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Ultrastructural study of spermiogenesis and the spermatozoon of Cavisoma magnum (Southwell, 1927) (Acanthocephala, Palaeacanthocephala, Cavisomidae), from Siganus lineatus (Pisces, Teleostei, Siganidae) (Valenciennes, 1835) in New Caledonia
Micron 43 (2012) 141–149
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Review
Ultrastructural study of spermiogenesis and the spermatozoon of Cavisoma magnum (Southwell, 1927) (Acanthocephala, Palaeacanthocephala, Cavisomidae), from Siganus lineatus (Pisces, Teleostei, Siganidae) (Valenciennes, 1835) in New Caledonia J. Foata a,∗ , Y. Quilichini a , J.-L. Justine b , R.A. Bray c , B. Marchand a a
CNRS UMR 6134, University of Corsica, “Parasites and Mediterranean Ecosystems” Laboratory, BP 52, 20250 Corte, France UMR 7138 Systématique, Adaptation, Évolution, Muséum National d‘Histoire Naturelle, Case postale 52, 57, rue Cuvier, 75231 Paris cedex 05, France c Department of Zoology, Natural History Museum, Cromwell Road, London SW7 5BD, UK b
a r t i c l e
i n f o
Article history: Received 25 August 2011 Received in revised form 12 October 2011 Accepted 25 October 2011 Keywords: Ultrastructure Spermatozoon Spermiogenesis Cavisoma magnum Acanthocephala TEM
a b s t r a c t This paper presents an ultrastructural study of Cavisoma magnum (Acanthocephala, Cavisomatidae) with a Transmission Electron Microscopy tool. This parasite of the fish Siganus lineatus is here reported for the first time from off New Caledonia, South Pacific. It is the first study describing the ultrastructure, spermiogenesis and spermatozoon of a species of the family Cavisomatidae. The young spermatid of C. magnum possesses a centriole constituted of doublets without a central element. After the elaboration of a flagellum of 9 + 2 pattern, the centriole migrates in a nuclear groove. Then the flagellum migration occurs and gives rise to a spermatozoon. The distribution and the size of the protein granules are reported and the posterior extremity appears like a chromatin lamina wave. Comparative ultrastructural data are presented on sperm and spermiogenesis of the Acanthocephala and Rotifers examined to date and the phylogenetic implications are discussed. © 2011 Elsevier Ltd. All rights reserved.
Contents 1. 2.
3.
4.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Fixation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Embedding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Ultramicrotomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Poststaining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Spermiogenesis (Figs. 1, 2 and 4) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Spermatozoon (Figs. 3 and 5) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Spermiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Spermatozoon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction The Acanthocephala are generally considered to be a small group of parasites, because they are rare. Nevertheless, there have been
∗ Corresponding author. E-mail address: [email protected] (J. Foata). 0968-4328/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2011.10.022
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some books devoted exclusively to them (e.g., Crompton, 1970; Crompton and Nickol, 1985, 2009; Kennedy, 2006; Petrochenko, 1956, 1958; Yamaguti, 1963). Currently, their phylogenetic position is debated (e.g., Sorensen and Giribet, 2006). Few studies have been conducted on the reproduction of acanthocephalans. The first light-microscopic studies were performed by Hyman (1951) and Guraya (1971) and the first ultrastructural study was Whitfield‘s work (1971) on Polymorphus minutus (Zeder, 1800). Our
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present-day knowledge of the ultrastructure of acanthocephalan reproduction is mainly the result of electron microscopical studies by (Marchand and Mattei, 1976a,b) with other recent contributions from Carcupino and Dezfuli (1995) and Foata et al. (2005, 2004). The biology of acanthocephalans was re-examined in a monograph published by Crompton and Nickol (1985) and their reproduction was reviewed by Carcupino and Dezfuli (1999). In the present work, we describe the ultrastructure of spermiogenesis and the spermatozoon of Cavisoma magnum (Southwell, 1927) (Paleacanthocephala), an intestinal parasite of the goldenlined spinefoot, Siganus lineatus (Valenciennes, 1835) from off New Caledonia. C. magnum has previously been described from other fish species in different localities: in the goldring surgeonfish Ctenochaetus strigosus and Serranus sp. in Indian Ocean (India and Sri Lanka), and in the milkfish Chanos chanos in the Pacific Ocean (Philippines) (Arthur et al., 1995). This work aims to present the first ultrastructural study of spermiogenesis and the spermatozoon of a cavisomatid acanthocephalan and draw a comparison with findings on the Rotifers, a group of mostly microscopic, aquatic invertebrates with about 2000 described species (Grinson et al., 2011). The Acanthocephala, previously considered to be a separate phylum, are considered as highly modified rotifers but the exact relationship to other members of the phylum is discussed. Spermatozoa are considered as an important source of characters for phylogenetic reconstructions. 2. Materials and methods Specimens of C. magnum (Southwell, 1927) were collected from the intestine of a naturally infected golden-lined spinefoot S. lineatus (Valenciennes, 1835) (Pisces, Teleostei) caught off Nouméa, New Caledonia (Pacific Ocean); prevalence was 16% (1/6), intensity 7. This is the first Acanthocephala identified from a fish off New Caledonia (Justine, 2010). The worms were removed alive from the digestive tract of their hosts. The parasites were carefully dissected, the entire genital apparatus of males were removed and the testes isolated. 2.1. Fixation The material was fixed in cold (4 ◦ C) 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer at pH 7.2 for 1 h, rinsed in 0.1 M sodium cacodylate buffer at pH 7.2 and post-fixed in cold (4 ◦ C) 1% osmium tetroxide in the same buffer for 1 h. 2.2. Embedding The material was dehydrated in a series of ethanol (from 70% to 100%) and propylene oxide (100%), embedded in Spurr (1969) and polymerised at 60 ◦ C for 24 h. 2.3. Ultramicrotomy Ultrathin sections (60–90 nm) of testis and seminal vesicle were cut on an ultramicrotome (Power tome PC, RMC Boeckeler® ). The sections were placed on 300 and 200-mesh copper grids. 2.4. Poststaining Grids were stained with ethanolic uranyl acetate (5% solution of uranyl acetate in 50% ethanol) for 10 min, rinsed in deionized water. Finally, they were stained with lead citrate (0.15 g lead citrate + 0.2 g sodium citrate in 50 mL of deionized water) for 5 min and rinced in deionized water.
Sections were examined on a Hitachi H-7650 transmission electron microscope, operating at an accelerating voltage of 80 kV, in the “Service d‘Étude et de Recherche en Microscopie Électronique” of the University of Corsica (Corte, France).
3. Results 3.1. Spermiogenesis (Figs. 1, 2 and 4) The young spermatid of C. magnum is a round cell which possesses a large nucleus in the central position and a centriole with 9 peripheral doublets without central elements (Fig. 1a). The beginning of spermiogenesis is characterized by a centriole with a “posterior bulb” abutted to the plasma membrane and perpendicular to the nuclear surface (Figs. 1b and 4a). This centriole gives rise to a flagellum (Figs. 1c and 4b) with a 9 + 2 pattern. The centriole then migrates into a nuclear groove towards the anterior part of the spermatid. The flagellum, surrounded by a plasma membrane, follows the centriole in its migration, and a cytoplasmic canal is formed (Figs. 1d, e and 4c, d). The flagellum stops its progression when the centriole reaches the anterior extremity of the nucleus (Fig. 4d). The centriole then continues its anterior migration, pushing the plasma membrane of the anterior extremity of the spermatid in front of it (Figs. 1f and 4e). Thus, it passes beyond the anterior extremity of the spermatid (Figs. 2a and 4f). During flagellar migration, the anterior and posterior parts of the flagellum are temporarily free (Fig. 2a). Migration stops when the posterior extremity of the flagellum passes into the spermatid (Fig. 4g and h). The posterior part of the flagellum is characterized by the disorganization of the axoneme marked by the transformation of doublets into singlets and a reduction in the number of microtubules (Fig. 2b–d). At the end of the flagellar migration, the nucleus extends towards the back of the spermatid and against the posterior part of the flagellum. At the same time, chromatin condenses in close contact to the nuclear groove, becomes progressively lamellar (Fig. 2e and f) and appears as a network of lamellar anastomoses. In the late spermatid, protein granules are elaborated, while chromatin reaches maximum condensation and appears as a thin lamina (Fig. 2g and h). By the end of spermiogenesis, the nuclear envelope opens and persists like a pentalaminate structure which corresponds to a remnant of the nuclear groove (Fig. 2h and i). Then, a nucleocytoplasmic derivative in is formed and the chromatin appears in close contact with the cytoplasm and the protein granules. At the same time, mitochondria are eliminated in residual cytoplasm.
3.2. Spermatozoon (Figs. 3 and 5) Spermatozoa are present in seminal vesicles. Transverse sections of the anterior extremity of the spermatozoon are characterized by the presence of a centriolar derivative made of 9 peripheral elements (Figs. 3a and 5a). This centriolar derivative is followed by a flagellum with a 9 + 2 pattern (Figs. 3b–d and 5b–d). The nucleocytoplasmic derivative (NCD) is reniform in section (Figs. 3f and 5d, e) with a thin layer of chromatin placed against the convex side of the spermatozoon (equivalent to a dorsal side). A pentalaminate remnant of nuclear envelope occurs on the concave side (equivalent to a ventral side). Many protein granules are arranged in two longitudinal and lateral rows between the remnant of the nuclear envelope and the chromatin (Figs. 3e–g and 5d, e). Their form and size are homogeneous. No mitochondria were observed in the spermatozoon of C.
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Fig. 1. (a–f) Spermiogenesis in Cavisoma magnum. (a) Cross-section showing a centriole (C). Bar = 0.4 m. (b) A subspherical nucleus (N) is present in the centre of the young spermatid. It is limited by 2 well individualized membranes (double arrow) and contains finely granular chromatin (Ch). Centriole (C), mitochondria (M), posterior bulb (arrow). Bar = 0.4 m. (c) The centriole gives rise to a flagellum (F). Bar = 0.2 m. (d) The centriole starts its migration and a cytoplasmic canal (cc) is formed. Flagellum (F). Bar = 0.2 m. (e) Cross section of a flagellum of 9 + 2 pattern with 9 peripherical doublets and 2 central singlets. Chromatin (Ch). This figure corresponds to a cross section at the level indicated in (d). Bar = 0.2 m. (f) The centriole reaches the anterior extremity of the nucleus. Anterior extremity of the cytoplasmic canal (arrow), chromatin (Ch), flagellum (F). Bar = 0.6 m.
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Fig. 2. (a–i) Spermiogenesis in Cavisoma magnum. (a) Longitudinal section of the spermatid. The flagellum (F) possesses two free extremities (arrows). Bar = 0.4 m. (b) Cross-section of the flagellum. All peripherical doublets and 2 central singlets are still present. Bar = 0.2 m. (c) Beginning of axoneme disorganization in cross-section: presence of 2 doublets, 2 partial doublets and 5 peripherical singlets and one central singlet. Bar = 0.2 m. (d) Flagellum disorganization in cross-section. Bar = 0.2 m. (e) Cross-section of a lengthening spermatid with different levels of chromatin condensation. Chromatin (Ch), nuclear envelope (Ne). Bar = 0.1 m. (f) Longitudinal section of a late spermatid with chromatin condensation (Ch). Bar = 0.4 m. (g) Cross-section of the flagellum in the posterior extremity at the level indicated in (i). Notice the reduction in the number of peripheral microtubules and transformation of some doublets into singlets, and absence of one central of the microtubules. Nuclear envelope (Ne). Bar = 0.1 m. (h) Cross-section of the flagellum at the level indicated in (i). Flagellum (F), pentalaminate remnant (Pr). Bar = 0.1 m. (i) Longitudinal section of a last stage of a late spermatid already showing the future form of the spermatozoon. Flagellum (F). Bar = 0.1 m.
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Fig. 3. (a–k) Mature spermatozoa in the seminal vesicle of Cavisoma magnum. (a) Transverse section of the anterior extremity of the spermatozoa (arrow); corresponds to level a in Fig. 5. Bar = 0.2 m. (b and c) Cross-sections of a flagellum (F) of the mature spermatozoa near the anterior extremity of the nucleocytoplasmic derivative (NCD); correspond respectively to levels b and c in Fig. 5. Bar = 0.2 m. (d) Cross-section of a flagellum of the mature spermatozoa. Bar = 0.2 m. (e) Longitudinal section of a mature spermatozoon which contains many protein granules (G). Bar = 0.2 m. (f and g) Transverse section of spermatozoon near the middle part of the nucleocytoplasmic derivative with a 9 + 2 pattern flagellum. Chromatin layer (arrow); G, protein granules; Pr, pentalaminate remnant of the nuclear envelope. Figure (f) corresponds to level d in Fig. 5. Bar = 0.2 m. (h and i) Transverse sections of the posterior part of the spermatozoon showing the extremity of the flagellum and the successive ending of the flagellar microtubules (arrows); correspond to level e in Fig. 5. Bar = 0.2 m. (j) Distal extremity of the flagellum (arrow) in cross section. The nucleocytoplasmic derivative is devoid of protein granules; corresponds to level f in Fig. 5. Bar = 0.2 m. (k) Cross sections of the posterior lamina of the spermatozoon (arrows); corresponds to level g in Fig. 5. Bar = 0.2 m.
magnum. The chromatin appears as a thin layer covering the dorsal side of the NCD (Figs. 3g–j and 4d, e). The posterior part of the spermatozoon is marked by the extremity of the flagellum recognizable by a reduction in the number
of microtubules, a transformation of the doublets into singlets (Figs. 3h, i and 5e) and a progressive interruption of all the microtubules (Figs. 3j, k and 5f, g). In the posterior extremity, the spermatozoon appears as a thin chromatin lamina (Figs. 3k and 5g).
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Fig. 4. (a–h) Diagram showing the main stages of spermiogenesis in Cavisoma magnum.
4. Discussion The phylogenetic position of Acanthocephala and the relationships between Rotifers and Acanthocephalans among metazoans have been a matter of debate for years (Ahlrichs, 1997; Garey et al., 1996, 1998; Giribet et al., 2004; Herlyn et al., 2003; Miquelis et al., 2000; Sorensen and Giribet, 2006; Winnepenninckx et al., 1995). 4.1. Spermiogenesis In C. magnum, the early spermatid possesses a single centriole, which gives rise to a flagellum. The presence of only one centriole in the spermatid of an acanthocephalan was first observed in Illiosentis furcatus (see Marchand and Mattei, 1976c), and was subsequently found regularly in several other acanthocephalans (e.g., Foata et al., 2005, 2004). During its migration, the centriole, followed by the flagellum, drags the posterior plasma membrane forward and a cytoplasmic canal is created. The axonemal migration continues until the posterior end of the flagellum passes beyond the posterior end of the nucleus. Our results are consistent with those of Marchand and Mattei (1976a) who were the first to describe an anterior
migration of the flagellum in some others acanthocephalan species (Foata et al., 2005). In C. magnum, during the elongation of the nucleus, the mitochondria stay in the cytoplasm which will be eliminated by sliding along the flagellum. In acanthocephalans, sliding and elimination of the residual cytoplasm mark the end of the elaboration of the nucleocytoplasmic derivative. Indeed, at the time of the sliding, the nuclear envelope opens, the protein granules are inserted between chromatin and nuclear envelope remnant, and the posterior lamina is created (e.g., Foata et al., 2005). In the rotifer Seison nebaliae some mitochondria are also present in the cytoplasm, but curiously at least one is always connected to the nucleus. The chromatin is highly condensed and assumes the shape of a small electrondense triangular prism close to the mitochondrion (Ferraguti and Melone, 1999). 4.2. Spermatozoon Today, most authors distinguish three subclades within each of the taxa: the Monogononta, Bdelloidea, and Seisonidea (Seison) within the Rotifera, and the Palaeacanthocephala, Eoacanthocephala, and Archiacanthocephala within the Acanthocephala
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Fig. 5. (a–g) Diagram showing the ultrastructural organization of the mature spermatozoon of Cavisoma magnum.
(e.g., Nielsen, 2001). Seison (Rotifera) have been placed into the Acanthocephala-Bdelloid subclade of the Syndermata, in agreement with other data on the spermatozoon (Ahlrichs, 1998; Ricci et al., 1993; Welch, 2000). In the acanthocephalan subclades (Eoacanthocephala, Paleacanthocephala, Archiacanthocephala), different flagellum patterns have been reported in spermatozoa: - In Eoacanthocephala a 9 + n pattern was described where n ranges between 0 and 5 (Marchand and Mattei, 1977).
- In Paleacanthocephala, the spermatozoon flagellum is usually of the 9 + 2 pattern, except in I. furcatus (Marchand and Mattei, 1976a) and Leptorhynchoides plagicephalus (Foata et al., 2004) where it was described as having a 9 + 0 pattern. C. magnum (present study) has a spermatozoon flagellum with a 9 + 2 pattern agreeing with most of the studied Paleacanthocephala. - In Archiacanthocephala, Moniliformis cestodiformis (Marchand and Mattei, 1978b) and Macracanthorhynchus hirudinaceus (Foata et al., 2005), the flagellum has a 9 + 2 pattern.
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The common character to both the Rotifera and Acanthocephala is reversed anatomy of the spermatozoon. In the mature spermatozoon of the palaeacanthocephalan C. magnum, protein granules occur regularly, in two longitudinal rows, between the chromatin and the pentalaminate remnant of the nuclear envelope. The forms of protein granules of C. magnum are homogeneous. It is the only palaeacanthocephalan to have simultaneously homogeneous protein granules and a regular and precise distribution of granules. In others Paleacanthocephala studied, either the distribution was ordered e.g., Centrorhynchus milvus (Marchand and Mattei, 1976c), or the granules had varied sizes such as in I. furcatus (Marchand and Mattei, 1976a). In Eoacanthocephala (Neoechynorhynchus agilis) and Archiacanthocephala (Moniliformis cestodiformis), protein granules form an unordered heterogeneous population (Marchand and Mattei, 1978a). Another feature, in C. magnum, is a thin chromatin layer which extends, from top to bottom, on the dorsal side of the nucleocytoplasmic derivative (NCD). Then, the NCD ends as a thin chromatin lamina. In the rotifer S. nebaliae, the general architecture of the spermatozoon exhibits similarities with acanthocephalan spermatozoa: an axoneme with a conventional 9 + 2 pattern, an elongate cell, in which there is, anteriorly, a flagellar portion with an anterior centriole, and posteriorly, a cell body with the nucleus, with irregularly condensed chromatin (Ferraguti and Melone, 1999). The spermatozoon of S. nebaliae shows a considerable number of vesicles reported as “accessory bodies” which occur regularly and appear full (Ferraguti and Melone, 1999). They look curiously like to protein granules observed in our study on C. magnum. This ultrastructural study carried out with a TEM (Transmission Electron Microscopy), on the general architecture of the C. magnum spermatozoon supplements former work carried out in other acanthocephalans and in the interrelationships acanthocephalans/rotifers (e.g., the reversed anatomy of the spermatozoon). Spermatozoan ultrastructure is a major character for phylogenetic reconstructions. In phylogenetic analyses, the Acanthocephala were formerly believed to be sister to the Rotifera, with the two groups together forming a clade that has been referred to as the Syndermata. A variety of analyses now strongly suggest that the Acanthocephala are in fact a clade of parasitic rotifers, most likely the sister-group to the free-living bdelloids (Garcia-Varela and Nadler, 2006; Min and Park, 2009; Witek et al., 2009), making the name Syndermata a junior synonym of Rotifera (Sorensen and Giribet, 2006). Based on a large-scale analysis of molecular data (amino acid sequences), Witek et al. (2009) concluded that Syndermata and Gnathostomulida together comprise a monophyletic clade known as the Gnathifera. The Gnathifera was originally proposed as a monophyletic clade based on jaw morphology and may also include the Micrognathozoa and, conceivably, the Cycliophora (Funch et al., 2005; Sorensen and Giribet, 2006). C. magnum has, however, distinct characteristics. Their spermatozoa are different in that the distribution and the size of protein granules are regular, and their posterior extremity becomes a wavelike chromatin lamina. These features have not been reported in spermatozoon in the other groups considered close to the Acanthocephala. Acknowledgement Rod Bray (NHM, London) kindly edited the English. References Ahlrichs, W.H., 1997. Epidermal ultrastructure of Seison nebaliae and Seison annulatus, and a comparison of epidermal structures within the Gnathifera. Zoomorphology 117, 41–48.
Ahlrichs, W.H., 1998. Spermatogenesis and ultrastructure of the spermatozoa of Seison nebaliae (Syndermata). Zoomorphology 118, 255–261. Arthur, J.R., Redigor, S.E., Albert, E., 1995. Redescription of Cavisoma magnum (Southwell 1927) (Acanthocephala: Cavisomatidae) from the Milkfish, Chanos chanos, in the Philippines. J. Helminthol. 62, 39–43. Carcupino, M., Dezfuli, B.S., 1995. Ultrastructural study of mature sperm of Pomphorhynchus laevis Müller (Acanthocephala Paleacanthocephala) a fish parasite. Invertebr. Reprod. Dev. 28, 25–32. Carcupino, M., Dezfuli, B.S., 1999. Acanthocephala. In: B.G.M, J. (Ed.), Reproductive Biology of Invertebrates. , pp. 229–242. Crompton, D.W.T., 1970. An Ecological Approach to Acanthocephalan Physiology. Cambridge at the University Press. Crompton, D.W.T., Nickol, B.B., 1985. Biology of the Acanthocephala. Cambridge University Press ed, Cambrige/New York/New Rochelle/Melbourne/Sydney. Crompton, D.W.T., Nickol, B.B., 2009. Biology of the Acanthocephala. Cambridge University Press ed, Cambrige/New York/New Rochelle/Melbourne/Sydney. Ferraguti, M., Melone, G., 1999. Spermiogenesis in Seison nebaliae (Rotifera Seisonidea): further evidence of a rotifer–acanthocephalan relationship. Tissue Cell 31, 428–440. Foata, J., Culioli, J.L., Marchand, B., 2005. Ultrastructure of spermiogenesis and the spermatozoon of Macracanthorhynchus hirudinaceus (Pallas, 1781) (Acanthocephala: Archiacanthocephala), a parasite of the wild boar Sus scrofa. J. Parasitol., 499–506. Foata, J., Dezfuli, B.S., Pinelli, B., Marchand, B., 2004. Ultrastructure of spermiogenesis and spermatozoon of Leptorhynchoides plagicephalus (Acanthocephala, Palaeacanthocephala), a parasite of the sturgeon Acipenser naccarii (Osteichthyes, Acipenseriformes). Parasitol. Res. 93, 582–589. Funch, P., Sorensen, M.V., Obst, M., 2005. On the phylogenetic position of Rotifera – have we come any further? Hydrobiologia 546, 11–28. Garcia-Varela, M., Nadler, S.A., 2006. Phylogenetic relationships among Syndermata inferred from nuclear and mitochondrial genes sequences. Mol. Phylogenet. Evol. 40, 61–72. Garey, J., Schmidt-Rhaesa, A., Near, T., Nadler, S., 1998. The evolutionary relationships of rotifers and acanthocephalans. Hydrobiologia 387, 83–91. Garey, J.R., Near, T.J., Nonnemacher, M.R., Nadler, S.A., 1996. Molecular evidence for acanthocephala as a subtaxon of Rotifera. J. Mol. Evol., 287–292. Giribet, G., Sorensen, M.V., Funch, P., Kristensen, R.M., Sterrer, W., 2004. Investigations into the phylogenetic position of Micrognathozoa using four molecular loci. Cladistics 20, 1–13. Grinson, G., Sreeraj, C.R., Dam Roy, S., 2011. Brachionid rotifer diversity in Andaman waters. Indian J. Geo-Mar. Sci. 40, 454–459. Guraya, S.S., 1971. Morphological and histochemical observations on the acanthocephalan spermatogenesis. Acta Morphol. Neerl. Scand. 9, 75–83. Herlyn, H., Piskurel, O., Schmitz, J., Ehlers, U., Zischler, H., 2003. The syndermatan phylogeny and evolution of acanthocephalan endoparasitism as inferred from 18S rDNA sequences. Mol. Phylogenet. Evol. 26, 155–164. Hyman, H.L., 1951. The invertebrates: Acanthocephala, Aschelminthes, and Entoprocta. In: The Pseudocoelompate Bilateria. McGraw-Hill, New York, pp. 459–531. Justine, J.L., 2010. Parasites of coral reef fish: how much do we know? With a bibliography of fish parasites in New Caledonia. Belgian J. Zool. 140, 155–190. Kennedy, C.R., 2006. Ecology of the Acanthocephala. Cambridge University Press, New York. Marchand, B., Mattei, X., 1976a. La Spermatogenèse des Acanthocéphales I. L’Appareil Centriolaire et Flagellaire au Cours de la Spermiogenèse d’Illiosentis furcatus var africana Golvan. 1956 (Paleacanthocephala, Rhadinorhynchidae). J. Ultra Res. 54, 347–358. Marchand, B., Mattei, X., 1976b. La Spermatogenèse des Acanthocéphales II. Variation du nombre de fibres centrales dans le flagelle spermatique d’Acanthosentis tilapiae Baylis 1947 (Eocanthocephala, Quadrigyridae). J. Ultra Res. 55, 391–399. Marchand, B., Mattei, X., 1976c. Ultrastructure du spermatozoïde de Centrorhynchus milvus Ward, 1956 (Paleacanthocephala, Polymorphidae). C. R. Seances Soc. Biol. 170, 237–240. Marchand, B., Mattei, X., 1977. La Spermatogenèse des Acanthocéphales III. Formation du dérivé centriolaire au cours de la spermiogenèse de Serrasentis socialis Van Cleava, 1924 (Paleacanthocephala, Gorgorhynchidae). J. Ultra Res. 59, 263–271. Marchand, B., Mattei, X., 1978a. La Spermatogenèse des Acanthocéphales IV. Le dérivé nucléocytoplasmique. Bio Cell 31, 79–90. Marchand, B., Mattei, X., 1978b. La Spermatogenèse des Acanthocéphales V. Flagellogenèse chez un Eoacanthocephala: Mise en place et Désorganisation de l’Axonème Spermatique. J. Ultra Res. 63, 41–50. Min, G.S., Park, J.K., 2009. Eurotatorian paraphyly: revisiting phylogenetic relationships based on the complete mitochondrial genome sequence of Rotatoria (Bdelloidea: Rotifera: Syndermata). BMC Genomics 10, 533. Miquelis, A., Martin, J., Carson, E., Brun, G., Gilles, A., 2000. Performance of 18S rDNA helix E23 for phylogenetic relationships within and between the RotiferaAcanthocephala clades. C. R. Acad. Sci. Ser. III-Sci. Vie-Life Sci. 323, 925–941. Nielsen, C., 2001. Animal Evolution, Interrelationships of the Living Phyla, 2nd ed. University Press, Oxford. Petrochenko, V.I., 1956. In: Skrjabin, K.I. (Ed.), Acanthocephala of Domestic and Wild Animals. Izdatel’stvo Akademii Nauk SSSR, Moscow. Petrochenko, V.I., 1958. In: Skrjabin, K.I. (Ed.), Acanthocephala of Domestic and Wild Animals. Izdatel’stvo Akademii Nauk SSSR, Moscow. Ricci, C., Melone, G., Sotgia, C., 1993. Old and new data on Seisonidea. Hydrobiologia 255–256, 495–511.
J. Foata et al. / Micron 43 (2012) 141–149 Sorensen, M.V., Giribet, G., 2006. A modern approach to rotiferan phylogeny: combining morphological and molecular data. Mol. Phylogenet. Evol. 40, 585–608. Spurr, A.R., 1969. A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultra Res. 26, 31–43. Welch, D., 2000. Evidence from a protein-coding gene that acanthocephalans are rotifers. Invertebr. Biol. 119, 17–26. Whitfield, P.J., 1971. Spermiogenesis and spermatozoa ultrastructure in Polymorphus minutus (Acanthocephala). Parasitology 62, 415–430.
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Review
Ultrastructural study of spermiogenesis and the spermatozoon of Cavisoma magnum (Southwell, 1927) (Acanthocephala, Palaeacanthocephala, Cavisomidae), from Siganus lineatus (Pisces, Teleostei, Siganidae) (Valenciennes, 1835) in New Caledonia J. Foata a,∗ , Y. Quilichini a , J.-L. Justine b , R.A. Bray c , B. Marchand a a
CNRS UMR 6134, University of Corsica, “Parasites and Mediterranean Ecosystems” Laboratory, BP 52, 20250 Corte, France UMR 7138 Systématique, Adaptation, Évolution, Muséum National d‘Histoire Naturelle, Case postale 52, 57, rue Cuvier, 75231 Paris cedex 05, France c Department of Zoology, Natural History Museum, Cromwell Road, London SW7 5BD, UK b
a r t i c l e
i n f o
Article history: Received 25 August 2011 Received in revised form 12 October 2011 Accepted 25 October 2011 Keywords: Ultrastructure Spermatozoon Spermiogenesis Cavisoma magnum Acanthocephala TEM
a b s t r a c t This paper presents an ultrastructural study of Cavisoma magnum (Acanthocephala, Cavisomatidae) with a Transmission Electron Microscopy tool. This parasite of the fish Siganus lineatus is here reported for the first time from off New Caledonia, South Pacific. It is the first study describing the ultrastructure, spermiogenesis and spermatozoon of a species of the family Cavisomatidae. The young spermatid of C. magnum possesses a centriole constituted of doublets without a central element. After the elaboration of a flagellum of 9 + 2 pattern, the centriole migrates in a nuclear groove. Then the flagellum migration occurs and gives rise to a spermatozoon. The distribution and the size of the protein granules are reported and the posterior extremity appears like a chromatin lamina wave. Comparative ultrastructural data are presented on sperm and spermiogenesis of the Acanthocephala and Rotifers examined to date and the phylogenetic implications are discussed. © 2011 Elsevier Ltd. All rights reserved.
Contents 1. 2.
3.
4.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Fixation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Embedding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Ultramicrotomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Poststaining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Spermiogenesis (Figs. 1, 2 and 4) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Spermatozoon (Figs. 3 and 5) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Spermiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Spermatozoon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction The Acanthocephala are generally considered to be a small group of parasites, because they are rare. Nevertheless, there have been
∗ Corresponding author. E-mail address: [email protected] (J. Foata). 0968-4328/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2011.10.022
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some books devoted exclusively to them (e.g., Crompton, 1970; Crompton and Nickol, 1985, 2009; Kennedy, 2006; Petrochenko, 1956, 1958; Yamaguti, 1963). Currently, their phylogenetic position is debated (e.g., Sorensen and Giribet, 2006). Few studies have been conducted on the reproduction of acanthocephalans. The first light-microscopic studies were performed by Hyman (1951) and Guraya (1971) and the first ultrastructural study was Whitfield‘s work (1971) on Polymorphus minutus (Zeder, 1800). Our
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present-day knowledge of the ultrastructure of acanthocephalan reproduction is mainly the result of electron microscopical studies by (Marchand and Mattei, 1976a,b) with other recent contributions from Carcupino and Dezfuli (1995) and Foata et al. (2005, 2004). The biology of acanthocephalans was re-examined in a monograph published by Crompton and Nickol (1985) and their reproduction was reviewed by Carcupino and Dezfuli (1999). In the present work, we describe the ultrastructure of spermiogenesis and the spermatozoon of Cavisoma magnum (Southwell, 1927) (Paleacanthocephala), an intestinal parasite of the goldenlined spinefoot, Siganus lineatus (Valenciennes, 1835) from off New Caledonia. C. magnum has previously been described from other fish species in different localities: in the goldring surgeonfish Ctenochaetus strigosus and Serranus sp. in Indian Ocean (India and Sri Lanka), and in the milkfish Chanos chanos in the Pacific Ocean (Philippines) (Arthur et al., 1995). This work aims to present the first ultrastructural study of spermiogenesis and the spermatozoon of a cavisomatid acanthocephalan and draw a comparison with findings on the Rotifers, a group of mostly microscopic, aquatic invertebrates with about 2000 described species (Grinson et al., 2011). The Acanthocephala, previously considered to be a separate phylum, are considered as highly modified rotifers but the exact relationship to other members of the phylum is discussed. Spermatozoa are considered as an important source of characters for phylogenetic reconstructions. 2. Materials and methods Specimens of C. magnum (Southwell, 1927) were collected from the intestine of a naturally infected golden-lined spinefoot S. lineatus (Valenciennes, 1835) (Pisces, Teleostei) caught off Nouméa, New Caledonia (Pacific Ocean); prevalence was 16% (1/6), intensity 7. This is the first Acanthocephala identified from a fish off New Caledonia (Justine, 2010). The worms were removed alive from the digestive tract of their hosts. The parasites were carefully dissected, the entire genital apparatus of males were removed and the testes isolated. 2.1. Fixation The material was fixed in cold (4 ◦ C) 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer at pH 7.2 for 1 h, rinsed in 0.1 M sodium cacodylate buffer at pH 7.2 and post-fixed in cold (4 ◦ C) 1% osmium tetroxide in the same buffer for 1 h. 2.2. Embedding The material was dehydrated in a series of ethanol (from 70% to 100%) and propylene oxide (100%), embedded in Spurr (1969) and polymerised at 60 ◦ C for 24 h. 2.3. Ultramicrotomy Ultrathin sections (60–90 nm) of testis and seminal vesicle were cut on an ultramicrotome (Power tome PC, RMC Boeckeler® ). The sections were placed on 300 and 200-mesh copper grids. 2.4. Poststaining Grids were stained with ethanolic uranyl acetate (5% solution of uranyl acetate in 50% ethanol) for 10 min, rinsed in deionized water. Finally, they were stained with lead citrate (0.15 g lead citrate + 0.2 g sodium citrate in 50 mL of deionized water) for 5 min and rinced in deionized water.
Sections were examined on a Hitachi H-7650 transmission electron microscope, operating at an accelerating voltage of 80 kV, in the “Service d‘Étude et de Recherche en Microscopie Électronique” of the University of Corsica (Corte, France).
3. Results 3.1. Spermiogenesis (Figs. 1, 2 and 4) The young spermatid of C. magnum is a round cell which possesses a large nucleus in the central position and a centriole with 9 peripheral doublets without central elements (Fig. 1a). The beginning of spermiogenesis is characterized by a centriole with a “posterior bulb” abutted to the plasma membrane and perpendicular to the nuclear surface (Figs. 1b and 4a). This centriole gives rise to a flagellum (Figs. 1c and 4b) with a 9 + 2 pattern. The centriole then migrates into a nuclear groove towards the anterior part of the spermatid. The flagellum, surrounded by a plasma membrane, follows the centriole in its migration, and a cytoplasmic canal is formed (Figs. 1d, e and 4c, d). The flagellum stops its progression when the centriole reaches the anterior extremity of the nucleus (Fig. 4d). The centriole then continues its anterior migration, pushing the plasma membrane of the anterior extremity of the spermatid in front of it (Figs. 1f and 4e). Thus, it passes beyond the anterior extremity of the spermatid (Figs. 2a and 4f). During flagellar migration, the anterior and posterior parts of the flagellum are temporarily free (Fig. 2a). Migration stops when the posterior extremity of the flagellum passes into the spermatid (Fig. 4g and h). The posterior part of the flagellum is characterized by the disorganization of the axoneme marked by the transformation of doublets into singlets and a reduction in the number of microtubules (Fig. 2b–d). At the end of the flagellar migration, the nucleus extends towards the back of the spermatid and against the posterior part of the flagellum. At the same time, chromatin condenses in close contact to the nuclear groove, becomes progressively lamellar (Fig. 2e and f) and appears as a network of lamellar anastomoses. In the late spermatid, protein granules are elaborated, while chromatin reaches maximum condensation and appears as a thin lamina (Fig. 2g and h). By the end of spermiogenesis, the nuclear envelope opens and persists like a pentalaminate structure which corresponds to a remnant of the nuclear groove (Fig. 2h and i). Then, a nucleocytoplasmic derivative in is formed and the chromatin appears in close contact with the cytoplasm and the protein granules. At the same time, mitochondria are eliminated in residual cytoplasm.
3.2. Spermatozoon (Figs. 3 and 5) Spermatozoa are present in seminal vesicles. Transverse sections of the anterior extremity of the spermatozoon are characterized by the presence of a centriolar derivative made of 9 peripheral elements (Figs. 3a and 5a). This centriolar derivative is followed by a flagellum with a 9 + 2 pattern (Figs. 3b–d and 5b–d). The nucleocytoplasmic derivative (NCD) is reniform in section (Figs. 3f and 5d, e) with a thin layer of chromatin placed against the convex side of the spermatozoon (equivalent to a dorsal side). A pentalaminate remnant of nuclear envelope occurs on the concave side (equivalent to a ventral side). Many protein granules are arranged in two longitudinal and lateral rows between the remnant of the nuclear envelope and the chromatin (Figs. 3e–g and 5d, e). Their form and size are homogeneous. No mitochondria were observed in the spermatozoon of C.
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Fig. 1. (a–f) Spermiogenesis in Cavisoma magnum. (a) Cross-section showing a centriole (C). Bar = 0.4 m. (b) A subspherical nucleus (N) is present in the centre of the young spermatid. It is limited by 2 well individualized membranes (double arrow) and contains finely granular chromatin (Ch). Centriole (C), mitochondria (M), posterior bulb (arrow). Bar = 0.4 m. (c) The centriole gives rise to a flagellum (F). Bar = 0.2 m. (d) The centriole starts its migration and a cytoplasmic canal (cc) is formed. Flagellum (F). Bar = 0.2 m. (e) Cross section of a flagellum of 9 + 2 pattern with 9 peripherical doublets and 2 central singlets. Chromatin (Ch). This figure corresponds to a cross section at the level indicated in (d). Bar = 0.2 m. (f) The centriole reaches the anterior extremity of the nucleus. Anterior extremity of the cytoplasmic canal (arrow), chromatin (Ch), flagellum (F). Bar = 0.6 m.
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Fig. 2. (a–i) Spermiogenesis in Cavisoma magnum. (a) Longitudinal section of the spermatid. The flagellum (F) possesses two free extremities (arrows). Bar = 0.4 m. (b) Cross-section of the flagellum. All peripherical doublets and 2 central singlets are still present. Bar = 0.2 m. (c) Beginning of axoneme disorganization in cross-section: presence of 2 doublets, 2 partial doublets and 5 peripherical singlets and one central singlet. Bar = 0.2 m. (d) Flagellum disorganization in cross-section. Bar = 0.2 m. (e) Cross-section of a lengthening spermatid with different levels of chromatin condensation. Chromatin (Ch), nuclear envelope (Ne). Bar = 0.1 m. (f) Longitudinal section of a late spermatid with chromatin condensation (Ch). Bar = 0.4 m. (g) Cross-section of the flagellum in the posterior extremity at the level indicated in (i). Notice the reduction in the number of peripheral microtubules and transformation of some doublets into singlets, and absence of one central of the microtubules. Nuclear envelope (Ne). Bar = 0.1 m. (h) Cross-section of the flagellum at the level indicated in (i). Flagellum (F), pentalaminate remnant (Pr). Bar = 0.1 m. (i) Longitudinal section of a last stage of a late spermatid already showing the future form of the spermatozoon. Flagellum (F). Bar = 0.1 m.
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Fig. 3. (a–k) Mature spermatozoa in the seminal vesicle of Cavisoma magnum. (a) Transverse section of the anterior extremity of the spermatozoa (arrow); corresponds to level a in Fig. 5. Bar = 0.2 m. (b and c) Cross-sections of a flagellum (F) of the mature spermatozoa near the anterior extremity of the nucleocytoplasmic derivative (NCD); correspond respectively to levels b and c in Fig. 5. Bar = 0.2 m. (d) Cross-section of a flagellum of the mature spermatozoa. Bar = 0.2 m. (e) Longitudinal section of a mature spermatozoon which contains many protein granules (G). Bar = 0.2 m. (f and g) Transverse section of spermatozoon near the middle part of the nucleocytoplasmic derivative with a 9 + 2 pattern flagellum. Chromatin layer (arrow); G, protein granules; Pr, pentalaminate remnant of the nuclear envelope. Figure (f) corresponds to level d in Fig. 5. Bar = 0.2 m. (h and i) Transverse sections of the posterior part of the spermatozoon showing the extremity of the flagellum and the successive ending of the flagellar microtubules (arrows); correspond to level e in Fig. 5. Bar = 0.2 m. (j) Distal extremity of the flagellum (arrow) in cross section. The nucleocytoplasmic derivative is devoid of protein granules; corresponds to level f in Fig. 5. Bar = 0.2 m. (k) Cross sections of the posterior lamina of the spermatozoon (arrows); corresponds to level g in Fig. 5. Bar = 0.2 m.
magnum. The chromatin appears as a thin layer covering the dorsal side of the NCD (Figs. 3g–j and 4d, e). The posterior part of the spermatozoon is marked by the extremity of the flagellum recognizable by a reduction in the number
of microtubules, a transformation of the doublets into singlets (Figs. 3h, i and 5e) and a progressive interruption of all the microtubules (Figs. 3j, k and 5f, g). In the posterior extremity, the spermatozoon appears as a thin chromatin lamina (Figs. 3k and 5g).
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Fig. 4. (a–h) Diagram showing the main stages of spermiogenesis in Cavisoma magnum.
4. Discussion The phylogenetic position of Acanthocephala and the relationships between Rotifers and Acanthocephalans among metazoans have been a matter of debate for years (Ahlrichs, 1997; Garey et al., 1996, 1998; Giribet et al., 2004; Herlyn et al., 2003; Miquelis et al., 2000; Sorensen and Giribet, 2006; Winnepenninckx et al., 1995). 4.1. Spermiogenesis In C. magnum, the early spermatid possesses a single centriole, which gives rise to a flagellum. The presence of only one centriole in the spermatid of an acanthocephalan was first observed in Illiosentis furcatus (see Marchand and Mattei, 1976c), and was subsequently found regularly in several other acanthocephalans (e.g., Foata et al., 2005, 2004). During its migration, the centriole, followed by the flagellum, drags the posterior plasma membrane forward and a cytoplasmic canal is created. The axonemal migration continues until the posterior end of the flagellum passes beyond the posterior end of the nucleus. Our results are consistent with those of Marchand and Mattei (1976a) who were the first to describe an anterior
migration of the flagellum in some others acanthocephalan species (Foata et al., 2005). In C. magnum, during the elongation of the nucleus, the mitochondria stay in the cytoplasm which will be eliminated by sliding along the flagellum. In acanthocephalans, sliding and elimination of the residual cytoplasm mark the end of the elaboration of the nucleocytoplasmic derivative. Indeed, at the time of the sliding, the nuclear envelope opens, the protein granules are inserted between chromatin and nuclear envelope remnant, and the posterior lamina is created (e.g., Foata et al., 2005). In the rotifer Seison nebaliae some mitochondria are also present in the cytoplasm, but curiously at least one is always connected to the nucleus. The chromatin is highly condensed and assumes the shape of a small electrondense triangular prism close to the mitochondrion (Ferraguti and Melone, 1999). 4.2. Spermatozoon Today, most authors distinguish three subclades within each of the taxa: the Monogononta, Bdelloidea, and Seisonidea (Seison) within the Rotifera, and the Palaeacanthocephala, Eoacanthocephala, and Archiacanthocephala within the Acanthocephala
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Fig. 5. (a–g) Diagram showing the ultrastructural organization of the mature spermatozoon of Cavisoma magnum.
(e.g., Nielsen, 2001). Seison (Rotifera) have been placed into the Acanthocephala-Bdelloid subclade of the Syndermata, in agreement with other data on the spermatozoon (Ahlrichs, 1998; Ricci et al., 1993; Welch, 2000). In the acanthocephalan subclades (Eoacanthocephala, Paleacanthocephala, Archiacanthocephala), different flagellum patterns have been reported in spermatozoa: - In Eoacanthocephala a 9 + n pattern was described where n ranges between 0 and 5 (Marchand and Mattei, 1977).
- In Paleacanthocephala, the spermatozoon flagellum is usually of the 9 + 2 pattern, except in I. furcatus (Marchand and Mattei, 1976a) and Leptorhynchoides plagicephalus (Foata et al., 2004) where it was described as having a 9 + 0 pattern. C. magnum (present study) has a spermatozoon flagellum with a 9 + 2 pattern agreeing with most of the studied Paleacanthocephala. - In Archiacanthocephala, Moniliformis cestodiformis (Marchand and Mattei, 1978b) and Macracanthorhynchus hirudinaceus (Foata et al., 2005), the flagellum has a 9 + 2 pattern.
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The common character to both the Rotifera and Acanthocephala is reversed anatomy of the spermatozoon. In the mature spermatozoon of the palaeacanthocephalan C. magnum, protein granules occur regularly, in two longitudinal rows, between the chromatin and the pentalaminate remnant of the nuclear envelope. The forms of protein granules of C. magnum are homogeneous. It is the only palaeacanthocephalan to have simultaneously homogeneous protein granules and a regular and precise distribution of granules. In others Paleacanthocephala studied, either the distribution was ordered e.g., Centrorhynchus milvus (Marchand and Mattei, 1976c), or the granules had varied sizes such as in I. furcatus (Marchand and Mattei, 1976a). In Eoacanthocephala (Neoechynorhynchus agilis) and Archiacanthocephala (Moniliformis cestodiformis), protein granules form an unordered heterogeneous population (Marchand and Mattei, 1978a). Another feature, in C. magnum, is a thin chromatin layer which extends, from top to bottom, on the dorsal side of the nucleocytoplasmic derivative (NCD). Then, the NCD ends as a thin chromatin lamina. In the rotifer S. nebaliae, the general architecture of the spermatozoon exhibits similarities with acanthocephalan spermatozoa: an axoneme with a conventional 9 + 2 pattern, an elongate cell, in which there is, anteriorly, a flagellar portion with an anterior centriole, and posteriorly, a cell body with the nucleus, with irregularly condensed chromatin (Ferraguti and Melone, 1999). The spermatozoon of S. nebaliae shows a considerable number of vesicles reported as “accessory bodies” which occur regularly and appear full (Ferraguti and Melone, 1999). They look curiously like to protein granules observed in our study on C. magnum. This ultrastructural study carried out with a TEM (Transmission Electron Microscopy), on the general architecture of the C. magnum spermatozoon supplements former work carried out in other acanthocephalans and in the interrelationships acanthocephalans/rotifers (e.g., the reversed anatomy of the spermatozoon). Spermatozoan ultrastructure is a major character for phylogenetic reconstructions. In phylogenetic analyses, the Acanthocephala were formerly believed to be sister to the Rotifera, with the two groups together forming a clade that has been referred to as the Syndermata. A variety of analyses now strongly suggest that the Acanthocephala are in fact a clade of parasitic rotifers, most likely the sister-group to the free-living bdelloids (Garcia-Varela and Nadler, 2006; Min and Park, 2009; Witek et al., 2009), making the name Syndermata a junior synonym of Rotifera (Sorensen and Giribet, 2006). Based on a large-scale analysis of molecular data (amino acid sequences), Witek et al. (2009) concluded that Syndermata and Gnathostomulida together comprise a monophyletic clade known as the Gnathifera. The Gnathifera was originally proposed as a monophyletic clade based on jaw morphology and may also include the Micrognathozoa and, conceivably, the Cycliophora (Funch et al., 2005; Sorensen and Giribet, 2006). C. magnum has, however, distinct characteristics. Their spermatozoa are different in that the distribution and the size of protein granules are regular, and their posterior extremity becomes a wavelike chromatin lamina. These features have not been reported in spermatozoon in the other groups considered close to the Acanthocephala. Acknowledgement Rod Bray (NHM, London) kindly edited the English. References Ahlrichs, W.H., 1997. Epidermal ultrastructure of Seison nebaliae and Seison annulatus, and a comparison of epidermal structures within the Gnathifera. Zoomorphology 117, 41–48.
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