Sensory specializations drive octopus and squid behaviour.

The evolution of new traits enables expansion into new ecological and behavioural niches. Nonetheless, demonstrated connections between divergence in protein structure, function and lineage-specific behaviours remain rare. Here we show that both octopus and squid use cephalopod-specific chemotactile receptors (CRs) to sense their respective marine environments, but structural adaptations in these receptors support the sensation of specific molecules suited to distinct physiological roles. We find that squid express ancient CRs that more closely resemble related nicotinic acetylcholine receptors, whereas octopuses exhibit a more recent expansion in CRs consistent with their elaborated 'taste by touch' sensory system. Using a combination of genetic profiling, physiology and behavioural analyses, we identify the founding member of squid CRs that detects soluble bitter molecules that are relevant in ambush predation. We present the cryo-electron microscopy structure of a squid CR and compare this with octopus CRs1 and nicotinic receptors2. These analyses demonstrate an evolutionary transition from an ancestral aromatic 'cage' that coordinates soluble neurotransmitters or tastants to a more recent octopus CR hydrophobic binding pocket that traps insoluble molecules to mediate contact-dependent chemosensation. Thus, our study provides a foundation for understanding how adaptation of protein structure drives the diversification of organismal traits and behaviour.

Structural basis of sensory receptor evolution in octopus.

Chemotactile receptors (CRs) are a cephalopod-specific innovation that allow octopuses to explore the seafloor via 'taste by touch'1. CRs diverged from nicotinic acetylcholine receptors to mediate contact-dependent chemosensation of insoluble molecules that do not readily diffuse in marine environments. Here we exploit octopus CRs to probe the structural basis of sensory receptor evolution. We present the cryo-electron microscopy structure of an octopus CR and compare it with nicotinic receptors to determine features that enable environmental sensation versus neurotransmission. Evolutionary, structural and biophysical analyses show that the channel architecture involved in cation permeation and signal transduction is conserved. By contrast, the orthosteric ligand-binding site is subject to diversifying selection, thereby mediating the detection of new molecules. Serendipitous findings in the cryo-electron microscopy structure reveal that the octopus CR ligand-binding pocket is exceptionally hydrophobic, enabling sensation of greasy compounds versus the small polar molecules detected by canonical neurotransmitter receptors. These discoveries provide a structural framework for understanding connections between evolutionary adaptations at the atomic level and the emergence of new organismal behaviour.

Process of cytoplasm elimination during spermiogenesis in Octopus tankahkeei: Polarized development of the spermatid and discarding of the residual body.

The elimination of the spermatid cytoplasm during spermiogenesis enables the sperm to acquire a streamlined architecture, which allows for unhindered swimming. While this process has been well described in vertebrates, it has rarely been reported in invertebrates. In this study, we observed the process of cytoplasm elimination during spermiogenesis in Octopus tankahkeei (Mollusca, Cephalopoda) using light microscopy, transmission electron microscopy, and immunofluorescence. In the early spermatid, the cell is circular, and the nucleus is centrally located. With spermatid development, the cell becomes polarized. The nucleus gradually elongates and moves toward the end of the cell where the tail is forming. As a result, the cytoplasm moves past the nucleus at the anterior region of the future sperm head (the foreside of the acrosome). Following this, during the late stage of spermiogenesis, the cytoplasm condenses and collects on the foreside of the acrosome until finally the residual body is discarded from the top of the sperm head. This represents a distinct directionality for the development of cytoplasmic polarity and discarding of residual body compared with that reported for vertebrates (in which the cytoplasm of the elongating spermatids is polarized toward the caudal region). The fact that the cytoplasm also becomes concentrated suggests that water pumps may be involved in the elimination of water from the cytoplasm before the residual body is discarded. Furthermore, we found that microtubules, forming a manchette-like structure, are involved not only in reshaping of the nucleus but also in the transport of mitochondria and vesicles to the foreside of the acrosome, subsequently allowing them to be discarded with the residual body. This study broadens our understanding of the development of polarization and elimination of cytoplasm from spermatids in animals.

Cryo-scanning electron microscopy investigation of the Octopus Vulgaris arm structures for the design of an octopus-like arm artefact.

Octopus vulgaris is a cephalopod of the Octopodidae family. It has four pairs of arms and two rows of suckers which perform many functions, including bending and elongation. For this reason the octopus was chosen as model to develop a new generation of soft-body robots. In order to explain some of the fine structures of the octopus arm in relation to its specific ability, we examined the external and internal structures of O. vulgaris arms in a frozen-hydrated state using cryo-scanning electron microscopy. The arms showed skin with a very complex design that is useful to elongation, and a pore pattern distribution on their surface which is functional to cutaneous oxygen uptake. The analysis of freeze-fractured frozen-hydrated arm samples allowed us to describe the developmental differences in the relative proportion of the areas of axial nerve cord, intrinsic and extrinsic musculature, in relation to the growth of the arms and of the increase in functional capability. In the suckers, we analyzed the shedding mechanisms in the outer part of the infundibulum and described the outer and inner characteristics of the denticles, showing in detail their pore system, which is fundamental for their ability to explore the environment. These results are discussed by considering their possible application in the design of new octopus-like artefacts, which will be able to take advantage of some of these ultrastructure characteristics and achieve advanced bioinspired functionalities.

Immunohistochemical observations of methionine-enkephalin and delta opioid receptor in the digestive system of Octopus ocellatus.

The study was designed to determine whether methionine-enkephalin (met-Enk) or delta opioid receptor was present in the digestive system of Octopus ocellatus. The results showed that they were both in the bulbus oris, esophagus, crop, stomach, gastric cecum, intestine, posterior salivary glands of O. ocellatus, one of them, met-Enk in the rectum, anterior salivary glands, digestive gland. And the distributions were extensive in the digestive system. Strong or general met-Enk immunoreactivity was observed in the inner epithelial cells of the bulbus oris, esophagus, stomach, gastric cecum, intestine, anterior salivary glands and the adventitia of the intestine and rectum, and so was the delta opioid receptor immunoreactivity in the inner epithelial cells of the bulbus oris, esophagus, and crop, however, they were weak in other parts. Combining with delta opioid receptor, met-Enk may be involved in the regulations of food intake, absorption, movement of gastrointestinal smooth muscle and secretion of digestive gland. The different densities of met-Enk and delta opioid receptor may be related to the different functions in the digestive system of O. ocellatus.

Molecular characterization of a KIF3B-like kinesin gene in the testis of Octopus tankahkeei (Cephalopoda, Octopus).

KIF3B is known for maintaining and assembling cilia and flagellum. To date, the function of KIF3B and its relationship with KIF3A during spermiogenesis in the cephalopod Octopus tankahkeei remains unknown. In the present study, we characterized a gene encoding a homologue of rat KIF3B in the O. tankahkeei testis and examined its temporal and spatial expression pattern during spermiogenesis. The cDNA of KIF3B was obtained with degenerate and RACE PCR and the distribution pattern of ot-kif3b were observed with RT-PCR. The morphological development during spermiogenesis was illustrated by histological and transmission electron microscopy and mRNA expression of ot-kif3b was observed by in situ hybridization. The 2,365 nucleotides cDNA consisted of a 102 bp 5' untranslated region (UTR), a 2,208 bp open reading frame (ORF) encoding a protein of 736 amino acids, and a 55 bp 3' UTR. Multiple alignments revealed that the putative Ot-KIF3B shared 68, 68, 69, 68, and 67% identity with that of Homo sapiens, Mus musculus, Gallus gallus, Danio rerio, and Xenopus laevis, respectively, along with high identities with Ot-KIF3A in fundamental structures. Ot-kif3b transcripts appeared gradually in early spermatids, increased in intermediate spermatids and maximized in drastically remodeled and final spermatids. The kif3b gene is identified and its expression pattern is demonstrated for the first time in O. tankahkeei. Compared to ot-kif3a reported by our laboratory before, our data suggested that the putative heterodimeric motor proteins Ot-KIF3A/B may be involved in intraspermatic transport and might contribute to structural changes during spermiogenesis.

Sperm ultrastructure of the hydrothermal vent octopod Vulcanoctopus hydrothermalis.

Sperm ultrastructure of the deep-sea hydrothermal vent octopod Vulcanoctopus hydrothermalis has been carried out by transmission electron microscopy. Spermatozoa of this species have the shortest head observed so far in octopodids. The acrosome possesses a helix with six gyres. The rod-shaped nucleus is short and wide in relation with other octopodids. Noteworthy features along the nucleus are the regularly disposed dense bands of cytoplasm, which have not been observed before in octopodids. The nuclear fossa is very short and wavy. Mitochondrial sheath has 10 elongated mitochondria running parallel to the axoneme-coarse fibers complex. Sperm morphology of V. hydrothermalis resembles that of Enteroctopus dofleini, suggesting a close phylogenetic relationship.

Acrosome reaction in Octopus tankahkeei induced by calcium ionophore A23187 and a possible role of the acrosomal screw.

The octopod sperm is unique especially in two aspects: the screw-shaped acrosome and its inner layered substructure (striation). The present study aims to investigate morphological changes of Octopus tankahkeei spermatozoa during the acrosome reaction (AR) and to pursue functions of the internal substructures revealed by inducing AR with the calcium ionophore A23187. Gradual changes of the spermatozoa were traced using fluorescence and electron microscopy. The AR process included the bulging, vesiculation, and dehiscence of the plasma membrane around the acrosome and the nucleus, as well as the vesiculation of the mitochondrial sheath. Membrane vesiculation outside the nucleus has never been reported in the order Octopoda. The rigid screw and the inner striation of the acrosome remained intact surmounting the nucleus, suggesting that these two structures have potential functions during fertilization. In addition, the detachment of the sperm head and the tail was commonly observed in this study, both in intact and acrosome-reacted sperm. Fluorescence microscopy revealed that the detached mitochondrial sheath usually gave weaker and more dispersive signals than the joint ones. This phenomenon implied that the intense energy release might promote the detachment of the mitochondrial sheath.

Sperm ultrastructure in Bathypolypus bairdii and B. sponsalis (Cephalopoda: Octopoda).

A morphological comparison of the spermatozoa of the octopods Bathypolypus bairdii and B. sponsalis has been carried out by electron microscopy. Although the mature spermatozoon of B. bairdii is longer and thinner than that of B. sponsalis, its general ultrastructure is similar except for some minor differences. Their characteristic acrosomes, described here for the first time, consist of a periodically banded cone surrounded by a double helix whose arrangement has been defined by a numeric expression. The plasma membrane of sponsalis that surrounds the acrosome has many projections filled with abundant granular cytoplasm, which appears as a star-shaped acrosome in cross-section. The mitochondrial sheath contains 9-11 mitochondria in bairdii but only 9 in sponsalis. A comparison with other Octopodidae shows that the nucleus of both species is the largest ever seen. Our results support the capability of sperm morphology to discriminate between species and could even discern at higher taxonomic levels.

An ultrastructural study of connective tissue in mollusc integument III. Cephalopoda.

We studied structure and ultrastructure of the subepidermal connective tissue (SEC) of the integument of three cephalopods (Sepia officinalis, Octopus vulgaris and Loligo pealii). In all species, three distinct regions of the SEC were recognised: (a) an outer zone (OZ) that included the dermal-epidermal junction, and consisted of a thin layer of connective tissue containing muscles, (b) an extensive middle zone (MZ) containing a compact network of collagen fibres and numerous cells, (c) an inner zone (IZ) of loose connective tissue that merged with muscular fascia. This arrangement differs from that in bivalves and gastropods and recalls vertebrate integument. The dermal-epidermal junction of cephalopods differed from that of bivalves, gastropods and mammals in that the epidermal cells did not possess hemidesmosomes, and their intermediate filaments terminated directly in the plasmamembrane. The thick (120-500 nm) basal membrane (BM) had a superficial zone containing a regular array of granules; a lamina densa composed of a compact network of small filaments and granules; and an IZ distinguished by expansions of granular material protruding into underlying structures. Collagen fibres contained fibroblast-derived cytoplasmic thread, running through their centres and were surrounded by granular material that joins them to adjacent fibres. The collagen fibrils were of medium diameter (30-80 nm) had the typical ultrastructure of fibrillar collagens, and were surrounded by abundant interfibrillar material. The hypodermis was loose, with a network of small bundles of collagen fibrils. Cephalopod integument appears to represent a major evolutionary step distinguishing this class of molluscs.

Chromatin condensation, cysteine-rich protamine, and establishment of disulphide interprotamine bonds during spermiogenesis of Eledone cirrhosa (Cephalopoda).

During spermiogenesis in Eledone cirrhosa a single protamine substitutes for histones in nuclei of developing spermatids. This protein displays a peculiar primary structure. It contains 22.6 mol% cysteine residues (19 cysteines in 84 residues). This makes it the most cysteine-rich protamine known. The proportion of basic residues is relatively low (arginine 36.9 mol%, lysine 19.0 mol%). The protamine of E. cirrhosa condenses spermiogenic chromatin in a pattern which comprises fibres with a progressively larger diameter and lamellae that finally undergo definitive coalescence. We have also performed a study that estimates the number of interprotamine disulphide bonds formed during the process of spermiogenic chromatin condensation by means of sequential disappearance of MMNA (monomaleimido-nanogold) labelling. During the first step of spermiogenesis, protamines are found spread over very slightly condensed chromatin with their cysteines in a reactive state (protamine-cys-SH). From this stage the interprotamine disulphide bonds are established in a progressive way. First they are formed inside the chromatin fibres. Subsequently, they participate in the mechanism of fibre coalescence and finally, in the last step of spermiogenesis, the remaining free reactive -SH groups of cysteine form disulphide bonds, thus promoting a definitive stabilization of the nucleoprotein complex in the ripe sperm nucleus.

Three-dimensional reconstruction of Sepia officinalis hemocyanin from frozen-hydrated specimens.

The three-dimensional reconstruction of Sepia hemocyanin from randomly oriented native molecules was carried out by the method of the random conical tilt series on a frozen-hydrated specimen. As other molluscan hemocyanins, the molecule resembles a hollow cylinder or pentahedron composed of five dimeric subunits. Each dimeric subunit, composed of 16 functional units, comprises one wall oblique unit made up of 12 functional units and one arch made up of four functional units. The five wall oblique units are separated from each other by five oblique clefts bridged by the five arches, formerly termed collar structures. Each arch is composed of two types of functional units that are probably Soe, a functional unit absent in Octopus hemocyanin, and Soh, the C-terminal functional unit of the polypeptide chain. The architecture of the arches and their intramolecular location in front of the edges of the pentahedron are strongly reminiscent of the arches of Octopus hemocyanin. The D5 point-group symmetry of the molecule suggests that the orientation of the polypeptide chains is antiparallel as in Octopus hemocyanin. Several models of architecture compatible with these results are designed.

The effect of L-glutamate on the afferent resting activity in the cephalopod statocyst.

The effects of bath application of L-glutamate and of excitatory amino acid agonists and antagonists on the resting activity of afferent crista fibers were studied in isolated preparations of the statocyst of the cuttlefish, Sepia officinalis. L-Glutamate (threshold 10(-5) M) and its agonists quisqualate and kainate (thresholds 10(-6) M) increased the resting activity in a dose-dependent manner. Glutamine (threshold 10(-5) M) was also excitatory, while D-glutamate had no effect. Also, no obvious excitatory effects were seen for NMDA and L-aspartate, nor was any antagonistic effect seen for the selective NMDA-receptor antagonist D-2-amino-5-phosphonovaleric acid (D-AP-5). The spider toxin Argiotoxin636 (threshold 10(-11) M), 2-amino-4-phosphonobutyric acid (AP-4), glutamic acid diethyl ester (GDEE), gamma-D-glutamylaminomethyl-sulfonic acid (GAMS), and kynurenic acid decreased the resting activity and effectively blocked or reversed the effect of L-glutamate and its non-NMDA agonists. Preliminary experiments with statocysts from the squid Sepioteuthis lessoniana and the octopod Octopus bimaculoides gave comparable results. All data show that in cephalopod statocysts L-glutamate, via non-NMDA receptors, has an excitatory effect on the activity of afferent fibers, an effect consistent with its possible function as a hair cell transmitter.

Electron-microscopic observations of the gravity receptor epithelia of normal and spinner juvenile Octopus maya.

Light and electron microscopy of the gravity receptor epithelia (maculae) of statocysts of normal and "spinner" juvenile Octopus maya showed differences between the structures of the hair cells, supporting cells, and afferent neurons of these cephalopods. The maculae of spinner animals were approximately 30% smaller in their surface area and had 40% fewer hair cells. Moreover, the average distance between randomly-chosen hair bundles in scanning electron micrographs of maculae of normal animals was significantly greater (4.33 +/- 6.47 microns) than those of maculae of spinner animals (3.38 +/- 4.90 microns; P less than 0.0001). The sectional area of the supporting cell's microvilli in spinner maculae was larger (0.16 +/- 0.18 microns) than those of normal (0.10 +/- 0.10 micron; P less than 0.0001) O. maya. The morphological differences observed between certain structural components of the maculae of normal and spinner O. maya may be related to the absence and/or malformation of the neuroepithelial suprastructures in spinners. This may have direct or indirect effects to their inability to orient to gravity with these organs.

Histochemical evidence for catecholamines as neurotransmitters in the statocyst of Octopus vulgaris.

Formaldehyde-induced fluorescence (Falck-Hillarp technique) provided histochemical evidence for the presence of catecholamines in the sensory epithelia (macula and crista) of the Octopus statocyst. A specific bright green fluorescence occurred in the neuronal plexus beneath the receptor cell layers of the epithelia and in the appropriate nerves. The histochemical findings are discussed with reference to the well-known neuronal and synaptic organization of the epithelia and to relevant results in cephalopods as well as in other molluscs. All data support the hypothesis that in the receptor systems of the Octopus statocyst catecholamines (probably dopamine and/or noradrenaline) act as neurotransmitters in the efferent fibre system.

Tissue channel morphology in Octopus.

The morphology of tissue channels in muscle and neural tissues of Octopus was investigated, at the ultrastructural level, with a technique involving the precipitation of ferrocyanide ions. The numbers, sizes and conductivities of the channels were estimated from quantitative data. No evidence was gained to indicate that the low microvascular density in Octopus is coupled to an especially extensive network of extravascular channels. The tissue channel system in Octopus appears to be broadly comparable with the mammalian system; a lack of information prevents more appropriate comparisons with marine fishes. Probable functions of tissue channels in Octopus and mammals, and reasons for apparent similarities and differences in the channel organization of these divergent groups, are discussed.

Octopus microvasculature: permeability to ferritin and carbon.

The permeability of Octopus microvasculature was investigated by intravascular injection of carbon and ferritin. Vessels were tight to carbon while ferritin penetrated the pericyte junction, and was found extravascularly 1-2 min after its introduction. Vesicles occurred rarely in pericytes; fenestrae were absent. The discontinuous endothelial layer did not consitute a permeability barrier. The basement membrane, although retarding the movement of ferritin, was permeable to it; carbon did not penetrate the basement membrane. Evidence indicated that ferritin, and thus similarly sized and smaller water soluble materials, traverse the pericyte junction as a result of bulk fluid flow. Comparisons are made with the convective (or junctional) and slower, diffusive (or vesicular) passage of materials known to occur across the endothelium of continuous capillaries in mammals. Previous macrophysiological determinations concerning the permeability of Octopus vessels are questioned in view of these findings. Possible reasons for some major structural differences in the microcirculatory systems of cephalopods and vertebrates are briefly discussed.

Uptake of ferritin by the cephalopod optic gland.

Horse spleen ferritin, injected into the blood of the octopus, leaves the capillaries via pericyte junctions and windows. One hour after the administration, ferritin has entered the optic gland main cells; three hours later, it is accumulated in dense-bodies. The evidence for resorption supports earlier papers reporting that the ultrastructure of the organ is unusual for an endocrine gland.

Observations on the olfactory organ of adult and juvenile Octopus joubini.

The olfactory organ has an epithelium containing many sense cells and a large subepithelial mass of receptor cells. The epithelium includes cells with cup-shaped, ciliated endings, and hollow, flask-shaped sense cells with ciliated cavities that open to the surface, through a small pore. Below the epithelium are large hollow cells with ciliated cavities and distal processes that either form patent connections between the ciliated cavity and the surface or have a ciliated ending at the surface. There are many synapses between processes in the olfactory nerve. The possible chemosensory function of the olfactory organ is discussed.