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

Avaliação da influência da intensidade de luz na expressão do gene mcyA e na produção de microcistina em Microcystis aeruginosa e Planktothrix agardhii

As cianobactérias são frequentemente associadas à produção de toxinas, nomeadamente microcistinas. A sua síntese é não ribossomal, e acontece utilizando complexos multienzimáticos (genes mcy). Diversos estudos têm demonstrado que os fatores ambientais podem influenciar a produção de toxina. O objetivo deste estudo foi avaliar a influência da intensidade da luz na transcrição do gene mcyA e correspondente produção de microcistina em isolados tóxicos de Microcystis aeruginosa e Planktothrix agardhii. Para esse fim, as culturas foram expostas a três diferentes intensidades de luz (4, 20 e 30 µmol fotões m-2 s-1) durante 18 dias a 20 ± 1ºC. O crescimento foi seguido diariamente espectrofotometricamente. O nível de transcritos foi quantificado por RT-qPCR e a expressão relativa determinada usando três genes de referência - rRNA 16S, gltA e rpoc1. Os resultados mostraram a existência de uma correspondência entre a taxa de crescimento e a intensidade de luz em ambas as espécies. As taxas de crescimento foram menores a 4 e maiores a 30 µmol fotões m-2 s-1. Em M. aeruginosa a concentração de microcistina por célula foi semelhante entre intensidades de luz e ao longo do tempo, enquanto que em P. agardhii a concentração foi mais elevada na fase estacionária a 4 µmol fotões m-2 s-1. Existiram diferenças na expressão de mcyA entre as duas espécies. Em M. aeruginosa, a expressão foi máxima a 4 µmol fotões m-2 s-1 na fase de adaptação, já em P. agardhii foi máxima a 4 µmol fotões m-2 s-1 na fase exponencial de crescimento

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/

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

Temporal variability of microcystin (mcyA) genotypes in a toxic cyanobacterial bloom

In this work a perennial bloom was monitored during two years (2012-2014) in order to characterize the genotype structure and succession.FCT-Ph.D research grant SFRH/BD65706/2009info:eu-repo/semantics/publishedVersio

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)