Ultrastructural characters of the spermatozoa in Digeneans of the genus Lecithochirium Lühe, 1901 (Digenea, Hemiuridae), parasites of fishes: comparative study of L. microstomum and L. musculus

This study provides the first ultrastructural data of spermatozoa in the genus Lecithochirium. The spermatozoa of L. microstomum (from Trichiurus lepturus in Senegal) and L. musculus (from Anguilla anguilla in Corsica) exhibit the general pattern described in the great majority of the Digenea, namely two axonemes with the 9 + “1” pattern typical of the Trepaxonemata, one mitochondrion, a nucleus, parallel cortical microtubules and external ornamentation of the plasma membrane. Spermatozoa of L. microstomum and L. musculus have some specific features such as the presence of a reduced number of cortical microtubules arranged on only one side of the spermatozoon, the lack of spine-like bodies and expansion of the plasma membrane. The external ornamentation of the plasma membrane entirely covers the anterior extremity of the spermatozoa. The ultrastructure of the posterior extremity of the spermatozoa corresponds to the pattern previously described in the Hemiuridae, characterized by only singlets of the second axoneme. A particularity of these spermatozoa is the organization of the microtubule doublets of the second axoneme around the nucleus in the posterior part of the spermatozoon.

Ultrastructural data of spermatozoa may provide a useful contribution to understanding of the phylogenetic relationships within the Hemiuridae. In the Platyhelminthes, most ultrastructural features of the spermatozoon proved to be valuable characters for phylogenetic purposes [3,4,[23][24][25][26][27]35]. The ultrastructural data, associated with the results of molecular phylogenetic studies, have greatly improved our understanding of the interrelationships in most groups of Platyhelminthes [9, 12, 14, 21, 31, 36-38, 46, 59]. In the Digenea, ultrastructural data of the spermatozoon are available for more than 75 species distributed among 45 families [6,53]. In the Hemiuroidea, ultrastructural data on spermiogenesis and/or spermatozoa exist for seven species belonging to four families [41]. In the Hemiuridae, such data exist for representatives of only two of the twelve currently recognized subfamilies, namely an elytrophalline, Lecithocladium excisum [42], and a hemiurine, Parahemiurus merus [41]. The present study is the first work of this kind in the Lecithochiriinae and describes the ultrastructural characteristics of the spermatozoon in two representatives of the genus Lecithochirium.      Two axonemes and cortical microtubules. (10). Two axonemes, cortical microtubules and the nucleus. (11). Two axonemes, cortical microtubules, the nucleus and the mitochondrion. (12). One axoneme completely formed, nucleus, mitochondrion, cortical microtubules and the disorganization of the first axoneme. Ax2 = second axoneme, Cm = cortical microtubules, Mt = mitochondrion, N = nucleus, Pae1 = posterior extremity of the first axoneme. 13-14. Cross-section of Region III of the spermatozoon of Lecithochirium microstomum. Scale bar = 0.2 lm. (13). One axoneme, nucleus, mitochondrion and cortical microtubules. (14). One axoneme, nucleus and cortical microtubules. Ax2 = second axoneme, Cm = cortical microtubules, Mt = mitochondrion, N = nucleus. 15-18. Cross-sections of Region IV of the spermatozoon of Lecithochirium microstomum. Scale bars = 0.2 lm. (15). The second axoneme and nucleus. (16). Disorganization of the second axoneme and nucleus. (17) The micrographs in Figures 19 and 37 show a positive result of the Thiéry test, exhibiting a reduced presence of glycogen granules along the mature spermatozoa in both species studied.

The anterior spermatozoon extremity
The spermatozoon extremity of the two species is filiform and exhibits a bulge described here for the first time in the Digenea. It is also covered by an external ornamentation of  the plasma membrane. In the other two Hemiuridae studied so far, namely Lecithocladium excisum and Parahemiurus merus [41,42], the anterior extremity of the spermatozoon has only one axoneme and external ornamentation of the plasma membrane. The unique feature of the spermatozoa of Lecithochirium is the presence of a few short cortical microtubules (6) in the anterior extremity of the spermatozoon in addition to the filamentous external ornamentation of the plasma membrane.
In the Hemiuroidea, this type of anterior spermatozoon extremity was previously described only in Gonapodasmius sp. by Justine and Mattei [28,30] (see Table 1) and in Prosorchis palinurichthi [45]. In 2011, Quilichini et al. [48] distinguished three types of digenean spermatozoa according to the localization of the external ornamentation: type 1 (external ornamentation in the anterior extremity of the spermatozoon), type 2 (external ornamentation at a more posterior level) and type 3 (absence of   external ornamentation). According to this criterion, the hemiuroidean spermatozoa can be classified as the first type (Table 1).
In the majority of digeneans studied up to now, the external ornamentation of the plasma membrane is associated with spine-like bodies [7,39,41,43,51]. However, these structures are absent in the spermatozoon of all studied Hemiuroidea.

Cortical microtubules
Cortical microtubules were described in spermatozoa of almost all of the digeneans studied so far. They are generally disposed in two bundles. One of the specific features of the spermatozoon of the Hemiuridae is the presence of only one reduced bundle of cortical microtubules on one side of the spermatozoon [41, 42 and present study]. Up to now, the maximum number of cortical microtubules encountered in Hemiuridae was eight in Lecithochirium excisum [42] and Lecithochirium microstomum (this study). The spermatozoon of Lecithochirium musculus exhibits only six cortical microtubules (this study). P. merus [41] has the smallest number of cortical microtubules (5) in Hemiuridae. The number of cortical microtubules is higher in other Hemiuroidea: 10 in Aponurus laguncula [49], 13 in Prosorchis ghanensis [25], 25 in Prosorchis palinurichthi [45] and 36 in Gonapodasmius sp. [28,30].
The principal difference between L. microstomum and L. musculus is in the number of cortical microtubules. In L. microstomum the maximum number of cortical microtubules is observed in the region of the spermatozoon with only the two axonemes, glycogen granules and nucleus. However, in L. musculus, the maximum number of cortical microtubules is six and is situated in a region of the spermatozoon with the simultaneous presence of the nucleus and the mitochondrion.

Mitochondrion
Similar to other taxa of the Hemiuroidea studied up to now (Table 1), the spermatozoa of L. microstomum and L. musculus have only one mitochondrion. Among more than 75 studied species of Digenea, spermatozoa with one mitochondrion were described in 40 species (see [6] completed by [40,41,45]). In the remaining species, spermatozoa with two or three mitochondria were described [1,2,20,34,44,51].

Posterior spermatozoon extremity
The posterior part of the spermatozoon is identical in both species of Lecithochirium examined in our study and characterized by the presence of only the nucleus and the posterior extremity of the second axoneme. The posterior extremity of the spermatozoa is particular and characterized by a disorganization of the axoneme associated with the migration of the posterior extremity of the nucleus in the central part of the disorganized axoneme. Thus, in the end of this posterior extremity, the nucleus disappears, and instead only the axonemal singlets are present in the posterior end of the spermatozoon. A posterior end of the spermatozoon with only axonemal singlets was described in all the Hemiuridae studied so far [41,42]. This suggests that this character can be a useful criterion for phylogenetic purposes. The presence of the nucleus in the posterior part of the spermatozoon was also described in all studied hemiurids [41, 42, present study]. In P. merus, some cortical microtubules are described in the posterior extremity of the spermatozoon. However, in L. microstomum and L. musculus (present study), cortical microtubules in the posterior extremity of the spermatozoon are absent. Similar to the other Hemiuridae studied to date [41,42], the spermatozoon in Lecithochirium presents the type III or Cryptogonimidean type characterized by the absence of cortical microtubules, and the sequence posterior extremity of the nucleus then posterior extremity of the second axoneme [50]. The terminal character is coincident in all Hemiuridae. However, the end of the cortical microtubules is different in Lecithocladium.

Glycogen
The presence of glycogen was described in most of the spermatozoa of digeneans described up to now. However, the particularity of the Hemiuridae is the presence of a reduced quantity of glycogen. We believe that it is necessary to perform more studies of this character to clarify its potential importance for the phylogeny of the Hemiuridae.