Girdle Bands Research Articles

QUANTITATIVE NANOMECHANICAL MAPPING OF MARINE DIATOM IN SEAWATER USING PEAK FORCE TAPPING ATOMIC FORCE MICROSCOPY(1).

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.

Infiltration of Silica Inside Fibrillar Collagen

Diatoms frustules are created under the control of biomolecules (silaffins, silacidins and long-chain polyamines) at close to physiologic conditions.[1–4] The mechanism of biosilica formation was traditionally based on the ability of zwitterionic water-soluble proteins to create macromolecular assemblies for silica polymerization.[5–7] Recent discoveries of water-insoluble collagen matrices within certain sponge biosilica spicules[8], chitin-based scaffolds in sponge and diatom biosilica formations[9,10], as well as cingulins within diatom girdle bands[4], revive the use of insoluble biomimetic organic templates for morphogenesis of non-porous silica structures. The use of fibrillar collagen as templates for biosilica synthesis was unsuccessful in the past as only extrafibrillar silica deposition was observed.[11,12] Intrafibrillar mineralization of collagen has important implications from a biophysical perspective.[13] Here, we report a collagen biosilicification scheme based on fusion of stabilized polysilicic acid into a fluidic precursor phase upon their infiltration into polyamine-enriched collagen. The latter serves as a template and catalyst for polymerization of the precursor phase into silica that faithfully reproduces the collagen tertiary architecture. Our findings provide a new concept in biosilica materials synthesis which does not require phosphate supplements.

Tabellaria vetteri, a New Diatom (Bacillariophyceae: Tabellariaceae) from Pennsylvania, USA.

ABSTRACT. A new diatom species, Tabellaria vetteri, is described from North-Central Pennsylvania. Unlike other representatives of the genus Tabellaria Ehrenberg, the new species lacks rimoportulae, the central part of its valve is internally thickened, and all girdle bands are open. Other characters, such as shape of the frustule, numerous ligulate girdle bands bearing septa, uniseriate striae with small poroid areolae, large apical pore fields, and simple spines fit the current concept of this genus. The new species is found so far only in two small soft-water streams and is especially abundant on sand.

Numerical Simulations of Drilling Mud Invasion into the Marine Gas Hydrate-Bearing Sediments

When drilling through the oceanic gas hydrate-bearing sediments, the water-based mud under overbalanced drilling condition will invade into the borehole sediments. The invasion behavior can influence the hydrate stability, wellbore stability and well logging evaluation. In this work, we performed the numerical simulations to study the effects of density (i.e., corresponding pressure), temperature and salinity of mud on the mud invasion and hydrate stability around borehole. The results show that the mud invasion will promote greatly the hydrate dissociation near wellbore sediments if the temperature of mud is higher than that of hydrate stability. Under certain conditions, the higher mud density, temperature and salinity, the greater degree of mud invasion and heat transfer, and the more hydrate dissociation. The gas produced from hydrate dissociation can reform hydrates again in the sediments, and even the hydrate saturation is higher than that in situ sediments due to the displacing effect of the mud invasion, which forms a high-saturation hydrate girdle band around the borehole.

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.

A description of the frustular morphology of Frustulia creuzburgensis (Krasske) Hustedt, with comments on its systematic position

Frustulia creuzburgensis was originally assigned to the genus Navicula and later transferred to Frustulia due to its unilaterally deflected external proximal raphe ends resembling those of F. weinholdii. Unlike most members of the genus, F. creuzburgensis is found in saline waters, and has been reported from Europe, North America, South Africa and Hawaii. Light and scanning electron microscopy of F. creuzburgensis from coastal streams in California indicate the presence of a ‘porte-crayon’ arrangement around the external distal raphe ends, a feature used to diagnose Frustulia. However, there is also a lack of ribs bordering the raphe system, which is inconsistent with other members of Frustulia. A notch in the girdle bands at about mid-valve, accompanied by girdle band protuberances, is described for the first time in this species. Populations of this taxon produce both cis and trans frustules in a 1:2.5 proportion, respectively. Enlarged external areolae openings near the external proximal raphe ends are also described for the first time. The possible relationships of this enigmatic taxon amongst close relatives and within the Naviculales is discussed.

Nanopatterned protein microrings from a diatom that direct silica morphogenesis

Diatoms are eukaryotic microalgae that produce species-specifically structured cell walls made of SiO(2) (silica). Formation of the intricate silica structures of diatoms is regarded as a paradigm for biomolecule-controlled self-assembly of three-dimensional, nano- to microscale-patterned inorganic materials. Silica formation involves long-chain polyamines and phosphoproteins (silaffins and silacidins), which are readily soluble in water, and spontaneously form dynamic supramolecular assemblies that accelerate silica deposition and influence silica morphogenesis in vitro. However, synthesis of diatom-like silica structure in vitro has not yet been accomplished, indicating that additional components are required. Here we describe the discovery and intracellular location of six novel proteins (cingulins) that are integral components of a silica-forming organic matrix (microrings) in the diatom Thalassiosira pseudonana. The cingulin-containing microrings are specifically associated with girdle bands, which constitute a substantial part of diatom biosilica. Remarkably, the microrings exhibit protein-based nanopatterns that closely resemble characteristic features of the girdle band silica nanopatterns. Upon the addition of silicic acid the microrings become rapidly mineralized in vitro generating nanopatterned silica replicas of the microring structures. A silica-forming organic matrix with characteristic nanopatterns was also discovered in the diatom Coscinodiscus wailesii, which suggests that preassembled protein-based templates might be general components of the cellular machinery for silica morphogenesis in diatoms. These data provide fundamentally new insight into the molecular mechanisms of biological silica morphogenesis, and may lead to the development of self-assembled 3D mineral forming protein scaffolds with designed nanopatterns for a host of applications in nanotechnology.

Separation of diatom valves and girdle bands from Coscinodiscus diatomite by settling method

Diatom valves and girdle bands are useful micro–nano materials in nanotechnology and micro manufacturing. A settling method is used to extract high-purity diatom valves and girdle bands from Coscinodiscus diatomite. The average models of diatom valves and girdle bands are established. Stokes Law and hydrokinetic theories are used to analyze the settling velocity of valves and girdle bands. Based on the calculation results, settling experiments are carried out, by which clean diatom valves with purity of 80% and girdle bands with purity of 90% are obtained. This method can be applied to other particles separation problem to separate micro-particles with similar radius but different sectional area.

Valve and girdle band morphogenesis in a bipolar centric diatom Plagiogrammopsis vanheurckii (Cymatosiraceae, Bacillariophyta)

Valve and girdle band morphogenesis in a bipolar centric diatom Plagiogrammopsis vanheurckii was examined under SEM. This is the first report demonstrating that the earliest stages of a bipolar centric valve exhibit a ring-shaped pattern centre, the annulus, although the presence of the annulus in mature valves has been known for a long time. Non-synchronous development of sibling valves was observed in the early stages of valve morphogenesis; however, it remains unknown whether this phenomenon is related to the heterovalvate nature of P. vanheurckii because the degree of the heterovalvy is slight in this species. The ocellulus was formed in the final stages of valve morphogenesis. The use of clonal cultures also allowed examination of girdle band formation. In early stages of morphogenesis, the girdle band had a framework similar to araphid diatom valves, in which the primary rib system comprises longitudinal and transverse ribs.

MICROMORPHOGENESIS DURING DIATOM WALL FORMATION PRODUCES SILICEOUS NANOSTRUCTURES WITH DIFFERENT PROPERTIES(1).

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.

Oomycete and chytrid infections of the marine diatom Pseudo-nitzschia pungens (Bacillariophyceae) from Prince Edward Island, Canada

Two eukaryotic parasites were found infecting the bloom-forming marine diatom Pseudo-nitzschia pungens (Grunow ex Cleve) Hasle in Prince Edward Island, Canada. The most common was an oomycete; the other, seen once, was a chytrid. The structure of the discharged sporangium of both is remarkably similar. The oomycete parasite caused the host cell to lay down several extra girdle bands as the parasite thallus grew and swelled to form a holocarpic, endobiotic, nonwalled multinucleate thallus within the host cytoplasm. At maturity the thallus formed a single discharge tube with a thickened base and a thin papillate apex. Many biflagaellate zoospores were formed that burst out from the discharge tube. Ultrastructural characteristics of the oomycete thallus include mitochondria with tubular cristae and vesicles with dense body inclusions, features common to the oomycetes. The morphological characteristics and biflagellate condition indicate a placement of this form in the genus Ectrogella . However, neither flagellar mastigonemes nor flagella flimmer vesicles were found. The absence of flimmers may indicate a closer phylogenetic relationship to Haptoglossa , an endoparasitic oomycete of nematodes, the zoospores of which lack flagellar mastigonemes. Cell infection frequencies ranged from 0.6%–15.9% during 1992–1995, at the four sampling sites.

Dynamics of silica cell wall morphogenesis in the diatom Cyclotella cryptica: Substructure formation and the role of microfilaments

Diatoms are unicellular algae that make cell walls out of silica with highly ornate features on the nano- to microscale. The complexity and variety of diatom cell wall structures exceeds those possible with synthetic materials chemistry approaches. Understanding the design and assembly processes involved in diatom silicification should provide insight into patterning on the unicellular level, and information for biomimetic approaches for materials synthesis. In this report we examine the formation of distinct cell wall structures (valves and girdle bands) in the diatom Cyclotella cryptica by high resolution imaging using SEM, AFM, and fluorescence microscopy. Special attention was paid to imaging structural intermediates, which provided insight into the underlying design and assembly principles involved. Distinct stages in valve formation were identified, indicating a transition from a fractally organized structure to a dynamic pathway-dependent process. Substructures in the valves appeared to be pre-positioned prior to complete silicification, suggesting that organics responsible for these structures were pre-assembled and put in place. Microtubules and microfilamentous actin play significant roles in the positioning process, and actin is also important in the pathway-dependent expansion of the front of silicification. Our results indicate that even though all silica structures in C. cryptica are made of assemblies of nanoparticulate silica, control of meso- and microscale structure occurs on a higher order. It is apparent that diatoms integrate bottom up and top down control and synthesis mechanisms to form the diversity of structures possible.

Diverse and conserved nano‐ and mesoscale structures of diatom silica revealed by atomic force microscopy

An outstanding example of biological pattern formation at the single cell level is the diversity of biomineral structures in the silica cell walls of the unicellular eukaryotic algae known as diatoms. We present a survey of cell wall silica structures of 16 diatom species, which included all major cell wall components (valves, girdle bands and setae), imaged across the nano-, meso- and microscales using atomic force microscopy. Because of atomic force microscopy's superior ability to image surface topology, this approach enabled visualization of the organization of possible underlying organic molecules involved in mineral structure formation. Diatom nanoscale silica structure varied greatly comparing the same feature in different species and different features within a single species, and frequently on different faces of the same object. These data indicate that there is not a strict relation between nanoscale silica morphology and the type of structure that contains it. On the mesoscale, there was a preponderance of linear structures regardless of the object imaged, suggesting that assembly or organization of linear organic molecules or subcellular assemblies that confine a linear space play an essential and conserved role in structure formation on that scale. Microscale structure imparted an overall influence over nano- and mesoscale structure, indicating that shaping of the silica deposition vesicle plays a key role in structure formation. These results provide insights into the design and assembly principles involved in diatom silica structure formation, facilitating an understanding of the native system and potentially aiding in development of biomimetic approaches.

Chitin in diatoms and its association with the cell wall.

Chitin is a globally abundant polymer widely distributed throughout eukaryotes that has been well characterized in only a few lineages. Diatoms are members of the eukaryotic lineage of stramenopiles. Of the hundreds of diatom genera, two produce long fibers of chitin that extrude through their cell walls of silica. We identify and describe here genes encoding putative chitin synthases in a variety of additional diatom genera, indicating that the ability to produce chitin is more widespread and likely plays a more central role in diatom biology than previously considered. Diatom chitin synthases fall into four phylogenetic clades. Protein domain predictions and differential gene expression patterns provide evidence that chitin synthases have multiple functions within a diatom cell. Thalassiosira pseudonana possesses six genes encoding three types of chitin synthases. Transcript abundance of the gene encoding one of these chitin synthase types increases when cells resume division after short-term silicic acid starvation and during short-term limitation by silicic acid or iron, two nutrient conditions connected in the environment and known to affect the cell wall. During long-term silicic acid starvation transcript abundance of this gene and one additional chitin synthase gene increased at the same time a chitin-binding lectin localized to the girdle band region of the cell wall. Together, these results suggest that the ability to produce chitin is more widespread in diatoms than previously thought and that a subset of the chitin produced by diatoms is associated with the cell wall.

HOMOTHALLIC AUXOSPORULATION IN PSEUDO-NITZSCHIA BRASILIANA (BACILLARIOPHYTA)(1).

Most pennate diatoms are allogamous, and various types of mating systems have been described. In Pseudo-nitzschia, reproductive stages have been identified in some species, and it is generally accepted that the genus is mainly heterothallic. Here we report homothallic auxosporulation of Pseudo-nitzschia brasiliana Lundholm, Hasle et G. A. Fryxell. To our knowledge, this is the first verified description of homothallic sexual reproduction in the genus. Auxospore formation was observed in all 16 subclones derived from three initial clonal cultures of P. brasiliana. Pairing was followed by production of two gametes per gametangium, which fused to give two zygotes. Each zygote (early auxospore) was initially spherical and adhered to one girdle band of the parental frustule. The two auxospores tended to expand parallel to each other and perpendicular to the parental frustule. Elongation was synchronous, slightly asynchronous, or totally asynchronous. The entire process of sexual reproduction, from gamete formation to the appearance of the initial vegetative cells, took 2-4 d. The occurrence of sex in a homothallic species seems an advantageous life strategy for this species in that any encounter between cells of the right size class is potentially sexual.

Physiological and Transcriptomic Evidence for a Close Coupling between Chloroplast Ontogeny and Cell Cycle Progression in the Pennate DiatomSeminavis robusta

Despite the growing interest in diatom genomics, detailed time series of gene expression in relation to key cellular processes are still lacking. Here, we investigated the relationships between the cell cycle and chloroplast development in the pennate diatom Seminavis robusta. This diatom possesses two chloroplasts with a well-orchestrated developmental cycle, common to many pennate diatoms. By assessing the effects of induced cell cycle arrest with microscopy and flow cytometry, we found that division and reorganization of the chloroplasts are initiated only after S-phase progression. Next, we quantified the expression of the S. robusta FtsZ homolog to address the division status of chloroplasts during synchronized growth and monitored microscopically their dynamics in relation to nuclear division and silicon deposition. We show that chloroplasts divide and relocate during the S/G2 phase, after which a girdle band is deposited to accommodate cell growth. Synchronized cultures of two genotypes were subsequently used for a cDNA-amplified fragment length polymorphism-based genome-wide transcript profiling, in which 917 reproducibly modulated transcripts were identified. We observed that genes involved in pigment biosynthesis and coding for light-harvesting proteins were up-regulated during G2/M phase and cell separation. Light and cell cycle progression were both found to affect fucoxanthin-chlorophyll a/c-binding protein expression and accumulation of fucoxanthin cell content. Because chloroplasts elongate at the stage of cytokinesis, cell cycle-modulated photosynthetic gene expression and synthesis of pigments in concert with cell division might balance chloroplast growth, which confirms that chloroplast biogenesis in S. robusta is tightly regulated.

Pseudostriatella (Bacillariophyta): a description of a new araphid diatom genus based on observations of frustule and auxospore structure and 18S rDNA phylogeny

S. Sato, D.G. Mann, S. Matsumoto and L.K. Medlin. 2008. Pseudostriatella (Bacillariophyta): a description of a new araphid diatom genus based on observations of frustule and auxospore structure and 18S rDNA phylogeny. Phycologia 47: 371–391. DOI: 10.2216/08-02.1Pseudostriatella oceanica gen et. sp. nov. is a marine benthic diatom that resembles Striatella unipunctata in gross morphology, attachment to the substratum by a mucilaginous stalk and possession of septate girdle bands. In light microscopy, P. oceanica can be distinguished from S. unipunctata by plastid shape, absence of truncation of the corners of the frustule, indiscernible striation and absence of polar rimoportulae. With scanning electron microscopy, P. oceanica can be distinguished by a prominent but unthickened longitudinal hyaline area, pegged areolae, multiple marginal rimoportulae and perforated septum. The hyaline area differs from the sterna of most pennate diatoms in being porous toward its expanded ends; in this respect, it resembles the elongate annuli of some centric diatoms, such as Attheya and Odontella. 18S rDNA phylogeny places P. oceanica among the pennate diatoms and supports a close relationship between P. oceanica and S. unipunctata, but the genetic distance between them, coupled with the morphological differences, justifies separation at genus level. However, the affinity of the P. oceanica – S. unipunctata clade remains unresolved both in molecular and in morphological study. Both genera are only distantly related to Hyalosira and Grammatophora, despite similarities in frustule structure and growth habit, arguing against their inclusion in the same family. The auxospore is covered with series of transverse and longitudinal bands, but the structure and arrangement of these bands appear to be more similar to the properizonia of some centric diatoms than to the classic type of perizonium seen in other pennate diatoms; a few scales are also present. The differences between properizonia and perizonia are discussed.

ULTRASTRUCTURE OF THE FRUSTULE OF UROSOLENIA SPECIES FROM THE COLOMBIAN AND PERUVIAN AMAZON: U. DELICATISSIMA SPEC. NOV., U. AMAZONICA SPEC. NOV. AND U. BRAUNII (HUSTEDT) ROTT & KLING

Fine morphology of the frustule and taxonomy of three Urosolenia species are discussed. The analysis with SEM of materials collected in lakes of the Colombian and Peruvian Amazon showed that these taxa differ in valve and girdle bands morphology and in morphometric parameters. One of the species in the conical part of the valve has 1 to 4 buttonhole shaped structures with external more or less developed labiate-like openings. This structure has previously not been described in Urosolenia or in the allied genera Rhizosolenia, Prosboscia, Neocalyptrella and Pseudosolenia. Two new species, Urosolenia delicatissima and U. amazonica are described and new details of the valve morphology of U. braunii (Hustedt) Rott & Kling are given.

The species concept in a marine diatom: LSU rDNA–based phylogenetic differentiation in Skeletonema marinoi/dohrnii (Bacillariophyceae) is not reflected in morphology

M. Ellegaard, A. Godhe, K. Härnström and M. McQuoid. 2008. The species concept in a marine diatom: LSU rDNA–based phylogenetic differentiation in Skeletonema marinoi/dohrnii (Bacillariophyceae) is not reflected in morphology. Phycologia 47: 156–167. DOI: 10.2216/07-09.1The ubiquitous marine diatom genus Skeletonema includes several newly described species. This paper presents a study of morphology and LSU sequence diversity of one of these new species, Skeletonema marinoi. The 15 monoclonal strains (from the Swedish west coast, the Swedish east coast, Portugal and Canada) included in the study showed differences in LSU rDNA sequence within the morphospecies, with differences seen even among clones established from a single plankton net sample. Morphologically, all clones were indistinguishable from each other and from the closely related species Skeletonema dohrnii. In the original description of these two species, they were differentiated by the structure of the girdle bands. However, the girdle band types of both species were found within single samples of almost all clones of S. marinoi in this study. The LSU-based phylogeny is consistent with the split into two species, and there may be a difference in their biogeographical distribution. We therefore do not at present suggest that the two species be merged. Questions regarding species delimitation and cryptic species within protists often arise from such studies that include data from both morphological and DNA sequence analysis, and such questions are addressed here.

Application of AFM in understanding biomineral formation in diatoms

We review previous work and present new data on the application of atomic force microscopy (AFM) to study biomineral formation in diatoms, unicellular algae that make cell walls of silica. Previous studies examined a small subset of mostly larger diatom species, identifying a prevalence of large particulate silica on the nanoscale. We survey different structures including valves, girdle bands, and elongated spines called setae, in a variety of species, and show a diversity of nano- and meso-scale silica morphologies, even on different portions of the same structure. A general trend of highly organized mesoscale silica structure on the proximal face of cell wall components was observed, with less organized structure occurring on the distal face. The highly organized structures have features suggestive of an underlying linear template, which defines the area of initial silica polymerization. Such features have not been imaged with such clarity previously, demonstrating the advantages of AFM to image small differences in surface morphology and providing new insights and confirming evidence for models of diatom silica structure formation. In addition to its imaging capability, more developed application of AFM to map locations of organic template components on the nanoscale will greatly aid in elucidating mechanisms of diatom biosilica synthesis.