Jorune Sakalauskaite
email: jorune.sakalauskaite@unito.it

Laurent Plasseraud

Jérôme Thomas

Marie Alberic

Mathieu Thoury

Jonathan Perrin

Frédéric Jamme

Cédric Broussard

Beatrice Demarchi

Frédéric Marin
email: frédéricmarin@u-bourgogne.fr

Marie Albéric

Affiliations

à la diffusion de documents scientifiques de niveau

Keywords: Biomineralization, Shell biochemistry, evolution, proteomics, liquid chromatography-tandem mass spectrometry

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

The shell matrix of the european thorny oyster, Spondylus gaederopus: microstructural and molecular characterization.

Introduction

Biomineralization is a biologically controlled process by which marine living systems precipitate mineral salts, predominantly calcium carbonate. In metazoans, this process emerged during the Precambrian-Cambrian transition in multiple lineages. Among these, molluscs represent one of the most diversified phyla, both from the morphological and ecological point of view. Their ability to biomineralize an exoskeleton, the shell, represented an innovative strategy in terms of support and protection for internal soft body parts, against predation and desiccation and as a reserve of calcium ions [START_REF] Kocot | Sea shell diversity and rapidly evolving secretomes: insights into the evolution of biomineralization[END_REF], explaining their evolutionary success in marine, freshwater and terrestrial environments. The biomineralization of the shell is controlled by a specialized organ, the mantle, a thin polarized epithelium that extrudes inorganic ions (mostly calcium and bicarbonate) via ion pumps and channels, and, at the same time, secretes a macromolecular matrix [START_REF] Marin | The formation and mineralization of mollusk shell[END_REF]. Both inorganic and organic components assemble in well defined microstructures at the nano-and microscales [START_REF] Marin | Shellome": Proteins involved in mollusk shell biomineralization-diversity, functions[END_REF] resulting in a diversity of micro-architecturesthe most common being the crossed-lamellar, foliated, prismatic and nacre (mother-of-pearl) [START_REF] Carter | Skeletal Biomineralization: Pattern, Processes, and Evolutionary Trends[END_REF][START_REF] Taylor | The shell structure and mineralogy of the Bivalvia. II. Lucinacea-Clavagellacea, Conclusions[END_REF][START_REF] Taylor | The shell structure and mineralogy of Bivalvia: introduction, Nuculacea-Trigonacea[END_REF].

The precise molecular mechanisms controlling shell biomineralization are still poorly understood. However, extensive research on the physico-chemical properties of calciumbased biominerals [START_REF] Lowenstam | Minerals formed by organisms[END_REF] and the biochemistry of several model organisms [START_REF] Addadi | A pavement of pearl[END_REF][START_REF] Evans | Tuning in" to mollusk shell nacreand prismatic-associated protein terminal sequences. Implications for biomineralization and the construction of high performance inorganic-organic composites[END_REF][START_REF] Kocot | Sea shell diversity and rapidly evolving secretomes: insights into the evolution of biomineralization[END_REF][START_REF] Marin | Shellome": Proteins involved in mollusk shell biomineralization-diversity, functions[END_REF], have shown that the secreted organic matrix, (i.e. a set of proteins, glycoproteins, polysaccharides and lipids), plays a crucial role in the mediation of crystal nucleation and growth [START_REF] Addadi | Mollusk shell formation: a source of new concepts for understanding biomineralization processes[END_REF][START_REF] Falini | Control of aragonite or calcite polymorphism by mollusk shell macromolecules[END_REF][START_REF] Jackson | Parallel evolution of nacre building gene sets in molluscs[END_REF][START_REF] Marin | Shellome": Proteins involved in mollusk shell biomineralization-diversity, functions[END_REF]. Very important components are the shell matrix proteins (referred to as SMPs), which get occluded inside the shell mineral during calcification. An increasing number of SMPs have been identified in the past decade using proteomics, transcriptomics and, to a lesser extent, genomics, which allowed researchers to identify and compare the shell proteomes ('shellomes') of different taxa. Most of these studies focused on mother-of-pearl shells [START_REF] Marie | Deep conservation of bivalve nacre proteins highlighted by shell matrix proteomics of the Unionoida Elliptio complanata and Villosa lienosa[END_REF](Marie et al., , 2013a;;[START_REF] Ramos-Silva | Novel molluskan biomineralization proteins retrieved from proteomics: a case study with Upsalin[END_REF][START_REF] Yarra | Characterization of the mantle transcriptome in bivalves: Pecten maximus, Mytilus edulis and Crassostrea gigas[END_REF], shedding light on some characteristics of shell proteins [START_REF] Arivalagan | Insights from the Shell Proteome: Biomineralization to Adaptation[END_REF][START_REF] Sleight | An Antarctic molluscan biomineralisation tool-kit[END_REF]. One interesting feature, is the widespread occurrence of low complexity domains/ repetitive low complexity domains (LCDs/RLCDs) [START_REF] Kocot | Sea shell diversity and rapidly evolving secretomes: insights into the evolution of biomineralization[END_REF][START_REF] Marin | Organic matrices in metazoan calcium carbonate skeletons: Composition, functions, evolution[END_REF][START_REF] Marin | Synthesis of calcium carbonate biological materials: how many proteins are needed?[END_REF]) -these were identified in proteins associated with nacre [START_REF] Jackson | Parallel evolution of nacre building gene sets in molluscs[END_REF][START_REF] Mcdougall | Rapid evolution of pearl oyster shell matrix proteins with repetitive, low-complexity domains[END_REF][START_REF] Sakalauskaite | Palaeoshellomics" reveals the use of freshwater motherof-pearl in prehistory[END_REF] but were also found in shell proteomes associated with other microstructures (Marie et al., 2013a), suggesting that these domains likely play a role in mineral binding and in providing flexibility [START_REF] Sarashina | Skeletal matrix proteins of invertebrate animals: Comparative analysis of their amino acid sequences[END_REF]. However, most importantly, 'shellomics' has also exposed the diversity of the SMP repertoires between shells that have similar microstructures or species that are phylogenetically close [START_REF] Jackson | Parallel evolution of nacre building gene sets in molluscs[END_REF]Marie et al., 2009aMarie et al., , 2009b)). This suggests that in shell forming proteins, beside a group of shared conserved functions, further adoptions of numerous lineage-dependent functions may have also occurred. In other words, independent evolution of SMPs may have occurred multiple times in different lineages.

Whilst there are numerous shell proteome studies of nacreous molluscs [START_REF] Gao | Layer-by-Layer Proteomic Analysis of Mytilus galloprovincialis Shell[END_REF][START_REF] Jackson | Parallel evolution of nacre building gene sets in molluscs[END_REF][START_REF] Marie | Deep conservation of bivalve nacre proteins highlighted by shell matrix proteomics of the Unionoida Elliptio complanata and Villosa lienosa[END_REF][START_REF] Marie | Different secretory repertoires control the biomineralization processes of prism and nacre deposition of the pearl oyster shell[END_REF][START_REF] Marie | Molecular evolution of mollusc shell proteins: insights from proteomic analysis of the edible mussel Mytilus[END_REF]Marie et al., , 2009a;;[START_REF] Marin | Caspartin and calprismin, two proteins of the shell calcitic prisms of the Mediterranean fan mussel Pinna nobilis[END_REF], only few studies have been performed on other shell microstructures, such as foliated [START_REF] Yarra | Characterization of the mantle transcriptome in bivalves: Pecten maximus, Mytilus edulis and Crassostrea gigas[END_REF] and cross-lamellar [START_REF] Osuna-Mascaró | The shell organic matrix of the crossed lamellar queen conch shell (Strombus gigas)[END_REF]. This can be explained by the fact that nacre displays remarkable mechanical properties, which make it a material of choice for biomimetics [START_REF] Espinosa | Merger of structure and material in nacre and bone -Perspectives on de novo biomimetic materials[END_REF][START_REF] Finnemore | Biomimetic layer-by-layer assembly of artificial nacre[END_REF][START_REF] Luz | Biomimetic design of materials and biomaterials inspired by the structure of nacre[END_REF], as well as to the commercial importance of nacreous molluscs, especially the pearl oyster and the edible mussel [START_REF] Gosling | Bivalve Molluscs -Biology, Ecology and Culture, Fishing New Books[END_REF]Watabe et al., 2011). In addition, nacre is considered to be an ancestral microstructural type and has been studied from an evolutionary point of view [START_REF] Jackson | Parallel evolution of nacre building gene sets in molluscs[END_REF]Marie et al., 2009a). On the contrary, the molecular information on the SMPs of crossed-lamellar shells is scarce [START_REF] Agbaje | Biomacromolecules in bivalve shells with crossed lamellar architecture[END_REF], despite that it is the most common microstructure in molluscs [START_REF] Carter | Skeletal Biomineralization: Pattern, Processes, and Evolutionary Trends[END_REF][START_REF] Wilmot | Electron microscopy of molluscan crossed-lamellar microstructure[END_REF] and possesses high fracture toughness [START_REF] Kamat | Structural basis for the fracture toughness of the shell of the conch Strombus gigas[END_REF].

The thorny oyster Spondylus gaederopus Linnaeus, 1758 represents a typical example of this bias in "shellomic" studies. It is a member of a small family of pteriomorphid bivalves, the Spondylidae. S. gaederopus is endemic to the Mediterranean sea, with a fixosessil life in water depths up to 50 m. The origin and evolution of spondylids is not well understood [START_REF] Yonge | Functional morphology with particular reference to hinge and ligament in Spondylus and Plicatula and a discussion on relations within the superfamily Pertinacea (Mollusca: Bivalvia)[END_REF]. Based on morphology and microstructure, the Spondylidae family was thought to be derived from a Pectinid ancestor in the middle Jurassic, possibly from the genus Spondylopecten [START_REF] Logan | Morphology and Life Habits of the Recent Cementing Bivalve Spondylus Americanus Hermann from the Bermuda Platform[END_REF][START_REF] Waller | Phylogeny of families in the Pectinoidea (Mollusca: Bivalvia): importance of the fossil record[END_REF]. This evolutionary process may have been driven by the increase of shell-crushing predators, which likely led to the development of more complex skeletal frameworks with improved density and strength [START_REF] Knoll | Biomineralization and Evolutionary History[END_REF]. Conversely, genetics studies suggest that Spondylidae is a sister group of the Pectinidae [START_REF] Barucca | Molecular phylogeny of the family Pectinidae (Mollusca: Bivalvia) based on mitochondrial 16S and 12S rRNA genes[END_REF][START_REF] Matsumoto | Phylogenetic analysis of the subclass Pteriomorphia (Bivalvia) from mtDNA COI sequences[END_REF][START_REF] Matsumoto | Phylogenetic analysis of the familyPectinidae (Bivalvia) based on mitochondrial cytochrome c oxidase subunit I[END_REF], albeit this issue has not been addressed thoroughly, since many of the molecular phylogenetic analyses use Spondylus as an outgroup to reconstruct the Pectinid family [START_REF] Barucca | Molecular phylogeny of the family Pectinidae (Mollusca: Bivalvia) based on mitochondrial 16S and 12S rRNA genes[END_REF][START_REF] Puslednik | Molecular phylogenetics of the Pectinidae (Mollusca: Bivalvia) and effect of increased taxon sampling and outgroup selection on tree topology[END_REF]. Considering that Pectinidae shells are mostly foliated (calcitic), whereas Spondylus is mostly crossed-lamellar (aragonitic), the question of evolution is puzzling and requires a careful reexamination.

Finally, Spondylus is of particular importance in the field of archaeology and has been regarded as an iconic shell in prehistoric times: its presence is well documented in the archaeological record and can be tracked all over Europe during the Neolithic period (Borrello andMicheli, 2011, 2004;[START_REF] Ifantidis | Spondylus in Prehistory: New Data and Approaches: Contributions to the Archaeology of Shell Technologies[END_REF][START_REF] Moshkovitz | Neolithic Spondylus[END_REF][START_REF] Shackleton | Strontium isotope dating of the source of Neolithic European Spondylus shell artefacts[END_REF][START_REF] Windler | From the Aegean Sea to the Parisian Basin: Spondylus shell exchange in Europe during the process of Neolithisation[END_REF], as well as in Pre-Columbian South America [START_REF] Paulsen | The Thorny Oyster and the Voice of God: Spondylus and Strombus in Andean Prehistory[END_REF][START_REF] Pillsbury | The Thorny Oyster and the Origins of Empire: Implications of Recently Uncovered Spondylus Imagery from Chan Chan, Peru[END_REF]. The possession of Spondylus was probably an indicator of wealth or high status, and therefore reified important ritual/symbolic messages.

In this context, our study has a dual goal: 1) characterizing the shell microstructure of Spondylus gaederopus and the molecular properties of the associated organic matrix, followed by an in-depth proteomics study; 2) establishing a biomolecular dataset for the Spondylus shell matrix, which will constitute a reference in future studies to identify the biological origin of fragments of prehistoric shell ornaments, where morphological identification is unattainable [START_REF] Demarchi | An integrated approach to the taxonomic identification of prehistoric shell ornaments[END_REF][START_REF] Sakalauskaite | Palaeoshellomics" reveals the use of freshwater motherof-pearl in prehistory[END_REF], taking into account the diagenetic processes.

Results

The microstructure of S. gaederopus shell

The shell of S. gaederopus displays a very distinctive appearance: characteristic hollow spines, a deep purple upper valve and a completely white lower valve (Fig. 1). When observed in cross-section (Fig. 2), it displays a complex microstructure that includes at least 3 different layers: prismatic (P), crossed-lamellar (CL) and foliated (F). At the basis of the shell spines, the microstructure is complex and irregular, but becomes crossed-foliated (calcitic CF) at later stages of spine growth (Fig. 2a,b). The outermost layer of the shell is calcitic foliated (F), similarly to what can be observed in pectinid and ostreid shells (Fig. 2c). The layer below is aragonitic crossed-lamellar (CL) and exhibits the typical oblique pattern that characterizes this microstructure (Fig. 2d,e). This layer is traversed by a thin prismatic (aragonitic) myostracal layer. The inner and uppermost CL layers, when viewed in cross-section, have different lamellar orientations. The crossed-lamellar layer dominates the hinge area and is very thick at the central part of the shell, but at the margin, the proportion changes, and the foliated becomes the dominant microstructure. The innermost layer is composed of thin aragonitic prisms, about 15-20 μm in diameter, that exhibit a fine layering perpendicular to the elongation axis.

We note that while the changes between the different underlying aragonitic microstructures are gradual, the transition between the aragonitic crossed-lamellar and the foliated calcitic structures is abrupt and well marked (Fig. 2c). This microstructural and mineralogical transition zone was also studied by UV photoluminescence imaging. The analysis was carried out at the DISCO Beamline (SOLEIL synchrotron, Paris) using an excitation wavelength of 275 nm. The spatial distribution of shell organics, was studied using a series of emission filters to detect the luminescence patterns. Fluorescence signal was detected in several different emission regions (327-353; 370-400; 420-480; 499-529), however, was the strongest in the regions of 456-481 and 535-607 nm (Fig. 3). An intense luminescenting layer was observed at the interface between the aragonitic crossed-lamellar and the foliated calcitic microstructures, likely arising due to the presence of organics (however exact fluorescent components are unknown). This indicates the existence of intercrystalline organic layer (OL) in the transitional zone between the two layers of different polymorphs. Extraction of the shell organic matrix

The organic matrices were extracted according to standard protocols (Marie et al., 2013b) from finely ground shell powders, which were prepared in different ways. First of all, shell valves were cleaned to remove any surface contamination (mechanically and by bleaching in NaOCl for 2 hrs, see Material and Methods for more details). Following this, we separated the coarsely crushed powders into subsamples from the upper (UV) red and lower (LV) white valves. To these, two different bleaching approaches (varying in intensity) were applied for both subsamples: a) "Mild: approach: coarsely crushed shell powders were bleached for 4 hrs (hereafter "2BL extracts"); these were further ground in fine powders (S1, S2, S3, S4). b) "Harsh" approach: obtained 2BL fine powders were bleached for extra 14 hrs (hereafter " 3BL extracts") (S5, S6,S7, S8). In total, eight shell matrices were obtained -four acid soluble (ASM) and four acid insoluble (AIM) organic fractions (Fig. 4; see also SI.1). After the 2BL treatment, the obtained organics represent ~1.5 -2 wt‰ (note: per mille) of the total shell powder; more than two thirds of this matrix is the AIM. The 14-hrs additional bleaching step resulted in a severe reduction in the amount of ASM (from ~0.5 wt‰ to ~0.04 wt‰) and in an even more drastic loss of AIM (from ~1.6 wt‰ to ~0.005 wt‰). We note that for both bleaching treatments, the upper valve yielded a slightly higher amount of matrix.

Figure 4: The scheme represents different approaches (bleaching pretreatments) by which shell organic matrix extracts were obtained (S1 -S8) and their subsequent yields. After initial bleach to remove superficial contaminants, all shell powders were bleached twice (2BL) or thrice (3BL). Bleaching procedure: 2BL -coarse powder bleached for 4 hrs; 3BL -fine powder bleached for additional 14 hrs. UV -upper valve, LV -lower valve. ASM -acid soluble matrix, AIM -acid insoluble matrix.

FT-IR analysis of organic matrices FTIR spectroscopy was used to characterize qualitatively the presence of functional groups in the shell organic matrix extracts. Due to low quantities of the 3BL matrices, the characterization was applied solely on the four 2BL extracts (Fig. 5). Distinctly, all samples exhibit absorption bands characteristic of proteins: the broad band between 3300 and 3260 cm -1 corresponding to amide A stretchings (𝝂N-H), the amide I band (𝝂C=O) at around 1640-1650 cm -1 and the amide II bands (𝝂C-N) found between 1510-1529 cm -1 . In addition, the four spectra present also 𝝂(C-O) absorption bands specific to carbohydrates, at around 1070 cm -1 , and another at around 1405-1460 cm -1 which can be related to carboxylate groups [𝝂 s (COO -)]; the latter appears clear in the two ASMs, while in the AIMs it is shifted towards higher wavenumbers and overlaps with another strong band at 1446-1460 cm -1 , only detected in the AIMs. A weak absorption band found in the four extracts at around 1227-1240 cm -1 can be assigned to the 𝝂S=O vibration and indicate the presence of sulphate groups [START_REF] Takeuchi | Biochemical characterization of the skeletal matrix of the massive coral, Porites australiensis -The saccharide moieties and their localization[END_REF]. Moreover, we note that the two ASM spectra (S1, S3) are almost completely superimposable. They mainly differ from the two AIMs (S2, S4) by the amide I and amide II band intensity ratios. The two AIMs spectra are also very similar, in particular exhibiting two close bands at 700-712 cm -1 associated with a more intense band at 854 cm -1 . These bands, including the intense peak at 1446-1460 cm -1 , slightly shifted from 1477 cm -1 , are characteristic of the aragonite fingerprint (internal vibration modes of CO 3 2-), suggesting that some mineral crystallites remain undissolved. In addition, the AIMs are characterized by the presence of 𝝂(C-H) stretching vibrations at around 2920-2852 cm -1 whereas in ASMs this signal is weaker and shifted to 3072-2943 cm -1 .

Figure 5: FT-IR (ATR) spectra of shell organic matrices collected in the range of 4000 -400 cm -1 : a,b) S1, S2 -ASM and AIM extracts from the shell upper valve; c,d) S3, S4 -ASM and AIM extracts from the shell lower valve. Peaks discussed in the text are marked in colour which correspond to: i) proteins; ii) organics rich in hydrocarbon groups; iii) glycosides; iv) sulphates; v) aragonite. ASM -acid soluble matrix, AIM -acid insoluble matrix.

SDS-PAGE

Figure 6: SDS-PAGE gels of Spondylus shell organic matrices: a) stained using silver staining; b) stained using Stains-all staining. Analysed samples are as indicated in Figure 3; STDstandard molecular weight markers.

All organic shell extracts were analysed by SDS-PAGE, and stained with silver nitrate and Stains-all (Fig. 6), except sample S8, which yielded a very low quantity of extract and was used only for proteomic analyses. Note that the AIM extracts correspond to the Laemmlisoluble AIMs (LS-AIMs). The silver stained gel (Fig. 6a) shows that all samples have a peculiar migration pattern, characterized by a smear from high to low molecular weights. There are no significant differences between the migration profiles from the upper (S1/2/5/6) and lower valves (S3/4/7/8); the 2BL and 3BL cleaning approaches (S1-4 and S5-7 respectively) produced similar patterns. In spite of the smear, several discrete diffuse bands can be observed in the intermediate molecular weight range: i) a set of three diffuse bands around 20, 17 and 13 kDa in all of the samples; ii) bands in the 70 -30 kDa range are shared by most of the samples. In the low molecular weight range, a thick band (<11 kDa) is strongly stained brown in only half of the samples (S2/4/6/7) which migrated faster than the migration front, suggesting the occurrence of very low molecular weight compounds. At last, around 11 kDa, a relatively sharp negatively-stained band is present in all samples. Stains-all (Fig. 6b) allows the detection of putative Ca 2+ binding proteins (metachromatic blue), but is also efficient in staining acidic proteins; in summary it stains sialoglycoproteins in blue, Ca 2+ binding proteins in deep blue to violet, proteins in red, and lipids in yellow-orange. All our preparations stained violet at different degrees of intensity, in contrast to the standards, which stained pink. Considering the two cleaning approaches (2BL vs 3BL), Stains-all shows that the signal is far less intense in the 3BL samples (S5-7). Contrary to silver staining, Stains-all does not reveal the blurred bands in the 70 -11 kDa range. However, it stains intensely violet in all lanes the sharp band at 11 kDa, which is precisely the one negatively stained with silver. We also find low molecular weight components (<11 kDa stained band) in all of the ASMs (S1/3/5/7), while very high molecular weight (>170 kDa) components are present in the AIMs (S2/4/6).

ELLA

Figure 7: Enzyme-linked lectin assay (ELLA) on two ASMs extracted from the upper (S1) and lower (S3) valves. ELLA was performed with 21 lectins, absorbance values at 405 nm were normalized to the highest value (Jacalin) corresponding to 100% reactivity (n = 4, means ± S.D). 21 lectins were tested for their affinity to saccharidic residues present in soluble shell matrices. The analysis was performed on two ASM samples extracted after 2BL treatments from the upper (S1) and lower (S3) valves and the resulting intensity of cross-reactivity is expressed as a percentage, the most reactive lectin giving 100% (Fig. 7). Jacalin was found to be the most reactive lectin with both extracts. This lectin binds preferentially D-galactose and oligosaccharides terminating with this sugar. It also targets the galactose-β(1-3)Nacetylgalactosamine dimer, and consequently, is specific of O-linked glycoproteins. A group of four lectins including concavalin A (conA), wheat germ agglutinin (WGA), Datura stramonium lectin (jimsonweed, DSL) and tomato lectin (Lycopersicon, LEL) gave reactivities between 60 and 40%. ConA is a mannose-binding lectin of N-linked glycopeptides, while WGA, DSL and LEL bind monomers to oligomers of N-acetylglucosamine, and thus, are usually considered as chitin-binding lectins. A second group of 6 lectins, including DBA, LCA, ECL, PHA-L, PNA and STL gave moderate reactivities, between 40 and 20%. DBA binds to N-acetyl-D-galactosamine, LCA, to complex α-linked mannose-containing sequences, ECL, to N-acetyllactosamine (dimers of β-galactose and N-acetylglucosamine), PHA-L, to N-linked oligosaccharides containing galactose, mannose and N-acetyl-glucosamine, PNA, to lactose (dimer of glucose and galactose), and finally, STL, to oligomers of N-acetylglucosamine (chitin-binding). The other ten lectins were considered as unreactive with the extracts (signals below 20%). The two soluble matrices exhibited very similar reactivity profiles with all the lectins, with the exception of ECL, DBA, PNA and PHA-I. In general, we noticed that in all of the cases S1 gave higher intensity than S3. The crystallization assay was used to check in which manner Spondylus ASMs (samples S1 and S3, obtained from the upper and lower valves of the shell respectively) interact with the formation of calcite crystals grown in vitro. Only the ASM fractions were tested due to the insolubility of their AIM counterparts and the results were obtained at concentrations of 0.5, 2, 4, 8 and 16 µg•mL -1 of each extract (Fig. 8). In blank conditions (no matrix), calcite crystals were typically rhombohedral with flat surfaces. The first signs of crystal shape modification could be observed already at low concentrations of ASMs (0.5 µg•mL -1 ), where aggregates were obtained. With both ASM extracts, the effects became more pronounced at 2 and 4 µg•mL -1 concentrations, at which crystals developed rounded edges. At higher concentrations (8 µg•mL -1 and above), crystal morphologies were completely altered. At 16 µg•mL -1 drop-shaped forms were produced, from the dense packing of numerous microcrystals. Overall, both ASMs exert similar effects on the crystallization of CaCO 3 and this effect is well pronounced already at low matrix concentrations.

In vitro CaCO3 crystallization assay

In situ lectin-gold localization of sugar moieties Figure 9: Lectin-gold in situ localization assay on Spondylus gaederopus shell sections. a) Western blot on Spondylus shell organic matrices (S1-4); the membrane was incubated with lectin Jacalin. b-g) In situ lectin-gold localization of the sugar moieties to which Jacalin binds in different zones. Images b) and f) corresponds to negative controls. The localizations were carried out on small polished sections, sampled in the near-margin region of the shell. Ffoliated calcite layer; CL -aragonitic crossed-lamellar; OL -organic layer.

Lectin-gold in situ tests were carried out to directly localize glycans that bind Jacalin along the shell structure. This lectin previously showed the strongest signal in ELLA assay and indicated the presence of galactose/N-acetylgalactosamine residues and/or oligosaccharides terminating with D-galactose. In situ localization experiments were performed on small fragments that were sampled in the region close to the margin, where foliated and crossedlamellar layers are in contact. Jacalin was also tested on Western blots to check its binding specificity to shell matrix components (Fig. 9a). We found that it reacted mostly with a thick discrete band located around 30-34 kDa in the two tested ASMs and it gave a strong, although polydisperse, signal at very high molecular weights in the two tested LS-AIMs. The experiments showed that Jacalin binds to the sugars groups (possibly glycoproteins) present only in the upper, foliated calcite layer (Fig. 9c) and not in the aragonitic cross-lamellar structure. A very intense signal is observed at the interface between the crossed-lamellar (CL) and foliated (F) microstructures, (Fig. 9g) and where irregular microstructure transits to well ordered CL layer (Fig. 9d,e). This suggests that such zones concentrate most of the glycosiderich organic matrix (marking the OL layer). This matrix is also enriched in proteins, as the same area gave a strong signal when stained with the antibody K5090 (elicited against the soluble matrix of the nacreous layer of Pinna nobilis) by using immunogold technique (for further details on this see the supplementary information SI.2).

ShellOmics

We characterized the proteome of Spondylus shell matrix, by analysing the eight organic extracts (Table 2). The tryptic digests (see Materials and Methods) were analysed by liquid chromatography coupled to a tandem mass spectrometer (LC-MS/MS). The raw mass spectrometry data were analysed using the software program PEAKS Studio 8.5 [START_REF] Ma | PEAKS: powerful software for peptide de novo sequencing by tandem mass spectrometry[END_REF], searching against a protein database obtained from NCBI and restricting the taxonomy to Mollusca. The number of identified proteins (with a minimum of 2 unique peptides) was higher in the AIM fractions than in the ASM (up to 21 vs 14 sequences respectively). This was also consistent with higher numbers of supporting peptides (up to 67 in AIM vs 18 in ASM) and the de novo tags (up to ~9100 vs 5600) (Table 1). Considering sequences identified with at least one unique peptide, a total of 46 proteins were found (SI Table 1). Many of these, while poorly covered, were detected in several of the eight extracts and some of them may potentially be relevant for calcification, e.g., alkaline phosphatase, mucin-5AC-like, insoluble matrix protein, extensin-like, ADAM family mig-17, fibronectin type III domain-containing protein 2-like, glycine-rich cell wall structural protein-like, testis-expressed protein 9-like, 15 kDa selenoprotein-like, brevican core protein, biotin-protein ligase-like. However, we discuss in detail only the identifications supported by a minimum of two unique peptides, in at least one of the matrices, hence restricting the pool to 6 proteins (Table 2). No significant differences were observed between the lower and the upper valves of the shell in terms of proteome composition. However, the duration of the bleaching treatment appears to have an effect on the quality of the identification: counterintuitively, we obtained more supporting peptides and higher coverages from the 3BL extracts than from the 2BL.

Table 1: Summary of proteomics results: number of identified proteins, total number of supporting peptides, total number of de novo only tags present in each sample. The thresholds for peptide and protein identification were set as follows: protein false discovery rate (FDR) = 0.5%, protein score 10lgP > 20, unique peptides 2, de novo sequences scores (ALC%) > 50.

Table 2: Skeletal matrix proteins identified in Spondylus shell. The presented sequences are identified with a minimum of 2 unique peptides (numbers in bold) in at least one of the eight matrices (S1-S8). Cell colour corresponds to the sequence coverage in the sample. The numbers indicate the total number of supporting peptides (Pep), unique peptides (Un) and the identified de novo tags (Dn.t) which share at least 6 amino acid residues (see legend for details).

Protein identifications

Five out of six shell proteins identified in Spondylus were originally identified from the japanese scallop Mizuhopecten yessoensis (family Pectinidae), for which the full genome has been published in 2017 [START_REF] Wang | Scallop genome provides insights into evolution of bilaterian karyotype and development[END_REF].

• Uncharacterized protein LOC110461617 (gi|1207960950|ref|XP_021370834.1) was found in all of the samples except S1, and with 9% coverage in sample S8 (30 supporting peptides and 234 identified de novo tags sharing at least 6 AA residues). It is a high molecular weight (289.152 kDa) and basic (theoretical pI 8.25) shell protein, enriched in glycine residues (20.1%), with a notable presence of serine (7.5%), glutamine (7.1%), threonine (6.2%), alanine (6%) and asparagine (6%). The protein exhibits four von Willebrand factor type A domains (VWA) and three chitin-binding domains. In sample S8, these domains are partially covered (Fig. 10). The protein shows the potential occurrence of glycosylation, phosphorylation and N-myristoylation sites (however only a few of the first two modifications have been actually identified in the peptides by the PEAKS software, using the Spider search); there are also predicted NHL repeats (commonly found in a variety of enzymes). Intrinsically disordered regions (IDRs) dominate the C-terminal half of the protein sequence (predicted by ANCHOR2 and IUPred2), but short disordered motifs are also located between the protein family (PFAM) domains (Fig. 10). Overall, an NCBI BLASTp search revealed that this sequence is similar to that of PIF protein (with a query coverage of 68%) and collagen alpha-6(VI) chain (both from Mizuhopecten yessoensis), collagen alpha-4(VI) chain (Crassostrea gigas) and BMSP (from Pinctada fucata and Mytilus galloprovincialis), all showing E-values of 0. The homologous BMSP protein from Mytilus galloprovincialis (gi|347800228|dbj|BAK86420.1|), a basic calcium-binding protein occurring in nacreous shells [START_REF] Suzuki | Identification and characterisation of a calcium carbonate-binding protein, blue mussel shell protein (BMSP), from the nacreous layer[END_REF], which similarly has four VWA and two chitinbinding domains interspersed by IDRs (SI Fig. 4), was also identified in three of our Spondylus AIM extracts (samples S2/6/8). • Uncharacterized protein LOC110452388 (gi|1207934318|ref|XP_021356561.1|; gi|1207934321|ref|XP_021356563.1|) is a small (280 AA, 30.58 kDa), very basic protein (theoretical pI 11.33) rich in proline (11.4%), arginine (9.6%), glycine (9.3%), serine (8.2%), threonine (8.2%) and asparagine (7.9%). This protein is predicted to have several glycosylation, phosphorylation and N-myristoylation sites (predicted with Interpro [START_REF] Mitchell | InterPro in 2019: improving coverage, classification and access to protein sequence annotations[END_REF]), but none of these modifications have been identified in our sequences by the PEAKS software. Most of the protein is predicted to be intrinsically disordered, with an ordered structure limited solely to the C-terminus (SI Fig. 4). A BLASTp search indicates that it is homologous to other shell proteins such as ubiquilin-like isoform X3 and heterogeneous nuclear ribonucleoprotein 87F-like isoform X2 proteins (both from Crassostrea virginica, E-values 2e-05 and 3e-05 respectively), although the maximum query coverage is only around 30%. • Laccase (gi|1205898913|gb|OWF48064.1|) and laccase-7-like isoforms X1, X2 (all from Mizuhopecten yessoensis) were identified in five of the samples (S1/2/6/7/8), albeit with a very weak coverage (1-3%). It is a slightly acidic protein (theoretical pI 6.5) with a molecular weight of 104.16 kDa and is enriched in serine (9.3%), glycine (8.1%), proline (7.4%) and histidine (7.3%). It is an enzyme which belongs to Lascorbate oxidase superfamily (E-value 7.44e-51) and has three cupredoxin domains (Cu-oxidase_3, Cu-oxidase and Cu-oxidase_2). This protein exhibits intrinsically disordered regions, located between the first two cupredoxin domains and at the C-terminal part of the sequence (SI Fig. 4). protein, with a composition dominated by serine (11.5%), glutamic (10.9%) and aspartic (8.4%) acids. Threonine (7.5%), leucine (7%), asparagine (6.7%) and isoleucine (6%) are also abundant. It belongs to the GPCR, family 2, secretin-like protein family, which is homologous to the C-type lectin-like superfamily. It possesses immunoglobulin-like domains, GPS motifs and GPCR domains. Most of its sequence is intrinsically disordered, except at the C-terminus (SI Fig. 4). The sequence shows similarities to several other uncharacterized shell proteins and adhesion G-protein coupled receptor G proteins from shells, such as Crassostrea virginica, Pomacea canaliculata, Lingula anatina.

• Acidic mammalian chitinase (gi|1205814209|gb|OWF36273.1|) and its isoforms from Mizuhopecten yessoensis were identified in sample S2 only. This is a basic protein (pI 9.03) with a molecular weight of 78.79 kDa. It has chitinase II and chitin-binding domains as well as predicted intrinsically disordered region in the last third part of the sequence (SI Fig. 4) and shows homology to the glycoside hydrolase superfamily proteins. The protein is homologous to many other molluscan chitinases, among them those from Hyriopsis cumingii (the triangle shell mussel), Crassostrea gigas (the Pacific edible oyster) and Octopus vulgaris (the common octopus). Chitinases are enzymes that cleave glycosidic bonds of chitin, and are consequently involved in the remodelling of chitinous scaffolds.

Peptidome analysis

Given the low number of proteins identified in the Spondylus shell organic matrix using standard database searches, we implemented a different characterisation strategy, focussing on the analysis of the several thousands peptide sequences which can be reconstructed by assisted de novo algorithms (using PEAKS Studio 8.5). First, all of the raw MS files were searched against a database of known common laboratory contaminants (cRAP) and such contaminant peptides were discarded. We then produced a list of genuine de novo peptides (ALC > 80 %) (SI Table2) and searched these "peptidomes" for any pattern emerging in the different extracts (Fig. 11). In agreement with previous findings we observed that the AIMs contain more peptides than the ASMs (~1930 vs ~1150). A number of these sequences display amino acid patterns that are typically observed in other shell proteins, such as poly-Gly (predominantly in AIMs), poly-Ser, poly-Gln (predominantly in ASMs) and acidic motifs (EE, DD), but also unusual motifs, like poly-Leu and poly-Pro.

Figure 11: Spondylus shell peptidomes (samples S1-S8) characterized by occuring motifs: a) heatmap that represents the occurrence of amino acid motifs found in peptides of samples S1-S8; b) example peptides with these motifs. The full table with peptidome sets can be found in SI Table 2.

Discussion

This study presents a comprehensive biomolecular and micro-structural investigation of the Mediterranean thorny oyster S. gaederopus. The bivalve, which underwent a relatively recent evolution, shows divergent molecular features compared to other well studied species. Our discussion examines different aspects, including microstructure, matrix biochemistry and proteomics.

Microstructure

Spondylus and the typical representatives of the Pectinidae family, e.g. Pecten, belong to the same superfamily (Pectinoidea), however their microstructures are different; the only shared characteristic being their upper foliated calcitic layers. Compared to other pectinids [START_REF] Carter | Skeletal Biomineralization: Pattern, Processes, and Evolutionary Trends[END_REF]) S. gaederopus shells are certainly more complex, especially with regard to the middle and inner layers, where at least three different microstructures are observed [START_REF] Logan | Morphology and Life Habits of the Recent Cementing Bivalve Spondylus Americanus Hermann from the Bermuda Platform[END_REF]: aragonitic prismatic, crossed lamellar (with also different lamellae orientations) and calcitic foliated. Furthermore, the thickness ratio of these three microstructures changes along the growth line, a likely thermally dependant effect [START_REF] Nishida | Thermal dependency of shell growth, microstructure, and stable isotopes in laboratory-reared Scapharca broughtonii (Mollusca: Bivalvia): Thermal control on shell microstructure[END_REF][START_REF] Nishida | Seasonal changes in the shell microstructure of the bloody clam, Scapharca broughtonii (Mollusca: Bivalvia: Arcidae)[END_REF]. We also observed that the change between different aragonitic microstructures is very gradual, while the switch between the layers of two different polymorphs (aragonitic cross-lamellar and calcitic foliated) is abrupt and well marked, confirming earlier descriptions [START_REF] Wilmot | Electron microscopy of molluscan crossed-lamellar microstructure[END_REF]. The long hollow spines, a characteristic feature of spondylids and which function both as an inducement for the settlement of epibionts on the shell and as antipredator-defense [START_REF] Feifarek | Spines and epibionts as antipredator defenses in the thorny oyster Spondylus americanus Hermann[END_REF][START_REF] Logan | Morphology and Life Habits of the Recent Cementing Bivalve Spondylus Americanus Hermann from the Bermuda Platform[END_REF], also display a complex microstructure, which is rather disordered at their basis, morphing into crossed-foliated. This microstructural feature has been characterized in gastropod shells [START_REF] Fuchigami | The shell structure of the Recent Patellogastropoda (Mollusca: Gastropoda)[END_REF][START_REF] Taylor | Shell microstructure and mineralogy of the Littorinidae: ecological and evolutionary significance[END_REF]), but, interestingly, has not been reported in bivalves.

Biochemistry

The organic fraction in S. gaederopus shell is very low and the amount of matrix that is extracted decreases even further with progressive bleaching times (in 3BL samples, only 0.04-0.25 wt‰ of organics, noted as 'intracrystalline', is obtained). The isolation of an "operational" intracrystalline fraction of organics is usually carried out using concentrated NaOCl (12% w/v) and exposure times of 48 hours or more [START_REF] Penkman | Closed-system behaviour of the intra-crystalline fraction of amino acids in mollusc shells[END_REF]. However, when considering the 2BL extracts, the reactivity of Jacalin (lectin that recognized the sugar moieties present in the shell organics) is comparable to that observed on 48 hrs bleached extracts (see supplementary information, SI.5); this indicates that the 3BL step isolates a fraction that is in all likelihood "intracrystalline".

The finding that Spondylus matrices are rather organic-poor is consistent with the data obtained on other cross-lamellar [START_REF] Agbaje | Biomacromolecules in bivalve shells with crossed lamellar architecture[END_REF][START_REF] Demarchi | Testing the effect of bleaching on the bivalve Glycymeris: A case study of amino acid geochronology on key Mediterranean raised beach deposits[END_REF]Marie et al., 2013a;[START_REF] Osuna-Mascaró | The shell organic matrix of the crossed lamellar queen conch shell (Strombus gigas)[END_REF] and foliated [START_REF] Pierini | Pecten as a new substrate for IcPD dating: the Quaternary raised beaches in the Gulf of Corinth, Greece[END_REF] shells; this contrasts to what is observed in organic-rich nacreous and prismatic microstructures (Marie et al., 2009b;[START_REF] Marin | Shellome": Proteins involved in mollusk shell biomineralization-diversity, functions[END_REF][START_REF] Sakalauskaite | Palaeoshellomics" reveals the use of freshwater motherof-pearl in prehistory[END_REF]. As spondylids represent a bivalve family that emerged rather recently, in the Middle Jurassic [START_REF] Waller | Phylogeny of families in the Pectinoidea (Mollusca: Bivalvia): importance of the fossil record[END_REF], our observations corroborate the pioneering idea of Palmer that, in molluscs, the "cost" for making a shell is mostly driven by the "cost" to synthesize the organic matrix [START_REF] Palmer | Calcification in marine molluscs: how costly is it?[END_REF]; we can argue that evolution was directed to strike a balance between the mechanical properties of a biomineral and the actual "cost" required to produce it. For example, nacre is very "expensive", while crossed-lamellar microstructures, albeit exhibiting only slightly inferior mechanical properties [START_REF] Kamat | Structural basis for the fracture toughness of the shell of the conch Strombus gigas[END_REF], are "cheaper" to produce. In the course of the Phanerozoic, mollusc evolution has favoured the latter solution; the Spondylidae may illustrate the emergence of very tough microstructures that do not require a lot of shell matrix for their synthesis.

The biochemical composition of Spondylus shell matrix is typical of mollusc shells, i.e. a mixture of proteins and saccharide moieties that display different solubilities in acidic conditions (hence, the distinction between AIM and ASM). In our study, no compelling differences were observed between the shell matrices of the upper red and lower white valves. On gels, the ASM set of macromolecules, with the exception of few, show mostly non-discrete band profiles -an observation that is similar to most, if not all, shell matrices [START_REF] Marin | Molluscan Shell Proteins: Primary Structure, Origin, and Evolution[END_REF]. While typical calcium-binding proteins (like calmodulin, which exhibits the EF-hand motif) stain blue with Stains-all, Spondylus ASMs stain purple. This feature had already been observed for other acidic shell matrices, such as that extracted from the calcitic prisms of the fan mussel Pinna nobilis [START_REF] Marin | Caspartin and calprismin, two proteins of the shell calcitic prisms of the Mediterranean fan mussel Pinna nobilis[END_REF]. We have also observed that Spondylus ASMs (which represent the sum of inter-and intracrystalline matrices) induced strong modifications of the crystal formation during the in vitro CaCO 3 crystallization; the effect was much more prominent compared to ASMs extracted from other aragonite shells, e.g. the land snail Helix aspersa maxima [START_REF] Pavat | The shell matrix of the pulmonate land snail Helix aspersa maxima[END_REF]. This result is interesting because in vitro assays typically show that acidic macromolecules (proteins and glycoproteins) induce the strongest effect and are usually thus considered to be intracrystalline [START_REF] Albeck | Regulation of calcite crystal morphology by intracrystalline acidic proteins and glycoproteins[END_REF][START_REF] Albeck | Interactions of various skeletal intracrystalline components with calcite crystals[END_REF]. Indeed, glycoproteins are identified in our Spondylus organic matrices, yet, more interestingly the proteomics data reveals that basic proteins are also abundant in the intracrystalline fraction, a finding that expands the knowledge on mineral-bound intracrystalline organics (Demarchi et al., 2016).

Skeletal shell matrices have their own saccharide signature [START_REF] Kanold | Spine and test skeletal matrices of the Mediterranean sea urchin Arbacia lixula--a comparative characterization of their sugar signature[END_REF] and these carbohydrates are suspected to perform several functions related to biomineralization [START_REF] Albeck | Regulation of calcite crystal morphology by intracrystalline acidic proteins and glycoproteins[END_REF][START_REF] Marie | The shell matrix of the freshwater mussel Unio pictorum (Paleoheterodonta, Unionoida). Involvement of acidic polysaccharides from glycoproteins in nacre mineralization[END_REF][START_REF] Samata | A novel phosphorylated glycoprotein in the shell matrix of the oyster Crassostrea nippona: A novel acidic glycoprotein from the oyster shells[END_REF][START_REF] Sarashina | The complete primary structure of molluscan shell protein 1 (MSP-1), an acidic glycoprotein in the shell matrix of the scallop Patinopecten yessoensis[END_REF]. However, they are largely ignored in biomineralization studies, whether they are present as free polysaccharides or glycoproteins. In our case, the lectin assay of the ASMs of S. gaederopus underlines the diversity of the sugar residues and of the glycosylation pattern:

1) The preponderance of O-linked glycoproteins and/or oligosaccharides with Gal, Gal-GalNAc dimers (as observed by the strong affinity to Jacalin); 2) The presence of N-linked mannose containing oligopeptides;

3) The presence of N-acetylglucosamine (or oligomers with this sugar residue), suggesting that chitin is most likely part of the shell organic matrix. As chitin is usually insoluble, it may be either that the glycans in the ASMs are constituted of oligomeric forms, or that this is a soluble form of chitin, partially cleaved by the bleaching treatment prior to the extraction.

It is interesting to note that Jacalin on Western blots shows different binding profiles for the ASM and AIM matrices (more precisely in the LC-AIM); this implies that either the glycans are different, or that they are identical but differently crossed-linked, i.e. the soluble monomeric form being present in the ASM while the crossed-linked one in the LC-AIM. Finally, in situ goldmapping shows that the glycans targeted with Jacalin are not evenly distributed throughout the shell cross-section, but are specific to the upper foliated calcitic layer and are particularly concentrated at the junction with the underlying aragonitic crossed-lamellar layer (Fig. 9), as seen also by luminescence imaging. The data suggest that this organic layer plays an important role in this structural and mineralogical transition -switching from foliated calcite to crossed-lamellar aragonite, while maintaining these two microstructures glued together [START_REF] Wilmot | Electron microscopy of molluscan crossed-lamellar microstructure[END_REF].

Proteomics

Shotgun proteomics was employed in order to characterise the shell proteome composition. It is however important to stress that the results obtained will be forcibly biased, due to several limitations, including the intrinsic peculiarities of shell matrix proteins and the paucity of -omics reference data for shell matrices, particularly evident in poorly studied organisms such as spondylids.

1) Shell matrix proteins are frequently characterized by the presence of repeated low complexity domains (LCDs or RLCDs, frequently found as long poly-Ala/Asp/Ser/Gly blocks [START_REF] Kocot | Sea shell diversity and rapidly evolving secretomes: insights into the evolution of biomineralization[END_REF][START_REF] Marie | Deep conservation of bivalve nacre proteins highlighted by shell matrix proteomics of the Unionoida Elliptio complanata and Villosa lienosa[END_REF][START_REF] Sakalauskaite | Palaeoshellomics" reveals the use of freshwater motherof-pearl in prehistory[END_REF], and post translational modifications such as phosphorylation [START_REF] Du | The role of phosphorylation and dephosphorylation of shell matrix proteins in shell formation: an in vivo and in vitro study[END_REF] and glycosylation [START_REF] Boskey | Intrinsically disordered proteins and biomineralization[END_REF][START_REF] Mann | The Lottia gigantea shell matrix proteome: re-analysis including MaxQuant iBAQ quantitation and phosphoproteome analysis[END_REF], making the protein sequences difficult to cleave using standard proteases and thus reducing the chance of identification.

2) The identification is database-dependent. Shell proteomes can be very diverse across taxa and different microstructures [START_REF] Jackson | Parallel evolution of nacre building gene sets in molluscs[END_REF]Marie et al., 2013a) but only a small part of such molluscan shell proteomes (mainly those of commercially-relevant species) have been studied (the phylum comprises between 85000 and 100000 shellforming species [START_REF] Feng | Identification of conserved proteins from diverse shell matrix proteome in Crassostrea gigas: characterization of genetic bases regulating shell formation[END_REF][START_REF] Marie | Different secretory repertoires control the biomineralization processes of prism and nacre deposition of the pearl oyster shell[END_REF][START_REF] Marie | Molecular evolution of mollusc shell proteins: insights from proteomic analysis of the edible mussel Mytilus[END_REF][START_REF] Yarra | Characterization of the mantle transcriptome in bivalves: Pecten maximus, Mytilus edulis and Crassostrea gigas[END_REF]).

Crossed-lamellar microstructures particularly common among bivalves, gastropods and scaphopods and yet, they are the most understudied.

In our study -the first on spondylids -we had to rely on the omics data available for closely related species (in particular pectinoid shells). However, it is likely that spondylids shell proteomes are much more diversified than presumed, given the complex evolution of Pectinoidea [START_REF] Smedley | Molecular phylogeny of the Pectinoidea (Bivalvia) indicates Propeamussiidae to be a nonmonophyletic family with one clade sister to the scallops (Pectinidae)[END_REF].

Comparison between shell extracts

Despite the low organics yield obtained from the bleached shell powders, high-resolution tandem mass spectrometry was successful in obtaining several thousands of product ion spectra for each sample; the sequences of these were reconstructed using the software PEAKS Studio 8.5. Spondylus ASMs and AIMs exhibit similar set of proteins, but more sequences were identified in the AIMs. Interestingly, this is particularly evident for the intracrystalline AIM matrix (the 3BL sample) indicating that by exposing shell to longer bleaching times and removing a large part of the intercrystalline organics, we can facilitate their detection. The use of PEAKS software resulted in the identification of between 5000-9000 de novo sequences for each sample (Table 1). Several of these reconstructed peptide sequences show interesting features, that are commonly observed in other shell proteomes (Fig. 11). These include glycine and serine repeats as well as acidic blocks (such as EE, DD) in the AIM extracts and polyQs in ASMs. Another motif, which is commonly encountered in nacreous shell proteins, i.e. poly-Ala blocks, was rare in Spondylus proteins.

While, at first sight, Spondylus peptidomes resemble "typical" shell protein sequences, the majority of de novo sequences could not be assigned to any known molluscan proteins. Five out of the six identified sequences belong to the pectinoid Mizuhopecten yessoensis, the japanese scallop, which is the phylogenetically closest relative that has a full genome analysed [START_REF] Wang | Scallop genome provides insights into evolution of bilaterian karyotype and development[END_REF]. The coverages were low for all of the identified sequences (1-9%), but most of these proteins possess features typical of shell biomineralization, such as WVA and chitin-binding domains present in Uncharacterized protein LOC110461617 [Misuhopecten yeonessis] and BSMP. Both of these proteins are homologues to PIF, suggesting these sequences found in Spondylus are important in calcium binding activity and possibly play a role in the biomineralization of the aragonitic structures [START_REF] Arivalagan | Insights from the Shell Proteome: Biomineralization to Adaptation[END_REF][START_REF] Feng | Identification of conserved proteins from diverse shell matrix proteome in Crassostrea gigas: characterization of genetic bases regulating shell formation[END_REF][START_REF] Suzuki | Identification and characterisation of a calcium carbonate-binding protein, blue mussel shell protein (BMSP), from the nacreous layer[END_REF].

The two enzymes identified also have functions related to shell formation: i) chitinase, a chitin binding protein that catalyses the cleavage of chitin, could be involved in the (re)modeling of the chitinous scaffold [START_REF] Yonezawa | Molecular cloning and functional analysis of chitinases in the fresh water snail, Lymnaea stagnalis[END_REF]; ii) laccase [Mizuhopecten yeonessis], a copper oxidase enzyme, could be involved in cross-linking of the matrix during biomineralization. In the prism matrix of the pearl oyster, an enzyme with similar function was identified (Marie et al., 2012), while laccase-like genes have been shown to be involved in shell immunity, biomineralization and pigmentation [START_REF] Mao | Transcriptional changes in the Japanese scallop (Mizuhopecten yessoensis) shellinfested by Polydora provide insights into the molecular mechanism of shell formation and immunomodulation[END_REF][START_REF] Yue | Identification of a laccase gene involved in shell periostracal tanning of the clam Meretrix petechialis[END_REF].

We note that all of the six identified shell matrix proteins are intrinsically disordered (Fig. 10, Fig. SI.4). An important characteristic of these intrinsically disordered regions (IDRs) lies in their flexibility, which enables matrix assembly and protein-mineral interaction [START_REF] Boskey | Intrinsically disordered proteins and biomineralization[END_REF][START_REF] Kalmar | Structural disorder in proteins brings order to crystal growth in biomineralization[END_REF]. This view is in agreement with the preponderance of these regions in many of skeletal matrix proteins [START_REF] Evans | Aragonite-associated biomineralization proteins are disordered and contain interactive motifs[END_REF]. The uncharacterized protein LOC110461617 identified in Spondylus matrices shows the presence of the IDRs located in between the functional domains (the WVAs in the N-terminal part of the sequence) and dominate almost half of the C-terminal part of the sequence (Fig. 10).

Interestingly, the highest coverage of this protein in our samples coincide with the conserved regions, while IDRs have very low and sporadic coverage; since IDRs are believed to evolve fast [START_REF] Kocot | Sea shell diversity and rapidly evolving secretomes: insights into the evolution of biomineralization[END_REF] and to be lineage-specific, this may explain why these domains are poorly covered, when taking the sequence data from other shells as reference.

Conclusion

Our paper describes the first molecular characterization of the shell of S. gaederopus, a representative of spondylid family, with a particularly complex microstructure. We found that Spondylus possesses a remarkably small fraction of its associated skeletal matrix -it is rich in glycosidic moieties and is mainly intercrystalline, in particular concentrated at the transition between different microstructural layers. We carried out proteomic analysis, which enabled to characterize (at least in part) the shell proteome of Spondylus. Even though almost all of the identified sequences were most closely matched to pectinoid shell proteins (belonging to Mizuhopecten yessoensis), the unusually low numbers of identified proteins and their scant coverages indicate that the S. gaederopus shellome is quite different from that of Pecten. Our key finding therefore is that S. gaederopus (and this might be extended to the whole spondylid family) is likely to have adopted distinctive molecular strategies to biomineralize its shell. In the future, this may result in a reassessment of the phylogenetic relationships of Spondylidae within the Bivalves clade. This unique molecular fingerprint , albeit puzzling from an evolutionary point of view, is rich in perspectives for "palaeoshellomics", i.e. the study of ancient shell proteins [START_REF] Sakalauskaite | Palaeoshellomics" reveals the use of freshwater motherof-pearl in prehistory[END_REF]. This is particularly interesting for biomolecular archaeologists aiming to obtain taxonomic identification of fragmented archaeological shell objects, where biological origin cannot be assigned via classical means (microstructural observations). Our findings suggest that the molecular identification of precious Spondylus shell fragments among many other shells in the palaeontological/ archaeological records, is possible.

Materials and Methods

Sampling and preparation

Spondylus gaederopus Linnaeus, 1758 (Mollusca; Bivalvia; Pteriomorphia; Pectinida; Pectinoidea; Spondylidae; Spondylus) is endemic to the Mediterranean sea and is not listed in the International Union for Conservation of Nature (IUCN's) list of threatened species. Adult shells (around 100 mm size) were purchased from Conchology, Inc ("Conchology, Inc," n.d.). The specimens were collected live at 15 m depth (by diving, as listed by the vendors), in Saronikos, Greece, in 2010. In the laboratory, the shell was cleaned using abrasive tools and immersed in diluted NaOCl (~1-1.5% active chlorine) for 2 hrs (we call this step as 1BL -1 st bleach treatment). The upper and lower valves were rinsed with water, air-dried and crushed into ∼2 mm fragments with a Jaw-crusher (Retsch BB200) separately, obtaining two sample subsets of the lower valve (LV) and the upper valve (UV). The obtained fragments were bleached in ~1-1.5% NaOCl for 4 hrs (2BL), rinsed with water (5x), ethanol (1x) and air-dried.

Coarsely crushed samples were powdered with a mortar grinder (Frisch Pulverisette 2) to 200 μm particle size. Half of both subsamples (UV and LV) were set aside (2BL samples) and the second batch was bleached for the third time in ~1-1.5% NaOCl for 14 hrs (3BL), thoroughly rinsed with water (5x) and air-dried. In total, 4 powdered samples were obtained: 2 batches of different cleaning approach (2Bl and 3 BL) each with 2 subsamples -upper and lower valves (UV and LV).

Extraction of shell organic matrix (SOM)

The extraction was carried out using a well-established protocol developed in our lab [START_REF] Ramos-Silva | Novel molluskan biomineralization proteins retrieved from proteomics: a case study with Upsalin[END_REF][START_REF] Takeuchi | Biochemical characterization of the skeletal matrix of the massive coral, Porites australiensis -The saccharide moieties and their localization[END_REF]. In short, powdered samples were decalcified using cold diluted acetic acid (10% v/v, 100 µL every 5 seconds) overnight and then centrifuged to separate the acid soluble (ASM) and acid insoluble matrices (AIM). The AIM matrix was cleaned with ultra pure water (5x) and freeze-dried. The ASM was ultrafiltered using 10 kDa cut-off membrane (Millipore, Ref. PLGC07610), dialysed against 1L of MiliQ water, with 6 changes over 2 days, and freeze-dried.

Biochemical characterization

FT-IR spectroscopy FT-IR analysis in Attenuated Total Reflectance (ATR) mode was used to check the overall composition of ASM and AIM matrices of the 2BL samples. The analysis was performed with a Bruker Vector 22 FT-IR spectrometer (BrukerOptics Sarl, France, Marne la Vallée) fitted with a GoldenGate ATR device (SpecacLtd, Orpington, UK) in the 4000-400 cm -1 range (twelve scans with a spectral resolution of 4 cm -1 ). Spectra analysis and assignment of absorption bands were performed by comparison with previously published data [START_REF] Kanold | Spine and test skeletal matrices of the Mediterranean sea urchin Arbacia lixula--a comparative characterization of their sugar signature[END_REF][START_REF] Takeuchi | Biochemical characterization of the skeletal matrix of the massive coral, Porites australiensis -The saccharide moieties and their localization[END_REF].

SDS-PAGE

ASMs were fully dissolved in ultrapure water to a final concentration of 4 µg/mL and 4x Laemmli buffer was added (ratio 3:1). AIMs were resuspended and treated similarly. Denaturation was carried out at 99 °C for 5 min, then samples were cooled on ice. Because of limited AIM solubility, only the Laemmli-soluble supernatants, referred to as LS-AIM, were analysed by SDS-PAGE. Proteins were run on precast (4-15 %) gradient (Bio-Rad) and 12% gels, then stained with silver nitrate and Stains-all [START_REF] Campbell | Staining of the Ca2+-binding proteins, calsequestrin, calmodulin, troponin C, and S-100, with the cationic carbocyanine dye "Stains-all[END_REF][START_REF] Takeuchi | Biochemical characterization of the skeletal matrix of the massive coral, Porites australiensis -The saccharide moieties and their localization[END_REF] respectively.

Western-blot

Samples S1 and S2 were run on a 12% SDS-PAGE gel as reported above, then electrotransferred on a PVDF Immobilon P membrane (Millipore) [START_REF] Matsudaira | Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes[END_REF]. To detect the glycosylated complex, the membrane was incubated in Carbo-Free blocking solution (Vector Laboratories, Peterborough, UK, ref. then in a solution that contained the biotinylated lectin Jacalin (Vector Labs. Ref. B-1155, diluted in TBS/Tween20, ratio 1:100) and finally, in a solution containing avidin-AP conjugate (Sigma A7294, dilution 1/70000). Thorough rinsing steps with TBS/Tween20 were performed. The membrane was stained with NBT/BCIP (Sigma B5655).

Enzyme-Linked Lectin Assay (ELLA)

The enzyme-linked immuno assay was carried out on two ASM fractions following previously described procedures [START_REF] Kanold | Spine and test skeletal matrices of the Mediterranean sea urchin Arbacia lixula--a comparative characterization of their sugar signature[END_REF][START_REF] Takeuchi | Biochemical characterization of the skeletal matrix of the massive coral, Porites australiensis -The saccharide moieties and their localization[END_REF]. In short, ASM aqueous solutions (100 µL per well, approx. ~200 ng/ well) were deposited in 96-well microplates (MaxiSorp, Nunc/Thermo Scientific, Nunc A/S, Roskilde, Denmark), incubated for 90 min at 37 °C, washed with TBS/Tween20 and blocked with Carbon-Free solution. Three sets of 7 biotinylated lectins were tested (Vector Laboratories, Peterborough, UK, Ref. BK-1000, BK-2000, BK-3000; diluted in TBS/Tween-20 with ratio 1:200 for kit 1,2 and 1:100 for kit 3); lectins were incubated for 90 min at 37 °C, washed, and a solution of alkaline phosphataseconjugated avidin (1:70000) was added for another 90 min at 37 °C. Microplates were washed and incubated with the ELISA (aqueous diethanolamine solution, pH 9.8) substrate solution, containing phosphatase substrate (p-nitrophenylphosphate) at 37 °C. Optical density was read using a BioRad Model 680 microplate reader at 405 nm; background values (blank tests) were subtracted and the values were converted to percentage of reactivity, with the highest OD values at 100%. A total of 4 replicates were performed.

In vitro CaCO 3 crystallization

The capacity of ASM to interact in vitro with CaCO 3 ASM was tested in the diffusion assay, as described previously [START_REF] Kanold | Spine and test skeletal matrices of the Mediterranean sea urchin Arbacia lixula--a comparative characterization of their sugar signature[END_REF][START_REF] Takeuchi | Biochemical characterization of the skeletal matrix of the massive coral, Porites australiensis -The saccharide moieties and their localization[END_REF]. 200 μL of 10 mM CaCl 2 solutions containing ASMs at increasing concentrations (0.25 -16 µg/ml) were placed in 16well culture slides (Lab-Tek, Nunc/ Thermo Scientific, Rochester, NY, USA). The plastic cover was pierced beforehand to allow the reaction with ammonium bicarbonate vapours, placed on top of the slide and sealed with parafilm. It was placed in a desiccator under vacuum which contained ammonium bicarbonate crystals and incubated at 4 °C for 72 hrs. Afterwards, the solution was carefully removed with a blunt-end needle and the slide was air-dried. The 16well culture slide was disassembled and CaCO 3 crystals were observed directly (without any carbon sputtering) using a tabletop scanning electron microscope (TM 1000, Hitachi).

In situ localization

Lectin-gold assay Using a diamond saw, a transverse section, perpendicular to the growth line, was cut from the upper valve of the shell (area closer to the margin). The section was polished using a fine-grain sandpaper, followed by an alumina suspension (0.05 µm) and cut into small square fragments. They were cleaned using sonication, then bleached for 10 min with NaOCl (~1-1.5 % active chlorine) and rinsed with ultrapure water. Fragments were etched with 1% EDTA (v/v) in the ultrasound bath for 5 min and rinsed again with ultrapure water. The rest of the procedure follows that reported in [START_REF] Takeuchi | Biochemical characterization of the skeletal matrix of the massive coral, Porites australiensis -The saccharide moieties and their localization[END_REF]: incubation in Carbo-Free blocking solution (1 hour); overnight incubation with Jacalin solution (prepared in TBS/Tween-20, diluted 1:10; plus NaNa 3 , 1/10000 to prevent bacterial growth); rinsing step with TBS/Tween-20 (6x times, 10 min each); incubation with goat anti-biotin antibody conjugated to ultra-small gold particles (0.8 nm) (GABio, Ultra Small, Ref. 800.088; Aurion, Wareningen, The Netherlands) for 2 hrs; after rinsing step and gentle drying, incubation in the silver enhancement solution (British Biocell International, Ref. SEKL15) for about 10-15 min; rinsing in milli-Q water and drying at 37 °C. All incubations were performed at room temperature with gentle shaking. Samples were observed with SEM (Hitachi, TM1000) under back-scattered electron mode To preclude false positive signals, 3 negative controls were performed in parallel using the same conditions but omitting one or two of the following steps: 1) incubation with Jacalin (negative control 1); 2) incubation with gold-coupled anti-biotin (negative control 2); 3) both incubations with Jacalin and the gold-coupled anti-biotin (negative control 3). All other steps were done similarly. Total of 4 replicas of the experiments were carried out to validate the observations.

Luminescence imaging by DUV synchrotron photoemission beamline

The Deep-Ultraviolet (DUV-visible) photoluminescence imaging was applied to trace the distribution of organics in the biomineral of a selected area. Fluorescence maps were recorded on the DISCO beamline [START_REF] Jamme | Deep UV autofluorescence microscopy for cell biology and tissue histology[END_REF] using the setup TELEMOS build around an Olympus IX71 inverted microscope stand and a Peltier iDus CCD detector with 26x26 µm pixel size. A small fragment was cut from the upper valve of the shell, that also includes a formed spine, and was mirror-polished. The area was scanned using a x40 objective and the luminescence maps were reconstructed and corrected using an in-house script in Matlab, developed at DISCO beamline by one of the authors (F.J.).

Proteomic analysis

For proteomic analyses, digestion with trypsin of each of the eight shell organic extracts were carried out in-gel after a short migration (around one centimeter in the separating gel) in an 8% acrylamide gel stained with Coomassie blue. In brief, after the gel runs, samples were destained twice with a mixture of 100 mM ammonium bicarbonate (ABC) and 50% (v/v) acetonitrile (ACN) for 30 min at 22 °C and then dehydrated using 100% ACN for 15 min, before being reduced with 25 mM ABC containing 10 mM DTT for 1 h at 56 °C and alkylated with 55 mM iodoacetamide in 25 mM ABC for 30 min in the dark at 22 °C. Gel pieces were washed twice with 25 mM ABC and dehydrated (twice, 20 min) with 100% ACN. Gel cubes were incubated with sequencing grade modified trypsin (Promega, USA; 12.5 ng/µl in 40 mM ABC with 10 % ACN, pH 8.0) overnight at 37 °C. After digestion, peptides were extracted twice with a mixture of 50% ACN -5% formic acid (FA) and then with 100% ACN. Extracts were dried using a vacuum centrifuge Concentrator plus. Resulting peptides were resuspended in 10 μL of 10% ACN -0.1 % trifluoroacetic acid (TFA) and subjected to analysis by LC-MS/MS. Mass spectrometry (MS) was performed using an Ultimate 3000 Rapid Separation Liquid Chromatographic (RSLC) system (Thermo Fisher Scientific) online with an Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific). 1μL of peptides was loaded, concentrated and washed on a C 18 reverse phase pre-column (3 µm particle size, 100 Å pore size, 75 µm i.d., 2 cm length, Thermo Fisher Scientific). The loading buffer contained 98% H 2 O, 2% ACN and 0.1% TFA. Peptides were then separated on a C 18 reverse phase resin (2 µm particle size, 100 Å pore size, 75 µm i.d., 25 cm length, Thermo Fisher Scientific) with a 1 hour gradient from 99% solvent A (0.1% FA and 100% H 2 O) to 90% solvent B (80% ACN, 0.085% FA and 20% H 2 O).

The mass spectrometer acquired data throughout the elution process and operated in a data dependent scheme with full MS scans acquired with the Orbitrap, followed by MS/MS HCD fragmentations acquired with the Ion Trap on the most abundant ions detected. Mass spectrometer settings were: full MS (AGC: 2x10e5, resolution: 6x10e4, m/z range 350-1500, maximum ion injection time: 60 ms) and MS/MS (HCD collision energy: 30%, AGC: 2x10e4, resolution:3x10e4, maximum injection time: 100 ms, isolation windows: 1.6m/z Da, dynamic exclusion time setting: 30 s). The fragmentation was permitted for precursors with a charge state of 2, 3, 4 and up.

Bioinformatic search

Product ion spectra were analysed using PEAKS Studio (v. 8.5, Bioinformatics Solutions Inc. (BSI) [START_REF] Ma | PEAKS: powerful software for peptide de novo sequencing by tandem mass spectrometry[END_REF]) and searched against a "Mollusca protein" database, downloaded from NCBI (https://www.ncbi.nlm.nih.gov/) on 19/12/2018, restricting the taxonomy to Mollusca, including the search of common laboratory contaminants (cRAP; common Repository of Adventitious Proteins: http://www.thegpm.org/crap/). Search parameters were defined assuming no enzyme digestion (in order to also detect naturally cleaved peptides, if any), fragment ion mass tolerance of 0.05 Da and a parent ion tolerance of 10 ppm. Results obtained by SPIDER searches (i.e. including all possible modifications) were used for peptide identification and protein characterization, choosing the following threshold values for acceptance of high-quality peptides: false discovery rate (FDR) threshold 0.5%, protein scores -10lgP ≥ 20, unique peptides ≥ 2, de novo sequences scores (ALC %) ≥ 50. An additional full list of protein sequences identified with just one unique peptide were also exported. The peptide sequences identified in shell proteins were individually checked using the Blastp tool (https://blast.ncbi.nlm.nih.gov/), and any sequences that were homologous to common laboratory contaminants were excluded from any further analysis.

Protein profiles

Protein physical and chemical parameters (MW, theoretical PI, amino acid composition) were assessed using ProtParam tool on the EXPASY server. Protein classification, prediction of domains and motifs was carried using Interpro database [START_REF] Mitchell | InterPro in 2019: improving coverage, classification and access to protein sequence annotations[END_REF] and Motif scan tool on the EXPASY server. Intrinsically disordered regions in proteins were predicted using IUPred2A [START_REF] Mészáros | IUPred2A: context-dependent prediction of protein disorder as a function of redox state and protein binding[END_REF] and the bLASTp tool was used to identify homologous proteins.

Peptidome analysis

For the shell peptidome analysis, the reconstructed de novo tags in PEAKS were searched against a database of common laboratory contaminants (cRAP, search parameters defined above). The results obtained by SPIDER search were used by selecting these threshold values: false discovery rate (FDR) threshold 0.5%, protein scores -10lgP ≥ 20, unique peptides ≥ 0. Only the very high quality de novo sequences were taken into account, by selecting the local confidence score ALC % ≥ 80; exported sequences were grouped in Supplementary Information Table 2.

Figure 1 :

 1 Figure 1: Spondylus gaederopus Linnaeus, 1758 shell, analysed in this study (Greece, Saronikos. Dived at 15 m., taken live in 2010).

Figure 2 :

 2 Figure 2: SEM micrographs of the microstructures observed along a polished transversal section of Spondylus gaederopus shell (upper valve): a) calcitic crossed-foliated (CF) structure of the spine; b) irregular calcitic CF microstructure observed at the point of spine formation; c) transition between the calcitic foliated (F) and the aragonitic crossed-lamellar (CL) structures; d) aragonitic crossed-lamellar (CL) structure in the upper part of the shell; e) aragonitic crossed-lamellar (CL) structure in the lower part of the section; f,g) aragonitic prismatic (P) layer in the bottom part of the shell and myostracum. Structural abbreviations: F -calcitic foliated; P -aragonitic prismatic; CL -aragonitic crossed-lamellar, CF -calcitic crossedfoliated.

Figure 3 .

 3 Figure 3. Micro-imaging luminescence of the organo-mineral interface between the foliated calcitic and aragonitic crossed-lamellar layers in the upper valve of Spondylus shell, obtained using a DUV photoemission beamline of 275 nm: a) area of interest, picture taken under optical microscope; b) false color luminescence images obtained using 456-481 nm and 535-607 nm filters for the detection. Marked layers: F -foliated, OL -organic, CL -crossed-lamellar.

Figure 8 :

 8 Figure 8: SEM images of the in vitro CaCO 3 crystallization assay in the presence of Spondylus ASMs (fractions S1 and S3) tested at different concentrations: the effect of ASMs was tested at increasing concentrations of 0.5, 2, 4, 8 and 16 µg•mL -1 .

Figure 10 :

 10 Figure 10: Prediction of intrinsically disordered regions (IDRs) for Uncharacterized protein LOC110461617 [Mizuhopecten yessoensis] and the coverage of this protein in sample S8.Structure prediction was obtained using the IUPred2A software (IUPred2 and ANCHOR2 tools). The graph shows the disorder tendency of the residues, where a higher score (y axis) corresponds to a higher probability of disorder (values from 0 to 1). The top graph shows this tendency throughout the full sequence; the regions where covering peptides have been identified (in sample S8) are marked in yellow and detailed in the figures below. Where present, protein domain families (PFAM) are indicated.

  It is homologous to other shell proteins including L-ascorbate oxidase [Crassostrea gigas], oxidoreductase OpS5-like [Crassostrea virginica] and other shell laccase-like proteins such as laccase-2-like [Crassostrea virginica], laccase-1-like [Pomacea canaliculata] or laccase-4-like [Lingula anatina].

	• G-protein	coupled	receptor	112	from	Mizuhopecten	yessoensis
	(gi|1205812537|gb|OWF34702.1|) was identified only in the AIM matrices (samples
	S2/4/6/8). It is a very acidic (theoretical pI 4.36) high molecular weight (302.80 kDa)

Acknowledgements JS, FM and BD would like to thank Emmanuel Fara (uB-FC) and Matthew Collins (Universities of Copenhagen and Cambridge) for valuable insights and discussions. JS, FM and BD are supported by the PHC Galilée programme, Italo-French University (UIF/UFI) (project G18-464 / 39612SB) and JS acknowledges the support of the Campus France fund obtained from French embassy in Italy (BGF) and program Eiffel. BD is funded by the "Giovani Ricercatori -Rita Levi Montalcini" Programme (MIUR; Ministero dell'Istruzione dell'Università e della Ricerca). The work performed at UMR CNRS 6282 Biogéosciences (JS, FM), Dijon was financed via the annual recurrent budget of this research unit and via extra CNRS funding (INTERRVIE). The work performed at SOLEIL synchrotron (JS, FM, MA, MT, JP, FJ) was financed via proposal no. 20180213 at DISCO IMAGING beamline.

Data deposition

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [START_REF] Vizcaíno | The Proteomics Identifications (PRIDE) database and associated tools: status in 2013[END_REF] partner repository with the dataset identifier PXD016760.

Competing interests statement

The authors declare that no competing interests exist.