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.     

Protein turnover in Azotobacter vinelandii during encystment and germination

Protein turnover occurs during differentiation of Azotobacter vinelandii 12837 to the extent of 50% during encystment and 7% during germination. The addition of rifampin at the initiation of encystment prevents encystment and inhibits turnover. In germinating cysts, protein turnover is essential owing to an apparent lack of certain amino acid biosynthetic enzymes. The capacity to synthesize sulfur-containing amino acids from inorganic precursors is regained about halfway through the germination process.     

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.     

Phytophthora parasitica biofilm formation: installation and organization of microcolonies on the surface of a host plant

Zoospores of the oomycete Phytophthora parasitica establish microbial spheroid microcolonies and biofilms on the surface of wounded leaves of their host, Nicotiana tabacum . The formation of microcolonies involves the movement of some zoospores towards attractants from wound sites, followed by their irreversible adsorption and the formation of a cluster of cells. These cells drive the migration of a second wave of zoospores (several hundreds cells) by setting up an external chemotactic gradient leading to massive zoospore encystment and cyst-orientated germination. Zoospores that are still swimming at this stage circulate within the nascent biofilm by opening channels. Concomitantly, the cell population secretes various substances to elaborate an extracellular mucilage. Embedded within the extracellular matrix, biofilm cells are organized into a structured community as coacervates. The granular surface is composed of individual cysts, located on the outside of the microcolony. Hyphae from these cysts plunge downwards towards the dense core formed by the founder cells. This report is the first to show the installation and organization of a biofilm formed by eukaryotic cells on plant surfaces. The P. parasitica microcolonies constitute heterogeneous microenvironments for the embedded and circulating cells. They may affect plant–pathogen interactions by serving as reservoirs for pathogenic microorganisms, as protecting niche against host defences or as structures for infecting populations.     

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.     

CHANGES IN CELL CHEMICAL COMPOSITION DURING THE LIFE CYCLE OF SCRIPPSIELLA TROCHOIDEA (DINOPHYCEAE)1

The cellular content of carbon, nitrogen, amino acids, polysaccharides, phosphorus and adenosine trtphosphate (ATP) was determined at several stages during the life cycle of the dinoflagellate Scrippsiella trochoidea (Stein) Loeblich. Carbon per cell decreased slightly between exponential and stationary phase growth in vegetative cells whereas nitrogen per cell did not change. Both of these cellular components increased markedly on encystment and then decreased to vegetative cell levels during dormancy and germination. C/N ratios increased gradually during cyst dormancy and activation, reflecting a more rapid decrease in N than in C pools, even though both decreased through time. Amino acid composition was relatively constant during the vegetative cell stages; glutamic acid was the dominant component. Arginine was notably higher in cysts than in vegetative cells but decreased significantly during germination, suggesting a role in nitrogen storage. The ratio of neutral ammo acids to total ammo acids (NAA/TAA) decreased as cysts were formed and then gradually increased during storage and germination. The ratio of basic ammo acids to total ammo acids (BAA/TAA) changed in the opposite direction of NAA/TAA, whereas the ratio of acidic acids to total amino adds (AAA/TAA) was generally invariant. Ammo acid pools were not static during the resting slate in the cysts: there was degradation or biosynthesis of certain, but not all, classes of these compounds. The monosacchande composition of cold and hot water extracted polysaccharides was quite different between cells and cysts. A high percentage of glucose in cysts suggests that the storage carbohydrate is probably in the form of glucan. Total cellular phosphorus was higher in all cyst stages than in vegetative cells. However, ATP-cell^?1 decreased as vegetative cells entered stationary phase and encysted, and continued to decrease in cysts during dark cold storage. ATP increased only as the cysts were activated at warm temperatures in the light and began to germinate. The above data demonstrate that dormancy and quiescence are not periods of inactive metabolism but instead are times when numerous biochemical transformations are occurring that permit prolonged survival in a resting state.     

Utilization of amino acids by Chromatium sp. strain D

Summary The duration of various morphologically distinct phases in the division cycle of the marine heterotroph Cryptothecodinium cohnii was measured in cultures initiated with synchronously excysted swarmer cells. Parent cysts were selectively isolated on plastic surfaces and progeny of a narrow age distribution harvested in a specifically conditioned medium. The swarmer phase, an interval of predivisional encystment, daughter cell formation and excystment were 5.0, 3.0 and 2.0 h respectively. Two major kinds of cytokinesis (production of 2 and 4 daughter cells) were observed resulting in a mean daughter cell number of 2.7 under these conditions. Other growth parameters for this dinoflagellate are described.     

Formation and germination of temporary cysts of Cochlodinium polykrikoides Margalef (Dinophyceae) and their ecological role in dense blooms

While the initiation and development of dense bloom of Cochlodinium polykrikoides have been shown to be related to some environmental factors, little is known about the ecological role of the formation and germination of temporary cysts, nor of their significance for the rapid expansion of dense regional-scale blooms. This study examined the factors affecting the formation and germination of temporary cysts of C. polykrikoides, and provides details about the germination process. In the laboratory experiments, C. polykrikoides produced the chain-forming temporary cysts that are immobile and surrounded by a hyaline membrane. The encystment experiment indicated that darkness induces the formation of chain-forming temporary cysts, consistent with field observation of morphology and fluxes of temporary cysts. Germination occurred twice from a single four-celled temporary cysts within 24 h after exposure to light, and the germlings appeared as two-celled chain-forming vegetative cells. The germination behavior of temporary cysts of C. polykrikoides differs from that of other dinoflagellates, and this may be a survival strategy for the maintenance of population size during dense blooms.     

ROLE OF ENCYSTMENT AND EXCYSTMENT OF PERIDINIUM BIPES F. OCCULATUM (DINOPHYCEAE) IN FRESH WATER RED TIDES IN LAKE KIZAKI,JAPAN1

The encystment flux of Peridinium bipes f. occulatum (Dinophyceae) was investigated with sediment traps from 1968 to 1990 in Lake Kazki. Cysts of P. bipes were formed throughout the blooms, Encystment flux of P. bipes in the pelagic zone was usually lower than those at shallow sites, and the density of P. bipes cysts in lake sediment was higher in the shallow region than in the pelagic zone. However, in the shallower region, The concentration of P. bipes cysts varied widely, possibly due to high rates of encystment and excystment. Peridinium bipes encystment occurred between 15° and 25° C in the laboratory, with very little cyst formation below 10°C. Though cyst formation was observed in continous darkness, the rate increased with irradiance. Under continuous darkness, no excystment was observed at any temperature from 5° to 25° C. Eighty-one percent of the cysts illuminated at 105 μE m^?2 s^?1 excysted after 13 days incubation at 15° C, and lower irradiances decreased germination success. Results from laboratory experiments suggest that light is a critical factor in the germination of P. bipes cysts. Bottom depth thus can have a significant effect on germination because cysts only can excyst from depths where light is sufficient. The shallow region of the lake is thus very important as a seed bed for P. bipes during early spring. Cyst deposited in deeper waters may not ever germinate unless they are resuspended and transported to shallow areas where light reaches the bottom.     

Characterising adhesiveness ofPhytophthora cinnamomi zoospores during encystment

During encystment,Phytophthora cinnamomi zoospores bind firmly to the host surface. We have developed a microassay to study adhesion of the zoospores to solid surfaces, both biological and non-biological. The results show that timing of the acquisition of adhesiveness during encystment correlates closely with the secretion of high molecular weight glycoproteins. The adhesive phase is short lived, occurring between 1 and 4 min after induction of encystment. During this period, cells that come into contact with a variety of surfaces (glass, plastic, and onion epidermis) become firmly attached, while cells that come into contact with one of these substrata after this period are unable to bind. Our results also show that EGTA inhibits cyst adhesion, while addition of calcium promotes cyst adhesion, especially of cysts more than 4 min old. To help identify the cyst surface component involved in adhesion we tested a number of lectins for their ability to block cyst adhesion. Soybean agglutinin andHelix pomatia agglutinin, lectins which bind to the secreted high molecular weight glycoproteins, both inhibit adhesion in the presence and absence of the hapten sugar, indicating that inhibition was non-specific. Wheatgerm agglutinin, a lectin which does not bind to the cyst surface, also blocked adhesion non-specifically.     

Defective zoospore encystment and suppressed cyst germination of Phytophthora palmivora caused by transient leaching treatments

The behaviour of encysting zoospores of Phytophthora palmivora during leaching conditions was studied. Zoospores encysted and germinated successfully on polycarbonate membranes after mechanical agitation. Transient (10 min) leaching treatments with nutrient-free buffer underneath the membranes resulted in abnormal encystment and poor germination. The disruption was greatest when leaching was applied during the first minutes after start of encystment and not observed after 20 min. The early sensitivity of cells to leaching coincided with the period when alkali-resistant cell walls were formed (2 – 6 min after mechanical agitation). Effects of calcium and organic nutrients on encystment during leaching and germination after these treatments were studied. The disruption of encystment by early leaching treatments, but not the suppression of cyst germination, was overcome by adding calcium chloride during mechanical agitation of zoospores. Leaching with calcium containing buffer resulted in suppressed cyst germination as was the case with buffer alone. Leaching with 0.1 % peptone containing buffer promoted consistently high encystment and germination. This revised version was published online in June 2006 with corrections to the Cover Date.     

Dynamic changes of the cell wall surface of Candida albicans associated with germination and adherence

The distribution of mannoproteins at the cell wall surface of Candida albicans was analyzed during the process of germination in conditions favoring adherence of germ tubes to a plastic matrix. Three cytochemical methods allowing the detection of concanavalin A binding sites, anionic sites and the enzyme acid phosphatase, respectively were used. All three methods gave similar results, indicating a spatial and temporal reorganization of some cell wall mannoproteins: a strong labeling was observed on blastoconidia; in contrast, as soon as the emergence of germ tubes took place, these reactions decreased dramatically at the surface of mother cells, whereas the germ tube surface was strongly stained. Some new components with multiple biological activities were detected at the germ-tube surface. Indeed, among mannoproteins responsible for an enhanced adhesion to plastic surfaces, two components with molecular weights of 68 and 60 to 62 kDa were shown to interact with laminin, fibrinogen, and C3d. This study therefore indicates that germination, and then adherence of germ tubes, imply a degradation of surface mannoproteins, and a simultaneous presentation of new molecules which can interact with their nonbiological materials or host proteins.     

Generalist Life Cycle Aids Persistence of Alexandrium ostenfeldii (Dinophyceae) in Seasonal Coastal Habitats of the Baltic Sea

In seasonal environments, strong gradients of environmental parameters can shape life cycles of phytoplankton. Depending on the rate of environmental fluctuation, specialist or generalist strategies may be favored, potentially affecting life cycle transitions. The present study examined life cycle transitions of the toxin producing Baltic dinoflagellate Alexandrium ostenfeldii and their regulation by environmental factors (temperature and nutrients). This investigation aimed to determine whether genetic recombination of different strains is required for resting cyst formation and whether newly formed cysts are dormant. Field data (temperature and salinity) and sediment surface samples were collected from a site with recurrent blooms and germination and encystment experiments were conducted under controlled laboratory conditions. Results indicate a lack of seasonal germination pattern, set by an endogenous rhythm, as commonly found with other dinoflagellates from the Baltic Sea. Germination of quiescent cysts was triggered by temperatures exceeding 10°C and combined nutrient limitation of nitrogen and phosphorus or a drop in temperature from 16 to 10°C triggered encystment most efficiently. Genetic recombination was not mandatory for the formation of resting cysts, but supported higher numbers of resistant cysts and enhanced germination capacity after a resting period. Findings from this study confirm that A. ostenfeldii follows a generalist germination and cyst formation strategy, driven by strong seasonality, which may support its persistence and possibly expansion in marginal environments in the future, if higher temperatures facilitate a longer growth season.     

Presence of a Nonhydrolyzable Biopolymer in the Cell Wall of Vegetative Cells and Astaxanthin-Rich Cysts of Haematococcus pluvialis (Chlorophyceae)

The green alga Haematococcus pluvialis accumulates massive amounts of the red pigment astaxanthin in response to stimuli inducing it to form cysts. During the encystment process the cell wall undergoes a clear hardening and thickening. In this work, a cell wall fraction withstanding successive acid and basic hydrolysis was isolated and proves to be algaenan by Fourier transform infrared spectroscopy. This compound is equally abundant in nonmotile vegetative cells and astaxanthin-rich cysts. This finding indicates that the synthesis of algaenan does not require the activation of the machinery for the massive production of secondary carotenoids. We conclude that algaenan cannot cause the changes occurring in the cell wall during the encystment process without the involvement of other cell wall components. Received November 7, 2000; accepted April 3, 2001.     

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.     

Adhesion of Phytophthora palmivora zoospores: detection and ultrastructural visualization of concanavalin A-receptor sites appearing during encystment.

The binding of concanavalin A (Con A) to the cell surface of zoospores and cysts of Phytophthora palmivora was studied by radiometry (125I-Con A), ultraviolet microscopy (fluorescein-Con A) and electron microscopy peroxidase-diaminobenzidine technique). Zoospores were found to secrete during the early stages of encystment a Con A-binding material susceptible to trypsin digestion. This glycoprotein is contained in the so-called peripheral vesicles and is probably responsible for the adhesion of the encysting zoospores to solid surfaces.     

Effect of biotic and abiotic factors on in vitro proliferation, encystment, and excystment of Pfiesteria piscicida

Pfiesteria spp. are mixotrophic armored dinoflagellates populating the Atlantic coastal waters of the United States. They have been a focus of intense research due to their reported association with several fish mortality events. We have now used a clonal culture of Pfiesteria piscicida and several new environmental isolates to describe growth characteristics, feeding, and factors contributing to the encystment and germination of the organism in both laboratory and environmental samples. We also discuss applied methods of detection of the different morphological forms of Pfiesteria in environmental samples. In summary, Pfiesteria, when grown with its algal prey, Rhodomonas sp., presents a typical growth curve with lag, exponential, and stationary phases, followed by encystment. The doubling time in exponential phase is about 12 h. The profiles of proliferation under a standard light cycle and in the dark were similar, although the peak cell densities were markedly lower when cells were grown in the dark. The addition of urea, chicken manure, and soil extracts did not enhance Pfiesteria proliferation, but crude unfiltered spent aquarium water did. Under conditions of food deprivation or cold (4 degrees C), Pfiesteria readily formed harvestable cysts that were further analyzed by PCR and scanning electron microscopy. The germination of Pfiesteria cysts in environmental sediment was enhanced by the presence of live fish: dinospores could be detected 13 to 15 days earlier and reached 5- to 10-times-higher peak cell densities with live fish than with artificial seawater or f/2 medium alone. The addition of ammonia, urea, nitrate, phosphate, or surprisingly, spent fish aquarium water had no effect.     

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.