Distribution and toxicity of Cylindrospermopsis raciborskii (Cyanobacteria) in Portuguese freshwaters

The cyanobacterium Cylindrospermopsis racborskii has become increasingly prevalent in freshwaters worldwide. This species is of concern from a water quality perspective due to its known ability to produce a potent hepatotoxic alkaloid cylindrospermopsin, which has been implicated in outbreaks of human sickness and cattle mortality. C. raciborskii isolates from Brazil have also been found to produce the highly toxic paralytic shellfish poisons (PSP's). In this paper we report the toxicity of four isolates of C. raciborskii taken from three reservoirs and one river in Portugal as well as the occurrence of this species in other water bodies used for potable and recreational purposes. All four isolates grown in pure culture in the laboratory were found to be toxic in the mouse bioassay at 8 - 24 hours after intraperitoneal administration of single doses ranging from 1337 to 1572 mg kg-1. Histological examination showed liver damage as the primary lesion, in addition to some inflammation in the intestine. HPLC/MS tests for the presence of cylindrospermopsin, microcystins and PSP toxins were negative. The available evidence suggests that another toxin may be present. This report constitutes the first report of toxic C. raciborskii in Europe and draws attention to our need for increased monitoring of this cyanobacterium in water bodies used for potable and recreational purposes.La cianobacteria Cylindrospermopsis racborskii ha incrementado su presencia en agua dulce por todo el mundo. Esta especie es de gran importancia debido a su conocida capacidad para producir un alcaloide hepatotóxico, cilindrospermopsina. Esta toxina es responsable de enfermedades en humanos y mortalidad en ganado. Cepas de C. raciborskii aisladas de Brasil se ha demostrado que tienen capacidad para producir la toxina paralytic shellfish poisons (PSP's). En este trabajo hemos estudiado la presencia de esta especie en agua dulce con usos recreacionales y de abastecimiento en Portugal. De las cuatro cepas C. raciborskii aisladas de tres embalses y de un río estudiamos la toxicidad con bioensayos en ratones. Todas presentaron toxicidad al cabo de las 8 - 24 horas tras inyección intraperitoneal, la dosis presenta un rango de concentración de 1337 a 1572 mg kg-1. La examinación histológica reveló daños en el hígado y señales inflamatorias en el intestino. Los análisis con HPLC/MS revelaron la ausencia de cilindrospermopsina, microcistinas y PSP, sugiriendo que otra toxina podría estar presente para las cepas aisladas y cultivadas en laboratorio. Este artículo es el primero acerca de la toxicidad de C. raciborskii en Europa y refleja la necesidad de aumentar el monitoreo de esta cyanobacteria en el agua potable y con fines recreativos

Metodologias para estudos integrados de recursos naturais: uma discussão a partir do zoneamento ecológico-econômico

This article examines methodological developments in integrated zoning, which are used in the environmental planning and land use in Brazil. In particular, it focuses on Ecological-Economic Zoning (EEZ), a methodology that has become the primary instrument of territorial planning in the Brazilian government. Throughout its historical development process, EEZ has become an interdisciplinary zoning method, which incorporates various environmental and socio-economic themes. However, there are still several central issues in constant discussion and many challenges to be resolved. Various sides of this discussion are analyzed, and new methodologies are suggested from other research areas, which may contribute to the efficiency of integrated environmental studies.Este artigo adentra-se no debate sobre as evoluções metodológicas dos zoneamentos integrados constantes nos instrumentos de planejamento ambiental e de ordenamento territorial do Brasil. Em especial, enfoca o debate promovido em torno da metodologia do Zoneamento Ecológico-Econômico – ZEE - brasileiro, instrumento basilar de planejamento territorial do país. Ao longo de seu processo histórico de desenvolvimento, o ZEE tornou-se um zoneamento amplo, que incorpora diversas temáticas ambientais e sócio-econômicas. Contudo, ainda existem diversos pontos nevrálgicos em constante discussão, bem como vários desafios a serem vencidos. O artigo pretende cobrir várias facetas desse debate, além de sugerir novas metodologias provenientes de outras áreas de estudo, e que podem contribuir para que os estudos integrados de meio ambiente tornem-se cada vez mais eficientes

Pigments extraction from Cyanobium sp. a comparison between pressure-based and electric fields-based technologies

Pigments from cyanobacteria, in special carotenoids and phycobiliproteins, have been seen with considerable interest for industrial applications due to their bioactive properties and their natural product characteristics. The extraction of these compounds is focused on the methodologies of cell disruption and on the chemical solubility of the compounds. In this study, two different methods were optimised and evaluated in terms of pigments´ extraction from the marine cyanobacterium Cyanobium sp.: a continuous pressurized solvent extraction (CPSE) system, and an electric fields-assisted extraction system based in ohmic heating (OH). For each method, a Central Composite Design (23) was performed. Optimal conditions for each extraction method were then compared to determine the best method for the extraction of pigments from Cyanobium sp. In both optimisation and comparison steps, two extracts were obtained from the same biomass: an ethanolic extract (carotenoids-targeted) and a successive water extract (phycobiliproteins-targeted). The content and profile of carotenoids and phycobiliproteins and the respective antioxidant capacity of extracts were evaluated. OH provided the best ethanolic extract, with a carotenoids content of 41.6 ± 1.7 mg gDW-1, and total antioxidant capacity of 8.0 ± 0.3 mgTE gDW-1, representing an increase of 1.3-fold and 2.5-fold respectively, when compared to CPSE. Regarding the aqueous extract, both methods led to the same content of phycobiliprotein (135 ± 10.0 mg gDW-1), although OH led to an antioxidant capacity of this extract of 8.3 ± 0.3 mgTE gDW-1, 3.6-fold higher when compared to CPSE. In terms of profile, no major variation was found between extraction methods, being lutein, zeaxanthin, echinenone and -carotene the major carotenoids (>60 % of total carotenoids), and phycocyanin and allophycocyanin the only present phycobiliproteins (in a 1:2 ratio). In addition to the productivity and composition of the extracts, the design and applicability of the system must be considered. Once again, OH overtook the other methods due to the scalability and possible continuous operation. Overall, OH proved to be the best of the two methodologies for pigments co-extraction from Cyanobium sp..A PhD fellowship (reference SFRH/BD/136767/2018) for author Fernando Pagels was granted by Fundação para a Ciência e Tecnologia (FCT, Portugal) under the auspices of Programa Operacional Capital Humano (POCH), supported by the European Social Fund and Portuguese funds (MECTES). This work was financially co-supported by the strategical funding from FCT UIDB/04423/2020, UIDP/04423/2020 and UIDB/04469/2020; and the project ALGAVALOR – MicroALGAs: produção integrada e VALORização da biomassa e das suas diversas aplicações (POCI-01-0247-FEDER-035234), supported by the European Regional Development Fund and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte.info:eu-repo/semantics/publishedVersio

OMICs approaches in diarrhetic shellfish toxins research

ReviewDiarrhetic shellfish toxins (DSTs) are among the most prevalent marine toxins in Europe’s and in other temperate coastal regions. These toxins are produced by several dinoflagellate species; however, the contamination of the marine trophic chain is often attributed to species of the genus Dinophysis. This group of toxins, constituted by okadaic acid (OA) and analogous molecules (dinophysistoxins, DTXs), are highly harmful to humans, causing severe poisoning symptoms caused by the ingestion of contaminated seafood. Knowledge on the mode of action and toxicology of OA and the chemical characterization and accumulation of DSTs in seafood species (bivalves, gastropods and crustaceans) has significantly contributed to understand the impacts of these toxins in humans. Considerable information is however missing, particularly at the molecular and metabolic levels involving toxin uptake, distribution, compartmentalization and biotransformation and the interaction of DSTs with aquatic organisms. Recent contributions to the knowledge of DSTs arise from transcriptomics and proteomics research. Indeed, OMICs constitute a research field dedicated to the systematic analysis on the organisms’ metabolisms. The methodologies used in OMICs are also highly e ective to identify critical metabolic pathways a ecting the physiology of the organisms. In this review, we analyze the main contributions provided so far by OMICs to DSTs research and discuss the prospects of OMICs with regard to the DSTs toxicology and the significance of these toxins to public health, food safety and aquacultureinfo:eu-repo/semantics/publishedVersio

Effects of Chrysosporum (Aphanizomenon) ovalisporum extracts containing cylindrospermopsin on growth, photosynthetic capacity, and mineral content of carrots (Daucus carota)

Natural toxins produced by freshwater cyanobacteria, such as cylindrospermopsin, have been regarded as an emergent environmental threat. Despite the risks for food safety, the impact of these water contaminants in agriculture is not yet fully understood. Carrots (Daucus carota) are root vegetables, extensively consumed worldwide with great importance for human nourishment and economy. It is, therefore, important to evaluate the possible effects of using water contaminated with cyanotoxins on carrot cultivation. The aim of this work was to investigate cylindrospermopsin effects on D. carota grown in soil and irrigated for 30 days, with a Chrysosporum ovalisporum extract containing environmentally relevant concentrations of cylindrospermopsin (10 and 50 μg/L). The parameters evaluated were plant growth, photosynthetic capacity, and nutritional value (mineral content) in roots of carrots, as these are the edible parts of this plant crop. The results show that, exposure to cylindrospermopsin did not have a clear negative effect on growth or photosynthesis of D. carota, even leading to an increase of both parameters. However, alterations in mineral contents were detected after exposure to crude extracts of C. ovalisporum containing cylindrospermopsin. A general decline was observed for most minerals (Ca, Mg, Na, Fe, Mn, Zn, Mo, and P), although an increase was shown in the case of K and Cu, pointing to a possible interference of the cyanobacterial extract in mineral uptake. This study is the first to evaluate the effects of C. ovalisporum extracts on a root vegetable, however, more research is necessary to understand the effects of this toxin in environmentally relevant scenarios.info:eu-repo/semantics/publishedVersio

Uncovering the Cyanobacterial Chemical Diversity: The Search for Novel Anticancer Compounds

This article belongs to the Proceedings of The 7th Iberian Congress on Cyanotoxins/3rd Iberoamerican Congress on Cyanotoxins[Abstract] Cancer has a tremendous negative socio-economic impact on our society. Thus, the discovery of new and more effective anticancer drugs is of utmost importance. To address this societal challenge, the main goal of the CYANCAN project was the discovery of anticancer compounds from cyanobacteria. These photosynthetic bacteria are considered among the most promising groups capable of producing metabolites with pharmaceutical applications. A valuable and underexplored natural resource that can underpin the discovery of promising compounds can be found in the Blue Biotechnology and Ecotoxicology Culture Collection (LEGE-CC) at CIIMAR (Interdisciplinary Centre of Marine and Environmental Research), comprising more than 700 different cyanobacterial strains. Herein, we present the recent advances implemented for finding robust anticancer lead compounds from LEGE-CC cyanobacteria. For this purpose, we developed a natural product library. Sixty cyanobacterial strains, representative of LEGE-CC biodiversity, were chromatographed to yield 480 fractions that were tested for their cytotoxic activity against 2D and 3D models of human colon carcinoma (HCT 116). The conjugation of monolayer assays and 3D cancer spheroids led to the selection of 11 active fractions, of which the chemical space was studied using an untargeted metabolomics approach. The putative annotation and identification of several compounds led to the selection of two marine strains for compound isolation: Leptothoe sp. and Lusitaniella coriacea. The isolation of the compounds was guided through bioactivity assays and mass spectrometry. These molecules were purified from the crudes by employing several chromatography methods, and the chemical structures were determined by means of NMR (nuclear magnetic resonance) and MS/MS (tandem mass spectrometry) experiments. Thus, a novel macrolide-type compound was isolated from Leptothoe sp., which presented a highly cytotoxic activity against our cancer cell models. Its effects on cancer vascularization and metastasis were studied using the zebrafish model. Moreover, from L. coriacea, five unprecedented salicyl-capped thiazol(in)e NRPs-PKs (nonribosomal peptides-polyketides) compounds were isolated. These compounds showed the potential to act as reversers of P-glycoprotein efflux activity.CIIMAR strategic funds UIDB/04423/2020 and UIDP/04423/2020; CYANCAN PTDC/MED-QUI/30944/2017, co-financed by NORTE 2020, Portugal 2020, and the European Union through the ERDF. EMERTOX: EU Horizon 2020 R&I programme under the Marie Skłodowska-Curie grant agreement No 778,069. Ribeiro, T: FCT grant SFRH/BD/139131/2018. Castelo-Branco, R: FCT grant SFRH/BD/136367/2018Centro Interdisciplinar de Investigação Marinha e Ambiental (Porto); UIDB/04423/2020Portugal. Fundação para a Ciência e a Tecnologia; PTDC/MED-QUI/30944/2017Portugal. Fundação para a Ciência e a Tecnologia; SFRH/BD/139131/2018Portugal. Fundação para a Ciência e a Tecnologia; SFRH/BD/136367/201

Bioactivity of Benthic and Picoplanktonic Estuarine Cyanobacteria on Growth of Photoautotrophs: Inhibition versus Stimulation

Understanding potential biochemical interactions and effects among cyanobacteria and other organisms is one of the main keys to a better knowledge of microbial population structuring and dynamics. In this study, the effects of cyanobacteria from benthos and plankton of estuaries on other cyanobacteria and green algae growth were evaluated. To understand how the estuarine cyanobacteria might influence the dynamics of phytoplankton, experiments were carried out with the freshwater species Microcystis aeruginosa and Chlorella sp., and the marine Synechocystis salina and Nannochloropsis sp. exposed to aqueous and organic (70% methanol) crude extracts of cyanobacteria for 96 h. The most pronounced effect observed was the growth stimulation. Growth inhibition was also observed for S. salina and M. aeruginosa target-species at the highest and lowest concentrations of cyanobacterial extracts. The methanolic crude extract of Phormidium cf. chalybeum LEGE06078 was effective against S. salina growth in a concentration-dependent manner after 96 h-exposure. All of the cyanobacterial isolates showed some bioactivity on the target-species growth, i.e., inhibitory or stimulating effects. These results indicate that the analyzed cyanobacterial isolates can potentially contribute to blooms’ proliferation of other cyanobacteria and to the abnormal growth of green algae disturbing the dynamic of estuarine phytoplankton communities. Since estuaries are transitional ecosystems, the benthic and picoplanktonic estuarine cyanobacteria can change both freshwater and marine phytoplankton succession, competition and bloom formation. Furthermore, a potential biotechnological application of these isolates as a tool to control cyanobacteria and microalgae proliferation can be feasible. This work is the first on the subject of growth responses of photoautotrophs to cyanobacteria from Atlantic estuarine environments

Structure of Hierridin C, Synthesis of Hierridins B and C, and Evidence for Prevalent Alkylresorcinol Biosynthesis in Picocyanobacteria

Small, single-celled planktonic cyanobacteria are ubiquitous in the world's oceans yet tend not to be perceived as secondary metabolite-rich organisms. Here we report the isolation and structure elucidation of hierridin C, a minor metabolite obtained from the cultured picocyanobacterium Cyanobium sp. LEGE 06113. We describe a simple, straightforward synthetic route to the scarcely produced hierridins that relies on a key regioselective halogenation step. In addition, we show that these compounds originate from a type III PKS pathway and that similar biosynthetic gene clusters are found in a variety of bacterial genomes, most notably those of the globally distributed picocyanobacteria genera Prochlorococcus, Cyanobium and Synechococcus.info:eu-repo/semantics/publishedVersio

Inhibition of Bacterial and Fungal Biofilm Formation by 675 Extracts from Microalgae and Cyanobacteria

Bacterial biofilms are complex biological systems that are difficult to eradicate at a medical, industrial, or environmental level. Biofilms confer bacteria protection against external factors and antimicrobial treatments. Taking into account that about 80% of human infections are caused by bacterial biofilms, the eradication of these structures is a great priority. Biofilms are resistant to old-generation antibiotics, which has led to the search for new antimicrobials from different sources, including deep oceans/seas. In this study, 675 extracts obtained from 225 cyanobacteria and microalgae species (11 phyla and 6 samples belonging to unknown group) were obtained from different culture collections: The Blue Biotechnology and Ecotoxicology Culture Collection (LEGE-CC), the Coimbra Collection of Algae (ACOI) from Portugal, and the Roscoff Culture Collection (RCC) from France. The largest number of samples was made up of the microalgae phylum Chlorophyta (270) followed by Cyanobacteria (261). To obtain a large range of new bioactive compounds, a method involving three consecutive extractions (hexane, ethyl acetate, and methanol) was used. The antibiofilm activity of extracts was determined against seven different bacterial species and two Candida strains in terms of minimal biofilm inhibitory concentration (MBIC). The highest biofilm inhibition rates (%) were achieved against Candida albicans and Enterobacter cloacae. Charophyta, Chlorophyta, and Cyanobacteria were the most effective against all microorganisms. In particular, extracts of Cercozoa phylum presented the lowest MBIC50 and MBIC90 values for all the strains except C. albicans