Aude Andouche

Stéphane Valera

Sébastien Baratte
email: sebastien.baratte@mnhn.fr

Exploration of Chemosensory Ionotropic Receptors in Cephalopods: the IR25 gene is expressed in the Olfactory Organs, Suckers, and Fins of Sepia officinalis

Keywords: cephalopod, ionotropic glutamate receptor, fin, olfactory organ, sucker

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INTRODUCTION

As active marine predators, cephalopods (Mollusca) are mostly renowned for their visual capacities and their ability for camouflage. Their eyes and the associated optic lobes reach a size and complexity only comparable to that of vertebrates [START_REF] Young | Evolution of the cephalopod brain[END_REF]. They even possess light sensors all over their body [START_REF] Ramirez | Eye-independent, light-activated chromatophore expansion (LACE) and expression of phototransduction genes in the skin of Octopus bimaculoides[END_REF][START_REF] Kingston | Visual phototransduction components in cephalopod chromatophores suggest dermal photoreception[END_REF]. But cephalopods are also good at chemoreception [START_REF] Cosmo | Cephalopod olfaction[END_REF]. In Sepia officinalis, the common cuttlefish of Europe and North Africa, olfaction is associated with prey and predator detection [START_REF] Boal | Distance chemoreception in the common cuttlefish, Sepia officinalis[END_REF]Romagny et al. 2012;[START_REF] Maselli | Sensorial Hierarchy in Octopus vulgaris's Food Choice: Chemical vs[END_REF], intraspecific communication [START_REF] Boal | Distance chemoreception in the common cuttlefish, Sepia officinalis[END_REF][START_REF] Boal | Social recognition using chemical cues in cuttlefish (Sepia officinalis Linnaeus, 1758)[END_REF][START_REF] Zatylny | ILME: a waterborne pheromonal peptide released by the eggs of Sepia officinalis[END_REF] including mating choice [START_REF] Boal | Female choice of males in cuttlefish (Mollusca: Cephalopoda)[END_REF]) and short-distance navigation [START_REF] Alves | Short-distance navigation in cephalopods: a review and synthesis[END_REF]. At the neuroendocrine level, olfaction is one of the key senses controlling reproductive behaviors in cephalopods [START_REF] Polese | Role of olfaction in Octopus vulgaris reproduction[END_REF]. Coleoid cephalopods (cuttlefish, octopuses, and squids) also possess two unique and fascinating features, namely their arms and suckers, which are not only motor organs able to touch, seize and catch objects but also able to discriminate different surfaces and tastes [START_REF] Wells | Taste by Touch: Some Experiments with Octopus[END_REF][START_REF] Wells | Tactile Discrimination of Surface Curvature and Shape by the Octopus[END_REF].

Similar to terrestrial animals, olfaction in cephalopods is mediated by sensory cells able to detect molecules arriving from a distance source at their specialized olfactory organs.

Rhinophores of nautiluses are quite similar to rhinophores and tentacles of gastropods, but olfactory organs of coleoids broadly resemble two nostrils located behind each eye (von Kölliker 1844). They contain well-described types of ciliated sensory cells [START_REF] Graziadei | Receptors in the sucker of the cuttlefish[END_REF]Graziadei and Gagne 1976;[START_REF] Wildenburg | Ultrastructure of the olfactory organ during embryonic development and at the hatching stage of Loligo vulgaris LAM (Cephalopoda)[END_REF][START_REF] Polese | Olfactory organ of Octopus vulgaris: Morphology, plasticity, turnover and sensory characterization[END_REF], whose role in chemoreception have been confirmed by physiological assays [START_REF] Mobley | Odorant responsiveness of squid olfactory receptor neurons[END_REF]). But we still have little information on how the diversity of odorant cues is perceived and integrated [START_REF] Mobley | Evidence for multiple signaling pathways in single squid olfactory receptor neurons[END_REF]. Behind the diversity of cell types, a large diversity of neurotransmitters Downloaded from https://academic.oup.com/chemse/advance-article/doi/10.1093/chemse/bjab047/6412677 by Museum National d'Histoire Naturelle user on 08 November 2021 A c c e p t e d M a n u s c r i p t 4 has been described [START_REF] Scaros | Immunohistochemical approach to understanding the organization of the olfactory system in the cuttlefish, Sepia officinalis[END_REF][START_REF] Scaros | Histamine and histidine decarboxylase in the olfactory system and brain of the common cuttlefish Sepia officinalis (Linnaeus, 1758)[END_REF]) and in the central nervous system, there is nothing resembling the vertebrate glomeruli that could potentialize odor discrimination [START_REF] Scaros | Immunohistochemical approach to understanding the organization of the olfactory system in the cuttlefish, Sepia officinalis[END_REF]. Finally, chemoreceptors for olfaction are still undescribed in cephalopods. Only recently, new receptors have been identified in the sucker rims, where cephalopods are able to "smell" or "taste" by touch. The sucker rims host several types of sensory neurons [START_REF] Graziadei | Receptors in the sucker of the cuttlefish[END_REF]Graziadei and Gagne 1976;[START_REF] Sakaue | Immunohistochemical localization of two types of choline acetyltransferase in neurons and sensory cells of the octopus arm[END_REF][START_REF] Bellier | Immunohistochemical and biochemical evidence for the presence of serotonin-containing neurons and nerve fibers in the octopus arm[END_REF], including chemo-tactile neurons expressing contact-dependent chemoreceptors belonging to the Acetylcholine Receptor family (van Geisan et al. 2020).

The objective of this paper is to identify olfactory chemoreceptors in Sepia officinalis and to validate their expression in chemosensory organs. In molluscs, olfactory receptors may belong to diverse protein families [START_REF] Derby | Molecular Mechanisms of Reception and Perireception in Crustacean Chemoreception: A Comparative Review[END_REF]). Among them, the ionotropic glutamate receptors (iGluRs) constitute a large family of ligand-gated ion channels (Figure 1), which are best characterized by their roles in synaptic communication in vertebrate nervous systems [START_REF] Mayer | Structure and function of glutamate receptor ion channels[END_REF]. The family is divided into diverse subtypes based on their ligand binding properties and sequence similarity: NMDA receptors, Epsilon receptors (lost in Protostomes) and receptors of the AKDF family (AKDF for AMPA, Kainate, Delta and Phi receptors). AMPA receptors mediate the majority of fast excitatory synaptic transmission in the vertebrate brain. Kainate receptors appear to have a modulatory role in this process and NMDA receptors are involved in synaptic and neuronal plasticity [START_REF] Croset | Ancient protostome origin of chemosensory ionotropic glutamate receptors and the evolution of insect taste and olfaction[END_REF].

Recently, a broad analysis of the iGluR family was conducted among metazoa, in which mollusc sequences from Crassostrea gigas (bivalve), Lottia gigantea (gastropod) and Octopus bimaculoides (cephalopod) were included (Ramos-Vicente et al. 2018).

A c c e p t e d M a n u s c r i p t 5

In protostomes, a variant subfamily of iGluRs, the Ionotropic Receptors (IRs), was first identified as a new class of chemosensory receptors in Drosophila melanogaster [START_REF] Benton | Variant ionotropic glutamate receptors as chemosensory receptors in Drosophila[END_REF]) and then in several other species. They are supposed to have evolved from an AKDF ancestor (Figure 1A), while acquiring a chemosensory function and the capacity to detect chemical signals from the external environment [START_REF] Croset | Ancient protostome origin of chemosensory ionotropic glutamate receptors and the evolution of insect taste and olfaction[END_REF]. In insects, for instance, a high diversification has led to a large family of antennal specific IRs [START_REF] Croset | Ancient protostome origin of chemosensory ionotropic glutamate receptors and the evolution of insect taste and olfaction[END_REF]. The IR25 receptor is supposed to be the oldest member of this family, since IR25a gene orthologs are found in molluscs, nematodes, crustaceans and insects, suggesting that this receptor may have fulfilled a chemosensory function in the protostome ancestor (Figure 1A).

It displays a broad expression in many olfactory and gustatory neurons in arthropods [START_REF] Corey | Ionotropic crustacean olfactory receptors[END_REF]Groh-Lunow et al. 2015;[START_REF] Guo | Variant ionotropic receptors are expressed in olfactory sensory neurons of coeloconic sensilla on the antenna of the desert locust (Schistocerca gregaria)[END_REF][START_REF] Zbinden | Comparative Study of Chemosensory Organs of Shrimp From Hydrothermal Vent and Coastal Environments[END_REF][START_REF] Rytz | Ionotropic Receptors (IRs): Chemosensory ionotropic glutamate receptors in Drosophila and beyond[END_REF] and gastropods [START_REF] Croset | Ancient protostome origin of chemosensory ionotropic glutamate receptors and the evolution of insect taste and olfaction[END_REF][START_REF] Liang | Ionotropic Receptors Identified within the Tentacle of the Freshwater Snail Biomphalaria glabrata, an Intermediate Host of Schistosoma mansoni[END_REF].

According to Ramos-Vicente et al. (2018), molluscs are likely to possess members of NMDA, AMPA, Kainate, and Delta subfamilies, as well as IR25 and specific IRs for chemoreception (Figure 1A). In order to explore the diversity of iGluRs in cephalopods and to identify IRs in Sepia officinalis, we realized a large survey of the iGluR family in molluscs, using all available sequences, from public databases, former studies in gastropods [START_REF] Croset | Ancient protostome origin of chemosensory ionotropic glutamate receptors and the evolution of insect taste and olfaction[END_REF][START_REF] Liang | Ionotropic Receptors Identified within the Tentacle of the Freshwater Snail Biomphalaria glabrata, an Intermediate Host of Schistosoma mansoni[END_REF]Ramos-Vicente et al., 2018) and transcriptomes of Sepia officinalis [START_REF] Bassaglia | ESTs library from embryonic stages reveals tubulin and reflectin diversity in Sepia officinalis (Mollusca -Cephalopoda)[END_REF]. Our phylogenetic analyses confirmed the existence of all expected iGluRs families in bivalves, gastropods and cephalopods. We identified one IR25 ortholog in cephalopods, belonging to a mollusc-specific IR25 subfamily. In S. officinalis, we synthesized a riboprobe of the IR25 gene (Soff-IR25) and performed in situ hybridizations (ISH). Our results strongly suggest the involvement of Soff-IR25 in chemoreception as we found it expressed in olfactory organs and suckers. The unexpected

Material and Methods

Ethic statements

All animal procedures were performed in compliance with the European Union (Directive 86/609) and French law (Decree 87/848) regulating animal experimentation. Efforts were made to minimize animal suffering and to reduce the number of animals used.

Animal rearing and tissue collection

Fertilized clutches of Sepia officinalis eggs were collected in the English Channel from the marine stations in Luc-sur-Mer (Université de Caen Basse-Normandie) and from Blainvillesur-Mer (SMEL) between April and July and kept at 18°C in oxygenated sea water in the laboratory. Specimens were sampled daily to obtain a complete collection of different stages over the approximate 30 days of development. The darkly pigmented egg capsule and chorion were removed in sea water. After viewing with a stereo dissecting microscope to determine stages of development according to [START_REF] Lemaire | Table de développement embryonnaire de Sepia officinalis L. (mollusque céphalopode)[END_REF], embryos were anesthetized by progressive temperature lowering on ice. For ISH, embryos were fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS), for different durations depending on the stage. After several washes in PBS, embryos were dehydrated in baths of increasing methanol concentration, and then stored at -20°C in 100% methanol until use. For RNA extraction, whole embryos or dissected embryos were immersed in RNAlater (Ambion, Austin, TX, USA).

Molluscan iGluR collection

Available protein sequences of different mollusc species were screened in NCBI nonredundant (nr) databases. Already published sequences (nucleic or amino acid) from Aplysia californica, Lottia gigantea [START_REF] Croset | Ancient protostome origin of chemosensory ionotropic glutamate receptors and the evolution of insect taste and olfaction[END_REF], Crassostrea gigas, Octopus bimaculoides (Ramos-Vicente et al., 2018) and Biomphalaria glabrata [START_REF] Liang | Ionotropic Receptors Identified within the Tentacle of the Freshwater Snail Biomphalaria glabrata, an Intermediate Host of Schistosoma mansoni[END_REF] were used as queries using either the BLASTx or tBLASTn algorithm, with standard parameters and with BLOSUM62 as a matrix of score. For each query, all significant hits (low primary cut-off with E-value=1.0) were collected until saturation of the collection. Our efforts were concentrated on the three main taxa of molluscs: bivalves with Crassostrea gigas (Cgig), Crassostrea virginica (Cvir), Mizuhopecten yessoensis (Myes), cephalopods with Octopus vulgaris (Ovul) and Octopus bimaculoides (Obim), gastropods with Aplysia californica (Acal), Lottia gigantea (Lgig), Biomphalaria glabrata (Bgla), Elysia cholorotica (Echl), Pomacea caniculata (Pcan). The same procedure was conducted with our own transcriptome database [START_REF] Bassaglia | ESTs library from embryonic stages reveals tubulin and reflectin diversity in Sepia officinalis (Mollusca -Cephalopoda)[END_REF] to extract a maximum of new sequences in Sepia officinalis.

Protein tree building and validation of molluscan iGluR

All collected amino acid sequences of putative mollusc iGluR-like proteins were aligned together with non-molluscan iGluR sequences from Ramos-Vicente et al. (2018). Alignment was performed using ClustalW Multiple Alignment [START_REF] Thompson | CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice[END_REF]) and examined in BioEdit Sequence Alignment Editor [START_REF] Hall | BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT[END_REF]. Maximum Likelihood (ML) trees were built under the Jones-Taylor-Thornton (JTT) model of substitution with 500 bootstrap replicates for node support (MEGA5, [START_REF] Tamura | MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods[END_REF], using a portion of 500 aligned residues covering the Ligand Binding Domain (LBD) and the Transmembrane Domain (TMD) (from domain S1 to domain M3, see Figure 1B,1C). Sequences that were too short and of too low 2018). Collected molluscan sequences that did not well cluster with other Metazoan iGluRs were considered as non-iGluR and were removed. The final collection of molluscan sequences was listed in Supplementary Data S1.

Gene and protein nomenclature

Among all the molluscan iGluR sequences we collected, some had annotations and names from previous studies, some had no annotation or not confirmed annotations (e.g. automated annotations on NCBI). According to the phylogeny we obtained and to the available annotations, we attributed a name to all molluscan sequences. This name was used, together with the accession number, to label sequences in our trees. All data about the sequences, their family assignment and the name we attributed, were summed up in Supplementary Data S1.

Names were given as follows. Receptor names are preceded by a four-letter species abbreviation consisting of an uppercase initial letter of the genus name and three lower case initial letters of the species name (see above). We respected the NC-IUPHAR nomenclature for iGluRs [START_REF] Collingridge | A nomenclature for ligand-gated ion channels[END_REF]): each species name is followed by ""Glu"" and a letter representing the subtype of the receptor (GluK for Kainate, GluA for AMPA, GluN for NMDA and GluD for delta) or IR for Ionotropic Receptors. Distinct subclades were annotated with additional numbers or letters: directly concatenated when orthology or paralogy was similar to that found outside molluscs (e.g. GluN1, GluN2 and GluN3, IR25a) and with a "-m" for mollusc-specific subclades (e.g. . Finally, if multiple copies of an ortholog existed for a species they are given the same name followed by a point and a number (e.g. Cvir-GluK-m7.1 and Cvir-GluK-m7.2).

Reverse Transcription PCR of Soff-IR25

Total RNA was extracted from whole S. officinalis embryos or from hand-dissected tissues (arms, brain, optic lobe, olfactory organs, funnel, mantle skin, gills, muscle, and fin) using NucleoSpin RNA set for NucleoZol (Machery-Nagel, Germany) according to manufacturer"s instructions. cDNA was synthesized using 500ng of these total RNA and a Omniscript® Reverse Transcription First-strand cDNA synthesis Kit (Qiagen,Valencia, CA, USA) providing a final 20µL volume of cDNA. A couple of primers were designed to amplify a 590 pb fragment of Soff-IR25 coding for most of the S2 and M3 conserved domains, Soff-

IR25-F

(5"-GGCAGTTTGGCACTACCCTA-3") and Soff-IR25-R (5"-TGGATTCACTGAAGGCAGGA-3"). PCR amplifications were performed with 1µL of cDNA template using REDTaq DNA polymerase (Eurogentec) in a C1000 Touch Thermocycler (BioRad) under the following conditions: 95°C for 5 min. + (95°C for 1 min., 55°C for 1 min., 72°C for 1 min.) for 30 cycles + 72°C for 5 min. The amplification of a 250 pb fragment of Sepia officinalis beta-actin (accession number HM157277.1) was used as a control for all cDNA templates: Soff-Act-F (5"-TTCCAGCCTTCCTTCTTGGG-3") and Soff-Act-R (5"-GGAGTATTTACGCTCGGGGG-3").

Soff-IR25 RNA in situ hybridization

The amplified fragment of Soff-IR25 was purified with a NucleoSpin PCR Clean-Up kit (Machery-Nagel, Germany), cloned into pDrive Cloning Vector (Qiagen,Valencia, CA, USA) and sequenced by Eurofin Biotech (Germany). RNA probes were then generated by in vitro transcription using digoxigenin-11-UTP (Dig RNA labelling mix kit, Roche, Meylan, France). Antisense and sense probes were obtained with T3 or T7 polymerase (Roche). RNA probes were purified by cold precipitation with lithium chloride and anhydrous alcohol.

Sense RNA probes were used as negative controls. In situ hybridizations were performed on MABT and left at 4°C in an AP buffer (100 mM Tris-HCl pH 9.5, 100 mM NaCl, 0.1% Tween-20) overnight. Finally, AP buffer, with 50 mM MgCl 2 , was added to the embryos twice for 30 min each before development of staining with the addition of 100 μg/mL 5bromo-4-chloro-3-indolyl phosphate (BCIP) and 80 μg/mL nitroblue tetrazolium chloride (NBT) until the desired contrast was reached. After several PBS rinses, embryos were postfixed with 3.7% formaldehyde for 24h, and finally rinsed with PBS. The total number of embryos used for ISH, including controls, was 6.

For histological visualization of staining, hybridized embryos were impregnated in 0.12M phosphate buffer pH 7.2 with 15% saccharose at 4°C for twice 24 h. Then, they were

Observation and Imaging

Whole embryos were viewed under a Leica M16 2F microscope and photographed using BK Plus digital imaging system (Dunn Inc, USA) with a Canon EOS 5DSR. Sections were viewed under a Leica DMLB microscope and imaged using a Leica MD 190 HD camera for color pictures. Final figures were constructed using Photoshop CC 2015 (Adobe Systems, Inc., San Jose, CA) with adjustments for size, contrast, and brightness only. Schematic diagrams were drawn using Adobe Photoshop CC 2015.

RESULTS

Phylogenetic survey of iGluRs in Molluscs

For an unambiguous identification of IRs sequences in S. officinalis, we first decided to construct a complete and informative molecular phylogeny of iGluR proteins from molluscs.

Previously described sequences of iGluR and IR proteins in molluscs (Croset et Inside the AKDF family, GluA (AMPA receptors) and GluK (Kainate receptors) are two sister groups (Figure 1A). Three molluscan subtrees gathered with other non-molluscan GluA (Figure 3). These trees contained sequences that were already annotated as GluA in Aplysia, Biomphalaria and Lottia [START_REF] Croset | Ancient protostome origin of chemosensory ionotropic glutamate receptors and the evolution of insect taste and olfaction[END_REF][START_REF] Liang | Ionotropic Receptors Identified within the Tentacle of the Freshwater Snail Biomphalaria glabrata, an Intermediate Host of Schistosoma mansoni[END_REF], Ramos-Vicente et al. 2018).

Since these sequences were diversely named in previous studies (R1 to R5, alpha to zeta), we decided to simply call these groups mA, mB and mC (m for mollusc). In the Kainate family, two molluscan subtrees were delineated and called m7 and m10 (in reference to two already annotated sequences GluR7 and GluR10 in Biomphalaria and Aplysia).

Inside the AKDF family, we also found monophyletic molluscan subtrees, with strong bootstraps (higher than 99) (Figure 4). Four of them were called "mollusc-specific GluR" since these sequences did not cluster with any other non-molluscan sequences. We called them GluR-m6 and GluR-m9 (according to previously annotated sequences Acal-GluR6, Acal-GluR9) and GluR-m11 and GluR-m12 (not previously described sequences). Another group contained several sequences that were already annotated as GluD, the Delta receptors that have been lost in most Protostomes (Ramos-Vicente et al. 2018). One of its remarkable features is the high number of paralogs that most species possess (e.g. up to 4 in O.

bimaculoides).

Description of IRs in Molluscs

The IR25 subfamily is shared by all protostomes and has been consistently found in molluscs in a monophyletic subtree with good bootstrap values (Figure 5A). However, two unexpected features were observed. First, the typical IR25a subfamily shared by all protostomes is lacking cephalopod sequences. Second, the IR25-like sequences we found in cephalopods belong to a well delineated group (bootstrap value of 100), close to the IR25a group and that we called IR25b in respect with the IR25b sequences previously described in Lottia [START_REF] Croset | Ancient protostome origin of chemosensory ionotropic glutamate receptors and the evolution of insect taste and olfaction[END_REF]). The arthropod-specific IR8 group is close but distinct from both IR25a and IR25b groups (high bootstrap value of 100). Interestingly, the IR25b group appears to be specific to molluscs. Consequently, gastropod and bivalve species possess two IR25 forms (paralogs IR25a and IR25b), while cephalopods only have genes from the IR25b-like group. A comparative multiple amino acid sequence alignment of all collected mollusc IR25 (together with arthropod IR8) is provided in Figure 5B. We point out some of the amino-acid positions that unambiguously discriminate sequences from the IR25a and IR25b groups, thereby eliminating any hypothesis of artifactual subdivision of the IR25 family.

Finally, inside the AKDF group, 93 sequences gathered into four large subtrees containing most of the already published IRs found in Lottia gigantea, Aplysia californica [START_REF] Croset | Ancient protostome origin of chemosensory ionotropic glutamate receptors and the evolution of insect taste and olfaction[END_REF]) and Biomphalaria glabrata [START_REF] Liang | Ionotropic Receptors Identified within the Tentacle of the Freshwater Snail Biomphalaria glabrata, an Intermediate Host of Schistosoma mansoni[END_REF]. We arbitrarily named them them IR-A, IR-B, IR-C and IR-D (Figure 6). IR-B and IR-C groups were respectively ). Conversely, two sequences annotated as IRs in L. gigantea turned out to be GluN (IR274, Figure 2) and mollusc-specific GluR (IR288, Figure 4).

Spatiotemporal expression patterns of Soff-IR25

We performed all our investigations on late embryos (stage 28, a few days before hatching, [START_REF] Lemaire | Table de développement embryonnaire de Sepia officinalis L. (mollusque céphalopode)[END_REF]. They possess all the organs of future juveniles and already react as such in their egg capsule. They also have the advantage of being small enough for whole-mount hybridization technique. RT-PCR of Soff-IR25 were conducted on cDNA from different dissected tissues of late embryos. Investigated tissues included potential chemosensory regions (arms, olfactory organs, skin), as well as non-sensory tissues (gills), central nervous structures (brain, optic lobe) and motor organs (mantle muscle, fin, funnel). Significant amplifications were obtained with cDNA of olfactory organs, arms and fins (Figure 7).

To further evaluate the expression of Soff-IR25 in chemosensory cells, we analyzed its cellular spatial distribution by whole-mount ISH in late embryos of S. officinalis (see Figure 8 for dorsal and ventral views). No staining was observed using a sense riboprobe for Soff-IR25 (Supplementary Figure S2A,B), while discrete staining was clearly visible in different parts of the animal bodies using an antisense riboprobe (Figure 8E). Expression at the cellular level was confirmed and determined following cryostat sectioning (Figure 9). Positive Soff-IR25 cells were observed in both olfactory organs of the embryos (Figure 8E, 9A, A", A"). At these stages of development, each olfactory organ is a roundish organ made of a sensory pseudostratified epithelium hosting OSNs [START_REF] Scaros | Immunohistochemical approach to understanding the organization of the olfactory system in the cuttlefish, Sepia officinalis[END_REF]. These placodes later invaginate to form a pit, like a nostril, behind the eyes. Thin sections have shown that staining is restricted to the outer layer of cells, which corresponds to the layer of olfactory sensory neurons (OSNs; Figure 9A",A"). Higher magnification confirmed the nature of these positive cells, as some of them showed the typical shape of olfactory organs OSNs in S.

officinalis [START_REF] Scaros | Immunohistochemical approach to understanding the organization of the olfactory system in the cuttlefish, Sepia officinalis[END_REF]. We also detected expression of Soff-IR25 in the developing suckers of all ten arms (Figure 8E, 9B, B", B"). At these stages of development, suckers are small bulges that later develop an inner rim [START_REF] Kimbara | Pattern of sucker development in cuttlefishes[END_REF]). Positive cells were detected at the edge of these developing rings, and on the lateral sides of the suckers (Figure 9B"), which corresponds to the location of the well-described sensory neurons of adult suckers [START_REF] Bellier | Immunohistochemical and biochemical evidence for the presence of serotonin-containing neurons and nerve fibers in the octopus arm[END_REF]). We also observed that Soff-IR25 was expressed in a discrete band all along the border of the fins (Figure 8E, 9C, C", C"). Thin sections showed that positive cells were located on the ventral face of the fins only (Figure 9C").

We found no evidence of staining in other parts of the embryos, neither from the external observation nor from thin sections of the entire embryos. Other colorations were confirmed as background noises (see Supplementary Data S4). They mostly correspond to cavities or sinuses (like eyes, shell or funnel) that typically retain compounds and provoke unspecific non-cellular staining.

Discussion iGluRs in Mollusc and Cephalopods

A good annotation of the iGluRs and IRs in molluscs was a prerequisite for the investigation of potential chemosensory IRs in cephalopods. Using non-molluscan sequences in addition to molluscan sequences collected from databases, the in silico phylogenetic analyses we subtrees of molluscan GluN (Figure 2), 3 subtrees of GluA and 2 subtrees of GluK sequences (Figure 3), one GluD subtree (Figure 4), 2 subtrees of IR25 sequences (IR25a, IR25b, Figure 5), 4 subtrees of unknown AKDF families (GluR-m6, m9, m11, m12, Figure 4) and 4

subfamilies we associated to IRs (Figure 6). Through this work, 68 "hypothetical" or "uncharacterized" sequences (22%) now have annotations and family assignments have been corrected for 44 sequences (14%) and refined for 61 "iGluR sequences" (19%) (See Supplementary Data S1).

Deep phylogenetic analyses have now clearly established that bivalves and gastropods are sister clades and that cephalopods are sister to them [START_REF] Kocot | Phylogenomics reveals deep molluscan relationships[END_REF][START_REF] Tanner | Molecular clocks indicate turnover and diversification of modern coleoid cephalopods during the Mesozoic Marine Revolution[END_REF]).

Here, with our phylogenetic analysis of molluscs iGluRs, we had too few and too short sequences to have high expectations of obtaining such a topology in our molluscan subtrees.

Nonetheless, cephalopod sequences were at the basal node of 8 out 15 molluscan subtrees (GluN1, GluN2, GluN3-mA, GluN3-mB, GluA-mB, GluK-m7, GluR-m6, m11, m12) (Figure 2, 3, 4). In other subfamilies (7 out of 15), the basal node was sometimes that of the gastropod sequences (GluA-mC, GluD) or that of bivalves (GluK-m10, GluA-mA, GluA-mB, GluR-m9, IR25b) (Figure 3, 4).

Regarding cephalopods, these are 57 sequences that have been analyzed (see Table 1), including 17 new sequences from Sepia officinalis, and 36 already published sequences whose annotations have been corrected or refined (Table 1). Some of these sequences are likely to code for the glutamate receptors that have been detected by immunochemistry and located in the central and peripheral nervous system of cephalopods: GluN in S. officinalis and O. vulgaris (Di [START_REF] Cosmo | N-methyl-D-aspartate receptor-like immunoreactivity in the brain of Sepia and Octopus[END_REF]), GluA in Loligo vulgaris [START_REF] Cosmo | Localization of Lglutamate and glutamate-like receptors at the squid giant synapse[END_REF][START_REF] Cosmo | L-glutamate and its ionotropic receptors in the nervous system of cephalopods[END_REF]. Alongside NMDA and AKDF receptors, we identified sequences of iGluRs variants, or IRs (including the IR25 subfamily), a group of iGluRs that is expected to have undergone high diversification as they gain a chemosensory function [START_REF] Croset | Ancient protostome origin of chemosensory ionotropic glutamate receptors and the evolution of insect taste and olfaction[END_REF]. Our analyses discriminated 64 IR sequences in gastropods, including 32 new sequences for L. gigantea and A. californica (Figure 6, Supplementary Data S1), as compared to those listed in [START_REF] Croset | Ancient protostome origin of chemosensory ionotropic glutamate receptors and the evolution of insect taste and olfaction[END_REF]. In bivalves, these are 24 sequences previously annotated as "GluR" that are now listed as IRs (Supplementary Data S1). In cephalopods, we found five IRs (Figure 6, Table 1) including two forms in S. officinalis (Soff-IRA1, Soff-IRA2). Thus, duplication of IRs seems to have been more frequent in gastropods than in bivalves and cephalopods, unless these numbers only reflect a lower effort of transcriptome sequencing of chemosensory organs in the two last groups.

Concerning the IR25 subfamily (Figure 6), the evolutionary story seems more complex than in other iGluRs. Actually, two groups of IR25-like sequences were delineated in Molluscs (Figure 5). First, we described an IR25a group that is close to the IR25a arthropods sequences (bootstrap value of 100 at the node) but that is surprisingly empty of cephalopod sequences (probably a specific loss early before cephalopod radiation). Second, we described an IR25blike group that contains sequences from all mollusc groups (including cephalopods) but with no equivalent in arthropods (probably a specific duplication of IR25a in molluscs or in lophotrochozoans). The arthropod-specific IR8 group is close to both IR25 groups (bootstrap value of 100 at the node) but is clearly distinct from them. We did not find any significant evidence for a closer relationship between the molluscan group IR25b and the arthropod group IR8 (bootstrap value of 41, Figure 5) but, with such a low resolution, the possibility that mollusc genes in IR25b are orthologs of arthropod IR8 genes can neither be rejected. For all these reasons, we retained the IR25b denomination for the molluscan sequences and reserved the "IR8" denomination to the arthropod subtree (consistently with [START_REF] Croset | Ancient protostome origin of chemosensory ionotropic glutamate receptors and the evolution of insect taste and olfaction[END_REF]).

If IR25a is known to be involved in chemosensory functions [START_REF] Croset | Ancient protostome origin of chemosensory ionotropic glutamate receptors and the evolution of insect taste and olfaction[END_REF], what about the IR25b-like form specifically found in molluscs? We cloned the Soff-IR25 gene and performed RT-PCR and ISH on late embryos of S. officinalis. Both techniques demonstrated that Soff-IR25 is significantly expressed in three different organs: the olfactory organs, the suckers and the fins (Figure 7, 8, 9). RT-PCR amplifications were also obtained at very low level in other tissues with no perceptible hybridization staining (Figure 7). Similar results in two gastropods have shown that IR25a can also be detected in non-sensory structures such as CNS and reproductive tracts [START_REF] Croset | Ancient protostome origin of chemosensory ionotropic glutamate receptors and the evolution of insect taste and olfaction[END_REF][START_REF] Liang | Ionotropic Receptors Identified within the Tentacle of the Freshwater Snail Biomphalaria glabrata, an Intermediate Host of Schistosoma mansoni[END_REF]) suggesting a possible role in endocrine-mediated signaling.

SoffIR25a is expressed in the olfactory organs

Our RT-PCR and ISH experiments have shown that Soff-IR25 is expressed in the olfactory organs of S. officinalis (Figure 7,8,9), supporting a conserved olfactory role of the IR25 receptor in cephalopods, even though it belongs to the IR25b-like group. In other protostomes, IR25a is detected in a variety of olfactory organs, such as antennae of insects (in the locust Schistocerca: [START_REF] Guo | Variant ionotropic receptors are expressed in olfactory sensory neurons of coeloconic sensilla on the antenna of the desert locust (Schistocerca gregaria)[END_REF]in Drosophila: Rytz et al. 2013), antennules and antennae of crustacea [START_REF] Corey | Ionotropic crustacean olfactory receptors[END_REF]Groh-Lunow et al. 2014;[START_REF] Zbinden | Comparative Study of Chemosensory Organs of Shrimp From Hydrothermal Vent and Coastal Environments[END_REF], rhinophores and tentacles of gastropods (in Aplysia: [START_REF] Croset | Ancient protostome origin of chemosensory ionotropic glutamate receptors and the evolution of insect taste and olfaction[END_REF]in Biomphalaria: Liang et al. 2016). The olfactory organ of cephalopods therefore represents a third type of organ in which IR25 may mediate the odorant detection.

Soff-IR25 expression is located at the surface of the olfactory organ (Figure 9A"), i.e.

in the epithelium hosting chemoreceptive neurons [START_REF] Wildenburg | Ultrastructure of the olfactory organ of Sepia officinalis at the end of embryonic development[END_REF][START_REF] Scaros | Immunohistochemical approach to understanding the organization of the olfactory system in the cuttlefish, Sepia officinalis[END_REF]. Up to five types of OSNs were described in the olfactory epithelium of cephalopods [START_REF] Emery | Observations on the olfactory organ of adult and juvenile Octopus joubini[END_REF][START_REF] Wildenburg | Structure of the so-called olfactory organ of octopods after hatching: evidence for its chemoreceptive function[END_REF][START_REF] Polese | Olfactory organ of Octopus vulgaris: Morphology, plasticity, turnover and sensory characterization[END_REF]), with at least three different neurotransmitters (Nitric Oxide, FMRFamides, histamine, Scaros et al. 2018) and specific odorant responsiveness and transduction pathways [START_REF] Mobley | Evidence for multiple signaling pathways in single squid olfactory receptor neurons[END_REF][START_REF] Mobley | Odorant responsiveness of squid olfactory receptor neurons[END_REF]. Since Soff-IR25 is not expressed in the whole epithelium, we hypothesize that IR25 is likely not the sole chemosensory receptor in the olfactory organs. Further studies will be required to determine whether Soff-IR25 positive cells are associated with a specific population of OSNs and/or with a specific neurotransmitter.

Soff-IR25 is expressed in suckers

Soff-IR25 positive cells were also found in some cells of the developing suckers (Figure 9B, B", B"). Suckers of cephalopods' arms are fascinating organs able to seize objects or prey but also to discriminate objects with different surfaces [START_REF] Wells | Tactile Discrimination of Surface Curvature and Shape by the Octopus[END_REF] or different tastes [START_REF] Wells | Taste by Touch: Some Experiments with Octopus[END_REF][START_REF] Wells | Some experiments on the chemotactile sense of octopuses[END_REF][START_REF] Anraku | Chemical stimuli and feeding behavior in octopus, Octopus vulgaris[END_REF]). In the late embryos we studied, suckers are small bulges in the center of which an invagination will form the future inner rim [START_REF] Kimbara | Pattern of sucker development in cuttlefishes[END_REF]. Soff-IR25 positive cells were observed at the periphery of the sucker bulge but also around the future rim (Figure 9B"), at the very same location and at the same stages that early sensory cells described by immunostaining [START_REF] Buresi | Emergence of sensory structures in the developing epidermis in Sepia officinalis and other coleoid cephalopods[END_REF]. This result suggests that these cells are one of the first chemosensory cells of the suckers [START_REF] Bellier | Immunohistochemical and biochemical evidence for the presence of serotonin-containing neurons and nerve fibers in the octopus arm[END_REF], ready to operate after hatching.

In 1963, Wells named "taste by touch" the capacity of Octopus vulgaris to discriminate objects with different chemical characteristics [START_REF] Wells | Taste by Touch: Some Experiments with Octopus[END_REF]. Recently, chemo- that these receptors are obviously note the sole to mediate odor detection. The IR25 receptor is also a possible actor of this capacity and it is interesting to note that, if so, the IR25 receptor would be both involved in "smell" (olfactory receptors) and "taste" (suckers). Arms and suckers are synapomorphies of cephalopods. Several molecular arguments now confirm that the arm crown surrounding the mouth opening in coleoid cephalopods is derived from the ancestral pedal area of molluscs [START_REF] Boletzky | Characteristics of cephalopod embryogenesis[END_REF][START_REF] Shigeno | Evolution of the cephalopod head complex by assembly of multiple molluscan body parts: Evidence from Nautilus embryonic development[END_REF][START_REF] Buresi | Nervous system development in cephalopods: How egg yolk-richness modifies the topology of the mediolateral patterning system[END_REF] and has evolved as efficient tools to catch prey but also to taste them. As such, they are probably not homologous to oral tentacles in gastropods. But similar selective pressures have operated in both types of organs, since in gastropods the IR25a receptor is also found both in olfactory rhinophores and in oral tentacles located in the mouth vicinity [START_REF] Croset | Ancient protostome origin of chemosensory ionotropic glutamate receptors and the evolution of insect taste and olfaction[END_REF][START_REF] Liang | Ionotropic Receptors Identified within the Tentacle of the Freshwater Snail Biomphalaria glabrata, an Intermediate Host of Schistosoma mansoni[END_REF].

Soff-IR25 is expressed in the fins

The discovery of a Soff-IR25 expression in the fins of Sepia officinalis was unexpected. Fins are motor appendages involved in the control of slow locomotion and fine motion. They contain fibres of motoneurons controlling the fin muscles, and fibres controlling the chromatophore structures, responsible for the versatile skin color patterns [START_REF] Gaston | Peripheral innervation patterns and central distribution of fin chromatophore motoneurons in the cuttlefish Sepia officinalis[END_REF]. A single study has suggested a sensory function in the cephalopod fins, with the description of mechanoreceptors in the fins of S. officinalis [START_REF] Kier | Mechanoreceptors in the fins of the cuttlefish, Sepia officinalis[END_REF]. Here, our in situ hybridizations detected expression of Soff-IR25 in epithelial cells of fins, which suggests that these positive cells may be primary sensory cells and probably chemosensory cells, maybe similar to that of the olfactory organs. Together with the suckers at the anterior side of

Summary and evolutionary perspectives

Soff-IR25 is the first chemosensory receptor whose expression has been investigated in a cephalopod. Its olfactory function is not formally demonstrated here but strongly suggested by the nature of the organs in which its expression has been detected, especially the olfactory organs (Figure 8, 9). Our results on late embryos suggest that juveniles hatch with a full array of odor detectors, anteriorly with their arms, laterally with their olfactory organs and posteriorly with their fins. Numerous features of Soff-IR25 yet remain to be unraveled: the molecules it can bind with or the other receptors it may operate with, the type of sensory cells expressing it, or the evidence of its involvement in olfaction in later juveniles and adults.

Soff-IR25 belongs to a large family of orthologous genes, whose relationship with chemosensation and olfaction is now deeply established from insects, and crustaceans, to gastropods [START_REF] Croset | Ancient protostome origin of chemosensory ionotropic glutamate receptors and the evolution of insect taste and olfaction[END_REF]) and now cephalopods. Regarding molluscs, bivalves remain the forgotten group to explore, and an effort must be undergone to understand the potential specific functions of the two groups of paralogous genes IR25a and IR25b we delineated in gastropods and bivalves (Figure 6). We also described several members of variant iGluR (IRs) in molluscs, including two forms in S. officinalis, Soff-IRA1 and Soff-IRA2. IR subunits have been found in the olfactory tissues of two divergent gastropod subclasses, pulmonates

  Soff-IR25 found in the fins is discussed, as it may provide evidence for a chemosensory role for these organs.

  . Trees were viewed and graphically edited with MEGA5. Sequences of plant iGluR (GLR) and of sponge iGluR (GluL) were used to root trees (Ramos-Vicente et al.

  S. officinalis at stage 28[START_REF] Lemaire | Table de développement embryonnaire de Sepia officinalis L. (mollusque céphalopode)[END_REF]. The protocol was adapted from[START_REF] Scaros | Immunohistochemical approach to understanding the organization of the olfactory system in the cuttlefish, Sepia officinalis[END_REF] and fromSinigalia et al. (2018). The embryos were rinsed in PTW (PBS, 0.1% Tween-20) at room temperature and permeabilized by 10 mg/mL proteinase K for 30 min. After two rinses in 2mg/mL glycine and two acetylation steps (in 0.1M Triethanolamine / 0.25% acetic anhydride and second with 0.90% acetic anhydride), the embryos were postfixed 1 hr in 4% PFA. After rinse in a saline sodium citrate buffer (2X SSC: 0.3 M sodium citrate, 3 M NaCl, pH 8.0), the embryos were incubated at 65°C for 5 hr in the prehybridization buffer (50% deionized formamide, 2X SSC, 0.1% Tween 20, 50 μg/ml heparin, 50 μg/mL tRNA, and 10% dextran sulfate). The hybridization buffer included the addition of 45 ng of probe/mL of pre-hybridization buffer and was left overnight at 65°C. After progressively stringent rinses in SSC (2X) and SSC (0.2X), at 65°C, embryos were then transferred to MABT (100 mM maleic acid, 150 mM NaCl, 100 mM NaOH, 0.1% Tween-20, pH 7.5) and incubated for 1 hour in a blocking solution (1% Blocking Reagent [Roche], 5% fetal bovine serum, in MABT) at room temperature. Embryos were then incubated in polyclonal sheep anti-DIG antibody (Roche) conjugated to alkaline phosphatase (AP) and diluted 1:1000 in blocking solution overnight at 4°C. Embryos were thoroughly rinsed with

  -50 TM embedding medium (Thermo Scientific) and blocks were frozen in 2 min at -60°C with PrestoCHILL (Milestone, USA). Sections of 20 μm were performed using cryostat HM 560 MV (Microm Microtech, France).

  s c r i p t 12 the general tree we obtained are provided in Supplementary Data S2 and S3. Inside this tree, molluscan sequences clustered into monophyletic subtrees with very good bootstrap values (often 100, never under 80), all containing clear clusters of sequences for each taxon we investigated (gastropods, bivalves and cephalopods). The following paragraphs detail how each of these subtrees have been annotated, mostly on the basis of their branching to other non-molluscan sequences but also according to annotations of already published sequences.Four molluscan subtrees gathered with GluN sequences (NMDA receptors) (Figure2). Since the Bilateria radiation, the NMDA receptor family is subdivided into three subfamilies GluN1, GluN2 and GluN3(Ramos-Vicente et al. 2018). This subdivision is conserved in molluscs: they possess a single GluN1 family, a single GluN2 family, but obviously two versions of GluN3 (we called mA and mB) according to the two molluscan subtrees we found inside the whole GluN3 branch.

  Figure6). Conversely, two sequences annotated as IRs in L. gigantea turned out to be GluN

  unambiguous mollusc-specific subtrees, with high bootstrap values: 4

  tactile receptors have been recently described in the sucker rim of Octopus species (van Geisan et al. 2020). They belong to a cephalopod-subfamily of Acetylcholine Receptors and seem able to detect poorly soluble natural products (van Geisan et al. 2020). Our results show Downloaded from https://academic.oup.com/chemse/advance-article/doi/10.1093/chemse/bjab047/6412677 by Museum National d'Histoire Naturelle user on 08 November 2021A c c e p t e d M a n u s c r i p t 20

  the animal and the two lateral olfactory organs close to the mantle opening, fins would add a posterior dimension to the detection of odors in S. officinalis. New fields of exploration are now open and future investigations must be conducted to describe the chemosensory cells, to Downloaded from https://academic.oup.com/chemse/advance-article/doi/10.1093/chemse/bjab047/6412677 by Museum National d'Histoire Naturelle user on 08 November 2021A c c e p t e d M a n u s c r i p t 21 validate any fin olfactory capacities by detecting behavioral or physiological reactions to odors and to unravel the sensory pathways to more central nervous structures, ganglia or brain lobes.

Figure 1 .

 1 Figure Legends

Figure 2 .

 2 Figure 2. Characterization of GluN sequences (NMDA receptors) in Molluscs.

Figure 3 .

 3 Figure 3. Characterization of GluA (AMPA receptors) and GluK (Kainate receptors) in

Figure 4 .

 4 Figure 4. Characterization of other AKDF subfamilies in Molluscs. Phylogenetic

Figure 5 .

 5 Figure 5. The IR25 family in Molluscs. A-Molecular phylogeny of IR25 receptors in

Figure 6 .

 6 Figure 6. Survey of the IRs in Molluscs. Phylogenetic construct obtained with AKDF

Figure 7 .

 7 Figure 7. Tissue expression of Soff-IR25. Top: schematic representation of a late embryo of

Figure 8 .

 8 Figure 8. Whole views of S. officinalis late embryos. A, B: dorsal views (drawing and

Figure 9 .

 9 Figure 9. Details of Soff-IR25 ISH staining in S. officinalis late embryos. A, A' and A":

AAAAAA

  Figure 1

A

  Figure 8

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	al. 2010;
	Liang et al. 2016; Ramos-Vicente et al., 2018) were used as initial queries to blast and mine
	available mollusc databases (NCBI) and our own S. officinalis transcriptome. A total of 312
	sequences was collected in molluscs (full list in Supplementary Data S1), all possessing the
	conserved Pfam domains (PF10613 and PF0060) coding for the iGluR-specific ligand-gated
	ion channel (see Figure 1B-C; Finn et al. 2008). To confirm their affiliation to the iGluR

family, a large phylogenetic analyze (ML algorithm) has been performed in addition with non-molluscan iGluR sequences (from

Ramos-Vicente et al. 2018)

. The alignment file and

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  [START_REF] Croset | Ancient protostome origin of chemosensory ionotropic glutamate receptors and the evolution of insect taste and olfaction[END_REF][START_REF] Liang | Ionotropic Receptors Identified within the Tentacle of the Freshwater Snail Biomphalaria glabrata, an Intermediate Host of Schistosoma mansoni[END_REF].Exploration of IRs in bivalves and cephalopods will probably demonstrate a similar general role for the IR family in chemosensory signaling in molluscs. More generally other potential chemosensory receptor families, like GPCRs, must be explored since they have been established as key receptors in olfaction for other mollusc species[START_REF] Cummins | Candidate chemoreceptor subfamilies differentially expressed in the chemosensory organs of the mollusc Aplysia[END_REF]) and may act in concert with IRs to contribute to the output of sensory neurons.

	Downloaded from https://academic.oup.com/chemse/advance-article/doi/10.1093/chemse/bjab047/6412677 by Museum National d'Histoire Naturelle user on 08 November 2021 (Biomphalaria) and opisthobranchs (Aplysia) (Downloaded from https://academic.oup.com/chemse/advance-article/doi/10.1093/chemse/bjab047/6412677 by Museum National d'Histoire Naturelle user on 08 November 2021 A c c e p t e d M a n u s c r i p t
	22

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Acknowledgments

We thank Olivier Basuyaux (SMEL, Blainville-sur-Mer) for providing us S. officinalis eggs, Marie-Pierre Dubos (PHYPAQ, BOREA) for maintaining them in the aquarium tanks at the Luc-sur-mer marine station (Université Caen Normandie) and the NEEC team (EthoS, UMR 6552) for our fruitful collaboration and discussions about S. officinalis behavior and olfaction. We express our deep gratitude to Alexia Scaros and Pr. Roger Croll (Dalhousie University, Halifax, NS, Canada) for their helpful comments on the manuscript, their English corrections and, above all, for introducing us to the world of olfaction. This work was supported by funding of the BOREA laboratory, through the CROCS olfactory project led by Dr. Magali Zbinden. Finally, thank you to Joël Henry and Celine Zatylny-Gaudin for their helpful confirmation of IR25 presence in their own S. officinalis transcriptomes. Downloaded from https://academic.oup.com/chemse/advance-article/doi/10.1093/chemse/bjab047/6412677 by Museum National d'Histoire Naturelle user on 08 November 2021

Data deposition

The new sequences of S. officinalis we reported and analyzed in this paper have been deposited in Genbank (18 sequences, from MW657339 to MW657356). See Dataset S1 for details.

Author Contributions

Conceived and designed the experiments: AA SB. Performed the experiments: AA, SV.

Analyzed the data: AA SB. Wrote the paper: AA SB.

Competing interests

The authors have declared that no competing interest exists.