Ecology of Benthic and Planktonic Dinoflagellates of Eastern Mediterranean Sea : a Focus on Ostreopsis and Ceratium Genus Along the Lebanese Coast Laury Açaf

Home , Ceratiaceae, Ceratium furca, Cochlodinium, Ostreopsis, Tripos (dinoflagellate)

Ecology of benthic and planktonic dinoflagellates of Eastern Mediterranean Sea : a focus on Ostreopsis and Ceratium genus along the Lebanese coast Laury Açaf

To cite this version:

Laury Açaf. Ecology of benthic and planktonic dinoflagellates of Eastern Mediterranean Sea :a focus on Ostreopsis and Ceratium genus along the Lebanese coast. Ecology, environment. Sorbonne Université, 2018. English. NNT : 2018SORUS263. tel-02868489

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Sorbonne Université

Ecole Doctorale des Sciences de l’Environnement d’Ile de France, ED 129 Laboratoire d’Océanographie de Villefranche-sur-mer (LOV)

“Ecology of benthic and planktonic dinoflagellates of Eastern Mediterranean Sea: a focus on Ostreopsis and Ceratium genus along the Lebanese coast”

Par Laury Açaf

Thèse de doctorat de Science du vivant et de l’environnement

Dirigée par Rodolphe Lemée et Marie Abboud-Abi Saab

Présentée et soutenue publiquement le 23 Mai 2018

Devant un jury composé de: BERDALET Elisa Docteur, Rapporteur MANGIALAJO Luisa Docteur, Rapporteur NIVAL Paul Professeur, Examinateur RABOUILLE Sophie Docteur, Examinateur ABBOUD-ABI SAAB Marie Professeur, Co-directeur de thèse LEMEE Rodolphe Docteur, Directeur de thèse Açaf Laury – Thèse de doctorat - 2018

To my parents, Alexandra and Elie,

and my sisters, Joelle and Yara,

I owe it all to you

Açaf Laury – Thèse de doctorat - 2018

Acknowledgements

I am indebted to Sorbonne Université and the Laboratoire d’Oceanologie de Villefranche (LOV) for welcoming me during my thesis. They offered a very rewarding work environment.

My gratitude also goes to the Lebanese National Council for Scientific Research CNRS-L for hosting me during the course of my thesis in its National Center for Marine Sciences (NCMS) in Batroun. I offer gratitude to my supervisor Dr. Rodolphe Lemée and co-supervisor Dr. Marie Abboud-Abi Saab because this thesis would not have been possible without their trust and support. I thank them both for their guidance and for granting their vast experience, advice and interpretations to help accomplish this work. It could not have been completed without their full commitment, patience and kindness.

I would like to offer my deepest gratitude to the jury members Dr. Luisa Mangialajo, Dr. Elisa Berdalet, Pr. Paul Nival, and Dr. Sophie Rabouille. They all gave me the honor and pleasure of judging my work and being present during my thesis defense and their remarks were very beneficial to me.

My thanks goes to Dr. Elisa Berdalet and Dr. Cécile Jauzein for their presence in my thesis committee.

I would also like to thank all the staff at the LOV, particularly at the “vieille forge” where I spent most of my stays, for their warm welcome and support in this new environment. My deepest gratitude goes to my friend and mentor, Dr. Cécile Jauzein, for her support, the passion and generosity in which she shares her knowledge, and above all, her friendship.

Similarly, I would like to thank all the staff of the CNRS-Lebanon for their warm welcome, kindness and confidence. I would like to thank all the members of the NCMS in Batroun and Jounieh, Lebanon. My gratituide goes to Ms. Nada Mattar and Marie-Thérèse Kassab for their help in lab analysis.

My gratitude then goes to all my colleagues turned dear friends: Myriam Lteif, Céline Mahfouz, Elissar Gemayel, Anthony Ouba, Amonda el Houssainy, Sherif Jemaa, Daniela Catania, Douglas Couet, Marin-Pierre Gémin, Mirella Khoury-Hanna, Abed El Rahman Hassoun, Ali Badereldinne, friends:

Açaf Laury – Thèse de doctorat - 2018

Despina El Hayek, Cécile Pinard, Sara Rachik, Fanny Sarfati, Ralph Zoughaib and my cousin and inspiration Dr. Rana Jreige, and all whom I could not mention for their continuous support at all times.

Last but not least, my deepest gratitude and love goes to my parents Alexandra Jreige and Elie Açaf and my sisters Joelle and Yara whose unconditional love, support and encouragement allowed me to complete this work.

Açaf Laury – Thèse de doctorat - 2018

Table of contents

Acknowledgements ...... 3 Table of contents ...... 5 Chapter I: General Introduction ...... 8 1. Dinoflagellates ...... 9 1.1. Generalities...... 9 1.2. Classification ...... 10 1.3. Morphology ...... 10 1.4. Distribution and Ecology ...... 12 1.4.1. Biogeography ...... 12 1.4.2. Habitat ...... 12 1.4.3. Nutrition ...... 13 1.4.4. Motility ...... 13 1.4.5. Reproduction and cysts ...... 13 2. Benthic Harmful Algal Blooms (BHABs). Target genus: Ostreopsis ...... 14 2.1. Generalities...... 14 2.2. Morphology and Identification...... 16 2.3. Ecology...... 17 2.3.1. Habitat ...... 17 2.3.2. Nutrition ...... 18 2.3.3. Effect of Environmental Parameters ...... 18 2.3.4. Reproduction ...... 19 2.3.5. Toxicity ...... 19 2.4. Previous Studies on Ostreopsis in Lebanese coastal waters ...... 19 2.5. Quantification of BHABs ...... 20 3. Dinoflagellates as Bio-Indicators. Target Genus: Ceratium ...... 21 3.1. Generalities...... 21 3.2. Morphology and Identification...... 21 3.3. Ecology...... 23 3.4. Role as biological models and bio-indicators ...... 23 4. Global change in the Eastern Mediterranean Sea ...... 24 5. Thesis objectives ...... 25 6. Valorization of the research done during this thesis ...... 26 Scientific articles ...... 27 Posters and Oral Communications: ...... 28 In International Conferences ...... 28 Chapter II: Quantification of Ostreopsis sp.: Optimization and intercalibration of new techniques ...... 29 Part 1: Optimization of sampling, cell collection and counting for the monitoring of benthic HAB: application to Ostreopsis spp. blooms in the Mediterranean Sea ...... 29 Part 2: Intercalibration of counting methods for Ostreopsis spp. blooms in the Mediterranean Sea 42 Chapter III: Bloom dynamics of the newly described toxic benthic dinoflagellate Ostreopsis fattorussoi along the Lebanese coast (Eastern Mediterranean) ...... 52 Abstract ...... 53 1. Introduction ...... 54 2. Material and methods ...... 56

Açaf Laury – Thèse de doctorat - 2018

2.1. Study area and sampling frequency ...... 56 2.2. Environmental parameters...... 58 2.3. Biological parameters ...... 58 2.3.1. Sampling ...... 58 2.3.2. Quantification of O. fattorussoi ...... 59 2.4. Statistical analysis ...... 60 3. Results ...... 61 3.1. Environmental parameters...... 61 3.1.1. Climatological parameters ...... 61 3.1.2. Hydrological parameters ...... 63 3.1.3. Nutrients ...... 65 3.2. Temporal and spatial variation in O. fattorussoi abundances ...... 66 3.2.1. Seasonal variation ...... 66 3.2.2. Spatial variation ...... 70 3.2.3. Relation between O. fattorussoi abundances and environmental parameters ...... 70 4. Discussion ...... 71 4.1. Temporal and spatial variation in O. fattorussoi abundances ...... 71 4.2. Influence of environmental factors on O. fattorussoi abundances ...... 73 4.2.1. Temperature ...... 73 4.2.2. Nutrients ...... 74 4.2.3. Cell Resuspension and Wind ...... 75 5. Conclusion ...... 76 Chapter IV: Nitrogen uptake of the benthic toxic dinoflagellates Ostreopsis cf. ovata and Ostreopsis fattorussoi of the Mediterranean Sea ...... 77 Nitrogen uptake of the benthic toxic dinoflagellates Ostreopsis cf. ovata and Ostreopsis fattorussoi of the Mediterranean Sea...... 78 Abstract ...... 78 1. Introduction ...... 79 2. Material and methods ...... 80 2.1. Kinetic experiments...... 80 2.1.1. Strains ...... 80 2.1.2. Medium and Culture conditions...... 80 2.1.3. Dilution and replicate cultures ...... 80 2.1.4. Resuspension in an N free medium ...... 81 2.1.5. Incubation ...... 81 2.1.6. Growth rate ...... 81 2.2. Toxin profile analysis for MCCV57 and MCC58 ...... 82 2.2.1. Toxin extraction ...... 82 2.2.2. LC-MS/MS analysis...... 82 3. Results ...... 83 3.1. Kinetic experiments...... 83 3.2. Toxin profiles for O. fattorussoi strains ...... 85 4. Discussion ...... 86 4.1. Competitiveness of Ostreopsis spp...... 87 4.2. General trends in N-uptake abilities between the studied strains ...... 88 4.3. Variability in N-uptake and toxin production ...... 89 5. Conclusion ...... 90

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Chapter V: Updated inventory of different taxa of the genus Ceratium in Lebanese waters (East. Mediterranean) and their use as bio-indicators for detecting environmental changes in the area ...... 92 Updated inventory of different taxa of the genus Ceratium in Lebanese waters (East. Mediterranean) and their use as bio-indicators for detecting environmental changes in the area ...... 93 Abstract ...... 93 1. Introduction ...... 94 2. Material and Methods ...... 94 2.1. Study site ...... 94 2.2. Environmental Parameters ...... 95 2.3. Plankton net sampling and species determination...... 95 2.4. Statistical analysis and biodiversity indices ...... 96 3. Results ...... 96 3.1. Environmental parameters...... 96 3.2. Variation in Ceratium abundance and diversity ...... 97 4. Discussion ...... 102 Chapter VI : General Discussion ...... 106 General discussion ...... 107 1. Benthic Harmful Algal Blooms: model genus Ostreopsis ...... 107 1.1. Ostreopsis of the Lebanese coast: what species? ...... 107 1.2. Ostreopsis sp. quantification: Optimization and Intercalibration ...... 107 1.2.1. Optimization of cells detachment in macroalgae sampling: ...... 108 1.2.2. Optimization of sampling using the deployment of artificial fiberglass screen (optimized set up described by Jauzein et al., 2016)...... 108 1.2.3. Optimization of counting of cells in the water column...... 109 1.2.4. Intercalibration of two innovative automated counting techniques of Ostreopsis spp. : RT- qPCR (MOL) and an opto-electronic device (OPR) ...... 109 1.3. Natural blooms of O. fattorussoi along the Lebanese coast and the role of environmental parameters ...... 110 1.3.1. Spatial and temporal distribution of O. fattorussoi ...... 110 1.3.2. Environmental parameters ...... 110 1.4. Ecological physiology ...... 111 1.5. Toxicity and potential risks associated to O. fattorussoi blooms ...... 113 2. Annual cycle of the genus Ceratium in Lebanese waters ...... 115 3. Blooms of Ostreopsis fattorussoi and temporal variation of Ceratium species in the Lebanese coast: an effect of global change in the Eastern Mediterranean? ...... 116 4. Perspectives...... 117 Table of Figures ...... 118 Table of tables ...... 121

Açaf Laury – Thèse de doctorat - 2018

Chapter I: General Introduction

Açaf Laury – Thèse de doctorat - 2018

General Introduction

1. Dinoflagellates

1.1. Generalities

Dinoflagellates are unicellular eukaryotic microorganisms, though some are pseudocolonial (coenocytic cells) or colonial (chain forming) c. They were previously known as Pyrrophyta (Greek Pyrro = fire) due to bioluminescent taxa. The name dinoflagellates (Greek δῖνος dinos "whirling" and Latin flagellum "whip, scourge") was proposed in 1885 by Butschli to designate this group, due to their spiraling forward movement using their flagella. They comprise of benthic, planktonic meroplanktonic, and tychoplanktonic species (Gómez, 2012a). Studies of planktonic dinoflagellates started in the 18th century, with the description of two species by Muller (Spector, 1984), whereas the first studies of benthic dinoflagellates did not take place till the early 20th century (Hoppenrath et al., 2014). An estimated number of 4500 species have been identified, including around 2500 fossils. Fossils are predominantly in the form of cysts, and have been dated back to the Upper Silurian, some 400 million years ago (Sarjeant, 1978). It is however believed that dinoflagellates existed considerably earlier (Butterfield and Rainbird, 1998), as most do not produce preservable cysts (Evitt, 1985; Head, 1996). Their long existence is evidence of their great adaptability to their environment (Spector, 1984), as reflected by their tremendous morphological and trophic diversity (Taylor, 1987). Similarly, 2337 living species distributed among 259 genera have been described (Gomez, 2012-b). They are common to abundant in all aquatic environments (83% marine and 17% fresh water), and have even been found in snow and sea ice (Taylor et al., 2008).

Typically, motile dinoflagellates are between 20 and 200 μm though molecular analyses in recent years has revealed the presence of picoplanktonic dinoflagellates, mostly undescribed (Moon-Van Der Staay et al., 2001; Lin et al., 2006). In addition, Noctiluca scintillans ranges in size between 200 μm and 2000 μm (https://botany.si.edu), making the extreme size range from less than 2 μm to 2 mm. At the ultrastructural level, the cell covering known as “amphiesma”, along with their unique nucleus and flagella are what differentiates them from other algal groups (Steidinger and Tangen, 1996) (See 1.3 Morphology).

Açaf Laury – Thèse de doctorat - 2018

1.2. Classification

The presence of alveoles unites the super-group alveolates, comprising mainly ciliates, apicomplexa, and dinoflagellates. Historically, dinoflagellates have been classified both as animals and as plants, due to the diversity of their trophic behaviors. They were also studied by paleontologists due to the presence of fossil cysts with a distinctive morphology. Some taxa were therefore described under the rules of the International Code for Zoological Nomenclatures, while others according to the International Code of Botanical Nomenclature. The classification was later on unified by Fensome et al. (1993) based on morphological features. It is now widely accepted that the ICBN should be used for their classification. They belong to the Empire Eukaryota, Kingdom: Chromista, Phylum: Miozoa, Subphylum: Myzozoa, Infraphylum: Dinozoa and the superclass Dinoflagellata (algaebase.org). With the new advances in molecular phylogeny, their classification is being continuously subjected to changes according to new findings, especially at the order level (Gomez et al., 2012-a).

1.3. Morphology

Dinoflagellates display a wide diversity in cell shapes such as spherical, ovoid, polygonal, anchor shaped, leaf-like, butterfly-like and medusa like (Taylor et al., 2008). Some display cell extensions such as lists, horns and spines (Steidinger and Tangen, 1996).

Surface morphology based on the cell covering or “amphiesma”, is also critical in dinoflagellates subdivision. It allows the distinction between naked = unarmoured and thecate = armoured = walled dinoflagellates. Thecate species are those that possess a theca = cell wall, which consists of closely fitted cellulosic plates within the alveola (a single layer of amphiesmal vesicles present beneath the cell membrane), whereas in athecate species the amphiesmal vesicles are devoid of cellulosic plates.

Most dinoflagellates are characterized by having, during at least one part of their life cycle, a motile stage with two dissimilar flagella. The placement of the flagella allows for distinction of two morphological cell types used in dinoflagellates identification: i- Desmokont type: where the two flagella are inserted apically (e.g. Prorocentrum) and ii- dinokont type: where they are inserted ventrally (e.g. Alexandrium). Desmokonts are divided into to a right and left valve. Dinokonts are divided by a transversal groove called the cingulum, into an apical (apex) and antapical parts. The apex is the anterior of the cell, in accordance with its moving direction. The antapex is in the posterior of the cell. The

Açaf Laury – Thèse de doctorat - 2018 anterior part is known as epitheca (thecate) or episome (naked), and the posterior part known as hypotheca (thecate) or hyposome (naked). A longitudinal groove, the sulcus, appears in the ventral side (by convention). The longitudinal flagellum emerges from the flagellar pore at the cingular-sulcal interface. The transverse flagellum emerges from a second flagellar pore just above that of which the longitudinal flagellum emerges (Spector, 1984). Thecate cells have an apical pore, whereas athecates might have an acrobase (Figure 1) (Steidinger and Tangen, 1996). The pattern, shape and size of the vesicles, containing or deprived of cellulose, known as tabulation are essential in taxonomy. The Kofoid system of tabulation is currently in universal use (Figure 2).

Figure 1 Figure 1- Overview of the external morphology and its terminology of athecate and thecate dinoflagellate cells. © Mona Hoppenrath from http://tolweb.org/Dinoflagellates/2445

Açaf Laury – Thèse de doctorat - 2018

Figure 2 Figure 2- Kofoid system of tabulation. © Mona Hoppenrath from http://tolweb.org/Dinoflagellates/2445

1.4. Distribution and Ecology

1.4.1. Biogeography

Dinoflagellates as a groups are cosmopolitan. Some taxa, such as the Alexandrium ostenfeldii complex are cosmopolitan themselves (Lundholm and Moestrup, 2006). Others however are confined to certain geographic zones. For instance, some species of Gymnodynium are confined to temperate and tropical waters (Granéli and Turner, 2006); Peridiniella catenata can bloom in the arctic (Granéli and Turner, 2006) whereas and Polarella glacialis is bipolar (Montresor et al., 2003). Their distribution into geographic zones is mainly based on temperature, current systems extending the temperature boundaries to the north. It has been termed “modified latitudinal cosmopolitanism”, meaning that the same morphospecies occur within similar climatic zones in both northern and southern hemispheres (Taylor and M. Saldarriaga, 2008).

1.4.2. Habitat

Their habitat distribution is dominated by marine species, including estuaries and brackish waters (1957 species = 82% of extant described species). 91% of marine species are planktonic, meaning they

Açaf Laury – Thèse de doctorat - 2018 float freely in the water column, whereas 9% are benthic, meaning they are attached to the bottom, mostly psammophilic (sand dwelling) or and phycophilic (epiphytic on macrophytes), or in tide pools, and on floating detritus and corals (Taylor et al., 2008; Gomez, 2012-b; Hoppenrath et al., 2014). This distinction is not clear-cut, as some benthic species are tychoplanktonic (accidentally present in the water column due to turbulence e.g. waves, human disturbance), while some planktonic are meroplanktonic (having a benthic stage e.g. cysts). Though most are free living, some are parasitic and a few are mutualistic symbionts, including the famous zooxanthellae Symbiodinium inhabiting reef building corals (Gomez, 2012-b).

1.4.3. Nutrition

About half of free living dinoflagellates are devoid of chloroplasts and are therefore strict heterotrophs. The other half contain chloroplasts, though they are not necessarily strict autotrophes. Many “autotrophes” are in fact mixotrophes, as they also combine osmotrophy (uptake of organic matter through osmosis) and phagotrophy (predation) along with photosynthesis as means to obtain nutrients (Taylor et al., 2008; Gomez, 2012-b) . Preys of dinoflagellates are often diatoms, and in turn they are grazed by zooplankton. It is widely believed that further studies will show most dinoflagellates are in fact mixotrophes (Spector, 1984).

1.4.4. Motility

Dinoflagellates are some of the fastest swimming protists, some exceeding a velocity of 1 mm.s-1 (e.g. Lim et al., 2014). The combined functions of a rotating transverse flagellum and a propelling longitudinal flagellum result in a helical swimming path. Their movement, including vertical migration, is directed and is influenced by gravity, light and chemical stimuli (chemosensory responses to prey) (Burkholder et al., 2001).

1.4.5. Reproduction and cysts

The dinokaryon is a unique nucleus with non-eukaryotic features. It is haploid, nucleosomal histones are absent, chromosomes are always condensed even during interphase and mitosis is closed with an external spindle. It contains a large amount of DNA (De la Espina et al., 2005). Most dinoflagellates reproduce asexually (mitosis) yielding haploid schizonts. A small percentage is known to have a sexual reproduction involving a diploid stage and might form a resting cyst as part of their sexual

Açaf Laury – Thèse de doctorat - 2018 life cycle before meiosis (Evitt, 1985). Some species can rapidly form temporary cysts in response to sudden adverse conditions as a survival mechanism (Parrow and Burkholder, 2003).

2. Benthic Harmful Algal Blooms (BHABs). Target genus: Ostreopsis

2.1. Generalities

Planktonic harmful dinoflagellates has long been studied (Spector, 1984). The study of benthic ones was delayed, possibly due to the complexity of their sampling and quantification. It did not start until the late seventies, (Yasumoto et al., 1977) attributed Ciguatera poisoning, the most common form of phycotoxin-borne seafood illness across the globe, to a benthic dinoflagellate. The culprit was later described as Gambierdiscus toxicus Adachi and Fukuyo (Adachi and Fukuyo, 1979). In the following years, around 30 of the 160 benthic species known (Taylor et al., 2008), were found to produce different toxic and bioactive chemical compounds, some responsible for major BHABs events such as species of Ostreopsis, Coolia, Alexandrium, Amphidinium, Prorocentrum and Vulcanodinium (Hoppenrath et al., 2014). The genus Ostreopsis received particular interest; Ostreopsis ovata Fukuyo (1981) was found to be toxic (Nakajima et al., 1981), producing palytoxin-like metabolites, which is among the most potent known naturally produced toxins of marine origin even at extremely low concentrations (Moore and Scheuer, 1971; Amzil et al., 2012a).

The dinoflagellate genus Ostreopsis belongs to the Class Dinophyceae, Order Gonyaulacales and the family Ostreopsidacea. The type species O. siamensis Schmidt was first identified in 1901 by Schmidt in the gulf of Siam Thailand. Currently, a total of eleven species have been described (Table 1), covering a global distribution from tropic to temperate areas (Rhodes et al., 2011).

Açaf Laury – Thèse de doctorat - 2018

Table 1 Table 1- A list of described Ostreopsis species along with their respecttive type locality, toxic molecules and examples of relevent refrences. *: non PLTX-like metabolite

Species Authority Type locality Toxins O. fattorussoi Accoroni, Batroun, Lebanon, (Accoroni et Isobaric (Accoroni et al., Romagnoli & Eastern Mediterranean al., 2016a) palytoxin 2016a) Totti, 2016 OVTX-a, d, e, i, j1, j2, k OVTX-a, d, e (Accoroni et al., 2016) O. rhodesiae Verma, Heron Reef Lagoon, (Verma et Chemical (Verma et al., Hoppenrath & southern Great Barrier al., 2016) formula not 2016) S. A. Murray, reef, Coral Sea, Australia known 2016 O. belizeana M. A. Faust, Belize, Central America, (Faust, - 1999 Caribbean Sea 1999) O. caribbeana M. A. Faust, La Parguera, Puerto (Faust, - 1999 Rico, Caribbean Sea 1999) O. marina M. A. Faust, Belize, Central America, (Faust, - 1999 Caribbean Sea 1999) O. labens M. A. Faust & Belize, Central America, (Faust and - S. L. Morton, Caribbean Sea Morton, 1995 1995) O. mascarenensis J. P. Quod, 1994 Reunion Island, West (Quod, Mascarenotoxin (Lenoir et al., Indian Ocean 1994) -a, b 2004) O. heptagona D. R. Norris, J. Florida Keys, Gulf of (Norris et Chemical (Norris et al., W. Bomber & Mexico al., 1985) formula not 1985) Balech, 1985 known O. lenticularis Y. Fukuyo, Gambier Islands and (Fukuyo, Ostreotoxin-1, (Mercado et al., 1981 Society Islands, French 1981) 3* 1994) Polynesia, South Pacific Ocean O. ovata Fukuyo, 1981 French Polynesia, New (Fukuyo, Isobaric (Onuma et al., Caledonia and Ryukyu 1981) palytoxin 1999; García- Islands, South and West Altares et al., Pacific Ocean 2015) Ovatoxin-a (Ciminiello et al., 2008) Ovatoxin-b, c, (Ciminiello et al., d,e 2010) Ovatoxin-f (Ciminiello et al., 2012-b) Ovatoxin-g (García-Altares et al., 2015) Ovatoxin-h (Brissard et al., 2015) Ovatoxin-l (Tartaglione et al., 2017) Mascarenotoxin (Scalco et al., -a, c 2012) Ostreol A* (Hwang et al., 2013) O. siamensis Johs. Schmidt, Trat Province, Gulf of (Schmidt, Ostreocin-d (Usami et al., (type species) 1901 Siam Thailand 1901) 1995; Ukena et al., 2002)

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In the Mediterranean Sea, three species are known to exist: O. siamensis, O. ovata and O. fattorussoi. Ostreopsis siamensis was the first to be noted in 1972 in the bay of Villefrance-sur-mer (North western Mediterranean) by Taylor (1979). A few years later, Abboud-Abi Saab (1989) reported the presence of Ostreopsis sp. in the water column in coastal waters of Lebanon (Eastern Mediterranean), where molecular analysis allowed the description of a new species Ostreopsis fattorussoi Accoroni, Romagnoli & Totti in 2016. Its presence in this area has gained increasing attention since the occurrence of the first BHAB of O. ovata in Italy in 1998 (Sansoni et al., 2003a). Since then, bloom occurrences especially of O. ovata have been a recurring summer event, increasing in frequency and intensity along the Mediterranean coasts, (e.g. Mangialajo et al., 2011; Accoroni and Totti, 2016), as expected for HABs in light of global environmental changes (Rhodes et al., 2011). This phenomenon is paralleled by an increase in the number of studies along the years (Shears and Ross, 2009). While no cases of human intoxication from clupeid fish consumption has been reported in this area, palytoxin equivalents have been found in shellfish and fish collected during an Ostreopsis bloom in Villefranche-sur-mer in concentrations exceeding the 30 μg·kg−1 in shellfish recommended by the European Food Safety Authority (EFSA) (Biré et al., 2013, 2015; Brissard et al., 2014). Several cases of human intoxication through direct contact or aerosols inhalation have been reported in the North Western Mediterranean Sea, presumably from palytoxin-like molecules produced by nearby blooming Ostreopsis (Masó and Garcés, 2006; Durando et al., 2007; Kermarec et al., 2008; Vila et al., 2008, 2016; Illoul et al., 2012; Pfannkuchen et al., 2012). Symptoms included skin irritations, fever, and respiratory difficulties, sometimes leading to hospitalization (Simoni et al., 2003; Ciminiello et al., 2006; Tichadou et al., 2010; Vila et al., 2016). In some cases, mass human intoxication i.e. involving more than 200 individuals occurred e.g. Genoa in 2005 (Durando et al., 2007) and Algeria in 2009 (Illoul et al., 2012). Several studies also demonstrated detrimental effects of O. ovata on marine invertebrates (e.g. Simonini et al., 2011; Blanfune et al., 2012; Privitera et al., 2012). In addition, blooms of Ostreopsis are often accompanied by closing of beaches, which can have serious economic impacts in touristic areas (Tichadou et al., 2010; Lemée et al., 2012).

2.2. Morphology and Identification

The genus Ostreopsis is an armoured dinoflagellate, with the typical dinokaryotic nucleus and two dissimilar flagella. The hypotheca and the epitheca are similar in size. All species have similar cell shape, and are described as “ovoid”, “tear shaped” or “lenticular” (Penna et al., 2005). Cells are

Açaf Laury – Thèse de doctorat - 2018 anteroposteriorly flattened and tapered ventrally, are therefore observed from the apical or antapical view point in light microscopy The smallest cells size were reported for the species O. ovata and O. rhodesae (Dorsoventral diameter (DV) ≈30µm) and the largest for O. mascarenensis (DV ≈198µm). Taxonomy was based on morphological features such as cell shape, cell size, dorsoventral diameter (DV), transdiameter (W), anteroposterior diameter (AP), and the ratio DV/AP as well as tabulation (thecal pattern, shape, size, ornamentations). However, many discrepancies exist around these features. For example, the high resemblance cell shape and the overlapping cell size ranges at the species level, as well as and the high similarity in thecal plate pattern making almost any Ostreopsis species could easily fit the original description of O. siamensis, with the exception of O. heptagona (Penna et al., 2005). These morphological discrepancies and the lack of genetic data for the holotype specimens have highlighted taxonomic issues and led to a trend in using genetic markers that can assuredly discriminate species, ideally down to strain level, in order to overcome the identification difficulties (Penna et al., 2005, 2010; Sato et al., 2011; Parsons et al., 2012). This has led authors to use the suffix confer (c.f.) when referring to the species O. cf. ovata and O. cf. siamensis, pending a complete revision of the taxonomy of the genus based on morphology and genetics (Penna et al., 2010).

2.3. Ecology

2.3.1. Habitat

Ostreopsis is found in shallow coastal water, mostly epi-benthic attached to macroalgae, and other biotic (marine phanerogams, benthic invertebrates) or abiotic substrate (rocks, pebbles, in the sand or in tide pools (Besada et al., 1982; Norris et al., 1985; Faust and Morton, 1995; Aligizaki and Nikolaidis, 2006; Totti et al., 2010; Accoroni et al., 2011). It can also be accidentally dislocated into the water column through movement e.g. wave action, human action, making it a tychoplanktonic species (Steidinger and Tangen, 1996). Their flattened cell shape is a common trait in benthic dinoflagellates (e.g. some Gambierdiscus species, Sinophysis, Planodinium, Amphidiniopsis) and is thought to allow them to attach more easily to surfaces or facilitate their movement in interstitial habitats. Alternatively, it has been proposed that the flattened surfaces may act to increase nutrient uptake in oligotrophic conditions, as they increase the surface/volume ratio compared to a spherical cell shape (Fraga et al., 2012; Hoppenrath et al., 2014).

Açaf Laury – Thèse de doctorat - 2018

2.3.2. Nutrition

Ecological physiology is greatly understudies for this genus. In terms of nutrition, the presence of golden chloroplast attests for their photosynthetic abilities. Mixotrophy through osmosis has been proven for North western Mediterranean strains of O. cf. ovata by Jauzein et al. (2017). In addition, Faust and Morton (1995) suggested that the ventral pore could be involved in phagotrophy through the release mucilaginous networks for prey capture. However, phagotrophy in Ostreopsis species is still elusive. The mucus could instead be an adaptation to the benthic environment (e.g. protection from for being dislocated by wave actions or desiccated at low tide (Hoppenrath et al., 2014), or to shade the cells when exposed to high intensities to avoid photodamage (Heil et al., 1993).

2.3.3. Effect of Environmental Parameters

Ostreopsis preference to shallow coastal waters is confirmed by several agreeing studies that report a significant decrease of abundances with depth, which could be linked to light intensity. The study of the effects of other environmental parameters such as hydrodynamism, water temperature, salinity and nutrients on bloom development of Ostreopsis have led to contrasting results. For instance, some studies reported that calm hydrodynamic conditions leads to higher concentrations of O. siamensis (Chang et al., 2000; Shears and Ross, 2009), whereas Ostreopsis sp.1 reportedly prefers slight agitation (Vila et al., 2001-b). While several suggest that Ostreopsis spp. need relatively high temperatures to proliferate (e.g. Mangialajo et al., 2008; Granéli et al., 2011; Cohu et al., 2013), others did not find a correlation between water temperatures and its abundances (Vila et al., 2001a; Totti et al., 2010; Accoroni et al., 2011, 2012a; Cohu et al., 2011; Mangialajo et al., 2011). Different ranges of salinity are reported during blooms of Ostreopsis as well (Accoroni and Totti, 2016). The contradictory results can be due to i- the studies involving different species of Ostreopsis ii- the synergic effects of several environmental parameters iii- the effect of biotic parameters such as bacteria, diatoms, allelopathic effects of biotic substrate which is often overlooked and iv- The existence of intra-specific differences. These differences further highlight the need for ecological studies, both in situ and under controlled experimental conditions. They also highlight the need for case by case studies, as different species or even strains of the same species have shown different responses to environmental drivers (e.g.Guerrini et al., 2010; Pezzolesi et al., 2012; Tawong et al., 2014).

Açaf Laury – Thèse de doctorat - 2018

2.3.4. Reproduction

The study of the life cycle of O. cf. ovata showed that they reproduce both sexually and asexually. Their life cycle involves both temporary and resting cysts (Bravo et al., 2012; Accoroni et al., 2014). Accoroni et al. (2014) suggested the presence of a temperature threshold affecting cyst germination.

2.3.5. Toxicity

The recent description of new species of Ostreopsis has increased the number of toxic species to 8 out of 11 (Table 1). Many of the toxins, including those produced by the Mediterranean species, (isobaric palytoxin, Ovatoxins (OVTX) -a through -h, OVTX-l, OVTX-i, j1, j2, k, mascarenotoxin and Ostreocin) belong to the Palytoxin (PLTX) group. PLTX is a large and very complex molecule, with a molecular formula of C129H221N3O54 and a molecular weight of 2680 Da (Moore and Bartolini, 1981) and contains both lipophilic and hydrophilic areas. It is one of the most potent marine toxins, causing the potentially fatal syndrome known as clupeotoxism, with a much higher mortality rate than ciguatera (Onuma et al., 1999). Symptoms include a sharp metallic taste, neurological symptoms, nausea, vomiting, abdominal pain, diarrhea, tingling sensations, dilated pupils, muscle cramps, paralysis and coma (Halstead, 1978 cited in Randall, 2005).

2.4. Previous Studies on Ostreopsis in Lebanese coastal waters

Contrary to the western Mediterranean Sea, studies of the genus Ostreopsis in our geographic area, the Eastern Mediterranean, are scarce. Nevertheless, presence of Ostreopsis sp. has been signaled in Lebanese coastal waters since 1979, as part of a quantitative and qualitative study of the coastal plankton (Abboud-Abi Saab, 1989). The author reported that the tabulation of the specimens did not correspond to any described species at the time, and suggested it could be a new species. One study published in 2013 specifically targeted Ostreopsis, referring to it as O. siamensis (Abboud-Abi Saab et al., 2013). This study suggested it to be a thermophilic species, present in shallow rocky sites along the Lebanese coast from spring to autumn, with densities reaching 10500 cells / L in planktonic samples, but epiphytic sampling was not performed. In 2016, morphological and molecular analysis of Ostreopsis cells from Batroun, Lebanon led to its description as a new species named O. fattorussoi Accoroni, Romagnoli & Totti. (Accoroni et al., 2016). The same genotype has also been reported prior to its

Açaf Laury – Thèse de doctorat - 2018 description in Cyprus (Tartaglione et al., 2016) in Greece, and outside the Eastern Mediterranean in Spain and in Puerto Rico (Accoroni et al., 2016 and references therein). Cells were described as ovate in shape and ventrally pointed with a DV of 42.5–72.5 lm, AP of 20– 32.5 µm and W 26.3–50 µm. The thecal plate formula is Po, 3’,7’’, 6c, 7s, 5’’’, 2’’’’, typical for the Ostreopsis genus. Unlike other Ostreopsis species (with the exception of O. heptagona), O. fattorussoi does show some morphological features that lead unequivocally to its identiﬁcation. These include: i- the curved suture between plate 1’ and 3’ which makes plates them approximately hexagonal ii- the 10 plate that lies in the left half of the epitheca and is obliquely orientated iii- the characteristic shape of plate 6″: its length: width ratio of 1.06 ± 0.11 (0.95–1.2) and the 6’’/5’’ suture length is almost twice as long as that of 6″/7’ (Accoroni et al., 2016). As for toxicity, three of the five strains of O. fattorussoi from Lebanon were found to be toxic, producing OVTX-a and structural isomers OVTX-d and -e, so far found only in O. cf. ovata (Accoroni et al., 2016). The same was found for other strains from Cyprus, as well as isobaric palytoxin, whereas others produce three exclusive palytoxin-like compounds (OVTX-i, OVTX-j1, OVTX-j2, and OVTX-k; (Tartaglione et al., 2016). The total toxin content the toxin content was relatively low (0.06–2.8 pg / cell) compared to that of O. cf. ovata. Eco-toxicological tests also revealed lower toxicity to Artemia salina nauplii (Tartaglione et al., 2016).

2.5. Quantification of BHABs

As a tychoplanktonic species, quantification of the genus Ostreopsis can be done simultaneously in the planktonic and benthic compartments (e.g. Mangialajo et al., 2011; Lemée et al., 2012). Benthic cells are however considered as the stock of available biomass (Mangialajo et al., 2011) and it is necessary to focus on the benthic compartment, as it is more conservative and more representative of the realistic risk (Lemée et al., 2012). Sampling methods in the planktonic compartment are well defined and allow meaningful comparisons to be made among different studies. Water is collected near the surface and the cells are fixed and quantified using the Utermöhl method (Utermöhl, 1958). The benthic compartment is far more complex; the substrates are diverse, and benthic species have a patchy distribution leading to high variability in results. Benthic sampling is therefore more challenging, and a number of different sampling methods were developed, each with its own advantages and limitations. Previously, these methods fell in three categories: vacuum collection, substrate sampling and artificial substrate (GEOHAB, 2012). Recently, another method has been developed such as the BEDI: Benthic Dinoflagellates Integrator (Mangialajo et al., 2017). A molecular method using quantitative PCR has

Açaf Laury – Thèse de doctorat - 2018 also been developed (Perini et al., 2011), however molecular techniques are not universally available. The most widespread method so far has been substrate sampling, where macroalgae is collected and benthic cells are quantified relative to the wet weight of macroalgae. The diversity of the methods and substrates make it difficult to establish a meaningful comparison among different studies, thus the need for the development of standardized sampling method.

3. Dinoflagellates as Bio-Indicators. Target Genus: Ceratium

3.1. Generalities

The dinoflagellate genus Ceratium Schrank 1793 is a planktonic dinoflagellate belonging to the Class Dinophyceae, Order Gonyaulacales and the family Ceratiaceae (algaebase.org). In recent years, Gómez et al. (2010) suggested splitting of Ceratium sensu lato into two genera based on molecular and morphological data: Ceratium sensu stricto would still designate freshwater species, whereas a new genus name, Neoceratium F. Gómez, D. Moreira & P. López-García would designate the marine species of Ceratium sensu lato. However, this nomenclature was contested (Calado and Huisman, 2010) and deemed illegitimate and nomenclatural priority was then given for the genus Tripos Bory (Gómez, 2013). In the present work, we chose to use the nomenclature by Sournia (1967) for the following reasons:

• The lack of consensus around this new nomenclature • Homogeneity with previous bibliographic references for the sake of the readers • The role of monographs and illustrations in simplification of the daunting task of identification

Basionyms and taxonomical synonyms can be checked in (Gómez et al., 2010) and (Gómez, 2012-a, 2013)

3.2. Morphology and Identification

Ceratium cells are motile due to the typical dissimilar flagella inserted in the cingulum and the sulcus. They have a typical dinokaryotic nucleus. They are large cells, somewhat dorsoventrally compressed and have a distinctive ventral area made out of fine platelets. They are easily recognizable in light microscopy thanks to their large size and distinctive outline often resembling a fork or an anchor

Açaf Laury – Thèse de doctorat - 2018 due to cell extensions referred to as “horns”. Horns are formed by extension of the thecal plates, the apical horn is formed by apical palates, the right and left antapical horns are formed by postcingular and antapical plates respectively. A fourth horn is atypical in marine species. Like Ostreopsis, but unlike most armoured dinoflagellate, tabulation is not a distinctive taxonomical character for this genus, as all marine taxa share the same plate pattern: Po, 4′, 6″, 5c, 2+s, 6″′, 2″″ and sulcal platelets (Steidinger and Tangen, 1996). Despite that, taxonomy and morphology are tightly linked for this genus. In fact, identification is based solely on morphological characteristics such as their shape, direction, length, and curvature of horns, total length of the cell, shape and width of the body, epitheca in relation to hypotheca, cell contour and surface markings. Four sub-genera are recognizable (Figure 3) through general direction of the horns (Tunin-Ley et al., 2012):

• Subg. Archaeceratium (no apical horn, or flattened apical horn) • Subg. Ceratium (antapical horns directed posteriorly) • Subg. Amphiceratium (cells elongated to the anteroposterior axis, right antapical horn absent or vestigial) • Subg. Tripoceratium (antapical horns curved forward, most diversified subgenus)

Figure 3 Figure 3- Representatives of the four sub-genera A: C. praelongum, Subg. Archaeceratium; B: C. furca, Subg. Ceratium, C: C.inflatum, Subg. AmphiCeratium; D: C. tripos, Subg. Tripoceratium

Açaf Laury – Thèse de doctorat - 2018

Identification at the species and subspecies level proves challenging, due to the vast morphological variability and subtle intraspecific morphological differences. In his monograph, Sournia (1967) provides detailed descriptions and illustrations at the subspecies level. He also came up with an unconventional nomenclature system which acknowledges the link between thermal preferences of the taxa with their morphology, leading other authors to designate “psychrophilic” and “thermophilic” varieties in their descriptions. Indeed, psychrophilic taxa are smaller in size with short horns but robust and commonly present crests, whereas thermophilic species usually have thinner longer horns and a slender appearance. Though the link between temperature and morphological variability and temperature was clear, the process behind it was uncertain. Sournia (1967) presented two hypothetical explanations: it is either an adaptation for floatability by increasing their surface and reducing their mass, or it was due to faster growth in warmer waters.

3.3. Ecology

Ceratium often represents a significant part of the microphytoplankton in terms of abundance and diversity, especially within the thecate dinoflagellates. They possess chloroplasts and contribute substantially to annual primary production (Nielsen, 1991; Dodge and Marshall, 1994), but can be heterotrophes or mixotrophes, as attested by the presence of food vacuoles (Steidinger and Tangen, 1996). The formation of resting cysts is rarely observed (Gómez et al., 2010). Some species are known to form red tides causing discoloration of the water, but harmful effects are limited to anoxic conditions. The genus has a rare taxonomic richness with exceptional morphological variability. It is found year round and covers a cosmopolitan distribution, from the arctics to the tropics both in neritic and oceanic waters (Graham, 1941; Sournia, 1967; Dodge and Marshall, 1994) and inhabits both marine (70 species) and freshwater environments (7 species) (Gómez, 2012-a). Species are essentially eurythermic and thermophile, with various tolerance concerning temperature (Sournia, 1967).

3.4. Role as biological models and bio-indicators

Beside the impact on morphology, temperature has a demonstrated effect on the biogeography of the genus. Many authors have proposed Ceratium as a biological indicator, either to water masses (Dodge, 1993; Okolodkov and Dodge, 1996; Ochoa and Gómez Caballero, 1997; Raine et al., 2002), marine currents (Dowidar, 1971), climate change and ocean warming (Dodge and Marshall, 1994; Johns

Açaf Laury – Thèse de doctorat - 2018 et al., 2003; Tunin-Ley and Lemée, 2013). Dodge and Marshall (1994) also demonstrated the effect of temperature on biogeography of this genus in the North Atlantic. Finally, (Tunin-Ley and Lemée, 2013) provided a list of features of the genus that match the prerequisites for ecological indicator validation (Table 2).

Table 2- Table 2- A list of features of Ceratium that match the prerequisites for ecological indicator validation Source: Tunin-Ley and Lemée, 2013

4. Global change in the Eastern Mediterranean Sea

Global environmental changes either due to anthropogenic activities or to natural variability, threaten both human and natural systems. Related drivers such as increasing temperature, ocean acidification, altered ocean circulation, and changes in nutrient availability can alter by distribution and phenology of organisms (Field et al., 2014). The Mediterranean Sea is a potential model of more global patterns (Lejeusne et al., 2010). It is one of the most responsive regions to global change and is considered as a prominent “climate change hotspot” (Giorgi, 2006). It is also considered a “marine biodiversity hotspot” (Bianchi and Morri, 2000; Boudouresque, 2004) as it harbors 14 to 18% of specific marine biodiversity (depending on the phylum considered) despite only accounting for 0.82% of the world’s ocean surface (Cartes et al., 2004). The eastern Mediterranean appears to be greatly affected by these changes. For instance, sea surface temperature has increased of 1°C to 3.5 °C in recent decades (Por, 2008) sea level rising by up to 11.9 mm/year (Fenoglio-Marc, 2002), compared to 1.1°C and 0.4 mm/year increase in the western Mediterranean Sea. Studies not specific to the eastern basin have shown a drop by 0.05 to 0.15 pH units (Hassoun et al., 2015) and rainfall (by 4 et 27 %), enhanced surface stratification (Mermex, 2011) and an increase in frequency and intensity of extreme weather events (GIEC et al., 2008). The prediction of coming changes in ecosystems structures and functioning as a

Açaf Laury – Thèse de doctorat - 2018 response to these changing environmental drivers is not a simple task. Changes in phytoplankton communities provide a sensitive early warning for climate-driven perturbations to marine ecosystems (Hallegraeff, 2010). According to Hallegraeff, (2010), the following changes are to be expected: i- range expansion of warm-water species at the expense of cold-water species, which are driven poleward; ii- species specific changes in the abundance and seasonal window of growth of HAB taxa; iii- earlier timing of peak production of some phytoplankton; iv- secondary effects for marine food webs, notably when individual zooplankton and fish grazers are differentially impacted (‘‘match-mismatch’’) by climate change. Few studies exist in the eastern Mediterranean, but some changes in phytoplankton have already been reported in the Lebanese coast: a continuous appearance of rare species, exceptional blooms of species normally present at low densities, and an increase in frequency and density of this study’s benthic model genus Ostreopsis (Abboud Abi-Saab, 2009). The planktonic model genus Ceratium is expected to reflect environmental changes in the offshore waters.

5. Thesis objectives

Dinoflagellates are a group of microscopic algae that are a major component of marine phytoplankton and o microphytobenthos. Microalgae can have huge impacts. Through photosynthesis, carbon sequestration, and their position at the basis of the food chain, they contribute to the production of 50 % of the oxygen that we breathe, removal of carbon dioxide that we produce, and are essential for the production of the (sea) food that we eat. Some can be bioluminescent, used in the food and pharmacology industries, and proliferate in large densities termed “blooms”. These blooms can simply cause discoloration of the water, or have harmful effects by depleting the oxygen, or even by the production of toxins. In this case, they are termed Harmful Algal Blooms (HABs). These HAB events are on an increasing trend in terms of geographic distribution, intensity and frequency, probably due to ocean warming and climate change (e.g. Hallegraeff, 2010). This trend has been observed in the Mediterranean Sea, considered a climate change hot spot (Giorgi, 2006), since the early 2000s through the emerging and recurring problem of HABs of the toxic genus Ostreopsis, causing mass human intoxication in several countries (e.g. Durando et al., 2007). In return, some taxa, such as the genus Ceratium can offer an accurate reflection of environmental changes and therefore can be used as biological indicators (Tunin-Ley and Lemée, 2013)

Açaf Laury – Thèse de doctorat - 2018

The present work regards ecology of benthic and planktonic dinoflagellates of the Eastern Mediterranean, which is simultaneously less investigated and greatly affected by sea surface temperature increase (Por, 2008). Its main interest is in the HABs of dinoflagellates through the toxic benthic species Ostreopsis fattorussoi, as it has been reported in high densities in Lebanese coastal waters. In addition, it takes interest in their use as biological indicators of environmental changes through the planktonic model genus Ceratium, benefiting from the availability of net samples previously collected off the coast of Batroun, Lebanon.

The specific objectives of the thesis were:

• Contributing to the optimization of sampling and quantification of BHABs: application to Ostreopsis spp. (see chapter II) • Studying the spatial and temporal distribution of Ostreopsis fattorussoi in Lebanese coastal waters in relation with environmental parameters (see chapter III) • Addressing ecological physiology by characterization of Nitrogen uptake by different strains of O. fattorussoi and O. cf. ovata (see chapter IV) • Detecting environmental changes in the eastern Mediterranean over the last few years through the use of the genus Ceratium as a biological indicator (see chapter V)

Following the present general introduction, constituting Chapter I, each of the thesis objectives is addressed in an article that represents a chapter (Chapters II, II, IV and V). Finally, the general discussion and perspectives of this work are presented in the last chapter (Chapter VI).

6. Valorization of the research done during this thesis

Each research theme studied during this thesis represents a chapter of this manuscript. The results of the studies are published or are to be published in international scientific journals. Below is a list of scientific articles as well as contributions to international and local seminars.

Açaf Laury – Thèse de doctorat - 2018

Scientific articles

Chapter II:

• Vassalli, M., Penna, A., Sbrana, F., Casabianca, S., Gjeci, N., Capellacci, S., Asnaghi, V., Ottaviani, E., Giussani, Pugliese, L., Jauzein, C., V., Lemée, R., Hachani, M.A., Turki, S., Açaf, L., Abboud-Abi Saab, M., Fricke, A., Mangialajo, L., Bertolotto, R., Totti, C.M., Accoroni, S., Berdalet, E., Vila, M. Chiantore, (2018). Intercalibration of counting methods for Ostreopsis spp. blooms in the Mediterranean Sea. Ecological Indicators, 85: 1092-1100. • Jauzein, C., Acaf, L., Asnaghi, V., Fricke, A., Hachani, A., Aboud-Abi Saab, M., Chiantore, C., Mangialajo, L., Totti, C. Turki, S. & R. Lemée. Optimization of sampling, cell collection and counting for the monitoring of benthic HAB: application to Ostreopsis spp. blooms in the Mediterranean Sea. Ecological Indicators, 91: 116-127.

Chapter III:

• Açaf, L., Abboud-Abi Saab, M., Khoury-Hanna, M. & R. Lemée. Developments of the newly described dinoflagellate Ostreopsis fattorussoi along the Lebanese coast (East Mediterranean) in relation with environmental factors. in revision in Harmful Algae.

Chapter IV:

• Açaf, L., Jauzein, C., Gémin, M.P., Marro, S., Blasco, T., Abboud Abi-Saab, M ., Amzil, Z. & R. Lemée. Nitrogen uptake of 3 potential nitrogen sources (Nitrate, ammonium and urea) in cultures of different strains of Ostreopsis cf. ovata and the newly described Ostreopsis fattorussoi. To be submitted to Marine Pollution Bulletin.

Chapter V:

• Açaf, L., Abboud-Abi Saab, M., & R. Lemée. In preparation. Updated inventory of different taxa of the genus Ceratium in Lebanese waters (East. Mediterranean) and their use as bio-indicators for detecting environmental changes in the area. To be finalized and submitted.

The results of this thesis were also valued through posters and oral presentations in international conferences and local seminars.

Açaf Laury – Thèse de doctorat - 2018

Posters and Oral Communications:

In International Conferences:

• Açaf, L., Abboud - Abi Saab, M. and Lemée, R. 2017. Benthic Harmful Algal Blooms of Ostreopsis fattorussoi in Beirut, Lebanon (Eastern Mediterranean) in relation with environmental factors. 11th International Conference On Modern And Fossil Dinoflagellates, 17 - 21 Juillet 2017, Bordeaux, France. • Açaf, L., Abboud - Abi Saab, M. and Lemée, R. 2016. Monitoring of Benthic Harmful Algal Blooms of Ostreopsis sp. in Naqoura South of Lebanon (Eastern Mediterranean). 41st CIESM Congress, 12 - 16 September 2016, Kiel, Germany. • Açaf, L., Abboud - Abi Saab, M. and Lemée, R. 2016. Preliminary results of the development of Benthic Harmful Algal Blooms in Lebanese coastal waters with emphasis on Ostreopsis species. The 22nd International Scientific Conference of the Lebanese Association for the Advancement of Science, Université Saint Esprit Kaslik, 14-15 April 2016, Kaslik, Liban.

In Local Seminars (Dissemination):

- Acaf, L., Sampling methodology of the dinoflagellate Ostreopsis sp., Seminar on Environmental Water and Toxic Algae in Lebanon, December 10, 2015, Jounieh, Lebanon

- Acaf, L., “Risk Monitoring, Modeling and Mitigation of Harmful Algal Blooms along benthic Mediterranean coasts: The Development of Harmful Algal Blooms of Ostreopsis sp. in Lebanese Coastal Waters”. Water – DROP Conference - Capitalization Meeting, Jounieh, Lebanon, May 23rd 2016.

Açaf Laury – Thèse de doctorat - 2018

Chapter II: Quantification of Ostreopsis sp.: Optimization and intercalibration of new techniques

Objective: Contributing to the optimization of sampling and quantification of BHABs: application to Ostreopsis spp.

Part 1: Optimization of sampling, cell collection and counting for the monitoring of benthic HAB: application to Ostreopsis spp. blooms in the Mediterranean Sea

To be cited as: Jauzein, C., Acaf, L., Asnaghi, V., Fricke, A., Hachani, A., Abboud-Abi Saab, M., Chiantore, C., Mangialajo, L., Totti, C. Turki, S. & R. Lemée. Optimization of sampling, cell collection and counting for the monitoring of benthic HAB: application to Ostreopsis spp. blooms in the Mediterranean Sea. Ecological Indicators, 91: 116-127.

Açaf Laury – Thèse de doctorat - 2018

Part 2: Intercalibration of counting methods for Ostreopsis spp. blooms in the Mediterranean Sea

To be cited as: Vassalli, M., Penna, A., Sbrana, F., Casabianca, S., Gjeci, N., Capellacci, S., Asnaghi, V., Ottaviani, E., Giussani, Pugliese, L., Jauzein, C., V., Lemée, R., Hachani, M.A., Turki, S., Açaf, L., Abboud-Abi Saab, M., Fricke, A., Mangialajo, L., Bertolotto, R., Totti, C.M., Accoroni, S., Berdalet, E., Vila, M. Chiantore, (2018). Intercalibration of counting methods for Ostreopsis spp. blooms in the Mediterranean Sea. Ecological Indicators, 85: 1092-1100.

Açaf Laury – Thèse de doctorat - 2018

Chapter III: Bloom dynamics of the newly described toxic benthic dinoflagellate Ostreopsis fattorussoi along the Lebanese coast (Eastern Mediterranean)

Objective: Studying the spatial and temporal distribution of Ostreopsis fattorussoi in Lebanese coastal waters in relation with environmental parameters

To be cited as: Açaf, L., Abboud-Abi Saab, M., Khoury-Hanna, M. & R. Lemée. Developments of the newly described dinoflagellate Ostreopsis fattorussoi along the Lebanese coast (East Mediterranean) in relation with environmental factors. In revision in Harmful Algae.

Açaf Laury – Thèse de doctorat - 2018

Bloom dynamics of the newly described toxic benthic dinoflagellate Ostreopsis fattorussoi along the Lebanese coast (Eastern Mediterranean)

Laury Açaf1, 2*, Marie Abboud-Abi Saab1, Mirella Khoury-Hanna1,3, Rodolphe Lemée2 1. National Council for Scientific Research, National Center for Marine Sciences, BP 534, Batroun, Lebanon 2. Sorbonne Université, CNRS-INSU, Laboratoire d’Océanographie de Villefranche, LOV UMR 7093, F-06230, Villefranche-sur-Mer cedex 3. Lebanese University, Faculty of Sciences II, Biodiversity: Management and Conservation of Natural Resources, Fanar, Lebanon *Email: [email protected]

Abstract

The present study involves monitoring of both benthic and planktonic abundances of the toxic dinoflagellate Ostreopsis fattorussoi in 5 stations along the Lebanese coast (Eastern Mediterranean); it extended from June 2014 to July 2016 and aims to study the dynamics of its development. Results revealed its presence year round along the coast, presenting seasonal blooms, both epiphytic and planktonic. A major bloom (maximum of 880 694 cells/g FW and 18 000 cells/L) occurred early in the summer (June-July), followed by a minor one in the fall (October-November) (≤ 50 000 cell/g FW). Its development seemed to be influenced by a combination of multiple environmental parameters. Water temperature seemed to play a major role in its development, as it appeared to be a thermophilic species, with maximum abundances per site reached between 27°C and 30.5°C. Its development could be facilitated by some agitation resulting from winds. Results concerning the role of nutrients and other environmental parameters remain inconclusive.

Keywords: Ostreopsis fattorussoi, Benthic HABs, Dinoflagellates, microphytobenthos, Lebanese coast, Eastern Mediterranean.

Açaf Laury – Thèse de doctorat - 2018

1. Introduction

The geographical distribution of the benthic dinoflagellate Ostreopsis spp. covers tropical and subtropical areas (Ballantine et al., 1988; Grzebyk et al., 1994; Tindall and Morton, 1998; Parsons and Preskitt, 2007) as well as temperate areas in New Zealand (Chang et al., 2000; Rhodes et al., 2000), Australia (Pearce et al., 2001), Japan (Taniyama et al., 2003) and the Mediterranean Sea (Vila et al., 2001b; Aligizaki and Nikolaidis, 2006; Mangialajo et al., 2011). Some studies suggest that it was possibly introduced from tropical to temperate areas via ship ballast water (Butrón et al., 2011), or as a result of the anthropogenic global climate change and warming of sea water (Masó and Garcés, 2006; Miraglia et al., 2009), whereas others suggest it could be indigenous to the Mediterranean Sea (Tognetto et al., 1995).

Two species, Ostreopsis cf. siamensis J. Schmidt and Ostreopsis cf. ovata Fukuyo, have been known for years in the North Western Mediterranean Sea; the latter in particular has bloomed in the area (Tognetto et al., 1995; Mangialajo et al., 2011) and is the subject in most available literature. Its presence was first reported in the Mediterranean Sea in the bay of Villefranche-sur-mer (France) in 1972 (Taylor, 1979). Both species are known to produce palytoxin-like metabolites. Palytoxin (PTX) is among the most potent known naturally produced toxins of marine origin even at extremely low concentrations (Moore and Scheuer, 1971; Amzil et al., 2012a) and has been linked to several human intoxications in tropical areas (Alcala et al., 1988).

Since the early 2000s, Ostreopsis spp. emerged as a threat to public health in the Mediterranean Sea. Many cases of human intoxication have been linked to this genus in the Western Mediterranean Sea (Masó and Garcés, 2006; Durando et al., 2007; Kermarec et al., 2008; Vila et al., 2008, 2016; Pfannkuchen et al., 2012). In some cases, more than 200 individuals were affected in one bloom episode, eg. Genoa in 2005 (Durando et al., 2007) and Algeria in 2009 (Illoul et al., 2012). The absence of reports of intoxication in the Eastern Mediterranean Sea could be due to undiagnosed cases or is simply a reflection of the meagerness of related studies in the area. The intoxication occurs either through direct contact with cells or through inhalation of aerosols (Sansoni et al., 2003b; Ciminiello et al., 2006, 2014; Tichadou et al., 2010). The affected population is therefore not limited to bathers and people involved in water activities, but also includes residents and people in the vicinity of the bloom area who are not in direct contact with the water. Reported symptoms in the Mediterranean Sea include

Açaf Laury – Thèse de doctorat - 2018 irritation to the eyes, nose, skin and respiratory system, fever and rarely respiratory distress (Simoni et al., 2003; Ciminiello et al., 2006; Tichadou et al., 2010; Vila et al., 2016) and have led to hospitalization (Durando et al., 2007). In addition, PTX and PTX-like metabolites have been found in edible marine organisms from different trophic levels (Biré et al., 2013, 2015). For these reasons, despite the fact that cases of intoxications through ingestion of seafood have not yet been reported in the Mediterranean Sea, they remain a possible risk. These toxins also represent a threat to the ecosystem itself, as they have been linked to a mass mortality (Illoul et al., 2012), decrease in population (Shears and Ross, 2009; Blanfune et al., 2012) and a negative impact on reproduction of some marine fauna (Guidi-Guilvard et al., 2012). As the occurrence of Ostreopsis blooms is accompanied by closing of beaches (Tichadou et al., 2010) and bad press, the consequences extend into the economy. An estimated “worst case scenario” could translate into losses of up to millions of euros in touristic areas such as the South East of France (Lemée et al., 2012) and this could be expected in other areas where the economy is based on fishing, aquaculture, ports and/or seasonal tourism attracted by coastal activities. As a result, Ostreopsis spp. has been studied extensively in the Western Mediterranean Sea in the last two decades. The biotic and abiotic factors driving its development are still not fully understood. In the Eastern Mediterranean Sea, related studies remain scarce. In Lebanon, Ostreopsis genus has been detected since 1979 (Abboud-Abi Saab, 1989). In a previous study, it was identified as Ostreopsis cf. siamensis, based on morphological characteristics (Abboud-Abi Saab et al., 2013). But in 2016, Accoroni et al. (2016) using molecular analysis, found mainly the new species named Ostreopsis fattorussoi Accoroni, Romagnoli & Totti and very few Ostreopsis cf. ovata. The presence of this last species was almost negligible (Casabianca S. and Penna A. pers. comm., in Accoroni et al., 2016). It was then likely, but not certain, that Ostreopsis cf. siamensis cited by Abboud-Abi Saab (1989) was a misidentification. Ostreopsis fattorussoi has also been reported prior to its description in Cyprus (Tartaglione et al., 2016), Greece, and outside the Eastern Mediterranean in Spain and in Puerto Rico (Accoroni et al., 2016)and references therein). As for toxicity, three of the five strains of O. fattorussoi from Lebanon were found to be toxic, producing OVTX-a and structural isomers OVTX-d and -e, so far found only in O. cf. ovata (Accoroni et al., 2016a). The same was found for other strains from Cyprus, whereas others produce three exclusive palytoxin-like compounds: (OVTX-i, OVTX-j1, OVTX-j2, and OVTX-k (Tartaglione et al., 2016). It is therefore crucial to study the ecology of this newly described species.

The present work is the first to study the ecology of O. fattorussoi in Lebanese coastal waters (Eastern Mediterranean) based on both epiphytic and planktonic abundances, through investigation of

Açaf Laury – Thèse de doctorat - 2018 their temporal and spatial distribution along the coast and their relation to environmental factors. It extends from June 2014 to July 2016, thus covering 3 summer bloom periods in 5 sites along 172 km of the coastline.

2. Material and methods

2.1. Study area and sampling frequency

Lebanon is situated in the East central part of the Levantine Basin (Eastern Mediterranean), with 220 km of coast, from Aarida in the north to Naqoura in the South. The continental shelf is very narrow (3 to 4 km; (Pfannenstiel, 1960) with a mean depth of 20 m to 40 m (Goedicke, 1972). The bottoms are rough with wide rocky patches (Majdalani, 2004), potentially providing support for proliferation of benthic biota. The general circulation along the coast of Lebanon is dominant northwards during most of the year along with the general counterclockwise current gyre of the Eastern Mediterranean (Goedicke, 1972). This current progressively loses force as it approaches eastwards (Nakhle, 2003), in addition, the tide is semidiurnal and is among lowest in the Mediterranean with a maximum range of 15 to 20 cm (Abboud-Abi Saab et al., 2004). The current is locally modified by the configuration of the coastline and the topography of the narrow continental shelf. Water movements along the coast are also strongly related to surface currents and seasonal meteorological factors (Goedicke, 1972). As a result, the small scale hydrodynamism is dominated by local winds. According to Abboud Abi-Saab (1985), wind of west direction is dominant during 2/3 of the year while wind from the north direction dominate during autumn and winter, whereas during this study wind of west direction was dominant on most sampling days (data not shown). All our sampling sites are not in a Bay and west winds can easily be associated to waves and then be a proxy of local agitation. Streams nearby BYB and TRI are seasonal as most of the water comes from rain and snow melting in the nearby mountains. They potentially bring in municipal wastewater and agricultural effluents rich with nutrients.

Preliminary sampling was conducted in May 2014 in 11 sites in order to scan most rocky beaches of the Lebanese Coast. It revealed the presence of O. fattorussoi in all 11 sites. The monitoring campaign that followed starting June 2014 was limited to five of the sites due to logistic restraints (such as site accessibility year round). The five sites were distributed from North to South and are thus representative of the entire coast. They are labeled as follows (North to South): TRI in Anfeh (close to Tripoli), BAT in

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Batroun, BYB in Byblos-Jbeil, BEY in Beirut and NAQ in Naquoura. They are represented in Figure 1 and described briefly in Table 1. Sampling was conducted on weekly basis in BAT, and on monthly basis in the remaining sites until December 2015. Additional sampling was conducted in TRI and BAT in the spring and summer of 2016 (Table 1). BYB is in proximity of a seasonal fresh water stream. In presence of Northern winds, BAT is affected by the discharges from a chemical plant located 3.9 Km its North (Abboud Abi-Saab, 1985) BYB and TRI are nearby beach resorts. NAQ and BEY are in proximity of small ports.

Figure 4 Figure 1 - A map showing the position of Lebanon on the Eastern Mediterranean coast as well as the location of the sampling sites during the study (preliminary & regular sampling sites)

Table 3- Table 1- Description and geographical coordinates (in decimal degrees) of the five regular sampling sites and of the timing and frequency of the sampling. Footnotes: * : Sampling frequency was on weekly basis in 2016. Site Sampling Sampling Dominant Site Latitude Longitude description end date frequency macroalgae TRI 34.3675 35.736 Rocky Jul 2016 Monthly* E. elongata BAT 34.2515 35.6568 Rocky Jul 2016 Weekly E. elongata BYB 34.1144 35.6482 Sandy/ Rocky Dec 2015 Monthly Variable BEY 33.902 35.4753 Rocky Dec 2015 Monthly E. elongata NAQ 33.1284 35.1458 Rocky Dec 2015 Monthly E. elongata

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2.2. Environmental parameters

Air temperature (Tair) was measured in situ using a mercury thermometer. Wind speed (WS) in m/s, and total precipitation in the week preceding sampling date (Rain) were obtained from the website windguru.cz which bases its forecasts on data produced by weather forecast models. Wind speed was used as a proxy of water agitation at the sampling date. Maximum wind speeds reached in the previous week (WSmax) of sampling were also used in each sampling site in order to analyze influence of previous conditions on Ostreopsis development. Water temperature (Twater) was measured in situ using a mercury thermometer at 10 cm depth. Salinity was measured using the Beckman © RS7-C salinometer. Samples destined for nutrients measurements were kept at -20˚C until they were analyzed within one month. A - single sample of 500 ml was collected for both Nitrites and Nitrates analysis. Nitrites (NO2 ) concentrations were measured following the methodology described by (Bendschneider and Robinson, - 1952) and Nitrates (NO3 ) were obtained according to the methodology described by (Strickland and Parsons, 1968) with a slight modification by (Grasshoff, 1964). A 250 ml sample was collected for

Orthophosphates (P-PO4) measurements. Orthophosphates concentrations were measured following the methodology described by (Murphy and Riley, 1962). N/P ratio (molar) is also calculated and corresponds to the Dissolved Inorganic Nitrogen (DIN) that were measured during the study (Nitrites + Nitrates in µmol/L) over total Orthophosphates (µmol/L).

2.3. Biological parameters

2.3.1. Sampling

Ostreopsis fattorussoi being a tychoplanktonic species, both benthic (macroalgae) and planktonic (sea water) samples were collected, in triplicates at three sampling stations assigned 10 m apart on each site. Planktonic samples were collected first, at 20 cm depth (30 cm above the macroalgae) using plastic bottles of 250 ml, with minimal disturbance to the substrate in order to avoid false results due to the resuspension of epiphytic O. fattorussoi into the water column. For the benthic samples, 9.5 ± 6.0 g of macroalgae were collected along with some surrounding water at the same spot at 50 cm depth, using plastic bottles of 250 ml. Typically, the red macroalgae Ellisolandia elongata (J.Ellis & Solander) K.R.Hind & G.W.Saunders was collected. The choice of macroalgae (Table 1) was based on dominance per site and availability on all sites, and was maintained for the most part throughout the sampling

Açaf Laury – Thèse de doctorat - 2018 period to limit the influence of different substrata that has been reported by several authors such as Vila et al. (2001-a) and Totti et al. (2010). Lugol solution at 1% vol/vol was added to the samples for preservation and they were stored at 4°C.

2.3.2. Quantification of O. fattorussoi

For planktonic samples, after homogenization, subsamples of 50 ml were settled in a counting chamber for 24 hours, according to Utermöhl’s sedimentation method (Utermöhl, 1958). Counting was performed using an inverted microscope Wild M40. Cells abundances were expressed in number of cells per liter (cells/L). For benthic samples, the sample was shaken vigorously for 10 seconds in order to dislodge the cells from the macroalgae. The macroalgae was then separated from the water using a 200 µm mesh sieve, and rinsed twice with 100 ml of filtrated sea water, until the complete removal of any remaining epiphytic O. fattorussoi cells. The total water (sample + rinsing water) was homogenized using a Motoda box. This box is a splitting apparatus (often used for zooplankton samples) allowing the separation of homogeneous subsamples (Motoda, 1959). One final 50 ml subsample was kept at 4°C. For counting, 1 ml of the subsample was placed in a 1ml Sedgwick-Rafter © counting chamber. The microscope used is the Motic BA 400. Cell abundance (C) was expressed in number of cells of O. fattorussoi per gram of fresh weighed macroalgae (cells/g FW). Identification of Ostreopsis cells at the species level was carried out in some samples through observation of thecal plates’ arrangements using calcofluor staining (Figure 2), since O. fattorussoi does show some morphological features that lead unequivocally to its identification (Accoroni et al., 2016). At least two samples per site were observed, selected from different bloom events belonging to different seasons. Only Ostreopsis fattorussoi species was found. Ostreopsis cf. ovata was never observed.

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Figure 5 Figure 2- Light micrographs of calcofluor stained Ostreopsis fattorussoi cells under UV light, showing the tabulation. Left: epitheca from TRI (October 2015). Right: hypotheca from BEY (October

2015). Apical pore Po (left image, under 2’) and 1’ plate forms are characteristic of O. fattorussoi (see

description in Accoroni et al., 2016). DV: left (75 µm) and right (65 µm).

2.4. Statistical analysis

All statistical analysis was applied using R programming language version 3.3.2 and its user interface R studio. It is to be noted that weekly data obtained in BAT were included in the graphs but excluded from statistical analysis as certain corresponding environmental data were not available; monthly data from this site were included in the analysis. As O. fattorussoi was sampled in triplicates, the site average was used in the dataset. Subsequently, the complete dataset consisted of 111 observations, of which 27 were from TRI, 25 from BAT, 17 from BYB, 21 from BEY, and 21 from NAQ. The Shapiro test of normality was applied to each variable (benthic and planktonic O. fattorussoi abundances as well as all available environmental variables). Accordingly, the non-parametric spearman rank correlation test was applied to a matrix including all the above mentioned variables in order to determine their relationships. The same test was repeated per site. A non-parametric independent measurement ANOVA (Kruskal–Wallis) was applied to both planktonic and benthic abundances of O. fattorussoi in order to evaluate possible spatial variations between the sites; a non-parametric repeated measurement ANOVA (Friedman) was also applied per site to determine temporal variability. A PCA

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(Principle Component Analysis) was applied using environmental variables as active variables, benthic and planktonic abundances of O. fattorussoi as quantitative illustrative variables, and sites as qualitative illustrative variable. A PERMANOVA was performed 999 random permutations on a dataset including the three stations’ individual measurements. Analyses were based on Euclidean distance-transformed data.

3. Results

3.1. Environmental parameters

3.1.1. Climatological parameters

Overall, measured air temperature (Tair) varied between a minimum of 14°C (BAT and BYB, Jan 2015) and a maximum of 36.3°C (NAQ, May 2015) (Table 2). It varied following a seasonal pattern and the graphs had similar shapes in all 5 stations. The highest temperatures were recorded in the summer and lowest in winter (Figure 3 A). Wind speed ranged between a minimum of 0 m/s (Dec 2015, BAT) and a maximum of 13 m/s (Feb

2015, NAQ) (Figure 3 B). Maximum Wind speed recorded in the previous week (WSmax) ranged between 3 m/s (Dec 2015, BEY) and 17 m/s (Jan 2015, BAT). WSmax was generally lower in the autumn months. WSmax was also highest in the most northern site TRI, (Figure 3 C). Total precipitation during the previous week reached a maximum of 125 mm/week (NAQ, Jan 2015), followed by a max of 79.9 mm/week in BAT (Jan 2015) (Figure 3 D). Summer was the dry season and maximum precipitation for each site was in January.

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Figure 6 Figure 3- Graph showing the variation of climatological parameters: A: Air temperature “Tair” (°C), B: Wind speed “WS” (m/s) and C: Maximum wind speed reached during the previous week

“WSmax” and D: Precipitation in the previous week “Rain WB” (mm)

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Table 4 Table 2 - Descriptive statistics (average ± Standard deviation and Minimum-Maximum) of all variables in the 5 sites

3.1.2. Hydrological parameters

Water temperature (Twater) ranged between a minimum of 17 °C (BAT, Mar 2015) and a maximum of 32°C (BAT, Aug 2014). In all 5 sites, Twater variation followed a typical seasonal pattern with the highest temperatures in the summer and lowest in winter (Figure 4 A). In TRI the autumn of 2014 water temperature remained high throughout autumn. High water temperatures also stand out in BAT in the summer of 2014 and spring of 2016. In each sites, water temperature corresponding to the maximum of abundance of O. fattorussoi (TCmax) never exceeds the maximum temperature (Tmax) reached on site (Figure 5 A). A gradient of TCmax vs. latitude was observed, as TCmax increases from south to north (Figure 5 B). The average measured salinity was 38.6 ± 1.3, ranging between a minimum of 34.5 (Jan 2015, BYB) and a maximum of 39.6 (Oct 2014, TRI). Salinity was higher in the most northern sites TRI and BAT as it rarely drops below 38.5; It was lower in NAQ (South) and BYB as it barely reaches 39.1 (Figure 4 B).

Açaf Laury – Thèse de doctorat - 2018

Figure 7 Figure 4- Graph showing the variation of hydrological parameters: A: Water temperature “Twater” (°C) and B: Salinity in the 5 sites during the sampling period

Figure 8 Figure 5 - Graph showing A: the variation of benthic O. fattorussoi abundances as a function of water temperature “Twater” (°C) and B: Distance between the sites as a function of water temperature corresponding to maximum abundances of O. fattorussoi (TCmax) in the 5 sites during the sampling period

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3.1.3. Nutrients

Nitrites concentration ranged between a minimum of 0.008 µmol/L (TRI, Jun 2015) and a maximum of 0.37 µmol/L (NAQ, Aug 2014) (Table 2). They were generally highest in BEY, and lowest in TRI and NAQ (Figure 6 A). Nitrates concentration ranged between a minimum of 0.06 µmol/L (TRI, Mar 2016) and a maximum of 25.12 µmol/L (NAQ, Jan 2015) (Table 2); it was highest in NAQ and BYB and lowest in BAT and TRI (Figure 6 B). Orthophosphates concentration ranged between a minimum of 0.01 µmol/L (BEY, Sep 2014) and a maximum of 3.53 µmol/L (NAQ, Aug 2015) (Table 2). They remained below 0.5 µmol/L in all sites except for occasional peaks in the summer (Figure 6 C). N/P ratio (molar ratio) ranged between a minimum 0.15 (TRI, Jun 2016) and a maximum of 964.05 (TRI, Feb 2016); it was highest in NAQ and lowest in BAT where it remained mostly below 16 throughout the sampling period (Figure 6 D).

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Figure 9 Figure 6 - Graph showing the variation of nutrients: A: Nitrites (µmol/L), B: Nitrates (µmol/L),

C: Orthophosphates (µmol/L) and D: N/P ratio in the 5 sites during the sampling period

3.2. Temporal and spatial variation in O. fattorussoi abundances

3.2.1. Seasonal variation

Cells of Ostreopsis fattorussoi were found year round in all the five sites in benthic samples and almost year round in planktonic samples. The most important benthic blooms (more than 200 000 cells/g FW) occurred almost exclusively during the summer. Less important blooms occurred in autumn, with maximum abundance around 50 000 cells/g FW, with the exception of one major autumn bloom of 2014

Açaf Laury – Thèse de doctorat - 2018 in NAQ (228 257 cells/g FW, October). In winter, an extreme drop in abundance was observed, and increases in abundance were noted starting early spring (Figure 8). In 2014, important benthic summer blooms occurred in all of the sites (often more than 200 000 cells/g FW), whereas in 2015, they were only detected in TRI, BYB and NAQ. In 2016, data was only available for TRI and BAT; in both cases, summer blooms of 2016 (TRI and BAT) were minor in terms of abundance (< 50 000 cells/g FW). In addition, minor spring blooms were detected in BAT (April and May). Planktonic abundances generally followed the same pattern as the benthic ones. In addition, planktonic blooms were observed in BAT and BEY in the summer of 2015, where benthic blooms were not detected (Figure 7). Yearly maximum abundances were variable; in 2014, they reached 880 694 cells/g FW (BEY, June) and 9 000 cells/L (BAT, June & July), as opposed to 397 242 cells/g FW (BYB, July) and 18 065 cells/ L (NAQ, June) in 2015, then 53 228 cells/g FW and 4 346 cells/L (BAT, ) in 2016. The ANOVA Friedman test showed no significant difference in cell abundances in either of the sites, neither in terms of seasons nor in terms of years (p > 0.05). Spearman rank correlation test showed a very strong significant correlation between benthic and planktonic O. fattorussoi (r = 0.88, p < 0.01) (Table 3).

Table 5 Table 3 - Spearman rank correlation between O. fattorussoi abundances and environmental variables. “OstreoB”: benthic O. fattorussoi abundances. “OstreoPl”: planktonic O. fattorussoi abundances. (* p < 0.05, and ** for p < 0.01)

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Figure 10 Figure 7 - Graph showing the variation of O. fattorussoi abundances (Cells/L) in planktonic sea water samples at 20 cm depth in the 5 sites during the sampling period

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Figure 11 Figure 8 - Graph showing the variation of epiphytic O. fattorussoi abundances (Cells/g FW) in benthic samples at 50 cm depth in the 5 sites during the sampling period

Açaf Laury – Thèse de doctorat - 2018

3.2.2. Spatial variation

Bloom occurrence also varied between the sites. TRI, BYB and NAQ all presented major planktonic and benthic summer blooms both in 2014, and with higher abundances in 2015. As for BAT and BEY, the summer blooms were minor in 2015 (less than 5 000 cells/g FW), whereas planktonic blooms were relatively important (often more than 2 500 cells/L). BEY had the highest abundances of O. fattorussoi during the summer bloom of 2014, reaching almost three times the maximum recorded on any other site (880 694 cells/g FW), and BYB in 2015 (397 242 cells/g FW). In 2014 and 2015, NAQ presented the most durable summer blooms that continued into autumn blooms with the highest abundances. In 2015, weekly sampling in BAT allowed the detection of two consecutive major blooms, during the second half of June then during the second half of July. In 2016, minor summer blooms occurred in both sampled sites, reaching their maximum abundances of 32 262 cells/g FW (BAT) and 45 398 cells/g FW (TRI) mid-June. Planktonic abundance followed the same pattern with a relatively high maximum of 4 346 cells/L (BAT). Spring blooms were detected in BAT starting in April and reached a maximum benthic abundance of around 50 000 cells / g FW both in April and in May. The Kruskal-Wallis test showed that no significant difference was noted between the 5 sites for both benthic (Kruskal-Wallis chi-squared = 104.04, p-value > 0.05) and planktonic abundances (Kruskal- Wallis chi-squared = 70.061, p > 0.05).

3.2.3. Relation between O. fattorussoi abundances and environmental parameters

Water temperature was strongly correlated with benthic abundances of O. fattorussoi (r = 0.61, p < 0.01) and moderately correlated with planktonic abundances (r = 0.49, p < 0.01) when considering all 5 sites; however, it varied between the sites from very strong in TRI (r =0.8, p < 0.05) to non-significant in NAQ (p > 0.05) (Table 3). A weak positive correlation was noted between wind speed and planktonic abundances (r = 0.26, p < 0.01), but none with WSmax. Strong negative correlation with precipitation was noted only in BEY (Table 3). No significant correlation was noted with the measured nutrients, with the exception of a moderate negative correlation between Nitrates and benthic abundances in BYB (r = - 0.53, p < 0.05) and BEY (r = -0.59, p < 0.05), and N/P BEY (Table 3). The first two principal components (PC) of the PCA analysis (Figure 9) amount to a cumulative percentage of the variance of

61.5%. The first PC (29.37%) is primarily a measure of Twater and Salinity, and is strongly but negatively correlated to Nitrates. The second PC (18.95%) mainly reflects wind speed, and is strongly correlated to

WSmax. The PCA did not reveal a significant influence of a specific environmental parameter on O.

Açaf Laury – Thèse de doctorat - 2018 fattorussoi abundances. The PERMANOVA test highlighted a significant effect of the interaction between different parameters such as of Twater*WSmax as well as the interaction of Wind speed*RainWB*N/P on O. fattorussoi abundances (p < 0.01).

Figure 12 Figure 9 - Principal Component Analysis Variable factor map (left) and individuals factor map (right) for environmental parameters (quantitative active variables), benthic and planktonic O. fattorussoi abundances (quantitative illustrative variables) and site (qualitative illustrative variable)

4. Discussion

4.1. Temporal and spatial variation in O. fattorussoi abundances

Major blooms of O. fattorussoi mostly occurred earlier in the summer, no later than July. Maximum yearly abundances per site were reached mostly in June and July (3 out of 9) although they could occur in August (1 out of 9) and in October (1 out of 9). Despite the blooms being site and year specific, a clear seasonal pattern was common to all sites. Ostreopsis fattorussoi cells were barely detectable in winter, but an increase in abundance was visible starting early spring. Yearly maximum abundances were reached in the summer, followed by less important peaks in the autumn, also reported in a previous study 2001 in the same area (Abboud-Abi Saab et al., 2013) (Table 4). Second blooms have also been reported in the North Western Mediterranean Sea, only a bit earlier (late summer-early fall) (Mangialajo et al., 2011). Summer blooms of O. fattorussoi occur early compared to all other

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Mediterranean sites. In addition, a couple of blooms were recorded as early as April and May, making them - along with one occurrence in the Aegean Sea in - the only spring blooms of Ostreopsis spp. reported in the Mediterranean Sea.

6 Table 4 - Maximum benthic (cells/g FW) and planktonic abundances (Cells/L) of Ostreopsis spp. reported in various areas in and outside the Mediterranean Sea. *: Sampling limited to these two months; **: Associated with toxic events; ***: Sporadic co-occurrence of O. siamensis is possible; Bold = maximum abundance; MS: Mediterranean Sea; Non MS: Non Mediterranean Areas; NW: North West; NA: North Adriatic; S: South; E: East; MaxB: Maximum benthic abundances; MaxPl: maximum planktonic abundances; n.a.: Not available

Maximum abundances reached during blooms of a species belonging to genus Ostreopsis in the Mediterranean Sea were variable. They were however similar in the Northern part of the Mediterranean Sea where the specie O. cf. ovata is predominant, as they reach millions of cells/g FW in benthic samples, and exceed 105 cells/L in planktonic samples (Table 4). In this study, maximum abundances remained below 400 000cells / g FW, with only one exception (880 694 x105 cells / g FW). Major blooms were absent on 2 of the sites for two years; instead, they were replaced by minor blooms, with abundances around or below 50 000 cells/g FW. Second blooms occurred in the autumn; these were

Açaf Laury – Thèse de doctorat - 2018 minor in terms of magnitude. Planktonic abundances reached 18x103 cells/L on one occasion, but remained below 9 000 cells/L for the most part of the sampling period. Both benthic and planktonic abundances remained considerably lower than those of Northern MS. It was worth indicating that as maximum abundances are year and site specific in NW MS, some sites presented lower maximum abundances than neighboring sites (Mangialajo et al., 2011), that were similar to those of O. fattorussoi in this study. In contrast, maximum O. fattorussoi abundances reached in this study exceed those reported in the Eastern and Southern Mediterranean. Outside the Mediterranean Sea, maximum abundances are variable. Compared to this study, they were much higher in New Zealand (Shears and Ross, 2009), and much lower in Mexico (Okolodkov et al., 2007) and in the Sea of Japan (Marina S Selina et al., 2014). Nevertheless, comparing maximum benthic abundances of Ostreopsis species is always difficult, since biotic substrates preferences can heavily affect this parameter, even at low spatial scale (Cohu et al., 2011).

Data from the more frequently sampled site (BAT) suggest that bloom duration is around one month, and bloom decline occurs within a week from reaching maximum abundances. Longer blooms such as the ones occurring in NAQ are not uncommon in the North western Mediterranean Sea (Mangialajo et al., 2011)

4.2. Influence of environmental factors on O. fattorussoi abundances

4.2.1. Temperature

Our data suggests that water temperature plays a major role in the development of O. fattorussoi. We found a moderate and strong positive correlation between water temperature and planktonic and benthic cell abundances, respectively. Maximum abundances per site were reached at relatively high water temperature (27°C < TCmax < 30.5°C). In the Mediterranean Sea, several studies have linked optimum growth of Ostreopsis spp. to relatively high temperatures (Sansoni et al., 2003b; Simoni et al., 2003; Mangialajo et al., 2008; Cohu et al., 2013a), whereas others did not find a correlation between temperatures and its abundances (Vila et al., 2001b; Totti et al., 2010; Accoroni et al., 2011, 2012b; Cohu et al., 2011; Mangialajo et al., 2011). In the present study, O. fattorussoi was found year round, thus in all the measured range of temperature during the study (17°C to 32°). We might consider 18°C (threshold temperature associated with cell abundances of ≥1 000 cell/g FW) triggers a swifter proliferation of available cells in their vegetative state. In the present study, maximum abundance was

Açaf Laury – Thèse de doctorat - 2018 recorded as water temperature was still increasing, whereas in Adriatic Sea, maximum abundances of O. cf. ovata were recorded as water temperature is decreasing (Totti et al., 2010; Accoroni et al., 2012;

Accoroni et al., 2015). In both cases, TCmax is lower than Tmax (in the present study in each of the 5 sites). The different TCmax per site could be due to the presence of different strains of O. fattorussoi on different sites; in fact this is not unusual for different species, as several studies have showed species, clades or subclades to respond differently in terms of temperature (Guerrini et al., 2010; Pezzolesi et al., 2012; Tawong et al., 2014).

4.2.2. Nutrients

In this study, taking into account the entire dataset, we found no correlation between O. fattorussoi abundances and any of the measured nutrients, nor with the N/P ratio. Orthophosphates concentrations in BAT did not seem to be affected by the nearby chemical factory. We did however find a moderate negative correlation when the correlation test was performed on site basis, between Nitrates and benthic abundances (in BEY and BYB), and with N/P ratio (in BEY). In both cases, the results are inconsistent with a previous study in the same area (3 sites in common) by Abboud -Abi Saab et al. (2013), that found a positive correlation of O. cf. siamensis abundances with Nitrite, Nitrates and N/P ratio, and a negative correlation with orthophosphate. Results were also conflicted in the Mediterranean, and the link between nutrients and Ostreopsis spp. has yet to be established. Several studies found no evidence of a role of nutrients in the bloom dynamics of Ostreopsis spp. (Vila et al., 2001; Accoroni et al., 2011; Accoroni et al., 2012). (Carnicer et al., 2015) found a negative correlation between O. cf. ovata and Nitrates, whereas, despite the absence of correlation, (Cohu et al., 2013) has reported a positive association between Phosphate concentration, (rather than nitrogen or silicate), and O. cf. ovata. Accoroni et al. (2015) found that N/P ratio around the Redfield value was necessary for cell proliferation of O. cf. ovata. The lack of a statistically significant correlation in these studies as well as the present one was not unexpected, as during the bloom cells were presumably consuming the nutrients (Accoroni et al., 2015).

Ammonium was not analyzed in the present study, while this nutrient was found to be important Ostreopsis species growth in a recent study (Jauzein et al., 2017). This could have influenced the present interpretation, mostly when using N/P ratio. More generally, analysis of every supplementary parameter, including organic sources, could be useful for interpretation when studying the ecology of Ostreopsis

Açaf Laury – Thèse de doctorat - 2018 species, since at least one species has been proved to be mixotrophic (Ostreopsis cf. ovata; (Jauzein et al., 2017).

4.2.3. Cell Resuspension and Wind

In this study we found a strong positive correlation between benthic and planktonic abundances of O. fattorussoi, as confirmed by (Vila et al., 2001-b) for Ostreopsis sp. also on samples of the macroalgae Ellisolandia elongata. A relatively good correlation between the two is also reported in the Mediterranean Sea for O. cf. ovata and O. cf. siamensis (Aligizaki and Nikolaidis, 2006; Mangialajo et al., 2008, 2011). This could be due to re-suspension of epiphytic cells into the water column caused by waves (Vila et al., 2001b; Totti et al., 2010; Mangialajo et al., 2011) as reflected by the positive correlation between planktonic abundances and wind speed, which in turn influences water agitation on a local level. Re-suspension is also brought on due to human activity causing movement of the substrata (Mangialajo et al., 2011), or due to daily cycle vertical migrations (Vila et al., 2008). Cell resuspension might partially explain the absence of a major benthic bloom in BAT and BEY in the summer of 2015, despite the occurrence of a planktonic one. Another explanation could be due to the heterogeneity of Ostreopsis sp. distribution on a small scale (within a few meters), revealed in this study by high standard deviation of O. fattorussoi abundance between triplicated samples (Figures 7 and 8). In addition, there was a positive correlation between wind speed and benthic abundances of O. fattorussoi in TRI; as water agitation at each site could be influenced by local winds, these results suggest that the species might prefer some agitation, as also observed for Ostreopsis spp. in other studies (Vila et al., 2001b; Marina S. Selina et al., 2014). The effects of water agitation on blooms of Ostreopsis spp. in the Mediterranean Sea might be variable, as it has been suggested that has a negative effect as higher abundances are found in sheltered or calm areas (Barone, 2007; Mangialajo et al., 2008; Totti et al., 2010; Accoroni et al., 2012b), as wind and waves could disperse the bloom (Cohu et al., 2011; Accoroni and Totti, 2016).

A single environmental parameter did not solely influence the development of O. fattorussoi, but it is a combination of several factors; the PCA did not highlight the influence of a specific environmental factor on its abundance, and the PERMANOVA showed a combined effect of water temperature, water agitation due to wind and Nitrates availability. This has also been reported for O. cf. ovata (Mangialajo et al., 2008; Accoroni et al., 2015)

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5. Conclusion

The present study found that O. fattorussoi is present year round in Lebanese coastal waters, with seasonal blooms along the coast. A major bloom occurs early in the summer (June-July), followed by a second minor one in the fall (October-November). Results showed that a combination of several environmental factors affect its development. It was revealed to be a thermophilic species, as the highest concentrations were reached between 27°C and 30.5°C depending on the site. A minimum of 18°C is suggested as a threshold temperature promoting proliferation of O. fattorussoi. Moreover, its development seemed slightly facilitated by some water agitation due to wind. Results concerning the role of nutrients and other environmental parameters remain inconclusive. Major blooms coincide with the summer where beaches are most frequented and minor blooms coincide with autumn, a busy period for recreational fishing (Sharif Jemaa, personal communication, June 6th, 2016). It is therefore recommended to maintain a monitoring program throughout the year, focusing on benthic samples as they are more conservative (Lemée et al., 2012). It should be intensified mid spring till the end summer, not only as a mitigation measure, but also in order to allow more thorough ecological studies of all phases of the bloom. It is also important to assess its toxins in the food chain, as it is customary for local inhabitants of the coastal zone to consume certain species of macroalgae and seafood including filtrating sea urchins and shellfish (Patella sp.). Cases of intoxication, if any, would have gone undiagnosed due to the lack of awareness on the issue in public health officials and medical practitioners as well as the general public in Lebanon.

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Chapter IV: Nitrogen uptake of the benthic toxic dinoflagellates Ostreopsis cf. ovata and Ostreopsis fattorussoi of the Mediterranean Sea

Objective: Addressing ecological physiology by characterization of Nitrogen uptake by different strains of O. fattorussoi and O. cf. ovata

To be cited as: Açaf, L., Jauzein, C., Gémin, M.P., Marro, S., Blasco, T., Abboud Abi Saab, M ., Amzil, Z. & R. Lemée. Nitrogen uptake of 3 potential nitrogen sources (Nitrate, ammonium and urea) in cultures of different strains of O. cf. ovata and the newly described O. fattorussoi. To be submitted to Marine Pollution Bulletin, pending co-authors approval.

Açaf Laury – Thèse de doctorat - 2018

Nitrogen uptake of the benthic toxic dinoflagellates Ostreopsis cf. ovata and Ostreopsis fattorussoi of the Mediterranean Sea

Laury Açaf 1, 2, Cécile Jauzein3, Marin-Pierre Gémin4, Thierry Blasco1, Sophie Marro1, Marie Abboud- Abi Saab2, Zouher Amzil4, Rodolphe Lemée1 1. Sorbonne Université, CNRS, Laboratoire d’Océanographie de Villefranche, 06234 Villefranche-sur- Mer, France 2. National Council for Scientific Research, National Center for Marine Sciences, BP 534, Batroun, Lebanon 3. Ifremer, Centre de Brest, DYNECO PELAGOS, F-29280 Plouzané, France 4. Ifremer, Centre de Nantes, DYNECO PHYC, Rue de l'île d'Yeu, Nantes 44311, France *Email: [email protected]

Abstract

Nitrogen availability could be a limiting factor of microalgae growth. It also has various effects on toxicity of HABs species. This study characterized N uptake abilities of two North Western strains of Ostreopsis cf. ovata as well as two Eastern Mediterranean strains of Ostreopsis fattorussoi, and the + potential ecological considerations. Three N potential N-sources were investigated: inorganic NH4 and - NO3 and organic N-urea. Results revealed mixotrophy in all the strains. Three of the strains showed the + - following order of preference for N-sources: NH4 > NO3 > N-urea, with the exception of one strain of + - O. fattorussoi that had similar uptake abilities for NH4 and NO3 . Some variability was also noted in uptake abilities between the four strains, including strains of O. fattorussoi isolated simultaneously in the same geographic area. The intraspecific variability often exceeded interspecific one. A combination + in variability in uptake of NH4 as well as in toxin production between the strains of O. fattorussoi led us + to suggest that summer NH4 availability would selectively favor the development of a toxic vs. a non- toxic strains of O. fattorussoi in Lebanese coastal waters. It also led to the hypothesis that a possible link between N-availability, N uptake ability with toxicity in O. fattorussoi.

Keywords: N-Uptake, dinoflagellate, O. fattorusosi, O. cf. ovata, kinetics, Mediterranean

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1. Introduction

Benthic Harmful Algal Blooms (B-HABs) of the genus Ostreopsis have increased in recent years in the Mediterranean Sea, both in intensity and in frequency. Since the early 2000s, Ostreopsis developments, mainly of Ostreopsis cf. ovata, have been linked to several cases of mass human intoxication on the Western coasts of the Mediterranean Sea. Cases of intoxication were reported for both swimmers, through direct contact with cells, and beaches users or residents, exposed to toxic bio- aerosols (Durando et al., 2007; Illoul et al., 2012; Ciminiello et al., 2014). For O. cf. ovata, these toxins have been identified as palytoxin (PLTX), one of the most potent toxins of natural marine origin, and its derivatives, ovatoxins (OVTX-a, OVTX-b, OVTX-c, OVTX-d, OVTX-e, OVTX-f, OVTX-g, OVTX-h, OVTX-l) of unknown toxicity (e.g. Ciminiello et al., 2006, 2008, 2014; Tartaglione et al., 2017). No cases of intoxication have been reported on the Eastern part of the Mediterranean Sea yet. The species found in Lebanon and Cyprus has been recently described as Ostreopsis fattorussoi (Accoroni et al., 2016a). Different strains of O. fattorussoi may display different toxicities, as some strains were revealed to be non-toxic, whereas others produce palytoxin, and OVTX-a, OVTX-d, and OVTX-e, or some exclusive ovatoxins, such as OVTX-i, OVTX-j1, OVTX-j2, and OVTX-k (Accoroni et al., 2016a; Tartaglione et al., 2016).

In light of the health, ecological and economic risks associated with these BHABs events (Lemée et al., 2012) , many studies have focused on bloom dynamics of Ostreopsis spp. and their control by environmental factors such as water temperature, hydrodynamism, salinity, light intensity and depth. Results from field studies remain conflicting, however, even for the same species (O. cf. ovata). While water temperature seems to be a leading factor, it appears that a synergy between water temperature, hydrodynamism and nutrients determines bloom dynamics (Accoroni et al., 2015). Conversely, ecological physiology of Ostreopsis sp. is greatly under-investigated, as for all benthic dinoflagellates.

In this study, nitrogen (N) uptake abilities of different strains of Ostreopsis were characterized for three inorganic and organic sources, ammonium, nitrates and N-urea. In addition, this study aims to look into the potential ecological outcomes, particularly, the potential effects of N availability conditions on the development and toxicity of Ostreopsis strains.

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2. Material and methods

2.1. Kinetic experiments

2.1.1. Strains

Four strains of the genus Ostreopsis were used in the present work: (i) two strains of Ostreopsis cf. ovata, MCCV48 that was isolated from Hercule port, Monaco (43° 44' 5.12" N, 07° 25' 33.48" E, August 2014), and MCCV51 isolated from Haliotis Nice, France (N 43° 40' 37.4''E 7° 13' 49'' E, September 2014) and (ii) Two strains of Ostreopsis fattorussoi, MCCV57 and MCCV58, that were isolated from Batroun Lebanon (34°15’4.32”, 35°39’25.08” E, February 2015). The cultures for the experiments were all monoclonal strains, obtained after single cell isolation, and maintained in the Mediterranean Culture Collection of Villefranche (MCCV).

2.1.2. Medium and Culture conditions

The medium used for maintaining the cultures and running the experiments consisted of a modified K/10 medium (Keller et al 1987), prepared with old filtrated and autoclaved seawater. Modifications of the original formulation were as followed: addition of silicate and Tris base was omitted, phosphorus was added as KH2PO4 (final concentration of 5 μM), ZnSO4 was added at a final concentration of 0.02 nM and urea was added at 5 μmol N/L final concentration in order to acclimate the strains to this specific organic N-source. Cultures were grown in flasks that were placed horizontally in order to maximize the growth surface and gas exchange. They were maintained in an incubator, at a fixed temperature of 23˚C, and exposed to light with an intensity of 200 μmol photons. m-2. s -1 and Light/ Dark (L/D) cycles of 16 h/8 h.

2.1.3. Dilution and replicate cultures

For each strain, a stock culture was grown in 15 ml of culture medium (flask s.a. of 25 cm2). A series of successive dilutions was made over several weeks, bulking up the stock cultures into 350 ml (flask of 410 ml and 300 cm2). This volume was then used to inoculate three replicate cultures of 350 ml, each destined for the characterization of uptake capabilities for one of the three studied nutrients - + )Nitrates NO3 , ammonium NH4 or urea CO(NH2)2(.

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2.1.4. Resuspension in an N free medium

Cells were re-suspended in an N-free medium in order to control the concentration of dissolved N available during the kinetic experiment and limit it to the labelled N. This was realized using gravity filtration on a 10 µm nylon membrane as described in Jauzein et al. (2017).

2.1.5. Incubation

Each culture was divided into a series of eight 40 ml flasks and served for the characterization of uptake kinetics for one of the three N sources. One labelled nutrient was added to each series following a gradient of 8 concentrations: 0.1, 0.2, 0.3, 0.5, 1, 2, 3, 5 and 10 µatg N/L. Incubation was stopped after one hour by filtration using a syringe and a pre-combusted and pre-weighed filter (A/E Life Science, 0.3 µm pores). They were rinsed with 20 ml of either 0.2 µm Filtrate Sea Water or N-free medium. Filters were then dried (24h at 60˚C), weighed and stored at room temperature until analysis. Measurements of particulate nitrogen (PN) and 15N/14N isotopic ratios were obtained by EA-IRMS (Elemental Analysis – Isotope Ratio Mass Spectrometry), using an Elementar Vario Pyro Cube analyzer in CN mode (combustion oven 920°C, reduction oven 600°C) coupled to an Isoprime 100 IRMS (Isotope Resolved Mass Spectrometer). Calibration of measurements was performed with certified caffeine (AIEA-600) and other laboratory standards (commercially available glycine (Sigma), acetinalide (Merck). Uptake rates (V in h-1) were calculated from the 15N enrichment of the samples, according to (Collos, 1987). These measurements produced saturating kinetics that were fitted to the Michaelis-Menten equation:

VN = Vmax × [N] / (Ks + [N])

-1 -1 - Where VN (h ) is the uptake rate at a concentration of [N] (μmol N L ), Vmax is the maximal velocity (h 1), and Ks is the half saturation constant (μmol N L-1). The slope α was also estimated at C = 0.5 μmol N L-1 using to the Michaelis-Menten model, as an indicator of N uptake abilities under low N conditions.

2.1.6. Growth rate

Cell abundances were estimated on subsamples of the cultures 48 h prior and the day of the incubation, to ensure that cultures were in exponential phase at the time of the experiment. Growth rates were calculated using the equation by (Guillard, 1973):

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where µ is the growth rate per day, C1 and C2 are cell concentrations (cell L-1), and t1 and t2 are the respective days of subsampling.

2.2. Toxin profile analysis for MCCV57 and MCC58

2.2.1. Toxin extraction

Toxin analysis was determined for the O. fattorussoi strains MCCV57 and MCCV58, at the end of exponential phase and during the stationary phase. Several ml of culture were filtered through GF/F filter (Whatmann). Palytoxin (PLTX) and ovatoxins (OVTXs) were extracted by addition of 5 mL of methanol/water (1:1 v/v), then sonicated twice for 40 minutes in an ice bath. The extract was centrifuged (4000 rpm, 10 min, 4°C) and the supernatant was collected. The pellet was rinsed with 5 mL of methanol/water (1:1 v/v) and centrifuged. The supernatant was discarded and the two extracts were pooled then filtrated through 0.22µm (Nanosep MF 0.2µm) for LC-MS/MS analysis.

2.2.2. LC-MS/MS analysis

Analysis of toxin content was performed using a LC system (UFLC XR, Shimadzu, Champs-sur- Marne, France) coupled to a hybrid triple quadrupole/linear ion-trap mass spectrometer (API 4000 Qtrap, AB SCIEX, Les Ulis, France) equipped with a turbospray. Toxins were separated at 40°C, in a Kinetex XB-C18 column (150 x 2.1mm, 2.6µm, Phenomex, Le Pecq, France) and eluted at 0.2mL/min flow rate. The mobile phase consisted of a mix of: water (A) and acetonitrile/water (95:5 v/v) (B) both containing 0.2% of acetic acid. The following gradient elution was used: 25-40% A over 20 min, 40- 90% A in 1 min and hold for 3 min before re-equilibration for 5 min. Injection volume was 5µL. Palytoxin (Wako Chemicals GmbH, Neuss, Germany) was used as a standard and concentration of OVTXs are expressed in equivalent of PLTX.

Mass spectrum detection was carried out in multiple reactions monitoring (MRM) using positive mode. The following source settings were used: curtain gas 30 psi, ion spray at 5000 V, turbogas temperature of 450°C, gas 1 and 2 set at 30 and 40 psi respectively. The energy collision of 31 eV was 2+, 2+ applied for the bi-charged ions [M+2H] [M + 2H - H2O] (declustering potential (DP) = 56 V) and 47

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3+ eV for tri-charged ions [M + 3H - H2O] (DP= 56 V) to produce characteristic fragments m/z 327, 343 and 371 (Fragment A). Transition for the detection of each toxin was summarized in Table 1.

Table 7 Table 1 - Transitions used for detection of palytoxin and ovatoxins

2+ Fragment [M + 2H - Fragment [M + 3H - Fragment Analogues [M+2H] → 2+ → 3+ → A H2O] A H2O] A PLTX 1340.3 → 327.3 1331.3 → 327.3 887.8 → 327.3 OVTX-a 1324.3 → 327.3 1315.3 → 327.3 877.2 → 327.3 OVTX-b 1346.3 → 371.2 1337.3 → 371.2 891.8 → 371.2 OVTX-c 1354.3 → 371.2 1345.3 → 371.2 987.2 → 371.2 OVTX-d 1332.3 → 327.3 1323.3 → 327.3 882.5 → 327.3 OVTX-e 1332.3 → 343.2 1323.3 → 343.2 882.5 → 343.2 OVTX-f 1338.3 → 327.3 1329.3 → 327.3 886.5 → 327.3

3. Results

3.1. Kinetic experiments

Average growth rates showed the cultures were in exponential phase at the time of the kinetic experiment (Table 2).

Table 8 Table 2 - Average growth rate µ (day-1) of the fours strains of Ostreopsis spp. preceding the kinetic experiments Geographic µ (day-1) Species Strain origin (average ± sd) O. cf. ovata Monaco MCCV48 0.49 ± 0.16 O. cf. ovata France MCCV51 0.66 ± 0.29 O. fattorussoi Lebanon MCCV57 0.55 ± 0.11 O. fattorussoi Lebanon MCCV58 0.53 ± 0.12

- + The four strains were capable of using the three N sources tested, NO3 , NH4 and N-urea (Figure 1). As a common characteristic between these Ostreopsis strains, poor abilities to take dissolved organic N in the form of urea was observed all along the concentration gradient tested. Maximal uptake rates -1 -1 (Vmax) for N-urea ranged between 0.0005 h and 0.0008 h . These parameter values were distinctly + - lower than Vmax obtained for NH4 or NO3 for each of the four strains tested (Figure 1, Table 3). Concerning uptake abilities under low N-conditions, estimations of the initial slope  also showed + - distinctly lower abilities to take up N-urea than the other N-sources, NH4 and NO3 . This parameter ranged between 0.0002 L μmol N-1 h-1 and 0.0006 L μmol N-1 h-1 (Figure 1, Table 3).

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Concerning uptake of inorganic N-sources, differences between strains were observed, including between strains of the same species that were often greater than interspecific ones. For O. cf. ovata strains isolated from the Western part of the Mediterranean Sea, N-uptake characteristics were close and + - clearly highlight a preference pattern following the ranking NH4 > NO3 > N-urea throughout the gradient tested (Figure 1 A and B, Table 3).

- For O. fattorussoi strains isolated along Lebanese coasts (Eastern Mediterranean Sea), NO3 uptake characteristics were close and similar to the strains of O. cf. ovata, in particular to the Monaco + strain MCCV48 (Figure 1 C and D, Table 3). Strong differences were noted for NH4 uptake. One strain MCCV58 showed higher uptake abilities than the O. cf. ovata strains throughout the tested gradient of + - concentration (higher alpha and higher Vmax), and maintained the NH4 > NO3 > N-urea (Figure 1 D). On the other hand, the strain MCCV57 showed similar uptake abilities for the two inorganic N sources - - in high N availability, and a slight preference to NO3 at low N conditions (similar Vmax and  NO3 >  + NH4 , Figure 1 C, Table 3).

-1 -1 -1 -1 Table 9 Table 3 - Values of kinetic parameters (Vmax in h , Ks in μmol N L , α in L μmol N h ), and mean N-uptake rates (h-1) obtained for the different strains of Ostreopsis cf. ovata and Ostreopsis cf. fattorussoi.

MCCV48 MCCV51 MCCV57 MCCV58 N- Strain source Monaco Nice Lebanon Lebanon

-1 Vmax (h ) 0.0171 0.0214 0.0175 0.0318 R2 (p-value) 0.9661 0.9524 0.9766 0.9724 + NH4 Ks (μmol N L-1) 2.3891 2.6151 6.2141 3.0258 α (L μmol N-1 h-1) 0.006 0.0069 0.0026 0.0091

-1 Vmax (h ) 0.0077 0.0105 0.0114 0.0105 R2 (p-value) 0.9855 0.9454 0.9629 0.9881 - NO3 Ks (μmol N L-1) 1.5785 1.304 2.2873 2.547 α (L μmol N-1 h-1) 0.0038 0.0058 0.0041 0.0035

-1 Vmax (h ) 0.0005 0.0008 0.0008 0.0006 N- R2 (p-value) 0.8528 0.9409 0.7551 0.9454 urea Ks (μmol N L-1) 0.5541 3.9179 0.6951 1.529 α (L μmol N-1 h-1) 0.0004 0.0002 0.0006 0.0003

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+ - Figure 13 Figure 1 - Kinetic curves of NH4 (in blue), NO3 (in red)_and N-urea (in green) uptake for different strains of Ostreopsis cf. ovata and Ostreopsis cf. fattorussoi. Data points show variations of + - NH4 , NO3 and N-urea uptake rates as a function of each respective nutrient concentration. Respective modeled curves correspond to Michaelis-Menten model and values of parameters (Vmax, Ks, α), as well as associated r² were listed in Table 3.

3.2. Toxin profiles for O. fattorussoi strains

For the strain MCCV58, the total toxin content per cell decreased by 55% between end of exponential phase (4.28 pg cell-1) and the stationary phase (1.69 pg cell-1) (Figure 2). The major analogue detected at the end of the exponential phase was OVTX-a making up 63% of the total toxin content. Other analogues, putative palytoxin (p-PLTX), OVTX-d, OVTX-e and OVTX-F were also found at 11%, 11%, 8% and 7% of the total toxin content, respectively. At the stationary phase, OVTX-a was also the major analogue 53% of the total toxin content. Ovatoxin-d and p-PLTX corresponded to

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24% and 22% of the total toxin content respectively, while OVTX-e and OVTX-f were not detected. As for the strain MCCV57, none of the toxins could be detected above the detection limit of 70 ng per ml of extract.

Figure 14 Figure 2 - Toxin profile in % (histogram bars) and total concentration in pg cell-1 (white dots) for the two Eastern Mediterranean strains of O. cf. fattorusoi MCCV57 and MCCV58 at the the end of exponential phase and in real stationary phase

4. Discussion

Nutrient enrichment and coastal eutrophication due to anthropogenic activities have been linked to the increase in occurrence of HABs in recent years (Glibert and Burkholder, 2006; Glibert et al., 2010). Though nutrient availability is considered an important factor in the development of blooms of Ostreopsis spp., a synergy between several environmental factors, that also include water temperature and hydrodynamism, seems to affect Ostreopsis bloom dynamics (Accoroni et al., 2015). Therefore, establishing a direct link between nutrients and Ostreopsis spp. abundances through in situ studies has proven to be a complex task. Field studies have produced conflicting results regarding the role of various nutrients (Abboud-Abi Saab et al., 2013; Cohu et al., 2013a; Carnicer et al., 2015), when others

Açaf Laury – Thèse de doctorat - 2018 report no link to Ostreopsis spp. abundances (Vila et al., 2001-b; Accoroni et al., 2011, 2012-a). The few existing experimental studies focused on the importance of N and P availability on growth of Ostreopsis ovata in cultures (Vanucci et al., 2012). While the role of N availability on growth of Ostreopsis spp. is undeniable, the quantification of a strain’s N uptake abilities allows an evaluation of species competitiveness and the potential ecological effects of its availability on its development in situ. The + - present study characterized N uptake from dissolved inorganic (NH4 , NO3 ) and organic (urea) sources by two Northwestern Mediterranean strains of O. cf. ovata (MCCV48 and MCCV51), as well as two strains of the newly described O. fattorussoi (MCCV57 and MCCV58) collected from Lebanon, in the Eastern Mediterranean Sea. As Jauzein et al. (2017) have already studied N uptake for two strains of Northwestern Mediterranean O. cf. ovata, data are now available in order to compare competitiveness between Ostreopsis species and strains, in addition to comparing between Ostreopsis and others microalgae.

4.1. Competitiveness of Ostreopsis spp.

Compared to other microalgal groups, competitive abilities of the studied strains were relatively -1 low in high N conditions. For ammonium, Vmax obtained for all four strains (min= 0.0171 μmol.h , max=0.0318 μmol.h-1) (Table 3) were within the range reported previously for harmful planktonic dinoflagellates (min= 0.002 μmol.h-1, max=0.075 μmol.h-1) (Lomas and Glibert, 2000; Cochlan et al., - -1 -1 2008). As for NO3 , Vmax for the four strains (min=0.0077 μmol.h , max=0.0114 μmol.h ) was lower than 0.05 μmol.h-1 reported for Prorocentrum minimum (Lomas and Glibert, 2000), and the range reported for planktonic diatoms (min=0.1 μmol.h-1, max=0.17 μmol.h-1) (Lomas and Glibert, 2000; Cochlan et al., 2008). These results are in line with the general trend of low competitive abilities for dissolved inorganic Nitrogen uptake by dinoflagellates compared to diatoms (Jauzein et al., 2017). The -1 same was observed for N-urea, as Vmax obtained for all 4 strains (min= 0.0005 μmol.h , max=0.0008 μmol.h-1) (Table 4) were within the lower range reported previously for harmful planktonic dinoflagellates (min= 0.0004 μmol.h-1, max=0.04 μmol.h-1) (Li et al., 2011; Hu et al., 2014). They were also lower than reported for Pseudo-Nitzchia australis (0.03 μmol.h-1) (Cochlan et al., 2008) and Heterosigma akashiwo (0.0029 μmol.h-1) (Herndon and Cochlan, 2007). At low concentrations, this - competitive disadvantage seems to be reversed a as Ks of NO3 was generally lower for Ostreopsis spp. + than it has been reported for diatoms (Lomas and Glibert, 2000) (except Liban 1 for NH4 and - Skeletonema costatum for NO3 ).

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4.2. General trends in N-uptake abilities between the studied strains

The ability to obtain N from all three N sources, both inorganic and organic, revealed an important common ecological trait for the fours strains: mixotrophy. Mixotrophy has been previously demonstrated by Jauzein et al. (2017) in other Northwestern Mediterranean strains of O. cf. ovata, but not yet for O. cf. fattorussoi. It is broadly defined as the diversification of potential nutrient sources to include both organic and inorganic ones. It presents a competitive advantage compared to obligate phototrophs/heterotrophs by increasing the total potential nutrient uptake and overcoming growth limitation by inorganic sources. This is of particular interest for Lebanon. The coastal area is overpopulated and different sources of organic nutrients are common with various inputs fresh water bodies bringing in litter, untreated sewage and agricultural and industrial effluents, as well as uncontrolled landfills along the seashore (Houri and El Jeblawi, 2007; Fakhri et al., 2008; MoE/UNDP/ECODIT, 2011).

In addition, the strains MCCV48 (Monaco), MCCV51 (France) and MCCV58 (Lebanon) all + - exhibited a clear pattern of preference in the order NH4 > NO3 > N-urea regardless of high or low N conditions. Jauzein et al. (2017) reported the same for two strains of O. cf. ovata collected in Villefranche. This order of preference was less evident for MCCV57 (Lebanon) at high N conditions + - - (Vm NH4 / Vm NO3 = 1.5). Conversely, this strain showed a slight preference to NO3 at low N conditions (Tables 3, Figure 2 C); this preference could possibly be overturned in field conditions, as + - NH4 been shown to inhibit the uptake of NO3 by strains of O. cf. ovata by up to 67% when both + nutrients are made available in the medium (Jauzein et al., 2017). NH4 preference is typical in microalgae due to advantageous energetic requirements of its uptake (Glibert et al., 2016). Being a regenerated nutrient, it is more available in the summer (Mulholland and Lomas, 2008). Its preference could therefore be a contributing factor in the development of blooms of O. cf. ovata as well as O. fattorussoi; as highest cell abundances reported for both species in their respective geographic areas occurred during the summer period (Cohu et al., 2011, 2013a; Mangialajo et al., 2011; Açaf et al., + 2016). These results further support the suggestion that NH4 is the main N source fueling blooms of Ostreopsis spp. (Jauzein et al., 2017).

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4.3. Variability in N-uptake and toxin production

The four strains had similar uptake abilities of NO3-, with a small advantage for the O. ovata + MCCV51 (France) at low N concentrations. The highest variability was observed in NH4 uptake + abilities; while all 4 strains appeared well adapted to a wide range of NH4 , MCCV58 (Lebanon) was clearly the most competitive both at high and low NH4+ availability (highest capacity and affinity respectively). It was followed by MCCV51, MCCV48, and then MCCV57. As for N-urea, MCCV51 had the highest capacity, while MCCV57 appeared to be the most adapted to low N-urea conditions. Results did not allow the designations of a more competitive species, as intraspecific variability exceeded interspecific variability at both high and low N availability conditions, regardless of the studied N source (Table 3). Strains of the same species were often on opposing ends of the range reported for a given Michaelis –Menten parameter (Figure 2, Table 3). The variability between strains of Ostreopsis spp. isolated from different geographic areas could be explained by acclimation and adaptation to the local N availability conditions. As for the variability between the strains MCVV57 and MCCV58 isolated simultaneously in the same site, we could consider two potential ecological strategies: either adaptability or efficiency, to be behind this variability, as suggested by Jauzein et al. (2008) when reporting a diversity in uptake abilities between strains of Alexandrium catenella. A molecular study of a bloom population would be needed verify the accuracy of this assumption.

Evidently, the ecological considerations of this variability are more relevant in the case of co- occurring strains as they would be in direct competition to obtain nutrients. For the two strains of O. + fattorussoi, the biggest variability was for the preferred N source NH4 , as MCCV58 was twice more + efficient in obtaining NH4 at high concentrations, and more than three times more efficient at low concentrations than MCCV57. This advantageous quality for MCCV58 would likely be translated in the + field in the summer period, where NH4 availability would favor its growth over MCCV57. In addition, another important metabolic difference was noted between the two strains. The toxin profile analysis revealed MCCV58 to be toxic, with a total toxin level 4.28 pg eqPLTX/cellat the end of the exponential phase and 1.69 pg eqPLTX/cell at the stationary phase due to its secretion of PLTX, as well as OVTX- d,-e,-f,-g, and -h (Figure 2). On the other hand, no toxins could be found in the case of MCCV57 above + the 70 ng per ml of extract limit of detection, and it was then considered non-toxic. Therefore, NH4 availability in the summer period would favor the development of MCCV58, and thus selectively favor the development of a toxic bloom of O. fattorussoi over a non-toxic one.

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Moreover, for many harmful algal species including some O. cf. ovata strains, toxin production is known to be affected by cell physiology such as growth stage, isolation site, environmental factors such as temperature, salinity, light and nutrients (Guerrini et al., 2010; Pistocchi et al., 2011; Pezzolesi et al., 2014; Carnicer et al., 2016). Few studies exist on the effect of nutrients on toxin production in O. cf. ovata. Vidyarathna and Granéli, (2013) found that nutrient deficiency, particularly N deficiency simultaneously reduced the growth and increased the toxin content of O. ovata cells. Those results are in line with the consensus that algal toxins are secondary metabolites produced under stress (disturbed physiology) (Granéli and Turner, 2006). On the other hand, both N and P limitation have been shown to decrease toxin production in O. cf. ovata, the first more than the latter (Vanucci et al., 2012; Accoroni et al., 2015; Pezzolesi et al., 2016). Authors attributed the decrease in toxins under N limitation to N content in the PLTX and OVTX molecules (Ciminiello et al., 2008), as reported for the N-containing saxitoxins (SXT) and domoic acid produced by Alexandrium spp. (Granéli and Turner, 2006; Pezzolesi et al., 2014 and references therein). Most observations also indicate that for SXT, cells synthesize more toxins when NH4+ is the N source instead of NO3-. In addition, in this study the strain MCCV57 + combined the two metabolic features of toxicity and superior abilities in obtaining NH4 than MCCV58. + This led us to hypothesize a possible link between NH4 and toxin production in O. fattorussoi. This hypothesis remains to be verified as metabolic pathways for biosynthesis of PLTX and OVTXs are still unknown.

5. Conclusion

This study showed N uptake abilities for the fours strains of Ostreopsis to be lower than other microalge groups for the three nutrients NH4+, NO3- and N-urea in the range of 0.1 µatg N .L-1 to 10 µatg N .L-1. All four strain were mixotrophs, which presents a competitive advantage compared to strict phototrophs/heterotrophs. Intraspecific variability exceeded interspecific one. The three strains + - MCCV48, MCCV51 and MCCV58 maintained an order of preference NH4 > NO3 > N-urea, whereas MCCV57 showed similar abilities for the two inorganic N sources in high N availability, and a slight - + preference to NO3 at low N conditions. Strongest variability was noted in uptake of NH4 between the two O. fattorussoi strains isolated in the same site, as MCCV57 was almost twice as competitive as MCCV58. Variablity in toxicity was also noted, as MCCV57 was toxic whereas MCCV58 was + considered non-toxic. This combination in variability in uptake of NH4 as well as in toxin production + led us to suggest that summer NH4 availability would selectively favor the development of a toxic vs. a

Açaf Laury – Thèse de doctorat - 2018 non-toxic strains of O. fattorussoi in Lebanese coastal waters. It also led to the hypothesis of a possible link between N-availability, N uptake ability with toxicity in O. fattorussoi.

Acknowledgments This work was supported by the European Union under the ENPI CBC Mediterranean Sea Basin Program, within the project M3-HABs, and the French ANR project OCEAN 2015. French authors are part of the national French GDR PHYCOTOX (CNRS and Ifremer). The MCCV (Mediterranean Culture Collection of Villefranche) is integrated to EMBRC (European Marine Biological Resources Center).

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Chapter V: Updated inventory of different taxa of the genus Ceratium in Lebanese waters (East. Mediterranean) and their use as bio-indicators for detecting environmental changes in the area

Objective: Detecting environmental changes in the eastern Mediterranean over the last few years through the use of the genus Ceratium as a biological indicator

Açaf, L., Abboud-Abi Saab, M., & R. Lemée. In preparation. Updated inventory of different taxa of the genus Ceratium in Lebanese waters (East. Mediterranean) and their use as bio-indicators for detecting environmental changes in the area. To be finalized and submitted.

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Updated inventory of different taxa of the genus Ceratium in Lebanese waters (East. Mediterranean) and their use as bio- indicators for detecting environmental changes in the area

Laury Açaf 1, 2, Marie Abboud-Abi Saab2, Rodolphe Lemée1 1. Sorbonne Université, CNRS, Laboratoire d’Océanographie de Villefranche, 06234 Villefranche-sur- Mer, France 2. National Council for Scientific Research, National Center for Marine Sciences, BP 534, Batroun, Lebanon *Email: [email protected]

Abstract

The EMT or Eastern Mediterranean Transient is a decadal event that consists of a shift in deep water source of the Levantine basin from the Adriatic to the Aegean Sea, bringing in warmer more saline waters. An EMT-like event occurred from 2005 to 2010 in the Eastern Mediterranean. It was reflected by changes in zooplankton communities in a site four miles off the coast of Lebanon (Point B2). This study aimed to find whether the planktonic dinoflagellates genus Ceratium, a well established biological model and bioindicator, could be used to detected the environmental event. Changes Ceratium abundance and diversity were documented in six annual cycles selected before, during and after the EMT-like event at Point B2. Ceratium population did not reflect environmental changes related to the EMT-like event. The annual cycle was characterized by seasonality predominantly influenced by temperature.

Keywords: Levantine basin, Lebanon, EMT-like, bioindicator, Ceratium

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1. Introduction

The Mediterranean Sea is one of the most responsive regions to global change and is considered as a prominent “climate change hotspot” (Giorgi, 2006). In the Lebanese coast in particular (Levantine basin) a study based in ¨Point B2¨, a long term monitoring site off the coast of North Lebanon, found a significant temporal trend in sea water temperature increase from the year 2000 to 2013, however it was limited to the spring season. Such a trend was not detected in annual sea water temperature (Ouba, 2015). This trend did not seem to affect the zooplankton population at Point B2. In addition, Ouba et al. (2016) found a significant increase in salinity between 2005 to 2010, after which salinity returned to previous conditions. This modification in salinity was accompanied by a significant increase in total zooplankton abundance. It was also concomitant with the EMT-like event occurring in the Eatern Mediterranean during this period (Theocharis et al., 2014a); The EMT or Eastern Mediterranean Transient is a decadal event that consists of a shift in deep water source of the Levantine basin from the Adriatic to the Aegean Sea, bringing in warmer more saline waters. An EMT-like event is similar to the EMT, with less intensity and consequences (Krokos et al., 2014; Theocharis et al., 2014).

In light of these findings, the present work aimed to find whether the genus Ceratium, a well established model genus and bioindicator, could be used to detect these documented environmental changes in the area, particularly the occurrence of the EMT-like event which had a demonstrated effect on several planktonic taxonomic groups (Ouba et al., 2016). The use of biological indicators allows the restraint of monitoring efforts to relevant responsive parameters. Variations in abundance and diversity the genus Ceratium were documented at Point B2 along with environmental parameters in selected years before, during and after the occurrence of the EMT-like event in Lebanese waters (Levantine basin).

2. Material and Methods

2.1. Study site

The sampling site Point B2 (N 34°14,856; E 35°36,067), is part of a long term monitoring program carried out since 1999 by the Lebanese National Center for Marine Sciences. It is located at around 4 km off the coast of Batroun, Lebanon (Levantine basin), with a depth of 500 m. It is considered to be an offshore station due to the narrowness and shallow depth of the continental shelf (Pfannenstiel,

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1960; Goedicke, 1972). Sampling was made on monthly basis. The choice of the study years was made to incorporate period before (2003-2004), during (2008-2009) and post (2012-2013) the EMT-like event which was marked by an increased salinity in Point B2 between 2005 and 2010 (Ouba et al., 2016).

2.2. Environmental Parameters

Water temperature, salinity, ortophosphates, Nitrates and nitrites were measured at 0 m, 20 m,40 m, and 60 m depths, and depth average values were used in this study. Water temperatures was measured in situ using a reversed thermometer (Richter & Wiese type, 0.05°C precision) attached to a Niskin bottle. Samples for salinity and nutrients were collected using said Niskin bottle. Salinity was later analyzed using a Beckman © RS7-C salinometer. Samples destined for nutrients measurements - were kept at -20˚C until they were analyzed. Nitrites (NO2 ) concentrations were measured following the - methodology described by (Bendschneider and Robinson, 1952) and Nitrates (NO3 ) were obtained according to the methodology described by (Strickland and Parsons, 1968) with a slight modification by Grasshoff (1964). Total nitrogen (Ntot) in this study is limited to the total concentrations of the two. Orthophosphates concentrations were measured following the methodology described by (Murphy and Riley, 1962).

2.3. Plankton net sampling and species determination

Ceratium samples were collected monthly through vertical hauls from 60 m to the surface, using a costume made conical net attached to a collector, both with 52 µm mesh size. The filtered volume ~ 8.7m3 was estimated according the dimensions of the net’s opening: V = π . r2 . h, where r is the radius of the opening (0.2 m) and h is the depth of the column (60 m). Samples were immediately fixed in formaldehyde (4% as a final concentration) buffered with borax (Sodium Borate) for subsequent analysis. For the sake of homogenization of calculations, each sample was adjusted to a final volume of 200 ml. Four 1 ml replicate sub-samples were counted in a calibrated Sedgwick-Rafter © counting chamber using a microscope. Identification was made at the species level based mainly on the nomenclature by (Sournia, 1967), in addition to standard taxonomical studies (Dowidar, 1971; Steidinger and Tangen, 1996).

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2.4. Statistical analysis and biodiversity indices

The Shannon’s biodiversity (H) and Pielou’s regularity index (J) calculations, as well as all statistical analysis were applied using R programming language version 3.3.2 and its user interface R studio. Specific richness was calculated through Microsoft excel. The complete dataset consisted of 70 observations as the samples from June 2008 and March 2012 were in unusable conditions. The significance level for all the tests was set to p = 0.05. A non-parametric repeated measurement ANOVA (Friedman) was also applied per site to determine significant difference between the years for total Ceratium abundance as well as each environmental parameter. A PCA (Principle Component Analysis) was applied using environmental variables as active variables, abundance of 12 selected species as illustrative qualitative variable, and years as qualitative illustrative variable. The choice of the 12 species was based on their dominance over the course of the study. A correspondence analysis was also performed using the same 12 species as active variables.

3. Results

3.1. Environmental parameters

Variations of environmental parameters are presented in Figure 1. Water temperature showed typical seasonal variation with no significant differences between the six years. The coldest were winter months (minimum of 16.9°C in March 2003) and the warmest were summer months (maximum of 27.8°C in July and August of 2012). It was consistently above 25°C from July till September in all years. Salinity showed little variation between 38.6 and 39.6. It was highest in 2008, 2009 and 2013, where it remained above 39 throughout the year, whereas it was below 39 in February, March and April of the remaining years. The Friedman test followed by a Nemenyi post-hoc showed significant difference in salinity between lowest and highest salinity years (p<0.005). Variations in nutrients concentrations did not seem to follow a seasonal pattern. Phosphate concentrations were below 0.2 µmol/L except some sporadic peaks. The Friedman test followed by a Neyemni post-hoc showed significant difference in phosphates concentrations between the year 2004 (highest) and the years 2003 and 2008 (lowest) (p<0.005). Nitrogen from Nitrites and Nitrates varied between a concentration of 0.07 µmol/L and 1.58 µmol/L, the highest yearly averages were noted in 2003 and 2013, and lowest in 2004 and 2008.

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Figure 15 Figure 1 – Monthly variation of environmental parameters : water temperature T (°C), Salinity S, Orthophosphates P (µmol/L) and Total Nitrogen Ntot (µmol/L) at Point B2 during the six years of the - - study. Ntot is limited to the total of two measured N nutrients: Nitrates NO3 and Nitrites NO2

3.2. Variation in Ceratium abundance and diversity

The genus Ceratium was among the most represented in all samples collected during this study. Total abundance ranged between 448 cells.m-3 (October 2013) and 5 666 cells.m-3 (June 2003) (Figure 2). Generally, consecutive peaks occurred throughout the year. Seasonality was therefore not very marked, with the exception of a noticeable drop in late summer/ early fall. In the year 2012, this drop was compensated by a bloom of C. trichoceros. Specific richness was positively correlated with total abundance and varied accordingly; the highest per year was usually observed in winter, and the lowest in late summer/early fall (Figure 3). A total of 40 species were encountered throughout the study (Table 1), but species richness did not exceed a maximum of 26 per sample (March 2003); the lowest was 11 species (September 2004). Overall, the 12 most abundant species accounted for at least 72% and up to 96% of the total in all samples (Figure 2). The Pielou’s regularity index (J) was high throughout the study period, and ranged between a minimum of 0.5 (August 2012) and a maximum of 0.93 (October 2013). It was negatively correlated with total abundance. The Shannon’s diversity index varied between a minimum of 1.42 (August 2012) and a maximum of 2.74 (February 2004) (Figure 3). The Friedman post-hoc test after Nemenyi showed significant difference in total abundance (p<0.05) only between 2003, where most monthly values are above 2 000 cells.m-3, and 2004 where they were below 1 000

Açaf Laury – Thèse de doctorat - 2018 cells.m-3. This test revealed the same when applied to species richness, but no significant difference was noted for the biodiversity indices J and H.

Figure 16 Figure 2 – Monthly variation in Ceratium abundances at Point B2 during the six years of the study (Total abundance of the genus Ceratium (Total) and of 12 dominant species (cell/m3)

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Table 10 Table 1 – A list of Ceratium species found at Point B2 during the six study years and their frequency of occurrence. The twelve dominant species are in bold font Species Fr (%) C. fusus (Ehrenberg) Dujardin, 1841 99 C. macroceros (Ehrenberg) Vanhöffen, 1897 99 C. horridum (Cleve) Gran, 1902 96 C. extensum (Gourret) Cleve, 1900 96 C. kofoidi Jörgensen, 1911 94 C. candelabrum (Ehrenberg) Stein, 1883 93 C. contrarium (Gourret) Pavillard, 1905 91 C. teres Kofoid, 1907 90 C. massiliense (Gourret) Jörgensen, 1911 90 C. tripos (Müller) Nitzsch, 1817 89 C. contrortum (Gourret) Cleve, 1900 89 C. symetricum Pavillard, 1905 83 C. trichoceros (Ehrenberg) Kofoid, 1908 73 C. euarcuatum Jörgensen, 1920 70 C. carriense Gourret, 1883 64 C. concilians Jörgensen, 1920 61 C. furca (Ehrenberg) Claparède & Lachmann, 1859 59 C. declinatum (Karsten) Jörgensen, 1911 56 C. arietinum Cleve, 1900 49 C. hexacanthum Gourret, 1883 46 C. inflatum (Kofoid) Jörgensen, 1911 36 C. paradoxides Cleve, 1900 30 C. ranipes Cleve, 1900 26 C. vultur Cleve, 1900 19 C. pentagonum Gourret, 1883 14 C. giberrum Gourret, 1883 13 C. breve (Ostenfeld & Schmidt) Schröder, 1906 7 C. incisum (Karsten) Jörgensen, 1911 7 C. lineatum (Ehrenberg) Cleve, 1899 6 C. longirostrum Gourret, 1883 6 C. belone Cleve, 1900 6 C. longissimum (Schröder) Kofoid, 1907 6 C. falcatiforme Jörgensen, 1920 4 C. ehrenbergii Kofoid 1907 4 C. limulus (Gourret ex Pouchet) Gourret, 1883 4 C. praelongum (Lemmermann) Kofoid ex Jörgensen, 1911 3 C. buceros Zacharias, 1906 1 C. gravidum Gourret, 1883 1 C. lunula (Schimper ex Karsten) Jörgensen, 1911 1 C. schroeteri Nie, 1936 1

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Figure 17 Figure 3 - Graph showing monthly variation in specific richness (S), Shannon’s diversity index (H) (bit.ind-1) and Pielou’s regularity index (J) at Point B2 during the 6 years of study.

The species C. fusus, C. macroceros, C. horridum, C. extensum, C. kofoidi, C. candelabrum, C. contrarium, C. teres and C. massiliense were present in high frequency (between 90 % and 99 % of samples) over the course of the study (Table 1). Ceratium fusus, C. kofoidi and C. horridum were also the most abundant, and at least one of them was dominant in all the samples (Figure 2, Figure 4). Particularly, C. fusus had the highest relative abundance in 50 % of the samples, and was dominant in all seasons except summer of 2003 and 2004. The winter months were characterized by a dominance of the association of C. fusus with C. horridum, whereas C. kofoidi had the highest relative abundance in the summer and fall. Ceratium trichoceros was also dominant in the fall of 2012 and winter (December) 2013. Ceratium teres appeared as a dominant species sporadically in the years 2003, 2004, 2008 and 2009, and C. furca in the years 2012 and 2013.

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. Figure 18 Figure 4 – Graph showing specific dominance index (%) and relevant abundance of dominant species of the genus Ceratium t Point B2 in the six study years. Sp.1, sp.2 and sp.3 are respectively the two (three) most common species observed. 101

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The correspondence analysis (CA) projected the species composition of each sampling date in an ordination space. The first two dimensions (axes) explained a cumulative 44.8% (24.4% and 20.4%) of the variance (figure 5). Ceratium trichoceros primarily contributed to the first factor (70%), while C. fusus and C. kofoidi primarily contributed to the second factor (58%). The asymmetric biplot distinguished C. trichoceros from other species and associated it with the fall of 2012. It reflected the association of C. fusus and C. horridum together with C. symetricum and C. contrarium (negative coordinated on dimension 1) to colder months of the years and opposed them C. kofoidi and C. tripos and C. candelabrum which were linked to summer months.

The first two principal components (PC) of the PCA analysis amounting to a cumulative percentage of the variance of 66.4% were maintained. Contributions of the variables to the PC are represented on the PCA correlation plot (Figure 6). Results were in accordance with those of the CA, as the species associated with warmer months in the CA correlated with Temperature of water, whereas those associated with colder month were opposed this variable. Ceratium fusus was positively associated 3- 3- with total abundance and Ntot, and negatively with PO4 . In turn, PO4 was associated to C. macroceros.

4. Discussion

The present study set out in the aim of using the genus Ceratium as a biological indicator of environmental changes at Point B2, a long term monitoring site off the Lebanese coast in the Leventine basin. Given its sensitivity to temperature, telling changes in Ceratium population were expected in response to global sea water warming, supported by a stretch of the six selected study years over a ten- year period (2003-2013). However, the Friedman test showed no significant interannual difference neither in water temperature, nor in total abundance of Ceratium. As for the effects of the EMT-like events, Ouba et al. (2016) reported significant changes in phenology, biomass and abundance of several phytoplankton taxonomic groups, including nanplankton, phytoplankton and zooplankton. In this study, there was no significant interannual difference in Ceratium abundance nor distinguishable trends in annual cycle.

Therefore, the study of the genus Ceratium at Point B2 failed to reflect the increasing trend in spring sea water temperature, most probably because Point B2 was not affected by this trend in the

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Açaf Laury – Thèse de doctorat - 2018 selected study years. Other explanations could be the use of average values of sea water temperature in the water column, despite a marked and long period of stratification reaching 60 m depth (Abboud-Abi Saab et al., 2004). In addition, the genus Ceratium did not reflect incoming water masses from the Agean sea during EMT-like years. Indeed, Ceratium is a well-established bioindicator of water masses water masses (Dodge, 1993; Okolodkov and Dodge, 1996; Ochoa and Gómez Caballero, 1997; Raine et al., 2002), marine currents (Dowidar, 1971), climate change and ocean warming (Dodge and Marshall, 1994; Johns et al., 2003; Tunin-Ley and Lemée, 2013), and biogeography (Dodge and Marshall, 1994). However, this quality is based on its sensitivity to temperature and not salinity. While an EMT event is characterized by warmer and more saline waters, the EMT-like event can exhibit lesser intensity and consequences. At point B2, characteristics of the 2005-2010 EMT-like event was limited to salinity. Therefore these results showed that contrary to nanoplankton, phytoplankton, zooplankton and other taxonomic groups (Ouba et al., 2016), Ceratium is not a good bioindicator to changes in salinity.

While the initial hypothesis of this study was not verified, some trends in the annual cycle of Ceratium could be linked to different environmental parameters. The range of total abundance of Ceratium per sample maxed at 5 666 cell/m3 is up to 50 fold lower than those previously reported in Lebanon. A study conducted throughout the year 1980 in Central coast of Lebanon found a maximum total abundance nearing 250.103 cell/m3 in an offshore station and around a 100.103 cell/m3 in coastal station (Abboud Abi-Saab, 1985). The large difference in total abundance compared to previous study in the same area is not necessarily and indicator of local environmental changes over the years. More likely, it was the result of patchiness in the vertical distribution of Ceratium, as the previous study was based on surface horizontal net sampling, whereas the present one was based on vertical sampling. Indeed, vertical migration due to phototaxis for example can lead euphotic species to accumulate in the first few meters near the surface; when said species are bloom forming, such as red tide species C. fusus and C. furca (Wandschneider, 1979; Baek et al., 2007), it could strongly affect total abundances vertical distribution. Results of this study were similar to those reported using vertical net sampling at similar depth in the NW Mediterranean Sea, particularly the Ligurian Sea, where total abundance of Ceratium spp. varied between from 834 to 3 734 cell/m3 (Lasternas et al, 2011). The latter study was conducted in an offshore station 45 km to the coast, over 1 month period. A study by Tunin-Ley et al. (2007) with more similarity to the present one (results over annual cycles, sampling site close to the coast) found total abundance of Ceratium below 5.103 cell/m3 in 50% of the samples, however the maximum reached 5 fold higher (26.103 cell/m3). This difference is in accordance with findings of the negative West-East

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Açaf Laury – Thèse de doctorat - 2018 gradient in nutrients concentration (Moutin and Raimbault, 2002; Moutin et al., 2012) and primary productivity in the Mediterranean Sea (Uitz et al., 2012).

In term of Ceratium diversity, the list of 40 species encountered over the course of 6 annual cycles was near identical to those found previously both in Lebanon (40 species) (Abboud Abi-Saab, 1985) and the Ligurian sea (43 species) (Tunin-Ley et al., 2007). The maximum species richness however did not exceed 26, reflecting the high rate of species that only appeared sporadically in low frequency (Table 1). The absence of one species in partic ular, Ceratium egyptiacum Halim, was noteworthy. This thermophilic species was identified as a regular component of Egyptian phytoplankton (Halim 1963, 1965). It reached the Lebanese waters along the currents from the Suez Canal and was found regularly from August to September in the early 1980s in net sample in an offshore station in Lebanon, at Twater > 25°C (Abboud Abi-Saab, 1985). Nevertheless, they were not found in this study despite suitable water temperatures that were above 25°C from July to September in all 6 years.

Evidently, several environmental parameters concurrently influence the development of Ceratium taxa and the species composition. The PCA confirmed the influence of all collected environmental parameters, although other factors such as depth, and biotic factors such as competition and predation (Tunin-Ley et al., 2007), would also play important roles. Water temperature in particular is expected to have significant influence.

The annual cycle was characterized by a seasonal trend with a marked contrast in specific richness between winter (highest values) and summer (lowest values). It was anticorrelated with water temperature, which showed typical season variation with lowest values in winter and highest in the summer. A decrease in specific richness as a result to sea water warming was also reported by (Tunin- Ley et al., 2009) in the NW Mediterranean Sea.

Seasonality was less noticeable in total abundance, but late summer/early fall period was characterized by a drop in total abundance. The PCA revealed that total abundance was anticorrelated to PO4.(Baek et al., 2008) did reveal that growth rates increased under P limitation of two species, C. fusus and C. furca. Ceratium fusus in particular maintained high relative abundance throughout this study and thus influenced total abundance. The regularity and diversity indices did not seem to follow a particular seasonal pattern, however the PCA showed that they were positively associated, which indicates that diversity was more influenced by species distribution rather than their totals. Species composition was

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Açaf Laury – Thèse de doctorat - 2018 clearly influenced by temperature. Few psychrophilic Ceratium species exist (Sournia, 1967), however, even in winter months, minimum water temperature did not drop below 17°C. Winter months were characterized by the association of C. fusus and C. horridum, both negatively associated with temperature (Figure 6). Both species are cosmopolite eurythermic, and have been found within a temperature range of 5°C to 30°C in the North Atlantic (Dodge and Marshall, 1994). Eurythermy and mixotrophy likely contributed to C. fusus maintaining high relative abundance throughout the year. In the remaining months, Water temperature was above 20°C. In addition C. kofoidii, C. teres and C. candelabrum were often dominant, associated with warmer months and temperature (figures 5 & 6). Some of these associations are in accordance with literature, as C. kofoidi and C. teres are considered to be tropical species. C. kofoidi was found in a small range of temperature (23 to 30 °C)(Dodge and Marshall, 1994); In addition, Sournia, (1967) has described thermophilic varieties for C. tripos and C. candelabrum.

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Chapter VI : General Discussion

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General discussion

1. Benthic Harmful Algal Blooms: model genus Ostreopsis

1.1. Ostreopsis of the Lebanese coast: what species?

The need for identification of Ostreopsis at the species level in a given study stems from the many contradictions reported by the different studies in recent years, in terms of seasonality, response to ecological factors, or most importantly, variability in toxicity. Indeed, the identification of Ostreopsis at the species level is a complicated task. Identification using solely optical microscopy is often indefinite as all species of this genus have the same outline and can be described as ovoid, oblong or tear shaped, and even size ranges of the different species often overlap (Penna et al., 2005; David et al., 2013). In culture, Ostreopsis cells are known to present changes in morphology, showing aberrant cells, or differences in shape and size of thecal plates or overall cell, sometimes linked to different vegetative stages (Aligizaki and Nikolaidis, 2006; Accoroni et al., 2014). Adding to this complexity is the fact that, unlike other dinoflagellates, most species of Ostreopsis share a similar tabulation pattern. Concerning Ostreopsis in Lebanese coastal waters, some skepticism was involved around species identification since it was first reported in the area in the late 70s, as the author suggested the possibility of it being a new species (Abboud Abi-Saab, 1985). A study in 2016 confirmed this suggestion and described new species named O. fattorussoi, but added to the controversy by citing the presence, though negligible, of a few O. cf. ovata cells (Accoroni et al., 2016a). Luckily, O. fattorussoi does possess a few discerning morphological features that lead to its unequivocal identification, based on shapes and measurements of thecal plates and sutures. This allowed for the identification of the blooming species as O. fattorussoi, using calcofluor and epifluorescence microscope for observation and measurements of cells selected randomly in samples from two different blooms per site.

1.2. Ostreopsis sp. quantification: Optimization and Intercalibration

As a step towards reaching reliable and standardized quantification protocols, the present work contributed to optimization and intercalibration of different quantification steps, applied to Ostreopsis spp., in the framework of the pan-mediterranean ENPI-CBCMED M3-HABs Project.

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1.2.1. Optimization of cells detachment in macroalgae sampling:

Experiments were conducted to determine the influence of three factors (the low or high level of abundance, the year of survey, and the type of fixative addition) on the proportion of O. cf. ovata cells detached from the macroalgal substrate during the different steps: addition of fixative solution, agitation, and 2 consecutive washings. A three way ANOVA determined the addition of fixative (acidic or non- acidic Lugol’s solution) does favor the detachment of Ostreopsis cells from macroalgae during the agitation steps. However, two successive agitation steps compensate for the poor detachment efficiency of benthic Ostreopsis cells without addition of a fixative. These results reveal an advantage for alert systems where time economy is a priority, as they prove the efficiency of a single washing, provided that a fixative is added before agitation.

1.2.2. Optimization of sampling using the deployment of artificial fiberglass screen (optimized set up described by Jauzein et al., 2016).

Macroalgae sampling is the most commonly used method for Ostreopsis sp. However, this method has some inherent disadvantages. In case of intense surveys, it can destructive to the site. In addition, the variability in macroalgae leads to difficulties in standardizing the results contributes to the patchiness of Ostreopsis spp. distribution (GEOHAB, 2012). Our results corroborate this last statement as they showed variability by a factor of 4 in 1 m² of seabed and on the same macroalgae. In the present work, an alternative sampling method- artificial substrate - independent from macroalgae, was tested in several coastal areas of the Mediterranean. Abundances of Ostreopsis cells collected from artificial substrates showed a positive correlation with benthic abundances collected using the traditional macroalgae sampling. Combining mean values obtained from the six different coastal zones of the Mediterranean Sea that were monitored, the final data set was modeled by Y = 0.641 X + 2.346 (r² = 0.55, p < 0.01), where the abundance collected from artificial substrates (Y, in cell.100 cm²) is expressed as a function of benthic abundance (X, in cells.g-1 fw). They also fitted well with the linear model from a study by Tester et al. (2014) in a lower concentration range. This adds reproducibility to the advantages of this method, that include eliminating the inherent disadvantages of macroalgae sampling, and the ability to integrate spatial and temporal variation (dial migrations) of this methods regardless of the coastal area, operator, or concentration level.

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In the case of alert systems, where quick results can be essential, the long incubation time could be an obstacle. However, testing different incubation times with the optimized set up by Jauzein et al. (2016) showed that when necessary, a shorter incubation time can provide a good estimation of population abundances on a shorter time scale, as collection efficiency reached 80% and more than 60% of total abundance over 8h and 6h respectively.

1.2.3. Optimization of counting of cells in the water column

A series of counts of O. cf. ovata planktonic cells was performed using different sedimentation volumes of either 10, 50 or 100 mL. Significant positive correlations were found between cell counts using a 10 mL, or a 100 mL column, and the reference counts, done with 50 mL. A threshold of 3,000 cells.L-1 (=150 cells in 50ml) was defined above which coefficient of variation was stable, meaning counting below this threshold using the Utermöhl method decreases its precision. Therefore, under lower concentrations, it is advisable to use higher sedimentation volumes. In this optimal range of concentration, when using a 100 mL column compared to the reference 50ml, results confirmed a significant underestimation (one-sample t-test, n = 27, p < 0.001), possibly due to increased cell adherence to the surface of the column. In this case, results highlighted the efficiency of using the 10 ml column particularly in alert systems, as it reduces incubation time to 4h (no significant difference between 50 ml and 10 ml counts one-sample t-test, n = 27, p= 0.88).

1.2.4. Intercalibration of two innovative automated counting techniques of Ostreopsis spp. : RT- qPCR (MOL) and an opto-electronic device (OPR)

An intercalibration campaign was set up in order to assess reliability of the automated counting methods in reference to the traditional to the traditional manual (MAN) counting techniques: Utermöhl for planktonic samples and macroalge sampling for the benthic samples. Counts using the different techniques were carried out on the same samples originating from different Mediterranean sites from. Ostreopsis cf. ovata was found in all sites except the Lebanese coast where only O. fattorussoi was present. A new qPCR assay was designed for specific O. fattorussoi quantification. The two proposed counting procedures MOL and OPR methods proved to be robust and reliable in a risk management perspective were in good agreement with manual results, with Pearson's coefficients higher than 0.5 in almost all situations. Overall, the MOL method presented a tendency to slightly over-estimate the count, while the OPR system typically under-estimated the guess. However, a posteriori normalization of the

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Açaf Laury – Thèse de doctorat - 2018 results could eliminate this disagreement, given its systematic nature. Interestingly for O. fattorussoi, the low correlation between (MAN) and (OPR) was obtained using the same template, designed for O. ovata, which was then less effective for O. fattorussoi quantification.

1.3. Natural blooms of O. fattorussoi along the Lebanese coast and the role of environmental parameters

1.3.1. Spatial and temporal distribution of O. fattorussoi

The present work was the first to follow the temporal and spatial distribution of O. fattorussoi along the Lebanese coast in such frequency (monthly/weekly) over three summer bloom periods, while investigating the effects of environmental factors. The species was present year round in both benthic and planktonic samples along the coast. Despite the blooms being site and year specific, a clear pattern was common to all sites: a marked seasonality with maximum densities occurring during summer blooms (max: 880 694 cells/g FW; 18 065 cells/ L), usually followed by less intense autumn blooms (max: <50 000 cells/g FW, 3 160 cells/L). Similar seasonality was reported previously in a study following planktonic Ostreopsis in the study area (Abboud-Abi Saab et al., 2013). Some minor spring blooms also occurred on some sites. Summer blooms occurred early (June-July) compared to those of Ostreopsis spp. in the western Mediterranean sea (late summer-early fall), and in lower intensity in some cases (Mangialajo et al., 2011), though we make this comparison cautiously due to effect of biotic substrates on quantification.

1.3.2. Environmental parameters

In the present work, statistical analyses (PCA, PERMANOVA) revealed that water temperature, wind speed and nutrients, co-influenced the development of natural blooms of O. fattorussoi. Such a synergic effect has previously been demonstrated for strains of O. cf. ovata (Accoroni et al., 2015). Ostreopsis fattorussoi was thermophilic as highest abundances per site were reached at relatively high water temperature (27°C < TCmax < 30.5°C). This result was in accordance with a previous study along the Lebanese coast (Abboud-Abi Saab et al., 2013) as well as several studies Ostreopsis sp. in the western Mediterranean (Mangialajo et al., 2008; Cohu et al., 2013b). Maximum abundances occurred while Twater was still increasing, resulting in a TCmax < Tmax, and inviting the hypothesis of a certain maximum temperature inducing bloom decline that need to be further studied. The differences in TCmax

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Açaf Laury – Thèse de doctorat - 2018 between the sites and the lack of a significant correlation with O. fattorussoi abundances on some sites can be due to the presence of different strains on different sites, as species, clades or subclades to respond differently in terms of temperature (Guerrini et al., 2010; Pezzolesi et al., 2012; Tawong et al., 2014)(Guerrini et al., 2010; Pezzolesi et al., 2012; Tawong et al., 2014). It could also be due to the interaction with the effects of the other environmental parameters. Warmer temperature of the Eastern Mediterranean could also explain the occurrence of spring blooms whereas they have not been reported in the western Mediterranean. In addition, as none of the sites are in sheltered areas, water agitation can be influenced by local winds. Therefore, correlation between wind speed and planktonic abundances reflects the effects of agitation on cells resuspension from the benthic compartment to the water column, which explains the absence of a major benthic bloom in BAT and BEY in the summer of 2015, despite the occurrence of planktonic ones. The correlation with benthic ones implies a possible preference to agitation, as also observed for Ostreopsis spp. in other studies (Vila et al., 2001b; Selina et al., 2014). The role of nutrients though confirmed, could not be determined, and results were variable between the sites. They were also inconsistent with a previous study by (Abboud-Abi Saab et al., 2013) in the area that reported a positive correlation with Nitrite, Nitrate and N/P ratio, and a negative one with orthophosphate. Both studies could have been erroneous as analysis of Ammonium is lacking. Ammonium is not often included in marine monitoring programs, despite being an important nutrient for microalgae, and for Ostreopsis particularly, as demonstrated by (Jauzein et al., 2017). More studies are needed to further clarify the role of agitation, nutrients and even temperature (e.g. threshold temperature, cyst germination) in growth and bloom dynamics of O. fattorussoi, as well as other biotic and abiotic factors known to influence Ostreopsis species that were not addressed in this study such as salinity, pH, depth/light intensity, macroalgae, bacteria and allelopathic interactions to name a few.

1.4. Ecological physiology

Attempting to infer a link between nutrients concentrations and O. fattorussoi abundances through monitoring failed to yield conclusive results, partly due to the co-influence of other environmental parameters in the natural conditions and the impossibility of monitoring all nutrients, particularly ammonium. An experiment was therefore put in place in an attempt to study certain aspects of O. fattorussoi’s nutrition in a controlled environment. The interest arose particularly from a recent study that proved mixotrophic abilities of NW strains of O. cf. ovata that were able to take up both inorganic as well as organic N sources (Jauzein et al., 2017). Particularly, we aimed to reveal whether

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Açaf Laury – Thèse de doctorat - 2018 there was some difference in nutritional strategies and competitiveness between the two species resulting in the lower intensity of O. fattorussoi blooms compared to those of O. cf. ovata reported in the western Mediterranean. N uptake from Nitrate, Nitrite and Urea were characterized individually for 4 strains: two of O. fattorussoi isolated from the same site at the same time, and two NW Mediterranean strains of O. cf. ovata from Monaco and Nice. The cultures were maintained in the same conditions where they were acclimated to Urea. All four strains were mixotrophic and were able to uptake all three nutrients. Three strains exhibited the same order of preference: NH4> NO3 > Urea, as previously reported for NW Mediterranean strains of O. cf. ovata (Jauzein et al., 2017). This preference was less evident for one strain of O. fattorussoi MCCV57. Ostreopsis cf. ovata did not have superior abilities compared to O. fattorussoi in terms of acquiring N nutrients, as intraspecific differences were often greater than interspecific ones. These physiological characteristics translate on an ecological level. Mixotrophy offers a competitive edge compared to strict autotrophs by diversifying their potential nutrient sources. It has long been recognized as a major mode of nutrition in oligotrophic waters such as the Mediterranean Sea, and more recently, in eutrophic environments. Oligotrophic nutrient levels have been reported in field in Monaco, Nice, and Batroun where the four studied strains were isolated, whereas eutrophic which can occur in some small bays on the Lebanese coast that receive high organic inputs from untreated sewage waters and leachates from uncontrolled dumps. The relatively high uptake + abilities of NH4 , added to it being a regenerated nutrient more available in the summer, Ostreopsis + bloom season, supports the suggestion that NH4 is the main N source fueling blooms of Ostreopsis spp. (Jauzein et al 2017). Incidentally, while some small differences were noted among all strains, the highest + was in terms of NH4 uptake abilities between the two strains of O. fattorussoi. It was also the one with the most ecological considerations, as the two strains co-occur in the natural environment. MCCV58 + was more efficient than MCCV57 in obtaining N from NH4 along the entire tested gradient of + concentrations. NH4 availability in the summer would therefore favor the development of MCCV58 over MCCV57. Another important metabolic difference was also noted between the two strains. Toxin analysis showed MCCV58 was toxic whereas MCCV57 was not, therefore it would selectively favor the development of a toxic bloom of O. fattorussoi over a non-toxic one. Toxin profile and the possible associated risk are addressed below.

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1.5. Toxicity and potential risks associated to O. fattorussoi blooms

Most species of Ostreopsis are known producers of various toxic molecules. So far, in the Mediterranean Sea, these toxic molecules have been identified as PLTX analogues, produced by O. cf. ovata in the west, and O. fattorussoi in the East (cf. Chapter I, Table 1), whereas O. cf. siamensis was non-toxic. Intoxication due to ingestion of PLTX contaminated seafood is rare but has a high mortality rate. Fatal cases have been reported in tropical seas through contaminated crab (Alcala et al., 1988), fish (Onuma et al., 1999). The great potency of PLTXs has led the European Food Safety Authority (EFSA), to derive an oral Acute Reference Dose (ARfD) of 0.2 μg/kg b.w. (body weight) for the sum of PLTX and its analogue ostreocin-D. In order for an adult with a body weight of 60 Kg to avoid exceeding the ARfD, a 400 g portion of shellfish meat should not contain more than 12 μg of the sum of PLTX and ostreocin-D, and shellfish meat toxin content should not exceed 30 µg/Kg of shellfish meat. For the sake of comparison, this limit ranges between 160 µg/Kg and 20 mg/Kg for other marine toxins (PSP, ASP, yessotoxins, okadaic acids and azaspiracids). Such cases, fatal or not, have not been reported in the Mediterranean Sea. However, the presence of PLTXs and its derivatives in edible marine organisms concentrations sometimes reaching 10 fold the threshold recommended by the EFSA (Aligizaki and Nikolaidis, 2006; Amzil et al., 2012b; Biré et al., 2013, 2015), coupled with the occurrence of human intoxication through other means (direct contact or inhalation, Vila et al., 2016 and references therein), have made this into a major public health concern. Similarly in Lebanon, the repeated bloom occurrences of the toxic O. fattorussoi over the course of this study suggests that it could present some threat to public health. We analyzed the toxin profile of two strains isolated in the same site at the same time, and maintained under the same culture conditions. For the strain MCCV57, toxin levels were below detection limits, and it was therefore considered as non-toxic, whereas the other strain MCCV58 produced isobaric PLTX and OVTXs, so far only produced by O. cf. ovata. Similar profiles have previously been found in strains of O. fattorussoi also isolated in BAT Lebanon (Accoroni et al., 2016), and three Cypriote strains (Tartaglione et al., 2016). Combined, these three studies characterized toxin profiles for 13 strains of O. fattorussoi, of which four different toxin profiles as well as not toxic strains (Table 1). This intraspecific variability is high compared to O. cf. ovata, as a recent study of the toxin profiles of a total of 55 Mediterranean strains came back to only for 4 different toxin profiles (Tartaglione et al., 2017). This high variability in terms of toxicity highlights the importance of further studies on blooming populations of O. fattorussoi. In addition, particularly in the case of co-occurring strains in the same site, differences in toxicity amplifies the role of environmental parameters. When a

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Açaf Laury – Thèse de doctorat - 2018 given environmental parameter favors the development of strain over another, it could be selectively favoring the development of a toxic/ non-toxic strain. Such a possibility was deduced when coupling our finding on ecophysiology with toxin profiles analysis for the two strains MCCV57 and MCCV58. The + toxic strain MCCV58 was also the most competitive in terms of NH4 uptake, meaning that the availability of this regenerated nutrient in the summer would likely favor the development of a toxic summer bloom over a non-toxic one.

Table 11 Table 1 - Geographic origin and toxin contents of different strains of O. fattorussoi Ratio of Total toxin Geographic toxic Toxins content References origin strains eq PLTX / cell Lebanon 1 : 2 isoPLTX, OVTX-a,d, e, f 1.69 - 4.28 the present work Lebanon 3 : 5 OVTX-a,d, e, 0.06 - 2.89 Accoroni et al ., 2016 isoPLTX, OVTX-a,d, e Cyprus 6 : 6 Or 0.28 - 0.94 Tartaglione et al., 2016 OVTX-i, j1, j2, k

In terms of quantification, our results showed the highest total toxin content per cell so far for O. fattorussoi at 4.28 pg eqPLTX / cell (Table 1). An estimation based on the maximal toxin content and maximum concentrations reached in the present work translates into 3 769 µg eqPLTX / g FW in benthic compartments respectively, which is considered like a stock for a tychoplanktonic species. In comparison to O. cf. ovata, this amount is close to some strains (e.g. 5.95 pg cell_1 to 15.77 pg cell_1, Scalco et al., 2012) but much lower than some others (e.g. 300 pg PLTX equivalent cell, (Brissard et al., 2014), which has led to the suggestion that it presents lower toxicity and sanitary risk than O. cf. ovata (Tartaglione et al., 2016). Given the lack of availability of pure standards of OVTXs to this day, this quantification is not an actual measure of toxicity of O. fattorussoi, but a mere comparative estimation to another species.

As for risks associated with the ecosystem itself, in the Western Mediterranean Sea, O. cf. ovata has been linked to detrimental effects on marine fauna, such negative impact on reproduction fauna (Guidi-Guilvard et al., 2012), decrease in population (Blanfune et al., 2012) and a mass mortality (Illoul et al., 2012), mortality in experimental conditions (Faimali et al., 2012). Given the recent description on O. fattorussoi, one study demonstrated an LC50-48 h values as low as 45 cells mL−1 on Artemia salina nauplii, using Cypriote strains of slightly less toxicity than ours (Tartaglione et al., 2016). Several

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Açaf Laury – Thèse de doctorat - 2018 aspects of toxicity are yet to be addressed, such as toxin production is known to be affected by cell physiology such as growth stage, isolation site, and environmental factors such as temperature, salinity, light and nutrients. Particularly, investigating the effect of different nutrients as N sources could yield interesting results.

2. Annual cycle of the genus Ceratium in Lebanese waters

Changes in Ceratium species were documented along with some environmental parameters in a regular monitoring site (Point B2) off the coast of Lebanon (Eastern Mediterranean). The study covered six annual cycles between 2003 and 2013. Specific diversity was nearly identical to that of the Ligurian Sea, however, maximum species abundance was five fold lower at Point B2, probably linked to the ultra-oligotrophy of the Eastern Mediterranean Sea. The annual cycle of the genus Ceratium at Point B2 was characterized by a low total abundance and specific richness in the summer period. A correspondence analysis and principal components analysis applied to 12 most important species distinguished two groups: i- those associated with “warm months”: C. kofoidi and C. tripos and C. candelabrum and ii- those associated with the “cold month” C. fusus and C. horridum together with C. symetricum and C. contrarium. Apart from C. kofoidi, a tropical species found in a small range of temperature (23°C to 30 °C), these two groups do not fall into the “thermophilic” and “psychrophilic” categories. In fact, many of the remaining species are eurythermic, and they all have been found in a range of temperature that includes the entire range of temperature covered by average water temperature in this study between 16.9°C and 27.8°C (Sournia, 1967; Dodge and Marshall, 1994). The tolerance to a wide range of temperature allowed these species to have near perennial presence in all six annual cycle, whereas temperature preference at the subspecies level, described in the monograph by Sournia (1967), led to the seasonality in their distribution. The use of average values for temperature in the water column and Ceartium abundance was dictated by the sampling method. Undoubtedly this has masked some variations related to temperature preference and vertical distribution, especially during the establishment of the thermocline (0 m to 80 m) from July till October/November (Ouba, 2015).

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3. Blooms of Ostreopsis fattorussoi and temporal variation of Ceratium species in the Lebanese coast: an effect of global change in the Eastern Mediterranean?

The increase in frequency and intensity of harmful algal bloom events are expected results of global environmental changes, as toxic warm water dinoflagellates species would benefit from sea water warming (Hallegraeff 2010). This appears to be the case for Ostreopsis fattorussoi in Lebanese coastal waters. Our results revealed that while blooms of O. fattorussoi were influenced by a combination of several environmental parameters, water temperature seemed to be the driving factor, influencing both timing (seasonality) and abundance of the blooms. Our results revealed that the species is thermophilic, with optimal in situ temperatures between 27°C and 30.5°C. In addition, compared with studies conducted in the late seventies, there seems to be an increase both in density and frequency of its appearance in planktonic coastal samples (Abboud-Abi Saab 1989), assuming that Ostreopsis sp. from old bibliographic references is indeed O. fattorussoi prior to its recent description.

Two environmental events that occurred in the Eastern Mediterranean in recent years are well documented: i- warming of seawater as a result of global climate change (Skliris et al., 2012; Shaltout and Omstedt, 2014) and ii- the EMT-like event from 2005 to 2010 (Krokos et al., 2014; Theocharis et al., 2014).

The initial hypothesis raised the following question: was the genus Ceratium an adequate bioindicator of these environmental changes in the Eastern Mediterranean Sea? By the end of this study, the short answer is no. The warming of sea water temperature in the Eastern Mediterranean was not reflected by any detectable changes in Ceratium, neither in total abundance nor in species composition. However, the lack of detectable change could in fact be considered an accurate reflection of the local temperature conditions at Point B2, which was not affected by sea water warming, at least in the study period and within the 0 m to 80 m depth surface layer.

It also proved to be a poor indicator of changes in salinity, as it did not reflect its significant increase in the Eastern Mediterranean nor at Point B2 during the EMT-like years. Conversely,

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Açaf Laury – Thèse de doctorat - 2018 significant changes in phenology, total abundance and biomass of several phytoplankton taxonomic groups were noted in response to this change (Ouba, 2015; Ouba et al., 2016).

4. Perspectives

The present work revealed interesting features of the ecology and ecophysiology of the newly described Ostreopsis fattorussoi and the genus Ceratium in Lebanese waters. Certainly, many remain to be uncovered. In future studies, it would be interesting to:

- Continue monitoring efforts to detect any potential risk to human health or marine fauna that may arise from blooms of O. fattorussoi and/or uncover any long term trends that may occur in response to future changes - Include additional biotic and abiotic parameters could influence bloom dynamics of O. fattorussoi in ecological studies such as light, depth, substrate, organic nutrients and biotic interactions. - Determine relationships between environmental parameters and growth stage on the development and toxicity of O. fattorussoi - Conduct long term studies of Ceratium variation at the sub-specific level at different depths in the water column - Couple Ceratium counts with zooplankton to assess the effect of predation

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Table of Figures

Chapter I: Figure 1- Overview of the external morphology and its terminology of athecate and thecate dinoflagellate cells. © Mona Hoppenrath from http://tolweb.org/Dinoflagellates/2445 ...... 11 Figure 2- Kofoid system of tabulation. © Mona Hoppenrath from http://tolweb.org/Dinoflagellates/2445 ...... 12 Figure 3- Representatives of the four sub-genera A: C. praelongum, Subg. Archaeceratium; B: C. furca, Subg. Ceratium, C: C.inflatum, Subg. AmphiCeratium; D: C. tripos, Subg. Tripoceratium ...... 22 Chapter II-Part 1 Figure 1 - Map showing the locations of the six sites sampled for the monitoring of Ostreopsis blooms in the Mediterranean Sea...... 31 Figure 2 - Experimental procedures used for estimating the role of the fixative addition and of the washing steps in the efficiency of the separation between epiphytic Ostreopsis cells and macroalgal substrate ...... 33 Figure 3- Abundances of Ostreopsis cells estimated in the planktonic fraction (A) and collected from the deployment of artificial substrates (B and C), expressed as a function of benthic abundances...... Error! Bookmark not defined. Figure 4- Kinetics of Ostreopsis cells’ accumulation on artificial substrates ...... 35 Figure 5- Contribution of fixative addition and of washing steps in the detachment ...... 36 Figure 6 - Efficiency of counting planktonic concentration of Ostreopsis cells using the Utermöhl method...... 37 Chapter II-Part 2 Figure 1 - Maximum abundance of Ostreopsis cells in seawater and on macroalgae during 2014 (a) and 2015 (b) summer season in the sampled locations along the Mediterranean Sea...... 46 Figure 2 - Comparison of automatic methods versus manual counting for planktonic samples. Blue and green dots represent optical recognition [OPR] and molecular [MOL] qPCR counts, respectively ...... 48 Figure 3 - Comparison of automatic methods versus manual counting for benthic samples. Blue and green dots represent optical recognition [OPR] and molecular [MOL] qPCR counts, respectively ...... 49 Figure 4 - Overall comparison of automatic methods versus manual counting for water (left) and benthic (right) samples. Optical recognition (OPR, blue) and molecular (MOL, green) counts, respectively ..... 50

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Chapter III Figure 1 - A map showing the position of Lebanon on the Eastern Mediterranean coast as well as the location of the sampling sites during the study (preliminary & regular sampling sites) ...... 57 Figure 2- Light micrographs of calcofluor stained Ostreopsis fattorussoi cells under UV light, showing the tabulation.)...... 60

Figure 3- Graph showing the variation of climatological parameters: A: Air temperature “Tair” (°C), B:

Wind speed “WS” (m/s) and C: Maximum wind speed reached during the previous week “WSmax” and D: Precipitation in the previous week “Rain WB” (mm) ...... 62

Figure 4- Graph showing the variation of hydrological parameters: A: Water temperature “Twater” (°C) and B: Salinity in the 5 sites during the sampling period ...... 64 Figure 5 - Graph showing A: the variation of benthic O. fattorussoi abundances as a function of water temperature “Twater” (°C) and B: Distance between the sites as a function of water temperature corresponding to maximum abundances of O. fattorussoi (TCmax) in the 5 sites during the sampling period ...... 64 Figure 6 - Graph showing the variation of nutrients: A: Nitrites (µmol/L), B: Nitrates (µmol/L), C: Orthophosphates (µmol/L) and D: N/P ratio in the 5 sites during the sampling period ...... 66 Figure 7 - Graph showing the variation of O. fattorussoi abundances (Cells/L) in planktonic sea water samples at 20 cm depth in the 5 sites during the sampling period ...... 68 Figure 8 - Graph showing the variation of epiphytic O. fattorussoi abundances (Cells/g FW) in benthic samples at 50 cm depth in the 5 sites during the sampling period ...... 69 Figure 9 - Principal Component Analysis Variable factor map (left) and individuals factor map (right) for environmental parameters (quantitative active variables), benthic and planktonic O. fattorussoi abundances (quantitative illustrative variables) and site (qualitative illustrative variable) ...... 71 Chapter IV + - Figure 1 - Kinetic curves of NH4 (in blue), NO3 (in red)_and N-urea (in green) uptake for different strains of Ostreopsis cf. ovata and Ostreopsis cf. fattorussoi...... 85 Figure 2 - Toxin profile in % (histogram bars) and total concentration in pg cell-1 (white dots) for the two Eastern Mediterranean strains of O. cf. fattorusoi MCCV57 and MCCV58 at the the end of exponential phase and in real stationary phase ...... 86

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Chapter V Figure 1 – Monthly variation of environmental parameters : water temperature T (°C), Salinity S, Orthophosphates P (µmol/L) and Total Nitrogen Ntot (µmol/L) at Point B2 during the six years of the - - study. Ntot is limited to the total of two measured N nutrients: Nitrates NO3 and Nitrites NO2 ...... 97 Figure 2 – Monthly variation in Ceratium abundances at Point B2 during the six years of the study (Total abundance of the genus Ceratium (Total) and of 12 dominant species (cell/m3) ...... 98 Figure 3 - Graph showing monthly variation in specific richness (S), Shannon’s diversity index (H) (bit.ind-1) and Pielou’s regularity index (J) at Point B2 during the 6 years of study...... 100 Figure 4 – Graph showing specific dominance index (%) and relevant abundance of dominant species of the genus Ceratium t Point B2 in the six study years...... 101

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Table of tables

Chapter I Table 1- A list of described Ostreopsis species along with their respecttive type locality, toxic molecules and examples of relevent refrences. *: non PLTX-like metabolite ...... 15 Table 2- A list of features of Ceratium that match the prerequisites for ecological indicator validation 24 Chapter II -Part 1 Table 1 - Main characteristics of the study sites and Ostreopsis blooms that were monitored ...... Error! Bookmark not defined. Table 2 - Statistical analyses of data obtained when characterizing conditions helping for the detachment of epiphytic Ostreopsis cells from macroalgal substrate during the ...... 37 Chapter II- Part 2 Table 1 - List of sampling stations...... 45 Chapter III Table 1- Description and geographical coordinates (in decimal degrees) of the five regular sampling sites and of the timing and frequency of the sampling...... 57 Table 2 - Descriptive statistics (average ± Standard deviation and Minimum-Maximum) of all variables in the 5 sites ...... 63 Table 3 - Spearman rank correlation between O. fattorussoi abundances and environmental variables. 67 Table 4 - Maximum benthic (cells/g FW) and planktonic abundances (Cells/L) of Ostreopsis spp. reported in various areas in and outside the Mediterranean Sea...... 72 Chapter IV Table 1 - Transitions used for detection of palytoxin and ovatoxins ...... 833 Table 2 - Average growth rate µ (day-1) of the fours strains of Ostreopsis spp. preceding the kinetic experiments ...... 83 -1 -1 -1 -1 Table 3 - Values of kinetic parameters (Vmax in h , Ks in μmol N L , α in L μmol N h ), and mean N- uptake rates (h-1) obtained for the different strains of Ostreopsis cf. ovata and Ostreopsis cf. fattorussoi...... 84 Chapter V Table 1 – A list of Ceratium species found at Point B2 during the six study years and their frequency of occurrence. The twelve dominant species are in bold font ...... 999

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Chapter VI Table 1 - Geographic origin and toxin contents of different strains of O. fattorussoi ...... 114

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