New records on toxic cyanobacteria from Brazil: Exploring their occurrence and geography.

Cyanobacterial Harmful Algal Blooms (CyanoHABs) pose a significant threat to communities globally, impacting ecosystems and public health. This study provides an in-depth review of the current state of cyanotoxins and the distribution of CyanoHABs species in Brazil, while also detailing the methods used for their detection. Four hundred and twenty-one incidents were analyzed from 1993 to 2021, compiling cyanotoxin records and toxic CyanoHABs occurrences. The investigation begins with the first detection of microcystins in 1994 and highlights pivotal moments, like the 1996 "Caruaru Syndrome" outbreak. This event encouraged research and updated cyanotoxin-monitoring guidelines. The Brazilian drought period of 2015-2016 exacerbated cyanobacterial growth and saxitoxin levels, coinciding with Zika-related microcephaly. This study delves into methods used for cyanotoxin analysis, including ELISA, bioassays, HPLC, and LC-MS. Additionally, we investigated the toxicity of 37 cyanobacterial strains isolated from various Brazilian environments. Extracts were tested against Artemia salina and analyzed by LC-MS. Results revealed toxicity in extracts from 49 % of cyanobacterial strains. LC-MS results were analyzed using GNPS MS/MS molecular networking for comparing experimental spectra with those of cyanotoxin standards against in-house databases and the existing literature. Our research underscores the variability in cyanotoxin production among species and over time, extending beyond microcystins. LC-MS results, interpreted through the GNPS platform, revealed six cyanotoxin groups in Brazilian strains. Yet, compounds present in 75 % of the toxic extracts remained unidentified. Further research is crucial for fully comprehending the impact of potentially harmful organisms on water quality and public health management strategies. The study highlights the urgent need for continuously monitoring cyanobacteria and the cyanotoxin inclusion of management in public health policies.

Cyanopeptides occurrence and diversity in a Brazilian tropical reservoir: Exploring relationships with water quality.

Microcystins (MCs) are a class of toxic secondary metabolites produced by some cyanobacteria strains that endanger aquatic and terrestrial organisms in various freshwater systems. Although patterns in MC occurrence are being recognized, divergences in the global data still hamper our ability to predict the toxicity of cyanobacterial blooms. This study aimed (i) to determine the dynamics of MCs and other cyanopeptides in a tropical reservoir, (ii) to investigate the correlation between peptides and potential cyanotoxin producers (iii) identifying the possible abiotic factors that influence the peptides. We analyzed, monthly, eight MC variants (MC-RR, -LA, -LF, -LR, -LW, -YR, [D-Asp3]-RR and [D-Asp3]-LR) and other peptides in 47 water samples collected monthly, all season long, from two sampling sites in a tropical eutrophic freshwater reservoir, in southeastern Brazil. The cyanopeptides were assessed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The biomass of potential cyanobacterial producers and water quality variables were measured. MCs were detected in both sampling sites year-round; the total MC concentration varied from 0.21 to 4.04 µg L-1, and three MC variants were identified and quantified (MC-RR, [D-Asp3]-RR, -LR). Additionally, we identified 28 compounds belonging to three other cyanopeptide classes: aeruginosin, microginin, and cyanopeptolin. As potential MC producers, Microcystis spp. and Dolichospermum circinalis were dominant during the study, representing up to 75% of the total phytoplankton. Correlational and redundancy analysis suggested positive effects of dissolved oxygen, nitrate, and total phosphorus on MC and microginins concentration, while water temperature appeared to favor aeruginosins. A comparison between our results and historical data showed a reduction in total phosphorus and cyanobacteria, suggesting increased water quality in the reservoir. However, the current MC concentrations indicate a rise in cyanobacterial toxicity over the last eight years. Moreover, our study underscores the pressing need to explore cyanopeptides other than MCs in tropical aquatic systems.

Metabolomics Applied to Cyanobacterial Toxins and Natural Products.

The biological and chemical diversity of Cyanobacteria is remarkable. These ancient prokaryotes are widespread in nature and can be found in virtually every habitat on Earth where there is light and water. They are producers of an array of secondary metabolites with important ecological roles, toxic effects, and biotechnological applications. The investigation of cyanobacterial metabolites has benefited from advances in analytical tools and bioinformatics that are employed in metabolomic analyses. In this chapter, we review selected articles highlighting the use of targeted and untargeted metabolomics in the analyses of secondary metabolites produced by cyanobacteria. Here, cyanobacterial secondary metabolites have been didactically divided into toxins and natural products according to their relevance to toxicological studies and drug discovery, respectively. This review illustrates how metabolomics has improved the chemical analysis of cyanobacteria in terms of speed, sensitivity, selectivity, and/or coverage, allowing for broader and more complex scientific questions.

A rapid LC-MS/MS method for multi-class identification and quantification of cyanotoxins.

Cyanobacteria can form harmful blooms in specific environmental conditions due to certain species producing toxic metabolites known as cyanotoxins. These toxins pose significant risks to public health and the environment, making it critical to identify and quantify them in food and water sources to avoid contamination. However, current screening methods only focus on a single class of cyanotoxins, limiting their effectiveness. Thus, fast and sensitive liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method was developed to analyze eighteen cyanotoxins simultaneously. A simplified extraction procedure using lyophilized samples of cyanobacterial biomass was also used, eliminating the need for traditional solid-phase extraction methods. This method uses multiple reaction monitoring and allows accurate determination and quantification of eighteen cyanotoxins, including anatoxin-a, homoanatoxin-a, cylindrospermopsin, deoxy-cylindrospermopsin, nodularin, guanitoxin, seven microcystins (RR, [D-Asp3] RR, LA, LR, LY, LW, and YR), and five saxitoxins (gonyautoxins - GTX-1&4, GTX-2&3, GTX-5), decarbamoylgonyautoxin (dcGTX-2&3), and N-Sulfocarbamoylgonyautoxin (C1&C2), all in a short acquisition time of 8 min. Therefore, this method provides a simple and efficient approach to identify and quantify harmful compounds produced by cyanobacteria. Hence, this represents the first method to detecting guanitoxin among cyanotoxins. By expanding the range of toxins analyzed, this method can help ensure high-quality food and drinking water and protect recreational users from exposure to cyanotoxins.

Ecotoxicological assessment of guanitoxin-producing cyanobacteria in Danio rerio and Daphnia similis.

Anthropogenic activity has dramatically deteriorated aquatic ecosystems in recent years. Such environmental alterations could change the primary producers' composition, exacerbating the proliferation of harmful microorganisms such as cyanobacteria. Cyanobacteria can produce several secondary metabolites, including guanitoxin, a potent neurotoxin and the only naturally occurring anticholinesterase organophosphate ever reported in the literature. Therefore, this study investigated the acute toxicity of guanitoxin-producing cyanobacteria Sphaerospermopsis torques-reginae (ITEP-024 strain) aqueous and 50% methanolic extracts in zebrafish (Danio rerio) hepatocytes (ZF-L cell line), zebrafish embryos (fish embryo toxicity - FET) and specimens of the microcrustacean Daphnia similis. For this, hepatocytes were exposed to 1-500 mg/L of the ITEP-024 extracts for 24 h, the embryos to 31.25-500 mg/L for 96 h, and D. similis to 10-3000 mg/L for 48 h. Non-target metabolomics was also performed to analyze secondary metabolites produced by the ITEP-024 using LC-MS/MS. Metabolomics indicated the guanitoxin presence just in the aqueous extract of the ITEP-024 and the presence of the cyanopeptides namalides, spumigins, and anabaenopeptins in the methanolic extract. The aqueous extract decreased the viability of zebrafish hepatocytes (EC(I)50(24h) = 366.46 mg/L), and the methanolic extract was not toxic. FET showed that the aqueous extract (LC50(96) = 353.55 mg/L) was more toxic than the methanolic extract (LC50(96) = 617.91 mg/L). However, the methanolic extract had more sublethal effects, such as abdominal and cardiac (cardiotoxicity) edema and deformation (spinal curvature of the larvae). Both extracts immobilized daphnids at the highest concentration analyzed. However, the aqueous extract was nine times more lethal (EC(I)50(48h) = 108.2 mg/L) than the methanolic extract (EC(I)50(48h) = 980.65 mg/L). Our results showed an imminent biological risk for aquatic fauna living in an ecosystem surrounded by ITEP-024 metabolites. Our findings thus highlight the urgency of understanding the effects of guanitoxin and cyanopeptides in aquatic animals.

Effect of ultraviolet radiation on the metabolomic profiles of potentially toxic cyanobacteria.

Interactions between climate change and ultraviolet radiation (UVR) have a substantial impact on aquatic ecosystems, especially on photosynthetic organisms. To counteract the damaging effects of UVR, cyanobacteria developed adaptive strategies such as the biosynthesis of secondary metabolites. This study aimed to evaluate the effects of UVR on the metabolomic profiles of potentially toxic cyanobacteria. Twelve strains were irradiated with ultraviolet A and ultraviolet B radiation and parabolic aluminized reflector lamps for 3 days, followed by liquid chromatography-tandem mass spectometry (LC-MS/MS) analysis to assess changes in metabolomic profiles. Matrices were used to generate principal component analysis biplots, and molecular networks were obtained using the Global Natural Products platform. Most strains showed significant changes in their metabolomic profiles after UVR exposure. On average, 7% of MS features were shown to be exclusive to metabolomic profiles before UVR exposure, while 9% were unique to metabolomic profiles after UVR exposure. The identified compounds included aeruginosins, spumigins, cyanopeptolins, microginins, namalides, pseudospumigins, anabaenopeptins, mycosporine-like amino acids, nodularins and microcystins. Data showed that cyanobacteria display broad metabolic plasticity upon UVR exposure, including the synthesis and differential expression of a variety of secondary metabolites. This could result in a competitive advantage, supporting cyanobacterial blooms under various UVR light exposures.

Identification and distribution of mycosporine-like amino acids in Brazilian cyanobacteria using ultrahigh-performance liquid chromatography with diode array detection coupled to quadrupole time-of-flight mass spectrometry.

RATIONALE: Mycosporine-like amino acids (MAAs) are UV-absorbing compounds produced by fungi, algae, lichens, and cyanobacteria when exposed to UV radiation. These compounds have photoprotective and antioxidant functions and have been widely studied for possible use in sunscreens and anti-aging products. This study aims to identify MAA-producing cyanobacteria with potential application in cosmetics. METHODS: A method for the identification of MAAs was developed using ultrahigh-performance liquid chromatography with diode array detection coupled to quadrupole time-of-flight mass spectrometry (UHPLC-DAD/QTOFMS). Chromatographic separation was carried out using a Synergi 4 µ Hydro-RP 80A column (150 × 2,0 mm) at 30°C with 0.1% formic acid aqueous solution + 2 mM ammonium formate and acetonitrile/water (8:2) + 0.1% formic acid as a mobile phase. RESULTS: Out of the 69 cyanobacteria studied, 26 strains (37%) synthesized MAAs. Nine different MAAs were identified using UHPLC-DAD/QTOFMS. Iminomycosporines were the major group detected (7 in 9 MAAs). In terms of abundance, the most representative genera for MAA production were heterocyte-forming groups. Oscilatoria sp. CMMA 1600, of homocyte type, produced the greatest diversity of MAAs. CONCLUSIONS: The UHPLC-DAD/QTOFMS method is a powerful tool for identification and screening of MAAs in cyanobacterial strains as well as in other organisms such as dinoflagellates, macroalgae, and microalgae. The different cyanobacterial genera isolated from diverse Brazilian biomes and environments are prolific sources of MAAs.