Parasitol Res (2011) 108:997–1005 DOI 10.1007/s00436-010-2144-z
ORIGINAL PAPER
Ultrastructure of spermiogenesis and mature spermatozoon of Breviscolex orientalis (Cestoda: Caryophyllidea) Aneta Yoneva & Céline Levron & Mikuláš Oros & Martina Orosová & Tomáš Scholz
Received: 23 September 2010 / Accepted: 28 October 2010 / Published online: 18 November 2010 # Springer-Verlag 2010
Abstract Spermiogenesis and spermatozoon ultrastructure of the caryophyllidean cestode Breviscolex orientalis Kulakovskaya, 1962, first member of the family Capingentidae studied, a parasite of cyprinid fish Abbottina rivularis, are described using transmission electron microscopy. Spermiogenesis in B. orientalis follows the Type II pattern described by Bâ and Marchand (Mém Mus Natl Hist Nat 166:87–95, 1995) for cestodes. It begins with the formation of a zone of differentiation containing a large nucleus and a pair of centrioles. The centrioles are separated from one another by an intercentriolar body composed of three electron-dense layers. Each centriole is associated with typical striated roots. At the beginning of the spermiogenesis, an electron-dense material is observed in the apical region of the differentiation zone. During the initial stage of spermiogenesis, one of the centrioles gives rise to a free flagellum, which then rotates and undergoes proximodistal fusion with the cytoplasmic protrusion of the differentiation zone. The A. Yoneva (*) Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, 2 Gagarin Street, 1113, Sofia, Bulgaria e-mail:
[email protected] A. Yoneva : C. Levron : T. Scholz Institute of Parasitology, Biology Centre of the Academy of Sciences of the Czech Republic, Branišovská 31, 370 05, České Budějovice, Czech Republic M. Oros : M. Orosová Parasitological Institute, Slovak Academy of Sciences, Hlinkova 3, 040 01, Košice, Slovak Republic
mature spermatozoon of B. orientalis corresponds to the Type III pattern described by Levron et al. (Biol Rev 85:523–543, 2010). It is characterized by the absence of mitochondrion and crested body. Five regions of the mature spermatozoon are differentiated. The main ultrastructural characteristics are: one axoneme of 9+“1” trepaxonematan pattern, cortical microtubules and nucleus. The comparison of the spermiogenesis of B. orientalis with those of the other caryophyllidean species demonstrates some variation within the order relative to the presence and morphology of the intercentriolar body, the presence of slight rotation of the flagellar bud and a complete proximodistal fusion of the free flagellum with a cytoplasmic protrusion.
Introduction Data on spermiogenesis and mature spermatozoa of parasitic Platyhelminthes, especially of tapeworms, have been used successfully in the interpretation of the phylogenetic relationships (Justine 1991, 1998, 2001; Bâ and Marchand 1995; Hoberg et al. 1997, 2001; Levron et al. 2010). The order Caryophyllidea comprises intestinal parasites of cypriniform and siluriform freshwater fishes. They are unique among Cestoda because their body lacks segmentation and have a single set of reproductive organs (Mackiewicz 1994). The order has been divided into four families: Balanotaeniidae, Capingentidae, Caryophyllaeidae and Lytocestidae, but recent data indicate that they are paraphyletic (Olson et al. 2008; Oros et al. 2008). Until now, only six species belonging to the families Caryophyllaeidae and Lytocestidae have been the subject of ultrastructural spermatological studies, but no data exist on members of remaining families, including Capingentidae
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(Kazacos and Mackiewicz 1972; Świderski 1986; Świderski and Mackiewicz 2002; Arafa and Hamada 2004; Bruňanská and Poddubnaya 2006; Gamil 2008; Miquel et al. 2008; Bruňanská 2009). The present study provides the first information of the spermiogenesis and the spermatozoon of a capingentid cestode, Breviscolex orientalis Kulakovskaya, 1962.
Materials and methods Live specimens of B. orientalis were collected from the intestine of naturally infected fish Abbottina rivularis (Basilewsky, 1855) (Cypriniformes, Cyprinidae) collected in March 2009 from the Yangtze River in Wuhan, Hubei Province, China. Live worms were rinsed in 0.9% NaCl solution and fixed with 1.5% glutaraldehyde and 1.5% paraformaldehyde in 0.1-M Hepes at pH 7.4, rinsed in 0.1M Hepes at pH 7.4, post-fixed in cold (4°C) 1% OsO4 in the same buffer for 1 h, dehydrated in graded series of acetone, and embedded in Spurr’s epoxy resin. Ultrathin sections (60–90 nm in thickness) were cut on a Leica Ultracut UCT ultramicrotome, placed on copper grids, and double-stained with uranyl acetate and lead citrate according to Reynolds (1963). Sections were examined under a JEOL 1010 transmission electron microscope at 80 kV.
Results Spermiogenesis Spermiogenesis of B. orientalis is illustrated in Figs. 1, 2 and 3. The beginning of spermiogenesis in B. orientalis is marked in each spermatid by the formation of a zone of differentiation. This zone is lined by a row of cortical microtubules and delimited by a ring of arching membranes. The zone of differentiation contains a large nucleus and two centrioles arranged in the same plane (Fig. 1a). The centrioles are separated from one another by an intercentriolar body (Fig. 1a–c). The latter is composed of five layers. Three of them are electron-dense with different thickness; the thicker layer is centrally located. They are separated by two electron-lucent layers (Figs. 1c, Inset; 3a). The centrioles are made up of nine triplets arranged in a circle and each centriole is additionally associated with typical striated roots (Fig. 1d, e). In the initial stage of spermiogenesis, the apical region of the zone of differentiation contains an electrondense material (Figs. 1a–c; 3b). During the process, one of the centrioles becomes elongated and develops a free flagellum (Fig. 1c). The other centriole produces a flagellar bud that also undergoes a slight rotation. It aborts later in the process (Figs. 1b, f; 3c). A cytoplasmic protrusion is formed
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distally to the centriolar area (Fig. 1f). Thereafter, the free flagellum rotates and becomes parallel to the elongated cytoplasmic protrusion of the differentiation zone (Fig. 1g–h). The end of this stage is illustrated on cross-sections where a single flagella and cytoplasmic protrusion can be seen in adjacent (Figs. 2a; 3d). The next step is the proximodistal fusion between the flagellum and the cytoplasmic protrusion. Two attachment zones are observed in the cytoplasmic protrusion corresponding to the area of the fusion with the flagellum (Fig. 2b). During the flagellar rotation, the nucleus elongates, becomes filiform, moves into the ring of arching membranes and begins its migration toward the cytoplasmic protrusion. At this stage, the chromatin is partially condensed and slightly electron-dense (Fig. 2c–f). The striated roots in the differentiation zone are still visible until the final stage of the process (Fig. 2e–f). The end of spermiogenesis is marked by the changes at the level of the arching membranes (Fig. 2g). Finally, the ring of arching membranes is constricted (Fig. 2g) and the mature spermatozoon becomes detached from the residual cytoplasm (Fig. 3e). Spermatozoon Mature spermatozoa of B. orientalis from seminal vesicle were observed (Fig. 4a). The ultrastructural organization of the spermatozoon is illustrated in Figs. 4 and 5. The male gamete is a long filiform cell, tapered at both extremities and lacks mitochondria. From the anterior to the posterior extremities of the gamete, it is possible to distinguish five regions with different ultrastructural characteristics. Region I (Figs. 4b; 5I) constitutes the anterior part of the spermatozoon. Cross-sections through this region have diameter of approximately 280 nm. This region contains the axoneme and a small number of cortical microtubules. The axoneme is of 9+“1” trepaxonematan type and is partially surrounded by a semi-arc of a few cortical microtubules. Region II (Figs. 4c–d; 5II) has a diameter of approximately 460 nm. Cross-sections through this region contain the axoneme and the cortical microtubules. The axoneme is surrounded by a layer of slightly electron-dense cytoplasm (Fig. 4c). The number of cortical microtubules lying beneath the plasma membrane increases. More distally, this region is characterized by a widening of the cell diameter due to the increasing of the volume of cytoplasm. Region III (Figs. 4e–g; 5III) corresponds to the nucleated portion of the mature spermatozoon. The diameter of the sperm cell is approximately 560 nm. The nucleus is electrondense and is localized in the center of the gamete. The diameter of the nucleus increases towards the distal part of the region. The cytoplasm is slightly electron-dense. At the end of the region, the nucleus comes into close contact with the plasma membrane (Fig. 4g). At this moment, only a few cortical microtubules are present.
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Fig. 1 Early stages of spermiogenesis in Breviscolex orientalis. a Longitudinal section of a zone of differentiation showing initial stage of spermiogenesis. C centrioles, Cm cortical microtubules, Dm electrondense material, Ib intercentriolar body, N nucleus. Scale bar=1 μm. b Longitudinal section of the zone of differentiation showing the disposition of the two centrioles and the intercentriolar body (Ib). B cytoplasmic bud, Dm electron-dense material, N nucleus. Scale bar= 0.5 μm. c Longitudinal section of the zone of differentiation showing the development of the free flagellum (F). Dm electron-dense material, Ib intercentriolar body, N nucleus. Scale bar=1 μm. Inset. Detail at the level of the intercentriolar body. Scale bar=0.2 μm. d Longitudinal section of a differentiation zone showing a centriole (C). N nucleus, n
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nucleolus, Sr striated roots. Scale bar=0.7 μm. e Cross-section of a differentiation zone through the striated roots (Sr) and the nucleus (N). Scale bar=0.4 μm. f Longitudinal section of a zone of differentiation showing the abortive centriole. Am arching membranes, B cytoplasmic bud, Cp cytoplasmic protrusion, N nucleus, Sr striated root. Scale bar= 0.5 μm. g Longitudinal section of a zone of differentiation showing the rotation of the free flagellum (F) with the cytoplasmic protrusion (Cp). B cytoplasmic bud. Scale bar=0.5 μm. h Longitudinal section of a zone of differentiation during the fusion of the flagellum (F) with the cytoplasmic protrusion (Cp). Am arching membranes, N nucleus, Sr striated roots. Scale bar=0.7 μm
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Fig. 2 Advanced stages of spermiogenesis in Breviscolex orientalis. a Cross-sections of spermatids before the proximodistal fusion of a free flagellum (F) with the cytoplasmic protrusion (Cp). Scale bar=0.5 μm. b Cross-sections showing the attachment zones of the cytoplasmic protrusion (Cp). Arrowheads indicate the two attachment zones; F free flagellum. Scale bar=0.3 μm. c Longitudinal section showing the migrating nucleus (N) with partially condensed chromatin. Cp cytoplasmic protrusion, F flagellum. Scale bar=0.6 μm. d Cross-sections of
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spermatids after the migration of the nucleus (N). Scale bar=0.5 μm. e Longitudinal section showing the migration of the nucleus (N). Am arching membranes, Ax axoneme, Sr striated roots. Scale bar=0.6 μm. f. Longitudinal section of a zone of differentiation in the final stage of nuclear migration. Note the disorganization of the striated roots (Sr). N nucleus. Scale bar=0.7 μm. g Longitudinal section of a zone of differentiation in a final stage of spermiogenesis. Am arching membranes, Ax axoneme. Scale bar=0.8 μm
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Fig. 3 a–e Schematic representation of the main stages of spermiogenesis in Breviscolex orientalis. Am arching membrane, Ax axoneme, Az attachment zones, B cytoplasmic bud, C centriole, Cm cortical
microtubules, Cp cytoplasmic protrusion, Dm dense material, F free flagellum, Ib intercentriolar body, N nucleus, Sr striated roots
Region IV (Figs. 4h; 5IV) represents the postnucleated part of the spermatozoon. It measures around 350 nm in diameter. This area is characterized by the lack of the nucleus. It contains only the axoneme surrounded by plasma membrane. In the posterior part of this region, the cortical microtubules progressively disappeared. Region V (Figs. 4e; 5V) corresponds to the posterior part of the spermatozoon. In the posterior area of this region, the axoneme loses its central element followed by transformation of the peripheral doublets into singlets.
Discussion Spermiogenesis Spermiogenesis in B. orientalis follows the general Type II pattern proposed by Bâ and Marchand (1995) for cestodes. This pattern is characterized by the presence of flagellar rotation and proximodistal fusion of the single flagellum with the cytoplasmic protrusion. Until now, it has been described for all studied members of the order Caryophyl-
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Fig. 4 Mature spermatozoon of Breviscolex orientalis. a Cross-section of seminal vesicle containing numerous spermatozoa. Scale bar=0.7 μm. b Cross-section of anterior spermatozoon extremity showing the axoneme (Ax). Cm cortical microtubules. Scale bar=0.1 μm. c Cross-section of region I with cortical microtubules (Cm). Ax axoneme. Scale bar=0.1 μm. d Cross-sections of regions II and III. Ax axoneme, Cm cortical microtubules. Scale bar=0.3 μm. e Cross-sections of region III and region
V of the mature spermatozoon. D doublets, N nucleus. Scale bar=0.3 μm. f Cross-sections of region III of mature spermatozoon. Scale bar=0.2 μm. g Cross-sections of distal part of region III of a mature spermatozoon. N nucleus. Scale bar=0.3 μm. h Cross-section of region IV of a mature spermatozoon containing the axoneme (Ax). The cytoplasm and the cortical microtubules decrease gradually. Scale bar=0.1 μm
lidea of the families Caryophyllaeidae and Lytocestidae, i.e., Glaridacris catostomi, Monobothrioides chalmersius, Khawia armeniaca, K. sinensis, and Wenyonia virilis (Świderski and Mackiewicz 2002; Arafa and Hamada 2004; Bruňanská and Poddubnaya 2006; Gamil 2008; Miquel et al. 2008; Bruňanská 2009). Apart from caryophyllideans, Type II of spermiogenesis has been observed in representatives of more evolved, but unrelated groups: Tetrabothriidea (Stoitsova et al. 1995), Diphyllidea (Azzouz-Draoui and Mokhtar-Maamouri 1986/88), Tetra-
phyllidea: Phyllobothriidae (Mokhtar-Maamouri 1979) and Mesocestoidea (Miquel et al. 1999, 2007a). However, differences between caryophyllideans were observed with respect to Type II spermiogenesis. In W. virilis, Miquel et al. (2008) mentioned the presence of rotation of the free flagellum more than 90°. This unique type of flagellar rotation was described for the first time in cestodes. In contrast, no evidence of rotation was reported for G. catostomi and M. chalmersius (Świderski and Mackiewicz 2002; Arafa and Hamada 2004). However,
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Fig. 5 I–V Schematic reconstruction of the mature spermatozoon of Breviscolex orientalis. Ase Anterior spermatozoon extremity, Ax Axoneme, Cm Cortical microtubules, N Nucleus, Pm Plasma membrane, Pse Posterior spermatozoon extremity
the reinvestigation of the description and illustrations of G. catostomi by Miquel et al. (2008) has shown the presence of flagellar rotation not described previously for that species (Świderski and Mackiewicz 2002). In K. armeniaca, W. virilis and K. sinensis, the free flagellum fuses completely with the cytoplasmic protrusion (Bruňanská and Poddubnaya 2006; Gamil 2008; Miquel et al. 2008; Bruňanská 2009). Similarly, our data on B. orientalis confirm complete proximodistal fusion between the flagellum and the cytoplasmic protrusion. Noncomplete fusion (the axoneme is not fully incorporated within the cytoplasmic protrusion) has been observed only in G. catostomi from North American suckers (Catostomidae) and M. chalmersius from African catfish (Świderski and Mackiewicz 2002; Arafa and Hamada 2004).
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In B. orientalis, we observed the presence of a slight rotation of the flagellar bud with the cytoplasmic protrusion, a character not reported previously for Type II spermiogenesis of Bâ and Marchand (1995). More recent data on K. armeniaca and K. sinensis (Bruňanská and Poddubnaya 2006; Bruňanská 2009) also provided evidence for the presence of such a rotation in these two species. The slight rotation of the flagellar bud seems to be a character typical for most caryophyllideans. The presence of striated roots and an intercentriolar body in the zone of differentiation are typical features of Type II spermiogenesis (Bâ and Marchand 1995). The intercentriolar body is considered as a plesiomorphic character within the Eucestoda, and its progressive reduction occurs in the higher cestodes (Justine 2001). In the order Caryophyllidea, as in the other cestode orders, variability exists to the presence and morphology of intercentriolar body (Levron et al. 2010). No intercentriolar body was described in the differentiation zone of M. chalmersius and W. virilis (Arafa and Hamada 2004; Gamil 2008). The intercentriolar body differs among the examined caryophyllideans by the number of the layers. In B. orientalis, the intercentriolar body is composed of three electron-dense layers separated by two, electron-lucent layers, similarly as in W. virilis and K. sinensis (Miquel et al. 2008; Bruňanská 2009). In K. armeniaca, a single electron-dense layer was described (Bruňanská and Poddubnaya 2006). Reduced intercentriolar body composed of only one electron-dense layer is also present in more evolved, acetabulate (“tetrafossate”) cestodes, i.e., Proteocephalidea, Lecanicephalidea, and one Mesocestoididae (Levron et al. 2010). Justine (2001) considered the presence of “reduced intercentriolar body” as an intermediate state between the welldeveloped intercentriolar body in the more primitive cestode orders and its absence in the orders Tetrabothriidea and Cyclophyllidea. B. orientalis possesses typical striated roots associated with each centriole during spermiogenesis. The striated roots associated with the centrioles in the differentiation zone are a variable character that may be present in some species, but absent in others (Justine 2001). In fact, the typical striated roots are generally present during spermiogenesis of the cestode orders presenting spermiogenesis of Type I and II (Bâ and Marchand 1995). Until now, typical striated roots associated with each centriole were observed in all studied caryophyllideans but this character has recently been observed also in the most evolved cestodes, i.e., cyclophyllideans (Miquel et al. 1999, 2007a; Ndiaye et al. 2003). Electron-dense material has been observed in the apical region of the zone of differentiation in the early stage of the spermiogenesis in B. orientalis. In the Caryophyllidea, a similar character has also been described in K. armeniaca, W. virilis and K. sinensis (Bruňanská and Poddubnaya 2006; Gamil 2008; Miquel et al. 2008; Bruňanská 2009) as well as
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in the spathebothriideans Cyathocephalus truncatus and Didymobothrium rudolphii, bothriocephalideans Eubothrium crassum, Triaenophorus nodulosus, Bothriocephalus scorpii, and Parabothriocephalus gracilis and diphyllobothriideans Diphylobothrium latum and Ligula intestinalis (Bruňanská et al. 2001, 2006; Levron et al. 2005, 2006a, b; Bruňanská and Poddubnaya 2010; Šipková et al. 2010). Spermatozoon Mature spermatozoon of four species of the Caryophyllidea has been described (Świderski and Mackiewicz 2002; Arafa and Hamada 2004; Gamil 2008), the most detailed description being that of K. sinensis provided by Bruňanská (2009). The mature spermatozoon of B. orientalis does not differ significantly from the general pattern known for other caryophyllideans of two families (no member of the Balanotaeniidae and Capingentidae was previously studied). It contains one axoneme, parallel cortical microtubules and nucleus, but devoid of a crested body and thus corresponds to the Type III pattern of Levron et al. (2010). The crested body or bodies characterize the anterior extremity of the gamete (Bâ et al. 1991) and their presence has been postulated as a synapomorphy for some eucestodes (Justine 2001). The crested body is absent in the mature spermatozoon of the Gyrocotylidea, Amphilinidea, Caryophyllidea, Haplobothriidea, Trypanorhyncha, Spathebothriidea and Diphyllobothriidea (Xylander 1989; Rohde and Watson 1986; Świderski and Mackiewicz 2002; Gamil 2008; Bruňanská 2009; MacKinnon and Burt 1985; Świderski 1976; McKerr 1985; Miquel and Świderski 2006; Miquel et al. 2007b; Bruňanská et al. 2006; Levron et al. 2006a, b, c, 2009). Comparing the ultrastructural characters of the spermatozoon of B. orientalis and the other caryophyllidean species, differences were found in the presence of glycogen inclusions. In G. catostomi and W. virilis, scattered glycogen granules were observed between the nucleus and the axoneme (Świderski and Mackiewicz 2002; Gamil 2008) whereas no glycogen inclusions were observed in B. orientalis and K. sinensis (Bruňanská 2009, present study). On the basis of the present results, we can conclude that the spermatological characters of B. orientalis (Capingentidae) are similar to those described for the representatives of Lytocestidae. The main differences concern the morphology of the intercentriolar body (Bruňanská and Poddubnaya 2006; Bruňanská 2009). Acknowledgements We are grateful to the staff of the Laboratory of Electron Microscopy, Institute of Parasitology, České Budějovice, Czech Republic, for technical assistance. This study was supported by the Grant Agency of the Czech Republic (project nos. 524/08/0885 and P506/10/1994), Institute of Parasitology (Z60220518), Research Centre of Ichthyoparasitology (LC 522), and Slovak Research and Development Agency (project no. LPP 0171-09).
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