Application of Real-Time PCR To Estimate Toxin Production by the Cyanobacterium Planktothrix sp.

Quantitative real-time PCR methods are increasingly being applied for the enumeration of toxic cyanobacteria in the environment. However, to justify the use of real-time PCR quantification as a monitoring tool, significant correlations between genotype abundance and actual toxin concentrations are required. In the present study, we aimed to explain the concentrations of three structural variants of the hepatotoxin microcystin (MC) produced by the filamentous cyanobacterium Planktothrix sp., [Asp, butyric acid (Dhb)]-microcystin-RR (where RR means two arginines), [Asp, methyl-dehydro-alanine (Mdha)]-microcystin-RR, and [Asp, Dhb]-microcystin-homotyrosine-arginine (HtyR), by the abundance of the microcystin genotypes encoding their synthesis. Three genotypes of microcystin-producing cyanobacteria (denoted the Dhb, Mdha, and Hty genotypes) in 12 lakes of the Alps in Austria, Germany, and Switzerland from 2005 to 2007 were quantified by means of real-time PCR. Their absolute and relative abundances were related to the concentration of the microcystin structural variants in aliquots determined by high-performance liquid chromatography (HPLC). The total microcystin concentrations varied from 0 to 6.2 μg liter⁻¹ (mean ± standard error [SE] of 0.6 ± 0.1 μg liter⁻¹) among the samples, in turn resulting in an average microcystin content in Planktothrix of 3.1 ± 0.7 μg mm⁻³ biovolume. Over a wide range of the population density (0.001 to 3.6 mm³ liter⁻¹ Planktothrix biovolume), the Dhb genotype and [Asp, Dhb]-MC-RR were most abundant, while the Hty genotype and MC-HtyR were found to be in the lowest proportion only. In general, there was a significant linear relationship between the abundance/proportion of specific microcystin genotypes and the concentration/proportion of the respective microcystin structural variants on a logarithmic scale. We conclude that estimating the abundance of specific microcystin genotypes by quantitative real-time PCR is useful for predicting the concentration of microcystin variants in water.During the last decade, genetic methods have significantly increased our understanding of the distribution of genes that are involved in the production of toxins within cyanobacteria that occur in fresh and brackish water (45). Although genetic methods can indicate only the potential risk of toxin synthesis and do not provide information about the actual toxin concentrations, quantitative real-time PCR has been increasingly applied for monitoring the toxin-producing genotypes of cyanobacteria in water (26, 33, 44). The development of real-time PCR methods was driven primarily by its potential (i) as an early-warning tool as well as to monitor toxin-producing cyanobacteria and (ii) to identify those factors that lead to a dominance/repression of toxin-producing genotypes versus nontoxic genotypes. For the first aim, it is essential that the abundance of toxin-producing cyanobacteria can be related to the concentration of the respective toxic substance in water. A few studies showed that the concentration of certain toxic genotypes was linearly related to the respective toxin concentrations, e.g., for the most common group of hepatotoxins, the microcystins (MCs) (7, 12, 14), and for the related nodularin (19). Both microcystins and nodularins are known to be potent inhibitors of eukaryotic protein phosphatases 1 and 2A, resulting in a health hazard to humans and the environment (9). In contrast, no correlation was found (37, 50), or even the opposite was reported, by other studies, i.e., that the measurement of microcystin-producing genotypes is not a satisfactory method for use in monitoring programs in order to predict the toxic risk associated with cyanobacterial proliferation (3). For microcystins, these contrasting results may be due to several reasons: (i) several genera producing microcystins frequently coexist in water bodies, and therefore, not all microcystin producers may have been identified; (ii) the semilogarithmic calibration curves limit the accuracy in estimations of genotype numbers and proportions (for example, the only laboratory comparison carried out so far revealed that among the three laboratories tested, the proportions of toxic genotypes were overestimated or underestimated by 0 to 72% and 0 to 50%, respectively [42]); and (iii) inactive mutants that contain the respective genes, however, which have been inactivated in toxin production through the insertion of transposable elements, may co-occur and decrease toxin production in a given population (6). Nevertheless, the real-time PCR technique is the only quantitative technique available for estimating the proportion of potential toxin-producing genotypes in water. The development of automated and field-applicable real-time PCR methods (e.g., see reference 35), in particular, may contribute to a more widespread integration of real-time PCR into routine monitoring programs in the future.In the present study, we attempted to quantify microcystin-producing genotypes in total as well as quantify the specific genotypes that were shown to encode different microcystin structural variants characterized for strains isolated from lakes in the Alps (23): (i) the methyl-dehydro-alanine residue (Mdha) genotype, which was found to synthesize structural variants containing only Mdha in position 7; (ii) the butyric acid (Dhb) genotype, which was found to contain Dhb instead of Mdha in the same position; and (iii) the homotyrosine (Hty) genotype, which was found to contain Hty and Leu in position 2 but never Arg. The Hty variant has always been found to co-occur with Dhb in position 7 of the molecule (24). Consequently, the Hty genotype forms a subgroup of the microcystin-producing population composed of the Mdha and Dhb genotypes. The following hypotheses were tested: (i) as only one microcystin-producing organism (Planktothrix sp.) is of quantitative importance in those lakes (32), the total microcystin concentration should be predictable from the sum of Mdha and Dhb genotypes; (ii) given that all Planktothrix genotypes are amenable to cultivation, all the structural microcystin variants found in the field samples should have been described for the strains isolated previously (23); and (iii) as, on average, the proportion of the inactive microcystin genotypes was found to be low and rather stable (<6.5% [32]), their occurrence should not reduce the ability to predict microcystin concentrations from genotype abundance. For this purpose, the phytoplankton in 12 lakes of the Alps in Austria, Germany, and Switzerland was monitored both with an inverted microscope as well as by means of real-time PCR over the course of 2 years (2005 to 2007). In parallel, microcystin concentrations in aliquots were determined by means of high-performance liquid chromatography (HPLC). We show that the abundance of specific microcystin genotypes can be related to the corresponding microcystin concentrations in water on a logarithmic scale over a range of trophic conditions. The proportion of certain genotypes encoding the synthesis of a specific microcystin variant significantly correlates with the concentration of the respective microcystin variant. We argue that these genotype-toxin concentration relationships are of great importance for the justification of real-time PCR use in monitoring programs.     

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.     

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

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

Diversity and dynamics of microcystin—Producing cyanobacteria in China's third largest lake, Lake Taihu

Microcystins (MC), the most prevalent group of harmful cyanobacterial hepatotoxins, are primarily produced by strains of cyanobacteria in Microcystis, Anabaena and Planktothrix. Lake Taihu, which is the third largest freshwater lake in China, is a hypertrophic shallow lake in eastern China that has experienced lake-wide cyanobacterial blooms annually during the last few decades. In this study, PCR-DGGE was used to evaluate the diversity of potential MC-producing cyanobacteria and real-time PCR was used to analyze the dynamics of this population based on the presence of the mcy gene in samples collected during a year long study. The results revealed that all MC-producing genotypes detected belonged to the genus Microcystis. In addition, the MC-producing genotype communities were more diverse during the bloom season than the non-bloom season, and the diversity in the late bloom period was lower than the diversity in the early bloom period. Furthermore, the abundance of MC-producing genotypes increased dramatically during the bloom development period, reaching its peak in late summer (September). The results also suggested that the highest mcy gene concentration lagged behind the highest MC concentration, and the potential MC-producing cyanobacterial community shift lagged behind the development of blooms.     

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

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

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

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

Application of Real-Time PCR for Quantification of Microcystin Genotypes in a Population of the Toxic Cyanobacterium Microcystis sp.

The cyanobacterium Microcystis sp. frequently develops water blooms consisting of organisms with different genotypes that either produce or lack the hepatotoxin microcystin. In order to monitor the development of microcystin (mcy) genotypes during the seasonal cycle of the total population, mcy genotypes were quantified by means of real-time PCR in Lake Wannsee (Berlin, Germany) from June 1999 to October 2000. Standard curves were established by relating cell concentrations to the threshold cycle (the PCR cycle number at which the fluorescence passes a set threshold level) determined by the Taq nuclease assay (TNA) for two gene regions, the intergenic spacer region within the phycocyanin (PC) operon to quantify the total population and the mcyB gene, which is indicative of microcystin synthesis. In laboratory batch cultures, the cell numbers inferred from the standard curve by TNA correlated significantly with the microscopically determined cell numbers on a logarithmic scale. The TNA analysis of 10 strains revealed identical amplification efficiencies for both genes. In the field, the proportion of mcy genotypes made up the smaller part of the PC genotypes, ranging from 1 to 38%. The number of mcyB genotypes was one-to-one related to the number of PC genotypes, and parallel relationships between cell numbers estimated via the inverted microscope technique and TNA were found for both genes. It is concluded that the mean proportion of microcystin genotypes is stable from winter to summer and that Microcystis cell numbers could be used to infer the mean proportion of mcy genotypes in Lake Wannsee.     

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

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

Environmental Conditions Determine the Course and Outcome of Phytoplankton Chytridiomycosis

Chytrid fungi are highly potent parasites of phytoplankton. They are thought to force phytoplankton organisms into an evolutionary arms race with high population diversity as the outcome. The underlying selection regime is known as Red Queen dynamics. However, our study suggests a more complex picture for chytrid parasitism in the cyanobacterium Planktothrix. Laboratory experiments identified a “cold thermal refuge”, inside which Planktothrix can grow without chytrid infection. A field study in two Norwegian lakes underlined the ecological significance of this finding. The study utilized sediment DNA as a biological archive in combination with existing monitoring data. In one lake, temperature and light conditions forced Planktothrix outside the thermal refuge for most of the growing season. This probably resulted in Red Queen dynamics as suggested by a high parasitic pressure exerted by chytrids, an increase in Planktothrix genotype diversity over time, and a correlation between Planktothrix genotype diversity and duration of bloom events. In the second lake, a colder climate allowed Planktothrix to largely stay inside the thermal refuge. The parasitic pressure exerted by chytrids and Planktothrix genotype diversity remained low, indicating that Planktothrix successfully evaded the Red Queen dynamics. Episodic Planktothrix blooms were observed during spring and autumn circulation, in the metalimnion or under the ice. Interestingly, both lakes were dominated by the same or related Planktothrix genotypes. Taken together, our data suggest that, depending on environmental conditions, chytrid parasitism can impose distinct selection regimes on conspecific phytoplankton populations with similar genotype composition, causing these populations to behave and perhaps to evolve differently.     

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

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

Distribution and Abundance of Nontoxic Mutants of Cyanobacteria in Lakes of the Alps

The filamentous cyanobacterium Planktothrix rubescens frequently occurs in deep and stratified lakes in the temperate region of the northern hemisphere and is a known producer of the hepatotoxic secondary metabolite microcystin. These cyclic heptapeptides are synthesized nonribosomally via large enzyme complexes encoded by the microcystin (mcy) synthetase gene cluster. The occurrence of cyanobacterial strains lacking microcystin, but containing the mcy gene cluster has been reported repeatedly; it was shown that this inactivation is due to mutations such as gene deletion events and the insertion of transposable elements. In the present study, 12 lakes in Austria, Germany, and Switzerland were sampled from July 2005 to October 2007, and the proportion of inactive mcy genotypes was quantified in relation to the total population of the red-pigmented filamentous cyanobacterium Planktothrix by means of quantitative polymerase chain reaction. In total, four different mutations were quantified, namely two insertions affecting mcyD, one insertion affecting mcyA, and a deletion within mcyH and mcyA. The mutations occurred over a wide range of population densities (40–570,000 filaments L⁻¹), and their abundance was found to be positively correlated with population density. However, on average, all nontoxic mutants were found in a low proportion only (min 0%, mean 6.5% ± 1.1 (SE), max 52% of the total population). The genotype containing the mcyHA deletion had a significantly higher proportion (min 0%, mean 3.7% ± 1, max 52%) when compared with all the genotypes containing insertions within the mcy gene cluster (min 0%, mean 2.8% ± 0.7, max 24%). The results demonstrate that the occurrence of inactive mcy genotypes is linearly related to the population density, and selective sweeps of nontoxic mutants did not occur during the transition from prebloom to bloom conditions. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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.     

Molecular Characterization of Potential Microcystin-Producing Cyanobacteria in Lake Ontario Embayments and Nearshore Waters

The distribution and genotypic variation of potential microcystin (MC) producers along the southern and eastern shores of Lake Ontario in 2001 and 2003 were examined using a suite of PCR primers. Cyanobacterial, Microcystis sp., and Microcystis-specific toxin primer sets identified shoreline distribution of cyanobacterial DNA (in 97% of the stations) and MC synthetase genes (in 50% of the stations). Sequence analysis of a partial mcyA amplicon targeting Microcystis, Anabaena, and Planktothrix species indicated that the Microcystis sp. genotype was the dominant MC genotype present and revealed a novel Microcystis-like sequence containing a 6-bp insert. Analysis of the same samples with genus-specific mcyE primers confirmed that the Microcystis sp. genotype was the dominant potential MC producer. Genotype compositions within embayments were relatively homogenous compared to those for shoreline and tributary samples. MC concentrations along the shoreline exhibited both temporal and spatial differences as evidenced by the protein phosphatase inhibition assay, at times exceeding the World Health Organization guideline value for drinking water of 1.0 μg MC-LR_eq liter⁻¹. MC genotypes are widespread along the New York State shoreline of Lake Ontario, appear to originate nearshore, and can be carried through the lake via wind and surface water current patterns.     

Estimating toxic cyanobacteria in a Brazilian reservoir by quantitative real-time PCR, based on the microcystin synthetase D gene

The production of microcystin toxins by cyanobacteria is an intrapopulation feature and the toxic and nontoxic genotypes can be separated only through molecular analyses targeting the mcy markers. Quantitative real-time PCR (qPCR) is a procedure that has been established, not only to detect but to specifically quantify these genotypes. In the present work, primers were designed for the mcyD region to estimate the number of cyanobacteria that are potential microcystin producers. Laboratory tests to verify the efficiency and the specificity of the primers were performed. The methodology was first established for single strain cultures and thereafter was applied in environmental water samples, from a reservoir located in the Brazilian savannah (“cerrado”). The results were very satisfactory, demonstrating the high efficiency and the specificity of the primers used, and their ability to detect different cyanobacteria genera. Of particular interest were the results showing a high proportion of toxic strains (as high as 100 %) in the environmental samples, as previously reported in another tropical system. Furthermore, the occurrence of a smaller fraction of toxic strains at high cyanobacteria densities, and of more toxic populations when fewer cyanobacteria were present, deserves further investigation. Although records of cyanobacteria blooms are very common in the tropics and suggest an increasing incidence of toxic populations, the present research is one of the few applying qPCR in a tropical environment. The results obtained here, by a technique that allows a more precise quantification and in situ follow-up of changes in toxicity, will make possible new observations of seasonal and spatial dynamics in these environments.     

PCR-based identification of microcystin-producing genotypes of different cyanobacterial genera

Microcystins are harmful hepatotoxins produced by many, but not all strains of the cyanobacterial genera Anabaena, Microcystis, Anabaena, Planktothrix, and Nostoc. Waterbodies have to be monitored for the mass development of toxic cyanobacteria; however, because of the close genetic relationship of microcystin-producing and non-producing strains within a genus, identification of microcystin-producers by morphological criteria is not possible. The genomes of microcystin-producing cells contain mcy genes coding for the microcystin synthetase complex. Based on the sequence information of mcy genes from Microcystis and Planktothrix, a primer pair for PCR amplification of a mcyA gene fragment was designed. PCR with this primer pair is a powerful means to identify microcystin-producing strains of the genera Anabaena, Microcystis, and Planktothrix. Moreover, subsequent RFLP analysis of the PCR products generated genus-specific fragments and allowed the genus of the toxin producer to be identified. The assay can be used with DNA from field samples.Abbreviations RFLP Restriction fragment length polymorphism - MALDI-TOF Matrix-assisted laser desorption/ionization-time of flight spectrometry - HPLC High performance liquid chromatography     

Detection of microcystin producing cyanobacteria in Spirulina dietary supplements using multiplex HRM quantitative PCR

Arthrospira species, under the name ‘Spirulina’, are used as food supplement for its protein, vitamins, and minerals which have several health benefits. Cyanobacterial toxins including microcystins can possibly contaminate these dietary supplements causing hepatotoxicity, tumour formation, and other disorders. The safe use of dietary supplements necessitates the need to assess such toxins in the algal food supplement. The methods which evaluate these dietary supplements should be highly sensitive, cost-effective, and rapid. In this study, multiplex HRM qPCR analysis was used to detect microcystin (MC)-producing cyanobacteria in Spirulina dietary supplements. The multiplex HRM qPCR detection limit was found to be 25 ag of mcyB spiked in a standard concentration of pcb (25 pg). Two distinct melt curves characteristic of pcb (Tm 82.8 ± 0.07 °C) and mcyB (Tm 77.9 ± 0.05 °C) were observed. Microcystin contamination was detected only in the fish food supplements and not in human dietary supplements of Spirulina. Liquid chromatography–high-resolution mass spectrometry analysis further confirmed the presence of the congeners of microcystin in the identified positive samples.     

Dynamics of toxic genotypes of Microcystis aeruginosa complex (MAC) through a wide freshwater to marine environmental gradient

Bloom-forming species belonging to Microcystis aeruginosa complex (MAC) are the most commonly reported worldwide. MAC blooms are composed by toxic and non-toxic genotypes and the environmental conditions favouring the dominance of toxic genotypes are still a matter of debate among the scientific community. In this study, we evaluated the distribution of toxic MAC genotypes along a seasonal cycle and over an environmental gradient spanning 800 km, from a eutrophic freshwater reservoir in Río Uruguay to marine water in the outer limit of Río de la Plata. Abundance of four mcy genes, mcyB, mcyD, mcyE and mcyJ was determined by qPCR and used as a proxy of abundance of toxic MAC genotypes. All the mcy genes were detected through the seasonal cycle at all sampling sites, being systematically higher in the freshwater reservoir and decreasing towards the marine site. The highest toxic genotype abundance was found during the austral summer months. According to generalized linear regressions and random forest models, temperature and conductivity were the most relevant explanatory variables. This suggests that although toxic MAC genotypes grow optimally in freshwater, they are also able to tolerate the high-salinity and low temperature conditions found in estuarine and marine waters. This ability to resist harsh conditions impose a health risk and a management challenge. To our knowledge, this is the first report addressing several mcy genes in a broad gradient that includes a wide array of different environmental conditions.     

Stability of toxin gene proportion in red-pigmented populations of the cyanobacterium <Emphasis Type="Italic">Planktothrix</Emphasis> during 29 years of re-oligotrophication of Lake Zürich

Background

Harmful algal blooms deteriorate the services of aquatic ecosystems. They are often formed by cyanobacteria composed of genotypes able to produce a certain toxin, for example, the hepatotoxin microcystin (MC), but also of nontoxic genotypes that either carry mutations in the genes encoding toxin synthesis or that lost those genes during evolution. In general, cyanobacterial blooms are favored by eutrophication. Very little is known about the stability of the toxic/nontoxic genotype composition during trophic change.

Results

Archived samples of preserved phytoplankton on filters from aquatic ecosystems that underwent changes in the trophic state provide a so far unrealized possibility to analyze the response of toxic/nontoxic genotype composition to the environment. During a period of 29 years of re-oligotrophication of the deep, physically stratified Lake Zürich (1980 to 2008), the population of the stratifying cyanobacterium Planktothrix was at a minimum during the most eutrophic years (1980 to 1984), but increased and dominated the phytoplankton during the past two decades. Quantitative polymerase chain reaction revealed that during the whole observation period the proportion of the toxic genotype was strikingly stable, that is, close to 100%. Inactive MC genotypes carrying mutations within the MC synthesis genes never became abundant. Unexpectedly, a nontoxic genotype, which lost its MC genes during evolution, and which could be shown to be dominant under eutrophic conditions in shallow polymictic lakes, also co-occurred in Lake Zürich but was never abundant. As it is most likely that this nontoxic genotype contains relatively weak gas vesicles unable to withstand the high water pressure in deep lakes, it is concluded that regular deep mixing selectively reduced its abundance through the destruction of gas vesicles.

Conclusions

The stability in toxic genotype dominance gives evidence for the adaptation to deep mixing of a genotype that retained the MC gene cluster during evolution. Such a long-term dominance of a toxic genotype draws attention to the need to integrate phylogenetics into ecological research as well as ecosystem management.

     

Use of qPCR for the study of hepatotoxic cyanobacteria population dynamics

Toxic cyanobacteria blooms are increasingly frequent and object of greater concern due to its ecological and health impacts. One important lack in the toxic cyanobacteria research field is to understand which parameters influence most and how they operate to regulate the overall levels of cyanotoxins in a body of water. MC concentration is believed to be influenced by changes in several seasonal environmental factors that influence the succession of toxic cyanobacteria. In the last years, qPCR (quantitative polymerase chain reaction) has been applied to determine the seasonal and temporal shifts in the proportions of MC-producing and non-MC-producing subpopulations by quantifying both mcy genotypes and total population numbers. We discuss the most prominent and recent studies using qPCR to address hepatotoxic cyanobacteria population dynamics and evaluate how they helped understanding the factors promoting the growth of toxic strains in situ and the succession of hepatotoxin-producing genera in natural populations.