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.     

DEOXYRIBONUCLEIC ACID CONTENT OF THE THREE CELL TYPES OF PHAEODACTYLUM TRICORNUTUM BOHLIN1

There is no significant difference in the DNA content of the oval, fusiform, and triradiate cell types of Phaeodactylum tricornutum, suggesting that they do not represent an alternation of haploid and diploid generations. The triradiate cells have a shorter generation time than the oval or fusiform cells.     

Characterization of the extracellular matrix of Phaeodactylum tricornutum (Bacillariophyceae): structure,composition, and adhesive characteristics

The extracellular matrix of the ovoid and fusiform morphotypes of Phaeodactylum tricornutum (Bohlin) was characterized in detail. The structural and nanophysical properties were analyzed by microscopy. Of the two morphotypes, only the ovoid form secretes adhesive mucilage; light microscopy and scanning electron microscopy images showed that the mucilage was secreted from the girdle band region of the cell as cell‐substratum tethers, accumulating on the surface forming a biofilm. After 7 d, the secreted mucilage became entangled, forming adhesive strands that crisscrossed the substratum surface. In the initial secreted mucilage atomic force microscopy identified a high proportion of adhesive molecules without regular retraction curves and some modular‐like adhesive molecules, in the 7 d old biofilm, the adhesive molecules were longer with fewer adhesive events but greater adhesive strength. Chemical characterization was carried out on extracted proteins and polysaccharides. Differences in protein composition, monosaccharide composition, and linkage analysis are discussed in relation to the composition of the frustule and secreted adhesive mucilage. Polysaccharide analysis showed a broad range of monosaccharides and linkages across all fractions with idiosyncratic enrichment of particular monosaccharides and linkages in each fraction. 3‐linked Mannan was highly enriched in the cell frustule fractions indicating a major structural role, while Rhamnose and Fucose derivatives were enriched in the secreted fractions of the ovoid morphotype suggesting involvement in cell adhesion. Comparison of SDS‐PAGE of extracellular proteins showed two major bands for the ovoid morphotype and four for the fusiform morphotype of which only one appeared to be common to both morphotypes.     

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.     

The cytoarchitecture of the torus semicircularis in the golden skink Mabuya multifasciata

The skink, Mabuya multifasciata, torus semicircularis was subdivided into the central (CN), the laminar (LN), and the superficial (SN) nuclei using Golgi and Nissl methods. The central nucleus consisted of small ovoid neurons surrounding a core of fewer large ovoid-triangular and fusiform neurons. The ovoid cells had scant cytoplasm and two to five dendritic trunks. Most of these processes were directed around the periphery of the central nucleus. The large neurons had clumped, darkly staining Nissl substance and a central nucleus. The sparse dendritic spine population on these cells increased distally on the three to five radiate dendrites. The laminar nucleus was present caudal and ventral to the central nucleus. At more rostral levels it was medial and dorsomedial to the central nucleus. The NL had three to five layers of ovoid and fusiform neurons. Scattered within these layers were a few ovoid-triangular neurons. Ovoid neurons had eccentric or central nuclei. The arborization of their dendrites was generally medial and lateral but was frequently oriented caudomedial and rostrolateral. Fusiform neurons had pale Nissl substance, central nuclei, and one to two dendritic processes. The ovoid-triangular neurons had dense, clumped Nissl substance and at least two dendritic trunks with few spines. The superficial nucleus was dorsal, lateral, and caudal to the central nucleus. Extending ventrolaterally around the central nucleus, the superficial nucleus became confluent with the laminar nucleus, ensheathing the central nucleus ventrally, laterally, and dorsally. Rostrally the central nucleus was covered by the layers of the laminar nucleus. Within the superficial nucleus were ovoid, fusiform and sparse ovoid-triangular neurons. The study indicated that the morphology of the torus semicircularis in the golden skink was similar to that in other lizards. This similarity correlates with the degree of development as it relates to the auditory function, but was independent of the type of inner ear restraint mechanism.     

Nanostructure and nanomechanics analysis of lymphocyte using AFM: From resting,activated to apoptosis

The ultrastructural and mechanical properties of single resting, activated and apoptosis lymphocyte have been investigated by atomic force microscopy (AFM). Using topographic imaging, we showed that the surface of the resting lymphocyte is smooth, while lymphocyte activation and apoptosis are often accompanied by changes in cell morphology. The apoptosis lymphocyte is rougher than those of the two other morphotypes, and coated with many big particles. Using spatially resolved force–distance curves, we found that the valve of the activated lymphocyte is about two to three times stiffer (Young's modulus of ～20 kPa) than those of the two other morphotypes (5–11 kPa). These results can improve our understanding of the mechanical properties of cells during growth and differentiation.     

Ectaquasperm-like parasperm in an internally fertilizing gastropod

Abstract. Pomacea canaliculata is an internally fertilizing gastropod that produces, besides fertilizing sperm (=eusperm), a large number of unfertile sperm (=parasperm) that have no chromatin, are fusiform, and have three to five flagella. Here, we report that this snail also produces another type of parasperm, which results from a peculiar spermiogenesis including an anterior cytoplasmic migration. The mature oligopyrene parasperm has: (1) a rounded head including a partly lysed nucleus, (2) a conical mid-piece with eight large mitochondrial structures, and (3) a single flagellum (∼20 μm). These characteristics, although not found in any other gastropod parasperm, are shared with the externally fertilizing "ectaquasperm" and with the early spermiogenic stages of internally fertilizing "introsperm" found among the Annelida and basal Mollusca. There are indications that this sperm type may be produced by a truncation of euspermiogenesis, as proposed by Buckland-Nicks & Scheltema for the expression of ectaquasperm in bilaterian evolution.     

Obligate costs of parental care to offspring: egg brooding-induced hypoxia creates smaller, slower and weaker python offspring

Python egg brooding typifies parental care because it consists of multiple behaviours that provide for multiple developmental needs. For example, tightly coiling around the eggs benefits embryonic water balance, but periodic female postural adjustments improve embryonic gas exchange. Regardless of these postural adjustments, egg brooding creates a hypoxic intra-clutch environment that constrains embryonic metabolism. We further examined this novel and useful parental care model to determine: (1) any fitness-related costs of egg brooding to offspring; (2) whether any long-term costs are alleviated by postural adjustments. We artificially incubated Children's python ( Antaresia childreni ) clutches and modulated oxygen partial pressure ( P O₂) to create three treatments: normoxic (NRM, 20.3 kPa O₂), brooding [BRD, P O₂ profile typical of clutch P O₂ ( P O_2clutch) in maternally brooded clutches, 15.8–19.3 kPa O₂] and low (LOW, predicted P O₂ profile of maternally brooded P O_2clutch if females did not make postural adjustments, 14.4–18.6 kPa O₂). Using various metrics from ∼12 days pre-hatching to 14 days post-hatching, we demonstrated that NRM offspring were larger, faster and stronger than BRD offspring. As only hatchling heart mass differed between BRD and LOW treatments (LOW > BRD), postural adjustments may not alleviate hypoxia-related costs to embryos. Our results demonstrate that parental care may represent a compromise between competing developmental needs and thus entails obligate costs to the offspring.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 98 , 414–421.     

Holocene carbon burial by lakes in SW Greenland

The role of the Arctic in future global change processes is predicted to be important because of the large carbon (C) stocks contained in frozen soils and peatlands. Lakes are an important component of arctic landscapes although their role in storing C is not well prescribed. The area around Kangerlussuaq, SW Greenland (66–68°N, 49–54°W) has extremely high lake density, with ∼20 000 lakes that cover about 14% of the land area. C accumulation rates and standing stock (kg C m⁻²), representing late- to mid-Holocene C burial, were calculated from AMS ¹⁴C-dated sediment cores from 11 lakes. Lake ages range from ∼10 000 cal yr  bp to ∼5400 cal yr  bp , and reflect the withdrawal of the ice sheet from west to east. Total standing stock of C accumulated in the studied lakes for the last ∼8000 years ranged from 28 to 71 kg C m⁻², (mean: ∼42 kg C m⁻²). These standing stock determinations yield organic C accumulation rates of 3.5–11.5 g C m⁻² yr⁻¹ (mean: ∼6 g C m⁻² yr⁻¹) for the last 4500 years. Mean C accumulation rates are not different for the periods 8–4.5 and 4.5–0 ka, despite cooling trends associated with the neoglacial period after 4.5 ka. We used the mean C standing stock to estimate the total C pool in small lakes (<100 ha) of the Kangerlussuaq region to be ∼4.9 × 10¹³ g C. This C stock is about half of that estimated for the soil pool in this region (but in 5% of the land area) and indicates the importance of incorporating lakes into models of regional C balance at high latitudes.     

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

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

Ultrastructure of the midgut and hindgut of Derocheilocaris remanei (Crustacea, Mystacocarida)

Ultrastructural features and structure of the midgut and hindgut of Derocheilocaris remanei were studied. The large endodermal midgut is differentiated into an anterior midgut and a posterior midgut separated by a conspicuous constriction. Both circular and longitudinal striated muscle bands surround the midgut, while the hindgut only presents longitudinal muscles. The limit between the midgut and the cuticle-lined hindgut is marked by a rectal valve. In cross-section, the short hindgut is triradiate and has a distinct Y-shaped lumen. The hindgut cuticular lining appears interrupted at the tip of every branch of the Y. Three different cell types are found in the midgut epithelium: basally located undifferentiated cells that give rise to the other two specialized cell types; secretory zymogen-like cells responsible for extracellular digestion and located mainly in the anterior midgut; and vacuolated cells, distributed all along the midgut and appearing to have several functions, including absorption, intracellular digestion, and nutrient transport. A single basic cell type forms the hindgut epithelium. The suggested function for the hindgut is the transport and ejection of waste products.     

Substrate stiffness affects the functional maturation of neonatal rat ventricular myocytes

Cardiac cells mature in the first postnatal week, concurrent with altered extracellular mechanical properties. To investigate the effects of extracellular stiffness on cardiomyocyte maturation, we plated neonatal rat ventricular myocytes for 7 days on collagen-coated polyacrylamide gels with varying elastic moduli. Cells on 10 kPa substrates developed aligned sarcomeres, whereas cells on stiffer substrates had unaligned sarcomeres and stress fibers, which are not observed in vivo. We found that cells generated greater mechanical force on gels with stiffness similar to the native myocardium, 10 kPa, than on stiffer or softer substrates. Cardiomyocytes on 10 kPa gels also had the largest calcium transients, sarcoplasmic calcium stores, and sarcoplasmic/endoplasmic reticular calcium ATPase2a expression, but no difference in contractile protein. We hypothesized that inhibition of stress fiber formation might allow myocyte maturation on stiffer substrates. Treatment of maturing cardiomyocytes with hydroxyfasudil, an inhibitor of RhoA kinase and stress fiber-formation, resulted in enhanced force generation on the stiffest gels. We conclude that extracellular stiffness near that of native myocardium significantly enhances neonatal rat ventricular myocytes maturation. Deviations from ideal stiffness result in lower expression of sarcoplasmic/endoplasmic reticular calcium ATPase, less stored calcium, smaller calcium transients, and lower force. On very stiff substrates, this adaptation seems to involve RhoA kinase.     

STARRIA ZIMBABWEËNSIS (CYANOPHYCEAE) GEN. NOV. ET SP. NOV.: A FILAMENT TRIRADIATE IN TRANVERSE SECTION1

A new filamentous bluegreen alga Starria zimbabweënsis gen. nov. et sp. nov. isolated from a soil sample collected near Zimbabwe, Southern Rhodesia, has a unique triradiate morphology. In transverse section each narrow cell possesses three arm-like projections, separated by 120° and in which pigment is concentrated. Filaments may be straight or twisted and various clonable biradiate forms have originated in cultures initiated from the triradiate type. Cell ultra-structure is typical of the Oscillatoriaceae except that 70 nm pit-like pores occur throughout the L-II layer of the longitudinal walls. Wall structure of deviant forms is identical to that of the triradiate “wild type.” The organism is assigned to the Oscillatoriaceae rather than to the Gomontiellaceae or to a new family.     

Multiparametric Analyses Reveal the pH-Dependence of Silicon Biomineralization in Diatoms

Diatoms, the major contributors of the global biogenic silica cycle in modern oceans, account for about 40% of global marine primary productivity. They are an important component of the biological pump in the ocean, and their assemblage can be used as useful climate proxies; it is therefore critical to better understand the changes induced by environmental pH on their physiology, silicification capability and morphology. Here, we show that external pH influences cell growth of the ubiquitous diatom Thalassiosira weissflogii, and modifies intracellular silicic acid and biogenic silica contents per cell. Measurements at the single-cell level reveal that extracellular pH modifications lead to intracellular acidosis. To further understand how variations of the acid-base balance affect silicon metabolism and theca formation, we developed novel imaging techniques to measure the dynamics of valve formation. We demonstrate that the kinetics of valve morphogenesis, at least in the early stages, depends on pH. Analytical modeling results suggest that acidic conditions alter the dynamics of the expansion of the vesicles within which silica polymerization occurs, and probably its internal pH. Morphological analysis of valve patterns reveals that acidification also reduces the dimension of the nanometric pores present on the valves, and concurrently overall valve porosity. Variations in the valve silica network seem to be more correlated to the dynamics and the regulation of the morphogenesis process than the silicon incorporation rate. These multiparametric analyses from single-cell to cell-population levels demonstrate that several higher-level processes are sensitive to the acid-base balance in diatoms, and its regulation is a key factor for the control of pattern formation and silicon metabolism.     

Polyaluminum chloride-enhanced concentration efficiency of poliovirus and f2 phage from sewage water

Aims:  To enhance the recovery of f₂ bacteriophage and poliovirus by an established method based on the adsorption to and elution from positively-charged Al(OH)₃-treated silica gel.
Methods and Results:  Polyaluminum Chloride (PAC) was added to water samples to neutralize the negatively charged materials, which can reduce virus recovery by providing a competing adsorption mode on the media surface. Using this improved process (PAC 30 mg l⁻¹, pH 6·5, temperature 20∼30°C), the recoveries of Poliovirus I and f₂ from small-volume sewage (100 ml) were 110·76 ± 36·0% and 92·06 ± 8·65%, respectively ( P  < 0·05 vs. traditional methods). Recovery from a 20-L volume of sewage averaged 85·65 ± 4·43% for f₂ and 88·73 ± 9·76% for poliovirus, significantly higher than the recoveries in the traditional methods ( P  < 0·05).
Conclusions:  PAC could enhance concentration efficiency of poliovirus and f₂ phage from sewage water.
Significance and Impact of the Study:  This method should significantly improve the recovery of viruses from sewage.     

Quantitative biogeography in the South America highlands—recognizing the Altoandina, Puna and Prepuna through the study of Poaceae

The distribution data of 340 grass species sampled in a region of 53.219 km² in the northwestern corner of Argentina (between ∼21°S and ∼24°S) were analyzed to search for concordance in species distributions by using the program NDM/VNDM. Here, the traditional biogeographic hypothesis proposed for the region is evaluated for the first time by using a quantitative method and an optimal criterion specifically developed within the context of areas of endemism. Three different grid sizes (0.5° × 0.5°, 0.35° × 0.35 ° and 0.2° × 0.2°) were used to analyze three nested data sets: species found in the Andes of Argentina, Bolivia and/or Chile; Andean distributed species; and all grass species found in the study region. The main areas supported by the analyses correspond generally to the traditional biogeographic hypothesis proposed for the region. Local distribution patterns defined by species restricted to the study region were best supported under the small grid sizes, while the bigger grid sizes recovered areas defined by species with a broader distribution. The local distribution patterns emerged in all the analyses even when widespread species were added to the data set.
 © The Willi Hennig Society 2009.     

Alpine biogeography of Parnassian butterflies during Quaternary climate cycles in North America

Growth of alpine glaciers during the Pleistocene had profound effects on montane landscapes in North America and the organisms now inhabiting alpine ecosystems. Biogeography of this region has often been viewed as a system of sky islands despite the fact that species richness patterns deviate from a strict island biogeographic model. One explanation is that alpine species are not in equilibrium because of late Quaternary geographic range shifts. Genetic data can provide evidence of nonequilibrium dynamics and the distributional shifts that occur during glaciation events in alpine landscapes. Using mitochondrial and nuclear sequence data, we examine the evolutionary history of butterflies in the Parnassius phoebus complex. We test explicit, alternative models of the biogeographic history of Parnassius smintheus and Parnassius behrii , including an equilibrium island model, ancestral radiation and fragmentation, an expanding alpine archipelago and an alpine archipelago refuge model. Our results support the alpine archipelago refuge model, in which alpine butterflies undergo population contraction during glacial climates followed by population expansion during interglacial phases. While butterflies can disperse between distant mountain ranges during glacial periods, gene flow is rare. We find evidence of recent connectivity between California and Colorado, population expansion events following deglaciation ∼20 000 years B.P., and small population sizes during the last glacial period. An analysis of lineage splitting suggests that morphological differences in P. smintheus and P. behrii are the result of late Pleistocene divergence (∼48 000 years B.P.) with limited gene flow. Our results demonstrate that spatially complex and nonequilibrium population dynamics influence alpine diversity patterns.     

Interaction of germanium (Ge) with biosilicification in the freshwater sponge Ephydatia mülleri: evidence of localized membrane domains in the silicalemma

In the presence of germanium (Ge) the needle-shaped silica spicules of the freshwater sponge Ephydatia m ulleri are very short and thin and possess bulbs with large spines. SEM-coupled X-ray analyses confirm the incorporation of Ge into the silica. A small number of bulbs are susceptible to erosion by HNO3 and hypochlorite and although the chemical basis of such erosion is presently unknown it suggests the presence of an organic matrix within the bulbs and/or an incomplete polymerization of the silica. Addition of Ge to control media in which silicification is newly initiated increases the incidence of erosion and results in centrally located eroded areas of the silica and discontinuities in its deposition. Removal of Ge from such newly forming structures results in a partial recovery of normal morphology (spine development and thickening of the silica) but only in the central region surrounding the bulbs. Both results establish the presence of a central, active region for silicification and further support the view that there is a distal spreading, away from this center, of transported forms of silica. Secondary centers may also be present. The newly assembled organic core of control structures is associated with tubular elements possibly derived from the surrounding membrane. In such newly silicifying structures the spicule tips contain oriented material in the form of "rays." Both of these new observations increase the likelihood of the presence of an organic matrix within the silica.     

Transmembrane redox control and proteolysis of PdeC,a novel type of c‐di‐GMP phosphodiesterase

The nucleotide second messenger c‐di‐GMP nearly ubiquitously promotes bacterial biofilm formation, with enzymes that synthesize and degrade c‐di‐GMP being controlled by diverse N‐terminal sensor domains. Here, we describe a novel class of widely occurring c‐di‐GMP phosphodiesterases (PDE) that feature a periplasmic “CSS domain” with two highly conserved cysteines that is flanked by two transmembrane regions (TM1 and TM2) and followed by a cytoplasmic EAL domain with PDE activity. Using PdeC, one of the five CSS domain PDEs of Escherichia coli K‐12, we show that DsbA/DsbB‐promoted disulfide bond formation in the CSS domain reduces PDE activity. By contrast, the free thiol form is enzymatically highly active, with the TM2 region promoting dimerization. Moreover, this form is processed by periplasmic proteases DegP and DegQ, yielding a highly active TM2 + EAL fragment that is slowly removed by further proteolysis. Similar redox control and proteolysis was also observed for a second CSS domain PDE, PdeB. At the physiological level, CSS domain PDEs modulate production and supracellular architecture of extracellular matrix polymers in the deeper layers of mature E. coli biofilms.