Mcy Gene Research Articles

Nostoc linckia (Bornet ex Bornet et Flahault, 1886) (Nostocales: Cyanobacteria) from the Sea of Azov: Morphology and Molecular Investigation of Toxigenicity

The Sea of Azov is a part of the Mediterranean basin and is the most continental sea in the world. But the phycological studies of the Sea of Azov were irregular. To assess the ecological safety of sea water, it is important not only to determine the presence of toxigenic species in the flora, but also to confirm the presence of strains capable of synthesizing toxins. The strain Nostoc linckia MZ–C21 was isolated from a planktonic sample collected near a seacoast of the Sivash (the bay of the Sea of Azov near Henichesk, Ukraine). PCR reactions demonstrated that the investigated strain has genes encoding all known groups of hepatotoxins: microcystin (the mcyE gene), nodularin (the ndaF gene), and cylindrospermopsin (the cyrB gene) synthetases. The novel strain MZ–C21 formed strongly supported phylogenetic lineages with other microcystin and nodularin producers. Thus, there is a high probability for the production of cyanotoxins by Nostoc linckia growths in the Sea of Azov near Henichesk.

Discovery of microcystin-producing Anagnostidinema pseudacutissimum from cryopreserved Antarctic cyanobacterial mats

Microcystins (MCs) are secondary metabolites produced by cyanobacteria and have been well-documented in temperate and tropical regions. However, knowledge of the production of MCs in extremely cold environments is still in its infancy. Recently, examination of 100-year-old Antarctic cyanobacterial mats collected from Ross Island and the McMurdo Ice Shelf during Captain R.F. Scott's Discovery Expedition revealed that the presence of MCs in Antarctica is not a new phenomenon. Here, morphological and molecular phylogenetic analyses are used to identify a new microcystin-producing freshwater cyanobacterium, Anagnostidinema pseudacutissimum. The strain was isolated from a deep-frozen (−15 °C) sample collected from a red-brown cyanobacterial mat in a frozen pond at Cape Crozier (Ross Island, continental Antarctica) in 1984–1985. Amplification of the mcyE gene fragment involved in microcystin biosynthesis from A. pseudacutissimum confirmed that it is identical to the sequence from other known microcystin-producing cyanobacteria. Analysis of extracts from this A. pseudacutissimum strain by HPLC-MS/MS confirmed the presence of MC-LR and -YR at concentrations of 0.60 μg/L and MC-RR at concentrations of 0.20 μg/L. This is the first report of microcystin production from a species of Anagnostidinema from Antarctica.

Effect of Zinc on Microcystis aeruginosa UTEX LB 2385 and Its Toxin Production

Cyanobacteria harmful algal blooms (CHABs) are primarily caused by man-made eutrophication and increasing climate-change conditions. The presence of heavy metal runoff in affected water systems may result in CHABs alteration to their ecological interactions. Certain CHABs produce by-products, such as microcystin (MC) cyanotoxins, that have detrimentally affected humans through contact via recreation activities within implicated water bodies, directly drinking contaminated water, ingesting biomagnified cyanotoxins in seafood, and/or contact through miscellaneous water treatment. Metallothionein (MT) is a small, metal-sequestration cysteine rich protein often upregulated within the stress response mechanism. This study focused on zinc metal resistance and stress response in a toxigenic cyanobacterium, Microcystis aeruginosa UTEX LB 2385, by monitoring cells with (0, 0.1, 0.25, and 0.5 mg/L) ZnCl2 treatment. Flow cytometry and phase contrast microscopy were used to evaluate physiological responses in cultures. Molecular assays and an immunosorbent assay were used to characterize the expression of MT and MC under zinc stress. The results showed that the half maximal inhibitory concentration (IC50) was 0.25 mg/L ZnCl2. Flow cytometry and phase contrast microscopy showed morphological changes occurred in cultures exposed to 0.25 and 0.5 mg/L ZnCl2. Quantitative PCR (qPCR) analysis of selected cDNA samples showed significant upregulation of Mmt through all time points, significant upregulation of mcyC at a later time point. ELISA MC-LR analysis showed extracellular MC-LR (µg/L) and intracellular MC-LR (µg/cell) quota measurements persisted through 15 days, although 0.25 mg/L ZnCl2 treatment produced half the normal cell biomass and 0.5 mg/L treatment largely inhibited growth. The 0.25 and 0.5 mg/L ZnCl2 treated cells demonstrated a ~40% and 33% increase of extracellular MC-LR(µg/L) equivalents, respectively, as early as Day 5 compared to control cells. The 0.5 mg/L ZnCl2 treated cells showed higher total MC-LR (µg/cell) quota yield by Day 8 than both 0 mg/L ZnCl2 control cells and 0.1 mg/L ZnCl2 treated cells, indicating release of MCs upon cell lysis. This study showed this Microcystis aeruginosa strain is able to survive in 0.25 mg/L ZnCl2 concentration. Certain morphological zinc stress responses and the upregulation of mt and mcy genes, as well as periodical increased extracellular MC-LR concentration with ZnCl2 treatment were observed.

Molecular and Immunological Methods to Confirm Toxiginicity (Microcystin Production) of Westiellopsis Prolifica Isolated from Tigris River – Iraq

Several toxigenic cyanobacteria produce the cyanotoxin (microcystin). Being a health and environmental hazard, screening of water sources for the presence of microcystin is increasingly becoming a recommended environmental procedure in many countries of the world. This study was conducted to assess the ability of freshwater cyanobacterial species Westiellopsis prolifica to produce microcystins in Iraqi freshwaters via using molecular and immunological tools. The toxigenicity of W. prolifica was compared via laboratory experiments with other dominant bloom-forming cyanobacteria isolated from the Tigris River: Microcystis aeruginosa, Chroococcus turigidus, Nostoc carneum, and Lyngbya sp. significant expression of mcyE gene and microcystin production was most evident in W. prolifica. Contrary to the prevailing concept that M. aeruginosa is a main microcystin producer in freshwaters around the world, no significant microcystin production was observed with this species throughout the time points studied in our laboratory methods. As for C. turigidus, N. carneum and Lyngbya sp., neither mcyE expression nor microcystin production was significant. Data from mcyE expression by RT-qPCR were generally in agreement with those obtained from microcystin quantification by ELISA. Interestingly, W. prolifica, which showed clear microcystin-producing ability in this study and which was not reported before in the literature to produce microcystin, can be added as a new microcystin producer to the list of toxigenic cyanobacteria.

The Effect of a Combined Hydrogen Peroxide-MlrA Treatment on the Phytoplankton Community and Microcystin Concentrations in a Mesocosm Experiment in Lake Ludoš.

Harmful cyanobacteria and their toxic metabolites constitute a big challenge for the production of safe drinking water. Microcystins (MC), chemically stable hepatotoxic heptapeptides, have often been involved in cyanobacterial poisoning incidents. A desirable solution for cyanobacterial management in lakes and ponds would eliminate both excess cyanobacteria and the MC that they potentially produce and release upon lysis. Hydrogen peroxide (H2O2) has recently been advocated as an efficient means of lysing cyanobacteria in lakes and ponds, however H2O2 (at least when used at typical concentrations) cannot degrade MC in environmental waters. Therefore, mesocosm experiments combining the cyanobacteria-lysing effect of H2O2 and the MC-degrading capacity of the enzyme MlrA were set up in the highly eutrophic Lake Ludoš (Serbia). The H2O2 treatment decreased the abundance of the dominant cyanobacterial taxa Limnothrix sp., Aphanizomenon flos-aquae, and Planktothrix agardhii. The intracellular concentration of MC was reduced/eliminated by H2O2, yet the reduction of the extracellular MC could only be accomplished by supplementation with MlrA. However, as H2O2 was found to induce the expression of mcyB and mcyE genes, which are involved in MC biosynthesis, the use of H2O2 as a safe cyanobacteriocide still requires further investigation. In conclusion, the experiments showed that the combined use of H2O2 and MlrA is promising in the elimination of both excess cyanobacteria and their MC in environmental waters.

A Multiplex Analysis of Potentially Toxic Cyanobacteria in Lake Winnipeg during the 2013 Bloom Season.

Lake Winnipeg (Manitoba, Canada), the world’s 12th largest lake by area, is host to yearly cyanobacterial harmful algal blooms (cHABs) dominated by Aphanizomenon and Dolichospermum. cHABs in Lake Winnipeg are primarily a result of eutrophication but may be exacerbated by the recent introduction of dreissenid mussels. Through multiple methods to monitor the potential for toxin production in Lake Winnipeg in conjunction with environmental measures, this study defined the baseline composition of a Lake Winnipeg cHAB to measure potential changes because of dreissenid colonization. Surface water samples were collected in 2013 from 23 sites during summer and from 18 sites in fall. Genetic data and mass spectrometry cyanotoxin profiles identified microcystins (MC) as the most abundant cyanotoxin across all stations, with MC concentrations highest in the north basin. In the fall, mcyA genes were sequenced to determine which species had the potential to produce MCs, and 12 of the 18 sites were a mix of both Planktothrix and Microcystis. Current blooms in Lake Winnipeg produce low levels of MCs, but the capacity to produce cyanotoxins is widespread across both basins. If dreissenid mussels continue to colonize Lake Winnipeg, a shift in physicochemical properties of the lake because of faster water column clearance rates may yield more toxic blooms potentially dominated by microcystin producers.

Cyanobacterial biodiversity of semiarid public drinking water supply reservoirs assessed via next-generation DNA sequencing technology.

Next-generation DNA sequencing technology was applied to generate molecular data from semiarid reservoirs during well-defined seasons. Target sequences of 16S-23S rRNA ITS and cpcBA-IGS were used to reveal the taxonomic groups of cyanobacteria present in the samples, and genes coding for cyanotoxins such as microcystins (mcyE), saxitoxins (sxtA), and cylindrospermopsins (cyrJ) were investigated. The presence of saxitoxins in the environmental samples was evaluated using ELISA kit. Taxonomic analyses of high-throughput DNA sequencing data showed the dominance of the genus Microcystis in Mundaú reservoir. Furthermore, it was the most abundant genus in the dry season in Ingazeira reservoir. In the rainy season, 16S-23S rRNA ITS analysis revealed that Cylindrospermopsis raciborskii comprised 46.8% of the cyanobacterial community in Ingazeira reservoir, while the cpcBAIGS region revealed that C. raciborskii (31.8%) was the most abundant taxon followed by Sphaerospermopsis aphanizomenoides (17.3%) and Planktothrix zahidii (16.6%). Despite the presence of other potential toxin-producing genera, the detected sxtA gene belonged to C. raciborskii, while the mcyE gene belonged to Microcystis in both reservoirs. The detected mcyE gene had good correlation with MC content, while the amplification of the sxtA gene was related to the presence of STX. The cyrJ gene was not detected in these samples. Using DNA analyses, our results showed that the cyanobacterial composition of Mundaú reservoir was similar in successive dry seasons, and it varied between seasons in Ingazeira reservoir. In addition, our data suggest that some biases of analysis influenced the cyanobacterial communities seen in the NGS output of Ingazeira reservoir.

The Biosynthesis of Rare Homo-Amino Acid Containing Variants of Microcystin by a Benthic Cyanobacterium.

Microcystins are a family of chemically diverse hepatotoxins produced by distantly related cyanobacteria and are potent inhibitors of eukaryotic protein phosphatases 1 and 2A. Here we provide evidence for the biosynthesis of rare variants of microcystin that contain a selection of homo-amino acids by the benthic cyanobacterium Phormidium sp. LP904c. This strain produces at least 16 microcystin chemical variants many of which contain homophenylalanine or homotyrosine. We retrieved the complete 54.2 kb microcystin (mcy) gene cluster from a draft genome assembly. Analysis of the substrate specificity of McyB1 and McyC adenylation domain binding pockets revealed divergent substrate specificity sequences, which could explain the activation of homo-amino acids which were present in 31% of the microcystins detected and included variants such as MC-LHty, MC-HphHty, MC-LHph and MC-HphHph. The mcy gene cluster did not encode enzymes for the synthesis of homo-amino acids but may instead activate homo-amino acids produced during the synthesis of anabaenopeptins. We observed the loss of microcystin during cultivation of a closely related strain, Phormidium sp. DVL1003c. This study increases the knowledge of benthic cyanobacterial strains that produce microcystin variants and broadens the structural diversity of known microcystins.

Mutagenesis of the Microcystin Tailoring and Transport Proteins in a Heterologous Cyanotoxin Expression System.

The microcystins are a large group of cyclic peptide hepatotoxins produced by several genera of freshwater cyanobacteria. The genes responsible for microcystin biosynthesis are encoded within a large (∼55 kbp) gene cluster, mcyA-J. The recent establishment of a cyanotoxin heterologous expression system in Escherichia coli has provided the means to study microcystin biosynthesis in a genetically tractable, rapidly growing host. Using this system, we demonstrate that deletion of the ABC-transporter, mcyH, and dehydrogenase, mcyI, abolishes microcystin production, while deletion of the O-methyltransferase, mcyJ, results in the production of the demethylated (DM) toxin [d-Asp3, DMAdda5]microcystin-LR. Both methylated and DM toxin variants were heterologously produced at high titers and efficiently exported into the extracellular medium, enabling easy purification. The results show that the mcy gene cluster can be engineered in E. coli to study the function of its individual components and direct the synthesis of particular microcystin variants. This technology could potentially be applied to other natural products of ecological and biomedical significance.

Unmasking the identity of toxigenic cyanobacteria driving a multi-toxin bloom by high-throughput sequencing of cyanotoxins genes and 16S rRNA metabarcoding

Cyanobacterial harmful algal blooms (CyanoHABs) are complex communities that include coexisting toxic and non-toxic strains only distinguishable by genetic methods. This study shows a water-management oriented use of next generation sequencing (NGS) to specifically pinpoint toxigenic cyanobacteria within a bloom simultaneously containing three of the most widespread cyanotoxins (the hepatotoxins microcystins, MCs; and the neurotoxins anatoxin-a, ATX, and saxitoxins, STXs). The 2013 summer bloom in Rosarito reservoir (Spain) comprised 33 cyanobacterial OTUs based on 16S rRNA metabarcoding, 7 of which accounted for as much as 96.6% of the community. Cyanotoxins and their respective biosynthesis genes were concurrently present throughout the entire bloom event including: MCs and mcyE gene; ATX and anaF gene; and STXs and sxtI gene. NGS applied to amplicons of cyanotoxin-biosynthesis genes unveiled 6 toxigenic OTUs, comprising 3 involved in MCs production (Planktothrix agardhii and 2 Microcystis spp.), 2 in ATX production (Cuspidothrix issatschenkoi and Phormidium/Tychonema spp.) and 1 in STXs production (Aphanizomenon gracile). These toxigenic taxa were also present in 16S rRNA OTUs list and their relative abundance was positively correlated with the respective toxin concentrations. Our results point at MC-producing P. agardhii and ATX-producing C. issatschenkoi as the main contributors to the moderate toxin concentrations observed, and suggest that their distribution in Southern Europe is broader than previously thought. Our findings also stress the need for monitoring low-abundance cyanobacteria (<1% relative abundance) in cyanotoxicity studies, and provide novel data on the presence of picocyanobacteria and potentially ATX-producing benthic taxa (e.g., Phormidium) in deep thermally-stratified water bodies. This study showcases a straightforward use of amplicon metagenomics of cyanotoxin biosynthesis genes in a multi-toxin bloom thus illustrating the broad applicability of NGS for water management in risk-oriented monitoring of CyanoHABs.

Deciphering the effects of nitrogen, phosphorus, and temperature on cyanobacterial bloom intensification, diversity, and toxicity in western Lake Erie

AbstractAlthough cyanobacterial harmful algal blooms (CHABs) are promoted by nutrient loading and elevated temperatures, the effects of these processes on bloom diversity are unclear. This study used traditional and next‐generation sequencing approaches to assess shifts in phytoplankton, cyanobacterial (16S rRNA), and microcystin‐producing (mcyE) communities during CHABs in western Lake Erie (Maumee and Sandusky Bays) in response to natural and experimental gradients of nitrogen (N), phosphorus (P), and temperature. CHABs were most intense near the Maumee and Sandusky Rivers and were dominated by Microcystis and Planktothrix, respectively. Sequencing of 16S amplicons revealed cryptic cyanobacterial diversity (47 genera) including high abundances of two distinct clades of Synechococcus in both bays and significant differences in community structure between nutrient‐rich nearshore sites and less eutrophic offshore sites. Sequencing of mcyE genes revealed low taxonomic (n = 3) but high genetic diversity (n = 807), with toxigenic strains of Planktothrix being more abundant than Microcystis and more closely paralleling microcystin concentrations. Cyanobacterial abundance significantly increased in response to elevated N, with the greatest increases in combined high N, P, and temperature treatments that concurrently suppressed green and brown algae. N significantly increased microcystin concentrations and the relative abundance of nondiazotrophic genera such as Planktothrix, while diazotrophic genera such as Dolichospermum and Aphanizomenon were, in some cases, enhanced by high P and temperature. While nutrients and elevated temperatures promote CHABs, differing combinations selectively promote individual cyanobacterial genera and strains, indicating management of both N and P will be required to control all cyanobacteria in Lake Erie, particularly as lake temperatures rise.

Cyanobacterial blooms in the central basin of Lake Erie: Potentials for cyanotoxins and environmental drivers

Abstract Lake Erie western basin (WB) cyanobacterial blooms are a yearly summer occurrence; however, blooms have also been reported in the offshore waters of the central basin (CB), and very little is known about what drives these blooms or their potential for cyanobacterial toxins. Cyanobacteria Index was quantified using MODIS and MERIS data for the CB between 2003 and 2017, and water samples were collected between 2013 and 2017. The goals were to 1) quantify cyanobacteria, 2) determine environmental drivers of CB blooms, and 3) determine the potential for cyanobacterial toxins in the CB. Dolichospermum (Anabaena) occurred in the CB during July before the onset of the WB bloom, and then in August and September, the cyanobacteria community shifted towards Microcystis. The largest Dolichospermum blooms (2003, 2012, 2013, and 2015) were associated with reduced water clarity (Secchi disk depth 20 μmol/L) and high nitrogen-to‑phosphorus ratios (>100), which indicate nutrient concentrations or ratios did not select for Dolichospermum. Additionally, the sxtA gene, but not mcyE or microcystins, were detected in the CB during July 2016 and 2017. The mcyE gene and microcystins were detected in the CB during August 2016 and 2017. The results indicate the CB's potential for cyanotoxins shifts from saxitoxins to microcystins throughout the summer. Continued monitoring of cyanobacteria and multiple cyanobacterial toxins is recommended to ensure safe drinking water for CB coastal communities.

Anabaena/Dolichospermum as the source of lethal microcystin levels responsible for a large cattle toxicosis event.

Thirty-two 14-month old steers died during a period of four days (19–23 June 2017) after drinking from Junipers Reservoir (southeastern Oregon, USA) during a cyanobacterial bloom. Clinical and histopathological findings were consistent with acute liver disease, and microcystin-LR was present at 3000 μg/L in a reservoir water sample and at 7100 μg/L in the rumen contents of one of the mortalities. Serum biochemistry and histological examination indicated severe liver damage consistent with microcystin toxicosis. Microscopic observation of reservoir water samples, limited to frozen or poorly stored and partially degraded samples, indicated the presence of abundant Anabaena/Dolichospermum, but the presence of other toxic cyanobacteria such as Microcystis could not be excluded. Metagenomic analysis showed the presence in these samples of a single cyanobacterium whose cpcBA, rpoB and rbcL genes indicated membership in the Anabaena/Dolichospermum genus. The sequence of a complete mcy gene cluster with homology to previously identified Anabaena mcy genes was recovered. These results emphasize the capacity for Anabaena/Dolichospermum blooms to produce lethal levels of microcystin, posing a danger to public health and livestock. Further, our findings indicate that such occurrences can occur outside the far-northern latitudes in which microcystin-producing Anabaena have typically been found.

PCR –Based Detection of Microcystin and Nodularin in both Freshwater and Bloom in Tigris River

Cyanobacterial blooms have a range of social, environmental and economic impacts due to their content of secondary metabolites involving toxins. Microcystins (MCs) is one of the cyanotoxins found in freshwater ecosystems. This study was aimed to adopt a rapid technique for determining the microcystin in blooms and freshwater by the detection of phycocyanin operon of cyanobacteria using PCβF and PCαR primers as well as mcyE gene encoding to microcystin/ nodularin using HEP primers. The molecular results showed that phycocyanin operon and mcyE gene were disclosed in the freshwater and bloom of samples at the studied sites. In conclusion, The PCR assay used in this study was helpful and rapid, in particular when the target organism concentration in the freshwater sample is very low.

First observation of microcystin- and anatoxin-a-producing cyanobacteria in the easternmost part of the Gulf of Finland (the Baltic Sea)

The aim of this study was to obtain the first data on the occurrence and distribution of potentially toxic cyanobacteria and cyanotoxins in the Russian Easternmost part of the Gulf of Finland of the Baltic Sea. Studied samples were collected from 2012 to 2017 and three independent approaches – HPLC-HRMS, PCR and light microscopy were applied for cyanotoxins analysis and detection of toxigenic cyanobacteria. Aphanizomenon flos-aquae Ralfs ex Born. et Flah., Planktothrix agardhii (Gom.) Anag. et Kom., Microcystis aeruginosa (Kütz.) Kütz. and Dolichospermum spp. dominated among cyanobacteria in collected samples. In 2012–2013 during research cruises, microcystins concentrations varied from below detection levels to low (0.01–0.6 μg L−1) values. In the autumn of 2015 and 2017, during cyanobacterial bloom events very high concentrations of microcystins (dissolved up to 49 μg L−1, intracellular up to 466 μg g−1) and dissolved anatoxin-a (1.4 μg L−1) were detected. The evaluated toxin profile was represented by most common arginine-containing variants of microcystins (MC-LR, MC-RR, MC-YR) and their desmethylated forms. Leucine-containing congeners (MC-LF; MC-LY; MC-LW for the biomass sample from the coast of Komarovo, 2015) were found at low concentrations. In environmental DNA from bloom samples, we identified mcy genes regions responsible for MC biosynthesis that are specific for Dolichospermum, Microcystis, and Planktothrix. This study is the first molecular evidence the ability of Microcystis aeruginosa and Planktothrix agardhii from the Gulf of Finland to produce microcystins. On the basis of the obtained data of genus-specific PCR and microscopy, we suppose the presence of anatoxin-a-producing Apanizomenon flos-aquae population in the phytoplankton of Russian part of the Gulf of Finland.

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.

A Systematic Literature Review for Evidence of Aphanizomenon flos-aquae Toxigenicity in Recreational Waters and Toxicity of Dietary Supplements: 2000⁻2017.

Previous studies of recreational waters and blue-green algae supplements (BGAS) demonstrated co-occurrence of Aphanizomenon flos-aquae (AFA) and cyanotoxins, presenting exposure risk. The authors conducted a systematic literature review using a GRADE PRISMA-p 27-item checklist to assess the evidence for toxigenicity of AFA in both fresh waters and BGAS. Studies have shown AFA can produce significant levels of cylindrospermopsin and saxitoxin in fresh waters. Toxicity studies evaluating AFA-based BGAS found some products carried the mcyE gene and tested positive for microcystins at levels ≤ 1 μg microcystin (MC)-LR equivalents/g dry weight. Further analysis discovered BGAS samples had cyanotoxins levels exceeding tolerable daily intake values. There is evidence that Aphanizomenon spp. are toxin producers and AFA has toxigenic genes such as mcyE that could lead to the production of MC under the right environmental conditions. Regardless of this ability, AFA commonly co-occur with known MC producers, which may contaminate BGAS. Toxin production by cyanobacteria is a health concern for both recreational water users and BGAS consumers. Recommendations include: limit harvesting of AFA to months when toxicity is lowest, include AFA in cell counts during visible blooms, and properly identify cyanobacteria species using 16S rRNA methods when toxicity levels are higher than advisory levels.

Molecular characterization and toxin quantification of Microcystis panniformis: A microcystin producer in Lake Taihu, China

Microcystis panniformis is a bloom forming species with flat panniform-like colonies. This species was recently found in Lake Taihu, China. To specifically characterize M. panniformis based on isolated strains, morphological examination on colonial transition and genetic examination are needed. Three M. panniformis strains isolated from a water bloom sample in Lake Taihu were characterized by molecular analysis and toxin quantification. Phylogenetic analysis based on both 16S rRNA gene and internal transcribed spacer (ITS) between 16S and 23S rRNA genes were performed and compared to facilitate easy identification of the species. Relatively high similarities (98%-99%) were shown in 16S rDNA sequences between the strains of M. panniformis and those of other Microcystis species, whereas the similarities for ITS sequences were 88%-95%. In the phylogenetic tree based on the 16S rDNA sequences, the M. panniformis and M. aeruginosa strains were intermixed together with no clear division, whereas all of the M. panniformis strains were clustered together in a single clade based on the ITS sequences based phylogenyetic tree. The mcyE gene was detected in all three strains, and microcystin was determined by high-performance liquid chromatography. The molecular detection and toxin production of M. panniformis strains are of great significance for the environmental risk assessment of Microcystis blooms.

Detección de poblaciones tóxicas de Microcystis spp. con distintas preferencias ambientales. Estudio de caso embalse de Salto Grande

In this work, the dynamics and toxicity of Microcystis spp. in Salto Grande reservoir (Uruguay river), as well as the genetic diversity of their toxic populations were assessed in winter and summer during 2013. Cell abundance of Microcystis spp. variated amongst different sites and between seasons, showing a signifcantly higher cell and toxic genotype abundances (copies of mcyE gene) and microcystin-LR concentration during summer. Furthermore, when genetic diversity of toxic Microcystis was analyzed by high resolution melting analysis of mcyJ amplicons, two genotypes exhibiting different environmental preferences were detected (cluster 1 and 2). Cluster 1 included samples found at water conditions of low temperature (lower than 17,6 °C) and slightly high conductivity (~52 to 58 µS cm-1), showing small Microcystis abundance and absence of microcystin-LR, whereas cluster 2 comprised samples from higher temperatures (between 17,6 and 25,9 °C) and lower conductivity (~50 to 52 µS cm-1) conditions, and with a higher cell abundance and microcystin-LR production. Our results suggest that toxic Microcystis spp. populations that bloom during summer are different from those that persist during winter and pointed to water temperature and conductivity as relevant variables that control their abundance and toxicity.

Preliminary Study to Find Occurrence and Dynamics of Toxic Cyanobacteria in Nuwara wewa and Nachchaduwa wewa, Two Man-made Reservoirs in Sri Lanka

Bloom of cyanobacteria in water bodies causes devastating problems to the ecosystem and humans when they depend on these waters for drinking purpose. Microcystin and nodularin, produced by some cyanobacteria, are toxic for human and many other aquatic organisms. Nuwara wewa and Nachchaduwa wewa are two important reservoirs supplying water for drinking purpose in addition to agricultural use. Therefore, preliminary study was conducted to evaluate these reservoirs for the presence of toxic cyanobacteria and their dynamics is of importance to take proper measures to control cyanobacterial growth and to ensure supply of quality water for human drinking purpose. Water samples were collected for 12 months from both reservoirs and physic-chemicals parameters of water were also recorded. Correlations between the physico-chemical parameters of water and the cyanobacterial density were established. Toxic cyanobacteria were isolated and identified using conventional and molecular methods. 16S rRNA gene and mcy-A gene were targeted for molecular identification using PCR. Cyanobacterial density had strong positive correlations with some physico-chemical parameters such as pH, temperature and dissolved oxygen in both reservoirs. These parameters can be used as good indicators to evaluate the condition these reservoirs with respect to cyanobacterial bloom. Some cyanobacterial isolates recovered from both reservoirs harboured mcy-A gene, showing the potential of microcystin production.