David L Ópez

Lora Oehlberg

Candemir Doger

Tobias Isenberg

Tablet-Based Interaction for Immersive 3D Data Exploration

INTRODUCTION

Effective visualization and interaction with 3D datasets that represent scans or simulations of the real world is at the heart of visualization research. Exploration and analysis are most strongly supported when both the best possible visual representations and the best possible interaction techniques are chosen. To present visual representations to users, the use of stereoscopy facilitates depth perception and thus high visual immersion. To facilitate an interactive exploration of data, the use of touch-based input for visualization provides high immersion through interaction due to its directness-the input and the affected data are at the same visual location (i. e., sticky interaction), resulting in users feeling "in control of the data" [START_REF] Yu | FI3D: Direct-touch interaction for the exploration of 3D scientific visualization spaces[END_REF].

Unfortunately, these two ways of achieving immersion are mutually exclusive. On the one hand, virtual objects in stereoscopic settings cannot be touched since they appear to float in empty space. Touch interaction, on the other hand, conflicts with stereoscopic display due to parallax issues as well as touch-through and invisible wall problems-it is far better suited for monoscopic displays.

Our overall vision is to enable researchers to explore 3D datasets with as much immersion as possible, arising both from visuals as well as from interaction. We therefore explore ways to combine an immersive large view of the 3D data with means to intuitively control this view with touch input on a separate mobile monoscopic tablet. This combination has the potential to increase people's acceptance of stereoscopic environments for 3D data visualization since-through touch-based interaction-it puts them in control of their data. Moreover, the indirect manipulation of (stereoscopically displayed) 3D data on a personal touch device has been shown to have potentially more efficient and precise interaction than interaction directly on a large display [START_REF] Knoedel | Multi-touch RST in 2D and 3D spaces: Studying the impact of directness on user performance[END_REF].

A SECOND SCREEN FOR VISUALIZATION CONTROL

Adding a second mobile display to an immersive visualization environment (Fig. 1) has the potential to provide additional visual feedback and thus facilitate flexible interactive data exploration scenarios. This setup is in contrast to an eyes-off, touch-only navigation with touch-enabled small devices, which has been explored in the past (e. g., [START_REF] Daiber | Designing gestures for mobile 3D gaming[END_REF][START_REF] Du | Tilt & touch: Mobile phone for 3D interaction[END_REF][START_REF] Liang | An investigation of suitable interactions for 3D manipulation of distant objects through a mobile device[END_REF]). The two-screen setup, however, requires us to consider a number of design goals and constraints to make both data views useful for the person exploring 3D data; in particular, the input on the mobile display needs to be as well-controlled and precise as possible. This implies that the tablet's view should be predominantly static such that small or large user movements in the environment do not result in ambiguous navigation commands. This relatively static tablet view exists in tandem with the stereoscopic view of the viewer which is updated based on 3D tracking to provide a true sense of immersion.

INTERACTION DESIGN FOR 3D NAVIGATION

Based on the static visualization displayed on the tablet (A in Fig. 3), we want to be able to initiate 3D navigation commands though touch input on the mobile device. For this pupose we are able to make use of existing touch-based 3D navigation techniques. We implemented * e-mail: david.lopezb@udea.edu.co † e-mail: {lora.oehlberg | tobias.isenberg}@inria.fr ‡ e-mail: cdoger@sabanciuniv.edu tBox [START_REF] Cohé | tBox: A 3D transformation widget designed for touch-screens[END_REF] and FI3D [START_REF] Yu | FI3D: Direct-touch interaction for the exploration of 3D scientific visualization spaces[END_REF] (Fig. 2) because they both deliver at least 7 DOF control and can be used with finger input and without an additional stylus (B in Fig. 3). We also implemented gyroscope control (C in Fig. 3) using Android's virtual rotation sensor that is based on the tablet's gyroscope, magnetometer, and acceleration sensors. It can be activated in a spring-loaded fashion (right button in Fig. 2) to directly map changes of the tablet's orientation to rotations of the depicted 3D dataset. The resulting navigation events are sent from the tablet to the PC driving the immersive view using a clientserver architecture. Also, in both interaction cases we automatically and without additional return to a 'frozen' projected view of the 3D data (A in Fig. 3) after the respective manipulations-a view that matches that seen on the immersive display.

The static nature of the mobile display, however, also implies that-due to movement of the interacting user in the visualization environment-the two views (immersive and tablet) can become outof-sync (Fig. 4; D in Fig. 3). This happens if the angle between the user's initial and current position with respect to the center location of the immersive display becomes too large-a common occurrence as the user moves around the immersive display to obtain a new view. We can thus expect a person exploring a visualization to have increasing difficulties with controlling the 3D datasets: A rotation around the x-axis, for instance, leads to a simple rolling action on the tablet's view of the dataset but is shown as rotation around a different axis in the stereoscopic view observed on the large display.

For such cases we implemented two dedicated and straightforward synchronization mechanisms: stereo view → tablet and tablet → stereo view. In the former (stereo view → tablet), the stereoscopic camera parameters from 3D tracking are used to define the tablet's virtual camera. As a result, the tablet shows the exact same view that the user sees stereoscopically at the time of the interaction. The latter (tablet → stereo view), supports the opposite effect: the stereo view is adjusted such that the interacting person sees the same VR view as was previously displayed only on the tablet. Since we cannot change the virtual camera that computes the stereoscopic view, we re-arrange the dataset such that the viewer camera shows the same view as previously shown only on the tablet.

USAGE SCENARIO

We implemented an interactive visualization environment based on the described two-screen setup (Fig. 1). While the combination with large-scale (wall-sized or CAVE-like) immersive environments is certainly possible, we experimented with a smaller-scale commercial 3DTV-based hardware with time-of-flight camera 3D tracking for simplicity and ease of maintenance reasons. With this setup we specifically target the exploration of scientific 3D datasets, such as from our collaborating domain experts in fluid dynamics and structural biology (see the visualizations in Fig. 2).

In our usage scenarios, we envision that a scientist would start by loading the dataset and initialize the views such that the tablet's view is synchronized based on the person's location in the room. Then, the scientist would be able to navigate in the data using the mechanisms provided by tBox and/or FI3D, shifting his or her attention between the tablet and the immersive display as necessary. The specific widget set depends on the type of intended interactions and the type of data, and the user can toggle between both modes. By engaging the gyroscope on-demand, the scientist can access rotations otherwise difficult to achieve with the two widgets. With the help of 3D tracking, the scientist would also be able to move in the room to get alternative views of the data, benefiting from the immersion resulting from a 3D-tracked stereoscopic depiction. To avoid the confusion that results from physically moving from the initial position in the room, the scientist would make use of the described synchronization mechanisms as needed.

In particular, the tablet → stereo view synchronization would allow him or her to return to a view that was initially found useful, thereby using the tablet as a way to store a good view of the dataset. Alternatively, if the scientist finds a good view through moving in the room, he or she can continue the data exploration from that perspective by using the stereo view → tablet mechanism.

Of course, simple 3D navigation is not sufficient for effective data exploration. Therefore, we envision that the described scenario is extended with additional visual data exploration mechanisms such as selection, filtering, and parameter adjustments to support higherlevel data analysis tasks. The advantage of our two-screen setup is that both displays show a representation of the data and that data exploration mechanisms can now be implemented directly on the tablet since it supports the directly-manipulative input.

CONCLUSION

We discussed our design and implementation of a system that allows people to view and explore immersive 3D data through tablet-based interactions. We demonstrated how to combine the stereoscopic view and input on the monoscopic tablet view in order to provide the user with both visual immersion and immersion through interaction. We showed that this setup can lead to situations where both views are out-of-sync, and showed that simple synchronization features can solve these problems. Our workflow can be adapted to a range of tablet interfaces-we implemented both FI3D and tBox on the monoscopic tablet interface to demonstrate a range of touch-based interfaces that could be applied to our interaction model. In the future we are interested, in particular, to examine additional data exploration techniques on the tablet.

Figure 1 :Figure 2 :

 12 Figure 1: Tablet navigation of stereoscopically displayed 3D data.

Fig. 3 :

 3 Fig. 3: Tablet-based navigation workflow for immersive visualizations.

Fig. 4 :

 4 Fig. 4: Due to physically moving in the room, the immersive and tablet views can become out-of-sync. This leads to confusion for the person exploring the visualization, in particular when rotating the dataset.


Charles Le Pabic
email: charles.lepabic@gmx.fr

Didier Goux

Maryline Guillamin

Georges Safi

Jean-Marc Lebel

Noussithé Koueta

Antoine Serpentini

Hemocyte morphology and phagocytic activity in the common cuttlefish (Sepia officinalis)

Introduction

Invertebrates resist pathogens despite their lack of adaptive immunity [START_REF] Loker | Invertebrate immune systems-not homogeneous, not simple, not well understood[END_REF]. The tremendous variety of invertebrate life histories and ecological niches suggests a great diversity of immune strategies [START_REF] Rowley | Invertebrate immune systems specific, quasi-specific, or nonspecific?[END_REF]. Among Molluscaone of the most diverse groups of animals [START_REF] Ponder | Molluscan evolution and phylogeny: an introduction[END_REF], studies of immunity have mostly focused on bivalves (e.g. [START_REF] Aladaileh | Characterization of phenoloxidase activity in Sydney rock oysters (Saccostrea glomerata)[END_REF][START_REF] Hong | Comparative study on the hemocytes of subtropical oysters Saccostrea kegaki (Torigoe & Inaba, 1981), Ostrea circumpicta (Pilsbry, 1904), and Hyotissa hyotis (Linnaeus, 1758) in Jeju Island, Korea: Morphology and functional aspec[END_REF][START_REF] Wootton | Structural and functional characterisation of the blood cells of the bivalve mollusc, Scrobicularia plana[END_REF][START_REF] Wang | Characterization of subpopulations and immune-related parameters of hemocytes in the green-lipped mussel Perna viridis[END_REF][START_REF] Salimi | Morphological characterization and size of hemocytes in Anodonta cygnea[END_REF][START_REF] Matozzo | Haemocytes of the cockle Cerastoderma glaucum: morphological characterisation and involvement in immune responses[END_REF][START_REF] López | Morphological characterization of the hemocytes of the clam, Ruditapes decussatus (Mollusca: Bivalvia)[END_REF][START_REF] Donaghy | Flow cytometry studies on the populations and immune parameters of the hemocytes of the Suminoe oyster, Crassostrea ariakensis[END_REF][START_REF] Donaghy | Functional and metabolic characterization of hemocytes of the green mussel, Perna viridis: in vitro impacts of temperature[END_REF][START_REF] Allam | Flow cytometric comparison of haemocytes from three species of bivalve molluscs[END_REF][START_REF] Chang | Morphological characterization via light and electron microscopy of two cultured bivalves: A comparison study between the Hard clam (Meretrix lusoria) and Pacific Oyster (Crassostrea gigas)[END_REF]) and gastropods (e.g. [START_REF] Travers | Morphologic, cytometric and functional characterisation of abalone (Haliotis tuberculata) haemocytes[END_REF][START_REF] Matricon-Gondran | Internal defenses of the snail Biomphalaria glabrata[END_REF][START_REF] Mahilini | Categorization of hemocytes of three gastropod species Trachea vittata (Muller), Pila globosa (Swainson) and Indoplanorbis exustus (Dehays)[END_REF][START_REF] Donaghy | First characterisation of the populations and immune-related activities of hemocytes from two edible gastropod species, the disk abalone, Haliotis discus discus and the spiny top shell, Turbo cornutus[END_REF][START_REF] Barracco | Morphological characterization of the hemocytes of the pulmonate snail Biomphalaria tenagophila[END_REF][START_REF] Accorsi | Comparative analysis of circulating hemocytes of the freshwater snail Pomacea canaliculata[END_REF]), while few studies have focused on cephalopods [START_REF] Ford | Host defense mechanisms of cephalopods[END_REF][START_REF] Castellanos-Martínez | Pathogens and immune response of cephalopods[END_REF].

Cephalopods are an interesting model because of their (1) vertebrate-like high-pressure closed circulatory system, (2) high sensitivity to environmental parameters, (3) short-life span and (4) elevated metabolic rate [START_REF] Melzner | Role of blood-oxygen transport in thermal tolerance of the cuttlefish, Sepia officinalis[END_REF][START_REF] Pierce | Geographic, seasonal and ontogenetic variation in cadmium and mercury concentrations in squid (Cephalopoda: Teuthoidea) from UK waters[END_REF]. Moreover, their economical importance has recently grown in terms of fisheries and aquaculture (e.g. [START_REF] Sykes | Cuttlefish culture -State of the art and future trends[END_REF][START_REF] Berger | Aquaculture of Octopus species : present status, problems and perspectives[END_REF][START_REF] Uriarte | Current Status and Bottle Neck of Octopod Aquaculture: The Case of American Species[END_REF][START_REF] Reid | Cephalopods of the world. An annotated and illustrated catalogue of species known to date. Volume 1. Chambered nautiluses and sepioids (Nautilidae, Sepiidae, Sepiolidae, Sepiadariidae, Idiosepiidae and Spirulidae)[END_REF][START_REF] Payne | Trends and assessment of cephalopod fisheries[END_REF]).

As in other invertebrates, the cephalopod immune system relies on humoral factors and cell-mediated mechanisms acting together to eliminate invading micro-organisms [START_REF] Soudant | Host-parasite interactions: Marine bivalve molluscs and protozoan parasites, Perkinsus species[END_REF][START_REF] Malham | Immunobiology of Eledone cirrhosa (Lamarck)[END_REF].

Humoral factors mainly include lectins (e.g. agglutinins, opsonins), antimicrobial factors (e.g. peptides, acid phosphatases, lysozymes), and several signaling pathways including prophenoloxidase (proPO) and proteolytic cascades [START_REF] Rowley | Invertebrate immune systems specific, quasi-specific, or nonspecific?[END_REF][START_REF] Wang | Research progress on the mollusc immunity in China[END_REF]. In contrast, cell-mediated defense mechanisms are primarly performed by hemocytes (Hcs)cells synthetized in white bodies and freely circulating in plasma and infiltrating in tissues [START_REF] Claes | Functional morphology of the white bodies of the cephalopod mollusc Sepia officinalis[END_REF][START_REF] Cowden | Cephalopods[END_REF]. Hcs are of central importance to invertebrates because of their involvement in numerous physiological functions [START_REF] Donaghy | Hemocytes of the carpet shell clam (Ruditapes decussatus) and the Manila clam (Ruditapes philippinarum): Current knowledge and future prospects[END_REF][START_REF] Beninger | Demonstration of nutrient pathway from the digestive system to oocytes in the gonad intestinal loop of the scallop Pecten maximus L[END_REF][START_REF] Matozzo | Effects of copper and cadmium exposure on functional responses of hemocytes in the clam, Tapes philippinarum[END_REF][START_REF] Montes | Cellular defence mechanism of the clam Tapes semidecussatus against infection by the protozoan Perkinsus sp[END_REF][START_REF] Mount | Hemocyte-mediated shell mineralization in the eastern oyster[END_REF], including their ability to phagocytose, encapsulate and destroy foreign particles [START_REF] Donaghy | First characterisation of the populations and immune-related activities of hemocytes from two edible gastropod species, the disk abalone, Haliotis discus discus and the spiny top shell, Turbo cornutus[END_REF][START_REF] Soudant | Host-parasite interactions: Marine bivalve molluscs and protozoan parasites, Perkinsus species[END_REF][START_REF] Rodríguez-Domínguez | Preliminary study on the phagocytic ability of Octopus vulgaris Cuvier, 1797 (Mollusca: Cephalopoda) haemocytes in vitro[END_REF]. In cephalopods, Hcs have mainly been described as a one cell-type population 1 1 Abbreviations: FCM: flow cytometry; Hc: hemocyte; Hcy: hemocyanin; MPS: molluscan physiological saline; NR: neutral red; PI: protease inhibitor; PO: phenoloxidase; proPO: prophenoloxidase; SEM: scanning electron microscopy; SD: standard deviation; TEM: transmission electron microscopy.

with large lobate nucleus and abundant granules, able to phagocytose [START_REF] Malham | Immunobiology of Eledone cirrhosa (Lamarck)[END_REF][START_REF] Cowden | Cephalopods[END_REF][START_REF] Rodríguez-Domínguez | Preliminary study on the phagocytic ability of Octopus vulgaris Cuvier, 1797 (Mollusca: Cephalopoda) haemocytes in vitro[END_REF][START_REF] Bolognari | struttura e funzione del "corpo bianco" dei cefalopodi. II. Struttura e funzione[END_REF][START_REF] Collins | Understanding the role of host hemocytes in a squid/Vibrio symbiosis using transcriptomics and proteomics[END_REF].

However, these Hc descriptions were mainly performed in Octopodidae as well as in the sepiolidae Euprymna scolopes and little is known about the immune cellular factors of other cephalopod species such as the sepiidae (cuttlefish) Sepia officinalis. Because of the distinct ecology of Sepiidae within Cephalopodia [START_REF] Uriarte | Current Status and Bottle Neck of Octopod Aquaculture: The Case of American Species[END_REF][START_REF] Boucaud-Camou | The migrations of the cuttlefish (Sepia officinalis L.) in the English Channel. The Cuttlefish[END_REF][START_REF] Wang | Spatial and temporal patterns of cuttlefish (Sepia officinalis) abundance and environmental influencesa case study using trawl fishery data in French Atlantic coastal , English Channel , and adjacent waters[END_REF], they may also have distinct immune requirements.

In this study, we performed cytological stainings, electron microscopy and flow cytometry (FCM) analysis to morphologically characterize S. officinalis Hcs. In addition, we investigated humoral factor localization between plasma and cells, and phagocytic reactions at several incubation times, temperatures and plasma concentrations. Our results highlighted one granulocyte population with various densities of acidophilic granules and unstained vesicles.

The Hcs, which contained acid phosphatase, lysozyme and proPO system enzymes, had high phagocytic ability, modulated by plasma in our assay conditions.

Material and methods

Animals

Thirty-one adult cuttlefish S. officinalis (21.5 ± 3.1 cm mantle length) were obtained from traps deployed during spring 2011 and 2012 along the Calvados coast (Normandy, France). Cuttlefish were then conditioned at the Centre de Recherches en Environnement Côtier (Luc-sur-Mer, Normandy, France) in 4500-L tanks in an open seawater circuit for at least 2 days, fed with crustaceans Crangon crangon and Carcinus maenas, and starved for 1 day before experimentation. The sex of each individual was determined.

Hemolymph collection

Before hemolymph sampling and following ethical procedures (Directive 2010/63/EU), cuttlefish were anesthetized as described by Andrews et al. [START_REF] Andrews | The identification and management of pain, suffering and distress in cephalopods, including anaesthesia, analgesia and humane killing[END_REF] through placement for 10 min in seawater containing 2% ethanol. Between 9 and 13 ml of hemolymph was then withdrawn from the anterior mantle vein [START_REF] King | Using ultrasound to understand vascular and mantle contributions to venous return in the cephalopod Sepia officinalis L[END_REF] using a syringe with 18-gauge needle.

The sample was transferred into a sterile tube, diluted or not with one volume of cooled sterile antiaggregative modified Alsever solution (115 mM glucose; 27 mM sodium citrate; 11.5 mM EDTA; 382 mM NaCl pH 7.5) [START_REF] Bachère | Separation of Crassostrea gigas hemocytes by density gradient centrifugation and counterflow centrifugal elutriation[END_REF], depending on the procedure (see below), and kept on ice to minimize cell clumping. Hc viability was checked by mixing one volume of Alseverdiluted hemolymph with one volume of trypan blue solution (0.4%) and Hc concentration was determined with non diluted hemolymph using a Thoma cell. Once sampling was completed, animals were euthanized by increasing ethanol concentration to 10% [START_REF] Oestmann | Special considerations for keeping cephalopods in laboratory facilities[END_REF].

Chemicals

Sodium chloride (NaCl), anhydrous and hexahydrate magnesium chloride (MgCl were obtained from Sigma-Aldrich (France). Halt Protease Inhibitor Cocktail, EDTA-Free (100X) was obtained from Thermo Fisher Scientific (Waltham, USA). Ethanol was obtained from Carlo erba (Milan, Italy). Hemacolor® staining kit was obtained from Merck Millipore (Darmstadt, Germany). Low melting point agar was obtained from Carl Roth (Lauterbourg, France). All chemicals used for electron microscopy i.e. glucose, sodium citrate, glutaraldehyde, sodium cacodylate, sucrose, osmium tetroxide (OsO 4 ), propylene oxide, araldite resin, uranyl acetate and lead citrate were obtained from Electron Microscopy Sciences (Hatfield, PA, USA).

Morphological characterization of S. officinalis Hcs

Hemolymph cell monolayer stainings

For Hc staining, one drop of hemolymph was placed on a Thermanox TM coverslip (Thermo Fisher Scientific, Waltham, USA) and allowed to adhere for 30 min at 15°C. Before staining, coverslips were rinsed in Molluscan Physiological Saline (MPS; 0.4 M NaCl, 0.1 M MgSO 4 , 20 mM HEPES, 10 mM CaCl 2 and 10 mM KCl modified after [START_REF] Lowe | Lysosomal membrane responses in the blood and digestive cells of mussels experimentally exposed to fluoranthene[END_REF]) to remove plasma. Hemacolor® staining was performed according to the manufacturer's recommendations. Wright staining was performed after 1 min air-drying by 1 min dehydration in absolute methanol, following by 1 min in Wright solution (0.66% in methanol), then diluted (1:4) in distilled water during 4 min and rinsed. To highlight the cell lysosomal system, neutral red (NR) uptake was performed by incubating cells for 30 min in NR solution (1:500; NR stock solution (20 mg NR/ml DMSO):MPS) in a moist chamber at 15°C, before observation.

Freshly spread Hcs were observed using an inverted binocular microscope (Leica® DM IRB, Leica Microsystems, Wetzlar, Germany). Stained Hc observations were carried out with a Nikon Eclipse 80i light microscope with computer-assisted microscopic image analysis system, NIS-elements D 2.30 software (Nikon®, Champigny-sur-Marne, France).

Electron microscopy

After 10 min 300 × g centrifugation, Hc pellets (N = 5) were rinsed with MPS and fixed in 3.2% glutaraldehyde in 0.31 M sodium cacodylate buffer, with 0.25 M sucrose (pH 7.4) during for 90 min at 4°C. The cells were washed 3 times in rinsing solution (0.4 M sodium cacodylate, 0.3 M sucrose, pH 7.4). Then, cells were post-fixed 1 h with 1% OsO 4 in cacodylate buffer 0.2 M, with 0.36 M sucrose (pH 7.4) at 4°C (protected from light), and washed in rinsing solution.

For scanning electron microscopy (SEM), cells were sedimented for 7 days on Thermanox TM coverslip coated with poly-l-lysine (Thermo Fisher Scientific, Waltham, USA).

They were then dehydrated in progressive bath of ethanol (70-100%) and critical point dried (Leica® EM CPD030). Finally, cells were sputtered with platinum and observed with scanning electron microscope JEOL 6400F. Freshly sampled cell diameters were determined by measuring 100 cells per cuttlefish (N = 5).

For transmission electron microscopy (TEM), cells were pellet in 2% low melting point agar at 40°C, and then dehydrated through increasing concentrations of ethanol (70-100%) and propylene oxide 100%, embedded in araldite resine and allowed to polymerise for 48 h at 60°C. Ultrathin sections were done and contrasted with 2.5% uranyl acetate diluted in 50% ethanol for 30 min and contrasted for 5 min in Reynold's lead citrate [START_REF] Venable | A simplified lead citrate stain for use in electron microscopy[END_REF]. Finally, cells were observed by transmission electron microscope JEOL 1011 and images were obtained with Camera Megaview 3 and Analysis Five software.

Biochemical analysis 2.5.1. Enzyme extraction

Hcs and plasma were separated in non diluted hemolymph by 500 × g centrifugation for 10 min at 4°C. Plasma was then removed, checked for absence of Hc microscopically, and stored in aliquots at -80°C until analysis. Upon complete plasma removal, Hc pellets were gently rinsed in either Tris buffer pH 7 (0.1 M Tris-HCl, 0.45 M NaCl, 26 mM MgCl 2 and 10 mM CaCl 2 ) for phenoloxidase (PO) assays [START_REF]Les phénoloxydases chez l'huître creuse Crassostrea gigas: biomarqueurs potentiels de stress environnemental[END_REF] or Tris buffer pH 8 (10 mM Tris-HCl and 150 mM NaCl) for phosphatase, lysozyme and protease inhibitor (PI) assays [START_REF] Safi | Etude de la variabilité spatio-temporelle des caractéristiques physiologiques des jeunes stades de vie de la seiche Sepia officinalis L . en Manche[END_REF]. Cells were resuspended at 10 × 10 6 cells ml -1 in same extraction buffer and sonicated at 60 W for 20 s.

Cell extracts were then centrifuged at 10,000 × g and their supernatant aliquoted and stored at -80°C until analysis.

Enzymatic assays

All activities were expressed in relation to protein concentration measured by the Bradford method [START_REF] Bradford | A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[END_REF] using BSA as standard.

Total acid phosphatase activity was determined according to Moyano et al. [START_REF] Moyano | Characterization of digestive enzyme activity during larval development of gilthead seabream (Sparus aurata)[END_REF] using p-nitrophenyl-phosphate 2% as substrate in a 1 M Tris buffer at pH 3. Then, 10 µl of supernatant was added to 10 µl of substrate in 96-well flat bottom plates (BD, USA). After 30 min incubation at 25°C, 100 µl of NaOH 1 M were added to stop the reaction. The absorbance was measured at 405 nm using a Mithras LB 940 luminometer (Berthold, Thoiry, France). Total acid phosphatase activity was expressed as specific activity (U mg -1 protein)

where one enzymatic unit corresponded to the absorbance recorded after incubation.

Lysozyme activity was quantified according to Malham et al. [START_REF] Malham | Lysozyme and antiprotease activity in the lesser octopus Eledone cirrhosa (Lam.) (Cephalopoda)[END_REF]. Fifty µl of HEW lysozyme (85 µg ml -1 in Tris buffer pH 8 described in section 2.5.1.) standard was serially diluted in triplicate in 96-well flat bottom plates (BD, USA). Fifty µl of each sample was added in triplicate to the 96-well plates as well as 50 µl of Tris buffer pH 8, as blanks. One hundred and fifty µl of the substrate, freeze-dried M. lysodeikticus ( The reductions in turbidity in the wells were read on Mithras LB 940 luminometer (Berthold, Thoiry, France) at 25°C for 5 minutes at 10 second intervals at 450 nm using negative kinetics. Lysozyme concentrations were calculated from the standard curve (µg HEW lysozyme equivalent ml -1 ). Final lysozyme-like activity was thus expressed as µg HEW lysozyme eq. mg protein -1 .

As described by Malham et al. [START_REF] Malham | Lysozyme and antiprotease activity in the lesser octopus Eledone cirrhosa (Lam.) (Cephalopoda)[END_REF] and Thompson et al. [START_REF] Thompson | The effect of dietary vitamin A and astaxanthin on the immunocompetence of rainbow trout[END_REF], PI activity was measured by transferring 20 µl of sample and 10 µl of trypsin TPCK (100 µg ml -1 of 0.05 M Tris buffer pH 8) in 96-well flat bottom plates (BD, USA), and mixed at room temperature for 5 minutes.

In parallel, intrinsic trypsin activity was measured by replacing 10 µl of trypsin by Tris buffer pH 8 described above (section 2.5.1.). A positive control was used by replacing the sample with Tris buffer pH 8. Two hundred µl of BAPNA substrate solution (5.2 mg BAPNA ml -1 DMSO) in 10 ml of 0.01 M trizma base buffer pH 7.4) was added to each well and incubated for 15 minutes at room temperature. Absorbance was read at 405 nm using Mithras LB 940 luminometer (Berthold, Thoiry, France), and PI activity was expressed as the percentage of trypsin sample inhibition (TI) compared to the positive control.

In order to partly discriminate PO synthesis and activation site, special care was taken to avoid unwanted activation of proPO during each step of the experiment. PO-like activity was measured spectrophotometrically by recording the formation of o-quinones, as described by Luna-Acosta [START_REF]Les phénoloxydases chez l'huître creuse Crassostrea gigas: biomarqueurs potentiels de stress environnemental[END_REF] with slight modifications to distinguish artificially activated PO (APO) (corresponding to PO-like activity resulting from zymogenic PO (proPO) activation plus already 'active' form) and in vivo 'active' PO form [START_REF] Pabic | Prophenoloxidase system, lysozyme and protease inhibitor distribution in the common cuttlefish Sepia officinalis[END_REF]. PO assays were conducted in 96well flat bottom plates (BD, USA). L-DOPA was used as substrate, at a final concentration of 10 mM [START_REF]Les phénoloxydases chez l'huître creuse Crassostrea gigas: biomarqueurs potentiels de stress environnemental[END_REF] and prepared extemporaneously in Tris buffer pH 7 described above (section 2.5.1.). Tropolone (10 mM) and trypsin TPCK (1 g l -1 ) were used respectively as PO inhibitor and elicitor as previously described in S. officinalis [START_REF] Pabic | Prophenoloxidase system, lysozyme and protease inhibitor distribution in the common cuttlefish Sepia officinalis[END_REF][START_REF] Lacoue-Labarthe | Phenoloxidase activation in the embryo of the common cuttlefish Sepia officinalis and responses to the Ag and Cu exposure[END_REF]. To avoid uncontrolled proPO activation by intrinsic proteases, Halt Protease Inhibitor Cocktail, EDTA-Free (1X) wide spectrum PI was used. For each sample, autoxidation of sample, 'basal', 'inhibited' and 'activated' PO-like activities were measured. For non-enzymatic sample autoxidation, 10 µl of sample was mixed with Tris buffer pH 7. For 'basal' PO-like activity, 10 µl of sample was firstly mixed during 10 min with 1.4 µl Halt Protease Inhibitor Cocktail (100X), followed by adapted volume of Tris buffer pH 7 and 80 µl L-DOPA. Similarly, for inhibited or APO-like activity, 10 µl of sample was mixed with 10 µl of tropolone (140 mM) or trypsin TPCK (14 g l -1 ), Tris buffer pH 7 and 80 µl L-DOPA. Each measurement was systematically controlled by replacing sample by buffer, always in a final volume reaction of 140 µl. Immediately after L-DOPA addition, PO-like activities were monitored at 25°C for 5 h using Mithras LB 940 luminometer (Berthold, Thoiry, France) at 490 nm [START_REF]Les phénoloxydases chez l'huître creuse Crassostrea gigas: biomarqueurs potentiels de stress environnemental[END_REF]. When an inhibited PO-like activity was measured, this value was subtracted from APO and PO-like activities. Tropolone, with its copper chelator and peroxidase substrate properties, ensured that PO-like activity alone was detected (and not peroxidase). Results were also systematically corrected for non-enzymatic autoxidation of the substrate and were expressed in enzyme unit (1 U) per mg of total protein.

One U corresponded to an increase of 0.001 in the absorbance per min at 25°C [START_REF] Pabic | Prophenoloxidase system, lysozyme and protease inhibitor distribution in the common cuttlefish Sepia officinalis[END_REF].

Flow cytometry (FCM) analysis

FCM analyses were performed using a Gallios flow cytometer (Beckman Coulter). In our study, excitation light was provided by a 22 mW blue diode (488 nm), and fluorescence was collected in the FL1 channel with a 525 nm bandpass filter. For each sample, about 20,000 events were acquired. Data were analysed using Kaluza software (Beckman Coulter).

Freshly sampled Hc cytomorphology

Hc morphology was based upon relative flow-cytometric parameters, Forward Scatter (FSC) and Side Scatter (SSC). FSC and SSC commonly measure particle size and internal complexity, respectively. Internal complexity, also reported as granularity, depends upon various inner components of the cells including shape of the nucleus, amount and types of cytoplasmic granules, cytoplasmic inclusions and membrane roughness [START_REF] Donaghy | First report of severe hemocytopenia and immunodepression in the sunray venus clam, Macrocallista nimbosa, a potential new aquaculture species in Florida[END_REF]. Freshly withdrawn Hc was pelleted by 300 × g centrifugation for 5 min at 4°C, rinsed in MPS, before to be fixed in 3.7% formaldehyde in MPS and kept in dark at 4°C until FCM analysis.

Phagocytosis assays

Evaluation of phagocytosis was based on the ingestion of carboxylate-modified

FluoSpheres® beads (1.0 µm, yellow-green carboxylate-modified FluoSpheres®, Molecular Probes) by Hcs. Phagocytosis was expressed as (i) the percentage of cells that had ingested three or more microbeads [START_REF] Hégaret | Flow cytometric analysis of haemocytes from eastern oysters, Crassostrea virginica, subjected to a sudden temperature elevation[END_REF][START_REF] Auffret | A multiparametric approach for monitoring immunotoxic responses in mussels from contaminated sites in Western Mediterranea[END_REF], and (ii) the average number of microbeads per phagocytic Hcs [START_REF] Donaghy | First characterisation of the populations and immune-related activities of hemocytes from two edible gastropod species, the disk abalone, Haliotis discus discus and the spiny top shell, Turbo cornutus[END_REF].

Hemolymph-diluted Alsever solution was used to plate Hcs at 1.0 × 10 6 cells per well in 6-well plates, into which three volumes of sterile artificial seawater (25.5 g l -1 NaCl, 6.4 g l - 1 MgSO 4 , 5.2 g l -1 Hepes, 1.1 g l -1 CaCl 2 , 0.75 g l -1 KCl pH 7.4) were added to allow cell adhesion. After 60 min of incubation in dark at 15°C, supernatant was removed and cells were covered with 1 ml modified sterile Leibovitz L-15 medium pH 7.6 (20.2 g l -1 NaCl, 0.54 g l -1 KCl, 0.6 g l -1 CaCl 2 , 1 g l -1 MgSO 4 •7H 2 O and 3.9 g l -1 MgCl 2 •6H 2 O) [START_REF] Domart-Coulon | Identification of media supplements that improve the viability of primarily cell cultures of Crassostrea gigas oysters[END_REF], supplemented with 2 mM L-glutamine, 100 µg ml -1 streptomycin and 60 µg ml -1 penicillin G into which FluoSpheres® were added at a ratio of 1:100 (Hc:beads). Hcs from same individual cuttlefish were analyzed at each time.

In vitro impact of time and temperature

Hcs were collected from 5 cuttlefish as described in section 2.2. To determine temperature-dependence of S. officinalis phagocytosis, three different temperatures were investigated; one physiological temperature (15°C) and two temperatures (4 and 25°C) out of the thermal window determined by Melzner et al. [START_REF] Melzner | Role of blood-oxygen transport in thermal tolerance of the cuttlefish, Sepia officinalis[END_REF] with 15°C-acclimated cuttlefish. In this context, media with FluoSpheres® were acclimated to different working temperatures before addition to Hcs. Then, 6-well plates were placed in dark at 4, 15 and 25°C CO 2 -free incubators, during 30, 60, 120 and 180 min incubation. Afterwards wells were rinsed with MPS to remove bead excess, following by gentle scrapping and centrifugation at 300 × g for 5 min at 4°C. Resulting supernatants were removed, pellets resuspended in 3.7% formaldehyde solution in MPS and stored in the dark at 4°C until FCM analysis.

In vitro impact of plasma concentrations

To study the plasma opsonization process, FluoSpheres® were added to modified L-incubated at 15°C during 1 h before homogenization and addition to cells. After 2 h incubation at 15°C, cells were treated as previously described (cf 2.6.2.1.) for FCM analysis.

In parallel, bead phagocytosis was performed in modified L-15 medium or 100% plasma, and Hcs analysed by TEM as described in section 2.4.2.

Data analysis

Residual distributions were tested for normality (Shapiro test) as well as homogeneity of variances (Levene test). Student's t-test was used to compare Hc concentrations between male and female individuals, specific activities between Hc and plasma compartments, PO-and APO-like activities in each compartment, and phagocytic parameters between control and several plasma treatments. One-way analysis of variance (ANOVA) followed by nonparametric pairwise permutational t-tests (N<30) was used to determine the impact of temperature on phagocytic parameters at each time. Analysis of covariance (ANCOVA) was used to compare phagocytic parameter time-evolution as function of the temperature. All results are expressed and displayed as mean ± standard deviation (SD). The statistical significance was designed as being at the level of p<0.05. R software was used for statistics and graphics.

Results and Discussion

Little is known about cephalopod Hcs, while Hc populations have been extensively studied in other mollusks (bivalves and gastropods). Our study provides the most comprehensive description of the circulating Hc population in the cuttlefish S. officinalis by using FCM, light and electron microscopy. We also report biochemical-and phagocytosis analysis of Hc interaction with plasma.

Hc viability and concentration

Hc viability in all cuttlefish used in this study was higher than 99% (data not shown), and no Hc concentration difference was observed between males (7.20 ± 5.70 × 10 6 cells ml -1 ; N=18) and females (7.27 ± 5.75 × 10 6 cells ml -1 ; N=13). Consequently, we obtained a mean Hc concentration in hemolymph of S. officinalis of 7.23 ± 5.62 × 10 6 cells ml -1 ranging from 0.92 to 20.31 × 10 6 cells ml -1 . This mean concentration and concentration variability are consistent with reports on cephalopod Hcs (Table 1). Although highly variable, the Hc concentration of S. officinalis appeared slightly higher than that of the loliginidae Sepioteuthis lessoniana, similar to that of the sepiolidae Euprymna scolopes, and lower than that of Octopodidae, where the highest Hc concentrations within cephalopods are found (10 × 10 6 cells ml -1 ) [START_REF] Rodríguez-Domínguez | Preliminary study on the phagocytic ability of Octopus vulgaris Cuvier, 1797 (Mollusca: Cephalopoda) haemocytes in vitro[END_REF][START_REF] Collins | Understanding the role of host hemocytes in a squid/Vibrio symbiosis using transcriptomics and proteomics[END_REF][START_REF] Castellanos-Martínez | Morphologic, cytometric and functional characterization of the common octopus (Octopus vulgaris) hemocytes[END_REF][START_REF] Heming | Cytoplasmic pH recovery in acidloaded haemocytes of squid (Sepioteuthis lessoniana)[END_REF][START_REF] Collins | Obtaining hemocytes from the Hawaiian bobtail squid Euprymna scolopes and observing their adherence to symbiotic and non-symbiotic bacteria[END_REF][START_REF] Malham | Effects of repeated sampling on the haemocytes and haemolymph of Eledone cirrhosa (Lam.)[END_REF][START_REF] Malham | A first insight into stressinduced neuroendocrine and immune changes in the octopus Eledone cirrhosa[END_REF]. Generally, Hcs of gastropod and bivalve mollusks appear less concentrated with most values ranging between 1 and 5 × 10 6 cells ml -1 (Table 1) [START_REF] Wootton | Structural and functional characterisation of the blood cells of the bivalve mollusc, Scrobicularia plana[END_REF][START_REF] Wang | Characterization of subpopulations and immune-related parameters of hemocytes in the green-lipped mussel Perna viridis[END_REF][START_REF] Matozzo | Haemocytes of the cockle Cerastoderma glaucum: morphological characterisation and involvement in immune responses[END_REF][START_REF] Donaghy | Flow cytometry studies on the populations and immune parameters of the hemocytes of the Suminoe oyster, Crassostrea ariakensis[END_REF][START_REF] Donaghy | Functional and metabolic characterization of hemocytes of the green mussel, Perna viridis: in vitro impacts of temperature[END_REF][START_REF] Donaghy | First characterisation of the populations and immune-related activities of hemocytes from two edible gastropod species, the disk abalone, Haliotis discus discus and the spiny top shell, Turbo cornutus[END_REF][START_REF] Barracco | Morphological characterization of the hemocytes of the pulmonate snail Biomphalaria tenagophila[END_REF][START_REF] Accorsi | Comparative analysis of circulating hemocytes of the freshwater snail Pomacea canaliculata[END_REF][START_REF] Donaghy | First report of severe hemocytopenia and immunodepression in the sunray venus clam, Macrocallista nimbosa, a potential new aquaculture species in Florida[END_REF][START_REF] Wootton | Bivalve immunity: comparisons between the marine mussel (Mytilus edulis), the edible cockle (Cerastoderma edule) and the razorshell (Ensis siliqua)[END_REF][START_REF] Barracco | Some haemato-immunological parameters in the mussel Perna perna[END_REF][START_REF] Goedken | Flow cytometry as a tool to quantify oyster defence mechanisms[END_REF][START_REF] Zhang | Morphological, structural, and functional characterization of the haemocytes of the scallop, Argopecten irradians[END_REF][START_REF] Nakayama | Morphological and functional characterization of hemocytes in the giant clam Tridacna crocea[END_REF][START_REF] Xie | Morphology and phagocytic ability of hemocytes from Cristaria plicata[END_REF][START_REF] Pampanin | Morphological and cytoenzymatic characterization of haemocytes of the venus clam Chamelea gallina[END_REF][START_REF] Jauzein | Flow cytometric characterization of hemocytes of the sunray venus clam Macrocallista nimbosa and influence of salinity variation[END_REF], albeit with population-dependent variation [START_REF] Wang | Characterization of subpopulations and immune-related parameters of hemocytes in the green-lipped mussel Perna viridis[END_REF][START_REF] Donaghy | Functional and metabolic characterization of hemocytes of the green mussel, Perna viridis: in vitro impacts of temperature[END_REF][START_REF] Santarém | Seasonal changes in hemocytes and serum defense factors in the blue mussel Mytilus galloprovincialis[END_REF].

Hc identification and characterization

Light microscopy

After adhesion, fresh cuttlefish Hcs rapidly displayed many thin pseudopodia, and most cells contained refringent and non-refringent granules of various densities in their cytoplasm (Fig. 1). Among different stains used, Wright's differential staining allowed the best observation of spontaneously adhering Hcs. A single cell type with large, lobate nucleus, slightly basophilic cytoplasm and acidophilic granules was identified (Fig. 2A). Lucent vesicles were also observed in the cytoplasm of some cells. NR uptake revealed dense lysosomal system in most cells (Fig. 2B). According to the classification of bivalve Hcs, 2 main classes are usually accepted: granulocytes with cytoplasmic granules and hyalinocytes with few or no granules [77]. Based on the high granule density found in most Hcs, we classify S. officinalis Hcs as granulocytes. These observations are consistent with the majority of studies performed in Octopodidae [START_REF] Malham | Immunobiology of Eledone cirrhosa (Lamarck)[END_REF][START_REF] Cowden | Cephalopods[END_REF][START_REF] Bolognari | struttura e funzione del "corpo bianco" dei cefalopodi. II. Struttura e funzione[END_REF][START_REF] Cowden | Observations on living cells dissociated from the leukopoietic organ of Octopus briareus[END_REF][START_REF] Kondo | Morphology of octopus hemocytes[END_REF], Sepiidae [START_REF] Claes | Functional morphology of the white bodies of the cephalopod mollusc Sepia officinalis[END_REF] and Sepiolidae [START_REF] Collins | Understanding the role of host hemocytes in a squid/Vibrio symbiosis using transcriptomics and proteomics[END_REF][START_REF] Heath-Heckman | The occurrence of chitin in the hemocytes of invertebrates[END_REF], which reported one granular cell population circulating in the hemolymph and also called macrophage-like Hc [START_REF] Cowden | Cephalopods[END_REF][START_REF] Nyholm | Sampling the light-organ microenvironment of Euprymna scolopes: description of a population of host cells in association with the bacterial symbiont Vibrio fischeri[END_REF].

Electron microscopy

SEM observations of circulating cuttlefish Hcs showed a single cell type of 10.3 ± 0.8 µm in diameter and able to form numerous pseudopodia (Fig. 3A-B), consistent with the above described light microscopy observations. This cell size is consistent with the 8-10 µm cell diameter measured on non-circulating mature Hcs in the cuttlefish white bodies (leukopoietic tissues) [START_REF] Claes | Functional morphology of the white bodies of the cephalopod mollusc Sepia officinalis[END_REF] and appeared slightly lower than octopod O. vulgaris and E. cirrhosa Hcs: 11.6 ± 1.2 and 12-15 µm, respectively [START_REF] Castellanos-Martínez | Morphologic, cytometric and functional characterization of the common octopus (Octopus vulgaris) hemocytes[END_REF][START_REF] Stuart | The reticulo-endothelial apparatus of the lesser octopus, Eledone cirrhosa[END_REF].

TEM observations of circulating Hcs highlighted a large lobate nucleus without nucleoli but highly condensed chromatin mainly localized along the inner surface of the nuclear membrane (Fig. 3C). The cytoplasm of most cells contained various sizes and densities of two distinct inclusion types (Fig. 3D): electron-dense granules corresponding to lysosomes [START_REF] Claes | Functional morphology of the white bodies of the cephalopod mollusc Sepia officinalis[END_REF],

and electron-lucent membrane-surrounded vesicles, containing various densities of molecules with similar size and circular shape as Hcy molecules (Fig. 3E-F; [START_REF] Bergmann | The hemocyanin from a living fossil, the cephalopod Nautilus pompilius: protein structure, gene organization, and evolution[END_REF][START_REF] Dilly | The branchial gland: a site of haemocyanin synthesis in Octopus[END_REF][START_REF] Muzii | Intracellular polymerized haemocyanin in the branchial gland of a cephalopod[END_REF][START_REF] Boisset | Sepia officinalis hemocyanin: A refined 3D structure from field emission gun cryoelectron microscopy[END_REF][START_REF] Martoja | Localisation cytologique du cuivre et de quelques autres métaux dans la glande digestive de la seiche, Sepia officinalis L. (Mollusque Céphalopode)[END_REF]). These molecules are referred to as Hcy-like hereafter. In contrast to E. cirrhosa and O. vulgaris Hcs, neither different electron-dense granules nor lipid droplets were observed [START_REF] Malham | Immunobiology of Eledone cirrhosa (Lamarck)[END_REF][START_REF] Cowden | Cephalopods[END_REF][START_REF] Castellanos-Martínez | Morphologic, cytometric and functional characterization of the common octopus (Octopus vulgaris) hemocytes[END_REF], suggesting different granule contents between these cephalopods. Mitochondria, Golgi apparatus and rough endoplasmic reticulum that often surrounded the nucleus were also observed in these cells (Fig. 3C inset).

Enzymatic assays

Hydrolytic enzyme lysozymes and acid phosphatases were detected in Hc extract alone (Table 2), consistent with their known lysosomal distribution [START_REF] Blasco | Acid and alkaline phosphatase activites in the clam Ruditapes philippinarum[END_REF][START_REF] Fiołka | Activity and immunodetection of lysozyme in earthworm Dendrobaena veneta (Annelida)[END_REF]. These data are in agreement with carbohydrates and acid phosphatases detected by stainings in E. cirrhosa Hcs [START_REF] Malham | Effects of repeated sampling on the haemocytes and haemolymph of Eledone cirrhosa (Lam.)[END_REF] and suggest their synthesis in S. officinalis Hcs or earlier during their maturation [START_REF] Malham | Effects of repeated sampling on the haemocytes and haemolymph of Eledone cirrhosa (Lam.)[END_REF][START_REF] Cowden | Some cytological and cytochemical observations on the leucopoietic organs, the "White Bodies", of Octopus vulgaris[END_REF]. PI activities were exclusively found in plasma (Table 2), corresponding at least in part to the already described α 2 -macroglobulin activitythe second most abundant protein in S.

officinalis plasma [START_REF] Thøgersen | Purification and characterization of an α-macroglobulin proteinase inhibitor from the mollusc Octopus vulgaris[END_REF][START_REF] Armstrong | Humoral immunity: α 2 -macroglobulin activity in the plasma of mollusks[END_REF][START_REF] Vanhoorelbeke | An α 2 -macroglobulin-like proteinase inhibitor in the haemolymph of the Decabrachia cephalopod Sepia officinalis[END_REF]. Interestingly, PIs were recently reported in E. cirrhosa and E.

scolopes Hcs [START_REF] Malham | Immunobiology of Eledone cirrhosa (Lamarck)[END_REF][START_REF] Collins | Understanding the role of host hemocytes in a squid/Vibrio symbiosis using transcriptomics and proteomics[END_REF], underlying the need for future investigations. PO-like activities were measured in both hemolymph compartments (Table 2). Siddiqui et al. [START_REF] Siddiqui | Location of intrinsic and inducible phenoloxidase activity in molluscan hemocyanin[END_REF] showed in S. officinalis that plasma-associated PO-activity resulted from Hcy, which molecule shares a structurally and functionally equivalent active site with POs [START_REF] Campello | Role of the tertiary structure in the diphenol oxidase activity of Octopus vulgaris hemocyanin[END_REF].

Therefore, PO-like activities found in Hc extracts probably result, at least in part, from Hcy presence in electron-lucent vesicles (TEM analysis). We note that PO activity was not previously detected in cephalopod Hcs. In contrast, proPO activationthe difference between PO-and APO-like activity, occurred in Hc extract alone (p=0.01).

FCM analysis

Fresh Hc cytogram

According to the criteria of cell size (FSC) and cell complexity (SSC), FCM supported the presence of a single Hc population in cuttlefish (Fig. 4A), albeit with variable internal complexity in some individuals (Fig. 4B), consistent with the above described TEM observations. Notably, recent O. vulgaris Hc characterization using TEM and FCM reported 2 circulating granulocyte morphs based on size, which were interpreted as different maturating stages of the same cell type [START_REF] Castellanos-Martínez | Morphologic, cytometric and functional characterization of the common octopus (Octopus vulgaris) hemocytes[END_REF]. Consistently, a study of S. officinalis Hc synthesis suggested a Hc maturation model with a single cell precursor [START_REF] Claes | Functional morphology of the white bodies of the cephalopod mollusc Sepia officinalis[END_REF]. This cephalopod organization is distinct from that of bivalves and gastropods, where most Hc studies reported several circulating Hc types derived from one or more cell precursors (e.g. [START_REF] Hong | Comparative study on the hemocytes of subtropical oysters Saccostrea kegaki (Torigoe & Inaba, 1981), Ostrea circumpicta (Pilsbry, 1904), and Hyotissa hyotis (Linnaeus, 1758) in Jeju Island, Korea: Morphology and functional aspec[END_REF][START_REF] Salimi | Morphological characterization and size of hemocytes in Anodonta cygnea[END_REF][START_REF] Matozzo | Haemocytes of the cockle Cerastoderma glaucum: morphological characterisation and involvement in immune responses[END_REF][START_REF] Donaghy | Flow cytometry studies on the populations and immune parameters of the hemocytes of the Suminoe oyster, Crassostrea ariakensis[END_REF][START_REF] Travers | Morphologic, cytometric and functional characterisation of abalone (Haliotis tuberculata) haemocytes[END_REF][START_REF] Mahilini | Categorization of hemocytes of three gastropod species Trachea vittata (Muller), Pila globosa (Swainson) and Indoplanorbis exustus (Dehays)[END_REF][START_REF] Donaghy | First characterisation of the populations and immune-related activities of hemocytes from two edible gastropod species, the disk abalone, Haliotis discus discus and the spiny top shell, Turbo cornutus[END_REF][START_REF] Accorsi | Comparative analysis of circulating hemocytes of the freshwater snail Pomacea canaliculata[END_REF][START_REF] Wang | Research progress on the mollusc immunity in China[END_REF][START_REF] Prado-Alvarez | Morphological characterization and functional immune response of the carpet shell clam (Ruditapes decussatus) haemocytes after bacterial stimulation[END_REF][START_REF] Rebelo | New insights from the oyster Crassostrea rhizophorae on bivalve circulating hemocytes[END_REF]). Our results further support the presence of a single mature circulating cell type in the Hc population of cephalopods.

Phagocytosis experiments

Time and temperature

Both phagocytosis parameters (i.e. percentage of phagocytic Hcs and quantity of ingested beads) appeared time-dependent at all temperatures tested (Fig. 5).

After 30 min, 35.5, 40.1, and 41.7% of cells were phagocytic at 4, 15 and 25°C, respectively, subsequently reaching 63.3, 70.6 and 69.8% after 180 min (Fig. 5A). These phagocytic percentages are consistent with previous observations of Hcs in O. vulgaris and E. cirrhosa where 50 and 70% of phagocytic cells were reported, respectively [START_REF] Rodríguez-Domínguez | Preliminary study on the phagocytic ability of Octopus vulgaris Cuvier, 1797 (Mollusca: Cephalopoda) haemocytes in vitro[END_REF][START_REF] Malham | Phagocytosis by haemocytes from the Lesser Octopus Eledone cirrhosa[END_REF]. In contrast, a lower phagocytic percentage (≤ 13%) was recently reported for O. vulgaris Hcs after 120 min incubation at 15°C [START_REF] Castellanos-Martínez | Morphologic, cytometric and functional characterization of the common octopus (Octopus vulgaris) hemocytes[END_REF]. The filtered seawater used as media during this in vitro experiment may account for this lower rate. Overall, our results are among the highest phagocytic percentages reported among mollusks, regardless of experimental conditions (e.g. [START_REF] Hong | Comparative study on the hemocytes of subtropical oysters Saccostrea kegaki (Torigoe & Inaba, 1981), Ostrea circumpicta (Pilsbry, 1904), and Hyotissa hyotis (Linnaeus, 1758) in Jeju Island, Korea: Morphology and functional aspec[END_REF][START_REF] Wootton | Structural and functional characterisation of the blood cells of the bivalve mollusc, Scrobicularia plana[END_REF][START_REF] Wang | Characterization of subpopulations and immune-related parameters of hemocytes in the green-lipped mussel Perna viridis[END_REF][START_REF] Donaghy | Flow cytometry studies on the populations and immune parameters of the hemocytes of the Suminoe oyster, Crassostrea ariakensis[END_REF][START_REF] Donaghy | Functional and metabolic characterization of hemocytes of the green mussel, Perna viridis: in vitro impacts of temperature[END_REF][START_REF] Travers | Morphologic, cytometric and functional characterisation of abalone (Haliotis tuberculata) haemocytes[END_REF][START_REF] Donaghy | First characterisation of the populations and immune-related activities of hemocytes from two edible gastropod species, the disk abalone, Haliotis discus discus and the spiny top shell, Turbo cornutus[END_REF][START_REF] Nakayama | Morphological and functional characterization of hemocytes in the giant clam Tridacna crocea[END_REF][START_REF] Jauzein | Flow cytometric characterization of hemocytes of the sunray venus clam Macrocallista nimbosa and influence of salinity variation[END_REF][START_REF] Prado-Alvarez | Morphological characterization and functional immune response of the carpet shell clam (Ruditapes decussatus) haemocytes after bacterial stimulation[END_REF][START_REF] Ordas | Phagocytosis inhibition of clam and mussel haemocytes by Perkinsus atlanticus secretion products[END_REF][START_REF] Carballal | In vitro study of phagocytic ability of Mytilus galloprovincialis Lmk. haemocytes[END_REF][START_REF] Di | Phagocytosis and respiratory burst activity of haemocytes from the ivory snail, Babylonia areolata[END_REF][START_REF] Cima | Haemocytes of the clam Tapes philippinarum (Adams & Reeve, 1850): morphofunctional characterisation[END_REF][START_REF] Xue | Flow cytometric assessment of haemocyte subpopulations in the European flat oyster, Ostrea edulis, haemolymph[END_REF][START_REF] Gorbushin | Functional characterization of Littorina littorea (Gastropoda: Prosobranchia) blood cells[END_REF].

The time-dependent evolution of phagocytosis measured in our study is consistent with previous findings on Hcs of mollusk bivalves [START_REF] Hong | Comparative study on the hemocytes of subtropical oysters Saccostrea kegaki (Torigoe & Inaba, 1981), Ostrea circumpicta (Pilsbry, 1904), and Hyotissa hyotis (Linnaeus, 1758) in Jeju Island, Korea: Morphology and functional aspec[END_REF][START_REF] Donaghy | Functional and metabolic characterization of hemocytes of the green mussel, Perna viridis: in vitro impacts of temperature[END_REF][START_REF] Ordas | Phagocytosis inhibition of clam and mussel haemocytes by Perkinsus atlanticus secretion products[END_REF][START_REF] Cima | Haemocytes of the clam Tapes philippinarum (Adams & Reeve, 1850): morphofunctional characterisation[END_REF][START_REF] Canesi | Bacteria-hemocyte interactions and phagocytosis in marine bivalves[END_REF] and gastropods [START_REF] Travers | Morphologic, cytometric and functional characterisation of abalone (Haliotis tuberculata) haemocytes[END_REF][START_REF] Donaghy | First characterisation of the populations and immune-related activities of hemocytes from two edible gastropod species, the disk abalone, Haliotis discus discus and the spiny top shell, Turbo cornutus[END_REF][START_REF] Adema | Generation of oxygen radicals in hemocytes of the snail Lymnaea stagnalis in relation to the rate of phagocytosis[END_REF], but also crustacean [START_REF] Raman | Agglutinin-mediated phagocytosisassociated generation of superoxide anion and nitric oxide by the hemocytes of the giant freshwater prawn Macrobrachium rosenbergii[END_REF] and fish blood cells [START_REF] Harford | Flow cytometric analysis and optimisation for measuring phagocytosis in three Australian freshwater fish[END_REF]. Within cephalopods, Malham et al. [START_REF] Malham | Phagocytosis by haemocytes from the Lesser Octopus Eledone cirrhosa[END_REF] showed similar evolution on E. cirrhosa Hcs, whereas Rodriguez-Dominguez et al. [START_REF] Rodríguez-Domínguez | Preliminary study on the phagocytic ability of Octopus vulgaris Cuvier, 1797 (Mollusca: Cephalopoda) haemocytes in vitro[END_REF] reported constant O. vulgaris Hc phagocytic percentages from 45 to 120 min incubation at 22°C.

Notably, temperature sensitivity appears species-specific in cephalopods, as no effects were observed in our study, whereas negative effects of low temperatures (4-10°C) were reported on Hc phagocytosis rate in O. vulgaris [START_REF] Rodríguez-Domínguez | Preliminary study on the phagocytic ability of Octopus vulgaris Cuvier, 1797 (Mollusca: Cephalopoda) haemocytes in vitro[END_REF], and E. cirrhosa [START_REF] Malham | Phagocytosis by haemocytes from the Lesser Octopus Eledone cirrhosa[END_REF]. Although temperatures used were extremes and out of the thermal window of 15°C-acclimated cuttlefish [START_REF] Melzner | Role of blood-oxygen transport in thermal tolerance of the cuttlefish, Sepia officinalis[END_REF] (25°C -inducing anaerobic metabolism, and 4°C -a lethal temperature [START_REF] Richard | Contribution à l'étude expérimentale de la croissance et de la maturation sexuelle de Sepia officinalis L. (Mollusque Céphalopode)[END_REF]), no significant effects on phagocytosis parameters were found in our study. This low effect of temperature on Hc phagocytosis ability is consistent with the eurythermy of S. officinalis.

The quantity of ingested beads measured by FCM evolved in similar ways as the percentage of phagocytic Hcs. Mean quantity of beads in Hcs increased during the entire experiment from 5.9, 7.0 and 6.8 beads cell -1 after 30 min to 16.4, 18.7 and 16.9 beads cell -1 after 180 min at 4, 15 and 25°C, respectively (Fig. 5B). Concurrent TEM observations confirmed the number of ingested beads registered by FCM (Fig. 6A) and visually demonstrated the engulfment process with (1) Hc-bead adhesion, (2) bead internalization by almost continuous close contact between membrane and particle, and (3) closing phagosome (Fig. 6B-C). Occasionally, we also observed lysosomal fusion with phagosome (Fig. 6D).

Our phagocytosis assay revealed similar rates of bead engulfment as those found in O. vulgaris [START_REF] Rodríguez-Domínguez | Preliminary study on the phagocytic ability of Octopus vulgaris Cuvier, 1797 (Mollusca: Cephalopoda) haemocytes in vitro[END_REF], but higher rates than in the mollusk bivalve Perna viridis and gastropods Lymnaea stagnalis, Haliotis discus discus and Turbo cornutus [START_REF] Donaghy | Functional and metabolic characterization of hemocytes of the green mussel, Perna viridis: in vitro impacts of temperature[END_REF][START_REF] Donaghy | First characterisation of the populations and immune-related activities of hemocytes from two edible gastropod species, the disk abalone, Haliotis discus discus and the spiny top shell, Turbo cornutus[END_REF][START_REF] Adema | Generation of oxygen radicals in hemocytes of the snail Lymnaea stagnalis in relation to the rate of phagocytosis[END_REF], highlighting higher engulfment capacity of cephalopod Hcs. Nevertheless, such differences could be explained by the high number of beads per cell used in our study (1:100) compared to those of previous ones (≤ 1:10).

Opsonization assay

As observed under FCM, opsonization experiments differently affected Hc phagocytosis percentage and engulfed bead number. While phagocytosis occurred without incubation of Hcs or beads in plasma, an increase in ingested bead number occurred after incubation in 1 or 10% plasma (p=0.008 and 0.045, respectively) (Fig. 7). In contrast, incubation of beads and Hcs in 50% plasma led to a decrease of phagocytosis percentage (p=0.04). TEM observations of phagocytosis conducted with plasma revealed a bead-coating by Hcy-like molecules (Fig. 8A-B) as previously suggested in cephalopods [START_REF] Stuart | The reticulo-endothelial apparatus of the lesser octopus, Eledone cirrhosa[END_REF][START_REF] Malham | Phagocytosis by haemocytes from the Lesser Octopus Eledone cirrhosa[END_REF][START_REF] Beuerlein | Hemocyanin and the branchial heart complex of Sepia officinalis: are the hemocytes involved in hemocyanin metabolism of coleoid cephalopods?[END_REF]. This observation could explain Hcy presence in Hc electron-lucent vesicles. Moreover, Hcy-like molecule presence in bridges between beads (Fig. 8C) suggested an agglutinating function, consistent with the increasing engulfed bead number observed at 1 and 10% plasma (Fig. 7). Such findings are consistent with a recent Octopus maya plasma agglutinin (OmA) characterization, showing homology between OmA subunits and one functional unit of Hcy from Octopus dolfeini [START_REF] Alpuche | Purification and partial characterization of an agglutinin from Octopus maya serum[END_REF].

Moreover, bead engulfment increased after as low as 1% plasma addition, underlying the high concentration of the molecule in charge of this process, and Hcy is known to represent more than 90% of cephalopod plasma proteins [START_REF] Malham | Phagocytosis by haemocytes from the Lesser Octopus Eledone cirrhosa[END_REF][START_REF] D'aniello | Electrolytes and nitrogen compounds of body fluids and tissues of Octopus vulgaris Lam[END_REF][START_REF] Ghiretti | Molluscan hemocyanins[END_REF][START_REF] Mangold | Appareils respiratoire et circulatoire : respiration et circulation[END_REF]. Such agglutinating function could also explain the phagocytic percentage decrease occurring with 50% plasma. Indeed, important bead agglutination might limit the availability of these particles for phagocytic process by increasing the size of particles to engulf. Ballarin et al. [START_REF] Ballarin | Phagocytosis in the colonial ascidian Botryllus schlosseri[END_REF] described a similar phagocytic decrease resulting from agglutinins in the ascidian Botryllus schlosseri after yeast pre-incubation in high plasma concentrations (≥ 50%). In cephalopods, this Hcy property could explain the decrease of phagocytosis by O. vulgaris Hcs measured after pre-incubation of zymosan in 100% plasma [START_REF] Rodríguez-Domínguez | Preliminary study on the phagocytic ability of Octopus vulgaris Cuvier, 1797 (Mollusca: Cephalopoda) haemocytes in vitro[END_REF], because of zymosan particle (about 3 µm) aggregation.

Malham et al. [START_REF] Malham | Phagocytosis by haemocytes from the Lesser Octopus Eledone cirrhosa[END_REF] and Ballarin et al. [START_REF] Ballarin | Phagocytosis in the colonial ascidian Botryllus schlosseri[END_REF] highlighted also the importance of phagocytose duration in this type of study, as phagocytosis enhancement after plasma incubation mainly occurred after 30 min. This is consistent with our study and that of Rodriguez-Dominguez et al. [START_REF] Rodríguez-Domínguez | Preliminary study on the phagocytic ability of Octopus vulgaris Cuvier, 1797 (Mollusca: Cephalopoda) haemocytes in vitro[END_REF] which report plasma-dependent phagocytosis decrease after 120 and 90 min of phagocytosis duration, respectively. Our results demonstrate the presence of a Hcy-like coating molecule with agglutinin function in S. officinalis plasma, which can modulate phagocytosis according to (1) pre-incubation plasma concentration and (2) phagocytosis duration.

Conclusions

Our study demonstrates, using FCM, light-and electron-microscopy, that a single granulocyte population with variable internal complexity circulates in the hemolymph of S. officinalis, as in other cephalopods. Acid phosphatase, lysozyme, and for the first time in cephalopods proPO system enzymes were detected in Hcs, but not in plasma [START_REF] Malham | Immunobiology of Eledone cirrhosa (Lamarck)[END_REF][START_REF] Grimaldi | Immune responses of Octopus vulgaris (Mollusca: Cephalopoda) exposed to titanium dioxide nanoparticles[END_REF]. These cells were able to recognize and incorporate foreign material at high rate independently of temperature and without need for plasma. Concurrent TEM and FCM analysis suggested a role for Hcy in foreign particle coating probably associated with its hypothesized agglutinin function. These data provide important information to understand the Hc-mediated immunity in the common cuttlefish, and a useful background for future studies of cephalopod Hcs.

  2 and MgCl 2 •6H 2 O), calcium chloride (CaCl 2 ), bovin serum albumin (BSA), Bradford reagent, trypsin TPCK (N-Tosyl-L-phenylalanine chloromethyl ketone), N-benzoyl-L-arginine 4nitroanilide hydrochloride (BAPNA), p-nitrophenyl-phosphate, dimethyl sulfoxide (DMSO), trizma hydrochloride (Tris-HCl), trizma base, 3,4-dihydroxy-L-phenylalanine (L-DOPA), tropolone, hen egg white (HEW) lysozyme, freeze-dried Micrococcus lysodeikticus, sodium phosphate dibasic dihydrate (Na 2 HPO 4 •2H 2 O), citric acid (C 6 H 8 O 7 ), Wright stain, neutral red, L-15 medium (Leibovitz), potassium chloride (KCl), magnesium sulphate heptahydrate (MgSO 4 •7H 2 O), formaldehyde solution, L-glutamine, streptomycin, sodium hydroxide (NaOH), ethylenediaminetetraacetic acid (EDTA), trypan blue solution, methanol and HEPES

Figure 1 :Figure 2 :

 12 Figure 1: Freshly adhesive Sepia officinalis Hcs presenting refringent (arrowhead) and nonrefringent (arrow) granules. Inset: Hcs completely spread

Figure 3 :

 3 Figure 3: Electron micrographs of S. officinalis circulating Hcs. (A) SEM micrograph of several Hcs showing similar aspect; (B) SEM micrograph of one hemocyte producing many pseudopodia; (C) Transmission electron micrograph of circulating Hcs presenting caracteristic lobate-nucleus (N) with highly condensed chromatin along the inner surface of the nuclear membrane, well-developped rough endosplasmic reticulum (rER), electron-dense lysosomal vesicles (arrow) and electron-lucent vesicles (arrowhead). Bar: 1 µm. Inset: high magnification of mitochondria (m) and Golgi apparatus (G). Bar: 0.1 µm; (D) Transmission electron micrograph of circulating Hcs presenting numerous vesicles; electron-dense lysosomal granules (arrow) and electron-lucent vesicles (arrowhead). Bar: 0.5 µm. (E-F) Electron-lucent vesicles showing inner Hcy-like molecules (small arrow). Bars: 100 nm.

Figure 4 :

 4 Figure 4: Flow cytometer bivariate plots showing distributions of particle size (FSC) vs internal complexity (SSC) of fresh Hcs of S. officinalis. Insets representing histogram of both variables. (A) Typical cuttlefish dot-plot; (B) Dot-plot showing wide internal complexity (SSC) distribution sometimes observed.

Figure 5 :

 5 Figure 5: Graphs representing evolution of phagocytic parameters function of time (30, 60, 120 and 180 min) at three temperatures (4, 15 and 25°C) (N = 5); (A) Phagocytic Hc percentages and (B) engulfed bead number.

Figure 6 :

 6 Figure 6: TEM micrographs of S. officinalis Hc. (A) Phagocytic Hc presenting several engulfed 1 µm latex beads (b). Bar: 1 µm. Sequential events of the internalization of beads (B-D); (B) Hc-bead adhesion. Bar: 0.1 µm; (C) bead engulfment. Bar: 0.2 µm; (D) and fusion between phagosomal and lysosomal compartments. Bar: 0.5 µm.

Figure

  Figure

Figure 7 :

 7 Figure 7: Graph representing phagocytic Hc percentage and mean engulfed bead number of S. officinalis Hcs (N ≥ 4; 2h incubation at 15°C) function of plasma concentration (%) treatments. Statistically significant differences were made against control (0% plasma concentration) at each plasma concentration for phagocytic cell percentage (º) and engulfed bead number (*); * and º p<0.05; ** p<0.01.

Figure 8 :

 8 Figure 8: TEM micrographs of 1 µm latex beads (b). (A) One bead after incubation in medium without plasma add; (B) one bead after incubation in plasma presenting important Hcy-coating (arrow). Bars: 100 nm. (C) Apparent Hcy-like molecule implication in bead aggregation (arrow). Bar: 200 nm.

Figure 1 Figure 2 Figure 3

 123 Figure 1

Figure 4

 4 Figure 4

Figure 5 Figure 6

 56 Figure 5

Figure 8

 8 Figure 7

  75 mg/100 ml of phosphate/citrate buffer pH 5.8 (Na 2 HPO 4 •2H 2 O, 4.45 g/250 ml distilled H 2 O; citric acid (C 6 H 8 O 7 ), 2.1 g/100 ml distilled H 2 O; NaCl, 0.09 g/100 ml buffer)), was added to each well.

Table 1 :

 1 Reported circulant Hc concentrations (mean ± SD and range, ×10 6 cell ml -1 ) in cephalopod, gastropod and bivalve mollusks In italic: data calculated from standard-error or 95% confident interval.

	Species	N Concentration Range

Table 2 :

 2 Specific activity repartition in hemolymph compartments: Hcs (10×10 6 cell ml -1 ) and plasma. Asterisk (*) indicates significance between PO-and APO-like activities in each

	compartment (p<0.05)
	Specific activity	N	Hc Hemolymph compartment Plasma
	Acid phosphatases (U mg prot -1 )	9	23.5 ± 10.6	0.1 ± 0.1
	Lysozymes (µg HEW lysozyme eq. mg prot -1 )	10	21.9 ± 9.0	0.1 ± 0.1
	PIs (trypsin inhibition %age)	9	0.0	31.8 ± 14.6
	PO-like (U mg prot -1 ) (U mg prot -1 ) APO-like	10	20.3 ± 9.5 30.5 ± 7.4*	2.6 ± 1.0 2.9 ± 0.9

Acknowledgments

This work was supported by the European, Interreg IV-A CHRONEXPO project. This study was partly conducted in the CREC (Centre de Recherche en Environnement Côtier) at Luc-sur-mer (Normandie, France). The authors thank Beatrice Adeline for technical support in cytological analysis and Dr Pierre Le Pabic for advices and help in English.