Digenea, Lepocreadiidae) Parasites of Fishes: a Comparative Study of Bianium Plicitum and Bianium Arabicum

Parasitol Res (2015) 114:3747–3757 DOI 10.1007/s00436-015-4604-y

ORIGINAL PAPER

Ultrastructural characters of the spermatozoa in Digeneans of the genus Bianium Stunkard, 1930 (Digenea, Lepocreadiidae) parasites of fishes: a comparative study of Bianium plicitum and Bianium arabicum

Yann Quilichini1 & Papa Ibnou Ndiaye2 & Aminata Sène2 & Jean-Lou Justine3 & Rodney A. Bray4 & Vasyl V. Tkach 5 & Cheikh Tidiane Bâ2 & Bernard Marchand1

Received: 12 June 2015 /Accepted: 19 June 2015 /Published online: 30 July 2015 # Springer-Verlag Berlin Heidelberg 2015

Abstract We present in this paper the first ultrastructural ornamentation were not located at the anterior extremity of study of the spermatozoon of species of the genus Bianium the spermatozoon but at a more posterior level, as in all the Stunkard, 1930, namely Bianium plicitum and Bianium species of Lepocreadioidea studied to date. Differences ob- arabicum belonging to the family Lepocreadiidae. Specimens served between spermatozoa of B. plicitum and B. arabicum are parasites of the tetraodontid teleosts Lagocephalus were observed in: (1) the location of the first mitochondrion, laevigatus caught in the Atlantic Ocean and Lagocephalus (2) size of cortical microtubules which were longer in sceleratus caught in Pacific Ocean, respectively. Live worms B. arabicum than in B. plicitum, and (3) the order of the dis- were fixed and processed for transmission electron microsco- appearance of the axonemes, mitochondrion, and cortical mi- py. Observations of numerous cross and longitudinal sections crotubules toward the posterior region of the spermatozoon. of the spermatozoa of B. plicitum and B. arabicum demon- Only the nucleus reaches into the posterior extremity of strated the general pattern described in all previously studied the spermatozoon in these two species. The general pattern Lepocreadioidea, namely two axonemes with 9+ B1^ pattern of the spermatozoon described in these two species exhibit of Trepaxonemata, two mitochondria, a nucleus, parallel cor- the general model in the genus Bianium. tical microtubules, and an external ornamentation of the plasma membrane associated with spine-like bodies. The maxi- Keywords Spermatozoon . Bianium spp. . Digenea . mum number of cortical microtubules and the external Ultrastructure . Electron microscopy

* Yann Quilichini Introduction [email protected] The Lepocreadiidae Odhner, 1905, is one of the six recog- 1 SERME Service d’Etude et de Recherche en Microscopie nized families of Lepocreadioidea and contains only fish par- Electronique, UMR 6134-SPE, CNRS – Università di Corsica, asites, overwhelmingly marine forms. They are of consider- Campus Grimaldi, 20250 Corte, Corsica, France able biological interest, as they comprise important groups of 2 Laboratory of Evolutionary Biology, Ecology and Management of worms in a range of marine habitats. According to Bray and Ecosystems, Faculty of Sciences and Techniques, Cheikh Anta Diop University of Dakar, BP 5055, Dakar, Senegal Justine (2012), the Lepocreadiidae is one of the largest digenean families with over 75 genera and over 200 species. 3 Institut de Systématique, Évolution, Biodiversité, ISYEB, UMR7205 CNRS, EPHE, MNHN, UPMC, Muséum National d’Histoire The phylogeny of this group has been the subject to many Naturelle, Sorbonne Universités, CP 51, 57 rue Cuvier, controversies. The forms now considered in the 75231 Paris, France Lepocreadioidea were initially placed in the superfamily 4 Department of Life Sciences, Natural History Museum, Cromwell Allocreadioidea (Cribb 2005). Thereafter, morphological con- Road, London SW7 5BD, United Kingdom siderations and molecular phylogenetic characters inferred 5 Department of Biology, University of North Dakota, 10 Cornell from nuclear and mitochondrial genes have permitted revision street, 101 Starcher hall, Grand Forks, ND 58202 – 9019, USA of the phylogeny of the Lepocreadioidea (Bray 2005a, b, c, d; 3748 Parasitol Res (2015) 114:3747–3757

Bray and Cribb 1997, 1998, 2001;Brayetal.2009). Morpho- study of the spermatozoon in the Lepocreadiidae and the first logical considerations were summarized in Bray (2005a, b, c, in the genus Bianium,fromspecimensofBianium plicitum d) and Hall and Cribb (2005). More recently, Bray and Cribb and Bianium arabicum. (2012) have proposed another reorganization of the superfamily Lepocreadioidea based on the molecular phylogeny pre- sented in Bray et al. (2009). Thus, six families are now recog- nized within the Lepocreadioidea, namely the Lepocreadiidae Materials and methods Odhner, 1905; Lepidapedidae Yamaguti, 1958; Aephnidiogenidae Yamaguti, 1934; Enenteridae Yamaguti, Adult specimens of B. plicitum were collected from the diges- 1958; Gorgocephalidae Manter, 1966; and Gyliauchenidae tive tract of a teleost Tetraodontidae Lagocephalus laevigatus Fukui, 1929. (Linnaeus, 1766) caught in the Atlantic Ocean, off Dakar At the moment, there are only six ultrastructural studies of (Senegal). Adult specimens of B. arabicum were collected the spermatozoon in the Lepocreadioidea: one from the digestive tract of another Tetraodontidae, Aephnidiogenidae, Holorchis micracanthum (Bâ et al. Lagocephalus sceleratus caught in the Pacific Ocean, off 2011); two Gyliauchenidae, Gyliauchen sp. (Quilichini et al. New Caledonia (see Bray et al. 2010). 2011)andRobphildollfusium fractum (Bakhoum et al. 2012); Live worms were rinsed with a 0.9 % NaCl solution and fixed and three Lepocreadiidae, Hypocreadium caputvadum in cold (4 °C) 2.5 % glutaraldehyde in a 0.1 M sodium (Kacem et al. 2012), Opechona bacillaris (Ndiaye et al. cacodylate buffer at pH 7.2, rinsed in 0.1 M sodium cacodylate 2015), and Neomultitestis aspidogastriformis (Bakhoum buffer at pH 7.2, post-fixed in cold (4 °C) 1 % osmium tetroxide et al. 2015). In this paper, we present the fourth ultrastructural in the same buffer for 1 h, dehydrated in ethanol and propylene

Fig. 1 Spermatozoon of Bianium plicitum. a Part of seminal vesicle of B. plicitum containing spermatozoa. Scale bar =1μm. b-f Cross sections of region I of the spermatozoon of B. plicitum. Scale bars =0.25μm. b Anterior extremity of the spermatozoon showing the anterior extremity of the first axoneme and the lateral expansion. c Anterior extremity of the spermatozoon showing only the axoneme 1 and an apical electron-dense material. d Anteri- or extremity of the spermatozoon showing the anterior extremity of the second axoneme. e-f Posterior part of region I exhibiting the two axonemes and some cortical microtubules. Aae1 anterior extremity of the first axoneme, Aae2 anterior extremity of the second axoneme, Adm apical electron- dense material, Ax1 first axoneme, Ax2 second axoneme, Cm cortical microtubules, Le lateral expansion, Spz spermatozoon, V seminal vesicle. Parasitol Res (2015) 114:3747–3757 3749 oxide, embedded in Spurr’s resin and polymerized at 60 °C for et de Recherche en Microscopie Électronique^ of the Univer- 24 h. sity of Corsica (Corte, France). Ultrathin sections (60–90 nm thick) were obtained using an Ultramicrotome (Power tome PC, RMC Boeckeler ®) with diamond knife. Sections placed on copper grids were Results double-stained with uranyl acetate and lead citrate. Sections placed on gold grids were stained with periodic acid, Observation of numerous longitudinal and transversal sec- thiocarbohydrazide, and silver proteinate to reveal the pres- tions of mature spermatozoa in seminal vesicles of ence of glycogen (Thiéry 1967). B. plicitum (Figs. 1, 2, 3, 4)andB. arabicum (Figs. 5, The grids were examined in a Hitachi H-7650 transmission 6, 7) allowed us to reveal the ultrastructural organization electron microscope operated at 80 kV,in the BService d'Étude of their spermatozoa. In both species, the spermatozoa

Fig. 2 Spermatozoon of Bianium plicitum. a-c Cross sections of region II and some cortical microtubules. h Two axonemes, nucleus, cortical mi- of the spermatozoon of B. plicitum. a Two axonemes, cortical crotubules, and the second mitochondrion. i-l Cross sections of region V microtubules, and the external ornamentation of the plasma membrane. of the spermatozoon of B. plicitum. i Nucleus, the second mitochondrion, b Two axonemes, cortical microtubules, the external ornamentation of the cortical microtubules, the axoneme 2, and the posterior extremity of the plasma membrane, and the first mitochondrion. c Two axonemes, cortical axoneme 1. j One axoneme, nucleus, cortical microtubules, and some microtubules, the external ornamentation of the plasma membrane, the singlets becoming to the disorganization of the first axoneme. k One first mitochondrion, and spine-like body. d-f Cross sections of region III axoneme, nucleus, and cortical microtubules. l Nucleus, cortical microtu- of the spermatozoon of B. plicitum. d Two axonemes, cortical microtubules, and the posterior extremity of the second axoneme. Cm cortical bules, and the posterior extremity of the first mitochondrion. e Two axo- microtubules, Eo external ornamentation, Mt1 first mitochondrion, Mt2 nemes and cortical microtubules disposed in two bundles. f Two axoneme the second mitochondrion, N nucleus, Pae1 posterior extremity of the first and a reduced number of cortical microtubules. g-h Cross sections of axoneme, Pae2 posterior extremity of the second axoneme, Sb spine-like region IVof the spermatozoon of B. plicitum. g Two axonemes, nucleus, body. Scale bars =0.25μm 3750 Parasitol Res (2015) 114:3747–3757 contained five regions (I–V) from the anterior to the pos- Region III (Figs. 2d–f, 4, 6a, b,and7) terior extremities. In B. arabicum, this region has only the two axonemes and cortical microtubules. In addition, in B. plicitum, in the ante- Region I (Figs. 1a–f, 4, 5b–e,and7) rior part of this region, we noted the presence of the posterior part of the first mitochondrion. In both species, the number of cortical microtubules is highest in the anterior part of this This region corresponds to the anterior extremity of the region (Figs. 2d, e and 5a) and decreases considerably up to spermatozoon. It exhibits an apical electron-dense material four in the posterior part of this region for the two species in a lateral expansion of cytoplasm, the first axoneme (Figs. 2f and 5b). (Figs. 1b, c and 5b, c) then the second axoneme (Figs. 1d and 5d, e). The lateral expansion of cytoplasm and the api- Region IV (Figs. 2g, h, 4, 6c–e,and7) cal electron-dense material disappear just after the appearance of the second axoneme and some cortical microtubules In B. plicitum, this region is characterized by the appearance in the posterior part of region I (Figs. 1e, f and 5e). of the nucleus, then the simultaneous presence of the nucleus and the second mitochondrion (Fig. 2g, h), in addition to the two axonemes and a progressive reduction of the number of Region II (Figs. 2a–c, 4, 5f–j,and7) cortical microtubules. However, the spermatozoon of B. arabicum lacks mitochondrion in region IV. In the latter In addition to the two axonemes and cortical microtubules, species, cross sections showed only the two axonemes, nucle- this region is characterized by the appearance of the external us, and the reduced number of cortical microtubules (Fig. 6c– ornamentation of the plasma membrane (Figs. 2a–c and 5f–j), e). We have also observed the disorganization of the first ax- the first mitochondrion (Figs. 2b, c and 5g–i) and spine-like oneme in the posterior part of this region IV (Fig. 6e). bodies (Figs. 2c and 5i). In the case of B. arabicum, the number of cortical microtubules increases considerably (from 16 Region V (Figs. 2i–l, 3a, b, 4, 6f–k,and7) cortical microtubules (Fig. 5f)to22(Fig.5j)) and reaches its maximum in this region. However, in B. plicitum,wenoted This region corresponds to the posterior part of the spermato- also an increase of the number of cortical microtubules in this zoon and contains all the structures described in region IV in region (15 or 16 cortical microtubules in the Fig. 2a–c), but B. plicitum (Fig. 2i). Sections showed a progressive disappear- their maximum number was observed in region III (Fig. 2e). ance of the second mitochondrion (Fig. 2j), the first axoneme Another noteworthy difference in the ultrastructural features (Fig. 2k) and then the second axoneme, and the cortical mi- of the spermatozoa of B. plicitum and B. arabicum was in the crotubules (Figs. 2l and 3a). Thus, the posterior extremity of location of the first mitochondrion. In B. arabicum,itoccupies the spermatozoon presents only the nucleus surrounded by only the middle part of region II (Figs. 5g–di an 7), while in glycogen granules (Fig. 3a, b). In B. arabicum, the second B. plicitum, it occupies almost the whole length of region II mitochondrion is located only in the anterior part of this region and a part of region III (Figs. 2b–d and 4). V(Fig.6f, g). The posterior extremity of the spermatozoon is

Fig. 3 Spermatozoon of Bianium plicitum. a, b Cross sections of region V of the spermatozoon of B. plicitum. c Transmission electron micrograph of spermatozoa of B. plicitum showing glycogen granules (G) revealed by the test of Thiéry. N nucleus, G glycogen granules. Scale bar =0.25μm Parasitol Res (2015) 114:3747–3757 3751

Fig. 4 Spermatozoon of Bianium Ase plicitum. Schematic reconstruction of the Aae1 spermatozoon in the species B. plicitum. Aae1 anterior extremity of the first axoneme, Adm Aae2 anterior extremity of the second axoneme, Adm apical Aae2 electron-dense material, Ase anterior spermatozoon extremity, Ax1 first axoneme, Ax2 second axo- I neme, Az attachment zone, Cm cortical microtubules, Eo external ornamentation, Mt1 first mito- Az chondrion, Mt2 second mitochondrion, N nucleus, Pae1 pos- Cm terior extremity of the first axo- Eo neme, Pae2 posterior extremity of the second axoneme, Pm plasma membrane, Pse posterior sperma- Mt1 tozoon extremity, Sb spine-like body Sb Eo Az II

Sb Mt1 Ax1 Cm Ax2 Mt1

III

Mt2

IV Mt2

N Pae1

Pae2 V

Pse characterized by the disappearance of the second axo- Discussion neme and cortical microtubules (Fig. 6h, i) to make way for the nucleus and glycogen granules in the posterior The mature spermatozoon of the genus Bianium is described extremity of the spermatozoon (Fig. 6j, k). The glyco- in this study in specimens of B. plicitum and B. arabicum.It gen granules are clearly highlighted with the Thiéry’s exhibits the general pattern of spermatozoon described in the method (Figs. 3c and 5l). Lepocreadiidae, namely presence of apical electron-dense 3752 Parasitol Res (2015) 114:3747–3757

Fig. 5 Spermatozoon of Bianium arabicum. a Part of seminal vesicle of membrane. g, h Two axonemes, cortical microtubules, the external orna- B. arabicum containing spermatozoa. Scale bar =1μm. b-e Cross mentation of the plasma membrane, and the first mitochondrion. i Two sections and longitudinal section (b) of region I of the spermatozoon of axonemes, cortical microtubules, the external ornamentation of the plas- B. arabicum. Scale bars =0.25μm. b Anterior extremity of the ma membrane, the first mitochondrion, and spine-like body. j Two axo- spermatozoon. c One axoneme and an apical electron-dense material. d nemes, cortical microtubules, and the external ornamentation of the plas- One axoneme, the anterior extremity of the second axoneme and a lateral ma membrane. Aae2 anterior extremity of the second axoneme, Adm expansion of cytoplasm containing the apical electron-dense material. e apical electron-dense material, Ax1 first axoneme, Cm cortical microtu- Two axonemes and cortical microtubules. f-j Cross sections of region II bules, Eo external ornamentation, Le lateral expansion, Mt1 first mito- of the spermatozoon of B. arabicum. Scale bars =0.25μm. f Two axo- chondrion, Sb spine-like body, Spz spermatozoon, Svw seminal vesicle nemes, cortical microtubules, and external ornamentation of the plasma wall material in the anterior extremity, two axonemes of the 9 + B1^ According to Bakhoum et al. (2015), this structure appears pattern of Trepaxonemata, two mitochondria, a nucleus, par- further posteriorly as a submembranous layer located around allel cortical microtubules, external ornamentation of the plas- the second axoneme. In other digeneans, a similar apical ma membrane, and spine-like bodies (Ndiaye et al. 2015; electron-dense material is described only in the pronocephalid Bakhoum et al. 2015). However, this spermatozoon presents Cricocephalus albus (Ndiaye et al. 2011)andinthe some peculiarities. mesometrid Elstia stossichianum (Bakhoum et al. 2013). How- ever, in these species, the apical electron-dense material is as- The anterior extremity of the spermatozoon sociated with external ornamentation of the plasma membrane and spine-like bodies. Cortical microtubules are also absent in Apical electron-dense material is described in the anterior ex- the anterior extremity of all the spermatozoa of Lepocreadiidae tremities of the spermatozoon of B. plicitum and B. arabicum described to date (Ndiaye et al. 2015;Bakhoumetal.2015). (this study). This structure is also described in spermatozoa of Thus, the simultaneous presence of the apical electron-dense all lepocreadioid species studied so far (Ndiaye et al. 2015). material (Adm) in anterior extremity of the spermatozoon Parasitol Res (2015) 114:3747–3757 3753

Fig. 6 Spermatozoon of Bianium arabicum. a, b Cross sections of region extremity of the second axoneme. i Nucleus and the posterior extremity III of the spermatozoon of B. arabicum showing two axonemes and of the second axoneme. j Posterior part of the spermatozoon with only cortical microtubules. c-e Cross sections of region IV of the nucleus and granules of glucogen. k Posterior extremity of the spermatozoon of B. arabicum. c, d Two axonemes, nucleus, and a spermatozoon with only granules of glycogen. l Transmission electron reduced number of cortical microtubules. e One axoneme, nucleus, a micrograph of spermatozoa of B. arabicum showing glycogen granules reduced number of cortical microtubules, and the posterior extremity of (G) revealed by the test of Thiéry. Ax1 axoneme 1, Ax2 axoneme 2, Cm the axoneme 1. f-k Cross sections of region V of the spermatozoon of cortical microtubules, G glycogen granules, Mt mitochondrion, N B. arabicum. f One axoneme, nucleus, cortical microtubules, and the nucleus, Pae1 posterior extremity of the axoneme 1, Pae2 posterior second mitochondrion. g One axoneme, nucleus, and cortical extremity of the axoneme 2. Scale bars =0.25μm microtubules. h Nucleus, cortical microtubules, and the posterior without external ornamentation of the plasma membrane and mitochondrion in the mature spermatozoon is an important spine-like body associated with the absence of cortical micro- characteristic of digeneans spermatozoa (Justine 1995, tubules seems to be a feature characteristic of the 1998). The number of mitochondria varies from one to three Lepocreadiidae and may be a useful tool for phylogenetic in the digenean spermatozoa (Bâ et al. 2011; Bakhoum et al. purposes. 2014; Ternengo et al. 2009;Quilichinietal.2007b). In the Similar to all other Lepocreadioidea studied so far (Ndiaye Lepocreadiidae, two mitochondria are described in all studied et al. 2015; Bakhoum et al. 2015), B. plicitum and B. arabicum species (Ndiaye et al. 2015; Bakhoum et al. 2015). The first lacked cortical microtubules in the anterior extremity of the mitochondrion is always located in the anterior part of the spermatozoon. spermatozoon and associated with external ornamentation of the plasma membrane and spine-like bodies in the case of The mitochondria and their associated structures O. bacillaris (Ndiaye et al. 2015), N. aspidogastriformis (Bakhoum et al. 2015), B. plicitum (present study), and The increase of the number of ultrastructural studies of the B. arabicum (present study). The mature spermatozoon of spermatozoa in the digeneans has helped to highlight numer- H. caputvadum is unique among the Lepocreadiidae in the ous peculiarities of the male gamete. Presence of at least one absence of spine-like bodies (Kacem et al. 2012). The 3754 Parasitol Res (2015) 114:3747–3757

Fig. 7 Spermatozoon of Bianium Ase arabicum. Schematic reconstruction of the Ax1 Aae1 spermatozoon in the species B. arabicum. Aae1 anterior Adm Aae2 extremity of the first axoneme, Aae2 anterior extremity of the Ax2 second axoneme, Adm apical Aae2 electron-dense material, Ase anterior spermatozoon extremity, Ax1 first axoneme, Ax2 second axoneme, Az attachment zone, Cm I cortical microtubules, Eo external ornamentation, Mt1 first mito- Az chondrion, Mt2 second mito- Eo chondrion, N nucleus, Pae1 posterior extremity of the first axo- Cm neme, Pae2 posterior extremity of the second axoneme, Pm plasma membrane, Pse posterior spermatozoon extremity, Sb spine-like Eo Mt1 body Sb Sb Mt1 II

Ax1 Cm Ax2

III

Pae1 N Mt2

Mt2

Pae2

Pse

gyliauchenid Gyliauchen sp. has spine-like bodies associated bodies in association with the external ornamentation of the with the part of the spermatozoon located posterior to the first plasma membrane in the mitochondrial region (Bakhoum mitochondrion that lacks external ornamentation of the plas- et al. 2012). The spermatozoon of the aephnidiogenid ma membrane (Quilichini et al. 2011). As in the H. micracanthum lacks spine-like bodies (Bâ et al. 2011). Lepocreadiidae, the gyliauchenid R. fractum has spine-like Thus, we think that more studies are needed to clarify the Parasitol Res (2015) 114:3747–3757 3755 phylogenetic value of the association external ornamentation, et al. 2010a, b) and can be used as a very interesting spine-like bodies, and mitochondrion in Lepocreadioidea in character for phylogenetic inference. The highest number general and particularly in the Lepocreadiidae. of microtubules can be located in the anterior extremity of The morphology, the length, and the shape of the first the spermatozoon or at a more posterior level and can mitochondrion also differ in all studied species of the inform us on the general organization of microtubules Lepocreadiidae. In the case of the genus Bianium, the first along the spermatozoon (Quilichini et al. 2010a, b). In mitochondrion of B. plicitum is relatively longer than that the case of Bianium, the two species studied show an in B. arabicum. In other Lepocreadioidea, one mitochon- increase of the number of cortical microtubules from the drion was described in the gyliauchenid Gyliauchen sp. anterior extremity toward the middle part of the spermato- (Quilichini et al. 2011), two mitochondria in another zoon and then a progressive decrease toward the posterior gyliauchenid, R. fractum (Bakhoum et al. 2012), and three extremity. This feature seems to be a typical characteristic mitochondria in the aephnidiogenid H. micracanthum (Bâ of the Lepocreadioidea (Table 1). et al. 2011).

The posterior extremity of the spermatozoon Location of external ornamentation and cortical microtubules The posterior part of the mature spermatozoon is characterized by the simultaneous presence of the two axonemes, the nucle- Quilichini et al. (2011) proposed three types of digenean us, a second mitochondrion, and the cortical microtubules in spermatozoa based on the position of the external orna- all the Lepocreadioidea studied. The spermatozoa of each spe- mentation of the plasma membrane in the anterior areas cies differ by the order of the disappearance of these structures of the mature spermatozoon. As in the other species of toward the posterior extremity of the spermatozoon. The sec- Lepocreadioidea, spermatozoa of B. plicitum and ond mitochondrion of B. arabicum is shorter than that in B. arabicum correspond to the type 2 characterized by B. plicitum and is located further toward the posterior end of the presence of external ornamentation further posterior- the spermatozoon. Table 1 summarizes all structures present in ly (Table 1), usually in the mitochondrial region. This the posterior region of the spermatozoon in the organization is also found in other digenean species Lepocreadioidea, their order of disappearance, and the type (Ndiaye et al. 2012;Quilichinietal.2007a, 2009). of posterior extremity. Table 1 shows that in the genus The external ornamentation and the maximum number Bianium, the posterior extremity of the spermatozoon exhibits of cortical microtubules are found at different levels in only the nucleus. The posterior extremity of the spermato- spermatozoa of the studied species. This feature has been zoon of the species of Lepocreadioidea studied to date noticed in the literature (Ndiaye et al. 2014; Quilichini exhibits only a nucleus or the posterior end of the second

Table 1 Ultrastructural characteristics of the spermatozoa of Lepocreadioidean species

Families and species Eo location CM max location Order of disappearance of structures toward PSE References the PSE

Aephnidiogenidae Holorchis micracanthum Not Ant Not Ant M2, Ax1, CM, N Ax2 Bâ et al. (2011) Lepocreadiidae Neomultitestis Not Ant Not Ant Ax1, M2, Ax2, CM N Bakhoum et al. (2015) aspidogastriformis Opechona bacillaris Not Ant Not Ant Ax1, M2, CM, N Ax2 Ndiaye et al. (2015) Hypocreadium caputvadum Not Ant Not Ant M2, CM, N Ax2 Kacem et al. (2012) Bianum plicitum Not Ant Not Ant M2, Ax1, CM, Ax2 N Present study Bianum arabicum Not Ant Not Ant Ax1, M2, CM, Ax2 N Present study Gyliauchenidae Gyliauchen sp. Not Ant Not Ant Ax1, CM, N Ax2 Quilichini et al. (2011) Robphildollfusium fractum M2, CM, Ax2 N Bakhoum et al. (2012)

Ax1 axoneme 1, Ax2 axoneme 2, CM cortical microtubules, CM max location location of the maximal number of cortical microtubules in the anterior extremity of the spermatozoon (Ant) or at a more posterior level (Not Ant), Eo location location of the external ornamentation in anterior extremity of the spermatozoon (Ant) or at a more posterior (Not Ant), M mitochondrion, N nucleus, PSE posterior spermatozoon extremity 3756 Parasitol Res (2015) 114:3747–3757 axoneme. Thus, the representatives of the Lepocreadiidae Bray RA, Cribb TH (2001) A review of the family Enenteridae Yamaguti, show two patterns: the posterior extremity with the nucle- 1958 (Digenea), with descriptions of species from Australian waters, including Koseiria huxleyi n. sp. Syst Parasitol 48:1– us only (Bakhoum et al. 2015; Ndiaye et al., the present 29 study) or the posterior extremity with the end of the sec- Bray RA, Cribb TH (2012) Reorganisation of the superfamily ond axoneme only (Kacem et al. 2012;Ndiayeetal. Lepocreadioidea Odhner, 1905 based on an inferred molecular phy- – 2015). Thus, more studies are needed to explore the pos- logeny. Syst Parasitol 83:169 177 Bray RA, Justine J-L (2012) A review of the Lepocreadiidae (Digenea, sible phylogenetic importance of the posterior extremity Lepocreadioidea) from fishes of the waters around New Caledonia. of spermatozoa in the Lepocreadioidea. Acta Parasitol 57:247–272 Bray RA, Waeschenbach A, Cribb TH, Weedall GD, Dyal P, Littlewood DTJ (2009) The phylogeny of the Lepocreadioidea (Platyhelminthes, Digenea) inferred from nuclear and mitochondrial genes: implications for their systematic and evolution. 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