Conférence nationale GdR Phycotox & GIS Cyano 2017 · 4 GdR Phycotox – GIS Cyano 2017,...

Conférence nationale GdR Phycotox & GIS Cyano 2017

Résumés des présentations et des posters

14 - 16 Mars 2017, Gif-sur-Yvette (France)

GdR Phycotox – GIS Cyano 2017, Gif-sur-Yvette

Comité scientifique d’organisation Romulo ARAOZ ......................................... CNRS – Gif-sur-Yvette

Hélène HEGARET ...................................... IUEM – LEMAR - Brest

Philipp HESS ............................................. IFREMER – Nantes DYNECO/PHYC

Christophe LAPLACE-TREYTURE ........... IRSTEA - Bordeaux

Delphine LATOUR ..................................... UNIVERSITE de Clermont-Ferrand

Benjamin MARIE ....................................... MNHN – Paris

Joël ROBIN ................................................ ISARA – Lyon

Raffaele SIANO .......................................... IFREMER – Brest DYNECO/Pelagos

Anne THEBAULT ....................................... ANSES – Maisons-Alfort.

Remerciements: Nous souhaitons remercier le CNRS de Gif-sur-Yvette d’avoir accueilli cette conférence dans ses

locaux. Que soit aussi remercié le CNRS, Irstea, l’Ifremer, l’ANSES et les sociétés Novakits et

Bionef pour leur soutien financier.

Enfin, nous remercions toutes les personnes qui se sont impliquées de près ou de loin dans

l’organisation de cette conférence et toutes celles qui apporteront leur aide précieuse pour en

assurer le bon déroulement le jour J.

Couverture : schéma réalisé par Romulo Araoz (CNRS – Gif/Yvette),

mise en page par Françoise Peyriguer (Irstea – Antony).

1


Program

Tuesday 14 March 2017

13:30-14:00 Welcome coffee and inscription

14:00-14:15 Introductory speech

14:15-15:00 Invited conference: The Integrated Pest Management Model as a Framework

for Aquaculture HAB Mitigation

Gary Wikfors

15:00-16:00 THEME: Effects of HABs on professional sectors and society at large: from

legal and socio-economic perspectives

Chairmans: Christophe Laplace-Treyture & Rodolphe Lemée

15:00-15:15 Coordinating the implementation of the interagency Global CFP Strategy

Mireille Chinain, Marie-Yasmine Dechraoui Bottein, Philipp Hess

15:15-15:30 Pinnatoxins, an emergent class of fast-acting marine toxins, are potential threat for

neurodevelopment and global health

Sophie Creuzet, Aurélie Couesnon, Vincent Dive, Armen Zakarian, Jordi Molgó, Denis

Servant

15:30-15:45 Eaters of oyster: From the risk of contamination by toxic algae to social

representations and practices of consumption

Elisabeth Guillou, Adeline Raymond

15:45-16:00 Thinking out of the box: finding new ways of regulating marine toxins

Marie Bonnin, Hélène Hégaret, Philippe Soudant, Philippe Fotso, Caroline Fabioux, Philipp

Hess

16:00-16:30 Coffee break

16:30-18:15 THEME: Tropics

Chairmans: Christophe Laplace-Treyture & Rodolphe Lemée

16:30-16:45 Ciguatera incidence in the Caribbean Islands

Jose Ernesto Mancera

16:45-17:00 Microalgae from French Polynesian lagoons: novel source of bioactive

compounds?

Mireille Chinain, Sebastien Longo, Kevin Henry, André Ung, Taiana Darius, Jérôme Viallon,

Martine Rodier, Bruno Delesalle, Alain Lo-Yat, Gilles Le Moullac, Romulo Araoz

17:00-17:15 Distribution and abundances of benthic toxic dinoflagellates in French West Indies

Aurélie Boisnoir, Pierre-Yves Pascal, Sophie Marro, Rodolphe Lemée

17:15-17:30 Taxonomic diversity of the dinoflagellate genus Ostreopsis in French Polynesia:

preliminary results from Marquesas Islands (Pacific Ocean)

Nicolas Chomérat, Clémence Gatti, Gwenael Bilien, André Ung, H. Taiana Darius, R.

Wayne Litaker, Patricia Tester, Mireille Chinain

17:30-17:45 Taxonomic characterization of Gambierdiscus species in Reunion Island, Indian

Ocean

Alina Tunin-Ley, Nicolas Chomérat, Gwenael Bilien, Jean Turquet

2


17:45 18:00 Environmental control and dynamics of blooms of the benthic toxic dinoflagellate

Ostreopsis cf. ovata in the Mediterranean Sea

Cécile Jauzein, Rodolphe Lemée

18h00 18h15 Ostreopsis blooms at the global scale: influence of macroalgal communities

Luisa Passeron Mangialajo, Rodolphe Lemée, Anna Fricke, Daniela Catania, Sarah Kulins,

Cécile Jauzein, Nick Shears

18:15-19:00 GdR Phycotox Annual general meeting

19:00-20:00 Welcome cocktail

Wednesday 15 March 2017

09:00-09:45 Invited conference: The diatom genus Pseudo-nitzschia in the Gulf of Naples

(Mediterranean Sea): exploring diversity and life history traits

Marina Montresor

09:45-10:45 THEME: Ecology of HAB

Chairmans: Delphine Latour & Mathilde Schapira

09:45-10:00 Genetic, morphological and ecological evidence of new oomycete parasites of the

toxic diatom genus Pseudo-nitzschia

Garvetto Andrea, Claire Gachon, Elisabeth Nézan, Raffaele Siano

10:00-10:15 Effects of salinity variation on the physiology and domoic acid production by two

strains of Pseudo-nitzschia australis

Nour Ayache, Amandine Caruana, Fabienne Hervé, Zouher Amzil

10:15-10:30 Interspecific and intraspecific variability in physiology among four Pseudo-nitzschia

species

Aurore Sauvey, Pascal Claquin, Juliette Fauchot

10:30-10:45 Nutrient ratios influence variability in Pseudo-nitzschia species diversity and

particulate domoic acid production in the Bay of Seine (France)

Juliette Fauchot, Maxine Thorel, Mathilde Schapira, Romain Le Gendre, Philippe Riou,

Didier Goux, Bertrand Le Roy, Virginie Raimbault, Anne-Flore Deton-Cabanillas, Pauline

Bazin, Valérie Kientz-Bouchart, Pascal Claquin


11:15-12:30 THEME: Diversity and theoretical ecological niches of HAB, synthesis and

transformation of metabolites, analytical challenges

Chairmans: Delphine Latour & Mathilde Schapira

11:15-11:30 Benthic Planktothrix and their antifungal natural products

Claire Pancrace, Jouni Jokela, Nathalie Sassoon, Christelle Ganneau, Marie Desnos-

Ollivier, Matti Wahlsten, Anu Humisto, Alexandra Calteau, Sylvie Bay, David P. Fewer,

Kaarina Sivonen, Muriel Gugger

11:30-11:45 From cyanobacteria to mammals: role of ergothionein, glutathione and its analogs

in stressors signalling and detoxication

Kinsley Narainsamy, Christophe Junot, Corinne Cassier-Chauvat, Franck Chauvat

3


11:45-12:00 Latest advances in two functional methods to detect ciguatoxins (CTXs): the

Receptor Binding Assay (RBA) & the Cell Based Assay (CBA-N2a)

Taiana Darius, Jérôme Viallon, Jennifer Mc Call, Taina Revel, Ransom Hardison, William

Holland, Patricia Tester, Wayne Litaker, Mireille Chinain

12:15-12:30 Rapid characterization of microcystin-producing cyanobacteria in freshwater lakes

by TSA-FISH (Tyramid Signal Amplification - Fluorescent In Situ Hybridization)

Isabelle Biegala, Luc Brient

12:30-14:00 Lunch

14:00-:14:45 Invited conference: The Užice Case

Jussy Meriluoto, Zorica Svirčev

14:45-16:15 THEME: Human and animal toxicology

Chairmans: Benjamin Marie & Ronel Biré

14:45-15:00 Mixtures of lipophilic phycotoxins: exposure data and in vitro toxicity assessment

Jimmy Alarcan, Ronel Biré, Ludovic Le Hegarat, Valérie Fessard

15:00-15:15 Ostreopsis cf. ovata blooms in the NW Mediterranean: Exploring the links between

toxins variability and impacts on human health

Elisa Berdalet, Rafael Abós-Herràndiz, Marta Estrada

15:15-15:30 Maitotoxin-4, a novel MTX analog produced by Gambierdiscus excentricus

Francesco Pisapia, Manoëlla Sibat, Christine Herrenknecht, Korian Lhaute, Greta Gaiani,

Pierre-Jean Ferron, Valérie Fessard, Catherine Roullier, Santiago Fraga, Silvia

Nascimento, Wayne Litaker, Philipp Hess

15:30-15:45 Transgenerational effects of Microcystin-LR on Daphnia magna

Claudia Wiegand, Rafael Ortiz-Rodríguez, Thanh Dao

15:45-16:00 Gambierol enhances quantal acetylcholine release from motor nerve terminals of

vertebrate neuromuscular junctions

Jordi Molgó, Sébastien Schlumberger, Makoto Sasaki, Haruhiko Fuwa, M. Louzao, Luis

Botana, Evelyne Benoit

16:00-16:15 Receptor-based lateral flow test for fast and early detection of neurotoxins acting

on nicotinic acetylcholine receptors

Romulo Araoz, Denis Servent


16:45-17:15 POSTER SESSION: My poster in 3 minutes

Chairman: Valérie Fessard

17:15-19:30 POSTER SESSION

18:30-19 :30 Réunion du comité de pilotage pour préparer la conférence ICHA 2018 à Nantes

19:30 Diner cocktail

4


Thursday 16 March 2017

09:00-09:45 Invited conference: Differential Effects of Algal Toxins on Grazing by the

Marine Copepod Acartia tonsa

Patricia Tester, Jiarong Hong, Rebecca Waggett, Joseph Katz, Allen Place,

Erik Davenport

09:45-10:45 THEME: Effects of HAB on marine organisms and ecosystems, trophic

transfer, etc.

Chairmans: Juliette Fauchot & Cécile Bernard

09:45-10:00 Interest of bivalves for the biosurvey of environmental contamination by

cyanobacterial toxins

Emilie Lance, Zouher Amzil, Alexandra Lepoutre, Luc Brient, Myriam Bormans

10:00-10:15 Impacts of Alexandrium minutum on the reproduction of the oyster Crassostrea

gigas: from gametogenesis to larval settlement

Justine Castrec, Philippe Soudant, Marianne Alunno Bruscia, Bruno Petton, Isabelle Queau,

Marc Suquet, Dominique Ratiskol, Jacqueline Le Grand, Christian Mingant, Nelly Le Goic,

Hélène Hégaret, Caroline Fabioux

10:15-10:30 Impact of the environmental toxin BMAA on the immune system of the zebra mussel

Dreissena polymorpha.

Alexandra Lepoutre, Nadia Milliote, Mélissa Palos Ladeiro, Marc Bonnard, Elisabeth

Faassen, Alain Geffard, Emilie Lance

10:30-10:45 Metabolomics for cyanobacteria ecotoxicology: insights from the Toxcyfish project

Benjamin Marie, Benoit Sotton, Séverine Le Manach, Alain Paris


11:15-11:30 Short term effects of the allelochemicals released by the toxic dinoflagellate

Alexandrium minutum upon the diatom Chaetoceros sp.

Marc Long, Kevin Tallec, Philippe Soudant, Fabienne Le Grand, Géraldine Sarthou, Dianne

Jolley, Hélène Hégaret

11:30-11:45 Phenolic compounds from the seagrasses Zostera marina and Zostera noltei affect

negatively the physiology of the neurotoxic dinoflagellate Alexandrium catenella

Mohamed Laabir

11:45-12:00 Paralytic Shellfish Toxin accumulation in oyster is predicted by gene expression

Audrey Mat, Christophe Klopp, Laura Payton, Céline Jeziorski, Morgane Chalopin, Zouher

Amzil, Damien Tran, Hélène Hégaret, Philippe Soudant, Caroline Fabioux, Arnaud Huvet

12:00-12:15 The Paralytic Shellfish Toxin, Saxitoxin, enters the cytoplasm and induces

apoptosis of oyster immune cells via a caspases-dependent pathway

Jean-Luc Rolland, Célina Abi-Khalil, Darren Finkelstein, Genevieve Conejero, Justin Du

Bois, Delphine Destoumieux-Garzon

12:15-12:30 Continued feeding with Gambierdiscus polynesiensis causes increasing

accumulation of ciguatoxin over 16 weeks in an herbivorous reef fish

Rachel Clausing, Barbara Losen, Taiana Darius, Philipp Hess, Manoella Sibat, Jerome

Viallon, Mireille Chinain, Marie-Yasmine Dechraoui Bottein

12:30-12:45 Molecular Characterization of Voltage-Gated Sodium Channels and Their Relations

with Paralytic Shellfish Toxin Bioaccumulation in the Pacific Oyster Crassostrea

5


gigas

Floriane Boullot, Justine Castrec, Adeline Bidault-Toffin, Natanael Dantas, Laura Payton,

Mickael Perrigault, Damien Tran, Zouher Amzil, Pierre BOUDRY, Philippe Soudant,

Hélène Hegaret, Caroline Fabioux

12:45-14:15 Lunch

14:15-15:00 Invited conference: Strategies to reduce toxins in shellfish

Ana Garcia Cabado

15:00-16:15 THEME: Diversity and theoretical ecological niches of HAB, synthesis and

transformation of metabolites, analytical challenges

Chairmans: Joël Robin & Elisa Berdalet

15:00-15:15 Monitoring and modelling cyanobacteria biomass in a small urban lake

Denis Plec, Lucas Jardim Porto, Bruno Lemaire, Philippe Dubois, Mohamed Saad, Cécile

Bernard, Laure Huguenard, Brigitte Vinçon-Leite

15:15-15:30 Temperature and stratification of the artificial lake and the proliferation of

cyanobacteria, case of the lake of the Eguzon dam - France

Zoubida Qsair, Laurent Touchart, Pascal Bartout

15:30-15:45 Genetic diversity of the toxic bloom-forming cyanobacterium Microcystis

aeruginosa: a meta-analysis at large spatial and temporal scales

Marion Sabart, Benjamin Misson, Marlène Jobard, Gaofei Song, Li Renhui, Delphine Latour

15:45-16:00 Wise use of metabolomics in the field of marine chemical ecology

Eva Ternon, Anne-Sophie Pavaux, Olivier Thomas

16:00-16:15 Metabolomics of ancient populations of DINoflAgellates in phosphorous-limited

conditions

Raffaele Siano, Florence Mondeguer, Marie Latimier, Julien Quéré, Manoella Sibat, Laure

Guillou, Philipp Hess

16:15 End of the symposium

6


Table of contents

Theme 1: Diversity and theoretical ecological niches of HAB, synthesis and transformation of metabolites, analytical challenges ..........................................................12

Rapid characterization of microcystin-producing cyanobacteria in freshwater lakes by TSA-FISH (Tyramid Signal Amplification - Fluorescent In Situ Hybridization), Isabelle Biegala [et al.] ......................................................................................................13

Latest advances in two functional methods to detect ciguatoxins (CTXs): the Receptor Binding Assay (RBA) & the Cell Based Assay (CBA-N2a),

Taiana Darius [et al.] ........................................................................................................14

From cyanobacteria to mammals: role of ergothionein, glutathione and its analogs in stressors signalling and detoxication,

Kinsley Narainsamy [et al.]. ..............................................................................................15

Benthic Planktothrix and their antifungal natural products, Claire Pancrace [et al.] ......................................................................................................16

Monitoring and modelling cyanobacteria biomass in a small urban lake, Denis Plec [et al.]. ............................................................................................................17

Temperature and stratification of the artificial lake and the proliferation of cyanobacteria, case of the lake of the Eguzon dam - France, Zoubida Qsair [et al.] ........................................................................................................18

Genetic diversity of the toxic bloom-forming cyanobacterium Microcystis aeruginosa: a meta-analysis at large spatial and temporal scales, Marion Sabart [et al.]. ......................................................................................................19

MEtabolomics of ancient populations of DINoflAgellates in phosphorous-limited conditions,

Raffaele Siano [et al.] ........................................................................................................20

Wise use of metabolomics in the field of marine chemical ecology, Eva Ternon [et al.]. ...........................................................................................................21

Theme 2: Ecology of HAB ....................................................................................................22

Genetic, morphological and ecological evidence of new oomycete parasites of the toxic diatom genus Pseudo-nitzschia,

Garvetto Andrea [et al.] ....................................................................................................23

Effects of salinity variation on the physiology and domoic acid production by two strains of Pseudo-nitzschia australis, Nour Ayache [et al.] ..........................................................................................................24

Nutrient ratios influence variability in Pseudo-nitzschia species diversity and particulate domoic acid production in the Bay of Seine (France), Juliette Fauchot [et al.] .....................................................................................................25

7


The diatom genus Pseudo-nitzschia in the Gulf of Naples (Mediterranean Sea): exploring diversity and life history traits, Marina Montresor ........................................................................................................................ 26

Interspecific and intraspecific variability in physiology among four Pseudo-nitzschia species, Aurore Sauvey [et al.] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

Theme 3: Effects of HAB on marine organisms and ecosystems, trophic transfer, etc. 28

Molecular Characterization of Voltage-Gated Sodium Channels and Their Relations with Paralytic Shellfish Toxin Bioaccumulation in the Pacific Oyster Crassostrea gigas, Floriane Boullot [et al.] .....................................................................................................29

Impacts of Alexandrium minutum on the reproduction of the oyster Crassostrea gigas: from gametogenesis to larval settlement,

Justine Castrec [et al.] ......................................................................................................30

Continued feeding with Gambierdiscus polynesiensis causes increasing accumulation of ciguatoxin over 16 weeks in an herbivorous reef fish, Rachel Clausing [et al.]. ....................................................................................................31

Phenolic compounds from the seagrasses Zostera marina and Zostera noltei affect negatively the physiology of the neurotoxic dinoflagellate Alexandrium catenella, Mohamed Laabir ...............................................................................................................32

Interest of bivalves for the biosurvey of environmental contamination by cyanobacterial toxins,

Emilie Lance [et al.] ..........................................................................................................33

Impact of the environmental toxin BMAA on the immune system of the zebra mussel Dreissena polymorpha, Alexandra Lepoutre [et al.] ...............................................................................................34

Short term effects of the allelochemicals released by the toxic dinoflagellate Alexandrium minutum upon the diatom Chaetoceros sp.. Marc Long [et al.] .............................................................................................................35

Metabolomics for cyanobacteria ecotoxicology: insights from the Toxcyfish project,

Benjamin Marie [et al.] .....................................................................................................36

Paralytic Shellfish Toxin accumulation in oyster is predicted by gene expression, Audrey Mat [et al.]. ..........................................................................................................37

The Paralytic Shellfish Toxin, Saxitoxin, enters the cytoplasm and induces apoptosis of oyster immune cells via a caspases-dependent pathway,

Jean-Luc Rolland [et al.] ..................................................................................................38

Differential Effects of Algal Toxins on Grazing by the Marine Copepod Acartia tonsa,

Patricia Tester [et al.] .......................................................................................................39

8


Theme 4: Human and animal toxicology ..............................................................................40

Receptor-based lateral flow test for fast and early detection of neurotoxins acting on nicotinic acetylcholine receptors., Romulo Araoz [et al.]........................................................................................................41 Mixtures of lipophilic phycotoxins: exposure data and in vitro toxicity assessment, Jimmy Alarcan [et al.] ......................................................................................................42

Ostreopsis cf. ovata blooms in the NW Mediterranean: Exploring the links between toxins variability and impacts on human health, Elisa Berdalet [et al.] ........................................................................................................43

The Užice Case, Jussi Meriluoto [et al.] .......................................................................................................................... 44

Gambierol enhances quantal acetylcholine release from motor nerve terminals of vertebrate neuromuscular junctions, Jordi Molgo [et al.] ...........................................................................................................45

Maitotoxin-4, a novel MTX analog produced by Gambierdiscus excentricus, Francesco Pisapia [et al.]. ................................................................................................46

Transgenerational effects of Microcystin-LR on Daphnia magna, Claudia Wiegand [et al.]. ..................................................................................................47

Theme 5: Effects of HABs on professional sectors and society at large: from legal and socio-economic perspectives ................................................................................................ 48

Thinking out of the box: finding news ways of regulating marine toxins, Marie Bonnin [et al.] .........................................................................................................49

Coordinating the implementation of the interagency Global CFP Strategy, Mireille Chinain [et al.]. ....................................................................................................50

Pinnatoxins, an emergent class of fast-acting marine toxins, are potential threat for neurodevelopment and global health, Sophie Creuzet [et al.]. ......................................................................................................51

Strategies to reduce toxins in shellfish, Ana Garcia-Cabado .........................................................................................................52 Eaters of oyster: From the risk of contamination by toxic algae to social representations and practices of consumption, Elisabeth Guillou [et al.] ...................................................................................................53

The Integrated Pest Management Model as a Framework for Aquaculture HAB Mitigation, Gary Wikfors ....................................................................................................................54

9


Theme 6: Tropics .................................................................................................................. 55

Distribution and abundances of benthic toxic dinoflagellates in French West Indies,

Aurelie Boisnoir [et al.] .....................................................................................................56

Microalgae from French Polynesian lagoons: novel source of bioactive compounds?, Mireille Chinain [et al.] .....................................................................................................57

Taxonomic diversity of the dinoflagellate genus Ostreopsis in French Polynesia: preliminary results from Marquesas Islands (Pacific Ocean),

Nicolas Chomerat [et al.]. ..................................................................................................58

Environmental control and dynamics of blooms of the benthic toxic dinoflagellate Ostreopsis cf. ovata in the Mediterranean Sea,

Cecile Jauzein [et al.] ........................................................................................................59

Ciguatera incidence in the Caribbean Islands, Jose Ernesto Mancera ......................................................................................................60

Ostreopsis blooms at the global scale: influence of macroalgal communities, Luisa Passeron Mangialajo [et al.]. ....................................................................................61

Taxonomic characterization of Gambierdiscus species in Reunion Island, Indian Ocean,

Alina Tunin-Ley [et al.] ....................................................................................................62

Poster: My poster in 3 minutes ............................................................................................. 63

Metabolic interactions between Daphnia and Cyanobacteria, Gorenka Bojadzija [et al.]. ...............................................................................................64

Transfer and fate of toxic cyanobacteria from freshwater to the marine environment, Maxime Georges Des Aulnois [et al.] .................................................................................65

Impact of the global warming on the production of toxins and other metabolites from Ostreopsis ovata,

Marin-Pierre Gemin [et al.] ...............................................................................................66

In vitro neurocutaneous effects of ciguatoxin P-CTX-2: evidence for a significant role for keratinocytes, Killian L’herondelle [et al.].................................................................................................67

Preliminary results of a quantitative risk assessment during blooms of Alexandrium minutum in Brittany, Mathias Lunghi [et al.] .....................................................................................................68

In vitro genotoxic and cytotoxic effects of microcystins (-LR, -RR and -LF) revealed by the micronucleus assay and high content analysis in the human HepaRG cell line, Rym Merabet [et al.] ........................................................................................................69

Monitoring cyanobacterial blooms using satellite remote sensing and modeling techniques. Application to Karaoun Reservoir, Lebanon, Najwa Sharaf ...................................................................................................................70

10


Poster ....................................................................................................................................... 71

First demonstration that Prorocentrolide A, from cultured Prorocentrum lima dinoflagellates collected in Japan, blocks nicotinic acetylcholine receptors, Muriel Amar [et al.] ............................................................................................................... 72

Physico-chemical and functional characterization of Portimine purified from Vulcanodinium rugosum strain IFR-VRU-01, Romulo Araoz [et al.] ............................................................................................................ 73

Electrophysiological evaluation of Pacific oyster (Crassostrea gigas) sensitivity to paralytic shellfish toxins, Floriane Boullot [et al.] .....................................................................................................74

Chemically mediated interactions between Microcystis and Planktothrix: impact on their physiology and metabolic profiles,

Enora Briand [et al.] .........................................................................................................75

Mechanisms of irreversible foam-forming blooms with cyanobacterias: a quantitative study, Philippe Brunet [et al.]. ....................................................................................................76

First report of Pseudo-nitzschia plurisecta in French Atlantic coastal waters, Amandine Caruana [et al.]. ...............................................................................................77

Identify toxic micro-algae. Longitudinal study of the representation of efflorescence through visual alerts, Simona Cojocariu [et al.] ..................................................................................................78

Pinnatoxins A and G block neuromuscular transmission and nicotinic acetylcholine receptors in vertebrate skeletal muscles, Aurélie Couesnon [et al.] ..................................................................................................79

ANOCIBIOTOX – Non targeted analysis of marine biotoxins in fishery products from mainland France and French overseas territories,

Ines Dom [et al.] ...............................................................................................................80

An in vitro approach to assess the impact of Dinophysis sp. on oyster Crassostrea gigas reproduction,

Helene Hegaret [et al.] ......................................................................................................81

Study of the relationship between toxin synthesis, photosynthesis and photoprotection mechanisms in order to better understand the regulation of microcystin synthesis and their possible protective role in different Planktothrix strains, Feilke Kathleen [et al.] ......................................................................................................82

The cyrtophora ciliates as potential regulators of filamentous cyanobacteria dynamics and models to study relationships between grazers and toxin-producing species, Benjamin Legrand [et al.] .................................................................................................83

Akinetes distribution and persistence trough the benthic phase in order to evaluate their potential of germination, Benjamin Legrand [et al.] .................................................................................................84

Development of New Biosensors to detect Ciguatoxins, Hélène Martin-Yken [et al.] ...............................................................................................85

11


Impact of the benthic toxic dinoflagellate Ostreopsis cf. ovata on coastal fauna in NW Mediterranean Sea, Anne-Sophie Pavaux [et al.] ...............................................................................................86

Detection of pacific ciguatoxins using liquid chromatography coupled to tandem low resolution mass spectrometry (LC-MS/MS), Manoella Sibat [et al.]. ......................................................................................................87

List of sponsors .................................................................................................................... 88

List of participants ..................................................................................................................90

12


Theme 1: Diversity and theoretical ecological niches of HAB, synthesis and transformation of metabolites,

analytical challenges

13


∗Speaker

Rapid characterization of microcystin-producing cyanobacteria in freshwater lakes by TSA-FISH (Tyramid Signal Amplification - Fluorescent In Situ

Hybridization)

Isabelle Biegala ∗ 1, Luc Brient 2

1 Institut Mediterraneen d’Oceanologie (M.I.O) – Institut de recherche pour le developpement [IRD] :

UMRUMR235, CNRS : UMR7249, Aix Marseille Universite, Universite de Toulon – Batiment Mediterranee, Campus de Luminy, 163 Avenue de Luminy, 13288 Marseille, France

2 Universite de Rennes-1 (Universite de Rennes-1) – Universitee de Rennes-1 – 2 rue du Thabor CS

46510 35065 Rennes cedex, France

Microcystin (MC) is a common and widespread toxin which represents a health hazard to

humans and animals. MC toxin concentrations are monitored by various direct or proxy techniques (HPLC, LC-MS/MS, ELISA PPIA) and potentially toxic strains by PCR. However, these techniques do not discriminate producing species from non-producing ones. In order to simultaneously provide the identity and activity of cyanotoxin producing species in freshwater lakes, we applied simple, and fully detailed, whole cell fluorescent in situ hybridization enhanced by tyramid signal amplification (TSA-FISH). DNA oligonucleotide probes MICR3 and MCYA were targeting 16S rRNA and mcyA-mRNA, respectively. The McyA gene is coding for the MC synthetase enzyme involved in MC synthesis. Controls were acquired with the general eubac- terial 16S rRNA probe EUB338, for TSA-FISH assay, and standard HPLC and LC-MS/MS as standard methods for the measurements of MC concentration. Results obtained from mono- clonal strains and natural samples demonstrated a specific identification of Microcystis species and were able to discriminate MC producing from non-producing ones. In addition, the MCYA probe allowed the specific detection of MC-synthetase mRNA within Planktothrix isothrix (Os- cillatoriale) filaments. Two kinds of mcyA-mRNA labeling were observed in these cells, spots like and plasmid like, which illustrates the well-known plasticity of microbial genome to adapt to environmental stresses. We demonstrated that a simple TSA-FISH assay allows acquiring rapidly dual information of the presence and abundance of potentially toxic species, while identifying species actively producing MC-synthetase mRNA, a proxy of MC toxin. This technique has the potential to be developed into an effective environmental monitoring tool in marine and fresh waters. In addition, detail visualization of cellular mRNAs is powerful for the acquisition of ecological and biomolecular studies of toxic cyanobacteria.

NOTE:

14


∗Speaker

Latest advances in two functional methods to detect ciguatoxins (CTXs): the Receptor

Binding Assay (RBA) & the Cell Based Assay (CBA-N2a)

Taiana Darius ∗ 1, Jerome Viallon 1, Jennifer Mc Call 2, Taina Revel 1,

Ransom Hardison 3, William Holland 3, Patricia Tester 4,

Wayne Litaker 3, Mireille Chinain 1

1 Institut Louis Malarde - UMR 241 EIO, Laboratoire des Micro-algues Toxiques – PO Box 30, 98713

Papeete, Tahiti, French Polynesia 2 University of North Carolina - Department of Biology and Marine Biology – 601 South College Road,

Wilmington, NC 28403-5915, United States 3 National Oceanic Atmospheric Administration, National Center for Coastal Ocean Science, Center for

Coastal Fisheries and Habitat Research (CCFHR) (CCFHR (NOAA)) – 101 Pivers Island Road, Beaufort, NC 28516, USA, United States

4 Ocean Tester, LLC – 381 Gillikin Road, Beaufort, North Carolina 28516, United States

The detection and quantification of ciguatoxins (CTX) remains difficult due to the variety of congener species present in trace amounts in contaminated seafood. Unlike other marine biotoxins, there is no official reference method for CTXs, and the mouse biological assay (MBA) is no longer recommended by the European Food Safety Authority (EFSA) since 2015. To date, only functional alternative methods based on the mode of action of CTXs allow quantification and high throughput screening of seafood. The radiolabeled receptor binding assay (RBAr) is based on competition binding between CTXs and tritiated labeled brevetoxin ([3H]PbTx) for voltage gated sodium channels (VGSCs) receptors at site 5. However, regulatory requirements related to the use of radioisotopes severely limit RBAr implantation in laboratories. The Cell Based Assay using neuroblastoma cells (CBA-N2a) allows determination of the overall toxicity of an extract and specifically detects the marine neurotoxins acting on VGSCs.

A comprehensive review of the literature shows the original protocol of CBA-N2a has undergone many significant changes in both the procedure and several key parameters making comparisons between studies difficult. CBA-N2a’s only advance was the use of differentiated concentrations of ouabain and veratridine to specifically detect toxins that inhibit or activate VGSCs. Addi- tionally, a fluorescently labeled brevetoxin (Bodipy-PbTx) allows the use of the RBA without the constraints inherent in using a radiolabeled competitor. When applied to CTX standards as well as fish samples, fluorescent RBAf results gave similar results to the radiolabeled RBAr. Here we propose to revisit the CBA-N2a protocol in order to obtain optimum cell viability, specific detection of activators versus inhibitors of VGSCs, standardized sample preparation and establishes objective controls of experimental conditions. Furthermore, CTXs’ detection in various in vitro strains of Gambierdiscus will be also presented showing that fluorescent RBAf is applicable to microalgae samples.

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∗Speaker

From cyanobacteria to mammals: role of ergothionein, glutathione and its analogs

in stressors signalling and detoxication

Kinsley Narainsamy 1, Christophe Junot∗ 2, Corinne Cassier-Chauvat 3,

Franck Chauvat 4

1 Commissariat a l’energie atomique (CEA) – CEA – Bat 142 CEA-Saclay, F-91191 Gif sur Yvette

cedex, France., France 2 Commissariat a l’energie atomique (CEA) – CEA – CEA, iBiTec-S, SPI, LEMM, Bat 136

CEA-Saclay, F-91191 Gif sur Yvette cedex, France., France 3 Institut de Biologie Integrative de la Cellule (I2BC, UMR9198) – CEA, Centre national de la

recherche scientifique - CNRS (France), Universite Paris-Sud, Universite Paris-Saclay – nstitute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ Paris-Sud, Universite, France

4 Institut de Biologie integrative de la cellule (I2BC) – CEA, CNRS : UMR9198, Universite Paris-Sud, Universite Paris-Saclay – Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ Paris-

Sud, Universite, France

Using a multidisciplinary approach (genetics, genomics, metabolomic) we found that the non- toxic model cyanobacterium Synechocystis PCC6803 synthetizes glutathione the evolutionary- conserved antioxidant tripeptide and its two cysteine-less analogues ophthalmate (OA) and norophthalmate (NOA), as well as another antioxidant molecule, ergothioneine (EGT). These findings are interesting because: i) ophthalmate (g-L-glutamyl-L-amino-n-butyryl-glycine) and norophthalmate (g -L-glutamyl- L-alanyl-glycine) were observed only in mammals so far, where ophthalmate is regarded as a biomarker of glutathione depletion and

ii) ergothioneine (2-mercaptohistidine trimethylbetaine) was reported to be biosynthetized ex- clusively by some fungi and actinobacteria. Mammals acquire EGT only from their diet and accumulate it up to millimolar levels in cells exposed to oxidative stress.

We also showed that the two cyanobacterial genes gshA and gshB encoding the enzymes for the synthesis of GSH also operate in the production of the two GSH analogues OA and NOA, and that EGT biosynthesis is dependent of GSH in Synechocystis PCC6803.

The role of EGT, OA and NOA in mammals and in cyanobacteria is still not well understood and will be discussed. We will also present our hypothesis concerning the EGT biosynthesis pathway in cyanobacteria.

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∗Speaker

Benthic Planktothrix and their antifungal natural products

Claire Pancrace ∗ 1,2, Jouni Jokela 3, Nathalie Sassoon 1, Christelle

Ganneau 4,5, Marie Desnos-Ollivier 6, Matti Wahlsten 3, Anu Humisto 3,

Alexandra Calteau 7, Sylvie Bay 4,5, David P. Fewer 3, Kaarina Sivonen 3,

Muriel Gugger 1

1 Collection des Cyanobacteries – Institut Pasteur – 25-28 rue du Docteur Roux F-75724 Paris Cedex

15, France 2 Sorbonne Universites, UPMC Univ Paris 06, UPEC, UDD, CNRS, INRA, IRD, IEES-Paris –

Universite Pierre et Marie Curie (UPMC) - Paris VI – 4, place Jussieu 75252 PARIS cedex 5, France 3 Microbiology and Biotechnology Division, Department of Food and Environmental Science, University

of Helsinki – Helsinki, Finland 4 Unit Chemistry of Biomolecules, Institut Pasteur – Institut Pasteur – 28 rue du Docteur Roux 75015

Paris, France 5 CNRS UMR 3523 – CNRS : UMR3523 – Paris, France

6 Institut Pasteur / CNRS URA3012, National Refence Center for Invasive Mycoses and Antifungals, Molecular Mycology Unit – CNRS : URA3012, Institut Pasteur – Paris, France

7 Commissariat a l’Energie Atomique et aux Energies Alternatives (CEA), Genoscope / CNRS, UMR

8030, Laboratoire d’Analyse Bioinformatique en Genomique et Metabolisme – CNRS : UMR8030 – Evry, France

Cyanobacteria are well-known producers of a wide range of natural products, including the widely studied microcystins. We studied Planktothrix, a dominant toxic genus in freshwater ecosystems and investigated its genetic potential dedicated to the biosynthesis of natural products. Diversity and distribution of natural products were established by in silico genomic analysis of newly sequenced strains of diverse origins. Biosynthetic gene clusters linked to known cyanotoxins and unexpected ones were found in this genus. Planktothrix appeared to exhibit different natural product gene clusters in function of its life style, planktic or benthic. Benthic Planktothrix strains presented natural products such as hassallidin, a glycolipopeptide with antifungal bioactivity previously only described in heterocystous cyanobacteria. The hassallidin gene cluster showed heavy rearrangement and characteristics suggesting its acquisition by horizontal gene transfer. Natural product characterization by mass spectrometry analyses permitted to detect a new variant of hassallidin, for which antifungal property was confirmed. Furthermore, the production levels of this compound were correlated with nitrogen availability. Recently, other antifungal compounds such as luminaolide B and tolytoxin were found in our benthic Planktothrix strains. Overall, these results raise questions on the cellular function and ecological relevance of antifungal cyanobacterial compounds.

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∗Speaker

Monitoring and modelling cyanobacteria biomass in a small urban lake

Denis Plec ∗ 1, Lucas Jardim Porto 2, Bruno Lemaire 3, Philippe Dubois 2, Mohamed Saad 2, Cecile Bernard 4, Laure Huguenard 5, Brigitte

Vincon-Leite 2

1 Ecole des Ponts ParisTech, AgroParisTech, UPEC, UPE (LEESU) – E cole des Ponts ParisTech

(ENPC) – 77455, Champs sur Marne, France 2 Ecole des Ponts ParisTech, AgroParisTech, UPEC, UPE (LEESU) – Ecole des Ponts ParisTech –

77455 Champs sur Marne, France 3 Ecole des Ponts ParisTech, AgroParisTech, UPEC, UPE (LEESU) – AgroParisTech – 77455 Champs

sur Marne, France 4 UMR 7245 MCAM MNHN - CNRS (MNHN - CNRS) – Museum National d’Histoire Naturelle

(MNHN), CNRS : UMR7245 – 57 Rue Cuvier 75231 Paris Cedex 05, France 5 conseil departemental de Seine Saint Denis (Direction de la Nature, des Paysages et de la

Biodiversite) – conseil departemental de Seine Saint Denis – Bobigny 93, France

Eutrophication of lakes often leads to potentially toxic cyanobacteria blooms and health

risks, which require a regular monitoring and warning systems when the human population is exposed. Due to the complexity of lake ecosystems, mathematical models combined with monitoring are valuable tools to better understand the variables driving the cyanobacteria blooms and to support decision-making and water management strategies. The objective of this research, which is part of the ANR project OSS-Cyano (Optical Sensors and drone system for the spatial Survey of Cyanobacteria in freshwater ecosystems), is to provide a modelling tool, able to forecast a short-term evolution of cyanobacteria biomass. The study lake is a small urban lake located in Champs sur Marne, 20 km east of Paris. It is inserted in a recreational park owned by the Conseil Departemental de Seine-Saint-Denis 93 (CD93). But, unfortunately swimming in the lake has been repeatedly prohibited each summer because of cyanobacteria blooms. We first present the database, which combines different types of data. Since 2006, in order to comply with the French bathing regulation, water samples collected near the bathing area arewere used for phytoplankton counting and chlorophyll-a analysis (MNHN and CD93). in order to comply with the French bathing regulation. Since May 2015, in the framework of OSS- Cyano project, a continuous monitoring includes high-frequency data, collected at 3 depths and 3 locations in the lake, of temperature, O2 and chlorophyll and fortnightly profiles of temperature and chlorophyll fluorescence. We first present the space and time evolution of the measured data and highlight their main patterns. We compare the total fluorescence measured by the probes and by laboratory analysis. We then present the coupled three-dimensional hydrodynamic and ecological model used for simulating the evolution the cyanobacteria biomass. The modelling results obtained for some summer short periods are compared with the measurements.

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∗Speaker

Temperature and stratification of the artificial lake and the proliferation of

cyanobacteria, case of the lake of the Eguzon dam. France

Zoubida Qsair ∗ 1, Laurent Touchart ∗ 2, Pascal Bartout ∗ 2

1 LABORATOIRE CEDETE – QSAIR – EA 1210 CEDETE, universite d’Orleans (France), France 2 LABORATOIRE CEDETE – Universite Orleans – EA 1210 CEDETE, universite d’Orleans (France),

France

Located 200 m above sea level on the first foothills of the Massif Central, Lake Chambon (or Lac d’Eguzon), created in 1926, partially flooded the Quaternary valley of the Creuse between Fresselines and Cuzion to form the highest dam d Europe of the time. Soon a century old, this reservoir shares with the natural lakes the contemporary problematic that is the proliferation of the cyanobacteria, question all the more crucial since the political authorities have founded the tourist attraction of the territory on the lake and They have been forced in recent years to manage various prohibitions (bathing, fishing ...). In previous work (Qsair et al., 2014), we were led to conduct a study in which 69% of those questioned on site complained about these proliferations and accused the dam of being responsible for this illness. As the limnic scientific literature emphasizes the thermal functioning of lakes in cyanobacterial proliferation analyzes (Jennifer et al., 2008) (Robarts and Zohary, 1987) (Reynolds and Walsby 1975, We wanted to explore this path by applying a scientific approach that takes into account both the periodicity and the extent (Sournia and Frontier, 1967), but also the spatial variation of the measurements. This study is based on 40,100 thermal measurements collected from 2014 to 2016, measures associated with cyanobacterial analyzes (partly carried out in the laboratory and others in situ with the Fluoroprobe probe). Correlations of temperature and cyanobacterial monitoring results helped to verify the link between the two criteria and to highlight some elements: (i) the effect of the dam is only a catalyst and can not be the determining factor Of the phenomenon; (Ii) cyanobacteria affect stagnant lake water, but they also adapt to waters with varying hydrody- namics.

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∗Speaker

Genetic diversity of the toxic bloom-forming cyanobacterium Microcystis aeruginosa: a

meta-analysis at large spatial and temporal scales

Marion Sabart ∗ 1, Benjamin Misson 2, Marlene Jobard 3, Gaofei Song 4,

Li Renhui 4, Delphine Latour 1

1 Microorganismes : genome et environnement (LMGE) – Universite d’Auvergne - Clermont-Ferrand I, CNRS : UMR6023, Universite Blaise Pascal - Clermont-Ferrand II – Universite Blaise Pascal, Campus

des Cezeaux, 24, avenue des Landais BP 80026 63 170 AUBIERE, France 2 Laboratoire PROTEE (PROTEE) – Universite du Sud - Toulon - Var : EA3819 – BP20132 83957 La

Garde, France 3 LMGE UMR 6023 – CNRS : UMR6023 – Laboratoire Microorganismes : Genome et Environnement

(LMGE) Universite Clermont Auvergne - UMR CNRS 6023 Campus Universitaire des Cezeaux 1

Impasse Amelie Murat TSA 60026 CS 60026 63178 AUBIERE CEDEX - FRANCE, France 4 Institute of Hydrobiology – The Chinese Academy of Sciences, Wuhan, 430072, China, China

Intraspecific genetic variability is of growing interest for present research on phytoplankton

ecology. In particular, elucidating the patterns of genetic diversity and population structure of bloom-forming species could contribute to better understand its population dynamics, life cycle and bloom formation, as well as to explain its ecological success. Among the HAB species, Microcystis aeruginosa is one of the most widespread in freshwaters. This cyanobacterium is known to produce thick and highly toxic scums worldwide which represent ecological, socio- economical and sanitary risks. For that reason, the understanding of its ecology represents a main objective of current research for improving ecosystem management. In recent years, few studies have been performed to decipher spatial and temporal variations of the genetic diversity at scales restricted in space (regional scale) and time (a bloom season). In order to investigate the genetic diversity and the population structuration at larger spatio-temporal scales, we performed a meta-analysis of several environmental datasets of the ITS 16S-23S obtained with the same methodology, i.e. cloning and sequencing of the ITS 16S-23S, a non- coding genomic region adapted to genetic investigations at the intraspecific level. The data originates from several freshwater ecosystems located in France and China during different years. Our analyses evidenced a high genetic diversity and the existence of ubiquitous genotypes at large spatial and temporal scales which could contribute to the adaptability and the ecological success of this cyanobacteria. Despite the ubiquity of some genotypes, the UniFrac analyses revealed a genetic structuration of the populations between ecosystems.

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∗Speaker

Metabolomics of ancient populations of DINoflAgellates in phosphorous-limited

conditions

Raffaele Siano ∗ 1, Florence Mondeguer 2, Marie Latimier 3, Julien Quere 3, Manoella Sibat 2, Laure Guillou 4, Philipp Hess 5

1 Dynamiques des Ecosystemes Cotiers (DYNECO Pelagos) – Institut Francais de Recherche pour l’Exploitation de la Mer (IFREMER) – Technopole Brest-Iroise, BP 70, 29280, Plouzane, France

2 Dynamiques des Ecosystemes Cotiers (DYNECO PHYC) – Institut Francais de Recherche pour

l’Exploitation de la Mer (IFREMER) – 44311 Cedex 03, Rue de l’Ile d’Yeu, Nantes., France 3 Dynamiques des Ecosystemes Cotiers (DYNECO Pelagos) – Institut Francais de Recherche pour l’Exploitation de la Mer (IFREMER) – Technopole Brest Iroise, BP 70, 29280, Plouzane, France.,

France 4 Station biologique de Roscoff [Roscoff ] (SBR) – Universite Pierre et Marie Curie (UPMC) - Paris VI,

CNRS : FR2424 – Place Georges Teissier - BP 74 29682 ROSCOFF CEDEX, France 5 Dynamiques des Ecosytemes Cotiers (DYNECO PHYC) – Institut Francais de Recherche pour

l’Exploitation de la Mer (IFREMER) – 44311 Cedex 03, Rue de l’Ile d’Yeu, Nantes., France

Given their physiological plasticity, phytoplankton organisms can adapt to the isotropic habitat in which they live. This capacity has likely been maximized by populations of coastal ecosystems which undergo important physical and biological variations through anthropogenic pressures since the industrialised era. Physiological adaptations can be studied by reviving cells from cysts buried in ancient sediments, supposing that resting stages have preserved their physiological adaptations to the ecosystem of the time in which they lived. We revived strains of the dinoflagellates Alexandrium minutum and Scrippsiella donghaienis from up to ca. 20 and ca. 30 years-old sediments of the bays of Morlaix and Brest (Brit- tany, France), respectively. Cultures (3 strains for each of 2 species, for 2 ages per ecosystem) were established to examine physiological responses and adaptations to phosphorous limitation. Phosphate was spiked in stationary phase to verify phosphorous (P) limitation. Metabolite profiles were analysed using liquid chromatography coupled to high resolution mass spectrometry at different growing phases.

Photophysiological cellular stress and cell-size increase were simultaneously observed after 8-11 days of culture in non-axenic, P-limited conditions, depending on strains and species. Contrarily to ancient strains (1986), modern strains (2006-2010) of S. donghaienis were coherently able to restart growth after the P spike. Except from one strain, neither modern (2006) nor ancient (1998) A. minutum strains were able to restart growing. The two species and the populations of different ages were significantly differentiated by 53 lipophilic compounds. Profiles of S. donghaienis of different ages and growth conditions were significantly different in 27 lipophilic compounds. These findings show inter- and intra-specific differences in physiological responses to P limitation for the studied dinoflagellates.

Further analyses may help to clarify which metabolites distinguish different growth phases and ages, and if a physiological profile of the strains depends on change in trophic strategy of these dinoflagellates.

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∗Speaker

Wise use of metabolomics in the field of marine chemical ecology

Eva Ternon ∗ 1, Anne-Sophie Pavaux 2, Olivier Thomas 3

1 Geoazur - Universite de Nice Sophia-Antipolis (UNICE) – CNRS : UMR7329 – 250 rue Albert

Einstein 06560 Valbonne, France 2 Laboratoire d’Oceanographie de Villefranche sur mer – Universite Pierre et Marie Curie [UPMC] -

Paris VI, CNRS : UMR7093 – Chemin du Lazaret 06230 Villefranche sur mer, France 3 National University of Ireland Galway – University Road, Galway, Ireland

Chemical ecology aims to study biotic interactions (predation, competition, pathogens ...)

controlled by chemical compounds. It is now well recognized that micro-algae call on chemical mediation although our knowledge regarding the nature of the mediators is very limited. During the past decade, the OMICS have contributed to an increasing number of publications within the field of marine chemical ecology. In particular, metabolomics focuses on the compounds of low-molecular weight synthesized by the specialized metabolism, including micro-algal toxins. The use of metabolomics recently enabled characterizing a kairomone involved in the sexual reproduction of a diatom species. However, and despite its great potential, metabolomics is constrained by the molecular characteristics of the chemical mediators and should be wisely used. As for illustration, two examples of chemical mediation involving bloom-forming dinoflagellates and other members of the ecosystem will be presented. A metabolomics approach was used to study the allelopathic interactions between the toxic dinoflagellate Ostreopsis cf. ovata and a co-occurring diatom species Licmophora paradoxa. On the contrary, the use of metabolomics was found to be irrelevant studying the chemical interactions between a bacterium of the genus Shewanella and the dinoflagellate Gyrodinium instriatum.

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Theme 2: Ecology of HAB

23


∗Speaker

Genetic, morphological and ecological evidence of new oomycete parasites of

the toxic diatom genus Pseudo-nitzschia

Garvetto Andrea ∗ 1, Claire Gachon 1, Elisabeth Nezan 2, Raffaele Siano 3

1 Scottish Association for Marine Science (SAMS) – SAMS, Scottish Marine Institute, Dunbeg, Oban, PA37 1QA, Scotland, UK, United Kingdom

2 IFREMER (LER BO) – Institut Francais de Recherche pour l’Exploitation de la Mer (IFREMER) –

Place de la Croix 29900 Concarneau, France 3 Dynamiques des Ecosystemes Cotiers (DYNECO Pelagos) – Institut Francais de Recherche pour l’Exploitation de la Mer (IFREMER) – Technopole Brest-Iroise, BP 70, 29280, Plouzane, France

Species-specific parasite infections reportedly play a major role in influencing species phenol- ogy both on land and in the sea. The diluted planktonic realm is not an exception and eukaryotic microparasites of phytoplankton are well known for fostering if not causing algal bloom demises. In fact, their role in controlling dinoflagellate toxic blooms has been acknowledged, but toxic diatom/parasites interactions have been barely studied. The existence of parasites of the toxigenic diatom genus Pseudo-nitzschia has been documented on the basis of morphological observations, but no phylogenetic affiliation has been provided so far, opening the debate on their systematic position. Furthermore ecological relevance and host-specificity for these organisms are still un- explored. Here we characterized intracellular eukaryotic parasites infecting three Pseudo-nitzschia taxa of the North Atlantic waters (Bay of Concarneau, Brittany, France and Lynn of Lorne, Scotland, UK) via microscopy, single-cell PCR amplifications of the 18srDNA and molecular phylogeny. These endoparasites branch into two different clades within the phylum Oomycota, previously composed of environmental sequences only. Those were obtained in the frame of non-targeted genetic diversity screening surveys.

We screened for the presence of the newly identified organisms in the world’s ocean using available and published metabarcoding databases and we provided their first worldwide distribution. Thanks to a high frequency sampling strategy and to the metabarcoding of the region V4 of the 18s rDNA, we were able to characterise the host-parasitic dynamics in the Bay of Concarneau, one of the areas in which the parasites have been isolated and genetically characterised. This study if the first comprehensive study addressing the problematic of the toxic Pseudo- nitzschia spp parasite interactions from both morphological, phylogenetic and ecological point of view and from a local to global spatial scale.

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∗Speaker

Effects of salinity variation on the physiology and domoic acid production

by two strains of Pseudo-nitzschia australis

Nour Ayache ∗ 1, Amandine Caruana 1, Fabienne Herve 1, Zouher Amzil 1

1 Laboratoire Phycotoxines (PHYC) – Institut Francais de Recherche pour l’Exploitation de la MER - IFREMER, Institut Francais de Recherche pour l’Exploitation de la Mer (IFREMER) – Rue de l’Ile

d’Yeu - BP 21105 - 44311 Nantes Cedex 03, France

The diatom Pseudo-nitzschia is a unicellular marine microalgae which includes some toxic species capable of producing domoic acid (DA). DA is a neurotoxin responsible for the amnesic shellfish poisoning syndrome (ASP). Impacts of these blooms present many risks ranging from serious ecological and economic damages to threats to public health. Based on literature, abundance and frequency of Pseudo-nitzschia blooms seem to be associated with eutrophication and climate change. In recent decades, several toxic Pseudo-nitzschia species are present repeatedly along the French coast in estuarine and coastal waters. These habitats are widely used as shellfish growing and harvesting areas and are often subject to extreme fluctuations in salinity. However, only three studies were conducted on the effect of salinity variation on the growth and DA production of Pseudo-nitzschia species. These studies showed varying results and this is usually justified by the wide inter- and intraspecies variability of Pseudo-nitzschia. Therefore, additional studies are needed to clarify the relationship between salinity and DA production of several Pseudo-nitzschia species and strains isolated in France. Consequently, the aim of these experiments is to determine the impact of salinity variation on the physiology and domoic acid production by two strains of Pseudo-nitzschia australis. The physiological state was investigated by measuring specific growth rates, photosynthetic efficiency and pigment profile throughout salinity stress and acclimation experiments. Results reported herein supply new data on the survival and adaptation of Pseudo-nitzschia and how the DA content of Pseudo-nitzschia may vary in salinity variable environments.

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∗Speaker

Nutrient ratios influence variability in

Pseudo-nitzschia species diversity and particulate domoic acid production in the

Bay of Seine (France)

Juliette Fauchot ∗ 1, Maxine Thorel 2, Mathilde Schapira 3, Romain Le

Gendre 3, Philippe Riou 3, Didier Goux 4, Bertrand Le Roy 1, Virginie

Raimbault 1, Anne-Flore Deton-Cabanillas 1, Pauline Bazin 1, Valerie

Kientz-Bouchart 5, Pascal Claquin 6

1 Universite de Caen Normandie - UMR BOREA (UNICAEN - BOREA) – CNRS : UMR7208, IRD-207,

Museum National d’Histoire Naturelle (MNHN), Universite Pierre et Marie Curie (UPMC) - Paris VI, UNICAEN, Universite Pierre et Marie Curie [UPMC] - Paris VI – Esplanade de la Paix CS

14032 14032 CAEN cedex 5, France 2 UMR BOREA – MNHN, UPMC, UCBN, CNRS-7208, IRD-207 – UNIVERSITE DE CAEN

BASSE-NORMANDIE Esplanade de la Paix CS 14032 14032 CAEN cedex 5, France 3 Ifremer LER/N – Institut Francais de Recherche pour l’Exploitation de la Mer (IFREMER) – Ifremer,

LER/N, 14520 Port en Bessin, France 4 CMAbio-SF 4206 ICORE UNICAEN – UNICAEN – Esplanade de la Paix CS 14032 14032 CAEN

cedex 5, France 5 LABEO Frank Duncombe – LABEO Frank Duncombe – Saint-Contest, 14053 Caen Cedex, France

6 Biologie des ORganismes et Ecosystemes Aquatiques (BOREA) – Universite de Caen

Basse-Normandie, Museum National d’Histoire Naturelle (MNHN), CNRS : UMR7208 – Institut de

Biologie Fondamentale et Appliquee Universite de Caen Basse-Normandie Esplanade de la Paix CS

14032 14032 CAEN cedex 5, France

The population dynamics of different Pseudo-nitzschia species and the temporal evolution of

particulate domoic acid (pDA) concentration were investigated from May 2012 to December 2013 in the Bay of Seine (English Channel, Normandy). This study describes the succession of Pseudo-nitzschia species during this period. While Pseudo-nitzschia spp. blooms occurred during the two years of study, Pseudo-nitzschia species diversity and particulate domoic acid concentrations varied greatly between both years. In 2012, three different species were identified during the spring bloom (P. australis, P. pungens and P. fraudulenta ) with high pDA concentrations ( ˜1400 ng l-1) resulting in an amnesic shellfish poisoning (ASP) event. In contrast, the 2013 spring was characterized by a P. delicatissima bloom and no ASP event. Above all, the results from the two years of survey show that high pDA concentrations coincided with the presence of P. australis and with potential silicate limitation (Si:N< 1), when nitrate concentrations were still replete. The contrasting environmental conditions between 2012 and 2013 highlight different environmental controls that might favour the development either of P. delicatissima or P. australis. This study points to the key role of Pseudo-nitzschia diversity and cellular toxicity in the control of particulate domoic acid variations and highlights the fact that diversity and toxicity are influenced by the nutrient situation, and especially nutrient ratios.

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∗Speaker

The diatom genus Pseudo-nitzschia in the Gulf of Naples (Mediterranean Sea):

exploring diversity and life history traits

Marina Montresor ∗ 1

1 Stazione Zoologica Anton Dohrn – Villa Comunale, 80121 - Napoli, Italy

The genus Pseudo-nitzschia includes about 45 species, half of which produce the neurotoxin domoic acid. Many of these species are present in the Mediterranean Sea, and domoic acid has been recorded in various areas linked to aquacultures activities. The monitoring of Harmful Algal Bloom (HAB) is complicated by the high diversity of species and the associated toxins, which calls for studies linking morphological, molecular and toxicological analyses of potentially harmful species. A total of 12 species have been recorded at the Long Term Ecological Research (LTER) station MareChiara in the Gulf of Naples based on morphological and molecular identifications. Clone libraries and High Throughput Sequencing DNA metabarcoding approaches further increased the number of recorded genotypes. A high level of genetic diversity has been reported also at the intra-specific level for several unicellular microalgae, but limited information is available on the genetic structure at a temporal scale. A study carried out over six years on Pseudo-nitzschia multistriata using microsatellite markers showed that the genetic structure of the population changed over time with a non-linear pattern. Moreover, and we could detect clear signatures of sexual recombination and clonal ex- pansions in our dataset. Sexual reproduction is a fundamental phase in the life cycle of diatoms, linked to the production of genotypic diversity and the formation of large-sized initial cells that ensure population persistence. The availability of genomic and transcriptomic resources for P.multistriata now allows decrypting the molecular mechanisms that regulate different life cycle phases. Ongoing research is shedding light on the signaling processes that mediate early-stage communication between the two mating types and on possible genes characterizing them.

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∗Speaker

Interspecific and intraspecific variability in physiology among four Pseudo-nitzschia

species

Aurore Sauvey ∗ 1, Pascal Claquin 1, Juliette Fauchot 1

1 Universite de Caen Normandie - UMR BOREA (UNICAEN - BOREA) – CNRS : UMR7208, IRD-207,

Museum National d’Histoire Naturelle (MNHN), Universite Pierre et Marie Curie (UPMC) - Paris VI, UNICAEN, Universite Pierre et Marie Curie [UPMC] - Paris VI – Esplanade de la Paix CS

14032 14032 CAEN cedex 5, France

Because they produce domoic acid, a neurotoxin that bioaccumulates in marine food webs,

the diatoms Pseudo-nitzschia spp are responsible for amnesic shellfish poisoning events on the French coasts. These events may have negative consequences on coastal ecosystem services especially through their impact on fisheries. In the Bay of Seine, the different species of Pseudo- nitzschia present distinct bloom phenologies and species diversity affect the toxicity of the blooms. However, the physiological responses of these species to environmental factors are still difficult to predict. A better understanding of the interspecific variability in Pseudo-nitzschia physiology is therefore a prerequisite to apprehend the determinism of toxic blooms. Toward this goal, the characterization of intraspecific variations in physiology is also essential and the peculiar life cycle of diatoms should be taken into account, especially through its influence on cell size. The experiments presented here aim to characterize physiological properties (growth rates, nutrient uptake kinetics, DA production dynamics) determined for different strains of four Pseudo-nitzschia species (P. australis, P. pungens, P. fraudulenta and P. delicatissima ) isolated in Atlantic (Iroise Sea and Arcachon Bay) and the Bay of Seine. The first results of this work show significant interspecific differences (e.g. DA production) and intraspecific differences were also observed (e.g. on uptake kinetics parameters and DA production dynamics). In particular, differences in DA production capacities between the four studied species were measured, but DA production was also impacted by cell size changes linked to life cycle. These results stress the importance of taking into account the interspecific and intraspecific diversity to better understand toxic bloom dynamics.

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Theme 3: Effects of HAB on marine organisms and ecosystems, trophic

transfer, etc.

29


∗Speaker

Molecular Characterization of

Voltage-Gated Sodium Channels and Their

Relations with Paralytic Shellfish Toxin

Bioaccumulation in the Pacific Oyster

Crassostrea gigas

Floriane Boullot 1, Justine Castrec 1, Adeline Bidault-Toffin 1, Natanael Dantas 2, Laura Payton 3, Mickael Perrigault 3, Damien Tran 3, Zouher

Amzil 4, Pierre Boudry 1, Philippe Soudant 1, Helene Hegaret 1, Caroline Fabioux ∗ 1

1 LEMAR – LEMAR – UMR 6539 (CNRS/UBO/IRD/Ifremer), Laboratoire des sciences de

l’Environnement MARin (LEMAR), Institut Universitaire Europeen de la Mer, rue Dumont d’Urville,

29280 Plouzane, France, France 2 Laboratory of Immunology and Pathology of Invertebrates, Department of Molecular Biology, Exact

and Natural Sciences Center, Federal University of Paraıba – Campus I, 58051-900 Joao Pessoa, Brazil 3 UMR 5805 Environnements et Paleoenvironnements Oceaniques et Continentaux (EPOC) – CNRS :

UMR5805, INSU, Universite Sciences et Technologies - Bordeaux I, E cole Pratique des Hautes E tudes

[EPHE], Observatoire Aquitain des Sciences de l’Univers – Station Marine d’Arcachon, Universite

Bordeaux 1, 2 Rue du Professeur Jolyet, 33120 Arcachon Cedex, France, France 4 Laboratoire Phycotoxines (PHYC) – Institut Francais de Recherche pour l’Exploitation de la MER -

IFREMER, Institut Francais de Recherche pour l’Exploitation de la Mer (IFREMER) – Rue de l’Ile


Paralytic shellfish toxins (PST) bind to voltage-gated sodium channels (Nav) and block

conduction of action potential in excitable cells. This study aimed to (i) characterise Nav

sequences in Crassostrea gigas and (ii) investigate a putative relation between Nav and PST-

bioaccumulation in oysters. The phylogenetic analysis highlighted two types of Nav in C. gi-

gas : a Nav1 (CgNav1 ) and a Nav2 (CgNav2 ) with sequence properties of sodium-selective

and sodium/calcium-selective channels, respectively. Three alternative splice transcripts of Cg-

Nav1 named A, B and C, were characterized. The expression of CgNav1, analyzed by in situ

hybridization, is specific to nervous cells and to structures corresponding to neuromuscular junc-

tions. Real-time PCR analyses showed a strong expression of CgNav1A in the striated muscle

while CgNav1B is mainly expressed in visceral ganglia. CgNav1C expression is ubiquitous. The

PST binding site (domain II) of CgNav1 variants possess an amino acid Q that could potentially

confer a partial saxitoxin (STX)-resistance to the channel. The CgNav1 genotype or alternative

splicing would not be the key point determining PST bioaccumulation level in oysters.

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∗Speaker

Impacts of Alexandrium minutum on the reproduction of the oyster Crassostrea

gigas: from gametogenesis to larval settlement

Justine Castrec ∗ 1, Philippe Soudant 1, Marianne Alunno Bruscia 2,

Bruno Petton 2, Isabelle Queau 2, Marc Suquet 2, Dominique Ratiskol 2,

Jacqueline Le Grand 2, Christian Mingant 2, Nelly Le Goic 1, Helene

Hegaret 1, Caroline Fabioux 1

1 Laboratoire des Sciences de l’Environnement Marin (LEMAR) – CNRS : UMR6539, Universite de

Bretagne Occidentale (UBO), Institut Universitaire Europeen de la Mer (IUEM), Institut de Recherche pour le Developpement, Institut Francais de Recherche pour l’Exploitation de la Mer (IFREMER) –

Technopole Brest-Iroise, Place Nicolas Copernic, 29280 Plouzane, France 2 Laboratoire de Physiologie des Invertebres (LPI) – Institut Francais de Recherche pour l’Exploitation

de la Mer (IFREMER) – 11, presqu’ıle du vivier, 29840 Argenton, France

Harmful algal blooms have increasingly disrupted coastal ecosystems for the last few decades with dinoflagellates of the genus Alexandrium being of global importance due to its widespread distribution and synthesis of Paralytic Shellfish Toxins (PSTs). While the effects of Alexandrium species upon the physiology of bivalves are now well documented, consequences on reproduction remain poorly studied. In France, Alexandrium minutum blooms have been recurrent for the last decades in Brittany. These toxic blooms appear generally during the season of the reproduction of most bivalves and could impact gametogenesis but also spawning, larval development or recruitment of juveniles, therefore causing real issues for economically important species, such as the oyster Crassostrea gigas. To assess the effect of toxic A. minutum blooms on the reproduction of C. gigas, adult oysters were experimentally exposed for two months, during a reproductive cycle to A. minutum, at concentrations close to those observed in the field. In order to determine the effects of direct A. minutum exposure on larvae, the subsequent embryo-larval development of their offspring was conducted in presence or absence of A. minutum. Effects at each stage of the reproduction were investigated on ecophysiological parameters, cellular responses, and offspring development. The results indicate that, although no impact on gonadal maturation and reproductive effort was observed, sperm motility was significantly reduced in A. minutum exposed oysters. When reared in control conditions, larval development of the offspring derived from exposed parents decreased compared to control offspring, suggesting a detrimental transgenerational effect of A. minutum. Moreover, A. minutum exposure during embryo-larval development of the offspring reduced growth of larvae derived from both exposed and control parents, compared to control offspring. This study provides evidence that A. minutum blooms can affect gamete quality and larval development of C. gigas, and could potentially impact oyster recruitment.

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∗Speaker

Continued feeding with Gambierdiscus polynesiensis causes increasing

accumulation of ciguatoxin over 16 weeks in an herbivorous reef fish

Rachel Clausing ∗ 1, Barbara Losen 1, Taiana Darius 2, Philipp Hess 3,

Manoella Sibat 3, Jerome Viallon 2, Mireille Chinain 2, Marie-Yasmine

Dechraoui Bottein 4

1 Environment Laboratories (IAEA) – 4a Quai Antoine Ier 98000 Monaco, Monaco


Papeete, Tahiti, French Polynesia 3 Institut Francais de Recherche pour l’Exploitation de la Mer - Nantes (IFREMER Nantes) –

Universite de Nantes – Rue de l’Ile d’Yeu - BP 21105 - 44311 Nantes Cedex 03, France 4 Environment Laboratories (IAEA) – 4 Quai Antoine Ier - 98000, Monaco

Here we present first evidence for experimental bioaccumulation of ciguatoxins (CTXs) in the

flesh of a coral reef herbivore over long-term consumption of Gambierdiscus cells. Juvenile Naso sp., a genus of fish distributed throughout Indo-Pacific reefs, were given a gel-food containing cells of Gambierdiscus polynesiensis (TB-92: 3.7 pg P-CTX-3C equiv. cell-1) five days per week for 2, 4, 8 and 16 weeks (N=5; 80 cells or 0.3 ng P-CTX-3C equiv. g-1 fish). Control fish (N=5) consumed gel-food without algal cells. Food toxicity was confirmed by LC-MS/MS (4.4 ng P-CTX-3C equiv. g food -1). Extracts of muscle tissue were analysed with the receptor binding assay using tritiated brevetoxin and demonstrated increasing accumulation of CTXs over time

in the flesh of Naso. After only 2 weeks, CTX concentrations in the flesh were above

thresholds for human intoxication (1.11 ng ± 0.18 SEM ng P-CTX-3C equiv g-1) and remained stable until 4 weeks (1.54 ng ± 0.66 SEM ng P-CTX-3C equiv g-1 muscle). By 8 weeks, however, concentrations had reached 3.24 ng ± 0.51 SEM, where they remained at 16 weeks (2.81 ng ± 0.51 SEM). Toxin presence was confirmed by CBA-N2A. Despite significant toxin retention, fish showed no behavioral signs of intoxication over the 16-week experiment. These toxin concentrations, the rate of accumulation ( ˜1 month lag-time from initial consumption), and the absence of signs of intoxication are consistent with findings on herbivorous fish in the wild and confirm the ecological relevance of this study. Furthermore, this work provides a good laboratory model for CTX toxicokinetic and trophic transfer studies.

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Phenolic compounds from the seagrasses

Zostera marina and Zostera noltei affect negatively the physiology of the neurotoxic

dinoflagellate Alexandrium catenella

Mohamed Laabir ∗ 1

1 Center for Marine Biodiversity, Exploitation and Conservation (MARBEC) – Universite Montpellier II - Sciences et Techniques du Languedoc – Universite de Montpellier, CNRS, IRD, Ifremer, Place Eugene

Bataillon, CC93, Montpellier Cedex 5 34095, France

Laboratory experiments were performed to test the inhibitory effect of the main pheno- lics (zosteric, rosmarinic, caffeic and coumaric acids, flavonoids and diosmetin, a sulfate free flavonoid), extracted and purified from fresh and detrital leaves of two seagrass species, Zostera marina and Zostera noltei collected in Thau lagoon (French Mediterranean) and Arcachon Bay (French Atlantic coast), on the growth of the toxic red tide dinoflagellate Alexandrium catenella. For the first time, exudates from Zostera leaves were successfully extracted from seawater, purified and their effect on the biology of A. catenella investigated. Results showed a significant reduction of the vegetative cells growth (at short and/or long term) of the exudates and of most of the molecules tested. Their effective concentrations (EC50) depend on the molecules and Zostera species. Exposition to the phenolic compounds induced severe structural anomalies in A. catenella whose membranes became lytic and cells necrotic. The growth inhibitions, as well as cellular degradations, were most often dose-and time-dependent. Altogether, these results showed that phenolic compounds produced by Zostera leaves have an important algicidal activity. These results indicate that Zostera beds have potentially the capacity to chemically mitigate Alexandrium harmful blooms and show the importance to preserve these ecosystems.

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∗Speaker

Interest of bivalves for the biosurvey of environmental contamination by

cyanobacterial toxins

Emilie Lance ∗ 1, Zouher Amzil 2, Alexandra Lepoutre 3, Luc Brient 4,

Myriam Bormans 4

1 UMR-I 02 INERIS-URCA-ULH SEBIO (SEBIO) – Universite Reims Champagne Ardennes – Campus

Moulin de la Housse - BP 1039 - 51687 Reims Cedex 2, France 2 Laboratoire Phycotoxines (ODE/UL/PHYC) – IFREMER – Centre Atlantique, Nantes, France

3 UMR-I 02 INERIS-URCA-ULH SEBIO (SEBIO) – Universite Reims – Campus Moulin de la Housse - BP 1039 - 51687 Reims Cedex 2, France

4 UMR 6553 Ecobio – universite Rennes 1 – UMR Eco-Bio, Bt 14 - p. 205 Campus de Beaulieu 35 042

RENNES CEDEX, France

The eutrophication of aquatic ecosystems, associated to the climate change, enhance the fre-

quency and the severity of cyanobacterial proliferations that produce endotoxins such as neurotoxins, hepatotoxins, dermatotoxins, and cytotoxins, threatening target organisms and humans. The bioaccumulation of the hepatotoxins microcystins (MCs) and cylindrospermopsins (CYN), and their effects on organisms is overall quite well documented. However, some cyanobacteria and diatoms strains may also produce the neurotoxin BMAA (β-N-methylamino-L-alanine), a non proteinogenic amino acid that may associated with neurodegenerative diseases like amyotrophic lateral sclerosis/Parkinsonism dementia complex (ALS-PDC). Bioaccumulation of BMAA in marine organisms (e.g., mussel, oyster, fish) is little documented. However, BMAA accumulation and impact on freshwater organism remains poorly investigated. Freshwater mussels are known to ingest phytoplankton and cyanobacteria and accumulate cyanotoxins. Therefore, the use of sentinel species to integrate and reveal MC and BMAA contamination, including at low cyanobacterial or phytoplankton densities, may allow a preventative management of contaminated sites. Our current research projects propose a dual approach in the laboratory and in situ with the objective to assess the suitability of various bivalves (Anodonta sp, Dreissena polymorpha and Mytilus edulis ) for representing the contamination of fresh and brackish waters by MCs and BMAA. The in situ approaches, both in freshwater sites and along a river continuum from fresh waters to interconnecting estuarine and coastal areas, consist in the evaluation of: 1) the frequency of cyanobacterial proliferations and of cyanotoxin production, 2) the cyanotoxin bioaccumulation in the three species of bivalves (natural or caged populations). These projects also propose laboratory studies on the kinetics of cyanotoxin accumulation and on their physiological effects. The results of these projects will facilitate the long-term tracking of the contamination of ecosystems by cyanotoxins, which will provide an advance in the knowledge about their ecodynamic.

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Impact of the environmental toxin BMAA on the immune system of the zebra mussel

Dreissena polymorpha

Alexandra Lepoutre ∗ 1, Nadia Milliote 1, Melissa Palos Ladeiro 1, Marc

Bonnard 1, Elisabeth Faassen 2, Alain Geffard 1, Emilie Lance 1

1 Universite de Reims Champagne-Ardenne (URCA) – Ministere de l’Enseignement Superieur et de la

Recherche Scientifique – 9 boulevard Paix - 51097 Reims cedex, France 2 Aquatic Ecology Water Quality Management Group – Wageningen University, P.O. Box 47,

Wageningen 6700 DD, The Netherlands, Netherlands

Among the toxins synthetized by phytoplankton, the BMAA (β-N-Methylamino-L-alanine), a non proteinogenic and neurotoxic amino acid, has the capacity to accumulate in the freshwater and marine food webs as well as in terrestrial vertebrates’ brain and could promote neurodegenerative pathologies such as the Amyotrophic Lateral Sclerosis (ALS). The zebra mussel Dreissena polymorpha is an efficient freshwater filter feeder organism well known for its capacity to bioaccumulate substances present in the water column among which cyanotoxins and BMAA. In bivalves, hemocytes are involved in various physiological mechanisms like shell repair or nutrient transport, and the immune defense of the organism. Thus studying the impact of BMAA on those cells brings information about zebra mussel’s individual state while exposed to this environmental toxin. This study aims to evaluate the bioaccumulation of BMAA in different organs (i.e., gills, mantle, digestive and genital glands, hemolymph), and the potential immunotoxicity (i.e, the phagocytosis capacity), cytotoxicity (i.e, viability) and genotoxicity (i.e, percentage of tail DNA) of BMAA on the hemocytes of the zebra mussel exposed to a constant concentration of dissolved BMAA (2,5 µg BMAA / individual / day) during a 3-week intoxication period followed by a 2-week depuration period.

The bioaccumulation data are currently being analyzed. The phagocytosis capacity of hemocytes did not vary between controls and individuals exposed to BMAA. However, a significant mortality of the hemocytes was observed on day 14 of exposure. Moreover, the degradation of the hemocytes’ DNA increased significantly from day 1 to 8 of exposure, suggesting a genotoxicity induced by BMAA. During the depuration period, no differences in those responses between the controls and the BMAA pre-exposed mussels were observed, suggesting a DNA repair mechanism.

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∗Speaker

Short term effects of the allelochemicals released by the toxic dinoflagellate

Alexandrium minutum upon the diatom Chaetoceros sp.

Marc Long ∗ 1,2, Kevin Tallec 1, Philippe Soudant 1, Fabienne Le Grand 1, Geraldine Sarthou 1, Dianne Jolley 2, Helene Hegaret 1



Technopole Brest-Iroise, Place Nicolas Copernic, 29280 Plouzane, France 2 School of Chemistry, University of Wollongong – NSW 2522, Australia

Dinoflagellates from the genus Alexandrium have the potential to produce paralytic shellfish

toxin (PST) and to release unknown extra-cellular compounds (ECC) with cytotoxic, hemolytic, ichtyotoxic and allelopathic activities. Impacts on organisms are widely reported, although the nature of these ECC remains unidentified. It is not even clear if one or more compounds with different properties are involved in the deleterious effects. In the literature, Alexandrium allelopathic activity is mainly reported to cause cell lysis or deleterious effects on the photosystem. However, the precise toxic allelopathic mechanisms are unknown. To better characterize these mechanisms, the allelopathic activity of A. minutum filtrate on the physiology of the diatom Chaetoceros sp. was investigated. When Chaetoceros sp. was exposed to A. minutum ECC, effects were observed upon the diatom photosystem II (PSII) within five minutes. PSII quan- tum yield (QY) increased within the first ten minutes. Along with QY inhibition, PSII energy fluxes changed with a large increase in dissipated energy. Photoinhibition of the PSII was ac- companied by an increase in non-photochemical quenching as well as changes in the pigment composition, especially those linked to the xanthophyll cycle. Along with an increase in reactive oxygen species production, modifications could be observed in composition of lipid membranes. Changes in lipid classes and a saturation of fatty acids were observed. Finally ECC decreased cell viability and induced cell lysis.

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∗Speaker

Metabolomics for cyanobacteria ecotoxicology:insights from the Toxcyfish

project

Benjamin Marie ∗ 1,2, Benoit Sotton 2, Severine Le Manach 2, Alain Paris 2,

1 Museum national d’histoire naturelle (MNHN) – Ministere de l’Ecologie, du Developpement Durable et de l’Energie, Ministere de l’Enseignement Superieur et de la Recherche, Museum National d’Histoire

Naturelle (MNHN) – 57, rue Cuvier - 75231 Paris Cedex 05, France 2 Musee National d’Histoire Naturelle - MNHN (France) – Musee National d’Histoire Naturelle -

MNHN (France) – 12 rue Buffon 75005 Paris, France

Cyanobacterial blooms are responsible of the production of a wide variety of potentially

toxic secondary metabolites that can impacts on the stability and the functioning of aquatic

ecosystems, and microcystins (MCs) and other cyanobacterial metabolites have been suspected to

induce various negative effects on various aquatic species. Recently, with the development of

”Omic” sciences, metabolomic analyses represent a powerful tool for the investigation of the

ecotoxicology of cyanobacterial, as developped in the Toxcyfish project:

1/ In order to better understand the variations in cyanotoxins production, such as microcystins

(MC), between clones of the same cyanobacterial blooms, we investigate in the present study the

diversity of several Microcystis strains isolated from different freshwater bloom-forming popula-

tions from various geographical area. Twenty four strains of Microcystis from the Paris’ Museum

Collection of cyanobacteria (PMC) were compared by an integrated approach combining geno-

typing and metabolites chemotyping.

2/ In order to identify the metabolic changes associated with cyanobacterial bloom exposure,

fish liver metabolome profiles were analyzed on perch, a typical fish species of freshwater Euro-

pean lakes, naturally exposed or not to cyanobacterial blooms, originating from field sampling in

Ile-de-France area.

These results originating from our observations will help us to better understand the genuine

ecotoxicological effects of cyanobacterial blooms producing a wide variety of potential noxious

metabolites on fish populations from natural ecosystems.

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∗Speaker

Paralytic Shellfish Toxin accumulation in oyster is predicted by gene expression

Audrey Mat ∗ 1, Christophe Klopp 2, Laura Payton 3, Celine Jeziorski 4,

Morgane Chalopin 1, Zouher Amzil 5, Damien Tran 3, Helene Hegaret 6,

Philippe Soudant 6, Caroline Fabioux 6, Arnaud Huvet 1

1 LEMAR UMR 6539 CNRS/UBO/IRD/Ifremer – Institut Francais de Recherche pour l’Exploitation de la Mer (IFREMER) – Centre de Bretagne, ZI de la Pointe du Diable, CS 10070, 29280 Plouzane, France

2 Plate-forme Genotoul Bioinfo – Institut national de la recherche agronomique (INRA) – UR875,

Auzeville, France 3 UMR 5805 EPOC – CNRS - Universite de Bordeaux – F-33120 Arcachon, France

4 GeT-PlaGe, Genotoul – Institut national de la recherche agronomique (INRA) – Auzeville, France 5 Laboratoire Phycotoxines – Institut Francais de Recherche pour l’Exploitation de la Mer (IFREMER)

– rue de l’Ile d’Yeu, BP 21105, F-44311 Nantes, France 6 LEMAR UMR 6539 – CNRS - UBO - IRD - Ifremer, CNRS - UBO - IRD - Ifremer – IUEM, 29280

Plouzane, France

Harmful Algal Blooms are an increasing international issue. They occur worldwide, are

related to poisoning events in humans and wildlife resources and induce economical losses estimated in billions of dollars. The genus Alexandrium is one of the most harmful genus, regularly causing Paralytic Shellfish Toxins (PST) events. Bivalves are the main vectors of PST, but they show a great inter-individual variability in toxin load. Understanding the determinism of toxin content is thus of critical importance for aquaculture and environmental health. The whole transcriptome of the digestive glands (DG) of Crassostrea gigas oysters fed on Alexandrium minutum for 6 days at environmentally realistic concentrations were explored to decipher the determinism of PST load. Differential expression analyses showed that the largest physiological differences appeared between oysters with high and low toxin contents (1098 differentially expressed genes), and not between exposed and control oysters (16 differentially expressed genes). Additionally, regression tools proved to be very efficient to model toxin load based on 70 genes. Two models were applied, showing 38 genes shared by both models, among which 23 were also differentially expressed. Real-time PCR data on 74 oysters sampled at 5 points over 48 h and studying 12 selected genes then revealed that differences in transcriptomes of exposed oysters was not determined initially but evolved with time and exposure to A. minutum. Finally, analyses of polymorphism revealed 441 SNP between oysters with low and high toxin load, impacting 528 genes. This study highlights i) that toxin content in oyster is at least partially related to their molecular functioning, ii) that there are associations between the genotype of oysters and their toxin content and iii) that a few genes and pathways are keys to understand interactions between A. minutum and C. gigas.

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∗Speaker

The Paralytic Shellfish Toxin, Saxitoxin, enters the cytoplasm and induces

apoptosis of oyster immune cells via a caspases-dependent pathway

Jean-Luc Rolland ∗ 1, Celina Abi-Khalil 1, Darren Finkelstein 2,

Genevieve Conejero 3, Justin Du Bois 2, Delphine Destoumieux-Garzon 1

1 laboratoire interactions hotes-pathogenes-environments (IHPE) – Institut Francais de Recherche pour

l’Exploitation de la Mer (IFREMER), CNRS : UMR5244, Universite de Montpellier : UMR5244 – , CNRS, Universite de Montpellier, Universite de Perpignan Via Domitia, IHPE, UMR 5244, F-34095

Montpellier, France, France 2 Department of Chemistry, Stanford University – 333 Campus Drive, Stanford, CA 94305-5080, United

States 3 Plant Cell Imaging platform PHIV – Institut national de la recherche agronomique (INRA) : UMR386, Centre National de la Recherche Scientifique - CNRS, Universite de Montpellier, Montpellier SupAgro –

UMR BPMP (INRA, CNRS, UM, SupAgro), Montpellier, France

Oyster exposure to the toxic dinoflagellate Alexandrium catenella was previously shown to result in apoptosis of Crassostrea gigas immune cells, the hemocytes. We investigated whether hemocytes were directly targeted by paralytic shellfish toxins (PSTs), and found that in vitro, coumarin-labeled saxitoxin localized in cytoplasmic granules of hemocytes, as determined by multiphoton microscopy. We also demonstrated that PSTs, including saxitoxin, were directly responsible for the apoptosis of hemocytes. Indeed, in vitro, STX and derivatives there of induced nuclear condensation, phosphatidylserine exposure, membrane permeability and DNA fragmentation of hemocytes, as determined by a series of labeling and microscopy experiments. The apoptosis pathway involved was dependent on caspase activation and independent of ROS production. Finally, the toxicity level of the main PSTs produced by A. catenella was evaluated. Unlike in vertebrates, gonyautoxin-5, which is present in large amounts in A. catenella was found to be more toxic than saxitoxin towards oyster immune cells. Collectively, our results show that PSTs produced by toxic dinoflagellates enter the cytoplasm and induce apoptosis of oyster immune cells through a caspase-dependent pathway. Because of the central role of hemocytes in mollusk immune defense, PST-induced death could negatively affect resistance of bivalve mollusks to microbial infections.

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∗Speaker

Differential Effects of Algal Toxins on

Grazing by the Marine Copepod Acartia tonsa

Patricia Tester ∗ 1, Jiarong Hong 2, Rebecca Waggett 3, Jospeh Katz 4,

Allen Place 5, Erik Davenport 6

1 Ocean Tester, LLC – 381 Gillikin Road, Beaufort, North Carolina 28516, United States

2 Department of Mechanical Engineering – University of Minnesota, United States 3 Department of Marine Science – University of Tampa, Tampa, Florida, United States

4 Johns Hopkins University (JHU) – Baltimore, USA, United States 5 University of Maryland – University of Maryland, College Park, MD 20742, USA, United States

6 National Oceanic Atmospheric Administration - NOAA (USA) (NOAA) – Silver Spring, Maryland, United States

Digital holographic cinematography was used to quantify and compare the grazing behavior of free-swimming Acartia tonsa exposed to toxic Karenia brevis and Karlodinium veneficum. Two grazing behaviors were recognized and quantified; both were log normally distributed.

”Sampling” behavior was characterized by short duration appendage beatings for less than 100 milliseconds (ms) while ”grazing” behavior generated feeding currents and persisted for up to 1,200 ms. In a filtered seawater control, A. tonsa only sampled at low frequencies. With the addition of non-toxic food (positive control), sampling increased slightly while grazing increased substantially. On diets of toxic food, sampling increased, but grazing decreased. This decreased grazing activity varied depending on the mode of action of the brevetoxins in K. brevis or karlotoxins from K. veneficum. These empirical findings were used to inform a stochastic model to evaluate how species-specific effects on grazing behavior influence top-down control of harmful algal blooms.

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Theme 4: Human toxicology

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∗Speaker

Receptor-based lateral flow test for fast and early detection of neurotoxins acting on

nicotinic acetylcholine receptors

Romulo Araoz ∗ 1,2, Denis Servent 2

1 CNRS/ Insitut de Neurosciences Paris- Saclay, UMR9197 – CNRS : UMR9197 – 1 avenue de la

Terrasse 91198 Gif sur Yvette, France 2 CEA/ DRF/ SIMOPRO/ LTMB/ Toxines recepteurs et canaux ioniques – CEA – CEA, Saclay, Bat.

152 91191 Gif sur Yvette, France

Muscle nicotinic acetylcholine receptors are evolutionary conserved pentameric transmem-

brane proteins that mediate fast neurotransmission at the neuromuscular junction necessary for

muscle contraction (scape from predation). Therefore, they constitute a primary target for a

wide array of toxins from diverse origin including cyanobacteria and dinoflagellates. The

assessment that ”fast acting” neurotoxins targeting nicotinic acetylcholine receptors could con-

taminate freshwater reservoirs (e.g. anatoxin-a) or shellfish (e.g. cyclic imine toxins), requests

the development of bioassays to detect these families of neurotoxins. Cyanobacterial anatoxins,

associated with fatal episodes of wild and stock animals, are agonists of nicotinic acetylcholine

receptors, while cyclic imine toxins are potent antagonist of nicotinic receptors. Shellfish accu-

mulate cyclic imine toxins following filter feeding on toxic dinoflagellates. We have developed

the lateral flow test for early detection of nicotinic neurotoxins directly in the field. ”NeuroTorp” is

based on a new concept for test strips: the high affinity of neurotoxins for their receptor targets.

We immobilized Torpedo marmorata electrocyte-membranes rich in nicotinic acetylcholine

receptors on the test-line. When a toxic sample is applied, the nicotinic toxin will displace a

biotinylated toxin tracer enabling its visual detection. Alternatively, we used fluorescent toxin

tracers simplifying toxin detection. In contrast to immunochromatographic tests that detect a

given toxin, NeuroTorp could be applied to the detection of an array of neurotoxins acting on

nicotinic acetylcholine receptors (e.g. anatoxin-a, pinnatoxins, spirolides, bungarotoxin, curare,

etc). NeuroTorp does provide the means for early, rapid and quantitative detection of nicotinic

neurotoxins in the field.

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∗Speaker

Mixtures of lipophilic phycotoxins: exposure data and in vitro toxicity assessment

Jimmy Alarcan ∗ 1, Ronel Bire 2, Ludovic Le Hegarat 1, Valerie Fessard 1

1 Unite de Toxicologie des Contaminants – Anses – 10 B rue Claude Bourgelat - 35306 Fougeres, France

2 Unite Biotoxines Marines – Anses – 14 rue Pierre et Marie Curie - 94706 Maisons-Alfort, France

Lipophilic phycotoxins are secondary metabolites produced by phytoplanktonic species.

They accumulate in filter feeding shellfish and can cause human intoxication. Regulatory limits have been set for toxin groups and toxicological features are well characterized for some of the individual toxins. Seafood contamination may also be related to co-exposure to phycotoxins but hazard assessment regarding mixture effects is rather scarce. Moreover, occurrence of phycotoxins can greatly vary from one region to another as well as concerning the groups involved. In this work, we first provided an overview on multi-toxins occurrence by a case study through analysis of 17 published papers. For each publication, ratio mixtures were calculated from raw data. Based on these calculated levels, we determined the most frequent mixtures (composition and ratio) found in seafood as well as the most frequent mixtures described depending on the shellfish type. Results showed that binary phycotoxin mixtures are the most common in shellfish. Okadaic acid (OA) is almost always the predominant toxin found in co-contaminated shellfish. Based on these established ratios, we investigated the mixture effects on in vitro human hepatic and intestinal cells. Different endpoints such as inflammation, oxidative stress or apoptosis are under screening. As recommended by EFSA, evaluation of mixtures toxicity is required to fully characterize the hazard related to shellfish phycotoxins contamination.

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∗Speaker

Ostreopsis cf. ovata blooms in the NW Mediterranean: Exploring the links

between toxins variability and impacts on human health

Elisa Berdalet ∗ 1, Rafael Abos-Herrandiz 2, Marta Estrada 3

1 Institute of Marine Sciences / Institut de Ciencies del Mar [Barcelona] (ICM-CSIC) – Passeig Maritim

de la Barceloneta 37-49 Barcelona 08003, Spain 2 Catalan Health Service (Department of Primary Health Care, Generalitat de Catalunya) – CAP

Carles I. Carrer Marina 168. E-08013 Barcelona, Spain 3 Institute of Marine Sciences / Institut de Ciencies del Mar [Barcelona] (ICM) – Passeig Maritim de la

Barceloneta 37-49 Barcelona 08003, Spain

Blooms of the benthic dinoflagellate Ostreopsis cf. ovata are recurrent in Sant Andreu de

Llavaneres beach (40 km North of Barcelona, NW Mediterranean) along the summer-fall season. In this locality, respiratory symptoms and general malaise in people exposed to marine aerosols are reported during the yearly microalgal bloom period. In addition, macrofauna seems also to be negatively affected by the presence of Ostreopsis. Within the OstreoRisk project (2015-2017) we are characterizing the variability of Ostreop- sis -related toxins (ovatoxins and palytoxins) along the bloom development period, in order to ascertain their role on the human and environmental health impacts in the area. Concurrently, epidemiological follow-up studies are conducted on the inhabitants of residences located in front of the Ostreopsis hot spot.

The blooms last for at least 2 months, with high numbers until the end of September. However, impacts on human health tend to peak at the transition from the exponential to the stationary phase of the bloom, usually between the end of July and early August. In the summer of 2015, total toxin intracellular concentrations maxima were also detected within that period (up to 300 - 700 pg·cell-1), while along the rest of the bloom season the values were below 30 pg·cell-1. On average, ovatoxin a (OVTX-a) accounted for 75% of the total toxin content, followed by OVTX-b (22%), while OVTXs c to g and isobaric palytoxin (PLTX) were minor components. PLTX (corresponding to < 0.5% of the total toxin content) was rarely detected, coinciding with the peaks in total toxin content and O. cf. ovata cell abundances (ca. 7-10·105 cells·g-1 of macroalgae fresh weight). With the technical approaches used, toxins have not been detected in the aerosol.

Our next steps include the chemical characterization of aerosols and ecotoxicology tests on model organisms.

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∗Speaker

The Uzice Case

Jussi Meriluoto ∗ 1,2, Zorica Svircev 2,1

1 Biochemistry, Faculty of Science and Engineering – Abo Akademi University, Turku, Finland, Finland 2 Laboratory for Paleoenvironmental Reconstruction. – University of Novi Sad, Serbia, Serbia

An intensive bloom of the cyanobacterium Planktothrix rubescens was observed in the Vrutci

reservoir in December 2013. The reservoir served as the drinking water source for the city of

Uzice, Serbia, with 70,000 inhabitants. Filaments of the cyanobacterium were found in the

treated water and there were complaints about discoloured tap water. The MC-LR concentra-

tion in drinking water was below the WHO provisional guideline value (1 µg/L) according to

the Institute of Public Health of Serbia.

Despite this, the Sanitary Inspectorate of the Republic of Serbia decided to ban the use of tap

water for drinking and cooking purposes to protect the health of the inhabitants of Uzice. The

situation soon resulted in unrest among the citizens. The toxicity of the cyanobacterial biomass

was first shown by Artemia salina bioassays. Microcystin(s) were later detected by LC-MS/MS in

samples of the reservoir and tap water, and in fish captured from the reservoir. Data about the

water use and the health of the inhabitants were collected with the help of a questionnaire which

clearly indicated a number of health issues. An epidemiological investigation also showed an

elevated occurrence of digestive tract and skin diseases compared to earlier years, possibly as a

result of exposure to the cyanobacterial mate- rial/metabolites. Based on the evidence found, it

is likely that a cyanobacterial bloom occurred in the reservoir already before 2013.

Lessons from the Uzice case are many. The presentation discusses i) the importance of interac-

tion between academia and authorities, ii) proper monitoring of cyanobacterial hazards, iii) the

necessity of adequate information and advice to the general public, iv) guidelines and legislation,

and v) collection of exposure and health data complementary to analytical results. Further, the

authors would like to underline the recurrent role of Planktothrix sp. in drinking water quality

problems.

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∗Speaker

Gambierol enhances quantal acetylcholine release from motor nerve terminals of

vertebrate neuromuscular junctions

Jordi Molgo ∗ 1, Sebastien Schlumberger 3,4, Makoto Sasaki 5,6,

Haruhiko Fuwa 5,6, M. Louzao 7, Luis Botana 7, Evelyne Benoit 1,7

1 Commissariat a l’Energie Atomique et aux energies alternatives (CEA) – CEA – Institut de Biologie et

Technologies de Saclay (IBITECS), Universite Paris-Saclay, Service d’Ingenierie Moleculaire des Proteines, batiment 152, 91191 Gif-sur-Yvette, France, France

3 Institut de Neurobiologie Alfred Fessard – FRC2118, Laboratoire de Neurobiologie et Developpement – UPR3294, CNRS, 91198 Gif sur Yvette cedex, France (CNRS) – CNRS : UMR3294 – Institut de

Neurobiologie Alfred Fessard – FRC2118, Laboratoire de Neurobiologie et Developpement – UPR3294, 91198 Gif sur Yvette cedex, France, France

4 Centre National de la Recherche Scientifique (CNRS) – CNRS : UPR3294 – CNRS, Laboratoire de Neurobiologie et Developpement, UPR3294, 91198 Gif sur Yvette cedex, France, France

5 Graduate School of Life Sciences – Graduate School of Life Sciences, Tohoku University, Aoba-ku, Sendai, Japan., Japan

6 Tohoku University – Graduate School of Life Sciences, Tohoku University, Aoba-ku, Sendai, Japan., Japan

7 Universidad de Santiago de Compostela – Departamento de Farmacologia, Facultad de Veterinaria, Universidad de Santiago de Compostela, Lugo, Spain., Spain

Gambierol is a polyether toxin that was first isolated with ciguatoxins from cultured Gam-

bierdiscus toxicus dinoflagellates, collected at Rangiroa (French Polynesia). Subsequently, the total chemical synthesis of gambierol was achieved by independent groups in Japan and the USA, allowing detailed analyses of its mode of action. Because gambierol has been reported to specifically inhibit some subtypes of native or expressed K+ channels in cells, we explored its actions on synaptic transmission at vertebrate neuromuscular junctions (NMJs). For this, we used synthetic gambierol, and conventional electrophysiological and intracellular spectrometric Ca2+ imaging techniques. Focal current-recordings at frog NMJs revealed that the duration of presynaptic currents was prolonged by gambierol (20 nM), so that the time elapsed between the peak of the presynaptic current and the beginning of the endplate currents was increased. Gambierol (0.5 to 2 nM), when applied to NMJs equilibrated in low-Ca2+ high-Mg2+ medium, increased the mean quantal content of endplate potentials. Also, gambierol (15 nM) increased the quantal content of endplate potentials in mouse NMJs previously poisoned by botulinum type-A toxin, bathed in standard Krebs-Ringer solution, by reducing the number of release- failures. In isolated mouse hemidiaphragm preparations, in which muscle Na+ channels have been blocked by µ-conotoxin GIIIB (to avoid muscle contraction), gambierol (1 to 20 nM) increased the amplitude of endplate potentials evoked by phrenic- nerve stimulation and reversed the effect of d-tubocurarine that blocks muscle nicotinic acetylcholine receptors. Selective load- ing of the Ca2+-sensitive dye Fluo-3/AM to frog nerve terminals of NMJs showed that gambierol in response to nerve stimulation at 1 or 10 Hz increased the relative intracellular Ca2+ fluorescence with respect to controls. It is concluded that gambierol blocks presynaptic K+ channels, greatly increases Ca2+ influx into motor nerve terminals and enhances both evoked and delayed quantal transmitter release at the NMJ.

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∗Speaker

Maitotoxin-4, a novel MTX analog

produced by Gambierdiscus excentricus

Francesco Pisapia ∗ 1, Manoella Sibat 1, Christine Herrenknecht 2, Korian

Lhaute 1, Greta Gaiani 3, Pierre-Jean Ferron 4, Valerie Fessard 4,

Catherine Roullier 2, Santiago Fraga 5, Silvia Nascimento 6, Wayne Litaker 7, Philipp Hess 1

1 Phycotoxins Laboratory (PHYC) – Institut Francais de Recherche pour l’Exploitation de la Mer

(IFREMER) – Rue de l’Ile d’Yeu - BP 21105 - 44311 Nantes Cedex 03, France 2 Mer, molecules et sante (MMS) – Universite de Nantes : EA2160 – UFR Sciences Pharmaceutiques et

Biologiques, 9 rue Bias, BP 53508, 44035, Nantes Cedex 1, France 3 University of Trieste – Department of Life Science, 34127 Trieste, Italy, Italy

4 French Agency for Food and Safety, Toxicology of Contaminants Unit (ANSES) – Anses – 10 B rue

Claude Bourgelat, 35133 Javene, France, France 5 Instituto Espanol de Oceanografıa, Centro Oceanografico de Vigo (IEO) – Subida a Radio Faro 50,

36390 Vigo, Spain, Spain 6 Universidade Federal do Estado do Rio de Janeiro, Departamento de Ecologia e Recursos Marinhos, Laboratorio de Microalgas Marinhas (UNIRIO) – Av. Pasteur, 458, 22.290-240, Rio de Janeiro, RJ,

Brazil, Brazil 7 National Oceanic Atmospheric Administration, National Center for Coastal Ocean Science, Center for

Coastal Fisheries and Habitat Research (CCFHR) (CCFHR (NOAA)) – 101 Pivers Island Road, Beaufort, NC 28516, USA, United States

Maitotoxins (MTXs) are among the most potent toxins known to date. They are produced by epi-benthic dinoflagellates of the genera Gambierdiscus and Fukuyoa and may potentially play a role in causing Ciguatera Fish Poisoning. A recent survey revealed G. excentricus as one of the most maitotoxic species examined to date. The aim of the present study was to characterize MTX-related compounds produced by different strains of G. excentricus. Crude extracts of the strains VGO791 and VGO792 (Canaries) were subjected to two sequential purification steps: (i) liquid-liquid partitioning between dichloromethane and aqueous methanol followed by (ii) size-exclusion chromatography. Fractions were screened for toxicity using two functional assays, both based on the neuro-2a cell model: the MTT cytotoxicity assay and the high content screening assay for Ca2+ influx. Liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS) analysis was performed to pinpoint potential MTX analogs. On the basis of cytotoxicity and Ca2+-related activity, non-targeted HRMS analysis permitted the identification of maitotoxin-4 (MTX4, M = 3292.4860 Da, as unsalted molecule) in the most toxic fractions. HRMS/MS spectra of MTX and MTX4 will be presented. In addition, five other strains of G. excentricus and 17 other strains of ten species of Gambierdiscus and Fukuyoa were screened for the presence of MTXs using low resolution tandem mass spectrometry (LRMS/MS) analysis. Neither the original MTX congener described nor MTX2 were present. Maitotoxin-3 appeared present from LRMS/MS but could not be confirmed using HRMS, suggesting potential interference for this compound in LRMS/MS. Maitotoxin-4 was detected in all seven strains of the species G. excentricus examined, independently of their origin (Canary Islands, Brazil and Caribbean), and was not detected in any other species. The chemical diversity of the MTX analogs produced by the genera Gambierdiscus and Fukuyoa appears higher than anticipated so far and may potentially be species-specific.

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∗Speaker

Transgenerational effects of Microcystin-LR on Daphnia magna

Claudia Wiegand ∗ 1, Rafael Ortiz-Rodrıguez 2, Thanh Dao 3

1 University Rennes1, UMR 6553 ECOBIO (ECOBIO) – Universite de Rennes I – Campus de Beaulieu,

35042 Rennes Cedex, France 2 Environmental Defense Fund – La Paz, Mexico

3 Duy Tan University, Institute of Research and Development – Da Nang, Vietnam

Eutrophication and climate factors increase the frequency of cyanobacterial blooms in fresh-

waters. Planktivorous organisms are affected in several ways: diminished food diversity, a lower food quality, and bioactive or toxic secondary metabolites. Cyanobacteria thus affect growth, survival and reproduction of zooplankton, amongst others Daphnia, which have a central position in pelagic food webs. One of the best studied toxic secondary metabolite are microcystins (MCs), interfering with protein-phosphatases type 1 and 2a, impacting cellular regulation processes. A possibility to ameliorate MC effects is to biotransform it via glutathione S transferase (GST) to a less toxic glutathione conjugate. This process was hypothesised to underlie the ability of Daphnia to withstand MC and to explain the enhanced tolerance in offspring from mothers exposed to toxic cyanobacteria. To proof this, two-generational experiments were conducted with D. magna, exposing the parental generation to MC for 1 or 7 days and determining the enzyme mediated tolerance to MC in their offspring. Acute effects of MC on biotransformation, antioxidant and metabolism enzymes were assessed within 24 hrs, and growth and toxicity in a chronic 21-day exposure. Seven days exposure of the mothers to MC induced higher activity of GST and malate dehydrogenase in the offspring and enables them to increase catalase activity when challenged again with MC whereas 1-day exposure of the mothers did not. Offspring from non-exposed and 1 day exposed mothers suffered higher mortalities when exposed to MC compared to the offspring from 7 day exposed mothers, which was correlated with the elevated activity of GST, malate dehydrogenase and catalase, suggesting maternal transfer of activation factors. The survival occurred at the expense of individual growth. These results prove transgenerational effects provoked by MC in Daphnia, which may explain the observed acquirement of enhanced tolerance over generations.

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Theme 5: Effects of HABs on professional sectors and society at

large: from legal and socio-economic perspectives

49 49


∗Speaker

Thinking out of the box: finding news ways of regulating marine toxins.

Marie Bonnin ∗ 1,2, Helene Hegaret 1, Philippe Soudant 3, Philippe Fotso 2, Caroline Fabioux 1, Philipp Hess 4



Technopole Brest-Iroise, Place Nicolas Copernic, 29280 Plouzane, France 2 LEMAR – LEMAR – UMR 6539 (CNRS/UBO/IRD/Ifremer), Laboratoire des sciences de

l’Environnement MARin (LEMAR), Institut Universitaire Europeen de la Mer, rue Dumont d’Urville,

29280 Plouzane, France, France 3 Laboratoire des Sciences de l’Environnement Marin (LEMAR) – CNRS : UMR6539, Universite de

Bretagne Occidentale (UBO), Institut Universitaire Europeen de la Mer (IUEM), Institut de Recherche pour le Developpement, Institut Francais de Recherche pour l’Exploitation de la Mer (IFREMER) – UMR

6539 (CNRS/UBO/IRD/Ifremer), Laboratoire des sciences de l’Environnement MARin (LEMAR), Institut Universitaire Europeen de la Mer, rue Dumont d’Urville, 29280 Plouzane, France, France

4 Dynamiques de l’Environnement Cotier (DYNECO) – Institut Francais de Recherche pour l’Exploitation de la Mer (IFREMER) – Technopole Brest-Iroise, BP 70, 29280, Plouzane, France

Currently there is no legislation on algal compounds that impact on food safety. Food safety legislation generally regulates harmful algae by setting maximum thresholds of the toxins they produce in shellfish. Nevertheless, there is no legal framework on micro-algal proliferation other than the Marine Strategy Framework Directive, which only limits microalgal proliferation in an indirect manner. In this presentation, legal concepts are proposed to allow for a limitation of algal proliferation or facilitate the mitigation of their impacts. There is two different perspectives to be considered to regulate harmful algae and/or their biotoxins by taking into account scientific knowledge raising the following questions: Could harmful algae be considered as invasive species in nature conservation law, or as contaminants in marine pollution law? What are the implications of considering different legal definitions of toxins? Are the definitions in natural sciences and in law compatible? Firstly, the existing legal framework for algal toxins is reviewed and secondly, the potential legal status are considered for algal species that produce toxins. This presentation is the product of positive crosstalk between different disciplines aimed at determining the legal status of algal toxins, at the example of saxitoxins and one of their biogenic origins, the genus Alexandrium.

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∗Speaker

Coordinating the implementation of the interagency Global CFP Strategy

Mireille Chinain ∗ 1, Marie-Yasmine Dechraoui Bottein ∗ 2, Philipp Hess ∗ 3


Papeete, Tahiti, French Polynesia 2 Environment Laboratories, Department of Nuclear Science and Application (IAEA) – 4a Quai

Antoine 1er 98000 Monaco, Monaco 3 Institut Francais de Recherche pour l’Exploitation de la Mer - Nantes (Laboratoire Phycotoxines) –

Universite de Nantes – Rue de l’Ile d’Yeu - BP 21105 - 44311 Nantes Cedex 03, France

Ciguatera Fish Poisoning (CFP) is a prominent non-bacterial seafood intoxication transmit- ted through fish and marine invertebrates. Originally endemic to the tropics, it has become a global problem due to the expansion of Gambierdiscus to temperate waters and recent reports of endogenous poisoning cases in European localities. Since 2013, a strategy to tackle CFP-related challenges was developed by the Inter-governmental Panel on Harmful Algal Blooms of the In- tergovernmental Oceanographic Commission of UNESCO. Since 2015, the initiative widened to interagency level, with adoption by the International Atomic Energy Agency, the Food and Agriculture Organization and the World Health Organization. Through technical cooperation and R&D programs by IAEA in synergy with other international organizations, efforts are being made to better define and address the gaps in risk assessment and needs for effective ciguatera management. Scientists from 35 countries received training in benthic HAB collection and identification, and 16 countries developed capacity for toxin analysis (receptor binding assay). Guidelines are also being developed, including surveillance of ciguatera cases, monitoring of toxic fish, benthic HABs and determination of associated ciguatoxins (CTXs).

At the Pacific level, efforts to develop a regional framework for CFP data compilation/sharing have been initiated through PaceNet+ initiative. Cigua-Watch, a program currently funded by the Fonds Pacifique, aims at strengthening the CFP epidemiological surveillance in the Pacific, while Cigua-Crimson will address the issue of the lack of a regional clinical case database to improve CFP biomedical studies. At European level, the program EuroCigua has been developed to monitor CFP in the Canaries, Azores and the Mediterranean, however, initiatives at French level need to be widened and further connected to international programmes. Coordinating the implementation of the CFP Strategy will require the organisation of work- shops to further structure research priorities (in the fields of epidemiology, risk assessment, CTXs detection) and attract funding.

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∗Speaker

Pinnatoxins, an emergent class of fast-acting marine toxins, are potential threat for neurodevelopment and global health

Sophie Creuzet ∗ 1, Aurelie Couesnon ∗ 1, Vincent Dive ∗ 2, Armen

Zakarian ∗ 3, Jordi Molgó ∗ 2, Denis Servant ∗ 2

1 Neuro-PSI (CNRS-UMR9197) – Nikon Imaging Centre@Institut Curie-Cnrs, CNRS : UMR9197 –

Institut des Neurosciences Paris Saclay Gif-sur-Yvette, France 2 Service d’Ingenierie Moleculaire des Proteines (SIMOPRO) – CEA – CEA Saclay 91191 Gif sur

Yvette cedex, France 3 UNIVERSITY OF CALIFORNIA SANTA BARBARA – University of California Santa Barbara,

Santa Barbara CA 93106-9510, United States

Pinnatoxins (PnTx) are selective potent antagonists of nicotinic acetylcholine receptors (nAChRs), and selective inhibitors of both muscle-types (α1 βγδ, α1 βδe) and neuronal (α7, α3β2 and α4β2) nAChRs.The dual tropism of theses toxins towards the muscular and neuronal nAChRs leads to question about the noxious downstream effects of single and chronic exposures on developing organisms.To this end, we have designed some experimental assays to determine whether PnTx could be toxic early in embryonic development, or had teratogenic effects on the embryo, and fetus. Due to the ease of access throughout ontogenesis, the chick embryo was used as a model for our functional assay. When administered during embryonic development, we show that PnTx are prone to block the neuromuscular junction. The PnTx rapidly impinges on the embryonic motility, and the spontaneous and stereotypical transverse flexion – axial ex- tension, and eventually leads to a global fetal ataxia. Notably, motor alterations coincide with defects in the maturation of the musculoskeletal system. Cartilage of the appendicular skeleton appears reduced in size and density, as embryos are significantly smaller when compared to controls. Furthermore, the activity of myogenic factors, required for the development and the maturation of the myogenic cells are misregulated, and confirm that PnTx-dependent blockade of the neuromuscular junction hampers the transcription activity of the myogenic master genes. In addition, the transcriptomic alterations in the musculoskeletal system coincide with aber- rant modifications in the programmed cell death of the spinal motor neurons, at the transverse level.Our results reveal how detrimental the PnTx are on the peripheral nervous system, hence preventing the maturation of the musculo-skeletal system during pre-natal life. Our data shed a new light on the potential risk of recurrent exposures to these environmental toxins during pregnancy for embryonic and fetal development.

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∗Speaker

Strategies to reduce toxins in shellfish

Ana Garcia-Cabado ∗ 1

1 ANFACO-CECOPESCA, Head of Food Safety Division – Crta. Colexio Universitario, 16 36310 Vigo

(Pontevedra), Spain

In Galicia (NW Spain), Mytilus galloprovincialis is mainly cultured in rafts supported by

floats since 1946. Within a few years mussels production in the Galician region increased sharply.

However, bivalves production worldwide is hampered by natural phenomena commonly known as

Harmful Algal Blooms (HABs).

These toxic episodes pose serious risks to human health, environment and aquatic organism

due to production of toxins by phytoplankton species. In addition to that, economic impacts

are suffered by producers, processors, restaurants, tourism and auxiliary industries due to closure

of shellfish production areas.

In this context, in order to minimize the effects of toxins in shellfish sector, several strategies

were defined, either in live or in processed mussels. In collaboration with depuration centers we have

carried out several trials by using compounds that increase the elimination rate of toxins in

contaminated shellfish. On the other hand, some experiments were performed by applying different

treatments and technologies in laboratory and processing industries.

This work was mostly funded by the research grants from Xunta de Galicia, FEDER

INNTERCONECTA 2013 (ITC-20133020) ”SinTox” and 2015 (ITC-20151273) ”MyTox”.

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∗Speaker

Eaters of oyster: from the risk of contamination by toxic algae to social

representations and practices of consumption

Elisabeth Guillou ∗ 1, Adeline Raymond ∗1

1 Laboratoire de Psychologie : Cognition, Comportement, Communication - EA 1285 (LP3C) – Universite de Bretagne Occidentale [UBO] : EA1285 – 20 rue Duquesne - CS 93837 29238 Brest cedex

3, France

Toxic algal blooms can have significant socio-economic and ecological impacts by making

oysters unfit for consumption. In order to better understand the phenomenon of accumulation of toxins in oysters, an interdisciplinary research project was initiated (ACCUTOX). It is interested in the differences between diploid and triploid oysters. It integrates a psychosociological approach that aims to analyze representations of the risks associated with oysters from the point of view of the consumers in order to understand the social stakes linked to this contamination. Therefore we questioned consumers on their actual practices in order to highlight the representation of the oyster, the methods of purchase or fishing, the preparation and consumption. Implicitly, it allows identifying the practices implemented to limit the possible risks linked to the purchase or the fishing as well as the consumption. A longitudinal study was conducted with 27 consumers over a period of eight months (July 2015-February 2016). Individuals were required to record in a logbook all practices of intention, purchase or fishing and consumption of oysters. Regular interviews were conducted on the basis of this logbook. 86 interviews were conducted and fully transcribed. A content analysis was conducted. Whether consumption is habitual or occasional, the results show that the oyster is associated with pleasure, and the notion of ”taking time”. In terms of risks, the choice of the oyster is governed by the taste, the type of oyster and its price. Some respondents spontaneously mention the triploids, but total trust is given to the oyster farmer. The risk of contamination is therefore not mentioned. The fisherman has knowledge of the health problems. Trust rests in his practice and experience.

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∗Speaker

The Integrated Pest Management Model as a Framework for Aquaculture HAB Mitigation

Gary Wikfors ∗ 1

1 National Oceanic Atmospheric Administration - NOAA (USA) (NOAA, NMFS, NEFSC) – 212

Rogers Avenue Milford, CT 06460, United States

Phytoplankton ”blooms” are natural phenomena that result from seasonal and other environ-

mental cycles that vary the availability of energy (light and turbulence) and materials (carbon and other nutrients) in space and time. As the geological epoch referred to as the Anthro- pocene proceeds, there is clear recognition that human activities are playing an increasing role in modifying environmental cycles that influence phytoplankton population dynamics, including those of taxa that are considered harmful, i.e., Harmful Algal Blooms, or HAB. Concurrently, as reliance of human populations upon advanced food-production systems is driving expansion of seafood cultivation – aquaculture – our reliance upon the quantity and quality of primary production becomes more specific and demanding of stability. Within the general context of direct trophic use of phytoplankton by cultivated shellfish, and considering the reliance of all aquaculture enterprises upon enabling environmental quality, HAB are correctly considered to be disruptions analogous to agricultural pests. Accordingly, one approach to developing a comprehensive strategy for managing aquaculture in seas increasingly experiencing HAB is to look toward agricultural models – for example Integrated Pest Management (IPM) – for applicable components. This presentation will explore the applicability of agricultural IPM models to aquaculture management of increasing HAB interactions.

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55


Theme 6: Tropics

56 56


∗Speaker

Distribution and abundances of benthic

toxic dinoflagellates in French West Indies

Aurelie Boisnoir ∗ 1, Pierre-Yves Pascal 2, Sophie Marro 3, Rodolphe

Lemee 4

1 CNRS-UPMC-UA – Universite Antilles Guyane (Pointe-a-Pitre, France) – (1) Laboratoire d’Oceanographie de Villefranche, UMR 7093 CNRS-UPMC, Observatoire Oceanologique de

Villefranche, France, (2) Laboratoire de Biologie Marine, UMR 7138 CNRS-UPMC, Universite des Antilles, Guadeloupe, France, France

2 CNRS-UPMC-UA – Universite Antilles Guyane (Pointe-a-Pitre, France) – Laboratoire de Biologie

Marine, UMR 7138 CNRS-UPMC, Universite des Antilles, Guadeloupe, France, France 3 CNRS-UPMC – Centre National de la Recherche Scientifique - CNRS – (1) Laboratoire

d’Oceanographie de Villefranche, UMR 7093 CNRS-UPMC, Observatoire Oceanologique de Villefranche, France, France

4 CNRS-UPMC – Universite Paris VI - Pierre et Marie Curie – (1) Laboratoire d’Oceanographie de

Villefranche, UMR 7093 CNRS-UPMC, Observatoire Oceanologique de Villefranche, France, France

Benthic Toxic Dinoflagellates (BTD) are often linked with health problems in tropical and

temperate areas. Species of genus Gambierdiscus are associated with Ciguetera Fish Poisoning (CFP) due to the accumulation of phycotoxins in the food web. Species of genus Ostreopsis are more often linked to health problem after direct contact with contaminated water or bioaerosols, even if phycotoxins were already found in seafood. The Caribbean area is the second region of the world affected by CFP and our bibliography analysis reveals few studies on ecology and distribution of BTD in the French West Indies. In order to evaluate the potential risk of BTD in this area, a first survey, followed by a more formal monitoring (18 months) of BTD has been undertaken in Guadeloupe and Martinique Islands.

Ostreopsis, Prorocentrum, Gambierdiscus, Amphidinium, Sinophysis, Coolia genera were found on both islands. Ostreopsis and Prorocentrum were the most abundant. Gambierdiscus, Coolia, Amphidinium and Sinophysis genera were present but in lower extent. In average, BTD were 5 times more abundant in Guadeloupe than in Martinique. Same Gambierdiscus spp. abundances were found in Guadeloupe and Martinique while those islands are respectively inside and outside of the ciguatera outbreak area. Moreover, species of this genus were more abundant at deep sites (1,50 m) than shallow sites (0,5 m). No clear season patterns of BTD were observed. Dinoflagellates variations were mainly controlled by water temperature but other parameters can interfere like wind force on planktonic cells and biotic substrates for benthic dinoflagellates.

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∗Speaker

Microalgae from French Polynesian lagoons:

novel source of bioactive compounds?

Mireille Chinain ∗ 1, Sebastien Longo 1, Kevin Henry 1, Andre Ung 1,

Taiana Darius 1, Jerome Viallon 1, Martine Rodier 2, Bruno Delesalle 3,

Alain Lo-Yat 4, Gilles Le Moullac 5, Romulo Araoz 6

1 Institut Louis Malarde (ILM) – BP30, 98713, Papeete Tahiti, French Polynesia

2 Institut de Recherche pour le Developpement (IRD) – BP 529, 98713 Papeete - Tahiti, French

Polynesia 3 E cole Pratique des Hautes E tudes (EPHE) – E cole Pratique des Hautes E tudes [EPHE] – 58 Avenue

Paul Alduy, 66860 Perpignan Cedex, France 4 Institut Francais de Recherche pour l’exploitation de la MER (IFREMER) – 7004, 98179 Taravao,

Tahiti, French Polynesia 5 Institut Francais de Recherche pour l’exploitation de la MER (IFREMER) – BP 49 - 98719 Taravao -

Tahiti, French Polynesia 6 L’Institut des Neurosciences Paris-Saclay (Neuro-PSI) – CNRS – CEA Saclay, Bat. 152, 91191

Gif-sur-Yvette, France

French Polynesian lagoons are often the site of phytoplankton blooms events involving both

benthic and pelagic (i.e. red-tide) species, that may occasionally lead to the production of toxic metabolites with potential deleterious effects on marine ecosystems and, in some instances, seafood safety issues for human populations (e.g. ciguatera outbreaks). As an example, an exceptionally long-lasting red tide event was reported from November 2013 until December 2014 on Takaroa atoll (Tuamotu archipelago, French Polynesia), resulting in significant mortalities among populations of the pearl oyster P. margaritifera. In order to characterize the algal taxa likely involved in past bloom events observed in Takaroa lagoon, two series of field experiments using various nutrient enrichment regimes were carried out in semi-controlled environmental conditions (i.e. in mesocosms) in 2015 and 2016. They led to the isolation of ˜50 strains belonging primarily to the dinoflagellates and diatoms groups. The EPSOM program aims at addressing the chemo-diversity characterizing these taxa. To this end, a toxicological screening of cell extracts obtained from in vitro cultures are currently underway using the Torpedo Microplate-Receptor Binding AssayQR and the neuroblastoma cell-based assay (CBA-N2a) which specifically target toxic compounds active on nicotinic acetylcholine receptors and voltage-gated sodium channels, respectively. The preliminary results of this work will be presented.

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∗Speaker

Taxonomic diversity of the dinoflagellate genus Ostreopsis in French Polynesia:

preliminary results from Marquesas Islands (Pacific Ocean)

Nicolas Chomerat ∗ 1, Clemence Gatti 2, Gwenael Bilien 1, Andre Ung 2, H.

Taiana Darius 2, R. Wayne Litaker 3, Patricia Tester 3, Mireille Chinain 2

1 IFREMER Laboratoire Environnement et Ressources Aquacoles de Bretagne Occidentale (IFREMER

LER BO) – Institut Francais de Recherche pour l’Exploitation de la Mer (IFREMER) – Station de Biologie Marine Place de la Croix 29900 Concarneau, France

2 Institut Louis Malarde (ILM) – Institut Louis Malarde – BP 30, 98713 Papeete Tahiti, French

Polynesia, France 3 National Oceanic Atmospheric Administration - NOAA (USA) – 101 Pivers Island Rd, Beaufort,

28516 North Carolina , USA, United States

The genus Ostreopsis is commonly found associated with Gambierdiscus in benthic HAB

assemblages of ciguateric biotopes of French Polynesia. In Rapa island (Australes archipelago), wild populations of this dinoflagellate were even found capable of producing palytoxin, emphasiz- ing the potential health risk associated with the proliferation of Ostreopsis in French Polynesian lagoons. Thanks to the wide use of the window-screens technique in field-campaigns conducted in various archipelagoes (i.e. Australes, Society, Tuamotu and Marquesas), a program aiming at the characterization of the taxonomic diversity of this genus was initiated in 2015. Whenever possible, cells were isolated and clonal cultures established, whereas wild samples were fixed immediately for taxonomic identification of dinoflagellates. Cell morphology was observed using light microscopy and high resolution field-emission scanning electron microscopy (FE-SEM) to study details of the thecal pattern and ornamentation while single-cell PCRs and sequencing of LSU rDNA (D1–D3 and D8-D10 domains) was used for molecular characterization. Here we present the preliminary results obtained from the samples collected in 2016 in Nuku-Hiva island (Marquesas archipelago). Samples collected from four distinct sites revealed the presence of three different Ostreopsis species. A large species was morphologically close to O. lenticularis but its sequences were identical to a yet undescribed species named ”sp. 5” in previous studies in the Pacific area. The two other species were smaller, tear-shaped, morphologically close to O. cf. ovata and difficult to differentiate in light microscopy, but rDNA sequences revealed two distinct genotypes, one being related to O. cf. ovata and the other corresponding to the recently described species O. rhodesae. Further toxicity studies on in vitro cultures of Ostreopsis will help clarify the potential contribution of this genus to the high toxicity risk level specific to Nuku-Hiva island.

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∗Speaker

Environmental control and dynamics of blooms of the benthic toxic

dinoflagellate Ostreopsis cf. ovata in the Mediterranean Sea

Cecile Jauzein ∗ 1,2, Rodolphe Lemee 3

1 Laboratoire d’oceanographie de Villefranche (LOV) – INSU, CNRS : UMR7093, Universite Pierre et Marie Curie (UPMC) - Paris VI, Universite Pierre et Marie Curie [UPMC] - Paris VI – Observatoire Oceanologique Station zoologique 181, chemin du lazaret BP 28 06230 VILLEFRANCHE SUR MER

Cedex, France 2 Laboratoire Ifremer DYNECO-Pelagos (Ifremer Dyneco-Pelagos) – Institut Francais de Recherche

pour l’Exploitation de la Mer (IFREMER) – Ifremer Centre Bretagne ZI de la Pointe du Diable CS

10070, 29280 Plouzane, France 3 Laboratoire d’oceanogrphie de Villefranche (LOV) – INSU, CNRS : UMR7093, Universite Pierre et Marie Curie [UPMC] - Paris VI, Universite Pierre et Marie Curie (UPMC) - Paris VI – Observatoire Oceanologique Station zoologique 181, chemin du lazaret BP 28 06230 VILLEFRANCHE SUR MER

Cedex, France

Benthic harmful algal blooms represent an increasing threat to human and environmental health worldwide. However, our knowledge of benthic HAB species is nascent: they have received considerably less attention than their planktonic counterparts. In particular, the processes that shape dynamics of benthic populations and facilitate the development of specific toxic species are still poorly understood. Along the North-western coasts of the Mediterranean Sea, recurrent toxic events associated with the development of the benthic dinoflagellate Ostreopsis cf. ovata have been observed over the last decade. They were associated with serious cases of human intoxication, with symptoms including skin irritations or broncho-constriction due to exposure to toxic marine aerosols. A better understanding of the environmental factors that control the growth of O. cf ovata populations can benefit both management strategies of alert systems and our understanding of ecosystem functioning in coastal waters of the Mediterranean Sea. Environmental conditions and bloom dynamics of O. cf. ovata were monitored during several years in the Bay of Villefranche-sur-mer (South of France). The main results coming from these field surveys will be presented. They include analysis of the potential influence of physical parameters (such as wind or temperature) in bloom dynamics, and consider the role of the inorganic and organic dissolved nutrient sources in fueling O. cf. ovata blooms. Interestingly, all these environmental factors appeared strongly linked during the summer season favorable to O. cf. ovata : strong wind conditions create sudden drops in water temperature and dissolved inorganic nutrient availability. Results showed a low dependence of O. cf. ovata growth on levels of concentration of inorganic nutrient sources. This suggests a potential importance of mixotrophic abilities of O. cf. ovata in the competitiveness of this species.

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∗Speaker

Ciguatera incidence in the Caribbean Islands

Jose Ernesto Mancera ∗ 1

1 ANCA Caribbean Network - Harmful Algae in the Caribbean and Adjacent Regions. Universidad

Nacional de Colombia (IOC-ANCA) – Universidad Nacional de Colombia Bogota, Colombia

The true extent of ciguatera and its impact on tourism and public health on the Caribbean is

poorly understood. For this reason, we analyze the incidence in the islands seeking spatial and temporal trends. We estimated per capita incidence of ciguatera through epidemiological reports obtained from the Caribbean Epidemiology Centre. The results show that over the period 1980- 2010, there were 10710 cases reported from 18 countries, with an average annual incidence of 42/100000. There was an increase between 1980-1990 and 2000-2010 with annual averages of 34.2 and 45.2 / 100 000, respectively. To determine the occurrence of epiphytic dinoflagellates in the coastal waters of Colombian Caribbean, we analyzed seagrass beds (Thalassia testudinium) in three marine reserved regions, founding 21 species belonging to seven genera of epiphytic dinoflagellates, some of these genera have been associated with toxins causing the Diarrehic Shellfish Poisoning and ciguatera. Prorocentrum lima, a cosmopolitan species distributed in tropical and temperate seas, characterized in different regions as toxin producers (okadaic acid and its analogs), is the most abundant and frequent of the 21 taxa observed in the epiphytic dinoflagellate community on the Turtle Grass. Considering that the development model of much of the region is based on the tourism industry and that fish is a major protein source for Caribbean communities, it can be concluded that ciguatera is a growing problem which is expected to increase in parallel with environmental change.

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∗Speaker

Ostreopsis blooms at the global scale: influence of macroalgal communities

Luisa Passeron Mangialajo ∗ 1,2, Rodolphe Lemee 2, Anna Fricke 1,

Daniela Catania 3, Sarah Kulins 4, Cecile Jauzein 5, Nick Shears 6

1 Universite Cote d’Azur (ECOMERS) – CNRS : FRE3729, Universite Cote d’Azur – Parc Valrose,

06108 Nice cedex 02, France 2 Laboratoire d’Oceanologie de Villefranche-sur-mer (LOV) – Sorbonne Universites, Universite Pierre et

Marie Curie (UPMC) - Paris VI, CNRS : UMR7093, INSU – Observatoire Oceanologique de Villefranche, 181 Chemin du Lazaret, 06230 Villefranche-sur-mer, France

3 Universite Cote d’Azur (ECOMERS) – Universit Cote dAzur, CNRS : FRE3729 – Parc Valrose,

06108 Nice cedex 02, France 4 Auckland University (Leigh Marine Laboratory) – Institute of Marine Science, The University of

Auckland, Auckland 0941, New Zealand 5 Laboratoire DYNECO-PELAGOS (DYNECO-PELAGOS) – Institut Francais de Recherche pour

l’Exploitation de la Mer (IFREMER) – Pointe du Diable, BP 70, 29280 Plouzane, France 6 Leigh Marine Laboratory – Institute of Marine Science, The University of Auckland, Auckland 0941,

New Zealand

Blooms of the genus Ostreopsis have been regularly recorded in recent decades in both hemispheres, with potential human health, economic and ecological consequences. Their ecology and ecosystem level impacts remain poorly known, also due to the difficulty of comparing data over wide spatial and temporal scales (the abundances of cells are generally expressed as cells/g of the host macroalgal species and therefore data are species-specific). Recently, new quantification methods have been conceived, allowing the quantification of benthic dinoflagellates abundances independently from the substrate (artificial substrates; BEDI, BEnthic Dinoflagellates Integrator).These new methods, together with the classical methods, have been applied in a coordinated way over the two hemispheres (Pacific Ocean and Mediter- ranean Sea), allowing the first comparison of blooms at the global scale. The quantification of Ostreopsis abundances independently from the substrates allowed testing the role of the complexity of macroalgal communities in facilitating blooms. Cell abundances were found to be highest in low complexity communities (foliose and turfing macroalgal habitats) and were considerably lower where dominant stands of fucoids and kelp (marine forests) were present. These results suggest a paramount role of macroalgal communities in facilitating blooms and that the loss of foundation canopy species may play a role in the increased occurrence of Ostreopsis blooms on temperate reefs at the global scale.

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∗Speaker

Taxonomic characterization of Gambierdiscus species in

Reunion Island, Indian Ocean

Alina Tunin-Ley∗ 1, Nicolas Chomerat 2, Gwenael Bilien 2, Jean

Turquet 1

1 HYDRO REUNION – HYDRO REUNION – BP 16 ZI Les Sables 97427 E TANG SALE , France

2 IFREMER (LER BO) – Institut Francais de Recherche pour l’Exploitation de la Mer (IFREMER) – Place de la Croix 29900 Concarneau, France

During the last decade, several phylogenetic studies have pointed out an unexpected species richness within the toxic dinoflagellate genus Gambierdiscus. They revealed that the genus exhibits in fact a great taxonomic diversity with distinctive biogeographical patterns, refuting the previously admitted idea of a single cosmopolite and largely predominant species, Gambierdiscus toxicus. Thus several new Gambierdiscus species were described these last years, and other ribotypes yet unnamed were identified. Nonetheless, the genus remains poorly studied in the Indian Ocean, where ciguatera is yet known in several endemic regions. Moreover the sparse works available were conducted before the description of several new species and the use of molecular tools. In order to improve our knowledge of the genus in this part of the world ocean, the present work aims to characterize the Gambierdiscus species in a French island of the Indian Ocean by additional taxonomic and phylogenetic analyses. In the present study, six strains have yet been collected from coral reefs lagoons in Reunion, and are successfully maintained in cultures in PHYTOBANK (Reunion). The morphology and thecal plate arrangement were investigated both with epifluorescence microscopy and scanning electron microscopy. In parallel, sequences of the large subunit of ribosomal DNA (D1-D3 and D8-D10 LSU rDNA) were successfully acquired for some of the strains. The preliminary results suggest that at least two distinct Gambierdiscus species are present in Reunion, genetically related to G. belizeanus and G. pacificus. In addition, the morphological characteristics of the strains, and more particularly the quite small size of the cells for most of them, seem to not match with G. toxicus features, according to Litaker’s revision of the genus.

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∗Speaker

Poster: My poster in 3 minutes

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∗Speaker

Metabolic interactions between Daphnia and Cyanobacteria

Gorenka Bojadzija ∗ 1, Enora Briand 1, Myriam Bormans 1, Linda

Lawton 2, Claudia Wiegand 1

1 Universite de Rennes 1, UMR ECOBIO 6553 (ECOBIO) – Universite de Rennes 1 – Campus de

Beaulieu 35042 Rennes, France 2 School of Pharmacy and Life Sciences, Sir Ian Wood Building Robert Gordon University – Garthdee

Road, Aberdeen AB10 7GJ, United Kingdom

Due to eutrophication freshwater ecosystems frequently experience cyanobacterial blooms,

many of which produce toxic and bioactive metabolites that can affect vertebrates and invertebrates life traits. While it has been suggested that cyanobacteria benefit from various secondary metabolites as a grazing defense against zooplankton, one focus of this project will be, if the presence of zooplankton will influence the production of cyanobacterial bioactive compounds. Furthermore, zooplankton are able to develop tolerance as a physiological response to cyanobacteria and their bioactive compounds, but only few of the mechanisms involved have been revealed so far. Qualitative changes in Daphnia detoxification and digestive enzymes contribute to the development of tolerance, however, this comes with energetic cost that in turn influence Daphnia life traits and may impair populations. The main aim of this project is a better understanding of the complexity of co-evolution, co-existence and mutual adaptation between cyanobacteria and their grazers. We will investigate the mutual physiological reactions of the common and well-studied Microcystis aeruginosa and its grazer D. magna. We hypothesize that: a) the presence of a predator (D. magna ) will induce a defense mechanism in both toxic and non-toxic M. aeruginosa via the production of specific bioactive compounds and b) the presence of toxic and non-toxic M. aeruginosa will affect physiological responses and life traits in D. magna. In order to disentangle mutual interactions, an innovative chamber that allows the exchange of the metabolites without direct contact will be used. Cyanobacterial growth, photosynthetic activity as well as metabolic profiling and the kinetics of cyanobacterial secondary metabolites will be monitored in the presence of Daphnia. In parallel, Daphnia ’s physiological and life trait responses will be recorded, including reproduction, biotransformation, oxidative stress, energy allocation and whole metabolomics response.

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∗Speaker

Transfer and fate of toxic cyanobacteria from freshwater to the marine

environment

Maxime Georges Des Aulnois ∗ 1, Amandine Caruana 1, Myriam Bormans 2, Zouher Amzil 1


d’Yeu - BP 21105 - 44311 Nantes Cedex 03, France 2 Ecosystemes, biodiversite, evolution (ECOBIO) – INEE, Universite de Rennes 1, CNRS : UMR6553,

Observatoire des Sciences de l’Univers de Rennes – Batiment 14 - Universite de Rennes 1 - Campus de

Beaulieu - CS 74205 - 35042 Rennes Cedex - France, France

Eutrophication of aquatic ecosystems combined with global warming lead to an increase

of the frequency and intensity of cyanobacterial blooms. These photosynthetic organisms produce a high number of bioactive molecules including a great diversity of cyanotoxins (microcystins, nodularins, anatoxins, cylindrospermopsins, saxitoxins and β-N-methylamino-L-alanine (BMAA)). Cyanobacterial harmful algal blooms (CyanoHABs) have been associated with cases of toxin bioaccumulation in animals and human poisonings. The ecology of freshwater cyanobacteria is well documented but less is known about the transfer and fate of freshwater CyanoHABs to the coastal environment. This thesis intends to assess the potential transfer of the toxic cyanobacteria risk via the river continuum into estuarine waters. It aims to better understand the survival of toxic freshwater cyanobacteria in estuarine waters and their impact on estuarine organisms. The objectives of the thesis are to: - Develop experiments under controlled conditions to determine the direct effects of abiotic factors (salinity, temperature) on growth and toxin production by toxic freshwater cyanobacteria in estuarine conditions. This will be performed with isolated strains of different genera and morphology. - Develop experiments to investigate the fate of toxic freshwater cyanobacteria and their toxins during the transfer from the freshwater to estuarine environment based on natural samples. - Develop experimental tests of marine bivalve contamination exposed to toxic freshwater cyanobacteria and assess the cyanotoxin bioaccumulation, in order to evaluate the potential contamination of estuarine organisms.

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∗Speaker

Impact of the global warming on the production of toxins and other

metabolites from Ostreopsis ovata

Marin-Pierre Gemin ∗ 1, Fabienne Herve 1, Rodolphe Lemee 2, Veronique

Sechet 1, Zouher Amzil 1


d’Yeu - BP 21105 - 44311 Nantes Cedex 03, France 2 Laboratoire d’oceanographie de Villefranche (LOV) – INSU, CNRS : UMR7093, Universite Pierre et

Marie Curie (UPMC) - Paris VI, Universite Pierre et Marie Curie [UPMC] - Paris VI – Observatoire Oceanologique Station zoologique 181, chemin du lazaret BP 28 06230 VILLEFRANCHE SUR MER

Cedex, France

During the last decade, recurrent proliferation of a toxic benthic dinoflagellate belonging to the genus Ostreopsis was observed along the Mediterranean coasts. The causative factors responsible for this proliferation have not yet been determined even if the global change is definitely involved. Indeed, the Mediterranean Sea is particularly susceptible to changes in temperature and light intensity which lead to variation of the water stratification and therefore alter the ecosystem functioning. Blooms of Ostreopsis generated public health problems with various symptoms: fever, rhinitis, sore throat, dry cough... Those symptoms are caused by palytoxin analogues, named ovatoxins (A to K) produced by Ostreopsis ovata. Otherwise, the metabolism of O.ovata is partially characterized and some unknown yet metabolites could be involved in the observed toxic effects. In this context, the first aim of the thesis is to optimize the extraction method of the ovatoxins and attempt to purify them from a first selected strain (MCCV 55) from Villefranche-sur-Mer (France). The second aim, is to isolate other active metabolites by fractionation, based on the use coupled method of biological test (Neuro-2a cytotoxic assay) and LCMS high resolution analysis. The third objective is to evaluate the influence of the temperature, light intensity and pH on growth, biomass production and intracellular content of a second selected strain (MCCV54) from the south of France. Here, the preliminary results of the optimization of ovatoxins extraction from culture cell then to determine the toxinic profile of different O.ovata strains.

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∗Speaker

In vitro neurocutaneous effects of ciguatoxin P-CTX-2: evidence for a significant role for

keratinocytes

Killian L’herondelle∗ 1, Reginald Philippe 2, Olivier Mignen 2, Richard J.

Lewis 3, Laurent Misery 1,4, Raphaele Le Garrec 1

1 Laboratoire des Interactions Epithelium-Neurone (LIEN) – Universite de Bretagne Occidentale

(UBO) : EA4685 – UFR medecine, 22 rue Camille Desmoulins, 29200 Brest, France 2 Genetique, genomique fonctionnelle et biotechnologies (U1078) – Inserm : U1078, Universite de

Bretagne Occidentale (UBO) – UFR medecine, 22 rue Camille Desmoulins, 29200 Brest, France 3 Institute for Molecular Bioscience (IMBQ) – The University of Queensland, St. Lucia, Queensland

4072, Australia 4 Service de Dermatologie – CHU Brest – 2 avenue Foch, 29200 Brest, France

Ciguatera is an intoxication caused by the ingestion of fish contaminated with ciguatoxins

(CTXs). Characteristic clinical signs include cutaneous sensory disorders such as cold-induced dysesthesia and a severe pruritus that persists for several weeks or even years. The main primary target of the CTXs is the voltage-gated sodium channel, especially in non-myelinated and thinly myelinated sensory fibers. By activating this channel at resting membrane potential, CTXs cause neuronal hyperexcitability which is the basis of clinical sensory disturbances. How- ever, the molecular events and mediators downstream of this activation, whose identification would allow to develop targeted treatments, are little known. Pruritus (or itch) is an abnormal sensation restricted to the skin that leads to the need to scratch. It is due to the activation of cutaneous sensory nerve endings through channels or receptors expressed on their surface, with the contribution of neighboring cells in the skin, including keratinocytes, in the promo- tion or facilitation especially of chronic pruritus. Recently, we showed that P-CTX-2 induces the release of the neuropeptides responsible for neurogenic inflammation, SP and CGRP, from an in vitro model of rat neurons cocultivated with human keratinocytes. Here, using calcium experiments, we show that keratinocytes can be directly activated by P-CTX-2 and that, in the coculture, they exert a synergistic effect on the P-CTX-2-induced SP release. In addition, pharmacological antagonists targeting receptors involved in pruritus pathophysiology were used to study the cellular and molecular mechanisms underlying P-CTX-2 effects. Our results point out keratinocytes as new cellular targets for CTXs. Moreover, they bring new molecular insights in the pathophysiology of sensory disorders in ciguatera with potential therapeutic applications.

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∗Speaker

Preliminary results of a quantitative risk assessment during blooms of Alexandrium

minutum in Brittany

Mathias Lunghi ∗ 1, Nathalie Arnich 2, Caroline Fabioux 3, Malwenn

Lassudrie 3, Philippe Soudant 3, Helene Hegaret 3, Anne Thebault 4

1 Direction Evaluation des Risques (ANSES) – Anses – 14 rue Pierre et Marie Curie batiment Copernic

94701 MAISONS-ALFORT Cedex, France 2 DER-UERALIM (ANSES) – Anses – 14 rue Pierre et Marie Curie batiment Copernic 94701

MAISONS-ALFORT Cedex, France 3 Laboratoire des Sciences de l’Environnement Marin (LEMAR) – CNRS : UMR6539, Universite de


Technopole Brest-Iroise, Place Nicolas Copernic, 29280 Plouzane, France 4 ANSES-DER-UME (ANSES) – Anses – 14 rue Pierre et Marie Curie batiment Copernic 94701

MAISONS-ALFORT Cedex, France

Shellfish areas of Brest (Brittany, France) are affected by blooms of Alexandrium minu-

tum and have been closed several times since 2012 due to levels above the regulatory limit. In order to better estimate the health risk associated with saxitoxin (STX) and its analogues in shellfish, we built a scenario to quantify the proportion of affected people from a population of 1000 exposed individuals if there were no management measures to avoid exposure to contaminated oysters. For that purpose, we studied one of the most contaminated areas in the bay of Brest. We considered Elisa results as a proxy of oysters toxic content during the blooms, human consumption data from three different databases and a dose-response relationship based on a systematic review of human cases. Variability between oysters contamination by STXs as well as the diploid/triploid status were taken into account in 2013, whereas only variability between diploids was considered during blooms in 2013 and 2014. A quantitative probabilistic model was carried out to estimate the health risk during the 2013 bloom for an adult coastal population of oyster consumers. This work is preliminary considering that it does not take into account: - uncertainty between Elisa results and reference methods such as mouse bioassay and LC- MS ; - decrease of the bloom, that was not covered by the sampling; - the fact that consumption data are based on old databases (INCA99, CALIPSO2005, France AgriMer 2008); - variability of exposure and severity of symptoms in the dose-response relationship.

This work is the first probabilistic modelling approach applied to STXs going from the environment to human cases.

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∗Speaker

In vitro genotoxic and cytotoxic effects of microcystins (-LR, -RR and -LF) revealed

by the micronucleus assay and high content analysis in the human HepaRG

cell line.

Rym Merabet ∗ 2,1, Kevin Hogeveen 2, Rachelle Lanceleur 2,

Djamel-Eddine Benouareth 3, Valerie Fessard 2

2 French Agency for Food and Safety, Toxicology of Contaminants Unit (ANSES) – Anses – 10 B rue

Claude Bourgelat, 35133 Javene, France, France 1 Department of Biology, SNVSTU faculty, LBEE- 8 Mai 1945 University Guelma – BP 401 GUELMA

24000, Algeria 3 Department of Biology, SNVSTU faculty, LBEE- 8 Mai 1945 University Guelma – BP 401 GUELMA

24000, Algeria

Microcystins (MCs) are cyclic hepatotoxins produced by various species of cyanobacteria.

Their structure includes two variable amino acids leading to more than 100 variants. While MC-LR is a potent tumor promoter by which inhibits protein phosphatase activities, increasing evidences suggest that the MC-LF variant might be even more toxic. The aim of the current study was to characterize the cytotoxicity and the genotoxicity of microcystins congeners MC- LR, -RR and -LF using high content analysis (HCA) and in vitro cytokinesis-block micronucleus (CBMN) assay in the human hepatoma cell line HepaRG. Undifferentiated HepaRG cells were processed for high content analysis after 24 hours of treatment with concentrations ranging from 0.39 to 50 µM and from 0.05 to 6.25 µM for MC-LF. A total of 6 cellular markers were evaluated for viability, apoptosis and DNA damage (cell count, active Caspase-3, γH2AX, phospho H3, p53 phospho S15 and ATM phospho S1981). A significant increase in each of these markers was observed which was correlated with the onset of cytotoxicity. The results also suggest that MC-LF elicited cytotoxic effects at lower concentrations compared to MC-LR and MC-RR. We also tested their potency for induction of micronuclei (MN). Following a 24 hour treatment of differentiated HepaRG cells with MC-LR and MC-RR to a dose range from 1.56 – 25 µM, a significant increase of MN was observed dose dependently with the two congeners. These findings provide key information in the mechanism of action of microcystin congeners.

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∗Speaker

Monitoring cyanobacterial blooms using satellite remote sensing and modeling

techniques. Application to Karaoun Reservoir, Lebanon

Najwa Sharaf ∗ 1,2,3

1 Ecole des Ponts ParisTech (ENPC) – E cole des Ponts ParisTech (ENPC) – E cole des Ponts ParisTech

6-8 avenue Blaise-Pascal Cite Descartes 77455 Champs-sur-Marne Marne-la-Vallee cedex 2, France 2 National Center for Remote Sensing, National Council for Scientific Research (CNRS) – P.O. Box

11-8281, Riad El Solh, 1107 2260 Beirut, Lebanon, Lebanon 3 Laboratory of Microorganisms and Food Irradiation, LAEC, Lebanese Atomic Energy Commission, National Council for Scientific Research (CNRS) – P.O. Box 11-8281, Riad El Solh, 1107 2260 Beirut,

Lebanon, Lebanon

Cyanobacterial blooms deteriorate the water quality and impair the use of lakes and reservoirs. They also threaten human health through toxin production. Therefore, continuous monitoring of cyanobacterial biomass is critical for bloom detection and further development of early warning systems. Satellite remote sensing was proven to be an effective tool for identifying and monitoring phytoplankton blooms across the whole lake surface. Recent research has focused on quantifying cyanobacterial biomass using the reflectance of phycocyanin, a unique cyanobacteria pigment. Karaoun Reservoir, the largest freshwater body in Lebanon, used mainly for hydroelectricity and irrigation, has been suffering from regular toxic cyanobacterial blooms, at least since 2009. Aiming at monitoring cyanobacterial biomass in space and time, we developed an algorithm for retrieving phycocyanin surface concentration in Karaoun Reservoir from the reflectance measured by Landsat 8 Operational Land Imager (OLI). To do this, we combined OLI images with simultaneous in situ surface phycocyanin concentration with a fluorescence TriOS probe. After validation, a dark-object-subtracted band ratio algorithm was found to be the most robust for predicting high and low phycocyanin concentrations (R2=0.87). It outper- formed previously published Landsat phycocyanin algorithms. It can be also applied to other eutrophic lakes, however with further validation. Modeling can complement the analysis of satellite imagery and makes it possible to estimate depth profiles and to interpolate temporal gaps in data derived from satellite and traditional water quality monitoring. Therefore, the objective of our current research is to integrate remote sensing and a coupled three dimensional hydrodynamic-ecological model (Delft3D suite, Deltares) in order to better monitor and understand the dynamics of cyanobacterial blooms in Karaoun Reservoir. This integration, rare for harmful algal blooms in freshwaters, will present a new perspective for water quality monitoring. It will also be implemented as a tool for building an early warning system.

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Poster

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∗Speaker

First demonstration that Prorocentrolide A, from cultured Prorocentrum lima

dinoflagellates collected in Japan, blocks nicotinic acetylcholine receptors

Muriel Amar ∗ 1, Romulo Araoz 1,2, Bogdan I. Iorga 3, Takeshi Yasumoto 4,

Denis Servent 1, Jordi Molgo ∗ 1,2

1 Institut de Biologie et de Technologies de Saclay (IBITECS) – CEA – CEA Saclay, bat 152, 91191 Gif

sur Yvette, France 2 Institut des Neurosciences Paris-Saclay (Neuro-PSI) – CNRS : UMR9197 – UMR 9197

CNRS/Universite Paris-Sud, 91198 Gif-sur-Yvette, France 3 Institut de Chimie des Substances Naturelles (ICSN) – CNRS : UPR2301 – CNRS, UPR 2301, Labex

LERMIT, 91198 Gif-sur-Yvette, France 4 Japan Food Research Laboratories – Nagayama, Tama, Tokyo, Japan

Several marine dinoflagellates species of the genus Prorocentrum are well known for produc-

ing lipid-soluble toxins that are responsible for diarrheic shellfish poisoning. Extracts obtained

from cultured Prorocentrum lima dinoflagellates, collected in Sesoko Island, Okinawa, Japan,

allowed the first chemical identification of a prorocentrolide. Further studies using bioassay-

guided fractionation of extracts of the dinoflagellate P. maculosum Faust, revealed the presence of

prorocentrolide B in those extracts. These prorocentrolides produced fast-toxicity in mouse

bioassays with symptoms that were completely different from those reported with diarrheic

toxins. Prorocentrolides are members of the cyclic imine phycotoxins family. Their chemical

structure includes a 26-membered carbo-macrocycle and a 28-membered macrocyclic lactone ar-

ranged around a hexahydroisoquinoline that incorporates the characteristic cyclic imine group.

Six prorocentrolides are already known which differ by their substituents. However, their mode of

action remains unknown. The aim of the present work was to determine whether prorocentrolide

A acts on nicotinic acetylcholine receptors (nAChRs), like other members of the cyclic imine

family of toxins. For this, we used conventional competition-binding assays and electro-

physiological techniques. Prorocentrolide A displaced [125I]α-bungarotoxin binding to Torpedo

membranes, expressing the muscle-type (α12β1γδ) nAChR, and in HEK-293 cells, expressing the

chimeric chick neuronal α7-5HT3 nAChR. Also, prorocentrolide A displaced [3H]epibatidine

binding to human α3β2 and α4β2 nAChRs. Functional studies revealed that prorocentrolide A

had no agonist action on nAChRs, but inhibited in a dose-dependent manner acetylcholine

(ACh)-induced currents in Xenopus oocytes that had incorporated to their membranes the Tor-

pedo α12β1γδ nAChR, or that expressed the human α7 nAChR, as revealed by voltage-clamp

recordings. In conclusion, this study is the first to show that prorocentrolide A acts on both

muscle and neuronal nAChRs, but with higher affinity for the muscle-type nAChR. Structural

factors responsible for activity are discussed based on molecular docking and modeling.

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∗Speaker

Physico-chemical and functional

characterization of Portimine purified from Vulcanodinium rugosum strain

IFR-VRU-01

Romulo Araoz ∗ 1,2, Claire Lamoise 1,2, Amandine Gaudin 2, Philipp Hess 3,

Denis Servent 2

1 CNRS/ Insitut de Neurosciences Paris- Saclay, UMR9197 – CNRS : UMR9197 – 1 avenue de la

Terrasse 91198 Gif sur Yvette, France 2 CEA/ DRF/ SIMOPRO/ LTMB/ Toxines recepteurs et canaux ioniques – CEA – CEA, Saclay, Bat.

152 91191 Gif sur Yvette, France 3 Ifremer, Laboratoire Phycotoxines, Centre Atlantique, 44311 Nantes Cedex, France. – Institut

Francais de Recherche pour l’Exploitation de la Mer (IFREMER) – France

Vulcanodinium rugosum, found in the Mediterraneen lagoon of Ingril (France) and in the

coastal zones of New Zealand, Australia Japan and China, is now spreading through maritime

commerce. While French V. rugosum isolate produces pinnatoxin-G and South Pacific V. ru-

gosum strains produce pinnatoxin-E, F, G and H, the novel V. rugosum isolate isolated from a

ship’s ballast tank in USA mainly produces portimine, a toxin that is common to several V.

rugosum isolates. Portimine is a polycyclic ether toxin containing a five-carbon imine ring (m/z

402.22804). Although less toxic to mouse by intraperitoneal injection than other cyclic imine

toxins, portimine exhibits potent cytotoxicity against different cell lines. We have found for the

first time that V. rugosum (French isolate) also produces portimine. Bio-guided purification of

portimine was performed using a non-radioactive receptor-binding assay. We have purified

Milligrams of portimine by preparative HPLC and characterized it by HRMS and 2D-NMR.

Portimine was quantified by NMR, using chloroform and benzene as internal standards. Two-

electrodes voltage clamp recordings on muscle and neuronal nAChRs showed that portimine

acts as antagonist of nicotinic acetylcholine receptors.

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∗Speaker

Electrophysiological evaluation of Pacific oyster (Crassostrea gigas) sensitivity to

paralytic shellfish toxins

Floriane Boullot 1, Caroline Fabioux 1, Helene Hegaret 1, Pierre Boudry 1,

Philippe Soudant 1, Evelyne Benoit ∗ 2

1 Laboratoire des Sciences de l’Environnement Marin (LEMAR) – Universite de Bretagne Occidentale

(UBO), Institut de Recherche pour le Developpement, CNRS : UMR6539, Institut Francais de Recherche pour l’Exploitation de la Mer (IFREMER) – Technopole Brest-Iroise, Place Nicolas

Copernic, 29280 Plouzane, France 2 DRF/iBiTec-S/SIMOPRO – CEA – CEA de Saclay, and Institut des Neurosciences Paris-Saclay

(Neuro-PSI), UMR 9197 CNRS/Universite Paris-Sud, 91190 Gif-sur-Yvette, France

Pacific oysters (Crassostrea gigas ) may bio-accumulate high levels of paralytic shellfish tox-

ins (PST) during harmful algal blooms of the genus Alexandrium that regularly occur in coastal waters, affecting their health and marketability. PST are composed of saxitoxin (STX) and analogues which, similarly to tetrodotoxin (TTX), block voltage-gated sodium channels and thus inhibit action potentials in excitable cells. The aim of our study was to analyse the PST- sensitivity of Pacific oysters in relation with their toxin bio-accumulation. The STX and TTX sensitivity of cerebrovisceral nerves isolated from oysters was evaluated using an electrophysiological approach, and the PST content in their digestive gland was measured using biochemical analyses. The compound nerve action potential (CNAP) of wild oysters collected on April 2014 was about 5-fold more sensitive to STX than that of oysters collected on October 2015, suggesting seasonal variability in STX sensitivity. Additionally, the STX-sensitivity of CNAP recorded from oysters experimentally fed A. minutum (toxic alga-exposed oysters) or Tisochrysis lutea, a non-toxic microalga (control oysters) revealed that they could be separated into two groups, resistant and sensitive, independently of their diet. Moreover, the percentage of toxin-sensitive nerves was lower and the STX concentration necessary to inhibit 50% of their CNAP was higher in toxic alga-exposed than control oysters. This supports the hypothesis that oysters exposed to PST are relatively more resistant to STX than non-exposed ones. However, no obvious correlation was observed between nerve sensitivity to STX and the PST content in oyster digestive gland, despite an individual variability in the level of bio-accumulated toxins. None of the nerves isolated from wild and farmed oysters was detected to be TTX-sensitive. In conclusion, C. gigas nerves are shown, for the first time, to have micromolar range of STX sensitivity, which shows seasonal variability and decreases when oysters are exposed to dinoflagellates producing PST.

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∗Speaker

Chemically mediated interactions between Microcystis and Planktothrix: impact on

their physiology and metabolic profiles

Enora Briand ∗ 1, Sebastien Reubrecht 1, Florence Mondeguer 2,

Manoella Sibat 2, Philipp Hess 2, Zouher Amzil 2, Myriam Bormans ∗ 1

1 ECOBIO – universite Rennes 1 – Rennes, France 2 Laboratoire Phycotoxines – Institut Francais de Recherche pour l’Exploitation de la Mer (IFREMER)

– Nantes, France

Freshwater cyanobacteria are well known for their ability to produce a wide variety of bioac-

tive compounds, some of which have been described as allelochemicals. There is growing evidence that these secondary metabolites play an important role in shaping community composition through biotic interactions; however, for the most part, their biological role and mode of regulation of the production are poorly understood. In temperate eutrophic freshwaters, Microcystis and Planktothrix often co-occur, with Planktothrix being an early colonizer and Microcystis ap- pearing subsequently. We tested if the production of a range of peptides by co-existing species could be regulated through interspecific interactions. Using a combined approach of co-cultures and analyses of metabolic profiles, we investigated chemically mediated interactions between two cyanobacteria, M. aeruginosa and P. agardhii. More precisely, we evaluated changes in growth, morphology and metabolites production and release by both interacting species. In- terestingly, culturing Microcystis cells with Planktothrix resulted in a reduction of the growth of Planktothrix together with a decrease of its filament size and alterations in the morphology of its cells. However, the production of specific intracellular compounds by Planktothrix was not different between mono and co-culture conditions. Concerning Microcystis, the number of specific intracellular compounds was higher under co-culture condition than under monoculture. In general, Microcystis produced a lower number of intracellular compounds under monoculture than Planktothrix, and a higher number of compounds than Planktothrix under co-culture condition. Our investigation did not allow us to identify specifically the compounds involved in the observed physiological and morphological changes of Planktothrix cells. However, altogether, these results suggest that specific compounds produced by Microcystis in the presence of Planktothrix have been specifically produced as potential allelochemicals.

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∗Speaker

Mechanisms of irreversible foam-forming blooms with cyanobacterias: a

quantitative study

Philippe Brunet ∗ 1, Julien Dervaux 1

1 Laboratoire Matiere et Systemes Complexes (MSC) – CNRS : UMR7057, Universite Paris Diderot -

Paris 7 – 10 rue Alice Domon et Leonie Duquet 75013 Paris, France

In response to natural or anthropocentric pollutions coupled to global climate changes, microorganisms from aquatic environments can suddenly accumulate on water surface. These dense suspensions, known as blooms, are harmful to ecosystems and significantly degrade the quality of water resources. In order to determine the physico-chemical parameters involved in their formation and quantitatively predict their appearance, we successfully reproduced irreversible cyanobacterial blooms in vitro. By combining chemical, biochemical and hydrodynamic evidences, we identify a mechanism, unrelated to the presence of internal gas vesicles, allowing the sudden collective upward migration in test tubes of several cyanobacterial strains (Microcystis aeruginosa PCC 7005, Microcystis aeruginosa PCC 7806 and Synechocystis sp. PCC 6803). The final state consists in a foamy layer of biomass at the air-liquid interface, in which microorganisms remain alive for weeks, the medium lying below being almost completely depleted of cyanobacteria. These ”laboratory blooms” start with the aggregation of cells at high ionic force in cyanobacterial strains that produce anionic extracellular polymeric substances (EPS). Under appropriate conditions of nutrients and light intensity, the high photosynthetic activity within cell clusters leads the dissolved oxygen (DO) to supersaturate and to nucleate into bubbles. Trapped within the EPS, these bubbles grow until their buoyancy pulls the biomass towards the free surface. By investigating a wide range of spatially homogeneous environmental conditions (illumination, salinity, cell and nutrient concentration) we identify species-dependent thresholds and timescales for bloom formation. We conclude on the relevance of such results for cyanobacterial bloom formation in the environment and we propose an efficient method for biomass harvesting in bioreactors.

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∗Speaker

First report of Pseudo-nitzschia plurisecta in French Atlantic coastal waters

Amandine Caruana ∗ 1, Nour Ayache 1, Virginie Raimbault 1, Michael

Retho 2, Fabienne Herve 1, Zouher Amzil 1, Nicolas Chomerat 3


d’Yeu - BP 21105 - 44311 Nantes Cedex 03, France 2 Laboratoire Laboratoire Environnement Ressources Morbihan Pays de Loire (LER/MPL) – Institut

Francais de Recherche pour l’Exploitation de la Mer (IFREMER) – 12, rue des resistants - B.P. 86

56470 La Trinite sur Mer, France 3 Laboratoire Environnement Ressources Bretagne Occidentale (LER BO) – Institut Francais de

Recherche pour l’Exploitation de la Mer (IFREMER) – Station de biologie marine Place de la Croix BP

40537 29185 CONCARNEAU CEDEX, France

The presence of Pseudo-nitzschiza plurisecta is reported for the first time in French Atlantic

coastal waters and the domoic acid production is directly shown. This species was described by Orive and co-workers in 2013 after isolation on the Spanish Atlantic coast. Prior to its formal identification, an American species belonging to the same clade was meanwhile isolated from the Gulf of Maine and was shown to produce domoic acid. P. plurisecta was later listed within domoic acid-producing species but with unclear reference due to the absence of its name. We isolated three strains of P. plurisecta from the Vilaine estuary in June 2015. They were all identified by molecular tools using Intergenic Transcribed Spacers (ITS) regions of the rDNA and transmission electron microscopy (TEM) was used on one strain to observe the fine morphological characters of the frustules and confirm the taxonomic identification. Batch cultures of P. plurisecta allowed investigating the pigment profile that is distinguished by a dominance of Chl c3 over c2. All the three strains of P. plurisecta were able to produce domoic acid in the range 3 - 59 fg cell-1. Consequently, we confirmed that P. plurisecta really represents a risk for Amnesic Shellfish Poisoning in human and thus, is worth being listed as a toxic Pseudo-nitzschia species.

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∗Speaker

Identify toxic micro-algae. Longitudinal study of the representation of

efflorescence through visual alerts

Simona Cojocariu ∗ 1, Elisabeth Guillou ∗ 2

1 Universite de Bretagne Occidentale (UBO) – Universite de Bretagne Occidentale [UBO] – 20 rue

Duquesne - CS 93837 29238 Brest cedex 3, France 2 Laboratoire de Psychologie : Cognition, Comportement, Communication - EA 1285 (LP3C) –

Universite de Bretagne Occidentale [UBO] : EA1285 – 20 rue Duquesne - CS 93837 29238 Brest cedex

3, France

In a participatory research program (Phenomer), we are interested in the phenomenon of

efflorescence. Since 2013, a multidisciplinary research, coordinated by Ifremer, has appealed to the public for signaling of all phenomena unusual in the appearance of seawater. In this program, the psychosocial approach aims to study the knowledge that the observers have of the phenomenon, the way in which they conceive it and possibly act toward it. This knowledge of ”common sense” comes from the appropriation of ”scientific” knowledge, and is then considered as a social representation. It is a form of social thought, anchored in a culture and associated with practices, which allows individuals to understand their environment, communicate and act towards it. In this research, a longitudinal study was carried out to. In the first year, 13 semi-directive interviews were conducted by telephone with people who reported and agreed to be re-contacted for an interview. The first results were used to build a questionnaire adapted to quantitative monitoring. It consists of 10 questions about the context in which the observation was made, the past experience of individuals about efflorescence, their feelings about this phenomenon, and how it is likely impact their environment. The results presented will cover three years of follow-up. In particular, they point out that observers are a well-informed public, the vast majority have already heard of the phenomenon of efflorescence. Nevertheless, and this is one of the main reasons for their participation, they know moderately the functioning of the phenomenon. Their description is therefore intended to help PHENOMER, but also to understand the observed phenomenon, its consequences on humans and environment. It is a targeted public, concerned about certain environmental issues, with an ecocentric profile, the consequences of the phenomenon being perceived more harmful for the environment than for man.

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∗Speaker

Pinnatoxins A and G block neuromuscular transmission and nicotinic acetylcholine receptors in vertebrate skeletal muscles

Aurélie Couesnon 1, Jirí Lindovsky 1, Romulo Araoz 1,2, Armen Zakarian 3, Sophie Creuzet 1, Denis Servent 2, Jordi Molgo 1,2, Evelyne Benoit 1,2

1 : Institut des Neurosciences Paris-Saclay (Neuro-PSI) CNRS : UMR 9197 CNRS/Université

Paris-Sud, 91198 Gif-sur-Yvette - France

2 : Service d'Ingénierie Moléculaire des Protéines (DRF/iBiTec-S/SIMOPRO) CEA de Saclay,

Université Paris-Saclay, 91191 Gif-sur-Yvette - France

3 : Department of Chemistry and Biochemistry University of California Santa Barbara, CA 93106 -

États-Unis

Pinnatoxins constitute an emerging class of neurotoxins belonging to the cyclic imine group that contaminates shellfish. In recent years, several approaches for the total synthesis of pinnatoxins were developed. We have analyzed the action of synthetic pinnatoxins A and G on nerve-evoked muscle contraction and synaptic transmission, using neuromuscular preparations isolated from adult mouse and chick embryos. In addition, the local effect of the toxins was investigated on the neuromuscular system of anesthetized mice. Pinnatoxins A and G, at nanomolar concentrations, reduced the amplitude of isometric contractions of adult mouse muscles following single or tetanic stimulation of the motor nerve. Similarly, pinnatoxin A efficiently blocked nerve-evoked tetanic contractions in the quadratus muscle of chick embryos. Also, the toxins reduced the amplitude of endplate potentials, in such a manner that they could not reach the threshold for opening voltage-gated sodium channels and trigger a muscle action potential. Consistent with such action, the amplitude of compound muscle action potentials recorded in anesthetized mice was reduced in a time- and dose-dependent manner and in a reversible way. In Xenopus oocytes that have incorporated muscle-type α12β1γδ nicotinic acetylcholine receptors on their membranes, pinnatoxins A and G decreased the peak amplitude of acetylcholine-elicited currents in a concentration-dependent manner with similar potency (mean IC50 values of 5.5 and 3.8 nM, respectively, as determined in voltage-clamp experiments; Araoz et al., 2011; Bourne et al., 2015). It is concluded that mature (α12β1δε) and embryonic (α12β1γδ) muscle-type nicotinic acetylcholine receptors are important targets of pinnatoxins. This research was supported in part by grant AQUANEUROTOX (ANR-12-ASTR-0037 to JM and DS) and by the National Institutes of Health (USA, grant NIGMS R01 GM077379 to AZ) NOTE:

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∗Speaker

ANOCIBIOTOX – Non targeted analysis of marine biotoxins in fishery products from

mainland France and French overseas territories

Ines Dom ∗ 1, Ronel Bire ∗ 1, Thierry Guerin 1

1 Laboratoire de securite des aliments de Maisons-Alfort – Anses – 14 rue Pierre et Marie Curie - 94701

Maisons-Alfort Cedex, France

Considering the phenomenon of geographical expansion of marine biotoxins, the emergence of

new toxins and the associated risk for human health, it is necessary to have versatile and efficient analytical methods able to detect a range, as wide as possible of known or emerging toxins. Technological advances make it possible to have more sensitive and faster equipment, with a better analytical specificity. This is the case of liquid chromatography-mass spectrometry. Due to the large number of marine biotoxins and their extensive range of polarity, current detection methods mainly focus on a toxin family or a group of toxins of the same polarity. In addition, these methods targeting known toxins are not able to detect emerging compounds. The ANOCIBIOTOX project aims to meet the expectations in terms of investigation of known or emerging phenomena, by implementing a non-targeted analytical approach to detect marine biotoxins. The rationale behind this approach also lays in its potential to enable retrospective analysis of acquisition data to detect contamination trends. The bigdata generated in high resolution mass spectrometry is analysed/treated using chemometric tools to compare the toxin profile of an unknown sample with that of a reference (sample, database) and thereby identify the compounds responsible for the difference in terms of toxin profile.

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∗Speaker

An in vitro approach to assess the impact of Dinophysis sp. on oyster Crassostrea gigas

reproduction

Helene Hegaret ∗ 1, Justine Castrec 1, Myrina Boulais ∗ 2, Nelly Le Goıc ∗ 1,

Veronique Sechet ∗ 3, Virginie Raimbault ∗ 3, Caroline Fabioux ∗ 1

1 Laboratoire des Sciences de l’Environnement Marin (LEMAR) – CNRS : UMR6539, Universite de Bretagne Occidentale (UBO), Institut Universitaire Europeen de la Mer (IUEM), Institut de Recherche

pour le Developpement, Institut Francais de Recherche pour l’Exploitation de la Mer (IFREMER) – Technopole Brest-Iroise, Place Nicolas Copernic, 29280 Plouzane, France

2 University of North Carolina Wilmington – Center for Marine Sciences, 5600 Marvin K. Moss Lane,

Wilmington, NC 28409, USA, United States 3 Institut Francais de Recherche pour l’Exploitation de la Mer - Laboratoire Phycotoxines Nantes

(IFREMER) – Institut Francais de Recherche pour l’Exploitation de la Mer (IFREMER) – Rue de l’Ile d’Yeu - BP 21105 - 44311 Nantes Cedex 03, France

Since the first toxic episode of 1983, the French coastal waters regularly experience proliferations of Dinophysis sp. Some species of the genus Dinophysis can synthesize lipophilic toxins responsible for Diarrheic Shellfish Poisoning (DSP) in consumers who ate contaminated shellfish. These toxic blooms generally occur during the reproduction season of most bivalves and may have an impact on the development of gametes, but also on spawning, fertilization success, larval development or recruitment of juveniles. The aim of this project was thus to investigate the effects of Dinophysis on cellular characteristics of gametes of an ecologically and economically important bivalve species: the Pacific oyster, Crassostrea gigas. Two species of Dinophysis, D. sacculus and D. accuminata were grown in L1 enriched natural seawater at concentrations close to those observed in the field, and then used for in vitro incuba- tion of oyster gametes (spermatozoa and oocytes). After 4 h of exposure, morphology, mortality and production of reactive oxygen species (ROS) of spermatozoa and oocytes were assessed by flow cytometry. Motility of spermatozoa, and activity of the ABC proteins of oocytes were also measured.

The results show an increased mortality and decreased percentage of motile spermatozoa and velocity of oyster spermatozoa directly exposed to both Dinophysis species, as well as a decrease in ROS production of oyster oocytes. This study clearly demonstrates the effects of two species of Dinophysis on the physiological responses of gametes and thus their quality, reflecting the potential impact that Dinophysis blooms can have on the recruitment of bivalves during the reproduction season.

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∗Speaker

Study of the relationship between toxin synthesis, photosynthesis and

photoprotection mechanisms in order to better understand the regulation of

microcystin synthesis and their possible protective role in different Planktothrix

strains

Feilke Kathleen ∗ 1, Diana Kirilovsky ∗1

1 I2BC – I2BC – Institut de Biologie Integrative de la Cellule (I2BC) Batiment 21 Avenue de la

Terrasse 91190 GIF-SUR-YVETTE, France

Cyanobacteria have developed physiological mechanisms that allow them to rapidly respond to

environmental changes and to acclimate to them leading under specific environmental conditions to toxic blooms hazardous for human and animal health. Among them, the microcystins (MC) are the most common toxins. A protectant role for MC in stress was suggested, but it remains unclear if the protection is a direct effect of the toxin or if a special photoprotective mechanism is up-regulated by the presence of toxins. This project studies the relationship between toxin synthesis, photosynthesis and photoprotection mechanisms to better understand their possible protective role. We focus on MC free and MC producing Planktothrix strains. Rapid acclimation processes to high light stress involve conformational changes and/or a reor- ganization of the photosynthetic complexes (e.g. orange carotenoid protein (OCP)-related non- photochemical quenching (NPQ) mechanism and the state transitions (ST)), while synthesis and/or degradation of proteins and protein complexes are slower responses (e.g. photoinhibition: photosystem II (PSII) D1 protein degradation). The rapid photoprotective mechanisms existing in Planktothrix rubescens and Planktothrix agardhii are studied concentrating on the two photoprotective mechanisms, OCP-related NPQ and state transitions. Preliminary results using fluorescence measurements show that the green Planktothrix agardhii strains show a faster recovery after OCP-induced NPQ compared to the phycoerythrin-rich red Planktothrix rubescens strains and a higher induction of state transitions independently of the presence or absence of cyanotoxins. This might be explained by the occurence of the green Planktothrix agardhii strains proliferating in eutrophic water bodies characterized by higher light intensities and fluctuations compared to red Planktothrix rubescens strains occuring in deep, stratified mesotrophic lakes. Moreover, preliminary results studying photsystem II photoinhibition during high light stress by measuring PSII activity with fluorescence and oxygen evolution show no significant differences between MC free and MC producing Planktothrix strains.

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∗Speaker

The cyrtophora ciliates as potential regulators of filamentous cyanobacteria

dynamics and models to study relationships between grazers and

toxin-producing species

Benjamin Legrand 1,2, Delphine Latour ∗ 1, Bernard Vigues 1


des Cezeaux, 24, avenue des Landais BP 80026 63 170 AUBIERE, France 2 Athos-environnement – Universite Blaise Pascal - Clermont-Ferrand II – 112 avenue du Brezet 63100

Clermont-Ferrand, France

Among cyanobacteria grazers, several species of ciliated Protozoans, comprising the cyr-

tophora, are highly specialized in feeding on the filamentous cyanobacteria. These ciliates are endowed with a cytopharyngeal basket adapted for attachment, ingestion and cutting long filaments. A model has been constructed to explain the way in which the filamentous cyanobacteria, enclosed in the food vacuole membrane, may be transported through the basket into the cytoplasm. According to this model, mechanical interaction between cytoskeletal elements of the basket and the food vacuole membrane, propels the latter through the basket. The food vacuole subsequently pinches-off from the cytopharynx, moves into the cytoplasm and becomes condensed by endocytosis of primary lysosomes. The ability of cyrtophora to ingest whole cyanobacterial filaments may be a direct way to limit trophic dead-ends induced by cyanobacterial filamentous proliferations. Cutting long filaments may also produce filaments with more suitable length for non-selective predators among zooplankton organisms. Furthermore, trophic interaction between ciliates and cyanobacteria raises interesting questions, especially regarding to the cyanotoxins control, as numerous filamentous cyanobacteria are able to produce these toxic molecules. In another hand, it might be of interest to know whether akinetes can constitute resistant cells even when ingested by the ciliates.

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∗Speaker

Akinetes distribution and persistence trough the benthic phase in order to evaluate their potential of germination

Benjamin Legrand 1,2, Anne-Helene Le Jeune 1, Jonathan Colombet 1,

Delphine Latour ∗ 1


des Cezeaux, 24, avenue des Landais BP 80026 63 170 AUBIERE, France 2 Athos-environnement – Universite Blaise Pascal - Clermont-Ferrand II – 112 avenue du Brezet 63100

Clermont-Ferrand, France

Autumnal blooms of Nostocalean cyanobacteria occur annually over vast areas of Aydat Lake

(France). One of the keys of this ecological success is based on the production of akinetes, specialized overwintering cells. Surface sediment sampling was performed in 14 points in December 2014 and April 2015 to highlight spatio-temporal distribution and viability of akinetes belonging to two species: Dolichospermun flos-aquae and D. macrosporum. This last one was dominant with maximal values reaching 867 000 akinetes.g-1 DW of sediment whereas D. flos-aquae not exceeded 36 000 akinetes.g-1 DW of sediment. Nevertheless, main akinetes concentrations of both species were located in the same hotspots such as deepest zone and lake outlet. This phenomenon is amplified during winter with a process of horizontal transport leading to a secondary sedimentation probably induced by lake mixing and wind. Benthic overwinter phase does not seem influence the percentage of intact akinetes which stay stable depending on the specie around values of 7% and 60% for D. macrosporum and D. flos-aquae respectively. So, these percentages may be the results of processes occurred in the water column such as para- sitism attack or impact of grazing. Laboratory experiments confirmed the high percentage of viability of these overwintering intact akinetes with over than 90% capable to germinate after 72h or 144h for D. flos-aquae and D. macrosporum respectively. The differential rates of intact and germinating akinetes according to the specie highlight different ecological strategies leading to extend germination window in time and in space and thus to optimize the water column colonization by Nostocalean. Keywords: benthic akinetes, spatial distribution, overwintering phase, potential of germination

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∗Speaker

Development of New Biosensors to detect

Ciguatoxins

Helene Martin-Yken ∗ 1, Sylvain Derick 2, Camille Gironde 2, Taiana

Darius 3, Christophe Furger 2, Dominique Laurent 4, Jean Marie

Francois 1, Mireille Chinain 3

1 Laboratoire d’Ingenierie des Systemes Biologiques et des Procedes (LISBP) – Institut National des

Sciences Appliquees [INSA], Institut national de la recherche agronomique (INRA) : UMR792, CNRS :

UMR5504 – 135 Avenue de rangueil 31077 Toulouse cedex 04, France 2 Led Engineering Developpment (L.E.D.) – CNRS : UPR8001 – 7 Rue Benedict Prevost, 82000

Montauban, France 3 Laboratoire des Micro-algues Toxiques, UMR 241 EIO, Institut Louis Malarde (ILM) – BP 30, 98713

Papeete, Tahiti, French Polynesia 4 Universite Toulouse 3 UMR 152 et IRD Polynesie francaise (IRD) – Institut de recherche pour le

developpement [IRD] : UMR152 – Universite Paul Sabatier Toulouse 3 UMR 152 et IRD Polynesie

francaise, BP 529 98713 Papeete – Tahiti, France

Ciguatoxins are lipid-soluble polyether compounds produced by dinoflagellates from the

genus Gambierdiscus spp.. Ciguatoxins are mostly found in tropical and subtropical zones;

however, within the last decade, they have been identified in fishes caught in European wa-

ters, notably in Madeira(1) and Canary Islands(2), while Gambierdiscus spp. have also been

found both in the NE Atlantic Ocean(3) and in the Mediterranean Sea(4). These toxins bind to

Voltage Gated Sodium Channels at the surface of human sensory neurons where they remain,

causing Ciguatera Fish Poisoning with a variety of gastrointestinal, cardiovascular and neuro-

logical symptoms (paresthesia, ataxia, cold allodynia), including persistent neurological effects.

Ciguatera is the major cause of food poisonings by seafood worldwide, with an estimated 50 000

to 500 000 victims per year. However, there is so far no simple and quick way of detecting these

toxins in contaminated samples. Currently, only heavy and expensive laboratory methods are

available to detect them: LC-MS/MS, receptor-binding assays by competition with radiolabeled

compounds, and neuroblastoma cell-based assays performed on mammalian neurons(5). We have

started to engineer biosensors based on the detection of a transcriptional signal in the yeast model

Saccharomyces cerevisiae. This unicellular eukaryotic model is well-known and easy to genetically

modify, grows fast and presents a very good conservation of signaling pathways with higher

eukaryotes. We present a series of genetically modified yeast strains which allow us to follow the

activation of specific signaling pathways responding linearly to ciguatoxin exposure. This pre-

exploratory project received a seed-funding by CNRS (PEPs project, Oceasafe).

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∗Speaker

Impact of the benthic toxic dinoflagellate Ostreopsis cf. ovata on coastal fauna in

NW Mediterranean Sea

Anne-Sophie Pavaux ∗ 1, Rodolphe Lemee 1, Stephane Gasparini 3,2

1 Laboratoire d’oceanographie de Villefranche (LOV) – INSU, CNRS : UMR7093, Universite Pierre et Marie Curie (UPMC) - Paris VI, Universite Pierre et Marie Curie [UPMC] - Paris VI – Observatoire Oceanologique Station zoologique 181, chemin du lazaret BP 28 06230 VILLEFRANCHE SUR MER

Cedex, France 3 Laboratoire d’oceanographie de Villefranche (LOV) – CNRS : UMR7093, INSU, Universite Paris VI -

Pierre et Marie Curie – BP 28 06234 VILLEFRANCHE SUR MER CEDEX, France 2 Universite Pierre et Marie Curie - Paris 6 (UPMC) – Universite Paris VI - Pierre et Marie Curie – 4

place Jussieu - 75005 Paris, France

Blooms of the benthic toxic dinoflagellate Ostreopsis cf. ovata have increased in frequency and intensity in temperate areas during past decades. Adverse effects on Human health of those blooms have been recorded with skin and eye irritations, fever, headaches and breath difficulties among main symptoms. The toxicity of this dinoflagellate is attributed to the synthesis of palytoxin analogs (ovatoxins), although the trophic transfer of those toxins remains poorly understood. Mortality of macrofauna was sometimes observed during (or just after) Ostreopsis cf. ovata blooms, but little work has been done to understand this phenomenon.

In the framework of my thesis, our work aims to assess the impact of toxins produced by Ostreopsis cf. ovata or other metabolites on survival, ingestion and fecundity rates of three models: holoplankton (calanoids copepods Acartia clausi and Temora stylifera ), macrofauna with meroplanktonic larvae (sea urchins Paracentrotus lividus ) and meiofauna (harpacticoids copepods). The different models will be reared in thermoregulated enclosures which permit to follow ingestion and survival rates in relation to the dinoflagellate concentrations. The potential allelopathic effect will also be evaluated. This study will be coupled with metabolomics studies to analyze dinoflagellates toxin profiles and eventually toxins transfer or bio-accumulation in fauna. In situ studies are also planned to evaluate direct impact of Ostreopsis cf. ovata on abundance and diversity of coastal fauna.

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∗Speaker

Detection of pacific ciguatoxins using liquid chromatography coupled to tandem low resolution mass spectrometry (LC-MS/MS).

Manoella Sibat ∗ 1, Christine Herrenknecht 2, Zouher Amzil 3, Philipp

Hess 1


d’Yeu - BP 21105 - 44311 Nantes Cedex 03, France 2 Mer, molecules et sante (MMS) – Universite de Nantes : EA2160 – UFR Sciences Pharmaceutiques et Biologiques, 9 rue Bias, BP 53508, 44035, Nantes Cedex 1, France



Ciguatera fish poisoning (CFP) is principally caused by consumption of tropical and subtropical fish contaminated by ciguatoxins (CTXs). These lipid-soluble, polyether neurotoxins are produced by dinoflagellates of the genera Gambierdiscus and Fukuyoa. While there is no regulatory level in Europe for CTXs, the United States adopted a guidance level of 0.01 µg CTX-1B equivalent Kg-1 of fish. This limit is extremely low and requires significant improvement in the detection of CTXs. We compared analytical protocols based on liquid chromatography coupled to tandem low resolution mass spectrometry LC-MS/MS to find the best conditions for either sensitivity and/or selectivity. Liquid chromatography was studied using different mobile phases (acetonitrile (ACN) and methanol (MeOH)) and different flow rates; the mass spectrometer was a Sciex API4000Qtrap, transitions included those of molecular ions and those of ammonium and sodium adducts.

The mobile phase with MeOH improved selectivity. Sensitivity was improved by lowering the flow rate in ACN- but not in MeOH-based mobile phase. Signal-to-noise ratios (S/N) indicated that detection limits with MeOH-based mobile phase were better for CTX-3C but not for CTX- 1B. Detection limits were always worse for the sodium adduct compared to either ammoniated adducts or molecular ions. For CTX-1B, despite 50% higher intensity of the signal for the transition [M+Na]+/[M+Na]+, the S/N ratio was twice as high for the water loss of the ammoniated adduct. For CTX-3C, the water loss of the ammoniated adduct gave both higher signal and higher S/N ratios, independent of the organic part of the mobile phase (ACN or MeOH). As transitions from molecular ions and ammoniated adducts can also give higher selectivity, pref- erence should be given to monitor these transitions. While our current LODs do not meet the US guidance level, the findings suggest careful evaluation of instrumental parameters for CTXs determination by LC-MS/MS.

NOTE:

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List of sponsors

CNRS : Centre national de la recherche scientifique

Le Centre national de la recherche scientifique est un organisme public de recherche

(Etablissement public a caractere scientifique et technologique, place sous la tutelle du Ministere

de l’E ducation nationale, de l’Enseignement superieur et de la Recherche). Il produit du savoir et met ce savoir au service de la societe.

Ifremer : Institut Francais de Recherche pour l’Exploitation de la Mer

L’Ifremer contribue, par ses travaux et expertises, a la connaissance des oceans et de leurs ressources, a la surveillance du milieu marin et du littoral et au developpement durable des

activites maritimes. A ces fins, il concoit et met en œuvre des outils d’observation, d’experimentation et de surveillance, et gere des bases de donnees oceanographiques.

Irstea : Institut national de recherche en sciences et technologies pour l’environnement et l’agriculture.

Irstea est un organisme de recherche qui, depuis plus de 30 ans, travaille sur les enjeux majeurs d’une agriculture responsable et de l’amenagement durable des territoires, la gestion de l’eau et

les risques associes, secheresse, crues, inondations, l’etude des ecosystemes complexes et de la biodiversite dans leurs interrelations avec les activites humaines. Recherche pluridisciplinaire,

expertise et appui aux politiques publiques agro-environnementales, partenariat avec les collectivites territoriales et les acteurs du monde economique, telles sont les caracteristiques

d’Irstea, labellise Institut Carnot

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ANSES : Agence nationale de securite sanitaire de l’alimentation, de l’environnement et du travail

L’Agence nationale de securite sanitaire de l’alimentation, de l’environnement et du travail (Anses) a ete creee le 1er juillet 2010. L’Anses est un etablissement public a caractere

administratif place sous la tutelle des ministeres charges de la Sante, de l’Agriculture, de l’Environnement, du Travail et de la Consommation.

Novakits propose les tests et standards pour l’analyse de toxines phycoplanctoniques et de cyanobacteries aux differents niveaux analytiques, du test de 1ere intention terrain et ELISA de

reference aux standards certifies et isotopiques pour l’analyse de confirmation. L’offre est completee par des materiels selectionnes, des essais inter-laboratoire. La proximite avec le monde

de la recherche permet d’identifier les besoins emergents et d’organiser leur disponibilite prochaine pour la communaute scientifique.

Bionef, est un fournisseur de systemes de mesure et de controle pour l’industrie et la recherche, pour le suivi de la qualite de l’eau, l’ecotoxicologie et la surveillance de l’environnement. Bionef vous propose des instruments et des equipements de haute qualite pour les processus industriels,

les sciences et technologies de l’environnement, la recherche scientifique.

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List of participants

LASTNAME Firstname E-MAIL LABORATORY

ABADIE Eric [email protected] Sète

ALARCAN Jimmy [email protected] Anses

AMAR Muriel [email protected] CEA Paris Saclay

AMZIL Zouher [email protected] IFREMER

ANDREA Garvetto [email protected] SAMS (Scottish Association for Marine Science)

ANFOSSO Marie [email protected]

ANNE Thebault [email protected] ANSES

ARAOZ Romulo [email protected] CNRS-NeuroPSI/ CEA-SIMOPRO

ARNICH Nathalie [email protected] ANSES / Direction de l'Evaluation des Risques

AYACHE Nour [email protected] Ifremer centre atlantique

BARBE Sabrina [email protected]

BENOIT Evelyne [email protected] CEA de Saclay

BERDALET Elisa [email protected] Institut de Ciències del Mar (CSIC)

BERNARD Cécile [email protected] MNHN

BIRÉ Ronel [email protected] Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (Anses)

BOISNOIR Aurélie [email protected] Université des Antilles - Guadeloupe

BOJADZIJA Gorenka [email protected] Université de Rennes 1

BORMANS Myriam [email protected] Université de Rennes 1

BRIENT Luc [email protected] Université de Rennes 1

CALABRO Kevin [email protected] National University of Ireland

CARUANA Amandine [email protected] Ifremer

CASSIER-CHAUVAT Corinne [email protected] CNRS

CASTREC Justine [email protected] IUEM

CHAUVAT Franck [email protected] CEA-Paris-Saclay

CHINAIN Mireille [email protected] Institut Louis Malardé

CHOMÉRAT Nicolas [email protected] IFREMER

CLAUSING Rachel [email protected]

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CREUZET Sophie [email protected] Institut des Neurosciences de Paris-Saclay

DALLE Caroline [email protected] Institut Pasteur

DECHRAOUI BOTTEIN Marie-Yasmine [email protected] IAEA

DJEDIAT Chakib [email protected] MNHN

DOM Inès [email protected] Anses

DURIMEL Claire [email protected]

DUVAL Charlotte [email protected] Museum National d'Histoire Naturelle

ENGUEHARD Sylvain [email protected] NOVAKITS

FABIOUX Caroline [email protected] IUEM_UBO

FAUCHOT Juliette [email protected] Université de Caen Normandie

FESSARD Valérie [email protected]

GÉMIN Marin-Pierre [email protected] IFREMER

GEORGES DES AULNOIS Maxime [email protected] IFREMER Nantes

GUGGER Muriel [email protected]

HEGARET Helene [email protected]

HERVE Jerome [email protected] CNRS

HERVE Fabienne [email protected] IFREMER

HESS Philipp [email protected] Ifremer

HUMBERT Jean-François [email protected] INRA

IORGA Bogdan [email protected] CNRS

JAUZEIN Cécile [email protected] Ifremer Centre Bretagne

KATHLEEN Feilke [email protected] CNRS

LAABIR Mohamed [email protected] Université Montpellier

LAFFORGUE Michel [email protected] Suez Consulting

LAMARQUE Amélie [email protected] Université Clermont Auvergne

LANCE Emilie [email protected] Université Reims Champagne Ardennes

LAPLACE-TREYTURE Christophe [email protected] Irstea - Unité Écosystèmes Aquatiques et Changements globaux (EABX)

LATOUR Delphine [email protected] Université Clermont Auvergne

LE JEUNE Anne-Hélène [email protected] UMR CNRS 6023

LE MANACH Séverine [email protected] Muséum National d'Histoire Naturelle

LEGRAND Benjamin [email protected] Université Clermont Auvergne

LEMÉE Rodolphe [email protected] UPMC-CNRS

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LEPOUTRE Alexandra [email protected] Université de Reims Champagne-Ardenne

LONG Marc [email protected] Institut Universitaire Européen de la Mer

LUNGHI Mathias [email protected] Anses (Agence nationale de sécurité sanitaire)

MALLET Clarisse [email protected] Université Clermont Auvergne

MANCERA Jose Ernesto [email protected] Universidad Nacional de Colombia

MANGIALAJO Luisa [email protected] 1) UCA; 2)OOV

MARIE Benjamin [email protected] CNRS

MARRO Sophie [email protected] CNRS

MARTIN-YKEN Hélène [email protected] INRA

MAT Audrey [email protected]

MERABET Rym [email protected] Université de Guelma

MOLGO Jordi [email protected] CNRS, CEA

MONTRESOR Marina [email protected] Stazione Zoologica Anton Dohrn

NICOLAS Marina [email protected]

PANCRACE Claire [email protected] Institut Pasteur

PAVAUX Anne-Sophie [email protected]

PERRIÈRE Fanny [email protected] Université Clermont Auvergne UCA

PISAPIA Francesco [email protected] IFREMER, Nantes

QIAO Qin [email protected]

QUIBLIER Catherine [email protected]

RAPHAELE Le Garrec [email protected] UBO

REALE Océane [email protected] ANSES

ROBIN Joel [email protected] ISARA-Lyon

ROLLAND Jean-Luc [email protected] IFREMER

SABART Marion [email protected] Université Clermont-Auvergne

SAUVEY Aurore [email protected] Université de Caen Normandie

SCHAPIRA Mathilde [email protected] Ifremer

SERVENT Denis [email protected] CEA

SHARAF Najwa [email protected] Ecole nationale de ponts et chaussées

SIANO Raffaele [email protected] Ifremer

SIBAT Manoella [email protected] Ifremer

TAMBOSCO Kevin [email protected] UPMC

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TERNON Eva [email protected]

TESTER Patricia [email protected] Ocean Tester, LLC

THOMAS Olivier [email protected] National University of Ireland Galway

VILLERIOT Nicolas [email protected] MUSÉUM NATIONAL D'HISTOIRE NATURELLE

VINÇON-LEITE Brigitte [email protected]

WALLET France [email protected] EDF

WIEGAND Claudia [email protected] Université Rennes1

WIKFORS Gary [email protected] NOAA Fisheries Service

ZOUBIDA Qsair [email protected] université d'Orléans

Conférence nationale GdR Phycotox & GIS Cyano 2017 · 4 GdR Phycotox – GIS Cyano 2017,...

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