Morphological and functional aspects of the epidermis of the sea lamprey Petromyzon marinus throughout development.

The development of the epidermis of sea lamprey Petromyzon marinus along the whole life cycle was studied using conventional staining techniques and lectin histochemistry. The epidermis undergoes variations in morphology and thickness throughout development. The simple cuboidal epithelium found in the epidermis of prolarvae becomes stratified cubic in the adult by increasing the number of cell layers. The cuticle thickness undergoes a steady increase during the larval period. There are changes in the glycoconjugate composition of the three main cell types of the P. marinus epidermis, mucous, granular and skein cells, which are more pronounced after metamorphosis. The Alcian blue-periodic acid Schiff (AB-PAS) histochemical method shows the presence of both acidic and neutral glycoconjugates in the mucous cells, indicating their secretory function. Moreover, lectin analysis reveals a mucous secretion containing glycoconjugates such as sulphated glycosaminoglycans (N-acetylglucosamine and N-acetylgalactosamine) and N-glycoproteins rich in mannose. Although granular cells are AB-PAS negative, they exhibit a similar glycoconjugate composition to the mucous cells. Moreover, granular cells show sialic acid positivity in larvae but this monosaccharide residue is not detected after metamorphosis. The skein cells, a unique cell of lampreys, are negative to AB-PAS staining but they mostly contain l-fucose and sialic acid residues, which also disappear after metamorphosis. The function of the granular and skein cells is still unknown but the role of their glycoconjugate composition is discussed. In addition, a different cellular origin is suggested for these two types of cells.

Contribution of gall microscopic structure to taxonomy of gallicolous aphids on Pistacia.

Aphids inducing galls on Pistacia plants belong to the tribe Fordini. According to the Heie & Wegierek classification, the genera are grouped into three subtribes. Previous microscopic studies showed that this taxonomy is not consistent with the histological characteristics of the galls. In this paper, galls induced by Aplonerura lentisci, Asiphonella cynodonti, Forda riccobonii, Slavun wertheimae and Smynthurodes betae were analyzed for the first time, as well as nine other galls previously described. Based on histological features three groups of galls can be establish: the first group comprises closed galls, induced by Baizongia pistaciae, Geoica utricularia, Rectinasus buxtoni and Slavun wertheimae; the second group includes two species of Geopemphigus (G. blackmani and G. torsus), and the third one is divided into two subgroups, the first comprises Aplonerura lentisci, Asiphonella cynodonti and Geopemphigus morral, and the second that includes Forda formicaria, F. marginata, F. riccobonii, Paracletus cimiciformis and Smynthurodes betae. An identification key of species based on microscopic features of galls is presented.

Food deprivation causes rapid changes in the abundance and glucidic composition of the cutaneous mucous cells of Atlantic salmon Salmo salar L.

Cutaneous mucus is the first physical and chemical barrier of fish. This slime layer is secreted by mucous cells located in the epidermis and is mainly composed of glycoproteins that have their origin in the diet. Therefore, food deprivation can potentially change the abundance and glucidic nature of skin mucous cells, thus changing the mucus properties. To test this hypothesis, we conducted an experiment with Atlantic salmon, Salmo salar L. Changes in the number and glucidic nature of epidermal mucus cells were analysed using standard techniques. The outcome of this study shows that food deprivation caused a rapid decrease in the density of epidermal mucous cells in Atlantic salmon. Lectin histochemistry revealed a change in the presence and stainability of some sugar residues in the mucous cells of unfed fish compared with fed fish. Given that the primary reason for mucus secretion in fish is for protection against infections, we speculate that the changes in the mucus properties caused by nutritional stress may affect their disease resistance. This fact is particularly important for fish that spend a period of time deprived of food, either as a part of their natural life cycle, or as part of farming practices.

A DiI-tracing study of the neural connections of the pineal organ in two elasmobranchs (Scyliorhinus canicula and Raja montagui) suggests a pineal projection to the midbrain GnRH-immunoreactive nucleus.

The pineal organ of elasmobranchs is an elongated photoreceptive organ. In order to investigate the afferent and efferent connections of the pineal organ of two elasmobranchs, the skate (Raja montagui) and the dogfish (Scyliorhinus canicula), a fluorescent carbocyanine (DiI) was applied to the pineal organ of paraformaldehyde-fixed brains. This application strongly labeled the pineal tract, which formed extensive bilateral projections. In both species, the pinealofugal fibers coursed to the dorsomedial thalamus, the medial pretectal area, the posterior tubercle, and the medial mesencephalic tegmentum and branched profusely in these areas. Application of DiI to the pineal organ also labeled occasional perikarya in the dorsomedial thalamus, posterior commissural region, posterior tubercle, and mesencephalic tegmentum. A comparison of these results with those of immunocytochemical analyses of the dogfish brain with an anti-salmon gonadotropin-releasing hormone (sGnRH) antiserum revealed a close topographical relation between the pineal projections and the midbrain sGnRH-immunoreactive (ir) nucleus, the only structure in the dogfish brain that contained sGnRHir neurons. This and the widespread distribution of sGnRHir fibers in the brain suggest that the midbrain sGnRHir nucleus is a part of the secondary pineal pathways and may be involved in light-mediated pineal regulation of brain function. Although GnRH distribution has not been studied in the skate, a midbrain GnRHir nucleus has been identified in three other elasmobranchs, including a skate relative. The probable existence of direct pineal projections to the GnRHir midbrain nucleus in elasmobranchs and other anamniotes is discussed.

Structural study of a possible neoplasia detected in Mytilus galloprovincialis collected from the Ria of Vigo (NW Spain).

Several specimens of Mytilus galloprovincialis, collected in the Ria of Vigo over a non-consecutive 2 yr period (1993 to 1994 and 1996 to 1997), presented a possible gonadal neoplasm, entailing morphologically abnormal germinal cells distributed throughout the follicle and invading the adjacent storage tissue. In some cases, affected cells were noted in gonoducts and in haemic sinusoids. Prevalence of this anomaly in the samples was 6%, and all affected individuals were found between April and June. During the rest of the year, individuals presented normal gonadal tissue.

Distribution of choline acetyltransferase immunoreactivity in the brain of an elasmobranch, the lesser spotted dogfish (Scyliorhinus canicula).

Although the distribution of cholinergic cells is remarkably similar across the vertebrate species, no data are available on more primitive species, such as cartilaginous fishes. To extend the evolutionary analysis of the cholinergic systems, we studied the distribution of cholinergic neurons in the brain and rostral spinal cord of Scyliorhinus canicula by immunocytochemistry using an antibody against the enzyme choline acetyltransferase (ChAT). Western blot analysis of brain extracts of dogfish, sturgeon, trout, and rat showed that this antibody recognized similar bands in the four species. Putative cholinergic neurons were observed in most brain regions, including the telencephalon, diencephalon, cerebellum, and brainstem. In the retrobulbar region and superficial dorsal pallium of the telencephalon, numerous small pallial cells were ChAT-like immunoreactive. In addition, tufted cells of the olfactory bulb and some cells in the lateral pallium showed faint immunoreactivity. In the preoptic-hypothalamic region, ChAT-immunoreactive (ChAT-ir) cells were found in the preoptic nucleus, the vascular organ of the terminal lamina, and a small population in the caudal tuber. In the epithalamus, the pineal photoreceptors were intensely positive. Many cells of the habenula were faintly ChAT-ir, but the neuropil of the interpeduncular nucleus showed intense ChAT immunoreactivity. In the pretectal region, ChAT-ir cells were observed only in the superficial pretectal nucleus. In the brainstem, the somatomotor and branchiomotor nuclei, the octavolateral efferent nucleus, and a cell group just rostral to the Edinger-Westphal (EW) nucleus contained ChAT-ir neurons. In addition, the trigeminal mesencephalic nucleus, the nucleus G of the isthmus, some locus coeruleus cells, and some cell populations of the vestibular nuclei and of the electroreceptive nucleus of the octavolateral region exhibited ChAT immunoreactivity. In the reticular areas of the brainstem, the nucleus of the medial longitudinal fascicle, many reticular neurons of the rhombencephalon, and cells of the nucleus of the lateral funiculus were immunoreactive to this antibody. In the cerebellum, Golgi cells of the granule cell layer and some cells of the cerebellar nucleus were also ChAT-ir. In the rostral spinal cord, ChAT immunoreactivity was observed in cells of the motor column, the dorsal horn, the marginal nucleus (a putative stretch-receptor organ), and in interstitial cells of the ventral funiculus. These results demonstrate for the first time that cholinergic neurons are distributed widely in the central nervous system of elasmobranchs and that their cholinergic systems have evolved several characteristics that are unique to this group.

Marginal cells in the spinal cord of four elasmobranchs (Torpedo marmorata, T. torpedo, Raja undulata and Scyliorhinus canicula): evidence for homology with lamprey intraspinal stretch receptor neurons.

This study reports for the first time the presence of marginal cells, probably with stretch receptor function, in the spinal cord of four elasmobranch species, two electric rays, Torpedo marmorata and T. torpedo, the skate Raja undulata and the dogfish Scyliorhinus canicula. In all four species, the marginal cells were located close to the lateroventral surface of the cord and possessed thick dendrites which formed part of characteristic glomerular structures. In vitro retrograde labelling of the spinal cord of the dogfish with horseradish peroxidase (HRP) showed that some of these cells have contralateral projections. Ultrastructural study of normal and retrograde HRP-labelled material showed that the glomerular dendrites of marginal cells give rise to numerous fingerlike structures and are associated with a rich plexus of nerve terminals. Characteristically, these dendrites contain numerous mitochondria. Immunocytochemical studies revealed a rich plexus of somatostatin- and GABA-immunoreactive fibres in the glomeruli. These results strongly suggest that the marginal cells of the elasmobranch spinal cord are stretch receptors homologous to lamprey edge cells and to the marginal nucleus cells of the spinal cord of urodeles and snakes. We discuss the possible role of these cells in the modulation of swimming movements.

Distribution of calcitonin gene-related peptide-like immunoreactivity in the brain of the small-spotted dogfish, Scyliorhinus canicula L.

The distribution of neuropeptides has been useful in comparing neuronal aggregates of elasmobranchs with those in other vertebrates. The distribution of calcitonin gene-related peptide (CGRP)-like immunoreactivity in the brain of the dogfish was examined with an antiserum to rat alpha-CGRP. Western blot analysis confirms that our antiserum recognizes a single peptide in the dogfish brain very similar to mammalian CGRP. CGRP-like immunoreactivity was located in discrete neuronal groups. CGRP-like-immunoreactive (CGRP-ir) neurons were found in the motor nuclei III, IV, V, VI, VII, IX, and X of the brainstem motor column and in the octavolateral efferent neurons. In the isthmal region, two groups of CGRP-ir neurons appeared in the parabrachial region and reticular substance. Three other CGRP-ir cell groups were observed in the mesencephalon: in the ventral tegmental area, in the substantia nigra, and one widely scattered but numerous population in superficial layers of the optic tectum. In the diencephalon, CGRP-ir cells were observed in the magnocellular preoptic nucleus and the organon vasculosum hypothalami. A population of CGRP-ir cells was also observed in the entopeduncular nucleus in the impar telencephalon. CGRP-ir fibers of central origin were widely distributed in the brain, but the most conspicuous areas were found in the ventral telencephalon, the hypothalamus, the mesencephalic lateral reticular area, and the dorsolateral isthmal region. The neurointermediate lobe of the hypophysis was also richly innervated by CGRP-ir fibers. CGRP-ir sensory fibers of cranial nerves IX and X and of dorsal spinal roots formed very conspicuous terminal fields in the lobus vagi and Cajal's nucleus commissuralis and in the dorsal region of the substantia gelatinosa, respectively. Comparison of the distribution of fibers and perikarya in dogfish and other vertebrates suggests that this CGRP-ir system has been well conserved during evolution.

Distribution of substance P-like immunoreactivity in the brain of the elasmobranch Scyliorhinus canicula.

Immunohistochemical methods were used to study the distribution of substance P in the brain of the small-spotted dogfish (Scyliorhinus canicula). Substance P-like immunoreactive (SP-IR) cell bodies and fibers were widely distributed. In the telencephalon, sparse populations of SP-IR neurons are present in the olfactory bulbs, pallium, and subpallium. In the subpallium numerous SP-IR boutons form unusual coats ("pericellular appositions") on SP-immunonegative neurons. In the diencephalon numerous SP-IR cerebrospinal fluid-contacting neurons are present in the preoptic recess organ and organon vasculosum hypothalami. Numerous SP-IR fibers also run in the hypothalamus, although no immunoreactivity was observed in the habenulo-interpeduncular system. A terminal field of SP-IR fibers is present in the median eminence. In the mesencephalic tegmentum, SP-IR neurons were observed in the Edinger-Westphal nucleus. SP-IR fibers are present at high density in the basal tegmentum, forming a conspicuous tract. In the hindbrain, numerous SP-IR fibers were observed in the isthmal region, the trigeminal descending root, the visceral sensory area and commissural nucleus, and the visceromotor column. SP-IR fibers occur at high density in the substantia gelatinosa of the rostral spinal cord.

Distribution and development of catecholaminergic neurons in the brain of the brown trout. A tyrosine hydroxylase immunohistochemical study.

We report the distribution of tyrosine hydroxylase immunoreactive (TH-ir) neurons in the brain of adult and developing trout. In the adult brain TH-ir neurons were observed in the telencephalon (in the olfactory bulbs and the ventral telencephalic region), the diencephalon (in both the parvicellular and magnocellular portions of the preoptic nucleus, the suprachiasmatic nucleus, the ventromedial thalamic nucleus, the posterior tuberal nucleus, the organon vasculosum hypothalami, the lateral recess nucleus and the dorsal periventricular region of the medial lobe recess), the pretectal region (nucleus pretectalis periventricularis), the isthmal region (locus coeruleus) and the medulla oblongata (in the area postrema, nucleus solitarius, the reticular area of the vagal region, and the reticular nucleus of the octaval region). The earliest TH-ir nuclei were observed in 10- to 11-min embryos in the posterior tuberal nucleus, locus coeruleus, nucleus solitarius and vagal reticular neurons. In the olfactory bulb, nucleus ventromedialis thalami and octaval reticular area, TH-ir cells a latter appeared at a later stage in development. In fry, juveniles and adults cerebrospinal fluid-contacting TH-ir cells were observed in circumventricular organs. The evolution of these TH-ir neuronal populations during development is described, and we discuss their significance for both comparative and developmental analysis.

Microspectrofluorimetric study of monoamines in the hypothalamus of Scyliorhinus stellaris L.

In a combined fluorescence histochemical and microspectrofluorimetrical study, the distribution, morphology and amino content of the hypothalamic circumventricular organs of an elasmobranch (Scyliorhinus stellaris) have been investigated. In the walls of the third ventricle, two aminergic circumventricular organs, viz. the preoptic recess organ and organon vasculosum hypothalami, were found to display an intense blue-green fluorescence. The emission maximum was registered at about 490 nm and the excitation spectra, conducted from 240 nm to 460 nm at neutral pH, displayed a main peak at 410 nm, with additional peaks at about 320 nm and 260 nm, values that are indicative of catecholamines. In neutral state, the quotient of the excitation peak ratio values 410/260 nm exceeded 1, thus determining the differentiation between noradrenaline and adrenaline, excluding the latter as a secondary monoamine. Recordings of hydrochloric acid (HCl)-treated sections showed a hypsochromic shift of the main excitation peak from 410 nm to 370 nm and a concurrent increase of the peak at 320 nm, with the mean peak ratio 370/320 nm being 0.98, pointing to the presence of dopamine. The fading curves indicated a photodecomposition of less than 20%, both in neutral and acidified sections, which is typical of dopamine. It can therefore be deduced that the blue-green fluorescent cells in the hypothalamic circumventricular organs of the dogfish contained mainly dopamine. Some cells expressed a yellowish fluorescence, showing rapid photodecomposition characteristic of indolethylamine fluorophores, pointing to the presence of 5-hydroxytryptamine (5-HT).

Organization of catecholaminergic systems in the hypothalamus of two elasmobranch species, Raja undulata and Scyliorhinus canicula. A histofluorescence and immunohistochemical study.

We examined the organization of catecholaminergic neurons in the hypothalamus of the painted ray, Raja undulata, and the small-spotted dogfish, Scyliorhinus canicula, with the use of formaldehyde-induced fluorescence (FIF) methods and tyrosine hydroxylase (TH) immunohistochemistry. In both species we identified distinct populations of catecholamine-containing neurons differing in a) their immunoreactivity to antibodies against the enzyme tyrosine hydroxylase (TH), b) their fluorescence in response to FIF methods for the detection of catecholamines, and c) their relationship with the third ventricle. One population is made up of FIF-positive and TH-negative neurons (most of which are CSF [cerebrospinal fluid]-contacting) and located in two circumventricular organs, the preoptic recess organ and the organon vasculosum hypothalami. Another population comprises TH-immunoreactive (TH-IR), FIF negative neurons that are located in the suprachiasmatic nucleus and the posterior tuberculum and are not related to the third ventricle recesses. A third population of TH-IR, CSF-contacting neurons is also present in the organon vasculosum hypothalami. The existence of three catecholaminergic populations suggests differences in the metabolism of catecholamines and/or different functions. The circumventricular neurons are not associated with the hypophysis and appear to accumulate catecholamine (dopamine) obtained from exogenous sources. In both Raja and Scyliorhinus the neurointermediate lobe is innervated by TH-IR fibres originating from dopamine-synthesizing neurons of the second catecholaminergic population.