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Toxin production in marine microalgae was previously shown to be tightly coupled with cellular stoichiometry. The highest values of cellular toxin are in fact mainly associated with a high carbon to nutrient cellular ratio. In particular, the cellular accumulation of C-rich toxins (i.e., with C:N > 6.6) can be stimulated by both N and P deficiency. Dinoflagellates are the main producers of C-rich toxins and may represent a serious threat for human health and the marine ecosystem. As such, the development of a numerical model able to predict how toxin production is stimulated by nutrient supply/deficiency is of primary utility for both scientific and management purposes. In this work we have developed a mechanistic model describing the stoichiometric regulation of C-rich toxins in marine dinoflagellates. To this purpose, a new formulation describing toxin production and fate was embedded in the European Regional Seas Ecosystem Model (ERSEM), here simplified to describe a monospecific batch culture. Toxin production was assumed to be composed by two distinct additive terms; the first is a constant fraction of algal production and is assumed to take place at any physiological conditions. The second term is assumed to be dependent on algal biomass and to be stimulated by internal nutrient deficiency. By using these assumptions, the model reproduced the concentrations and temporal evolution of toxins observed in cultures of Ostreopsis cf. ovata, a benthic/epiphytic dinoflagellate producing C-rich toxins named ovatoxins. The analysis of simulations and their comparison with experimental data provided a conceptual model linking toxin production and nutritional status in this species. The model was also qualitatively validated by using independent literature data, and the results indicate that our formulation can be also used to simulate toxin dynamics in other dinoflagellates. Our model represents an important step towards the simulation and prediction of marine algal toxicity.

Blooms of the benthic toxic dinoflagellate genus Ostreopsis have been recorded more frequently during the last two decades, particularly in warm temperate areas such as the Mediterranean Sea. The proliferation of Ostreopsis species may cause deleterious effects on ecosystems and can impact human health through skin contact or aerosol inhalation. In the eastern Atlantic Ocean, the toxic O. cf. ovata has not yet been reported to the north of Portugal, and the only species present further north was O. cf. siamensis, for which the toxic risk is considered low. During summer blooms of unidentified Ostreopsis species on the French Basque coast (Atlantic) in 2020 and 2021, people suffered from irritations and respiratory disorders, and the number of analyzed cases reached 674 in 2021. In order to investigate the causes, sampling was carried out during summer 2021 to (i) taxonomically identify Ostreopsis species present using a molecular approach, (ii) isolate strains from the bloom and culture them, and (iii) characterize the presence of known toxins which may be involved. For the first time, this study reports the presence of both O. cf. siamensis and O. cf. ovata, for which the French Basque coast is a new upper distribution limit. Furthermore, the presence of ovatoxins a, b, c, and d in the environmental sample and in a cultivated strain in culture confirmed the toxic nature of the bloom and allowed identifying O. cf. ovata as the producer. The present data identify a new health risk in the area and highlight the extended distribution of some harmful dinoflagellates, presumably in relation to climate change.

The marine dinoflagellate genera Coolia Meunier and Ostreopsis Schmidt have been reported in the Western Indian Ocean and include potentially harmful species. However, no comprehensive observations have been reported in Mauritian waters. The primary aim of this study was to isolate, identify and characterize potentially toxic epiphytic Coolia and Ostreopsis species from the coastal waters of Mauritius. Morphological characteristics were examined using light/fluorescence microscopy and scanning electron microscopy. The morphologies of the Coolia and Ostreopsis strains were similar to those of Coolia canariensis and Ostreopsis ovata. The phylogenetic analyses (large subunit ribosomal [LSU] rDNA D1/D2) revealed that the Mauritian strains of Coolia canariensis and Ostreopsis ovata clustered within the clades of these species complexes with other isolates from different areas. This represents the first record of Coolia canariensis in Mauritius, ascribed to C. canariensis phylogroups I and V, with the latter representing a new lineage of this species complex. The findings broaden the current body of knowledge of Coolia canariensis lineages, while the additional information of Ostreopsis ovata further supports the presence of an Indo-Pacific lineage.

The genus of benthic dinoflagellates Ostreopsis is of particular interest because some species negatively impact human health and coastal marine ecosystems. Ostreopsis populations from a remote area, such as the Galapagos Marine Reserve with its unique biodiversity, can provide significant data. Samples of epibionthic dinoflagellates were collected from two islands (Santa Cruz and Santa Fé) in 2017. Species of the genera Gambierdiscus, Amphidinium, Coolia and Ostreopsis were found. Ostreopsis strains were isolated to characterize their morphology, molecular biology and toxicity. Three dif­ferent morphotypes of Ostreopsis based on dorsoventral and width diameters (n=369) were distinguished. The small cell morphotype was dominant in ten samples, with abundances of up to 33405 cells g-1 fresh weight of macroalgae. A total of 16 strains were isolated from field samples with subsequent polymerase chain reaction amplifications of rDNA, 5.8S rDNA and internal transcribed space regions; 13 strains (small cell morphotype) clustered in the O. cf. ovata Atlantic/Indian/Pacific clade; and 3 strains (large cell morphotype) clustered in the Ostreopsis lenticularis genotype from the type locality. The strains proved to be non-toxic. The presence of these genera/species represents a potential threat to marine ecosystems, and it is thus important to consider benthic species in the surveillance of harmful algae blooms in the reserve.

Harmful benthic dinoflagellates, usually developing in tropical areas, are expanding to temperate ecosystems facing water warming. Reports on harmful benthic species are particularly scarce in the Southern Mediterranean Sea. For the first time, three thermophilic benthic dinoflagellates (Ostreopsis cf. ovata, Prorocentrum lima and Coolia monotis) were isolated from Bizerte Bay (Tunisia, Mediterranean) and monoclonal cultures established. The ribotyping confirmed the morphological identification of the three species. Maximum growth rates were 0.59 ± 0.08 d−1 for O. cf. ovata, 0.35 ± 0.01 d−1 for C. monotis and 0.33 ± 0.04 d−1 for P. lima. Toxin analyses revealed the presence of ovatoxin-a and ovatoxin-b in O. cf. ovata cells. Okadaic acid and dinophysistoxin-1 were detected in P. lima cultures. For C. monotis, a chromatographic peak at 5.6 min with a mass m/z = 1061.768 was observed, but did not correspond to a mono-sulfated analogue of the yessotoxin. A comparison of the toxicity and growth characteristics of these dinoflagellates, distributed worldwide, is proposed.

This study aimed to improve the understanding of the nutrient modulation of Ostreopsis cf. ovata toxin content. During the 2018 natural bloom in the NW Mediterranean, the total toxin content (up to ca. 57.6 ± 7.0 pg toxin cell−1) varied markedly. The highest values often coincided with elevated O. cf. ovata cell abundance and with low inorganic nutrient concentrations. The first culture experiment with a strain isolated from that bloom showed that cell toxin content was higher in the stationary than in the exponential phase of the cultures; phosphate- and nitrate-deficient cells exhibited similar cell toxin variability patterns. The second experiment with different conditions of nitrogen concentration and source (nitrate, urea, ammonium, and fertilizer) presented the highest cellular toxin content in the high-nitrogen cultures; among these, urea induced a significantly lower cellular toxin content than the other nutrient sources. Under both high- and low-nitrogen concentrations, cell toxin content was also higher in the stationary than in the exponential phase. The toxin profile of the field and cultured cells included ovatoxin (OVTX) analogues -a to -g and isobaric PLTX (isoPLTX). OVTX-a and -b were dominant while OVTX-f, -g, and isoPLTX contributed less than 1-2%. Overall, the data suggest that although nutrients determine the intensity of the O. cf. ovata bloom, the relationship of major nutrient concentrations, sources and stoichiometry with cellular toxin production is not straightforward.

Ostreopsis is a benthic and epiphytic dinoflagellate producing potent toxins widespread in tropical and warm temperate coastal areas world-wide. We tested the hypothesis that as it is benthic, it would show distinct biogeographical patterns in comparison with planktonic species. Here, we analyse sequence variability in ribosomal DNA markers to provide the first phylogeographical study of this toxic benthic dinoflagellate. Mediterranean Sea, Atlantic Ocean, Pacific Ocean. Ribosomal DNA sequence data from partial nuclear LSU (D1/D2 domains) and 5.8S genes and non-coding internal transcribed spacer (ITS) regions were obtained from 82 isolates of Ostreopsis species, collected at 26 localities throughout the world. Molecular sequence data were analysed using maximum parsimony, maximum likelihood and Bayesian methods for phylogenetic inference. A statistical parsimony network was obtained based on concatenated LSU and 5.8S rDNA-ITS region sequences of the Mediterranean/Atlantic Ostreopsis cf. ovata isolates to infer haplotype distribution over their geographical range. Light epifluorescence microscopy analyses were performed on cultured and field Ostreopsis material for taxonomic identification, while laboratory experiments for encystment induction were carried out on selected O. cf. ovata isolates. Toxin assays of Ostreopsis species isolates were carried out using the haemolytic-based method. Analyses based on single and concatenated ribosomal genes gave substantially similar results. The rDNA phylogeny revealed different clades corresponding to different species within the genus Ostreopsis. In the species O. cf. ovata, different genetic lineages were correlated with macrogeographical distribution. A network of haplotypes inferred from the Atlantic and Mediterranean isolates of O. cf. ovata revealed that these two areas might host a single panmictic population. The Atlantic/Mediterranean population of O. cf. ovata was differentiated considerably from the Indo-Pacific populations. Other species of Ostreopsis were found, but they turned out to be restricted to just one of the two main warm-water oceanic basins, the Mediterranean/Atlantic and the Indo-Pacific. Ostreopsis cf. ovata was found to be widely dispersed throughout the coastal areas of tropical and some warm temperate seas. In the Atlantic/Mediterranean region it may constitute a panmictic population that is highly distinct from Indo-Pacific populations. Ostreopsis cf. siamensis was found only in the Mediterranean Sea, and strains identified as Ostreopsis lenticularis and Ostreopsis labens were found only in the Indo-Pacific region.

In September 2015, a massive occurrence of the Ostreopsis species was recorded in central Adriatic Kaštela 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).

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 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.

Dinoflagellates of the genus Ostreopsis are known to cause (often fatal) food poisoning in tropical coastal areas following the accumulation of palytoxin (PLTX) and/or its analogues (PLTX group) in crabs, sea urchins or fish. Ostreopsis spp. occurrence is presently increasing in the northern to north western Mediterranean Sea (Italy, Spain, Greece and France), probably in response to climate change. In France, Ostreopsis. cf. ovata has been associated with toxic events during summer 2006, at Morgiret, off the coast of Marseille, and a specific monitoring has been designed and implemented since 2007. Results from 2008 and 2009 showed that there is a real danger of human poisoning, as these demonstrated bioaccumulation of the PLTX group (PLTX and ovatoxin-a) in both filter-feeding bivalve molluscs (mussels) and herbivorous echinoderms (sea urchins). The total content accumulated in urchins reached 450 µg PLTX eq/kg total flesh (summer 2008). In mussels, the maximum was 230 µg eq PLTX/kg (summer 2009) compared with a maximum of 360 µg found in sea urchins during the same period at the same site. This publication brings together scientific knowledge obtained about the summer development of Ostreopsis spp. in France during 2007, 2008 and 2009.