Fine-Structural Aspects of Macrogametogenesis in Eimeria magna * †

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GAMETOGENESIS IN Eimeria magna

ding methods. J . Biophys. Biochem. Cytol. ( J . Cell Biol.) 9, 40914. 18. MacMillan, W. G. 1970. Some aspects of the biology of Nematocystis magna Schmidt. Ph.D. Thesis, University of Glasgow, Scotland. 19. __ 1972. Structure and permeability of an interspecific cell junction. Parasitology, in press. 20. , Brocklesby, D. W. & Purnell, R. E. 1971. The fine structure of tick stages of Theileria parva. J . Parasit. 57, 1 1 28-9. 21. Miles, H. B. 1966. The contractile system of the acephaline gregarine Nematocystis magna : observations by electron microscope. Rev. Zber. Parasit. 26, 361-8. 22. _ _ 1968. The fine structure of the epicyte of the acephaline gregarines Monocystis lumbrici-olidi and Nematocystis magna : observations by electron microscope. Rev. Iber. Parasit. 28, 455-65. 23. Phillips, N. E. & MacKinnon, D. L. 1945. Observations on a monocystid gregarine Apolocystis elongata n. sp. in seminal vesicles of Eisenia foetida (Sav.) . Parasitology 36, 65-74. 24. Pitelka, D. R. 1963. Electron Microscopic Structure of Protozoa, Pergamon Press, Oxford. 25. Porter, J. F. 1897. Two new Gregarina. 1. Morph. 14, 1-20. 26. de Puytorac, P. 1957. L’argyrome chez les grCgarines Monocystinae. C . R . Assoc. Anat., 43rd Reunion, Lisbon, 1956, 694-706. 27. Rener. 1. F. 1967. The fine structure of the rrreearine Pyxinoides balani parasitic in the barnacle Balanus tintin&bYulum. J . Protozoal. 14, 488-97. 28. , Barnett. A. & Poger. M. P. 1967. Observation on an unusual. membrane complex found in gregarines parasitic in the barnacle Balanus tintinnabulum. J. Ultrastruct. Res. 18, 422-7. 29. Reynolds, E. S. 1963. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J . Cell Biol. 17, 208-13.

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30. Richardson, K. C., Jarret, L. & Finke, E. H. 1960. Embedding in epoxy resins for ultra-thin sectioning in electron microsCODY. Stain Techn. 35. 313-23. 31. Roots, B. I. 1955. The water relations of earthworms. J . Exp. Biol. 32, 766-74. 32. Roskin. G. & Levinson. L. B. 1929. Die Kontraktilen und die Skelettelemente der Protozoen. I. Der kontraktile und der Skelettapparat der Gregarinen (Monocystidae) . Arch. Protistenk. 66, 355-401. 33. Sabatini, D. D., Bensch, K. & Barnett, R. J. 1963. Cytochemistry and electron microscopy. The preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation. J . Cell Biol. 17, 19-58. 34. SchrCvel, J. & Vivier, E. 1966. Etude de l’ultrastructure et du r61e de la rCgion antCrieure (mucron et CpimCrite) de gr6garines parasites d’AnnClides polychttes. Protistologica 2, 17-28. 35. Stempak, J. G. & Ward, R. T. 1964. An improved staining method for electron microscopy. J . Cell Biol. 22, 697-9. 36. Vinckier, D. 1969. Organisation ultrastructurale corticale de quelques monocystidCes parasites du ver oligochete Lumbricus terrestris L. Protistologica 5, 505-17. 37. -& Vivier, E. 1968. Organisation ultrastructurale corticale de la grCgarine Monocystis herculea. C . R . Acad. Sci., Paris, 266, 1737-9. 38. Vivier, E. 1967. Contributions des recherches ultrastructurales A la connaissance du mCcanisme de la locomotion chez les GrCgarines. J . Micros. 6, 87a. 39. - 1968. L’organisation ultrastructurale corticale de la grCgarine Lecudina pellucida ; ses rapports avec l’alimentation et la locomotion. J . Protozool. 15, 230-46. 40. Warner, F. D. 1968. The fine structure of Rhynchocystis pilosa (Sporozoa, Eugregarinida). J . Protozool. 15, 59-73. 41. Watson, M. E. 1916. Studies on gregarines. Illinois Biol. Monogr. 2, 3-258. 42. Watters, C. D. 1962. Analysis of motility in a new species of gregarine. Biol. Bull. 123, 514. ‘

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J. PROTOZOOL. 2 0 ( 2 ) , 274-281 (1973).

Fine-Structural Aspects of Macrogametogenesis in Eimeria magnu* t CLARENCE A. SPEER,* DATUS M. HAMMOND, NABIL N. YOUSSEF and HARRY D. DANFORTH

Department

of

Zoology, Utah State University, Logan, Utah 8 0 2 1

SYNOPSIS. Macrogamonts in tissues from rabbits killed 5% days after inoculation with Eimeria magna oocysts were studied with the electron microscope. In young macrogamonts, parts of cytoplasm, sometimes including micronemes, were pinched off into the parasitophorous vacuole. I n all stages of development, small segments of the inner membrane complex were present beneath the limiting membrane. Micropores also were seen in all stages, and some apparently functional ones were present in mature macrogametes. Wall-forming bodies of Type I and Type I1 were observed in relatively early stages. The former were less numerous than the latter, which had a more compact appearance than in other species. Usually, several Golgi complexes were present and several Golgi adjuncts occurred in the vicinity of the nucleus in all stages of development. Microgametes were observed in the cytoplasm of host cells harboring immature macrogametes. Index Key Words: Eimeria magna; rabbits; macrogamonts ; electron microscopy.

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LTHOUGH several fine-structural studies of macrogametogenesis in Eimeria (12) have been made, a number of unresolved questions concerning this process remain. We report herein on the development of macrogametes in Eimeria magna. MATERIALS AND METHODS Infected tissues were obtained by scraping the mucosa of the lower 2/3 of the small intestine of 3- to 4-month-old rabbits which had been inoculated orally 5% days earlier with approximately 2 x 105 oocysts of Eimeria magna. These scrapings were placed in Karnovsky’s fixative ( 4 ) for 2-4 hr, rinsed in cacoThis investigation was supported in part by Research Grant AI-07488 from the NIAID, U.S. Public Health Service. 1 Paper No. 1299, Utah Agricultural Experiment Station. 3: Present address: Department of Histology, Dental Branch, University of Texas, Texas Medical Center, Houston 77025.

dylate buffer for 12 hr, and postfixed for 1 hr with 5.0% ( w / v ) osmium tetroxide. Tissues were washed in 2 changes of cacodylate buffer, partially dehydrated in a graded series of ethanol, and prestained with 1% ( w / v ) uranyl acetate and 1% ( w / v ) phosphotungstic acid in the 70% ethanol a t 4 C for 12 hr. They were further dehydrated in ethanol followed by 2 changes of propylene oxide, embedded in Epon, sectioned, placed on 200mesh copper grids, stained with lead citrate for 5 min, and examined in a Zeiss EM 9S2 electron microscope. RESULTS Young Macrogamonts Young macrogamonts were distinguishable from microgamonts by their relatively large nucleus and prominent nucleolus as well as a t least one large mitochondrion (Fig. 1 ) . In young microgamonts, the mitochondria were usually small and more nurner-

GAIWETCGENESIS I N Eimeria magna

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All figures are electronmicrographs of Eimeria magna macrogamonts fixed in Karnovsky’s fluid (4). Figs. 1-3. [Young macrogamonts. G , Golgi complex; M, mitochondrion; Nu, parasite nucleolus.] 1. Spherical mass of cytoplasm (arrow) is seen pinching off from the surface of the gamont lodged in a parasitophorous vacuole (Pv), which contains also the cytoplasmic bodies ( B ) . OB, osmiophilic body; Mp, micropore. X 24,000. 2. Peripheral segment of a gamont. Micronemes ( M n ) are seen within the spherical cytoplasm mass still attached to the parasite’s surface and in a cytoplasmic body ( B ) free in the parasitophorous vacuole. The inner membrane complex ( I m ) is seen in some of the cytoplasmic bodies and in a r e a of the parasite. x 26,500. 3. In the gamont lodged in the host cell note the wall-forming bodies of Types I and 11 (WfI, WfII). One of the latter is in a cisterna of the endoplasmic reticulum (parallel arrows). Ga, Golgi adjunct; Hn, host cell nucleus; NP, nuclear pore. x 16,500.

ous. Some mitochondria were greatly elongated, with a narrow region consisting chiefly of the limiting membranes. T h e earliest stages seen had several osmiophilic bodies (Fig. 1 ) Granular endoplasmic reticulum, Golgi complexes, and numerous free ribosomes were present in the cytoplasm. Usually, only a single unit membrane was present at the surface (Figs. 1, 3, 4 ) , but in some specimens, short segments of an underlying membrane complex were observed (Figs. 2, 5 ) . The parasitophorous vacuole usually

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had several spherical masses of cytoplasm, which were similar in appearance to gamont cytoplasm and limited by a single membrane, or had also an incomplete underlying membrane complex (Fig. 2 ) . Some of these bodies were seen pinching off from the surface of the parasite (Figs. 1, 2 ) ; a few had micronemes (Fig. 2 ) . Initially, the macrogainont nucleus had a prominent nucleolus and several clumps of chromatin, some of which were peripheral (Fig. l ) . In more advanced stages, little chromatin

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GAMETOGENESIS IN Eimeria magna Golgi-adjuncts were distributed randomly around the nucleus (Figs. 6, 8 ) . Portions of microgametes were observed in the cytoplasm of a host cell harboring an intermediate macrogamont (Fig. 8). These portions, located close to the parasitophorous vacuole around the macrogamont, included cross sections of several flagella lying free in the cytoplasm, as well as a flagellum and part of the body of a microgamete with the characteristic mitochondrion, apparently lodged in a vacuole.

was observed (Fig. 3 ) . Thick-walled vesicles similar to those termed “Golgi adjuncts” by Ogino & Yoneda ( 6 ) in Toxoplasma usually were located close to the nucleus (Fig. 3 ) . Occasionally, outpocketings of the nuclear envelope appeared to extend for a considerable distance into the cytoplasm (Fig. 4 ) and were associated with indentations of the cytoplasm into the nucleus. I n some intermediate macrogametes, such indentations appeared in certain sections as intranuclear masses of membrane-bound cytoplasm (Fig. 6). In other specimens, the nucleus had large, fingerlike outpocketings from the nucleus (Fig. 5 ) . Lipid inclusions and several small, electron dense wall-forming bodies also were present in this stage. The latter appeared to develop within cisternae of the endoplasmic reticulum (Fig. 3 ) . Such wall-forming bodies were found also to develop between the membranes of the nuclear envelope (Fig. 7 ) . In some specimens, a fine honeycomb-like substructure was visible in these wall-forming bodies, resembling that of Type I1 ( WfII) bodies of other species ( 1 2 ) . Less dense, smaller, peripherally-located bodies (Fig. 4),with a distinct limiting membrane, were similar to the wall-forming bodies of Type I of other species. A channel of rough endoplasmic reticulum or several of the channels were present immediately below the cell surface (Fig. 4). Typical micropores were seen (Fig. 1 ) .

Mature Macrogamonts Mature macrogamonts contained larger and more numerous amylopectin bodies and wall-forming bodies of both types than were present in earlier stages. Instead of the elongate mitochondrion found earlier, these macrogamonts had several smaller ones, with a relatively electron-dense matrix (Fig. 9 ) , usually in a peripheral location. Some mitochondria were dumbbell-shaped. Most of the canaliculi were located near the pellicle (Fig. 9 ) . Lipid bodies, several Golgi adjuncts, and peripherally-located Golgi complexes were observed. Some specimens had several micropores, some in close proximity to one another, and some apparently enlarged. Fusion of the WfII bodies evidently was occurring in some specimens, in which additional membranes were present beneath the outermost membrane in some areas (Figs. 10-12). These organisms might have represented early zygotes, with the beginning of oocyst wall formation. Relatively dense mitochondria were concentrated at the periphery of such stages. Lipid inchsions occupied a more interior location.

Intermediate Macrogamonts Intermediate macrogamonts were considered to be immature stages in which amylopectin could be seen (Fig. 6 ) . In such organisms, the wall-forming bodies, especially those of Type 11, were larger and more numerous than in the young macrogamonts. Also present at various places in the cytoplasm were the canaliculi, the so-called ‘canal system’ of endoplasmic reticulum (Fig. 6 ) , similar to those observed in E. tenella macrogamonts ( 1 1 ) . In some specimens, these canaliculi appeared to consist of a number of parallel membranes, interspersed with electron-transparent spaces (Fig. 6 ) . At 1 pole of the Golgi complex there were relatively large and somewhat flattened vesicles (Fig. 8 ) , which apparently gave rise to large vesicles containing particulate matter. Lipid bodies were associated with these latter vesicles, suggesting that they may be formed from the Golgi complex. As in an earlier stage, several Golgi complexes were found near the periphery of the gamont (Figs. 6, 8 ) . Usually, several mitochondria were present, some of which were unusually long (Fig. 8 ) ; others had a terminal loop (Fig. 7). In more advanced stages, amylopectin bodies were larger and more numerous than in earlier stages (Fig. 6 ) and were usually associated with endoplasmic reticulum or the surface of lipid bodies (Fig. 8). Several ~

DISCUSSION T h e earliest macrogamonts have micronemes, which are lost during the early stages of the development of the macrogamont. The pinching off of parts of cytoplasm into the parasitophorous vacuole by young macrogamonts suggests that this may be one method for getting rid of certain of the merozoite organelles, such as micronemes. Although we saw no inner membrane complex in some early macrogametes, it probably was present in some areas of all developmental stages; such areas could easily be missing in certain sections. Snigirevskaya (14) reported that early macrogamonts of E. intestinalis had a single limiting membrane, but the membrane was double in mature stages. I n the present study, we found apparently enlarged micropores in mature macrogametes, indicating that intake of nutrients may be accomplished by this method. According to Snigirevskaya (14) the micropores evidently were functional in immature macrogametes of E. intestinalis, but not in mature ones, in which ~~

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Figs. 4, 5. [Young macrogamonts in parasitophorous vacuoles (Pv). G, Golgi complex; Hn, host cell nucleus; L, lipid body; M, mitochondrion; Nu, parasite nucleolus; WfI and WfII, wall-forming bodies of Types I and 11.1 4. Outpocketing of the nuclear envelope (arrows) and the connection (parallel short arrows) between the granular and agranular endoplasmic reticulum (Er ) are evident in this section. X 22,750. 5. Finger-like protrusions (arrows) of the parasite nucleus should be noted. Ga, Golgi adjunct. x 20,930. Figs. 6-8. [Intermediate macrogamonts. A, amylopectin; Er, endoplasmic reticulum ; G, Golgi complex; Ga, Golgi adjunct ; Hn, host cell nucleus; L, lipid body; M, mitochondrion; N, parasite nucleus; Ne, nuclear envelope; Nu, parasite nucleolus; WfI and WfII, wall-forming bodies of Types I and 11.1 6. Cross section of cytoplasm indentation (arrow) into the nucleus is seen in the organism, which contains a variety of cytoplasmic inclusions. C, canaliculi. x 22,750. 7. Terminal loop (parallel arrows) of the mitochondrion and wall-forming Type I1 body (arrow), the latter within the perinuclear space, are evident in a portion of the gamont. Im, inner membrane complex. x 27,300. 8. In this section, note the relatively large vesicles (arrows) apparently arising from the Golgi complex ( G ) . Flagella ( F ) of the microgametes lie free in the host cell cytoplasm which contains also a part of a microgamete [with the mitochondrion (Mi) evidently enclosed in a vacuole.] X 13,286. Fig. 9. Nearly mature macrogamont; note especially the micropores ( M p ) and mitochondria ( M ) . A, amylopectin; Ga, Golgi adjunct; L, lipid body; N, parasite nucleus; WfI and WfII, wall-forming bodies. x 23,595. Fig. 10. Zygote, apparently soon after fertilization. I n this stage, with numerous amylopectin ( A ) and lipid ( L ) inclusions, the Type I1 wall-forming bodies (WfII) are in the process of fusion (arrow). Areas with segments of the inner membrane complex (parallel arrows), canaliculi ( C ) , mitochondria (M), and a micropore ( M p ) also are visible, Nu, parasite nucleolus; WfI, wall-forming bodies of Type I. x 13,350.

GAMETCMXNESIS I N Eimeria wiagna

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GABIETOGENESIS I N

Eimeria magna

G.wi:.TocmF.srs I N

Eimeria magna

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GAMETOGENESIS I N Eimeria magna

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Figs. 11, 12. Parts of zygotes, with numerous amylopectin inclusions ( A ) . The outer parasite membrane (Om) and the inner membrane complex (Im) are seen in Fig. 11, which includes also longitudinal sections of canaliculi ( C ) and discrete wall-forming bodies of Types I and I1 (WfI and WfII). Fusion of WfII bodies (arrows) is seen in Fig. 12. L, lipid body; M, mitochondrion; N, parasite nucleus. x 21,000. intake of nutrients was thought to occur by pinocytosis in association with invaginations of the surface, similar to those reported for E. auburnensis macrogametes ( 3 ) . Such invaginations were not observed in the macrogametes of E. magna. The wall-forming bodies in E . magna differ somewhat from those of other Eimeria species. The WfII bodies have smaller internal spaces than is typical for others species, such as E . perforans, in which these bodies have a loose, sponge-like structure ( 9 ) ; however, this difference may be associated with the fixatives and other technics used in preparation of the material for electronmicroscopic examination. The WfII bodies in E. magna resemble those of other species in that they occur in membranebound spaces connected with the endoplasmic reticulum or Golgi complex (12). The development of the WfII bodies in association with the nuclear envelope has not previously been reported. The WfI bodies resemble those of E. tenella, E. maxima, E. intestinalis, E. perjorans, and E. auburnensis in having a limiting membrane-like layer. They also resemble those of E. bouis and E. auburnensis in being smaller than the WfII bodies ( 1 2 ) . In all these species, the WfI bodies were reported to appear later than the WfII bodies, but in E. magna, the former were observed as early as were the latter. I n mature and immature macrogametes, the WfI bodies were much less numerous than the WfII bodies. Whereas segments of the inner membrane complex are retained in the macrogametes of E. magna, the gametocytes of Plasmodium spp. originate from merozoites in which the inner membrane and microtubules of the pellicle do not dedifferentiate ( 1 ) The occurrence of Golgi adjuncts in macrogamonts of different Eimeria has not been reported previously. They occur in the asexual stages of several species, such as E . alabamensis (8),E. magna ( 2 ) , E. callospermophili (7), Toxoplasma gondii ( 6 , 13)

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and Frenkelia sp. ( 5 ) . T h e function of the adjuncts is unknown, but they appear to be associated with formation of new merozoites in the asexual stages in which they occur. T h e finding of these organelles in macrogametes indicates that they occur over a greater range in the life cycle than had been thought previously. The finding of microgametes in the cytoplasm of host cells harboring apparently immature macrogamonts is of considerable interest, because little is known about fertilization in the Coccidia. The microgametes must enter and pass through the host cell to fertilize the macrogamete. Evidently, microgametes may invade the host cell before the macrogamete reaches maturity. Scholtyseck and Hammond (10) observed a microgamete within a macrogamete of E. bouis, and a number of microgametes were seen also in the parasitophorous vacuole and in the cytoplasm of the host cell containing the macrogamete. LITERATURE CITED 1. Aikawa, M., Huff, C. G . & Sprinz, H. 1969. Comparative fine structure study of the gametocytes of avian, reptilian, and mammalian malarial parasites. J . Ultrastruct. Res. 26, 316-31. 2. Danforth, H. D. & Hammond, D. M. 1972. Stages of merogony in multinucleate merozoites of Eimeria magna Perard, 1925. J . Protozoal. 19,454-7. 3. Hammond, D. M., Scholtyseck, E. & Chobotar, B. 1967. Fine structures associated with nutrition of the intracellular parasite Eimeria auburnensis. J . Protorool. 14, 678-83. 4. Karnovsky, M. J. 1965. A formaldehyde-glutaraldehyde fixative of a high osmolality for use in electron microscopy. J . Cell Biol. 27, 137A-8A. 5. Kepka, 0. & Scholtyseck, E. 1970. Weitere Untersuchungen der Feinstruktur von Frenkelia spec. (M-Organismus, Sporozoa) . Protistologica 6, 249-66. 6. Ogino, N. & Yoneda, C. 1966. The fine structure of reproducing Toxoplasma gondii. Parasitology 53, 643-9.

CILIARYRESORPTION I N Cyathodinium

7. Roberts, W. L., Hammond, D. M., Anderson, L. C. & Speer, C. A. 1970. Ultrastructural study of schizogony in Eimeria callospermophili. J . Protozool. 17, 584-92. 8. Sampson, J. R. & Hammond, D. M. 1972. Fine structural aspects of development of Eimeria alabamensis schizonts in cell cultures. J . Parasit. 58, 311-22. 9. Scholtyseck, E. 1962. Electron microscope studies on Eimeria perforans (Sporozoa). J . Protozool. 9, 407-14. 10. _ _ & Hammond, D. M. 1970. Electron microscope studies of macrogametes and fertilization in Eimeria bouis. Z . Parasitenk. 34, 310-8.

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11. , Gonnert, R. & Haberkorn, A. 1969. Die Feinstruktur der Makrogameten des Hiihnercoccids Eimeria tenella. Z . Parasitenk. 33,31-43. 12. , Mehlhorn, H. & Hammond, D. M. 1971. Fine structure of macrogametes and oocysts of coccidia and related organisms. Z . Parasitenk. 37, 1-43. 13. Sheffield, H. & Melton, M. L. 1968. The fine structure 209-26. and reproduction of Toxoplasma gondii. I. Parasit. 14. Snigirevskaya, E. S. 1969. Electron microscope study of the macrogametes of Eimeria intestinalis (Coccidia) [in Russian]. Acta Protozool. 11, 700-06.

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The Resorption of Cilia in Cyathodinium pirijorme* JEROME J. PAULIN

Department of Zoology, University of Georgia, Athens, Georgia 30601

SYNOPSIS. The fine structure of the cilium, kinetosome, kinetodesmal fiber, and basal microtubules has been described in Cyathodinium piriforme. The ciliary axoneme is encased in an electron-dense jacket termed the axonemal jacket. This jacket surrounds the axoneme and is found midway between the axoneme and the ciliary membrane when viewed in cross section. Before division or reorganization the cilia are withdrawn into the cell. Intact cilia surrounded by their jackets are found in the cytoplasm during the early phases of retraction. Degradation of the axonemal microtubules precedes the dissolution of the axonemal jacket. Profiles of the jackets are observed after the microtubules have been resorbed. The cilia appear to detach from the kinetosomes. Barren kinetosomes are seen below the cell surface frequently with kinetodesmal fibers still attached. Whether all or some of these barren kinetosomes contribute to the formation of the new ciliary anlage cannot be ascertained.

Index Key Words:

Cyathodinium pyriforme ; cilia ; kinetosomes; kinetodesmal fibers; basal microtubules ; axonemal jacket; morphogenesis; electron microscopy.

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UCAS (8) described reorganization and division in the ciliate Cyathodinium piriforme. She observed that these curious ciliates symbiotic in the cecum of the guinea pig transformed from pyriform to conical individuals. Following this transformation cilia were lost (resorbed?), endospritsl resorbed, and a new ciliary anlage (= endocellular cyst) arose d e nouo in the endoplasm; 2 anlagen appeared in the case of dividing forms. These anlagen gave rise to the new ciliature. Da Cunha & De Freitas ( 5 , 6 ) , and later Nie ( l o ) , found Lucas’s observations on the transformation to be partially incorrect,2 but loss of cilia, resorption of endosprits, and development of the endocytoplasmic anlage( n ) were indeed unique to these symbiotic organisms. Paulin & Corliss (18) postulated that this mode of anlage formation was reminiscent of a form of endogenous budding characteristic of some suctorian ciliates (Discophrya). They also called attention to the fact that the withdrawal of cilia preceded the anlage phase producing a “naked” quiescent stage. Paulin (14, 16) had drscribed these early stages, but a detailed study had not been published. The purpose of this paper is to describe the early stages of ciliary resorption. The emphasis is placed on the following: ( a ) the unique structure of the cilium and its withdrawal into the cell; (b) the degradation of the axonemal microtubules and ancillary structures; and ( c ) explore the possibility that continuity of kinetosoines exists in differentiating Cyathodinium.

* The author wishes to acknowledge with gratitude the kindness of Dr. John 0. Corliss, University of Maryland, for reviewing this manuscript. On the fine-structural level the endosprits are similar to suctorian tentacles consisting of a ring of microtubules that surrounds 4 crescent-shaped groups of microtubules ( 18). * Lucas’s pyriform organism is Cyathodinium piriforme and the conical form is a separate species, C . cunhai (5, 6, 13, 15). Both species may inhabit the same host.

MATERIAL AND METHODS Methods of obtaining C . piriforme from the guinea pig and detailed fixation and embedding technics for electron microscopy have been described elsewhere (15). A variety of fixatives were used, therefore a brief account of each will be presented. Material for Figs. 1-4 was fixed in phosphate-buffered 3% ( v / v ) glutaraldehyde with postfixation in 2% ( w / v ) OsO,; for Figs. 5 and 7 in acetate-veronal-buffered 2% ( w / v ) OsO,; and for Fig. 6 in ~ . sections were s-collidine-buffered 1.33% ( w / v ) 0 ~ 0 Thin stained with saturated uranyl acetate (aqueous) and/or lead citrate. They were examined in a Hitachi 11A or Siemens 101 electron microscope operating at 75, 80, or 100 Kv. OBSERVATIONS

Light Microscopy Detailed descriptions of C. piriforme have been published elsewhere (11, 13, 17), therefore only a general description will be given, The ciliate is pyriform, with the posterior pole tapering to a small caudal tip. I t measures -30-20 p m and has a large depression occupying -% of its anterio-ventral surface. This depression contains 8 rows of cilia while the remainder of the body has 9 rows, circumventing the depression; 2 posterior somatic rows completely encircle the organism. T h e endosprits are arranged perpendicular to, and along the left lip of the depression. Their fine structure has been described ( 18).

Electron Microscopy T h e somatic cilia are -6 pni long. Each cilium projects from a depression (-0.5 pin deep) formed by the invagination of the outer pellicular membrane (Figs. 1, 4 ) . A parasomal sac ( P S ) is found in each depression (Figs. 4, 7 ) . T h e pellicular membrane is continuous and forms the ciliary membrane (CM,

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