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.     

Rapid Biotic and Abiotic Transformation of Toxins produced by Ostreopsis. cf. ovata

The dinoflagellate Ostreopsis cf. ovata produces several families of toxic polyketides. Despite only a few field measurements of these phycotoxins in seawater and aerosols, they are believed to be responsible for dermatitis and the toxic inhalations reported during blooms of this species. Therefore, the stability of these compounds in seawater is essential to understanding the causes of these symptoms, however, this has never been assessed. In the current study, the optimization of a solid phase extraction (SPE) procedure was first performed to ensure the most efficient extraction of all phycotoxins known to be produced by this strain, including the recently described liguriatoxins. The SPE cartridge SDBL^® under non acidified conditions offered the best option. The stability of the ovatoxins and the liguriatoxins under biotic and abiotic stress was assessed by exposing the spent medium of a culture of Ostreopsis cf. ovata to its bacterial consortium and natural sunlight. A rapid biotic transformation was detected for both families of compounds. When exposed to bacteria, the half-lives of the ovatoxins were reached before 10 h and at 36 h, 97% of these toxins had been transformed. The half-lives of the liguriatoxins were 10 h under these conditions. Photolysis (abiotic degradation) of the ovatoxins (T_1/2 < 36 h) was faster than for the liguriatoxins (T_1/2 > 62 h). Although none of the catabolites of these phycotoxins were thoroughly identified, an untargeted metabolomics approach combined with molecular networking highlighted the presence of several compounds exhibiting structural similarities with the ovatoxins. Additional work should confirm the preliminary findings on these potential ovatoxins’ catabolites and their biological properties. The rapid transformation of O. cf. ovata’s phycotoxins introduces questions concerning their presence in seawater and their dispersion in the sea spray aerosols. The compounds involved in the toxic inhalations and dermatitis often experienced by beachgoers may stem from the catabolites of these toxins or even unrelated and as yet unidentified compounds.     

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.     

Intrabody Tetrodotoxin Distribution and Possible Hypothesis for Its Migration in Ribbon Worms Cephalothrix cf. simula (Palaeonemertea,Nemertea)

Tetrodotoxin (TTX) is a potent neurotoxin found in many marine and terrestrial animals, but only a few species, such as the ribbon worms of the genus Cephalothrix, accumulate it in extremely high concentrations. The intrabody distribution of TTX in highly toxic organisms is of great interest because it helps researchers to understand the pathways by which the toxin migrates, accumulates, and functions in tissues. Using immunohistochemistry with anti-TTX antibodies, the authors of this study investigated the toxin’s distribution inside the organs, tissues, and cells of Cephalothrix cf. simula. The cell types of TTX-positive tissues were identified by light microscopy. The main sites of TTX accumulation occurred in the secretory cells of the integuments, the microvilli of the epidermal ciliary cells, cephalic glands, the glandular epithelia of the proboscises, the enterocytes of the digestive systems, and nephridia. Obtained data suggest the toxin migrates from the digestive system through blood vessels to target organs. TTX is excreted from the body through the nephridia and mucus of epidermal cells.