The mature spermatozoon of G. phycidis exhibits the general pattern described in most hemiuroideans species: 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 the most Hemiuroideans species studied, 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). In the Hemiuroidea, mature spermatozoa exhibiting two axonemes in their anterior extremity are reported only in two species namely the Didymozoidae Gonapodasmius sp. (Justine and Mattei, 1982) and the Sclerodistomoididae Sclerodistomoides pacificus (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 species, cortical microtubules are absent in the anterior tip of the spermatozoon (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 five superfamilies of Digenea. These are 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 ornamentations. This type of lateral expansion is described here for the first time in the anterior extremity of the digenean spermatozoa.
An extramembranous ornamentation 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. which lack external ornamentation (see table 1). The mature spermatozoon of G. phycidis displays two types of external ornamentations 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 is not associated with cortical microtubules.
To date, the presence of filamentous ornamentation is reported only in the superfamily of Hemiuroidea and has been observed in many species namely Aphanurus stossichii, H. appendiculatus, L. microstomum, P. palinurichthi, S. italicum (Ndiaye et al. 2013b, 2014, 2017; Dione et al. 2016; Kacem et al. 2020). Their presence is also demonstrated in micrographs of Gonapodasmius sp. (Justine and Mattei 1982).
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 species 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, 2017), Sclerodistomoididae (Bâ et al. 2020), Didymozoidae (Justine and Mattei, 1982), and Gonocercidae (present study).
The location of the maximum number of these cortical microtubules is also variable according to the species. Quilichini et al. (2007) propose for the first time two groups of digenean species 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 hemiuroidean species studied except Gonapodasmius sp. which exhibits maximum number in the anterior region and S. pacificus in the posterior one (see table1).
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 also in the didymozoid Gonapodasmius sp. (see table 1). In contrast, in the remaining Hemiuroidea described to date, only one field of cortical microtubules has been described (see table 1).
Mitochondria
In the digenean spermatozoan, 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 other hemiuroidean species studied up to now (see table 1). On the other hand, the morphology of this 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 (see Bâ et al. 2020 and table 1). 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 (Bakhoum et al. 2015), the aephnidiogenids Holorchis micracanthum and H. pycnoporus (Bâ et al. 2011; 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 namely U-shaped posterior extremity has been also described in the opecoelid A. 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 “axoneme, nucleus and cortical microtubules”, type 2 or fasciolidean type with the sequence “cortical microtubules, axoneme and nucleus” and type 3 or cryptogonimidean type with the sequence “cortical microtubules, nucleus and axoneme”. Following this criterion, the mature spermatozoon of G. phycidis exhibits type 3. This type has been observed in nearly all digenean species belonging to the Hemiuroidea (see Table 1). On the other hand, in the lecithasteridae Aponurus laguncula (Quilichini et al. 2010a) the posterior part of the spermatozoon is characterized by the persistence of both two axonemes and the mitochondrion.
Spermatozoa models in the Hemiuroidea
The ultrastructural study in the Hemiuroidea reveals similarities in their mature spermatozoa. According to Bakhoum et al. (2017a), the hemiuroidean species described until now show the type II of sperm models. 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 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 of sperm models. Several characters present in the male gamete of G. phycidis are absent in the mature spermatozoa of other hemiuroidean species. Those are:
- the lateral expansion associated with external ornamentations and without cortical microtubules,
- 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 area 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, 2018, 2019). According to these authors, Gonocerca spp. are phylogenetically distant from other hemiuroid trematodes, including Derogenes varicus (Müller, 1784), 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 is important to perform ultrastructural studies of spermatozoon not only in representatives of the family Derogenidae, but also in the remaining genera of Gonocercidae.