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.     

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.     

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.     

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.     

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.     

Encystment of Peridinium gatunense: occurrence, favourable environmental conditions and its role in the dinoflagellate life cycle in a subtropical lake

1. The abundance of cysts of the bloom‐forming dinoflagellate Peridinium gatunense in the sediments of Lake Kinneret and the effects of environmental conditions on encystment were studied in relation to bloom dynamics. Peak cyst formation coincided with the highest growth rate of the population, prior to bloom peak. 2. Peridinium cysts were counted in water and sediment corer samples from 2000 to 2003 and in archived sediment trap samples collected during 1993–94. The cyst data were examined in relation to ambient temperature and nutrient records, and revealed no direct correlation. 3. In laboratory encystment experiments with Peridinium cells collected from the lake, 0.2–3% of the vegetative cells encysted. Temperature, light and cell density had no significant effect on the percentage of encystment. 4. Cysts were always present in the lake sediments but their abundance in ‘non Peridinium’ years was much lower than after a massive bloom. Vegetative cells were always present in the water column after the collapse of the annual dinoflagellate bloom, potentially serving as the inoculum for the next bloom. We propose that the hardy cysts serve as an emergency ‘gene bank’ to initiate population build up following catastrophic die outs.     

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.     

The encystment of a freshwater dinoflagellate: A light and electron-microscopical study

The process of encystment, or resting spore formation, in a freshwater dinoflagellate (Woloszynskia tylota nov. comb.) has been studied with both light and electron microscopy. The main features of the process are as follows: (i) the replacement of the theca by a thin, amorphous outer wall, which gradually thickens by the deposition of material on its inner face; (ii) the appearance of a layer of closely-packed lipid droplets at the cytoplasmic margin of the mature cyst, resembling a granular ‘inner wall’ in the light microscope; (iii) the reduction in size or disappearance of cytoplasmic structures such as chloroplasts, Golgi bodies and pusule; and (iv) the enlargement of a central ‘accumulation body’ and cytoplasmic vacuoles containing crystals. Comparisons are made with light-microscope studies of encystment of other dinoflagellates, with ultrastructural studies of non-motile division stages, with zooxanthellae and with fossil dinoflagellate cysts or hystrichospheres.     

Balamuthia mandrillaris: staining properties of cysts and trophozoites and the effect of 2,6-dichlorobenzonitrile and calcofluor white on encystment

Here, we determined the staining properties of Balamuthia mandrillaris cysts, and assessed the effect of 2, 6-dichlorobenzonitrile (DCB), a cellulose synthesis inhibitor, and calcofluor white, a brightening agent, on its encystment. Periodic acid-Schiff reagent stained the inner wall intensely and middle and outer walls weakly suggesting that the cyst wall of B. mandrillaris may contain glycans. Furthermore, cysts, but not trophozoites, fluoresced when stained with calcofluor white. Calcofluor white and DCB, a cellulose synthesis inhibitor, inhibited B. mandrillaris encystment. This is the first report suggesting possible glycan biosynthesis in B. mandrillaris encystment, and this pathwaymay provide a potentially useful drug target and help improve treatment.     

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.     

Wall formation in the saprolegniales

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

Identification of Differentially Expressed Water‐insoluble Proteins in the Encystment Process of Colpoda cucullus by Two‐dimensional Electrophoresis and LC‐MS/MS Analysis

In the encystment process of the ciliate protist Colpoda cucullus, we observed that the cell total protein abundance was reduced at 12 h–1 d after the onset of encystment induction subsequent to the reduction in mRNA abundance. We analyzed the alteration of the expression levels of water‐insoluble proteins by two‐dimensional polyacrylamide gel electrophoresis using polyoxyethylene (20) sorbitan monooleate (Tween‐80), and we identified proteins whose expression levels were altered in the encystment process by a liquid chromatography tandem mass spectrometry analysis. The expression level of a 60‐kDa protein (p60; heat shock protein 60) was temporarily enhanced and that of a 55‐kDa protein (p55; actin) and a 49‐kDa protein (p49; actin) was enhanced in the Colpoda encystment process. In mature cysts, the expression level of p55 and p49 tended to be reduced, whereas the expression level of a 50‐kDa protein (p50d; α‐tubulin), a 25‐kDa protein (p25; α‐tubulin) and a 52‐kDa protein (p52c; β‐tubulin) was enhanced.     

The outer cyst wall ofBdellovibrio bdellocysts is made de novo and not from preformed units from the prey wall

Bdellovibrio sp. strain W bdellocysts were produced inEscherichia coli using three sources of³H-diaminopimelic acid (DAP) for incorporation into the cyst wall peptidoglycan: (a) labeledE. coli peptidoglycan, (b) labeledBdellovibrio peptidoglycan, and (c) exogenous³H-DAP in the encystment medium. After cysts were produced, they were either sonicated to remove the prey cell wall, or germinated to solubilize the cyst wall. The results show that label was incorporated into the cyst wall preferentially from the exogenous DAP in the medium, and not from the bdellovibrio or bdelloplast peptidoglycan. The encysting bdellovibrio does not therefore incorporate existing peptidoglycan units from the bdelloplast for synthesis of the cyst wall.     

SEXUALITY AND CYST FORMATION IN THE DINOFLAGELLATE GONYAULAX TAMARENSIS: CYST YIELD IN BATCH CULTURES1

Encystment of the toxic dinoflagellate Gonyaulax tamarensis Lebour (var. excavata) was monitored in batch cultures exposed to a variety of nutritional and environmental treatments. Limitation by nitrogen (as ammonium or nitrate) or phosphorus (as phosphate) resulted in cyst formation. When the initial concentration of limiting nutrient was varied, total cyst yield (mL^?1) was directly proportional to the cell yield at all but the highest nutrient concentrations (where encystment was minimal). Encystment efficiency was relatively constant (0.1–0.2 cysts · cell^?1) over a 5-fold range of cell densities, indicating that 20 to 40% of the vegetative populations successfully encysted. Cyst formation was negligible in nutrient-replete medium, even with a significant reduction in growth rate due to non-optimal light, temperature, or to high batch culture cell densities. Low light levels did decrease cyst yield once encystment was initiated by nutrient limitation, but this was probably linked to smaller motile cell yield and not to a specific inhibition of encystment. In contrast, encystment was more sensitive to temperature than was growth rate: optimal cyst production occurred over a relatively narrow temperature range and no cysts were formed at [Page missing]     

Entamoeba invadens: the requirement for galactose ligands during encystment

During periods of stress, trophozoites of Entamoeba invadens (strain IP-1) undergo a process of differentiation (encystment) that results in a dormant cyst with a chitin-containing cyst wall. Encystment can be induced by resuspension of trophozoites from growth medium into a diluted glucose-free medium (47% LG) containing 5% adult bovine serum (ABS). ABS is thought to be a source of gal-terminated ligands that are required for high levels of encystment. After resuspension of trophozoites in 47% LG, encystment cultures were examined every 2h for responses to the (i) addition of 10mM free-galactose, (ii) resuspension of cells to serum-free medium, (iii) and dilution of encysting cultures to cell densities below that known to support full encystment (from 5 x 10(5) to 1 x 10(4)cells/ml). The role of serum components (and the gal-terminated ligand asialofetuin; ASF) adsorbed onto the surface upon which encystment proceeds, and their effect on the multi-cellular aggregation patterns formed during encystment, were also investigated. The addition of free-galactose reduced the levels of encystment (compared with the control) even when added at 10h after resuspension of trophozoites in 47% LG. The requirement for the presence of ABS during encystment was lost within 6h, with levels of encystment of cells washed free of serum reaching 80% of the control. The ability of cells to encyst when diluted to a cell density below that normally thought to support encystment reached over 50% by 8h. Efficient encystment could be obtained in 47% LG in the absence of ABS or ASF using pre-treated glass culture tubes. Encystment (47% LG; 5% ABS) using ultra low attachment plates was poor, suggesting attachment of cells to a surface via gal-terminated ligands was important for efficient encystment. The results suggest that ABS is probably not the only source of gal-terminated ligands necessary for high levels of encystment in 47% LG. While serum may provide a source of ligands which enhance the levels of encystment initially, other gal-terminated ligands possibly released by the encysting cells are still required for the completion of the encystment process and the formation of mature cysts. In addition, the gal-terminated ligands necessary for encystment efficiency may be adsorbed onto the glass surface of culture tubes and aid the initial aggregation process, as well as be involved in cell signaling during the encystment process.     

Host-specific plant signal and G-protein activator, mastoparan, trigger differentiation of zoospores of the phytopathogenic oomycete Aphanomyces cochlioides

We found that the gradient of a host-specific attractant, cochliophilin A (5-hydroxy-6,7-methylenedioxyflavone) isolated from the roots of spinach triggered encystment followed by germination of zoospores of Aphanomyces cochlioidesat a concentration less than micromolar order. This compound did not affect the growth and reproduction of this phytopathogen up to 10^–6 M concentration in the culture medium. We also observed that mastoparan, an activator of heterotrimeric G-protein could inhibit the motility of zoospores and then strikingly effect encystment followed by 60–80% germination of cysts. Concomitant application of cochliophilin A and mastoparan showed stronger encystment followed by 100% germination of cysts. In addition, we have observed that chemicals interfering with phospholipase C activity (neomycin) and Ca²⁺ influx/release (EGTA and loperamide) suppress cochliophilin A or mastoparan induced encystment and germination. These results suggest that G-protein mediated signal transduction mechanism may be involved in the differentiation of the A. cochlioides zoospores. This is the first report on the differentiation of oomycete zoospores initiated by a host-specific plant signal or a G-protein activator.     

Ultrastructure and Composition of the Nannochloropsis gaditana Cell Wall

Marine algae of the genus Nannochloropsis are promising producers of biofuel precursors and nutraceuticals and are also harvested commercially for aquaculture feed. We have used quick-freeze, deep-etch electron microscopy, Fourier transform infrared spectroscopy, and carbohydrate analyses to characterize the architecture of the Nannochloropsis gaditana (strain CCMP 526) cell wall, whose recalcitrance presents a significant barrier to biocommodity extraction. The data indicate a bilayer structure consisting of a cellulosic inner wall (∼75% of the mass balance) protected by an outer hydrophobic algaenan layer. Cellulase treatment of walls purified after cell lysis generates highly enriched algaenan preparations without using the harsh chemical treatments typically used in algaenan isolation and characterization. Nannochloropsis algaenan was determined to comprise long, straight-chain, saturated aliphatics with ether cross-links, which closely resembles the cutan of vascular plants. Chemical identification of >85% of the isolated cell wall mass is detailed, and genome analysis is used to identify candidate biosynthetic enzymes.     

Characterization of zoospore and cyst surface structure in saprophytic and fish pathogenicSaprolegnia species (oomycete fungal protists)

Summary The importance of the surface structure and chemistry in zoospores and cysts of oomycetes is briefly reviewed and the organelle systems associated with encystment described. The surface structure and chemistry of primary and secondary zoospores and cysts ofSaprolegnia diclina (a representative saprophytic species) andS. parasitica (a representative salmonid fish pathogen) were explored using the lectins concanavilin A (Con A) and wheat germ agglutinin (WGA) and monoclonal antibodies (MAbs) raised against a mixed zoospore and cyst suspension ofS. parasitica. The binding of lectins and antibodies to spores was determined using immunofluorescence microscopy with fluorescein isothiocyanate-labelled probes and with electron microscopy with gold-conjugated probes applied to spore suspensions post-fixation. In both species Con A, which is specific for glucose and mannose sugars, bound to both the surface of primary and secondary zoospores (the surface glycocalyx) and their cyst coats and readily induced zoospore encystment. The binding to the cysts appeared to be mainly associated with the matrix material released from the primary and secondary encystment vesicles and which appeared to diminish with time. No binding to germ tube walls was observed with this lectin. The MAb labelling showed a generally similar binding pattern to the primary and secondary cysts to that observed with Con A, although the binding to zoospores was more variable. Primary zoospores bound the antibodies but secondary zoospores appeared less reactive. It is suggested that the MAbs share a common epitope with one or more of the Con A-binding components. In both species WGA, which is specific for amongst other things the sugar N-acetyl glucosamine, bound to localised apical patches on the primary zoospores. This lectin also binds to the ventral groove region of secondary zoospores ofS. diclina, which were induced to encyst by this lectin. In contrast secondary zoospores ofS. parasitica were not induced to encyst by the addition of WGA and showed a patchy dorsal binding with this lectin. WGA also binds to both the inner wall of discharged primary cysts and the young germ tube walls of both species. These observations are discussed both in relation to other oomycete spores and to their possible functional and ecological significance.Abbreviations BSA bovine serum albumin - Con A Concanavalin A - DBA Dolichos biflorus agglutinin - ELISA enzyme-linked immunosorbent assay - EM electron microscope - EV encystment vesicles - FCS foetal calf serum - FITC Fluorescein isothiocyanate - FV peripheral fibrillar vesicles - G+F 0.2% glutaraldehyde and 2.0% formaldehyde primary fixative solution - 2G 2% glutaraldehyde primary fixative - LM light microscopy - MAbs monoclonal antibodies - LPV large peripheral vesicles - PBS phosphate buffered saline - PCV flattened peripheral cisternae - PEV primary encystment vesicle - PIPES piperazine-N,N1-bis(2-ethane sulfonic acid) - PNA Ricinus communis agglutinin - RAM-FITC/Au_10–20 Fluorescein isothiocyanate/gold (10 or 20 nm) labelled rabbit anti-mouse immunoglobulin - RCA Ricinus communis agglutinin - SEM scanning electron micrograph - SBA soybean agglutinin - SEV secondary encystment vesicles - TEM transmission electron micrograph - UEA I Ulex europaeus agglutinin - WGA wheat germ agglutinin     

Cellular, biochemical, and molecular changes during encystment of free-living amoebae

Free-living amoebae are protozoa found in soil and water. Among them, some are pathogenic and many have been described as potential reservoirs of pathogenic bacteria. Their cell cycle is divided into at least two forms, the trophozoite and the cyst, and the differentiation process is named encystment. As cysts are more resistant to disinfection treatments than trophozoites, many studies focused on encystment, but until recently, little was known about cellular, biochemical, and molecular modifications operating during this process. Important signals and signaling pathways at play during encystment, as well as cell responses at the molecular level, have been described. This review summarizes our knowledge and focuses on new findings.