Acta path. microbial. scand. Sect. B, 85: 363-373, 1977
T H E ULTRASTRUCTURE AND DISTRIBUTION OF MICROPORES IN T H E VARIOUS DEVELOPMENTAL FORMS O F EIMERIA BRUNETTI D. J. P. FERGUSON~? *, A. BIRCH-ANDERSGN~, W. M. HUTCH IS ON^ and J.
&R.
SIIM’
FAO/WHO Collaborating Centre for Research and Reference i n Toxoplasmosisl and Department 01 Biophysicsz, Statens Senuninstitut, Copenhagen,Denmark, and D e p a r t m m of Biologys, University of Strathclyde, Glasgow, Scotland
Ferguson, D. J. P., Birch-Andersen, A., Hutchison, W. M. & Siim, J. Chr. The ultrastructure and distribution of micropores i n the various developmental forms of Eimcria brunctti. Acta path. m*icrobiol.xand. Sect. B, 85: 363-373, 1977. The structure and distribution of micropores in the various developmental stages of Eimcria brunctti was examined. Micropores were observed in all the endogenous forms with the exception of the microgamete. Oocysts from chicken faeces were also examined at various stages of sporulation and micropores were demonstrated in zygotes, spomblasts, ~porozoites, and the residual cytoplasmic masses. The number of micropores per organism appeared to be correlated with the surface area of the orgaruisms irrespective of whether these were endogenous or sporulating forms. The increase in the number of micropores did not appear to be related to micropore activity because seemingly acbive micropares were observed only in the trophozoites, i n the early multinucleate forms (early schizmts and microgamonts), and in the early macrogamonts. All these forms, however, possessed relatively few micropores. No active micropores were ever observed within the sporulating oocysts. Key words: Eimcriu brunctti; micropores; endogenous forms; oocysts; chickens. Statens Seruminstitut, D. J. P. Ferguson, Department of Toxoplas&s, Amager Boulevard 80, DK-2300 Copenhagen S, Denmark.
Received 8.vi,i.77
Accepted 8.A.77
The micropore was first observed in the sporozoite of Plasmodium falciparum (12) and has subsequently been reported as a characteristic organelle of the Sporozoa. Detailed reviews on the occurrence of micropores within the S$orozoa have been publish-
*
Work initiated while a Wellcome Trust Travelling Research Fellow, and completed as a Danish Medical Research Council Fellow.
ed by Scholtyseck & Mehlhorn (27) and Senaud et a!. (29). Within the genus Eimeria, the presence of a micropore was first reported in the merozoites of Eimeria intestinalis by Cheissin & Snigirevskaya ( 5 ) and they suggested that it possibly functioned as an “ultracytostome”. Within the haemarpolridians there is a great deal of evidence for the micropore acting as a cytostome and participating in the ingestion of host material (1, 2). I t has been more difficult to find support
363
for the ultracytostome hypothesis among the non-haemosporidian members of the Sporozoa, but the available evidence has recently been reviewed by Senaud et al. (29). In all the species of the genus Eimeria which have been studied, micropores have been observed in the sporozoites and in all the endogenous forms, with the exception of the microgametes. Until recently the ultrastructural details of the stages within the oocyst were unknown, but in a previous brief communication ( 11) we have noted the presence of a micropore in the late sporoblast of Eimeria brunetti. In the present study, for the first time, all the stages in the complete life cycle of E. brunetti are examined for the presence of micropores. The relative distribution and the apparent functional status in the various developmental forms will be discussed.
Figures 1-15 are all micrographs which illustrate the presence of micropores in the various stages of the endogenous development of E . brunetti within the epithelial cells of the d l intestines of infected chickens. F i g u m 16-23 are all micrographs which illustrate the presence of micropores in the various stages of the sporulation of oocy-sts of E . brunetti in the external environment. A double bar ( = ) on a micrograph represents 1 q and a single bar (-) represents 100 nm. The following abbreviations are used thmughout: AL = amorphous layer; B = basal body; C = collar of dense material; DB = dense body (anlage of refractde body) ; ER = rough endoplasmic reticulum; EV = electron translucent vacuole; FL = flagellum; G = Golgi body; LM = limiting membrane; M = micmneme; ME = m e m i t e ; MI = mitochondrion; M P = micropre; MV = mdtimembraneous vacuole; N = nucleus; 0 = outer layer of the oocyst w d ; OW = oocyst wall; PG =-polysaccharide granule; PL = pellicle; PV = parasitophorous vacuole; R = ribosome; R H = rhoptry; R M = residual cytoplasmic mass; S H = schizont; SP = sporozoitte; WFBI = wall forming bodies of type I ; WFB I1 = wall forming bodies of type 11.
Fig. 1. A section through part of a multinucleate stage showing two nuclei and an active micropore.
30,000 x . Fig. 2. An enlargement of the active micropore in Fig. 1 showing that it conskts of an invagination
364
MATERIALS AND METHODS
Examination of the endogenous forms: Small intestines of young domestic fowls infected with E. brunetti were examined. The methods were similar to those previously described (7), but can be summarized as follows: Pieces of the small intestine were fixed i n glutaraldehyde and osmium tetroxide and embedded in Vestopal W. Thin sections were examined in the electron microscope after staining with magnesium uranyl acetate and lead citrate. Examination of oocysts: The oocysts were obtained from chicken faeces and were allowed to sporulate for various time intervals before being h a t e d by 'the double sectioning technique described previously ( 4 ) . This involved pre-embedding of oocysts in crossed-licnked bovine serum albumin and deep f d n g in liquid nitrogen prior to cryo-sectioning, which was followed by primary fixation. Further treatments for ultramicrotomy and electron microscopy were as summarized above, and the observations were based on the examination of approximately 4000 electron micrographs.
of the limiting membrane with a collar of dense material round the neck (arrows). 90,000 x .
Fig. 3. Part of a fully formed merozoite showing the typical anterior organelles (rhoptries and micronemes) . Note the inactive micropore situated just anterior to the nucleus. 30,000 X . Fig. 4 . A section through an inactive micropore situated on the pellicle of a m e m i t e . The m i c m pore is formed by an invagination of the outer membrane of the pellicle and the collar (arrows) by an invagination of the inner layer of the pellide. 90,000 x . Fig. 5. A section through part of a schizont showing a m e m i t e as it is formed by a protrusion from the surface of the schimnt. Note the inactive micropore on the limiting membrane of the schizont. 45,000 x . Fig. 6. A section through the periphery of a microgamont. A group of three cross-sectioned micropores is present ( m w s ) . 90,000 x
.
Fig. 7 . Part of an early microgamont showing the basal body and flagellum of a developing m i c m gamete. An inactive micropore is present on the limiting membrane of the cytoplasmic mass of the organism. 45,000 x . Fig. 8. A section through a mature microgamont showing the nuclei and flagella of some of the microgametes. Note the inactive micropore on the limiting membrane of the residual cytoplasmk m a s of the microgamont. 45,000 x .
365
366
RESULTS
Ultrastructure: The micropore was found to consist of a circular invagination (70-80 nm in diameter) of the limiting membrane of the organism (Figs 6, 13 and 18). I n forms which possessed a pellicle, the inner layer of the pellicle was also seen to invaginate for a short distance (60-70 nm) (Figs 3 and 4), and thus a dense collar (120-130 nm in diameter) was present round the neck of the micropore (Figs 6, 13 and 18). This collar was also present in organisms which were limited only by a single unit membrane (Figs 2, 10, 12, 14 and 17). Based on the depth of the invagination of the limiting membrane, the micropores could be divided into two types, probably of different functional significance. The first consisted of seemingly
Fig. 9. Part of an early macrogamont showing the nucleus, some multimembraneous vacuoles, some developing W F B I and a Golgi body. The organism is limited by a single membrane o n which an active micmpore is present. 30,000 X . Fig. 10. An enlargement of the active micropore in Fig. 9. Note that the micropore consists of an invagination of the limimting membrane. with a collar of dense material round the neck region (arrows). 90,000 x
.
Fig. 11. Part of an oblique section through a late macrogamont. In addition to the three crass sectioned micropores (arrows),some WFB I and some WFB I1 are shown together with a number of polysaccharide granules. 30,000 X . Fig. 12. Part of a section of a macrogamete on which the amorphous layer exterior to the limiting membrane is well ilelustrated. An inactive micropore is also pment. 90,000 X . Fig. 13. A cross sectioned micropore present in an oblique section through the periphery of an organism in which oocyst wall' formation is occurring. 90,000 x. Fig. 14. Part of a section of a forming oocyst. Note the presence of the inactive micropore on the limiting membrane of the cytoplasmic mass below the outer layer of the oocyst wall. 9O,OO(E x . Fig. 15. Part of a section through an organism in which the outer layer of the oocyst wall is present. Note the five micropores (arrows) a t the periphery of the cytoplasmic mass below the outer oocyst wa1.l. 15,OOO x .
inactive micropores in which the invagination was only 90-140 nm deep (Figs 4, 5, 7, 8, 12, 14, 17, 20, 21 and 22). The second consisted of the seemingly active micropores in which the invagination was much deeper (370-420 nm) and which, in addition, presented a bulbous appearance (Figs 1, 2, 9 and 10). The structural characteristics of the active and inactive micropores are diagrammatically presented in Text Fig. 1. Distribution The endogenous forms. Active micropores were observed only in the trophozoites, in the early multinucleate stages (Fig. 1), which develop into schzonts or microgamonts, and in the early macrogamont (Figs 9 and 10). The number of active micropores observed in these stages was low. If present, normally only one was observed per section, but occasionally two could be found in a single section. In the later developmental stages only inactive micropores were observed. This applied to organisms which had developed to the stage where scEzonts could be differentiated from microgamonts because of the initiation of merozoite or microgamete formation and also to organisms throughout the later stages of schizogony and microgametogenesis (Figs 5, 6 and 7). In the schizonts, the number of micropores was low, although a somewhat higher number was observed in the large 1st generation schizonts. In the microgamont, which possessed deep invaginations of the limiting membrane of the organism, the number of micropores appeared relatively higher. In this stage the micropores were often observed in groups with as many as three present in a n area of 0.15 pm2 (Fig. 6). In the merozoite formed by schizogony only a single micropore was observed per organism. This micropore was of the inactive type and was situated on the pellicle just anterior to the nucleus (Figs 3 and 4). No micropores were observed in the microgametes. 367
Fig. 16. A section through part of a zygote within an oocyst. The cytoplasm contains a nucleus, some polysaccharide granules, some electron translucent vacuoles, some mitochondria, and some rough emdoplasmic reticulum. Note the three inactive micmpores (arrows) on the hmciting membrane of the zygote.
15,000 x . Fig. 17. A field of view which shows part of the limiting membrane of a zygote. Note the presence of an inactive micropore. 90,000 X .
Fig. 18. A section through the periphery of a zygote. Note the presence of a cross sectioned micropore. 90,000 x .
In the later developmental stages of the macrogamont and in the fully formed macrogamete the number of micropores was relatively higher (Fig. 11 ) . Here as many as six micropores have been observed in two adjacent sections of one organism. After the initial developmental stages of the macrogamont, these micropores were all of the inactive type (Fig. 12). In organisms in which the oocyst wall had started to form it was possib!e to find large numbers of inactive micropores (Figs 13, 14 and 15). In a single thin section, as many as six were observed situated on the limiting
36%
membrane of the cytoplasmic mass below the developing oocyst wall. Oocysts. In the initial cytoplasmic mass, the zygote, it was possible to find inactive micropores (Figs 17 and 18).I n this case as many as three have been observed in a single thin section (Fig. 16). Inactive micropores were also observed in the early sporoblasts which are formed by the division of the zygote. The late spomblast was found to be limited by two unit membranes, and inactive micropores were observed associated with the inner of these membranes (Fig. 20), i.e. with the one which
Fig. 19. Part of a section through a late spomblast with two inactive micropores (arrows) at the periphery of the organism. The cytaplasm contains dense bodies, eleotron translucent vacuoles, and some rough endoplasmic reticulum. 30,000 X . Fig. 20. A detail of an inactive micropore from a late Jporoblast. Note that it is the inner of the two limiting membranes which invaginates to form the mficropore. The collar of dense material is present in the neck region (arrow). 90,000 X. Fig. 22. A field of view showing part of a developing sporocyst in which a sporozoite is being formed. Note the inactive micropore on the l h i t i n g membrane of the cytoplasmic mass from which the sporomite is being formed. 30,000 X . Fig. 22. A section through a mature spomyst in which parts of the two sporozoites can be seen. An inactive micropore is present on the limiting membrane of the residual crtoplasmic mass. 30,000 X . Fig. 23. A section through part of a mature sporocyst in which an inactive micropore is present on the pellicle of one of the sporozoites. 30,000 x .
369
constitutes the plasmalemma and not the outer membrane which is related to the formation of the sporocyst wall. Two micropores have been observed in a single thin section through a sporoblast (Fig. 19). In the sporocyst, inactive micropores were also observed on the limiting membrane of the cytoplasmic mass as sporodte formation was occurring (Fig. 2 1 ) . In addition, micropores could be observed on the residual mass, which was left after completion of sporozoite formation (Fig. 22). The sporozoites ,formed within the sporocyst each possessed an inactive micropore situated just anterior to their nuclei (Fig. 23). DISCUSSION
In our previous studies on the endogenous development of E . brunetti we have referred to the presence of micropores without relating this to their functional status (7, 8, 9, 10). I n this study we have made the assump tion, based on the evidence reviewed by Senaud et al. (29), that the micropore is functioning as a cytostome giving rise to food vacuoles and that this activity can be related to its structural appearance. We have used the terms active and inactive to describe functional and non-functional micropores, respectively. This is similar to the terminology used by Michael (18) and a diagrammatical representation of the two types of micropores is given in our Text Fig. 1. The basic ultrastructure of the micropore of E. brunetti is similar to that reported for the Eimeria spp. and other members of the Sporotoa, (for references see 27, 29 and 35). The exact number of micropores in the vanious developmental stages cannot be ascertained unless complete serial sections through individual organisms can be examined; this was not carried out. We have examined the relative number of micropores, i.e. the frequency with which one or more micropores were observed within the sections that were available through the different stages in the life cycle of the organism. It was found that the micropores were less frequent370
A
C
Text Fig. 1. A diagrammatical representatim of longitudidy sectioned inactive ( A ) and d v e (C) micmpores. The cross sectional appearance through the neck region is shown in (B). The average dimensions are given in m.
ly observed in the small stages ( i x . merozoite, sporozoite and trophozoite) and that the relative number appeared to increase with increased size of the developmental stage examined. The increase in number of micropores per organism was apparently related to the surface area of the organism so that the largest numbers were observed in organisms with the largest surface area (i.e. 1st generation schizonts, microgamonts and macrogamonts) . Similar observations have also been reported for the endogenous forms of other Eirneria, spp. (13, 16, 27, 31, 33). I t would appear that formation of the micropore is somehow related to the synthesis of the limiting membrane of the organism. Hypothetically, the genome for the synthesis of the plasmalemma could be related to the genome for micropore formation in such a manner that when the surface area of the organism increases the number of micropores would also increase. This would explain the presence of micropores under the forming oocyst wall which has been reported in the present study and also for other Eimeria spp. (6, 15, 35). In this situation it is difficult to attribute a functional significance to the micropore. In addition, the absence of micropores in the microgamete could be due to the very small size of this form. Because microgametes have a very small surface area, formation of their plasmalemma is limited and probably 'insufficient to trigger off any micropore formation. This is in accordance with reported observations in which micropores
are absent in members of the Sporotoa which produce small microgametes (see review by Scholtyseck et al. ( % ) ) , but have been reported to be present in the large microgametes produced by other members such as Coelotropha durchoni (20) and Aggregata eberthi (24). I n E. brunetti active micropores are limited to the early developmental stages of the endogenous forms. These early stages are the actively growing stages and apparently active micropores have only been observed during this growth phase. In the later stages when the organisms are undergoing differentiation (formation of merozoites, microgametes or macrogametes) , although the micropores are relatively more abundant, they are all of the inactive type. I t would thus appear that in E. brunetti the endogenous forms do not feed continually but satisfy their macromolecular requirement during the growth phase. The presence of active micropores in the early stages only, corresponds well to their presence during macrogametogenesis in E. intestinalis as earlier reported (32). In a number of studies providing evidence for the cytostome function of the micropore in Eimeria. spp., the micropores were reported to be present in the early developmental stages (14, 26, 29, 34). For E. acervulina, (18) and E . magna (33), however, the presence of both active and inactive micropores has been reported in the later stages of macrogametogenesis. It is possible that the differences noted are related to variations between eimerian species. I n E. brunetti, as observed in this study, an increase in the number of micropores does not appear to be related to increased nutritional requirements but more to an increase in the surface area of the organism. Micropores were also found in the stages within the oocyst. This was not completely unexpected since one example of micropores on a developing sporoblastoid of Plasmodium vivux has been described (30), although micropores have never been observed in the oocysts of the haemosporidia (3, and Garnham, personal communication). In addition other parasites belonging to families and
genera related to the Eimeria have been reported to possess micropores during their sporulation (17, 19, 21, 22, 23, 25, 36). In these previous studies, the micropores were all of the inactive type. This was also the case for those found within the oocysts of E. brunetti in our study. Within the oocysts of E. brunetti the largest number of micropores were observed in the stage with the largest surface area (the zygote). Thus the micropore in E . brunetti is present in all stages of the life cycle except the microgamete. In the majority of cases, though, the micropore is present as a vestige rather than as an active micropore which is found only on actively growing organisms of the endogenous phase. We are indebted to Ithe Central Veterinary Laboratory, Ministry of Agriculture, Fisheries and Food, New Haw, Weybridge, Surrey, England, for supplying a pure sample of oocysts of E. brunetti, and to K . L. Fennestad, V.M.D., Statens Serumimtitut, for provision and maintenan- of the chickens. We gratefully acknowledge Mrs. H . Ravn and Mrs. J . Berg for technical assistance, and Miss A . Overgaard and Mr. F. Laursen far photographic assistance. The work was supported by grants from the World Hea!th Organization, Geneva, the Wellcome Trust, the British Council, the Danish Medical Research Council, and the W . H . Ross Foundation.
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salivary glands, with particular reference to the incidence of the micropore. Trans,roy. Soc. trop. Me&. Hyg. 67: 631-637, 1973. 31. Snigirevskaya, E. S.: The occurrence of micropores in schizonts, microgametocytes and mauogametes of Eimeria intestinalis. Acta Protozoal. 5: 381-386, 1968. 32. Snigirevskaya, E. S.: Electron microscope study of the macrogametes of Eimeria intestinalis (Coccidia): Tsitologiya I 1 : 700-706, 1969. (In Russian). 33. Speer, C. A., Hammond, D . M., Youssef, N . N . & Danfortk, H . D.: Fine struotural aspects of macrogametogemsis in Eimeria magna. J. Protozoal. 20: 274-281, 1973,.
24 Act. path. microbial. rond. Sect. B, 85, 6
34. Strout, R . G. & Scholtyseek, E.: The ultrastructure of f i t generation development of Eimeria tenelfa (Railliet & Lucet 1891) Fantham, 1909, in cell cultures. Z. Parasitenk 35: 87-96, 1970. 35. Varghese, T.: The fine structure of the emdgenous stages of Eimeria labbsana. 3. Feeding organelles. Z. Parasitenk. 49: 25-32, 1976. 36. Vivier, E., Petiprez, A . & Landau, I.: Observations ultrastructurales sur la sporoblast~ genhse de I’h6mogdgarine Hepatozoon domerquei coccidie Adeleidea. Protistologica VZZZ: 315-333, 1972.
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T H E ULTRASTRUCTURE AND DISTRIBUTION OF MICROPORES IN T H E VARIOUS DEVELOPMENTAL FORMS O F EIMERIA BRUNETTI D. J. P. FERGUSON~? *, A. BIRCH-ANDERSGN~, W. M. HUTCH IS ON^ and J.
&R.
SIIM’
FAO/WHO Collaborating Centre for Research and Reference i n Toxoplasmosisl and Department 01 Biophysicsz, Statens Senuninstitut, Copenhagen,Denmark, and D e p a r t m m of Biologys, University of Strathclyde, Glasgow, Scotland
Ferguson, D. J. P., Birch-Andersen, A., Hutchison, W. M. & Siim, J. Chr. The ultrastructure and distribution of micropores i n the various developmental forms of Eimcria brunctti. Acta path. m*icrobiol.xand. Sect. B, 85: 363-373, 1977. The structure and distribution of micropores in the various developmental stages of Eimcria brunctti was examined. Micropores were observed in all the endogenous forms with the exception of the microgamete. Oocysts from chicken faeces were also examined at various stages of sporulation and micropores were demonstrated in zygotes, spomblasts, ~porozoites, and the residual cytoplasmic masses. The number of micropores per organism appeared to be correlated with the surface area of the orgaruisms irrespective of whether these were endogenous or sporulating forms. The increase in the number of micropores did not appear to be related to micropore activity because seemingly acbive micropares were observed only in the trophozoites, i n the early multinucleate forms (early schizmts and microgamonts), and in the early macrogamonts. All these forms, however, possessed relatively few micropores. No active micropores were ever observed within the sporulating oocysts. Key words: Eimcriu brunctti; micropores; endogenous forms; oocysts; chickens. Statens Seruminstitut, D. J. P. Ferguson, Department of Toxoplas&s, Amager Boulevard 80, DK-2300 Copenhagen S, Denmark.
Received 8.vi,i.77
Accepted 8.A.77
The micropore was first observed in the sporozoite of Plasmodium falciparum (12) and has subsequently been reported as a characteristic organelle of the Sporozoa. Detailed reviews on the occurrence of micropores within the S$orozoa have been publish-
*
Work initiated while a Wellcome Trust Travelling Research Fellow, and completed as a Danish Medical Research Council Fellow.
ed by Scholtyseck & Mehlhorn (27) and Senaud et a!. (29). Within the genus Eimeria, the presence of a micropore was first reported in the merozoites of Eimeria intestinalis by Cheissin & Snigirevskaya ( 5 ) and they suggested that it possibly functioned as an “ultracytostome”. Within the haemarpolridians there is a great deal of evidence for the micropore acting as a cytostome and participating in the ingestion of host material (1, 2). I t has been more difficult to find support
363
for the ultracytostome hypothesis among the non-haemosporidian members of the Sporozoa, but the available evidence has recently been reviewed by Senaud et al. (29). In all the species of the genus Eimeria which have been studied, micropores have been observed in the sporozoites and in all the endogenous forms, with the exception of the microgametes. Until recently the ultrastructural details of the stages within the oocyst were unknown, but in a previous brief communication ( 11) we have noted the presence of a micropore in the late sporoblast of Eimeria brunetti. In the present study, for the first time, all the stages in the complete life cycle of E. brunetti are examined for the presence of micropores. The relative distribution and the apparent functional status in the various developmental forms will be discussed.
Figures 1-15 are all micrographs which illustrate the presence of micropores in the various stages of the endogenous development of E . brunetti within the epithelial cells of the d l intestines of infected chickens. F i g u m 16-23 are all micrographs which illustrate the presence of micropores in the various stages of the sporulation of oocy-sts of E . brunetti in the external environment. A double bar ( = ) on a micrograph represents 1 q and a single bar (-) represents 100 nm. The following abbreviations are used thmughout: AL = amorphous layer; B = basal body; C = collar of dense material; DB = dense body (anlage of refractde body) ; ER = rough endoplasmic reticulum; EV = electron translucent vacuole; FL = flagellum; G = Golgi body; LM = limiting membrane; M = micmneme; ME = m e m i t e ; MI = mitochondrion; M P = micropre; MV = mdtimembraneous vacuole; N = nucleus; 0 = outer layer of the oocyst w d ; OW = oocyst wall; PG =-polysaccharide granule; PL = pellicle; PV = parasitophorous vacuole; R = ribosome; R H = rhoptry; R M = residual cytoplasmic mass; S H = schizont; SP = sporozoitte; WFBI = wall forming bodies of type I ; WFB I1 = wall forming bodies of type 11.
Fig. 1. A section through part of a multinucleate stage showing two nuclei and an active micropore.
30,000 x . Fig. 2. An enlargement of the active micropore in Fig. 1 showing that it conskts of an invagination
364
MATERIALS AND METHODS
Examination of the endogenous forms: Small intestines of young domestic fowls infected with E. brunetti were examined. The methods were similar to those previously described (7), but can be summarized as follows: Pieces of the small intestine were fixed i n glutaraldehyde and osmium tetroxide and embedded in Vestopal W. Thin sections were examined in the electron microscope after staining with magnesium uranyl acetate and lead citrate. Examination of oocysts: The oocysts were obtained from chicken faeces and were allowed to sporulate for various time intervals before being h a t e d by 'the double sectioning technique described previously ( 4 ) . This involved pre-embedding of oocysts in crossed-licnked bovine serum albumin and deep f d n g in liquid nitrogen prior to cryo-sectioning, which was followed by primary fixation. Further treatments for ultramicrotomy and electron microscopy were as summarized above, and the observations were based on the examination of approximately 4000 electron micrographs.
of the limiting membrane with a collar of dense material round the neck (arrows). 90,000 x .
Fig. 3. Part of a fully formed merozoite showing the typical anterior organelles (rhoptries and micronemes) . Note the inactive micropore situated just anterior to the nucleus. 30,000 X . Fig. 4 . A section through an inactive micropore situated on the pellicle of a m e m i t e . The m i c m pore is formed by an invagination of the outer membrane of the pellicle and the collar (arrows) by an invagination of the inner layer of the pellide. 90,000 x . Fig. 5. A section through part of a schizont showing a m e m i t e as it is formed by a protrusion from the surface of the schimnt. Note the inactive micropore on the limiting membrane of the schizont. 45,000 x . Fig. 6. A section through the periphery of a microgamont. A group of three cross-sectioned micropores is present ( m w s ) . 90,000 x
.
Fig. 7 . Part of an early microgamont showing the basal body and flagellum of a developing m i c m gamete. An inactive micropore is present on the limiting membrane of the cytoplasmic mass of the organism. 45,000 x . Fig. 8. A section through a mature microgamont showing the nuclei and flagella of some of the microgametes. Note the inactive micropore on the limiting membrane of the residual cytoplasmk m a s of the microgamont. 45,000 x .
365
366
RESULTS
Ultrastructure: The micropore was found to consist of a circular invagination (70-80 nm in diameter) of the limiting membrane of the organism (Figs 6, 13 and 18). I n forms which possessed a pellicle, the inner layer of the pellicle was also seen to invaginate for a short distance (60-70 nm) (Figs 3 and 4), and thus a dense collar (120-130 nm in diameter) was present round the neck of the micropore (Figs 6, 13 and 18). This collar was also present in organisms which were limited only by a single unit membrane (Figs 2, 10, 12, 14 and 17). Based on the depth of the invagination of the limiting membrane, the micropores could be divided into two types, probably of different functional significance. The first consisted of seemingly
Fig. 9. Part of an early macrogamont showing the nucleus, some multimembraneous vacuoles, some developing W F B I and a Golgi body. The organism is limited by a single membrane o n which an active micmpore is present. 30,000 X . Fig. 10. An enlargement of the active micropore in Fig. 9. Note that the micropore consists of an invagination of the limimting membrane. with a collar of dense material round the neck region (arrows). 90,000 x
.
Fig. 11. Part of an oblique section through a late macrogamont. In addition to the three crass sectioned micropores (arrows),some WFB I and some WFB I1 are shown together with a number of polysaccharide granules. 30,000 X . Fig. 12. Part of a section of a macrogamete on which the amorphous layer exterior to the limiting membrane is well ilelustrated. An inactive micropore is also pment. 90,000 X . Fig. 13. A cross sectioned micropore present in an oblique section through the periphery of an organism in which oocyst wall' formation is occurring. 90,000 x. Fig. 14. Part of a section of a forming oocyst. Note the presence of the inactive micropore on the limiting membrane of the cytoplasmic mass below the outer layer of the oocyst wall. 9O,OO(E x . Fig. 15. Part of a section through an organism in which the outer layer of the oocyst wall is present. Note the five micropores (arrows) a t the periphery of the cytoplasmic mass below the outer oocyst wa1.l. 15,OOO x .
inactive micropores in which the invagination was only 90-140 nm deep (Figs 4, 5, 7, 8, 12, 14, 17, 20, 21 and 22). The second consisted of the seemingly active micropores in which the invagination was much deeper (370-420 nm) and which, in addition, presented a bulbous appearance (Figs 1, 2, 9 and 10). The structural characteristics of the active and inactive micropores are diagrammatically presented in Text Fig. 1. Distribution The endogenous forms. Active micropores were observed only in the trophozoites, in the early multinucleate stages (Fig. 1), which develop into schzonts or microgamonts, and in the early macrogamont (Figs 9 and 10). The number of active micropores observed in these stages was low. If present, normally only one was observed per section, but occasionally two could be found in a single section. In the later developmental stages only inactive micropores were observed. This applied to organisms which had developed to the stage where scEzonts could be differentiated from microgamonts because of the initiation of merozoite or microgamete formation and also to organisms throughout the later stages of schizogony and microgametogenesis (Figs 5, 6 and 7). In the schizonts, the number of micropores was low, although a somewhat higher number was observed in the large 1st generation schizonts. In the microgamont, which possessed deep invaginations of the limiting membrane of the organism, the number of micropores appeared relatively higher. In this stage the micropores were often observed in groups with as many as three present in a n area of 0.15 pm2 (Fig. 6). In the merozoite formed by schizogony only a single micropore was observed per organism. This micropore was of the inactive type and was situated on the pellicle just anterior to the nucleus (Figs 3 and 4). No micropores were observed in the microgametes. 367
Fig. 16. A section through part of a zygote within an oocyst. The cytoplasm contains a nucleus, some polysaccharide granules, some electron translucent vacuoles, some mitochondria, and some rough emdoplasmic reticulum. Note the three inactive micmpores (arrows) on the hmciting membrane of the zygote.
15,000 x . Fig. 17. A field of view which shows part of the limiting membrane of a zygote. Note the presence of an inactive micropore. 90,000 X .
Fig. 18. A section through the periphery of a zygote. Note the presence of a cross sectioned micropore. 90,000 x .
In the later developmental stages of the macrogamont and in the fully formed macrogamete the number of micropores was relatively higher (Fig. 11 ) . Here as many as six micropores have been observed in two adjacent sections of one organism. After the initial developmental stages of the macrogamont, these micropores were all of the inactive type (Fig. 12). In organisms in which the oocyst wall had started to form it was possib!e to find large numbers of inactive micropores (Figs 13, 14 and 15). In a single thin section, as many as six were observed situated on the limiting
36%
membrane of the cytoplasmic mass below the developing oocyst wall. Oocysts. In the initial cytoplasmic mass, the zygote, it was possible to find inactive micropores (Figs 17 and 18).I n this case as many as three have been observed in a single thin section (Fig. 16). Inactive micropores were also observed in the early sporoblasts which are formed by the division of the zygote. The late spomblast was found to be limited by two unit membranes, and inactive micropores were observed associated with the inner of these membranes (Fig. 20), i.e. with the one which
Fig. 19. Part of a section through a late spomblast with two inactive micropores (arrows) at the periphery of the organism. The cytaplasm contains dense bodies, eleotron translucent vacuoles, and some rough endoplasmic reticulum. 30,000 X . Fig. 20. A detail of an inactive micropore from a late Jporoblast. Note that it is the inner of the two limiting membranes which invaginates to form the mficropore. The collar of dense material is present in the neck region (arrow). 90,000 X. Fig. 22. A field of view showing part of a developing sporocyst in which a sporozoite is being formed. Note the inactive micropore on the l h i t i n g membrane of the cytoplasmic mass from which the sporomite is being formed. 30,000 X . Fig. 22. A section through a mature spomyst in which parts of the two sporozoites can be seen. An inactive micropore is present on the limiting membrane of the residual crtoplasmic mass. 30,000 X . Fig. 23. A section through part of a mature sporocyst in which an inactive micropore is present on the pellicle of one of the sporozoites. 30,000 x .
369
constitutes the plasmalemma and not the outer membrane which is related to the formation of the sporocyst wall. Two micropores have been observed in a single thin section through a sporoblast (Fig. 19). In the sporocyst, inactive micropores were also observed on the limiting membrane of the cytoplasmic mass as sporodte formation was occurring (Fig. 2 1 ) . In addition, micropores could be observed on the residual mass, which was left after completion of sporozoite formation (Fig. 22). The sporozoites ,formed within the sporocyst each possessed an inactive micropore situated just anterior to their nuclei (Fig. 23). DISCUSSION
In our previous studies on the endogenous development of E . brunetti we have referred to the presence of micropores without relating this to their functional status (7, 8, 9, 10). I n this study we have made the assump tion, based on the evidence reviewed by Senaud et al. (29), that the micropore is functioning as a cytostome giving rise to food vacuoles and that this activity can be related to its structural appearance. We have used the terms active and inactive to describe functional and non-functional micropores, respectively. This is similar to the terminology used by Michael (18) and a diagrammatical representation of the two types of micropores is given in our Text Fig. 1. The basic ultrastructure of the micropore of E. brunetti is similar to that reported for the Eimeria spp. and other members of the Sporotoa, (for references see 27, 29 and 35). The exact number of micropores in the vanious developmental stages cannot be ascertained unless complete serial sections through individual organisms can be examined; this was not carried out. We have examined the relative number of micropores, i.e. the frequency with which one or more micropores were observed within the sections that were available through the different stages in the life cycle of the organism. It was found that the micropores were less frequent370
A
C
Text Fig. 1. A diagrammatical representatim of longitudidy sectioned inactive ( A ) and d v e (C) micmpores. The cross sectional appearance through the neck region is shown in (B). The average dimensions are given in m.
ly observed in the small stages ( i x . merozoite, sporozoite and trophozoite) and that the relative number appeared to increase with increased size of the developmental stage examined. The increase in number of micropores per organism was apparently related to the surface area of the organism so that the largest numbers were observed in organisms with the largest surface area (i.e. 1st generation schizonts, microgamonts and macrogamonts) . Similar observations have also been reported for the endogenous forms of other Eirneria, spp. (13, 16, 27, 31, 33). I t would appear that formation of the micropore is somehow related to the synthesis of the limiting membrane of the organism. Hypothetically, the genome for the synthesis of the plasmalemma could be related to the genome for micropore formation in such a manner that when the surface area of the organism increases the number of micropores would also increase. This would explain the presence of micropores under the forming oocyst wall which has been reported in the present study and also for other Eimeria spp. (6, 15, 35). In this situation it is difficult to attribute a functional significance to the micropore. In addition, the absence of micropores in the microgamete could be due to the very small size of this form. Because microgametes have a very small surface area, formation of their plasmalemma is limited and probably 'insufficient to trigger off any micropore formation. This is in accordance with reported observations in which micropores
are absent in members of the Sporotoa which produce small microgametes (see review by Scholtyseck et al. ( % ) ) , but have been reported to be present in the large microgametes produced by other members such as Coelotropha durchoni (20) and Aggregata eberthi (24). I n E. brunetti active micropores are limited to the early developmental stages of the endogenous forms. These early stages are the actively growing stages and apparently active micropores have only been observed during this growth phase. In the later stages when the organisms are undergoing differentiation (formation of merozoites, microgametes or macrogametes) , although the micropores are relatively more abundant, they are all of the inactive type. I t would thus appear that in E. brunetti the endogenous forms do not feed continually but satisfy their macromolecular requirement during the growth phase. The presence of active micropores in the early stages only, corresponds well to their presence during macrogametogenesis in E. intestinalis as earlier reported (32). In a number of studies providing evidence for the cytostome function of the micropore in Eimeria. spp., the micropores were reported to be present in the early developmental stages (14, 26, 29, 34). For E. acervulina, (18) and E . magna (33), however, the presence of both active and inactive micropores has been reported in the later stages of macrogametogenesis. It is possible that the differences noted are related to variations between eimerian species. I n E. brunetti, as observed in this study, an increase in the number of micropores does not appear to be related to increased nutritional requirements but more to an increase in the surface area of the organism. Micropores were also found in the stages within the oocyst. This was not completely unexpected since one example of micropores on a developing sporoblastoid of Plasmodium vivux has been described (30), although micropores have never been observed in the oocysts of the haemosporidia (3, and Garnham, personal communication). In addition other parasites belonging to families and
genera related to the Eimeria have been reported to possess micropores during their sporulation (17, 19, 21, 22, 23, 25, 36). In these previous studies, the micropores were all of the inactive type. This was also the case for those found within the oocysts of E. brunetti in our study. Within the oocysts of E. brunetti the largest number of micropores were observed in the stage with the largest surface area (the zygote). Thus the micropore in E . brunetti is present in all stages of the life cycle except the microgamete. In the majority of cases, though, the micropore is present as a vestige rather than as an active micropore which is found only on actively growing organisms of the endogenous phase. We are indebted to Ithe Central Veterinary Laboratory, Ministry of Agriculture, Fisheries and Food, New Haw, Weybridge, Surrey, England, for supplying a pure sample of oocysts of E. brunetti, and to K . L. Fennestad, V.M.D., Statens Serumimtitut, for provision and maintenan- of the chickens. We gratefully acknowledge Mrs. H . Ravn and Mrs. J . Berg for technical assistance, and Miss A . Overgaard and Mr. F. Laursen far photographic assistance. The work was supported by grants from the World Hea!th Organization, Geneva, the Wellcome Trust, the British Council, the Danish Medical Research Council, and the W . H . Ross Foundation.
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