Papers by Francesco Pisapia
Les dinoflagelles epi-benthiques Gambierdiscus et Fukuyoa produisent les ciguatoxines (CTXs) et l... more Les dinoflagelles epi-benthiques Gambierdiscus et Fukuyoa produisent les ciguatoxines (CTXs) et les maitotoxines (MTXs), qui sont parmi les toxines marines les plus puissantes connues. Les CTXs sont bioaccumulees et bio-transformees le long de la chaine alimentaire marine et provoquent la ciguatera, l'intoxication alimentaire non bacterienne la plus commune au monde. Recemment, la ciguatera a ete rapporte dans des zones non endemiques, notamment les iles Canaries, et un grand nombre d'especes a ete decouvert au sein de ces genres. Peu de donnees sont disponibles sur la toxicite relative entre les differentes especes et sur les congeneres de toxines produits par ces dinoflagelles, en particulier par des souches en dehors de l'ocean Pacifique. Plusieurs souches de Gambierdiscus et Fukuyoa ont ete examinees pour leur cigua- et maito-toxicite via les tests neuro-2a et hemolytique, respectivement. G. excentricus a montre une forte toxicite et deux souches des iles Canaries ont ete selectionnees pour la purification des toxines via une approche de fractionnement bioguide. L'analyse non-ciblee de spectrometrie de masse (SM) a haute resolution a permis la decouverte d'un nouveau congenere de MTX, la maitotoxine-4 (MTX4). La SM a basse resolution de plusieurs souches de Gambierdiscus et Fukuyoa n’a revele la presence de MTX4 que dans G. excentricus, y compris des souches provenant des iles Canaries, du Bresil et du Golfe du Mexique. La MTX4 pourrait donc servir de biomarqueur pour l’espece hautement toxique G. excentricus. Les travaux futurs porteront sur l'isolement et l'elucidation de la structure de la MTX4 et l'identification des congeneres de CTX produits par G. excentricus.
Rapid Communications in Mass Spectrometry, Aug 26, 2020
Rationale The dinoflagellate genera Gambierdiscus and Fukuyoa are producers of toxins responsible... more Rationale The dinoflagellate genera Gambierdiscus and Fukuyoa are producers of toxins responsible for Ciguatera Poisoning (CP). Although having very low oral potency, maitotoxins (MTXs) are very toxic following intraperitoneal injection and feeding studies have shown they may accumulate in fish muscle. To date, six MTX congeners have been described but two congeners (MTX2 and MTX4) have not yet been structurally elucidated. The aim of the present study was to further characterize MTX4. Methods Chemical analysis was performed using Liquid Chromatography coupled to a Diode Array Detector (DAD) and positive ionization mode High Resolution Mass Spectrometry (LC/HRMS) on partially purified extracts of G. excentricus (strain VGO792). HRMS/MS studies were also carried out to tentatively explain the fragmentation pathways of MTX and MTX4. Results The comparison of UV and HRMS (ESI+) spectra between MTX and MTX4 led us to propose the elemental formula of MTX4 (C157H241NO68S2, as unsalted molecule). The comparison of the theoretical and measured m/z values of the doubly charged ions of the isotopic profile in ESI+ were coherent with the proposed elemental formula of MTX4. The study of HRMS/MS spectra on the triammoniated adduct ([M-H+3NH4]2+) of both molecules gave additional information about structural features. The cleavage observed, probably located at C99-C100 in both MTX and MTX4, highlighted the same Aside product ion shared by the two molecules. Conclusion Please note that this is an author-produced PDF of an article accepted for publication following peer review. The definitive publisher-authenticated version is available on the publisher Web site. All these investigations on the characterization of MTX4 contribute to highlighting that MTX4 belongs to the same structural family of MTXs. However to accomplish a complete structural elucidation of MTX4, NMR-based study and LC/HRMSn investigation will have to be carried out.
Zenodo (CERN European Organization for Nuclear Research), Apr 24, 2019
Manual de Cultivo de Gambierdiscus y Fukuyoa spp. • 7 ES Manual de Cultivo de Gambierdiscus y Fuk... more Manual de Cultivo de Gambierdiscus y Fukuyoa spp. • 7 ES Manual de Cultivo de Gambierdiscus y Fukuyoa spp. • 13 ES Manual de Cultivo de Gambierdiscus y Fukuyoa spp. • 21 ES
<p>This figure shows the estimated toxin production (fg CTX3C eq. cell<sup>-1</sup... more <p>This figure shows the estimated toxin production (fg CTX3C eq. cell<sup>-1</sup> d<sup>-1</sup>) rate for each species.</p
<p><i>Gambierdiscus excentricus</i> and <i>G</i>. <i>silvae&l... more <p><i>Gambierdiscus excentricus</i> and <i>G</i>. <i>silvae</i> were excluded from the analysis because only a single clone was examined. Abbreviations: n = sample size, M = median toxicity (fg CTX3C eq. cell<sup>-1</sup>), <i>H</i> = Kruskal-Wallis test statistic, <i>df</i> = degrees of freedom. Brackets denote result of the Dunn’s follow up test. The statistic is designed to estimate median toxicities to determine if the species partitioned into distinct groups.</p
Les dinoflagellés epi-benthiques Gambierdiscus et Fukuyoa produisent les ciguatoxines (CTXs) et l... more Les dinoflagellés epi-benthiques Gambierdiscus et Fukuyoa produisent les ciguatoxines (CTXs) et les maïtotoxines (MTXs), qui sont parmi les toxines marines les plus puissantes connues. Les CTXs sont bioaccumulées et bio-transformées le long de la chaîne alimentaire marine et provoquent la ciguatéra, l'intoxication alimentaire non bactérienne la plus commune au monde. Récemment, la ciguatéra a été rapporté dans des zones non endémiques, notamment les îles Canaries, et un grand nombre d'espèces a été découvert au sein de ces genres. Peu de données sont disponibles sur la toxicité relative entre les différentes espèces et sur les congénères de toxines produits par ces dinoflagellés, en particulier par des souches en dehors de l'océan Pacifique. Plusieurs souches de Gambierdiscus et Fukuyoa ont été examinées pour leur cigua- et maïto-toxicité via les tests neuro-2a et hémolytique, respectivement. G. excentricus a montré une forte toxicité et deux souches des îles Canaries on...
<p>The citations in the species column indicate where the species was described. The refere... more <p>The citations in the species column indicate where the species was described. The reference(s) under the strain designation indicate other publications where the strain has been studied. Many of the strains analyzed for CTX-like activity in this study were also assayed for maitotoxicity in separate investigations [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185776#pone.0185776.ref032" target="_blank">32</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185776#pone.0185776.ref033" target="_blank">33</a>]. The strain growth rates (± standard deviation) were determined from triplicate, independent cultures started for each isolate. Mean species growth rates and average toxicities were determined by averaging all replicate culture data for a given species. Toxicity was normalized both as femtograms (fg) CTX3C equivalents [eq.] cell<sup>-1</sup> and per biovolume attograms (ag) CTX3C eq. μm<sup>-3</sup>. Numbers in parentheses in the data cells of the last three columns = coefficient of variation. Correlation coefficients (R<sup>2</sup>) for the time versus cell number relationships used to the calculate growth rates for each of the cultures exceeded 0.98.</p
<p>Exponential growth was achieved by acclimating cells to optimal temperature, light and n... more <p>Exponential growth was achieved by acclimating cells to optimal temperature, light and nutrient conditions and maintained in exponential growth phase by periodic dilution with nutrient rich media.</p
<p>Natural log of cellular toxicity versus growth rate for each of the <i>Gambierdisc... more <p>Natural log of cellular toxicity versus growth rate for each of the <i>Gambierdiscus</i> and <i>Fukuyoa</i> species normalized (A) to femtograms (fg) CTX3C eq. cell<sup>-1</sup> and (B) attograms (ag) CTX3C eq. per μm<sup>-3</sup> biovolume. Error bars = ± 1 standard deviation. The red arrows indicate data for <i>F</i>. <i>ruetzleri</i>, which had a higher toxicity than the <i>Gambierdiscus</i> species growing at a similar rate.</p
Les dinoflagelles epi-benthiques Gambierdiscus et Fukuyoa produisent les ciguatoxines (CTXs) et l... more Les dinoflagelles epi-benthiques Gambierdiscus et Fukuyoa produisent les ciguatoxines (CTXs) et les maitotoxines (MTXs), qui sont parmi les toxines marines les plus puissantes connues. Les CTXs sont bioaccumulees et bio-transformees le long de la chaine alimentaire marine et provoquent la ciguatera, l'intoxication alimentaire non bacterienne la plus commune au monde. Recemment, la ciguatera a ete rapporte dans des zones non endemiques, notamment les iles Canaries, et un grand nombre d'especes a ete decouvert au sein de ces genres. Peu de donnees sont disponibles sur la toxicite relative entre les differentes especes et sur les congeneres de toxines produits par ces dinoflagelles, en particulier par des souches en dehors de l'ocean Pacifique. Plusieurs souches de Gambierdiscus et Fukuyoa ont ete examinees pour leur cigua- et maito-toxicite via les tests neuro-2a et hemolytique, respectivement. G. excentricus a montre une forte toxicite et deux souches des iles Canaries on...
Rapid Communications in Mass Spectrometry, 2020
RationaleThe dinoflagellate genera Gambierdiscus and Fukuyoa are producers of toxins responsible ... more RationaleThe dinoflagellate genera Gambierdiscus and Fukuyoa are producers of toxins responsible for Ciguatera Poisoning (CP). Although having very low oral potency, maitotoxins (MTXs) are very toxic following intraperitoneal injection and feeding studies have shown they may accumulate in fish muscle. To date, six MTX congeners have been described but two congeners (MTX2 and MTX4) have not yet been structurally elucidated. The aim of the present study was to further characterize MTX4.MethodsChemical analysis was performed using liquid chromatography coupled to a diode‐array detector (DAD) and positive ion mode high‐resolution mass spectrometry (LC/HRMS) on partially purified extracts of G. excentricus (strain VGO792). HRMS/MS studies were also carried out to tentatively explain the fragmentation pathways of MTX and MTX4.ResultsThe comparison of UV and HRMS (ESI+) spectra between MTX and MTX4 led us to propose the elemental formula of MTX4 (C157H241NO68S2, as the unsalted molecule). ...
PloS one, 2017
Dinoflagellate species belonging to the genera Gambierdiscus and Fukuyoa produce ciguatoxins (CTX... more Dinoflagellate species belonging to the genera Gambierdiscus and Fukuyoa produce ciguatoxins (CTXs), potent neurotoxins that concentrate in fish causing ciguatera fish poisoning (CFP) in humans. While the structures and toxicities of ciguatoxins isolated from fish in the Pacific and Caribbean are known, there are few data on the variation in toxicity between and among species of Gambierdiscus and Fukuyoa. Quantifying the differences in species-specific toxicity is especially important to developing an effective cell-based risk assessment strategy for CFP. This study analyzed the ciguatoxicity of 33 strains representing seven Gambierdiscus and one Fukuyoa species using a cell based Neuro-2a cytotoxicity assay. All strains were isolated from either the Caribbean or Gulf of Mexico. The average toxicity of each species was inversely proportional to growth rate, suggesting an evolutionary trade-off between an investment in growth versus the production of defensive compounds. While there ...
Marine drugs, Jan 11, 2017
Maitotoxins (MTXs) are among the most potent toxins known. These toxins are produced by epi-benth... more Maitotoxins (MTXs) are among the most potent toxins known. These toxins are produced by epi-benthic dinoflagellates of the genera Gambierdiscus and Fukuyoa and may play a role in causing the symptoms associated with Ciguatera Fish Poisoning. A recent survey revealed that, of the species tested, the newly described species from the Canary Islands, G. excentricus, is one of the most maitotoxic. The goal of the present study was to characterize MTX-related compounds produced by this species. Initially, lysates of cells from two Canary Island G. excentricus strains VGO791 and VGO792 were partially purified by (i) liquid-liquid partitioning between dichloromethane and aqueous methanol followed by (ii) size-exclusion chromatography. Fractions from chromatographic separation were screened for MTX toxicity using both the neuroblastoma neuro-2a (N2a) cytotoxicity and Ca(2+) flux functional assays. Fractions containing MTX activity were analyzed using liquid chromatography coupled to high-res...
Harmful algae, Mar 1, 2017
Species in the epi-benthic dinoflagellate genus Gambierdiscus produce ciguatoxins (CTXs) and mait... more Species in the epi-benthic dinoflagellate genus Gambierdiscus produce ciguatoxins (CTXs) and maitotoxins (MTXs), which are among the most potent marine toxins known. Consumption of fish contaminated with sufficient quantities of CTXs causes Ciguatera Fish Poisoning (CFP), the largest cause of non-bacterial food poisoning worldwide. Maitotoxins, which can be found in the digestive system of fish, could also contribute to CFP if such tissues are consumed. Recently, an increasing number of Gambierdiscus species have been identified; yet, little is known about the variation in toxicity among Gambierdiscus strains or species. This study is the first assessment of relative CTX- and MTX-toxicity of Gambierdiscus species from areas as widespread as the North-Eastern Atlantic Ocean, Pacific Ocean and the Mediterranean Sea. A total of 13 strains were screened: (i) seven Pacific strains of G. australes, G. balechii, G. caribaeus, G. carpenteri, G. pacificus, G. scabrosus and one strain of an u...
Ciguatera Fish Poisoning (CFP) is a food-borne illness caused by consumption of fish contaminated... more Ciguatera Fish Poisoning (CFP) is a food-borne illness caused by consumption of fish contaminated with polyether toxins known as ciguatoxins (CTXs). Originally known as a tropical disease, CFP has recently been reported from areas previously not considered endemic. The benthic dinoflagellate Gambierdiscus is considered to be the primary producer of CTXs. An increasing number of species have recently been discovered in this genus, even in areas where it had previously not been observed. This genus also produces other types of potent toxins, namely maitotoxins. CTXs are bio-accumulated and transformed in herbivorous and carnivorous fish along the marine trophic chain. This study is part of a PhD project aiming at the elucidation of hitherto unidentified ciguatoxins in recently discovered Gambierdiscus species, especially strains isolated in the Caribbean and North East Atlantic seas. Cultures have been obtained from culture collections and from international collaborators for evaluati...
Marine Drugs, 2014
Ostreopsis cf. ovata produces palytoxin analogues including ovatoxins (OVTXs) and a putative paly... more Ostreopsis cf. ovata produces palytoxin analogues including ovatoxins (OVTXs) and a putative palytoxin (p-PLTX), which can accumulate in marine organisms and may possibly lead to food intoxication. However, purified ovatoxins are not widely available and their toxicities are still unknown. The aim of this study was to improve understanding of the ecophysiology of Ostreopsis cf. ovata and its toxin production as well as to optimize the purification process for ovatoxin. During Ostreopsis blooms in 2011 and 2012 in Villefranche-sur-Mer (France, NW Mediterranean Sea), microalgae epiphytic cells and marine organisms were collected and analyzed both by LC-MS/MS and hemolysis OPEN ACCESS Mar. Drugs 2014, 12 2852 assay. Results obtained with these two methods were comparable, suggesting ovatoxins have hemolytic properties. An average of 223 μg• kg −1 of palytoxin equivalent of whole flesh was found, thus exceeding the threshold of 30 μg• kg −1 in shellfish recommended by the European Food Safety Authority (EFSA). Ostreopsis cells showed the same toxin profile both in situ and in laboratory culture, with ovatoxin-a (OVTX-a) being the most abundant analogue (~50%), followed by OVTX-b (~15%), p-PLTX (12%), OVTX-d (8%), OVTX-c (5%) and OVTX-e (4%). Ostreopsis cf. ovata produced up to 2 g of biomass per L of culture, with a maximum concentration of 300 pg PLTX equivalent cell −1. Thus, an approximate amount of 10 mg of PLTX-group toxins may be produced with 10 L of this strain. Toxin extracts obtained from collected biomass were purified using different techniques such as liquid-liquid partition or size exclusion. Among these methods, open-column chromatography with Sephadex LH20 phase yielded the best results with a cleanup efficiency of 93% and recovery of about 85%, representing an increase of toxin percentage by 13 fold. Hence, this purification step should be incorporated into future isolation exercises.
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Papers by Francesco Pisapia