MICROMORPHOGENESIS DURING DIATOM WALL FORMATION PRODUCES SILICEOUS NANOSTRUCTURES WITH DIFFERENT PROPERTIES1

Micromorphogenesis within the silica deposition vesicle (SDV) of the diatom Pinnularia viridis (Nitzsh) Ehrenb. resulted in distinct silica nanostructures and layers within forming valves and girdle bands. These siliceous components were similarly disclosed following alkaline etching of mature valves/girdle bands, where their different susceptibilities to dissolution over time resulted from apparent differences in silica density and/or chemistry. The bulk of silica appeared to be deposited at the interface of the forming valve or girdle band with the silicalemma and occurred by the outward expansion of microfibrils of silica that aligned perpendicularly to the silicalemma. Microfibrils originated from both sides of the “silica lamella,” the first nanostructure formed within the SDV, and several silica species of distinct nanostructure and density resulted, including distinctive inner and outermost silica “coverings” of mature valves/girdle bands and the central and terminal nodules. Not all silica deposition and micromorphogenesis occurred in contact with the expanding silicalemma, but was somehow directed within the SDV cavity, and resulted in the distinct silica layers that lined the raphe fissures and poroids. Following alkaline etching, the inner surfaces of valves/girdle bands, as well as the silica layers lining the raphes, poroids, and slits, were determined to be significantly more resistant to alkaline etching than the exterior surfaces, while the outer silica coating and the nodules were quickly dissolved. The processes of micromorphogenesis must have exerted precise control over the chemical nature of the silica formed at different positions within the SDV and affected the overall structure and function of the diatom wall.     

Studies on the biochemistry and fine structure of silica shell formation in diatoms

Summary The sequential wall formation in the centric diatom,Ditylum brightwellii (West) Grunow, is described. The silica deposition vesicle is formed by the coalescence of small vesicles. Silicification of the new valve starts from the central labiate process area prior to the completion of cytokinesis, and the developing valve grows in a centrifugal direction. The initiation of the structures on the valve follows the sequence: labiate process, marginal ridge, and rota. A novel labiate process apparatus, which is situated in the cytoplasm close to the developing labiate process, appears prior to the initiation of the labiate process and disappears upon its maturation. Segments of the girdle bands are formed in individual silica deposition vesicles after the valve matures and is exocytosed. Three morphological forms of deposited silica have been determined: thin base layers, microfibrils, and hexagonal columns. The involvement of cytoplasmic structures in the patterning of the siliceous wall is discussed.     

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.     

Valve morphogenesis in Diploneis smithii (Bacillariophyta)

Diploneis species have perhaps the most complex valve structure among pennate diatoms. The development of this structure was studied in Diploneis smithii and begins with the formation of a primary band, which then develops secondary arms at both poles and the center, as in the classic Chiappino–Volcani model of raphid diatom ontogeny. Spine‐like projections grow out from the primary band and secondary arms to establish the transapical ribs (virgae) of the mature valve and themselves develop spines, which are spaced first oppositely and then alternately and fuse with each other to delimit the stria pores. Subsequently, new pattern and structures develop both externally (formation of bifurcating projections that fuse to delimit the outer, sieve‐like layer of the valve) and internally (growth and fusion of flanges from the first‐formed ribs to create the longitudinal canals and deposition of a hymenate strip over the internal face of each stria). Comparisons are made with morphogenesis in other diatoms. Diploneis smithii ontogeny suggests how very slight developmental changes might have created the very variable external morphology of Diploneis species. It also indicates that the longitudinal canals of Diploneis and Fallacia have different origins, since the porous external wall is not formed as a unilaterally attached flap in Diploneis and the canal is internal to the first‐formed rib–stria system in Diploneis, but external to it in Fallacia.     

Filament formation in the diatomSkeletonema costatum

Summary Skeletonema costatum consists of cells joined by open gutter-like connecting spines (strutted tubuli) which form from the margin of the valve face. The intercellular spaces occupied by tubuli are alternately enclosed by finely perforate bands of wall material. The intercellular bands are in the form of overlapping, open ended cylinders attached to the epitheca in the region of the cell girdle. A model is proposed for filament formation explaining the origin of alternating walled and unwalled intercellular spaces.     

Morphological investigations of the frustule, perizonium and initial valves of the freshwater diatom Achnanthes crenulata Grunow (Bacillariophyceae)

The present study clarifies the fine structure of the vegetative frustules, initial valves and perizonium of Achnanthes crenulata Grunow. The valves of the vegetative cell are distinctly linear‐lanceolate with an undulate margin. The valve face is quite flat and in girdle view is smoothly curved as in species of Gephyria (Bacillariophyceae). However, the valve face of the initial cells is slightly rounded and does not have an undulate margin. Furthermore, the rapheless sternum is centrally positioned along the apical axis of the araphid initial valve. As this taxon develops from auxospore to initial valve, it forms only longitudinal perizonial bands; no transverse bands arise. The perizonium consists of three silicified bands: one large, central longitudinal plate and two bands that underlie this plate; these two bands are either open or closed. This taxon has several conspicuous structures compared to other marine species of Achnanthes, but the structure of the perizonium supports the position of A. crenulata within Achnanthes sensu stricto.     

Observations on girdle bands during cell division in the diatom Stephanodiscus

The fate of old girdle bands and the formation of new bands in the centric diatom Stephanodiscus are discussed. The use of scanning electron microscopy is a great aid in such studies since it enables observation of complete cells and determination of the position and overlap of girdle bands. It appears that both parent girdle bands are lost during cell division and two new bands formed around each daughter cell.     

OVERLOOKED HETEROPOLARITY IN SURIRELLA CF. FASTUOSA (BACILLARIOPHYTA) AND RELATIONSHIPS BETWEEN VALVE MORPHOGENESIS AND AUXOSPORE DEVELOPMENT

Surirella cf. fastuosa is an apparently isopolar elliptic marine raphid diatom. We observed cells before and after sexual reproduction in monoclonal cultures using light and scanning electron microscopy (LM and SEM). After sexual reproduction cells were approximately twice as large as before, in valve length and width. The stria and infundibula densities were stable during the life cycle. Subtle morphological differences were detectable between the two poles of the frustule. One pole (pole A) was characterized by endings of the external raphe fissure that turned toward the valve face, continuity of the domed wall of the raphe canal externally, an elliptic chamber visible internally, a shallow nick in the interior of the valvocopula. The other pole (pole B) was with the following: straight endings of the external raphe fissures, a dent in the domed wall of the raphe canal externally, a double chamber internally, presence of the open ends of the valvocopula nearby, a deep nick in the valvocopula. Furthermore, at pole A virgae developed at an early stage in morphogenesis, whereas at pole B they were not formed. In the auxospores, pole A was situated beneath the primary transverse perizonial band. Pole A is suggested to be homologous with the head pole in heteropolar Surirella and is the “protopole” likely equivalent to the central nodule in naviculoid taxa. Pole B is homologous with the foot pole in heteropolar Surirella and is the “synaptic pole” formed by fusion of components equivalent to both poles of naviculoid taxa.     

QUANTITATIVE NANOMECHANICAL MAPPING OF MARINE DIATOM IN SEAWATER USING PEAK FORCE TAPPING ATOMIC FORCE MICROSCOPY1

It is generally accepted that a diatom cell wall is characterized by a siliceous skeleton covered by an organic envelope essentially composed of polysaccharides and proteins. Understanding of how the organic component is associated with the silica structure provides an important insight into the biomineralization process and patterning on the cellular level. Using a novel atomic force microscopy (AFM) imaging technique (Peak Force Tapping), we characterized nanomechanical properties (elasticity and deformation) of a weakly silicified marine diatom Cylindrotheca closterium (Ehrenb.) Reimann et J. C. Lewin (strain CCNA1). The nanomechanical properties were measured over the entire cell surface in seawater at a resolution that was not achieved previously. The fibulae were the stiffest (200 MPa) and the least deformable (only 1 nm). Girdle band region appeared as a series of parallel stripes characterized by two sets of values of Young’s modulus and deformation: one for silica stripes (43.7 Mpa, 3.7 nm) and the other between the stripes (21.3 MPa, 13.4 nm). The valve region was complex with average values of Young’s modulus (29.8 MPa) and deformation (10.2 nm) with high standard deviations. After acid treatment, we identified 15 nm sized silica spheres in the valve region connecting raphe with the girdle bands. The silica spheres were neither fused together nor forming a nanopattern. A cell wall model is proposed with individual silica nanoparticles incorporated in an organic matrix. Such organization of girdle band and valve regions enables the high flexibility needed for movement and adaptation to different environments while maintaining the integrity of the cell.     

A preliminary list of sublittoral marine algae from the west of Scotland

The valves and girdle bands of Melosira varians C. A. Agardh have been shown to possess greater detail than had been supposed from light microscopy. Spines, pores and projections on the surface of the loculate valve are described, and the precise relationships of the girdle bands to each other and to the valves have been elucidated.     

CELL DIVISION AND MORPHOGENESIS OF THE CENTRIC DIATOM CHAETOCEROS DECIPIENS (BACILLARIOPHYCEAE) II. ELECTRON MICROSCOPY AND A NEW PARADIGM FOR TIP GROWTH

Valve morphogenesis starts when the silica deposition vesicle (SDV) expands across a cleavage furrow covered by an unidentified layer, which may aid in its shaping. A labiate process (LP) is present only in the outer valve of terminal cells in the filament. Before these particular cells form setae, a layered "labiate process apparatus" (LPA) appears on the SDV in the exact center of the forming valve, near the microtubule center arising after cleavage. The LPA thereafter surmounts the lips of the LP as it forms. After the girdle bands separate slightly, two lateral protrusions develop in the corners of the cell. These nascent setae are lined internally by a cylindrical, fibrous band (sleeve), which assembles immediately ahead of the expanding edge of the SDV, very close to the plasmalemma. Then these protrusions, lined by the fibrous band, the SDV, and the forming silica wall, grow through two gaps in the girdle bands. The cytoplasm at the tip of the growing seta is naked. Immediately behind the tip, this fibrous band is adpressed to the plasmalemma and thereby apparently defines the diameter of the seta; it extends to internally ensheath the tipmost edge of the SDV for a short distance, like a tight-fitting inner sleeve. This structure is considered the major organelle involved in seta morphogenesis. Microtubules (MTs), while present, are variable in extent and disposition within the seta. Turgor pressure is considered irrelevant in driving seta growth. Instead, a new paradigm proposed for tip-growing cells generally, may apply to seta morphogenesis, as follows. If, as is suspected, the fibrous band contains actin, cycling of this actin (as in animal cells undergoing ruffling or filopodial extension) could drive seta extension via attachment of the band to the just-formed silica wall. The band is visualized as a molecular treadmill whose support base, the new wall, is being continually extended; extension is controlled and generated strictly at the tip.     

NANOSTRUCTURE OF THE DIATOM FRUSTULE AS REVEALED BY ATOMIC FORCE AND SCANNING ELECTRON MICROSCOPY

The cell wall (frustule) of the freshwater diatom Pinnularia viridis (Nitzsch) Ehrenberg is composed of an assembly of highly silicified components and associated organic layers. We used atomic force microscopy (AFM) to investigate the nanostructure and relationship between the outermost surface organics and the siliceous frustule components of live diatoms under natural hydrated conditions. Contact mode AFM imaging revealed that the walls were coated in a thick mucilaginous material that was interrupted only in the vicinity of the raphe fissure. Analysis of this mucilage by force mode AFM demonstrated it to be a nonadhesive, soft, and compressible material. Application of greater force to the sample during repeated scanning enabled the mucilage to be swept from the hard underlying siliceous components and piled into columns on either side of the scan area by the scanning action of the tip. The mucilage columns remained intact for several hours without dissolving or settling back onto the cleaned valve surface, thereby revealing a cohesiveness that suggested a degree of cross-linking. The hard silicified surfaces of the diatom frustule appeared to be relatively smooth when living cells were imaged by AFM or when field-emission SEM was used to image chemically cleaned walls. AFM analysis of P. viridis frustules cleaved in cross-section revealed the nanostructure of the valve silica to be composed of a conglomerate of packed silica spheres that were 44.8 ± 0.7 nm in diameter. The silica spheres that comprised the girdle band biosilica were 40.3 ± 0.8 nm in diameter. Analysis of another heavily silicified diatom, Hantzschia amphioxys (Ehrenberg) Grunow, showed that the valve biosilica was composed of packed silica spheres that were 37.1 ± 1.4 nm and that silica particles from the girdle bands were 38.1 ± 0.5 nm. These results showed little variation in the size range of the silica particles within a particular frustule component (valve or girdle band), but there may be differences in particle size between these components within a diatom frustule and significant differences are found between species.     

Electron microscopical observations onPseudaphelidium drebesii Schweikert and Schnepf, a parasite of the centric diatomThalassiosira punctigera

Summary Ultrastructural observations revealed details of the infection process and the fine structure ofPseudaphelidium drebesii Schweikert and Schnepf, a parasite of the marine centric diatomThalassiosira punctigera (Castracane) Hasle. After attachment and encystment of the zoospore an intracellular infection tube is generated. This structure is everted between the overlap of the girdle bands or the gap between the valve and the girdle band of the host diatom. The bulk of thePseudaphelidium protoplast enters the silica shell of the diatom via the infection tube but does not pierce the host plasma membrane. Parts of the host cytoplasm are phagocytized with microfilament involvement.Pseudaphelidium drebesii is characterized by unusual microbodies containing tubular inclusions and by closed mitosis with a perinuclear spindle. The taxonomic position ofP. drebesii is discussed.     

WALL MORPHOGENESIS IN COSCINODISCUS WAILESII GRAN AND ANGST. I. VALVE MORPHOLOGY AND DEVELOPMENT OF ITS ARCHITECTURE1

The morphology of the valve of Coscinodiscus wailesii and the development of its siliceous architecture, studied in the SEM and TEM, is compared with valve formation in Thalassiosira eccentrica (Ehrenberg.) Cleve. Though the areolae-architecture of these two species differs in such that the cribrum is proximal and the foramen distal in T. eccentrica, and in C. wailesii the cribrum is distal and the foramen proximal, the formation of their complex loculate system is similar, displacing a centrifugal growth pattern with respect to the valve, and a proximal to distal, sequentially. During base layer formation a hitherto undescribed rib system outlines the prospective areolae. The vertical differentiation is in principle the same as in T. eccentrica and also the cribra are formed centripetally in relation to the areolae in both species. The location of the cribra at the proximal or distal side, therefore, seems to be of minor importance for the sequence of silica deposition. Variation in girdle bands is discussed with respect to cell division. The prophasic nuclear migration from the interphase position to the girdle bands, where mitosis is performed, seems to be necessary for triggering the formation of the unilateral cleavage furrow that later forms a cleavage ring with excentric position. The divided nuclei migrate with the ingrowing cleavage furrow to the center of the newly created protoplasmic surfaces to initiate valve formation.     

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.     

VALVE AND BAND MORPHOLOGY OF SOME FRESHWATER DIATOMS. III. PRE- AND POST-AUXOSPORE FRUSTULES AND THE INITIAL CELL OF MELOSIRA ROESEANA1

Frustules of a clonal culture of Melosira roeseana Rabenh. were examined with light and scanning electron microscopy. Vegetative valves in the post-auxospore (full size) stage exhibit a larger width/length ratio than those in the pre-auxospore (size-reduced) stage. Cells form chains by linking spines of adjacent valves which occur at the periphery of the valve face-mantle junction. Three or jour large pores occur at the center of the valve face, with the diameter of each pore tapering from the inner to the outer valve surface; these pores are often occluded by siliceous processes. Features of M. roeseana, not shown previously for Melosira, include a “stepped” mantle, on only one of the two valves resulting from the same cell division, flattened processes attached to short siliceous stalks on the valve face, disk-like processes on the mantle, and an open girdle band with up to eight antiligulae. Siliceous scales on the surface of the initial cell are remnants of the auxospore wall. The epivalve of the initial cell is larger in diameter than the hypovalve, and both valves lack linking spines and a step on the valve surface. The initial, cell epicingulum consists of only two bands; the hypocingulum has up to seven. Initial cells with four or more hypocingular bands divide to form new post-auxospore filaments. Melosira roeseana should not be included in the genus Melosira as it is presently defined by the type species, M. nurnmuloides C. Ag. Major differences include irregular linking spines, a closed pseudoloculate valve construction, and labiate processes on the valve face and mantle of M. nummuloides, compared with well-defined linking spines, a valve constructed of a basal siliceous layer perforated by poroid areolae, and labiate processes lacking on the valve of M. roeseana.     

Frustule morphogenesis of raphid pennate diatom <Emphasis Type="Italic">Encyonema ventricosum</Emphasis> (Agardh) Grunow

Diatoms stand out among other microalgae due to the high diversity of species-specific silica frustules whose components (valves and girdle bands) are formed within the cell in special organelles called silica deposition vesicles (SDVs). Research on cell structure and morphogenesis of frustule elements in diatoms of different taxonomic groups has been carried out since the 1950s but is still relevant today. Here, cytological features and valve morphogenesis in the freshwater raphid pennate diatom Encyonema ventricosum (Agardh) Grunow have been studied using light and transmission electron microscopy of cleaned frustules and ultrathin sections of cells, and scanning electron and atomic force microscopy of the frustule surface. Data have been obtained on chloroplast structure: the pyrenoid is spherical, penetrated by a lamella (a stack of two thylakoids); the girdle lamella consists of several short lamellae. The basic stages of frustule morphogenesis characteristic of raphid pennate diatoms have been traced, with the presence of cytoskeletal elements near SDVs being observed throughout this process. Degradation of the plasmalemma and silicalemma is shown to take place when the newly formed valve is released into the space between sister cells. The role of vesicular transport and exocytosis in the gliding of pennate diatoms is discussed.     

SCALY INCUNABULA,AUXOSPORE DEVELOPMENT,AND GIRDLE POLYMORPHISM IN SELLAPHORA MARVANII SP. NOV. (BACILLARIOPHYCEAE)1

Uniparental auxosporulation was observed in a monoclonal culture of a Sellaphora clone isolated from the epipelon of a fishpond in the Czech Republic. The cox1 sequence for the clone confirmed that it belonged to the Sellaphora pupula–bacillum species complex but showed significant differences from all previously characterized Sellaphora species, and it is therefore described as S. marvanii sp. nov. Protoplast, valve, and girdle structure resembled those of other Sellaphora species, but a novel finding for all diatoms was a change in girdle structure during the life cycle: the most advalvar girdle band (valvocopula) bore a single line of pores in enlarged postauxospore cells but was entirely plain in small cells and gametangia. The young auxospores were covered by incunabula containing large, delicate, ± circular scales, resembling those of centric diatom auxospores; similar scales have been reported in a few other raphid diatoms (Pseudo‐nitzschia multiseries, Diploneis sp.) but contrast with the strip incunabula of some Nitzschia and Pinnularia and the helmet‐like caps of Neidium. The scales persisted during auxospore expansion, mostly as two caps over the auxospore poles. The transverse perizonium comprised a very wide, closed primary band, flanked by numerous secondary bands whose open ends were strongly incurved toward the center. Initial valves were differentiated from their immediate descendants by the very strong external demarcation of the raphe sternum, irregular shape, and curved transapical profile.     

The Fine Structure of the Cinctum of Ellobiophrya conviva (Ciliophora: Peritricha)

Ellobiophrya conviva clasps tentacles of the bryozoan Bugula neritina with a ring-like structure formed from aboral extensions of its body that taper into two slender arms. The tips of the arms overlap and join to form a unique organelle, the bouton. Each arm contains a massive myoneme that splays out at the bouton. The bouton consists of the cupped tips of the arms and a cavity, which is filled with dense homogeneous material. Long digitations containing longitudinal microtubules at their periphery project from the inner surface of the tip of each arm into the cavity. Deep folds of pellicle with pores opening into their depths line the wall of the cavity. Conventional kinetosomes are not visible in the bouton, but circular or elliptical arrays of microtubules are found at the bases of digitations. The nonfunctional scopula of the adult is in a depression enclosed by pellicular folds. The bouton is distant from the scopula, but its fine structure somewhat resembles it, supporting Chatton and Lwoff's hypothesis that the cinctal arms carry parts of the scopula at their tips. The fine structure of the cinctum supports their suggestion that the cinctal arms are homologous to the spasmonemes of vorticellid peritrichs.