Histochemical and ultrastructural studies on the metacercarial cyst of Echinostoma revolutum (Trematoda).

Histochemical and ultrastructural studies were made on the metacercarial cyst of Echinostoma revolutum obtained from the kidney of experimentally infected Physa and Lymnaea snails. Ultrastructural studies revealed three cyst walls, an outer, middle and inner. The outer wall was more electron-dense than the middle, and contained coarser granules than those found in the middle layer. The inner wall was lamellated and contained membranous whorls. Collagenous fibers presumably of host origin surrounded the outer cyst wall. The outer and middle cyst walls stained identically with all histochemical procedures used. These walls contained acid mucopolysaccharides and glycoprotein, whereas the inner cyst wall contained glycoprotein. All cyst walls stained positively with a variety of protein stains.     

The structure and composition of the cyst wall of the metacercaria of Cloacitrema narrabeenensis (Howell & Bearup, 1967) (Digenea: Philophthalmidae).

The mstacercarial cyst of Cloacitrema narrabeenensis which is formed in the open is composed of four layers: an outermost layer of acid mucopolysaccharide, a layer of protein which is presumed to be tanned, a layer of neutral mucopolysaccharide and an innermost layer of keratinized protein. The two layers which together form the outer cyst wall can be split off by slight pressure from the two remaining layers which together form the inner cyst wall. In the centre of the ventral side of the inner cyst wall, the keratinized layer is incomplete and this ventral plug region is composed of neutral mucopolysaccharide. The cyst wall is therefore very similar to that of Fasciola hepatica, the main difference being that the order of the two layers of the outer cyst is reversed. General evolutionary and functional relationships of metacercarial cysts are discussed.     

Strategies To Discover the Structural Components of Cyst and Oocyst Walls

Cysts of Giardia lamblia and Entamoeba histolytica and oocysts of Toxoplasma gondii and Cryptosporidium parvum are the infectious and sometimes diagnostic forms of these parasites. To discover the structural components of cyst and oocyst walls, we have developed strategies based upon a few simple assumptions. Briefly, the most abundant wall proteins are identified by monoclonal antibodies or mass spectrometry. Structural components include a sugar polysaccharide (chitin for Entamoeba, β-1,3-linked glucose for Toxoplasma, and β-1,3-linked GalNAc for Giardia) and/or acid-fast lipids (Toxoplasma and Cryptosporidium). Because Entamoeba cysts and Toxoplasma oocysts are difficult to obtain, studies of walls of nonhuman pathogens (E. invadens and Eimeria, respectively) accelerate discovery. Biochemical methods to dissect fungal walls work well for cyst and oocyst walls, although the results are often unexpected. For example, echinocandins, which inhibit glucan synthases and kill fungi, arrest the development of oocyst walls and block their release into the intestinal lumen. Candida walls are coated with mannans, while Entamoeba cysts are coated in a dextran-like glucose polymer. Models for cyst and oocyst walls derive from their structural components and organization within the wall. Cyst walls are composed of chitin fibrils and lectins that bind chitin (Entamoeba) or fibrils of the β-1,3-GalNAc polymer and lectins that bind the polymer (Giardia). Oocyst walls of Toxoplasma have two distinct layers that resemble those of fungi (β-1,3-glucan in the inner layer) or mycobacteria (acid-fast lipids in the outer layer). Oocyst walls of Cryptosporidium have a rigid bilayer of acid-fast lipids and inner layer of oocyst wall proteins.     

Digestive System of Trichodorus porosus

The onchiostyle of Trichodorus porosus has an anterior outer portion, a fine inner spear and a posterior onchiostyle extension. The extension has a ventral lumen and is fused to the pharynx wall. The inner spear enters the dorsal wall of the outer onchiostyle posterior to the guide ring and extends anteriorly inside the anterior portion of the onchiostyle. Muscle cells are absent in the basal position of the esophagus. The glandular portion of the basal part of the esophagus consists mainly of endoplasmic reticulum lined with ribosomes. A sinus empties into the lumen through the dorsal esophageal gland orifice. The configuration of the intesinal lumen is highly variable. The rectum is attached to the dorsal and ventral walls of the body cavity by striated rectal muscle cells.     

A histochemical study of the secretory gland cells of Cercaria shikokuensis and their role during development from cercaria to metacercaria.

The roles of secretory glands during the developmental process from an immature cercaria to a metacercaria in Cercaria shikokuensis were studied. Four types of secretory cells were identified in this species. On maturation of the cercaria in redia, the products of ventral gland cells and mucoid gland cells formed a thick surface coat on the mature cercaria, and the products of cephalic gland cells also formed a thin cover on the surface coat. In the process leading to the formation of a metacercaria, the surface coat constituted the outer layer of the cyst, mucoid gland cells secreted mucous substances inside the wall, and then cystogenous gland cells discharged their products to the inner wall. The cyst wall was composed of four layers, and it was thought that the outermost surface layer helped the cyst wall to adhere to the matrix and the intermediate layers helped to put together outer and inner walls.     

On the blue stem colour in Plagiochila longispina Lindenb. & Gottsche (Plagiochilaceae)

The blue colour of the shoot tips of Plagiochila (sect. Glaucescentes) longispina is caused by the presence of blue oil bodies in the outer cell layers of the stem. Oil bodies disintegrate in microscopical preparations after a few minutes and blue-coloured drops accumulate under the cover glass. It is suggested that the substances of these drops become associated with cortex cell walls during drying of plant material, leading to the blue colour of these walls in herbarium specimens.     

Wall formation in the saprolegniales

Summary The primary and secondary cysts of Saprolegnia ferax and the secondary cysts of Dictyuchus sterile have a two layered wall structure, the outer layer of which bears various types of spines. These spines, and the outer wall layer are derived from preformed structures (bars) found in the cytoplasm prior to encystment. Golgi derived vesicles appear to contribute to the inner layer of the primary cyst wall of S. ferax. The outer surface of the secondary cyst walls of this species has fibrils which are not embedded in matrix material.     

Systematic relevance of seed coat anatomy in the European heathers (Ericeae, Ericaceae)

The anatomy of the seed coat of the European species of tribe Ericeae (Calluna, Daboecia and Erica) of the Ericaceae family was studied, and the taxonomic importance of their characters was analyzed. The seed coat is mostly formed by a one-cell layer with thick, pitted inner walls and thin outer walls that collapse at maturity over the inner walls. The cell junctions are either raised with anticlinal walls up to four times the height of the periclinal walls or are not raised with similar values for the height of both the anticlinal and periclinal walls. Three main cell junction types were found and described. The thickness of the inner walls is variable, but there is a large overlap among the results for different species. Calluna vulgaris is the only species with no pits, and E. multiflora has a pitted pattern on its inner walls, which is distinctive from the rest of the species. Our main results agree with the external seed morphology, and valuable new data were obtained for certain groups such as the E. cinerea-E. terminalis or the E. scoparia complex. The similarities that are found in seed coat characters are not in accordance with the classical taxonomic delimitation of infrageneric groups within Erica.     

Studies on metacercarial excystment in Himasthla leptosoma (Trematoda: Echinostomatidae) and newly emerged metacercariae

Metacercarial cysts of Himasthla leptosoma were subjected to a solution containing bile salts, trypsin and l cysteine at 41°C. The treatment induced intense metacercarial activity and after 20 min metacercariae burst through the cyst walls and emerged. Electron microscopy demonstrated that organisms burst through a small area of cyst wall which was devoid of a layer of lamellae present elsewhere towards the innermost surface. The appearance of ruptured cyst walls indicated that they had been softened by the excystment medium. Newly emerged metacercariae possessed a reniform collar of 29 cephalic spines and these were sometimes withdrawn into pits, presumably by action of a muscle complex in the head region. Sensory papillae were distributed in a bilaterally symmetrical arrangement around the anterior sucker and none were visible on the surface of the ventral sucker. Tegumental spines were found only from a point some distance behind the head collar to the region of the ventral sucker. The most anterior spines were simple and peg-like and they quickly merged posteriorly into more complex palmate forms.     

In vitro excystment of the metacercaria of Maritrema arenaria (Digenea: Microphallidae)

Metacercarial cysts of Mantrema arenaria were subjected to a solution containing trypsin and bile salts at 41°C. This treatment induced intense metacercarial activity and after 15 min metacercariae burst through their cyst walls and emerged. Electron microscopy demonstrated that during the process of excystment the inner layer of the cyst wall changed from a compact to a loose fibrous state. Experiments showed that only cysts containing viable metacercariae underwent this change whereas cysts which had been forcibly vacated before treatment did not. This indicated that the structural change of the inner layer of the cyst wall could not be attributed to the excystment medium. Also there was much less acid phosphatase activity in and on the surface of newly excysted metacercariae compared with encapsulated specimens. It was concluded that the excystment medium induced physical activity in, and the release of enzymic material by, the metacercariae. Together these activities rendered the cyst wall soft and susceptible to rupture by physical pressure.     

Carbohydrate analysis of Acanthamoeba castellanii

We analyzed biochemically Acanthamoeba castellanii trophozoites, intact cysts and cyst walls belonging to the T4 genotype using gas chromatography combined with mass spectrometry. Cyst walls were prepared by removing intracellular material from cysts by pre-treating them with sodium dodecyl sulphate (SDS) containing dithiothreitol, and then subjecting these to a series of sequential enzymatic digestions using amyloglucosidase, papain, DNase, RNase and proteinase K. The resulting “cyst wall” material was subsequently lyophilized and subjected to glycosyl composition analysis. Transmission electron microscopy confirmed the removal of intracystic material following enzymatic treatment. Our results showed that treated A. castellanii trophozoites, intact cysts and cyst walls contained various sugar moieties, of which a high percentage was galactose and glucose, in addition to small amounts of mannose, and xylose. Linkage analysis revealed several types of glycosidic linkages including the 1,4-linked glucosyl conformation, indicative of cellulose. Inhibitor studies suggested that, beside sugar synthesis, cytoskeletal re-arrangement and mitogen-activated protein kinase-mediated pathways are involved in A. castellanii encystment.     

Ultrastructural Changes During Encystment of Schizopyrenus russelli*

Ultrastructural changes associated with the encystment of Schizopyrenus russelli have been studied by electron microscopy. Before encystment small “black bodies” appear in the cytoplasm and later migrate toward the periphery. The outer cyst wall is secreted at this stage as a thin discontinuous layer which thickens and subsequently becomes continuous. Concomitant with this, the endoplasmic reticulum surrounds the mitochondria. The inner cyst wall later appears as a multilayered structure which presumably is cast off from the plasma membrane. Between the inner and outer layers of the cyst wall, there is a middle, less electron-dense layer wherein extruded cytoplasmic material is found embedded at certain places.     

Einfluß von Tricyclazol auf die Entwicklung und Melaningehalte der Sklerotien von Botrytis cinerea

Effect of tricyclazole on production and melanin contents of sclerotia of Botrytis cinerea Tricyclazole retarded production of sclerotia of Botrytis cinerea on agar medium more severely than mycelium growth. At a concentration range (50–200 mg/l) that did not control Botrytis on grape leaves, sclerotia production was significantly reduced. There was a negative relation between the bleaching duration of sclerotia and the tricyclazole concentrations in the medium on which they were formed. Light microscopical studies showed that sclerotia from tricyclazole-containing medium contained a significantly poorer developed outer melanin layer than that from the control medium. Ultrastructural studies with 5 days old untreated sclerotia revealed intense electron impermeable deposits in the intercellular spaces and a small electron dense layer in the outer cell walls, on the other hand treated sclerotia of the same age showed only sporadic small pigmented deposits between the cells and the pigmentation of the outer cell wall was absent.     

Detection of antigenic cyst wall elements in Colpoda inflata: an immunoelectron microscopic study and immunoblotting identification of cyst wall polypeptides

By using an antiserum against isolated cyst walls from resting cysts of the ciliate Colpoda inflata, cyst wall polypeptides have been identified by immunoblotting test. Likewise, an immunoelectron microscopical study on both complete resting cysts and isolated cyst walls to localize the cyst wall proteins recognized by the antiserum, has been carried out. The immunoblotting test showed that three main polypeptide bands were recognized by the antiserum, with tentative molecular weights of 61, 66 and 70 kDa respectively. This methodology provides a better identification of cyst wall proteins after electrophoretic separation of cyst wall samples from ciliate resting cysts.     

Down-Regulation of Cellulose Synthase Inhibits the Formation of Endocysts in Acanthamoeba

Acanthamoeba cysts are resistant to unfavorable physiological conditions and various disinfectants. Acanthamoeba cysts have 2 walls containing various sugar moieties, and in particular, one third of the inner wall is composed of cellulose. In this study, it has been shown that down-regulation of cellulose synthase by small interfering RNA (siRNA) significantly inhibits the formation of mature Acanthamoeba castellanii cysts. Calcofluor white staining and transmission electron microscopy revealed that siRNA transfected amoeba failed to form an inner wall during encystation and thus are likely to be more vulnerable. In addition, the expression of xylose isomerase, which is involved in cyst wall formation, was not altered in cellulose synthase down-regulated amoeba, indicating that cellulose synthase is a crucial factor for inner wall formation by Acanthamoeba during encystation.     

Stereoscan studies of rediae, cercariae, cysts, excysted metacercariae and migratory stages of Fasciola hepatica

The external surface of the redial body of Fasciola hepatica is provided with microvillus-like projections or short lamellae, and short cilium-like structures are common anteriorly. The anterior part of the cercarial body possesses a pattern of regularly arranged small depressions each containing a spine. Both long and short cilium-like structures occur anteriorly. The tail is spineless and provided with dorsolateral folds. The outer cyst wall is formed by granules secreted from the tegument all over the body apart from the ventral sucker. Most granules transform into fibrillae which form the thick outer spongy layer. The precursor of the inner cyst wall is at the beginning closely attached to the metacercarial surface, but later the membrane-like cyst wall extends, and when fully formed the metacercaria lies free in the flattened circular inner cyst. The ventral plug is formed by the ventral sucker. The tegument of newly excysted metacercariae is provided with simple pointed spines, but later during migration in the mouse the spines become flattened and multipointed. Very young migratory stages may be attached with host cells.     

Sites of accumulation and composition of hydrocarbons in Botryococcus braunii

Raman spectrometry and electron microscopy show that, in the hydrocarbon-rich alga Botryococcus braunii, hydrocarbons accumulate in two distinct sites; internally in cytoplasmic inclusions and externally in successive outer walls and derived globules. No other classes of lipid are present in noticeable amounts in the cytoplasmic inclusions and in the external globules. The same hydrocarbons are observed in the internal and external pools but with different relative abundances, the shorter hydrocarbons being more abundant in the internal pool. The bulk of B. braunii hydrocarbons (ca 95%) is located in the external pool. Such an extracellular location allows this species to exhibit both an unusually high hydrocarbon content (15% of dry wt) and a normal level (0.75%) within the cells. The hydrocarbon pattern and location of B. braunii were compared with that of other organisms; a close relation appears between higher plant epidermal cells and this green alga. The trilaminar outer walls of B. braunii, at whose contact external hydrocarbon globules accumulate, contain a sporopollenin-like compound.     

Morphology and ultrastructure of Devonian prasinophycean algae (Chlorophyta)

The ultrastructure of three Devonian prasinophycean species (green algae): Inderites reticulatus (Naumova) Telnova, I. devonicus (Naumova) Telnova, and Tasmanites domanicus (Naumova) Telnova is described. Members of the genus Inderites are shown to have double-layer walls (outer homogeneous layer, and inner finely porous layer). The genus Tasmanites is characterized by single-layer (homogeneous) walls. All of the described Paleozoic Tasmanites have a similar ultrastructure. Species differ in shell thickness and distributional pattern of pores.     

Effects of Lysozyme and Inorganic Anions on the Morphology of Streptococcus mutans BHT: Electron Microscopic Examination

The effects of hen egg white lysozyme and the inorganic salt sodium thiocyanate on the integrity of Streptococcus mutans BHT were studied by transmission electron microscopy. Both control cells and cells exposed to NaSCN possessed thick outer cell walls and densely staining inner cell walls juxtaposed to the plasma membranes. In the presence of NaSCN, however, the S. mutans BHT nucleoid was coagulated into thick electron-dense filaments. Exposure of S. mutans BHT to 150 μg of hen egg white lysozyme per ml resulted in the progressive destruction of both the cell walls and the plasma membranes. The enzyme appeared to affect the region of the cell wall septum, and exposure to 150 μg of hen egg white lysozyme per ml for as short a time as 10 min resulted in visible morphological cell wall alterations. At 30 min, ultrastructural observations revealed that the majority of the cells were in the process of expelling a portion of their cytoplasmic contents from the septal and other regions of the cells at the time of fixation. After 3 h of incubation in the presence of this high lysozyme concentration, gelled protoplasmic masses, which were free from the cells, were evident. In addition, extensive damage to the outer and inner cell walls and to the plasma membranes was apparent, although the cells maintained their shape. On some areas of the cell surface, the outer cell wall and plasma membrane were completely absent, whereas at other locations the outer cell wall was either split away from the inner cell wall and plasma membrane or distended from an area free of inner cell wall and plasma membrane. Upon addition of NaSCN to the hen egg white lysozyme-treated cells, both the gelled protoplasmic masses and the damaged cells exhibited an exploded appearance and existed as membrane ghosts, cell wall fragments, or dense aggregates of cytoplasmic components. The effects of a low lysozyme concentration (22.5 μg/ml) on S. mutans morphology were less pronounced at short incubation times (i.e., 10 and 30 min) than those that were observed with a high enzyme concentration; however, breaks in the cell walls and dissolution of the plasma membranes with resulting cell lysis were visible after a prolonged (3-h) incubation and after subsequent addition of NaSCN.     

Toxoplasma gondii: ultrastructure and antigenicity of purified tachyzoite pellicle.

When purified Toxoplasma gondii tachyzoites were treated with hemolysin, DNase, and RNase, the organisms yielded a three-component system containing the outer membrane (pellicle), microtubules, and conoid in relatively normal morphological configuration. Further treatment of this preparation with protease digested all but the pellicle which was more collapsed in appearance. These two preparations were used in rabbit anti-toxoplasma and goat anti-rabbit ferritin labeling experiments. The three-component system showed ferritin label on the conoid and equal ferritin label on the outer and inner surfaces of the pellicle. The microtubules were unlabeled. The pellicle after protease treatment was labeled equally on its outer and inner surfaces, which indicated that the rabbit anti-toxoplasma serum contained antibodies against antigens on the outer and inner surfaces of the pellicle.