Live adult specimens of R. fractum were collected from the digestive tract of a naturally infected Sarpa salpa (Teleostei, Sparidae) captured off the coast of Dakar (Senegal).

After dissection, live digeneans were routinely processed for TEM examination. 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, postfixed in cold (4 °C) 1% osmium tetroxide in the same buffer for 1 h, rinsed in a 0.1 M sodium cacodylate buffer at pH 7.4, dehydrated in an ethanol series and propylene oxide, and finally embedded in Spurr resin. After localization of testes and seminal vesicle in semithin sections, ultrathin sections were made using a Reichert-Jung Ultracut E ultramicrotome, placed on copper grids and double-stained with uranyl acetate and lead citrate according to the Reynolds [12] technique. To evidence glycogen, gold grids were also processed according to the Thiéry [13] test. They were treated in periodic acid, thiocarbohydrazide, and silver proteinate (PA-TCH-SP) as follows: 30 min in 10% PA, rinsed in milli-Q water, 24 h in TCH, rinsed in acetic solutions and milli-Q water, 30 min in 1% SP in the dark, and rinsed in milli-Q water. All ultrathin sections were examined using a JEOL 1010 transmission electron microscope operating at 80 kv.

The main stages of spermiogenesis in R. fractum are illustrated in Figs. 1–9.

Spermiogenesis begins by the formation of the differentiation zone in which each centriole, associated with a striated rootlet, originates a free flagellum that grows orthogonally to a median cytoplasmic process (Figs. 1, 3 and 9a). Within this differentiation zone, there is also an intercentriolar body with seven electron-dense layers (Fig. 2). Later, both flagella begin their rotation to become nearly parallel to the median cytoplasmic process (Figs. 4 and 9b). In this stage cross and longitudinal sections of the proximal area of the differentiation zone show the nucleus in migration into the median cytoplasmic process (Figs. 4 and 5) while sections of the distal area show only the median cytoplasmic process (Fig. 6). Moreover, in this stage four attachment zones are already present in the median cytoplasmic process (Figs. 5 and 6). After the proximodistal fusion of the flagella with the median cytoplasmic process, the ring of arched membranes initiates its constriction (Figs. 7–9c,d). In an early stage of this progressive constriction, the striated rootlets are still present in the spermatid body and a pear shaped electron-dense material is observed as a central element of both centrioles (Figs. 7 and 9c). In the final stage of the constriction of the ring of arched membranes, the striated rootlets disappear and it is noticeable that the mitochondrion is still migrating (Figs. 8 and 9d).

The interpretation of several cross and longitudinal sections of the mature spermatozoon of R. fractum allows us to establish three distinctive regions from the anterior to the posterior spermatozoon extremity (Figs. 10–30).

Region I or anterior spermatozoon extremity (Figs. 10–20 and 30I) is characterized by the presence of two axonemes of the 9 + ‘1’ trepaxonematan pattern, a pear shaped electron-dense material at the level of centrioles, an electron-dense material at the periphery of spermatozoon, external ornamentations of the plasma membrane, parallel cortical microtubules, spine-like bodies and the first mitochondrion. This region could be subdivided in three parts:

• • the anterior part of region I exhibits the anterior spermatozoon extremity with a sharp tip (Figs. 10 and 11). The two axonemes are slightly displaced longitudinally to each other (Fig. 12). In the area containing the two axonemes, there is an electron-dense material beneath the plasma membrane surrounding one of the axonemes and only two cortical microtubules (Figs. 12 and 13). It is also remarkable the presence of only two attachment zones (Figs. 12 and 13);
• • in the middle part of region I the number of cortical microtubules increases and show its typical pattern into two fields, each limited by the two attachment zones (Figs. 14 and 15). This area exhibits the maximum number of cortical microtubules, six on the dorsal side and 15 on the ventral side (Figs. 14 and 15). This area also shows spine-like bodies (Figs. 15 and 16);
• • the posterior part of region I is characterized by the presence of the first mitochondrion and the reduction in the number of cortical microtubules (Figs. 17–20). External ornamentations of the plasma membrane, spine-like bodies and granules of glycogen are also present (Figs. 17–20). It is interesting to note that spine-like bodies probably mark the transition from the non-ornamented to the ornamented portion of region I (Figs. 15–17).

Region II (Figs. 21–24, 29 and 30II) corresponds to a transitional region before the nuclear area. Cross-sections in this region show the two axonemes, a reduced number of parallel cortical microtubules divided into two submembranous bundles and granules of glycogen (Figs. 21 and 22). Note the disorganization of one axoneme before the appearance of a second mitochondrion located between the disorganized area of the axoneme and the appearance of the nucleus (Figs. 22–24).

Region III (Figs. 24–29 and 30III) represents the nuclear region and the posterior spermatozoon extremity. In its proximal area, cross-sections show the nucleus, one axoneme and granules of glycogen and cortical microtubules with progressive reduction (Figs. 24 and 25). When cortical microtubules disappear, cross-sections are characterized only by the presence of nucleus and the disorganized axoneme, which exhibits the nine doublets and the central core (Fig. 26). Additionally, during the disorganization of the second axoneme, doublets are located around the nucleus (Fig. 27). Finally, the posterior spermatozoon tip exhibits only the nucleus (Fig. 28). Glycogen was evidenced by the test of Thiéry (Fig. 29).

Concerning the superfamily Lepocreadioidea, spermiogenesis has been described in two species: the apocreadiid Neoapocreadium chabaudi [14] and the deropristid Deropristis inflata [15]. Spermiogenesis in these species is similar to that observed in R. fractum in the present study and in most digenean species. It is characterized by the formation of the differentiation zone surrounded by cortical microtubules, delimited at its base by a ring of arched membranes and containing a nucleus, numerous mitochondria, and two centrioles associated with striated rootlets and with an intercentriolar body. The centrioles originate two free flagella that grow orthogonally to the median cytoplasmic process. A flagellar rotation of 90° is also reported in most digeneans [5,6,16–19], although a flagellar rotation greater than 90° is described in some species [7,20–24].

Concerning the intercentriolar body, seven electron-dense layers are observed in R. fractum and N. chabaudi [14] as described in general in Digenea [9,10]. In the case of D. inflata, the intercentriolar body is constituted of six electron-dense plates [15]. Other digeneans exhibit an intercentriolar body formed by five to seven [21] or nine electron-dense layers [7].

Both flagellar rotation and the number of electron-dense layers of the intercentriolar body have been regarded to be interesting arguments in the interpretation of digenean relationships [9,17].

Most ultrastructural characters described previously in digeneans are present in the mature spermatozoon of R. fractum. These are: two axonemes of the 9 + ‘1’ pattern of the Trepaxonemata [25], two parallel bundles of cortical microtubules, two mitochondria, a nucleus and granules of glycogen. In addition to these characters, the presence/absence of an external ornamentation of the plasma membrane, a pear shaped electron-dense material, spine-like bodies, an electron-dense material beneath the plasma membrane, and the morphologies of both anterior and posterior spermatozoon extremities are also compared, particularly with the Lepocreadioidea.

An anterior spermatozoon extremity containing two axonemes very slightly longitudinally displaced to one another has been reported in R. fractum and in all the lepocreadioid species described until now (Table 1). However, some differences were noted within the studied Lepocreadioidea. In fact, an electron-dense material appears around the second axoneme in the lepocreadiid Holorchis micracanthum [26] and in the gyliauchenid Gyliauchen sp. [27] as observed in R. fractum (present study). However, this electron-dense material was absent in the apocreadiid N. chabaudi [14] and in the deropristid D. inflata [15]. With respect to N. chabaudi [14], the authors observed cortical microtubules accompanied by external ornamentation of the plasma membrane in the tip of the spermatozoon.

Concerning the pear shaped electron-dense material, it has previously been observed during spermiogenesis in D. inflata, Echinostoma caproni and Brachycoelium salamandrae [15,28,29]. However, the present study constitutes the first description of this character in the mature spermatozoon.

An association of external ornamentation and spine-like bodies was also observed in the anterior area of the spermatozoon in most digeneans [4,6,10,30,31]. However, there are other digeneans presenting spermatozoa with external ornamentation but lacking spine-like bodies. This is the case of the faustulid Pronoprymna ventricosa [32], the phaneropsolid Postorchigenes gymnesicus [33], or the zoogonid Diphterostomum brusinae [34]. Moreover, in several older studies previous to the description of spine-like bodies [4], these structures could have been misinterpreted, considered as artefacts or omitted, as occurred in Haematoloechus sp. [35] and Paragonimus ohirai [36]. Within the Lepocreadioidea, spine-like bodies are absent in the mature sperm of D. inflata [15] and H. micracanthum [26] (Table 1), while in N. chabaudi [14] and Gyliauchen sp. [27], they are present but not in the ornamented area of the spermatozoon. Concerning R. fractum (present study), spine-like bodies have been observed in both areas. Thus, these structures mark the transition between ornamented and not ornamented area. The particular disposition of spine-like bodies in relation to the ornamented area needs more attention in phylogenetic analyses within the Lepocreadioidea.

Variability of the number of mitochondria is observed within the digeneans in general and in the studied lepocreadioid species in particular (Table 1). Although the determination of the number of mitochondria depends on each author's considerations [11,17,37], at least one mitochondrion is observed in all digenean species studied until now. This criterion would be of great interest in phylogenetic interpretations within the Platyhelminthes considering that the absence of mitochondrion is a recognized synapomorphy for the Eucestoda [10,27].

The posterior spermatozoon extremity of R. fractum containing the nucleus would correspond to a type 2 posterior spermatozoon extremity [38], according to the terminal character in the posterior tip. This morphology of posterior spermatozoon extremity has been described in N. chabaudi [14] and in most digenean spermatozoa [10]. In the remaining Lepocreadioidea studied until now, the posterior spermatozoon extremity contains one axoneme (Table 1). Additionally to these morphologies, a posterior spermatozoon extremity ended by cortical microtubules has been reported in other digeneans, particularly those belonging to the family Opecoelidae [4,5,8,23,30]. Moreover, a posterior spermatozoon extremity containing a mitochondrion has been described recently in the lecithasterid Aponurus laguncula [38]. All these morphologies concerning the posterior spermatozoon extremity emphasise the usefulness of this criterion when establishing spermatozoon models within the Digenea.

The spermatozoon of R. fractum possesses a short axoneme that does not reach the nuclear region. Indeed, several cross-sections in the nuclear region show only an axoneme accompanied by the nucleus and cortical microtubules. This fact could distinguish the mature spermatozoon of R. fractum from those of the remaining lepocreadioids and in particular from the sperm cell of Gyliauchen sp., which belongs to the same family [27].

Comparing molecular and ultrastructural results, some congruencies could be supported. Indeed, the Gyliauchenidae are regarded to be close to the lepocreadiids and are nested in a clade named Lepocreadiata in a molecular study [39], as also adopted by Bray [40], who placed gyliauchenids in the Lepocreadioidea. The genus Robphildollfusium has been established as a gyliauchenid although its status is problematic [41]. These viewpoints could be supported by spermatological characters observed in some species belonging to the Lepocreadioidea. In fact, many similarities are observed between the spermatological characters of the two gyliauchenids and those of other lepocreadioid species, especially the lepocreadiid H. micracanthum [26]. This fact would allow us to support the inclusion of the Gyliauchenidae in the superfamily Lepocreadioidea. Concerning the problematic genus Robphildollfusium [41], similarities between the mature spermatozoon of R. fractum and Gyliauchen sp. could be valuable arguments to consider R. fractum as a gyliauchenid. However, more ultrastructural studies are required in the remaining gyliauchenid species and also in the other unexplored lepocreadioid families considering that, out of the ten families that compose this superfamily [40], only four have been studied until now.