Ultrastructure,encystment and cyst wall composition of the resting cyst of the peritrich ciliate Opisthonecta henneguyi

The cyst wall of Opisthonecta henneguyi has been studied ultrastructurally and cytochemically by light and electron microscopy, as well as by chemical and electrophoretic analyses, to examine the structure of the cyst wall and its composition. The cyst wall consists of four morphologically distinct layers. The ectocyst is a thin dense layer. The mesocyst is the thickest layer and is composed of a compact material. The endocyst is a thin layer like the ectocyst, but less dense. The granular layer varies in thickness and is composed of a granular material. In the resting cyst, kinetosomes of both oral apparatus and trochal band as well as the myoneme system are maintained, and only cilia are resorbed. The sugars present in the cyst wall are predominantly N-acetylglucosamine (90%) and glucose (10%). The mesocyst is composed of chitin, and the endocyst includes glycoproteins and acid mucopolysaccharides. During secretion of the cyst wall, the endocyst and granular layer are secreted from precursors synthesized "de novo". No cytoplasmic precursors of ectocyst and mesocyst have been detected.     

Cytochemical and Electrophoretic Studies on the Cyst Wall of a Ciliate, Histriculus muscorum Kahl

The localization of proteins and polysaccharides in the cyst wall of a ciliate, Histriculus muscorum, was examined by light and electron microscope cytochemistry and by using an enzyme digestion test on thin sections. The endocyst and ectocyst were digested by treatment with pepsin or protease VI. The endocyst was intensely PAS-positive and alcian blue-positive. The ectocyst was also PAS- and alcian blue-positive, but the reaction was weaker than that of the endocyst. At the electron microscope level, an intensely positive reaction to methenamine silver was observed in the endocyst and a weak reaction in the ectocyst of the mature cyst. In the ectocyst of the encysting organism, however, the reaction to methenamine silver was more intense than that of the mature cyst. These results demonstrate the possible presence of glycoproteins in the ectocyst and endocyst. The mesocyst was negative to all cytochemical and enzyme digestion tests examined. The cyst wall, isolated by sonication, was analyzed by SDS-polyacrylamide gel electrophoresis. Two bands, 190 and 140 kilodaltons, were specific for the cyst wall. The 190 kilodalton band was the only PAS-positive band and its localization in the cyst was was discussed.     

The Fine Structure of the Cyst Wall of the Ciliated Protozoon Didinium nasutum1

SYNOPSIS. Electron microscope observations of the complex cyst wall of Didinium nasutum are reported. The cyst wall is composed of 3 major coats. The outermost coat, the ectocyst, consists of short strands of filamentous material which forms a diffuse, amorphous layer approximately 8–9 μ thick. Culture debris, bacteria and unidentified inclusions have been observed adhering to the outer coat. The mesocyst, approximately 2.5 μ thick, has 2 distinct regions. The outer region is differentiated into several slightly thicker layers which in face view have a honeycomb appearance. The deeper region of the mesocyst consists of compact lamellae lacking the obvious honeycomb appearance of the layers of the outer region. Finally, the endocyst (0.3 μ thick), which arises in the mature cyst in the space that develops between the pellicle and the mesocyst, consists of delicate fibrils in a compact matrix. Both mesocyst and endocyst may be undulant and folded. The structure, origin and possible relationships of the various coats composing the cyst wall are discussed. The present study also contributes information on the role and fate of mucocysts and other cytoplasmic structures during the formation of the cyst wall.     

Structure of Cyst Wall Precursors and Kinetics of Their Appearance During the Encystment of Laurentiella acuminata (Hypotrichida,Oxytrichidae)1

The encystment of Laurenliella acuminata was divided into five stages: stage A (precystic semitransparent cell with dark-globules), stage B (precystic transparent cell), stage C (precystic pigmented cell), stage D (spherical shape without cyst wall) and stage E (young resting cyst), on the basis of observations of changes in morphology and pigmentation during encystment. The duration of these stages was also established. Observations by electron microscopy confirmed that the cyst wall, composed of four layers, is derived from different kinds of precursors which are synthesized “de novo.” The ectocyst precursors are composed of stacks of between 5 and 12 small thin plates or discs; these stacks are about 0.9 μm in length and 0.06 μm in height. The mesocyst precursors are fibrillar bodies of variable shapes, about 2.4 μm in maximum length and 0.12–0.16 μm in diameter. These precursors appear in the cytoplasm of the precystic cell during the first precystic stage (stage A). The endocyst precursors are rounded bodies surrounded by a fine membrane, and their contents appeared similar to the endocyst. The granular layer precursors are spherical bodies about 0.1–0.2 μm in diameter, surrounded by a double membrane presenting ribosomes adhering to its outer membrane. Both endocyst and granular layer precursors are observed in the precystic cytoplasm from stage B. On the basis of ultrastructural studies, a formation and growth model of the cyst wall of the hypotrichous ciliate Laurentiella acuminata is proposed.     

Production and characterization of three polyclonal antibodies raised against cyst wall proteins of a hypotrichous ciliate.

Three polyclonal antibodies raised against Paraurostyla sp. cyst wall polypeptides of molecular weight 110,000 (p110), 66,000 (p66) and 52,000 (p52) have been obtained. The specificity of the antisera was tested by immunoblotting. Anti-p110 antibody detected five bands of 300, 170, 135, 110 and 40 kDa, respectively. Antiserum obtained against p66 recognized only this protein. Anti-p52 antiserum showed reaction for two different bands of 52 and 44 kDa, respectively. The precise localization of these proteins in the cyst wall was assessed by light microscope immunocytochemistry. Anti-p110 antiserum produced a strong positive reaction in both the ectocyst and endocyst. Both anti-p66 and anti-p52 antibodies recognized the ectocyst.     

The Excystment Process in the Ciliate Nassula ornata Ehrbg.

SYNOPSIS. The ciliate was cultured in natural lake water on Oscillatoria curviceps. Encystment was induced by lack of food. The resting cyst has 2 membranes—a faceted, lamellate ectocyst and a thin endocyst. The kinetosomal rows and the cytopharynx are retained in the cyst, and the position of the cytopharynx with respect to the rows remains unchanged. Thus, the usual polarity of the organism is preserved. Distilled water and plant infusions were relatively ineffective excystment-inducing agents, but a 0.1% (w/v) aqueous solution of peptone, pre-inoculated with wild bacteria, was highly effective. At 21 C it induced the excystment of 97% of the cysts within 2-3.5 hr. During excystment, the contractile vacuole enlarges, thereby producing pressure which ruptures the membranes. The ectocyst tears first—always at the posterior end of the cyst—the endocyst somewhat later. Emergence is largely by cytoplasmic streaming through a relatively small slit in the ectocyst, and the posterior end of the ciliate always emerges first.     

The differentiation of cellular structure during encystment in the soil hypotrichous ciliate Australocirrus cf. australis (Protista,Ciliophora)

Ciliates are able to form resting cysts as a survival strategy in response to stressful environmental factors. Studies on the characteristics of cellular structure during encystment may provide useful information for further understanding of the regulatory mechanism of cellular patterns and supply new clues regarding the phylogeny of ciliates. Scanning and transmission electron microscopies were used to observe the ultrastructure of cells during encystment of the soil ciliate Australocirrus cf. australis. The dedifferentiation of ciliature was revealed for the first time. Ciliary shafts first shortened, and the remaining ciliature, including basal bodies and the fibrillar cirral basket, retracted into the cytoplasm and was surrounded by the autophagic vacuoles and then gradually digested. A large number of autophagic vacuoles were observed in mature resting cysts. Autophagy might not only be necessary for the differentiation of cellular structures during encystment but might also be important to sustain the basic life activities in the resting stage. Australocirrus cf. australis formed a kinetosome-resorbing cyst and contained four layers in the cyst wall: the ectocyst, mesocyst, endocyst and granular layer. The ciliature resorbing state and the number of layers in the cyst wall were consistent with those found in other oxytrichous ciliates. However, the phenomenon wherein the two macronuclear nodules are not fused during encystment is not commonly observed among oxytrichids. Additionally, the octahedral granules in the mesocyst of this species exhibit different morphology from the congeners.     

The Fine Structure of Secretion in Hyalophysa chattoni: Formation of the Attachment Peduncle and the Chitinous Phoretic Cyst Wall

ABSTRACT. The settling tomite stage of the apostome Hyalophysa chattoni secretes a phoretic cyst wall composed of chitin, mucopolysaccharides, and protein. Within 1 1/2 h after settling, an electron-dense proteinaceous cyst layer (the outer layer) is formed from secretions originating at the base of the kineties and from the thick pellicular layer between the kineties. The inner cyst layer, composed primarily of chitin (acidic and neutral polysaccharides are also present), is secreted across the entire cell surface. Cyst wall formation is completed within 6 h. The fine structure of endocyst secretion resembles stages in the secretion of chitin by fungi, yeasts, and arthropods. A proteinaceous attachment peduncle is secreted to anchor the cell to a shrimp host and is formed by the release of electron-dense secretory bodies from the cell's ventral surface.     

Production and Characterization of Three Polyclonal Antibodies Raised Against Cyst Wall Proteins of a Hypotrichous Ciliate

ABSTRACT Three polyclonal antibodies raised against Paraurostyla sp. cyst wall polypeptides of molecular weight 110,000 (p110), 66,000 (p66) and 52,000 (p52) have been obtained. The specificity of the antisera was tested by immunoblotting. Anti-p110 antibody detected five bands of 300, 170, 135, 110 and 40 kDa, respectively. Antiserum obtained against p66 recognized only this protein. Anti-p52 antiserum showed reaction for two different bands of 52 and 44 kDa, respectively. The precise localization of these proteins in the cyst wall was assessed by light microscope immunocytochemistry. Anti-p110 antiserum produced a strong positive reaction in both the ectocyst and endocyst. Both anti-p66 and anti-p52 antibodies recognized the ectocyst.     

冠突伪尾柱虫营养期和形成包囊期间细胞的超微结构

冠突伪尾柱虫营养细胞中含有轴杆样结构。细胞形成包囊期间,胞质内发生自噬作用,并产生由这聚集在一起组成的高尔基体,在高尔基体内其分泌物质聚集成高电子密度的嗜锇晶体。细胞分化的结果形成含膜粒层,内层壁和外层壁的“尾柱虫类包囊”。     

Resting cysts of Parentocirrus hortualis Voß, 1997 (Ciliophora,Hypotrichia), with preliminary notes on encystation and various types of excystation

Resting cysts of Parentocirrus hortualis were investigated, using live observation, SEM and TEM. Processes during encystation and excystation were observed in vivo under the light microscope. During encystation, the trophic body becomes globular, the ciliature is resorbed in an anterior direction, the macronuclear nodules fuse into an elongated mass, and finally a cyst wall develops. As typical for oxytrichids, the resting cysts of P. hortualis are of the kinetosome-resorbing type and their wall is made of four layers: ectocyst, mesocyst, endocyst, and metacyst. The beginning of excystation is indicated by the formation of an excystation vacuole that helps the regenerating specimen to break the cyst wall. The excysting specimen leaves the resting cyst in a thin membrane that is gradually resorbed in the outer environment. Also two other excystation modes were observed. During the rare mode, the excystation vacuole breaks the thin membrane instead of the cyst wall that ruptures under the pressure of the body of the regenerating specimen. During the reproduction mode, the regenerating specimen divides within the resting cyst, producing two to four tomites. This is the first report of division in resting cysts of oxytrichids, but reproduction in division cysts was already described in keronopsids.     

Characterization of Selenaion koniopes n. gen., n. sp., an Amoeba that Represents a New Major Lineage within Heterolobosea,Isolated from the Wieliczka Salt Mine

A new heterolobosean amoeba, Selenaion koniopes n. gen., n. sp., was isolated from 73‰ saline water in the Wieliczka salt mine, Poland. The amoeba had eruptive pseudopodia, a prominent uroid, and a nucleus without central nucleolus. Cysts had multiple crater‐like pore plugs. No flagellates were observed. Transmission electron microscopy revealed several typical heterolobosean features: flattened mitochondrial cristae, mitochondria associated with endoplasmic reticulum, and an absence of obvious Golgi dictyosomes. Two types of larger and smaller granules were sometimes abundant in the cytoplasm—these may be involved in cyst formation. Mature cysts had a fibrous endocyst that could be thick, plus an ectocyst that was covered with small granules. Pore plugs had a flattened dome shape, were bipartite, and penetrated only the endocyst. Phylogenies based on the 18S rRNA gene and the presence of 18S rRNA helix 17_1 strongly confirmed assignment to Heterolobosea. The organism was not closely related to any described genus, and instead formed the deepest branch within the Heterolobosea clade after Pharyngomonas, with support for this deep‐branching position being moderate (i.e. maximum likelihood bootstrap support—67%; posterior probability—0.98). Cells grew at 15–150‰ salinity. Thus, S. koniopes is a halotolerant, probably moderately halophilic heterolobosean, with a potentially pivotal evolutionary position within this large eukaryote group.     

The fine structure of secretion in Hyalophysa chattoni: formation of the attachment peduncle and the chitinous phoretic cyst wall.

The settling tomite stage of the apostome Hyalophysa chattoni secretes a phoretic cyst wall composed of chitin, mucopolysaccharides, and protein. Within 1 1/2 h after settling, an electron-dense proteinaceous cyst layer (the outer layer) is formed from secretions originating at the base of the kineties and from the thick pellicular layer between the kineties. The inner cyst layer, composed primarily of chitin (acidic and neutral polysaccharides are also present), is secreted across the entire cell surface. Cyst wall formation is completed within 6 h. The fine structure of endocyst secretion resembles stages in the secretion of chitin by fungi, yeasts, and arthropods. A proteinaceous attachment peduncle is secreted to anchor the cell to a shrimp host and is formed by the release of electrondense dense secretory bodies from the cell's ventral surface.     

Encystment and Excystment of the Heterotrichous Ciliate Blepharisma stoltei Isquith*

SYNOPSIS. The structure and cytochemistry of encystment and excystment of Blepharisma stoltei Isquith are described. The encystment process may be subdivided into 4 stages: (i) in the precystic stage the buccal apparatus overlaps about the posterior, (ii) in early encystment, the buccal apparatus is resorbed and an ectocyst is secreted, (iii) an interwall space, endocyst, and plug are secreted during late encystment, and (iv) the resting cyst stage typically has disc-like structures on the ectocyst, and a vacuole in the macronucleus. In excystment, 6 distinct stages may be defined: (i) partial kineties are formed in early excystment, (ii) permanent kineties give rise to anlagen of the buccal apparatus during stomatogenesis, (iii) the organism elongates and reforms the vegetative shape in late excystment, (iv) some cysts then divide, (v) the redeveloped organism is liberated thru the plug pore, and (vi) the postcystic stage resembles the vegetative form except for its size and lack of pigmentation. Cortical structures, extracellular membranes, and the macronuclear membrane are composed of protein-lipids. Unbound protein and RNA are found in the cytoplasm thruout the cystic cycle. DNA is present only in the nuclei. Polysaccharides, 1st found in the cytoplasm, are shifted to the plug in encystment. The plug material disappears during excystment, while PAS positive granules appear in the cytoplasm.     

Ultrastructural study of encystation and excystation in Acanthamoeba castellanii

Encystation and excystation of Acanthamoeba castellanii were studied by transmission electron microscopy. The differentiation process was induced in asynchronous cultures grown axenically. Cytoplasmic vesicles containing a dense fibrous material very similar in appearance to the cyst wall were observed in trophozoites induced to encyst. When these trophozoites were incubated with calcofluor white m2r, fluorescence was observed in cytoplasmic vesicles, suggesting that the material contained in these vesicles corresponded to cyst wall precursors. Semithin cryosections of mature cysts with the same treatment showed fluorescence in the ectocyst and a less intense fluorescence in the endocyst, suggesting the presence of cellulose in both structures of the cyst wall. In mature cysts induced to excystation, small structures very similar to electron-dense granules (EDG) previously described in other amoebae were frequently observed. The EDGs were either sparsely distributed in the cytoplasm or associated with the cytoplasmic face of the plasma membrane. Many of them were located near the ostiole. In advanced phases of excystation, endocytic activity was suggested by the formation of endocytic structures and the presence of vacuoles with fibrous content similar to that of the cyst wall. Electron-dense granules in the process of dissolution were also observed in these vacuoles. Furthermore, the formation of a pseudopod suggests a displacement of the amoeba toward the ostiole.     

Ultrastructure of the Amoeboflagellate Tetramitus rostratus1

The life-cycle of the amoeboflagellate Tetramitus rostratus includes amoeboid, cyst, and flagellate stages. The ultrastructure of these three stages is illustrated, with particular emphasis on flagellate morphology. Amoeba morphology is typical of that of limax amoebas. Cysts, forming from trophic amoebas, are enclosed by a wall made up of two layers: ectocyst (ca. 70 nm), and endocyst (200 nm). The wall apparently forms from precursor material present in vesicles in the pre-cyst stage cytoplasm. Flagellate morphology is characterized by a well-defined top-shaped profile, maintained by microtubules under the plasma membrane. The flagellar apparatus or mastigont consists of four flagella, their basal bodies, sheaves of microtubules associated with two of the basal bodies, and several rhizoplasts (periodicity 20 nm). A deep, microtubule-supported, ventral invagination appears to function as a gullet. A small number of mitotic stages observed in amoeboid and flagellate individuals suggests similarity in the division process in both stages: intranuclear mitotic apparatus, nucleolus persisting through mitosis, no centrioles or basal bodies functioning as centrioles, difficulty in resolving chromosomes. The text compares ultrastructures of several amoeboflagellate organisms and evaluates the phylogenetic significance of those features common to different species. On the basis of this study, Tetramitus most closely resembles Naegleria spp.     

异毛虫形成包囊过程中细胞皮层及纤毛结构分化的扫描电镜观察

在纤毛虫无性生殖中,生命活动受阻时经常会发生形成包囊的现象。研究纤毛虫的包囊现象,已成为揭示真核细胞的结构与功能、细胞模式形成与控制机理的一个重要方面。目前,对腹毛目纤毛虫中游仆虫类包囊的形态及其生理生化特征已进行了较系统的研究,积累了较多的资料,但对其他类     

Paravahlkampfia francinae n. sp. Masquerading as an Agent of Primary Amoebic Meningoencephalitis

ABSTRACT. Paravahlkampfia francinae n. sp., a new species of the free-living amoeba genus Paravahlkampfia , designated as CDC:V595, was isolated from the cerebrospinal fluid of a patient with headache, sore throat, and vomiting, typical symptoms of primary amoebic meningoencephalitis (PAM) caused by Naegleria fowleri . The isolate grew at 33 °C, 37 °C, 40 °C, and 42 °C and destroyed mammalian cell cultures. However, it did not kill young mice upon intranasal inoculation. P. francinae does not produce flagellates and does not grow on agar plates coated with Gram-negative bacteria such as Escherichia coli , the usual food source of Paravahlkampfia ustiana , the type species of the genus. The trophozoite at light microscopy exhibited eruptive locomotion and possessed a single vesicular nucleus. Ultrastructurally, the trophozoites had numerous mitochondria with discoidal cristae but did not have a Golgi apparatus. The trophozoites differentiated into cysts after consuming most of the monolayer. The cyst had an inner well-differentiated endocyst and an outer thin, wrinkled, and wavy ectocyst with no pores. During excystation trophozoites ruptured the cyst wall and emerged from the cysts. A unique feature seen in the cysts was the presence of bacterial endosymbionts, both in the endoplasm and within the cyst wall. Full-length sequencing analysis of the 18S and 5.8S RNA genes of P. francinae showed that they were distinct from those of other Paravahlkampfia species. The patient recovered within a few days indicating that some of the previously reported cases of PAM that survived may have been due to P. francinae .     

The body wall of cheilostome Bryozoa. II. Interzoidal communication organs

Communication organs (septulae) of cheilostome Bryozoa are more complex than perviously believed. Annuli, present only in lateral septulae, are thickenings of the intercalary cuticle. Each communication pore is filled with a ring-like “pore cincture,” through which project a pair of “special cells.” Septulae of all species examined (10 species from 6 families) can be considered modifications of the same structure, varying only in degree of calcification and number of communication pores. External walls, including basal and lateral walls, are best defined as reinforcements of the ectocyst, which is derived by intussusception from the primary cuticle of the ancestrula. The lateral ectocyst must be considered a double layer formed by invagination of the distal ectocyst. Internal walls are developed by apposition from inner parts of the ectocyst; they include pore plates and transverse walls. External walls are laid down first. Lenticular masses develop unilaterally on the uncalcified lateral ectocyst; the pore plate develops by apposition from the interior part of the ectocyst. Depending on the species, the pore plate may or may not be calcified at the time of its formation. Communication pores are formed when the developing pore plate abuts against embryonic special cells. The septular ectocyst never calcifies; it breaks down when the pore plate is complete. Some ascophorans undergo “reparative budding,” in which new zoids are formed within dead zoecia. Hollow, ectocyst-covered buds lined with blastemic epithelia are produced from septulae of live zoids; adjacent buds may fuse. These findings are consistent with the view that lateral septulae are aborted zoids and that pore plates represent transverse walls.     

A light and electron microscopic study of tissue interactions between a parasitic copepod, Scolecodes huntsmani (Henderson), and its host ascidian, Styela gibbsii (Stimpson)

The structure of the cellular cyst which encapsulates the parasitic copepod, Scolecodes huntsmani, in the subendostylar blood vessel of the ascidian, Styela gibbsii, is described from light and electron microscopic studies. The cells comprising the cyst are contributed by the ascidian. The cells are columnar, contain large central reservoirs of glycogen and lipid, and have a conspicuous Golgi apparatus, many small cisternae of smooth endoplasmic reticulum and peripheral mitochondria. The cells are held together by complex basal interdigitations and a short apical zonula occludens. Long cilia emerge in circular clusters from the cell apices and beat in the lumen of the cyst. As atypical of a columnar epithelial layer, the nuclei are staggered in position in the cells and there is no basal lamina. One end of the cyst is blind, but the other end, which may be either anterior or posterior with respect to the longitudinal axis of the host, narrows to a profusely ciliated duct which opens through the wall of the blood vessel to the atrium of the ascidian by a ciliated funnel. The effective beat of the cilia of the duct and the funnel is outward toward the atrium. The first nauplii of the copepod emerge from the incubatory pouch of the adult and pass to the exterior sea water through the cyst funnel and the atrium and atrial siphon of the ascidian. As in other notodelphyid copepods, the life cycle of this incarcerated form also involves free-living naupliar stages followed by two free-living copepodid stages. The provision of an egress for the first nauplii is, therefore, important to the survival of the species. The adult females of Scolecodes, which range in length from 2 to 14.6 mm, are sluggish when removed from the cyst and fail to survive in sea water for more than 24 hours. The males, which have only been obtained when parasitic fifth copepodids molt in culture, are much smaller, averaging 0.8 mm, and are very active. Since one dead male has been found inside the cyst of an adult female and females are often found with attached spermatophores, it is suggested that the funnel of the cyst may also serve as an entrance for the males. Evidence is presented for the formation of the cyst as an accumulation of totipotent lymphocytes around the copepod. Cysts of parasitic developmental stages (third through fifth copepodids) are also described. All of these cysts and those of immature adult females lack funnels to the atrium. The funnel of the cyst of mature females is formed, in part, by modified cells of the wall of the blood vessel, but is induced after the major portion of the cellular cyst has been formed. Cells in the general circulation of the ascidian and those inside the lumen of the cyst are compared. The cells in the lumen of the mature cyst do not arise by diapedesis of blood cells from the subendostylar blood vessel, but by conversion and migration of cells composing the cyst proper. These cells have been found in the guts of the copepods and they may serve as a nutritive source. The ascidian appears not to be harmed by the association, but the copepod gains in many ways.