The ultrastructure of the extrusomes in <Emphasis Type="Italic">Pseudourostyla cristata</Emphasis>, a hypotrichous ciliated protozoan

By using scanning and transmission electron microscopy, the present study demonstrates a great number of trichocyst-like extrusomes distributed in the cortical cytoplasm of the protozoan Pseudourostyla cristata, a hypotrichous ciliate. Of these, the mature organelles are rod-shaped with a cap consisting of tubular structures, a tip located at the apex of the cap, a body consisting of strateform structures of uneven electron density and an elongated shaft located along the longitudinal central axis of the body. The electron microscopic observations suggest that the extrusive organelles in P. cristata might undergo a morphogenetic process including the following sequential events: the occurrence of the vesicles in the cytoplasm, the condensation of the fibrous substances within the vesicles, the appearance of the electron-dense shaft, and the formation of the cap. In contrast with a large quantity of extrusomes in trophozoit P. cristata, there are no such extrusive organelles in the encysted cells of the ciliate. The phenomena that P. cristata ciliates can readily enter physiological reorganization or encysting phases and discharge a great number of their extrusomes when prepared for SEM and TEM observation suggest that the extrusive process of the extrusomes in P. cristata might have an important influence on the life activity of the ciliate and could be one of the causes leading to the physiological reorganization and the encysting of the ciliate. These reactions of P. cristata might be a protective or defensive response to the environmental changes.     

Discotricha papillifera: Structure and Morphogenesis of a Marine Interstitial Ciliate*

SYNOPSIS. Discotricha papillifera Tuffrau, a marine interstitial ciliate, is redescribed with the aid of light, scanning, and transmission electron microscopy from cells collected at a New Hampshire beach. Presence of primitive membranelles as well an an advanced stomatogenesis is demonstrated. The ultrastructure, including a unique membrane-bounded septate structure, is described. The cell is tentatively placed in the nassulid suborder Microthoracina, but affinities with other groups are discussed.     

A re-description of the ciliate genus and type species, Balantidium entozoon

Members of the ciliate genus Balantidium possess a specialized “Villeneuve-Brachon's” field of somatic cilia to the right of the vestibule, or in a dextroral location. Specimens of the type species were collected in Italy and fixed for study by light microscopy, and scanning and transmission electron microscopy. The presence of the field in the type species and several other species of the genus indicates a need to re-describe the genus by including details of the ultrastructure of that field. Scanning electron microscopy shows that the field consists of one row of relatively short cilia of uniform length flanked on each side by 2–3 rows, or more, of very short cilia. Their kinetids have typical litostome structure in transmission electron micrographs. We speculate on a possible function for the Villeneuve-Brachon's field and also present morphometric data on the type species. The base sequence of the small subunit ribosomal RNA gene of Balantidium entozoon has been determined and found to differ by 5% from that of B. coli. Based on the location and ultrastructure, organelles found around the somatic kinetosomes and within inter-kinetal ridges of B. entozoon were identified as hydrogenosomes.     

Hydrogenosomes in the rumen entodiniomorphid ciliate Polyplastron multivesiculatum

The rumen entodiniomorphid ciliate protozoon Polyplastron multivesiculatum was shown, by biochemical and electron microscopic techniques, to possess hydrogenosomes. After differential centrifugation of whole cell homogenates the hydrogenosomal marker enzymes pyruvate:ferredoxin oxidoreductase and hydrogenase were recovered predominantly (61% and 70% of activity respectively) in the large granular fractions that were sedimented by centrifugation for 10(4) g-min (fraction P1) and 10(5) g-min (fraction P2). These subcellular fractions contained membrane-bound organelles that were approximately 0.4-0.6 microns in diameter and which had a mean equilibrium density of 1.22-1.24 g ml-1 after isopycnic centrifugation in sucrose gradients. Malate dehydrogenase (decarboxylating) activity, however, was predominantly non-sedimentable after centrifugation for 6 x 10(6) g-min. Numerous hydrogenosome-like organelles were present in the ectoplasm and endoplasm of the cell. Hydrogenase activity was demonstrated and localized in the protozoan cell using a novel staining procedure with distyryl nitroblue tetrazolium chloride (DSNBT).     

The Fine Structure of Saprodinium dentatum Lauterborn, 1908 as a Representative of the Odontostomatida (Ciliophora)

The fine structure of the sapropelic ciliate Saprodinium dentatum is described based on phase-contrast microscopy, silver-staining techniques, cryo-fracture scanning electron microscopy, and thin sections. The study concentrates on a detailed analysis of the somatic cortex and the oral ciliature of this highly asymmetric, laterally compressed ciliate. The cell shape is dominated by a number of site-specific spines and the curving course of 10 somatic kineties (SK 1–10). The SK, composed of dikinetids, show an intrakinety differentiation that seems characteristic for other odontostomes as well. The anterior segment of the SK is mostly ciliated, followed by a non-ciliated segment in which the kinetosomes lack all typical fiber systems. Except for SK 4–6, the posterior segment is ciliated again, forming the spine kinetics associated with particular caudal spines. The anterior segment of SK 3 through SK 7 form the frontal band, which together with the two frontal kineties constitutes the main locomotory organelle for a ciliate that creeps on the substratum. A short kinety with inverse polarity, not seen in earlier light microscopical studies, was observed near the oral spine. We made particular effort to find a logical explanation for the observed association of the SK with the various caudal spines. The oral ciliature consists of nine adoral organelles located in a tripartite oral cavity. The absence of a paroral ciliature together with the position of the cytostome anterior to the adoral organelles may be the result of rotational movement of the oral apparatus during the evolution of these bizarre ciliates. Results are discussed with special reference to the phylogenetic relationship of the Odontostomatida to the Heterotrichida and no conclusive answer was found in this first electron microscopical study of an odontostomatid ciliate.     

New views of tapetum ultrastructure and pollen exine development in Arabidopsis thaliana

Background and Aims

The Arabidopsis thaliana pollen cell wall is a complex structure consisting of an outer sporopollenin framework and lipid-rich coat, as well as an inner cellulosic wall. Although mutant analysis has been a useful tool to study pollen cell walls, the ultrastructure of the arabidopsis anther has proved to be challenging to preserve for electron microscopy.

Methods

In this work, high-pressure freezing/freeze substitution and transmission electron microscopy were used to examine the sequence of developmental events in the anther that lead to sporopollenin deposition to form the exine and the dramatic differentiation and death of the tapetum, which produces the pollen coat.

Key Results

Cryo-fixation revealed a new view of the interplay between sporophytic anther tissues and gametophytic microspores over the course of pollen development, especially with respect to the intact microspore/pollen wall and the continuous tapetum epithelium. These data reveal the ultrastructure of tapetosomes and elaioplasts, highly specialized tapetum organelles that accumulate pollen coat components. The tapetum and middle layer of the anther also remain intact into the tricellular pollen and late uninucleate microspore stages, respectively.

Conclusions

This high-quality structural information, interpreted in the context of recent functional studies, provides the groundwork for future mutant studies where tapetum and microspore ultrastructure is assessed.     

An anaerobic protozoon, with symbiotic methanogens, living in municipal landfill material

Abstract We have established that anaerobic protozoa do live in municipal landfill material although they probably spend much of the time encysted, especially in the drier (< 40% water) site. At least eight species were observed; they were readily isolated by adding anoxic water to dry landfill samples. The ciliate Metopus palaeformis was frequently i isolated; it appears to be ubiquitous in anaerobic landfills. It has a polymorphic life cycle, it is positive for hydrogenase, each ciliate contains about 500 bromoethanesulfonate-sensitive methanogen symbionts (probably Methanobacterium formicicum ), and maximum cell densities in culture exceed 3000 per ml. The methanogens are not attached to the hydrogenosomes, neither do they undergo morphological transformation; the ciliate receives no measurable energetic advantage from its symbionts. The ciliate encysts in response to a shortage of food or water, and the methanogens remain viable within the cysts. When the protozoon excysts, the methanogens resume growth and cell division within the trophic form of the ciliate. Unlike free-living methanogens, the M. palaeformis -methanogen consortium is not particularly sensitive to oxygen; the symbiotic methanogens remain viable following exposure of the consortium to atmospheric oxygen for several days. Dispersal of methanogen-bearing protozoan cysts through oxygenated environments is a potential mechanism of transfer between landfill sites and other anaerobic environments. Anaerobic protozoan consortia are theoretically capable of making a significant contribution to methane generation from wet landfill sites.     

Electron microscopic studies on the macronuclear development in the ciliate Chilodonella uncinata.

The development of macronuclear anlage in the ciliate Chilodonella uncinata has been studied through electron microscopy. The ultrastructure of macro- and micronuclei is described for comparison. During the first stage of development, when the DNA content of the macronuclear anage increases from 2 C to 32 C, chromosomes are visible as distinct osmiophilic bodies inside the anlage. At the end of the initial polyploidization phase, the chromosomes despiralize, giving rise to long filamentous structures 25 to 50 nm in diameter. The latter show a singular banding pattern, with dense bands 12 to 25 nm thick alternating regularly with less dense interbands. Such an organization has not yet been observed in any other type of nucleus. These filamentous structures have been interpreted as highly despiralized oligotenic chromosomes. During the final stage of macronuclear development, these structures condense into thin chromatin strands and small dense granules; the number of granules increases progressively as the chromatin strands disappear. These small granules very likely fuse amongst themselves to form the chromatin granules of the vegetative macronucleus. No evidence has yet been found for a fragmentation of chromatin in this ciliate, but this problem needs further study. The old macronucleus maintains a normal ultrastructure until a late stage of development of the macronuclear anlage, becoming pycnotic only towards the very end of the latter process.     

THE EFFECT OF STRYCHNINE AND LIGHT ON PIGMENTATION IN BLEPHARISMA UNDULANS STEIN

The effect of strychnine sulfate and light on pigmentation in the ciliate protozoan Blepharisma undulans has been determined. Upon exposure of cells to strychnine, the pigment granules become loosened from their surrounding membranes. Eventually these membranes break and the granules are simultaneously released from the cell. At the cell surface, a fusion occurs between adjacent membraneless granules with the incorporation of membrane fragments. This fusion of granules and membrane fragments results in the formation of a pigmented "capsule" around the organism. After elimination of the pigment, the granule membranes remaining in the cytoplasm fuse to form apparently empty vesicles. Other cell organelles are generally undisturbed. A similar situation occurs upon exposure of cells to artificial light for 12 to 18 hr, however, the slow elimination of granules from the cells under these conditions does not result in the formation of a pigmented "capsule." The possible mechanisms of these reactions are discussed.     

Ultrastructure and Ecology of Perispira ovum (Ciliophora: Litostomatea): An Aerobic, Planktonic Ciliate that Sequesters the Chloroplasts, Mitochondria, and Paramylon of Euglena proxima in a Micro-oxic Habitat

ABSTRACT. High resolution sampling of the stratified water column in a fjord-like ecosystem revealed a green-pigmented planktonic ciliate that was found to be a ravenous predator of Euglena proxima. The vertical distributions of both predator and prey were coincident, and maximum populations occurred across the transition from oxic to anoxic water. This ciliate was identified as Perispira ovum (family Spathidiidae; Order Haptorida). P. ovum was observed by transmission electron microscopy to retain not only the chloroplasts, but also the mitochondria and paramylon reserve of its algal prey. A mechanism of sequestration of algal organelles is demonstrated for the first time. This mechanism includes: recognition, capture, and ingestion of prey; rupture and release of algal cell contents; and enrobing of individual organelles and paramylon by the host vacuolar membrane. The structural integrity, peripheral location, and association with host endoplasmic reticulum suggests the sequestered organelles may be functional within P. ovum. The occurrence and high biomass of this aerobic ciliate in an oxygen-limited environment also suggests that the sequestered chloroplasts are photosynthetically active and may provide additional substrates (such as oxygen) and metabolic capabilities that are crucial for its survival.     

The Fine Structure of the Cytostome of the Apostomatous Ciliate Hyalophysa chattoni*

SYNOPSIS. The fine structure of the organelles concerned with the ingestion of exuvial fluid by the trophont of the apostome ciliate, Hyalophysa chattoni, has been examined. One of the taxonomic characteristics of the order Apostomatida is that cytostomes of ciliates within the taxon are reduced and evolving toward astomy. When examined by electron microscopy the cytostome of H. chattoni appears as a small region of active pinocytosis which is continuous with a very large cortical area, the extended cytostome. The fine structure of the extended cytostome resembles that of the cytostomes of ciliates from other orders in that it is covered by a single membrane underlain with microtubular ribs. Beneath the extended cytostome are accumulations of peculiar organelles that may represent stored membrane for recycling during food vacuole formation. Associated with the site of pinocytosis is a complex fiber that may be contractile.     

Subcellular distribution of marker enzymes in cells of a minute fungus, Fusidium sp. 100-3

An electron microscope cytochemical technique was used to determine the subcellular distribution of marker enzymes in Fusidium sp. 100-3 cells. Nucleoside diphosphatase was found in the nuclear envelope and intracytoplasmic membrane segment. Thiamine pyrophosphatase was found to be associated with the mesosomes. Cytochrome c (oxidase) activity was found only in the mitochondrial cristae. Strong alkaline phosphatase activity was present in the vacuole; in addition, the enzyme activity was discretely dispersed throughout the cytoplasm without any association with any membrane material. The overall characteristics of the cell ultrastructure and subcellular enzyme distribution of Fusidium sp. 100-3 cells compare fairly well with those of a fungal cell. But there are considerable differences from the characteristics of higher eucaryotic cells. Detailed data on the marker enzymes distribution in a variety of fungal cells are not available. Therefore, it is not possible to conclude whether the marker enzyme distribution of Fusidium sp. 100-3 cells is unique or is typical of any fungal organism. Detailed studies of cell ultrastructure of and marker enzyme distribution in minute fungal cells and their comparison to the ultrastructure of and marker enzyme distribution in other fungal organisms may be helpful in understanding the phylogenetic and ontogenic development of subcellular organelles.     

Studies in the electron microscope of the adoral zone of membranelles of the rumen ciliate entodinium caudatum using the technique of negative staining

The structure of the organelles situated beneath the kinetosomes in the adoral zone of membranelles of the rumen ciliate Entodinium caudatum have been investigated in the electron microscope using the technique of negative staining. Each kinetosome was joined to a sub-kinetosomal plate and these plates were joined together in rows which in turn were more loosely linked to form a sheet. The structure or these plates is described and their relationship to similar structures in other protozoa is discussed. The nature of the argentophilic structures observed in the light microscope at the anterior end of Entodinium caudatum has also been investigated in the electron microscope.     

The Excystment Process in the Ciliate Euplotes muscicola: An Integrated Light and Scanning Electron Microscopic Study1

Resting cysts and the excystment process in the freshwater ciliate Euplotes muscicola were studied by both light and scanning electron microscopy. Groups of distinctly crested resting cysts adhere to the substrate. Silver-stained preparations reveal surface conservation of dorsal kinetosomes and dorsal argyrome while ventral organelles are directed inward. Excystment involves the development of an expanding excystment vacuole concurrent with a localized thinning on the dorsal cyst wall surface. Cells exit through the pre-formed ostiole, mid-dorsal region first, initially by the force of cytoplasmic streaming, but later aided by cirral movement. Newly emerged cells retain the excystment vacuole and show no dorsal ridging. As the cell expels its excystment vacuole and partially unfolds, normal trophont morphology is re-established. Both cyst structure and cyst typology have implications for hypotrich taxonomy.     

Lessons from tomographic studies of the mammalian Golgi

Basic structure studies of the biosynthetic machinery of the cell by electron microscopy (EM) have underpinned much of our fundamental knowledge in the areas of molecular cell biology and membrane traffic. Driven by our collective desire to understand how changes in the complex and dynamic structure of this enigmatic organelle relate to its pivotal roles in the cell, the comparatively high-resolution glimpses of the Golgi and other compartments of the secretory pathway offered to us through EM have helped to inspire the development and application of some of our most informative, complimentary (molecular, biochemical and genetic) approaches. Even so, no one has yet even come close to relating the basic molecular mechanisms of transport, through and from the Golgi, to its ultrastructure, to everybody's satisfaction. Over the past decade, EM tomography has afforded new insights into structure-function relationships of the Golgi and provoked a re-evaluation of older paradigms. By providing a set of tools for structurally dissecting cells at high-resolution in three-dimensions (3D), EM tomography has emerged as a method for studying molecular cell biology in situ. As we move rapidly toward the establishment of molecular atlases of organelles through advances in proteomics and genomics, tomographic studies of the Golgi offer the tantalizing possibility that one day, we will be able to map the spatio-temporal coordinates of Golgi-related proteins and lipids accurately in the context of 4D cellular space.     

Cellular and body scale morphology of Heterocapsa huensis sp. nov. (Peridiniales, Dinophyceae) found in Hue, Vietnam

Cellular and body scale structure of a new armored dinoflagellate Heterocapsa huensis , collected from Hue, Vietnam were investigated. Morphology of motile cell was observed by light, fluorescent and scanning electron microscopy, and body scale structure was examined by whole mounts of transmission electron microscopy. Cells of H. huensis were ellipsoid with a spherical nucleus located in the posterior and multiple pyrenoids located above the nucleus; this arrangement was similar to that of Heterocapsa pygmaea . Transmission electron microscopy revealed ultrastructure of the body scales consisted of a rounded triangular basal plate and three-dimensional ornaments. Structure of the basal plate resembles that of Heterocapsa illdefina ; however, the number of the peripheral spine is different from that of H. illdefina and this structure has never been reported from Heterocapsa species. A new Heterocapsa species, H. huensis Iwataki et Matsuoka sp. nov., is described based on positions of organelles and body scale ultrastructure.     

The vasculature of the rat embryo: an electron microscope study

The ultrastructure of portions of the arterial and venous systems of the 11.5 day old Wistar rat embryos has been studied by scanning and transmission electron microscopy. The vessels at this stage of development are in the form of capillaries, and the arterial and venous types can be distinguished by the morphology of the endothelial cells by SEM. The endothelial cells of the arterial vessels gave prominent nuclear bulges and numerous microvilli apart from their spindle shape, whilst those of the veins appear flattened, are polygonal in shape, and have few microvilli. Transmission electron microscopy shows that the endothelial cells of the arteries and veins are identical in structure. The ultrastructure of these cells resembles that of endothelial cells at later stages of development including the adult type in that mature forms of cytoplasmic organelles are obtained. In studies on the intercellular junctions and fenestrations with lanthanum nitrate, the impression is formed that the vessels at this stage are impermeable to small molecular size particles, compared with adult capillaries. This suggests that cytoplasmic vesicles must play a major role in the transport of macromolecules in the 11.5 day embryonic vessels.     

Synchronous Division of an Endosymbiotic Methanogenic Bacterium In the Anaerobic Ciliate Plagiopyla Frontata Kahl

ABSTRACT The life cycle of a methanogenic bacterium, symbiotic within the marine, free-living anaerobic ciliate Plagiopyla frontata , was studied using light microscopy and transmission electron microscopy (TEM). the bacteria are disc-shaped. During the growth phase of the host, bacteria and hydrogenosomes (organelles which ferment pyruvate into acetate and hydrogen) are arranged in conglomerates resembling stacks of coins in which bacteria and hydrogenosomes alternate; hydrogenosomes always cap the ends of the stacks. During the growth phase, numbers of hydrogenosomes and bacteria remain constant (about 5,000 and 3,500 per cell, respectively). Hydrogenosomes increase in volume shortly after cell division. Methanogens increase in volume slowly during the growth phase of the ciliate and rapidly when the ciliate begins to divide. the hydrogenosomes divide mainly during the initial phases of cell division while the methanogens divide synchronously during the last phase of ciliate division. the timing of reproduction of the symbionts is controlled by the host-cell cycle. the ciliate is known to receive an energetic advantage from its symbionts. the suppression of continuous bacterial reproduction may trigger the secretion of excess bacterial production as soluble organic compounds, for use by the ciliate.     

Cellular architecture of Plasmodium falciparum-infected erythrocytes

Plasmodium falciparum is a protozoan parasite that is responsible for the most pathogenic form of human malaria. The particular virulence of this parasite derives from its ability to develop within the erythrocytes of its host and to subvert their function. The intraerythrocytic parasite devours haemoglobin, and remodels its host cell to cause adhesion to blood vessel walls. Ultrastructural studies of P. falciparum have played a major role in defining its cell architecture and in resolving cell biology controversies. Here we review some of the early studies and describe some recent developments in electron microscopy techniques that have revealed information about the organization of the parasite in the blood stage of development. We present images of P. falciparum at different stages of the life cycle and highlight some of the plasmodium-specific organelles, the haemoglobin digestive apparatus and the membrane structures that are elaborated in the host cell cytoplasm to traffic virulence proteins to the erythrocyte surface. We describe methods for whole cell ultrastructural imaging that can provide three-dimensional views of intraerythrocytic development.     

Electron microscopic observation of cytoskeletal frame structures and detection of tubulin on the apical region of Cryptosporidium parvum sporozoites

Cryptosporidium parvum is an intracellular protozoan parasite belonging to the phylum Apicomplexa, and a major cause of waterborne gastroenteritis throughout the world. Invasive zoites of apicomplexan parasites, including C. parvum, are thought to have characteristic organelles on the apical apex; however, compared with other parasites, the cytoskeletal ultrastructure of C. parvum zoites is poorly understood. Thus, in the present study, we ultrastructurally examined C. parvum sporozoites using electron microscopy to clarify the framework of invasive stages. Consequently, at the apical end of sporozoites, 3 apical rings and an electron-dense collar were seen. Two thick central microtubules were seen further inside sporozoites and extended to the posterior region. Using anti-alpha and -beta tubulin antibodies generated from sea urchin and rat brain, both antibodies cross-reacted at the apical region of sporozoites in immunofluorescent morphology. The molecular mass of C. parvum alpha tubulin antigen was 50 kDa by Western blotting and the observed apical cytoskeletal structures were shown to be composed of alpha tubulin by immunoelectron microscopy. These results suggested that C. parvum sporozoites were clearly different in their cytoskeletal structure from those of other apicomplexan parasites.