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Ultrastructure of spermiogenesis and the spermatozoon of Aporina delafondi (Cyclophyllidea, anoplocephalidae), intestinal parasite of turtle doves in senegal
Internorional
Pergamon
Journalfor Parasitology. Vol. 24, No. 2, pp. 225-235, 1994 Copyright 0 1994 Australian Society for Parasitology Elsetier Science Ltd Printed in Great Britain. All rights resewed 002&7519/94 S7.00+ 0.00
ULTRASTRUCTURE OF SPERMIOGENESIS AND THE SPERMATOZOON OF APORINA DELAFONDI (CYCLOPHYLLIDEA, ANOPLOCEPHALIDAE), INTESTINAL PARASITE OF TURTLE DOVES IN SENEGAL CHEIKH TIDIANE BA* and BERNARD
MARCHAND
Laboratory of Parasitology, Department of Animal Biology, Faculty of Sciences, Cheikh Anta Diop University of Dakar, Dakar, Senegal (Received 6 April 1993; accepted 26 August 1993)
Abstract-B.4 C. T. and MARCHAND B. 1994. Ultrastructure of spermiogenesis and the spermatozoon of Aporina delafondi (Cyclophyllidea, Anoplocephalidae), intestinal parasite of turtle doves in Senegal. International Journal for Parasitology 24: 22>235. Spermiogenesis in Aporina delafondi begins with the formation of a differentiation zone bordered by cortical microtubules and containing from the beginning a portion of nucleus and two parallel centrioles. One of the centrioles aborts, the other gives rise to a flagellum. The cortical microtubules elongate and spiralize while the nucleus migrates along the axoneme and crest-like bodies form at the level of the differentiation zone. The old spermatid separates from the residual cytoplasm by strangulation of the ring of arched membranes. The mature spermatozoon lacks mitochondria, is tiliform and tapered at both its extremities. Its anterior extremity is capped by an apical cone of electron-dense material and exhibits five crest-like bodies of unequal lengths on its periphery. Its cortical microtubules are regularly spiralized except at their posterior extremity where they become parallel to the spermatozoon axis. The cytoplasm is slightly dense in the anterior regions (I and II) and exhibits many protein granules and patches of electron-lucent material in the middle (III) and posterior zones (IV and V). The nucleus is an electron-dense cord coiled in a spiral around the middle region (III) of the axoneme. This is of the 9 + “1” pattern and ends before the posterior extremity of the gamete. Spermiogenesis in Aporina delafondi differs from that of the other Cyclophyllidea by the very early movement of the nucleus into the differentiation zone, the formation of a ring of arched membranes in the distal part of the differentiation zone, the appearance of crest-like bodies during migration of the nucleus and the formation of a cytoplasmic bud which contains the abortive centriole and develops to temporarily form a large lateral extension. The mature spermatozoon differs from that of the other Cyclophyllidea in the presence of lucent patches in its cytoplasm and of five helicoidal crest-like bodies. The systematic position of the genus Aporina is also debated. INDEX KEY WORDS: Aporina aklafondi; spermatozoon; Cyclophyllidea; Anoplocephalidae; Senegal
INTRODUCTION ONLY a few authors to date have taken an interest in spermiogenesis of the Cyclophyllidea (Rosario, 1964; Featherston, 1971; Kelsoe, Ubelakker & Allison, 1977; Robinson & Bogitsh, 1978; Mokhtar-Maamouri & Azzouz-Draoui, 1990; BB, Marchand & Mattei, 1991; BP & Marchand, 1992b, in press b). In Thysaniezia ovilla it was demonstrated by BB et al. (1991) that the crest-like body or bodies always mark the anterior extremity of the cestode spermatozoon. A
*To whom all correspondence should be addressed at: Laboratoire de Parasitologie, Departement de Biologie animale, Facultt des Sciences, Universite Ch. A. Diop de Dakar, Senegal.
spermiogenesis; ultrastructure; turtle doves;
study of spermiogenesis and the spermatozoon of another Anoplocephalidae, Mathevotaenia herpestis by Bb & Marchand (in press b) revealed for the first time the existence of centrioles flanked by ciliary roots in a Platyhelminth and a nucleus with an annular cross section in a Cestode. In the present work we describe the ultrastructure of spermiogenesis and the spermatozoon of A. delafondi. MATERIALS AND METHODS The specimens of A. delafondi were gathered from the small intestine of Streptopelia senegalensis (Laughing Dove) and kept alive for several hours in a 9%oNaCl solution. Seminal vesicles and testes were removed under a binocular microscope, then fixed for 24 h at 4°C with 2.5% glutaraldehyde in a 0.1 M-sodium cacodylate buffer at pH 7.2, 225
C. T. B.+iand B.
226 post-fixed
for 1 h at 4°C with 1% osmium tetroxide in the same buffer, then dehydrated with ethanol and propylene oxide before being embedded in epon. Ultrathin sections were cut on a Reichert-Jung Ultracut E ultramicrotome, then stained with uranyl acetate and lead citrate. They were examined in Siemens Elmiskop 101 and JEOL 100 C X II electron microscopes. Digestions by pronase to show up the protein, were carried out, according to the Monneron & Bernhard technique (1966) on sections of material embeded in epon. The sections were floated for 20 mn on 1% periodic acid, for 24 h at 37°C on a 1% pronase solution in a 0.1 M phosphate buffer at pH 7.2, before being stained with uranyl acetate and lead citrate.
RESULTS Spermiogenesis Spermiogenesis
in A. delafondi
begins
in each
spermatid with the formation of a differentiation zone bordered by sub-membranous cortical microtubules and containing two parallel centrioles (Figs. 1, 2 and 19a). One of the centrioles becomes orientated in a lateral cytoplasmic bud (Fig. 1) while the other remains in the axis of the differentiation zone and rapidly gives rise to a flagellum (Fig. 3). Very early on the nucleus forms a cone which interposes itself between the centrioles. At this point it contains a large, finely granular nucleolus (Fig. 2). From the beginning of the formation of the flagellum a transitory ring of arched membranes appears at the distal extremity of the differentiation zone (Figs. 3 and 19b). It disappears rapidly while a new ring appears encircling the anterior extremity of the differentiation zone. The cortical microtubules rapidly elongate and spiralize, while the lateral cytoplasmic bud grows, producing a large cytoplasmic extension. The lateral centriole never forms a flagellum. It stays in the spermatid cytoplasm, opposite the lateral cytoplasmic extension, always outside the ring of arched membranes (Figs. 4 and 19~). Later on the nucleus, which undergoes considerable elongation, becomes filiform (Fig. 5), moves into the new ring of arched membranes, then migrates along the flagellum, in the spermatid body (Figs. 6 and 19d). At this stage the chromatin is partially condensed and still only slightly electrondense, and the nucleus has a kidney-shaped cross section (Fig. 7). From the start of migration of the nucleus five fine cords of electron-dense material, of unequal length, which correspond to the crest-like bodies, appear in the anterior part of the differentiation zone, between the cortical microtubules (Figs. 6 and 7). Towards the end of spermiogenesis the ring formed by the new arched membranes narrows (Fig. 6) until it causes the residual cytoplasm to become detached from the old spermatid.
MARCHAND
Spermatozoon
From anterior to posterior we were able to distinguish five regions in the spermatozoon. There is no clear morphological discontinuity between them, but they nevertheless exhibit ultrastructural characters enabling them to be differentiated. Region I (Figs. 8, 11 and 201) is 0.15 to 0.25 m wide. It is capped by an apical cone of electron-dense material with a base of roughly 0.15 m and a length of 0.30 m (Fig. 8). The axoneme is in a central position and is surrounded by a fine layer of slightly electrondense material. The cortical microtubules are spiralized. They are accompanied on the outside by five helicoidal crest-like bodies of unequal length, roughly 15 to 40 q thick. In cross-section there thus appear one to five crest-like bodies associated with the subjacent layer of cortical microtubules. Region II (Figs. 12 and 2011) is between 0.25 and 0.30 m wide. Like the preceding one it exhibits spiralized cortical microtubules and a central axoneme surrounded by a fine layer of slightly dense cytoplasm; but it does not exhibit any crest-like bodies. Region III (Figs. 9, 13 and 20111), which is 0.300.50 p wide, is characterized by the presence of the nucleus. This is a compact cord coiled in a spiral round the axoneme (Fig. 9). In cross-section it appears in the shape of a horseshoe with regularly smooth internal and external surfaces (Fig. 13). The cytoplasm contains numerous small electron-dense granules and exhibits in places patches of electron-lucent material. The cortical microtubules are spiralized and appear in longitudinal section as a continuous layer of dense sub-membranous material (Fig. 9). Region IV (Figs. 10, 14, 15 and 201V) is 0.40 to 0.50 ,um wide. The axoneme is slightly eccentric (Fig. 14). The cytoplasm exhibits areas of electronlucent material (Fig. 14) and small granules of proteinaceous material (Fig. 15). The cortical microtubules describe a helix making an angle of about 20 with the spermatozoon axis (Fig. 10). Region V (Figs. 10, 16, 17, 18 and 20V) has a maximum width of 0.50 m. It is marked by the progressive disorganization of the axoneme. The central element disappears first (Fig. 16), then we see a simplification of the doublets into singlets (Fig. 17). The cytoplasm exhibits a few protein granules (Fig. 16), patches of electron-lucent material (Figs. 16 and 17) and a densification at the posterior extremity of the spermatozoon (Fig. 10). The cortical microtubules become parallel to the spermatozoon axis (Fig. 18) and then stop.
The presence
DISCUSSION of one or two centrioles
has been
Spertniogenesis
and spermatozoon
of A. delafondi
FIG. 1. Longitudinal section of a differentiation zone of Aporinu delafondi. One of the two centrioles (C) is oriented in a lateral cytoplasmic bud (B). Cm=cortical microtubules. Bar=05 p. FIG. 2. Longitudinal section of a differentiation zone. At the beginning of spermiogenesis, no ring of arched membranes can be seen at the anterior part of the differentiation zone (arrowheads). C = centriole; Cm = cortical microtubules; N = nucleus, n = nucleolus. Bar = 0.5 m. FIG. 3. Longitudinal section of a differentiation zone. Am= transitory ring of arched membranes; C = centriole; Cm = cortical microtubules; F = flagellum. Bar = 0.5 m.
221
C. T. BA and
B. MARCHAND
Spermiogenesis and spermatozoon reported in the differentiation zones of young cestode spermatids (B1 & Marchand, 1992b). At the very beginning of spermiogenesis the position of the nucleus in relation to these centrioles is very variable. In the Cyclophyllidea (Swiderski, Z. 1986. Abstract in Proceedings of the XZth International Congress of Electron Microscopy, Kyoto 2959-2960) the Pseudo-
phyllidea (Swiderski & Mokhtar-Maamouri, 1980; Swiderski, Z. 1986. Abstracts in Proceedings of the XZth International Kyoto 2959-2960)
Congress of Electron Microscopy,
the Diphyllidea (Azzouz-Draoui & Mokhtar-Maamouri, 1986) and a few Tetraphyllidea (Mokhtar-Maamouri & Swiderski, 1975; MokhtarMaamouri, 1982; Mahendrasingam, Fairweather 8~ Halton, 1989) the nucleus is still in the spermatid cytoplasm, outside the differentiation zone. On the other hand, as shown in a Haplobothrioidean, Haplobothrium globultforme by Mackinnon & Burt (1985) another Tetraphyllidean, Phyllobothrium gratile by Mokhtar-Maamouri (1979), a Proteocephalidean, Proteocephalus longicollis by Swiderski, Z. (1985. Abstract in Proceedings of the EIectron Microscopy Society of Southern Africa 15: 181-182) and two Cyclophyllidea, Nematotaenia chantalae by Mokhtar-Maamouri & Azzouz-Draoui (1990) and Thysaniezia ovilla by Bd et al. (199 1) the nucleus moves into the differentiation zone but still remains above the centrioles. In the particular case of Aporina delafondi the nucleus forms a cone, right from the start of spermiogenesis, which moves in, between the centrioles, in the differentiation zone. Rings of arched membranes have been described at the anterior end of the differentiation zones of the spermatids of a species of the Cestodaria-Amphilinidea, Austramphihna elongata by Rohde & Watson (1986) various Cestodes (Mokhtar-Maamouri & Swiderski, 1975; Mokhtar-Maamouri, 1979, 1982; Swiderski & Mokhtar-Maamouri, 1980; Mackinnon & Burt, 1985; Azzouz-Draoui & Mokhtar-Maamouri, 1986; Swiderski, Z. 1986. Abstract in Proceedings of the XZth International Congress of Electron Microscopy, Kyoto 2959-2960; Mahendrasingam et al., 1989;
FIG. 4. Longitudinal
of A. delafondi
229
Mokhtar-Maamouri & Azzouz-Draoui, 1990; Bd & Marchand, 1992b, in press b), Trematodes (Justine, 1991; Justine & Mattei, 1982a,b) and Monogenea (Justine & Mattei, 1983a,b, 1985). However, as shown in a few Turbellaria (Sopott-Ehlers, 1989; SopottEhlers, 1990) and in a Trematode, Schistosoma bovis by Justine (1982) arched membranes are not formed during spermiogenesis. In S. bovis in particular, the spermatozoa are formed after fragmentation of the cytoplasm of the old spermatid. In the particular case of A. delafondi, contrary to what happens in the other Platyhelminthes, the arched membranes first form at the posterior end of the differentiation zone. A cytoplasmic bud associated with a centriole, in the young Platyhelminth spermatids, has already been described in a Monogenean, Heterocotyie sp. by Justine KcMattei (1986) a species of the Cestodaria, Austramphilina elongata by Rohde & Watson (1986) and a few Cestodes, a Diphyllidean, Echinobothrium harfordi by Azzouz-Draoui & Mokhtar-Maamouri (1986) a Tetraphyllidean, Phyllobothrium gracile by Mokhtar-Maamouri (1979), and three Cyclophyllidea, Nematotaenia chantalae by Mokhtar-Maamouri & Azzouz-Draoui (1990), Thysaniezia ovifla by Bb et al. (1991) and Mathevotaenia herpestis by Bb & Marchand (in press b). In Austramphilina elongata and Heterocotyle sp. this centriole gives rise to a flagellum. In the Cestodes, on the other hand, when a centriole is situated in a cytoplasmic bud of the spermatid, it never forms a flagellum. In E. harfordi and N. chantalae it migrates at the same time as the nucleus in the spermatid body, where it becomes disorganized at the end of spermiogenesis. In Th. ovilla and M. herpestis the proximal centriole along with the cytoplasmic bud that is associated with it abort in the old spermatid, outside the ring of arched membranes. Here again A. delafondi is distinguished from the other Platyhelminthes by the substantial development of the cytoplasmic bud which persists right to the end of spermiogenesis. A single crest-like body has been described during
section of a differentiation zone of Aporina delafondi. Am = new ring of arched membranes; axoneme; B = cytoplasmic bud; C = centriole; Cm = cortical microtubules. Bar = 1 m.
Ax =
FIG. 5. Longitudinal section of a spermatid. At the time of migration, the nucleus becomes filiform and its chromatin is electron-lucent. Am = new ring of arched membranes; Ax = axoneme; Cm = cortical microtubules; N = nucleus. Bar = 0.5 /sn. FIG. 6. Longitudinal section of a spermatid. During the migration of the nucleus (N) around the axoneme (Ax), crest-like bodies (Cb) appear between the cortical microtubules (Cm) and the plasma membrane of the differentiation zone. The bent arrows show the strangulation of arched membranes at the end of spermiogenesis. Bar = 1 m. FIG. 7. Cross-section
of a differentiation
zone during
the migration of the nucleus (N). Ax = axoneme; Bar=O.S p.
Cb = crest-like
body.
230
C.T. BA and B.MARCHAND
Spermiogenesis
and spermatozoon
spermiogenesis of the Monogenea, Calceostoma by Justine & Mattei (1986). In the Cestodes the crest-like body (or bodies) forms after the migration of the nucleus in the differentiation zone of the old spermatids and usually marks the end of spermiogenesis as shown by Mokhtar-Maamouri, F. (unpublished thesis, University of Montpellier II, 1976), MokhtarMaamouri (1979, 1982), Mokhtar-Maamouri & Swiderski (1975), Azzouz-Draoui & MokhtarMamouri (1986), Mahendrasingam et al. (1989), Mokhtar-Maamouri & Azzouz-Draoui (1990), BB et al. (1991), and Bd & Marchand (in press b). However, in one cestode, Hymenolepis nana, we were able to observe crest-like bodies from the start of spermiogenesis (Ba & Marchand, 1992b). A. delafondi is therefore again distinguished from the other Platyhelminthes by the formation of its crest-like bodies during the migration of the nucleus. We demonstrated previously that the crest-like bodies (l-12) are always situated at the anterior extremity of the Cestode spermatozoon (Ba et al., 1991). The extremity with crest-like bodies (region I) of the Aporina delafondi spermatozoon corresponds therefore, as in the other Cestodes, to its anterior extremity, and the one without crest-like bodies to its posterior extremity. Spermatozoa possessing a single crest-like body
of A. delafondi
231
have been described in a Pseudophyllidean by Justine (1986), a few Diphyllidea (Azzouz-Draoui, N., unpublished thesis, University of Tunis, 1985; Azzouz-Draoui & Mokhtar-Maamouri, 1986), all the Tetraphyllidea studied (Mokhtar-Maamouri, F. unpublished thesis, University of Montpellier II, 1976; Mokhtar-Maamouri, 1979, 1982; Mokhtar-Maamouri & Swiderski, 1975; Mackinnon & Burt, 1984; Mahendrasingam et al., 1989) and a few Cyclophyllidea (Mokhtar-Maamouri & Azzouz-Draoui, 1990; BB & Marchand, 1992a,c). Spermatozoa exhibiting two crest-like bodies have been observed in some Cyclophyllidea (Bd et al., 1991; Bd & Marchand, 1992~). In two Cyclophyllidea Hymenolepididae, six crest-like bodies have been found in the Retinometra serrata spermatozoon (Bb & Marchand, 1993) and twelve in that of Hymenolepis nana by Bb & Marchand (1992b). Anterioly in the A. delafondi spermatozoon we counted five of different lengths. To our knowledge such a large number of crest-like bodies has never before been reported in an Anoplocephalidae. The maximum number was two, found in Thysaniezia ovilla by BB et al. (1991), Moniezia expansa and Moniezia benedeni by Bb & Marchand (1992~). The crest-like bodies measure roughly 30 to 60 nm in thickness in Moniezia expansa and 30 to 40 nm in M. benedeni as shown by BB & Marchand (1992~). The
FIG. 8. Longitudinal section of region I of the mature Aporina delafondi spermatozoon. AC= apical cone; Ax = axoneme; Cb = crest-like body; Cm = cortical microtubules. Bar = 1 jnn. FIG. 9. Longitudinal section of region III microtubules; G = granules of electron-dense FIG. 10. Longitudinal
section
FIG. 11. Cross-section FIG. 12. Cross-section FIG. 13. Cross-section
FIG. 14. Cross-section
of regions
of region
of the mature A. delafondi spermatozoon. Ax =axoneme; material; N = nucleus; P = patch of electron-lucent material.
IV and V of the mature A. delafondi spermatozoon. Bar= 1 p.
I of the mature
of region II of the mature of region
A. delafondi spermatozoon.
III of the mature
of region IV of the mature
A. delafondi spermatozoon.
Cb =crest-like Cm = cortical
A. delafondi spermatozoon. Bar=0.5 m.
A. delufondi spermatozoon. w.
Cm = cortical
body.
microtubules;
P = patch of electron-lucent
microtubules.
Bar = 0.5 m.
microtubules.
Cm = cortical
Cm=cortical Bar = 0.5 m.
Bar = 0.5 m. N = nucleus.
material.
Bar = 0.5
FIG. 15. Cross-section of region IV of the mature A. delafondi spermatozoon. Digestion by pronase for 6 h at 37°C of the electron-dense granules (G) observed in Fig. 9 in region III of the spermatozoon. Bar=0.5 p. FIG. 16. Cross-section
of region V of the mature A. delafondi spermatozoon. element; P = patch of electron-lucent material.
FIG. 17. Cross-section
of region V of the mature
A. delafondispermatozoon.
D = axonemal Bar = 0.5 p.
doublet;
E = axonemal
central
P = patch of electron-lucent material; S = singlet.
Bar=0.5 m. FIG. 18. Cross-section
of region V of the mature
A. dehfondi spermatozoon. w.
Cm = cortical microtubules;
S = singlet. Bar = 0.5
232
C. T. BA and B. MARCHAND
_-__________________--____ ___
---____ _______________-____ ___--_
yg( )U/ N
N
Cm
B
I
/I
I
Am2
C
a
B Cm
C
_________________________ _--
Y&J N
Cm
C
B
II
Am1
-F
L
/
b
d
FIG. 19(a-d). Attempted reconstruction of the main stages of spermiogenesis of Aporina delafondi. (a) Organization of the differentiation zone at the beginning of spermiogenesis. (b) Rapid formation of a flagellum and a transitory ring of arched membranes at the distal part of the differentiation zone. (c) Formation of a new ring of arched membranes at the proximal part of the differentiation zone, rapid elongation of cortical microtubules and growth of the lateral cytoplasmic bud. (d) Migration of the nucleus in the body of the spermatid and appearance of crest-like bodies followed by the constriction of the new ring of arched membranes (arrows), then the separation of the old spermatid from the residual cytoplasm. To make the diagram clearer, the spiral coil of the cortical microtubules has not been shown. Am1 = transitory ring of arched membranes; Am2 = new ring of arched membranes; B = cytoplasmic bud; C = centriole; Cb = crest-like bodies; Cm = cortical microtubules; F = flagellum; N = nucleus.
Spermiogenesis and spermatozoon
of A. delafondi
233
thickest reach 200 nm and have been described in Avitellina centripunctata by B1& Marchand (in press a). In A. delafondi the crest-like bodies are 15 to 40 nm
I
HiCm
II
thick. Spiralization of the cortical microtubules was recently considered to be a synapomorphy of the Cyclophyllidea (Justine, 1991). The angle of spiralization of these microtubules is more or less marked. It has been estimated as 50” in Stilesia globipunctata by Bb & Marchand (1992a), 40” in M. expansa by Swiderski (1968) and Ba & Marchand (1992c), 40” in M. herpestis by Bb & Marchand (in press b), 35” in A. centripunctata by Bb & Marchand (in press a), 25” in R. serrata by Bb & Marchand (1993) and 15” in H. nana by BB & Marchand (1992b). In A. delafondi the cortical microtubules are also spiralized, with the coil making a relatively small angle of roughly 20”. The cytoplasm of the cestode spermatozoon is generally of low electron density. However, we have already revealed electron-dense material at the posterior extremity of the spermatozoa of A. centripunctata, M. herpestis and R. serrata. In A. centripunctata, only the apical cone and region IV of the spermatozoon are fairly electron-dense. In H. nana, on the other hand, the cytoplasm is electron-lucent in regions I and II of the spermatozoon and slightly electron-dense over all the rest. The cytoplasm of the A. delafondi spermatozoon is fairly electron-dense in regions I and II whereas in regions III, IV and V it exhibits protein granules and patches of electron-lucent material. As far as we know these lucent patches have never been reported before in the spermatozoon of a Cestode. According to Schmidt (1986) the Hymenolepididae make up a family “closely related to Dilepididae and Anoplocephalidae, the three grading together so much that it sometimes is arbitrary to decide which family should receive certain genera”. The crest-like bodies of the A. delafondi spermatozoon, by their position outside the cortical microtubules, the angle of spiralization, their morphology, thickness and even their number, are closer to those of the Hymenolepididae (Bb & Marchand, 1992b, 1993) than those of the Anoplocephalidae already studied (Ba & Marchand,
Ax
FIG. 20. Attempted reconstruction of the mature spermatozoon of Aporinadelafondiin which we distinguish five regions (I-V) from anterior to posterior. To make the diagram clearer, the spiral coil of the cortical microtubules has not been shown. Aae = axonemal anterior extremity; AC = apical cone; Ape = axonemal posterior extremity; Ax = axoneme; Cb = crest-like bodies; Cm = cortical microtubules; Dm = electron-dense material; G = granular electron-dense material; N = nucleus; P = patch of electron-lucent material.
234
C. T. BA and B. MARCHAND
1992a,c; in press a, b). This ultrastructural quasisimilitude between the A. delufondi spermatozoa and those of the Hymenolepididae poses the problem of whether the genus Aporina belongs to the family Anoplocephalidae or that of Hymenolepididae. A study of the spermatozoon and spermiogenesis of a greater number of representatives of these two families should enable us to answer this question. REFERENCES
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JUSTINEJ. L. & MA~EI X. 1986. Comparative ultrastructural study of spermiogenesis in monogeneans (Flatworms). 5. Calceostoma (Monopisthocotylea, Calceostomatidae). Journal of Ultrastructure and Molecular Structure Research 96: 54-63.
JUSTINEJ. L. 1991. Phylogeny of Parasitic Platyhelminthes: a critical study of synapomorphies proposed on the basis of the ultrastructure of spermiogenesis and spermatozoa. Canadian Journal of Zoology 69: 1421-1440.
KELSOEG. H., UBELAKKER J. E. &ALLISONV. F. 1977. The fine structure of spermatogenesis in Hymenolepis diminuta (Cestoda) with a description of the mature spermatozoon. Zeitschrijt fui Parasitenkunde 54: 175-187. MACKINNON B. M. & BURTM. D. B. 1984. The comparative ultrastructure of spermatozoa from Bothrimonus sturionis Duv. 1842 (Pseudophyllidea), Pseudanthobothrium hanseni Baer, 1956 (Teraphyllidea) and Monoecocestus americanus Stiles, 1895 (Cyclophyllidea). Canadian Journal of Zoology 62: 1059-10696.
MACKINNONB. M. &BURT M. D. B. 1985. Ultrastructure of spermatogenesis and the mature spermatozoon of Haplobothrium globuliforme Cooper, 1914 (Cestoda: Haplobothrioidea). Canadian Journal of Zoology 63: 1478-1487. MAHENDRASINGHAM S., FAIRWEATHER I. & HALTOND. W. 1989. Spermatogenesis and the fine structure of the mature spermatozoon in the free proglottis of Trilocularia acanthiaevulgaris (Cestoda, Tetraphyllidea). Parasitology Research 75: 287-298.
MOKHTAR-MAAMOURIF. 1979. Etude en microscopic Clectronique de la spermiogentse et du spermatozoide de Phyllobothrium gracile Weld, 1955 (Cestoda, Tetraphyllidea, Phyllobothriidae). Zeitschrift fir Parasitenkunde 59: 245-258.
MOKHTAR-MAAMOURI F. 1982. Etude ultrastructurale de la spermiogenese de Acanthobothrium jilicolle var. Jilicolle Zschokke, 1888 (Cestoda, Tetraphyllidea, Onchobothriidae). Annales de Parasitologie Humaine et Compar& 57: 429442.
MOKHTAR-MAAMOURIF. & Azzouz-DR~OUI N. 1990. Etude de la spcrmioge&se et de l’ultrastructure du
Spetmiogenesis
and spermatozoon
spermatozoide de Nematotaenia chantaiae Dollfus, 1957 (Cestoda, Cyclophyllidea, Nematotaeniidae). Annales de Parasitologie
Humaine et Comparee 65: 221-228.
MOKHTAR-MAAMOURI F. & SWIDERSKI2. 1975. Etude en microscopic electronique de la spermatogentse de deux cestodes Acanthobothrium filicolie benedenii Loennberg, 1889et Onchobothrium uncinatum (Rud., 1819)(Tetraphyllidea, Onchobothriidae). Zeitschrzfi fur Parasitenkunde 47: 269-281. MONNERONA. KLBERNHARDW. 1966. Action de certaines enzymes sur des tissus inclus en Epon. Journal de Microscopic 5: 697-714. ROHDED. & WATSONN. 1986. Ultrastructure of spermiogenesis and sperm of Austramphiiina elongata (Platyhelminthes, Amphilinidea). Journal of Submicroscopic Cytology 18: 361-374. ROBINSON J. M. & BCKXTSH B. J. 1978. A morphological and cytochemical study of sperm development in Hymenolepis diminuta. Zeitschrift fur Parasitenkunde 56: 81-92. ROSARIO B. 1964. An electron microscope study of
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SCHMIDT G. D. 1986. CRC Handbook of Tapeworm Identz3cation. CRC Press, Boca Raton, FL. SOFQT~-EHLERS B. 1989. On the spermiogenesis of Invenusta aestus (Platyhelminthes, Proseriata) An ulstrastructural study with implications for platyhelminth phylogeny. Zoomorphology
109: 145-152.
SOPOTT-EHLERS B. 1990. Functional aspects of the intercentriolar body in the spermiogenesis of Nematoplana coeiogynoporoides (Platyhelminthes, Proseriata). Zoomorphology 109: 245-249.
SWIDERSKI Z. 1968. The fine structure ofthe spermatozoon of sheep tapeworm, Moniezia expansa (Rud., 1810) Cyclophyllidea, Anoplocephalidae. Zoologica Poloniae 18:475 486.
SWIDERSKI Z & MOKHTAR-MAAMOURI F. 1980. Etude de la spennatogenese de Bothriocephalus clavibothrium Ariola, 1899 (Cestoda, Pseudophyllidea). Archives de I’lnstitut Pasteur de Tunis 57: 323-347.
Pergamon
Journalfor Parasitology. Vol. 24, No. 2, pp. 225-235, 1994 Copyright 0 1994 Australian Society for Parasitology Elsetier Science Ltd Printed in Great Britain. All rights resewed 002&7519/94 S7.00+ 0.00
ULTRASTRUCTURE OF SPERMIOGENESIS AND THE SPERMATOZOON OF APORINA DELAFONDI (CYCLOPHYLLIDEA, ANOPLOCEPHALIDAE), INTESTINAL PARASITE OF TURTLE DOVES IN SENEGAL CHEIKH TIDIANE BA* and BERNARD
MARCHAND
Laboratory of Parasitology, Department of Animal Biology, Faculty of Sciences, Cheikh Anta Diop University of Dakar, Dakar, Senegal (Received 6 April 1993; accepted 26 August 1993)
Abstract-B.4 C. T. and MARCHAND B. 1994. Ultrastructure of spermiogenesis and the spermatozoon of Aporina delafondi (Cyclophyllidea, Anoplocephalidae), intestinal parasite of turtle doves in Senegal. International Journal for Parasitology 24: 22>235. Spermiogenesis in Aporina delafondi begins with the formation of a differentiation zone bordered by cortical microtubules and containing from the beginning a portion of nucleus and two parallel centrioles. One of the centrioles aborts, the other gives rise to a flagellum. The cortical microtubules elongate and spiralize while the nucleus migrates along the axoneme and crest-like bodies form at the level of the differentiation zone. The old spermatid separates from the residual cytoplasm by strangulation of the ring of arched membranes. The mature spermatozoon lacks mitochondria, is tiliform and tapered at both its extremities. Its anterior extremity is capped by an apical cone of electron-dense material and exhibits five crest-like bodies of unequal lengths on its periphery. Its cortical microtubules are regularly spiralized except at their posterior extremity where they become parallel to the spermatozoon axis. The cytoplasm is slightly dense in the anterior regions (I and II) and exhibits many protein granules and patches of electron-lucent material in the middle (III) and posterior zones (IV and V). The nucleus is an electron-dense cord coiled in a spiral around the middle region (III) of the axoneme. This is of the 9 + “1” pattern and ends before the posterior extremity of the gamete. Spermiogenesis in Aporina delafondi differs from that of the other Cyclophyllidea by the very early movement of the nucleus into the differentiation zone, the formation of a ring of arched membranes in the distal part of the differentiation zone, the appearance of crest-like bodies during migration of the nucleus and the formation of a cytoplasmic bud which contains the abortive centriole and develops to temporarily form a large lateral extension. The mature spermatozoon differs from that of the other Cyclophyllidea in the presence of lucent patches in its cytoplasm and of five helicoidal crest-like bodies. The systematic position of the genus Aporina is also debated. INDEX KEY WORDS: Aporina aklafondi; spermatozoon; Cyclophyllidea; Anoplocephalidae; Senegal
INTRODUCTION ONLY a few authors to date have taken an interest in spermiogenesis of the Cyclophyllidea (Rosario, 1964; Featherston, 1971; Kelsoe, Ubelakker & Allison, 1977; Robinson & Bogitsh, 1978; Mokhtar-Maamouri & Azzouz-Draoui, 1990; BB, Marchand & Mattei, 1991; BP & Marchand, 1992b, in press b). In Thysaniezia ovilla it was demonstrated by BB et al. (1991) that the crest-like body or bodies always mark the anterior extremity of the cestode spermatozoon. A
*To whom all correspondence should be addressed at: Laboratoire de Parasitologie, Departement de Biologie animale, Facultt des Sciences, Universite Ch. A. Diop de Dakar, Senegal.
spermiogenesis; ultrastructure; turtle doves;
study of spermiogenesis and the spermatozoon of another Anoplocephalidae, Mathevotaenia herpestis by Bb & Marchand (in press b) revealed for the first time the existence of centrioles flanked by ciliary roots in a Platyhelminth and a nucleus with an annular cross section in a Cestode. In the present work we describe the ultrastructure of spermiogenesis and the spermatozoon of A. delafondi. MATERIALS AND METHODS The specimens of A. delafondi were gathered from the small intestine of Streptopelia senegalensis (Laughing Dove) and kept alive for several hours in a 9%oNaCl solution. Seminal vesicles and testes were removed under a binocular microscope, then fixed for 24 h at 4°C with 2.5% glutaraldehyde in a 0.1 M-sodium cacodylate buffer at pH 7.2, 225
C. T. B.+iand B.
226 post-fixed
for 1 h at 4°C with 1% osmium tetroxide in the same buffer, then dehydrated with ethanol and propylene oxide before being embedded in epon. Ultrathin sections were cut on a Reichert-Jung Ultracut E ultramicrotome, then stained with uranyl acetate and lead citrate. They were examined in Siemens Elmiskop 101 and JEOL 100 C X II electron microscopes. Digestions by pronase to show up the protein, were carried out, according to the Monneron & Bernhard technique (1966) on sections of material embeded in epon. The sections were floated for 20 mn on 1% periodic acid, for 24 h at 37°C on a 1% pronase solution in a 0.1 M phosphate buffer at pH 7.2, before being stained with uranyl acetate and lead citrate.
RESULTS Spermiogenesis Spermiogenesis
in A. delafondi
begins
in each
spermatid with the formation of a differentiation zone bordered by sub-membranous cortical microtubules and containing two parallel centrioles (Figs. 1, 2 and 19a). One of the centrioles becomes orientated in a lateral cytoplasmic bud (Fig. 1) while the other remains in the axis of the differentiation zone and rapidly gives rise to a flagellum (Fig. 3). Very early on the nucleus forms a cone which interposes itself between the centrioles. At this point it contains a large, finely granular nucleolus (Fig. 2). From the beginning of the formation of the flagellum a transitory ring of arched membranes appears at the distal extremity of the differentiation zone (Figs. 3 and 19b). It disappears rapidly while a new ring appears encircling the anterior extremity of the differentiation zone. The cortical microtubules rapidly elongate and spiralize, while the lateral cytoplasmic bud grows, producing a large cytoplasmic extension. The lateral centriole never forms a flagellum. It stays in the spermatid cytoplasm, opposite the lateral cytoplasmic extension, always outside the ring of arched membranes (Figs. 4 and 19~). Later on the nucleus, which undergoes considerable elongation, becomes filiform (Fig. 5), moves into the new ring of arched membranes, then migrates along the flagellum, in the spermatid body (Figs. 6 and 19d). At this stage the chromatin is partially condensed and still only slightly electrondense, and the nucleus has a kidney-shaped cross section (Fig. 7). From the start of migration of the nucleus five fine cords of electron-dense material, of unequal length, which correspond to the crest-like bodies, appear in the anterior part of the differentiation zone, between the cortical microtubules (Figs. 6 and 7). Towards the end of spermiogenesis the ring formed by the new arched membranes narrows (Fig. 6) until it causes the residual cytoplasm to become detached from the old spermatid.
MARCHAND
Spermatozoon
From anterior to posterior we were able to distinguish five regions in the spermatozoon. There is no clear morphological discontinuity between them, but they nevertheless exhibit ultrastructural characters enabling them to be differentiated. Region I (Figs. 8, 11 and 201) is 0.15 to 0.25 m wide. It is capped by an apical cone of electron-dense material with a base of roughly 0.15 m and a length of 0.30 m (Fig. 8). The axoneme is in a central position and is surrounded by a fine layer of slightly electrondense material. The cortical microtubules are spiralized. They are accompanied on the outside by five helicoidal crest-like bodies of unequal length, roughly 15 to 40 q thick. In cross-section there thus appear one to five crest-like bodies associated with the subjacent layer of cortical microtubules. Region II (Figs. 12 and 2011) is between 0.25 and 0.30 m wide. Like the preceding one it exhibits spiralized cortical microtubules and a central axoneme surrounded by a fine layer of slightly dense cytoplasm; but it does not exhibit any crest-like bodies. Region III (Figs. 9, 13 and 20111), which is 0.300.50 p wide, is characterized by the presence of the nucleus. This is a compact cord coiled in a spiral round the axoneme (Fig. 9). In cross-section it appears in the shape of a horseshoe with regularly smooth internal and external surfaces (Fig. 13). The cytoplasm contains numerous small electron-dense granules and exhibits in places patches of electron-lucent material. The cortical microtubules are spiralized and appear in longitudinal section as a continuous layer of dense sub-membranous material (Fig. 9). Region IV (Figs. 10, 14, 15 and 201V) is 0.40 to 0.50 ,um wide. The axoneme is slightly eccentric (Fig. 14). The cytoplasm exhibits areas of electronlucent material (Fig. 14) and small granules of proteinaceous material (Fig. 15). The cortical microtubules describe a helix making an angle of about 20 with the spermatozoon axis (Fig. 10). Region V (Figs. 10, 16, 17, 18 and 20V) has a maximum width of 0.50 m. It is marked by the progressive disorganization of the axoneme. The central element disappears first (Fig. 16), then we see a simplification of the doublets into singlets (Fig. 17). The cytoplasm exhibits a few protein granules (Fig. 16), patches of electron-lucent material (Figs. 16 and 17) and a densification at the posterior extremity of the spermatozoon (Fig. 10). The cortical microtubules become parallel to the spermatozoon axis (Fig. 18) and then stop.
The presence
DISCUSSION of one or two centrioles
has been
Spertniogenesis
and spermatozoon
of A. delafondi
FIG. 1. Longitudinal section of a differentiation zone of Aporinu delafondi. One of the two centrioles (C) is oriented in a lateral cytoplasmic bud (B). Cm=cortical microtubules. Bar=05 p. FIG. 2. Longitudinal section of a differentiation zone. At the beginning of spermiogenesis, no ring of arched membranes can be seen at the anterior part of the differentiation zone (arrowheads). C = centriole; Cm = cortical microtubules; N = nucleus, n = nucleolus. Bar = 0.5 m. FIG. 3. Longitudinal section of a differentiation zone. Am= transitory ring of arched membranes; C = centriole; Cm = cortical microtubules; F = flagellum. Bar = 0.5 m.
221
C. T. BA and
B. MARCHAND
Spermiogenesis and spermatozoon reported in the differentiation zones of young cestode spermatids (B1 & Marchand, 1992b). At the very beginning of spermiogenesis the position of the nucleus in relation to these centrioles is very variable. In the Cyclophyllidea (Swiderski, Z. 1986. Abstract in Proceedings of the XZth International Congress of Electron Microscopy, Kyoto 2959-2960) the Pseudo-
phyllidea (Swiderski & Mokhtar-Maamouri, 1980; Swiderski, Z. 1986. Abstracts in Proceedings of the XZth International Kyoto 2959-2960)
Congress of Electron Microscopy,
the Diphyllidea (Azzouz-Draoui & Mokhtar-Maamouri, 1986) and a few Tetraphyllidea (Mokhtar-Maamouri & Swiderski, 1975; MokhtarMaamouri, 1982; Mahendrasingam, Fairweather 8~ Halton, 1989) the nucleus is still in the spermatid cytoplasm, outside the differentiation zone. On the other hand, as shown in a Haplobothrioidean, Haplobothrium globultforme by Mackinnon & Burt (1985) another Tetraphyllidean, Phyllobothrium gratile by Mokhtar-Maamouri (1979), a Proteocephalidean, Proteocephalus longicollis by Swiderski, Z. (1985. Abstract in Proceedings of the EIectron Microscopy Society of Southern Africa 15: 181-182) and two Cyclophyllidea, Nematotaenia chantalae by Mokhtar-Maamouri & Azzouz-Draoui (1990) and Thysaniezia ovilla by Bd et al. (199 1) the nucleus moves into the differentiation zone but still remains above the centrioles. In the particular case of Aporina delafondi the nucleus forms a cone, right from the start of spermiogenesis, which moves in, between the centrioles, in the differentiation zone. Rings of arched membranes have been described at the anterior end of the differentiation zones of the spermatids of a species of the Cestodaria-Amphilinidea, Austramphihna elongata by Rohde & Watson (1986) various Cestodes (Mokhtar-Maamouri & Swiderski, 1975; Mokhtar-Maamouri, 1979, 1982; Swiderski & Mokhtar-Maamouri, 1980; Mackinnon & Burt, 1985; Azzouz-Draoui & Mokhtar-Maamouri, 1986; Swiderski, Z. 1986. Abstract in Proceedings of the XZth International Congress of Electron Microscopy, Kyoto 2959-2960; Mahendrasingam et al., 1989;
FIG. 4. Longitudinal
of A. delafondi
229
Mokhtar-Maamouri & Azzouz-Draoui, 1990; Bd & Marchand, 1992b, in press b), Trematodes (Justine, 1991; Justine & Mattei, 1982a,b) and Monogenea (Justine & Mattei, 1983a,b, 1985). However, as shown in a few Turbellaria (Sopott-Ehlers, 1989; SopottEhlers, 1990) and in a Trematode, Schistosoma bovis by Justine (1982) arched membranes are not formed during spermiogenesis. In S. bovis in particular, the spermatozoa are formed after fragmentation of the cytoplasm of the old spermatid. In the particular case of A. delafondi, contrary to what happens in the other Platyhelminthes, the arched membranes first form at the posterior end of the differentiation zone. A cytoplasmic bud associated with a centriole, in the young Platyhelminth spermatids, has already been described in a Monogenean, Heterocotyie sp. by Justine KcMattei (1986) a species of the Cestodaria, Austramphilina elongata by Rohde & Watson (1986) and a few Cestodes, a Diphyllidean, Echinobothrium harfordi by Azzouz-Draoui & Mokhtar-Maamouri (1986) a Tetraphyllidean, Phyllobothrium gracile by Mokhtar-Maamouri (1979), and three Cyclophyllidea, Nematotaenia chantalae by Mokhtar-Maamouri & Azzouz-Draoui (1990), Thysaniezia ovifla by Bb et al. (1991) and Mathevotaenia herpestis by Bb & Marchand (in press b). In Austramphilina elongata and Heterocotyle sp. this centriole gives rise to a flagellum. In the Cestodes, on the other hand, when a centriole is situated in a cytoplasmic bud of the spermatid, it never forms a flagellum. In E. harfordi and N. chantalae it migrates at the same time as the nucleus in the spermatid body, where it becomes disorganized at the end of spermiogenesis. In Th. ovilla and M. herpestis the proximal centriole along with the cytoplasmic bud that is associated with it abort in the old spermatid, outside the ring of arched membranes. Here again A. delafondi is distinguished from the other Platyhelminthes by the substantial development of the cytoplasmic bud which persists right to the end of spermiogenesis. A single crest-like body has been described during
section of a differentiation zone of Aporina delafondi. Am = new ring of arched membranes; axoneme; B = cytoplasmic bud; C = centriole; Cm = cortical microtubules. Bar = 1 m.
Ax =
FIG. 5. Longitudinal section of a spermatid. At the time of migration, the nucleus becomes filiform and its chromatin is electron-lucent. Am = new ring of arched membranes; Ax = axoneme; Cm = cortical microtubules; N = nucleus. Bar = 0.5 /sn. FIG. 6. Longitudinal section of a spermatid. During the migration of the nucleus (N) around the axoneme (Ax), crest-like bodies (Cb) appear between the cortical microtubules (Cm) and the plasma membrane of the differentiation zone. The bent arrows show the strangulation of arched membranes at the end of spermiogenesis. Bar = 1 m. FIG. 7. Cross-section
of a differentiation
zone during
the migration of the nucleus (N). Ax = axoneme; Bar=O.S p.
Cb = crest-like
body.
230
C.T. BA and B.MARCHAND
Spermiogenesis
and spermatozoon
spermiogenesis of the Monogenea, Calceostoma by Justine & Mattei (1986). In the Cestodes the crest-like body (or bodies) forms after the migration of the nucleus in the differentiation zone of the old spermatids and usually marks the end of spermiogenesis as shown by Mokhtar-Maamouri, F. (unpublished thesis, University of Montpellier II, 1976), MokhtarMaamouri (1979, 1982), Mokhtar-Maamouri & Swiderski (1975), Azzouz-Draoui & MokhtarMamouri (1986), Mahendrasingam et al. (1989), Mokhtar-Maamouri & Azzouz-Draoui (1990), BB et al. (1991), and Bd & Marchand (in press b). However, in one cestode, Hymenolepis nana, we were able to observe crest-like bodies from the start of spermiogenesis (Ba & Marchand, 1992b). A. delafondi is therefore again distinguished from the other Platyhelminthes by the formation of its crest-like bodies during the migration of the nucleus. We demonstrated previously that the crest-like bodies (l-12) are always situated at the anterior extremity of the Cestode spermatozoon (Ba et al., 1991). The extremity with crest-like bodies (region I) of the Aporina delafondi spermatozoon corresponds therefore, as in the other Cestodes, to its anterior extremity, and the one without crest-like bodies to its posterior extremity. Spermatozoa possessing a single crest-like body
of A. delafondi
231
have been described in a Pseudophyllidean by Justine (1986), a few Diphyllidea (Azzouz-Draoui, N., unpublished thesis, University of Tunis, 1985; Azzouz-Draoui & Mokhtar-Maamouri, 1986), all the Tetraphyllidea studied (Mokhtar-Maamouri, F. unpublished thesis, University of Montpellier II, 1976; Mokhtar-Maamouri, 1979, 1982; Mokhtar-Maamouri & Swiderski, 1975; Mackinnon & Burt, 1984; Mahendrasingam et al., 1989) and a few Cyclophyllidea (Mokhtar-Maamouri & Azzouz-Draoui, 1990; BB & Marchand, 1992a,c). Spermatozoa exhibiting two crest-like bodies have been observed in some Cyclophyllidea (Bd et al., 1991; Bd & Marchand, 1992~). In two Cyclophyllidea Hymenolepididae, six crest-like bodies have been found in the Retinometra serrata spermatozoon (Bb & Marchand, 1993) and twelve in that of Hymenolepis nana by Bb & Marchand (1992b). Anterioly in the A. delafondi spermatozoon we counted five of different lengths. To our knowledge such a large number of crest-like bodies has never before been reported in an Anoplocephalidae. The maximum number was two, found in Thysaniezia ovilla by BB et al. (1991), Moniezia expansa and Moniezia benedeni by Bb & Marchand (1992~). The crest-like bodies measure roughly 30 to 60 nm in thickness in Moniezia expansa and 30 to 40 nm in M. benedeni as shown by BB & Marchand (1992~). The
FIG. 8. Longitudinal section of region I of the mature Aporina delafondi spermatozoon. AC= apical cone; Ax = axoneme; Cb = crest-like body; Cm = cortical microtubules. Bar = 1 jnn. FIG. 9. Longitudinal section of region III microtubules; G = granules of electron-dense FIG. 10. Longitudinal
section
FIG. 11. Cross-section FIG. 12. Cross-section FIG. 13. Cross-section
FIG. 14. Cross-section
of regions
of region
of the mature A. delafondi spermatozoon. Ax =axoneme; material; N = nucleus; P = patch of electron-lucent material.
IV and V of the mature A. delafondi spermatozoon. Bar= 1 p.
I of the mature
of region II of the mature of region
A. delafondi spermatozoon.
III of the mature
of region IV of the mature
A. delafondi spermatozoon.
Cb =crest-like Cm = cortical
A. delafondi spermatozoon. Bar=0.5 m.
A. delufondi spermatozoon. w.
Cm = cortical
body.
microtubules;
P = patch of electron-lucent
microtubules.
Bar = 0.5 m.
microtubules.
Cm = cortical
Cm=cortical Bar = 0.5 m.
Bar = 0.5 m. N = nucleus.
material.
Bar = 0.5
FIG. 15. Cross-section of region IV of the mature A. delafondi spermatozoon. Digestion by pronase for 6 h at 37°C of the electron-dense granules (G) observed in Fig. 9 in region III of the spermatozoon. Bar=0.5 p. FIG. 16. Cross-section
of region V of the mature A. delafondi spermatozoon. element; P = patch of electron-lucent material.
FIG. 17. Cross-section
of region V of the mature
A. delafondispermatozoon.
D = axonemal Bar = 0.5 p.
doublet;
E = axonemal
central
P = patch of electron-lucent material; S = singlet.
Bar=0.5 m. FIG. 18. Cross-section
of region V of the mature
A. dehfondi spermatozoon. w.
Cm = cortical microtubules;
S = singlet. Bar = 0.5
232
C. T. BA and B. MARCHAND
_-__________________--____ ___
---____ _______________-____ ___--_
yg( )U/ N
N
Cm
B
I
/I
I
Am2
C
a
B Cm
C
_________________________ _--
Y&J N
Cm
C
B
II
Am1
-F
L
/
b
d
FIG. 19(a-d). Attempted reconstruction of the main stages of spermiogenesis of Aporina delafondi. (a) Organization of the differentiation zone at the beginning of spermiogenesis. (b) Rapid formation of a flagellum and a transitory ring of arched membranes at the distal part of the differentiation zone. (c) Formation of a new ring of arched membranes at the proximal part of the differentiation zone, rapid elongation of cortical microtubules and growth of the lateral cytoplasmic bud. (d) Migration of the nucleus in the body of the spermatid and appearance of crest-like bodies followed by the constriction of the new ring of arched membranes (arrows), then the separation of the old spermatid from the residual cytoplasm. To make the diagram clearer, the spiral coil of the cortical microtubules has not been shown. Am1 = transitory ring of arched membranes; Am2 = new ring of arched membranes; B = cytoplasmic bud; C = centriole; Cb = crest-like bodies; Cm = cortical microtubules; F = flagellum; N = nucleus.
Spermiogenesis and spermatozoon
of A. delafondi
233
thickest reach 200 nm and have been described in Avitellina centripunctata by B1& Marchand (in press a). In A. delafondi the crest-like bodies are 15 to 40 nm
I
HiCm
II
thick. Spiralization of the cortical microtubules was recently considered to be a synapomorphy of the Cyclophyllidea (Justine, 1991). The angle of spiralization of these microtubules is more or less marked. It has been estimated as 50” in Stilesia globipunctata by Bb & Marchand (1992a), 40” in M. expansa by Swiderski (1968) and Ba & Marchand (1992c), 40” in M. herpestis by Bb & Marchand (in press b), 35” in A. centripunctata by Bb & Marchand (in press a), 25” in R. serrata by Bb & Marchand (1993) and 15” in H. nana by BB & Marchand (1992b). In A. delafondi the cortical microtubules are also spiralized, with the coil making a relatively small angle of roughly 20”. The cytoplasm of the cestode spermatozoon is generally of low electron density. However, we have already revealed electron-dense material at the posterior extremity of the spermatozoa of A. centripunctata, M. herpestis and R. serrata. In A. centripunctata, only the apical cone and region IV of the spermatozoon are fairly electron-dense. In H. nana, on the other hand, the cytoplasm is electron-lucent in regions I and II of the spermatozoon and slightly electron-dense over all the rest. The cytoplasm of the A. delafondi spermatozoon is fairly electron-dense in regions I and II whereas in regions III, IV and V it exhibits protein granules and patches of electron-lucent material. As far as we know these lucent patches have never been reported before in the spermatozoon of a Cestode. According to Schmidt (1986) the Hymenolepididae make up a family “closely related to Dilepididae and Anoplocephalidae, the three grading together so much that it sometimes is arbitrary to decide which family should receive certain genera”. The crest-like bodies of the A. delafondi spermatozoon, by their position outside the cortical microtubules, the angle of spiralization, their morphology, thickness and even their number, are closer to those of the Hymenolepididae (Bb & Marchand, 1992b, 1993) than those of the Anoplocephalidae already studied (Ba & Marchand,
Ax
FIG. 20. Attempted reconstruction of the mature spermatozoon of Aporinadelafondiin which we distinguish five regions (I-V) from anterior to posterior. To make the diagram clearer, the spiral coil of the cortical microtubules has not been shown. Aae = axonemal anterior extremity; AC = apical cone; Ape = axonemal posterior extremity; Ax = axoneme; Cb = crest-like bodies; Cm = cortical microtubules; Dm = electron-dense material; G = granular electron-dense material; N = nucleus; P = patch of electron-lucent material.
234
C. T. BA and B. MARCHAND
1992a,c; in press a, b). This ultrastructural quasisimilitude between the A. delufondi spermatozoa and those of the Hymenolepididae poses the problem of whether the genus Aporina belongs to the family Anoplocephalidae or that of Hymenolepididae. A study of the spermatozoon and spermiogenesis of a greater number of representatives of these two families should enable us to answer this question. REFERENCES
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