Toward Isolation of Palytoxins: Liquid Chromatography Coupled to Low- or High-Resolution Mass Spectrometry for the Study on the Impact of Drying Techniques, Solvents and Materials.

Palytoxin (PLTX) and its congeners are emerging toxins held responsible for a number of human poisonings following the inhalation of toxic aerosols, skin contact, or the ingestion of contaminated seafood. Despite the strong structural analogies, the relative toxic potencies of PLTX congeners are quite different, making it necessary to isolate them individually in sufficient amounts for toxicological and analytical purposes. Previous studies showed poor PLTX recoveries with a dramatic decrease in PLTX yield throughout each purification step. In view of a large-scale preparative work aimed at the preparation of PLTX reference material, we have investigated evaporation as a critical-although unavoidable-step that heavily affects overall recoveries. The experiments were carried out in two laboratories using different liquid chromatography-mass spectrometry (LC-MS) instruments, with either unit or high resolution. Palytoxin behaved differently when concentrated to a minimum volume rather than when evaporated to complete dryness. The recoveries strongly depended on the solubility as well as on the material of the used container. The LC-MS analyses of PLTX dissolved in aqueous organic blends proved to give a peak intensity higher then when dissolved in pure water. After drying, the PLTX adsorption appeared stronger on glass surfaces than on plastic materials. However, both the solvents used to dilute PLTX and that used for re-dissolution had an important role. A quantitative recovery (97%) was achieved when completely drying 80% aqueous EtOH solutions of PLTX under N2-stream in Teflon. The stability of PLTX in acids was also investigated. Although PLTX was quite stable in 0.2% acetic acid solutions, upon exposure to stronger acids (pH < 2.66), degradation products were observed, among which a PLTX methyl-ester was identified.

Variability in Toxin Profiles of the Mediterranean Ostreopsis cf. ovata and in Structural Features of the Produced Ovatoxins.

Fifty-five strains of Ostreopsis were collected in the Mediterranean Sea and analyzed to characterize their toxin profiles. All the strains were grown in culture under the same experimental conditions and identified by molecular PCR assay based on the ITS-5.8S rDNA. A liquid chromatography-high resolution multiple stage mass spectrometry (LC-HRMSn) approach was used to analyze toxin profiles and to structurally characterize the detected toxins. Despite morphological and molecular characterization being consistent within the species O. cf. ovata, a certain degree of toxin variability was observed. All the strains produced ovatoxins (OVTXs), with the exception of only one strain. Toxin profiles were quite different from both qualitative and quantitative standpoints: 67% of the strains contained OVTX-a to -e, OVTX-g, and isobaric PLTX, in 25% of them only OVTX-a, -d, -e and isobaric PLTX were present, while 4% produced only OVTX-b and -c. None of the strains showed a previously identified profile, featuring OVTX-f as dominant toxin, whereas OVTX-f was a minor component of very few strains. Toxin content was mostly in the range 4-70 pg/cell with higher levels (up to 238 pg/cell) being found in strains from the Ligurian and South Adriatic Sea. Structural insights into OVTX-b, -c, -d, and -e were gained, and the new OVTX-l was detected in 36 strains.

Mediterranean Azadinium dexteroporum (Dinophyceae) produces six novel azaspiracids and azaspiracid-35: a structural study by a multi-platform mass spectrometry approach.

Azadinium dexteroporum is the first species of the genus described from the Mediterranean Sea and it produces different azaspiracids (AZA). The aims of this work were to characterize the toxin profile of the species and gain structural information on azaspiracids produced by the A. dexteroporum strain SZN-B848 isolated from the Gulf of Naples. Liquid chromatography-mass spectrometry (LC-MS) analyses were carried out on three MS systems having different ion source geometries (ESI, TurboIonSpray®, ESI ION MAX) and different MS analyzers operating either at unit resolution or at high resolution, namely a hybrid triple quadrupole-linear ion trap (Q-Trap MS), a time of flight (TOF MS), and a hybrid linear ion trap Orbitrap XL Fourier transform mass spectrometer (LTQ Orbitrap XL FTMS). As a combined result of these different analyses, A. dexteroporum showed to produce AZA-35, previously reported from Azadinium spinosum, and six compounds that represent new additions to the AZA-group of toxins, including AZA-54 to AZA-58 and 3-epiAZA-7, a stereoisomer of the shellfish metabolite AZA-7. Based on the interpretation of fragmentation patterns, we propose that all these molecules, except AZA-55, have the same A to I ring system as AZA-1, with structural modifications all located in the carboxylic side chain. Considering that none of the azaspiracids produced by the Mediterranean strain of A. dexteroporum is currently regulated by European food safety authorities, monitoring programs of marine biotoxins in the Mediterranean area should take into account the occurrence of the new analogues to avoid an underestimation of the AZA-related risk for seafood consumers. Graphical Abstract A multi-platform MS approach reveals known and new azaspiracids in a Mediterranean strain of Azadinium dexteroporum.

Ostreopsis fattorussoi sp. nov. (Dinophyceae), a new benthic toxic Ostreopsis species from the eastern Mediterranean Sea.

The new benthic toxic dinoflagellate, Ostreopsis fattorussoi sp. nov., is described from the Eastern Mediterranean Sea, Lebanon and Cyprus coasts, and is supported by morphological and molecular data. The plate formula, Po, 3', 7″, 6c, 7s, 5‴, 2'''', is typical for the Ostreopsis genus. It differs from all other Ostreopsis species in that (i) the curved suture between plates 1' and 3' makes them approximately hexagonal, (ii) the 1' plate lies in the left half of the epitheca and is obliquely orientated leading to a characteristic shape of plate 6″. The round thecal pores are bigger than the other two Mediterranean species (O. cf. ovata and O. cf. siamensis). O. fattorussoi is among the smallest species of the genus (DV: 60.07 ± 5.63 µm, AP: 25.66 ± 2.97 µm, W: 39.81 ± 5.05 µm) along with O. ovata. Phylogenetic analyses based on the LSU and internal transcribed spacer rDNA shows that O. fattorussoi belongs to the Atlantic/Mediterranean Ostreopsis spp. clade separated from the other Ostreopsis species. Ostreopsis fattorussoi produces OVTX-a and structural isomers OVTX-d and -e, O. cf. ovata is the only other species of this genus known to produce these toxins. The Lebanese O. fattorussoi did not produce the new palytoxin-like compounds (ovatoxin-i, ovatoxin-j1 , ovatoxin-j2 , and ovatoxin-k) that were previously found in O. fattorussoi from Cyprus. The toxin content was in the range of 0.28-0.94 pg · cell-1 . On the Lebanon coast, O. fattorussoi was recorded throughout the year 2015 (temperature range 18°C-31.5°C), with peaks in June and August.

An aquarium hobbyist poisoning: Identification of new palytoxins in Palythoa cf. toxica and complete detoxification of the aquarium water by activated carbon.

Palytoxin (PLTX) is a lethal natural toxin often found in Palythoa zoantharians that, together with its congeners, may induce adverse effects in humans after inhalation of toxic aerosols both in open-air and domestic environments, namely in the vicinity of public and private aquaria. In this study, we describe a poisoning of an aquarium hobbyist who was hospitalized after handling a PLTXs-containing zoantharian hexacoral. Furthermore, we provide evidence for water detoxification. The zoantharian was morphologically and genetically identified as Palythoa cf. toxica (Cnidaria: Anthozoa). Palytoxin itself and two new PLTX congeners, a hydroxyPLTX and a deoxyPLTX, were detected and structurally identified by liquid chromatography high resolution multiple stage mass spectrometry (LC-HRMSn, n = 1, 2). Total and individual toxins were quantified by LC-HRMS and sandwich ELISA both in the zoantharian (93.4 and 96.80 µg/g, respectively) and in the transport water (48.3 and 42.56 µg/mL, respectively), with an excellent mean bias of 1.3% between the techniques. Activated carbon adsorbed 99.7% of PLTXs contained in the seawater and this represents a good strategy for preventing aquarium hobbyist poisonings.

NMR-based identification of the phenolic profile of fruits of Lycium barbarum (goji berries). Isolation and structural determination of a novel N-feruloyl tyramine dimer as the most abundant antioxidant polyphenol of goji berries.

Biological properties of fruits of Lycium barbarum (goji berries) have been ascribed to their high content of nutrients and phenolics. Comprehensive studies aimed at unambiguously identifying the phenolic components in goji berries are still lacking. In this paper, we report on the isolation and NMR-based identification of the major phenolics in commercially available goji berries. Together with already known phenolics, including caffeic acid, p-coumaric acid, rutin, scopoletin, N-trans-feruloyl tyramine, and N-cis-feruloyl tyramine, an unreported N-feruloyl tyramine dimer was characterized as the most abundant polyphenol isolated from the berries. Usually divalent molecules show enhanced biological activities than their corresponding monomers.

Chemical, molecular, and eco-toxicological investigation of Ostreopsis sp. from Cyprus Island: structural insights into four new ovatoxins by LC-HRMS/MS.

Blooms of benthic dinoflagellates of the genus Ostreopsis (mainly O. cf. ovata and occasionally O. cf. siamensis) represent a serious concern for humans in the Mediterranean area, due to production of palytoxin-like compounds listed among the most potent marine toxins known. In this work, six strains of Ostreopsis sp. from Cyprus Island were analyzed through an integrated approach based on molecular, chemical, and eco-toxicological methods. Cypriot Ostreopsis sp. was found to be a species distinct from O. cf. ovata and O. cf. siamensis, belonging to the Atlantic/Mediterranean Ostreopsis spp. clade. Some variability in toxin profiles emerged: three strains produced ovatoxin-a (OVTX-a), OVTX-d, OVTX-e, and isobaric palytoxin, so far found only in O. cf. ovata; the other three strains produced only new palytoxin-like compounds, which we named ovatoxin-i, ovatoxin-j1, ovatoxin-j2, and ovatoxin-k. The new ovatoxins present the same carbon skeleton as ovatoxin-a, differing primarily in an additional C2H2O2 moiety and an unsaturation in the region C49-C52. Other minor structural differences were found, including the presence of a hydroxyl group at C44 (in OVTX-j1 and OVTX-k) and the lack of a hydroxyl group in the region C53-C78 (in OVTX-i and OVTX-j1). The toxin content of the analyzed Ostreopsis sp. strains was in the range 0.06-2.8 pg cell(-1), definitely lower than that of a Ligurian O. cf. ovata strain cultured under the same conditions. Accordingly, an eco-toxicological test on Artemia salina nauplii demonstrated that Ostreopsis sp. presents a very low toxicity compared to O. cf. ovata. The whole of these data suggest that Ostreopsis sp. from Cyprus Island poses a relatively low risk to humans.

Determination of Palytoxins in Soft Coral and Seawater from a Home Aquarium. Comparison between Palythoa- and Ostreopsis-Related Inhalatory Poisonings.

Anecdotal reports exist of aquarium hobbyists that experienced severe respiratory distress and/or skin injury following cleaning operation of home aquaria containing Palythoa sp. soft corals. Hundreds of cases of respiratory illness and/or dermatitis have been recorded in proximity to the sea concomitantly with algal blooms of Ostreopsis spp. in the Mediterranean area. Both Palythoa spp. and Ostreopsis spp. contain congeners of palytoxin, a highly potent toxin whose inhalation hazard is however unknown. In this study, we demonstrate the presence of high levels of palytoxins (palytoxin and hydroxypalytoxin) in both soft coral and seawater from a home marine aquarium involved in the poisoning of a whole family. Due to the high toxin levels found in seawater, a procedure for a rapid and efficient determination of palytoxin in seawater was setup. A comparison of symptoms of Palythoa- and Ostreopsis-related inhalatory poisonings showed many similarities including fever, respiratory distress, nausea, and flu-like symptoms. From the chemical and symptomatological data reported herein it is reasonable to hold palytoxins responsible for respiratory disorders following inhalation. Although the exact mechanism through which palytoxin congeners exert their inhalatory toxicity is still unknown, this represents a step toward demonstrating that palytoxin congeners exert toxic effects through inhalation both in natural environments and in the surroundings of private and public aquaria.

Ovatoxin-a, A Palytoxin Analogue Isolated from Ostreopsis cf. ovata Fukuyo: Cytotoxic Activity and ELISA Detection.

This study provides the first evaluation of the cytotoxic effects of the recently identified palytoxin (PLTX) analog, ovatoxin-a (OVTX-a), the major toxin produced by Ostreopsis cf. ovata in the Mediterranean Sea. Its increasing detection during Ostreopsis blooms and in seafood highlights the need to characterize its toxic effects and to set up appropriate detection methods. OVTX-a is about 100 fold less potent than PLTX in reducing HaCaT cells viability (EC50 = 1.1 × 10(-9) M vs 1.8 × 10(-11) M, MTT test) in agreement with a reduced binding affinity (Kd = 1.2 × 10(-9) vs 2.7 × 10(-11) M, saturation experiments on intact cells). Similarly, OVTX-a hemolytic effect is lower than that of the reference PLTX compound. Ost-D shows the lowest cytotoxicity toward HaCaT keratinocytes, suggesting the lack of a hydroxyl group at C44 as a critical feature for PLTXs cytotoxic effects. A sandwich ELISA developed for PLTX detects also OVTX-a in a sensitive (LOD = 4.2 and LOQ = 5.6 ng/mL) and accurate manner (Bias = 0.3%), also in O. cf. ovata extracts and contaminated mussels. Although in vitro OVTX-a appears less toxic than PLTX, its cytotoxicity at nanomolar concentrations after short exposure time rises some concern for human health. The sandwich ELISA can be a viable screening method for OVTXs detection in monitoring program.

Ostreopsis cf. ovata from western Mediterranean Sea: Physiological responses under different temperature and salinity conditions.

The dinoflagellate Ostreopsis cf. ovata proliferates seasonally in the Mediterranean Sea, producing palytoxin-like compounds (ovatoxins) which are considered among the most potent marine toxins. Blooms have been related to several toxic events in which respiratory problems in humans and mortality of benthic marine organisms have been observed. In the coming decades, an increase in temperature and salinity is predicted in the Mediterranean Sea as a consequence of global warming that may provoke alterations in the dynamics of marine microorganisms. In this study, the physiological effects of changes in water temperature and salinity were analyzed, and their interaction through a multi-factorial experiment using two strains of O. cf. ovata in culture that had been isolated from the western Mediterranean Sea. In order to perform an accurate and reliable estimation of cell abundance, hydrochloric acid and sodium-ethylenediaminetetraacetic acid treatments were evaluated for the purpose of disaggregating cell clumps, with the former providing lower counting errors, especially after the stationary phase. Results of the physiological study showed that growth was inhibited at 19°C for all salinities. The highest growth rates were registered at 24°C for both strains (0.48±0.05divday-1), and a significant variability in growth rate was found among salinities at 24°C and 28°C. Two groups were distinguished by cell size in all high temperature conditions and a positive correlation was found between the amount of small cells and growth rate. The concentration of palytoxin-like compounds in the cultures increased with time and significantly higher amounts of toxin were found at 28°C in comparison to 24°C. The results suggest that climate change may not affect intensity of blooms, but their toxicity may be enhanced.

The sxt Gene and Paralytic Shellfish Poisoning Toxins as Markers for the Monitoring of Toxic Alexandrium Species Blooms.

Paralytic shellfish poisoning (PSP) is a serious human illness caused by the ingestion of seafood contaminated with saxitoxin and its derivatives (STXs). These toxins are produced by some species of marine dinoflagellates within the genus Alexandrium. In the Mediterranean Sea, toxic Alexandrium spp. blooms, especially of A. minutum, are frequent and intense with negative impact to coastal ecosystem, aquaculture practices and other economic activities. We conducted a large scale study on the sxt gene and toxin distribution and content in toxic dinoflagellate A. minutum of the Mediterranean Sea using both quantitative PCR (qPCR) and HILIC-HRMS techniques. We developed a new qPCR assay for the estimation of the sxtA1 gene copy number in seawater samples during a bloom event in Syracuse Bay (Mediterranean Sea) with an analytical sensitivity of 2.0 × 10° sxtA1 gene copy number per reaction. The linear correlation between sxtA1 gene copy number and microalgal abundance and between the sxtA1 gene and STX content allowed us to rapidly determine the STX-producing cell concentrations of two Alexandrium species in environmental samples. In these samples, the amount of sxtA1 gene was in the range of 1.38 × 10(5) - 2.55 × 10(8) copies/L and the STX concentrations ranged from 41-201 nmol/L. This study described a potential PSP scenario in the Mediterranean Sea.

(1S,3R,4S,5R)5-O-Caffeoylquinic acid: isolation, stereo-structure characterization and biological activity.

Sorbus domestica fruits (sorbs) are commonly harvested and consumed for their nutritious qualities and have long been used as natural remedy against diabetes in the popular medicine. Recently, sorbs have been described as sources of antioxidant compounds including polyphenols. Chemical analyses carried out on sorbs collected in Southern Italy have led to the isolation of a chlorogenic acid isomer as the main antioxidant compound contained in the fruit butanol extract. NMR studies have identified the isolated compound as (1S,3R,4S,5R)5-O-Caffeoylquinic acid. In comparison to chlorogenic acid, the isolated isomer features an inverted configuration at C4. Such configurational inversion causes the chlorogenic acid stereoisomer acid to assume a preferential conformation remarkably different from that of chlorogenic acid. This seems to account for the enhanced potency of (1S,3R,4S,5R)5-O-Caffeoylquinic acid to reduce both the glucose and cholesterol uptakes by the cell line HepG2 when compared to chlorogenic acid.

The novel ovatoxin-g and isobaric palytoxin (so far referred to as putative palytoxin) from Ostreopsis cf. ovata (NW Mediterranean Sea): structural insights by LC-high resolution MS(n.).

Blooms of the benthic dinoflagellate Ostreopsis cf. ovata are a concern in the Mediterranean Sea, since this species produces a wide range of palytoxin-like compounds listed among the most potent marine toxins. This study focused on two analogs of palytoxin found in cultures of six strains of O. cf. ovata isolated from the south of Catalonia (NW Mediterranean Sea). In addition to some already known ovatoxins, our strains produced two minor compounds, ovatoxin-g and the so far called putative palytoxin, whose structures had not been elucidated before. Insufficient quantity of these compounds impeded a full nuclear magnetic resonance (NMR)-based structural elucidation; thus, we studied their structure in crude algal extracts through liquid chromatography-electrospray ionization high-resolution mass spectrometry(n) (LC-ESI-HRMS(n)) in positive ion mode. Under the used MS conditions, the molecules underwent fragmentation at many sites of their backbone and a large number of diagnostic fragment ions were identified. As a result, tentative structures were assigned to both ovatoxin-g and the putative palytoxin, the latter being identified as a palytoxin isomer and re-named as  isobaric palytoxin.

Liquid chromatography-high-resolution mass spectrometry for palytoxins in mussels.

Palytoxins from Ostreopsis cf. ovata (a putative palytoxin and ovatoxins) are emerging toxins in the Mediterranean basin and are not yet regulated, although there is evidence that they can accumulate in seafood and thus enter the human food chain. This poses serious concerns for human health, because palytoxin itself is among the most potent marine toxins known. In 2009, the European Food Safety Authority (EFSA) announced the need for optimization of efficient analytical methods for detecting palytoxins and for preparing standards. Herein, we propose a procedure including a one-step extraction, solid-phase-extraction (SPE) clean-up, and liquid chromatography-high resolution mass spectrometry (LC-HRMS) detection of individual palytoxins in mussels. The method enabled efficient chromatographic separation of individual compounds, including structural isomers, with good sensitivity, reproducibility, and linearity in a large dynamic range (14-1000 ng mL(-1) in matrix). As a result, the putative palytoxin from Ostreopsis cf. ovata was identified as an isomer of palytoxin itself and re-named isobaric palytoxin. The whole procedure (sample preparation and LC-HRMS analysis) proved able to detect palytoxins in both spiked and natural mussel samples at levels as low as 70 µg kg(-1) in crude mussel extracts and 15 µg kg(-1) after SPE clean-up. Although full validation of the method is currently prevented by the unavailability of palytoxin(s) certified standards and reference material, this study constitutes a first step towards achieving this.

SxtA and sxtG gene expression and toxin production in the Mediterranean Alexandrium minutum (Dinophyceae).

The dinoflagellate Alexandrium minutum is known for the production of potent neurotoxins affecting the health of human seafood consumers via paralytic shellfish poisoning (PSP). The aim of this study was to investigate the relationship between the toxin content and the expression level of the genes involved in paralytic shellfish toxin (PST) production. The algal cultures were grown both in standard f/2 medium and in phosphorus/nitrogen limitation. In our study, LC-HRMS analyses of PST profile and content in different Mediterranean A. minutum strains confirmed that this species was able to synthesize mainly the saxitoxin analogues Gonyautoxin-1 (GTX1) and Gonyautoxin-4 (GTX4). The average cellular toxin content varied among different strains, and between growth phases, highlighting a decreasing trend from exponential to stationary phase in all culture conditions tested. The absolute quantities of intracellular sxtA1 and sxtG mRNA were not correlated with the amount of intracellular toxins in the analysed A. minutum suggesting that the production of toxins may be regulated by post-transcriptional mechanisms and/or by the concerted actions of alternative genes belonging to the PST biosynthesis gene cluster. Therefore, it is likely that the sxtA1 and sxtG gene expression could not reflect the PST accumulation in the Mediterranean A. minutum populations under the examined standard and nutrient limiting conditions.

Stereoisomers of 42-hydroxy palytoxin from Hawaiian Palythoa toxica and P. tuberculosa: stereostructure elucidation, detection, and biological activities.

Palytoxin ranks among the most potent marine biotoxins. Its lethality was well known to native Hawaiians that used to smear a "moss" containing the toxin on their spears to cause instant death to their victims. Human intoxications due to exposure to palytoxin and to its many congeners have been reported worldwide. Currently, palytoxins constitute the main threat to public health across the Mediterranean Sea. In the present work we report on the isolation and stereostructural determination of a new palytoxin analogue from a Hawaiian Palythoa tuberculosa sample. This new toxin is a stereoisomer of 42-hydroxypalytoxin isolated from Palythoa toxica. The whole absolute configuration of this latter toxin is also reported in the paper. Interestingly, the two 42-hydroxypalytoxins do not share the same biological activity. The stereoisomer from P. tuberculosa showed cytotoxicity toward skin HaCaT keratinocytes approximately 1 order of magnitude lower than that of 42-hydroxypalytoxin from P. toxica and about 2 orders of magnitude lower than that of palytoxin itself. This finding holds the prospect of interesting structure-activity relationship evaluations in the future.

First finding of Ostreopsis cf. ovata toxins in marine aerosols.

Since the late 1990s, a respiratory syndrome has been repetitively observed in humans concomitant with Ostreopsis spp. blooms (mainly O. cf. ovata) in the Mediterranean area. Previous studies have demonstrated that O. cf. ovata produces analogues of palytoxin (ovatoxins and a putative palytoxin), one of the most potent marine toxins. On the basis of the observed association between O. cf. ovata blooms, respiratory illness in people, and detection of palytoxin complex in algal samples, toxic aerosols, containing Ostreopsis cells and/or the toxins they produce, were postulated to be the cause of human illness. A small scale monitoring study of marine aerosol carried out along the Tuscan coasts (Italy) in 2009 and 2010 is reported. Aerosols were collected concomitantly with O. cf. ovata blooms, and they were analyzed by both PCR assays and LC-HRMS. The results, besides confirming the presence of O. cf. ovata cells, demonstrated for the first time the occurrence of ovatoxins in the aerosol at levels of 2.4 pg of ovatoxins per liter of air. Given the lack of toxicological data on palytoxins by inhalation exposure, our results are only a first step toward a more comprehensive understanding of the Ostreopsis-related respiratory syndrome.

Identification of palytoxin-Ca2+ complex by NMR and molecular modeling techniques.

More than 40 years after its isolation, the understanding of how palytoxin interacts with biological systems has yet to be fully determined. The Na(+),K(+)-ATPase pump constitutes a molecular receptor for palytoxin that is able to convert the pump into an open channel, with consequent loss of cellular K(+) and remarkable rise of cytosolic Na(+) levels. In addition, a slight permeability to Ca(2+) is detected when palytoxin binds to the pump. It has been demonstrated that the increase of cytosolic free Ca(2+) concentration gives rise to downstream events ultimately leading to cell death. The widely accepted recognition of the dependence of important cellular events on calcium ion concentration propelled us to investigate the occurrence of palytoxin-Ca(2+) complex in aqueous solution by NMR- and molecular modeling-based approach. We identified two specific regions of palytoxin where Ca(2+) is preferentially coordinated. This study constitutes the first characterization of a calcium complex with palytoxin and, as such, is expected to support the investigation of the toxin molecular bioactivity.

New insights on cytological and metabolic features of Ostreopsis cf. ovata Fukuyo (Dinophyceae): a multidisciplinary approach.

The harmful dinoflagellate Ostreopsis cf. ovata has been causing toxic events along the Mediterranean coasts and other temperate and tropical areas, with increasing frequency during the last decade. Despite many studies, important biological features of this species are still poorly known. An integrated study, using different microscopy and molecular techniques, Raman microspectroscopy and high resolution liquid chromatography-mass spectrometry (HR LC-MS), was undertaken to elucidate cytological aspects, and identify main metabolites including toxins. The species was genetically identified as O. cf. ovata, Atlantic-Mediterranean clade. The ultrastructural results show unique features of the mucilage network abundantly produced by this species to colonize benthic substrates, with a new role of trichocysts, never described before. The amorphous polysaccharidic component of mucilage appears to derive from pusule fibrous material and mucocysts. In all stages of growth, the cells show an abundant production of lipids. Different developmental stages of chloroplasts are found in the peripheral cytoplasm and in the centre of cell. In vivo Raman microspectroscopy confirms the presence of the carotenoid peridinin in O. cf. ovata, and detects in several specimen the abundant presence of unsaturated lipids structurally related to docosahexaenoic acid. The HR LC-MS analysis reveals that ovatoxin-a is the predominant toxin, together with decreasing amounts of ovatoxin-b, -d/e, -c and putative palytoxin. Toxins concentration on a per cell basis increases from exponential to senescent phase. The results suggest that benthic blooms of this species are probably related to features such as the ability to create a unique mucilaginous sheath covering the sea bottom, associated with the production of potent toxins as palytoxin-like compounds. In this way, O. cf. ovata may be able to rapidly colonize benthic substrates outcompeting other species.