effect of cycloheximide on the encystment and ultrastructure of the ciliate, histriculus

7
PURIFIED EXOERYTHROCYTIC MEROZOITES 619 from day to day and is reflected in the wide range of recovery of each ionic strength. Buffers of different ionic strengths were tested in pairs. Parasites from 6 flasks were pooled, divided into 2 equal parts and applied to 2 columns of different ionic strength under study. As ionic strength increased so did recov- ery. Even though the range of recoveries from various ionic strengths in Table 1 do not appear different, when values of pairs tested on the same day are compared, the differences become clearly apparent. For example, in one experiment re- covery at ionic strength of 0.220 it was 36% and at 0.250, 51%. Comparison of ionic strengths 0.250 with 0.280 gave recoveries of 46% and 56%, respectively, comparison of 0.280 with 0.320 gave recoveries of 45% and 62%, respectively, and those of 0.320 with 0.350, 48% and 58% respectively. Adequate elution and purity of merozoites was obtained at ionic strength of 0.22 as judged by electron microscopy (Fig. 2). At the higher ionic strengths tested, recovery was much greater, but so was the degree of contamination with host cells (Fig. 3). The purity of a sample of merozoites eluted at ionic strength of 0.22 was further confirmed by PAGE (Fig. 4). Major bands present in the host cell sample (arrows in Fig. 4) are absent in the mero- zoite sample, indicating that most host cell material was re- tained on the column. The elution profile of merozoites in one such experiment is shown in Fig. 5. Most of the sample eluted in the first 9 ml and tapered off by 20 ml. For subsequent ex- periments 2 0 4 eluate was collected. The procedure does not separate degenerated from viable merozoites and eluates reflect the quality of parasite samples applied to the column. In pre- viously published investigations, parasites have been allowed to accumulate for 2 days before collection. In our experience, degeneration of merozoites is considerably increased if samples are allowed to accumulate in culture medium for more than a 24-h period. From 6 of 150-cm square flasks of heavily parasit- ized almost confluent monolayers, 25- 100 million merozoites can be recovered. Our findings now allow biochemical and immunologic studies with exoerythrocytic merozoites of P. lophurae. LITERATURE CITED 1. Aikawa M. 1971. Plasmodium: the fine structure of malarial par- asites. Exp. Parasitol. 30, 284-320. 2. Beaudoin RL, Strome CPA. 1973. Plasmodium lophurae: the ultrastructure of the exoerythrocytic stages. Exp. Parasitol. 34, 3 13- 36. 3. Davis AG, Huff CG, Palmer TT. 1966. Procedures for maximum production of exoerythrocytic stages of Plasmodium fallax in tissue culture. Exp. Parasifol. 19, 1-8. 4. Graham HA, Stauber LA, Palczuk NC, Barnes WD. 1973. Im- munity to exoerythrocytic forms of malaria. 11. Passive transfer of im- munity to exoerythrocytic forms. Exp. Parasitol. 34, 372-81. 5. Holbrook TW, Palczuk NC, Stauber LA. 1974. Immunity to exo- erythrocytic forms of Plasmodium fallax. J. Parasitol. 60, 348-54. 6. Huff CG. 1969. Exoerythrocytic stages of avian and reptilian malarial parasites. Exp. Parasitol. 24, 383-42 1. 7. Kilejian A. 1974. A unique histidine-rich polypeptide from the malarial parasite, Plasmodium lophurae. J. Bid. Chem. 249, 4650-5. 8. Laemmli UK. 1970. Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature 227, 680-5. 9. Mack SR, Vanderberg JP, Nawrot R. 1978. Column separation of Plasmodium berghei sporozoites. J. Parasitol. 64, 166-8. 10. Trosper JH. 1975. The effects of exposure to the mosquito he- mocoel on exoerythrocytic Plasmodium fallax and Plasmodium lophu- rae. Dissert. Abst. Int. 35B, 4929-30. J. Prorozool., 26(4), 1919, pp. 619425 0 1979 by the Society of Protozoologists Effect of Cycloheximide on the Encystment and Ultrastructure of the Ciliate, Histriculus TADAO MATSUSAKA Department of Biology, Faculty of Science, Kumamoto University, Kumamoto 860, Japan SYNOPSIS. The effect of cycloheximide on the experimentally induced synchronous encystment of a hypotrich ciliate, Hisfriculus, was examined and the cytoplasmic ultrastructures of the control and the inhibitor-treated organisms were compared. Encystment was divided into the following 5 stages based on observations of living cells: stage I, cells with brown cytoplasm; stage 2, cells with smooth, transparent cytoplasm; stage 3, spindle-shaped cells; stage 4, spherical cells without cyst walls; and stage 5, young cysts with cyst walls. When cycloheximide treatment was preceded by stage 2, encystment and transformation into the next stages were totally blocked, whereas even in the presence of the inhibitor, stage 4 and stage 5 cells encysted normally, and stage 3 cells transformed into stage 4 although they did not form cyst walls. On the basis of ultrastructural studies it is suggested that the formation and excretion of ectocyst precursors are severely inhibited by cycloheximide and that polysome formation, active in stages I and 2, might be almost finished by stage 3. The role of lysosomal enzymes in the early stages of encystment are discussed. Index Key Words: Histriculus; encystment; cytodifferentiation; cycloheximide; polysomes; protein synthesis. ROTOZOAN encystment has been considered a good and P rather simple model system for the study of cytodifferen- tiation, because the process leads to only one type of cell, the dormant cyst (12). Since the establishment of the technic for inducing synchronous encystment in Acunthamoebn castellanii (12), biochemical information has been accumulated on amebic encystment (10, 11, 19, 21-26). Several of these reports have indicated that this process is accompanied by RNA and protein synthesis (1 1, 19, 24-26). Ciliate encystment is also an attractive model system for studies on cytodifferentiation, and may be more suitable than the amebic system, especially for compar- ative studies between structure and biochemistry, since the cil- iate system contains several morphologically distinct stages. Only a few publications, however, have appeared on the bio- chemical aspects of ciliate encystment (16-18), although there are numerous morphological papers (3, 8, 27; for review see Ref. 1). Furthermore, there have been no reports concerning protein synthesis in relation to ciliate encystment, although it was shown in several papers dealing with fine structure (3, 8, 27) that there were numerous polysomes which suggested that

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Page 1: Effect of Cycloheximide on the Encystment and Ultrastructure of the Ciliate, Histriculus

PURIFIED EXOERYTHROCYTIC MEROZOITES 619

from day to day and is reflected in the wide range of recovery of each ionic strength. Buffers of different ionic strengths were tested in pairs. Parasites from 6 flasks were pooled, divided into 2 equal parts and applied to 2 columns of different ionic strength under study. As ionic strength increased so did recov- ery. Even though the range of recoveries from various ionic strengths in Table 1 do not appear different, when values of pairs tested on the same day are compared, the differences become clearly apparent. For example, in one experiment re- covery at ionic strength of 0.220 it was 36% and at 0.250, 51%. Comparison of ionic strengths 0.250 with 0.280 gave recoveries of 46% and 56%, respectively, comparison of 0.280 with 0.320 gave recoveries of 45% and 62%, respectively, and those of 0.320 with 0.350, 48% and 58% respectively. Adequate elution and purity of merozoites was obtained at ionic strength of 0.22 as judged by electron microscopy (Fig. 2). At the higher ionic strengths tested, recovery was much greater, but so was the degree of contamination with host cells (Fig. 3). The purity of a sample of merozoites eluted at ionic strength of 0.22 was further confirmed by PAGE (Fig. 4). Major bands present in the host cell sample (arrows in Fig. 4) are absent in the mero- zoite sample, indicating that most host cell material was re- tained on the column. The elution profile of merozoites in one such experiment is shown in Fig. 5. Most of the sample eluted in the first 9 ml and tapered off by 20 ml. For subsequent ex- periments 2 0 4 eluate was collected. The procedure does not separate degenerated from viable merozoites and eluates reflect the quality of parasite samples applied to the column. In pre- viously published investigations, parasites have been allowed to accumulate for 2 days before collection. In our experience,

degeneration of merozoites is considerably increased if samples are allowed to accumulate in culture medium for more than a 24-h period. From 6 of 150-cm square flasks of heavily parasit- ized almost confluent monolayers, 25- 100 million merozoites can be recovered.

Our findings now allow biochemical and immunologic studies with exoerythrocytic merozoites of P. lophurae.

LITERATURE CITED 1. Aikawa M. 1971. Plasmodium: the fine structure of malarial par-

asites. Exp. Parasitol. 30, 284-320. 2. Beaudoin RL, Strome CPA. 1973. Plasmodium lophurae: the

ultrastructure of the exoerythrocytic stages. Exp. Parasitol. 34, 3 13- 36.

3. Davis AG, Huff CG, Palmer TT. 1966. Procedures for maximum production of exoerythrocytic stages of Plasmodium fallax in tissue culture. Exp. Parasifol. 19, 1-8.

4. Graham HA, Stauber LA, Palczuk NC, Barnes WD. 1973. Im- munity to exoerythrocytic forms of malaria. 11. Passive transfer of im- munity to exoerythrocytic forms. Exp. Parasitol. 34, 372-81.

5. Holbrook TW, Palczuk NC, Stauber LA. 1974. Immunity to exo- erythrocytic forms of Plasmodium fallax. J . Parasitol. 60, 348-54.

6. Huff CG. 1969. Exoerythrocytic stages of avian and reptilian malarial parasites. Exp. Parasitol. 24, 383-42 1.

7. Kilejian A. 1974. A unique histidine-rich polypeptide from the malarial parasite, Plasmodium lophurae. J . B i d . Chem. 249, 4650-5.

8. Laemmli UK. 1970. Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature 227, 680-5.

9. Mack SR, Vanderberg JP, Nawrot R. 1978. Column separation of Plasmodium berghei sporozoites. J. Parasitol. 64, 166-8.

10. Trosper JH. 1975. The effects of exposure to the mosquito he- mocoel on exoerythrocytic Plasmodium fallax and Plasmodium lophu- rae. Dissert. Abst. Int. 35B, 4929-30.

J . Prorozool., 26(4), 1919, pp. 619425 0 1979 by the Society of Protozoologists

Effect of Cycloheximide on the Encystment and Ultrastructure of the Ciliate, Histriculus

TADAO MATSUSAKA Department of Biology, Faculty of Science, Kumamoto University,

Kumamoto 860, Japan

SYNOPSIS. The effect of cycloheximide on the experimentally induced synchronous encystment of a hypotrich ciliate, Hisfriculus, was examined and the cytoplasmic ultrastructures of the control and the inhibitor-treated organisms were compared. Encystment was divided into the following 5 stages based on observations of living cells: stage I , cells with brown cytoplasm; stage 2, cells with smooth, transparent cytoplasm; stage 3, spindle-shaped cells; stage 4, spherical cells without cyst walls; and stage 5, young cysts with cyst walls. When cycloheximide treatment was preceded by stage 2, encystment and transformation into the next stages were totally blocked, whereas even in the presence of the inhibitor, stage 4 and stage 5 cells encysted normally, and stage 3 cells transformed into stage 4 although they did not form cyst walls. On the basis of ultrastructural studies it is suggested that the formation and excretion of ectocyst precursors are severely inhibited by cycloheximide and that polysome formation, active in stages I and 2, might be almost finished by stage 3. The role of lysosomal enzymes in the early stages of encystment are discussed.

Index Key Words: Histriculus; encystment; cytodifferentiation; cycloheximide; polysomes; protein synthesis.

ROTOZOAN encystment has been considered a good and P rather simple model system for the study of cytodifferen- tiation, because the process leads to only one type of cell, the dormant cyst (12). Since the establishment of the technic for inducing synchronous encystment in Acunthamoebn castellanii (12), biochemical information has been accumulated on amebic encystment (10, 11, 19, 21-26). Several of these reports have indicated that this process is accompanied by RNA and protein synthesis (1 1, 19, 24-26). Ciliate encystment is also an attractive model system for studies on cytodifferentiation, and may be

more suitable than the amebic system, especially for compar- ative studies between structure and biochemistry, since the cil- iate system contains several morphologically distinct stages. Only a few publications, however, have appeared on the bio- chemical aspects of ciliate encystment (16-18), although there are numerous morphological papers (3, 8, 27; for review see Ref. 1). Furthermore, there have been no reports concerning protein synthesis in relation to ciliate encystment, although it was shown in several papers dealing with fine structure (3, 8, 27) that there were numerous polysomes which suggested that

Page 2: Effect of Cycloheximide on the Encystment and Ultrastructure of the Ciliate, Histriculus

620 INHIBITION OF CILIATE ENCYSTMENT BY CYCLOHEXIMIDE

All figures are of Histriculus sp.

Figs. 1-5. [Photomicrographs of living organisms in sequential stages of encystment. ~ 3 4 0 . 1 1. Stationary phase. 2. Early (a) to late (d) stage 2 cells. 3. Stage 3. 4. Stage 4. 5. Stage 5 .

active protein synthesis might be taking place in the process. Recently, a method for inducing synchronous encystment in the hypotrichous ciliate. Histriculus, has been introduced (9). It makes biochemical studies on ciliate encystment possible. In the present paper the participation of protein synthesis in ciliate encystment is described.

MATERIALS AND METHODS The organism used in the present study was Histriculus sp.

stock I. which was used earlier (9). The species seemed to be H . muscorutn Kahl both in its habitat and in number and ar- rangement of the ventral cirri. It differed, however, from Kahl’s description in macronuclear number [2 in Kahl’s original de- scription (5); 4 in the organism employed in the present study]. Ciliates were fed Tetrahymena sp. in 0.1% (w/v) lettuce extract at 25 C. Synchronous encystment was induced by washing the stationary-phase organisms and suspending them in a lox Os- terhout’s medium (encysting medium, EM) at 20 C, as de- scribed earlier (9).

To examine whether protein synthesis is essential for en- cystment, the stationary-phase ciliates from a culture were di- vided into 2 groups: experimental and control. In the experi- mental group, cycloheximide (Nakarai Chemical Ltd.) was added to the EM at 20 C, to a final concentration of 50 pdml , at several stages of encystment. In the control group, the cil- iates were allowed to encyst synchronously in the EM at 20 C. The encystment rate was determined by differential cell counts, as described earlier (9), and the percentage of cells in each stage was calculated. For accurate determination of the encystment stages sensitive to cycloheximide, 30-40 ciliates of each stage were transferred into the EM containing 50 pglml cyclohex- imide, and the fate of these treated cells was followed.

To compare the ultrastructure of the control and the cyclo- heximide-treated ciliates, the organisms, except for the mature cysts, were fixed in a 1:3 mixture of 5% (v/v) glutaraldehyde in 0.05 M Na-cacodylate (pH 7.3), and 2% (wiv) OsO,. Mature cysts were fixed in 1% (v/v) glutaraldehyde in 0.05 M Na-cac- odylate (pH 7.3), followed by post-fixation in 2% (w/v) OsO,. Fixation was carried out at each stage in the control group, and at -5 h after the first wash with the EM in the experimental group. Fixed specimens were dehydrated through graded series of ethanols, embedded in Epon (3, and sectioned on a Porter- Blum MT-I ultramicrotome. The sections were picked up on Formvar-carbon-coated single hole grids, double-stained with

uranyl acetate and lead citrate (20), and examined in a JEM IOOC electron microscope operated at 80 kV.

RESULTS Encystment Stages

For description of experimental results, the definitions of the encystment stages given in an earlier report (9) were modified. The following 5 stages were chosen on the basis of observations of living cells. Stationary phase organisms are dorso-ventrally flattened ellipsoids, measuring -130 x SO x 20 pm (Fig. 1); they appear granulated and brownish in transmitted light. Four ellipsoidal macronuclei are oriented along the central longitu- dinal axis of the cells, and 2-6 spherical micronuclei are seen close to the macronuclei.

Stage 1 . This stage starts just after the first wash with the EM, lasts for -2 h at 20 C, and has the same morphologic characteristics as the stationary phase organisms. Stage 2. Many black granules appear in the cytoplasm (Fig. 2a). They gradually concentrate in the posterior end of the cell forming large black globules (Figs. 2b,c). The globules are expelled in several steps, resulting in a smooth transparent cytoplasm (Fig. 2d). It usually takes -3 h after the first wash with the EM for almost all ciliates to transform into the last phase of this stage. Stage 3 . The posterior end of the cell becomes pointed. This is followed by antero-posterior contraction and dorso-ventral swelling, the cell becoming spindle-shaped (Fig. 3). The organ- isms remain in this stage for a short time, usually 10-15 min. This stage is usually found in the synchronously encysting pop- ulation at -3-3.5 h after the first wash. Stage 4 . Cells are spher- ical, with a functional contractile vacuole (Fig. 4), but they have no cyst wall. They remain in this stage for only a few minutes. Stage 5 . This stage starts soon after the cell assumes spherical shape, and forms the cyst wall (Fig. 5 ) . In the earlier phase of this stage, a contractile vacuole is still functional, but subsequently it becomes inconspicuous and finally disappears. The young cysts appear first -3.5 h after the first wash with the EM, almost all cells transforming into this stage at -4-4.5 h after the first wash. Cells in the later phase of this stage are difficult to distinguish from mature cysts, which have a semi- transparent cytoplasm. The rather opaque cytoplasm of the young cysts is their only apparent difference from the mature cysts.

The mature cysts are spherical, measuring -50 pm in di-

Page 3: Effect of Cycloheximide on the Encystment and Ultrastructure of the Ciliate, Histriculus

INHIBITION OF CILIATE ENCYSTMENT BY CYCLOHEXIMIDE

TABLE I . EffPct of cycloheximide on the several encystment stages of Histriculus.

~

No. No. cells attaining stage* Stages cells treated treated 1 Late 2 3 4 5

1 37 37 0 0 0 0 2 (early) 43 0 43 0 0 0 2 (late) 39 0 39 0 0 0 3 43 0 0 3 40 0 4 40 0 0 0 0 40

* 5 h after treatment.

ameter. They have a cyst wall in which 3 layers, ecto-, meso-, and endocyst, can be distinguished by electron microscopy.

Fate of Cycloheximide Treated Cells The rate data of encystment are shown in Fig. 6. The time

scale of Fig. 6, however, was adjusted to the time of the first appearance of stage 2 cells (a) or the time of the addition of cycloheximide (b, c, d) of a typical experiment, since rate data of individual experiments showed -30-min variations. In Fig. 6, stages 4 and 5 are grouped as stage 5 because of the technical difficulty in differentiating them by low power light microscopy. Arrows in Fig. 6 indicate the times when cycloheximide was added, and 0 time indicates the end of the first wash with the EM. As shown in Fig. 6a, when cycloheximide was added at 0 time, all cells remained in stage 1 (brown cytoplasm). They neither encysted nor transformed into stage 2 . When population in which most cells were in late stage 1 and a few in early stage 2 were treated with the inhibitor, -50% of the ciliates remained in stage 1 and the remainder transformed into stage 2, but none encysted (Fig. 6b). When the treatment was at stage 2 , all cells remained in stage 2 and did not encyst (Fig. 6c). When cyclo- heximide was added at the phase in which -70% of the ciliates were in late stage 2 and -30% of the cells in stage 3 , -60% of the ciliates seemed to encyst normally but the remainder did not encyst remaining in stage 2 (Fig. 6d).

The foregoing results seem to indicate that cycloheximide acts as an encystment inhibitor when applied at early stages of encystment and that it does not affect the later stages of en- cystment. To time the cycloheximide-sensitive stages more pre- cisely, 30-40 cells of each stage were transferred into the EM, containing 50 pmg/ml cycloheximide, and their fate was fol- lowed. The results are summarized in Table 1 . It is evident that, in the presence of cycloheximide, stage 1 and 2 cells nei- ther encysted nor transformed into the following stages, al- though early stage 2 cells continued to differentiate until they reached the last phase of stage 2 (smooth, transparent cyto- plasm). Stage 3 cells with pointed posterior ends became spher- ical even in the presence of cycloheximide. Observation of the spherical cells revealed that they were in stage 4. Although some of the cells had an abnormally swollen ectocyst, almost all lacked a cyst wall and underwent cytolysis during the next 12 h. The inhibitory effect of cycloheximide on encystment was reversible, since the inhibitor-treated cells encysted normally after having been washed and resuspended in the fresh EM provided, if they were washed within 5 h of exposure to cy- cioheximide. The process, however, was no longer synchro- nous. Furthermore, the trophozoites emerged from the result- ing cysts multiplied and re-encysted normally. Vegetative cells also excysted from the cysts formed in the presence of the inhibitor administered at stages later than stage 4. They too proliferated and re-encysted normally.

2 3

03 4 TIME (HRS)

3 4

t

0 4 5 TIME (HRS)

62 I

Fig. 6. Encystment rates of control (-) and cycloheximide-treat- ed (-----) cells. Each point indicates mean value of 5 (a, b, d) or 6 (c) independent experiments with S.E. Arrows indicate the times at which the inhibitor was added to the system. 0 & ., stage 1 ; 0 & 0, stage 2 ; 0 & 0 , stage 3; (3 & 0 , stage 5.

Electron Microscope Observations Since the ultrastructure of Histriculus during encystment was

similar to that described previously for Oxytricha fallax (3), Styfonychia mytilus (27), and Pfeurotricha sp. (8), only some of the cytoplasmic events are detailed in the present paper. The cytoplasm of the normal encysting cells is shown in Figs. 7-10. In stages 1 to 3, most ribosomes were in the polysomal state (Figs. 7, 8). The polysomes began to disintegrate into mono- somes from stage 4 to early stage 5 (Fig. 9). In mature cysts, all ribosomes were monosomes (Fig. 10). Many electron-dense, spherical granules were located in the cytoplasmic vacuolar

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622 I N H I B I T I O N OF CILIATE ENCYSTMENT BY CYCLOHEXIMIDE

Page 5: Effect of Cycloheximide on the Encystment and Ultrastructure of the Ciliate, Histriculus

INHIBITION OF CILIATE ENCYSTMENT B Y CYCLOHEXIMIDE 623

Figs. 11-13. [Electronmicrographs of ultrathin sections through cycloheximide-treated organisms. x40,OOO.] 11. In the cytoplasm of a cell that was exposed to the inhibitor at 0 time, note the granules resembling bacteria (bg), ribosomal masses (rm), mitochondria (mt), and starch-like granules (st). Ribosomes not in the masses are monosomes. 12. Transparent cytoplasm of a cell treated with cycloheximide in stage 2. Except for the ribosomal mass (rm) in the center of the Figure, most ribosomes are in the monosomal state. An ectocyst precursor (ep) and a lipid droplet are also evident in this section. 13. Cortical cytoplasm of a cell exposed to the inhibitor in stage 3. Note the presence of numerous polysomes and cilia (lower right comer of the Figure). ep, ectocyst precursor.

t

Figs. 7-10. [Electronmicrographs of ultrathin sections through normally encysting ciliates. x40,OOO.] 7. Stage 1. Polysomes and electron- dense granules which resemble bacteria (bg) are evident in the cytoplasm. Note also mitochondria (mt) and a lipid droplet (Id). 8. Late stage 2. Polysomes and ectocyst precursors (ep) are seen in the cytoplasm which contains also a starch-like granule (st). 9. Stage 5. Disintegration of polysomes marks this stage. Id, Lipid droplet; st, starch-like granule. 10. Mature cyst. Most ribosomes are in the monosomal state.

Page 6: Effect of Cycloheximide on the Encystment and Ultrastructure of the Ciliate, Histriculus

624 INHIBITION OF CILIATE ENCYSTMENT BY CYCLOHEXIMIDE

regions of the stationary phase cells, stage 1 and early stage 2 (Fig. 7) (cytoplasm brown in color). These granules were con- centrated in the small vacuoles of early stage 2 cells. The cy- toplasm of the late stage 2 cells. with smooth, transparent cy- toplasm, stages 3-5. and the mature cysts did not contain the electron-dense spherical granules (Figs. 8- 10). In the late stage 2 cells, which had transparent cytoplasm, ectocyst precursors began to be formed in the cytoplasm (Fig. 8). They first ap- peared as small, spherical granules filled with electron-dense granular substances; thin disks subsequently appeared within the spherical granules (Fig. 8). The formation and the shape of the ectocyst precursors were similar to those described for 0.

fullax (3) and Pleurotricha sp. (8). Autolysosomes were first found in late stage I to stage 2 cells. Stage 3 and 4 cells con- tained many autolysosomes. Ciliary organelles began to be de- differentiated in late stage 3. and the mature cysts had neither these organelles nor kinetosomes.

The cytoplasm of organisms treated with cycloheximide is shown in Figs. 11-13. The cells in various stages of encystment were fixed at -5 h after the first wash, when most control cells were in stage 5. It is shown in Figs. 1 1 and 12 that when treat- ment with the inhibitor was performed at a stage earlier than 2 , the cytoplasm was rather translucent as compared with con- trols; most ribosomes were in the monosomal state, and poly- somes were rare. However, several masses of presumed ribo- somes and structures resembling vesicular, rough endoplasmic reticulum, were often present (Figs. I I , 12). In contrast, when stage 3 cells were subjected to the inhibitor, most ribosomes were in the polysomal state (Fig. 13), even though the cells were spherical and indistinguishable from normal stage 4 or- ganisms, in which polysomes begin to disintegrate into mono- somes (see Fig. 9). Electron-dense, spherical granules were found in the cytoplasmic vacuoles of the brown cells exposed to the inhibitor at 0 time or in stage 1 (Fig. 1 I ) . Ectocyst pre- cursors, observed in the late stage 2 cells, were not seen when the organisms were treated with cycloheximide at 0 time or at stage 1. When the inhibitor was added to late stage 2 cells, the ectocyst precursors were found in the cytoplasm, but they were fewer in number than in the non-treated control organisms and their formation was not observed. When cycloheximide was added to stage 3 ciliates, the cells came to contain many ec- tocyst precursors in the peripheral cytoplasm (Fig. 13); they had. however, no cyst wall. Many ciliary organelles were evi- dent, although the cell shape was indistinguishable from that of normal stage 4 cells. Autolysosomes, abundant in stage 3 and 4 cells, were not found in organisms treated with the inhibitor in stages earlier than stage 2.

DISCUSSION Protein synthesis has been demonstrated to be involved in

amebic encystment (1 1, 19, 26) and in microcyst differentiation of a cellular slime mold, Polysphondyliurn pallidurn (2, 13-15). In these organisms, however, morphological stages of cyst for- mation which require protein synthesis are not evident. From the present experiments it became clear that protein synthesis might be essential for progress to encystment in the stage 1 and 2 cells. Even in the presence of cycloheximide, however, en- cystment could proceed normally if the cells were in a stage later than stage 4. Furthermore, the transformation from stage 3 to 4 could occur, even though the cells did not form a cyst wall and died within 12 h. These results may indicate that the presently described encystment stages. which are based on cell structure, are also representative of the physiological states of the encysting cells.

As described for 0. fullrix (3) and Pleurotricha sp. (8), His- triculus ribosomes were in the polysomal state in the stage 1 to

3 cells, suggesting active protein synthesis. In contrast, after cycloheximide treatment at stages preceding 2, most ribosomes were in the monosomal state. Ectocyst precursors were found to be formed at stage 2 in organisms encysting normally, but were not found in the inhibitor-treated cells when the treatment was made at 0 time or at stage 1. This may suggest a close association between ectocyst precursor formation and protein synthesis, although it is not clear at present whether protein synthesis and ectocyst precursor formation are directly related, because the chemical nature of the ectocyst is not known. Ec- tocyst precursor excretion also seemed to require protein syn- thesis, because many of these precursors remained in the cy- toplasm of stage 4 cells which were pretreated with the inhibitor at stage 3 . In these cells, most ribosomes were in the polysomal state, although in stage 4 control cells the polysomes began to disintegrate into monosomes. Since cycloheximide has been known to inhibit the translocation of ribosomes ( 6 ) , resulting in the inhibition of formation and dissociation of polysomes, electronmicrographs included in this paper may picture the ef- fect of cycloheximide on ribosomes. Absence of polysomes in the cells treated with this inhibitor may indicate that they are formed normally in stages preceding stage 2 . The presence of numerous polysomes in stage 3 cells subjected to cyclohexi- mide may suggest that enough polysomes have been formed before the treatment to support the encystment process.

It was observed by light microscope that many black granules began to appear in early stage 2 organisms, followed by their concentration into large black globules, which were eventually expelled. This phenomenon has also been reported for 0. fallax (4) and Pleurotricha sp. (8). Many electron-dense spherical granules were observed by electron microscopy in stationary phase stage 1 and in early stage 2 cells, which have granular brownish cytoplasm. These granules were not found in the late stage 2 cells with smooth transparent cytoplasm or in cells in the more advanced stages of encystment. These granules were also frequently observed in the brown cells which were kept from transforming into stage 2 cells by exposure to cyclohex- imide. The foregoing observations suggest that the black gran- ules observed by light microscopy may correspond to the elec- tron-dense granules observed by electron microscopy. Accumulation and discharge of the black granules were not observed after the addition of cycloheximide at 0 time or at stage 1 . This observation suggests that the accumulation or discharge of the black granules is prevented by the inhibitor. Even in the presence of cycloheximide, however, discharge of the black globules occurred when the inhibitor was added to early stage 2 cells. Perhaps protein synthesis may be a prereq- uisite for the accumulation of the black granules but i s not required for the discharge of the globules.

It has been reported that lysosomal enzyme activities are elevated in the early phase of encystment of P . pallidurn and that this enhancement of enzymic activities can be blocked by cycloheximide treatment ( 2 , 13-15). In the present investiga- tion, autophagic activities were first detected from late stage 1 to early stage 2 cells, and autolysosomes, abundant in control stage 3 and stage 4 cells, were not found in the cells after cy- cloheximide treatment at 0 time or at stage I . This might suggest that lysosomal enzyme synthesis is inhibited by this compound and that the enzymic activities are related to the process of accumulation of the black granules, which began at early stage 2 . Furthermore, since in our system there was no exogenous nutrient supply, the lysosomal degradation of preexisting ma- terials might be a prerequisite for the subsequent formation of new macromolecules, and the inhibition of lysosomal enzyme synthesis could result in the inhibition of encystment, at least in its early stages.

Page 7: Effect of Cycloheximide on the Encystment and Ultrastructure of the Ciliate, Histriculus

INHJBITION OF CILIATE ENCYSTMENT BY CYCLOHEXIMIDE 625

ACKNOWLEDGEMENT The author wishes to thank Mr. S. Kimura for his aid in the

preparation of Figs. 1-5. The help of the reviewers and editors in putting this report into proper English is acknowledged with gratitude.

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CORRECTION to Sherman, I . W. &Jones, L. A. 1979. Plasmodium lophurue: membrane proteins of erythrocyte-free plasmodia and malaria-infected red cells. J . Protozool. 26,

On page 495, legends for Figures 9 and 10 should read: Analyses by SDS-PAGE (densitometric scans) of plasma membranes from normal duckling (Fig. 9) and human

stained (glycoprotein) material, - - - - -.

489-501.

(Fig. 10) erythrocytes. Coomassie brilliant blue-stained (protein) material, - ; PAS-