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Zoologischer Anzeiger 252 (2013) 404–409

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Zoologischer Anzeiger

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rosorchis palinurichthi Kurochkin, Parukhin & Korotaeva, 1971 (Digenea,clerodistomidae): Ultrastructure of the mature spermatozoon

apa Ibnou Ndiayea, Yann Quilichinib,∗, Aminata Sènea, Rodney A. Brayc, Cheikh Tidiane Bâa,ernard Marchandb

Laboratory of Evolutionary Biology, Ecology and Management of Ecosystems, Faculty of Sciences and Techniques, Cheikh Anta Diop University of Dakar, BP 5055, Dakar, SenegalCNRS UMR 6134, University of Corsica, Laboratory “Parasites and Mediterranean Ecosystems”, 20250 Corte, Corsica, FranceDepartment of Zoology, Natural History Museum, Cromwell Road, London SW7 5BD, UK

r t i c l e i n f o

rticle history:eceived 24 August 2012eceived in revised form 7 November 2012ccepted 7 November 2012vailable online 20 December 2012orresponding Editor: Martin V. Sørensen.

eywords:

a b s t r a c t

This study presents the ultrastructure of the mature spermatozoon of Prosorchis palinurichthi (Digenea:Hemiuroidea: Sclerodistomidae), parasite of the digestive tract of the teleost fish Schedophilus velaini(Sauvage, 1879) (Centrolophidae) captured in the Atlantic Ocean, near Dakar (Senegal). This is a first reportof P. palinurichthi from Senegal and from this host species. The mature spermatozoon of P. palinurichthi isfiliform, tapered at both ends and exhibits the general pattern described in the hemiuroids: two axonemesof the 9 + ‘1’ pattern of trepaxonematans, a nucleus, a mitochondrion, external ornamentation of theplasma membrane in the anterior region of the spermatozoon and parallel cortical microtubules disposed

latyhelminthesrepaxonemataemiuroideaEM

in one side of the spermatozoon except in the anterior spermatozoon extremity and in the mitochondrialregion. However, the particularities of the spermatozoon of P. palinurichthi are the presence of corticalmicrotubules at the anterior extremity, a great number of cortical microtubules (up to 25) in the regionII of the spermatozoon, an elongate nucleus longer than the mitochondrion and the absence of spinelikebodies. Our results are discussed in context with those of previous studies in the Digenea in general andparticularly in the Hemiuroidea.

. Introduction

The family Sclerodistomidae is a small group of trematodes,enerally parasites of marine teleosts, with only four subfam-lies (Sclerodistominae, Kenmackenziinae, Prosogonotrematinaend Prosorchiinae) and five genera (Gibson, 2002). This family istill subject to multiple controversies (Dollfus, 1932; Yamaguti,934, 1958, 1971; Vigueras, 1940; Skrjabin and Guschanskaja,956; Gibson and Bray, 1977, 1979; Gibson, 1983).

Ultrastructural data of spermatozoa have produced many use-ul characters for phylogenetic purposes in Platyhelminthes (Euzett al., 1981; Jamieson and Daddow, 1982; Justine, 1991, 1995, 1998,001; Bâ and Marchand, 1995; Hoberg et al., 1997, 1999, 2001;evron et al., 2010). To our knowledge, there are no ultrastructuralata of the mature spermatozoon of a member of the Sclerodisto-idae. Only spermiogenesis was studied in Prosorchis ghanensis by

ustine (1995). Here we present the first ultrastructural study of theature spermatozoon of Prosorchis palinurichthi, a species belong-

ng to Sclerodistomidae. P. palinurichthi was previously described

∗ Corresponding author. Tel.: +33 495 450 006; fax: +33 495 450 045.E-mail address: quilichini@univ-corse.fr (Y. Quilichini).

044-5231/$ – see front matter © 2012 Elsevier GmbH. All rights reserved.ttp://dx.doi.org/10.1016/j.jcz.2012.11.001

© 2012 Elsevier GmbH. All rights reserved.

as a parasite of black butterfish Hyperoglyphe pringlei (Centrolophi-dae) from the South Atlantic. Thus, our finding of P. palinurichthi inthe centrolophid violet warehou Schedophilus velaini in Senegal isa new host and geographic locality record for this parasite.

2. Materials and methods

Adult specimens of P. palinurichthi (Kurochkin et al., 1971) werecollected from the stomach of a teleost Centrolophidae S. velaini(Sauvage, 1879) caught in the Atlantic Ocean, near Dakar (Senegal).Live worms 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 pH7.2, rinsed in 0.1 M sodium cacodylate buffer at pH 7.2, post-fixed incold (4 ◦C) 1% osmium tetroxide in the same buffer for 1 h, rinsed ina 0.1 M sodium cacodylate buffer at pH 7.2, dehydrated in ethanoland propylene oxide, embedded in Spurr’s resin and polymerizedat 60 ◦C for 24 h.

Ultrathin sections (60–90 nm thick) were obtained using anUltramicrotome (Power tome PC, RMC Boeckeler®) with diamond

knife. Sections placed on copper grids were double-stained withuranyl acetate and lead citrate. Sections placed on gold grids werestained with periodic acid, thiocarbohydrazide and silver pro-teinate to reveal the presence of glycogen (Thiéry, 1967).

P.I. Ndiaye et al. / Zoologischer Anzeiger 252 (2013) 404–409 405

Figs. 1–7. Region I of the mature spermatozoon of Prosorchis palinurichthi. (1) Longitudinal section of anterior extremity of the spermatozoon (Ase) showing the two axonemes(Ax1 and Ax2). (2) Cross section in the anterior extremity of the spermatozoon showing the centriole of the axoneme 1 (C1). (3 and 4) Cross section in the anterior extremityof the spermatozoon showing the centrioles of the two axonemes (C1 and C2), cortical microtubules (Cm) and the external ornamentation of the plasma membrane (Eo)in Fig. 4. (5) Cross section in the anterior part of the spermatozoon with two axonemes (Ax1 and Ax2), cortical microtubules and external ornamentation of the plasmam es, exm

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embrane (Eo). (6 and 7) Sections of the spermatozoon showing the two axonemicrotubules (Cm). Scale bar = 0.2 �m.

The grids were examined in a Hitachi H-7650 transmission elec-ron microscope operated at 80 kV, in the “Service d’Étude et deecherche en Microscopie Électronique” of the University of CorsicaCorte, France).

. Results

Observations of a great number of longitudinal and cross-ections of the mature spermatozoon of P. palinurichthi enabled uso reconstitute its ultrastructural organization and distinguish fouregions (I–IV).

Region I (Figs. 1–7 and 21I) corresponds to the anterior part ofhe spermatozoon. It exhibits two axonemes with a small offsetith respect to each other (Figs. 1–4), cortical microtubules under

he plasma membrane (Figs. 3 and 4) and an external ornamenta-ion of the plasma membrane (Figs. 4–7). However, the appearancef the external ornamentation of the plasma membrane is accom-

anied by a reduction of the submembranous layer of corticalicrotubules (Figs. 4 and 7). In the posterior part of this region, the

xternal ornamentation of the plasma membrane covers almost thentire outer surface of the spermatozoon (Figs. 6 and 7).

ternal ornamentation of the plasma membrane and a reduced number of cortical

Region II (Figs. 8–11 and 21II) is characterized by the disappear-ance of the external ornamentation of the plasma membrane, theappearance of the nucleus, and the concentration of the corticalmicrotubules on only one side of the spermatozoon. The number ofcortical microtubules is reduced in the posterior part of the region I(6 in Fig. 7), but increases progressively from 6 (Fig. 8) to 12 (Fig. 9),then to 17 (Fig. 10) and finally to 25 (Fig. 11). This corresponds toits maximum number in the entire length of the spermatozoon.Another particularity of this region is the presence of some (five)microtubules free within the cytoplasm and not associated with theplasma membrane (Fig. 11). The four attachment zones are visiblein this entire region (Fig. 8).

Region III (Figs. 12, 13 and 21III) corresponds to the mitochon-drial region of the spermatozoon. This region is characterized bythe simultaneous presence of the mitochondrion and the nucleusand the disappearance of the microtubules free in the cytoplasm.

Region IV (Figs. 14–19 and 21IV) is characterized by the disap-pearance of the mitochondrion (Fig. 14), the nucleus reaches its

maximum width (Fig. 15) and a distinct reduction of the numberof cortical microtubules (3 in Fig. 15 and 1 in Fig. 16) which thendisappear in the posterior part of the spermatozoon (Figs. 17–19).This posterior extremity of the spermatozoon contains only

406 P.I. Ndiaye et al. / Zoologischer Anzeiger 252 (2013) 404–409

Figs. 8–13. Cross sections in region II (Figs. 8–11) of the mature spermatozoon of P. palinurichthi. (8–10) Cross section of the spermatozoon with two axonemes, corticalmicrotubules (Cm) disposed in one side of the spermatozoon and a small size of the nucleus (N). The arrowheads indicate the attachment zones. (11) In addition to thestructures observed in Figs. 8–10, this section of the spermatozoon presents unattached cortical microtubules within the cytoplasm. (12–13) Cross sections in the regionI onec 0.2 �m

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II of the spermatozoon with two axonemes, cortical microtubules (Cm) disposed inortical microtubule is observed in the dorsal side of the spermatozoon. Scale bar =

he extremity of the second axoneme which soon becomesisorganized.

Cross-sections stained by the method of Thiéry (Fig. 20) revealhe presence of glycogen granules, precisely in the regions II, III andV.

. Discussion

The mature spermatozoon of P. palinurichthi exhibits the generalattern described from other hemiuroideans so far: two axonemesf the 9 + ‘1’ pattern of trepaxonematans (Ehlers, 1984), nucleus,itochondrion, an external ornamentation of the plasma mem-

rane in the anterior region of the spermatozoon and parallelortical microtubules disposed generally on one side of the sper-atozoon (Justine and Mattei, 1982, 1983, 1984a; Quilichini et al.,

010b; Ndiaye et al., 2012a). A peculiarity of the P. palinurichthipermatozoon is the presence of cortical microtubules in the ante-ior extremity. So far this character has been described from onepecies of Hemiuroidea, the didymozoid Gonapodasmius sp. (Justinend Mattei, 1982, 1984a). In this species, Justine and Mattei (1984a)escribed a complete submembranous layer of cortical micro-ubules in the anterior extremity of the spermatozoon. In the otherpecies of Hemiuroidea studied, authors have described an ante-ior extremity of the spermatozoon without cortical microtubulesJustine and Mattei, 1982, 1983, 1984b; Justine, 1995; Pamplona-asilio et al., 2001; Quilichini et al., 2010b; Ndiaye et al., 2012a).hus, we think that additional data of the spermatozoon of specieselonging to the Sclerodistomidae are needed to confirm if thisharacter is peculiar to this family. In others regions of the sper-

atozoon of P. palinurichthi, cortical microtubules are disposed

n one side only, except in the mitochondrial region, as in allpecies of Hemiuroidea studied to date, namely in Gonapodas-ius sp. (Justine and Mattei, 1982, 1984a), P. ghanensis (Justine,

side and a simultaneous presence of nucleus (N) and mitochondrion (M). Only one.

1995), Aponurus laguncula (Quilichini et al., 2010b). However, in thedidymozoid Didymozoon sp. (Justine and Mattei, 1983) the maturespermatozoon lacks cortical microtubules. We also observed corti-cal microtubules within the cell cytoplasm in the region II (regionof the anterior extremity of the nucleus) of the spermatozoon of P.palinurichthi. Also this characteristic has previously been describedfrom a single species of Hemiuroidea only, namely Gonapodasmiussp. (see Justine and Mattei, 1982). In comparison to the generalcase described in the detailed studies of the spermatozoon of somespecies of hemiuroideans (Quilichini et al., 2010b; Ndiaye et al.,2012a), the spermatozoon of P. palinurichthi presents an increasednumber of cortical microtubules, reaching the maximum number(25) in the middle and the posterior part of the region II (cor-responding to the anterior extremity of the nucleus). Then thenumber of cortical microtubules decreases and they disappeartowards the posterior extremity of the spermatozoon.

The extramembranous ornamentation of the plasma mem-brane described from all the hemiuroids species studied so far,is also observed in the anterior region of the spermatozoon ofP. palinurichthi but it does not reach the anterior extremity as inthe didymozoid Gonapodasmius sp. (Justine and Mattei, 1982), thelecithasterid A. laguncula (Quilichini et al., 2010b) and the hemiuridLecithocladium excisum (Ndiaye et al., 2012a). The structure of theexternal ornamentation of the plasma membrane of hemiuroidsis different to that observed in the other digeneans. In addition,this external ornamentation of the plasma membrane is usuallyassociated with a submembranous layer of cortical microtubulesand is interspersed with spinelike bodies in most of the digeneans,for example, the liver fluke Fasciola gigantica (Ndiaye et al., 2004),

Nicolla testiobliquum (Quilichini et al., 2007), Adlardia novaecaledo-niae [as Siphoderina elongata] (Quilichini et al., 2009), Diplodiscussubclavatus (Bakhoum et al., 2011), Cricocephalus albus (Ndiayeet al., 2011), Pleurogonius truncatus (Ndiaye et al., 2012b) and

P.I. Ndiaye et al. / Zoologischer Anzeiger 252 (2013) 404–409 407

Figs. 14–20. Cross sections in the posterior region of the mature spermatozoon of P. palinurichthi showing (14) the disappearance of the mitochondrion, (15) disorganizationof the first axoneme. (15–18) The nucleus reaches its great size and decreases progressively up to disappearance. (19) Posterior extremity of the spermatozoon with only adisorganized axoneme. (20) TEM photomicrograph of mature spermatozoa of P. palinurichthi showing granules of glycogen (G) revealed by the test of Thiéry. Ax2, axoneme2; Cc, central core; Cm, cortical microtubule; D, doublet of microtubules; N, nucleus; Pse, posterior spermatozoon extremity. Scale bar = 0.2 �m.

408 P.I. Ndiaye et al. / Zoologischer Anzeiger 252 (2013) 404–409

F n of tA axonem ma m

Ws(hcpolcoAtmi

ig. 21. Diagram showing the four regions (I–IV) of the ultrastructural organizatioae2, anterior axoneme extremity 2; Ase, anterior spermatozoon extremity; Ax1,icrotubules; Eo, external ornamentation; M, mitochondrion; N, nucleus; Pm, plas

ardula capitellata (Bakhoum et al., 2012). In most hemiuroideanstudied, namely A. laguncula (Quilichini et al., 2010b), L. excisumNdiaye et al., 2012a) and P. palinurichthi (present study) authorsave described the absence of this submembranous layer ofortical microtubules. The mature spermatozoon of P. palinurichthiresents 6 cortical microtubules in the area with the externalrnamentation. However, in other hemiuroids species (i.e. A.

aguncula, L. excisum) authors have described the absence ofortical microtubules associated with the external ornamentationf the plasma membrane, with the exception of Gonapodasmius sp.

s with the other hemiuroids studied so far, the mature sperma-

ozoon of P. palinurichthi lacks the spinelike bodies described inost digeneans. Thus, we interpret the absence of spinelike bodies

n Hemiuroidea as a plesiomorphy of this superfamily.

he mature spermatozoon of P. palinurichthi. Aae1, anterior axoneme extremity 1;me 1; Ax2, axoneme 2; Cc, central core; Cm, cortical microtubules; D, doublet of

embrane; Pse, posterior spermatozoon extremity.

Another peculiarity of the mature spermatozoon of P. palin-urichthi is the length of the nucleus that occupies the entire lengthof the regions II and III. It is longer than the mitochondrion. Thischaracter has never been described previously in the Hemiuroidea.In A. laguncula, Quilichini et al. (2010b) described an elongate mito-chondrion located in regions II and III and a small nucleus locatedin the posterior part of region II and the beginning of region III.

The posterior extremity of the spermatozoon of P. palinurichthi,posterior to the end of the first axoneme, exhibits the modeldescribed from the hemiurid L. excisum (Ndiaye et al., 2012a),

with only one axoneme and nucleus. Later the nucleus disappearsand then the second axoneme disorganizes into doublets and sin-glets. This extremity corresponds to the type 3 (Quilichini et al.,2010a) characterized by: absence of cortical microtubules, and the

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P.I. Ndiaye et al. / Zoologisch

equence – posterior extremity of the nucleus then the posteriorxtremity of the second axoneme. In the Lecithasteridae species. laguncula (Quilichini et al., 2010b), the posterior part of thepermatozoon is different. Indeed in A. laguncula this part of thepermatozoon exhibits a mitochondrion associated with the twoxonemes which then become disorganized progressively towardshe posterior end of the spermatozoon. Only the doublets and sin-lets resulting from the disorganization of the second axonemeeach the posterior end of the spermatozoon.

Thus, we think that more studies in all the families of hemi-roids are needed to elucidate the models of the spermatozoon inhe Hemiuroidea and the characters of the spermatozoa which arealuable for phylogenetic utility.

cknowledgements

Authors are thank to Online Access to Research in the Environ-ent (OARE) for facilitate us access to party of the bibliography.

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