Structure of Monocercomonas sp. as Revealed by Electron Microscopy*

SYNOPSIS. Monocercomonas shares many fine-structural features with all other trichomonads. These include the basic arrangement of the kinetosomes as well as of the recurrent and 3 anterior flagella. The pelta-axostyle complex and the parabasal apparatus, i.e. the Golgi complex and the periodic filaments, also conform to the trichomonad pattern. Of interest with regard to the crucial evolutionary position of Monocercomonas, considered to represent the most primitive trichomonad type, is the fact that it has some structures in common with other Monocercomonadidae and Trichomonadinae and others in common with Devescovinidae and Tritrichomonadinae. Among the former organelles are the marginal lamella and the costal base, and among the latter, the comb-like organelle situated between the infrakinetosomal body and parabasal filament 2 as well as the infrakinetosomal body. No traces of either costa or undulating membrane have been noted, but a complex structure homologous to the marginal lamella of Hypotrichomonas and Trichomonadinae is found underlying the short anteriormost portion of the recurrent flagellum that is attached to the body surface. Observations of sections of selected division stages indicate the potential of parental kinetosomes #1 and #3 to become daughter kinetosome #2.     

Structure of Trichomonads as Revealed by Scanning Electron Microscopy*†

SYNOPSIS.
A scanning electron microscope (SEM) study of Hypotrichomonas acosta (Moskowitz), Trichomonas vaginalis Donné. Pentatrichomonas hominis (Davaine), and Tritrichomonas foetus (Riedmüller) provided new information about the structure of the periflagellar canal: emergence of the flagella from the cell body; structure of the undulating membrane; and position, shape, and size of the pelta. Of special interest were the spatial relationships of the attached part of the recurrent flagellum and the accessory filament in Hypotrichomonas and in the members of Trichomonadinae, i.e. Trichomonas and Pentatrichomonas.     

Structure of Hypotrichomonas acosta (Moskowitz) (Monocercomonadidae,Trichomonadida) as Revealed by Electron Microscopy*

SYNOPSIS. The fine structure of Hypotrichomonas acosta resembles in many respects that of Trichomonadidae, and especially of members of the sub-family Trichomonadinae which have been examined to date by electron microscopy. In addition, the flagellate has certain ultrastructural differences from the latter organisms, some of which are of phylogenetic significance. Among these, the structure of the undulating membrane and the apparently occasional presence of a fine filament which may be considered as homologous to the costa of Trichomonadidae are the most important. The undulating membrane is represented by a rather low and otherwise poorly developed dorsal cytoplasmic fold with an ill-defined distal marginal lamella; the recurrent flagellum is applied near the dorsum of the fold. In a very few preparations a relatively short filament, of a diameter falling below the resolution limits of light microscope, is seen in a position which corresponds to that of the costa of Trichomonadidae. The identity of the filament as a probable rudimentary costa is supported also by the character of its periodicity. The rare appearance of the rudimentary costa among hundreds of sections may be explained either by its minute dimensions or by its absence from many hypotrichomonads. Other structures recorded for the first time in trichomonads are: the fine filamentous connections of the axostylar microtubules; the branching of parabasal filament 2; and the unusually organized, perhaps helical, polysomes, which are found in addition to the ribosomal complexes associated with the endoplasmic reticulum and commonly found in trichomonads. A detailed analysis of interconnections among various mastigont structures is presented and several kinds of cytoplasmic inclusions are described. H. acosta is of interest in the study of the nuclear envelope and presence of nuclear pores, which are numerous and conspicuous in this flagellate. The fine structure of the hypotrichomonad is discussed in relation to that of other trichomonads and in some instances to that of other protozoa.     

The Mastigont System of Trichomonas gallinae (Rivolta) as Revealed by Electron Microscopy

SYNOPSIS. The fine structure of Trichomonas gallinae has been examined by electron microscopy and correlated with previous light microscope observations. A composite diagram of the flagellate, derived from both types of examination, is presented. Details of relationships of various mastigont organelles are documented by electron micrographs. The extent of the pelta and its connection to the capitulum of the axostyle have been determined. Four types of kinetosome rootlets have been described. One consists of superficial “filaments” radiating from each of the 9 triplet microtubules of kinetosomes #1, #2 and #3. A 2nd type of rootlet structure is represented by single comma-shaped filaments emerging clockwise from kinetosomes #1 and #3. The filament from kinetosome #1 has a periodic structure similar to that of the marginal lamella with which it is believed to connect. A 3rd type of rootlet emerges from kinetosome #2 as a sheet of about 9 filaments which traverse a sigmoid course and terminate on the inner surface of the microtubules of the pelta near the peltar-axostylar junction. The 4th set of structures consists of the costa and parabasal filaments. These structures have major periodicities of similar dimension but have readily differentiable repeating units. The costa appears to originate at the kinetosome of the recurrent flagellum, but its origin is also contiguous with that of parabasal filament 2 which has some continuity with kinetosomes #2 and #3. Parabasal filament 1, on the other hand, arises solely from or near kinetosome #2. Occasional observations of a costa and a parabasal filament in juxtaposition over a great part of their length has led to the suggestion that the parabasal filament may play a role in the development of the costa. Periodic and filamentous structures have been observed in paraxostylar and paracostal granules and in nearby cytoplasm. Their possible role in providing substance for the developing axostyle and the costa is discussed. The results are discussed in the light of available information pertaining to structure of various trichomonad species as revealed by light and electron microscopy.     

Fertilization Cone of Carp Eggs as Revealed by Scanning Electron Microscopy

The process of formation of the fertilization cone in carp eggs was examined by scanning electron microscopy. The fertilized eggs responded to penetration of one sperm by primary and secondary steps of formation of a fertilization cone of unique morphology. In the primary step, the earliest fertilization cone was seen at the superior or anterosuperior part of a fused sperm head in inseminated eggs fixed 20 sec after immersion in fresh water. The cone reached a maximum of more than 10 μm in length and 3–4 μm in thickness by 40 sec, resulting in a transient plugging of the micropylar canal. In the secondary step, usually seen at 105–120 sec, a conformation reminiscent of a very small caldera volcano was formed, with the shortened earlier cone and part of the sperm tail at its top. By 2.5 min, the fertilization cone had become conical, and the sperm tail still extended from its top. At 3 min, the sperm tail was often not detectable, but a cytoplasmic eminence was still seen as a trace of the fertilization cone. The role of the earlier fertilization cone in blocking polyspermy is discussed.     

Ultrastructure of the Squid Axon Membrane as Revealed by Freeze-Fracture Electron Microscopy

The structure of the axolemma of the squid giant axon was studied by freeze-fracture electron microscopy. Three types of preparations were examined: intact axons, axons with their Schwann cell sheaths stripped off prior to freezing, and axons with their Schwann cell sheaths chemically detached but not mechanically removed. Because of a problem of cross-fracturing, the first two types of preparations revealed very few membrane faces of the axolemma. This cross-fracturing problem, however, was eliminated when we used a complementary replication method to fracture the third type of preparation. We found that the E-face of the axon membrane was smooth relative to the P-face, which showed many prominent intramembrane particles (IMP). The diameters of the typical IMP range from 6 to 15 nm. The P-face of the adjacent Schwann cells also showed many large IMP. The sizes and heights of the Schwann-cell IMP, however, appear to be more homogeneous than the P-face axolemma.     

Surface Features of Bacillus polymyxa Spores as Revealed by Scanning Electron Microscopy

The surface features of Bacillus polymyxa spores were compared by use of thin sections, carbon replicas, and the scanning electron microscope. Some features of the characteristic ridges, previously reported in ultrathin sections and carbon replicas of spores of this species, were more clearly revealed with the scanning electron microscope. A three-dimensional image is provided because of the greater depth of focus possible with this instrument. End-on views of B. polymyxa spores readily illustrate the polygonal porelike structure present.     

Compartmentalization of Pancreatic Secretory Zymogen Granules as Revealed by Low-Voltage Transmission Electron Microscopy

Low-voltage (5-kV) transmission electron microscopy revealed a novel aspect of the pancreatic acinar cell secretory granules not previously detected by conventional (80-kV) transmission electron microscopy. Examination of ultra-thin (30-nm) sections of non-osmicated, stain-free pancreatic tissue sections by low-voltage electron microscopy revealed the existence of granules with non-homogeneous matrix and sub-compartments having circular or oval profiles of different electron densities and sizes. Such partition is completely masked when observing tissues after postfixation with osmium tetroxide by low-voltage transmission electron microscopy at 5 kV and/or when thicker sections (70 nm) are examined at 80 kV. This morphological partition reflects an internal compartmentalization of the granule content that was previously predicted by morphological, physiological, and biochemical means. It corresponds to the segregation of the different secretory proteins inside the granule as demonstrated by high-resolution immunocytochemistry and reflects a well-organized aggregation of the secretory proteins at the time of granule formation in the trans-Golgi. Such partition of the granule matrix undergoes changes under experimental conditions known to alter the secretory process such as stimulation of secretion or diabetes.     

Inclusion Bodies in Xenosomes Purified on Percoll Gradients as Revealed by Electron Microscopy

A method is described for the isolation and purification of xenosomes, intracytoplasmic bacterial symbionts of the marine hymenostome Parauronema acutum , using percoll gradients. Xenosomes isolated by this procedure retained both their ability to kill susceptible Uronema strains and to infect homologous and heterologous P. acutum strains. Unexpectedly, both killer and non-killer xenosomes were found to contain inclusion bodies, heretofore unseen in fixed whole cell preparations, in the form of double helices, which we have termed H-bodies. The nature and function of these bodies is unknown.     

Three-Dimensional Structure of Macronuclei from Paramecium Determined by Scanning Electron Microscopy*

SYNOPSIS. Macronuclei of Paramecium primaurelia were isolated and examined by scanning electron microscopy. These nuclei consisted of a closely packed array of chromatin bodies measuring ～ 0.2 μm in diameter. We estimated there were ～ 30,000 such bodies/macronucleus, 20 times more than the number of unit genome equivalents. This suggests that a unit genome is physically shared by several chromatin bodies.     

Preliminary Observations on the Fine Structure of the Pansporoblast of Thelohania bracteata (Strickland, 1913) (Microsporida, Nosematidae) as Revealed by Freeze-Etching Electron Microscopy

SYNOPSIS. The ultrastructure of a microsporidan pansporoblast was observed with freeze-etching electron microscopy. The cross-fractured face of ovoid mature spores, with the upper part of the spore coat fractured off, revealed the spore membrane; the convex face had many small depressions and the concave face bore fine particles. In cross-section the spore-coat was highly laminated and about 0.5 μ in diameter.
In the cytoplasm of the pansporoblast, fluid-filled and finger-print-life profiles of vesicles were observed. The vesicles were approximately 180 nm in diameter and laminated, each lamella being about 15–18 nm thick. In addition to these vesicles, a population of elevations, each with an average diameter of 40 nm, was evenly distributed in the pansporoblast among the spores. No other cytoplasmic organelles were observed within the pansporoblast. The pansporoblast wall was about 15–19 nm thick with particles 15–18 nm in diameter on its outer surface.     

Electron Microscopy of Small Cells: Mycoplasma hominis

The size, ultrastructure, and reproduction of Mycoplasma hominis species H39 were studied by electron microscopy. These are the smallest known cells.     

Mode of Infection of Phyllosticta maculata on Banana as Revealed by Scanning Electron Microscopy

The initial infection stages of Phyllosticta maculata on banana were studied using scanning electron microscopy. Conidial germination on the banana leaf surface commenced within 3 h postinoculation to produce a long and slender germ tube. The hyphae developed secondary branches and mostly grew randomly across the leaf surface. Appressoria were formed at the apex of the germ tubes within 18 h postinoculation and were variable in shape. A layer of an extracellular matrix surrounded the appressoria at the pathogen–host interface. On the fruit surface, conidia germinated to produce predominantly swollen germ tubes which functioned as lateral appressoria together with some slender ones. These germ tubes were formed within 3 h postinoculation. There was no stomatal penetration apparent on the leaf; instead, direct penetration through the cuticle with and without the formation of appressoria was observed. Cuticular degradation on the leaf surface was evident with a circular, darkened area around the point of penetration by hyphae or appressoria. The significant role of pycnidia and conidia in the epidemiology of the disease was further demonstrated in naturally infected leaf samples.     

The Ultrastructure of Chlorobium tepidum Chlorosomes Revealed by Electron Microscopy

Chlorosomes are the light harvesting structures of green photosynthetic bacteria. Each chlorosome from green sulfur bacteria houses hundreds of thousands of bacteriochlorophyll molecules in addition to smaller amounts of chlorobiumquinone and carotenoids. In electron microscopy studies, chlorosomes exhibit different appearances depending on the fixation method used. Fixation with osmium tetroxide results in electron-transparent chlorosomes. Fixation with potassium permanganate results in clearly delineated electron-dense chlorosomes. This fixation method features an electron-transparent area in the interior of the chlorosome. In addition to electron density patterns that can be considered compositions of rod-shaped elements, chlorosomes exhibit a striation pattern that is oriented parallel to the longitudinal axis. Treatment with osmium tetroxide followed by potassium permanganate treatment results in a more diffused density distribution that outlines connecting elements between the chlorosome and the cytoplasmic membrane, and connecting elements between the cytoplasmic membrane and the outer membrane, which act as a diffusion barrier for electron density.     

Detection of Pentatrichomonas hominis DNA in biological specimens by PCR

AIM: To develop a sensitive and specific polymerase chain reaction for the detection of Pentatrichomonas hominis in biological specimens. METHODS: Three primers, associated in two primer pairs, were designed to amplify a sequence from the SSU rRNA gene of P. hominis. The specificity of both primer pairs was established by testing DNA extractions of different Trichomonad species, protozoa, bacteria, yeasts, and human leucocytes. The analytical sensitivity was determined through testing dilutions of P. hominis trophozoites. The clinical specificity and applicability of the assay was evaluated on stool samples and self-administered vaginal swabs. CONCLUSIONS: A highly specific and sensitive PCR assay was developed. Both primer pairs performed equally well. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of P. hominis in vaginal specimens has not been reported before.     

Macromolecular Organization of the Cellulolytic Enzyme Complex of Clostridium thermocellum as Revealed by Electron Microscopy

Clostridium thermocellum JW20 and YM4 both synthesize cellulolytic enzyme complexes, cellulosomes, when grown on medium containing cellulose. Electron microscopic studies showed that, in the early stages of growth of strain JW20, clusters of tightly packed cellulosomes, i.e., polycellulosomes, were located on the cell surface and were bound to cellulose. The polycellulosome was estimated to have a particle mass of 50 × 10⁶ to 80 × 10⁶ daltons (Da), while that of the cellulosome was estimated to be 2 × 10⁶ to 2.5 × 10⁶ Da and to contain about 35 polypeptides ranging from 20 to 200 kDa. The cellulosome produced by strain YM4 was found to be somewhat larger, with the estimated particle mass being 3.5 × 10⁶ Da, and the number of polypeptides was counted to be 45 to 50, ranging from 20 to 200 kDa. In the early stages of cultivation, the cellulosomes from both species exist as tightly packed complexes (tight cellulosomes). These subsequently decompose to loosely packed complexes (loose cellulosomes) and ultimately to free polypeptides. Examination of the loose cellulosomal particles showed that they contain rows of equidistantly spaced, similarly sized polypeptide subunits, with an apparently identical orientation arranged parallel to the major axis of the cellulosome. It is postulated that on binding of a cellulose chain alongside such a row of subunits a simultaneous multicutting event occurs that leads to the release of cellooligosaccharides of four cellobiose units in length (C₄). Rows of smaller-sized subunits with lower center-to-center distances, which are also present in the cellulosome, subsequently cleave the C₄ fragments (or cellulose) to C₂ (cellotetraose) or C₁ (cellobiose). In this way the cellulosome can catalyze the complete hydrolysis of cellulose.     

Cytoskeletal Asymmetrical Dumbbell Structure of a Gliding Mycoplasma,Mycoplasma gallisepticum,Revealed by Negative-Staining Electron Microscopy

Several mycoplasma species feature a membrane protrusion at a cell pole, and unknown mechanisms provide gliding motility in the direction of the pole defined by the protrusion. Mycoplasma gallisepticum, an avian pathogen, is known to form a membrane protrusion composed of bleb and infrableb and to glide. Here, we analyzed the gliding motility of M. gallisepticum cells in detail. They glided in the direction of the bleb at an average speed of 0.4 μm/s and remained attached around the bleb to a glass surface, suggesting that the gliding mechanism is similar to that of a related species, Mycoplasma pneumoniae. Next, to elucidate the cytoskeletal structure of M. gallisepticum, we stripped the envelopes by treatment with Triton X-100 under various conditions and observed the remaining structure by negative-staining transmission electron microscopy. A unique cytoskeletal structure, about 300 nm long and 100 nm wide, was found in the bleb and infrableb. The structure, resembling an asymmetrical dumbbell, is composed of five major parts from the distal end: a cap, a small oval, a rod, a large oval, and a bowl. Sonication likely divided the asymmetrical dumbbell into a core and other structures. The cytoskeletal structures of M. gallisepticum were compared with those of M. pneumoniae in detail, and the possible protein components of these structures were considered.Mycoplasmas are commensal and occasionally pathogenic bacteria that lack a peptidoglycan layer (50). Several species feature a membrane protrusion at a pole; for Mycoplasma mobile, this protrusion is called the head, and for Mycoplasma pneumoniae, it is called the attachment organelle (25, 34-37, 52, 54, 58). These species bind to solid surfaces, such as glass and animal cell surfaces, and exhibit gliding motility in the direction of the protrusion (34-37). This motility is believed to be essential for the mycoplasmas'' pathogenicity (4, 22, 27, 36). Recently, the proteins directly involved in the gliding mechanisms of mycoplasmas were identified and were found to have no similarities to those of known motility systems, including bacterial flagellum, pilus, and slime motility systems (25, 34-37).Mycoplasma gallisepticum is an avian pathogen that causes serious damage to the production of eggs for human consumption (50). The cells are pear-shaped and have a membrane protrusion, consisting of the so-called bleb and infrableb (29), and gliding motility (8, 14, 22). Their putative cytoskeletal structures may maintain this characteristic morphology because M. gallisepticum, like other mycoplasma species, does not have a cell wall (50). In sectioning electron microscopy (EM) studies of M. gallisepticum, an intracellular electron-dense structure in the bleb and infrableb was observed, suggesting the existence of a cytoskeletal structure (7, 24, 29, 37, 58). Recently, the existence of such a structure has been confirmed by scanning EM of the structure remaining after Triton X-100 extraction (13), although the details are still unclear.A human pathogen, M. pneumoniae, has a rod-shaped cytoskeletal structure in the attachment organelle (9, 15, 16, 31, 37, 57). M. gallisepticum is related to M. pneumoniae (63, 64), as represented by 90.3% identity between the 16S rRNA sequences, and it has some open reading frames (ORFs) homologous to the component proteins of the cytoskeletal structures of M. pneumoniae (6, 17, 48). Therefore, the cytoskeletal structures of M. gallisepticum are expected to be similar to those of M. pneumoniae, as scanning EM images also suggest (13).The fastest-gliding species, M. mobile, is more distantly related to M. gallisepticum; it has novel cytoskeletal structures that have been analyzed through negative-staining transmission EM after extraction by Triton X-100 with image averaging (45). This method of transmission EM following Triton X-100 extraction clearly showed a cytoskeletal “jellyfish” structure. In this structure, a solid oval “bell,” about 235 nm wide and 155 nm long, is filled with a 12-nm hexagonal lattice. Connected to this bell structure are dozens of flexible “tentacles” that are covered with particles 20 nm in diameter at intervals of about 30 nm. The particles appear to have 180° rotational symmetry and a dimple at the center. The involvement of this cytoskeletal structure in the gliding mechanism was suggested by its cellular localization and by analyses of mutants lacking proteins essential for gliding.In the present study, we applied this method to M. gallisepticum and analyzed its unique cytoskeletal structure, and we then compared it with that of M. pneumoniae.