Taxonomic diversity and toxicological assessment of Cyanobacteria in Moroccan inland waters

Research on the ecology, biodiversity and toxicology of cyanobacteria in Moroccan inland waters has been carried out since 1994. The results demonstrate the existence of several taxa of cyanobacteria. Most of them are toxic, bloom‑forming species present in various water bodies of the country. The present study follows upon this earlier work and spans the 2003-2006 period. The major aim was to update and supplement the existing national cyanobacteria inventory and to isolate new toxic strains. During the study period, more than 40 aquatic environments were visited and sampled.Almost 300 taxa of cyanobacteria were recorded. They belonged to 3 orders, 14 families and 46 genera. Among these, about 78 taxa are recorded for the first time in Morocco; 29 strains of cyanobacteria were successfully isolated and cultured in the laboratory. All the collected cyanobacteria, including natural blooms, mats, and cultured strains, were analyzed for toxicity and hepatotoxins (microcystins) were quantified. Using the High-performance liquid chromatography technique coupled to photodiode array (PDA) detector (HPLC-PDA), four samples of Microcystis blooms showed the presence of microcystins (MCs), with a concentration ranging between 1.87 and 64.4 µg•g‑1 MC‑LR eq (microcystin-LR equivalents). A total of five different structural variants of MCs were detected (MC-LR, -RR, -YR, -FR, -WR). Furthermore, 3 of 29 isolates were confirmed as MCs producing strains.The results show that the widening of the survey led to a better knowledge of the diversity of cyanobacteria. The taxonomic inventory was greatly increased and several cyanobacteria strains were characterized for their toxicity. The results should be useful as a database for the identification of various aquatic environments contaminated by cyanobacterial toxins (microcystins), which represent a potent sanitary risk for human and animals.Au Maroc, les recherches sur l’écologie, la biodiversité et la toxicologie des cyanobactéries des eaux continentales ont été entamées à partir de 1994. Les résultats obtenus ont montré l’existence de plusieurs taxons de cyanobactéries, dont certains sont responsables de la formation de blooms toxiques. Faisant suite aux précédents travaux, la présente étude, réalisée lors de la période 2003-2006, où plus de 40 milieux aquatiques ont été prospectés, a pour objectif de compléter et d’actualiser l’inventaire national des cyanobactéries d’eau douce du Maroc et d’isoler de nouvelles souches toxiques.Plus de 300 taxons de cyanobactéries appartenant à 3 ordres, 14 familles et 46 genres ont été inventoriés. À notre connaissance, 78 taxons sont cités pour la première fois au Maroc et 29 souches de cyanobactéries ont pu être isolées et cultivées en laboratoire. Le matériel cyanobactérien planctonique ou benthique collecté sur le terrain (blooms, écumes, films benthiques, etc.) et la biomasse des souches isolées produite en culture au laboratoire, ont été analysés pour l’évaluation de la toxicité et la quantification des cyanotoxines (microcystines).L’utilisation de la technique HPLC-PDA (High-performance liquid chromatography technique coupled to photodiode array (PDA) detector) a permis d’identifier quatre blooms toxiques à Microcystis et la détection de microcystines (MCs) à des concentrations variant entre 1,87 et 64,4 µg•g‑1 eq MC-LR (microcystin-LR equivalents). Cinq variantes structurales de MCs ont pu être détectées (MC-LR, -RR, -YR, -FR, -WR). Parmi les 29 souches isolées et produites au laboratoire, trois seulement ont confirmé la production de microcystines.Les résultats obtenus constituent un apport substantiel à la taxonomie des cyanobactéries et à l’évaluation de la biodiversité des cyanobactéries du Maroc. Ces données peuvent être utilisées comme base pour identifier les milieux aquatiques potentiellement contaminés par les cyanobactéries et capables de générer un haut risque sanitaire pour les hommes et les animaux

Preclinical Characterization of Antioxidant Quinolyl Nitrone QN23 as a New Candidate for the Treatment of Ischemic Stroke

Nitrones are encouraging drug candidates for the treatment of oxidative stress-driven diseases such as acute ischemic stroke (AIS). In a previous study, we found a promising quinolylnitrone, QN23, which exerted a neuroprotective effect in neuronal cell cultures subjected to oxygen–glucose deprivation and in experimental models of cerebral ischemia. In this paper, we update the biological and pharmacological characterization of QN23. We describe the suitability of intravenous administration of QN23 to induce neuroprotection in transitory four-vessel occlusion (4VO) and middle cerebral artery occlusion (tMCAO) experimental models of brain ischemia by assessing neuronal death, apoptosis induction, and infarct area, as well as neurofunctional outcomes. QN23 significantly decreased the neuronal death and apoptosis induced by the ischemic episode in a dose-dependent manner and showed a therapeutic effect when administered up to 3 h after post-ischemic reperfusion onset, effects that remained 11 weeks after the ischemic episode. In addition, QN23 significantly reduced infarct volume, thus recovering the motor function in a tMCAO model. Remarkably, we assessed the antioxidant activity of QN23 in vivo using dihydroethidium as a molecular probe for radical species. Finally, we describe QN23 pharmacokinetic parameters. All these results pointing to QN23 as an interesting and promising preclinical candidate for the treatment of AIS.This work was supported by the Instituto de Salud Carlos III and co-financed by the European Development Regional Fund (FEDER) through grants PI18/00255, RD16/0019/0006, and RD21/0006/0019 to J.M. and A.A., and RD16/0019/0008 and RD21/0006/0014 to J.B.S.; the MINECO grant SAF2015-65586-R to J.M.-C.; and the Comunidad de Madrid Neurocentro project B2017/BMD-3760 to D.G.-N

Compensatory Growth Induced in Zebrafish Larvae after Pre-Exposure to a Microcystis aeruginosa Natural Bloom Extract Containing Microcystins

Early life stage tests with zebrafish (Danio rerio) were used to detect toxic effects of compounds from a Microcystis aeruginosa natural bloom extract on their embryolarval development. We carried out the exposure of developing stages of fish to complex cyanobacterial blooms containing hepatotoxic molecules - microcystins. Fish embryo tests performed with the bloom extract containing 3 mg·L−1 Eq microcystin-LR showed that after 24 h of exposure all fish embryos died. The same tests performed with other diluted extracts (containing 0.3, 0.1 and 0.03 mg·L−1 Eq microcystin-LR) were shown to have an influence on zebrafish development and a large number of embryos showed malformation signs (edema, bent and curving tail). After hatching the larvae were transferred to a medium without toxins to follow the larval development under the new conditions. The specific growth of the pre-exposed larvae was significantly more important than that of the control larvae. This may represent a compensatory growth used to reduce the difference in size with the control fish noted after hatching

Evolución a corto y largo plazo de la biomasa y microcistinas en cianobacterias del Embalse de Pinilla: Identificación y caracterización de Microcystis productoras y no productoras de microcistinas = Short and long-term evolution of cyanobacteria and biomass and its microcystin content in the drinking-water supply ; identification and characterization of microcystin producing and non-producing Microcystis strains

Tesis doctoral inédita. Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología. Fecha de lectura: 03-07-200

Caractérisation biochimique et moléculaire d’efflorescences à cyanobactéries toxiques dans le réservoir Lalla Takerkoust (Maroc)

En complément aux travaux réalisés depuis 1994 sur la toxicologie des cyanobactéries dans différents lacs-réservoirs du Maroc, le présent travail se propose d'apporter des informations supplémentaires en se fixant trois principaux objectifs: 1) Mise à jour de la base de données de la variation temporelle des teneurs en cyanotoxines intracellulaires (microcystines) d'efflorescences cyanobactériennes fréquemment occasionnés dans le lac réservoir Lalla Takerkoust; 2) Évaluation de la contamination des eaux brutes du lac par les microcystines (MC) (quantification des MC extracellulaires); 3) Caractérisation de la diversité moléculaire des souches cyanobactériennes par la détection des gènes de la synthèse des cyanotoxines (MC) en utilisant les deux méthodes multiplex‑PCR et RFLP (Restriction Fragment Length Polymorphism). L'analyse par HPLC des échantillons 2005 et 2006 d'efflorescences cyanobactériennes a montré qu'il y a une variation qualitative et quantitative des microcystines intracellulaires (MC). L'évaluation des teneurs en MC et dissoutes dans l'eau brute, par ELISA, a révélé des quantités très importantes de MC extracellulaires avec un maximum de 95,4 μg•L‑1 durant le mois de décembre 2005 (phase de déclin du développement des cyanobactéries). En général, durant l'année, les concentrations des MC dissoutes restent toujours au‑dessus de la valeur guide recommandée par l'OMS pour l'eau de boisson (1 μg•L‑1). La caractérisation moléculaire, recherche du gène de synthèse des MC, a confirmé que seule Microcystis aeruginosa est la souche productrice des MC au sein de la fraction phytoplanctonique. Ce travail a pour mérite de confirmer pour la première fois au Maroc que, lors de proliférations d'efflorescences cyanobactériennes à Microcystis, les teneurs en MC dans les eaux brutes du lac sont si importantes qu'il est fortement recommandé de prendre en compte les divers risques sanitaires potentiellement engendrés par cette contamination lors de l'utilisation de cette eau (eau d'alimentation sans traitement, eau récréative, eau d'irrigation, etc.). D'après ces résultats, nous concluons que la mise en place d'un programme de surveillance des cyanobactéries et de contrôle des cyanotoxines (MC) doit être basée sur la caractérisation biochimique (détermination de la nature et de la quantité de toxines produites) et complémentée par la caractérisation génétique des souches potentiellement productrices de ces cyanotoxines.Complementary to earlier research carried out since 1994 related to toxic cyanobacteria in different Moroccan lake-reservoirs, this work furnishes new data relating to this research topic. Three major objectives were established: 1) update knowledge concerning the temporal variation of the intracellular contents of cyanotoxines (microcystines, MCs) in cyanobacteria blooms, which frequently occur in the Lalla Takerkoust lake-reservoir; 2) evaluate the MC contamination of raw waters (quantification of extracellular MCs); 3) molecular characterization of cyanobacteria MC producing strains (detection of genes involved in MC synthesis using two methods, multiplex-PCR and RFLP (Restriction fragment length polymorphism)). For bloom samples collected during 2005-2006, the HPLC analysis showed quantitative and qualitative temporal variation in intracellular MCs. Evaluation of the extracellular MC content, determined by ELISA analysis, revealed a maximum concentration of 95.4 μg•L-1 in raw water. This value was measured during December 2005, corresponding to the decline of a Microcystis bloom (cell lysis and MC release into the surrounding water). In general, the dissolved MC concentrations in raw water always remained above the guideline value recommended by the World Health Organization for drinking water (1 μg•L-1). The genetic analysis, based on the detection of the MC synthetase gene ( mcyA gene), confirmed that among the bloom-forming phytoplanktonic fraction, Microcystis aeruginosa is the only MC producing strain. This work thus confirms for the first time in Morocco that during Microcystis blooms, the MC extracellular content greatly exceeds WHO guidelines. It is strongly recommended that the various potential sanitary risks involved in the use of such untreated water (drinking, recreation and irrigation waters) be taken into account. Based on these results, we conclude that the establishment of any successful monitoring programs for cyanobacteria and MC should be based first on biochemical characterization (identification and determination of the amount of produced toxins) and be supplemented by the genetic characterization of the toxin-producing strains