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The polar ring complex in ookinetes of Leucocytozoon simondi (Apicomplexa: Haemosporina) and evidence for a conoid in haemosporidian ookinetes
European Journal of
Europ.J.Protistol. 24, 244-251 (1989)
PROTISTOLOGY
The Polar Ring Complex in Ookinetes of Leucocytozoon simondi (Apicomplexa: Haemosporina) and Evidence for a Conoid in Haemosporidian Ookinetes W. Brockley Paterson and Sherwin S. Desser Department of Zoology, University of Toronto, Canada
SUMMARY Ookinetes of Leucocytozoon simondi have a Polar Ring Complex (PRe) which is composed of a stout electron-lucent polar ring with about 34 posteriorly extending tines. The latter are subtended by about 76 subpellicular microtubules. A broad collar of electron-dense material is associated with the cytoplasmic surface of the inner double membrane complex of the pellicle. Additional particulate material of varying density occupies the space between tines and the dense material associated with the pellicle.A conoid and two preconoidal rings are present. We conclude that a PRC and a typical apicomplexan conoid, although often unrecognized, occur in ookinetes of all the haemosporidians examined thusfar.
Abbreviations B Bm Ca Co Cr Dm Ep Er He Hm Kc Mn Mp Mt Mu Np Nu Oc Pm Pp Pr Rp Sm Tn Tr
= Basal lamina
= Blood meal
= Dense material coating cytoplasmic surface of Dm
= Conoid
= Crystalloid = Inner double membrane complex of pellicle = Gut epithelium
= Endoplasmic reticulum
= Heterochromatin
= Haemocoel = Kinetochore = Microneme
= Particulate material between Dm and Tn = Subpellicular microtubules = Muscle cell
= Nuclear plaque = Nucleus
Introduction In a recent study, it was hypothesized that the gamonts of Haemogregarina magna [18] were structurally homologous to the ookinetes of the Haemosporina and the kinetes of the Piroplasmea. It was further suggested that the apical structures which comprise the Polar Ring Complex (PRC) commonly occur in apicomplexans in those stages that are motile in their invertebrate hosts. Verification of this hypothesis will require the careful examination of the appropriate stages of several apicomplexans. The presence of a PRC and conoid in ookinetes of Leucocytozoon simondi is demonstrated in this study. Previously unrecognized evidence in the literature for a PRC and conoid in ookinetes of Plasmodium, Haemoproteus and Parahaemoproteus species is discussed.
= Oocyst
= Plasmalemma = Precipitation within Mp
Material and Methods
= Polar ring
= = = =
Preconoidal rings Spindle microtubule Tine Dense material between Tn and Mt
0932-4739/89/0024-0244$3.50/0
Engorged female blackflies (Simulium Tugglesi) were collected from Pekin ducks whose blood contained mature gametocytes of L. simondi. The flies were maintained at 20 C. At approximately 15 h post-feeding, their midguts were dissected and fixed for elec© 1989 by Gustav FischerVerlag,Stuttgart
Polar Ring Complex of Leucocytozoon simondi . 245 tron microscopy using 2.5% glutaraldehyde in Sorensen's phosphate buffer (0.2 M, pH 7.0) or in sodium cacodylate buffer (0.1 M, pH 7.0). Calcium chloride (0.5% w/v) was added to each buffer (0.5 mU10 rnl). Tissue blocks were post-fixed in 1% OS04 in the appropriate buffer. K3FE(CN)6 (6.4% w/v) was added to the postfixative (0.5 mU4.5 ml), Intact midguts were initially placed in the fixative and after 15 min were cut into quarters. The pieces were returned to the fixative for an additional hour. Following postfixation, the blocks were washed in buffers, rinsed in dH 20 , en bloc stained in 0.5% (w/v) aqueous uranyl acetate, and dehydrated in an ascending series of ethanol. The blocks were embedded in Spurr's epoxy resin [23]. Ultrathin sections were stained in uranyl acetate and lead citrate [18] and were examined using a Philips EM201C operated at an accelerating voltage of 60kV. Midguts also were prepared for light microscopy at hourly intervals following feeding. Gut contents and epithelium were smeared out on glass slides, air-dried, fixed in absolute methanol and stained with Giemsa stain (1 : 4 in phosphate buffer at pH 7.2 for 10 min).
Results Since ultrastructural data on post-zygotic development of Leucocytozoon species have been published previously [3,5-7,28], our results will be restricted to novel observations with emphasis on the detailed structure of the PRe.
1. Zygote to Ookinete The earliest stages observed were young zygotes prior to formation of the pellicle of the ookinete. Microtubules of the division spindle appeared to extend uninterrupted throughout the length of the nucleus of these zygotes. The nuclear plaque consisted of electron-dense material which spanned the nuclear envelope at the pole (Fig. 1). A compact sphere of dense heterochromatin was observed in a young ookinete (Fig. 2) at one end of a nucleus, which contained a division spindle in adjacent serial sections.
2. Mature Ookinete Light micrographs (Figs. 3, 4) of ookinetes revealed condensed areas of chromatin within the nucleus at nine hours post-feeding. The PRC is apparent in Giemsa stained specimens (Fig. 3). The mature ookinete possessed the polar ring and associated tines, which comprise the Polar Ring Complex, and a conoid. The conoid is located at the apex of the cell immediately posterior to two preconoidal rings (Figs. 9, 11, 17). The cone-shape is evident in oblique section (Fig. 12a-d) as is its tubular substructure (Fig. 10). Surrounding the posterior aspect of the conoid and lying between the inner double membrane complex of the pellicle (Dm) and the subpellicular microtubules was a stout, electron-lucent polar ring with closely spaced, radially arranged and posteriorly directed tines (Figs. 5-7). The tines were composed of a similar lucent material. The subpellicular microtubules lay immediately under the Dm except in the region of the PRe. Approximately 76 (74-78) subpellicular microtubules were associated with the inner surface of the polar ring. These microtubules extended posteriorly from the ring, lying against a band of electron-dense material that was apposed to the inner surface of the tines (Figs. 5-7). Thirty-four tines fused anteriorly (Figs. 6, 7) to form the polar ring. The central portion of the polar ring was radially reinforced with dense material (Figs. 5, 9-11). Between the tines and the dense material associated with the Dm was a region of irregularly electron-dense particulate material (Figs. 5-7). The zone of particulate material was extracted in specimens that were sub-optimally fixed (Fig. 11). A diagrammatic representation of the PRC and associated apical structures of ookinetes of 1. simondi is shown in Fig. 17. The pellicle consisted of an outer plasmalemma and an inner double membrane complex (Dm). The latter terminated anteriorly at the region of the conoid (Figs. 5, 9-12). Ducts of the micronemes traversed the conoid and two preconoidal rings (Figs. 5, 9). A wide band of dense material lined the cytoplasmic face of the Dm (Figs. 5-11).
Figs. 1-7. - Figs. 1-2. Zygote. - Fig. 1. Spindlemicrotubules (Sm) extend the length of the nucleus. the nuclear plaque (Np) is evident as dense polar material that spans the nuclear envelope. (x 48000). - Fig. 2. The dense heterochromatin (He) from the microgamete persists as a discrete lobe of the nucleus of the young zygote. Dense material (arrows) occupies the nuclear pores. (x 25000). Figs. 3,4. Light micrographs of mature ookinetes at nine hours post-feeding. The region of the PRC (arrow) is evident with Giemsa stain. Densely stained chromatin, probably chromosomes, is evident in the nucleus (Nu) of the ookinete. (X 1800). - Figs. 5-17. Electron micrographs of mature ookinetes. - Fig. 5. Anterior end of a mature ookinete. The pellicle consists of a plasmalemma (Pm) and an inner double membrane complex (Dm).A thickened, electron lucent polar ring (Pr) has posteriorly projecting tines (Tn) which are situated exterior to the subpellicular microtubules (Mt). The interior of the polar ring is radially reinforced by dense material. Additional dense material (Tr) is interposed between the microtubules and the tines. In the region of the tines, particulate material of varying density (Mp) is situated between the inner double membrane complex and the tines. Lines of precipitation may occur in this region (Pp).The cytoplasmic face of the inner membrane of the pellicularcomplex is broadly coated with electron dense material (Ca). Micronemes (Mn) are evident. A conoid (Co) is situated atop the polar ring. The inner double membrane complex terminates near the conoid. (x 51400). - Fig. 6. The tines which project posteriorly from the polar ring are subtended by microtubules. (x 51400). Fig. 7. An oblique section at the levelof the polar ring. Individual tines fuse into the polar ring. Dense material radially reinforcesthe centre of the ring. There are about 76 microtubules. (x 47300).
~
246 . W. B. Paterson and S. S. Desser
Figs. 8-12. - Fig. 8. An ookinete within the midgut epithelium of the blackfly. (x 15000). - Fig. 9. Adjacent serial section to Fig. 8. The apex of the ookinete is extracellular at the basal lamina. The ducts of the micronemes pass through the conoid. The ookinete is separated from the epithelial cells' cytoplasm by membranes (arrows). (x 55000). - Fig. 10. Tubular substructure of the conoid (arrow) as seen in longitudinal section. (x 43300). - Fig. 11. Two preconoidal rings (Rp) are situated anteriorto the conoid. Material presumed to be secretion is at the apex of the cell. With suboptimal fixation, the dense material between the coated inner pellicular membrane and the polar ring and tines is extracted allowing the conoid (Co) and preconoidal rings to be readily apparent. (X 45500). - Figs. 12a-d. Oblique sections through apical region of ookinetes revealing the presence and position of the conoid (arrows). Note its conical nature. (x 22800).
248 . W. B. Paterson and S. S. Desser
Figs. 13-16. Young Oocysts. - Fig. 13. The first indication of oocyst wall formation is the presence of dense subspherical particles (arrowheads) external to the parasite in the area beneath the basal lamina adjacent to the pellicle. (x 26200). - Fig. 14. Spherical particles outside the oocyst are membrane-bound and occur in various sizes. Tines of the PRC and associated subpellicular microtubules are observed in cross section. (x 63700). - Fig. 15. The polar ring and tines (Tn) persist in young oocysts. A nuclear division spindle is again evident. Nu = nucleus. (x 16600).Fig. 16. The nuclear plaque (Np) traverses the nuclear envelope. A kinetochore (Kc) is seen on a spindle microtubule (Sm). Densities (arrows) associated with the chromatin-facing side of the inner membrane of the nuclear envelope may represent sites of chromosomal attachment. (X 13 800).
Ca Tn
Tr
Mt
Fig. 17. Diagrammatic representation of the features of the Polar Ring Complex of the ookinete of Leucocytozoon simondi. The diagram illustrates the components of the PRC and their relationship to the other apical structures, and is not drawn to scale.
Polar Ring Complex of Leucocytozoon simondi . 249
Blebs of electron dense material which appeared to be secretions were occassionally observed exterior to the apex of ookinetes in the blood meal (Fig. 11).
3. Young oocysts Intraepithelial ookinetes were enclosed by host cell membranes (Fig. 9). Young oocysts were observed beneath the basal lamina and between neighbouring epithelial cells (Figs. 8, 9). The first indications of oocystic wall formation was the appearance of tiny, dense subspherical, membrane-bound particles surrounding the oocyst (Figs. 13, 14). The tines of the PRC were still apparent in young oocysts (Figs. 14, 15). Spindle microtubules and nuclear plaques were once again evident in young oocysts (Figs. 15, 16). The dense material of the nuclear plaque was observed on both sides of the nuclear envelope (Fig. 16). Kinetochores were observed infrequently on the spindle. Periodically arranged densities not associated with the nuclear pores were seen on the chromatin-facing side of the inner nuclear membrane (Fig. 16).
Discussion The chromosome-like condensations observed in light micrographs of young ookinetes of L. simondi are suggestive of a reduction division at this stage. With the recent observation of synaptinemal complexes in ookinetes of Plasmodium falciparum [21-23] and the presence of these complexes in ookinetes of Parahaemoproteus velans (illustrated, but not recognized by the author at that time [4]), it is likely that meiosis occurs in the ookinetes of all haemosporidians. As in Plasmodium spp. [23], spindle microtubules were not present in mature ookinetes of L. simondi. Thus, those in young oocysts were formed anew. It therefore seems likely that the first division observed in oocysts was the second meiotic division, however, a mitotic division indicating the start of sporogony cannot be ruled out on present evidence. Spindle microtubules have been recorded previously for several haemosporidians both in the zygote [1, 8, 14,
21-23] and in the young oocyst [2, 4, 14], however, the presence of densities on the chromatin side of the inner membrane of the nuclear envelope of the young oocyst is novel. Perhaps these represent a membrane-association of the chromosomes as they separate in the second meiotic division. Ookinetes of L. simondi were enclosed by membrane during their passage through the midgut epithelium en route to their final extracellular position beneath the basal lamina. There has been recent debate as to whether ookinetes of Plasmodium species penetrate through [13] or between [15, 16] mosquito midgut cells and whether they are enclosed by membranes during their passage through cells. Although ookinetes of L. simondi were clearly membrane-bound while passing through the midgut epithelium, it was not possible to determine whether the enclosing membranes comprised a parasitophorous vac-
uole or whether the parasites were in an intercellular position. Oocysts observed in this study were at an earlier stage of development than those of other haemosporidian species described previously [13, 23, 25, 26]. The dense, subspherical, membrane-bound particles seen around recently transformed ookinetes of L. simondi were the first sign of oocystic wall formation. These particles were present prior to the disaggregation of the double inner membrane complex of the ookinete. We agree with Mehlhorn et al. [14] that the wall of the oocyst is not derived from bodies within the ookinete, and that the parasite apparently induces the host to contribute to the formation of the oocystic wall. The apical complex of ookinetes of L. simondi is strikingly similar to that recorded in gamonts of Haemogregarina magna [18]. Both parasites share the following features: a conoid and two preconoidal rings, subpellicular microtubules which subtend the inner face of the polar ring that has posteriorly projecting tines, and dense material interposed between these microtubules and the tines. A major difference is that in H. magna, the tines of the PRC are closely apposed to the Dm while in haemosporidian ookinetes, the two structures are separated by a zane of irregularly-dense particulate material and the Dm is thickly coated on its inner surface with electron dense material [1,5,7-10, 13, 14, 16]. In haemosporidians, the opaque, pellicle-associated material usually has been referred to as the "canopy" or "collar" [1,2,8, 10]. The region of particulate material of varying density occasionally contains lines of precipitation between the tines of the polar ring and the coating of the Dm. Variation in the width of this zone of particulate material as observed in Figs. 7-12 demonstrates that the anterior end of the ookinete, in the region of the PRC, is capable of deformation, presumably because microtubules do not support the pellicle here. This region of particulate material is readily extracted when it is not optimally fixed and we believe that it corresponds to the subpellicular space which has been described in the anterior end of certain haemosporidian ookinetes [1,3]. In other instances, the polar ring itself has been mistaken for a space [9], because of its lucidity. We believe that the PRC is an ancestral feature of coccidians. This structure is commonly observed in: gregarines; adeleids (where it is found in the gamonts of Haemogregarina and Hepatozoon spp. and the sporokinetes of Karyolysus spp.); the zygote and kinetes of piroplasms; and the ookinetes of haemosporidians. Galucci [8] catalogued differing interpretations of the apical structures of haemosporidian ookinetes. It seems likely that the various terminologies used previously led to confusion and contributed to the failure to recognize the PRC as a structure common to the adeleids, piroplasms, gregarines, and haemosporidians. We urge the adoption of a standard terminology to describe these structures. Nichols and Chiappino [17] recently employed the term "Polar Ring Complex" to describe the polar ring and associated subpellicular microtubules and the anterior region of the Dm of the pellicle of zoites of Toxoplasma gondii. Coccidian zaites in vertebrate hosts are distinct
250 . W. B. Paterson and S. S. Desser from haemosporidian ookinetes, adeleid gamonts and sporokinetes, and kinetes of piroplasms in arthropod hosts. The latter group has far more subpellicular microtubules (20s versus 70s) which subtend the posteriorly projecting tines of a stout, well-defined polar ring. We propose a more restrictive definition for the PRC than that of Nichols and Chiappino [17]. We define the PRC as the polar ring and its associated tines. The former serves as the microtubular organizing centre for the subpellicular microtubules [20]. Although the PRC comprises a basic component of the apical complex of the motile stages of all apicomplexans, it is reduced in certain stages. Tines, for example, have not been recorded in merozoites or sporozoites of eimeriid coccidians and haemosporidians. Data from the present study and a careful examination of the literature indicate that a similar PRC occurs in ookinetes of all haemosporidians examined thusfar [e.g. Leucocytozoon tawaki ([6], Fig. 1), Haemoproteus columbae ([8], Figs. 21, 23-30), Parahaemoproteus velans ([4], Figs. 3, 4), Plasmodium gallinaceum ([1], Figs. 19, 20), ([10], Figs. 7, 8), ([14], Fig. 13) and Plasmodium [alciparum ([22], Fig. 6A) and Plasmodium berghei ([2] Fig.1A)]. The conoid was difficult to demonstrate in ookinetes of L. simondi despite a directed search for this structure. It invariably appeared less electron dense and therefore was less well defined than its counterparts in other coccidians. However, its typical location and ultrastructure, consisting of a truncated cone of tubules clearly indicates that this organelle is a conoid as defined by Gustafson et al. [11]. A similar conoid was demonstrated (loc. cit. Figs. 21-23) in ookinetes of H. columbae [8] and is also evident (loc. cit. Fig. 13) in P. gallinaceum [14]. Unfortunately, the conoid was not recognized as such in the latter study. This oversight led to the proposal to create a new class Aconoidea to include the haemosporidians and the piroplasms. This classification has been perpetuated by Levine [12] in his recent review. We believe that that PRC represents an ancestral feature that occurs commonly in members of the class Sporozoea having a stage that penetrates through the tissues of an invertebrate host. The kinetes of Babesia bigemina and B. avis were shown to contain a PRC [27], but this stage should be re-examined for more detailed information and to ascertain whether a conoid occurs in the subclass Piroplasmia. Study of the appropriate motile stages of the piroplasms and gregarines may reveal a greater structural homology among the coccidians than is currently recognized.
Acknowledgements The authors are grateful to the Ontario Ministry of Natural Resources for the use of their facilities at the Wildlife Research Station in Algonquin Park. The assistance of the Electron Microscopy Unit of the Department of Zoology, University of Toronto is appreciated. We thank John Barta, Don Martin and Henry Hong for their constructive reviews of the manuscript. Research was funded by an NSERC operating grant awarded to S.S.D. (A6596).
References 1 Aikawa M., Carter R., Ito Y. and Nijhout M. (1984): New observations on gametocytogenesis, fertilization and zygote transformation in Plasmodium gallinaceum. J. Protozool., 31,403-413. 2 Canning E. U. and Sinden R. E. (1973): The organization of the ookinete and observations on nuclear division in oocysts of Plasmodium berghei. Parasitology, 67, 29-40. 3 Desser S. S. (1970): The fine structure of Leucocytozoon simondi. III. The ookinete and mature sporozoite. Can. J. Zool., 48, 641-646. 4 Desser S. S. (1972): The fine structure of the ookinete of Parahaemoproteus velans (= Haemoproteus velans Coatney and Roudabush) (Haemosporidia: Haemoproteidae). Can. J. Zool., 50, 477-480. 5 Desser S. S. (1972): The fine structure of Leucocytozoon simondi. V. The oocyst. Can. J. Zool., 50, 707-711. 6 Desser S. S. and Allison F. (1979): Aspects of the sporogonic development of Leucocytozoon tawaki of the Fiordland crested penguin in its primary vector, Austrosimulium ungulatum: an ultrastructural study. J. Parasitol., 65, 737-744. 7 Desser S. S. and Wright K. A. (1968): A preliminary study of the fine structure of the ookinete, oocyst and sporozoite formation of Leucocytozoon simondi Mathis and Leger. Can. J. Zool., 46, 303-307. 8 Galucci B. B. (1974): Fine structure of Haemoproteus columbae Kruse during differentiation of the ookinete. J. Protozool., 21, 264-275. 9 Garnham P. C. c., Bird R. G., Baker J. R., DesserS. S. andEINahal H. M. S. (1969): Electron microscopic studies on motile stages of malaria parasites. VI. The ookinete of Plasmodium berghei yoelii and its transformation into the early oocyst. Trans. Roy. Soc. Trop. Med. Hyg., 63, 187-194. 10 Gass R. F. (1979): Ultrastructure of cultured Plasmodium gallinaceum ookinetes: a comparison of intact stages with forms damaged from blood fed susceptible Aedes aegypti. Acta Tropica, 36, 323-334. 11 Gustafson P. V., Agar H. D. and Cramer D. J. (1954): An electron microscope study of Toxoplasma. Am. J. Trop. Med. Hyg., 3, 1008-1021. 12 LevineN. D. (1988): Blood parasites: the malaria and related parasites. In: The protozoan phylum Apicomplexa, vol. 2. CRC Press, Boca Raton, Fla. 13 Maier W. A., Becker-Feldman H. and Seitz H. M. (1987): Pathology of malaria-infected mosquitoes. Parasitology Today, 3, 216-217. 14 Mehlhorn H., Peters W. and Haberkorn A. (1980): The formation of kinetes and oocysts in Plasmodium gallinaceum (Haemosporidia) and considerations on phylogenetic relationships between Haemosporidia, Piroplasmida and other coccidia. Protistologica, 16, 135-154. 15 Meis J. F. G. M. and Ponndurai T. (1987): Interaction of malaria with mosquitoes. Parasitology Today, 3, 369. 16 Meis J. F. G. M. and Ponndurai T. (1987): Ultrastructural studies on the interaction of Plasmodium falciparum ookinetes with the midgut epithelium of Anopheles stephensi mosquitoes. Parasitology Research, 73, 500-506. 17 Nichols B. A. and Chiappino M. L. (1987): Cytoskeleton of Toxoplasma gondii. J. Protozool., 34, 217-226. 18 Paterson W. B., Desser S. S. and Barta J. R. (1988): Ultrastructural features of the apical complex, pellicle, and membranes investing the gamonts of Haemogregarina magna (Apicomplexa: Adeleina). J. Protozool., 35, 73-80. 19 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.
Polar Ring Complex of Leucocytozoon simondi . 251 20 Russell D. G. and Burns R. G. (1984): The polar ring of coccidian zoites: a unique microtubular-organizing centre. J. Cell Sci., 65, 193-207. 21 Sinden R. E. (1985): Gametocytogenesis in Plasmodium spp. and observations on the meiotic division. Ann. Soc. Belge Med. Trop., 65, 21-33. 22 Sinden R. E. and Hartley R. H. (1985): Identification of the meiotic division of malarial parasites. J. Protozool., 32, 742-744. 23 Sinden R. E., Hartley R. H. and Winger L. (1985): The development of Plasmodium ookinetes in vitro: an ultrastructural study including a description of meiotic division. Parasitology, 91, 227-244. 24 Spurr A. R. (1969): A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ulstrastruct. Res., 26, 31.
25 Sterling C. R. and DeGiusti D. L. (1974): Fine structure of differentiating oocysts and mature sporozoites of Haemoproteus metchnikovi in its intermediate host Chrysops callidus. J. Protozool., 21, 276-283. 26 Terzakis J. A. (1971): Transformation of the Plasmodium cynomolgi oocyst. J. Protozool., 18, 62-73. 27 Weber G. (1980): Ultrastrukturen und Cytochemie der PeIlikula und des Apikalkomplexes der Kineten von Babesia bigemina und Babesia ovis in Hamolymphe und Ovar von Zecken. J. Protozool., 27, 59-71. 28 Wong S. T. C. and Desser S. S. (1976): Fine structure of oocyst transformation and the sporozoites of Leucocytozoon dubreuili. J. Protozool., 23, 115-126.
Key words: Leucocytozoon simondi - Ultrastructure - Ookinetes - Polar Ring Complex - Conoid
W. Brockley Paterson and Sherwin S. Desser, Department of Zoology, University of Toronto, Toronto, Ontario, Canada M5S 1A1
Europ.J.Protistol. 24, 244-251 (1989)
PROTISTOLOGY
The Polar Ring Complex in Ookinetes of Leucocytozoon simondi (Apicomplexa: Haemosporina) and Evidence for a Conoid in Haemosporidian Ookinetes W. Brockley Paterson and Sherwin S. Desser Department of Zoology, University of Toronto, Canada
SUMMARY Ookinetes of Leucocytozoon simondi have a Polar Ring Complex (PRe) which is composed of a stout electron-lucent polar ring with about 34 posteriorly extending tines. The latter are subtended by about 76 subpellicular microtubules. A broad collar of electron-dense material is associated with the cytoplasmic surface of the inner double membrane complex of the pellicle. Additional particulate material of varying density occupies the space between tines and the dense material associated with the pellicle.A conoid and two preconoidal rings are present. We conclude that a PRC and a typical apicomplexan conoid, although often unrecognized, occur in ookinetes of all the haemosporidians examined thusfar.
Abbreviations B Bm Ca Co Cr Dm Ep Er He Hm Kc Mn Mp Mt Mu Np Nu Oc Pm Pp Pr Rp Sm Tn Tr
= Basal lamina
= Blood meal
= Dense material coating cytoplasmic surface of Dm
= Conoid
= Crystalloid = Inner double membrane complex of pellicle = Gut epithelium
= Endoplasmic reticulum
= Heterochromatin
= Haemocoel = Kinetochore = Microneme
= Particulate material between Dm and Tn = Subpellicular microtubules = Muscle cell
= Nuclear plaque = Nucleus
Introduction In a recent study, it was hypothesized that the gamonts of Haemogregarina magna [18] were structurally homologous to the ookinetes of the Haemosporina and the kinetes of the Piroplasmea. It was further suggested that the apical structures which comprise the Polar Ring Complex (PRC) commonly occur in apicomplexans in those stages that are motile in their invertebrate hosts. Verification of this hypothesis will require the careful examination of the appropriate stages of several apicomplexans. The presence of a PRC and conoid in ookinetes of Leucocytozoon simondi is demonstrated in this study. Previously unrecognized evidence in the literature for a PRC and conoid in ookinetes of Plasmodium, Haemoproteus and Parahaemoproteus species is discussed.
= Oocyst
= Plasmalemma = Precipitation within Mp
Material and Methods
= Polar ring
= = = =
Preconoidal rings Spindle microtubule Tine Dense material between Tn and Mt
0932-4739/89/0024-0244$3.50/0
Engorged female blackflies (Simulium Tugglesi) were collected from Pekin ducks whose blood contained mature gametocytes of L. simondi. The flies were maintained at 20 C. At approximately 15 h post-feeding, their midguts were dissected and fixed for elec© 1989 by Gustav FischerVerlag,Stuttgart
Polar Ring Complex of Leucocytozoon simondi . 245 tron microscopy using 2.5% glutaraldehyde in Sorensen's phosphate buffer (0.2 M, pH 7.0) or in sodium cacodylate buffer (0.1 M, pH 7.0). Calcium chloride (0.5% w/v) was added to each buffer (0.5 mU10 rnl). Tissue blocks were post-fixed in 1% OS04 in the appropriate buffer. K3FE(CN)6 (6.4% w/v) was added to the postfixative (0.5 mU4.5 ml), Intact midguts were initially placed in the fixative and after 15 min were cut into quarters. The pieces were returned to the fixative for an additional hour. Following postfixation, the blocks were washed in buffers, rinsed in dH 20 , en bloc stained in 0.5% (w/v) aqueous uranyl acetate, and dehydrated in an ascending series of ethanol. The blocks were embedded in Spurr's epoxy resin [23]. Ultrathin sections were stained in uranyl acetate and lead citrate [18] and were examined using a Philips EM201C operated at an accelerating voltage of 60kV. Midguts also were prepared for light microscopy at hourly intervals following feeding. Gut contents and epithelium were smeared out on glass slides, air-dried, fixed in absolute methanol and stained with Giemsa stain (1 : 4 in phosphate buffer at pH 7.2 for 10 min).
Results Since ultrastructural data on post-zygotic development of Leucocytozoon species have been published previously [3,5-7,28], our results will be restricted to novel observations with emphasis on the detailed structure of the PRe.
1. Zygote to Ookinete The earliest stages observed were young zygotes prior to formation of the pellicle of the ookinete. Microtubules of the division spindle appeared to extend uninterrupted throughout the length of the nucleus of these zygotes. The nuclear plaque consisted of electron-dense material which spanned the nuclear envelope at the pole (Fig. 1). A compact sphere of dense heterochromatin was observed in a young ookinete (Fig. 2) at one end of a nucleus, which contained a division spindle in adjacent serial sections.
2. Mature Ookinete Light micrographs (Figs. 3, 4) of ookinetes revealed condensed areas of chromatin within the nucleus at nine hours post-feeding. The PRC is apparent in Giemsa stained specimens (Fig. 3). The mature ookinete possessed the polar ring and associated tines, which comprise the Polar Ring Complex, and a conoid. The conoid is located at the apex of the cell immediately posterior to two preconoidal rings (Figs. 9, 11, 17). The cone-shape is evident in oblique section (Fig. 12a-d) as is its tubular substructure (Fig. 10). Surrounding the posterior aspect of the conoid and lying between the inner double membrane complex of the pellicle (Dm) and the subpellicular microtubules was a stout, electron-lucent polar ring with closely spaced, radially arranged and posteriorly directed tines (Figs. 5-7). The tines were composed of a similar lucent material. The subpellicular microtubules lay immediately under the Dm except in the region of the PRe. Approximately 76 (74-78) subpellicular microtubules were associated with the inner surface of the polar ring. These microtubules extended posteriorly from the ring, lying against a band of electron-dense material that was apposed to the inner surface of the tines (Figs. 5-7). Thirty-four tines fused anteriorly (Figs. 6, 7) to form the polar ring. The central portion of the polar ring was radially reinforced with dense material (Figs. 5, 9-11). Between the tines and the dense material associated with the Dm was a region of irregularly electron-dense particulate material (Figs. 5-7). The zone of particulate material was extracted in specimens that were sub-optimally fixed (Fig. 11). A diagrammatic representation of the PRC and associated apical structures of ookinetes of 1. simondi is shown in Fig. 17. The pellicle consisted of an outer plasmalemma and an inner double membrane complex (Dm). The latter terminated anteriorly at the region of the conoid (Figs. 5, 9-12). Ducts of the micronemes traversed the conoid and two preconoidal rings (Figs. 5, 9). A wide band of dense material lined the cytoplasmic face of the Dm (Figs. 5-11).
Figs. 1-7. - Figs. 1-2. Zygote. - Fig. 1. Spindlemicrotubules (Sm) extend the length of the nucleus. the nuclear plaque (Np) is evident as dense polar material that spans the nuclear envelope. (x 48000). - Fig. 2. The dense heterochromatin (He) from the microgamete persists as a discrete lobe of the nucleus of the young zygote. Dense material (arrows) occupies the nuclear pores. (x 25000). Figs. 3,4. Light micrographs of mature ookinetes at nine hours post-feeding. The region of the PRC (arrow) is evident with Giemsa stain. Densely stained chromatin, probably chromosomes, is evident in the nucleus (Nu) of the ookinete. (X 1800). - Figs. 5-17. Electron micrographs of mature ookinetes. - Fig. 5. Anterior end of a mature ookinete. The pellicle consists of a plasmalemma (Pm) and an inner double membrane complex (Dm).A thickened, electron lucent polar ring (Pr) has posteriorly projecting tines (Tn) which are situated exterior to the subpellicular microtubules (Mt). The interior of the polar ring is radially reinforced by dense material. Additional dense material (Tr) is interposed between the microtubules and the tines. In the region of the tines, particulate material of varying density (Mp) is situated between the inner double membrane complex and the tines. Lines of precipitation may occur in this region (Pp).The cytoplasmic face of the inner membrane of the pellicularcomplex is broadly coated with electron dense material (Ca). Micronemes (Mn) are evident. A conoid (Co) is situated atop the polar ring. The inner double membrane complex terminates near the conoid. (x 51400). - Fig. 6. The tines which project posteriorly from the polar ring are subtended by microtubules. (x 51400). Fig. 7. An oblique section at the levelof the polar ring. Individual tines fuse into the polar ring. Dense material radially reinforcesthe centre of the ring. There are about 76 microtubules. (x 47300).
~
246 . W. B. Paterson and S. S. Desser
Figs. 8-12. - Fig. 8. An ookinete within the midgut epithelium of the blackfly. (x 15000). - Fig. 9. Adjacent serial section to Fig. 8. The apex of the ookinete is extracellular at the basal lamina. The ducts of the micronemes pass through the conoid. The ookinete is separated from the epithelial cells' cytoplasm by membranes (arrows). (x 55000). - Fig. 10. Tubular substructure of the conoid (arrow) as seen in longitudinal section. (x 43300). - Fig. 11. Two preconoidal rings (Rp) are situated anteriorto the conoid. Material presumed to be secretion is at the apex of the cell. With suboptimal fixation, the dense material between the coated inner pellicular membrane and the polar ring and tines is extracted allowing the conoid (Co) and preconoidal rings to be readily apparent. (X 45500). - Figs. 12a-d. Oblique sections through apical region of ookinetes revealing the presence and position of the conoid (arrows). Note its conical nature. (x 22800).
248 . W. B. Paterson and S. S. Desser
Figs. 13-16. Young Oocysts. - Fig. 13. The first indication of oocyst wall formation is the presence of dense subspherical particles (arrowheads) external to the parasite in the area beneath the basal lamina adjacent to the pellicle. (x 26200). - Fig. 14. Spherical particles outside the oocyst are membrane-bound and occur in various sizes. Tines of the PRC and associated subpellicular microtubules are observed in cross section. (x 63700). - Fig. 15. The polar ring and tines (Tn) persist in young oocysts. A nuclear division spindle is again evident. Nu = nucleus. (x 16600).Fig. 16. The nuclear plaque (Np) traverses the nuclear envelope. A kinetochore (Kc) is seen on a spindle microtubule (Sm). Densities (arrows) associated with the chromatin-facing side of the inner membrane of the nuclear envelope may represent sites of chromosomal attachment. (X 13 800).
Ca Tn
Tr
Mt
Fig. 17. Diagrammatic representation of the features of the Polar Ring Complex of the ookinete of Leucocytozoon simondi. The diagram illustrates the components of the PRC and their relationship to the other apical structures, and is not drawn to scale.
Polar Ring Complex of Leucocytozoon simondi . 249
Blebs of electron dense material which appeared to be secretions were occassionally observed exterior to the apex of ookinetes in the blood meal (Fig. 11).
3. Young oocysts Intraepithelial ookinetes were enclosed by host cell membranes (Fig. 9). Young oocysts were observed beneath the basal lamina and between neighbouring epithelial cells (Figs. 8, 9). The first indications of oocystic wall formation was the appearance of tiny, dense subspherical, membrane-bound particles surrounding the oocyst (Figs. 13, 14). The tines of the PRC were still apparent in young oocysts (Figs. 14, 15). Spindle microtubules and nuclear plaques were once again evident in young oocysts (Figs. 15, 16). The dense material of the nuclear plaque was observed on both sides of the nuclear envelope (Fig. 16). Kinetochores were observed infrequently on the spindle. Periodically arranged densities not associated with the nuclear pores were seen on the chromatin-facing side of the inner nuclear membrane (Fig. 16).
Discussion The chromosome-like condensations observed in light micrographs of young ookinetes of L. simondi are suggestive of a reduction division at this stage. With the recent observation of synaptinemal complexes in ookinetes of Plasmodium falciparum [21-23] and the presence of these complexes in ookinetes of Parahaemoproteus velans (illustrated, but not recognized by the author at that time [4]), it is likely that meiosis occurs in the ookinetes of all haemosporidians. As in Plasmodium spp. [23], spindle microtubules were not present in mature ookinetes of L. simondi. Thus, those in young oocysts were formed anew. It therefore seems likely that the first division observed in oocysts was the second meiotic division, however, a mitotic division indicating the start of sporogony cannot be ruled out on present evidence. Spindle microtubules have been recorded previously for several haemosporidians both in the zygote [1, 8, 14,
21-23] and in the young oocyst [2, 4, 14], however, the presence of densities on the chromatin side of the inner membrane of the nuclear envelope of the young oocyst is novel. Perhaps these represent a membrane-association of the chromosomes as they separate in the second meiotic division. Ookinetes of L. simondi were enclosed by membrane during their passage through the midgut epithelium en route to their final extracellular position beneath the basal lamina. There has been recent debate as to whether ookinetes of Plasmodium species penetrate through [13] or between [15, 16] mosquito midgut cells and whether they are enclosed by membranes during their passage through cells. Although ookinetes of L. simondi were clearly membrane-bound while passing through the midgut epithelium, it was not possible to determine whether the enclosing membranes comprised a parasitophorous vac-
uole or whether the parasites were in an intercellular position. Oocysts observed in this study were at an earlier stage of development than those of other haemosporidian species described previously [13, 23, 25, 26]. The dense, subspherical, membrane-bound particles seen around recently transformed ookinetes of L. simondi were the first sign of oocystic wall formation. These particles were present prior to the disaggregation of the double inner membrane complex of the ookinete. We agree with Mehlhorn et al. [14] that the wall of the oocyst is not derived from bodies within the ookinete, and that the parasite apparently induces the host to contribute to the formation of the oocystic wall. The apical complex of ookinetes of L. simondi is strikingly similar to that recorded in gamonts of Haemogregarina magna [18]. Both parasites share the following features: a conoid and two preconoidal rings, subpellicular microtubules which subtend the inner face of the polar ring that has posteriorly projecting tines, and dense material interposed between these microtubules and the tines. A major difference is that in H. magna, the tines of the PRC are closely apposed to the Dm while in haemosporidian ookinetes, the two structures are separated by a zane of irregularly-dense particulate material and the Dm is thickly coated on its inner surface with electron dense material [1,5,7-10, 13, 14, 16]. In haemosporidians, the opaque, pellicle-associated material usually has been referred to as the "canopy" or "collar" [1,2,8, 10]. The region of particulate material of varying density occasionally contains lines of precipitation between the tines of the polar ring and the coating of the Dm. Variation in the width of this zone of particulate material as observed in Figs. 7-12 demonstrates that the anterior end of the ookinete, in the region of the PRC, is capable of deformation, presumably because microtubules do not support the pellicle here. This region of particulate material is readily extracted when it is not optimally fixed and we believe that it corresponds to the subpellicular space which has been described in the anterior end of certain haemosporidian ookinetes [1,3]. In other instances, the polar ring itself has been mistaken for a space [9], because of its lucidity. We believe that the PRC is an ancestral feature of coccidians. This structure is commonly observed in: gregarines; adeleids (where it is found in the gamonts of Haemogregarina and Hepatozoon spp. and the sporokinetes of Karyolysus spp.); the zygote and kinetes of piroplasms; and the ookinetes of haemosporidians. Galucci [8] catalogued differing interpretations of the apical structures of haemosporidian ookinetes. It seems likely that the various terminologies used previously led to confusion and contributed to the failure to recognize the PRC as a structure common to the adeleids, piroplasms, gregarines, and haemosporidians. We urge the adoption of a standard terminology to describe these structures. Nichols and Chiappino [17] recently employed the term "Polar Ring Complex" to describe the polar ring and associated subpellicular microtubules and the anterior region of the Dm of the pellicle of zoites of Toxoplasma gondii. Coccidian zaites in vertebrate hosts are distinct
250 . W. B. Paterson and S. S. Desser from haemosporidian ookinetes, adeleid gamonts and sporokinetes, and kinetes of piroplasms in arthropod hosts. The latter group has far more subpellicular microtubules (20s versus 70s) which subtend the posteriorly projecting tines of a stout, well-defined polar ring. We propose a more restrictive definition for the PRC than that of Nichols and Chiappino [17]. We define the PRC as the polar ring and its associated tines. The former serves as the microtubular organizing centre for the subpellicular microtubules [20]. Although the PRC comprises a basic component of the apical complex of the motile stages of all apicomplexans, it is reduced in certain stages. Tines, for example, have not been recorded in merozoites or sporozoites of eimeriid coccidians and haemosporidians. Data from the present study and a careful examination of the literature indicate that a similar PRC occurs in ookinetes of all haemosporidians examined thusfar [e.g. Leucocytozoon tawaki ([6], Fig. 1), Haemoproteus columbae ([8], Figs. 21, 23-30), Parahaemoproteus velans ([4], Figs. 3, 4), Plasmodium gallinaceum ([1], Figs. 19, 20), ([10], Figs. 7, 8), ([14], Fig. 13) and Plasmodium [alciparum ([22], Fig. 6A) and Plasmodium berghei ([2] Fig.1A)]. The conoid was difficult to demonstrate in ookinetes of L. simondi despite a directed search for this structure. It invariably appeared less electron dense and therefore was less well defined than its counterparts in other coccidians. However, its typical location and ultrastructure, consisting of a truncated cone of tubules clearly indicates that this organelle is a conoid as defined by Gustafson et al. [11]. A similar conoid was demonstrated (loc. cit. Figs. 21-23) in ookinetes of H. columbae [8] and is also evident (loc. cit. Fig. 13) in P. gallinaceum [14]. Unfortunately, the conoid was not recognized as such in the latter study. This oversight led to the proposal to create a new class Aconoidea to include the haemosporidians and the piroplasms. This classification has been perpetuated by Levine [12] in his recent review. We believe that that PRC represents an ancestral feature that occurs commonly in members of the class Sporozoea having a stage that penetrates through the tissues of an invertebrate host. The kinetes of Babesia bigemina and B. avis were shown to contain a PRC [27], but this stage should be re-examined for more detailed information and to ascertain whether a conoid occurs in the subclass Piroplasmia. Study of the appropriate motile stages of the piroplasms and gregarines may reveal a greater structural homology among the coccidians than is currently recognized.
Acknowledgements The authors are grateful to the Ontario Ministry of Natural Resources for the use of their facilities at the Wildlife Research Station in Algonquin Park. The assistance of the Electron Microscopy Unit of the Department of Zoology, University of Toronto is appreciated. We thank John Barta, Don Martin and Henry Hong for their constructive reviews of the manuscript. Research was funded by an NSERC operating grant awarded to S.S.D. (A6596).
References 1 Aikawa M., Carter R., Ito Y. and Nijhout M. (1984): New observations on gametocytogenesis, fertilization and zygote transformation in Plasmodium gallinaceum. J. Protozool., 31,403-413. 2 Canning E. U. and Sinden R. E. (1973): The organization of the ookinete and observations on nuclear division in oocysts of Plasmodium berghei. Parasitology, 67, 29-40. 3 Desser S. S. (1970): The fine structure of Leucocytozoon simondi. III. The ookinete and mature sporozoite. Can. J. Zool., 48, 641-646. 4 Desser S. S. (1972): The fine structure of the ookinete of Parahaemoproteus velans (= Haemoproteus velans Coatney and Roudabush) (Haemosporidia: Haemoproteidae). Can. J. Zool., 50, 477-480. 5 Desser S. S. (1972): The fine structure of Leucocytozoon simondi. V. The oocyst. Can. J. Zool., 50, 707-711. 6 Desser S. S. and Allison F. (1979): Aspects of the sporogonic development of Leucocytozoon tawaki of the Fiordland crested penguin in its primary vector, Austrosimulium ungulatum: an ultrastructural study. J. Parasitol., 65, 737-744. 7 Desser S. S. and Wright K. A. (1968): A preliminary study of the fine structure of the ookinete, oocyst and sporozoite formation of Leucocytozoon simondi Mathis and Leger. Can. J. Zool., 46, 303-307. 8 Galucci B. B. (1974): Fine structure of Haemoproteus columbae Kruse during differentiation of the ookinete. J. Protozool., 21, 264-275. 9 Garnham P. C. c., Bird R. G., Baker J. R., DesserS. S. andEINahal H. M. S. (1969): Electron microscopic studies on motile stages of malaria parasites. VI. The ookinete of Plasmodium berghei yoelii and its transformation into the early oocyst. Trans. Roy. Soc. Trop. Med. Hyg., 63, 187-194. 10 Gass R. F. (1979): Ultrastructure of cultured Plasmodium gallinaceum ookinetes: a comparison of intact stages with forms damaged from blood fed susceptible Aedes aegypti. Acta Tropica, 36, 323-334. 11 Gustafson P. V., Agar H. D. and Cramer D. J. (1954): An electron microscope study of Toxoplasma. Am. J. Trop. Med. Hyg., 3, 1008-1021. 12 LevineN. D. (1988): Blood parasites: the malaria and related parasites. In: The protozoan phylum Apicomplexa, vol. 2. CRC Press, Boca Raton, Fla. 13 Maier W. A., Becker-Feldman H. and Seitz H. M. (1987): Pathology of malaria-infected mosquitoes. Parasitology Today, 3, 216-217. 14 Mehlhorn H., Peters W. and Haberkorn A. (1980): The formation of kinetes and oocysts in Plasmodium gallinaceum (Haemosporidia) and considerations on phylogenetic relationships between Haemosporidia, Piroplasmida and other coccidia. Protistologica, 16, 135-154. 15 Meis J. F. G. M. and Ponndurai T. (1987): Interaction of malaria with mosquitoes. Parasitology Today, 3, 369. 16 Meis J. F. G. M. and Ponndurai T. (1987): Ultrastructural studies on the interaction of Plasmodium falciparum ookinetes with the midgut epithelium of Anopheles stephensi mosquitoes. Parasitology Research, 73, 500-506. 17 Nichols B. A. and Chiappino M. L. (1987): Cytoskeleton of Toxoplasma gondii. J. Protozool., 34, 217-226. 18 Paterson W. B., Desser S. S. and Barta J. R. (1988): Ultrastructural features of the apical complex, pellicle, and membranes investing the gamonts of Haemogregarina magna (Apicomplexa: Adeleina). J. Protozool., 35, 73-80. 19 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.
Polar Ring Complex of Leucocytozoon simondi . 251 20 Russell D. G. and Burns R. G. (1984): The polar ring of coccidian zoites: a unique microtubular-organizing centre. J. Cell Sci., 65, 193-207. 21 Sinden R. E. (1985): Gametocytogenesis in Plasmodium spp. and observations on the meiotic division. Ann. Soc. Belge Med. Trop., 65, 21-33. 22 Sinden R. E. and Hartley R. H. (1985): Identification of the meiotic division of malarial parasites. J. Protozool., 32, 742-744. 23 Sinden R. E., Hartley R. H. and Winger L. (1985): The development of Plasmodium ookinetes in vitro: an ultrastructural study including a description of meiotic division. Parasitology, 91, 227-244. 24 Spurr A. R. (1969): A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ulstrastruct. Res., 26, 31.
25 Sterling C. R. and DeGiusti D. L. (1974): Fine structure of differentiating oocysts and mature sporozoites of Haemoproteus metchnikovi in its intermediate host Chrysops callidus. J. Protozool., 21, 276-283. 26 Terzakis J. A. (1971): Transformation of the Plasmodium cynomolgi oocyst. J. Protozool., 18, 62-73. 27 Weber G. (1980): Ultrastrukturen und Cytochemie der PeIlikula und des Apikalkomplexes der Kineten von Babesia bigemina und Babesia ovis in Hamolymphe und Ovar von Zecken. J. Protozool., 27, 59-71. 28 Wong S. T. C. and Desser S. S. (1976): Fine structure of oocyst transformation and the sporozoites of Leucocytozoon dubreuili. J. Protozool., 23, 115-126.
Key words: Leucocytozoon simondi - Ultrastructure - Ookinetes - Polar Ring Complex - Conoid
W. Brockley Paterson and Sherwin S. Desser, Department of Zoology, University of Toronto, Toronto, Ontario, Canada M5S 1A1