Yannicke Dauphin
email: yannickedauphin@upmc.fr

Gilles Luquet

Aline Percot

Laure Bonnaud-Ponticelli

Comparison of embryonic and adult shells of Sepia officinalis (Cephalopoda, Mollusca)

Keywords:

Development and evolution of the shell in cephalopods is difficult to establish as there is few species with a calcified shell that could be fossilized (stable in geological time). Internal cuttlebone of sepiids is so particular that homologies are difficult to find. The developmental sequence in embryos give some response elements by comparison with adult cuttlebone. The macro and microstructure of adult shell is well known but an approach at nanostructural level allows to determine structure and composition of the two main parts, the dorsal shield and chambered part. We evidence in the embryonic shell, mainly organic, a light calcification of the shell, which occurs directly as aragonite, as it is all along the formation of the shell and whatever the parts. In embryonic shell, the prismatic and/or lamellar layers, present in adult, are not differentiated and the dorsal shield grows progressively, from posterior to anterior. Despite microstructural differences, all layers of both chambered part and dorsal shield are composed of rounded nanogranules (between 50 and 100 nm), similar to what is found in other mollusc shells.

Finally, the presence of pillars evidenced in embryo suggests either that their absence in extinct

Introduction

Mollusks are a major group of marine organisms. Among them, cephalopods received a peculiar attention in different fields of research. They are able to synthesize inner or outer shells and are consequently of interest in biomineralization [START_REF] Mutvei | On the shells of Nautilus and Spirula with notes on the shell secretion in non cephalopod mollusks[END_REF][START_REF] Dauphin | Organisation ultrastructurale de l'os de seiche (Cephalopoda-Dibranchiata)[END_REF][START_REF] Dauphin | Microstructures des coquilles de Céphalopodes. II-La seiche (Dibranchiata, Decapoda)[END_REF][START_REF] Lowenstam | On Biomineralization[END_REF][START_REF] Bandel | Cephalopod shell structure and general mechanisms of shell formation[END_REF][START_REF] Marie | Evolution of nacre: biochemistry and proteomics of the shell organic matrix of the cephalopod Nautilus macromphalus[END_REF][START_REF] Cuif | Synchrotron-based photoelectron spectroscopy provides evidence for a molecular bond between calcium and mineralizing organic phases in invertebrate calcareous skeletons[END_REF][START_REF] Checa | The cuttlefish Sepia officinalis (Sepiidae, Cephalopoda) constructs cuttlebone from a liquid-crystal precursor[END_REF][START_REF] Pabic | Overview of the shell development of the common cuttlefish Sepia officinalis during early-life stages[END_REF] as well as from an evolutionary point of view [START_REF] Haas | Trends in the evolution of the Decabrachia[END_REF][START_REF] Kröger | Cephalopod origin and evolution: A congruent picture emerging from fossils, development and molecules[END_REF][START_REF] Allcock | The contribution of molecular data to our understanding of cephalopod evolution and systematics: a review[END_REF]. Known since the Paleozoic era, they are used in palaeontology as good stratigraphic markers because of calcified shells, fast evolution and large dispersion of major extinct species. Only three extant cephalopod genera are able to build a calcified shell: external in Nautilus, internal in Spirula and Sepia. For this particularity, they are used in biomimetic and biomedical research [START_REF] Ogasawara | Template mineralization of ordered macroporous chitin-silica composites using a cuttlebone-derived organic matrix[END_REF][START_REF] Kim | Cellular attachment and osteoblast differentiation of mesenchymal stem cells on natural cuttlefish bone[END_REF][START_REF] Karthika | Structural characterization and in vitro biomedical activities of sulfated chitosan from Sepia pharaonis[END_REF]. Recently, calcified processes have been studied in the context of global change and ocean acidification [START_REF] Gutowska | Growth and calcification in the cephalopod Sepia officinalis under elevated seawater pCO2[END_REF][START_REF] Dorey | Ocean acidification and temperature rise: effects on calcification during early development of the cuttlefish Sepia officinalis[END_REF][START_REF] Sigwart | Elevated pCO2 drives lower growth and yet increased calcification in the early life history of the cuttlefish Sepia officinalis (Mollusca: Cephalopoda)[END_REF].

Sepia officinalis [START_REF] Linnaeus | Systema Naturae per regna tria naturae, secundum classes, ordines, genera, species, cum characteribus, differentiis, synonymis, locis. Editio decima[END_REF] and Sepia pharaonis Ehrenberg, 1931 are the most studied species, notably because of their abundance and commercial interest as seafood [START_REF] Bettencourt | Age studies on daily growth increments in statoliths and growth lamellae in cuttlebone of cultured Sepia officinalis[END_REF][START_REF] Sykes | Sepia officinalis[END_REF].

One of the first detailed microstructural analyses of the shell and the living tissues of Sepia officinalis was that of [START_REF] Appellöf | Die Schalen von Sepia, Spirula und Nautilus. Studien über den Bau und das Wachstum[END_REF]. The proostracum (dorsal shield component), the ventral part with pillars and lamellae, the fork, the siphonal tube and the rostrum were identified by [START_REF] Naef | Die fossilen Tintenfische[END_REF]. The continuity of the layers in dorsal shield and rostrum was described, as well as the presence of chitin [START_REF] Appellöf | Die Schalen von Sepia, Spirula und Nautilus. Studien über den Bau und das Wachstum[END_REF][START_REF] Naef | Die fossilen Tintenfische[END_REF]. Numerous comparative studies have been done to establish homologies of the different parts of the shell of present versus fossil coleoids.

In his comprehensive work on the structure and mineralogy of mollusc shells, [START_REF] Boggild | The shell structure of the molluscs[END_REF] noted that the dorsal shield is composed of three layers, the median layer being fully organic in the lateral part, and more or less calcified in the middle of the cuttlebone. He also showed that all the layers of Sepia are aragonitic, the dorsal shield and the rostrum being prismatic. As other cephalopod shells, the aragonite has a low Sr content [START_REF] Dauphin | Aragonitic rostra of the Turonian belemnitid Goniocamax: Arguments from diagenesis[END_REF][START_REF] Florek | Complementary microstructural and chemical analyses of Sepia officinalis endoskeleton[END_REF].

The structure of the chambered part and the labyrinthine ("meanderlike") organization of the pillars/walls were also described and illustrated. [START_REF] Barskov | Microstructure of the skeletal layers of Sepia and Spirula compared with the shell layers of other molluscs[END_REF] used SEM to observe the minute structure of the cuttlebone and described the microstructure of the septa. A comparison of some Sepia species has shown that despite differences in the morphology of the cuttlebone, their arrangements and microstructures are similar [START_REF] Dauphin | Microstructures des coquilles de Céphalopodes. II-La seiche (Dibranchiata, Decapoda)[END_REF]. The regular layered structure of the septa of Spirula was described by [START_REF] Grégoire | Sur la structure de la nacre septale des Spirulidae, étudiée au microscope électronique[END_REF][START_REF] Grégoire | Sur la structure des matrices organiques des coquilles de mollusques[END_REF], who called it "nacre" despite the absence of tablets. The same structure was also called "lamellar fibrillar structure" [START_REF] Erben | Über die Bildung und das Wachstum von Perlmutt[END_REF] or "type II nacre" [START_REF] Mutvei | Ultrastructure of the mineral and organic components of molluscan nacreous layers[END_REF], then lamello-fibrillar nacre [START_REF] Mutvei | Siphuncular structure in the extant Spirula and other Coleoids (Cephalopoda)[END_REF]. Such a structure was also found in Sepia [START_REF] Dauphin | Mise en évidence d'un type microstructural coquillier spécifique des Céphalopodes dibranchiaux[END_REF][START_REF] Doguzhaeva | Siphonal zone structure in the cuttlebone of Sepia officinalis[END_REF].

Recently an analysis of the cuttlebone using for the first time CT-scan has drawn a dynamic 3D image of the development and growth of the shell from embryo to adult (Le [START_REF] Pabic | Three-dimensional structural evolution of the cuttlefish Sepia officinalis shell from embryo to adult stages[END_REF].

Most studies dealing with the microstructure and composition of the cuttlebones were performed on adult samples. For example, the presence of chitin and proteins in the adult cuttlebone has been shown by [START_REF] Degens | Paleobiochemistry of molluscan shell proteins[END_REF], [START_REF] Drozdova | Chemical composition and infra-red absorption spectra of the organic matrix of the shell in the squid Sepia pharaonis[END_REF], [START_REF] Hunt | A comparative study of protein composition in the chitin-protein complexes of the beak, pen, sucker disc, radula and oesophageal cuticle of cephalopods[END_REF].

The first studies on embryonic shell of S. officinalis and S. pharaonis were done by [START_REF] Bandel | A comparative study of the structure, development and morphological relationships of chambered cephalopod shells[END_REF], then detailed by [START_REF] Sen | The cuttlebone development of common cuttlefish [Sepia officinalis[END_REF] and [START_REF] Pabic | Overview of the shell development of the common cuttlefish Sepia officinalis during early-life stages[END_REF][START_REF] Pabic | Three-dimensional structural evolution of the cuttlefish Sepia officinalis shell from embryo to adult stages[END_REF], but the microstructures and compositions of the calcified parts of Sepia embryonic shells remained to be more accurately characterized.

In this study, we therefore examined the microstructures and nanostructures of the calcified parts of Sepia officinalis embryonic shells, with some data on their composition, in comparison to the adult cuttlebone and other mineralized structures synthesized by mollusks. In addition to the understanding of variation in building the shell during development and growth of animal, our study brings new information on shell evolution within mollusks.

Material and methods

Materials

Sepia officinalis adult shells were collected on beaches in Normandy (France). The maximal length of the cuttlebones was 30 cm. S. officinalis eggs were collected in Luc-sur-Mer (Normandie, France) and kept in the lab in artificial sea water (salinity: 35 ppt -closed circulatory system), at 18°C. Eggs were opened and the embryos (whose stage is determined by morphological characteristics) were immerged in sea-water with 5% Ethanol, following recommendations of Buttler-Struben et al. (2018) and [START_REF] Winlow | Sense and Insensibility -An appraisal of the effects of clinical anesthetics on Gastropod and Cephalopod molluscs as a Step to Improve welfare of Cephalopods[END_REF] and according to directive 2010-63-UE and French decree 2013-118.

Cuttlebones were obtained by dissection. All soft tissues were removed and the cuttlebones were kept dried. [START_REF] Naef | Die fossilen Tintenfische[END_REF]Naef ( , 1928aNaef ( , 1928b) ) has divided the embryonic period into stages, specified later by [START_REF] Lemaire | Table de développement embryonnaire de Sepia officinalis L. (Mollusque, Céphalopode)[END_REF] and [START_REF] Boletzky S Von | A developmental table of embryogenesis in Sepia officinalis[END_REF]. We used the Lemaire and Boletzky et al. staging in this study. Stages from the beginning of matrix shell formation (stage 24), beginning of mineralisation (and first chamber, stage 25) to early final stage 30 (6 to 8 chambers) were analysed.

Crab chitin (Sigma Aldrich) and Bovine serum albumin (BSA, Eurobio) were used as references for the organic components. A marine non biogenic aragonite (botryoidal aragonite) was selected as a mineral reference.

Scanning electron microscopy (SEM)

A Philips XL30 and a Zeiss Gemini LEO 1550 were used on coated samples. Secondary electron images are indicated as SE-SEM in the legends. A FEI FESEM Quanta F600 and a Phenom Pro X were used for images in back scattered electron mode (BSE-SEM). In this technique, imaging contrast depends on the atomic number of the atoms composing the substrate material: the organic structures appear in black, in contrast to the mineral units with heavier chemical elements (Ca, Sr). The sample does not need to be covered by a conductive layer, so that the same sample can be used for SEM and AFM observations. Fractured shells and polished samples were observed. Samples embedded in resin were polished using various grades of diamond paste down to a final 0.25 mm grade. The polished surfaces were then cleaned with a detergent mixed with hot water for 1 min under ultrasonication to remove any oil residue from the pastes and rinsed with water. Then, according to the technique subsequently employed, additional preparative procedures were undertaken, the details of which are given in the figure captions.

Figure S1 displays the used terminology for the orientation of the sections.

Atomic Force Microscopy (AFM)

Samples were studied using a Nanoscope IIIa multi-mode scanning probe microscope operating in tapping mode. The tapping mode AFM utilizes an oscillating tip with amplitude of approximately several tens of nm when the tip is not in contact with the surface. The resolution of tapping mode AFM is in the order of a few nm. Phase imaging is a powerful extension to detect variations in chemical composition, friction and other physical properties. The AFM sensor oscillates over the surface sample, and the amplitude of the oscillation is used to control the scanner. An image can be formed from this amplitude signal that emphasizes the details of the sample surface.

Compared with conventional secondary electron imaging SEM (SE-SEM), AFM provides topographic direct height measurements and views of surface features since no coating is necessary. Moreover, three-dimensional nanoscale resolution AFM images are obtained without the time-consuming sample preparation (e.g. decalcification, thinning...) required for similar resolution Transmission Electron Microscopy examination.

Fourier Transform Infrared Spectrometry (FTIR)

All spectra were recorded at 4 cm -1 resolution with 16 scans on a Perkin-Elmer Frontier Fourier transform infrared spectrometer (FTIR), in the wavenumber range 4000 to 450 cm -1 . The spectrometer was equipped with a Diffuse Reflectance accessory, which permits DRIFT measurements with high sensitivity on powders. All spectra were corrected by the Kubelka-Munk function. A background spectrum was measured for pure KBr. Sample spectra were automatically ratioed against background to minimize CO2 and H2O bands. Correlation coefficients between two spectra of the same samples were about 99%. The dorsal shield and the ventral zone were mechanically separated in adult samples, immersed in 3% NaClO for 1 h to remove organic contaminants, rinsed with Milli-Q water, dried, and ground into powder.

Micro-Raman spectroscopy

The Raman spectra were recorded with a LabRam HR 800 spectrometer (Horiba Jobin Yvon) equipped with a Peltier cooled CCD detector and using the 514 nm line of a water-cooled Ar+ laser (Innova 90C, Coherent Inc.). Raman scattering is collected via a microscope (Olympus) equipped with a 50x long working distance objective, allowing a laser spot size of about 5 μm.

The laser power at the sample is between 2 and 3 mW. The spectrometer is equipped with a 600 lines/mm grating and allowing a 2 cm -1 spectral resolution on the range 100-1700 cm -1 .

Calibration has been checked with respect to the 520.7 cm -1 band of silicon. Typical recording times consist of 3 accumulations of 10 s.

Calcein labelling

Stage 25/26 and 30 embryos were incubated in vitro in calcein-containing seawater (30 mg/L; CNAM, Sigma) during 24h followed by 48h incubation in seawater without dye. Then shells were removed from frozen embryos and observed using a Leica DM-IRB microscope.

Results

Morphology -terminology

The descriptive terminology of the cuttlebone and the comparisons with external shells differ according to the authors. Here, we used the main terms defined by [START_REF] Barskov | Microstructure of the skeletal layers of Sepia and Spirula compared with the shell layers of other molluscs[END_REF]. The adult cuttlebone is divided into two main parts: the compact dorsal shield and the porous ventral zone, called chambered part (Fig. 1A). The ventral zone contains the septa, the siphuncular zone and the fork (Fig. 1A); the posterior part of the cuttlebone is sharp and called rostrum or spine (Fig. 1B). In embryo, the dorsal shield is thin, and thanks to transparency, the complex outlines of the first septa are visible (Fig. 1C). The growth occurs anteriorly by a ventral addition of new septa, delimitating chambers sustained by pillars, all being slightly mineralized (Fig. 1D-F). The fork, wings and rostrum are absent in the embryo cuttlebones (Fig. 1C,D). At hatching, the shell is composed of 6 to 8 chambers (Fig. 1F).

The organization of the adult cuttlebone has been repeatedly described, so that only the main features are here shown. The microstructure and nanostructure are detailed and illustrated.

Dorsal shield (= outer wall)

Adult shell

Three main layers are present in the dorsal shield (Fig. 2A,B): a thick prismatic outer spherulitic layer, a layered median layer (type II nacre or lamello-fibrillar nacre) and a thin prismatic inner layer. The outer spherulitic prismatic layer is composed of several sublayers (Fig. 2A,B). The thickness of the sublayers is irregular, because of the spherulites seen on the outer surface and described as tubercles [START_REF] Appellöf | Die Schalen von Sepia, Spirula und Nautilus. Studien über den Bau und das Wachstum[END_REF]. Growth layers are visible, made of elongated crystallites perpendicular to the sublayers (Fig. 2C). Etched and polished surfaces show the small diameter (< 1 µm) of the crystallites of the outer layer, so that they are more acicular than prismatic (Fig. 2D-E). Unetched oblique fractures in these acicular crystallites show the non-geometric shapes of granules (50-100 nm diameter) (Fig. 2F). The surface of the growth layers of the spherulitic prismatic layer reveals that the granules are not adjacent (Fig. 2G), and the large content of organic components is shown by AFM phase image contrast, as homogenous dark "flat" zones (Fig. 2H). The shades of colour in AFM phase image within a granule are indicative of a heterogeneous composition (Fig. 2I).

The median layer is thinner and shows overlapped sheets. The orientation of the acicular crystallites differs in every sheet (Fig. 3A). A cross-section shows that the sheets are stacked, parallel and thin (Fig. 3B,C) and some substructures perpendicular to the sheets are visible (Fig. 3C, stars). AFM height images show that a sheet is composed of rounded granules (Fig. 3D,E), and that the granules have a heterogeneous composition, as shown by shades of colour in phase contrast image (Fig. 3F).

The inner layer is prismatic and thin (Fig. 3G). Elongated crystallites are more or less divergent towards the ventral side of the shell (Fig. 3G). At larger magnifications, granules are irregular and do not seem aligned (Fig. 3H). They are composite (Fig. 3I), as shown by AFM phase image.

Embryonic shell

The embryonic dorsal shield begins to be organized and grows from posterior to anterior.

At stage 24, the shell is still organic and the mineralization, even low, starts at stage 25 (Fig. 4A and see below, Raman analysis). At stage 27, the thickness of the dorsal shield is about 15 microns (Fig. 4B-C). The prismatic structure of the outer layer (OP) is not well defined (Fig. 4D), and the elongated crystallites are not distinct (Fig. 4D). No sublayer is visible. The median layer seems to be entirely organic (Fig. 4E) and no inner arrangement is evidenced. The inner layer, IP, is similar to the outer layer, OP (Fig. 4D), with a somewhat indistinct arrangement.

Nevertheless, in some samples (the oldest one, i.e. stage 30), the elongated crystallites perpendicular to the boundary of the layer are visible (Fig. 4E,F), so that this layer is said to be prismatic. AFM phase images of the dorsal shield show rounded granules, and parallel elongated structures (Fig. 4G). Elongated structures are composed of several parallel units, the diameter of each one rangin4 from 20 to 50 nm (Fig. 4H). In the three layers of the dorsal shield, similar rounded granules are present. They are surrounded by a cortex, as shown by amplitude, phase and height images (Fig. 4I-K, c). In phase image (Fig. 4J), the cortex is darker than the inner part of the granules, so that it contains more organic components. The presence of amorphous calcium carbonate cannot be excluded. The maximal observed diameter of the granules is about 100 nm.

If the adult dorsal shield comprises three layers, at stages 27-30 the layers and structures are not very distinct. At stage 28, near the growing edge and on the lateral parts of the dorsal surface, a banding pattern of about 40-50 microns (Fig. 5A,B) and another one with thinner growth lines are present (Fig. 5C). The first tubercles resulting from the irregular thickness of the growth layer of the dorsal shield are visible (Fig. 5D). The posterior limit of every septum corresponding to the aperture is easily identified on the ventral face (Fig. 5E-G). The most posterior is a circular aperture corresponding to the open siphuncle (Fig. 5G).

After 24h in vitro incubation of the embryo in a calcein-containing sea water, and observations using light and fluorescence microscopy, the progressive antero-posterior development of the shield covering progressively the growing septum by irregular waves (Fig. 5 H-K) is well visible as well as the development of the pillars in each chamber (Fig. 5 I-K).

Ventral zone: chambered zone, septa

Adult shell

The ventral zone is porous. Longitudinal sections show parallel septa on which vertical pillars are inserted (Fig. 6A, Fig. S2A) so that chambers are reinforced. Pillars are covered by an organic membrane (Fig. 6B,C,m) with extensions parallel to the septa (Fig. 6B). In the adult chambers, minor growth zones are visible on the pillars (Fig. 6D,E). When the organic membranes on the surface of the pillars are removed, a granular structure is revealed, the granules being more or less arranged in columns (Fig. 6F, Fig. S2B). The average diameter of the granules is about 50 nm (Fig. 6G). Fractures parallel to the long axis or perpendicular to this axis show that pillars are not homogenous structures: the central part is hollow or soft (Fig. S2C,D).

Septa are composed of two layers (Fig. 6B, 6H: L). A closer examination shows a thin prismatic layer with crystallites perpendicular to the septa, and a lamello-fibrillar nacreous layer (Fig. 6C, H-I). Sections of the lamello-fibrillar nacre of the lamellae show the parallel arrangement of the superimposed sheets and lamellae seem to be well mineralized (Fig. 6J). The lamello-fibrillar nacre is composed of overlapped sheets and the orientation of acicular crystallites differs in two successive sublayers (Fig. 6K-L). The structure of the thin prismatic layer is similar to that of the inner layer of the dorsal shield.

Embryonic shell

As for the dorsal shield, the chambered part is not fully calcified so that the detailed structure is difficult to decipher. The outlines of septa are emphasized by the presence of more or less calcified zones, as shown by BSE images (Fig. 5E-G).

Septa comprise two parts: the pillars, perpendicular to the outer surface of the shell, and thin lamellae parallel to the outer surface of the shell. Pillars are not arranged in walls as in the adult shell, but they are isolated and their outline shows multiple folds (Fig. 7A,B). At the insertion of a pillar on a lamella, the basal part of the pillar is enlarged (Fig. 7C). Pillars are often branched (Fig. 7D,E, Fig. S2E). The surface of the lamellae between the pillars shows small granules (Fig. 7F, Fig. S2F). They are probably covered by a thin organic membrane, but no horizontal extensions are present. The pillars are composed of successive growth layers (Fig. 7D,E), and transverse sections show that the outer peripheral layer is more calcified (Fig. 7F).

The septum building starts with the secretion of a chitinous layer visible on the edge of the ventral zone (Fig. 5F, star). Lamellae on which pillars are inserted are composed of two layers, a prismatic layer and a lamello-fibrillar nacre, as observed in adult (Fig. 7G,H: L). The insertion of a septum on the dorsal shield in an embryonic shell does not show distinct structures (Fig. 7I). The two layers of the lamellae between the pillars are nanogranular (Fig. 7J), granules being embedded in an abundant matrix, probably organic as shown by AFM phase image (Fig. 7K,L). It must be noted that in adult shell, pillars merged in a zig zag pattern, while they are clearly separated and curly in embryonic shells (Fig. S2G).

Incubation of embryos in calcein-containing seawater reveals that calcification occurs in the pillars as soon as they start to grow from a dorsal septum to the future ventral one. The calcification is particularly located at the distal branching ends of each pillar until they connect with a new septum in formation (Fig. S2H,I).

Rostrum

In the adult shell, the rostrum is clearly visible as a sharp curved spine (Fig. 8A,B). Such sections also show the topographic relationships between the ventral fold, the fork, the dorsal shield and the first septa (Fig. 8A-C). The concentric structure is displayed in a transverse fracture (Fig. 8D) whereas the layered arrangement is shown in longitudinal sections (Fig. 8E,F). Despite the continuity between the dorsal shield and the rostrum, the growth layers of the rostrum are thinner and more regular than those of the dorsal shield (Fig. 8F), the rostrum being smaller and smoother than the dorsal surface of the shell. No tubercle is present on the rostrum.

The thickness of the growth layers is variable, and these layers are composed of parallel elongated crystallites, similar to those of the dorsal shield (Fig. 8G). Depending on the orientation of the section and the thickness of the growth layers, the crystallites are more or less visible.

The rostrum is not developed at the studied embryonic stages (Fig. 1D-F, Fig. 8H-I). It starts to differentiate from one month, after hatching.

Fork -ventral process

Fork is in contact and inserted on the inner prismatic layer of the dorsal shield (Fig. 8C,9A). The fork is composed of parallel lamellae, the thickness of which is similar to those of the septa (Fig. 9B). Depending on the orientation of the fracture, the aspect of the lamellae differs (Fig. 9B,C), but a closer examination shows that the compact lamellae comprise two sublayers: one sublayer is "prismatic" (Fig. 9D,1), the second one being more "lamellar" (Fig. 9D: 2). The boundary between these two sublayers is not distinct, and their thickness is irregular. Thus, in some fractures, one of these sublayers is not pulled out (Fig. 9C).

The ventral fold, also called ventral process (Fig. 8A-B), shows a radial structure, composed of a series of thin organic and mineralized layers (Fig. 9E). The prismatic structure of the mineralized layers is similar to that of the outer layer of the dorsal shield (Fig. 9F).

Fork and ventral process are not differentiated in the embryonic shells (Fig. 1C,4A,E).

Composition of adult and embryonic samples

The spectra of the calcite and aragonite groups are characterized by three major bands attributed to the carbonate ion CO3 2-: 3 at 1429 cm -1 , the 2 doublet 877 -848 cm -1 , and 4 at 713 cm -1 for the calcite group; 3 at 1471 cm -1 and two doublets: 2 at 858 -844 cm -1 and 4 at 713 -700 cm - 1 for the aragonite group [START_REF] Adler | Infrared absorption frequency trends for anhydrous normal carbonates[END_REF]. The weak 1 band is at 1012 cm -1 for calcite and at 1083 cm -1 for aragonite [START_REF] Jones | Infrared transmission spectra of carbonate minerals[END_REF]. According to [START_REF] Ylmen | Carbonation of Portland cement studied by diffuse reflection Fourier transform infrared spectroscopy[END_REF], the 3 asymmetric stretching of CO3 2-has a larger wavenumber range from 1425 and 1590 cm -1 .

The FTIR spectrum of the geological aragonite used as standard shows these main features (Fig. 10).

The dorsal shield and ventral chambered zone of adult shells also displayed the main bands assigned to aragonite: 1 at 1083 cm -1 , 2 doublet at 860-878 cm -1 and a doublet 4 at 700-713 cm -1 . Strong bands are visible respectively at 1512 and 1514 cm -1 . Unfortunately, in this region (1400-1600 cm -1 ), protein and chitin have characteristic amide I and II bands, and aragonite has an intense 3 vibration band at 1483 cm -1 . The spectral pattern between 1644 and 1510 cm -1 shows 2 intense bands probably indicative of a mixture of organic and mineral (CO3 2-) components. The bands at 1657 and 1644 cm -1 could be attributed to amide I vibration, whereas, the bands at 1512 and 1514 cm -1 are probably a mixture of amide II and 3 CO3 2-vibrations.

Sugars are common components of the organic matrices extracted from mollusc shells. Bands between 1050 and 1150 cm -1 are known to be characteristic for sugars. Although these bands are not clearly observed on both spectra, previous results (Le [START_REF] Pabic | First proteomic analyses of the dorsal and ventral parts of the Sepia officinalis cuttlebone[END_REF] and Fig S3 confirm the presence of sugars (large CO vibration band around 1030-1070 cm -1 ). The main part of the dorsal shield is the prismatic outer layer, low in organic components. Organic matrices in the form of abundant lamellae exist in the ventral part, so that chitin is more abundant.

The size of embryonic samples does not allow to separate the dorsal shield and the septa, so that the whole samples were powdered for FTIR analyses. The "mineral bands" ( to Fig. 10) are weak. As observed in the adult spectra, 1456 and 1639 cm -1 bands can be assigned to amide vibrations from both proteins and chitin. The characteristic sugar band at 1030-1070 cm -1 is probably hidden by the spectrum background (as also observed for the adult spectra).

To sum up, in all non decalcified samples, bands assigned to organic components are present, but they represent a mixture of proteins, chitin and lipids, so that a precise assignment is not possible. Spectra of the embryonic (green) and adult (orange and blue) shells are indicative of a mixed mineral -organic composition, with very low mineral content in the case of the juvenile shell.

One of the advantages of Raman spectroscopy is the small size of the beam (~5 µm with the green laser), allowing precise in situ punctual analyses. Regarding the first appearance of mineralisation, this was detected by micro-Raman spectroscopy at stage 25. The spectrum obtained (Fig. 11) is characteristic of a crystalline polymorph of calcium carbonate, more precisely aragonite, with main peaks at 152, 206, 701-705 and 1085 cm -1 [START_REF] Urmos | Characterization of some biogenic carbonates with Raman spectroscopy[END_REF][START_REF] Wehrmeister | Amorphous, nanocrystalline and crystalline calcium carbonates in biological materials[END_REF]. The strongest band at 1085 cm -1 is an internal mode that derives from the symmetric stretching mode (1) of carbonate ions. The peaks visible at 152 and 206 cm -1 correspond to translational and rotational lattice modes of aragonite carbonate ions, respectively.

The doublet 701-705 cm -1 , specific for aragonite, corresponds to the in-plane bending (4) mode of aragonite carbonate ions.

Moreover, aragonite is already present whatever the parts of the cuttlebone analysed (pillar, septa, even at the level of the chamber in formation), suggesting that the calcification is concomitant to the growth process. Note that ACC (amorphous calcium carbonate, precursor of any crystalline polymorph) was never evidenced. The mineralization seems to be happening directly as aragonite.

Discussion

Anatomy and structure

The anatomy and organisation of the adult cuttlebone of Sepia are known since the work of [START_REF] Appellöf | Die Schalen von Sepia, Spirula und Nautilus. Studien über den Bau und das Wachstum[END_REF]. Since, the relationships between the different elements (rostrum, fork...) have been more precisely defined [START_REF] Dauphin | Organisation ultrastructurale de l'os de seiche (Cephalopoda-Dibranchiata)[END_REF][START_REF] Dauphin | Microstructures des coquilles de Céphalopodes. II-La seiche (Dibranchiata, Decapoda)[END_REF][START_REF] Cadež | Formation and morphogenesis of a cuttlebone's aragonite biomineral structures for the common cuttlefish (Sepia officinalis) on the nanoscale: Revisited[END_REF].

In Sepia officinalis, the septa as well as the dorsal shield are made of three layers, a thick "prismatic" outer layer, a lamello-fibrillar "nacre" and a thin "prismatic" inner layer. All layers are built with elongated acicular crystallites, parallel to the surface of the shell in the lamellofibrillar "nacre", perpendicular in the prismatic layers. These last ones, from a morphological point of view and despite they are called "prismatic", clearly differ from other aragonitic prismatic layers known in other mollusc shells (e.g., Unionids: [START_REF] Dauphin | Structure and composition of Unio pictorum shell: arguments for the diversity of the nacroprismatic arrangement in molluscs[END_REF][START_REF] Schoeppler | Biomineralization as a paradigm of directional solidification: A physical model for Molluscan shell ultrastructural morphogenesis[END_REF]). The crystallites are not organized in large prisms, the reason why these layers are called "fibres" in some taxa. These prismatic layers are more similar to the aragonitic "fibres" from coral skeletons ( [START_REF] Cuif | The two-step mode of growth in the scleractinian coral skeletons from the micrometre to the overall scale[END_REF][START_REF] Cuif | Calcification in the Cnidaria through time: an overview of their skeletal patterns from individual to evolutionary viewpoints[END_REF]). The absence of tablets in the lamellofibrillar "nacre" is also confirmed. The lamellar aspect observed for all the layers and the presence of parallel elongated crystallites were described by [START_REF] Grégoire | Sur la structure de la nacre septale des Spirulidae, étudiée au microscope électronique[END_REF], then [START_REF] Mutvei | On the shells of Nautilus and Spirula with notes on the shell secretion in non cephalopod mollusks[END_REF] The changes in the crystallite orientation at every lamella as well as the absence of tablets were also observed by these authors.

Since, SEM observations have confirmed that such a structure is present in Spirula [START_REF] Dauphin | Microstructure des coquilles de Céphalopodes: I. Spirula spirula L. (Dibranchiata, Decapoda). Bull Mus natn hist nat, Paris, 3è sér[END_REF][START_REF] Bandel | A comparative study of the structure, development and morphological relationships of chambered cephalopod shells[END_REF], and belemnites [START_REF] Doguzhaeva | The pro-ostracum and primordial rostrum at early ontogeny of lower Jurassic belemnites from North-Western Germany[END_REF].

All the modern cephalopod shells are aragonitic, with different structures. The prismatic layers are diverse in structure and thickness. For example, in modern shells, the lamellar layer is thick and calcified (as here in Sepia but also in Nautilus) or very thin and organic (outer wall in Spirula). Note that in Spirula, the nacreous layer is the thickest part of the septa and is strongly mineralized [START_REF] Mutvei | On the shells of Nautilus and Spirula with notes on the shell secretion in non cephalopod mollusks[END_REF].

The outer surface of the dorsal shield of Sepia is not smooth, and the presence of tubercles is due to irregular spherulites. In the outer layer of the shell of Nautilus, irregular spherulites also exist, so that this layer is said to be spherulitic prismatic [START_REF] Mutvei | On the shells of Nautilus and Spirula with notes on the shell secretion in non cephalopod mollusks[END_REF]. Similar structures also exist in fossil genera: Belosepia, Beloptera and Belopterina [START_REF] Dauphin | Microstructures des coquilles de Céphalopodes. IV-Le "rostre" de Belosepia (Dibranchiata)[END_REF][START_REF] Dauphin | Microstructural studies on cephalopod shells. V-the apical part of Beloptera (Dibranchiata, Tertiary)[END_REF][START_REF] Dauphin | Microstructure des coquilles de Céphalopodes: la partie apicale de Belopterina (Coleoidea)[END_REF]. Despite these different microstructures, high-resolution SEM and especially AFM reveal, at the nanolevel, that all the aragonitic layers are made of rounded heterogeneous granules (diameter < 100 nm). The abundance of the organic matrices, around (and probably within) the granules, is shown by AFM phase images and BSE-SEM images.

Another feature is the complex structure of the pillars. Not only they show an organic envelope, growth zones and granules, but transversal sections reveal that their inner structure is heterogeneous: the central zone is sometimes empty or is more organic that the outer layer. (Fig. S2C,D). It must be added that the shape of the pillars is not constant: curly and isolated in the embryo, they are connected in zig zag wall in the adult shell.

All the morphological elements found in the adult shell exist in the embryonic shell but only the dorsal shield and the septa with pillars are visible at the hatching phase. The anatomy of the first stages of the cuttlebone has been described but most studies are focused on the number of septa and shape of the pillars. The cuttlebone of Sepia officinalis appears at stage 24 as a fully organic structure in a closed shell sac, the first calcified layers appear between stage 24 and 25 and the first pillars are built as soon as the formation of the second septa with the formation of the first chamber [START_REF] Bandel | Cephalopod shell structure and general mechanisms of shell formation[END_REF]Boletzky 1979, Le Pabic et al. 2016).

The three layers of the dorsal shield are visible, despite the microstructural elements (prisms or lamello-fibrillar nacre) are not so well differentiated. Embryonic shell shows that growth lines are visible in the pillars of the ventral part as soon as stage 29 (Le [START_REF] Pabic | Overview of the shell development of the common cuttlefish Sepia officinalis during early-life stages[END_REF].

They are also clearly visible in SEM images, as well as the prismatic structure. Rostrum is a weak bump at stage 30, with a structure similar to that of the dorsal shield. Fork and ventral process are not differentiated in the newly hatched shell, and "wings" are absent or not developed. They are lateral extensions of the dorsal shield [START_REF] Barskov | Microstructure of the skeletal layers of Sepia and Spirula compared with the shell layers of other molluscs[END_REF][START_REF] Dauphin | Microstructures des coquilles de Céphalopodes. II-La seiche (Dibranchiata, Decapoda)[END_REF], and their surface is granular (Fig. S2F).

In most fossil sepiids, the ventral region and the fragile structure of the pillars are not preserved. Lamellae and pillars are visible in Miocene Sepiids from Australia, but their inner structure is not displayed [START_REF] Košt'ák M, Ruman | Miocene sepiids (Cephalopoda, Coleoidea) from Australia[END_REF]. A change in wall/pillar configuration between the different regions of the cuttlebone (lateral areas vs median and latero-median areas) has been reported both in Sepia (extant species) and Belosaepia (fossil species) [START_REF] Sherrard | Cuttlebone morphology limits habitat depth in eleven species of Sepia (Cephalopoda: Sepiidae)[END_REF][START_REF] Yancey | The middle Eocene Belosaepia ungula (Cephalopoda: Coleoida) from Texas: structure, ontogeny and function[END_REF]. A recent study performed using CT-scan study did not evidenced such a difference in adult Sepia cuttlebone (Le [START_REF] Pabic | Three-dimensional structural evolution of the cuttlefish Sepia officinalis shell from embryo to adult stages[END_REF]). Actually Košt'ák et al. (2016) have not evidenced any imprints of pillar in the exceptionally preserved embryonic part of the holotype of S. vindobonensis, or in specimen NHMW 2004z0076/0039 of S. aff. sanctacrucensis. In this case, the development of pillars in the embryonic shell of extant cephalopods could represent an evolutionary novelty. Nevertheless, our results have shown that calcification of the embryonic shell is very light so that it cannot be excluded that these very small embryonic pillars have disappeared during fossilisation.

Composition

FTIR analyses show clearly that both the embryonic and the adult Sepia cuttlebones are composed of biogenic aragonite (result confirmed by Raman spectroscopy), which means they are made of a mixture of a crystalline polymorph of CaCO3 and an inner organic matrix. The spectra obtained from the powdered dorsal shield and the ventral chambered part exhibit bands corresponding mainly to aragonite, proteins, chitin and other carbohydrates. FTIR spectra of the soluble and insoluble matrices resulting from the decalcification of the ventral zone and dorsal shield have been provided by [START_REF] Pabic | First proteomic analyses of the dorsal and ventral parts of the Sepia officinalis cuttlebone[END_REF]. Whereas bands associated with proteins (vibrational amide bands) appear in the two spectra, bands associated with carbohydrates are more important in the insoluble matrix (but stronger in the chambered part than in the dorsal shield probably linked to the presence of an abundant matrix inside the chambers).

Mineralization

Raman analyses allowed deciphering the calcification process during the development of the cuttlebone from the embryo to the adult. Until stage 24 embryo, the shell in formation appears fully organic. From stage 25, calcification is detected by the presence of bands characteristic of CaCO3, wherever the area analysed, which means that this process starts in all the cuttlebone at the same time. From this stage, the cuttlebone can then be considered as a biomineral, mixture of mineral and organic components.

Raman and FTIR spectroscopy techniques confirm that the CaCO3 polymorph of the adult is aragonite as already reported. As for this adult shell, although the polymorph is crystalline for all the layers, the nanostructure reveals that they are composed of irregular rounded granules as observed for other biogenic crystalline CaCO3 polymorph [START_REF] Cuif | The two-step mode of growth in the scleractinian coral skeletons from the micrometre to the overall scale[END_REF]Cuif et al. 2012). The maximal observed diameter of the granules is about 100 nm. They are not homogenous from a compositional point of view, as shown by AFM phase images, and they are surrounded by a cortex, probably a mixture of organic matrix and amorphous CaCO3 (although this polymorph was not detected by Raman or FTIR methods).

In embryos, despite the transparency and fragility of the shell structures, using Raman spectroscopy we demonstrate for the first time that a light mineralization appears at stage 25 directly as aragonite. Mineralization remains light until hatching, occurring from this stage concomitantly to the growth of the shell. For example, the calcein-labelling method evidenced that the growth and calcification of the pillars occur simultaneously from a new septum towards the branched distal ends of these pillars.

Inside the egg, the embryo feeds only with maternal yolk and the perivitelline fluid surrounding the embryo has more or less the same composition than the seawater, with ions passing through the envelope. But as the embryo is in a protected environment, the shell has not a real function. The main source of calcium comes from food after hatching and the position in the water column changes, leading to an environmental medium richer in calcium. As a consequence, the developing shell becomes hardly mineralized. The Raman spectra show that there is no modification of the mineral type between the embryonic shell inside the egg and the adult shell: mineral is aragonite as soon as it could be detected in embryo. It is not excluded that mineralization could start earlier as ACC (amorphous calcium carbonate) but this was not detected.

Conclusion

The examination of adult shells of Sepia officinalis shows that despite a particular morphology, the main layers of the cephalopod shell are present. The dorsal shield comprises three layers, the thickness and mineralization degree of which differs. The ventral part (pillars and lamellae) represents the septa and the siphuncular zone.

In both septa and dorsal shield, the middle layer is lamellar, with acicular crystallites, as the classical nacreous layer but the tablets are absent. Pillars of the ventral zone are not homogeneous: the inner part is more organic than the outer part, as displayed by longitudinal and transverse sections. Rostrum, absent in embryo, is an extension of the dorsal shield.

Despite differences in the structure of the layers, and despite the crystalline structure of the constitutive mineral, all microstructural units are composed of more or less rounded granules (diameter between 50 and 100 nm). The organic matrix contains mainly chitin and proteins, but also, in a lesser extent, other carbohydrates and lipids, as in all the calcified biominerals.

In the embryonic shell, an early mineralization, directly as aragonite, is detected at stage 25. At stages 28 and 29, before hatching, the shell has about 6 chambers septa, and is still weakly mineralized. The micro-and nanostructures of the pillars and lamellae of the ventral zone are similar to those of adult shells, despite the pillars are branching, and not yet linked to form undulating walls. Growth lines are visible, as well as granules. Fork, ventral process and rostrum

are not yet differentiated. Chitin is probably the main component of the embryonic shell, and FTIR profile is more similar to the ventral zone of an adult shell (less mineralized) than to the dorsal shield. The dorsal shield and the ventral zone were mechanically separated in adult samples, immersed in 3% NaClO for 1 h to remove organic contaminants, rinsed with Milli-Q water, dried, and ground into powder by grinding with an electric mortar for 10 min to obtain homogeneous granulometry. Powdered samples were immersed in Milli-Q water and decalcified by progressive addition of 50% acetic acid so that the pH (automatically controlled with a titrimeter) is above 4.

A-B

The entire extract was centrifuged at 21,000 g for 15 min, which separated the supernatant (soluble) and precipitated (insoluble) fractions. The soluble fraction was desalted by exchange with Milli-Q water on a Microconcentrator (Filtron) using a 3-kDa cut-off membrane and lyophilized. Powdered samples and KBr were oven-dried at 38°C overnight. Then, they were mixed (about 5% powdered samples in KBr) and loaded into the sample cup.

Fig. 2

 2 photographs, E-F optical images, D X-ray image)

Fig. 3

 3 Fig. 3 Micro-and nanostructure of the lamello-fibrillar nacre (A-F) and the inner prismatic layer (G-I) of the dorsal shield of the adult shell.

Fig. 4 A

 4 Fig. 4 Micro-and nanostructures of the dorsal shield of embryonic shells. A stage 24, B-K stage 27.

Fig. 5 A

 5 Fig. 5 Morphology of embryonic shell. A-G stage 28, H-J stage 26, K stage 30

Fig. 6

 6 Fig. 6 Micro-and nanostructure of the ventral part of the adult shell.

Fig. 7

 7 Fig. 7 Micro-and nanostructure of the ventral zone of the embryonic stage 28 shell.

Fig. 8

 8 Fig. 8 Rostrum of the adult shell (SE-SEM).

Fig. 9

 9 Fig. 9 Fork and ventral process, adult shell (SE-SEM)

Fig. 10

 10 Fig. 10 Fourier Transform Infrared spectra of a geological aragonite, proteins (BSA), polysaccharides (chitin), embryonic and adult Sepia shells.

Fig. 11

 11 Fig. 11 Raman spectrosopy analysis of Sepia shell showing the presence of aragonite.

Fig. S1

 S1 Fig. S1 Terminology used for the orientation of the fractures and polished sections studied using SEM and AFM.

Fig. S2 A

 S2 Fig. S2 Variations in morphology and structure of the chambered zone of adult and embryonic shells. A Enlargement of the basal part of a pillar of an adult shell; fracture fixed and etched (chromium sulphate pH 3.5 for 4 h.); SE -SEM. B Pillars are covered by an organic membrane (upper part); under the membrane, the granular structure of the pillar is visible; adult sample. SE -SEM. C Etched fracture trough a pillar of an adult shell showing the empty middle part (NaOH 0.5M for 4h at 100°C, pronase pH 7.6 for 6 h at 34°C, chitinase pH 5.6 for 22 h at 24°C); SE -SEM. D Etched vertical section through the pillar of an adult shell showing the different behaviour of the middle and external parts (NaOH 1 M for 4h 30 at 100°C, then 4 days at 20°C, lipase 1 mg/ml pH 9 for 28 h at 35°C); SE -SEM. E Pillars in the first septa of an adult shell. SE -SEM. F Unetched granular surface of the lateral growing edge of a larval shell; BSE -SEM. G Unetched tangential fracture showing the zig zag "wall" made by coalescent pillars in an adult shell. H-I Stage 25/26 embryonic shell after 24h in vitro incubation of the embryo in a calcein-containing sea water (30 mg/L; CNAM, Sigma) followed by 48h incubation in sea water, H Optical image,

Fig. S3

 S3 Fig.S3FTIR spectra of the soluble and insoluble organic matrices extracted from the dorsal shield and ventral zone of an adult shell, showing the similarity between the insoluble matrix and the crab chitin.

Figure

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 2 Figure 2

Figure 3

 3 Figure 3

Figure 4

 4 Figure 4

Figure 5

 5 Figure 5

Figure 6

 6 Figure 6

Figure 7

 7 Figure 7

Figure 8

 8 Figure 8

Figure 9

 9 Figure 9

Figure 10

 10 Figure 10

Figure 11

 11 Figure 11

Acknowledgements

This work was financially supported by the ATM "Interactions Minéral -Vivant" funding of the Muséum national d'Histoire naturelle (SEPIOM project). The authors thank all the members of the Max Planck Institute for Interfaces and Colloids (Golm, Germany) for their help. We thank C. Jozet-Alves and the CREC (University of Caen) for providing eggs of Sepia officinalis. LBP thanks G. Patriache and L. Largeau (CNRS-LPN) for the first tests on mineral composition of embryonic shell several years ago.

Compliance with ethical standards

Animal protocols were carried out in accordance with European legislation (directive 2010-63-UE and French decree 2013-118).

Conflict of interest

Authors declare that they have no conflict of interest.

Research involving human and animal participants

We neither used endangered species nor were the investigated animals collected in protected areas.


Maria Teresa Caccamo

Vincenzo Zammuto

Concetta Gugliandolo

Claire Madeleine-Perdrillat

Antonio Spanò

Salvatore Magazù

Claire Madeleine- Perdrillat

Thermal restraint of a bacterial exopolysaccharide of shallow vent origin

Keywords: Extremophiles, Thermal analysis, Attenuated-Total-Reflectance Infra-Red spectroscopy, Spectral distance, Wavelet cross-correlation analysis

Introduction

Biomolecules, such as complex polysaccharides and exopolysaccharides (EPSs), from extremophiles inhabiting hot environments are expected to possess unique characteristics, mainly the thermostability, useful in responding to the increasing demand of new attractive bio-products in medical and pharmaceutical applications. Thermophilic bacteria, with optimal growth at temperature ranging from 45°C to 70°C, offer interesting features for biotechnological applications, as they are able to produce their biomolecules faster than those from mesophilic and psychrophilic counterparts [START_REF] Gugliandolo | New bacilli from shallow hydrothermal vents of Panarea Island (Italy) and their biotechnological potential[END_REF]. New thermophilic and thermotolerant bacteria, isolated from the shallow hydrothermal vents of Eolian Islands (Italy), were able to produce novel EPSs potentially useful in various applicative fields [START_REF] Gugliandolo | New bacilli from shallow hydrothermal vents of Panarea Island (Italy) and their biotechnological potential[END_REF][START_REF] Maugeri | A halophilic thermotolerant Bacillus isolated from a marine hot spring able to produce a new exopolysaccharide[END_REF][START_REF] Maugeri | Three novel halotolerant and thermophilic Geobacillus strains from shallow marine vents[END_REF][START_REF] Nicolaus | A thermophilic Bacillus isolated from an Eolian shallow hydrothermal vent, able to produce exopolysaccharides[END_REF]. More recently, a novel alkalophilic and thermophilic Bacillus licheniformis strain T14, isolated from a submarine vent off Panarea Island (Italy), has been reported as producer of a new exopolysaccharide (EPS1-T14), by using relatively simple purification process [START_REF] Spanò | A novel EPS-producing strain of Bacillus licheniformis isolated from a shallow vent off Panarea Island (Italy)[END_REF]. In details, EPS1-T14, containing fructose, fucose and glucose as major monosaccharides, displayed structural characteristics (high carbohydrate content, 99% and high molecular weight, 1,000 KDa), and interesting rheological properties (i.e. viscoelasticity) (Tab. 1) for which it could be used as a convenient thickener in industrial products [START_REF] Spanò | A novel EPS-producing strain of Bacillus licheniformis isolated from a shallow vent off Panarea Island (Italy)[END_REF]. Moreover, EPS1-T14 is soluble in water, non-cytotoxic up to 400g ml -1 , and it was also able to inhibit the cytotoxic effects of Avarol in an brine shrimp assay, and therefore, applications of the biopolymer in the development of anti-cytotoxic drugs could be envisaged [START_REF] Spanò | A novel EPS-producing strain of Bacillus licheniformis isolated from a shallow vent off Panarea Island (Italy)[END_REF]. Furthermore, it represents a promising exopolymer in pharmaceutical applications, as natural antiviral, immunostimulant and immunomodulator [START_REF] Gugliandolo | Antiviral and immunomodulatory effects of a novel bacterial exopolysaccharide of shallow marine vent origin[END_REF] and with anti-adhesive properties able to prevent biofilm formation by pathogenic bacteria [START_REF] Spanò | In vitro antibiofilm activity of an exopolysaccharide from the marine thermophilic Bacillus licheniformis T14[END_REF]. Many EPSs from thermophilic bacteria have been reported to be thermostable, as determined using the thermo-gravimetrical analysis (TGA) technique, with the highest thermostability registered for the exopolymer from Geobacillus tepidamans V246 (280 °C) [START_REF] Kambourova | Production and characterization of a microbial glucan, synthesized by Geobacillus tepidamans V264 isolated from Bulgarian hot spring[END_REF], followed by those from G. thermodenitrificans B3-72 (240 °C) [START_REF] Arena | An exopolysaccharide produced by Geobacillus thermodenitrificans strain B3-72: antiviral activity on immunocompetent cells[END_REF] and by EPS1-T14 from B. licheniformis T14, that was very stable up to 210 °C and started to decompose at 240 °C, yielding a weight loss of the half at 260 °C [START_REF] Spanò | A novel EPS-producing strain of Bacillus licheniformis isolated from a shallow vent off Panarea Island (Italy)[END_REF].

However, further information about the influence of the temperature on the molecular structure of EPS1-T14 are needed to address its potential use in biotechnological areas requiring high-temperature processes, such as food and cosmetic industries, and also for novel prospectives in medical and pharmaceutical applications, especially for drug delivery [START_REF] Xu | Synthesis and characterization of nanoparticles based on negatively charged xanthan gum and lysozyme[END_REF].

The changing of molecular structure is quantitatively measurable by InfraRed (IR) spectroscopy, providing a unique label-free tool for studying molecular composition and dynamics [START_REF] Branca | Destructuring effect of trehalose on the tetrahedral network of water: A Raman and neutron diffraction comparison[END_REF][START_REF] Branca | Tetrahedral order in homologous disaccharide-water mixtures[END_REF][START_REF] Ballone | Structural and vibrational properties of trehalose: A density functional study[END_REF][START_REF] Magazù | Structural and dynamical properties of water in sugar mixture[END_REF]. Among the IR techniques, the Fourier transform IR (FTIR) in the Attenuated Total Reflectance-FTIR (ATR-FTIR) mode represents a fast analytical method that permits to determine changes in molecular structure, using simultaneous analyses of multiple components from the same sample in a single instrumental measurement [START_REF] Deepa | FTIR Investigation on ion-ion interaction in liquid and gel polymeric electrolytes-LiCF3SO3-PC-PMMA[END_REF][START_REF] Magazù | FTIR spectroscopy studies on the bioprotective effectiveness of trehalose on human hemoglobin aqueous solutions under 50 Hz electromagnetic field exposure[END_REF]. ATR-FTIR technique is widely employed to characterize the elemental structures and the molecular interactions among components in polymers [START_REF] Barker | Review article: tolevamer, a novel toxin-binding polymer: overview of preclinical pharmacology and physicochemical properties[END_REF]. Furthermore, FTIR technique is complementary to Raman scattering, inelastic neutron scattering and density function simulations, these techniques furnishing valuable information with different probe-system coupling [START_REF] Hennet | Aerodynamic levitation and laser heating: Applications at synchrotron and neutron sources[END_REF][START_REF] Magazù | Innovative wavelet protocols in analyzing elastic incoherent neutron scattering[END_REF][START_REF] Magazù | Investigations of homologous disaccharides by Elastic Incoherent Neutron Scattering and Wavelet Multiresolution Analysis[END_REF][START_REF] Marchese | New generation non-stationary portable neutron generators for biophysical applications of Neutron Activation Analysis[END_REF]. Although FTIR analysis if used alone has been reported to be unable to ascertain the composition of polymorphic forms, the empowering of vibrational spectroscopy, by association with mathematical tools for the extraction of valuable information from complex evolving data, has demonstrated to be highly successful [START_REF] Migliardo | Infrared, Raman and INS studies of poly-ethylene oxide oligomers[END_REF]. Only a few recent studies reported ATR-FTIR technique as a useful tool for monitoring the thermal behavior of pharmaceutical products [START_REF] Calvo | A dynamic thermal ATR-FTIR/chemometric approach to the analysis of polymorphic interconversions: Cimetidine as a model drug[END_REF][START_REF] Migliardo | Elastic incoherent neutron scatterings wavevector and thermal analysis on glass-forming homologous disaccharides[END_REF][START_REF] Migliardo | Thermal analysis on bioprotectant disaccharides by elastic incoherent neutron scattering[END_REF].

In this work, the thermal restrain of the whole biopolymer EPS1-T14 and of its main monosaccharidic components (i.e. fructose, fucose and glucose) was investigated using a biophysical approach able to provide a measure of the

A C C E P T E D M A N U S C R I P T

3 system sensitivity to temperature changes [START_REF] Caccamo | Multiscaling Wavelet analysis of Infrared and Raman data on polyethylene glycol 1000 aqueous solutions[END_REF]. The Attenuated Total Reflectance Fourier Transform Infra-Red (ATR-FTIR) spectroscopy was applied over a temperature range from 20 °C to 80 °C, and each spectrum was analyzed by using innovative mathematical tools: i) non-ideal spectral deviation; ii) OH-stretching band frequency center shift; iii) spectral distance; and iv) wavelet cross-correlation analysis.

Material and Methods

The EPS-T14 production and characterization

The isolation of the halophilic and thermophilic Bacillus licheniformis T14 from a thermal fluid sample, emitted at a shallow hydrothermal vent of Panarea Island (Eolian Islands, Italy), and the production of a new exopolysaccharide (EPS1-T14) have been previously reported [START_REF] Spanò | A novel EPS-producing strain of Bacillus licheniformis isolated from a shallow vent off Panarea Island (Italy)[END_REF]. Briefly, characteristics of the emitted fluid were: temperature 50 °C, pH 5.42, and conductivity 42.90 mS m -1 . Strain T14 grew aerobically from 25 to 60 °C and its optimal temperature occurred at 50 °C. The pH range for growth was 4-10, with optimum at pH 8. Strain T14 grew in a range 2-10 % NaCl with optimal growth at 5 %.

The exopolysaccharide was produced (366 mg l -1 yield) at the beginning of the stationary growth phase of strain T14 in the minimal medium SWY (yeast extract, 0.1 %, sucrose 5 %, in seawater) after 48 h of incubation in bioreactor, under optimal growth condition (temperature 50 °C, pH 8, and NaCl 5 %) and air flow at 1 vvm. After purification, EPS1-T14 fraction displayed the highest carbohydrates content (99%), with a molecular weight estimated about 1,000 kDa (Table 1). Monosaccharide analysis revealed the presence of fructose/fucose/glucose composing the saccharide unit. NMR suggested a β-manno-pyranosidic configuration (Fig 1). Thermogravimetrical analysis (TGA) was performed using a Mettler-Toledo Star System apparatus, and a sample (5 mg) was heated rpm 30 to 400 °C at a heating rate of 20 °C min -1 under nitrogen. Measurements of viscosity () were performed with different EPS concentrations in aqueous solution at 30 °C using a Cannon-Ubbelohde 75 suspended-level viscosimeter.

Experimental setup

ATR-FTIRVertex 70V spectrometer (Bruker Optics) using Platinum diamond ATR was employed to collect spectra on EPS1-T14, fructose, fucose and glucose, in the temperatures range from 20° C to 80 °C. All the spectra were obtained by collecting 48 scans with a resolution of 4 cm -1 in the 4000 to 400 cm -1 wavenumber range. The following data pre-processing procedures were applied: i) a baseline correction, in order to diminish the dissimilarities between spectra due to baseline shift; ii) a smoothing treatment, to reduce the instrumental noise; iii) a first derivative treatment, to correct baseline shift together, and a second derivative treatment, to better discriminate features associated with spectra; iv) a spectral normalization, to correct the path-length variation and to reduce the differences among single measurements [START_REF] Al-Qadiri | Studying of the bacterial growth phases using fourier transform infrared spectroscopy and multivariate analysis[END_REF][START_REF] Davis | Fourier Transform Infrared (FT-IR) Spectroscopy: a rapid tool for detection and analysis of foodborne pathogenic bacteria[END_REF][START_REF] Gautam | Review of multidimensional data processing approaches for Raman and infrared spectroscopy[END_REF].

The intramolecular OH-stretching band frequency shift evaluation and the wavelet cross-correlation analysis were performed by MATLAB 2016a software (Mathworks, Natick, USA). The spectral distance calculation was performed by Origin 9 software package (OriginLab Co., Northampton, USA).

A C C E P T E D

M A N U S C R I P T 4 2.

Non ideal spectral deviation

In order to characterize the interaction mechanisms among the three major components of EPS1-T14, the difference between the experimental spectrum and the ideal spectrum of EPS1-T14, obtained as a weighed sum of the three component spectra, according to Eq. ( 1), was calculated using Eq. ( 2):

𝐼 𝑖𝑑𝑒𝑎𝑙 (𝜔) = 𝛼𝐼 𝑓𝑢𝑐𝑜𝑠𝑒 (𝜔) + 𝛽𝐼 𝑓𝑟𝑢𝑐𝑡𝑜𝑠𝑒 (𝜔) + 𝛾𝐼 𝑔𝑙𝑢𝑐𝑜𝑠𝑒 (𝜔) (1)
where 𝛼, 𝛽 and 𝛾 represent the weight factors of the three different components, i.e. fructose, fucose and glucose, respectively.

∆𝐼(𝜔) = 𝐼 𝑒𝑥𝑝𝑒𝑟𝑖𝑚𝑒𝑛𝑡𝑎𝑙 (𝜔) -𝐼 𝑖𝑑𝑒𝑎𝑙 (𝜔) (2)

Hypsochromic frequency shift

A first and the second-order derivative spectra treatments were employed to calculate the band center frequency [START_REF] Gallignani | Transmission FTIR derivative spectroscopy for estimation of furosemide in raw material and tablet dosage form[END_REF][START_REF] Lee | Determination of primary spectral bands for remote sensing of aquatic environments[END_REF]. The hypsochromic shift of the band maximum to higher frequencies was used to highlight the change of Hbond crosslinks among the structural components of EPS1-T14 at increasing temperature. The thermal properties of EPS1-T14 were evaluated by analyzing the OH stretching contribution shift.

Spectral Distance (SD)

To investigate the temperature-induced structural rearrangements of EPS1-T14 H-bond network, the Spectral Distance (SD) of the intramolecular OH stretching band contribution was calculated in the temperature interval (from 20 °C to 80 °C) using Eq. ( 3):

𝑆𝐷 = {∑ [𝐼(𝜔, 𝑇) -𝐼(𝜔, 𝑇 = 20°𝐶)] 2 Δ𝜔 𝑣 } 1 2 ⁄ ( 3
)
where 𝐼(𝜔) is the absorbance at frequency 𝜔 and Δ𝜔 is the instrumental frequency resolution.

In order to extract further quantitative information on the whole EPS1-T14 thermal stability, for the SD versus temperature data fit, the following Eq. ( 4) has been applied:

𝑆𝐷(𝑇) = 𝐴 (1 - 1 1+𝑒 -𝐵(𝑇-𝑇 0 ) ) + (𝐶 -𝐷𝑇) (4)
Where A represents the sigmoid amplitude, whose inverse A -1 is connected to the thermal restraint. B is the sigmoid steepness; T0 represents the temperature value of the sigmoid inflection point and, finally, C-DT takes into account the low temperature vibrational contribution.

Wavelet Cross-Correlation Analysis

In order to quantitatively evaluate the correlation degree among the registered spectra at different scales, a Wavelet Cross-Correlation (XWT) analysis between the different couples of spectra was performed [START_REF] Caccamo | Multiscaling Wavelet analysis of Infrared and Raman data on polyethylene glycol 1000 aqueous solutions[END_REF][START_REF] Caccamo | Tagging the oligomer-to-polymer crossover on EG and PEGs by infrared and Raman spectroscopies and by wavelet cross-correlation spectral analysis[END_REF][START_REF] Grinsted | Application of the cross wavelet transform and wavelet coherence to geophysical time series[END_REF][START_REF] Li | Wavelet cross-correlation analysis and its application to a plane turbulent jet[END_REF][START_REF] Magazù | Investigations of homologous disaccharides by Elastic Incoherent Neutron Scattering and Wavelet Multiresolution Analysis[END_REF][START_REF] Veleda | Cross-Wavelet bias corrected by normalizing scales[END_REF].

A C C E P T E D M A N U S C R I P T 5

Results and discussion

Attenuated Total Reflectance Fourier Transform Infra-Red (ATR-FTIR) spectroscopy

The spectra obtained by means of ATR-FTIR technique on EPS1-T14 and on its main components (i.e. fructose, fucose and glucose) (Fig. 2), according to the peak wavenumber assignment (Table 2), showed the following main features: i) an intense band in the region between 950 cm -1 and 1200 cm -1 , corresponding to the -C-O, C-CH stretching and to the -CO and C-O-C bending of the glycoside ring [START_REF] Coates | The interpretation of Infrared Spectra: published reference sources[END_REF]; ii) bands related to the C-CH, O-CH stretching and -CO stretching in the 1300 cm -1 -1450 cm -1 spectral region, [START_REF] Szymanski | Infrared Handbook[END_REF]; iii) a broad and intense band in the region between 2500 cm -1 and 3600 cm -1 , which is composed by several bands ascribed to the OH stretching of the polymer glycoside ring, and to the OH stretching of free water and of water involved in hydrogen bonds [START_REF] Magazù | Hydration and transport properties of aqueous solutions of α-α-trehalose[END_REF][START_REF] Pagnotta | Water structure around trehalose[END_REF].

The only detectable change in the reported spectra ( d). In detail, the spectrum of EPS1-T14 (fig. 2 a) in the region 1700-2000 cm -1 shows a lower thermal variation in respect with that of glucose (fig. 2 d), but similar to that of fructose (fig. 2 b) and fucose (fig. 2c). These observations demonstrate that the thermal restrain of EPS1-T14 mainly depends from fructose than glucose.

Non ideal spectral deviation

The EPS1-T14 ideal spectrum, reconstructed starting from the spectra of its main constituents, is shown in Fig. 3.

The spectral deviation, i.e. the difference between the experimental spectrum and the ideal spectrum is reported in Fig. 4, where marked deviations from the ideal behavior are registered in the whole investigated spectral range. Such a circumstance suggests that remarkable interactions take place among the constituents. In particular, although in the 1800cm -1 to 2800cm -1 spectral range the evaluated ideal spectrum is almost coincident to the experimental one, remarkable differences occur in the OH-stretching region, which is connected with the system H-bond network.

The non-ideal spectral deviation revealed that the real spectrum of EPS1-T14 is not the simple sum of its components, but it seems to acquire new properties and different responses at increasing temperature. This different thermal behavior could be ascribed to the interaction of its three main monosaccharidic components (i.e. fructose, fucose and glucose), that contribute differently to create the complex unique matrix of EPS1-T14.

Hypsochromic frequency shift

The 3D-FTIR spectra of EPS1-T14 in the 3000 <  < 3600 cm -1 range, as a function of temperature are reported in Fig. 5a. The EPS1-T14 OH stretching band frequency shift, in the temperature range, changes from 3271 cm -1 to 3313 cm -1 . Furthermore, this registered hypsochromic frequency shift changes linearly [START_REF] Spanò | A novel EPS-producing strain of Bacillus licheniformis isolated from a shallow vent off Panarea Island (Italy)[END_REF] with the increasing of the temperature, as shown in Fig. 5b. In order to fit the data the following Eq. ( 5) was employed:

𝜔(𝑇) = 𝐴 + 𝐵𝑇 (5)
The thermal restraint determined by hypsochromic frequency center shift was equal to 1.49.

The spectral analyses (hypsochromic frequency center shift and spectral distance) convey coherent information on the thermal evolution of EPS1-T14, both showing that the whole biopolymer was stable at the maximum value of the tested temperature (80 °C). The high resistance of the biopolymer at the increasing temperature could be explained by

A C C E P T E D M A N U S C R I P T 6
the increasing H-bounded connectivity. Since the frequency center linearly shifted with the increasing temperature, we may suppose that all the rheological properties (i.e. viscosity, density, etc) of EPS1-T14 could be maintained at 80 °C.

Spectral Distance (SD)

Spectral distance analysis of the spectra of EPS1-T14 at increasing temperatures showed a sigmoid trend, with an inflection point at T=50 °C (Fig. 6). The SD versus temperature behavior followed a logistic function trend allowing to extract quantitative information on the relaxation amplitude, as well as on its temperature localization.

Wavelet Cross-Correlation

The analysis by wavelet tools revealed different pathways of EPS1-T14 and its components (Fig. 7), where the shaded region indicates the Cone Of Influence (COI). This advanced investigation indicated that the spectrum of fucose is more closely correlated to that of the whole EPS1-T14, whereas glucose is the less correlated. This finding suggests that fucose plays a key role in the EPS1-T14 structure, and it may be also involved in the biological properties of the exopolysaccharide. The fucose-containing polysaccharides have an increased market value, because fucose is one of the rare sugars difficult to be obtained, and it is also involved in numerous biological activities, such as antioxidant, immune-stimulator and anti-aging agent [START_REF] Freitas | Advances in bacterial exopolysaccharides: from production to biotechnological applications[END_REF][START_REF] Cescutti | Structure of the exopolysaccharide produced by Enterobacter amnigenus[END_REF]. Fucoidan is the most well-known fucose-rich polysaccharide produced by brown seaweeds, and used in many human activities [START_REF] Li | Fucoidan: structure and bioactivity[END_REF]. Based on results previously obtained using TGA analysis, the decomposition temperature of EPS1-T14 (240 °C) appears higher than that of fucoidan (210 °C) [START_REF] Saravana | Structural, antioxidant, and emulsifying activities of fucoidan from Saccharina japonica using pressurized liquid extraction[END_REF]. For its thermal stability and thermal restraint, EPS1-T14 could be more cooking stable than other fucose polysaccharides, such as fucoidan, actually commercially available as nutritional supplements [START_REF] Moroney | Seaweed polysaccharides (laminarin and fucoidan) as functional ingredients in pork meat: an evaluation of anti-oxidative potential, thermal stability and bioaccessibility[END_REF]. However, to our knowledge, no data on the thermal behavior of fucoidan have been until now reported for further comparison.

The thermal characterization is also required in the drug deliveries system, a novel pharmaceutical field that allows to use lower doses of drugs and to develop drug or food carriers. Indeed, polymeric nanoparticles can be fabricated from polysaccharides, due to their low bio-toxicity and high adaptability [START_REF] Magazù | Investigations of homologous disaccharides by Elastic Incoherent Neutron Scattering and Wavelet Multiresolution Analysis[END_REF]. Sodium carboxymethyl cellulose [START_REF] Chang | Cellulose-based hydrogels: Present status and application prospects[END_REF], pectin [START_REF] Chittasupho | Pectin nanoparticle enhances cytotoxicity of methotrexate against hepG2 cells[END_REF], chitosan [START_REF] Pramanik | A novel drug "copper acetylacetonate" loaded in folic acid-tagged chitosan nanoparticle for efficient cancer cell targeting[END_REF] and xanthan gum [START_REF] Xu | Synthesis and characterization of nanoparticles based on negatively charged xanthan gum and lysozyme[END_REF] are among polysaccharides until now studied for delivering drugs, nutrients and bioactive substances. Nowadays, chitosan is the most characterized polysaccharide in the field of drug and food deliveries, however its uses are still limited, due to its relatively lower thermal stability (below 40 °C), as spectroscopically determined [START_REF] Szymańsk | Stability of chitosan a challenge for pharmaceutical and biomedical applications[END_REF]. For its physicochemical properties and thermo-stability EPS1-T14 could be also considered as useful biopolymer for nanoparticle composites.

Conclusion

The fructose-rich exopolysaccharide EPS1-T14, produced by the marine thermophilic Bacillus licheniformis T14, has been previously reported to possess interesting physicochemical properties and biological activities, as anticytotoxic, antiviral, immunostimulant, and antibiofilm agent, that make it potentially useful in different biotechnological applications.

Since thermostability represents a prerequisite in applicative areas that require high temperature processes, in this study the structural changes of EPS1-T14 at increasing temperatures have been monitored by using the ATR-FTIR spectroscopic technique, coupled with innovative tools (non-ideal spectral deviation, hypsocromic frequency center shift, spectral distance and wavelet cross correlation analysis). The thermal restraint analysis revealed that the whole EPS1-T14 system possessed high stability until 80 °C, and suggested that fucose was mainly involved in the EPS1-T14 thermal stability, whereas glucose was responsible for its molecular flexibility.

A C C E P T E D M A N U S C R I P T 7

Our findings provide new insights on the thermal stability of EPS1-T14, enabling us to consider it as a promising biopolymer useful for traditional biotechnological areas, including food, cosmetic and pharmaceutical applications, and novel biomedicine areas that require high-temperature processes, such as drug delivery system and nanoparticle composites.

A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T

10

Fig 2 )

 2 is relative to the temperature effects in the region around 3000-3500 cm -1 of EPS1-T14 (Fig 2a), and in the region around 1700-2000 cm -1 of fructose, fucose, glucose (Fig 2 b-

Fig. 1 .

 1 Fig.1.1 H-NMR (a) and13 C-NMR (b) spectra of EPS1-T14 from Bacillus licheniformis strain T14[START_REF] Spanò | A novel EPS-producing strain of Bacillus licheniformis isolated from a shallow vent off Panarea Island (Italy)[END_REF].

Fig. 2 .

 2 Fig. 2. ATR-FTIR spectra of whole EPS1-T14 a) and its main monosaccharidic components: b) fructose, c) fucose and d) glucose in the 400<∆<4000 cm -1 spectral range for positive thermal scans from 20 to 80 °C.

Fig. 3 .

 3 Fig.3. Ideal spectrum of EPS1-T14 reconstructed from the spectra of its main constituents, obtained using ATR-FTIR in the 400<∆<4000 cm -1 spectral range at 25 °C.

Fig. 4 .

 4 Fig. 4. Difference spectrum obtained by subtracting ideal spectrum to experimental spectrum of EPS1-T14.

Fig. 5 .

 5 Fig. 5. Hypsochromic frequency shift: a) 3D FTIR spectra of EPS1-T14 in the 3000 <  < 3600 cm -1 range, as a function of temperature and intensity, and b) the center frequency of OH stretching band of EPS1-T14, as a function of temperature. Black dots: experimental data.

Fig. 6 .

 6 Fig. 6. Spectral Distance of the intramolecular OH stretching bands of the EPS1-T14 at the increasing temperature.

Fig. 7 . 15 Fig. 3 .

 7153 Fig. 7. Wavelet Cross Correlation between the whole EPS1-T14 and a) fructose, b) fucose and c) glucose at 25 °C.

Fig. 4 . 17 Fig. 5 .Fig

 4175 Fig. 4.

Table 1

 1 Characteristics of EPS1-T14 from B. licheniformis strain T14[START_REF] Spanò | A novel EPS-producing strain of Bacillus licheniformis isolated from a shallow vent off Panarea Island (Italy)[END_REF].

		EPS1-T14
	Carbohydrate (%/v)	99
	Protein (%/v)	1.2
	Uronic acid (%/v)	0.5
	Molecular weight	1000 kDa
	Monosaccharide composition	Fructose/fucose /glucose/galactosamine/mannose
	(ratio of relative proportion)	(1.0:0.75:0.28:tr:tr)
	Saccharide repeating unit	Trisaccharide
	Anomeric configuration	β-manno pyranoside
	Specific viscosity	0.58η
	Thermogravimetrical analysis	240 °C
	Brine shrimp assay	Positive

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors

Conflict of interest

The authors declare that they have no conflict of interest.


C S Bianchini

A Di Renzo

T Lucioli

F Borgia

G Gianfreda

P Rossini
email: 6paolo.rossini@istc.cnr.it

Lexematic units and highly iconic structures in Italian Sign Language (LIS): new methods of analysis aided by ad hoc software

5) Università di Roma I Signs, both written or transcribed, are annotated using the SignWriting system (SW) of Sutton (1995), which allows to represent the manual components as well as the non manual components, thanks to a set of symbols called glyphs. This writing system has the advantage of being global and iconic, thus allowing an easy reading of the represented signs.

The notation system: SignWriting The annotation system: SignManager Given the need to annotate texts written in SW, an ad hoc software, called SignManager, has been developed by [START_REF] Borgia | SWift: SignWriting Improved Fast Transcriber[END_REF]; even in its early stages, this program allows to add tags to the images created in SW, and therefore easier ranging and analysis.

In this work, we make use of LIS texts coded in SignWriting (SW); we analyzed the typology, the stability and the variability of signed forms and the form-meaning correspondence. We examined 5 LIS narrative texts produced by 3 signing deaf people after they watched the same video (the "Pear Story" by [START_REF] Chafe | The Pear Stories. Cognitive, cultural, and linguistics aspects of narrative productions[END_REF]): 2 texts have been produced face-to-face and then transcribed; 3 texts have been composed directly in written form.

Materials and methods

The presence of such REs urges to investigate the constituent units in SL lexicon and their comparability to VL units. We will deal with this question, keeping in mind the lack of appropriate forms of written representation for these languages [START_REF] Antinoro Pizzuto | Deixis, anaphora and Higly Iconic Structures: Cross-linguistic evidenze on American (ASL), French (LSF), and Italian (LIS) Sing Languages: spinning and unravelling the past, present and future[END_REF], affecting all the research about LS lexicon carried out so far.

Aim of the study

Two classes of Referential Expressions (REs) can be identified in Sign Language (SL) discourse and, more specifically, in LIS. These units have been acknowledged by most researchers, but they are still named and classified in different ways; according to [START_REF] Cuxac | Emergence, norme et variation dans les langues des signes: vers une redéfinition notionnelle[END_REF] we call them: -Lexematic Unit (LU): the LU is somehow comparable to a "word" in verbal language (VL) and it is often included in SL dictionaries;

-Highly Iconic Structure (HIS): HIS forms a complex class of REs, recurring very often in signed discourse; HIS is formed by strongly iconic elements; some researchers consider it a nonlinguistic or a partially-linguistic element therefore they claim that HIS cannot be included in dictionaries; one important consequence of this lexicographic decision is that SL dictionaries contains limited amount of items. We wish to thank Dr. Giulia Petitta for her suggestions.

The "to look" action might be expressed by manual and non manual components, the last ones "rappresenting iconically the eye-gaze of entity simbolized" (Antinoro Pizzuto, 2009: 145)

Signed history videotaped

Aided by the SignManager [START_REF] Borgia | SWift: SignWriting Improved Fast Transcriber[END_REF], we identified the expressive forms used for a common set of entities and actions mentioned in the texts. Eight of the more frequent expressions in the texts (man, child, tree, pear, basket, bicycle, to move, and to look) were chosen for more detailed analysis.

In this presentation we focus the analysis of the action "to look".

Transcription (using SW)

The results show recurring patterns in the identified expressive forms including both LU and HIS. Referring to the same units of meaning, HISs were used more frequently than LUs, in narrative texts. Contrary to the conclusions of most papers, we observed strong regularities among the HIS elements, and among the composition choices. In addition, HIS recurring elements were more heavily contextualized than the LUs. This finding suggests the need to include these elements among the constituent units of LIS.

Overall, results suggest the need to deeply review a model of LIS lexicon, now conceived on the basis of VL lexicon; it also suggests the need to identify more appropriate forms in order to take into account the particular iconic and multilinear SL traits.

In the future, the SignManager instrument should be improved for better performance and for integrated data management with traditional annotation softwares (i.e. Elan, Anvil…)

Coll. "Formal and experimental advances in sign language theory" (FEAST), Venezia 20-22/06/11 1 C.S. Bianchini 1 *°^, A. Di Renzo 2 *, T. Lucioli 3 *, F. Borgia 4+# , G. Gianfreda 5 *, P. Rossini 6 *

LEXEMATIC UNITS AND HIGHLY ICONIC STRUCTURES IN ITALIAN SIGN LANGUAGE: NEW METHODS OF ANALYSIS AIDED BY AD HOC SOFTWARE

Two classes of Referential Expressions (RE) can be identified in Italian Sign Language (LIS) discourse and in Sign Language (SL) discourse generally: according to [START_REF] Cuxac | La Langue des Signes Française; les Voies de l'Iconicité[END_REF] and [START_REF] Cuxac | Emergence, norme et variation dans les langues des signes: vers une redéfinition notionnelle[END_REF] they are named Lexematic Unit (LU) and Highly Iconic Structures (HIS). These units have been acknowledged by the whole academic community, but they are still named and classified in different ways. The LU are somehow comparable to "words" in Verbal Language (VL) and they are often included in SL dictionaries; on the other hand, HIS form a more complex class of RE, recurring very often in signed discourse (30% to 70% of recognizable RE, see [START_REF] Russo | Iconicity and productivity in sign language discourse: an analysis of three LIS discourse registers[END_REF][START_REF] Sallandre | Les unités du discourse en Langue des Signes Française (LSF) -Tentative de categorisation dans le cadre d'une grammaire de l'iconicité[END_REF][START_REF] Antinoro Pizzuto | Deixis, anaphora and Higly Iconic Structures: Cross-linguistic evidenze on American (ASL), French (LSF), and Italian (LIS) Sing Languages: spinning and unravelling the past, present and future[END_REF]. HIS are formed by strongly iconical elements, many academics consider them non-linguistic or partially-linguistic elements therefore they claim HIS can't be included in dictionaries (e.g. see [START_REF] Johnston | Corpus linguistics and signed languages: no lemmata, no corpus[END_REF]. One important consequence of this lexicographic decision is the seeming "poorness" of SL dictionaries, which contain from 2500 up to 5000 terms on average.

The presence of such ER urges to investigate about the constituent units in SL lexicon and about their comparability to VL units. We will deal with this interrogative, keeping in mind the lack of appropriate forms of written representation for these languages (see [START_REF] Russo | Un lessico di frequenza della LIS[END_REF][START_REF] Antinoro Pizzuto | Deixis, anaphora and Higly Iconic Structures: Cross-linguistic evidenze on American (ASL), French (LSF), and Italian (LIS) Sing Languages: spinning and unravelling the past, present and future[END_REF], affecting all the research about LS lexicon carried out so far. Experiences conducted with competent LIS signers has shown that SignWriting (SW), a SL representation system developed by Sutton years ago (1999), but little used in research, could be effective tool to transcribe signed face-to-face productions (video recorded data) and to compose texts conceived in written LIS [START_REF] Di Renzo | Italian Sign Language: can we write and transcribe it with Sign Writing[END_REF][START_REF] Gianfreda | Dalla modalità faccia-a-faccia ad una lingua scritta emergente: nuove prospettive su trascrizione e scrittura della Lingua dei Segni Italiana (LIS)[END_REF].

In this work, we make use of LIS texts coded in SW; we analyzed the typology, the stability and the variability of signed forms and the form-meaning correspondence. We examined 5 LIS narrative texts produced (at different times) by 3 signing deaf people after they watched the same video (the "Pear Story" by Chafe, 1980): 2 texts have been produced face-to-face and then transcribed and 3 texts have been composed directly in written form. Aided by the SignManager (v. 1.1), an annotation software developed ad hoc for SW text analysis [START_REF] Borgia | SWift: SignWriting Improved Fast Transcriber[END_REF], we identified the expressive forms used for a common set of entities (animate and inanimate) and actions mentioned in the texts. Eight out of the more frequent expressions in the 5 texts (more specifically: man, child, tree, pear, basket, bicycle, to move, and to look) were chosen for more detailed analysis.

Together with the LIS signers who produced the texts we investigated (starting from the SW representation in their text) the nature of the chosen expressions, later we discussed about the eventual citation forms, analyzing the similarities and the differences between the latter and the expression produced in the text.

The results show recurring patterns in the identified expressive forms including both LU and HIS. Referring to the same units of meaning HIS were used more frequently than LU. Unlike the conclusions of most research, we observed strong regularities among the HIS elements, and among the composition choices. In addition HIS recurring elements were more heavily contextualized than the LU. This finding suggests the need to include these elements among the LIS constituent units.

Overall, the results suggest the need to deeply review the modeling of the LIS lexicon (and SL in general) based on forms conceived for the VL, it also suggests the need to identify more appropriate forms in order to take into account the particular iconic and multilinear SL traits.

  E., Rossini, P., Sallandre, M.-A. Wilkinson, E. (2008), Deixis, anaphora and Highly Iconic Structures. In TISLR9 (online): 475-495. Antinoro Pizzuto, E. (2009). Meccanismi di coesione testuale e SGI nella LIS e altre LS. In C.Bertone & A.Cardinaletti (eds.), Alcuni capitoli della grammatica della LIS. Venezia: Cafoscarina,: 137-158, Borgia, F. (2010). Swift: SignWriting Improved Fast Transcriber. Tesi, Uni. Roma "La Sapienza". Chafe, W. (1980). The Pear Stories. Norwood, Ablex Pub. Corp. Cuxac, C., Antinoro Pizzuto, E. (2010), Emergence, norme et variation dans les LS. In B.Garcia & M.Derycke (eds.), Sourds et LS, "Langage et Societé", n.131 (3/2010): 37-53. Sallandre, M.-A. (2003), Les unités du discourse en LSF. Thèse, Université Paris 8. Sutton, V. (1995). Lessons in SignWriting. La Jolla, DAC. This work has been partially funded by the projects MIUR-FIRB VISEL (RBNE074T5L) (2009-2012) and DEAL-TOI (CEE Leonardo) (2009-2010).