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Reversibility of encystment in the ciliate, Histriculus cavicola (kahl, 1935) (ciliophora: stichotrichia)
Europ.]. Protisto!' 28, 51-55 (1992) February 21, 1992
European Journal of
PROTISTOLOGY
Reversibility of Encystment in the Ciliate, Histriculus cavicola (Kahl, 1935) (Ciliophora: Stichotrichia) Takahiro Nakamura and Tadao Matsusaka* Graduate School of Science and Technology and *Department of Biological Science, Faculty of Science, Kumamoto University, Kumamoto, Japan
SUMMARY An early phase of encystment of the ciliate, Histriculus cavicola is subdivided into 5 stages, stages A to E. Resorption of feeding organelles, undulating membranes (UM) and the adoral zone of membranelles (AZM) start from stage B. In stage E cells, resorption ofUM is completed and that of the AZM reaches to the level of anterior end of left marginal cirral row from its posterior end. When stage A cells are transferred in culture medium with or without food organisms, or in normal or 10 times concentrated Osterhout's solutions with food, most cells do not encyst. More than half of stage C cells do not encyst at 20°C after transfer in culture medium. All stage E cells encyst, irrespective of the medium or of the presence or the absence of food into which they are transferred. About half of stage A cells and all stage B to E cells contain ectocyst precursors. Our results indicate that initiation of ectocyst precursor formation does not necessarily direct the cells to encyst but cells in stage E are determined to encyst.
Introduction Many factors are supposed to induce ciliate encystment [1, 17]. Of these, the most universal inducing factor for ciliate encystment may be starvation or deficiency of essential nutrients in the medium [3]. In stichotrich ciliates, resorption of feeding organelles, the undulating membranes (UM) and the adoral zone of membranelles (AZM), is the first morphological sign of encystment [2, 6]. In Oxytricha fallax, when an early encysting cell is cut in two, the anterior fragment, which still has UM and AZM, regenerates all ciliary organelles and does not encyst, but the encystment of the posterior fragment, which has neither UM nor AZM, is accelerated [6]. The results suggest that the presence of feeding organelles may inhibit encystment of the ciliate. The formation of cyst wall precursors is another ultrastructural characteristic of encysting ciliates [2,5,7-9,11, 15, 16, 18]. Precursors of the outermost cyst wall, the ectocyst, are known to be formed first during encystment in several stichotrich ciliates [2, 5, 8, 9, 11, 16, 18]. Therefore, the presence of ectocyst precursors is one of the indicators of encysting cells in these ciliates. © 1992 by Gustav Fischer Verlag, Stuttgart
In the present study, an early phase of encystment of the stichotrich ciliate, Histriculus cavicola (late stage 1 and stage 2 of Matsusaka, [11]), is subdivided into 5 stages determined by the extent of resorptions of UM and AZM observable with a phase-contrast mciroscope. The following 2 problems are then examined: 1) whether the cells in each stage of encystment continue encysting after their transfer in different environmental conditions (different media, different temperatures, and presence or absence of food organisms) and 2) whether the cells in each stage contain ectocyst precursors.
Material and Methods The organism used in the present study was the KM-1 strain of the stichotrich ciliate, Histriculus cavicola. The ciliates were cultivated at 25°C in 9 ern (diameter) Petri dishes containing 30 ml of 1: 1 mixture of 0.1 % (w/v) lettuce extract and 0.1 % (w/v) wheat extract (culture medium, CM), into which Chlorogonium elongatum was introduced as a food source as described earlier [14]. In this culture condition, the ciliates attained a stationary phase of growth within 3-4 days after inoculation. 0932-4739/92/0028-0051 $3.50/0
52 . T. Nakamura and T. Matsusaka Encystment was induced by washing the cells three times with 10 times concentrated Osterhout's balanced salt solution (10 X OS) by low speed centrifugation (about 50 g) while they were in the stationary phase of growth. Then the cells were suspended in 10 X OS and maintained in 6 cm (diameter) Petri dishes at 20°C [10]. When the population is observed every 15 min after induction of encystment, synchronous encystment ensues and almost all cells transform into cysts within 5 h [10]. When observations were made less frequently, however, the synchrony of encystment was poor even after the same treatment for induction of encystment and encystment of almost all cells required ca. 8 h. To determine the stages of encystment, a single cell was isolated from the encysting population between 3 and 7 h after induction of encystment. The cell was kept in a hanging drop under a cover
slip and observed with a phase-contrast microscope using a X 40 objective lens. Subdivision of an early phase of encystment (late stage 1 and stage 2, Matsusaka, [11]) was made on the basis of the extent of resorptions of UM and AZM. After determination of the encystment stage, the cell was transferred in a well of a 96-well microplate containing 100 ftl of normal Osterhout's balanced salt solution (1 X 05),10 X OS, or CM, and maintained at 20 or 25°C. To determine whether the presence or the absence of food organisms affect encystment, C. elongatum was introduced into some wells. The encystment of each cell was examined at 2, 4, 6, and 24 h after transfer. About 50 to 150 cells were examined for each determination. The cells in each stage were fixed in a 1 : 1 mixture of 5% (w/v) glutaraldehyde in 0.05 M cacodylate buffer (pH 7.3) and 2% (w/v) aqueous OS04 at room temperature for 15 min. They were
Fig. 1. Phase-contrast photomicrographs (a-e) of encysting Histriculus cavicola. The anterior parts were line drawn (a' -e') to show the criteria of each stage. a and a': A cell in stage A, showing no change in ciliary organelles (arrow head, UM; arrow, AZM). band b': A cell in stage B, showing resorption of the anterior part of the UM (arrow head). Arrow indicates AZM. c and c': A cell in stage C, showing unresorbed short posterior UM (arrow head) and beginning of resorption of the AZM from its posterior end (arrow). d and d': A cell in stage D, showing absence ofUM (arrow head) and resorption of a few membranelles of the posterior part of AZM (arrow). e and e': A cell in stage E, showing absence of UM (arrow head) and the resorption of membranelJes of AZM extending to the level of the anterior end of left marginal cirri (arrow). Bar = 50 urn,
Reversibility of Ciliate Encystment . 53 then dehydrated thro ugh an etha nol series and embedded in Qu erol 65 1. Serial sections of a whole cell were made with a Reichert -Jun g Ultracut E ultram icroto me using a diamond knife; the sections were observed with a J EM 100 C UEOL) electron microscope operated at 80 kY.
were presented (Fig. 1). However, it was not difficult to differentiate each stage in the living cells because ciliary movements could be seen. Reversibility of Encys tme nt
Results Subdivision of Early Stages of Encys tment
The late stage 1 and stage 2 of encystment recognized earlier [11] were subdivided into the following 5 stages. Stage A (Fig. l a, a '): this stage corre spon ds to late stage 1 of the earlier study [11]. Cells of this stage occurred in the popu lation 3 h after indu ction of encystment. It was determined by low power observation with a light microscope that the cells in this stage did not contain distinct food vacuoles or cytoplasmic granules (which were commonly observable in the troph ozoites). Resorption of feeding organelles did not begin, however, and the disposition of the ciliary organelles was the same as in the trophic form. Stage B (Fig. 1b, b' ): this stage corre spond s to the intermediate stage between stages 1 and 2 of the earlier study [11]. A part of the UM was resorbed from its anterior end but the AZM remained intact. Stage C (Fig. 1c, c' ): this stage also corresponds to the intermediate stage between stages 1 and 2. Onl y the posteriormost part of the UM remained unr esorbed and resorp tion of the AZM began from its posterior end. Stage D (Fig. 1d, d'): th is stage corresponds to mid-stage 2 of the earlier study [11]. Resorpti on of UM was completed and a few membranelles were resorb ed from the posterior end of AZM. Stage E (Fig. Le, e') : this stage corresponds to late stage 2. The membranelles of the AZM were resorbe d from its posterior end to the level of the anterior end of the left marginal cirral row . Duri ng stages A through E, no somatic cirri were resor bed. Because recognition of each stage is somewhat difficult from the pre sent photomicrographs, line drawings of the anterior part of encysting cells
Th e encystment of individual cells in each stage was exa mined after transfer of one cell into each well of a 96-well microp late conta ining different media, with or witho ut food organisms. The results at 24 h after transfer were tabulated and expressed as percentages of encysted cells (Ta ble 1). Stage A: The fate of the cells in this stage depended essentially upon the presence or the absence of food orga nisms, regardless of the kinds of media and temperatures maintained. When the cells were transferred into salt solutions with food organ isms or into CM with or without food, around 80% or more of the cells did not encyst, irrespective of the maintaining temperatures. There were, however, two exceptions; 1) the case transferred in 1 X OS with food and maintained at 25 °C (66.7% of the cells did not encyst), and 2) the case transferred in CM without food at 25 °C (47% of the cells did no t encyst). When the cells in this stage were transferred in salt solutions without food s, on the cont rary, more than 90% of cells encysted, irrespective of the maintaining temper ature . The only exception was the case tr ansferred into 10 x OS and maintained at 20 °C (18.5% of cells did not encyst), in spite of the same conditions to indu ce encystment. Stage B: The fate of the cells in this stage depended essentially upon the kinds of media and temper ature s into which the cells were tran sferred . When the cells were transferred into CM with or with out food and maintained at 20 °C, 70- 80% of the cells did not encyst. Conversely, 70-80% of the cells encysted at 25 °C even after transfer into the same medium. When the tran sfer was mad e into salt solutions, encystment ra tes were generally high, especially in the case maintained witho ut food (100% ). Even in the presence of food, more than 90% of cells
Table 1. Percentages of encysted cells Solution 10 X 10 X 1X I X CM CM 10 x 10 X 1X 1X CM CM
OS OS OS OS OS OS OS OS
Conditio ns Temp. (0C) Food 20 25 20 25 20 25 20 25 20 25 20 25
+ + + + + +
A
B
81.5 92.6 91.1 92.6 10.4 53.0 20.3 12.9 4.7 33.3
100 100 100 100 32.9 76.1 92.5 90.7 63.2 93.6 19.4 66.7
a
16.6
Stages of encystment C 100 100 100 100 48.1 89.7 100 100 94.4 95.8 38.2 78.4
D
E
100 100 100 100 80.0 100 100 100 95.2 96 .8 79.0 100
100 100 100 100 100 100 100 100 100 100 100 100
Cells in each stage of encystment were transferred individually in wells of a 96 -well microp late containing 1 X OS, 10 X OS, or CM with (+) or without (-) food orga nisms (Ch!orogonium e!ongatum), and were main tained at 20 or 25 "C. Percenta ges of encysted cells were determined at 24 h after transfer. Abou t 50 to 150 cells were coun ted for each determination.
54 . T. Nakamura and T. Matsusaka
encysted in the salt solutions. The case transferred i~to 1 X OS and maintained at 20°C was the only exception (36.8% of the cells did not encyst). Stage C: Most cells in this stage encysted after transfer into salt solutions, regardless of the presence or the absence of food organisms and of the temperatures maintained. When the transfer was made into CM, however, the encystment was dependent mostly upon temperatures, irrespective of the presence or the absence of food; 80-90% of the cells encysted at 25°C, but 50 to 60% of the cells did not encyst at 20 "C. Stage D: Almost all cells encysted after transfer into any medium, regardless of the presence or absence of food and of the temperatures. However, when the cells were transferred into CM and maintained at 20°C, about 20% of the cells still did not encyst. Stage E: All the cells encysted wit??ut except.ion after transfer into all environmental conditions examined, The cells which failed to encyst in the medium without food usually did not divide, but rarely divided only once within 24 h after transfer. When the cells were transferred into medium containing food organisms, the cells divided 1-3 times within 24 h after transfer. Under most conditions examined, cells encysted more readily at 25°C than at 20°e. Examination of Ectocyst Precursors
Within the cytoplasm of stage B cells, the presence of small bodies (ca. 1 urn long and ca. 0.3 urn wide), which contained stacks of thin, electron dense plates, was demonstrated electron microscopically (Fig. 2). These bodies showed the same ultrastructural characteristics as the ectocyst precursors described earlier [2, 5, 8, 9, 11, 16,18]. The ectocyst precursors were identified in 4 out of 10 cells in stage A and in all cells in stages B to E (Table 2).
Fig. 2. A part of the cytoplasm of a stage B cell showing ectocyst precursors (arrows). Bar = 0.5 urn.
Table 2. Presence of ectocyst precursors Stages
A
B
c
D
E
alb
4/10
11/11
8/8
5/5
4/4
a: Numbers of cells containing ectocyst precursors. b: Numbers of cells observed.
Discussion Almost all cells encysted within 8 h after induction of encystment in 10 X OS at 20°C, unless the .encysting population was disturbed. However, wh~n c~lls m stageoA were transferred into 10 X OS and maintained at 20 C without food, about 20% of the cells did not encyst within 24 h. This result may be caused by the disturbance of the process by the transfer. The encystment rates after transfer of the cells were generally higher at 25°C than at 20 °C (Table 1). The results may indicate acceleration of encystment at higher temperatures once the process had started, supporting the earlier reports that encystment of Laurentiella acuminata [4] and Oxytricha fallax [6] proceeds faster at higher temperatures. When environmental conditions were experimentally changed, some encysting cells did not comple~e encystment (Table 1). It has been shown [6] that the antenor fragments of encysting cells which possess UM and AZM as regenerated ciliary organelles do not encyst, whereas the encystment of the posterior fragments which have neither UM nor AZM is accelerated. In the present experiments, 50-60% of stage C cells and about 20% of stage D cells did not encyst at 20°C after transfer into CM. All stage E cells, however, encysted after any treatment to which they were subjected in the present study. In stage C, the UM, though only a part, remained unresorbed and the resorption of the AZM just began. The resorption of UM was completed in both stages D and E but that of the AZM was slightly more advanced in stage E cells than in stage D cells. These findings suggest that the retention of UM and AZM inhibits encystment and that the inhibition may result from the retention of feeding ability of the cells. If this is true, the presence of an almost intact AZM supports feeding to some extent even in the absence of UM, or the cells regenerated UM and AZM before their resumption of feeding, but it is not clear. This supposition supports the earlier result [6], showing inhibition of the encystment of the anterior half. Acceleration of the encystment of the posterior half [6] can be explained by supposing the presence of an "encystment promoting factor" in the posterior part of the cell. This idea, however, may not be true because in the present study whole cells were used and lower encystment rates resulted from the media containing food organisms. When the cells were transferred in CM, lower encystment rates generally resulted. In these cases, it may be that proliferation of bacteria occurred in the CM and the bacteria may have served as a food source. Transcription and translation are known to be involved in the encystment of several species of ciliates [4, 11, 13, 15, 19]. In Histriculus cavicola, protein is one of the
Reversibility of Ciliate Encystment . 55
components of the ectocyst [12] and cycloheximide blocks the formation of ectocyst precursors and encystment [11]. Th e present experiments indicate that about half of the cells in stage A and all cells in stages B to E contain ectocyst precursors in their cytoplasm (Table 2), and that some of these encysting cells do not encyst after transfer into different media, especially those containing food organisms (Ta ble 1). This suggests that structural genes for the ectocyst precursors must be expressed at least by late stage A, but the expression of the genes does not necessarily mean the continuation of the encystment process. This is consistent with the earlier findings [13] that actinomycin D inhibits encystment of the same species when the drug is applied before mid-stage 1, because stage A corresponds to late stage 1 of the earlier study [11]. Stage E cells encysted without exception after any treatment in the present exper iments, indicating that stage E cells are determined to encyst. These results suggest that major regulating genes for encystment may be expressed at least by two steps and that the second gene expre ssion may be decisive for encystment. Acknowledgement The authors would like to express their sincere thanks to Prof. John F. Tibb s (Division of Biological Sciences, University of Montana, USA) for his kind help in editing the manu script.
References Corliss J. O. and Esser S. C. (1974): Comments on the role of the cyst in the life cycle and survival of free-living protozoa. Tr ans. Amer. Micros . Soc., 93, 578 -593. 2 Grimes G. W. (1973): Differentiation during encystment and excystment in Oxytricha [allax, J. Protozool., 20, 92-104. 3 Gutierrez J. c., Martin-Gonzalez A. and Matsusaka T. (1990): Toward a generalized model of encystment (cryptobiosis) in ciliates: a review and hypothesis. BioSystems, 24, 17-24. 4 Gutierrez J. c., Torres A. and Perez-Silva J. (1981): Excystment cortical morphogenesis and nuclear processes during encystment and excystment in Lau rentiel/a acuminata (H ypotr ichida, Oxytrichidae). Acta Prorozool., 20, 145-152.
5 Guti errez J. c., Torres A. and Perez-Silva J. (1983): Structure of the cyst wall precursors and kinetics of their appearance durin g the encystment of Laure ntiel/a acum inata (Hypotrichida, Oxytrichidae). J. Protozool., 30, 226-233. 6 Hashimoto K. (1962): Relation ships between feeding organelles and encystment in Oxytri cha (aI/ax Stein. J. Prot ozool., 9, 161-169. 7 Holt P. A. and Chapman G. B. (1971): The fine stru ctur e of the cyst wall of the ciliated protozoon Didinium nasutum. J. Protozool., 18, 604- 614. 8 Jareiio M. A. (1985): Etude ultr astru cturale de l'enk ystement et du dekystement chez Onycho dro mu s acum inatus (Ciliata, Hypot richida). Protist ologica, 21, 313- 321. 9 Matsusaka T. (1976): An ultras tructural study of encystment in the hypotrichous ciliate Pleurotr icha sp. Kumamoto J. Sci., Biol., 13, 13-26. 10 Matsusaka T. (1977): Indu ction of synchronous encystment in a hypotrichous ciliate, H istriculus sp. Exp. Cell Res., 110 , 459-462. 11 Matsusaka T. (1979): Effect of cycloheximide on the encystment and ultrastructure of the ciliate, Histriculus. J. Proto zool., 26, 619- 625. 12 Matsusaka T. and Hon go F. (1984): Cytochemical and electrophoretic studies on the cyst wa ll of a ciliate, H istriculu s muscorum Kahl. J. Protozool., 3 1, 471-475. 13 Matsusaka T. and Kimura S. (1981): Changes in macronuclear ultra structure dur ing encystment in a ciliate, Histriculus muscorum. Kumamoto J. Sci., Biol., 15, 49-58. 14 Nakamura T. and Matsusaka T. (1991): Effects of cyst age on excystment of the ciliate, Hist riculu s cavicola (Kahl, 1935) (Ciliophora: Stichotri chia). Europ. J. Protisto!., 27, 375-38 0. 15 Ruthm ann A. and Kuck A. (1985): Form ation of the cyst wall of the ciliate Co lpoda steinii. J. Protozool. , 31, 677-682. 16 Verni F., Rosati G. and Ricci N. (1984): The cyst of Oxytricha bi(aria (Ciliata Hypotri chida). II. The ultra structure. Prot istologica, 20, 87-95 . 17 Wagtendonk W. J. van (1955): Encystment and excystment of pro tozoa. In: Hutner S. H. and Lwoff A. (eds.): Biochemistry and physiology of protozoa, vol. II, pp. 85-90. Academic Press, New York. 18 Walker G. K., Maugel T. K. and Good D. (1980): Encystment and excystment in hypotrich ciliates. I. Gastrostyla steinii. Protistologica, 16, 511-524. 19 Yonezawa F. (1985): Effect of actinomycin D, RNas e and protein synthesis inhibitors on encystment in Eupl otes encysticus (Ciliophora). J. Sci. Hiro shima Univ., Ser. B., Div. 1., 32, 73-82.
Key words: Ciliate - Reversibility - Encystment - Histriculus Tadao Ma tsusaka , Department of Biological Science, Faculty of Science, Kumamoto University, Kumamoto 860, Japan
European Journal of
PROTISTOLOGY
Reversibility of Encystment in the Ciliate, Histriculus cavicola (Kahl, 1935) (Ciliophora: Stichotrichia) Takahiro Nakamura and Tadao Matsusaka* Graduate School of Science and Technology and *Department of Biological Science, Faculty of Science, Kumamoto University, Kumamoto, Japan
SUMMARY An early phase of encystment of the ciliate, Histriculus cavicola is subdivided into 5 stages, stages A to E. Resorption of feeding organelles, undulating membranes (UM) and the adoral zone of membranelles (AZM) start from stage B. In stage E cells, resorption ofUM is completed and that of the AZM reaches to the level of anterior end of left marginal cirral row from its posterior end. When stage A cells are transferred in culture medium with or without food organisms, or in normal or 10 times concentrated Osterhout's solutions with food, most cells do not encyst. More than half of stage C cells do not encyst at 20°C after transfer in culture medium. All stage E cells encyst, irrespective of the medium or of the presence or the absence of food into which they are transferred. About half of stage A cells and all stage B to E cells contain ectocyst precursors. Our results indicate that initiation of ectocyst precursor formation does not necessarily direct the cells to encyst but cells in stage E are determined to encyst.
Introduction Many factors are supposed to induce ciliate encystment [1, 17]. Of these, the most universal inducing factor for ciliate encystment may be starvation or deficiency of essential nutrients in the medium [3]. In stichotrich ciliates, resorption of feeding organelles, the undulating membranes (UM) and the adoral zone of membranelles (AZM), is the first morphological sign of encystment [2, 6]. In Oxytricha fallax, when an early encysting cell is cut in two, the anterior fragment, which still has UM and AZM, regenerates all ciliary organelles and does not encyst, but the encystment of the posterior fragment, which has neither UM nor AZM, is accelerated [6]. The results suggest that the presence of feeding organelles may inhibit encystment of the ciliate. The formation of cyst wall precursors is another ultrastructural characteristic of encysting ciliates [2,5,7-9,11, 15, 16, 18]. Precursors of the outermost cyst wall, the ectocyst, are known to be formed first during encystment in several stichotrich ciliates [2, 5, 8, 9, 11, 16, 18]. Therefore, the presence of ectocyst precursors is one of the indicators of encysting cells in these ciliates. © 1992 by Gustav Fischer Verlag, Stuttgart
In the present study, an early phase of encystment of the stichotrich ciliate, Histriculus cavicola (late stage 1 and stage 2 of Matsusaka, [11]), is subdivided into 5 stages determined by the extent of resorptions of UM and AZM observable with a phase-contrast mciroscope. The following 2 problems are then examined: 1) whether the cells in each stage of encystment continue encysting after their transfer in different environmental conditions (different media, different temperatures, and presence or absence of food organisms) and 2) whether the cells in each stage contain ectocyst precursors.
Material and Methods The organism used in the present study was the KM-1 strain of the stichotrich ciliate, Histriculus cavicola. The ciliates were cultivated at 25°C in 9 ern (diameter) Petri dishes containing 30 ml of 1: 1 mixture of 0.1 % (w/v) lettuce extract and 0.1 % (w/v) wheat extract (culture medium, CM), into which Chlorogonium elongatum was introduced as a food source as described earlier [14]. In this culture condition, the ciliates attained a stationary phase of growth within 3-4 days after inoculation. 0932-4739/92/0028-0051 $3.50/0
52 . T. Nakamura and T. Matsusaka Encystment was induced by washing the cells three times with 10 times concentrated Osterhout's balanced salt solution (10 X OS) by low speed centrifugation (about 50 g) while they were in the stationary phase of growth. Then the cells were suspended in 10 X OS and maintained in 6 cm (diameter) Petri dishes at 20°C [10]. When the population is observed every 15 min after induction of encystment, synchronous encystment ensues and almost all cells transform into cysts within 5 h [10]. When observations were made less frequently, however, the synchrony of encystment was poor even after the same treatment for induction of encystment and encystment of almost all cells required ca. 8 h. To determine the stages of encystment, a single cell was isolated from the encysting population between 3 and 7 h after induction of encystment. The cell was kept in a hanging drop under a cover
slip and observed with a phase-contrast microscope using a X 40 objective lens. Subdivision of an early phase of encystment (late stage 1 and stage 2, Matsusaka, [11]) was made on the basis of the extent of resorptions of UM and AZM. After determination of the encystment stage, the cell was transferred in a well of a 96-well microplate containing 100 ftl of normal Osterhout's balanced salt solution (1 X 05),10 X OS, or CM, and maintained at 20 or 25°C. To determine whether the presence or the absence of food organisms affect encystment, C. elongatum was introduced into some wells. The encystment of each cell was examined at 2, 4, 6, and 24 h after transfer. About 50 to 150 cells were examined for each determination. The cells in each stage were fixed in a 1 : 1 mixture of 5% (w/v) glutaraldehyde in 0.05 M cacodylate buffer (pH 7.3) and 2% (w/v) aqueous OS04 at room temperature for 15 min. They were
Fig. 1. Phase-contrast photomicrographs (a-e) of encysting Histriculus cavicola. The anterior parts were line drawn (a' -e') to show the criteria of each stage. a and a': A cell in stage A, showing no change in ciliary organelles (arrow head, UM; arrow, AZM). band b': A cell in stage B, showing resorption of the anterior part of the UM (arrow head). Arrow indicates AZM. c and c': A cell in stage C, showing unresorbed short posterior UM (arrow head) and beginning of resorption of the AZM from its posterior end (arrow). d and d': A cell in stage D, showing absence ofUM (arrow head) and resorption of a few membranelles of the posterior part of AZM (arrow). e and e': A cell in stage E, showing absence of UM (arrow head) and the resorption of membranelJes of AZM extending to the level of the anterior end of left marginal cirri (arrow). Bar = 50 urn,
Reversibility of Ciliate Encystment . 53 then dehydrated thro ugh an etha nol series and embedded in Qu erol 65 1. Serial sections of a whole cell were made with a Reichert -Jun g Ultracut E ultram icroto me using a diamond knife; the sections were observed with a J EM 100 C UEOL) electron microscope operated at 80 kY.
were presented (Fig. 1). However, it was not difficult to differentiate each stage in the living cells because ciliary movements could be seen. Reversibility of Encys tme nt
Results Subdivision of Early Stages of Encys tment
The late stage 1 and stage 2 of encystment recognized earlier [11] were subdivided into the following 5 stages. Stage A (Fig. l a, a '): this stage corre spon ds to late stage 1 of the earlier study [11]. Cells of this stage occurred in the popu lation 3 h after indu ction of encystment. It was determined by low power observation with a light microscope that the cells in this stage did not contain distinct food vacuoles or cytoplasmic granules (which were commonly observable in the troph ozoites). Resorption of feeding organelles did not begin, however, and the disposition of the ciliary organelles was the same as in the trophic form. Stage B (Fig. 1b, b' ): this stage corre spond s to the intermediate stage between stages 1 and 2 of the earlier study [11]. A part of the UM was resorbed from its anterior end but the AZM remained intact. Stage C (Fig. 1c, c' ): this stage also corresponds to the intermediate stage between stages 1 and 2. Onl y the posteriormost part of the UM remained unr esorbed and resorp tion of the AZM began from its posterior end. Stage D (Fig. 1d, d'): th is stage corresponds to mid-stage 2 of the earlier study [11]. Resorpti on of UM was completed and a few membranelles were resorb ed from the posterior end of AZM. Stage E (Fig. Le, e') : this stage corresponds to late stage 2. The membranelles of the AZM were resorbe d from its posterior end to the level of the anterior end of the left marginal cirral row . Duri ng stages A through E, no somatic cirri were resor bed. Because recognition of each stage is somewhat difficult from the pre sent photomicrographs, line drawings of the anterior part of encysting cells
Th e encystment of individual cells in each stage was exa mined after transfer of one cell into each well of a 96-well microp late conta ining different media, with or witho ut food organisms. The results at 24 h after transfer were tabulated and expressed as percentages of encysted cells (Ta ble 1). Stage A: The fate of the cells in this stage depended essentially upon the presence or the absence of food orga nisms, regardless of the kinds of media and temperatures maintained. When the cells were transferred into salt solutions with food organ isms or into CM with or without food, around 80% or more of the cells did not encyst, irrespective of the maintaining temperatures. There were, however, two exceptions; 1) the case transferred in 1 X OS with food and maintained at 25 °C (66.7% of the cells did not encyst), and 2) the case transferred in CM without food at 25 °C (47% of the cells did no t encyst). When the cells in this stage were transferred in salt solutions without food s, on the cont rary, more than 90% of cells encysted, irrespective of the maintaining temper ature . The only exception was the case tr ansferred into 10 x OS and maintained at 20 °C (18.5% of cells did not encyst), in spite of the same conditions to indu ce encystment. Stage B: The fate of the cells in this stage depended essentially upon the kinds of media and temper ature s into which the cells were tran sferred . When the cells were transferred into CM with or with out food and maintained at 20 °C, 70- 80% of the cells did not encyst. Conversely, 70-80% of the cells encysted at 25 °C even after transfer into the same medium. When the tran sfer was mad e into salt solutions, encystment ra tes were generally high, especially in the case maintained witho ut food (100% ). Even in the presence of food, more than 90% of cells
Table 1. Percentages of encysted cells Solution 10 X 10 X 1X I X CM CM 10 x 10 X 1X 1X CM CM
OS OS OS OS OS OS OS OS
Conditio ns Temp. (0C) Food 20 25 20 25 20 25 20 25 20 25 20 25
+ + + + + +
A
B
81.5 92.6 91.1 92.6 10.4 53.0 20.3 12.9 4.7 33.3
100 100 100 100 32.9 76.1 92.5 90.7 63.2 93.6 19.4 66.7
a
16.6
Stages of encystment C 100 100 100 100 48.1 89.7 100 100 94.4 95.8 38.2 78.4
D
E
100 100 100 100 80.0 100 100 100 95.2 96 .8 79.0 100
100 100 100 100 100 100 100 100 100 100 100 100
Cells in each stage of encystment were transferred individually in wells of a 96 -well microp late containing 1 X OS, 10 X OS, or CM with (+) or without (-) food orga nisms (Ch!orogonium e!ongatum), and were main tained at 20 or 25 "C. Percenta ges of encysted cells were determined at 24 h after transfer. Abou t 50 to 150 cells were coun ted for each determination.
54 . T. Nakamura and T. Matsusaka
encysted in the salt solutions. The case transferred i~to 1 X OS and maintained at 20°C was the only exception (36.8% of the cells did not encyst). Stage C: Most cells in this stage encysted after transfer into salt solutions, regardless of the presence or the absence of food organisms and of the temperatures maintained. When the transfer was made into CM, however, the encystment was dependent mostly upon temperatures, irrespective of the presence or the absence of food; 80-90% of the cells encysted at 25°C, but 50 to 60% of the cells did not encyst at 20 "C. Stage D: Almost all cells encysted after transfer into any medium, regardless of the presence or absence of food and of the temperatures. However, when the cells were transferred into CM and maintained at 20°C, about 20% of the cells still did not encyst. Stage E: All the cells encysted wit??ut except.ion after transfer into all environmental conditions examined, The cells which failed to encyst in the medium without food usually did not divide, but rarely divided only once within 24 h after transfer. When the cells were transferred into medium containing food organisms, the cells divided 1-3 times within 24 h after transfer. Under most conditions examined, cells encysted more readily at 25°C than at 20°e. Examination of Ectocyst Precursors
Within the cytoplasm of stage B cells, the presence of small bodies (ca. 1 urn long and ca. 0.3 urn wide), which contained stacks of thin, electron dense plates, was demonstrated electron microscopically (Fig. 2). These bodies showed the same ultrastructural characteristics as the ectocyst precursors described earlier [2, 5, 8, 9, 11, 16,18]. The ectocyst precursors were identified in 4 out of 10 cells in stage A and in all cells in stages B to E (Table 2).
Fig. 2. A part of the cytoplasm of a stage B cell showing ectocyst precursors (arrows). Bar = 0.5 urn.
Table 2. Presence of ectocyst precursors Stages
A
B
c
D
E
alb
4/10
11/11
8/8
5/5
4/4
a: Numbers of cells containing ectocyst precursors. b: Numbers of cells observed.
Discussion Almost all cells encysted within 8 h after induction of encystment in 10 X OS at 20°C, unless the .encysting population was disturbed. However, wh~n c~lls m stageoA were transferred into 10 X OS and maintained at 20 C without food, about 20% of the cells did not encyst within 24 h. This result may be caused by the disturbance of the process by the transfer. The encystment rates after transfer of the cells were generally higher at 25°C than at 20 °C (Table 1). The results may indicate acceleration of encystment at higher temperatures once the process had started, supporting the earlier reports that encystment of Laurentiella acuminata [4] and Oxytricha fallax [6] proceeds faster at higher temperatures. When environmental conditions were experimentally changed, some encysting cells did not comple~e encystment (Table 1). It has been shown [6] that the antenor fragments of encysting cells which possess UM and AZM as regenerated ciliary organelles do not encyst, whereas the encystment of the posterior fragments which have neither UM nor AZM is accelerated. In the present experiments, 50-60% of stage C cells and about 20% of stage D cells did not encyst at 20°C after transfer into CM. All stage E cells, however, encysted after any treatment to which they were subjected in the present study. In stage C, the UM, though only a part, remained unresorbed and the resorption of the AZM just began. The resorption of UM was completed in both stages D and E but that of the AZM was slightly more advanced in stage E cells than in stage D cells. These findings suggest that the retention of UM and AZM inhibits encystment and that the inhibition may result from the retention of feeding ability of the cells. If this is true, the presence of an almost intact AZM supports feeding to some extent even in the absence of UM, or the cells regenerated UM and AZM before their resumption of feeding, but it is not clear. This supposition supports the earlier result [6], showing inhibition of the encystment of the anterior half. Acceleration of the encystment of the posterior half [6] can be explained by supposing the presence of an "encystment promoting factor" in the posterior part of the cell. This idea, however, may not be true because in the present study whole cells were used and lower encystment rates resulted from the media containing food organisms. When the cells were transferred in CM, lower encystment rates generally resulted. In these cases, it may be that proliferation of bacteria occurred in the CM and the bacteria may have served as a food source. Transcription and translation are known to be involved in the encystment of several species of ciliates [4, 11, 13, 15, 19]. In Histriculus cavicola, protein is one of the
Reversibility of Ciliate Encystment . 55
components of the ectocyst [12] and cycloheximide blocks the formation of ectocyst precursors and encystment [11]. Th e present experiments indicate that about half of the cells in stage A and all cells in stages B to E contain ectocyst precursors in their cytoplasm (Table 2), and that some of these encysting cells do not encyst after transfer into different media, especially those containing food organisms (Ta ble 1). This suggests that structural genes for the ectocyst precursors must be expressed at least by late stage A, but the expression of the genes does not necessarily mean the continuation of the encystment process. This is consistent with the earlier findings [13] that actinomycin D inhibits encystment of the same species when the drug is applied before mid-stage 1, because stage A corresponds to late stage 1 of the earlier study [11]. Stage E cells encysted without exception after any treatment in the present exper iments, indicating that stage E cells are determined to encyst. These results suggest that major regulating genes for encystment may be expressed at least by two steps and that the second gene expre ssion may be decisive for encystment. Acknowledgement The authors would like to express their sincere thanks to Prof. John F. Tibb s (Division of Biological Sciences, University of Montana, USA) for his kind help in editing the manu script.
References Corliss J. O. and Esser S. C. (1974): Comments on the role of the cyst in the life cycle and survival of free-living protozoa. Tr ans. Amer. Micros . Soc., 93, 578 -593. 2 Grimes G. W. (1973): Differentiation during encystment and excystment in Oxytricha [allax, J. Protozool., 20, 92-104. 3 Gutierrez J. c., Martin-Gonzalez A. and Matsusaka T. (1990): Toward a generalized model of encystment (cryptobiosis) in ciliates: a review and hypothesis. BioSystems, 24, 17-24. 4 Gutierrez J. c., Torres A. and Perez-Silva J. (1981): Excystment cortical morphogenesis and nuclear processes during encystment and excystment in Lau rentiel/a acuminata (H ypotr ichida, Oxytrichidae). Acta Prorozool., 20, 145-152.
5 Guti errez J. c., Torres A. and Perez-Silva J. (1983): Structure of the cyst wall precursors and kinetics of their appearance durin g the encystment of Laure ntiel/a acum inata (Hypotrichida, Oxytrichidae). J. Protozool., 30, 226-233. 6 Hashimoto K. (1962): Relation ships between feeding organelles and encystment in Oxytri cha (aI/ax Stein. J. Prot ozool., 9, 161-169. 7 Holt P. A. and Chapman G. B. (1971): The fine stru ctur e of the cyst wall of the ciliated protozoon Didinium nasutum. J. Protozool., 18, 604- 614. 8 Jareiio M. A. (1985): Etude ultr astru cturale de l'enk ystement et du dekystement chez Onycho dro mu s acum inatus (Ciliata, Hypot richida). Protist ologica, 21, 313- 321. 9 Matsusaka T. (1976): An ultras tructural study of encystment in the hypotrichous ciliate Pleurotr icha sp. Kumamoto J. Sci., Biol., 13, 13-26. 10 Matsusaka T. (1977): Indu ction of synchronous encystment in a hypotrichous ciliate, H istriculus sp. Exp. Cell Res., 110 , 459-462. 11 Matsusaka T. (1979): Effect of cycloheximide on the encystment and ultrastructure of the ciliate, Histriculus. J. Proto zool., 26, 619- 625. 12 Matsusaka T. and Hon go F. (1984): Cytochemical and electrophoretic studies on the cyst wa ll of a ciliate, H istriculu s muscorum Kahl. J. Protozool., 3 1, 471-475. 13 Matsusaka T. and Kimura S. (1981): Changes in macronuclear ultra structure dur ing encystment in a ciliate, Histriculus muscorum. Kumamoto J. Sci., Biol., 15, 49-58. 14 Nakamura T. and Matsusaka T. (1991): Effects of cyst age on excystment of the ciliate, Hist riculu s cavicola (Kahl, 1935) (Ciliophora: Stichotri chia). Europ. J. Protisto!., 27, 375-38 0. 15 Ruthm ann A. and Kuck A. (1985): Form ation of the cyst wall of the ciliate Co lpoda steinii. J. Protozool. , 31, 677-682. 16 Verni F., Rosati G. and Ricci N. (1984): The cyst of Oxytricha bi(aria (Ciliata Hypotri chida). II. The ultra structure. Prot istologica, 20, 87-95 . 17 Wagtendonk W. J. van (1955): Encystment and excystment of pro tozoa. In: Hutner S. H. and Lwoff A. (eds.): Biochemistry and physiology of protozoa, vol. II, pp. 85-90. Academic Press, New York. 18 Walker G. K., Maugel T. K. and Good D. (1980): Encystment and excystment in hypotrich ciliates. I. Gastrostyla steinii. Protistologica, 16, 511-524. 19 Yonezawa F. (1985): Effect of actinomycin D, RNas e and protein synthesis inhibitors on encystment in Eupl otes encysticus (Ciliophora). J. Sci. Hiro shima Univ., Ser. B., Div. 1., 32, 73-82.
Key words: Ciliate - Reversibility - Encystment - Histriculus Tadao Ma tsusaka , Department of Biological Science, Faculty of Science, Kumamoto University, Kumamoto 860, Japan