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

Ocorrência e disseminação da microalga Gonyostomum semen em albufeiras portuguesas

Objetivos: Neste artigo pretende-se descrever a deteção de G. semen em amostras de água doce colhidas em albufeiras portuguesas. São ainda apresentados dados preliminares relativos à sua distribuição geográfica em território continental, no período entre 2009 e 2014

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

Successful isolation and cultivation of Cylindrospermopsis raciborskii strains isolated from finished drinking water samples

This work presents the successful establishment of Cylindrospermosis raciborskii cultures isolated from water samples collected at the exit point of Water Treatment Plant (WTP). An intense bloom dominated by filamentous cyanobacteria (Aphanizomenon spp., Planktothrix spp., Cylindrospermosis raciborskii, Anabaena spp.) occurred in the summer of 2015 in Roxo reservoir (south Portugal). Several cyanotoxins (microcystins, saxitoxins, cylindrospermopsin) were detected in raw and treated water, although at levels below the corresponding regulatory and/or guideline values. Nevertheless, this bloom caused intense unpleasant odour and taste in the water supplied to the populations and cyanobacterial cells (up to 1000 cells.mL-1) were detected in finished water samples collected at the exit point of WTP. Treated water samples collected at the WTP and at the city water deposit were inoculated in Z8 culture medium and cyanobacterial growth was followed by optical microscopy. After 30 days, cyanobacterial growth was observed showing resistance to the treatment processes and maintenance of reprodution capacity. Interestingly, morphometric and molecular analysis revealed the presence of C. raciborskii. Three isolates of this species were obtained and none were cylindrospermopsin- or microcystins-producers, as confirmed by Enzyme Linked Immunosorbent Assays (ELISA) and by amplification of genes (PS, PKS, mcyA-cd, mcyAB, mcyB) involved in those cyanotoxin synthesis. However, the ELISA for saxitoxins was positive for the 3 isolates and confirmation of this toxin production is in progress. To our knowledge, this is the first report on the establishment of successful cultures of C. raciborskii that survived to conventional water treatment processes.N/

The Kidney Vero-E6 Cell Line: A Suitable Model to Study the Toxicity of Microcystins

Microcystins (MCs) are toxins produced by cyanobacteria from water environments that can induce acute and chronic effects on humans and animals, after ingestion/contact with contaminated water. This group of cyclic heptapeptides comprises approximately 80 variants, being microcystin-LR (MCLR) the most frequent and toxic variant. The studies of MCs effects on cell lines often lead to contradictory results, given the fact that distinct MC toxicity endpoints (mainly cytotoxicity and genotoxicity) have been studied in diverse cell lines (and cell clones) under distinct exposure conditions (different doses-ranges, time of exposure, MCs variants, etc). In our work with Vero-E6 cells we tested MCLR (both pure toxin and from cyanobacterial extracts of M. aeruginosa) within a wide range of concentrations (1 nM- 200 μM), using several endpoints and methodologies (cytotoxicity, morphology, genotoxicity, protein expression). In this chapter we will summarize our results and discuss the utility of Vero-E6 cell line to evaluate the toxicological properties of MCLR

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

Isolation and characterization of cylindrospermopsis raciborskii strains from finished drinking water

In the summer of 2015, an intense cyanobacterial bloom producing geosmin/2- methylisoborneol (MIB) occurred in the Roxo freshwater reservoir in Alentejo, Portugal. The drinking water supplied from the Roxo water treatment plant (WTP) exhibited an unpleasant odor/taste and a significant cyanobacteria density was detected in the finished water at the exit of the WTP. Cyanobacteria were not evaluated downstream of the WTP, namely, at the city reservoir. The aim of this work was to isolate and characterize viable cyanobacteria present in finished water (exit of the WTP and city reservoir) that withstand conventional water treatment. Treated water samples collected at both sites were inoculated in Z8 culture medium to provide the conditions for putative cyanobacterial growth. After 30 days, filamentous cyanobacteria were observed in cultures inoculated with samples from the exit point of the WTP. Viable trichomes were isolated and identified as Cylindrospermopsis raciborskii by morphometric and molecular analysis. None of the isolates were cylindrospermopsin/microcystin producers, as confirmed by ELISA and amplification of corresponding genes (PS/PKS and mcyA-cd/mcyAB/mcyB). ELISA results were positive for saxitoxin, but saxitoxin and derivatives were not detected by liquid chromatography with fluorescence detection (LC-FLD), nor were their related genes (sxtA/sxtA4/sxtB/sxtM/sxtPer/sxtI). To our knowledge, this is the first report on the establishment of cultures of C. raciborskii that resisted water treatment processes.Portuguese Foundation for Science and Technology (FCT) for the grant SFRH/BPD/77981/2011 attributed to E.D. This work was partially supported by FCT/MCTES through national funds for the research project “Exploring the Aquatic Resistome” (PTDC/BIA-BMA/31451/2017) and the project UIDB/00211/2020.info: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)