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1 Reinvestigation of the sperm ultrastructure of Hypoderaeum conoideum (: 1 2 2 ) 3 4 5 3 6 7 4 Jordi Miquel1,2,*, Magalie René Martellet3, Lucrecia Acosta4, Rafael Toledo5, Anne- 8 9 6 10 5 Françoise Pétavy 11 12 6 13 14 7 1 Secció de Parasitologia, Departament de Biologia, Sanitat i Medi ambient, Facultat de 15 16 17 8 Farmàcia i Ciències de l’Alimentació, Universitat de Barcelona, Av. Joan XXIII, sn, 08028 18 19 9 Barcelona, Spain 20 21 2 22 10 Institut de Recerca de la Biodiversitat (IRBio), Universitat de Barcelona, Av. Diagonal, 645, 23 24 11 08028 Barcelona, Spain 25 26 3 27 12 Université Clermont Auvergne, INRA, VetAgro Sup, UMR EPIA Epidémiologie des 28 29 13 maladies animales et zoonotiques, 63122 Saint-Genès-Champanelle, France 30 31 14 4 Área de Parasitología del Departamento de Agroquímica y Medioambiente, Universidad 32 33 34 15 Miguel Hernández de Elche, Alicante, Spain 35 36 16 5 Departament de Farmàcia i Tecnologia Farmacèutica i Parasitologia, Facultat de Farmàcia, 37 38 39 17 Universitat de València, 46100 Burjassot, València, Spain 40 41 18 6 Laboratoire de Parasitologie et Mycologie Médicale, Faculté de Pharmacie, Université Claude 42 43 44 19 Bernard-Lyon 1, 8 Av. Rockefeller, 69373 Lyon Cedex 08, France 45 46 20 47 48 49 21 *Corresponding author: 50 51 22 Jordi Miquel 52 53 23 Secció de Parasitologia, Departament de Biologia, Sanitat i Medi Ambient, Facultat de 54 55 56 24 Farmàcia i Ciències de l’Alimentació, Universitat de Barcelona, Av. Joan XXIII, sn, 08028 57 58 25 Barcelona, Spain. 59 60 61 62 63 1 64 65 26 e-mail: [email protected] 1 2 27 Telephone: +34 93 4024500 3 4 5 28 Fax: +34 93 4024504 6 7 29 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 2 64 65 30 Abstract 1 2 31 Spermatological characteristics of the digenean Hypoderaeum conoideum (Echinostomatidae) 3 4 5 32 collected from Anas platyrhynchos in the Lac d'Annecy (France) were reinvestigated using 6 7 33 transmission electron microscopy. The previous study on this only describes the 8 9 10 34 presence of two axonemes of unequal lengths, a mitochondrion, a posterior nucleus and the 11 12 35 disposition of cortical microtubules. The present ultrastructural study reveals that the mature 13 14 36 spermatozoon of H. conoideum is a filiform cell, tapered at both extremities. The sperm cell 15 16 17 37 exhibits the characteristics of digenean spermatozoa type V namely two axonemes of the 9+'1' 18 19 38 pattern of trepaxonematan Platyhelminthes, external ornamentation of the plasma membrane 20 21 22 39 associated with cortical microtubules and located in the anterior part of the proximal region of 23 24 40 the sperm cell, lateral expansions, two bundles of parallel cortical microtubules, maximum 25 26 27 41 number of cortical microtubules in the anterior part of the spermatozoon and presence of two 28 29 42 mitochondria. In addition, the sperm cell of H. conoideum shows spine-like bodies and a 30 31 43 posterior extremity with only the nucleus. The ultrastructural characters of the spermatozoon of 32 33 34 44 H. conoideum are compared with those of other digeneans belonging to the superfamily 35 36 45 Echinostomatoidea. 37 38 39 46 40 41 47 Keywords: Hypoderaeum conoideum; Echinostomatidae; Digenea; Sperm characters; 42 43 44 48 Ultrastructure. 45 46 49 47 48 49 50 Introduction 50 51 51 The Echinostomatidae constitutes a large family of digeneans currently including ten 52 53 52 subfamilies and numerous genera basically due to the large range of vertebrates acting as 54 55 56 53 definitive hosts and to their wide geographical distribution (Kostadinova 2005). Hypoderaeum 57 58 54 is a genus included within the subfamily Echinostomatinae. Individuals in this subfamily are 59 60 61 62 63 3 64 65 55 mainly characterized by the presence of a circumoral head collar ventrally interrupted but 1 2 56 uninterrupted at a dorsal level. The present study describes the sperm ultrastructure of 3 4 5 57 Hypoderaeum conoideum, which is the type species of this genus, and parasitizes the intestine 6 7 58 of birds and mammals (Kostadinova 2005). 8 9 10 59 The study of the sperm ultrastructure provides information on a large number of 11 12 60 characters potentially useful to the interpretation of relationships within the Platyhelminthes. 13 14 61 Thus, several researchers have largely studied these characters in different groups such as 15 16 17 62 monogeneans or cestodes and more recently digeneans (Bâ and Marchand 1995; Justine 1991a, 18 19 63 b, 1998, 2001; Levron et al. 2010; Quilichini et al. 2010a, 2011; Bakhoum et al. 2017; Justine 20 21 22 64 and Poddubnaya 2018). 23 24 65 The sperm ultrastructure of Echinostomatoidea is one of the most poorly analysed among 25 26 27 66 Digenean superfamilies. There are only four studies on the spermatozoon, two of them on the 28 29 67 family Fasciolidae (Fasciola gigantica and Fasciola hepatica by Ndiaye et al. 2003, 2004), and 30 31 68 the remaining on the Echinostomatidae (Echinostoma caproni by Iomini and Justine 1997 and 32 33 34 69 Hypoderaeum conoideum by Chen et al. 1996). However, Chen et al. (1996) did not describe 35 36 70 the full ultrastructural organization of H. conoideum spermatozoa, their results were poorly 37 38 39 71 illustrated. In fact, Chen et al. (1996) describes only general characters such as the presence of 40 41 72 two 9+'1' axonemes of unequal lengths, mitochondrion, nucleus and parallel cortical 42 43 44 73 microtubules. Therefore, the aim of the present study is to provide the first complete study on 45 46 74 the spermatozoon of H. conoideum through an accurate analysis of different ultrastructural 47 48 49 75 characters and their organization. 50 51 76 52 53 77 Materials and methods 54 55 56 78 Materials 57 58 59 60 61 62 63 4 64 65 79 Live adult specimens of Hypoderaeum conoideum (Bloch, 1782) were collected from the 1 2 80 digestive tract of a naturally infected mallard duck Anas platyrhynchos Linnaeus, 1758 3 4 5 81 (Anseriformes: Anatidae) captured in the Lac d'Annecy (Saint Jorioz, France) during November 6 7 82 2017. 8 9 10 83 11 12 84 Molecular analyses and specific diagnosis 13 14 85 The specific identification of the adult worms studied herein was performed by molecular 15 16 17 86 analysis as described previously by Georgieva et al. (2014). Genomic DNA was isolated from 18 19 87 an individual worm stored in absolute ethanol using Tteh Realpure Spin Kit (Durviz, Valencia, 20 21 22 88 Spain), according to the manufacturer’s instructions. A partial sequence of the NADH 23 24 89 dehydrogenase subunit 1 (ND1) gene was amplified using the primers NDJ11 (forward; 5'- 25 26 27 90 AGA TTC GTA AGG GGC CTA ATA-3') and NDJ2A (reverse; 5'-CTT CAGCCT CAG CAT 28 29 91 AAT-3') (Morgan and Blair 1995; Kostadinova et al. 2003). The PCR thermocycling profile 30 31 92 comprised initial denaturation at 95 °C for 5 min, followed by 35 cycles (30 s denaturation at 32 33 34 93 94 °C, 20 s primer annealing at 48 °C, and 45 s at 72 °C for primer extension), with a final 35 36 94 extension step of 4 min at 72 °C as described by Georgieva et al. (2014). All PCR reactions 37 38 39 95 were performed with a T100™ Thermal Cycle (Biorad, Hercules, California, USA). PCR 40 41 96 products were visualized on a 1% agarose gel. Sanger sequencing was performed, and 42 43 44 97 chromatograms were inspected visually to resolve any ambiguities. Search of similarities was 45 46 98 performed using BLASTn 47 48 49 99 (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch) and forward and reverse 50 51 100 sequences were aligned using EMBOSS needle tool 52 53 101 (https://www.ebi.ac.uk/Tools/psa/emboss_needle/nucleotide.html). 54 55 56 102 57 58 103 Transmission electron microscopy (TEM) 59 60 61 62 63 5 64 65 104 For the present TEM study, several live adult worms were immediately rinsed with a 0.9% 1 2 105 NaCl solution and fixed in cold (4 °C) 2.5% glutaraldehyde in a 0.1 M sodium cacodylate buffer 3 4 5 106 at pH 7.4 for a minimum of 2 h, rinsed in 0.1 M sodium cacodylate buffer at pH 7.4, post-fixed 6 7 107 in cold (4 °C) 1% osmium tetroxide with 0.9% potassium ferricyanide in the same buffer for 1 8 9 10 108 h, rinsed in Milli-Q water (Millipore Gradient A10), dehydrated in an ethanol series and 11 12 109 propylene oxide, embedded in Spurr's resin and polymerized at 60 °C for 72 h. Ultrathin 13 14 110 sections (60–90 nm thick) at the level of the seminal vesicle were obtained using a Reichert- 15 16 17 111 Jung Ultracut E ultramicrotome. Sections were placed on 200-mesh copper and gold grids. 18 19 112 Sections placed on copper grids were double-stained with uranyl acetate and lead citrate 20 21 22 113 according to the Reynolds (1963) procedure. Copper grids were examined in a JEOL 1010 23 24 114 transmission electron microscope operated at an accelerating voltage of 80 kV, in the 'Centres 25 26 27 115 Científics i Tecnològics' of the University of Barcelona (CCiTUB). 28 29 116 30 31 117 Cytochemistry 32 33 34 118 Sections placed on gold grids were treated according to the Thiéry (1967) test to reveal the 35 36 119 presence of glycogen. Thus, they were treated in periodic acid (PA), thiocarbohydrazide (TCH) 37 38 39 120 and silver proteinate (SP) as follows: 30 min in 10% PA, rinsed in Milli-Q water, 24 h in TCH, 40 41 121 rinsed in acetic solutions and Milli-Q water, 30 min in 1% SP in the dark and rinsed in Milli-Q 42 43 44 122 water. Sections were examined in a JEOL 1010 transmission electron microscope in the 45 46 123 CCiTUB. 47 48 49 124 50 51 125 Results 52 53 126 The general morphological and morphometric data of the adult worms collected were consistent 54 55 56 127 with previous reports of Hypoderaeum conoideum (Toledo et al. 1996) and molecular analysis 57 58 128 confirmed this diagnosis (Fig. 1). The 419 bp obtained herein (GenBank accession number: 59 60 61 62 63 6 64 65 129 MH282580) showed a 99.8% of homology with the previously published GenBank 497 bp long 1 2 130 NADH dehydrogenase subunit 1 (ND1) gene of H. conoideum (Accession number: 3 4 5 131 AY168949.1), with a score of 2086.0 and without gaps. 6 7 132 The mature spermatozoon of H. conoideum is a filiform cell, tapered at both extremities 8 9 10 133 and exhibiting two axonemes of the 9+'1' pattern of trepaxonematan Platyhelminthes, parallel 11 12 134 cortical microtubules, two mitochondria, nucleus, external ornamentation of the plasma 13 14 135 membrane, two lateral expansions, spine-like bodies, and a large amount of glycogen granules. 15 16 17 136 Based on the organization and location of these structures from the anterior to the posterior 18 19 137 sperm extremities (Figs. 2, 3, and 4), the sperm cell was divided into three regions (I to III). 20 21 22 138 Region I (Figs. 2a-m, 4I) corresponds to the anterior region of the spermatozoon. The 23 24 139 anterior tip of the spermatozoon is filiform and sharp (Fig 2a). Cross-sections of the anterior 25 26 27 140 area of region I exhibit the presence of parallel cortical microtubules and the two axonemes that 28 29 141 seem to appear simultaneously (Fig. 2b-e). The ornamented area is observed when the two 9+'1' 30 31 142 axonemes are already formed (Fig. 2f-m). The ornamented area also surrounds two differently 32 33 34 143 shaped lateral expansions (Fig. 2g, h). One of them consists in a simple lateral cytoplasmic 35 36 144 expansion that progressively reduces and disappears before the second one. The second 37 38 39 145 expansion is hook-shaped and in a dorsolateral position (Fig. 2h, i). Thus, there is an area of the 40 41 146 spermatozoon exhibiting two lateral expansions (Fig. 2g, h) and another with only the hook- 42 43 44 147 shaped expansion (Fig. 2i-k). The first mitochondrion appears in the posterior part of region I 45 46 148 (Fig. 2h, i). The transition toward the posterior area of region I is marked by the appearance of 47 48 49 149 spine-like bodies (Fig. 2j, l), the reduction and disappearance of the hook-shaped dorsolateral 50 51 150 expansion (Fig. 2j-l) and the progressive increase of granular material (Fig. 2j-m). Cross- 52 53 151 sections in the posterior part of region I show the reduction of external ornamentation to only 54 55 56 152 cover one side of the male gamete and the arrangement of cortical microtubules in two fields 57 58 153 (Fig. 2m). 59 60 61 62 63 7 64 65 154 Region II (Figs. 2n, o, 3a, b and 4II) is the middle region of the spermatozoon and exhibits 1 2 155 the two axonemes, two fields of parallel cortical microtubules and granular material (Fig. 2n, 3 4 5 156 o), whereas posterior areas are characterized by the presence of the second mitochondrion (Fig. 6 7 157 3a, b). 8 9 10 158 Region III (Figs. 3c-h) is the posterior and nuclear region of the spermatozoon. The main 11 12 159 characteristic of this region is the presence of the nucleus from its anterior area to the posterior 13 14 160 extremity of the sperm cell (Fig. 3c-h). The transition of characters along this region is: (a) the 15 16 17 161 disappearance of the first axoneme (Fig. 3c), (b) the disappearance of one of the bundles of 18 19 162 cortical microtubules (Fig. 3c, d), (c) the disappearance of the second mitochondrion and the 20 21 22 163 remaining cortical microtubules (Fig. 3d, e), and (d) the disorganization and disappearance of 23 24 164 the second axoneme (Fig. 3f-h). A particularity of this region is the presence of the nucleus 25 26 27 165 throughout its length (Fig. 3h). 28 29 166 All the granular material observed in the spermatozoon of H. conoideum was identified 30 31 167 as glycogen by means of the test of Thiéry (Fig. 3i). 32 33 34 168 35 36 169 Discussion 37 38 39 170 Adult H. conoideum specimens were accurately identified by molecular and morphology-based 40 41 171 studies. The ultrastructural study of their mature spermatozoa revealed the main characters 42 43 44 172 usually found in most digeneans, namely two axonemes of the 9+'1' pattern of the 45 46 173 Trepaxonemata (Ehlers 1984), parallel cortical microtubules arranged in two fields, external 47 48 49 174 ornamentation of the plasma membrane, mitochondria, nucleus and granules of glycogen. 50 51 175 However, it also presents other less frequent characters in digeneans such as the lateral 52 53 176 expansions and spine-like bodies. 54 55 56 177 57 58 178 Lateral expansion 59 60 61 62 63 8 64 65 179 The lateral expansion is a character present in the anterior area of the spermatozoa of certain 1 2 180 digeneans (see Bakhoum et al. 2017 for a review). The lateral expansion is usually associated 3 4 5 181 with other characters, namely cortical microtubules, external ornamentation of the plasma 6 7 182 membrane and spine-like bodies. The presence/absence of a lateral expansion in the 8 9 10 183 spermatozoon was recently considered a criterion for the establishment of sperm models in the 11 12 184 Digenea (Bakhoum et al. 2017). The latter authors consider spermatozoa exhibiting a lateral 13 14 185 expansion as the type V spermatozoon. To date, all digeneans presenting spermatozoa with 15 16 17 186 lateral expansion exhibited only one expansion except for the mesometrid Mesometra 18 19 187 brachycoelia, which shows two lateral expansions (Bakhoum and Miquel 2011). In the present 20 21 22 188 study, we describe the second digenean species presenting two expansions in the male gamete. 23 24 189 In the Echinostomatoidea, both fasciolids and echinostomatids have lateral expansions in 25 26 27 190 their spermatozoa (Iomini and Justine 1997; Ndiaye et al. 2003, 2004). Moreover, the 28 29 191 morphology of the lateral expansions in these groups is quite similar: they consist in the so- 30 31 192 called hook-shaped dorsolateral expansion. This type of lateral expansion is also present in the 32 33 34 193 sperm cell of the troglotrematid Troglotrema acutum (Miquel et al. 2006). In H. conoideum 35 36 194 (present study), one of the lateral expansions is also hook-shaped, while the other consists in a 37 38 39 195 simple lateral elongation of cytoplasm. 40 41 196 42 43 44 197 External ornamentation and cortical microtubules 45 46 198 The external ornamentation of the plasma membrane is present in the spermatozoon of most 47 48 49 199 digeneans. Considering the data on the external ornamentation of the plasma membrane 50 51 200 available at the time, Quilichini et al. (2011) established three types of anterior regions of 52 53 201 spermatozoa in the Digenea; the anterior region of the spermatozoon either presented (i) 54 55 56 202 ornamentation more anteriorly, (ii) more posteriorly or (iii) no ornamentation. More recently, 57 58 203 Bakhoum et al. (2017) considered that (i) the presence/absence of external ornamentation and 59 60 61 62 63 9 64 65 204 its location, along with (ii) its association or not with cortical microtubules are potentially useful 1 2 205 for clarifying relationships within digeneans. Although the external ornamentation of the 3 4 5 206 plasma membrane is usually associated with cortical microtubules (see Bakhoum et al. 2017 6 7 207 for a review), in some cases ornamentation was not found to be associated with cortical 8 9 10 208 microtubules. This is the case of the sperm cell of the Faustulidae Pronoprymna ventricosa 11 12 209 (Quilichini et al. 2007) and other digeneans such as hemiurids, lecithasterids and 13 14 210 sclerodistomids (Quilichini et al. 2010b; Dione et al. 2016; Ndiaye et al. 2017). 15 16 17 211 In the Echinostomatoidea, all species studied to date have external ornamentation of the 18 19 212 plasma membrane associated with cortical microtubules and located in the anterior region of 20 21 22 213 the male gamete. Moreover, this ornamentation occurs in the sperm area presenting lateral 23 24 214 expansion or expansions and the maximum number of cortical microtubules. 25 26 27 215 Regarding cortical microtubules, two characteristics are important for phylogenetic 28 29 216 inference: (i) the presence/absence and arrangement in one or two bundles and (ii) the location 30 31 217 of their maximum number (see Bakhoum et al. 2017). Only certain didymozoids lack cortical 32 33 34 218 microtubules (Justine and Mattei 1983; Pamplona-Basilio et al. 2001). With respect to the 35 36 219 arrangement in one/two bundles, as most digeneans, all the echinostomatids and fasciolids 37 38 39 220 studied to date present two bundles of parallel cortical microtubules (see Table 1). There are 40 41 221 only a few digeneans with a single bundle of cortical microtubules, the Faustulidae P. 42 43 44 222 ventricosa and some hemiuroideans (Quilichini et al. 2007, 2010b; Dione et al. 2016). The 45 46 223 location of the maximum number of cortical microtubules is anterior in all the studied 47 48 49 224 Echinostomatoidea, concretely in the area of expansion/expansions (see Table 1). 50 51 225 52 53 226 Spine-like bodies 54 55 56 227 These structures have been generally found in the male gamete of numerous digeneans (see 57 58 228 Bakhoum et al. 2017 for a review). However, according to some authors (Bakhoum et al. 2017) 59 60 61 62 63 10 64 65 229 its usefulness for phylogenetic inference within the Digenea is unknown since in some older 1 2 230 studies these bodies were probably misinterpreted as artefacts in the descriptions of sperm cell 3 4 5 231 (Justine and Mattei 1982; Orido 1988). 6 7 232 The spine-like bodies are prominent, submembranous electron-dense structures that 8 9 10 233 contain a sort of vesicle (Miquel et al. 2000). For some digeneans, a periodicity between spine- 11 12 234 like bodies has been described, while in other species these ultrastructural elements are 13 14 235 irregularly distributed along the sperm cell (see Miquel et al. 2006 and Kacem et al. 2010 for a 15 16 17 236 review). Moreover, spine-like bodies are observed as isolated structures in most digeneans that 18 19 237 exhibit these elements in their spermatozoa. Nevertheless, some bucephalids show numerous 20 21 22 238 spine-like bodies at a same level in cross-sections (Miquel et al. 2017; Kacem and Miquel 23 24 239 2018). 25 26 27 240 According to the available data on the Echinostomatoidea these structures have been 28 29 241 described in the two fasciolids studied to date (Fasciola gigantica and Fasciola hepatica, 30 31 242 Ndiaye et al. 2003, 2004), whereas in the echinostomatids there is variation in the 32 33 34 243 presence/absence of this character. Spine-like bodies have been observed in the present study 35 36 244 whereas Echinostoma caproni lacks these elements in its sperm cells (Iomini and Justine 1997). 37 38 39 245 40 41 246 Transition of characters toward the posterior spermatozoon extremity 42 43 44 247 The transition sequence of cortical microtubules, second axoneme and nucleus toward the 45 46 248 posterior extremity of the spermatozoon was proposed as a potential feature to evaluate 47 48 49 249 interrelationships within digeneans establishing three models of posterior spermatozoon 50 51 250 extremities in the Digenea, namely opecoelidean, fasciolidean and cryptogonimidean 52 53 251 (Quilichini et al. 2010a). Nevertheless, incongruences found in some species, e.g. 54 55 56 252 Scaphiostomum palaearcticum or Aponurus laguncula (Ndiaye et al. 2002; Quilichini et al. 57 58 253 2010b), led several authors to propose using exclusively the terminal character instead of the 59 60 61 62 63 11 64 65 254 sequence of characters towards the posterior spermatozoon tip as a potential feature to evaluate 1 2 255 interrelationships within digeneans (Bakhoum et al. 2017). Thus, in the Echinostomatoidea both 3 4 5 256 fasciolids (Ndiaye et al. 2003, 2004) and H. conoideum (present study) exhibit a transition of 6 7 257 characters in the posterior extremity of their spermatozoa coincident with the fasciolidean type, 8 9 10 258 characterized by the following sequence of characters: posterior end of cortical microtubules 11 12 259 followed by the disappearance of the second axoneme and finally the nucleus. The sole doubtful 13 14 260 data concerns E. caproni (Iomini and Justine 1997), species for which published TEM 15 16 17 261 micrographs do not show clearly the posterior spermatozoon character (see Table 1). 18 19 262 20 21 22 263 Concluding remarks 23 24 264 Mature spermatozoa of H. conoideum correspond to type V of Bakhoum et al. (2017) and this 25 26 27 265 is also the model for all the studied species of the Echinostomatoidea. According to these 28 29 266 authors, this type is characterised by the following principal characters: (i) the presence of two 30 31 267 axonemes of the 9+'1' trepaxonematan pattern, (ii) lateral expansion, (iii) external 32 33 34 268 ornamentation of the plasma membrane associated with cortical microtubules that are located 35 36 269 in the anterior part of the proximal region of the sperm cell, (iv) maximum number of cortical 37 38 39 270 microtubules in the anterior part of the spermatozoon, (v) organisation of cortical microtubules 40 41 271 into two bundles and (vi) presence of one mitochondrion in general. The sperm cell of H. 42 43 44 272 conoideum presents two lateral expansions and two mitochondria, while fasciolids and E. 45 46 273 caproni have only one lateral expansion and a single mitochondrion. A secondary character 47 48 49 274 (not considered for the establishment of digenean sperm models of Bakhoum et al. 2017) such 50 51 275 as the presence/absence of spine-like bodies shows variability between the two studied species 52 53 276 of the family Echinostomatidae. 54 55 56 277 57 58 278 Acknowledgments 59 60 61 62 63 12 64 65 279 We thank the "Fédération des Chasseurs de Haute-Savoie", the "Office National de la Chasse 1 2 280 et de la Faune Sauvage" and the "Syndicat Intercommunal du Lac d'Annecy", and especially 3 4 5 281 Mr. Jean Jacques Pasquier for obtaining permissions and also for their help during the field 6 7 282 work. The authors are grateful to the staff of the "Centres Científics i Tecnològics" of the 8 9 10 283 University of Barcelona (CCTiUB) for their assistance in the preparation of samples. JM is a 11 12 284 member of the 2017-SGR-1008 research group. 13 14 285 15 16 17 286 Funding information 18 19 20 287 This work was partly supported by "Ministerio de Economía y Competitividad" (Madrid, Spain; 21 22 288 grant number BFU2016-75639-P). 23 24 25 26 289 27 28 29 290 Conflict of interest 30 31 32 291 The authors declare that they have no conflict of interest. 33 34 35 292 36 37 293 References 38 39 40 294 Bâ CT, Marchand B (1995) Spermiogenesis, spermatozoa and phyletic affinities in the Cestoda. 41 42 295 Mém Mus Natn Hist Nat, Paris 166:87–95 43 44 45 296 Bakhoum AJS, Miquel J (2011) Caracteres ultraestructurales del espermatozoide en la familia 46 47 297 Mesometridae Poche, 1926 (, Digenea). 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J Microsc 6:987–1018 23 24 407 Toledo R, Muñoz-Antolí C, Pérez M, Esteban JG (1996) Redescription of the adult stage of 25 26 27 408 Hypoderaeum conoideum (Bloch, 1782) (Trematoda: Echinostomatidae) and a new record 28 29 409 in Spain. Res Rev Parasitol 56:195–201 30 31 410 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 18 64 65 411 Legends of figures 1 2 412 Fig. 1. Nucleotide alignment of Hypoderaeum conoideum partial NADH dehydrogenase 3 4 5 413 subunit 1 (ND1) gene sequence (Accession number: MH282580) with the previously GenBank 6 7 414 published (Accession number: AY168949.1). 8 9 10 415 Fig. 2. Spermatozoon of Hypoderaeum conoideum (Regions I and II). a Longitudinal section 11 12 416 of the anterior spermatozoon extremity (ASE). b–e Correlative cross-sections showing the 13 14 417 progressive appearance of axonemes. f–m Correlative cross-sections of the ornamented area of 15 16 17 418 Region I. Note the presence of lateral expansions (LE1 and LE2), spine-like bodies (SB) and 18 19 419 the first mitochondrion (M1) along this area. A large amount of glycogen (G) also appears in 20 21 22 420 the posterior area of Region I. n, o Cross-sections of Region II showing the decrease in the 23 24 421 number of cortical microtubules (CM). Ax axoneme, Ax1 first axoneme, C1 and C2 centrioles 25 26 27 422 of the first and second axonemes, EO external ornamentation of the plasma membrane. Scale 28 29 423 bars = 300 nm. 30 31 424 Fig. 3. Spermatozoon of Hypoderaeum conoideum (Regions II and III). a, b Cross-sections of 32 33 34 425 Region II showing the appearance of the second mitochondrion (M2). c-h Correlative cross- 35 36 426 sections of the nuclear region or Region III showing the transition of characters toward the 37 38 39 427 posterior extremity of the spermatozoon. i Positive test of Thiéry for glycogen (G). CM cortical 40 41 428 microtubules, D doublets, N nucleus, S singlets. Scale bars = 300 nm. 42 43 44 429 Fig. 4. Schematic reconstruction of the spermatozoon of Hypoderaeum conoideum. In order to 45 46 430 make the diagram clearer, granules of glycogen were omitted in the longitudinal section. ASE 47 48 49 431 anterior spermatozoon extremity, Ax1 and Ax2 first and second axoneme, C1 and C2 centrioles 50 51 432 of the first and second axoneme, CM cortical microtubules, D doublets, EO external 52 53 433 ornamentation of the plasma membrane, G glycogen, LE1 and LE2 lateral expansions, M1 and 54 55 56 434 M2 first and second mitochondrion, N nucleus, PM plasma membrane, PSE posterior 57 58 435 spermatozoon extremity, S singlets, SB spine-like bodies. 59 60 61 62 63 19 64 65 Table Click here to download Table Hypoderaeum conoideum Table ParasitolRes REV.docx

Table 1. Available data on the ultrastructure of the spermatozoon in the Echinostomatoidea.

Families and species Spermatozoon characters References TS TAx LE EO EO+CM LEO BCM LMCM M SB PSC ECHINOSTOMATIDAE Echinostoma caproni V 9+'1' + + + AntA 2 AntS 1 - Ax/N? Iomini and Justine (1997) Hypoderaeum conoideum V 9+'1' + + + AntA 2 AntS 2 + N Present study FASCIOLIDAE Fasciola gigantica V 9+'1' + + + AntA 2 AntS 1 + N Ndiaye et al. (2004) Fasciola hepatica V 9+'1' + + + AntA 2 AntS 1 + N Ndiaye et al. (2003)

AntA, anterior part of the anterior region; AntS, anterior region of the spermatozoon; Ax, axoneme; BCM, number of bundles of cortical microtubules; EO, external ornamentation of plasma membrane; EO+CM, association of external ornamentation with cortical microtubules; LE, lateral expansion; LEO, location of external ornamentation; LMCM, location of maximum number of cortical microtubules; M, number of mitochondria; N, nucleus; PSC, posterior spermatozoon character, SB, spine-like bodies; TAx, type of axoneme; TS, type of spermatozoon; +/-, presence/absence of considered character; ?, doubtful data. Figure 1 Click here to download Figure Hypoderaeum conoideum Fig 1.tif Figure 2 Click here to download Figure Hypoderaeum conoideum Fig2.png Figure 3 Click here to download Figure Hypoderaeum conoideum Fig3.png Figure 4 Click here to download Figure Hypoderaeum conoideum Fig4 REV.pdf

ASE I

C1 C2 LE1

LE2

EO LE2 LE1 EO M1 SB M1 SB

Ax1

Ax2 II

M2 G CM PM

M2

III

N

N D

S PSE