Spermatological characteristics of Pleurogenidae (Digenea) inferred from the ultrastructural study of Pleurogenes claviger, Pleurogenoides medians and Prosotocus confusus

The present work constitutes the first ultrastructural analysis of the spermatozoon in the Pleurogenidae, with the study of three species belonging to three of the 16 genera included in this family, namely Pleurogenes claviger, Pleurogenoides medians and Prosotocus confusus. The mature spermatozoa of these pleurogenids present two axonemes of the 9+“1” trepaxonematan pattern, a nucleus, two mitochondria, two bundles of parallel cortical microtubules, external ornamentation, spine-like bodies and granules of glycogen. The organization of these characters in the sperm cell is similar in the three species. Thus, the anterior spermatozoon extremity is filiform and a continuous and submembranous layer of parallel cortical microtubules surrounds the axonemes at their anterior end. The posterior spermatozoon extremity exhibits the second axoneme and corresponds to the Cryptogonimidean type of Quilichini et al. (2010). Slight differences were noted between the spermatozoon of P. confusus and those of the two remaining species in the location of mitochondria.


Introduction
Ultrastructural characteristics of spermatozoa were proved to be valuable tools for the analysis of phylogenetic relationships within the Platyhelminthes, particularly for the Eucestoda but also for the Monogenea [3, 22, 31-36, 46, 83]. It is gener-ally accepted that this source of characters contributes to the establishment of a more robust phylogeny when they are combined with morphological, biological and/or molecular data [28,29,35,46,66,89]. Considering digeneans, during the last years there was an important increase of ultrastructural studies on the spermatozoon, as reviewed by Bakhoum [5]. However, numerous groups such as the Pleurogenidae have been neglected and no data were available until now.
The family Pleurogenidae includes species parasitizing mainly amphibians, but some species have also been reported in reptiles and sometimes in fish and mammals, probably as accidental infections [51]. The present study includes data about three (Pleurogenes, Pleurogenoides and Prosotocus) of the 16 genera included in the Pleurogenidae [51]. Thus, we present the first spermatological data of this family, with the study of the ultrastructural organization of the mature spermatozoon of Pleurogenes claviger, Pleurogenoides medians and Prosotocus confusus.
After dissection, live digeneans were routinely processed for TEM examination. Therefore, 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 (OsO 4 ) with 0.9% potassium ferricyanide [K 3 Fe(CN) 6 ] in the same buffer for 1 h, rinsed in milliQ water, dehydrated in an ethanol series and propylene oxide, embedded in Spurr's resin and finally polymerized at 60°C for 72 h. Ultrathin sections (50-60 nm thick) were obtained using a Reichert-Jung Ultracut E ultramicrotome, placed on copper grids and double-stained with uranyl acetate and lead citrate according to Reynolds [78] methodology. Ultrathin sections were examined using a JEOL 1010 TEM operated at an accelerating voltage of 80 kV.
The Thiéry [86] technique was used to locate glycogen. Gold grids were treated in periodic acid, thiocarbohydrazide and silver proteinate (PA-TCH-SP) as follows: 30 min in 10% PA, rinsed in milliQ water, 24 h in TCH, rinsed in acetic solutions and milliQ water, 30 min in 1% SP in the dark and rinsed in milliQ water.

Results
The observation of numerous cross-and longitudinal sections allows us to distinguish three different regions from the anterior to the posterior extremity of mature spermatozoa of the three studied pleurogenids (Pleurogenes claviger, Pleurogenoides medians and Prosotocus confusus). Each of these three regions exhibits distinctive ultrastructural characteristics shown in Figures 1-8. The usual characters found in the mature spermatozoon of most digeneans are also present in the male gamete of these pleurogenids, i.e. two axonemes of the 9+''1'' trepaxonematan pattern, external ornamentation of the plasma membrane, nucleus, mitochondrion, parallel cortical microtubules and granules of glycogen. Other particular features as the morphology of both extremities and the presence of spine-like bodies also characterize the spermatozoon of these species.
(I) Anterior part or Region I (Figures 1a-g  The glycogenic nature of the granular material observed in Regions II and III of the mature spermatozoa of the three studied pleurogenids was evidenced by the test of Thiéry ( Figure 7).

Discussion
The mature spermatozoa of Pleurogenes claviger, Pleurogenoides medians and Prosotocus confusus exhibit the same

Spine-like bodies
Spine-like bodies consist in prominent electron-dense structures that seem to contain a spherical vesicle and are usually located in the ornamented area of the spermatozoon. These elements were described only in the Digenea. Miquel et al. [52] discussed the similarities and differences between spine-like bodies of digeneans and crested bodies present in most cestodes. In cross-sections spine-like bodies and crested bodies resemble each other. However, spine-like bodies are isolated elements while crested bodies consist in one or more helical cords that surround the spermatozoon.
A periodicity in the presence of spine-like bodies was described in some species. For example, periodicity was estimated as 1 lm in F. gigantica, O. furcatus and N. testiobliquum  [53,57,74], 0.7 lm in P. furcatum [47] or 0.6 lm in N. wisniewskii [77]. Their distribution is irregular in numerous species, e.g. R. exasperatum or W. capitellata [7,11]. In the case of P. claviger the location of spine-like bodies seems to follow a periodicity of 0.5-0.6 lm. Nevertheless, in most of the previously cited species spine-like body periodicity is not evident because of the difficulty in observing longitudinal sections containing successive spine-like bodies.
Thus, external ornamentation and/or spine-like bodies are present in the spermatozoon of the great majority of digeneans studied until now. The role of these elements remains unknown, but they probably constitute important structures in the process of fertilization considering their location in the male gamete. In fact, the external elements associated to the plasma membrane are always anterior to the nuclear region and in most cases they are located in anterior areas of the sperm cell. During fertilization in digeneans but also in monogeneans, the anterior part of sperm cell coils around the oocyte, penetrating it by lateral fusion [42,43]. Moreover, several authors hypothesized that the external ornamentation participates in the fusion of the spermatozoon and oocyte membranes during the fertilization process [39]. In addition to their aberrant morphology, schistosomes lack these anterior structures [37]. Certain cestodes also exhibit anterior external structures in the male gamete such as the helical crested bodies that probably also play an important role during fertilization [34,36,46].

Posterior spermatozoon extremity
The posterior tip of digenean spermatozoa is morphologically variable. Quilichini et al. [70] distinguished three types of posterior parts of the spermatozoon (the Opecoelidean type, the Fasciolidean type and the Cryptogonimidean type). These types are characterized by the sequence of characters towards the posterior spermatozoon tip. The type 1 or Opecoelidean type is characterized by the sequence ''axoneme, nucleus and cortical microtubules''. The type 2 or Fasciolidean type presents the sequence ''cortical microtubules, axoneme and nucleus''. Finally, the type 3 or Cryptogonimidean type follows the sequence ''cortical microtubules, nucleus and axoneme''. According to these authors, there is a possibility of a fourth group characterized by a different sequence: posterior extremity of the first axoneme, posterior extremity of cortical microtubules and posterior extremity of the second axoneme. This group would be represented by the brachylaimid S. palaearcticum [56], the deropristid D. inflata [24] and the lecithasterid A. laguncula [71]. However, A. laguncula does not coincide exactly with this pattern because cortical microtubules end before the axonemes do. Moreover, this species exhibits a unique morphological pattern, in which the mitochondrion reaches the posterior spermatozoon extremity [71]. In our study, each of the three Pleurogenidae studied species exhibits the second axoneme in its posterior spermatozoon tip, an arrangement which corresponds to the Cryptogonimidean type or type 3 posterior spermatozoon extremity. Therefore, according to other authors [7] in order to minimize the existing variations in these sequences, it would be more interesting to consider only the terminal characteristic rather than the succession of characters towards the posterior spermatozoon extremity.

Perspectives
In the future, the present study can contribute to a better knowledge of relationships between the Digenea and particularly between the Microphalloidea. The taxonomic status of pleurogenids is controversial. This group of digeneans was initially established as a subfamily included in the Brachycoeliidae or in the Lecithodendriidae depending on the authors [84]. More recently, it was raised to family rank, although some taxonomists do not accept this familial status. All these controversies demonstrate that the classical morphological characters alone cannot resolve the existing systematic confusion in numerous taxa such as the pleurogenids [65,87,88]. Thus, as numerous authors advocate, a multidisciplinary approach to the systematics and phylogeny of Platyhelminthes is crucial, for example by integrating molecular and ultrastructural tools [27][28][29]. The molecular analysis of Digenea by Olson et al. [65] shows the Microphalloidea as composed of two clades: the first constituted by the Pachypsolidae, the Renicolidae and the Eucotylidae, and the second formed by the Zoogoni-dae+Faustulidae, the Lecithodendriidae, the Microphallidae, the Pleurogenidae and the Prosthogonimidae. Unfortunately, there are no spermatological data on the families nested in the first clade. On the other hand, in another molecular study, Tkach et al. [87] considered that the Microphalloidea were composed of three clades corresponding to diverse families, the lecithodendriids, the microphallids and the prosthogoni-mids+pleurogenids. Moreover, these authors established several subclades within the Pleurogenidae. The analysis of the available spermatological data shown in Table 1 for microphalloideans emphasizes the necessity to complete additional studies on the spermatozoon of other families, thus allowing for comparisons with molecular data. Presently, sperm ultrastructural data confirm the proximity of pleurogenids and prosthogonimids as evidenced in molecular analysis [87]. Unlike the remaining families, in pleurogenids and prosthogonimids the two axonemes and spine-like bodies are present in the anterior spermatozoon extremity (see Table 1). Moreover, data concerning ultrastructural studies of the spermatozoon in some microphalloideans [18,26] are not included in Table 1 because they do not show the complete ultrastructural organization of the male gamete and they also contain numerous misinterpretations. This emphasizes the need for a thorough analysis of the spermatozoon in order to (1) avoid missing data on any of the characters and (2) allow for comparisons among all available studies.