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Ultrastructure of the spermatozoon of Parahemiurus merus (Linton, 1910) (Digenea: Hemiuroidea: Hemiuridae), a parasite of Sardinella auritaValenciennes, 1847 and S. maderensis (Lowe, 1838) (Teleostei: Clupeidae) in the Senegalese coast
Zoologischer Anzeiger 252 (2013) 572–578
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Ultrastructure of the spermatozoon of Parahemiurus merus (Linton, 1910) (Digenea: Hemiuroidea: Hemiuridae), a parasite of Sardinella aurita Valenciennes, 1847 and S. maderensis (Lowe, 1838) (Teleostei: Clupeidae) in the Senegalese coast Papa Ibnou Ndiaye a , Abdoulaye J.S. Bakhoum b,c , Aminata Sène a , Jordi Miquel b,c,∗ a
Laboratory of Evolutionary Biology, Ecology and Management of Ecosystems, Faculty of Sciences and Techniques, Cheikh Anta Diop University of Dakar, BP 5055, Dakar, Senegal Laboratori de Parasitologia, Departament de Microbiologia i Parasitologia Sanitàries, Facultat de Farmàcia, Universitat de Barcelona, Av. Joan XXIII, s/n, E-08028 Barcelona, Spain c Institut de Recerca de la Biodiversitat, Facultat de Biologia, Universitat de Barcelona, Av. Diagonal, E-645, 08028 Barcelona, Spain b
a r t i c l e
i n f o
Article history: Received 18 September 2012 Received in revised form 21 November 2012 Accepted 26 November 2012 Available online 29 December 2012 Corresponding Editor: Martin V. Sørensen. Keywords: TEM Dakar coast Senegal
a b s t r a c t The mature spermatozoon of Parahemiurus merus (Digenea: Hemiuroidea: Hemiuridae) is filiform, tapered in both ends and exhibits the general pattern described in most digeneans so far: two axonemes of the 9+‘1’ pattern of trepaxonematan Platyhelminthes, nucleus, mitochondrion, external ornamentation of the plasma membrane and parallel cortical microtubules. However, the spermatozoon of P. merus presents some peculiarities. The presence of an external ornamentation of the plasma membrane not associated with cortical microtubules in the anterior part of the spermatozoon is described for a second genus in the family Hemiuridae. Cortical microtubules are disposed in only one side of the spermatozoon as described in other species of Hemiuroidea but, in P. merus, the number of cortical microtubules is much lower. The results obtained are compared with the available data on Hemiuroidea in order to improve the understanding of phylogenetic relationships of hemiuroids. Similarities found between spermatozoa of hemiurids and lecithasterids are in accordance with molecular evidence that group these two families in a same clade. © 2012 Elsevier GmbH. All rights reserved.
1. Introduction Digeneans of the superfamily Hemiuroidea Looss, 1899 are usually parasitic of the digestive tract of fishes, particularly the stomach of marine teleosts, although they also occur in freshwater teleosts, elasmobranchs and occasionally in amphibians and reptiles (Gibson, 2002). This superfamily has been subject of controversies, but in the most extensive revision of Hemiuroidea, Gibson and Bray (1979) recognize 14 families within the group. However, Gibson (2002), considering molecular evidences of Blair et al. (1998), excludes the family Azygiidae Lühe, 1909 from the Hemiuroidea. This exclusion was confirmed recently in another study, which places the Azygioidea Lühe, 1909 as a sister group of Hemiuroidea (Olson et al., 2003). On the other hand, these molecular analyses strongly support the retention of the remaining families within the Hemiuroidea, including the enigmatic family
∗ Corresponding author at: Laboratori de Parasitologia, Departament de Microbiologia i Parasitologia Sanitàries, Facultat de Farmàcia, Universitat de Barcelona, Av. Joan XXIII, s/n, E-08028 Barcelona, Spain. Tel.: +34 93 4024500; fax: +34 93 4024504. E-mail address: [email protected] (J. Miquel). 0044-5231/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.jcz.2012.11.005
Didymozoidae Monticelli, 1888. Gibson (2002) therefore considers the Hemiuroidea to accommodate 13 families, including the Hemiuridae Looss, 1899, which in turn includes the species Parahemiurus merus (Linton, 1910) studied in the present work. However, the phylogenetic relationships between some families remain controversial (see Cribb et al., 2001; Olson et al., 2003). In this context, the study of the spermatozoon of species belonging to all these families represent an important source of ultrastructural characters that can be a great contribution for elucidating phylogenetic relationships within this group. To our knowledge, in the Hemiuroidea, ultrastructural data on spermiogenesis and/or spermatozoa exist for only six species belonging to four families, namely the didymozoids Didymocystis wedli Ariola, 1902 (see Pamplona-Basilio et al., 2001), Didymozoon sp. (see Justine and Mattei, 1983, 1984a) and Gonapodasmius sp. (see Justine and Mattei, 1982, 1984b), the sclerodistomid Prosorchis ghanensis Fischtal & Thomas, 1972 (see Justine, 1995), the lecithasterid Aponurus laguncula Looss, 1907 (see Quilichini et al., 2010a) and the hemiurid Lecithocladium excisum (Rudolphi, 1819) (see Ndiaye et al., 2012a). Thus, the aim of the present study is to provide new ultrastructural spermiological data on a second genus of the family Hemiuridae.
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Fig. 1. (a–j) Spermatozoon of Parahemiurus merus. (a–g) Consecutive cross-sections of the anterior region of the spermatozoon (region I). Note the presence of an external ornamentation of the plasma membrane (EO) not associated with cortical microtubules (CM) and the appearance of a single field of cortical microtubules in the posterior area of this region. Figure d shows a detail of the external ornamentation of the plasma membrane. PM, plasma membrane; S, singlets. Scale bars (a–c, e–g) = 0.2 m; (d) = 0.1 m. (h–j) Consecutive cross-sections of the mitochondrial or middle region of the spermatozoon (region II) characterized by the presence of the mitochondrion (M) and the anterior nucleus extremity. This region contains the maximum number of cortical microtubules (CM) (five). N, nucleus; scale bars = 0.2 m.
2. Materials and methods Live adult specimens of P. merus (Linton, 1910) (Digenea: Hemiuroidea: Hemiuridae) were collected from the digestive tract of two marine teleosts, the Round sardinella Sardinella aurita Valenciennes, 1847 and the Madeiran sardinella S. maderensis (Lowe, 1838) (Clupeidae), both captured in the Atlantic Ocean, off Dakar (Senegal) during April 2011. Worms were rinsed with a 0.9% NaCl solution and fixed in cold (4 ◦ C) 2.5% glutaraldehyde in a 0.1 M sodium cacodylate buffer at pH 7.4, rinsed in 0.1 M sodium cacodylate buffer at pH 7.4, postfixed in cold (4 ◦ C) 1% osmium tetroxide with K4 FeCn6 in the same buffer for 1 h, rinsed in a 0.1 M sodium cacodylate buffer at pH 7.4, dehydrated in an ethanol series and propylene oxide, embedded in Spurr’s resin and polymerized at 60 ◦ C for 72 h. Ultrathin sections (60–90 nm thick) of areas containing seminal ducts and seminal vesicle were made in a Reichert-Jung Ultracut E ultramicrotome using a diamond knife. Then, sections were placed on copper and gold grids. Those sections placed on copper grids were double-stained with uranyl acetate and lead citrate according to the Reynolds (1963) procedure. The sections placed on gold grids were treated according to the Thiéry (1967) test to reveal the presence of glycogen. Thus, they were treated in periodic acid (PA), thiocarbohydrazide (TCH) and silver proteinate (SP) as follows:
30 min in 10% PA, rinsed in milliQ water, 24 h in TCH, rinsed in acetic solutions and milliQ water, 30 min in 1% SP in the dark, and rinsed in milliQ water. The grids were examined in a JEOL 1010 transmission electron microscope operated at 80 kV, in the “Centres Científics i Tecnològics de la Universitat de Barcelona (CCiTUB)”.
3. Results The observation of transversal and longitudinal sections of the mature spermatozoon permitted us to distinguish three regions (I–III) with different ultrastructural characters. Region I (Figs. 1a–g and 3I) corresponds to the anterior part of the spermatozoon. Consecutive transverse sections from anterior to posterior areas of region I exhibit progressively: (i) singlets of axonemes (Fig. 1a–c), (ii) the external ornamentation of the plasma membrane (Fig. 1c and d), (iii) the appearance of axonemes (Fig. 1c and e), (iv) the progressive reduction and disappearance of the external ornamentation (Fig. 1f and g), and (v) the appearance of a single field (ventral) of parallel cortical microtubules (Fig. 1g). Region II (Fig. 1h–j, 2a and 3II) is the middle region or mitochondrial area of the spermatozoon. In addition to the structures observed in the posterior part of region I (two axonemes and
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Fig. 2. (a–h) Spermatozoon of Parahemiurus merus. (a) Cross-sections showing the transition between regions II and III. M, mitochondrion; N, nucleus; scale bar = 0.2 m. (b–g) Consecutive cross-sections of the posterior part (region III) characterized by the presence of the posterior nucleus extremity. In this region there is the disorganization of the two axonemes and the disappearance of cortical microtubules (CM). CC, central core; D, doublets; N, nucleus; S, singlets; scale bars = 0.2 m. (h) Revelation of glycogen (G) by the test of Thiéry. Scale bar = 0.2 m.
cortical microtubules), a mitochondrion is present in region II (Fig. 1h). The nucleus coincides with an area where the layer of cortical microtubules is formed by the maximum number of elements (five) (Fig. 1i). Then, the mitochondrion and the nucleus increase in size and the layer of cortical microtubules decreases to four elements (Fig. 1j). The posterior part of this region is characterized by a decrease in the size of the mitochondrion and a large-sized nucleus, while the layer of cortical microtubules is formed by only three elements (Fig. 2a). Region III (Figs. 2a–g and 3III) corresponds to the posterior part of the spermatozoon. It is characterized by: (i) disappearance of the mitochondrion (Fig. 2a), (ii) disorganization of the first axoneme (Fig. 2b and c), (iii) progressive reduction of the nucleus size until its disappearance (Fig. 2d and e), and (iv) loss of cortical microtubules and disorganization of the second axoneme into doublets and singlets (Fig. 2e–g).
Fig. 2h shows the positive result of the Thiéry test exhibiting a very reduced amount of granules of glycogen. 4. Discussion The mature spermatozoon of P. merus exhibits the general pattern of most digeneans described so far: two axonemes of the 9+‘1’ pattern of trepaxonematans (Ehlers, 1984), nucleus, mitochondrion, external ornamentation of the plasma membrane and parallel cortical microtubules (Jamieson and Daddow, 1982; Iomini and Justine, 1997; Tang et al., 1998; Miquel et al., 2000; Ndiaye et al., 2003a; Quilichini et al., 2007a, 2009, 2010a). However, it also presents some peculiarities. The anterior part of the spermatozoon is characterized by an external ornamentation of the plasma membrane not associated with cortical microtubules. In the Hemiuridae, L. excisum also exhibits this organization of the external
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Fig. 3. (I–III) Schematic reconstruction of the spermatozoon of Parahemiurus merus showing the ultrastructural organization of the three considered regions. ANE, anterior nucleus extremity; ASE, anterior spermatozoon extremity; Ax1, first axoneme; Ax2, second axoneme; CC, central core; CM, cortical microtubules; D, doublets; EO, external ornamentation of the plasma membrane; G, granules of glycogen; M, mitochondrion; N, nucleus; PM, plasma membrane; PNE, posterior nucleus extremity; PSE, posterior spermatozoon extremity; S, singlets.
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Table 1 Available data on the spermatozoon ultrastructure in the Hemiuroidea. Families and species
Ultrastructural spermatozoon characters Ax
Didymozoidae Didymocystis wedli Didymozoon sp. Gonapodasmius sp. Hemiuridae Lecithocladium excisum Parahemiurus merus Lecithasteridae Aponurus laguncula Sclerodistomidae Prosorchis ghanensis
ASE
References
EO
CM
PSE
+/–
loc
EO/CM
bund
n
9+‘1’ 9+0 9+‘1’
? ? 1Ax-EO-CM
– – +
NA NA RAnt
NA NA +
– – 2
0 0 36a
? ? 2F
Pamplona-Basilio et al. (2001) Justine and Mattei (1983, 1984a) Justine and Mattei (1982, 1984b)
9+‘1’ 9+‘1’
1Ax-EO 1Ax-EO
+ +
RAnt RAnt
– –
1 1
8 5
1Ax 1Ax
Ndiaye et al. (2012a) Present study
9+‘1’
1Ax-EO
+
RAnt
–
1
10
D+S
Quilichini et al. (2010a)
9+‘1’
?
?
NA
NA
1–2b
13–15b
?
Justine (1995)
Spermatozoon characters: ASE, anterior spermatozoon extremity; Ax, axoneme; bund, number of bundles of cortical microtubules; CM, cortical microtubules; D, doublets; EO, external ornamentations of the plasma membrane; EO/CM, association of external ornamentations and cortical microtubules; F, flagellum; loc, location of external ornamentations; n, maximum number of cortical microtubules; NA, not applicable; PSE, posterior spermatozoon extremity; RAnt, anterior region; S, singlets; +/−, presence/absence;?, unknown data. a The maximum number of cortical microtubules occurs in the anterior spermatozoon area in a continuous submembranous layer. b During spermiogenesis there are 2 bundles of 1 and 15 microtubules in the median cytoplasmic process. After the proximodistal fusion there is only 1 bundle of 13 microtubules.
ornamentation (see Ndiaye et al., 2012a). In this family, there are ultrastructural studies of the spermatozoon only in P. merus and L. excisum (Ndiaye et al., 2012a). However, in the latter the external ornamentation covers entirely the anterior extremity of the spermatozoon. In all of the remaining hemiuroids studied to date, only the lecithasterid A. laguncula exhibits this pattern (Quilichini et al., 2010a). Moreover, in the Hemiuridae and Lecithasteridae the external ornamentation is observed as a continuous electron-dense material, whereas in other digeneans that exhibit the ornamentation associated with cortical microtubules, in both anterior and mitochondrial areas of the sperm cell the external ornamentation seems to be constituted by a layer of isolated electron-dense elements (see Miquel et al., 2000, 2006). With respect to the didymozoid Gonapodasmius sp., Justine and Mattei (1982, 1984b) described an external ornamentation of the plasma membrane associated with a continuous bundle of cortical microtubules in the anterior spermatozoon extremity. Contrarily, other didymozoids namely D. wedli and Didymozoon sp. lack external ornamentation of the plasma membrane and cortical microtubules (Justine and Mattei, 1983, 1984a; Pamplona-Basilio et al., 2001). Like other hemiuroids studied so far, the mature spermatozoon of P. merus presents a low number of cortical microtubules with different lengths disposed only in the ventral side. In the Hemiuroidea, the maximum number of cortical microtubules is described in the anterior region of the spermatozoon of the didymozoid Gonapodasmius sp. with 36 elements surrounding the sperm cell beneath the plasma membrane (Justine and Mattei, 1982) while other didymozoids lack cortical microtubules (Justine and Mattei, 1983, 1984a; Pamplona-Basilio et al., 2001). In the remaining studied species the number of cortical microtubules is low and distributed in a single bundle as occurs in P. merus. The hemiurid L. excisum exhibits a maximum of eight microtubules and the lecithasterid A. laguncula a maximum of ten, located in the mitochondrial region of the spermatozoon in both cases (Quilichini et al., 2010a; Ndiaye et al., 2012a). In the case of P. merus there is a maximum of five microtubules, also in the mitochondrial area. A low number of cortical microtubules of different lengths and their disposition in only the ventral side of the spermatozoon seem to be characteristics of the male gamete of species belonging to the families Hemiuridae and Lecithasteridae Odhner, 1905 (Quilichini et al., 2010a; Ndiaye et al., 2012a). With respect to the remaining hemiuroids, apart from the enigmatic group of didymozoids (Justine and Mattei, 1982, 1983, 1984a,b; Pamplona-Basilio et al., 2001), there are only observations
on the spermiogenesis of the Sclerodistomidae P. ghanensis (see Justine, 1995). Spine-like bodies have been described in the spermatozoon of numerous digeneans belonging to different families and superfamilies, e.g. echinostomatoideans, microscaphidioideans, paramphistomoideans, pronocephaloideans, allocreadioideans, gorgoderoideans, lepocreadioideans, opisthorchioideans and plagiorchioideans (Miquel et al., 2000, 2006; Ndiaye et al., 2003a,b, 2012b,c; Agostini et al., 2005; Quilichini et al., 2007b, 2010b; Seck et al., 2008a,b; Kacem et al., 2010; Bakhoum et al., 2011a,b, 2012a,b, in press; Foata et al., 2012). Contrarily, they have not been observed in any hemiuroid. Unlike the great majority of digeneans (Jamieson and Daddow, 1982; Iomini and Justine, 1997; Miquel et al., 2000, 2006; Ndiaye et al., 2002, 2011; Kacem et al., 2010; Bakhoum et al., 2011b, 2012a), the mature spermatozoon of P. merus has a small quantity of glycogen granules as occurs also in the hemiurid, L. excisum (Ndiaye et al., 2012a). Regarding the lecithasterid A. laguncula, even though Quilichini et al. (2010a) did not perform the test of Thiéry, the published TEM micrographs (see Figs. 2C–G, I, K and 3B, C, G in Quilichini et al., 2010a) may indicate a scarce presence of glycogen, as occurs in the hemiurids. The posterior extremity of the spermatozoon of P. merus is characterized by the presence of only one axoneme that disorganizes into doublets and singlets. This type of posterior extremity is also described in L. excisum (Ndiaye et al., 2012a). In spite of the limited hemiurids studied until now, the species of this family appear to possess a posterior spermatozoon extremity of type III or the cryptogonimidean type, characterized by the terminal sequence: absence of cortical microtubules, posterior nuclear extremity and posterior extremity of the second axoneme (see Quilichini et al., 2010b). Nevertheless, in the case of P. merus a single cortical microtubule is observed after the posterior extremity of the nucleus. In the lecithasterid A. laguncula Quilichini et al. (2010a) describe the presence of mitochondrion and the disorganization of both axonemes in the posterior extremity of the spermatozoon. However, some doublets and singlets of the axonemes reach the posterior spermatozoon tip, and this fact is considered in Table 1. In a recent molecular analysis of Digenea Carus, 1863 made by Olson et al. (2003), lecithasteridids are nested within the Hemiuridae as also observed in a previous work of Cribb et al. (2001). Thus, these authors support the monophyly of the Hemiuridae proposed by Gibson and Bray (1979). Table 1 shows the available
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ultrastructural data on the spermatozoon of hemiuroids. It is interesting to remark the similarities between hemiurids and lecithasterids in diverse aspects such as: (i) the morphology of the anterior spermatozoon extremity, (ii) the presence of a single bundle with a low number of cortical microtubules, and (iii) the presence of an external ornamentation of the plasma membrane not associated with cortical microtubules and located in the anterior part of the sperm cell. These similarities are indicative of the proximity between both families and support the results of the above-mentioned molecular analysis. Acknowledgements Authors wish to thank Núria Cortadellas and Almudena García from the “Unitat de Microscòpia, Facultat de Medicina, Centres Científics i Tecnològics de la Universitat de Barcelona (CCiTUB)” for their support in the preparation of samples. Authors also thank Online Access to Research in the Environment (OARE) for facilitating access to literature. The present study was partly supported by the PCI projects (no. A/023428/09 and no. A/030039/10) of the ˜ “Agencia Espanola de Cooperación Internacional para el Desarrollo (AECID)”. A.J.S. Bakhoum benefits from MAEC-AECID doctoral grant (2010–11, no. 0000538055). References Agostini, S., Miquel, J., Ndiaye, P.I., Marchand, B., 2005. Dicrocoelium hospes Looss, 1907 (Digenea, Dicrocoeliidae): spermiogenesis, mature spermatozoon and ultrastructural comparative study. Parasitology Research 96, 38–48. Ariola, V., 1902. Contributo per una monografia dei Didymozoon. I. Didymozoon parassiti del tonno. Archives de Parasitologie 6, 1–11. Bakhoum, A.J.S., Bâ, C.T., Shimalov, V.V., Torres, J., Miquel, J., 2011a. Spermatological characters of the digenean Rubenstrema exasperatum (Rudolphi, 1819) (Plagiorchioidea, Omphalometridae). Parasitology Research 108, 1283–1293. Bakhoum, A.J.S., Ndiaye, P.I., Sène, A., Bâ, C.T., Miquel, J., 2012a. Spermiogenesis and ultrastructure of the spermatozoon of Wardula capitellata (Digenea, Mesometridae), an intestinal parasite of the sparid teleost Sarpa salpa in Senegal. Acta Parasitologica 57, 34–45. Bakhoum, A.J.S., Ribas, A., Eira, C., Bâ, C.T., Miquel, J. Brachycoelium salamandrae (Frölich, 1789) (Digenea: Brachycoeliidae): ultrastructural study of spermiogenesis and the mature spermatozoon. Zoologischer Anzeiger, http://dx.doi.org/ 10.1016/j.jcz.2012.05.003, in press. Bakhoum, A.J.S., Sène, A., Ndiaye, P.I., Bâ, C.T., Miquel, J., 2012b. Spermiogenesis and the spermatozoon ultrastructure of Robphildollfusium fractum (Digenea: Gyliauchenidae), an intestinal parasite of Sarpa salpa (Pisces: Teleostei). Comptes Rendus Biologies 335, 435–444. Bakhoum, A.J.S., Torres, J., Shimalov, V.V., Bâ, C.T., Miquel, J., 2011b. Spermiogenesis and spermatozoon ultrastructure of Diplodiscus subclavatus (Pallas, 1760) (Paramphistomoidea, Diplodiscidae), an intestinal fluke of the pool frog Rana lessonae. Parasitology International 60, 64–74. Carus, J.V., 1863. Vermes. In: Peters, W.C.H., Carus, J.V., Gerstaecker, C.E.A. (Eds.), Handbuch der Zoologie. Verlag von Wilhelms Engelmann, Leipzig, pp. 422–484. Blair, D., Bray, R.A., Barker, S.C., 1998. Molecules and morphology in phylogenetic studies of the Hemiuroidea (Digenea: Trematoda: Platyhelminthes). Molecular Phylogenetics and Evolution 9, 15–25. Cribb, T.H., Bray, R.A., Littlewood, D.T.J., Pichelin, S.P., Herniou, E.A., 2001. The Digenea. In: Littlewood, D.T.J., Bray, R.A. (Eds.), Interrelationships of the Platyhelminthes. Taylor and Francis, London, pp. 168–185. Ehlers, U., 1984. Phylogenetisches System der Plathelminthes. Verhandlungen des Naturwissenschaftlichen Vereins zu Hamburg (NF) 27, 291–294. Fischtal, J.H., Thomas, J.D., 1972. Additional hemiurid and other trematodes of fishes from Ghana. Bulletin de l’Institut Fondametal d’Afrique Noire 34A, 9–25. Foata, J., Quilichini, Y., Greani, S., Marchand, B., 2012. Sperm ultrastructure of the Digenean Aphallus tubarium (Rudolphi, 1819) Poche, 1926 (Platyhelminthes, Cryptogonimidae) intestinal parasite of Dentex dentex (Pisces, Teleostei). Tissue & Cell 44, 15–21. Gibson, D.I., 2002. Superfamily Hemiuroidea Looss, 1899. In: Gibson, D.I., Jones, A., Bray, R.A. (Eds.), Keys to the Trematoda, vol. 1. CABI Publishing and The Natural History Museum, London, pp. 299–304. Gibson, D.I., Bray, R.A., 1979. The Hemiuroidea: terminology, systematic and evolution. Bulletin of the British Museum (Natural History) (Zoology) 36, 35–146. Iomini, C., Justine, J.-L., 1997. Spermiogenesis and spermatozoon of Echinostoma caproni (Platyhelminthes, Digenea): transmission and scanning electron microscopy, and tubulin immunocytochemistry. Tissue & Cell 29, 107–118. Jamieson, B.G.M., Daddow, L.M., 1982. The ultrastructure of the spermatozoon of Neochasmus sp. (Cryptogonimidae, Digenea, Trematoda) and its phylogenetic significance. International Journal for Parasitology 12, 547–559.
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Spermiogenesis and sperm ultrastructure of the liver fluke Fasciola hepatica L., 1758 (Digenea, Fasciolidae): scanning and transmission electron microscopy, and tubulin immunocytochemistry. Acta Parasitologica 48, 182–194. Ndiaye, P.I., Miquel, J., Bâ, C.T., Feliu, C., Marchand, B., 2002. Spermiogenesis and sperm ultrastructure of Scaphiostomum palaearcticum Mas-Coma, Esteban et Valero, 1986 (Trematoda, Digenea, Brachylaimidae). Acta Parasitologica 47, 259–271. Ndiaye, P.I., Miquel, J., Feliu, C., Marchand, B., 2003b. Ultrastructure of spermiogenesis and spermatozoa of Notocotylus neyrai González Castro, 1945 (Digenea, Notocotylidae), intestinal parasite of Microtus agrestis (Rodentia: Arvicolidae) in Spain. Invertebrate Reproduction and Development 43, 105–115. Ndiaye, P.I., Quilichini, Y., Sène, A., Bâ, C.T., Marchand, B., 2011. Ultrastructure of the spermatozoon of the digenean Cricocephalus albus (Kuhl & van Hasselt, 1822) Looss, 1899 (Platyhelminthes, Pronocephaloidea, Pronocephalidae), parasite of “the hawksbill sea turtle” Eretmochelys imbricata (Linnaeus, 1766) in Senegal. Zoologischer Anzeiger 250, 215–222. Ndiaye, P.I., Quilichini, Y., Sène, A., Tkach, V.V., Bâ, C.T., Marchand, B., 2012b. Ultrastructural study of the male gamete of Pleurogonius truncatus Prudhoe, 1944 (Platyhelminthes, Digenea, Pronocephalidae) parasite of Eretmochelys imbricata (Linnaeus, 1766). Comptes Rendus Biologies 335, 239–246. Ndiaye, P.I., Quilichini, Y., Sène, A., Tkach, V.V., Bâ, C.T., Marchand, B., 2012c. Ultrastructural study of the spermatozoon of the digenean Enodiotrema reductum Looss, 1901 (Platyhelminthes, Plagiorchioidea, Plagiorchiidae), parasite of the green turtle Chelonia mydas (Linnaeus, 1758) in Senegal. Parasitology Research 111, 859–864. Odhner, T., 1905. Die Trematoden de arktischen Gebietes. Fauna Arctica 4, 289–372. Olson, P.D., Cribb, T.H., Tkach, V.V., Bray, R.A., Littlewood, D.T.J., 2003. Phylogeny and classification of the Digenea (Platyhelminthes: Trematoda). International Journal for Parasitology 33, 733–755. Pamplona-Basilio, M.C., Baptista-Farias, M.F.D., Kohn, A., 2001. Spermatogenesis and spermiogenesis in Didymocystis wedli Ariola, 1902 (Didymozoidae, Digenea). Memorias do Instituto Oswaldo Cruz 96, 1153–1159. Quilichini, Y., Foata, J., Justine, J.-L., Bray, R.A., Marchand, B., 2009. Sperm ultrastructure of the digenean Siphoderina elongata (Platyhelminthes, Cryptogonimidae) intestinal parasite of Nemipterus furcosus (Pisces, Teleostei). Parasitology Research 105, 87–95. Quilichini, Y., Foata, J., Justine, J.-L., Bray, R.A., Marchand, B., 2010a. Spermatozoon ultrastructure of Aponurus laguncula (Digenea: Lecithasteridae), a parasite of Aluterus monoceros (Pisces, Teleostei). Parasitology International 59, 22–28. Quilichini, Y., Foata, J., Justine, J.-L., Bray, R.A., Marchand, B., 2010b. Ultrastructural study of the spermatozoon of Heterolebes maculosus (Digenea, Opistholebetidae), a parasite of the porcupinefish Diodon hystrix (Pisces, Teleostei). Parasitology International 59, 427–434.
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Quilichini, Y., Foata, J., Marchand, B., 2007a. Ultrastructural study of the spermatozoon of Nicolla testiobliquum (Digenea, Opecoelidae) parasite of brown trout Salmo trutta (Pisces, Teleostei). Parasitology Research 101, 1295–1301. Quilichini, Y., Foata, J., Orsini, A., Marchand, B., 2007b. Ultrastructural study of spermiogenesis and the spermatozoon of Crepidostomum metoecus (Digenea: Allocreadiidae), a parasite of Salmo trutta (Pisces: Teleostei). Journal of Parasitology 93, 458–468. Reynolds, E.S., 1963. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. Journal of Cell Biology 17, 208–212. Rudolphi, C.A., 1819. Entozoorum synopsis cui accedunt mantissa duplex et indices locupletissimi. August Rücker, Berolini, 811 pp.
Seck, M.T., Marchand, B., Bâ, C.T., 2008a. Spermiogenesis and sperm ultrastructure of Cotylophoron cotylophorum (Trematoda, Digenea, Paramphistomidae), a parasite of Bos taurus in Senegal. Parasitology Research 103, 157–166. Seck, M.T., Marchand, B., Bâ, C.T., 2008b. Spermiogenesis and sperm ultrastructure of Carmyerius endopapillatus (Digenea, Gastrothylacidae), a parasite of Bos taurus in Senegal. Acta Parasitologica 53, 9–18. Tang, J., Wang, W., Wang, G., 1998. Studies on ultrastructure of spermatogenesis and sperm in Pseudorhipidocotyle elpichthys. Acta Hydrobiologica Sinica 22, 168–173. Thiéry, J.P., 1967. Mise en évidence des polysaccharides sur coupes fines en microscopie électronique. Journal of Microscopy 6, 987–1018. Valenciennes, A., 1847. In: Cuvier, G., Valenciennes, A. (Eds.), Histoire naturelle des poissons, vol. 20, livre 21, De la famille des Clupéoïdes. 263 pp., 594 pl.
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Ultrastructure of the spermatozoon of Parahemiurus merus (Linton, 1910) (Digenea: Hemiuroidea: Hemiuridae), a parasite of Sardinella aurita Valenciennes, 1847 and S. maderensis (Lowe, 1838) (Teleostei: Clupeidae) in the Senegalese coast Papa Ibnou Ndiaye a , Abdoulaye J.S. Bakhoum b,c , Aminata Sène a , Jordi Miquel b,c,∗ a
Laboratory of Evolutionary Biology, Ecology and Management of Ecosystems, Faculty of Sciences and Techniques, Cheikh Anta Diop University of Dakar, BP 5055, Dakar, Senegal Laboratori de Parasitologia, Departament de Microbiologia i Parasitologia Sanitàries, Facultat de Farmàcia, Universitat de Barcelona, Av. Joan XXIII, s/n, E-08028 Barcelona, Spain c Institut de Recerca de la Biodiversitat, Facultat de Biologia, Universitat de Barcelona, Av. Diagonal, E-645, 08028 Barcelona, Spain b
a r t i c l e
i n f o
Article history: Received 18 September 2012 Received in revised form 21 November 2012 Accepted 26 November 2012 Available online 29 December 2012 Corresponding Editor: Martin V. Sørensen. Keywords: TEM Dakar coast Senegal
a b s t r a c t The mature spermatozoon of Parahemiurus merus (Digenea: Hemiuroidea: Hemiuridae) is filiform, tapered in both ends and exhibits the general pattern described in most digeneans so far: two axonemes of the 9+‘1’ pattern of trepaxonematan Platyhelminthes, nucleus, mitochondrion, external ornamentation of the plasma membrane and parallel cortical microtubules. However, the spermatozoon of P. merus presents some peculiarities. The presence of an external ornamentation of the plasma membrane not associated with cortical microtubules in the anterior part of the spermatozoon is described for a second genus in the family Hemiuridae. Cortical microtubules are disposed in only one side of the spermatozoon as described in other species of Hemiuroidea but, in P. merus, the number of cortical microtubules is much lower. The results obtained are compared with the available data on Hemiuroidea in order to improve the understanding of phylogenetic relationships of hemiuroids. Similarities found between spermatozoa of hemiurids and lecithasterids are in accordance with molecular evidence that group these two families in a same clade. © 2012 Elsevier GmbH. All rights reserved.
1. Introduction Digeneans of the superfamily Hemiuroidea Looss, 1899 are usually parasitic of the digestive tract of fishes, particularly the stomach of marine teleosts, although they also occur in freshwater teleosts, elasmobranchs and occasionally in amphibians and reptiles (Gibson, 2002). This superfamily has been subject of controversies, but in the most extensive revision of Hemiuroidea, Gibson and Bray (1979) recognize 14 families within the group. However, Gibson (2002), considering molecular evidences of Blair et al. (1998), excludes the family Azygiidae Lühe, 1909 from the Hemiuroidea. This exclusion was confirmed recently in another study, which places the Azygioidea Lühe, 1909 as a sister group of Hemiuroidea (Olson et al., 2003). On the other hand, these molecular analyses strongly support the retention of the remaining families within the Hemiuroidea, including the enigmatic family
∗ Corresponding author at: Laboratori de Parasitologia, Departament de Microbiologia i Parasitologia Sanitàries, Facultat de Farmàcia, Universitat de Barcelona, Av. Joan XXIII, s/n, E-08028 Barcelona, Spain. Tel.: +34 93 4024500; fax: +34 93 4024504. E-mail address: [email protected] (J. Miquel). 0044-5231/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.jcz.2012.11.005
Didymozoidae Monticelli, 1888. Gibson (2002) therefore considers the Hemiuroidea to accommodate 13 families, including the Hemiuridae Looss, 1899, which in turn includes the species Parahemiurus merus (Linton, 1910) studied in the present work. However, the phylogenetic relationships between some families remain controversial (see Cribb et al., 2001; Olson et al., 2003). In this context, the study of the spermatozoon of species belonging to all these families represent an important source of ultrastructural characters that can be a great contribution for elucidating phylogenetic relationships within this group. To our knowledge, in the Hemiuroidea, ultrastructural data on spermiogenesis and/or spermatozoa exist for only six species belonging to four families, namely the didymozoids Didymocystis wedli Ariola, 1902 (see Pamplona-Basilio et al., 2001), Didymozoon sp. (see Justine and Mattei, 1983, 1984a) and Gonapodasmius sp. (see Justine and Mattei, 1982, 1984b), the sclerodistomid Prosorchis ghanensis Fischtal & Thomas, 1972 (see Justine, 1995), the lecithasterid Aponurus laguncula Looss, 1907 (see Quilichini et al., 2010a) and the hemiurid Lecithocladium excisum (Rudolphi, 1819) (see Ndiaye et al., 2012a). Thus, the aim of the present study is to provide new ultrastructural spermiological data on a second genus of the family Hemiuridae.
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Fig. 1. (a–j) Spermatozoon of Parahemiurus merus. (a–g) Consecutive cross-sections of the anterior region of the spermatozoon (region I). Note the presence of an external ornamentation of the plasma membrane (EO) not associated with cortical microtubules (CM) and the appearance of a single field of cortical microtubules in the posterior area of this region. Figure d shows a detail of the external ornamentation of the plasma membrane. PM, plasma membrane; S, singlets. Scale bars (a–c, e–g) = 0.2 m; (d) = 0.1 m. (h–j) Consecutive cross-sections of the mitochondrial or middle region of the spermatozoon (region II) characterized by the presence of the mitochondrion (M) and the anterior nucleus extremity. This region contains the maximum number of cortical microtubules (CM) (five). N, nucleus; scale bars = 0.2 m.
2. Materials and methods Live adult specimens of P. merus (Linton, 1910) (Digenea: Hemiuroidea: Hemiuridae) were collected from the digestive tract of two marine teleosts, the Round sardinella Sardinella aurita Valenciennes, 1847 and the Madeiran sardinella S. maderensis (Lowe, 1838) (Clupeidae), both captured in the Atlantic Ocean, off Dakar (Senegal) during April 2011. Worms were rinsed with a 0.9% NaCl solution and fixed in cold (4 ◦ C) 2.5% glutaraldehyde in a 0.1 M sodium cacodylate buffer at pH 7.4, rinsed in 0.1 M sodium cacodylate buffer at pH 7.4, postfixed in cold (4 ◦ C) 1% osmium tetroxide with K4 FeCn6 in the same buffer for 1 h, rinsed in a 0.1 M sodium cacodylate buffer at pH 7.4, dehydrated in an ethanol series and propylene oxide, embedded in Spurr’s resin and polymerized at 60 ◦ C for 72 h. Ultrathin sections (60–90 nm thick) of areas containing seminal ducts and seminal vesicle were made in a Reichert-Jung Ultracut E ultramicrotome using a diamond knife. Then, sections were placed on copper and gold grids. Those sections placed on copper grids were double-stained with uranyl acetate and lead citrate according to the Reynolds (1963) procedure. The sections placed on gold grids were treated according to the Thiéry (1967) test to reveal the presence of glycogen. Thus, they were treated in periodic acid (PA), thiocarbohydrazide (TCH) and silver proteinate (SP) as follows:
30 min in 10% PA, rinsed in milliQ water, 24 h in TCH, rinsed in acetic solutions and milliQ water, 30 min in 1% SP in the dark, and rinsed in milliQ water. The grids were examined in a JEOL 1010 transmission electron microscope operated at 80 kV, in the “Centres Científics i Tecnològics de la Universitat de Barcelona (CCiTUB)”.
3. Results The observation of transversal and longitudinal sections of the mature spermatozoon permitted us to distinguish three regions (I–III) with different ultrastructural characters. Region I (Figs. 1a–g and 3I) corresponds to the anterior part of the spermatozoon. Consecutive transverse sections from anterior to posterior areas of region I exhibit progressively: (i) singlets of axonemes (Fig. 1a–c), (ii) the external ornamentation of the plasma membrane (Fig. 1c and d), (iii) the appearance of axonemes (Fig. 1c and e), (iv) the progressive reduction and disappearance of the external ornamentation (Fig. 1f and g), and (v) the appearance of a single field (ventral) of parallel cortical microtubules (Fig. 1g). Region II (Fig. 1h–j, 2a and 3II) is the middle region or mitochondrial area of the spermatozoon. In addition to the structures observed in the posterior part of region I (two axonemes and
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Fig. 2. (a–h) Spermatozoon of Parahemiurus merus. (a) Cross-sections showing the transition between regions II and III. M, mitochondrion; N, nucleus; scale bar = 0.2 m. (b–g) Consecutive cross-sections of the posterior part (region III) characterized by the presence of the posterior nucleus extremity. In this region there is the disorganization of the two axonemes and the disappearance of cortical microtubules (CM). CC, central core; D, doublets; N, nucleus; S, singlets; scale bars = 0.2 m. (h) Revelation of glycogen (G) by the test of Thiéry. Scale bar = 0.2 m.
cortical microtubules), a mitochondrion is present in region II (Fig. 1h). The nucleus coincides with an area where the layer of cortical microtubules is formed by the maximum number of elements (five) (Fig. 1i). Then, the mitochondrion and the nucleus increase in size and the layer of cortical microtubules decreases to four elements (Fig. 1j). The posterior part of this region is characterized by a decrease in the size of the mitochondrion and a large-sized nucleus, while the layer of cortical microtubules is formed by only three elements (Fig. 2a). Region III (Figs. 2a–g and 3III) corresponds to the posterior part of the spermatozoon. It is characterized by: (i) disappearance of the mitochondrion (Fig. 2a), (ii) disorganization of the first axoneme (Fig. 2b and c), (iii) progressive reduction of the nucleus size until its disappearance (Fig. 2d and e), and (iv) loss of cortical microtubules and disorganization of the second axoneme into doublets and singlets (Fig. 2e–g).
Fig. 2h shows the positive result of the Thiéry test exhibiting a very reduced amount of granules of glycogen. 4. Discussion The mature spermatozoon of P. merus exhibits the general pattern of most digeneans described so far: two axonemes of the 9+‘1’ pattern of trepaxonematans (Ehlers, 1984), nucleus, mitochondrion, external ornamentation of the plasma membrane and parallel cortical microtubules (Jamieson and Daddow, 1982; Iomini and Justine, 1997; Tang et al., 1998; Miquel et al., 2000; Ndiaye et al., 2003a; Quilichini et al., 2007a, 2009, 2010a). However, it also presents some peculiarities. The anterior part of the spermatozoon is characterized by an external ornamentation of the plasma membrane not associated with cortical microtubules. In the Hemiuridae, L. excisum also exhibits this organization of the external
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Fig. 3. (I–III) Schematic reconstruction of the spermatozoon of Parahemiurus merus showing the ultrastructural organization of the three considered regions. ANE, anterior nucleus extremity; ASE, anterior spermatozoon extremity; Ax1, first axoneme; Ax2, second axoneme; CC, central core; CM, cortical microtubules; D, doublets; EO, external ornamentation of the plasma membrane; G, granules of glycogen; M, mitochondrion; N, nucleus; PM, plasma membrane; PNE, posterior nucleus extremity; PSE, posterior spermatozoon extremity; S, singlets.
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Table 1 Available data on the spermatozoon ultrastructure in the Hemiuroidea. Families and species
Ultrastructural spermatozoon characters Ax
Didymozoidae Didymocystis wedli Didymozoon sp. Gonapodasmius sp. Hemiuridae Lecithocladium excisum Parahemiurus merus Lecithasteridae Aponurus laguncula Sclerodistomidae Prosorchis ghanensis
ASE
References
EO
CM
PSE
+/–
loc
EO/CM
bund
n
9+‘1’ 9+0 9+‘1’
? ? 1Ax-EO-CM
– – +
NA NA RAnt
NA NA +
– – 2
0 0 36a
? ? 2F
Pamplona-Basilio et al. (2001) Justine and Mattei (1983, 1984a) Justine and Mattei (1982, 1984b)
9+‘1’ 9+‘1’
1Ax-EO 1Ax-EO
+ +
RAnt RAnt
– –
1 1
8 5
1Ax 1Ax
Ndiaye et al. (2012a) Present study
9+‘1’
1Ax-EO
+
RAnt
–
1
10
D+S
Quilichini et al. (2010a)
9+‘1’
?
?
NA
NA
1–2b
13–15b
?
Justine (1995)
Spermatozoon characters: ASE, anterior spermatozoon extremity; Ax, axoneme; bund, number of bundles of cortical microtubules; CM, cortical microtubules; D, doublets; EO, external ornamentations of the plasma membrane; EO/CM, association of external ornamentations and cortical microtubules; F, flagellum; loc, location of external ornamentations; n, maximum number of cortical microtubules; NA, not applicable; PSE, posterior spermatozoon extremity; RAnt, anterior region; S, singlets; +/−, presence/absence;?, unknown data. a The maximum number of cortical microtubules occurs in the anterior spermatozoon area in a continuous submembranous layer. b During spermiogenesis there are 2 bundles of 1 and 15 microtubules in the median cytoplasmic process. After the proximodistal fusion there is only 1 bundle of 13 microtubules.
ornamentation (see Ndiaye et al., 2012a). In this family, there are ultrastructural studies of the spermatozoon only in P. merus and L. excisum (Ndiaye et al., 2012a). However, in the latter the external ornamentation covers entirely the anterior extremity of the spermatozoon. In all of the remaining hemiuroids studied to date, only the lecithasterid A. laguncula exhibits this pattern (Quilichini et al., 2010a). Moreover, in the Hemiuridae and Lecithasteridae the external ornamentation is observed as a continuous electron-dense material, whereas in other digeneans that exhibit the ornamentation associated with cortical microtubules, in both anterior and mitochondrial areas of the sperm cell the external ornamentation seems to be constituted by a layer of isolated electron-dense elements (see Miquel et al., 2000, 2006). With respect to the didymozoid Gonapodasmius sp., Justine and Mattei (1982, 1984b) described an external ornamentation of the plasma membrane associated with a continuous bundle of cortical microtubules in the anterior spermatozoon extremity. Contrarily, other didymozoids namely D. wedli and Didymozoon sp. lack external ornamentation of the plasma membrane and cortical microtubules (Justine and Mattei, 1983, 1984a; Pamplona-Basilio et al., 2001). Like other hemiuroids studied so far, the mature spermatozoon of P. merus presents a low number of cortical microtubules with different lengths disposed only in the ventral side. In the Hemiuroidea, the maximum number of cortical microtubules is described in the anterior region of the spermatozoon of the didymozoid Gonapodasmius sp. with 36 elements surrounding the sperm cell beneath the plasma membrane (Justine and Mattei, 1982) while other didymozoids lack cortical microtubules (Justine and Mattei, 1983, 1984a; Pamplona-Basilio et al., 2001). In the remaining studied species the number of cortical microtubules is low and distributed in a single bundle as occurs in P. merus. The hemiurid L. excisum exhibits a maximum of eight microtubules and the lecithasterid A. laguncula a maximum of ten, located in the mitochondrial region of the spermatozoon in both cases (Quilichini et al., 2010a; Ndiaye et al., 2012a). In the case of P. merus there is a maximum of five microtubules, also in the mitochondrial area. A low number of cortical microtubules of different lengths and their disposition in only the ventral side of the spermatozoon seem to be characteristics of the male gamete of species belonging to the families Hemiuridae and Lecithasteridae Odhner, 1905 (Quilichini et al., 2010a; Ndiaye et al., 2012a). With respect to the remaining hemiuroids, apart from the enigmatic group of didymozoids (Justine and Mattei, 1982, 1983, 1984a,b; Pamplona-Basilio et al., 2001), there are only observations
on the spermiogenesis of the Sclerodistomidae P. ghanensis (see Justine, 1995). Spine-like bodies have been described in the spermatozoon of numerous digeneans belonging to different families and superfamilies, e.g. echinostomatoideans, microscaphidioideans, paramphistomoideans, pronocephaloideans, allocreadioideans, gorgoderoideans, lepocreadioideans, opisthorchioideans and plagiorchioideans (Miquel et al., 2000, 2006; Ndiaye et al., 2003a,b, 2012b,c; Agostini et al., 2005; Quilichini et al., 2007b, 2010b; Seck et al., 2008a,b; Kacem et al., 2010; Bakhoum et al., 2011a,b, 2012a,b, in press; Foata et al., 2012). Contrarily, they have not been observed in any hemiuroid. Unlike the great majority of digeneans (Jamieson and Daddow, 1982; Iomini and Justine, 1997; Miquel et al., 2000, 2006; Ndiaye et al., 2002, 2011; Kacem et al., 2010; Bakhoum et al., 2011b, 2012a), the mature spermatozoon of P. merus has a small quantity of glycogen granules as occurs also in the hemiurid, L. excisum (Ndiaye et al., 2012a). Regarding the lecithasterid A. laguncula, even though Quilichini et al. (2010a) did not perform the test of Thiéry, the published TEM micrographs (see Figs. 2C–G, I, K and 3B, C, G in Quilichini et al., 2010a) may indicate a scarce presence of glycogen, as occurs in the hemiurids. The posterior extremity of the spermatozoon of P. merus is characterized by the presence of only one axoneme that disorganizes into doublets and singlets. This type of posterior extremity is also described in L. excisum (Ndiaye et al., 2012a). In spite of the limited hemiurids studied until now, the species of this family appear to possess a posterior spermatozoon extremity of type III or the cryptogonimidean type, characterized by the terminal sequence: absence of cortical microtubules, posterior nuclear extremity and posterior extremity of the second axoneme (see Quilichini et al., 2010b). Nevertheless, in the case of P. merus a single cortical microtubule is observed after the posterior extremity of the nucleus. In the lecithasterid A. laguncula Quilichini et al. (2010a) describe the presence of mitochondrion and the disorganization of both axonemes in the posterior extremity of the spermatozoon. However, some doublets and singlets of the axonemes reach the posterior spermatozoon tip, and this fact is considered in Table 1. In a recent molecular analysis of Digenea Carus, 1863 made by Olson et al. (2003), lecithasteridids are nested within the Hemiuridae as also observed in a previous work of Cribb et al. (2001). Thus, these authors support the monophyly of the Hemiuridae proposed by Gibson and Bray (1979). Table 1 shows the available
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ultrastructural data on the spermatozoon of hemiuroids. It is interesting to remark the similarities between hemiurids and lecithasterids in diverse aspects such as: (i) the morphology of the anterior spermatozoon extremity, (ii) the presence of a single bundle with a low number of cortical microtubules, and (iii) the presence of an external ornamentation of the plasma membrane not associated with cortical microtubules and located in the anterior part of the sperm cell. These similarities are indicative of the proximity between both families and support the results of the above-mentioned molecular analysis. Acknowledgements Authors wish to thank Núria Cortadellas and Almudena García from the “Unitat de Microscòpia, Facultat de Medicina, Centres Científics i Tecnològics de la Universitat de Barcelona (CCiTUB)” for their support in the preparation of samples. Authors also thank Online Access to Research in the Environment (OARE) for facilitating access to literature. The present study was partly supported by the PCI projects (no. A/023428/09 and no. A/030039/10) of the ˜ “Agencia Espanola de Cooperación Internacional para el Desarrollo (AECID)”. A.J.S. Bakhoum benefits from MAEC-AECID doctoral grant (2010–11, no. 0000538055). References Agostini, S., Miquel, J., Ndiaye, P.I., Marchand, B., 2005. Dicrocoelium hospes Looss, 1907 (Digenea, Dicrocoeliidae): spermiogenesis, mature spermatozoon and ultrastructural comparative study. Parasitology Research 96, 38–48. Ariola, V., 1902. Contributo per una monografia dei Didymozoon. I. Didymozoon parassiti del tonno. Archives de Parasitologie 6, 1–11. Bakhoum, A.J.S., Bâ, C.T., Shimalov, V.V., Torres, J., Miquel, J., 2011a. Spermatological characters of the digenean Rubenstrema exasperatum (Rudolphi, 1819) (Plagiorchioidea, Omphalometridae). Parasitology Research 108, 1283–1293. Bakhoum, A.J.S., Ndiaye, P.I., Sène, A., Bâ, C.T., Miquel, J., 2012a. Spermiogenesis and ultrastructure of the spermatozoon of Wardula capitellata (Digenea, Mesometridae), an intestinal parasite of the sparid teleost Sarpa salpa in Senegal. Acta Parasitologica 57, 34–45. Bakhoum, A.J.S., Ribas, A., Eira, C., Bâ, C.T., Miquel, J. Brachycoelium salamandrae (Frölich, 1789) (Digenea: Brachycoeliidae): ultrastructural study of spermiogenesis and the mature spermatozoon. Zoologischer Anzeiger, http://dx.doi.org/ 10.1016/j.jcz.2012.05.003, in press. Bakhoum, A.J.S., Sène, A., Ndiaye, P.I., Bâ, C.T., Miquel, J., 2012b. Spermiogenesis and the spermatozoon ultrastructure of Robphildollfusium fractum (Digenea: Gyliauchenidae), an intestinal parasite of Sarpa salpa (Pisces: Teleostei). Comptes Rendus Biologies 335, 435–444. Bakhoum, A.J.S., Torres, J., Shimalov, V.V., Bâ, C.T., Miquel, J., 2011b. Spermiogenesis and spermatozoon ultrastructure of Diplodiscus subclavatus (Pallas, 1760) (Paramphistomoidea, Diplodiscidae), an intestinal fluke of the pool frog Rana lessonae. Parasitology International 60, 64–74. Carus, J.V., 1863. Vermes. In: Peters, W.C.H., Carus, J.V., Gerstaecker, C.E.A. (Eds.), Handbuch der Zoologie. Verlag von Wilhelms Engelmann, Leipzig, pp. 422–484. Blair, D., Bray, R.A., Barker, S.C., 1998. Molecules and morphology in phylogenetic studies of the Hemiuroidea (Digenea: Trematoda: Platyhelminthes). Molecular Phylogenetics and Evolution 9, 15–25. Cribb, T.H., Bray, R.A., Littlewood, D.T.J., Pichelin, S.P., Herniou, E.A., 2001. The Digenea. In: Littlewood, D.T.J., Bray, R.A. (Eds.), Interrelationships of the Platyhelminthes. Taylor and Francis, London, pp. 168–185. Ehlers, U., 1984. Phylogenetisches System der Plathelminthes. Verhandlungen des Naturwissenschaftlichen Vereins zu Hamburg (NF) 27, 291–294. Fischtal, J.H., Thomas, J.D., 1972. Additional hemiurid and other trematodes of fishes from Ghana. Bulletin de l’Institut Fondametal d’Afrique Noire 34A, 9–25. Foata, J., Quilichini, Y., Greani, S., Marchand, B., 2012. Sperm ultrastructure of the Digenean Aphallus tubarium (Rudolphi, 1819) Poche, 1926 (Platyhelminthes, Cryptogonimidae) intestinal parasite of Dentex dentex (Pisces, Teleostei). Tissue & Cell 44, 15–21. Gibson, D.I., 2002. Superfamily Hemiuroidea Looss, 1899. In: Gibson, D.I., Jones, A., Bray, R.A. (Eds.), Keys to the Trematoda, vol. 1. CABI Publishing and The Natural History Museum, London, pp. 299–304. Gibson, D.I., Bray, R.A., 1979. The Hemiuroidea: terminology, systematic and evolution. Bulletin of the British Museum (Natural History) (Zoology) 36, 35–146. Iomini, C., Justine, J.-L., 1997. Spermiogenesis and spermatozoon of Echinostoma caproni (Platyhelminthes, Digenea): transmission and scanning electron microscopy, and tubulin immunocytochemistry. Tissue & Cell 29, 107–118. Jamieson, B.G.M., Daddow, L.M., 1982. The ultrastructure of the spermatozoon of Neochasmus sp. (Cryptogonimidae, Digenea, Trematoda) and its phylogenetic significance. International Journal for Parasitology 12, 547–559.
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