Dr Maria Kitsara
email: kitsara.m@gmail.com

Dr Dimitrios Kontziampasis
email: dkontziampasis001@dundee.ac.uk

Efi Bolomiti

Alexandre Simon

Panagiotis Dimitrakis

Antoine Miche

Georgios Kokkoris

Onnik Agbulut
email: prof.onnikagbulut@sorbonne-universite.fr

Vincent Humblot

Surfaces for hearts: Establishing the optimum plasma surface engineering methodology on polystyrene for cardiac cell engineering

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Polystyrene (PS) is currently one of the most widely used polymers, mainly due to its advanced mechanical properties and low cost of production [START_REF] Maul | Polystyrene and Styrene Copolymers[END_REF][START_REF] Imbert-Laurenceau | Surface modification of polystyrene particles for specific antibody adsorption[END_REF][START_REF] Capricho | Upcycling Polystyrene[END_REF]. PS can be naturally transparent and nontoxic to cells, and for these properties it has prevailed as a biomaterial that is used in biomedical research [START_REF] Wang | Surface modification of polystyrene with atomic oxygen radical anions-dissolved solution[END_REF][START_REF] Rytlewski | Laser-induced surface modification of polystyrene[END_REF]. Even though the use of PS in standard clinical practices is not widespread, it poses as the material of choice for petri dishes, test tubes, and well plates for cell, tissue, and bacterial growth [START_REF] Trentin | N2/H2 plasma surface modifications of polystyrene inhibit the adhesion of multidrug resistant bacteria[END_REF]. As a polymeric material however, PS has an intrinsic hydrophobicity and low surface energy, while simultaneously lacks the desired surface chemistry that favours cell attachment, spreading, and proliferation.

In order to maintain all the desired properties for the material and solve the previously mentioned problems in cell affinity and final compatibility, the route of surface modification is typically followed. In recent studies, chemical (for example grafting, oxidation, sulfonation, crosslinking, etching) and physical (for example electron or ion beam, laser treatment, sputtering) techniques are utilised for the surface modification of polymers [START_REF] Deng | Developments and new applications of UV-induced surface graft polymerizations[END_REF][START_REF] Siqueira-Petri | Surface modification of thin polystyrene films[END_REF][START_REF] Xue | A simple technique of constructing nano-roughened polydimethylsiloxane surface to enhance mesenchymal stem cell adhesion and proliferation[END_REF][START_REF] Kim | Surface reformed anisotropic polystyreneblock -poly(ethyleneran -butylene)block -polystyrenegraft -maleic anhydride layer via ion-beam irradiation for liquid crystals[END_REF][START_REF] Slepička | Surface Modification of Polymer Substrates for Biomedical Applications[END_REF][START_REF] Wang | Morphology-controllable gold hierarchically micro/nanostructured arrays prepared by electrodeposition on colloidal monolayer and their structurally related wettability[END_REF][START_REF] Park | Photo-Cross-Linkable Thermoresponsive Star Polymers Designed for Control of Cell-Surface Interactions[END_REF][START_REF] Pertile | Surface modification of bacterial cellulose by nitrogen-containing plasma for improved interaction with cells[END_REF][START_REF] Desmet | Nonthermal Plasma Technology as a Versatile Strategy for Polymeric Biomaterials Surface Modification: A Review[END_REF][START_REF] Pfleging | Laser-assisted modification of polystyrene surfaces for cell culture applications[END_REF][START_REF] Günther | Copper Ions Absorbed on Acrylic-Acid-Grafted Polystyrene Enable Direct Bonding with Tunable Bonding Strength and Debonding on Demand[END_REF][START_REF] Zheng | Acute effects of three surface-modified nanoplastics against Microcystis aeruginosa: Growth, microcystin production, and mechanisms[END_REF][START_REF] Golubenko | Effect of current density, concentration of ternary electrolyte and type of cations on the monovalent ion selectivity of surface-sulfonated graft anion-exchange membranes: modelling and experiment[END_REF][START_REF] Schmitt | Polyethylene Glycol Coatings on Plastic Substrates for Chemically Defined Stem Cell Culture[END_REF]. Comparing to all the aforementioned approaches, plasma surface modification shows a unique dual capability of homogeneous and simultaneous modification of both surface chemistry and topography [START_REF] Gogolides | Controlling roughness: from etching to nanotexturing and plasma-directed organization on organic and inorganic materials[END_REF][START_REF] Kontziampasis | Biomimetic, antireflective, superhydrophobic and oleophobic PMMA and PMMA-coated glass surfaces fabricated by plasma processing[END_REF]. Plasma, in numerous studies, has been used as a tool to alter and enhance the surface properties of a plethora of polymers in order to promote their use for biological and biomedical applications, either via removing or by depositing material on the surface [START_REF] Bourkoula | Roughness threshold for cell attachment and proliferation on plasma micro-nanotextured polymeric surfaces: the case of primary human skin fibroblasts and mouse immortalized 3T3 fibroblasts[END_REF][START_REF] Kontziampasis | Effects of Ar and O 2 Plasma Etching on Parylene C: Topography versus Surface Chemistry and the Impact on Cell Viability[END_REF][START_REF] Kitsara | Permanently hydrophilic, piezoelectric PVDF nanofibrous scaffolds promoting unaided electromechanical stimulation on osteoblasts[END_REF][START_REF] Oberländer | Temperature, concentration, and surface modification influence the cellular uptake and the protein corona of polystyrene nanoparticles[END_REF]. In order to provide the material under treatment with the necessary chemical cues and allow it to promote specific cell functionalities, two routes are the most widely used in plasmas. The first and most used one is O2 plasma treatment (or etching), which increases the -OH, =O and other oxygen groups, while additionally it creates structures on the polymer surface as proven in the literature [START_REF] Bourkoula | Roughness threshold for cell attachment and proliferation on plasma micro-nanotextured polymeric surfaces: the case of primary human skin fibroblasts and mouse immortalized 3T3 fibroblasts[END_REF][START_REF] Singh | Chitin and carbon nanotube composites as biocompatible scaffolds for neuron growth[END_REF][START_REF] Arinda | The characteristics of surface morphology on the polystyrene thin film using DC-bias oxygen plasma treatment[END_REF][START_REF] Vesel | Surface modification of PS polymer by oxygenatom treatment from remote plasma: Initial kinetics of functional groups formation[END_REF]. N2 plasma poses as the second most used route, and its use increases the -NH2, =NH, ≡N, and amines on the surface. The latter treatment appears less invasive in terms of modifying the topography of the material [START_REF] Wang | Acid and basic functionalities of nitrogen and carbon dioxide plasma-treated polystyrene[END_REF][START_REF] Novi | The Effect of Substrate Temperature on Surface Modification of Polystyrene by using Nitrogen Plasma[END_REF]. Especially for PS, various studies have provided insights on how different plasmas invoke different changes on its surface properties, i.e. surface chemistry and topography [START_REF] Trentin | N2/H2 plasma surface modifications of polystyrene inhibit the adhesion of multidrug resistant bacteria[END_REF] , [START_REF] Chen | Applied Surface Science Surface modification and biocompatible improvement of polystyrene film by Ar , O 2 and Ar + O 2 plasma[END_REF][START_REF] Li | Surface Modification of Polystyrene with O Atoms Produced Downstream from an Ar/O2 Microwave Plasma[END_REF][START_REF] Ramsey | Surface Treatments and cell attachment[END_REF][START_REF] Luan | Effect of water vapor on plasma processing at atmospheric pressure: Polymer etching and surface modification by an Ar/H 2 O plasma jet[END_REF][START_REF] Kong | Surface modifications of polystyrene and their stability: A comparison of DBD plasma deposition and direct fluorination[END_REF][START_REF] Chiper | Tailoring the working gas flow to improve the surface modification of plasmatreated polymers[END_REF][START_REF] Chen | A study of plasma power effects on surface activation of polystyrene[END_REF][START_REF] Amorim | Effects of cold SF6 plasma treatment on a-C:H, polypropylene and polystyrene[END_REF][START_REF] Surya Arinda | Stability of Polystyrene Film Surface Wettability Modified Using Oxygen Plasma[END_REF]. Recent initial studies, have started to investigate the effect of plasma surface modification on PS for controlling cultured cell functions [START_REF] Slepička | Surface Modification of Polymer Substrates for Biomedical Applications[END_REF][START_REF] Suzuki | UV/ozone surface modification combined with atmospheric pressure plasma irradiation for cell culture plastics to improve pluripotent stem cell culture[END_REF].

Haubenwallner et al. suggested that there is a direct connection of cell fate to the polymer niche [START_REF] Haubenwallner | Effects of the polymeric niche on neural stem cell characteristics during primary culturing[END_REF].

In their study, they reveal that the use of a specific type of polymer with a tailored microenvironment can be considered as an effective tool for promoting essential cell characteristics for subsequent approaches and point out the necessity of using a cell type specific screening approach when referring to synthetic materials and material treatments for particular cell types. This indicates that previously described treatments for a specific type of cell will unlikely be appropriate for another type, or for triggering a similar behaviour of the cell, showcasing that there is no global solution. They point out that every type of cell and tissue prefers a different microenvironment.

Cardiac cell engineering has also signalled the need of identifying and decoding an optimum plasma surface modification in order to obtain a microenvironment that shows improved attachment, morphology, and protein expression. This is essential for understanding and investigating the pathologies of the heart in culturing plates (disease modelling), as well as the effect of drugs on normal behaving cells and tissues, and it is a vital step towards the 'heart on a chip' integration [START_REF] Kitsara | Fibers for hearts: A critical review on electrospinning for cardiac tissue engineering[END_REF][START_REF] Kitsara | Heart on a chip: Micro-nanofabrication and microfluidics steering the future of cardiac tissue engineering[END_REF].

The current study aims to address this knowledge gap, and tailor an optimum surface for cardiac cell engineering, via modification of PS. This will be achieved by bringing together the two most widely used components in terms of material (PS) and surface modification method (plasma). The aspiration is not only to characterise the topographical or chemical changes induced by the plasma treatments on the surface of PS, but to additionally investigate the cardiac cells' response to these changes. This will allow for an understanding of which route favours the normal function of the cardiac cells. The focus of the current study is on cardiac cell culture, and the ultimate goal is to provide an integrated systematic study from both the materials and biological perspective, when using the three most common plasma treatments to modify the favourite material in biological research. These treatments are namely O2, N2, and Ar plasma.

Since PS poses as the main material for cell culture dishes and plates, its cytocompatibility is very well known. Even though it can be used as is for several types of cells, this is not the case for cardiac cells. Cardiac cells are very selective and require a pre-treatment with coating before culture, which is based on extracellular matrix proteins. In addition, to the best of our knowledge, there is no report in literature that has studied the effect of plasma treatment of PS on primary cardiac cell function. Herein, we culture two different types of cells on numerous plasma-treated PS surfaces without any pre-treatment with extracellular matrix proteins. Initially, the cell line H9c2 was studied, which is derived from an embryonic rat heart. This was necessary for acquiring useful information on the viability and cell stress using a big batch of cells, which are easier to culture. Subsequently, we moved forward to the culture of primary cardiomyocytes that were derived from neonatal rat hearts, culturing both non-purified and purified cardiomyocytes. The former culture was a mixture and contained other types of cardiac cells, i.e. fibroblasts, endothelial cells, and smooth muscle cells.

Materials and methods

Materials

PS slides were purchased from Thermo Fisher Scientific®, (nunc, 960004), which were pre-treated for cell culture by the manufacturer.

Plasma treatment

PS slides were plasma-treated using O2, N2, O2+N2 and Ar+N2 gas mixtures in a reactive ion etching (RIE) tool (Alcatel Nextral NE330). This reactor is producing by the application of a radio frequency (RF) power, a capacitively coupled plasma discharge. The chamber of the plasma reactor (see Fig. S1 † , ESI), which is 420mm in diameter, was cleaned by O2 plasma before the PS samples were introduced for treatment to avoid any contamination from previous treatments. Taking into account the chamber size, a set of 10 slides with the same interspace distance were treated in each experiment. The RF biased electrode, on top of which the slides are put, carries a quartz cover plate (see Fig. S1 † , ESI).

The feed gas mixture and the operating conditions were chosen in accordance with treatments reported in literature [START_REF] Gogolides | Controlling roughness: from etching to nanotexturing and plasma-directed organization on organic and inorganic materials[END_REF][START_REF] Kontziampasis | Biomimetic, antireflective, superhydrophobic and oleophobic PMMA and PMMA-coated glass surfaces fabricated by plasma processing[END_REF]. O2, N2, and mixtures of O2 with N2 and Ar with N2 were utilised. The feed of the reactor was 100 sccm for O2, 50 sccm for N2, and 100+50 sccm for O2+N2 and Ar+N2, respectively. The pressure in the reactor was kept at 20 mTorr and the generator power was 200 W. The treatment time was 20 and 30 min, and the reasons for choosing the are the following. In the case of oxygen plasma, we are awar that in order to achieve the dual scale structures, which will have the desired effects in this specific reactor (Nextral 330, RIE from Alcatel), we needed to etch for long times, in the region of tens of minutes, so 20 and 30 minutes fell into that window of values [START_REF] Ellinas | Superhydrophobic Fabrics with Mechanical Durability Prepared by a Two-Step Plasma Processing Method[END_REF][START_REF] Ellinas | Ultra-low friction, superhydrophobic, plasma micro-nanotextured fluorinated ethylene propylene (FEP) surfaces[END_REF][START_REF] Tsougeni | Plasma Nanotextured PMMA Surfaces for Protein Arrays : Increased Protein Binding and Enhanced Detection Sensitivity[END_REF].

Additionally, we have chosen to use 20 and 30 minutes of treatment in order to achieve surfaces with stable surface chemistry over time. Indeed, we have found that the 30 min of nitrogen plasma treatment led to the creation of stably hydrophilic surfaces. This is important for the current research, as having samples that are stable over the time of the study is necessary, especially since there are repeated cell culture studies that need to be performed at different time periods and at different intervals from the plasma treatment of the samples.

In parallel, for the case of nitrogen plasma, these times allow for "big enough" nanostructures to be created with plasma. It was found in initial experiments that for approximately 10-15 minutes the corresponding nanostructures created on the surface were not sufficient to invoke the desired effect on wettability, as it was with the case of oxygen. Combining all of the above, it was chosen to use 20 and 30 minutes etching times for all the plasma treatments in this study.

It should be stressed that every 3 min the plasma process was stopped for 2 min in order to prevent the heating of the quartz substrate of the plasma reactor. Heating οf the quartz substrate originates from the power deposited by the plasma generated charged species (ions and electrons). The power density deposited depends on the ion and electron energies and flux and is measured or estimated from hundreds of mW/cm2 to 1 W/cm 2 [48,49]. After plasma treatment, the samples were kept in a sealed box in a room with controlled temperature and humidity (20 o C, relative humidity (RH) 40%).

Scanning electron microscopy (SEM)

SEM was used to visualise the structures of the plasma-treated PS (Gemini SEM 500, Zeiss). The samples were sputtered with a thin platinum layer prior to SEM observation using the ACE600, Leica. Images at different magnifications were obtained on at least five different areas of the same sample and in three different samples with the same conditions for statistical analysis. The images were acquired by applying beam voltage of 3 kV and using InLens detector.

Wettability assessment

Water contact angle (WCA) measurements were performed using the DSA 30 system from Krüss. The sessile drop method was used, and the measurements took place at room temperature (20 o C). The volume of the applied drops of deionised water was 1 μl, and the WCAs were measured 10 seconds after drop deposition. WCA data were obtained by averaging over several measurements in different areas on the sample's surface.

X-ray Photoelectron Spectroscopy (XPS)

XPS analyses were performed using an Omicron Argus X-ray photoelectron spectrometer, equipped with a monochromated AlKα radiation source (hν = 1486.6 eV), and a 280 W electron beam power. The emission of photoelectrons from the sample was analysed at a takeoff angle of 45° under ultrahigh vacuum conditions (≤10-9 mBar). Spectra were carried out with a 100 eV pass energy for the survey scan and 20 eV pass energy for the C 1s, O 1s, and N 1s regions. Binding energies were calibrated against the C 1s (C-C) binding energy at 284.8 eV and element peak intensities were corrected by Scofield factors. The peak areas were determined after subtraction of a shirley background. The spectra were fitted using Casa XPS v.2.3.15 software (Casa Software Ltd, U.K.) and applying a gaussian/lorentzian ratio equal to 70/30.

H9c2 cell culture

The H9c2 cell line, derived from embryonic rat heart tissue, was cultured in high glucose DMEM + Glutamax supplemented with 10% foetal bovine serum and 1% penicillin/streptomycin. Cells (1 x scaffolds were washed with PBS and the cells were fixed with 4% paraformaldehyde solution.

Isolation of neonatal rat cardiomyocytes

Newborn rat cardiomyocytes (NRCMs) were obtained from 1-day-old Wistar RjHan rat (Janvier Labs, Saint-Berthevin, France). The hearts were cut in small pieces and the cardiac cells were isolated, using a neonatal heart dissociation kit (Miltenyi Biotec, Paris, France) following the manufacturer's instructions. Cardiomyocytes were purified by depletion of non-target cells (such as fibroblasts, endothelial cells), using a neonatal cardiomyocyte isolation kit (Miltenyi Biotec, Paris, France). 5 x 10 5 cells were seeded on 2cm 2 -PS pieces and cultured with DMEM medium (with 4.5g/L D-Glucose and without Na Pyruvate) supplemented with L-glutamine, penicillin-streptomycin, 10% horse serum and 5% fetal bovine serum (FBS) at 37 °C and 5% CO2 for 3 days. All animal studies were approved by our institutional Ethics Committee "Charles Darwin" and conducted according to the French and European laws, directives, and regulations on animal care (European Commission Directive 2010/ 63/EU) under the license B75.13.20.

Viability and oxidative stress assay

The viability of both H9c2 and NRCMs was assessed by using the Muse® Count & Viability Kit. The kit differentially stains viable and non-viable cells based on their permeability to the two DNA binding dyes present in the reagent. The oxidative stress of the H9c2 cells was calculated by using the Muse® Oxidative Stress Kit, which allows for the quantitative measurements of Reactive Oxygen Species (ROS), namely superoxide radicals in cells undergoing oxidative stress. The assay provides the relative percentage of cells that are ROS negative and positive. The measurements were repeated 3 times, using 2 samples per case.

Immunostaining and cell morphology analysis

All cells were washed with PBS and fixed with 4% paraformaldehyde solution. After 3 washing steps, they were incubated with 5% bovine serum albumin (BSA) for 1 hour. H9c2 cells were incubated for 90 minutes at room temperature with an antibody against vinculin (mouse IgG1, dilution 1:200, Sigma-Aldrich, Saint-Quentin-Fallavier, France). After 3 washes in PBS, the H9c2 cells were incubated in the secondary antibody (Alexa 488 goat-anti-mouse, dilution 1:1000, Life Technologies) and Alexa Fluor® 647 Phalloidin (Life technologies). NRCMs were incubated for 90 minutes at room temperature with antibodies against α-actinin (mouse IgG, dilution 1:300, Sigma-Aldrich, Saint-Quentin-Fallavier, France) and fibronectin (rabbit IgG antibody, dilution 1:50, Santa Cruz Biotechnology, Heidelberg, Germany). After 3 washes in PBS, NRCMs were incubated in the presence of secondary antibodies (Alexa 488 goat-anti-mouse and Alexa 568 goat-anti-rabbit, dilution 1:1000; Life Technologies). All the samples were mounted with mowiol containing 5 µg/ml Hoescht 33342 (Life Technologies). Images were captured using a motorised confocal laser scanning microscope (Leica TCS SP5).

Statistical analyses

Groups were statistically compared with GraphPad Prism 7 using ordinary one-way analysis of variance. The normality was checked using the Shapiro-Wilk normality test. If the normality of distribution assumption was not met, the nonparametric Kruskal-Wallis test was used instead of an ordinary ANOVA test. If a significant difference was found, then multiple comparison tests were performed to compare the different groups analysed (Dunn's or Tukey multiple comparison test following a nonparametric or parametric test, respectively). A p-value ≤ 0.05 was considered significant. Values are given as the means ± standard error of the mean (SEM).

Results and discussion

Assessment of roughness and hydrophilicity changes to PS by plasma treatment

As it is mentioned in the introduction, plasma treatment of polymers leads to the formation of structures on their surface. The reasons behind this formation of structures, have been analysed in literature extensively [50][51][52][53][54]. In this work, we validate differences on these structures via using SEM to examine samples that have undergone different plasma treatments. Since the focus of this work is to identify the optimum plasma treatment for cardiac cell culture, one can clearly distinguish different morphologies of topography on the surface of PS for the different plasma treatments, comparing the average size of structures as they appear in the acquired SEM images.

Electron microscopy of the samples (Fig. 1) shows that in the case of O2 plasma treatment, there is dual scale roughness created. Islands of flower-like structures, having a size in the region of several hundred nm to a few μm size, can be identified. Additionally, one can clearly notice the presence of round and oval-shaped nanostructures with 20-30 nm diameters, scattered uniformly across the surface. The aforementioned flower-like morphology is similar (although not identical in shape) to the one reported for O2 plasma treatment of PMMA (poly-(methyl methacrylate)) when using the same reactor [START_REF] Kontziampasis | Biomimetic, antireflective, superhydrophobic and oleophobic PMMA and PMMA-coated glass surfaces fabricated by plasma processing[END_REF]. In the case of O2 plasma treatment, the size of the large nanostructures increases from average maximum dimensions of ~115 nm width and ~120 nm length to 170 nm width and 200 nm width when plasma treatment time increases from 20 to 30 min. The underlying small nanostructures that appear in the background are not affected from the increase in treatment time. When a mixture of N2 and O2 is used for the plasma treatment of PS, microscopy indicates the same type of dual scale roughness on its surface, i.e. the formation of flower-like islands and small underlying nanostructures. The size of the islands increases to ~160 nm width and ~180 nm length to 270 nm width and 280 nm width for the 20 min and 30 min plasma treatment samples, respectively. Taking into consideration the trend of increasing dimensions of the structures on the PS surface when increasing time, and adding the fact that O2 plasma treatment leads to the formation of an identically shaped morphology, one can hypothesise that the nitrogen ions act in a catalytic fashion on the reactive oxygen ion etching in the case of PS.

In the case of N2 plasma treatment, SEM shows a different type of structures on the PS surface (Fig. S2 † contains images at higher magnifications for better visualisation). There, we can identify closely packed round and oval-shaped nanostructures present over the whole polymer surface, which have a diameter of ~35 nm and ~45 nm for 20 min and 30 min treatment times respectively. When N2 and Ar mixture is used for the treatment of PS, SEM images show that there is a minor change in the topography of the surface. Even at high magnifications, (Fig. S3 † ), there is no measurable change on the surface. Only the nanosized structures due to sputtering before SEM imaging appear, similarly with the untreated PS SEM image (Fig. S4 † ). SEM of untreated PS showed a flat surface (with some stochastic nanostructures due to sputtering before SEM), showcasing that the structuring of the PS surface resulted from the effect of plasma etching.

If one were aiming to illuminate the mechanisms behind the formation of the different surface morphology, a multiscale modeling framework linking with predictive capabilities would have been required. This framework should link the operating parameters of the plasma reactor or the plasma parameters with the surface morphology [53]; critical for this framework is the accuracy and the validity of the model used for the description of the surface morphology (e.g., cell based description of a Monte Carlo model). However, this would have diverted this research away from its aim, and is not considered further in the current study, and will be followed up in a future publication. The wettability of the plasma-treated PS was evaluated by WCA measurements (Fig. 2). The WCA of the commercial PS slides was found to be ~ 70 o , a value that is lower than that of pure PS (~90 0 ) [55], but still not hydrophilic. Following plasma treatment, all the samples rendered to superhydrophilic immediately, with the exception of the Ar-N2 treated sample. Subsequently, the ageing of the plasma-induced surface wettability was examined for a period of more than one month of storage under controlled conditions (20 o C, RH 40%). It was observed that most of the samples started gradually to become less hydrophilic over time. Only the N2-treated samples were an exception and remained stably superhydrophilic for a period of more than one month. It is very interesting to note that the 20min O2-treated samples exhibited a highly progressive recovery of the WCA. The WCA measurements also showcased that in all samplesexcept for the N2 ones -there is a clear difference between the 20 and 30 mins of treatment with plasma. The longest plasma treatment assisted to a longer-term hydrophilicity, which in the case of N2 led to a "stably-hydrophilic" surface (see also Fig. S5 † and movie M1).

Evaluation of PS surface chemistry after modification with various plasma gases

XPS measurements were also performed in order to assess the validity of plasma treatments. Figure S6 † presents the survey spectra of the untreated PS sample (control) versus the treated ones with different plasma conditions. On the control spectrum, one can clearly see the strong C1s contribution at 285.0 eV, together with a non-negligible O1s contribution at 530.0 eV, representing almost 14 % in elementary composition (Table 1). The presence of oxygen in the control strongly suggests that the slides surface does not contain only pure PS. This is expected as according to the manufacturer the slides were treated for cell culture. However, further information on the type of treatment is not provided. Also, the fact that the samples were not kept under vacuum increased a possible contamination from air.

After 30 minutes of pure O2 plasma treatment, the atomic percentage of the O1s contribution rises to 38.3 % (Table 1), which is within the values reported for similar treatments and the equivalent time of plasma processing [56,57]. Regarding N2 plasma treatments, several combinations were tested, pure N2, O2+N2 and Ar+N2. All three conditions show the presence of N1s on the survey spectra (Fig. 6 † ) within different proportions. When nitrogen is mixed with oxygen for the plasma, due to oxygen being more reactive than nitrogen, only a small quantity of nitrogen is found on the surface after activation (1.1 %) and the resulting spectrum is very similar to the one obtained for standalone O2 plasma treatment. However, the use of either pure N2 or the one mixed with Ar, leads to up to 10.0 % of N2 present on the PS surface in the best-case scenario (Fig. 6 † and Table 1). It should be noted that also in the case of using Ar, the measured amount of oxygen is higher than the non-treated sample.

For the cell culture experiments, it was decided to concentrate only on the pure plasma treatments, either O2 or N2. Therefore, the high-resolution XPS spectra obtained for C1s, N1s and O1s for these conditions are presented on Figure 3. The high-resolution XPS spectra of Ar-N2 are quite similar to the N2 ones and have added at the EIS (Fig. S7 † ). As mentioned previously, the C1S region for the control sample exhibits few contributions from pure PS (284.8 eV for aromatic carbon atoms and C_C, C_H bonds of the lateral chains of the polymer and 291.5 eV corresponding to the aromatic shake-up contributions representing around 5-10 % of the total C1s intensity) [58], but also some contributions due to the treatment by the manufacturer and the storage conditions [57]. After (O2 or N2) plasma treatments, the associated spectra exhibit differences both in shape but also in chemical composition. Following O2 plasma, the spectrum is dominated by 2 strong contributions at low binding energy, assigned to the C-C, C-H and aromatic carbon atoms (284.9 eV, 61.1 at. %), and to carbon atoms linked oxygen species (285.7 eV, 22.4 at. %). These results are in good agreement with previous studies [58]. We noted that the related O1s spectrum (Figure 3b), exhibits a thin sharp peak centred at 533.0 eV, suggesting oxygen species of the same chemical nature.

For N2 treatment, the C1s spectrum exhibits a more enlarged peak with several contributions arising from either N species or O species for binding energies contributions encompassed between 285.0 and 288.0 eV (Table 2) [57,58]. This spectrum is much more complicated to analyse due to the presence of multiple possible species, confirmed by a wide O1s peak (Figure 3b). In fact, the different contributions are linked to the chemical environment of a given atom and the associated chemical bound. Therefore, the more different chemical environments are present, the larger the peak will appear. As a result, starting with a complex surface chemistry as observed for the PS control, after plasma reaction, multiple new species will be formed upon this chemical reaction. This will have a consequence of enlarging peaks' contributions (the full width at half maximum increases) and also of energy shifts of the contributions due to difference in electronegativity of the atomic environments. This is also visible on the N1s spectrum (Figure 3c) with a large peak in the 401.0 eV region, composed of two equivalent contributions for charge and neutral nitrogen species [57], but also with the presence of a small contribution at higher binding energy (408.0 eV) usually assigned to NOx species [59].

Summarising, the XPS results that are obtained for the different untreated and treated PS surfaces, appear in agreement with the wettability measurements that are presented above, and can confirm the initial hypothesis that activation by plasma treatment is a promising treatment method for the creation of PS biocompatible surfaces. S2 † presents both element by element 100 % and the whole elements.)

Samples Assign. C-C, C-H C-O, C-N, C-O-C C=O, N=C O=C-O, O=C-N CO3 -π-π* N-CO + N-C NOx *C=O, C-O,

Influence of plasma gas on H9c2 cells viability and oxidative stress

Even though PS cytocompatibility is well known, to the best of our knowledge, there is no report scrutinising the effect of plasma treatment of PS on cardiac cells function. In order to evaluate the impact of plasma treatment of PS on cardiomyocyte viability and morphology, we firstly utilised the H9c2 cell line, which is isolated from the embryonic rat heart tissue. This well-characterised cell line was used for acquiring useful information on the viability and cell stress, via using a big batch of cells [60]. H9c2 cells viability was determined after 2 days in culture on different plasma treated PS samples. Quantification of live cells using the Muse® Count & Viability Kit showed that the number of viable cells was lower for O2-treated surfaces, compared to N2 or Ar+N2 treated surfaces although the difference did not reach statistical significance (Fig. 4). In parallel to this measurement, the oxidative stress of the H9c2 cells was calculated by using the Muse® Oxidative Stress Kit, which allows for the quantitative measurements of ROS. As demonstrated in Figure S8 † , the relative percentage of ROS positive cells were increased in O2-treated surfaces.

The morphology of H9c2 that were cultured on plasma-treated PS was evaluated using immunofluorescence imaging. Figure 5 shows confocal microscopic images after immunostaining for vinculin and staining for F-actin, and nuclei of H9c2 cells on the PS samples (see supplementary movies in the ESI). Evidently, the cells were spread all over the samples and show a nice structure of their actin filaments in the control, as well as the N2 and Ar+N2 treated samples, but not in the O2 treated samples. The vinculin immunostaining indicates the focal adhesion contacts of the cells to the substrate. Vinculin is a cytoplasmic actin-binding protein enriched at both cell-cell and cell-matrix adhesions [61,62]. It is evident in the images that cells adherent to the N2-treated samples displayed prominent vinculin staining in focal contacts, which is more intense in the 30-min treated ones. In contrast, the cells that were cultured on the O2-treated substrates displayed much less organisation of vinculin to focal contacts and an increased diffuse cytoplasmic vinculin staining, accompanied by short and disrupted actin bundles. The rest of the surfaces exhibit vinculin focal contacts, but do not appear as strong as the N2-treated ones. These results are in agreement with a previous study that reported the effect of nanotopography on H9c2 cell viability in the case of tantalum-based nanodot arrays [63]. There, they observed that vinculin immunostaining was widely distributed within cells grown on flat surfaces and up to 50-nm nanodot arrays, whereas it was dramatically decreased for the 200-nm nanodot samples. In the current study, the N2-treated surfaces have nanostructures that are at the range of 45 nm, while the O2 and O2+N2 have quite larger ones (more than 100 nm).

N2 plasma treatment favours cardiomyocytes viability and morphology

To confirm that the optimum plasma treatment of PS for cardiomyocyte culture is with N2 gas, primary cardiomyocytes from neonatal rat hearts were cultured for three days on the plasma-treated PS surfaces. In these experiments, we used both non-purified and purified cardiomyocytes, and their viability was determined after 3 days in culture.

Initially, we characterised the PS surfaces without plasma treatment in order to assess the cardiomyocytes' viability and function. Quantification of live cells using the Muse® Count & Viability Kit showed that the number of live cells was lower in the case of the purified cardiomyocytes (Fig. 6a). These results are expected due to the proliferation capability of some cells that are present in this case, and mainly of fibroblasts. It must be noted that cardiomyocytes do not proliferate and their culture is characterised as quite selective in comparison to other types of cells, due to their difficulty to adhere and spread in vitro. For this experiment, we performed double immunostaining of α-actinin and F-actin in order to distinguish the cardiomyocytes in the non-purified cells (Fig. 6b,c). Actin fibers are present in all types of cells, while α-actinin exists exclusively in the cardiomyocytes. These images show clearly that the presence of the other cell populations aids significantly in the survival of the cardiomyocytes, which happens even in the case of the O2-treated samples. The sarcomeres have better structure as it is revealed by the immunostaining with α-actinin in the Fig. 6b that in the Fig. 6c. The microscopy analysis is in total agreement with the viability results as can be seen in the figures. Quantification of live cells using the Muse® Count & Viability Kit showed that the number of viable cells was higher for N2-treated surfaces but the difference did not reach statistically significant level with non-purified cardiomyocytes (Fig. 7a). Similarly, the control did not show statistical significance in the case of non-purified cardiomyocytes (Fig. 7a). This difference becomes more apparent in the case of the purified cardiomyocytes. As shown in Figure 7b, the number of viable cells was lower for O2-treated samples, as well as the control, while the highest number of cells was calculated on the N2-30 min treated surfaces. The number of viable cells remains similar even after 30 mins of treatment for N2 samples, with a slight increase. Contrarily, the number of viable cells decrease for O2 samples when 30 min plasma treatment samples are used in comparison to 20 min of treatment samples.

Fig. 7. Cell viability monitoring of primary cardiomyocytes cultured on different plasma treated PS. Non-purifed (A) and purified (B) neonatal rat cardiomyocytes cultured for 3 days on various plasma-treated PS, and live cells were quantified using the Muse® Count & Viability Kit. Statistical analyses were realised using one-way ANOVA. If a significant difference was found, Tukey's post hoc tests for multiple comparisons were used. *p < 0.05, **p < 0.01.

The shape of the cardiomyocytes that are growing on the plasma-treated PS samples was further analysed using confocal microscopy and SEM. The confocal microscopic images after immunostaining for α-actinin and nuclei of the purified cardiomyocytes, showed cells with normal shape, with well-organised sarcomeric structure, and well defined striations of their sarcomeres on the N2-treated PS samples, which are much better than those cultured on the control (Fig. 8a,b). However, cells grown on the O2-treated surfaces presented a round shape morphology without any sign of organisation of their α-actinin. It can also be observed that the higher O2 plasma treatment time, further undermines the survival of the cardiomyocytes as it is shown in Figure 8c,d. In line with the confocal microscopy results, cell morphology analysis by SEM indicated that cells grown on the N2 surfaces had an elongated cylindrical shape (Fig. 9). However, cells grown on O2 treated surfaces presented a round shape. The magnified images in the right column of Fig. 9 reveal in more detail the cell-substrate interaction. Details on how the pseudopodia of cardiomyocytes are attached to the PS surface can be clearly seen. It is evident in the images that the structured O2treated PS aided to the adhesion of cardiomyocytes as it allowed the pseudopodia to find increased surface to attach, but this fact was proven to be insufficient to allow them to spread and have their normal shape. Overall, these results clearly showcase the specificity of purified primary cardiomyocytes and their difficulty to adhere and expand in in vitro cultures. Indeed, the non-purified cardiomyocytes can adhere to all of the surfaces that were used in this study, even to the O2-treated ones (Fig. S9 † ). It appears that the N2 plasma treatment favours cardiomyocytes' survival and spreading, via providing a combination of favourable nanotopography in the range of 50nm, as well as surface chemistry for the sample.

Conclusion

In this work, a combined characterisation of both the morphology and surface chemistry is presented for numerous popular plasma treatments on PS. SEM results show that O2 plasma creates dual scale roughness on the surface while N2 plasma creates oval-shaped nanostructures. Mixtures of gasses showed that the resulting topography is mostly affected by the most chemical reactive ions, i.e. oxygen for oxygen mixtures and nitrogen for nitrogen mixtures. WCA measurements showed superhydrophilisation of all surfaces. The hydrophobic recovery though was much slower in the N2 plasma treated samples. XPS shows interestingly oxygen peaks for both oxygen and N2 treated samples, and as expected nitrogen peaks for N2 treated samples. Increasing the plasma treatment time led to the increase of both oxygen and nitrogen species.

When H9c2 cells are cultured on the samples, analysis showed that the N2 treated samples displayed a better behaviour than all the other plasma treatments both in terms of viability and morphology. Longer plasma treated samples promoted these findings even further. In the case of primary purified and non-purified cardiomyocytes, the viability of the cells is drastically improved in the case of the N2 plasma treatment comparing to O2 plasma treated surfaces. For the control the purified cells indicated a worse viability comparing to the N2 treated samples. The O2 plasma treated samples provided a 'hostile' environment towards the cardiomyocytes, as both in the case of purified and not purified cardiomyocytes the number of cells dramatically decreased, especially in the case of the 30 minutes O2 plasma treatment sample. Confocal microscopy showed the well-defined structure of striations of the sarcomeres of the cells only in the case of the N2 treated sample, whereas in the case of O2 treated sample as well as the control sample, the cell showed a circular morphology and loss of organisation of α-actinin. SEM images confirmed the same morphology of the cardiomyocytes with an elongated and spherical one for N2 and O2 plasma treated surfaces respectively.

Our results provide strong evidence on what is the desirable plasma treatment in the case of using PS for cardiac cells. Additionally, the outcome of our analysis overturns the common belief that O2 plasma treatment provides an optimum tool to construct an appropriate microenvironment and conditions for cell culture in general. Although favourable towards surface hydrophilisation, here oxygen species are proven to not be able to provide the expected environment for cells, as viability, shape, and striations are not ideal for cardiac cell culture. Contrarily, N2 plasma allows cardiac cells to perform their normal functions, leading to the conclusion that it produces a friendlier cellular environment. Moreover, the stability in hydrophilicity, thus in surface chemistry, of N2 treated PS is an additional factor for concluding that this treatment is preferable in the case of culture studies. From all the above and from the SEM images that show pseudopodia and attachment of cells on all cases, one can clearly see that the cellular behaviour is mostly affected and led by the surface chemistry and that topography plays an ancillary role in the case of primary cardiomyocyte culture.

Our research can lead to the control of numerous cellular mechanisms by minimising effects of the culture surface and allowing the cardiac cells to develop in their natural shape, promoting their normal behaviour when cultured.

Fig. 1 .

 1 Fig. 1. Morphology of plasma-treated PS slides. SEM images of PS slides, undergone various plasma treatments after 20 min (left column), and 30 min of exposure (right column).

Fig. 2 .

 2 Fig.2. Hydrophilicity of plasma-treated PS slides. Mean apparent water contact angle WCA of plasma-treated PS slides at different periods. dx stands for the measurement at day x after plasma treatment.

Fig. 3 .

 3 Fig. 3. Evaluation of plasma-treated PS surface chemistry. High Resolution XPS spectra of untreated PS (control) and treated with O2 and N2 plasma for 30 minutes (O2 30 and N2 30, respectively): (a) C1s, (b) O1s and N1s.

Fig. 4 .Fig. 5 .

 45 Fig. 4. Cell viability monitoring of H9c2 cells cultured on different plasma treated PS. Two days after cells seeding on various plasma-treated PS, live cells were quantified using the Muse® Count & Viability Kit. Statistical analyses were realised using non-parametric Kruskal-Wallis test.

Fig. 6 .

 6 Fig. 6. Cell viability and morphology assessment of primary cardiomyocytes cultured on untreated PS serving as control. Viable cells (a) and fluorescent images of non-purified (b) and purified (c) neonatal rat cardiomyocytes cultured on the control PS for 3 days. Green: α-actinin, red: actin, blue: nuclei. Statistical analyses were realised using parametric t-test. **p < 0.01.

Fig. 8 .

 8 Fig. 8. Morphology of primary cardiomyocytes cultured on plasma-treated PS. Fluorescent images of purified neonatal rat cardiomyocytes cultured on plasma-treated PS for 3 days: (a)-(b) 20 and 30min of N2 treatment, and (c)-(d) 20 and 30min of O2 treatment, respectively. Green: 𝛼-actinin and blue: nuclei.

Fig. 9 .

 9 Fig. 9. SEM images of primary cardiomyocytes cultured on plasma-treated PS. Primary cardiomyocytes were cultured for three days on non-treated or on O2 or N2-treated PS.

Table 1

 1 Summary of PS atomic composition derived from XPS analyses.

		N 2 30	Intensity (CPS, a.u.)	N 2 30
	Intensity (CPS, a.u.)	O 2 30	544 542 540 538 536 534 532 530 528 526 Binding Energy (eV) control O 2 30
		control	Intensity (CPS, a.u.)	N 2 30
				O 2 30
				control
	296 294 292 290 288 286 284 282 280	412 410 408 406 404 402 400 398 396 394
	Binding Energy (eV)		Binding Energy (eV)

Table 2

 2 Summary of binding energies (BEs) and functional groups from the high resolution XPS scan results (C1s, N1s, O1s) of the PS before and after O2 and N2 plasma treatments. For the O1s region, the eV values are given for the apex of the spectrum. (Table

Acknowledgements

This work was supported by the LabEx REVIVE (ANR-10-LABX-73), the Ile-de-France Region in the framework of the DIM Respore, the Île-de-France network of Excellence in Porous Solids and the AFM-Téléthon (contract number: 22142). MK acknowledges personal funding from the LabEx REVIVE and DIM Respore too. The authors also acknowledge IMPC from Sorbonne University (Institut des Matériaux de Paris Centre, FR CNRS 2482) and the C'Nano projects of the Region Ilede-France, for Omicron XPS apparatus funding. The authors thank the personnel of the Photon Microscopy Facility of IBPS for helpful advice and technical assistance during microscopy image acquisition and analysis.

Declaration of Competing Interest

The authors declared that there is no conflict of interest.


Rosa Rafael
email: rafael.d.rosa@ufsc.br

Alonso Pascal

Santini Adrien

Vergnes Agnes

Bachere Evelyne

Rafael D Rosa

High polymorphism in big defensin gene expression reveals presence-absence gene variability (PAV) in the oyster Crassostrea gigas

Keywords: mollusk bivalve, antimicrobial peptide, polymorphism, gene copy number variation, highthroughput microfluidic RT-qPCR, Vibrio infection

We report here the first evidence in an invertebrate, the oyster Crassostrea gigas, of a phenomenon of Presence-Absence Variation (PAV) affecting immune-related genes. We previously evidenced an extraordinary interindividual variability in the basal mRNA abundances of oyster immune genes including those coding for a family of antimicrobial peptides, the big defensins (Cg-BigDef). Cg-BigDef is a diverse family composed of three members: Cg-BigDef1 to -3. Here, we show that besides a high polymorphism in Cg-BigDef mRNA expression, not all individual oysters express simultaneously the three Cg-BigDefs. Moreover, in numerous individuals, no expression of Cg-BigDefs could be detected. Further investigation at the genomic level revealed that in individuals in which the transcription of one or all Cg-BigDefs was absent the corresponding Cg-bigdef gene was missing. In our experiments, no correlation was found between Cg-bigdef PAV and oyster capacity to survive Vibrio infections. The discovery of P-A immune genes in oysters leads to reconsider the role that plays the immune system in the individual adaptation to survive environmental, biotic and abiotic stresses.

Highlights

► Cg-BigDefs display a high interindividual polymorphism in basal mRNA expression. ► All three Cg-BigDefs are not simultaneously expressed in a single oyster. ► Lack of Cg-BigDef mRNA expression is likely due to the absence of the corresponding gene. ► First report in an invertebrate of Presence-Absence Variation affecting immune genes.

Introduction

Understanding the mechanisms of resistance or susceptibility to infections in the oyster

Crassostrea gigas should contribute to identify the causes of mass mortalities that affect this marine mollusk species worldwide. In this context of multifactorial disease, significant progresses were made in characterizing the oyster immune system and responses to biotic and abiotic environment factors. Particular attention has been paid to the mechanisms involved in the capability of the oyster to overcome infections [START_REF] De Lorgeril | Whole transcriptome profiling of successful immune response to Vibrio infections in the oyster Crassostrea gigas by digital gene expression analysis[END_REF][START_REF] Schmitt | The antimicrobial defense of the Pacific oyster, Crassostrea gigas. How diversity may compensate for scarcity in the regulation of resident/pathogenic microflora[END_REF] but also to the genetic bases that govern oyster survival [START_REF] Rosa | A hemocyte gene expression signature correlated with predictive capacity of oysters to survive Vibrio infections[END_REF].

High-throughput transcriptomic studies have revealed in C. gigas great individual polymorphism in the expression of many immune-related genes [START_REF] Rosa | A hemocyte gene expression signature correlated with predictive capacity of oysters to survive Vibrio infections[END_REF]. This supported previous evidence that C. gigas is a highly polymorphic and intron-rich species in terms of number of SNPs and repetitive DNA sequences [START_REF] Kim | Development of type I genetic markers from expressed sequence tags in highly polymorphic species[END_REF][START_REF] Saavedra | Bivalve genomics[END_REF][START_REF] Sauvage | Single Nucleotide polymorphisms and their relationship to codon usage bias in the Pacific oyster Crassostrea gigas[END_REF]. Thus, besides sequence polymorphism, the oyster displays great polymorphism of expression that can be related to gene Copy Number Variation (CNV) in genome contents between different individuals [START_REF] Rosa | A hemocyte gene expression signature correlated with predictive capacity of oysters to survive Vibrio infections[END_REF][START_REF] Schmitt | Sequence polymorphism and expression variability of Crassostrea gigas immune related genes discriminate two oyster lines contrasted in term of resistance to summer mortalities[END_REF]. CNV are known to be of fundamental importance for genetic variation in populations, evolution of genome complexity and evolution of genes with novel functions.

Many studies have looked for associations between the copy number of genes and disease susceptibility (reviewed in [START_REF] Cantsilieris | Correlating multiallelic copy number polymorphisms with disease susceptibility[END_REF]. For instance, in human, whereas extensive copy number of β-defensin antimicrobial peptide has been associated to the risk of psoriasis [START_REF] Hollox | Psoriasis is associated with increased beta-defensin genomic copy number[END_REF], low copy number of β-defensin has been reported to predispose to Crohn disease [START_REF] Fellermann | A chromosome 8 gene-cluster polymorphism with low human beta-defensin 2 gene copy number predisposes to Crohn disease of the colon[END_REF]. Surprisingly, our high-throughput transcriptomic studies have also revealed numerous genes whose basal levels of expression could not be detected in some individuals [START_REF] Rosa | A hemocyte gene expression signature correlated with predictive capacity of oysters to survive Vibrio infections[END_REF]. Those genes were shown to be involved in immune responses such as genes encoding members of the big defensin antimicrobial peptide family, Cg-BigDef [START_REF] Rosa | Big defensins, a diverse family of antimicrobial peptides that follows different patterns of expression in hemocytes of the oyster Crassostrea gigas[END_REF].

Oyster big defensins (Cg-BigDef) have been shown to be a diverse antimicrobial peptide family composed of three members (Cg-BigDef1, Cg-BigDef2 and Cg-BigDef3) that are encoded by distinct genomic sequences [START_REF] Rosa | Big defensins, a diverse family of antimicrobial peptides that follows different patterns of expression in hemocytes of the oyster Crassostrea gigas[END_REF]. An interesting particularity of the oyster Cg-BigDef family is that their members follow different patterns of gene regulation upon microbial challenge. Indeed, whereas Cg-BigDef1 and Cg-BigDef2 are up-regulated in circulating hemocytes in response to pathogenic Vibrio infection, Cg-BigDef3 is constitutively expressed and non-regulated [START_REF] Rosa | Big defensins, a diverse family of antimicrobial peptides that follows different patterns of expression in hemocytes of the oyster Crassostrea gigas[END_REF]. In this study, an unusual variability in mRNA expression was evidenced in oyster sampling for all Cg-BigDefs. As Cg-BigDef1 and -2 transcriptions are highly induced by microbial molecular patterns [START_REF] Rosa | Big defensins, a diverse family of antimicrobial peptides that follows different patterns of expression in hemocytes of the oyster Crassostrea gigas[END_REF], it was reasonable to expect that these molecules could be implicated in preventing from development infectious processes. This prompted us to further analyze such polymorphism of expression in C. gigas.

Here, a detailed analysis of Cg-BigDef mRNA expression at individual level confirmed firstly a high interindividual variability in basal gene expression for the three Cg-BigDef forms. In addition, we found that the three Cg-BigDefs are not simultaneously transcribed in a single oyster. More surprisingly, in some individual oysters, basal mRNA levels were not detected for any Cg-BigDefs. Besides, we evidenced that the absence of Cg-BigDef transcription is likely associated to the lack of Cg-bigdef genes, revealing the extreme form of CNV named Presence-Absence Variation (PAV). Finally, the Presence-Absence Variation of Cg-bigdefs in the oyster genome was shown to be not related to a better capacity of the oysters to survive experimental Vibrio infections.

Materials and Methods

Oysters and experimental Vibrio infections

Adult two-year old Crassostrea gigas oysters were purchased from a local oyster farm in Mèze (Gulf of Lion, France) and kept in aquaria containing filtered seawater at 15°C at the Aquaculture Experimental Platform of Ifremer, Palavas-Les-Flots. During two weeks, animals were fed twice with a mixture of live microalgae (Nannochloropsis oculata: 2.5×10 5 cells/ml; Tetraselmis suecica: 2.5×10 2 cells/ml). Following the acclimatation period, 90 oysters were individually tagged and hemolymph (~500 µl per animal) was withdrawn from the adductor muscle without causing the death of the animals. Hemocyte samples were individually collected by centrifugation (1,500 ×g, 15 min, 4°C), homogenized in 1 ml of TRIzol ® reagent (Invitrogen) and frozen at -20°C until RNA extraction. After hemolymph collection, animals were placed in glass tanks (20 animals per tank) and allowed to recover for 8 days prior to experimental infections. Then, animals were intramuscularly injected with 5×10 8 CFU/animal of the virulent V. tasmaniensis LGP32 (=V. splendidus LGP32). Mortalities were monitored daily and individually tagged animals that did not survive were noted and discarded. After the end of acute mortalities (96 h post-infection), stored hemolymph samples were categorized according to oyster survival (surviving versus non-surviving). The standardization of the experimental infections and the preparation of the bacterial inoculum were performed as previously described [START_REF] Duperthuy | The major outer membrane protein OmpU of Vibrio splendidus contributes to host antimicrobial peptide resistance and is required for virulence in the oyster Crassostrea gigas[END_REF]. All experimentations were performed according to the Ifremer animal care guideline and policy.

Hemocyte samples from 73 additional oysters (naive animals) were obtained as described above, homogenized in 1 ml of TRIzol ® reagent (Invitrogen) and frozen at -20°C until RNA extraction. From those, 23 oysters were randomly chosen, removed from their shells and immediately frozen in liquid nitrogen. Frozen whole-body oysters were individually ground to fine powder for further extractions of total RNA and genomic DNA in same individuals.

Genomic DNA isolation

Genomic DNA (gDNA) was isolated individually as previously described [START_REF] Schmitt | Molecular diversity of antimicrobial effectors in the oyster Crassostrea gigas[END_REF]. Briefly, 50-100 mg of oyster powder were incubated overnight at 55°C in 500 µl of DNA extraction buffer (100 mM NaCl, 10 mM Tris-HCl pH 8, 25 mM EDTA pH 8, 0.5% SDS, 0.1 mg/ml protease K). Following a step of phenol/chloroform extraction, gDNA samples were precipitated by addition of cold ethanol and treated with 50 µg/ml RNase A (Invitrogen) for 30 min at 37°C to eliminate contaminating RNA. Quantification and quality of gDNA samples were assessed by spectrophotometry (NanoDrop ND-1000 Thermo Scientific) and 0.8% agarose gel electrophoresis, respectively.

Total RNA extraction and cDNA synthesis

Total RNA was extracted individually by using TRIzol ® reagent (Invitrogen) according to the manufacturer's instructions. RNA samples were then treated with DNase I (Invitrogen) for 15 min at room temperature to eliminate contaminating genomic DNA. After DNase I inactivation (10 min at 65°C), samples were precipitated with 0.3 M sodium acetate. RNA amount and quality were assessed by spectrophotometric analysis and the integrity of total RNA was analyzed by 0.8% agarose gel electrophoresis.

Following heat denaturation (70°C for 5 min), reverse transcription was performed using 250 ng of purified total RNA with 50 ng/µl oligo(dT) 12-18 in a 20-µl reaction volume containing 1 mM dNTPs, 1 unit of RNaseOUT Ribonuclease and 200 units M-MLV reverse transcriptase in reverse transcriptase buffer according to the manufacturer's instructions (Invitrogen). Attempting to increase the number of cDNA templates, a supplementary step of preamplification was performed using 1.25 μl of cDNA, 1.25 μl of a mixture of forward and reverse primers (200 nM each) and 2.5 μl of 2× TaqMan ® PreAmpMaster Mix (Applied Biosystems). The specific primer pairs for each Cg-BigDef form are listed in Table 1. PCR cycling conditions were as follows: polymerase activation at 95°C for 10 min, followed by 14 cycles of 95°C for 15 s and 60°C for 4 min. Preamplification products were diluted to 1:5 in TE buffer (10 mM Tris, 1 mM EDTA, pH 7.5) and stored at -20°C.

High-throughput RT-qPCR using 96.96 microfluidic dynamic arrays

The relative gene expression was measured by using the high-throughput microfluidic RT-qPCR platform BioMark TM (Fluidigm) [START_REF] Jang | Quantitative miRNA expression analysis using fluidigm microfluidics dynamic arrays[END_REF] The cycling program used consisted of 10 min at 95°C followed by 40 cycles of 95°C for 15 s and 1 min at 60°C. Melting curves analysis was performed after completed RT-qPCR collecting fluorescence between 60-95°C at 0.5°C increments. Data were analyzed using the BioMark TM Real-time PCR analysis software to obtain Cq values. Results are presented as changes in relative expression normalized with the Cq geometric mean value of the eukaryotic translation elongation factor 1-alpha (Cg-ef1α: AB122066), the ribosomal protein L40 (Cg-rpl40: FP004478) and the 40S ribosomal protein S6 (Cg-rps6: CU686508) as endogenous reference genes [START_REF] Livak | Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method[END_REF]. Statistical significance between expression detection of Cg-BigDefs and oyster capacity to survive V. tasmaniensis LGP32 infections was determined by ANOVA test at p<0.05.

Genotyping of Cg-bigdefs

PCR amplifications for Cg-bigdef genotyping were conducted in a 25-µl reaction volume using 1 µl of synthesized cDNA or 30 ng of purified gDNA as template. Primer sequences are listed in Table 1. PCR conditions were as follows: 10 min at 94°C, then 30 cycles at 94°C for 1 min, 56°C for 1 min, 72°C for 3 min and a final elongation step of 72°C for 10 min. PCR products were analyzed by electrophoresis on 1.5% agarose gel stained with ethidium bromide. The amplification products were cloned into a pCR ® -Blunt II-TOPO ® cloning vector using a Zero Blunt ® TOPO ® PCR cloning kit (Invitrogen). The positive recombinant clones were identified by colony PCR and were sequenced in both directions.

Sequence data analysis

Obtained sequences were analyzed for similarities using BLASTX at the National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov). The multiple alignments were generated by using the ClustalW2 Multiple Alignment Program (ClustalW2; http://www.ebi.ac.uk/Tools/msa/clustalw2/). The phylogenetic analyses based on the fulllength cDNA and genomic sequences of C. gigas big defensins were performed using the Neighbour-Joining method with the software MEGA version 4 [START_REF] Tamura | MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0[END_REF].

Bootstrap sampling was reiterated 1,000 times.

Results

Oyster big defensins display a high interindividual polymorphism in basal gene expression

The basal expression levels of the three big defensins were first analyzed by a highthroughput RT-qPCR device in 163 individual oysters (Fig. 1 2A). Besides, the three Cg-BigDef forms were never found to be simultaneously expressed among the 163 oysters analyzed (Table 2). Interestingly, only one or two forms of Cg-BigDefs were shown to be expressed in a same individual. Cg-BigDef1 and Cg-BigDef2 were seen in 81 and 47 individuals, respectively, while Cg-BigDef3 was expressed in 21 oysters only. Similar results were obtained when using different specific primer pairs for each Cg-BigDef form (Table 1).

More surprisingly, among the 163 oysters, 39 did not express any Cg-BigDefs (Table 2). This lack of expression was further confirmed by RT-qPCR analyses using two universal primer pairs common to all Cg-BigDef sequences. As a control to these RT-qPCR analyses, the expression of three reference genes (Cg-rpl40, Cg-ef1α and Cg-rps6) was found to be uniform across all tested individuals (Appendix A). These results evidenced a high interindividual variability in gene expression of C. gigas big defensins, including cases where their basal expression is fully absent in some individuals.

Lack of Cg-BigDef expression is likely associated to the absence of encoding genes in the oyster genome

In light of the degree of polymorphism in gene expression observed for Cg-BigDefs, we further investigated the relationships between the expression and presence of the three Cgbigdef genes in 23 individuals from which we have extracted both mRNA and gDNA.

Detection of transcripts was performed by RT-qPCR and detection of corresponding genes by conventional PCR with specific primer pairs for each form (Table 1). The expression profiles obtained for these 23 individuals confirmed the high variability in basal expression levels observed above by high-throughput RT-qPCR analyses (Fig. 2A; Appendix A). Figure 2B shows the amplification of Cg-bigdef genes in ten individual oysters which are representatives of the 23 oysters analyzed. Interestingly, genes encoding a given Cg-BigDef were only amplified in individuals expressing the corresponding form at basal levels (Fig. 2B). The accuracy of these results was further confirmed by using an additional primer pair specific for the different Cg-bigdefs and two universal primer pairs (common to all forms). Besides, as a control of the integrity of extracted gDNA samples, Cg-ef1α gene was successfully amplified in all oysters analyzed (Fig. 2B). Indeed, whereas Cg-bigdef1 and Cg-bigdef2 genes included two exons interrupted by a single intron, an additional intron and exon were observed in Cg-bigdef3 upstream the first exon common to the other Cg-bigdef genes (Fig. 3). Interestingly, in one individual in which both Cg-BigDef1 and -2 were expressed, two different Cg-bigdef1 sequences were identified in addition to the Cg-bigdef2 gene (Fig. 4). In this oyster, the Cg-bigdef1 genes differed in the length of the intron sequence and by the presence of two nucleotide substitutions (Fig. 4).

Cg-BigDef

Moreover, for the other individuals analyzed, single nucleotide polymorphisms (SNP) were found between transcripts and genes, which was evocative of two different alleles of a same Cg-bigdef gene. Thus, each Cg-bigdef would correspond rather to a distinct gene than to alleles from a same Cg-bigdef locus.

Sequence analysis revealed that, for each Cg-bigdef gene, the length of exons was well conserved among individuals whereas the introns lengths were variable. According to individuals, SNP were identified in both intron and exon sequences for a same Cg-bigdef gene. Additionally, introns from all Cg-bigdef genes included some microsatellite sequences (such as CTAT, CT and CA) and the canonical GT/AG splice site junctions (Fig. 3). The intron sequences of Cg-bigdef3 appeared to differ considerably in both length and nucleotide composition from those of Cg-bigdef1 and Cg-bigdef2 genes (Fig. 3).

A phylogenetic tree was built with the nucleotide sequence of six Cg-bigdef gene sequences obtained here and with Cg-bigdef gene sequences previously deposited in GenBank [START_REF] Rosa | Big defensins, a diverse family of antimicrobial peptides that follows different patterns of expression in hemocytes of the oyster Crassostrea gigas[END_REF]. In this tree, Cg-bigdef1 and Cg-bigdef2 genes clustered in a same clade distinct from the Cg-bigdef3 gene (Fig. 5). An additional phylogenetic tree constructed from cDNA sequence data confirmed that Cg-BigDef1 and Cg-BigDef2 forms are more related to each other than to Cg-BigDef3 (Fig. 5). Altogether, results from sequencing and phylogenetic analyses strongly suggest that Cg-BigDefs constitute a multigenic family of antimicrobial peptides in C. gigas oysters. 3). No significant differences were found in the detection of Cg-BigDefs according to the S and NS phenotypes, namely according to the oyster capability to survive virulent Vibrio infections (p>0.05). Additionally, no significant differences in basal gene expression levels of the three Cg-BigDefs were found between individuals from both survival capacity phenotypes.

Presence-absence variation (PAV) in

Discussion

Taking advantages of the development of high-throughput genomic technologies, we have explored the genetic determinants for Crassostrea gigas capacity to survive infections at the individual level. These analyses highlighted an extraordinary interindividual polymorphism in the basal expression of genes in this species [START_REF] Rosa | A hemocyte gene expression signature correlated with predictive capacity of oysters to survive Vibrio infections[END_REF]. Indeed, most of the transcriptome profilings are performed with oyster sample pools, which hides great information about oyster genomic diversity and complexity. Here, high-throughput RT-qPCR analyses at the individual level revealed a high variability in basal gene expression regulation of antimicrobial peptides belonging to Cg-BigDef family. Basal mRNA amounts of both the inducibly expressed Cg-BigDef1 and Cg-BigDef2 and the constitutively-expressed Cg-BigDef3 forms appeared to be highly variable among individual oysters. For instance, variations up to 30-fold were found in Cg-BigDef1 expression according to individuals. This interindividual polymorphism in Cg-BigDef transcription levels may reflect genetic structure variation in gene copy numbers (CNV) as recently reported for other oyster AMPs, Cg-Defs and Cg-Prps [START_REF] Schmitt | Sequence polymorphism and expression variability of Crassostrea gigas immune related genes discriminate two oyster lines contrasted in term of resistance to summer mortalities[END_REF].

When analyzing the heterogeneity of transcription profiles of the big defensins, we showed that no expression was detected in some oysters and that the lack of basal expression was likely associated to the absence of the corresponding encoding sequences at the genome level. Such results highlight structural variations in oyster genomes and particularly gene Presence-Absence variation (PAV), a phenomenon described as the extreme case of Copy Number Variation (CNV) in which a large DNA segment present in some individuals can be entirely missing in others. To our knowledge, this is the first evidence of PAV in oysters.

Considering that C. gigas oyster displays a high degree of DNA sequence polymorphism and heterozygosity [START_REF] Sauvage | Single Nucleotide polymorphisms and their relationship to codon usage bias in the Pacific oyster Crassostrea gigas[END_REF][START_REF] Schmitt | Molecular diversity of antimicrobial effectors in the oyster Crassostrea gigas[END_REF][START_REF] Taris | Sequence polymorphism can produce serious artefacts in real-time PCR assays: hard lessons from Pacific oysters[END_REF], a rigorous accuracy has been required in the choice of the primers used in this study.

Two primer pairs specific for each of the three Cg-BigDefs have been designed as well as two primer pairs common for all three forms. The relationship between the presence-absence (P-A) of transcripts and the presence-absence of corresponding encoding gene has been confirmed in 23 individuals by different PCR analyses using eight different primer pairs spread across the entire sequences. Besides, the correspondence between the different genes and their corresponding transcripts amplified has been assessed by nucleotide sequencing.

Thus, the non-amplification of any Cg-BigDef is likely due to the lack of genes instead of the existence of null alleles commonly reported in oysters [START_REF] Reece | Microsatellite marker development and analysis in the eastern oyster (Crassostrea virginica): confirmation of null alleles and non-Mendelian segregation ratios[END_REF].

Here, we have improved also knowledge about oyster big defensin diversity by the identification of novel variants for each Cg-BigDef form. Interestingly, in single individuals, we evidenced the presence of different variants for a same Cg-bigdef gene, suggesting the presence of different isoforms of a given Cg-BigDef in a same oyster. Likewise, the human DEF locus has been shown to be highly variable among individuals [START_REF] Taudien | Polymorphic segmental duplications at 8p23.1 challenge the determination of individual defensin gene repertoires and the assembly of a contiguous human reference sequence[END_REF].

Variations in the human DEF locus include the presence of different alleles for a same DEF gene, and both CNV and PAV phenomena.

PAV appeared to concern all Cg-bigdefs but more particularly the constitutively expressed Cg-bigdef3 gene that was recorded in less than 13% of the analyzed oyster population. Importantly, since we sampled animals from a same oyster hatchery, we cannot presume that the obtained frequency distribution of Cg-bigdef genes is representative of natural oyster populations or representative of the species. The three Cg-BigDefs were never seen simultaneously expressed in a single oyster, probably due to the relatively limited oyster sampling size. Indeed, based on the occurrence frequency of the three Cg-bigdefs in this oyster population, the probability of one individual carrying the three loci was calculated to be less than 2%. Any of the three big defensin forms were detected in 23.9% ( 39 [START_REF] Cuthbertson | Diversity of the penaeidin antimicrobial peptides in two shrimp species[END_REF][START_REF] O'leary | Genomic structure and transcriptional regulation of the penaeidin gene family from Litopenaeus vannamei[END_REF][START_REF] Rosa | Functional divergence in shrimp anti-lipopolysaccharide factors (ALFs): from recognition of cell wall components to antimicrobial activity[END_REF]. Each penaeidin member is encoded by a separated gene and the interindividual molecular diversity is mainly generated by DNA polymorphism within each PEN gene locus (O' Leary and Gross, 2006). The simultaneous expression of different members of a given antimicrobial peptide family in a single animal has been shown to improve its antimicrobial defenses [START_REF] Cuthbertson | Diversity of the penaeidin antimicrobial peptides in two shrimp species[END_REF][START_REF] Rosa | Functional divergence in shrimp anti-lipopolysaccharide factors (ALFs): from recognition of cell wall components to antimicrobial activity[END_REF]. [START_REF] Schmitt | Molecular diversity of antimicrobial effectors in the oyster Crassostrea gigas[END_REF]. Thus far, no transcription profiling of Cg-Defs and Cg-PRPs have been performed at the individual level but it can be assumed that PAV can also affect these effectors in oysters.

Analyses of the

To date, intraspecific variations in gene contents such as PAV have never been reported in oysters and any other non-model invertebrate species, probably because few genome sequences are available. Indeed, whole C. gigas genome has been recently sequenced from one individual [START_REF] Zhang | The oyster genome reveals stress adaptation and complexity of shell formation[END_REF]. It is noteworthy that we failed to evidence in the published database any full sequences of big defensin encoding genes [START_REF] Zhang | The oyster genome reveals stress adaptation and complexity of shell formation[END_REF].

The PAV phenomenon for big defensins or any other genes in C. gigas deserves to be fully characterized by genetic mapping or through population-scale genome sequencing.

PAV is essentially known in human and particularly in plants in which this genetic diversity is exploited in domestication programs. In human, PAV has been described for the DEFA3 gene encoding human neutrophil AMP HNP-3 [START_REF] Linzmeier | Human defensin gene copy number polymorphisms: comprehensive analysis of independent variation in alpha-and beta-defensin regions at 8p22-p23[END_REF].

Interestingly, the occurrence of DEFA3 gene is variable depending on the human geographical populations, and it has been suspected to be implicated with differences in infectious disease susceptibility [START_REF] Ballana | Inter-population variability of DEFA3 gene absence: correlation with haplotype structure and population variability[END_REF]. In our study, no correlation has been shown between the P-A prevalence of the big defensins and the intrinsic capacity of the oysters to survive experimental Vibrio infections. However, one can expect that P-A genes others than those genes encoding AMP could have substantial involvement on oyster fitness and health in the context of multifactorial disease that currently affects this species worldwide.

In plants, regions of the genome encompassing PAV have been shown to contain gene clusters involved in disease resistance [START_REF] Zhang | Genome-wide patterns of large-size presence/absence variants in sorghum[END_REF]. Thus, it is assumed that PAV may play important role in generating new pathogen resistances and they are now considered as polymorphic markers for genetic studies and breeding [START_REF] Wang | Exploration of presence/absence variation and corresponding polymorphic markers in soybean genome[END_REF][START_REF] Zhang | Genome-wide patterns of large-size presence/absence variants in sorghum[END_REF].

Overall, the great number of P-A genes observed in plants is recognized to play important roles in individual adaptation to external pressure and environmental stresses [START_REF] Haun | The composition and origins of genomic variation among individuals of the soybean reference cultivar Williams 82[END_REF][START_REF] Springer | Maize inbreds exhibit high levels of copy number variation (CNV) and presence/absence variation (PAV) in genome content[END_REF][START_REF] Swanson-Wagner | Pervasive gene content variation and copy number variation in maize and its undomesticated progenitor[END_REF]. Indeed, such structural variations imply that the missing genes in some individuals are not necessary for survival but rather contribute to the genome evolution and to the interplay with the environment [START_REF] Marroni | Structural variation and genome complexity: is dispensable really dispensable?[END_REF]. Undoubtedly, the first discovery of PAV that affect immune-related genes in oyster open questions about the functional consequences of such genome structural variation on the oyster immune responses but also on the individual capability to survive environmental, biotic and abiotic stresses. Tables Table 1. Nucleotide sequence of primers.

  . The sample reaction mixtures were performed in a final volume of 5 μl containing 1.25 μl of preamplified cDNA (diluted 1:5), 2.5 μl of 2× TaqMan ® Gene Expression Master Mix (Applied Biosystems), 0.25 μl of 20× DNA Binding Dye Sample Loading Reagent (Fluidigm), 0.25 μl of 20× EvaGreen (Biotium) and 0.75 μl of TE buffer. Primer reaction mixtures were made in the same volume of 5 μl containing 2.5 μl of 2× Assay Loading Reagent (Fluidigm), 1.25 μl of 20 μM of forward and reverse primer mix and 1.25 μl of TE buffer. Both sample and primer reaction mixtures were loaded into the dynamic array chip that was subsequently placed on the NanoFlex TM 4-IFC Controller for loading and mixing. After approximately 50 min, the chip was transferred to the BioMark TM Real-Time PCR System.

  ; Appendix A). Specific primers were used for each Cg-BigDef form. The RT-qPCR analyses evidenced a high interindividual variability for the three Cg-BigDef transcripts. The Cg-bigdef1 and Cg-bigdef2 forms whose expression is inducible showed lower basal expression compared to the Cg-bigdef3 form, constitutively expressed. Figure 2A shows the relative mRNA abundance of Cg-BigDefs in ten individual oysters which are representatives of individual differences in basal gene expression of the 163 oysters analyzed in this study. Depending on individuals, differences reached up to 30, 27 and 7-fold for Cg-BigDef1, Cg-BigDef2 and Cg-BigDef3, respectively (Fig. 1; Fig.

  forms are likely encoded by three distinct genes We performed an exhaustive gDNA and cDNA cloning and sequencing of the Cgbigdefs from four individuals expressing one or two forms, first to ensure the authenticity of the PCR amplifications but also to gain insights into the genomic organization of the big defensin family. We sampled one individual that transcribed simultaneously Cg-BigDef1 and -2 as well as one individual that was shown to transcribe both Cg-BigDef1 and -3. Two additional individuals expressing only Cg-BigDef2 or -3 were also sampled. Both gene and cDNA sequences were obtained from these four individuals where the Cg-BigDefs were detected at the level of transcripts and genes. The obtained sequences were deposited in GenBank under the accession numbers JN251121 to JN251132. Multiple alignments of the Cg-BigDef gDNA and cDNA sequences obtained in single individuals confirmed that each oyster big defensin form is transcribed by a separated gene.

  Cg-BigDef gene expression is not associated to oyster capacity to survive virulent Vibrio infections In order to investigate whether the presence or absence of Cg-BigDefs was associated to an inherent capacity of oysters to survive infections, we have quantified the mRNA levels of all three Cg-BigDefs (Cg-BigDef1, Cg-BigDef2 and Cg-BigDef3) in 90 individual animals before a challenge with an oyster pathogen. By using a nonlethal sampling method prior to the experimental infections (Rosa et al., 2012), we have assessed the basal transcription profile of all three Cg-BigDefs in oysters able (S) and not able (NS) to survive infections by the virulent V. tasmaniensis LGP32 (=V. splendidus LGP32). Three independent experimental infections were conducted and hemolymph samples from 45 oysters of each survival capacity phenotype were obtained. The detection of Cg-BigDef transcripts and their mRNA abundance were compared in individual oysters from both S and NS phenotypes by high-throughput microfluidic RT-qPCR analyses. Transcripts of Cg-BigDef1 were detected in 25 individuals from both S and NS phenotypes whereas transcripts of Cg-BigDef2 were detected in 10 and 12 individuals from S and NS phenotypes, respectively (Table 3). Cg-BigDef3 transcripts were detected in 4 individuals from the S phenotype and in 2 from the NS phenotype. For the S phenotype, 27 and 6 individuals expressed, respectively, only one or two Cg-BigDefs (Cg-BigDef1 and Cg-BigDef2 or Cg-BigDef1 and Cg-BigDef3) and 12 oysters did not express any Cg-BigDefs (Table 3). For the NS phenotype, 4 and 31 oysters expressed, respectively, only one or two Cg-BigDefs (Cg-BigDef1 and Cg-BigDef2 or Cg-BigDef1 and Cg-BigDef3) and 10 oysters did not express any Cg-BigDefs. No oysters expressing simultaneously Cg-BigDef2 and Cg-BigDef3 or all three Cg-BigDefs were found in both S and NS phenotypes (Table

  /163) of the individuals examined. A single oyster can express only one or up two Cg-BigDefs concomitantly. Conversely, in Litopenaeus shrimp species all three members of the penaeidin family (Litvan PEN2, Litvan PEN3 and Litvan PEN4) and all four members of the antilipopolysaccharide factor (ALF) family (Litvan ALF-A, Litvan ALF-B, Litvan ALF-C and Litvan ALF-D) are simultaneously transcribed in a single individual

  Cg-bigdef gene sequences obtained from a single individual suggest that each Cg-BigDef form is encoded by a separate gene and not by different alleles from a same Cg-bigdef locus. Interestingly, the intron sequences of Cg-bigdef3 were shown to be different from those of Cg-bigdef1 and Cg-bigdef2 genes, suggesting an ancient divergence between the constitutively and the inducibly expressed forms. Likewise to Cg-BigDefs, Cg-Defs and Cg-Prps are also multigenic families whose the different members are encoded by separate genes displaying distinct gene structures

Figure captions Figure 1 .

 captions1 Figure captions

Figure 2 .

 2 Figure 2. The lack of detection of Cg-BigDef expression is likely due to the lack of the

Figure 3 .

 3 Figure 3. Each Cg-BigDef form is encoded by a separated gene. (A) A not-to-scale

Figure 4 .

 4 Figure 4. Nucleotide alignment of the first exon and intron sequences from alleles of Cg-

Figure 5 .

 5 Figure 5. Phylogeny of the oyster big defensin family. Phylogenetic trees were constructed

  Figure 1

Acknowledgments

We are in debt with Frédérique Cerqueira, Jean-Luc Rolland and Marc Leroy for their excellent technical assistance. We are also grateful to François Bonhomme for helpful comments and Delphine Destoumieux-Garzón for critical reading of the manuscript. This study received financial support from Ifremer and CNRS. RD Rosa was supported by a doctoral founding from CNPq-Brazil. Data used in this work were partly produced through molecular genetic analysis technical facilities of the SFR "Montpellier Environnement Biodiversité" and the "Platform qPHD UM2/Montpellier GenomiX".


Parvovirus 4 in Blood Donors, France

To the Editor: In the past few years, several novel parvoviruses have been identifi ed, including human parvovirus B19-related strains V9 and A6, and bocavirus. In 2005, parvovirus 4 (PARV4), a new putative member of the family Parvoviridae, was identifi ed in the plasma of a patient in North America who had an acute virus infection [START_REF] Jones | New DNA viruses identifi ed in patients with acute viral infection syndrome[END_REF]. This virus had limited sequence homology with parvovirus B19 (<30% aa similarity) despite a conserved genomic organization showing 2 large nonoverlapping open reading frames (ORF). Phylogenetic studies performed with near-complete sequences have proposed that human PARV4 can be described by ≥3 genogroups (2).

Little information is available about the natural history of the virus, and its clinical role remains unknown. Initial studies showed that PARV4 was present in the blood of febrile patients, intravenous drug users, and persons positive for hepatitis C virus or HIV at prevalences of 6%-30% [START_REF] Fryer | Parvoviruses PARV4/5 in hepatitis C virus-infected persons[END_REF][START_REF] Fryer | Frequent detection of the parvoviruses, PARV4 and PARV5, in plasma from blood donors and symptomatic individuals[END_REF][START_REF] Lurcharchaiwong | Parvovirus 4 (PARV4) in serum of intravenous drug users and blood donors[END_REF]. Two recent studies also showed the virus in cohorts of kidney transplant patients [START_REF] Vallerini | Parvoviruses in blood donors and transplant patients, Italy[END_REF][START_REF] Biagini | Human parvovirus 4 in kidney transplant patients, France[END_REF]. PARV4 has also been identifi ed in persons without apparent pathologic changes, such as blood donors (1.0%-3.95%), and in blood products negative for parvovirus B19 DNA [START_REF] Fryer | Frequent detection of the parvoviruses, PARV4 and PARV5, in plasma from blood donors and symptomatic individuals[END_REF][START_REF] Lurcharchaiwong | Parvovirus 4 (PARV4) in serum of intravenous drug users and blood donors[END_REF][START_REF] Vallerini | Parvoviruses in blood donors and transplant patients, Italy[END_REF]. Virus DNA also was found in bone marrow and various tissues or organs, suggesting possible dispersion of the virus in diverse biologic locations [START_REF] Schneider | Persistence of novel human parvovirus PARV4 in liver tissue of adults[END_REF].

To provide new insights into the dispersion of PARV4 in healthy persons, we assessed the frequency of PARV4 viremia in a cohort of blood donors by using a dual real-time assay. A total of 304 volunteer blood donors (154 men; mean age of all volunteers 40 years) living in southeastern France entered the study. The sex ratio (men:women: 1.03) and age distribution (19-65 years) of the cohort were considered representative of the population of blood donors at that time. Blood samples were collected in vacuum tubes (Vacutainer, SST, Becton Dickinson, Meylan, France) and centrifuged, and 1-mL plasma aliquots were stored at -80°C until use. Nucleic acids were extracted as described previously [START_REF] Biagini | Human parvovirus 4 in kidney transplant patients, France[END_REF] and tested for PARV4 genomic DNA immediately after extraction to avoid freezing/thawing cycles.

Five microliters of the eluted material served for PARV4 DNA detection using real-time PCR TaqMan methods (StepOne Plus, Applied Biosystems, Courtaboeuf, France) and primers (ORF2) compatible with the detection of the 3 virus genogroups described at this time [START_REF] Simmonds | A third genotype of the human parvovirus PARV4 in sub-Saharan Africa[END_REF][START_REF] Fryer | Frequent detection of the parvoviruses, PARV4 and PARV5, in plasma from blood donors and symptomatic individuals[END_REF][START_REF] Biagini | Human parvovirus 4 in kidney transplant patients, France[END_REF]. Two probes were tested in separate amplifi cation assays: PARV4-O (5′-FAM-TGTTCAACTTTCTCAGGTCCT ACCGCCC-TAMRA-3′) (4,7) and PARV4-N (5′-FAM-TCCTACYGCC CSCTCCTCCTTCTT-TAMRA-3′). The second primer was designed after identifi cation and sequencing of several in-house real-time PCR products (GenBank accession nos. FJ883557-61), and more particularly those detectable on agarose gels but showing negative signal with PARV4-O Taq-Man assay because of mismatches identifi ed on the probe recognition site (Figure). Amplifi cation reactions were performed as described previously [START_REF] Biagini | Human parvovirus 4 in kidney transplant patients, France[END_REF]. Both TaqMan assays were estimated to detect as few as 10 copies of PARV4 DNA per reaction using dilutions of a synthetic template [START_REF] Biagini | Human parvovirus 4 in kidney transplant patients, France[END_REF].

Fifteen (4.9%) blood donors gave positive signal with probe PARV4-O; 62 (20.4%) were positive with probe PARV4-N; 6 (2.0%) samples were positive in both assays. Overall prevalence for PARV4 DNA was 24.0% in the blood donors tested.

PARV4 origin of randomly selected PCR products from PARV4-O and PARV4-N TaqMan assays ( 5 samples in age groups highlighted a relative homogeneity throughout the corresponding cohort.

Our study shows that PARV4 infection is readily detectable in French blood donors. Prevalence results using probe PARV4-O were comparable to those obtained in previous studies involving healthy persons originating from various countries (4-6). Conversely, the high prevalence obtained by using probe PARV4-N was unexpected because only 1 study demonstrated a higher value (45.7%) after the investigation of PARV4 DNA in bone marrow aspirates of AIDS patients from Italy [START_REF] Longhi | Human parvovirus in the bone marrow of Italian patients with AIDS[END_REF].

This fi nding suggests a larger dispersion of PARV4 than expected initially in the general population and highlights the need for improvement in detection systems directed toward PARV4 DNA, particularly by interlaboratory collaborations, in direct connection with studies investigating PARV4 genetic diversity. These considerations are consistent with the recent description of a new PARV4 genogroup in humans and characterization of highly divergent variants in bovine and porcine species [START_REF] Lau | Identifi cation of novel porcine and bovine parvoviruses closely related to human parvovirus 4[END_REF]. In addition, such data raise the question of the consequent persistence of PARV4 infection in healthy persons. Future studies need to explore both dispersion and potential clinical impact of PARV4 on infected hosts. To the Editor: Nontuberculous mycobacteria (NTM) are increasingly recognized as human pathogens [START_REF] Jones | New DNA viruses identifi ed in patients with acute viral infection syndrome[END_REF]. Otomastoiditis is a rare extrapulmonary NTM disease type fi rst described in 1976; Mycobacterium chelonae-M. abscessus group bacteria, which are rapidly growing NTM, are the most frequent causative agents and the disease mostly affects children (1-3). In the Netherlands, M. chelonae-M. abscessus group isolates have been reported from the otologic samples of an average of 2 patients annually since 2006, as compared to 6 patients in the preceding 10 years. This emergence is not a likely result of improved laboratory facilities or awareness in clinicians because liquid culture and molecular identifi cation techniques predate the rise in notifi cation and Dutch guidelines advise against performing cultures for chronic otorrhea.

Mhammed

We identifi ed 10 patients from the national reference laboratory database with otologic samples yielding M. chelonae-M. abscessus group bacteria during January 1995-June 2007. We resubjected these isolates to molecular identifi cation by rpoB gene sequencing (4) and performed a medical fi le review.

  Figure. Alignment of parvovirus 4 (PARV4) sequences showing the location of the 2 probes used in the real-time experiments. A) Partial sequences used for the design of probe PARV4-N: PARV4 prototype isolate (AY622943) and in-house PCR products characterized initially (GenBank accession nos. FJ883557-61). B) Examples of point mutations located on the PARV4-O recognition site identifi ed in amplicons originating from samples positive with PARV4-N assay. Mismatches identifi ed in the alignments are underlined. Nucleotides shown in lowercase letters correspond to 5′/3′ ends of the real-time primers. Mismatches identifi ed in the alignments are underlined and in boldface.