Ultrastructure of mature spermatozoa of three Bucephalidae (Prosorhynchus longisaccatus, Rhipidocotyle khalili and Bucephalus margaritae) and phylogenetic implications

We describe here the mature spermatozoa of three species of bucephalids, namely Bucephalus margaritae, Rhipidocotyle khalili and Prosorhynchus longisaccatus. This study provides the first ultrastructural data on the genera Bucephalus and Rhipidocotyle and enabled us to confirm the model of the mature spermatozoon in the Bucephalinae. The spermatozoon exhibits two axonemes with the 9 + “1” pattern of the Trepaxonemata, one of which is very short, lateral expansion, external ornamentation of the plasma membrane located in the anterior extremity of the spermatozoon and associated with cortical microtubules, spine-like bodies, a mitochondrion, and a nucleus. The maximum number of cortical microtubules is located in the anterior part of the spermatozoon. However, more studies are needed to elucidate if spine-like bodies are present in all the Bucephalinae or not. In the Prosorhynchinae, the mature spermatozoon exhibits a similar ultrastructural pattern. Some differences are observed, particularly the axoneme lengths and the arrangement of the spine-like bodies. The posterior extremity of the spermatozoon in the Bucephalinae exhibits only the nucleus, but prosorhynchines have microtubules.


Introduction
The Bucephalidae Poche, 1907 (Platyhelminthes: Digenea) is a large cosmopolitan family that is parasitic in marine, brackish-water and freshwater fishes. It currently comprises 25 genera and 380 nominal species. It is now considered to be the single family within the superfamily Bucephaloidea [19,21]. These parasites have been reported from various fish species from over one hundred families. The systematic placement of the Bucephalidae within the Digenea has been debated since early studies considered the ''gasterostomes'' to be distinct from most other digenean groups. Later studies on life-cycles (e.g. [11]), and particularly molecular studies (e.g. [20]), have clearly shown that they are not basal, but closely related to other digenean groups, most notably the superfamily Gymnophalloidea. Nevertheless, more morphological and molecular studies are needed to improve our knowledge on the taxonomic and phylogenetic relationships among the bucephalids.
Today, ultrastructural studies of the mature spermatozoon of Platyhelminthes have provided useful characters for the understanding of phylogenetic relationships between Platyhelminthes [1,9,12]. In the Bucephalidae, ultrastructural data on the mature spermatozoon exist for four species belonging to three genera, namely the bucephalines Prosorhynchoides arcuatus [10], Prosorhynchoides gracilescens published as Bucephaloides gracilescens [5], Pseudorhipidocotyle elopichthys [26] and the prosorhynchine Prosorhynchus aculeatus [13]. We agree with Kacem and Miquel [10] that the ultrastructural studies of the spermatozoa of B. gracilescens [5] and P. elopichthys [26] provide only limited information. In this study, we provide ultrastructural data on the mature spermatozoon in the Bucephalidae with a study of two bucephalines, Bucephalus margaritae and Rhipidocotyle khalili and one prosorhynchine, Prosorhynchus longisaccatus. This is the first ultrastructural study of spermatozoa in the genera Bucephalus and Rhipidocotyle.

Transmission electron microscopy
Live worms were rinsed with a 0.9% NaCl solution and fixed in cold (4°C) 2.5% glutaraldehyde in a 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, embedded in Spurr's resin and polymerized at 60°C for 24 h.
Ultrathin sections (60-90 nm thick) were obtained using an Ultramicrotome (Power tome PC, RMC Boeckeler Ò ) with a diamond knife. Sections placed on copper grids were double-stained with uranyl acetate and lead citrate, according to the method described by Reynolds [25]. The grids were examined in a Hitachi H-7650 transmission electron microscope operated 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).

Cytochemistry
Sections placed on gold grids were stained according to the method developed by Thiéry [27] to detect the presence of glycogen. To do this, they were treated with periodic acid (PA), thiocarbohydrazide (TCH) and silver proteinate (SP) as follow: 30 min in 10% PA, rinsed in Milli-Q water, 2 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. Gold grids were examined as above. Region I (Figs. 1a-1g, 2I, 3a-3f, 4I, 5a-5d, 6I) corresponds to the anterior extremity of the mature spermatozoon. The anterior extremity of this region is characterized by the presence of an external ornamentation of the plasma membrane, a lateral expansion of the plasma membrane and cortical microtubules under the plasma membrane, respectively in P. longisaccatus (Fig. 1a), R. khalili (Fig. 3a), and B. margaritae (Fig. 5a). Then, the first and the second axoneme appear progressively (Figs. 1b-1d, 3b-3c, 5b). The posterior extremity of this region is characterized by the presence of two axonemes, external ornamentation of the plasma membrane and cortical microtubules under the plasma membrane (Figs. 1e-1f, 3d-3e, 5c). Then, the external ornamentation disappears progressively (Figs. 1g, 3f, 5c). In these three bucephalids, the maximum number of cortical microtubules is observed in the anterior extremities of the spermatozoon, respectively 52 in B. margaritae (Figs. 5a, 5d), 48 in P. longisaccatus (Fig. 1a), and 38 in R. khalili (Fig. 3a). The presence of undeveloped spine-like bodies was also observed along the anterior extremity of the spermatozoon of B. margaritae (Fig. 5a), in the posterior extremity of region I and in the anterior part of region II of the spermatozoon of P. longisaccatus (Figs. 1g, 1h, 1j). The number of cortical microtubules decreases toward the posterior extremities of the spermatozoon.

Observations
Region II (Figs. 1h-1n, 2II, 3g-3m, 4II, 5e-5i, 6II) corresponds to the middle part of the spermatozoon. There is no external ornamentation of the plasma membrane and the number of cortical microtubules under the plasma membrane is reduced in all three species. In P. longisaccatus, the mature spermatozoon also exhibits the undeveloped spine-like bodies in this region (Figs. 1h, 1j), the appearance of the mitochondrion      (Figs. 1l-1n), and the disappearance of the first axoneme (Fig. 1n). The posterior extremity of this region exhibits only one axoneme, the disorganization of the axoneme 1, a reduced number of cortical microtubules (3), the mitochondrion, and the nucleus (Fig. 1n). In R. khalili, one can see the presence of only the two axonemes and cortical microtubules in the anterior part (Figs. 3g-3h) and then the disappearance of the first axoneme (Figs. 3i-3k), and the appearance of the mitochondrion (Fig. 3l) and nucleus (Fig. 3m). Thus, the posterior extremity exhibits only one axoneme, a reduced number of cortical microtubules, mitochondrion and nucleus (Fig. 3m). In B. margaritae, we also observed the presence of the two axonemes and cortical microtubules in the anterior part (Fig. 5e), the reduction of the number of cortical microtubules towards this region, the disorganization and disappearance of the first axoneme (Figs. 5f, 5g), and the   appearance of the mitochondrion and the nucleus (Figs. 5h, 5i). The posterior extremity of this region contains only one axoneme, mitochondrion, nucleus and six cortical microtubules (Fig. 5i). Region III (Figs. 1o-1r, 2III, 3n-3r, 4III, 5j-5n, 6III) contains, in its anterior part, one axoneme, the mitochondrion, the nucleus and similar numbers of cortical microtubules (4,7,8) in P. longisaccatus, R. khalili and B. margaritae, respectively (Figs. 1o, 3n, 5j). Then, one can see the disappearance of the mitochondrion, the second axoneme and the nucleus toward the posterior extremity of the spermatozoon of P. longisaccatus (Figs. 1p-1r). In this species, the posterior extremity of the mature spermatozoon exhibits only microtubules (Fig. 1r). In R. khalili, one can also see the disappearance of the mitochondrion, then the disorganization of the second axoneme (Figs. 3o-3q). The posterior extremity of the spermatozoon exhibits only the nucleus (Fig. 3r). In B. margaritae the posterior part of the spermatozoon is characterized by the disorganization of axoneme 2 and the disappearance of the mitochondrion (Figs. 5k-5m). The posterior extremity of the spermatozoon exhibits only the nucleus (Fig. 5n).
The glycogen granules are clearly highlighted with the Thiéry method, and micrographs (Figs. 7a, 7b) show their presence in regions II and III of the mature spermatozoon of Prosorhynchus longisaccatus.

Discussion
Mature spermatozoa of Prosorhynchus longisaccatus, Rhipidocotyle khalili and Bucephalus margaritae exhibit the general pattern of the spermatozoon as described in most of the digeneans [4,6,7,[15][16][17][18][22][23][24]. However, new ultrastructural data provided in this study allowed us to have an overview of the pattern in the Bucephalinae and Prosorhynchinae. In the Bucephalinae, the descriptions of the mature spermatozoa of R. khalili and B. margaritae (this study) and Prosorhynchoides arcuatus [10] show that the sperm cell structure is homogeneous in this subfamily. Indeed, we agree that the mature spermatozoon of the Bucephalinae contains two axonemes (including one which is shorter), external ornamentation placed in the anterior part of the spermatozoon, numerous cortical microtubules decreasing in number from the anterior to posterior extremity of the spermatozoon, one mitochondrion and a nucleus in the posterior part of the spermatozoon, which exhibits a posterior extremity with only the nucleus. The presence of spine-like bodies associated with the external ornamentation in the anterior part of the spermatozoon was described only in Prosorhynchoides arcuatus [10] and in B. margaritae (this study). The type of spine-like bodies observed in the TEM micrographs of B. margaritae seems to be less developed than in most digeneans [14,17], and their absence in R. khalili (this study) and P. gracilescens [5] could be interpreted as a plesiomorphy within the Bucephalidae. Nevertheless, based on the variability of the number and the frequency of these elements from one species to another [22,24], we believe that further studies are needed to elucidate the status of this character in the mature spermatozoa of the Bucephalinae. We described in R. khalili and B. margaritae an anterior extremity of the mature spermatozoon with only external membrane ornamentation and a row constituted by a high number of cortical microtubules under the plasma membrane. TEM micrographs of P. gracilescens [5] show the same structures. However, Kacem and Miquel [10] also described in Prosorhynchoides arcuatus the presence of one axoneme in the anterior extremity of the spermatozoon. The presence of a lateral expansion of the plasma membrane in the anterior extremity of the mature spermatozoon described in Prosorhynchoides arcuatus by Kacem and Miquel [10] is also confirmed in R. khalili and B. margaritae (this study). In P. gracilescens, Erwin and Halton [5] described an anterior extremity of the mature spermatozoon with a single axoneme, mitochondrion and nucleus. However, we believe that these authors were mistaken when they described the presence of a nucleus and mitochondrion in the anterior extremity of the mature spermatozoon.
Concerning the Prosorhynchinae, Prosorhynchoides aculeatus [13] and Prosorhynchus longisaccatus (this study) present the same type of anterior extremity of the spermatozoon as the Bucephalinae. A unique difference is described in P. longisaccatus with the presence of spine-like bodies only in the posterior part of its anterior extremity and in the median part of the spermatozoon. Until now, the presence of spine-like bodies in the median part of the spermatozoon in Bucephalidae was described only in P. longisaccatus and Prosorhynchoides arcuatus.
The middle region of the mature spermatozoon of the studied Prosorhynchinae presents two axonemes. However, in the Bucephalinae it exhibits one axoneme and the disorganization of the first axoneme in its anterior part (this study). In Prosorhynchoides arcuatus, this middle part of the spermatozoon exhibits only one axoneme. Such a short axoneme in digeneans was described for the first time in Bucephalinae by Kacem and Miquel [10] in Prosorhynchoides arcuatus, then in two other Bucephalinae: B. margaritae and R. khalili (this study).
The posterior extremity of the mature spermatozoon of the Bucephalidae exhibits a nucleus in Bucephalinae ( [10], this study) or microtubules in Prosorhynchinae ( [13], this study). These microtubules could be cortical microtubules or singlets from the second axoneme.
Mature spermatozoa of the Bucephalidae present numerous similarities with the type V described by Bakhoum et al. [1]. This typology enables these authors to classify digeneans in five types of spermatozoa according to the state of eight principal ultrastructural characters. Type V is characterized by: (1) two axonemes with the 9 + ''1'' pattern of the Trepaxonemata, (2) lateral expansion, (3) presence of external ornamentation, (4) association ''external ornamentation + cortical microtubules'', (5) external ornamentation located in the anterior part of the spermatozoon, (6) two bundles of parallel cortical microtubules, (7) maximum number of cortical microtubules located in the anterior part of the spermatozoon, and (8) one mitochondrion in general. Table 1 shows the homogeneity of the sperm structure of the Bucephalidae.

Conclusion
Our study increases the ultrastructural data on the mature spermatozoon in the Bucephalidae, and provides characters that might be useful for phylogenetic purposes. However, the lack of ultrastructural data on the mature spermatozoon in the other three recognized subfamilies of Bucephalidae (Dolichoenterinae, Heterobucephalopsinae and Paurorhynchinae) justifies the need for more ultrastructural studies to define a spermatozoon model for the Bucephaloidea superfamily. Nevertheless, we conclude that mature spermatozoa of Prosorhynchus longisaccatus, Rhipidocotyle khalili and Bucephalus margaritae exhibit all the structures described to date in the Bucephalidae, namely (i) two axonemes of the 9 + ''1'' pattern of Trepaxonemata with different lengths; the difference in length is variable according to the genus; (ii) the anterior extremity of the mature spermatozoon exhibits only the anterior extremity of the axoneme 1, a lateral expansion and extramembranar ornamentation of the plasma membrane and a complete row of cortical microtubules under the plasma membrane; (iii) the mitochondrion and nucleus are located in the posterior part of the spermatozoon; and (iv) there is variation in the type of posterior extremity of mature spermatozoa according to the subfamily.

Conflict of interest
The Editor-in-Chief of Parasite is one of the authors of this manuscript. COPE (Committee on Publication Ethics, https:// publicationethics.org/), to which Parasite adheres, advises special treatment in these cases. In this case, the peer-review process was handled by an Invited Editor, Jérôme Depaquit.