Sperm characteristics in the digenean Diplodiscus amphichrus (Paramphistomoidea, Diplodiscidae), a parasite of the Chinese edible frog Hoplobatrachus rugulosus

The ultrastructural characteristics of the mature spermatozoon of Diplodiscus amphichrus (Digenea, Paramphistomoidea, Diplodiscidae) and their ultrastructural organisation were examined by means of transmission electron microscopy. Live digeneans were collected from the Chinese edible frog Hoplobatrachus rugulosus in Thailand. The male gamete of D. amphichrus is a filiform cell with two axonemes of the 9 + ’1’ trepaxonematan pattern, nucleus, one mitochondrion, parallel cortical microtubules, a well-developed lateral expansion, external ornamentation of the plasma membrane, spine-like bodies, and granules of glycogen. These ultrastructural characteristics have already been described in other paramphistomoids studied so far except for the cladorchiids, which present some differences. Two characteristics of the male gamete of D. amphichrus were found for the first time in a digenean: (i) the appearance of the initial part of the lateral expansion before the axonemes in the anterior extremity and (ii) the separation between the second axoneme and the nucleus in the posterior tip. Our results are compared with the available data in the Digenea and particularly with other paramphistomoids.


Introduction
Diplodiscus amphichrus Tubangui, 1949 is a digenean that belongs to the family Diplodiscidae. This family, included in the superfamily Paramphistomoidea, constitutes a cosmopolitan and small group of digeneans with only six valid genera, namely Australodiscus, Catadiscus, Dermatemytrema, Diplodiscus, Progonimodiscus, and Pseudodiplodiscus. Representatives of Diplodiscidae are parasites of the digestive tract, and they are predominantly found parasitizing amphibians but also recorded in reptiles and fish (see Jones 2005).
The usefulness of sperm characters and their organisation in the male gamete for the interpretation of relationships between Platyhelminthes have been demonstrated for different groups by several authors (Justine 1991a(Justine , b, 1998(Justine , 2001Bâ and Marchand 1995;Miquel et al. 1999;Levron et al. 2010;Quilichini et al. 2010Quilichini et al. , 2011Bakhoum et al. 2017a;Justine and Poddubnaya 2018). Regarding the digenean superfamily Paramphistomoidea, spermatological studies have been done on seven species belonging to four families: the Cladorchiidae, Diplodiscidae, Gastrothylacidae, and Paramphistomidae (Ashour et al. 2007;Seck et al. 2007Seck et al. , 2008aSwarnakar 2010;Bakhoum et al. 2011). In the Diplodiscidae, there is only an ultrastructural study of the spermatozoon of Diplodiscus subclavatus (Bakhoum et al. 2011). The present study aims to describe the sperm characters and their organisation along the mature spermatozoon of a second species of the family Diplodiscidae, D. amphichrus. Our results are also compared with the available data in other digeneans, particularly paramphistomoids, to highlight the most characteristic features and their phylogenetic importance.  Sey (1985Sey ( , 1991 and were previously reported in northeast Thailand (Wongsawad et al. 1998).

Transmission electron microscopy
For the present TEM study, several flukes 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 7.4 for a minimum of 2 h, rinsed in 0.1 M sodium cacodylate buffer at pH 7.4, post-fixed in cold (4 °C) 1% osmium tetroxide with 0.9% potassium ferricyanide in the same buffer for 1 h, rinsed in Milli-Q water (Millipore Gradient A10), dehydrated in an ethanol series and propylene oxide, embedded in Spurr's resin, and polymerised at 60 °C for 72 h. Ultrathin sections (60-90 nm thick) at the level of the seminal vesicle were obtained using a Reichert-Jung Ultracut E ultramicrotome. Sections were placed on 200-mesh copper and gold grids. Sections placed on copper grids were double-stained with uranyl acetate and lead citrate according to Reynolds (1963). Copper grids were examined in a JEOL 1010 transmission electron microscope operated at an accelerating voltage of 80 kV, in the 'Serveis Científics i Tecnològics de la Universitat de Barcelona (CCiTUB)'.

Cytochemistry
Sections placed on gold grids were treated according to the specific cytochemical test of Thiéry (1967) to reveal the presence of glycogen. Thus, they were treated in periodic acid (PA), thiocarbohydrazide (TCH), and silver proteinate (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. Sections were examined in a JEOL1010 transmission electron microscope at an accelerating voltage of 80 kV, in the CCiTUB.

Results
The observation of numerous ultrathin sections at the level of the seminal vesicle allowed us to distinguish three regions in the mature spermatozoon of Diplodiscus amphichrus (Figs. 1, 2, 3). These three regions exhibit different ultrastructural characteristics and organisation as follows: Region I or anterior region of the spermatozoon (Figs. 1a-i and 3I): The main characteristics of this region are the simultaneous presence of external ornamentation of the plasma membrane and a well-developed lateral expansion (Figs. 1a-i and 3I). The anterior spermatozoon extremity is formed by a large section with a continuous and submembranous layer of parallel cortical microtubules associated with the external ornamentation of the plasma membrane (Fig. 1a). The two axonemes of the 9 + '1' pattern appear almost simultaneously ( Fig. 1b-d). The maximum number of cortical microtubules (around 63) is observed in the part of the sperm cell exhibiting the maximum development of the lateral expansion ( Fig. 1d-f). In the posterior part of region I, the lateral expansion reduces and disappears ( Fig. 1g, h) and the external ornamentation progressively reduces ( Fig. 1h, i). The cortical microtubules form a continuous and submembranous layer along region I except in the posterior part where their arrangement into two bundles is observed (Fig. 1i). Another ultrastructural character of the region I is the presence of spine-like bodies irregularly distributed ( Fig. 1d, f, i). Finally, electron-dense granules appear in the cytoplasm in the posterior part of region I ( Fig. 1g-i). This granular material was identified as glycogen by the application of the specific cytochemical test of Thiéry (see Fig. 2l for the remaining regions).
Region II or middle region of the spermatozoon (Figs. 1j-k, 2l, and 3II): It is mainly characterised by the absence of most of the above-mentioned characters. Thus, region II lacks the lateral expansion, the external ornamentation of the plasma membrane, and spine-like bodies (Figs. 1j-k and 3II). Thereby, the middle region just presents shows two separate sections containing the second axoneme and the nucleus. j Disorganisation of the second axoneme. k Posterior spermatozoon tip presenting only the nucleus. l Positive result for glycogen using the test of Thiéry. Ax2 s axoneme, CC1 central core of the first axoneme, CM cortical microtubules, G granules of glycogen, M mitochondrion, N nucleus, S1 and S2 singlets of the first and second axonemes. Scale bars 300 nm the two 9 + '1' axonemes, two bundles of parallel cortical microtubules and a large amount of granules of glycogen ( Fig. 1j-k).
Region III or posterior region of the spermatozoon (Figs. 2a-l and 3III): This region corresponds with the nuclear and also mitochondrial part of the sperm cell. The anterior part of region III only has the nucleus (Fig. 2a). Later, the mitochondrion appears when the nucleus is already present (Fig. 2b, c). After the disappearance of the mitochondrion, the first axoneme becomes disorganised and disappears ( Fig. 2d-f). In the posterior part of region III, the second axoneme exhibits a lateral cytoplasmic protrusion (Fig. 2g, h). Finally, the posterior extremity of the sperm cell is characterised by a separation between the second axoneme and the nucleus by a plasma membrane cytokinesis (Fig. 2i, j). That way, the posterior tip contains only the nucleus (Fig. 2k).

Discussion
The mature spermatozoon of Diplodiscus amphichrus exhibits many ultrastructural characters previously described in most digenean species. These are two axonemes of the 9 + '1' trepaxonematan pattern (Ehlers 1984), a nucleus, a mitochondrion, parallel cortical microtubules, a lateral expansion, external ornamentation of the plasma membrane, spine-like bodies, and granules of glycogen. The presence/ absence and organisation of these features along the sperm cell show many similarities with the remaining studied paramphistomoids when they are compared (see Table 1). However, the spermatozoon of D. amphichrus exhibits two ultrastructural particularities: (i) the anterior extremity formed by the proximal part of the lateral expansion before the appearance of the two axonemes and (ii) the posterior extremity showing a separation between the second axoneme and the nucleus by a plasma membrane cytokinesis. To our knowledge, the present study constitutes the first finding of such characteristics.

Anterior spermatozoon region: lateral expansion, external ornamentation, and spine-like bodies
The most characteristic features present in the anterior region of the spermatozoon of D. amphichrus are a welldeveloped lateral expansion, the external ornamentation of the plasma membrane, and spine-like bodies.
Lateral expansions are present in the sperm cell of diverse digeneans belonging to the Bucephalidae, Echinostomatidae, Fasciolidae, Mesometridae, and Troglotrematidae (Ndiaye et al. 2003;Miquel et al. 2006Miquel et al. , 2018Bakhoum et al. 2013;Kacem and Miquel 2018). In the Paramphistomoidea, all the studied species exhibit this feature. Therefore, a lateral To make the diagram clearer, granules of glycogen are not shown in longitudinal sections. ASE anterior spermatozoon extremity, Ax1 and Ax2 first and second axonemes, CC1 central core of the first axoneme, CM cortical microtubules, EO external ornamentation of the plasma membrane, G granules of glycogen, LE lateral expansion, M mitochondrion, N nucleus, PM plasma membrane, PSE posterior spermatozoon extremity, S1 singlets of the first axoneme, SB spine-like bodies expansion has been reported by Ashour et al. (2007) in Basidiodiscus ectorchis and Sandonia sudanensis (Cladorchiidae), by Bakhoum et al. (2011) in Diplodiscus subclavatus (Diplodiscidae), by Seck et al. (2008a) in Carmyerius endopapillatus (Gastrothylacidae), and by Seck et al. (2007Seck et al. ( , 2008b and Swarnakar (2010) in Paramphistomum microbothrium, Cotylophoron cotylophorum, and Orthocoelium scoliocoelium (Paramphistomidae). These lateral expansions, as in D. amphichrus (the present study), are associated with external ornamentation of the plasma membrane and submembranous cortical microtubules (see Table 1). However, the morphology of lateral expansion is not similar in all these digeneans. Among digeneans, the morphology of lateral expansions is variable; e.g., there are reduced lateral expansions or hook-shaped lateral expansions. Only D. subclavatus (Bakhoum et al. 2011) presents a well-developed lateral expansion as its congener D. amphichrus. To our knowledge, only aspidogastreans and some monogeneans exhibit in their spermatozoa lateral expansions so developed (Justine and Mattei 1985;Giese et al. 2020). Both spermatozoa of monogeneans and digeneans have numerous cortical microtubules in their lateral expansions. In contrast, in the lateral expansions of aspidogastreans, there are also internal microtubules additionally to the peripheral ones.
The external ornamentation of the plasma membrane is usually associated with cortical microtubules and is frequent in digenean spermatozoa (see Bakhoum et al. 2017a). This is the case for D. amphichrus and also for all the currently studied species of the Paramphistomoidea (see Table 1). However, in other cases, the ornamentation of the plasma membrane is not observed in association with cortical microtubules, e.g., in Pronoprymna ventricosa (Faustulidae) and hemiuroideans (Quilichini et al. 2007;Kacem et al. 2020).
Recently, both lateral expansion and external ornamentation have been considered useful criteria to establish different models of spermatozoa in the Digenea (see Bakhoum et al. 2017a). These authors consider the type V of spermatozoon as the characteristic for the Paramphistomoidea, but also present in other digenean species such as brachylaimoideans, echinostomatoideans, microscaphidioideans, or pronocephaloideans.
Additionally, the majority of paramphistomoids also exhibit spine-like bodies in this region. Only the two studied cladorchiids (Ashour et al. 2007) lack these ultrastructural elements (see Table 1). Spine-like bodies, described for the first time in Opecoeloides furcatus (Opecoelidae) by Miquel et al. (2000), are prominent electron-dense structures usually present in the ornamented region of the spermatozoon. Only the apocreadiid Neoapocreadium chabaudi has spine-like bodies not associated with the external ornamentation of the plasma membrane (Kacem et al. 2010).

Table 1
Ultrastructural characteristics of the spermatozoon in the Paramphistomoidea

Cortical microtubules: number of bundles, the maximum number, and their location
Another interesting characteristic concerns cortical microtubules. These submembranous ultrastructural elements are present in the sperm cells of the majority of digeneans with a parallel disposition in contrast with the more evolved cestodes (Justine et al. 2001). Only some didymozoids lack cortical microtubules in their spermatozoa (Justine and Mattei 1983;Pamplona-Basilio et al. 2001). When present, cortical microtubules are normally arranged into two fields in the principal region of the spermatozoon (mitochondrial and nuclear regions). However, species of the Hemiuridae and the faustulid P. ventricosa have male gametes with cortical microtubules arranged into a single bundle (Quilichini et al. 2007;Kacem et al. 2020). An interesting aspect is that related to the high number of cortical microtubules in D. amphichrus (63 elements). The remaining studied paramphistomoids also present a high number (about 50 or more). Other taxa with a comparably high number of cortical microtubules (around 40-50) are included in the Mesometridae, Notocotylidae, Pleurogenidae, or Pronocephalidae (Ndiaye et al. 2012a(Ndiaye et al. , 2015aBakhoum et al. 2013;Miquel et al. 2013). The location along the spermatozoon of the maximum number of cortical microtubules has also been considered as an interesting aspect in the above-mentioned review of Bakhoum et al. (2017a). As occurs in D. amphichrus, all the paramphistomoids present the maximum number of these structural elements in the anterior region of the spermatozoon, specifically in the lateral expansion.

Mitochondria: number and morphology
The mitochondrion is another structure present in the spermatozoon of the Digenea. The number and morphology of the mitochondria vary according to the species. The number of mitochondria varies between 1 and 3 (see Bakhoum et al. 2017a). In the Paramphistomoidea, all the species analysed until now have one mitochondrion except the cladorchiids B. ectorchus and S. sudanensis (Ashour et al. 2007) in which they are three mitochondria (see Table 1). The remaining digeneans have one or two mitochondria in the spermatozoon and only two species, namely Euryhelmis squamula (Heterophyidae) and Anisocoelium capitellatum (Cryptogonimidae), contain three mitochondria in their spermatozoa (Bakhoum et al. 2009;Ternengo et al. 2009). Considering the morphology, variability is also described in the sperm cells of digenean species. A filiform mitochondrion has been reported in the majority of species (see Bakhoum et al. 2017a). A second type, a moniliform mitochondria constituted by several mitochondrial bulges joined by a mitochondrial cord, was described for the first time in Holorchis micracanthum by Bâ et al. (2011), and posteriorly, it has been reported in the male gamete of some digeneans such as the cryptogonimids Aphallus tubarium and Timoniella imbutiforme (Foata et al. 2012;Kacem et al. 2017a), the acanthocolpid Stephanostomoides tenuis (Bakhoum et al. 2015), the opecoelids Allopodocotyle pedicellata and Macvicaria obovata (Bakhoum et al. 2017b;Kacem et al. 2017b), the lepocreadiid Opechona bacillaris (Ndiaye et al. 2015b), the plagiorchiid Enodiotrema reductum (Ndiaye et al. 2012b), and the sclerodistomoidid Sclerodistomoides pacificus (Bâ et al. 2020). Finally, Kacem et al. (2019) have recently described in the opecoelid Allopodocotyle tunisiensis a U-shaped mitochondrion characterised by the presence of a circular fold in its posterior part. In the Paramphistomoidea, all the species exhibit a filiform type of mitochondrion.

Posterior spermatozoon extremity
As the anterior extremity, the posterior one shows variable characters depending on the species. Thus, several authors have proposed the use of posterior spermatozoon morphology as phylogenetic characters (Quilichini et al. 2010;Bakhoum et al. 2017a). Quilichini et al. (2010) considered the sequence of the disappearance of principal characters (nucleus, second axoneme, and cortical microtubules) in the posterior extremity of the spermatozoon. They distinguished three types of posterior spermatozoon extremities: type 1 or opecoelidean type characterised by the sequence 'second axoneme, nucleus, and cortical microtubules', type 2 or fasciolidean type with the sequence 'cortical microtubules, second axoneme, and nucleus', and type 3 or cryptogonimidean type with the sequence 'cortical microtubules, nucleus, and second axoneme'. However, several years later, due to several inconsistencies with these three types (Quilichini et al. 2010) in some digeneans, Bakhoum et al. (2017a) proposed the consideration of only the last spermatozoon character. Thus, as occurs in D. amphichrus, the posterior character in the sperm cells of the majority of paramphistomoids is the nucleus (see Table 1).

Concluding remarks
All the currently studied paramphistomoids share several features in their sperm cells that follow the type V of Bakhoum et al. (2017a). This model of spermatozoon is mainly characterised by (i) two 9 + '1' axonemes, (ii) a lateral expansion, (iii) external ornamentation associated with cortical microtubules and located in the anterior part of the anterior region, (iv) the parallel cortical microtubules arranged into two bundles, (v) the maximum number of cortical microtubules located in the anterior part, and (vi) generally one mitochondrion.