Skeletal Ultrastructure in the Cyclostome Bryozoan Hornera

Abstract Scanning electron microscopy of calcified walls in two species of the cyclostome bryozoan Hornera has revealed previously undescribed details of skeletal morphology and growth. The calcitic interior walls of both H. robusta MacGillivray and H. squamosa Hutton have a laminated structure. Walls are extended at distal growing edges where the formation of new crystallites is concentrated and wall fabric is nacreous or semi-nacreous. New crystallites are seeded on the surface of existing crystallites as six-sided rhombs. At the centres of the rhombs in H. robusta there are often three ‘spikes' which point towards alternate sides of the rhomb. Screw dislocations resulting in spiral overgrowths are also common at these distal wall edges. Wall thickening occurs further proximally where walls develop a regularly foliated structure of imbricated laths growing towards the colony base. Although often thought to be ubiquitous in cyclostomes, the division of walls into three layers (an inner, primary layer flanked on both sides by secondary layers) is absent in Hornera. Wall ultrastructure contrasts strongly with the lamellar–fibrous–lamellar structure recently described from cinctiporid cyclostomes. The c-axes of the crystallites are orientated perpendicular to the wall surface in Hornera, unlike cinctiporids in which they are orientated within the plane of the wall. Apparent similarities in ultrastructure suggest that Hornera may provide a good model for wall growth in extinct trepostome bryozoans.     

Electron diffraction studies of the calcareous skeletons of bryozoans

Electron microscopy and electron diffraction were used to investigate mineral crystallites dissociated from the skeletal walls of six species belonging to the Bryozoa, a phylum of predominantly marine colony-forming invertebrate animals. Four cheilostome bryozoans (Flustra foliacea, Membranipora membranacea, Thalamoporella novaehollandiae and Cellarinella foveolata) and two cyclostomes (Fasciculipora ramosa and Hornera robusta) were analysed. In each case, an attempt was made to relate the crystal morphology imaged in situ by scanning electron microscopy with the crystallographic orientation of isolated crystals determined by electron diffraction analysis in the transmission electron microscope. The results showed that the calcitic cheilostome and cyclostome skeletons consisted of closely packed arrays of plate-like Mg-containing calcite crystallites, and that the crystallographic a-axis was preferentially aligned perpendicular to the top and bottom surfaces of the flattened particles. The results suggest that calcite biomineralization occurs under similar crystallographic constraints in the five species studied even though the origins of cheilostomes and cyclostomes are separated by over 300 million years in the fossil record of the bryozoans. Similar studies for the aragonite crystallites in skeletons of M. membranacea indicated that the crystallographic b-axis was preferentially oriented perpendicular to the basal surfaces of irregular plate-like particles.     

The Ultramicrostructure of Tubiphyte Walls

An ultramicroscopic study of tubiphytes—tubular formations, composed of pelitomorphic calcite from the Lower Permian Tra-Tau reef (Cis-Urals)—was carried out. A multilayer structure of the walls was recognized. The inner layer is an ultrathin layer of acicular calcite crystals, which often has aragonite habit; the next layer is a relatively thick one represented by a dense pelitomorphic mass, which is often encrusted with the calcite crystals. Between the first and second layers are traces of mineralized biofilms, “lace” secretions of mineralized glycocalyx. It is assumed the inner layer is the tubiphyte wall and the pelitomorphic one is the result of biochemogenic carbonate precipitation as a consequence of ability of epiphytic bacterial activity, which colonized the walls of an original organism. A relatively high C/Ca ratio in the glycocalyx relics, its consequent decrease from the acicular wall towards the pelitomorphic one, and finally to the purely chemogenic crystalline formations of crustified rims is an indirect proof of such a formation mechanism of tubiphytes.     

Modification of cambial cell wall architecture during cambium periodicity in Populus tomentosa Carr.

Cambium periodicity is correlated with changes in the cambial cell wall, but the heterogeneity of cell wall structure and composition makes it difficult to give an accurate interpretation, especially for complex secondary vascular tissues. A combination of different methods is necessary to reveal the structure of this complex cell wall. In this study, the cell wall architecture and composition of active and dormant cambial cells in Populus tomentosa were investigated by a combination of light microscopy, rapid-freezing and deep-etching electron microscopy, Fourier-transform infrared microspectroscopy and immuno-histochemistry. The results showed that the architecture of dormant cambial cell walls displayed a multi-layered structure, denser fibril network, smaller pore size, and fewer crosslinks between microfibrils than active cambial cell walls. The FTIR spectra of cell walls from active and dormant cambium showed differences in the intensity of bands near 1,740, 1,629, 1,537, 1,240, and 830 cm⁻¹, which reflected differences in cell wall composition. Immuno-labeling indicated that high methyl-esterified homogalacturonan and (1 → 4)-β-d-galactan epitopes were abundant and distributed in active cambial cell walls, and relatively de-esterified homogalacturonan and (1 → 5)-α-l-arabinan epitopes had weak labeling in the active cambium, while almost no labeling or very weak labeling for high methyl-esterified homogalacturonan, (1 → 4)-β-d-galactan and (1 → 5)-α-l-arabinan epitopes occurred in dormant cambial cells, except for the de-esterified homogalacturonan epitope, which was abundant in dormant cambial cells. These results demonstrate that there are great differences, both in structure and composition, between active and dormant cambial cell walls, which reflect their dynamic changes during cambium activity.     

Quantifying the importance of galactofuranose in Aspergillus nidulans hyphal wall surface organization by atomic force microscopy

The fungal wall mediates cell-environment interactions. Galactofuranose (Galf), the five-member ring form of galactose, has a relatively low abundance in Aspergillus walls yet is important for fungal growth and fitness. Aspergillus nidulans strains deleted for Galf biosynthesis enzymes UgeA (UDP-glucose-4-epimerase) and UgmA (UDP-galactopyranose mutase) lacked immunolocalizable Galf, had growth and sporulation defects, and had abnormal wall architecture. We used atomic force microscopy and force spectroscopy to image and quantify cell wall viscoelasticity and surface adhesion of ugeAΔ and ugmAΔ strains. We compared the results for ugeAΔ and ugmAΔ strains with the results for a wild-type strain (AAE1) and the ugeB deletion strain, which has wild-type growth and sporulation. Our results suggest that UgeA and UgmA are important for cell wall surface subunit organization and wall viscoelasticity. The ugeAΔ and ugmAΔ strains had significantly larger surface subunits and lower cell wall viscoelastic moduli than those of AAE1 or ugeBΔ hyphae. Double deletion strains (ugeAΔ ugeBΔ and ugeAΔ ugmAΔ) had more-disorganized surface subunits than single deletion strains. Changes in wall surface structure correlated with changes in its viscoelastic modulus for both fixed and living hyphae. Wild-type walls had the largest viscoelastic modulus, while the walls of the double deletion strains had the smallest. The ugmAΔ strain and particularly the ugeAΔ ugmAΔ double deletion strain were more adhesive to hydrophilic surfaces than the wild type, consistent with changes in wall viscoelasticity and surface organization. We propose that Galf is necessary for full maturation of A. nidulans walls during hyphal extension.     

A new model for cellulose architecture in some plant cell walls

Summary A new model of rotating fibre components (helicoidal model) is proposed to explain the architecture of some plant cell walls. On the basis of tilting observations under the electron microscope, we establish the validity of this model for the cell wall ofChara vulgaris oospores. We suggest that this model explains the architecture seen in a number of published micrographs from a variety of different plant cell walls. Helicoidal architecture is shown to be distinct from the previously established crossed polylamellate architecture. The diagnostic features of helicoidal architecture are given. Morphogenesis of plant cell walls is discussed, with particular reference to self assembly in cholesteric liquid crystals.     

WALL STRUCTURE AND ACANTHOPORES IN THE BRYOZOAN LEIOCLEMA ASPERUM

Leioclema asperum (Hall), a typical trepostomatous bryozoan, shows well developed acanthopores which are modified parts of orthodox zooecial walls and not separate skeletal entities. They resulted from locally accelerated forward growth of the wall, hence their distinctive cone-in-cone structure. The comparative absence of growth lines in the axial region of each acanthopore suggests that deposition of calcite in that situation was virtually continuous. There is no reason to believe that the acanthopores were originally hollow and they cannot have housed kenozooids, as is commonly supposed.     

<Emphasis Type="Italic">Solenomeris:</Emphasis> from biomineralization patterns to diagenesis

Microstructural and diagenetic patterns in acervulinid foraminifers are investigated through detailed petrographical, SEM, and geochemical analyses of Recent and fossil Acervulina and Eocene Solenomeris. The acervulinid test is composed of an optically radial hyaline magnesian calcite. The chessboard-chamber arrangement represents an efficient way for minimizing the volume accretion rate of cytoplasm, this being probably an important requirement for foraminifera with no predesigned external shape, such as the encrusting forms. Several newly described skeletal features reflect directly adaptation and responses to environmental constraints, such as microborer pressure, water energy, adaptation to substrate shape, skeleton repair, and compartmentalization of cytoplasm. The Eocene Solenomeris from the Pyrenees have recorded in their skeletons several diagenetic processes comprising two different phases of cementation followed by recrystallization. These events are accompanied by a marked loss of magnesium content and preceded or followed by an alteration of the Primary Organic Membrane. These diagenetic processes, in particular the early cementation, alter the overall layered pattern typical of foraminifera and produce a pseudo-file arrangement, mimicking an algal structure.     

Individual chitin synthase enzymes synthesize microfibrils of differing structure at specific locations in the Candida albicans cell wall

The shape and integrity of fungal cells is dependent on the skeletal polysaccharides in their cell walls of which beta(1,3)-glucan and chitin are of principle importance. The human pathogenic fungus Candida albicans has four genes, CHS1, CHS2, CHS3 and CHS8, which encode chitin synthase isoenzymes with different biochemical properties and physiological functions. Analysis of the morphology of chitin in cell wall ghosts revealed two distinct forms of chitin microfibrils: short microcrystalline rodlets that comprised the bulk of the cell wall; and a network of longer interlaced microfibrils in the bud scars and primary septa. Analysis of chitin ghosts of chs mutant strains by shadow-cast transmission electron microscopy showed that the long-chitin microfibrils were absent in chs8 mutants and the short-chitin rodlets were absent in chs3 mutants. The inferred site of chitin microfibril synthesis of these Chs enzymes was corroborated by their localization determined in Chsp-YFP-expressing strains. These results suggest that Chs8p synthesizes the long-chitin microfibrils, and Chs3p synthesizes the short-chitin rodlets at the same cellular location. Therefore the architecture of the chitin skeleton of C. albicans is shaped by the action of more than one chitin synthase at the site of cell wall synthesis.     

Observations of the tissue-skeleton interface in the scleractinian coral <Emphasis Type="Italic">Stylophora pistillata</Emphasis>

Recent micro-analytical studies of coral skeletons have led to the discovery that the effects of biology on the skeletal chemical and isotopic composition are not uniform over the skeleton. The aim of the present work was to provide histological observations of the coral tissue at the interface with the skeleton, using Stylophora pistillata as a model, and to discuss these observations in the context of skeletal ultra-structural organization and composition. Several important observations are reported: (1) At all scales of observation, there was a precise morphological correspondence between the tissues and the skeleton. The morphological features of the calicoblastic ectoderm correspond exactly to the shape of individual crystal fiber bundles in the underlying skeleton, indicating that the calicoblastic cell layer is in direct physical contact with the skeletal surface. This is consistent with the previously observed chemical and isotopic composition of the ultra-structural components in the skeleton. (2) The distribution and density of desmocyte cells, which anchor the calicoblastic ectoderm to the skeletal surface, vary spatially and temporally during skeletal growth. (3) The tissue above the coenosteal spines lack endoderm and consists only of ectodermal cell-layers separated by mesoglea. These findings have important implications for models of vital effects in coral skeletal chemistry and isotope composition.     

Concise and informative title: innovative geotextile materials for the extension of urban green space—contribution to urban sustainability

Modern Movement architecture employed new materials to deal with social challenges and industrial production. Today environmental problems are the challenge. Resilience to natural hazards, including climate change, is such one. Geotextiles are employed in large landscape (ash/garbage deposits, river sides), but planting on building surfaces is possible. We focus on green walls/roofs for emergency housing, including reshaping of temporary propping systems. Green walls allow integrating parcels remaining empty after the collapse of buildings through Pocket Parks. The earthquake impact becomes urban restructuration occasion. A special support skeleton for the earth&seeds&geotextile in green wall installations will be designed, and later pattented.     

Multiple phases of mg‐calcite in crustose coralline algae suggest caution for temperature proxy and ocean acidification assessment: lessons from the ultrastructure and biomineralization in Phymatolithon (Rhodophyta,Corallinales)1

Magnesium content, strongly correlated with temperature, has been developed as a climate archive for the late Holocene without considering anatomical controls on Mg content. In this paper, we explore the ultrastructure and cellular scale Mg‐content variations within four species of North Atlantic crust‐forming Phymatolithon. The cell wall has radial grains of Mg‐calcite, whereas the interfilament (middle lamella) has grains aligned parallel to the filament axis. The proportion of interfilament and cell wall carbonate varies by tissue and species. Three distinct primary phases of Mg‐calcite were identified: interfilament Mg‐calcite (mean 8.9 mol% MgCO₃), perithallial cell walls Mg‐calcite (mean 13.4 mol% MgCO₃), and hypothallium Mg‐calcite (mean 17.1 mol% MgCO₃). Magnesium content for the bulk crust, an average of all phases present, showed a strongly correlated (R² = 0.975) increase of 0.31 mol% MgCO₃ per °C. Of concern for climate reconstructions is the potential for false warming signals from undetected postgrazing wound repair carbonate that is substantially enriched in Mg, unrelated to temperature. Within a single crust, Mg‐content of component carbonates ranged from 8 to 20 mol% MgCO₃, representing theoretical thermodynamic stabilities from aragonite‐equivalent to unstable higher‐Mg‐calcite. It is unlikely that existing current predictions of ocean acidification impact on coralline algae, based on saturation states calculated using average Mg contents, provide an environmentally relevant estimate.     

Skeletal Ultrastructure in some Tubuliporine Cyclostome Bryozoans

The ultrastructure of the calcareous skeleton is described in twenty–one species of recent tubuliporine cyclostome bryozoans, using field emission SEM. The succession of skeletal fabrics in interior walls may be classified into four different fabric suites. The first–formed part of the calcitic skeleton in all species for which it has been observed is a precursory fabric of tiny, wedge–shaped crystallites. This is succeeded in about half of the species studied by a fabric of transverse fibres, followed by foliated fabric and often semi–nacre (fabric suite 1). Most of the remaining species lack transverse fibres and have interior walls largely comprising semi–nacre (fabric suite 2). A few species have skeletons consisting of predominantly distally–oriented, irregularly or regularly foliated fabric (fabric suite 3). A single species has a skeleton of proximally–oriented foliated fabric (fabric suite 4). Basal exterior walls in all species have a precursory fabric of tiny wedge–shaped crystallites without a strong preferred orientation, deposited directly upon the organic cuticle, followed by a layer of planar spherulitic structure, which in turn is succeeded by a similar fabric to that developed in the interior wall of the species concerned. Outermost layers of frontal exterior walls exhibit one of the following combinations of three fabrics: an outer layer of (1) finely granular or wedge–shaped crystallites; a thin dense granular layer followed by (2) distally accreting planar spherulitic fabric., or (3) obliquely accreting planar spherulitic fabric growing partly towards the midline of the frontal wall. Terminal diaphragms usually have outer layers dominated by planar spherulitic ultrastructure with centripetal growth directions. The fabric suites present in tubuliporines encompass most known fabrics found in the other cyclostome suborders and support the notion that this species–rich suborder occupies a central position in cyclostome evolution.     

Wall architecture with high porosity is established at the tip and maintained in growing pollen tubes of Nicotiana tabacum

A major question in pollen tube growth in planta remains: do the pollen tube walls form a barrier to interaction with the environment? Using cryo‐FESEM, we directly assessed the 3D construction and porosity of tobacco pollen tube walls. Fractured mature primary walls showed a 40–50 nm spaced lattice of continuous fibers interconnected by short rods in the primary wall. These observations agree with TEM observations of sectioned walls. In the secondary callose wall, for which no structure is visible using TEM, cryo‐FESEM also revealed a 50 nm lattice consisting of longer fibers, approximately 10–15 nm wide, with rod‐like, thinner interconnections at angles of approximately 90° with the longer fibers. Such architecture may reflect functional needs with respect to porosity and mechanical strength. The wall does not form a mechanical barrier to interaction with the environment and is gained at low cost. Cryo‐FESEM additionally revealed another special feature of the wall: the tubes were tiled with scales or rings that were highly conspicuous after pectin extraction with EDTA. These rings cause the typical banding patterns of pectin that are commonly seen in pollen tubes during oscillatory growth, as confirmed by staining with toluidine blue as well as by DIC microscopy. Growth analysis by VEC‐LM showed that the ring‐ or scale‐like structures of the primary wall consist of material deposited prior to the growth pulses. The alternating band pattern seen in the callose wall is probably imposed by constrictions resulting from the rings of the primary wall.     

Skeletal formation in the modern but ultraconservative chaetetid spongeSpirastrella (Acanthochaetetes) wellsi (demospongiae,porifera)

Summary The modern hadromerid coralline spongeSpirastrella (Acanthochaetetes) wellsi exhibits a unique secondary high-Mg calcite (>19 mol % MgCO₃) basal skeleton. The basal skeleton is constructed of bundles of elongated crystals more or less tangentially orientated. The initial formation of these crystals is controlled by soluble highly acidic aspartic and glutamic-rich (40%) macromolecules. The skeletal mineralization occurs in four different loci: in the top of the calicles, at the tabulae, on collagenous anchor fibres, and within closed spaces between the tabulae. The clicle walls are formed on the uppermost top of the basal skeleton as a continuous process. Based on long term stainings with Ca²⁺-chelating fluorochroms (calcein, chlorotetracyclines) the growth rate of this sponge is extremely low with ca. 50–100μm/a. The skeletal formation takes places outside the sponge, within a narrow zone (300–500 nm) between the basopinacoderm and the mature basal skeleton. The sponge produces thread-like folded templates (‘spaghetti fibres’) of 0,5–2 μm size, the shape controlling insoluble organic matrix. These templates become mineralized in a first step as MgCO₃, then are stretched. A soluble organic matrix is also secreted, and remains are included inside the mineralized skeleton. This organic matrix consists of in a complex mixture containing small very acidic proteins (5, 13, 31 KD; 40% Asp and Glu and therefore most probably Ca²⁺-binding) and high molecular weight glycoproteins among several other organic compounds. The mature crystals are high-Mg calcites. During calcification large cells with large reserve granules (LCG) are always present in a tight connection with the basopinacoderm. These cells form also the collagenous anchor fibres. Primary tabulae are formed by a non-collagenous organic sheet. Calcification happens only when LCG cells are enriched on the organic sheet. Randomly oriented high-Mg calcite crystals are growing on the collagenous anchor fibres. The same type of the mineralization is observed within the spaces of the tabulae. This particular case of mineralization is controlled by decaying sponge tissue (ammonification). The δ¹³C values are in equilibrium with the ambient sea water and vary between +3.2 and +2.8 ‰. The mode of mineralization of the basal skeleton can be described as biologically induced resp. matrix mediated.     

Microstructure, growth banding and age determination of a primnoid gorgonian skeleton (Octocorallia) from the late Younger Dryas to earliest Holocene of the Bay of Biscay

A fossil primnoid gorgonian skeleton (Octocorallia) was recovered on the eastern Galician Massif in the Bay of Biscay (NE Atlantic) from 720 m water depth. The skeleton shows a growth banding of alternating Mg–calcitic and organic (gorgonin) increments in the inner part, surrounded by a ring of massive fibrous calcite. Three calcite-dominated cycles, bounded by thick organic layers, consist of five light-dark couplets of calcite and gorgonin. Two AMS-¹⁴C datings of the fossil skeleton give ages of 10,880 and 10,820 ± 45 ¹⁴C years before present (BP). We arrive at a calibrated age range of 11,829–10,072 cal. years BP (two σ), which comprises the late Younger Dryas to the earliest part of the Holocene. The cyclic calcitic–organic growth banding may be controlled by a constant rate of calcite secretion with a fluctuating rate of gorgonin production, possibly related to productivity cycles. The skeletal fabric change of alternating calcitic–organic increments to massive fibrous calcite may be the result of hydrographic changes during the deglaciation as reflected by preliminary stable isotope data. If this hypothesis proves to be correct, primnoid gorgonians are able to match with varying hydrodynamic conditions by changing their biomineralisation mode.     

Origin and phylogeny of Guyniidae (Scleractinia) in the light of microstructural data

The set of skeletal characters of the Recent azooxanthellate coral Guynia annulata Duncan, 1872 is unique among extant scleractinians and encompasses: (a) undifferentiated septal calcification centers (in most extant scleractinians calcification centers are clearly separated); (b) completely smooth septal faces (septa of almost all extant scleractinians bear granular ornamentation); (c) deeply recessed septa in respect to the epithecal rim in the adult coralla (in adults of the majority of extant scleractinians the relationships between septa and wall are the reverse); and (d) an aseptal part of the initial ontogenetic stage, just above the basal plate (almost all known scleractinians have a septate initial coralla). Skeletal features of five other extant traditional guyniids are typical of other caryophylliines (and of Scleractinia). However, the wall types present in different species of traditional guyniids exceed limits traditionally attributed to one caryophylliine family: i.e., Stenocyathus and Truncatoguynia have a marginothecal wall like the Flabellidae, whereas Schizocyathus and Temnotrochus usually have an entirely epithecal wall, as in Gardineriidae (Volzeioidea). Moreover, Pourtalocyathus and Schizocyathus show intraspecific variation in distribution of septal calcification centers (separated vs. non-separated) and in wall types (epithecal vs. consisting of large spherulite-like bodies). These major differences in skeletal architecture form the basis for a new, threefold taxonomical subdivision of the traditional guyniids: (1) Guyniidae Hickson, 1910, containing only monospecific Guynia with an epithecal wall, and septa with non-separated calcification centers; (2) Schizocyathidae fam.n., groups Microsmilia Schizocyathus, Pourtalocyathus, Temnotrochus, which have an epithecal wall and septa with usually well-separated calcification centers; and (3) Stenocyathidae fam.n. with Stenocyathus and Truncatoguynia which have a marginothecal wall and septa with well-separated calcification centers. Despite differences in the basic architecture of the skeleton, all taxa attributed to these families have 'thecal pores' formed by selective dissolution of the skeleton. I propose two hypotheses for evolutionary relationships among Guyniidae, Schizocyathidae, and Stenocyathidae: (1) Hypothesis A: the three families are not phylogenetically related and 'pores' originated independently in different scleractinian lineages: e.g., Guyniidae may represent distant zardinophyllid or gigantostyliid descendants, Schizocyathidae may be a volzeioid offshoot, whereas Stenocyathidae may be a flabellid descendant; (2) Hypothesis B: the three families are phylogenetically related and 'thecal pores' are synapomorphic for the clade (superfamily Guynioidea). Additional approaches, such as anatomical observations, molecular studies on guyniid DNA sequences, and in-depth studies on scleractinian biomineralization will be necessary to test these hypotheses.     

Changes in Pectin Structure during Epidermal Cell Elongation in Pea (Pisum sativum) and Its Implications for Cell Wall Architecture

Changes in cell wall architecture during elongation of epidermalcells in pea epicotyls were visualized by rapid-freezing anddeep-etching (RFDE) techniques. The abundant network structurecomposed of the association of granular substances disappearedfrom the cell wall during elongation. The granular substanceswere demonstrated to be pectic polysaccharides by their disappearanceupon EDTA treatment and by chemical analysis of the EDTA-ex-tractablesubstances. Labeling with the monoclonal antibody JIM5, whichrecognizes unesterified pectins, was much more extensive inthe cell walls of the non-elongating region than in those ofthe elongating region. The pore size of the cell wall was largerin the non-elongating region than in the elongating region.These observations suggest that the formation of the pecticgel itself is not involved in the control of the wall porosity.We proposed that the association of the granular substancesis involved in the swelling of the cell walls in the elongatingregion. (Received May 18, 1998; Accepted September 25, 1998)     

Microstructural roughness of skeletal calcite in ophiuroid vertebral ossicles: evidence of wear?

Local areas of roughened skeletal calcite are reported from the otherwise smooth, imperforate skeletal articulations of the ophiuroid vertebral ossicle (Echinodermata: Ophiuroidea). Complementary patterns of roughness on both proximal and distal articulating surfaces suggest local points of wear between adjacent ossicles, presumably caused by repeated rotation of the intervertebral joint. This surface feature is discussed with respect to its possible origin (mechanical action, resorption of skeletal material, experimental artifact) and the functional morphology of the ophiuroid arm skeleton.