THE FINE STRUCTURE OF ACANTHAMOEBA CASTELLANII (NEFF STRAIN) : II. Encystment

Encysting cells of Acanthamoeba castellanii, Neff strain, have been examined with the electron microscope. The wall structure and cytoplasmic changes during encystment are described. The cyst wall is composed of two major layers: a laminar, fibrous exocyst with a variable amount of matrix material, and an endocyst of fine fibrils in a granular matrix. The two layers are normally separated by a space except where they form opercula in the center of ostioles (exits for excysting amebae). An additional amorphous layer is probably present between the wall and the protoplast in the mature cyst. Early in encystment the Golgi complex is enlarged and contains a densely staining material that appears to contribute to wall formation. Vacuoles containing cytoplasmic debris (autolysosomes) are present in encysting cells and the contents of some of the vacuoles are deposited in the developing cyst wall. Lamellate bodies develop in the mitochondria and appear in the cytoplasm. Several changes are associated with the mitochondrial intracristate granule. The nucleus releases small buds into the cytoplasm, and the nucleolus decreases to less than half its original volume. The cytoplasm increases in electron density and its volume is reduced by about 80%. The water expulsion vesicle is the only cellular compartment without dense content in the mature cyst. The volume fractions of lipid droplets, Golgi complex, mitochondria, digestive vacuoles, and autolysosomes have been determined at different stages of encystment by stereological analysis of electron micrographs. By chemical analyses, dry weight, protein, phospholipid, and glycogen are lower and neutral lipid is higher in the mature cyst than in the trophozoite.     

Correlation of cellulose synthesis in vivo and in vitro during the encystment of Acanthamoeba

Acanthamoeba castellanii differentiates when placed in a starvation medium. The mature cysts formed are characterized by a cellulosic wall synthesized from endogenous sources during encystment. A particulate enzyme system whose specific activity increases some 30-fold during encystment catalyzes the formation of an alkali-soluble and an alkali-insoluble β-(1 → 4)-glucan (cellulose). The activity in vitro of this enzyme extracted from populations of cells during encystment correlates with the formation in vivo of the mature cyst and the alkali-insoluble β-glucan of the cyst wall. The conclusion is based on the following observations:

1.

1. Both alkali-soluble and alkali-insoluble β-glucans similar to the enzymatic products of the isolated β-glucan synthetase occur in cyst walls.     

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 resistance of cysts of Stictodora lari (Trematoda: Heterophyidae) to encapsulation by cells of the fish host

Whereas glass beads are encapsulated by cells within 3 days after implantation into the abdominal cavity of Gambusia affinis, cysts of Stictodora lari in the same site are not encapsulated until 21–23 days after infection. The achievement of encystment in vitro demonstrated that the initial cyst wall is of parasite origin and fluorescent antibody methods showed that it does not mimic fish tissue in composition. Cysts formed in vivo have material, presumably of fish origin, associated with the cyst wall, as do living and fixed in vitro cysts following implantation.It is considered that cysts are not encapsulated for some weeks after infection because they are disguised as host tissue by material of fish origin associated with the cyst wall. An alternative explanation is proposed if the fish material does not have this functional role; the presence of spikes on the initial cyst wall may form an unsatisfactory substrate for the attachment of cells.     

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.     

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.     

阔口尖毛虫形成包囊期间细胞超微结构的观察

阔口尖毛虫形成囊期间,细胞质内出现条带状或管产产的内质网和由不同大小的囊泡组成的包囊壁前体。并且,前体的产生与内质网有关;细胞质内发生自噬泡消化现象,这是细胞将原有结构和能量进行贮存,利用的一种重要形式;大核向细胞质突出形成阿米巴形结构,这与大核向细胞质排出部分核物质有关。     

Evaluation of membrane-bound black bodies in trophozoites and cysts of Naegleria spp

Various Naegleria strains were examined to determine the possible origin and significance of membrane-bound black bodies that were found in all exponentially growing cell populations. The bodies, 40–80 nm in diameter, were distributed randomly in the cytoplasm of Naegleria with ultrastructural features typical of trophozoites. No evidence was obtained that the contents of the black bodies were synthesized in the rough endoplasmic reticulum (ER) and packaged by membranous components, which could be a primitive “Golgi complex” in these amoebae. Examination of cells in various stages of encystment indicated that at least some of the cyst wall material was synthesized and packaged by the rough ER. After condensation into amorphous granules in the cisternae, the cyst wall material appeared in vesicles of the rough ER; these were frequently seen in close proximity to the cell membrane in the vicinity of developing cyst wall. Amorphous granules (～100 nm in diameter), which had variable densities and did not appear to be membrane bound, were seen in the cytoplasm of encysting cells. The substance of these granules also seemed to be incorporated into the cyst wall. The membrane-bound black bodies appeared to be destroyed in lysosomal elements during encystment. The membrane-bound black bodies were concluded to be characteristic of trophozoites and unrelated to encytment of Naegleria.     

A chemical and autoradiographic study of cellulose synthesis during the encystment of Acanthamoeba castellanii

When cells of Acanthamoeba castellanii are placed in a non-nutrient medium, they differentiate into cysts which possess cellulosic walls. In the present study, the source of the glucosyl unit for cyst wall cellulose was investigated by following the encystment of trophozoites grown in the presence of ¹⁴C-labeled fatty acids (uniformly labeled palmitate or oleate) or [3-³H]glucose. Cells were fractionated at the beginning and after 30 hr of encystment using a modified Schmidt-Tannhauser procedure. In cells grown on fatty acids, 90% of the labeled material was in the lipid fractions both before and after encystment with the total amount of label/cell changing very little. Both partial and complete acid hydrolysis of the glycogen of the acidsoluble fraction and the alkali-insoluble residue of the cyst wall indicated that the glucose of both fractions was not radioactive, although Acanthamoeba is known to have a functional glyoxylate pathway.Fractionation data of cells grown on [³H]glucose indicated a sevenfold increase in radioactivity in the wall insoluble fraction and a fivefold decrease in the acid-soluble fraction with the cpm/cell of the other fractions changing very little after 30 hr of encystment. Approximately 70% of the ³H-labeled material was recovered as glucose from the 30-hr wall insoluble fraction following complete acid hydrolysis. The specific radioactivity of glucose in the cyst wall insoluble fraction was the same as that of glycogen glucose isolated from the acid soluble fraction of trophozoites. Electron microscopic autoradiography showed that the majority of nonlipid radioactivity was due to glycogen in trophozoites. Autoradiograms failed to reveal Golgi bodies or any particular region of the cell as being the specialized site of cellulose synthesis. The results of the fractionation and autoradiographic studies are consistent with the concept that glycogen is a precursor of cyst wall cellulose, and that glucosyl units of glycogen and/or other glucose derivatives are converted to cellulose without significant dilution under the experimental conditions used.     

Formation of the Giardia Cyst Wall: Studies on Extracellular Assembly Using Immunogold Labeling and High Resolution Field Emission SEM

Encystment of the intestinal protozoan, Giardia, is a key step in the life cycle that enables this parasite to be transmitted from host to host via either fecal oral, waterborne, or foodborne transmission. The process of encystment was studied by localizing cyst wall specific antigens with immunofluorescence for light microscopy and immunogold staining for field emission scanning electron microscopy. Chronological sampling of Giardia cultures stimulated with endogenous bile permitted identification of an intracellular and extracellular phase in cyst wall formation, a process which required a total of 14-16 h. The intracellular phase lasted for 8-10 h, while the extracellular phase, involved the appearance of cyst wall antigen on the trophozoite membrane, and the assembly of the filamentous layer, a process requiring an additional 4-6 h for completion of mature cysts. The extracellular phase was initiated with the appearance of cyst wall antigen on small protrusions of the trophozoite membrane (-15 nm), which became enlarged with time to caplike structures ranging up to 100 nm in diameter. Caplike structures involved with filament growth were detected over the entire surface of the trophozoite including the adhesive disc and flagella. Encysting cells rounded up, lost attachment to the substratum, and became enclosed in a layer of filaments. Late stages in encystment included a “tailed” cyst in which flagella were not fully retracted into the cyst. Clusters of cysts were seen in which filaments at the surface of one cyst were connected with the surface of adjacent cysts or the “tailed” processes of adjacent cysts, suggesting that the growth of cyst wall filaments may be at the terminal end. In conclusion, the process of encystment has been shown to consist of two morphologically different stages (intracellular and extracellular) which requires 16 h for completion. Further investigation of the extracellular stage with regard to assembly of the filamentous layer of the cyst wall may lead to innovative methods for interfering with production of an intact functional cyst wall, and thereby, regulation of viable Giardia cyst release from the host.     

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     

High-resolution electron microscopic evidence for the filamentous structure of the cyst wall in Giardia muris and Giardia duodenalis

High-resolution morphological studies of the cyst wall of Giardia spp. were performed using low-voltage scanning electron microscopy (LVSEM) and transmission electron microscopy (TEM). The cyst wall was composed of membranous and filamentous layers. The membranous layer consisted of an inner and an outer cyst membrane separated by a thin layer of cytoplasm. The filamentous layer contained individual filaments that ranged from 7 to 20 nm in diameter when measured by LVSEM, formed a dense meshwork with branches or interconnections, and were occasionally arranged on the surface in whorled patterns. Cysts of Giardia muris from mice, Giardia duodenalis from dogs, pigs, voles, beavers, muskrats, and humans, and Giardia psittaci from a bird (parakeet), possessed an essentially identical wall composed of filaments. Inducement of excystation in viable Giardia cysts produced a dramatic increase in the interfilament spacing over an entire cyst, but none was observed in heat-killed or chemically fixed control cysts. These results demonstrated that the cyst wall of Giardia spp. was composed of a complex arrangement of filaments, presumably formed during the process of encystment.     

Identification of Precystic Stages and Encystment Kinetics Analysis of Colpoda inflata by Using an Immunofluorescent Method

ABSTRACT. The application of an immunocytochemical method to identify precystic stages and to analyse the encystment kinetics, by using a polyclonal antiserum against isolated cyst walls from the ciliate Colpoda inflata , is reported for the first time. Three different precystic phases were chosen on the basis of morphological changes and degree of cyst wall formation. By using this procedure a better identification of mature resting cysts with regard to precystic cells or young cysts is provided. An average consensus encystment kinetics of C. inflata , by using an accumulated class frequency analysis, is reported.     

MULTIPLE ROUTES OF SEXUALITY IN ALEXANDRIUM TAYLORI (DINOPHYCEAE) IN CULTURE1

Alexandrium taylori Balech is a cyst‐forming dinoflagellate species responsible for recurrent blooms in Mediterranean coastal waters. The nuclear development of the cells during the sexual cycle and the effect of different external nitrate and phosphate levels were studied. Nuclear fusion of gametes occurred 6–12 h after the complete cytoplasmic fusion. The U‐shaped nuclei fused through the end of one nucleus and the mid‐area of the other. The mobile and biflagellated zygote had a large, U‐shaped nucleus and may follow three different fates: direct division, short‐term encystment (ecdysal), and long‐term encystment (resting). Ecdysal cysts may divide in >24–96 h into two, four, six, or eight cells before germinating. Meiosis presumably occurred in three locations: in the planozygote, within the ecdysal cyst, and in the planomeiocyte (germling) liberated either from ecdysal or resting cysts. The effects of nutrients on these routes were studied in individually isolated sexual stages. (1) Direct divisions occurred mainly under replete conditions (L1), whereas no direct planozygote divisions were recorded in media with no phosphate added (L‐P). (2) Short‐term encystment was larger in media lacking phosphate (L‐P and L/30) than in medium with no nitrate added (L‐N) or under replete conditions (L1). (3) Long‐term encystment was only observed in medium with no nitrate added (L‐N). The long‐lived resting cyst, not previously described for this species, had a clear double wall, an irregular shape, a flat morphology, and a middle orange spot. No cysts germinated in 1–2 months, whereas 86% of the cysts germinated 2–3 months after being formed. A flow cytometry analysis showed that sexual induction and zygote formation were very fast and highly common processes, zygotes being nearly half of the population at days 3 and 5 after the induction of sexuality in the cultures.     

Encystment and alkylresorcinol production by<Emphasis Type="Italic"> Azotobacter vinelandii</Emphasis> strains impaired in poly-β-hydroxybutyrate synthesis

The lipids poly-beta-hydroxybutyrate (PHB) and alkylresorcinols are the major metabolic products of Azotobacter vinelandii cysts. Cysts are formed in less than 0.01% of late stationary phase cells grown on sucrose. Culturing vegetative cells in n-butanol or beta-hydroxybutyrate induces encystment. After induction of encystment, PHB rapidly accumulates in large granules. Then, the cells begin the synthesis of alkylresorcinols that replace the phospholipids in the membranes and are components of the exine, the outer layer of the cyst envelope. Vegetative cells do not synthesize alkylresorcinols. We report here the effect of mutations in the phbBAC operon, coding for the enzymes of the PHB biosynthetic pathway, on the synthesis of alkylresorcinols and cyst formation. The phb mutations did not impair the capacity to form mature cysts. However, the cysts formed by these strains posses a thicker exine layer and a higher content of alkylresorcinols than the cysts formed by the wild-type strain. A blockage of PHB synthesis caused by phb mutations resulted in the synthesis of alkylresorcinols and encystment even under non-inducing conditions. We propose that, as a consequence of the blockage in the PHB biosynthetic pathway, the acetyl-CoA and reducing power pools are increased causing the shift to lipid metabolism required for the synthesis of alkylresorcinols and cyst formation.     

Three-Dimensional Ultrastructural Study of Oil and Astaxanthin Accumulation during Encystment in the Green Alga Haematococcus pluvialis

Haematococcus pluvialis is a freshwater species of green algae and is well known for its accumulation of the strong antioxidant astaxanthin, which is used in aquaculture, various pharmaceuticals, and cosmetics. High levels of astaxanthin are present in cysts, which rapidly accumulate when the environmental conditions become unfavorable for normal cell growth. It is not understood, however, how accumulation of high levels of astaxanthin, which is soluble in oil, becomes possible during encystment. Here, we performed ultrastructural 3D reconstruction based on over 350 serial sections per cell to visualize the dynamics of astaxanthin accumulation and subcellular changes during the encystment of H. pluvialis. This study showcases the marked changes in subcellular elements, such as chloroplast degeneration, in the transition from green coccoid cells to red cyst cells during encystment. In green coccoid cells, chloroplasts accounted for 41.7% of the total cell volume, whereas the relative volume of astaxanthin was very low (0.2%). In contrast, oil droplets containing astaxanthin predominated in cyst cells (52.2%), in which the total chloroplast volume was markedly decreased (9.7%). Volumetric observations also demonstrated that the relative volumes of the cell wall, starch grains, pyrenoids, mitochondria, the Golgi apparatus, and the nucleus in a cyst cell are smaller than those in green coccid cells. Our data indicated that chloroplasts are degraded, resulting in a net-like morphology, but do not completely disappear, even at the red cyst stage.     

ENCYSTMENT OF THE DINOFLAGELLATE GYRODINIUM UNCATENUM: TEMPERATURE AND NUTRIENT EFFECTS1

Sexual reproduction and encystment of the marine dinoflagellate Gyrodinium uncatenum Hulburt were induced in nitrogen and phosphorus-limited batch cultures. Sexuality did not occur under nutrient-replete conditions even when growth rate was reduced by non-optimal temperatures. Growth was optimal over a broader temperature range than encystment and virtually no cysts were produced at some low and high temperatures where growth occurred. Most cells initiated sexuality as intracellular pools of each limiting nutrient reached minimum or subsistence levels as much as four days after extracellular nutrients were exhausted. High nitrogen cell quotas during the phosphorus experiment indicate that sexuality was induced by a shortage of phosphorus and not by an indirect effect on nitrogen uptake. Total cyst yield corresponded to successful encystment of 9–13% of the motile populations, yet 60–85% of the plateau-phase motile cells were planozygotes (swimming zygotes formed from fusing gametes). Batch culture studies monitoring total cyst yield may thus seriously underestimate the extent of sexuality. More importantly, the number of cysts produced in a dinoflagellate population may be significantly reduced by environmental factors acting on the cells after sexual induction and fusion.     

LOCALIZATION OF CYTOPLASMIC NUCLEIC ACID DURING GROWTH AND ENCYSTMENT OF ENTAMOEBA INVADENS

With a modified color-translating ultraviolet microscope, the distribution of material showing an absorption maximum at 265 mµ was studied in samples from whole cultures of Entamoeba invadens at intervals during growth and from cysts allowed to mature under controlled conditions. Absorption by the cytoplasm in general gradually increased as trophozoites approached the period of maximum encystment. In late trophozoites and precystic forms, the absorbing material was concentrated into small bodies which coalesced to form large crystalloids of very high specific absorption. Maximum crystallization occurred in early cysts, where cytochemical tests have shown the large crystalloids to be ribonucleoprotein. Electron micrographs show that the crystalloids are composed of particles 200 to 300 A in diameter. During cyst maturation the amount of absorbing material per cyst is not visibly reduced, but the large bodies fragment into smaller units until finally there is only a very high diffuse absorption over the entire cyst. From these and other results the hypothesis is advanced that the large crystalloids ("chromatoid bodies") are a manifestation of a special parasite-host adaptive mechanism; ribonucleoprotein is synthesized under favorable conditions, crystallized in the resistant cyst stage, and dispersed in the newly excysted amebae thereby enabling them to establish themselves in a new host by a period of quick growth.     

DNA synthesis in growth and encystment of Acanthamoeba castellanii.

Differentiation of Acanthamoeba castellanii into dormant cysts occurs spontaneously in stationary phase cultures, or can be induced experimentally by starvation. Although no further increase in cell density occurred after induction in either case, incorporation of [H]thymidine into DNA continued at a reduced rate through the period when differentiated products (cyst wall components) were formed. No net accumulation of DNA occurred during differentiation, indicating that the DNA synthesis occurring at this time was balanced by breakdown. When either 5-fluorodeoxyuridine (FUdR) or hydroxyurea was added to exponentially growing cultures, growth was terminated and the subsequent spontaneous encystment was delayed in comparison with untreated stationary phase cultures. A similar delay was observed for experimentally induced encystment of FUdR-pretreated cells. In all cases, delay of encystment was correlated with inhibition of 32PO4 incorporation into DNA, and unexpectedly also into RNA. Addition of FUdR at zero-time of experimental induction of cells not previously exposed to FUdR, on the other hand, had no effect on encystment or on 32PO4 incorporation. The delay of encystment produced by FUdR and hydroxyurea, therefore, appeared to reflect a requirement for normal synthesis of DNA and/or RNA not only during encystment, but also during the period of exponential growth just before encystment induction.