SILICATE UPTAKE AND SILICA SHELL FORMATION BY SYNCHRONOUSLY DIVIDING CELLS OF THE DIATOM NAVICULA PELLICULOSA (BRÉB.) HILSE1,2,3

Cells of the freshwater diatom Navicula pelliculosa were induced to divide synchronously by the addition of excess silicate (20 mg Si/liter) to a culture which had been silicate-starved for 14 hr. After a 3- to 4-hr lag period, characterized by rapid silicate uptake (6.5 mg Si/liter/4 hr) and silica shell formation, the cell number then doubled in the succeeding 2-3 hr. This system promises to have significance in the study of silicon metabolism, transport phenomena, and silica deposition.     

STUDIES ON THE BIOCHEMISTRY AND FINE STRUCTURE OF SILICA SHELL FORMATION IN DIATOMS : I. The Structure of the Cell Wall of Cylindrotheca fusiformis Reimann and Lewin

An electron microscope study on the cell wall of the diatom Cylindrotheca fusiformis was carried out using stereoscopic and sectioning techniques. Material prepared by an enzyme treatment or by a mechanical method showed that the wall consists of two major components: a silica shell and organic material. Vapor of hydrofluoric acid was employed to remove the silica and thereby reveal the arrangement of the organic material. An attempt was made to increase the contrast of the organic component by "staining." Uranylacetate not only increased the electron opacity of the organic material but also apparently decreased the electron opacity of the silica shell. In ultrathin sections of complete cells, the structure as revealed by stereoscopy could be confirmed and extended. Every part of the silica shell is tightly enclosed by organic material. In the valve region the silica enclosed in this way is located between other layers of organic material. The whole cell wall is surrounded by a mucilaginous substance which stains with ruthenium red.     

OBSERVATIONS ON THE FINE STRUCTURE OF CALLIXYLON WOOD

Secondary wood of three species of Callixylon of Lower Mississippian age, preserved by three different modes (fusinization, silicification, and phosphatization), have been studied and characterized in detail. Problems of interpretation at the SEM level of permineralized woods, both containing (silicified wood of Callixylon erianum) and essentially lacking (phosphatized wood of C. arnoldii) original organic cell wall material, are analyzed and discussed. In particular, it is concluded that the flat to curved surfaces showing no evidence of apertures, observed in bordered pit pairs, commonly represent pit membrane surfaces. It is accepted, however, that some concave surfaces might be the mineral accretion surfaces of incomplete pit cavity casts as proposed by Leo and Barghoorn (1976). Regions between groups of pits, previously interpreted as crassulae, may be artifacts of preservation. The fusinized wood has the general appearance of charcoal, but unlike commercially produced charcoal provides evidence of its original microfibrillar structure. The origin of fusain in the fossil record is discussed, and it is concluded that it probably had several origins, including forest fire. Since charcoal can be produced naturally in the absence of O₂ (Brown and Davis, 1973), the suggestion that fusain (charcoal) in the geologic column provides a basis for “assessing oxygen levels in paleoatmospheres” (Cope and Chaloner, 1980) is not supported. Natural sites of fusain production in the absence of O₂ are regions of vulcanism and organic sediments inhabited by anaerobic microorganisms. A circular pattern of crystal orientation in the pit borders of C. arnoldii is interpreted to represent the original microfibrillar pattern. Pit apertures in C. arnoldii are shown to be circular to slightly elliptical. Interpretive evidence of heterogeneous pit membranes in C. arnoldii suggests but does not prove the presence of a torus. The distinctive central region in some pit membranes of the fusinized wood of Callixylon sp. might represent accumulations of waste metabolites. It is argued that a torus would be highly adaptive in large pits with circular apertures.     

FINE STRUCTURE OF THE DIATOM AMPHIPLEURA PELLUCIDA. II. CYTOPLASMIC FINE STRUCTURE AND FRUSTULE FORMATION

The general arrangement of cytoplasmic organelles in Amphipleura pellucida Kutz. is similar to that in other naviculoid diatoms. The chromatophores are parietal with a single, non-membrane-limited, pyrenoid. The pyrenoid is crossed by several double-disc lamellar bands which are occasionally interrupted by less dense areas containing convoluted tubules. Similar areas also interrupt the three-disc bands of the chromatophores. The nucleus is irregular in shape. The outer membrane of the porous nuclear envelope outfolds around the chromatophore. A perinuclear dictyosome complex is present. Amorphous dense bodies are formed in elaborations of the dictyosomes. Vesicles, both with and without dense inclusions, are formed by the dictyosomes during cell division and a role is suggested for these vesicles in both cytokinesis and frustule development. The first evidence of frustule formation is the deposition of the siliceous median rib within a membranous sac. This sac expands laterally to form the silica deposition vesicle which appears to serve as a mold for the formation of the valve. After the valve is formed, the membranes and the small amount of cytoplasm external to it are sloughed off.     

ELECTRON MICROSCOPE STUDIES OF EPIDERMAL MELANOCYTES, AND THE FINE STRUCTURE OF MELANIN GRANULES

Melanocytes and melanin granules have been studied by electron microscopy in normal human and cat skin, and in hyperplastic human skin lesions. The melanocytes have always been found as free cells within the epidermis,i.e., on the epidermal side of the dermal membrane. Melanocytes frequently rest on the dermal membrane or bulge towards the dermis. In such cases the uninterrupted dermal membrane is uniformly thin and smooth in appearance, in contrast with the regions alongside Malpighian cells, where it appears appreciably thicker and seemingly anchored to the basal cell layer. Two types of melanin granules have been distinguished according to their location in the melanocytes and to morphological characteristics which may only express different stages in the maturation of the granules: (a) light melanin granules in which a structure resembling a fine network is apparent; (b) dense melanin granules which, in osmium-fixed preparations, appear as uniformly dense masses surrounded by a coarsely granular, intensely osmiophilic shell. Treatment of sections of osmium-fixed tissues with potassium permanganate has revealed within the dense granules the existence of an organized framework in the form of a regular, crystalline-like lattice. It is suggested that this basic structure is protein in nature and may include the enzymatic system capable of producing melanin. The existence is reported of fine filaments located in the cytoplasm of melanocytes and morphologically distinct from the tonofilaments found in Malpighian cells.     

SILICEOUS SCALE PRODUCTION IN CHRYSOPHYTE AND SYNUROPHYTE ALGAE. I. EFFECTS OF SILICA-LIMITED GROWTH ON CELL SILICA CONTENT,SCALE MORPHOLOGY,AND THE CONSTRUCTION OF THE SCALE LAYER OF SYNURA PETERSENII1

The cells of synurophyte flagellates (algal class Synurophyceae, formerly included in the Chrysophyceae) are enclosed within a regularly imbricate layer of ornamented siliceous scales. Scale morphology is of critical taxonomic importance within this group of algae, and the scales are valuable indicator microfossils in paleolimnological studies. The data presented here demonstrate that scale morphology and the integrity of the scale layer can exhibit extreme variability in culture as a function of the cellular quota of silica under silica-limited growth. Silica-limited, steady-state populations of the colonial flagellate Synura petersenii Korsh. were maintained over a range of specific growth rates (μ= 0.11–0.69 days^?1) and silica cell quotas (Q_si= 0.13–2.40 pmoles Si · cell¹). Scale morphology and the organization of the scale layer became increasingly aberrant as silica stress increased. Under severe stress, scale deposition was completely suppressed so that cells appeared scale-free. This depression of scale deposition was reversible; populations of silica-starved, scale-free cells rapidly regenerated new scale layers when placed in batch culture and spiked with dissolved silica. During recovery from silica stress, cell division was repressed for 24 h while mean cell silica quota increased 25-fold. The first new scales appeared within 2 h after the silica addition, and development of the new scale layer proceeded in an approximately synchronous manner, residting in normal scale layers on virtually all cells after 48 h of recovery in Sirich medium. Silica content of silica-replete Synura cells is comparable to freshwater diatoms of siynilar size, but Synura has much greater potential quota variability than diatoms and no apparent threshold silica requirement. Silica-limited growth kinetics and competition between diatoms and Synura for silica are discussed. The results suggest that morphological variability of siliceous scales in natural populations of synurophyte flagellates may result from silica stress and that the experimental approach developed here has great potential value as a means for circumscribing ecotypic variation in scale morphology. Results also demonstrate that scale production can be uncoupled from cell division, suggesting that cell cycle regulation of silica biomineralization in the Synurophyceae may be fundamentally different from that of diatoms (algal class Bacillariophyceae). This experimental system has application in the future study of the intracellular membrane systems and the regulatory processes involved in silica biomineralization.     

LOCATION AND EXPRESSION OF FRUSTULINS IN THE PENNATE DIATOMS CYLINDROTHECA FUSIFORMIS, NAVICULA PELLICULOSA, AND NAVICULA SALINARUM (BACILLARIOPHYCEAE)

A detailed immunocytochemical and biochemical study of the location and expression of frustulins, a family of proteins associated with the frustules of diatoms, has been performed for Cylindrotheca fusiformis Reimann et Lewin, Navicula pelliculosa (Brébisson et Kützing) Hilse, and Navicula salinarum (Grunow) Husted. Immunocytochemistry revealed that frustulins, which share homologous epitopes but are different in size, were predominantly located in the organic casing. Based on timed immunolocalization experiments and Western blotting analysis of cell extracts obtained sequentially after repleting silicate to Si-synchronized cells, the continuous presence of the frustulins in the mature and parental organic casing of the examined species was observed. The frustulins of N. pelliculosa appeared as proteins similar to those of C. fusiformis, sharing identical epitopes. The extractions, however, yielded a markedly lower abundance of frustulins in N. pelliculosa. Peak concentrations of extracted frustulins appeared to be expressed just ahead of the silicification process in C. fusiformis, whereas the level of expression in N. pelliculosa increased along with maturation of the new valves. For N. salinarum, the presence of the frustulins could not be confirmed properly by Western blotting, most probably because of the small sample volumes, inefficient extraction, and a lower amount of homologous frustulins in the casing of this species. It is concluded that the frustulins of these species are not associated with the silicalemma of the newly formed silica deposition vesicles and therefore do not seem to be involved in the silicification process itself. Overall, the results imply a structural role of the frustulins in the casing of diatoms rather than a regulation of the silicification process.     

MORPHOLOGY,FINE STRUCTURE,AND ONTOGENY OF THE STINGING EMERGENCE OF URTICA DIOICA

Stinging emergences in Urtica dioica L. characteristically possess an elongate stinging cell and a multicellular pedestal. The emergence is derived from the epidermal and subepidermal cell layers. The apical wall of the stinging cell is composed of silica bodies which decrease basipetally in concentration. The basal portion of the cell wall of the stinging cell is devoid of silica bodies and lacks primary pit fields or pits between it and the pedestal cells. X-ray microanalysis of electron dense particles located in the stinging cell ER-golgi complex indicate that these particles contain silicon. There is no ultrastructural evidence for the presence of a toxin synthesizing system or a toxin itself.     

THE POLYMORPHIC DIATOM PHAEODACTYLUM TRICORNUTUM: ULTRASTRUCTURE OF ITS MORPHOTYPES1,2

The ultrastructure of the oval, fusiform and triradiate morphotypes of Phaeodactylum tricornutum Bohlin is described. The organization and structure of the cytoplasmic organelles is similar in all three morphotypes, except that the vacuoles occupy the extra volume created by the arms of the fusiform and triradiate cells. The frustule in fusiform and triradiate cells is organic; in the oval type it may be organic or one of the valves may have a silica frustule surrounded by an organic wall. In all cells, the organic cell wall has up to 10 silica bands (13 nm wide) embedded in its surface in the girdle region, lacks girdle bands, and has an outer corrugated cell wall layer, except in the girdle region. Cell division, organic wall formation and silica deposition are described in detail. Four types of oval cells are also described. The relation to other diatoms is discussed.     

THE FINE STRUCTURE OF THE GALL BLADDER EPITHELIUM OF THE MOUSE

Sections of mouse gall bladder epithelium fixed by perfusion with buffered osmium tetroxide have been studied in the electron microscope as an example of simple columnar epithelium. The free surface presents many microvilli, each presenting a dense tip, the capitulum, and displaying a radiating corona of delicate filaments, the antennulae microvillares. Very small pit-like depressions, representing caveolae intracellulares, are encountered along the cell membrane of the microvilli. The free cell surface between microvilli shows larger cave-like depressions, likewise representing caveolae intracellulares, containing a dense material. The lateral cell borders are extensively folded into pleats, which do not interdigitate extensively with corresponding folds of the adjacent cell membrane. The terminal bars are shown to consist of thickened densities of the cell membrane itself in the region of insertion of the lateral cell wall with the free cell surface. This thickening is associated with an accumulation of dense cytoplasmic material in the immediate vicinity. The terminal bar is thus largely a cytoplasmic and cell membrane structure, rather than being primarily intercellular in nature. The basal cell membrane is relatively straight except for a conical eminence near the center of the cell, projecting slightly into the underlying tunica propria. The basal cell membrane itself is overlain by a delicate limiting membrane, which does not follow the lateral contours of the cell. Unmyelinated intercellular nerve terminals with synaptic vesicles have been encountered between the lateral walls of epithelial cells. A division of the gall bladder epithelial cell into five zones according to Ferner has been found to be convenient for this study. The following cytoplasmic components have been noted, and their distribution and appearance described: dense absorption granules, mitochondria, Golgi or agranular membranes, endoplasmic reticulum or ergastoplasm, ring figures, and irregular dense bodies, perhaps lipoid in nature. The nucleus of these cells is also described.     

MUCILAGE-PRODUCING CELLS IN THE SEED COAT OF PLANTAGO OVATA: DEVELOPMENTAL FINE STRUCTURE

As the ovule of Plantago ovata matures into a seed its epidermal cells are transformed from undifferentiated parenchyma to thin-walled containers of almost pure mucilage. During this process the volume of the cells increases 60–80 fold, and the protoplast degenerates to a remnant. Rapid cell expansion begins with pollination and is accompanied by an increase in the size of the nucleus and nucleolus, a change in the random arrangement of ribosomes, a decrease in the thickness of cell walls, and synthesis of starch. Deposition of mucilage inside vacuoles and between the plasma membrane and cell wall accompanies a marked increase in the number and size of Golgi vesicles. Histochemical evidence using the thiocarbohydrazide-osmium vapor method shows polysaccharide to be present within Golgi vesicles while they are still attached to the Golgi apparatus. Mucilage deposition is associated with further cell expansion, separation of the protoplast from the cell wall, fusion of vacuoles and extra protoplasmic space, and the disappearance of starch.     

Fine Structure of Silica Deposition and the Origin of Shell Components in a Testate Amoeba Netzelia tuberculata1

ABSTRACT Netzelia tuberculata secretes a test composed of siliceous particles cemented together by organic plaques forming a single-layered spheroidal shell. The siliceous particles are produced within cytoplasmic vacuoles by three mechanisms: 1) synthesis de novo by deposition of the silica on a matrix; 2) deposition of silica on particles remaining in digestive vacuoles, including starch grains and undigested walls of yeast cells; and 3) secretion of silica as a hollow sphere at the periphery of vacuoles enclosed by the silicasecreting membrane. The silicalemma (silica-secreting membrane) originates as fibril-containing vesicles (GFV) secreted by the Golgi body. Fusion of these vesicles with membranes surrounding digestive vacuoles or with membranes surrounding specialized vacuoles containing a silica-binding matrix apparently converts the vacuole into a silica-depositing organelle. Small spherules of silica occur on the vacuolar side of the membrane surrounding the developing test granules, marking the presence of silicalemma activity. These colloidal spherules become aggregated into larger spherules that condense to form the siliceous surface of the developing test particle. Other Golgi vesicles, designated Golgi plaque vesicles (GPV), produce the organic plaques that are deposited among the siliceous particles at the periphery of the cell during new test construction during cell division. The fine structure of the GFV and GPV and their role in test wall deposition are discussed in relation to other silica-biomineralizing protozoa, including radiolaria.     

STRUCTURE OF THE PERIPLAST OF CRYPTOMONAS OVATA VAR. PALUSTRIS1,2

The periplast of Cryptomonas ovata var. palustris is composed of polygonal plates which are delineated by shallow ridges. A small ejectosome is located at each corner of the plate area. The plate areas vary in size; they are smallest at the anterior and posterior ends and are largest in the middle of the cell with an average length of 0.5 μ and of width 0.4 μ. In cross section a plate area is composed of 2 distinct layers, an outer plasma membrane layer with a fine particulate, appearance, and an inner layer consisting of two sheets. The sheets of the inner layer have a striated lattice pattern with a periodicity of about 20 nm. In negatively stained preparations one lattice appears to underlie another at certain angles. Protease digestion removed polygonal shaped inner layer.     

THE FINE STRUCTURE AND SCALE FORMATION OF CHRYSOLEPIDOMONAS DENDROLEPIDOTA GEN. ET. SP. NOV.(CHRYSOLEPIDOMONADACEAE FAM. NOV., CHRYSOPHYCEAE)1

Chrysolepidomonas gen. nov. is described for single-celled monads with two flagella, a single chloroplast, and distinctive canistrate and dendritic scales. The type species, Chrysolepidomonas dendrolepidota sp. nov., is described for the first time. The canistrate scales bear eight “bumps” on the top surface, and the dendriticscales have a tapered base with a quatrifid tip. These organic scales are formed in the Golgi apparatus and storred in a scale reservoir. The scale reservoir is bounded on two sides by the R₁ and R₂ in microtubular roots of the basal apparatus. The cyst (=stomatocyst, statospore) forms endogenously by means of a silica deposition vesicle. The outer cyst surface is smooth, and the pore region is unornamented. Two other organisms bearing canistrate and dendritic scales, previously assigned to the genus Sphaleromants, are transferred to the genus Chrysolepidomonas. They are C.angalica sp. nov. and C. marine(Pienaar) comb. nov. The distinguishing features of Chrysolepidomonas and Sphaleromantis are discussed. A new family, Chrysolepidomonadceae fam. noc., is described for flagellates covered with organic scales.     

BIOCHEMICAL TAXONOMY OF MARINE PHYTOPLANKTON BY ELECTROPHORESIS OF ENZYMES. I. THE CENTRIC DIATOMS THALASSIOSIRA PSEUDONANA AND T. FLUVIATILIS1,2

Diatom systematics depends almost entirely upon structure of the silica shell. It is not known to what extent the taxonomic species, as defined by shell structure, corresponds to the genetic species—i.e., to the reproductively isolated population. As an approach to this problem, we report here a comparison of enzymes by electrophoresis. We have examined the genetic constitution of a number of clones of (presumably) the same species for each of 2 closely related, centric diatom species: Thalassiosira pseudonana Hasle and Heimdal and T. fluviatilis Hustedt. The 4 clones of T. fluviatilis form a distinct group, clearly separated from all the T. pseudonana clones. Within T. pseudonana, 4 estuarine clones and one reef clone form a group that is distinctly different from 4 oceanic clones. A single clone of T. pseudonana from the Continental Slope waters is intermediate between these 2 groups and probably shares genes with both groups, indicating that the 2 T. pseudonana groups are not genetically isolated. We conclude that i) within groups, isolates are closely related even though they originated from different continents; and, ii) T. pseudonana is subdivided into ecological races.     

THE FINE STRUCTURE OF THE FLAGELLAR APPARATUS OF CARTERIA1,2

The fine structure of the flagellar apparatus of 5 species of the green quadriflagellate alga Carteria is described. The 5 species can be morphologically separated into 2 groups on the bases of cell shape and ultrastructure of the pyrenoid and flagellar apparatus. Group I cells are spherical, possess many pyrenoid thylakoids, and retain a flagellar apparatus similar to that of Chlamydomonas reinhardi. The flagellar bases are oriented at approximately 90° to one another, have distal and proximal fibers, and are associated with 4 cruciately arranged microtubule bands. Cells of group II are ellipsoid, possess few pyrenoid thylakoids, and show a complex system of microtubule bands and sigmoid-shaped, electron dense rods which extend between opposite pairs of basal bodies. The basal bodies of group II cells are directed inward in a circular pattern rather than outward as in group I cells. Unlike Chlamydomonas, the distal fiber of the Carteria species is nonstriated. The proximal fiber is striated, and both distal and proximal fibers are composed of 60–80 Å diameter microfibrils.     

TISSUE PREPARATION AND FINE STRUCTURE OF THE RADICLE APEX FROM COTTON SEEDS

A comparative methodological study was made of the fine structure of apical cortical cells in excised radicles from cotton (Gossypium hirsutum L. var M-8) seeds. Radicles from dry seed had 12% moisture content and were prepared for electron microscopy using several different techniques. These included different methods of chemical fixation or freeze-fracture and etching of unfixed tissue for transmission electron microscopy (TEM) and cryofracturing of fixed and dehydrated radicles for scanning electron microscopy (SEM). Cortical cells had a similar appearance regardless of the method used in tissue preparation. Cell walls had a pronounced waviness which was particularly evident in SEM images of cells lining the elongated intercellular air spaces. The plasma membrane (PM) delimited the cytoplasm of each cell as an intact unit membrane. Single layers of tightly-packed lipid bodies (LB) were apposed to the PM and protein bodies (PB). Distension of cells, membranous organelles and LB was observed in radicles fixed by immersion in aqueous solutions, suggesting that a certain amount of hydration occurred during fixation. This interpretation was supported by the compact appearance of cells and organelles in tissue prepared by freeze-etch or vapor fixation. We conclude that freeze-fracture and etching of unfixed tissue provided the best information for cell morphology and structure of membranes and organelles in dry tissue. Complementary data on the fine details of nuclei and cytoplasmic organelles were best observed with TEM of fixed tissue. These data when viewed collectively indicate the advantage of using several techniques to obtain analogous and complementary information essential for establishing a baseline level of information on the fine structure of cells in dry tissue.     

OBSERVATIONS ON THE STRUCTURE OF SOME FORMS OF GOMPHONEMA PARVULUM KÜTZ. III. FRUSTULE FORMATION1

Gomphonema parvulum Kütz. was investigated by electron microscopy for details of frustule formation. An expansion of the cell along the pervalvar plane occurs prior to cell division. After nuclear division the organelles are, separated into 2 entities, either by division or by dispersion. The cell divides into 2 halves by the invagination of the plasmalemma which is derived from Golgi vesicular activity. When cytoplasmic cleavage, is complete, the Golgi actively produces electronlucent vesicles which collect and coalesce beneath the. plasmalemma to form the silicalemma around the silicon deposition vesicle. The endoplasmic reticulum is also closely associated with this vesicular activity. The vesicle gradually expands and becomes extremely electron dense as silica is deposited within it—first in the region, followed by the mantle edge. When the valve is mature, Golgi vesicles collect and fuse to form the silicalemma of the first girdle band. The first girdle band becomes aligned against the mantle edge on completion, by the “sloughing off” of the external silicalemma and plasmalemma. The second and third bands are formed, individually in a similar manner. Separation of the 2 daughter cells commences at the apical pole and progresses to the basal pole. The plasmalemma and external silicalemma are “sloughed off” so that the 2 cells can separate. The inner segment of the silicalemma becomes the new plasmalemma of the daughter cell.     

Selective isolation of bacteria from soil with hydrophobic materials

Bacterial strains having a hydrophobic cell surface have often been considered as degraders of hydrophobic organic pollutants in soil. In this study, bacterial strains were isolated using hydrophobic materials from 12 soil samples, and their cell surface hydrophobicity was determined by evaluating their adherence to n-hexane. Bacterial strains isolated using polytetrafluoroethylene (PTFE) membrane were more hydrophobic on an average than those isolated with styrene–divinylbenzene (DVB) particles or octadecylsilyl silica gel (ODS) particles. Strains closely related to Burkholderia cepacia could be selectively isolated using the PTFE membrane; those closely related to Ralstonia pickettii, using ODS and DVB particles; and those closely related to B. fungorum, using DVB. These results indicate that bacterial strains having a hydrophobic cell surface or within certain phyla can be selectively isolated from soils using hydrophobic materials, and that this isolation method would be useful for collecting candidates for bioremediation of hydrophobic pollutants.