Action of Ultraviolet Radiation on Excystment and Concomitant Macromolecular Syntheses in Schizopyrenus russelli*

SYNOPSIS. Excystment of Schizopyrenus russelli was associated with increase in DNA, RNA, and protein. Both excystment and these macromolecular increases were sensitive to ultraviolet radiation. The effect of UV was partly reversed by incubation in the dark at 28 C, but not at 4 C. Excystment from cysts preincubated in the excystment medium (before UV treatment) was relatively resistant to radiation.     

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.     

Behavior of Peridinium bipes (Dinophyceae) resting cysts in the Asahi Reservoir

The role of resting cysts includes short- and long-term survival under extreme conditions, bloom initiation, species dispersal, reproduction, and preservation of genetic variation. Accordingly, it is important to understand their behavior in a water environment, especially in lakes and reservoirs where dinoflagellate blooms are observed. In this study, we estimated the behavior of the Peridinium bipes cysts in the Asahi Reservoir using laboratory experiments and field surveys. It was observed that the amount of light strongly influenced excystment, and few cysts germinated under dark conditions in the laboratory experiment. The minimum temperature on excystment was inferred from the laboratory experiment and field surveys to be about 5°C. Although the frequency of excystment did not depend on the water temperature from 10° to 20°C, the average preparation period for excystment decreased with the increase of water temperature. In the Asahi Reservoir, the excystment was estimated to occur during the months of March and April. If excystment did not occur in spring, the dinoflagellate bloom was not be observed until about July, although the bloom often began to appear in about May in the Asahi Reservoir. Consequently, the blooming season in the Asahi Reservoir is affected by the biomass of the germinated cysts in spring. Received: August 31, 2000 / Accepted: March 1, 2001     

Duration of the parasitic phase determines subsequent performance in juvenile freshwater pearl mussels (Margaritifera margaritifera)

Host–parasite systems have been useful in understanding coevolutionary patterns in sympatric species. Based on the exceptional interaction of the long‐lived and highly host‐specific freshwater pearl mussel (FPM; Margaritifera margaritifera) with its much shorter‐lived host fish (Salmo trutta or Salmo salar), we tested the hypotheses that a longer duration of the parasitic phase increases fitness‐related performance of mussels in their subsequent post parasitic phase, and that temperature is the main factor governing the duration of the parasitic phase. We collected juvenile mussels from naturally and artificially infested fish from eight rivers in Norway. Excysted juvenile mussels were maintained separately for each collection day, under similar temperature and food regimes, for up to 56 days. We recorded size at excystment, post excystment growth, and survival as indicators of juvenile fitness in relation to the duration of the parasitic phase. We also recorded the daily average temperatures for the entire excystment period. We observed strong positive relationships between the length of the parasitic phase and the post parasitic growth rate, size at excystment and post parasitic survival. Temperature was identified as an important factor governing excystment, with higher temperatures decreasing the duration of the parasitic phase. Our results indicate that juvenile mussels with the longest parasitic phase have better resources (larger size and better growth rate) to start their benthic developmental phase and therefore to survive their first winter. Consequently, the parasitic phase is crucial in determining subsequent survival. The temperature dependence of this interaction suggests that climate change may affect the sensitive relationship between endangered FPMs and their fish hosts.     

Seasonality in the excystment of Alexandrium minutum and Alexandrium tamarense in Irish coastal waters

Excystment experiments were carried out on cysts of Alexandrium minutum and A. tamarense (Group III) taken from Cork Harbour, Ireland. Freshly sampled cysts were isolated into well plates and their excystment was monitored over a 30 day period. A pronounced seasonality was observed in the excystment behaviour in both species when measurements were carried out seasonally. Between 80 and 100% of the isolated cysts excysted within 30 days when samples were taken between March and June. However, between only 0 and 15% excysted in samples taken between August and early February. This seasonal characteristic was observed repeatedly over a 3 year period (2004–2007). The effect was observed in cysts which had been sampled from both inter-tidal and sub-tidal locations. No endogenous clock was evident in the germination of A. tamarense and A. minutum cysts taken from Cork Harbour which had been stored cool and in the dark under anoxic conditions for up to 18 months. The seasonal effect observed was independent of water temperature, although temperature did affect the rate of excystment. Temperature also affected the maturation of cysts which had been freshly formed in the laboratory. The significance of incorporating seasonality in excystment of Alexandrium into models describing its growth is also discussed.     

Role of Riboflavin and Certain Amino Acids in the Excystment of Schizopyrenus russelli*

SYNOPSIS. A simple medium, consisting of riboflavin and a mixture of L-glutamic acid, L-tryptophan, L-isoleucine, L-serine, and L-proline has been shown to induce rapid and mass scale excystment in Schizopyrenus russelli. Whereas percentage excystment was found to depend on the concentration of riboflavin and amino acids, more than 80% of cysts were found to excyst within 4 hr, when adequate amounts of these were supplied. Several individual amino acids, particularly L-glutamic acid, L-tryptophan, and L-proline, also supported considerable excystment, but riboflavin was always indispensable.     

The Excystment Process in the Ciliate Euplotes muscicola: An Integrated Light and Scanning Electron Microscopic Study1

Resting cysts and the excystment process in the freshwater ciliate Euplotes muscicola were studied by both light and scanning electron microscopy. Groups of distinctly crested resting cysts adhere to the substrate. Silver-stained preparations reveal surface conservation of dorsal kinetosomes and dorsal argyrome while ventral organelles are directed inward. Excystment involves the development of an expanding excystment vacuole concurrent with a localized thinning on the dorsal cyst wall surface. Cells exit through the pre-formed ostiole, mid-dorsal region first, initially by the force of cytoplasmic streaming, but later aided by cirral movement. Newly emerged cells retain the excystment vacuole and show no dorsal ridging. As the cell expels its excystment vacuole and partially unfolds, normal trophont morphology is re-established. Both cyst structure and cyst typology have implications for hypotrich taxonomy.     

Fine structure of excystment of the quadriflagellate algaPolytomella agilis

Summary Populations of mature resting cysts of the algaPolytomella agilis were purified from asynchronously encysting cultures and incubated in fresh culture medium to promote excystment. Up to 90 percent of the cysts germinated, with approximately 50 percent excysting between 3 and 7 hours of incubation. Each germinating cyst releases a single, fully differentiated, swimming cell. The entire excystment process of individual cysts was followed by light microscopy to establish the time course of release and cells at comparable stages of excystment were examined by electron microscopy. During the first 3 hours of incubation the cysts increase in size, presumably due to uptake of water, and a polarity is established in the cytoplasm which makes it possible to identify the site of subsequent release. Release involves a selective degradation of a portion of the cyst wall at this site followed by a physical rupturing of the weakened area. Details of the structural alterations in the wall and cytoplasm are described. The cytoplasmic organelles observed to dedifferentiate during encystment (preceding paper) are completely redifferentiated during excystment. The emergent cell is flagellated and possesses the elongate form typical of the swimming cell.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.     

THE KINETICS OF EXCYSTMENT IN COLPODA DUODENARIA

1. Extensive experimental data have been collected on the time required for the excystment process of the small ciliate Colpoda duodenaria throughout a range of temperatures of 8° through 32°C. and a range of concentrations of yeast extract excystment media of 0.08 through 22.4 gm./liter. 2. The excystment process has been separated into two periods, the first inversely proportional to the concentration of the yeast extract and the second independent of its concentration. 3. The first excystment period has been found to depend on the time for diffusion through the protoplasm of a compound from the yeast extract and on the time for a chemical reaction with the extremely high energy of activation of 220,000 calories/mole. 4. The changes in viscosity with temperature for this Colpoda, inferred from diffusion rate changes, have been found to be almost the same as those found by Heilbrunn for Amoeba dubia by the direct method of centrifuging granules. 5. The second excystment period is shown to be controlled by reactions whose apparent activation energies are 44,000 calories/mole below 15°C. and 18,000 calories/mole above 15°C.; above 25°C. this period is independent of temperature. 6. The distribution of the log excystment times of individual organisms about the mean log excystment time is found to be independent of temperature except in the range where the reaction with highest activation energy takes a significant length of time, and to increase rapidly with decreasing temperatures in this range.     

Differentiation During Encystment and Excystment in Oxytricha fallax*

During encystment of Oxytricha fallax, a wall composed of 4 distinct layers, each derived from a different kind of endoplasmic vesicle, is formed between the 2 unit membranes that cover the vegetative cell. Numerous autophagic vacuoles arise in the endoplasm and later (during excystment undergo internal changes comparable to those characteristic of food vacuoles. Mitochondria aggregate into a band. The 2 macronuclei fuse and their nucleoli become homogeneous. Except for the 2 cell membranes, all visible cortical structures, including cilia, kinetosomes, and microtubules, disappear. Despite the absence of visible ciliature in the mature cyst, the various primordia of the normal vegetative ciliature arise during excystment in the same positional relations to one another as is characteristic of developments during cell division.     

Effect of water temperature on the excystment of the dinoflagellate Ceratium hirundinella (O. F. Müller) Bergh

The effect of water temperature on the excystment of the dinoflagellate Ceratium hirundinella (O. F. Müller) Bergh was studied in the laboratory. Excystment was observed between 15–30 °C and was 2% at an optimum water temperature of 20 °C–25°C. Little excystment occurred between 5 and 10 °C. The results at low temperatures are not in accordance with those obtained in an English lake. This disagreement suggests an adaptability of excystment to the temperature regime of the lake.     

Scale Formation in an Amoeba, Cochliopodium sp.

During excystment of an amoeba, Cochliopodium sp., scale formation was examined with light and electron microscopy. This amoeba was covered with scales. When the amoeba encysted, the scales remained on the external surface of the cyst wall. Soon after the induction of excystment the Golgi complex began to develop. Many vesicles were extruded from it and changed into vacuoles. Scales were observed first in the vacuole adjacent to the Golgi complex and later in inside the cyst wall. When the amoeba excysted it had been coated by the newly formed scales. It is suggested that the scale formation is dependent on the activity of the Golgi complex.     

Factors regulating excystment of Alexandrium in Puget Sound,WA, USA

Factors regulating excystment of a toxic dinoflagellate in the genus Alexandrium were investigated in cysts from Puget Sound, Washington State, USA. Experiments were carried out in the laboratory using cysts collected from benthic seedbeds to determine if excystment is controlled by internal or environmental factors. The results suggest that the timing of germination is not tightly controlled by an endogenous clock, though there is a suggestion of a cyclical pattern. This was explored using cysts that had been stored under cold (4 °C), anoxic conditions in the dark and then incubated for 6 weeks at constant favorable environmental conditions. Excystment occurred during all months of the year, with variable excystment success ranging from 31–90%. When cysts were isolated directly from freshly collected sediments every month and incubated at the in situ bottom water temperature, a seasonal pattern in excystment was observed that was independent of temperature. This pattern may be consistent with secondary dormancy, an externally modulated pattern that prevents excystment during periods that are not favorable for sustained vegetative growth. However, observation over more annual cycles is required and the duration of the mandatory dormancy period of these cysts must be determined before the seasonality of germination can be fully characterized in Alexandrium from Puget Sound. Both temperature and light were found to be important environmental factors regulating excystment, with the highest rates of excystment observed for the warmest temperature treatment (20 °C) and in the light.     

BIOCHEMICAL COMPOSITION AND METABOLIC ACTIVITY OF SCRIPPSIELLA TROCHOIDEA (DINOPHYCEAE) RESTING CYSTS1

The composition and metabolic activity of cysts of the marine dinoflagellate Scrippsiella trochoidea (Stein) Loeblich were examined during dormancy, quiescence, and germination. On a per cell basis, newly formed cysts contained an order of magnitude more carbohydrate but significantly less protein and chlorophyll a than did exponentially growing vegetative cells. Loss of lipid and carbohydrate from cysts during the initial dormancy period reflected a respiration rate estimated to be 10% of the respiratory activity in vegetative cells. Among older, quiescent cysts the calculated respiration rate decreased further to approximately 1.5% of the vegetative rate and appeared to proceed largely at the expense of carbohydrate reserves. These estimated rates of respiration were in good agreement with direct measurements of cyst oxygen consumption. The transfer of quiescent cysts to conditions permissive for germination resulted in a rapid increase in respiration rate, as evidenced by carbohydrate loss and O₂ consumption. The increased respiratory activity was followed by an increase in protein content and, later, by an increase in chlorophyll a content and photosynthetic capacity. Just prior to germination the P/R ratio became greater than 1, and the estimated chlorophyll-specific photosynthetic activity reached 75% of the rate in vegetative cells. Complete restoration of photosynthetic and respiratory capacity apparently was not achieved until after excystment. These data confirm the common assumption that dinoflagellate cysts represent true “resting” cells, containing extensive energy reserves and displaying greatly reduced metabolic activity.     

包囊游仆虫包囊形成和解脱过程中大、小核的研究

包囊游仆虫形成包囊时大核除经历形态大小的变化外,大核DNA含量也低于正常大核水平;细胞脱包囊前,在大核一端或两端发生染色质粗浓集,是大核DNA复制的结果;染色质粒在整个大核内浓集时,大核DNA含量已达到正常大核水平,此时DNA复制结束,细胞脱包囊。小核在游仆虫形成包囊和脱包囊过程中,其形态大小、DNA含量等无明显变化。     

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.     

Ultrastructure of Cysts of Naegleria spp: A Comparative Study*

SYNOPSIS. Ultrastructure of cysts of Naegleria gruberi, Naegleria fowleri, and Naegleria jadini was compared by transmission electron microscopy. Pores in the cyst wall were observed in all 3 species. In N. gruberi they were surrounded by a collar and sealed with a relatively large mucoid plug; no such collar was seen around the pores in the other 2 species, in which the plug was smaller than that in N. gruberi. An electron-dense plaque serving as an additional pore closure was present in all 3 species. In N. gruberi, the cyst wall consisted of an inner thick and an outer thin layer; however, only the inner component was present in cysts of N. fowleri and N. jadini, which had a smooth appearance. At the ultrastructural level, excystment of N. fowleri involved digestion of the mucoid plug and emergence of the trophozoite through the pore. Some digestion of the cyst wall also appeared to take place during excystment.     

Effects of nitrogen deficiency and light of high intensity onCryptomonas rufescens (Cryptophyceae)

Summary C.rufescens excystment, experimentally induced, corresponds to a general metabolism recovery of the cell, previously in a resting phase. The cytoplasm changes without any polarity, and organelles like gullet and flagella redifferentiate. The thylakoids develop mainly from the stored lipidic compounds which then disappear. Phycoerythrin immediately fills the intrathylakoidal lumen. Pigment synthesis seems closely associated with the development of membranes. The activated cell divides and the cyst wall breaks down. The destruction of the wall begins in the median layer and is followed by a mechanical rupture of the external and internal layers. Each germinative cyst releases two or four fully differentiated cells. There is an exact symmetry between excystment and encystment, all the transformations of theC. rufescens cell being reversible.     

Fasciola hepatica: Simple,large-scale, in vitro excystment of metacercariae and subsequent isolation of juvenile liver flukes

A simple method has been developed for the in vitro excystment of metacercariae of Fasciola hepatica, and for the isolation of large numbers of juvenile liver flukes free from intact metacercariae and from cyst-wall material. In this method, the outer cyst wall was removed by gently grinding the metacercariae between small glass plates. The metacercariae were activated by incubation for 1 hr under $\text{60\%}\text{CO}{}_2\text{40\%}\text{N}{}_2$ and excysted by the addition of 10% sterilized sheep bile or an equivalent amount of taurocholic acid. Excystment was accomplished in an experimental apparatus allowing the newly excysted juveniles to escape from the bile-containing excystment medium into a medium with low bile content. The yield of isolated liver flukes was 60–80%; their protein content was about 125 ng. Both bile and taurocholic acid, though necessary for excystment, were detrimental to the survival of the juvenile liver flukes. The presence of bile in the host intestine may be a stimulus for juveniles to leave the gut and enter the abdominal cavity.     

Algal cyst dormancy: a temporal escape from herbivory

Many phytoplankton species form resting cysts and remain dormant for part of the year. The subsequent excystment is regulated by the external environment and internal maturation processes. Here we assessed the excystment of the dinoflagellates Ceratium hirundinella and Peridinium aciculiferum in relation to herbivores and temperature in laboratory and field studies. C. hirundinella, which has a grazer-resistant morphology, forms summer blooms, whereas P. aciculiferum, which is vulnerable to grazers, grows underneath the ice during winter. In our study, herbivore abundance, and thereby grazing pressure, was low during periods when water temperatures were low, and the abundance of P. aciculiferum was high. In the laboratory experiment, excystment of C. hirundinella occurred at high temperatures irrespective of whether zooplankton exudate was added or not, whereas at intermediate temperatures, excystment was lower if zooplankton exudate was added. Germination of P. aciculiferum cysts was lower in the presence of exudate from a zooplankton culture than in controls at all temperatures. Our studies suggest that dinoflagellates use the presence of zooplankton in addition to temperature as a cue to determine when to excyst. Consequently, not only abiotic factors, but also the composition of the food web, may determine succession and composition of phytoplankton communities.