Dereplication of Gambierdiscusbalechii extract by LC-HRMS and in vitro assay: First description of a putative ciguatoxin and confirmation of 44-methylgambierone.

Marine toxins have a significant impact on seafood resources and human health. Up to date, mainly based on bioassays results, two genera of toxic microalgae, Gambierdiscus and Fukuyoa have been hypothesized to produce a suite of biologically active compounds, including maitotoxins (MTXs) and ciguatoxins (CTXs) with the latter causing ciguatera poisoning (CP) in humans. The global ubiquity of these microalgae and their ability to produce (un-)known bioactive compounds, necessitates strategies for screening, identifying, and reducing the number of target algal species and compounds selected for structural elucidation. To accomplish this task, a dereplication process is necessary to screen and profile algal extracts, identify target compounds, and support the discovery of novel bioactive chemotypes. Herein, a dereplication strategy was applied to a crude extract of a G. balechii culture to investigate for bioactive compounds with relevance to CP using liquid chromatography-high resolution mass spectrometry, in vitro cell-based bioassay, and a combination thereof via a bioassay-guided micro-fractionation. Three biologically active fractions exhibiting CTX-like and MTX-like toxicity were identified. A naturally incurred fish extract (Sphyraena barracuda) was used for confirmation where standards were unavailable. Using this approach, a putative I/C-CTX congener in G. balechii was identified for the first time, 44-methylgambierone was confirmed at 8.6 pg cell-1, and MTX-like compounds were purported. This investigative approach can be applied towards other harmful algal species of interest. The identification of a microalgal species herein, G. balechii (VGO920) which was found capable of producing a putative I/C-CTX in culture is an impactful advancement for global CP research. The large-scale culturing of G. balechii could be used as a source of I/C-CTX reference material not yet commercially available, thus, fulfilling an analytical gap that currently hampers the routine determination of CTXs in various environmental and human health-relevant matrices.

Untargeted and targeted LC-MS and data processing workflow for the comprehensive analysis of oligopeptides from cyanobacteria.

Cyanobacteria produce a plethora of structurally diverse bioactive secondary metabolites, including cyanotoxins which pose a serious threat to humans and other living organisms worldwide. Currently, a wide variety of mass spectrometry-based methods for determination of microcystins (MCs), the most commonly occurring and studied class of cyanotoxins, have been developed and employed for research and monitoring purposes. The scarcity of commercially available reference materials, together with the ever-growing range of mass spectrometers and analytical approaches, make the accuracy of quantitative analyses a critical point to be carefully investigated in view of a reliable risk evaluation. This study reports, a comparative investigation of the qualitative and quantitative MCs profile obtained using targeted and untargeted liquid chromatography-mass spectrometry approaches for the analyses of cyanobacterial biomass from Lake Kastoria, Greece. Comparison of the total MCs content measured by the two approaches showed good correlation, with variations in the range of 3.8-13.2%. In addition, the implementation of an analytical workflow on a hybrid linear ion trap/orbitrap mass spectrometer is described, based on combining data-dependent acquisition and a powerful database of cyanobacterial metabolites (CyanoMetDB) for the annotation of known and discovery of new cyanopeptides. This untargeted strategy proved highly effective for the identification of MCs, microginins, anabaenopeptins, and micropeptins. The systematic interpretation of the acquired fragmentation patterns allowed the elucidation of two new MC structural variants, MC-PrhcysR and MC-Prhcys(O)R, and proposal of structures for two new microginins, isomeric cyanostatin B and MG 821A, and three isomeric micropeptins at m/z 846.4715, 846.4711 and 846.4723.

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.

Improving in vitro ciguatoxin and brevetoxin detection: selecting neuroblastoma (Neuro-2a) cells with lower sensitivity to ouabain and veratridine (OV-LS).

Marine biotoxins accumulating in seafood products pose a risk to human health. These toxins are often potent in minute amounts and contained within complex matrices; requiring sensitive, reliable, and robust methods for their detection. The mouse neuroblastoma (Neuro-2a) cytotoxicity assay (N2a-assay) is a sensitive, high-throughput, in vitro method effective for detecting sodium channel-specific marine biotoxins. The N2a-assay can be conducted to distinguish between specific effects on voltage-gated sodium (NaV) channels, caused by toxins that activate (e.g., ciguatoxins (CTXs), brevetoxins (PbTxs)) or block (e.g., tetrodotoxins, saxitoxins) the target NaV. The sensitivity and specificity of the assay to compounds activating the NaV are achieved through the addition of the pharmaceuticals ouabain (O) and veratridine (V). However, these compounds can be toxic to Neuro-2a cells and their application at insufficient or excessive concentrations can reduce the effectiveness of this assay for marine toxin detection. Therefore, during growth incubation, Neuro-2a cells were exposed to O and V, and surviving cells exhibiting a lower sensitivity to O and V (OV-LS) were propagated. OV-LS Neuro-2a cells were selected for 60-80% survival when exposed to 0.22/0.022 mM O/V during the cytotoxicity assay. At these conditions, OV-LS N2a cells demonstrated a 3.5-fold higher survival rate 71% ± 7.9 SD (n = 232), and lower sensitivity to O/V, compared to the original Neuro-2a cells 20% ± 9.0 SD (n = 16). Additionally, OV-LS N2a cells were 1.3-2.6-fold more sensitive for detecting CTX3C 1.35 pg/ml, CTX1B 2.06 pg/ml, and PbTx-3 3.04 ng/ml compared to Neuro-2a cells using 0.1/0.01 mM O/V. Detection of CTX3C in a complex fish matrix using OV-LS cells was 0.0048 pg CTX3C/mg fish tissue equivalent. This work shows the potential for a significant improvement in sensitivity for CTX3C, CTX1B, and PbTx-3 using the OV-LS N2a-assay.

Ciguatera Mini Review: 21st Century Environmental Challenges and the Interdisciplinary Research Efforts Rising to Meet Them.

Globally, the livelihoods of over a billion people are affected by changes to marine ecosystems, both structurally and systematically. Resources and ecosystem services, provided by the marine environment, contribute nutrition, income, and health benefits for communities. One threat to these securities is ciguatera poisoning; worldwide, the most commonly reported non-bacterial seafood-related illness. Ciguatera is caused by the consumption of (primarily) finfish contaminated with ciguatoxins, potent neurotoxins produced by benthic single-cell microalgae. When consumed, ciguatoxins are biotransformed and can bioaccumulate throughout the food-web via complex pathways. Ciguatera-derived food insecurity is particularly extreme for small island-nations, where fear of intoxication can lead to fishing restrictions by region, species, or size. Exacerbating these complexities are anthropogenic or natural changes occurring in global marine habitats, e.g., climate change, greenhouse-gas induced physical oceanic changes, overfishing, invasive species, and even the international seafood trade. Here we provide an overview of the challenges and opportunities of the 21st century regarding the many facets of ciguatera, including the complex nature of this illness, the biological/environmental factors affecting the causative organisms, their toxins, vectors, detection methods, human-health oriented responses, and ultimately an outlook towards the future. Ciguatera research efforts face many social and environmental challenges this century. However, several future-oriented goals are within reach, including digital solutions for seafood supply chains, identifying novel compounds and methods with the potential for advanced diagnostics, treatments, and prediction capabilities. The advances described herein provide confidence that the tools are now available to answer many of the remaining questions surrounding ciguatera and therefore protection measures can become more accurate and routine.

CyanoMetDB, a comprehensive public database of secondary metabolites from cyanobacteria.

Harmful cyanobacterial blooms, which frequently contain toxic secondary metabolites, are reported in aquatic environments around the world. More than two thousand cyanobacterial secondary metabolites have been reported from diverse sources over the past fifty years. A comprehensive, publically-accessible database detailing these secondary metabolites would facilitate research into their occurrence, functions and toxicological risks. To address this need we created CyanoMetDB, a highly curated, flat-file, openly-accessible database of cyanobacterial secondary metabolites collated from 850 peer-reviewed articles published between 1967 and 2020. CyanoMetDB contains 2010 cyanobacterial metabolites and 99 structurally related compounds. This has nearly doubled the number of entries with complete literature metadata and structural composition information compared to previously available open access databases. The dataset includes microcytsins, cyanopeptolins, other depsipeptides, anabaenopeptins, microginins, aeruginosins, cyclamides, cryptophycins, saxitoxins, spumigins, microviridins, and anatoxins among other metabolite classes. A comprehensive database dedicated to cyanobacterial secondary metabolites facilitates: (1) the detection and dereplication of known cyanobacterial toxins and secondary metabolites; (2) the identification of novel natural products from cyanobacteria; (3) research on biosynthesis of cyanobacterial secondary metabolites, including substructure searches; and (4) the investigation of their abundance, persistence, and toxicity in natural environments.

Development of a data dependent acquisition-based approach for the identification of unknown fast-acting toxins and their ester metabolites.

Phycotoxins in the marine food-web represent a serious threat to human health. Consumption of contaminated shellfish and/or finfish poses risk to consumer safety: several cases of toxins-related seafood poisoning have been recorded so far worldwide. Cyclic imines are emerging lipophilic toxins, which have been detected in shellfish from different European countries. Currently, they are not regulated due to the lack of toxicological comprehensive data and hence the European Food Safety Authority has required more scientific efforts before establishing a maximum permitted level in seafood. In this work, a novel data dependent liquid chromatography - high resolution mass spectrometry (LC-HRMS) approach has been successfully applied and combined with targeted studies for an in-depth investigation of the metabolic profile of shellfish samples. The proposed analytical methodology has allowed: i) to discover a plethora of unknown fatty acid esters of gymnodimines and ii) to conceive a brand new MS-based strategy, termed as backward analysis, for discovery and identification of new analogues. In particular, the implemented analytical workflow has broadened the structural diversity of cyclic imine family through the inclusion of five new congeners, namely gymnodimine -F, -G, -H, -I and -J. In addition, gymnodimine A (376.5 µg/kg), 13-desmethyl spirolide C (11.0-29.0 µg/kg) and pinnatoxin G (3.1-7.7 µg/kg) have been detected in shellfish from different sites of the Mediterranean basin (Tunisia and Italy) and the Atlantic coast of Spain, with the confirmation of the first finding of pinnatoxin G in mussels harvested in Sardinia (Tyrrhenian Sea, Italy).

HPLC-Based Analysis of Impurities in Sapropterin Branded and Generic Tablets.

This work was aimed at the definition of a chromatographic method able to separate and quantify impurities present in sapropterin-containing drugs during an accelerated stability study. The chromatographic method was applied to the orphan drug Kuvan® and to its corresponding generic sapropterin Dipharma (Diterin®), both of which are approved for the treatment of hyperphenylalaninemia-induced symptoms. The two products tested had a similar manufacture date and both had an approved stability shelf-life of three years. Samples were analyzed by HPLC at T = 0 and after six months of storage at 40 °C and 75% relative humidity. Identification of the impurities was supported by a detailed mass spectrometry and MS/MS profile. The analysis demonstrated an overall higher stability for the Diterin® formulation, which was related to a lower increase of some impurities compared to Kuvan®.

Exploring the Photodynamic Properties of Two Antiproliferative Benzodiazopyrrole Derivatives.

The identification of molecules whose biological activity can be properly modulated by light is a promising therapeutic approach aimed to improve drug selectivity and efficacy on the molecular target and to limit the side effects compared to traditional drugs. Recently, two photo-switchable diastereomeric benzodiazopyrrole derivatives 1RR and 1RS have been reported as microtubules targeting agents (MTAs) on human colorectal carcinoma p53 null cell line (HCT 116 p53-/-). Their IC50 was enhanced upon Light Emitting Diode (LED) irradiation at 435 nm and was related to their cis form. Here we have investigated the photo-responsive behavior of the acid derivatives of 1RR and 1RS, namely, d1RR and d1RS, in phosphate buffer solutions at different pH. The comparison of the UV spectra, acquired before and after LED irradiation, indicated that the trans→cis conversion of d1RR and d1RS is affected by the degree of ionization. The apparent rate constants were calculated from the kinetic data by means of fast UV spectroscopy and the conformers of the putative ionic species present in solution (pH range: 5.7-8.0) were modelled. Taken together, our experimental and theoretical results suggest that the photo-conversions of trans d1RR/d1RS into the corresponding cis forms and the thermal decay of cis d1RR/d1RS are dependent on the presence of diazonium form of d1RR/d1RS. Finally, a photo-reaction was detected only for d1RR after prolonged LED irradiation in acidic medium, and the resulting product was characterized by means of Liquid Chromatography coupled to High resolution Mass Spectrometry (LC-HRMS) and Nuclear Magnetic Resonance (NMR) spectroscopy.

Massive Occurrence of the Harmful Benthic Dinoflagellate Ostreopsis cf. ovata in the Eastern Adriatic Sea.

In September 2015, a massive occurrence of the Ostreopsis species was recorded in central Adriatic Kastela Bay. In order to taxonomically identify the Ostreopsis species responsible for this event and determine their toxin profile, cells collected in seawater and from benthic macroalgae were analyzed. Conservative taxonomic methods (light microscopy and SEM) and molecular methods (PCR-based assay) allowed the identification of the species Ostreopsis cf. ovata associated with Coolia monotis. The abundance of O. cf. ovata reached 2.9 × 104 cells L-1 in seawater, while on macroalgae, it was estimated to be up to 2.67 × 106 cells g-1 of macroalgae fresh weight and 14.4 × 106 cells g-1 of macroalgae dry weight. An indirect sandwich immunoenzymatic assay (ELISA) and liquid chromatography-high-resolution mass spectrometry (LC-HRMS) were used to determine the toxin profile. The ELISA assay revealed the presence of 5.6 pg palytoxin (PLTX) equivalents per O. cf. ovata cell. LC-HRMS was used for further characterization of the toxin profile, which showed that there were 6.3 pg of the sum of ovatoxins (OVTXs) and isobaric PLTX per O. cf. ovata cell, with a prevalence of OVTXs (6.2 pg cell-1), while the isobaric PLTX concentration was very low (0.1 pg cell-1). Among OVTXs, the highest concentration was recorded for OVTX-a (3.6 pg cell-1), followed by OVTX-b (1.3 pg cell-1), OVTX-d (1.1 pg cell-1), and OVTX-c (0.2 pg cell-1).

First detection of tetrodotoxin and high levels of paralytic shellfish poisoning toxins in shellfish from Sicily (Italy) by three different analytical methods.

Paralytic shellfish toxins (PST) and tetrodotoxin (TTX) are naturally-occurring toxins that may contaminate the food chain, inducing similar neurological symptoms in humans. They are co-extracted under the same conditions and thus their combined detection is desirable. Whilst PST are regulated and officially monitored in Europe, more data on TTX occurrence in bivalves and gastropods are needed before meaningful regulations can be established. In this study, we used three separate analytical methods - pre-column oxidation with liquid chromatography and fluorescence detection, ultrahigh performance hydrophilic interaction liquid chromatography (HILIC) tandem mass spectrometry (MS/MS) and HILIC high resolution (HR) MS/MS - to investigate the presence of PST and TTX in seawater and shellfish (mussels, clams) collected in spring summer 2015 to 2017 in the Mediterranean Sea. Samples were collected at 10 sites in the Syracuse Bay (Sicily, Italy) in concomitance with a mixed bloom of Alexandrium minutum and A. pacificum. A very high PST contamination in mussels emerged, unprecedentedly found in Italy, with maximum total concentration of 10851 µg saxitoxin equivalents per kg of shellfish tissue measured in 2016. In addition, for the first time TTX was detected in Italy in most of the analysed samples in the range 0.8-6.4 µg TTX eq/kg. The recurring blooms of PST-producing species over the 3-year period, the high PST levels and the first finding of TTX in mussels from the Syracuse bay, suggest that monitoring programmes of PST and TTX in seafood should be activated in this geographical area.

Structural studies and biological evaluation of T30695 variants modified with single chiral glycerol-T reveal the importance of LEDGF/p75 for the aptamer anti-HIV-integrase activities.

Some G-quadruplex (GQ) forming aptamers, such as T30695, exhibit particularly promising properties among the potential anti-HIV drugs. T30695 G-quadruplex binds to HIV-1 integrase (IN) and inhibits its activity during 3'-end processing at nanomolar concentrations. Herein we report a study concerning six T30695-GQ variants, in which the R or S chiral glycerol T, singly replaced the thymine residues at the T30695 G-quadruplex loops. CD melting, EMSA and HMRS experiments provided information about the thermal stability and the stoichiometry of T30695-GQ variants, whereas CD and 1H NMR studies were performed to evaluate the effects of the modifications on T30695-GQ topology. Furthermore, LEDGF/p75 dependent and independent integration assays were carried out to evaluate how T loop modifications impact T30695-GQ biological activities. The obtained results showed that LEDGF/p75 adversely affects the potencies of T30695 and its variants. The IN inhibitory activities of the modified aptamers also depended on the position and on the chirality (R or S) of glycerol T loop in the GQ, mostly regardless of the G-quadruplex stabilities. In view of our and literature data, we suggest that the allosteric modulation of IN tetramer conformations by LEDGF/p75 alters the interactions between the aptamers and the enzyme. Therefore, the new T30695 variants could be suitable tools in studies aimed to clarify the HIV-1 IN tetramers allostery and its role in the integration activity.

Plastic-associated harmful microalgal assemblages in marine environment.

Plastic debris carry fouling a variety of class-size organisms, among them harmful microorganisms that potentially play a role in the dispersal of allochthonous species and toxic compounds with ecological impacts on the marine environment and human health. We analyzed samples of marine plastics floating at the sea surface using a molecular qPCR assay to quantify the attached microalgal taxa, in particular, harmful species. Diatoms were the most abundant group of plastic colonizers with maximum abundance of 8.2 × 104 cells cm-2 of plastics, the maximum abundance of dinoflagellates amounted to 1.1 × 103 cells cm-2 of plastics. The most abundant harmful microalgal taxon was the diatom Pseudo-nitzschia spp., including at least 12 toxic species, and the dinoflagellate Ostreopsis cf. ovata with 6606 and 259 cells cm-2, respectively. The abundance of other harmful microalgal species including the toxic allochthonous dinoflagellate Alexandrium pacificum ranged from 1 to 73 cells cm-2. In the present study, a direct relationship between the abundance of harmful algal species colonizing the plastic substrates and their toxin production was found. The levels of potential toxins on plastic samples ranged from 101 to 102 ng cm-2, considering the various toxin families produced by the colonized harmful microalgal species. We also measured the rate of adhesion by several target microalgal species. It ranged from 1.8 to 0.3 day-1 demonstrating the capacity of plastic substrate colonizing rapidly by microalgae. The present study reports the first estimates of molecular quantification of microorganisms including toxin producing species that can colonize plastics. Such findings provide important insights for improving the monitoring practice of plastics and illustrate how the epi-plastic community can exacerbate the harmful effects of plastics by dispersal, acting as an alien and toxic species carrier and potentially being ingested through the marine trophic web.

Role of temperature and nutrients on the growth and toxin production of Prorocentrum hoffmannianum (Dinophyceae) from the Florida Keys.

The benthic dinoflagellate Prorocentrum hoffmannianum M.A. Faust is typical of tropical warm waters and produces biotoxins responsible for diarrhetic shellfish poisoning (DSP). In this study, the effect of temperature and nutrient limitation on growth and toxin production of P. hoffmannianum isolated from field samples collected in the Florida Keys was investigated. Batch culture experiments were ran at two temperatures (i.e. 21 ± 0.1 and 27 ± 0.1 °C) and under nitrogen-limited (14.7 µmol L-1 N-NO3- and 18.1 µmol L-1 P-PO43-) and phosphorus-limited (441 µmol L-1 N-NO3- and 0.6 µmol L-1 P-PO43-) levels with respect to control nutrient conditions (441 µmol L-1 N-NO3-and 18.1 µmol L-1 P-PO43-). Both temperature and nutrient conditions significantly affected growth rates and maximum yield of P. hoffmannianum with the maximum values being recorded at the higher temperature and in the replete medium. Production of okadaic acid was induced under all conditions (from 13.5 to 859.8 pg cell-1), with values up to one order of magnitude higher than those observed in other DSP toxin producing species. Toxin production was enhanced under P limitation at 27 °C, corroborating the theory that toxin production is modulated by cell physiological conditions, which are in turn affected by a wide spectrum of factors, including environmental stressors such as nutrient availability. Toxin fraction released in the growth medium was negligible. No okadaic acid esters were detected in this strain of P. hoffmannianum.

Identification of a Sorbicillinoid-Producing Aspergillus Strain with Antimicrobial Activity Against Staphylococcus aureus: a New Polyextremophilic Marine Fungus from Barents Sea.

The exploration of poorly studied areas of Earth can highly increase the possibility to discover novel bioactive compounds. In this study, the cultivable fraction of fungi and bacteria from Barents Sea sediments has been studied to mine new bioactive molecules with antibacterial activity against a panel of human pathogens. We isolated diverse strains of psychrophilic and halophilic bacteria and fungi from a collection of nine samples from sea sediment. Following a full bioassay-guided approach, we isolated a new promising polyextremophilic marine fungus strain 8Na, identified as Aspergillus protuberus MUT 3638, possessing the potential to produce antimicrobial agents. This fungus, isolated from cold seawater, was able to grow in a wide range of salinity, pH and temperatures. The growth conditions were optimised and scaled to fermentation, and its produced extract was subjected to chemical analysis. The active component was identified as bisvertinolone, a member of sorbicillonoid family that was found to display significant activity against Staphylococcus aureus with a minimum inhibitory concentration (MIC) of 30 µg/mL.

NMR-based phytochemical analysis of Vitis vinifera cv Falanghina leaves. Characterization of a previously undescribed biflavonoid with antiproliferative activity.

Vitis vinifera cv Falanghina is an ancient grape variety of Southern Italy. A thorough phytochemical analysis of the Falanghina leaves was conducted to investigate its specialised metabolite content. Along with already known molecules, such as caftaric acid, quercetin-3-O-ß-d-glucopyranoside, quercetin-3-O-ß-d-glucuronide, kaempferol-3-O-ß-d-glucopyranoside and kaempferol-3-O-ß-d-glucuronide, a previously undescribed biflavonoid was identified. For this last compound, a moderate bioactivity against metastatic melanoma cells proliferation was discovered. This datum can be of some interest to researchers studying human melanoma. The high content in antioxidant glycosylated flavonoids supports the exploitation of grape vine leaves as an inexpensive source of natural products for the food industry and for both pharmaceutical and nutraceutical companies. Additionally, this study offers important insights into the plant physiology, thus prompting possible technological researches of genetic selection based on the vine adaptation to specific pedo-climatic environments.

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.

Influence of environmental factors on the toxin production of Ostreopsis cf. ovata during bloom events.

Intense blooms of the toxic dinoflagellate Ostreopsis have been a recurrent phenomenon along several Mediterranean coasts. Blooms have been associated with noxious effects on human health and mortality of marine organisms, due to the production of palytoxin-like compounds. We analyzed the toxin concentrations throughout an O. cf. ovata bloom to highlight their relationships with environmental parameters in the Conero Riviera, northern Adriatic Sea. High temperature and balanced nutrient conditions were the optimal environmental conditions to start and sustain blooms as well as to maximize toxin production. Ostreopsis showed a gradual decrease of toxin content throughout the bloom ascribed to the occurring of the same non-optimal conditions that led to the bloom decline. Moreover, our results suggest that toxin fraction released during bloom could be higher than that released in batch culture. Results from this study pointed out that the first bloom phase is potentially the most dangerous to human health.

Toxin Variability Estimations of 68 Alexandrium ostenfeldii (Dinophyceae) Strains from The Netherlands Reveal a Novel Abundant Gymnodimine.

Alexandrium ostenfeldii is a toxic dinoflagellate that has recently bloomed in Ouwerkerkse Kreek, The Netherlands, and which is able to cause a serious threat to shellfish consumers and aquacultures. We used a large set of 68 strains to the aim of fully characterizing the toxin profiles of the Dutch A. ostenfeldii in consideration of recent reports of novel toxins. Alexandrium ostenfeldii is known as a causative species of paralytic shellfish poisoning, and consistently in the Dutch population we determined the presence of several paralytic shellfish toxins (PST) including saxitoxin (STX), GTX2/3 (gonyautoxins), B1 and C1/C2. We also examined the production of spiroimine toxins by the Dutch A. ostenfeldii strains. An extensive liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed a high intraspecific variability of spirolides (SPX) and gymnodimines (GYM). Spirolides included 13-desMethyl-spirolide C generally as the major compound and several other mostly unknown SPX-like compounds that were detected and characterized. Besides spirolides, the presence of gymnodimine A and 12-Methyl-gymnodimine A was confirmed, together with two new gymnodimines. One of these was tentatively identified as an analogue of gymnodimine D and was the most abundant gymnodimine (calculated cell quota up to 274 pg cell-1, expressed as GYM A equivalents). Our multi-clonal approach adds new analogues to the increasing number of compounds in these toxin classes and revealed a high strain variability in cell quota and in toxin profile of toxic compounds within a single population.

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.