The bulbus arteriosus of stenothermal and temperate teleosts: a morphological approach

The bulbus arteriosus, 'windkessel', of several species of stenothermal and temperate teleosts has been studied by conventional light microscopy and electron microscopy. The bulbus wall is divided into an endocardium, ridges, and middle and external layers. The endocardium of all species shows moderately-dense bodies, which vary widely although the significance is not known. The endocardium in Antarctic teleosts invaginates into the ridge tissue to form solid epithelial cords that show signs of active secretion related to protective substances. Cords also form in serranidic and sparidic species, but signs of active secretion are not evident. The ridges consist of cells within a filamentous meshwork. Ridge cells appear to be smooth muscle cells that undergo a phenotypic transition from the endocardium toward the middle layer. Middle layer cells are typical smooth muscle cells surrounded by a filamentous matrix. The appearance and composition of the extracellular matrix varies widely among species, with those from the Antarctic lacking collagen and elastin fibres. The external layer is a collagenous matrix that contains fibroblasts, blood vessels and nerves. In most Antarctic teleost species this layer lacks blood vessels, but contains nerve fibres. Some of these fibres could have a sensory function to control bulbus dilatation. The external layer of Trematomus bernacchii has the appearance of a germinal centre and may be involved in the immune humoral response. The epicardium is atight epithelium that may control passage of substances with the pericardial cavity.     

Fine structure and cytochemistry of the endothelial cells and rodlet cells in the bulbus arteriosus in species of Cichlidae (Teleostei)

The ultrastructure of endothelial cells and rodlet cells in the bulbus arteriosus of specimens representing six genera of Cichlidae is described. The former are very closely packed by membrane–bound and mainly electron–dense inclusion bodies (0.3–0.7μm).
In Apistogramma ramirezi I observed numerous subendothelial rodlet cells throughout the entire length of the bulbus arteriosus. These cells penetrate the endothelium and connect to the latter by desmosomes and tight junctions. The luminal part of the cell contains numerous vesicles and tubules (width 50–100 nm), whereas the basal part is occupied by a number of membrane–bound, club–like inclusions (length ≤ 5 μm). Between these two layers there occurs a layer of small, elongated mitochondria. Peripherally, these cells consist of a filamentous wall, except in the apical area.
The endothelial and rodlet cell inclusion bodies do not react with phosphotungstic acid (pH 1) or Sudan black B stain. The endothelial cells react strongly with periodic acid–Schiff (PAS) stain, whereas the rodlet cells are only moderately coloured by this stain.
The present results are discussed and compared with those reported previously for endothelial/ endocardial cells and rodlet cells in bony fish.     

Ultrastructural and ultrahistochemical studies on ventricular endocardial cells in teleosts (Pisces)

The ultrastructure of the ventricular endocardial cells in 17 bony-fish species representing eight families, is described. In species of Characidae, Cobitidae, Cyprinidae, and Gyrinocheilidae these cells are flat (1–2 µm at nucleus) and contain numerous ribosomes, some few bristle-coated vesicles (BCV) and small (0.243 pm) electron dense inclusion bodies. However, in species of Cichlidae, Gadidae, and Poeciliidae most endocardial cells appear relatively thicker (2–5 µm at nucleus) and contain numerous BCV, tubules of agranular endoplasmic reticulum, and large (0.5-1.5 µm) moderately electron dense bodies (MDB). In the MDB occur a number of small (20–150 nm) electron dense granules. Within the family Anabantidae, most endocardial cells seem to be of the first type in Colisa laliu and Trichogaster leeri. whereas in Helosioma iemmincki there are numerous cells of the second type. When glutaraldehyde/tannic acid fixed heart tissue of Pollachius virens is treated with ferrous chloride, ferric chloride, or osmium tetroxide, and grid stained by uranyl and lead solutions, damaged endocardial cells appear highly electron dense, whereas undamaged ones are electron lucent. Further, when glutaraldehyde fixed tissue is treated with osmium tetroxide/potassium ferrocyanide the subendocardial space is filled by a highly electron dense material. The methods described in this study make it possible to distinguish between those endocardial vacuoles having structural contact with the cell membrane, and those lacking such contact, and also to determine whether the lumen of the former is continuous with the subendocardial space, or with the intertrabecular lumen.     

Histological, histochemical and ultrastructural studies on the bulbus arteriosus of the sticklebacks, Gasterosteus aculeatus and Pungitius pungitius (Pisces: Teleostei)

The bulbar wall has three layers. Its lining consists of squamous-columnar endothelial cells that store neutral mucopolysaccharides and are PAS-positive. They do not contain large amounts of acid phosphatase, acid mucopolysaccharides, glycogen or lipids. A morphometric analysis shows that 32% of the cell volume in Pungitius and 12% in Gasterosteus is occupied by specific granules, 100–600 nm in diameter. According to X-ray probe micro-analysis, these granules bind chromium ions, even though the endothelial cells do not contain catecholamines. Rootlets, packed with plasmalemmal vesicles, extend from the endothelial cells into the middle layer of the bulbus. Here, smooth muscle cells alternate with elastic fibres. The staining reactions of bulbar elastica are compared with those in the mammalian aorta and the ligamentum nuchae. The outer layer of the bulbus is visceral pericardium and beneath its covering mesothelial cells are numerous collagen fibres, non-myelinated nerves, occasional fibroblasts and melanocytes. Scanning electron microscopy shows that the bulbar lining is thrown into longitudinal folds, but that there are no trabeculae subdividing the lumen.
Many features of the bulbus arteriosus may be related to the low systolic pressures of teleosts and to the proximity of their heart and gills. In contrast to mammals, only a small part of the arterial system can act as a windkessel. The bulbus is thus more distensible than the mammalian aorta and must lie within the pericardial cavity so that its greater excursions can be accommodated. Perhaps because the bulbus is so distensible, it has elastic fibres rather than lamellae. This in turn may affect the organization of the smooth muscle cells which do not form "span muscles" as in some mammalian aortae. Like most cells in the bulbus, they are joined to others by desmosomes. Evidently, firm cohesion is important in highly distensible vessels.     

Functional morphology of the bulbus arteriosus of rainbow trout (Salmo gairdneri Richardson)

The rate of expansion in volume of the bulbus arteriosus with increase in pressure is measured. From this it is calculated the elastic rebound of the bulbus can account for approximately 25 % of blood flow in the ventral aorta; this proportion decreases as cardiac output increases. The structure of the wall of the bulbus is shown to consist of a compact outer layer with a series of separate longitudinal elements on the inner surface. These elements are connected to the compact layer by numerous radial fibres. This structure equalizes strain in all the structural elements of the wall during large changes in volume. Evidence is discussed which shows that the bulbus arteriosus in teleosts is morphologically and biochemically distinct from the ventral aorta. The bulbus is probably of cardiac origin rather than an expansion of the posterior end of the aorta as generally supposed.     

A comparative study on the innervation and the vascularization of the bulbus arteriosus in teleost fish

Summary The structure of the bulbus arteriosus of a wide range of teleost fish is described with particular reference to the vascularization and innervation. The adventitia of the organ consists of blood vessels and large nerve bundles in a collagen matrix. The nerve bundles contain monoamines, and fluorescence studies show small terminal bundles penetrating the muscular media; this is confirmed by electron microscopy. The media consists of an extensive elastic tissue matrix with a spiral arrangement of smooth muscle cells joined end to end by desmosomes and presumed electrotonic junctions. The muscle cells are innervated only at the adventitia/media boundary and the significance of this innervation is discussed. It is proposed that there is a correlation between the degree of vascularization and innervation and the activity of a particular species offish.     

A review of the bacterial flora of teleosts and elasmobranchs, including methods for its analysis

Techniques for the quantitative and qualitative analysis of the commensal bacterial flora of fish are reviewed. The literature concerning the commensal bacterial flora of fish indicates that teleost skin often features 10²–0⁷ organisms per cm² of skin, representing the following taxa, in descending numerical order, Achromobacter, Pseudomonas, Flavo-bacterium and Cytophaga species also coryneform organisms. Actively feeding teleosts appear to have an alimentary canal bacterial flora similar to that of the skin and gills, often there are from 10³–10⁸ organisms per 1.0 g, wet weight, of tissue. According to the limited data available for elasmobranchs Gram–positive bacteria are the predominant component of the bacterial flora on the skin of sharks, though the bacterial flora of North Sea skate ( Raja spp.) is reported as being similar to that of teleosts. The commensal flora of fish, particularly of teleosts, is related to their environment and is influenced by environmental changes that affect the quality of the water. Regular monitoring of the water of commercial fish ponds and aquaria for bacterial quality, temperature, dissolved oxygen concentration and pH, combined with a base–line knowledge of the water's quality, may provide an advanced indication of the presence of bacteria potentially able to produce diseased conditions, and factors that will enhance their growth.     

Characterization of the effects of vasoactive substances on the bulbus arteriosus of the eel,Anguilla rostrata

The fish bulbus arteriosus (BA) smooths cardiac output by expanding during cardiac systole and rebounding during diastole, thereby providing constant perfusion of the gills downstream. Published data have demonstrated innervation of the teleost BA and shown that the tension and compliance of the BA responded to vasoactive agonists, such as epinephrine and acetylcholine, suggesting that the BA was more than a mere "windkessel." To examine vasoactivity in the BA more directly, we measured the responses of isolated tissue rings from the BA of the eel, Anguilla rostrata to a suite of putative vasoactive agonists, which had been shown to affect vascular smooth muscle in a variety of teleosts. The BA of the eel was insensitive to acetylcholine but constricted when endothelin (ET-1) was applied. Nitric oxide, sodium nitroprusside (SNP; NO donor), natriuretic peptides (NP), and prostaglandin E1 (but not the prostacyclin agonist carbaprostacyclin) produced significant dilation in the BA. Since both ET-1 and sarafotoxin S6c produced concentration-dependent constriction, it appears that endothelin receptor B-type (ETB) receptors (and possibly ETA receptors) are present. The dilation produced by SNP was also concentration dependent, as were the dilations produced by porcine C-type natriuretic peptide, eel atrial natriuretic peptide (NP receptor agonists), Sulprostone and Butaprost (PGE receptor agonists). Our data demonstrate that the BA of eel is responsive to a variety of vasoactive agonists, suggesting that the BA is under neurohumoral control. The role of agonist-induced changes in BA tension in fish cardiovascular physiology remains to be determined, as do the specific receptor types involved.     

Morphological and biometric revision of the cleithra, opercular and pharyngeal bones of Iberian teleosts belonging to the genus Barbus (Pisces, Cyprinidae)

Species of the genus Barbus present in the Iberian Peninsula are an interesting study group, due to its diversity and complex taxonomic position. In this work, 299 specimens of eight species were studied. All of them are endemic to the Iberian Peninsula, except for Barbus meridionalis. Cleithra, opercular and pharyngeal bones were extracted from each specimen. These bones were morphologically compared and measured, obtaining biometric indices. From these indices, the biometric differences of each bone were analysed with a principal component analysis. Later, a principal component analysis and a discriminant analysis were performed considering the three bones together. The morphological differences and similarities are congruous with the biometric results. In addition, this osteological comparison partially agrees with the present taxonomic position of these species, being a contribution to the systematics and phylogeny of genus.     

Ultrastructure of the peritoneal exudate cells of seawater teleosts,seabream (Sparus aurata) and sea bass (Dicentrarchus labrax)

The peritoneal exudates of seabream and sea bass consist of granulocytes, lymphocytes and macrophages. These cells show conspicuous ultrastructural differences from the same cell-types of blood and head-kidney, which have not been reported previously. Peritoneal exudate granulocytes differ from their corresponding circulating or head-kidney forms in the following way: (a) they are larger in size, and (b) their abundant cytoplasmic granules have some new ultrastructural features, and a new granule population might also be present. Likewise, lymphocytes also show a noticeable difference; they contain a sparse population of small dense cytoplasmic granules. Monocytes, macrophages, and transitional forms between these two cell-types, are also found. The percentage of peritoneal exudate cell-types is different in seabream and sea bass. Macrophages in sea bass represent the most abundant peritoneal exudate cell-type. However, seabream shows lower percentages of macrophages than granulocytes.     

Gonadotropin-releasing hormone (GnRH) pathways and reproductive control in elasmobranchs

Synopsis Immunoreactive (ir) gonadotropin-releasing hormone (GnRH) is localized in many neurons of the terminal nerve (TN) and midbrain tegmentum, while few ir-cells are observed in the preoptic area and ventral hypothalamus. The paucity of preoptic ir-cells may relate to an unusual feature of the elasmobranch pituitary, i.e. a lack of portal control of gonadotropin-producing cells. TN and midbrain GnRH-ir neurons may be major sources of GnRH used to modulate or otherwise control both pituitary and brain cells via delivery through the systemic circulation. These ir-nuclei also appear to directly innervate CNS regions (the preoptic area, habenula and clasper control area of the spinal cord) involved in sexual functions. Important regulatory mechanisms, represented by interactions between GnRH pathways and sex-steroid concentrating neurons, are likely to occur in the preoptic area, habenula and midbrain tegmentum.     

Expression of the giant protein AHNAK (desmoyokin) in muscle and lining epithelial cells.

Here we report a detailed analysis of the expression and localization of the giant protein AHNAK in adult mouse tissues. We show that AHNAK is widely expressed in muscle cells, including cardiomyocytes, smooth muscle cells, skeletal muscle, myoepithelium, and myofibroblasts. AHNAK is also specifically expressed in epithelial cells of most lining epithelium, but is absent in epithelium with more specialized secretory or absorptive functions. In all adult tissues, the main localization of AHNAK is at the plasma membrane. A role for AHNAK in the specific organization and the structural support of the plasma membrane common to muscle and lining epithelium is discussed.     

Responses of MSH and prolactin cells to 5-hydroxytryptophan (5-HTP) in amphibians and teleosts

Summary Injection of 5-hydroxytryptophan (5-HTP), a precursor of serotonin, induces dispersion of melanin in the amphibians, Pleurodeles waltlii (Urodela) and Xenopus laevis (Anura), in the goldfish, Carassius auratus, and in the carp, Cyprinus carpio. It is accompanied by a dispersion of erythrophore pigments. In the pituitaries of Pleurodeles and goldfish, a stimulation of MSH cells, characterized by a significant nuclear hypertrophy, is also observed; in Carassius, MSH cells may become degranulated. Serotonin appears to exert a stimulating effect on MSH release in lower vertebrates. Swimming behavior is disturbed in the goldfish and the carp; gaseous metabolism in the swim-bladder may be affected by injection of 5-HTP, as previously reported in the eel. Prolactin (PRL) cells appear activated, but remain granulated in the treated goldfish. No clear response of PRL cells to injection of 5-HTP can be observed in Pleurodeles. A possible role of serotonin in Pleurodeles submitted to an experimental aeroionization is briefly discussed.     

Cytogenetic studies of Poecilia (Pisces)

Natural populations of triploid females resembling the gynogenetic teleost, Poecilia formosa (Girard), occur in northeastern Mexico where they intermingle with diploid populations of this species and the members of congeneric bisexual species such as P. mexicana or P. latipinna. Mitotic configurations from gill epithelial cells show 46 chromosomes for the diploid fishes, but 69 chromosomes for members of the triploid clones associated with P. formosa. Triploid females have erythrocytes that are significantly larger than those from diploid specimens and also show a roughly 50% elevation in the average DNA content of their somatic nuclei. Similar analyses of two functionally incompetent males of P. formosa, of a number of bisexual F₁ and F₂ hybrid offpsring from P. latipinna x P. mexicana, and of females from several other poeciliid species consistently show only diploid DNA levels and somatic chromosome complements where 22N=46. Demonstration of cytogenetic criteria by which females from triploid clones may be clearly distinguished from sympatric diploid specimens of P. formosa or P. mexicana leaves unresolved, for the present, problems of an appropriate systematic designation for natural populations of triploid gynogenetic fishes. The role of sympatric speciation in the evolution of poeciliid genomes is discussed in terms of alternative mechanisms to account for the persistence in nature of a vertebrate triploid of hybrid origin.This work was supported by grants from the National Science Foundation (GB 7393) and from the U.S. Public Health Service (GM 14644).Recipient of a Research Career Development Award from the U.S. Public Health Service (1 K3 GM 3455).