X-ray microanalysis of the mineral components in the scales of an amoeba,Cochliopodium sp. (Testacea)

Summary Mineral components of the scales in an amoeba, Cochliopodium sp., were examined by energy-dispersive X-ray microanalysis. The precipitates in potassium antimonate-treated material detected calcium in the scales. Calcium was also clearly detected in freeze-substituted thin sections. Similar deposits of calcium antimonate were detected in scales in formation within vacuoles, and also in Golgi cisternae, Golgi vesicles and special granules near the nucleus. There were only minute amounts of magnesium and potassium. This suggests that calcium is the main mineral component of the scales and that it is added in the Golgi complex during scale formation.     

Phylogenetic position and morphology of Raphidiophrys elongata sp. nov. (Haptista: Centroplasthelida) with notes on cyst wall structure and evolution

The majority of centrohelids bear external coverings consisting of organic spicules or siliceous scales. Cyst coverings are usually reinforced with additional layers of modified scales. The cyst wall of Raphidiophrys heterophryoidea has an unusual and complex structure. It consists of three different types of scales and includes the mosaic scale layer not known in other centrohelids. During excystment, the cyst wall fragments along the sutures of the mosaic layer. For other Raphidiophrys species, cyst coverings are not studied. The present paper describes a new Raphidiophrys species, R. elongata, belonging to the NC7 environmental clade. Trophozoites bore thin plate scales with reduced upper plate. Under starvation, cysts emerged in clonal cultures. Cyst coverings of R. elongata and R. heterophryoidea were studied in comparison with the use of FIB-SEM. Cyst wall of R. elongata was significantly thinner than in R. heterophryoidea and was formed with 3–5 layers of uniform overlapping scales. No mosaic scale layer was present. During excystment, trophozoite exited cyst shell through random fissure. Possible evolutionary events and driving forces behind the complication of cyst wall within Raphidiophrys were discussed.     

OBSERVATIONS ON “VAMPYRELLA PENULA-STYLODINIUM SPHAERA” AND THE ULTRASTRUCTURE OF THE REPRODUCTIVE CYST

“Vampyrella-Stylodinium,” an artificial name for a predaceous organism of uncertain taxonomic position, has at least three distinct phases in its life history: the amoeboid phase, both free-floating and attached; the feeding cyst or immobile phase; and flagellated gymnodinoid swarmers. The orange free-floating amoeba has unbranched, filose pseudopodia and several contractile vacuoles. When feeding on the filamentous green alga Oedogonium, the pseudopodia shorten and rearrange. After dissolution of part of the Oedogonium cell wall, the amoeba ingests the host protoplast. Then a stalked reproductive cyst may form. This cyst changes color from green to light orange as it matures. At the time of excystment, the cyst has a smooth outer wall, a spinose inner wall, and a well-delineated phagocytic vacuole. As this vacuole moves from its central position to the cyst's periphery, the walls rupture and 2-4 amoebulae emerge. With TEM observations, the reproductive cyst is shown to be multinucleate. Each nucleus is eukaryotic in organization and possesses one nucleolus. Mitochondria have tubular cristae and no structures unique to the division Pyrrhophyta are observed. Although this stage of the life history does not have a dinokaryotic nucleus, the gymnodinoid swarmers that can emerge from the reproductive cyst, do. Like other parasites which have been assigned to the division Pyrrhophyta, “Vampyrella-Stylodinium” does not conform well to the generalized concept of a dinoflagellate.     

Morphological Study of the Encystment and Excystment of Vermamoeba vermiformis Revealed Original Traits

Free‐living amoebae are ubiquitous protozoa commonly found in water. Among them, Acanthamoeba and Vermamoeba (formerly Hartmannella) are the most represented genera. In case of stress, such as nutrient deprivation or osmotic stress, these amoebae initiate a differentiation process, named encystment. It leads to the cyst form, which is a resistant form enabling amoebae to survive in harsh conditions and resist disinfection treatments. Encystment has been thoroughly described in Acanthamoeba but poorly in Vermamoeba. Our study was aimed to follow the encystment/excystment processes by microscopic observations. We show that encystment is quite rapid, as mature cysts were obtained in 9 h, and that cyst wall is composed of two layers. A video shows that a locomotive form is likely involved in clustering cysts together during encystment. As for Acanthamoeba, autophagy is likely active during this process. Specific vesicles, possibly involved in ribophagy, were observed within the cytoplasm. Remarkably, mitochondria rearranged around the nucleus within the cyst, suggesting high needs in energy. Unlike Acanthamoeba and Naegleria, no ostioles were observed in the cyst wall suggesting that excystment is original. During excystment, large vesicles, likely filled with hydrolases, were found in close proximity to cyst wall and digest it. Trophozoite moves inside its cyst wall before exiting during excystment. In conclusion, Vermamoeba encystment/excystment displays original trends as compare to Acanthamoeba.     

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

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

A Light and Electron Microscopical Study of a New,Polymorphic Free-Living Amoeba,Phreatamoeba balamuthi n. g., n. sp.1

ABSTRACT. A new amoeba, isolated from well water in Gambia, West Africa, is described and named Phreatamoeba balamuthi n. g., n. sp. Requiring anaerobic conditions for growth, it is easily cultured monoxenically with Escherichia coli or axenically in complex, undefined organic media. Three phenotypes have been observed in the life cycle: an amoeba, a flagellate, and a cyst. The amoeba moves by monopodia, is predominantly multinucleate, and varies from 11 to 160 μm in length. The flagellate has a single flagellum and is from 6 to 50 μm long. The cyst is surrounded by a resistant wall that lacks pores and ranges from 9 to 18 μm in diameter. The transformation from amoeba to flagellate can be induced nutritionally, the exact inducing factor(s) being unknown. Sexual reproduction has not been observed.     

Fine structure of excystment of the quadriflagellate algaPolytomella agilis

Summary Populations of mature resting cysts of the algaPolytomella agilis were purified from asynchronously encysting cultures and incubated in fresh culture medium to promote excystment. Up to 90 percent of the cysts germinated, with approximately 50 percent excysting between 3 and 7 hours of incubation. Each germinating cyst releases a single, fully differentiated, swimming cell. The entire excystment process of individual cysts was followed by light microscopy to establish the time course of release and cells at comparable stages of excystment were examined by electron microscopy. During the first 3 hours of incubation the cysts increase in size, presumably due to uptake of water, and a polarity is established in the cytoplasm which makes it possible to identify the site of subsequent release. Release involves a selective degradation of a portion of the cyst wall at this site followed by a physical rupturing of the weakened area. Details of the structural alterations in the wall and cytoplasm are described. The cytoplasmic organelles observed to dedifferentiate during encystment (preceding paper) are completely redifferentiated during excystment. The emergent cell is flagellated and possesses the elongate form typical of the swimming cell.This work was supported by grant A6353 from the National Research Council of Canada to D. L.Brown and by the Inland Waters Directorate of Environment Canada.     

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.     

Ultrastructure of Cysts of Naegleria spp: A Comparative Study*

SYNOPSIS. Ultrastructure of cysts of Naegleria gruberi, Naegleria fowleri, and Naegleria jadini was compared by transmission electron microscopy. Pores in the cyst wall were observed in all 3 species. In N. gruberi they were surrounded by a collar and sealed with a relatively large mucoid plug; no such collar was seen around the pores in the other 2 species, in which the plug was smaller than that in N. gruberi. An electron-dense plaque serving as an additional pore closure was present in all 3 species. In N. gruberi, the cyst wall consisted of an inner thick and an outer thin layer; however, only the inner component was present in cysts of N. fowleri and N. jadini, which had a smooth appearance. At the ultrastructural level, excystment of N. fowleri involved digestion of the mucoid plug and emergence of the trophozoite through the pore. Some digestion of the cyst wall also appeared to take place during excystment.     

Fine structure of encystment of the quadriflagellate alga,Polytomella agilis

Summary The differentiation of resting cysts of the algaPolytomella agilis was examined by electron microscopy. During encystment the free-swimming, quadriflagellate unicells lose their flagella, sink to the bottom of the culture, and form a thick cell wall. Populations of cells at various stages of encystment were collected on microscope slides placed at the bottom of the culture flasks. The mature cyst wall consists of four layers which are laid down sequentially next to the plasma membrane. Freeze-etching has shown that the first layer of wall deposited consists of fibrils which are formed partly embedded within the plasma membrane. A proliferation of rough endoplasmic reticulum and Golgi bodies is seen in early stages of encystment followed by a reduction in size or number of these organelles and of plastids in the maturing cyst. Microtubular structures, including the basal bodies, dedifferentiate and are not observed in the later stages of encystment. The redifferentiation of the swimming cell during excystment is described in the companion paper.This work was supported by grant A6353 from the National Research Council of Canada to D. L.Brown and by the Inland Waters Directorate of Environment Canada.     

Effects of nitrogen deficiency and light of high intensity onCryptomonas rufescens (Cryptophyceae)

Summary C.rufescens excystment, experimentally induced, corresponds to a general metabolism recovery of the cell, previously in a resting phase. The cytoplasm changes without any polarity, and organelles like gullet and flagella redifferentiate. The thylakoids develop mainly from the stored lipidic compounds which then disappear. Phycoerythrin immediately fills the intrathylakoidal lumen. Pigment synthesis seems closely associated with the development of membranes. The activated cell divides and the cyst wall breaks down. The destruction of the wall begins in the median layer and is followed by a mechanical rupture of the external and internal layers. Each germinative cyst releases two or four fully differentiated cells. There is an exact symmetry between excystment and encystment, all the transformations of theC. rufescens cell being reversible.     

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.     

AN ENCYSTMENT STAGE,BEARING A NEW SCALE TYPE,OF THE ANTARCTIC PRASINOPHYTE PYRAMIMONAS GELIDICOLA AND ITS PALEOLIMNOLOGICAL AND TAXONOMIC SIGNIFICANCE1

Cysts of the Antarctic prasinophyte Pyramimonas gelidicola McFadden were found in water samples from a fjord and a saline lake in the Vestfold Hills, Antarctica Unialgal cultures of P. gelidicola from Ace Lake produced cysts. After ca. five weeks, tile cysts settled and adhered to the bottom of the culture flask. The cyst wall was covered by a scale type not seen on the flagellated cells; however, the base of the cyst scale was similar to the box scales of P. gelidicola motile cells. Cyst scales were also found off the continental shelf in Prydz Bay. In a 1.7 m sediment core taken from Ace Lake, both cyst scales and box scales of P. gelidicola occurred at most depths. Differences in the ratio of these two scale types at different depths in the core may indicate past ecological changes in the lake. Upper sediments of the core were dated at 5310 ± 90 yrs B.P., indicating that prasinophyte scales may be recognizably preserved for extended periods. P. gelidicola was widely distributed in saline lakes of the Vestfold Hills with salinities of 3.2–133% and temperatures ranging from – 5.0 to 10.4°C. This is the first report of encystment of P. gelidicola and, to our knowledge, is the first record of a prasinophyte with two distinctly different scale types occurring on cells during different stages of the life history.     

Fine Structure of the Cyst and Some Sporulation Stages of Ichthyosporidium (Microsporida)*

SYNOPSIS. Ichthyosporidium sp. Schwartz, 1963, apparently identical with the type species, I. giganteum (Thélohan, 1895) Swarczewsky, 1914, was studied with the electron microscope. Only late stages, a mature cyst containing sporulation stages and a cyst in the terminal (necrotic) stage were observed. The cyst, originating from host tissue, is a highly organized structure that is integrated with the surrounding connective tissue by means of numerous conspicuous processes. It is interpreted as essentially a manifestation of a defensive reaction of the host that is elicited by the parasite and then used to its advantage. Eventually the cyst dies and disintegrates. This type of cyst, peculiar among those associated with microsporidia, may be regarded as a distinctive character of the poorly defined genus Ichthyosporidium. Other observations let to an hypothesis which reconciles several different views regarding the identity of the Golgi complex. According to this new interpretation, these different views concern different aspects af the total complex. When all such views are integrated, a “classical Golgi” can be recognized in the presporoblastic stages and the “primitive Golgi” concept disappears. This “classical Golgi” then becomes highly modified during spore morphogenesis, giving rise to many of the internal organelles that are peculiar to the spore.     

Structure,composition, and biogenesis of prasinophyte cell coverings

Summary The cell body and flagellar surfaces of certain green flagellates are covered by non-mineralized scales. Scale structure has been widely used in the systematics of this group of algae commonly known as the Prasinophyceae. The special importance of the flagellar hairs as a taxonomic marker is discussed. We summarize current knowledge about the structure and chemical composition of these scales with emphasis on thecate flagellates. Scales consist mainly of acidic polysaccharides involving unusual 2-keto sugar acids. Glycoproteins as minor components are mainly involved in mediating scale subunit and scale-membrane interactions and species specific glycosylation patterns exist. In thecate prasinophytes the elaboration of 3-deoxy-manno-2-octulosonic acid and galacturonic acid side chains presumably favours a complex of thecal scales with calcium ions and thus extracellular coalescence of the scales to a rigid cell wall. Scales are formed within the Golgi apparatus (GA) and especially in thecate prasinophytes scale formation (i.e., during flagellar regeneration) represents an excellent model system for GA function. Movement of developing scales through the GA requires cisternal progression. Biogenesis of scales involves mainly polysaccharide synthesis, whereas about 50% of the scale-associated glycoproteins are added from a pre-existing pool. Possible functions of prasinophyte scales are briefly discussed.Abbreviations Dha 3-deoxy-lyxo-2-heptulosaric acid - DSA Datura stramonium agglutinin - ER endoplasmic reticulum - GA Golgi apparatus - GNA Galanthus nivalis agglutinin - Kdo 3-deoxy-manno-2-octulosonic acid - MeKdo 3-deoxy-5-O-methyl-manno-2-octulosonic acid - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis     

THE FINE STRUCTURE AND SCALE FORMATION OF CHRYSOLEPIDOMONAS DENDROLEPIDOTA GEN. ET. SP. NOV.(CHRYSOLEPIDOMONADACEAE FAM. NOV., CHRYSOPHYCEAE)1

Chrysolepidomonas gen. nov. is described for single-celled monads with two flagella, a single chloroplast, and distinctive canistrate and dendritic scales. The type species, Chrysolepidomonas dendrolepidota sp. nov., is described for the first time. The canistrate scales bear eight “bumps” on the top surface, and the dendriticscales have a tapered base with a quatrifid tip. These organic scales are formed in the Golgi apparatus and storred in a scale reservoir. The scale reservoir is bounded on two sides by the R₁ and R₂ in microtubular roots of the basal apparatus. The cyst (=stomatocyst, statospore) forms endogenously by means of a silica deposition vesicle. The outer cyst surface is smooth, and the pore region is unornamented. Two other organisms bearing canistrate and dendritic scales, previously assigned to the genus Sphaleromants, are transferred to the genus Chrysolepidomonas. They are C.angalica sp. nov. and C. marine(Pienaar) comb. nov. The distinguishing features of Chrysolepidomonas and Sphaleromantis are discussed. A new family, Chrysolepidomonadceae fam. noc., is described for flagellates covered with organic scales.     

A light and electron microscopical study of a new, polymorphic free-living amoeba, Phreatamoeba balamuthi n. g., n. sp

A new amoeba, isolated from well water in Gambia, West Africa, is described and named Phreatamoeba balamuthi n. g., n. sp. Requiring anaerobic conditions for growth, it is easily cultured monoxenically with Escherichia coli or axenically in complex, undefined organic media. Three phenotypes have been observed in the life cycle: an amoeba, a flagellate, and a cyst. The amoeba moves by monopodia, is predominantly multinucleate, and varies from 11 to 160 microns in length. The flagellate has a single flagellum and is from 6 to 50 microns long. The cyst is surrounded by a resistant wall that lacks pores and ranges from 9 to 18 microns in diameter. The transformation from amoeba to flagellate can be induced nutritionally, the exact inducing factor(s) being unknown. Sexual reproduction has not been observed.     

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.     

The Fine Structure of Acanthamoeba astronyxis,with Special Emphasis on Encystment1

ABSTRACT. The fine structure of the trophozoite, encysting cells, and the cyst of Acanthamoeba astronyxis has been examined. In the trophic form a microtubule organizing center was associated with a well developed Golgi complex. During encystment the organelles of the amoeba changed considerably. The profiles of rough endoplasmic reticulum elongated and were often arranged in circles of multilayered concentric systems, enclosing mitochondria, the nucleus, or other inclusions. The mitochondria showed a tendency toward elongation and constriction. One or two nucleolus-like bodies appeared in the nucleus. Lipid droplets increased considerably in amount and were distributed individually or as aggregates. The mature cyst was star-shaped and surrounded by an almost circular exocyst and an endocyst that was closely apposed to the cell membrane. Both walls differed in their thickness and granulation. The exocyst was continuous over the entire cyst, while the endocyst was interrupted by gaps, ostioles. in the region of the rays. Within the ostioles was a bell-shaped structure, the operculum. The latter was composed of a granular material comparable in electron density to that of the endocyst.     

The Formation and Fate of Tetrahymena patula Cryptostomes

A reliable method for producing reproductive cysts in Tetrahymena patula is described. The procedure involves the isolation of macrostomes without cytopharyngeal pouches in microdrops of distilled water under oil. The study of silver-impregnated specimens has shown that a complex pattern of oral resorption and reformation occurs within the cyst that leads to the formation of a group of small cells with recessed oral apparatuses. These cells, called “cryptostomes,” swim very rapidly on excystment and are incapable of either feeding or reproducing. They are presumably dispersal forms. Oral morphogenesis during the transformation of excysted cryptostomes into microstomes and macrostomes is also described.