Histological and scanning electron microscope observations on the developing retina of the cuttlefish (Sepia officinalis Linnaeus, 1758).

In this work we present a detailed study of the major events during retinal histogenesis of the cuttlefish Sepia officinalis from early embryos to newly hatched animals and juveniles. For this purpose, we carried out morphometric and histological analyses using light and scanning electron microscopy. From St19, the first embryonic stage analysed, to St23/24 the embryonic retina is composed of a pseudostratified epithelium showing abundant mitotic figures in the more internal surface. At St24 the first photoreceptor nuclei appear in the presumptive inner segment layer, while an incipient layer of apical processes of the future rhabdomeric layer become visible at St25. From this stage onwards, both the rhabdomeric layer and the inner segment layer increase in size until postnatal ages. In contrast, the width of the supporting cell layer progressively decreases from St25/26 until postnatal ages. S. officinalis embryos hatched in a morphologically advanced state, showing a differentiated retina even in the last stages of the embryonic period. However, features of immaturity are still observable in the retinal tissue during the first postnatal weeks of life, such as the existence of mitotic figures in the apical region of the supporting cell layer and migrating nuclei of differentiating photoreceptors crossing the basal membrane to reach their final location in the inner segment layer. Therefore, postnatal retinal neurogenesis is present in juvenile specimens of S. officinalis.

Combined metabolomics and histological analysis of the tissues from cuttlefish Sepia pharaonis exposed to inking stress.

Inking is part of a defensive stress response in cephalopods (cuttlefish, squid, and octopus). Some individual cuttlefish (Sepia pharaonis) die after continued stress and inking; however, the physiological effects of cephalopods in response to stress and inking remain unknown. The present study investigated the metabolic profile and discussed the physiological roles of S. pharaonis tissues in response to continuous inking using the 1H NMR spectroscopy coupled with multivariate data analysis. A total of 50 metabolites, including amino acids, organic osmolytes, nucleotides, energy storage compounds, and obvious tissue-specific metabolites induced by inking stress, were identified in S. pharaonis tissues. Exposure to inking stress had different effects on the levels of the studied metabolites, for example, the levels of isoleucine, trimethylamine-N-oxide, and betaine increased, but those of arginine and ATP decreased in the liver; inosine and lactate were accumulated whereas glutamate and choline were depleted in the gill; the levels of lactate and isoleucine were elevated but those of arginine and glycogen were depleted in the muscle tissue. Furthermore, the corresponding metabolic pathways of the characteristic metabolites indicated major changes in the functions of these metabolites. Histological changes in the studied tissues revealed liver lobule damage immediately after inking, with the presence of disordered epithelial cells and partial cell necrosis in the gill. Our results demonstrated that a combination of metabolomics and histological analyses could provide molecular-level insights for elucidating the defense response of cuttlefish against predators.

Morphology of reproductive accessory glands in female Sepia pharaonis (Cephalopoda: Sepiidae) sheds light on egg encapsulation.

The pharaoh cuttlefish, Sepia pharaonis, is an important cephalopod fishery species in southeastern Asia, with understudied reproductive physiology. The present study aimed to investigate the cellular characteristics of epithelial cells found in the nidamental glands (NGs) and accessory NGs (ANGs), as well as the structural connections between these two glands in mature female S. pharaonis. A histological analysis revealed two types of epithelial cells in NGs: Alcian blue-positive, PAS-negative mucosubstance-secreting cells and eosinophilic, PAS-positive granule-secreting cells. Using transmission electron microscopy, three types of epithelial cells were identified: cells with electron-dense granules, cells with electron-lucent granules, and cells with both cilia and microvilli in the apex. Mature ANGs contain an abundance of tubular units composed of epithelial cells resting on a thin layer of basal lamina. Innervated muscle cells are tightly adhered to the basal lamina. In addition, we observed epithelial canalization of ANG tubules penetrating through the connective tissue linking NGs and the walls of the tubules in ANGs, which allows the contents of the ANG tubules to be transported to the NGs. Our results suggest that ANGs participate in the encapsulation of the ova via the same pathway as NGs, which provides an important basis for future studies on the mechanism of protection provided by NGs and ANGs during embryonic development in S. pharaonis.

Microanatomy and ultrastructure of outer mantle epidermis of the cuttlefish, Sepia esculenta (Cephalopoda: Sepiidae).

This study describes the ultrastructural characteristics of external epidermis of mantle of Sepia esculenta using light and electron microscopy. The epidermis was thicker on the ventral surface than on the dorsal surface, with a higher secretory cell distribution on the ventral surface than on the dorsal surface. The epidermis was a single layer composed of epithelial cells, secretory cells, ciliated cells and neuroglial cells. Epithelial cells were columnar with well-developed microvilli on the free surface, and the microvilli were covered with glycocalyx. The epithelial cells were connected to the neighboring cells by tight junctions and membrane interdigitations of the apico-frontal surface. Well-developed microfilaments were arranged in a vertical direction in the cortical cytoplasm. The secretory cells were categorized into three types (A, B and C) in accordance with the light microscopical characteristics and ultrastructures of the secretory granules. The distribution of these cells was in the following order: Type A>Type B>Type C. SEM observation revealed that the secretory pore size of the Type A secretory cells was approximately 8.6 µm×12.2 µm. Cytoplasm displayed a red color as the result of Masson's trichrome stain and H-E stain, and contained polygonal granules of approximately 1.2 µm2 with a high electron density. The secretory pore size of the Type B secretory cells was approximately 10.1 µm×12.1 µm. As the results of AB-PAS (pH 2.5) and AF-AB (pH 2.5) reactions, the cytoplasm displayed a red color. The cells contained membrane bounded secretory granules with very low electron density. The secretory pore of the Type C secretory cells was circular shape, and approximately 5.5 µm×5.5 µm. Cytoplasm was found to be homogeneous under H-E stain and Masson's trichrome stain, and displayed a red color. As the result of AB-PAS (pH 2.5) reaction, the cytoplasm displayed a red color. The electron density of the secretory substance was the highest among the three types of secretory cells. The ciliated cells had a ciliary tuft on the free surface and were distributed throughout the mantle with the exception of the adhesive organs. Neuroglial cells were connected to the basal membrane, epithelial cells, secretory cells and nerve fibers through cytoplasmic process, and contained neurosecretory granules with high electron density within the cytoplasm.

Cuttlefish skin papilla morphology suggests a muscular hydrostatic function for rapid changeability.

Coleoid cephalopods adaptively change their body patterns (color, contrast, locomotion, posture, and texture) for camouflage and signaling. Benthic octopuses and cuttlefish possess the capability, unique in the animal kingdom, to dramatically and quickly change their skin from smooth and flat to rugose and three-dimensional. The organs responsible for this physical change are the skin papillae, whose biomechanics have not been investigated. In this study, small dorsal papillae from cuttlefish (Sepia officinalis) were preserved in their retracted or extended state, and examined with a variety of histological techniques including brightfield, confocal, and scanning electron microscopy. Analyses revealed that papillae are composed of an extensive network of dermal erector muscles, some of which are arranged in concentric rings while others extend across each papilla's diameter. Like cephalopod arms, tentacles, and suckers, skin papillae appear to function as muscular hydrostats. The collective action of dermal erector muscles provides both movement and structural support in the absence of rigid supporting elements. Specifically, concentric circular dermal erector muscles near the papilla's base contract and push the overlying tissue upward and away from the mantle surface, while horizontally arranged dermal erector muscles pull the papilla's perimeter toward its center and determine its shape. Each papilla has a white tip, which is produced by structural light reflectors (leucophores and iridophores) that lie between the papilla's muscular core and the skin layer that contains the pigmented chromatophores. In extended papillae, the connective tissue layer appeared thinner above the papilla's apex than in surrounding areas. This result suggests that papilla extension might create tension in the overlying connective tissue and chromatophore layers, storing energy for elastic retraction. Numerous, thin subepidermal muscles form a meshwork between the chromatophore layer and the epidermis and putatively provide active papillary retraction.

Characterization of organics consistent with ß-chitin preserved in the Late Eocene cuttlefish Mississaepia mississippiensis.

BACKGROUND: Preservation of original organic components in fossils across geological time is controversial, but the potential such molecules have for elucidating evolutionary processes and phylogenetic relationships is invaluable. Chitin is one such molecule. Ancient chitin has been recovered from both terrestrial and marine arthropods, but prior to this study had not been recovered from fossil marine mollusks. METHODOLOGY/PRINCIPAL FINDINGS: Organics consistent with ß-chitin are recovered in cuttlebones of Mississaepia mississippiensis from the Late Eocene (34.36 million years ago) marine clays of Hinds County, Mississippi, USA. These organics were determined and characterized through comparisons with extant taxa using Scanning Electron Microscopy/Energy Dispersive Spectrometry (SEM/EDS), Field Emission Scanning Electron Microscopy (Hyperprobe), Fourier Transmission Infrared Spectroscopy (FTIR) and Immunohistochemistry (IHC). CONCLUSIONS/SIGNIFICANCE: Our study presents the first evidence for organics consistent with chitin from an ancient marine mollusk and discusses how these organics have been degraded over time. As mechanisms for their preservation, we propose that the inorganic/organic lamination of the cuttlebone, combined with a suboxic depositional environment with available free Fe(2+) ions, inhibited microbial or enzymatic degradation.

Spectral and spatial properties of polarized light reflections from the arms of squid (Loligo pealeii) and cuttlefish (Sepia officinalis L.).

On every arm of cuttlefish and squid there is a stripe of high-reflectance iridophores that reflects highly polarized light. Since cephalopods possess polarization vision, it has been hypothesized that these polarized stripes could serve an intraspecific communication function. We determined how polarization changes when these boneless arms move. By measuring the spectral and polarizing properties of the reflected light from samples at various angles of tilt and rotation, we found that the actual posture of the arm has little or no effect on partial polarization or the e-vector angle of the reflected light. However, when the illumination angle changed, the partial polarization of the reflected light also changed. The spectral reflections of the signals were also affected by the angle of illumination but not by the orientation of the sample. Electron microscope samples showed that these stripes are composed of several groups of multilayer platelets within the iridophores. The surface normal to each group is oriented at a different angle, which produces essentially constant reflection of polarized light over a range of viewing angles. These results demonstrate that cuttlefish and squid could send out reliable polarization signals to a receiver regardless of arm orientation.

Sperm nucleomorphogenesis in the cephalopod Sepia officinalis.

Sperm nucleomorphogenesis in the cephalopod Sepia officinalis is the product of the interaction between perinuclear microtubules and condensing chromatin. This interaction occurs during spermiogenesis and is established through the nuclear membrane. As in other cephalopod species, the perinuclear microtubules are transient structures. In the case of S. officinalis, they begin to appear in the basal area of the early spermatid and progress from there, establishing contact with the external nuclear membrane and follow a defined, but not symmetric, geometry. Thus, the microtubules accumulate preferentially in one area of the nuclear membrane which we refer to here as the "dorsal zone". Later, the microtubules will be eliminated before the mature spermatid migrates to the epidydimis. The chromatin is condensed within the nucleus following a complex pattern, beginning as fibro-granular structures until forming fibres of approximately 45 nm diameter (patterning phases). From this stage on, an increase in the chemical basicity of DNA-interacting proteins is produced, and chromatin fibres coalesce together, being recruited to the dorsal zone of the membrane, where there is a higher density of microtubules. This last step (condensation phases) allows the chromatin fibres to be arranged parallel to the axis of the elongating nucleus, and more importantly, is deduced to cause a lateral compression of the nucleus. This lateral compression is in fact a recruitment of the ventral zone toward the dorsal zone, which brings about an important reduction in nuclear volume. The detailed observations which comprise this work complement previous studies of spermiogenesis of Sepia and other cephalopods, and will help to better understand the process of cellular morphology implicated in the evolution of sperm nuclear shape in this taxonomic group.

Adhesive mechanisms in cephalopods: a review.

Several genera of cephalopods (Nautilus, Sepia, Euprymna and Idiosepius) produce adhesive secretions, which are used for attachment to the substratum, for mating and to capture prey. These adhesive structures are located in different parts of the body, viz. in the digital tentacles (Nautilus), in the ventral surface of the mantle and fourth arm pair (Sepia), in the dorsal epidermis (Euprymna), or in the dorsal mantle side and partly on the fins (Idiosepius). Adhesion in Sepia is induced by suction of dermal structures on the mantle, while for Nautilus, Euprymna and Idiosepius adhesion is probably achieved by chemical substances. Histochemical studies indicate that in Nautilus and Idiosepius secretory cells that appear to be involved in adhesion stain for carbohydrates and protein, whilst in Euprymna only carbohydrates are detectable. De-adhesion is either achieved by muscle contraction of the tentacles and mantle (Nautilus and Sepia) or by secretion of substances (Euprymna). The de-adhesive mechanism used by Idiosepius remains unknown.