Olfactory-like neurons are present in the forehead of common cuttlefish, Sepia officinalis Linnaeus, 1758 (Cephalopoda: Sepiidae)

Olfactory-like neurons are present in the forehead of common cuttlefish, Sepia officinalis... Abstract According to the literature, the cuttlefish, Sepia officinalis, possesses a specialized olfactory organ and cells, located in olfactory ventral pits. In this study, the location of olfactory receptor neurons (ORNs) at the cellular level was determined using cellular morphology and immunohistochemistry. An antiserum against PBP3 was used as a marker to identify ORN-like cells in cuttlefish after validation for specificity to cephalopod ORN cells in the common octopus, Octopus vulgaris. The results show that ORN-like cells in S. officinalis were not found in the ventral pits, suckers or the mouth lips. Instead, ORN-like cells were found scattered in the forehead, between the eyes. The absence of ORN-like cells in a pit in S. officinalis and the sharing of four similar types of ORN cells with the squid and octopus lineages suggest that this might be a later innovation in olfaction and is probably associated with the specialized lifestyle of these later evolved cephalopods. Together, this evidence suggests a diversification of ORN cell types in Coleoidea, which did not occur in Nautiloidea, which might have preceded the diversification of the Coleoidea. cellular morphology, cephalopod, European cuttlefish, Sepia officinalis, olfaction, olfactory receptor neurons (ORNs) INTRODUCTION Cephalopods are a class in the phylum Mollusca, with over 700 species currently described. They have evolved from their early gastropod ancestor to efficient marine predators, diverging from the typical molluscan body plan and converging with vertebrates in locomotion, camouflage and sensory functions (Packard, 1972; Budelmann, 1995; Buresi et al., 2014). The coleoid cephalopods (squids, octopuses and cuttlefishes) have a large, centralized and complex nervous system (Buresi et al., 2016) that rivals the complexity of vertebrate nervous systems and is considered the most sophisticated of the invertebrates’ (Budelmann, 1996). The sensory system in cephalopods includes highly specialized eyes, ‘lateral lines’ equilibrium receptor systems (neck proprioceptors), touch, and chemical-sensitive tentacles (Budelmann, 1980; Sundermann, 1983; Budelmann & Bleckmann, 1988; Budelmann, 1994; Lenz, 1997; Stubbs & Stubbs, 2016). Moreover, squid and octopus have a well-developed and active olfactory organ located in olfactory pits, posterior to the eye but anterior to the mantle (Woodhams & Messenger, 1974; Emery, 1975, 1976; Braby, 1998; Shigeno et al., 2001; Villanueva & Norman, 2008; Polese, Bertapelle & Di Cosmo, 2016). In these organisms, the olfactory organ is composed of several epidermal folds containing odorant-sensitive cells. In squid, five morphologically and functionally distinct olfactory receptor neurons (ORNs) have been identified (Emery, 1975; Braby, 1998; Lucero, Horrigan & Gilly, 1992; Mobley, Michel & Lucero, 2008). Signalling in squid ORNs involves G-protein coupled receptors and Ca2+, cyclic AMP and PLC signalling (Piper & Lucero, 1999; Lucero, Huang & Dang, 2000; Mobley, Mahendra & Lucero, 2007; Mobley et al., 2008). Cuttlefish occupy an ancestral position in the Coleoidea lineage, and chemosensory cells have been identified in the arms and tentacles and in the lip/mouth regions (Graziadei, 1964, 1965). According to Budelmann (1996) and Nixon & Mangold (1998), a pair of olfactory organs exists in the late embryo of Sepia officinalis (Naef, 1928), each of the pair being located posterior to the eye and in front of the inhalant channel of the mantle, where two types of sensory cell and one type of ciliated epithelial cell are present. Although Nixon & Dilly (1977) related the existing sucker sensory receptors with mechanoreception, feeding behaviour experiments have suggested that the location of the olfactory organ might be in the arms and tentacles of cuttlefish (Boal & Golden, 1999; Archdale & Anraku, 2005). In Nautilus, a modified tentacle, the rhinophore, acts as a specialized olfactory organ (Young, 1965; Barber & Wright, 1969), further supporting the idea that the arms and/or tentacles might be a potential location for an olfactory organ in cuttlefish. More recently, Buresi et al. (2014) identified a pair of ventral pits in the anterior part of the head as the putative olfactory organ in S. officinalis. However, these same authors reported that although the structure of these cuttlefish olfactory pits resembles those of squid and octopus, the nervous cell types found in cuttlefish do not resemble any of the ORNs described in other advanced cephalopods. Therefore, despite published information on the existence of a putative olfactory organ (with similar ORN cells to other cephalopods) in the cuttlefish, S. officinalis, there is no scientific basis for confirming its function. This leads to two hypotheses: (1) S. officinalis does not have ORN cells similar to those reported in other cephalopods and the ventral pits are true olfactory organs with ORN cells still undescribed or (2) the ventral pits do not function as olfactory organs and the already-described ORN cells are found elsewhere. This study aimed to establish the existence and possible location of ORN-like cells in S. officinalis using cellular morphology and immunohistochemistry. MATERIAL AND METHODS Ethical statement Experiments were performed under project SEPIABREED (PTDC/MAR/120876/2010), which was approved before Directive 2010/63/EU (EU, 2010) was transposed into national legislation in Portugal. Cephalopod origin and sample preparation One-day-old hatchlings of S. officinalis (0.086 ± 0.011 g) were obtained from eggs laid by F2 and F3 captive stocks cultured at CCMAR facilities, as described in Sykes, Domingues & Andrade (2014). Eggs were individually sorted, according to the colour and morphology (only black ink-stained eggs with a flask-shaped morphology were used), and incubated for 30 days inside a 250 l fibreglass tank in a flow-through system (Sykes et al., 2006) with the following seawater quality conditions: 19 ± 1 °C, 35 ± 1 PSU, dissolved oxygen saturation above 90% and 100 lux light intensity. Cuttlefish developed, hatched without any apparent malformations and behaved normally. Hatchlings were euthanized (n = 3) by immersing in a cold seawater (4 °C) solution containing 5% of MgCl2 for 20 min (Sykes et al., 2012) and afterwards were fixed in 4% paraformaldehyde (PFA) in 1× phosphate-buffered saline (PBS; pH 7) at 4 °C overnight. Samples were then rinsed in 1× PBS/0.1% Tween20 (PBT) and transferred to 100% methanol through a grade series of PBS/methanol. Fixed samples were then stored in methanol at −20 °C. Samples were processed in a Leica TP 1020 (Leica Microsystems GmbH, Germany) tissue processor, through a graded series of ethanol (70–100%), followed by ethanol:xylene (1:1 v/v), xylene (100%) and then embedded in low-melting-point paraffin wax. Sagittal and transversal serial sections (10 µm) of hatchlings were prepared with a rotary microtome (Microm HM340E, Microm International GmbH, Germany) and mounted on poly-l-lysine-coated glass slides. For cryo-microtome sections (100–200 µm), the cuttlefish were transferred through graded methanol/1× PBS and then washed in 1× PBS. Samples were incubated on 1× PBS/30% sucrose for c. 4 h at room temperature, followed by embedding in OCT medium (VWR, Portugal) and freezing at −80 °C. Samples were sagittally or transversely sectioned using a Leica CM3050S cryo-microtome (Leica Microsystems GmbH, Germany), mounted on slides and stored at −20 °C until use. One adult S. officinalis (520 g) and one Octopus vulgaris (780 g) were used for the sampling of tissue from the olfactory pit. While the cuttlefish was obtained from a CCMAR culture stock, the octopus was purchased alive from the local fish market in Quarteira (southern Portugal). For both, olfactory pit tissue samples were collected after euthanasia (cuttlefish – 5% MgCl2; octopus a mixture of 5% pure ethanol and 5% of MgCl2; both compensated for salinity changes derived from the use of the salt) in seawater (Fiorito et al., 2015). The tissues were fixed in 4%PFA/1× PBS overnight at 4 °C. Afterwards, the tissues were rinsed in PBT, transferred to 100% methanol and kept at −20 °C until inclusion. Paraffin inclusion was carried out as described above, and a transversal section of the olfactory pit was cut and collected onto glass slides. Immunohistochemistry Paraffin sections (10 µm) were dewaxed in xylene and rehydrated through a graded ethanol series (100, 75, 50, 25% ethanol:1× PBS) into 1× PBS. Prior to the incubation with an acetylate α-tubulin antibody (T6793 Sigma–Aldrich, Spain), samples were immersed in acetone, cooled at −20 °C (30 min) and rinsed in 1× PBS. The slides were blocked in 1× PBS/10% sheep serum/0.5% Triton X100 for 2 h at room temperature and incubated overnight at 4 °C with an anti-acetylated α-tubulin antibody (1:1000; Sigma–Aldrich) or an anti-pheromone binding protein antibody (1:1 PBP3 of Manduca sexta moth antennae; Developmental Studies Hybridoma Bank, USA). A goat anti-mouse IgG H+L conjugated with Dylite 488 (1:400, AnaSpec, Belgium) was used for labelling after overnight incubation at 4 °C. Slides were rinsed in PBT with 0.7 nM 4′,6-diamidino-2-phenylindole (DAPI; Carl Roth GmbH + Co. KG, Germany) for 5 min, washed in PBS and mounted in glycerol + 2.4% 1,4-diazabicyclo-octane (DABCO; Carl Roth GmbH + Co. KG, Germany). For double immunohistochemistry, after the detection of PBP3 with goat anti-mouse IgG H+L conjugated with Dylite 596 (1:400, AnaSpec, Belgium), the slides were incubated in 1× PBS/10% sheep serum/0.5% Triton X100 and an anti-acetylated β-tubulin antibody (1:1000) overnight at 4 °C. Tubulin fluorescent detection was carried out overnight at 4 °C, after incubation with goat anti-mouse IgG2 Gamma conjugated with Alexa488 (1:200; Life Technologies). The slides were rinsed in PBT with 0.7 nM DAPI for 5 min, washed in PBS and mounted in glycerol + 2.4% DABCO. Negative controls (same procedure but omitting the primary antibody: 10% sheep serum buffer) were conducted to assess the specificity of labelling. For cryo-microtome sections, the slides were dried for 1 h at 37 °C in an incubator and hydrated in 1× PBS. The immunohistochemical procedures carried out for these sections were identical with those for the paraffin sections. Stained sections were examined with an Axio Imager Z2 fluorescence microscope and z-stacks were developed (Carl Zeiss AG, Oberkochen, Germany). The acquired images were deconvoluted using Huygens 4.3 software (Scientific Volume Imaging B.V., Hilversum, The Netherlands), and maximum projections were generated after FIJI analysis (Schindelin et al., 2012). For consistency, the head was considered anterior, the mantle apex posterior, the funnel ventral and the opposite side dorsal. The nomenclature adopted for the central nervous system of S. officinalis was in agreement with the previous studies (Boycott, 1961). Immunohistochemistry of O. vulgaris olfactory pit sections for PBP3 and acetylated α-tubulin was carried out as described above. These sections were used to validate the PBP3 antiserum as specific for olfactory neurons. RESULTS The PBP3 antiserum was chosen because it detects odorant cells in invertebrates. The identification of putative ORN-like cells in S. officinalis and O. vulgaris was attained with a PBP3 antiserum that labelled olfactory cells in M. sexta (Nardi et al., 2003). This antiserum was validated with the identification of ORN cells present in the olfactory pits in O. vulgaris. The staining of PBP3 only in ORN-like neurons in the olfactory pit of this species (as described by Polese et al., 2016 with an olfactory marking protein) confirmed that this antiserum only detected odorant-specific neurons (Fig. 1A–C). No staining for PBP3 was detected when the serum was omitted from the immunohistochemical procedure. This confirmed that the PBP3 antiserum is a reliable tool for identifying ORN-like neurons in cephalopods. Figure 1. View largeDownload slide PBP3 is only specific to ORN cells in octopus olfactory pits. Composite maximum projection of 50-µm image stacks after immunostaining in transversal paraffin sections of octopus olfactory pits for PBP3 (cyan), acetylated alfa-tubulin (green) and nuclear staining (DAPI, magenta). A–C, different ORN cells in octopus olfactory system. Different nervous cells are found in the olfactory pit region, but only ORN (white outlined cells) are positive for both PBP3 and tubulin (A–C). In parallel control sections (PBP3 serum omitted), no PBP3 signal is found (D). Scale bar = 10 µm. Figure 1. View largeDownload slide PBP3 is only specific to ORN cells in octopus olfactory pits. Composite maximum projection of 50-µm image stacks after immunostaining in transversal paraffin sections of octopus olfactory pits for PBP3 (cyan), acetylated alfa-tubulin (green) and nuclear staining (DAPI, magenta). A–C, different ORN cells in octopus olfactory system. Different nervous cells are found in the olfactory pit region, but only ORN (white outlined cells) are positive for both PBP3 and tubulin (A–C). In parallel control sections (PBP3 serum omitted), no PBP3 signal is found (D). Scale bar = 10 µm. We tested whether the olfactory organ was located in the arms, lips or tentacles (Fig. 2A), using immunohistochemistry to examine whether ORN-like cells existed in those locations. Staining sagittal sections of arm tips in S. officinalis hatchlings with anti-tubulin revealed several different nervous-like cells (Fig. 2B). These cells were present at the surface, below and transversely in the skin at the tip of the arm (Fig. 2B) but did not resemble the morphology of the ORNs described in squid (Emery, 1975; Lucero et al., 2000; Mobley et al., 2008) or octopus olfactory pits (Polese et al., 2016). In addition, no signal for PBP3 was detected in S. officinalis arms or tentacles tips (Fig. 2B). Also, all cells stained with α-tubulin in the proximal part of the arms lacked the morphology characteristic of ORN-like cells (Fig. 2C). In contrast, a PBP3-specific signal was detected in the most anterior part of the mouth/lips (Fig. 2C). Notably, the PBP3 signal co-localized with the α-tubulin staining in some, but not all, structures (Fig. 2C). This co-staining was subepidermal, and the cells did not have an ORN-like morphology (Fig. 2C). Figure 2. View largeDownload slide Olfactory-like neurons are not found in tentacles, arms, funnel and mouth of cuttlefish. Bright field sagittal section of cuttlefish hatchling showing regions that were analysed for the presence of ORN-like cells (coloured boxes; A). Maximum projections of 20-µm-deep stack after double immunofluorescent staining for acetylated alfa-tubulin (green), PBP3 (red) and nuclear staining (DAPI, magenta) on the tip of the tentacle (B), mouth (C) and funnel (D). Scale bar in (A) = 100 µm; scale bar in (B) = 10 µm; scale bar in (C) = 50 µm. Figure 2. View largeDownload slide Olfactory-like neurons are not found in tentacles, arms, funnel and mouth of cuttlefish. Bright field sagittal section of cuttlefish hatchling showing regions that were analysed for the presence of ORN-like cells (coloured boxes; A). Maximum projections of 20-µm-deep stack after double immunofluorescent staining for acetylated alfa-tubulin (green), PBP3 (red) and nuclear staining (DAPI, magenta) on the tip of the tentacle (B), mouth (C) and funnel (D). Scale bar in (A) = 100 µm; scale bar in (B) = 10 µm; scale bar in (C) = 50 µm. We also tested whether ORN-like cells were found in the ventral pits of adult cuttlefish, described by Buresi et al. (2014), as the olfactory organ with a similar capability and cells present in both squid and octopus. After extracting these structures from an adult cuttlefish, two distinct regions were identified (Fig. 3A). The ventral cavities had a folded (red square in Fig. 3A) and irregular flat surface (blue square in Fig. 3A) and contained anti-tubulin-positive nerve cells (Fig. 3A, B). However, none of the identified cells resembled the ORN cell types described previously in squid or octopus. Instead, a group of tubulin-positive cells that projected fibril-like structures into the pocket lumen (Fig. 3B), or were intensely stained with tubulin apically on the lumen-facing side of the cell, were observed (Fig. 3C). In both regions, nerve projections were observed (Fig. 3B, C) and the morphology resembled that reported by Buresi et al. (2014), but not any type described for other Coleoidea. Figure 3. View largeDownload slide Olfactory-like neurons are not found in ventral head pits of cuttlefish. Bright field image of excised ventral head pits (A). Red and blue squares in (A) denote regions in (B) and (C), respectively. Composite maximum projection of 20-µm stacks of folded pit region [red square in (A)] after fluorescent immunostaining for acetylated alfa-tubulin (green) and nuclear staining (DAPI, magenta) (B). Composite maximum projection of 20-µm stacks of flat pit region [blue square in (A)] after fluorescent immunostaining for acetylated alfa-tubulin (green) and nuclear staining (DAPI, magenta) (C). Scale bar = 50 µm. Figure 3. View largeDownload slide Olfactory-like neurons are not found in ventral head pits of cuttlefish. Bright field image of excised ventral head pits (A). Red and blue squares in (A) denote regions in (B) and (C), respectively. Composite maximum projection of 20-µm stacks of folded pit region [red square in (A)] after fluorescent immunostaining for acetylated alfa-tubulin (green) and nuclear staining (DAPI, magenta) (B). Composite maximum projection of 20-µm stacks of flat pit region [blue square in (A)] after fluorescent immunostaining for acetylated alfa-tubulin (green) and nuclear staining (DAPI, magenta) (C). Scale bar = 50 µm. The PBP3 staining was used as a marker for regions with putative olfactory detection capability in cuttlefish. Using this approach, a high concentration of structures stained positive for PBP3 in the forehead, between the eyes, of S. officinalis hatchlings (Fig. 4A). These structures were found between the dorsal posterior end of the head, just before the mantle, and the most anterior part of the face, just before the beginning of the arms (Fig. 4A). Since the PBP3 serum was raised against an olfactory binding protein from M. sexta (Nardi et al., 2003), the high concentration of PBP3-positive structures is probably related to olfaction. To further explore this hypothesis, the presence of nervous cells in the same region of the head was investigated with α-tubulin staining. This revealed a great number of nervous cells in the dorsal epidermis that seem to project directly to the brain (Fig. 4B). Nonetheless, the presence of nervous cells cannot be fully proven using just histological sections. The double detection of PBP3 and α-tubulin revealed that both proteins were present in the same ciliated cells at the top of the epidermis and in some sub-epithelial structures (Fig. 4C). However, we could not determine the exact nature of the sub-epithelial structures labelled for both markers; we can only infer that they are probably nervous system structures. Moreover, stained epithelial nerve cells with PBP3 were only observed in this region of the head of S. officinalis. A more detailed analysis revealed that these cells had ciliated structures at their most apical end that projected outside the skin (Fig. 4D–F). Furthermore, these cells were always found in clusters and shared morphology with ORN in other cephalopods (Fig. 4D–F). The putative S. officinalis ORN cells (Fig. 4D, E) resembled octopus and squid types 1 (Fig. 4E), 3 (Fig. 4D), 4 (Fig. 4F) and 5 (Fig. 4D, F) (Emery, 1975; Lucero et al., 2000; Mobley et al., 2007, 2008; Polese et al., 2016). Figure 4. View largeDownload slide Olfactory-like neurons are only found in the cuttlefish dorsal forehead, between the eyes. Composite image of bright field, nuclear staining (DAPI, blue) and immunostaining of PBP3 (red) in the dorsal forehead region between the eyes (A). Composite maximum projection of 200-µm stack of dorsal forehead between the eyes after fluorescent immunostaining for acetylated alfa-tubulin (green) and nuclear staining (DAPI, magenta; B). Composite maximum projection of 50-µm stack from the dorsal forehead after double fluorescent immunostaining for acetylated alfa-tubulin (green), PBP3 (red) and nuclear staining (DAPI, magenta; C). Composite maximum projection of 50-µm stack from the dorsal forehead after fluorescent immunostaining for acetylated alfa-tubulin (green) and nuclear staining (DAPI, magenta; D–F). D–F, presumed ORN-like neurons are outlined to enhance their morphology and numbers next to cells indicate squid and octopus ORN classes previously identified (Emery, 1975). Scale bar in (A) = 100 µm; scale bar in (B, C) = 50 µm; scale bar in (D–F) = 10 µm. Figure 4. View largeDownload slide Olfactory-like neurons are only found in the cuttlefish dorsal forehead, between the eyes. Composite image of bright field, nuclear staining (DAPI, blue) and immunostaining of PBP3 (red) in the dorsal forehead region between the eyes (A). Composite maximum projection of 200-µm stack of dorsal forehead between the eyes after fluorescent immunostaining for acetylated alfa-tubulin (green) and nuclear staining (DAPI, magenta; B). Composite maximum projection of 50-µm stack from the dorsal forehead after double fluorescent immunostaining for acetylated alfa-tubulin (green), PBP3 (red) and nuclear staining (DAPI, magenta; C). Composite maximum projection of 50-µm stack from the dorsal forehead after fluorescent immunostaining for acetylated alfa-tubulin (green) and nuclear staining (DAPI, magenta; D–F). D–F, presumed ORN-like neurons are outlined to enhance their morphology and numbers next to cells indicate squid and octopus ORN classes previously identified (Emery, 1975). Scale bar in (A) = 100 µm; scale bar in (B, C) = 50 µm; scale bar in (D–F) = 10 µm. DISCUSSION Well-differentiated olfactory organs have been reported for most cephalopods. Nautilus has a specialized tentacle (rhinophore) (Young, 1965), and squid and octopus have a pair of active olfactory organs located immediately posterior to the eye (Woodhams & Messenger, 1974; Emery, 1975; Braby, 1998; Villanueva & Norman, 2008; Polese et al., 2016). Recently, Buresi et al. (2014) described the existence of octopus-like olfactory pits in S. officinalis, but the cells identified in those structures did not have a cellular morphology resembling any other cephalopod olfactory cells in the literature. Olfactory organs or capacities were initially though to be possibly located in the arms and/or in tentacle suckers of cuttlefish because these structures possess ciliated cells (Graziadei, 1964; Boal & Golden, 1999; Hanlon & Messenger, 1996; Archdale & Anraku, 2005). In this study, nerve cells were identified in the tip of the arms and tentacles of S. officinalis, but none of these cells either resembled ORN-like cells described previously for other cephalopods (Emery, 1975; Polese et al., 2016) or were immunoreactive for PBP3 (Fig. 2B–D). Therefore, it is unlikely that these structures are involved in S. officinalis olfaction. This result is remarkably different from what is reported for Nautilus, where the rhinophore (a modified tentacle) assumes the olfactory role (Young, 1965; Barber & Wright, 1969). This study suggests that the development of the rhinophore (containing an ORN cell intermediate to types 4 and 5 in squid) might represent a Nautilus lineage-specific organ and adaptation to the habitat rather than an ancestral feature common to the Nautiloidea and Coleoidea lineages. We identified two small ventral cavities, located in the posterior head region just before the mantle that have an ultrastructure reminiscent of squid and octopus olfactory pits, as previously reported by Buresi et al. (2014) (Fig. 2A). Although the presence of tubulin staining in these pockets indicates that they might have sensory capacity, these cells have not been reported previously, as ORN and their function are cryptic. In fact, none of the cells identified in the sensory epithelia of the pit-like pocket in our study or in Buresi et al. (2014) resembled any type of ORN cell described in other cephalopods (Woodhams & Messenger, 1974; Emery, 1975; Braby, 1998; Mobley et al., 2008; Polese et al., 2016). The PBP3 antiserum, which labels olfactory-like neurons in M. sexta (Nardi et al., 2003) and the ORNs in O. vulgaris (Fig. 1), enabled the discovery of immunoreactive cells to both PBP3 and acetylated α-tubulin in the forehead of S. officinalis (Fig. 4), which suggests the presence of putative ORN-like cells. A more detailed analysis showed that these PBP3/tubulin-positive cells were morphologically identical to cephalopod ORN cells (namely, types 1, 3, 4 and 5), further suggesting that they are true olfactory neurons. Furthermore, these cells were always found in clusters in the forehead between the eyes. Sundermann (1983) reported the presence of type 1 ciliated neurons in the head of S. officinalis. Moreover, Messenger (1967) reported that the S. officinalis ventral pits were no more sensitive to chemical stimuli than other areas of the body (such as arms, fin, head and back) and that the species seemed to be more sensitive to chemical stimuli when the olfactory pit was removed, specifically in its wound. Together with our data, these previous results suggest that S. officinalis most likely has the ability to detect odorant molecules by a diffuse olfactory system scattered across the forehead. Full confirmation of the true nature and function of the ORN-like cells of the species will require electrophysiological characterization. From an evolutionary perspective, the evidence suggests that in the two extant cephalopod subclasses (Nautiloidea and Coleoidea), olfaction evolved independently with more advanced members of the two classes having highly specialized olfactory organs (Woodhams & Messenger, 1974; Emery, 1975; Braby, 1998). It is not clear whether (1) the diffuse olfactory ‘organ’ in S. officinalis constitutes the primordial condition from which more advanced cephalopods evolved a dedicated, fully differentiated olfactory organ or (2) this organ has been lost due to adaptation (Lindgren et al., 2012) since the species typically buries itself for concealment in the wild, leaving only the forehead and eyes slightly above the substrate (Hanlon & Messenger, 1996). Only one ORN-like cell type was found in the Nautilus rhinophore (Young, 1965; Emery, 1975), contrasting with the presence of at least four different ORN-like cells found in the dorsal forehead of S. officinalis (present study) and five different cell types in squid and octopus (Woodhams & Messenger, 1974; Emery, 1975; Braby, 1998; Lucero et al., 2000; Mobley et al., 2008). This suggests that there was a diversification of ORN cell types in Coleoidea that did not occur in the Nautiloidea, which might have preceded the diversification of Coleoidea. However, it is also possible that convergent evolution of olfactory capacity in cephalopods led to the development of a distinct olfactory organ in squids and octopuses. Convergent evolution of different traits has been shown to be common in cephalopods (Lindgren et al., 2012). A particularly well-documented case is the cornea, which evolved in a convergent manner in both squid and octopus lineages. Deeper-level relationships between cephalopod orders and relationships between taxa within these orders are still poorly understood. Genomic studies are needed to increase the current knowledge and understanding of cephalopod evolution and systematics (Allcock, Lindgren & Strugnell, 2015). In summary, this study reveals that the forehead of S. officinalis, between the eyes, contains cells with ORN-like characteristics, probably with the ability to detect odorant molecules. Moreover, these cuttlefish ORN-like cells have similar morphology to distinct classes of ORN cells found in squid and octopus. Functional confirmation of these observations and definition of the cellular role will require electrophysiological/functional studies. ACKNOWLEDGEMENTS We thank Prof. Deborah M. Power for reagents, equipment and critical comments on the manuscript. A.V.S. and M.A.C. were funded by Fundação para a Ciência e Tecnologia (FCT) Post-Doc Grant (SFRH/BPD/36100/2007 and SFRH/BPD/66808/2009, respectively) and FCT Investigator Grant (IF/00576/2014 and IF/01274/2014, respectively). FCT funded this study through project SEPIABREED (PTDC/MAR/120876/2010) and Pluriannual funding to CCMAR (UID/Multi/04326/2013). REFERENCES Allcock AL , Lindgren A , Strugnell JM . 2015 . The contribution of molecular data to our understanding of cephalopod evolution and systematics: a review . Journal of Natural History 49 : 1373 – 1421 . 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Google Scholar CrossRef Search ADS © 2017 The Linnean Society of London, Zoological Journal of the Linnean Society This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Zoological Journal of the Linnean Society Oxford University Press

Olfactory-like neurons are present in the forehead of common cuttlefish, Sepia officinalis Linnaeus, 1758 (Cephalopoda: Sepiidae)

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© 2017 The Linnean Society of London, Zoological Journal of the Linnean Society
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0024-4082
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Abstract

Abstract According to the literature, the cuttlefish, Sepia officinalis, possesses a specialized olfactory organ and cells, located in olfactory ventral pits. In this study, the location of olfactory receptor neurons (ORNs) at the cellular level was determined using cellular morphology and immunohistochemistry. An antiserum against PBP3 was used as a marker to identify ORN-like cells in cuttlefish after validation for specificity to cephalopod ORN cells in the common octopus, Octopus vulgaris. The results show that ORN-like cells in S. officinalis were not found in the ventral pits, suckers or the mouth lips. Instead, ORN-like cells were found scattered in the forehead, between the eyes. The absence of ORN-like cells in a pit in S. officinalis and the sharing of four similar types of ORN cells with the squid and octopus lineages suggest that this might be a later innovation in olfaction and is probably associated with the specialized lifestyle of these later evolved cephalopods. Together, this evidence suggests a diversification of ORN cell types in Coleoidea, which did not occur in Nautiloidea, which might have preceded the diversification of the Coleoidea. cellular morphology, cephalopod, European cuttlefish, Sepia officinalis, olfaction, olfactory receptor neurons (ORNs) INTRODUCTION Cephalopods are a class in the phylum Mollusca, with over 700 species currently described. They have evolved from their early gastropod ancestor to efficient marine predators, diverging from the typical molluscan body plan and converging with vertebrates in locomotion, camouflage and sensory functions (Packard, 1972; Budelmann, 1995; Buresi et al., 2014). The coleoid cephalopods (squids, octopuses and cuttlefishes) have a large, centralized and complex nervous system (Buresi et al., 2016) that rivals the complexity of vertebrate nervous systems and is considered the most sophisticated of the invertebrates’ (Budelmann, 1996). The sensory system in cephalopods includes highly specialized eyes, ‘lateral lines’ equilibrium receptor systems (neck proprioceptors), touch, and chemical-sensitive tentacles (Budelmann, 1980; Sundermann, 1983; Budelmann & Bleckmann, 1988; Budelmann, 1994; Lenz, 1997; Stubbs & Stubbs, 2016). Moreover, squid and octopus have a well-developed and active olfactory organ located in olfactory pits, posterior to the eye but anterior to the mantle (Woodhams & Messenger, 1974; Emery, 1975, 1976; Braby, 1998; Shigeno et al., 2001; Villanueva & Norman, 2008; Polese, Bertapelle & Di Cosmo, 2016). In these organisms, the olfactory organ is composed of several epidermal folds containing odorant-sensitive cells. In squid, five morphologically and functionally distinct olfactory receptor neurons (ORNs) have been identified (Emery, 1975; Braby, 1998; Lucero, Horrigan & Gilly, 1992; Mobley, Michel & Lucero, 2008). Signalling in squid ORNs involves G-protein coupled receptors and Ca2+, cyclic AMP and PLC signalling (Piper & Lucero, 1999; Lucero, Huang & Dang, 2000; Mobley, Mahendra & Lucero, 2007; Mobley et al., 2008). Cuttlefish occupy an ancestral position in the Coleoidea lineage, and chemosensory cells have been identified in the arms and tentacles and in the lip/mouth regions (Graziadei, 1964, 1965). According to Budelmann (1996) and Nixon & Mangold (1998), a pair of olfactory organs exists in the late embryo of Sepia officinalis (Naef, 1928), each of the pair being located posterior to the eye and in front of the inhalant channel of the mantle, where two types of sensory cell and one type of ciliated epithelial cell are present. Although Nixon & Dilly (1977) related the existing sucker sensory receptors with mechanoreception, feeding behaviour experiments have suggested that the location of the olfactory organ might be in the arms and tentacles of cuttlefish (Boal & Golden, 1999; Archdale & Anraku, 2005). In Nautilus, a modified tentacle, the rhinophore, acts as a specialized olfactory organ (Young, 1965; Barber & Wright, 1969), further supporting the idea that the arms and/or tentacles might be a potential location for an olfactory organ in cuttlefish. More recently, Buresi et al. (2014) identified a pair of ventral pits in the anterior part of the head as the putative olfactory organ in S. officinalis. However, these same authors reported that although the structure of these cuttlefish olfactory pits resembles those of squid and octopus, the nervous cell types found in cuttlefish do not resemble any of the ORNs described in other advanced cephalopods. Therefore, despite published information on the existence of a putative olfactory organ (with similar ORN cells to other cephalopods) in the cuttlefish, S. officinalis, there is no scientific basis for confirming its function. This leads to two hypotheses: (1) S. officinalis does not have ORN cells similar to those reported in other cephalopods and the ventral pits are true olfactory organs with ORN cells still undescribed or (2) the ventral pits do not function as olfactory organs and the already-described ORN cells are found elsewhere. This study aimed to establish the existence and possible location of ORN-like cells in S. officinalis using cellular morphology and immunohistochemistry. MATERIAL AND METHODS Ethical statement Experiments were performed under project SEPIABREED (PTDC/MAR/120876/2010), which was approved before Directive 2010/63/EU (EU, 2010) was transposed into national legislation in Portugal. Cephalopod origin and sample preparation One-day-old hatchlings of S. officinalis (0.086 ± 0.011 g) were obtained from eggs laid by F2 and F3 captive stocks cultured at CCMAR facilities, as described in Sykes, Domingues & Andrade (2014). Eggs were individually sorted, according to the colour and morphology (only black ink-stained eggs with a flask-shaped morphology were used), and incubated for 30 days inside a 250 l fibreglass tank in a flow-through system (Sykes et al., 2006) with the following seawater quality conditions: 19 ± 1 °C, 35 ± 1 PSU, dissolved oxygen saturation above 90% and 100 lux light intensity. Cuttlefish developed, hatched without any apparent malformations and behaved normally. Hatchlings were euthanized (n = 3) by immersing in a cold seawater (4 °C) solution containing 5% of MgCl2 for 20 min (Sykes et al., 2012) and afterwards were fixed in 4% paraformaldehyde (PFA) in 1× phosphate-buffered saline (PBS; pH 7) at 4 °C overnight. Samples were then rinsed in 1× PBS/0.1% Tween20 (PBT) and transferred to 100% methanol through a grade series of PBS/methanol. Fixed samples were then stored in methanol at −20 °C. Samples were processed in a Leica TP 1020 (Leica Microsystems GmbH, Germany) tissue processor, through a graded series of ethanol (70–100%), followed by ethanol:xylene (1:1 v/v), xylene (100%) and then embedded in low-melting-point paraffin wax. Sagittal and transversal serial sections (10 µm) of hatchlings were prepared with a rotary microtome (Microm HM340E, Microm International GmbH, Germany) and mounted on poly-l-lysine-coated glass slides. For cryo-microtome sections (100–200 µm), the cuttlefish were transferred through graded methanol/1× PBS and then washed in 1× PBS. Samples were incubated on 1× PBS/30% sucrose for c. 4 h at room temperature, followed by embedding in OCT medium (VWR, Portugal) and freezing at −80 °C. Samples were sagittally or transversely sectioned using a Leica CM3050S cryo-microtome (Leica Microsystems GmbH, Germany), mounted on slides and stored at −20 °C until use. One adult S. officinalis (520 g) and one Octopus vulgaris (780 g) were used for the sampling of tissue from the olfactory pit. While the cuttlefish was obtained from a CCMAR culture stock, the octopus was purchased alive from the local fish market in Quarteira (southern Portugal). For both, olfactory pit tissue samples were collected after euthanasia (cuttlefish – 5% MgCl2; octopus a mixture of 5% pure ethanol and 5% of MgCl2; both compensated for salinity changes derived from the use of the salt) in seawater (Fiorito et al., 2015). The tissues were fixed in 4%PFA/1× PBS overnight at 4 °C. Afterwards, the tissues were rinsed in PBT, transferred to 100% methanol and kept at −20 °C until inclusion. Paraffin inclusion was carried out as described above, and a transversal section of the olfactory pit was cut and collected onto glass slides. Immunohistochemistry Paraffin sections (10 µm) were dewaxed in xylene and rehydrated through a graded ethanol series (100, 75, 50, 25% ethanol:1× PBS) into 1× PBS. Prior to the incubation with an acetylate α-tubulin antibody (T6793 Sigma–Aldrich, Spain), samples were immersed in acetone, cooled at −20 °C (30 min) and rinsed in 1× PBS. The slides were blocked in 1× PBS/10% sheep serum/0.5% Triton X100 for 2 h at room temperature and incubated overnight at 4 °C with an anti-acetylated α-tubulin antibody (1:1000; Sigma–Aldrich) or an anti-pheromone binding protein antibody (1:1 PBP3 of Manduca sexta moth antennae; Developmental Studies Hybridoma Bank, USA). A goat anti-mouse IgG H+L conjugated with Dylite 488 (1:400, AnaSpec, Belgium) was used for labelling after overnight incubation at 4 °C. Slides were rinsed in PBT with 0.7 nM 4′,6-diamidino-2-phenylindole (DAPI; Carl Roth GmbH + Co. KG, Germany) for 5 min, washed in PBS and mounted in glycerol + 2.4% 1,4-diazabicyclo-octane (DABCO; Carl Roth GmbH + Co. KG, Germany). For double immunohistochemistry, after the detection of PBP3 with goat anti-mouse IgG H+L conjugated with Dylite 596 (1:400, AnaSpec, Belgium), the slides were incubated in 1× PBS/10% sheep serum/0.5% Triton X100 and an anti-acetylated β-tubulin antibody (1:1000) overnight at 4 °C. Tubulin fluorescent detection was carried out overnight at 4 °C, after incubation with goat anti-mouse IgG2 Gamma conjugated with Alexa488 (1:200; Life Technologies). The slides were rinsed in PBT with 0.7 nM DAPI for 5 min, washed in PBS and mounted in glycerol + 2.4% DABCO. Negative controls (same procedure but omitting the primary antibody: 10% sheep serum buffer) were conducted to assess the specificity of labelling. For cryo-microtome sections, the slides were dried for 1 h at 37 °C in an incubator and hydrated in 1× PBS. The immunohistochemical procedures carried out for these sections were identical with those for the paraffin sections. Stained sections were examined with an Axio Imager Z2 fluorescence microscope and z-stacks were developed (Carl Zeiss AG, Oberkochen, Germany). The acquired images were deconvoluted using Huygens 4.3 software (Scientific Volume Imaging B.V., Hilversum, The Netherlands), and maximum projections were generated after FIJI analysis (Schindelin et al., 2012). For consistency, the head was considered anterior, the mantle apex posterior, the funnel ventral and the opposite side dorsal. The nomenclature adopted for the central nervous system of S. officinalis was in agreement with the previous studies (Boycott, 1961). Immunohistochemistry of O. vulgaris olfactory pit sections for PBP3 and acetylated α-tubulin was carried out as described above. These sections were used to validate the PBP3 antiserum as specific for olfactory neurons. RESULTS The PBP3 antiserum was chosen because it detects odorant cells in invertebrates. The identification of putative ORN-like cells in S. officinalis and O. vulgaris was attained with a PBP3 antiserum that labelled olfactory cells in M. sexta (Nardi et al., 2003). This antiserum was validated with the identification of ORN cells present in the olfactory pits in O. vulgaris. The staining of PBP3 only in ORN-like neurons in the olfactory pit of this species (as described by Polese et al., 2016 with an olfactory marking protein) confirmed that this antiserum only detected odorant-specific neurons (Fig. 1A–C). No staining for PBP3 was detected when the serum was omitted from the immunohistochemical procedure. This confirmed that the PBP3 antiserum is a reliable tool for identifying ORN-like neurons in cephalopods. Figure 1. View largeDownload slide PBP3 is only specific to ORN cells in octopus olfactory pits. Composite maximum projection of 50-µm image stacks after immunostaining in transversal paraffin sections of octopus olfactory pits for PBP3 (cyan), acetylated alfa-tubulin (green) and nuclear staining (DAPI, magenta). A–C, different ORN cells in octopus olfactory system. Different nervous cells are found in the olfactory pit region, but only ORN (white outlined cells) are positive for both PBP3 and tubulin (A–C). In parallel control sections (PBP3 serum omitted), no PBP3 signal is found (D). Scale bar = 10 µm. Figure 1. View largeDownload slide PBP3 is only specific to ORN cells in octopus olfactory pits. Composite maximum projection of 50-µm image stacks after immunostaining in transversal paraffin sections of octopus olfactory pits for PBP3 (cyan), acetylated alfa-tubulin (green) and nuclear staining (DAPI, magenta). A–C, different ORN cells in octopus olfactory system. Different nervous cells are found in the olfactory pit region, but only ORN (white outlined cells) are positive for both PBP3 and tubulin (A–C). In parallel control sections (PBP3 serum omitted), no PBP3 signal is found (D). Scale bar = 10 µm. We tested whether the olfactory organ was located in the arms, lips or tentacles (Fig. 2A), using immunohistochemistry to examine whether ORN-like cells existed in those locations. Staining sagittal sections of arm tips in S. officinalis hatchlings with anti-tubulin revealed several different nervous-like cells (Fig. 2B). These cells were present at the surface, below and transversely in the skin at the tip of the arm (Fig. 2B) but did not resemble the morphology of the ORNs described in squid (Emery, 1975; Lucero et al., 2000; Mobley et al., 2008) or octopus olfactory pits (Polese et al., 2016). In addition, no signal for PBP3 was detected in S. officinalis arms or tentacles tips (Fig. 2B). Also, all cells stained with α-tubulin in the proximal part of the arms lacked the morphology characteristic of ORN-like cells (Fig. 2C). In contrast, a PBP3-specific signal was detected in the most anterior part of the mouth/lips (Fig. 2C). Notably, the PBP3 signal co-localized with the α-tubulin staining in some, but not all, structures (Fig. 2C). This co-staining was subepidermal, and the cells did not have an ORN-like morphology (Fig. 2C). Figure 2. View largeDownload slide Olfactory-like neurons are not found in tentacles, arms, funnel and mouth of cuttlefish. Bright field sagittal section of cuttlefish hatchling showing regions that were analysed for the presence of ORN-like cells (coloured boxes; A). Maximum projections of 20-µm-deep stack after double immunofluorescent staining for acetylated alfa-tubulin (green), PBP3 (red) and nuclear staining (DAPI, magenta) on the tip of the tentacle (B), mouth (C) and funnel (D). Scale bar in (A) = 100 µm; scale bar in (B) = 10 µm; scale bar in (C) = 50 µm. Figure 2. View largeDownload slide Olfactory-like neurons are not found in tentacles, arms, funnel and mouth of cuttlefish. Bright field sagittal section of cuttlefish hatchling showing regions that were analysed for the presence of ORN-like cells (coloured boxes; A). Maximum projections of 20-µm-deep stack after double immunofluorescent staining for acetylated alfa-tubulin (green), PBP3 (red) and nuclear staining (DAPI, magenta) on the tip of the tentacle (B), mouth (C) and funnel (D). Scale bar in (A) = 100 µm; scale bar in (B) = 10 µm; scale bar in (C) = 50 µm. We also tested whether ORN-like cells were found in the ventral pits of adult cuttlefish, described by Buresi et al. (2014), as the olfactory organ with a similar capability and cells present in both squid and octopus. After extracting these structures from an adult cuttlefish, two distinct regions were identified (Fig. 3A). The ventral cavities had a folded (red square in Fig. 3A) and irregular flat surface (blue square in Fig. 3A) and contained anti-tubulin-positive nerve cells (Fig. 3A, B). However, none of the identified cells resembled the ORN cell types described previously in squid or octopus. Instead, a group of tubulin-positive cells that projected fibril-like structures into the pocket lumen (Fig. 3B), or were intensely stained with tubulin apically on the lumen-facing side of the cell, were observed (Fig. 3C). In both regions, nerve projections were observed (Fig. 3B, C) and the morphology resembled that reported by Buresi et al. (2014), but not any type described for other Coleoidea. Figure 3. View largeDownload slide Olfactory-like neurons are not found in ventral head pits of cuttlefish. Bright field image of excised ventral head pits (A). Red and blue squares in (A) denote regions in (B) and (C), respectively. Composite maximum projection of 20-µm stacks of folded pit region [red square in (A)] after fluorescent immunostaining for acetylated alfa-tubulin (green) and nuclear staining (DAPI, magenta) (B). Composite maximum projection of 20-µm stacks of flat pit region [blue square in (A)] after fluorescent immunostaining for acetylated alfa-tubulin (green) and nuclear staining (DAPI, magenta) (C). Scale bar = 50 µm. Figure 3. View largeDownload slide Olfactory-like neurons are not found in ventral head pits of cuttlefish. Bright field image of excised ventral head pits (A). Red and blue squares in (A) denote regions in (B) and (C), respectively. Composite maximum projection of 20-µm stacks of folded pit region [red square in (A)] after fluorescent immunostaining for acetylated alfa-tubulin (green) and nuclear staining (DAPI, magenta) (B). Composite maximum projection of 20-µm stacks of flat pit region [blue square in (A)] after fluorescent immunostaining for acetylated alfa-tubulin (green) and nuclear staining (DAPI, magenta) (C). Scale bar = 50 µm. The PBP3 staining was used as a marker for regions with putative olfactory detection capability in cuttlefish. Using this approach, a high concentration of structures stained positive for PBP3 in the forehead, between the eyes, of S. officinalis hatchlings (Fig. 4A). These structures were found between the dorsal posterior end of the head, just before the mantle, and the most anterior part of the face, just before the beginning of the arms (Fig. 4A). Since the PBP3 serum was raised against an olfactory binding protein from M. sexta (Nardi et al., 2003), the high concentration of PBP3-positive structures is probably related to olfaction. To further explore this hypothesis, the presence of nervous cells in the same region of the head was investigated with α-tubulin staining. This revealed a great number of nervous cells in the dorsal epidermis that seem to project directly to the brain (Fig. 4B). Nonetheless, the presence of nervous cells cannot be fully proven using just histological sections. The double detection of PBP3 and α-tubulin revealed that both proteins were present in the same ciliated cells at the top of the epidermis and in some sub-epithelial structures (Fig. 4C). However, we could not determine the exact nature of the sub-epithelial structures labelled for both markers; we can only infer that they are probably nervous system structures. Moreover, stained epithelial nerve cells with PBP3 were only observed in this region of the head of S. officinalis. A more detailed analysis revealed that these cells had ciliated structures at their most apical end that projected outside the skin (Fig. 4D–F). Furthermore, these cells were always found in clusters and shared morphology with ORN in other cephalopods (Fig. 4D–F). The putative S. officinalis ORN cells (Fig. 4D, E) resembled octopus and squid types 1 (Fig. 4E), 3 (Fig. 4D), 4 (Fig. 4F) and 5 (Fig. 4D, F) (Emery, 1975; Lucero et al., 2000; Mobley et al., 2007, 2008; Polese et al., 2016). Figure 4. View largeDownload slide Olfactory-like neurons are only found in the cuttlefish dorsal forehead, between the eyes. Composite image of bright field, nuclear staining (DAPI, blue) and immunostaining of PBP3 (red) in the dorsal forehead region between the eyes (A). Composite maximum projection of 200-µm stack of dorsal forehead between the eyes after fluorescent immunostaining for acetylated alfa-tubulin (green) and nuclear staining (DAPI, magenta; B). Composite maximum projection of 50-µm stack from the dorsal forehead after double fluorescent immunostaining for acetylated alfa-tubulin (green), PBP3 (red) and nuclear staining (DAPI, magenta; C). Composite maximum projection of 50-µm stack from the dorsal forehead after fluorescent immunostaining for acetylated alfa-tubulin (green) and nuclear staining (DAPI, magenta; D–F). D–F, presumed ORN-like neurons are outlined to enhance their morphology and numbers next to cells indicate squid and octopus ORN classes previously identified (Emery, 1975). Scale bar in (A) = 100 µm; scale bar in (B, C) = 50 µm; scale bar in (D–F) = 10 µm. Figure 4. View largeDownload slide Olfactory-like neurons are only found in the cuttlefish dorsal forehead, between the eyes. Composite image of bright field, nuclear staining (DAPI, blue) and immunostaining of PBP3 (red) in the dorsal forehead region between the eyes (A). Composite maximum projection of 200-µm stack of dorsal forehead between the eyes after fluorescent immunostaining for acetylated alfa-tubulin (green) and nuclear staining (DAPI, magenta; B). Composite maximum projection of 50-µm stack from the dorsal forehead after double fluorescent immunostaining for acetylated alfa-tubulin (green), PBP3 (red) and nuclear staining (DAPI, magenta; C). Composite maximum projection of 50-µm stack from the dorsal forehead after fluorescent immunostaining for acetylated alfa-tubulin (green) and nuclear staining (DAPI, magenta; D–F). D–F, presumed ORN-like neurons are outlined to enhance their morphology and numbers next to cells indicate squid and octopus ORN classes previously identified (Emery, 1975). Scale bar in (A) = 100 µm; scale bar in (B, C) = 50 µm; scale bar in (D–F) = 10 µm. DISCUSSION Well-differentiated olfactory organs have been reported for most cephalopods. Nautilus has a specialized tentacle (rhinophore) (Young, 1965), and squid and octopus have a pair of active olfactory organs located immediately posterior to the eye (Woodhams & Messenger, 1974; Emery, 1975; Braby, 1998; Villanueva & Norman, 2008; Polese et al., 2016). Recently, Buresi et al. (2014) described the existence of octopus-like olfactory pits in S. officinalis, but the cells identified in those structures did not have a cellular morphology resembling any other cephalopod olfactory cells in the literature. Olfactory organs or capacities were initially though to be possibly located in the arms and/or in tentacle suckers of cuttlefish because these structures possess ciliated cells (Graziadei, 1964; Boal & Golden, 1999; Hanlon & Messenger, 1996; Archdale & Anraku, 2005). In this study, nerve cells were identified in the tip of the arms and tentacles of S. officinalis, but none of these cells either resembled ORN-like cells described previously for other cephalopods (Emery, 1975; Polese et al., 2016) or were immunoreactive for PBP3 (Fig. 2B–D). Therefore, it is unlikely that these structures are involved in S. officinalis olfaction. This result is remarkably different from what is reported for Nautilus, where the rhinophore (a modified tentacle) assumes the olfactory role (Young, 1965; Barber & Wright, 1969). This study suggests that the development of the rhinophore (containing an ORN cell intermediate to types 4 and 5 in squid) might represent a Nautilus lineage-specific organ and adaptation to the habitat rather than an ancestral feature common to the Nautiloidea and Coleoidea lineages. We identified two small ventral cavities, located in the posterior head region just before the mantle that have an ultrastructure reminiscent of squid and octopus olfactory pits, as previously reported by Buresi et al. (2014) (Fig. 2A). Although the presence of tubulin staining in these pockets indicates that they might have sensory capacity, these cells have not been reported previously, as ORN and their function are cryptic. In fact, none of the cells identified in the sensory epithelia of the pit-like pocket in our study or in Buresi et al. (2014) resembled any type of ORN cell described in other cephalopods (Woodhams & Messenger, 1974; Emery, 1975; Braby, 1998; Mobley et al., 2008; Polese et al., 2016). The PBP3 antiserum, which labels olfactory-like neurons in M. sexta (Nardi et al., 2003) and the ORNs in O. vulgaris (Fig. 1), enabled the discovery of immunoreactive cells to both PBP3 and acetylated α-tubulin in the forehead of S. officinalis (Fig. 4), which suggests the presence of putative ORN-like cells. A more detailed analysis showed that these PBP3/tubulin-positive cells were morphologically identical to cephalopod ORN cells (namely, types 1, 3, 4 and 5), further suggesting that they are true olfactory neurons. Furthermore, these cells were always found in clusters in the forehead between the eyes. Sundermann (1983) reported the presence of type 1 ciliated neurons in the head of S. officinalis. Moreover, Messenger (1967) reported that the S. officinalis ventral pits were no more sensitive to chemical stimuli than other areas of the body (such as arms, fin, head and back) and that the species seemed to be more sensitive to chemical stimuli when the olfactory pit was removed, specifically in its wound. Together with our data, these previous results suggest that S. officinalis most likely has the ability to detect odorant molecules by a diffuse olfactory system scattered across the forehead. Full confirmation of the true nature and function of the ORN-like cells of the species will require electrophysiological characterization. From an evolutionary perspective, the evidence suggests that in the two extant cephalopod subclasses (Nautiloidea and Coleoidea), olfaction evolved independently with more advanced members of the two classes having highly specialized olfactory organs (Woodhams & Messenger, 1974; Emery, 1975; Braby, 1998). It is not clear whether (1) the diffuse olfactory ‘organ’ in S. officinalis constitutes the primordial condition from which more advanced cephalopods evolved a dedicated, fully differentiated olfactory organ or (2) this organ has been lost due to adaptation (Lindgren et al., 2012) since the species typically buries itself for concealment in the wild, leaving only the forehead and eyes slightly above the substrate (Hanlon & Messenger, 1996). Only one ORN-like cell type was found in the Nautilus rhinophore (Young, 1965; Emery, 1975), contrasting with the presence of at least four different ORN-like cells found in the dorsal forehead of S. officinalis (present study) and five different cell types in squid and octopus (Woodhams & Messenger, 1974; Emery, 1975; Braby, 1998; Lucero et al., 2000; Mobley et al., 2008). This suggests that there was a diversification of ORN cell types in Coleoidea that did not occur in the Nautiloidea, which might have preceded the diversification of Coleoidea. However, it is also possible that convergent evolution of olfactory capacity in cephalopods led to the development of a distinct olfactory organ in squids and octopuses. Convergent evolution of different traits has been shown to be common in cephalopods (Lindgren et al., 2012). A particularly well-documented case is the cornea, which evolved in a convergent manner in both squid and octopus lineages. Deeper-level relationships between cephalopod orders and relationships between taxa within these orders are still poorly understood. Genomic studies are needed to increase the current knowledge and understanding of cephalopod evolution and systematics (Allcock, Lindgren & Strugnell, 2015). In summary, this study reveals that the forehead of S. officinalis, between the eyes, contains cells with ORN-like characteristics, probably with the ability to detect odorant molecules. 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Published: Nov 11, 2017

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