New spermatological characteristics in the Hemiuroidea inferred from the ultrastructure of the spermatozoon of Gonocerca phycidis a stomach parasite of Gephyroberyx darwinii (Teleostei: Trachichthyidae) off the Senegal coast

The present study provides the first ultrastructural data on the digenean Gonocerca phycidis belonging to the family Gonocercidae. Live adults digenean were collected from the stomach of the marine fish Gephyroberyx darwinii (Teleostei: Trachichthyidae) caught in the Atlantic Ocean, off Dakar (Senegal). This is also the first report of G. phycidis from Senegal and from this fish. The mature spermatozoon exhibits (i) two axonemes with the 9 + ‘1’ pattern of the Trepaxonemata, (ii) a lateral expansion associated with external ornamentations and without cortical microtubules, (iii) two types of external ornamentation of the plasma membrane located in the anterior region of the spermatozoon, (iv) two bundles of cortical microtubules in the mitochondrial and nuclear areas of the spermatozoon, (v) one mitochondrion and (vi) a nucleus. The maximum number of cortical microtubules is located in the median part of the spermatozoon. Moreover, the mature spermatozoon of G. phycidis presents some peculiarities. The presence of a lateral expansion without cortical microtubules associated with external ornamentations is described here, for the first time, in digenean spermatozoa. The presence of two elongated bundles of cortical microtubules is also reported for the first time in the superfamily of Hemiuroidea. However, the ultrastructure of the posterior spermatozoon extremity confirms that already described in other hemiuroidean species.


Introduction
The systematic position of the genus Gonocerca has been the subject of many controversies. It was considered as a member of the subfamily Gonocercinae Skrjabin and Guschanskaja 1955, within the family Derogenidae in the Superfamily of Hemiuroidea (Gibson and Bray 1979;Gibson 2002). However, based on molecular studies, Olson et al. (2003) and Panklov et al. (2006) were the first to propose the reconsideration of the Gonocercinae as an independent family. According to these authors, the family Derogenidae is polyphyletic. Likewise, Hemipera manteri, a member of the Gonocercinae, and Derogenes varicus, the type genus of the Derogenidae, are phylogenetically distant from others. This fact was also supported posteriorly by Sokolov et al. (2016). More recently, based on molecular studies carried out on four gonocercids including the type species G. phycidis, Sokolov et al. (2018) have considered the Gonocercidae as a separate family from the Derogenidae. Molecular studies show also that this family is basal to the remaining families of the Hemiuroidea (Sokolov et al. 2019). To date, the Gonorcercidae includes only two genera namely Gonocerca (the type genus) and Hemipera. However, according to Sokolov et al. (2018) the generic composition of this new family requires further clarification as the molecular data do not support the inclusion of the genus Hemipera in the Gonocercidae.
In this context, the ultrastructural studies of species belonging to the family Gonocercidae are of great importance to bring additional information to complement the existing molecular data. Gonocerca phycidis is the type species of the genus Gonocerca and the first member of the Gonocercidae family to be studied by transmission electron microscopy.
To increase the data on spermatological studies in the Hemiuroidea, the present study provides, for the first time, the spermatological characteristics of the genus Gonocerca and the family Gonocercidae. We compare our results with those obtained in other hemiuroideans previously described.

Material and methods
Adult specimens of Gonocerca phycidis Manter, 1925 were collected from the stomach of Gephyroberyx darwinii (Trachichtyidae) caught off the coast of Dakar (Atlantic Ocean). The adult specimens were washed in 0.9% NaCl solution and fixed in cold (4 °C) 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer at pH 7.2, post-fixed in cold (4 °C) 1% osmium tetroxide in the same buffer for 1 h, dehydrated in ethanol and propylene oxide, embedded in Spurr's resin and finally polymerized at 60 °C for 48 h.
Ultrathin Sects. (60-90 nm thick) were cut on an ultramicrotome (Power tome PC, RMC Boeckeler) with a diamond knife, placed on copper and gold grids. Sections on copper grids were double-stained with uranyl acetate and lead citrate. The ultrathin sections were examined using a Hitachi H-7650 electron microscope operated at 80 kV, in the ''Service d'Étude et de Recherche en Microscopie Électronique'' of the University of Corsica (Corte, France).

Results
The interpretation of several cross and longitudinal sections of the mature spermatozoon of G. phycidis allows us to establish three distinctive regions (I-III) from the anterior to the posterior spermatozoon extremity.
Region I (Figs. 1A-H and 3I) corresponds to the anterior extremity of the spermatozoon. Cross-sections of the anterior tip show axonemal microtubules surrounded by a continuous and submembranous layer of parallel cortical microtubules with a maximum number around 24 (Fig. 1A, B). Then, the two centrioles appear almost simultaneously and they are covered entirely by filamentous ornamentation associated with the layer of cortical microtubules (Fig. 1C, D). A well-developed lateral expansion associated only with external ornamentation of the plasma membrane appears before the complete formation of the second axoneme ( Fig. 1E). At this level the layer of cortical microtubules becomes discontinuous and their number decreases progressively (Fig. 2E, H). The transition toward region II is characterized by the disappearance of lateral expansion.
Region II (Figs. 2A-F and 3II) corresponds to the middle region of the spermatozoon. In the anterior part of this region, cross-sections show only two axonemes, their attachment zones, and four cortical microtubules ( Fig. 2A). The number of cortical microtubules progressively increases in this region by 4 ( Fig. 2A)

Discussion
The mature spermatozoon of G. phycidis exhibits the general pattern described in most hemiuroideans: two axonemes of the 9 + '1' pattern of Trepaxonemata (Ehlers 1984), nucleus, mitochondria, external ornamentation of the plasma membrane, and parallel cortical microtubules (Kacem et al. 2020). However, compared with hemiuroideans studied to date, the mature spermatozoon of G. phycidis presents some unusual characteristics (Table 1).

Anterior spermatozoon morphology
The mature spermatozoon of G. phycidis exhibits in its anterior tip two centrioles corresponding to the two axonemes of 9 + '1' trepaxonematan pattern (Ehlers 1984).  (Bâ et al. 2020), whereas in the remaining studied species there is only one axoneme in their anterior tip (see Table 1). In this anterior tip, there is also the presence of a continuous and submembranous layer of cortical microtubules. Within the Hemiuroidea, this feature was previously observed only in the didymozoid Gonapodasmius sp. by Justine and Mattei (1982). However, some cortical microtubules were also observed in the anterior spermatozoon extremity of other hemiuroideans namely the Hemiuridae Hemiurus appendiculatus and Lecithochirium microstomum (Ndiaye et al. 2014;Dione et al. 2016) and the Sclerodistomidae Prosorchis palinurichthi and Sclerodistomum italicum (Ndiaye et al. 2013b(Ndiaye et al. , 2017 whereas in the remaining hemiuroideans, cortical microtubules are absent in the anterior tip  (Table 1). Nevertheless, their appearance is noted only when both axonemes are already formed. However, it is important to remark that in the didymozoids Didymocystis wedli and Didymozoon sp. (Justine and Mattei 1983;Pamplona-Basilio et al. 2001), the sperm cell lacks cortical microtubules.
Another peculiarity observed in the anterior spermatozoon region is the presence of a well-developed lateral expansion that distinguishes the mature spermatozoon of G. phycidis from those of the remaining hemiuroids studied until now. A lateral expansion has been described at the anterior extremity of the spermatozoon of digeneans belonging to five superfamilies: the Echinostomatoidea, the Microscaphidioidea, the Paramphistomoidea, the Pronocephaloidea and the Bucephaloidea (Bakhoum et al. 2017a;Kacem and Miquel 2018;Ndiaye et al. 2019). In the present work, we describe for the first time its presence in a Hemiuroidea. Its morphology is variable according to the species and it is generally associated with external ornamentation, cortical microtubules, and sometimes with spine-like bodies. In G. phycidis the lateral expansion is associated with only external ornamentation. This type of lateral expansion is described here for the first time in the anterior extremity of the digenean spermatozoa.
An external ornamentation of the plasma membrane is described in the anterior region of the spermatozoon from most species of Digenea in general and in particular from all Hemiuroidea studied to date except in the Didymozoids Didymocystis wedli and Didymozoon sp. (Pamplona-Basilio et al. 2001;Justine and Mattei 1983) which lack external ornamentation (see Table 1). The mature spermatozoon of G. phycidis displays two types of external ornamentation in its anterior region. The first type is filamentous and appears as a continuous layer associated with cortical microtubules while the second type is the classic one found in the majority of digeneans and usually is associated with cortical microtubules. However, in the Hemiuroidea this classical external ornamentation, when present, is not associated with cortical microtubules. To date, the presence of filamentous ornamentation is reported only in species of the superfamily Hemiuroidea, namely Aphanurus stossichii, H. appendiculatus, L. microstomum, P. palinurichthi, S. italicum and   Gonapodasmius sp. (Justine and Mattei 1982;Ndiaye et al. 2013bNdiaye et al. , 2014Ndiaye et al. , 2017Dione et al. 2016;Kacem et al. 2020).

Cortical microtubules
In the Digenea, the number of cortical microtubules in the mature spermatozoon varies according to the species from zero in the Didymozoidae Didymozoon sp. and Didymocystis wedli (Justine and Mattei 1983;Pamplona-Basilio et al. 2001) to 73 in Diplodiscus subclavatus (Bakhoum et al. 2011). In most hemiuroids this number is between 5 to 11 as described in the Hemiuridae and the Lecithasteridae (Quilichini et al. 2010a; Kacem et al. 2020). However, about 24 to 36 cortical microtubules have been reported in Sclerodistomidae (Ndiaye et al. 2013b(Ndiaye et al. , 2017, Sclerodistomoididae (Bâ et al. 2020), Didymozoidae (Justine and Mattei, 1982), and Gonocercidae (present study). The location of the maximum number of cortical microtubules is also variable according to the species. Quilichini et al. (2007) proposed for the first time two groups of digeneans according to the location of the maximum number of cortical microtubules in the spermatozoa: type 1 with the maximum number of cortical microtubules in the anterior part and type 2 in the median part of the spermatozoon. In G. phycidis, the maximum number of cortical microtubules is located in the median part of the spermatozoon and corresponds to the type 2 sensu Quilichini et al. (2007). This type 2 is also present in all hemiuroideans studied until now, except Gonapodasmius sp. which exhibits the maximum number in the anterior region and S. pacificus in the posterior one (see Table 1).
Most digeneans show an arrangement of cortical microtubules into two fields in the mitochondrial and nuclear regions of the spermatozoon as occurs in G. phycidis (present study) and in the didymozoid Gonapodasmius sp. (see Table 1). In contrast, in the remaining hemiuroideans studied to date, only one field of cortical microtubules has been described (see Table 1).

Mitochondria
In the digenean spermatozoa, the number of mitochondria varies from one to three, depending on the species (Bakhoum et al. 2017a). The mature spermatozoon of G. phycidis exhibits one mitochondrion as described in the remaining hemiuroideans studied up to now (see Table 1). On the other hand, the morphology of the mitochondrion is variable according to the species. Recently, a mitochondrial matrix granule has been observed in the hemiurid Aphanurus stossichii (Kacem et al. 2020). Moreover, a moniliform mitochondrion has been described in the sclerodistomoidid S. pacificus (Bâ et al. 2020). The morphological particularity of this type of mitochondrion has been described by the authors as a succession of bulges and cords. This type was also described in the male gamete of some digeneans such as the acanthocolpid Stephanostomoides tenuis , the aephnidiogenids Holorchis micracanthum and H. pycnoporus Kacem and Miquel 2020a), the cryptogonimids Aphallus tubarium and Timoniella imbutiformis (Foata et al. 2012;Kacem et al. 2017a), the lepocreadids Opechona bacilliaris and Prodistomum polonii (Ndiaye et al. 2015;Kacem and Miquel 2020b), the opecoelids Allopodocotyle pedicellata and Macvicaria obovata (Bakhoum et al. 2017b;Kacem et al. 2017b) and the plagiorchiid Enodiotrema reductum (Ndiaye et al. 2012b). Another morphology of mitochondrion with a U-shaped posterior extremity has been described in the opecoelid Allopodocotyle tunisiensis by Kacem et al. (2019).

Posterior spermatozoon morphology
The posterior spermatozoon extremity is morphologically variable within digeneans. Quilichini et al. (2010b) were the first authors to propose three types of posterior spermatozoon extremities according to the disappearance of characters toward the posterior tip. Type 1 or opecoelidean type with the sequence "second axoneme, nucleus and cortical microtubules", type 2 or fasciolidean type with the sequence "cortical microtubules, second axoneme and nucleus" and type 3 or cryptogonimidean type with the sequence "cortical microtubules, nucleus and second axoneme". Following this criterion, the mature spermatozoon of G. phycidis exhibits type 3 of posterior spermatozoon extremity. This type has been observed in nearly all digenean species belonging to the Hemiuroidea (see Table 1).

Spermatozoa models in the Hemiuroidea
The ultrastructural study of species belonging to the Hemiuroidea reveals similarities in their mature spermatozoa. According to Bakhoum et al. (2017a), the hemiuroideans described until now show the type II sperm model. The latter is characterized by the presence of two 9 + '1' axonemes, external ornamentation of the plasma membrane not associated with cortical microtubules and located in the anterior part of the spermatozoon, one bundle of cortical microtubules of which the maximum number is located in a middle part of the spermatozoon and one mitochondrion. However, the mature spermatozoon described in G. phycidis seems not to follow this type II sperm model. Several characters present in the male gamete of G. phycidis are absent in the mature spermatozoa of other hemiuroideans. Those are: -The lateral expansion associated with external ornamentation of the plasma membrane and without cortical microtubules, 1 3 -The two types of external ornamentation of the plasma membrane located in the anterior extremity of the spermatozoon, -The two bundles of cortical microtubules in the mitochondrial and nuclear areas of the spermatozoon.
The position of the Gonocercidae within the Hemiuroidea seems to be confirmed. Our study confirms the previous molecular studies regarding the position of Gonocercidae compared with the rest of the hemiuroidean species (Sokolov et al. 2016(Sokolov et al. , 2018(Sokolov et al. , 2019. According to these authors, Gonocerca spp. are phylogenetically distant from other hemiuroid trematodes, including Derogenes varicus, representative of the type genus of the family Derogenidae. Unfortunately, the family Derogenidae remains unexplored from the spermatological point of view. In the future, it would be important to perform ultrastructural studies of spermatozoon not only in representatives of the family Derogenidae but also in the remaining genera of Gonocercidae.

Conclusion
The present study enlarges the data on ultrastructural studies in the Digenea providing, for the first time, spermatological characteristics in the Gonocercidae. The most interesting findings are the presence of a lateral expansion associated only with external ornamentation and the presence of two fields of cortical microtubules in the mitochondrial and nuclear regions of the male gamete of G. phycidis. These features distinguish the mature spermatozoon of G. phycidis from those reported in other hemiuroideans. However, additional spermatological studies are needed on this family, but especially on the Derogenidae from which the Gonocercidae has been separated to confirm and complete the results of the molecular studies.