Effects of UV-Radiation on Kinetics of Encystment and on Morphology of Resting Cysts of the Ciliate Laurentiella acuminata1

The effects of ultraviolet radiation (λ= 254 nm) on the kinetics of encystment of the hypotrichous ciliate Laurentiella acuminata and the structure of resting cysts obtained from irradiated precystic cells are reported. High doses of UV-radiation caused a delay of encystment with a linear increase in the average time for obtaining 50% of encystment (EN₅₀). Resting cysts with abnormal cyst walls were obtained when precystic cells were irradiated in the exposure range 720 to 960 J/m². The cystic layer (mesocyst) was approximately twice as thick (6.5 μ m) as normal (3.7 μ m). Microscopical observations of abnormal cysts revealed the presence of two complete mesocysts, and the absence of the spines characteristic of the ectocyst. The UV-dependent effects on the cyst wall were gradually corrected in successive generations of the irradiated cells.     

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.     

Induction of synchronous encystment in a hypotrichous ciliate, Histriculus sp

A method for the induction of synchronous encystment in a hypotrichous ciliate,Histriculus sp. is described. When the ciliates were transferred into 10× Osterhout's solution at around 7 h after disappearance of the food organisms, Tetrahymena, and were maintained at 20 °C, the organisms underwent synchronous encystment. Phase 1 cells (precystic cells) appeared at around 2 h after transfer into the 10× Osterhout solution, and almost all organisms had transformed into phase 1 cells approx. 3 h after transfer. The first cyst appeared at around 3 h after transfer into the 10× solution and essentially 100% of the organisms had transformed into cysts within approx. 4.5 h after transfer.     

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.     

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.     

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.     

Structural Changes in Tetrahymena rostrata during Induced Encystment*

SYNOPSIS. Experimentally induced precystic stages and mature cysts from 3 clones of Tetrahymena rostrata were examined by light and electron microscopy. It was demonstrated by cytochemical staining and fine-structural observations that precystic stages release mucocyst material that provides for the production of a cyst wall. Early and late cysts also contain numerous autophagous vacuoles. In late cysts there is a replacement of depleted mucocyst organelles. The developmental evidence obtained from sampling of sequential developmental stages suggests an ～24-h timetable of cytoplasmic events associated with encystment in this organism.     

EXOCYTOSIS OF LATEX BEADS DURING THE ENCYSTMENT OF ACANTHAMOEBA

Cells of Acanthamoeba castellanii (Neff) are known to form mature cysts characterized by a cellulose-containing cell wall when transferred to a nonnutrient medium. Amebas which engulfed latex beads before encystment formed mature cysts essentially devoid of bead material. The encystment of bead-containing cells appeared to be similar to that of control cells since no important differences between the two were observed with respect to cellular levels of glycogen or protein, cellulose synthetase activity, the amount of cyst wall polysaccharide formed, or the percentage of cysts formed. Actinomycin D and cycloheximide inhibited encystment as well as bead expulsion. Ultrastructural analysis revealed that the beads, which initially were contained in phagocytic vesicles, were released from the cell by fusion of vesicular membranes with the plasma membrane. Exocytosis was observed in cells after 3 hr of encystment, with most of the beads being lost before cyst wall formation. Each bead-containing vesicle involved in expulsion was conspicuously demarcated by an area of concentrated cytoplasm, which was more homogeneously granular than the surrounding cytoplasm. Beads were not observed in the cytoplasm of mature cysts but were occasionally found in the cyst wall.     

Giant Cysts and Cysts with Multiple Central Bodies in Azotobacter vinelandii

Cyst germination in Azotobacter vinelandii ATCC 12837 was studied by using phase contrast and electron microscopy. Germination in this organism was accompanied by the formation of large cyst forms of two different types: giant cysts and cysts containing multiple central bodies. Previously, these two types have been reported only when yeast extract was added to the encystment medium. In this study, we observed giant cysts and cysts with multiple central bodies in nitrogen-free liquid medium. The germination of "normal" cysts is often preceded by enlargement to the giant form and division of the central body to produce cysts with multiple central bodies. Structures similar in appearance to ribosomal aggregates were observed only in cysts undergoing pregermination transformations.     

Cortical and Nuclear Events During Cell Division and Resting Cyst Formation in Colpoda inflata

Nuclear and cortical phenomena during dividing and resting cyst formation of Colpoda inflata are described. Cell division forms a cyst and produces two or four tomites. In each tomite, the right oral field results from the proliferation of the anterior extreme of a single kinety, and the left oral field results from the proliferation of four, five, or six somatic kineties. After macronuclear division, each macronuclear mass undergoes a chromatinic extrusion process. During resting cyst formation, the oral infraciliature of the vegetative cell is resorbed. The somatic kineties dispose in a radial way and some pairs of kinetosomes disappear. As in cell division, there is an extrusion process. From these results we conclude that the resting cysts of Colpoda inflata cannot be included in any group of the previous classifications for hypotrich resting cysts. Thus, we propose a new additional group to Walker and Maugel's classification called PKR (partial-kinetosome-resorbing) cysts.     

Effects of copper sulphate on in vitro encystment of the cercariae of Echinostoma caproni

The effects of various concentrations of copper sulphate were studied on in vitro encystment of Echinostoma caproni in a Locke's-artificial spring water (ASW) (1:1) medium. Cercariae were killed in 10,000 mg l(-1) CuSO4 in Locke's-ASW (1:1) within 24 h and extruded cystogenous material to produce an abnormal cyst wall. The 'emergency response' of encystment to high concentrations of copper reported for Parorchis acanthus cercariae did not occur in E. caproni. Concentrations of 1000 mg l(-1) and 100 mg l(-1) CuSO4 in Locke's-ASW (1:1) also killed the cercariae without encystment by 48 h. A concentration of 10 mg l(-1) CuSO4 in Locke's-ASW (1:1) allowed for normal in vitro encystment within 48 h and these cysts were capable of excystation in a trypsin-bile salts medium.     

The primary and secondary spore cyst of Aphanomyces (Oomycetes, Saprolegniales)

The ultrastructural organization of the primary (1°) and secondary (2°) cysts of Aphanomyces astaci and A. laevis is extremely similar, and similar to that of the 1° and 2° cysts of A. eutekhes as presented earlier by Hoch and Mitchell. Synchronous populations of 2° cysts can be induced by mechanical shock and encystment appears to be essentially instantaneous. The cyst coat–wall appears to be formed extremely rapidly from material from the peripheral vesicles with flocculent content. After encystment the microtubule cytoskeleton found in the zoospore is maintained in the 1° and 2° cyst (i.e. the single microtubules which extend along the pyriform nucleus from the ki–netosomes–centrioles and the bundles of closely appressed microtubules are retained). The peripheral vesicles with granular content found in the zoospore are not seen in the 1° or 2° cyst. Multivesicular bodies and lomasomes are observed in the 1° and 2° cyst which are not found in the zoospore. The peripheral cisternae of the zoospore are lost upon encystment and may be formed from dictyosome–derived vesicles during excystment of the 1° and 2° cyst. The U–body of A. astaci has a paracrystalline content while the U–body of A laevis and A eutekhes has a tubular content. A microbody–lipid body complex (sensu Powell) is found in the 1° and 2° cysts of A laevis but not in A astaci or A eutekhes. The significance of the presence of a microbody–lipid body complex in a biflagellate zoospore is discussed.     

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.     

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.     

HIGH ENCYSTMENT SUCCESS OF THE DINOFLAGELLATE SCRIPPSIELLA CF. LACHRYMOSA IN CULTURE EXPERIMENTS 1

Close to 100% encystment efficiency and a yield above 10⁵ cysts·mL ^{? 1} were routinely achieved in full strength f/2 medium‐based batch cultures (883 μM NO₃ ^? and 36 μM PO₄ ^{? 3}) of the marine dinoflagellate Scrippsiella cf. lachrymosa Lewis. Increases in cell density led to nutrient depletion in this enriched medium, which was the most likely cause for initiation of cyst formation. Lowering the concentration of either nutrient to 1/10 the initial levels decreased the encystment efficiency, whereas use of ammonium as the N source resulted in both low cell yield and low encystment efficiency. The mandatory dormancy period was ca. 60 days and was not affected by cold dark storage of the cysts. Cysts produced in the initial phase of sexual reproduction were relatively large (length 47 μm, width 31 μm) with a heavy calcareous cover. Cysts produced thereafter lacked apparent calcareous cover and were smaller (length 29 μm, width 19 μm). The decrease of cyst volume (by a factor of 0.24–0.4) suggested strong resource limitation during the course of encystment. However, after the mandatory dormancy period, germination success of the smaller cysts was higher (80%), compared with the larger cysts that had been produced initially (50%). Germling survival (74%) was independent of cyst type but was enhanced by higher nutrient concentration during incubation. The ratio of initial nutrient concentration in the medium to the cyst yield was used as a proxy to estimate the cellular nutrient quota. The conservative estimates of 9 pmol N·cyst ^{? 1} and 0.4 pmol P·cyst ^{? 1} obtained in this manner are at the low end of the range of previous published estimates for other dinoflagellate cysts. Given the high encystment observed in laboratory experiments, we have no reason to assume an inherently lower encystment success in dinoflagellate field populations. Our results do not challenge the low nutrient paradigm for dinoflagellate sexuality. We believe that the high encystment success and cyst yield of this particular species is at least partly due to its ability to achieve very high cell densities in cultures, which evidently leads to nutrient depletion even in f/2 medium.     

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.     

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.     

First record of cysts in the tidal tardigrade Echiniscoides sigismundi

Tardigrades are microscopic metazoans that withstand environmental extremes by entering dormant states, such as cryptobiosis (latent life). In addition, they may also form cysts. Here, we present the first report of cyst formation in a marine heterotardigrade, i.e., Echiniscoides sigismundi, which constitutes a cryptic species complex present worldwide in tidal zones. The cysts were initially discovered during experimental series constructed to investigate osmotic stress tolerance. The animals, which eventually formed cysts, showed signs of an imminent molt at the beginning of experimentation. We use the term “cyst” for stages, where a total of three or more cuticles have been synthesized. Our observations show that encystment in E. sigismundi involves synthesizing of at least two new cuticle layers. Legs with discharged claws are present in connection with the first outer cuticle, as well as the second cuticular layer. In the most developed cyst, a third cuticle lacking claws seems to surround the animal, which is delineated by a fourth cuticle. Many features are shared with the well-studied cysts of eutardigrades. The cysts of E. sigismundi, however, lack pigmentation and have an extra set of claws, and the animal inside retains buccopharyngeal sclerified parts, until discharging the third cuticle. The finding of cysts in a marine heterotardigrade is novel and confirms that encystment also occurs within this major evolutionary lineage.     

The effect of penicillin on the encystment of Bdellovibrio sp. strain W

When penicillin, and other inhibitors of peptidoglycan synthesis were added to encysting cultures of Bdellovibrio strain W, the encysting process continued, resulting in the production of cysts which were spherical in shape. Transmission electron micrographs of these spherical bdellocysts revealed the absence of an outer cyst wall. These cysts, devoid of cyst wall, were capable of germination under appropriate condition with the emergence from the prey ghost of highly motile spheroplasts. Withdrawl of the antibiotics after encystment had begun led to the production of spherical cysts that were surrounded by an outer cyst wall.