Morphogenesis in the Heterotrich Ciliate Climacostomum virens. I. Oral Development During Cell Division

The principal characteristics of stomatogenesis during division in Climacostomum virens are: (A) Kinetosomal proliferation on the left side of a variable number of kineties in the ventral somatic cortex forms an oral primordium consisting of several kinetosomal fields, which then fuse to form a single anarchic field. (B) A constant topographical relationship exists between the primordium and a well defined cortical pattern, the zone of discontinuity. (C) The anarchic field primordium divides into 2 unequal parts—to the left, the AZM primordium, and to the right, the paroral primordium, which differentiates into the apical membranelles, the peristomial field, and the buccal tube. (D) Preoral and oblique kineties of the somatic cortex form along the right side of the paroral primordium. (E) Parental oral structures are partially dedifferentiated. Stomatogenesis in C. virens and other heterotrichs is compared.     

Simple Synthesis of Climacostol,a Defensive Secretion by the Ciliate Climacostomum virens

Climacostol {1,3-dihydroxy-5-[(Z)-2'-nonenyl]benzene,1}, a defensive secretion by the protozoan ciliate Climacostomum virens against predators, was synthesized in a 43% overall yield in three steps by starting from methyl 1,3-bis(tert-butyldimethylsilyloxy) phenylacetate (3).     

A Redescription of Climacostomum virens (Ehrenberg) Stein and Proposal of a New Heterotrich Ciliate Family, Climacostomidae fam. n.

SYNOPSIS. Structure of the vegetative and asexually dividing forms of the large ciliate Climacostomum virens is redescribed with emphasis on stomatogenesis. These findings are discussed in relation to the taxonomic and possible evolutionary position of Climacostomum among heterotrichous ciliates. Comparative considerations of the buccal and somatic structures as well as of the stomatogenic patterns in this and other closely related ciliate genera indicate the need for placing Climacostomum and Fabrea in a new family, CLIMACOSTOMIDAE . The morphologic evidence suggests that Climacostomum may represent a line linking Fabrea and Stentor.     

Light and Electron Microscopic Observations on Encystment of Acanthamoeba palestinensis, Reich

SYNOPSIS. The ultrastructural changes occurring during encystment of Acanthamoeba palestinensis have been investigated. The cyst wall consists of endocyst and exocyst, both having the same fine structure. At irregular intervals in the cyst wall ostioles occupied by opercula are present. The nuclear membrane forms bulb-shaped projections and releases vesicles bounded by double membranes into the cytoplasm. Dense nucleolus-like bodies of different sizes and variable numbers are found in the nucleus of every cyst. The importance of the cyst structure as a taxonomic criterion is discussed.     

Rapid Three-Dimensional Reconstruction at the Light Microscopic Level and a Technique for Re-Embedding the Same Semithin Sections for Electron Microscopic Examination

Rapid three-dimensional reconstruction of serial sections at the light microscopic and ultrastructural levels was accomplished using a two-step technique. Fixed specimens were embedded in Epon and 1 μm sections were cut and placed on glass slides. One of every four sections was drawn onto transparency film for rapid three-dimensional reconstruction. The semi-thin sections were re-embedded in Epon and sectioned at 90 nm for examination in the electron microscopy.     

Caractéristiques De La Stomatogenèse Et Des Ultrastructures Corticale Et Buccale Du Cilié Colpodidea Bursaria Truncatella O. F. Müller, 1773

RESUME. Chacun des 45–80 organelles adoraux de Bursaria truncatella O. F. Müller est constitué de 3 rangées de cinétosomes et l'aire buccale droite est couverte de nombreuses doubles rangées de cinétosomes. La stomatogenèse débute par la désorganisation et la résorption des organelles buccaux postérieurs. Puis, il y a désorganisation des rangées parorales de cinétosomes et multiplication des cinétosomes sur l'aire orale droite, en měme temps que sont rompues, selon une ligne oblique, un certain nombre de cinéties somatiques. La prolifération des cinétosomes aux extrémités des cinéties. de part et d'autre de la ligne de rupture, aboutit, d'une part, à la formation d'un champ anarchique qui est le primordium oral droit de l'opisthe, d'autre part, à la formation de nombreux doublets qui constituent chacun le primordium de chaque organelle adoral. Après la séparation des tomites, les cinétosomes de l'aire droite s'ordonnent en doubles rangées et les organelles adoraux se complètent par addition d'une 3ème rangée de cinétosomes. Les cinétosomes somatiques sont jumelés, reliés par 2 desmoses. Les fibres transverses postérieures et les fibres postciliaires forment de longs rubans de microtubules dirigés vers l'arrière et juxtaposés dans les crětes intercinétiennes. Les doubles rangées droites de cinétosomes buccaux sont assimilables à des stichodyades. Les organelles des cinétosomes adoraux portent des rideaux de fibres postciliaires convergents ou divergents. La rangée postérieure de chaque organelle est non ciliée. Par son type de stomatogenèse, par sa structure corticale, par l'ultrastructure des organelles adoraux, Bursaria appartient aux Colpodidea, ce qui suggère des remarques de plusieurs types. SYNOPSIS. In Bursaria truncatella O. F. Müller, each of the 45–80 adoral organelles is composed of 3 rows of kinetosomes, and the right buccal area is covered by many double rows of kinetosomes. Stomatogenesis begins by disorganization and disappearance of the posterior buccal organelles. Next, there is disorganization of the paroral rows of kinetosomes and multiplication of kinetosomes in the right oral area; at the same time, some somatic kineties are disrupted along an oblique line. Multiplication of kinetosomes at the extremities of the kineties, on both sides of the disruption, leads to the formation of an anarchic field which is the right oral primordium of the opisthe and the formation of doublets each of which constitutes an adoral organelle. After the separation of the tomites. the kinetosomes in the right buccal area position themselves, and the adoral organelles are completed by the addition of a 3rd row of kinetosomes. Somatic kineties are formed by successive pairs of ciliated kinetosomes united by 2 desmoses. the long posterior transverse ribbons and the postciliary ribbons extend posteriad, overlapping in the pellicular ridges. Oral rows of kinetosomes on the right can be compared with stichodyads. the adoral kinetosomes have convergent or divergent postciliary ribbons. the posterior row of kinetosomes in each organelle is not ciliated. By the type of stomatogenesis, the cortical ultrastructure, the ultrastructure adoral of its organelles, Bursaria belongs to the Colpodidea.     

Light and Electron Microsopic Observations on the Pathogenesis of Naegleria fowleri in Mouse Brain and Tissue Culture

Two strains of pathogenic Naegleria were employed to infect mice and monkey kidney (Vero line) cell cultures. Mice were infected intranasally. Moribund mice were sacrificed and their brains processed for light and electron microscopy. The normal architecture of the infected brain was completely destroyed; the olfactory lobes and the cerebral cortex showed the heaviest damage. The inflammatory response was mainly in the form of neutrophil polymorphs (PMN) and was confined to the olfactory lobes and the superficial regions of cerebral cortex. Numerous amebas were seen interspersed with the degenerating neurons, glial processes, and PMN. Most conspicuous were the food vacuoles which contained host tissue in various stages of digestion. Amebas in the brain tissue also produced many micropinocytotic vesicles from the surface of the plasma membrane. These vesicles are interpreted as vehicles of transport of nutritive materials from the host tissue. The infected cell culture showed the characteristic cytopathic effect (CPE). The CPE was chiefly in the form of cell shrinkage, nuclear pycnosis and discontinuity of cell sheet. Amebas were often seen in an intracellular location. The Vero cells produced many fuzzy pinocytotic vesicles at these loci where the ameba plasma membrane and Vero cell membrane were in close apposition; the probable significance of this is discussed. Most impressive, however, were the pseudopodial formation and capturing of the host material which indicated the great phagocytic activity of the amebas. This was confirmed further by the presence of large numbers of food vacuoles containing host material in various stages of digestion. These observations show that the amebas invade and destroy the brain tissue by active phagocytosis.     

Angiostrongylus cantonensis: Scanning Electron Microscopic Observations on the Cuticle of Moulting Larvae

Angiostrongylus cantonensis is a parasitic nematode that needs to develop in different hosts in different larval stages. Freshwater snails, such as Pomacea canaliculata, are the intermediate host, and rats are the definitive host. Periodic shedding of the cuticle (moulting) is an important biological process for the survival and development of the parasite in the intermediate and definitive hosts. However, there are few studies on the cuticle alterations between different stages of this parasite. In this study, we observed the ultrastructural appearance and changes of the cuticle of the 2nd/3rd stage larvae (L2/L3) and the 3rd/4th stage larvae (L3/L4) using a scanning electron microscope. We also first divided L2/L3 into late L2 and early L3. The late L2 lacked alae, but possessed a pull-chain-like fissure. Irregular alignment of spherical particles on the cuticle were noted compared to the L3. Alae appeared in the early L3. The old cuticle turned into a thin film-like structure which adhered to the new cuticle, and spherical particles were seen regularly arranged on the surface of this structure. Regular rectangular cavities were found on the surface of L3/L4. The caudal structure of L3/L4 was much larger than that of L3, but caudal inflation, such as seen in L4, was not observed. These results are the first to reveal the ultrastructural changes of the cuticle of A. cantonensis before and after moulting of L2/L3 and L3/L4.     

Microscopic Observations on the Filopodia of Entamoeba histolytica*

Living Entamoeba histolytica trophozoites were examined by phase-contrast microscopy. Intact critical point dried trophozoites were examined by transmission electron microscopy at an accelerating voltage of 1000 kV (HVEM) and by scanning electron microscopy (SEM). Half and quarter m? thick sections of epoxy-embedded trophozoites were examined by HVEM. Many of the trophozoites of 2 strains examined had surface filopodia, 1 to over 100 pan in length. The cytoplasm of filopodia was continuous with the cytoplasm and bounded by surface plasmalemma bearing a glycocalyx. Structures called “surface-active lysosomes with trigger,”“dendritic plasmalemmal extensions,” and “extra-amebic vesicles” by previous investigators probably represent portions of filopodia demonstrated in the present study. Filopodia appear to be of frequent normal occurrence in E. histolytica and may function in: (a) endocytosis or pinocytosis; (b) exocytosis; (c) attachment to substratum; (d) penetration of tissue; (e) release of cytotoxic substances; or (f) contact cytolysis of host cells.     

Cortical Ultrastructure of the Scuticociliates Dexiotricha media and Dexiotricha colpidiopsis (Hymenostomata)*†

SYNOPSIS. Cortical ultrastructure of the scuticociliates Dexiotricha media and Dexiotricha colpidiopsis was investigated. The following elements of the somatic cortex were studied: the cell membrane, alveolar membranes and the epiplasm, kinetodesmal fibers, postciliary and transverse microtubular ribbons, and transverse fibers associated with single and paired kinetosomes; mitochondria and single microtubules located in interkinetal ridges; mature and early extrusion stages of mucocysts: the expulsion vacuole pore and tube, the nephridioplasm and the cytoproct. In the buccal cortex, the paroral kinety-ribbed wall complex, the 3 polykineties, and the cytostome-cytopharynx were investigated. Comparative survey of ciliate ultrastructure indicates 2 principal orientation patterns for kinetodesmal and postciliary fibers, recognition of which leads to reevaluation of the theory of paroral kinety formation and the ideas of homology based on this theory. Ultrastructurally, the scuticociliates are not distinct from tetrahymenines and peniculines; the 3 groups appear to be 1 assemblage.     

A Study of Criteria Permitting the Use of Plastinated Specimens for Light and Electron Microscopy

Plastination permits the preservation of anatomical specimens in a physical state approaching that of the living condition. We studied the possibility of using silicone plastinated fragments of spleen and pancreas for optical and electron microscopy, and found that with an adequate fixation protocol, plastinated specimens can be used for both light microscopy and ultra-structural studies. Deplastination with sodium methoxide permitted production of clean sections. Artifacts produced by plastination/deplastination could be nearly eliminated by glutaraldehyde/formaldehyde fixation. The (Biodur) silicone S10 polymer is transparent and stable in an electron beam, and plastinated tissues can be contrasted or colored similar to tissues embedded in Epon 812. In addition to being very life-like, plastinated tissues are stable and easy to handle. They can also be used for electron and light microscopic studies. This technique may also allow retrospective epidemiological studies of archived pathology specimens.     

Recent Developments in Correlative Super-Resolution Fluorescence Microscopy and Electron Microscopy

The recently developed correlative super-resolution fluorescence microscopy (SRM) and electron microscopy (EM) is a hybrid technique that simultaneously obtains the spatial locations of specific molecules with SRM and the context of the cellular ultrastructure by EM. Although the combination of SRM and EM remains challenging owing to the incompatibility of samples prepared for these techniques, the increasing research attention on these methods has led to drastic improvements in their performances and resulted in wide applications. Here, we review the development of correlative SRM and EM (sCLEM) with a focus on the correlation of EM with different SRM techniques. We discuss the limitations of the integration of these two microscopy techniques and how these challenges can be addressed to improve the quality of correlative images. Finally, we address possible future improvements and advances in the continued development and wide application of sCLEM approaches.     

中海拔地区慢性暴露大鼠心肌及肝脏的光镜及电镜观察

目的 探讨中度海拔高度地区慢性低氧大鼠心肌、肝的组织学及超微结构变化。方法 本实验用Wistar大鼠20只,雌雄各半,六日内从海拔5米运至海拔3418米饲养,8周后断头处死大鼠,留取心肝组织作光电镜观察,同时高原暴露前后测定血RBC数及Hb含量。结果 心肝组织学改变主要为细胞水肿,即心肌颗粒变性,肝细胞疏松化,其次为心肌、肝细胞嗜酸性变。心肌组织有少量小灶状坏死,肝组织中未见坏死。超微结构主要有肌浆网扩张,线粒体肿胀,糖元颗粒减少,未见不可逆性损伤如线粒体出现杆状嵴、三膜嵴及核染色质边聚现象。毛细血管内皮细胞多有突起伸向管腔,胞质空泡变性,微饮泡较少。另外,高原暴露后RBC数及Hb含量明显升高。结论 该海拔地区慢性低氧大鼠心肌、肝组织及毛细血管的病变是可逆性的; 左右心室病变程度无显著性差异; 肝组织的病变程度明显轻于心肌组织。     

Scanning Electron Microscopy of the Cortex of the Ciliate Stentor coeruleus. A View from the Inside*

SYNOPSIS. A microdissection procedure was developed which permits the viewing of the inside surface of the cortex of Stentor coeruleus with scanning electron microscopy. Parallel bands of myonemes cover the entire inner surface of the cortex. The myonemes of the stalk region are ribbon-shaped and lack cross connections. The myonemes of the anterior cortex are flattened against the surface and are interconnected by an extensive system of cross branches. The inner surface of the frontal field is covered with a regularly cross-branched myoneme system which follows the curved pattern of frontal field kinety. The observed branching patterns and shapes of the myonemes support the hypothesis that they cause contraction of the cell. The membranellar root system was examined. Each membranellar root makes a 90° counterclockwise twist along its vertical axis (viewed from the inside) as it descends into the cell. The outer edge of each root fuses with the inner edge of the adjacent one, forming a continuous fiber sheet linking the roots together.     

New Observations on the Ciliate Genus Vestibulongum (Pycnotrichidae): Vestibular Ultrastructure,Macronuclear Endosymbiotic Bacteria,Biogeography, and Evidence for Host Specificity

Two isolates of the pycnotrichid ciliate genus, Vestibulongum, were collected from the host fish, Acanthurus xanthopterus, from two locations in the Southern Pacific Ocean. One was from the Great Barrier Reef (GBR), and a second from Papua New Guinea. These sites are thousands of km from the type locality, off the coast of South Africa. New data were collected from protargol‐stained samples to more fully characterize the general form and light microscopic structures of the ciliate. Specimens from all three sites had a long vestibule, characteristic of most members of the family. Data suggest that specimens from each site are the same genus. The kinetids of the Vestibulongum isolated from the GBR contained the typical components of postciliary, transverse, and nemodesmatal microtubules, and Kd fibrils. Also, two quite different forms of endomacronuclear bacteria were observed and are described. One of those has distinct endospores, which are similar to endospores in nuclear endosymbiotic bacteria in a species of Balantidium from the gut of another species of surgeonfish.     

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.     

Fluorescence and Electron Microscopic Localization of F-actin in the Ependymocytes

The organization of F-actin in the ventricular system has been reported to display pronounced regional differences with respect to shape, size, and development. However, the real roles played by F-actin in these cells cannot be understood unless the precise localization of F-actin is defined. In the present study, we used double-fluorescence labeling to further examine the localization of F-actin in the ependymocytes and its spatial relation to the other two cytoskeletal components, microtubules and intermediate filaments. Then we converted fluorescence signals for F-actin to peroxidase/DAB reaction products by use of a phalloidin-based FITC-anti-FITC system. This detection technique provided an overview of the distribution of F-actin in the ependymocytes at the ultrastructural level, and has been proven to be helpful in correlating light and electron microscopic investigations. (J Histochem Cytochem 57:741–751, 2009)     

Correlative Light and Scanning Electron Microscopy for Observing the Three-Dimensional Ultrastructure of Membranous Cell Organelles in Relation to Their Molecular Components

Although the osmium maceration method has been used to observe three-dimensional (3D) structures of membranous cell organelles with scanning electron microscopy (SEM), the use of osmium tetroxide for membrane fixation and the removal of cytosolic soluble proteins largely impairs the antigenicity of molecules in the specimens. In the present study, we developed a novel method to combine cryosectioning with the maceration method for correlative immunocytochemical analysis. We first immunocytochemically stained a semi-thin cryosection cut from a pituitary tissue block with a cryo-ultramicrotome, according to the Tokuyasu method, before preparing an osmium-macerated specimen from the remaining tissue block. Correlative microscopy was performed by observing the same area between the immunostained section and the adjacent face of the tissue block. Using this correlative method, we could accurately identify the gonadotropes of pituitary glands in various experimental conditions with SEM. At 4 weeks after castration, dilated cisternae of rough endoplasmic reticulum (RER) were distributed throughout the cytoplasm. On the other hand, an extremely dilated cisterna of the RER occupied the large region of the cytoplasm at 12 weeks after castration. This novel method has the potential to analyze the relationship between the distribution of functional molecules and the 3D ultrastructure in different composite tissues.     

Sublethal Cytotoxic Effects of Mercuric Chloride on the Ciliate Tetrahymena pyriformis*

SYNOPSIS. The behavior and ultrastructure of Tetrahymena pyriformis was assessed after exposure to dosages of 8 and 16% of the lethal concentration of HgCl² (TLm 96 hr). The lower dosage caused no abnormal changes in cell motility, activity of the water explusion vesicles, or cell shape; the higher dosage caused deleterious changes in these parameters. The higher sublethal HgCl² concentration (0.50 mg/liter) elicited damage of several cell structures. This damage persisted and accumulated with time up to 24 hr. At the lower HgCl² dosage (0.25 mg liter) there were extensive changes after 1-hr exposure involving primarily mitochondria; however, all major changes were repaired after 24 hr of constant exposure to the HgCl², indicating adaptation to the toxicant. Based solely on cytotoxic evidence an attempt is made to apply the findings defining what constitutes a “safe'’concentration of HgCl₂ in the cell's environment.     

Assessment of the Acrylic Resin Technovit 7200 VLC for Studying the Gingival Mucosa by Light and Electron Microscopy

Technovit 7200 VLC is an acrylic resin formulated for embedding undecalcified hard tissues which are prepared for light microscopy according to a cutting-grinding technique. To employ this resin for embedding and cutting soft tissues by ultramicrotomy, we carried out a qualitative study on biopsies of canine gingival mucosa using light and transmission electron microscopy. For a critical evaluation of this resin, some biopsies were embedded in Agar 100, an epoxy resin widely used in morphological studies. At the light microscopic level the samples embedded in Technovit 7200 VLC showed good morphology and excellent toluidine blue staining of different cell types and extracellular matrix. At the ultrastrueturallevel, nuclei, cytoplasmic organelles, collagen fibrils and ground substance appeared well preserved and showed high electron density. The acrylic resin was stable under the electron beam and its degree of shrinkage appeared to be very low. We conclude that Technovit 7200 VLC can be employed for ultramicrotomy for both light and electron microscopic investigation of soft tissues.