Encystment Rate Research Articles

Echinostome-Induced Mortality Varies Across Amphibian Species in the Field

Echinostomes are receiving increased attention because of their emerging parasite status in landscapes associated with human development and their ability to infect and kill many North American larval amphibians. While laboratory experiments have shown that echinostomes can cause extensive mortality in their amphibian hosts, their effect on tadpoles in the field is less clear. I conducted a controlled-infection field-enclosure experiment in 4 ponds to compare the effects of echinostomes on green frog (Rana clamitans) and gray tree frog (Hyla versicolor) tadpoles in the field. I measured tadpole growth, development, mortality, and infection intensity. Echinostome infection resulted in high mortality in green frog tadpoles and less mortality in gray tree frogs. However, metacercariae encystment rates were higher in gray tree frog tadpoles than in green frog tadpoles. The effect of echinostomes on mortality varies across amphibian species, with the result that some species may experience more extensive echinostome-induced mortality than others. Mortality as a result of echinostome infection in green frog tadpoles was similar to mortality observed in predation experiments.

Disinfection Efficacy and Encystment Rate of Soft Contact Lens Multipurpose Solutions Against Acanthamoeba

To assess the ability of commercial and experimental soft contact lens multipurpose solutions (MPS) to promote Acanthamoeba trophozoite encystment and their biocidal efficacy against Acanthamoeba trophozoites and cysts. The effects on encystment and disinfection efficacy by the incorporation of propylene glycol (PG) in the formulation of MPS were also investigated. Acanthamoeba trophozoites (Acanthamoeba castellanii ATCC 50730 and Acanthamoeba polyphaga CCAP 1501/3G) were inoculated into MeniCare Soft, MeniCare Soft (-PG), Epica Cold II, OptiFree Plus, and Rohto C cube Softone-Moist MPS, and the percentage of encystment induced by each solution was determined after 24 hr. In addition, Acanthamoeba trophozoites and cysts (A. castellanii ATCC 50730 and A. polyphaga CCAP 1501/3G and Ros) were also inoculated into each of the five MPS, and their log reduction determined after 0, 1, 4, 6, and 24 hr of incubation using stand-alone assays. Significantly higher encystment rates were found with Epica Cold II for A. polyphaga CCAP 1501/3G and Rohto C cube Softone-Moist for A. polyphaga and A. castellanii compared with the other MPS assessed (P<0.05). MeniCare Soft, MeniCare Soft (-PG), and Opti-Free Plus produced little or no encystment, with mean encystment values ranging from 0.0% to 2.0%. A significantly higher disinfection efficacy was found with MeniCare Soft, MeniCare Soft (-PG), and Epica Cold II compared with Opti-Free Plus and Rohto C cube Softone-Moist (P<0.05). Significant differences in encystment rate and disinfection efficacy between MPS were found. The presence of PG in the formulation of MeniCare Soft did not affect the disinfection efficacy or the encystment rate. The latter indicates that other factors play a role in the induction of Acanthamoeba encystment after inoculation into MPS.

Eco-fingerprinting of the dinoflagellate Borghiella dodgei: experimental evidence of a specific environmental niche

In Lake Tovel, an oligotrophic and weakly stratified lake, the dinoflagellate Borghiella dodgei Moestrup, Hansen et Daugbjerg, showed a peculiar spatial–temporal pattern with highest abundances in the bottom of the shallow side bay (4 m) along with remarkable abundance variations from year to year. We investigated B. dodgei’s growth in laboratory cultures and related results to their implication for bloom formation. B. dodgei was cultivated under different temperature, nutrient and light conditions. Growth rates, cell biovolume, cyst formation and pigment and mycosporine-like amino acids (MAAs) concentrations were determined. Experiments showed that this alga (i) had higher growth rates at low temperatures ( 7°C) and (vii) had high encystment rates with temperatures >7°C. These laboratory fingerprints allowed us to construct a theoretical model defining the species’ niche. Borghiella needed a mixture of low temperatures, high irradiance levels and sufficient quantities of dissolved organic nitrogen to form blooms. Such a strict combination was probably a transient situation and occurred in oligotrophic Lake Tovel only in early summers followed by heavy spring rains.

The role of resting cysts in Alexandrium minutum population dynamics

Abstract The role of resting cysts on the development of Alexandrium minutum blooms in a typical Mediterranean semi-enclosed water body (Arenys de Mar Harbor, NW Mediterranean) was studied by means of matrix and dynamic population models. We used a series of scenarios, constrained when possible by experimentally measured parameters to test whether excystment and encystment fluxes and changes in the dormancy period had a major effect on bloom intensity and duration. The results of the simulations highlighted the importance of knowing not only the magnitude and variability of growth and life-cycle transition rates, but also those of loss rates (both in the water column and in the sediment) due to physical or biological factors. Given the maximum encystment rates determined for A. minutum in the study area (0.01 d −1 ), this process contributed to reduce the peak concentrations of vegetative cells but did not have a dominant effect on bloom termination. Excystment fluxes could contribute to enhance population densities of vegetative cells during times or low or negative net growth rate and during the initial phases of a bloom, but once exponential growth had started, additional excystment had negligible effect on bloom magnitude. However, even if cysts did not contribute to larger blooms, they could represent a safety mechanism for reintroduction of the species when the vegetative cell population went extinct due to unfavorable environmental conditions. Increasing the dormancy time exposed newly formed cysts to a longer period of losses in the sediment that reduced the concentration of excystment-ready sediment cysts and decreased excystment fluxes. More complex models will be needed to explore the implications of different life-cycle strategies in a wider natural ecological context.

Acanthamoeba castellanii : growth on human cell layers reactivates attenuated properties after prolonged axenic culture

The free-living, but potentially pathogenic, bacteriovorous amoebae of the genus Acanthamoeba can be easily grown axenically in a laboratory culture. This, however, often leads to considerable losses in virulence, and encystment capacity, and to changes in drug susceptibility. We evaluated potential options for a reactivation of a number of physiological properties, attenuated by prolonged axenic laboratory culture, including encystment potential, protease activity, heat resistance, growth rates and drug susceptibility against N-chlorotaurine (NCT). Toward this end, a strain that had been grown axenically for 10 years was repeatedly passaged on human HEp-2 cell monolayers or treated with 5′-azacytidine (AzaC), a methyltransferase inhibitor, and trichostatin A (TSA), a histone deacetylase inhibitor, in order to uplift epigenetic gene regulation. Culture on human cell monolayers resulted in significantly enhanced encystment potentials and protease activities, and higher susceptibility against NCT, whereas the resistance against heat shock was not altered. Treatment with AzaC/TSA resulted in increased encystment rates and protease activities, indicating the participation of epigenetic mechanisms. However, lowered resistances against heat shock indicate that possible stress responses to AzaC/TSA have to be taken into account. Repeated growth on human cell monolayers appears to be a potential method to reactivate attenuated characteristics in Acanthamoeba.

Effects of malathion on embryonic development and latent susceptibility to trematode parasites in ranid tadpoles

We investigated the effects of embryonic exposure to the widely used organophosphate malathion (15-600 micro/L) on the early development and latent susceptibility of pickerel frog (Rana palustris) tadpoles to the trematode parasite Echinostoma trivolvis. The latent effects of contaminant exposure are rarely examined but could have important implications for individual survival and population viability. Malathion decreased hatching success by 6.5% and viability rates by 17% at 600 microg/L, which is a lower concentration than previously documented for anuran embryos. Incidence of malformations increased from 0.5% in controls to 11.2% in the 600-microg/L malathion treatment. The primary malformations documented in the two highest pesticide concentrations were ventralization and axial shortening. After seven weeks of development in water with no malathion, tadpoles previously exposed as embryos for only 96 h to 60 and 600 microg/L malathion suffered increased parasite encystment rates when compared to controls. Our research identifies embryonic development as a sensitive window for establishing latent susceptibility to infection in later developmental stages.

LIFE-HISTORY TRADE-OFFS INFLUENCE DISEASE IN CHANGING CLIMATES: STRATEGIES OF AN AMPHIBIAN PATHOGEN

Life-history trade-offs allow many animals to maintain reproductive fitness across a range of climatic conditions. When used by parasites and pathogens, these strategies may influence patterns of disease in changing climates. The chytrid fungus, Batrachochytrium dendrobatidis, is linked to global declines of amphibian populations. Short-term growth in culture is maximal at 17 degrees-25 degrees C. This has been used in an argument that global warming, which increases the time that amphibians spend at these temperatures in cloud-covered montane environments, has led to extinctions. Here we show that the amphibian chytrid responds to decreasing temperatures with trade-offs that increase fecundity as maturation rate slows and increase infectivity as growth decreases. At 17 degrees-25 degrees C, infectious zoospores encyst (settle and develop a cell wall) and develop into the zoospore-producing stage (zoosporangium) faster, while at 7 degrees-10 degrees C, greater numbers of zoospores are produced per zoosporangium; these remain infectious for a longer period of time. We modeled the population growth of B. dendrobatidis through time at various temperatures using delayed differential equations and observational data for four parameters: developmental rate of thalli, fecundity, rate of zoospore encystment, and rate of zoospore survival. From the models, it is clear that life-history trade-offs allow B. dendrobatidis to maintain a relatively high long-term growth rate at low temperatures, so that it maintains high fitness across a range of temperatures. When a seven-day cold shock is simulated, the outcome is intermediate between the two constant temperature regimes, and in culture, a sudden drop in temperature induces zoospore release. These trade-offs can be ecologically important for a variety of organisms with complex life histories, including pathogenic microorganisms. The effect of temperature on amphibian mortality will depend on the interaction between fungal growth and host immune function and will be modified by host ecology, behavior, and life history. These results demonstrate that B. dendrobatidis populations can grow at high rates across a broad range of environmental temperatures and help to explain why it is so successful in cold montane environments.

Relative Toxicity of Malathion to Trematode-Infected and Noninfected Rana palustris Tadpoles

Amphibian populations around the world are facing threats that include disease and pollution. Although the effect of environmental contaminants on susceptibility to infection has been demonstrated for several amphibian species, to our knowledge, the opposite interaction, infection status affecting contaminant susceptibility, has not been studied. We conducted standard 48-h toxicity tests to compare susceptibility to malathion, a widely used organophosphate insecticide, of uninfected pickerel frog (Rana palustris) tadpoles and tadpoles infected with two levels (10 or 30 cercariae) of the trematode Echinostoma trivolvis. Trematode encystment rates were high (>90%) in both trematode treatment groups. LC(50) values ranged from 16.5 to 17.4 mg/L, within the range reported for other amphibian species. However, we found no differences in susceptibility to malathion among parasite treatments. Although we detected no effect of parasites on pesticide susceptibility in this system, it is important to investigate this question using other pesticides, parasites, and amphibian hosts before dismissing this potentially threatening interaction.

Modelling vegetative growth, gamete production and encystment of dinoflagellates in batch culture

An intriguing case of encystment of the dinoflagellate Scrippsiella lachrymosa in culture has recently been presented: cyst concentrations still increased after the abundance of the vegetative cells had decreased to very low numbers. To account for this apparent time lag, and using a model with an encystment rate equation in which vegetative growth and encystment rates were coupled, the calculated growth and encystment rates had to be 8 to 12 d -1 , values which were considered 'not biologically meaningful'. Here a new model of dinoflagellate growth and encystment is presented in which the mitotic cycle (vegetative growth) is coupled quantitatively to the sexual cycle (cyst forma- tion) by having 4 gametes emanate from 1 vegetative cell, but without directly coupling the rates of vegetative growth and encystment. Calibrated on literature data of S. lachrymosa cultured in f/4 medium, this model satisfactorily describes motile cell (vegetative cells and gametes) and cyst devel- opment with correlations between log-transformed model and experimental data of r 2 = 0.80 (motile cells) and r 2 = 0.94 (cysts) and with typical maximum rates in the exponential growth phase of μcell = 0.55 d -1 (gross vegetative cell rate), μgamete+cell = 0.38 d -1 (net motile cell growth rate), e = 0.42 d -1 (encystment rate). All these rates declined in the stationary growth phase. Sample sonication is sug- gested as the cause for low motile cell concentrations in the original experiment when cyst produc- tion was high. Inorganic carbon limitation due to low inorganic carbon to nitrogen concentrations in the growth media is probably the reason why cysts in f/4 medium stopped making calcite covers in later stages of the experiment and why cyst yield in f/2 medium was not double the yield in f/4. A new method for measuring in situ encystment rates of dinoflagellate populations with a phased sexual cycle is proposed.

Encystment probability and encystment rate: new terms to quantitatively describe formation of resting cysts in planktonic microbial populations

Many dinoflagellates and other groups of phytoplankton have benthic resting cysts as part of their life cycle. Details of transitions among life cycle stages are few in the literature and often do not meet the rigorous standards needed for across-species generalisations or model parameterisation. One regularly reported but poorly understood aspect is the cyst yield, a quantitative characterisation of cyst formation in relation to the size of the vegetative population. The literature provides various formulae for calculating cyst yield; however, not all of these give biologically meaningful results. Here we intro- duce 2 new terms, 'encystment probability' and 'encystment rate' to quantitatively describe and easily calculate the average cyst formation potential of a population during a given time interval. Encystment probability (φ) is defined as the average probability of vegetative cells in a population switching to sexual reproduction (i.e. transforming into gametes which subsequently fuse to form planozygotes) as opposed to continuing vegetative growth through binary fission. Encystment rate (e) is an exponential loss rate from the vegetative population; it is the difference between the instantaneous growth rate of the population (µ) and the apparent increase of the vegetative cell population (µ - e), provided no other losses take place. We propose a method of calculating encystment rate and encystment probability from readily available variables such as the number of vegetative cells at the beginning and end of a time interval and the number of resting cysts formed during the same period.

Quantification of Pfiesteria piscicida growth and encystment parameters using a numerical model

In the past decade, there has been growing interest in understanding the physiological ecology and life cycle of toxic forms of Pfiesteria piscicida. However, transformations among non- inducible (NON-IND; formerly described as nontoxic) stages have received less attention despite the fact that NON-IND stages are found in nature and may be ecologically important as prey and preda- tors. NON-IND stages are also mixotrophic and have the ability to retain and utilize prey chloroplasts in a process termed 'kleptoplastidic mixotrophy'. Quantifying growth, grazing and encystment rates from P. piscicida laboratory experiments is confounded by the interrelationship between mixotrophy and life stage transformations. By fitting a numerical model to a laboratory experiment on NON-IND P. piscicida, we were able to isolate the potential mechanisms that cause encystment and speculate on the interrelationship between adverse conditions (i.e. low light and limiting prey) and life stage transformations. The structure of the laboratory experiment allowed for the estimation of several growth and encystment parameters including grazing rates, gross growth and assimilation efficien- cies, as well as the retention time of chloroplasts. Model results suggest a link between encystment and mixotrophic ability. Furthermore, the model results suggest that encystment rates and gross growth and assimilation efficiencies calculated from the model are lower than expected.

Toxicity of anti-fouling biocides to Parorchis acanthus (Digenea: Philophthalmidae) cercarial encystment.

The toxicity of the anti-fouling biocides tributyltin (TBTO), copper, and Irgarol 1051 (irgarol) at nominal concentrations ranging from 10 to 10,000 microg l(-1) was investigated against the speed of encystment and successful formation of a protective cyst of the cercariae of Parorchis acanthus. For all biocide exposures, cercariae had a much slower rate of encystment and reduced cyst formation than controls. Exposure of the snail host Nucella lapillus for 7 d caused complete cessation of cercarial shedding in irgarol-exposed snails but had no effect on cercarial encystment from TBTO and copper-exposed snails. The mechanisms of toxicity of the biocides are briefly discussed.

Formation of two types of cysts by a mixotrophic dinoflagellate, Pfiesteria piscicida

ABSTRACT: Despite the widespread occurrence of mixotrophic dinoflagellates, most research oncyst formation in dinoflagellates has focused on phototrophic organisms or on factors affecting photo-trophic growth (i.e. light intensity and nutrient supply). Presumably, factors that stimulate cyst for-mation in mixotrophic organisms would be combinations of those factors that affect phototrophic and phagotrophic growth (such as limiting light intensity and limiting prey concentrations). The toxicdinoflagellate Pfiesteria piscicida is an interesting test case because it has a complex life history andis considered mixotrophic. Recently, a form of P. piscicida has been described that does not have theability to produce toxins (termed ‘non-inducible’). The objectives of this study were to identify whichlife stages are likely to be present in a mixotrophic culture of ‘non-inducible’ P. piscicida and to deter-mine the morphological differences based on fluorescent stain uptake. Also, we examined whichcombinations of adverse environmental factors (low light intensity and low prey concentrations)affect life stage transformations. In this culture, we observed 3 distinct life stages (a zoospore stage,and 2 cysts). One of the cysts (termed Cyst A) has a thick cell wall and appears to form from activelyfeeding zoospores regardless of light intensity. The second type of cyst (Cyst B) has a much thinnercell wall and only forms from recently fed zoospores maintained in complete darkness. During theexperiment, encystment rates to either cyst were low, suggesting that encystment will not dramati-cally affect bloom dynamics on small timescales.KEY WORDS:

Encystment-inducing factors in the ciliate Euplotes elegans.

Although starvation is considered one of the most important induces of ciliate encystment, its nature has been unclear. Euplotes is a well-known ciliate genus, but the relationship in Euplotes between encystment and food has not been reported. The encystment of Euplotes elegans is facilitated when it is transferred to Chalkley's solution without bacteria as food. A higher ciliate density also facilitates encystment. Thus, starvation and ciliate density needed to be examined. Ciliates were inoculated into 3 treatments: Chalkley's solution with formaldehyde-fixed bacteria as nutritive particles (FFB group), with polystyrene latex particles as non-nutritive particles (PLP group), and without particles (control group). Cysts appeared fastest and ciliate numbers increased in the FFB group. Although the encystment kinetics of the PLP group was similar to that of the control group, the encystment rate of the PLP group was lower than that of the control group in the earliest phase. This suggests that the ciliates were temporarily deceived into feeding on PLP, because they had food vacuoles containing PLP during the earliest phase of incubation. A cell-free old culture solution from a stationary phase, which probably contained excreted substances from high-density ciliates, also facilitated encystment.

Role of temporary cysts in the population dynamics of Alexandrium taylori (Dinophyceae)

Although temporary cyst stages are common in dinoflagellates, their role remains unclear. Every year Alexandrium taylori (Dinophyceae) forms dense patches (10 6 cells l -1 ) along La Fosca beach (Spain, northwest Mediterranean), which last for 2 months (July, August). One of the characteristics of the life history of A. taylori is the shift from a vegetative motile stage to non-motile temporary cysts. Here we present the temporal changes in the abundance of temporary cysts in sediments and their in situ encystment and excystment rates. The in situ encystment rate of temporary cysts from the water column to the sediment ranged from 1.8 X 10 6 to 4.4 X 10 6 cysts m -2 day -1, whereas the excystment rate was between 0.9 X 10 6 to 2. 7 X 10 6 cysts m -2 day -1 during the bloom period. Some of the temporary cysts in the sediment took more than 1 day to produce vegetative cells and remained viable for at least 4 days. We propose that temporary cyst formation in this species is a tool for reducing population losses. The production of temporary cysts can be an advantage since part of the population is stored in the sediments.

Population dynamics of active and total ciliate populations in arable soil amended with wheat.

Soil protozoa are characterized by their ability to produce cysts, which allows them to survive unfavorable conditions (e.g., desiccation) for extended periods. Under favorable conditions, they may rapidly excyst and begin feeding, but even under optimal conditions, a large proportion of the population may be encysted. The factors governing the dynamics of active and encysted cells in the soil are not well understood. Our objective was to determine the dynamics of active and encysted populations of ciliates during the decomposition of freshly added organic material. We monitored, in soil microcosms, the active and total populations of ciliates, their potential prey (bacteria and small protozoa), their potential competitors (amoebae, flagellates, and nematodes), and their potential predators (nematodes). We sampled with short time intervals (2 to 6 days) and generated a data set, suitable for mathematical modeling. Following the addition of fresh organic material, bacterial numbers increased more than 1,400-fold. There was a temporary increase in the number of active ciliates, followed by a rapid decline, although the size of the bacterial prey populations remained high. During this initial burst of ciliate growth, the population of cystic ciliates increased 100-fold. We suggest that internal population regulation is the major factor governing ciliate encystment and that the rate of encystment depends on ciliate density. This model provides a quantitative explanation of ciliatostasis and can explain why protozoan growth in soil is less than that in aquatic systems. Internally governed encystment may be an essential adaptation to an unpredictable environment in which individual protozoa cannot predict when the soil will dry out and will survive desiccation only if they have encysted in time.

Red rot resistance in interspecific protoplast fusion product progeny of Porphyra yezoensis and P. tenuipedalis (Bangiales, Rhodophyta)

SUMMARY Nine primary regenerants were recovered by interspecific protoplast fusion of Porphyra yezoensis Ueda T-14 (Py) (cultivated Porphyra) and Porphyra tenuipedalis Miura (Pt). This combination is difficult to achieve with conventional sexual hybridization, yet is important in that non-cultivated P. tenuipedalis is partially resistant (PR) to red rot disease, caused by the microbial pathogen, Pythium porphyrae Takahashi et Sasaki. Out of the nine primary regenerants, two strains (Py-Pt-4 and Py-Pt-7) were like the parent, P. tenuipedalis, while the rest were like the other cultivated parent P. yezoensis T-14 in their life cycle. Red rot resistance was assessed in parents and interspecific fusion product progeny (FPP) by exposing the foliose thalli to equivalent infection and measuring two parameters of the host-pathogen interactions: supported fungal biomass and amount of disease produced. Intermediate resistance between P. yezoensis T-14 (1.00) and P. tenuipedalis (0.13) was observed in two of the Py-type FPP, Py-Pt-2F2 (0.25) and Py-Pt-5F2 (0.23). Stable inheritance of resistance was observed through two subsequent generations. The morphologic and reproductive characteristics of the regenerated foliose thalli, and nature of host-pathogen interactions were used to further verify the hybrid origin of the FPP. Host-pathogen interactions were followed using epi-fluorescence and scanning electron microscopy (SEM). The zoospores encysted at higher rates on the susceptible cultivated parent (P. yezoensis T-14) germinated immediately and the short germ tubes formed appres-soria and penetrated the algal cells near the site of encystment. While on the PR parental (P. tenuipedalis) and partially resistant FPP (PRFPP) progeny (Py-Pt-2F2 and Py-Pt-5F2) the low rate of zoospore encystment was followed by cyst germination, but only a few of the germ tubes formed appressoria and penetrated the thallus surface. Long germ tubes (with no appressoria) were seen growing on the thallus surface without host penetration. The minimal rate of encystment concomitant with low rate of appressorium formation on the PR parent and PRFPP was observed as the major factor responsible for the partial resistance in these thalli.

CHRYSOPHYTE POPULATION AND ENCYSTMENT PATTERNS IN TWO CANADIAN LAKES1

ABSTRACT The dynamics of chrysophyte populations and the onset of encystment in relation to several physical, chemical, and biological variables were studied in two Canadian lakes, Lake Opinicon (LO) and Upper Rock Lake (URL). LO is shallow and polymictic during the ice‐free season, whereas URL is deep and dimictic. Seasonal patterns of many limnological variables, as well as species composition and seasonal dynamics of chrysophyte assemblages, were broadly similar in both lakes. Three groups of chrysophytes were recognized: 1) taxa that were more abundant during ice cover and/or early spring and occasionally in late fall (Dinobryon spp. Ehrenberg and Synura spinosa Korsh), 2) taxa that seemed to prefer moderately warm water proglena americana Calkins and Ochromonas sp. Wyssotzki) and had maximum densities in late summer and occasionally in the fall, and 3) taxa that occurred in low numbers and did not exhibit any discernible seasonality in the two lakes (Mallomonas spp. Perty). Encystment rarely occurred during ice cover in both lakes. Stomatocyst concentrations were highest shortly after ice breakup (late April or early May), declined to trace levels by June, and remained at those levels until the end of the sampling season (late November). Encystment rates (cyst concentrations) were higher in dimictic URL. Encystment in both lakes coincided with concurrent changes in physical and chemical variables and chrysophyte vegetative cell densities, but it was not possible to pinpoint the exact conditions responsible for triggering encystment. Only a small proportion of chrysophytes encysted. Cyst production within a lake may vary from year to year.

Rapid growth of Acanthamoeba in defined media; induction of encystment by glucose-acetate starvation.

Defined media are described that support 14-20 h generation times for Acanthamoeba castellanii and A. rhysodes in monolayer cultures. The media differ in minor ways from previously described media, but the growth rates are greatly improved over previously reported values. Maximum growth rates were observed for A. castellanii in a complex medium containing 21 amino acids, but near-maximum rates could be achieved in relatively simple media containing 9 amino acids. Growth occurred with 6 amino acids, as reported by others, but generation times exceeded 30 h. Amitosis was a common problem during early subcultures in defined media, defined media by glucose and acetate starvation. The rate of encystment varied with cell density at the time of starvation and was optimal at initial densities of 400-800 amebae/mm2.

Biochemical changes during growth and encystment of the cellular slime mold Polysphondylium pallidum.

The growth of the cellular slime mold, Polysphondylium pallidum, was studied on a semidefined medium in shaken suspension. When the medium contained large quantities of particulate material, growth was more rapid and the cellular size and protein content were smaller than when growth occurred on a medium containing less particulate material. The cellular levels of DNA, RNA, and protein; of lysosomal enzymes (acid phosphatase, acid proteinase); and of peroxisomal enzymes (catalase) were assayed during growth and the subsequent stationary phase that led eventually to encystment. Only DNA remained at a constant cellular level. Encystment of exponentially growing cells could also be initiated by washing them and introducing them into a soluble peptone medium. The rate of encystment was proportional to the osmolarity of this medium. The encystment process was followed with respect to the cellular levels of DNA, RNA, protein, carbohydrates, acid phosphatase, acid beta-N-Ac-glucosaminidase, and catalase. The most dramatic change occurred in the cellular cellulose content, which increased by at least an order of magnitude by the time encystment was morphologically complete. It was concluded that the encystment of this slime mold in suspension exhibits a number of biochemical similarities to the development of this and other cellular slime molds on a surface.