Ultrastructure of the spermatozoon of Macvicaria obovata (Digenea: Opecoelidae), a parasite of Sparus aurata (Pisces: Teleostei) from the Gulf of Gabès, Mediterranean Sea

The ultrastructural organization of the spermatozoon of the digenean Macvicaria obovata (Opecoelidae) is described by transmission electron microscopy. Alive digeneans were collected from the digestive tract of Sparus aurata (Teleostei, Sparidae), caught from the Gulf of Gabès in Chebba, Tunisia (Eastern Mediterranean Sea). The male gamete of M. obovata is a filiform cell, tapered at both extremities and exhibits typical characters such as two axonemes of different lengths showing the 9+‘1’ trepaxonematan pattern, a nucleus, mitochondria, two bundles of parallel cortical microtubules, external ornamentation of the plasma membrane, spine-like bodies and granules of glycogen. Cortical microtubules are absent in the anterior spermatozoon extremity and appear after the disappearance of the electron-dense material. The first mitochondrion is of moniliform type and it is associated with the external ornamentation of the plasma membrane and spine-like bodies. The second mitochondrion is more posteriorly located, reaching the nuclear region. The present study provides new data on the mature male gamete of M. obovata that may be useful for the understanding of digenean relationships and phylogenetic studies.


Introduction
The Opecoelidae is a large, cosmopolitan family of digeneans, with over 90 genera and nearly 900 species, almost solely found in marine and freshwater teleost fishes (Bray et al. 2016).
According to Cribb (2005) this family was divided into four subfamilies, namely the Opecoelinae, Opecoelininae, Plagioporinae and Stenakrinae. Recently, a phylogenetic study of the Opecoelidae carried on the molecular analysis of 41 species of this family revealed that the Opecoelidae is monophyletic (Bray et al. 2016). Moreover, based on this analysis and in the characters of eggs and uterus the new subfamily Helicometrinae was erected for the genera Helicometra, Helicometrina and Neohelicometra (Bray et al. 2016).
In this context, the ultrastructural studies of species belonging to the family Opecoelidae are of great importance to bring additional information that complements the molecular results. In this sense, it has been demonstrated that ultrastructural study of the mature spermatozoon provides numerous characters, which are useful for phylogenetic inference in parasitic Platyhelminthes (Justine 1991(Justine , 1995(Justine , 1998(Justine , 2001Levron et al. 2010;Quilichini et al. 2010Quilichini et al. , 2011a.

Results
The interpretation of numerous cross-and longitudinal sections of the mature spermatozoon of M. obovata allows us to establish three distinctive regions (I, II and III) from the anterior to the posterior extremities of the male gamete exhibiting different ultrastructural characteristics.
The mature spermatozoon of M. obovata exhibits the usual structures found in most of digeneans. Indeed, it contains two axonemes of the 9+'1' trepaxonematan pattern, external ornamentation of the plasma membrane, spine-like bodies, nucleus, two mitochondria, two bundles of parallel cortical microtubules and granules of glycogen. The granular material observed in all the regions of the mature spermatozoon is evidenced as glycogen using the test of Thiéry (1967) (Fig. 3).

Discussion
The mature spermatozoon of M. obovata exhibits the usual ultrastructural elements as most species of digeneans described so far: two axonemes, nucleus, mitochondrion, two sets of parallel cortical microtubules, external ornamentation of the plasma membrane and granules of glycogen. Additional aspects include the presence of spine-like bodies in the ornamented area of the sperm cell and the electron-dense material in the anterior extremity of the spermatozoon. Some of these characters as well as the morphology of both extremities could be potential candidates for phylogenetic analysis.

Anterior spermatozoon extremity
The anterior extremity of the mature spermatozoon of M. obovata presents two slightly longitudinally displaced centrioles corresponding to both axonemes of the 9+'1' trepaxonematan pattern (Ehlers 1984). This is the typical structure of axonemes observed in all digeneans except for the species of the genus Schistosoma with a special 9+'1' pattern with a poorly contrasted central element (Justine et al. 1993) and species of Didymozoon with a 9 + 0 pattern (Justine and Mattei 1983).
The presence of a submembranous electron-dense material in the anterior extremity of the mature spermatozoon of M. obovata was also observed in several digeneans such as Holorchis micracanthum (Bâ et al. 2011), Gyliauchen sp. (Quilichini et al. 2011a), Robphildollfusium fractum , Hypocreadium caputvadum (Kacem et al. (Ndiaye et al. 2015) or Neomultitestis aspidogastriformis (Bakhoum et al. 2015a). Concerning the Opecoelidae, an electron-dense material is present in the anterior extremity of the sperm cell of six of the nine studied species, namely A. pedicellata, H. epinepheli, H. fasciata, M. ovobata, P. magnatestis and P. furcatum (Levron et al. 2003(Levron et al. , 2004Quilichini et al. 2011b;Diagne et al. 2016;Bakhoum et al. 2017;present study -see Table I). For three of these species, authors do not mention its presence, but they are clearly visible in the published TEM micrographs. This is the case of H. fasciata and P.
Another peculiarity observed in the anterior spermatozoon extremity of M. obovata is the absence of cortical microtubules. In fact, the appearance of cortical microtubules is noted only when the anterior electron-dense material disappears.

Spine-like bodies
Spine-like bodies consist of a submembranous electron-dense elements usually present in the  Table I Table I). Thus, the presence or absence of external ornamentation and their location in the spermatozoon may provide additional characters useful for a phylogenetic analysis of the Digenea.

Posterior spermatozoon extremity
The posterior spermatozoon extremity is morphologically variable within digeneans.  Table I). Other particularities concern H. epinepheli (Quilichini et al. 2011b) andP. magnatestis (Diagne et al. 2016) and these two species present a second mitochondrion that reaches the level of the disorganization of the second axoneme.     ADM-anterior electron-dense material; CM-cortical microtubules; EO-external ornamentation of the plasma membrane; EO+CM-association external ornamentation-cortical microtubules; LEO-location of the external ornamentation; M-number of mitochondria; N-nucleus; NA-not applicable; Post-posterior; PSC-posterior spermatozoon character; SB-spine-like bodies; +/--presence/absence of considered character.