Relation between the relative abundance and collapse of Aphanizomenon flos-aquae and microbial antagonism in Upper Klamath Lake, Oregon.

Aphanizomenon flos-aquae (AFA) is the dominant filamentous cyanobacterium that develops into blooms in Upper Klamath Lake, Oregon, each year. During AFA bloom and collapse, ecosystem conditions for endangered Lost River and shortnose suckers deteriorate, thus motivating the need to identify processes that limit AFA abundance and decline. Here, we investigate the relations between AFA and other members of the microbial community (photosynthetic and nonphotosynthetic bacteria and archaea), how those relations impact abundance and collapse of AFA, and the types of microbial conditions that suppress AFA. We found significant spatial variation in AFA relative abundance during the 2016 bloom period using 16S rRNA sequencing. The Pelican Marina site had the lowest AFA relative abundance, and this was coincident with increased relative abundance of Candidatus Sericytochromatia, Flavobacterium, and Rheinheimera, some of which are known AFA antagonists. The AFA collapse coincided with phosphorus limitation relative to nitrogen and the increased relative abundance of Cyanobium and Candidatus Sericytochromatia, which outcompete AFA when dissolved inorganic nitrogen is available. The data collected in this study indicate the importance of dissolved inorganic nitrogen combined with microbial community structure in suppressing AFA abundance.

Aphanizomenon flos-aquae (AFA) Extract Prevents Neurodegeneration in the HFD Mouse Model by Modulating Astrocytes and Microglia Activation.

Obesity and related metabolic dysfunctions are associated with neurodegenerative diseases, such as Alzheimer's disease. Aphanizomenon flos-aquae (AFA) is a cyanobacterium considered a suitable supplement for its nutritional profile and beneficial properties. The potential neuroprotective effect of an AFA extract, commercialized as KlamExtra®, including the two AFA extracts Klamin® and AphaMax®, in High-Fat Diet (HFD)-fed mice was explored. Three groups of mice were provided with a standard diet (Lean), HFD or HFD supplemented with AFA extract (HFD + AFA) for 28 weeks. Metabolic parameters, brain insulin resistance, expression of apoptosis biomarkers, modulation of astrocytes and microglia activation markers, and Aß deposition were analyzed and compared in the brains of different groups. AFA extract treatment attenuated HFD-induced neurodegeneration by reducing insulin resistance and loss of neurons. AFA supplementation improved the expression of synaptic proteins and reduced the HFD-induced astrocytes and microglia activation, and Aß plaques accumulation. Together, these outcomes indicate that regular intake of AFA extract could benefit the metabolic and neuronal dysfunction caused by HFD, decreasing neuroinflammation and promoting Aß plaques clearance.

Spasmolytic Effects of Aphanizomenon Flos Aquae (AFA) Extract on the Human Colon Contractility.

The blue-green algae Aphanizomenon flos aquae (AFA), rich in beneficial nutrients, exerts various beneficial effects, acting in different organs including the gut. Klamin® is an AFA extract particularly rich in ß-PEA, a trace-amine considered a neuromodulator in the central nervous system. To date, it is not clear if ß-PEA exerts a role in the enteric nervous system. The aims of the present study were to investigate the effects induced by Klamin® on the human distal colon mechanical activity, to analyze the mechanism of action, and to verify a ß-PEA involvement. The organ bath technique, RT-PCR, and immunohistochemistry (IHC) were used. Klamin® reduced, in a concentration-dependent manner, the amplitude of the spontaneous contractions. EPPTB, a trace-amine receptor (TAAR1) antagonist, significantly antagonized the inhibitory effects of both Klamin® and exogenous ß-PEA, suggesting a trace-amine involvement in the Klamin® effects. Accordingly, AphaMax®, an AFA extract containing lesser amount of ß-PEA, failed to modify colon contractility. Moreover, the Klamin® effects were abolished by tetrodotoxin, a neural blocker, but not by L-NAME, a nitric oxide-synthase inhibitor. On the contrary methysergide, a serotonin receptor antagonist, significantly antagonized the Klamin® effects, as well as the contractility reduction induced by 5-HT. The RT-PCR analysis revealed TAAR1 gene expression in the colon and the IHC experiments showed that 5-HT-positive neurons are co-expressed with TAAR1 positive neurons. In conclusion, the results of this study suggest that Klamin® exerts spasmolytic effects in human colon contractility through ß-PEA, that, by activating neural TAAR1, induce serotonin release from serotoninergic neurons of the myenteric plexus.

HEPN-MNT Toxin-Antitoxin System: The HEPN Ribonuclease Is Neutralized by OligoAMPylation.

Prokaryotic toxin-antitoxin (TA) systems are composed of a toxin capable of interfering with key cellular processes and its neutralizing antidote, the antitoxin. Here, we focus on the HEPN-MNT TA system encoded in the vicinity of a subtype I-D CRISPR-Cas system in the cyanobacterium Aphanizomenon flos-aquae. We show that HEPN acts as a toxic RNase, which cleaves off 4 nt from the 3' end in a subset of tRNAs, thereby interfering with translation. Surprisingly, we find that the MNT (minimal nucleotidyltransferase) antitoxin inhibits HEPN RNase through covalent di-AMPylation (diadenylylation) of a conserved tyrosine residue, Y109, in the active site loop. Furthermore, we present crystallographic snapshots of the di-AMPylation reaction at different stages that explain the mechanism of HEPN RNase inactivation. Finally, we propose that the HEPN-MNT system functions as a cellular ATP sensor that monitors ATP homeostasis and, at low ATP levels, releases active HEPN toxin.

Physiological and morphological response of Aphanizomenon flos-aquae to watermelon (Citrullus lanatus) peel aqueous extract.

Natural algaecides are more likely to be specific and biodegradable, and may offer an environmentally friendly method for control of cyanobacterial blooms. We explored, for the first time, the potential for watermelon peel aqueous extract (WMPAE) to control the growth of the harmful blue-green alga Aphanizomenon flos-aquae. The growth inhibition and several physiological parameters of A. flos-aquae, in response to WMPAE, were analyzed. Results showed that WMPAE significantly inhibited the growth of A. flos-aquae in a concentration-dependent way. The highest inhibition reached 94 % after 3 days' treatment with 6 g L-1 of WMPAE and a significant effect was obtained with lower doses and shorter times as well. The cell viability decreased quickly, cell shape changed, and intracellular structural damage occurred. At the same time, the antioxidant enzymes (superoxide dismutase SOD, catalase CAT and peroxidase POD) and malondialdehyde (MDA) levels all increased significantly, indicating that WMPAE between 2-6 g L-1 induced severe oxidative stress and damage to A. flos-aquae. Moreover, production of the four pigments chlorophyll a (Chl a), carotenoids, phycocyanin (PC), and allophycocyanin (APC) were all stimulated, though photosynthesis of A. flos-aquae was clearly inhibited. The maximum quantum yield of photosystem II (Fv/Fm) and the effective quantum yield of photosystem II ( Fv'/Fm') declined sharply, suggesting the decreased photosystem capacity of A. flos-aquae to convert light energy into chemical energy. In addition, non-photochemical quenching (NPQ) of A. flos-aquae increased after a very short time exposure to WMPAE, and decreased significantly with prolonged exposure time, which indicated the failure of photo protection mechanisms. These results suggest that the loss of cell viability, and increases in oxidative stress, and damage to intracellular structure and photosynthetic systems might be the mechanisms for the inhibitory effects. Our results suggested that WMPAE could be a novel and effective approach for controlling the growth of A. flos-aquae in aquatic environments.

Ultrasound-Assisted Extraction and Assessment of Biological Activity of Phycobiliprotein-Rich Aqueous Extracts from Wild Cyanobacteria (Aphanizomenon flos-aquae).

Cyanobacteria are photosynthetic microorganisms that are considered as an important source of bioactive metabolites, among which phycobiliproteins (PBPs) are a class of water-soluble macromolecules of cyanobacteria with a wide range of applications. Massive proliferation of cyanobacteria can lead to excessive surface water blooms, of which removal, as a management measure, should be prioritized. In this study, the utilization of wild cyanobacteria biomass (Aphanizomenon flos-aquae) for extraction of phycobiliproteins is reported. Extraction of phycobiliproteins by conventional methods, such as homogenization, freeze-thaw cycles, and solid-liquid extraction, were optimized prior to ultrasound-assisted extraction. Standardization of ultrasonication for different parameters, such as ultrasonication amplitude (38, 114, and 190 µm) and ultrasonication time (1, 5.5, and 10 min), was carried out using a central composite design and response surface methodology for each of the primary techniques. A substantial increase on the individual and total phycobiliprotein yields was observed after ultrasonic treatment. The highest total PBP yield (115.37 mg/g of dry weight) was observed with samples treated with a homogenizer (30 min, 30 °C, and 1 cycle) combined with ultrasound treatment (8.7 min at 179 µm). Moreover, in vitro antioxidant capacity was observed for the obtained extracts in the Folin-Ciocalteu and ABTS* + assays. In addition, a cytotoxic effect against C6 glioma cells was observed for A. flos-aquae PBPs. Conclusively, wild cyanobacteria could be considered as an alternative feedstock for recovery of PBPs.

Heat-Resistant Aphanizomenon flos-aquae (AFA) Extract (Klamin®) as a Functional Ingredient in Food Strategy for Prevention of Oxidative Stress.

Microalgae are generally considered an excellent source of vitamins, minerals, and bioactive molecules that make them suitable to be introduced in cosmetics, pharmaceuticals, and food industries. Aphanizomenon flos-aquae (AFA), an edible microalga, contains numerous biomolecules potentially able to prevent some pathologies including age-related disorders. With the aim to include an AFA extract (Klamin®) as a functional ingredient in baked products, we investigated if its bioactive molecules are destroyed or inactivated after standard cooking temperature. The AFA extract was exposed to heat stress (AFA-HS), and no significant decrease in pigment, polyphenol, and carotenoid content was detected by spectroscopic analysis. Thermal stability of AFA-HS extract was demonstrated by thermogravimetric analysis (TGA), and no change in the morphology of the granules of the powder was noticed by SEM microscopic observation. By Folin-Ciocalteu, ORAC, and ABTS assays, no change in the antioxidant activity and polyphenol contents was found after high-temperature exposition. When added in cell culture, solubilized AFA-HS lost neither its scavenging ability against ROS generation nor its protective role against Abeta, the main peptide involved in Alzheimer's disease. Prebiotic and antioxidant activities of AFA extract that are not lost after thermal stress were verified on E. coli bacteria. Finally, AFA-HS cookies, containing the extract as one of their ingredients, showed increased polyphenols. Here, we evaluate the possibility to use the AFA extract to produce functional food and prevent metabolic and age-related diseases.

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.

A study of polymethoxy-1-alkenes in Raphidiopsis (Cylindrospermopsis) raciborskii and Aphanizomenon gracile isolated in Poland.

Previous studies indicated that teratogenic polymethoxy-1-alkenes (PMAs) are produced by phylogenetically diverse cyanobacteria taxa, however corresponding studies on the occurrence of PMAs in European cyanobacteria are lacking. Herein, the presence of PMAs in strains of Raphidiopsis raciborskii and Aphanizomenon gracile isolated from surface waters in Poland was studied using nuclear magnetic resonance and mass spectrometry. No PMAs were detected in any of the strains investigated, indicating that production of these compounds may be geographically diversified. Further studies are necessary to elucidate mechanisms of cyanobacterial PMAs synthesis.

Responses of pro- and anti-inflammatory cytokines in zebrafish liver exposed to sublethal doses of Aphanizomenon flosaquae DC-1 aphantoxins.

Blooms of the dominant cyanobacterium Aphanizomenon flosaquae are frequently encountered in natural waters, and their secretion of neurotoxic paralytic shellfish toxins called aphantoxins threatens environmental safety and human health worldwide. The liver is the primary detoxification organ in animals, and its pro- and anti-inflammatory responses are important functions in the detoxification of toxins. Therefore, we investigated the response of these inflammatory factors to aphantoxins in the liver of zebrafish (Danio rerio). A. flosaquae DC-1 was sampled during blooms in Dianchi Lake, China and cultured, and the toxin was extracted and analyzed using high performance liquid chromatography. The primary constituents were gonyautoxins 1 (34.04%) and 5 (21.28%) and neosaxitoxin (12.77%). Zebrafish were injected intraperitoneally with 5.3 µg (low dose) or 7.61 µg (high dose) of saxitoxin equivalents [equivalents (eq.)]/kg body weight of A. flosaquae DC-1 aphantoxins. Hyperemia, the hepatosomatic index (HSI), and physiological and molecular responses of pro- and anti-inflammatory cytokines in the zebrafish liver were investigated at different time points 1-24 h post-exposure. Aphantoxins significantly enhanced hepatic hyperemia and altered the HSI 3-24 h post-exposure, suggesting that inflammation caused morphological changes. Subsequent investigations using the enzyme-linked immunosorbent assay showed that the pro-inflammatory cytokines tumor necrosis factor-α, interleukin-1ß (IL-1ß), IL-6, and IL-8 and anti-inflammatory cytokines IL-10 and transforming growth factor ß were higher in the liver of zebrafish exposed to aphantoxins, which indicated physiological inflammatory responses. Further analysis by real-time fluorescence quantitative polymerase chain reaction demonstrated upregulated mRNA expression of these cytokines, suggesting molecular inflammatory responses in the zebrafish liver. These changes showed dose- and time-dependent patterns. These results indicated that aphantoxins induced hyperemia and altered the HSI, and subsequently increased the levels of proinflammatory cytokines TNF-α, IL-1ß, IL-6 and IL-8 to induce physiological inflammatory responses. These changes activated the anti-inflammatory cytokines IL-10 and TGF-ß to suppress inflammatory damage. The induced changes were the result of upregulated mRNA expression of these inflammatory cytokines caused by aphantoxins. Aphantoxins resulted in hepatic immunotoxicity and response by inducing pro-inflammatory cytokines. Zebrafish liver in turn suppressed the inflammatory damage by upregulating the activities of anti-inflammatory cytokines. In the future, these pro- and anti-inflammatory cytokines in the zebrafish liver may be prove to be useful biomarkers of aphantoxins and blooms in nature.

Effects of the Aphanizomenon flos-aquae Extract (Klamin®) on a Neurodegeneration Cellular Model.

Cyanobacteria have been recognized as a source of bioactive molecules to be employed in nutraceuticals, pharmaceuticals, and functional foods. An extract of Aphanizomenon flos-aquae (AFA), commercialized as Klamin®, was subjected to chemical analysis to determine its compounds. The AFA extract Klamin® resulted to be nontoxic, also at high doses, when administered onto LAN5 neuronal cells. Its scavenging properties against ROS generation were evaluated by using DCFH-DA assay, and its mitochondrial protective role was determined by JC-1 and MitoSOX assays. Klamin® exerts a protective role against beta amyloid- (Aß-) induced toxicity and against oxidative stress. Anti-inflammatory properties were demonstrated by NFßB nuclear localization and activation of IL-6 and IL-1ß inflammatory cytokines through ELISA. Finally, by using thioflavin T (ThT) and fluorimetric measures, we found that Klamin® interferes with Aß aggregation kinetics, supporting the formation of smaller and nontoxic structures compared to toxic Aß aggregates alone. Altogether, these data indicate that the AFA extract may play a protective role against mechanisms leading to neurodegeneration.

Paralytic shellfish toxin producing Aphanizomenon gracile strains isolated from Lake Iznik, Turkey.

Aphanizomenon gracile is one of the most widespread Paralytic Shellfish Toxin (PST) producing cyanobacteria in freshwater bodies in the Northern Hemisphere. It has been shown to produce various PST congeners, including saxitoxin (STX), neosaxitoxin (NEO), decarbamoylsaxitoxin (dcSTX) and gonyautoxin 5 (GTX5) in Europe, North America and Asia. Three cyanobacteria strains were isolated in Lake Iznik in northwestern Turkey. Morphological characterization of these strains suggested all three strains conformed to classical taxonomic identification of A. gracile with some differences such as clumping of filaments, partially hyaline cells in some filaments and longer than usual vegetative cells. Sequences of 16S rRNA gene of these strains were placed within an A. gracile cluster including the majority of PST producing strains, confirming the identification of these strains as A. gracile. These new strains possessed saxitoxin biosynthesis genes sxtA, sxtG and their sequences clustered with those of other A. gracile. Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis demonstrated the presence of NEO, STX, dcSTX and decarbamoylneosaxitoxin (dcNEO) in all strains. This is the first report of a PST producer in any water body in Turkey and first observation of dcNEO in an A. gracile culture.

Mycosporine-like Amino Acids and Other Phytochemicals Directly Detected by High-Resolution NMR on Klamath (Aphanizomenon flos-aquae) Blue-Green Algae.

This study describes for the first time the use of high-resolution nuclear magnetic resonance (NMR) on Klamath (Aphanizomenon flos-aquae, AFA) blue-green algae directly on powder suspension. These algae are considered to be a "superfood", due to their complete nutritional profile that has proved to have important therapeutic effects. The main advantage of NMR spectroscopy is that it permits the detection of a number of metabolites all at once. The Klamath alga metabolome was revealed to be quite complex, and the most peculiar phytochemicals that can be detected directly on algae by NMR are mycosporine-like amino acids (porphyra-334, P334; shinorine, Shi) and low molecular weight glycosides (glyceryl ß-d-galactopyranoside, GalpG; glyceryl 6-amino-6-deoxy-α-d-glucopyranoside, ADG), all compounds with a high nutraceutical value. The presence of cis-3,4-DhLys was revealed for the first time. This molecule could be involved in the anticancer properties ascribed to AFA.

Inhibitory effects of Aphanizomenon flos-aquae constituents on human UDP-glucose dehydrogenase activity.

OBJECTIVE: The purpose of this study was to investigate the in vitro inhibitory effects of the edible microalga Aphanizomenon flos-aquae (AFA) on human UDP-α-d-glucose 6-dehydrogenase (UGDH) activity, a cytosolic enzyme involved both in tumor progression and in phytochemical bioavailability. METHODS: Both the hydrophilic and ethanolic AFA extracts as well as the constitutive active principles phycocyanin (PC), phycocyanobilin (PCB) and mycosporine-like amino acids (MAAs) were tested. RESULTS: Among AFA components, PCB presented the strongest inhibitory effect on UGDH activity, acting as a competitive inhibitor with respect to UDP-glucose and a non-competitive inhibitor with respect to NAD(+). In preliminary experiments, AFA PCB was also effective in reducing the colony formation capacity of PC-3 prostate cancer cells and FTC-133 thyroid cancer cells. CONCLUSIONS: Overall, these findings confirmed that AFA and its active principles are natural compounds with high biological activity. Further studies evaluating the effects of AFA PCB in reducing tumor cell growth and phytochemical glucuronidation are encouraged.

Extracellular Polymeric Substances of Aphanizomenon flos-aquae (EPS-A) Induced Apoptosis in Astrocytes of Zebrafish.

In this study, extracellular polymeric substances of Aphanizomenon flos-aquae (EPS-A) were investigated in order to explore their effect on astrocytes of zebrafish and potential risk for environment. Astrocytes were treated with varying concentrations of EPS-A, the results showed that EPS-A inhibited astrocytes growth in a dose-and time-dependent manner. With the concentrations of EPS-A increasing, the adherent ability of astrocytes decreased and the number of astrocytes floating in the culture medium increased. When treated with 2.35 µg/mL EPS-A, EPS-A induced cell cycle arrest and made the collapse of mitochondrial membrane potential and then led to astrocytes apoptosis. The results suggested that EPS-A could pose a threat to zebrafish and represent risk for environment, so regularly monitoring the presence of EPS-A was very important in nutrient-rich freshwaters when A. flos-aquae blooms broke out.

The response and detoxification strategies of three freshwater phytoplankton species, Aphanizomenon flos-aquae, Pediastrum simplex, and Synedra acus, to cadmium.

The response and detoxification mechanisms of three freshwater phytoplankton species (the cyanobacterium Aphanizomenon flos-aquae, the green alga Pediastrum simplex, and the diatom Synedra acus) to cadmium (Cd) were investigated. The cell growth of each species was measured over 10 days, and chlorophyll a fluorescence, Cd bioaccumulation (including surface-adsorbed and intracellular Cd), and phytochelatin (PC) synthesis were determined after 96-h exposures. The growth of the three phytoplankton species was significantly inhibited when Cd concentrations were ≥5 mg L(-1). Compared with P. simplex, greater growth inhibition in S. acus and A. flos-aquae occurred. The changes in chlorophyll fluorescence parameters including the maximal quantum yield of PSII (Fv/Fm) and relative variable fluorescence of the J point (Vj) demonstrated that the increase in Cd concentration damaged PSII in all three species. After 96-h exposures, the accumulation of surface-adsorbed Cd and intracellular Cd increased significantly in all three species, with the increase of Cd concentrations in the media; total cadmium accumulation was 245, 658, and 1670 times greater than that of the control in A. flos-aquae, P. simplex, and S. acus, respectively, after exposure to 10 mg L(-1). Total thiols exhibited a similar trend to that of Cd accumulation. PC3 was found in A. flos-aquae and P. simplex in all Cd treatments. Glutathione (GSH) and PC2 were also produced in response to exposure to high concentrations of Cd. PC4 was only discovered at exposure concentrations of 10 mg L(-1) Cd and only in S. acus. The intracellular Cd/PCs ratio increased in all three phytoplankton with an increase in Cd concentrations, and a linear relationship between the ratio and the growth inhibition rates was observed with P. simplex and S. acus. Our results have demonstrated that metal detoxification mechanisms were dependent on the species. This study suggested that the variance of metal detoxification strategies, such as cadmium accumulation and PCs, might be an explanation why algal species have different sensitivity to Cd at various levels.

Biochemical and growth performance of the aquatic macrophyte Azolla filiculoides to sub-chronic exposure to cylindrospermopsin.

Physiological and biochemical effects of cylindrospermopsin (CYN), a cyanobacterial toxin that inhibits protein synthesis and released during a harmful cyanobacterial bloom, has been overlooked in plants. Therefore, at the present research, the toxic effects (physiological and biochemical) of a crude extract containing CYN were assessed in the aquatic fern Azolla filiculoides exposed to three concentrations (0.05, 0.5 and 5 µg CYN mL(-1)). At 5 µg CYN mL(-1), fern growth rate has showed a drastic decrease (0.001 g g(-1) day(-1)) corresponding to a 99.8% inhibition, but at the concentrations of 0.05 and 0.5 µg CYN mL(-1) the growth rate was similar to the control plants. Growth rate also indicated a IC50 of 2.9 µg CYN mL(-1). Those data point to the presence of other compounds in the crude extract may stimulate the fern growth and/or the fern is tolerant to CYN. Chlorophyll (a and b), carotenoids and protein content as well as the activities of glutathione reductase (GR) and glutathione-S-transferase (GST) has increased at 5 µg CYN mL(-1) which may indicate that photosynthesis and protein synthesis are not affected by CYN and the probable activation of defense and detoxifying mechanisms to overcome the effects induced by the presence of CYN. Low uptake of cylindrospermopsin (1.314 µg CYN g(-1) FW) and low bioconcentration factor (0.401) point towards to a safe use of A. filiculoides as biofertilizer and as food source, but also indicate that the fern is not suitable for CYN phytoremediation.

Cylindrospermopsin induces neurotoxicity in tilapia fish (Oreochromis niloticus) exposed to Aphanizomenon ovalisporum.

Cylindrospermopsin (CYN) is a cytotoxic cyanotoxin produced by several species of freshwater cyanobacteria, such as Aphanizomenon ovalisporum. CYN is a tricyclic alkaloid known for its ability to inhibit both protein and glutathione synthesis, and the alteration of different oxidative stress biomarkers in mammals and vertebrates. Although the liver and kidney appear to be the main CYN targets for this toxin, it also affects other organs. In fish, there is no evidence about the neurotoxicity of CYN yet. In the present study, we aimed to study the potential neurotoxicity of CYN, based on the measure of Acetylcholinesterase (AChE) activity, lipid peroxidation (LPO) levels and histopathological studies in brain of tilapia (Oreochromis niloticus) subchronically exposed to repeated concentrations of 10µg CYN/L by immersion in an A.ovalisporum culture for 14 days. The results showed significant inhibition of AChE activity and increases in LPO levels, as well as relevant histopathological alterations in the brain of fish (O. niloticus) subchronically exposed to the toxin. Moreover, we also investigated the potential recovery of these parameters by subjecting the fish to two depuration periods (3 and 7 days) in clean uncontaminated water, showing a recovery of the biochemical parameters since 3 days of depuration, and being necessary 7 days to recover the histopathological changes. In order to support these results, CYN was detected and quantified by enzyme-linked immunosorbent assay (ELISA) in brain of all the exposed fish and the effects of the depuration periods were also observed. Based on these results, it was demonstrated for the first time the neurotoxicity of CYN and its presence in brain of tilapia fish subchronically exposed to CYN.

Endocrine, teratogenic and neurotoxic effects of cyanobacteria detected by cellular in vitro and zebrafish embryos assays.

Cyanobacteria contain various types of bioactive compounds, which could cause adverse effects on organisms. They are released into surface waters during cyanobacterial blooms, but there is little information on their potential relevance for effects in vivo. In this study presence of bioactive compounds was characterized in cyanobacteria Microcystis aeruginosa (Chroococcales), Planktothrix agardhii (Oscillatoriales) and Aphanizomenon gracile (Nostocales) with selected in vitro assays. The in vivo relevance of detected bioactivities was analysed using transgenic zebrafish embryos tg(cyp19a1b-GFP). Teratogenic potency was assessed by analysis of developmental disorders and effects on functions of the neuromuscular system by video tracking of locomotion. Estrogenicity in vitro corresponded to 0.95-54.6 ng estradiol equivalent(g dry weight (dw))(-1). In zebrafish embryos, estrogenic effects could not be detected potentially because they were masked by high toxicity. There was no detectable (anti)androgenic/glucocorticoid activity in any sample. Retinoid-like activity was determined at 1-1.3 µg all-trans-retinoic acid equivalent(g dw)(-1). Corresponding to the retinoid-like activity A. gracile extract also caused teratogenic effects in zebrafish embryos. Furthermore, exposure to biomass extracts at 0.3 gd wL(-1) caused increase of body length in embryos. There were minor effects on locomotion caused by 0.3 gd wL(-1)M. aeruginosa and P. agardhii extracts. The traditionally measured cyanotoxins microcystins did not seem to play significant role in observed effects. This indicates importance of other cyanobacterial compounds at least towards some species or their developmental phases. More attention should be paid to activity of retinoids, estrogens and other bioactive substances in phytoplankton using in vitro and in vivo bioassays.