Spermatozoon ultrastructure in two monorchiid digeneans

Introduction

The description of ultrastructural characteristics of the mature spermatozoon of the Platyhelminthes, and in particular the Digenea, has recently proved to be an interesting tool for phylogenetic purposes (Justine, 1995; Levron et al., 2010; Miquel et al., 2013; Bakhoum et al., 2012a; Bakhoum et al., 2012b; Bakhoum et al., 2015a; Bakhoum et al., 2015b; Ndiaye et al., 2015a; Ndiaye et al., 2015b). The Monorchioidea comprise two families of trematodes namely, the Lissorchiidae and the Monorchiidae (Bray, 2008). Among the latter, which includes 40 valid genera (Madhavi, 2008), only two species, Monorchis parvus and Helicometroides atlanticus (Levron, Ternengo & Marchand, 2004a; Diagne et al., 2015) have been explored for spermatological characteristics. Moreover, the mature spermatozoon of M. parvus and H. atlanticus exhibit some differences especially in their respective anterior extremity.

The aim of this work was to bring more spermatological descriptions from two additional genera, Opisthomonorchis and Paramonorcheides, in order to elucidate the general morphology of the spermatozoon in the Monorchiidae. In addition, the mature spermatozoon of the monorchiids was compared to those of the other digeneans with a brief comment on its phylogenetic relevance.

Materials and Methods

Fish were bought dead, but very fresh, at the fishmarket in Nouméa, New Caledonia. Specimens of Opisthomonorchis dinema Bray & Justine, 2013 were collected from the digestive tract of Carangoides dinema Bleeker (Perciformes, Carangidae) caught off Nouméa, New Caledonia, from the same fish specimen (MNHN JNC3224) as the paratypes of the species (Bray & Justine, 2013). Specimens of Paramonorcheides selaris Lakshmi & Madhavi, 2009 were collected from the digestive tract of Selar crumenophthalmus (Bloch, 1793) (Perciformes, Carangidae) caught off Nouméa, New Caledonia, on 10 Sept. 2009 (fish specimen MNHN JNC 3043).

Digenean specimens were fixed in cold (4°C) 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer at pH 7.2, rinsed 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 series, embedded in Spurr resin and polymerized at 60°C for 24 h. Ultrathin sections (60–90 nm) of the seminal vesicle were obtained on an ultramicrotome (Power tome PC, RMC Boeckeler^®). The sections were placed on 300 and 200 mesh copper grids and stained with uranyl acetate and lead citrate according to Reynolds (1963) methodology. To locate glycogen granules, the Thiéry technique (1967) was also used in several sections placed on gold grids.

All grids were examined on a Hitachi H-7650 transmission electron microscope, operating at an accelerating voltage of 80 kV, in the “Service d’Étude et de Recherche en Microscopie Électronique” of the University of Corsica (Corte, France).

Results

The organization of the mature spermatozoon of Opisthomonorchis dinema and Paramonorcheides selaris is described after observations of cross- and longitudinal sections (Figs. 1–4). Thus, in both monorchiid species four regions, with distinctive ultrastructural characteristics, are evident, from the anterior to the posterior spermatozoon extremities. The granules of glycogen present in O. dinema and P. selaris spem cells were detected by the cytochemical test of Thiéry (Fig. 3E for P. salaris).

Region I, corresponding to the anterior extremity of the mature spermatozoon, is characterized by the presence of the first axoneme accompanied by the anterior extremity of the second axoneme, cortical microtubules and external ornamentation of the plasma membrane. In O. dinema the anterior spermatozoon tip exhibits both anterior extremities of the axonemes, surrounded by a layer of cortical microtubules (about 26) and external ornamentation (Figs. 1A, 1B and 4I). In P. selaris longitudinal and cross-sections of the anterior spermatozoon tip show that it tapers to a point (Fig. 2A), and that it contains the anterior extremities of both axonemes and the cortical microtubules (about 26) (Fig. 2C) as well as exhibiting external ornamentation of the plasma membrane (Figs. 2B, 2C and Fig. 4I). In the middle part of the region I, when both axonemes are formed, spine-like bodies appear interrupting the external ornamentation of the spermatozoa of O. dinema and P. selaris (Figs. 1C, 2D and 4I). The posterior part of the region I is characterized, for each species, by the presence of external ornamentation of the plasma membrane associated with cortical microtubules and the first mitochondrion (Figs. 1D, 1E, 2E and 4I). No glycogen granule have been highlighted by the Thiéry’s method in this region.

Region II corresponds to the transitional area before the nuclear region. For both O. dinema and P. selaris spermatozoa, it is characterized by the absence of external ornamentation and the presence of the axonemes and the first mitochondrion (Figs. 1F, 2F and Fig. 4II). In addition, for each monorchiid species, the posterior part of the region II contains a reduced number of cortical microtubules: about 5 in O. dinema (Fig. 1G) and 13 in P. selaris (Fig. 2G). Only few granules of glycogen are observed in this region.

Region III exhibits several ultrastructural characteristics that distinguish the mature spermatozoa of O. dinema (Figs. 3A and 4IIIA) from those of P. selaris (Figs. 3B, 4IIIB):

• In the anterior part of the region III, the spermatozoon is characterized by the disorganization of the first axoneme in O. dinema (Figs. 1H, 4IIIA), whereas in P. selaris the two axonemes and the second mitochondrion are observed (Figs. 2H, 4IIIB).

• The middle part of the region III is characterized in O. dinema by the presence of only one axoneme, the nucleus, few cortical microtubules and the second mitochondrion (Figs. 1I and 4IIIA). In contrast, in P. selaris, the second mitochondrion, the nucleus and few cortical microtubules were accompanied by the two axonemes (Figs. 2I, 4IIIB). Note that this part corresponds to the disorganization of the first axoneme in P. selaris (Figs. 2J and 4IIIB).

• In the posterior part of the region III, the second mitochondrion, accompanied by one axoneme, the nucleus and cortical microtubules is observed in both O. dinema (Figs. 1I and 4III) and in P. selaris (Fig. 3A, 4III).

This region is characterized, for the two species, by a high number of glycogen granules.

Region IV represents the posterior spermatozoon extremity. When the second mitochondrion disappears in both O. dinema and P. selaris mature spermatozoa, cross-sections exhibit only one axoneme, the nucleus and very few cortical microtubules (Figs. 1J, 3B and 4IV). Moreover, for each species, the second axoneme disorganizes exhibiting doublets and singlets of microtubules (Figs. 1K, 3C). Consequently, the posterior spermatozoon tips in O. dinema and P. selaris show only the nucleus (Figs. 1L, 3D and 4IV).

For the two species, only few glycogen granules have been observed in the anterior part of this region.

Discussion

The mature spermatozoon described in Opisthomonorchis dinema and Paramonorcheides selaris possesses two axonemes of the 9+“1” trepaxonematan pattern, two mitochondria, a nucleus, two bundles of parallel cortical microtubules, an external ornamentation of the plasma membrane, spine-like bodies and granules of glycogen. In addition to these ultrastructural characters, the male gamete of O. dinema and P. selaris exhibits similar anterior and posterior spermatozoon morphologies.

Within the Monorchiidae most of these spermatological characters were described previously in Monarchus parvus and Helicometroides atlanticus (Levron, Ternengo & Marchand, 2004a; Diagne et al., 2015). Thus, the characters of the mature spermatozoon, its anterior and posterior morphologies were used here in comparative studies between the different species (Table 1).