Jordi Miquel1,2, Eulàlia Delgado3, Lídia Sarra3, Jordi Torres1,2
Sperm characters of the digenean Prosorhynchus aculeatus Odhner, 1905 (Bucephalidae), a 
parasite of the marine fish Conger conger (Linnaeus, 1758) (Congridae) 
1 Secció de Parasitologia, Departament de Biologia, Sanitat i Medi ambient, Facultat de 
Barcelona, Spain 
2 Institut de Recerca de la Biodiversitat (IRBio), Universitat de Barcelona, Av. Diagonal, 645, 
08028 Barcelona, Spain 
3 Departament de Ciències Ambientals, Facultat de Ciències, Universitat de Girona, Campus 
de Montilivi, sn, 17071 Girona, Spain 
* Corresponding author: Jordi Miquel 
Secció de Parasitologia, Departament de Biologia, Sanitat i Medi ambient, Facultat de 
Barcelona, Spain. 
E-mail: jordimiquel@ub.edu
ORCID: 0000-0003-1132-3772 
Abstract 
Within the Digenea, the family Bucephalidae includes numerous species parasitizing mainly 
marine and freshwater fishes. This family includes five recognized subfamilies, and
ultrastructural data on their sperm cells are very scarce. The existing data are restricted to the 
subfamily Bucephalinae. Thus, the present study is the first complete analysis of the sperm 
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cell of a bucephalid belonging to the subfamily Prosorhynchinae. Herein, we describe the 
ultrastructure of the mature spermatozoon of Prosorhynchus aculeatus, a parasite of the 
conger eel Conger conger, assessed by means of transmission electron microscopy (TEM). 
The spermatozoon of P. aculeatus is a filiform cell that presents 
pattern of trepaxonematan Platyhelminthes, parallel cortical microtubules, mitochondrion, 
nucleus, external ornamentation of the plasma membrane, spine-like bodies and a large 
amount of glycogen granules. According to the anterior location of the external ornamentation 
of the plasma membrane, P. aculeatus presents a
With respect to the posterior extremity, the sperm cell of P. aculeatus corresponds to the 
 cryptogonimidean type. Our results are compared with those of the two 
previously studied bucephalids (Bucephalinae), Prosorhynchoides gracilescens and 
Pseudorhipidocotyle elopichthys.
Key words: Prosorhynchus aculeatus, Bucephalidae, Digenea, sperm characters, 
ultrastructure 
Introduction 
The study of sperm characters has been proven as a useful tool in the analysis of relationships 
of Platyhelminthes, particularly in the case of monogeneans and cestodes (Justine 1991a, b, 
1995, 1998, 2001; Bâ and Marchand 1995; Levron et al. 2010). With respect to the Digenea, 
some authors have attempted to analyse the potential usefulness of diverse ultrastructural 
characteristics of sperm cells (see Quilichini et al. 2010, 2011; Bakhoum 2012). However, 
ultrastructural descriptions of sperm cell characters are still missing concerning many 
digenean families, such as the Bucephalidae. In fact, the available information on the 
Bucephalidae concerns only two species of the subfamily Bucephalinae: Prosorhynchoides 
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gracilescens and Pseudorhipidocotyle elopichthys (Erwin and Halton 1983; Tang et al. 1998). 
Furthermore, the study on P. gracilescens (published as Bucephaloides gracilescens) contains 
several misinterpretations of TEM micrographs and the study on P. elopichthys presents a 
very scarce amount of data.  
The Bucephalidae is a complex family, which includes about 25 genera and 380 species 
parasitizing the swim-bladder, the body cavity, the stomach or the intestine of mainly marine 
and freshwater fishes (Nolan et al. 2015). This family was formerly known as 
Gasterostomatidae, but the genus Gasterostomum was synonymized with Bucephalus and 
thus Bucephalidae was retained as the correct family name. The Bucephalidae includes five 
subfamilies, namely the Bucephalinae, the Dolichoenterinae, the Heterobucephalopsinae, the 
Paurorhynchinae and the Prosorhynchinae (see Overstreet and Curran 2002; Nolan et al. 
2015). According to the recent combined analysis of morphology and genetic data, the 
subfamily Heterobucephalopsinae is basal to the remaining four subfamilies. The 
Dolichoenterinae and the Prosorhynchinae are monophyletic sister clades, basal to the 
Bucephalinae and to the Paurorhynchinae. Contrarily, bucephalines appear as a paraphyletic 
group (see Nolan et al. 2015). Within the subfamily Prosorhynchinae there is the 
cosmopolitan genus Prosorhynchus, including species parasitizing the intestine of marine 
fishes (see Overstreet and Curran 2002).
In order to increase datasets on the ultrastructural organization of spermatozoa characters in 
the Bucephalidae, the present study provides information on Prosorhynchus aculeatus
(Prosorhynchinae). Together with future ultrastructural studies on the spermatozoon of 
bucephalids, the present results will contribute to a better understanding of relationships 
within this digenean family.  
Materials and methods 
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Live adult specimens of Prosorhynchus aculeatus were collected during May 2013 from the 
digestive tract of several conger eels Conger conger (Linnaeus, 1758) (Pisces: Congridae) 
captured in the Mediterranean Sea, off Palamós (Girona, Spain).
Several 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 pH 7.4 for a minimum of 2 h, rinsed in 
0.1 M sodium cacodylate buffer at pH 7.4, post-fixed in cold (4 °C) 1% osmium tetroxide 
with 0.9% potassium ferricyanide [K3Fe(CN)6] in the same buffer for 1 h, rinsed in Milli-Q
water (Millipore Gradient A10), dehydrated in an ethanol series and propylene oxide, 
 60 °C for 72 h. Ultrathin sections (60 90 nm 
thick) of the seminal vesicle were obtained using a Reichert-Jung Ultracut E ultramicrotome.
Sections were placed on 200-mesh copper and gold grids. Sections placed on copper grids 
were double-stained with uranyl acetate and lead citrate according to the Reynolds (1963) 
procedure. Copper grids were examined in a JEOL 1010 transmission electron microscope 
University of Barcelona (CCiTUB). 
Sections placed on gold grids were treated according to the Thiéry (1967) test to reveal 
the presence of glycogen. Thus, they were treated in periodic acid (PA), thiocarbohydrazide 
(TCH) and silver proteinate (SP) as follows: 30 min in 10% PA, rinsed in MilliQ water, 24 h 
in TCH, rinsed in acetic solutions and MilliQ water, 30 min in 1% SP in the dark, and rinsed 
in MilliQ water. Sections were examined in a JEOL 1010 transmission electron microscope in 
the CCiTUB.
Results 
The mature spermatozoon of Prosorhynchus aculeatus is a filiform cell exhibiting the main 
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trepaxonematan Platyhelminthes, parallel cortical microtubules, mitochondrion, nucleus,
external ornamentation of the plasma membrane, spine-like bodies and large amount of 
glycogen granules. The organization and location of all these characters along the male 
gamete permit us to establish three regions (I to III) from the anterior to the posterior sperm 
extremities (Figs. 1 to 4). 
Region I (Figs. 1a g and 4I) corresponds to the anterior region of the spermatozoon. This 
region is mainly characterized by the presence of external ornamentation of the plasma 
membrane (Figs. 1a g and 4I), surrounding nearly all the perimeter of the sperm cell,
progressively disappearing toward the posterior area of region I (Fig. 1g). In fact, the 
complete disappearance of the external ornamentation marks the transition between regions I 
and II. Spine-like bodies are also present along region I (Figs. 1c, e g and 4I). The two 
axonemes are longitudinally displaced in relation to one another (Figs. 1b e and 4I). Cortical 
microtubules are parallel to the hypothetical longitudinal sperm axis and they consist in a 
submembraneous and continuous layer that totally surrounds the cell in the anterior area of 
region I (Figs. 1a d and 4I), while in more posterior area they are discontinuous (Figs. 1e g 
and 4I). 
Region II (Figs. 1h l, 3 and 4II) corresponds to the middle region of the spermatozoon. 
The external ornamentation of the plasma membrane and spine-like bodies are no longer 
present in the anterior part of region II. This region is characterized by the presence of the two 
axonemes, by the progressive organization of cortical microtubules into two bundles (Fig. 1h, 
i, k, l) and by the appearance of the mitochondrion in the posterior area (Figs. 1l and 4II). A
large amount of granular material also appears along this region (Figs. 1j l). The glycogenic 
nature of this granular material has been determined by the Thiéry test (Fig. 3). Notice the 
presence of glycogen between the central core and peripheral doublets of axonemes (Fig. 1j
l).
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Region III (Figs. 2a j, 3 and 4III) corresponds to the nuclear and posterior extremity of 
the spermatozoon. The presence of the nucleus is the main characteristic of this region (Figs. 
2a h and 4III). As in region II, glycogen is present between the central core and the peripheral 
doublets of the axonemes in the anterior areas of region III (Figs. 2a and 3). Along region III, 
the nucleus progressively increases in size as the mitochondrion disappears (Fig. 2a e). The 
posterior extremity of the sperm cell is marked by (i) the disorganization of one axoneme 
(Figs. 2d and 4III), followed by (ii) the disappearance of the mitochondrion (Figs. 2f and 
4III), and by (iii) the disorganization of the other axoneme (Figs. 1g and 4III). During the 
progressive disorganization of the second axoneme there is a reduction in nucleus size (Fig. 
2g, h) and in the number of cortical microtubules (Fig. 2g i). Thus, the posterior 
spermatozoon tip is characterized by the presence of doublets and singlets of the second 
axoneme (Fig. 2j).
Discussion 
The mature spermatozoon of Prosorhynchus aculeatus is a filiform cell, which exhibits a 
similar ultrastructural organization to that reported for most digenean species. However, the 
present study allowed describing the particular arrangement of several characters in all 3 
regions of the sperm cell.  
Anterior spermatozoon region 
The anterior region of the spermatozoon of digeneans shows a number of characters 
potentially useful for phylogenetic inference (see Quilichini et al. 2011; Bakhoum et al. 
2017). 
The character of external ornamentation of the plasma membrane is usually present in the 
spermatozoon of digeneans. According to Quilichini et al. (2011), there are three groups of 
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digenean spermatozoa defined according to the presence/absence and location of the external 
ornamentation of the plasma membrane. Thus, a first group is characterized by the presence 
of the external ornamentation in the proximal area of the sperm cell, a second group exhibits 
the ornamentation in a more distal area of the gamete, and finally a third group includes 
spermatozoa that lack ornamentation. The sperm cell of P. aculeatus is clearly included in the 
first group, presenting the external ornamentation of the plasma membrane in a large area of 
the anterior spermatozoon extremity. The other studies on the ultrastructure of bucephalid 
spermatozoa (Prosorhynchoides gracilescens and Pseudorhipidocotyle elopichthys -Erwin 
and Halton 1983; Tang et al. 1998), present cross-sections exhibiting external ornamentation 
associated only with cortical microtubules, which compares well with the organization 
observed in the sperm cell of P. aculeatus in the present study. We suggest that the 
ornamented sections described in P. gracilescens and P. elopichthys (Erwin and Halton 1983; 
Tang et al. 1998) correspond to anterior areas of the gamete. 
Spine-like bodies, which are usually associated with the external ornamentation of the 
plasma membrane, were described for the first time in the opecoelid Opecoeloides furcatus
(Miquel et al. 2000). Since then, spine-like bodies have been generally described in many 
studies on the ultrastructure of spermatozoa. These structures can be observed in TEM 
micrographs of older works on male gametes, but authors failed to mention their presence and 
probably considered them to be artefacts (Justine and Mattei 1982; Orido 1988). In our 
opinion, at the present state of knowledge provided by the ultrastructural studies of the 
spermatozoon in bucephalids, no conclusion can be drawn concerning spine-like bodies. 
Thus, the absence of spine-like bodies in previous studies on bucephalids (P. gracilescens and 
P. elopichthys -Erwin and Halton 1983; Tang et al. 1998) and its presence in P. aculeatus 
highlight the need for future ultrastructural works, particularly on this character, in species 
belonging to the Bucephalidae.  
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Mitochondrial region 
As in all digeneans, the spermatozoon of P. aculeatus presents a mitochondrion. There are 
different viewpoints concerning the number of mitochondria present in the mature 
spermatozoon of digeneans. According to Burton (1972), during spermiogenesis, several 
mitochondria are present in the zone of differentiation, penetrate into the spermatid body and 
fuse to form a long mitochondrion. Indeed, most of the ultrastructural studies of digeneans 
describe the presence of a single mitochondrion in the male gamete. However, several authors 
have considered the presence of two mitochondria, and even three (see Bakhoum 2012 for a 
review). In the spermatozoon of P. aculeatus there is only one mitochondrion and for the 
remaining bucephalids studied until now, P. gracilescens and P. elopichthys, TEM 
micrographs seem to show a similar situation (Erwin and Halton 1983; Tang et al. 1998). As 
in most digeneans, including P. aculeatus, when a single mitochondrion is present, it is 
located in the middle region of the sperm cell and it usually overlaps the anterior part of the 
nucleus. 
Posterior spermatozoon region 
Several authors have considered the nucleus as a typical character of the anterior region. This 
is the case of the studies on the two bucephalids studied until now, P. gracilescens and P.
elopichthys (Erwin and Halton 1983; Tang et al. 1998). However, the fertilization study in the 
digenean Gonapodasmius sp. (Justine and Mattei 1984) determined that the extremity of the 
spermatozoon containing the nucleus is the last region of the male gamete to fuse with the 
oocyte. Since then, it has been universally accepted that the region containing the nucleus is 
the posterior extremity of the sperm cell.  
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With respect to the posterior region of digenean spermatozoa, the disappearing sequence 
of the principal characters (second axoneme, cortical microtubules and nucleus) toward the 
posterior spermatozoon tip is another interesting aspect. Quilichini et al. (2010) consider three 
types of posterior spermatozoon extremities in the Digenea. A first type (opecoelidean) is 
characterise or axonemal extremity-posterior nuclear extremity-
. A second type (fasciolidean) is characterised by the cortical 
microtubules-posterior axonemal extremity-posterior nuclear extremity hird type 
(cryptogonimidean) is characterised by the cortical microtubules-posterior nuclear 
extremity-posterior axonemal extremity  The posterior spermatozoon extremity of P.
aculeatus seems to correspond to the cryptogonimidean type, even though cortical 
microtubules are still present when the nucleus is no longer observed. A similar situation is 
observed in the two studied bucephalids P. gracilescens and P. elopichthys (Erwin and Halton 
1983; Tang et al. 1998), for which authors show TEM micrographs of the posterior 
spermatozoon extremity sections with a single axoneme and few cortical microtubules.
Considering that some species do not completely fit the character disappearing sequences 
described by Quilichini et al. (2010), other authors have recently proposed the use of only the 
posterior spermatozoon character to identify spermatozoon posterior extremity types 
(Bakhoum et al. 2017).  The use of only one character avoids the existing discrepancies for 
some species in the transition of characters toward the posterior spermatozoon extremity. 
Thus, the cryptogonimidean type of posterior sperm extremity should consider only the 
axoneme as the terminal character. In fact, the presently available ultrastructural data on the 
spermatozoon of three bucephalid genera (Prosorhynchus, Prosorhynchoides and 
Pseudorhipidocotyle) indicate the cryptogonimidean type as characteristic for bucephalids. 
This is also the case of numerous species included in other taxa (for a review see Bakhoum 
2012). Among these taxa, we emphasise the families Hemiuridae with studies on four species
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from three different genera (Ndiaye et al. 2012, 2013, 2014) and Pleurogenidae with four 
studied genera (Miquel et al. 2013; 2014).
Concluding remarks 
The ultrastructural organization of the mature spermatozoon in bucephalids corresponds to the 
classical pattern found in most digeneans. However, some characters should be highlighted in 
this family, including (1) the presence of the external ornamentation in the anterior region of 
the spermatozoon, (2) the presence of a single mitochondrion and (3) the second axoneme as 
the most terminal character of the sperm cell. Nevertheless, these facts should be considered 
with caution due to the scarce ultrastructural data on bucephalids. Also, other characters such 
as the spine-like bodies should be carefully studied in future works on the Bucephalidae. 
Acknowledgements 
The authors are grateful to the fishermen Mr. Miquel Mir and Mr. Francesc Subirats. We are 
also grateful 
for her assistance in the preparation of samples.  
Conflict of interest 
The authors declare that they have no conflict of interest. 
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Legends to figures 
Fig. 1 Prenuclear or anterior regions (I and II) of the spermatozoon of Prosorhynchus 
aculeatus. (a g) Consecutive cross-sections of region I showing the appearance of both 
axonemes and the progressive reduction in the number of cortical microtubules and in the 
external ornamentation of the plasma membrane. (h, i) Cross-sections of anterior area of 
region II. (j, k) Cross- and longitudinal sections of middle area of region II characterized by 
the presence of glycogen. Note the particular location of glycogen between the central core 
and peripheral doublets of axonemes. (l) Cross-section of a posterior area of region II 
characterized by the presence of the mitochondrion. Ax1 first axoneme, C2 centriole of the 
second axoneme, CC1 central core of the first axoneme, CC2 central core of the second 
axoneme, CM cortical microtubules, EO external ornamentation of plasma membrane, G
glycogen, M mitochondrion, SB spine-like bodies. Scale bars 300 nm. 
Fig. 2 Nuclear or posterior region (III) of the spermatozoon of Prosorhynchus aculeatus. (a
e) Cross-sections showing the progressively increasing nucleus size. Note the disorganization 
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of the first axoneme in d. (f) Cross-section without mitochondrion. (g, h) Consecutive cross-
sections toward the disorganization of the second axoneme. (i, j) Cross-sections of the 
posterior spermatozoon extremity containing some cortical microtubules and microtubules of 
the second axoneme. CM cortical microtubules, D doublets, G glycogen, M mitochondrion, N
nucleus, S singlets. Scale bars 300 nm. 
Fig. 3 Glycogen labelling by means of Thiéry test. G glycogen, M mitochondrion, N nucleus. 
Scale bar 300 nm. 
Fig. 4 Schematic reconstruction of the spermatozoon of Prosorhynchus aculeatus. In order to 
make the diagram clearer, the granules of glycogen were omitted in the longitudinal section. 
ASE anterior spermatozoon extremity, Ax1 first axoneme, Ax2 second axoneme, C1 centriole 
of the first axoneme, C2 centriole of the second axoneme, CM cortical microtubules, D
doublets, EO external ornamentation of plasma membrane, G glycogen, M mitochondrion, N
nucleus, PM plasma membrane, PSE posterior spermatozoon extremity, S singlets, SB spine-
like bodies. 
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