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Spermatological characters of the digenean Lecithostaphylus retroflexus (Molin, 1859) (Microphalloidea: Zoogonidae), a parasite of the teleost fish Belone belone gracilis
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Spermatological characters of the digenean Lecithostaphylus retroflexus (Molin, 1859) (Microphalloidea: Zoogonidae), a parasite of the teleost fish Belone belone gracilis H. Kacem a,∗ , P.I. Ndiaye b , L. Neifar a , J. Torres c,d , J. Miquel c,d a
Laboratoire de Biodiversité et Ecosystèmes Aquatiques, Département des Sciences de la Vie, Faculté des Sciences de Sfax, BP 1171, 3000 Sfax, Tunisia Laboratory of Evolutionary Biology, Ecology and Management of Ecosystems, Faculty of Sciences and Techniques, Cheikh Anta Diop University of Dakar, Senegal c Laboratori de Parasitologia, Departament de Microbiologia i Parasitologia Sanitàries, Facultat de Farmàcia, Universitat de Barcelona, Av. Joan XXIII, s/n, 08028 Barcelona, Spain d Institut de Recerca de la Biodiversitat, Facultat de Biologia, Universitat de Barcelona, Av. Diagonal, 645, E-08028 Barcelona, Spain b
a r t i c l e
i n f o
Article history: Received 16 March 2015 Received in revised form 15 April 2015 Accepted 12 May 2015 Available online xxx Keywords: Lecithostaphylus retroflexus Zoogonidae Microphalloidea Digenea Ultrastructure Spermatozoon
a b s t r a c t The ultrastructural organization of the spermatozoon of the digenean Lecithostaphylus retroflexus (Microphalloidea: Zoogonidae) was described. Alive digeneans were collected from Belone belone gracilis (Teleostei: Belonidae), caught from the Gulf of Gabès in Chebba (Tunisia). The mature spermatozoon of L. retroflexus exhibits two axonemes of different lengths with the 9 + ‘1’ Trepaxonematan pattern, a nucleus, two mitochondria, two bundles of parallel cortical microtubules and granules of glycogen. Additionally, the spermatozoon of L. retroflexus shows type 2 of the external ornamentation according to Quilichini et al. (2011), spine-like bodies and a continuous and submembranous layer of parallel cortical microtubules surrounding the axonemes at their anterior end. Moreover, the morphology of the posterior spermatozoon extremity in L. retroflexus corresponds to the fasciolidean type according to Quilichini et al. (2010). © 2015 Elsevier Ltd. All rights reserved.
1. Introduction The family Zoogonidae includes digenean species mainly parasitizing marine fish. According to a recent review (Bray and Justine, 2014), this family contains 33 genera with a total of 159 species. Its placement as a microphalloidean has been demonstrated in several molecular analyses. The Microphalloidea includes two major clades. Three families are placed in the first clade (Pachypsolidae, Renicolidae and Eucotylidae). The Zoogonidae that was found closely related and probably paraphyletic to the Faustudidae is placed in the second clade. Thus, the second clade includes the Zoogonidae + Faustulidae as the most basal taxon with other taxa (Lecithodendriidae, Microphallidae, Pleurogenidae and Prosthogonimidae) as progressively more derived (see Olson et al., 2003; Bray and Justine, 2014).
∗ Corresponding author. Tel.: +216 98 48 34 26; fax: +216 74 27 64 00. E-mail address: [email protected] (H. Kacem).
In this context, the ultrastructural studies of species belonging to the Zoogonidae family are of great importance to bring additional information that complements the molecular results. The ultrastructural study of the mature spermatozoon provides numerous characters, which are useful for phylogenetic inference in parasitic Platyhelminthes (Justine, 1991, 1995; Levron et al., 2010; Quilichini et al., 2010, 2011). Digenean trematodes have been the subject of numerous ultrastructural studies on spermatology. In fact, to our knowledge there are ultrastructural data of the spermatozoon for more than 75 species distributed among 45 families (Ndiaye et al., 2014). With respect to the Zoogonidae family and to the best of our knowledge, there is only one ultrastructural study on the sperm of Diphterostomum brusinae (Levron et al., 2004a). The aim of the present study is to produce the first complete description of the ultrastructure of the spermatozoon of Lecithostaphylus retroflexus (Molin, 1859), contributing to the ultrastructural database concerning the Digenea. Our results were also compared with the available data on digenean spermatology, in particular, with those species belonging to the Microphalloidea
http://dx.doi.org/10.1016/j.tice.2015.05.003 0040-8166/© 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Kacem, H., et al., Spermatological characters of the digenean Lecithostaphylus retroflexus (Molin, 1859) (Microphalloidea: Zoogonidae), a parasite of the teleost fish Belone belone gracilis. Tissue Cell (2015), http://dx.doi.org/10.1016/j.tice.2015.05.003
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Fig. 1. Spermatozoon of Lecithostaphylus retroflexus: (a–d) cross-sections of region I showing the appearance of both axonemes; (e–h) cross-sections of the ornamented areas of region I; (i–k) cross-sections of region II showing the appearance of glycogen; (i) cross-section of posterior area of region II showing the second mitochondrion. Arrowheads: attachment zones; AxM, axonemal microtubules; C, centriole; CM, cortical microtubules; EO, external ornamentation of the plasma membrane; G, granules of glycogen; M1, first mitochondrion; M2, second mitochondrion; SB, spine-like bodies. Bars = 0.3 m.
superfamily in order to select the possible criteria useful for phylogeny. 2. Materials and methods Specimens of L. retroflexus (Molin, 1859) were collected from the digestive tract of Belone belone gracilis (Teleostei, Belonidae) caught from the Gulf of Gabès off Chebba (34◦ 14 N, 11◦ 06 E) (Tunisia). The worms were isolated from their host. They were fixed in cold (4 ◦ C) 2.5% glutaraldehyde in a 0.1 M sodium cacodylate buffer at pH 7.4 for a minimum of 2 h, rinsed in a 0.1 M sodium cacodylate buffer at pH 7.4. They were then postfixed in cold (4 ◦ C) 1% osmium tetroxide (OsO4 ) with 0.9% potassium ferricyanide [K3 Fe(CN)6 ] in the same buffer for 1 h, rinsed in Milli-Q water (Millipore Gradient A10), dehydrated in an ethanol series and propylene oxide, embedded in Spurr resin and finally polymerized at 60 ◦ C for 72 h. Ultrathin sections were obtained using a Reichert-Jung Ultracut-E ultramicrotome, placed on copper grids and double-stained with uranyl
acetate and lead citrate according to Reynolds (1963) methodology. Finally, all stained grids were studied with a JEOL 1010 transmission electron microscope operated at 80 kV. The Thiéry (1967) technique was used for glycogen localization. Gold grids with ultrathin sections were treated in periodic acid, thiocarbohydrazide and silver proteinate (PA-TCH-SP) as follows: 30 min in 10% PA, rinsed in milliQ water, 24 h in TCH, rinsed in acetic solutions and milliQ water, 30 min in 1% SP in the dark, and rinsed in Milli-Q water. 3. Results The observation of numerous ultrathin sections in the seminal vesicle of L. retroflexus allows the distinction of three different regions, I, II and III, from the anterior to the posterior spermatozoon extremities, considering the presence of different ultrastructural characteristics (Figs. 1–4). The mature spermatozoon of L. retroflexus exhibits the usual structures found in
Please cite this article in press as: Kacem, H., et al., Spermatological characters of the digenean Lecithostaphylus retroflexus (Molin, 1859) (Microphalloidea: Zoogonidae), a parasite of the teleost fish Belone belone gracilis. Tissue Cell (2015), http://dx.doi.org/10.1016/j.tice.2015.05.003
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Fig. 2. Spermatozoon of Lecithostaphylus retroflexus: (a–f) cross-sections of the region III from the appearance of nucleus to the posterior spermatozoon extremity. CM, cortical microtubules; D, doublets; G, granules of glycogen; M2, second mitochondrion; N, nucleus. Bars = 0.3 m.
numerous digeneans. Indeed, it contains two axonemes of the 9 + ‘1 trepaxonematan pattern, external ornamentation of the plasma membrane, spine-like bodies, nucleus, two mitochondria, two parallel bundles of cortical microtubules and granules of glycogen. Region I (Figs.1a–h and 4I) corresponds to the anterior extremity of the spermatozoon. Cross-sections through the anterior tip show axonemal microtubules forming the centrioles surrounded by a continuous and submembranous layer of parallel cortical microtubules, with a maximum of 33 when both axonemes of the 9 + ‘1’ trepaxonematan pattern are completely formed (Fig. 1a–c). At this level no attachment zones are observed (Fig. 1a–c). Two and posteriorly four attachment zones appear (Fig. 1d–f). In the middle part of this region, the external ornamentation of the plasma membrane as well as the reduction of cortical microtubules are noted and the first mitochondrion appear in the side containing the great number of cortical microtubules (Fig. 1e–g). In the distal part of region I, we notice the disappearance of the first mitochondrion and the parallel cortical microtubules become clearly arranged into two fields with a reduction in their number. It is worthy to mention that an external ornamentation of the plasma membrane and spine-like bodies are observed in association with cortical microtubules (Fig. 1h). The disappearance of
the external ornamentation marks the transition between regions I and II. Region II (Figs. 1i–l, 3 and 4II) corresponds to the middle region of the spermatozoon, which is mainly characterized by the simultaneous presence of two axonemes, two bundles of cortical microtubules and granules of glycogen in its anterior portion (Figs. 1i–k and 4II). The same characters are present towards the posterior portion of region II together with the appearance of the second mitochondrion (Fig. 1l). In this region, the number of cortical microtubules decreases up to about five. Region III (Figs. 2a–f, 3 and 4III) corresponds to the posterior spermatozoon extremity. It begins with the appearance of the nucleus (Fig. 2a). The cross-sections in its anterior part show two axonemes, nucleus, the second mitochondrion, cortical microtubules and granules of glycogen (Fig. 2a,b). Posteriorly, we notice the increasing size of the nucleus, the disappearance of the second mitochondrion, the reduction and disappearance of cortical microtubules and the disorganization and disappearance of the first axoneme (Fig. 2c,d). Then, near the posterior spermatozoon extremity, the second axoneme disappears and only the nucleus surrounded by few granules of glycogen reaches the posterior spermatozoon tip (Fig. 2e,f). The granular material observed in all the
Please cite this article in press as: Kacem, H., et al., Spermatological characters of the digenean Lecithostaphylus retroflexus (Molin, 1859) (Microphalloidea: Zoogonidae), a parasite of the teleost fish Belone belone gracilis. Tissue Cell (2015), http://dx.doi.org/10.1016/j.tice.2015.05.003
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median cytoplasmic process in most basal or proximal areas of the differentiation zone during spermiogenesis. Such organization occurring in several species is clearly illustrated in the case of Troglotrema acutum (Miquel et al., 2006). 4.2. Spine-like bodies
Fig. 3. Transmission electron micrograph showing the positive test of Thiéry. Bar = 0.3 m.
regions of the mature spermatozoon is evidenced as glycogen using the test of Thiéry (1967) (Fig. 3). 4. Discussion The mature spermatozoon of L. retroflexus shows the usual pattern described previously in the male gamete of digeneans so far: two axonemes of the 9 + ‘1 pattern of trepaxonematan Platyhelminthes (Ehlers, 1984), nucleus, mitochondria, granules of glycogen, external ornamentation and two sets of parallel cortical microtubules. Additional aspects include the presence of spine-like bodies in the ornamented area of the sperm cell and the continuous distribution of cortical microtubules in the anterior extremity of the spermatozoon. It is interesting to note that some of these characters as well as the morphology of both extremities could be potential candidates for phylogenetic analysis. 4.1. Anterior spermatozoon extremity The anterior spermatozoon extremity of the L. retroflexus exhibits two centrioles corresponding to two axonemes of the 9 + ‘1 trepaxonematan pattern (Ehlers, 1984). This is the typical structure of axonemes observed in all digeneans except for the species of the genus Schistosoma with a special 9 + ‘1 pattern with a poorly contrasted central element (Justine et al., 1993) and species of Didymozoon with a 9 + 0 pattern (Justine and Mattei, 1983). Anterior spermatozoon extremities with two axonemes have been reported in the spermatozoon of microphalloideans such as Microphallus primas (Castilho and Barandela, 1990) and Mediogonimus jourdanei (Bakhoum et al., 2012) contrary to the remaining microphalloideans that present only a single axoneme (see Table 1). It is also worthy to note that the axonemes of the L. retroflexus are surrounded by a continuous and submembranous layer of parallel cortical microtubules at their anterior end, whose organization is also reported in D. brusinae (Levron et al., 2004a), M. jourdanei (Bakhoum et al., 2012), Pleurogenes claviger, Pleurogenoides medians and Prosotocus confusus (Miquel et al., 2013), Brandesia turgida ˇ et al., 2014) or Postorchigenes gymnesicus (Gracenea (Brunanská et al., 1997). A similar structure was reported in numerous digenean species (Bakhoum, 2012). The presence of cortical microtubules without attachment zones indicates the absence of fusion between the free flagella and the
Spine-like bodies consist in prominent electron-dense structures that seem to contain a spherical vesicle and were described for the first time in the spermatozoon of Opecoeloides furcatus (Opecoelidae) by Miquel et al. (2000). These structures were observed in the ornamented area of the mature spermatozoon of L. retroflexus. Concerning the species belonging to the Microphalloidea superfamily studied until now, spine-like bodies have been described in four species (see Table 1). In most digeneans in which these structures were observed, they are present in the anterior areas of the spermatozoon and associated with the external ornamentation of the plasma membrane, and usually in the mitochondrial region of the male gamete (Quilichini et al., 2011). However, in the case of N. chabaudi (Kacem et al., 2010), spine-like bodies appear posteriorly to the ornamented area of the spermatozoon. Another interesting aspect dealing with the spine-like bodies is the presence or absence of periodicity. Besides, their number is not always evident because of the difficulty in observing longitudinal sections containing successive spine-like bodies. A periodicity of these elements along the sperm cell is observed and estimated as 1 m in O. furcatus, Fasciola gigantica and Nicolla testiobliquum (Miquel et al., 2000; Ndiaye et al., 2004; Quilichini et al., 2007a), 0.7 m in Poracanthium furcatum (Levron et al., 2004b) or 0.6 m in Nicolla wisniewskii (Quilichini et al., 2007c) or 0.5–0.6 m P. claviger (Miquel et al., 2013). However, in L. retroflexus like other species (Miquel et al., 2013) it was not possible to evaluate the periodicity spine-like bodies. Considering all these aspects, this character will be of great interest to the elucidation of the relationships between digeneans at the family level. 4.3. External ornamentation The spermatozoon of L. retroflexus displays external ornamentation of the plasma membrane localized in the anterior spermatozoon region as many species belonging to the superfamily Microphalloidea, namely Pronoprymna ventricosa, M. primas, P. gymnesicus, M. jourdanei; D. brusinae; B. turgida (Quilichini et al., 2007b; Castilho and Barandela, 1990; Gracenea et al., 1997; ˇ Bakhoum et al., 2012; Levron et al., 2004a; Brunanská et al., 2014). This structure has also been observed in the majority of digenean families (Bakhoum, 2012). The common presence and variability in the location of external ornamentation in the spermatozoon of digeneans is of particular interest for phylogenetic purposes. Thus, according to Quilichini et al. (2011), there are three types of digenean anterior spermatozoon regions according to the localization of the external ornamentation: (i) type 1 presents external ornamentation in the anterior extremity of the spermatozoon; (ii) type 2 presents external ornamentation at a more posterior level, usually in the mitochondrial region; and (iii) type 3 lacks external ornamentation. This attempt of classification could be important at different taxonomic levels. Indeed, all species belonging to the Microphalloidea superfamily studied so far have external ornamentation (Group 1 or 2) with the exception of Maritrema linguilla (Hendow and James, 1988) and Ganeo tigrinum (Sharma and Rai, 1995) which apparently lacks external ornamentation (Group 3). However, these two old studies show a scarce number of micrographs and thus, these results should be considered with caution. Ultrastructural findings are consistent
Please cite this article in press as: Kacem, H., et al., Spermatological characters of the digenean Lecithostaphylus retroflexus (Molin, 1859) (Microphalloidea: Zoogonidae), a parasite of the teleost fish Belone belone gracilis. Tissue Cell (2015), http://dx.doi.org/10.1016/j.tice.2015.05.003
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Fig. 4. Schematic reconstruction of the mature spermatozoon of Lecithostaphylus retroflexus. The sperm cell is organized in three different regions: region I or anterior part, region II or middle part and region III or posterior part. ASE, anterior spermatozoon extremity; AxM, axonemal microbubules; C, centrioles; CM, cortical microtubules; D, doublets; EO, external ornamentation of the plasma membrane; G, granules of glycogen; M1, first mitochondrion; M2, second mitochondrion; N, nucleus; PM, plasma membrane; PSE, posterior spermatozoon extremity; SB, spine-like bodies.
with the phylogenetic studies of Tkach et al. (2003) based on molecular data that challenge the status of genus Maritrema, and the one ´ (2008) to review the status of genus Ganeo proposed by Pojmanska among Lecithodendridae.
presents two mitochondria with a thin one in the ornamented area, and a second one that is more posteriorly located, reaching the nuclear region. In the remaining species of this superfamily, spermatozoa have only one mitochondrion (Table 1).
4.4. Number of mitochondria
4.5. Posterior spermatozoon extremity
Similar to other Microphalloidea (Hendow and James, 1988; Gracenea et al., 1997), the mature spermatozoon of L. retroflexus
As occurs with the anterior spermatozoon tip, the posterior spermatozoon extremity is morphologically variable within
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Table 1 Spermatological characters in the Microphalloidea superfamily. Families and species
Collyriclidae Collyricloides massanae Faustulidae Pronoprymna ventricosa Lecithodendriidae Ganeo tigrinuma Microphallidae Microphallus primas Maritrema linguillaa Phaneropsolidae Postorchigenes gymnesicus Pleurogenidae Pleurogenes claviger Pleurogenoides medians Prosotocus confusus Brandesia turgida Prosthogonimidae Mediogonimus jourdanei Zoogonidae Diphterostomum brusinae Lecithostaphylus retroflexus
Spermatological characters
References
ASE
EO
M
SB
PSE
2Ax
+
1
+
1Ax
Bakhoum et al. (2014)
1Ax
+
1
–
1Ax
Quilichini et al. (2007b)
?
–
1
–
1Ax
Sharma and Rai (1995)
2Ax ?
+ –
1 2
– –
1Ax 1Ax
Castilho and Barandela (1990) Hendow and James (1988)
1Ax
+
2
–
1Ax + G
Gracenea et al. (1997)
2Ax 2Ax 2Ax 2Ax
+ + + +
2 2 2 1
+ + + –
1Ax 1Ax 1Ax 1Ax
Miquel et al. (2013) Miquel et al. (2013) Miquel et al. (2013) ˇ Brunanská et al. (2014)
2Ax
+
1
+
1Ax
Bakhoum et al. (2012)
1Ax 2Ax
+ +
1 2
– +
N+G N+G
Levron et al. (2004a) Present study
ASE, anterior spermatozoon extremity; Ax, axoneme; EO, external ornamentation; G, granules of glycogen; M, mitochondrion; N, nucleus; PSE, posterior spermatozoon extremity; SB, spine-like body; +/−, presence/absence of considered character. a Data of these species should be considered with caution.
digeneans. Quilichini et al. (2010) distinguished three types of posterior parts of the spermatozoon (opecoelidean type, fasciolidean type and cryptogonimidean type). In Microphalloidean species, two posterior spermatozoon patterns have been observed. The first morphology concerns species with posterior spermatozoon tip belonging to the fasciolidean type of Quilichini et al. (2010). This type is characterized by the sequence of cortical microtubules, posterior end of the second axoneme and the posterior end of the nucleus as observed in L. retroflexus and D. brusinae (Levron et al., 2004a – see Table 1). The second morphology species groups belong to the cryptogonimidean type of Quilichini et al. (2010) with a posterior spermatozoon tip. The latter is characterized by the absence of cortical microtubules and the sequence posterior extremity of the nucleus, the posterior extremity of the second axoneme as reported in P. ventricosa, G. tigrinum, M. primas, M. linguilla, P. gymnesicus, M. jourdanei (Quilichini et al., 2007b; Sharma and Rai, 1995; Castilho and Barandela, 1990; Hendow and James, 1988; Gracenea et al., 1997; Bakhoum et al., 2012). Thus, the pattern of the posterior spermatozoon extremity appears of great interest for understanding the relationships within the digeneans. Further studies of this character would be of importance for a phylogenetic analysis.
Acknowledgements The authors wish to thank the staff of the “Centres Científics i Tecnològics” of the University of Barcelona (CCTiUB) for their assistance. The study was partly supported by AECID (A/023585/09) and AGAUR (2014 SGR 1241).
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Please cite this article in press as: Kacem, H., et al., Spermatological characters of the digenean Lecithostaphylus retroflexus (Molin, 1859) (Microphalloidea: Zoogonidae), a parasite of the teleost fish Belone belone gracilis. Tissue Cell (2015), http://dx.doi.org/10.1016/j.tice.2015.05.003
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Spermatological characters of the digenean Lecithostaphylus retroflexus (Molin, 1859) (Microphalloidea: Zoogonidae), a parasite of the teleost fish Belone belone gracilis H. Kacem a,∗ , P.I. Ndiaye b , L. Neifar a , J. Torres c,d , J. Miquel c,d a
Laboratoire de Biodiversité et Ecosystèmes Aquatiques, Département des Sciences de la Vie, Faculté des Sciences de Sfax, BP 1171, 3000 Sfax, Tunisia Laboratory of Evolutionary Biology, Ecology and Management of Ecosystems, Faculty of Sciences and Techniques, Cheikh Anta Diop University of Dakar, Senegal c Laboratori de Parasitologia, Departament de Microbiologia i Parasitologia Sanitàries, Facultat de Farmàcia, Universitat de Barcelona, Av. Joan XXIII, s/n, 08028 Barcelona, Spain d Institut de Recerca de la Biodiversitat, Facultat de Biologia, Universitat de Barcelona, Av. Diagonal, 645, E-08028 Barcelona, Spain b
a r t i c l e
i n f o
Article history: Received 16 March 2015 Received in revised form 15 April 2015 Accepted 12 May 2015 Available online xxx Keywords: Lecithostaphylus retroflexus Zoogonidae Microphalloidea Digenea Ultrastructure Spermatozoon
a b s t r a c t The ultrastructural organization of the spermatozoon of the digenean Lecithostaphylus retroflexus (Microphalloidea: Zoogonidae) was described. Alive digeneans were collected from Belone belone gracilis (Teleostei: Belonidae), caught from the Gulf of Gabès in Chebba (Tunisia). The mature spermatozoon of L. retroflexus exhibits two axonemes of different lengths with the 9 + ‘1’ Trepaxonematan pattern, a nucleus, two mitochondria, two bundles of parallel cortical microtubules and granules of glycogen. Additionally, the spermatozoon of L. retroflexus shows type 2 of the external ornamentation according to Quilichini et al. (2011), spine-like bodies and a continuous and submembranous layer of parallel cortical microtubules surrounding the axonemes at their anterior end. Moreover, the morphology of the posterior spermatozoon extremity in L. retroflexus corresponds to the fasciolidean type according to Quilichini et al. (2010). © 2015 Elsevier Ltd. All rights reserved.
1. Introduction The family Zoogonidae includes digenean species mainly parasitizing marine fish. According to a recent review (Bray and Justine, 2014), this family contains 33 genera with a total of 159 species. Its placement as a microphalloidean has been demonstrated in several molecular analyses. The Microphalloidea includes two major clades. Three families are placed in the first clade (Pachypsolidae, Renicolidae and Eucotylidae). The Zoogonidae that was found closely related and probably paraphyletic to the Faustudidae is placed in the second clade. Thus, the second clade includes the Zoogonidae + Faustulidae as the most basal taxon with other taxa (Lecithodendriidae, Microphallidae, Pleurogenidae and Prosthogonimidae) as progressively more derived (see Olson et al., 2003; Bray and Justine, 2014).
∗ Corresponding author. Tel.: +216 98 48 34 26; fax: +216 74 27 64 00. E-mail address: [email protected] (H. Kacem).
In this context, the ultrastructural studies of species belonging to the Zoogonidae family are of great importance to bring additional information that complements the molecular results. The ultrastructural study of the mature spermatozoon provides numerous characters, which are useful for phylogenetic inference in parasitic Platyhelminthes (Justine, 1991, 1995; Levron et al., 2010; Quilichini et al., 2010, 2011). Digenean trematodes have been the subject of numerous ultrastructural studies on spermatology. In fact, to our knowledge there are ultrastructural data of the spermatozoon for more than 75 species distributed among 45 families (Ndiaye et al., 2014). With respect to the Zoogonidae family and to the best of our knowledge, there is only one ultrastructural study on the sperm of Diphterostomum brusinae (Levron et al., 2004a). The aim of the present study is to produce the first complete description of the ultrastructure of the spermatozoon of Lecithostaphylus retroflexus (Molin, 1859), contributing to the ultrastructural database concerning the Digenea. Our results were also compared with the available data on digenean spermatology, in particular, with those species belonging to the Microphalloidea
http://dx.doi.org/10.1016/j.tice.2015.05.003 0040-8166/© 2015 Elsevier Ltd. All rights reserved.
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Fig. 1. Spermatozoon of Lecithostaphylus retroflexus: (a–d) cross-sections of region I showing the appearance of both axonemes; (e–h) cross-sections of the ornamented areas of region I; (i–k) cross-sections of region II showing the appearance of glycogen; (i) cross-section of posterior area of region II showing the second mitochondrion. Arrowheads: attachment zones; AxM, axonemal microtubules; C, centriole; CM, cortical microtubules; EO, external ornamentation of the plasma membrane; G, granules of glycogen; M1, first mitochondrion; M2, second mitochondrion; SB, spine-like bodies. Bars = 0.3 m.
superfamily in order to select the possible criteria useful for phylogeny. 2. Materials and methods Specimens of L. retroflexus (Molin, 1859) were collected from the digestive tract of Belone belone gracilis (Teleostei, Belonidae) caught from the Gulf of Gabès off Chebba (34◦ 14 N, 11◦ 06 E) (Tunisia). The worms were isolated from their host. They were fixed in cold (4 ◦ C) 2.5% glutaraldehyde in a 0.1 M sodium cacodylate buffer at pH 7.4 for a minimum of 2 h, rinsed in a 0.1 M sodium cacodylate buffer at pH 7.4. They were then postfixed in cold (4 ◦ C) 1% osmium tetroxide (OsO4 ) with 0.9% potassium ferricyanide [K3 Fe(CN)6 ] in the same buffer for 1 h, rinsed in Milli-Q water (Millipore Gradient A10), dehydrated in an ethanol series and propylene oxide, embedded in Spurr resin and finally polymerized at 60 ◦ C for 72 h. Ultrathin sections were obtained using a Reichert-Jung Ultracut-E ultramicrotome, placed on copper grids and double-stained with uranyl
acetate and lead citrate according to Reynolds (1963) methodology. Finally, all stained grids were studied with a JEOL 1010 transmission electron microscope operated at 80 kV. The Thiéry (1967) technique was used for glycogen localization. Gold grids with ultrathin sections were treated in periodic acid, thiocarbohydrazide and silver proteinate (PA-TCH-SP) as follows: 30 min in 10% PA, rinsed in milliQ water, 24 h in TCH, rinsed in acetic solutions and milliQ water, 30 min in 1% SP in the dark, and rinsed in Milli-Q water. 3. Results The observation of numerous ultrathin sections in the seminal vesicle of L. retroflexus allows the distinction of three different regions, I, II and III, from the anterior to the posterior spermatozoon extremities, considering the presence of different ultrastructural characteristics (Figs. 1–4). The mature spermatozoon of L. retroflexus exhibits the usual structures found in
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Fig. 2. Spermatozoon of Lecithostaphylus retroflexus: (a–f) cross-sections of the region III from the appearance of nucleus to the posterior spermatozoon extremity. CM, cortical microtubules; D, doublets; G, granules of glycogen; M2, second mitochondrion; N, nucleus. Bars = 0.3 m.
numerous digeneans. Indeed, it contains two axonemes of the 9 + ‘1 trepaxonematan pattern, external ornamentation of the plasma membrane, spine-like bodies, nucleus, two mitochondria, two parallel bundles of cortical microtubules and granules of glycogen. Region I (Figs.1a–h and 4I) corresponds to the anterior extremity of the spermatozoon. Cross-sections through the anterior tip show axonemal microtubules forming the centrioles surrounded by a continuous and submembranous layer of parallel cortical microtubules, with a maximum of 33 when both axonemes of the 9 + ‘1’ trepaxonematan pattern are completely formed (Fig. 1a–c). At this level no attachment zones are observed (Fig. 1a–c). Two and posteriorly four attachment zones appear (Fig. 1d–f). In the middle part of this region, the external ornamentation of the plasma membrane as well as the reduction of cortical microtubules are noted and the first mitochondrion appear in the side containing the great number of cortical microtubules (Fig. 1e–g). In the distal part of region I, we notice the disappearance of the first mitochondrion and the parallel cortical microtubules become clearly arranged into two fields with a reduction in their number. It is worthy to mention that an external ornamentation of the plasma membrane and spine-like bodies are observed in association with cortical microtubules (Fig. 1h). The disappearance of
the external ornamentation marks the transition between regions I and II. Region II (Figs. 1i–l, 3 and 4II) corresponds to the middle region of the spermatozoon, which is mainly characterized by the simultaneous presence of two axonemes, two bundles of cortical microtubules and granules of glycogen in its anterior portion (Figs. 1i–k and 4II). The same characters are present towards the posterior portion of region II together with the appearance of the second mitochondrion (Fig. 1l). In this region, the number of cortical microtubules decreases up to about five. Region III (Figs. 2a–f, 3 and 4III) corresponds to the posterior spermatozoon extremity. It begins with the appearance of the nucleus (Fig. 2a). The cross-sections in its anterior part show two axonemes, nucleus, the second mitochondrion, cortical microtubules and granules of glycogen (Fig. 2a,b). Posteriorly, we notice the increasing size of the nucleus, the disappearance of the second mitochondrion, the reduction and disappearance of cortical microtubules and the disorganization and disappearance of the first axoneme (Fig. 2c,d). Then, near the posterior spermatozoon extremity, the second axoneme disappears and only the nucleus surrounded by few granules of glycogen reaches the posterior spermatozoon tip (Fig. 2e,f). The granular material observed in all the
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median cytoplasmic process in most basal or proximal areas of the differentiation zone during spermiogenesis. Such organization occurring in several species is clearly illustrated in the case of Troglotrema acutum (Miquel et al., 2006). 4.2. Spine-like bodies
Fig. 3. Transmission electron micrograph showing the positive test of Thiéry. Bar = 0.3 m.
regions of the mature spermatozoon is evidenced as glycogen using the test of Thiéry (1967) (Fig. 3). 4. Discussion The mature spermatozoon of L. retroflexus shows the usual pattern described previously in the male gamete of digeneans so far: two axonemes of the 9 + ‘1 pattern of trepaxonematan Platyhelminthes (Ehlers, 1984), nucleus, mitochondria, granules of glycogen, external ornamentation and two sets of parallel cortical microtubules. Additional aspects include the presence of spine-like bodies in the ornamented area of the sperm cell and the continuous distribution of cortical microtubules in the anterior extremity of the spermatozoon. It is interesting to note that some of these characters as well as the morphology of both extremities could be potential candidates for phylogenetic analysis. 4.1. Anterior spermatozoon extremity The anterior spermatozoon extremity of the L. retroflexus exhibits two centrioles corresponding to two axonemes of the 9 + ‘1 trepaxonematan pattern (Ehlers, 1984). This is the typical structure of axonemes observed in all digeneans except for the species of the genus Schistosoma with a special 9 + ‘1 pattern with a poorly contrasted central element (Justine et al., 1993) and species of Didymozoon with a 9 + 0 pattern (Justine and Mattei, 1983). Anterior spermatozoon extremities with two axonemes have been reported in the spermatozoon of microphalloideans such as Microphallus primas (Castilho and Barandela, 1990) and Mediogonimus jourdanei (Bakhoum et al., 2012) contrary to the remaining microphalloideans that present only a single axoneme (see Table 1). It is also worthy to note that the axonemes of the L. retroflexus are surrounded by a continuous and submembranous layer of parallel cortical microtubules at their anterior end, whose organization is also reported in D. brusinae (Levron et al., 2004a), M. jourdanei (Bakhoum et al., 2012), Pleurogenes claviger, Pleurogenoides medians and Prosotocus confusus (Miquel et al., 2013), Brandesia turgida ˇ et al., 2014) or Postorchigenes gymnesicus (Gracenea (Brunanská et al., 1997). A similar structure was reported in numerous digenean species (Bakhoum, 2012). The presence of cortical microtubules without attachment zones indicates the absence of fusion between the free flagella and the
Spine-like bodies consist in prominent electron-dense structures that seem to contain a spherical vesicle and were described for the first time in the spermatozoon of Opecoeloides furcatus (Opecoelidae) by Miquel et al. (2000). These structures were observed in the ornamented area of the mature spermatozoon of L. retroflexus. Concerning the species belonging to the Microphalloidea superfamily studied until now, spine-like bodies have been described in four species (see Table 1). In most digeneans in which these structures were observed, they are present in the anterior areas of the spermatozoon and associated with the external ornamentation of the plasma membrane, and usually in the mitochondrial region of the male gamete (Quilichini et al., 2011). However, in the case of N. chabaudi (Kacem et al., 2010), spine-like bodies appear posteriorly to the ornamented area of the spermatozoon. Another interesting aspect dealing with the spine-like bodies is the presence or absence of periodicity. Besides, their number is not always evident because of the difficulty in observing longitudinal sections containing successive spine-like bodies. A periodicity of these elements along the sperm cell is observed and estimated as 1 m in O. furcatus, Fasciola gigantica and Nicolla testiobliquum (Miquel et al., 2000; Ndiaye et al., 2004; Quilichini et al., 2007a), 0.7 m in Poracanthium furcatum (Levron et al., 2004b) or 0.6 m in Nicolla wisniewskii (Quilichini et al., 2007c) or 0.5–0.6 m P. claviger (Miquel et al., 2013). However, in L. retroflexus like other species (Miquel et al., 2013) it was not possible to evaluate the periodicity spine-like bodies. Considering all these aspects, this character will be of great interest to the elucidation of the relationships between digeneans at the family level. 4.3. External ornamentation The spermatozoon of L. retroflexus displays external ornamentation of the plasma membrane localized in the anterior spermatozoon region as many species belonging to the superfamily Microphalloidea, namely Pronoprymna ventricosa, M. primas, P. gymnesicus, M. jourdanei; D. brusinae; B. turgida (Quilichini et al., 2007b; Castilho and Barandela, 1990; Gracenea et al., 1997; ˇ Bakhoum et al., 2012; Levron et al., 2004a; Brunanská et al., 2014). This structure has also been observed in the majority of digenean families (Bakhoum, 2012). The common presence and variability in the location of external ornamentation in the spermatozoon of digeneans is of particular interest for phylogenetic purposes. Thus, according to Quilichini et al. (2011), there are three types of digenean anterior spermatozoon regions according to the localization of the external ornamentation: (i) type 1 presents external ornamentation in the anterior extremity of the spermatozoon; (ii) type 2 presents external ornamentation at a more posterior level, usually in the mitochondrial region; and (iii) type 3 lacks external ornamentation. This attempt of classification could be important at different taxonomic levels. Indeed, all species belonging to the Microphalloidea superfamily studied so far have external ornamentation (Group 1 or 2) with the exception of Maritrema linguilla (Hendow and James, 1988) and Ganeo tigrinum (Sharma and Rai, 1995) which apparently lacks external ornamentation (Group 3). However, these two old studies show a scarce number of micrographs and thus, these results should be considered with caution. Ultrastructural findings are consistent
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Fig. 4. Schematic reconstruction of the mature spermatozoon of Lecithostaphylus retroflexus. The sperm cell is organized in three different regions: region I or anterior part, region II or middle part and region III or posterior part. ASE, anterior spermatozoon extremity; AxM, axonemal microbubules; C, centrioles; CM, cortical microtubules; D, doublets; EO, external ornamentation of the plasma membrane; G, granules of glycogen; M1, first mitochondrion; M2, second mitochondrion; N, nucleus; PM, plasma membrane; PSE, posterior spermatozoon extremity; SB, spine-like bodies.
with the phylogenetic studies of Tkach et al. (2003) based on molecular data that challenge the status of genus Maritrema, and the one ´ (2008) to review the status of genus Ganeo proposed by Pojmanska among Lecithodendridae.
presents two mitochondria with a thin one in the ornamented area, and a second one that is more posteriorly located, reaching the nuclear region. In the remaining species of this superfamily, spermatozoa have only one mitochondrion (Table 1).
4.4. Number of mitochondria
4.5. Posterior spermatozoon extremity
Similar to other Microphalloidea (Hendow and James, 1988; Gracenea et al., 1997), the mature spermatozoon of L. retroflexus
As occurs with the anterior spermatozoon tip, the posterior spermatozoon extremity is morphologically variable within
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Table 1 Spermatological characters in the Microphalloidea superfamily. Families and species
Collyriclidae Collyricloides massanae Faustulidae Pronoprymna ventricosa Lecithodendriidae Ganeo tigrinuma Microphallidae Microphallus primas Maritrema linguillaa Phaneropsolidae Postorchigenes gymnesicus Pleurogenidae Pleurogenes claviger Pleurogenoides medians Prosotocus confusus Brandesia turgida Prosthogonimidae Mediogonimus jourdanei Zoogonidae Diphterostomum brusinae Lecithostaphylus retroflexus
Spermatological characters
References
ASE
EO
M
SB
PSE
2Ax
+
1
+
1Ax
Bakhoum et al. (2014)
1Ax
+
1
–
1Ax
Quilichini et al. (2007b)
?
–
1
–
1Ax
Sharma and Rai (1995)
2Ax ?
+ –
1 2
– –
1Ax 1Ax
Castilho and Barandela (1990) Hendow and James (1988)
1Ax
+
2
–
1Ax + G
Gracenea et al. (1997)
2Ax 2Ax 2Ax 2Ax
+ + + +
2 2 2 1
+ + + –
1Ax 1Ax 1Ax 1Ax
Miquel et al. (2013) Miquel et al. (2013) Miquel et al. (2013) ˇ Brunanská et al. (2014)
2Ax
+
1
+
1Ax
Bakhoum et al. (2012)
1Ax 2Ax
+ +
1 2
– +
N+G N+G
Levron et al. (2004a) Present study
ASE, anterior spermatozoon extremity; Ax, axoneme; EO, external ornamentation; G, granules of glycogen; M, mitochondrion; N, nucleus; PSE, posterior spermatozoon extremity; SB, spine-like body; +/−, presence/absence of considered character. a Data of these species should be considered with caution.
digeneans. Quilichini et al. (2010) distinguished three types of posterior parts of the spermatozoon (opecoelidean type, fasciolidean type and cryptogonimidean type). In Microphalloidean species, two posterior spermatozoon patterns have been observed. The first morphology concerns species with posterior spermatozoon tip belonging to the fasciolidean type of Quilichini et al. (2010). This type is characterized by the sequence of cortical microtubules, posterior end of the second axoneme and the posterior end of the nucleus as observed in L. retroflexus and D. brusinae (Levron et al., 2004a – see Table 1). The second morphology species groups belong to the cryptogonimidean type of Quilichini et al. (2010) with a posterior spermatozoon tip. The latter is characterized by the absence of cortical microtubules and the sequence posterior extremity of the nucleus, the posterior extremity of the second axoneme as reported in P. ventricosa, G. tigrinum, M. primas, M. linguilla, P. gymnesicus, M. jourdanei (Quilichini et al., 2007b; Sharma and Rai, 1995; Castilho and Barandela, 1990; Hendow and James, 1988; Gracenea et al., 1997; Bakhoum et al., 2012). Thus, the pattern of the posterior spermatozoon extremity appears of great interest for understanding the relationships within the digeneans. Further studies of this character would be of importance for a phylogenetic analysis.
Acknowledgements The authors wish to thank the staff of the “Centres Científics i Tecnològics” of the University of Barcelona (CCTiUB) for their assistance. The study was partly supported by AECID (A/023585/09) and AGAUR (2014 SGR 1241).
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