Ultrastructural study of sperm cells in Acanthocolpidae: the case of Stephanostomum murielae and Stephanostomoides tenuis (Digenea)

Anterior spermatozoon extremity

The anterior spermatozoon extremity of Stephanostomum murielae contains one axoneme and some cortical microtubules (about one or two) whereas in the anterior spermatozoon extremity of Stephanostomoides tenuis two axonemes slightly longitudinally displaced one to another were observed, surrounded by about 20 cortical microtubules. Spermatozoa showing two axonemes in their anterior extremities have also been reported in Deropristis inflata (Foata, Quilichini & Marchand, 2007) and Neoapocreadium chabaudi (Kacem et al., 2010). However, in the latter, the anterior spermatozoon extremity also contains external ornamentation of the plasma membrane. In the lepocreadioidean species studied so far, one axoneme accompanied by electron dense material in the anterior spermatozoon extremities was described in the Aephnidiogenidae Holorchis micracanthum (Bâ et al., 2011), the Gyliauchenidae Gyliauchen sp. and Robphildollfusium fractum (Quilichini et al., 2011; Bakhoum et al., 2012) and the Lepocreadiidae Hypocreadium caputvadum and Opechona bacillaris (Kacem et al., 2012; Ndiaye et al., 2015).

The distinction in the anterior spermatozoon tip of the two acanthocolpids studied here, compared to lepocreadioidean species, is the presence of cortical microtubules in the anterior tip (Table 1). In fact, some cortical microtubules are observed in the anterior spermatozoon tip in S. murielae (about 2) and S. tenuis (about 20), whereas in all the lepocreadioids species described until now the cortical microtubules are absent in the anterior spermatozoon tip. Nevertheless, their appearance is noted only when both axonemes are already formed (see Bâ et al., 2011; Quilichini et al., 2011; Bakhoum et al., 2012; Kacem et al., 2012; Ndiaye et al., 2015).

The variability of the anterior spermatozoon extremity may be an important criterion for phylogenetic analysis and would also be interesting when establishing spermatozoa models in digeneans.

Antero-lateral electron-dense material

Electron dense material is located in the anterior spermatozoon extremity partially surrounding the second axoneme beneath the plasma membrane. In fact, the simultaneous presence of this antero-lateral electron-dense material and the absence of cortical microtubules in the anterior tip of the mature spermatozoon are described in some Lepocreadioidea studied (see Ndiaye et al., 2015, Table 1). In agreement with Ndiaye et al. (2015), these features may characterize the Lepocreadioidea, distinguishing their mature spermatozoa from those of other species of digeneans. Moreover, this anterolateral electron dense material could be an apomorphic character considering its absence in most digenean spermatozoa.

In the mature spermatozoa of S. murielae and S. tenuis antero-lateral electron-dense material is not observed. The absence of antero-lateral electron-dense material is also reported in the spermatozoon of the apocreadiid N. chabaudi (Kacem et al., 2010) and deropristid D. inflata (Foata, Quilichini & Marchand, 2007). It is interesting to remark that these three families (Acanthocolpidae, Apocreadiidae and Deropristidae) are placed outside of the Lepocreadioidea by Bray & Cribb (2012) in their recent re-organisation of the superfamily. The absence of antero-lateral electron-dense material in the mature spermatozoon of the studied species belonging to Acanthocolpidae (present study), Apocreadiidae (Kacem et al., 2012) and Deropristidae (Foata, Quilichini & Marchand, 2007) are an ultrastructural argument supporting the exclusion of these families from the Lepocreadioidea (Table 1). Moreover, more spermatological studies are needed on all families, but particularly the Acanthocolpidae, Apocreadiidae and Deropristidae, considering that only one study has been carried out in each family.

External ornamentations and spine-like bodies

The presence of an association “external ornamentation + cortical microtubules + spine-like bodies” has been observed in the anterior area of the spermatozoon of most digeneans species (Justine & Mattei, 1983; Justine, Jamieson & Southgate, 1993; Miquel et al., 2006; Miquel et al., 2013; Quilichini, Foata & Marchand, 2007; Bakhoum et al., 2011b; Bakhoum, 2012). In the spermatozoon of S. murielae and in S. tenuis such association is observed in the area containing the first mitochondrion. The location of external ornamentations in both acanthocolpids corresponds to the type 2 of digenean spermatozoa, according to the localization of the external ornamentation established by Quilichini et al. (2011) (Table 1).

In the mature spermatozoon of the deropristid D. inflata (Foata, Quilichini & Marchand, 2007), external ornamentations are present associated with cortical microtubules but spine-like bodies are absent. In the apocreadiid N. chabaudi (Kacem et al., 2010), spine-like bodies are present but not in the ornamented area of the spermatozoon.

Within the superfamily Lepocreadioidea as recognised by Bray & Cribb (2012) the association “external ornamentation + cortical microtubules” is present in all species studied (see Ndiaye et al., 2015, Table 1). However, spine-like bodies are absent in H. micracanthum (Bâ et al., 2011) and H. caputvadum (Kacem et al., 2012), while they are present in Gyliauchen sp. (Quilichini et al., 2011), R. fractum (Bakhoum et al., 2012) and O. bacillaris (Ndiaye et al., 2015).

What we refer to as ‘spine-likes bodies,’ are reported, to our knowledge, only in the spermatozoa of digeneans (see Bakhoum, 2012; Bruňanská et al., 2014) and consequently need more attention in phylogenetic analyses.

Number and morphology of mitochondria

The mature spermatozoa of Stephanostomum murielae and Stephanostomoides tenuis possess two mitochondria. The first mitochondrion is located at the level of the external ornamentation of the plasma membrane, and the second one in the area containing the nucleus. In the spermatozoon of Stephanostomoides tenuis a particular anterior mitochondrion is observed, appearing as successive bulges interconnected by a cord. This type of mitochondrion was named “moniliform” by Bâ et al. (2011) and was also reported in O. bacillaris by Ndiaye et al. (2015) (Table 1). The presence of two mitochondria has been described in several species, including the apocreadiid N. chabaudi (Kacem et al., 2010), the deropristid D. inflata (Foata, Quilichini & Marchand, 2007), the gyliauchenid R. fractum (Bakhoum et al., 2012), the lepocreadiids H. caputvadum (Kacem et al., 2012) and O. bacillaris (Ndiaye et al., 2015), the fasciolid Fasciola hepatica (Ndiaye et al., 2003), and the troglotrematid Troglotrema acutum (Miquel et al., 2006). However, in most digenean spermatozoa studied until now only one mitochondrion is reported (see Bakhoum, 2012).

It is also important to notice that three mitochondria were also described in the mature spermatozoon of the paramphistomids Basidiodiscus ectorchus and Sandonia sudanensis (Ashour, Garo & Gamil, 2007), the cryptogonimid Anisocoelium capitellatum (Ternengo et al., 2009) and the heterophyid Euryhelmis squamula (Bakhoum et al., 2009).

In the present study, the morphology of the first mitochondrion is the main ultrastructural characteristic that distinguishes the mature spermatozoa of Stephanostomum murielae from those of Stephanostomoides tenuis.

Morphology of posterior spermatozoon extremity

The morphology of the posterior spermatozoon extremity presents a great variability that emphasise the usefulness of this criterion when establishing spermatozoon models in Digenea (Quilichini et al., 2010b; Bakhoum, 2012). In S. murielae and S. tenuis the posterior spermatozoon extremity is devoid of cortical microtubules and exhibits only the nucleus. This morphology corresponds to the fasciolidean type or type 2 according to Quilichini et al. (2010b). A posterior spermatozoon extremity showing only a nucleus is reported in most digeneans (Ndiaye et al., 2003; Ndiaye et al., 2012; Quilichini et al., 2010b; Bakhoum et al., 2011a; Bakhoum et al., 2011b). In the superfamily Lepocreadioidea (Bray & Cribb, 2012), a posterior spermatozoon extremity containing only a nucleus is observed in Robphildollfusium fractum (Bakhoum et al., 2012) while in the remaining species only one axoneme is present as described in Holorchis micracanthum (Bâ et al., 2011), Gyliauchen sp. (Quilichini et al., 2011), Hypocreadium caputvadum (Kacem et al., 2012) and Opechona bacillaris (Ndiaye et al., 2015) (Table 1).

Additionally, a posterior spermatozoon extremity containing cortical microtubules has been reported in other digeneans, particularly in the families Opecoelidae and Opistholebetidae (Miquel, Nourrisson & Marchand, 2000; Levron, Ternengo & Marchand, 2004; Quilichini et al., 2007). Moreover, in the lecithasterid Aponurus laguncula (Quilichini et al., 2010a) the posterior spermatozoon extremity containing a mitochondrion has been described.

These different types of posterior spermatozoon extremity could be used when establishing spermatozoon models considering that this criterion may allow distinguishing families or superfamilies within the Digenea.

Phylogenetical significances

The mature spermatozoon of Stephanostomum murielae and Stephanostomoides tenuis share several ultrastructural features (Table 1). The main differences in their mature spermatozoon are the morphology of the anterior spermatozoon extremity and that of the first mitochondrion. Thus, the spermatological similarities between the best known genus Stephanostomum with over 100 nominal species and the genus Stephanostomoides with only 2 nominal species confirm that the later belongs to the same family Acanthocolpidae. Moreover, Stephanostomoides is considered as the genuine genus of acanthocolpid based on its morphological similarity to Acanthocolpus and Stephanostomum (Bray et al., 2005). However, spermatological evidence from the type-genus Acanthocolpus would improve our understanding of the ultrastructural characteristics in the Acanthocolpidae.

Molecular evidence (lsrDNA) of the position of the Acanthocolpidae presented by Curran, Tkach & Overstreet (2006) suggested that it should be placed in the superfamily Brachycladioidea, along with the Opecoelidae, Opistholebetidae and Brachycladiidae. Bray et al. (2009) using lsrDNA + nad1 sequences presented evidence that the Acanthocolpidae and Brachycladiidae were, at least, closely related, and were distinct from the Lepocreadioidea. Littlewood, Bray & Waeschenbach (in press) utilised 202 ssrDNA and 556 lsrDNA trematode sequences to produce an estimate of the phylogeny of the group. These genes were found to be useful in defining the constituents of superfamilies, but much less satisfactory for assessing the relationships between superfamilies. Their results indicated that of the families placed in the Brachycladioidea by Curran, Tkach & Overstreet (2006) only the Brachycladiidae and Acanthocolpidae were consistently resolved together, and that the Opecoelidae and Opistholebetidae are best considered in a separate superfamily, the Opecoeloidea. Unfortunately, ultrastructural data are lacking for the Brachycladiidae to corroborate the close relationships between acanthocolpids and brachycladiids. However, spermatological characters observed in some species belonging to the Opecoelidae and Opistholebetidae reveal several differences between mature spermatozoa of opecoelids + opistholebetids and those of the two acanthocolpids described here. One of the main differences between these clades concerns the morphology of the posterior spermatozoon extremity as stated above. These spermatological differences corroborate the molecular findings that the Acanthocolpidae is not close to the clade Opecoelidae + Opistholebetidae.