Abordagem multidisciplinar na identificação e monitorização de cianobactérias potencialmente tóxicas

O risco que as florescências cianobacterianas representam para a saúde humana advêm do facto destes desenvolvimentos excessivos estarem frequentemente associados à produção de cianotoxinas. As principais vias de exposição para o homem são através de água potável contaminada, diálise, consumo de peixe e marisco contaminado e atividades recreativas. A toxicidade destes compostos é elevada, como pode ser constatado no gráfico 1 em que está representada a comparação da toxicidade, com base na dose-letal (LD50%) em murganhos, entre as cianotoxinas e algumas das toxinas mais conhecidas em relação ao cianeto

Applicability of the real-time PCR assay in the amplification of cyanobacterial DNA from preserved samples

Publicaciones del III Congreso Ibérico de Cianotoxinas (Blanes, 2013)The study and monitoring of cyanobacterial blooms often involves the use of preserved samples to avoid cellular degradation. However, preserved samples may not be suitable for molecular biology studies because preservation methods can interfere with DNA quality/quantity. Real-time quantitative PCR analysis (qPCR) has been widely applied in molecular analysis and is considered a promising method for monitoring purposes. This study intended to evaluate the applicability of the real-time qPCR technique in samples that were subjected to different methods of preservation: (1) 15% Lugol’s iodine solution (2) 4% formaldehyde and (3) 25% glutaraldehyde. The ability to amplify and quantify DNA extracted from Planktothrix agardhii was assessed using the rpoC1 gene as the target fragment in both raw water samples and in vitro cultures. No reliable DNA amplification was obtained from glutaraldehyde-preserved samples. Successful amplification was obtained from Lugol’s and formaldehyde-preserved samples. In this case, however, the quantification that was achieved by real-time PCR cannot be used to infer cell numbers, because the Ct values that were obtained from the Lugol’s and formaldehydepreserved samples were significantly higher than the Ct values that were obtained from the unpreserved samples. Therefore real-time PCR can be used for the detection and identification of cyanobacteria in preserved samples but no reliable cell quantification can be performed using this method.This research was supported by the Fundação para Ciência e Tecnologia, Portugal (FCT) through the project PPCDT/AMB/67075/2006. The authors also acknowledge the PhD research grant SFRH/BD65706/2009 to C. Churro and the Post-Doc research grant SFRH/BPD/75922/2011 to E. Valério

Risk levels of toxic cyanobacteria in portuguese recreational freshwaters

Portuguese surface freshwaters are widely used as a source of drinking water as well as bathing water. Cyanobacterial blooming in these water resources are common and are often associated with cyanotoxin production. The Portuguese legislation for drinking water (Decreto-Lei nº306/2007) establishes the regulatory level of 1 μg/L for total microcystins in treated water. This parameter is determined when eutrophication of water is suspected and when the number of potentially toxic cyanobacteria exceeds 2000 cells/mL. Conversely, the Portuguese legislation concerning the quality of bathing water (Decreto-Lei nº 135/2009), that was transposed form the European Directive nº2006/7/CE, do not include any guideline for cyanobacterial cells nor microcystins concentrations. It only recommends that when the bathing water profile indicates a potential for cyanobacterial proliferation, appropriate monitoring shall be carried out to enable timely identification of health risks. When cyanobacterial proliferation is detected visually, it is the responsibility of the local health delegate to evaluate the associated risk. If any risk has been identified or presumed, health and environmental authorities should implement the adequate management measures to prevent exposure, including information to the public. According to national specificities, some European countries complemented the European Directive nº2006/7/CE and implemented their own guidance or regulations, based on cyanobacterial cell numbers, biovolumes, pigments and/or cyanotoxin concentrations (Ibelings et al., 2015; Chorus, 2012). Prior to establishing regulatory or guideline values, it will be fundamental to characterize Portuguese inland bathing waters concerning the frequency, density, specie composition and toxicity of cyanobacterial blooms. These data are available but not systematized at a national scale. In this work we present the results of the monitoring of cyanobacteria and microcystins in 8 freshwater reservoirs located in the centre of Portugal largely used for bathing and recreational activities. These results will contribute to identify the cyanobacterial blooms profile and to assess the risk level of toxic cyanobacteria in Portuguese recreational freshwaters.N/

Monitorização do gene mcyA e de microcistina numa florescência de Planktothrix agardhii – Que papel desempenha o parasitismo quitrídeo na dinâmica destas florescências?

A cianobactéria Planktothrix agardhii forma blooms persistentes em reservatórios de água doce superficiais e está frequentemente associada à presença de microcistinas. No entanto, densidades celulares elevadas de P.agardhii nem sempre correspondem a níveis elevados de microcistinas e vice-versa. As florescências de Planktothrix sp. são constituídas por estirpes tóxicas e não-tóxicas que são visualmente indistinguíveis. Contudo, as estirpes tóxicas podem ser quantificadas molecularmente uma vez que possuem um conjunto de genes envolvidos na síntese de microcistinas, onde se inclui o gene mcyA. Neste trabalho, foi monitorizada uma florescência perene de P.agardhii durante dois anos (2012-2014), com o objectivo de caracterizar a variabilidade temporal dos genótipos tóxicos e concentração de microcistinas. Foram também medidos vários parâmetros físico-químicos (nitratos, fósforo total, pH e condutividade) e biológicos (presença de quitrídeos parasitas nos tricomas de P.agardhii). A concentração total de microcistina na água foi medida por ELISA. O nº de cópias do gene mcyA e do gene 18SrDNA de fungos quitrídeos foi quantificado por PCR em tempo-real. Os resultados demonstraram que a quantidade do gene mcyA e a concentração de microcistina total variam ao longo do tempo estando ambos correlacionados (coeficiente de correlação de Spearman de 0,84). O período em que a concentração do gene mcyA e de microcistina foi mais elevado coincidiu com a presença de parasitas quitrídeos da cianbactéria P.agardhii. A quantidade do gene 18SrDNA correlaciona-se com o gene mcyA (coeficiente de correlação de Spearman de 0,83) e com a concentração de microcistina (coeficiente de correlação de Spearman de 0,82). Não houve qualquer correlação entre os parâmetros físico-químicos e a concentração do gene mcyA e de microcistinas. Face aos resultados obtidos colocam-se questões que interessa explorar: Qual será a influencia dos parasitas quitrídeos na modelação da densidade e toxicidade das florescências de P.agardhii? Será que a sua presença favorece o aparecimento de florescências tóxicas? Que factores influenciam a relação parasitas quitrídeos- Planktothrix

Evaluation of phylogenetic markers suitable for Planktothrix spp. discrimination

In Portugal, potentially toxic cyanobacteria from Planktothrix genus have become frequently observed in freshwater reservoirs. Identification of Planktothrix species through optical microscopy is complicated due to limited morphological differences among them. The aim of this work was to determine the most suitable phylogenetic markers that could be used for the molecular identification of Planktothrix species. In order to do so, several genes of interest were selected: rpoB, rpoC1, cpcA, cpcB, rbcX, 16S rRNA genes and 16S rRNA–tRNAIle–tRNAAla-23S rRNA internal transcribed spacer (ITS), and their sequences retrieved from public databases. Phylogenetic analysis showed that 16S rRNA, cpcA, rbcX genes and ITS region trees do not allow a clear discrimination of Planktothrix species, however cpcB and rpoB seemed putative suitable phylogenetic markers for Planktothrix species identification. The applicability of these markers was then evaluated in 20 Planktothrix isolates, isolated over the years from several Portuguese freshwater reservoirs and maintained in the Estela Sousa e Silva Algae Culture Collection (ESSACC). The selected genes, cpcB and rpoB, were amplified by PCR and sequenced and the resulting trees compared with the phylogenetic clustering obtained with our previously characterized rpoC1 phylogenies. The phylogenetic analyses, based on the three gene regions, revealed that Planktothrix isolates analyzed in this study could be phylogenetically resolved into their corresponding species. This work contributes for the discussion of the appropriate genes that can be used in phylogenetic identification of Planktothrix species.The authors would like to thank the Ph.D research grant SFRH/BD65706/2009 to Catarina Churro and the financial support through project PPCDT/AMB/60675/2006 both given by Fundação para a Ciência e Tecnologia (Portugal)

Coleção de culturas de algas Estela Sousa e Silva

A coleção de culturas de algas Estela Sousa e Silva (ESSACC) foi criada em 1956 e reside atualmente no Laboratório de Biologia e Ecotoxicologia no Instituto Nacional de Saúde Dr. Ricardo Jorge. A ESSACC foi implementada em resposta à necessidade de um repositório de material biológico para investigação na área do fitoplâncton. Este importante recurso biológico contém culturas monoclonais de algas eucarióticas e cianobactérias provenientes de águas costeiras e albufeiras portuguesas. Atualmente, a coleção mantém acima de 176 isolados vivos, dos quais 151 são cianobactérias de água doce e 25 são dinoflagelados marinhos. Adicionalmente são também mantidos alguns isolados pertencentes a outros grupos de fitoflagelados. Esta coleção permitiu até agora a identificação e caracterização de espécies assim como a produção e purificação de toxinas para aplicação em estudos toxicológicos entre outras diversas áreas de investigação. Deste modo, a ESSACC constitui uma ferramenta importante no fornecimento de culturas de algas a investigadores na área do fitoplâncton, particularmente no estudo de espécies nocivas

Detection of a Planktothrix agardhii Bloom in Portuguese Marine Coastal Waters

Cyanobacteria blooms are frequent in freshwaters and are responsible for water quality deterioration and human intoxication. Although, not a new phenomenon, concern exists on the increasing persistence, scale, and toxicity of these blooms. There is evidence, in recent years, of the transfer of these toxins from inland to marine waters through freshwater outflow. However, the true impact of these blooms in marine habitats has been overlooked. In the present work, we describe the detection of Planktothrix agardhii, which is a common microcystin producer, in the Portuguese marine coastal waters nearby a river outfall in an area used for shellfish harvesting and recreational activities. P. agardhii was first observed in November of 2016 in seawater samples that are in the scope of the national shellfish monitoring system. This occurrence was followed closely between November and December of 2016 by a weekly sampling of mussels and water from the sea pier and adjacent river mouth with salinity ranging from 35 to 3. High cell densities were found in the water from both sea pier and river outfall, reaching concentrations of 4,960,608 cellsL(-1) and 6810.3 x 10(6) cellsL(-1) respectively. Cultures were also established with success from the environment and microplate salinity growth assays showed that the isolates grew at salinity 10. HPLC-PDA analysis of total microcystin content in mussel tissue, water biomass, and P. agardhii cultures did not retrieve a positive result. In addition, microcystin related genes were not detected in the water nor cultures. So, the P. agardhii present in the environment was probably a non-toxic strain. This is, to our knowledge, the first report on a P. agardhii bloom reaching the sea and points to the relevance to also monitoring freshwater harmful phytoplankton and related toxins in seafood harvesting and recreational coastal areas, particularly under the influence of river plumes.project SNMB-INOV: Innovation for a more competitive shellfish sector - Portuguese Government, Operational Program (OP), Portugal; European Union through European Structural Funds and Investment Funds (FEEI); European Maritime and Fisheries Fund (EMFF); Centre of Marine Sciences (CCMAR), University of Algarve and Interdisciplinary Centre of Marine and Environmental Research (CIIMAR) of the University of Porto [UID/Multi/04326/2013]; RD Units Strategic Plan from the Portuguese Foundation for Science and Technology (FCT) [UID/Multi/04423/2013]info:eu-repo/semantics/publishedVersio

Development of Toxicological Risk Assessment Models for Acute and Chronic Exposure to Pollutants

Alert level frameworks advise agencies on a sequence of monitoring and management actions, and are implemented so as to reduce the risk of the public coming into contact with hazardous substances. Their effectiveness relies on the detection of the hazard, but with many systems not receiving any regular monitoring, pollution events often go undetected. We developed toxicological risk assessment models for acute and chronic exposure to pollutants that incorporate the probabilities that the public will come into contact with undetected pollution events, to identify the level of risk a system poses in regards to the pollutant. As a proof of concept, we successfully demonstrated that the models could be applied to determine probabilities of acute and chronic illness types related to recreational activities in waterbodies containing cyanotoxins. Using the acute model, we identified lakes that present a ‘high’ risk to develop Day Away From Work illness, and lakes that present a ‘low’ or ‘medium’ risk to develop First Aid Cases when used for swimming. The developed risk models succeeded in categorising lakes according to their risk level to the public in an objective way. Modelling by how much the probability of public exposure has to decrease to lower the risks to acceptable levels will enable authorities to identify suitable control measures and monitoring strategies. We suggest broadening the application of these models to other contaminants