Introduction

Among taeniid cestodes, the genus Echinococcus includes species with great medical and veterinary importance, causing important zoonotic infections, namely cystic and alveolar echinococcosis. Within this genus, Echinococcus multilocularis is the zoonotic agent of human alveolar echinococcosis or alveolar hydatid disease. It is present in the northern hemisphere, and its indirect life cycle includes wild canids (mainly foxes and wolfs) and also dogs as definitive hosts, harbouring the adult tapeworm, whereas some micromammals, particularly Arvicola terrestris and Microtus spp., act as intermediate hosts, harbouring the larval stage. Humans may also be infected by the metacestode of E. multilocularis and develop the alveolar hydatid disease.

The usefulness of ultrastructural characters of the spermatozoon for phylogenetic inference has been clearly demonstrated within Platyhelminthes, particularly in cestodes (Justine 1991a, b, 1998, 2001; Bâ and Marchand 1995; Miquel et al. 1999, 2007; Levron et al. 2010). Presently, there is a consensus concerning the necessity of integrating morphology, molecular and biological data for a better knowledge of systematics and evolution of Platyhelminthes (Hoberg et al. 1997, 1999; Littlewood et al. 1998; Olson et al. 2001; Waeschenbach et al. 2007, 2012). In the last years, numerous ultrastructural studies have been published concerning spermiogenesis and/or the spermatozoon of cestodes (see Marigo 2011). Most of them refer to the order Cyclophyllidea for which more than 60 species were analysed (Marigo 2011; Yoneva et al. 2012; Bâ et al. 2014; Miquel et al. 2015). Concerning tapeworms of the family Taeniidae, there are ultrastructural and spermatological studies for ten species, namely Echinococcus granulosus (Morseth 1969), E. multilocularis (Barrett and Smyth 1983; Shi et al. 1994), Taenia crassiceps (Willms et al. 2004; Willms and Robert 2007), T. hydatigena (Featherston 1971; Miquel et al. 2015), T. mustelae (Miquel et al. 2000), T. parva (Ndiaye et al. 2003), T. pisiformis (Tian et al. 1998a, b), T. saginata (Tian et al. 1998a, b; Bâ et al. 2011), T. solium (Tian et al. 1998a, b; Willms et al. 2003) and T. taeniaeformis (Miquel et al. 2009a, b). However, studies on the spermatozoon ultrastructure of the genus Echinococcus have been rather neglected; the available results are limited to briefly illustrated papers or conference proceedings presenting incomplete ultrastructural data on the organisation of their sperm cells.

The aim of the present work concerns the ultrastructural study of E. multilocularis in order to draw a complete description of the spermatozoon for comparison with other cyclophyllideans, particularly taeniids. The present work constitutes the first complete study of the ultrastructural organisation of the male gamete in a species of the genus Echinococcus. In the framework of the Paravac project, the present study on sperm ultrastructure is essential for future works with both E. granulosus and E. multilocularis concerning the immunohistochemical investigation of the expression sites of some antigenic proteins during sperm development.

Materials and methods

Live specimens of Echinococcus multilocularis were isolated from the intestine of a naturally infected red fox (Vulpes vulpes L.) from La Roche sur Foron (France) captured in June 2014.

Adult recovered tapeworms were immediately rinsed with a 0.9 % NaCl solution. Later, 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 0.1 M sodium cacodylate buffer at pH 7.4, post-fixed in cold (4 °C) 1 % osmium tetroxide with 0.9 % potassium ferricyanide in the same buffer for 1 h, rinsed in MilliQ water (Millipore Gradient A10), dehydrated in an ethanol series and propylene oxide, embedded in Spurr’s resin and polymerised at 60 °C for 72 h. Ultrathin sections (60–90-nm thick) of mature segments at the level of the vas deferens were obtained in a Reichert-Jung Ultracut E ultramicrotome. Sections were placed on 200-μm mesh copper grids and double-stained with uranyl acetate and lead citrate according to the Reynolds (1963) methodology. The grids were examined in a JEOL 1010 transmission electron microscope operated at 80 kV, in the “Centres Científics i Tecnològics” of the University of Barcelona (CCiTUB).

Results

The observation of numerous ultrathin sections has enabled us to establish the main ultrastructural characteristics of the mature spermatozoon of Echinococcus multilocularis (Figs. 1, 2 and 3). The spermatozoon is a filiform cell, tapered at both extremities and lacking mitochondria. Four consecutive regions (I–IV) with differential ultrastructural features can be distinguished.

Fig. 1
figure 1

Spermatozoon of Echinococcus multilocularis. a Longitudinal section of region I. b and c Two details of the anterior spermatozoon extremity showing the apical cone. d and e Cross-sections of region I at the level of centriole and axoneme, respectively. f Transitional area between regions I and II. g and h Cross-sections of region II, anterior and posterior part, respectively. AC apical cone, ASE anterior spermatozoon extremity, Ax axoneme, C centriole, CB crested body, CM cortical microtubules, IW intracytoplasmic walls, PS periaxonemal sheath

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Fig. 2
figure 2

Spermatozoon of Echinococcus multilocularis. a Longitudinal section of region II. b and c Longitudinal sections of region III, anterior part and posterior part, respectively. d–f Cross-sections of region III showing the nuclear morphology evolving from horseshoe (anterior part of region, level * in a) to annular shape (posterior part of region, level ** in c). g Longitudinal section of region IV showing the posterior spermatozoon extremity. h–j Cross-sections of region IV showing the progressive disorganisation of the axoneme. Ax axoneme, C centriole, CB crested body, CM cortical microtubules, D doublets, IW intracytoplasmic walls, N nucleus, PS periaxonemal sheath, PSE posterior spermatozoon extremity, S singlets

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Fig. 3
figure 3

Schematic reconstruction of the spermatozoon of Echinococcus multilocularis. AC apical cone, ASE anterior spermatozoon extremity, Ax axoneme, C centriole, CB crested body, CM cortical microtubules, D doublets, IW intracytoplasmic walls, N nucleus, PM plasma membrane, PS periaxonemal sheath, PSE posterior spermatozoon extremity

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Region I (Figs. 1a–f and 3i) constitutes the anterior extremity of the spermatozoon. This region is mainly characterised by the presence of an apical cone and two crested bodies. From the anterior tip of the spermatozoon to the centriole, there is an electron-dense apical cone (Fig. 1a–c) more than 900-nm long and about 200-nm wide. The crested bodies are formed by two helical cords very close to one another that externally surround the sperm cell throughout region I (Fig. 1a–f). The maximum thickness of crested bodies is about 75 nm at the level of the centriole (Fig. 1b–d). The thickness of the crested bodies decreases progressively toward the end of region I (Fig. 1f). The axoneme, of the 9 + ‘1’ trepaxonematan pattern (Fig. 1e), is centrally located and surrounded by a thin layer of electron lucent cytoplasm and by an electron-dense submembranous layer of cortical microtubules spirally arranged at an angle of about 45° in relation to the hypothetical longitudinal axis of the spermatozoon (Fig. 1b, c).

Region II (Figs. 1f–h, 2a and 3II) is devoid of crested body being characterised by the progressive appearance of both periaxonemal sheath and intracytoplasmic walls (Fig. 1g–h). In fact, both periaxonemal sheath and intracytoplasmic walls are absent in the anterior part of region II (Fig. 1g). The periaxonemal sheath is a striated layer that surrounds the axoneme, and the intracytoplasmic walls consist in transverse structures that connect the periaxonemal sheath with the peripheral layer of spiralled cortical microtubules.

Region III (Figs. 2b–f and 3III) corresponds to the nuclear area of the mature spermatozoon. The nucleus forms a loose spiral around the axoneme, presenting a horseshoe shape in the anterior part of region III (Fig. 2b, d, e) and an annular shape in the posterior part (Fig. 2c, f). The nucleus is localised between the rods of the periaxonemal sheath and the axoneme (Fig. 2b). In the posterior part of this region, the nucleus encircles totally the axoneme and periaxonemal sheath, while the intracytoplasmic walls disappear (Fig. 2f).

Region IV (Figs. 2g–j and 3IV) constitutes the posterior spermatozoon extremity. It is characterised by the sole presence of the axoneme surrounded by the plasma membrane (Fig. 2h). Near the posterior spermatozoon extremity, the axoneme becomes disorganised (Fig. 2i, j), and the posterior tip of the male gamete consists of electron-dense material surrounded by the plasma membrane (Fig. 2g).

Discussion

The spermatozoon of Echinococcus multilocularis is a filiform cell, which lacks mitochondrion as in all the studied cestoidean (polyzoic) cestodes (Justine 1998, 2001). Contrarily, spermatozoa of monozoic cestodes (Gyrocotylidea and Amphilinidea), as in all other non-cestode platyhelminths, exhibit mitochondrion (Xylander 1989). Thus, the absence of mitochondrion in the sperm cells was postulated as a synapomorphy for the Eucestoda (Justine 1998, 2001).

According to Levron et al. (2010), four different patterns of spermatozoa were described in cyclophyllideans (IV to VII). Type IV spermatozoa can be differentiated from the remaining three due to the presence of parallel cortical microtubules. The latter are present in phyllobothriids and lecanicephalideans and also in mesocestoidids, representing a particular case within cyclophyllideans (Miquel et al. 1999, 2007). The three remaining spermatozoon types (V to VII) exhibit spiralled cortical microtubules and are observed in different families of cyclophyllideans. Type V spermatozoa are present in some anoplocephalids, in hymenolepidids and in nematotaeniids. Spermatozoa of type VI are observed in other anoplocephalids, in catenotaeniids, in dilepidids, in dipylidiids and in gryporhynchids. Finally, type VII spermatozoa are present in some anoplocephalids, in davaineids, in metadilepidids, in paruterinids and in taeniids, and also in tetrabothriideans (see Levron et al. 2010). The ultrastructural organisation of the sperm cell of Echinococcus multilocularis follows the type VII pattern. It corresponds to a uniflagellate spermatozoon that presents crested bodies, periaxonemal sheath and intracytoplasmic walls, spiralled cortical microtubules and nucleus spiralled around the axoneme.

The existing ultrastructural studies of spermatozoa in the family Taeniidae concern eight species of the genus Taenia and two of the genus Echinococcus (see Table 1). For Echinococcus, the present study is the first comprehensive work describing the complete ultrastructural organisation of the sperm cell. With respect to Taenia species, it is interesting to remark that the published results on T. pisiformis are jointly presented with those of T. saginata and T. solium by Tian et al. (1998a), and the authors do not identify the species corresponding to each TEM micrograph. For the two human Taenia species, there are additional papers showing some aspects concerning the ultrastructural organisation of their sperm cells (Willms et al. 2003; Bâ et al. 2011). Unfortunately, there are no other papers on T. pisiformis, and consequently, this species is not included in Table 1.

Table 1 Spermatological characteristics of the species of the family Taeniidae
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Within taeniids, it is crucial to perform an exhaustive comparison of sperm cell ultrastructure in species of Echinococcus with those of Taenia. To date, the ultrastructural knowledge concerning the male gamete of species of the genus Echinococcus is confined to a few papers or proceedings containing a reduced amount of information. For E. granulosus, Morseth (1969) refers the particular 9 + ‘1’ pattern of the axoneme, the absence of mitochondrion, and the spiralled disposition of both cortical microtubules and nucleus. Also, the published TEM micrographs show the horseshoe shape of the nucleus in cross-sections. For E. multilocularis, Barrett and Smyth (1983) describe the early abortion of one basal body and associated axoneme during spermiogenesis and, consequently, the presence of a single axoneme in the sperm cell. Also, Shi et al. (1994) show the presence of periaxonemal sheath in the spermatozoon of E. multilocularis. With respect to the genus Taenia, even though information on the spermatozoa ultrastructure is available for eight Taenia species (see Table 1), complete descriptions are available for only five of them, namely T. hydatigena, T. mustelae, T. parva, T. saginata and T. taeniaeformis (Miquel et al. 2000, 2009a, 2015; Ndiaye et al. 2003; Bâ et al. 2011). Considering the need to fully understand the sperm cell ultrastructural organisation within taeniids, the present paper provides a full description of the ultrastructural organisation of the sperm cell of an Echinococcus species. The most relevant results concern the crested bodies. These structures are helical cords that externally surround the spermatozoon of most cestodes, and, if present, they always characterise the anterior spermatozoon region. All of the taeniids studied to date exhibit a single crested body, whereas two crested bodies were observed in the mature spermatozoon of E. multilocularis. Previous ultrastructural studies on both E. granulosus and E. multilocularis sperm cells do not remark any information concerning crested bodies (Morseth 1969; Barrett and Smyth 1983; Shi et al. 1994). As for the remaining ultrastructural characters found in the sperm cell of E. multilocularis, they are present in the remaining studied taeniids (see Table 1), emphasising the potential importance of finding two crested bodies in E. multilocularis sperm cells in the present work. A complete ultrastructural study of other congener species would be of particular interest in order to confirm whether or not the presence of two crested bodies is a characteristic of the genus Echinococcus.