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Spermatozoon ultrastructure of Aponurus laguncula (Digenea: Lecithasteridae), a parasite of Aluterus monoceros (Pisces: Teleostei) ARTICLE in PARASITOLOGY INTERNATIONAL · JULY 2009 Impact Factor: 1.86 · DOI: 10.1016/j.parint.2009.06.007 · Source: PubMed
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Spermatozoon ultrastructure of Aponurus laguncula (Digenea: Lecithasteridae), a parasite of Aluterus monoceros (Pisces: Teleostei) Y. Quilichini a,⁎, J. Foata 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 Équipe Biogéographie Marine Tropicale, Unité Systématique, Adaptation, Évolution (CNRS, UPMC, MNHN, IRD), Institut de Recherche pour le Développement, BP A5, 98848 Nouméa Cedex, Nouvelle-Calédonie, 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 23 February 2009 Received in revised form 15 June 2009 Accepted 16 June 2009 Available online 24 June 2009 Keywords: Spermatozoon Aponurus laguncula Lecithasteridae Digenea Ultrastructure TEM
a b s t r a c t The mature spermatozoon of Aponurus laguncula, a parasite of the unicorn leatherjacket Aluterus monoceros, was studied by transmission electron microscopy. The spermatozoon possesses 2 axonemes of the 9 + “1” trepaxonematan pattern, attachment zones, a nucleus, a mitochondrion, external ornamentation of the plasma membrane and cortical microtubules. The major features are the presence of: 1) external ornamentation in the anterior part of the spermatozoon not associated with cortical microtubules; 2) one mitochondrion; and 3) cortical microtubules arranged as a single field in the ventral side. The maximum number of microtubules is in the nuclear region. The extremities of the axonemes are characterized by the disappearance of the central core and the presence of microtubule doublets or singlets. This study is the first undertaken with a member of the Lecithasteridae and exemplifies the sperm ultrastructure for the superfamily Hemiuroidea. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Ultrastructural features of male gametes are valuable tools in the analysis of Platyhelminthes relationships in general [1–3] and especially for the parasitic Platyhelminthes [4–6]. Although significant differences have been found, such as the presence of one or two axonemes in monogeneans [7] and cestodes [8] and the lack of mitochondria in the Eucestoda [5], the spermatozoon ultrastructure of digeneans appears relatively homogeneous. Nevertheless, recent ultrastructural studies of spermiogenesis and the spermatozoon have highlighted some potential phylogenetic criteria, e.g. the arrangement and number of cortical microtubules [9] and the location of the external ornamentation of the plasma membrane [10]. However, so many digenean families have not been studied that conclusions on the importance of these criteria cannot yet be drawn. Indeed, only about forty species have been studied, whereas the Digenea comprise 25 superfamilies, 148 families, nearly 2800 nominal genera and about 18,000 nominal species [11]. The present study describes for the first time the spermatozoon of a lecithasterid fluke, Aponurus laguncula, a parasite of the unicorn leatherjacket Aluterus monoceros. Moreover, among the 13 families [12], including the lecithasterids, which constitute the superfamily
⁎ Corresponding author. Tel.: +33 495 450 006; fax: +33 495 450 045. E-mail address:
[email protected] (Y. Quilichini). 1383-5769/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.parint.2009.06.007
Hemiuroidea Looss, 1899, spermatozoon ultrastructure was only known in one family, the Didymozoidae [13–16]. The aim of this study is to compare the ultrastructure of the spermatozoon of A. laguncula to that of other digeneans in order to highlight criteria possibly useful for phylogeny. 2. Materials and methods Seven specimens of A. laguncula Looss, 1907 were collected live from the stomach and intestine of a freshly killed unicorn leatherjacket A. monoceros (Linnaeus, 1758) (Monacanthidae), caught off Nouméa, New Caledonia, South Pacific (22°21′S, 166°15′E, 1 August 2007). For systematics, five specimens were fixed by being pipetted into nearly boiling saline and immediately preserved in formalin or 70% ethanol, and whole-mounts were stained with Mayer's paracarmine, cleared in beechwood creosote and mounted in Canada balsam. The identification of these specimens as A. laguncula is based mostly on the observations of Machida and Kuramochi [17] who reported the species from A. monoceros from Japanese waters and synonymised some of the Aponurus species reported from Tetraodontiformes. Voucher specimens were identified and are deposited in the collections of the Muséum National d'Histoire Naturelle (Paris) as MNHN JNC2262 and in the British Museum (Natural History) Collection at the Natural History Museum, London, UK (BMNH 2009.2.19.1-2.). For ultrastructural study, two specimens were fixed in cold (4 °C) 2.5% glutaraldehyde in a 0.1 M sodium cacodylate buffer at pH 7.2, rinsed in 0.1 M sodium cacodylate buffer at pH 7.2, post
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fixed in cold (4 °C) 1% osmium tetroxide in the same buffer for 1 h, dehydrated in ethanol and propylene oxide, embedded in Spurr's resin [18], and polymerized at 60 °C for 48 h. Ultrathin sections (60–90 nm) in seminal vesicle were cut on an ultramicrotome (Power tome PC, RMC Boeckeler®). The sections were placed on 300-mesh copper grid and stained with uranyl acetate and lead citrate according to Reynolds [19]. Sections were examined on a Hitachi H-600 transmission electron microscope, operating at an accelerating voltage of 75 kV. 3. Results Observation of numerous sections in the seminal vesicle of A. laguncula enabled us to reconstitute the ultrastructural organization of the spermatozoon. We noticed the presence of two axonemes, attachment zones, a mitochondrion, a nucleus, external ornamentation of the plasma membrane, cortical microtubules and the absence of spine-like bodies. Three regions were recognized from the anterior to the posterior extremity of the spermatozoon. 3.1. Region I Region I (Figs. 1 and 4) is the anterior region of the spermatozoon. The anterior part of this region is characterized by a complete axoneme, some microtubules and an external ornamentation of the plasma membrane. The complete axoneme is defined by the presence of nine microtubule doublets associated with a central core (Fig. 1A). The microtubules observed in this zone are organized in singlets and represent the anterior extremity of the second axoneme (Fig. 1A). The external ornamentation is only present around the first axoneme (Fig. 1A). In the middle part of this region the second axoneme is present and characterized by a central core and microtubules organized in singlets or in doublets (Fig. 1B). In the posterior part of this region, the two axonemes are complete (9 microtubule doublets and a central core) and the external ornamentation is present on most of the periphery of the plasma membrane except on a small part between the two axonemes (Fig. 1C). 3.2. Region II Region II (Figs. 2 and 4) is the middle region of the spermatozoon. The external ornamentation of the plasma membrane has not been observed in this zone. The anterior part of this region is characterized by only two axonemes and their four attachment zones (Fig. 2A). These attachment zones are small electron-dense zones on both sides of each axoneme. Posteriorly we noticed an increase of
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the number of cortical microtubules: 1 (Fig. 2B), 2 (Fig. 2C), 3 (Fig. 2D). The anterior extremity of the mitochondrion is associated with the presence of 3 cortical microtubules (Fig. 2E). The middle part of this region is characterized by the presence of 4 cortical microtubules associated with the anterior extremity of the nucleus (Fig. 2F). The simultaneous presence of the mitochondrion and of the nucleus allows the discrimination of the ventral (mitochondrial) and dorsal (nuclear) sides (Fig. 2G). The cortical microtubules are organized in a single field arranged along the plasma membrane of the ventral side. The number of cortical microtubules continues to increase in the posterior part of this region: 6 (Fig. 2H), 7 (Fig. 2I), 8 (Fig. 2J) and 10 (Fig. 2K). The ten cortical microtubules counted in this zone constitute the maximum number counted in the entire spermatozoon. 3.3. Region III This is the posterior region of the spermatozoon (Figs. 3 and 4). In the anterior part of this region the two axonemes, their attachment zones, the nucleus, the mitochondrion and cortical microtubules are present (Fig. 3A). The number of cortical microtubules progressively decreases: 9 (Fig. 3A), 8 (Fig. 3B), 7 (Fig. 3C). Observation of cross-sections showing 7 cortical microtubules with a nucleus (Fig. 3C) and without a nucleus (Fig. 3D) enabled us to associate the posterior extremity of the nucleus with 7 cortical microtubules. Posteriorly, the number of cortical microtubules continues to decrease: 7 (Fig. 3D), 6 (Fig. 3E), 5 (Fig. 3F), 4 (Fig. 3G), 3 (Fig. 3H), 2 (Fig. 3I), 0 (Fig. 3J). In the middle part of this region there are two complete axonemes and the mitochondrion (Fig. 3J). The posterior part of the axonemes is characterized by the disappearance of the central core and the disorganisation of the microtubule doublets (Fig. 3K). The posterior extremities of the central cores of the two axonemes do not occur at the same level (Fig. 3K and L). The posterior extremity of the mitochondrion is associated on both sides with the posterior extremity of the two axonemes characterized by 9 microtubule doublets (Fig. 3M). The posterior extremity of the spermatozoon is characterized by the distal end of the axonemes with microtubule doublets and singlets (Fig. 3N) and finally by only a few singlets (Fig. 3O). 4. Discussion 4.1. The axonemes The mature spermatozoon of A. laguncula possesses two axonemes with 9 + “1” pattern of trepaxonematan Platyhelminthes. This structure
Fig. 1. TEM photomicrographs of cross-sections of region I of the mature spermatozoon of Aponurus laguncula. (A) Anterior extremity of region I showing the central core and the microtubule doublets of the first axoneme surrounded by the external ornamentation of the plasma membrane and the microtubule singlets of the second axoneme. (B) Middle part of the region I showing the complete axoneme 1 and the incomplete axoneme 2 characterized by microtubule singlets and doublets and a central core. (C) Posterior part of region I showing the two complete axonemes surrounded by the external ornamentation. Ax1, axoneme 1; Ax2, axoneme 2; Cc, central core; Dm, microtubule doublet; Eo, external ornamentation; Sm, microtubule singlet. (Bar = 0.2 μm).
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Fig. 2. TEM photomicrographs of cross-sections of region II of the mature spermatozoon of Aponurus laguncula. (A) Anterior part of region II showing the two axonemes and their four attachment zones. (B) 1 cortical microtubule. (C) 2 cortical microtubules. (D) 3 cortical microtubules. (E) Anterior extremity of the mitochondrion associated with 3 cortical microtubules. (F) Middle part of region II showing the anterior part of the nucleus associated with 4 cortical microtubules. (G) Ventral (mitochondrial) side and dorsal (nuclear) side of the spermatozoon. (H) 6 cortical microtubules. (I) 7 cortical microtubules. (J) 8 cortical microtubules. (K) 10 cortical microtubules. Arrowheads indicate attachment zones; Ax1, axoneme 1; Ax2, axoneme 2; Cm, cortical microtubule; Ds, dorsal side; M, mitochondrion; N, nucleus; Vs, ventral side. (Bar = 0.2 μm). Fig. 3. TEM photomicrographs of cross-sections of region III of the mature spermatozoon of Aponurus laguncula. (A) Anterior part of region III showing the mitochondrion, the nucleus, 9 cortical microtubules, the two axonemes and their attachment zones. (B) 8 cortical microtubules. (C) Posterior extremity of the nucleus associated with 7 cortical microtubules. (D) 7 cortical microtubules without nucleus. (E) 6 cortical microtubules. (F) 5 cortical microtubules. (G) 4 cortical microtubules. (H) 3 cortical microtubules. (I) 2 cortical microtubules. (J) Two complete axonemes and the mitochondrion without cortical microtubules. (K) Posterior extremity of the first axoneme characterized by 9 microtubule doublets. (L) Disorganisation of the second axoneme. (M) Posterior extremity of the mitochondrion with 9 microtubule doublets in both sides. (N) Only microtubule doublets and singlets. (O) Posterior extremity of the spermatozoon showing a few microtubule singlets. Arrowheads indicate attachment zones; Ax1, axoneme 1; Ax2, axoneme 2; Cm, cortical microtubule; Dm, microtubule doublet; M, mitochondrion; N, nucleus; Sm, microtubule singlet. (Bar = 0.2 μm).
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ventricosa [10], Troglotrema acutum [25] but not in Nicolla wisniewskii [26] which possesses two axonemes characterized by the central core at their extremities. The attachment zones observed in both sides of each axoneme correspond to the fusion lines of flagella with the median cytoplasmic expansion during the spermiogenesis process. These elements have been observed in numerous digeneans: Helicometra fasciata [27], Diphterostomum brusinae [28], Poracanthium furcatum [29] and T. acutum [25]. The absence of attachment zones in the region I enabled us to confirm the orientation of the spermatozoon. Indeed during the spermiogenesis process the fusion of the flagella is proximo-distal and the fusion does not occur in proximal area of spermatid. Thus, in the anterior extremity of the spermatozoon the attachment zones are absent. 4.2. The external ornamentation of the plasma membrane
Fig. 4. Diagram showing the ultrastructural organisation of the mature spermatozoon of Aponurus laguncula. Ase, anterior spermatozoon extremity; Ax1, axoneme 1; Ax2, axoneme 2; Az, attachment zone; Cc, central core; Cm, cortical microtubule; Dm, microtubule doublet; Ds, dorsal side; Eo, external ornamentation; M, mitochondrion; N, nucleus; Pm, plasma membrane; Pse, posterior spermatozoon extremity; Sm, microtubule singlet; Vs, ventral side.
has been described in the spermatozoon of digenean species and is constituted by nine radial spokes, which join the central core to the outer microtubule doublets [20–22]. Within the Platyhelminthes, this structure is considered as a synapomorphy of the Trepaxonemata [1] and thus is found in the principal groups of parasitic Platyhelminthes, i.e. Cestoda, Monogenea and Trematoda. The structure of the axonemes of A. laguncula, observed in crosssections, confirms the homogeneity of this criterion in the parasitic Platyhelminthes in general and the Digenea in particular. In the spermatozoon of A. laguncula the anterior and posterior axoneme extremities are characterized by a few microtubule singlets. The central core is present only when the microtubules are organized in doublets. This is the case observed in most digenean spermatozoa: Crepidostomum metoecus [23], Deropristis inflata [24], Pronoprymna
The role of this element is unknown. Justine and Mattei [30] tentatively proposed that they play a role the fusion of the spermatozoon and ovum membranes at fertilization. The external ornamentation of the plasma membrane has been observed in the anterior part of the spermatozoon (region I). Quilichini et al. [10] distinguished two groups of digenean spermatozoa according to its location: a first group with the ornamentation in the proximal part and a second group with the ornamentation in the distal part of the spermatozoon. The spermatozoon of A. laguncula thus belongs in the first group. A particularity of the spermatozoon of A. laguncula is the absence of cortical microtubules associated with the ornamentation. Hitherto, this organisation has only been observed in one digenean species, P. ventricosa [10]. Spine-like bodies have not been observed on the plasma membrane of the spermatozoon of A. laguncula. These elements, described as triangular prominences containing an electron-dense spherical vesicle [31], are always associated with the ornamentation, e.g.: Opecoeloides furcatus [31], Notocotylus neyrai [32], Fasciola gigantica [33], Nicolla testiobliquum [9] and Carmyerius endopapillatus [34]. The external ornamentation of the plasma membrane has also been observed in several monogeneans such as the monopisthocotyleans Calicotyle kroyeri [35], Cleitharticus sp. [36], Heterocotyle sp. [36,37], Loimosina wilsoni [38], Neoheterocotyle rhinobatidis [39], Pseudodactylogyrus bini [40], Troglocephalus rhinobatidis [39] and the polyopisthocotyleans Microcotyle sp. [41] and Pseudomazocraes sp. [41]. This structure was designated as ‘flocular material outside the plasma membrane’ in Concinnocotyla australensis [42] and ‘knob-like protrusions’ in Protomicrocotyle ivoriensis and in Gastrocotyle sp. [43]. According to Watson et al. [42] the common presence of external ornamentation in monogeneans and digeneans reinforces the view that these groups have a common ancestry. 4.3. The mitochondrion The spermatozoon of A. laguncula possesses an elongate mitochondrion located in the regions II and III. Its length is greater than that of the nucleus. The number of mitochondria varies in digenean spermatozoa. Species possess either 1 mitochondrion: D. brusinae [28], P. ventricosa [10], C. endopapillatus [34], A. laguncula (present study), 2 mitochondria: Postorchigenes gymnesicus [44], Dicrocoelium hospes [45], D. inflata [24], C. metoecus [23], or 3 mitochondria: Anisocoelium capitellatum [46]. A particularity of the posterior part of the A. laguncula spermatozoon is the persistence of both two axonemes and mitochondrion whereas in the majority of digenean spermatozoa this posterior zone is characterized by the presence of the nucleus and only one axoneme e.g.: D. brusinae [28], Monorchis parvus [47], F. gigantica [33],
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P. ventricosa [10], C. metoecus [23] and C. endopapillatus [34] or of the nucleus and cortical microtubules e.g. P. furcatum [29], N. wisniewskii [26], N. testiobliquum [9] and Cotylophoron cotylophorum [48].
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Dr Jack Randall (Bishop Museum, Hawai'i) identified the fish (from photographs). References
4.4. The cortical microtubules The cortical microtubules of the spermatozoon of A. laguncula are arranged in a single field in the ventral (mitochondrial) side. This organisation has previously been described in a single digenean species, the faustulid P. ventricosa [10]. In the other digenean spermatozoa, two fields of cortical microtubules, one ventral and one dorsal, are observed. This is the case in allocreadiids [23], bucephalids [49], cryptogonimids [46,50], didymozoids [14], haematoloechids [30], troglotrematids [25] and zoogonids [28]. We noticed that the number of cortical microtubules increases from the anterior extremity of the spermatozoon to the nuclear zone and then decreases to the posterior extremity of the spermatozoon. Such an organisation was observed in faustulids [10] and opecoelids [9,26,27,29,31]. However, in certain digeneans, the maximum number of cortical microtubules is in the anterior part and this number decreases along the spermatozoon towards the posterior extremity. This is the case in allocreadiids [23], deropristids [24], fasciolids [33,51,52], gastrothylacids [34], notocotylids [32] and paramphistomids [48,53]. The maximum number of cortical microtubules in digenean spermatozoa is highly variable: 8 in P. ventricosa [10], 10 in A. laguncula (present study), 20 in Anisocoelium capitellatum [46], 25 in N. testiobliquum [9], and 32 in C. metoecus [23]. 4.5. Lecithasterids, and not didymozoids, as typical hemiuroideans Among the 13 families of the Hemiuroidea [12], ultrastructural studies of the spermatozoon have dealt only with two families, the lecithasterids (present study) and the didymozoids. Three didymozoid species have been studied: Didymocystis wedli [16], Didymozoon sp. [13,15] and Gonapodasmius sp. [14]. The spermatozoon of Gonapodasmius sp. has cortical microtubules and external ornamentation [14] whereas the spermatozoa of Didymozoon sp. and D. wedli do not possess these elements [13,16]. Moreover Didymozoon sp. is the only digenean (and even the single platyhelminth) which has two axonemes of the 9 + 0 type. Didymozoids are an enigmatic group because of their atypical morphology and atypical life-cycle [12]. In addition, they exhibit a distinct reproductive biology, often being encysted in pairs in closed cysts within the host organs. In each cyst, a female and a male, or two hermaphrodites, are present. This certainly decreases sperm competition and has probably had a major impact on sperm morphology: didymozoid spermatozoa are exceptionally short, and the absence of the central core in the 9 + 0 axonemes of Didymozoon is a major modification. Major modifications of sperm structure in response to atypical fertilization biology have also been found in other parasitic Platyhelminthes such as the monogenean Diplozoon [54,55], in which the two hermaphroditic members of the pair are permanently fused, and the schistosomes [56,57], in which the female is held in the gynaecophoric canal of the male and which are, with the didymozoids, amongst the few cases of separate sexes within the parasitic flatworms. For these reasons, we consider that didymozoid spermatozoa are highly modified by their special biology and that the spermatozoon of the lecithasterid A. laguncula should be considered as the ‘typical’ spermatozoon of the Hemiuroidea, hence allowing future comparisons with other superfamilies. This should be confirmed by studies of other members of the Hemiuroidea. Acknowledgements Miguel Clarque, Claire Goiran, Aude Sigura and Angelo Di Matteo participated in the fishing expedition and the parasitological survey.
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