A comparative study of the ultrastructural characteristics of the mature spermatozoa of two fellodistomids Tergestia clonacantha and T. laticollis and contribution to the phylogenetic knowledge of the Gymnophalloidea

The ultrastructure of the mature spermatozoa of Tergestia clonacantha and T. laticollis collected from the digestive tracts of fishes from New Caledonia is described using transmission electron microscopy and compared to that of related species. The spermatozoa of the two species exhibit the general pattern described in most digeneans, namely two axonemes with the 9 + “1” pattern of the Trepaxonemata, nucleus, mitochondrion, cortical microtubules, an external ornamentation of the plasma membrane, spine-like bodies and granules of glycogen. The spermatozoa of T. clonacantha and T. laticollis show the same ultrastructural model with some specificities in each case, particularly in the disposition of the structures in the posterior extremities of the spermatozoon. This study confirms that ultrastructural characters of the mature spermatozoon are useful tools for the phylogenetic analysis of the Digenea.


Introduction
It is now recognized that the ultrastructure of mature spermatozoa constitutes a useful complement to molecular, morphological and biological data in the understanding of the phylogeny of the Platyhelminthes. Studies of sperm structure in the poorly known orders and additional comparative studies are expected to provide new information for elucidation of phylogenetic relationships [2,7,11,17,25,28]. Characters of special interest are: (1) presence or absence of mitochondria in the mature spermatozoon and their number, (2) the type of spermiogenesis, (3) presence or absence of the intercentriolar body during spermiogenesis, (4) the striated roots, (5) the periaxonemal sheath, (6) apical electron dense material in the spermatozoon, (7) the apical cone, (8) the ultrastructure of the anterior or posterior extremities, (9) the form and disposition of the nucleus, (10) the number and distribution of cortical microtubules, (11) the spine-like bodies, (12) the external ornamentation of the plasma membrane, (13) the disposition of the glycogen granules, and (14) the expansion of the plasma membrane.
In this study, we describe the ultrastructural features of the mature spermatozoa of Tergestia clonacantha and T. laticollis. Only the fellodistomid Tergestia acanthocephala [13] has been studied among the five families of Gymnophalloidea. The aim of this work was to add spermatological data for the Fellodistomidae and the Gymnophalloidea. Moreover, we aimed to confirm the stability of ultrastructural sperm characters at a low taxonomic level (species) to emphasize their usefulness to discriminate higher taxonomic groups (orders or superfamilies).

Material and methods
Live adult specimens of Tergestia clonacantha Manter, 1963 and T. laticollis (Rudolphi, 1819) Stossich, 1899 were collected from the digestive tracts of Hemirhamphus far and Carangoides chrysophrys, respectively, off Nouméa (New Caledonia). Specimens were rinsed with 0.9% NaCl solution and fixed in cold (4°C) 2.5% glutaraldehyde in 0.1M sodium cacodylate buffer at pH 7.2, postfixed 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 24 h. Ultrathin sections (60-90 nm thick) were cut on an ultramicrotome (Power tome PC, RMC Boeckeler) with diamond knife and placed on copper and gold grids. Copper grids were double stained with uranyl acetate and lead citrate. To reveal the presence of glycogen, golds grids were stained according to the method of Thiéry [31] with periodic acid (PA), thiocarbohydrazide (TCH) and silver proteinate (SP) as follows: 30 min in 10% PA, rinsed in Milli-Q water, 2 h in TCH, rinsed in acetic solution and Milli-Q, 30 min in 1% SP in the dark and rinsed in Milli-Q water. Then, copper and gold grids 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 observation of numerous transverse sections in the seminal vesicle of the spermatozoa of Tergestia clonacantha and T. laticollis under the transmission electron microscope permitted us to distinguish four regions (I-IV) from the anterior to the posterior parts of mature spermatozoa of these species. The spermatozoa exhibit the main ultrastructural characteristics generally described in digeneans, namely two axonemes with the 9 + "1" pattern of the Trepaxonemata, nucleus, mitochondria, cortical microtubules, an external ornamentation of the plasma membrane, spine-like bodies, and granules of glycogen. However, the spermatozoa of T. clonacantha and T. laticollis present some peculiarities that we describe below.
Region I (Figs. 1A-1C, 3A-3F and 5I). This region corresponds to the anterior extremity of the mature spermatozoon characterized by the presence of a first axoneme in T. clonacantha (Fig. 1A)  In T. clonacantha, it is characterized by the appearance of the second mitochondrion in addition to the axoneme and cortical microtubules ( Fig. 2A). The nucleus appears posterior to the anterior end of the mitochondrion (Fig. 2B). The second mitochondrion disappears at the posterior extremity of the spermatozoon (Fig. 2C), then the cortical microtubules, then the axoneme (Figs. 2D-2E). Thus, the cross-section of the posterior extremity of the spermatozoon shows the presence of the nucleus only (Fig. 2E).
In the case of T. laticollis, cross-sections in the anterior part of this region exhibit the appearance of the nucleus in addition

Discussion
The ultrastructure of the mature spermatozoon of Tergestia clonacantha and T. laticollis shows the general features described in the spermatozoon of most digeneans, namely: two axonemes with the 9 + "1" pattern of the Trepaxonemata, a nucleus, a mitochondrion or two, cortical microtubules, external ornamentation of the plasma membrane, spine-like bodies, and granules of glycogen. However, the mature spermatozoa of T. clonacantha and T. laticollis exhibit some peculiarities and give additional spermatological data for a better understanding of the relationships between the Gymnophalloidea and other digenean superfamilies. As is the case in many species of the superfamilies Gorgoderoidea, Lepocreadioidea, Opecoeloidea and Plagiorchioidea [7] with spermatozoon of type III or IV, we also found two mitochondria in the spermatozoa of T. clonacantha and T. laticollis. These are located in the posterior part of the mature spermatozoon in T. clonacantha and T. laticollis, in contrast to T. acanthocephala [13] which has the first mitochondrion in the anterior part of the spermatozoon. In addition, the mature spermatozoon of T. acanthocephala was described as similar to type IV proposed by Bakhoum et al. [7] but spermatozoa of T. clonacantha and T. laticollis (present study) exhibit type III. Thus, the assignation of type IV for spermatozoa of Gymnophalloidea in Bakhoum et al. [7] should be revised. This demonstrates the need for more studies in this superfamily to elucidate ultrastructural characteristics of the spermatozoa and their relationships. The ultrastructural model of the mature spermatozoon described in T. clonacantha and T. laticollis by the present study highlights some differences between them, mainly in the posterior region of the spermatozoa. The posterior extremity of the spermatozoon is characterized in the two species by the presence of the nucleus only, as in T. acanthocephala [13]. Nevertheless, in the case of T. clonacantha, before this extremity, we described successively the appearance of the second mitochondrion, then the nucleus, then the disappearance of cortical microtubules, then the extremity of the second axoneme, and finally the posterior extremity of the nucleus. In parallel and successively, in T. laticollis, the nucleus appears, then the second  Comparative ultrastructural studies of the mature spermatozoon have been performed in several species belonging to the same genus, within the Digenea. Overall, the authors found a similar general pattern. Table 1 shows that the same spermatozoon type (according to Bakhoum et al. [7]) has usually been found for species of the same genus: type III for Holorchis micracanthum [3] and H. pycnoporus [12]; type V for Prosorhynchus aculeatus and [18] P. longisaccatus [26]; type V for the Diplodiscidae Diplodiscus amphichrus [10] and D. subclavatus [5]; type V for Fasciola gigantica [22] and F. hepatica [21]; type II for Lecithochirium microstomum [23] and L. musculus [23]; type III for the Lepocreadiidae Bianium arabicum [30] and B. plicitum [30]; type IV for the Opisthorchiidae Opisthorchis felineus [32] and O. viverrini [19]; type IV for the Opecoelidae Allopodocotyle pedicellata [6] and A. tunisiensis [15], and type III for the Opecoelidae Nicolla testiobliquum [27] and N. wisniewskii [28]. Nevertheless, some differences have been observed between spermatozoa of species belonging to the same genus like the Notocotylidae Notocotylus neyrai [20] (undefined type) and N. noyeri [24] (type IV) concerning the location of the    [14] (type IV) concerning the location of the external ornamentation. The data shown in Table 1 indicate that that the general model of the spermatozoon is usually the same when the species belong to the same genus but can, in some case, differ according to one ultrastructural character.

Conflict of interest
The Editor-in-Chief of Parasite is one of the authors of this manuscript. COPE (Committee on Publication Ethics, http:// publicationethics.org), to which Parasite adheres, advises special treatment in these cases. In this case, the peer-review process was handled by an Invited Editor, Jérôme Depaquit. Adm, anterior electron-dense material; AntA, anterior part of the anterior region; AntS, anterior region of the spermatozoon; Ax, axoneme; BCm, number of bundles of cortical microtubules; Cm, cortical microtubules; Cob, cytoplasmic ornamented buttons; Eo, external ornamentation of the plasma membrane; Eo + Cm, association of external ornamentation with cortical microtubules; Le, lateral expansion; LEo, location of the external ornamentation; LMCm, location of maximum number of cortical microtubules; M, number of mitochondria; MedS, median part of the spermatozoon; N, nucleus; PostA, posterior to the anterior region; Psc, posterior spermatozoon character; Sb, spinelike bodies; TAx, type of axoneme; +/À, presence/absence of considered character; ?, doubtful or unknown data.