Micro‐photoluminescence of single living diatom cells

Diatoms are single‐celled microalgae that possess a nanostructured, porous biosilica shell called a frustule. This study characterized the micro‐photoluminescence (μ‐PL) emission of single living cells of the photosynthetic marine diatom Thalassiosira pseudonana in response to UV laser irradiation at 325 nm using a confocal Raman microscope. The photoluminescence (PL) spectrum had two primary peaks, one centered at 500–510 nm, which was attributed to the frustule biosilica, and a second peak at 680 nm, which was attributed to auto‐fluorescence of photosynthetic pigments. The portion of the μ‐PL emission spectrum associated with biosilica frustule in the single living diatom cell was similar to that from single biosilica frustules isolated from these diatom cells. The PL emission by the biosilica frustule in the living cell emerged only after cells were cultivated to silicon depletion. The discovery of the discovery of PL emission by the frustule biosilica within a single living diatom itself, not just its isolated frustule, opens up future possibilities for living biosensor applications, where the interaction of diatom cells with other molecules can be probed by μ‐PL spectroscopy. Copyright © 2016 John Wiley & Sons, Ltd.     

A new diatom (Bacillariophyceae) genus with two new species from New Mexico,USA

A new diatom (Bacillariophyceae) genus and two new species are described from the arid region of the North American southwest. The new genus, Playaensis, and new species, P. circumfimbria and P. furtiva, are distinguished by frustule morphology and autecology. Playaensis possesses unique, narrowly lunate longitudinal ribs bordering the raphe and spathulate spines, but their evolutionary relationship to known genera is unclear. In Payton Lake, New Mexico, P. circumfimbria and P. furtiva are rare in the diatom community, and their geographic and geological extent are not known, but they may be restricted to alkaline, slightly saline lakes of the region.     

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.     

Substratum adhesion and gliding in a diatom are mediated by extracellular proteoglycans

Diatoms are unicellular microalgae encased in a siliceous cell wall, or frustule. Pennate diatoms, which possess bilateral symmetry, attach to the substratum at a slit in the frustule called the raphe. These diatoms not only adhere, but glide across surfaces whilst maintaining their attachment, secreting a sticky mucilage that forms a trail behind the gliding cells. We have raised monoclonal antibodies to the major cell surface proteoglycans of the marine raphid diatom Stauroneis decipiens Hustedt. The antibody StF.H4 binds to the cell surface, in the raphe and to adhesive trails and inhibits the ability of living diatoms to adhere to the substratum and to glide. Moreover, StF.H4 binds to a periodate-insensitive epitope on four frustule-associated proteoglycans (relative molecular masses 87, 112, and >200 kDa). Another monoclonal antibody, StF.D5, binds to a carbohydrate epitope on the same set of proteoglycans, although the antibody binds only to the outer surface of the frustule and does not inhibit cell motility and adhesion. Received: 2 December 1996 / Accepted: 6 March 1997     

EXTRACELLULAR MATRIX PROTEO‐GLYCANS INVOLVED IN DIATOM ADHESION AND MOTILITY

Although diatom extracellular matricies are usually thought of exclusively in terms of the beautiful, architecturally complex silicious frustule, polymers exuded through the frustule are critical mediators of interactions with the external environment. In several species, complex proteoglycans appear to be the primary components involved in adhesion and motility. When viewed with high‐resolution cryo‐scanning electron microscopy methods, the ubiquity and pervasiveness of these polymers was revealed in both freshwater and marine taxa. Monoclonal antibody mapping of carbohydrate epitopes characterized by NMR, methylation and monosaccharide analysis and correlated with structural observations by EM revealed an organizational pattern far more complex than previously proposed. Modeling assembly of extracellular “stalks” in the marine biofouling diatom Achnanthes longipes involves intracellular sequestering of multiple components, deposition at the protoplasmic membrane/diatotepum interface, transport through the multilayered diatotepum and holes in the silica, extrusion from the frustule, and assembly into a very complex multi‐laminate biocomposite structure. The mechanism of extracellular polymer participation in motility is complex in a different way, as some current models of raphe associated motility involve cytoskeletal interactions and molecular motors.     

管壳缝类硅藻中国新记录属种——非洲南氏藻

利用光学和电子显微镜对采自黄海水域的1个管壳缝类硅藻——非洲南氏藻进行了形态学研究,并对其地理分布进行了讨论.结果表明:(1)该种壳体带面呈矩形,壳面窄椭圆形,具有钝圆的末端.(2)壳缝居中,由两条等长的分支组成.(3)管壳缝由复杂、接合的肋突支撑,但无龙骨.(4)每条线纹仅有1个孔纹,壳套上最多有1列孔纹.(5)目前...     

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.     

THE “PARADOX” DIATOM BACILLARIA PAXILLIFER (BACILLARIOPHYTA) REVISITED1

Defined by its unique colonial locomotion, Bacillaria paxillifer (O. F. Müll.) Hendey was recognized as a single, pandemic species by many phycologists. However, reinvestigation of colonies from different habitats revealed three distinct groups: (A) brackish/freshwater, (B) marine littoral, and (C) marine planktonic taxa. Groups differed in colony and cell form, raphe flanges (RFs), shape and position of transapical ribs (Tr's), and morphogenesis. Linear‐shaped species were restricted to group A: Tr's thickened principally to the interior. Lanceolate forms were confined to groups B and C: valve formation proceeded from an internal base layer to the exterior. The planktonic species differed in the shape of its raphe slit, and the transformation of girdle bands (GBs) into “winglets.” Taxa also differed in chloroplast shape and number. All species formed motile colonies. Siblings adhered via elastic fibrils secreted through their raphe. Raphe ribs were held in position by siliceous clamps (fibulae), anchored in an extra pair of axial ribs (fibular ribs) parallel to the raphe ribs. This raphe system resembled that of Cylindrotheca rather than the “canal raphe” of Nitzschia. Many valves were asymmetric along the apical axis due to protruding RFs shuttling in a 1:1 ratio within a colony, but raphe slits were mirror images, as were the growth direction of fibulae and position of plastids, with pyrenoids tilted in the same direction. Species possessed four open GBs per epitheca; the third band invariably bore an internal, organic ridge to aid in adhesion of the plasmalemma during cleavage. The results suggested that these taxa are a natural phylogenetic group, requiring precise determination of their taxonomic position.     

PYRENOIDS,RAPHES, AND OTHER FINE STRUCTURE IN DIATOMS

Pyrenoids of 6 diatoms, Acnanthes minutissima, Cyclotella meneghiniania, Cymbella affinis, Gomphonema parvulum, Nitzschia sp., and Surirella ovalis, were examined comparatively with the electron microscope. All except those of G. parvulum were membrane-limited. The pyrenoid membrane forms a ridge around the pyrenoid of S. ovalis, C. meneghiniania, and Nitzschia sp. Distended double-disc bands cross the pyrenoids of C. affinis, G. parvulum, S. ovalis, and A. minutissima; single-disc bands occur in C. meneghiniania, and 3 disc bands in Nitzschia sp. The raphe fissures of A. minutissima, Nitzschia sp., and S. ovalis did not contain any cytoplasm or obvious organs of locomotion. In G. parvulum, membranous profiles extend outward from the raphe fissure, but these probably are not responsible for movement. In this diatom 2 membranes usually cross the raphe, and the distance between them fluctuates indicating that they may undulate to force water through the raphe. A 7–10 mμ subfrustular zone is associated with the inner surface of the silica, but separate from the cytoplasmic membrane, in Nitzschia sp. and G. parvulum. In the latter, it is attached to siliceous knobs protruding from the inner surface of the silica. It may join the 2 halves of the frustale, which are otherwise not connected either to each other or to the cytoplasm. Pores 80–100 mμ in diameter occur in the nuclear envelopes of all diatoms examined. Dictyosomes are always perinuclear. In S. ovalis the vesicular complex is a convoluted, single-membrane-limited structure containing a variety of vesicular profiles, and occupying much of the central cytoplasm. Internally it resembles leucosin bodies of a chrysophyte, but the arrangement and morphology of the internal vesicles suggests that intracellular pinocytosis may occur.     

The effect of drugs on diatom valve morphogenesis

Summary The effects of various drugs on cell wall (valve) morphogenesis was investigated in three species of diatoms (Pinnularia spp., Surirella robusta, andHantzschia amphioxys) using light microscopy (LM) and scanning electron microscopy (SEM). Treatment ofSurirella with the microtubule (MT) disrupting agent colchicine during early valve formation results in a characteristic malformation of the valve, whereby part of the normally circumferential raphe canal forms as an abnormal protruding lip on the valve surface, located up to 20 m from the edge of the valve. The position of this malformed lip coincides with the location of a microtubule center (MC) at the time of colchicine addition, suggesting that the MC may play a direct role in positioning the tip of the raphe canal during valve formation. The migration of this MC to the tip of the cell during early valve morphogenesis is reversibly inhibited by the metabolic inhibitor 2-4-dinitrophenol (DNP). The effect of colchicine onPinnularia valve formation is less severe, causing occasional malformation of the raphe, but little if any lateral displacement. InHantzschia, colchicine has no effect on the positioning of the raphe, but prolonged exposure causes fusion of the raphe canal with the valve face. Cochicine treatment also results in the absence of the normal curvature at the central interruption in the raphe, as well as abnormal pore formation in this central area. Addition of cytochalasin D during early valve formation inHantzschia causes the raphe canal to form in the center of the valve face, suggesting that the normal translocation of the raphe canal to the valve edge is actindependent. Comparison of valves from control and cytochalasintreatmentHantzschia suggest that the pore spacing within the valve is determined by the position relative to the raphe, and does not depend on whether to pores form on the side (mantle) or the face of the mature valve.Abbreviations DM diatom medium - DNP dinitrophenol - MT microtubule - MC microtubule center - PSS primary silicification site - SDV silica deposition vesicle     

Diversity and distribution of epibiotic bacteria on Pseudo-nitzschia multiseries (Bacillariophyceae) in culture, and comparison with those on diatoms in native seawater

Previous studies on the interaction between bacteria and harmful algal bloom species have mostly considered the bacteria in the bulk solution. Here, we document the abundance and mode of attachment of bacteria growing on the cell surface of the domoic acid-producing diatom Pseudo-nitzschia multiseries (Hasle) Hasle in culture, compared with diatoms in field samples. The epiphytic bacteria were examined by scanning electron microscopy to visualize their morphology and mode of attachment. Two P. multiseries cultures were studied: clone CLN-1 and sub-clone CLN-1-NRC; the latter had been maintained in another laboratory for 2 years. Each of these P. multiseries cultures exhibited a clearly different assemblage of epibiotic bacteria, even though both originated from the same parent culture. The bacterial diversity was greater in clone CLN-1 (nine distinct morphotypes seen) than in sub-clone CLN-1-NRC (six morphotypes). The former clone also produced more domoic acid than the latter. There was a succession of bacterial morphotypes as well as an increase in the number of epiphytic bacteria per diatom cell during the progression from exponential to stationary phase. The most diverse and common morphotypes were rod-shaped cells (e.g. a Caulobacter-like bacterium attached by a discoid holdfast). Epibionts showed a preference for attachment at specific regions of the host diatom frustule, e.g. the raphe or cingulum, locations where organic matter may be extruding from the diatom cell. Most diatom cells carried only one to five bacteria, and up to ca. 60% of the intact diatom cells (although intact cells themselves were infrequent) were still free of epibiotic bacteria at the end of the 31-day batch culture experiment. Sequencing of the SSU rRNA gene showed that five of the eight bacterial strains isolated from the P. multiseries cultures were members of the Alphaproteobacteria, three of the Gammaproteobacteria and one of the Bacteroidetes. A morphologically diverse assemblage of epibiotic bacteria was also found on both centric and pennate planktonic diatoms in natural coastal waters. Of the eight morphotypes recorded, all but two were also found in the cultures. Relatively fewer wild diatom cells carried bacteria compared to cells in culture. We hypothesize that the diversity and abundance of epiphytic bacteria may explain some of the variability seen in the production of DA by different P. multiseries clones, and should be considered as another important and controllable variable that influences diatom cell physiology.     

OBSERVATIONS OF A TUBE-DWELLING DIATOM: NAVICULA HAMULIFERA (BACILLARIOPHYCEAE)1

Colonies of the tube-dwelling diatom Navicula hamulifera Grunow living on mangrove prop roots in Indian River. Florida and at La Parguera, Puerto Rico, were studied using light and electron microscopy. Observations of the tube morphology and cell structure of this diatom from fresh samples and cultures are described, as well as the ultrastructural morphology of its frustule. The formation of tubes by this diatom is reported for the first time. Comparisons are made with the closest species; Navicula delognei V.H. and Navicula pseudocomoides Hendey.     

CULTURES OF CHYTRIDS - PARASITES OF ALGAE IN BIOLOGICAL INSTITUTE OF PETERSBURG UNIVERSITY

Although diatom extracellular matricies are usually thought of exclusively in terms of the beautiful, architecturally complex silicious frustule, polymers exuded through the frustule are critical mediators of interactions with the external environment. In several species, complex proteoglycans appear to be the primary components involved in adhesion and motility. When viewed with high-resolution cryo-scanning electron microscopy methods, the ubiquity and pervasiveness of these polymers was revealed in both freshwater and marine taxa. Monoclonal antibody mapping of carbohydrate epitopes characterized by NMR, methylation and monosaccharide analysis and correlated with structural observations by EM revealed an organizational pattern far more complex than previously proposed. Modeling assembly of extracellular "stalks" in the marine biofouling diatom Achnanthes longipes involves intracellular sequestering of multiple components, deposition at the protoplasmic membrane/diatotepum interface, transport through the multilayered diatotepum and holes in the silica, extrusion from the frustule, and assembly into a very complex multi-laminate biocomposite structure. The mechanism of extracellular polymer participation in motility is complex in a different way, as some current models of raphe associated motility involve cytoskeletal interactions and molecular motors.     

Influence of environmental factors on the development of the valve in diatoms

Summary The species-specific form and structure of the diatom shell is variable within a given genetical reaction-norm, depending on the dynamic interrelation between cell and environment. The appearing modifications—based on quantitative disarrangement of construction-units as well as on a change in size and outline—can be understood as the morphological expression of a changed metabolism which has become necessary for adaption to adverse conditions. The diatoms react very sensitively, especially to the salinity factor, whereby actually two alternatives of adaption occur: a vegetative, in building resting spores (f.i., Navicula cuspidata) and a generative (f.i., Anomoeoneis sphaerophora, Surirella peisonis).Teratologies have been found in totally unbalanced surroundings (especially under conditions of ion unbalance), where the usually symmetrical forms have lost the coordination of the construction-units to each other (f.i., Surirella peisonis). They supply good criteria in clarifying the problems concerning pattern development.Dedicated to Univ.-Prof. Dr. H. A. v.Stosch's 70th Birthday.     

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.     

Spatial heterogeneity of diatom silicification and growth in a eutrophic reservoir

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Morphology of the marine epiphytic diatom Cocconeis heteroidea (Bacillariophyceae)

The morphology and fine valve structure of the marine epiphytic diatom Cocconeis heteroidea Hantzsch have been investigated. The entire frustule, including the internal and external structure of the raphid valve (RV) and araphid valve (AV), and the complete cingulum, are described using light microscopy and scanning and transmission electron microscopy, using a bleaching method. The strongly sigmoid raphe terminates in elongate hooked helictoglossae internally. The hymenes, with perforations arranged in a centric array, are located near the internal openings of the areolae in the RV. The striae in the AV consist of alveoli occluded by hymenes, that have perforations arranged in a parallel array and are located near the outer surface. The complete cingulum of AV consists of three open bands without fimbriae: a valvocopula, a copula with a ligula and a pleura with a small ligula. The RV has only a valvocopula which is open type and not fimbriate.