Clémentine Un Bisou À Baptiste

Valeria J'adresse

Laura

Mag

Vanessa Milica

Cette Thèse

Soumis

Mies, M Chloé A Pupier

* Pupier

Fine, M., Rottier, C J A J M Van De Water

Vanessa N Bednarz

Renaud Grover

Maoz Fine

Jean-François Maguer

Christine Ferrier-Pagès

En préparation

Mon odyssée monégasque touche à sa fin et je souhaite remercier toutes les personnes qui ont contribué à son accomplissement. Trois années si vite passées… placées sous le signe du partage

Liste des communications orales

 Pupier, C. A., Fine, M., Bednarz, V. N., Rottier, C., Grover, R., Ferrier-Pagès, C. (2018). Carbon acquisition of two octocorals from shallow and mesophotic coral reef ecosystems. 5 th Young Reef Scientists Meeting, 5-7 septembre, Munich (Allemagne). Pupier, C. A., Grover, R., Fine, M., Rottier, C., van de Water, J. A. J. M., Ferrier-Pagès, C. (2021). Assimilation of dissolved nitrogen by soft and hard corals: a multi-species comparison. 14th International Coral Reef Symposium (ICRS), reporté 18-23 juillet, Brême (Allemagne).

Les octocoralliaires représentent l'un des principaux groupes formant la communauté macrobenthique des récifs coralliens tropicaux. Ils sont notamment abondants au sein des écosystèmes perturbés où les changements environnementaux entraînent le déclin des coraux constructeurs de récifs. Bien que la nutrition joue un rôle fondamental dans la régulation de l'abondance d'une population, l'acquisition de nutriments par les octocoralliaires reste à ce jour peu connue. Dans ce contexte, les objectifs de cette thèse étaient 1) de caractériser l'acquisition et l'assimilation de carbone et d'azote, par différentes espèces d'octocoralliaires de mer Rouge, et 2) d'évaluer les changements de nutrition le long d'un gradient de profondeur, depuis la surface (5 m) jusqu'à la zone récifale mésophotique supérieure (50 m). Les résultats démontrent que les octocoralliaires forment une symbiose nutritionnelle avec leurs dinoflagellés, dont le fonctionnement diffère largement de celui de la symbiose dinoflagellésscléractiniaire. La symbiose des octocoralliaires se démarque particulièrement par un apport de carbone autotrophe stable le long du gradient de profondeur, alors que les scléractiniaires connaissent un approvisionnement réduit avec l'augmentation de profondeur. De plus, l'assimilation de composés azotés dissous (par les dinoflagellés ou les symbiotes microbiens) par les octocoralliaires est très inférieure à celle des scléractiniaires. Ces résultats suggèrent que les octocoralliaires dépendent largement de sources alimentaires hétérotrophes pour satisfaire leurs besoins nutritionnels. L'importance de l'hétérotrophie est confirmée par de fortes concentrations tissulaires en biomarqueurs lipidiques spécifiques du zooplancton aux deux profondeurs, avec une augmentation en milieu mésophotique chez certaines espèces. Une telle mixotrophie confère aux octocoralliaires une grande plasticité trophique, ce qui pourrait contribuer à une plus grande résistance aux changements environnementaux en cours.

Mots clés : octocoralliaires | symbiose | autotrophie | hétérotrophie | mésophotique

Octocorals are one of the major groups forming the macrobenthic community of tropical coral reefs. They are notably abundant within disturbed ecosystems where environmental changes have led to the decline of reef-building corals. Although nutrition plays a fundamental role in regulating the abundance of a population, the acquisition of nutrients by octocorals has received little attention to date. In this context, the aims of this thesis were to 1) characterize the acquisition and assimilation of carbon and nitrogen by several octocoral species from the Red Sea, and 2) investigate the nutritional changes along a depth gradient, from the shallow (5 m) down to the upper mesophotic (50 m) reef zone. The results show that octocorals form a nutritional symbiosis with dinoflagellates, but the functioning differs significantly as compared to the scleractinian-dinoflagellate symbiosis. Particularly, the octocoral symbiosis is characterized by a stable supply of autotrophic carbon along the depth gradient, whereas scleractinian corals experience a reduced supply with increase in water depth. In addition, octocorals assimilate along the entire depth gradient significantly less dissolved nitrogen compounds (from dinoflagellates or microbial symbionts) as compared to scleractinian corals. These results suggest that octocorals strongly depend on heterotrophic food sources to meet their nutritional requirements. The importance of heterotrophy is confirmed by high concentrations of lipid biomarkers specific to zooplankton in the octocoral tissue at shallow and mesophotic depths, with an increased concentration for some species in the mesophotic environment. Such mixotrophy provides octocorals with a wide trophic plasticity, which may contribute to their higher resistance to cope with already on-going environmental changes. The longitudinal section highlights the epithelial layers and internal body organization. For clarity, pinnules are only represented on one tentacle and solenia are limited to a little part of the coenenchyme. This is a simplified scheme, which should comprise eight mesenteries (i.e. that alternate with the eight tentacles) and eight mesenterial filaments. The gastrovascular cavity ends in a dead-end canal. Retractor septal muscles and transverse fibers of mesenteries, which play a role in polyp retraction and movement of tentacles are not displayed here. Drawing by C. Pupier, based on [START_REF] Fabricius | Soft corals and sea fans: a comprehensive guide to the tropical shallow-water genera of the Central-West Pacific, the Indian Ocean and the Red Sea[END_REF] [START_REF] Bayer | Illustrated trilingual glossary of morphological and anatomical terms applied to Octocorallia[END_REF]. C: Sclerite of Xenia sp. seen under an electron microscope (× 1750, from [START_REF] Bayer | Illustrated trilingual glossary of morphological and anatomical terms applied to Octocorallia[END_REF]. ............ 9 ). Significant differences between depths are displayed with an asterisk. Since there was no similar assimilation rates between hard and soft corals, Δ shows the species that differentiate from the other two within its group (i.e. either hard or soft corals, for a single N source). AFDW = ash-free dry weight. DFAA = dissolved free amino acids. ns = not sampled. . ). Significant differences between temperatures are displayed with an asterisk. Δ shows the species that differentiate from the other two within its group (i.e. either host or Symbiodiniaceae fraction, for a single N source). AFDW = ash-free dry weight. DFAA = dissolved free amino acids. ns = not sampled. ...... 104 . Contrast between the functioning of soft coral versus hard coral nutritional symbioses from shallow to mesophotic reef zones in Eilat (Red Sea). The range of rates corresponding to nutrient acquisition and fluxes is indicated below the nutrient acronym. These rates are not comparable between each other but between groups (soft vs hard corals) and depths (shallow vs mesophotic) only. Chl = Chlorophyll concentration (µg Chl (a + c2) g -1 AFDW). Pg = Autotrophic carbon acquisition (µg C µg -1 Chl). Ts = Carbon translocated from the symbionts to the host (µg C g -1 AFDW h -1 ). AFDW = ash-free dry weight. DIN = Dissolved inorganic nitrogen (including NH4 and NO3, in µg N g -1 AFDW h -1 ). DON = Dissolved organic nitrogen (including DFAA, in µg N g -1 AFDW h -1 ). DFAA = Dissolved free amino acids. DDN = Diazotrophically-derived nitrogen (ng N mg -1 DW h -1 ). DW = Dry weight. Rate of N2 fixation in seawater are in ng N L -1 d -1 . DIC = Dissolved inorganic carbon (µg C g -1 AFDW h -1 ). POM = Particulate organic matter. The assimilation of POM has been investigated with a lipid biomarker specific to crustacean zooplankton (cis-gondoic acid (CGA, 20:19), in µg g -1 AFDW). Lipid biomarkers specific to autotrophy, stearidonic acid (SDA, 18:43) and docosapentaenoic acid (DPA, 22:53), do not appear on the figure. Concentrations of SDA were 1-70 and 1-146 µg g -1 AFDW in shallow and mesophotic corals, respectively, and decreased from 11 to 3 µg g -1 AFDW with depth in the hard coral. Concentrations of DPA were 3-7 and 3-5 µg g -1 AFDW in shallow and mesophotic corals, respectively, and decreased from 5 to 2 µg g -1 AFDW with depth in the hard coral.

Liste des tableaux

Table 1. Description of the different symbioses. One example is provided for each type of symbiosis. The species that benefits from the association (B) may obtain nutrients, shelter, support or even locomotion from the host species (A). 3. Summary about the range of metrics used in previous studies to normalize physiological processes in soft corals. AFDW = ash-free dry weight. DW = total dry weight. BW = buoyant weight. DMSP = dimethylsulfopropionate. P/A pigments = photosynthetic and accessory pigments. ETS = electron transport system. DOM = dissolved organic matter. The group 'biochemical composition of tissues' gathers measured parameters such as amino acid, protein, carbohydrate and/or lipid content. The asterisk notifies a study for which the normalizing metric (i.e., dry weight of decalcified samples) has been considered as ash-free dry weight by the authors. . [START_REF] Flügel | Patterns of phanerozoic reef crises[END_REF]. The first reef-like carbonate structures were formed by stromatolites and are among the earliest evidences of life on Earth [START_REF] Allwood | 3.43 billion-year-old stromatolite reef from the Pilbara Craton of Western Australia: Ecosystem-scale insights to early life on Earth[END_REF]. Nowadays, the most common and well-known reefs are those formed by hard corals (Cnidaria, Scleractinia), which are the most emblematic builders.

Liste des abréviations

Coral reefs thus result from the deposition of calcium carbonate by corals over millennia. One of them, the Great Barrier Reef, is one of the world's biggest structures ever built by living organisms. Reef formation depends on the speed of skeletal deposition by hard corals and the extent of skeleton erosion [START_REF] Rohwer | The destruction of the "animal forests" in the oceans: Towards an oversimplification of the benthic ecosystems[END_REF]. Reefs are found whenever coral larvae can settle on hard substrate, such as in the deep ocean floor, volcanic islands and continental coastlines. Large reef ecosystems, built by the so-called cold-water corals, exist in the deep zones of all oceans [START_REF] Roberts | Reefs of the deep: the biology and geology of cold-water coral ecosystems[END_REF]. This thesis will however focus on tropical reefs, built by scleractinian corals living in symbiosis with dinoflagellate algae. These corals need optimal living environmental conditions and are thus limited to the photic zone, within latitudes ranging from 30°N to 30°S (Figure 1).

Tropical coral reefs are ecologically important because they constitute complex, diverse, and productive ecosystems. Their extent (260,000-600,000 km²) represents less than 0.2% of the ocean's surface, which equates to a smaller land area than France. Yet, they shelter from one-quarter to one-third of the world's marine species [START_REF] Knowlton | Coral reef biodiversity[END_REF]. The epicenter of the planet's marine biodiversity is called the Coral Triangle. Located in Indo-Pacific, this area gathers more species diversity than anywhere else in the world (Figure 1). Secondary centers are identified in the Red Sea and North of Madagascar [START_REF] Veron | Overview of distribution patterns of zooxanthellate Scleractinia[END_REF]. Due to this exceptional biodiversity, coral reefs are often referred to as the 'rainforests of the sea' [START_REF] Connell | Diversity in tropical rain forests and coral reefs high diversity of trees and corals is maintained[END_REF]. Coral reefs, and their associated mangrove wetlands and seagrass meadows are also among the most productive ecosystems per unit surface area [START_REF] Costanza | The value of ecosystem services: Putting the issues in perspective[END_REF]. These three habitats are tightly interlinked, especially for providing nurseries and feeding grounds to reef fishes [START_REF] Dorenbosch | Indo-Pacific seagrass beds and mangroves contribute to fish density and diversity on adjacent coral reefs[END_REF]. Besides their ecological role, coral reefs provide humans with multiple essential goods and services. For example, they protect shorelines from erosion, are an important source of food for the population living nearby, generate incomes thanks to fisheries, tourism, and molecules of interest for the pharmaceutical industry [START_REF] Moberg | Ecological goods and services of coral reef ecosystems[END_REF][START_REF] Wells | Assessing the effectiveness of marine protected areas as a tool for improving coral reef management[END_REF][START_REF] Laurans | Economic valuation of ecosystem services from coral reefs in the South Pacific: Taking stock of recent experience[END_REF]. Reefs generate around thirty billion dollars per year [START_REF] Spalding | World atlas of coral reefs[END_REF][START_REF] Stoeckl | The economic value of ecosystem services in the Great Barrier Reef: our state of knowledge[END_REF]) and 500 million people directly benefit from the reefs [START_REF] Wilkinson | Status of coral reefs of the world: 2008[END_REF]).

1.1.2 Major benthic groups: the bricks  , the cement  and the stabilizers Although hard corals are considered as the bricks of the reef framework, they are not capable of building reefs on their own. The coral reef ecosystem includes a mosaic of ecosystem engineers such as crustose coralline algae (CCA), sponges and octocorals, which contribute to the three-dimensional structure of the reef (Figure 2). All together, they function similarly to terrestrial forests providing architectural complexity, food and shelter to other reef organisms.

As trees in the forest, they also contribute significantly to the reef primary productivity and biogeochemical cycles, either directly or through symbiotic associations (Rossi 2013).

 Designation from Dr. Maggie D. Johnson

Crustose coralline algae (CCA) form hard crusts on rocky surfaces and cement the coral bricks together. The CCA crusts provide a settlement substrate for marine invertebrate larvae of corals, abalones and sea urchins among others [START_REF] Heyward | Natural inducers for coral larval metamorphosis[END_REF][START_REF] Nelson | Calcified macroalgae critical to coastal ecosystems and vulnerable to change: a review[END_REF].

Fleshy algae, encompassing turfs and macroalgae, are not considered as reef engineers, but are however common components of the reefscape. They are the primary food source of herbivores and the most common competitors with hard corals. Sponges act as reef stabilizers through their impact on benthic substrates. On the one hand, they are one of the primary bioeroders of coral reefs, processing solid carbonate into smaller fragments and fine sediments [START_REF] Bell | The functional roles of marine sponges[END_REF]) and references therein). On the other hand, they are known to bind coral fragments or rubble together, thereby increasing coral survival by enhancing settlement of carbonate secreting organisms [START_REF] Wulff | Do sponges help hold coral reefs together?[END_REF][START_REF] Wulff | Sponge-mediated coral reef growth and rejuvenation[END_REF].

Octocorals, which are the main subject of this thesis, do contribute to the habitat complexity of the reef but are not considered as reef-builders as such [START_REF] Fabricius | Octocorallia[END_REF]Schubert et al. 2017 and references therein). Only few exceptions produce a solid skeleton (e.g., Heliopora coerulea, Tubipora musica and Corallium spp.) or massive trunks of consolidated sclerites (some species of the genus Sinularia, [START_REF] Schuhmacher | Soft corals as reef builders[END_REF][START_REF] Jeng | Sclerite calcification and reefbuilding in the fleshy octocoral genus Sinularia (Octocorallia: Alcyonacea)[END_REF], thus contributing to the reef construction and growth. Most of the species contribute to substrate stabilization by releasing sclerites after their death.

Ecophysiology of soft corals

Classification and anatomy

Soft corals belong to the phylum Cnidaria, class Anthozoa, subclass Octocorallia and order Alcyonacea (Figure 3). Cnidarian are tentacle-bearing invertebrates, distinguished by stinging structures called nematocysts and a radial symmetry. They are also characterized by an internal cavity that opens through a single orifice (i.e. the mouth, which serves for nutrient uptake and excretion). Members of the class Anthozoa, also named 'flower animals', are represented at the adult stage by small units called polyps. Both hard and soft corals belong to this class and, except few single-polyp species, form a colonial arrangement made of a myriad of polyps. While soft corals belong to the sub-class Octocorallia, reef-building corals belong to the Hexacorallia. The most pronounced characteristic that differentiates these subclasses is that members of Octocorallia have an eight-fold symmetry (i.e. eight tentacles on each polyp) fringed on both sides by one or several rows of pinnules, whereas Hexacorallia tentacules have a six-fold symmetry without pinnules [START_REF] Fabricius | Soft corals and sea fans: a comprehensive guide to the tropical shallow-water genera of the Central-West Pacific, the Indian Ocean and the Red Sea[END_REF].

At present, Octocorallia consists of three orders, which are Heliporacea (commonly called blue corals), Pennatulacea (commonly called sea pens) and Alcyonacea (Figure 3). The main groups of the order Alcyonacea are sea fans, sea whips, sea rods, sea plumes (all globally termed gorgonians) and soft corals. Unlike 'gorgonians', soft corals do not present a solid internal axis. Throughout this thesis, the term octocoral will qualify any member of Alcyonacea family, without further distinction between taxa. Soft coral growth forms are divided into categories ranging from encrusting to arborescent [START_REF] Bayer | Illustrated trilingual glossary of morphological and anatomical terms applied to Octocorallia[END_REF]. The shape and size of soft coral colonies can thus widely vary from one species to another. Even within the same species, these features can considerably vary in response to the environmental conditions. In massive and erect forms, it is often possible to distinguish parts such as stalk, stem, branches, or capitulum (Figure 4). Soft corals species are called monomorphic or dimorphic depending whether the colony bears one or two polyp categories, respectively. The typical type of polyp is the autozooid, responsible for food capture and reproduction. In addition, some species can have another polyp category called siphonozooid, which ensures seawater irrigation through the colony.

Autozooids are cylindrical with a mouth and tentacles at one end whereas siphonozooids are smaller and have no or rudimentary tentacles [START_REF] Fabricius | Soft corals and sea fans: a comprehensive guide to the tropical shallow-water genera of the Central-West Pacific, the Indian Ocean and the Red Sea[END_REF]. Tentacles and their pinnules are mobile, contractile and hollow. An octocoral polyp consists of two main parts.

The portion that extends above the colony surface or completely retracts is called anthocodia (cylindrical body with terminal mouth and tentacles). The lower part of the polyp extends from the mouth into the gastrovascular cavity and includes the pharynx (Figure 5). The pharynx is the place where digestion of food particles starts and it encompasses a gutter lined with cilia, which ensures water circulation inside the gastrovascular cavity. From there, can be distinguished eight protruding tissue plates, the mesenteries, which have an important function in the hydraulic system of the polyps. They thicken into mesenterial filaments, including two that create an upward current in the gastrovascular cavity, and six that contain digestive glands and gonads (they complete the digestion of food particles that has started in the pharynx). At the base of a colony, desmocytes are extensions originating from the ectoderm, which play an active role in anchoring tissue to substrate [START_REF] Barneah | Attachment to the substrate by soft coral fragments: desmocyte development, structure, and function[END_REF].

Soft corals are diploblastic animals, namely they comprise two epithelial cell layers (Figure 5). The outer tissue layer (i.e. epidermis) is the interface with seawater, which extends over the entire colony. It gathers mucocysts, the cells that produce mucus, along with sensory cells such as nematocysts. The inner tissue layer (i.e. gastrodermis) lines the tentacles, pharynx and gastrovascular cavity. In soft corals living in symbiosis with Symbiodiniaceae dinoflagellates (see 1.2.4), most of the symbionts are localized in the cytoplasm of the gastrodermic coral cells, within a symbiosome [START_REF] Roth | Homology in endosymbiotic systems: the term "symbiosome[END_REF]. In between these two cell layers stands an acellular tissue called coenenchyme. It consists of mesoglea usually containing fibrous proteins like collagen, sclerites, along with amoeboid cells that play a role in fighting infections and phagocytosing debris and bacteria. The coenenchyme is thin in the tentacles but makes the main bulk of the colony in most species. It is crossed by a network of canals (solenia), which connect the gastrovascular cavity of each polyp to the others.

Although mostly studied in other Cnidaria taxa, soft corals may have a basic and diffused nervous system [START_REF] Watanabe | Cnidarians and the evolutionary origin of the nervous system[END_REF], with muscle bundles and nerve fibers located in the coenenchyme and other nerve and muscle cells lying under the epithelial layers [START_REF] Josephson | The neural control of behavior in sea anemones[END_REF]). Sclerites are an important feature used for the identification of soft corals since their shape, size and coloration vary widely among species. For example, spindle-shaped sclerites of up to 3 mm long are observed in Dendronephthya sp. while tiny oval corpuscules of 0.02 mm long are rather found in Xenia sp. (Figure 6).

Life history features and habitat requirements

Octocorals are the second most common group of macrobenthic animals on many Indo-Pacific, Caribbean and Red Sea reefs after hard corals [START_REF] Benayahu | Competition for space among coral-reef sessile organisms at Eilat, Red Sea[END_REF][START_REF] Fabricius | Soft corals and sea fans: a comprehensive guide to the tropical shallow-water genera of the Central-West Pacific, the Indian Ocean and the Red Sea[END_REF][START_REF] Benayahu | Controlling the false discovery rate: a practical and powerful approach to multiple testing[END_REF]. Some soft coral species of the family Xeniidae and Nephtheidae present high growth rates and asexual reproduction by colony fission [START_REF] Benayahu | Faunistic composition and patterns in the distribution of soft corals (Octocorallia, Alcyonacea) along the coral reefs of Sinai peninsula[END_REF]Loya 1985, 1987;Fabricius et al. 1995a;[START_REF] Karlson | Density-dependent dynamics of soft coral aggregations: the significance of clonal growth and form[END_REF]. Such features can allow them to be pioneer colonizers of cleared substrates and also increase their chances to successfully compete for space. Species like Xenia macrospiculata even have the ability to actively migrate for settling on living colonies of hard corals or natural surfaces [START_REF] Benayahu | Faunistic composition and patterns in the distribution of soft corals (Octocorallia, Alcyonacea) along the coral reefs of Sinai peninsula[END_REF]Loya 1981, 1985). Conversely, most Alcyoniidae are characterized by slow growth, low reproduction rates and greater persistence [START_REF] Fabricius | Slow population turnover in the soft coral genera Sinularia and Sarcophyton on mid-and outer-shelf reefs of the Great Barrier Reef[END_REF]. They are able to perform fission too, though operating slower than for the other families, with asexual recruits making a greater contribution to the population than sexual recruits [START_REF] Bastidas | Demographic aspects of the soft coral Sinularia flexibilis leading to local dominance on coral reefs[END_REF]). These life history features can result in the formation of large aggregations over the long-term [START_REF] Bastidas | Demographic aspects of the soft coral Sinularia flexibilis leading to local dominance on coral reefs[END_REF]). Moreover, some soft coral species, probably introduced through aquarium leak, have been reported as invasive species and overgrew coral communities in Venezuela and Brazil [START_REF] Lages | Chemical defense of an exotic coral as invasion strategy[END_REF][START_REF] Ruiz Allais | The first incidence of an alien soft coral of the family Xeniidae in the Caribbean, an invasion in eastern Venezuelan coral communities[END_REF][START_REF] Mantelatto | Invasion of aquarium origin soft corals on a tropical rocky reef in the southwest Atlantic, Brazil[END_REF].

Most octocorals occur in high abundance in wave-protected areas because they are susceptible to abrasion and dislodgement. They also search for strong currents for maximizing food encounter [START_REF] Fabricius | Soft corals and sea fans: a comprehensive guide to the tropical shallow-water genera of the Central-West Pacific, the Indian Ocean and the Red Sea[END_REF]. For symbiotic soft coral species, the presence of algal endosymbionts limits them to the photic zone, or deeper depending on the slope and water quality [START_REF] Schubert | Symbiotic versus non-symbiotic octocorals: physiological and ecological implications[END_REF]. Up to 75% of octocoral species however occur in depths greater than 50 m, and are mostly dominated by non-symbiotic species.

Octocorals and reef organisms

Soft corals are well known for their secondary metabolites, which are of particular interest for medical bioprospecting (reviewed in van de [START_REF] Van De Water | Host-microbe interactions in octocoral holobionts -recent advances and perspectives[END_REF]). In the reef environment, these substances are allelopathic and can act as a barrier to recruitment, growth and survival of hard corals [START_REF] Sammarco | Competitive strategies of soft corals (Coelenterata: Octocorallia): allelopathic effects on selected scleractinian corals[END_REF][START_REF] Fleury | Complementary (secondary) metabolites in a soft coral: sex-specific variability, inter-clonal variability, and competition[END_REF]). However, octocorals can host many macro organisms such as sponges, hydrozoans and decapods (e.g. [START_REF] Nakano | The soft-coral associated pistol shrimp Synalpheus neomeris (De Man) (Decapoda: Alpheidae) defends its host against nudibranchs in Okinawa, Japan[END_REF][START_REF] Maggioni | A mesophotic hotel: the octocoral Bebryce cf grandicalyx as a host[END_REF]. They provide essential resources for fishes and nudibranchs, including food, shelter and protection (e.g., [START_REF] Avila | An association between a dendronotid nudibranch (Mollusca, Opisthobranchia) and a soft coral (Octocorallia, Alcyonaria) from the red sea[END_REF][START_REF] Pratchett | Dietary overlap among coral-feeding butterflyfishes (Chaetodontidae) at Lizard Island, northern Great Barrier Reef[END_REF][START_REF] Fitzpatrick | Symbiodinium diversity in the soft coral Heteroxenia sp. and its nudibranch predator Phyllodesmium lizardensis[END_REF][START_REF] Epstein | Are soft coral habitats unfavourable? A closer look at the association between reef fishes and their habitat[END_REF]. As all multicellular organisms, octocorals are holobiont entities associated with many microorganism assemblages (van de [START_REF] Van De Water | Host-microbe interactions in octocoral holobionts -recent advances and perspectives[END_REF]. In other words, they are complex meta-organisms, which comprise the coral animal host, its symbiotic dinoflagellates and other microorganisms, including fungi, bacteria, archaea and viruses [START_REF] Knowlton | Multispecies microbial mutualisms on coral reefs: The host as a habitat[END_REF].

Symbiosis with Symbiodiniaceae: a nutritional-based partnership

 Symbiosis: definition A symbiosis is the "living together" of two unlike organisms in a long-term relationship.

It is a biological interaction that can span from mutualism to commensalism and parasitism (Table 1). However, the nature of these associations is not completely fixed and can shift from mutualism to commensalism or parasitism depending on environmental conditions. Symbioses can be either obligate, meaning that one or both of the partners entirely depend on each other for survival, or facultative (they can generally live independently). The bigger organism is called host while the smaller is the symbiont. In addition, the symbioses can be designated depending on the location of the symbionts. They can be referred as intra-or extracellular endosymbiosis (symbionts inside host cells or tissue, respectively), or ectosymbiosis (symbionts outside the host tissue).

Table 1. Description of the different symbioses. One example is provided for each type of symbiosis. The species that benefits from the association (B) may obtain nutrients, shelter, support or even locomotion from the host species (A).

Symbiotic associations are ubiquitous and play important roles in ecological and evolutionary processes [START_REF] Thrall | Coevolution of symbiotic mutualists and parasites in a community context[END_REF]. The symbiotic association between soft or hard corals and dinoflagellates of the family Symbiodiniaceae (see below) is qualified of mutualistic endosymbiosis. Symbionts are localized inside endodermic host cells (in the tentacles and periphery of the flesh, in the shallow gastrodermis, mostly not deeper than about 100 µm, Y.

Benayahu pers. com.) and both partners benefit from this relationship, through nutritional interactions described in detail in the next chapters. In addition, the coral host provides its symbionts with a protected habitat (as compared to free-living Symbiodiniaceae that are more likely grazed). Environmental conditions can create disparity in benefits and costs of the two partners, which rather promotes a hybrid model straddling between mutualism and parasitism (e.g., [START_REF] Baker | Climate change promotes parasitism in a coral symbiosis[END_REF]). In addition to photosynthetic dinoflagellates, corals harbor a diverse array of microorganisms such as bacteria, archaea, fungi and viruses [START_REF] Knowlton | Multispecies microbial mutualisms on coral reefs: The host as a habitat[END_REF]. The metabolic interactions within this symbiotic consortium are fundamental and drive coral's response to environmental changes (e.g., reviews of [START_REF] Ainsworth | The other microeukaryotes of the coral reef microbiome[END_REF][START_REF] Benavides | Diazotrophs: overlooked key players within the coral symbiosis and tropical reef ecosystems?[END_REF][START_REF] Bednarz | Diazotrophic community and associated dinitrogen fixation within the temperate coral Oculina patagonica[END_REF][START_REF] Pernice | Down to the bone: the role of overlooked endolithic microbiomes in reef coral health[END_REF].

 Symbiodiniaceae

Dinoflagellates of the family Symbiodiniaceae (kingdom Chromista, infrakingdom Alveolata, class Dinophyceae, order Suessiales) are of particular interest in tropical oligotrophic waters, because they can form mutualistic symbioses with a wide array of benthic metazoan and protist hosts (Figure 7). Dinoflagellates are unicellular microeukaryotes, characterized by two flagella, which allow them to migrate through the water column when they are free living or released in the water by their host. The genetic diversity within and between Symbiodiniaceae genera also reflects a physiological diversity, explaining their different response to environmental changes. For example, Durusdinium sp. was characterized as tenacious symbiont concerning thermal stress, meaning that it undergoes photo-damage but is not expelled by the host [START_REF] Silverstein | Tenacious D: Symbiodinium in clade D remain in reef corals at both high and low temperature extremes despite impairment[END_REF].

Due to the physiological diversity of the symbionts, some hard coral species can shuffle or switch their symbionts (sensu [START_REF] Baker | Flexibility and specificity in coral-algal symbiosis: diversity, ecology, and biogeography of Symbiodinium[END_REF] to acclimate to changing environmental conditions (but see [START_REF] Stat | Stability of coralendosymbiont associations during and after a thermal stress event in the southern Great Barrier Reef[END_REF]. Octocorals are however known to present more stable relationships with their dinoflagellates overtime [START_REF] Goulet | Stability of an octocoral-algal symbiosis over time and space[END_REF][START_REF] Kirk | Stable Symbiodinium composition in the sea fan Gorgonia ventalina during temperature and disease stress[END_REF][START_REF] Goulet | Most corals may not change their symbionts[END_REF][START_REF] Goulet | Most scleractinian corals and octocorals host a single symbiotic zooxanthella clade[END_REF].

 A nutritional-based partnership
Symbiotic corals are considered as mixotrophic organisms because they can use both inorganic and organic sources of nutrients. This dual mode of nutrition enables them to reap the maximum benefit in oligotrophic waters, where each of the partner would hardly persist alone due to the low concentration of inorganic nutrients and prey availability [START_REF] Stoecker | Mixotrophy in the marine plankton[END_REF].

Most of the knowledge on the nutritional ecology of corals is from hard corals, as described in the next two paragraphs. Corals are animals capable of heterotrophic feeding (i.e., the capture of particulate food in suspension in seawater, [START_REF] Houlbrèque | Heterotrophy in tropical scleractinian corals[END_REF].

Heterotrophy is especially important for supplying the host with nitrogen (N), phosphorus (P), amino-acids, or trace elements (e.g., iron and vitamins) for tissue, skeletal growth and reserve stocks [START_REF] Ferrier-Pagès | Effect of natural zooplankton feeding on the tissue and skeletal growth of the scleractinian coral Stylophora pistillata[END_REF], 2010, 2018;[START_REF] Seemann | The use of lipids and fatty acids to measure the trophic plasticity of the coral Stylophora subseriata[END_REF][START_REF] Tremblay | Heterotrophy promotes the re-establishment of photosynthate translocation in a symbiotic coral after heat stress[END_REF].

Corals can capture food directly, with their tentacles, or via suspension feeding [START_REF] Bak | Bacterial suspension feeding by coral reef benthic organisms[END_REF][START_REF] Anthony | Coral suspension feeding on fine particulate matter[END_REF]). The coral host can transfer a fraction of its heterotrophically-acquired nutrients to the symbionts, for their own needs [START_REF] Tremblay | Trophic dynamics of scleractinian corals: stable isotope evidence[END_REF]. It also transfers large amounts of dissolved inorganic carbon (DIC) resulting from its respiration, along with metabolic waste products [START_REF] Muscatine | Reef corals: Mutualistic symbioses adapted to nutrient-poor environments[END_REF].

Corals are also autotrophic organisms thanks to the presence of their photosynthetic symbionts, which transform inorganic into organic nutrients. The chloroplasts of Symbiodiniaceae cells possess photosynthetic pigments (chlorophyll a and c2, carotenoids) that enable the symbionts to catch light energy. As a result of cascading chemical reactions, the symbionts produce, from DIC, low molecular weight organic compounds called photosynthates [START_REF] Muscatine | Estimating the daily contribution of carbon from zooxanthellae to coral animal respiration[END_REF]. These organic molecules are rich in carbon and are generally in the form of sugars (e.g., glucose, [START_REF] Burke | Evidence that glucose is the major transferred metabolite in dinoflagellate--cnidarian symbiosis[END_REF]. Besides DIC, symbionts are likely the first site of assimilation of other nutrients dissolved in seawater, such as dissolved inorganic (ammonium, nitrate) and organic (urea, DFAA) nitrogen (DIN and DON, respectively) [START_REF] Grover | Uptake of ammonium by the scleractinian coral Stylophora pistillata: Effect of feeding, light, and ammonium concentrations[END_REF][START_REF] Grover | Nitrate uptake in the scleractinian coral Stylophora pistillata[END_REF][START_REF] Grover | Urea uptake by the scleractinian coral Stylophora pistillata[END_REF][START_REF] Grover | Uptake of dissolved free amino acids by the scleractinian coral Stylophora pistillata[END_REF][START_REF] Pernice | A single-cell view of ammonium assimilation in coral-dinoflagellate symbiosis[END_REF] or dissolved inorganic and organic phosphorus (DIP and DOP, respectively) (see [START_REF] Ferrier-Pagès | Phosphorus metabolism of reef organisms with algal symbionts[END_REF]) and references therein)

for the two major macronutrients. These nutrients can also be transferred to the coral host in the form of amino acids and other phosphorylated nutrients [START_REF] Wang | Essential amino acid synthesis and nitrogen recycling in an algainvertebrate symbiosis[END_REF]. Since the dinoflagellates are embedded in the animal tissue, dissolved inorganic nutrients first transit through the host tissues, which have developed particular adaptations [START_REF] Furla | Physiological adaptation to symbiosis in cnidarians[END_REF]. Most of the autotrophically-acquired nutrients, which are not used directly by the symbionts for their own needs and growth, are transferred to the host [START_REF] Muscatine | Fate of photosynthetic fixed carbon in light-and shade-adapted colonies of the symbiotic coral Stylophora pistillata[END_REF][START_REF] Tremblay | Autotrophic carbon budget in coral tissue: a new 13 C-based model of photosynthate translocation[END_REF].

A large percentage of the photosynthates is either immediately respired by the host to cover its metabolic needs, or released as dissolved and particulate organic matter [START_REF] Tremblay | Autotrophic carbon budget in coral tissue: a new 13 C-based model of photosynthate translocation[END_REF]).

The remaining photosynthates can be used for growth and energy reserves [START_REF] Grottoli | Heterotrophic plasticity and resilience in bleached corals[END_REF]).

In addition, corals are associated with a large diversity of microorganisms, which can contribute to providing nutrients to the coral host and symbionts. For example, diazotrophs are specialized bacteria able to transform dinitrogen (N2) gas into a bioavailable form of N. They have been discovered within the surface mucus layer, the coral tissue and skeleton [START_REF] Lema | Corals form characteristic associations with symbiotic nitrogen-fixing bacteria[END_REF][START_REF] Ainsworth | The coral core microbiome identifies rare bacterial taxa as ubiquitous endosymbionts[END_REF]. Studies that used the labelled gas 15 N2 demonstrated that diazotrophically-derived N (DDN) was assimilated by the coral host and symbionts (reviewed by [START_REF] Benavides | Diazotrophs: overlooked key players within the coral symbiosis and tropical reef ecosystems?[END_REF]. Although this form of N contributes only to ca. 1% to the N needs of the coral holobiont compared to the other N forms (inorganic N dissolved in seawater or from the host metabolic wastes), it may help corals recovering faster from bleaching [START_REF] Bednarz | The assimilation of diazotroph-derived nitrogen by scleractinian corals depends on their metabolic status[END_REF].

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In octocorals, the relative contribution of autotrophy and heterotrophy to the holobiont's nutritional requirements is poorly known (reviewed in [START_REF] Schubert | Symbiotic versus non-symbiotic octocorals: physiological and ecological implications[END_REF]. The common belief is that octocorals have low rates of primary productivity and depend on both autotrophy and heterotrophy to meet their metabolic needs [START_REF] Sorokin | Biomass, metabolic rates and feeding of some common reef zoantharians and octocorals[END_REF][START_REF] Fabricius | Widespread mixotrophy in reef-inhabiting soft corals: the influence of depth, and colony expansion and contraction on photosynthesis[END_REF].

However, the few reports investigating the contribution of photosynthesis to host respiration (P:R ratio) rather support a reliance on autotrophy for the respiratory needs [START_REF] Mergner | Productivity and seasonal changes in selected reef areas in the Gulf of Aqaba (Red Sea)[END_REF][START_REF] Sorokin | Biomass, metabolic rates and feeding of some common reef zoantharians and octocorals[END_REF][START_REF] Fabricius | Widespread mixotrophy in reef-inhabiting soft corals: the influence of depth, and colony expansion and contraction on photosynthesis[END_REF][START_REF] Riegl | Effects of sediment on the energy budgets of four scleractinian (Bourne 1900) and five alcyonacean (Lamouroux 1816) corals[END_REF][START_REF] Kremien | Beneft of pulsation in soft corals[END_REF][START_REF] Bednarz | Seasonal variation in dinitrogen fixation and oxygen fluxes associated with two dominant zooxanthellate soft corals from the northern Red Sea[END_REF][START_REF] Rossi | Linking host morphology and symbiont performance in octocorals[END_REF][START_REF] Rossi | Trophic ecology of Caribbean octocorals: autotrophic and heterotrophic seasonal trends[END_REF], except for few species [START_REF] Ramsby | Symbiodinium photosynthesis in Caribbean octocorals[END_REF][START_REF] Baker | Productivity links morphology, symbiont specificity and bleaching in the evolution of Caribbean octocoral symbioses[END_REF]. The small number of studies, which have addressed a carbon budget observed high interspecies variation, with photosynthate translocation rates ranging from 10%

in Capnella gabonensis to 75% in Sinularia flexibilis [START_REF] Schlichter | Functional autotrophy of Heteroxenia fuscescens (Anthozoa: Alcyonaria): carbon assimilation and translocation of photosynthates from symbionts to host[END_REF][START_REF] Farrant | Nutrition of the temperate Australian soft coral Capnella gaboensis I. Photosynthesis and carbon fixation[END_REF][START_REF] Sorokin | Biomass, metabolic rates and feeding of some common reef zoantharians and octocorals[END_REF][START_REF] Khalesi | Energy budget for the cultured, zooxanthellate octocoral Sinularia flexibilis[END_REF]. In gorgonian octocorals, the symbionts performance is linked to the host morphology because species with thin branches and small polyps are more autotrophic than those with a massive shape and large polyps [START_REF] Baker | Productivity links morphology, symbiont specificity and bleaching in the evolution of Caribbean octocoral symbioses[END_REF][START_REF] Rossi | Linking host morphology and symbiont performance in octocorals[END_REF]). The colony morphology may also play an important role in the nutrition of soft corals.

The ratio of body surface area to volume, which determines both gas exchange through the epidermal tissue and photosynthetically active radiation (PAR) exposure, is low in soft corals as compared to hard corals [START_REF] Fabricius | Widespread mixotrophy in reef-inhabiting soft corals: the influence of depth, and colony expansion and contraction on photosynthesis[END_REF]. Any increase of this ratio, through the expansion of colony, may enhance primary productivity. More investigations are therefore needed to better understand the autotrophic capacities of octocorals in general, and soft corals in particular.

Regarding heterotrophy, soft corals have long been considered as mainly suspension feeders, because their nematocysts are small, simply structured and inefficient for paralyzing actively swimming prey [START_REF] Mariscal | A comparison of putative sensory receptors associated with nematocysts in an anthozoan and a scyphozoan[END_REF]. However, the recent discovery of a novel type of nematocysts could potentially highlight a more diverse arsenal of stinging structures than previously thought [START_REF] Yoffe | A modified view on octocorals: Heteroxenia fuscescens nematocysts are diverse, featuring both an ancestral and a novel type[END_REF]. Moreover, microvilii densely cover the epidermis of soft corals and likely plays a role in the absorption of dissolved matter (Schlichter 1982b, a).

The diet of octocorals has been determined via field and feeding experiments, from temperate [START_REF] Coma | Feeding and prey capture cycles in the aposymbiontic gorgonian Paramuricea clavata[END_REF][START_REF] Coma | An energetic approach to the study of life-history traits of two modular colonial benthic invertebrates[END_REF][START_REF] Coma | Natural heterotrophic feeding by a temperate octocoral with symbiotic zooxanthellae: a contribution to understanding the mechanisms of die-off events[END_REF][START_REF] Orejas | Role of small-plankton communities in the diet of two antarctic octocorals (Primnoisis antarctica and Primnoella sp.)[END_REF][START_REF] Rossi | Temporal variability in zooplankton prey capture rate of the passive suspension feeder Leptogorgia sarmentosa (Cnidaria: Octocorallia), a case study[END_REF]) and tropical species [START_REF] Lewis | Feeding behaviour and feeding ecology of the Octocorallia (Coelenterata: Anthozoa)[END_REF][START_REF] Sebens | Predation on zooplankton by the benthic anthozoans Alcyonium siderium (Alcyonacea) and Metridium senile (Actiniaria) in the New England subtidal[END_REF][START_REF] Sorokin | Biomass, metabolic rates and feeding of some common reef zoantharians and octocorals[END_REF][START_REF] Fabricius | Depletion of suspended particulate matter over coastal reef communities dominated by zooxanthellate soft corals[END_REF][START_REF] Rossi | Trophic ecology of Caribbean octocorals: autotrophic and heterotrophic seasonal trends[END_REF].

It is composed of small zooplankton (Figure 9). In addition, and on the contrary to many hard corals, soft corals can also largely prey on phytoplankton (Figure 9), whose ingestion has been described in both asymbiotic (Fabricius et al. 1995a(Fabricius et al. , b, 1998) ) and symbiotic species (Ribes et al. 1998;[START_REF] Leal | Coral feeding on microalgae assessed with molecular trophic markers[END_REF][START_REF] Piccinetti | Herbivory in the soft coral Sinularia flexibilis (Alcyoniidae)[END_REF]. A particular behavior called 'deposit feeding' has even been observed in the cold-water soft coral Gersemia Antarctica, which bends against the substrate to supplement its capture of prey on sediment [START_REF] Slattery | Deposit feeding: a novel mode of nutrition in the Antarctic colonial soft coral Gersemia antarctica[END_REF]. The relative contribution of auto-and heterotrophy to soft coral nutrition may not be fixed and depend on environmental conditions and/or on the availability of resources (i.e. depth and season; [START_REF] Rossi | Trophic ecology of Caribbean octocorals: autotrophic and heterotrophic seasonal trends[END_REF]. However, this remains to be further investigated.

Major threats to coral reefs and soft corals

Existing coral reefs are not the result of continuous growth. They have faced both natural and human-induced disturbances that have affected and still affect their integrity periodically or over the long-term. Reefs are generally able to recover from natural disturbances in a few years to decades (e.g., Harmelin-Vivien 1994). However, human activities add up local and global stressors to the natural disturbances, which act together to weaken coral reefs and eventually drive major declines.

Global disturbances

Anthropogenic activities such as fossil fuel combustion, industrialization and deforestation, have raised the concentrations of greenhouse gases in the atmosphere since the beginning of the industrial era [START_REF] Sabine | The oceanic sink for anthropogenic CO2[END_REF]). Such emissions are the cause of global climate changes including ocean warming and acidification. As a result, marine ecosystems are changing and coral reefs are among the most threatened [START_REF] Hoegh-Guldberg | The Ocean[END_REF][START_REF] Gattuso | Contrasting futures for ocean and society from different anthropogenic CO2 emissions scenarios[END_REF].

 Ocean warming

All coral live close to their upper thermal maximum and can suffer from thermal stress when sea surface temperatures increase by ≥ 1°C above the average summer maximum [START_REF] Wooldridge | Breakdown of the coral-algae symbiosis: Towards formalising a linkage between warm-water bleaching thresholds and the growth rate of the intracellular zooxanthellae[END_REF]. When corals experience extreme and prolonged heat stress, the symbiosis with their photosynthetic dinoflagellates is disrupted, resulting in the expulsion of the symbionts, a phenomenon called bleaching [START_REF] Brown | Coral bleaching: causes and consequences[END_REF]. Bleached corals are physiologically damaged, and prolonged bleaching often leads to high levels of coral mortality [START_REF] Hughes | Global warming transforms coral reef assemblages[END_REF]. Although positive thermal anomalies are the main cause for bleaching, it can also be induced by other stress factors such as bacterial infections, burial under sediment or high irradiance [START_REF] Fabricius | Soft corals and sea fans: a comprehensive guide to the tropical shallow-water genera of the Central-West Pacific, the Indian Ocean and the Red Sea[END_REF]. According to the World Meteorological Organization, the last decade was the warmest one on record. Reefs worldwide have therefore encountered unprecedented and recurrent coral mass bleaching events [START_REF] Hughes | Global warming and recurrent mass bleaching of corals[END_REF]

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Mass bleaching of tropical octocorals has been reported in many reefs worldwide [START_REF] Slattery | Population structure and feeding deterrence in three shallowwater antartic soft corals[END_REF][START_REF] Paulay | Patterns and consequences of coral bleaching in Micronesia (Majuro and Guam) in 1992-1994[END_REF][START_REF] Marshall | Bleaching of corals on the Great Barrier Reef: differential susceptibilities among taxa[END_REF][START_REF] Arceo | Coral bleaching in Philippine reefs: Coincident evidences with mesoscale thermal anomalies[END_REF][START_REF] Loya | Coral bleaching : the winners and the losers[END_REF][START_REF] Mcclanahan | Coral and algal changes after the 1998 coral bleaching: Interaction with reef management and herbivores on Kenyan reefs[END_REF][START_REF] Celliers | Coral bleaching on high-latitude marginal reefs at Sodwana Bay, South Africa[END_REF]Goulet et al. 2008a;[START_REF] Chavanich | Mass bleaching of soft coral, sarcophyton spp. in Thailand and the role of temperature and salinity stress[END_REF][START_REF] Prada | Octocoral bleaching during unusual thermal stress[END_REF][START_REF] Van Woesik | Revisiting the winners and the losers a decade after coral bleaching[END_REF][START_REF] Dias | Bleaching in scleractinians, hydrocorals, and octocorals during thermal stress in a northeastern Brazilian reef[END_REF][START_REF] Slattery | Annual thermal stress increases a soft coral's susceptibility to bleaching[END_REF]. Similarly to other coral groups, their susceptibility to thermal stress is heterogeneous and, some species or individuals exhibit a higher tolerance, depending on the host or symbiont genus or on the general health state, energetic reserves and acclimatization of the colonies [START_REF] Loya | Coral bleaching : the winners and the losers[END_REF][START_REF] Lasker | Zooxanthella densities within a Caribbean octocoral during bleaching and non-bleaching years[END_REF][START_REF] Strychar | Loss of Symbiodinium from bleached soft corals Sarcophyton ehrenbergi, Sinularia sp. and Xenia sp[END_REF]Goulet et al. 2008a;[START_REF] Van Woesik | Revisiting the winners and the losers a decade after coral bleaching[END_REF][START_REF] Slattery | Annual thermal stress increases a soft coral's susceptibility to bleaching[END_REF]). Records of bleaching may also be biased towards greater numbers of bleached persistent species (e.g. Alcyoniidae), as opposed to the ones that die and disintegrate (e.g. Xeniidae) within several days [START_REF] Fabricius | Tissue loss and mortality in soft corals following mass-bleaching[END_REF]. [START_REF] Sammarco | Responses to high seawater temperatures in zooxanthellate octocorals[END_REF][START_REF] Netherton | Physiological correlates of symbiont migration during bleaching of two octocoral species[END_REF]). The exception is a species of the family Xeniidae (Bayerxenia spp.), which did not display changes neither related to the symbionts or host cells, after being exposed to +6°C during one week [START_REF] Ziegler | Thermal stress response in a dinoflagellate-bearing nudibranch and the octocoral on which it feeds[END_REF]. Some species also mitigated bleaching through symbionts migration and accumulation in the stolons [START_REF] Parrin | Withincolony migration of symbionts during bleaching of octocorals[END_REF][START_REF] Parrin | Symbiodinium migration mitigates bleaching in three octocoral species[END_REF]. Therefore, octocorals seem to be as affected as hard corals by ocean warming. The overall stability of octocoral populations, while hard corals have steadily declined (e.g., [START_REF] Ruzicka | Temporal changes in benthic assemblages on Florida Keys reefs 11 years after the 1997/1998 El Niño[END_REF], may involve other processes than resistance (i.e.

resilience, acclimatization, and life history features).

 Ocean acidification (OA)

OA is an on-going process, which induces several changes in the seawater carbonate chemistry (e.g. [START_REF] Feely | Impact of anthropogenic CO2 on the CaCO3 system in the oceans[END_REF]. In particular, it decreases seawater pH and the concentration of carbonate ions. Such modifications therefore negatively affect the biogenic calcification (e.g.

of aragonite and calcite) and can also drive the net dissolution of carbonate structures. Thus, OA poses significant problems to marine organisms that form calcium carbonate shells, skeletons, or internal structures [START_REF] Orr | Anthropogenic ocean acidification over the twenty-first century and its impact on calcifying organisms[END_REF][START_REF] Gattuso | Ocean acidification[END_REF]. For example, present-day conditions have been shown to dissolve exoskeletons of Dungeness crab larvae [START_REF] Bednaršek | Exoskeleton dissolution with mechanoreceptor damage in larval Dungeness crab related to severity of present-day ocean acidification vertical gradients[END_REF]. On reefs, OA can lead to reduced structural complexity and resilience due to decreased coral abundance and calcification rates [START_REF] Kroeker | Impacts of ocean acidification on marine organisms: Quantifying sensitivities and interaction with warming[END_REF][START_REF] Smith | Shifts in coralline algae, macroalgae, and coral juveniles in the Great Barrier Reef associated with present-day ocean acidification[END_REF].

Observations of acidified natural environments such as carbon dioxide seeps have shown a loss of key habitat-forming species and major simplification of the ecosystem [START_REF] Sunday | Ocean acidification can mediate biodiversity shifts by changing biogenic habitat[END_REF][START_REF] Agostini | 2 La symbiose, à la base de la productivité des récifs coralliens Les coraux constructeurs de récifs (scléractiniaires), ainsi que de nombreux octocoralliaires, sont des méta-organismes complexes, constitués par le corail animal hôte[END_REF]. Overall, reefs acclimatized to acidified waters demonstrate a clear dominance of non-calcareous macroalgae (pH < 7.7, [START_REF] Enochs | Shift from coral to macroalgae dominance on a volcanically acidified reef[END_REF], followed by monospecific aggregations of hard corals (e.g. Porites sp., [START_REF] Fabricius | Losers and winners in coral reefs acclimatized to elevated carbon dioxide concentrations[END_REF] or soft corals (e.g., Sarcophyton sp., [START_REF] Inoue | Spatial community shift from hard to soft corals in acidified water[END_REF]) under moderate conditions (pH > 7.8). Mesocosm experiments have shown that the effects of OA are highly context dependent and will differ greatly between habitats, and depending on species composition (e.g. [START_REF] Comeau | Coral reef calcifiers buffer their response to ocean acidification using both bicarbonate and carbonate[END_REF][START_REF] Comeau | Water flow modulates the response of coral reef communities to ocean acidification[END_REF][START_REF] Comeau | Flow-driven micro-scale pH variability affects the physiology of corals and coralline algae under ocean acidification[END_REF].

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While in situ observations suggest shifts from hard coral to octocoral dominance under levels of pH predicted by the end of the century [START_REF] Inoue | Spatial community shift from hard to soft corals in acidified water[END_REF], only few experimental studies have addressed octocoral's response to OA. They showed that octocoral tissue descriptors and physiological parameters (i.e. symbiont cell density, pigmentation, sclerite structure, polyp pulsation rate and growth) were not affected by OA [START_REF] Gabay | Does elevated pCO2 affect reef octocorals?[END_REF][START_REF] Gabay | Octocoral tissue provides protection from declining oceanic pH[END_REF][START_REF] Enochs | Micro-CT analysis of the Caribbean octocoral Eunicea flexuosa subjected to elevated pCO2[END_REF]. Only one study showed a decreased calcification, but the pH tested (7.1) exceeded the values projected by the end of the century [START_REF] Gómez | Responses of the tropical gorgonian coral Eunicea fusca to ocean acidification conditions[END_REF]. OA mitigation by octocorals could be explained by their thick tissue, which can protect sclerites [START_REF] Gabay | Does elevated pCO2 affect reef octocorals?[END_REF][START_REF] Gabay | Octocoral tissue provides protection from declining oceanic pH[END_REF], and the process of calcification itself, which may be more stable than the one of hard corals despite altered carbonate chemistry [START_REF] Enochs | Micro-CT analysis of the Caribbean octocoral Eunicea flexuosa subjected to elevated pCO2[END_REF]. Although there is a paucity of data on the combined effects of OA and other threats on octocorals, it has been suggested that hard corals, which are already stressed by OA alone, will be more affected than octocorals with the combination of several stressors (e.g., eutrophication, [START_REF] Januar | Tropical coral reef coral patterns in Indonesian shallow water areas close to underwater volcanic vents at Minahasa Seashore, and Mahengetang and Gunung Api Islands[END_REF]).

Local stressors

Coral reefs can be affected by local anthropogenic pressures such as overfishing, invasive species and various sources of pollution (sediment, nutrients, and pesticides loads) due to coastal and agriculture development [START_REF] Burke | Evidence that glucose is the major transferred metabolite in dinoflagellate--cnidarian symbiosis[END_REF][START_REF] Anthony | Operationalizing resilience for adaptive coral reef management under global environmental change[END_REF][START_REF] Wear | Sewage pollution: mitigation is key for coral reef stewardship[END_REF]. At the ecosystem level, local stressors may be the cause of structural impairment and mortality of most organisms (e.g. destructive fishing). They may also impact reefs indirectly by inducing cascading effects across the ecosystem. For example, overfishing (alteration in top-down controls) depletes stocks of large predators and grazers, and leads to reduced herbivory and subsequent algal overgrowth [START_REF] Hughes | Catastrophes, phase shifts, and large-scale degradation of a Caribbean coral reef[END_REF][START_REF] Jackson | Historical overfishing and the recent collapse of coastal ecosystems[END_REF][START_REF] Scheffer | Cascading effects of overfishing marine systems[END_REF]. Finally, reduced water quality, due to overloads of nutrients and sediments (alteration of bottom-up controls), can stimulate the growth of algae [START_REF] Mccook | Competition between corals and algae on coral reefs: a review of evidence and mechanisms[END_REF], the development of diseases [START_REF] Pollock | Sediment and turbidity associated with offshore dredging increase coral disease prevalence on nearby reefs[END_REF]) and can favor the emergence of corallivorous

Acanthaster planci outbreaks [START_REF] Birkeland | Terrestrial runoff as a cause of outbreaks of Acanthaster planci (Echinodermata: Asteroidea)[END_REF][START_REF] Brodie | Are increased nutrient inputs responsible for more outbreaks of crown-of-thorns starfish? An appraisal of the evidence[END_REF][START_REF] Fabricius | Three lines of evidence to link outbreaks of the crownof-thorns seastar Acanthaster planci to the release of larval food limitation[END_REF]. At the organism level, the severity of water pollution on hard corals depends on the intensity, duration and frequency of exposure. They contribute to the impairment of coral fitness and to lessen

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Reports on the effects of declining water quality on the abundance of soft corals are not conclusive. They either show a negative impact on the cover of both hard and soft corals [START_REF] Fabricius | Identifying ecological change and its causes: a case study on coral reefs[END_REF], no effect [START_REF] Fabricius | Biodiversity on the Great Barrier Reef: large-scale patterns and turbidity-related local loss of soft coral taxa[END_REF], or a relative dominance of soft corals in areas affected by sedimentation [START_REF] Mcclanahan | Sedimentation effects on shallow coral communitiesin Kenya[END_REF][START_REF] Schleyer | Coral dominance at the reef-sediment interface in marginal coral communities at Sodwana Bay, South Africa[END_REF], moderate to high concentrations of suspended particulate matter [START_REF] Fabricius | Depletion of suspended particulate matter over coastal reef communities dominated by zooxanthellate soft corals[END_REF][START_REF] Fabricius | Effects of terrestrial runoff on the ecology of corals and coral reefs: Review and synthesis[END_REF], or a combination of particulate and dissolved matter [START_REF] Baum | Abundance and physiology of dominant soft corals linked to water quality in Jakarta Bay, Indonesia[END_REF]. Data from the Great Barrier Reef, Hong-Kong and Palau suggest that a poor water quality is rather related to a decline in species diversity [START_REF] Fabricius | Identifying ecological change and its causes: a case study on coral reefs[END_REF][START_REF] Fabricius | Changes in algal, coral and fish assemblages along water quality gradients on the inshore Great Barrier Reef[END_REF][START_REF] Fabricius | Octocorallia in Palau, Micronesia: effects of biogeography and coastal influences on local and regional biodiversity[END_REF][START_REF] Fabricius | Changes in octocoral communities and benthic cover along a water quality gradient in the reefs of Hong Kong[END_REF]. Further research is thus needed to explain the confounding effects of water quality on octocorals. 1.3.3 Changes in the reef community structure: phase shifts to octocoraldominated reefs

Intense competition continuously takes place between benthic reef organisms for the access to light and space. Intra-or interspecific competitive interactions can have major effects on the growth, recruitment and survivorship of benthic competitors [START_REF] Chadwick | Competition among sessile organisms on coral reefs[END_REF]. Human activities may unbalance the dynamic of these interactions, by favoring the decline of an organism or providing the competitive advantage to another. Phase shifts are community changes in response to a persistent modification in environmental conditions [START_REF] Done | Phase shifts in coral reef communities and their ecological significance[END_REF][START_REF] Dudgeon | Phase shifts and stable states on coral reefs[END_REF]). On coral reefs, a phase shift results in the transition from a hard coral-dominated reef to a reef dominated by other benthic organisms such as algae, sponges, octocorals, or corallimorphs [START_REF] Mccook | Macroalgae, nutrients and phase shifts on coral reefs: scientific issues and management consequences for the Great Barrier Reef[END_REF][START_REF] Norström | Alternative states on coral reefs: beyond coral-macroalgal phase shifts[END_REF][START_REF] Barott | Unseen players shape benthic competition on coral reefs[END_REF].

This process is often associated with reef degradation, due to the loss of the 'bricks' of the ecosystem and associated functions. Unless few exceptions, a community shift may be difficult to reverse to pre-disturbance conditions (e.g., [START_REF] Diaz-Pulido | Doom and Boom on a Resilient Reef: Climate Change, Algal Overgrowth and Coral Recovery[END_REF].

Although shifts to algae-dominated reefs are prominent worldwide, shifts to other major benthic groups, such as octocorals and sponges, have also been reported [START_REF] Norström | Alternative states on coral reefs: beyond coral-macroalgal phase shifts[END_REF].

Changes in the community structure ending up towards the dominance of octocorals can occur following COTS outbreaks, anthropogenic pollution, storms, bleaching, diseases and dynamite fishing (Figure 10 and Table 2). These disturbances led to a decreased cover of hard corals whereas soft corals maintained or increased their abundance. The colonization of the reef by octocorals occurs when the right conditions regarding their recruitment and growth are met. For example, [START_REF] Wood | What is the future for extensive areas of reef impacted by fish blasting and coral bleaching and now dominated by soft corals? A case study from Malaysia[END_REF] recorded that, under similar environmental conditions, some fields of coral rubble have provided suitable settlement substrate for soft corals whereas some others did not. The successfully colonized part of the reef underwent a phase shift from hard corals to xeniids-Clavularia assemblage and the other part remained unoccupied, likely not stable enough. Moreover, [START_REF] Fabricius | Soft coral abundance on the central Great Barrier Reef: Effects of Acanthaster planci, space availability, and aspects of the physical environment[END_REF] observed that there was no difference in soft coral cover before and after a COTS outbreak that removed a large proportion of hard corals. Even eight years after the event, soft corals showed little tendency to take advantage of increased available space due to the environmental conditions that were not suitable for them [START_REF] Fabricius | Soft corals and sea fans: a comprehensive guide to the tropical shallow-water genera of the Central-West Pacific, the Indian Ocean and the Red Sea[END_REF].

Soft corals of the Northern Red Sea

Red Sea coral reefs, like other marine ecosystems, experience global warming and ocean acidification [START_REF] Fine | Coral reefs of the Red Sea -Challenges and potential solutions[END_REF]. However, hard corals in the Gulf of Aqaba have an unusually high thermal tolerance and rarely experience bleaching, despite rapidly rising sea surface temperatures and recurrent positive temperature anomalies. They withstand water temperature anomalies that cause severe bleaching or mortality in most hard corals elsewhere when the water temperature exceeds 1-2°C above the local maximum monthly mean (e.g., [START_REF] Bellworthy | Beyond peak summer temperatures, branching corals in the Gulf of Aqaba are resilient to thermal stress but sensitive to high light[END_REF][START_REF] Grottoli | Physiological and biogeochemical responses of super-corals to thermal stress from the northern gulf of Aqaba, Red Sea[END_REF]). As a consequence, the Gulf of Aqaba has been identified as a coral refuge from climate change [START_REF] Fine | A coral reef refuge in the Red Sea[END_REF][START_REF] Kleinhaus | Science, diplomacy, and the Red Sea's unique coral reef: It's time for action[END_REF]).

Study site

Most of the experiments of this thesis were conducted on corals collected in Eilat, in the reef adjacent to the Interuniversity Institute for Marine Sciences (Figure 11). This site is of particular interest for giving access to shallow and mesophotic reef zones. Mesophotic coral reef ecosystems comprise the light-dependent communities of corals and other organisms found at depths below 30 to 150 m in tropical and subtropical regions [START_REF] Puglise | Mesophotic coral ecosystem research strategy International Workshop to Prioritize Research and Management Needs for Mesophotic Coral Ecosystems[END_REF]. Based on measurements of light intensity and spectrum [START_REF] Tamir | Light environment drives the shallow to mesophotic coral community transition[END_REF], combined with concomitant changes in benthic composition, the mesophotic reefs of Eilat have been classified into two distinctive light habitats: the upper-mesophotic zone (30-80 m), which is characterized by a high diversity of depth-generalist coral species, and the lower mesophotic zone (80-160 m), dominated by a single species of depth-generalist [START_REF] Eyal | The Red Sea: Israel[END_REF]). For our work, corals were collected at depths of 8-10 m (shallow) and 40-50 m (upper-mesophotic). Light attenuation at these depths is described in Appendix II. Characteristics of the water are stable along this gradient, including a salinity over 41‰, temperatures ranging from 20.7°C to 30.3°C at 10 m depth and 20.9-27.8°C at 50 m depth over the year, as well as low concentrations of dissolved nutrients (<0.5 µM dissolved nitrogen and <0.2 µM dissolved phosphorus) (data from the Israel National Monitoring Program, [START_REF] Eyal | The Red Sea: Israel[END_REF]. Soft corals are the second most abundant faunal group in the shallow and mesophotic reefs of Eilat, after scleractinians. They have a moderate cover of 3-10% along the depth gradient but rarely cover more than 20% of the substrate [START_REF] Eyal | The Red Sea: Israel[END_REF]. Two distinct communities of octocorals have been identified at shallow and upper-mesophotic (30-45 m) depths, which only shared 16% of species in common [START_REF] Shoham | Higher species richness of octocorals in the upper mesophotic zone in Eilat (Gulf of Aqaba) compared to shallower reef zones[END_REF]. The upper-mesophotic octocoral community features a higher species richness, but a similar diversity, as compared to the shallow one. More particularly, the upper-mesophotic octocoral community is characterized by a predominance of symbiotic species [START_REF] Shoham | Higher species richness of octocorals in the upper mesophotic zone in Eilat (Gulf of Aqaba) compared to shallower reef zones[END_REF]).

Biological material

Several species of soft and hard corals were studied in this thesis, chosen depending on their morphology and physiological characteristics (Figure 12 and Figure 13).

Dendronephthya sp. (Kükenthal, 1905) is the only species that does not harbor any Symbiodiniaceae and have ca. 180-200 polyps per g DW, of a diameter of 3-4 mm [START_REF] Lewis | Feeding behaviour and feeding ecology of the Octocorallia (Coelenterata: Anthozoa)[END_REF][START_REF] Sorokin | Morphology and ecological physiology of corals[END_REF]. Heteroxenia fuscescens (Kölliker, 1874) have polyps of 12-14 mm [START_REF] Lewis | Feeding behaviour and feeding ecology of the Octocorallia (Coelenterata: Anthozoa)[END_REF]. Members of the family Xeniidae exhibit a unique, rhythmic pulsation of their tentacles.

Such feature induces an upward motion and enables the mixing of the coral boundary layer, which prevents refiltration of water by neighboring polyps and enhances the coral's photosynthesis [START_REF] Kremien | Beneft of pulsation in soft corals[END_REF]

Aims of the thesis

1.5.1 Why a focus on soft corals?

As described in the above parts, octocorals are important components of reefs:

-They are the second main group of macrobenthic animals on reefs (part 1.2.2).

-They are ecosystem engineers, and thus provide essential habitat and food to reefassociated organisms (parts 1.1.2 and 1.2.3).

-They are often abundant within disturbed reef systems, where they can exhibit a higher survivorship than hard corals in a wide range of environmental conditions (part 1.3).

However, the accumulated data on the physiological characteristics of soft corals, or on their trophic ecology and relationship with endosymbionts is highly fragmented and not exhaustive [START_REF] Schubert | Symbiotic versus non-symbiotic octocorals: physiological and ecological implications[END_REF][START_REF] Van De Water | Host-microbe interactions in octocoral holobionts -recent advances and perspectives[END_REF]. It does not help having a holistic view of the functional role and ecological importance of these organisms. Research on octocorals indeed lags well behind the research on hard corals (Figure 14) and important knowledge gaps related to their physiology, particularly to their nutrition, still need to be addressed.

Figure 14. Number of publications obtained in a Google scholar search. Key words used are "Scleractinian corals" and "physiology" versus "Octocorals" and "physiology".

Aims of the thesis

Nutrition is one of the most important factors regulating the abundance and distribution of a community within an ecosystem, along with environmental conditions and interactions with other organisms (e.g., competition and predation). Particularly, nutrient acquisition supplies energy for coral growth, reproduction and can promote coral resistance or resilience to environmental changes.

In the oligotrophic reefs of Eilat (Gulf of Aqaba, northern Red Sea), two distinct communities of octocorals have been identified at shallow and upper-mesophotic depths.

Interestingly, the upper-mesophotic octocoral community is characterized by a higher species richness, along with a predominance of symbiotic species. While these characteristics suggest an adequate light regime for coral's photosynthesis to occur there [START_REF] Shoham | Higher species richness of octocorals in the upper mesophotic zone in Eilat (Gulf of Aqaba) compared to shallower reef zones[END_REF], the nutritional strategies of soft corals have received little attention so far.

This thesis therefore focuses on the functioning of the dinoflagellate-soft coral symbiosis and aims to bring insights into the nutritional interactions between different partners of the holobiont. The respective contributions of auto-and heterotrophy in the metabolic needs of soft corals along the depth gradient were of particular interest. For this purpose, the acquisition and allocation of several forms of carbon and nitrogen were assessed. Four objectives corresponding to the following chapters were identified to explore these nutrient fluxes under different environmental conditions and to compare them to processes already known in hard corals.

 Aim 1 -Chapter 2: Normalization metric and processing protocol Normalization is a fundamental step in data treatment that will deeply affect the outcome of an experiment. Several problems have been identified for soft corals: 1) The gelatinous and leather-like nature of soft coral tissue do not allow an easy extraction of tissue descriptors, such as symbiont density, pigment and protein concentration, 2) the most widely used normalization metric in scleractinian corals, the skeletal surface area, is not relevant for soft corals since they do not possess any skeleton, 3) surface area of a nubbin is difficult to estimate due to their highly variable hydroskeleton, which can change size by several fold depending on environmental conditions, and 4) physiological parameters and processes of soft corals need to be compared with other coral species, such as scleractinians.

An experiment was thus designed to 1) develop a fast protocol for an easy tissue homogenization and 2) identify a normalization metric that can be used to perform interstudies and inter-species comparisons.

For this purpose, the effect of different tissue sample states and media used for homogenization were tested on the measurement of tissue descriptors in the soft coral Heteroxenia fuscescens. Moreover, the suitability of several normalizing metrics was investigated in Dendronephthya sp., Heteroxenia fuscescens, Litophyton arboreum and Rhytisma fulvum fulvum.

 Aim 2 -Chapter 3: Acquisition of dissolved inorganic carbon

The autotrophic carbon acquisition contributes largely to the metabolic needs of hard corals, which receive 90% of the photosynthates produced by the symbionts. In octocorals, the contribution of the symbionts to the energetic requirements of their host is still poorly understood. First of all, studies addressing the productivity of soft corals are scarce, limited to a depth of 20 m, and the results obtained present a high variability (see supplementary information in [START_REF] Rossi | Linking host morphology and symbiont performance in octocorals[END_REF]. There are several identified sources for such variability: 1) soft coral colonies can reach various degrees of expansion depending on the environmental conditions, that confers a high biological variability to the measurements (e.g., [START_REF] Fabricius | Widespread mixotrophy in reef-inhabiting soft corals: the influence of depth, and colony expansion and contraction on photosynthesis[END_REF]; 2) the morphology of octocoral colony and polyps, notably their surface area to volume ratio, can be more or less suited for autotrophy [START_REF] Baker | Productivity links morphology, symbiont specificity and bleaching in the evolution of Caribbean octocoral symbioses[END_REF][START_REF] Rossi | Linking host morphology and symbiont performance in octocorals[END_REF];

and 3) the use of different methods and normalization metrics (see Chapter 2 for the latter) hardly enables comparisons. In addition, the quantification of photosynthate translocation from the symbionts to the host has been investigated in only one tropical (Heteroxenia fuscecens)

and one temperate species (Capnella gaboensis) using 14 C [START_REF] Schlichter | Functional autotrophy of Heteroxenia fuscescens (Anthozoa: Alcyonaria): carbon assimilation and translocation of photosynthates from symbionts to host[END_REF][START_REF] Farrant | Nutrition of the temperate Australian soft coral Capnella gaboensis I. Photosynthesis and carbon fixation[END_REF]).

An experiment was conducted to estimate the importance of autotrophically acquired carbon for the host requirements in soft corals.

For this purpose, bicarbonate labeled with the stable isotope 13 C was used and its fate was traced within the host-symbionts association. We tested the effect of light availability Nitrogen is considered as one of the limiting nutrients in the oceans due to the scarcity of its dissolved forms. As described in this chapter, corals can acquire almost all forms of dissolved nitrogen, the dinitrogen gas, the dissolved inorganic forms (DIN) such as ammonium and nitrate, as well as the dissolved organic forms (DON). In this thesis, we have tackled the assimilation of all these forms by soft corals, and compared the assimilation rates obtained with those measured in scleractinian coral species.

i) Diazotrophy

The most abundant dissolved N form, the dinitrogen, is only bioavailable to corals through the action of diazotrophs. Only two publications have investigated the rates of dinitrogen fixation in the presence of soft corals (e.g., [START_REF] Bednarz | Seasonal variation in dinitrogen fixation and oxygen fluxes associated with two dominant zooxanthellate soft corals from the northern Red Sea[END_REF][START_REF] Cardini | Budget of primary production and dinitrogen fixation in a highly seasonal Red Sea coral reef[END_REF].

However, these studies have used the acetylene reduction assay, which only quantifies gross dinitrogen fixation occurring in the seawater without providing insights into who-among the seawater or coral associated microorganisms, the coral host or the symbionts, is actually assimilating the fixed nitrogen. Only the use of 15 N2 gas, allows tracing the fate of diazotrophically-acquired nitrogen (DDN) in the different compartments.

An experiment was thus performed to assess the importance of dinitrogen fixation

for soft coral nutrition. The point arising from this objective notably questions whether soft corals are associated with active diazotrophs. The influence of the trophic status (i.e.

strict heterotroph versus mixotroph) and increased heterotrophic capacity with depth on

this process were tested. Moreover, a comparison with hard corals was undertaken.

This experiment was designed to measure net dinitrogen fixation and distinguish between the different compartments (i.e., seawater particles, coral tissue, dinoflagellate symbionts) for the very first time in soft corals, using labelled 15 N2 gas. For this purpose, experiments were performed 1) in the laboratory, to assess whether soft and hard corals have the same capacities to assimilate DDN, and 2) to assess dinitrogen fixation in corals collected from the field. For 1), the soft Sarcophyton sp. and hard Stylophora pistillata corals were used. As corals have the ability to capture preys, heterotrophy can represent a significant acquisition of carbon, nitrogen and other nutrients. Literature on the diet of tropical soft corals is however very sparse (e.g., [START_REF] Lewis | Feeding behaviour and feeding ecology of the Octocorallia (Coelenterata: Anthozoa)[END_REF]Fabricius et al. 1995a;[START_REF] Sorokin | Morphology and ecological physiology of corals[END_REF] and the heterotrophic capacity of corals has been mostly estimated through laboratory studies, or studies undertaken with incubation chambers. The heterotrophic status of corals under in situ conditions is thus still poorly understood, in all coral species.

An experiment was designed to investigate the autotrophic and heterotrophic

status of soft corals in situ, using their fatty acid composition. The effect of depth was tested and a comparison with a hard coral species was made.

For this purpose, the concentrations of fatty acids specific to autotrophy (stearidonic and docosapentaenoic acids) and heterotrophy (cis-gondoic acid) were measured in soft and hard corals collected in the field. Species investigated were Dendronephthya sp., Litophyton sp., Rhytisma fulvum fulvum, and Sarcophyton sp. for the soft corals and Stylophora pistillata for the scleractinian. They were collected in the Red Sea at two depths (8-10 m and 40-50 m depth).

These species harbor different Symbiodiniaceae genera and different morphological shapes.

The depths investigated enable the assessment of the effect of putative increased heterotrophic capacity.

Chapter 2 | Normalization metric and processing protocol

Chapter 2 | Normalization metric and processing protocol

Publication n°1

Chloé A. Pupier

Abstract

Soft corals (Octocorallia) often constitute the second most abundant macrobenthic group on many tropical and temperate reefs. However, the gelatinous and leather-like nature of their tissue and their variable hydroskeleton entails a number of problems for tissue homogenization and data normalization. An easy and fast protocol for tissue homogenization, as well as a normalization metric that can be used to perform inter-studies or inter-species comparisons, are thus needed. In this study, we tested whether the tissue sample state before processing (frozen vs freeze-dried samples) and the media used for tissue homogenization (0.2 µm filtered seawater; FSW vs milliQ water; DI) affects the quantitative measurements of tissue descriptors (chlorophyll, protein and Symbiodinium concentrations) in the model species Heteroxenia fuscescens. Furthermore, the suitability of dry weight (DW) and ash-free dry weight (AFDW) as size-normalizing metric was investigated across different soft coral species.

Our results reveal that freeze-drying the samples and homogenizing them in DI water exhibited several benefits, namely enhancing chlorophyll and protein concentrations up to 50%, saving processing time and providing a more accurate determination of DW and AFDW. Overall, this optimized tissue processing protocol offers a more reliable quantification of tissue descriptors and reduces the chance of underestimating these parameters in soft corals. Finally, since the

Studies with soft corals -recommendations on sample processing and normalization metrics

contribution of sclerites to the total DW of the colony can highly differ between species, we demonstrate that AFDW is a reliable metric for normalizing soft coral data, particularly when inter-species comparisons are made.

Introduction

Soft corals (Octocorallia: Alcyonacea) represent, after reef-building scleractinian corals, the second most abundant macrobenthic group on many tropical and temperate reefs [START_REF] Fabricius | Soft corals and sea fans: a comprehensive guide to the tropical shallow-water genera of the Central-West Pacific, the Indian Ocean and the Red Sea[END_REF]. They notably become often dominant within disturbed reef systems [START_REF] Inoue | Spatial community shift from hard to soft corals in acidified water[END_REF]. High abundances of octocorals (soft corals or gorgonians) have thus been observed in Caribbean and Indo-Pacific eutrophicated reefs, where a reduced water quality does not favour the growth of scleractinian corals [START_REF] Fabricius | Changes in algal, coral and fish assemblages along water quality gradients on the inshore Great Barrier Reef[END_REF][START_REF] Baum | Abundance and physiology of dominant soft corals linked to water quality in Jakarta Bay, Indonesia[END_REF]. Despite the fact that soft corals are increasingly recognized as key taxa on reefs, two major issues regarding physiological and ecological studies remain however to be clarified.

First, the coenenchyme (colonial tissue) of soft corals consists of thick gelatinous material containing fibres, amoeboid cells, scleroblast cells and calcareous sclerites [START_REF] Fabricius | Soft corals and sea fans: a comprehensive guide to the tropical shallow-water genera of the Central-West Pacific, the Indian Ocean and the Red Sea[END_REF]. Altogether, these leather-like tissue elements make cell layers difficult to break apart and tissue homogenization challenging. Therefore, measurements of tissue descriptors (Symbiodinium, chlorophyll and protein concentrations) are difficult to obtain from frozen or fresh soft coral samples, and a reliable and easy tissue homogenization protocol remains to be implemented.

Second, a common normalization metric to perform inter-species comparisons of physiological and/or ecological processes is required. For example, apprehending functional processes that occur at the reef-ecosystem level (e.g., primary production) implies to understand how soft corals operate over space and time compared to scleractinian corals. Comparative measurements related to these sub-classes are therefore necessary, but are only enabled if they are normalized to the same metric. Normalization of tissue parameters or physiological measurements requires a metric that reflects the size of the organisms and is stable across variable environmental conditions. In scleractinian corals, skeletal surface area is the most commonly used metric for normalizing data to the size of the colony [START_REF] Edmunds | Normalizing physiological data for scleractinian corals[END_REF]. However, soft corals lack a calcium carbonate skeleton and rely on hydrostatic pressure for body shape maintenance [START_REF] Fabricius | Soft corals and sea fans: a comprehensive guide to the tropical shallow-water genera of the Central-West Pacific, the Indian Ocean and the Red Sea[END_REF]. Although this hydroskeleton can vary over time depending on environmental fluctuations [START_REF] Fabricius | Slow population turnover in the soft coral genera Sinularia and Sarcophyton on mid-and outer-shelf reefs of the Great Barrier Reef[END_REF][START_REF] Hellström | Robustness of size measurement in soft corals[END_REF], few studies have approximated soft coral surface area based on geometric body measurements (e.g., [START_REF] Bednarz | Inorganic nutrient availability affects organic matter fluxes and metabolic activity in the soft coral genus Xenia[END_REF][START_REF] Bednarz | Seasonal variation in dinitrogen fixation and oxygen fluxes associated with two dominant zooxanthellate soft corals from the northern Red Sea[END_REF][START_REF] Kremien | Beneft of pulsation in soft corals[END_REF] or linear projections [START_REF] Fabricius | Depletion of suspended particulate matter over coastal reef communities dominated by zooxanthellate soft corals[END_REF]. Such normalization metric is critical when studying processes (e.g., photosynthesis) that strongly depend on a vectorial parameter (e.g., light) as it allows understanding the effectiveness of these processes [START_REF] Kremien | Beneft of pulsation in soft corals[END_REF]. For other measurements, such as the quantification of structural tissue descriptors or the production of metabolites, the variable size of the hydroskeleton excludes the possibility to accurately normalize to surface area at different time intervals [START_REF] Haydon | Soft corals are significant DMSP producers in tropical and temperate reefs[END_REF]. Several other normalization metrics have thus been applied to soft corals and are summarized in Figure 15, Table 3 and Appendix I -Table S1. Table 3. Summary about the range of metrics used in previous studies to normalize physiological processes in soft corals. AFDW = ashfree dry weight. DW = total dry weight. BW = buoyant weight. DMSP = dimethylsulfopropionate. P/A pigments = photosynthetic and accessory pigments. ETS = electron transport system. DOM = dissolved organic matter. The group 'biochemical composition of tissues' gathers measured parameters such as amino acid, protein, carbohydrate and/or lipid content. The asterisk notifies a study for which the normalizing metric (i.e., dry weight of decalcified samples) has been considered as ash-free dry weight by the authors.

Normalization metric Family Measured parameter Reference

AFDW

Alcyoniidae

Biochemical composition of tissues, DMSP concentration, feeding rates, mucus production, nutrient flux, oxygen flux [START_REF] Fabricius | Widespread mixotrophy in reef-inhabiting soft corals: the influence of depth, and colony expansion and contraction on photosynthesis[END_REF][START_REF] Riegl | Effects of sediment on the energy budgets of four scleractinian (Bourne 1900) and five alcyonacean (Lamouroux 1816) corals[END_REF]Migné andDavoult 1997a, b, 2002;[START_REF] Van Alstyne | The distribution of dimethylsulfoniopropionate in tropical Pacific coral reef invertebrates[END_REF][START_REF] Slattery | Direct and indirect effects of a new disease of alcyonacean soft corals[END_REF]) Briareidae

Oxygen flux [START_REF] Fabricius | Widespread mixotrophy in reef-inhabiting soft corals: the influence of depth, and colony expansion and contraction on photosynthesis[END_REF] Nephtheidae Feeding rates, oxygen flux [START_REF] Fabricius | Widespread mixotrophy in reef-inhabiting soft corals: the influence of depth, and colony expansion and contraction on photosynthesis[END_REF]Fabricius et al. 1995a[START_REF] Fabricius | In situ depletion of phytoplankton by an azooxanthellate soft coral[END_REF]

Xeniidae

Oxygen flux (Fabricius and Klumpp 1995)

DW

Alcyoniidae

Biochemical composition of tissues, caloric content, metabolite concentration, P/A pigments, Symbiodinium density [START_REF] Slattery | Population structure and feeding deterrence in three shallowwater antartic soft corals[END_REF][START_REF] Ben-David-Zaslow | Competence and longevity in planulae of several species of soft corals[END_REF][START_REF] Khalesi | Flow-dependent growth in the zooxanthellate soft coral Sinularia flexibilis[END_REF][START_REF] Khalesi | Light-dependency of growth and secondary metabolite production in the captive zooxanthellate soft coral Sinularia flexibilis[END_REF]Rocha et al. 2013b, b;[START_REF] Leal | Coral feeding on microalgae assessed with molecular trophic markers[END_REF][START_REF] Costa | The effect of mixotrophy in the ex situ culture of the soft coral Sarcophyton cf. glaucum[END_REF]) Clavulariidae Biochemical composition of tissues [START_REF] Slattery | Population structure and feeding deterrence in three shallowwater antartic soft corals[END_REF] Nephtheidae Biochemical composition of tissues, inorganic carbon, oxygen flux, P/A pigments, Symbiodinium density [START_REF] Farrant | Nutrition of the temperate Australian soft coral Capnella gaboensis I. Photosynthesis and carbon fixation[END_REF][START_REF] Slattery | Population structure and feeding deterrence in three shallowwater antartic soft corals[END_REF] Xeniidae Biochemical composition of tissues, caloric content, inorganic carbon, P/A pigments [START_REF] Schlichter | Functional autotrophy of Heteroxenia fuscescens (Anthozoa: Alcyonaria): carbon assimilation and translocation of photosynthates from symbionts to host[END_REF][START_REF] Schlichter | Zooxanthellae providing assimilatory power for the incorporation of exogenous acetate in Heteroxenia fuscescens (Cnidaria: Alcyonaria)[END_REF]Ben-David-Zaslow andBenayahu 1998, 1999;Bednarz et Biochemical composition of tissues, calcification rate, cell growth, DMSP concentration, ETS activity, inorganic carbon, oxygen flux, P/A pigments, Symbiodinium density [START_REF] Farrant | Nutrition of the temperate Australian soft coral Capnella gaboensis I. Photosynthesis and carbon fixation[END_REF][START_REF] Tentori | Cell growth and calcification result from uncoupled physiological processes in the soft coral Litophyton arboreum[END_REF][START_REF] Baum | Abundance and physiology of dominant soft corals linked to water quality in Jakarta Bay, Indonesia[END_REF][START_REF] Haydon | Soft corals are significant DMSP producers in tropical and temperate reefs[END_REF] Among these metrics, those involving wet weight and buoyant weight may also be sources of error as soft coral tissue withholds a lot of water and the density of a colony may be similar to that of seawater [START_REF] Davies | Short-term growth measurements of corals using an accurate buoyant weighing technique[END_REF]. For example, normalization of dimethylsulphoniopropionate (DMSP) production to soft coral fresh weight [START_REF] Van Alstyne | The distribution of dimethylsulfoniopropionate in tropical Pacific coral reef invertebrates[END_REF]) led to significant lower values compared to the normalization to protein content, underestimating the functional role of soft compared to hard corals [START_REF] Haydon | Soft corals are significant DMSP producers in tropical and temperate reefs[END_REF]. Coral biomass may therefore offer a greater potential to accurately reflect the size of a soft coral colony. Protein content has been widely used to normalize physiological data in corals.

However it can encounter great variability across environmental conditions, and may thus interfere with the intrinsic pattern of the parameter of interest [START_REF] Edmunds | Normalizing physiological data for scleractinian corals[END_REF]. Ashfree dry weight (AFDW) is another common proxy for tissue biomass of many benthic reef organisms such as scleractinian corals, coralline algae, sponges [START_REF] Kötter | In situ feeding rates of obligate and facultative coelobite (cavitydwelling) sponges in a Caribbean coral reef[END_REF][START_REF] Schoepf | Coral energy reserves and calcification in a high-CO2 world at two temperatures[END_REF][START_REF] Comeau | Water flow modulates the response of coral reef communities to ocean acidification[END_REF]) and soft corals (Figure 15). AFDW quantification relies on a completely destructive method, whereas dry weight (DW) allows for potential analysis of tissue parameters before burning the sample. Although tissue energy reserves (i.e., lipid, protein and carbohydrate) of soft corals have been determined from dried material (Ben-David-Zaslow and Benayahu 1999), the possibility to obtain reliable measurements involving Symbiodinium (cell density and chlorophyll content) still needs to be tested.

Based on these issues the present study aimed 1) to optimize a protocol for soft coral tissue processing and 2) to find the normalization metric most relevant for physiological experiments involving interspecies comparisons. We tested whether the tissue sample state before processing (frozen vs freeze-dried samples) and the media used for tissue homogenization (0.2 µm filtered seawater; FSW vs milliQ water; DI) affects the quantitative measurements of tissue descriptors in the model species Heteroxenia fuscescens (Ehrenberg, 1834). Furthermore, the suitability of DW and AFDW as size-normalizing metric was investigated across different soft coral species (H. fuscescens, Dendronephthya sp.

( Kuekenthal, 1905), Litophyton arboreum (Forskål, 1775) and Rhytisma fulvum (Forskål, 1775), which are key components of Red Sea reefs. These species particularly exhibit different growth forms ranging from arborescent (Dendronephthya sp. and L. arboreum) to capitate (H.

fuscescens) and encrusting (R. fulvum).

Materials and methods

Biological material

Nineteen colonies of H. fuscescens with 20 polyps and four colonies of Dendronephthya sp., L. arboreum and R. fulvum, collected in the Red Sea were used for the experiments. They were equally allocated to five 20 L tanks and maintained under the same conditions for eight weeks prior to starting the experiments. Tanks were continuously supplied with seawater, at a renewal rate of 10 L h -1 . HQI lamps (Tiger E40, Faeber, Italy) above the tanks provided photosynthetically active radiation (PAR) of 200 µmol photons m -2 s -1 .

Optimized protocol for Symbiodinium, chlorophyll and protein determination

A first experiment, performed with H. fuscescens, aimed to compare Symbiodinium, chlorophyll and protein concentrations in frozen or freeze-dried samples, homogenized in FSW or DI. Four combinations were thus considered, with 5 replicates per condition: (i) freeze-dried samples homogenized in FSW, (ii) freeze-dried samples homogenized in DI, (iii) frozen samples homogenized in FSW and (iv) frozen samples homogenized in DI. For this purpose, 20 tubes were prepared, each containing one freshly cut polyp of ten colonies of H. fuscescens randomly chosen in the tanks. All tubes were flash-frozen in liquid nitrogen. Half of them were processed directly whereas the other half was freeze-dried (Alpha 2-4 LDplus freeze-dryer, Martin Christ, Germany), until obtaining a dried powder. Frozen and freeze-dried samples were ground with a potter tissue grinder and solubilized in 10 mL FSW or DI. This procedure was rapid for the freeze-dried samples (5 minutes) but took more than 30 minutes for the frozen samples. From each homogenate, 500 µL was collected for total protein measurement (see following section). The remaining homogenate was centrifuged for 10 min (at 11,000 g at 4°C)

to pellet the Symbiodinium. Light microscopy confirmed the total removal of Symbiodinium from the supernatant (i.e., host fraction), which was freeze-dried again. This was done to determine the host DW and to subsequently compare the tissue parameters of frozen and freezedried samples normalized to the same metric. Symbiodinium pellets were rinsed twice to eliminate any remaining host cells [START_REF] Tremblay | Autotrophic carbon budget in coral tissue: a new 13 C-based model of photosynthate translocation[END_REF], and re-suspended in 10 mL FSW or DI. After mixing, 100 µL and 5 mL were subsampled for the determination of Symbiodinium density and total chlorophyll concentration, respectively (see following section).

Tissue parameter measurements

Proteins were extracted from the 500 µL subsamples in a sodium hydroxide solution (0.5 M) for 5 h at 60°C. Thereafter, protein content was measured using a BC assay kit (Interchim, France) [START_REF] Smith | Measurement of protein using bicinchoninic acid[END_REF], and protein standards were prepared using bovine serum albumin (Interchim, France). Absorbance of subsamples was measured at 562 nm by an UVmc² spectrophotometer (SAFAS, Monaco). Symbiodinium density was quantified microscopically via eight replicate haemocytometer counts in the 100 µL subsamples (Neubauer haemocytometer, Marienfeld, Germany). For chlorophyll analysis, the 5 mL subsamples were centrifuged for 10 min (at 8,000 g at 4°C). The supernatant was discarded and 5 mL of acetone (100%) amended with magnesium chloride (Sigma Aldrich, Germany) were added to the Symbiodinium pellet to extract the chlorophyll over 24h in the dark (at 4°C). After centrifugation at 11,000 g for 15 min at 4°C, absorbances were measured at 750 nm, 663 nm and 630 nm using a Xenius spectrophotometer (SAFAS, Monaco) and chlorophyll concentrations calculated according to [START_REF] Jeffrey | New spectrophotometric equations for determining chlorophylls a, b, c1 and c2 in higher plants, algae and natural phytoplankton[END_REF].

Normalization parameters

An experiment was performed with H. fuscescens to highlight any correlation between biomass (DW), protein content and colony's body volume. The body volume of 5 colonies with different polyp numbers was estimated using the water displacement technique [START_REF] Benayahu | Sexual reproduction of a soft coral: synchronous and brief annual spawning of Sarcophyton glaucum (Quoy & Gaimard, 1833)[END_REF]), before colonies were freeze-dried and homogenized in 10 mL DI. A subsample of 500 µL was used for protein content determination. We also compared the DW and ash weight (AW) of the four different coral species listed above. For this purpose, colonies of each species were freeze-dried, crushed and split in five powder heaps of different weights (DW). They were combusted at 450°C for 4 h in a muffle furnace (Thermolyne 62700, Thermo Fischer Scientific, the United States). Such combustion process avoids losses of sclerites (Harvell and Fenical, 1989) and allows estimating the DW of sclerites.

Statistical analyses

Analyses were performed using R software (R Foundation for Statistical Computing)

and assumptions of normality and homoscedasticity of variance were evaluated through Shapiro's and Bartlett's tests along with graphical analyses of residuals. Protein, Symbiodinium and chlorophyll concentrations were analyzed using a two-way ANOVA, in which the sample state (frozen vs freeze-dried) and type of water (FSW vs DI) were fixed effects. Regression equations were established between colony DW and protein content, volume or AW. Pearson's product moment correlation coefficients were calculated to test the strength of the linear associations.

Results and discussion

This study shows that both the sample state (i.e., frozen and freeze-dried) and the homogenization media (i.e., DI and FSW) significantly influence the quantification of tissue parameters in soft corals, while no effect was observed for Symbiodinium counts (Figure 16 and Table 4). Sample state mainly affects chlorophyll determinations with two-fold higher concentrations in freeze-dried as compared to frozen samples (Figure 16b). A previous work on reef sands also demonstrated a 27 to 39 % increased chlorophyll concentration in freezedried over frozen samples, likely due to the removal of mucous matrices surrounding the cells and a better exposure of pigments to the solvent in the former [START_REF] Hannides | Extraction and quantification of microphytobenthic Chl a within calcareous reef sands[END_REF]. Here, the reduced chlorophyll concentration in frozen samples likely results from pigment damage due to the longer homogenization period of frozen fragments (30 min) as compared to the freezedried fragments (5 min). Although samples were kept on ice during homogenization, this caution did not prevent tissue slurry from mechanic heating with the Potter grinder. Similarly, [START_REF] Hannides | Extraction and quantification of microphytobenthic Chl a within calcareous reef sands[END_REF]) highlighted that longer sonication periods caused a significant decline in chlorophyll content, due to pigment degradation [START_REF] Metaxatos | Seasonality of algal pigments in the sea water and interstitial water/sediment system of an eastern Mediterranean coastal area[END_REF]. Variations in the length of the homogenization procedure can occur for coral samples with different tissue thickness, and may amplify variance in the chlorophyll measurements. Future studies on corals should thus take into account such tissue processing flaws and compare the efficacy of the usual tissue extraction procedure with the freeze-drying one. Care must also be taken when comparing studies that use photosynthetic pigments as normalization metric for other parameters (e.g, coral productivity). As highlighted in this study, a 50% reduced photosynthetic pigment extraction in frozen as compared to freeze-dried samples can lead to a significant overestimation of the productivity in the former, an issue that can be extended to scleractinian corals too. Finally, saving time is another benefit when processing freeze-dried over frozen samples, which is important in experiments involving numerous samples. Although the homogenization media has an overall low effect on tissue processing, the use of DI compared to FSW significantly increases the protein content measurable in tissue samples (Figure 16c and Table 4). This could have important implications when comparing physiological processes across different coral species and environmental conditions. The use of DI also reduces the amount of salts in homogenates, thereby providing a higher accuracy for the determination of absolute host and Symbiodinium DW as well as avoiding salt crystals entering instruments such as mass spectrometers [START_REF] Baker | Productivity links morphology, symbiont specificity and bleaching in the evolution of Caribbean octocoral symbioses[END_REF]. As highlighted in the introduction, a literature review identified 36 physiological studies on soft corals, which display a wide variety of normalization metrics (Figure 15, Table 3 andTable S1). Although the choice of the metrics certainly depends on the process under study and the information provided, a consensual normalization metric is important to allow better comparability of results among studies. For a given soft coral species maintained under a constant environmental condition, all metrics can be proxies for biomass and colony size.

Results obtained with H. fuscescens indeed exhibit significant linear relationships between total DW and body volume or protein concentration (Figure 17a and Table 5). Other studies have also shown significant correlations between AFDW or DW and polyp number, colony height, or carbon and nitrogen content [START_REF] Migné | Relations" taille-poids" chez quelques Cnidaires coloniaux[END_REF]Fabricius et al. 1995a;[START_REF] Migné | Biometrics, carbon and nitrogen content in two cnidarians: Urticina Felina and Alcyonium Digitatum[END_REF]. However, the use of several metrics listed above can be unsatisfactory when comparing processes between different species or different environmental conditions. We demonstrate that DW does not provide a reliable normalization metric to compare tissue parameters across coral species (Figure 17b and Table 5), although dried samples enable better tissue processing in soft corals. Linear regression analyses between DW and AW of different soft coral species show species-specific linear relationships; this is due to the presence of sclerites, which can differ in size, shape and abundance within a colony and between species [START_REF] Van Alstyne | Antipredator defenses in tropical Pacific soft corals (Coelenterata: Alcyonacea). I. Sclerites as defenses against generalist carnivorous fishes[END_REF].

Sclerites are frequently used as taxonomic indicator, and species-specific differences likely depend on the necessity to accumulate sclerites for structural defence against predators and/or skeletal support against wave action [START_REF] Van Alstyne | Antipredator defenses in tropical Pacific soft corals (Coelenterata: Alcyonacea). I. Sclerites as defenses against generalist carnivorous fishes[END_REF]). Overall, our results show that the contribution of sclerites to the total DW of the colony can highly differ between species, and thus, we recommend the use of AFDW over DW for normalizing physiological processes in soft corals, particularly when inter-species comparisons are made. In conclusion, we recommend to dry the biological material for biochemical and tissue parameter analyses and to use a subset of the dried powder for AFDW determination as final normalization metric (Figure 18). AFDW stands for a useful metric to normalize tissue parameters in soft corals, benefiting of the aforementioned advantages owed from its freezedried state and allowing best comparability among species. Other non-destructive normalization metrics, such as surface area, can however be used in parallel to provide a different information on the physiological processes studied.

Abstract

Soft corals often constitute one of the major benthic groups of coral reefs. Although they have been documented to outcompete reef-building corals following environmental disturbances, their physiological performance and thus their functional importance in reefs are still poorly understood. In particular, the acclimatization to depth of soft corals harboring dinoflagellate symbionts and the metabolic interactions between these two partners have received little attention. We performed stable isotope tracer experiments on two soft coral species (Litophyton sp. and Rhytisma fulvum fulvum) from shallow and upper mesophotic Red Sea coral reefs to quantify the acquisition and allocation of autotrophic carbon within the symbiotic association. Carbon acquisition and respiration measurements distinguish Litophyton sp. as mainly autotrophic and Rhytisma fulvum fulvum as rather heterotrophic species. In both species, carbon acquisition was constant at the two investigated depths. This is a major difference from scleractinian corals, whose carbon acquisition decreases with depth. In

Productivity and carbon fluxes depend on species and symbiont density in soft coral symbioses addition, carbon acquisition and photosynthate translocation to the host decreased with an increase in symbiont density, suggesting that nutrient provision to octocoral symbionts can quickly become a limiting factor of their productivity. These findings improve our understanding of the biology of soft corals at the organism-scale and further highlight the need to investigate how their nutrition will be affected under changing environmental conditions.

Introduction

Mutualistic symbioses between cnidarians and photosynthetic dinoflagellates of the family Symbiodiniaceae are widespread in marine environments [START_REF] Venables | Photosynthetic symbioses in animals[END_REF]. In coral reef ecosystems, this association is the backbone for the growth and survival of corals surrounded by oligotrophic waters that hardly provide any exogenous nutrients. Indeed, the dinoflagellate symbionts of scleractinian corals translocate a large proportion of their photosynthetically fixed carbon compounds to the coral host for its own nutrition, growth, reproduction and energetic needs [START_REF] Muscatine | Fate of photosynthetic fixed carbon in light-and shade-adapted colonies of the symbiotic coral Stylophora pistillata[END_REF]. The percentage of carbon translocation can be as high as 90% in well-lit shallow waters [START_REF] Falkowski | Light and the bioenergetics of a symbiotic coral[END_REF][START_REF] Tremblay | Autotrophic carbon budget in coral tissue: a new 13 C-based model of photosynthate translocation[END_REF], and can remain high in mesophotic environments [START_REF] Ezzat | Carbon and nitrogen acquisition in shallow and deep holobionts of the scleractinian coral S. pistillata[END_REF]). However, the total amount of autotrophically-acquired carbon generally decreases from shallow to deep reef environments through a reduced productivity of dinoflagellate symbionts [START_REF] Ezzat | Carbon and nitrogen acquisition in shallow and deep holobionts of the scleractinian coral S. pistillata[END_REF].

Simultaneously, the coral host becomes more dependent on heterotrophic food sources and it is the plasticity between host heterotrophy and symbiont autotrophy that allows the association to thrive in such contrasting reef environments [START_REF] Houlbrèque | Heterotrophy in tropical scleractinian corals[END_REF]. So far, our understanding of coral holobiont performance and host-symbiont metabolic interactions under different environmental conditions is limited to studies on scleractinian corals, while there is very little information available for other prominent members of reef systems such as soft corals (Alcyonacea).

Soft corals often constitute the second major benthic group of reef ecosystems [START_REF] Schubert | Symbiotic versus non-symbiotic octocorals: physiological and ecological implications[END_REF]. They can thrive with relative high abundance and diversity under very different environmental conditions ranging from turbid to clear-water [START_REF] Fabricius | Depletion of suspended particulate matter over coastal reef communities dominated by zooxanthellate soft corals[END_REF][START_REF] Fabricius | Photosynthetic symbionts and energy supply determine octocoral biodiversity in coral reefs[END_REF] or from shallow to mesophotic reef environments [START_REF] Shoham | Higher species richness of octocorals in the upper mesophotic zone in Eilat (Gulf of Aqaba) compared to shallower reef zones[END_REF]. Similarly to scleractinian corals, a large proportion (≥ 50%) of soft coral taxa is associated with Symbiodiniaceae in the Eastern Pacific, Caribbean, Red Sea and Great Barrier Reef [START_REF] Schubert | Symbiotic versus non-symbiotic octocorals: physiological and ecological implications[END_REF]. There is evidence showing that the abundance of soft coral populations maintained or increased in most regions worldwide whereas scleractinian coral cover generally declined over the last decades [START_REF] Stobart | Coral recovery at Aldabra Atoll, Seychelles: Five years after the 1998 bleaching event[END_REF][START_REF] Lenz | Long-term variation of octocoral populations in St. John, US Virgin Islands[END_REF], due to increased sea surface temperatures, eutrophication and pollution. This is usually explained by a greater nutritional plasticity of soft corals, which are considered as mixotrophic species [START_REF] Fabricius | Widespread mixotrophy in reef-inhabiting soft corals: the influence of depth, and colony expansion and contraction on photosynthesis[END_REF]. They can therefore acquire nutrients through the autotrophic activity of the symbionts, but also through the heterotrophic capacity of the host [START_REF] Fabricius | Widespread mixotrophy in reef-inhabiting soft corals: the influence of depth, and colony expansion and contraction on photosynthesis[END_REF][START_REF] Fabricius | Depletion of suspended particulate matter over coastal reef communities dominated by zooxanthellate soft corals[END_REF]. Evidence in the literature demonstrates that feeding has a positive effect on coral tissue, enhancing the growth of both partners of the symbiosis (Ferrier-Pagès et al. 2011). In addition, feeding can play a central role in maintaining physiological function when autotrophy is reduced [START_REF] Anthony | Energetics approach to predicting mortality risk from environmental stress: a case study of coral bleaching[END_REF]). The lower dependency of soft corals on autotrophy, compared to scleractinian corals, has been deduced from few measurements performed in shallow water conditions (0-20 m) [START_REF] Fabricius | Widespread mixotrophy in reef-inhabiting soft corals: the influence of depth, and colony expansion and contraction on photosynthesis[END_REF][START_REF] Ramsby | Symbiodinium photosynthesis in Caribbean octocorals[END_REF][START_REF] Baker | Productivity links morphology, symbiont specificity and bleaching in the evolution of Caribbean octocoral symbioses[END_REF][START_REF] Schubert | Symbiotic versus non-symbiotic octocorals: physiological and ecological implications[END_REF][START_REF] Rossi | Linking host morphology and symbiont performance in octocorals[END_REF].

Therefore, knowledge gaps exist for the main autotrophic physiological processes in soft corals thriving under different environmental conditions. The few studies which have dealt with their carbon budget observed highly variable contributions of photosynthetically fixed carbon provided by the symbionts to the host, with carbon translocation rates ranging from 10% in Capnella gabonensis to 75% in Sinularia flexibilis [START_REF] Schlichter | Functional autotrophy of Heteroxenia fuscescens (Anthozoa: Alcyonaria): carbon assimilation and translocation of photosynthates from symbionts to host[END_REF][START_REF] Farrant | Nutrition of the temperate Australian soft coral Capnella gaboensis I. Photosynthesis and carbon fixation[END_REF][START_REF] Sorokin | Biomass, metabolic rates and feeding of some common reef zoantharians and octocorals[END_REF][START_REF] Khalesi | Energy budget for the cultured, zooxanthellate octocoral Sinularia flexibilis[END_REF]). In addition, different normalization metrics for physiological processes in soft and scleractinian corals often hinder comparisons of the trophic characteristics between the two groups.

While tropical soft corals are considered reef engineers which create habitats for other reef species [START_REF] Benayahu | Faunistic composition and patterns in the distribution of soft corals (Octocorallia, Alcyonacea) along the coral reefs of Sinai peninsula[END_REF][START_REF] Schubert | Symbiotic versus non-symbiotic octocorals: physiological and ecological implications[END_REF], little comprehensive data exist on their trophic ecology, and therefore on their functional role as primary producers and carbon sinks within the reef ecosystem [START_REF] Fabricius | Widespread mixotrophy in reef-inhabiting soft corals: the influence of depth, and colony expansion and contraction on photosynthesis[END_REF]Fabricius et al. 1995a;[START_REF] Fabricius | Depletion of suspended particulate matter over coastal reef communities dominated by zooxanthellate soft corals[END_REF][START_REF] Bednarz | Seasonal variation in dinitrogen fixation and oxygen fluxes associated with two dominant zooxanthellate soft corals from the northern Red Sea[END_REF]. Since autotrophic carbon acquisition is a key factor shaping coral productivity, physiology, and ecology, and partly explains coral success or failure under changing environmental conditions, more studies should be dedicated to better understand the autotrophic capacity of soft corals. Therefore, the present study aims to assess the carbon fluxes between dinoflagellate symbionts and host of two common soft coral species (Litophyton sp. and Rhytisma fulvum fulvum) from shallow and mesophotic reefs of the Gulf of Eilat. For this purpose, we used stable isotope tracers ( 13 C-bicarbonate) to measure the rates of carbon fixed, exchanged and lost by the shallow and mesophotic symbiotic associations under their natural photosynthetically active irradiance (PAR) levels. Importantly, we also compare our results with those of scleractinian coral species by applying a standardized normalization metric for all parameters [START_REF] Haydon | Soft corals are significant DMSP producers in tropical and temperate reefs[END_REF][START_REF] Pupier | Studies with soft corals -recommendations on sample processing and normalization metrics[END_REF].

Material and methods

Soft coral collection and experimental setup

The water PAR reached 1200 µmoles photon m -2 s -1 at midday (Appendix II -Figure S1). The PAR received at 10 m and 40 m depth peaked at ca. 550 µmoles photon m -2 s -1 and 50 µmoles photon m -2 s -1 respectively, considering attenuation coefficients of 0.072-0.1 m -1 at this precise location [START_REF] Akkaynak | What is the space of attenuation coefficients in underwater computer vision[END_REF][START_REF] Tamir | Light environment drives the shallow to mesophotic coral community transition[END_REF]). These values are in agreement with one-off measurements performed with a data logger during coral collection. The in situ seawater temperature (24°C) and nutrient concentrations (< 0.5 µM dissolved nitrogen, and < 0.2 µM dissolved phosphorus) were stable throughout the investigated depth gradient and period of time (http://www.iui-eilat.ac.il/Research/NMPMeteoData.aspx). Coral nubbins were maintained for one day under these conditions before the following measurements were performed in the outside aquaria under exactly the same environmental conditions.

NaH 13 CO 3 incubations

The rates at which carbon was fixed by the shallow and mesophotic coral colonies under their natural PAR levels were estimated using 13 C-labelled bicarbonate according to [START_REF] Tremblay | Autotrophic carbon budget in coral tissue: a new 13 C-based model of photosynthate translocation[END_REF]. To take into account variation in PAR levels during the day, corals were incubated for 5h, between 10 am and 3 pm, to cover the maximal daily irradiance [START_REF] Stambler | Light and picophytoplankton in the Gulf of Eilat (Aqaba)[END_REF] (Appendix II -Figure S1). For each species and depth condition, 10 coral nubbins, from 10 different colonies were placed in individual beakers filled with 200 mL FSW enriched with 0.6 mM NaH 13 CO3 (98 atom % 13 C, #372382, Sigma-Aldrich, St-Louis, MO, USA). After this "pulse" period of 5h, half of the corals were sampled and stored frozen at -20°C until further analysis (T0). The other half was transferred into 200 mL of non-enriched FSW for a chase period of 19 h (T24). The determination of % 13 C enrichment, and total carbon content in the symbionts and host compartments were performed with a Delta plus Mass spectrometer coupled to a C/N analyser (Thermo Fischer Scientific, Bremen, Germany). The natural isotopic abundance of each species at each depth was determined from the corals used for the respiration measurements. Detailed calculations are described in [START_REF] Tremblay | Autotrophic carbon budget in coral tissue: a new 13 C-based model of photosynthate translocation[END_REF]. Data were normalized to ash-free dry weight of the organisms [START_REF] Pupier | Studies with soft corals -recommendations on sample processing and normalization metrics[END_REF]).

Physiological and tissue descriptor measurements

Estimation of the dark respiration rates of the whole colony, as well as of the isolated symbionts, was needed for the establishment of the daily carbon budget. Dark respiration rates were measured on four nubbins per species and depth (from four different colonies), as well as on freshly isolated symbionts from four other nubbins according to [START_REF] Tremblay | Autotrophic carbon budget in coral tissue: a new 13 C-based model of photosynthate translocation[END_REF].

Dark-acclimated nubbins/symbionts were individually placed in stirred incubation chambers filled with 0.45 µM filtered seawater and maintained at 24°C. Changes in dissolved oxygen were monitored over 30 minutes using optodes connected to an Oxy-4 (PreSense, Regensburg, Germany). Oxygen fluxes were converted into carbon equivalents (RC) [START_REF] Tremblay | Autotrophic carbon budget in coral tissue: a new 13 C-based model of photosynthate translocation[END_REF].

The autotrophic carbon acquisition to respiration ratios (PC/RC) were estimated considering a maximal autotrophic carbon acquisition rate for 6h, and a half rate for the remaining 6h. At the end of the incubations, nubbins were flash-frozen in liquid nitrogen, freeze-dried, weighed for the total DW determination and processed as described in [START_REF] Pupier | Studies with soft corals -recommendations on sample processing and normalization metrics[END_REF] (Appendix II) for the further determination of the symbiont density, total chlorophyll concentration and ash-free dry weight (AFDW). The genus of the symbionts hosted by the shallow and mesophotic populations of Litophyton sp. and R. f. fulvum was investigated following the protocol of Santos et al. [START_REF] Santos | Molecular phylogeny of symbiotic dinoflagellates inferred from partial chloroplast large subunit (23S)-rDNA sequences[END_REF].

Interspecies comparisons

To compare our data on soft corals, normalized to AFDW, with those found in the literature on scleractinian corals, often normalized to skeletal surface area, it was necessary to use the same normalization metric. As surface area could not be measured with accuracy on soft corals (since they have a highly variable hydroskeleton which expands and shrinks the colony size by several folds depending on the environmental conditions), we normalized all data to AFDW [START_REF] Pupier | Studies with soft corals -recommendations on sample processing and normalization metrics[END_REF]. For this purpose, we chose eight nubbins (skeletal surface area ranging from 4 to 29 cm²) of the scleractinian coral Stylophora pistillata originating from the Red Sea and grown at the Monaco Scientific Center, as it is one of the dominant species in the Gulf of Eilat and many data have been acquired on this species. AFDW was determined as described above after separating the tissue from the skeleton and combusting it. Surface area was determined using the wax technique [START_REF] Veal | Increasing the accuracy of surface area estimation using single wax dipping of coral fragments[END_REF]. A conversion factor (255.65 ± 8.98, Table S5), estimated from the ratio between skeletal surface area (in cm²) and AFDW (in g) of these nubbins, was used to transform data normalized to surface area (Appendix II -Table S4) into data normalized to AFDW.

Statistical analyses

Analyses were performed using R software (R Foundation for Statistical Computing).

All data were expressed as mean ± standard error. Prior to analyses, outlier values were identified using Grubb's test and were excluded when p-values were significant (p < 0.05).

Assumptions of normality and homoscedasticity of variance were evaluated through Shapiro's and Bartlett's tests along with graphical analyses of residuals. A two-way analysis of variance (ANOVA) was performed to test the effect of species and depth on symbiont density, chlorophyll concentrations and carbon acquisition. The rates at which carbon was assimilated, translocated and lost were analysed separately between the two time points (i.e., pulse and chase periods) using 2-way ANOVAs with species and depth as fixed effects. When the interaction was significant pairwise, Tukey tests were performed as a posteriori testing.

Results

Physiological measurements

The dinoflagellate-host association was species-specific. Litophyton sp. than in R. f. fulvum at both depths and was for both species always higher in shallow than in mesophotic con-specifics (Figure 19a, Appendix II -Table S2). For both species, total chlorophyll concentrations (per AFDW) were significantly higher for mesophotic corals (Figure 19b, Appendix II -Table S2). Also, symbionts contained significantly more chlorophyll per cell in mesophotic corals (2.8•10 -6 ± 3.2•10 -7 µg cell -1 for Litophyton sp. and 7.5•10 -6 ± 4.9•10 -7 µg cell -1 for R. f. fulvum, Appendix II -Table S2) as compared to shallow corals (1.6•10 -6 ± 1.5•10 -7 µg cell -1 for Litophyton sp. and 4.2•10 -6 ± 1.2•10 -6 µg cell -1 for R. f. fulvum, Appendix II -Table S2). Autotrophic carbon acquisition, estimated using 13 C-bicarbonate incorporation over 5h under maximal irradiance (PC, expressed in µg C g -1 AFDW h -1 ), was significantly different between the two coral species, with Litophyton sp. exhibiting higher Pc rates than R. f. fulvum (Figure 19c, Appendix II -Table S2). When normalized to symbiont cells, Pc rates were significantly different between depths, with higher values obtained in mesophotic conditions (Figure 19d, Appendix II -Table S2). Respiration rates (Rc, expressed in µg C g -1 AFDW h -1 )

were similar between depths for R. f. fulvum (347 ± 45 and 489 ± 47 in shallow and mesophotic conditions, respectively), whereas they were two-fold lower for mesophotic (148 ± 47) than shallow (406 ± 18) nubbins of Litophyton sp. (Appendix II -Table S2, Tukey HSD p < 0.01).

While symbiont respiration accounted for less than 1% of the holobiont respiration for Litophyton sp. in both depths, symbiont respiration accounted ca. 6% and 4% to holobiont respiration for mesophotic and shallow R. f. fulvum, respectively.

Carbon budget

After the pulse period (5h), both species exhibited significant differences in their carbon budget between depths (Figure 20a, b, c, d, Appendix II -Tables S2 andS3). Overall, rates of photosynthate translocation were lower in Litophyton sp. at both depths (36% to 60% of the total fixed carbon) compared to R. f. fulvum (85%), and symbionts of Litophyton sp. retained four to five times more carbon than those of R. f. fulvum (Figure 20b, d, Appendix II -Table S2). However, no difference was observed in the rate of carbon assimilated by the host between depths and species (134-150 µg C g -1 AFDW h -1 ). For Litophyton sp. from mesophotic depth, symbionts translocated less carbon to the host (36% versus 60%) and less carbon was lost by the holobiont (19% versus 44%) as compared to nubbins from shallow depth. On the contrary, photosynthate translocation (85%) and carbon loss (64-70%) were comparable between shallow and mesophotic R. f. fulvum corals.

After the chase period (24h), the carbon budget of both species was clearly speciesspecific but without depth-specific differences (Figure 21a, b, c, d, Appendix II -Table S2). In Litophyton sp., symbiont cells assimilated half of the photosynthesized carbon and translocated only 47% to the coral host in both shallow and mesophotic colonies (Figure 21a, b, c, d, Appendix II -Table S2). On the contrary, symbionts in R. f. fulvum translocated 86-92% of the photosynthetically fixed carbon to the host and assimilated only 4-11% of this carbon for their own energetic needs. Consequently, significant differences were evident between the two species for the rates of carbon 1) translocated (up to one third lower in Litophyton sp. than in R. f. fulvum), 2) retained in the symbionts (ten-fold higher in Litophyton sp. than in R. f. fulvum)

and 3) lost from the symbiotic association (two-fold lower in Litophyton sp. than in R. f. fulvum) S2).

(Figure 21a, b, c, d, Appendix II -Table

Relationship between Symbiodiniaceae density and carbon flux

Our results show that in the two coral species at both depths the carbon acquisition per symbiont cell exponentially decreased with increasing symbiont density (Figure 22a). In addition, we found a positive linear correlation between symbiont cell carbon acquisition and translocation (Figure 22b).

Discussion

This study shows that the functioning of the coral-dinoflagellate symbiosis in soft corals holds some different characteristics to that in many scleractinian corals, and that the two groups are differently acclimatized to depth. The two investigated soft coral species maintained an equivalent carbon acquisition from shallow to poorly-lit mesophotic habitats, while carbon acquisition of scleractinian corals generally decreases with depth.

This autotrophic efficiency, combined with putative high heterotrophic capacities, may provide soft corals with an ecological advantage in the upper Red Sea mesophotic reefs [START_REF] Shoham | Higher species richness of octocorals in the upper mesophotic zone in Eilat (Gulf of Aqaba) compared to shallower reef zones[END_REF]. In addition, the results show that carbon acquisition and translocation rates per symbiont cell were negatively correlated with the symbiont density within the host tissue, with a higher assimilation of carbon in symbiont biomass under high symbiont density. However, the rates at which carbon was translocated to and assimilated by the host were unaffected, indicating that dense symbiont populations can remain mutualistic.

Our findings indicate that the mean autotrophic carbon acquisition in both soft coral species (measured during 5h and using 13 C-labelling) remained stable from shallow to mesophotic environments, which suggests that soft corals are either photo-limited in shallow waters or well photo-acclimatized to depth (Figure 23). This is in agreement with previous observations [START_REF] Fabricius | Widespread mixotrophy in reef-inhabiting soft corals: the influence of depth, and colony expansion and contraction on photosynthesis[END_REF]) that maximal photosynthetic rates of several soft coral species from the Great Barrier Reef were measured at 20 m depth rather than in shallower waters (Table 6). Such constant carbon acquisition along the depth gradient is surprising in comparison to the general patterns observed in scleractinian corals. Most scleractinian corals experience a significant decrease in rates of carbon acquisition with depth, although this physiological parameter has been poorly investigated in mesophotic environments [START_REF] Lesser | Photoacclimatization by the coral Montastraea cavernosa in the mesophotic zone: light, food, and genetics[END_REF][START_REF] Einbinder | Novel adaptive photosynthetic characteristics of mesophotic symbiotic microalgae within the reefbuilding coral, Stylophora pistillata[END_REF][START_REF] Ezzat | Carbon and nitrogen acquisition in shallow and deep holobionts of the scleractinian coral S. pistillata[END_REF]. However, a recent study, which investigated the photosynthetic performance of the mesophotic coral Euphyllia paradivisa, found a high photosynthetic capacity of this coral species under low light conditions [START_REF] Eyal | Euphyllia paradivisa, a successful mesophotic coral in the northern Gulf of Eilat/Aqaba, Red Sea[END_REF]. Pigment content, symbiont density and symbiont genus are generally involved in shaping the photo-acclimatization of the coral symbiosis from shallow to mesophotic environments. Our results highlight for the two soft coral species a host-specific Symbiodiniaceae genus that remained stable along the depth profile up to 40 m depth.

Litophyton sp. was associated with Symbiodinium sp., as previously reported (formerly Symbiodinium clade A, [START_REF] Barneah | Diversity of dinoflagellate symbionts in Red Sea soft corals: mode of symbiont acquisition matters[END_REF][START_REF] Lajeunesse | Do introduced endosymbiotic dinoflagellates 'take'' to new hosts?[END_REF]), while R. f. fulvum was rather associated with Cladocopium sp., in agreement with studies performed in the Red Sea (formerly Symbiodinium clade C, [START_REF] Barneah | Diversity of dinoflagellate symbionts in Red Sea soft corals: mode of symbiont acquisition matters[END_REF]), or the Great Barrier Reef [START_REF] Van Oppen | Diversity of algal endosymbionts (zooxanthellae) in octocorals: the roles of geography and host relationships[END_REF]Goulet et al. 2008b). Such clade specificity is assumed to be persistent both in space and time in octocorals [START_REF] Goulet | Stability of an octocoral-algal symbiosis over time and space[END_REF]Baker et al. 2013a). Thus, acclimatization of the soft corals to low light levels was independent of the symbiont genus, but potentially driven by higher concentrations of total chlorophyll per AFDW and per symbiont cell in mesophotic corals (Figure 19b), which maximize the light harvesting capacity of the cells under low light levels [START_REF] Mass | Photoacclimation of Stylophora pistillata to light extremes: metabolism and calcification[END_REF][START_REF] Ezzat | Carbon and nitrogen acquisition in shallow and deep holobionts of the scleractinian coral S. pistillata[END_REF]). In addition, both species exhibited a decreased symbiont density in mesophotic as compared to shallow colonies which is likely another adaptation to depth by reducing self-shading under reduced PAR [START_REF] Cohen | Long term photoacclimation responses of the coral Stylophora pistillata to reciprocal deep to shallow transplantation: photosynthesis and calcification[END_REF][START_REF] Ziegler | Mesophotic coral depth acclimatization is a function of host-specific symbiont physiology[END_REF]. A lower symbiont density also reduces the competition for inorganic nutrient supply [START_REF] Cunning | A dynamic bioenergetic model for coral-Symbiodinium symbioses and coral bleaching as an alternate stable state[END_REF] The autotrophic carbon acquisition to respiration ratios (PC/RC) of Litophyton sp.

ranged from 1 to 2 from shallow to mesophotic depths, the latter value being above the conservative threshold for net autotrophy (PC/RC>1.5). Considering that the solar radiation level in November is one of the lowest annual levels [START_REF] Stambler | Light and picophytoplankton in the Gulf of Eilat (Aqaba)[END_REF], the data suggest that Litophyton sp. is mostly an autotrophic species. On the contrary, PC/RC ratios of R. f. fulvum in November were always below compensation (PC/RC = 1) even when considering a maximal autotrophic carbon acquisition rate sustained for 12h. This strongly suggests that this species relies, at least in winter, on heterotrophy to sustain its daily respiratory needs.

The difference in PC/RC between the two species may be due to their different growth shapes and different micro-morphological features of the polyps. Two recent studies on gorgonian octocorals, indeed highlighted a correlation between host morphology, polyp size and productivity [START_REF] Baker | Productivity links morphology, symbiont specificity and bleaching in the evolution of Caribbean octocoral symbioses[END_REF][START_REF] Rossi | Linking host morphology and symbiont performance in octocorals[END_REF], thereby corroborating the first observations of [START_REF] Porter | Autotrophy, heterotrophy, and resource partitioning in Caribbean reefbuilding corals[END_REF].

Symbiont cell carbon acquisition was negatively correlated with symbiont density in both investigated soft coral species and decreased exponentially as soon as the symbiont density increased. Although this pattern is first described on soft corals in this study, similar observations were reported with gorgonian octocorals [START_REF] Rossi | Linking host morphology and symbiont performance in octocorals[END_REF]) and scleractinian corals [START_REF] Scheufen | Seasonal variation modulates coral sensibility to heat-stress and explains annual changes in coral productivity[END_REF], and might be due to a host-dependent regulation of light and nutrient ressources [START_REF] Scheufen | Seasonal variation modulates coral sensibility to heat-stress and explains annual changes in coral productivity[END_REF], or a self-shading of the symbionts (Cunning et al. 2017).

Our results also highlight a positive correlation between carbon acquisition and carbon translocation rates per symbiont cell, similarly to a previous study on scleractinian corals [START_REF] Leal | Symbiont type influences trophic plasticity of a model cnidarian-dinoflagellate symbiosis[END_REF]. In Litophyton sp., a low carbon acquisition per cell corresponded to a high symbiont density and consequently, symbiont cells assimilated more carbon for their own metabolism rather than translocating it to the host (50% translocation). In contrast, the symbionts in R. f. fulvum translocated significantly more carbon (80 to 90% translocation) to the host, likely due to their lower abundancy and increased rates of carbon acquisition per cell. Carbon translocation rates of >80% have also been reported for other coral species [START_REF] Falkowski | Light and the bioenergetics of a symbiotic coral[END_REF][START_REF] Muscatine | Fate of photosynthetic fixed carbon in light-and shade-adapted colonies of the symbiotic coral Stylophora pistillata[END_REF][START_REF] Tremblay | Autotrophic carbon budget in coral tissue: a new 13 C-based model of photosynthate translocation[END_REF]. For example, Scheufen et al. ( 2017) measured highest productivity rates in corals during the very oligotrophic summer season despite seasonally reduced symbiont density. This may also imply a high carbon translocation to the host that is essential when exogenous food sources are scarce.

Differences in cell-specific carbon assimilation and translocation rates may not only be related to the symbiont density but also to the symbiont genus. Indeed, Symbiodinium sp. (associated with Litophyton sp. here) is known to translocate less carbon to its host as compared to Cladocopium sp. (associated with R. fulvum fulvum here) [START_REF] Starzak | The influence of symbiont type on photosynthetic carbon flux in a model cnidarian-dinoflagellate symbiosis[END_REF][START_REF] Leal | Symbiont type influences trophic plasticity of a model cnidarian-dinoflagellate symbiosis[END_REF][START_REF] Baker | Climate change promotes parasitism in a coral symbiosis[END_REF]). However, an important observation is that the lower percentage of carbon translocation in Litophyton sp. is not indicative of a shift towards symbiont parasitism [START_REF] Baker | Climate change promotes parasitism in a coral symbiosis[END_REF], as the carbon translocation to the host still exceeded the metabolic costs for holobiont respiration. In R. f. fulvum, due to the low symbiont density, an increased translocation rate per cell was needed to transfer the same amount of carbon to the host. In addition, the percentage of carbon lost by the host was lower in Litophyton sp. than in R. f. fulvum, compensating for the lower percentage of carbon translocated from the symbionts. As a consequence, the rates at which carbon was retained in the host were constant between species and depths (between 13% and 19% of the photosynthesized carbon, or 93 to 138 µg C g -1 AFDW h -1 ). Assimilation rates of 10 to 20% of the photosynthesized carbon by the animal host seems to be a general feature in corals [START_REF] Tremblay | Autotrophic carbon budget in coral tissue: a new 13 C-based model of photosynthate translocation[END_REF][START_REF] Ezzat | Carbon and nitrogen acquisition in shallow and deep holobionts of the scleractinian coral S. pistillata[END_REF].

Soft and scleractinian corals therefore seem to exhibit a different trend of carbon acquisition along the depth gradient, although more investigations with different scleractinian and soft coral species are still needed. In order to compare carbon fixation per biomass between soft and scleractinian corals, we used data previously obtained on the scleractinian coral Stylophora pistillata sampled at the exact same location, depths and month [START_REF] Ezzat | Carbon and nitrogen acquisition in shallow and deep holobionts of the scleractinian coral S. pistillata[END_REF]) (Appendix II -Table S4) and normalized the rates to AFDW.

The estimates we obtained show that soft corals fix less carbon per biomass as compared to S. pistillata at shallow depth in November (estimation of 2426 µg C g -1 AFDW h -1 for S. pistillata versus 554-958 µg C g -1 AFDW h -1 ), although soft corals contain comparable or higher dinoflagellate density [START_REF] Thornhill | A connection between colony biomass and death in Caribbean reef-building corals[END_REF]) and chlorophyll content per biomass (Appendix II -Table S4) than scleractinian corals. However, in deeper environments, soft corals tend to fix carbon per biomass at similar or even higher rates (estimation of 555 µg C g -1 AFDW h -1 for S. pistillata versus 710-829 µg C g -1 AFDW h -1 for R. f. fulvum and Litophyton sp.). This may indicate that in shallow waters, soft corals have particularly less fixed carbon available to maintain or grow their biomass as compared to scleractinian corals. Morphological characteristics of the host, which have an impact on the diffusion and transmission of light to the symbionts, may explain differences in carbon acquisition in shallow water corals. Soft corals lack a calcium carbonate skeleton, contrary to scleractinian corals, and only possess sclerites as calcified structures. However, skeletons present light scattering abilities, which enhance light absorption efficiency of the symbionts (e.g., [START_REF] Enríquez | Key functional role of the optical properties of coral skeletons in coral ecology and evolution[END_REF]. Although characteristics of sclerites and tissues remain to be investigated to understand the potential light amplification and dispersion in soft corals, we hypothesized that they would affect the light environment of the symbionts to a lesser extent than skeletons do in scleractinian corals. In addition, soft corals have a thick coenenchyme and exhibit a low ratio of colony surface area to volume, which does not favor light exposure and gas or nutrient exchange through the epidermal tissue [START_REF] Wangpraseurt | Lateral light transfer ensures efficient resource distribution in symbiont-bearing corals[END_REF]. Finally, the variability of their hydroskeleton allows soft corals to contract and expand their tissue. Tissue contraction removes symbionts from the tissue surface, thereby higher irradiance is required to reach the symbionts and to achieve photosynthetic compensation and saturation.

Understanding nutritional ecology of octocorals is still in its infancy. The common belief is that octocorals are more heterotrophic than scleractinian corals, because of their low carbon acquisition in surface waters, and because their morphology is more suited for heterotrophy [START_REF] Schubert | Symbiotic versus non-symbiotic octocorals: physiological and ecological implications[END_REF]. Two recent studies on Caribbean gorgonian octocorals have observed a possible correlation between host morphology and symbiont performance [START_REF] Baker | Productivity links morphology, symbiont specificity and bleaching in the evolution of Caribbean octocoral symbioses[END_REF][START_REF] Rossi | Linking host morphology and symbiont performance in octocorals[END_REF]. Octocorals with thin branches and small polyps can be more autotrophic as compared to those with a massive shape and large polyps. In our work, a different degree of autotrophy was demonstrated for the two investigated soft coral species, which might be linked to morphological traits. The arborescent shape of Litophyton sp. may enhance the exposure of symbionts to light and thus favour autotrophy compared to the mat-forming shape of R. f. fulvum. In addition, our results strongly support the view that high symbiont density results in lower cell-specific carbon translocation and acquisition. However, soft corals with high symbiont densities (Litophyton sp.) reached similar carbon translocation than those with low symbiont density. Further work involving various soft coral species, growth-shapes and symbiont-specificities will be essential to provide more insight into the physiology and trophic ecology of octocorals thriving along environmental gradients.

Chapter 4 | Acquisition of dissolved inorganic and organic nitrogen dinoflagellate symbionts), but it is still poorly known to what extent and under which conditions corals profit from DDN.

Most of the research using the 15 N2 method to assess DDN assimilation in adult tropical corals has been directed towards only one species, Stylophora pistillata, belonging to the Pocilloporidae family. These studies showed that S. pistillata can acquire DDN via grazing on a culture of diazotrophic cells [START_REF] Benavides | Diazotrophs: a non-negligible source of nitrogen for the tropical coral Stylophora pistillata[END_REF]). However, under natural conditions, this coral species has variable capacities to stimulate diazotrophic activity in seawater as well as variable DDN assimilation rates into the coral holobiont [START_REF] Grover | Nitrogen fixation in the mucus of Red Sea corals[END_REF][START_REF] Bednarz | The assimilation of diazotroph-derived nitrogen by scleractinian corals depends on their metabolic status[END_REF][START_REF] Lesser | Diazotroph diversity and nitrogen fixation in the coral Stylophora pistillata from the Great Barrier Reef[END_REF]. As DDN can play a major role in supplying N to corals [START_REF] Cardini | Budget of primary production and dinitrogen fixation in a highly seasonal Red Sea coral reef[END_REF][START_REF] Bednarz | The assimilation of diazotroph-derived nitrogen by scleractinian corals depends on their metabolic status[END_REF]

Materials and methods

Laboratory experiment: DDN assimilation from external diazotrophs

To assess whether scleractinian and soft corals have the same capacities to assimilate DDN from external active diazotrophs in the seawater, an experiment was performed at the Monaco Scientific Centre. We used the scleractinian coral S. pistillata and the soft coral Sarcophyton sp., which have been maintained in aquaria for years, and are not associated with active diazotrophs (V. Bednarz, unpublished data; [START_REF] Kooperman | Coral mucusassociated bacterial communities from natural and aquarium environments[END_REF]. All colonies were maintained under the same light (200 µmoles photons m -2 s - 1 ) and temperature (25°C) conditions to ensure comparability between the species during the following experiment. For this purpose, a culture of diazotrophs (Crocosphaera watsonii) was prepared and diluted to obtain two cell concentrations of 100 and 1000 cells mL -1 , respectively, matching concentrations of 3-20 µm size class phycoerythrincontaining unicellular cyanobacteria under non-bloom and bloom conditions [START_REF] Campbell | Annual variability of phytoplankton and bacteria in the subtropical ENSO event[END_REF]. C. watsonii concentrations were determined using a hemocytometer (Z1 particles counter, Beckman Coulter, USA). Four nubbins of each coral species per C. watsonii concentration were then individually placed in 320 mL gas tight bottles completely filled with 280 mL of 0.22 µm-filtered seawater amended with the adequate concentration of diazotrophs and 40 mL of 15 N2 enriched seawater. In addition, three nubbins of each species were incubated under the same above conditions but without C. watsonii cells. At the end of the 24h incubation, corals were removed and stored frozen until subsequent analysis.

They were then processed as described below. The 15 N2 seawater addition method was used to assess N2 fixation rates. For this purpose, 15 N2-enriched seawater was produced prior to the incubation experiment by injection of 10 mL of 15 N2 gas (98% Eurisotop) into gas-tight 250 mL bottles completely filled with degassed, 0.2 µm filtered seawater, followed by vigorous shaking for 12 h to ensure 100% 15 N2 equilibration [START_REF] Mohr | Methodological underestimation of oceanic nitrogen fixation rates[END_REF]). In order to test which coral species can assimilate DDN or can enrich the seawater in DDN, collected nubbins from each coral species were individually placed in gas-tight bottles of 600 mL. Bottles were completely filled with 120 µm-filtered seawater, directly pumped from the reef, with 10% replaced by 15 N2-enriched seawater (resulting in theoretical enrichment of ~9.8 atom%). A first set of three control bottles was prepared as described above but without corals to measure the baseline of N2 fixation by planktonic diazotrophs. To evaluate the natural 15 N abundance of the corals, a second set of controls consisted in incubating three coral nubbins from each species (only two for Cynarina sp.) in 120 µm-filtered seawater incubations without 15 N2 addition. All bottles were incubated for 24 h in several outdoor aquaria. The seawater temperature was kept constant at in situ temperature (~ 24°C) by using continuous supply of seawater in the aquaria. Corals were exposed to the natural diel light cycle and shaded to the corresponding irradiance of their collection site by applying layers of black mesh above the aquaria and considering an attenuation coefficient equal to 0.072-0.1 m -1 [START_REF] Akkaynak | What is the space of attenuation coefficients in underwater computer vision[END_REF][START_REF] Tamir | Light environment drives the shallow to mesophotic coral community transition[END_REF]. At the end of the incubations, corals were removed from seawater and stored frozen until subsequent analysis. Incubation water of all bottles was filtered onto pre-combusted (450°C for 5 h) GF/F filters which were dried at 60°C for 48h. Corals and filters were processed as described in the sample analysis section.

Sample analysis

For scleractinian corals, the tissue was removed from the skeleton using an air brush and homogenized with a potter tissue grinder. The host tissue and zooxanthellae were separated through a series of centrifugations according to [START_REF] Grover | Nitrate uptake in the scleractinian coral Stylophora pistillata[END_REF] and each fraction was freeze-dried. Soft corals were treated as described in [START_REF] Pupier | Studies with soft corals -recommendations on sample processing and normalization metrics[END_REF].

Briefly, they were freeze-dried, the resulting powder homogenized in distilled water and then separated in several centrifugation steps into the host tissue and zooxanthellae fractions as described above. Each fraction was subsequently freeze-dried again. The 15 N enrichment, as well as particulate organic carbon (POC) and particulate N content (PN) of each sample and filter were determined using a mass spectrometer (Delta Plus; Thermo Fisher Scientific, Germany) coupled to a C/N analyzer (Flash EA; Thermo Fisher Scientific, Germany). To calculate N2 fixation in particles of the incubation water or DDN assimilation by corals (host and/or zooxanthellae), the equation of [START_REF] Montoya | A simple, high-precision, high-sensitivity tracer assay for N2 fixation[END_REF] was used:

N2 fixation or DDN assimilation= atom% 15 Nexcess × PN sample t × 9.8

Where: t is the incubation time, 9.8 the initial 15 N enrichment of the incubation water, and PNsample the particulate N content of the samples. For each sample, the atom% 15 Nexcess enrichment was calculated by subtracting the natural 15 N enrichment of control samples without 15 N2 exposure (atom% 15 Ncontrol) from the 15 N enrichment of samples after exposure to 15 N2 enriched seawater (atom% 15 Nsample). The atom% 15 Nsample was considered significant when it was at least three fold higher than the standard deviation of the atom% 15 Ncontrol. N2 fixation and DDN assimilation values were normalized to the volume of water filtered or to the total dry weight of the sample. For comparison with previous studies, scleractinian corals data were also normalized to the skeletal surface area determined using the wax technique [START_REF] Veal | Increasing the accuracy of surface area estimation using single wax dipping of coral fragments[END_REF]).

Statistical analyses

Analyses were performed using R software (R Foundation for Statistical Computing). All data were expressed as mean ± standard error. Prior to analyses, outlier values were identified using Grubb's test and were excluded when p-values were significant (p < 0.05). Assumptions of normality and homoscedasticity of variance were evaluated through Shapiro's and Bartlett's tests. A non-parametric Kruskal-Wallis test was used to test for differences between groups (hard vs soft corals) and depths (shallow vs deep soft corals) on POC and POC:PN. Analyses of variance (ANOVA) were performed to respectively test the effect of species and morphologies on DDN transfer and assimilation. Tukey tests were performed as a posteriori testing.

Results

In the laboratory experiment, no N2 fixation (neither in seawater nor in coral tissue)

was measured when corals were incubated without the addition of Crocophaera watsonii cells to the seawater. Both S. pistillata and Sarcophyton sp. however demonstrated abilities to assimilate DDN in the presense of active diazotrophs (i.e. Crocosphaera watsonii) in seawater (Figure 24). Overall, Sarcophyton sp. assimilated 65% more DDN in its tissue than S. pistillata. While there was no difference between the DDN assimilations of S.

pistillata exposed at the two cell concentrations (Tukey HSD: p = 0.389), Sarcophyton sp.

significantly increased its DDN assimilation under higher cell concentration (Tukey HSD: p = 0.020). In the field experiment, a very low level of N2 fixation was measured in seawater with natural populations of diazotrophs, in the absence of corals (Figure 25a). All coral species enriched the seawater in mucus-containing particles during the 24 h incubation (Figure 25a). Particulate organic carbon, which is a proxy for living and detrital particles present in the water and being released by the corals, was two to seven fold higher in chambers containing scleractinian corals compared to those containing soft corals (Kruskal-Wallis: p < 0.001). There was no difference in POC release between shallow and mesophotic soft corals (Kruskal-Wallis: p = 0.138). Particles released by scleractinian corals presented a higher POC:PN ratio (between 11.9 and 16.6) compared to those of soft corals (between 4.9 and 7.5) (Figure 25b, Kruskal-Wallis: p < 0.001). While there was no N2 fixation in the incubation water containing all soft coral species sampled in shallow or mesophotic environments, a significant fixation was observed in the incubation water of all four scleractinian species (Figure 26a,b). Fixation rates ranged from 34 to 211 ng N L - 1 over the 24 h incubation, or between 6 and 135 10 -3 ng N mg DW -1 h -1 when expressed per coral biomass. For both normalizations, rates were significantly different between species (ANOVA: p = 0.003 and p = 0.002, respectively) and morphologies (branching vs mounding, ANOVA: p < 0.001) with higher rates obtained for A. eurystoma and P.

damicornis. Significant assimilation of DDN was observed in the host tissue and zooxanthellae of scleractinian corals, but not for the soft coral species from either shallow or mesophotic environments (Figure 26c). The DDN assimilation in host tissue was equal or higher than the assimilation in the zooxanthellae fraction. Moreover, an inverse trend was observed between N2 fixation occurring in incubation water and DDN assimilated by the whole symbiotic association (Figure 26d).

Discussion

Divergent capacity of coral mucus to enrich seawater with DDN

This study first highlights contrasting capacities of scleractinian and soft corals to promote N2 fixation in seawater. Freshly fixed N2 was traced in the incubation water containing scleractinian coral species, while no fixation in the seawater occurred in presence of soft corals.

A previous study, which has used the ARA method has also recorded much lower rates of gross N2 fixation in the presence of soft compared to scleractinian corals [START_REF] Bednarz | Seasonal variation in dinitrogen fixation and oxygen fluxes associated with two dominant zooxanthellate soft corals from the northern Red Sea[END_REF]. A difference in the quality and/or quantity of mucus (particulate and dissolved organic carbon, POC and DOC) released by the two coral groups as observed here, together with a different amount of mucus-associated bacteria most likely explain differences in seawater diazotroph abundance or activity. While the sugar composition of the mucus can be similar between soft and scleractinian corals [START_REF] Meikle | Structural investigations on the mucus from six species of coral[END_REF][START_REF] Hadaidi | Carbohydrate composition of mucus from scleractinian corals from the central Red Sea[END_REF], both the amount and POC:PN content of mucus is often species-dependent [START_REF] Naumann | Organic matter release by dominant hermatypic corals of the Northern Red Sea[END_REF][START_REF] Hadaidi | Carbohydrate composition of mucus from scleractinian corals from the central Red Sea[END_REF]. [START_REF] Pogoreutz | Sugar enrichment provides evidence for a role of nitrogen fixation in coral bleaching[END_REF] highlighted that DOC enrichment, in the form of sugars, can significantly stimulate diazotrophic N2 fixation. In our study, scleractinian corals released between 1.9 and 8.9 mg POC m -2 h -1 , which is in the range of what has been reported for Red Sea scleractinian corals (between 0.3 to 6.5 mg POC m -2 h -1 ) by [START_REF] Naumann | Organic matter release by dominant hermatypic corals of the Northern Red Sea[END_REF]. Soft corals however released POC at two-to ten-fold lower rates. This is in agreement with previous measurements performed on another Red Sea soft coral belonging to the Xeniidae family, for which no POC release was observed [START_REF] Bednarz | Inorganic nutrient availability affects organic matter fluxes and metabolic activity in the soft coral genus Xenia[END_REF]). As POC is a proxy for detrital and living particles, the lower particle content in the soft coral surrounding seawater can be explained by the well-known antimicrobial properties of soft coral mucus [START_REF] Kelman | Antimicrobial activity of Red Sea corals[END_REF][START_REF] Nakajima | Release of dissolved and particulate organic matter by the soft coral Lobophytum and subsequent microbial degradation[END_REF]). These authors indeed demonstrated that little antimicrobial activity was measured for scleractinian coral mucus whereas soft corals, including those studied in this work, significantly inhibited the growth of co-occurring seawater bacteria through the production of antibiotic compounds. Hence, scleractinian corals release mucus with a large number of bacteria into the surrounding seawater that can influence the activity and diversity of planktonic diazotroph populations. This feature may be greatly reduced in soft corals, as their high antimicrobial activity constitutes a defense strategy against invading pathogens and fouling organisms for the holobiont, thus possibly assisting in competition over space and nutrition [START_REF] Kelman | Chemical warfare in the sea: The search for antibiotics from Red Sea corals and sponges[END_REF]). Finally, the organic matter (OM) released by soft corals presented a particularly low POC:PN content (of 5 to 7, i.e. particles enriched in N) compared to the OM released by scleractinian corals (from 12 to 17 in this study; range in agreement with [START_REF] Naumann | Organic matter release by dominant hermatypic corals of the Northern Red Sea[END_REF]. This is also in agreement with the findings of two previous studies [START_REF] Meikle | Structural investigations on the mucus from six species of coral[END_REF][START_REF] Nakajima | Release of dissolved and particulate organic matter by the soft coral Lobophytum and subsequent microbial degradation[END_REF], which observed a higher protein and lower carbohydrate composition of soft coral mucus compared to scleractinian coral mucus. Since N2 fixation is rather promoted by N deprivation [START_REF] Knapp | The sensitivity of marine N2 fixation to dissolved inorganic nitrogen[END_REF], a low POC:PN ratio is not in favour of N2 fixation. Put together, our results suggest that the lack of N2 fixation in seawater surrounding soft corals is potentially due to the antimicrobial properties of soft coral mucus and the N repletion of the released particles. On the contrary to soft corals, seawater N2 fixation was recorded in presence of scleractinian corals. Measured rates (0.01-0.13 ng N mg -1 DW h -1 or 0.03 nmol N cm -2 h -1 or 2 to 15 nmol N L -1 d -1 ) are however significantly lower than those recorded for other benthic substrates such as sediment, sands or microbial mats (reviewed in [START_REF] Benavides | Diazotrophs: overlooked key players within the coral symbiosis and tropical reef ecosystems?[END_REF]. Nevertheless, they are in the same range as those previously measured with seawater diazotrophs of the Great Barrier Reef (from 5 to 70 nmol N L -1 d -1 , [START_REF] Messer | Diversity and activity of diazotrophs in Great Barrier Reef surface waters[END_REF].

Divergent capacity of corals to assimilate DDN

Our results demonstrate that all investigated scleractinian species assimilated DDN, since 15 N enrichment was observed in both host tissue and symbionts. In contrast, no 15 N enrichment of soft coral tissue was detected either in corals collected from shallow or mesophotic reefs. The fact that even mesophotic soft coral colonies did not assimilate DDN stands in contrast to the scleractinian species S. pistillata, which shows higher assimilation rates in deep compared to shallow waters [START_REF] Bednarz | The assimilation of diazotroph-derived nitrogen by scleractinian corals depends on their metabolic status[END_REF]). Here, DDN assimilation rates of scleractinian corals (ca. 0.3 to 1 ng cm -2 h -1 or 0.6 to 1.7 nmol cm -2 d -1 ) are in agreement with those measured for the species S. pistillata sampled in shallow waters of the Red Sea or the Great Barrier Reef [START_REF] Bednarz | The assimilation of diazotroph-derived nitrogen by scleractinian corals depends on their metabolic status[END_REF][START_REF] Lesser | Diazotroph diversity and nitrogen fixation in the coral Stylophora pistillata from the Great Barrier Reef[END_REF]). However, they were 6 to 10 times lower than those measured for corals depending more on heterotrophy, such as bleached corals or those living in mesophotic and temperate environments [START_REF] Bednarz | The assimilation of diazotroph-derived nitrogen by scleractinian corals depends on their metabolic status[END_REF][START_REF] Bednarz | Diazotrophic community and associated dinitrogen fixation within the temperate coral Oculina patagonica[END_REF]. These observations suggest that the contribution of DDN to the N requirements of corals increases during nutrient deprivation, or when the uptake of other inorganic N forms by dinoflagellate symbionts is reduced [START_REF] Bednarz | Diazotrophic community and associated dinitrogen fixation within the temperate coral Oculina patagonica[END_REF]). The inverse trend observed in this study between seawater N2 fixation and DDN assimilation rates in corals suggests that a substantial part of the DDN assimilated by corals is obtained from heterotrophic feeding on fixed N compounds and/or from diazotrophic cells growing in the mucus layer [START_REF] Bednarz | The assimilation of diazotroph-derived nitrogen by scleractinian corals depends on their metabolic status[END_REF]. Therefore, the lack of N2 fixation in the surrounding seawater of soft corals (highlighting the absence of active diazotrophs in their mucus) may also explain why we did not detect any DDN assimilation by shallow and mesophotic soft corals. Furthermore, coral polyps with a mounding morphology show generally higher heterotrophic feedings rates as compared to those with a branching morphology [START_REF] Palardy | The effect of morphology, polyp size, depth, and temperture on feeding in three species of Panamanian corals[END_REF]). This corresponds to the higher DDN assimilation rates observed for Cynarina sp. and Goniastrea sp. (mounding morphology) as compared to A. cervicornis and P. damicornis (branching morphology). Corals can receive DDN not only from mucus-associated but also from pelagic diazotrophs [START_REF] Camps | Released coral mucus does not enhance planktonic N2 fixation rates[END_REF]). In our experiment on C. watsonii cells, both corals assimilated DDN (i.e. in the form of N2 fixing C. watsonii cells or DDN compounds released by C. watsonii cells) from the surrounding seawater with rates similar to those observed for our corals from the Red Sea. Moreover, Sarcophyton sp. assimilated more DDN in the high versus the low C. watsonii concentration suggesting that the assimilation capacity of this species was not saturated even under simulated bloom conditions.

This indeed indicates that soft corals also have the capacity to assimilate diazotrophs/DDN from the seawater. Thus, the lack in DDN assimilation by Red Sea soft corals sampled in the field, including the Symbiodiniaceae-free and heterotrophic genus Dendronephthya sp., is likely not due to a lower heterotrophy as compared to scleractinian corals, but rather linked to the absence of active diazotrophs in the soft coral mucus. For example, strains of putative diazotrophs (Vibrio campbellii and Vibrio parahaemolyticus; [START_REF] Chimetto | Vibrios dominate as culturable nitrogen-fixing bacteria of the Brazilian coral Mussismilia hispida[END_REF] have been isolated from Dendronephthya sp. [START_REF] Harder | A distinctive epibiotic bacterial community on the soft coral Dendronephthya sp. and antibacterial activity of coral tissue extracts suggest a chemical mechanism against bacterial epibiosis[END_REF]. We therefore hypothesize that even if diazotrophs can be present, the mucus of soft corals does not represent a favorable habitat for diazotrophic activity. Moreover, non-negligible DDN assimilation rates have been detected in the skeleton of scleractinian corals due to the activity of endolithic diazotrophs [START_REF] Sangsawang | 13 C and 15 N assimilation and organic matter translocation by the endolithic community in the massive coral Porites lutea[END_REF][START_REF] Bednarz | Diazotrophic community and associated dinitrogen fixation within the temperate coral Oculina patagonica[END_REF]. Such contribution to the total N budget of the holobiont cannot be observed in soft corals as they lack a calcium carbonate skeleton. In situ experiments remain to be investigated to assess whether soft corals benefit from any other external source of N on reefs that could enhance their opportunistic life history features.

Conclusion

It is widely accepted that N is one of the most limiting nutrients for reef primary productivity [START_REF] Eyre | Mass coral spawning: A natural large-scale nutrient addition experiment[END_REF], and that benthic N2 fixation plays an important role in supplying bioavailable N within benthic and pelagic reef habitats [START_REF] Cardini | Budget of primary production and dinitrogen fixation in a highly seasonal Red Sea coral reef[END_REF][START_REF] Messer | Diversity and activity of diazotrophs in Great Barrier Reef surface waters[END_REF].

The surface structure of benthic organisms and substrates provides an important habitat for the colonization by diazotrophs, but the abundance, composition and activity of the diazotrophic community may depend on the type of organism/substrate. Here, we suggest that soft coral mucus represents likely a less favorable habitat for microbes as compared to scleractinian coral mucus due to its relatively low C but high N content along with antimicrobial properties. The limitation. This may ultimately affect primary production, carbon sequestration and the functioning of coral reef ecosystems and could be an interesting topic for future studies.

contrasting capacities to take advantage of this nutrient source can help in understanding how soft and scleractinian corals cope with different nutrient loads.

Introduction

Nitrogen (N) is essential for the life of all organisms on Earth, as it is required for the biosynthesis of key cellular components. However, N concentrations are low in many marine ecosystems such as coral reefs, with the most abundant N form, dinitrogen gas (N2), being only available to nitrogen-fixing microorganisms. N is therefore a growth-limiting nutrient for most organisms living in oligotrophic environments. Animals are only able to assimilate particulate and dissolved organic N (DON), such as free amino acids (DFAA) and urea, whereas algae and microorganisms can also assimilate the inorganic forms (DIN) such as nitrate (NO3 -) and ammonium (NH4 + ) [START_REF] Muscatine | Ammonium uptake by symbiotic and aposymbiotic reef corals[END_REF]). Consequently, many reef animals have evolved as symbiotic meta-organisms (i.e. holobionts), in which the host is associated with microorganism assemblages, in order to efficiently take up, conserve and recycle the few available nutrients.

Consequently, many reef animals have evolved as symbiotic metaorganisms (i.e., holobionts), in which the host is associated with microalgae and/or nitrogen-fixing bacteria to survive in their nutrient-poor environment.

Symbiotic corals are the most iconic reef holobionts. They are indeed associated with a wide range of microorganisms such as diazotrophs and dinoflagellates of the family Symbiodiniaceae (LaJeunesse et al. 2018). As a result, they can use almost all N forms available in their surrounding environment, with the symbionts primarily assimilating DIN [START_REF] Grover | Uptake of ammonium by the scleractinian coral Stylophora pistillata: Effect of feeding, light, and ammonium concentrations[END_REF][START_REF] Grover | Nitrate uptake in the scleractinian coral Stylophora pistillata[END_REF][START_REF] Pernice | A single-cell view of ammonium assimilation in coral-dinoflagellate symbiosis[END_REF], and the host DON [START_REF] Grover | Urea uptake by the scleractinian coral Stylophora pistillata[END_REF][START_REF] Grover | Uptake of dissolved free amino acids by the scleractinian coral Stylophora pistillata[END_REF]. Therefore, when corals bleach (i.e. loss of dinoflagellate symbionts due to environmental stress), they are depleted of a major N source and their chances of recovery are partly determined by their ability to restore the functions of their symbionts [START_REF] Suggett | Symbiotic dinoflagellate functional diversity mediates coral survival under ecological crisis[END_REF]. Assimilation of dissolved N also depends on the environmental conditions to which the corals are exposed. For example, elevated seawater temperatures, which reduce nutrient availability through water column stratification [START_REF] Behrenfeld | Climate-driven trends in contemporary ocean productivity[END_REF], lessens the ability of the coral holobiont to take up DIN [START_REF] Godinot | Coral uptake of inorganic phosphorus and nitrogen negatively affected by simultaneous changes in temperature and pH[END_REF][START_REF] Krueger | Temperature and feeding induce tissue level changes in autotrophic and heterotrophic nutrient allocation in the coral symbiosis -A NanoSIMS study[END_REF]. Finally, metabolic compatibility between coral hosts and their algal symbionts also determine the nutritional performance of the coral holobiont [START_REF] Leal | Symbiont type influences trophic plasticity of a model cnidarian-dinoflagellate symbiosis[END_REF][START_REF] Pernice | A nanoscale secondary ion mass spectrometry study of dinoflagellate functional diversity in reef-building corals[END_REF]. Overall, the nutritional status of corals and their capacity to efficiently exploit nutrients from the surrounding environment are major functional traits influencing their distribution and abundance in a given environment.

However, the nutritional ecology of coral-dinoflagellate symbioses, regarding their N metabolism, has most exclusively concerned few species of hard corals, as they are the main reef builders [START_REF] Muscatine | The effect of external nutrient resources on the population dynamics of zooxanthellae in a reef coral[END_REF][START_REF] Hoegh-Guldberg | Availability of two forms of dissolved nitrogen to the coral Pocillopora damicornis and its symbiotic zooxanthellae[END_REF][START_REF] Mills | Particulate matter ingestion and associated nitrogen uptake by four species of scleractinian corals[END_REF]Baker et al. 2013b;[START_REF] Ezzat | Carbon and nitrogen acquisition in shallow and deep holobionts of the scleractinian coral S. pistillata[END_REF]. Other major benthic reef organisms such as soft corals have been overlooked (qualitative assimilation of NH4 + in Lobophyton sp., [START_REF] Burris | Uptake and assimilation of 15 NH4 + by a variety of corals[END_REF]; uptake of DFAA by Heteroxenia fuscescens , Schlichter 1982b). This lack of data, together with different normalization metrics between the two coral groups prevent any predictions on the nutritional conditions that favour the growth of either hard or soft corals. Soft corals have however been observed in higher abundance in areas with high concentrations of dissolved inorganic nutrients [START_REF] Baum | Abundance and physiology of dominant soft corals linked to water quality in Jakarta Bay, Indonesia[END_REF]. This observation suggests that they might have a different nutrient metabolism than hard corals, or a different nutritional relationship with their symbionts, but these hypotheses remain to be investigated. Therefore, this study assessed dissolved N assimilation (i.e. NH4 + , NO3 -, and DFAA) in soft corals, addressed which forms present the highest assimilation rates, and compared assimilation rates of soft and hard corals.

For this purpose, a multi-species comparison was performed using the 15 N stable isotope tracer to follow N incorporation into the tissues of several soft and hard corals sampled in shallow and upper mesophotic reefs of the Red Sea. This study thus allows a better understanding of the functional ecology of both soft and hard corals.

Material and methods

Biological material

The To assess the assimilation rates of N under natural living conditions, twenty nubbins per coral species were collected from different colonies at the shallow and mesophotic depths (except for L. arboreum which was only found in shallow waters). Five nubbins were immediately frozen for the determination of Symbiodiniaceae genus and natural isotopic abundance, while the others were brought back to the Red Sea Simulator facility (described in Bellworthy and Fine 2018) to recover for one week prior to starting the incubations with the 15 N stable isotope tracers (see below). For this purpose, nubbins were allocated to 12 outdoor aquaria (one per species and depth), which were continuously supplied with seawater directly pumped from the reef. Corals were maintained under the natural light cycle at levels corresponding to their in situ depth (ca. 450 and 30 µmole photons m -2 s -1 at midday, for shallow and mesophotic environments respectively). These levels were adjusted using mesh clothes.

In addition to the conditions described above, 3 additional aquaria were prepared, each containing 5 nubbins per species of soft corals collected from the shallow reef (5 m). Aquaria were maintained under the exact same conditions as above, except that temperature was gradually increased (+ 0.5°C per day) up to 30°C (+5°C than the control condition). Corals were kept at this higher temperature for one week prior to the incubations with 15 N tracers, run at the same time as the other incubations.

Incubations

15 N stable isotope tracers were used to assess the assimilation rates of dissolved inorganic and organic N sources by hard and soft corals. To this end, 15 L of 0.45 µm-filtered seawater were enriched with either 15 NH4Cl, 15 NO3Cl and 15 N-DFAA (all at 98 atom % 15 N, Sigma-Aldrich, St-Louis, USA) to reach a final N concentration of 3 µM. Five 200 mL beakers were prepared per species, depth and N compound and entirely filled with the 15 N-enriched solution. Incubations were performed in the Red Sea simulator under the natural irradiance of shallow and mesophotic depths. Corals were incubated at 25°C or at 30°C for five hours, between 11:00 am and 04:00 pm to cover the maximal daily irradiance. At the end of the incubations, coral nubbins were rinsed in 0.2 µm-filtered seawater (FSW) and frozen at -80°C until further analysis.

Samples processing

Soft coral samples were processed as described in [START_REF] Pupier | Studies with soft corals -recommendations on sample processing and normalization metrics[END_REF]. Briefly, they were freeze-dried, weighed and crushed into powder. For hard corals, the tissue was first removed from the skeleton using an air-brush and homogenized with a Potter-Elvehjem tissue grinder, before being freeze-dried, weighed and crushed into powder. The skeleton was kept for the determination of the skeletal surface area using the wax dipping technique [START_REF] Veal | Increasing the accuracy of surface area estimation using single wax dipping of coral fragments[END_REF]. A fraction of the powder (i.e. ca. 30 mg) was used for the determination of the ash-free dry weight (AFDW) as described below and the remaining tissue was homogenized in 10 mL distilled water with a Potter-Elvehjem tissue grinder. The host and symbiont fractions of each sample were separated through a series of centrifugations [START_REF] Grover | Nitrate uptake in the scleractinian coral Stylophora pistillata[END_REF]. The symbiont pellet was re-dissolved in 10 mL distilled water. A 500 µL subsample was used for the determination of algal symbiont density via haemocytometer counts (Neubauer-improved haemocytometer, Marienfeld, Germany) using a light microscope. In addition, a 2.5 mL subsample from the symbiont fraction was used for the determination of the chlorophyll concentration according to [START_REF] Jeffrey | New spectrophotometric equations for determining chlorophylls a, b, c1 and c2 in higher plants, algae and natural phytoplankton[END_REF], using a spectrophotometer (SAFAS, Monaco). The remaining fractions of the host and symbionts were subsequently freeze-dried and weighed. Approximately 500 µg of host and symbiont powder were transferred in tin caps for analysis of the 15 N enrichment, N content and the natural  15 N signatures using a mass spectrometer (Delta Plus, Thermo Fisher Scientific, Germany) coupled to a C/N analyzer (Flash EA, Thermo Fisher Scientific, Germany). The total amount of N assimilated in each compartment was then determined using the modified equations of [START_REF] Dugdale | The use of 15 N to measure nitrogen uptake in eutrophic oceans; experimental considerations1,2[END_REF] according to [START_REF] Grover | Uptake of ammonium by the scleractinian coral Stylophora pistillata: Effect of feeding, light, and ammonium concentrations[END_REF][START_REF] Grover | Nitrate uptake in the scleractinian coral Stylophora pistillata[END_REF][START_REF] Grover | Uptake of dissolved free amino acids by the scleractinian coral Stylophora pistillata[END_REF]. These equations compare the N enrichment obtained in the incubations with 15 N with the natural isotopic values of control corals, sampled at the beginning of the experiment.

All data were normalized to the AFDW of the nubbins, which was used as a proxy for biomass. The subsample used for the determination of the AFDW was first weighed, and then combusted at 450°C for 4 h in a muffle furnace (Thermolyne 62700, Thermo Fischer Scientific, the United States). AFDW was determined as the difference between the dry weight and ash weight of the subsample and extrapolated to the total weight of the nubbin. AFDW was chosen for normalization because the surface area of soft corals cannot be accurately determined, as besides polyp expansion, their fleshy body can extend or retract extensively depending on environmental conditions (e.g., [START_REF] Fabricius | Widespread mixotrophy in reef-inhabiting soft corals: the influence of depth, and colony expansion and contraction on photosynthesis[END_REF]. Moreover, since N is assimilated into coral biomass, it is therefore more relevant to estimate the assimilation rates per biomass. This is in agreement with the observations of [START_REF] Edmunds | Normalizing physiological data for scleractinian corals[END_REF], who also concluded that normalizing data to biomass is most effective in accounting for coral size in comparative studies.

Statistical analyses

Analyses were performed using the R environment for statistical computing and graphics (R Core Team, 2017 Generalized linear models were used to test: 1) the effect of species, depth and N source on the assimilation rates of N by the host or the dinoflagellate fractions at 25°C, 2) the effect of species, temperature and N source on the assimilation rates of N by the host or dinoflagellate fractions in shallow soft corals, and 3) the effect of species and depth on tissue descriptors. We did not differentiate between the three N sources tissue descriptor data, as we assumed that the incubation time and N concentration used here would not affect symbiont density and chlorophyll concentration (Hoegh-Guldberg 1994), and the N assimilated represented less than 0.5% of the total N content. Generalized linear models (family = Gaussian) were fitted on data transformed using the Box-Cox transformation procedure ((x λ- 1 )/λ, Appendix III -Table S6) as implemented in the R-package MASS [START_REF] Venables | Photosynthetic symbioses in animals[END_REF]. Compliance with the assumptions of a normal distribution of the model residuals and homoscedasticity was verified using, respectively, the Shapiro-Wilk test and Levene's test as implemented in the R-package car (Fox and Weisberg, 2019). The emmeans package [START_REF] Lenth | emmeans: Estimated Marginal Means, aka least-squares means[END_REF]) was used to calculate estimated marginal means and compute biologically relevant contrasts (i.e. between depths for each species and between species for each depth). The Benjamini-Hochberg procedure (Benjamini and Hochberg, 1995) was used for multiplicity adjustments of p-values.

Results

For clarity, the results of the statistical treatments are reported in Appendix III (Table S7) only. Shallow nubbins of L. arboreum and S. pistillata harbored Symbiodinium sp.

(formerly clade A), while nubbins from all other species and depths were associated with Cladocopium sp. (formerly clade C). Symbiodiniaceae densities per tissue biomass (Figure 27)

were overall similar between hard and soft corals. They were significantly different between depths only for G. fascicularis, S. hystrix (lower at mesophotic depth) and R. f. fulvum (higher at mesophotic depth). Total chlorophyll concentration per tissue biomass was significantly higher in R. f. fulvum at shallow depth, compared to the other species (Appendix III -Figure S2a). Mesophotic colonies presented a higher chlorophyll concentration per biomass than shallow colonies in S. hystrix and S. pistillata (Appendix III -Figure S2a). At shallow depth, R.

f. fulvum and Sarcophyton sp. contained higher chlorophyll concentrations per symbiont cell than hard corals (Appendix III -Figure S2b). All hard coral species, as well as Sarcophyton sp, showed significant higher concentrations in chlorophyll at the mesophotic depth as compared to shallow (Appendix III -Figure S2b). Significant differences between depths are displayed with an asterisk. Significant differences between species are distinguished with letters by depth. AFDW = ash-free dry weight. ns = not sampled.

Concerning the elemental composition of the host tissue, the C:N ratios increased from ca. 5 in scleractinian corals and L. arboreum from shallow and mesophotic depths to ca. 8 in shallow and mesophotic Sarcophyton sp, and to more than 15 in R. fulvum fulvum (Figure 28a).

Such C:N increase in the last two species was explained by a higher C content rather than a lower N content, which was not different between species (Figure 28b). The C:N ratio decreased with depth in G. fascicularis and S. hystrix. Again, these changes were not entirely due to changes in the N content of the coral tissue, which increased in S. hystrix only. The Symbiodiniaceae fraction presented variable C:N ratios (Figure 29a), which were however higher in shallow colonies of R. f. fulvum, due to a lower N content compared to the other corals (Figure 29b). Overall, the C:N ratios increased with depth except in R. f. fulvum, due to a decrease in N content. ) are soft corals. Significant differences between depths are displayed with an asterisk. Significant differences between species are distinguished with letters by depth. AFDW = ash-free dry weight. ns = not sampled.

The assimilation of N per tissue biomass in the host is ca. ten-fold higher in hard compared to soft corals for all investigated N sources (Figure 30). At each depth, scleractinian species had similar assimilation rates of NH4, NO3 and DFAA into host tissue, except G.

fasicularis, which had higher NO3 assimilation compared to the other species at shallow depth (Table S2, Figure 30a). Rates of DFAA assimilation into host tissue were consistently higher at mesophotic compared to shallow depth (Figure 30a and Appendix III -Table S7). The same depth effect was observed on the rates of NO3 assimilation into the host tissue of S. hystrix and S. pistillata. No effect of depth was observed for NH4 assimilation rates. Finally, NH4 was assimilated at higher rates than the other N sources in shallow hard corals, whereas there was no difference in assimilation rates between sources in mesophotic colonies (Appendix III -Table S7). Concerning the soft coral species, and at each depth, NH4 and DFAA assimilation rates were significantly lower in Sarcophyton sp. than in the two other species, while NO3 assimilation rates were higher in R. fulvum fulvum (Figure 30b and Appendix III -Table S7).

There was no difference in the assimilation rates of NH4, NO3 and DFAA between shallow and mesophotic soft corals, except for NO3 assimilation rate in R. fulvum fulvum, which was higher at mesophotic depth (Appendix III -Table S7). Soft corals assimilated NH4 at higher rates at both depths, as compared to NO3 and DFAA (Figure 30b).

Assimilation rates in algal symbionts were also overall higher in hard compared to soft corals, with the following exceptions (Figure 31): symbionts of shallow colonies of L. arboreum and mesophotic colonies of R. f. fulvum had similar NH4 assimilation rates than symbionts of G. fascicularis at the corresponding depths. DFAA assimilation rates were not different between the symbionts of shallow colonies of L. arboreum, S. pistillata and G. fascicularis.

Mesophotic depth decreased the assimilation rates of NH4 and NO3 in Sarcophyton sp, as well as NO3 in R. fulvum fulvum. However, it increased DFAA assimilation in R. fulvum fulvum.

There was no preferential assimilation of a nitrogen source by the symbionts of soft corals.

After exposing shallow soft corals to +5°C during one week (Figure 32), we observed, at 30°C, higher assimilation rates of NH4 in the host tissue and symbionts of R. f. fulvum as well as in the host tissue of L. arboreum. Assimilation rates of NO3 were enhanced in the host tissue and symbionts of R. f. fulvum while assimilation rates of DFAA were higher in both the host tissue and symbionts of R. f. fulvum and Sarcophyton sp. at 30°C. At both 25°C and 30°C, Sarcophyton sp. assimilated NH4 (both host and symbiont fractions) and DFAA (host fraction) at lower rates than the other species. Assimilation rates of NO3 were significantly higher in R.

f. fulvum at the two temperatures (both fractions) and the assimilation of DFAA was higher in ). Significant differences between depths are displayed with an asterisk. Since there was no similar assimilation rates between hard and soft corals, Δ shows the species that differentiate from the other two within its group (i.e. either hard or soft corals, for a single N source). AFDW = ash-free dry weight. DFAA = dissolved free amino acids. ns = not sampled. ). Significant differences between temperatures are displayed with an asterisk. Δ shows the species that differentiate from the other two within its group (i.e. either host or Symbiodiniaceae fraction, for a single N source). AFDW = ash-free dry weight. DFAA = dissolved free amino acids. ns = not sampled.

Discussion

N availability is a major factor determining coral growth and productivity, and its acquisition is thus among one of the major processes affecting coral fitness [START_REF] Rädecker | Nitrogen cycling in corals: the key to understanding holobiont functioning?[END_REF]. Therefore, the capacity of different coral species to efficiently exploit nutrients from the surrounding environment, or compete for nutrients with other species, can explain coral distribution and response to environmental stress. We thus compared the assimilation rates of different forms of N in soft and hard corals, sampled at shallow and mesophotic depths, in order to better understand their ecological strategies and the conditions favouring their growth. This study first highlights a different capacity of soft and hard corals to assimilate dissolved N forms.

The lower assimilation rates per unit biomass observed in soft corals are likely due to divergent nutritional strategies, along with different morphological traits. In addition, we observed, for most but not all species, an increased assimilation of N with increasing depth or seawater temperature.

Differences in N assimilation between soft and hard corals

This study first highlights ten-fold lower rates of DIN and DON assimilation per unit biomass in soft compared to hard corals, whose uptake rates are comparable to previous studies (Figure 30, Figure 31, Figure 33, Appendix III -Figure S2; [START_REF] Grover | Uptake of ammonium by the scleractinian coral Stylophora pistillata: Effect of feeding, light, and ammonium concentrations[END_REF][START_REF] Pernice | A single-cell view of ammonium assimilation in coral-dinoflagellate symbiosis[END_REF][START_REF] Ezzat | Carbon and nitrogen acquisition in shallow and deep holobionts of the scleractinian coral S. pistillata[END_REF]. These lower assimilation rates of DIN/DON in soft corals suggest that they have lower needs in these N sources for tissue growth or alternatively, lower uptake capacities than hard corals, due to differences in Symbiodiniaceae characteristics. Uptake rates of dissolved nutrients have been linked to the Symbiodiniaceae densities in host tissue, or Symbiodiniaceae species, some being less efficient than others in nutrient acquisition (Baker et al. 2013a;[START_REF] Ezzat | Carbon and nitrogen acquisition in shallow and deep holobionts of the scleractinian coral S. pistillata[END_REF]). In our study, however, the genus and density of Symbiodiniaceae were similar between the soft and hard corals investigated and can thus not explain the differences observed in N assimilation rates.

The similar N contents per unit biomass in soft and hard corals (Figure 28) do not support lower N needs in soft corals. Therefore, a different nutritional strategy of the soft and hard corals is the most likely explanation for the lower assimilation of DN by soft corals. Since N2 fixation does not occur in many soft corals (Pupier et al. 2019a), they should likely rely on the heterotrophic feeding on particulate organic matter (POM) to meet their N requirements [START_REF] Fabricius | Depletion of suspended particulate matter over coastal reef communities dominated by zooxanthellate soft corals[END_REF]. Soft corals, and octocorals in general, are known to capture high amounts of phyto-and zooplankton, as well as other forms of POM suspended in the surrounding seawater [START_REF] Sebens | Predation on zooplankton by the benthic anthozoans Alcyonium siderium (Alcyonacea) and Metridium senile (Actiniaria) in the New England subtidal[END_REF]Fabricius et al. 1995aFabricius et al. , b, 1998;;[START_REF] Fabricius | Depletion of suspended particulate matter over coastal reef communities dominated by zooxanthellate soft corals[END_REF][START_REF] Migné | Experimental nutrition in the soft coral Alcyonium digitatum (Cnidaria: Octocorallia): Removal rate of phytoplankton and zooplankton[END_REF]. Ingestion of diatoms has also been observed in soft coral species such as Sinularia flexibilis [START_REF] Piccinetti | Herbivory in the soft coral Sinularia flexibilis (Alcyoniidae)[END_REF].

Several morphological and metabolic characteristics can contribute to higher N assimilation rates in hard corals compared to soft corals. For example, photosynthesis of hard corals living in shallow waters is higher than the one of soft corals (e.g., [START_REF] Fabricius | Widespread mixotrophy in reef-inhabiting soft corals: the influence of depth, and colony expansion and contraction on photosynthesis[END_REF]. As N uptake is proportional to symbiont's photosynthetic activity (e.g., [START_REF] Grover | Uptake of ammonium by the scleractinian coral Stylophora pistillata: Effect of feeding, light, and ammonium concentrations[END_REF], the higher photosynthesis of hard corals can explain their higher rates of N assimilation.

In addition, soft corals are primarily characterized by the absence of a calcium carbonate skeleton and only possess sclerites as calcified structures [START_REF] Fabricius | Soft corals and sea fans: a comprehensive guide to the tropical shallow-water genera of the Central-West Pacific, the Indian Ocean and the Red Sea[END_REF]. In hard corals, the deposition of a hard skeleton (calcification) generates protons that have to be neutralized to avoid tissue acidification [START_REF] Comeau | Coral reef calcifiers buffer their response to ocean acidification using both bicarbonate and carbonate[END_REF]. [START_REF] Crossland | The role of metabolic nitrogen in coral calcification[END_REF] but also [START_REF] Biscéré | Enhancement of coral calcification via the interplay of nickel and urease[END_REF], suggested that ammonia may be involved in proton neutralization, through the ornithine cycle and urea production, therefore possibly promoting the uptake of DIN by the holobiont. Moreover, coral skeletons harbor endolithic communities, which can assimilate nitrogenous compounds and translocate them back to the coral host (see review by [START_REF] Pernice | Down to the bone: the role of overlooked endolithic microbiomes in reef coral health[END_REF]. Finally, soft corals comprise a large coenenchyme mostly constituted of mesoglea, which includes collagen fibers, sclerites and free amoeboid cells [START_REF] Helman | Extracellular matrix production and calcium carbonate precipitation by coral cells in vitro[END_REF][START_REF] Orgel | Molecular and ultrastructural studies of a fibrillar collagen from octocoral (Cnidaria)[END_REF]. Although they have a large gastrovascular system allowing efficient transport of metabolites within the colony [START_REF] Gateño | Gastrovascular circulation in an octocoral: Evidence of significant transport of coral and symbiont cells[END_REF], several experiments on sea anemones suggested that the mesoglea can represent a significant barrier to transepithelial diffusion of molecules (oxygen in Bradfield and Chapman 1983); bicarbonate in [START_REF] Fox | Diffusional permeability of dissolved inorganic carbon through the isolated oral epithelial layers of the sea anemone, Anemonia viridis[END_REF]). The expansion state, together with the low surface area to body volume ratio, usually found for soft corals exhibiting fleshy and massive morphologies, are also not in favor of exchanges through the epidermal tissue [START_REF] Kirschner | Water and ions[END_REF][START_REF] Shick | Energy metabolism and respiratory gas exchange[END_REF][START_REF] Fabricius | Widespread mixotrophy in reef-inhabiting soft corals: the influence of depth, and colony expansion and contraction on photosynthesis[END_REF]. 4.9.2 Effect of the N form, depth and seawater temperature on N assimilation rates

The higher assimilation rates of NH4, over the other N forms, in both hard and soft corals sampled in shallow waters is in agreement with previous studies [START_REF] Grover | Uptake of ammonium by the scleractinian coral Stylophora pistillata: Effect of feeding, light, and ammonium concentrations[END_REF][START_REF] Grover | Nitrate uptake in the scleractinian coral Stylophora pistillata[END_REF][START_REF] Grover | Uptake of dissolved free amino acids by the scleractinian coral Stylophora pistillata[END_REF]. As NH4 assimilation does not involve a redox reaction, it requires less energy than the assimilation of NO3. As NH4 is also a smaller molecule compared to DFAA, and can be absorbed more easily, it is likely the preferred dissolved N source. At mesophotic depths, however, the increased needs in N with depth (see below) caused corals to assimilate all available forms of N in seawater equally.

Both soft and hard corals increased either their chlorophyll content per unit biomass or per symbiont cell, which is a likely a strategy to increase the capture of light energy [START_REF] Kahng | Light, temperature, photosynthesis, heterotrophy, and the lower depth limits of mesophotic coral ecosystems[END_REF] for acclimation to the lower light levels at mesophotic depth. Depth had however a different effect on the assimilation rates of DN and the tissue N content of hard and soft corals.

In hard corals, the overall assimilation rates of N in host tissue (especially DFAA, and NO3), were enhanced at mesophotic depth. This finding is in contrast with previous observations that light stimulates N uptake and assimilation [START_REF] Grover | Uptake of ammonium by the scleractinian coral Stylophora pistillata: Effect of feeding, light, and ammonium concentrations[END_REF]. Such discrepancy may be due to the fact that previous measurements of assimilation rates were normalized to surface area whereas our data is normalized to tissue biomass, which decreases with depth. The higher assimilation of organic N (DFAA) corresponds with a reduced reliance on autotrophy due to reducing light levels with increasing depth in mesophotic hard corals. Consequently, they may rely more on heterotrophy [START_REF] Williams | Biophysical drivers of coral trophic depth zonation[END_REF], as well as obtain more N from diazotrophic bacteria [START_REF] Bednarz | Contrasting seasonal responses in dinitrogen fixation between shallow and deepwater colonies of the model coral Stylophora pistillata in the northern Red Sea[END_REF]. Overall, the enhanced assimilation rates of N at mesophotic depth suggest higher N needs in mesophotic colonies to sustain their metabolism, but this hypothesis remains to be further investigated. In contrast to hard corals, there were consistent assimilation rates of N throughout the depth gradient in soft corals, suggesting that low rates of N supply may be sufficient for all basal requirements of soft corals. Increased temperatures, however, tended to enhance the assimilation of N in both the host tissue and symbiont fractions of soft corals, which is in agreement with a previous study performed on N assimilation rates of S. pistillata at high temperature (29°C) [START_REF] Godinot | Coral uptake of inorganic phosphorus and nitrogen negatively affected by simultaneous changes in temperature and pH[END_REF]. Such increase in N assimilation can be of a mechanistic nature, for example a thermal optimum reached for the enzymes involved in N assimilation. It could also be linked to an increased metabolism at high temperature [START_REF] Gillooly | Effects of size and temperature on metabolic rate[END_REF], but this hypothesis remains to be tested.

Conclusion

This study corroborates the dependency of soft corals on another N source than dissolved compounds to meet their N requirements for tissue growth. This observation suggests that soft corals rely primarily on heterotrophy, especially for species bearing polyps with low surface area to volume ratios, which were shown to have lower photosynthetic rates and rely less on autotrophy than polyps with high surface area to volume ratios [START_REF] Baker | Productivity links morphology, symbiont specificity and bleaching in the evolution of Caribbean octocoral symbioses[END_REF][START_REF] Rossi | Linking host morphology and symbiont performance in octocorals[END_REF]. A higher reliance on heterotrophy can help soft corals to cope with higher turbidity or sedimentation regimes, two conditions, which decrease the amount of light received by corals (decreased autotrophy), but increase the amount of particles in suspension in seawater [START_REF] Mcclanahan | Sedimentation effects on shallow coral communitiesin Kenya[END_REF]. Further, soft corals may benefit from lower DN assimilation rates in areas with low water quality. Indeed, under excess N availability, symbionts can shift from an N limited to a phosphorus-starved state [START_REF] Wiedenmann | Nutrient enrichment can increase the susceptibility of reef corals to bleaching[END_REF], which then causes the substitution of phospholipids with sulpholipids in the chloroplast thylakoid membranes.

Since phospholipids are essential to the stability of the membranes under heat-stress [START_REF] Tchernov | Membrane lipids of symbiotic algae are diagnostic of sensitivity to thermal bleaching in corals[END_REF]), increased N availability may increase the bleaching susceptibility of corals [START_REF] Wiedenmann | Nutrient enrichment can increase the susceptibility of reef corals to bleaching[END_REF]. In addition, it was shown that excess N can reduce photosynthate translocation rates by Symbiodiniaceae [START_REF] Ezzat | New insights into carbon acquisition and exchanges within the coral-dinoflagellate symbiosis under NH4 + and NO3supply[END_REF], leading to the starvation of the coral host. Since soft corals have low assimilation rates of N, it is unlikely that excess N enters the tissue and disrupts the symbiosis. Soft corals have thus been shown to be more abundant than hard corals in reefs, which suffer from high concentrations of dissolved inorganic nutrients [START_REF] Baum | Abundance and physiology of dominant soft corals linked to water quality in Jakarta Bay, Indonesia[END_REF]. Further studies investigating the N and phosphorus budget of soft versus hard corals along a eutrophication gradient are needed to investigate how nutrients may explain the ecological niches of these two coral groups.

Chapter 5 | In situ acquisition of autotrophic and heterotrophic nutrients mixotrophy for all dinoflagellate-bearing species, with a significant input of heterotrophic feeding on crustacean zooplankton at both depths. Although octocorals maintained similar concentrations of autotrophic markers with increasing depth, the scleractinian species exhibited a decrease in autotrophy with depth, in agreement with previous measurements of photosynthetically-fixed carbon acquisition. The increase in heterotrophic capacity with depth was species-specific, likely related to physiological and morphological characteristics. The relatively high level of the heterotrophic marker in all symbiotic species in shallow conditions does not corroborate the common idea that corals rely mostly on autotrophy in shallow waters.

This study thus highlights the importance of heterotrophy across the euphotic-upper mesophotic depth gradient and brings major advances on our understanding of the ecological significance of feeding for reef corals. Furthermore, it allows for a better understanding of the capacity of different octocoral species to exploit nutrient resources and help improving predictions on the impacts of climate and environmental changes on this important reef-dwelling taxon.

Introduction

Mixotrophs are some of the most widespread organisms on Earth [START_REF] Selosse | Mixotrophy in mycorrhizal plants: Extracting carbon from mycorrhizal networks[END_REF][START_REF] Stoecker | Mixotrophy in the marine plankton[END_REF] because their dietary flexibility often allows them to use both inorganic and organic nutrients [START_REF] Berge | Modeling succession of key resourceharvesting traits of mixotrophic plankton[END_REF]). This dual mode of feeding is particularly advantageous in oligotrophic ecosystems with low concentrations of inorganic nutrients and prey availability [START_REF] Stoecker | Mixotrophy in the marine plankton[END_REF]. Mixotrophic species can span from predominantly autotrophic to predominantly heterotrophic [START_REF] Rottberger | Influence of nutrients and light on autotrophic, mixotrophic and heterotrophic freshwater chrysophytes[END_REF], although it is often difficult to disentangle the relative contributions of each feeding modes to the diet. Therefore, the trophic ecology of mixotrophic species is still not fully understood.

Symbiotic corals are some of the most well-known mixotrophic organisms, because they build the framework and form the basis of coral reefs, which are among the most productive and biologically diverse ecosystems in the planet [START_REF] Knowlton | Coral reef biodiversity[END_REF][START_REF] Sheppard | The biology of coral reefs[END_REF].

Corals display a tripartite trophic feeding strategy: (i) photoautotrophy performed by their dinoflagellate endosymbionts (Symbiodiniaceaesee [START_REF] Lajeunesse | Systematic revision of Symbiodiniaceae highlights the antiquity and diversity of coral endosymbionts[END_REF])) which translocate photosynthates to the coral host, (ii) heterotrophy by actively feeding on plankton and suspended particles, and (iii) osmotrophy, which is a form of heterotrophy that is based on the absorption of dissolved organic matter [START_REF] Muscatine | The role of symbiotic algae in carbon and energy flux in reef corals[END_REF][START_REF] Sorokin | Biomass, metabolic rates and feeding of some common reef zoantharians and octocorals[END_REF][START_REF] Al-Moghrabi | Valine uptake by the scleractinian coral Galaxea fascicularis: characterization and effect of light and nutritional status[END_REF][START_REF] Ferrier-Pagès | Effect of natural zooplankton feeding on the tissue and skeletal growth of the scleractinian coral Stylophora pistillata[END_REF][START_REF] Houlbrèque | Heterotrophy in tropical scleractinian corals[END_REF]. Most studies on coral nutritional ecology have focused on the scleractinian (reef-building) coral group. Symbiotic scleractinian corals living in shallow waters largely rely on the symbiontderived autotrophic supply of nutrients for most of their immediate metabolic needs [START_REF] Muscatine | Fate of photosynthetic fixed carbon in light-and shade-adapted colonies of the symbiotic coral Stylophora pistillata[END_REF][START_REF] Tremblay | Carbon translocation from symbiont to host depends on irradiance and food availability in the tropical coral Stylophora pistillata[END_REF]). These corals can however switch to a dominant heterotrophic feeding mode whenever their autotrophic nutrition is impacted. For example, heterotrophy is enhanced during bleaching (loss of the dinoflagellate symbiontssee [START_REF] Glynn | Coral reef bleaching: ecological perspectives[END_REF], or in deep waters where zooplankton availability is higher and light levels are too low for effective photosynthesis to occur [START_REF] Muscatine | The effect of external nutrient resources on the population dynamics of zooxanthellae in a reef coral[END_REF][START_REF] Hughes | Heterotrophic compensation: a possible mechanism for resilience of coral reefs to global warming or a sign of prolonged stress[END_REF][START_REF] Tremblay | Heterotrophy promotes the re-establishment of photosynthate translocation in a symbiotic coral after heat stress[END_REF]. Therefore, heterotrophy is a key factor that contributes to the resistance and resilience of corals to environmental stress. Independently of a stressful situation, a few studies have also suggested that coral heterotrophy can be significant even when corals receive a large nutritional input from their autotrophic symbionts [START_REF] Radice | Evaluating coral trophic strategies using fatty acid composition and indices[END_REF], but this aspect remains to be further investigated. Since nutrition is an important driver of coral population dynamics, further studies are needed to quantify variation in coral nutrition in the field, both for scleractinians and other important but understudied taxa, such as octocorals (soft corals).

Octocorals are important members of shallow and mesophotic coral reef communities [START_REF] Schubert | Symbiotic versus non-symbiotic octocorals: physiological and ecological implications[END_REF][START_REF] Benayahu | Controlling the false discovery rate: a practical and powerful approach to multiple testing[END_REF]) and create complex three-dimensional habitats for other reef species (i.e. marine forestssee [START_REF] Rossi | An overview of the animal forests of the world[END_REF]. Although some symbiotic octocoral species have been reported as mixotrophic in shallow waters [START_REF] Fabricius | Widespread mixotrophy in reef-inhabiting soft corals: the influence of depth, and colony expansion and contraction on photosynthesis[END_REF], two recent studies performed on gorgonian octocorals have concluded that the extent of autotrophy and heterotrophy depends on host morphology (e.g., colony shape and polyp size) and symbiont specificity [START_REF] Baker | Productivity links morphology, symbiont specificity and bleaching in the evolution of Caribbean octocoral symbioses[END_REF][START_REF] Rossi | Linking host morphology and symbiont performance in octocorals[END_REF]. Therefore, octocorals may differ from scleractinians in the extent to which they depend on heterotrophy for nutrition. These differences remain however poorly defined, with a small number of studies dedicated to their trophic ecology (but see [START_REF] Rossi | Trophic ecology of Caribbean octocorals: autotrophic and heterotrophic seasonal trends[END_REF]. With natural and anthropogenic impacts on coral reefs leading to the overall decline in scleractinian coral cover [START_REF] Hoegh-Guldberg | Coral reefs under rapid climate change and ocean acidification[END_REF][START_REF] De'ath | The 27-year decline of coral cover on the Great Barrier Reef and its causes[END_REF][START_REF] Hughes | Global warming transforms coral reef assemblages[END_REF], octocorals may become one of the dominant reef taxa in the next decades [START_REF] Lasker | Resilience of octocoral forests to catastrophic storms[END_REF]. It is therefore urgent to understand the conditions that favor octocoral growth and survivorship. In particular, their nutritional ecology may explain their diversity and abundance in locations where those of scleractinian corals are lower [START_REF] Schubert | Symbiotic versus non-symbiotic octocorals: physiological and ecological implications[END_REF].

Lipids such as fatty acids (FA) have been widely applied in marine food webs as markers of predator-prey interactions. Since FA composition is often specific to particular groups of organisms [START_REF] Volkman | A review of sterol markers for marine and terrigenous organic matter[END_REF][START_REF] Volkman | Fatty acid and lipid composition of 10 species of microalgae used in mariculture[END_REF]) and animals are generally unable to produce all necessary FA [START_REF] Dalsgaard | Fatty acid trophic markers in the pelagic marine environment[END_REF][START_REF] Ruess | Carbon stable isotope fractionation and trophic transfer of fatty acids in fungal based soil food chains[END_REF][START_REF] Budge | Studying trophic ecology in marine ecosystems using fatty acids: a primer on analysis and interpretation[END_REF], their content in coral tissue can give useful information on their autotrophic or heterotrophic origin. While specific FA have been validated as tracers of the photosynthetic contribution of the symbionts, such as stearidonic acid (SDA, 18:43) and docosapentaenoic acid (DPA, 22:53) [START_REF] Papina | Symbiotic zooxanthellae provide the host-coral Montipora digitata with polyunsaturated fatty acids[END_REF][START_REF] Treignier | Effect of light and feeding on the fatty acid and sterol composition of zooxanthellae and host tissue isolated from the scleractinian coral Turbinaria reniformis[END_REF][START_REF] Imbs | Diversity of fatty acid composition of symbiotic dinoflagellates in corals: evidence for the transfer of host PUFAs to the symbionts[END_REF][START_REF] Mies | Production of three symbiosis-related fatty acids by Symbiodinium types in clades A-F associated with marine invertebrate larvae[END_REF][START_REF] Mies | In situ shifts of predominance between autotrophic and heterotrophic feeding in the reef-building coral Mussismilia hispida: an approach using fatty acid trophic markers[END_REF], other FA may be specific to crustacean zooplankton and therefore heterotrophy, such as the cis-gondoic acid (CGA, 20:19) [START_REF] Dalsgaard | Fatty acid trophic markers in the pelagic marine environment[END_REF][START_REF] Dodds | Lipid biomarkers reveal geographical differences in food supply to the cold-water coral Lophelia pertusa (Scleractinia)[END_REF][START_REF] Naumann | Trophic ecology of two cold-water coral species from the Mediterranean Sea revealed by lipid biomarkers and compound-specific isotope analyses[END_REF][START_REF] Mies | In situ shifts of predominance between autotrophic and heterotrophic feeding in the reef-building coral Mussismilia hispida: an approach using fatty acid trophic markers[END_REF].

In this study, we quantified autotrophy-(SDA, DPA) and heterotrophy-specific (CGA) markers to investigate whether symbiotic octocorals of the upper mesophotic reefs (40-50 m)

of the Northern Red Sea rely more on heterotrophy than those living at shallow depths. For this purpose, four octocoral species (Dendronephthya sp., Litophyton arboreum, Rhytisma fulvum fulvum, and Sarcophyton sp.) and one scleractinian coral species (Stylophora pistillata) were sampled. Our results shed light on the nutritional ecology of octocorals, which are one of the less studied coral groups. In addition, because nutrition is an important factor involved in coral resistance and resilience, studies on this subject may contribute to understanding coral reef response to environmental changes.

Materials and methods

Collection and sampling procedures

Laboratory experimental controls

Although SDA, DPA and CGA have been validated as precise and specific trophic markers for corals and other marine taxa [START_REF] Dalsgaard | Fatty acid trophic markers in the pelagic marine environment[END_REF][START_REF] Mies | Production of three symbiosis-related fatty acids by Symbiodinium types in clades A-F associated with marine invertebrate larvae[END_REF][START_REF] Mies | In situ shifts of predominance between autotrophic and heterotrophic feeding in the reef-building coral Mussismilia hispida: an approach using fatty acid trophic markers[END_REF], four controls were performed to further confirm their specificity, especially for octocorals: (i) symbiont cells (isolated from S. pistillata grown in the laboratory), to confirm the presence of SDA and DPA and the absence of CGA; (ii) S. pistillata and Sarcophyton sp. colonies, originating from the Red Sea and grown in the laboratory, maintained in strict autotrophy to confirm presence of SDA and DPA and, most importantly, absence of CGA; (iii) S. pistillata and Sarcophyton sp. colonies fed with zooplankton (to confirm presence of CGA); and (iv) a mixture of zooplankton to confirm the absence of SDA and DPA and the presence of CGA. For this purpose, ten nubbins per species (S. pistillata and Sarcophyton sp.) were kept in 20-L tanks for one month prior to starting the experiment. The tanks received a continuous supply of oligotrophic seawater (< 0.5 µM dissolved inorganic nitrogen and < 0.2 µM inorganic phosphate) at a flow rate of 20 L h -1 . Water was mixed using mini-pumps (404 L h -1 , Newa, Loreggia, Italy). Seawater temperature was kept at 25ºC with the use of submersible resistance heaters (300 W Titanium, Schego, Offenbach, Germany) and light level was maintained at 200 µmol photons m -2 s -1 (12:12) using metal halide lamps (Philips, HPIT 400 W, Distrilamp, Bossée, France). Nubbins were then separated in two groups, one unfed (i.e. strict autotrophy) and the other fed (i.e. mixotrophy). Sarcophyton sp. was fed for two months with a mixture of rotifers and copepods (Ocean Nutrition, San Diego, USA) three days a week, and Artemia salina nauplii (Ocean Nutrition, San Diego, USA) twice a week. The scleractinian S. pistillata was only fed twice a week with A. salina nauplii. At the end of the experiment, nubbins were frozen at -80°C and freeze-dried for subsequent analyses. Each sample was crushed into powder using a mortar and pestle and then divided in two subsamples. A subsample was kept for lipid extraction and gas chromatography analysis, while the other was weighed and combusted at 450°C for 4 h in a muffle furnace (Thermolyne 62700, Thermo Fischer Scientific, USA). Ashfree dry weight (AFDW) was determined as the difference between the dry weight (DW) and ash weight, and extrapolated to the total weight of powder used for lipid extraction (Appendix IV -Table S8).

Symbiont identification

The predominant symbiont phylotype had been previously identified (C. Pupier, unpublished data) for all sampled coral species through gDNA extraction and standard PCR and sequencing protocols [START_REF] Sambrook | Molecular cloning: a laboratory manual[END_REF]. The primer set (taken from [START_REF] Santos | Molecular phylogeny of symbiotic dinoflagellates inferred from partial chloroplast large subunit (23S)-rDNA sequences[END_REF] used for PCR amplification targeted amplicons for the chloroplast large subunit (cp23S-rDNA), a marker that is widely used for assessments of Symbiodiniaceae diversity [START_REF] Santos | Molecular phylogeny of symbiotic dinoflagellates inferred from partial chloroplast large subunit (23S)-rDNA sequences[END_REF][START_REF] Lajeunesse | Systematic revision of Symbiodiniaceae highlights the antiquity and diversity of coral endosymbionts[END_REF]).

Lipid extraction and gas chromatography analysis

Lipid extraction was performed in similar fashion to [START_REF] Bligh | Diffusion of oxygen through the mesogloea of the sea anemone Calliactis parasitica[END_REF], with minor modifications. One milliliter of a solution of methanol and dichloromethane (1:1) was added to each sample tube together with 125 µL of the internal standard (C13:0 triacylglyceride, T3882, Sigma) solution (5 mg mL -1 in hexane). Thereafter, the tubes were agitated for 1 min and centrifuged at 10,000 X g (-4 o C). The solvent was dried in pure N2 gas, then the samples were methylated by dissolving in 500 µL of 5% HCl in methanol, before heating to 100ºC for 2 h.

Fatty acid methyl esters were then extracted with hexane.

Gas chromatography procedures were nearly identical to [START_REF] Martins | Biotechnological potential of benthic marine algae collected along the Brazilian coast[END_REF]. Fatty acid methyl esters were analysed by gas chromatography coupled with mass spectrometry (QP2010, Shimadzu, Japan). A 30-m fused silica capillary column (VF-Max, 0.25 µm film, Agilent) was utilized. One microliter of each sample was injected at 220ºC in split mode. The carrier gas was helium, at a flow rate of 1 mL min -1 . Initial temperature was 60ºC with an increase of 5ºC per minute until it reached 260ºC, which was then maintained for 10 min. The fatty acid methyl esters of the three polyunsaturated fatty acids, including two autotrophy markers (SDA and DPA) and one heterotrophy marker (CGA), were identified by comparison of their retention times against those in the certified reference standard 47033 PUFA No. 1

Marine Source (Sigma).

Statistical analyses

The following analysis was performed using the R environment for statistical computing and graphics (R Core Team 2017). Generalized linear models (GLM) were fitted to test for differences in SDA, DPA and CGA concentrations between species, depths, and the interaction of these factors. Data were transformed using the Box-Cox transformation procedure ((x λ-1 )/λ, with λ = -0.060606 for SDA, λ = -0.343434 for DPA, and λ = 0.101010 for CGA), as implemented in [START_REF] Venables | Photosynthetic symbioses in animals[END_REF] to comply with assumptions of normality of residuals (tested for with the Shapiro-Wilk test) and homoscedasticity (tested for with Levene's test; [START_REF] Fox | Diffusional permeability of dissolved inorganic carbon through the isolated oral epithelial layers of the sea anemone, Anemonia viridis[END_REF]. Biologically relevant pairwise comparisons (i.e., between depths for each species and between species for each depth) were made for species with detectable fatty acid concentrations based on estimated marginal means [START_REF] Lenth | emmeans: Estimated Marginal Means, aka least-squares means[END_REF]) and simultaneous general linear hypothesis testing (glht() function of the multcomp package; [START_REF] Hothorn | Simultaneous inference in general parametric models[END_REF]. P-values were adjusted using the Benjamini-Hochberg procedure (Benjamini and Hochberg 1995).

To address multivariate differences in markers' concentration between the five species and the two depth strata we used a non-metric multidimensional scaling (nMDS) ordination to summarize similarities (Bray-Curtis) on SDA, DPA and CGA content. The Spearman nonparametric correlation coefficient, based on ranks, was used to investigate the relationship between variables and the MDS axes, with results illustrated as superimposed vectors in the MDS diagram. In addition, a 2-way crossed analysis of similarities (ANOSIM) was used to evaluate significant differences according to species and depth levels. The MDS and ANOSIM analyses were performed using the PRIMER 6 software [START_REF] Clarke | Primer v6.: user manual/tutorial[END_REF].

Results

The identities of Symbiodiniaceae phylotypes associated with the four symbiotic species are summarized in Table 8. While the three octocorals were associated with the same symbiont phylotype in both shallow and mesophotic depths (Symbiodinium sp. A10 or Cladocopium goreaui), S. pistillata displayed a different association between depths (Symbiodinium microadriaticum in the shallow and Cladocopium goreaui at mesophotic depths). The first laboratory experimental control confirmed the presence of both SDA and DPA and the absence of CGA in the symbiont cells (Figure 35). The exact opposite was detected for the plankton mixture, with presence of CGA and absence of SDA and DPA. Colonies of Sarcophyton sp. kept in strict autotrophy also displayed higher concentration of the autotrophic markers and absence of the heterotrophy marker. The same was observed for S. pistillata, although with trace amounts of CGA. The last controls, S. pistillata and Sarcophyton sp.

colonies fed with the plankton mix, displayed similar SDA and DPA content to those unfed, but also displayed up to 20 times more CGA. In addition, in situ nubbins of the non-symbiotic octocoral Dendronephthya sp. contained CGA, while SDA was found in trace concentrations. arboreum) and, in the other side, species showing lower CGA and SDA content and higher DPA content (e.g. Sarcophyton sp.) (Figure 36). Figure 37 shows that all corals sampled in situ contained the three markers at different concentrations. Pairwise comparisons highlighted significant differences between depths in SDA for L. arboreum (decrease with depth), in DPA for S. pistillata (decrease with depth) and in CGA for Dendronephthya sp. and R. f. fulvum (increase with depth) (Appendix IV -Table S9).

Discussion

This study is one of the first to evaluate the shift in autotrophic/heterotrophic nutrition along the depth gradient for several species of octocorals sampled in the Northern Red Sea and to compare their nutritional ecology with the most abundant scleractinian coral species (Stylophora pistillata) of this oligotrophic area. Octocorals are indeed one of the most abundant groups of organisms in the upper mesophotic zone of the Red Sea reefs, often displaying higher species richness than in shallow reefs [START_REF] Shoham | Higher species richness of octocorals in the upper mesophotic zone in Eilat (Gulf of Aqaba) compared to shallower reef zones[END_REF][START_REF] Eyal | The Red Sea: Israel[END_REF]. A better knowledge of their trophic ecology along the depth gradient is therefore of prime importance to understand their distribution and growth in nutrient-poor conditions. Our findings highlight several species-specific aspects of the nutritional ecology of octocorals. The analysis of specific fatty acid markers shows that soft corals did not reduce their autotrophic capacity in mesophotic reefs, and that both symbiotic and asymbiotic species can display an increased heterotrophy with increasing depth. The symbiotic reef-building coral, on the contrary, showed significant decrease in autotrophic activity with depth. However, it displayed a high heterotrophic capacity throughout the depth gradient, and even at very shallow depths.

The fatty acids SDA, DPA and CGA had been previously validated as specific markers of feeding strategies for reef corals [START_REF] Mies | Production of three symbiosis-related fatty acids by Symbiodinium types in clades A-F associated with marine invertebrate larvae[END_REF][START_REF] Mies | In situ shifts of predominance between autotrophic and heterotrophic feeding in the reef-building coral Mussismilia hispida: an approach using fatty acid trophic markers[END_REF][START_REF] Marangoni Lf De | Peroxynitrite generation and increased heterotrophic capacity are linked to the disruption of the coral-dinoflagellate symbiosis in a scleractinian and hydrocoral species[END_REF]). The additional experimental controls produced here all confirmed their specificity. Although nearly undetectable amounts of CGA were found in S. pistillata maintained in strict autotrophy, such residual trace concentrations are not unusual for corals that have been kept under starvation for short periods [START_REF] Mies | In situ shifts of predominance between autotrophic and heterotrophic feeding in the reef-building coral Mussismilia hispida: an approach using fatty acid trophic markers[END_REF]. Therefore, our controls agree with previous experiments and validations. It is important to note that CGA concentration reflects predation on crustacean zooplankton only; therefore, although copepods may comprise the most abundant and ecologically relevant group of zooplankton in coral reef systems [START_REF] Hamner | Copepod swarms: attributes and role in coral reef ecosystems[END_REF], uptake of other dietary items such as salps is not detected. Thus, CGA is a valid proxy, but likely an underestimation of the total heterotrophic capacity [START_REF] Mies | In situ shifts of predominance between autotrophic and heterotrophic feeding in the reef-building coral Mussismilia hispida: an approach using fatty acid trophic markers[END_REF].

Field analyses show that R. f. fulvum is the species with the highest concentration of autotrophy markers at both shallow and deep sites, likely being the most autotrophic of all octocorals investigated. Although there may be some correlation between the concentration of autotrophic markers and symbiont density in host tissue, it is often weak because of variations in fatty acid production, storage and translocation by Symbiodiniaceae [START_REF] Zhukova | Fatty acid variations in symbiotic dinoflagellates from Okinawan corals[END_REF][START_REF] Imbs | Diversity of fatty acid composition of symbiotic dinoflagellates in corals: evidence for the transfer of host PUFAs to the symbionts[END_REF][START_REF] Mies | Production of three symbiosis-related fatty acids by Symbiodinium types in clades A-F associated with marine invertebrate larvae[END_REF][START_REF] Mies | In situ shifts of predominance between autotrophic and heterotrophic feeding in the reef-building coral Mussismilia hispida: an approach using fatty acid trophic markers[END_REF], as well as environmental influence on these processes [START_REF] Kneeland | Lipid biomarkers in Symbiodinium dinoflagellates: new indicators of thermal stress[END_REF]. Further evidence for this is that the highest symbiont density for the five coral species investigated was observed in L. arboreum (Pupier et al. 2019b), which did not have the highest concentration of autotrophy markers. Symbiont genotypic diversity can also affect the quantity of translocated material to the host [START_REF] Loram | Functional significance of genetically different symbiotic algae Symbiodinium in a coral reef symbiosis[END_REF][START_REF] Starzak | The influence of symbiont type on photosynthetic carbon flux in a model cnidarian-dinoflagellate symbiosis[END_REF], although no clear relationship between CGA levels and symbiont identity could be observed in the present study. The concentration of autotrophy markers did not significantly decrease with depth for any of the octocoral species investigated, in agreement with a recent finding that carbon acquisition in octocorals is constant from shallow to at least 40 m depth (Pupier et al. 2019b). For S. pistillata, however, autotrophic markers were found in higher concentration in the shallow colonies, which is also in agreement with findings that photosynthesis and carbon acquisition in this species decrease along the depth gradient [START_REF] Mass | Photoacclimation of Stylophora pistillata to light extremes: metabolism and calcification[END_REF][START_REF] Einbinder | Novel adaptive photosynthetic characteristics of mesophotic symbiotic microalgae within the reefbuilding coral, Stylophora pistillata[END_REF][START_REF] Ezzat | Carbon and nitrogen acquisition in shallow and deep holobionts of the scleractinian coral S. pistillata[END_REF]. Altogether, these results suggest that octocorals may have a greater ability to transform light in energy at deep reefs, which might explain their relatively high abundance/diversity at mesophotic depths.

The bathymetric stability of the symbiont-host association might explain the constant level of autotrophic markers in octocoral species. Symbiodiniaceae is a functionally-diverse group, with species displaying differences in adaptation to light intensity, thermal stress, and many other environmental conditions [START_REF] Baker | Flexibility and specificity in coral-algal symbiosis: diversity, ecology, and biogeography of Symbiodinium[END_REF][START_REF] Robison | Differential impacts of photoacclimation and thermal stress on the photobiology of four different phylotypes of Symbiodinium (Pyrrhophyta)[END_REF]Stat et al. 2008;[START_REF] Swain | Consensus thermotolerance ranking for 110 Symbiodinium phylotypes: an exemplar utilization of a novel iterative partial-rank aggregation tool with broad application potential[END_REF]. All octocoral species investigated here were associated with the same symbiont phylotype at shallow and mesophotic depths, as often observed for octocorals [START_REF] Van Oppen | Diversity of algal endosymbionts (zooxanthellae) in octocorals: the roles of geography and host relationships[END_REF][START_REF] Goulet | Most scleractinian corals and octocorals host a single symbiotic zooxanthella clade[END_REF]). However, our findings show that S. pistillata shifts from Symbiodinium microadriaticum to Cladocopium goreaui at mesophotic depths. Symbiodinium microadriaticum has been reported to assimilate less autotrophic carbon and have a lower capacity of photosynthate translocation than C. goreaui (Stat et al. 2008;[START_REF] Ezzat | Carbon and nitrogen acquisition in shallow and deep holobionts of the scleractinian coral S. pistillata[END_REF],

indicating that plasticity in symbiont association may explain between-species differences in bathymetric distribution.

Photosynthetic products are carbon-rich but may be deficient in other critical elements for growth including nitrogen and phosphorus [START_REF] Battey | Glycerol translocation in Condylactis gigantea[END_REF][START_REF] Titlyanov | Photo-acclimation dynamics of the coral Stylophora pistillata to low and extremely low light[END_REF][START_REF] Palardy | Effect of naturally changing zooplankton concentrations on feeding rates of two coral species in the Eastern Pacific[END_REF]. Therefore, heterotrophic feeding is critical for fulfilling such deficiency [START_REF] Al-Moghrabi | Valine uptake by the scleractinian coral Galaxea fascicularis: characterization and effect of light and nutritional status[END_REF][START_REF] Anthony | Shifting roles of heterotrophy and autotrophy in coral energetics under varying turbidity[END_REF][START_REF] Ferrier-Pagès | Effect of natural zooplankton feeding on the tissue and skeletal growth of the scleractinian coral Stylophora pistillata[END_REF][START_REF] Houlbrèque | Heterotrophy in tropical scleractinian corals[END_REF]. Most octocorals are known to ingest organic matter, bacteria, phytoplankton and zooplankton (Fabricius et al. 1995a[START_REF] Fabricius | In situ depletion of phytoplankton by an azooxanthellate soft coral[END_REF][START_REF] Widdig | Phytoplankton: a significant trophic source for soft corals?[END_REF][START_REF] Ribes | Natural feeding of the temperate asymbiotic octocoralgorgonian Leptogorgia sarmentosa (Cnidaria: Octocorallia)[END_REF][START_REF] Coma | Natural heterotrophic feeding by a temperate octocoral with symbiotic zooxanthellae: a contribution to understanding the mechanisms of die-off events[END_REF][START_REF] Piccinetti | Herbivory in the soft coral Sinularia flexibilis (Alcyoniidae)[END_REF]. Our findings show that the heterotrophic input in Red Sea octocorals is species-and depth-specific. Rhytisma f. fulvum presented the highest CGA content at both shallow and mesophotic depths, suggesting that it is also the most effective heterotrophic species, out of the four ones evaluated here. CGA values for R. f. fulvum were even higher than for Dendronephthya sp., an asymbiotic and obligate heterotroph. Combined with its high autotrophic capacity, this might explain why R. f. fulvum has high resilience, efficiently colonizing reefs and forming large carpets after major environmental disturbances [START_REF] Benayahu | Space partitioning by stony corals soft corals and benthic algae on the coral reefs of the northern Gulf of Eilat (Red Sea)[END_REF][START_REF] Buckley | Report on the 2005 Aldabra Marine Programme Research[END_REF][START_REF] Buckley | Was the 1998 coral bleaching in the Southern Seychelles a catastrophic disturbance? 1999-2006 Reef fish responses to coral substrate changes[END_REF][START_REF] Stobart | Coral recovery at Aldabra Atoll, Seychelles: Five years after the 1998 bleaching event[END_REF].

The scleractinian S. pistillata showed a relatively high degree of heterotrophy (i.e high CGA content) even at shallow depth. This is contrary to the common belief that shallow scleractinian corals rely almost exclusively on autotrophy [START_REF] Muscatine | The effect of external nutrient resources on the population dynamics of zooxanthellae in a reef coral[END_REF][START_REF] Muscatine | Resource partitioning by reef corals as determined from stable isotope composition -II. δ 15 N of zooxanthellae and animal tissue versus depth[END_REF][START_REF] Lesser | Photoacclimatization by the coral Montastraea cavernosa in the mesophotic zone: light, food, and genetics[END_REF]. In fact, our findings suggest that heterotrophy may not compensate for a lack of autotrophy, but rather complement it. A more profound investigation of CGA content in other scleractinian coral species should confirm whether this heterotrophic capacity is restricted to S. pistillata, to Eilat reefs, or a general feature of shallow scleractinian corals. It is important to note that there is a strong diel pattern of increased zooplankton biomass at dusk and dawn in the Gulf of Aqaba/Eilat, often ten-fold higher during the night than during the day [START_REF] Yahel | Diel pattern with abrupt crepuscular changes of zooplankton over a coral reef[END_REF]). In addition, the region is characterized by convective mixing of approximately 300 m in autumn and winter (when our collections were performed) [START_REF] Labiosa | The interplay between upwelling and deep convective mixing in determining the seasonal phytoplankton dynamics in the Gulf of Aqaba: Evidence from SeaWiFS and MODIS[END_REF], which may have enhanced coral heterotrophy in shallow waters [START_REF] Gove | Temporal variability of current-driven upwelling at Jarvis Island[END_REF][START_REF] Roder | Trophic response of corals to large amplitude internal waves[END_REF][START_REF] Sevadjian | Shoreward advection of phytoplankton and vertical re-distribution of zooplankton by episodic near-bottom water pulses on an insular shelf: Oahu, Hawaii[END_REF][START_REF] Williams | Biophysical drivers of coral trophic depth zonation[END_REF]). If most coral species are as heterotrophic as S. pistillata in shallow areas, zooplankton abundance should then be considered as a major driver of scleractinian abundance and health and further explorations on this topic are warranted.

A bathymetric trophic zonation was evident for R. f. fulvum and Dendronephthya sp., which displayed CGA increase at mesophotic depths. This pattern may be explained by an increase in predation pressure and/or increased zooplankton availability (reviewed in [START_REF] Houlbrèque | Heterotrophy in tropical scleractinian corals[END_REF]. However, CGA content for L. arboreum and Sarcophyton sp. did not differ between shallow and deep sites. This, together with the lower CGA content in these two species, suggest that they either have a limited prey capture ability or rely on other resources such as bacteria and/or dissolved organic matter. This agrees with previous reports that species within Litophyton and Sarcophyton genera are poor zooplankton-feeders and rely more on bacterioplankton [START_REF] Sorokin | Biomass, metabolic rates and feeding of some common reef zoantharians and octocorals[END_REF]. Nonetheless, our results reinforce that depth is a critical factor in shaping Red Sea octocoral communities also because species diversity increases significantly at 20-60 m [START_REF] Shoham | Higher species richness of octocorals in the upper mesophotic zone in Eilat (Gulf of Aqaba) compared to shallower reef zones[END_REF][START_REF] Eyal | The Red Sea: Israel[END_REF]).

The species-specificity in heterotrophic capacity for the five coral species studied here can be related to the identity and/or functioning of the symbionts hosted by each species, as it has been demonstrated that heterotrophy was positively correlated with the amount of translocated carbon [START_REF] Leal | Symbiont type influences trophic plasticity of a model cnidarian-dinoflagellate symbiosis[END_REF]. This finding matches with the observation that the most autotrophic species (Rhytisma f. fulvum) is also the most heterotrophic one. Alternatively, heterotrophy can be associated with the morphophysiological characteristics of the species. Increased heterotrophy and predation capacities have been linked to polyp and/or corallite size [START_REF] Anthony | Shifting roles of heterotrophy and autotrophy in coral energetics under varying turbidity[END_REF][START_REF] Marangoni Lf De | Peroxynitrite generation and increased heterotrophic capacity are linked to the disruption of the coral-dinoflagellate symbiosis in a scleractinian and hydrocoral species[END_REF]. Rhytisma f. fulvum, a species that presented high CGA content, has a polyp diameter similar to Sarcophyton sp. (2.4-4 mm; [START_REF] Lewis | Feeding behaviour and feeding ecology of the Octocorallia (Coelenterata: Anthozoa)[END_REF][START_REF] Sorokin | Biomass, metabolic rates and feeding of some common reef zoantharians and octocorals[END_REF], while L. arboreum, a low-CGA species, features polyps almost ten times smaller [START_REF] Ofwegen | The genus Litophyton Forskål, 1775 (Octocorallia, Alcyonacea, Nephtheidae) in the Red Sea and the western Indian Ocean[END_REF]. Species-specificity is also evident for the autotrophic markers. Rhytisma f. fulvum presented unusually high SDA and absence of DPA, which, as of now, is unique for all symbiotic octocorals/scleractinians investigated [START_REF] Mies | In situ shifts of predominance between autotrophic and heterotrophic feeding in the reef-building coral Mussismilia hispida: an approach using fatty acid trophic markers[END_REF][START_REF] Marangoni Lf De | Peroxynitrite generation and increased heterotrophic capacity are linked to the disruption of the coral-dinoflagellate symbiosis in a scleractinian and hydrocoral species[END_REF]. It is unclear whether the host, symbiont or the environment, controls the concentration of specific autotrophy markers, but R. f. fulvum has been reported as a species with unusual biochemical composition and processes [START_REF] Trifman | Bisdioxycalamenene: a bis-sesquiterpene from the soft coral Rhytisma fulvum fulvum[END_REF]. Therefore, the cause for variations in trophic behavior is likely a multifactorial interaction between holobiont physiology and environmental conditions.

This study shows that Red Sea octocorals are functional mixotrophs in both shallow and mesophotic reefs. In addition, our findings show that heterotrophy becomes even more relevant at mesophotic reefs, and without a reduction in autotrophic input. This later finding was unexpected, as soft corals display a different pattern than the one known for scleractinians. This consistent autotrophic capacity may have major implications for explaining differences in bathymetric distribution/dominance of species across depth gradients. In addition, it suggests that soft corals may find more efficient thermal-stress refugia in deep reefs than scleractinians, and may become the dominant group in some coral reefs in the future. This study helps understanding coral functional and trophic ecology, especially in mesophotic reefs, which are conservation-dependent areas [START_REF] Rocha | Mesophotic coral ecosystems are threatened and ecologically distinct from shallow water reefs[END_REF][START_REF] Eyal | The Red Sea: Israel[END_REF].

Chapter 6 | General discussion and perspectives Chapter 6 | General discussion and perspectives

General discussion

The main objective of this thesis was to provide insights into the nutritional ecology of several symbiotic octocoral species from the Northern Red Sea, down to mesophotic depths.

The dinoflagellate-octocoral symbiosis is known to be different from the one of scleractinian corals, in terms of diversity and stability. While octocorals exhibit low symbiont diversity, scleractinian corals generally host multiple symbiont types and are more flexible depending on the prevalent environmental conditions [START_REF] Baker | Multiple symbiotic partnerships are common in scleractinian corals, but not in octocorals: Comment on Goulet (2006)[END_REF]. This difference has been attributed to octocorals having a greater dependence on heterotrophy and therefore having less to gain from retaining flexibility as a symbiotic trait [START_REF] Van Oppen | Diversity of algal endosymbionts (zooxanthellae) in octocorals: the roles of geography and host relationships[END_REF]. However, the importance of autotrophy and heterotrophy and their respective contribution to the host's energy budget has been scarcely studied in octocorals, and mainly tested in gorgonians [START_REF] Rossi | Trophic ecology of Caribbean octocorals: autotrophic and heterotrophic seasonal trends[END_REF]. Advancing knowledge on the nutritional ecology of soft coral symbioses is though of increasing importance, considering that soft corals are one of the main components of reef ecosystems and that climate change and local disturbances are predicted to increasingly induce transitions from hard coral-to octocoral-dominated reefscapes (e.g., [START_REF] Tsounis | Three decades of coral reef community dynamics in St. John, USVI: A contrast of scleractinians and octocorals[END_REF].

Therefore, in this thesis, we investigated the acquisition and fluxes of organic and inorganic sources of nutrients, relative to carbon and nitrogen, in several soft coral hosts, their dinoflagellates symbionts, and other possibly associated microorganisms such as diazotrophs.

Comparisons with scleractinian-dinoflagellate symbioses highlighted major functional differences between the two groups and allowed the discussion of our results in a broader context.

6.1.1 Soft and scleractinian corals form different functional symbioses from the shallow down to the upper mesophotic reef zone  Autotrophy remains stable along the depth gradient in soft corals whereas it decreases in scleractinian corals

The autotrophic acquisition of carbon by the coral symbionts was assessed in this thesis in three different soft coral species via two different but complementary approaches. The first one (Chapter 3) consisted in measuring the acquisition and assimilation of dissolved inorganic carbon into the coral host tissue and symbionts, using a 13 C-bicarbonate tracer. The second technique consisted in measuring the concentration of two fatty acids (stearidonic and docosapentaenoic acids) specific to autotrophy (Chapter 5) in the coral tissue. A comparison was made with the scleractinian coral Stylophora pistillata, using the same techniques.

The two approaches showed, and for all soft coral species investigated, that the short and long-term assimilation of carbon in the coral host tissue was stable along the depth gradient or even increased at mesophotic depths. The stability in autotrophic nutrient supply along the depth gradient suggests that soft corals are either photo-limited in shallow waters and/or well photo-acclimatized to depth. This is in agreement with previous observations that maximal photosynthetic rates of several soft coral species from the Great Barrier Reef were measured at 20 m depth rather than in shallower waters [START_REF] Fabricius | Widespread mixotrophy in reef-inhabiting soft corals: the influence of depth, and colony expansion and contraction on photosynthesis[END_REF]. Strategies of acclimatization to low light levels were noticed in the symbionts (i.e., reduced self-shading and increased light harvesting capacity of the symbiont cells; [START_REF] Cohen | Long term photoacclimation responses of the coral Stylophora pistillata to reciprocal deep to shallow transplantation: photosynthesis and calcification[END_REF][START_REF] Ziegler | Mesophotic coral depth acclimatization is a function of host-specific symbiont physiology[END_REF][START_REF] Einbinder | Novel adaptive photosynthetic characteristics of mesophotic symbiotic microalgae within the reefbuilding coral, Stylophora pistillata[END_REF]). However, the autotrophic stability was independent of the Symbiodiniaceae genus harbored by the soft coral host, since the associations were speciesspecific and are prone to be stable over space and time [START_REF] Goulet | Stability of an octocoral-algal symbiosis over time and space[END_REF]. On the contrary, the hard coral S. pistillata experienced a two-fold decrease in autotrophic carbon acquisition with depth and shifted from Symbiodinium (previously clade A) to Cladocopium (previously clade C) from shallow to deep reefs [START_REF] Mass | Photoacclimation of Stylophora pistillata to light extremes: metabolism and calcification[END_REF][START_REF] Ezzat | Carbon and nitrogen acquisition in shallow and deep holobionts of the scleractinian coral S. pistillata[END_REF].

The comparison of the carbon budget in soft and scleractinian corals indicates that the rates of autotrophic carbon acquisition is at least two-fold higher in S. pistillata at shallow depth, as compared to the soft coral species investigated. However, the carbon acquired was also lost (as respiration and mucus production) twice faster in scleractinian corals, suggesting a different functioning of the symbioses and a different metabolic demand between soft and hard coral species. The higher metabolic demand in scleractinian corals is likely due to calcification, since most of the carbon respired is used for calcium carbonate deposition. Indeed, almost 70-75% of the carbon used for calcification in hard corals comes from respiration, and the remaining 25-30% comes from external seawater carbon [START_REF] Erez | Vital effect on stable-isotope composition seen in foraminifera and coral skeletons[END_REF][START_REF] Furla | Sources and mechanisms of inorganic carbon transport for coral calcification and photosynthesis[END_REF][START_REF] Hughes | Acquisition and assimilation of carbon in non-bleached and bleached corals[END_REF]. Moreover, the presence of skeleton enhances coral productivity through internal light scattering, which may also contribute to explain the differences in carbon acquisition between soft and hard corals.

 Heterotrophy is important at all depths and increases in the mesophotic reef for several species

Another main finding of this thesis points out that symbiotic soft corals from both shallow and mesophotic depths are most likely to depend more on heterotrophy than autotrophy for nutrient acquisition, as compared to hard corals. This finding is supported by the large amount of lipid biomarkers, specific to crustacean zooplankton, measured in the soft coral tissue from shallow and mesophotic reefs (Chapter 5). For some species, the amount of biomarker significantly increased with depth, suggesting that this type of nutrition is favored in the mesophotic reef. In addition, we found in Chapter 4 that soft corals have a low assimilation of dissolved inorganic or organic nitrogen (DIN and DON) and therefore need to acquire nitrogen from particulate feeding.

Soft and scleractinian corals form different functional symbioses

with their dinoflagellates, which might explain the greater resistance of soft corals to eutrophication

We observed that soft corals have a lower capacity than scleractinian corals to assimilate inorganic and organic nitrogen dissolved in seawater. As low amounts of inorganic nutrients enter the soft coral-dinoflagellate symbiosis, it is unlikely that changes in water quality related to nutrients (e.g., eutrophication) disrupt the symbiosis.

First of all, we showed that soft coral mucus likely represents an unfavorable habitat for the colonization and activity of diazotrophs, as it is known to present antimicrobial properties and contains particles with a relatively high N content [START_REF] Kelman | Antimicrobial activity of Red Sea corals[END_REF][START_REF] Nakajima | Release of dissolved and particulate organic matter by the soft coral Lobophytum and subsequent microbial degradation[END_REF]). As a result, soft corals neither promoted N2 fixation in their surrounding seawater nor assimilated DDN. Moreover, up to ten-fold lower assimilation rates of dissolved N (NH4, NO3, DFAA) were observed in soft corals as compared to hard corals, with no clear difference between depths. This could not be explained by i) a different capacity for nutrient acquisition related to the dinoflagellate symbionts (Baker et al. 2013a;[START_REF] Ezzat | Carbon and nitrogen acquisition in shallow and deep holobionts of the scleractinian coral S. pistillata[END_REF], since both groups shared the same symbiont genotypes and/or density or ii) a different need in structural N, since soft and hard corals exhibited similar N tissue content. Soft corals therefore likely rely on heterotrophic feeding to meet their N requirements.

Overall, due to the poor capacity of soft coral symbionts to acquire dissolved nutrients, it is unlikely that changes in water quality related to nutrients (e.g., eutrophication) disrupt the soft coral-dinoflagellate symbiosis. Although this finding has not been experimentally tested in this thesis and remains hypothetical, it could be mechanistically supported by the conceptual model on unfavorable ratios of dissolved inorganic nutrients, introduced by Wiedenmann et al. Therefore, since less dissolved nitrogen enters the tissue of soft corals, the scenario described above may be restricted to hard corals.

As a matter of fact, the relative dominance of soft corals has been reported in areas affected by high concentrations of dissolved and particulate matter [START_REF] Mcclanahan | Sedimentation effects on shallow coral communitiesin Kenya[END_REF][START_REF] Fabricius | Depletion of suspended particulate matter over coastal reef communities dominated by zooxanthellate soft corals[END_REF][START_REF] Schleyer | Coral dominance at the reef-sediment interface in marginal coral communities at Sodwana Bay, South Africa[END_REF][START_REF] Fabricius | Effects of terrestrial runoff on the ecology of corals and coral reefs: Review and synthesis[END_REF][START_REF] Baum | Abundance and physiology of dominant soft corals linked to water quality in Jakarta Bay, Indonesia[END_REF]). In addition, the capacity of soft corals to thrive under environmental conditions ranging from turbid to clear-water [START_REF] Fabricius | Depletion of suspended particulate matter over coastal reef communities dominated by zooxanthellate soft corals[END_REF][START_REF] Fabricius | Photosynthetic symbionts and energy supply determine octocoral biodiversity in coral reefs[END_REF] may also be attributed to their nutritional flexibility. For example, under high turbidity or sedimentation regimes, a lower reliance on the symbiosis could help soft corals to cope with reduced light availability, while suspended particles can represent a potential source of nutrients for heterotrophic feeding.

Graphical summary

The main results of this thesis are summarized in Figure 38. The stability of the octocoral symbiosis along the depth gradient is ensured by a stable association (one symbiont genus), and the maximization of nutrient acquisition and conservation at the two depths by both autotrophy and heterotrophy. This is supported by a low assimilation of DIN and DON, a stable acquisition of DIC (per AFDW and per µg chlorophyll) a large amount of POM assimilated at both depths and no DDN assimilation nor promotion of N2 fixation in the surrounding seawater. Moreover, the same amounts of carbon acquired through photosynthesis were translocated from the symbionts to the host at the two depths. In comparison to soft corals, scleractinian corals exhibited a higher assimilation of DIN and DON (with increased rates of DON assimilated with depth), an acquisition of DIC higher in the shallow but similar (per µg chlorophyll), perhaps lower (per AFDW), one at mesophotic depths, significant assimilation of DDN and promotion of N2 fixation in the surrounding seawater (although not investigated in the deeper corals). They also had a higher carbon loss in the shallow but lower carbon loss than soft corals at the mesophotic depth and symbionts transferred much lower amounts of carbon with depth. The presence of a calcium carbonate skeleton in hard corals likely plays an important role in the differences of metabolism (i.e., energy requirements) and productivity highlighted here.

Finally, species-specific differences in nutrient acquisition and allocation may be due to i) the ratio of colony surface area to volume (a low ratio do not favor light exposure and gas or nutrient exchange through epidermal tissue), ii) micro-morphological features of the polyps (thin branches, small polyps, and high polyp surface area to volume ratio being more suited for autotrophy; big polyps for heterotrophy), iii) the symbiont species hosted (influencing cellspecific carbon assimilation and translocation), iv) the symbiont density (influencing cellspecific carbon assimilation and translocation), and v) the nutritional status (higher heterotrophic efficiency being correlated with higher rates of translocated carbon).

Figure 38. Contrast between the functioning of soft coral versus hard coral nutritional symbioses from shallow to mesophotic reef zones in Eilat (Red Sea). The range of rates corresponding to nutrient acquisition and fluxes is indicated below the nutrient acronym. These rates are not comparable between each other but between groups (soft vs hard corals) and depths (shallow vs mesophotic) only. Chl = Chlorophyll concentration (µg Chl (a + c2) g -1 AFDW). Pg = Autotrophic carbon acquisition (µg C µg -1 Chl). Ts = Carbon translocated from the symbionts to the host (µg C g -1 AFDW h -1 ). AFDW = ash-free dry weight. DIN = Dissolved inorganic nitrogen (including NH4 and NO3, in µg N g -1 AFDW h -1 ). DON = Dissolved organic nitrogen (including DFAA, in µg N g -1 AFDW h -1 ). DFAA = Dissolved free amino acids. DDN = Diazotrophically-derived nitrogen (ng N mg -1 DW h -1 ). DW = Dry weight. Rate of N2 fixation in seawater are in ng N L -1 d -1 . DIC = Dissolved inorganic carbon (µg C g -1 AFDW h -1 ). POM = Particulate organic matter. The assimilation of POM has been investigated with a lipid biomarker specific to crustacean zooplankton (cis-gondoic acid (CGA, 20:19), in µg g -1 AFDW). Lipid biomarkers specific to autotrophy, stearidonic acid (SDA, 18:43) and docosapentaenoic acid (DPA, 22:53), do not appear on the figure. Concentrations of SDA were 1-70 and 1-146 µg g -1 AFDW in shallow and mesophotic corals, respectively, and decreased from 11 to 3 µg g -1 AFDW with depth in the hard coral. Concentrations of DPA were 3-7 and 3-5 µg g -1 AFDW in shallow and mesophotic corals, respectively, and decreased from 5 to 2 µg g -1 AFDW with depth in the hard coral.

Conclusion

A functional mixotrophy was observed throughout the depth gradient for all symbiotic soft coral species, with a significant input of heterotrophic feeding on crustacean zooplankton at all depth. Mixotrophy was also observed for the only scleractinian species investigated in this thesis (Stylophora pistillata), suggesting that all coral species need to fulfill the deficiency of photosynthetic products in nitrogen and phosphorus through heterotrophy (see Houlbrèque and Ferrier-Pagès 2009). Moreover, heterotrophic feeding on zooplankton significantly increased with depth for several species, thereby emphasizing an increase in predation pressure and/or zooplankton availability with depth.

This thesis therefore highlights the importance of both autotrophic and heterotrophic inputs in soft coral nutrition, which provides them with a trophic plasticity along the depth gradient. Such dual mode of nutrition surely represents an asset for the successful settlement in different habitats and may provide them with an adaptive mechanism to sustain growth under stressful conditions.

Perspectives

The findings obtained in each chapter paved the way for future experiments, allowing a deeper understanding of the processes observed here at the organism scale. Some examples are given below. In scleractinian corals, it has been shown that the nitrogen to phosphorus ratio (N:P) plays a major role in the stability of the symbiosis. Mainly, an increase in nitrogen availability (mostly in the form of nitrate) without a similar increase in phosphorus, can enhance the population growth of the dinoflagellates and subsequently enhance coral bleaching. Since soft corals are less prone to take up dissolved inorganic nitrogen, it would be interesting to test i) the levels of nitrate inducing a disruption of the symbiosis, 2) the role of phosphorus availability for the maintenance of such symbiosis under diverse nitrogen and/or environmental conditions.

The results could bring information on how soft corals cope with eutrophication and thermal stress.

Investigate the oxidative stress in soft corals

The presence of symbionts in corals is often beneficial due to the energetic input supplied from photosynthesis. However, it can also impose restrictions (e.g., bathymetric distribution) and risks (e.g., higher increase in reactive oxygen species released under stressful conditions through photosynthesis). At the cellular level, the production of reactive oxygen and nitrogen species represents a pivotal factor responsible for coral bleaching. Since the results of this thesis show different nutritional strategies displayed by soft and hard corals along the depth gradient (especially regarding nitrogen), a different reliance on autotrophy, along with an overall flexibility towards the symbiosis, it could be interesting to investigate mechanisms possibly leading to a breakdown of the symbiosis in soft corals, in a context of thermal stress.

Heterotrophy

As heterotrophic nutrition was only assessed through a lipid biomarker specific to zooplankton, experiments involving incubations with a wide size range of labelled prey (picoand nano-plankton) would help to further characterize the diet of soft corals. Particularly, the use of stable isotope labelling would confirm the effective ingestion of the prey, since the mucus of soft corals is known to have antimicrobial properties, which could eventually affect the concentrations of plankton measured in the chambers.

Contribution of autotrophy versus heterotrophy in the nutritional budget of soft corals

Finally, this thesis could not quantitatively provide any relative contribution of auto-and heterotrophic nutrient supply in the metabolic needs of the holobiont. Another experiment using lipid biomarkers specific to auto-and heterotrophy could be designed for this purpose and must involve different nutritional situations encountered by the same colonies (e.g., seasonal followup). Another possibility would be to use a multi-biomarkers approach, by combining stable isotope ( 13 C and  15 N) signatures, along with total lipids (discriminate between neutral and polar lipid's fatty acids).

dinoflagellés photosynthétiques et un large éventail d'autres microorganismes tels que des bactéries, des archées, des champignons et des virus [START_REF] Knowlton | Multispecies microbial mutualisms on coral reefs: The host as a habitat[END_REF][START_REF] Van De Water | Host-microbe interactions in octocoral holobionts -recent advances and perspectives[END_REF] Chez les octocoralliaires, la contribution relative de l'autotrophie et de l'hétérotrophie aux besoins nutritionnels de l'holobionte est mal connue (revue dans [START_REF] Schubert | Symbiotic versus non-symbiotic octocorals: physiological and ecological implications[END_REF]. On pense généralement que les octocoralliaires ont un faible taux de productivité primaire et qu'ils dépendent à la fois de l'autotrophie et de l'hétérotrophie pour satisfaire leurs besoins métaboliques [START_REF] Sorokin | Biomass, metabolic rates and feeding of some common reef zoantharians and octocorals[END_REF][START_REF] Fabricius | Widespread mixotrophy in reef-inhabiting soft corals: the influence of depth, and colony expansion and contraction on photosynthesis[END_REF]. Cependant, les quelques études portant sur la contribution de la photosynthèse à la respiration de l'hôte (rapport P:R) soutiennent plutôt une dépendance à l'autotrophie pour satisfaire les besoins respiratoires [START_REF] Mergner | Productivity and seasonal changes in selected reef areas in the Gulf of Aqaba (Red Sea)[END_REF][START_REF] Sorokin | Biomass, metabolic rates and feeding of some common reef zoantharians and octocorals[END_REF][START_REF] Riegl | Effects of sediment on the energy budgets of four scleractinian (Bourne 1900) and five alcyonacean (Lamouroux 1816) corals[END_REF][START_REF] Fabricius | Widespread mixotrophy in reef-inhabiting soft corals: the influence of depth, and colony expansion and contraction on photosynthesis[END_REF][START_REF] Kremien | Beneft of pulsation in soft corals[END_REF][START_REF] Bednarz | Seasonal variation in dinitrogen fixation and oxygen fluxes associated with two dominant zooxanthellate soft corals from the northern Red Sea[END_REF][START_REF] Rossi | An overview of the animal forests of the world[END_REF][START_REF] Rossi | Trophic ecology of Caribbean octocorals: autotrophic and heterotrophic seasonal trends[END_REF], excepté pour quelques espèces [START_REF] Ramsby | Symbiodinium photosynthesis in Caribbean octocorals[END_REF][START_REF] Baker | Productivity links morphology, symbiont specificity and bleaching in the evolution of Caribbean octocoral symbioses[END_REF]. Le peu d'études qui ont porté sur un bilan de carbone montrent une forte variation inter-espèces, avec des taux de transfert de photosynthétats allant de 10 % chez Capnella gabonensis à 75 % chez Sinularia flexibilis [START_REF] Schlichter | Functional autotrophy of Heteroxenia fuscescens (Anthozoa: Alcyonaria): carbon assimilation and translocation of photosynthates from symbionts to host[END_REF][START_REF] Farrant | Nutrition of the temperate Australian soft coral Capnella gaboensis I. Photosynthesis and carbon fixation[END_REF][START_REF] Sorokin | Biomass, metabolic rates and feeding of some common reef zoantharians and octocorals[END_REF][START_REF] Khalesi | Energy budget for the cultured, zooxanthellate octocoral Sinularia flexibilis[END_REF]. La performance des symbiotes semble liée à la morphologie de l'hôte, les espèces ayant des branches fines et de petits polypes étant plus autotrophes que celles ayant une forme massive et de gros polypes [START_REF] Baker | Productivity links morphology, symbiont specificity and bleaching in the evolution of Caribbean octocoral symbioses[END_REF][START_REF] Rossi | Linking host morphology and symbiont performance in octocorals[END_REF]. La morphologie de la colonie entière peut également jouer un rôle important dans la nutrition des coraux mous : le rapport entre la surface corporelle et le volume, qui détermine à la fois l'échange gazeux à travers le tissu épidermique et l'exposition au rayonnement photosynthétiquement actif, est faible chez les coraux mous par rapport aux coraux durs [START_REF] Fabricius | Widespread mixotrophy in reef-inhabiting soft corals: the influence of depth, and colony expansion and contraction on photosynthesis[END_REF]. Toute augmentation de ce rapport, induit par l'expansion de la colonie, peut améliorer la productivité primaire. Des recherches supplémentaires sont donc nécessaires pour mieux comprendre les capacités autotrophes des octocoralliaires en général, et des coraux mous en particulier.

En ce qui concerne l'hétérotrophie, les coraux mous ont longtemps été considérés comme étant principalement des suspensivores, car leurs nématocystes sont petits, simplement structurés et inefficaces pour paralyser des proies nageant activement (Mariscal et Bigger, 1977). Toutefois, la découverte récente d'un nouveau type de nématocystes pourrait potentiellement mettre en évidence un arsenal de structures urticantes plus diversifiées qu'on ne le pensait auparavant [START_REF] Yoffe | A modified view on octocorals: Heteroxenia fuscescens nematocysts are diverse, featuring both an ancestral and a novel type[END_REF]. De plus, des microvillosités recouvrent l'épiderme des coraux mous de manière dense et jouent probablement un rôle dans l'absorption de la matière dissoute (Schlichter, 1982a(Schlichter, , 1982b)). Le régime alimentaire des octocoralliaires a été déterminé par des expériences impliquant des espèces tempérées [START_REF] Coma | Feeding and prey capture cycles in the aposymbiontic gorgonian Paramuricea clavata[END_REF]1998 ;2015 ;[START_REF] Orejas | Role of small-plankton communities in the diet of two antarctic octocorals (Primnoisis antarctica and Primnoella sp.)[END_REF][START_REF] Rossi | Temporal variability in zooplankton prey capture rate of the passive suspension feeder Leptogorgia sarmentosa (Cnidaria: Octocorallia), a case study[END_REF] et tropicales [START_REF] Lewis | Feeding behaviour and feeding ecology of the Octocorallia (Coelenterata: Anthozoa)[END_REF][START_REF] Sebens | Predation on zooplankton by the benthic anthozoans Alcyonium siderium (Alcyonacea) and Metridium senile (Actiniaria) in the New England subtidal[END_REF][START_REF] Sorokin | Biomass, metabolic rates and feeding of some common reef zoantharians and octocorals[END_REF]Fabricius et Domisse 2000 ;[START_REF] Rossi | Trophic ecology of Caribbean octocorals: autotrophic and heterotrophic seasonal trends[END_REF], et comprend du zooplancton de petite taille, ainsi que phytoplancton (Fabricius et al., 1995a(Fabricius et al., , 1995b ; ;1998 ;[START_REF] Ribes | Seasonal variation of in situ feeding rates by the temperate ascidian Halocynthia papillosa[END_REF] ; Leal et al. 2013 ;[START_REF] Piccinetti | Herbivory in the soft coral Sinularia flexibilis (Alcyoniidae)[END_REF]. La contribution relative de l'autotrophie et de Les activités anthropiques telles que la combustion d'énergies fossiles, l'industrialisation et la déforestation, ont augmenté les concentrations de gaz à effet de serre dans l'atmosphère depuis le début de l'ère industrielle [START_REF] Sabine | The oceanic sink for anthropogenic CO2[END_REF]. Ces émissions sont la cause de changements climatiques à l'échelle mondiale, notamment le réchauffement et l'acidification des océans.

 Le réchauffement de l'océan

Les coraux vivent dans des conditions environnementales proches de leur maximum thermique et peuvent souffrir de stress thermique lorsque la température de la surface de la mer augmente de ≥ 1°C au-dessus du maximum estival moyen [START_REF] Wooldridge | Breakdown of the coral-algae symbiosis: Towards formalising a linkage between warm-water bleaching thresholds and the growth rate of the intracellular zooxanthellae[END_REF]. Lorsque les coraux subissent un stress thermique extrême et prolongé, la symbiose avec leurs dinoflagellés photosynthétiques est perturbée, ce qui entraîne généralement l'expulsion des symbiotes, un phénomène appelé blanchissement [START_REF] Brown | Coral bleaching: causes and consequences[END_REF]. Les coraux blanchis sont physiologiquement endommagés, et un blanchissement prolongé entraîne souvent des niveaux élevés de mortalité chez les coraux [START_REF] Hughes | Global warming transforms coral reef assemblages[END_REF].

Ce phénomène aujourd'hui généralisé a été signalé dans de nombreux récifs du monde entier et n'épargne pas les octocoralliaires [START_REF] Paulay | Patterns and consequences of coral bleaching in Micronesia (Majuro and Guam) in 1992-1994[END_REF][START_REF] Marshall | Bleaching of corals on the Great Barrier Reef: differential susceptibilities among taxa[END_REF][START_REF] Arceo | Coral bleaching in Philippine reefs: Coincident evidences with mesoscale thermal anomalies[END_REF][START_REF] Loya | Coral bleaching : the winners and the losers[END_REF], McClahanan et al. 2001, Cellier et Schleyer 2002, Goulet et al. 2008[START_REF] Chavanich | Mass bleaching of soft coral, sarcophyton spp. in Thailand and the role of temperature and salinity stress[END_REF], Prada et al. 2009[START_REF] Van Woesik | Revisiting the winners and the losers a decade after coral bleaching[END_REF][START_REF] Dias | Bleaching in scleractinians, hydrocorals, and octocorals during thermal stress in a northeastern Brazilian reef[END_REF][START_REF] Slattery | Annual thermal stress increases a soft coral's susceptibility to bleaching[END_REF]. Tout comme pour les coraux durs, il a été montré que la sensibilité des octocoralliaires au stress thermique n'est pas uniforme et qu'il existe une tolérance plus élevée chez certaines espèces, voire entre individus d'une même espèce [START_REF] Loya | Coral bleaching : the winners and the losers[END_REF][START_REF] Lasker | Zooxanthella densities within a Caribbean octocoral during bleaching and non-bleaching years[END_REF][START_REF] Strychar | Loss of Symbiodinium from bleached soft corals Sarcophyton ehrenbergi, Sinularia sp. and Xenia sp[END_REF]Goulet et al. 2008 ;[START_REF] Van Woesik | Revisiting the winners and the losers a decade after coral bleaching[END_REF][START_REF] Slattery | Annual thermal stress increases a soft coral's susceptibility to bleaching[END_REF]. Les quelques études expérimentales qui portent sur les réponses des octocoralliaires tropicaux au stress thermique ont montré une majorité de réponses négatives, impliquant notamment une réduction de la photosynthèse, un blanchissement, une diminution des réserves énergétiques et de la production reproductive, ainsi que de la nécrose [START_REF] Michalek-Wagner | Impacts of bleaching on the soft coral Lobophytum compactum. Ii. Biochemical changes in adults and their eggs[END_REF][START_REF] Drohan | Synergistic effect of high temperature and ultraviolet-B radiation on the gorgonian Eunicea tourneforti (Octocorallia: Alcyonacea: Plexauridae)[END_REF][START_REF] Strychar | Loss of Symbiodinium from bleached soft corals Sarcophyton ehrenbergi, Sinularia sp. and Xenia sp[END_REF]Imbs et Yakatova, 2012 ;[START_REF] Sammarco | Responses to high seawater temperatures in zooxanthellate octocorals[END_REF][START_REF] Netherton | Physiological correlates of symbiont migration during bleaching of two octocoral species[END_REF]. Par conséquent, les octocoralliaires semblent être aussi affectés que les coraux durs par le réchauffement de l'océan.

 Acidification des océans L'acidification des océans est un processus se produisant actuellement de manière continue, qui induit plusieurs changements dans la chimie des carbonates de l'eau de mer (e.g., [START_REF] Feely | Impact of anthropogenic CO2 on the CaCO3 system in the oceans[END_REF]. En particulier, il diminue le pH de l'eau de mer et la concentration des ions carbonate. Ces modifications ont donc un effet négatif sur la calcification biogénique (e.g., de l'aragonite et de la calcite) et peuvent également entraîner la dissolution nette des structures carbonatées. Ainsi, l'acidification des océans pose d'importants problèmes aux organismes marins qui forment des coquilles, des squelettes ou des structures internes en carbonate de calcium [START_REF] Gattuso | Ocean acidification[END_REF][START_REF] Orr | Anthropogenic ocean acidification over the twenty-first century and its impact on calcifying organisms[END_REF].

Alors que les observations in situ suggèrent qu'une baisse du pH de l'eau de mer dans les années à venir devrait induire une transition de la dominance des coraux scléractiniaires vers une prépondérance des coraux mous et autres espèces non-calcifiantes [START_REF] Inoue | Spatial community shift from hard to soft corals in acidified water[END_REF], seules quelques études expérimentales ont abordé la réponse des octocoralliaires à l'acidification des océans. Elles montrent que les descripteurs tissulaires et les paramètres physiologiques (c'est-à-dire la densité des cellules des symbiotes, la pigmentation, la structure des sclérites, le taux de pulsation des polypes et la croissance) ne sont pas affectés par l'acidification [START_REF] Gabay | Does elevated pCO2 affect reef octocorals?[END_REF][START_REF] Wooldridge | Differential thermal bleaching susceptibilities amongst coral taxa: Reposing the role of the host[END_REF], Enochs et al. 2015b). Une seule étude a montré une diminution de la calcification, mais le pH testé (7,1) dépasse les valeurs prévues pour la fin du siècle (Gomez et al., 2014). L'atténuation de l'acidification par les octocoralliaires pourrait s'expliquer par leur tissu épais, qui peut protéger les sclérites [START_REF] Gabay | Does elevated pCO2 affect reef octocorals?[END_REF][START_REF] Wooldridge | Differential thermal bleaching susceptibilities amongst coral taxa: Reposing the role of the host[END_REF], et par le processus de calcification lui-même, qui pourrait être moins affecté que celui des coraux durs malgré l'altération de la chimie des carbonates (Enochs et al., 2015b). Bien qu'il existe peu de données sur les effets combinés de l'acidification et d'autres menaces sur les octocoralliaires, il a été suggéré que les coraux durs, qui sont déjà stressés par l'acidification seule, seront plus touchés que les octocoralliaires s'ils sont déjà confrontés à d'autres facteurs de stress tels que l'eutrophisation [START_REF] Januar | Tropical coral reef coral patterns in Indonesian shallow water areas close to underwater volcanic vents at Minahasa Seashore, and Mahengetang and Gunung Api Islands[END_REF].

 Eutrophisation et autres facteurs de stress locaux L'eutrophisation de l'eau est un phénomène complexe qui implique une augmentation de la concentration en sels nutritifs dissous (nitrates, phosphates), une augmentation des particules en suspension et une réduction de la luminosité. Les récifs côtiers sont les plus impactés par l'eutrophisation, qui résulte d'une agriculture et/ou urbanisation intensive, ainsi que du rejet de polluants inorganiques dans l'eau. Les effets de l'eutrophisation sur l'abondance des coraux mous ne sont pas concluants. En effet, les études portant sur le taux de couverture des coraux durs et mous montrent un impact négatif [START_REF] Fabricius | Identifying ecological change and its causes: a case study on coral reefs[END_REF], un effet nul (Fabricius et De'ath, 2001), ou bien une dominance relative des coraux mous dans des zones affectées par la sédimentation [START_REF] Mcclanahan | Sedimentation effects on shallow coral communitiesin Kenya[END_REF][START_REF] Schleyer | Coral dominance at the reef-sediment interface in marginal coral communities at Sodwana Bay, South Africa[END_REF] ou soumises à de fortes concentrations de particules en suspension et matières dissoutes [START_REF] Fabricius | Effects of terrestrial runoff on the ecology of corals and coral reefs: Review and synthesis[END_REF][START_REF] Fabricius | Depletion of suspended particulate matter over coastal reef communities dominated by zooxanthellate soft corals[END_REF][START_REF] Baum | Abundance and physiology of dominant soft corals linked to water quality in Jakarta Bay, Indonesia[END_REF]. Des données provenant de la Grande Barrière de Corail, de Hong-Kong et de Palau suggèrent qu'une pauvre qualité d'eau (incluant tous les paramètres cités ci-dessus) serait plutôt liée à une diminution de la diversité des espèces [START_REF] Fabricius | Identifying ecological change and its causes: a case study on coral reefs[END_REF][START_REF] Fabricius | Changes in octocoral communities and benthic cover along a water quality gradient in the reefs of Hong Kong[END_REF][START_REF] Fabricius | Changes in algal, coral and fish assemblages along water quality gradients on the inshore Great Barrier Reef[END_REF][START_REF] Goulet | Most scleractinian corals and octocorals host a single symbiotic zooxanthella clade[END_REF] [START_REF] Baker | Productivity links morphology, symbiont specificity and bleaching in the evolution of Caribbean octocoral symbioses[END_REF][START_REF] Rossi | Linking host morphology and symbiont performance in octocorals[END_REF] ; et 3) l'utilisation de différentes méthodes et mesures de normalisation ne permet guère de faire des comparaisons.

En outre, la quantification du transfert de photosynthétats des symbiotes vers l'hôte a été étudiée chez une seule espèce tropicale (Heteroxenia fuscecens) et une seule espèce tempérée (Capnella gaboensis) en utilisant le 14 C [START_REF] Schlichter | Functional autotrophy of Heteroxenia fuscescens (Anthozoa: Alcyonaria): carbon assimilation and translocation of photosynthates from symbionts to host[END_REF][START_REF] Farrant | Nutrition of the temperate Australian soft coral Capnella gaboensis I. Photosynthesis and carbon fixation[END_REF].

Nous avons donc réalisé des expériences de traçage du carbone inorganique acquis par voie autotrophe au moyen de bicarbonate enrichi en 13 [START_REF] Lewis | Feeding behaviour and feeding ecology of the Octocorallia (Coelenterata: Anthozoa)[END_REF][START_REF] Sorokin | Biomass, metabolic rates and feeding of some common reef zoantharians and octocorals[END_REF]Fabricius et al., 1995) Corail [START_REF] Fabricius | Widespread mixotrophy in reef-inhabiting soft corals: the influence of depth, and colony expansion and contraction on photosynthesis[END_REF]. Des stratégies d'acclimatation à de faibles niveaux de lumière ont été observées chez les symbiotes (c'est-à-dire une réduction de l'auto-ombrage et une augmentation de la capacité de captage de la lumière des cellules des symbiotes ; [START_REF] Cohen | Long term photoacclimation responses of the coral Stylophora pistillata to reciprocal deep to shallow transplantation: photosynthesis and calcification[END_REF][START_REF] Ziegler | Mesophotic coral depth acclimatization is a function of host-specific symbiont physiology[END_REF][START_REF] Einbinder | Novel adaptive photosynthetic characteristics of mesophotic symbiotic microalgae within the reefbuilding coral, Stylophora pistillata[END_REF]. Cependant, la stabilité de l'autotrophie le long du gradient de profondeur était indépendante du genre de Symbiodiniaceae hébergé par le corail hôte, puisque les associations étaient spécifiques à l'espèce et sont susceptibles d'être stables dans l'espace et le temps [START_REF] Goulet | Stability of an octocoral-algal symbiosis over time and space[END_REF] [START_REF] Erez | Vital effect on stable-isotope composition seen in foraminifera and coral skeletons[END_REF][START_REF] Furla | Sources and mechanisms of inorganic carbon transport for coral calcification and photosynthesis[END_REF][START_REF] Hughes | Acquisition and assimilation of carbon in non-bleached and bleached corals[END_REF] (coraux mous contre coraux durs) et les profondeurs (peu profond contre mésophotique). Chl = concentration en chlorophylles (μg Chl (a + c2) g -1 AFDW). Pg = Acquisition de carbone autotrophe (μg C μg -1 Chl). Ts = Carbone transféré des symbiotes vers l'hôte (μg C g -1 AFDW h -1 ). AFDW = poids sec sans cendres. DIN = Azote inorganique dissous (comprenant NH4 et NO3, en μg N g -1 AFDW h -1 ). DON = Azote organique dissous (comprenant les acides aminés libres dissous, en μg N g -1 AFDW h -1 ). DFAA = Acides aminés libres dissous. DDN = Azote provenant des diazotrophes (ng N mg -1 DW h -1 ). DW = Poids sec. Les taux de fixation de N2 dans l'eau de mer sont exprimés en ng N L -1 d -1 . DIC = Carbone inorganique dissous (μg C g -1 AFDW h -1 ). POM = Matière organique particulaire. L'assimilation de POM a été étudiée avec un biomarqueur lipidique spécifique du zooplancton (acide cis-gondoïque (CGA, 20:1ω9), en μg g - Table S5: Coefficients estimated to convert data normalized to surface area of the skeleton into data normalized to ash-free dry weight (mean ± standard error = 255.65 ± 8.98). The nubbins (n = 8) were fragmented from Stylophora pistillata colonies grown at the Monaco Scientific Center. DW = dry weight. AFDW = ash-free dry weight. Calculated coefficients are the ratio between surface area of the skeleton and ash-free dry weight.

Références

Les octocoralliaires représentent l'un des principaux groupes formant la communauté macrobenthique des récifs coralliens tropicaux. Ils sont notamment abondants au sein des écosystèmes perturbés où les changements environnementaux entraînent le déclin des coraux constructeurs de récifs. Bien que la nutrition joue un rôle fondamental dans la régulation de l'abondance d'une population, l'acquisition de nutriments par les octocoralliaires reste à ce jour peu connue. Dans ce contexte, les objectifs de cette thèse étaient 1) de caractériser l'acquisition et l'assimilation de carbone et d'azote, par différentes espèces d'octocoralliaires de mer Rouge, et 2) d'évaluer les changements de nutrition le long d'un gradient de profondeur, depuis la surface (5 m) jusqu'à la zone récifale mésophotique supérieure (50 m). Les résultats démontrent que les octocoralliaires forment une symbiose nutritionnelle avec leurs dinoflagellés, dont le fonctionnement diffère largement de celui de la symbiose dinoflagellésscléractiniaire. La symbiose des octocoralliaires se démarque particulièrement par un apport de carbone autotrophe stable le long du gradient de profondeur, alors que les scléractiniaires connaissent un approvisionnement réduit avec l'augmentation de profondeur. De plus, l'assimilation de composés azotés dissous (par les dinoflagellés ou les symbiotes microbiens) par les octocoralliaires est très inférieure à celle des scléractiniaires. Ces résultats suggèrent que les octocoralliaires dépendent largement de sources alimentaires hétérotrophes pour satisfaire leurs besoins nutritionnels. L'importance de l'hétérotrophie est confirmée par de fortes concentrations tissulaires en biomarqueurs lipidiques spécifiques du zooplancton aux deux profondeurs, avec une augmentation en milieu mésophotique chez certaines espèces. Une telle mixotrophie confère aux octocoralliaires une grande plasticité trophique, ce qui pourrait contribuer à une plus grande résistance aux changements environnementaux en cours.

Mots clés : octocoralliaires | symbiose | autotrophie | hétérotrophie | mésophotique

Octocorals are one of the major groups forming the macrobenthic community of tropical coral reefs. They are notably abundant within disturbed ecosystems where environmental changes have led to the decline of reef-building corals. Although nutrition plays a fundamental role in regulating the abundance of a population, the acquisition of nutrients by octocorals has received little attention to date. In this context, the aims of this thesis were to 1) characterize the acquisition and assimilation of carbon and nitrogen by several octocoral species from the Red Sea, and 2) investigate the nutritional changes along a depth gradient, from the shallow (5 m) down to the upper mesophotic (50 m) reef zone. The results show that octocorals form a nutritional symbiosis with dinoflagellates, but the functioning differs significantly as compared to the scleractinian-dinoflagellate symbiosis. Particularly, the octocoral symbiosis is characterized by a stable supply of autotrophic carbon along the depth gradient, whereas scleractinian corals experience a reduced supply with increase in water depth. In addition, octocorals assimilate along the entire depth gradient significantly less dissolved nitrogen compounds (from dinoflagellates or microbial symbionts) as compared to scleractinian corals. These results suggest that octocorals strongly depend on heterotrophic food sources to meet their nutritional requirements. The importance of heterotrophy is confirmed by high concentrations of lipid biomarkers specific to zooplankton in the octocoral tissue at shallow and mesophotic depths, with an increased concentration for some species in the mesophotic environment. Such mixotrophy provides octocorals with a wide trophic plasticity, which may contribute to their higher resistance to cope with already on-going environmental changes.

  Key words: octocoral | symbiosis | autotrophy | heterotrophy | mesophotic XII XIII

Figure 1 .

 1 Figure 1. Global diversity of symbiotic hard coral species, indicated by records of occurrences. Modified from Veron et al. (2015).......................................................................................................................................... 4 Figure 2. Painting representing some of the major benthic groups on reefs (hard coral, octocoral, sponge and algae). Modified from Creartgraphic. .............................................................................................................. 5 Figure 3. Simplified phylogeny of Cnidaria. Taxonomic position of studied species appears in blue. Adapted from http://www.marinespecies.org/. Picture credits: (top) (Leclère and Röttinger, 2017) and (middle) https://ian.umces.edu/. ............................................................................................................................................. 6 Figure 4. Morphological parts of soft coral (left) and gorgonian (right) colonies. The following terms have been described by Bayer et al. (1983). Capitulum: more or less disk-shaped or hemispherical, polypiferous part of soft coral colony. Stalk: barren basal part of the colony, in Alcyonacea and Pennatulacea; narrow proximal part of a non-retractile polyp in Nephtheidae. Stem: basal part of the colony from which branches may or may not arise, in gorgonians; polypiferous part of the colony usually giving rise to branches in soft corals. Photos credit: C. Pupier. ..................................................................................................................................................................... 7 Figure 5. Autozooid polyp of octocoral. The longitudinal section highlights the epithelial layers and internal body organization. For clarity, pinnules are only represented on one tentacle and solenia are limited to a little part of the coenenchyme. This is a simplified scheme, which should comprise eight mesenteries (i.e. that alternate with the eight tentacles) and eight mesenterial filaments. The gastrovascular cavity ends in a dead-end canal. Retractor septal muscles and transverse fibers of mesenteries, which play a role in polyp retraction and movement of tentacles are not displayed here. Drawing by C. Pupier, based on Fabricius and Alderslade (2001). ..................... 8 Figure 6. Sclerites of soft corals. (a) Sclerites are visible on the surface of branches of Dendronephthya sp. (photo credit: M. Lanini). (b) Sclerites of Dendronephthya sp. seen under an electron microscope (× 250, from Bayer et al., 1983). C: Sclerite of Xenia sp. seen under an electron microscope (× 1750, from Bayer et al., 1983). ............ 9 Figure 7. Octocoral polyps and close-up on their Symbiodiniaceae endosymbionts. Photo credits: B. Ping (left) and S. Santos (right). .................................................................................................................................... 12 Figure 8. Relative abundance of Symbiodiniaceae genera in octocorals of several biogeographic regions. Genera are indicated in the bottom-left corner, followed by the former clade designation in brackets. A large number of octocoral species does not possess algal symbionts (≤ 50%), similarly to hard corals. Unreported are Effrenium sp. (formerly clade E) and Fugacium sp. (formerly clade F), which can be found associated with hard corals. Modified from van de Water al. (2018). .................................................................................................... 13 Figure 9. Plankton size classes included in the diet of octocorals. The classes highlighted in blue indicate the microbial fraction. Scanning electron micrographs of A: Prochlorococcus sp. (0.6 µm) and B: Synechococcus sp. (1 µm). C: epifluorescence microscope image showing one nanoflagellate cell indicated by the arrow. D: ciliates (100-200 µm) taken under phase contrast microscope. E: crab zoea (1000 µm). Modified from Houlbrèque and Ferrier-Pagès (2009). ............................................................................................................................................ 16 Figure 10. High cover of soft corals. Local dominance of Sarcophyton sp. observed in the Seychelles (left, photo credit: M. Boussion) and high abundance of multi-species assemblage of soft corals observed in Indonesia (right, photo credit: C. Pupier). ........................................................................................................................................ 22 Figure 11. Study site in the Red Sea (Eilat, Israel, Gulf of Eilat/Aqaba). IUI: Interuniversity Institute for Marine Sciences. The three bathymetric zones are represented (shallow water corresponding to 0-30 m depth, in blue; mesophotic zone corresponding to 30-150 m depth, in red; and deep waters corresponding to 150-800 m depth, in green). Modified from Shoham and Benayahu (2016). ......................................................................................... 25 Figure 12. Soft corals used during this thesis. Corresponding family, Symbiodiniaceae associated with the coral and growth form of the colony are mentioned. Photo credits are attributed to M. Boussion for Dendronephthya sp. and H. fuscescens (right), G. Banc-Prandi for Litophyton sp. (left), P. Colla for Sarcophyton sp. (right), and C. Pupier for H. fuscescens (left), R. f. fulvum (left) and Sarcophyton sp. (left). ..................................................... 27 Figure 13. Hard corals used during this thesis. Corresponding family, growth form of the colony and Symbiodiniaceae associated with the coral are mentioned. Photo credits are attributed to E. Turak for A. eurystoma, N. Coleman for Cynarina sp., G. Paulay for Goniastrea sp., C. Veron for S. hystrix, and C. Pupier for G. fascicularis, P. damicornis and S. pistillata. ......................................................................................................... 28

  Figure 1. Global diversity of symbiotic hard coral species, indicated by records of occurrences. Modified from Veron et al. (2015).......................................................................................................................................... 4 Figure 2. Painting representing some of the major benthic groups on reefs (hard coral, octocoral, sponge and algae). Modified from Creartgraphic. .............................................................................................................. 5 Figure 3. Simplified phylogeny of Cnidaria. Taxonomic position of studied species appears in blue. Adapted from http://www.marinespecies.org/. Picture credits: (top) (Leclère and Röttinger, 2017) and (middle) https://ian.umces.edu/. ............................................................................................................................................. 6 Figure 4. Morphological parts of soft coral (left) and gorgonian (right) colonies. The following terms have been described by Bayer et al. (1983). Capitulum: more or less disk-shaped or hemispherical, polypiferous part of soft coral colony. Stalk: barren basal part of the colony, in Alcyonacea and Pennatulacea; narrow proximal part of a non-retractile polyp in Nephtheidae. Stem: basal part of the colony from which branches may or may not arise, in gorgonians; polypiferous part of the colony usually giving rise to branches in soft corals. Photos credit: C. Pupier. ..................................................................................................................................................................... 7 Figure 5. Autozooid polyp of octocoral. The longitudinal section highlights the epithelial layers and internal body organization. For clarity, pinnules are only represented on one tentacle and solenia are limited to a little part of the coenenchyme. This is a simplified scheme, which should comprise eight mesenteries (i.e. that alternate with the eight tentacles) and eight mesenterial filaments. The gastrovascular cavity ends in a dead-end canal. Retractor septal muscles and transverse fibers of mesenteries, which play a role in polyp retraction and movement of tentacles are not displayed here. Drawing by C. Pupier, based on Fabricius and Alderslade (2001). ..................... 8 Figure 6. Sclerites of soft corals. (a) Sclerites are visible on the surface of branches of Dendronephthya sp. (photo credit: M. Lanini). (b) Sclerites of Dendronephthya sp. seen under an electron microscope (× 250, from Bayer et al., 1983). C: Sclerite of Xenia sp. seen under an electron microscope (× 1750, from Bayer et al., 1983). ............ 9 Figure 7. Octocoral polyps and close-up on their Symbiodiniaceae endosymbionts. Photo credits: B. Ping (left) and S. Santos (right). .................................................................................................................................... 12 Figure 8. Relative abundance of Symbiodiniaceae genera in octocorals of several biogeographic regions. Genera are indicated in the bottom-left corner, followed by the former clade designation in brackets. A large number of octocoral species does not possess algal symbionts (≤ 50%), similarly to hard corals. Unreported are Effrenium sp. (formerly clade E) and Fugacium sp. (formerly clade F), which can be found associated with hard corals. Modified from van de Water al. (2018). .................................................................................................... 13 Figure 9. Plankton size classes included in the diet of octocorals. The classes highlighted in blue indicate the microbial fraction. Scanning electron micrographs of A: Prochlorococcus sp. (0.6 µm) and B: Synechococcus sp. (1 µm). C: epifluorescence microscope image showing one nanoflagellate cell indicated by the arrow. D: ciliates (100-200 µm) taken under phase contrast microscope. E: crab zoea (1000 µm). Modified from Houlbrèque and Ferrier-Pagès (2009). ............................................................................................................................................ 16 Figure 10. High cover of soft corals. Local dominance of Sarcophyton sp. observed in the Seychelles (left, photo credit: M. Boussion) and high abundance of multi-species assemblage of soft corals observed in Indonesia (right, photo credit: C. Pupier). ........................................................................................................................................ 22 Figure 11. Study site in the Red Sea (Eilat, Israel, Gulf of Eilat/Aqaba). IUI: Interuniversity Institute for Marine Sciences. The three bathymetric zones are represented (shallow water corresponding to 0-30 m depth, in blue; mesophotic zone corresponding to 30-150 m depth, in red; and deep waters corresponding to 150-800 m depth, in green). Modified from Shoham and Benayahu (2016). ......................................................................................... 25 Figure 12. Soft corals used during this thesis. Corresponding family, Symbiodiniaceae associated with the coral and growth form of the colony are mentioned. Photo credits are attributed to M. Boussion for Dendronephthya sp. and H. fuscescens (right), G. Banc-Prandi for Litophyton sp. (left), P. Colla for Sarcophyton sp. (right), and C. Pupier for H. fuscescens (left), R. f. fulvum (left) and Sarcophyton sp. (left). ..................................................... 27 Figure 13. Hard corals used during this thesis. Corresponding family, growth form of the colony and Symbiodiniaceae associated with the coral are mentioned. Photo credits are attributed to E. Turak for A. eurystoma, N. Coleman for Cynarina sp., G. Paulay for Goniastrea sp., C. Veron for S. hystrix, and C. Pupier for G. fascicularis, P. damicornis and S. pistillata. ......................................................................................................... 28

Figure 7 .Figure 14 .

 714 Figure 1. Global diversity of symbiotic hard coral species, indicated by records of occurrences. Modified from Veron et al. (2015).......................................................................................................................................... 4 Figure 2. Painting representing some of the major benthic groups on reefs (hard coral, octocoral, sponge and algae). Modified from Creartgraphic. .............................................................................................................. 5 Figure 3. Simplified phylogeny of Cnidaria. Taxonomic position of studied species appears in blue. Adapted from http://www.marinespecies.org/. Picture credits: (top) (Leclère and Röttinger, 2017) and (middle) https://ian.umces.edu/. ............................................................................................................................................. 6 Figure 4. Morphological parts of soft coral (left) and gorgonian (right) colonies. The following terms have been described by Bayer et al. (1983). Capitulum: more or less disk-shaped or hemispherical, polypiferous part of soft coral colony. Stalk: barren basal part of the colony, in Alcyonacea and Pennatulacea; narrow proximal part of a non-retractile polyp in Nephtheidae. Stem: basal part of the colony from which branches may or may not arise, in gorgonians; polypiferous part of the colony usually giving rise to branches in soft corals. Photos credit: C. Pupier. ..................................................................................................................................................................... 7 Figure 5. Autozooid polyp of octocoral. The longitudinal section highlights the epithelial layers and internal body organization. For clarity, pinnules are only represented on one tentacle and solenia are limited to a little part of the coenenchyme. This is a simplified scheme, which should comprise eight mesenteries (i.e. that alternate with the eight tentacles) and eight mesenterial filaments. The gastrovascular cavity ends in a dead-end canal. Retractor septal muscles and transverse fibers of mesenteries, which play a role in polyp retraction and movement of tentacles are not displayed here. Drawing by C. Pupier, based on Fabricius and Alderslade (2001). ..................... 8 Figure 6. Sclerites of soft corals. (a) Sclerites are visible on the surface of branches of Dendronephthya sp. (photo credit: M. Lanini). (b) Sclerites of Dendronephthya sp. seen under an electron microscope (× 250, from Bayer et al., 1983). C: Sclerite of Xenia sp. seen under an electron microscope (× 1750, from Bayer et al., 1983). ............ 9 Figure 7. Octocoral polyps and close-up on their Symbiodiniaceae endosymbionts. Photo credits: B. Ping (left) and S. Santos (right). .................................................................................................................................... 12 Figure 8. Relative abundance of Symbiodiniaceae genera in octocorals of several biogeographic regions. Genera are indicated in the bottom-left corner, followed by the former clade designation in brackets. A large number of octocoral species does not possess algal symbionts (≤ 50%), similarly to hard corals. Unreported are Effrenium sp. (formerly clade E) and Fugacium sp. (formerly clade F), which can be found associated with hard corals. Modified from van de Water al. (2018). .................................................................................................... 13 Figure 9. Plankton size classes included in the diet of octocorals. The classes highlighted in blue indicate the microbial fraction. Scanning electron micrographs of A: Prochlorococcus sp. (0.6 µm) and B: Synechococcus sp. (1 µm). C: epifluorescence microscope image showing one nanoflagellate cell indicated by the arrow. D: ciliates (100-200 µm) taken under phase contrast microscope. E: crab zoea (1000 µm). Modified from Houlbrèque and Ferrier-Pagès (2009). ............................................................................................................................................ 16 Figure 10. High cover of soft corals. Local dominance of Sarcophyton sp. observed in the Seychelles (left, photo credit: M. Boussion) and high abundance of multi-species assemblage of soft corals observed in Indonesia (right, photo credit: C. Pupier). ........................................................................................................................................ 22 Figure 11. Study site in the Red Sea (Eilat, Israel, Gulf of Eilat/Aqaba). IUI: Interuniversity Institute for Marine Sciences. The three bathymetric zones are represented (shallow water corresponding to 0-30 m depth, in blue; mesophotic zone corresponding to 30-150 m depth, in red; and deep waters corresponding to 150-800 m depth, in green). Modified from Shoham and Benayahu (2016). ......................................................................................... 25 Figure 12. Soft corals used during this thesis. Corresponding family, Symbiodiniaceae associated with the coral and growth form of the colony are mentioned. Photo credits are attributed to M. Boussion for Dendronephthya sp. and H. fuscescens (right), G. Banc-Prandi for Litophyton sp. (left), P. Colla for Sarcophyton sp. (right), and C. Pupier for H. fuscescens (left), R. f. fulvum (left) and Sarcophyton sp. (left). ..................................................... 27 Figure 13. Hard corals used during this thesis. Corresponding family, growth form of the colony and Symbiodiniaceae associated with the coral are mentioned. Photo credits are attributed to E. Turak for A. eurystoma, N. Coleman for Cynarina sp., G. Paulay for Goniastrea sp., C. Veron for S. hystrix, and C. Pupier for G. fascicularis, P. damicornis and S. pistillata. ......................................................................................................... 28

  .......................................................................................................................... 102 Figure 31. Assimilation rates of dissolved N in the Symbiodiniaceae fraction in (a) hard corals (Galaxea fascicularis, Seriatopora hystrix, Stylophora pistillata) and (b) soft corals (Litophyton arboreum, Rhytisma fulvum fulvum, Sarcophyton sp.). Significant differences between depths are displayed with an asterisk. Significant differences between species are distinguished with letters by depth. Blue bold letters highlight a similar rate between hard and soft coral species. AFDW = ash-free dry weight. ns = not sampled. DFAA = dissolved free amino acids. ns = not sampled. ........................................................................................................................... 103 Figure 32. Assimilation rates of dissolved N in shallow soft corals exposed at two temperatures. (a) gathers rates related to the host tissue and (b) to the Symbiodiniaceae fraction. (Litophyton arboreum, Rhytisma fulvum fulvum, Sarcophyton sp.

Figure 33 .

 33 Figure 36. Non-metric multidimensional scaling (MDS) ordination used to assess similarities in the concentration (µg g -1 ash-free dry weight) of the fatty acids SDA, DPA (autotrophy markers) and CGA (heterotrophy marker) in the tissues of Dendronephthya sp., Litophyton arboreum, Rhytisma fulvum fulvum, Sarcophyton sp., and Stylophora pistillata, sampled in both shallow (8-10 m) and mesophotic (40-50 m) sections of the reef adjacent to the Inter-University Institute for Marine Sciences, in Eilat, Israel. ......... 120 Figure 37. Concentration (µg g -1 ash-free dry weight) of the fatty acid trophic markers (a) SDA, (b) DPA (stearidonic acid and docosapentaenoic acid, autotrophy markers) and (c) CGA (cis-gondoic acid, heterotrophy marker) from the tissues of Dendronephthya sp. (asymbiotic octocoral, Den), Litophyton arboreum (symbiotic octocoral, Lit), Rhytisma fulvum fulvum (symbiotic octocoral, Rhy), Sarcophyton sp.

  ................................................................................................................... 134

  ........................................................................................................................................................... 11 Table 2. Reported shifts from hard corals to soft corals dominance. COTS: crown-of-thorn starfishes. From van de Water et al. (2018). .................................................................................................................................... 23 Table

  ................................................................................................................................................................. 40 Table 4. Statistical results about the effect of sample state and homogenization media on Symbiodinium density, total chlorophyll and protein concentrations measured in Heteroxenia fuscescens. ....................... 48 Table 5. Significant correlations between dry weight (DW), protein content, body volume and ash weight (AW) of Heteroxenia fuscescens, Rhytisma fulvum, Dendronephthya sp. and Litophyton arboreum. ........... 50 Table 6. Tissues descriptors and autotrophic carbon acquisition reported for different octocorals, normalized to ash-free dry weight (AFDW). PC = autotrophic carbon acquisition. .......................................... 68 Table 7. Scleractinian and soft coral species investigated in the study. .......................................................... 79 Table 8. Predominant symbiont species associated with the four symbiotic Red Sea coral species sampled at shallow and mesophotic depths of the reef adjacent to the Inter-University Institute for Marine Sciences in Eilat, Israel. ........................................................................................................................................................ 118

Figure 1 .

 1 Figure 1. Global diversity of symbiotic hard coral species, indicated by records of occurrences. Modified from Veron et al. (2015).

Figure 2 .

 2 Figure 2. Painting representing some of the major benthic groups on reefs (hard coral, octocoral, sponge and algae). Modified from Creartgraphic.

Figure 3 .

 3 Figure 3. Simplified phylogeny of Cnidaria. Taxonomic position of studied species appears in blue. Adapted from http://www.marinespecies.org/. Picture credits: (top) (Leclère and Röttinger 2017) and (middle) https://ian.umces.edu/.

Figure 4 .

 4 Figure 4. Morphological parts of soft coral (left) and gorgonian (right) colonies. The following terms have been described by Bayer et al. (1983). Capitulum: more or less diskshaped or hemispherical, polypiferous part of soft coral colony. Stalk: barren basal part of the colony, in Alcyonacea and Pennatulacea; narrow proximal part of a non-retractile polyp in Nephtheidae. Stem: basal part of the colony from which branches may or may not arise, in gorgonians; polypiferous part of the colony usually giving rise to branches in soft corals. Photos credit: C. Pupier.

Figure 5 .

 5 Figure 5. Autozooid polyp of octocoral. The longitudinal section highlights the epithelial layers and internal body organization. For clarity, pinnules are only represented on one tentacle and solenia are limited to a little part of the coenenchyme. This is a simplified scheme, which

Figure 6 .

 6 Figure 6. Sclerites of soft corals. (a) Sclerites are visible on the surface of branches of Dendronephthya sp. (photo credit: M. Lanini). (b) Sclerites of Dendronephthya sp. seen under an electron microscope (× 250, from[START_REF] Bayer | Illustrated trilingual glossary of morphological and anatomical terms applied to Octocorallia[END_REF]. C: Sclerite of Xenia sp. seen under an electron microscope (× 1750, from[START_REF] Bayer | Illustrated trilingual glossary of morphological and anatomical terms applied to Octocorallia[END_REF].

Figure 7 .

 7 Figure 7. Octocoral polyps and close-up on their Symbiodiniaceae endosymbionts. Photo credits: B. Ping (left) and S. Santos (right).

Figure 8 .

 8 Figure 8. Relative abundance of Symbiodiniaceae genera in octocorals of several biogeographic regions. Genera are indicated in the bottom-left corner, followed by the former clade designation in brackets. A large number of octocoral species does not possess algal symbionts (≤ 50%), similarly to hard corals. Unreported are Effrenium sp. (formerly clade E) and Fugacium sp. (formerly clade F), which can be found associated with hard corals. Modified from van de Water al. (2018).

Figure 9 .

 9 Figure 9. Plankton size classes included in the diet of octocorals. The classes highlighted in blue indicate the microbial fraction. Scanning electron micrographs of A: Prochlorococcus sp. (0.6 µm) and B: Synechococcus sp. (1 µm). C: epifluorescence microscope image showing one nanoflagellate cell indicated by the arrow. D: ciliates (100-200 µm) taken under phase contrast microscope. E: crab zoea (1000 µm). Modified from Houlbrèque and Ferrier-Pagès (2009).

Figure 10 .

 10 Figure 10. High cover of soft corals. Local dominance of Sarcophyton sp. observed in the Seychelles (left, photo credit: M. Boussion) and high abundance of multi-species assemblage of soft corals observed in Indonesia (right, photo credit: C. Pupier).

Figure 11 .

 11 Figure 11. Study site in the Red Sea (Eilat, Israel, Gulf of Eilat/Aqaba). IUI: Interuniversity Institute for Marine Sciences. The three bathymetric zones are represented (shallow water corresponding to 0-30 m depth, in blue; mesophotic zone corresponding to 30-150 m depth, in red; and deep waters corresponding to 150-800 m depth, in green). Modified from Shoham and Benayahu (2016).

Figure 12 .

 12 Figure 12. Soft corals used during this thesis. Corresponding family, Symbiodiniaceae associated with the coral and growth form of the colony are mentioned. Photo credits are attributed to M. Boussion for Dendronephthya sp. and H. fuscescens (right), G. Banc-Prandi for Litophyton sp. (left), P. Colla for Sarcophyton sp. (right), and C. Pupier for H. fuscescens (left), R. f. fulvum (left) and Sarcophyton sp. (left).

Figure 13 .

 13 Figure 13. Hard corals used during this thesis. Corresponding family, growth form of the colony and Symbiodiniaceae associated with the coral are mentioned. Photo credits are attributed to E. Turak for A. eurystoma, N. Coleman for Cynarina sp., G. Paulay for Goniastrea sp., C. Veron for S. hystrix, and C. Pupier for G. fascicularis, P. damicornis and S. pistillata.

(

  depth) and Symbiodiniaceae genus on two soft coral species (Litophyton sp. and Rhytisma fulvum fulvum), sampled in the Red Sea at two depths (8 m and 40 m depth). These species harbor two different Symbiodiniaceae genera and different morphological shapes. The two depths tested enabled the assessment of the effect of light availability on carbon fluxes and the comparison with the carbon acquisition and allocation in the scleractinian coral Stylophora pistillata living at the same location and depths.  Aim 3 -Chapter 4: Acquisition of dissolved inorganic/organic nitrogen

For 2 )

 2 soft coral (Dendronephthya sp., Litophyton sp. and Rhytisma fulvum fulvum) and hard coral (Acropora eurystoma, Pocillopora damicornis, Goniastrea sp. and Cynarina sp.) species were sampled in the Red Sea at two depths (8-10 m and 40-45 m depth). These species harbor different Symbiodiniaceae genera (or not at all) and different morphological shapes. The depths investigated enable the assessment of the effect of increased heterotrophic capacity on dinitrogen fixation. ii) Uptake of DIN and DON A second experiment on nitrogen assimilation investigated the acquisition of dissolved inorganic and organic nitrogen by the soft coral-Symbiodiniaceae symbiosis. It aimed to determine which sources of dissolved nitrogen are acquired by soft corals. The effects of light availability (depth), Symbiodiniaceae genus and increased temperature were tested. As for the previous experiment, a comparison with hard corals was made to evaluate the efficiency of nitrogen assimilation among coral species. This experiment was designed to measure the assimilation of NH4 + , NO3 -, and DFAA in soft and hard corals, using stable isotope labelling. For this purpose, several species of soft corals (Litophyton arboreum, Rhytisma fulvum fulvum and Sarcophyton sp.) and hard corals (Galaxea fascicularis, Stylophora pistillata and Seriatopora hystrix) were sampled in the Red Sea at two depths (8-10 m and 40-50 m depth). These species harbor different Symbiodiniaceae genera and present different morphological shapes.  Aim 4 -Chapter 5: In situ acquisition of autotrophic and heterotrophic nutrients in soft corals

Figure 15 .

 15 Figure 15. Normalization metrics used in physiological studies on soft corals. Percentages are based on the occurrence of the studies using the metric of interest. DW = dry weight. AFDW = ash-free dry weight.

Figure 16 .

 16 Figure 16. Tissue parameter quantification in soft corals using different sample processing techniques. Samples were either processed frozen or after freeze-drying and homogenized in filtered seawater (FSW) or MilliQ water (DI). All tissue parameters (Symbiodinium density, (a); chlorophyll, (b) and protein content, (c)) are normalized to host dry weight. Error bars represent standard deviation of five samples.

Figure 17 .

 17 Figure 17. Correlations between the normalizing metrics. (a) Total dry weight with protein content and body volume of five Heteroxenia fuscescens colonies. (b) Linear regressions between total dry weight and ash of four colonies of Rhytisma fulvum, Dendronephthya sp., H. fuscescens and Litophyton arboreum. Grey areas display confidence interval of 0.95 around the regression lines.

Figure 18 .

 18 Figure 18. Optimized workflow for processing soft coral tissue samples. DW = dry weight. AFDW = ash-free dry weight. DI = MilliQ water.

  study was conducted in November 2017 at the Inter-University Institute for Marine Science (IUI), Gulf of Eilat. Experiments were performed with two soft coral species harboring dinoflagellate symbionts, Litophyton sp. (Forskål, 1775) and Rhytisma fulvum fulvum (Forskål, 1775) belonging to the Alcyoniidae family.Ten colonies per species were sampled on the reef adjacent to the IUI (29°51'N, 34°94'E) at 8 m and 40 m depth, respectively, to generate a total of 40 nubbins per species (two nubbins per colony). Nubbins were allowed to recover for ten days in open-water cages placed at the original collection site before being brought back to the Red Sea Simulator facility[START_REF] Bellworthy | The Red Sea Simulator: A high-precision climate change mesocosm with automated monitoring for the long-term study of coral reef organisms[END_REF]. There, nubbins were allocated to three different outside aquaria per species and depth. All aquaria were continuously supplied with water directly pumped from the reef and were exposed to the natural diurnal light cycle. Furthermore, aquaria were shaded with several layers of mesh clothes to adjust daily maximum light levels to the maximum irradiance measured at the corresponding depth. Thus, all nubbins were maintained under natural diurnal variation in irradiance and received the depth-corresponding in situ maximum irradiance at noon. Levels of photosynthetically active radiation (PAR) were obtained from the Israel National Monitoring program of the Gulf of Eilat (http://www.iuieilat.ac.il/Research/NMPMeteoData.aspx). During the five days of our experiment, surface

Figure 19 .

 19 Figure 19. Physiological and tissue descriptors measured in the soft coral species investigated. For panels (a), (b) and (c), data are normalized to ash-free dry weight. (a) Symbiodiniaceae density. (b) Total chlorophyll concentration. (c) Autotrophic carbon acquisition. (d) Autotrophic carbon acquisition per Symbiodiniaceae cell. Error bars represent standard error. There were significant differences between species and depths for (a), between depths for (b) and (d), and between species for (c) (* = p < 0.05, ** = p < 0.01, *** = p < 0.001).

Figure 20 .

 20 Figure 20. Carbon budgets obtained after the 5 hours pulse period in the soft coral species investigated. (a) Shallow Litophyton sp.. (b) Mesophotic Litophyton sp.. (c) Shallow Rhytisma fulvum fulvum. (d) Mesophotic Rhytisma fulvum fulvum. Symbionts are represented by a green circle. PC = autotrophic carbon acquisition. ρS = carbon assimilated in Symbiodiniaceae. ρH = carbon assimilated by the coral host. TS = translocation of photosynthates. CLS = carbon lost by the symbionts through respiration. CLH = carbon lost by the host through respiration. PC and carbon fluxes are expressed in µg C g -1 AFDW h -1 .

Figure 21 .

 21 Figure 21. Carbon budgets obtained after the 24 hours chase period in the soft coral species investigated. (a) Shallow Litophyton sp.. (b) Mesophotic Litophyton sp.. (c) Shallow Rhytisma fulvum fulvum. (d) Mesophotic Rhytisma fulvum fulvum. Symbionts are represented by a green circle. ρS = carbon assimilated in Symbiodiniaceae. ρH = carbon assimilated by the coral host. TS = translocation of photosynthetates. CLS = carbon lost by the symbionts through respiration. CLH = carbon lost by the host through respiration and mucus production. Carbon fluxes are expressed in µg C g -1 AFDW h -1 .

Figure 22 .

 22 Figure 22. Relationships between Symbiodiniaceae density and carbon fluxes of the soft coral species investigated. (a) Exponential relationship between carbon acquisition per cell and density of Symbiodiniaceae cells of Litophyton sp. and Rhytisma fulvum fulvum (R² = 0.78, y = 4.7087x -0.744 ). (b) Orthogonal regressions between carbon acquisition per cell and photosynthates translocation of Symbiodiniaceae cells of Litophyton sp. (R² = 0.60, y = 4.8964x -1.7547) and Rhytisma fulvum fulvum (R² = 0.99, y = 1.1818x -0.0091).

Figure 23 .

 23 Figure 23. Metabolic interactions between soft corals and their dinoflagellate symbionts. (a) Shallow Litophyton sp.. (b) Mesophotic Litophyton sp.. (c) Shallow Rhytisma fulvum fulvum. (d) Mesophotic Rhytisma fulvum fulvum. Boxes and arrows are indicative of differences in density and fluxes. Color intensity of symbionts corresponds to chlorophyll concentration levels.

  . It has been demonstrated that mesophotic scleractinian corals can display several strategies to acclimatize to low light levels, such as modifications in the organization of photosynthetic apparatus (shift to a PSII-based system, including additional photosynthetic antenna, Einbinder et al. 2016), modifications in the organization of antenna (growth of specialized paracrystalline light-harvesting antenna domains, Scheuring and Sturgis 2005), or the synthesis of fluorescent pigments by the host (that can perform wavelength transformation to facilitate light penetration, Smith et al. 2017); however, these strategies remain to be further investigated in soft corals.

  , the estimation of the capacity of different coral species to take advantage of this nutrient source can help in understanding how they cope with nutrient limitation. In addition, no study so far investigated DDN assimilation by soft corals, although they represent together with scleractinian corals the two most dominant benthic groups on many reefs worldwide. Soft corals can quickly colonize open space due to their fast growth rates, high fecundity and asexual reproduction and these opportunistic life history features may be facilitated by diazotrophs as additional N source (i.e. DDN). The aim of this study was to perform a multi-species comparison and to investigate the capacity of different scleractinian and soft coral species to either assimilate DDN/diazotrophic cells and/or transfer it to the surrounding seawater.

4. 2 . 2

 22 Field experiment: DDN/diazotrophic cells assimilation and transfer to seawater  Biological materialThe study was conducted during November 2017 at the Interuniversity Institute (IUI) of Marine Sciences, Eilat, Red Sea. Coral fragments were collected by SCUBA diving from different colonies of the reef adjacent to the IUI and were brought back to the Red Sea Simulator facility (see[START_REF] Bellworthy | The Red Sea Simulator: A high-precision climate change mesocosm with automated monitoring for the long-term study of coral reef organisms[END_REF] for more details regarding the system) to recover for one day prior to starting the incubations. Scleractinian corals (Acropora eurystoma, n = 6; Pocillopora damicornis, n = 3; Goniastrea sp., n = 3; and Cynarina sp., n = 2) were sampled in shallow waters (8-10 m depth) and soft corals (n = 5 for all species; Dendronephthya sp., Rhytisma fulvum fulvum and Litophyton sp.) in shallow waters and at upper mesophotic depths (40-45 m). Biological replicates for each species were derived from individual colonies and the different species were chosen in order to cover a broad range of families and morphological traits (Table7). Three additional nubbins per species per depth were collected to measure natural 15 N abundance of the corals. At the collection time, the site was characterized by stable temperature (24-25°C) and nutrient levels (< 0.5 µM dissolved N and < 0.2 µM dissolved phosphorus) along the depth gradient (data from the Israel National Monitoring program of the Gulf of Eilat, http://www.iui-eilat.ac.il/Research/NMPMeteoData.aspx).

Figure 24 .

 24 Figure 24. Scleractinian and soft corals DDN assimilation from two concentrations of active diazotrophs (Crocosphaera watsonii) in seawater. Investigated species are Stylophora pistillata (scleractinian coral) and Sarcophyton sp. (soft coral). Error bars indicate standard error. Letters refer to Tukey HSD testing. DDN = diazotrophicallyderived nitrogen. DW = dry weight.

Figure 25 .

 25 Figure 25. POC (a) and POC:PN ratio (b) measured in the incubation water of corals. POC = Particulate Organic Carbon; PN = Particulate Nitrogen. DW = dry weight. Asterisks indicate a significant difference between the two groups (scleractinian vs soft corals, p < 0.001). From left to right: Acr. = Acropora cervicornis, Cyn. = Cynarina sp., Gon. = Goniastrea sp., Poc. = Pocillopora damicornis, Den. = Dendronephthya sp., Lit. = Litophyton sp., Rhy. = Rhytisma fulvum fulvum.

Figure 26 .

 26 Figure 26. N2 fixation in seawater (a,b), assimilation in coral tissues (c), and relationship between the two (d). The white line indicates N2 fixation rate occurring in 120 µm-filtered seawater incubated without corals. Error bars indicate standard error. n.d. = not detected. DDN = diazotrophically-derived nitrogen. DW = dry weight. From left to right: Acr. = Acropora cervicornis, Cyn. = Cynarina sp., Gon. = Goniastrea sp., Poc. = Pocillopora damicornis, Den. = Dendronephthya sp., Lit. = Litophyton sp., Rhy. = Rhytisma fulvum fulvum.

  resulting different capacity of scleractinian and soft corals to promote active diazotroph populations and N2 fixation in reef waters may have several implications for N availability and reef biogeochemistry in the future. Particularly coral reefs that have experienced phase shifts from hard to soft coral dominance[START_REF] Stobart | Coral recovery at Aldabra Atoll, Seychelles: Five years after the 1998 bleaching event[END_REF][START_REF] Norström | Alternative states on coral reefs: beyond coral-macroalgal phase shifts[END_REF][START_REF] Pratchett | Changes in coral assemblages during an outbreak of Acanthaster planci at Lizard Island, northern Great Barrier Reef (1995-1999)[END_REF][START_REF] Inoue | Spatial community shift from hard to soft corals in acidified water[END_REF]) may suffer from a significant decrease in N2 fixation and subsequent N

  study was conducted at the Inter-University Institute (IUI) for Marine Sciences (Eilat, Israel) in November 2018. The experiment was performed with 3 species of hard corals (Galaxea fascicularis, Stylophora pistillata and Seriatopora hystrix) and 3 species of soft corals (Litophyton arboreum, Rhytisma fulvum fulvum and Sarcophyton sp.). Coral fragments were collected by SCUBA diving on the shallow (8-10 m depth) and mesophotic (40-50 m depth) reefs adjacent to the IUI. Temperature and nutrient levels at the time of sampling were not different between the shallow and mesophotic reefs. Temperature ranged from 24.7 to 25.4°Cduring the length of the experiment. Nitrate concentrations ranged from 0.17 to 0.25 µM, whereas dissolved inorganic phosphorus and ammonium were below 0.2 µM (data obtained from the National Monitoring Program of the Gulf of Eilat (http://www.iuieilat.ac.il/Research/NMPMeteoData.aspx). The genus of the symbionts hosted by the corals was investigated following the protocol of[START_REF] Santos | Molecular phylogeny of symbiotic dinoflagellates inferred from partial chloroplast large subunit (23S)-rDNA sequences[END_REF], using chloroplastic ribosomal deoxyribonucleic acid sequences.

Figure 27 .

 27 Figure 27. Symbiodiniaceae density in the species investigated. The first three species (Galaxea fascicularis, Seriatopora hystrix, Stylophora pistillata) are hard corals and the last three (Litophyton arboreum, Rhytisma fulvum fulvum, Sarcophyton sp.) are soft corals.Significant differences between depths are displayed with an asterisk. Significant differences between species are distinguished with letters by depth. AFDW = ash-free dry weight. ns = not sampled.

Figure 28 .

 28 Figure 28. Elemental composition of the host tissue in the species investigated. (a) C:N ratio. (b) N content. The first three species (Galaxea fascicularis, Seriatopora hystrix, Stylophora pistillata) are hard corals and the last three (Litophyton arboreum, Rhytisma fulvum fulvum, Sarcophyton sp.) are soft corals. Significant differences between depths are displayed with an asterisk. Significant differences between species are distinguished with letters by depth. AFDW = ash-free dry weight. ns = not sampled.

Figure 29 .

 29 Figure 29. Elemental composition of the Symbiodiniaceae fraction in the species investigated. (a) C:N ratio. (b) N content. The first three species (Galaxea fascicularis, Seriatopora hystrix, Stylophora pistillata) are hard corals and the last three (Litophyton arboreum, Rhytisma fulvum fulvum, Sarcophyton sp.) are soft corals. Significant differences between depths are displayed with an asterisk. Significant differences between species are distinguished with letters by depth. AFDW = ash-free dry weight. ns = not sampled.

Figure 30 .

 30 Figure 30. Assimilation rates of dissolved N in the host tissue in (a) hard corals (Galaxea fascicularis, Seriatopora hystrix, Stylophora pistillata) and (b) soft corals (Litophyton arboreum, Rhytisma fulvum fulvum, Sarcophyton sp.). Significant differences between depths are displayed with an asterisk. Since there was no similar assimilation rates between hard and soft corals, Δ shows the species that differentiate from the other two within its group (i.e. either hard or soft corals, for a single N source). AFDW = ash-free dry weight. DFAA = dissolved free amino acids. ns = not sampled.

Figure 31 .

 31 Figure 31. Assimilation rates of dissolved N in the Symbiodiniaceae fraction in (a) hard corals (Galaxea fascicularis, Seriatopora hystrix, Stylophora pistillata) and (b) soft corals (Litophyton arboreum, Rhytisma fulvum fulvum, Sarcophyton sp.). Significant differences between depths are displayed with an asterisk. Significant differences between species are distinguished with letters by depth. Blue bold letters highlight a similar rate between hard and soft coral species. AFDW = ash-free dry weight. ns = not sampled. DFAA = dissolved free amino acids. ns = not sampled.

Figure 32 .

 32 Figure 32. Assimilation rates of dissolved N in shallow soft corals exposed at two temperatures. (a) gathers rates related to the host tissue and (b) to the Symbiodiniaceae fraction. (Litophyton arboreum, Rhytisma fulvum fulvum, Sarcophyton sp.). Significant differences between temperatures are displayed with an asterisk. Δ shows the species that differentiate from the other two within its group (i.e. either host or Symbiodiniaceae fraction, for a single N source). AFDW = ash-free dry weight. DFAA = dissolved free amino acids. ns = not sampled.

Figure 33 .

 33 Figure 33. Total N assimilation rates in the species investigated. Values displayed are means ± standard error. (a) gathers hard corals (Gal = Galaxea fascicularis, Ser = Seriatopora hystrix, Sty = Stylophora pistillata) and (b) soft corals (Lit = Litophyton arboreum, Rhy = Rhytisma fulvum fulvum, Sar = Sarcophyton sp.). Sh = shallow. Dp = Deep. AFDW = ash-free dry weight. DFAA = Dissolved free amino acids.

  Colonies of Dendronephthya sp. (a non-symbiotic species), L. arboreum, R. f. fulvum, Sarcophyton sp. and S. pistillata (Figure34) were sampled by scuba diving on the reef adjacent to the Inter-University Institute for Marine Science (IUI) (29°51'N, 34°94'E), Gulf of Eilat, Israel, in November 2018. Five nubbins of approximately 3 cm were collected from different colonies for each of the five species. Samples were directly frozen at -80°C and freeze-dried after one week. These procedures were performed at both shallow (8-10 m) and upper mesophotic (40-50 m) sections of the reef.

Figure 34 .

 34 Figure 34. The five Red Sea coral species investigated for their nutritional ecology. From left to right: the asymbiotic octocoral Dendronephthya sp. (photo credit: M. Boussion) and symbiotic octocorals Litophyton arboreum (photo credit: G. Banc-Prandi), Rhytisma fulvum fulvum (photo credit: C. Pupier) and Sarcophyton sp. (photo credit: C. Pupier), along with the symbiotic scleractinian Stylophora pistillata (photo credit: E. Tambutté).

Figure 35 .

 35 Figure 35. Controls used for validation of the specificity of the fatty acid trophic markers SDA, DPA (stearidonic acid and docosapentaenoic acid, autotrophy markers) and CGA (cis-gondoic acid, heterotrophy marker). Sty and Sar respectively refer to Stylophora pistillata and Sarcophyton sp. grown at the laboratory. auto = autotrophy. mixo = mixotrophy. (i) Isolated symbiont cells from Stylophora pistillata, (ii) Stylophora pistillata and Sarcophyton sp. tissue from nubbins maintained under strict autotrophy, (iii) Stylophora pistillata and Sarcophyton sp. tissue from nubbins fed with zooplankton, and (iv) zooplankton mix, comprising rotifers, copepods and Artemia salina nauplii, used to feed the coral nubbins. Fatty acid concentration (µg g -1 ash-free dry weight) is expressed as mean  standard error.

Figure 36 .

 36 Figure 36. Non-metric multidimensional scaling (MDS) ordination used to assess similarities in the concentration (µg g -1 ash-free dry weight) of the fatty acids SDA, DPA (autotrophy markers) and CGA (heterotrophy marker) in the tissues of Dendronephthya sp., Litophyton arboreum, Rhytisma fulvum fulvum, Sarcophyton sp., and Stylophora pistillata, sampled in both shallow (8-10 m) and mesophotic (40-50 m) sections of the reef adjacent to the Inter-University Institute for Marine Sciences, in Eilat, Israel.

Figure 37 .

 37 Figure 37. Concentration (µg g -1 ash-free dry weight) of the fatty acid trophic markers (a) SDA, (b) DPA (stearidonic acid and docosapentaenoic acid, autotrophy markers) and (c) CGA (cis-gondoic acid, heterotrophy marker) from the tissues of Dendronephthya sp. (asymbiotic octocoral, Den), Litophyton arboreum (symbiotic octocoral, Lit), Rhytisma fulvum fulvum (symbiotic octocoral, Rhy), Sarcophyton sp. (symbiotic octocoral, Sar), and Stylophora pistillata (symbiotic scleractinian coral, Sty), sampled in both shallow (8-10 m) and mesophotic (40-50 m) sections of the reef adjacent to the Inter-University Institute for Marine Sciences, in Eilat, Israel. Data are expressed as mean  standard error.

(

  2012). In a nutshell, in hard corals, increased dissolved inorganic nitrogen concentrations promote a fast population growth of dinoflagellates. A proliferating symbiont population can induce an enhanced cellular demand, thereby leading to an undersupply in phosphorus or other essential nutrients, and ultimately to the starvation of symbiont cells. Such condition alters the lipid composition of their membranes, which renders corals more susceptible to bleaching through the production of reactive oxygen species (D'Angelo and Wiedenmann 2014).

6. 3 . 1

 31 Investigate the internal light environment of soft corals: the role of tissue and sclerites Scleractinian corals have the ability to regulate the light environment perceived by their symbionts in hospite. For example, they can filter out the excess of light that can be harmful to the algae or enhance their internal light diffusion and scattering due to unique optical properties of their tissue and skeleton. Such amplification in the local light field of the symbionts notably improves photosynthesis. While soft corals do not have a skeleton, which might explain their lower rates of carbon acquisition and assimilation compared to scleractinian corals, they possess a large amount of sclerites. Sclerites could act as light diffusers, and their species-specific size, shape and color could help explaining differences in photosynthetic efficiencies. It would thus be interesting to investigate the internal light environment of soft corals as well as the possible role of sclerites in light scattering, using light microsensors. The hydroskeleton of soft corals may however entail some technical problems as it is likely to shrink on microsensors. 6.3.2 Importance of phosphorus acquisition for the stability of the soft coral symbiosis

  l'hétérotrophie à la nutrition des coraux mous n'est pas fixe et dépend des conditions environnementales et/ou de la disponibilité des ressources (par exemple de la profondeur et de la saison ;[START_REF] Rossi | Trophic ecology of Caribbean octocorals: autotrophic and heterotrophic seasonal trends[END_REF]. Cependant, cette notion reste à étudier plus en détails.1.4 Les principales menaces pesant sur les récifs coralliens et les coraux mousLes récifs coralliens sont généralement capables de se remettre des perturbations naturelles en quelques années ou décennies s'ils ne subissent pas d'autres dommages entre temps. Cependant, les activités humaines ajoutent aux perturbations naturelles des facteurs de stress locaux et globaux, qui agissent ensemble pour affaiblir les récifs coralliens et finissent par entraîner des déclins importants. En conséquence, les écosystèmes marins se modifient et les récifs coralliens sont parmi les plus menacés.

Figure récapitulative .

 récapitulative Figure récapitulative. Différence de fonctionnement des symbioses nutritionnelles du corail mou et du corail dur des zones récifales peu profondes à mésophotiques en mer Rouge. La gamme des taux correspondant à l'acquisition et aux flux de nutriments est indiquée sous l'acronyme des nutriments. Ces taux ne sont pas comparables entre eux mais uniquement entre les groupes

Fabricius

  Photosynthetic and accessory pigments (chlorophyll a, phaeopigments and total photopigments)Fabricius et al. (1995b) Nephtheidae Dendronephthya hemprichiFeeding rates (phytoplankton)Nephtheidae Dendronephthya sinaiensis Feeding rates (phytoplankton)Nephtheidae Scleronephthya corymbosa Feeding rates (phytoplankton)Nephtheidae Dendronephthya hemprichi Feeding rates (phytoplankton)Nephtheidae Dendronephthya hemprichi Feeding rates (phytoplankton)[START_REF] Fabricius | In situ depletion of phytoplankton by an azooxanthellate soft coral[END_REF]

Figure S1 :

 S1 Figure S1: Levels of photosynthetically active radiation (PAR) received at the seasurface, 10 m depth and 40 m depth in Eilat during the five days of our experiment. Surface data was provided by the Israel National Monitoring program of the Gulf of Eilat and levels along the depth gradient were calculated according to Beer-Lambert law, with an attenuation coefficient of 0.1 m -1[START_REF] Akkaynak | What is the space of attenuation coefficients in underwater computer vision[END_REF][START_REF] Tamir | Light environment drives the shallow to mesophotic coral community transition[END_REF]).

Figure S2 .

 S2 Figure S2. Total chlorophyll concentration in the species investigated. Normalized to biomass in the top panel (AFDW = ash-free dry weight) and normalized to Symbiodiniaceae cell in the bottom panel. The first three species (Galaxea fascicularis, Seriatopora hystrix, Stylophora pistillata) are hard corals and the last three (Litophyton arboreum, Rhytisma fulvum fulvum, Sarcophyton sp.) are soft corals. Significant differences between depths are displayed with an asterisk. Significant differences between species are distinguished with letters by depth. ns = not sampled.

Figure S3 .

 S3 Figure S3. Assimilation rates of the three N sources in hard corals, normalized to surface area of the skeleton, in the host tissue (top panel) and in the Symbiodiniaceae fraction (bottom panel). Significant differences between depths are displayed with an asterisk. Significant differences between species are distinguished with letters by depth. Gal = Galaxea fascicularis. Ser = Seriatopora hystrix. Sty = Stylophora pistillata.

  Key words: octocoral | symbiosis | autotrophy | heterotrophy | mesophotic

Table des illustrations

 des Liste des figures

  This is a simplified scheme, which should comprise eight mesenteries (i.e. that alternate with the eight tentacles) and eight mesenterial filaments. The gastrovascular cavity ends in a dead-end canal. Retractor septal muscles and transverse fibers of mesenteries, which play a role in polyp retraction and movement of tentacles are not displayed here. Drawing by C. Pupier, based on[START_REF] Fabricius | Soft corals and sea fans: a comprehensive guide to the tropical shallow-water genera of the Central-West Pacific, the Indian Ocean and the Red Sea[END_REF].Conversely to hard corals, soft corals do not produce a calcium carbonate skeleton.Instead, they rely on hydrostatic pressure for body support. Most soft corals however possess small isolated calcium carbonate structures called sclerites. They are formed by specialized cells (i.e. scleroblasts) in the coenenchyme and are composed of magnesium-enriched calcite.

Table 2 . Reported shifts from hard corals to soft corals dominance. COTS

 2 : crown-of-thorn starfishes. From van de[START_REF] Van De Water | Host-microbe interactions in octocoral holobionts -recent advances and perspectives[END_REF].

		Location	Cause	Abundance of octocoral	References
				(cover)
				From (%) Up to (%)
	Red Sea	North and Center	Anthropogenic pollution,	5-10	30-50	(Al-Zibdah et al. 2007; Tilot et al.
			COTS outbreak			2008; Wilkinson 2008)
		South	Anthropogenic pollution,	4	30	(Reinicke et al. 2003; Bruckner and
			bleaching, storms			Dempsey 2015)
	Caribbean	Florida Keys	Bleaching	6	14	(Ruzicka et al. 2013)
		US Virgin Islands	Diseases			(Lenz et al. 2015)
	Indian Ocean Madagascar/Seychelles	Bleaching	1.3	2	(Stobart et al. 2005)
		Seychelles	Anthropogenic pollution,			(Wilkinson 2008)
			bleaching
	Pacific Ocean	Malaysia	Bleaching, COTS outbreak,	1	30	(Wood and Dipper 2008)
			dynamite fishing
		Indonesia	Anthropogenic pollution	7	13	(Baum et al. 2016)
			Dynamite fishing			(Fox et al. 2003)
		Australia	Bleaching, COTS outbreak	10	16	(Wakeford et al. 2008)
		Fiji	Anthropogenic pollution			(Hoffmann 2002)

  1,2 , Vanessa N. Bednarz 1 , Christine Ferrier-Pagès 1

1 Centre Scientifique de Monaco, Marine Department, 8 Quai Antoine Ier, MC-98000 Monaco, Principality of Monaco 2 Sorbonne Université, Collège doctoral, F-75005 Paris, France

Table 4 . Statistical results about the effect of sample state and homogenization media on Symbiodinium density, total chlorophyll and protein concentrations measured in Heteroxenia fuscescens.

 4

	Factor	df	F values p values
	Symbiodinium density
	Sample state	1	0.801	0.384
	Homogenization media	1	1.121	0.305
	Sample state × homogenization media	1	0.026	0.873
	Residuals	16
	Total chlorophyll concentration
	Sample state	1	80.478	<0.01
	Homogenization media	1	3.058	0.100
	Sample state × homogenization media	1	0.284	0.602
	Residuals	16
	Protein concentration
	Sample state	1	1.251	0.280
	Homogenization media	1	11.925	<0.01
	Sample state × homogenization media	1	0.005	0.947
	Residuals	16

Table 5 . Significant correlations between dry weight (DW), protein content, body volume and ash weight (AW) of Heteroxenia fuscescens, Rhytisma fulvum, Dendronephthya sp. and Litophyton arboreum.

 5

	Species	Tested correlation	R	t	df	p values
	H. fuscescens	DW × protein content DW × body volume	0.9682 6.7005 0.9964 20.341	3 3	<0.01 <0.01
	R. fulvum	DW × AW	0.9997 161.28	18	<0.01
	Dendronephthya sp. DW × AW	0.9996 157.53	18	<0.01
	H. fuscescens	DW × AW	0.9868 25.866	18	<0.01
	L. arboreum	DW × AW	0.9648 15.575	18	<0.01

  1,2 , Maoz Fine 3,4 , Vanessa N. Bednarz 1 , Cécile Rottier 1 ,

Renaud Grover 1 ǂ , Christine Ferrier-Pagès 1 ǂ 1 Centre Scientifique de Monaco, Marine Department, 8 Quai Antoine Ier, MC-98000 Monaco, Principality of Monaco 2 Sorbonne Université, Collège doctoral, F-75005 Paris, France 3 The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel 4 The Interuniversity Institute for Marine Science in Eilat, PO Box 469, Eilat 88103, Israel ǂ equal contribution

  Litophyton sp. harbored Symbiodinium sp. (formerly Symbiodinium clade A, LaJeunesse et al. 2018) whereas R. f.

	fulvum was associated with Cladocopium thermophilum (formerly Symbiodinium
	thermophilum clade C3, Hume et al. 2015; LaJeunesse et al. 2018), regardless of the depth
	investigated. Symbiodiniaceae density, normalized to AFDW, was two-fold higher in

Table 6 . Tissues descriptors and autotrophic carbon acquisition reported for different octocorals, normalized to ash-free dry weight (AFDW

 6 ). PC = autotrophic carbon acquisition.

			OCTOCORALS
	Species	Depth	Symbiont density	Total chlorophyll (a+c2)	PC	Reference
		(m)	(10 8 cell g -1 AFDW)	(µg g -1 AFDW)	(µg C g -1 AFDW h -1 )
	Great Barrier Reef
	Asterospicularia sp.	20			530
	Briareum stechei	5			419-452
		20			707-838
	Capnella lacertiliensis	5			949-1047
		20			511
	Efflatounaria sp.	5			975-1375
		20			772-1191
	Lobophytum spp.	5			367
		20			419
	Nephthea sp.	5			1145
		10			1100
		20			956	Fabricius and
	Paralemnalia sp.	5			838-923	Klumpp (1995)
		10			792-1191
		20			884-956
	Sarcophyton spp.	5			367-445
		10			1034-3076
		20			511-707
	Sinularia spp.	5			713-825
		10			772
		20			406-903
	Xenia spp.	5			890-1231
		10			1165-1872
		20			812
	Caribbean
	Antillogorgia sp.	2-8	1-3	1300-2120	1105-2291	Baker et al. (2015); Rossi et al. (2018)
	Briareum asbestinum	3-8			249	Baker et al. (2015)
	Eunicea sp.	2-8	1-4	760-2150	51-382
	Gorgonia ventalina	2-8	1	730	457-3284	Baker et al. (2015);
	Plexaura sp.	2-8	0.5	1280	20-742	Rossi et al. (2018)
	Pterogorgia anceps	2-8	2	3490-4050	184-3338
	Red Sea
	Litophyton sp.	8	9.4	1591	958
		40	6.4	2240	829	This study
	Rhytisma f. fulvum	8	4.5	1222	554
		40	2.9	2075	710

Table 7 . Scleractinian and soft coral species investigated in the study.

 7

	Coral group Species	Family	Morphology
	Scleractinian Acropora eurystoma	Acroporidae	Branching
		Cynarina sp.	Lobophyliidae	Mounding
		Goniastrea sp.	Merulinidae	Mounding
		Pocillopora damicornis	Pocilloporidae	Branching
	Soft	Dendronephthya sp.	Nephtheidae	Arborescent
		Litophyton sp.	Nephtheidae	Arborescent
		Rhytisma fulvum fulvum	Alcyoniidae	Mat-forming
	 N2 fixation measurements

Table 8 . Predominant symbiont species associated with the four symbiotic Red Sea coral species sampled at shallow and mesophotic depths of the reef adjacent to the Inter- University Institute for Marine Sciences in Eilat, Israel.

 8

	Host coral species	Shallow (8-10 m)	Mesophotic (40-50 m)
	Litophyton arboreum	Symbiodinium sp. A10	Symbiodinium sp. A10
	Rhytisma fulvum fulvum	Cladocopium goreaui C1	Cladocopium goreaui C1
	Sarcophyton sp.	Cladocopium goreaui C1	Cladocopium goreaui C1
	Stylophora pistillata	Symbiodinium microadriaticum A1	Cladocopium goreaui C1

  . La taxonomie des Symbiodiniaceae a été récemment révisée, avec une description formelle de sept genres[START_REF] Lajeunesse | Systematic revision of Symbiodiniaceae highlights the antiquity and diversity of coral endosymbionts[END_REF]. L'association entre les coraux et les dinoflagellés est appelée symbiose mutualiste et se présente comme un partenariat basé sur la nutrition. Les coraux symbiotiques sont ainsi considérés comme des organismes mixotrophes car ils peuvent utiliser des sources de nutriments à la fois inorganiques et organiques. Alors que l'animal est capable de capturer des proies en suspension dans le milieu, les dinoflagellés fixent le carbone inorganique via la photosynthèse et sont également capables d'absorber d'autres nutriments dissous, comme l'ammonium, les nitrates et les phosphates. Certaines bactéries telles que les diazotrophes fixent quant à elles le diazote. Chaque partenaire échange des nutriments avec les autres partenaires pour soutenir sa propre nutrition et subvenir à ses propres besoins métaboliques. Ainsi, les dinoflagellés transfèrent plus de 90% de leurs photosynthétats à l'animal (dans le cas des coraux scléractiniaires) dans les récifs de surface. Ce mode de nutrition multiple permet aux coraux de tirer le meilleur parti des eaux oligotrophes, où chacun des partenaires ne persisterait guère seul en raison de la faible concentration de nutriments inorganiques et de la faible disponibilité des proies[START_REF] Stoecker | Mixotrophy in the marine plankton[END_REF].Le principal trait qui distingue les octocoralliaires des scléractiniaires (coraux durs), outre une symétrie d'ordre huit, est l'absence d'un squelette de carbonate de calcium. De ce fait, les coraux mous dépendent, pour le soutien de leur corps, à la fois de la pression hydrostatique et d'une « micro-ossature » constituée d'une multitude de petites structures isolées de carbonate de calcium appelées sclérites. Les octocoralliaires constituent le deuxième groupe d'animaux macrobenthiques le plus abondant sur de nombreux récifs de l'Indo-Pacifique, des Caraïbes et de la mer Rouge, après les coraux durs[START_REF] Benayahu | Competition for space among coral-reef sessile organisms at Eilat, Red Sea[END_REF]

	Fabricius et Alderslade, 2001 ; Benayahu et al., 2019 et références dans ce document). Les
	octocoralliaires peuvent être associés à cinq genres de Symbiodiniaceae selon les régions
	biogéographiques. La diversité génétique au sein des genres de la famille Symbiodiniaceae
	reflète également une diversité physiologique, expliquant différentes réponses aux
	changements environnementaux.
	Les interactions
	métaboliques au sein de ce consortium symbiotique, qui sont nombreuses et diverses,
	conditionnent également la réponse du corail aux changements environnementaux (par
	exemple, voir Ainsworth et al. 2017 ; Benavides et al. 2017 ; van de Water et al. 2018 ; Pernice
	et al. 2019).
	1.3 Zoom sur la symbiose et la nutrition des octocoralliaires
	Les octocoralliaires, qui sont le sujet principal de cette thèse, appartiennent au phylum
	Cnidaria et à la classe Anthozoa. Actuellement, les octocoralliaires se composent de trois
	ordres : Helioporacea (communément appelés « coraux bleus »), Pennatulacea (communément
	appelées « plumes de mer ») et Alcyonacea. Les principaux groupes de l'ordre Alcyonacea
	incluent les gorgones, le corail rouge et les coraux mous. Contrairement aux gorgones et au
	corail rouge, les coraux mous ne présentent pas d'axe interne solide. Tout au long de ce résumé,
	le terme « octocoralliaire » qualifie donc tout membre de cette sous-classe sans distinction entre
	taxons, alors le terme « coraux mous » se réfère uniquement aux octocoralliaires ne disposant
	pas d'un axe interne solide.

  Aqaba, nord de la mer Rouge), deux communautés distinctes d'octocoralliaires ont été identifiées, l'une en surface et l'autre dans la zone mésophotique supérieure(30-45 m). Il est intéressant de noter que la communauté d'octocoralliaires qui se développe en profondeur est caractérisée par une plus grande richesse en espèces, ainsi que par une prédominance d'espèces symbiotiques. Bien que ces caractéristiques suggèrent un régime lumineux adéquat pour que la photosynthèse du corail s'y déroule[START_REF] Shoham | Higher species richness of octocorals in the upper mesophotic zone in Eilat (Gulf of Aqaba) compared to shallower reef zones[END_REF], les stratégies nutritionnelles de ces coraux mous ont fait l'objet de peu d'attention jusqu'à présent. La nature gélatineuse, ou semblable au cuir, des tissus des coraux mous ne permet pas une extraction facile des descripteurs tissulaires, tels que la densité des symbiotes, la concentration de pigments et de protéines, 2) la métrique de normalisation la plus utilisée chez les coraux scléractiniaires, la surface du squelette, n'est pas pertinente pour les coraux mous puisqu'ils ne possèdent pas de squelette, 3) la surface d'un fragment de corail mou est difficile à estimer en raison de leur hydrosquelette très variable, qui peut rapidement changer de taille en fonction des conditions environnementales et 4) ils ne permettent pas la comparaison de paramètres et processus physiologiques des coraux mous à ceux d'autres espèces de coraux comme les scléractiniaires. poids sec sans cendres. Dans l'ensemble, ce protocole optimisé offre une quantification plus fiable des descripteurs tissulaires et réduit le risque de sous-estimation de ces paramètres chez les coraux mous. Enfin, étant donné que la contribution des sclérites au poids sec total de la colonie peut être très différente d'une espèce à l'autre, nous avons démontré que la différence entre le poids sec et le poids sec sans cendre, qui correspond à la quantité de matière organique de l'échantillon, est une mesure fiable pour normaliser les données relatives aux coraux mous,

	2. Résultats
	2.1 Métrique de normalisation et protocole de traitement des échantillons
	(Chapitre 2)
	. Des
	recherches supplémentaires sont donc nécessaires pour expliquer les effets de la qualité de l'eau sur les octocoralliaires. En conséquence, les octocoralliaires sont souvent abondants dans les systèmes récifaux perturbés où ils peuvent présenter un taux de survie plus élevé que les coraux durs sous un large éventail de conditions environnementales. Des changements dans la structure de la communauté aboutissant à la dominance des octocoralliaires ont été observés suite à des épidémies d'étoiles de mer corallivores (Acanthaster planci), à la pollution anthropique, à des tempêtes, au blanchissement, à des maladies et à la pêche à la dynamite. Ces perturbations ont conduit à une diminution de la couverture des coraux durs alors que les coraux mous ont maintenu ou augmenté leur abondance. 1.5 Objectifs de la thèse La communauté scientifique n'a encore que très peu de données sur les caractéristiques physiologiques des coraux mous, ou sur leur écologie trophique et leur relation avec leurs symbiotes (Schubert et al., 2017, van de Water et al., 2018). La recherche sur les octocoralliaires est en effet très en retard par rapport à la recherche sur les coraux durs et d'importantes lacunes dans les connaissances relatives à leur physiologie, en particulier à leur nutrition, doivent encore être comblées. La nutrition est l'un des facteurs les plus importants impliqués dans la régulation de l'abondance et la distribution d'une communauté au sein d'un écosystème, au même titre que les conditions environnementales et les interactions avec d'autres organismes (par exemple la compétition et la prédation). En particulier, l'acquisition de nutriments fournit l'énergie nécessaire à la croissance et à la reproduction des coraux et peut favoriser la résistance ou la résilience des coraux aux changements environnementaux. Dans les récifs oligotrophes d'Eilat (golfe d'Cette thèse se concentre donc sur le fonctionnement de la symbiose dinoflagellé-corail mou et vise à apporter des connaissances sur l'écologie nutritionnelle de plusieurs espèces d'octocoralliaires symbiotiques du nord de la mer Rouge, jusqu'à des profondeurs mésophotiques. La symbiose dinoflagellé-octocoralliaire est connue pour être différente de celle des coraux scléractiniaires, en termes de diversité et de stabilité. Alors que les octocoralliaires présentent une faible diversité de symbiotes, les coraux scléractiniaires hébergent généralement plusieurs types de symbiotes et sont plus flexibles en fonction des conditions environnementales dominantes (Baker et Romanski, 2006). Cette différence a été attribuée au fait que les octocoralliaires dépendent davantage de l'hétérotrophie et ont donc moins à gagner d'une telle flexibilité symbiotique (van Oppen et al., 2005). Cependant, l'importance de l'autotrophie et de l'hétérotrophie, ainsi que leur contribution respective aux besoins métaboliques de l'hôte ont été peu étudiées chez les octocoralliaires et principalement testées chez les gorgones (Rossi et al., 2020). Les coraux mous étant l'une des principales composantes des écosystèmes récifaux et le changement climatique et les perturbations locales étant susceptibles d'induire des transitions de plus en plus importantes vers des récifs dominés par les octocoralliaires (par exemple Tsounis et Edmunds, 2017), il devient primordial de faire progresser les connaissances sur l'écologie nutritionnelle de leur symbiose. Par conséquent, dans cette thèse, nous avons étudié l'acquisition et les flux de nutriments organiques et inorganiques, chez plusieurs hôtes de corail mou, leurs symbiotes dinoflagellés, et d'autres micro-organismes avec lesquels ils seraient éventuellement associés tels que les diazotrophes. Les comparaisons avec les symbioses scléractiniaires-dinoflagellés ont mis en évidence des différences fonctionnelles majeures entre les deux groupes et ont permis de discuter nos résultats dans un contexte plus large. La normalisation est une étape fondamentale dans le traitement des données, dont la pertinence influencera profondément le résultat d'une expérience et son interprétation. Chez les coraux durs, ce choix se porte généralement sur la surface du squelette, ainsi que la quantité de chlorophylles ou de protéines après extraction des tissus. Cependant, la structure des coraux mous entraîne un certain nombre de problèmes pour l'homogénéisation des tissus et la normalisation des données : 1) Un protocole simple et rapide pour l'homogénéisation des tissus, ainsi qu'une métrique de normalisation pouvant être utilisée pour effectuer des études ou des comparaisons inter-espèces, sont donc nécessaires. Dans cette première étude, nous avons vérifié si l'état de l'échantillon de tissu avant traitement (échantillons congelés vs lyophilisés) et le milieu utilisé pour l'homogénéisation des tissus (eau de mer filtrée à 0,2 μm vs eau distillée) affectent les mesures des descripteurs de tissu (concentrations en chlorophylles, protéines et dinoflagellés) chez l'espèce modèle Heteroxenia fuscescens. En outre, la pertinence du poids sec (après lyophilisation) et du poids sec sans cendres (après calcination à 450°C pendant plusieurs heures) comme métrique de normalisation a été étudiée chez différentes espèces de coraux mous. Nos résultats ont révélé que la lyophilisation des échantillons et leur homogénéisation dans de l'eau distillée présentent plusieurs avantages, à savoir la minimalisation des pertes de chlorophylles et protéines (jusqu'à 50%), un gain de temps, ainsi qu'une détermination plus précise du poids 2.2 Acquisition du carbone inorganique dissous (Chapitre 3) L'acquisition autotrophe de carbone contribue largement aux besoins métaboliques des coraux durs, qui reçoivent 90% des photosynthétats produits par les symbiotes. Chez les octocoralliaires, la contribution des symbiotes aux besoins énergétiques de leur hôte est encore mal comprise. Tout d'abord, les études portant sur la productivité (primaire) des coraux mous sont rares, limitées à une profondeur de 20 m et les résultats obtenus présentent une forte variabilité (voir les informations supplémentaires dans Rossi et al., 2018). Il existe plusieurs sources identifiées pour cette variabilité : 1) les colonies de coraux mous peuvent atteindre différents degrés d'expansion en fonction des conditions environnementales, ce qui confère une grande variabilité biologique aux mesures (voir par exemple Fabricius et Klumpp, 1995) ; 2) la morphologie de la colonie et des polypes, notamment le rapport surface/volume, peut être plus sec et du notamment lorsque des comparaisons entre espèces sont effectuées. ou moins adaptée à l'autotrophie

  C, qui est un isotope stable, sur deux espèces de coraux mous (Litophyton sp. et Rhytisma fulvum fulvum), provenant des récifs coralliens de la mer Rouge de surface et de la zone mésophotique supérieure (40-50 m), afin de quantifier l'acquisition et l'allocation de carbone autotrophe au sein de l'association symbiotique. Les mesures de l'acquisition et de la respiration du carbone distinguent Litophyton sp. comme une espèce principalement autotrophe et Rhytisma fulvum fulvum comme une espèce plutôt hétérotrophe. Chez les deux espèces, l'acquisition du carbone était constante aux deux profondeurs étudiées. Cela représente une différence majeure avec les coraux scléractiniaires, dont l'acquisition de carbone diminue avec la profondeur. De plus, l'acquisition du carbone et le transfert de photosynthétats vers l'hôte diminuent avec l'augmentation de la densité des symbiotes, ce qui suggère qu'à forte densité, les symbiotes conservent les nutriments pour leurs propres besoins au lieu de les transférer à leur hôte.2.3 Acquisition de l'azote inorganique/organique dissous (Chapitre 4) La présence des dinoflagellés symbiotiques procure de nombreux avantages aux organismes qui les accueillent au sein de leurs cellules. Parmi ces avantages figurent la capacité de survivre dans des environnements oligotrophes grâce à une absorption, une conservation et un recyclage efficaces des nutriments, réalisés essentiellement par les symbiotes. L'azote est un élément nutritif essentiel au fonctionnement des holobiontes coralliens, mais sa disponibilité est souvent faible dans les eaux récifales du fait de la rareté de ses formes dissoutes. Des études récentes ont fait progresser notre compréhension du cycle de l'azote chez les coraux scléractiniaires. Il a été montré que ces coraux peuvent acquérir presque toutes les formes 'indications sur les bénéficiaires de l'azote fixé parmi les constituants de l'holobionte. Seule l'utilisation du gaz 15 N2, permet de suivre le devenir de l'azote diazotrophe (DDN) dans les différents compartiments de l'holobionte corallien.Une première étude a donc été réalisée pour évaluer l'importance de la fixation d'azote pour la nutrition des coraux mous. L'objectif de cette étude était notamment de savoir si les coraux mous sont associés à des diazotrophes actifs. Nous avons quantifié les taux d'assimilation de DDN chez quatre espèces de scléractiniaires et trois espèces de coraux mous des eaux peu profondes de la mer Rouge en utilisant la technique du traceur 15 N2. Toutes les espèces de scléractiniaires ont stimulé de manière significative la fixation de N2 dans l'eau de mer (et le mucus) entourant le corail et ont assimilé le DDN dans leurs tissus. Au contraire, la fixation de N2 n'a pas été détectée dans l'eau d'incubation des coraux mous, et aucune assimilation de DDN n'a été mesurée dans les tissus de ces coraux. Cependant, des incubations au laboratoire de coraux mous avec des cultures de diazotrophes ont montré que les coraux chez plusieurs espèces de coraux mous et scléractiniaires échantillonnés dans la mer Rouge (Eilat, Israël) à deux profondeurs différentes (8-10 m et 40-50 m). Le principal résultat montre que pour toutes les sources d'azote, les coraux scléractiniaires présentent des taux d'assimilation par biomasse plus élevés que les coraux mous (jusqu'à dix fois), bien qu'ils abritent les mêmes genres de Symbiodiniaceae en densités similaires. Cela suggère que les

	coraux mous doivent compléter leur faible acquisition d'azote dissous par l'acquisition d'autres
	formes d'azote, s'appuyant ainsi plutôt sur une alimentation hétérotrophe pour satisfaire leurs
	besoins en azote structurel. Cette découverte met en évidence une acquisition d'azote très
	différente entre les symbioses de coraux mous et scléractiniaires avec leurs dinoflagellés. Un
	tel contraste dans la capacité à tirer parti de cette source de nutriments peut aider à comprendre
	comment les coraux mous et scléractiniaires répondent à différents degré d'enrichissement
	d'eau de mer en nutriments.
	2.4 Acquisition in situ de nutriments autotrophes et hétérotrophes chez les coraux
	mous (Chapitre 5)
	Étant donné que l'azote est souvent un nutriment limitant la productivité primaire dans les eaux
	 Diazotrophie oligotrophes des récifs, la capacité divergente des coraux scléractiniaires et des coraux mous à
	favoriser la fixation de l'azote peut avoir des implications sur la disponibilité de l'azote et la
	La forme la plus abondante d'azote dissous, le diazote atmosphérique, ne peut être biogéochimie des récifs dominés par des scléractiniaires par rapport aux récifs dominés par les
	rendue biologiquement disponible que par l'action de bactéries fixatrices de diazote appelées coraux mous.
	diazotrophes (qui réduisent le diazote en ammonium). En milieu marin, ces bactéries
	diazotrophes existent sous forme libre ou forment une association symbiotique avec de  Assimilation du DIN et du DON
	nombreux invertébrés qui vont tirer profit de leur activité fixatrice. Les quelques études portant
	sur l'assimilation de l'azote dérivé des diazotrophes (DDN) par les coraux tropicaux se limitent La capacité des coraux mous à assimiler l'azote dissous à l'échelle de l'organisme restant
	à quelques espèces de scléractiniaires (e.g. Stylophora pistillata) et seules deux publications ont inexplorée, une deuxième expérience sur l'assimilation d'azote a eu pour objet l'étude de

d'azote dissous, dont le diazote gazeux, les formes inorganiques telles que l'ammonium et le nitrate, ainsi que des composés organiques dissous tels que des acides aminés. Malgré ces connaissances obtenues sur les coraux scléractiniaires, la capacité des autres groupes coralliens, tels que les octocoralliaires, à assimiler les différentes formes d'azote n'a pratiquement jamais été évaluée. Dans cette thèse, nous avons donc abordé l'assimilation de toutes ces formes d'azote par les coraux mous, et comparé les taux d'assimilation obtenus avec ceux mesurés chez les espèces de coraux scléractiniaires.

étudié les taux de fixation du diazote en présence de coraux mous

[START_REF] Bednarz | Seasonal variation in dinitrogen fixation and oxygen fluxes associated with two dominant zooxanthellate soft corals from the northern Red Sea[END_REF][START_REF] Cardini | Budget of primary production and dinitrogen fixation in a highly seasonal Red Sea coral reef[END_REF]

. Cependant, ces études ont utilisé la technique de réduction de l'acétylène, qui ne quantifie que la fixation de diazote se produisant dans l'eau de mer brute, sans fournir dmous étaient capables d'absorber le DDN provenant de diazotrophes libres. Le mucus des coraux mous représente probablement un habitat défavorable pour la colonisation et l'activité des diazotrophes car il contient une faible quantité de matière organique particulaire, avec une teneur en azote relativement élevée, par rapport au mucus des coraux scléractiniaires. De plus, il est connu pour présenter des propriétés antimicrobiennes. Dans l'ensemble, cette étude suggère que l'assimilation du DDN dans les tissus du corail dépend de la présence de diazotrophes actifs dans la couche de mucus du corail et/ou dans l'eau de mer environnante. l'acquisition de l'azote inorganique et organique dissous par la symbiose coraux mous-Symbiodiniaceae. En utilisant le marquage avec des isotopes stables, nous avons effectué une comparaison multi-espèces pour étudier l'assimilation d'ammonium, de nitrate et d'acides aminés libres Les octocoralliaires sont d'importants habitants des récifs, de la surface aux profondeurs mésophotiques et au-delà. Cependant, l'acquisition autotrophe ou hétérotrophe de nutriments est mal connue chez les coraux mous symbiotiques. Comme les coraux ont la capacité de capturer des proies, l'hétérotrophie peut représenter une acquisition importante de carbone, d'azote et d'autres nutriments. La littérature sur le régime alimentaire des coraux mous tropicaux est cependant très rare (par exemple

  et la capacité hétérotrophe des coraux a été principalement estimée par des études en laboratoire. Le statut hétérotrophe des coraux en conditions in situ est donc encore mal compris, et ce pour toutes les espèces de coraux. de l'autotrophie avec la profondeur, en accord avec les mesures précédentes d'acquisition de carbone par la photosynthèse. L'augmentation de la capacité hétérotrophe avec la profondeur était spécifique à l'espèce, probablement liée aux caractéristiques physiologiques et morphologiques. Le niveau relativement élevé du marqueur hétérotrophe chez toutes les

	espèces symbiotiques en eaux peu profondes ne corrobore pas l'idée reçue selon laquelle les
	coraux dépendent principalement de l'autotrophie dans les eaux peu profondes. Cette étude
	souligne donc l'importance de l'hétérotrophie à travers le gradient de profondeur euphotique-
	mésophotique supérieur et apporte des avancées majeures dans notre compréhension de
	l'importance écologique de l'alimentation chez les coraux. En outre, elle permet de mieux
	comprendre la capacité des différentes espèces d'octocoralliaires à exploiter les ressources en
	nutriments et contribue à améliorer les prévisions concernant les effets des changements
	climatiques et environnementaux sur cet important taxon vivant dans les récifs.
	3. Discussion générale et perspectives
	3.1 Les coraux mous et scléractiniaires forment des symbioses fonctionnelles
	différentes avec leurs dinoflagellés tant dans les récifs de surface que dans
	les récifs de la zone mésophotique supérieure
	 L'autotrophie reste stable le long du gradient de profondeur chez les coraux mous alors
	qu'elle diminue chez les coraux scléractiniaires
	L'acquisition autotrophe de carbone par les symbiotes du corail a été évaluée dans cette
	thèse par deux approches complémentaires (traceur bicarbonate 13 C et acides gras spécifiques
	à l'autotrophie, Chapitres 3 et 5). Les deux approches ont montré, pour toutes les espèces de
	coraux mous étudiées, que l'assimilation à court et à long terme du carbone dans le tissu hôte
	du corail était stable le long du gradient de profondeur ou même augmentée en profondeur
	Une expérience a été conçue pour étudier le statut autotrophe et hétérotrophe des coraux mésophotique. La stabilité de l'apport en nutriments autotrophes le long du gradient de
	mous in situ. À cette fin, l'abondance de trois biomarqueurs lipidiques, spécifiques de profondeur suggère que les coraux mous sont soit photo-limités dans les eaux peu profondes
	l'autotrophie et de l'hétérotrophie, a été étudiée dans les tissus de quatre espèces et/ou photo-acclimatés en profondeur. Ceci est en accord avec de précédentes observations
	d'octocoralliaires, et une de scléractiniaire, prélevées au sein de récifs peu profond et indiquant des taux de photosynthèse maximaux mesurés à 20 m de profondeur (en comparaison
	mésophotique supérieur de mer Rouge. Nos résultats montrent une mixotrophie fonctionnelle avec des eaux de surface) chez plusieurs espèces de coraux mous de la Grande Barrière de

pour toutes les espèces symbiotiques, avec un apport significatif de zooplancton aux deux profondeurs. Bien que les octocoralliaires aient maintenu des concentrations similaires de marqueurs autotrophes avec l'augmentation de la profondeur, l'espèce scléractiniaire a montré une diminution

  . Au contraire, chez le corail dur S. pistillata, on observe une diminution de l'acquisition de carbone autotrophe avec la profondeur et le remplacement du genre de symbiote Symbiodinium (anciennement clade A) au genre Cladocopium (anciennement clade C) avec la profondeur[START_REF] Mass | Photoacclimation of Stylophora pistillata to light extremes: metabolism and calcification[END_REF][START_REF] Ezzat | Carbon and nitrogen acquisition in shallow and deep holobionts of the scleractinian coral S. pistillata[END_REF].La comparaison du bilan carbone chez les coraux mous et scléractiniaires indique que le taux d'acquisition du carbone autotrophe est au moins deux fois plus élevé chez S. pistillata à faible profondeur par rapport aux espèces de coraux mous étudiées. Cependant, le carbone acquis est également perdu (sous forme de respiration et de production de mucus) deux fois

plus vite chez les coraux scléractiniaires, ce qui suggère un fonctionnement différent des symbioses, ainsi qu'une demande métabolique différente entre les espèces de coraux mous et durs. La demande métabolique plus élevée chez les coraux scléractiniaires est probablement due à la calcification, puisque la majeure partie du carbone respiré est utilisée pour le dépôt de carbonate de calcium. En effet, près de 70-75% du carbone utilisé pour la calcification chez les coraux durs provient de la respiration, et les 25-30% restants du carbone externe de l'eau de mer

  qui suggère que ce type de nutrition est favorisé dans le récif mésophotique. En outre, nous avons constaté au Chapitre 4 que les coraux mous ont une faible assimilation d'azote inorganique ou organique dissous et ont donc besoin d'acquérir de l'azote à partir d'une autre source telle que l'alimentation particulaire. Tout d'abord, nous avons montré que le mucus des coraux mous représente un habitat probablement défavorable pour la colonisation et l'activité des diazotrophes, car il est connu pour présenter des propriétés antimicrobiennes et contient des particules à teneur relativement élevée en azote[START_REF] Kelman | Antimicrobial activity of Red Sea corals[END_REF][START_REF] Nakajima | Release of dissolved and particulate organic matter by the soft coral Lobophytum and subsequent microbial degradation[END_REF]. En conséquence, les coraux mous ne favorisent pas la fixation de diazote dans l'eau de mer qui les entoure et n'assimilent pas le autres nutriments essentiels. Un apport insuffisant en phosphore entraîne une modification de la composition lipidique des membranes des symbiotes (qui passent des phospholipides aux sulpholipides), ce qui les rend plus sensibles au blanchissement (D'Angelo et Wiedenmann, 2014). La théorie selon laquelle ce modèle ne serait applicable qu'aux coraux durs, et donc que la symbiose des coraux mous ne serait perturbée qu'en moindre mesure par rôle important dans les différences de métabolisme (c'est-à-dire de besoins énergétiques) et de productivité mises en évidence ici. Enfin, les différences spécifiques aux espèces en matière d'acquisition et de répartition des nutriments peuvent être dues 1) au rapport entre la surface et le volume de la colonie (un faible rapport ne favorise pas l'exposition à la

	lumière et l'échange de gaz ou de nutriments à travers le tissu épidermique), 2) aux
	3.2 Les coraux mous et scléractiniaires forment des symbioses fonctionnelles différentes une augmentation de nutriments, est soutenue par des observations de terrain : une dominance caractéristiques micro-morphologiques des polypes (les branches fines, les petits polypes et le
	avec leurs dinoflagellés, ce qui pourrait expliquer la plus grande résistance des relative des coraux mous a été signalée dans les zones impactées par de fortes concentrations rapport élevé entre la surface et le volume des polypes étant plus adaptés à l'autotrophie ; grands
	coraux mous à l'eutrophisation de matières dissoutes et particulaires (McClanahan et Obura, 1997, Fabricius et Dommisse, polypes pour l'hétérotrophie), 3) l'espèce de symbiote hébergée (influence l'assimilation et le
	2000 ; Schleyer et Celliers, 2003 ; Fabricius, 2005 ; Baum et al., 2016). De plus, d'autres transfert de carbone), 4) la densité des symbiotes (influence l'assimilation et le transfert du
	propriétés physiologiques pourraient s'ajouter à leur faible capacité à assimiler l'azote dissous carbone), et 5) l'état nutritionnel (une efficacité hétérotrophe plus élevée étant corrélée à des
	pour expliquer ce phénomène : 1) l'eutrophisation entraînant une plus forte turbidité de l'eau, taux plus élevés de carbone transféré).
	une moindre dépendance des coraux mous sur leur symbiose avec leurs dinoflagellés pourrait
	les aider à faire face à une diminution de la quantité de lumière disponible pour la photosynthèse
	et 2) les particules en suspensions pourraient représenter une source potentielle de nutriments
	pour l'alimentation hétérotrophe.
	3.3 Synthèse graphique
	Les principaux résultats de cette thèse sont résumés dans la figure ci-après. La stabilité de la
	symbiose des octocoralliaires le long du gradient de profondeur est assurée par la maximisation
	de l'acquisition et de la conservation des nutriments aux deux profondeurs par autotrophie et
	hétérotrophie. Ceci est soutenu par une faible assimilation d'azote dissous, une acquisition
	stable de carbone inorganique dissous, une grande quantité de matière organique particulaire
	Du fait de la faible capacité des coraux mous à assimiler l'azote dissous dans l'eau de assimilée aux deux profondeurs et aucune assimilation de DDN. De plus, les mêmes quantités
	mer, de faibles quantités de nutriments inorganiques pénètrent dans leurs tissus. Il est donc peu de carbone acquises par photosynthèse ont été transférées des symbiotes à l'hôte aux deux
	 L'hétérotrophie est importante à toutes les profondeurs et augmente en zone probable que les changements de la qualité de l'eau liés aux nutriments (e.g., eutrophisation) profondeurs. Par rapport aux coraux mous, les coraux scléractiniaires ont une assimilation plus
	mésophotique pour plusieurs espèces perturbent la symbiose des coraux mous, contrairement aux coraux durs. Bien que cette élevée d'azote dissous (avec des taux d'assimilation d'azote organique dissous plus élevés en
	conclusion n'ait pas été testée expérimentalement dans le cadre de cette thèse et reste profondeur), une acquisition de carbone inorganique dissous plus élevée dans les eaux peu
	Une autre conclusion principale de cette thèse est que les coraux mous symbiotiques de hypothétique, elle pourrait être illustrée par le modèle conceptuel des rapports défavorables des profondes mais similaire (voire plus faible) en profondeur, une assimilation significative de
	surface et de profondeur mésophotique sont plus susceptibles de dépendre de l'hétérotrophie nutriments inorganiques dissous, introduit par Wiedenmann et al. (2012). En résumé, les coraux DDN et une stimulation de la fixation de diazote dans l'eau de mer environnante. Ils subissent
	que de l'autotrophie pour l'acquisition de nutriments, par rapport aux coraux durs. Cette durs ont de fortes capacités d'assimilation d'azote dissous, ainsi de grandes quantités d'azote également une perte de carbone plus importante en eaux peu profondes, mais plus faible que

. En outre, la présence du squelette améliore la productivité du corail grâce à la diffusion interne de la lumière, ce qui peut également contribuer à expliquer les différences d'acquisition du carbone entre les coraux mous et les coraux durs. conclusion est étayée par la grande quantité de biomarqueurs lipidiques, spécifiques au zooplancton, mesurée dans les tissus des coraux mous aux deux profondeurs (Chapitre 5). Pour certaines espèces, la quantité de biomarqueurs augmente significativement avec la profondeur, ce DDN. De plus, des taux d'assimilation de l'azote dissous jusqu'à dix fois inférieurs ont été observés chez les coraux mous par rapport aux coraux durs, sans différence nette entre les profondeurs. Cela ne peut pas s'expliquer par 1) une capacité différente d'acquisition des nutriments liée aux symbiotes dinoflagellés

(Baker et al., 2013 ;[START_REF] Ezzat | Carbon and nitrogen acquisition in shallow and deep holobionts of the scleractinian coral S. pistillata[END_REF]

, puisque les deux groupes partagent les mêmes génotypes et/ou densité de symbiotes ou 2) un besoin différent en azote structurel, puisque les coraux mous et durs présentent une teneur en azote similaire. Les coraux mous dépendent donc probablement d'une alimentation hétérotrophe pour satisfaire leurs besoins en azote. pénètrent au sein de leurs tissus lorsque l'azote est disponible en fortes concentrations dans l'eau de mer. Cela favorise une croissance rapide de la population de dinoflagellés, donc une demande cellulaire accrue, à laquelle ne peuvent subvenir de faibles concentrations en phosphore ou les coraux mous à la profondeur mésophotique. Les symbiotes des coraux scléractiniaires ont transféré des quantités de carbone beaucoup plus faibles avec la profondeur que ceux des coraux mous. La présence d'un squelette de carbonate de calcium chez les coraux durs joue probablement un

  Les sclérites pourraient agir comme diffuseurs de lumière. Leur taille, leur forme et leur couleur spécifiques à l'espèce pourraient notamment contribuer à expliquer les différences d'efficacité de la photosynthèse. Il serait donc intéressant d'étudier l'environnement lumineux interne des coraux mous, ainsi que le rôle possible des sclérites dans la diffusion de la lumière, en utilisant des microcapteurs de lumière. L'hydrosquelette des coraux mous peut cependant poser quelques problèmes techniques car il est susceptible de se contracter sur les microcapteurs. Importance de l'acquisition du phosphore pour la stabilité de la symbiose des coraux mous Chez les coraux scléractiniaires, il a été démontré que le rapport azote/phosphore joue un rôle majeur dans la stabilité de la symbiose. Principalement, une augmentation de la disponibilité en azote (majoritairement sous forme de nitrate) sans une augmentation similaire de phosphore, peut augmenter la croissance de la population de dinoflagellés et par conséquent

	 Hétérotrophie Sclerites DW	Alcyoniidae Nephtheidae Dendronephthya hemprichi Alcyonium digitatum Nephtheidae Capnella gaboensis Alcyoniidae Sarcophyton cf. glaucum Alcyoniidae Cladellia sp. Alcyoniidae Sinularia flexibilis	Oxygen flux Feeding rates (phytoplankton) Photosynthetic and accessory pigments (chlorophyll content) Symbiodinium density Calcification rate Photosynthetic and accessory pigments (chlorophyll a content)	Migné and Davoult (1997b) Fabricius et al. (1998) Tentori and Allemand (2006)
	Alcyoniidae Nephtheidae Xeniidae Alcyoniidae La nutrition hétérotrophe n'ayant été évaluée qu'à l'aide d'un biomarqueur lipidique Alcyonium digitatum Feeding rates (phyto-and zooplankton) Capnella gaboensis Symbiodinium density Heteroxenia fuscescens DOM flux Sinularia flexibilis Symbiodinium density spécifique au zooplancton dans cette thèse, des expériences impliquant des incubations avec Alcyoniidae Sarcophyton trochellophorum DMSP concentration Alcyoniidae Alcyonium paessleri Biochemical composition of tissues (carbohydrates, lipids, protein, refractory, Xeniidae Heteroxenia fuscescens Inorganic carbon Xeniidae Heteroxenia fuscescens Amino acids and glucose Alcyoniidae Sinularia sp. Biochemical composition of tissues (lipids and fatty acids)	Migné and Davoult (2002) Schlichter (1982) Imbs et al. (2010)
	1 AFDW). Les biomarqueurs lipidiques spécifiques de l'autotrophie, l'acide stéaridonique (SDA, 18:4 ω3) et l'acide docosapentaénoïque (DPA, 22:5 ω3), n'apparaissent pas sur la figure. Les concentrations de SDA étaient de 1-70 et de 1-146 μg g -1 AFDW dans les coraux peu profonds et mésophotiques, respectivement, et ont diminué de 11 à 3 μg g -1 AFDW avec la profondeur dans le corail dur. Les concentrations de DPA étaient de 3-7 et 3-5 μg g -1 AFDW dans les coraux peu profonds et les coraux mésophotiques, respectivement, et diminuaient de 5 à 2 μg g -1 d'AFDW avec la profondeur dans le corail dur. une large gamme de proies marquées (picoplancton et nanoplancton) permettraient de mieux Alcyoniidae Sinularia macropodia DMSP concentration Alcyoniidae Sinularia maxima DMSP concentration ash) Biochemical composition of tissues Nephtheidae Capnella gaboensis Inorganic carbon Nephtheidae Capnella gaboensis Oxygen flux Nephtheidae Nephthea spp. Electron transport system activity Alcyoniidae Sarcophyton spp. Electron transport system activity caractériser le régime alimentaire des coraux mous. En particulier, l'utilisation d'un marquage à l'aide d'isotopes stables confirmerait l'efficacité de l'ingestion des proies, puisque le mucus des coraux mous est connu pour avoir des propriétés antimicrobiennes et pourrait éventuellement affecter les concentrations de plancton mesurées dans les chambres. Alcyoniidae Sinularia pauli DMSP concentration Alcyoniidae Sinularia polydactyla DMSP concentration Alcyoniidae Biochemical composition of tissues (protein, Clavulariidae Clavularia frankliniana (carbohydrates, lipids, protein, refractory, ash) Biochemical composition of tissues Nephtheidae Photosynthetic and accessory pigments Alcyoniidae Sinularia sp. DMSP concentration Capnella gaboensis (chlorophyll content) Nephtheidae Capnella gaboensis Symbiodinium density Alcyoniidae Sarcophyton sp. DMSP concentration Nephtheidae Capnella gaboensis DMSP concentration Sinularia maxima carbohydrate and lipid content) Alcyoniidae Biochemical composition of tissues (protein, Nephtheidae Gersemia antartica (carbohydrates, lipids, protein, refractory, ash) Alcyoniidae Lobophytum compactum Anthothelidae Erythropodium hicksoni DMSP concentration Symbiodinium density Alcyoniidae Sinularia flexibilis Budding rate Sinularia polydactyla carbohydrate and lipid content) Sinularia polydactyla x Biochemical composition of tissues (protein, Xeniidae Xenia umbellata Caloric content Alcyoniidae Rhytisma fulvum Caloric content Nephtheidae Litophyton arboreum Biochemical composition of tissues (protein content) Buoyant weight Alcyoniidae Sinularia flexibilis Energy expenditure Alcyoniidae Sinularia flexibilis Oxygen flux Alcyoniidae maxima hybrid carbohydrate and lipid content) Alcyoniidae Lobophytum patulum Oxygen flux and mucus production Xeniidae Heteroxenia fuscescens Biochemical composition of tissues (carbohydrates, lipids, protein, ash) Wet weight Nephtheidae Litophyton arboreum Calcification rate Colony Xeniidae Heteroxenia fuscescens Oxygen flux Nephtheidae Litophyton arboreum Cell growth Photosynthetic and accessory pigments	Van Alstyne et al. (2006) Baum et al. (2016) Slattery and McClintock (1995) Farrant et al. (1987) Haydon et al. (2018) Slattery et al. (2013) Michalek-Wagner and Willis (2001) Khalesi et al. (2009) Ben-David-Zaslow and Benayahu (1998) Khalesi et al. (2011) Ben-David-Zaslow and Benayahu (1999) Kremien et al. (2013) Tentori et al (2004)
		Alcyoniidae Alcyoniidae Alcyoniidae Alcyoniidae Nephtheidae Nephtheidae Dendronephthya hemprichi Lobophytum depressum Sinularia dura Sinularia flexibilis Sinularia flexibilis Litophyton arboreum	Oxygen flux and mucus production Oxygen flux and mucus production Symbiodinium density (DW) Metabolite concentration (flexibilide) Biochemical composition of tissues (protein (chlorophyll a, phaeopigments and total content) photopigments)	Riegl and Branch (1995)* Khalesi et al. (2007) Khalesi et al. (2009)
	Polyp number	Alcyoniidae Alcyoniidae Xeniidae Xeniidae Xeniidae Alcyoniidae Alcyoniidae Alcyoniidae Alcyoniidae Alcyoniidae Alcyoniidae Alcyoniidae	Sinularia leptoclados Sarcophyton glaucum Heteroxenia fuscescens Heteroxenia fuscescens Xenia Cladellia sp. Sinularia flexibilis Sarcophyton trocheliophorum Sinularia flexibilis Sarcophyton cf. glaucum Sinularia pauli Sinularia maxima	Oxygen flux and mucus production Oxygen flux and mucus production Biochemical composition of tissues (protein content) Inorganic carbon Photosynthetic and accessory pigments Calcification rate (chlorophyll a content) Photosynthetic and accessory pigments DMSP concentration Symbiodinium density Symbiodinium density DMSP concentration DMSP concentration	Schlichter et al (1983) Tentori and Allemand (2006) Bednarz et al. (2012) Rocha et al. (2013a) Rocha et al. (2013b) Van Alstyne et al. (2006)
	DW	Xeniidae Xeniidae Nephtheidae Nephtheidae Alcyoniidae Alcyoniidae Alcyoniidae Alcyoniidae Alcyoniidae	Heteroxenia fuscescens Heteroxenia fuscescens Capnella gaboensis Capnella gaboensis Sinualria polydactyla Sinularia flexibilis Sinularia macropodia Sarcophyton cf. glaucum Sinularia flexibilis	Photosynthetic and accessory pigments Inorganic carbon Inorganic carbon Oxygen flux Photosynthetic and accessory pigments DMSP concentration (chlorophyll a content) DMSP concentration Photosynthetic and accessory pigments (chlorophyll a content) Biochemical composition of tissues (protein content)	Schlichter et al (1984) Farrant et al. (1987) Leal et al. (2014) Khalesi et al. (2007)
	 Étudier le stress oxydant chez les coraux mous Alcyoniidae Sarcophyton cf. glaucum	Photosynthetic and accessory pigments	Costa et al. (2016)
	 Étudier l'environnement lumineux interne des coraux mous : le rôle des tissus et des La présence de symbiotes chez les coraux est souvent bénéfique en raison de l'apport
	sclérites énergétique fourni par la photosynthèse. Toutefois, elle peut également imposer des restrictions
	(e.g., la distribution bathymétrique) et des risques (e.g., une augmentation plus importante des
	Les coraux scléractiniaires ont la capacité de réguler l'environnement lumineux perçu espèces réactives à l'oxygène libérées dans des conditions de stress par la photosynthèse). Au
	par leurs symbiotes à l'intérieur de leurs tissus. Par exemple, ils peuvent filtrer l'excès de niveau cellulaire, la production d'espèces réactives d'oxygène et d'azote représente un facteur
	lumière qui peut être nocif pour les algues ou améliorer la diffusion et la dispersion interne de responsable du blanchissement des coraux. Puisque les résultats de cette thèse montrent
	la lumière grâce aux propriétés optiques uniques de leurs tissus et de leur squelette. Une telle différentes stratégies nutritionnelles affichées par les coraux mous et durs le long du gradient
	amplification dans le champ lumineux local des symbiotes améliore notablement la de profondeur (en particulier en ce qui concerne l'azote), une dépendance différente à

3.4 Conclusion et perspectives

Cette thèse souligne l'importance des apports autotrophes et hétérotrophes dans la nutrition des coraux mous, ce qui leur confère une plasticité trophique le long du gradient de profondeur. Ce double mode de nutrition représente certainement un atout pour la colonisation de différents habitats et peut leur fournir un mécanisme d'adaptation pour soutenir leur croissance en conditions stressantes. Les résultats obtenus dans chaque chapitre ont ouvert la voie à de futures expériences, qui permettraient une compréhension plus approfondie des processus observés ici à l'échelle de l'organisme. Quelques exemples sont donnés ci-dessous. photosynthèse. Bien que les coraux mous n'aient pas de squelette, ce qui pourrait expliquer leur taux d'acquisition et d'assimilation du carbone plus faible par rapport à ceux des coraux scléractiniaires, ils possèdent une grande quantité de sclérites. augmenter le blanchissement des coraux. Comme les coraux mous sont moins enclins à absorber l'azote inorganique dissous, il serait intéressant de tester 1) les niveaux de nitrate induisant une rupture de la symbiose et 2) le rôle de la disponibilité en phosphore pour le maintien de cette symbiose dans diverses conditions d'azote et/ou environnementales. Les résultats pourraient apporter des informations sur la manière dont les coraux mous font face à l'eutrophisation et au stress thermique. l'autotrophie, ainsi qu'une flexibilité générale envers la symbiose, il pourrait être intéressant d'étudier les mécanismes pouvant conduire à une rupture de la symbiose chez les coraux mous, dans un contexte de stress thermique.  Contribution de l'autotrophie par rapport à l'hétérotrophie dans le budget nutritionnel des coraux mous Enfin, cette thèse n'a pu fournir d'information sur la contribution relative de l'apport en nutriments autotrophes et hétérotrophes dans les besoins métaboliques de l'holobionte. Une autre expérience utilisant des biomarqueurs lipidiques spécifiques de l'autotrophie et de l'hétérotrophie pourrait être conçue à cette fin et devrait impliquer différentes situations nutritionnelles rencontrées par les mêmes colonies (e.g., un suivi saisonnier). Une autre possibilité serait d'utiliser une approche multi-biomarqueurs, en combinant les signatures d'isotopes stables avec les lipides totaux.

Table S3 Atom% 13 C of studied corals (Host) and their Symbiodiniaceae (Symbionts) after a 5h pulse of H 13 CO3 ¯ and a 19h chase in filtered seawater.

 S3 Data represent the mean ± standard error of five replicates collected from the shallow (8 m depth) and mesophotic (40 m depth) reef in Eilat, Israel. Nat. ab. = Natural abundance; reflects the natural abundance isotope composition prior to tracer incubation.

	Residuals		15
	ρS (µg C g -1 AFDW h -1 )
	Species		1	27.360	< 0.01
	Depth		1	1.285	0.274752
	Depth:Species		1	0.623	0.442087
	Residuals		15	Host			Symbionts
	TS (µg C g -1 AFDW h -1 )	Nat. ab.		Pulse	Chase	Nat. ab	Pulse	Chase
	Species	(%)	1	15.25 (%)	< 0.01 (%)	(%)	(%)	(%)
	Df TISSUE DESCRIPTORS 1 1 Species:Depth Symbiodiniaceae density Species Depth 1 Residuals 35 Depth 1 Depth:Species 1 Litophyton sp. 1.0816 ± Shallow 0.0003 Residuals 15 1.0803 ± Mesophotic 0.0003 CL (µg C g -1 AFDW h -1 ) Species 1 Depth 1 Depth:Species 1 Rhytisma fulvum 1.0835 ± Shallow 0.0003 fulvum 1.0830 ± Mesophotic 0.0001	F-value 31.230 9.783 0.996 7.95 52.82 1.2272 ± 0.0205 1.2449 ± 0.0175 78.426 6.758 14.945 1.1730 ± 0.0065 1.1617 ± 0.0142	p-value < 0.01 < 0.01 0.32518 0.01294 1.2216 ± 0.0198 < 0.01 1.2348 ± 0.0094 < 0.01 0.0201 < 0.01 1.1734 ± 0.0132 1.1532 ± 0.0092	1.0844 ± 0.0002 1.0823 ± 0.0005 1.0904 ± 0.0008 1.0957 ± 0.0018	1.2645 ± 0.0346 1.3123 ± 0.0287 1.1245 ± 0.0055 1.1324 ± 0.0025	1.2986 ± 0.0325 1.3021 ± 0.0279 1.1026 ± 0.0029 1.1242 ± 0.0066
	Chlorophyll concentration (µg g -1 AFDW) Residuals	15
	Species	CHASE	1	2.326	0.135965
	Depth ρH (µg C g -1 AFDW h -1 )		1	18.395	< 0.01
	Species:Depth Species		1 1	0.341 0.908	0.563096 0.357
	Residuals Depth		36 1	1.309	0.272
	Chlorophyll concentration (µg symbiont cell -1 ) Depth:Species	1	0.515	0.485
	Species Residuals		1 14	27.996	< 0.01
	Depth ρS (µg C g -1 AFDW h -1 )		1	10.260	< 0.01
	Species:Depth Species		1 1	2.188 51.692	0.14806 < 0.01
	Residuals Depth		35 1	0.052	0.823
	Pc (µg C g -1 AFDW h -1 ) Depth:Species		1	1.147	0.302
	Species Residuals		1 14	9.619	< 0.01
	Depth TS (µg C g -1 AFDW h -1 )		1	0.004	0.9524
	Species:Depth Species		1 1	2.654 5.850	0.1241 0.0298
	Residuals Depth		15 1	0.049	0.8277
	Pc (µg C symbiont cell -1 ) Depth:Species		1	1.935	0.1859
	Species Residuals		1 14	3.231	0.0938
	Depth CL (µg C g -1 AFDW h -1 )		1	6.124	0.0267
	Species:Depth Species		1 1	0.978 6.785	0.3395 0.0208
	Residuals Depth		14 1	0.033	0.8584
	Rc (µg C g -1 AFDW h -1 ) Depth:Species		1	0.866	0.3678
	Species Residuals		1 14	13.504	< 0.01
	Depth		1	3.151	0.10352
	Species:Depth		1	23.537	< 0.01
	Residuals		11
		PULSE
	ρH (µg C g -1 AFDW h -1 )
	Species		1	0.020	0.888
	Depth		1	0.177	0.680
	Depth:Species		1	0.008	0.931

Table S4 : Total chlorophyll and autotrophic carbon acquisition of Stylophora pistillata along the depth gradient

 S4 . (a) Parameters normalized to surface area of the skeleton[START_REF] Ezzat | Carbon and nitrogen acquisition in shallow and deep holobionts of the scleractinian coral S. pistillata[END_REF]. (b) Parameters normalized to ash-free dry weight. The values of this table represent estimates calculated from A using a conversion coefficient(255.65 ± 8.98) determined in this study. PC = autotrophic carbon acquisition.

	a	Total chlorophyll	PC
		(µg cm -2 )	(µg C cm -2 h -1 )
	Depth (m)	November	November
	5	3.61	9.49
	50	6.87	2.17
	b	Total chlorophyll	PC
		(µg g -1 AFDW)	(µg C g -1 AFDW h -1 )
	Depth (m)	November	November
	5	923	2,426
	50	1,756	555

Table S7 . Statistical differences estimated between sources in the host and Symbiodiniaceae fractions. DN assimilation in the host fraction (per AFDW) -Figure 4

 S7

		Source Source	emmean emmean	SE SE	df df	asymp.LCL asymp.UCL .group asymp.LCL asymp.UCL .group
		DFAA Nitrate	-2.4887 1.8189 Depth = Shallow, Species = Rhytisma: 0.167 Inf -2.81578 0.167 Inf 1.49184	-2.162 2.146	a a
		Nitrate DFAA Source	-0.8421 2.2423 emmean	0.167 0.167 SE	Inf Inf df	-1.16916 1.91523 asymp.LCL asymp.UCL .group -0.515 b 2.569 a
		Ammonium Ammonium DFAA	-0.1498 3.3253 -0.8722	0.167 0.167 0.199	Inf Inf Inf	-0.47686 2.99819 -1.2619	0.177 3.652 -0.483	c b a
		Nitrate	0.521		0.222	Inf		0.0853	0.957	b
	Depth = Deep, Species = Seriatopora: emmean SE df asymp.LCL asymp.UCL .group DN assimilation in the Symbionidinaceae fraction -Figure 5 Source Ammonium 0.8382 0.222 Inf 0.4025 1.274 b
		Nitrate	2.9323 Depth = Shallow, Species = Sarcophyton: 0.167 Inf 2.60519 Depth = Deep, Species = Galaxea:	3.259	a
		DFAA Source Source	3.3656 emmean emmean	0.167 SE SE	Inf df df	3.03852 asymp.LCL asymp.UCL .group 3.693 a asymp.LCL asymp.UCL .group
		Ammonium Nitrate DFAA	3.4852 1.6264 -1.5362	0.167 0.199 0.199	Inf Inf Inf		3.15813 1.2367 -1.9259	3.812 2.016 -1.147	a a a
		Ammonium Nitrate	1.8664 -1.2347	0.199 0.199	Inf Inf		1.4766 -1.6245	2.256 -0.845	a a
		DFAA Ammonium	Depth = Deep, Species = Stylophora: 0.222 Inf -0.0254 1.9423 0.199 Inf	1.5066 -0.4151	2.378 0.364	a b
		Source	emmean	SE	df	asymp.LCL asymp.UCL .group
		Nitrate	2.6151 Depth = Shallow, Species = Seriatopora: 0.167 Inf 2.28801 Depth = Deep, Species = Rhytisma:	2.942	a
		Ammonium Source Source	3.2699 emmean emmean	0.167 SE SE	Inf df df	2.9428 asymp.LCL asymp.UCL .group 3.597 b asymp.LCL asymp.UCL .group
		DFAA Nitrate DFAA	3.3586 -0.1784 2.1631	0.167 0.199 0.222	Inf Inf Inf		3.03148 -0.5682 1.7274	3.686 0.211 2.599	b a a
		DFAA Nitrate	0.0133 2.4711		0.199 0.222	Inf Inf		-0.3764 2.0353	0.403 2.907	a ab
		Ammonium Ammonium	1.284 2.8849	Depth = Shallow, Species = Galaxea: 0.199 Inf 0.199 Inf	0.8943 2.4952	1.674 3.275	b b
		Source	emmean	SE	df	asymp.LCL asymp.UCL .group
		DFAA	2.4936 Depth = Deep, Species = Sarcophyton: 0.167 Inf 2.16654 Depth = Shallow, Species = Stylophora:	2.821	a
		Nitrate Source Source	3.1077 emmean emmean	0.167 SE SE	Inf df df	2.78057 asymp.LCL asymp.UCL .group 3.435 b asymp.LCL asymp.UCL .group
		Ammonium Nitrate DFAA	3.2491 -1.9264 1.8023	0.167 0.199 0.199	Inf Inf Inf		2.92197 -2.3161 1.4126	3.576 -1.537 2.192	b a a
		DFAA Nitrate	-1.3339 2.5301	0.199 0.222	Inf Inf		-1.7236 2.0944	-0.944 2.966	ab b
		Ammonium Ammonium	Depth = Shallow, Species = Litophyton: -0.781 0.199 Inf -1.1707 3.1161 0.199 Inf 2.7264	-0.391 3.506	b b
		Source	emmean	SE	df	asymp.LCL asymp.UCL .group
	DN	Nitrate DFAA Source	-1.388 -0.1899 Depth = Deep, Species = Seriatopora: 0.167 Inf 0.167 Inf -0.51702 -1.7151 emmean SE df asymp.LCL asymp.UCL .group -1.061 a 0.137 b
		Ammonium DFAA	1.2232 1.915		0.187 0.222	Inf Inf		0.85749 1.4793	1.589 2.351	c a
		Nitrate	2.6506		0.199	Inf		2.2609	3.04	b
		Ammonium	Depth = Shallow, Species = Rhytisma: 2.8001 0.199 Inf	2.4104	3.19	b
		Source	emmean	SE	df	asymp.LCL asymp.UCL .group
		DFAA	-0.5066 Depth = Deep, Species = Stylophora: 0.167 Inf -0.83364	-0.179	a
		Nitrate Source	0.3197 emmean	0.167 SE	Inf df	-0.00742 asymp.LCL asymp.UCL .group 0.647 b
		Ammonium Nitrate	1.4321 2.2872		0.187 0.222	Inf Inf		1.06641 1.8515	1.798 2.723	c a
		DFAA	2.5943		0.199	Inf		2.2046	2.984	a
		Ammonium	Depth = Deep, Species = Galaxea: Depth = Shallow, Species = Sarcophyton: 2.7448 0.199 Inf 2.3551	3.135	a
		Source Source	emmean emmean	SE SE	df df	asymp.LCL asymp.UCL .group asymp.LCL asymp.UCL .group
		Nitrate DFAA	2.8788 -2.3626 Depth = Shallow, Species = Galaxea: 0.167 Inf 0.167 Inf -2.68966 2.5517	3.206 -2.035	a a
		Ammonium Nitrate Source	3.1948 -1.0863 emmean	0.167 0.167 SE	Inf Inf df	2.86767 -1.41334 asymp.LCL asymp.UCL .group 3.522 a -0.759 b
		DFAA Ammonium DFAA	3.3961 -0.015 1.477		0.167 0.187 0.199	Inf Inf Inf	3.06897 -0.38067 1.0873	3.723 0.351 1.867	a c a
		Nitrate	2.2978		0.199	Inf		1.9081	2.688	b
		Ammonium	Depth = Deep, Species = Rhytisma: Depth = Shallow, Species = Seriatopora: 2.3215 0.199 Inf 1.9318	2.711	b
		Source Source	emmean emmean	SE SE	df df	asymp.LCL asymp.UCL .group asymp.LCL asymp.UCL .group
		DFAA DFAA	-0.0938 2.0527 Depth = Shallow, Species = Litophyton: 0.167 Inf -0.42085 0.167 Inf 1.72558	0.233 2.38	a a
		Nitrate Nitrate Source	1.0336 2.2123 emmean	0.167 0.167 SE	Inf Inf df	0.70652 1.88516 asymp.LCL asymp.UCL .group 1.361 b 2.539 a
		Ammonium Ammonium Nitrate	1.76 3.4938 -1.3415	0.167 0.167 0.199	Inf Inf Inf		1.43296 3.1667 -1.7312	2.087 3.821 -0.952	c b a
		DFAA	1.1549		0.199	Inf		0.7652	1.545	b
		Ammonium	Depth = Deep, Species = Sarcophyton: Depth = Shallow, Species = Stylophora: 1.734 0.199 Inf 1.3443	2.124	b

assimilation in the host fraction (per AFDW) -Figure 6 Temperature

	DFAA	0.0221	0.157	Inf	-0.2854	0.33	b
	Ammonium	1.826	0.157	Inf	1.5185	2.134	c
		Temperature = 30, Species = Rhytisma:
	Source	emmean	SE	df	asymp.LCL asymp.UCL .group
	DFAA	0.1935	0.175	Inf	-0.1503	0.537	a
	Nitrate	0.8811	0.175	Inf	0.5372	1.225	b
	Ammonium	2.1178	0.157	Inf	1.8103	2.425	c
		Temperature = 30, Species = Sarcophyton:
	Source	emmean	SE	df	asymp.LCL asymp.UCL .group
	DFAA	-1.6453	0.157	Inf	-1.9528	-1.338	a
	Nitrate	-1.3083	0.157	Inf	-1.6158	-1.001	a
	Ammonium	-0.512	0.175	Inf	-0.8558	-0.168	b
	DN
		Temperature = 25, Species = Litophyton:
	Source	emmean	SE	df	asymp.LCL asymp.UCL .group
	Nitrate	-1.35	0.157	Inf	-1.6575	-1.043	a
	DFAA	-0.1886	0.157	Inf	-0.4961	0.119	b
	Ammonium	1.2564	0.175	Inf	0.9125	1.6	c
		Temperature = 25, Species = Rhytisma:
	Source	emmean	SE	df	asymp.LCL asymp.UCL .group
	DFAA	-0.4983	0.157	Inf	-0.8058	-0.191	a
	Nitrate	0.3227	0.157	Inf	0.0152	0.63	b
	Ammonium	1.4785	0.175	Inf	1.1347	1.822	c
		Temperature = 25, Species = Sarcophyton:
	Source	emmean	SE	df	asymp.LCL asymp.UCL .group
	DFAA	-2.2549	0.157	Inf	-2.5624	-1.947	a
	Nitrate	-1.0623	0.157	Inf	-1.3698	-0.755	b
	Ammonium	-0.0132	0.175	Inf	-0.357	0.331	c
		Temperature = 30, Species = Litophyton:
	Source	emmean	SE	df	asymp.LCL asymp.UCL .group
	Nitrate	-1.1274	0.157	Inf	-1.4349	-0.82	a

assimilation in the Symbiodiniaceae fraction (per AFDW) -Figure 6 Temperature

	Source	Temperature = 30, Species = Sarcophyton: emmean SE df asymp.LCL asymp.UCL .group
	Source DFAA	emmean 3.2	SE 0.292	df Inf	asymp.LCL asymp.UCL .group 2.63 3.77 a
	Nitrate Ammonium	-1.0584 3.37	0.211 0.292	Inf Inf	-1.472 2.8	-0.645 3.94	a a
	DFAA Nitrate	-0.7463 3.8	0.211 0.292	Inf Inf	-1.16 3.23	-0.332 4.38	a a
	Ammonium	0.0184		0.211	Inf	-0.395	0.432	b
		Depth = Shallow, Species = Seriatopora:
	Source DN assimilation in the host fraction (per surface) -Figure S2 emmean SE df DFAA 3.96 0.292 Inf	asymp.LCL asymp.UCL .group 3.39 4.53 a
	Nitrate	4.21	Depth = Shallow, Species = Galaxea: 0.326 Inf	3.57	4.85	ab
	Source Ammonium	emmean 4.97	SE 0.292	df Inf	asymp.LCL asymp.UCL .group 4.4 5.54 b
	DFAA	11.52		0.829	Inf		9.89	13.14	a
	Nitrate	14.54 Depth = Shallow, Species = Stylophora: 0.742 Inf 13.09	15.99	b
	Ammonium Source	15.09 emmean	0.829 SE	Inf df	13.46 asymp.LCL asymp.UCL .group 16.71 b
	DFAA	2.48		0.292	Inf		1.91	3.05	a
	Nitrate	Depth = Shallow, Species = Seriatopora: 3.07 0.292 Inf	2.5	3.64	a
	Source Ammonium	emmean 4.27	SE 0.292	df Inf	asymp.LCL asymp.UCL .group 3.7 4.84 b
	Nitrate	7.81		0.742	Inf		6.36	9.27	a
	DFAA Source Ammonium Source Nitrate DFAA DFAA Nitrate Ammonium Nitrate Source Ammonium	9.61 Temperature = 25, Species = Litophyton: 0.829 Inf 7.99 Depth = Deep, Species = Galaxea: emmean SE df 19.77 0.742 Inf 18.32 emmean SE df asymp.LCL asymp.UCL .group 11.24 a 21.23 b asymp.LCL asymp.UCL .group -1.2491 0.236 Inf -1.712 -0.786 1.99 0.326 Inf 1.35 2.63 a a 1.212 0.211 Inf 0.798 1.626 Depth = Shallow, Species = Stylophora: 2.15 0.292 Inf 1.58 2.73 a b 1.8732 0.211 Inf 1.459 2.287 4.05 0.742 Inf 2.6 5.5 a b emmean SE df asymp.LCL asymp.UCL .group 2.62 0.292 Inf 2.05 3.19 a
	DFAA Source Ammonium Source DFAA DFAA Nitrate Nitrate Ammonium Nitrate Source Ammonium	5.82 emmean Depth = Deep, Species = Seriatopora: 0.742 Inf Temperature = 25, Species = Rhytisma: SE df 11.23 0.742 Inf emmean SE df asymp.LCL asymp.UCL .group 4.37 7.28 a 9.78 12.68 b asymp.LCL asymp.UCL .group -0.834 0.211 Inf -1.248 -0.42 2.43 0.326 Inf 1.79 3.07 a a 0.5358 0.236 Inf 0.073 0.998 Depth = Deep, Species = Galaxea: 3.2 0.292 Inf 2.63 3.77 a b 0.8691 0.236 Inf 0.406 1.332 9.41 0.742 Inf 7.95 10.86 a b emmean SE df asymp.LCL asymp.UCL .group 4.6 0.292 Inf 4.03 5.17 b
	DFAA Source Ammonium Source DFAA Nitrate Nitrate DFAA Ammonium Nitrate Source Ammonium	9.84 emmean Depth = Deep, Species = Stylophora: 0.742 Inf Temperature = 25, Species = Rhytisma: SE df 12.82 0.742 Inf emmean SE df asymp.LCL asymp.UCL .group 8.39 11.29 a 11.37 14.28 b asymp.LCL asymp.UCL .group -1.4385 0.211 Inf -1.852 -1.025 2.78 0.326 Inf 2.14 3.42 a a -1.0351 0.211 Inf -1.449 -0.621 Depth = Deep, Species = Seriatopora: 2.88 0.292 Inf 2.31 3.45 a a -0.0202 0.211 Inf -0.434 0.394 7.37 0.742 Inf 5.91 8.82 a b emmean SE df asymp.LCL asymp.UCL .group 3.37 0.292 Inf 2.8 3.94 a
	DFAA Source Ammonium	13.75 Temperature = 30, Species = Litophyton: 0.742 Inf 12.3 emmean SE df asymp.LCL asymp.UCL .group 15.21 b 16.3 0.829 Inf 14.67 17.92 b
	Nitrate DFAA Ammonium Source Nitrate	-1.3204 1.0603 Depth = Deep, Species = Stylophora: 0.211 Inf 0.211 Inf 1.6943 0.273 Inf emmean SE df asymp.LCL asymp.UCL .group -1.734 -0.907 a 0.646 1.474 b 1.16 2.229 b 5.57 0.829 Inf 3.94 7.2 a
	DFAA Source Ammonium	9.67 emmean Temperature = 30, Species = Rhytisma: 0.742 Inf SE df asymp.LCL asymp.UCL .group 8.21 11.12 b 12.67 0.829 Inf 11.04 14.3 c
	DFAA	-0.1224	0.236	Inf	-0.585	0.34	a
	Nitrate DN assimilation in the Symbiodiniaceae fraction (per surface) -Figure S2 1.324 0.211 Inf 0.91	1.738	b
	Ammonium	1.7141		0.211	Inf		1.3	2.128	b
			Depth = Shallow, Species = Galaxea:

Table S9 . Biologically relevant pairwise comparisons based on estimated marginal means and simultaneous general linear hypothesis testing.

 S9 Data are p-values adjusted using Benjamini-Hochberg procedure. Significant values (p < 0.05) are displayed in bold. Species are Dendronephthya sp., Litophyton arboreum, Rhystima fulvum fulvum and Sarcophyton sp. for the octocorals, and Stylophora pistillata for the scleractinian.

Remerciements

Acknowledgements

We thank D. Allemand for scientific support, M. Boussion and C. Rottier for their contribution in sample processing, and the ecophysiology team for useful comments. Two anonymous reviewers greatly contributed to improve this manuscript. This study was funded by the Centre Scientifique de Monaco.

Acknowledgements

We thank D. Allemand, Director of the Centre Scientifique de Monaco, for scientific support and the staff of the IUI for assistance on the field. This study was funded by the Centre Scientifique de Monaco and CFP was funded by "Les Explorations de Monaco".

Acknowledgements

We thank D. Allemand, Director of the Centre Scientifique de Monaco, for scientific support, the staff of the IUI for assistance on the field, and S. Rabouille, from the Laboratoire d'Océanographie de Villefranche-sur-mer, for the culture of diazotrophs. This study was funded by the Centre Scientifique de Monaco.

Acknowledgements

We thank D. Allemand, director of the Centre Scientifique de Monaco, for support and the staff of the IUI for assistance on the field. CFP would like to thank Monaco Exploration Society for promoting this research, through the ENCOR project in the Red Sea. This study is part of the RTPI Nutress.

into their tissue. Interestingly, N2 fixation was not detected in the tissue and surrounding seawater of soft corals, despite the fact that soft corals were able to take up DDN from a culture of free-living diazotrophs. Soft coral mucus likely represents an unfavorable habitat for the colonization and activity of diazotrophs as it contains a low amount of particulate organic matter, with a relatively high N content, compared to the mucus of scleractinian corals. In addition, it is known to present antimicrobial properties. Overall, this study suggests that DDN assimilation into coral tissues depends on the presence of active diazotrophs in the coral's mucus layer and/or surrounding seawater. Since N is often a limiting nutrient for primary productivity in oligotrophic reef waters, the divergent capacity of scleractinian and soft corals to promote N2 fixation may have implications for N availability and reef biogeochemistry in scleractinian versus soft coral dominated reefs.

Introduction

Coral reefs are highly productive ecosystems despite thriving in oligotrophic waters, which contain low levels of essential dissolved inorganic nutrients such as nitrogen (N) [START_REF] De Goeij | Surviving in a marine desert: the sponge loop retains resources within coral reefs[END_REF]. Corals, which are the main reef builders, can achieve high productivity thanks to their association with endosymbiotic dinoflagellates, which recycle the animal waste products and are efficient in scavenging inorganic N dissolved in seawater [START_REF] Wang | Essential amino acid synthesis and nitrogen recycling in an algainvertebrate symbiosis[END_REF][START_REF] Grover | Nitrate uptake in the scleractinian coral Stylophora pistillata[END_REF]. Corals also profit from high rates of N recycling in the water column and sediments via microbial processes (Carpenter and [START_REF] Capone | Nitrogen in the marine environment[END_REF], and from biological dinitrogen (N2) fixation of diazotrophic symbionts [START_REF] Bednarz | The assimilation of diazotroph-derived nitrogen by scleractinian corals depends on their metabolic status[END_REF], which are heterotrophic bacteria and cyanobacteria that convert atmospheric N2 into bioavailable N. In addition to being associated with benthic organisms (reviewed in [START_REF] Benavides | Diazotrophs: overlooked key players within the coral symbiosis and tropical reef ecosystems?[END_REF], diazotrophs can live pelagic, and colonize reef substrates (e.g., sediments, coral rubble). Many studies using the acetylene reduction assay (ARA) method have recorded high rates of N2 fixation either in reef waters or in the presence of corals (i.e., [START_REF] Bednarz | Seasonal variation in dinitrogen fixation and oxygen fluxes associated with two dominant zooxanthellate soft corals from the northern Red Sea[END_REF][START_REF] Cardini | Budget of primary production and dinitrogen fixation in a highly seasonal Red Sea coral reef[END_REF][START_REF] Rädecker | Nitrogen cycling in corals: the key to understanding holobiont functioning?[END_REF].

The ARA technique quantifies gross N2 fixation without providing insights into DDN (diazotrophically-derived nitrogen) assimilation by the coral-dinoflagellate symbiosis.

Instead, the use of labelled 15 N2 gas measures DDN assimilation (net N2 fixation) and distinguishes between the different compartments (i.e. seawater particles, tissue,

Publication n°5

Chloé A. Pupier 1,2 , Renaud Grover 1 , Maoz Fine

Abstract

Symbioses between marine organisms and photosynthetic dinoflagellates of the family Symbiodiniaceae are vital for reef ecosystems. Coral holobionts are able to grow and survive in oligotrophic environments through the efficient uptake, conservation and recycling of nutrients. Nitrogen (N) is one essential nutrient for coral holobiont functioning but nitrogen availability is often low in reef waters. Recent studies have advanced our understanding of nitrogen cycling in scleractinian corals but the nutrient metabolism of other major benthic organisms on reefs still needs to be addressed. For example, the capacity of soft corals to assimilate dissolved nitrogen at the organism level remains unexplored. Here, using stable isotope labeling, we performed a multi-species comparison to investigate dissolved nitrogen (i.e. ammonium, nitrate, and free amino acids) assimilation in several species of soft and scleractinian corals sampled in the Red Sea (Eilat, Israel) at two different depths (8-10 m and 40-50 m depth). The main result shows that for all nitrogen sources, scleractinian corals exhibited higher assimilation rates per tissue biomass than soft corals (up to ten fold), despite harboring the same genera of Symbiodiniaceae in similar densities. Soft corals may thus complement their low acquisition of DN by the acquisition of other N forms, thereby rather relying on heterotrophic feeding to meet their N requirements. This finding highlights a very different acquisition of N between the soft and scleractinan-dinoflagellate associations. Such

Abstract

Octocorals are important reef inhabitants, from the shallow to mesophotic depths and beyond. However information regarding their nutritional ecology along the depth gradient is limited, despite the fact that nutrient acquisition is a fundamental process explaining the health condition and distribution of reef organisms. In particular, the contribution of the autotrophic versus heterotrophic acquisition of nutrients in symbiotic soft corals is poorly known. Here, the abundance of three lipid biomarkers, specific for autotrophy and heterotrophy, was investigated in the tissue of four octocoral species and one scleractinian sampled in shallow and upper mesophotic reefs of the oligotrophic Northern Red Sea. Our findings show functional

Lipid biomarkers reveal the trophic plasticity of octocorals along a depth gradient

Appendices

Appendix I -Supplementary material of publication n°1 (Chapter 2) Appendix II -Supplementary material of publication n°3 (Chapter 3)

Material and Methods

Physiological and tissue descriptor measurements

For the determination of the symbiont density, total chlorophyll concentration and ash-free dry weight (AFDW) from the experimental nubbins, we followed the protocol of Pupier et al. [START_REF] Pupier | Studies with soft corals -recommendations on sample processing and normalization metrics[END_REF]. Briefly, freeze-dried samples were homogenized in a Potter tissue grinder. A subsample was weighed, and then combusted at 450°C for 4 h in a muffle furnace (Thermolyne 62700, Thermo Fischer Scientific, the United States). AFDW was determined as the difference between the DW and ash weight (AW) of the subsample and extrapolated to the total weight of the nubbin. The remaining sample was also weighed, and then homogenized with 10 mL distilled water (DI). The homogenate was centrifuged for 10 min (at 11,000 g at 4°C) to separate the animal host (supernatant) from the symbionts (pellet). Light microscopy confirmed the total removal of symbionts from the supernatant. The symbiont pellet was rinsed twice to eliminate any remaining host cells [START_REF] Tremblay | Autotrophic carbon budget in coral tissue: a new 13 C-based model of photosynthate translocation[END_REF], and re-suspended in 10 mL DI. After mixing, 500 µL and 2.5 mL were subsampled for the determination of symbiont density and total chlorophyll concentration, respectively. Symbiont density was quantified microscopically via eight replicate haemocytometer counts (Neubauer-improved haemocytometer, Marienfeld, Germany). For chlorophyll analysis, the 2.5 mL subsamples were centrifuged for 10 min (at 8,000 g at 4°C) and the supernatant was discarded. Pellets were resuspended into 4 mL of acetone (100%) amended with magnesium chloride (Sigma Aldrich, where Cpulse and Cchase are the percent 13 C enrichment of the enriched and non-enriched incubation media, respectively (Cchase=1.1%).

The amount of carbon acquired through photosynthesis, translocated from the symbionts to the host (TS) was calculated as:

where PC is the total amount of autotrophic carbon produced by the symbionts; ρs is the carbon assimilation rate and RS the respiration rate of the symbionts.

Finally, the amount of carbon lost (CL) through respiration (RC) and through particulate and dissolved carbon release (ρPOC/DOC) is:

Appendix III -Supplementary material of publication n°5 (Chapter 4)