Studies on formation and resorption of fish scales

Summary Scale formation in Cyprinodon variegatus was found to be initiated at about 26 to 30 days after hatching. Ultrastructural investigation revealed that within 4 to 6 h in the first-formed scales the marginal cells begin to flatten and differentiate into osteogenic cells, which later change to osteoblasts and fibroblasts. These cells are separated from the surrounding epithelial cells by a basal lamina. The osteoid is formed by the marginal and osteogenic cells; the osseous layer by the osteoblasts; and the fibrillary plate by the fibroblasts.The osteoid is formed within 2 to 3 h after the initiation of the scale, and within 20 to 24 h the osseous layer is formed. Hydroxyapatite crystals are deposited in the matrix of the osseous layer without apparent association with collagen fibers. No matrix vesicles or dense bodies are evident at the sites of calcification. The fibrillary plate arises 18 to 20 h after the initiation of the scale. It is also partially calcified, but not before the third week of scale formation. The crystals develop almost exclusively between the collagen fibers at the extreme edge of the calcifying front, but solid calcification of the fibers results with further growth of the crystals. The fibroblasts appear to participate in calcification of the fibrillary plate.Contribution No. 332, Belle W. Baruch Institute for Marine Biology and Coastal Research, University of South Carolina, Columbia, South Carolina, 29208, USA     

The histology and calcification of regenerating scales in the blackspotted topminnow, Fundulus olivaceus (Storer)

The regenerating scale and tissues comprising the scale pocket of Fundulus olivaceus were examined microscopically at specific intervals. Scale removal resulted in a thickening of the epidermis which persisted through the early stages of regeneration. This thickening was due in part to the appearance of columnar basal cells which divided producing cells that became mucous cells and squamous cells. The scale regenerated as a relatively large plate of bone which first appeared between layers of scleroblasts on the floor of the scale pocket and then grew producing circuli and radii. By the fourth day of regeneration, calcium was observed in the cytoplasm of the scleroblasts and at randomly distributed foci in the osseous portion of the scale. The osseous layer was completely calcified by 15 days of regeneration.     

In vitro differentiation of the human osteosarcoma cell lines, HOS and KHOS.

The osteogenic potential of the two human osteosarcoma cell lines HOS and KHOS; a cell line produced by the transformation of the HOS cells by the Kirsten murine sarcoma virus, was studied in vitro. HOS cells cultured more than 2 weeks formed nodules composed of two morphologically distinct layers, an epithelial-like surface cell layer and a collagen-rich inner cell layer. Alkaline phosphatase (ALPase) activity occurred in the plasma membrane of the surface cell layer, and calcified substances developing along collagen fibers were detected in the collagen-rich inner cell layer. The calcified substances were further examined by analytical electron microscopy and were shown to be hydroxyapatite crystals. In contrast, there was neither ALPase nor the deposition of a calcified substance in the KHOS cells.     

CONCEPTACLE STRUCTURE OF THE PARASITIC CORALLINE RED ALGA CHOREONEMA THURETII (CORALLINALES) AND ITS TAXONOMIC IMPLICATIONS1

The major diagnostic features for erecting the red algal subfamily Choreonematoideae (Corallinales) were a combination of 1) absence of both cell fusions and secondary pit connections, 2) conceptacle roof and wall comprised of a single cell layer, and 3) presence of tetrasporangial pore plugs within a uniporate conceptacle in the monotypic taxon Choreonema thuretii (Bornet) Schmitz. Because this alga is a parasite, the absence of secondary cell connections is most likely an adaptation to a reduced thallus. This study shows that all conceptacles are not composed of a file of cells but rather a single layer of epithallial cells that are underlain by a thick layer of calcified acellular material; both epithallial cells and the calcified layer are produced by peripheral sterile cells. Although the outermost tetrasporangial pore canal is uniporate, there is a calcified acellular multiporate plate recessed just below the rim. The plate is produced by interspersed sterile cells and is continuous with the calcified layer supporting the conceptacle. These unique structures are likely due to parasitism rather than to the ancestral state. Based on these results and a reexamination of published micrographs depicting lenticular cells in Austrolithon intumescens Harvey et Woelkerling, we propose that both subfamily Choreonematoideae and Austrolithoideae are closely allied with subfamily Melobesioideae. This distant relationship to its host (Corallinoideae) plus a combination of unique conceptacle and unusual type of parasitism indicates that C. thuretii is an alloparasite and that it is likely the most ancient red algal parasite studied to date.     

Opercular regeneration in Pomatoceros lamarckii Quatrefages (Polychaeta: Serpulidae). Differentiation of the operculum and deposition of the calcareous opercular plate

Following opercular amputation, stages in opercular regeneration in Pomatoceros lamarckii have been described by light, transmission and scanning electron microscopy. Two to three days after amputation, the rudimentary opercular filament is invested with a delicate cuticle composed of an outer filamentous layer and an inner thicker component composed of orthogonally-arranged layers of small fibril bundles. The opercular plate is uncalcified and composed of two major components, an outer, thin, electron-dense layer and an inner, thicker component which structurally resembles that of the opercular filament cuticle. Between five and eight days, opercular plate calcification is initiated as needle-like crystallites. The structural organization of the organic components of the opercular plate show changes which are related to the onset of calcification. From 13–17 days, the opercular plate becomes heavily calcified and is composed of highly-ordered, prism-like crystals. X-ray diffraction shows these crystals to be aragonite. The structure of the cuticle remains unchanged except that the orthogonally-arranged fibril bundles aggregate into thicker fibres. Amino acid analysis of the regenerated cuticle and organic components of the opercular plate show that they differ from one another and from the normal cuticle and opercular plate. During opercular regeneration, the differentiation of the cuticle and opercular plate-secreting cells are described and the mechanisms of cuticle and calcareous opercular plate secretion are discussed.     

Structure of comblike teeth of the ayu sweetfish,Plecoglossus altivelis (Teleostei: Isospondyli): I. Denticles and tooth attachment

The structure and tooth attachment of the comblike teeth and denticles of the ayu sweetfish, Plecoglossus altivelis, were examined by light and scanning electron microscopy. The denticle is composed of a spoonlike crown with a spine pointed anteriorly, a triangular plate in the cervical region, and a root that curves laterally and tapers off to a point. The root apex is fused with a long thin pedicle that turns abruptly anteriad toward the jaw bone. Planes of the spine, the spoonlike crown, the triangle plate and the root of the denticle are varied, and the denticle is twisted in the region of the triangle plane. The superficial layer of the dentine is homogeneously calcified and is considered to be enameloid, because some of the inner dentinal epithelial cells in the tooth germ are columnar and possess cellular processes at their apical ends. The dentine is fibrous and fine dentinal tubules are visible in dentine treated with sodium hydroxide and observed by scanning electron microscopy. The upper half of the root is surrounded by a dense layer of collagen fibers running parallel to the tooth axis, and the lower half is encompassed by interlaced collagen fibers. The lower part of the root is open on its lingual side. The pedicle is a long rod which is homogeneously calcified and enmeshed by interlaced collagen fibers, and it curves mediad as it nears the jaw bone. The pedicles are interposed between a layer of gelatinous connective tissue and the jaw bone and terminate on the periosteum. Comparative aspects of ayu tooth morphology are discussed. © 1993 Wiley-Liss, Inc.     

The histological structure of the calcified lung of the fossil coelacanth Axelrodichthys araripensis (Actinistia: Mawsoniidae)

Abstract: The palaeohistological study of the calcified internal organ of Axelrodichthys araripensis Maisey, 1986, a coelacanthiform from the Lower Cretaceous of Brazil (Crato (Aptian) and Santana (Albian) formations of the Araripe Basin), shows that the walls of this organ consist of osseous blades of variable thickness separated from each other by the matrix. This indicates that, in the living individuals, the walls were reinforced by ossified plates, probably separated by conjunctive tissue. This calcified sheath present in Axelrodichthys, as well as in other fossil coelacanths, lies in ventral position relative to the gut and its single anterior opening is located under the opercle, suggesting a direct connection with the pharynx or the oesophagus. The calcified organ of Axelrodichthys, like that of other fossil coelacanths, is here regarded as an ‘ossified lung’ and compared with the ‘fatty lung’ of the extant coelacanth Latimeria. The reinforcement of the pulmonary walls by the overlying osseous blades could be interpreted as a means of adapting volumetric changes in the manner of bellows, a necessary function for ventilation in pulmonary respiration. Other functional hypotheses such as hydrostatic and/or acoustic functions are also discussed.     

Fine Structure of the Developing Scale in the Cichlid Hemichromis bimaculatus (Pisces,Teleostei, Perciformes)

The study of the formation and structure of the early teleost scale and its associated cells has been carried out on Hemichromis bimaculatus fry using in toto staining with alizarin and transmission electron microscopy techniques. Results of the study show very rapid scale formation in Hemichromis. The papilla of the scale differentiates a little in advance of the bone scale formation. No epidermal cells are involved in the constitution of the scale pocket made up of scleroblasts. In Hemichromis, as in other teleost scales, the osseous layer is the first one to be secreted by, presumably, only the scleroblasts. Then the scleroblasts specialize in their functions. Superficial ones are involved in the formation of osseous circuli; marginal scleroblasts are responsible for growth in diameter of the scale; while deep scleroblasts allow the scales to thicken owing to the progressive addition of collagen fibrils organized in a “plywood-like” structure which constitutes the fibrillary plate of the scale. Mineralization occurs very rapidly within the osseous layer in the form of hydroxyapatite-like crystal deposits. The fibrillary plate is not yet mineralized in Hemichromis at the stages studied here, but presumably is later. Results obtained in Hemichromis are discussed against similar data available in the literature on teleost scale formation.     

Effect of Coccolith Polysaccharides Isolated from the Coccolithophorid, <Emphasis Type="Italic">Emiliania huxleyi</Emphasis>, on Calcite Crystal Formation in In Vitro CaCO<Subscript>3</Subscript> Crystallization

Marine coccolithophorids (Haptophyceae) produce calcified scales “coccoliths” which are composed of CaCO₃ and coccolith polysaccharides (CP) in the coccolith vesicles. CP was previously reported to be composed of uronic acids and sulfated residues, etc. attached to the polymannose main chain. Although anionic polymers are generally known to play key roles in biomineralization process, there is no experimental data how CP contributes to calcite crystal formation in the coccolithophorids. CP used was isolated from the most abundant coccolithophorid, Emiliania huxleyi. CaCO₃ crystallization experiment was performed on agar template layered onto a plastic plate that was dipped in the CaCO₃ crystallization solution. The typical rhombohedral calcite crystals were formed in the absence of CP. CaCO₃ crystals formed on the naked plastic plate were obviously changed to stick-like shapes when CP was present in the solution. EBSD analysis proved that the crystal is calcite of which c-axis was elongated. CP in the solution stimulated the formation of tabular crystals with flat edge in the agarose gel. SEM and FIB-TEM observations showed that the calcite crystals were formed in the gel. The formation of crystals without flat edge was stimulated when CP was preliminarily added in the gel. These observations suggest that CP has two functions: namely, one is to elongate the calcite crystal along c-axis and another is to induce tabular calcite crystal formation in the agarose gel. Thus, CP may function for the formation of highly elaborate species-specific structures of coccoliths in coccolithophorids.     

Process of Carbonate Precipitation by Deleya halophila

Scanning electron microscopy and X-ray dispersive energy microanalysis were used to investigate the formation of carbonate crystals by Deleya halophila. The formation of calcium carbonate crystals (polymorphous aragonite) by D. halophila is a sequential process that commences with a nucleus formed by the aggregation of a few calcified bacterial cells and the subsequent accumulation of more calcified cells and carbonate, which acts to weld the bacteria together. The process leads to the formation of spherical bioliths measuring approximately 50 μm in diameter. The mechanism of carbonate precipitation by D. halophila under our working conditions represents a process of induced biomineralization.     

A comparative study of the ultrastructure and mineralogy of calcified land snail eggs (Pulmonata: Stylommatophora)

This study indicates that eggs containing calcium carbonate crystals occur in at least 36 of the 65 known families of the land snails (class Gastropoda: order Stylommatophora). Eggs from 22 of these families were available for examination. The x-ray diffraction data, available for the first time for 21 of these families, shows that these egg shells are all made of calcite only, or of a combination of calcite with smaller amounts of aragonite. All of the snail (body) shells examined were made of aragonite only. This is the first ultrastructural investigation of these egg shells, and it indicates that the eggs exhibit enough structural diversity to allow identification of parental animals to genus, and often to species level solely on the basis of egg shell ultrastructure. All of the calcified eggs may be divided into two groups: (1) partly calcified, with discrete crystals of CaCo₃ dispersed in the jelly layer, and (2) heavily calcified, with a hard, brittle egg shell made of fused crystals of CaCO₃ much like an avian egg. Both types of calcified eggs occur in oviparous as well as in ovoviviparous snails. Because of the wide distribution of calcified eggs in the Stylommatophora, and because of the occurrence of heavily calcified eggs in ancient families such as Partulidae, Endodontidae, and Zonitidae, the calcified egg is viewed as a primitive land snail trait associated with terrestrial adaptation. The function of the calcified egg shell, in addition to mechanical support of egg contents, is to supply the developing embryo with enough calcium to form the embryonic shell by the time of hatching.     

Fine structure of regenerating scales and their associated cells in the cichlid Hemichromis bimaculatus (Gill)

Summary Scale regeneration has been studied in Hemichromis bimaculatus. The removed scale, which serves as a control, is covered by its surrounding scleroblasts as can be seen with scanning electron microscopy. Subsequently, during regeneration, a population of scleroblasts arises in the empty dermal pocket as shown with transmission electron microscopy. At first, an elongated papilla of regeneration forms, probably from the differentiation of dermal fibroblasts. A scale anlage composed of the osseous layer appears in the middle of the papilla, which becomes a regenerating bag. All the surrounding large scleroblasts are involved in scale formation, although later three populations of scleroblasts specialize according to their location around the scale. Superficial scleroblasts flatten when the final thickness of the osseous layer of the scale is attained; the deep scleroblasts are responsible for the formation of the basal plate whereas marginal scleroblasts increase the diameter of the osseous layer of the scale.During scale regeneration, scleroblasts are more numerous and larger than during scale ontogenesis. In particular, deep scleroblasts form a columnar epithelium when the basal plate is laid down, a feature which is not found during scale ontogenesis. Moreover, the regenerated basal plate exhibits an orthogonal plywood arrangement that is never seen in the embryonic scale where the plywood is of the intermediate type.     

Polymorphism of Crystals of Salmonella minnesota Re and Ra Lipopolysaccharides

Salmonella minnesota Re and Ra lipopolysaccharides (LPSs) formed three-dimensional crystals when they were precipitated by the addition of 2 volumes of 95% ethanol containing 375 mM MgCl₂ and incubated in 70% ethanol containing 250 mM MgCl₂ at 4 C. Besides typical shapes of crystals, hexagonal plates and solid columns, which were already reported (J. Bacteriol. 172: 1516–1528 (1990)), the LPSs thus treated formed crystals possessing various shapes such as square or rectangular plate, lozenge plate, discoid, and truncated hexangular pyramid forms. Electron diffraction patterns from all these crystals except square or rectangular plate crystals obtained by electron irradiation from the direction perpendicular to the basal plane were essentially the same as those from hexagonal plate crystals, indicating that they consist of hexagonal lattices with the lattice constant of 4.62 Å. From these results as well as the results of electron microscopic observations of these crystals, it was concluded that all these crystals except square or rectangular plate crystals are composed of hexagonal plate sheets as the basic structural units. Square or rectangular crystals were assumed to correspond to the {1011} planes of solid hexagonal column crystals.     

Siphuncular structure in the orders Tarphycerida and Barrandeocerida (Cephalopoda: Nautiloidea)

Abstract: The siphuncular structure is described in two Silurian taxa, Boionautilus tyrannus and Cumingsoceras complanatus, currently placed in the Tarpycerida. Tarphycerids have the Nautilus type of connecting ring that is composed of an outer, thick, spherulitic‐prismatic layer and an inner glycoprotein layer, the latter was destroyed by diagenesis. However, both Silurian specimens have the connecting ring of the calcified‐perforate type, previously known to occur in orthocerids, actinocerids, plectronocerids and now also in barrandeocerids. In this type, the inner layer of the connecting ring is calcified and perforated by pore canals. Boionautilus and Cumingsoceras are therefore classified with barrandeocerids and not with tarphycerids.     

Bone histology of claudiosaurus germaini (reptilia,claudiosauridae) and the problem of pachyostosis in aquatic tetrapods

Histological study of the skeleton of Claudiosaurus germaini reveals extensive pachyostosis. This feature results from the filling of intra‐osseous cavities by centripetal, endosteal deposits and occurs in conjunction with an intense remodelling of the bones by resorption and re‐deposition. Epiphyseal calcified cartilages are rapidly and entirely resorbed. Extensive pachyostosis suggests that Claudiosaurus was an aquatic reptile. However, the pachyostotic condition in this genus appears histogenetically quite different from the common type of pachyostosis observed in other aquatic tetrapods. Hence, it probably had a distinct physiological significance.     

Crystallization and Analyses of Crystals of Various Chemotypes of R-Form Lipopolysaccharides from Salmonella Spp.

Various chemotypes (Re, Rd₂, Rd₁P^–, Rd₁, RcP^–, Rc, Rb₃, Rb₂, Rb₁, and Ra) of R-form lipopolysaccharides (LPSs) of Salmonella spp. were crystallized by treatment with 70% ethanol containing 250 mM MgCl₂, and crystals of the LPSs were observed electron microscopically and analyzed by electron diffraction and synchrotron X-ray diffraction. All the LPSs tested formed three-dimensional crystals showing very similar shapes; hexagonal plate, solid column, discoid, square or rectangular plate, lozenge plate and truncated hexangular or rectangular pyramid forms. Electron diffraction patterns from the hexagonal plate crystals of all these LPSs obtained by electron irradiation from the direction perpendicular to the basal plane showed that they consist of hexagonal lattices with the lattice constant of 4.62 Å. The crystals of all the LPSs thus formed gave ring-like X-ray diffraction patterns because of their small sizes. The long-axis values were calculated from the X-ray diffraction patterns from crystals of all the LPSs in the low-angle region and they corresponded roughly to the length of the proposed primary chemical structures of the R cores of the LPSs. The volume occupied by a single molecule of all the LPSs were calculated from the molecular weights based on the proposed structures and the crystallographic data obtained by electron diffraction, X-ray diffraction, and density determination.     

The body wall of cheilostome Bryozoa. I. The ectocyst of Watersipora nigra (Canu and Bassler)

The cuticle of Watersipora nigra is at first translucent, but it later becomes black and differentiates into two layers. It is composed, at least in part, of a protein-polysaccharide complex. Calcified parts are three-layered: (1) an outer, cuticular layer, (2) a calcium carbonate skeleton deposited on a matrix of acid mucopolysaccharide, and (3) a “skeletal membrane.” The relationships of these layers indicate that the skeleton is intracuticular. A layer of cuticular material, the “intercalary cuticle” is present in lateral walls, but not transverse walls; it may become calcified in some species. The cuticles of calcified and uncalcified parts of cheilostomes are not necessarily homologous.     

Studies on scale structure in the common goby Pomatoschistus microps Krøyer

A histological study on scale structure in the common goby, Pomatoschistus microps (Krøyer) has established a similarity to other teleost scales, but yielded two new findings. First, the osseous layer of the scale was formed of successive and parallel calcified bands that may reflect a rhythm in scale growth. Second, extracellular matrix vesicles (100–200 nm) were identified by electron microscopy, at the calcifying area in the anterior growing edge of the scale; distribution of these vesicles corresponded closely to the pattern of scale calcification. A proposal on the mechanism of growth and calcification of scale has been made. The surface topography of the scale has also been examined by scanning electron microscopy. The raised edge of the circulus is formed of minute knobs, which may be used as criterion in fish taxonomy.     

Scales in young Polypterus senegalus are elasmoid: new phylogenetic implications

A component of the basal plate which has a plywood-like organization similar to that of the elasmoid scales of teleosts is described in the scales of Polypterus senegalus for the first time. The origin and development of this structure is studied in young (50-117 mm, standard length) and adult (225 and 240 mm) specimens using light and electron microscopy. In 50 mm fish, the scales are imbricated and composed mainly of a succession of orthogonal collagen layers forming a plywood-like structure, the isopedin. The outer surface of the scale is ornamented locally by irregular patches of collagenous material. The layers are not mineralized, whereas the superficial patches are well calcified. The isopedin thickens until it has 12-15 layers and then stops growing (88 mm fish). It mineralizes irregularly from its upper part, and two vascular regions, surrounded by woven-fibered osseous material, form on the outer and deeper surfaces of the isopedin. These regions thicken while the vascular canals close by centripetal deposition of parallel-fibered osseous tissue. The outer region is the superficial part of the mature scale (called here osteodentin), which is covered by the ganoine deposited by the epidermal cells. The deeper part constitutes the definitive basal plate, composed of parallel-fibered osseous tissue. The results show 1) that the young ganoid scales of Polypterus senegalus have a structure similar to that of typical elasmoid scales; and 2) that the isopedin structure does not change during ontogeny and so represents a permanent record of the first ontogenetic stages. The phylogenetic implication of these results is that the elasmoid scales of teleosts arose by a process of paedomorphosis.     

The ultrastructure and calcification of the scales of Tilapia mossambica (Peters)

Summary The scales of Tilapia are surrounded by an envelope of scleroblasts responsible for the production of layers of collagen that constitute the bulk of the scale. The scleroblasts adjoining the lateral face of the oldest scale region gradually atrophy. New collagen layers are deposited against the inner face of the scale, the adjoining scleroblasts showing evidence of high metabolic activity. Calcification occurs by inotropic deposition of crystals alongside the fibres. There is no sharp demarcation between calcified and non-calcified scale regions, a calcification front gradually moving towards newly formed collagen layers. It is felt that fish scales should be considered as calcified derivatives of dermal collagen layers.