Marco Gerdol

Nicolas Puillandre

Gianluca De Moro

Corrado Guarnaccia

Marianna Lucafò

Monica Benincasa

Ventislav Zlatev

Chiara Manfrin

Valentina Torboli

Piero Giulio Giulianini

Gianni Sava

Paola Venier

Alberto Pallavicini
email: pallavic@units.it

Identification and characterization of a novel family of cysteine-rich peptides (MgCRP-I) from Mytilus galloprovincialis

Keywords: toxin, antimicrobial peptide, bivalve mollusc, mussel, transcriptome Toxin, antimicrobial peptide, bivalve mollusc, mussel, transcriptome

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Introduction

Marine ecosystems are characterized by an astonishing species diversity, with over 2 million different eukaryotic species belonging to various phyla estimated to compose the marine fauna [START_REF] Mora | How Many Species Are There on Earth and in the Ocean?[END_REF]. Thus, marine organisms and environments can be regarded as a virtually unlimited source of bioactive compounds, either produced through complex biochemical synthetic reactions or gene-encoded peptides [START_REF] Mayer | Marine pharmacology in 2007-8: Marine compounds with antibacterial, anticoagulant, antifungal, anti-inflammatory, antimalarial, antiprotozoal, antituberculosis, and antiviral activities; Affecting the immune and nervous system, and other miscellaneous mechanisms of action[END_REF].

Nowadays, computer-assisted data mining coupled with the advent of next-generation sequencing (NGS) technologies allows the in silico identification of bioactive molecules also in non-model marine organisms [START_REF] Li | Molluscan antimicrobial peptides, a review from activity-based evidences to computer-assisted sequences[END_REF][START_REF] Sperstad | Antimicrobial peptides from marine invertebrates: Challenges and perspectives in marine antimicrobial peptide discovery[END_REF]. The quick increase of bivalve transcriptome datasets (Suárez-Ulloa, et al. 2013b) and the recent genome sequencing of the oysters Crassostrea gigas [START_REF] Zhang | The oyster genome reveals stress adaptation and complexity of shell formation[END_REF] and Pinctada fucata [START_REF] Takeuchi | Draft genome of the pearl oyster Pinctada fucata: A platform for understanding bivalve biology[END_REF]) further broadens the horizons of genetic and genomic studies in bivalve mollusks. Due to their relevance as sea food and sentinel organisms, significant RNA sequencing efforts, both with 454 and Illumina technologies, have been performed on Mytilus spp. [START_REF] Bassim | Ontogeny of bivalve immunity: assessing the potential of next-generation sequencing techniques[END_REF][START_REF] Craft | Pyrosequencing of Mytilus galloprovincialis cDNAs: Tissue-specific expression patterns[END_REF][START_REF] Freer | Biomineral proteins from Mytilus edulis mantle tissue transcriptome[END_REF][START_REF] Gerdol | RNA sequencing and de novo assembly of the digestive gland transcriptome in Mytilus galloprovincialis fed with toxinogenic and non-toxic strains of Alexandrium minutum[END_REF][START_REF] González | A phylogenetic backbone for Bivalvia: an RNA-seq approach[END_REF][START_REF] Philipp | Massively parallel rna sequencing identifies a complex immune gene repertoire in the lophotrochozoan Mytilus edulis[END_REF][START_REF] Romiguier | Comparative population genomics in animals uncovers the determinants of genetic diversity[END_REF]Suárez-Ulloa, et al. 2013a). Moreover, an unrefined genome of the Mediterranean mussel Mytilus galloprovincialis recently released, further extends the molecular data available for this species [START_REF] Nguyen | Genetic parameters and response to selection in blue mussel (Mytilus galloprovincialis) using a SNP-based pedigree[END_REF]. The bioinformatic analysis of the mussel data has already contributed to the discovery of important immune-related molecules, including pathogen-recognition receptors, signaling intermediates and antimicrobial peptides (AMPs) [START_REF] Gerdol | Big defensins and mytimacins, new AMP families of the Mediterranean mussel Mytilus galloprovincialis[END_REF][START_REF] Gerdol | The C1q domain containing proteins of the Mediterranean mussel Mytilus galloprovincialis: A widespread and diverse family of immune-related molecules[END_REF]Gerdol and Venier ;[START_REF] Rosani | Massively parallel amplicon sequencing reveals isotype-specific variability of antimicrobial peptide transcripts in Mytilus galloprovincialis[END_REF][START_REF] Toubiana | Toll-like receptors and MyD88 adaptors in Mytilus: Complete cds and gene expression levels[END_REF][START_REF] Toubiana | Toll signal transduction pathway in bivalves: Complete cds of intermediate elements and related gene transcription levels in hemocytes of immune stimulated Mytilus galloprovincialis[END_REF]. As reported in this paper, large-scale bioinformatic analyses can also drive the discovery of novel gene families encoding peptides with unique chemico-physical properties and/or sequence patterns.

Actually, cysteine-rich peptides (CRPs) encompass a large and widespread group of secreted bioactive molecules, heterogeneous in primary sequence and structural arrangement, with different functional roles and present in almost all living organisms, from bacteria to fungi, animals and plants [START_REF] Gruber | Insecticidal plant cyclotides and related cystine knot toxins[END_REF][START_REF] Marshall | Cysteine-Rich Peptides (CRPs) mediate diverse aspects of cell-cell communication in plant reproduction and development[END_REF][START_REF] Taylor | Review: Structure-activity relationships in β-defensin peptides[END_REF]. Invertebrate CRPs are particularly abundant and they have been frequently related to the immune defense against potential pathogens (Mitta, et al. 2000b). According to the number of cysteine residues and their arrangement in the tridimensional space, many families of cysteine-rich AMPs have been described in invertebrates, as for instance in crustaceans [START_REF] Bartlett | Crustins, homologues of an 11.5-kDa antibacterial peptide, from two species of penaeid shrimp, Litopenaeus vannamei and Litopenaeus setiferus[END_REF][START_REF] Destoumieux | Penaeidins, a new family of antimicrobial peptides isolated from the shrimp Penaeus vannamei (Decapoda)[END_REF], insects [START_REF] Bulet | Insect antimicrobial peptides: Structures, properties and gene regulation[END_REF] and arachnids [START_REF] Ehret-Sabatier | Characterization of novel cysteine-rich antimicrobial peptides from scorpion blood[END_REF][START_REF] Fogaça | Cysteine-rich antimicrobial peptides of the cattle tick Boophilus microplus: Isolation, structural characterization and tissue expression profile[END_REF]). In Mytilus spp., different families of CRPs have been progressively discovered: defensins [START_REF] Charlet | Innate immunity: Isolation of several cysteine-rich antimicrobial peptides from the blood of a mollusc, Mytilus edulis[END_REF], mytilins (Mitta, et al. 2000a), myticins [START_REF] Mitta | Myticin, a novel cysteine-rich antimicrobial peptide isolated from haemocytes and plasma of the mussel Mytilus galloprovincialis[END_REF], mytimycins [START_REF] Charlet | Innate immunity: Isolation of several cysteine-rich antimicrobial peptides from the blood of a mollusc, Mytilus edulis[END_REF], macins, big defensins [START_REF] Gerdol | Big defensins and mytimacins, new AMP families of the Mediterranean mussel Mytilus galloprovincialis[END_REF]) and myticusins [START_REF] Liao | Molecular characterization of a novel antimicrobial peptide from Mytilus coruscus[END_REF].

Other evolutionarily related CRPs are animal venom components which possess neurotoxic properties, since they can selectively block various types of ion channels for predation or defense [START_REF] Froy | Arthropod defensins illuminate the divergence of scorpion neurotoxins[END_REF][START_REF] De | On the evolution of invertebrate defensins[END_REF]. Notably, spider and scorpion venoms contain an extraordinary mixture of cysteine-rich peptides whose complexity has been only recently fully appreciated by 'omics approaches [START_REF] Ma | Transcriptome analysis of the venom gland of the scorpion Scorpiops jendeki: Implication for the evolution of the scorpion venom arsenal[END_REF][START_REF] Zhang | Transcriptome analysis of the venom glands of the Chinese wolf spider Lycosa singoriensis[END_REF]. Even within the Mollusca phylum some species have developed a lethal venom arsenal to be used for predation: marine gastropods of the genus Conus indeed use a modified radula as a sting to inject and paralyze their prey with a powerful venom cocktail, mostly of peptidic nature (Olivera, et al. 2012). Due to their biological properties, many CRPs have been studied to guide the development of new drugs for therapeutic applications in both human and veterinary medicine [START_REF] Adams | Conotoxins and their potential pharmaceutical applications[END_REF][START_REF] Otero-González | Antimicrobial peptides from marine invertebrates as a new frontier for microbial infection control[END_REF][START_REF] Saez | Spider-venom peptides as therapeutics[END_REF].

Despite having physico-chemical properties similar to cysteine-rich AMPs and toxins, certain animal CRPs lack the expected activities and are instead involved in diverse functions: among these, Kunitz-type [START_REF] Ranasinghe | Structure and function of invertebrate Kunitz serine protease inhibitors[END_REF] and Kazal-type [START_REF] Rimphanitchayakit | Structure and function of invertebrate Kazal-type serine proteinase inhibitors[END_REF] proteinase inhibitors represent two widespread groups. The abundance and diversity of the CRPs described in protostomes is remarkable and, given the poor genomic knowledge of many taxonomic groups, a large part of these peptides probably still remain to be uncovered. In this paper, we report the application of a genome-and transcriptomescale approach to the identification of novel cysteine-rich peptides the Mediterranean mussel M.

galloprovincialis. In agreement to nomenclature criteria reported elsewhere (Gerdol and Venier), we present the new MgCRP-I family, characterized by a conserved pre-pro region and an highly variable mature peptide with six conserved cysteine residues organized in the consensus C(X 3- 6 )C(X 1-7 )CC(X 3-4 )C(x 3-5 )C. We investigated the organization and evolution of mussel MgCRP-I genes, the main features and possible functional roles of the encoded peptides, and finally we discuss their possible involvement in innate immune defense.

Materials and methods

Identification of MgCRP-I sequences in mussel transcriptomes

The M. galloprovincialis Illumina transcriptomes available at the NCBI Sequence Reads Archive (retrieved in February, 2015) were assembled with Trinity v.2014-07-17 [START_REF] Grabherr | Full-length transcriptome assembly from RNA-Seq data without a reference genome[END_REF]) and with the CLC Genomics Workbench 7.5 (CLC Bio, Aarhus, Denmark), using default parameters. Following translation of the assembled contigs into the 6 possible reading frames with EMBOSS TranSeq [START_REF] Rice | EMBOSS: The European Molecular Biology Open Software Suite[END_REF] we investigated the virtual mussel proteins for the presence of the C(X 1-10 )C(X 1-10 )CC(X 1-10 )C(X 1-10 )C signature, using a Perl script developed in-house (available upon request to the corresponding author). Matching sequences were aligned with MUSCLE [START_REF] Edgar | MUSCLE: Multiple sequence alignment with high accuracy and high throughput[END_REF]) to generate a HMMER v3.0 profile [START_REF] Eddy | Accelerated profile HMM searches[END_REF] which was then used to retrieve partialmatching cases within the assembly (e-value cutoff 1x10 -5 ). The procedure was re-iterated until no additional matches could be retrieved. Sequences showing an identity higher than 95% t the nucleotide level were considered as redundant and collapsed in a single consensus sequence, unless they were confirmed by genomic evidence (see section below).

Identification of MgCRP-I genes in the mussel genome

The M. galloprovincialis genomic contigs [START_REF] Nguyen | Genetic parameters and response to selection in blue mussel (Mytilus galloprovincialis) using a SNP-based pedigree[END_REF] were downloaded from GenBank and scanned for the presence of MgCRP-I genes as follows: (i) genes were identified based on BLASTn identity [START_REF] Altschul | Basic local alignment search tool[END_REF]) to the previously identified MgCRP-I transcripts (e-value threshold of 1x10 -5 ); (ii) genomic scaffolds were translated into the six possible reading frames with the EMBOSS Transeq tool [START_REF] Rice | EMBOSS: The European Molecular Biology Open Software Suite[END_REF] and novel MgCRP-I loci were identified with HMMERv 3.0 [START_REF] Eddy | Accelerated profile HMM searches[END_REF].

The genes identified were manually annotated with mRNA and CDS traces, based on: (i) MUSCLE alignment between genomic contigs and the corresponding assembled transcripts, whenever available; (ii) mapping of the available M. galloprovincialis sequencing reads (see [START_REF] Reese | Improved Splice Site Detection in Genie[END_REF]). An example of the results of the annotation procedure is shown in Supplementary Figure 1 (Supplementary material online). Results obtained from the genome and transcriptome analyses were compared and redundant results (identity percentage higher than 95%) were removed, unless multiple gene copies were confirmed in the mussel genome (e.g. the presence of paralogous genes was tolerated).

MgCRP-I protein sequence analysis

Protein translations of mussel genes and transcripts matching the sequence pattern mentioned above were further analyzed as follows: the presence of a signal peptide was detected with SignalP 4.0 [START_REF] Petersen | SignalP 4.0: discriminating signal peptides from transmembrane regions[END_REF], and discriminated from transmembrane domains with Phobius [START_REF] Käll | A combined transmembrane topology and signal peptide prediction method[END_REF]. Potential sites of post-translational proprotein convertase cleavage were identified with ProP 1.0 [START_REF] Duckert | Prediction of proprotein convertase cleavage sites[END_REF]. Possible post-translational C-terminal cleavage sites by carboxypeptidase E or by peptidylglycine, α-amidating monooxygenase were detected with ELM [START_REF] Dinkel | ELM-the database of eukaryotic linear motifs[END_REF]). The subcellular localization was predicted (for full-length peptides only) with TargetP 1.1 [START_REF] Emanuelsson | Locating proteins in the cell using TargetP, SignalP and related tools[END_REF]. Isoelectric point and molecular weight of the predicted mature peptides was calculated at ExPASy (http://web.expasy.org/compute_pi/). Structural homologies with proteins deposited in the RCSB Protein DataBank database were investigated by Phyre2 [START_REF] Kelley | Protein structure prediction on the Web: a case study using the Phyre server[END_REF].

The probabilities of codon bias for the six conserved cysteines and for the arginine residue responsible of the post-translational pro region cleavage were calculated assuming a binomial distribution, based on the codon usage inferred from the M. galloprovincialis transcriptome [START_REF] Gerdol | RNA sequencing and de novo assembly of the digestive gland transcriptome in Mytilus galloprovincialis fed with toxinogenic and non-toxic strains of Alexandrium minutum[END_REF]) and using the tool cusp included in the EMBOSS package [START_REF] Rice | EMBOSS: The European Molecular Biology Open Software Suite[END_REF].

We used PAML 4.7 [START_REF] Yang | PAML 4: Phylogenetic analysis by maximum likelihood[END_REF]) and the graphical interface PAMLX 1.2 [START_REF] Xu | pamlX: A Graphical User Interface for PAML[END_REF] to test whether some sites in the codon-based alignments of MgCRP-I nucleotide sequences were under positive selection. In detail, only full-length coding sequences were processed and two site F o r P e e r R e v i e w 8 models were compared: M1, which assumes that the dN/dS ratio along the sequence ranges from 0 to 1 (purifying selection to neutral drift), and M2, which assumes that a few sites have a dN/dS ratio (i.e., ω > 1; positive selection). The likelihoods of the two models were compared using a likelihood ratio test (LRT) with a χ 2 distribution, with 2 degrees of freedom. The Empirical Bayes approach was used to calculate the posterior probabilities (PP) for site classes. Positive selection was concluded at PP > 0.95.

Comparative genomics analyses

The NGS Illumina transcriptome data available for 70 bivalve species were downloaded from the Sequence Read Archive (SRA). The full list and the corresponding Bioproject accession IDs are shown in Supplementary Table S1, supplementary material online. The bivalve sequence datasets were independently de novo assembled with the CLC Genomic Workbench 7.5 (CLC Bio, Aarhus, Denmark). All transcriptomes were translated into the six possible open reading frames with EMBOSS Transeq [START_REF] Rice | EMBOSS: The European Molecular Biology Open Software Suite[END_REF] and significant similarity with MgCRP-I proteins was assessed with BLASTp (e-value threshold 0.01) and HMMER v 3.0 using the protein profile mentioned above (p-value threshold 0.01).

The complete UniProtKB/Swiss-Prot protein sequence database and the whole set of peptides predicted from the fully sequenced genomes of C. gigas (release 9) [START_REF] Wang | The oyster genome reveals stress adaptation and complexity of shell formation[END_REF] and P. fucata (v 1.0) [START_REF] Takeuchi | Draft genome of the pearl oyster Pinctada fucata: A platform for understanding bivalve biology[END_REF] were screened for the presence of the C-C-CC-C-C pattern with a custom Perl script, without any constraint about the spacing between cysteine residues. Only full-length sequences shorter than 100 amino acids and showing a signal peptide by SignalP 4.0 [START_REF] Petersen | SignalP 4.0: discriminating signal peptides from transmembrane regions[END_REF])) were selected. Sequences bearing more than 7 cysteine residues within the mature region were considered as characterized by more complex disulfide arrays and discarded.

The possible presence of mis-annotated CRP-I-like genes in C. gigas and P. fucata was evaluated by performing the same analyses on the genomic scaffolds translated into the 6 possible reading frames with EMBOSS TranSeq.

Phylogeny and evolutionary tests

We used all the available MgCRP-I proteins, their orthologous sequences identified in Mytilus edulis (MeCRP-I), Mytilus californianus (McCRP-I) and Mytilus trossulus (MtCRP-I), and the positive hits resulting from the data mining (see Materials and methods), to infer the phylogenetic relationships among sequences bearing a similar cysteine signature. Given the high sequence diversity, only the signal peptides, as predicted using SignalP 4.0 [START_REF] Petersen | SignalP 4.0: discriminating signal peptides from transmembrane regions[END_REF], were retained to facilitate sequence alignment. Following alignment with Muscle [START_REF] Edgar | MUSCLE: Multiple sequence alignment with high accuracy and high throughput[END_REF]) and manual refinement, maximum likelihood analyses were performed with RaxML [START_REF] Stamatakis | RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models[END_REF] as implemented on the CYPRES Portal (www.phylo.org/portal2), using the RAxML-HPC2 on TG Tool. The robustness of the nodes was assessed with a bootstrapping procedure of 100 replicates.

Because the evolutionary relationships of the peptides included in the analysis with other peptides were unknown, no outgroup was considered and a mid-point rooting strategy was applied. A similar analysis was performed with a dataset that included only the M. galloprovincialis CRP-I peptides.

Partial MgCRP-I peptides with an incomplete signal peptide were excluded from the analyses.

Peptide synthesis, oxidative folding and disulfide mapping

MgCRP-I 7 (26 aa) and MgCRP-I 9 (26 aa) peptides were synthesized according standard solid-phase Fmoc chemistry using 4 equivalents of HCTU/Fmoc-Xaa-OH/DIEA (0.95/1.00/1.90) with respect to the resin loading (Tentagel S-Trt, 0.2mmol/g (Sigma-Aldrich, St. Louis, MO)). The synthesis was semi-automatically performed with a customized Gilson Aspec XL peptide synthesizer (Middleton, WI) on a 0.05 mmol scale. Cysteines were manually added as N-α-Fmoc-S-trityl-L-cysteine pentafluorophenyl ester in order to minimize racemization. After cleavage from the resin the peptides were precipitated with diethylether, washed and freeze-dried. The peptides reduced by TCEP treatment were purified by RP-HPLC on a semipreparative Zorbax 300SB-C18 9.4x250mm column (Agilent, Santa Clara, CA) using a gradient from water/0.1% TFA to acetonitrile/0.1% TFA.

Pure peptide fractions from RP-HPLC were checked by electrospray mass spectrometry (amaZonSL iontrap, Bruker (Billerica, MA)) and immediately diluted at 0.1 mg/ml in either of the following refolding buffers: (i) RefoldA: 0.2 M Tris-HCl, 2 mM EDTA, 10 mM GSH, 1 mM GSSG, pH 8, previously degassed with argon bubbling; (ii) RefoldB: 50 mM NaOAc, 1 mM EDTA, 1 mM GSH, 0.1 mM GSSG, 2M (NH 4 ) 2 SO 4 , pH 7.7. The oxidative refolding proceeded for 18 h at 4°C, was quenched by TFA addition and finally checked by LC-MS analysis.

All proteolysis reactions were carried out at 37 °C for 18-48 h in sodium acetate buffer (100 mM, pH 5.5) containing 1 M GuHCl and 5 mM CaCl 2 . The purified MgCRP-I 7 peptide (60 µg) was dissolved in 90 µl of buffer and trypsin (3 µg) was added. A second aliquot of MgCRP-I 7 (60µg in 90µl) was incubated for 48 h at 37 °C in the presence of chymotrypsin (6 µg). Digestions of MgCRP-I 9 with trypsin and chymotrypsin were carried out in the same conditions. The digestions were quenched using formic acid (1% final) and the proteolytic fragments were fractionated by RP-HPLC (column Jupiter C18, 1x50mm, Phenomenex (Torrance, CA) using a gradient from water/0.1% formic acid to 60% acetonitrile and analyzed by LC-MS/MS (amaZonSL, Bruker (Billerica, MA)).

Gene expression analysis

The expression levels of selected MgCRP-I genes were evaluated in samples representing hemolymph, digestive gland, inner mantle, mantle rim, gills, foot and posterior adductor muscle.

Total RNA was extracted from the tissues of 30 adult specimens (5-7 cm shell length) collected from the Gulf of Trieste, Italy, homogenized in equal quantity in Trizol® (Life Technologies, Carlsbad, CA) according to the manufacturer's protocol. RNA quality was assessed by electrophoresis on denaturing agarose gel and its quantity was estimated by UV-spectrophotometry. cDNAs were prepared using a qScript™ cDNA Synthesis Kit (Quanta BioSciences Inc., Gaithersburg, MD) according to the manufacturer's instructions. Primer pairs were designed to obtain the specific PCR amplicons (Table 1), with the exception of the primer pairs co-amplifying the paralogous sequences MgCRP-I 3/25 and MgCRP-I 10/26. The 15 µL PCR reaction mix comprised 7.5 µL of SsoAdvanced™ SYBR® Green Supermix (Bio-rad, Hercules, CA), 0.3 µL of each of the two 10 µM primers and 2 µL of a 1:20 cDNA dilution.

The following thermal profile was used for qPCR amplification in a C1000 thermal cycler (Bio-Rad, Hercules, CA): an initial denaturation step at 95° C for 3', followed by 40 cycles at 95° for 5" and 55° for 30". The products of amplification were analyzed with a 65°/95° C melting curve. The expression of the selected genes was calculated with the delta Ct method; Ct values were corrected based on primer pairs PCR efficiencies using Lin-RegPCR [START_REF] Ramakers | Assumption-free analysis of quantitative realtime polymerase chain reaction (PCR) data[END_REF]) and expression values were normalized using the elongation factor EF-1 as a housekeeping gene.

Results are shown as the mean with standard deviation of three technical replicates.

Cytotoxicity assays

Human colorectal carcinoma (HT-29), human neuroblastoma (SHSY5Y) and breast cancer (MDAMB231) cell lines were used for the cytotoxicity assays. HT-29 was maintained in RPMI-1640 and MDAMB231 was maintained in Dulbecco's Modified Eagle's Medium (DMEM): the culture medium was supplemented with 10% (v/v) fetal bovine serum (FBS), penicillin (100 U/mL), streptomycin (100 µg/mL), and L-glutamine 2 mM. SHSY5Y was cultured in DMEM medium supplemented with penicillin (100 U/mL), streptomycin (100 µg/mL), L-glutamine 2 mM and with 10% heat-inactivated fetal bovine serum. Cells were grown at 37 °C in a 95 % air and 5 % CO 2 humidified incubator.

HT-29, SHSY5Y and MDAMB231 were harvested by trypsinization and plated into 96-well culture plates at a density of approximately 1.5 × 10 4 cells per well. After 24 hrs of incubation, different concentrations of MgCRP-I 7 and 9 (10, 1, 0.1 and 0.01 µM) dissolved in culture medium were added to each well. Then, the samples were incubated 24 hrs at 37 °C in the humidified atmosphere (5 % CO 2 ). The colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was performed to assess the metabolic activity of cells treated as described above. Aliquots of 20 µl stock MTT (5 mg/mL) were added to each well, and cells were then incubated for 4 hrs at 37 °C. Cells were lysed with isopropanol HCl 0.04 N. Absorbance was measured at 540 and 630 nm using a microplate reader (Automated Microplate Reader EL311, BIOTEK® Instruments, Vermont, USA). All measurements were done in six technical replicates, and three independent experiments were carried out.

Insecticidal test

The synthetic peptides MgCRP-I 7 and 9 were dissolved in PBS and injected with a sterile syringe in Zophobas morio larvae (~50 mm long, weighting ~500 mg). The control group (n = 10) was injected with a volume of 50 µl PBS. Two experimental groups of larvae for each peptide (n = 10) were injected with 30 and 300 µg peptide/Kg body weight respectively, for an injection volume of 50 µl. Larvae were monitored for signs of neurotoxicity for 48 hours, including lack of movement, twitching and death. During the experimental time course, larvae were not fed and kept at room temperature. The median lethal dose (LD50) for the two mussel peptides was calculated according to Tedford and colleagues (2001).

Bacterial/fungal strains and minimum inhibitory concentration (MIC assay)

The growth inhibitory effect of MgCRP-I 7 and 9 was tested on Escherichia coli ATCC were diluted 1:30 in fresh MHB and incubated at 37°C with shaking for approximately two hours to obtain a mid-logarithmic phase bacterial culture. Fungi were grown on Sabouraud agar (Difco) plates at 30°C for 48 hours. Fungal suspensions were prepared by picking and suspending five colonies in 5 ml of sterile PBS. The turbidity of the bacterial or fungal suspensions was measured at 600 nm and was adjusted to obtain the appropriate inoculum according to previously derived curves relating the number of colony forming units (CFUs) with absorbance.

The antimicrobial activity was evaluated by the broth micro-dilution susceptibility assay performed according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI) and previously described [START_REF] Benincasa | Antifungal activity of Amphotericin B conjugated to carbon nanotubes[END_REF][START_REF] Benincasa | Antimicrobial activity of Bac7 fragments against drug-resistant clinical isolates[END_REF]). Briefly, two-fold serial dilutions of MgCRP-I 7 and MgCRP-I 9 were prepared in 96-well microplates in the appropriate medium, to a final volume of 50 µl. Fifty µl of bacterial suspension in MHB, or fungal suspension in Sabouraud, were added to each well to a final concentration of 1-5x10 5 cells/ml for bacteria, and 5x10 4 cells/ml for fungi. Bacterial and fungal samples were then incubated at 37°C for 24h or 30°C for 48h, respectively. The MIC (Minimum Inhibitory Concentration) was taken as the lowest concentration of peptide resulting in the complete inhibition of visible growth after incubation. All tests were performed in triplicate.

Results and discussion

MgCRP-I sequence features

Overall, we identified 67 different MgCRP-I sequences (Table 2). More in detail, 48 sequences could be identified in over 2 million genomic contig sequences, which provide a preliminary view of the mussel genome [START_REF] Nguyen | Genetic parameters and response to selection in blue mussel (Mytilus galloprovincialis) using a SNP-based pedigree[END_REF]. Twenty-three of them were also detected as expressed transcripts in publicly available RNA-seq data. In addition, 19 expressed sequences with no match in the genomic contigs were identified, likely corresponding to genes located in genomic regions which are still not covered by the assembly. Overall, 41 sequences can be considered of full length, as the entire CDS from the initial ATG to the STOP codon was represented. The remaining partial sequences (Table 2) were lacking either the 5' or the 3' end, due to truncated genomic contigs or low read coverage from RNA-seq data.

The MgCRP-I peptides are secreted pro-peptides characterized by the highly conserved cysteine array C-C-CC-C-C: all the members of this family display an unambiguous N-terminal signal peptide cleavage site, followed by a ~15 residues long pro-region ending with a highly conserved dibasic cleavage site for proprotein convertases (KR or, more rarely, RR). While the signal peptide and pro-peptide regions show low sequence variability, the C-terminal region of MgCRP-I corresponding to the putative mature peptide, appears hypervariable (Figure 1). The six invariant cysteine residues involved into the formation of intramolecular disulfide bridges are embedded within this highly variable region. The two central cysteine residues (Cys3 and Cys4) are directly linked with a peptidic bond. As a result, the basic consensus of these peptides can be defined as C(X 3-6 )C(X 1-7 )CC(X 3-4 )C(x 3-5 )C (Figure 1 and 2).

Most MgCRP-I peptides present a short C-terminal extension and, after the 6 th cysteine, they often display dibasic amino-acidic motifs which might be the target of post-translational cleavage by carboxypeptidase E, one of the most common modifications observed in neurotoxic peptides 15 from invertebrates such as scorpion venoms [START_REF] Xiong | The cDNA and genomic DNA sequences of a mammalian neurotoxin from the scorpion Buthus martensii Karsch[END_REF]) and conotoxins [START_REF] Fan | A novel conotoxin from Conus betulinus, κ-BtX, unique in cysteine pattern and in function as a specific BK channel modulator[END_REF][START_REF] Wang | cDNA cloning of two A-superfamily conotoxins from Conus striatus[END_REF]. Secreted cysteine-rich peptides are among the molecules undergoing the largest amount of different post-translational modifications, as demonstrated by the case of conotoxins [START_REF] Bergeron | A 'conovenomic' analysis of the milked venom from the mollusk-hunting cone snail Conus textile-The pharmacological importance of post-translational modifications[END_REF][START_REF] Craig | Post-translationally modified neuropeptides from Conus venoms[END_REF]) and the case of MgCRP-I peptides could be similar; given the difficulty of obtaining purified peptides from mussel tissues due to their low expression levels, in the absence of proteomic studies we had to rely on in silico prediction of the most likely modification sites.

Based on the predicted proteolytic cleavages, and with a few exceptions, the virtual MgCRP-I mature peptides are 25-38 amino acids long with estimated molecular weight of 2.5-4 KDa. Almost invariably, the mature peptides have a basic isoelectric point (mostly between 8 and 9.5), indicative of positive net charge at physiological pH, which might be balanced by the presence of conserved negatively charged residues in the pro-region (Figure 1); this feature might be important for the biological activity of MgCRP-I peptides, as it is maintained in all sequences despite their remarkable sequence variability.

In addition to the standard pre-pro-peptide organization described above, several peculiar transcripts, named "multi-MgCRP-I" encoding precursors characterized by multiple cysteine-rich modules were also identified (Table 2). The modules, ranging from 2 to 4 in number, are structurally close to each other, as each Cys6 of the N-terminal module is separated by just 2-4 residues from the Cys1 of the following one. Cysteine-rich domains of multi-MgCRP-I do not show any peculiarity compared to those of regular, mono-domain peptides (see supplementary figure S3, supplementary material online) and the different domains within the same sequence are likely derived by the duplication of a single original module. Although the functional significance of the multi-MgCRP-I peptides is still unknown, the maintenance of cysteine pattern and isoelectric points within each single domain (data not shown) suggest that these long precursors might be posttranslationally cleaved into smaller functional peptides. In this case, such a process would be an interesting strategy adopted to achieve the co-expression of different variants, in a similar fashion to other invertebrate AMPs [START_REF] Herbst | Antimicrobial and pore-forming peptides of free-living and potentially highly pathogenic Naegleria fowleri are released from the same precursor molecule[END_REF][START_REF] Ratzka | Molecular Characterization of antimicrobial peptide genes of the carpenter ant Camponotus floridanus[END_REF].

Structure of MgCRP-I genes

Despite the low average size of the assembled genomics contigs of M. galloprovincialis, we could identify eleven MgCRP-I gene regions corresponding to a full-length coding sequence, from the initial ATG to the STOP codon 14,18,24,28,40,45,11,12 and 13) and several partial matches, either to the 3' or to the 5' region of the CDS (Table 2).

The structure of MgCRP-I genes is conserved, with four exons and three introns (Figure 2). The first exon, which could only be annotated in five sequences thanks to the alignment with RNA-seq data, partially includes the 5'UTR region. In most cases, the second exon encodes the first 17 amino acids of the precursor protein, thus comprising most of the signal peptide. A phase-1 intron separates the second and the third exon. The third exon is ~100 nt long and covers the signal [START_REF] Nguyen | Genetic parameters and response to selection in blue mussel (Mytilus galloprovincialis) using a SNP-based pedigree[END_REF], this has to be considered as 2). For the same reason, the presence of common regulatory regions and transposable elements which could have driven the expansion of this gene family is matter of future investigations.

Gene duplication is not sufficient by itself to explain some peculiar features of MgCRP-I genes: indeed, the amino acid diversity of the peptide precursors is strikingly higher within the mature peptide region compared to the signal peptide and pro-region which are, in turn, highly conserved (Figure 1). This observation suggests an increased rate of mutations within the 4 th exon, and an accelerated evolutionary rate. The likelihood ratio tests we performed to assess this hypothesis strongly support positive selection of the MgCRP-I genes (p-value = 1.717x10 -8 ). In fact, we could identify nine positively selected sites (PP > 0.95), all located within the mature peptide region, after the cleavage site of the pro-peptide (Figure 1). Five out of the six invariable cysteines engaged in disulfide bridges, buried within this hypervariable and positively selected region, undergo site-specific codon preservation (Figure 4). This peculiar phenomenon, which was also observed for the arginine responsible of the pro-peptide cleavage, is likely driven by the unique properties of these residues for the maintenance of the tridimensional structure and an efficient biosynthesis and folding of the mature peptide, as suggested for many other protein families, including conotoxins [START_REF] Conticello | Mechanisms for evolving hypervariability: The case of conopeptides[END_REF][START_REF] Steiner | On the importance of oxidative folding in the evolution of conotoxins: cysteine codon preservation through gene duplication and adaptation[END_REF].

Although structurally important codons appear to be somehow protected from variation, the high selective pressure acting on the fourth exon (encoding the entire mature peptide) of MgCRP-I

CRP-I sequences are only found in the order Mytiloida

Following BLAST searches, the MgCRP-I peptides did not show significant sequence similarity with any other sequence deposited in public databases and the prediction of their tridimensional structure was considered unreliable by Phyre 2, due to the absence of models sharing sufficient sequence affinity within the PDB database. For this reason, we investigated the presence of MgCRP-I-like sequences in genomic and transcriptomic,datasets which are increasingly available also for bivalve molluscs (Suárez-Ulloa, et al. 2013b).

Looking for short secreted CRP-I-like peptides in the transcriptomes of 70 different species (see Materials and methods and supplementary table S1, supplementary material online), we could find the CRP-I signature only in the mussels M. edulis, M. trossulus, and M. californianus.

The first two species and M. galloprovincialis are widespread and genetically close to each other, as evidenced by the presence of natural hybrid populations [START_REF] Beaumont | Hybridisations between Mytilus edulis and Mytilus galloprovincialis and performance of pure species and hybrid veliger larvae at different temperatures[END_REF]) whereas M. californianus is distributed in the Pacific coast of North America and is more distantly related to the other mussel ecotypes [START_REF] Hilbish | Origin of the antitropical distribution pattern in marine mussels (Mytilus spp.): routes and timing of transequatorial migration[END_REF]. The full length CRP-I sequences identified, here named McCRP-I, MeCRP-I or MtCRP-I on the basis of the species name, are reported in supplementary material online. No CRP-I-like sequence was detected in any of the other bivalve transcriptomes and we hypothesize their complete absence or no detectable expression in the analyzed bivalve species. Due to the high depth of NGS technologies, the latter hypothesis is unlikely and the genomic analysis of the oysters C. gigas and P. fucata strongly supports the absence of CRP-I-like genes, thus ruling out the possibility of a missed detection of poorly expressed transcripts. In addition, no evidence of CRP-I-like peptides was found in Limnoperna fortunei and Perna viridis, the only two species among the over 50 different genera of the order Mytiloida, beside Mytilus spp., which have been subjected to Illumina RNA-sequencing so far.

Based on the available data, CRP-Is are certainly present in Mytilus spp. and appear to be absent in other mytiloids. Since the C-C-CC-C-C array is not common in bivalves and nothing similar was found in the fully sequenced genomes of C. gigas (order Ostreoida) and P. fucata (order Pterioida), the CRP-I-like sequences, appear to have a narrow taxonomical distribution, comparable to that of other mussel cysteine-rich peptides (i.e. mytilins, myticins and mytimycins which cannot be found outside Mytiloida).

Occurrence of the C-C-CC-C-C array in protein databases and relationship with CRP-I peptides

Large-scale bioinformatic analyses revealed the presence of a CRP-I-like cysteine pattern mostly in animals and, among them, almost exclusively in invertebrates (see Table 3). More in detail, within the UniProtKB/Swiss-Prot database we found 452 peptides (279 if considering nonredundant peptides based on a 95% sequence identity criterion) mostly belonging to invertebrate animals: cone snails, turrids and terebrids (grouped as Conoidea in Figure 5), spiders, scorpions, pancrustaceans (with a single horseshoe crab and 35 insect sequences) and nematodes. Almost the totality of these peptides display a neurotoxic activity due to their high affinity to ion channels, like in the case of conotoxins, turritoxins and augertoxins [START_REF] Aguilar | Peptide pal9a from the venom of the turrid snail Polystira albida from the Gulf of Mexico: Purification, characterization, and comparison with P-conotoxin-like (framework IX) conoidean peptides[END_REF][START_REF] Imperial | The augertoxins: Biochemical characterization of venom components from the toxoglossate gastropod Terebra subulata[END_REF][START_REF] Terlau | Conus Venoms: A Rich Source of Novel Ion Channel-Targeted Peptides[END_REF]) and peptides produced in the venom gland of spiders (Zhang, et [START_REF] Ma | Transcriptome analysis of the venom gland of the scorpion Scorpiops jendeki: Implication for the evolution of the scorpion venom arsenal[END_REF]. With the exception of a wasp toxin, all the entries from insects were related to bombyxin, a prothoracicotropic hormone involved in morphogenesis [START_REF] Nijhout | Bombyxin is a growth factor for wing imaginal disks in lepidoptera[END_REF].

No CRP-I-like cysteine pattern was found in chordates, with the exception of 6 peptides (5 intestinal trefoil factors, with unclear function, and veswaprin, a snake antimicrobial peptide). A limited number of such peptides were found in Fungi, with the positive hits corresponding to uncharacterized proteins, and in green plants, with all cases representing antimicrobial peptides.

The CRP-I-like array was also found in Baculoviruses (in a viral family of conotoxin-like peptides) [START_REF] Eldridge | Characterization of a baculovirus gene encoding a small conotoxinlike polypeptide[END_REF]. The full list of these peptides with their taxonomic origin and reported function are shown in supplementary table 2, supplementary material online.

Given the great sequence diversity, likely dependent on a fast evolutionary rate of molecular substitutions, the relationships among the MgCRP-I peptides (Figure 3) and with other CRPs peptides from a single taxon were found in different clusters (e.g. conopeptides typically cluster in different groups that correspond to different superfamilies [START_REF] Kaas | Conopeptide characterization and classifications: An analysis using ConoServer[END_REF][START_REF] Puillandre | Molecular phylogeny, classification and evolution of conopeptides[END_REF])); (ii) some peptides, characterized by a long branch in the tree, clustered in a group mostly composed by peptides from another taxon (as an example, two conopeptides clustered within the group of mussel CRP-I peptides, with long branches, suggesting that they may belong to completely different structural classes).

Overall, no distinct traits can unequivocally link the evolutionary history of CRP-I peptides with those of other protein families characterized by the same cysteine array. As CRP-I-like peptides are absent in bivalves other than mussels, the only other molluscan group where the C-C-

Oxidative refolding and disulfide bond topology of synthetic MgCRP-I peptides

Optimization of the oxidative folding yield for peptides with disulfide bridges is still an empiric exercise. Various parameters affect folding yields such as temperature, additives, redox couples, peptide concentration and duration of the folding reaction [START_REF] Bulaj | Formation of disulfide bonds in proteins and peptides[END_REF][START_REF] Bulaj | Folding of conotoxins: Formation of the native disulfide bridges during chemical synthesis and biosynthesis of Conus peptides[END_REF]. We synthetized the MgCRP-I 7 and MgCRP 9 peptides and their purified fractions were subjected to oxidative folding reactions in the presence of redox reagents GSH and GSSG (10:1) at 4°C buffered at pH 8 8 (RefoldA, see Materials and methods). This protocol, with slight modifications, has been commonly used in the refolding of disulfide rich proteins and, for example, it yielded good amounts (~90%) of the cystine knot peptide Huwentoxin-IV in its native structure [START_REF] Deng | Synthesis and biological characterization of synthetic analogs of Huwentoxin-IV (Mutheraphotoxin-Hh2a), a neuronal tetrodotoxin-sensitive sodium channel inhibitor[END_REF] and also in the synthesis of ω-conotoxin MVIIC (~50%). We tested also a high salt refolding mixture (RefoldB) which had shown improved yields of the same conotoxin [START_REF] Kubo | Oxidative folding of ω-conotoxin MVIIC: Effects of temperature and salt[END_REF].

In our hands each peptide refolding mixture (RefoldA) produced one major component, with yields of 85% and 79% for MgCRP-I 7 and MgCRP-I 9, respectively, as determined by RP-HPLC peak area integration (area of the purified reduced peptide before folding = 100%) (see supplementary figure S6 andS7, supplementary material online). Refold B gave similar results but with slightly lower yields (data not shown). According to ESI-MS, the molecular mass of folded MgCRP-I 7 and folded MgCRP-I 9 were in good agreement with those of the fully oxidized products (see supplementary Figure S3 and S4, Supplementary Material online): 3,160.0 (calc. ,090.1 (calc. mono. 3,090.2), respectively. The evidence of one dominant product in the refolding of both peptides is consistent with the assumption of native conformation but, on the other hand, no comparison is currently possible between the synthetic products and the native counterparts eventually present in Mytilus.

We performed a disulfide connectivity prediction using the DiANNA (DiAminoacid Neural Network Application) Web Server of the Boston college [START_REF] Ferrè | DiANNA: A web server for disulfide connectivity prediction[END_REF]; the algorithm predicted a 1-2, 3-4, 5-6 topology for MgCRP-I 9 and a 1-4, 2-6, 3-5 topology for MgCRP-I 7 but with a very high score (0.76 on a maximum of 1) in favor of a Cys14-Cys15 disulfide. At this point we experimentally determined the disulfide bond geometry using enzymatic fragmentation (trypsin and chymotrypsin) and LC/MS/MS. The analysis of both peptides revealed that the cysteine connectivity follows the nearest-neighbor pattern (1-2, 3-4, 5-6), namely Cys3-Cys8, Cys14-Cys15 and Cys20-Cys25 (see supplementary tables S3 and S4, supplementary material online). In a published detailed disulfide classification based on SwissProt and PFam databases the topology 1-2, 3-4, 5-6 is largely represented and contains a very heterogeneous ensemble of protein families [START_REF] Gupta | A classification of disulfide patterns and its relationship to protein structure and function[END_REF]); notably, the vicinal disulfide bond present in our MgCRP-I peptides and formed between the side chains of adjacent cysteines (Cys14-Cys15) represents a rare structural element. The vicinal disulfide, due to its intrinsic constrained nature, is usually described to be accompanied by the formation of a tight turn of the protein backbone [START_REF] Carugo | Vicinal disulfide turns[END_REF]; additionally, the oxidized and reduced states of this bond present very different structural features suggesting a possible role as conformational switch [START_REF] Carugo | Vicinal disulfide turns[END_REF]. At the present time, we do not know the significance that this vicinal bond could have on the activity of the MgCRP-I peptides but the observed 1-2, 3-4, 5-6 topology is distinctively different from the 1-4, 2-5, 3-6 topology common to knottins, which comprise conopeptides and most of the peptides represented in Figure 5 with an experimentally determined tridimensional structure [START_REF] Hartig | Intramolecular disulphide bond arrangements in nonhomologous proteins[END_REF]. The number of sequences related to the MgCRP-I family identified in the many mussel transcriptome datasets analyzed was extremely low (Table 4) and suggests a very limited basal expression of these genes in different tissues under physiological conditions. More in detail, no evidence of MgCRP-Is was found in Sanger sequencing-based EST collections, with the exception of a single M. edulis sequence detected in a SSH library (digestive gland of mussels exposed to styrene). The number of CRP-I sequences detected in the pyrosequencing-based datasets increased, even though in many cases MgCRP-I transcripts could not be detected. Finally, the analysis of Illumina sequencing-based transcriptomes clearly pointed out the high sequencing depth necessary to detect MgCRP-I messenger RNAs, which can be estimated to cumulatively contribute to less than 0.01% (but often to even less than 0.001%) of the total gene expression in most tissues.

MgCRP-I transcript levels

To better evaluate the MgCRP-I tissue-specificity, we analyzed the expression levels of 17 MgCRP-I transcripts by quantitative PCR (qPCR) in different tissues (hemolymph, digestive gland, inner mantle, mantle rim, foot, posterior adductor muscle and gills) of a pool of 30 naïve adult mussels (M. galloprovincialis). We found large variability among the expression profiles of individual CRP-I sequences, with the overall expression levels being almost invariably very low in all tissues. However, three tissues emerged as main sites of MgCRP-I expression, namely the digestive gland, the inner mantle and the mantle rim. Several MgCRP-I displayed, at least to some extent, a certain degree of tissue specificity (Figure 6). In most cases these genes were not expressed at all in hemolymph, foot, gills and posterior adductor muscle, indicating that these are not the primary sites of production of MgCRP-I peptides, which is consistent with RNA-seq data (Table 4).

Overall, the gene expression data leave room to different hypotheses which need to be tested in future experiments: (i) the expression of these peptides may be induced by still unknown specific stimuli; (ii) MgCRP-I are expressed by a low number of highly specialized cells and therefore the global contribution to mRNAs extracted from macro-tissues is low; (iii) MgCRP-I are not expressed in adult individuals, but they play an important role in the early developmental stages (but this hypothesis seems to be disproved by the analysis of M. edulis larvae RNA-seq data, see Table 4).

MgCRP-I synthetic peptides do not show any significant cytotoxic, insecticidal, antifungal and antimicrobial activity

In an attempt to characterize the biological activity of the synthetic peptides MgCRP-I 7 and MgCRP-I 9, we evaluated their cytotoxicity on human tumor cell lines and insect larvae, and their antimicrobial activity on the bacteria E. coli and S. aureus, and on fungal strains of C. albicans and C. neoformans (see Materials and methods). The MTT assays indicated that both synthetic peptides were not cytotoxic on the HT-29, SHSY5Y and MDAMB231 cell lines up to 10 µM concentration.

Similarly, no insecticidal effect was observed in Z. morio larvae 48 hours after the injection of 300 µg peptide/Kg body weight, a quantity much higher than those determining visible neurotoxic effects, or even death, for other invertebrate toxins [START_REF] Yang | Chemical punch packed in venoms makes centipedes excellent predators[END_REF][START_REF] Zhong | A novel neurotoxin from venom of the spider, Brachypelma albopilosum[END_REF]. Finally, the antimicrobial activity assay evidenced that both MgCRP-I 7 and 9 had a MIC > 32 µM on the selected bacterial and fungal strains.

Although these results indicate that MgCRP-I synthetic peptides did not display antimicrobial or cytotoxic activity in the tested conditions, their involvement in defense processes cannot be ruled out. In fact, post-translational modifications might occur in mussel cells but this can hardly be investigated due to the low expression levels of MgCRP-I genes which, in turn, makes the purification of native peptides difficult. Hence, the absence of biological effects could depend on a variety of modifications not present in the synthetic MgCRP-Is but often reported as fundamental for the antimicrobial or toxic activity of short cysteine-rich peptides [START_REF] Bergeron | A 'conovenomic' analysis of the milked venom from the mollusk-hunting cone snail Conus textile-The pharmacological importance of post-translational modifications[END_REF]Buczek, et al. 2005a;Buczek, et al. 2005b;[START_REF] Guder | Posttranslationally modified bacteriocins -The lantibiotics[END_REF].

Other factors could also play a role in the evaluation of MgCRP-Is, such as the possible peptide interactions with the culture medium, and in particular with the serum components. Although these

Conclusions

Thanks to an exploratory bioinformatics approach applied to the NGS sequencing data, we could identify a novel family of cysteine rich peptides, named MgCRP-I, which appears to be exclusively present in Mytiloida, an order of marine filter-feeding mussels. The MgCRP-I gene family and the encoded peptides share a number of structural and evolutionary traits in common with other families of CRPs, which almost invariably have an antimicrobial or toxic function. These marked similarities initially suggested that MgCRP-I peptides could have similar biological functions, thus making them intriguing targets for possible future biotechnological and pharmacological applications. However, all the tests performed on two synthetic MgCRP-I peptides led to inconclusive results, leaving their biological role still puzzling. In addition, the biological targets (both at the molecular and at the species level) of MgCRP-I peptides are still unknown and the events triggering the expression of these molecules are still elusive. In absence of further indications, these questions remain unsolved.

Overall, we have provided a preliminary overview on MgCRP-I peptides, which is intended as starting point for further investigations on their possible action on prokaryotic or eukaryotic cells.

Our work also highlights the possibility of identifying previously uncharacterized, potentially bioactive, peptides from whole genomes and transcriptomes of non-model organisms without any previous knowledge about their primary sequence, an experimental approach which could speed up the discovery or the design of novel molecules with potential biotechnological applications. Due to their still limited genomic knowledge, marine invertebrates in particular represent a virtually unlimited and almost unexplored source of novel bioactive compounds. codon usage for the Arg residue responsible of the pro-peptide cleavage site and for the 6 cysteine residues involved in the disulfide bridges, calculated on M. galloprovincialis MgCRP-I peptides. The probabilities of finding the observed codon biases were calculated assuming a binomial distribution and the codon usage (a priori probabilities) inferred from the tanscriptome published by Gerdol et al. 2014 (75.1%-24.9% for TGT-TGC, encoding Cys, 49.3%-16.3%-13.3%-12.5%-4.7%-3.8% for AGA-AGG-CGA-CGT-CGG-CGC, encoding Arg). Significant (p<0.05) and highly significant (p<0.01) deviations from the expected distributions (p<0,01) are marked by * and ** respectively. NS = not significant.

  with the CLC Genomics Workbench large gap mapping tool; (iii) refinement of splice site positions with Genie

25922,

  Staphylococcus aureus ATCC 25923, two strains of Candida albicans (ATCC 90029 and a clinical isolate), and on four strains of Cryptococcus neoformans (ATCC 90112, ATCC 52816, ATCC 52817 and a clinical isolate). The bacterial inoculum was incubated overnight in Mueller-Hinton Broth (MHB, Difco) at 37°C with shaking. For the assays, the overnight bacterial cultures

  peptide cleavage site and most of the propeptide region. The open reading frame is interrupted by a phase-2 intron, which separates the third and the fourth exon. The last exon invariably comprises the final 10 nucleotides of the pro-region, including the highly conserved dibasic cleavage site, the complete cysteine-rich C-terminal region and the entire 3'UTR region. No additional exons are present in multi-MgCRP-I genes, whose multiple cysteine-rich arrays are encoded by the fourth exon. A schematic representation of the structural organization of MgCRP-I genes and encoded precursor proteins is shown in Figure 2. Gene duplication and positive selection are driving the evolution of the the MgCRP-I gene family The combined genomic/transcriptomic analyses indicate the presence of at least 67 different potentially functional CRP-I loci in the genome of the Mediterranean mussel. Owing to the preliminary nature of the released mussel genome

  . The phylogenetic analysis of the CRP-I signal peptide regions (Figure3), evidenced the existence of several highly similar paralogous genes, highlighting the important role of gene duplication events in the evolution of the MgCRP-I gene family, which in some cases appear to have occurred very recently (supplementary figure S4, supplementary material online). A number of MgCRP-I pseudogenes with frame-shift or missense mutations were identified in the mussel genome (data not shown) and, at the same time, the sequence truncation caused by the small size of the genomic contigs makes impossible to infer how many of the incomplete MgCRP-I loci are fully functional (see Table

  cases introduced loss-of-function mutations, and resulted in the disruption of disulfide bonds (supplementary figure S5, supplementary material online). MgCRP-I 12 and MgCRP-I 23 represent two instructive examples, since the deduced proteins maintain the highly conserved signal peptide and pro-peptide regions, features clearly identifying them as CRP-I related sequences in a phylogenetic analysis though lacking the canonical cysteine array. Indeed, MgCRP-I 12 lacks four out of the six conserved cysteine residues and just the two adjacent residues Cys3 and Cys4 are retained; on the other hand, MgCRP-I 23 is an even more extreme case, as completely devoid of cysteines.

(Figure 5 )

 5 remain unresolved. Nevertheless, several features could be underlined by the phylogenetic analyses. All MgCRP-I sequences and the orthologous sequences from M. edulis, M. trossulus and M. californianus clustered together in a single clade. Most of the other CRPs sequences clustered in distinct groups which included peptides from the same taxon (Pancrustacea, Conoidea, Spiders, Scorpions, Nematodes, Chordates, Plants, Fungi and Viruses), even if: (i)

  -C array is present is represented by Conoidea (Gastropoda). Nevertheless the absence of this molecular motif in the fully sequenced genomes of other gastropods (Aplysia californica, Lottia gigantea and Biomphalaria glabrata) suggests that it might have been acquired independently in these two molluscan groups. Even though the large sequence divergence prevents conclusions, the study of the disulfide bonds topology in the MgCRP-I peptides has provided further support to this hypothesis, as reported in the next section.

  in perspective, a detailed characterization of the MgCRP-Is is beyond the scope of this paper.

Figure 1 :

 1 Figure legends

Figure 2 .

 2 Figure 2. Exon/intron structure of the complete coding regions of the MgCRP-I 13, 14, 28, 45 and

Figure 3 :

 3 Figure 3: Maximum likelihood tree obtained with the MgCRP-I peptides based on the alignment of

Figure 4 :

 4 Figure 4: codon usage for the Arg residue responsible of the pro-peptide cleavage site and for the 6

Figure 5 :

 5 Figure 5: Maximum likelihood tree obtained with the MgCRP-I, the orthologous sequences from

Figure 6 :

 6 Figure 6: gene expression of 17 selected MgCRP-I genes in six tissues (H: hemolymph; DG:

  Sequence variability of MgCRP-I sequences; variability index W is plotted in the upper panel, while the sequence consensus, obtained with Weblogo (http://weblogo.berkeley.edu) is shown in the lower panel. Only positions covered by at least 50% sequences in the global alignment of MgCRP-I peptides are shown. Sites under positive selection are indicated by an asterisk.

  structure of the complete coding regions of the MgCRP-I 13, 14, 28, 45 and multi-MgCRP-I 2 genes (panel A) and corresponding organization of the encoded peptide precursors (panel B). The positions of the signal peptide, pro-region and mature peptide regions are highlighted and each cysteine-rich module is marked by a box.

  Maximum likelihood tree obtained with the MgCRP-I peptides based on the alignment of the signal peptide only. Only Booststraps values > 75 are shown. Some sequences were not considered in this analysis as their N-terminal region was incomplete (see Table2). Arrows indicate two MgCRP-I peptides

  Maximum likelihood tree obtained with the MgCRP-I, the orthologous sequences from other mussel species and all the CRPs mined from UniProtKB/Swiss-Prot (see Materials and methods). Peptides from pancrustaceans, conoideans, spiders, scorpions, nematodes, chordates, plants, fungi, viruses and mussels are shown. Mussel CRP-I peptides are highlighted in a grey background.

  17 selected MgCRP-I genes in six tissues (H: hemolymph; DG: digestive gland; IM: inner mantle; MR: mantle rim; F: foot; G: gills; AM: posterior adductor muscle); primers for MgCRP-I 3 also target MgCRP-I 25, primers for MgCRP-I 10 also target MgCRP-I 26. Bars represent the expression relative to EF-1 alpha; results are mean ± standard deviation of three replicates. MgCRP-I sequences are divided into three panels based on their expression level: panel A -genes with maximum relative expression value comprised between 0.05 and 0.25; panel B -genes with maximum relative expression value comprised between 0.004 and 0.01; panel C -with maximum relative expression value lower than 0.003. Panel D shows a schematic representation of a M. galloprovincialis anatomical features, highlighting the sampled tissues.

Table 1 :

 1 primers designed for assessing the tissue-specific expression levels of MgCRP-I genes by real-time PCR.

Table 2 :

 2 list of the MgCRP-I sequences identified in the present work. *complete sequence correspond to a full-length coding sequence, from the initial ATG to the STOP codon; **T:

	transcriptome; G: genome.

Table 3 :

 3 Number of peptides with a MgCRP-I-like array found in the UniProtKB/Swiss-Prot protein sequence database, listed per taxonomic group (function is indicated if available).

	Taxonomic

group Number of identified CRPs in UniprotKB* Molecular function

	F o
	r
	P
	e e r
	Cone snails Spiders Fungi	163 223 2 R e v i e w uncharacterized conotoxins venom toxins
	Viruses
	(Buculoviridae)	4	uncharaxterized
	Insects	35	hormones/venom toxins
	Green Plants	5	antimicrobial peptides
	Scorpions	8	venom toxins
	Terebrids	2	augertoxins
	Horseshoe crabs	1	antimicrobial peptides
	Nematodes	1	Insulin-like
	Vertebrates	6	unnknown/antimicrobial peptides
	Turrids	2	turritoxins

Table 4 :

 4 Number of CRP-I sequencing reads identified in the publicly available transcriptome datasets from Mytilus spp. (retrieved from NCBI SRA, February 2015).

	Page 37 of 43
	Database	REF	tissue	sequencing strategy	total number of sequences	sequences related to MgCRP-I
	M. galloprovincialis (Venier, et al. 2009)	mixed tissues	Sanger	19,617	0	0
	M. californianus	NA	mixed tissues	Sanger	42,354	0	0
	M. coruscus	NA	foot	Sanger	719	0	0
	M. galloprovincialis (Craft, et al. 2010)	foot	454	31,227	0	0
	M. galloprovincialis (Craft, et al. 2010)	mantle	454	52,057	0	0
	M. galloprovincialis (Suárez-Ulloa, et al. 2013a)	digestive gland	454	2,206,478	0	0
	M. trossulus	(Romiguier, et al. 2014)	mixed tissues	Illumina	~58 million	142	<0
	M. galloprovincialis NA	hemocytes	Illumina	~106 million	490	<0
	M. galloprovincialis NA	gills	Illumina	~52 million	182	<0
	M. edulis	(Philipp, et al. 2012)	hemocytes	454	407,061	2	<0
	M. edulis	(Bassim, et al. 2014)	larvae	Illumina	~295 million	3,423	0.0
	M. californianus	(Romiguier, et al. 2014)	mixed tissues	Illumina	~78 million	644	0.0
	M. edulis	(Philipp, et al. 2012)	mixed tissues	454	365,626	3	0.0
	M. edulis	(Freer, et al. 2014)	mantle	454	494,391	8	0.0
	M. galloprovincialis (Craft, et al. 2010)	gill	454	58,271	1	0.0
	M. galloprovincialis NA	gills	Illumina	~120 million	3,103	0.0
	M. edulis	(Philipp, et al. 2012)	digestive gland	454	1,112,061	30	0.0
	M. galloprovincialis (Gerdol, et al. 2014)	digestive gland	Illumina	~54 million	3,269	0.0
	M. galloprovincialis NA	posterior adductor muscle	Illumina	~103 million	9,429	0.0
	M. galloprovincialis (Romiguier, et al. 2014)	mixed tissues	Illumina	~108 million	11,695	0.0
	M. edulis	(Philipp, et al. 2012)	inner mantle	454	323,482	46	0.0
	M. edulis	(Romiguier, et al. 2014)	mixed tissues	Illumina	~103 million	15,086	0.0
	M. edulis	(González, et al. 2015)	mantle/foot	Illumina	~49 million	8,492	0.0
	M. edulis	NA	mixed tissues	Sanger	5,300	1	0.0
	M. galloprovincialis NA	mantle	Illumina	~108 million	30,802	0.0
	M. galloprovincialis (Craft, et al. 2010)	digestive gland	454	33,992	2	0.0
	M. edulis	(Philipp, et al. 2012)	mantle rim	454	324,592	299	0.0

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Acknowledgements

This work was supported by BIVALIFE (FP7-KBBE-2010-4) and PRIN 2010-11 (20109XZEPR).