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Ultrastructural characteristics of the mature spermatozoon of the digenean Sclerodistomum italicum (Stossich, 1893) (Hemiuroidea, Sclerodistomidae) intestinal parasite of Hypocanthus amia (Teleostei, Carangidae)
Accepted Manuscript Title: Ultrastructural characteristics of the mature spermatozoon of the digenean Sclerodistomum italicum (Stossich, 1893) (Hemiuroidea, Sclerodistomidae) intestinal parasite of Hypocanthus amia (Teleostei, Carangidae) Author: Papa Ibnou Ndiaye Yann Quilichini Adji Mama Marigo Cheikh Tidiane Bˆa Vasyl V. Tkach Bernard Marchand PII: DOI: Reference:
S0040-8166(16)30229-4 http://dx.doi.org/doi:10.1016/j.tice.2016.12.007 YTICE 1065
To appear in:
Tissue and Cell
Received date: Revised date: Accepted date:
16-9-2016 18-12-2016 18-12-2016
Please cite this article as: Ndiaye, Papa Ibnou, Quilichini, Yann, Marigo, Adji Mama, Bˆa, Cheikh Tidiane, Tkach, Vasyl V., Marchand, Bernard, Ultrastructural characteristics of the mature spermatozoon of the digenean Sclerodistomum italicum (Stossich, 1893) (Hemiuroidea, Sclerodistomidae) intestinal parasite of Hypocanthus amia (Teleostei, Carangidae).Tissue and Cell http://dx.doi.org/10.1016/j.tice.2016.12.007
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Ultrastructural characteristics of the mature spermatozoon of the digenean Sclerodistomum italicum (Stossich, 1893) (Hemiuroidea, Sclerodistomidae) intestinal parasite of Hypocanthus amia (Teleostei, Carangidae) Papa Ibnou Ndiayea*, Yann Quilichinib, Adji Mama Marigoa, Cheikh Tidiane Bâa, Vasyl V. Tkachc, Bernard Marchandb
a
Laboratory of Evolutionary Biology, Ecology and Management of Ecosystems, Faculty
of Sciences and Techniques, Cheikh Anta Diop University of Dakar, BP 5055, Dakar, Republic of Senegal b
UMR SPE 6134, CNRS – Università di Corsica, SERME Service d’Etude et de
Recherche en Microscopie Electronique, Campus Grimaldi, 20250 Corte, Corsica, France c
Department of Biology, University of North Dakota, Grand Forks, North Dakota, USA
Running title: Spermatozoon ultrastructure of Sclerodistomum italicum
(*): Corresponding author Name: Papa Ibnou Ndiaye Postal address: Laboratory of Evolutionary Biology, Ecology and Management of Ecosystems, Faculty of Sciences and Techniques, Cheikh Anta Diop University of Dakar, BP 5055, Dakar, Republic of Senegal E-mail address: [email protected] Phone number: 00221 778142834
Highlights
2
This paper provide the first ultrastructural data of the spermatozoon of a species becoming to the genus Sclerodistomum,
It’s described for first time in this work, the presence of two types of extramembranous ornamentations in the mature spermatozoon of a digenean,
The presence of short cortical microtubules in the anterior part of the spermatozoon in addition to the presence of only one bundle of cortical microtubules in the median part of the spermatozoon,
In accordance to the others mature spermatozoa described in Hemiuroidea, the spermatozoon of Sclerodistomum italicum lack spine-like body.
Abstract The mature spermatozoon of Sclerodistomum italicum is filiform, tapered at both ends and shows the following features: 2 axonemes of the 9 + “1” pattern of the Trepaxonemata, mitochondrion, nucleus and parallel cortical microtubules. The specific features of the spermatozoon of S. italicum include the simultaneous presence of two types of extramembranous ornamentations, the presence of short cortical microtubules in the anterior part of the spermatozoon and the presence of only one bundle of cortical microtubules in the median part of the spermatozoon. Thus far these structures are known only in the Hemiuroidea. The presence of filamentous ornamentation in the anterior extremity of the spermatozoon has not previously been described in the Sclerodistomidae. Similar to spermatozoa of other hemiuroideans, S. italicum lack spine-like bodies described in spermatozoa of many digenean taxa. The posterior extremity of the spermatozoon exhibits the same ultrastructural characteristics typical of the Hemiuroidea. Keywords: Digenea; Sclerodistomidae; Sclerodistomum italicum; Sperm; ultrastructure.
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1. Introduction The Digenea is the largest group of internal metazoan parasites. They are an extremely ubiquitous group, parasitizing all major vertebrate groups as definitive host, gastropods and other mollusks as first intermediate hosts and representatives of several animal phyla as second intermediate host, when it is present. Diseases caused by digeneans in humans, domestic animals and wildlife receive considerable attention from researchers across a broad range of disciplines. On the other hand, due to their broad distribution and complex life cycles, digeneans have long been an important model in evolutionary, ecological and biodiversity studies. Both applied and basic research of digeneans benefit from molecular phylogenetic analyses of digeneans which have been published at an accelerating rate in the last 20-25 years (Barker et al. 1993; Tkach and Pawlowski 1999; Tkach et al. 2000; Cribb et al. 2001; Olson and Littlewood 2002; Olson et al. 2003; Levron et al. 2010). As a part of this global effort, Blair et al. (1998) have conducted a molecular phylogenetic study of the Hemiuroidea, a large group of digeneans parasitizing primarily marine teleosts, but also occurring in freshwater teleosts, elasmobranchs, amphibians, and reptiles. These authors have concluded that previous phylogenies of hemiurids based on characters derived from morphology and life cycles have been controversial. The relationships within the hemiuroids, and exact number of groups that should be included in the taxon, have been debated (Blair et al. 1998). The current system recognizes at least the families Hemiuridae, Accacoeliidae, Bathycotylidae,
Derogenidae,
Dictysarcidae,
Lecithasteridae,
Ptychogonimidae,
Hirudinellidae,
Sclerodistomidae,
Isoparorchiidae,
Sclerodistomoididae
and
Syncoeliidae within the Hemiuroidea (Gibson et al. 2002). However, most authors agree that the phylogenetic relationships among hemiuroids remain controversial and require a combination of morphological, molecular and ultrastructural data (Pamplona-Basilio et al. 2001; Levron et al. 2010; Ndiaye et al. 2013a, 2014). To our knowledge, ultrastructural data of the spermatozoon of hemiuroids exist only for the following taxa: the Didymozoidae: Gonapodasmius (Justine and Mattei 1982, 1984b), Didymozoon sp. (Justine and Mattei 1983, 1984a), Didymocystis wedli (Pamplona-Basilio et al. 2001); the Hemiuridae: Lecithocladium excisum (Ndiaye et al. 2012), Parahemiurus merus (Ndiaye et al. 2013a), Lecithochirium microstomum (Ndiaye et al. 2014), L. musculus (Ndiaye et al. 2014), Hemiurus appendiculatus (Dione et al. 2016); the Lecithasteridae: Aponurus laguncula (Quilichini et al. 2010); the
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Sclerodistomidae: Prosorchis palinurichthi (Ndiaye et al. 2013b). Sclerodistomids are normally parasites of the stomach of marine teleosts. The Sclerodistomidae currently includes only three subfamilies: Sclerodistominae, Kenmackenziinae and Prosorchiinae. Ultrastructural data on the spermatozoon of sclerodistomids exist only for the Prosorchiinae: Prosorchis palinurichthi. Herein, we present results of ultrastructural study of spermatozoon in Sclerodistomum italicum, the type species of the genus Sclerodistomum.
2.
Materials and Methods Adult specimens of Sclerodistomum italicum (Stossich, 1893) were collected from
the digestive tract of a teleost Holocentridae Hypocanthus amia (Linnaeus, 1758) caught in the Atlantic Ocean, near Dakar, Senegal. Live worms were rinsed with a 0.9 % NaCl solution and fixed in cold (4ºC) 2.5 % glutaraldehyde in 0.1 M sodium cacodylate buffer at pH 7.2, then rinsed in 0.1 M sodium cacodylate buffer at pH 7.2, post-fixed in cold (4ºC) 1 % osmium tetroxide in the same buffer for 1 h, dehydrated in ethanol and propylene oxide, embedded in Spurr’s resin and finally polymerized at 60 ºC for 24 h. Ultrathin sections (60-90 nm thick) were obtained using an Ultramicrotome (Power tome PC, RMC Boeckeler ®) with diamond knife. Sections placed on copper grids were double-stained with uranyl acetate and lead citrate. Sections placed on gold grids were stained with periodic acid, thiocarbohydrazide and silver proteinate to reveal the presence of glycogen (Thiéry 1967). The grids were examined in a Hitachi H-7650 transmission electron microscope operated at 80kV, at the “Service d'Étude et de Recherche en Microscopie Électronique” of the University of Corsica (Corte, France).
3. Results Observation and analyses of numerous cross and longitudinal sections of mature spermatozoa enabled us to distinguish, from anterior to posterior extremity, four regions in the mature spermatozoon of Sclerodistomum italicum.
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3. 1.
Region I (Figures. 1a-e and 3I)
This region corresponds to the anterior extremity of the spermatozoon. It is entirely covered by extramembranous ornamentations (Figure 1a). Cross-sections in different parts of this region demonstrate that the anterior extremity of the first axoneme appears first (Figure 1b) followed by the anterior end of the second axoneme (Figure 1c). Later, both axonemes are visible (Figure 1d-e). Cross-sections in this region have permitted us to distinguish two types of extramembranous ornamentations based on the differences in their structure. The type I (Eo1) is a discontinuous layer associated with short cortical microtubules. It appears as a filamentous structure in front of each cortical microtubule (Figure 1c). The type II (Eo2) is a continuous row in the external surface of the plasma membrane (Figure 1d-e). Extramembranous ornamentations are present only in this party of the mature spermatozoon of S. italicum. 3. 2.
Region II (Figures 1f-i and 3II)
This region is characterized by the appearance of long cortical microtubules (Figure 1f), the increase of the number of these cortical microtubule to 25-28 (Figure 1g) and the appearance of the mitochondrion in the posterior part of this region (Figure 1h-i). 3. 3.
Region III (Figures 2a-b and 3III)
Cross-sections in this region exhibit the appearance of the nucleus in addition to the mitochondrion, the two axonemes and cortical microtubules (Figure 2a). However, crosssections in the posterior part of this region show the disappearance of the mitochondrion and the disorganization of the first axoneme (Figure 2b and Figure 3III). 3. 4.
Region IV (Figures 2c-d and 3IV)
This region corresponds to the posterior part of the spermatozoon. It is characterized by the presence of only the second axoneme, the nucleus and cortical microtubules (Figure 2c). Cross-sections in the anterior part of this region reveal the progressive disappearance of the cortical microtubules followed by disappearance of the nucleus. The posterior extremity of the spermatozoon contains only the extremity of the second axoneme (Figure 2d). The glycogen granules are clearly highlighted with the Thiéry’s method and micrographs show their abundance in the region II of the mature spermatozoon of S. italicum (Figure 2e).
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4. Discussion The mature spermatozoon of Sclerodistomum italicum has characteristics usually described in the mature spermatozoa of the majority of the Digenea, namely the presence of two axonemes of the 9 + “1” pattern of the Trepaxonemata, mitochondrion, nucleus and parallel cortical microtubules (Jamieson and Daddow 1982; Ehlers 1986; Iomini and Justine 1997; Miquel et al. 2000; Quilichini et al. 2007; Ndiaye et al. 2011; Dione et al. 2016). However, the mature spermatozoon of S. italicum also shows some specific features. The simultaneous presence of two types of extramembranous ornamentations is described only in hemiuroids (Justine and Mattei 1982, 1984b; Ndiaye et al. 2013a, 2014). Usually, literature describes only one type of extramembranous ornamentation of the plasma membrane in the spermatozoa (Iomini and Justine 1997; Agostini et al. 2005; Ndiaye et al. 2011, 2015). Another distinguishing feature is the presence of short cortical microtubules in the anterior part of the spermatozoon and longer cortical microtubules in the middle and posterior regions of the spermatozoon. Among digeneans this character is known only in rerpesentatives of the Hemiuroidea. It is demonstrated in micrographs of Gonapodasmius sp. (Justine and Mattei 1982), Prosorchis palinurichthi (Ndiaye et al. 2013b) and Lecithochirium microstomum and L. musculus (Ndiaye et al. 2014) but none of these authors commented on it except in Gonapodasmius sp. In this species, Justine and Mattei (1982) have described an anterior region with peripheral microtubules, an intermediate region without cortical microtubules and a middle region with only ventral microtubules. The number of cortical microtubules reported in the hemiuroids is usually low, from 5 to 10 in the Hemiuroidea and the Lecithasteridae. In contrast, Gonapodasmius sp. (Justine and Mattei 1982) and in the Sclerodistomidae Prosorchis palinurichthi (Ndiaye et al. 2013b) and in S. italicum (present study) have respectively 28, 25 and 28 cortical microtubules in the mature spermatozoon. Thus, this feature requires additional attention from the viewpoint of its phylogenetic utility. Spine-like bodies, previously described in the spermatozoa of most digeneans (Quilichini et al. 2009; Ternengo et al. 2009; Foata et al. 2012) are absent in S. italicum, similarly to the condition in other hemiuroids. The Table 1 summarizes the main characters found in the spermatozoon of all hemiuroids studied so far. The table show significant similarity in the ultrastructural features of the spermatozoa in this superfamily, namely the presence of filamentous ornamentation in the anterior extremity of the spermatozoon which becomes the external ornamentation of the plasma membrane; the
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presence of only a single mitochondrion, the presence of only one bundle of cortical microtubules in the middle part of the spermatozoon and the posterior extremity of the spermatozoon with only singlets becoming to the disorganization of doublets of the second axoneme. The presence of only one bundle of cortical microtubules in the mature spermatozoon is known currently only in hemiuroids and is likely a synapomorphy of this group. 5. Author contributions All authors have participated in the consolidation and development of the manuscript.
6. Acknowledgments The authors are grateful to the anonymous referees for theirs comments, Online Acces to Research in the Environment (OARE) and the Ministère de l’Enseignement Supérieur et de la Recherche Scientifique et Technique du Sénégal for facilitating access to literature.
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Levron C, Miquel J, Oros M, Scholz T (2010) Spermatozoa of tapeworms (Platyhelminthes, Eucestoda): advances in ultrastructural and phylogenetic studies. Biol Rev 85: 523 - 543. doi: 10.1111/j.1469-185X.2009.00114.x. Miquel J, Hidalgo C, Feliu C, Marchand B (2000) Sperm ultrastructure of Taenia mustelae (Cestoda, Taeniidae), an intestinal parasite of the weasel, Mustela nivalis (Carnivora). Invert Reprod Dev 38: 43-51. Ndiaye PI, Bakhoum AJS, Sène A, Diagne PM, Miquel J (2015) The ultrastructural characters of the spermatozoon of Opechona bacillaris (Molin, 1859) (Digenea, Lepocreadiidae) a parasite of Scomber colias Gmelin, 1789 (Scombridae) off the coast of Dakar (Senegal). Acta Zool 96: 91-98. doi: 10.1111/azo.12054. Ndiaye PI, Bakhoum AJS, Sène A, Miquel J (2013a) Ultrastructure of the spermatozoon of Parahemiurus merus (Linton, 1910) (Digenea: Hemiuroidea: Hemiuridae), a parasite of Sardinella aurita Valenciennes, 1847 and S. maderensis (Lowe, 1838) (Teleostei: Clupeidae)
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org/10.1016/j.jcz.2012.11.005. Ndiaye PI, Diagne PMB, Sène A, Bakhoum AJS, Miquel J (2012) Ultrastructure of the spermatozoon of the digenean Lecithocladium excisum (Rudolphi, 1819) (Hemiuroidea, Hemiuridae), a parasite of marine teleost in Senegal. Folia Parasitol 59: 173-178. Ndiaye PI, Quilichini Y, Sène A, Ba CT, Marchand B (2011) Ultrastructure of the spermatozoon of the digenean Cricocephalus albus (Kuhl & van Hasselt 1822) Loss, 1899 (Platyhelminthe, Pronocephaloidea, Pronocephalidae) parasite of the “hawksbill sea turtle” Eretmochelis imbricate (Linnaeus, 1766) in Senegal. Zool Anz 250: 215-222. doi: 10.1016/j.jcz.2011.04.002. Ndiaye PI, Quilichini Y, Sène A, Bray RA, Bâ CT, Marchand B (2013b) Prosorchis palinurichthi Kurochkin & Korotaeva, 1971 (Digenea, Sclerodistomidae): ultrastructure of the mature spermatozoon. Zool Anz 252: 404-409. doi: org/10.1016/j.jcz.2012.11.001. Ndiaye PI, Quilichini Y, Sène A, Tkach VV, Bâ CT, Marchand B (2014) Ultrastructural characters of the spermatozoa in Digeneans of the genus Lecithochirium Lühe, 1901 (Digenea, Hemiuridae), parasites of fishes: comparatives study of L. microstomum and L. musculus. Parasite 21: 1:12. doi: 10.1051/parasite/2014050.
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Olson PD, Cribb TH, Tkach VV, Bray RA, Littlewood DTJ (2003) Phylogeny and classification of the Digenea (Platyhelminthes: Trematoda). Int J Parasitol 30: 89-93. doi: 10.1016/S0020-7519 (03)00049-3. Olson PD, Littlewood DTJ (2002) Phylogenetics of the Monogenea – evidence from a medley of molecules. Int J Parasitol 32: 233-244. doi: org/10.1016/S00207519(01)00328-9. Pamplona-Basilio MC, Baptista-Farias MFD, Kohn A (2001) Spermatogenesis and spermiogenesis in Didymocystis wedli Ariola, 1902 (Didymozoidae, Digenea). Mem Inst Oswaldo Cruz 96: 1153-1159. Quilichini Y, Foata J, Marchand B (2007) Ultrastructural study of the spermatozoon of Nicolla testiobliquum (Digenea, Opecoelidae) parasite of brown trout Salmo trutta (Pisces, Teleostei). Parasitol Res 101: 1295-1301. doi: 10.1007/s00436-007-0636-2. Quilichini Y, Foata J, Justine J-L, Bray RA, Marchand B (2009) Sperm ultrastructure of the digenean Siphoderina elongata (Platyhelminthes, Cryptogonimidae) intestinal parasite of Nemipterus furcosus (Pisces, Teleostei). Parasitol Res 105: 87-95. doi: 10.1007/s00436-009-1366-4. Quilichini Y, Foata J, Justine J-L, Bray RA, Marchand B (2010) Spermatozoon ultrastructure of Aponurus laguncula (Digenea: Lecithasteridae), a parasite of Aluterus monoceros (Pisces, Teleostei). Parasitol Int 59: 22-28. doi: 10.1016/j.parint2009.06.007. Ternengo S, Quilichini Y, Katharios P, Marchand B (2009) Sperm ultrastructure of the gall bladder fluke Anisocoelium capitellatum (Digenea: Cryptogonimidae), a parasite of Uranoscopus scaber (Pisces: Uranoscopidae). Parasitol Res 104: 801-807. doi : 10.1007/s00436-008-1259-y. Thiéry JP (1967) Mise en évidence des polysaccharides sur coupes fines en microscopie électronique. J Microsc 6:987–1018. Tkach VV, Pawlowski J (1999) A new method of DNA extraction from the ethanol-fixed parasitic worms. Acta Parasitol 44:147–148. Tkach VV, Pawlowski J, Mariaux J (2000) Phylogenetic analysis of the suborder Plagiorchiata (Platyhelminthes, Digenea) based on partial 1srDNA sequences. Int J Parasitol 30: 89-93. doi: org/10.1016/S0020-7519(99)00163-0.
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8. Legend of figures
Figure 1. Mature spermatozoon of Sclerodistomum italicum. a- e Region I of the spermatozoon showing (a) Longitudinal section in the anterior extremity showing the first axoneme and the extramembranous ornamentation of the plasma membrane. (b) Crosssection in the anterior extremity of the spermatozoon showing only the anterior extremity of the first axoneme. (c) Cross-section in the anterior part of the spermatozoon with the first axoneme and the anterior extremity of the second axoneme. Please also note the type
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I of extramembranous ornamentation and short cortical microtubules of type 1. (d) Crosssection in the posterior extremity of the spermatozoon showing the two axoneme and absence of cortical microtubules. (e) Cross-section in the posterior extremity of the spermatozoon showing the two axonemes and the type II of extramembranous ornamentation. f-i Cross-sections in the region II of the spermatozoon showing (f-g) the two axonemes, the longer cortical microtubules of type 2 and attachment zones. (h-i) Sections showing two axonemes, cortical microtubules and mitochondrion. Aae1 = anterior extremity of the first axoneme, Aae2 = anterior extremity of the second axoneme, Ax1 = first axoneme, Ax2 = second axoneme, Az = attachment zones, Cm1 = cortical microtubules of type 1, Cm2 = cortical microtubules of type 2 Eo = extramembranous ornamentation, Eo1 = extramembranous ornamentation of type 1, Eo2 = extramembranous ornamentation of type 2, M = mitochondrion. Scale Bar = 0.2 µm.
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Figure 2. Mature spermatozoon of Sclerodistomum italicum.a-b Cross-sections in the region III of the mature spermatozoon showing the two axonemes, cortical microtubules of type 2, mitochondrion and nucleus (a). Then the two axonemes, nucleus and cortical microtubules of type 2 (b). c-d Cross-sections in the posterior region of the spermatozoon
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showing only one axoneme, cortical microtubules of type 2 and nucleus (c). (d) Crosssection in the posterior extremity of the spermatozoon with only one axoneme. (e) Transmission electron micrograph of spermatozoa of Sclerodistomum italicum showing glycogen granules (G) revealed by the test of Thiéry. Ax= axoneme, Cm2 = cortical microtubules of type 2, G = glycogen granules, M = mitochondrion, N = nucleus. Scale Bar = 0.2 µm.
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Figure 3: A schematic reconstruction of the mature spermatozoon of Sclerodistomum italicum. Arrowheads indicate attachment zones. Aae1 = anterior extremity of the axoneme 1, Aae2 = anterior extremity of the axoneme 2, Ax1 = axoneme 1, Ax2 = second axoneme, Ase = anterior spermatozoon extremity,Cm1 = cortical microtubule of type 1, Cm2 = cortical microtubule of type 2, Eo1 = external ornamentation of type 1, Eo2 = external ornamentation of type 2, M = mitochondrion, N = nucleus, Pm = plasma membrane, Pae1
16
= posterior extremity of the axoneme 1, Pae2 = posterior extremity of the axoneme 2, Pse = posterior spermatozoon extremity.
17
Table 1: Some ultrastructural characteristics of the spermatozoon in the species of Hemiuroidea studied Ase
Number of mitochondria
Eo
Aae1 + Cm Aae1 + Eo + Cm
1 1 1
Hemiuridae Lecithochirium microstomum Lecithochirium musculus Lecithocladium excisum Parahemiurus merus Hemiurus appendiculatus
Aae1 + Eo + Cm Aae1 + Eo + Cm Aae1 + Eo Aae1 + Eo + Cm Aae1 + Eo + Cm
Lecithasteridae Aponurus laguncula Sclerodistomidae Prosorchis palinurichthi Sclerodistomum italicum
Families and species Didymozoidae Didymocystis wedli Didymozoon sp. Gonapodasmius sp.
Cm
Pse
References
28
Pae2
Pamplona-Basilio et al. (2001) Justine and Mattei (1983, 1984a) Justine and Mattei (1982a, 1984b)
1 1 1 1 1
8 6 8 5 7
Pae2 Pae2 Pae2 Pae2 Pae2
Ndiaye et al. (2014) Ndiaye et al. (2014) Ndiaye et al. (2012) Ndiaye et al. (2013a) Dione et al. (2016)
Eo
1
10
Pae2
Quilichini et al. (2010)
FEo+Eo FEo+Eo
1 1
25 28
Pae2 Pae2
Ndiaye et al. (2013b) Present study
Bund
n
FEo+Eo
1
1 1 1 1 1
FEo+Eo FEo+Eo Eo Eo FEo + Eo
Aae1 + Eo
1
Aae1 + Cm Aae1 + Cm +Eo
1 1
Aae1 = anterior extremity of the first axoneme, Ase = anterior spermatozoon extremity, Bund = number of bundle of cortical microtubules, Cm = cortical microtubules, Eo = external ornamentation, FEo = filamentous external ornamentation, n = maximum number of cortical microtubules, Pae2 = posterior extremity of the second axoneme, Pse = posterior spermatozoon extremity, - = absence of data.
S0040-8166(16)30229-4 http://dx.doi.org/doi:10.1016/j.tice.2016.12.007 YTICE 1065
To appear in:
Tissue and Cell
Received date: Revised date: Accepted date:
16-9-2016 18-12-2016 18-12-2016
Please cite this article as: Ndiaye, Papa Ibnou, Quilichini, Yann, Marigo, Adji Mama, Bˆa, Cheikh Tidiane, Tkach, Vasyl V., Marchand, Bernard, Ultrastructural characteristics of the mature spermatozoon of the digenean Sclerodistomum italicum (Stossich, 1893) (Hemiuroidea, Sclerodistomidae) intestinal parasite of Hypocanthus amia (Teleostei, Carangidae).Tissue and Cell http://dx.doi.org/10.1016/j.tice.2016.12.007
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Ultrastructural characteristics of the mature spermatozoon of the digenean Sclerodistomum italicum (Stossich, 1893) (Hemiuroidea, Sclerodistomidae) intestinal parasite of Hypocanthus amia (Teleostei, Carangidae) Papa Ibnou Ndiayea*, Yann Quilichinib, Adji Mama Marigoa, Cheikh Tidiane Bâa, Vasyl V. Tkachc, Bernard Marchandb
a
Laboratory of Evolutionary Biology, Ecology and Management of Ecosystems, Faculty
of Sciences and Techniques, Cheikh Anta Diop University of Dakar, BP 5055, Dakar, Republic of Senegal b
UMR SPE 6134, CNRS – Università di Corsica, SERME Service d’Etude et de
Recherche en Microscopie Electronique, Campus Grimaldi, 20250 Corte, Corsica, France c
Department of Biology, University of North Dakota, Grand Forks, North Dakota, USA
Running title: Spermatozoon ultrastructure of Sclerodistomum italicum
(*): Corresponding author Name: Papa Ibnou Ndiaye Postal address: Laboratory of Evolutionary Biology, Ecology and Management of Ecosystems, Faculty of Sciences and Techniques, Cheikh Anta Diop University of Dakar, BP 5055, Dakar, Republic of Senegal E-mail address: [email protected] Phone number: 00221 778142834
Highlights
2
This paper provide the first ultrastructural data of the spermatozoon of a species becoming to the genus Sclerodistomum,
It’s described for first time in this work, the presence of two types of extramembranous ornamentations in the mature spermatozoon of a digenean,
The presence of short cortical microtubules in the anterior part of the spermatozoon in addition to the presence of only one bundle of cortical microtubules in the median part of the spermatozoon,
In accordance to the others mature spermatozoa described in Hemiuroidea, the spermatozoon of Sclerodistomum italicum lack spine-like body.
Abstract The mature spermatozoon of Sclerodistomum italicum is filiform, tapered at both ends and shows the following features: 2 axonemes of the 9 + “1” pattern of the Trepaxonemata, mitochondrion, nucleus and parallel cortical microtubules. The specific features of the spermatozoon of S. italicum include the simultaneous presence of two types of extramembranous ornamentations, the presence of short cortical microtubules in the anterior part of the spermatozoon and the presence of only one bundle of cortical microtubules in the median part of the spermatozoon. Thus far these structures are known only in the Hemiuroidea. The presence of filamentous ornamentation in the anterior extremity of the spermatozoon has not previously been described in the Sclerodistomidae. Similar to spermatozoa of other hemiuroideans, S. italicum lack spine-like bodies described in spermatozoa of many digenean taxa. The posterior extremity of the spermatozoon exhibits the same ultrastructural characteristics typical of the Hemiuroidea. Keywords: Digenea; Sclerodistomidae; Sclerodistomum italicum; Sperm; ultrastructure.
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1. Introduction The Digenea is the largest group of internal metazoan parasites. They are an extremely ubiquitous group, parasitizing all major vertebrate groups as definitive host, gastropods and other mollusks as first intermediate hosts and representatives of several animal phyla as second intermediate host, when it is present. Diseases caused by digeneans in humans, domestic animals and wildlife receive considerable attention from researchers across a broad range of disciplines. On the other hand, due to their broad distribution and complex life cycles, digeneans have long been an important model in evolutionary, ecological and biodiversity studies. Both applied and basic research of digeneans benefit from molecular phylogenetic analyses of digeneans which have been published at an accelerating rate in the last 20-25 years (Barker et al. 1993; Tkach and Pawlowski 1999; Tkach et al. 2000; Cribb et al. 2001; Olson and Littlewood 2002; Olson et al. 2003; Levron et al. 2010). As a part of this global effort, Blair et al. (1998) have conducted a molecular phylogenetic study of the Hemiuroidea, a large group of digeneans parasitizing primarily marine teleosts, but also occurring in freshwater teleosts, elasmobranchs, amphibians, and reptiles. These authors have concluded that previous phylogenies of hemiurids based on characters derived from morphology and life cycles have been controversial. The relationships within the hemiuroids, and exact number of groups that should be included in the taxon, have been debated (Blair et al. 1998). The current system recognizes at least the families Hemiuridae, Accacoeliidae, Bathycotylidae,
Derogenidae,
Dictysarcidae,
Lecithasteridae,
Ptychogonimidae,
Hirudinellidae,
Sclerodistomidae,
Isoparorchiidae,
Sclerodistomoididae
and
Syncoeliidae within the Hemiuroidea (Gibson et al. 2002). However, most authors agree that the phylogenetic relationships among hemiuroids remain controversial and require a combination of morphological, molecular and ultrastructural data (Pamplona-Basilio et al. 2001; Levron et al. 2010; Ndiaye et al. 2013a, 2014). To our knowledge, ultrastructural data of the spermatozoon of hemiuroids exist only for the following taxa: the Didymozoidae: Gonapodasmius (Justine and Mattei 1982, 1984b), Didymozoon sp. (Justine and Mattei 1983, 1984a), Didymocystis wedli (Pamplona-Basilio et al. 2001); the Hemiuridae: Lecithocladium excisum (Ndiaye et al. 2012), Parahemiurus merus (Ndiaye et al. 2013a), Lecithochirium microstomum (Ndiaye et al. 2014), L. musculus (Ndiaye et al. 2014), Hemiurus appendiculatus (Dione et al. 2016); the Lecithasteridae: Aponurus laguncula (Quilichini et al. 2010); the
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Sclerodistomidae: Prosorchis palinurichthi (Ndiaye et al. 2013b). Sclerodistomids are normally parasites of the stomach of marine teleosts. The Sclerodistomidae currently includes only three subfamilies: Sclerodistominae, Kenmackenziinae and Prosorchiinae. Ultrastructural data on the spermatozoon of sclerodistomids exist only for the Prosorchiinae: Prosorchis palinurichthi. Herein, we present results of ultrastructural study of spermatozoon in Sclerodistomum italicum, the type species of the genus Sclerodistomum.
2.
Materials and Methods Adult specimens of Sclerodistomum italicum (Stossich, 1893) were collected from
the digestive tract of a teleost Holocentridae Hypocanthus amia (Linnaeus, 1758) caught in the Atlantic Ocean, near Dakar, Senegal. Live worms were rinsed with a 0.9 % NaCl solution and fixed in cold (4ºC) 2.5 % glutaraldehyde in 0.1 M sodium cacodylate buffer at pH 7.2, then rinsed in 0.1 M sodium cacodylate buffer at pH 7.2, post-fixed in cold (4ºC) 1 % osmium tetroxide in the same buffer for 1 h, dehydrated in ethanol and propylene oxide, embedded in Spurr’s resin and finally polymerized at 60 ºC for 24 h. Ultrathin sections (60-90 nm thick) were obtained using an Ultramicrotome (Power tome PC, RMC Boeckeler ®) with diamond knife. Sections placed on copper grids were double-stained with uranyl acetate and lead citrate. Sections placed on gold grids were stained with periodic acid, thiocarbohydrazide and silver proteinate to reveal the presence of glycogen (Thiéry 1967). The grids were examined in a Hitachi H-7650 transmission electron microscope operated at 80kV, at the “Service d'Étude et de Recherche en Microscopie Électronique” of the University of Corsica (Corte, France).
3. Results Observation and analyses of numerous cross and longitudinal sections of mature spermatozoa enabled us to distinguish, from anterior to posterior extremity, four regions in the mature spermatozoon of Sclerodistomum italicum.
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3. 1.
Region I (Figures. 1a-e and 3I)
This region corresponds to the anterior extremity of the spermatozoon. It is entirely covered by extramembranous ornamentations (Figure 1a). Cross-sections in different parts of this region demonstrate that the anterior extremity of the first axoneme appears first (Figure 1b) followed by the anterior end of the second axoneme (Figure 1c). Later, both axonemes are visible (Figure 1d-e). Cross-sections in this region have permitted us to distinguish two types of extramembranous ornamentations based on the differences in their structure. The type I (Eo1) is a discontinuous layer associated with short cortical microtubules. It appears as a filamentous structure in front of each cortical microtubule (Figure 1c). The type II (Eo2) is a continuous row in the external surface of the plasma membrane (Figure 1d-e). Extramembranous ornamentations are present only in this party of the mature spermatozoon of S. italicum. 3. 2.
Region II (Figures 1f-i and 3II)
This region is characterized by the appearance of long cortical microtubules (Figure 1f), the increase of the number of these cortical microtubule to 25-28 (Figure 1g) and the appearance of the mitochondrion in the posterior part of this region (Figure 1h-i). 3. 3.
Region III (Figures 2a-b and 3III)
Cross-sections in this region exhibit the appearance of the nucleus in addition to the mitochondrion, the two axonemes and cortical microtubules (Figure 2a). However, crosssections in the posterior part of this region show the disappearance of the mitochondrion and the disorganization of the first axoneme (Figure 2b and Figure 3III). 3. 4.
Region IV (Figures 2c-d and 3IV)
This region corresponds to the posterior part of the spermatozoon. It is characterized by the presence of only the second axoneme, the nucleus and cortical microtubules (Figure 2c). Cross-sections in the anterior part of this region reveal the progressive disappearance of the cortical microtubules followed by disappearance of the nucleus. The posterior extremity of the spermatozoon contains only the extremity of the second axoneme (Figure 2d). The glycogen granules are clearly highlighted with the Thiéry’s method and micrographs show their abundance in the region II of the mature spermatozoon of S. italicum (Figure 2e).
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4. Discussion The mature spermatozoon of Sclerodistomum italicum has characteristics usually described in the mature spermatozoa of the majority of the Digenea, namely the presence of two axonemes of the 9 + “1” pattern of the Trepaxonemata, mitochondrion, nucleus and parallel cortical microtubules (Jamieson and Daddow 1982; Ehlers 1986; Iomini and Justine 1997; Miquel et al. 2000; Quilichini et al. 2007; Ndiaye et al. 2011; Dione et al. 2016). However, the mature spermatozoon of S. italicum also shows some specific features. The simultaneous presence of two types of extramembranous ornamentations is described only in hemiuroids (Justine and Mattei 1982, 1984b; Ndiaye et al. 2013a, 2014). Usually, literature describes only one type of extramembranous ornamentation of the plasma membrane in the spermatozoa (Iomini and Justine 1997; Agostini et al. 2005; Ndiaye et al. 2011, 2015). Another distinguishing feature is the presence of short cortical microtubules in the anterior part of the spermatozoon and longer cortical microtubules in the middle and posterior regions of the spermatozoon. Among digeneans this character is known only in rerpesentatives of the Hemiuroidea. It is demonstrated in micrographs of Gonapodasmius sp. (Justine and Mattei 1982), Prosorchis palinurichthi (Ndiaye et al. 2013b) and Lecithochirium microstomum and L. musculus (Ndiaye et al. 2014) but none of these authors commented on it except in Gonapodasmius sp. In this species, Justine and Mattei (1982) have described an anterior region with peripheral microtubules, an intermediate region without cortical microtubules and a middle region with only ventral microtubules. The number of cortical microtubules reported in the hemiuroids is usually low, from 5 to 10 in the Hemiuroidea and the Lecithasteridae. In contrast, Gonapodasmius sp. (Justine and Mattei 1982) and in the Sclerodistomidae Prosorchis palinurichthi (Ndiaye et al. 2013b) and in S. italicum (present study) have respectively 28, 25 and 28 cortical microtubules in the mature spermatozoon. Thus, this feature requires additional attention from the viewpoint of its phylogenetic utility. Spine-like bodies, previously described in the spermatozoa of most digeneans (Quilichini et al. 2009; Ternengo et al. 2009; Foata et al. 2012) are absent in S. italicum, similarly to the condition in other hemiuroids. The Table 1 summarizes the main characters found in the spermatozoon of all hemiuroids studied so far. The table show significant similarity in the ultrastructural features of the spermatozoa in this superfamily, namely the presence of filamentous ornamentation in the anterior extremity of the spermatozoon which becomes the external ornamentation of the plasma membrane; the
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presence of only a single mitochondrion, the presence of only one bundle of cortical microtubules in the middle part of the spermatozoon and the posterior extremity of the spermatozoon with only singlets becoming to the disorganization of doublets of the second axoneme. The presence of only one bundle of cortical microtubules in the mature spermatozoon is known currently only in hemiuroids and is likely a synapomorphy of this group. 5. Author contributions All authors have participated in the consolidation and development of the manuscript.
6. Acknowledgments The authors are grateful to the anonymous referees for theirs comments, Online Acces to Research in the Environment (OARE) and the Ministère de l’Enseignement Supérieur et de la Recherche Scientifique et Technique du Sénégal for facilitating access to literature.
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Levron C, Miquel J, Oros M, Scholz T (2010) Spermatozoa of tapeworms (Platyhelminthes, Eucestoda): advances in ultrastructural and phylogenetic studies. Biol Rev 85: 523 - 543. doi: 10.1111/j.1469-185X.2009.00114.x. Miquel J, Hidalgo C, Feliu C, Marchand B (2000) Sperm ultrastructure of Taenia mustelae (Cestoda, Taeniidae), an intestinal parasite of the weasel, Mustela nivalis (Carnivora). Invert Reprod Dev 38: 43-51. Ndiaye PI, Bakhoum AJS, Sène A, Diagne PM, Miquel J (2015) The ultrastructural characters of the spermatozoon of Opechona bacillaris (Molin, 1859) (Digenea, Lepocreadiidae) a parasite of Scomber colias Gmelin, 1789 (Scombridae) off the coast of Dakar (Senegal). Acta Zool 96: 91-98. doi: 10.1111/azo.12054. Ndiaye PI, Bakhoum AJS, Sène A, Miquel J (2013a) Ultrastructure of the spermatozoon of Parahemiurus merus (Linton, 1910) (Digenea: Hemiuroidea: Hemiuridae), a parasite of Sardinella aurita Valenciennes, 1847 and S. maderensis (Lowe, 1838) (Teleostei: Clupeidae)
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Olson PD, Cribb TH, Tkach VV, Bray RA, Littlewood DTJ (2003) Phylogeny and classification of the Digenea (Platyhelminthes: Trematoda). Int J Parasitol 30: 89-93. doi: 10.1016/S0020-7519 (03)00049-3. Olson PD, Littlewood DTJ (2002) Phylogenetics of the Monogenea – evidence from a medley of molecules. Int J Parasitol 32: 233-244. doi: org/10.1016/S00207519(01)00328-9. Pamplona-Basilio MC, Baptista-Farias MFD, Kohn A (2001) Spermatogenesis and spermiogenesis in Didymocystis wedli Ariola, 1902 (Didymozoidae, Digenea). Mem Inst Oswaldo Cruz 96: 1153-1159. Quilichini Y, Foata J, Marchand B (2007) Ultrastructural study of the spermatozoon of Nicolla testiobliquum (Digenea, Opecoelidae) parasite of brown trout Salmo trutta (Pisces, Teleostei). Parasitol Res 101: 1295-1301. doi: 10.1007/s00436-007-0636-2. Quilichini Y, Foata J, Justine J-L, Bray RA, Marchand B (2009) Sperm ultrastructure of the digenean Siphoderina elongata (Platyhelminthes, Cryptogonimidae) intestinal parasite of Nemipterus furcosus (Pisces, Teleostei). Parasitol Res 105: 87-95. doi: 10.1007/s00436-009-1366-4. Quilichini Y, Foata J, Justine J-L, Bray RA, Marchand B (2010) Spermatozoon ultrastructure of Aponurus laguncula (Digenea: Lecithasteridae), a parasite of Aluterus monoceros (Pisces, Teleostei). Parasitol Int 59: 22-28. doi: 10.1016/j.parint2009.06.007. Ternengo S, Quilichini Y, Katharios P, Marchand B (2009) Sperm ultrastructure of the gall bladder fluke Anisocoelium capitellatum (Digenea: Cryptogonimidae), a parasite of Uranoscopus scaber (Pisces: Uranoscopidae). Parasitol Res 104: 801-807. doi : 10.1007/s00436-008-1259-y. Thiéry JP (1967) Mise en évidence des polysaccharides sur coupes fines en microscopie électronique. J Microsc 6:987–1018. Tkach VV, Pawlowski J (1999) A new method of DNA extraction from the ethanol-fixed parasitic worms. Acta Parasitol 44:147–148. Tkach VV, Pawlowski J, Mariaux J (2000) Phylogenetic analysis of the suborder Plagiorchiata (Platyhelminthes, Digenea) based on partial 1srDNA sequences. Int J Parasitol 30: 89-93. doi: org/10.1016/S0020-7519(99)00163-0.
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8. Legend of figures
Figure 1. Mature spermatozoon of Sclerodistomum italicum. a- e Region I of the spermatozoon showing (a) Longitudinal section in the anterior extremity showing the first axoneme and the extramembranous ornamentation of the plasma membrane. (b) Crosssection in the anterior extremity of the spermatozoon showing only the anterior extremity of the first axoneme. (c) Cross-section in the anterior part of the spermatozoon with the first axoneme and the anterior extremity of the second axoneme. Please also note the type
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I of extramembranous ornamentation and short cortical microtubules of type 1. (d) Crosssection in the posterior extremity of the spermatozoon showing the two axoneme and absence of cortical microtubules. (e) Cross-section in the posterior extremity of the spermatozoon showing the two axonemes and the type II of extramembranous ornamentation. f-i Cross-sections in the region II of the spermatozoon showing (f-g) the two axonemes, the longer cortical microtubules of type 2 and attachment zones. (h-i) Sections showing two axonemes, cortical microtubules and mitochondrion. Aae1 = anterior extremity of the first axoneme, Aae2 = anterior extremity of the second axoneme, Ax1 = first axoneme, Ax2 = second axoneme, Az = attachment zones, Cm1 = cortical microtubules of type 1, Cm2 = cortical microtubules of type 2 Eo = extramembranous ornamentation, Eo1 = extramembranous ornamentation of type 1, Eo2 = extramembranous ornamentation of type 2, M = mitochondrion. Scale Bar = 0.2 µm.
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Figure 2. Mature spermatozoon of Sclerodistomum italicum.a-b Cross-sections in the region III of the mature spermatozoon showing the two axonemes, cortical microtubules of type 2, mitochondrion and nucleus (a). Then the two axonemes, nucleus and cortical microtubules of type 2 (b). c-d Cross-sections in the posterior region of the spermatozoon
14
showing only one axoneme, cortical microtubules of type 2 and nucleus (c). (d) Crosssection in the posterior extremity of the spermatozoon with only one axoneme. (e) Transmission electron micrograph of spermatozoa of Sclerodistomum italicum showing glycogen granules (G) revealed by the test of Thiéry. Ax= axoneme, Cm2 = cortical microtubules of type 2, G = glycogen granules, M = mitochondrion, N = nucleus. Scale Bar = 0.2 µm.
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Figure 3: A schematic reconstruction of the mature spermatozoon of Sclerodistomum italicum. Arrowheads indicate attachment zones. Aae1 = anterior extremity of the axoneme 1, Aae2 = anterior extremity of the axoneme 2, Ax1 = axoneme 1, Ax2 = second axoneme, Ase = anterior spermatozoon extremity,Cm1 = cortical microtubule of type 1, Cm2 = cortical microtubule of type 2, Eo1 = external ornamentation of type 1, Eo2 = external ornamentation of type 2, M = mitochondrion, N = nucleus, Pm = plasma membrane, Pae1
16
= posterior extremity of the axoneme 1, Pae2 = posterior extremity of the axoneme 2, Pse = posterior spermatozoon extremity.
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Table 1: Some ultrastructural characteristics of the spermatozoon in the species of Hemiuroidea studied Ase
Number of mitochondria
Eo
Aae1 + Cm Aae1 + Eo + Cm
1 1 1
Hemiuridae Lecithochirium microstomum Lecithochirium musculus Lecithocladium excisum Parahemiurus merus Hemiurus appendiculatus
Aae1 + Eo + Cm Aae1 + Eo + Cm Aae1 + Eo Aae1 + Eo + Cm Aae1 + Eo + Cm
Lecithasteridae Aponurus laguncula Sclerodistomidae Prosorchis palinurichthi Sclerodistomum italicum
Families and species Didymozoidae Didymocystis wedli Didymozoon sp. Gonapodasmius sp.
Cm
Pse
References
28
Pae2
Pamplona-Basilio et al. (2001) Justine and Mattei (1983, 1984a) Justine and Mattei (1982a, 1984b)
1 1 1 1 1
8 6 8 5 7
Pae2 Pae2 Pae2 Pae2 Pae2
Ndiaye et al. (2014) Ndiaye et al. (2014) Ndiaye et al. (2012) Ndiaye et al. (2013a) Dione et al. (2016)
Eo
1
10
Pae2
Quilichini et al. (2010)
FEo+Eo FEo+Eo
1 1
25 28
Pae2 Pae2
Ndiaye et al. (2013b) Present study
Bund
n
FEo+Eo
1
1 1 1 1 1
FEo+Eo FEo+Eo Eo Eo FEo + Eo
Aae1 + Eo
1
Aae1 + Cm Aae1 + Cm +Eo
1 1
Aae1 = anterior extremity of the first axoneme, Ase = anterior spermatozoon extremity, Bund = number of bundle of cortical microtubules, Cm = cortical microtubules, Eo = external ornamentation, FEo = filamentous external ornamentation, n = maximum number of cortical microtubules, Pae2 = posterior extremity of the second axoneme, Pse = posterior spermatozoon extremity, - = absence of data.