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.     

Analysis of environmental drivers influencing interspecific variations and associations among bloom-forming cyanobacteria in large,shallow eutrophic lakes

Non-diazotrophic Microcystis and filamentous N₂-fixing Aphanizomenon and Dolichospermum (formerly Anabaena) co-occur or successively dominate freshwaters globally. Previous studies indicate that dual nitrogen (N) and phosphorus (P) reduction is needed to control cyanobacterial blooms; however, N limitation may cause replacement of non-N₂-fixing by N₂-fixing taxa. To evaluate potentially counterproductive scenarios, the effects of temperature, nutrients, and zooplankton on the spatio-temporal variations of cyanobacteria were investigated in three large, shallow eutrophic lakes in China. The results illustrate that the community composition of cyanobacteria is primarily driven by physical factors and the zooplankton community, and their interactions. Niche differentiation between Microcystis and two N₂-fixing taxa in Lake Taihu and Lake Chaohu was observed, whereas small temperature fluctuations in Lake Dianchi supported co-dominance. Through structural equation modelling, predictor variables were aggregated into ‘composites’ representing their combined effects on species-specific biomass. The model results showed that Microcystis biomass was affected by water temperature and P concentrations across the studied lakes. The biomass of two filamentous taxa, by contrast, exhibited lake-specific responses. Understanding of driving forces of the succession and competition among bloom-forming cyanobacteria will help to guide lake restoration in the context of climate warming and N:P stoichiometry imbalances.     

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.     

Occurrence of dinoflagellate Alexandrium tamarense, a causative organism of paralytic shellfish poisoning in Chinhae Bay, Korea

A paralytic shellfish poisoning (PSP) incident caused by consumptionof the mussel Mytilus edulis occurred for the first time inKorea in April 1986. Weekly water samplings were carried Outduring the period from 7 March to 21 April 1989 in Chinhae Bay,Korea, in order to identify the causative organism. The temperaturecharacteristics of the water column indicated three differenthydrological regimes: well mixed (up to 7 March), weakly stratified(17–31 March) and stratified (7–21 April). Toxicityof the phytoplankton was detected during the weakly stratifiedperiod, but only in the 10–50 p.m phytoplankton size fraction.This study presents the occurrence of the toxigenic dinoflagellateAlexandrium tamarense, which is a causative organism of PSP,in Korean coastal waters. Its biomass varied at different depthsin the water column, ranging from 200 to 8000 cells 1^–1in the water column. The weekly fluctuation of A.tamarense toxicitywas similar to that of mussel toxicity. ¹ Present address: Department of Biology, College of NaturalSciences, Hanyang University, Seoul 133-791, Korea     

Quantitative Real-Time PCR for Determination of Microcystin Synthetase E Copy Numbers for Microcystis and Anabaena in Lakes

Cyanobacterial mass occurrences in freshwater lakes are generally formed by Anabaena, Microcystis, and Planktothrix, which may produce cyclic heptapeptide hepatotoxins, microcystins. Thus far, identification of the most potent microcystin producer in a lake has not been possible due to a lack of quantitative methods. The aim of this study was to identify the microcystin-producing genera and to determine the copy numbers of microcystin synthetase gene E (mcyE) in Lake Tuusulanjärvi and Lake Hiidenvesi in Finland by quantitative real-time PCR. The microcystin concentrations and cyanobacterial cell densities of these lakes were also determined. The microcystin concentrations correlated positively with the sum of Microcystis and Anabaena mcyE copy numbers from both Lake Tuusulanjärvi and Lake Hiidenvesi, indicating that mcyE gene copy numbers can be used as surrogates for hepatotoxic Microcystis and Anabaena. The main microcystin producer in Lake Tuusulanjärvi was Microcystis spp., since average Microcystis mcyE copy numbers were >30 times more abundant than those of Anabaena. Lake Hiidenvesi seemed to contain both nontoxic and toxic Anabaena as well as toxic Microcystis strains. Identifying the most potent microcystin producer in a lake could be valuable for designing lake restoration strategies, among other uses.     

Seasonal changes of phytoplankton and cyanobacteria/cyanotoxin risk in two shallow morphologically altered lakes: Effects of water level manipulation (Wieprz-Krzna Canal System,Eastern Poland)

Seasonal development of phytoplankton was compared over two years in two eutrophic, morphologically altered lakes. During study, the water level in Lake Tomaszne were periodically regulated whereas in Lake Mytycze, the water level manipulation was not performed. We assumed that the supply with fertile waters and subsequent discharge can be reflected in seasonal changes of phytoplankton taxa composition and biomass, especially of toxigenic cyanobacteria. In Lake Tomaszne, the development of toxigenic Nostocales (Aphanizomenon gracile and Dolichospermum planctonicum) was supported by the entrance of water from a canal (due to supply of water containing NH₄⁺-N). After water discharge, the replacement of Nostocales by the toxigenic Planktothrix agardhii was associated with low light conditions and a supply of phosphates from the bottom sediments. In Lake Mytycze, microalgae (Chlorococcales) were predominant throughout the study period. The rapid growth of toxigenic cyanobacteria (A. gracile, Planktolyngbya limnetica and Microcystis spp.) occurred only at extremely low water levels. In Lake Tomaszne, the lake with periodic water level regulation, the high cyanobacteria/cyanotoxin risk and decline of its ecological status were more conspicuous than in Lake Mytycze, the lake with natural water level changes.     

Isolation of viable cell mass from frozen <Emphasis Type="Italic">Microcystis viridis</Emphasis> bloom containing microcystin-RR

Cyanobacterial species commonly occur in the phytoplankton of freshwater lakes and sometimes develop as toxin-producing blooms. Microcystis is one of the most common genera of freshwater cyanobacteria and is often the dominating phytoplankton of eutrophic lakes all over the world. In eutrophic lakes, large amounts of Microcystis may overwinter in the sediment and re-inoculate the water column in spring. In most cases, the overwintering pelagic population—if it exists—is small, and its role in re-inoculation has not been clear yet. In December 2005, we found large amounts of Microcystis on the surface, frozen in the ice cover in a eutrophic pond (Pond Hármashegy, Hungary). We identified the Microcystis species and investigated the viability and the toxicity of the frozen cells. The dominant species in the bloom samples was Microcystis viridis. Viability tests showed that the colonies isolated from the ice cover were composed of living cells. The isolated strain was found toxic, we analyzed the microcystin composition in the frozen planktonic Microcystis mass; in the investigated samples microcystin-RR was the main cyanotoxin.     

Phylogenies of Microcystin-Producing Cyanobacteria in the Lower Laurentian Great Lakes Suggest Extensive Genetic Connectivity

Lake St. Clair is the smallest lake in the Laurentian Great Lakes system. MODIS satellite imagery suggests that high algal biomass events have occurred annually along the southern shore during late summer. In this study, we evaluated these events and tested the hypothesis that summer bloom material derived from Lake St. Clair may enter Lake Erie via the Detroit River and represent an overlooked source of potentially toxic Microcystis biomass to the western basin of Lake Erie. We conducted a seasonally and spatially resolved study carried out in the summer of 2013. Our goals were to: 1) track the development of the 2013 summer south-east shore bloom 2) conduct a spatial survey to characterize the extent of toxicity, taxonomic diversity of the total phytoplankton population and the phylogenetic diversity of potential MC-producing cyanobacteria (Microcystis, Planktothrix and Anabaena) during a high biomass event, and 3) compare the strains of potential MC-producers in Lake St. Clair with strains from Lake Erie and Lake Ontario. Our results demonstrated a clear predominance of cyanobacteria during a late August bloom event, primarily dominated by Microcystis, which we traced along the Lake St. Clair coastline downstream to the Detroit River''s outflow at Lake Erie. Microcystin levels exceeded the Province of Ontario Drinking Water Quality Standard (1.5 µg L⁻¹) for safe drinking water at most sites, reaching up to five times this level in some areas. Microcystis was the predominant microcystin producer, and all toxic Microcystis strains found in Lake St. Clair were genetically similar to toxic Microcystis strains found in lakes Erie and Ontario. These findings suggest extensive genetic connectivity among the three systems.     

Diurnal changes in the vertical distribution of phytoplankton in hypertrophic Lake Kasumigaura,Japan

The daily vertical migration of five species;Microcystis aeruginosa (Kütz.) Trevis,Anabaena spiroides Klebahn f.crassa (L.) Elenkin,Aphanizomenon flos-aquae (L.) Ralfs,Melosira granulata (E). Ralfs, andCoscinodiscus lacustris Grun. was studied using a close-interval water sampler on a calm summer day in Lake Kasumigaura. Many colonies ofMicrocystis were observed at the middle of the water column (approx. 1.5 m depth) in the afternoon, and at the surface in the early morning.Anabaena occurred mostly in the upper layer whileAphanizomenon tended to be uniformly distributed. The difference in migration patterns suggests thatMicrocystis is superior toAnabaena andAphanizomenon in obtaining both light and nutrients from this lake. Among diatoms,Melosira remained at the bottom of the water column throughout day and night, but Coscinodiscus was uniformly distributed.     

Relationships between microcystins and environmental parameters in 30 subtropical shallow lakes along the Yangtze River, China

1. A survey of 30 subtropical shallow lakes in the middle and lower reaches of the Yangtze River area in China was conducted during July–September in 2003–2004 to study how environmental and biological variables were associated with the concentration of the cyanobacterial toxin microcystin (MC). 2. Mean MC concentration in seasonally river‐connected lakes (SL) was nearly 33 times that in permanently river‐connected lakes (RL), and more than six times that in city lakes (NC) and non‐urban lakes (NE) which were not connected to the Yangtze River. The highest MC (8.574 μg L^?1) was detected in Dianshan Lake. 3. MC‐RR and MC‐LR were the primary toxin variants in our data. MC‐RR, MC‐YR and MC‐LR were significantly correlated with Chl a, biomass of cyanobacteria, Microcystis and Anabaena, indicating that microcystins were mainly produced by Microcystis and Anabaena sp. in these lakes. 4. Nonlinear interval maxima regression indicated that the relationships of Secchi depth, total nitrogen (TN) : total phosphorus (TP) and NH with MC were characterised by negative exponential curves. The relationships between MC and TN, TP, NO + NO were fitted well with a unimodal curve. 5. Multivariate analyses by principal component and classifying analysis indicated that MC was mainly affected by Microcystis among the biological factors, and was closely related with temperature among physicochemical factors.     

Diet selection by copepods in the presence of cyanobacteria

Boeckella, the dominant calanoid in many Southern Hemispherelakes, can survive, grow and reproduce to varying extents onmonocultures of cyanobacteria. In this study, we determinedthe effects of algal and cyanobacterial foods of different nutritionalvalue and concentration on food preferences of adult femaleBoeckella trianiculata and Boeckella hamata. Four species ofcyanobacteria (Anabaena flos-aquae, Nostoc sp. 2, OscillatoriatenuisandMicrocystis aeruginosa) were offered alone and mixedwith equal biomasses of Cryptomonas sp., Choricystis or a cyanobacterium.Food preferences were calculated as ratios of the rates at whichthe copepods removed each food at high and low food concentrations.In high-concentration mixtures with cyanobacteria, Cryptomonaswas consistently preferred by both Boeckella spp. In low-concentrationmixtures, both Boeckella spp. preferred Anabaena and Nostoc,which they removed at high rates(81–142 ml mg^–1h^–1), although Cryptomonas was selected in preferenceto Oscillatoria and Microcystis. When fed mixtures of filamentouscyanobacteria, both species of Boeckella showed invariant discriminationagainst Nostoc, andshifts in preference between Anabaena andOscillatoria that were related to food concentration. Microcystis,the least favouredfood, appeared to have a toxic effect on B.triarticulata. ¹Present address: Nursing and Midwifery Department, Otago Polytechnic,Forth Street, Dunedin, New Zealand     

Diurnal changes in buoyancy and vertical distribution in populations of Microcystisin two shallow lakes

Changes in the buoyancy of Microcystis populations were followedover 24 h periods in two shallow well-mixed lakes, Lake Vinkeveen(area 0 6 km²) and Lake IJsselmeer (1190 km²), in the NetherlandsThe Microcystis colonies collected from the surface layers inboth lakes showed a buoyancy decrease during the day and anincrease at night The buoyant colonies, and especially the faster-movinglarge ones, became concentrated by flotation into the surfacemixed layers As a result the mean position of the cyanobacterialpopulation became located nearer the surface than that of othernon-buoyant phytoplankton, such as Scencdesmus. The cyanobacteriawould, therefore, have received a higher average irradianceThe Microcystis in these shallow lakes had weaker gas vesiclesthan those found previously in deeper lakes but it was demonstratedthat the loss of buoyancy, which occurred at high irradiances,resulted from an increase in carbohydrate ballast rather thanthrough turgoi-driven gas vesicle collapse     

Role of Environmental Factors and Toxic Genotypes in the Regulation of Microcystins-Producing Cyanobacterial Blooms

The aim of this study was to understand: (1) how environmental conditions can contribute to formation of Microcystis-dominated blooms in lowland, dam reservoirs in temperate climate—with the use of quantitative molecular monitoring, and (2) what is the role of toxic Microcystis genotypes in the bloom functioning. Monitoring of the Sulejow Reservoir in 2009 and 2010 in two sites Tresta (TR) and Bronislawow BR), which have different morphometry, showed that physicochemical conditions were always favorable for cyanobacterial bloom formation. In 2009, the average biomass of cyanobacteria reached 13 mg L^?1 (TR) and 8 mg L^?1 (BR), and in the second year, it decreased to approximately 1 mg L^?1 (TR and BR). In turns, the mean number of toxic Microcystis genotypes in the total Microcystis reached 1 % in 2009, both in TR and BR, and in 2010, the number increased to 70 % in TR and 14 % in BR. Despite significant differences in the biomass of cyanobacteria in 2009 and 2010, the mean microcystins (MCs) concentration and toxicity stayed at a similar level of approximately 1 μg L^?1. Statistical analysis indicated that water retention time was a factor that provided a significant difference between the two monitoring seasons and was considered a driver of the changes occurring in the Sulejow Reservoir. Hydrologic differences, which occurred between two studied years due to heavy flooding in Poland in 2010, influenced the decrease in number of Microcystis biomass by causing water disturbances and by lowering water temperature. Statistical analysis showed that Microcystis aeruginosa biomass and 16S rRNA gene copy number representing Microcystis genotypes in both years of monitoring could be predicted on the basis of total and dissolved phosphorus concentrations and water temperature. In present study, the number of mcyA gene copies representing toxic Microcystis genotypes could be predicted based on the biomass of M. aeruginosa. Moreover, MCs toxicity and concentration could be predicted on the basic of mcyA gene copy number and M. aeruginosa (biomass, 16S rRNA), respectively. Present findings may indicate that Microcystis can regulate the number of toxic genotypes, and in this way adjust the whole bloom to be able to produce MCs at the level which is necessary for its maintenance in the Sulejow Reservoir under stressful hydrological conditions.     

Grazing on colonial and filamentous, toxic and non-toxic cyanobacteria by the zebra mussel Dreissena polymorpha

Colony forming and toxic cyanobacteria form a problem in surfacewaters of shallow lakes, both for recreation and wildlife. Zebramussels, Dreissena polymorpha, have been employed to help torestore shallow lakes in the Netherlands, dominated by cyanobacteria,to their former clear state. Zebra mussels have been presentin these lakes since they were created in the 19th century bythe excavation of peat and are usually not considered to bean invasive species. Most grazing experiments using Dreissenahave been performed with uni-cellular phytoplankton laboratorystrains and information on grazing of larger phytoplankton taxahardly exists. To gain more insight in to whether D. polymorphais indeed able to decrease cyanobacteria in the phytoplankton,we therefore performed grazing experiments with zebra musselsand two species of cyanobacteria, that greatly differ in shape:colony forming strains of Microcystis aeruginosa and the filamentousspecies Planktothrix agardhii. For both species a toxic anda non-toxic strain was selected. We found that zebra musselscleared toxic Planktothrix at a higher rate than non-toxic Planktothrix,toxic or non-toxic Microcystis. Clearance rates between theother strains were not significantly different. Both phytoplanktonspecies, regardless of toxicity, size and shape, were foundin equal amounts (based on chlorophyll concentrations) in theexcreted products of the mussels (pseudofaeces). The resultsshow that zebra mussels are capable of removing colonial andfilamentous cyanobacteria from the water, regardless of whetherthe cyanobacteria are toxic or not. This implies that the musselsmay be used as a biofilter for the removal of harmful cyanobacterialblooms in shallow (Dutch) lakes where the mussels are alreadypresent and not a nuisance. Providing more suitable substratefor zebra mussel attachment may lead to appropriate mussel densitiescapable of filtering large quantities of cyanobacteria.     

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

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

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.     

Effect of cyanobacterial blooms on thermal stratification

Enclosure experiments were performed at Akanoi Bay, Lake Biwa, in 1995 to determine whether the blooms of cyanobacterial algae changed thermal stratification in the lake. We used four rectangular enclosures, each 10 m × 10 m, with a volume of 200 m³, which were open to the sediments. Two enclosures, A and B, were mixed artificially by aquatic pumps from 1000 to 1400 every day, and the other two enclosures, C and D, were controls with no mixing. The experiment was conducted during late summer from August 3 to September 27. Chlorophyll a concentrations were highest in enclosure D, followed by enclosure C, both of which were controls without mixing. Enclosure A had lower concentrations than enclosures C and D, and enclosure B had the lowest concentrations. No large cyanobacterial algae blooms of Anabaena sp. and Microcystis sp. were seen in the mixed enclosures A and B. In enclosures C and D, blooms of Anabaena sp. occurred in the middle of August, and Microcystis sp. later became dominant in enclosure D at the end of August. In enclosure D, the water temperature changed over the diel cycle before August 17, with thermal stratification during the day and complete mixing at night. After August 17, as Anabaena sp. and Microcystis sp. became dominant, the temperature at the bottom of the enclosure did not change clearly over the 24-h cycle. The APE (available potential energy) density (a measure of water column stability) in the enclosures increased by almost 100% when the biovolume of Anabaena sp. + Microcystis sp. exceeded 20 mm³ l⁻¹. These results indicate that blooms of Anabaena sp. and Microcystis sp. can increase the available potential energy in the water column and create more stable stratification for their growth. Received: September 25, 1999 / Accepted: January 6, 2000     

Growth interactions among blue-green (Anabaena Oscillarioides,Microcystis aeruginosa) and green (Chlorella sp.) algae

The growth interactions amongst the blue-green algal species Anabaena oscillarioides, Microcystis aeruginosa and the green alga, Chlorella sp. were studied both in mixed cultures and in filter cultures separated by a membrane filter in the two arms of an interaction U-tube. The role of nutrients especially phosphate upon the interaction has also been studied. Anabaena and Microcystis both inhibited the growth of Chlorella while Microcystis also inhibited the growth of Anabaena. The inhibitory effect of Microcystis was found to be dependent on high concentrations of the initial algal inocula and independent of the initial concentration of nutrients such as inorganic phosphate, indicating that the nature of the inhibition is probably due to the production of inhibitory extracellular products by Microcystis. On the other hand, the inhibitory effect of Anabaena on Chlorella is the consequence of nutrient competition with Anabaena competing more effectively for the available phosphate.     

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.     

Molecular techniques for the early warning of toxic cyanobacteria blooms in freshwater lakes and rivers

The aim of this work was to test the efficacy of molecular techniques for detecting toxigenic cyanobacteria in environmental water samples collected from freshwater lakes, rivers and reservoirs in Portugal. Of 26 environmental samples tested, 21 were found to contain Microcystis using a genus-specific polymerase chain reaction (PCR). Another primer pair was applied to the same DNA template to test for the presence of microcystin synthetase genes. This primer pair resulted in the formation of a PCR product in 15 of the samples containing Microcystis and one sample that did not give a positive result in the Microcystis genus-specific PCR. A restriction assay using the enzyme EcoRV was then applied to show that in most cases, the gene fragment was from toxigenic strains of Microcystis and, in one above-mentioned case, from a microcystin-producing strain of Planktothrix. All environmental samples were examined microscopically to confirm the presence of cyanobacteria species. Samples were also tested for the presence of microcystins using the ELISA plate assay. There was good agreement between the results obtained with molecular techniques and those obtained from microscopy and chemical methods. The PCR techniques applied in this paper were found to be useful, particularly when the concentration of the target organism was very low compared with other organisms. This technique can be used to detect inocula for cyanobacterial populations and therefore provide a useful tool for assessing under which conditions particular species can grow into bloom populations.