Eutrophication, toxic cyanobacteria and cyanotoxins: when ecosystems cry for help

Eutrophication of freshwater resources has been studied worldwide and its consequences are of concern especially in waters used for recreation or drinking. The occurrence of cyanobacteria blooms is nowadays much better documented and among the major impacts we may point out the decrease in water transparency and oxygen levels, the production of off-flavours and the production of toxins. Among the most common toxins, microcystins, anatoxin-a and saxitoxins are the most common in Portugal. A diversity of microcystins has been identified so far but microcystin-LR is the most common. Many aquatic organisms are capable to resist certain levels of cyanotoxins making them vectors for toxins. We analysed this possibility using a diversity of cyanotoxins and organisms and found that microcystins and bivalves seem to be the combination that maximizes toxin transfer. New challenges are presented today in this research area, by using new and more sensitive chemical, biochemical and molecular techniques. This allows us to better understand these organisms that can produce both potent toxins and innovative drugs.La eutrofización de las aguas dulces ha sido estudiada por todo el mundo y sus consecuencias son una preocupación en aguas utilizadas para ocio o para beber. La ocurrencia de florecimientos de cianobacterias esta hoy mucho mejor documentada y entre los impactos más grandes podemos destacar la disminución de la transparencia del agua y de los niveles de oxigeno disuelto, la producción de olores y sabores y de toxinas. De entre las toxinas más comunes, las microcistinas, anatoxina-a y saxitoxinas son las más comunes en Portugal. Hasta ahora identificamos una gran diversidad de microcistinas pero la microcistina-LR es la más común. Muchos organismos acuáticos pueden resistir a ciertos niveles de cianotoxinas, haciendo que sean vectores de toxinas. Analizamos esta posibilidad utilizando una diversidad de cyanotoxinas y de organismos y encontramos que las microcistinas y los bivalvos parecen ser la combinación que maximiza la transferencia de toxinas. Hoy se nos presentan nuevos desafíos en esta área de investigación, utilizando técnicas químicas, bioquímicas y moleculares nuevas y más sensibles. Esto nos permite entender mejor estos organismos que pueden producir potentes toxinas y nueva drogas

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

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

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

Avaliação do efeito da microcistina-LR no crescimento, sistema antioxidante e indução de apoptose em Saccharomyces cerevisiae

Neste trabalho pretendeu-se usar a levedura Saccharomyces cerevisiae como modelo para compreender melhor os mecanismos de toxicidade induzidos pela microcistina-LR. Este é o organismo eucariótico mais simples e estudado que tem sido amplamente utilizado como modelo no estudo de mecanismos de toxicidade, devido à facilidade de acesso à informação sobre o seu genoma (totalmente sequenciado e anotado), proteoma e processos bioquímicos correspondentes.Este trabalho foi parcialmente financiado pelo Programa Operacional Potencial Humano, pela Fundação para a Ciência e a Tecnologia através da Bolsa de pós-doc SFRH/BPD/75922/2011 e o Projeto PEst-C/MAR/LA0015/2013

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)

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

The Role of Gene Duplication and Unconstrained Selective Pressures in the Melanopsin Gene Family Evolution and Vertebrate Circadian Rhythm Regulation

Melanopsin is a photosensitive cell protein involved in regulating circadian rhythms and other non-visual responses to light. The melanopsin gene family is represented by two paralogs,OPN4x and OPN4m, which originated through gene duplication early in the emergence of vertebrates. Here we studied the melanopsin gene family using an integrated gene/protein evolutionary approach, which revealed that the rhabdomeric urbilaterian ancestor had the same amino acid patterns (DRY motif and the Y and E conterions) as extant vertebrate species, suggesting that the mechanism for light detection and regulation is similar to rhabdomeric rhodopsins. Both OPN4m and OPN4x paralogs are found in vertebrate genomic paralogons, suggesting that they diverged following this duplication event about 600 million years ago, when the complex eye emerged in the vertebrate ancestor. Melanopsins generally evolved under negative selection (ω = 0.171) with some minor episodes of positive selection (proportion of sites = 25%) and functional divergence (θI = 0.349 and θII = 0.126). The OPN4m and OPN4x melanopsin paralogs show evidence of spectral divergence at sites likely involved in melanopsin light absorbance (200F, 273S and 276A). Also, following the teleost lineage-specific whole genome duplication (3R) that prompted the teleost fish radiation, type I divergence (θI = 0.181) and positive selection (affecting 11% of sites) contributed to amino acid variability that we related with the photo-activation stability of melanopsin. The melanopsin intracellular regions had unexpectedly high variability in their coupling specificity of G-proteins and we propose that Gq/11 and Gi/o are the two G-proteins most-likely to mediate the melanopsin phototransduction pathway. The selection signatures were mainly observed on retinal-related sites and the third and second intracellular loops, demonstrating the physiological plasticity of the melanopsin protein group. Our results provide new insights on the phototransduction process and additional tools for disentangling and understanding the links between melanopsin gene evolution and the specializations observed in vertebrates, especially in teleost fish

New Method for Simultaneous Determination of Microcystins and Cylindrospermopsin in Vegetable Matrices by SPE-UPLC-MS/MS

Cyanotoxins are a large group of noxious metabolites with different chemical structure and mechanisms of action, with a worldwide distribution, producing effects in animals, humans, and crop plants. When cyanotoxin-contaminated waters are used for the irrigation of edible vegetables, humans can be in contact with these toxins through the food chain. In this work, a method for the simultaneous detection of Microcystin-LR (MC-LR), Microcystin-RR (MC-RR), Microcystin-YR (MC-YR), and Cylindrospermopsin (CYN) in lettuce has been optimized and validated, using a dual solid phase extraction (SPE) system for toxin extraction and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) for analysis. Results showed linear ranges (5–50 ng g−1 f.w.), low values for limit of detection (LOD) (0.06–0.42 ng g−1 f.w.), and limit of quantification (LOQ) (0.16–0.91 ng g−1 f.w.), acceptable recoveries (41–93%), and %RSDIP values for the four toxins. The method proved to be robust for the three variables tested. Finally, it was successfully applied to detect these cyanotoxins in edible vegetables exposed to cyanobacterial extracts under laboratory conditions, and it could be useful for monitoring these toxins in edible vegetables for better exposure estimation in terms of risk assessment.España MINECO AGL2015-64558-

Mammalian Keratin Associated Proteins (KRTAPs) Subgenomes: Disentangling Hair Diversity and Adaptation to Terrestrial and Aquatic Environments

Background: Adaptation of mammals to terrestrial life was facilitated by the unique vertebrate trait of body hair, which occurs in a range of morphological patterns. Keratin associated proteins (KRTAPs), the major structural hair shaft proteins, are largely responsible for hair variation. Results: We exhaustively characterized the KRTAP gene family in 22 mammalian genomes, confirming the existence of 30 KRTAP subfamilies evolving at different rates with varying degrees of diversification and homogenization. Within the two major classes of KRTAPs, the high cysteine (HS) subfamily experienced strong concerted evolution, high rates of gene conversion/recombination and high GC content. In contrast, high glycine-tyrosine (HGT) KRTAPs showed evidence of positive selection and low rates of gene conversion/recombination. Species with more hair and of higher complexity tended to have more KRATP genes (gene expansion). The sloth, with long and coarse hair, had the most KRTAP genes (175 with 141 being intact). By contrast, the hairless dolphin had 35 KRTAPs and the highest pseudogenization rate (74% relative to the 19% mammalian average). Unique hair-related phenotypes, such as scales (armadillo) and spines (hedgehog), were correlated with changes in KRTAPs. Gene expression variation probably also influences hair diversification patterns, for example human have an identical KRTAP repertoire as apes, but much less hair. Conclusions: We hypothesize that differences in KRTAP gene repertoire and gene expression, together with distinct rates of gene conversion/recombination, pseudogenization and positive selection, are likely responsible for micro and macro-phenotypic hair diversification among mammals in response to adaptations to ecological pressures