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.     

Structural studies on the thymus of the dogfish, Scyliorhinus canicula L.

Histological and ultrastructural studies of the thymus of the dogfish, Scyliorhinus canicula L., revealed paired lobular masses above the first two gill arches of embryonic and newly hatched dogfish, involuting at approximately 3 weeks. It did not have defined cortex and medullary regions and ultrastructural investigations showed the tissue to be composed mainly of a range of different sized lymphocytes within a connective tissue capsule and reticular epithelial cell framework. Lymphoblasts often in mitosis were also present being more electron lucent and larger than lymphocytes. Macrophages contained cellular debris and frequently whole small lymphocytes. A cell with cytoplasmic granules was occasionally observed.     

Preliminary studies on trypanospmes from the dogfish, Scyliorhinus canicula L.

In a sample of 83 dogfish, Scyliorhinus canicula , 30% were found to be infected with trypanosomes during the summer of 1981 at Plymouth. Infected fish were over 53.0 cm total length and slightly anaemic, reflected in a 4.6% reduction in circulating erythrocytes with a small (0.6%) increase in leucocytes. Individual differences in serum proteins did not correlate with infection, although spleens from infected fish showed lymphoid aggregations around blood vessels. Parasitaemias were consistently low, ranging between 1.5×l0³ and 6.O×10³ml⁻¹. No dividing forms were found in the peripheral blood or lymphomyeloid tissues, although two size forms were found in the peripheral blood. The trypanosome, Trypanosoma scyllii , is described from living and stained preparations and has been successfully maintained in laboratory culture.     

Studies on the reticulo-endothelial system of the dogfish,Scyliorhinus canicula

Summary Clearance and subsequent localisation of a range of materials, including colloidal carbon, latex beads, sheep erythrocytes, bacteria and dextran were followed in the lesser spotted dogfish, Scyliorhinus canicula. It was found that two populations of peripheral blood leucocytes — monocytes and thrombocytes, but not granulocytes — were involved in clearance of the circulation. In the case of carbon, this material was cleared from the plasma after 12 h, and both the colloid-containing thrombocytes and monocytes disappeared from circulation by 8 weeks post injection. Upon injection of some of the materials, and particularly bacteria, a settling out of monocytes containing phagocytosed material was seen in the secondary lamellae and cavernous bodies of the gills. Large clumps of monocytes were found in the gills as early as 30 min post injection and these increased in size for up to one week, after which they gradually dispersed. The lining cells of the cavernous body, known as CB cells, were also responsible for the sequestration of carbon, latex beads and probably erythrocytes, but dextran and bacteria were not internalised. The origin, functions and phylogenetic significance of the CB cells are discussed.     

Structural studies on lymphomyeloid tissues of the dogfish,Scyliorhinus canicula L.

The structure and ultrastructure of the lymphomyeloid epigonal and Leydig organs of the dogfish Scyliorhinus canicula L. were investigated. The tissues produce leucocytes, mainly granulocytic cells, but also some lymphocytes and plasma cells. Lymphocytes sometimes form nodule-like aggregations, especially in the epigonal organ. Granulocytes are probably stored within the lymphomyeloid tissues and enter the circulation on maturation. They may also release biologically active substances into the blood.     

Distribution of monoamines in the diencephalon and pituitary of the dogfish,Scyliorhinus canicula L.

Summary The distribution of monoamines in the diencephalon and pituitary of the dogfish, Scyliorhinus canicula, has been investigated using the histochemical fluorescence technique of Falck and Hillarp (Falck and Owman, 1965). Terminals of monoamine-containing axons were found in the neurointermediate lobe of the pituitary and the axons were traced, by means of nialamide and L-dopa treatment and lesions, to the nucleus medius hypothalamicus. A separate hypothalamic system converging on the anterior median eminence and the occurrence of aminergic cells in the nuclei lobi inferiores and nucleus medius hypothalamicus were similarly demonstrated. Normal fish show a bilateral uncrossed tegmental tract and two areas of catecholamine-containing neurones in modified ependymal organs. The organum vasculosum hypothalami includes both primary catecholamine and 5-hydroxytryptamine-containing cell types whilst the organum vasculosum praeopticum has only the former type. Both organs contain cells which send club-like processes into the third ventricle. The subcommissural organ does not contain monoamines.The role of hypothalamic catecholamine systems in the regulation of pituitary function is discussed.     

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.     

Metal accumulation and metallothionein induction in the spotted dogfish Scyliorhinus canicula

Recent studies indicate that elasmobranch fish respond differently to metal exposure than marine teleosts. Accumulation rates can be high, which despite the fact that normal background levels for metals in the marine environment are low, is worrying due to the long life span and late fecundity of most shark. The goals of the present study were to examine differences in accumulation rates and toxicity of a range of metals at equimolar concentrations (10 µM) in the Mediterranean or spotted dogfish, Scyliorhinus canicula. For this purpose, we exposed the dogfish to Ni (587 µg/L), Cd (1124 µg/L), Pb (2072 µg/L), Cu (635 µg/L), and Ag (1079 µg/L and two additional exposures at 10 µg/L and 1 µg/L) for one week and measured total metal accumulation, metallothionein induction, and parameters related to osmoregulation. Our study confirms the high toxicity and accumulation rates of Ag for elasmobranch fish, even at levels 100 to 1000 times lower than exposure levels of other metals. Also Pb accumulated readily in all organs, but did not cause any osmoregulatory disturbance at the exposure levels used. Ni and Cd seem to accumulate primarily in the kidney while Cu mainly accumulated in liver. In contrast to Ni and Cd, the three other metals Ag, Cu and Pb accumulated in the rectal gland, an important organ for osmoregulation and possible target organ for metal toxicity. Only Cu succeeded in initiating a protective response by inducing MT synthesis in liver and gills.     

Characterization of a chymosin-like pepsin from the dogfish Scyliorhinus canicula

1. Pepsin II extracted from the gastric mucosa of Scyliorhinus canicula has been characterized and compared to calf chymosin. 2. The kcat and Km of the dogfish enzyme for the synthetic hexapeptide Leu-Ser-Phe(NO2)-Nle-Ala-Leu-OMe have been determined. The kcat/Km ratio is close to that of calf chymosin. Its milk-clotting efficiency is however 21-fold lower than that of calf chymosin. 3. The proteolytic activity against haemoglobin is optimal at pH 2.5. It clots the milk up to pH 6.8. 4. The dogfish pepsin II shows relatively better activity at low temperatures than calf chymosin.     

Light and electron microscopic studies on the pineal organ of the dogfish,Scyliorhinus canicula L.

Summary The pineal organ of the dogfish,Scyliorhinus canicula, is a long, thin, tubular structure consisting of an end-vesicle and a stalk. The pineal parenchyma consists of receptor cells, glycogen-storing cells, supporting cells, cells containing dense granules of 1,500–3,000 Å diameter, cytosome-rich cells, and ganglion cells. The latter alledgedly give rise to the diffusely distributed pineal tract which runs to the posterior commissure. A few pineal fibres diverge to the habenular commissure. The receptor cells have well-developed outer segments with morphological features characteristic of the retinal cone. Interaction between receptor cells and ganglion cells take place in neuropil-like areas. Boutons are found which are believed to belong to the receptor cells because of the presence of occasional synaptic rods. The few synapses observed always display synaptic vesicles both pre- and post-synaptically. The functional significance of the reported morphological features is discussed with the aid of the pertinent literature and it is postulated that the pineal organ of the dogfish is a photosensitive organ.Work done with the aid of a research scolarship from the Alexander von Humboldt Foundation, Bad Godesberg, Germany. — The animal material was provided by the Stazione Zoologica di Napoli, Italy, and by the Biologische Anstalt, Helgoland, Germany. — The electron microscope used in this study was placed at the disposal of Prof.Oksche by the Deutsche Forschungsgemeinschaft.     

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.     

Effect of temperature and photoperiod on the growth of the embryonic dogfish, Scyliorhinus canicula

Photoperiod has no effect on the growth of embryonic dogfish. Conversely, developmental rates are highly susceptible to temperature variation. Embryos maintained at 16°C grew faster and ecloded earlier than those maintained at 10°C. Increased temperature had no effect on size at eclosion.     

Fatty acid composition and lipoxygenase metabolism in blood cells of the lesser spotted dogfish, Scyliorhinus canicula.

1. The fatty acid composition of erythrocytes and leucocytes of the elasmobranch, Scyliorhinus canicula, was determined so as to indicate substrate availability for eicosanoid formation. 2. Leucocytes showed a greater degree of fatty acid unsaturation than the erythrocytes, with particularly high levels of docosahexaenoic acid (22:6,n-3). 3. The major eicosanoid precursors, arachidonic acid (20:4,n-6) and eicosapentaenoic acid (20:5,n-3), represented 13.9% and 5.2% of the total fatty acid, respectively, in erythrocytes compared with 10.7% and 6% in leucocytes. 4. Whole blood and isolated leucocytes were stimulated with calcium ionophore, A23187 and the resulting lipoxygenase products separated by reverse phase high performance liquid chromatography. 5. The main lipoxygenase products formed were 6-trans-leukotriene B4, 6-trans-12-epi-leukotriene B4, 5(S),6(R) dihydroxyeicosatetraenoic acid and 5- and 15-hydroxyeicosatetraenoic acid. 6. No leukotriene B4, leukotriene B5, or lipoxins were detected.     

Proteomic analysis of the spermatogonial stem cell compartment in dogfish Scyliorhinus canicula L

In the dogfish (Scyliorhinus canicula L.) the testicular germinative zone (GZ), composed of large isolated spermatogonia surrounded by elongating pre-Sertoli cells, is located between the albuginea and the ventrolateral intratesticular vessel. During the spermatogenic wave, cysts radiate in maturational order forming distinct testicular zones. In this study, soluble proteins of the GZ and of the zone containing cysts with spermatocytes were separated by two-dimensional electrophoresis. Gel images were matched and then evaluated for GZ-specific proteins. From the1400 protein spots identified, 680 were found to be apparently specific to this zone. Using MALDI-TOF/TOF mass spectrometry, de novo sequences were obtained for 33 proteins out of the 169 selected for identification by mass spectrometry, but only 16 of these 169 proteins were identified. One of them, proteasome subunit alpha-6, was analyzed further by immunohistochemistry. This study demonstrates the utility of the dogfish as a model for proteome analysis of the spermatogonial stem cell niche, even if it remains restricted by the lack of genomic data available on Elasmobranchs.     

On the diel rhythms in metabolism and activity of post-hatching lesser spotted dogfish, Scyliorhinus canicula

The diel rhythms in metabolic rate ( M_R ) and activity level ( A_L ) were measured for single post-hatching dogfish (weight range, 2.76–10.61 g) at 15° C by the indirect calorimetric method of rate of oxygen consumption ( V O₂) and by video-observation respectively, over a period of 72 b. The mean VO ₂ increased from 62.0 (s.e. 2.9) mg O₂ kg⁻¹ h⁻¹ in the daylight hours to 85.5 (s.e. 3.1) mg O₂ kg⁻¹ h⁻¹ during the dark (light regíme, 12 h L: 12 h D). The simultaneous measurement of A _L also showed mean night elevation from 0.6 (s.e. 0.2) min h⁻¹ in the light phase to 14.5 (s.e. 1.6) min h⁻¹ during the darkness. Bimodal nocturnal activity (BNA) was exhibited by the post-hatching dogfish within the 12 h dark period, with V O₂ increasing from 71.4 (s.e. 2.8) mg O₂ kg⁻¹ h⁻¹ before 01.00 hours to 99.5 (s.e. 4.2) mg O₂ kg⁻¹ h⁻¹ after 01.00 hours. Similarly, A _L also increased from 8.9 (s.e. I.7)min h⁻¹ before 01.00 hours to 21.1 (s.e. 2.8) min h⁻¹ after 01.00 hours. The importance of the results presented to the natural behavioural ecology of the hatching dogfish are discussed.     

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.     

Some observations on the collagen fibrils of the egg capsule of the dogfish, Scyliorhinus canicula

The egg capsule of the dogfish Scyliorhinus canicula is a collagenous material with a laminated, plywood (orthogonal) construction. The collagen fibrils which constitute the bulk of the egg capsule wall have a unique, highly ordered structure (Knight and Hunt, 1974; 1976, 1986; Gathercole et al., 1993) which is thought to represent a smectic A liquid crystalline phase (Knight et al., 1993). The egg capsule is extremely strong and chemically inert (Hunt, 1985). It is stored, secreted and formed by the nidamental gland (Rusaou?n 1976, 1990 a, b; Knight and Feng, 1992). During intracellular storage, secretion and fibrillogenesis, the dogfish egg capsule collagen appears to pass through a remarkable series of textures within a lyotropic liquid crystalline phase diagram (Knight et al., 1993). In the present communication, further observations on the ultrastructure of the collagen fibrils and their arrangement within the laminae of the fully-formed egg capsule are reported. The effect of tilting ultrathin sections of fibrils in the goniometer stage of a transmission electron microscope are described, demonstrating that the crystalline lattice within the fibril appeared twisted more or less regularly into a long pitch helix. Other observations indicated that some of the fibrils were in turn twisted round one another to form fibres which therefore had a coiled-coil structure. The fibres are arranged parallel to one another in the laminae which are stacked to give an orthogonal plywood construction. The effects of staining fibrils with cuprolinic blue and with tannic acid are reported. Reduction in the water content of the fibrils before fixation appeared to move some of the fibrils through the part of the lyotropic phase transition diagram converting them from smectic A to smectic C. Finally, evidence is presented that the fibrils shrank, but remarkably, still retained a longitudinally-ordered but modified, molecular arrangement even after boiling in water for periods of up to 10 min. These observations are discussed in relation to other collagens.