Morphology, Systematics and Life Cycle of Ozanamia fimbriatus (Haptista: Centroplasthelida), With Notes on Evolution of Organic Skeleton in Centrohelids.

On the phylogenetic position of the genus Raphidocystis (Haptista: Centroplasthelida) with notes on the dimorphism in centrohelid life cycle

21. The emerging role of nanotechnology in anatomical pathology

Systems biology in 3D space--enter the morphome.

Raphidocystis pallida, a centrohelid heliozoan with unusually shaped tubular siliceous scales, was reisolated from Jamor river, Portugal, and studied with the use of light and electron microscopy. In enriched cultures, the cells were naked, devoid of siliceous external skeleton with the exception of several scales present in one cell. Instead, such cells were covered with a layer of rod-shaped bacteria. In clonal cultures, the cells gradually acquired siliceous coverings typical for this species and retained them in next generations. Phylogenetic position of R. pallida was clarified with SSU rDNA-based molecular phylogenetics, and its placement within the genus Raphidocystis despite unusual coverings structure was confirmed. The implications of phylogenetic placement of R. pallida and possible origins of the previously undescribed naked form were discussed.

Phylogenetic position and morphology of Raphidiophrys elongata sp. nov. (Haptista: Centroplasthelida) with notes on cyst wall structure and evolution

Hansen G. and Daugbjerg N. 2011. Moestrupia oblonga gen. et comb. nov. (syn.: Gyrodinium oblongum), a new marine dinoflagellate genus characterized by light and electron microscopy, photosynthetic pigments and LSU rDNA sequence. Phycologia 50: 583–599. DOI: 10.2216/11-11.1A small-sized, peridinin-containing, athecate dinoflagellate (13–17 µm long) was isolated into clonal culture from a water sample collected at a nearshore location in Tenerife, Spain (October 2004). Based on phenotypic characters (size, shape, pyrenoid and nucleus position), the culture was identified as Gyrodinium oblongum. However, a detailed ultrastructural examination revealed a number of features that did not fit into the current delineation of the genus Gyrodinium or any other dinoflagellate. Likewise, the molecular phylogeny and sequence divergence estimates based on partial LSU rDNA sequences indicated that this isolate has a taxonomically isolated position, and we therefore propose the new genus Moestrupia to accommodate this species within the Dinophyceae. The ultrastructural study uncovered a number of unique features for Moestrupia, for example, a microtubule supported ventral flange situated along the right cingular border and onto the episome. Also, the exit point of the peduncle, through a lip-like protrusion situated in a cavity on the episome, is new in dinoflagellates. The transverse flagellum appeared less coiled compared to other dinoflagellates, and its distal end terminated some distance within the wide sulcus. The upper cingulum border had a distinct rim supported by microtubules; whereas, the lower cingulum border was indistinct. The apical groove was short and curved and bordered on each side by a delicate rim, which may represent amphiesmal vesicles. It ran from the anterior part of the ventral flange to the middorsal side of the episome. Thus, it differs markedly from that in species of Gyrodinium and emphasizes it being distantly related to this genus.

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

Silicate scaling during high pH Alkaline Surfactant Polymer (ASP) flooding is known to adversely affect oil production. The silicate scale occurs as a result of the dissolution of silicates under high pH conditions and where the fluids subsequently flow into a region of lower pH where they then precipitate. The precipitation of magnesium silicate strongly depends on solution pH and temperature and is affected by the kinetics of the silicate scaling reaction. In this paper, the effect of pH on the stoichiometry and morphology of silicate scale is studied. A range of spectroscopic techniques, including Environmental Scanning Electron Microscope/ Energy Dispersive X-Ray Spectroscopy (ESEM/EDX), Fourier Transform Infrared (FTIR) and X-Ray Powder Diffraction (XRD) are applied in order to analyse the precipitated silicate scales in the laboratory. These spectroscopic techniques, when used along with reference sample spectra, yield a number of interpretive clues as to the nature of the silicate precipitates which are formed. The further analysis of the solution and precipitate by Inductively Coupled Plasma-Optical Emission Spectroscopy (ICP-OES) and ESEM/EDX also gave complementary information which was consistent with the results obtained from the other spectral methods above. The approach used in this work has enabled us to establish the composition and morphology of the silicate scales formed under different pH condition. There is relationship between the pH conditions and the compounds appearing in the precipitate. Results obtained can be used to help determine the most appropriate type of scale inhibitor for silicate scale mitigation in future.

The role of an organic matrix during the formation of siliceous scales in the heliozoon Actinophrys sol (actinophryida, protista)

K. Lindberg, Ø. Moestrup and N. Daugbjerg. 2005. Studies on woloszynskioid dinoflagellates I: Woloszynskia coronata re-examined using light and electron microscopy and partial LSU rDNA sequences, with description of Tovellia gen. nov. and Jadwigia gen. nov. (Tovelliaceae fam. nov.). Phycologia 44: 416–440.Sediment samples were collected from a small pond in southern Sweden. Several cysts from the samples germinated into clonal cultures, identified as Woloszynskia coronata (Wolosz.) R.H. Thompson 1951. They were compared with other species of Woloszynskia established in culture, using scanning electron microscopy, transmission electron microscopy, partial large subunit ribosomal DNA (LSU rDNA) and morphology of the resting cysts. Significant differences were found, and we conclude that the genus Woloszynskia as presently circumscribed is artificial, and comprises at least four genera. In this first paper we transfer W. coronata to a new genus; Tovellia gen. nov., type species: Tovellia coronata (Wolosz.) comb. nov. Previous studies on ultrastructure and DNA sequencing referring to Woloszynskia coronata are based on W. coronata var. glabra, which is raised to species level as Tovellia glabra sp. nov. Other species included in the new genus are Tovellia apiculata (basionym Woloszynskia apiculata Stosch) and Tovellia stoschii (basionym Woloszynskia stoschii R. Shyam & Sarma). Two identical cultures presently identified as Woloszynskia limnetica Bursa (from University of Washington Culture Collection, Seattle) and W. pseudopalustris (J. Schiller) Kiselev [from Culture Collection of Algae at the University of Cologne, Cologne] differ from Tovellia in LSU rDNA sequences and in cyst type and are transferred to Jadwigia gen. nov., as J. applanata sp. nov. The most striking feature of Tovellia and Jadwigia is the anatomy of the eyespot, which is extraplastidial, and composed of nonmembrane bound lipid globules. This type of eyespot is also present in Katodinium campylops (T.M. Harris) A.R. Loebl., a species undoubtedly related to Tovellia, and in ‘Glenodinium sp.’ sensu Kreimer 1999, and together they form a distinct family, Tovelliaceae fam. nov.

Species belonging to the genus Bysmatrum are peridinoid, thecate, photosynthetic dinoflagellates. The plate formula of Bysmatrum spp., arranged in a Kofoidian series, is almost identical to that of Scrippsiella spp. Bysmatrum spp., which were originally classified as Scrippsiella spp., but were transferred to the genus Bysmatrum spp. because of separation of the intercalary plates 2a and 3a by plate 3′. Whether this transfer from Scrippsiella spp. to Bysmatrum spp. is reasonable should be genetically confirmed. Dinoflagellates were isolated from 2 solar saltons located in western Korea in 2009–2010 and 3 clonal cultures from Sooseong solar saltons and 2 clonal cultures from Garolim solar saltons were successfully established. All of these dinoflagellates were identified as Bysmatrum caponii based on morphology analysis by light and electron microscopy. The plates of all Korean strains of B. caponii were arranged in a Kofoidian series of Po, X, 4′, 3a, 7″, 6c, 4s, 5‴, 0 (p), and 24’. When properly aligned, the ribosomal DNA (rDNA) sequences of the 3 Sooseong strains of B. caponii were identical, as were those of the 2 Garolim strains. Furthermore, the sequences of the 3 Sooseong strains were 0.01% different from those of the Garolim strains. However, the sequences of SSU rDNA of these Korean B. caponii strains were 9% different from that of Bysmatrum subsalsum and > 10% from that of any other dinoflagellate thus far reported. In the phylogenetic trees generated using SSU and LSU rDNA sequences, these Korean B. caponii strains formed a clade with B. subsalsum which was clearly divergent from the Scrippsiella clade. However, this Bysmatrum clade was phylogenetically close to the Protoperidinium and/or Peridinium clades. The results of the present study suggest that Bysmatrum spp. are markedly different genetically from Scrippsiella spp..

Correlative light and electron microscopy (CLEM) is a method used to investigate the exact same region in both light and electron microscopy (EM) in order to add ultrastructural information to a light microscopic (usually fluorescent) signal. Workflows combining optical or fluorescent data with electron microscopic images are complex, hence there is a need to communicate detailed protocols and share tips & tricks for successful application of these methods. With the development of volume-EM techniques such as serial blockface scanning electron microscopy (SBF-SEM) and Focussed Ion Beam-SEM, correlation in three dimensions has become more efficient. Volume electron microscopy allows automated acquisition of serial section imaging data that can be reconstructed in three dimensions (3D) to provide a detailed, geometrically accurate view of cellular ultrastructure. In addition, combining volume-EM with high-resolution light microscopy (LM) techniques decreases the resolution gap between LM and EM, making retracing of a region of interest and eventual overlays more straightforward. Here, we present a workflow for 3D CLEM on mouse liver, combining high-resolution confocal microscopy with SBF-SEM. In this workflow, we have made use of two types of landmarks: (1) near infrared laser branding marks to find back the region imaged in LM in the electron microscope and (2) landmarks present in the tissue but independent of the cell or structure of interest to make overlay images of LM and EM data. Using this approach, we were able to make accurate 3D-CLEM overlays of liver tissue and correlate the fluorescent signal to the ultrastructural detail provided by the electron microscope. This workflow can be adapted for other dense cellular tissues and thus act as a guide for other three-dimensional correlative studies. LAY DESCRIPTION: As cells and tissues exist in three dimensions, microscopy techniques have been developed to image samples, in 3D, at the highest possible detail. In light microscopy, fluorescent probes are used to identify specific proteins or structures either in live samples, (providing dynamic information), or in fixed slices of tissue. A disadvantage of fluorescence microscopy is that only the labeled proteins/structures are visible, while their cellular context remains hidden. Electron microscopy is able to image biological samples at high resolution and has the advantage that all structures in the tissue are visible at nanometer (10-9 m) resolution. Disadvantages of this technique are that it is more difficult to label a single structure and that the samples must be imaged under high vacuum, so biological samples need to be fixed and embedded in a plastic resin to stay as close to their natural state as possible inside the microscope. Correlative Light and Electron Microscopy aims to combine the advantages of both light and electron microscopy on the same sample. This results in datasets where fluorescent labels can be combined with the high-resolution contextual information provided by the electron microscope. In this study we present a workflow to guide a tissue sample from the light microscope to the electron microscope and image the ultra-structure of a specific cell type in the liver. In particular we focus on the incorporation of fiducial markers during the sample preparation to help navigate through the tissue in 3D in both microscopes. One sample is followed throughout the workflow to visualize the important steps in the process, showing the final result; a dataset combining fluorescent labels with ultra-structural detail.

ABSTRACTThe genus Mallomonas consists of single-celled flagellates covered with siliceous scales and bristles and is well known in freshwater environments. Two new marine Mallomonas species were collected from Dongho Beach, Jeollabukdo, Korea. To fully understand the taxonomy of the new species, we performed molecular phylogenetic analysis based on a concatenated dataset and observed morphological features using light and electron microscopy. For the phylogenetic analysis, we used a combined dataset from five gene sequences: nuclear small subunit (SSU) and large subunit (LSU) rDNA and plastid LSU rDNA, rbcL and psaA genes. The new species M. cuspis sp. nov. grouped with M. heterospina and M. oviformis in the section Planae. It had scales with a broad shield marked with V-shaped internal ridge that lacked submarginal ribs and a dome. The other new species, M. marina sp. nov., clustered with M. cratis, M. pseudocratis, M. asmundiae and M. striata var. serrata in section Striatae and had scale characters that matched the group, including a well-developed posterior submarginal rib and series of transverse ribs on the shield and dome. In addition, we investigated the effect of salinity on growth rate and cell morphology. The two marine species represented high growth rate between salinities of 10 and 30 psu, suggesting that both species are well adapted in marine environments. When exposed to salinities of 0 and 5 psu, they underwent cell enlargement.

Ophiocordyceps unituberculata, a new insect pathogenic fungus from southwestern China, is described using molecular phylogenetic and morphological data. This fungus differs from other Ophiocordyceps species by its enormously long monophialidic conidiogenous cells and a large periclinal protuberance often growing near the apex of conidiogenous cells, single conidia (lanceolate to fusiform) embedded in mucous sheath, and much larger conidial dimensions. Four-locus (nrSSU, nrLSU, tef-1α and rpb1) and ITS data phylogenetic analyses show that O. unituberculata belongs to the Hirsutella nodulosa clade within the genus Ophiocordyceps of Ophiocordycipitaceae and is a separate clade from other allied species. Molecular phylogeny and morphology both strongly support the distinctiveness of this taxon. The interspecific relationships in the H. nodulosa clade are discussed.

The symbiont Capsaspora owczarzaki, nov. gen. nov. sp., isolated from three strains of the pulmonate snail Biomphalaria glabrata is related to members of the Mesomycetozoea