Complex Toxin Profile of French Mediterranean Ostreopsis cf. ovata Strains,Seafood Accumulation and Ovatoxins Prepurification

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.     

Exposure to the Neurotoxic Dinoflagellate,Alexandrium catenella,Induces Apoptosis of the Hemocytes of the Oyster,Crassostrea gigas

This study assessed the apoptotic process occurring in the hemocytes of the Pacific oyster, Crassostrea gigas, exposed to Alexandrium catenella, a paralytic shellfish toxins (PSTs) producer. Oysters were experimentally exposed during 48 h to the toxic algae. PSTs accumulation, the expression of 12 key apoptotic-related genes, as well as the variation of the number of hemocytes in apoptosis was measured at time intervals during the experiment. Results show a significant increase of the number of hemocytes in apoptosis after 29 h of exposure. Two pro-apoptotic genes (Bax and Bax-like) implicated in the mitochondrial pathway were significantly upregulated at 21 h followed by the overexpression of two caspase executor genes (caspase-3 and caspase-7) at 29 h, suggesting that the intrinsic pathway was activated. No modulation of the expression of genes implicated in the cell signaling Fas-Associated protein with Death Domain (FADD) and initiation-phase (caspase-2) was observed, suggesting that only the extrinsic pathway was not activated. Moreover, the clear time-dependent upregulation of five (Bcl2, BI-1, IAP1, IAP7B and Hsp70) inhibitors of apoptosis-related genes associated with the return to the initial number of hemocytes in apoptosis at 48 h of exposure suggests the involvement of strong regulatory mechanisms of apoptosis occurring in the hemocytes of the Pacific oyster.     

Effects of Organic and Inorganic Nitrogen on the Growth and Production of Domoic Acid by Pseudo-nitzschia multiseries and P. australis (Bacillariophyceae) in Culture

Over the last century, human activities have altered the global nitrogen cycle, and anthropogenic inputs of both inorganic and organic nitrogen species have increased around the world, causing significant changes to the functioning of aquatic ecosystems. The increasing frequency of Pseudo-nitzschia spp. in estuarine and coastal waters reinforces the need to understand better the environmental control of its growth and domoic acid (DA) production. Here, we document Pseudo-nitzschia spp. growth and toxicity on a large set of inorganic and organic nitrogen (nitrate, ammonium, urea, glutamate, glutamine, arginine and taurine). Our study focused on two species isolated from European coastal waters: P. multiseries CCL70 and P. australis PNC1. The nitrogen sources induced broad differences between the two species with respect to growth rate, biomass and cellular DA, but no specific variation could be attributed to any of the inorganic or organic nitrogen substrates. Enrichment with ammonium resulted in an enhanced growth rate and cell yield, whereas glutamate did not support the growth of P. multiseries. Arginine, glutamine and taurine enabled good growth of P. australis, but without toxin production. The highest DA content was produced when P. multiseries grew with urea and P. australis grew with glutamate. For both species, growth rate was not correlated with DA content but more toxin was produced when the nitrogen source could not sustain a high biomass. A significant negative correlation was found between cell biomass and DA content in P. australis. This study shows that Pseudo-nitzschia can readily utilize organic nitrogen in the form of amino acids, and confirms that both inorganic and organic nitrogen affect growth and DA production. Our results contribute to our understanding of the ecophysiology of Pseudo-nitzschia spp. and may help to predict toxic events in the natural environment.     

Ovatoxin-a and palytoxin accumulation in seafood in relation to Ostreopsis cf. ovata blooms on the French Mediterranean coast

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.     

A Feedback Mechanism to Control Apoptosis Occurs in the Digestive Gland of the Oyster Crassostrea gigas Exposed to the Paralytic Shellfish Toxins Producer Alexandrium catenella

To better understand the effect of Paralytic Shellfish Toxins (PSTs) accumulation in the digestive gland of the Pacific oyster, Crassostrea gigas, we experimentally exposed individual oysters for 48 h to a PSTs producer, the dinoflagellate Alexandrium catenella. In comparison to the effect of the non-toxic Alexandrium tamarense, on the eight apoptotic related genes tested, Bax and BI.1 were significantly upregulated in oysters exposed 48 h to A. catenella. Among the five detoxification related genes tested, the expression of cytochrome P450 (CYP1A) was shown to be correlated with toxin concentration in the digestive gland of oysters exposed to the toxic dinoflagellate. Beside this, we observed a significant increase in ROS production, a decrease in caspase-3/7 activity and normal percentage of apoptotic cells in this tissue. Taken together, these results suggest a feedback mechanism, which may occur in the digestive gland where BI.1 could play a key role in preventing the induction of apoptosis by PSTs. Moreover, the expression of CYP1A, Bax and BI.1 were found to be significantly correlated to the occurrence of natural toxic events, suggesting that the expression of these genes together could be used as biomarker to assess the biological responses of oysters to stress caused by PSTs.     

First evidence of palytoxin and 42-hydroxy-palytoxin in the marine cyanobacterium Trichodesmium

Marine pelagic diazotrophic cyanobacteria of the genus Trichodesmium (Oscillatoriales) are widespread throughout the tropics and subtropics, and are particularly common in the waters of New Caledonia. Blooms of Trichodesmium are suspected to be a potential source of toxins in the ciguatera food chain and were previously reported to contain several types of paralyzing toxins. The toxicity of water-soluble extracts of Trichodesmium spp. were analyzed by mouse bioassay and Neuroblastoma assay and their toxic compounds characterized using liquid chromatography coupled with tandem mass spectrometry techniques. Here, we report the first identification of palytoxin and one of its derivatives, 42-hydroxy-palytoxin, in field samples of Trichodesmium collected in the New Caledonian lagoon. The possible role played by Trichodesmium blooms in the development of clupeotoxism, this human intoxication following the ingestion of plankton-eating fish and classically associated with Ostreopsis blooms, is also discussed.     

Report on the first detection of pectenotoxin-2, spirolide-a and their derivatives in French shellfish

In the context of the French Phytoplankton and Phycotoxins Monitoring Network (REPHY) programme, shellfish samples were harvested from different locations where harmful algae blooms were known to have occurred. For all shellfish samples found positive by the mouse bioassay for diarrhetic shellfish poisoning (DSP) toxins, liquid chromatography (LC) coupled with mass spectrometry (MS) was used to search for the following lipophilic toxins: okadaic acid (OA), dinophysistoxins (DTXs), pectenotoxins (PTXs), azaspiracids (AZAs), yessotoxins (YTXs), spirolides (SPXs) and gymnodimines (GYMs). In order to investigate the presence of acyl-OAs and/or acyl-DTX-1,-2 (DTX-3), alkaline hydrolysis was performed on all samples, and LC/MS analyses were carried out on the samples before and after hydrolysis. The results revealed different lipophilic toxin profiles as a function of the shellfish sampling location. The primary finding was that all of the samples contained OA and acyl-OA. In addition, other lipophilic toxins were found in shellfish samples: DTX-2, acyl-DTX-2 and SPXs (SPX-A, SPX-desMeC) on the Atlantic coast (Southern Brittany, Arcachon), and pectenotoxins (PTX-2, PTX-2-seco-acid and 7-epi-PTX-2-seco-acid) on the Mediterranean coast (Thau lagoon, the island of Corsica). This paper reports on the first detection of PTX-2, SPX-A and their derivatives in French shellfish.     

First Characterization of Ostreopsis cf. ovata (Dinophyceae) and Detection of Ovatoxins during a Multispecific and Toxic Ostreopsis Bloom on French Atlantic Coast

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.     

Monitoring the Emergence of Algal Toxins in Shellfish: First Report on Detection of Brevetoxins in French Mediterranean Mussels

In France, four groups of lipophilic toxins are currently regulated: okadaic acid/dinophysistoxins, pectenotoxins, yessotoxins and azaspiracids. However, many other families of toxins exist, which can be emerging toxins. Emerging toxins include both toxins recently detected in a specific area of France but not regulated yet (e.g., cyclic imines, ovatoxins) or toxins only detected outside of France (e.g., brevetoxins). To anticipate the introduction to France of these emerging toxins, a monitoring program called EMERGTOX was set up along the French coasts in 2018. The single-laboratory validation of this approach was performed according to the NF V03-110 guidelines by building an accuracy profile. Our specific, reliable and sensitive approach allowed us to detect brevetoxins (BTX-2 and/or BTX-3) in addition to the lipophilic toxins already regulated in France. Brevetoxins were detected for the first time in French Mediterranean mussels (Diana Lagoon, Corsica) in autumn 2018, and regularly every year since during the same seasons (autumn, winter). The maximum content found was 345 µg (BTX-2 + BTX-3)/kg in mussel digestive glands in November 2020. None were detected in oysters sampled at the same site. In addition, a retroactive analysis of preserved mussels demonstrated the presence of BTX-3 in mussels from the same site sampled in November 2015. The detection of BTX could be related to the presence in situ at the same period of four Karenia species and two raphidophytes, which all could be potential producers of these toxins. Further investigations are necessary to understand the origin of these toxins.     

Guidance Level for Brevetoxins in French Shellfish

Brevetoxins (BTXs) are marine biotoxins responsible for neurotoxic shellfish poisoning (NSP) after ingestion of contaminated shellfish. NSP is characterized by neurological, gastrointestinal and/or cardiovascular symptoms. The main known producer of BTXs is the dinoflagellate Karenia brevis, but other microalgae are also suspected to synthesize BTX-like compounds. BTXs are currently not regulated in France and in Europe. In November 2018, they have been detected for the first time in France in mussels from a lagoon in the Corsica Island (Mediterranean Sea), as part of the network for monitoring the emergence of marine biotoxins in shellfish. To prevent health risks associated with the consumption of shellfish contaminated with BTXs in France, a working group was set up by the French Agency for Food, Environmental and Occupational Health & Safety (Anses). One of the aims of this working group was to propose a guidance level for the presence of BTXs in shellfish. Toxicological data were too limited to derive an acute oral reference dose (ARfD). Based on human case reports, we identified two lowest-observed-adverse-effect levels (LOAELs). A guidance level of 180 µg BTX-3 eq./kg shellfish meat is proposed, considering a protective default portion size of 400 g shellfish meat.