Mcy Gene Research Articles

Dynamics and polyphasic characterization of odor-producing cyanobacterium Tychonema bourrellyi from Lake Erhai, China.

The previous studies indicated that Tychonema-like strains from Lake Erhai could release geosmin so that the species was listed as the potential harmful cyanobacteria influencing the drinking water safety around Lake Erhai. But, the dynamics and biological information of this species were too limited. In this study, the polyphasic approach was used to reveal its biological characterization and the dynamics in Lake Erhai. The characters of trichomes, including filaments with solitary or bundle state, reddish-brown or blue-green color, planktonic habitat, and presence of keritomized content, were examined by the microscopic method. The 16S rDNA sequences of these strains were used for phylogenetic analysis and molecular identification. The strains were morphologically classified as Tychonema bourrellyi, and geosmin and β-ionone were identified as the major volatile substances using gas chromatography-mass spectrometry (GC-MS) analysis. No strains of T. bourrellyi were found to produce microcystin by the HPLC and mcy gene approaches. Cell numbers at 12 sampling sites in Lake Erhai were shown as an average of 3 × 10(4) cells L(-1) in 2009 and 2010. The obvious peaks occurred in July and August each year. This was the first report on occurrence of T. bourrellyi from outside of Europe, and T. bourrellyi was also a newly recorded species in China. Such a result demonstrated that T. bourrellyi could distribute extending from cold waters in North Europe to the warm waters in subtropical regions. It was interesting to observe the coincidence of the occurrence of T. bourrellyi with slightly eutrophicated waters since Lake Erhai had been regarded as an early phase of eutrophicated lake.

Molecular Analysis of the Cyanobacterial Community in Gastric Contents of Egrets with Symptoms of Steatitis.

Many deaths of wild birds that have drunk water contaminated with hepatotoxic microcystin-producing cyanobacteria have been reported. A mass death of egrets and herons with steatitis were found at the agricultural reservoir occurring cyanobacterial waterblooms. This study aimed to verify a hypothesis that the egrets and herons which died in the reservoir drink microcystin-producing cyanobacteria and microcystin involves in the cause of death as well as the symptoms of steatitis. The cyanobacterial community in gastric contents of egrets and herons that died from steatitis was assessed using cyanobacterial 16S rRNA-based terminal-restriction fragment length polymorphism (T-RFLP) profiling and a cyanobacterial 16S rRNA-based clone library analysis. In addition, PCR amplification of the mcyB–C region and the mcyG gene, involved in microcystin biosynthesis, was examined. The cyanobacterial community in the gastric contents of two birds showed a simplistic composition. A comparison of cyanobacterial T-RFLP profiling and cloned sequences suggested that the genus Microcystis predominated in both samples of egrets died. Although we confirmed that two egrets which died in the reservoir have taken in cyanobacterial waterblooms containing the genus Microcystis, no mcy gene was detected in both samples according to the mcy gene-based PCR analysis. This study is the first to show the profiling and traceability of a cyanobacterial community in the gastric contents of wild birds by molecular analysis. Additionally, we consider causing symptoms of steatitis in the dead egrets.

Seasonal patterns of microcystin-producing and non-producing Planktothrix rubescens genotypes in a deep pre-alpine lake

The filamentous cyanobacterium Planktothrix rubescens produces secondary metabolites called microcystins (MC) that are potent toxins for most eukaryotes, including zooplankton grazers, cattle and humans. P. rubescens occurs in many deep and thermally stratified lakes throughout Europe. In Lake Zurich (Switzerland), it re-appeared in the 1970s concomitant with decreasing eutrophication. Since then, P. rubescens has become the dominant species in this major drinking water reservoir, where it forms massive metalimnetic blooms during late summer. These cyanobacteria harbor subpopulations of non-MC producers, but little is known about the environmental factors affecting the success of such genotypes. The non-MC-producing subpopulation of P. rubescens was studied using a quantitative real-time PCR (qPCR) assay on the MC synthetase (mcy) gene cluster that targets a deletion on the mcyH and mcyA genes, which inactivates MC biosynthesis. Two complementary qPCR assays were used to assess the total population abundance (based on the 16S rDNA gene) and the mcy gene copy number (based on a conserved region in the adenylation domain of the mcyB gene). The objective was to evaluate the seasonal patterns of the share of non-MC-producing filaments in the total P. rubescens population. The mcyHA mutants were present in low proportions (up to 14%) throughout the year. Their highest relative abundances occurred during the winter mixis, when total concentrations of P. rubescens were minimal. The MC deficient mutants seemed to better survive in sparse populations, possibly because of lower grazing pressure and a consequently reduced need for MC-mediated protection. Alternatively, the mutants might cope better with the sub-optimal, stressful pressure and light conditions during the winter mixis. Altogether, our results suggest that subtle trade-offs might seasonally determine the proportions of non-MC producers within P. rubescens populations.

UV-B Exposure Affects the Biosynthesis of Microcystin in Toxic Microcystis aeruginosa Cells and Its Degradation in the Extracellular Space.

Microcystins (MCs) are cyclic hepatotoxic heptapeptides produced by cyanobacteria that can be toxic to aquatic and terrestrial organisms. MC synthesis and degradation are thought to be influenced by several different physical and environmental parameters. In this study, the effects of different intensities of UV-B radiation on MC biosynthesis in Microcystis cells and on its extracellular degradation were investigated by mRNA analysis and degradation experiments. Exposure to UV-B at intensities of 1.02 and 1.45 W/m2 not only remarkably inhibited the growth of Microcystis, but also led to a decrease in the MC concentration. In addition, mcyD transcription was decreased under the same UV-B intensities. These results demonstrated that the effects of UV-B exposure on the biosynthesis of MCs in Microcystis cells could be attributed to the regulation of mcy gene transcription. Moreover, the MC concentration was decreased significantly after exposure to different intensities of UV-B radiation. Of the three MC variants (MC-LR, -RR and -YR, L, R and Y are abbreviations of leucine, arginine and tyrosine), MC-LR and MC-YR were sensitive to UV-B radiation, whereas MC-RR was not. In summary, our results showed that UV-B radiation had a negative effect on MC production in Microcystis cells and MC persistence in the extracellular space.

Co-occurrence of microcystin and anatoxin-a in the freshwater lake Aydat (France): Analytical and molecular approaches during a three-year survey

Cyanobacterial mass occurrence is becoming a growing concern worldwide. They notably pose a threat to water users when cyanotoxins are produced. The aim of this study was to evaluate the occurrence and the dynamics of two cyanotoxins: microcystin (MC) and anatoxin-a (ANTX-a), and of two of the genes responsible for their production (respectively mcyA and anaC) during three consecutive bloom periods (2011, 2012 and 2013) in Lake Aydat (Auvergne, France). MC was detected at all sampling dates, but its concentration showed strong inter- and intra-annual variations. MC content did not correlate with cyanobacterial abundance, nor with any genera taken individually, but it significantly correlated with mcyA gene abundance (R2=0.51; p=0.042). MC content and mcyA gene abundance were maximal when cyanobacterial abundance was low, either at the onset of the bloom or during a trough of biomass. The LC-MS/MS analysis showed the presence of ANTX-a in the 2011 samples. To our knowledge, this is the first report of the presence of this neurotoxin in a French lake. The presence of ANTX-a corresponded to the only year for which Anabaena did not dominate the cyanobacterial community alone, and several cyanobacterial genera were present, including notably Aphanizomenon. anaC gene detection by PCR was not coherent with ANTX-a presence, both gene and toxin were never found for a same sample. This implies that molecular tools to study genes responsible for the production of anatoxin-a are still imperfect and the development of new primers is needed. This study also highlights the need for better monitoring practices that would not necessarily focus only on the peak of cyanobacterial abundance and that would take cyanotoxins other than MC into account.

Microcystin mcyA and mcyE Gene Abundances Are Not Appropriate Indicators of Microcystin Concentrations in Lakes.

Cyanobacterial harmful algal blooms (cyanoHABs) are a primary source of water quality degradation in eutrophic lakes. The occurrence of cyanoHABs is ubiquitous and expected to increase with current climate and land use change scenarios. However, it is currently unknown what environmental parameters are important for indicating the presence of cyanoHAB toxins making them difficult to predict or even monitor on time-scales relevant to protecting public health. Using qPCR, we aimed to quantify genes within the microcystin operon (mcy) to determine which cyanobacterial taxa, and what percentage of the total cyanobacterial community, were responsible for microcystin production in four eutrophic lakes. We targeted Microcystis-16S, mcyA, and Microcystis, Planktothrix, and Anabaena-specific mcyE genes. We also measured microcystins and several biological, chemical, and physical parameters—such as temperature, lake stability, nutrients, pigments and cyanobacterial community composition (CCC)—to search for possible correlations to gene copy abundance and MC production. All four lakes contained Microcystis-mcyE genes and high percentages of toxic Microcystis, suggesting Microcystis was the dominant microcystin producer. However, all genes were highly variable temporally, and in few cases, correlated with increased temperature and nutrients as the summer progressed. Interestingly, toxin gene abundances (and biomass indicators) were anti-correlated with microcystin in all lakes except the largest lake, Lake Mendota. Similarly, gene abundance and microcystins differentially correlated to CCC in all lakes. Thus, we conclude that the presence of microcystin genes are not a useful tool for eliciting an ecological role for toxins in the environment, nor are microcystin genes (e.g. DNA) a good indicator of toxins in the environment.

Microcystin-producing and non-producing cyanobacterial blooms collected from the Central India harbor potentially pathogenic Vibrio cholerae

On the basis of relative abundance, frequency and biovolume, the important value index ranks were assigned to individual cyanobacteria in phytoplankton samples collected from fourteen water resources of Central India. The mcyABDE genes were detected in all the blooms with Microcystis (-aeruginosa, –viridis, -panniformis, –botrys) as being the major constituent morphospecies. On the other hand, blooms composed of primarily Oscillatoria (-limosa,-agardhii, -laetevirens) along with Anabaena, Nostoc, Phormidium and Spirulina as sub-dominant forms exhibited quite a patchy distribution of one or the other mcy genes. Fifty percent of Microcystis- but none of the Oscillatoria dominant blooms produced microcystins–RR and desmethyl–RR at 0.03–0.41mgg−1 bloom dry mass. Traces of dissolved microcystin was detected in lake water, which is well below the WHO guideline. Irrespective of cyanobacterial composition and microcystin production ability, during the study period 43–64% of the cyanobacterial bloom samples exhibited association of viable but nonculturable forms of Vibrio cholerae O1 and O139, as evident from amplification of the antigen genes. We believe that spread of endemic cholera is the major threat associated with harmful algal blooms.

First report of an Anabaena Bory strain containing microcystin-LR in a freshwater body in Africa

In South Africa, little is known about the production of microcystin by the genus Anabaena Bory. In April 2012, during a cyanobacterial bloom event in Theewaterskloof Dam, Western Cape province, the plankton was sampled on 10 occasions. The dominant algae belonged to the genus Anabaena, a family of filamentous cyanobacteria known to produce cyanotoxins such as anatoxin-a, harmful to humans and the aquatic foodweb. The specimens isolated lacked the characteristic akinetes and/or heterocysts associated with this genus. Therefore the 16S rRNA gene was Sanger sequenced and a maximum parsimony tree was constructed, confirming its identity as Anabaena ucrainica (Schkorbatow) M. Watanabe. Enzyme-linked immunosorbent assay (ELISA) confirmed the presence of microcystin-LR in the isolated A. ucrainica field sample, while PCR analysis and sequencing further confirmed the presence of mcy genes in this species. It was speculated from the data that prevailing low water-column temperatures and strong gusty winds may have resulted in the lack of akinete or heterocyst production. The Anabaena strain isolated from Theewaterskloof Dam is the first report of a strain containing microcystin-LR belonging to this genus in a freshwater body in Africa.

Toxic Microcystis novacekii T20-3 from Phakalane Ponds, Botswana: PCR Amplifications of Microcystin Synthetase (mcy) Genes, Extraction and LCESI- MS Identification of Microcystins

Treated water effluent from Phakalane waste water secondary maturation ponds in Gaborone City (Botswana) enters the Limpopo River via Notwane River. Effluent samples from these ponds were collected and investigated using molecular and analytical methods to determine presence of mcy genes and microcystins (MCs), respectively. It was observed that, potentially toxic algal blooms were present in this effluent and therefore algal toxins. Using Polymerase Chain Reaction (PCR) method; cyanobacterial 16S-rRNA and mcyA, -B, -C, -D, -E and -G genes were amplified and PCR products separated by gel electrophoresis and visualized after ethidium bromide staining. DNA sequence for mcyA gene was obtained using PCR products from a newly designed primer pair for the amplification of mcyA gene. BLAST results of the obtained DNA sequence were evaluated and aligned to NCBI database for species identification. The alignment gave the highest similarity (100%) in nucleotide sequence that was aligned to the DNA sequence of a toxigenic M. novacekii T20-3 based on the published data. Microcystin-RR, -YR, -LR and -WR were chromatographed, identified and quantified from M. novacekii T20-3 cell extracts using LC-ESI-MS technique after liquid-partitioning (LP) and solid-phase extraction (SPE) steps complementing PCR findings. Higher quantities of MC-RR, -LR and YR: 53.620 ± 0.063, 12.114 ± 0.024 and 5.280 ± 0.035 μg/g DW were observed, respectively.

Toxic Microcystis novacekii T20-3 from Phakalane Ponds, Botswana: PCR Amplifications of Microcystin Synthetase (mcy) Genes, Extraction and LCESI-MS Identification of Microcystins

Toxic Microcystis novacekii T20-3 from Phakalane Ponds, Botswana: PCR Amplifications of Microcystin Synthetase (mcy) Genes, Extraction and LCESI-MS Identification of Microcystins

一种快速检测微囊藻毒素mcyG基因的新技术——环介导恒温扩增

PDF HTML阅读 XML下载 导出引用 引用提醒 一种快速检测微囊藻毒素mcyG基因的新技术——环介导恒温扩增 DOI: 10.5846/stxb201308112059 作者: 作者单位: 宁波大学,宁波大学,宁波大学,宁波大学,宁波大学,宁波大学 作者简介: 通讯作者: 中图分类号: 基金项目: 浙江省重大科技专项重点农业项目(2012C12009-4); 宁波市海洋藻类资源高效开发利用创新团队项目(2011B81007) A novel assay for rapid detection of microcystin mcyG gene: loop-mediated isothermal amplification Author: Affiliation: Ningbo University,Ningbo University,,,, Fund Project: 摘要 | 图/表 | 访问统计 | 参考文献 | 相似文献 | 引证文献 | 资源附件 | 文章评论 摘要:利用环介导恒温扩增(LAMP)技术,以微囊藻毒素(Microcystins,MCs)合成基因簇中的mcyG基因为靶序列,设计了1 套LAMP引物,建立了LAMP反应体系并进行灵敏度和特异性实验。结果表明mcyG基因的最低检测限为:24 cfu/mL,远低于常规PCR(Polymerase Chain Reaction)。整个检测过程仅需40 min,且可直接目测结果。特异性实验中, 13 株淡水常见水华蓝藻分属:色球藻属(Chroococcus)、念珠藻属(Nostoc)、鱼腥藻属(Anabaena)、束丝藻属(Aphanizomenon)、微囊藻属(Microcystis),其中10 株呈阳性反应, 3 株为阴性。在野外样品检测中,来自太湖与黄庆苗池塘的水样PCR检测显示阴性反应,而LAMP检测均呈阳性反应,提示此两处水样中可能含有产毒微囊藻,显示出了LAMP检测方法良好的野外检测和预警能力。综合上述,LAMP检测方法能够快速检测产微囊藻毒素的关键基因,且结果可视化。该方法简便、快捷、不依赖特殊检测设备,极具推广前景。 Abstract:Microcystin-producing cyanobacteria blooms, as well as their increasing global occurrence, are of worldwide concern because they have been implicated in the death of animals and humans. Currently, many techniques are used to evaluate whether toxins are present in water samples, including high-performance liquid chromatography (HPLC), mouse bioassays, protein phosphatase inhibition assays (PPIA), enzyme linked immunosorbent assay (ELISA), and mass spectrometry. While some of these techniques are quite sensitive, they are of use only once the toxin is present in the water source. However, microcystins remain within cyanobacterial cells until being released in high concentrations during cell lysis. Hence, identifying potential microcystin-producing strains in the environment prior to the release of toxins into the water body is more useful than detection of toxins. Many microcystin-producing strains cannot be differentiated from non-toxic strains by traditional microscopy or with ribosomal gene sequences. Synthetase genes (mcy) are responsible for the biosynthesis of microcystin and are only present in the genome of toxic strains. Polymerase chain reaction (PCR) assays based on mcy gene sequences have been reported. Though these assays for rapid, sensitive, and specific detection appear to be promising, PCR applied as a rapid diagnostic test in the field has been seriously constrained owing to the complexity of the amplification conditions and the need for specialized high-cost instruments. Recently, loop-mediated isothermal amplification (LAMP), a new technology for amplifying DNA, has been developed. Unlike PCR, which requires special instruments to control reaction temperature, LAMP requires only a temperature-controlled water bath. In this manuscript, we designed a set of LAMP primers to target the sequence of the mcyG gene, which is responsible for the first step in the biosynthesis of Adda (3-amino-9-methoxy-2, 6, 8-trim ethyl-10-phenyldeca-4, 6-dienoic acid), an important part of microcystin. The conditions of amplification were optimized to implement the sensitivity and specificity experiments. We found that 61 ℃ for 40 min was the most suitable reaction condition. We then selected thirteen cyanobacterial strains, including Microcystis, Aphanizomenon, Chroococcus, Nostoc, Oscillatoria, and Anabaena, to determine the specificity and sensitivity of the primers in the LAMP reactions. The results of amplification were positive with ten microcystin-producing strains and negative with three non-toxic strains, which demonstrated that the primers had suitable levels of specificity. The sensitivity of LAMP, with a detection limit of 24 cfu/ml, was 1000-fold higher than that of PCR. For verifying the application of this LAMP assay in the aquatic ecosystem, seven environmental samples from ponds and lakes in Ningbo and one from Tai Lake were analyzed using both the LAMP assay targeting the mcyG gene and a PCR assay. The LAMP assay detected amplification in two water samples that the PCR assay did not detect, demonstrating that the LAMP assay is more effective and more suitable for field testing. The LAMP method is fast, simple, and low in cost making it advantageous for the detection of potential toxic cyanobacterial blooms. By employing the LAMP assay, workers at the Environmental Monitoring Station will be able to detect toxic cyanobacterial cells at low levels, before they reach an unsafe concentration. This will allow the workers, through routine monitoring of potential toxin status in the freshwater, to have enough time to implement a series of control strategies for avoiding human exposure to cyanobacterial hepatotoxins at dangerous levels. 参考文献 相似文献 引证文献

Temporal variations in microcystin-producing cells and microcystin concentrations in two fresh water ponds

The relationship between microcystin production, microcystin-producing cyanobacteria, including Microcystis spp., and various biological and physicochemical parameters in Sankuldhara and Lakshmikund, situated in the same geographical area was studied over a period of 1.5 years. Seasonal variation in cyanobacterial 16S rRNA, Microcystis spp. 16S rRNA, mcyA and mcyB genes were quantitatively determined by real-time PCR. Microcystis was the dominant microcystin producer in both study sites constituting 67% and 97% of the total microcystin-producing cyanobacteria at Sankuldhara and Lakshmikund, respectively. Microcystin concentrations were 2.19–39.60 μg/L and 15.22–128.14 μg/L at Sankuldhara and Lakshmikund, respectively, as determined by LC-MS. Principal component analysis revealed a strong positive correlation between microcystin concentration and the copy number of mcyA and mcyB, chlorophyll a and cyanobacterial biomass at both sites. The higher microcystin concentrations in Lakshmikund pond were attributed to the high copy number of mcy genes present coupled with the pond's eutrophication status, as indicated by high total algal biomass, high chlorophyll a content, high nutrient load and low DO. Therefore, a significant difference in microcystin concentrations, correlating with these various biological and physicochemical parameters, confirms the importance of local environmental variables in the overall regulation of microcystins production.

Why Cyanobacteria Produce Toxins? Evolutionary Game Theory Suggests the Key

Cyanobacteria are a source of potent toxins among which the microcystin (a hepatotoxic peptide encoded by the mcy gene cluster of Microcystis spp. ) is a frequent cause of poisoning in inland waters worldwide. Although the molecular basis of microcystin production is known, its role is still unknown. It was suggested that microcystin production have a metabolic cost that could be offset by some benefit (e.g. protection from grazing). We check that: i) microcystin-producing and non-producing strains occurs simultaneously in the Microcystis spp. blooms, ii) evolutionary forces (mutation, genetic drift) control frequencies of microcystin production and non-production strains, and iii) microcystin producing strains have diminished fitness compared with non-producing strains. We employ evolutionary game theory to explain the maintaining of microcystin-producing genotypes in natural populations of Microcystis spp . A two-strategy (to produce or not microcystin), two-players game of cooperators (microcystin-producing genotypes) and cheaters (non-producing genotypes) explains the coexistence of both genotypes in the same bloom. A bloom composed mostly by the microcystin-producing “cooperators” genotype, the “temptation of defection” (increase of non-producing genotypes) is counteracted by kin selection, which enable that natural selection can favour the cooperators. The closest related individuals occur within cyanobacteria blooms, cyanobacteria reproduces asexually providing sets of clones.

Integrating phylogeny, geographic niche partitioning and secondary metabolite synthesis in bloom-forming Planktothrix.

Toxic freshwater cyanobacteria form harmful algal blooms that can cause acute toxicity to humans and livestock. Globally distributed, bloom-forming cyanobacteria Planktothrix either retain or lose the mcy gene cluster (encoding the synthesis of the secondary metabolite hepatotoxin microcystin or MC), resulting in a variable spatial/temporal distribution of (non)toxic genotypes. Despite their importance to human well-being, such genotype diversity is not being mapped at scales relevant to nature. We aimed to reveal the factors influencing the dispersal of those genotypes by analyzing 138 strains (from Europe, Russia, North America and East Africa) for their (i) mcy gene cluster composition, (ii) phylogeny and adaptation to their habitat and (iii) ribosomally and nonribosomally synthesized oligopeptide products. Although all the strains from different species contained at least remnants of the mcy gene cluster, various phylogenetic lineages evolved and adapted to rather specific ecological niches (for example, through pigmentation and gas vesicle protein size). No evidence for an increased abundance of specific peptides in the absence of MC was found. MC and peptide distribution rather depended on phylogeny, ecophysiological adaptation and geographic distance. Together, these findings provide evidence that MC and peptide production are primarily related to speciation processes, while within a phylogenetic lineage the probability that strains differ in peptide composition increases with geographic distance.

Variability of microcystins and its synthetase gene cluster in Microcystis and Planktothrix waterblooms in shallow lakes of Hungary.

Waterbloom samples of Microcystis aeruginosa and Planktothrix agardhii were collected from a variety of ponds, lakes and reservoirs in Hungary. Samples were tested with matrix-assisted laser desorption/ionization - time-of-flight mass spectrometry (MALDI-TOF MS) to identify the microcystin forms. The concentration of the microcystins was measured with capillary electrophoresis and the toxicity was tested by sinapis test. DNA was extracted from the samples and tested using a range of primers linked to the biosynthesis of microcystin. All of the fourteen collected samples gave positive results for the presence of the mcy genes with PCR products with sizes between of 425 and 955 bp, respectively, indicating the presence of the genes implicated in the production of microcystins. The results showed that a wide range of microcystin (MC) forms were detected in the Microcystis containing samples, among which MC-LR, -RR, and -YR were the most common. The highest MC concentration was 15,701 mg g-1, which was detected in an angling pond. The samples containing Planktothrix agardhii were less toxic, and the most common form in this species was the Asp3-MC-LR.

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.

Comparison of cyanobacterial microcystin synthetase (mcy) E gene transcript levels, mcy E gene copies, and biomass as indicators of microcystin risk under laboratory and field conditions.

Increased incidences of mixed assemblages of microcystin-producing and nonproducing cyanobacterial strains in freshwater bodies necessitate development of reliable proxies for cyanotoxin risk assessment. Detection of microcystin biosynthetic genes in water blooms of cyanobacteria is generally indicative of the presence of potentially toxic cyanobacterial strains. Although much effort has been devoted toward elucidating the microcystin biosynthesis mechanisms in many cyanobacteria genera, little is known about the impacts of co-occurring cyanobacteria on cellular growth, mcy gene expression, or mcy gene copy distribution. The present study utilized conventional microscopy, qPCR assays, and enzyme-linked immunosorbent assay to study how competition between microcystin-producing Microcystis aeruginosa CPCC 299 and Planktothrix agardhii NIVA-CYA 126 impacts mcyE gene expression, mcyE gene copies, and microcystin concentration under controlled laboratory conditions. Furthermore, analyses of environmental water samples from the Missisquoi Bay, Quebec, enabled us to determine how the various potential toxigenic cyanobacterial biomass proxies correlated with cellular microcystin concentrations in a freshwater lake. Results from our laboratory study indicated significant downregulation of mcyE gene expression in mixed cultures of M. aeruginosa plus P. agardhii on most sampling days in agreement with depressed growth recorded in the mixed cultures, suggesting that interaction between the two species probably resulted in suppressed growth and mcyE gene expression in the mixed cultures. Furthermore, although mcyE gene copies and McyE transcripts were detected in all laboratory and field samples with measureable microcystin levels, only mcyE gene copies showed significant positive correlations (R2 > 0.7) with microcystin concentrations, while McyE transcript levels did not. These results suggest that mcyE gene copies are better indicators of potential risks from microcystins than McyE transcript levels or conventional biomass proxies, especially in water bodies comprising mixed assemblages of toxic and nontoxic cyanobacteria.

Planktic cyanobacteria from freshwater localities in Thuathien-Hue province, Vietnam. III. Phylogenetic inference based on partial phycocyanin sequences, morphological and toxicological characters

The morphology, toxicity and partial sequences of beta and alpha subunits including the intergenic spacer (cpcBA-IGS) of the phycocyanin operon were studied in 21 strains of nonaxenic cyanobacterial cultures. The cultures were established from water samples collected in the Thuathien Hue province in Vietnam, 2004. Based on testing for the presence of the mcyA gene, microcystin-production of Microcystis aeruginosa, M. botrys and M. panniformis was confirmed. The presence of the mcyA gene in a strain of Microcystis flos-aquae and a strain of Planktothrix spiroides (HA308) revealed their toxic potential. The phylogeny of the Vietnamese cyanobacteria was inferred using sequences encoding the cpcBA genes. There was no correlation between toxic characters based on ELISA, HPLC and mcyA gene and the phylogeny based on partial cpcBA sequences. Although the resulting tree topology assisted in resolving the phylogeny of some strains of Anabaena, Dolichospermum and Planktothrix, taxonomical problems still exist at the species level for Lyngbya and Microcystis. Future morphological and molecular studies will have to solve these issues.

Quantification and genetic diversity of total and microcystin-producing Microcystis during blooming season in Tai and Yang-cheng lakes, China

The aims of present study were to evaluate the abundances, genetic diversity of total and microcystin-producing Microcystis over temporal and spatial scales, and to investigate relationships among Microcystis and water parameters in Tai and Yang-cheng lakes. Abundances of total and microcystin-producing Microcystis varied across sampling periods and locations, which were assessed using qPCR with primers specific to Microcystis 16S rDNA and mcyA genes. The 16S rDNA from two lakes were relatively diverse. However, mcyA genes were rather conservative and were >97% identical to reference sequences. The highly positive correlations between mcyA and microcystin presence (r = 0·671 in Tai; r = 0·799 in Yang-cheng) suggested that mcyA can be used as a good biomarker for microcystin productions. The results demonstrated that Microcystis were genetically diverse between these conjunctive lakes; however, mcyA genes were relatively conservative in two lakes. Quantifying mcyA by qPCR was an efficient tool for monitoring toxic Microcystis. This study has improved our understanding of observable differences within and between each lake on spatial and temporal scales. And the discovery of new mcyA sequences in natural water enriched the understanding of phylogenetic diversity of Microcystis and toxin-production-related mcy gene.

Técnicas moleculares para la detección de cianobacterias en los embalses Riogrande II y La Fe, Colombia

In lentic water bodies as reservoirs occur eutrophication processes, originated mainly from human activities (i.e. agriculture, animal exploitation). This influx of nutrients in aquatic ecosystems could promote blooms of potentially toxic cyanobacteria. The purpose of this work is to detect the presence of cyanobacteria strains in water samples, using molecular techniques to help in preventive management of reservoirs dedicated to water purification. We used two molecular techniques to detect genes implied with the synthesis of hepatotoxic microcystins from potentially toxic cyanobacteria strains, and to evaluate the molecular diversity of cyanobacteria in water samples from two high-mountain reservoirs used for purification of drinking water for the metropolitan area of Medellin, Colombia. Between 2010-2011 collections of 12 water samples were taken and DNA extraction together with PCR and DGGE analyses where carried out. We amplified 22 sequences between 250-300bp of the genes mcyA and mcyE, and these sequences were related with several strains and cyanobacteria genera accessions from NCBI-GenBank databases. Moreover, sequence amplifications of the 16S small ribosomal RNA subunit - 16S rRNA- between 400-800bp were also performed in order to use them for the DGGE technique. The amplification products of DGGE were set in polyacrilamide gel with posterior denaturing electrophoresis, and the scanned images of the gel bands were analysed with the software GelCompar II. For Riogrande II and La Fe reservoirs we found 35 and 30 different DGGE bands, respectively, as a measurement of molecular diversity in these artificial ecosystems. Here, we demonstrated the utility of two molecular techniques for the detection of genes associated with toxicity and molecular diversity of cyanobacteria in reservoirs destined for drinking water in urban centers. We recommend strongly following with periodically molecular biology studies in these ecosystems combined with limnological and ecological data, as new tools for management of plants of water purification and for the prevention of potentially public health concerns.