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Papa Mbagnick Diagne1,2, Alexis Ribas2,3, Srisupaph Poonlaphdecha3, Jordi Miquel2,3,* 1 2 3 4 5 Sperm characteristics in the digenean Diplodiscus amphichrus (Paramphistomoidea, 6 7 Diplodiscidae), a parasite of the Chinese edible frog Hoplobatrachus rugulosus 8 9 10 11 12 1 Laboratoire de Biologie Evolutive, Ecologie et Gestion des Ecosystèmes, Département de 13 14 Biologie animale, Faculté des Sciences et Techniques, Université Cheikh Anta Diop de Dakar, 15 16 17 Dakar, Sénégal 18 19 2 Secció de Parasitologia, Departament de Biologia, Sanitat i Medi Ambient, Facultat de 20 21 22 Farmàcia i Ciències de l'Alimentació, Universitat de Barcelona, Av. Joan XXIII, sn, 08028 23 24 Barcelona, Spain 25 26 3 27 Institut de Recerca de la Biodiversitat (IRBio), Universitat de Barcelona, Av. Diagonal, 645, 28 29 08028 Barcelona, Spain 30 31 32 33 34 *Corresponding author: 35 36 Jordi Miquel 37 38 39 email: [email protected] 40 41 42 43 44 ORCID of the authors: 45 46 Papa Mbagnick Diagne: 0000-0003-1526-7501 47 48 Alexis Ribas: 0000-0002-1256-3316 49 50 51 Srisupaph Poonlaphdecha: 0000-0002-9434-3703 52 53 Jordi Miquel: 0000-0003-1132-3772 54 55 56 57 58 59 60 61 62 63 64 1 65 Abstract 1 2 The ultrastructural characteristics of the mature spermatozoon of Diplodiscus amphichrus 3 4 5 (, Paramphistomoidea, Diplodiscidae) and their ultrastructural organisation were 6 7 examined by means of transmission electron microscopy. Live digeneans were collected from 8 9 10 the Chinese edible frog Hoplobatrachus rugulosus in Thailand. The male gamete of D. 11 12 amphichrus is a filiform cell with two axonemes of the 9+'1' trepaxonematan pattern, nucleus, 13 14 one mitochondrion, parallel cortical microtubules, a well-developed lateral expansion, external 15 16 17 ornamentation of the plasma membrane, spine-like bodies and granules of glycogen. These 18 19 ultrastructural characteristics have already been described in other paramphistomoids studied 20 21 22 so far except for the cladorchiids, which present some differences. Two characteristics of the 23 24 male gamete of D. amphichrus were found for the first time in a digenean: (i) the appearance 25 26 27 of the initial part of the lateral expansion before the axonemes in the anterior extremity and (ii) 28 29 the separation between the second axoneme and the nucleus in the posterior tip. Our results are 30 31 compared with the available data in the Digenea and particularly with other paramphistomoids. 32 33 34 35 36 Keywords: Diplodiscus amphichrus; Diplodiscidae; Paramphistomoidea; Digenea; Sperm 37 38 39 characters; Ultrastructure 40 41 42 43 44 Introduction 45 46 Diplodiscus amphichrus Tubangui, 1949 is a digenean that belongs to the family Diplodiscidae. 47 48 This family, included in the superfamily Paramphistomoidea, constitutes a cosmopolitan and 49 50 51 small group of digeneans with only six valid genera, namely Australodiscus, Catadiscus, 52 53 Dermatemytrema, Diplodiscus, Progonimodiscus and Pseudodiplodiscus. Representatives of 54 55 56 Diplodiscidae are parasites of the digestive tract and they are predominantly found parasitizing 57 58 amphibians but also recorded in reptiles and fish (see Jones, 2005). 59 60 61 62 63 64 2 65 The usefulness of sperm characters and their organisation in the male gamete for the 1 2 interpretation of relationships between Platyhelminthes have been demonstrated for different 3 4 5 groups by several authors (Justine 1991a, 1991b, 1998, 2001; Bâ and Marchand 1995; Miquel 6 7 et al. 1999; Levron et al. 2010; Quilichini et al. 2010a, 2011; Bakhoum et al. 2017a; Justine and 8 9 10 Poddubnaya 2018). Regarding the digenean superfamily Paramphistomoidea, spermatological 11 12 studies have been done on seven species belonging to four families: the Cladorchiidae, 13 14 Diplodiscidae, Gastrothylacidae and Paramphistomidae (Ashour et al. 2007; Seck et al. 2007, 15 16 17 2008a, 2008b; Swarnakar 2010; Bakhoum et al. 2011). In the Diplodiscidae, there is only an 18 19 ultrastructural study of the spermatozoon of Diplodiscus subclavatus (Bakhoum et al. 2011). 20 21 22 The present study aims to describe the sperm characters and their organisation along the mature 23 24 spermatozoon of a second species of the family Diplodiscidae, D. amphichrus. Our results are 25 26 27 also compared with the available data in other digeneans, particularly paramphistomoids, to 28 29 highlight the most characteristic features and their phylogenetic importance. 30 31 32 33 34 Materials and methods 35 36 Specimens 37 38 39 Live adult specimens of Diplodiscus amphichrus Tubangui, 1949 were isolated from a Chinese 40 41 edible frog Hoplobatrachus rugulosus (Wiegmann, 1834) collected in May 2014 by hand from 42 43 44 a frog farm (N 17° 49.21'; E 102° 76.62'; 173m asl) in Udon Thani Province, Thailand. The 45 46 collected amphibian was immediately transported alive to the laboratory at Udon Thani 47 48 Rajabhat University. The frog was anesthetised and subsequently killed using MS222 (ethyl-4- 49 50 51 aminobenzoate). After dissection, digeneans were isolated and fixed for transmission electron 52 53 microscopy (TEM). This study was approved by the Udon Thani Rajabhat University 54 55 56 Care and Ethical Use Committee. Specimens of D. amphichrus were identified according to 57 58 Sey (1985, 1991) and were previously reported in northeast Thailand (Wongsawad et al. 1998). 59 60 61 62 63 64 3 65

1 2 Transmission electron microscopy 3 4 5 For the present TEM study, several flukes were rinsed with a 0.9% NaCl solution and fixed in 6 7 cold (4 °C) 2.5% glutaraldehyde in a 0.1M sodium cacodylate buffer at pH 7.4 for a minimum 8 9 10 of 2 h, rinsed in 0.1M sodium cacodylate buffer at pH 7.4, post-fixed in cold (4 °C) 1% osmium 11 12 tetroxide with 0.9% potassium ferricyanide in the same buffer for 1 h, rinsed in Milli-Q water 13 14 (Millipore Gradient A10), dehydrated in an ethanol series and propylene oxide, embedded in 15 16 17 Spurr's resin and polymerised at 60 °C for 72 h. Ultrathin sections (60–90 nm thick) at the level 18 19 of the seminal vesicle were obtained using a Reichert-Jung Ultracut E ultramicrotome. Sections 20 21 22 were placed on 200-mesh copper and gold grids. Sections placed on copper grids were double- 23 24 stained with uranyl acetate and lead citrate according to Reynolds (1963). Copper grids were 25 26 27 examined in a JEOL 1010 transmission electron microscope operated at an accelerating voltage 28 29 of 80 kV, in the 'Serveis Científics i Tècnics de la Universitat de Barcelona (CCiTUB)'. 30 31 32 33 34 Cytochemistry 35 36 Sections placed on gold grids were treated according to the specific cytochemical test of Thiéry 37 38 39 (1967) to reveal the presence of glycogen. Thus, they were treated in periodic acid (PA), 40 41 thiocarbohydrazide (TCH) and silver proteinate (SP) as follows: 30 min in 10% PA, rinsed in 42 43 44 Milli-Q water, 24 h in TCH, rinsed in acetic solutions and Milli-Q water, 30 min in 1% SP in 45 46 the dark, and rinsed in Milli-Q water. Sections were examined in a JEOL1010 transmission 47 48 electron microscope at an accelerating voltage of 80 kV, in the CCiTUB. 49 50 51 52 53 Results 54 55 56 The observation of numerous ultrathin sections at the level of the seminal vesicle allowed us to 57 58 distinguish three regions in the mature spermatozoon of Diplodiscus amphichrus (Figs. 1-3). 59 60 61 62 63 64 4 65 These three regions exhibit different ultrastructural characteristics and organisation as follows: 1 2 Region I or anterior region of the spermatozoon (Figs. 1a-i and 3I). The main 3 4 5 characteristics of this region are the simultaneous presence of external ornamentation of the 6 7 plasma membrane and a well-developed lateral expansion (Figs. 1a-i and 3I). The anterior 8 9 10 spermatozoon extremity is formed by a large section with a continuous and submembranous 11 12 layer of parallel cortical microtubules associated with the external ornamentation of the plasma 13 14 membrane (Fig. 1a). The two axonemes of the 9+'1' pattern appear almost simultaneously (Fig. 15 16 17 1b-d). The maximum number of cortical microtubules (around 63) is observed in the part of the 18 19 sperm cell exhibiting the maximum development of the lateral expansion (Fig. 1d-f). In the 20 21 22 posterior part of region I the lateral expansion reduces and disappears (Fig. 1g, h) and the 23 24 external ornamentation progressively reduces (Fig. 1h, i). The cortical microtubules form a 25 26 27 continuous and submembranous layer along region I except in the posterior part where their 28 29 arrangement into two bundles is observed (Fig. 1i). Another ultrastructural character of the 30 31 region I is the presence of spine-like bodies irregularly distributed (Fig. 1d, f, i). Finally, 32 33 34 electron-dense granules appear in the cytoplasm in the posterior part of region I (Fig. 1g-i). This 35 36 granular material was identified as glycogen by the application of the specific cytochemical test 37 38 39 of Thiéry (see Fig. 2l for the remaining regions). 40 41 Region II or middle region of the spermatozoon (Figs. 1j-k, 2l and 3II). It is mainly 42 43 44 characterised by the absence of most of the above-mentioned characters. Thus, region II lacks 45 46 the lateral expansion, the external ornamentation of the plasma membrane and spine-like bodies 47 48 (Figs.1j-k and 3II). Thereby, the middle region just presents the two 9+'1' axonemes, two 49 50 51 bundles of parallel cortical microtubules and a large amount of granules of glycogen (Fig. 1j- 52 53 k). 54 55 56 Region III or posterior region of the spermatozoon (Figs. 2a-l and 3III). This region 57 58 corresponds with the nuclear and also mitochondrial part of the sperm cell. The anterior part of 59 60 61 62 63 64 5 65 region III only has the nucleus (Fig. 2a). Later, the mitochondrion appears when the nucleus is 1 2 already present (Fig. 2b, c). After the disappearance of the mitochondrion, the first axoneme 3 4 5 becomes disorganised and disappears (Fig. 2d-f). In the posterior part of region III, the second 6 7 axoneme exhibits a lateral cytoplasmic protrusion (Fig. 2g, h). Finally, the posterior extremity 8 9 10 of the sperm cell is characterised by a separation between the second axoneme and the nucleus 11 12 by a plasma membrane cytokinesis (Fig. 2i, j). That way, the posterior tip contains only the 13 14 nucleus (Fig. 2k). 15 16 17 18 19 Discussion 20 21 22 The mature spermatozoon of Diplodiscus amphichrus exhibits many ultrastructural characters 23 24 previously described in most digenean species. These are two axonemes of the 9+'1' 25 26 27 trepaxonematan pattern (Ehlers 1984), a nucleus, a mitochondrion, parallel cortical 28 29 microtubules, a lateral expansion, external ornamentation of the plasma membrane, spine-like 30 31 bodies and granules of glycogen. The presence/absence and organisation of these features along 32 33 34 the sperm cell show many similarities with the remaining studied paramphistomoids when they 35 36 are compared (see Table 1). However, the spermatozoon of D. amphichrus exhibits two 37 38 39 ultrastructural particularities: (i) the anterior extremity formed by the proximal part of the lateral 40 41 expansion before the appearance of the two axonemes and (ii) the posterior extremity showing 42 43 44 a separation between the second axoneme and the nucleus by a plasma membrane cytokinesis. 45 46 To our knowledge, the present study constitutes the first finding of such characteristics. 47 48 49 50 51 Anterior spermatozoon region: lateral expansion, external ornamentation and spine-like bodies 52 53 The most characteristic features present in the anterior region of the spermatozoon of D. 54 55 56 amphichrus are a well-developed lateral expansion, the external ornamentation of the plasma 57 58 membrane and spine-like bodies. 59 60 61 62 63 64 6 65 Lateral expansions are present in the sperm cell of diverse digeneans belonging to the 1 2 Bucephalidae, Echinostomatidae, Fasciolidae, Mesometridae and Troglotrematidae (Ndiaye et 3 4 5 al. 2003; Miquel et al. 2006, 2018; Bakhoum et al. 2013; Kacem and Miquel 2018). In the 6 7 Paramphistomoidea, all the studied species exhibit this feature. So, a lateral expansion has been 8 9 10 reported by Ashour et al. (2007) in Basidiodiscus ectorchis and Sandonia sudanensis 11 12 (Cladorchiidae), by Bakhoum et al. (2011) in Diplodiscus subclavatus (Diplodiscidae), by Seck 13 14 et al. (2008a) in Carmyerius endopapillatus (Gastrothylacidae), and by Seck et al. (2007, 15 16 17 2008b) and Swarnakar (2010) in Paramphistomum microbothrium, Cotylophoron 18 19 cotylophorum and Orthocoelium scoliocoelium (Paramphistomidae). These lateral expansions, 20 21 22 as in D. amphichrus (present study) are associated with external ornamentation of the plasma 23 24 membrane and submembranous cortical microtubules (see Table 1). However, the morphology 25 26 27 of lateral expansion is not similar in all these digeneans. Among digeneans the morphology of 28 29 lateral expansions is variable; e.g. there is reduced lateral expansions or hook-shaped lateral 30 31 expansions. Only D. subclavatus (Bakhoum et al. 2011) presents a well-developed lateral 32 33 34 expansion as its congener D. amphichrus. To our knowledge only aspidogastreans and some 35 36 monogeneans exhibit in their spermatozoa lateral expansions so developed (Justine and Mattei 37 38 39 1985; Giese et al. 2020). Both spermatozoa of monogeneans and digeneans have numerous 40 41 cortical microtubules in their lateral expansions. In contrast, in the lateral expansions of 42 43 44 aspidogastreans there is also internal microtubules additionally to the peripheral ones. 45 46 The external ornamentation of the plasma membrane is usually associated with cortical 47 48 microtubules and are frequent in digenean spermatozoa (see Bakhoum et al. 2017a). This is the 49 50 51 case for D. amphichrus and also for all the currently studied species of the Paramphistomoidea 52 53 (see Table 1). However, in other cases, the ornamentation of the plasma membrane is not 54 55 56 observed in association with cortical microtubules, e.g. in Pronoprymna ventricosa 57 58 (Faustulidae) and hemiuroideans (Quilichini et al. 2007; Kacem et al. 2020). 59 60 61 62 63 64 7 65 Recently, both lateral expansion and external ornamentation have been considered useful 1 2 criteria to establish different models of spermatozoa in the Digenea (see Bakhoum et al. 2017a). 3 4 5 These authors consider the type V of spermatozoon as the characteristic for the 6 7 Paramphistomoidea, but also present in other digenean species such as brachylaimoideans, 8 9 10 echinostomatoideans, microscaphidioideans or pronocephaloideans. 11 12 Additionally, the majority of paramphistomoids also exhibit spine-like bodies in this 13 14 region. Only the two studied cladorchiids (Ashour et al. 2007) lack these ultrastructural 15 16 17 elements (see Table 1). Spine-like bodies, described for the first time in Opecoeloides furcatus 18 19 (Opecoelidae) by Miquel et al (2000), are prominent electron-dense structures usually present 20 21 22 in the ornamented region of the spermatozoon. Only the apocreadiid Neoapocreadium chabaudi 23 24 has spine-like bodies not associated with the external ornamentation of the plasma membrane 25 26 27 (Kacem et al. 2010). 28 29 30 31 Cortical microtubules: number of bundles, the maximum number and their location 32 33 34 Another interesting characteristic concerns cortical microtubules. These submembranous 35 36 ultrastructural elements are present in the sperm cells of the majority of digeneans with a 37 38 39 parallel disposition in contrast with the more evolved cestodes (Justine et al. 2001). Only some 40 41 didymozoids lack cortical microtubules in their spermatozoa (Justine and Mattei 1983; 42 43 44 Pamplona-Basilio et al. 2001). When present, cortical microtubules are normally arranged into 45 46 two fields in the principal region of the spermatozoon (mitochondrial and nuclear regions). 47 48 However, species of the Hemiuridae and the faustulid P. ventricosa have male gametes with 49 50 51 cortical microtubules arranged into a single bundle (Quilichini et al. 2007; Kacem et al. 2020). 52 53 An interesting aspect is that related to the high number of cortical microtubules in D. 54 55 56 amphichrus (63 elements). The remaining studied paramphistomoids also present a high 57 58 number (about 50 or more). Other taxa with a comparably high number of cortical microtubules 59 60 61 62 63 64 8 65 (around 40 to 50) are included in the Mesometridae, Notocotylidae, Pleurogenidae or 1 2 Pronocephalidae (Ndiaye et al. 2012a, 2015a; Bakhoum et al. 2013; Miquel et al 2013). The 3 4 5 location along the spermatozoon of the maximum number of cortical microtubules has also 6 7 been considered as an interesting aspect in the above-mentioned review of Bakhoum et al. 8 9 10 (2017a). As occurs in D. amphichrus, all the paramphistomoids present the maximum number 11 12 of these structural elements in the anterior region of the spermatozoon, specifically in the lateral 13 14 expansion. 15 16 17 18 19 Mitochondria: number and morphology 20 21 22 The mitochondrion is another structure present in the spermatozoon of the Digenea. The 23 24 number and morphology of the mitochondria vary according to the species. The number of 25 26 27 mitochondria varies between one and three (see Bakhoum et al. 2017a). In the 28 29 Paramphistomoidea, all the species analysed until now have one mitochondrion except the 30 31 cladorchiids B. ectorchus and S. sudanensis (Ashour et al. 2007) in which they are three 32 33 34 mitochondria (see Table I). The majority of digeneans have one or two mitochondria in the 35 36 spermatozoon and only two species, namely Euryhelmis squamula (Heterophyidae) and 37 38 39 Anisocoelium capitellatum (Cryptogonimidae) contain three mitochondria in their spermatozoa 40 41 (Bakhoum et al. 2009; Ternengo et al. 2009). 42 43 44 Considering the morphology, variability is also described in the sperm cells of digenean 45 46 species. A filiform mitochondrion has been reported in the majority of species (see Bakhoum 47 48 et al. 2017a). A second type, a moniliform mitochondria constituted by several mitochondrial 49 50 51 bulges joined by a mitochondrial cord, was described for the first time in Holorchis 52 53 micracanthum by Bâ et al. (2011) and posteriorly it has been reported in the male gamete of 54 55 56 some digeneans such as the cryptogonimids Aphallus tubarium and Timoniella imbutiforme 57 58 (Foata et al. 2012; Kacem et al. 2017a), the acanthocolpid Stephanostomoides tenuis (Bakhoum 59 60 61 62 63 64 9 65 et al. 2015), the opecoelids Allopodocotyle pedicellata and Macvicaria obovata (Bakhoum et 1 2 al. 2017b; Kacem et al. 2017b), the lepocreadiid Opechona bacillaris (Ndiaye et al. 2015b), the 3 4 5 plagiorchiid Enodiotrema reductum (Ndiaye et al. 2012b) and the sclerodistomoidid 6 7 Sclerodistomoides pacificus (Bâ et al. 2020). Finally, Kacem et al. (2019) have recently 8 9 10 described in the opecoelid Allopodocotyle tunisiensis a U-shaped mitochondrion characterised 11 12 by the presence of a circular fold in its posterior part. In the Paramphistomoidea all the species 13 14 exhibit a filiform type of mitochondrion. 15 16 17 18 19 Posterior spermatozoon extremity 20 21 22 As the anterior extremity, the posterior one shows variable characters depending on the species. 23 24 Thus, several authors have proposed the use of posterior spermatozoon morphology as 25 26 27 phylogenetic characters (Quilichini et al. 2010; Bakhoum et al. 2017a). Quilichini et al. (2010) 28 29 considered the sequence of the disappearance of principal characters (nucleus, second axoneme 30 31 and cortical microtubules) in the posterior extremity of the spermatozoon. They distinguished 32 33 34 three types of posterior spermatozoon extremities: type 1 or opecoelidean type characterised by 35 36 the sequence 'second axoneme, nucleus and cortical microtubules', type 2 or fasciolidean type 37 38 39 with the sequence 'cortical microtubules, second axoneme and nucleus' and type 3 or 40 41 cryptogonimidean type with the sequence 'cortical microtubules, nucleus and second axoneme'. 42 43 44 However, several years later, due to several inconsistencies with these three types (Quilichini 45 46 et al. 2010) in some digeneans, Bakhoum et al. (2007a) proposed the consideration of only the 47 48 last spermatozoon character. Thus, as occurs in D. amphichrus, the posterior character in the 49 50 51 sperm cells of the majority of paramphistomoids is the nucleus (see Table 1). 52 53 54 55 56 Concluding remarks 57 58 All the currently studied paramphistomoids share several features in their sperm cells that 59 60 61 62 63 64 10 65 follow the type V of Bakhoum et al. (2017a). This model of spermatozoon is mainly 1 2 characterised by (i) two 9+'1' axonemes, (ii) a lateral expansion, (iii) external ornamentation 3 4 5 associated with cortical microtubules and located in the anterior part of the anterior region, (iv) 6 7 the parallel cortical microtubules arranged into two bundles, (v) the maximum number of 8 9 10 cortical microtubules located in the anterior part, and (vi) generally one mitochondrion. 11 12 13 14 Acknowledgements 15 16 17 We thank Renu Sirimongkonthaworn from Sakon Nakhon Provincial Fisheries Office and 18 19 Tiwarat Thalernkietleela from Biodiversity Research Group, Inland Fisheries Research and 20 21 22 Development Division, Department of Fisheries, both gave us valuable advice in first steps of 23 24 this study. We are also grateful to the staff of the 'Centres Científics i Tecnològics' of the 25 26 27 University of Barcelona (CCTiUB) for their assistance in the preparation of samples. 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Note the presence of 51 52 external ornamentation of the plasma membrane associated with cortical microtubules and 53 54 55 spine-like bodies. h, i Cross-sections of the posterior part of region I lacking lateral expansion. 56 57 j, k Cross-sections of the region II lacking external ornamentation of the plasma membrane. 58 59 Ax1 first axoneme, C1 and C2 centrioles of the first and second axonemes, CM cortical 60 61 62 63 64 18 65 microtubules, EO external ornamentation of the plasma membrane, G granules of glycogen, LE 1 2 lateral expansion, SB spine-like bodies. Scale bars 300 nm. 3 4 5 Fig. 2. Spermatozoon of Diplodiscus amphichrus: region III. a-d Correlative cross-sections of 6 7 the anterior part of nuclear region before the disorganisation of the first axoneme. Note the 8 9 10 presence of the mitochondrion in this part. e Cross-section at the level of the posterior extremity 11 12 of first axoneme. f-h Correlative cross-sections showing the appearance of a cytoplasmic 13 14 extension near the second axoneme. i Cytokinesis of plasma membrane (arrows); the sperm cell 15 16 17 shows two separate sections containing the second axoneme and the nucleus. j Disorganisation 18 19 of the second axoneme. k Posterior spermatozoon tip presenting only the nucleus. l Positive 20 21 22 result for glycogen using the test of Thiéry. Ax2 second axoneme, CC1 central core of the first 23 24 axoneme, CM cortical microtubules, G granules of glycogen, M mitochondrion, N nucleus, S1 25 26 27 and S2 singlets of the first and second axonemes. Scale bars 300 nm. 28 29 Fig. 3. Schematic reconstruction of the mature spermatozoon of Diplodiscus amphichrus. The 30 31 sperm cell is organised in three different regions (I, II and III). To make the diagram clearer, 32 33 34 granules of glycogen are not shown in longitudinal sections. ASE anterior spermatozoon 35 36 extremity, Ax1 and Ax2 first and second axonemes, CC1 central core of the first axoneme, CM 37 38 39 cortical microtubules, EO external ornamentation of the plasma membrane, G granules of 40 41 glycogen, LE lateral expansion, M mitochondrion, N nucleus, PM plasma membrane, PSE 42 43 44 posterior spermatozoon extremity, S1 singlets of the first axoneme, SB spine-like bodies. 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 19 65 Figure 1 Click here to access/download;Figure;Diplodiscus amphichrus Fig1 Zoomorphology.tif Figure 2 Click here to access/download;Figure;Diplodiscus amphichrus Fig2 Zoomorphology.tif Figure 3

ASE

Ax1 LE LE

EO SB SB EO I

CM CM

PM

G II

III

M

M

S1

CC1

N N

Ax2

PSE Table

Table I: Ultrastructural characteristics of the spermatozoon in the Paramphistomoidea

Families/Species ASE LE EO EO+CM LEO MCM BCM LMCM M SB PSC References Cladorchiidae Basidiodiscus ectorchus 1Ax? + + + AntA ? 2 AntS 3 - N? Ashour et al. (2007) Sandonia sudanensis 1Ax? + + + AntA ≈ 90? 2 AntS 3 - N? Ashour et al. (2007) Diplodiscidae Diplodiscus subclavatus 1Ax + + + AntA 75 2 AntS 1 + N Bakhoum et al. (2011) Diplodiscus amphichrus 2Ax-LE+CM+EO + + + AntA 63 2 AntS 1 + N Present study Gastrothylacidae Carmyerius endopapillatus 1Ax + + + AntA ≈ 50? 2 AntS 1 + N Seck et al. (2008a) Paramphistomidae Cotylophoron cotylophorum 1Ax + + + AntA ? 2 AntS 1 + N Seck et al. (2008b) Orthocoelium scoliocoelium ? + + + AntA ? 2 AntS 1 ? 1Ax? Swarnakar (2010) Paramphistomum microbothrium 1Ax + + + AntA ≈ 50? 2 AntS 1 + N Seck et al. (2007) AntA anterior part of the anterior region, AntS anterior region of the spermatozoon, ASE anterior spermatozoon extremity, Ax axoneme, BCM number of bundles of cortical microtubules, EO external ornamentation of the plasma membrane, EO+CM association between the external ornamentation and cortical microtubules, LE lateral expansion, LEO location of the external ornamentation, LMCM location of maximum number of cortical microtubules, M number of mitochondria, MCM maximum number of cortical microtubules, N nucleus, NA not applicable, PSC posterior spermatozoon character SB spine-like bodies, +/- presence/absence of the considered character, ? doubtful or unknown data.