Fine structure of the central brain in the octopod Eledone cirrhosa (Lamarck, 1798) (Mollusca-Octopoda).

This study aims to investigate the fine structure of the different cell types in the central brain of Eledone cirrhosa; the organelles in the neurons and the glial cells; the glial hemolymph-brain barrier; the neuro-secretions and the relationships between glial and nerve cells. The brain is surrounded by a non-cellular neurilemma followed by a single layer of perilemmal cells. Ependymal cells, highly prismatic glial cells, astrocytes, oligodendrocytes and epithelial processes were observed. The perikarya of the neurons are filled with slightly oval nuclei with heterochromatin, a strongly tortuous ER, numerous mitochondria and Golgi apparatus with two types of vesicles. In the cellular cortex, glial cells are much less numerous than the neurons and they are located preferably at the border between perikarya and neuropil. Furthermore, they send many branching shoots between the surrounding neuron perikarya and the axons. The glial cytoplasmic matrix appears more electrodense than that of the neurons. Only few ribosomes are attached to the membranes of the ER; the vast majorities are free. In the perikarya of the glial cells, mitochondria, multi-vesicular bodies, various vacuoles and vesicles are present. The essential elements of the hemolymph-brain barrier are the endothelial cells with their tight junctions. The cytoplasm contains various vesicles and mitochondria. However, two other cell types are present, the pericytes and the astrocytes, which are of great importance for the function of the hemolymph-brain barrier. The cell-cell interactions between endothelial cells, pericytes and astrocytes are as close as no other cells.

The role of hairs in the adhesion of octopus suckers: a hierarchical peeling approach.

Organisms like the octopus or the clingfish are a precious source of inspiration for the design of innovative adhesive systems based on suction cups, but a complete mechanical description of their attachment process is still lacking. In this paper, we exploit the recent discovery of the presence of hairs in the acetabulum roof of octopus suction cups to revise the current model for its adhesion to the acetabulum wall. We show how this additional feature, which can be considered an example of a hierarchical structure, can lead to an increase of adhesive strength, based on the analysis of the cases of a simple tape and an axisymmetrical membrane adhering to a substrate. Using peeling theory, we discuss in both cases the influence of hierarchical structure and the resulting variation of geometry on the adhesive energy, highlighting how an increase in number of hierarchical levels contributes to its increment, with a corresponding improvement in functionality for the octopus suckers.

Octopus vulgaris: An Alternative in Evolution.

Octopus vulgaris underwent a radical modification to cope with the benthic lifestyle. It diverged from other cephalopods in terms of body plan, anatomy, behavior, and intelligence. It independently evolved the largest and most complex nervous system and sophisticated behaviors among invertebrates in a separate evolutionary lineage. It is equipped with unusual traits that confer it an incredible evolutionary success: arms capable of a wide range of movements with no skeletal support; developed eyes with a complex visual behavior; vestibular system; primitive "hearing" system; chemoreceptors located in epidermis, suckers, and mouth; and a discrete olfactory organ. As if these were not enough, the occurrence of recently discovered adult neurogenesis and the high level of RNA editing give it a master key to face environmental challenges. Here we provide an overview of some of the winning evolutionary inventions that octopus puts in place such as the capacity to see color, smell by touch, edit own genes, and rejuvenate own brain.

Nerve regeneration in the cephalopod mollusc Octopus vulgaris: label-free multiphoton microscopy as a tool for investigation.

Octopus and cephalopods are able to regenerate injured tissues. Recent advancements in the study of regeneration in cephalopods appear promising encompassing different approaches helping to decipher cellular and molecular machinery involved in the process. However, lack of specific markers to investigate degenerative/regenerative phenomena and inflammatory events occurring after damage is limiting these studies. Label-free multiphoton microscopy is applied for the first time to the transected pallial nerve of Octopus vulgaris Various optical contrast methods including coherent anti-Stokes Raman scattering (CARS), endogenous two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) have been used. We detected cells and structures often not revealed with classical staining methods. CARS highlighted the involvement of haemocytes in building up scar tissue; CARS and TPEF facilitated the identification of degenerating fibres; SHG allowed visualization of fibrillary collagen, revealing the formation of a connective tissue bridge between the nerve stumps, likely involved in axon guidance. Using label-free multiphoton microscopy, we studied the regenerative events in octopus without using any other labelling techniques. These imaging methods provided extremely helpful morpho-chemical information to describe regeneration events. The techniques applied here are species-specific independent and should facilitate the comparison among various animal species.

De novo transcriptome sequencing of the Octopus vulgaris hemocytes using Illumina RNA-Seq technology: response to the infection by the gastrointestinal parasite Aggregata octopiana.

BACKGROUND: Octopus vulgaris is a highly valuable species of great commercial interest and excellent candidate for aquaculture diversification; however, the octopus' well-being is impaired by pathogens, of which the gastrointestinal coccidian parasite Aggregata octopiana is one of the most important. The knowledge of the molecular mechanisms of the immune response in cephalopods, especially in octopus is scarce. The transcriptome of the hemocytes of O. vulgaris was de novo sequenced using the high-throughput paired-end Illumina technology to identify genes involved in immune defense and to understand the molecular basis of octopus tolerance/resistance to coccidiosis. RESULTS: A bi-directional mRNA library was constructed from hemocytes of two groups of octopus according to the infection by A. octopiana, sick octopus, suffering coccidiosis, and healthy octopus, and reads were de novo assembled together. The differential expression of transcripts was analysed using the general assembly as a reference for mapping the reads from each condition. After sequencing, a total of 75,571,280 high quality reads were obtained from the sick octopus group and 74,731,646 from the healthy group. The general transcriptome of the O. vulgaris hemocytes was assembled in 254,506 contigs. A total of 48,225 contigs were successfully identified, and 538 transcripts exhibited differential expression between groups of infection. The general transcriptome revealed genes involved in pathways like NF-kB, TLR and Complement. Differential expression of TLR-2, PGRP, C1q and PRDX genes due to infection was validated using RT-qPCR. In sick octopuses, only TLR-2 was up-regulated in hemocytes, but all of them were up-regulated in caecum and gills. CONCLUSION: The transcriptome reported here de novo establishes the first molecular clues to understand how the octopus immune system works and interacts with a highly pathogenic coccidian. The data provided here will contribute to identification of biomarkers for octopus resistance against pathogens, which could improve octopus farming in the near future.

Immunolocalization of choline acetyltransferase of common type in the central brain mass of Octopus vulgaris.

Acetylcholine, the first neurotransmitter to be identified in the vertebrate frog, is widely distributed among the animal kingdom. The presence of a large amount of acetylcholine in the nervous system of cephalopods is well known from several biochemical and physiological studies. However, little is known about the precise distribution of cholinergic structures due to a lack of a suitable histochemical technique for detecting acetylcholine. The most reliable method to visualize the cholinergic neurons is the immunohistochemical localization of the enzyme choline acetyltransferase, the synthetic enzyme of acetylcholine. Following our previous study on the distribution patterns of cholinergic neurons in the Octopus vulgaris visual system, using a novel antibody that recognizes choline acetyltransferase of the common type (cChAT), now we extend our investigation on the octopus central brain mass. When applied on sections of octopus central ganglia, immunoreactivity for cChAT was detected in cell bodies of all central brain mass lobes with the notable exception of the subfrontal and subvertical lobes. Positive varicosed nerves fibers where observed in the neuropil of all central brain mass lobes.

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.

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.

Post-hatching development of the brain in Octopus ocellatus.

To investigate the post-hatching development of the brain in a benthic octopod, Octopus ocellatus, we performed volumetric analyses of the brain. The brain consisting of the supra- and subesophageal masses was divided into 5 regions according to the functions suggested for the brain of another benthic octopod Octopus vulgaris, and the volume of each region was estimated at three post-hatching ages. We found that the inferior frontal lobe system and the brachial lobe increased in relative volume as the animals grew, while the basal lobe system decreased in relative volume. This result suggests that increasing demand for processing tactile information after hatching is reflected in the higher developmental rate in the centers devoted for tactile sense and related learning. We also found that the inner neuropile layer mainly consisting of dendrites, synapses and axons showed great increases in volume compared with the outer neural-cell-body layer. Although the increase in volume of the inner layer was marked during 1 month after hatching in all brain regions examined, the extent of the increase varied among brain regions. Developmental changes in cell densities in the outer layer also varied among the regions. The present results suggest that the post-hatching development of the brain in O. ocellatus is not homogeneous but varies among brain regions depending on different roles in controlling the behavior.

Evolution of octopod sperm I: comparison of nuclear morphogenesis in Eledone and Octopus.

Morphogenesis of the Eledone cirrhosa sperm nucleus, as studied by electron microscopic techniques, is compared with that of Octopus vulgaris. Both species of cephalopods belong to the family Octopodidae. The results indicate that extensive nuclear helicoidization during E. cirrhosa spermiogenesis is brought about by modifications of the function of structural components already present in the late steps of O. vulgaris spermiogenesis. In particular, changes in the regulation of perinuclear microtubule contraction in E. cirrhosa spermatids, as well as a decrease in basicity of protamines, promote nuclear helicoidization. Disulphide bond formation between protamine molecules fixes the completely helicoidal shape of the nucleus in mature sperm of E. cirrhosa.

Evolution of octopod sperm II: comparison of acrosomal morphogenesis in Eledone and Octopus.

The first stages of acrosome development during Eledone cirrhosa spermiogenesis are similar to that in Octopus vulgaris, and comprise the initial elongation of both organelles. However, the acrosome in E. cirrhosa does not continue its elongation as it does in O. vulgaris. Instead, its length remains fixed and it undergoes a process of helicoidization that includes the entire organelle. In each spermatid, helicoidization of the E. cirrhosa acrosome occurs simultaneously with helicoidization of the nucleus. The acrosome is associated with special structures that probably are involved in the helical torsion of the organelle. We propose a hypothesis to explain the evolutionary relationship between the acrosomes of O. vulgaris and E. cirrhosa, particularly as it is influenced by nucleomorphogenesis and microtubular contraction.

Centrifugal neurons of the octopus optic lobe cortex are immunopositive for calcitonin gene-related peptide.

Distribution of calcitonin gene-related peptide (CGRP)-like substance in the optic lobe cortex and retina of the octopus was examined immunohistochemically. Wheat germ agglutinin (WGA), a retrograde-transporting marker, was also used to label the centrifugal neurons. CGRP-immunoreactive (CGRP-IR) somata were seen in the inner granular cell layer, but not in the outer granular cell layer or the retina. CGRP-IR fibers were seen not only in the optic lobe cortex, but also in the retinal nerve plexus. Retrogradely labeled somata were seen in the inner granular cell layer, but not in the outer granular cell layer. Immunohistochemical double staining indicated that WGA-labeled centrifugal neurons were immunopositive for CGRP. These results suggested that the centrifugal neurons in the octopus optic lobe cortex are CGRP-like peptide-containing neurons, and that the peptide may modulate photoreceptor cell functions.

Adapting a compact confocal microscope system to a two-photon excitation fluorescence imaging architecture.

Within the framework of a national National Institute of Physics of Matter (INFM) project, we have realised a two-photon excitation (TPE) fluorescence microscope based on a new generation commercial confocal scanning head. The core of the architecture is a mode-locked Ti:Sapphire laser (Tsunami 3960, Spectra Physics Inc., Mountain View, CA) pumped by a high-power (5 W, 532 nm) laser (Millennia V, Spectra Physics Inc.) and an ultracompact confocal scanning head, Nikon PCM2000 (Nikon Instruments, Florence, Italy) using a single-pinhole design. Three-dimensional point-spread function has been measured to define spatial resolution performances. The TPE microscope has been used with a wide range of excitable fluorescent molecules (DAPI, Fura-2, Indo-1, DiOC(6)(3), fluoresceine, Texas red) covering a single photon spectral range from UV to green. An example is reported on 3D imaging of the helical structure of the sperm head of the Octopus Eledone cirrhosa labelled with an UV excitable dye, i.e., DAPI. The system can be easily switched for operating both in conventional and two-photon mode.

Reflector cells in the skin of Octopus dofleini.

The cells that form the reflecting layer beneath the chromatophore organs of the octopus are conspicuous elements of its dermal chromatic system. Each flattened, ellipsoidal reflector cell in this layer bears thousands of peripherally radiating, discoidal, reflecting lamellae. Each lamella consists of a proteinaceous reflecting platelet enveloped by the plasmalemma. The lamellae average 90 nm in thickness and have variable diameters with a maximum of about 1.7 micrometer. Sets of reflecting lamellae are organized into functional units called reflectosomes. The lamellae in each reflectosome form a parallel array - similar to a stack of coins. The average number of lamellae in a reflectosome is 11. Adjacent lamellae are uniformly separated by an extracellular gap of about 60 nm in embedded specimens. The reflectosomes are randomly disposed over the surface of the reflector cell. The observed organization of the reflectosomes is compatible with its role as a quarter-wave thin-film interference device. The alternating reflecting lamellae and intelamellar spaces constitute layers of high and low refractive indices. Using measurements of the thicknesses and refractive indices of the platelets and interlamellar spaces, we have calculated that the color of reflected light should be blue - green, as seen in vivo. The sequence of events leading to the definitive arrangement of the reflectosomes is uncertain. The reflector cells of O. dofleini are compared and contrasted with the iridophores of squid.

An unusual receptor in the octopus.

The epithelium of the rim of the octopus sucker in the site of several different types of primary receptors. One is a non-ciliated cell with unusual characteristics. (1) The surface of the cell is extremely irregular with finger-like extension of cytoplasm, especially far reaching in the basal region. (2) The slender neck contains a canal whose apical opening is in contact with the environment. This canal is lined with microvilli and contains granular material in an electron-dense matrix. (3) Patches of presumed glycogen granules occur throughout the cell, being especially abundant in the outer reaches of the cytoplasmic extensions. Their presence, together with numerous mitochondria and free ribosomes, indicate a high intrinsic metabolism. (4) Small fascicles of microtubules are randomly situated throughout the perikaryon. They gather into a coherent system of larger and larger bundles which ultimately enter the axon leading from the cell. This axon extends some distance in the basal region of the epithelium before crossing the subepithelial space to enter the infundibular muscle. Possible functions of this cell are discussed. On the basis of its specific position on the sucker and its intrinsic morphology we suggest that it is a mechanoreceptor involved in shape and/or negative pressure discrimination.

Ciliated sensory cells and associated neurons in the lip of Octopus joubini Robson.

The lip of Octopus joubini is a fleshy fold around the beak that is subdivided distally into finger-like papillae and overlayed by an uninterrupted noncellular cuticle. The muscular core of the lip has a high proportion of nervous tissue. The simple epithelium contains numerous ciliated sensory cells, especially in the papillae. In many of these cells the cilia lie deep within the cytoplasm and usually appear to extend toward the surface. Receptors with intracellular cilia also lie below the epithelium and send dendrites bearing cilia to the surface. Large unipolar interneurons that may receive synapses from the ciliated receptors lie in the musculature near the papillae. The sensory system of the octopus lip is more advanced than that of the squid, and it is very similar to that of Sepia. The relationship of these findings to the phylogeny and ecology of cephalopods is discussed.