J. Parasitol., 92(3), 2006, pp. 441–453 䉷 American Society of Parasitologists 2006
SPERMIOGENESIS AND SPERMATOZOON ULTRASTRUCTURE OF THE CRANIAL DIGENEAN TROGLOTREMA ACUTUM (LEUCKART, 1842) Jordi Miquel, Christine Fournier-Chambrillon*, Pascal Fournier*, and Jordi Torres Laboratori de Parasitologia, Departament de Microbiologia i Parasitologia Sanita`ries, Facultat de Farma`cia, Universitat de Barcelona, Av. Joan XXIII, sn, E-08028 Barcelona, Spain. e-mail:
[email protected] ABSTRACT: Ultrastructure of spermiogenesis and the main characters of the mature spermatozoon of Troglotrema acutum are described by means of transmission electron microscopy. Specimens were obtained from the nasolacrimal sinuses of an American mink (Mustela vison). Spermiogenesis in T. acutum follows the general pattern of digeneans. The zone of differentiation is a conical-shaped area bordered by cortical microtubules and delimited at its base by a ring of arched membranes. This area contains 2 centrioles associated with striated rootlets and an intercentriolar body between them. The centrioles develop 2 free flagella that grow ortogonally to the median cytoplasmic process. The posterior flagellar rotation and proximodistal fusion of the free flagella with the median cytoplasmic process originate the spermatozoon. The mature spermatozoon of T. acutum is characterized by the presence of 2 axonemes of different lengths presenting the 9⫹‘1’ trepaxonematan pattern, 2 bundles of parallel cortical microtubules, 2 mitochondria, a nucleus, and granules of glycogen. These ultrastructural characters are compared with other digenean species previously studied and the importance of different spermatological features is discussed.
Troglotrema acutum (Leuckart, 1842) (Troglotrematidae) is a recognized cranial pathogenic fluke that usually infects polecats (Mustela putorius), although it also has been reported in other carnivores (mainly mustelids and also canids) (Koubek et al., 2004). It is mainly distributed in eastern and central European countries, but it also has been recovered from the polecat in Spain (Torres et al., 1996). No information regarding spermiogenesis or sperm structure is available for the Troglotrematidae. In recent years, there has been a significant consensus regarding the use of spermatological characters for phylogenetic inference in the Platyhelminthes (Justine, 1991, 1995, 1998, 2001; Baˆ and Marchand, 1995; Hoberg et al., 1997, 2001; S´widerski and Mackiewicz, 2002). However, when referring to digeneans, despite the high number of studies on the ultrastructure of sperm features (Levron et al., 2004a, 2004b; Ndiaye, 2003; Agostini et al., 2005), most are incomplete and include misinterpretations. In reality, the high potential of these data and observations for phylogenetic purposes is yet to be clarified. This is particularly evident in the digenean spermiogenesis processes that show a reduced variability between digeneans, thus leading to a very different situation in comparison with cestodes, in which different patterns have been accurately described (S´widerski, 1986; Baˆ and Marchand, 1995; S´widerski and Mackiewicz, 2002). In the future, it is contended that a relatively large number of ultrastructural characters dealing with spermiogenesis and spermatozoon structure will become available for phylogenetic purposes. The most interesting features should be (1) the extremities (anterior and posterior), (2) the cytoplasmic expansions, (3) the external ornamentation of the plasma membrane, and (4) the spinelike bodies. The aim of the present study is to elucidate the principal characteristics of spermiogenesis and the ultrastructural organization of the mature spermatozoon of T. acutum, providing data on the Troglotrematidae for the first time. MATERIALS AND METHODS Adult specimens of T. acutum analyzed in the present study were obtained alive from the nasolacrimal sinuses of a naturally infected, Received 14 September 2005; revised 23 November 2005; accepted 28 November 2005. * GREGE, Route de Pre´chac, F-33730 Villandraut, France. 441
free-ranging female American mink, Mustela vison Schreber, 1777, captured in April 2005 in Cazaubon (Gers, France). American minks were trapped as part of the control program of the southwestern French feral population, one of the main actions included in the conservation action plan for the European mink, Mustela lutreola (Linnaeus, 1761), in France. The living digeneans were placed in a 0.9% NaCl solution. After dissection, different portions containing testes and seminal vesicle were routinely processed for transmission electron microscopy (TEM) examination. Specimens were fixed in cold (4 C) 2.5% glutaraldehyde in a 0.1 M sodium cacodylate buffer at pH 7.2 for 2 hr, rinsed in a 0.1 M sodium cacodylate buffer at pH 7.2, postfixed in cold (4 C) 1% osmium tetroxide in the same buffer for 1 hr, rinsed in a 0.1 M sodium cacodylate buffer at pH 7.2, dehydrated in an ethanol series and propylene oxide, and finally embedded in Spurr’s resin. Ultrathin sections were obtained using a Reichert-Jung Ultracut E ultramicrotome, placed on copper grids, and double stained with uranyl acetate and lead citrate. Ultrathin sections were examined using a JEOL 1010 transmission electron microscope.
RESULTS Spermiogenesis The ultrastructural aspects of spermiogenesis in T. acutum are illustrated in Figures 1–20. Spermiogenesis begins with the formation of the zone of differentiation. This is a conicalshaped region delimited at its base by a ring of arched membranes and bordered by cortical microtubules (Figs. 1–3). This area contains 2 centrioles associated with striated rootlets and an intercentriolar body between them (Figs. 3–4). The intercentriolar body consists of a cylindrical structure made up of several electron-dense and electron-lucent layers. Sections of the intercentriolar body (Figs. 3–5) show 7 electron-dense layers; there is a thin and central electron-dense plate and 3 parallel external plates on each side of the central plate. The 2 most external pairs are coarse and striated and are also the thickest electron-dense layers. The 2 centrioles develop into 2 free flagella that grow orthogonally to the median cytoplasmic process (Figs. 2, 3). Later, the 2 free flagella rotate (flagellar rotation) and fuse (proximodistal fusion) with this median cytoplasmic process (Figs. 6, 7). In the median cytoplasmic process, 4 electron-dense marks (the attachment zones) occur near the area of fusion with the 2 flagella (Fig. 8). The nucleus initiates its migration followed by the mitochondria along the median cytoplasmic process before the proximodistal fusion of flagella (Figs. 6, 9–11). Nevertheless, the complete migration of mito-
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chondria occurs in the final stage of spermiogenesis (Figs. 14, 15, 18, 19). The processes of flagellar rotation and proximodistal fusion are asynchronous (Fig. 12). The formation of external ornamentation on the plasma membrane and of spinelike bodies is observed in advanced stages of spermiogenesis, after the fusion of flagella with the median cytoplasmic process (Figs. 13, 14). Finally, the ring of arched membranes constricts, and the spermatozoon is liberated from the residual cytoplasm (Figs. 16, 17, 19). Longitudinal and cross-sections of the basal area of the spermatid, at the level of the ring of arched membranes, show a continuous layer of parallel cortical microtubules (Figs. 17–19). Spermatozoon The ultrastructure of the mature spermatozoon of T. acutum is illustrated in Figures 21–42. The observation of several longitudinal and cross-sections by means of transmission electron microscopy has enabled to establish 5 (I–V) different regions in the mature spermatozoon. The mature sperm contains 2 axonemes of different lengths of the 9⫹‘1’ pattern of trepaxonematan Platyhelminthes, 2 bundles of parallel cortical microtubules, 2 mitochondria, the nucleus, and granules of glycogen. Region I corresponds to the anterior part of the spermatozoon. The anterior tip is filiform (Figs. 21, 22). At the base of this filiform structure the section enlarges, and the cortical microtubules occur before the first axoneme (Figs. 21, 23). The second axoneme occurs soon after the first one (Figs. 24, 25). When both axonemes are present, they are surrounded by a continuous and submembranous layer of cortical microtubules and at this level the spermatozoon lacks attachment zones (Fig. 25). In the distal area of region I, the mature sperm presents a hook-shaped cytoplasmic expansion in its dorsal face, spinelike bodies, and external ornamentation of the plasma membrane covering the totality of its surface, except for the area occupied by the second axoneme and the internal region of the dorsal cytoplasmic expansion (Figs. 26–29). These cross-sections pre-
sent 2 bundles of submembranous cortical microtubules and 4 attachment zones as a result of the proximodistal fusion during spermiogenesis. Granules of glycogen are irregularly distributed along this distal area of region I (Figs. 26, 29). Region II is characterized by the presence of 2 axonemes and 2 mitochondria. The anterior area of this region presents a mitochondrion, the dorsal cytoplasmic expansion, external ornamentation of the plasma membrane, and spinelike bodies (Figs. 31, 32). The posterior area of region II contains a second mitochondrion (Figs. 33, 34), which extends up to the nuclear region. Between the 2 mitochondria there is an intermediate zone with only the 2 axonemes (Fig. 30). Cortical microtubules form 2 (ventral and dorsal) submembranous bundles (Figs. 32– 34). A large amount of granules of glycogen are irregularly distributed along this region (Figs. 30, 31, 33). Region III contains the axonemes and both mitochondrion and nucleus (Fig. 36). Cortical microtubules are also present in the form of ventral and dorsal bundles (Fig. 36). Granules of glycogen also are observed (Fig. 36). Region IV is characterized by the presence of the axonemes and the nucleus (Fig. 37). The second axoneme disorganizes and disappears in this region (Fig. 38); therefore, in the posterior areas of the cell only 1 axoneme remains (Fig. 39), along with the nucleus that decreases in its section up to its end (Fig. 40). Cortical microtubules progressively disappear in this region (Figs. 37–40). Granules of glycogen also are observed (Figs. 35, 37–39). Region V constitutes the posterior part of the spermatozoon and contains only the first axoneme (Fig. 41). DISCUSSION Spermiogenesis pattern The spermiogenesis process is very homogeneous in all the digeneans investigated until now (Ndiaye, 2003). As observed in the present study, most of the digeneans present a spermio-
→ FIGURES 1–10. Spermiogenesis in T. acutum. (1) General view of testes tissue showing numerous zones of differentiation (arrowheads). Bar ⫽ 5 m. (2) Zone of differentiation showing the development of the first flagellum (F1). Am, arched membranes; N, nucleus. Bar ⫽ 1 m. (3) Zone of differentiation with 2 centrioles (C), intercentriolar body (Ib), striated rootlets (Sr), and nucleus (N). Bar ⫽ 0.5 m. (4) Cross-section of a differentiation zone. C, centrioles; Ib, intercentriolar body; N, nucleus. Bar ⫽ 0.5 m. (5) Detail of the intercentriolar body showing 7 electrondense layers (arrowheads). Bar ⫽ 0.3 m. (6) Longitudinal section of a differentiation zone after the flagellar rotation of the first flagellum (F1). The nucleus (N) migrates along the median cytoplasmic process. Am, arched membranes; Sr, striated rootlets. Bar ⫽ 1 m. (7) Longitudinal section of a differentiation zone before the proximodistal fusion of the 2 free flagella (F1 and F2). Ib, intercentriolar body; Mcp, median cytoplasmic process; Mt, mitochondrion; N, nucleus; Sr, striated rootlets. Bar ⫽ 1 m. (8, 9, 10) Cross-sections of spermatids at 3 different developmental stages. Note the presence of 4 attachment zones (arrowheads) in the median cytoplasmic process (Mcp). Cm, cortical microtubules; Mt, mitochondrion; N, nucleus. Bars ⫽ 0.5 m. FIGURES 11–19. Spermiogenesis in T. acutum. (11) Cross-section of a spermatid before the proximodistal fusion showing the presence of both nucleus (N) and mitochondrion (Mt). Bar ⫽ 0.5 m. (12) Cross-section of a differentiation zone in a previous stage to the proximodistal fusion of the second flagellum (F2). N, nucleus. Bar ⫽ 0.5 m. (13, 14) Cross-sections of spermatids showing the appearance of the spinelike body (Sb), the external ornamentation of the plasma membrane (Eo), and glycogen (G). Arrowheads indicate a peripheric electron-dense material. Cm, cortical microtubules; Mt, mitochondrion. Bars ⫽ 0.5 m. (15) Longitudinal section of a differentiation zone after the proximodistal fusion of the 2 flagella. Am, arched membranes; Ax1, first axoneme; Ax2, second axoneme; Mt, mitochondrion. Bar ⫽ 1 m. (16) General view of testes tissue showing numerous zones of differentiation (arrowheads) in an advanced stage of spermiogenesis. Bar ⫽ 2 m. (17) Longitudinal section of a differentiation zone in a final stage of spermiogenesis. The level ‘‘a’’ is showed in cross-section in Figure 18. Cm, cortical microtubules. Bar ⫽ 1 m. (18) Cross-section of the level ‘‘a’’ marked in 17. Bar ⫽ 0.3 m. (19) Longitudinal section of a differentiation zone in a final stage of spermiogenesis. Note the constriction of the ring of arched membranes (arrows). Mt, mitochondrion. Bar ⫽ 1 m. FIGURE 20. Schematic drawing showing the main stages of spermiogenesis in T. acutum. Am, arched membranes; C1, centriole of the first flagellum; C2, centriole of the second flagellum; Cm, cortical microtubules; F1, first flagellum; F2, second flagellum; Ib, intercentriolar body; Mcp, median cytoplasmic process; Mt, mitochondria; N, nucleus; Sr, striated rootlets.
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genesis process in which 2 free flagella grow orthogonally to the median cytoplasmic process. However, recent studies (Levron et al., 2003, 2004c; Ndiaye, Miquel, Fons et al., 2003; Agostini et al., 2005) described a growth of free flagella having an angle of up to 120⬚ with respect to the hypothetical longitudinal axis of the median cytoplasmic process in digeneans belonging to 4 families (Dicrocoeliidae, Fasciolidae, Monorchiidae, and Opecoelidae). It is also interesting to notice the situation during flagellar rotation in certain cestodes, which present an angle of rotation of ⬍90⬚. This is the case for 2 cyclophyllideans, the catenotaeniid Catenotaenia pusilla (Hidalgo et al., 2000) and the taeniid Taenia parva (Ndiaye, Miquel, and Marchand, 2003), in which rotations of about 45⬚ have been described. Within cestodes, this aspect represents an intermediate state between the plesiomorphic flagellar rotation of 90⬚ and the absence of flagellar rotation. Considering all these examples, it may be possible to interpret this variability as a gradual reduction of the angle of rotation of free flagellum(a) from primitive to more evolved Platyhelminthes. Another general aspect in spermiogenesis of the digeneans is the asynchronic flagellar rotation of the 2 free flagella. Thus, 1 of the flagella fuses with the median cytoplasmic process before the other during the proximodistal fusion process. This is also observed in T. acutum. Other aspects as nuclear and mitochondrial migration along the spermatid probably represent minor variations in the general pattern of digeneans, although this assertion cannot be made with total certainty at the moment. Intercentriolar body The intercentriolar body is a plesiomorphic character which is present in the Digenea and also in most of the Cestoda, excepting the Tetrabothriidea and Cyclophyllidea (Justine, 1998, 2001). The most frequent morphology of the intercentriolar body in digeneans shows 7 electron-dense layers as occurs in our study in T. acutum. The morphology and disposition of
these layers in T. acutum are similar to the description of intercentriolar body of Corrigia vitta (Robinson and Halton, 1982). However, Rees (1979) for Cryptocotyle lingua, Castilho and Barandela (1990) for Microphallus primas, and Levron et al. (2004c) for Monorchis parvus describe this structure as constituted by 9 electron-dense layers. Furthermore, Levron et al. (2003) describe an intercentriolar body constituted by 5 electron-dense layers in Helicometra fasciata. Another particular case was observed in the didymozoids Didymozoon sp. (Justine and Mattei, 1984) and Didymocystis wedli (Pamplona-Basilio et al., 2001), in which the zone of differentiation lacks intercentriolar body. Considering the reduction of this character in certain cestodes, such as pseudophyllideans (3 layers) (Brunˇanska´ et al., 2001), proteocephalideans (a single layer) (Se`ne et al., 1997; Brunˇanska´ et al., 2003, 2004, 2005), and mesocestoidids (a single layer) (Miquel et al., 1999), it would be very interesting to conduct more detailed studies on this particular structure for phylogenetic analysis of digeneans in particular and also of Platyhelminthes in general. Anterior spermatozoon extremity There is a reduced morphological variability in the anterior tip of the mature spermatozoon in the Digenea. The most frequent situation is the presence of a single axoneme as occurs in T. acutum. Nevertheless, in Echinostoma caproni, the anterior spermatozoon extremity is constituted by 2 axonemes (Iomini and Justine, 1997). Interestingly, in numerous studies crosssections with a single axoneme are always interpreted as a posterior tip of sperm (Castilho and Barandela, 1990). The observation of longitudinal sections of anterior and posterior extremities and also the application of scanning electron microscopy would present evidence of the real morphology of the anterior extremity. The morphology of the anterior extremity of the mature spermatozoon of M. parvus is also noticeable (Levron et al., 2004c). These authors show micrographs in which
→ FIGURES 21–31. Mature spermatozoon of T. acutum. (21) Longitudinal section of region I showing the anterior extremity of the spermatozoon (Ase). The levels ‘‘a, b, c, and d’’ are shown in cross-section in 22, 23, 24, and 25, respectively. Bar ⫽ 0.5 m. (22–25) Cross-sections of different levels (‘‘a, b, c and d’’) of region I marked in 21. Ax1, first axoneme; Cm, cortical microtubules. Bars (22, 23) ⫽ 0.2 m; (24, 25) ⫽ 0.3 m. (26) Longitudinal section of region II showing spinelike body (Sb) and the external ornamentation of the plasma membrane (Eo). Note the absence of cortical microtubules (Cm) around the second axoneme (Ax2). The level ‘‘e’’ is shown in cross-section in 27. Ax1, first axoneme; G, glycogen; Pm, plasma membrane. Bar ⫽ 0.5 m. (27) Cross-section of level ‘‘e’’ of region I marked in 26. Dce, dorsal cytoplasmic expansion; Eo, external ornamentation of plasma membrane. Bar ⫽ 0.3 m. (28, 29) Cross-sections of posterior areas of region I. Dce, dorsal cytoplasmic expansion; Eo, external ornamentation of plasma membrane; Sb, spinelike body. Bars ⫽ 0.3 m. (30) Cross-section of the intermitochondrial area of region II. Az, attachment zones; G, glycogen. Bar ⫽ 0.5 m. (31) Longitudinal section of region II. The level ‘‘f’’ is shown in cross-section in 32. Eo, external ornamentation of plasma membrane; G, glycogen; Mt1, first mitochondrion. Bar ⫽ 0.5 m. FIGURES 32–41. Mature spermatozoon of T. acutum. (32) Cross-section of region II at the level of the first mitochondrion (Mt1) (level ‘‘f’’ marked in Fig. 31). Eo, external ornamentation of plasma membrane; G, glycogen. Bar ⫽ 0.3 m. (33, 34) Cross-sections of region II at the level of the second mitochondrion (Mt2). Cm, cortical microtubules; G, glycogen. Bars ⫽ 0.3 m. (35) Longitudinal section of region IV. The level ‘‘g’’ is shown in cross-section in Figure 37. G, glycogen; N, nucleus. Bar ⫽ 1 m. (36) Cross-section of region III showing the simultaneous presence of mitochondrion (Mt) and nucleus (N). G, glycogen. Bar ⫽ 0.3 m. (37) Cross-section of the biflagellar area of region IV, marked as level ‘‘g’’ in Figure 35. Cm, cortical microtubules; G, glycogen. Bar ⫽ 0.3 m. (38) Cross-section of region IV showing the disorganization of the second axoneme. D, doublets; N, nucleus. Bar ⫽ 0.3 m. (39, 40) Cross-sections of the posterior area of region IV showing the progressive reduction in the diameter of nucleus. Az, attachment zones; Cm, cortical microtubules; G, glycogen. Bars ⫽ 0.3 m. (41) Cross-section of region V showing the presence of the first axoneme. Bar ⫽ 0.3 m. FIGURE 42. Schematic reconstruction of the mature spermatozoon of T. acutum. To simplify the diagram, glycogen granules are not shown in the longitudinal section. Ase, anterior spermatozoon extremity; Ax1, first axoneme; Ax2, second axoneme; Az, attachment zones; Cm, cortical microtubules; D, doublets; Dce, dorsal cytoplasmic expansion; Eo, external ornamentation of plasma membrane; G, glycogen; Mt1, first mitochondrion; Mt2, second mitochondrion; N, nucleus; Pm, plasma membrane; Pse, posterior spermatozoon extremity; Sb, spinelike body.
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only the central elements of the axonemes extend beyond the anterior extremity of the peripheral doublets. Cytoplasmic expansions The cytoplasmic expansions are present in anterior areas of the spermatozoa of some Digenea (Table I). The morphology of these structures is variable according to the species. Certain digeneans present simple lateral expansions, e.g., Ndiaye et al. (2002) for Scaphiostomum palaearcticum, Levron et al. (2003) for H. fasciata, and Levron et al. (2004a) for Poracanthium furcatum, whereas others show a hook-shaped dorsolateral expansion, e.g., Iomini and Justine (1997) for E. caproni, Ndiaye, Miquel, Fons et al. (2003) for F. hepatica, and Ndiaye et al. (2004) for Fasciola gigantica. Morphologically, the cytoplasmic expansion present in the mature spermatozoon of T. acutum is also hook-shaped as occurs in the sperm cells of the aforementioned species, but it exhibits a more dorsal location, similar to that observed in the male gamete of Saccocoelioides godoyi and Dicrocoelium hospes (Baptista-Farias et al., 2001; Agostini et al., 2005). In contrast, the complete analysis of the available data confirms a concordance between the presence of cytoplasmic expansion and the external ornamentation of the plasma membrane (Table I). All the species with cytoplasmic expansions also present ornamentations in their spermatozoa with the only exception (Levron et al., 2003) being H. fasciata. In these areas, the presence of attachment zones indicates the region of the spermatid where these expansions have probably originated. For F. hepatica and F. gigantica, the proximodistal fusion of the second free flagellum forms a hook-shaped dorsolateral expansion (Ndiaye, Miquel, Fons et al., 2003; Ndiaye et al., 2004). External ornamentation of plasma membrane Numerous flukes present an external ornamentation in the plasma membrane of their spermatozoa (Table I). These ornamentations are normally present in the anterior areas of the spermatozoon and usually they do not cover all the perimeter of the cell. The presence of attachment zones in this area, as occurs in cytoplasmic expansions, indicates that they occur after the proximodistal fusion of the 2 free flagella with the median cytoplasmic process. Ornamentations of the membrane have never been observed in the median cytoplasmic process; consequently, the appearance of this ornamentation probably occurs in the final stages of spermiogenesis when flagella fusion has already occurred. Nevertheless, Justine and Mattei (1982a) describe the presence of external ornamentation coincident with cortical microtubules in the zone of differentiation of Haematoloechus sp. According to these authors, the zone of differentiation is preserved in the mature sperm cell. However, in the spermatozoon of digeneans, 2 different regions with external ornamentation have been described: (1) anterior areas, probably originated from the zone of differentiation, where cortical microtubules surround totally the sperm cell body; and (2) a more posterior area, originated after the fusion of flagella, in which attachment zones are observed. In Haematoloechus sp., these 2 regions with external ornamentation of the plasma membrane were observed but, as occurs in T. acutum and in most digeneans, the external ornamentation is restricted to areas of the
sperm cell that have originated after the proximodistal fusion of 1 or 2 flagella (with 2 or 4 attachment zones, respectively). The presence or absence and the variability in the disposition of the external ornamentation of plasma membrane are other aspects to consider for a future phylogenetic analysis in this group of Platyhelminthes. Spinelike bodies This character was reported for the first time by Miquel et al. (2000) in the spermatozoon of Opecoeloides furcatus (Opecoelidae). Posteriorly, spinelike bodies have been detected in the spermatozoa of other digeneans, e.g., Agostini et al. (2005) for D. hospes, Ndiaye, Miquel, Fons et al. (2003) for F. hepatica, Ndiaye et al. (2004) for F. gigantica, Ndiaye, Miquel, Feliu et al. (2003) for Notocotylus neyrai, and Levron et al. (2004a) for P. furcatum. The accurate examination of TEM micrographs published in older articles (Justine and Mattei, 1982a; Orido, 1988) seems to show the presence of very similar structures in the spermatozoa of 2 other digeneans, Haematoloechus sp. (Haematoloechidae) and Paragonimus ohirai (Paragonimidae), respectively. Nevertheless, these authors did not mention the presence of spinelike bodies. It is possible that in older studies, observations of similar structures have been misinterpreted as artifacts. In all the digeneans in which these structures were detected, they are always present in the anterior areas of the spermatozoon and associated with the external ornamentation of the plasma membrane, usually in the mitochondrial region of the gamete. Additionally, some of these studies show a periodicity in the appearance of spinelike bodies along the spermatozoon body, e.g., O. furcatus (periodicity of 1 m; Miquel et al., 2000), P. furcatum (periodicity of 700 nm; Levron et al., 2004a), and F. gigantica (periodicity of 1 m; Ndiaye et al., 2004). In the mature spermatozoon of T. acutum, sections presenting a sufficient length to establish the periodicity of spinelike bodies were not observed. The present study corroborates the origin of spinelike bodies in the spermatid after the proximodistal fusion of flagella. Indeed, observations of T. acutum showing the appearance of spinelike bodies during spermiogenesis are in agreement with the original description (Miquel et al., 2000) of this character in O. furcatus. The spinelike bodies observed in the mature spermatozoon of T. acutum and in other digeneans (Miquel et al., 2000; Ndiaye, Miquel, Feliu et al., 2003; Ndiaye, Miquel, Fons et al., 2003; Ndiaye et al., 2004; Levron et al., 2004a; Agostini et al., 2005) resemble the crested bodies present in most cestodes, although several differences must be emphasized: (1) its distribution along the sperm cell considering that, unlike crested bodies, the spinelike bodies are not present in the anterior extremity of the spermatozoon; (2) its morphology, taking into account that spinelike bodies seem to contain an spherical vesicle; and (3) although spinelike bodies exhibit a periodicity similar to that of crested bodies, they consist of several isolated structures, whereas crested bodies are helical cords that surround the sperm cell. This character will probably be of great interest in the future in what refers to the elucidation of relationships between digeneans at the family level.
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TABLE I. Some ultrastructural characteristics of the spermatozoon in Digenea species. Family and species of Digenea
Ce*
Eo*
Sb*
Brachylaimidae S. palaearcticum
⫹
⫹
⫺
Ndiaye et al. (2002)
Bucephalidae Bucephaloides gracilescens Pseudorhipidocotyle elpichthys
⫺ ⫹
⫹ ⫹
⫺ ⫺
Erwin and Halton (1983) Tang et al. (1998)
Cryptogonimidae Neochasmus sp.
⫺
⫹
⫺
Jamieson and Daddow (1982)
Dicrocoeliidae Dicrocoelium dendriticum Dicrocoelium hospes Dicrocoelium chinensis C. vitta
⫺ ⫹ ⫺ ⫺
⫺ ⫹ ⫺ ⫺
⫺ ⫹ ⫺ ⫺
Cifrian et al. (1993) Agostini et al. (2005) Tang (1996) Robinson and Halton (1982)
Didymozoidae Didymozoon sp. Gonapodasmius sp. D. wedli
⫺ ⫺ ⫺
⫺ ⫹ ⫺
⫺ ⫺ ⫺
Justine and Mattei (1983) Justine and Mattei (1982b) Pamplona-Basilio et al. (2001)
Diplostomatidae Pharyngostomoides procyonis
⫺
⫺
⫺
Grant et al. (1976)
Echinostomatidae E. caproni
⫹
⫹
⫺
Iomini and Justine (1997)
Fasciolidae F. hepatica F. gigantica
⫹ ⫹
⫹ ⫹
⫹ ⫹
Ndiaye, Miquel, Fons et al. (2003) Ndiaye et al. (2004)
Haematoloechidae Haematoloechus sp.
⫹
⫹
⫹
Justine and Mattei (1982a)
Haploporidae S. godoyi
⫹
⫹
⫺
Baptista-Farias et al. (2001)
Heterophyidae C. lingua
⫺
⫺
⫺
Rees (1979)
Lecithodendriidae P. gymnesicus
⫺
⫹
⫺
Gracenea et al. (1997)
Microphallidae M. linguilla M. primas
⫺ ⫺
⫺ ⫹
⫺ ⫺
Hendow and James (1988) Castilho and Barandela (1990)
Monorchiidae M. parvus
⫺
⫹
⫺
Levron et al. (2004c)
Notocotylidae N. neyrai
⫺
⫹
⫹
Ndiaye, Miquel, Feliu et al. (2003)
Opecoeliidae H. fasciata O. furcatus P. furcatum
⫹ ⫺ ⫹
⫺ ⫹ ⫹
⫺ ⫹ ⫹
Levron et al. (2003) Miquel et al. (2000) Levron et al. (2004a)
Paragonimidae P. ohirai
⫹
⫹
⫹
Orido (1988)
Troglotrematidae T. acutum
⫹
⫹
⫹
Present study
Zoogonidae Diphterostomum brusinae
⫺
⫹
⫺
Levron et al. (2004b)
* Ce, cytoplasmic expansions; Eo, external ornamentation of plasma membrane; Sb, spinelike bodies.
Reference
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Posterior spermatozoon extremity As occurs with the anterior extremity of the spermatozoon, its posterior tip is morphologically variable (Agostini et al., 2005). There are digeneans with a posterior extremity of spermatozoa comprising (1) the 2 axonemes; (2) a single axoneme, with or without a nucleus; (3) only the nucleus; and (4) granules of glycogen. As occurs in numerous digeneans, in T. acutum the posterior extremity of the mature spermatozoa includes only a single axoneme, whereas the second axoneme, cortical microtubules, and granules of glycogen disappear in the nuclear region of sperm. The length of cortical microtubules is also variable. In certain species, they stop before the terminal region of cell where usually 1 of the axonemes and nucleus are present, e.g., F. hepatica, F. gigantica, and D. hospes and also T. acutum (Ndiaye, Miquel, Fons et al., 2003; Ndiaye et al., 2004; Agostini et al., 2005). In the posterior areas of the nuclear region of the mature spermatozoon of T. acutum, the cortical microtubules are not present. In other species, e.g., O. furcatus, S. palaearcticum, and P. furcatum, cortical microtubules reach the posterior tip of spermatozoon (Miquel et al., 2000; Ndiaye et al., 2002; Levron et al., 2004a). Number of mitochondria There are different viewpoints in the number of mitochondria present in the mature spermatozoon of Digenea. According to Burton (1972), several mitochondria are present in the differentiation zone, and, during spermiogenesis, they penetrate into the spermatid body. They accompany the nucleus in its migration along the spermatid, and posteriorly these multiple mitochondria fuse to form a long mitochondrion. Indeed, most of the studies on the sperm of digeneans describe the presence of a single mitochondrion in the male gamete. However, several authors, to make a logical interpretation in the disposition of ultrastructural characters along the mature spermatozoon, have considered the presence of 2 mitochondria, as for Neochasmus sp. (Cryptogonimidae) (Jamieson and Daddow, 1982), D. hospes (Dicrocoeliidae) (Agostini et al., 2005), F. hepatica (Fasciolidae) (Ndiaye, Miquel, Fons et al., 2003), Postorchigenes gymnesicus (Lecithodendriidae) (Gracenea et al., 1997), Maritrema linguilla (Microphallidae) (Hendow and James, 1988), M. parvus (Monorchiidae) (Levron et al., 2004c), N. neyrai (Notocotylidae) (Ndiaye, Miquel, Feliu et al., 2003), P. furcatum (Opecoelidae) (Levron et al., 2004a), and P. ohirai (Paragonimidae) (Orido, 1988). This is also true for the mature sperm cell of T. acutum in which there is an anterior mitochondrion and a posterior mitochondrion that coincides partially with the nuclear region. However, only Agostini et al. (2005) in D. hospes have demonstrated the presence of 2 parallel mitochondria in the same region of sperm. Considering that it is very difficult to observe longitudinal sections showing the transition between mitochondria, it is only possible to infer about the number of mitochondria after observing a very large number of transverse sections. Thus, research on this subject is very time-consuming. Application of some techniques of labeling of mitochondria also would be very useful to corroborate the results of these studies and to elucidate the real variability in the number of mitochondria.
ACKNOWLEDGMENTS We thank the ‘‘Serveis Cientı´fics i Te`cnics’’ of the University of Barcelona for support in the preparation of samples and the French ‘‘American mink trapping network.’’ This study was partially supported by ‘‘DURSI, Generalitat de Catalunya’’ (Spain).
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