Parasitol Res (2012) 110:19–30 DOI 10.1007/s00436-011-2446-9 ORIGINAL PAPER Spermiogenesis and spermatozoon ultrastructure of the bothriocephalidean cestode Clestobothrium crassiceps (Rudolphi, 1819), a parasite of the teleost fish Merluccius merluccius (Gadiformes: Merlucciidae) Adji Mama Marigo & Eulàlia Delgado & Jordi Torres & Cheikh Tidiane Bâ & Jordi Miquel Received: 17 March 2011 /Accepted: 4 May 2011 /Published online: 17 May 2011 # Springer-Verlag 2011 Abstract Spermiogenesis and the ultrastructure of the process. Flagella then undergo a rotation of 90° until they spermatozoon of the bothriocephalidean cestode Clesto- become parallel to the median cytoplasmic process, bothrium crassiceps (Rudolphi, 1819), a parasite of the followed by the proximodistal fusion of the flagella with teleost fish Merluccius merluccius (Linnaeus, 1758), have the median cytoplasmic process. The nucleus elongates and been studied by means of transmission electron microscopy. afterwards it migrates along the spermatid body. Spermio- Spermiogenesis involves firstly the formation of a differ- genesis finishes with the appearance of the apical cone entiation zone. It is characterized by the presence of two surrounded by the single helical crested body at the base of centrioles associated with striated rootlets, an intercentriolar the spermatid. Finally, the narrowing of the ring of arched body and an electron-dense material in the apical region of membranes detaches the fully formed spermatozoon. The this zone. Later, two flagella develop from the centrioles, mature spermatozoon of C. crassiceps is filiform and growing orthogonally in relation to the median cytoplasmic contains two axonemes of the 9+“1” trepaxonematan pattern, a parallel nucleus, parallel cortical microtubules, and electron-dense granules of glycogen. The anterior : : extremity of the gamete exhibits a short electron-dense A. M. Marigo J. Torres J. Miquel apical cone and one crested body, which turns once around Laboratori de Parasitologia, Departament de Microbiologia i Parasitologia Sanitàries, Facultat de Farmàcia, the sperm cell. The first axoneme is surrounded by a ring of Universitat de Barcelona, thick cortical microtubules that persist until the appearance Av. Joan XXIII, sn, of the second axoneme. Later, these thick cortical micro- E08028 Barcelona, Spain tubules disappear and thus, the mature spermatozoon A. M. Marigo : J. Torres : J. Miquel (*) exhibits two bundles of thin cortical microtubules. The Institut de Recerca de la Biodiversitat, Facultat de Biologia, posterior extremity of the male gamete presents only the Universitat de Barcelona, nucleus. Results are discussed and compared particularly Av. Diagonal, 645, with the available ultrastructural data on the former E08028 Barcelona, Spain “ ” e-mail: [email protected] pseudophyllideans . Two differences can be established between spermatozoa of Bothriocephalidea and Diphyllo- E. Delgado bothriidea, the type of spermatozoon (II vs I) and the Departament de Ciències Ambientals, Facultat de Ciències, presence/absence of the ring of cortical microtubules. Universitat de Girona, Campus de Montilivi, sn, E17071 Girona, Spain Introduction C. T. Bâ Laboratoire de Parasitologie, Département de Biologie animale, Faculté des Sciences, Université Cheikh Anta Diop de Dakar, The genus Clestobothrium is included in the recent Dakar, Senegal tapeworm order Bothriocephalidea, which had formerly 20 Parasitol Res (2012) 110:19–30 been included in the suppressed order “Pseudophyllidea” crassiceps, with the aim of providing new data on this (Kuchta et al. 2008a). In their study, Kuchta et al. (2008a) genus potentially useful for phylogenetic analyses. showed that the order “Pseudophyllidea” consists of two unrelated clades using molecular, morphological, and ecological approaches. The ultrastructural spermatological Materials and methods data available to date support these results and confirm the existence of important ultrastructural differences between Live adult specimens of C. crassiceps were collected from the species of both orders (Levron et al. 2005, 2006a, b, c, the intestine of the teleost fish Merluccius merluccius 2009; Bâ et al. 2007; Bruňanská et al. 2010; Šípková et al. (Gadiformes: Merlucciidae) caught in Roses (Girona, Spain). 2010, 2011). Bothriocephalideans are intestinal parasites of Live cestodes were first placed in a 0.9% NaCl solution. teleost fishes including 46 genera distributed into four Later the mature proglottids were fixed in cold (4°C) 2.5% families (Bothriocephalidae, Echinophallidae, Philobythii- glutaraldehyde in a 0.1-M sodium cacodylate buffer at pH 7.4 dae, and Triaenophoridae). The genus Clestobothrium is for a minimum of 2 h, rinsed in a 0.1-M sodium cacodylate included in the Bothriocephalidae along with another 13 buffer at pH 7.4, postfixed in cold (4°C) 1% osmium tetroxide valid genera (Kuchta et al. 2008a, b). Morphologically, in the same buffer for 1 h, rinsed in a 0.1-M sodium individuals belonging to the genus Clestobothrium differ cacodylate buffer at pH 7.4, dehydrated in an ethanol series from other bothriocephalids because they possess a sphinc- and propylene oxide, and finally embedded in Spurr’sresin. ter surrounding the anterior aperture of bothria (Schmidt Ultrathin sections were obtained using a Reichert-Jung Ultra- 1986; Bray et al. 1994). To date, this genus comprises only cut E ultramicrotome, placed on copper grids, and double- three valid species: (1) Clestobothrium crassiceps,the stained with uranyl acetate and lead citrate (Reynolds 1963). genus type species that was initially described as Bothrio- Ultrathin sections were examined using a JEOL 1010 TEM cephalus crassiceps, (2) Clestobothrium gibsoni, formerly operated at an accelerating voltage of 80 kV. described as Bathygadus macrops, and (3) Clestobothrium The presence of glycogen was detected using the method neglectum, described as Raniceps raninus (Kuchta et al. of Thiéry (1967). Gold grids were treated in periodic acid, 2008b). The remaining families of Bothriocephalidea thiocarbohydrazide, and silver proteinate (PA-TCH-SP) as include eight genera in the Echinophallidae, two genera in follows: 30 min in 10% PA, rinsed in distilled water, 24 h in the Philobythiidae, and 22 genera in the Triaenophoridae TCH, rinsed in acetic solutions, and distilled water, 30 min (Kuchta et al. 2008a, b). in 1% SP in the dark, and rinsed in distilled water. The usefulness of ultrastructural data of spermiogenesis and the spermatozoon to elucidate the phylogenetic relationships within the Platyhelminthes has been demon- Results strated by several authors (Euzet et al. 1981; Justine 1991, 1998, 2001; Bâ and Marchand 1995;Levronetal.2010). Spermiogenesis To our knowledge, the spermatological characters of only 13 species of the former “Pseudophyllidea” have been Spermiogenesis in C. crassiceps is described in Figs. 1, 2, 3 studied, including 10 bothriocephalideans and three diphyl- and 4. In the testes, spermatids are grouped in rosettes and lobothriideans. Concerning the bothriocephalideans, the are interconnected to a central cytophore by cytoplasmic analyzed species are the bothriocephalids Bothriocephalus bridges (Fig. 1a). Each spermatid contains a large nucleus clavibothrium, B. claviceps, B. scorpii, Oncodiscus sau- with scattered chromatin and numerous mitochondria ridae,andSenga sp. (Świderski and Mokhtar-Maamouri (Fig. 1a). Spermiogenesis starts by the formation of a zone 1980;Levronetal.2006b;Bâetal.2007; Šípková et al. of differentiation situated at the periphery of each spermatid 2011); the echinophallids Parabothriocephalus gracilis (Fig. 1b). In the very early stages of spermiogenesis it is and Paraechinophallus japonicus (Levron et al. 2006a; possible to observe an electron-dense material in the Šípková et al. 2010); and the triaenophorids Eubothrium peripheral region of the zone of differentiation (Fig. 1b). crassum, E. rugosum,andTriaenophorus nodulosus The differentiation zone also contains two centrioles (Bruňanská et al. 2001, 2002, 2010;Levronetal.2005). associated with striated rootlets and an intercentriolar body In what is referred to as the diphyllobothriideans, the (Fig. 1b–d). The intercentriolar body is a cylindrical three studied species are the scyphocephalid Duthiersia structure and consists of three electron-dense plates: one fimbriata (Justine 1986), and the diphyllobothriids Diphyl- central thicker electron-dense layer bordered by two thin lobothrium latum and Ligula intestinalis (Levron et al. electron-dense layers (Fig. 1c). The differentiation zone is 2006c, 2009). lined by a layer of submembranous cortical microtubules The present study describes for the first time the and delimited at its base by a ring of arched membranes spermiogenesis and the spermatozoon ulstrastructure of C. (Fig. 1d). Cross-sections of this zone show that cortical Parasitol Res (2012) 110:19–30 21 Fig. 1 Spermiogenesis in C. crassiceps. a Rosette showing numerous peripheric spermatids attached to the central cytophore (Cy). M mitochondria, N nucle- us. Bar 5 μm. b Longitudinal section of an early stage of spermiogenesis showing the appearance of the electron-dense material (DM). IB intercentriolar body, N nucleus, SR striated rootlets. Bar 0.5 μm. c Cross- section of the differentiation zone showing the intercentriolar body and the cortical microtu- bules (CM) associated with the electron-dense material (DM). Arrowheads indicate the three electron-dense layers forming the intercentriolar body. SR stri- ated rootlets.