Molecular tools to detect anatoxin-a genes in aquatic ecosystems: Toward a new nested PCR-based method

Over the last few decades, cyanobacterial mass occurrence has become a recurrent feature of aquatic ecosystems. This has led to ecosystem exposure and health hazards associated with cyanotoxin production. The neurotoxin anatoxin-a and its homologs can be synthesized by benthic cyanobacterial species in lotic systems, but also by planktonic lacustrine species such as Dolichospermum (also known as Anabaena). However, only a few studies have focused on anatoxin-a occurrence and its biosynthesis genes in freshwater lakes. The initial aim of this study was to evaluate the molecular tools available in the literature to detect anatoxin-a biosynthesis genes in lacustrine environments. Having tested different sets of PCR primers, we found that that some sets of primers, such as anxC, were too specific and did not amplify anatoxin-a biosynthesis genes in all producing strains. On the other hand, some sets of primers, such as atxoa, seemed not to be specific enough, amplifying numerous non-specific bands in environmental samples, especially those from sediments. Furthermore, anaC and anaF amplification exhibited different band intensities during electrophoresis, suggesting a high variation in number of gene copies between samples. As a result, we proposed a new nested PCR-based method which considerably improved the amplification of the anaC gene in our environmental samples, eliminating non-specific bands and weak detections. Using this tool, our study also highlighted that anatoxin-a genes are widely distributed throughout freshwater lakes. This suggests the need for further ecological investigations into anatoxin-a in these ecosystems.     

Sediment and phytoplankton records of the cyanobacterial genus Anabaena in boreal Lake Pyhäjärvi

Past occurrence and quantities of Anabaena cyanobacteria in Lake Pyhäjärvi, SW Finland, were investigated using sediment and phytoplankton records. A short sediment core covering the past 20 years was examined for Anabaena resting spores (akinetes) in order to assess the utility of akinetes as a paleolimnological proxy. Sedimentary akinetes confirmed the past existence of Anabaena in water, but did not show a direct correlation with the amount of Anabaena spp. in water samples. The amount of planktonic Anabaena spp. correlated with total phosphorus and nitrogen concentrations in Lake Pyhäjärvi, but the number of akinetes was considered to have a relationship with low nutrient concentrations and dominant bloom-forming cyanobacteria. Akinetes are probably suitable for low-resolution and long time scale paleolimnological investigations, where they provide information of past cyanobacteria that cannot be directly attained otherwise.     

Expansion of bloom-forming Dolichospermum lemmermannii (Nostocales,Cyanobacteria) to the deep lakes south of the Alps: Colonization patterns,driving forces and implications for water use

Since the beginning of the 1990s, the largest lakes south of the Alps (Garda, Iseo, Como and Maggiore) showed a progressive colonization of Dolichospermum lemmermannii (Cyanobacteria). The appearance of surface blooms of this species raised serious concerns because of the impacts on the tourist economy and the potential toxigenic effects. Nevertheless, no detailed investigations were done to clarify the taxonomic position, ecology and toxicity of this species. In this work, phylogenetic analyses based on the 16S rRNA and rpoB genes demonstrated how the strains isolated from the Italian lakes were clustered together with other D. lemmermannii strains isolated in Northern Europe. The expansion of this species in the southern subalpine lakes contrasted with the prevailing south to north dispersion paths typical of other Nostocales (e.g. Cylindrospermopsis and Aphanizomenon). Conversely, the spread was consistent with the geographical areal of this cyanobacterium, which appears circumscribed between the 40th parallel and the Arctic Circle. This aspect highlights the ecological heterogeneity that characterizes the order Nostocales. In Lake Garda, D. lemmermannii always developed in the warmest months (>15 °C) with low abundances (generally <200 cell mL⁻¹). Nevertheless, owing to its ability to form surface water blooms, this species is considered as one of the most nuisance algae in the subalpine lake district. From the other side, different strains isolated from these large lakes tested negative for the biosynthesis of microcystins, anatoxin-a, nodularins and cylindrospermopsins, and for the presence of mcyE and anaC genes of the microcystins and anatoxin-a gene clusters.     

The diversity and distribution of toxigenic Microcystis spp. in present day and archived pelagic and sediment samples from Lake Erie

The reoccurrence of significant cyanobacterial blooms in Lake Erie during the last 13 years has raised questions concerning the long-term persistence of microcystin-producing cyanobacteria and the presence of natural sediment reservoirs for potentially toxic cyanobacteria in this large lake system. To address these questions, we analyzed phytoplankton and sediment samples which were collected and preserved in the 1970s as well as samples collected in 2004 from locations within Lake Erie. The identification of microcystin-producing cyanobacteria in Lake Erie was examined via PCR amplification of the mcyA gene fragment. Based on the high % sequence similarity, the mcyA sequences from all 1970s phytoplankton and sediment samples were determined to belong to Microcystis spp., in spite of reports suggesting that Lake Erie was dominated by filamentous cyanobacteria in the 1970s. In sediment samples from 2004, signature genes for Microcystis were distributed and preserved not only in the surface sediments but also up to 10–12 cm in depth. Based on cell quantities determined by a quantitative polymerase chain reaction (qPCR) method, 0.18% of eubacteria in the sediments were Microcystis cells, of which 4.8% were potential microcystin producers. In combination with experiments showing that Microcystis cells can be cultured from Lake Erie surface sediments, this paper demonstrates the potential for these sediments to act as a reservoir for pelagic Microcystis populations and that the composition of the population of microcystin-producing cyanobacteria in Lake Erie has not changed remarkably since the 1970s.     

Paleolimnological evidence of change in a shallow, hypereutrophic lake: Upper Klamath Lake, Oregon, USA

Sediment cores were collected from Upper Klamath Lake in October, 1998 and analyzed for ²¹⁰Pb, ¹⁴C, ¹⁵N, N, P, C, Ti, Al, diatoms, Pediastrum, and cyanobacterial akinetes. These results were used to reconstruct changes in water quality in Upper Klamath Lake over the last 150 years. The results showed that there was substantial mixing of the upper 10 cm of sediment, representing the previous 20 to 30 years. However, below that, ²¹⁰Pb activity declined monotonically, allowing reasonable dating for the period from about 1850 to 1970. The sediment accumulation rates (SAR) showed a substantial increase in the 20th century. The increase in SAR corresponded with increases in erosional input from the watershed as represented by the increases in sediment concentrations of Ti and Al. The upper 20 cm of sediment, representing the last 150 years, also showed increases in C, N, P, and ¹⁵N. The increases in nutrient concentrations may be affected to various degrees by diagenetic reactions within the sediments, although the changes in concentrations also were marked by changes in the N:P ratio and in a qualitative change in the source of N as reflected in increasing δ¹⁵N. The diatoms showed modest changes in the 20th century, with increases in Asterionella formosa, Stephanodiscus hantzschii, and S. parvus. Pediastrum, a green alga, was well-preserved in the sediments and exhibited a sharp decline in relative abundance in the upper sediments. Total cyanobacteria, as represented by preserved akinetes, exhibited only minor changes in the last 1000 years. However, Aphanizomenon flos-aquae, a taxon which was formerly not present in the lake 150 years ago, but that now dominates the summer phytoplankton, has shown major increases over the past 100 years. The changes in sediment composition are consistent with activities including timber harvest, drainage of wetlands, and agricultural activities associated with livestock grazing, irrigated cropland, and hydrologic modifications.     

Biodegradability and adsorption on lake sediments of cyanobacterial hepatotoxins and anatoxin-a

Cyanobacterial hepatotoxins and anatoxin-a, a neurotoxin, were shown to be degraded when crude extracts of lysed toxic laboratory strains of cyanobacteria were exposed to natural populations of micro-organisms from lakes. While anatoxin-a decayed equally fast with all the inocula from lake sediment and water, the degradation rate of hepatotoxins was higher with inocula from places at which cyanobacterial water blooms had occurred than with inocula from places with no known mass occurrences of cyanobacteria. Degradation was slowest when an inoculum from a humic lake was used. A part of the loss of the toxins was shown to be due to adsorption on lake sediments.     

Isolation and Characterization of Toxic Cyanobacteria from Different Natural Sources

We isolated seven algologically and five bacteriologically pure cultures of toxin-producing cyanobacteria from Turgen gorge (Kazakhstan), Karlovy Vary (Czech Republic), and Shar-Nuur Lake, Bayan Ulgiiregion (Mongolia) springs. According to the Daphnia magna test, Desertifilum sp. and Nostoc sp. strains were the most toxic in the test of isolated strains (complete death of all test organisms was detected after 48 h). These strains possessed the highest inhibitory effect on proliferation of the HeLa cancer cell line. The Anabaena sp. 35 and Nostoc sp. 4 strains were also high toxic. Model strains Synechocystis PCC 6803 and Synechococcus elongates PCC 7942, as well as the strain isolated in the present work, Synechococcus sp. 55, were less toxic. Mass spectrometry made it possible to assign cyanobacterial toxins to cyclic depsipeptides. Two cyclic depsipeptides, micropeptin T and oscillapeptin, were detected in Desertifilum sp. extracts. Cryptophycin and small amounts of cyclic depsipeptide micropeptin SD were detected in Nostoc sp. extract.     

Toxin-Producing Anabaena flos-aquae Induces Settling of Chlamydomonas reinhardtii,a Competing Motile Alga

Toxin production is an adaptation that allows cyanobacteria in resource-limiting environments to ameliorate the effects of herbivory and competition with other phototrophs. We demonstrate that the cyanobacterial toxins anatoxin-a and microcystin-LR paralyze the motile green alga Chlamydomonas reinhardtii. In addition, both purified toxins and cyanobacterial extracellular products containing these toxins cause the alga to settle faster than in nontoxic media. In microcosm experiments, the presence of either the cyanobacterium or its extracellular products induce settling in the alga, similar to the response observed with the addition of both anatoxin-a and microcystin-LR. The cyanobacterial production of paralyzing toxins represents a novel mechanism for phytoplankton settling. This prokaryotic/eukaryotic chemical interaction may create a competitor-free zone for cyanobacteria in lake environments, predicating optimal conditions for a toxic cyanobacterial bloom.     

Fungal Parasitism: Life Cycle,Dynamics and Impact on Cyanobacterial Blooms

Many species of phytoplankton are susceptible to parasitism by fungi from the phylum Chytridiomycota (i.e. chytrids). However, few studies have reported the effects of fungal parasites on filamentous cyanobacterial blooms. To investigate the missing components of bloom ecosystems, we examined an entire field bloom of the cyanobacterium Anabaena macrospora for evidence of chytrid infection in a productive freshwater lake, using a high resolution sampling strategy. A. macrospora was infected by two species of the genus Rhizosiphon which have similar life cycles but differed in their infective regimes depending on the cellular niches offered by their host. R. crassum infected both vegetative cells and akinetes while R. akinetum infected only akinetes. A tentative reconstruction of the developmental stages suggested that the life cycle of R. crassum was completed in about 3 days. The infection affected 6% of total cells (and 4% of akinètes), spread over a maximum of 17% of the filaments of cyanobacteria, in which 60% of the cells could be parasitized. Furthermore, chytrids may reduce the length of filaments of Anabaena macrospora significantly by “mechanistic fragmentation” following infection. All these results suggest that chytrid parasitism is one of the driving factors involved in the decline of a cyanobacteria blooms, by direct mortality of parasitized cells and indirectly by the mechanistic fragmentation, which could weaken the resistance of A. macrospora to grazing.     

Assessment of rapid bioassays for detecting cyanobacterial toxicity

Simple and easy-to-use bioassays with Artemia salina (brine shrimp) larvae, luminescent bacteria and Pseudomonas putida were evaluated for the detection of toxicity due to cyanobacterial hepato-and neurotoxins. The hepatotoxins and a neurotoxin, anatoxin-a, were extracted from laboratory-grown cultures and natural bloom samples by the solid phase fractionation method and dissolved in diluent for different bioassays. The toxin concentration of cyanobacterial extracts was determined with HPLC. The Artemia biotest appeared to be quite sensitive to cyanobacterial hepatotoxins, with LC 50 values of 3–17 mg l^-1. The Artemia test was also shown to be of value for the detection of toxicity caused by anatoxin-a. The fractionated extract of anatoxin-a was not lethal to Artemia but it disturbed the ability of the larvae to move forwards. Filtered cyanobacterial cultures with anatoxin-a, on the other hand, caused mortality of Artemia larvae at concentrations of 2–14 mg l^-1. With the solid phase fractionation of cyanobacterial samples, no non-specific toxicity due to compounds other than hepato- and neurotoxins was observed. In the luminescent bacteria test, the inhibition of luminescence did not correlate with the abundance of hepatotoxins or anatoxin-a. The growth of Ps. putida was enhanced, rather than inhibited by cyanobacterial toxin fractions.     

Targeted deep sequencing reveals high diversity and variable dominance of bloom-forming cyanobacteria in eutrophic lakes

Cyanobacterial blooms in eutrophic lakes are severe environmental problems worldwide. To characterize the spatiotemporal heterogeneity of cyanobacterial blooms, a high-throughput method is necessary for the specific detection of cyanobacteria. In this study, the cyanobacterial composition of three eutrophic waters in China (Taihu Lake, Dongqian Lake, and Dongzhen Reservoir) was determined by pyrosequencing the cpcBA intergenic spacer (cpcBA-IGS) of cyanobacteria. A total of 2585 OTUs were obtained from the normalized cpcBA-IGS sequence dataset at a distance of 0.05. The 238 most abundant OTUs contained 92% of the total sequences and were classified into six cyanobacterial groups. The water samples of Taihu Lake were dominated by Microcystis, mixed Nostocales species, Synechococcus, and unclassified cyanobacteria. Besides, all the samples from Taihu Lake were clustered together in the dendrogram based on shared abundant OTUs. The cyanobacterial diversity in Dongqian Lake was dramatically decreased after sediment dredging and Synechococcus became exclusively dominant in this lake. The genus Synechococcus was also dominant in the surface water of Dongzhen Reservoir, while phylogenetically diverse cyanobacteria coexisted at a depth of 10 m in this reservoir. In summary, targeted deep sequencing based on cpcBA-IGS revealed a large diversity of bloom-forming cyanobacteria in eutrophic lakes and spatiotemporal changes in the composition of cyanobacterial communities. The genus Microcystis was the most abundant bloom-forming cyanobacteria in eutrophic lakes, while Synechococcus could be exclusively dominant under appropriate environmental conditions.     

Mesozooplankton and microzooplankton grazing during cyanobacterial blooms in the western basin of Lake Erie

Lake Erie is the most socioeconomically important and productive of the Laurentian (North American) Great Lakes. Since the mid-1990s cyanobacterial blooms dominated primarily by Microcystis have emerged to become annual, late summer events in the western basin of Lake Erie yet the effects of these blooms on food web dynamics and zooplankton grazing are unclear. From 2005 to 2007, grazing rates of cultured (Daphnia pulex) and natural assemblages of mesozooplankton and microzooplankton on five autotrophic populations were quantified during cyanobacterial blooms in western Lake Erie. While all groups of zooplankton grazed on all prey groups investigated, the grazing rates of natural and cultured mesozooplankton were inversely correlated with abundances of potentially toxic cyanobacteria (Microcystis, Anabaena, and Cylindrospermopsis; p < 0.05) while those of the in situ microzooplankton community were not. Microzooplankton grazed more rapidly and consistently on all groups of phytoplankton, including cyanobacteria, compared to both groups of mesozooplankton. Cyanobacteria displayed more rapid intrinsic cellular growth rates than other phytoplankton groups under enhanced nutrient concentrations suggesting that future nutrient loading to Lake Erie could exacerbate cyanobacterial blooms. In sum, while grazing rates of mesozooplankton are slowed by cyanobacterial blooms in the western basin of Lake Erie, microzooplankton are likely to play an important role in the top-down control of these blooms; this control could be weakened by any future increases in nutrient loads to Lake Erie.     

Cyanobacteria and cyanobacterial toxins in the alkaline crater lakes Sonachi and Simbi, Kenya

The phytoplankton communities and the production of cyanobacterial toxins were investigated in two alkaline Kenyan crater lakes, Lake Sonachi and Lake Simbi. Lake Sonachi was mainly dominated by the cyanobacterium Arthrospira fusiformis, Lake Simbi by A. fusiformis and Anabaenopsis abijatae. The phytoplankton biomasses measured were high, reaching up to 3159 mg l⁻¹ in L. Sonachi and up to 348 mg l⁻¹ in L. Simbi. Using HPLC techniques, one structural variant of the hepatotoxin microcystin (microcystin-RR) was found in L. Sonachi and four variants (microcystin-LR, -RR, -LA and -YR) were identified in L. Simbi. The neurotoxin anatoxin-a was found in both lakes. To our knowledge this is the first evidence of cyanobacterial toxins in L. Sonachi and L. Simbi. Total microcystin concentrations varied from 1.6 to 12.0 μg microcystin-LR equivalents g⁻¹ DW in L. Sonachi and from 19.7 to 39.0 μg microcystin-LR equivalents g⁻¹ DW in L. Simbi. Anatoxin-a concentrations ranged from 0.5 to 2.0 μg g⁻¹ DW in L. Sonachi and from 0 to 1.4 μg g⁻¹ DW in L. Simbi. In a monocyanobacterial strain of A. fusiformis, isolated from L. Sonachi, microcystin-YR and anatoxin-a were produced. The concentrations found were 2.2 μg microcystin g⁻¹ DW and 0.3 μg anatoxin-a g⁻¹ DW. This is the first study showing A. fusiformis as producer of microcystins and anatoxin-a. Since A. fusiformis occurs in mass developments in both lakes, a health risk for wildlife can be expected.     

A novel method to produce Anabaena-free Azolla by in vitro fertilization of micromanipulated megasporocarps

In the Azolla-Anabaena azollae symbiotic system, Anabaena akinetes get entrapped between the indusium and the apical cap of the megaspore apparatus during megasporocarp development, thus maintaining the continuity of the cyanobacterial association throughout the life cycle of the fern. The entrapped akinetes serve as the source of inoculum for infecting the new sporophyte when it is emerging from the megaspore apparatus. A procedure to generate Anabaena-free Azolla was developed by fertilizing the germinating megasporocarps in which the indusium along with the akinetes were removed by micromanipulation. This method has the advantage of not requiring drastic treatments of Azolla with antibiotics to eliminate the endosymbiotic cyanobacterial cells. Details of this new method and its usefulness in studies aimed at recombination of Azolla with Anabaena azollae are discussed.Abbreviations IRRI International Rice Research Institute - I^– IRRI medium devoid of combined nitrogen - I⁺ IRRI medium containing combined nitrogen - SDS sodium dodecyl sulfate     

Molecular detection of potentially toxic cyanobacteria and their associated bacteria in lake water column and sediment

We investigated the molecular diversity of cyanobacteria and bacteria during a water bloom in a lake with a long history of toxic cyanobacterial blooms (Lake Kastoria, Greece). We also tested the hypothesis whether bloom-forming cyanobacteria are preserved in the lake’s sediment 2 years after the bloom. The dominant cyanobacteria during the bloom included the potentially toxin-producing Microcystis aeruginosa and several other Chroococcales forms closely related to the genus Microcystis. This suggests that the use of cyanobacterial-specific primers seems to be very informative in describing the cyanobacteria during the water blooms. The bacterial community showed high diversity, consisting mostly of singleton and doubleton phylotypes. The majority of the phylotypes were typical lake bacteria including some potential pathogens and toxin metabolising bacteria, suggesting that the dominant toxic cyanobacteria did not have any significant effect on the bacterial community structure. In the sediment, 2 years after the water bloom, no bloom-forming cyanobacteria were retrieved, suggesting that they cannot be preserved in the sediment. Similar to the water column, sediment bacterial diversity was also high, consisting mostly of yet-uncultured bacteria that are related to environments where organic matter degradation takes place.     

Benthic fauna affects recruitment from sediments of the harmful cyanobacterium Nodularia spumigena

Physical disturbance and feeding by macrofauna in the sediment can potentially affect bloom initiation of phytoplankton species that have benthic stages in their life cycle. In this experimental study, we investigated how different species of macrozoobenthos can affect the recruitment of Nodularia spumigena from the sediment to the water column. N. spumigena is a toxic, nitrogen-fixing filamentous cyanobacterium, which forms large summer blooms in the Baltic Sea. Benthic recruitment from resting stages (akinetes) and vegetative cells deposited on the seafloor have long been suspected to initiate the blooms. We found that, depending on species-specific traits, deposit-feeding macrofauna (an amphipod, Monoporeia affinis, a bivalve, Macoma balthica and an invasive polychaete, Marenzelleria cf. arctia) has the potential to either reduce or facilitate recruitment of this cyanobacterium. Shorter filament length in treatments with fauna than in the treatment without indicates feeding on or mechanical destruction of N. spumigena by the animals. Our results show the importance of an often overlooked aspect of phytoplankton bloom initiation, the role of macrozoobenthos.     

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.     

Nodularia (Cyanobacteriaceae) Akinetes in the Sediments of the Peel-Harvey Estuary, Western Australia: Potential Inoculum Source for Nodularia Blooms

The populations of viable Nodularia (Cyanobacteriaceae) propagules in the sediments of the Peel-Harvey Estuary in Western Australia were assessed over location, time, and depth. The sediments of the Harvey Estuary had greater numbers of Nodularia propagules than those of the contiguous Peel Inlet. This was consistent with the distribution of Nodularia blooms in the system. The sediment populations of Nodularia propagules has increased up to 100-fold at the study sites over a 4-year period during which three blooms have occurred. It is considered that the majority of the propagules are akinetes. The significance of the sediment akinetes in providing the inoculum for rapid onset of Nodularia blooms is discussed. The population of akinetes decreased with depth, but viable akinetes were still found at 35 cm, the maximum depth sampled. Bioturbation by polychaete worms is likely to be significant in the distribution of akinetes to these depths.     

Morphological and habitat evolution in the Cyanobacteria using a compartmentalization approach

A phylogenomic approach was used to study the evolution of traits in the Cyanobacteria. A cyanobacterial backbone tree was constructed using multiple concatenated sequences from whole genome sequences. Additional taxa were added using a separate alignment that contained morphological characters, SSU (small subunit) and LSU (large subunit) rDNA, rpoC, rpoD, tufA, and gyrB genes. A compartmentalization approach was then used to construct a robust phylogeny with resolved deep branches. Additional morphological characters (e.g. unicellular or filamentous growth, presence or absence of heterocysts) were coded, mapped onto the backbone cyanobacterial tree, and the ancestral character states inferred. Our analyses show that the earliest cyanobacterial lineages were likely unicellular coccoid/ellipsoidal/short rods that lived in terrestrial/freshwater environments. Later cyanobacterial lineages independently gained the ability to colonize brackish, marine, and hypersaline environments while acquiring a large number of more complex traits: sheath, filamentous growth, nitrogen fixation, thermophily, motility, and use of sulphide as an electron donor. Many of these adaptations would have been important in the appearance of dense microbial mats early in Earth's history. Complex traits such as hormogonia, heterocysts, and akinetes had a single ancestor. Within the Nostocales, hormogonia and heterocysts arose before akinetes.     

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.