Ostreopsis Chemical Ecology and Allelopathy Network – OCEAN-15

Task 1: The method of extraction and the analytical conditions were optimised in LC-MS for the determination of the toxinic profiles of all collected samples. The cytotoxicity assay using Neuro-2A cells (Mice neuroblastoma) was adapted and optimised for the culture extracts analyses of O. ovata containing the ovatoxins but also other metabolites. This method is used in the bioguided fractionation of the extract on LH20 column where the fractions are tested for their N-2A bioactivity but also LC-MS/MS and LC-HRMS analyses.
The biosynthetic experiments were performed in June 2017 at the IAEA Monaco by feeding of radioactive putative precursors of the ovatoxins.
Task 2: Data obtained during the first field trip (summer 2016) allowed the identification of the conditions for the study of global change on the ecophysiology and metabolism of Ostreopsis (T C: 20, 25, 30 C; Light: 100, 250, 400 µmol.m-2.s-1 ). The collection of a large quantity of Ostreopsis were also performed in situ to provide more biological material.
Task 3: A study on the putative allelopathic effects of Ostreopsis was performed by coculture on competitive benthic microalgae. A study of the effects on other associated fauna like copepods was also undertaken.
Task 4: On the toxicology side, the effects of palytoxin were studied in vitro on model cells of intestinal and nasal epithelia. In the absence of other purified toxins, we started our work with palytoxin, a close and commercially available analogue of the ovatoxins produced by Ostreopsis ovata. The first results showed that ovatoxins may be less toxic than palytoxin.

Task 1: On the basis of the toxinic profiles obtained on in situ strains of O. ovata two strains were selected for culturing and further analyses. The strain with the most complex toxinic profile was selected for the studies on task 2 and 3 (MCCV54) for the studies on the metabolome while the second strain (MCCV55) was dedicated to the purifications of the metabolites of task 1. The analytical method by LC-MS was also developed for the toxins.
For the biosynthetic experiment, two out of the 4 tested precursors were clearly incorporated in the ovatoxins (beta-alanine and SAM)
Task 2: The monitoring undertaken in the summer 2016 showed a relationship between the development of Ostreopsis and the environmental parameters. Unlike several publications, higher temperature have a negative effect on its development.
Task 3: The allelopathic study showed an effect of Ostreopsis but only by contact. An inhibitory effect of O. ovata on several primary producers (7 species of diatoms and dinoflagellates) was also evidenced. Some unknown secondary metabolites would be involved in this interaction. The consumption of the toxic microalga by some copepods seems related to an important effect on the reproduction of the crustaceans.
Task 4: The results in toxicology showed that after 24 h of exposure palytoxin induces apoptotic effects on human intestinal cells of enterocyte type Caco2 and on a coculture of enterocytes with mucus (75% Caco2/25% HT29-MTX). An additional inflammatory response was also observed. The effects of palytoxin were also investigated on a 3D culture system of human nasal epithelium. After 24 h of exposure results do not show any effect of palytoxin between 0.5 and 10 nM.

Task 1: Culture of the strain MCCV55 at larger scale (20 L) is still in an optimization process to provide a large biomass for the purification and isolation of metabolites (Task 1). The study of the exometabolome is currently ongoing.
About the biosynthetic experiment, the assessment of the specific activity of the ovatoxins produced by O. ovata cultures fed with radiolabeled precursors is still ongoing after purification by HPLC and counting on a beta imager. It will allow us to propose some key biosynthetic steps of this complex toxin.
Task 2: Colelction of additional samples of O. ovata will be performed in summer 2017 and the first studies on the effects of changes in the environmental parameters on the chemical profiles will be performed.
Task 3: the identification of the metabolites responsible for the behaviour observed with other microalgae in the allelopathic experiment will be undertaken. The study of the interaction of O. ovata and macroalgal species is also ongoing.
Task 4: Some cell extracts obtained in Task 1 are under toxicological evaluation since may 2017 and the study of the effects of palytoxin are still ongoing.

1. Jauzein, C., Couet, D., Blasco, T. & R. Lemée (2017). Uptake of dissolved inorganic and organic nitrogen by the benthic toxic dinoflagellate Ostreopsis cf. ovata. Harmflul Algae, 65: 9-18.
2. Berdalet, E., Tester, P.A., Chinain, M., Fraga, S., Lemée, R., Litaker, W., Penna, A., Usup, G., Vila, M. & A. Zingone (2017). Harmful Algal Blooms in Benthic Systems: Recent progress and future research. Oceanography 30(1): 36-45.

Several species of microalgae have developed a complex specialized metabolism yielding to the production of toxic compounds. When highly concentrated and quickly multiplying, these toxic microalgae are likely to induce negative environmental or toxicological effects, by forming Harmful Algal Blooms (HABs). During the past decade, a toxic benthic dinoflagellate belonging to the genus Ostreopsis has bloomed repetitively along the Mediterranean coastline. The causes implied in the increasing incidence of these toxic blooms have not yet been determined even if global change has been pointed out. Indeed, due to its geographical features, the Mediterranean Sea is particularly sensitive to increasing temperatures and solar irradiance that would lead to enhanced thermal stratification and therefore to alterations in ecosystem functioning.
Blooms of Ostreopsis were associated to human afflictions in Italy and France, such as fever, water rhinorrhea, pharyngeal pain, dry or mildly productive cough, headache, nausea/vomiting, and bronchoconstriction. Adverse effects on benthic communities of bivalves, gastropods and echinoderms were also observed in some cases. These deleterious effects on both the humans and ecosystem health were attributed to analogs of the potent palytoxin, namely ovatoxins (a to h) produced by Ostreopsis cf. ovata. However, the specialized metabolism of O. cf. ovata has been partially identified and other metabolites involved in the toxic effects are likely to be produced by the microalgae and require additional studies. Specialized metabolites are also involved in the chemical mediation between organisms and, up to now, the influence of chemical cues on the development of several benthic organisms has rarely been investigated. Therefore, any answers coming from this field named chemical ecology will be of high added value. In this context, the overall objective of OCEAN-15 is to investigate the effects of climate change on the specialized metabolism of these microalgae in order to anticipate the potential modification of its toxic behavior as well as the subsequent ecological interactions that would alter marine ecosystems. This objective fits the societal challenge 1 listed in the ANR 2015 Work Program and more specifically its axis 2 through an interdisciplinary research on "health risks facing environmental changes" bringing useful knowledge to integrative policy in public health. The project OCEAN-15 was subdivided in four main tasks addressing several aspects of the chemical ecology of O. cf. ovata: (1) study of the specialized metabolism, (2) effects of global change on this metabolism, (3) ecological impacts of the metabolism; and (4) toxicological effects and mechanisms associated to the metabolites.
A truly collaborative and multidisciplinary effort will help reaching the proposed objectives. In this project, we will combine some of the leading groups in France in the field of marine chemical ecology and metabolomics (ICN), phycotoxin chemistry and ecotoxicology (IFREMER), phytoplanktonic ecology (LOV) as well as human toxicology (ANSES). The capability and success of this consortium has already been demonstrated through the joint participation of the different partners to diverse research groups supported by the CNRS (GdR Phycotox, GdR MediatEC) as well as to international consortium (ISSHA, International Society for the Study of Harmful Algae). Thus, through this synergistic project, we are convinced to bring answers on the impact of global change on Ostreopsis cf. ovata specialized metabolism and thus allelopathy and toxicity. Any answers coming from this project would benefit to the society, as they will help further monitoring of HABs and protecting human health along the touristic Mediterranean coastline.