Nitrogen flux into metabolites and microcystins changes in response to different nitrogen sources in Microcystis aeruginosa NIES-843.

The over-enrichment of nitrogen (N) in the environment has contributed to severe and recurring harmful cyanobacterial blooms, especially by the non-N2 -fixing Microcystis spp. N chemical speciation influences cyanobacterial growth, persistence and the production of the hepatotoxin microcystin, but the physiological mechanisms to explain these observations remain unresolved. Stable-labelled isotopes and metabolomics were employed to address the influence of nitrate, ammonium, and urea on cellular physiology and production of microcystins in Microcystis aeruginosa NIES-843. Global metabolic changes were driven by both N speciation and diel cycling. Tracing 15 N-labelled nitrate, ammonium, and urea through the metabolome revealed N uptake, regardless of species, was linked to C assimilation. The production of amino acids, like arginine, and other N-rich compounds corresponded with greater turnover of microcystins in cells grown on urea compared to nitrate and ammonium. However, 15 N was incorporated into microcystins from all N sources. The differences in N flux were attributed to the energetic efficiency of growth on each N source. While N in general plays an important role in sustaining biomass, these data show that N-speciation induces physiological changes that culminate in differences in global metabolism, cellular microcystin quotas and congener composition.

Physical drivers facilitating a toxigenic cyanobacterial bloom in a major Great Lakes tributary.

The Maumee River is the primary source for nutrients fueling seasonal Microcystis-dominated blooms in western Lake Erie's open waters though such blooms in the river are infrequent. The river also serves as source water for multiple public water systems and a large food services facility in northwest Ohio, USA. On 20 September 2017, an unprecedented bloom was reported in the Maumee River estuary within the Toledo metropolitan area, which triggered a recreational water advisory. Here we (1) explore physical drivers likely contributing to the bloom's occurrence, and (2) describe the toxin concentration and bacterioplankton taxonomic composition. A historical analysis using ten-years of seasonal river discharge, water level, and local wind data identified two instances when high-retention conditions occurred over ≥10 days in the Maumee River estuary: in 2016 and during the 2017 bloom. Observation by remote sensing imagery supported the advection of cyanobacterial cells into the estuary from the lake during 2017 and the lack of an estuary bloom in 2016 due to a weak cyanobacterial bloom in the lake. A rapid-response survey during the 2017 bloom determined levels of the cyanotoxins, specifically microcystins, in excess of recreational contact limits at sites within the lower 20 km of the river while amplicon sequencing found these sites were dominated by Microcystis. These results highlight the need to broaden our understanding of physical drivers of cyanobacterial blooms within the interface between riverine and lacustrine systems, particularly as such blooms are expected to become more prominent in response to a changing climate.

Seasonal Gene Expression and the Ecophysiological Implications of Toxic Microcystis aeruginosa Blooms in Lake Taihu.

Harmful cyanobacterial blooms represent an increasing threat to freshwater resources globally. Despite increased research, the physiological basis of how the dominant bloom-forming cyanobacteria, Microcystis spp., proliferate and then maintain high population densities through changing environmental conditions is poorly understood. In this study, we examined the transcriptional profiles of the microbial community in Lake Taihu, China at 9 stations sampled monthly from June to October in 2014. To target Microcystis populations, we collected metatranscriptomic data and mapped reads to the M. aeruginosa NIES 843 genome. Our results revealed significant temporal gene expression patterns, with many genes separating into either early or late bloom clusters. About one-third of genes observed from M. aeruginosa were differentially expressed between these two clusters. Conductivity and nutrient availability appeared to be the environmental factors most strongly associated with these temporal gene expression shifts. Compared with the early bloom season (June and July), genes involved in N and P transport, energy metabolism, translation, and amino acid biosynthesis were down-regulated during the later season (August to October). In parallel, genes involved in regulatory functions as well as transposases and the production of microcystin and extracellular polysaccharides were up-regulated in the later season. Our observation indicates an eco-physiological shift occurs within the Microcystis spp. transcriptome as cells move from the rapid growth of early summer to bloom maintenance in late summer and autumn.

Seasonally Relevant Cool Temperatures Interact with N Chemistry to Increase Microcystins Produced in Lab Cultures of Microcystis aeruginosa NIES-843.

Freshwater cyanobacterial blooms are regularly formed by Microcystis spp., which are well-known producers of the hepatotoxin microcystin. The environmental factors that regulate microcystin synthesis remain unclear. We used reverse transcription-quantitative PCR (RT-qPCR), metabolomics, and toxin profiling (both by LC-MS) to measure the response of Microcystis aeruginosa NIES-843 to nitrogen (N) concentration, N chemistry (nitrate versus urea), and a range of seasonally relevant temperatures. Growth rates at lower temperatures were slower but resulted in increased cellular microcystin content (quota), and at these lower temperatures, N concentration had no effect on toxin production. In contrast, at warmer temperatures, reduction in N concentration increased toxin production, especially when urea was supplied as the nitrogen source. Our culture results demonstrate how temperature may lead to physiological responses ranging from slow growing yet very toxic cells at cool temperatures, to faster growing but less-toxic cells at warmer temperatures. This response represents a key interaction in bloom dynamics. Capturing this phenomenon as a temperature-driven toxin phenotype incorporated into models might improve the ability to predict microcystin biosynthesis during cyanobacterial blooms.

Ecophysiological Examination of the Lake Erie Microcystis Bloom in 2014: Linkages between Biology and the Water Supply Shutdown of Toledo, OH.

Annual cyanobacterial blooms dominated by Microcystis have occurred in western Lake Erie (U.S./Canada) during summer months since 1995. The production of toxins by bloom-forming cyanobacteria can lead to drinking water crises, such as the one experienced by the city of Toledo in August of 2014, when the city was rendered without drinking water for >2 days. It is important to understand the conditions and environmental cues that were driving this specific bloom to provide a scientific framework for management of future bloom events. To this end, samples were collected and metatranscriptomes generated coincident with the collection of environmental metrics for eight sites located in the western basin of Lake Erie, including a station proximal to the water intake for the city of Toledo. These data were used to generate a basin-wide ecophysiological fingerprint of Lake Erie Microcystis populations in August 2014 for comparison to previous bloom communities. Our observations and analyses indicate that, at the time of sample collection, Microcystis populations were under dual nitrogen (N) and phosphorus (P) stress, as genes involved in scavenging of these nutrients were being actively transcribed. Targeted analysis of urea transport and hydrolysis suggests a potentially important role for exogenous urea as a nitrogen source during the 2014 event. Finally, simulation data suggest a wind event caused microcystin-rich water from Maumee Bay to be transported east along the southern shoreline past the Toledo water intake. Coupled with a significant cyanophage infection, these results reveal that a combination of biological and environmental factors led to the disruption of the Toledo water supply. This scenario was not atypical of reoccurring Lake Erie blooms and thus may reoccur in the future.

Joint analysis of stressors and ecosystem services to enhance restoration effectiveness.

With increasing pressure placed on natural systems by growing human populations, both scientists and resource managers need a better understanding of the relationships between cumulative stress from human activities and valued ecosystem services. Societies often seek to mitigate threats to these services through large-scale, costly restoration projects, such as the over one billion dollar Great Lakes Restoration Initiative currently underway. To help inform these efforts, we merged high-resolution spatial analyses of environmental stressors with mapping of ecosystem services for all five Great Lakes. Cumulative ecosystem stress is highest in near-shore habitats, but also extends offshore in Lakes Erie, Ontario, and Michigan. Variation in cumulative stress is driven largely by spatial concordance among multiple stressors, indicating the importance of considering all stressors when planning restoration activities. In addition, highly stressed areas reflect numerous different combinations of stressors rather than a single suite of problems, suggesting that a detailed understanding of the stressors needing alleviation could improve restoration planning. We also find that many important areas for fisheries and recreation are subject to high stress, indicating that ecosystem degradation could be threatening key services. Current restoration efforts have targeted high-stress sites almost exclusively, but generally without knowledge of the full range of stressors affecting these locations or differences among sites in service provisioning. Our results demonstrate that joint spatial analysis of stressors and ecosystem services can provide a critical foundation for maximizing social and ecological benefits from restoration investments.

Metatranscriptomic evidence for co-occurring top-down and bottom-up controls on toxic cyanobacterial communities.

Little is known about the molecular and physiological function of co-occurring microbes within freshwater cyanobacterial harmful algal blooms (cHABs). To address this, community metatranscriptomes collected from the western basin of Lake Erie during August 2012 were examined. Using sequence data, we tested the hypothesis that the activity of the microbial community members is independent of community structure. Predicted metabolic and physiological functional profiles from spatially distinct metatranscriptomes were determined to be ≥90% similar between sites. Targeted analysis of Microcystis aeruginosa, the historical causative agent of cyanobacterial harmful algal blooms over the past ∼20 years, as well as analysis of Planktothrix agardhii and Anabaena cylindrica, revealed ongoing transcription of genes involved in microcystin toxin synthesis as well as the acquisition of both nitrogen and phosphorus, nutrients often implicated as independent bottom-up drivers of eutrophication in aquatic systems. Transcription of genes involved in carbon dioxide (CO2) concentration and metabolism also provided support for the alternate hypothesis that high-pH conditions and dense algal biomass result in CO2-limiting conditions that further favor cyanobacterial dominance. Additionally, the presence of Microcystis-specific cyanophage sequences provided preliminary evidence of possible top-down virus-mediated control of cHAB populations. Overall, these data provide insight into the complex series of constraints associated with Microcystis blooms that dominate the western basin of Lake Erie during summer months, demonstrating that multiple environmental factors work to shape the microbial community.

Rating impacts in a multi-stressor world: a quantitative assessment of 50 stressors affecting the Great Lakes.

Ecosystems often experience multiple environmental stressors simultaneously that can differ widely in their pathways and strengths of impact. Differences in the relative impact of environmental stressors can guide restoration and management prioritization, but few studies have empirically assessed a comprehensive suite of stressors acting on a given ecosystem. To fill this gap in the Laurentian Great Lakes, where considerable restoration investments are currently underway, we used expert elicitation via a detailed online survey to develop ratings of the relative impacts of 50 potential stressors. Highlighting the multiplicity of stressors in this system, experts assessed all 50 stressors as having some impact on ecosystem condition, but ratings differed greatly among stressors. Individual stressors related to invasive and nuisance species (e.g., dreissenid mussels and ballast invasion risk) and climate change were assessed as having the greatest potential impacts. These results mark a shift away from the longstanding emphasis on nonpoint phosphorus and persistent bioaccumulative toxic substances in the Great Lakes. Differences in impact ratings among lakes and ecosystem zones were weak, and experts exhibited surprisingly high levels of agreement on the relative impacts of most stressors. Our results provide a basin-wide, quantitative summary of expert opinion on the present-day influence of all major Great Lakes stressors. The resulting ratings can facilitate prioritizing stressors to achieve management objectives in a given location, as well as providing a baseline for future stressor impact assessments in the Great Lakes and elsewhere.

Microcystin aids in cold temperature acclimation: Differences between a toxic Microcystis wildtype and non-toxic mutant.

For Microcystis aeruginosa PCC 7806, temperature decreases from 26 °C to 19 °C double the microcystin quota per cell during growth in continuous culture. Here we tested whether this increase in microcystin provided M. aeruginosa PCC 7806 with a fitness advantage during colder-temperature growth by comparing cell concentration, cellular physiology, reactive oxygen species damage, and the transcriptomics-inferred metabolism to a non-toxigenic mutant strain M. aeruginosa PCC 7806 ΔmcyB. Photo-physiological data combined with transcriptomic data revealed metabolic changes in the mutant strain during growth at 19 °C, which included increased electron sinks and non-photochemical quenching. Increased gene expression was observed for a glutathione-dependent peroxiredoxin during cold treatment, suggesting compensatory mechanisms to defend against reactive oxygen species are employed in the absence of microcystin in the mutant. Our observations highlight the potential selective advantages of a longer-term defensive strategy in management of oxidative stress (i.e., making microcystin) vs the shorter-term proactive strategy of producing cellular components to actively dissipate or degrade oxidative stress agents.

Cool temperature acclimation in toxigenic Microcystis aeruginosa PCC 7806 and its non-toxigenic mutant.

For Microcystis aeruginosa PCC 7806, temperature decreases from 26° C to 19° C double the microcystin quota per cell during growth in continuous culture. Here we tested whether this increase in microcystin provided M. aeruginosa PCC 7806 with a fitness advantage during colder-temperature growth by comparing cell concentration, cellular physiology, and the transcriptomics-inferred metabolism to a non-toxigenic mutant strain M. aeruginosa PCC 7806 ΔmcyB. Photo-physiological data combined with transcriptomic data revealed metabolic changes in the mutant strain during growth at 19° C, which included increased electron sinks and non-photochemical quenching. Increased gene expression was observed for a glutathione-dependent peroxiredoxin during cold treatment, suggesting compensatory mechanisms to defend against reactive oxygen species are employed in the absence of microcystin in the mutant. Our observations highlight the potential selective advantages of a longer-term defensive strategy in management of oxidative stress (i.e., making microcystin) vs the shorter-term proactive strategy of producing cellular components to actively dissipate or degrade oxidative stress agents.

Are little brown bats (Myotis lucifugus) impacted by dietary exposure to microcystin?✰.

The cyanobacterium, Microcystis aeruginosa, can produce the hepatotoxin microcystin. When toxic M. aeruginosa overwinters in the sediments of lakes, it may be ingested by aquatic insects and bioaccumulate in nymphs of Hexagenia mayflies. When volant Hexagenia emerge from lakes to reproduce, they provide an abundant, albeit temporary, food source for many terrestrial organisms including bats. Little brown bats, Myotis lucifugus, feed opportunistically on aquatic insects including Hexagenia. To determine if microcystin moves from aquatic to terrestrial ecosystems via trophic transfer, we combined a dietary analysis with the quantification of microcystin in bat livers and feces. In June 2014, coincident with the local Hexagenia emergence, bat feces were collected from underneath a maternity roost near Little Traverse Lake (Leelanau County, Michigan, USA). Insects in the diet were identified via molecular analyses of fecal pellets from the roost and from individual bats. Livers and feces were collected from 19 female M. lucifugus, and the concentrations of microcystin in these liver tissues and feces were measured using an enzyme-linked immunosorbent assay (ELISA) and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). We show that the majority of the bats' diets consisted of aquatic insects and that microcystin was detected in high concentrations (up to 129.9 µg/kg dw) in the bat feces by ELISA. Histopathological examination of three bat livers with the highest concentrations of microcystin showed no evidence of phycotoxicosis, indicating that M. lucifugus may not be immediately affected by the ingestion of microcystin. Future work could examine whether bats suffer delayed physiological effects from ingestion of microcystin.

Metagenome-Assembled Genome of a Pseudanabaena sp. from a Crimson Cyanobacterial Bloom in Lake Salubria, New York, USA.

Pseudanabaena spp. are filamentous cyanobacteria widely distributed in temperate lakes. Though infrequent, they can form harmful algal blooms. Here, we present a high-quality metagenome-assembled genome of a Pseudanabaena sp. from a toxic, crimson cyanobacterial bloom in Lake Salubria, NY.

Changes in Microbiome Activity and Sporadic Viral Infection Help Explain Observed Variability in Microcosm Studies.

The environmental conditions experienced by microbial communities are rarely fully simulated in the laboratory. Researchers use experimental containers ("bottles"), where natural samples can be manipulated and evaluated. However, container-based methods are subject to "bottle effects": changes that occur when enclosing the plankton community that are often times unexplained by standard measures like pigment and nutrient concentrations. We noted variability in a short-term, nutrient amendment experiment during a 2019 Lake Erie, Microcystis spp. bloom. We observed changes in heterotrophic bacteria activity (transcription) on a time-frame consistent with a response to experimental changes in nutrient availability, demonstrating how the often overlooked microbiome of cyanobacterial blooms can be altered. Samples processed at the time of collection (T0) contained abundant transcripts from Bacteroidetes, which reduced in abundance during incubation in all bottles, including controls. Significant biological variability in the expression of Microcystis-infecting phage was observed between replicates, with phosphate-amended treatments showing a 10-fold variation. The expression patterns of Microcystis-infecting phage were significantly correlated with ∼35% of Microcystis-specific functional genes and ∼45% of the cellular-metabolites measured across the entire microbial community, suggesting phage activity not only influenced Microcystis dynamics, but the biochemistry of the microbiome. Our observations demonstrate how natural heterogeneity among replicates can be harnessed to provide further insight on virus and host ecology.

Global gene expression profiling in larval zebrafish exposed to microcystin-LR and microcystis reveals endocrine disrupting effects of Cyanobacteria.

Microcystis blooms occur worldwide and threaten aquatic ecosystems and human health. Sublethal effects on early developmental stages of fish are largely unknown, and research has mainly focused on microcystin toxins (such as MC-LR) rather than Microcystis cells. We exposed (96 h) zebrafish larvae to purified MC-LR (0-1000 µg/L) or lyophilized Microcystis aeruginosa containing 4.5 µg/L MC-LR and evaluated changes in global gene expression (Affymetrix GeneChip zebrafish genome arrays). Significant changes in gene expression (≥ 1.7-fold change, p < 0.0001) were determined with Rosetta Resolver 7.0, and ontology analysis was conducted with the DAVID bioinformatics tool. The number of differentially expressed genes relative to control increased with MC-LR concentration and included genes related to known mechanisms of action for MC-LR in mammals and older life stages of fish, as well as genes unique to larval zebrafish. Up-regulation of vitellogenin genes (vtg) (19.2-fold to >100-fold on arrays; 619.3-fold confirmed by quantitative PCR) was observed in Microcystis-exposed larvae but not in larvae exposed to MC-LR. Up-regulation of vtg indicates exposure to estrogenic substance(s) and suggests that Microcystis may be a natural source of environmental estrogens. Concerns about effects of Microcystis blooms may extend beyond those associated with the microcystin toxin.

Dark adaptation and ability of pulse-amplitude modulated (PAM) fluorometry to identify nutrient limitation in the bloom-forming cyanobacterium, Microcystis aeruginosa (Kützing).

Harmful algal blooms in inland waters are widely linked to excess phosphorus (P) loading, but increasing evidence shows that their growth and formation can also be influenced by nitrogen (N) and iron (Fe). Deficiency in N, P, and Fe differentially affects cellular photosystems and is manifested as changes in photosynthetic yield (Fv/Fm). While Fv/Fm has been increasingly used as a rapid and convenient in situ gauge of nutrient deficiency, there are few rigorous comparisons of instrument sensitivity and ability to resolve specific nutrient stresses. This study evaluated the application of Fv/Fm to cyanobacteria using controlled experiments on a single isolate and tested three hypotheses: i) single Fv/Fm measurements taken with different PAM fluorometers can distinguish among limitation by different nutrients, ii) measurements of Fv/Fm made by the addition of DCMU are comparable to PAM fluorometers, and iii) dark adaptation is not necessary for reliable Fv/Fm measurements. We compared Fv/Fm taken from the bloom-forming Microcystis aeruginosa (UTEX LB 3037) grown in nutrient-replete treatment (R) and N-, P-, and Fe-limited treatments (LN, LP, LFe, respectively), using three pulse-amplitude modulated (PAM) fluorometers and the chemical photosynthesis inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), and evaluated the effects of dark adaptation prior to PAM measurement. There were significant differences in Fv/Fm estimates among PAM fluorometers for light- versus dark-adapted cell suspensions over the whole experiment (21 days), which were all significantly higher than the DCMU-based measurements. However, dark adaptation had no effect on Fv/Fm when comparing PAM-based values across a single nutrient treatment. All Fv/Fm methods could distinguish LN and LP from R and LFe treatments but none were able to resolve LFe from R, or LN from LP cultures. These results indicated that for most PAM applications, dark adaptation is not necessary, and furthermore that single measurements of Fv/Fm do not provide a robust measurement of nutrient limitation in Microcystis aeruginosa UTEX LB 3037, and potentially other, common freshwater cyanobacteria.

Metagenome-Assembled Genome Sequences of Raphidiopsis raciborskii and Planktothrix agardhii from a Cyanobacterial Bloom in Kissena Lake, New York, USA.

Raphidiopsis raciborskii and Planktothrix agardhii are filamentous, potentially toxin-producing cyanobacteria that form nuisance blooms in fresh waters. Here, we report high-quality metagenome-assembled genome sequences of R. raciborskii and P. agardhii collected from a bloom in Kissena Lake, New York.

Roles of Nutrient Limitation on Western Lake Erie CyanoHAB Toxin Production.

Cyanobacterial harmful algal bloom (CyanoHAB) proliferation is a global problem impacting ecosystem and human health. Western Lake Erie (WLE) typically endures two highly toxic CyanoHABs during summer: a Microcystis spp. bloom in Maumee Bay that extends throughout the western basin, and a Planktothrix spp. bloom in Sandusky Bay. Recently, the USA and Canada agreed to a 40% phosphorus (P) load reduction to lessen the severity of the WLE blooms. To investigate phosphorus and nitrogen (N) limitation of biomass and toxin production in WLE CyanoHABs, we conducted in situ nutrient addition and 40% dilution microcosm bioassays in June and August 2019. During the June Sandusky Bay bloom, biomass production as well as hepatotoxic microcystin and neurotoxic anatoxin production were N and P co-limited with microcystin production becoming nutrient deplete under 40% dilution. During August, the Maumee Bay bloom produced microcystin under nutrient repletion with slight induced P limitation under 40% dilution, and the Sandusky Bay bloom produced anatoxin under N limitation in both dilution treatments. The results demonstrate the importance of nutrient limitation effects on microcystin and anatoxin production. To properly combat cyanotoxin and cyanobacterial biomass production in WLE, both N and P reduction efforts should be implemented in its watershed.

Possible mechanism for the foodweb transfer of covalently bound microcystins.

Microcystins (MCs) are cyanobacterial toxins that inhibit protein phosphatases 1 and 2A (PP1, PP2A) within an animal through both reversible and covalent interactions. Only MCs that have accumulated in animal tissue in reversible interactions are currently considered when estimating risk to higher trophic levels and humans through food web exposure. However, the majority of MCs is likely covalently bound to target proteins in tissues and these MCs are not quantified or included in these assessments. These covalently bound MCs may be made bioavailable in the digestive system of a consumer through the digestion of their attached protein phosphatase. Three common digestive enzymes, pepsin, chymotrypsin, and trypsin, did not digest cyclic MC-LR and MC-LY, but were very active against a control peptide with typical linkages and standard amino acids in "L" conformation, supporting the possibility for MC-peptide formation during gut passage. To test if digestion products could be biologically active in the consumer, four predicted MC-peptides were synthesized and assayed for activity against PP1 by the protein phosphatase inhibition assay (PPIA). All four MC-peptides were active against PP1 and comparably half (58%) as inhibitory as the parent toxin. This in vitro study demonstrated that MCs covalently bound to proteins may represent a reservoir of potential toxicity for consumers.

Limnological Differences in a Two-Basin Lake Help to Explain the Occurrence of Anatoxin-a, Paralytic Shellfish Poisoning Toxins, and Microcystins.

Chautauqua Lake, New York, is a two-basin lake with a deeper, cooler, and less nutrient-rich Northern Basin, and a warmer, shallower, nutrient-replete Southern Basin. The lake is populated by a complex mixture of cyanobacteria, with toxigenic strains that produce microcystins, anatoxins, and paralytic shellfish poisoning toxins (PSTs). Samples collected from 24 sites were analyzed for these three toxin classes over four years spanning 2014-2017. Concentrations of the three toxin groups varied widely both within and between years. During the study, the mean and median concentrations of microcystins, anatoxin-a, and PSTs were 91 and 4.0 µg/L, 0.62 and 0.33 µg/L, and 32 and 16 µg/L, respectively. Dihydro-anatoxin was only detected once in Chautauqua Lake, while homo-anatoxin was never detected. The Northern Basin had larger basin-wide higher biomass blooms with higher concentrations of toxins relative to the more eutrophied Southern Basin, however blooms in the North Basin were infrequent. Chlorophyll concentrations and toxins in the two basins were correlated with different sets of environmental and physical parameters, suggesting that implementing controls to reduce toxin loads may require applications focused on more than reductions in cyanobacterial bloom density (e.g., reduction of phosphorus inputs), and that lake limnological factors and morphology are important determinants in the selection of an appropriate management strategy. Chautauqua Lake is a drinking water source and is also heavily used for recreation. Drinking water from Chautauqua Lake is unlikely to be a significant source of exposure to cyanotoxins due to the location of the intakes in the deeper North Basin, where there were generally low concentrations of toxins in open water; however, toxin levels in many blooms exceeded the US Environmental Protection Agency's recreational guidelines for exposure to cyanotoxins. Current cyanotoxin monitoring in Chautauqua Lake is focused on microcystins. However, the occurrence of blooms containing neurotoxic cyanotoxins in the absence of the microcystins indicates this restricted monitoring may not be sufficient when aiming to protect against exposure to cyanotoxins. The lake has a large number of tourist visitors; thus, special care should be taken to prevent recreational exposure within this group.

Molecular and morphological characterization of a novel dihydroanatoxin-a producing Microcoleus species (cyanobacteria) from the Russian River, California, USA.

Reports of anatoxins poisoning of wildlife and domestic animals by toxigenic cyanobacteria in streams and rivers are increasing globally. Little is known about the taxonomy, morphology and genomics of anatoxins producing species, limiting our knowledge about their environmental preferences. We isolated three benthic non-heterocystous filamentous cyanobacterial strains from the Russian River in Northern California (USA), which produce anatoxin-a and dihydroanatoxin-a. Both 16S rRNA and protein sequence phylogenetic analyses showed that the strains represent a distinct new member of the cyanobacterial genus Microcoleus (Oscillatoriales). A novel species, Microcoleus anatoxicus is described and accompanied with light microscope photomicrographs, toxin profiles and the complete anatoxin-a gene cassette with the first description of the anaK gene in Microcoleus.