Positive Impacts of Aphanizomenon Flos Aquae Extract on Obesity-Related Dysmetabolism in Mice with Diet-Induced Obesity.

The present study evaluated the ability of KlamExtra®, an Aphanizomenon flos aquae (AFA) extract, to counteract metabolic dysfunctions due to a high fat diet (HFD) or to accelerate their reversion induced by switching an HFD to a normocaloric diet in mice with diet-induced obesity. A group of HFD mice was fed with an HFD supplemented with AFA (HFD-AFA) and another one was fed with regular chow (standard diet-STD) alone or supplemented with AFA (STD-AFA). AFA was able to significantly reduce body weight, hypertriglyceridemia, liver fat accumulation and adipocyte size in HFD mice. AFA also reduced hyperglycaemia, insulinaemia, HOMA-IR and ameliorated the glucose tolerance and the insulin response of obese mice. Furthermore, in obese mice AFA normalised the gene and the protein expression of factors involved in lipid metabolism (FAS, PPAR-γ, SREBP-1c and FAT-P mRNA), inflammation (TNF-α and IL-6 mRNA, NFkB and IL-10 proteins) and oxidative stress (ROS levels and SOD activity). Interestingly, AFA accelerated the STD-induced reversion of glucose dysmetabolism, hepatic and VAT inflammation and oxidative stress. In conclusion, AFA supplementation prevents HFD-induced dysmetabolism and accelerates the STD-dependent recovery of glucose dysmetabolism by positively modulating oxidative stress, inflammation and the expression of the genes linked to lipid metabolism.

Transcriptionally active nitrogen fixation and biosynthesis of diverse secondary metabolites by Dolichospermum and Aphanizomenon-like Cyanobacteria in western Lake Erie Microcystis blooms.

Cyanobacterial harmful algal blooms (cyanoHABs) in the western basin of Lake Erie are dominated by microcystin producing Microcystis spp., but other cyanobacterial taxa that coexist in these communities may play important roles in production of toxins and shaping bloom dynamics and community function. In this study, we used metagenomic and metatranscriptomic data from the 2014 western Lake Erie cyanoHAB to explore the genetic diversity and biosynthetic potential of cyanobacteria belonging to the Anabaena, Dolichospermum, Aphanizomenon (ADA) clade. We reconstructed two near-complete metagenome-assembled genomes from two distinct ADA clade species, each containing biosynthetic gene clusters that encode novel and known secondary metabolites, including those with toxic and/or known taste and odor properties, that were transcriptionally active. However, neither ADA metagenome-assembled genome contained genes encoding guanitoxins, anatoxins, or saxitoxins, which are known to be produced by ADA. The ADA cyanobacteria accounted for most of the metagenomic and metatranscriptomic reads from nitrogen fixation genes, suggesting they were the dominant N-fixers at the times and stations sampled. Despite their relatively low abundance, our results highlight the possibility that ADA taxa could influence the water quality and ecology of Microcystis blooms, although the extent of these impacts remains to be quantified.

Biomarkers in bivalve mollusks and amphipods for assessment of effects linked to cyanobacteria and elodea: Mesocosm study.

The effects of cyanobacteria (Aphanizomenon flos-aquae (90%), Microcystis aeruginosa) and dense Elodea canadensis beds on the health endpoints of the amphipod Gmelinoides fasciatus and bivalve mollusc Unio pictorum were examined in mesocosms with simulated summer conditions (July-August 2018) in the environment of the Rybinsk Reservoir (Volga River Basin, Russia). Four treatments were conducted, including one control and three treatments with influencing factors, cyanobacteria and dense elodea beds (separately and combined). After 20 days of exposure, we evaluated the frequency of malformed and dead embryos in amphipods, heart rate (HR) and its recovery (HRR) after stress tests in molluscs as well as heat tolerance (critical thermal maximum or CTMax) in both amphipods and molluscs. The significant effect, such as elevated number of malformed embryos, was recorded after exposure with cyanobacteria (separately and combined with elodea) and presence of microcystins (MC) in water (0.17 µg/l, 40% of the most toxic MC-LR contribution). This study provided evidence that an elevated number (>5% of the total number per female) of malformed embryos in amphipods showed noticeable toxicity effects in the presence of cyanobacteria. The decreased oxygen under the influence of dense elodea beds led to a decrease in HR (and an increase in HRR) in molluscs. The notable effects on all studied biomarkers, embryo malformation frequency and heat tolerance in the amphipod G. fasciatus, as well as the heat tolerance and heart rate in the mollusc U. pictorum, were found when both factors (elodea and cyanobacteria) were combined. The applied endpoints could be further developed for environmental monitoring, but the obtained results support the importance of the combined use of several biomarkers and species, especially in the case of multi-factor environmental stress.

In Vitro Toxicological Screening of Stable and Senescing Cultures of Aphanizomenon, Planktothrix, and Raphidiopsis.

Toxicity of cyanobacteria is the subject of ongoing research, and a number of toxic metabolites have been described, their biosynthesis pathways have been elucidated, and the mechanism of their action has been established. However, several knowledge gaps still exist, e.g., some strains produce hitherto unknown toxic compounds, while the exact dynamics of exerted toxicity during cyanobacterial growth still requires further exploration. Therefore, the present study investigated the toxicity of extracts of nine freshwater strains of Aphanizomenon gracile, an Aphanizomenon sp. strain isolated from the Baltic Sea, a freshwater strain of Planktothrix agardhii, and two strains of Raphidiopsis raciborskii obtained from 25- and 70-day-old cultures. An in vitro experimental model based on Cyprinus carpio hepatocytes (oxidative stress markers, DNA fragmentation, and serine/threonine protein activity) and brain homogenate (cholinesterase activity) was employed. The studied extracts demonstrated toxicity to fish cells, and in general, all examined extracts altered at least one or more of considered parameters, indicating that they possess, to some degree, toxic potency. Although the time from which the extracts were obtained had a significant importance for the response of fish cells, we observed strong variability between the different strains and species. In some strains, extracts that originated from 25-day-old cultures triggered more harmful effects on fish cells compared to those obtained from 70-day-old cultures, whereas in other strains, we observed the opposite effect or a lack of a significant change. Our study revealed that there was no clear or common pattern regarding the degree of cyanobacterial bloom toxicity at a given stage of development. This means that young cyanobacterial blooms that are just forming can pose an equally toxic threat to aquatic vertebrates and ecosystem functioning as those that are stable or old with a tendency to collapse. This might be largely due to a high variability of strains in the bloom.

Phylogenomic Analysis of Secondary Metabolism in the Toxic Cyanobacterial Genera Anabaena, Dolichospermum and Aphanizomenon.

Cyanobacteria produce an array of toxins that pose serious health risks to humans and animals. The closely related diazotrophic genera, Anabaena, Dolichospermum, and Aphanizomenon, frequently form poisonous blooms in lakes and brackish waters around the world. These genera form a complex now termed the Anabaena, Dolichospermum, and Aphanizomenon (ADA) clade and produce a greater array of toxins than any other cyanobacteria group. However, taxonomic confusion masks the distribution of toxin biosynthetic pathways in cyanobacteria. Here we obtained 11 new draft genomes to improve the understanding of toxin production in these genera. Comparison of secondary metabolite pathways in all available 31 genomes for these three genera suggests that the ability to produce microcystin, anatoxin-a, and saxitoxin is associated with specific subgroups. Each toxin gene cluster was concentrated or even limited to a certain subgroup within the ADA clade. Our results indicate that members of the ADA clade encode a variety of secondary metabolites following the phylogenetic clustering of constituent species. The newly sequenced members of the ADA clade show that phylogenetic separation of planktonic Dolichospermum and benthic Anabaena is not complete. This underscores the importance of taxonomic revision of Anabaena, Dolichospermum, and Aphanizomenon genera to reflect current phylogenomic understanding.

Nucleoside 2',3'-Cyclic Monophosphates in Aphanizomenon flos-aquae Detected through Nuclear Magnetic Resonance and Mass Spectrometry.

Aphanizomenon flos-aquae (AFA) cyanobacteria from Klamath Lake (Oregon) are considered a "superfood" due to their broad nutritional profile that has proved to have health-enhancing properties. The AFA metabolome is quite complex. Here, we present a study that, combining multinuclear 1H, 31P, and 13C Nuclear Magnetic Resonance (NMR) spectroscopy and high-resolution mass spectrometry, led to the detection of uncommon phosphorylated metabolites in AFA. We focused our attention on 31P NMR signals at 20 ppm, a chemical shift that usually points to the presence of phosphonates. The molecules contributing to 20 ppm 31P NMR signals revealed, instead, to be nucleoside 2',3'-cyclic monophosphates. These metabolites were fully characterized by multinuclear 1H, 31P, and 13C NMR spectroscopy and high-resolution mass spectrometry.

Effects of typical algae species (Aphanizomenon flosaquae and Microcystis aeruginosa) on photoreduction of Hg2+ in water body.

Photoreduction characteristics of divalent inorganic mercury (Hg2+) in the presence of specific algae species are still not well known. Laboratory experiments were conducted in the present study to identify the effects of different concentrations of living/dead algae species, including Aphanizomenon flosaquae (AF) and Microcystis aeruginosa (MA), on the photoreduction rate of Hg2+ under various light conditions. The experimental results showed that percentage reduction of Hg2+ was significantly influenced by radiation wavelengths, and dramatically decreased with the presence of algae. The highest percentage reduction of Hg2+ was induced by UV-A, followed by UV-B, visible light and dark for both living and dead AF, and the order was dark > UV-A > UV-B > visible light for both living and dead MA. There were two aspects, i.e., energy and attenuation rate of light radiation and excrementitious generated from algae metabolisms, were involved in the processes of Hg2+ photoreduction with the presence of algae under different light conditions. The percentage reduction of Hg2+ decreased from 15% to 11% when living and dead AF concentrations increased by 10 times (from 106 to 105 cells/mL), and decreased from11% to ~9% in the case of living and dead MA increased. Algae can adsorb Hg2+ and decrease the concentration of free Hg2+, thus inhibiting Hg2+ photoreduction, especially under the conditions with high concentrations of algae. No significant differences were found in percentage reduction of Hg2+ between living and dead treatments of algae species. The results are of great importance for understanding the role of algae in Hg2+ photoreduction.

Phosphate availability affects fixed nitrogen transfer from diazotrophs to their epibionts.

Dinitrogen (N2) fixation is a major source of external nitrogen (N) to aquatic ecosystems and therefore exerts control over productivity. Studies have shown that N2 -fixers release freshly fixed N into the environment, but the causes for this N release are largely unclear. Here, we show that the availability of phosphate can directly affect the transfer of freshly fixed N to epibionts in filamentous, diazotrophic cyanobacteria. Stable-isotope incubations coupled to single-cell analyses showed that <1% and ~15% of freshly fixed N was transferred to epibionts of Aphanizomenon and Nodularia, respectively, at phosphate scarcity during a summer bloom in the Baltic Sea. When phosphate was added, the transfer of freshly fixed N to epibionts dropped to about half for Nodularia, whereas the release from Aphanizomenon increased slightly. At the same time, the growth rate of Nodularia roughly doubled, indicating that less freshly fixed N was released and was used for biomass production instead. Phosphate scarcity and the resulting release of freshly fixed N could explain the heavy colonization of Nodularia filaments by microorganisms during summer blooms. As such, the availability of phosphate may directly affect the partitioning of fixed N2 in colonies of diazotrophic cyanobacteria and may impact the interactions with their microbiome.

In vitro antioxidant, anti-glycation and immunomodulation activities of fermented blue-green algae Aphanizomenon flos-aquae.

To clarify the antioxidant, anti-glycation and immunomodulatory capacities of fermented blue-green algae Aphanizomenon flos-aquae (AFA), hot aqueous extract suspensions made from 10% AFA were fermented by Lactobacillus plantarum AN7 and Lactococcus lactis subsp. lactis Kushiro-L2 strains isolated from a coastal region of Japan. The DPPH and O2- radical scavenging capacities and Fe-reducing power were increased in the fermented AFA. The increased DPPH radical scavenging capacity of the fermented AFA was fractionated to mainly < 3 kDa and 30-100 kDa. The increased O2- radical scavenging capacities were fractionated to mainly < 3 kDa. Anti-glycation activity in BSA-fructose model rather than BSA-methylglyoxal model was increased by the fermentation. The increased anti-glycation activity was fractionated to mainly 30-100 kDa. The NO concentration in the murine macrophage RAW264.7 culture media was high with the fermented AFA. The increased immunomodulation capacity was also fractionated to mainly 30-100 kDa. These results suggest that the fermented AFA is a more useful material for health foods and supplements.

Single-cell imaging of phosphorus uptake shows that key harmful algae rely on different phosphorus sources for growth.

Single-cell measurements of biochemical processes have advanced our understanding of cellular physiology in individual microbes and microbial populations. Due to methodological limitations, little is known about single-cell phosphorus (P) uptake and its importance for microbial growth within mixed field populations. Here, we developed a nanometer-scale secondary ion mass spectrometry (nanoSIMS)-based approach to quantify single-cell P uptake in combination with cellular CO2 and N2 fixation. Applying this approach during a harmful algal bloom (HAB), we found that the toxin-producer Nodularia almost exclusively used phosphate for growth at very low phosphate concentrations in the Baltic Sea. In contrast, the non-toxic Aphanizomenon acquired only 15% of its cellular P-demand from phosphate and ~85% from organic P. When phosphate concentrations were raised, Nodularia thrived indicating that this toxin-producer directly benefits from phosphate inputs. The phosphate availability in the Baltic Sea is projected to rise and therefore might foster more frequent and intense Nodularia blooms with a concomitant rise in the overall toxicity of HABs in the Baltic Sea. With a projected increase in HABs worldwide, the capability to use organic P may be a critical factor that not only determines the microbial community structure, but the overall harmfulness and associated costs of algal blooms.

Influence of Glycine and Arginine on Cylindrospermopsin Production and aoa Gene Expression in Aphanizomenon ovalisporum.

Arginine (Arg) and glycine (Gly) seem to be the only substrates accepted by the amidinotransferase that catalyze the first step of the synthesis pathway of the cyanotoxin cylindrospermopsin (CYN), leading to guanidinoacetate (GAA). Here, the effect of these amino acids on the production of CYN in cultures of the cylindrospermopsin-producing strain, Aphanizomenon ovalisporum UAM-MAO, has been studied. Arg clearly increased CYN content, the increment appearing triphasic along the culture. On the contrary, Gly caused a decrease of CYN, observable from the first day on. Interestingly, the transcript of the gene ntcA, key in nitrogen metabolism control, was also enhanced in the presence of Arg and/or Gly, the trend of the transcript oscillations being like that of aoa/cyr. The inhibitory effect of Gly in CYN production seems not to result from diminishing the activity of genes considered involved in CYN synthesis, since Gly, as Arg, enhance the transcription of genes aoaA-C and cyrJ. On the other hand, culture growth is affected by Arg and Gly in a similar way to CYN production, with Arg stimulating and Gly impairing it. Taken together, our data show that the influence of both Arg and Gly on CYN changes seems not to be due to a specific effect on the first step of CYN synthesis; it rather appears to be the result of changes in the physiological cell status.

Temperature Influences the Production and Transport of Saxitoxin and the Expression of sxt Genes in the Cyanobacterium Aphanizomenon gracile.

The cyanobacterium Aphanizomenon gracile is the most widely distributed producer of the potent neurotoxin saxitoxin in freshwaters. In this work, total and extracellular saxitoxin and the transcriptional response of three genes linked to saxitoxin biosynthesis (sxtA) and transport (sxtM, sxtPer) were assessed in Aphanizomenon gracile UAM529 cultures under temperatures covering its annual cycle (12 °C, 23 °C, and 30 °C). Temperature influenced saxitoxin production being maximum at high temperatures (30 °C) above the growth optimum (23 °C), concurring with a 4.3-fold increased sxtA expression at 30 °C. Extracellular saxitoxin transport was temperature-dependent, with maxima at extremes of temperature (12 °C with 16.9% extracellular saxitoxin; and especially 30 °C with 53.8%) outside the growth optimum (23 °C), coinciding with a clear upregulation of sxtM at both 12 °C and 30 °C (3.8-4.1 fold respectively), and yet with just a slight upregulation of sxtPer at 30 °C (2.1-fold). Nitrate depletion also induced a high extracellular saxitoxin release (51.2%), although without variations of sxtM and sxtPer transcription, and showing evidence of membrane damage. This is the first study analysing the transcriptional response of sxtPer under environmental gradients, as well as the effect of temperature on putative saxitoxin transporters (sxtM and sxtPer) in cyanobacteria in general.

Dolichospermum and Aphanizomenon as neurotoxins producers in some Russian freshwaters.

Last decades, cyanobacterial blooms have been commonly reported in Russia. Among the boom-forming species, potential toxin producers have been identified. The aim of this paper was to study the presence of neurotoxic compounds - saxitoxins and anatoxin-a - in water bodies from different regions of Russia. We also made attempts to identify the neurotoxin-producing genera. The good convergence of the results obtained by light microscopy, PCR and LC-MS/MS analyses indicated the presence of active neurotoxin producing species in all investigated water bodies. Saxitoxin was detected in phytoplankton from 4 water bodies in Central European Russia and West Siberia, including lake and reservoirs used as a source for potable water. The water bodies differed with the respect of saxitoxin producers which belonged to Aphanizomenon and/or Dolichospermum genera. For the first time, we obtained quantitative data on the intracellular saxitoxin concentration in Russian freshwaters using LC-MS/MS. Anatoxin-a was detected only in lakes of Northwestern Russia. In the eutrophic shallow Lower Suzdal Lake, Aphanizomenon was the stated anatoxin-a-producing genus. In the large shallow artificial hypertrophic Sestroretskij Razliv Lake, it was very likely that both dominant species - Aphanizomenon flos-aquae and Dolichospermum planctonicum - were anatoxin-a producers.

Dietary l-carnitine prevents histopathological changes in tilapia (Oreochromis Niloticus) exposed to cylindrospermopsin.

Cylindrospermopsin (CYN) is a cytotoxin highly water-soluble, which is easily taken up by several aquatic organisms. CYN acts as a potent protein and glutathione synthesis inhibitor, as well as inducing genotoxicity, oxidative stress, and histopathological alterations. This is the first study reporting the protective effect of a l-carnitine (LC) pretreatment (400 or 880 mg LC/kg bw fish/day, for 21 days) on the histopathological alterations induced by pure CYN or Aphanizomenon ovalisporum lyophilized cells (400 µg CYN/kg bw fish) in liver, kidney, heart, intestines, and gills of tilapia (Oreochromis niloticus) acutely exposed to the toxin by oral route. The main histopathological changes induced by CYN were disorganized parenchyma with presence of glycogen and lipids in the cytoplasm (liver), glomerulonephritis, glomerular atrophy, and dilatation of Bowman's capsule (kidney), myofibrolysis, loss of myofibrils, with edema and hemorrhage (heart), intestinal villi with necrotic enterocytes and partial loss of microvilli (gastrointestinal tract), and hyperemia and hemorrhage (gills). LC pretreatment was able to totally prevent those CYN-induced alterations from 400 mg LC/kg bw fish/day in almost all organs, except in the heart, where 880 mg LC/kg bw fish/day were needed. In addition, the morphometric study indicated that LC managed to recover totally the affectation in the cross sections of the proximal and distal convoluted tubules in CYN-exposed fish. © 2015 Wiley Periodicals, Inc. Environ Toxicol 32: 241-254, 2017.

Physiological Responses of Aphanizomenon flos-aquae Under the Stress of Sagittaria sagittifolia Extract.

The algal growth and physiological characters of Aphanizomenon flos-aquae were studied under the stress of Sagittaria sagittifolia extract. The results showed that the growth of A. flos-aquae was significantly inhibited by S. sagittifolia extract. The exopolysaccharide (EPS), total soluble protein, intracellular phosphorus (o-PO4-P) contents and malondialdehyde (MDA) contents in A. flos-aquae cells increased significantly. These results suggested that A. flos-aquae can adapt to stress by increasing its normal metabolic activity. The algal cellular antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD), were triggered to different degrees when exposed to S. sagittifolia extract. The MDA contents and activities of SOD, CAT and POD in algal cells suggested that oxidative damage induced by S. sagittifolia extract via the oxidation of ROS (O2·-) might be an important factor responsible for the inhibition of the growth of A. flos-aquae. In addition, SOD may be an important site for the inhibition of S. sagittifolia extract on A. flos-aquae cells. These results indicate that S. sagittifolia may be a good candidate for controlling A. flos-aquae blooms.

Cell-specific nitrogen- and carbon-fixation of cyanobacteria in a temperate marine system (Baltic Sea).

We analysed N2 - and carbon (C) fixation in individual cells of Baltic Sea cyanobacteria by combining stable isotope incubations with secondary ion mass spectrometry (SIMS). Specific growth rates based on N2 - and C-fixation were higher for cells of Dolichospermum spp. than for Aphanizomenon sp. and Nodularia spumigena. The cyanobacterial biomass, however, was dominated by Aphanizomenon sp., which contributed most to total N2 -fixation in surface waters of the Northern Baltic Proper. N2 -fixation by Pseudanabaena sp. and colonial picocyanobacteria was not detectable. N2 -fixation by Aphanizomenon sp., Dolichospermum spp. and N. spumigena populations summed up to total N2 -fixation, thus these genera appeared as sole diazotrophs within the Baltic Sea's euphotic zone, while their mean contribution to total C-fixation was 21%. Intriguingly, cell-specific N2 -fixation was eightfold higher at a coastal station compared to an offshore station, revealing coastal zones as habitats with substantial N2 -fixation. At the coastal station, the cell-specific C- to N2 -fixation ratio was below the cellular C:N ratio, i.e. N2 was assimilated in excess to C-fixation, whereas the C- to N2 -fixation ratio exceeded the C:N ratio in offshore sampled diazotrophs. Our findings highlight SIMS as a powerful tool not only for qualitative but also for quantitative N2 -fixation assays in aquatic environments.

In vivo effects of Aphanizomenon flos-aquae DC-1 aphantoxins on gas exchange and ion equilibrium in the zebrafish gill.

Aphantoxins, neurotoxins or paralytic shellfish poisons (PSPs) generated by Aphanizomenon flos-aquae, are a threat to environmental safety and human health in eutrophic waters worldwide. The molecular mechanisms of neurotoxin function have been studied; however, the effects of these neurotoxins on oxidative stress, ion transport, gas exchange, and branchial ultrastructure in fish gills are not fully understood. Aphantoxins extracted from A. flos-aquae DC-1 were detected by high-performance liquid chromatography. The major ingredients were gonyautoxins 1 and 5 and neosaxitoxin, which comprised 34.04%, 21.28%, and 12.77% of the total, respectively. Zebrafish (Danio rerio) were administered A. flos-aquae DC-1 aphantoxins at 5.3 or 7.61µg saxitoxin equivalents (eq)/kg (low and high doses, respectively) by intraperitoneal injection. The activities of Na(+)-K(+)-ATPase (NKA), carbonic anhydrase (CA), and lactate dehydrogenase (LDH), ultrastructural alterations in chloride and epithelial cells, and reactive oxygen species (ROS) and total antioxidative capacity (T-AOC) were investigated in the gills during the first 24h after exposure. Aphantoxins significantly increased the level of ROS and decreased the T-AOC in zebrafish gills from 3 to 12h post-exposure, suggesting an induction of oxidative stress and inhibition of antioxidant capacity. Reduced activities of NKA and CA demonstrated abnormal ion transport and gas exchange in the gills of aphantoxin-treated fish. Toxin administration also resulted in increased LDH activity and ultrastructural alterations in chloride and epithelial cells, suggesting a disruption of function and structure in zebrafish gills. The observed abnormalities in zebrafish gills occurred in a time- and dose-dependent manner. These findings demonstrate that aphantoxins or PSPs may inhibit ion transport and gas exchange, increase LDH activity, and result in ultrastructural damage to the gills through elevations in oxidative stress and reduced antioxidant capacity. These effects of aphantoxins in the gills of zebrafish suggest an induction of respiratory toxicity. The parameters investigated in this study may be also considered as biomarkers for studying aphantoxin/PSP exposure and cyanobacterial blooms in nature.

A review of the phylogeny, ecology and toxin production of bloom-forming Aphanizomenon spp. and related species within the Nostocales (cyanobacteria).

The traditional genus Aphanizomenon comprises a group of filamentous nitrogen-fixing cyanobacteria of which several memebers are able to develop blooms and to produce toxic metabolites (cyanotoxins), including hepatotoxins (microcystins), neurotoxins (anatoxins and saxitoxins) and cytotoxins (cylindrospermopsin). This genus, representing geographically widespread and extensively studied cyanobacteria, is in fact heterogeneous and composed of at least five phylogenetically distant groups (Aphanizomenon, Anabaena/Aphanizomenon like cluster A, Cuspidothrix, Sphaerospermopsis and Chrysosporum) whose taxonomy is still under revision. This review provides a thorough insight into the phylogeny, ecology, biogeography and toxicogenomics (cyr, sxt, and ana genes) of the five best documented "Aphanizomenon" species with special relevance for water risk assessment: Aphanizomenon flos-aquae, Aphanizomenon gracile, Cuspidothrix issatschenkoi, Sphaerospermopsis aphanizomenoides and Chrysosporum ovalisporum. Aph. flos-aquae, Aph. gracile and C. issatschenkoi have been reported from temperate areas only whereas S. aphanizomenoides shows the widest distribution from the tropics to temperate areas. Ch. ovalisporum is found in tropical, subtropical and Mediterranean areas. While all five species show moderate growth rates (0.1-0.4day-1) within a wide range of temperatures (15-30°C), Aph. gracile and A. flos-aquae can grow from around (or below) 10°C, whereas Ch. ovalisporum and S. aphanizomenoides are much better competitors at high temperatures over 30°C or even close to 35°C. A. gracile has been confirmed as the producer of saxitoxins and cylindrospermopsin, C. issatschenkoi of anatoxins and saxitoxins and Ch. ovalisporum of cylindrospermopsin. The suspected cylindrospermopsin or anatoxin-a production of A. flos-aquae or microcystin production of S. aphanizomenoides is still uncertain. This review includes a critical discussion on the the reliability of toxicity reports and on the invasive potential of "Aphanizomenon" species in a climate change scenario, together with derived knowledge gaps and research needs. As a whole, this work is intended to represent a key reference for scientists and water managers involved in the major challenges of identifying, preventing and mitigating toxic Aphanizomenon blooms.

N2-fixation, ammonium release and N-transfer to the microbial and classical food web within a plankton community.

We investigated the role of N2-fixation by the colony-forming cyanobacterium, Aphanizomenon spp., for the plankton community and N-budget of the N-limited Baltic Sea during summer by using stable isotope tracers combined with novel secondary ion mass spectrometry, conventional mass spectrometry and nutrient analysis. When incubated with (15)N2, Aphanizomenon spp. showed a strong (15)N-enrichment implying substantial (15)N2-fixation. Intriguingly, Aphanizomenon did not assimilate tracers of (15)NH4(+) from the surrounding water. These findings are in line with model calculations that confirmed a negligible N-source by diffusion-limited NH4(+) fluxes to Aphanizomenon colonies at low bulk concentrations (<250 nm) as compared with N2-fixation within colonies. No N2-fixation was detected in autotrophic microorganisms <5 µm, which relied on NH4(+) uptake from the surrounding water. Aphanizomenon released about 50% of its newly fixed N2 as NH4(+). However, NH4(+) did not accumulate in the water but was transferred to heterotrophic and autotrophic microorganisms as well as to diatoms (Chaetoceros sp.) and copepods with a turnover time of ~5 h. We provide direct quantitative evidence that colony-forming Aphanizomenon releases about half of its recently fixed N2 as NH4(+), which is transferred to the prokaryotic and eukaryotic plankton forming the basis of the food web in the plankton community. Transfer of newly fixed nitrogen to diatoms and copepods furthermore implies a fast export to shallow sediments via fast-sinking fecal pellets and aggregates. Hence, N2-fixing colony-forming cyanobacteria can have profound impact on ecosystem productivity and biogeochemical processes at shorter time scales (hours to days) than previously thought.

Effects of Cylindrospermopsin Producing Cyanobacterium and Its Crude Extracts on a Benthic Green Alga-Competition or Allelopathy?

Cylindrospermopsin (CYN) is a toxic secondary metabolite produced by filamentous cyanobacteria which could work as an allelopathic substance, although its ecological role in cyanobacterial-algal assemblages is mostly unclear. The competition between the CYN-producing cyanobacterium Chrysosporum (Aphanizomenon) ovalisporum, and the benthic green alga Chlorococcum sp. was investigated in mixed cultures, and the effects of CYN-containing cyanobacterial crude extract on Chlorococcum sp. were tested by treatments with crude extracts containing total cell debris, and with cell debris free crude extracts, modelling the collapse of a cyanobacterial water bloom. The growth inhibition of Chlorococcum sp. increased with the increasing ratio of the cyanobacterium in mixed cultures (inhibition ranged from 26% to 87% compared to control). Interestingly, inhibition of the cyanobacterium growth also occurred in mixed cultures, and it was more pronounced than it was expected. The inhibitory effects of cyanobacterial crude extracts on Chlorococcum cultures were concentration-dependent. The presence of C. ovalisporum in mixed cultures did not cause significant differences in nutrient content compared to Chlorococcum control culture, so the growth inhibition of the green alga could be linked to the presence of CYN and/or other bioactive compounds.