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Parasitology International j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p a r i n t
Ultrastructural study of the spermatozoon of Heterolebes maculosus (Digenea, Opistholebetidae), a parasite of the porcupinefish Diodon hystrix (Pisces, Teleostei) Y. Quilichini a,⁎, J. Foata a, J.L. Justine b,c, R.A. Bray d, 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 Aquarium des Lagons, B.P. 8185, 98807 Nouméa, Nouvelle-Calédonie, France d Department of Zoology, Natural History Museum, Cromwell Road, London SW7 5BD, UK b c
a r t i c l e
i n f o
Article history: Received 25 February 2010 Received in revised form 1 June 2010 Accepted 2 June 2010 Available online 9 June 2010 Keywords: Spermatozoon Heterolebes maculosus Opistholebetidae Digenea Ultrastructure TEM
a b s t r a c t This paper describes the ultrastructure of the mature spermatozoon of Heterolebes maculosus. It is the first study of this kind concerning the Opistholebetidae (Platyhelminthes, Digenea). The ultrastructural elements observed in the spermatozoon are: two axonemes with 9+“1” pattern of Trepaxonemata and their attachment zones, two mitochondria, a nucleus, cortical microtubules, external ornamentation of the plasma membrane and spine-like bodies. The number and the disposition of cortical microtubules, the organisation of 11 cortical microtubules disposed in semi-circle around the first mitochondrion in the external ornamentation region and the organisation of the posterior part of the spermatozoon are discussed. Three principal types of posterior part of digenean spermatozoa are proposed. The similarity between the spermatozoon of the Opistholebetidae H. maculosus and Opecoelidae enables us to confirm that these two families are closely related. © 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
2. Materials and methods
For 50 years, ultrastructural studies of platyhelminth spermatozoa in general and of digenean in particular have been published [1,2], and for the last 25 years or so the male reproductive system has been used as an indicator of the phylogeny of the Platyhelminthes [3–7]. In this study we describe the spermatozoon ultrastructure of Heterolebes maculosus, a parasite of Diodon hystrix (Pisces, Teleostei). It is the first study of this kind concerning an opistholebetid species. The Opistholebetidae is a small family of trematodes from the intestine of marine fishes which comprises only three genera: Opistholebes Nicoll, 1915, Maculifer Nicoll, 1915 and Heterolebes Ozaki, 1935 [8]. This family is generally considered to be closely related to the Opecoelidae Ozaki, 1925 because all the elements of opistholebetid morphology are found within the Opecoelidae [9]. This resemblance has been confirmed by molecular analysis [10,11]. We perform here a comparison of the ultrastructural elements and their organisation in the spermatozoon of H. maculosus with those of the other digeneans and more especially with those of the spermatozoa of five Opecoelidae studied up to now: Helicometra fasciata [12], Nicolla testiobliquum [13], Nicolla wisniewskii [14], Opecoeloides furcatus [15] and Poracanthium furcatum [16].
Specimens of H. maculosus Ozaki, 1935 were collected live from the intestine of naturally infected porcupinefish D. hystrix Linnaeus, 1758 (Pisces, Teleostei) caught off New Caledonia (Pacific Ocean). Voucher specimens are deposited under the numbers: MNHN JNC JNC2344 and BMNH 2009.12.22.1. The worms were removed from their hosts, fixed in cold (4 °C) 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer at pH 7.2, rinsed in 0.1 M sodium cacodylate buffer at pH 7.2, post-fixed in cold (4 °C) 1% osmium tetroxide in the same buffer for 1 h, dehydrated in ethanol and propylene oxide, embedded in Spurr [17] and polymerised at 60 °C for 24 h. Ultra-thin sections (60–90 nm) in the seminal vesicle were cut on an ultramicrotome (Power tome PC, RMC Boeckeler ®). The sections were placed on 300 and 200-mesh copper grids and stained with uranyl acetate and lead citrate [18]. Sections were examined on a Hitachi H-600 transmission electron microscope, operating at an accelerating voltage of 75 kV, in the “Service d'Étude et de Recherche en Microscopie Électronique” (Corte, France). 3. Results
⁎ Corresponding author. Tel.: +33 495 450 006; fax: +33 495 450 045. E-mail address:
[email protected] (Y. Quilichini). 1383-5769/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.parint.2010.06.002
The observation of a great numbers of sections of seminal vesicles of H. maculosus enabled us to reconstitute the ultrastructure of its
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spermatozoon. This reconstitution is based on the arrangement of ultrastructural elements observed in transverse sections: two axonemes, four attachment zones, cortical microtubules, a nucleus, two mitochondria, external ornamentation of the plasma membrane and spine-like bodies. Four regions have been defined according to the organisation of these elements from the anterior to the posterior extremity of the spermatozoon.
3.1. Region I Region I (Figs. 1a–g and 4I) corresponds to the anterior part of the spermatozoon. The anterior extremity of this region is characterised by the anterior extremity of the first axoneme constituted by a central core and doublets of microtubules without arms and some singlets of microtubules of the second axoneme. We noticed also a thin
Fig. 1. TEM photomicrographs of cross sections of region I (a–g) and of the region II (h–k) of the mature spermatozoon of Heterolebes maculosus. (a) Anterior extremity of region I showing the anterior extremity of the first axoneme, some singlets of microtubules of the second axoneme and the cytoplamic expansion. (b and c) Cytoplasmic expansion undulated. (d) Anterior part of region I showing the complete first axoneme, the anterior extremity of the second axoneme and the four attachment zones. (e) Middle part of region I showing the two axonemes completely formed with a central core, nine doublets of microtubules with arms and connection filaments and 4 cortical microtubules. (f) 7 cortical microtubules. (g) Posterior part of the region I showing two rows of 5 and 8 cortical microtubules. (h) Anterior part of the region II showing the first mitochondrion, the external ornamentation of the plasma membrane and 13 cortical microtubules. (i) Observation of a spine-like body associated with the external ornamentation observed in longitudinal section. (j) Ventral side (mitochondrial side) with 11 cortical microtubules disposed in semi-circle around the first mitochondrion and associated with external ornamentation of the plasma membrane and dorsal side with a row of 3 cortical microtubules. (h) Posterior part of the region II showing 15 cortical microtubules and the presence of the four attachment zones. Arrowheads indicate attachment zones; Ar, arms of microtubule doublet; Ax1, axoneme 1; Ax2, axoneme 2; Cc, central core; Ce, cytoplasmic expansion; Cf, connection filament between doublet of microtubules and central core; Cm, cortical microtubule; D, dorsal side; Dm, doublet of microtubules; Eo, external ornamentation; M1, mitochondrion 1; Sb, spine-like body; Sm, singlet of microtubule; V, ventral side. (Bar = 0.2 μm).
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cytoplasmic expansion undulated and mobile in the extremity of the spermatozoon (Fig. 1a–c). The shift of the second axoneme is also observed in the Fig. 1d. Indeed, the first axoneme is completely formed with the nine doublets of microtubule with arms, a central core and filaments connecting the peripheral doublets with the central core whereas the second axoneme presents only nine doublets of microtubules and a central core. Four attachment zones which appear as electron-dense zones are observed between both axonemes. On Fig. 1e the two axonemes are completely formed. The attachment zones are present and four cortical microtubules are counted. The number of these elements increases to the posterior end of this region: 4 (Fig. 1e), 7 (Fig. 1f) and 13 (Fig. 1g). 3.2. Region II In region II (Figs. 1h–k and 4II) the two axonemes and their four attachment zones are still present. The characteristics of this region are the presence of the first mitochondrion (Fig. 1h–k), the external ornamentation of the plasma membrane (Fig. 1h–k) and spine-like bodies (Fig. 1i). The presence and the disposition of the mitochondrion enabled us to distinguish the ventral side (mitochondrial side) of the spermatozoon (Fig. 1j). The number of cortical microtubules in this region increases from the anterior part to the posterior part: 13 (Fig. 1h), 14 (Fig. 1j) and 15
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(Fig. 1k). This number increases only in the dorsal side (from 2 to 4) of the spermatozoon and is constant (11) in the ventral side. Moreover the 11 cortical microtubules observed in the ventral side are arranged as a semi-circle around the mitochondrion (Fig. 1h,j,k). The external ornamentation of the plasma membrane is only observed on the ventral side, associated with the 11 cortical microtubules (Fig. 1h,j,k). The spine-like bodies are associated with the external ornamentation (Fig. 1i). 3.3. Region III In region III (Figs. 2 and 4III) the two axonemes and their four attachment zones are present (excepted in the posterior part of this region). The anterior part of this region is characterised by the posterior extremity of the first mitochondrion (Fig. 1a) and the absence of external ornamentation of the plasma membrane and of the spinelike bodies. The microtubules are arranged as two fields located between the two axonemes (Fig. 2a–g) except in the posterior part of this region where they are disposed around the two axonemes (Fig. 2h,i). When two fields are present, their number of cortical microtubules is unequal. The number of cortical microtubules increases (the number of cortical microtubules by field is indicated between brackets) in the
Fig. 2. TEM micrographs of cross sections of region III of the mature spermatozoon of Heterolebes maculosus. (a) Anterior part of region III showing the posterior extremity of the first mitochondrion and 17 cortical microtubules disposed in two fields of 3 and 14. (b) 18 cortical microtubules disposed in two fields of 3 and 15. (c) 19 cortical microtubules disposed in two fields of 3 and 16. (d) 23 cortical microtubules disposed in two fields of 4 and 19. (e) 20 cortical microtubules disposed in two fields of 4 and 20. (f) 25 cortical microtubules disposed in two fields of 5 and 20. (g) 27 cortical microtubules disposed in two fields of 5 and 22. (h) 28 cortical microtubules (maximal number counted in this region and in the entire spermatozoon) disposed around the two axonemes. (i) Posterior extremity of region III showing 24 cortical microtubules disposed around the two axonemes. Arrowheads indicate attachment zones; Cm, cortical microtubule; F1, field 1 of cortical microtubules; F2, field 2 of cortical microtubules; M1, mitochondrion 1. (Bar = 0.2 μm).
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anterior and middle part of this region: 17 (3 + 14) (Fig. 2a), 18 (3 + 15) (Fig. 2b), 19 (3 + 16) (Fig. 2c), 23 (4 + 19) (Fig. 2d), 24 (4 + 20) (Fig. 2e), 25 (5 + 20) (Fig. 2f), 27 (5 + 22) (Fig. 2g), 28 (Fig. 2h); and decreases in the posterior end of this region: 24 (Fig. 2i). The maximal number of cortical microtubules observed in this region (28) is also the maximal number counted in the transverse sections of the entire spermatozoon. The attachment zones are not observed in the posterior part of this region (Fig. 2h,i).
and the posterior extremity of this mitochondrion is associated with 15 cortical microtubules (Fig. 3e). Fig. 3f–i presents 14 cortical microtubules and show the posterior extremity of the first axoneme (Fig. 3f), of the second axoneme (Fig. 3g) and of the nucleus (Fig. 3h). The posterior extremities of the second mitochondrion and of the first axoneme are almost at the same level. The attachment zones remain present after the posterior end of the axonemes (Fig. 3i). The posterior extremity of the spermatozoon is characterised by only some cortical microtubules (Fig. 3j and k).
3.4. Region IV
4. Discussion
Region IV (Figs. 3 and 4IV) corresponds to the posterior part of the spermatozoon. The anterior part of this region is characterised by the presence of the two axonemes, their attachment zones and two fields of cortical microtubules located between the two axonemes (Fig. 3a–c). The number of cortical microtubules decreases progressively: 23 (Fig. 3a), 21 (Fig. 3b), 20 (Fig. 3c), 18 (Fig. 3d), 15 (Fig. 3e), 14 (Fig. 3f–i), 9 (Fig. 3j) and 3 (Fig. 3k). The anterior extremity of the second mitochondrion and of the nucleus is associated with the presence of 18 microtubules (Fig. 3d)
In this study we reconstitute the ultrastructure of the spermatozoon of Heterolebes maculosus. It is the first study of this kind concerning a digenean of the family Opistholebetidae. 4.1. The axonemes The spermatozoon of H. maculosus possesses two axonemes with a 9+“1” pattern of Trepaxonemata [3]. This structure is that always found in the digenean spermatozoa except in Didymozoon sp. [19,20]; this difference could be explained by the atypical biology of the
Fig. 3. TEM micrographs of cross sections of region IV of the mature spermatozoon of Heterolebes maculosus. (a) Anterior part of region IV showing 23 cortical microtubules disposed in two fields between the two axonemes. (b) 21 cortical microtubules disposed in two fields. (c) 20 cortical microtubules disposed in two fields. (d) Anterior extremity of the nucleus and of the second mitochondrion associated with 18 cortical microtubules. (e) Posterior extremity of the second mitochondrion associated with 15 cortical microtubules. (f) Posterior extremity of the first axoneme associated with 14 cortical microtubules. (g) Posterior extremity of the second axoneme associated with 14 cortical microtubules. (h) Posterior extremity of the nucleus associated with 14 cortical microtubules. (i) 14 cortical microtubules and attachment zones. (j) 9 cortical microtubules. (k) Posterior extremity of the spermatozoon showing some cortical microtubules. Arrowheads indicate attachment zones; Ax1, axoneme 1; Ax2, axoneme 2; Cm, cortical microtubules; F1, field 1 of cortical microtubules; F2, field 2 of cortical micrtotubules; M2, mitochondrion 2; N, nucleus. (Bar = 0.2 μm).
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Fig. 4. Diagram showing the ultrastructural organisation of the mature spermatozoon of Heterolebes maculosus. (I) Region I. (II) Region II. (III) Region III. (IV) Region IV. Aae1, anterior axoneme extremity 1; Aae2, anterior axoneme extremity 2; Ase, anterior spermatozoon extremity; Ax1, axoneme 1; Ax2, axoneme 2; Az, attachment zone; Cm, cortical microtubule; D, dorsal side; Eo, external ornamentation; M1, mitochondrion 1; M2, mitochondrion 2; N, nucleus; Pae1, posterior axoneme extremity 1; Pae2, posterior axoneme extremity 2; Pm, plasma membrane; Pse, posterior spermatozoon extremity; Sb, spine-like body; V, ventral side.
didymozoids [21,22]. If it is difficult to establish the length of the two axonemes, we can nevertheless detect a small shift between them, characterised by a different location of their anterior and posterior extremities. This shift has been observed in numerous digenean spermatozoa.
Moreover the extremities of the axonemes present nine peripheral doublets of microtubules devoid of arms and do not possess filaments connecting these doublets with the central core. The four attachment zones associated with the two axonemes are considered as the fusion lines of the flagella and the median cytoplasmic
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process which occurs during the spermiogenesis. These structures are along the entire spermatozoon excepted in the anterior and posterior extremity of the spermatozoon and in the posterior part of the region III which correspond to the disposition of the cortical microtubules around the two axonemes. This discontinuity cannot be explained but we notice that the attachment zones observed during the proximo-distal fusion are not necessarily observed in the spermatozoon of several monogenea (e.g. [23]). 4.2. The cortical microtubules The number and arrangement of the cortical microtubules have been the subject of detailed attention in this study. First, the number of cortical microtubules increases from the anterior extremity of the spermatozoon to the posterior part of the region III and then decreases to the extremity of the spermatozoon. This organisation has been found in the spermatozoa of the Opecoelidae studied so far: Helicometra fasciata [12], Nicolla testiobliquum [13], Nicolla wisniewskii [14], Opecoeloides furcatus [15], Poracanthium furcatum [16] and in the Cryptogonimidae Anisocoelium capitellatum [24], Adlardia novaecaledoniae (as Siphoderina elongata) [25] [26] and Neochasmus sp. [27] whereas in the Allocreadiidae Crepidostomum metoecus [28], Deropristidae Deropristis inflata [29], Gastrothylacidae Carmyerius endopapillatus [30] and the Notocotylidae Notocotylus neyrai [31] the number of cortical microtubules decreases from the anterior extremity to the posterior extremity of the spermatozoon. Secondly, the two fields of cortical microtubules located between the two axonemes present in the spermatozoon of H. maculosus have been also observed in the Opecoelidae (listed above) and in other digenean families (e.g. Heterophyidae [32], Lecithodendriidae [33],
and Troglotrematidae [34]) whereas only one field of cortical microtubules has been observed in the Faustulidae Pronoprymna ventricosa [35] and in the Lecithasteridae Aponurus laguncula [36], and no cortical microtubule has been observed in the Didymozoidae Didymozoon sp. [19,20]. The disposition and the number of cortical microtubules have been demonstrated to be highly useful for our understanding of the phylogeny of parasitic Platyhelminthes [5,37], and especially monogeneans [38,39] and cestodes [40]; they could certainly be of interest for the phylogeny of the Digenea. 4.3. The external ornamentation region The external ornamentation of the plasma membrane is located in region II of the spermatozoon of H. maculosus. This ornamentation is located on the ventral side of the spermatozoon and is associated with 11 cortical microtubules disposed in semi-circle around the mitochondrion. Such organisation has been described in opecoelids and cryptogonimids and compared by Quilichini et al. [25]. Concerning this region, the difference between the spermatozoa of these species is the number of cortical microtubules disposed in semi-circle: 8 in Neochasmus sp. (Cryptogonimidae) [27], 11 in Adlardia novaecaledoniae (Cryptogonimidae) [25] and in H. maculosus (Opistholebetidae) (present study), 12 in Opecoeloides furcatus (Opecoelidae) [15] and in Poracanthium furcatum (Opecoelidae) [16], 15 in Nicolla testiobliquum (Opecoelidae) [13] and Nicolla wisniewskii (Opecoelidae) [14]. The only spermatozoon of Opecoelidae which does not possess this organisation is Helicometra fasciata [12]. The spine-like bodies, described for the first time by Miquel et al. [15], have been found since in several families of digeneans: Allocreadidae
Fig. 5. Diagram showing the three principal types of the spermatozoa posterior part of digenea. (a) Type 1: Opecoeloidean type found in Opecoelidae (Helicometra fasciata, Nicolla testiobliquum, Nicolla wisniewskii, Opecoeloides furcatus and Poracanthium furcatum) and in Opistholebetidae (Heterolebes maculosus). (b) Type 2: Fasciolidean type found in Dicrocoelidae (Dicrocoelium hospes), in Fasciolidae (Fasciola gigantica and Fasciola Hepatica), in Gastrothylacidae (Carmyerius endopapillatus) and in Zoogonidae (Diphterostomum brusinae). (c) Type 3: Cryptogonomidean type found in Allocreadiidae (Crepidostomum metoecus), in Cryptogonimidae (Adlardia novaecaledoniae, Anisocoelium capitellatum and Neochasmus sp.), in Faustulidae (Pronoprymna ventricosa), in Lecithodendriidae (Postorchigenes gymnesicus), in Notocotylidae (Notocotylus neyrai), and in Troglotrematidae (Troglotrema acutum). Cm, cortical microtubule; N, nucleus; Pae, posterior axoneme extremity; Pse, posterior spermatozoon extremity.
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[28], Cryptogonimidae [24,25], Dicrocoeliidae [40], Echinostomatidae [42], Fasciolidae [43,44], Gastrothylacidae [30], Notocotylidae [31], Opecoelidae [13–16], Paragonimidae [45], Paramphistomidae [46,47], Troglotrematidae [34]. These elements are not easily quantifiable and we notice that the spermatozoon of Adlardia novaecaledoniae [25] presents several spine-like bodies on the same transverse sections. In all the cases these elements are associated with the external ornamentation.
4.4. The posterior part of the spermatozoon The posterior part of the spermatozoon of H. maculosus is characterised by the succession: posterior extremity of the second axoneme, posterior extremity of the nucleus and cortical microtubules in the posterior extremity. Fig. 5 presents a comparison of the principal sequences of the posterior parts found in the digenean spermatozoa. Among the 12 families and 19 species considered (Table 1), we distinguish three types of spermatozoa posterior parts: – Type 1, or opecoelidean type (Fig. 5a and Table 1), characterised by the sequence: posterior axoneme extremity, posterior nucleus extremity and cortical microtubules. – Type 2, or fasciolidean type (Fig. 5b and Table 1), characterised by the absence of cortical microtubules, and the sequence posterior extremity of the second axoneme then the posterior extremity of the nucleus. – Type 3, or cryptogonimidean type (Fig. 5c and Table 1), characterised by the absence of cortical microtubules, and the sequence posterior extremity of the nucleus then the posterior extremity of the second axoneme.
Table 1 3 types of spermatozoa posterior parts of digenean. Families and digenean species Allocreadiidae Crepidostomum metoecus Cryptogonimidae Adlardia novaecaledoniae Anisocoelium capitellatum Neochasmus sp. Dicrocoeliidae Dicrocoelium hospes Fasciolidae Fasciola gigantica Fasciola hepatica Faustulidae Pronoprymna ventricosa Gastrothylacidae Carmyerius endopapillatus Lecithodendriidae Postorchigenes gymnesicus Notocotylidae Notocotylus neyrai Opistholebetidae Heterolebes maculosus Opecoelidae Helicometra fasciata Nicolla testiobliquum Nicolla wisniewskii Opecoeloides furcatus Poracanthium furcatum Troglotrematidae Troglotrema acutum Zoogonidae Diphterostomum brusinae
Type 1
Type 2
Type 3
References
x
[28]
x x x
[25] [24] [27]
x
[41]
x x
[44] [43] x
x
[35] [30]
x
[33]
x
[31]
x
Present study
x x x x x
[12] [13] [14] [15] [16] x x
[34] [48]
Types 1, 2 and 3 correspond to the sequence of spermatozoa posterior parts of digenean. Type1: posterior axoneme extremity, posterior nucleus extremity and cortical microtubules; type 2: posterior extremity of the second axoneme and posterior extremity of the nucleus (without cortical microtubules); type 3: posterior extremity of the nucleus and posterior extremity of the second axoneme (without cortical microtubules).
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In Table 1 we notice that all the Opecoelidae studied until now and the Opistholebetidae Heterolebes maculosus possess the same type (1) of spermatozoon posterior part. This homogeneity is also found within the Fasciolidae (type 2) and within the Cryptogonomidae (type 3). These observations enable us to consider this criterion as potentially interesting for phylogeny and to confirm that the Opecoelidae and the Opistholebetidae are closely related. Some recent references have not been included in this comparative study because the description of the posterior part of the spermatozoon was not sufficient to distinguish the last element of the spermatozoon. It is the case of the Paramphistomidae Cotylophoron cotylophorum [47] and Paramphistomum microbothrium [46] which could correspond to type 1, or the Monorchidae Monorchis parvus [49] which could correspond to type 3. Finally a fourth group, represented by the Deropristidae Deropristis inflata [29] (and maybe the Brachylaimidae Scaphiostomum palaearticum [50] and the Lecithasteridae Aponurus laguncula [36]), could be characterized by the sequence: posterior extremity of the first axoneme, posterior extremity of cortical microtubules and posterior extremity of the second axoneme. More investigations will enable us to confirm if it is a real group or only an exception. 5. Conclusion The ultrastructure of the spermatozoon of H. maculosus has revealed the presence of several elements found in other digenean spermatozoa: two axonemes of 9+“1” pattern of Trepaxonemata and their attachment zones, two mitochondria, a nucleus, cortical microtubules, external ornamentations of the plasma membrane and spine-like bodies. However, the organisation of these elements, like the number and the arrangement of cortical microtubules, the organisation of the external ornamentation region and the type of the posterior part of the spermatozoon, has revealed a similar structure of the spermatozoon of the Opistholebetidae H. maculosus with that of members of the Opecoelidae. The conclusion of this ultrastructural study is the same as that obtained by morphological [9] or molecular approaches [10,11] and confirms that the Opistholebetidae is closely related to the Opecoelidae [8,51] or should be included within the Opecoelidae [10,52]. References [1] Gresson RAR, Perry MM. Electron microscope studies of spermateleosis in Fasciola hepatica L. Exp Cell Res 1961;22:1–8. [2] Burton PR. Fine structure of the unique central region of the axial unit of lung-fluke spermatozoa. J Ultrastruct Res 1967;19:166–72. [3] Ehlers U. Phylogenetisches system der Plathelminthes, Vol. 27. Verhandlungen des Naturwissenschaftlichen Verein in Hamburg (NF); 1984. p. 291–4. [4] Ehlers U. Comments on a phylogenetic system of the Platyhelminthes. Hydrobiologia 1986;132:1–12. [5] Justine JL. Phylogeny of parasitic Platyhelminthes: a critical study of synapomorphies proposed on the basis of the ultrastructure of spermiogenesis and spermatozoa. Can J Zool 1991;69:1421–40. [6] Bâ CT, Marchand B. Spermiogenesis, spermatozoa and phyletic affinities in the Cestoda. Mém Mus Natn Hist Nat 1995;166:87–95. [7] Justine JL. La classification générale des Plathelminthes parasites: changements récents et utilisation des caractères ultrastructuraux, en particulier des spermatozoïdes. Bull Soc Zool France 1997;122:269–77. [8] Cribb TH. Family Opistholebetidae Fukui. In: Jones A, Bray RA, Gibson DI, editors. Key to the Trematoda. Volume 2, 2005. Wallingford: CABI Publishing and the Natural History Museum; 1929. p. 533–9. [9] Cribb TH. Family Opecoelidae Ozaki. In: Jones A, Bray RA, Gibson DI, editors. Keys to the Trematoda. Volume 2, 2005. Wallingford: CABI Publishing and the Natural History Museum; 1925. p. 443–531. [10] Olson PD, Cribb TH, Tkach VV, Bray RA, Littlewood DTJ. Phylogeny and classification of the Digenea (Platyhelminthes: Trematoda). Int J Parasitol 2003;33:733–55. [11] Olson PD, Tkach VV. Advances and trends in the molecular systematics of the parasitic platyhelminthes. Adv Parasitol 2005;60:165–243. [12] Levron C, Ternengo S, Marchand B. Ultrastructure of spermiogenesis and the spermatozoon of Helicometra fasciata (Digenea, Opecoelidae), a parasite of Labrus merula (Pisces, Teleostei). Acta Parasitol 2003;48:255–64. [13] Quilichini Y, Foata J, Marchand B. Ultrastructural study of the spermatozoon of Nicolla testiobliquum (Digenea, Opecoelidae) parasite of brown trout Salmo trutta (Pisces, Teleostei). Parasitol Res 2007;101:1295–301.
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