J. PRUTOZOOL. 15(4), 725-732 (1968).

Fine Structure of Colpoda steinii During Encystment and Excystment

JACK TIBBS

Dept. of Biochemistry, Univ. of Dundee, Scotland

SYNOPSIS. The encystment and excystment of Colpoda steinii was examined by electron microscopy. Cellular organelles including cilia are retained in the cyst without any fundamental alteration in structure. During encystmen’t, the cell becomes surrounded by 2 coats, the inner of which is the more substanial and regular and is about 1600 A or more thick. It is probably formed in the main from material contained in bodies which have no obvious structure and

HE protozoon Colpoda stein& exists as a motile ciliate and as a cyst. In the latter form the organism is

dormant, can exist without food, and respires a t a low rate. The encystment process is one in which cellular material is used to produce a thick protein coat rich in glutamic acid (1 1) and when conditions are favorable the dormant organ- ism digests this away and regains its free-swimming form. Besides providing information on the mechanisms by which this coat is formed and degraded, t& possibility that drastic cytoplasmic reorganisations accompany these trans- formations made an electron microscopic study of them appear well worth while.

MATERIALS AND METHODS

The isolation and general method of culture of the organism have been described (11). For the study of excystment, the organism was grown in petri dishes and cultures allowed to stand for several days after encystment. The cysts which adhered to the glass surface were rinsed several times with water before adding hay infusion. Excyst- ment was normally complete within 90 min a t 25 C and cultures at various stages of excystment were taken for electron microscopy. For studying encystment, cysts from culture were excysted and the motile cells concentrated by centrifugation and suspended in hay infusion. Debris was removed by centrifugation for 1 min at 50 g and the ciliates in the supernatant then sedimented (2 min at 5 0 0 g and washed several times with water. After making sure they were still motile, they were spread on petri dishes. Within 2 hr the cells stopped swimming and started to encyst. Cultures were taken a t various stages for electron microscopy.

Material was fixed with cold 4% glutaraldehyde in p H 7.3 0.1 M phosphate buffer for at least 2 hr (9). After thoro washing, it was then post-fixed with 1% osmium tetroxide in pH 7.3 veronal-acetate buffer (4) for 1 hr, washed and dehydrated thru increasing concen- trations of ethanol before embedding in Araldite (3) and sectioping. A diamond knife was used for sectioning, since with cysts and‘par- tially encysted cells glass knives became scored too readily to be‘of much use. Sections were mounted on uncoated copper grids and stained with 2% uranyl acetate followed by lead citrate (7) prior to microscopy and photography.

RESULTS

Ciliate pellicle. The complexity of the ciliate pellicle may be seen in Fig. 1 but the details of its structure are clearer in the excysting cell shown in Figs. 8, 9. A unit membrane (Fig. 9) with an overall width of about 80 A appears to overlie a series of flattened alveoli which form the boundary to a distinct layer of ectoplasm which is fairly free from

72 5

which may be seen in the cell during cyst formation. Discharging vacuoles containing sheet-like material, probably derived from ingested bacteria, are particularly visible during encystment but probably play no direct role in the formation of the main cyst coat. During excystment, this coat is eroded away and, when it becomes thin enough, the motile cell bursts its way out.

ribosomes. This is separated from the endoplasm by vesicles of endoplasmic reticulum. Similar structures have been described in the tetrahymenids ( 5 , 6, 13) and in Colpoda maupasi (8).

Surface layers of the cyst. In the mature cyst the pellicle of the organism appears to be retained intact but is exten- sively folded due to shrinkage of the cell on encystment (Fig. 2 ) . Outside the pellicle there are usually 2 additional coats, the inner of which is 1600 A or more thick. It touches only the “high spots” of the corngoluted pellicle and itself shows no folds. Presumably then it is laid down after the organism has expelled its excess water prior to encyst- ment. It shows no regular fine structure and appears to be composed of fibers rather than sheets of material at least in its early stages of formation (Fig. 3 ) . This coat does not appear to be completely homogeneous, however, for the outer region appears to stain more heavily (Fig. 2 ) and there is occasional evidence of a layered structure (Fig. 4). The outer coat is not always present and presumably plays no essential role in the encystment and excystment pro- cesses. I t is more voluminous than the inner coat, immedi- ately adjacent parts may vary considerably in thickness (Figs. 2, 5) and no fine structure is apparent. Associated with, and external to this layer, may be seen numerous vesicle3 which appear to have a double membrane wall (Fig. 5 ) and are presumably the myelin-like figures which occur in old food vacuoles and which have been excreted by the cell.

Cilia. During the cyst stage, the cilia of the organism appear still to be present and lie in grooves in the pellicle. Fig. 6 shows cilia in a cell which had been stationary and encysting for about 5 hr. After this time, the cyst coat is well formed and oxygen uptake has decreased to a level almost as low as that of the mature cyst ( 1 2 ) . I t appears unlikely that drastic cytoplasmic reorganization occurs after this time but electron micrographs of older cysts unambig- uously revealing the presence of unaltered cilia have not been obtained.

Mitochondria. The tubular system of the mitochondria of the motile cell is extensively developed and some of these tubules appear to be double (Fig. 1, inset). On encystment, the mitochondria show some modification in structure. Colpoda cysts respire a t only a very low rate (12), but in contrast to the mitochondria of the amoeba Hartmannella

726 FINE STRUCTURE OF C. steinii

castellani, which lose much of their internal organization when the organism encysts ( 2 ) , the mitochondria of Colpo- da retain their tubular system. The tubules become packed together and, in many cases, form a hexagonal array (Fig. 2 ) . Measurements made on the center-to-center spacing in 23 such mitochondria showed it to vary between 320 A and 520 A. The mitochondria appear to be surrounded by a sac of endoplasmic reticulum and associated ribosomes men

tho, at this stage, there is little obvious endoplasmic reticu- lum elsewhere in the cell (Fig. 7 ) . There is no suggestion that mitochondria are reduced in number in the cyst. With sections of cysts formed from cells encysting for the 1st time, a mitochondrion could be observed for every 1.23 (S.E.M. 0.12) p2 of section area whilst in the ciliates derived from these cysts by treatment with hay, infusion, the figure was 2.26 (S.E.M. 0.05). In sections of cysts

Fig. 1. Starved cell of Colpoda steinii, settling down to encyst, showing vacuolated cytoplasm and small vacuoles Y containing bac-

terial remnants and other membrane material. X24,OOO. Fig. 1 (in- set). Mitochondrion of Fig. 1 showing double tubules. X64,OOO.

FINE STRUCTURE OF C. steinii 72 7

Fig. 2. Three-day-old cyst encysted from culture showing the 2 Fig. 5 . The 2 cyst coats together with double membrane material cyst coats, the crenated pellicle, and hexagonal packing of tubules in one of the mitochondria. X48,OOO. Fig. 6. Cell 5 hours after settling down to encyst showing coats

Fig. 3. A cell, 2 hours after it had settled down to encyst. The newly-formed coat seems to be fibrous in nature. x80,ooO. Fig. 7. Cyst mitochondrion surrounded by sac of endoplasmic

Fig. 4. Isolated cyst coat showing layered nature. X24,000.

outside the external coat. X96,OOO.

well formed but cilia still present. X64,ooO.

reticulum. X 104,000.

728 FINE STRUCTURE OF C. steinii

formed from these ciliates, the figure was again down to 1.27 (S.E.M. 0.09). Thus, there are more mitochondria per unit area in the cyst than in the trophozoite, but since the ciliate swells on excystment this is to be expected.

Ribosomes and endoplasmic reticulum. Ribosomes are obvious at all stages of cellular development but endoplas- mic reticulum appears more obvious during excystment (Fig. 8) .

Food vacuoles. Rudzinska et al. (8) described the food vacuoles of C. maupasi; those of C. steinii are similar. In well-fed cells the cytoplasm is packed with food vacuoles containing partially digested bacteria. The limiting mem- branes of the bacterial cells appear to resist digestion and in vacuoles of the type shown in Fig. 10 may be seen as double structures of width about 70 A. The cytoplasm of the ingested bacteria has been digested away and the walls have collapsed to produce myelin-like figures. The content of these vacuoles, whilst still appearing as membranes may become further degraded (Fig. 11). Alternatively, since myelin-like figures may frequently be observed outside en- cysting cells (Figs. 2 , 5, 1 3 ) , some vacuoles may discharge at this stage of development. Bv disruption they may also give rise to small vacuoles such as those marked “v” on Fig. 1, and intracellular membrane material.

Formation o f the cyst coats. The processes by which these surface lavers are formed are not clear. As the cell settles down prior to encystment, it becomes extensively vacuolated and many of these vacuoles contain membranes (Figs. 1, 1 2 ) . The crucial point, not completely resolved. is whether these membranes are derived entirely from old food vacuoles or whether freshlv synthesized material is appearing. The more opaque of these vacuoles undoubtedlv contain bacterial remnants but there exists a large amount of membrane material in the cell which is not associated with vacuoles and could be arising from biosynthetic activi- tv ( m on Fig. 12) . Spherical bodies without any obvious ultrastructure, possibly analogous to muciqenic bodies or mucocvcts (1. 1 3 ) , may be seen for the 1st time at this stage (Fig. 1 2 ) . They are not found in the motile cell itself h i t become quite common as encystment proceeds (Fig. 14) .

The outer cyst coat appears and, since it is often seen alow with material similar in apnearance to that nresent in old vacuoles. it may in fact be formed from their contents (Fig. 1 3 ) . Whilst the main coat is beinn formed, mem- brane containing vacuoles are apparently discharging from the cell (Figs. 17, 18. 19, 20). As discussed later, thew vacuoles Drobably contain food residues rather than coat material. The photoqraphs provide no evidence as to wheth- er or not this discharge is taking place thru the cytoproct ( 10). The spherical bodies become more common as encyst- ment proceeds and may contain major coat components. They have not been observed discharging directly thru the pellicle of the organism but may be seen in close proximity to i t , apparently fusing with the surface laver of ectoplasm (Fig. 14). Several times they have been observed a5 com- ponents of apparently discharging vacuoles (Fig. 19).

Excystment. During excystment the inner cyst coat is

degraded away. As it becomes thinner, particulate material, which may represent the products of wall degradation, appears on the cell side of this wall (Fig. 15). The fact that the partially digested inner coat may split into more than one layer (Fig. 16) suggests that it is not homogeneous. The coats do not become completely degraded for when they become weak enough the organism bursts out and the remains then collapse.

Shrinkage and swelling of organism during encystment u,nd excystment. Measurements were made on photomicro- graphs of C. steinii in order to determine the size of the organism at various stages. Cysts, encysted from the culture in which they had grown, had a volume of 1990 (S.E.M. 90) p3. Only cysts containing single cells were included for these measurements. Ciliates prepared from this batch of cysts had a volume of 3040 (S.E.M. 180) p3. This figure was obtained by taking the maximum and minimum di- mensions of individual ciliates and assuminq that the shape of the ciliate was that of an oblate sDheroid. Cysts formed from these cells had a volume of 1250 (S.E.M. 90) p3.

DISCUSSION

Some difficulty was experienced in obtaininq good quality electron micrographs of cysts. Typically such photographs were rather ‘muddy’, probably due to the increased density of the organism in this form. On sectioning, cysts had a persistent tendency to tear out of the polymer in which they were embedded. This may have been due to lack of penetration or possibly lack of polymer continuity across the dense cyst coat. On those occasions when the orpankm remained in the Araldite, sectioning usually deformed the surface layers and made the clear identification of associ- ated cilia impossible. Nevertheless. cilia were clearlv ceen in 5 hr cysts at a stage when wall formation appears largely complete. Ribosomes and unaltered macro- and micro- nuclei are present in the cyst. Mitochondria are obvious but the tubular system appears to be more tightlv packed and organized than in the motile cell. Presumably this i q the result of the extrusion of water and is in line with the general shrinkage of the cell taking place on encystment.

The resting cell does not require the maiority of its mitochondria and cell material must be used to provide material for cyst coat synthesis. However, mitochondria are present in the cyst in numbers which exceed, per unit volume, those of the motile cell. Without making assump- tions about mitochondria1 size in cysts and ciliates, it is not possible to see how closely these figures may be correlated with cell volume changes but they give no support to the idea that mitochondria increase in number on excystment or decrease significantly during encystment. They strongly suggest that the mitochondria in a cyst can adequately account for the respiration of the ciliate derived from it. In accordance with the smaller size of cells in the 2nd encyst- ment compared with the 1st. some small reduction in mito- chondrial size or number no doubt does take place when a cell encysts and this is reflected in the similar distribution, per unit section area, of mitochondria in such cysts.

Fig. 8. Excysting cell showing obvious endoplasmic reticulum and

Fig. 9. Enlargement of part of Fig. 8 showing ciliate pellicle and

Fig. 10. A food vacuole with contents in an advanced stage of

Fig. 11. Food vacuole at a stige later than that shown in Fig. 10. details of pellicle. X32,OOO.

double nature of outside membrane. X128,OOO.

digestion. x80,OOO.

X 32,000.

930 FINE STRUCTURE OF C. steinii

A large proportion of cyst ribosomes do not appear to be associated with endoplasmic reticulum. Of those which are, some surround the mitochondria and it may be that these are concerned with the basal synthetic activity of the rest- ing cell. Probably associated with increased biosynthetic activity is the more obvious endoplasmic reticulum of the excysting cell.

During encystment vacuoles appear to eject their mem- brane contents from the cell with a fairly high frequency. Altho this ejection may be occurring thru the cytoproct (lo), this is uncertain and encysting cells may well be suitable subjects for studying vacuolar discharge. The mem- branes, which are probably food residues, might well be incorporated into the coat but several points indicate that

Fig. 12. Ciliate settling down prior to encystment and showing vacuolated cytoplasm with membrane material m, vacuole u contain- Fig. 15. Excysting cell. The main coat is reduced in thickness and ing bacterial remnants and structureless bodies s. X32,OOO. particulate material, which may represent the products of wll

Fig. 13. Cyst coats forming on encysting cell; the outer coat is closely associated with material similar in appearance to that present Fig. 16. The partially digested main coat of an excysting cell in old vacuoles. X40,OOO.

Fig. 14. Structureless bodies possibly analogous to mucocysts asso-

ciated with the pellicle of an encysting cell. X40,OOO.

digestion, may he seen between it and the ciliate pellicle. X40,OOO.

splitting into 2. X40,000.

FINE STRUCTURE OF C. steinii 73 1

Fig. 17. A vacuole containing membranes apparently being dis- X80,OOO. charged from an encysting cell. X80,ooO.

Fig. 18. As for Fig. 17. X40,ooO. Fig. 19. A vacuole apparently being discharged from an encysting

cell; 2 structureless bodies appear to be components of this vacuole.

Fig. 20. Section thru an encysting cell. The vacuole is probably discharging outside the plane of the section since membrane material is present between the partially formed cyst coat and the ciliate pellick. X25,ooO.

732 LIFE CYCLE OF Eimeria bila.mellata

they are not major coat components. The material which is packing together to form the main coat is more fibrous. Discharging vacuoles and unidentified cytoplasmic mem- branes are seen most frequently when the cell is settling down to encyst and less frequently a few hours after encyst- ment has started but whilst the coat is still quite thin. Structureless bodies, probably similar to mucocysts, may be voided as components of vacuoles or discharged directly thru the pellicle, and there is a good correlation in time between the appearance of these bodies in the cell and the formation of the cyst coat. The coat itself is of uniform thickness and of a rigid nature. This probably implies cross-linking or binding together of wall components outside the cell and such bonding could be spontaneous or en- zyme-catalyzed. It could be oxidative in nature or cross- linking may be prevented from taking place in the cell either by the physical separation of the components or by enzyme inhibition.

During excystment, the main cyst coat is eroded away. The secretion of a degradative enzyme may be takinq place as the result of an excystment trigger molecule passing thru the surface layers of the cell. Alternatively, the wall may contain degradative enzymes which are activated by trigger molecules.

Thanks are due to the Medical Research Council of Great Britain for some financial support and to Professor R. E. Coupland, until

recently of the Department of Anatomy in this University, for electron microscope facilities in that Department.

REFERENCES

1. Cheissin, E. M & Mosevich, T. N. 1962. An electron microscope study of ColMdium colpoda (Ciliata, Holotricha) . Arch. Protistenk. 106,~181-200. - 2. Griffiths, A. J., Lloyd, D., Roach, G. I. & Hughes, D. E. 1967.

Metabolic changes in Hartmanella castelhni during encystment. Bio- chem. J . 103, 21P.

methods. I . Biophys. Biochem. Cytol. 9,409-14. 3. Luft, J. H. 1961. Improvements in epoxy resin embedding

4. Palade. G. E. 1952. A studv of fixation for electron microscopy. J . Exp . Me&. 95, 285-97.

svstems of three tetrahvmenid ciliates. J. Protozool. 8, 75-89. 5. Pitelka, D. R. 1961. Fine structure of the silverline and fibrillar

6. - 1963. Electron microscopy of protozoa.. Pergamon Press,

7. Reynolds, E. S. 1963. The use of lead citrate at high DH as an electron-opaque stain in electron microscopy. J . Cell Biol. IS, 208-12. 8. Rudzinska, M. A., Jackson, G. J. & Tuffrau, M. 1966. The

fine structure of Colpodu maupasi with special emphasis on food vacuoles. J. Protozool. 13,440-59. 9. Sabatini, D. D.. Bensch, K. & Barrnett. R. J. 1963. Cytochemis-

try and electron microscopy. The preservation of cellular ultrastruc- ture and enzymatic activity by aldehyde fixation. J . Cell. BioZ. 17, 19-58. 10. Schneider, L. 1964. Electronenmikroskopische Untersuchungen

an den Ernahrungsorganellen von Paramecium. 11. Die Nahrungsvac- nolen und die Cytopyge. Z . Zellforsch. 62, 225-45. 11. Tibbs, J. 1966. The cyst wall of Colpoda steinii. A substance

rich in dutamic acid residues. Biochem. J. 98, 645-651. 12. Tibbs, J. & Marshall, B. J. Unpublished work. 13. Tokuyasu, R. & Scherbaum, 0. H. 1965. Ultrastructure of

mucocysts and pellicle of Tetrahymena Pyrijormis. J . Cell Biol. 27,

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J. PROTOZOOL. 15(4), 732-740 (1968).

Observations on the Life Cycle of Eimeria bilamellata Henry, 1932 in the Uinta Ground Squirrel Spermophilus armatus*

KENNETH S. TODD, JR.,t DATUS M. HAMMOND, and LARRY C. ANDERSON

Dept. of Zoology, Utah State Univ., Logan, Utah

SYNOPSIS. Oocysts of Eimeria bilamellata were found in Spcrmo- phiZus armatus, Utah and Wyoming; S. beecheyi, California; and S variegatus, Utah. Oocysts were not found in S. lateralis, S . richard- soni, S . cokmbianus, S . tridecemlimatus, or the white-tailed prairie dog, Cynomys leucurus. Experimental infections were established in S. armatus, S. variegatus, and S. lateralis, but not in S. tichardsoni, least chipmunks Eutamius minimus, laboratory rats Rottus norvegi- cus, or Mongolian gerbils Meriones unguicuhtus. After one experi- mental infection S. armatus and S. variegatus were immune to further infections. Spermophilus lateralis could be infected 3 or 4 times before the animals were immune. However, individuals of S. armatus in a natural population had more than one infection with E. bilomellata; probably infections must be of a certain level before immunity develops. When S. ormatus were inoculated with about 100,OOO oocysts, the animals usually died on the Yth day after incoculation.

E found previously (20, 21) that 2 species of Eimeria from the ground squirrel, Spermophilus (syn., Citel-

lus)armatus, had less host specificity and were less immuno- genic than is true of most coccidia. In studies of a 3rd species from this host, E. bilamellata Henry, 1932, we found that this species resembled the other 2 in host-

Oocysts were 33-37 by 25-31 p (mean 34.5 by 28.2 p ) . The oocyst wall was brown and composed of 2 layers. A distinct micropyle was present. Sporocysts were 18-23 by 9-12 p (mean 19.9 by 10.3 a). In experimental infections, the prepatent period was 10 days and the patent period 5-21 (mean 9) days. Schizonts were 1st seen 7 days after inoculation. They were located above the host cell nuclei of epithelial cells at the tips of the villi of the jejunum and ileum. One or more earlier generations of schizonts were thought to occur. but these were not observed. Gametogony took place in epithelial cells of the jejunum and ileum. Shortly after ,the merozoites entered the cells, the cells became enlarged and were displaced into the lamina propria. The microgametocytes were considerably larger than the macrogametes and contained a central residual body. Macrogametes had a peripheral eosinophilic layer as we11 as cytoplasmic granules; both apparently participated in formation of the oocyst wall.

specificity but was more immunogenic. The cytoplasmic granules of the macrogametes of E. bilamellata are of spe- cial interest. In the present paper we report out findings on

*Supported in part by a grant from the Utah State University Research Council and by NSF Grant GB 5667X.

t Present address: Dept. of Veterinary Pathology and Hygiene, College of Veterinary Medicine, Univ. of Illinois, Urbana, Illinois.