Development of the Epicardium in the Dogfish (Scyliorhinus canicula)

The development of the epicardium has been described in mammals, including man, birds and amphibians. However, there is no information concerning this morphogenetic process in fishes. A study carried out in embryos of the dogfish ( Scyliorhinus canicula ) showed that, in this elasmobranch species, the precursors of the epicardium originate from two mesothelial anlagen, the right and left, that initially lie at the ventrolateral parts of the liver. These two anlagen, which will be referred to as the proepicardium, later shift to the right and left parts of the pericardial aspect of the transverse septum. The proepicardium comprises numerous spheric, smooth-contoured cells and a relatively small amount of extracellular matrix. The proepicardium is not covered by an epithelial layer.
Cells detaching from the proepicardium adhere to the surface of the heart and develop into epicardial cells. They firstly ensheathe the atrioventricular groove as well as the dorsal and lateral aspects of the ventricle, and the ventral and lateral aspects of the atrium. Both the sinus venosus and conus arteriosus become lined later.
In spite of the phylogenetic distance between elasmobranchs and mammals, the mechanism by which the epicardium develops is similar in both groups. This similarity relies principally on the arrangement and location of the proepicardium and the way in which the epicardial precursors reach and invest the heart.     

Immunological Responses of the Dogfish (Scyliorhinus canicula L.) to Cellular Antigens

Humoral and cellular aspects of the immune response of the common dogfish (Scyliorhinus canicula L.) to a variety of cellular antigens have been examined. The fish made a relatively slow but positive antibody response to injected Salmonella typhi. Using the haemolytic plaque technique, antibody synthesis was shown to occur in the spleen after fish were challenged with sheep red blood cells, suggesting that this organ is a major site of antibody synthesis. Intraperitoneal and intravenous injections of allogeneic leucocytes stimulated marked histological changes in the spleens of recipients, indicative of a host-vs-graft reaction and suggesting that the fish are capable of alloimmune reactions.     

Macrophages and Reticulum Cells in the Spleen of the Dogfish,Scyliorhinus canicula

The presence and ultrastructural features of reticulum cells and macrophages were studied in the spleen of the dogfish Scyliorhinus canicula. Three morphologically distinguishable regions of the spleen were identified: the white pulp, the red pulp and the ellipsoids. In all three, the splenic parenchyma was a meshwork supported by reticulum cells and fibres. Reticulum cells in both the white and the red pulp are irregular elements, the processes of which are joined by cell junctions and embrace developing reticular fibres. The ellipsoids of the dogfish spleen are terminal branches of the splenic arteries of the white pulp, with a sheath consisting of reticulum cells, reticular fibres, ground substance, macrophages and occasional lymphocytes. Isolated melanomacrophages also occur in the ellipsoid walls as well as in the red pulp. In both the white and the red pulp phagocytic reticulum cells, and macrophages appear frequently forming cell associations with surrounding blood cells, mainly lymphocytes. The functional significance of the ellipsoids and the cell-cell clusters of the white and the red pulp is discussed in relation to the immune capacities demonstrated in elasmobranchs.     

Phagocytic Cells in the Peripheral Blood of the Dogfish Scyliorhinus canicula L. I. In Vitro Studies

In vitro phagocytosis by peripheral blood leucocytes of the dogfish Scyliorhinus canicula L. was examined by exposing adherent cells to a variety of particulate and soluble antigens and inert material. Their subsequent uptake was monitored by light, scanning and transmission electron microscopy. The monocyte and the neutrophil-like granulocyte were found to be the major phagocytic cells. Larger particles like yeasts and erythrocytes were the most avidly phagocytosed. From studies on the effects of pH, temperature and the presence of plasma, metabolic inhibitors and divalent ions, it appeared that optimum phagocytosis occurred at pH 7.0 and between 10 and 20°C. Serum factors did not enhance the process in this species. Finally, the in vitro clearance of 5 bacterial species indicated that the presence of blood phagocytes had little or no effect on bacterial numbers.     

Phagocytic Cells in the Peripheral Blood of the Dogfish Scyliorhinus canicula L. II. In Vivo Studies

In vivo phagocytosis by peripheral blood leucocytes of the dogfish Scyliorhinus canicula L. was examined by monitoring the fate of a variety of injected materials, both particulate and soluble, in normal and immunised fish. Carbon, yeasts and bacteria were phagocytosed by monocytes, thrombocytes and type 1 granulocytes (neutrophils). Quantitative in vivo antigen clearance studies employed five species of bacteria, yeast and KLH. After an initial significant decrease of these antigens in the circulation, low numbers of viable bacteria and yeasts and low concentrations of KLH persisted for long periods after injection. Previous exposure to several of these antigens had little or no effect.     

Immunohistochemical mapping of galanin-like immunoreactivity in the brain of the dogfish Scyliorhinus canicula.

The distribution of galanin-like immunoreactivity in the brain of the dogfish Scyliorhinus canicula was investigated using the indirect immunofluorescence technique. In the telencephalon, positive cells and fibers were located in the mid-caudal part of the area superficialis basalis, the n. septi caudoventralis and in the n. interstitialis commissurae anterioris. Most of the galanin-containing neurons observed in the hypothalamus were located in the magnocellular preoptic nucleus. Positive perikarya were also found in the n. lobi lateralis hypothalami and in the n. lateralis tuberis. A dense network of positive nerve processes was noted in the caudal part of the median eminence. In the dorso-caudal part of the diencephalon numerous immunoreactive neurons were seen in the recessus posterioris. A large bundle of galanin-containing fibers, which divided in two branches, was observed in the basal midbrain tegmentum. The widespread distribution of galanin-like material suggests that, in the dogfish, galanin may be involved in various brain functions including neuroendocrine regulations.     

Functional Morphology of the Olfactory Organs in the Spiny Dogfish (Squalus acanthias L.) and the Small-spotted Catshark (Scyliorhinus canicula (L.))

The morphology of the olfactory organs in two sharks, the spiny dogfish and the small-spotted catshark, was studied by light microscopy and electron microscopy (TEM and SEM). The olfactory epithelium is arranged on olfactory lamellae which are provided with secondary folds. The epithelium mainly consists of microvillous receptor cells, multiciliated supporting cells and basal cells. The find of only one type of receptor cells, the microvillous type, is discussed and the condition considered a derived (apomorphic) character. The route of the water current through the olfactory organ and the different driving forces of the ventilation process are subject to discussion. In both the pelagic dogfish and the bottom-dwelling catshark the pressure difference between the incurrent and excurrent nostrils achieved by active swimming appears to be the driving force, whereas the role of the beating of the non-sensory cilia is not evident. In the bottom-dwelling catshark the ventilation of the olfactory organ is also supported by the respiratory activity.     

Tachykinin-like immunoreactivity in the sinus venosus of the dogfish (Scyliorhinus canicula)

The sinus venosus of the elasmobranch heart is characterized by the presence of large bundles of unmyelinated nerve fibres that bulge into the cardiac lumen, below the endocardium. In the dogfish (Scyliorhinus canicula), these fibres contain numerous dense-core membrane-bounded granules of about 200 nm in diameter. Most intramural ganglion cells of the sinus venosus also show densely packed granules similar to those found in the subendocardial fibres. We have observed strong substance-P-like immunoreactivity in the large fibre bundles and in the perikarya of the ganglion cells. Preabsorption of the antisera with fragment 7–11 of substance P has shown that the antisera recognize the tachykinin canonic sequence. Our findings suggest that an undetermined tachykinin is secreted in the elasmobranch heart, and that it is probably released into the blood stream in the context of a little-known neuroendocrine system.     

Immunohistochemical localization of delta sleep-inducing peptide (DSIP) in the brain and pituitary of the cartilaginous fish Scyliorhinus canicula.

The distribution of delta sleep-inducing peptide (DSIP) in the brain and pituitary of the cartilaginous fish Scyliorhinus canicula was investigated using the indirect immunofluorescence technique. Delta sleep-inducing peptide-like immunoreactive cell bodies were mainly observed in the nucleus lateralis tuberis of the hypothalamus. Immunolabeled perikarya were also distributed in the nucleus lobi lateralis hypothalami and in the dorso-lateral wall of the recessus posterioris. Most of these cells, located in the subependymal layers of the infundibulum and lateral lobes, had the typical aspect of cerebrospinal fluid-contacting elements. The DSIP-like immunoreactive fibers were localized in the basal telencephalon, within the regions of the nucleus interstitialis commissurae anterioris and the nucleus entopeduncularis. A dense network of DSIP-positive fibers was seen throughout the midcaudal hypothalamus, the lateral lobes, and the posterior lobe. In the pituitary, numerous DSIP-like immunoreactive cells were detected in the median lobe of the pars distalis. In particular, a high concentration of cells was seen in the dorsal wall of the median lobe, an area which is known to contain melanin-concentrating hormone (MCH)-producing cells. Comparison of the distribution of DSIP- and MCH-like immunoreactive cells revealed that the two neuropeptides are stored in the same cells of the median lobe of the pituitary. These findings provide the first evidence for the presence of a DSIP-related peptide in fish. The distribution of the immunoreactive material supports the view that DSIP may act as a neuromodulator and/or a hypophysiotropic factor. Moreover, the presence of DSIP-like immunoreactive cells in the pars distalis suggests that this peptide may exert autocrine or paracrine effect in the pituitary.     

The functional anatomy of the gills of the dogfish (Scyliorhinus canicula)

Vascular casting techniques combined with scanning electron microscopy of fixed tissue have been used to describe the vascular anatomy of the gills of the dogfish ( Scyliorhinus canicula ). Two distinct blood pathways were revealed: a 'respiratory' blood pathway which conveys deoxygenated blood from the heart to the gas exchange surface of the gills and then carries oxygenated blood away to the systemic circulation, and a 'non-respiratory' blood pathway which interconnects with the respiratory blood pathway and which diverts blood into the venous drainage from the gills.
The physiological function of this complex circulation in the gills of elasmobranchs is discussed in terms of both the osmotic constraints upon the animal, and the possible control of blood flow.     

Diet of the small-spotted catshark Scyliorhinus canicula in the Aegean Sea (eastern Mediterranean)

The diet of the small-spotted catshark Scyliorhinus canicula, captured in the Aegean Sea by bottom-trawl from 2006 to 2012, was investigated with respect to sex, maturity condition, sampling location and season. The stomach contents of 432 specimens, measuring from 144 to 517?mm in total length, were analysed. The cumulative prey curve showed that the sample size was adequate to describe the species’ diet, which was quantified using the percentage gravimetric composition (%W). The identified prey items belonged to eight major groups: Teleostei, Chondrichthyes, Crustacea, Cephalopoda, Annelida, Echinodermata, phanerogams and macroalgae, with Teleostei, Crustacea and Cephalopoda being the most consumed in both females (%W?=?48.1, 16.0 and 31.4, respectively) and males (%W?=?33.9, 31.6 and 29.8, respectively). Higher diet diversity was observed in males than females, in immature individuals than mature ones, regardless of sex, as well as in spring in comparison to autumn and winter. Feeding intensity seemed to be influenced mainly by sex and maturity condition. No significant dietary overlap was observed for all possible combinations of the factors examined. Gut indices were compared between the two sexes with females showing statistically significantly higher median relative gut length, as well as a longer gut than males of the same length. Based on the diet composition, S. canicula can be considered a generalist predator consuming, with geographical differentiation, a wide variety of benthic taxa. The estimated fractional trophic level (τ?=?4.22) classified the species as a carnivore with a preference for Teleostei and Cephalopoda, thus confirming its key role in the food web.     

Primary structure of glucagon from the gut of the common dogfish (Scyliorhinus canicula)

The primary structure of glucagon isolated from the intestine of the common dogfish, Scyliorhinus canicula, was established as H S E G T F T S D Y S K Y M D N R R A K D F V Q W L M N T. The peptide shows four substitutions compared with human glucagon: Glu-3 for Gln, Met-14 for Leu, Asn-16 for Ser and Lys-20 for Gln. Glucagon represented the predominant molecular form of the glucagon-like immunoreactivity in the dogfish gut extracts demonstrating that the pathway of posttranslational processing of proglucagon in the gut of this fish differs markedly from the pathway in the mammalian gut.     

Immunohistochemical localization of calbindin-D28K in the brain of a cartilaginous fish, the dogfish (Scyliorhinus canicula L.)

The occurrence and distribution of the vitamin-D-induced calcium-binding protein, calbindin-D28K, has been studied in the brain of a cartilaginous fish using immunohistochemical techniques. A strong immunoreactivity was found in the perikarya, dendrites and axons of neurons located in the nucleus interstitialis commissurae anterioris, the nucleus medialis of the left habenula, the thalamus dorsalis, the thalamus ventralis, the nucleus lobi lateralis, the nucleus interpeduncularis, the lobus vagi and the medial reticular zone. Fibre tracts associated with some of these neuronal groups, such as the fasciculus retroflexus, the stria medullaris and the commissura habenulae, also contained immunopositive fibres. Only a minor immunoreactivity could be detected in other brain areas such as the tectum mesencephali and some telencephalic zones. Interestingly, the cerebellum did not show any immunoreactivity in Purkinje cells nor in other neurons. The distribution of calbindin-D28K in the dogfish brain appears to be mainly related to the viscerosensory centres.     

Metabolism of glucosamine in the liver of Scyliorhinus canicula (selachian)]

(1) The metabolism of the glucosamine was studied in the liver of S. canicula, after injection of D-(1-14C) glucosamine into the animal. (2) The labelled acid-soluble derivatives were separated by ion exchange columns and characterized by chromatography and electrophoresis, and were identified as glucosamine, glucosamine 6-P, N-acetylglucosamine, N-acetylmannosamine, N-acetylneuraminic acid, N-acetylglucosamine 6-P, N-acetylglucosamine 1-P, N-acetylmannosamine 6-P, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine. (3) The variation with time after glucosamine injection of the radioactivity of the fractions separated on Dowex 1-X4 was investigated. This study showed a decrease of the radioactivity in the fraction 1 (glucosamine and glucosamine 6-P), an increase in the fraction II (N-acetylneuraminic acid) and the fraction IV (UDP-N-acetylhexosamines), and a stability in the fraction III (phosphorylated N-acetylhexosamines). (4) The absence of label in neutral hexoses and their phosphorylated derivatives was interpretated as due to the weak activity of the glucosamine 6-P isomerase, which is positively modulated by the N-acetylglucosamine 6-P.     

Metabolic effects after short-term sublethal cadmium exposure to dogfish (Scyliorhinus canicula)

1. Four groups of dogfish were subjected to 50 ppm of cadmium for 1, 2, 3 and 4 days and haematological and metabolic parameters analyzed from blood and tissue samples.2. A significant increase of blood glucose and lactate was observed.3. No differences were found in the majority of haematological parameters except for the red blood cell counts that showed a significant increase during the second and third day of treatment.4. Comparing liver and muscle metabolism a decrease of protein levels are observed in both tissues and reductions of glycogen in liver and lactate in muscle were detected.5. The results of the different parameters indicate a pattern of dual response depending on the time of exposure; a first phase of toxic impact during the first two days, followed by a second phase of recovery at the end of 4 days treatment.     

The developmental expression of foxl2 in the dogfish Scyliorhinus canicula

The FoxL2 genes are a subfamily of the Fox (forkhead box) gene family. FOXL2 is mutated in the disorder Blepharophimosis, Ptosis, and Epicanthus Inversus Syndrome (BPES), which is characterized by eyelid malformations, and Premature Ovarian Failure (POF). In the mouse expression is seen in the perioptic mesenchyme, developing eyelids, ovary and pituitary. We have isolated a foxl2 cDNA from the dogfish Scyliorhinus canicula (also known as the lesser spotted catshark), allowing the characterisation of this gene's sequence and expression from a lineage that diverged early in the evolution of gnathostomes. Molecular phylogenetic analysis strongly grouped this sequence with the gnathostomes within the FoxL2 subfamily. We demonstrate the early expression of Scyliorhinus canicula foxl2 in the mandibular head mesoderm and later in continuous populations of mandibular arch cells and mandibular head mesenchyme cells around the developing pituitary. As development proceeds expression decreases in the mesenchyme of the head but is seen in the mesenchyme around the eye and later in the developing eyelids. Additionally expression is seen in regions of pharyngeal arch mesoderm and in ectoderm from which gill buds will form. This expression is maintained in the developing and elongating gill buds. Thus, S. canicula foxl2 is a marker for the mandibular mesoderm and gill buds and its expression is conserved in the perioptic mesenchyme, developing eyelids and pituitary.     

Ciliated cells on the surface of embryos of Scyliorhinus canicula

Monociliated surface cells were found for the first time in an elasmobranch and were common on embryos of Scyliorhinus canicula . They occurred mainly near the anterior end, until Ballard stage 30, but disappeared soon thereafter. The functions of surface ciliated cells suggested in other embryos ( e.g. surface currents in lungfishes and amphibians, and determination of left–right patterns in amniotes) seem unlikely in elasmobranchs.