Spermatozoon ultrastructure in two monorchiid digeneans

Spermatological characteristics of species from two monorchiid genera, Opisthomonorchis and Paramonorcheides, have been investigated, for the first time, by means of transmission electron microscopy. The ultrastructural study reveals that the mature spermatozoon of Opisthomonorchis dinema and Paramonorcheides selaris share several characters such as the presence of two axonemes of different lengths showing the 9+“1” pattern of the Trepaxonemata, a nucleus, two mitochondria, two bundles of parallel cortical microtubules, external ornamentation of the plasma membrane, spine-like bodies, granules of glycogen and similar morphologies of the anterior and posterior extremities. The slight differences between the male gamete of O. dinema and P. selaris are the length of the first axoneme and the position of the second mitochondrion. This study also elucidates the general morphology of the spermatozoon in all monorchiid species described so far, which corresponds to a unique spermatozoon type. Other interesting finds concern the spermatological similarities between monorchiid spermatozoa and the mature spermatozoon reported in the apocreadiid Neoapocreadium chabaudi. These similarities allow us to suggest a close phylogenetical relationship between the Monorchiidae and the Apocreadiidae, although more studies are needed, especially in the unexplored taxa.

The aim of this work was to bring more spermatological descriptions from two additional genera, Opisthomonorchis and Paramonorcheides, in order to elucidate the general morphology of the spermatozoon in the Monorchiidae. In addition, the mature spermatozoon of the monorchiids was compared to those of the other digeneans with a brief comment on its phylogenetic relevance.
Digenean specimens were fixed in cold (4 • C) 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer at pH 7.2, rinsed in 0.1 M sodium cacodylate buffer at pH 7.2, post-fixed in cold (4 • C) 1% osmium tetroxide in the same buffer for 1 h, dehydrated in ethanol and propylene oxide series, embedded in Spurr resin and polymerized at 60 • C for 24 h. Ultrathin sections (60-90 nm) of the seminal vesicle were obtained on an ultramicrotome (Power tome PC, RMC Boeckeler R ). The sections were placed on 300 and 200 mesh copper grids and stained with uranyl acetate and lead citrate according to Reynolds (1963) methodology. To locate glycogen granules, the Thiéry technique (1967) was also used in several sections placed on gold grids.
All grids were examined on a Hitachi H-7650 transmission electron microscope, operating at an accelerating voltage of 80 kV, in the ''Service d'Étude et de Recherche en Microscopie Électronique'' of the University of Corsica (Corte, France).

RESULTS
The organization of the mature spermatozoon of Opisthomonorchis dinema and Paramonorcheides selaris is described after observations of cross-and longitudinal sections . Thus, in both monorchiid species four regions, with distinctive ultrastructural characteristics, are evident, from the anterior to the posterior spermatozoon extremities. The granules of glycogen present in O. dinema and P. selaris spem cells were detected by the cytochemical test of Thiéry ( Fig. 3E for P. salaris).
Region I, corresponding to the anterior extremity of the mature spermatozoon, is characterized by the presence of the first axoneme accompanied by the anterior extremity of the second axoneme, cortical microtubules and external ornamentation of the plasma For the two species, only few glycogen granules have been observed in the anterior part of this region.

DISCUSSION
The mature spermatozoon described in Opisthomonorchis dinema and Paramonorcheides selaris possesses two axonemes of the 9+''1'' trepaxonematan pattern, two mitochondria, a nucleus, two bundles of parallel cortical microtubules, an external ornamentation of the plasma membrane, spine-like bodies and granules of glycogen. In addition to these ultrastructural characters, the male gamete of O. dinema and P. selaris exhibits similar anterior and posterior spermatozoon morphologies.
Within the Monorchiidae most of these spermatological characters were described previously in Monarchus parvus and Helicometroides atlanticus (Levron, Ternengo & Marchand, 2004a;Diagne et al., 2015). Thus, the characters of the mature spermatozoon, its anterior and posterior morphologies were used here in comparative studies between the different species (Table 1). Table 1 Comparative ultrastructural characteristics of the spermatozoon in the Monorchiidae. Ase, anterior spermatozoon extremity; Ax, axoneme; Cm, cortical microtubules; Edm, electron-dense material; Eo, external ornamentation of the plasma membrane; M, number of mitochondria; MCm, maximum number of cortical microtubules; N, nucleus; TAx, type of axoneme; TEo, type of external ornamentation according to Quilichini et al., (2011a); Psc, posterior spermatozoon character; Sb, spine-like bodies; +/−, 2013, presence/absence of considered character.

General organisation of the mature spermatozoon in the Monorchiidae
From the anterior part to the posterior part of the gametes, six characteristics are presented here to describe the architecture of the mature spermatozoa in Monorchiidae. Moreover, monorchiid spermatozoa are compared to those reported from the other digenean species.

Type of axoneme
The structure of the axoneme with 9+''1'' pattern of Trepaxonemata (Ehlers, 1984) has been observed in O. dinema and P. selaris as described previously in M. parvus and H. atlanticus (Table 1). A similar morphology of the axoneme has been reported in most digenean species (Quilichini et al., 2011a;Foata et al., 2012;Miquel et al., 2013;Ndiaye et al., 2012;Ndiaye et al., 2015a) with the exception of schistosomes in which the 9+''1'' special pattern was described and some didymozoid species showing the 9+0 pattern (Justine & Mattei, 1983;Justine, Jamieson & Southgate, 1993;Yang, Dong & Jiang, 2003). Their different length is another aspect related to axonemes. In both O. dinema and P. selaris, the posterior extremity of the axoneme 2 is longer than that of the first one. Moreover, the spermatozoon of O. dinema is distinguished by the first axoneme that does not reach the nuclear region, while in P. selaris the first axoneme is observed in the nuclear area like in M. parvus and H. atlanticus (Levron, Ternengo & Marchand, 2004a;Diagne et al., 2015).

Anterior spermatozoon morphology
The anterior spermatozoon extremity of O. dinema and P. selaris contains two axonemes, cortical microtubules and external ornamentation of the plasma membrane. Within the Monorchiidae, an anterior spermatozoon extremity showing two axonemes, cortical microtubules and external ornamentation has been reported in Monorchis parvus (Levron, Ternengo & Marchand, 2004a). Whereas in the other monorchiid species studied so far, namely H. atlanticus (Diagne et al., 2015), only electron-dense material and two axonemes have been observed in the anterior spermatozoon extremity.
Mature spermatozoa showing two axonemes and external ornamentation in their anterior extremities have also been reported in spermatozoa belonging to some digenean species such as the apocreadiid Neoapocreadium chabaudi (Kacem et al., 2010), the pronocephalids Pleurogonius truncatus and Cricocephalus albus (Ndiaye et al., 2011;Ndiaye et al., 2012) and the mesometrids Centroderma spinosissima and Wardula capitellata (Bakhoum et al., 2012b;Bakhoum et al., 2013). In the two latter families, the presence of a lateral expansion is described in the anterior spermatozoon extremity of the studied species.

External ornamentation and its location
In all monorchiid species studied, the presence of external ornamentation associated with cortical microtubules has been observed in the anterior region of the spermatozoon as in most digenean species (Justine & Mattei, 1982;Miquel et al., 2006;Bâ et al., 2011;Ndiaye et al., 2012;Ndiaye et al., 2015a;Ndiaye et al., 2015b;Bakhoum et al., 2015a;Bakhoum et al., 2015b). However, the location of the external ornamentation distinguishes the spermatozoa of O. dinema and P. selaris from those of other digenean species. In fact, based on the diagram of the localization of the external ornamentation established by Quilichini et al. (2011a), the mature spermatozoon of O. dinema and P. selaris presents the type 1 of external ornamentation, i.e., located in proximal part of the anterior spermatozoon region. It is also interesting to note that in both O. dinema and P. selaris, the external ornamentation is extended from centriole level to the area containing the first mitochondrion. Such disposition of the external ornamentation is also reported in the monorchiid M. parvus but not in H. atlanticus (Table 1). This latter species seems to exhibit type 2 external ornamentation according to the diagram of Quilichini et al. (2011a).

Spine-like bodies
Since their first description by Miquel, Nourrisson & Marchand (2000) the spine-like bodies have frequently been reported in the mature spermatozoon especially in its anterior part. In the male gamete of O. dinema and P. selaris, spine-like bodies are present in the ornamented area associated with cortical microtubules. Moreover, in both monorchiid species the morphology of the spine-like bodies follows that reported in most digenean species i.e., appearance of a small vesicle interrupting the external ornamentation of the plasma membrane (Miquel et al., 2006;Quilichini et al., 2011b;Bakhoum et al., 2015b;Ndiaye et al., 2015a;Ndiaye et al., 2015b).
Within the Monorchiidae, the absence of spine-like bodies was mentioned in the male gamete of M. parvus and H. atlanticus (Levron, Ternengo & Marchand, 2004a;Diagne et al., 2015) (Table 1) hence the spine-like bodies are described here, for the first time, in the mature spermatozoon of monorchiid species.
Taking into account all these aspects, the presence or absence of spine-like bodies in the Monorchiidae needs more ultrastructural investigations.

Number of mitochondria
The presence of mitochondria in the mature spermatozoon is considered as a plesiomorphic character in the Digenea (Bakhoum et al., 2014) whereas in other groups, such as the Eucestoda, the absence of mitochondria has been highlighted as a synapomorphy (Justine, 1991a). Besides its interest in phylogenetic relationships, the other criterion related to mitochondria is their number.
In the present study, the position of the second mitochondrion is an ultrastructural characteristic that distinguishes the mature spermatozoa of O. dinema from those of P. selaris, H. atlanticus and M. parvus. The second mitochondrion appears after the disorganization of the first axoneme in O. dinema, whereas in the remaining monorchiid the appearance of the second mitochondrion is noted before the posterior extremity of the first axoneme.

Phylogenetic perspectives of ultrastructural characteristics
The ultrastructural differences observed in the spermatozoa are phylogenetically informative in the Neodermata, particularly in the Cestoda and Monogenea (Justine, 1991b;Justine, 1998;Levron et al., 2010). In the Digenea a large amount of spermatological data accumulated over the recent years could serve as a complementary resource for understanding their phylogenetic relationships (Quilichini et al., 2010;Miquel et al., 2013;Bakhoum et al., 2015a;Diagne et al., 2015).
The phylogenetic relationships in the Monorchiidae and its systematic placement within the Digenea have been controversial. The Monorchiidae has been considered polyphyletic because of the great diversity exhibited in the morphology of its members (Madhavi, 2008). The most recent classification based on DNA sequences includes the Monorchiidae and Lissorchiidae in the superfamily Monorchioidea (Bray, 2008), which was included in the new suborder Monorchiata (Olson et al., 2003).
Here, the understanding of the relationships and phylogenetic affinities in Monorchiidae are attempted with ultrastructural characteristics of the spermatozoa, although there still remain some unexplored groups. The morphology of the spermatozoa described in O. dinema, P. selaris and other monorchiid species is similar in several points to that reported in the apocreadiid Neoapocreadium chabaudi (Kacem et al., 2010). The main similarities concern (1) the type of axoneme, (2) the morphology of both anterior and posterior spermatozoon extremities, (3) the presence of type 1 external ornamentation according to Quilichini et al. (2011a), (4) the two bundles of parallel cortical microtubules and (5) the presence of spine-like bodies.
Besides these similarities, it is interesting to note that the systematic position of the Apocreadiidae is unresolved. Some authors have grouped this family with the Haploporoidea and Monorchioidea (Cribb et al., 2001). Whereas other researchers suggested the removal of the Apocreadiidae from the Lepocreadioidea (see Bray & Cribb, 2012). Thus, to our knowledge no robust data are available to validate the phylogenetic position of the Apocreadiidae.
The present spermatological findings suggest, for the first time, a close relationship between the Monorchiidae and the Apocreadiidae based on ultrastructural characteristics of their mature spermatozoa. However, further studies are needed in order to support our hypothesis. Moreover, ultrastructural studies in the Lissorchiidae (probably a sister group) are also needed to test their close relationships with the Monorchiidae.

Conclusion
The mature spermatozoa of Opisthomonorchis dinema and Paramonorcheides selaris share several ultrastructural features with those reported in Monorchis parvus and Helicometroides atlanticus (Table 1). These similarities allow us to define the type of spermatozoon in the Monorchiidae, which exhibits the following features: -Presence of an anterior extremity showing two axonemes accompanied by cortical microtubules and external ornamentation, -Presence of the association ''external ornamentation+cortical microtubules'', -Location of the external ornamentation corresponding to the type 1 according Quilichini et al. (2011a), -Presence of two bundles of parallel cortical microtubules and two mitochondria, -Posterior spermatozoon extremity containing only the nucleus according to Quilichini et al. (2010). The model of mature spermatozoon described here for the monorchiid species is relatively similar to that reported to the apocreadiid Neoapocreadium chabaudi (Kacem et al., 2010). These similarities allow us to suggest a close relationship between the Monorchiidae and Apocreadiidae, although additional spermatological evidences are needed especially, from the unexplored taxa belonging to the families mentioned above and the Lissorchiidae.
The ultrastructural characteristics described in digenean and in particular, the Monorchiidae could be used as phylogenetic tools and when establishing spermatozoon models, given that they may allow the distinguishing of genera, families or superfamilies within the Digenea.

ADDITIONAL INFORMATION AND DECLARATIONS Funding
A.J.S. Bakhoum has a post-doctoral fellowship (No. CE/01/2014) from the ''Collectivité Territoriale de Corse -Direction de l'Enseignement Supérieur et de la Recherche''. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.