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Spermatozoon ultrastructure of Gyliauchen sp. (digenea: Gyliauchenidae), an intestinal parasite of Siganus fuscescens (Pisces: Teleostei).

Introduction

A peculiarity of Platyhelminthes in general and parasitic flatworms in particular is the absence of fossils. Therefore, to understand the relationships between species and their phylogeny, research can be carried out only on extant taxa, and has included both morphological and ultrastructural data (Rohde, 1971, 1990; Brooks et al., 1985; Ehlers, 1985; Brooks, 1989; Brooks and McLennan, 1993; Hoberg et al., 1997), and more recently, molecular analysis (Littlewood et al., 1998; Olson et al., 2003; Olson and Tkach, 2005).

The ultrastructural characters of the male reproductive system, and especially the spermatozoon (e.g., the structure [the 9+"1" pattern of Trepaxonemata], the number of axonemes, the number of mitochondria, and the spiralled or parallel disposition of cortical microtubules), have been utilized for the estimation of phylogenies based only on these characters (Euzet et al., 1981; Justine et al., 1985; Justine, 1991a, 1997; Ba and Marchand, 1995; Levron et al., 2010).

Although the number of ultrastructural studies of digenean spermatozoa has increased during the last 10 years, several important species and families have not yet been examined. Indeed, among the 18,000 nominal species that have been described (Bray et al., 2008), fewer than 1% have been studied. In this present work, the first study within the Gyliauchenidae, we describe the spermatozoon of Gyliauchen sp.

According to Jones et al. (2005), the Gyliauchenidae is one of the 10 families that constitute the Lepocreadioidea. To our knowledge, the spermatozoa of only one species of Apocreadiidae (Neoapocreadium chabaudi) (Kacem et al., 2010), one species of Deropristidae (Deropristis inflata) (Foata et al., 2007), and one species of Lepocreadiidae (Holorchis micracanthum) (Ba et al., 2011) have been described within this superfamily.

The aim of this study was to compare the ultrastructure of the spermatozoon of Gyliauchen sp. with that of the other parasitic flatworms in general and digeneans in particular in terms of the principal criteria used in the phylogeny of the Platyhelminthes. In addition, we hoped to distinguish ultra-structural characteristics that could be used in digenean phylogeny.

Materials and Methods

Adult specimens of an unknown species of Gyliauchen Nicoll, 1915, were collected live with the gut-wash method (Cribb and Bray, 2010) from the intestine of naturally infected specimens of the dusky rabbitfish Siganus fuscescens (Houttuyn, 1782) (Pisces, Teleostei) caught near Reed Snark (22[degrees]26'S, 166[degrees]25'E) off Noumea, New Caledonia (South West Pacific Ocean). Voucher specimens on slides are deposited under the number MNHN JNC2541.

Worms were fixed in cold (4[degrees]C) 2.5% glutaraldehyde in 0.1 mol [1.sub.-1] sodium cacodylate buffer at pH 7.2, rinsed in 0.1 mol [1.sub.-1] sodium cacodylate buffer at pH 7.2, post-fixed in cold (4[degrees]C) 1% osmium tetroxide in the same buffer for 1 h, dehydrated in ethanol and propylene oxide, embedded in Spurr (Spurr, 1969), and polymerized at 60[degrees]C for 24 h.

Ultrathin sections (60-90 mu) of the seminal vesicle were cut on an ultramicrotome (Power tome PC, RMC Boeckeler). Sections were placed on 300- and 200-mesh copper grids and stained with uranyl acetate and lead citrate (Reynolds, 1963). The sections were examined on a Hitachi H-7650 transmission electron microscope, operating at an accelerating voltage of 80 kV, in the "Service d' Etude et de Recherche en Microscopic Electronique" of the University of Corsica (Corte, France).

Results

The observation of a great number of sections of seminal vesicles of Gyliauchen sp. enabled us to reconstruct the ultrastructure of its spermatozoon. This reconstruction is based on the presence or absence of ultrastructural elements observed in cross- and longitudinal sections--two axonemes, four attachment zones, a mitochondrion, a nucleus, external ornamentation of the plasma membrane, and spine-like bodies--and on the number of cortical microtubules observed in cross-sections.

Four regions have been defined from the anterior to the posterior extremity of the spermatozoon: Region I, anterior region of the spermatozoon; Region II, mitochondrial region of the spermatozoon; Region III, median region of the spermatozoon without mitochondrion or nucleus; Region IV, posterior region of the spermatozoon (or nuclear region).

Region I (Figs. 1-6 and 321)

Region I corresponds to the anterior part of the spermatozoon. The anterior extremity of this region is characterized by a thin cytoplasm termination associated with the anterior extremity of the first axoneme (Figs. 1, 2). A shift of the second axoneme is observed (Figs. 2-4). Indeed, the first axoneme is completely formed, whereas the second axoneme presents only nine doublets of microtubules with no central core (Fig. 3) or arms (Fig. 4). The four attachment zones are clearly visible when the central core of the second axoneme is present (Fig. 4). In this region the second axoneme is associated with a lateral expansion of the plasma filled with electron-dense material more concentrated in the periphery (Figs. 3-5). In the posterior part of this region, only the electron-dense material in the periphery is observed between the two attachment zones of the second axoneme (Fig. 6).

The number of cortical microtubules in this region increases from the anterior to the posterior part: 0 (Figs. 2, 3), 2 (Fig. 4), 3 (Fig. 5), 7 (Fig. 6), always arranged as a single row.

Region II (Figs. 7-9 and 3211)

In this region the two axonemes, their four attachment zones, and the cortical microtubules are still present. Nevertheless, this region possesses two characteristics: (i) the presence of external ornamentation of the plasma membrane in the anterior part of this region (Fig. 7); and (ii) the presence of a mitochondrion observed throughout the region (Figs. 7-9).

The number of cortical microtubules increases in this entire region: 13 (Fig. 7), 14 (Fig. 8), and 16 (Fig. 9). They are arranged on the mitochondria! side (12-15 cortical microtubules) and on the opposite side (always one cortical microtubule).

Region III (Figs. 10-19 and 300)

In this region the two axonemes, their four attachment zones, and the cortical microtubules are still present, but the mitochondrion and the external ornamentation of the plasma membrane are absent. This region presents two main characteristics: (i) The first characteristic is the presence of spine-like bodies in the anterior part of this region (Fig. 10). These elements are located in the middle of the side which possesses the highest number of cortical microtubules. The distance between two spine-like bodies varies from 150 nm to 450 nm (Fig. II). Cross-sections of this zone show an alternation of spermatozoon with or without spine-like bodies (Figs. 10, 12-16). (ii) The second characteristic is the arrangement of the cortical microtubules. In the anterior part of this zone, which can be defined as the zone with spine-like bodies (Figs. 10-16), the cortical microtubules are arranged as two fields: a field on the side of the spine-like bodies (14 to 16 elements) and a field on the opposite side (2 or 3 elements). In the posterior part of this region (without spine-like bodies) the cortical microtubules are arranged as a single field around one or two axonemes (Figs. 17-19).

The number of cortical microtubules increases throughout the region-16 (Fig. 10), 17 (Figs. 12, 13), 18 (Figs. 14, 15), 19 (Fig. 16), 25 (Fig. 17), 26 (Fig. 18)--except in its posterior extremity, where this number starts to decrease: 22 (Fig. 19).

[FIGURE OMITTED]

Region IV (Figs. 20-31 and 32IV)

This region corresponds to the posterior part of the spermatozoon. It can be defined as the nuclear region and is characterized by the presence of two axonemes, their four attachment zones, cortical microtubules, and, of course, the nucleus.

In the anterior part of this region, the nucleus has a central position and the cortical microtubules are arranged as two almost equal fields: 10 and 9 (Fig. 20); 9 and 7 (Fig. 21); 8 and 6 (Fig. 22).

The nucleus has an excentric position in the median part of this region. The cortical microtubules are arranged as a single field, except in Figure 23, where two fields are still present. Their number decreases: 8 (Fig. 23), 7 (Fig. 24), 3 (Fig. 25), 2 (Figs. 27, 28). The posterior extremity of the second axoneme is characterized by the absence of filaments (connecting the central core to the peripheral doublets of microtubules) (Fig. 25) and of the central core (Figs. 26, 27). Two cortical microtubules and only two attachment zones associated with the first axoneme are still present after the posterior extremity of the second axoneme (Fig. 28).

[FIGURE OMITTED]

In the posterior part of this region the cortical microtubules are absent (Figs. 29-31). The posterior extremity of the nucleus is associated with the posterior extremity of the central core of the first axoneme (Figs. 29, 30). At this level attachment zones are not visible. The posterior extremity of the spermatozoon is characterized by a diminution of the cytoplasm and the presence of only peripheral doublets of microtubules of the first axoneme (Figs. 30, 31).

Discussion

The discussion below is based on the location and the organization of the ultrastructural elements observed in the mature spermatozoon of Gyliauchen sp.

The axonemes

The number of axonemes--two in Gyliauchen sp. and homogeneous in digenean spermatology, with the single exception of schistosomes (Justine and Mattei, 1981)-- can vary from one to two in other groups of parasitic Platyhelminthes such as Monogenea (Justine et al., 1985) or Cestoda (Euzet et al., 1981; Levron et al., 2010).

The structure of these axonemes (9 +"1" pattern of Trepaxonemata; Ehlers, 1984) is found in all other digenean spermatozoa except Didymozoon sp. (Justine and Mattei, 1983), which possesses two axonemes with a 9+0 pattern, and the schistosomes, which have a unique non-trepaxonematan pattern (Justine and Mattei, 1981; Justine et al., 1993).

The lengths of these two axonemes are unequal, and their anterior and posterior extremities do not end at the same level in the spermatozoon, which is also true for the digeneans Adlardia novaecaledoniae (as Siphoderina elongata) (Miller et al., 2009; Quilichini et al., 2009), Diplodiscus subclavatus (Bakhoum et al., 2011), Heterolebes maculosus (Quilichini et al., 2010b), and Anisocoelium capitellatum (Ternengo et al., 2009).

The posterior extremities of these axonemes are characterized by the presence of doublets of microtubules without arms. Such organization is observed in other digenean spermatozoa: Notocotylus neyrai (Ndiaye et al., 2003b), Poracanthium furcatum (Levron et al., 2004b), Crepidostomum metoecus (Quilichini et al., 2007d), Aponurus laguncula (Quilichini et al., 2010a), and Heterolebes maculosus (Quilichini et al., 2010b). Nevertheless, in several digenean spermatozoa this posterior extremity is characterized by the presence of the central core as in Nicolla wisniewskii (Quilichini et al., 2007c) and Anisocoelium capitellatum (Ternengo et al., 2009).

The four attachment zones, associated with these two axonemes, are considered as remnants of the fusion of the flagella and median cytoplasmic process, which occurs during spermiogenesis.

The cortical microtubules

The parallel organization of these elements is found in all digenean spermatozoa known to date except the Didymozoidae Didymozoon sp. (Justine and Mattei, 1983), which has no cortical microtubules. If we compare this parallel disposition to the principal groups of parasitic flatworms, we notice that it is also the case in the Monogenea (except in certain monopisthocotyleans; Justine et al., 1985; Justine, 1991b) and in the Eucestoda (except in the Cyclophyllidea and Tetrabothriidea, which have spiralled cortical microtubules; Levron et al., 2010).

The arrangement of cortical microtubules as two fields located between the two axonemes has also been observed in numerous digenean families--for example, Apocreadiidae (Kacem et al., 2010), Deropristidae (Foata et al., 2007), Lepocreadiidae (Ba et al., 2011), Notocotylidae (Ndiaye et al., 2003b), Troglotrematidae (Miguel et al., 2006), and Zoogonidae (Levron et al., 2004a). In contrast, only one field of cortical microtubules has been observed in the Faustulidae Pronoprymna ventricosa (Quilichini et al., 2007b) and the Lecithasteridae Aponurus laguncula (Quilichini et al., 2010a).

The mitochondria

The presence of mitochondria is always observed in digenean spermatozoa, but their numbers vary. Some species possess one mitochondrion (e.g., Haematoloechus medioplexus; Justine and Mattei, 1982), two mitochondria (e.g., Troglotrema acutum; Miguel et al., 2006), or three mitochondria (e.g., Anisocoelium capitellatum (Ternengo et al., 2009). This variability is also observed within the superfamily Lepocreadioidea (Table 1). Recently, an ultra-structural study of the spermatozoon of Holorchis micracanthum (Ba et al., 2011) has revealed the presence of a unique moniliform mitochondrion. The mitochondrion, observed in longitudinal sections, appears to form successive bulges connected by a fine mitochondrial cord, and extends almost the whole length of the gamete. In the past, the mitochondrial cord could have been misinterpreted as a second mitochondrion or a nuclear membrane.

Nevertheless, the presence of at least one mitochondrion confirms the homogeneity of the Digenea for this criterion and justifies interest in this element for the phylogeny of the Platyhelminthes. Indeed, the absence of a mitochondrion is considered a synapomorphy for the Eucestoda (Ehlers, 1985).

The external ornamentation

Quilichini et al. (2007b) distinguished two groups of digenean spermatozoa according to the location of the external ornamentation: a first group with the ornamentation in the proximal part, and a second group with the ornamentation in a more distal part of the spermatozoon. The spermatozoon of Gyliauchen sp. can be classed in the second group. A third group can be added, including the species for which the ornamentation is not described in the description of their spermatozoa (Fig. 33).
Table 1

Some spermatozoon ultrastructural characteristics in
the Lepocreadioidea

Families and     Ase    Eo  Sb  M  Pse   References
species of
Lepocreadioidea

Apocreadiidae    Eo-Cm  +   +   2  Ax-N  Kacem et
Neoapocreadium                           al., 2010
chabaudi
Deropristidae    Ax-Cm  +   -   2  Ax    Foata et
Deropristis                              al., 2007
inflate
Lepocreadiidae   Ax     +   -   1  Ax    Ba et al.,
Holorchis                                2011
micracanthum
Gyliauchenidae   Ax     +   +   1  Ax    Present
Gyliauchen sp.                           study

+/--, presence/absence of considered character;
Ase, anterior spermatozoon extremity; Ax, axoneme;
Cm, cortical microtubules; Eo, external ornamentation;
N, nucleus; M, number of mitochondria; Pse, posterior
spermatozoon extremity; Sb, spine-like body.


Within the Lepocreadioidea, the three other species studied until now possess this external ornamentation (Table 1). It is noticeable that Deropristis inflata (Foata et al., 2007) and Holorchis micracanthum (Ba et al., 2011) are also in the second group, but that Neoapocreadium chabaudi (Kacem et al., 2010) is in the first group. Molecular results indicated that apocreadiids may not be lepocreadioids, and the two families have been classified in two independent suborders (Olson et al., 2003); our spermatological result confirm this interpretation.

The spine-like bodies

These ultrastructural elements were described for the first time by Miguel et al. (2000). Subsequently, spine-like bodies have been observed in the spermatozoa of several digenean families: Allocreadiidae (Quilichini et al., 2007d), Apocreadiidae (Kacem et al., 2010), Cryptogonimidae (Quilichini et al., 2009; Ternengo et al., 2009), Diplodiscidae (Bakhoum et al., 2011), Fasciolidae (Ndiaye et al., 2003a, 2004), Gastrothylacidae (Seck et al., 2008a), Notocotylidae (Ndiaye et al., 2003b), Opecoelidae (Miguel et al., 2000; Levron et al., 2004b; Quilichini et al., 2007a, c), Opistholebetidae (Quilichini et al., 2010b), and Paramphistomidae (Seck et al., 2007, 2008b).

The number and the periodicity of these elements vary from one species to another. Indeed in some species only one element is observed in transverse section (e.g., Gyliauchen sp.), whereas in other species several elements can be observed (e.g., Neoapocreadium chabaudi) (Kacem et al., 2010).

[FIGURE 32 OMITTED]

The distance between two spine-like bodies measured in longitudinal section can vary between species: I Am in Opecoeloides furcatus (Miguel et al., 2000) and in Nicolla testiobliquum (Quilichini et al., 2007a), compared to 0.6 um in Nicolla wisniewskii (Quilichini et al., 2007c). This distance can also vary within the same species: 1 um to 1.8 um in Adlardia novaecaledoniae (Quilichini et al., 2009) or 0.15 to 0.45 um in the present study.

[FIGURE 33 OMITTED]

In the spermatozoon of Gyliauchen sp. the spine-like bodies are not associated with the external ornamentation. This organization has been found in only one species before: Neoapocreadium chabaudi (Kacem et al., 2010). It is notable that this is also a member of the Lepocreadioidea. However, these ultrastructural elements have not been observed in the other species of this superfamily studied to date-Deropristis inflata (Foata et al., 2007) and Holorchis micracanthum (BA et al., 2011) (Table 1).

The extremities of the spermatozoon (Table 1)

The thin undulated anterior cytoplasm expansion has already been observed in several digenean spermatozoa: Heterolebes maculosus (Quilichini et al., 2010b) and Nicolla wisniewskii (Quilichini et al., 2007c), and it is described as flexible and sharp in Dicrocoelium hospes (Agostini et al., 2005).

The sequence of the posterior extremity of the spermatozoon of Gyliauchen sp. corresponds to the type 3 posterior part of the digenean spermatozoan (Quilichini et al., 2010b).

Further studies might confirm the value of these structures for the understanding of phylogenetic relationships within this superfamily.

Acknowledgments

Gerard Mou-Tham, John Butcher, Aude Sigura, and Cyndie Dupoux participated in the fishing expedition and parasitological survey.

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Received 20 April 2011; accepted 9 August 2011.

* To whom correspondence should be addressed. E-mail: quilichini[AT]univ-corse.fr

Y. QUILICHINI (1)*, J. FOATA (1), J.-L. JUSTINE (2,3), R. A. BRAY (4), AND B. MARCHAND (1)

(1.) CNRS UMR 6134, University of Corsica, "Parasites and Mediterranean Ecosystems" Laboratory, BP 52, 20250 Corte, France;

(2.) UMR 7138 Systematique, Adaptation, Evolution, Museum National d'Histoire Naturelle, 57, rue Cuvier, 75231 Paris cedex 05, France;

(3.) Aquarium des Lagons, B.P. 8185, 98807 Noumea, New Caledonia; and

(4.) Department of Zoology, Natural History Museum, Cromwell Road, London SW7 5BD, UK
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Author:Quilichini, Y.; Foata, J.; Justine, J.-L.; Bray, R.A.; Marchand, B.
Publication:The Biological Bulletin
Article Type:Report
Geographic Code:4EUFR
Date:Oct 1, 2011
Words:4351
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