediterranean Europe and highlights the need for including morphological, chemotypical and physiological criteria to address the sedimentation of complex Microcystis populations.