Parasitol Res (2005) 96: 38–48 DOI 10.1007/s00436-005-1318-6
O R I GI N A L P A P E R
Sylvia Agostini Æ Jordi Miquel Æ Papa Ibnou Ndiaye Bernard Marchand
Dicrocoelium hospes Looss, 1907 (Digenea, Dicrocoeliidae): spermiogenesis, mature spermatozoon and ultrastructural comparative study Received: 13 December 2004 / Accepted: 21 December 2004 / Published online: 17 March 2005 Ó Springer-Verlag 2005
Introduction
knowledge of the phylogeny in several groups (Justine 1991, 1995, 1998). In the present work, we aimed at undertaking an ultrastructural study of spermiogenesis and of the mature spermatozoon in a digenean species, Dicrocoelium hospes Looss, 1907, a parasite collected in Bos indicus from Senegal (Africa) which can be also accidentally found in humans. Digenean trematodes have been the subject of numerous ultrastructural studies on spermatology (see Ndiaye 2003), including 72 species (see Ndiaye 2003; Levron 2004). However, relatively few ultrastructural investigations have been made on spermatogenesis and spermatozoon in Dicrocoeliidae digeneans. These referred to D. dendriticum (Morseth 1969; Cifrian et al. 1993), Eurytrema pancreaticum (Fujino et al. 1977), Corrigia vitta (Robinson and Halton 1982) and D. chinensis (Tang 1996). The family Dicrocoeliidae Odhner, 1910 contains species with a noticeable human and veterinary interest (Mas-Coma and Bargues 1997). This is the case of the species object of the present investigation, D. hospes. The present study describes for the first time the major ultrastructural events involved in spermiogenesis in a Dicrocoeliidae digenean, D. hospes, and details the fine ultrastructural organisation of the mature spermatozoon. Findings are compared with those reported for other species among the same Dicrocoeliidae family.
The use of ultrastructural characters on the reproduction of Platyhelminthes has shed some light on the
Materials and methods
Abstract This work describes the first ultrastructural results on spermiogenesis and on the mature spermatozoon of Dicrocoelium hospes (Trematoda, Digenea) collected in Bos indicus from Senegal (Africa). Examination of this species was processed by TEM. Spermiogenesis follows the general pattern found in the digenean, but reveals a particularity consisting of the appearance of glycogen granules in the late spermatids within the testes. The mature spermatozoon possesses five distinct regions and presents all features found in Digenea gametes: two axonemes, mitochondria, nucleus and parallel cortical microtubules. However, several characters allow us to distinguish D. hospes from other digenetic trematodes within the Dicrocoeliidae family. In fact, we observed several structures that are absent in the other species of Dicrocoeliidae studied until now, such as: a cytoplasmic expansion, extramembranar ornamentation, spine-like bodies and two parallel mitochondria in the mature sperm. Moreover, additional particular characteristics were observed in this species in both extremities of the spermatozoon. This work produced new data on the ultrastructure of this trematode family which may be useful for phylogenetic purposes.
S. Agostini Æ J. Miquel Æ P. I. Ndiaye Laboratori de Parasitologia, Departament de Microbiologia i Parasitologia Sanita`ries, Facultat de Farma`cia, Universitat de Barcelona, Av. Joan XXIII, sn, 08028 Barcelona, Spain S. Agostini (&) Æ B. Marchand Laboratoire Parasites et E´cosyste`mes Medite´rrane´ens, Faculte´ des Sciences et Techniques, Universite´ de Corse, BP 52, 20250 Corte, France E-mail:
[email protected] Fax: +33-4-95716700
Dicrocoelium hospes specimens were collected alive from B. indicus in Senegal (Africa). After its extraction from the liver, adults were kept in a 0.9% NaCl solution. Several portions of these trematodes were dissected and fixed in cold (4°C) 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer at pH 7.2 for 2 h, rinsed in 0.1 M sodium cacodylate at pH 7.2, postfixed in cold (4°C) 1% osmium tetroxide in the same buffer for 1 h, rinsed in 0.1 M sodium cacodylate buffer at pH 7.2, dehydrated in an ethanol series and propylene oxide, embedded in
39
Fig. 1 Initial stage of spermiogenesis showing the growth of the first free flagellum with an angle of about 120°. F1 Free flagellum 1, Ib intercentriolar body, N nucleus. Bar=0.5 lm Fig. 2 Differentiation zone showing two free flagella. Ib Intercentriolar body, N nucleus. Bar=0.5 lm Fig. 3 Detail of a differentiation zone in a cross section. Mt Mitochondrion, N nucleus, Sr striated rootlet. Bar=0.5 lm Fig. 4 Cross-section of a differentiation zone at the level of the ring of arched membranes (Am). Notice the cisternae of rough endoplasmic reticulum (arrowheads). Mt Mitochondrion, N nucleus. Bar=0.5 lm Fig. 5 Longitudinal section of a differentiation zone showing the striated rootlets (Sr) and the initial migration of the mitochondria (Mt) during the flagellar rotation. Arrowheads show the RER cisternae. Am Arched membranes, Ib intercentriolar body, Mcp median cytoplasmic process. Bar=1 lm
Fig. 6 Longitudinal section of a differentiation zone before the proximodistal fusion. Notice the initial nuclear migration (N) and the parallel disposition of the free flagellum. Arrowheads show the RER cisternae. Am Arched membranes, Mcp median cytoplasmic process, Sr striated rootlet. Bar=1 lm Fig. 7 Cross-section of the median cytoplasmic process (Mcp) showing the nuclear migration (N) before the proximodistal fusion of the free flagella. Az Attachment zones. Bar=0.3 lm Fig. 8 Cross-section of the median cytoplasmic process (Mcp) showing the attachment zones (Az) before the proximodistal fusion. Cm Cortical microtubules. Bar=0.2 lm Fig. 9 Median cytoplasmic process cross-section showing the migration of the two parallel mitochondria (Mt) before the proximodistal fusion. Az Attachment zones. Bar=0.3 lm Fig. 10 Another cross-section showing the migration of the nucleus (N) and the mitochondrion (Mt) before the fusion of the flagella. Az Attachment zones. Bar=0.3 lm
Spurr and polymerised at 60°C for 48 h. Ultrathin sections (60–90 nm) of testes, seminal ducts and seminal vesicle were obtained using a Reichert-Jung Ultracut E ultramicrotome, placed on copper grids and double-
stained with uranyl acetate (30 min) and lead citrate (15 min) following Reynolds (1963) methodology. Copper grids were examined using a Jeol 1010 electron microscope at 75 kV.
40
Fig. 11 Spermiogenesis of Dicrocoelium hospes. Longitudinal section of spermatid showing the migration of three mitochondria (Mt). Bar=1 lm Fig. 12 Longitudinal section of spermatid showing the asynchronous proximodistal fusion. Ax Axoneme, F2 free flagellum 2, N nucleus. Bar=1 lm Fig. 13 Cross-section of spermatid showing the appearance of two spine-like bodies (Sb) after the proximodistal fusion. Bar=0.2 lm Fig. 14 Longitudinal section of spermatid showing the nuclear migration (N) after the proximodistal fusion of the two flagella. Am Arched membranes, Ax axoneme, Ib intercentriolar body, Sr striated rootlet. Bar=1 lm Fig. 15 Cross-section of spermatid showing the presence of spinelike body (Sb) and extramembranar ornamentation (Eo). Mt Mitochondrion. Bar=0.2 lm Fig. 16 Longitudinal section of spermatid in a final stage of spermiogenesis showing the migration of mitochondria (Mt).
Notice the appearance of glycogen (G) and the relative position of the ring of arched membranes (Am) and the axonemes (Ax). Bar=2 lm Fig. 17 Cross-section of a late spermatid showing the development of the RER cisternae (arrowheads). Am Arched membranes. Bar=0.5 lm Fig. 18 Another cross-section of a late spermatid showing a more basal area whithout axoneme. Arrowheads show the development of the RER cisternae. Am Arched membranes. Bar=1 lm Fig. 19 Section of a late spermatid. Arrowheads show the development of the RER cisternae. Striated rootlets (Sr) and intercentriolar body (Ib) remain in the residual cytoplasm (Rc). Am Arched membranes. Bar=1 lm Fig. 20 Longitudinal section of a late spermatid showing the development of the RER cisternae (arrowheads) previous to the liberation of the young spermatozoon. Am Arched membranes, Rc residual cytoplasm. Bar=1 lm
41
Fig. 21 Mature spermatozoon of Dicrocoelium hospes. Longitudinal section of region I showing the anterior spermatozoon extremity (Ase). Adm Anterior dense material, Ax axoneme. Bar=0.2 lm Fig. 22 Detail of the anterior spermatozoon extremity. Bar=0.2 lm Fig. 23 Tangential section of the anterior spermatozoon extremity showing the anterior dense material (Adm). Bar=0.1 lm Fig. 24 Cross-section of region I showing the area of spermatozoon with a single axoneme. Bar=0.2 lm Fig. 25 Cross-section of region I showing two axonemes and cortical microtubules (Cm). Bar=0.2 lm Fig. 26 Cross-section of region II showing the presence of two parallel mitochondria (Mt), cytoplasmic expansion (Ce) and extramembranar ornamentation (Eo). Bar=0.2 lm Fig. 27 Cross-section of region II showing the presence of spine-like body (Sb) in the ventral side of the sperm. Bar=0.2 lm
Fig. 28 Longitudinal section of region II showing the presence of spine-like bodies (Sb) in the same side of the sperm and extramembranar ornamentation (Eo). Ax Axonemes. Bar=0.2 lm Fig. 29 Cross-section of region II showing the presence of spinelike body (Sb) in the dorsal side of the sperm. Eo Extramembranar ornamentation, Mt mitochondrion. Bar=0.2 lm Fig. 30 Longitudinal section of region II showing the presence of spine-like bodies (Sb) in each side of the sperm. Eo Extramembranar ornamentation, G glycogen, Mt mitochondrion. Bar=0.2 lm Fig. 31 Cross-section of region II showing the presence of glycogen (G) and two bundles of cortical microtubules. Mt Mitochondrion. Bar=0.2 lm Fig. 32 Cross-section of region III showing the simultaneous presence of nucleus (N) and mitochondrion (Mt). Cm Cortical microtubules, G glycogen. Bar=0.2 lm Fig. 33 Another cross-section of region III showing the reduction of the dorsal cortical microtubules (Cm) number. Mt Mitochondrion. Bar=0.2 lm
42
Results Spermiogenesis Spermiogenesis in D. hospes begins in the spermatid with the formation of a differentiation zone (Figs. 1, 2, 3), which elongates into a long cone to form the median cytoplasmic process (Figs. 4, 5, 6, 7, 8, 9, 10). This conical area is characterized by the presence of arched membranes and bordered by submembranous cortical Fig. 34 Mature spermatozoon of Dicrocoelium hospes. Longitudinal section of region III showing the nucleus (N), mitochondrion (Mt) and glycogen (G). Bar=0.5 lm Fig. 35 Cross-section of region III showing the simultaneous presence of nucleus (N), mitochondrion (Mt) and the two bundles of cortical microtubules. G Glycogen. Bar=0.2 lm Fig. 36 Cross-section of region IV showing the disappearance of the mitochondrion and the diminution of the ventral cortical microtubules (Cm) number. N Nucleus. Bar=0.2 lm Fig. 37 Cross-section of region IV showing the total disappearance of the cortical microtubules. N Nucleus. Bar=0.2 lm Fig. 38 Cross-section of the transition area between regions IV and V showing the disappearance of the first axoneme. Cc Central core, N nucleus. Bar=0.2 lm Fig. 39 Cross-section of region V showing the presence of nucleus (N) with a single axoneme. Notice the thick irregular nuclear envelope. Bar=0.2 lm Fig. 40 Cross-section of region V showing the nucleus (N) with a smaller diameter and the single axoneme. Bar=0.2 lm Fig. 41 Cross-section of the posterior part of the spermatozoon (region V) after the disappearance of the second axoneme. N Nucleus. Bar=0.2 lm
microtubules. It contains the nucleus, mitochondria and two centrioles associated with two parallel striated roots (Figs. 3, 5, 6) presenting an intercentriolar body between them (Figs. 1, 2, 5). In the ring of arched membranes, it is possible to observe submembranous cisternae of rough endoplasmic reticulum (RER) (Figs. 4, 5, 6). The cortical microtubules initiate their migration along this median process and each centriole develops an externally growing flagellum (Figs. 1, 2, 5, 6, 8). Thereafter, a flagellar rotation of about 120° (at least for one of them, Fig. 1) occurs and both flagella become parallel to the cytoplasmic extension followed by the fusion of flagella with the cytoplasmic extension (Figs. 6, 12). The rotation and proximodistal fusion of the free flagella are asynchronous (Figs. 6, 12). During flagellar rotation, most of the mitochondria migrate toward the median cytoplasmic process (Figs. 5, 9, 11). Before proximodistal fusion, the nucleus also initiates a migration toward the median cytoplasmic process overtaking the mitochondria (Figs. 4, 6, 7, 10, 12). Attachment zones emphasise the fusion of the free flagella with the median cytoplasmic process (Figs. 7, 8, 9, 10). Flagellar rotation and posterior fusion, which is also asynchronic, determine the appearance of two sets of cortical microtubules: a dorsal bundle that corresponds to the nuclear area and a ventral one that corresponds to the mitochondrial side of the sperm (Figs. 7, 8, 9, 10). The ring of arched membranes is perfectly visible at the base of the spermatid (Figs. 5, 6). When flagellar fusion is completed, the nucleus continues its migration (Fig. 14).
43 Fig. 42 a–f Diagram showing the main stages of spermiogenesis in Dicrocoelium hospes: a-Initial stage with each centriole forming a free flagellum and showing the beginning of the asynchronous flagellar rotation. bDifferentiation zone showing flagellar rotation and the beginning of the nuclear and mitochondrial migration. cDifferentiation zone showing the migration of numerous mitochondria to the median cytoplasmic process during flagellar rotation. d-Stage showing the nuclear migration to the median cytoplasmic process before the fusion; notice the parallel position of the first flagellum in relation to this structure. e-Stage showing the asynchronous proximodistal fusion. f-Final stage of spermiogenesis; notice the RER cisternae development and the progressive displacement of the striated rootlets towards the base of the spermatid. Am arched membranes, Ax1 axoneme 1, Ax2 axoneme 2, Az attachment zones, C1 centriole 1, C2 centriole 2, Cm cortical microtubules, F1 flagellum 1, F2 flagellum 2, Fr flagellar rotation, G granules of glycogen, Ib intercentriolar body, Mcp median cytoplasmic process, Mt mitochondrion, Mt1 mitochondrion 1, Mt2 mitochondrion 2, N nucleus, Pf proximodistal fusion, Rc residual cytoplasm, RERc rough endoplasmic reticulum cisternae, Sr striated rootlets
Cross-sections of late spermatids reveal extramembranous ornamentations and spine-like bodies (Figs. 13, 15). We can also notice the presence of small amounts of glycogen granules in the spermatid during the final stage of spermiogenesis (Fig. 16). Furthermore, in this stage, the RER cisternae become expanded (Figs. 17, 18, 19, 20) and both striated rootlets and intercentriolar body are progressively displaced towards the base of the spermatid (Fig. 19). Finally, the ring of arched membranes is strangled and the young spermatozoon becomes detached from the residual cytoplasm (Fig. 20).
Spermatozoon The mature spermatozoon is described by longitudinal and transverse sections which are arranged in an anteroposterior sequence according to the morphological information acquired by transmission electron microscopy. It exhibits the usual structure previously observed in the Digenea: nucleus, mitochondrion, parallel cortical microtubules and two axonemes of the 9+‘1’ pattern of trepaxonematan Platyhelminthes. The spermatozoon is filiform, tapered at both ends, and from its anterior to
44 Fig. 43 i–v Diagram showing the ultrastructural organization of the mature spermatozoon of Dicrocoelium hospes. Adm anterior dense material, Ase anterior spermatozoon extremity, Ax1 axoneme 1, Ax2 axoneme 2, Ce cytoplasmic expansion, Cm cortical microtubules, Eo extramembranar ornamentation, G granules of glycogen, Mt1 mitochondrion 1, Mt2 mitochondrion 2, N nucleus, Pm plasma membrane, Pse posterior spermatozoon extremity, Sb spine-like body
posterior extremities it is possible to distinguish five regions (I–V) presenting different ultrastructural features. Region I (Figs. 21, 22, 23, 24, 25) corresponds to the anterior end of the spermatozoon. It is characterized by the presence of two axonemes showing the 9+‘1’ pattern of the Trepaxonemata, a submembranous layer of parallel cortical microtubules and spine-like bodies. At the beginning of the region, the anterior extremity of the spermatozoon is sharp (Figs. 21, 22) and it usually shows a flexible shape (Fig. 22). This extremity contains electron-dense filiform structures (the anterior dense material; Figs. 21, 23). Soon, a first axoneme appears (Fig. 24) followed by the appearance of a second one. It
is also possible to observe a continuous layer of cortical microtubules (Fig. 25). Region II is characterized by the presence of two axonemes, mitochondria in the ventral part of the spermatozoon, two bundles of cortical microtubules, cytoplasmic expansion, spine-like bodies and extramembranous ornamentation (Figs. 26, 27, 28, 29, 30, 31). Transverse sections of the anterior part of this region reveal the presence of a cytoplasmic expansion, extramembranous ornamentation and two parallel mitochondria (a small one and an elongated one which extends up until the beginning of the Region III, Fig. 26). In the posterior part of this region, it is possible
45 Table 1 Available data on the spermatozoon ultrastructure of digenean trematodes Families and species of Digenea
Ce
Eo
Brachylaimidae Scaphiostomum palaearticum Bucephalidae Bucephaloides gracilescens Pseudorhipidocotyle elpichthys Cryptogonimidae Neochasmus sp. Dicrocoeliidae Corrigia vitta Dicrocoelium chinensis Dicrocoelium dendriticum
+
Dicrocoelium hospes Echinostomatidae Echinostoma caproni Fasciolidae Fasciola gigantica Fasciola hepatica Heterophyidae Cryptocotyle lingua Lecithodendriidae Ganeo tigrinum Postorchigenes gymnesicus Microphallidae Maritrema linguilla Microphallus primas Monorchiidae Monorchis parvus Notocotylidae Notocotylus neyrai Opecoelidae Helicometra fasciata Opecoeloides furcatus Paragonimidae Paragonimus ohirai
Mt
Ase
Pse
References
+
1
1 Ax
Cm
Ndiaye et al. (2002)
+ +
1 1 2
Cm 1Ax D
1 Ax+Cm Cm D
Erwin and Halton (1983) Tang et al. (1998) Jamieson and Daddow (1982)
+ +
+ +
+
1 1 N 2 2 1
1 1 1 1 1 2
Ax Ax Ax Ax+Cm Ax Ax+Cm
1 Ax 1 Ax 1 Ax 1 Ax N 1 Ax+N
Robinson and Halton (1982) Tang (1996) Morseth (1969) Cifrian et al. (1993) Present study Iomini and Justine (1997)
+ + +
+
+
+
+
1 2 1 1
1 1 1 1
Ax Ax Ax Ax+Cm
N 1 Ax N 1 Ax
Ndiaye et al. (2004) Stitt and Fairweather (1990) Ndiaye et al. (2003b) Rees (1979)
+
1 2
Cm 1 Ax+Cm
Cm 1 Ax+G
Sharma and Rai (1995) Gracenea et al. (1997)
+ + +
+
2 1 2 2
1 Ax+Cm 2Ax Cm 1 Ax
1 Ax+Cm 1 Ax+N N+D D
Hendow and James (1988) Castilho and Barandela (1990) Levron et al. (2004) Ndiaye et al. (2003a)
+
+
1 1 n
1 Ax 1 Ax 2 Ax
G Cm N
Levron et al. (2003) Miquel et al. (2000) Fujino et al. (1977)
+ +
Sb
+
Ase anterior spermatozoon extremity, Ax axoneme, Ce cytoplasmic expansion, Cm cortical microtubules, D doublets, Eo extramembranar ornamentation, G granules of glycogen, Mt (number) mitochondria, N nucleus, n several, Pse posterior spermatozoon extremity, Sb spine like bodies. +/ presence/absence of considered character
to observe that the cortical microtubules form two bundles. In the same region, it is also possible to notice that the spine-like bodies and the external ornamentation disappear while glycogen granules are present in abundance (Fig. 31). Region III is characterized by the simultaneous presence of mitochondrion and nucleus (Figs. 32, 33, 34, 35). The mitochondrion is located between the axonemes, the nucleus appears in the dorsal part of the spermatozoon and there are abundant glycogen granules (Figs. 32, 33, 35). The cortical microtubules are arranged in two small bundles along each axoneme (Figs. 32, 33, 35). While the nucleus increases in diameter, the number of cortical microtubules progressively decreases until the ventral bundle of cortical microtubules disappears (Fig.33). Region IV (Figs. 36, 37, 38) corresponds to the nuclear area of the mature spermatozoon. It is characterized by the disappearance of both the mitochondrion and the dorsal bundle of cortical microtubules that seems to lead the axonemes to approach each other (Figs. 36, 37). The nucleus is covered by a thick and irregular nuclear envelope (Figs. 36, 37). In addition to a large nucleus, we detected a reduced quantity of glycogen granules (Fig. 37). In the posterior area of this region one axoneme becomes disorganized and disappears (Fig. 38).
Region V (Figs. 39, 40, 41) corresponds to the posterior part of the spermatozoon. It is characterized by the presence of a single axoneme and nucleus (Figs. 39, 40). This axoneme becomes disorganized and the posterior extremity of this region presents a smaller diameter as it contains only the nucleus (Fig. 41). Schematic diagrams Figures 42 and 43 correspond to schematic diagrams showing, respectively the main stages of spermiogenesis and the ultrastructural organization of the mature spermatozoon.
Discussion Spermiogenesis Spermiogenesis in D. hospes follows the general pattern described for other digenean species (Mohandas 1983; Orido 1988; Stitt and Fairweather 1990; Gracenea et al. 1997; Miquel et al. 2000; Baptista-Farias et al. 2001; Ndiaye et al. 2003a). The presence of two centrioles and striated rootlets and also a proximodistal fusion are general characteristics of parasitic Platyhelminthes
46
(Justine 1991, 1995). Nuclear elongation and migration at least into the proximal median process is a universal phenomenon in the Digenea (Gresson and Perry 1961; Sato et al. 1967; Rees 1979). The single noteworthy characteristic concerns the sequence in the migration process of nucleus and mitochondria along the spermatid body. In fact, the present study shows a mitochondrial migration toward the median cytoplasmic process which occurs before the nuclear migration, as found only for some digeneans (Postorchigenes gymnesicus, Gracenea et al. 1997; D. dendriticum, Cifrian et al. 1993) while in most digeneans the reverse process has been observed (C. vitta, Robinson and Halton 1982; Microphallus primas, Castilho and Barandela 1990; Opecoeloides furcatus, Miquel et al. 2000; Scaphiostomum palaearcticum, Ndiaye et al. 2002; Fasciola hepatica, Ndiaye et al. 2003a; Notocotylus neyrai, Ndiaye et al. 2003b; Helicometra fasciata, Levron et al. 2003; F. gigantica, Ndiaye et al. 2004; Monorchis parvus, Levron et al. 2004). Furthermore, in D. hospes migration of the nucleus and mitochondria occurs before the fusion of the first flagellum unlike the results concerning D. dendriticum spermiogenesis (Cifrian et al. 1993) where the nucleus migrates after the proximodistal fusion and also unlike the results concerning S. palaearcticum (Ndiaye et al. 2002) and N. neyrai (Ndiaye et al. 2003b) where the mitochondrion migrates after the proximodistal fusion. Our results corroborate those of Burton (1972), who suggested that nuclear migration is necessary for the fusion of the median process with flagella, and also that the nucleus is always present when fusion occurs. However, we must hold into account that fusion occurs without nuclear migration in some species of Digenea such as Neochasmus sp. (Daddow and Jamieson 1983). An interesting feature observed in the present study refers to the development of the free flagella at the initial stage of spermiogenesis. In D. hospes, at least one of the two free flagella grows externally to the median cytoplasmic process with an angle of about 120°. This fact has also been observed in other digeneans such as F. hepatica (Ndiaye et al. 2003a), H. fasciata (Levron et al. 2003) and M. parvus (Levron et al. 2004). Moreover concerning the latter species, Levron et al. (2004) describe an angle of 120° for both free flagella. The present work enlarges the number of families of Digenea, in which this aspect has been described. In fact, to date, only four species belonging to four families (Fasciolidae, Opecoelidae, Monorchiidae and now Dicrocoeliidae) show this growth pattern for the free flagella. This is a differentiating pattern concerning spermiogenesis when D. hospes is compared with the other dicrocoeliids studied until now, and particularly considering the congeneric D. dendriticum (Cifrian et al. 1993). As in all Digenea studied, the striated rootlets and intercentriolar body disappear at the end of spermiogenesis. They are not visible in the mature spermatozoon as reported by other authors (Erwin and Halton 1983; Justine 1995; Ndiaye et al. 2002). Thus, it is assumed that these structures remain in the residual cytoplasm
and degenerate (Rees 1979; Erwin and Halton 1983; Cifrian et al. 1993; Brunanska et al. 2001). Additionally, in our study the great development of the RER cisternae in the final stage of spermiogenesis demonstrates the probable involvement of this process in the constriction of the ring of arched membranes and the posterior liberation of the young spermatozoon. Spermatozoon The mature spermatozoon of D. hospes is filiform as most parasitic Platyhelminthes. It exhibits the usual structure observed in the Digenea so far: nucleus, mitochondria, parallel cortical microtubules and two axonemes of the 9+‘1’ pattern of trepaxonematan Platyhelminthes (Ehlers 1985, 1986). However, it presents some ultrastructural particularities, which allow us to distinguish this species from other Dicrocoeliidae digeneans. In fact, the mature spermatozoon of D. hospes is characterized by several features: (1) a cytoplasmic expansion, (2) an extramembranar ornamentation disrupted in some parts, (3) spine-like bodies located on a broad part of the spermatozoon, and (4) the presence of two parallel mitochondria in Region II. These structures are absent in the other species of Dicrocoeliidae studied until now (see Table 1). Both cytoplasmic expansions and extramembranar ornamentations have been observed in the mature spermatozoon of several digeneans (see Table 1). Nevertheless, the present study constitutes the first finding of these structures in the spermatozoon of a representative of the family Dicrocoeliidae. In fact, cytoplasmic expansions and/or extramembranar ornamentations in spermatozoa were not detected in D. dendriticum, D. chinensis or C. vitta (Morseth 1969; Robinson and Halton (1982; Cifrian et al. 1993; Tang 1996). Concerning the presence of spine-like bodies (Table 1), certain authors detect these structures for other digenean species (O. furcatus, Miquel et al. 2000; N. neyrai, Ndiaye et al. 2003b; F. hepatica, Ndiaye et al. 2003a; F. gigantica, Ndiaye et al. 2004), but only on one side of the spermatozoon. Our study shows a wider localization of these structures on each side of the spermatozoon. Moreover, in the present study, we detect for the first time this structure in a Dicrocoeliidae species. Our work also highlights the presence of two parallel mitochondria appearing in the anterior part of the spermatozoon. In fact, although a single mitochondrion was generally observed in many species of Digenea (Table 1), the presence of several mitochondria had already been reported for digeneans such as D. dendriticum (Morseth 1969; Cifrian et al. 1993), Haematoloechus medioplexus (Burton 1972), Pharyngostomoides procyonis (Grant et al. 1976), Cryptocotyle lingua (Rees 1979), Paragonimus ohirai (Fujino et al. 1977; Orido 1988), Neochasmus sp. (Jamieson and Daddow 1982), Maritrema linguilla (Hendow and James 1988), F. hepatica (Stitt and Fairweather 1990), P. gymnesicus (Gracenea et al. 1997), N. neyrai (Ndiaye et al. 2003b),
47
and M. parvus (Levron et al. 2004). However, they had never been observed in a parallel position. Therefore, our study brings new information, considering that in the other studied species the mitochondria are placed one in the anterior part and the other in the posterior part of the sperm, while in D. hospes they are placed side by side and they are both located in the anterior part of the spermatozoon. According to Robinson and Halton 1982, during spermiogenesis mitochondria would merge thus forming only one large mitochondrion. However our study relates to mature spermatozoa met within the seminal vesicle, and suggests that the presence of several mitochondria would not be necessarily related with the maturation stage of the sperm but rather with a question of energy requirements. This assumption implies more investigations on this subject. Additional particular characteristics are observed for this species concerning both extremities of the spermatozoon. The anterior extremity presents only one axoneme as shown for C. vitta (Robinson and Halton 1982), D. chinensis (Tang 1996), D. dendriticum (Morseth 1969; Cifrian et al. 1993). In what refers to the posterior extremity, the presence of nucleus exclusively contrasts with the ultrastructural results found for the other dicrocoeliids. This posterior part of the mature sperm is characterized by the great development of the nucleus, as in other digeneans like D. dendriticum (Cifrian et al. 1993), Mesocoelium monas (Iomini et al. 1997), Echinostoma caproni (Iomini and Justine 1997), P. gymnesicus (Gracenea et al. 1997), O. furcatus (Miquel et al. 2000), S. palaearcticum (Ndiaye et al. 2002), N. neyrai (Ndiaye et al. 2003b), F. hepatica and F. gigantica (Ndiaye et al. 2003a, 2004) (see Table 1). This work enables us to see that the marked differences in the ultrastructure of the spermatozoon of D. dendriticum and D. hospes constitute additional data supporting the specific identity of these two species. Several authors have made studies on the variability range and frequency of occurrence of different morphological types of D. dendriticum (Macko and Pacenovsky 1987) concluding on the possibility of interpretation of D. hospes as an intrapopulation of D. dendriticum. In our opinion complementary studies on the ultrastructure of the reproduction of different morphotypes of Dicrocoelium spp. should enable us to bring new and additional information on this subject. Acknowledgements We thank the ‘‘Serveis Cientı´ fics i Te`cnics’’ of the University of Barcelona for their support in the preparation of material. The study was supported by the Spanish grant 2001SGR-00088 of the ‘‘Departament d’Universitats Recerca i Societat de la Informacio´ (Generalitat de Catalunya)’’, and by INTERREG III.
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