Mature spermatozoa of the Eucestoda show a high variability and provide important characters useful for phylogenetic studies (Justine, 1998, 2001; Levron et al., 2010). In general, they contain one or two axonemes, cortical microtubules, a nucleus, and various granules in the cytoplasm (Justine, 2003). Male gametes of the order Caryophyllidea are of special interest, as phylogenetic relationships within this group and its interrelationships with other tapeworms remain unclear (Waeschenbach et al., 2012; Caira & Littlewood, 2013).
The Caryophyllidea is considered to comprise 42 genera and 122 valid species arranged in four families (Scholz & Oros, 2017). Uptodate, sperm characters of 12 caryophyllidean taxa of 3 families have been described (see Table 1). Of these taxa, 1 species (
Variation of the maximum number of cortical microtubules (CM) in the spermatozoa of the Caryophyllidea.
Families | Genus Species | CM | References |
---|---|---|---|
Capingentidae | 15 | Yoneva et al., 2011 | |
Caryophyllaeidae | 11 | Bruňanská et al., 2019 | |
15 | Bruňanská & Kostič, 2012 | ||
Świderski & Mackiewicz, 2002 | |||
10 | Yoneva et al., 2012a | ||
30 | Gamil, 2008 | ||
Lytocestidae | Bruňanská et al., 2011 | ||
19 | Matoušková et al., 2018 | ||
35 | present study | ||
22 | Matoušková et al., 2019 | ||
15 | Bruňanská, 2009 | ||
25 | Yoneva et al., 2012b | ||
40 | Arafa & Hamada, 2004 |
Therefore, the present study aims to provide missing ultrastructural characters of the mature spermatozoa of
Adult specimens of
The tapeworms were cut into small pieces, fixed immediately in ice cold 3 % glutaraldehyde in sodium cacodylate buffer (pH 7.4) for 3 – 8 h, followed by three changes of cacodylate buffer and postfixed in 1 % OsO4 for 1 h. The material was dehydrated in a graded alcohol series, acetone, and embedded in Araldite. The ultrathin sections (90 nm) were cut on diamond knife using a Leica Ultracut UCT ultramicrotome, placed on copper grids, and double-stained with uranyl acetate (30 min.) and lead citrate (20 min.). The grids were examined in a JEOL 1010 transmission electron microscope operated at 80 kV.
Cytochemical technique of Thiéry (1967) with periodic acid-thiosemicarbazide-silver proteinate (PA-TSC-SP) was used for visualisation of glycogen in the mature spermatozooa. Ultrathin sections were placed on gold grids, treated in 1 % PA (20 – 25 min.), washed in destilled water, processed with 1 % TSC (40 min.), washed in 10 % acetic acid and destilled water, treated in 1 % SP (30 min.) and finally washed in destilled water. The grids were observed in JEOL 1010 transmission electron microscope.
The research related to animals has been complied with all the relevant national regulations and institutional policies for the care and use of animals.
A large number of cross- and longitudinal sections of the mature spermatozoa from vasa deferentia of
The vas deferens is situated between vasa eferentia and the ejaculatory duct of the cirrus pouch. The wall of convoluted vas deferens is formed by a thin syncytial epithelial layer, the luminal surface of which is lined by numerous sinuous lamellae and/or rare cilia (measuring up to 3.5 μm) (Fig. 1a).
Male gametes are long, filiform cells, tapered at both extremities, with one incorporated axoneme of the 9+‘1’ trepaxonematan structure, cortical microtubules and a nucleus which are situated parallel to the longitudinal axis of the spermatozoon, and granules of glycogen. Five different regions (I – V) with specific ultrastructural organisation can be recognized (Figs. 1, 2, 3).
Region I (Figs. 1a, b; 3I) corresponds to the anterior extremity of the spermatozoon. It contains one axoneme which is surrounded by a semiarc of up to 5 cortical microtubules (CM) located under the plasma membrane. The diameter of the spermatozoon is about 300 nm.
Region II (Figs. 1a, c; 2a, d; 3II) exhibits an increase of both the volume of cytoplasm and the number of CM. The CM are arranged in one continuous row composed of up to 23 elements (Fig. 1c). Granules of glycogen (Figs. 2a, d), can be detected in the cytoplasm using Thiéry method (1967). One pair of attachment zones illustrates the points of fusion of the free flagellum with the median cytoplasmic process during spermiogenesis (Figs. 1c; 3II). The diameter of the spermatozoon reaches up to 460 nm.
Region III (Figs. 1a, d, e, f, g; 2b, 3III) corresponds to the middle part of the mature spermatozoon, containing the nucleus. The diameter of the nucleus is about 40 nm at the beginning (Fig. 1d), gradually enlarges (Fig. 1e) up to 500 nm in its middle region (Fig. 1f). Posteriorly, the nucles diameter diminishes again, having about 160 nm near its posterior extremity (Fig. 1g). The CM are arranged in a semicircle at first (Fig. 1d). Subsequently, an enlarged nucleus is approaching the plasma membrane of the spermatozoon, thus dividing originally continuous semicircle of CM in two opposite parts (Fig. 1e, f, g), each consisting of 12 – 15 elements. One pair of attachment zones can be recognized (Fig. 1f). Based on examination of cross sections, the maximum number of CM in the spermatozoon of
Region IV (Figs. 1a, i, j, 2c, 3IV) is the postnuclear part of the spermatozoon. The CM are arranged at first in two opposite rows, each having 9 elements, and one single microtubule is situated in the middle between the two opposite rows (Fig. 1h). More posteriorly, a strong reduction of the cytoplasm volume, and only a few CM (up to 4) are observed (Fig. 1i). Here, the diameter of the spermatozoon is diminished to 380 nm. At the end of Region IV only one axoneme is present (Fig. 1j).
Region V (Figs. 1a, k, l, 3V), or the posterior part of the spermatozoon, contains only one axoneme which undergoes major disorganization: the central core unit disappears at first (Fig. 1k), and is followed by further disorganization of peripheral doublets (Figs. 1l, 3V). The very posterior extremity of the
This study provides an evidence that ultrastructural architecture of the mature spermatozoon of
The parallel alignment of CM in a longitudinal axis occurrs in most Eucestoda (except for the Cyclophyllidea and Tetrabothriidea, which have spiralled CM), Digenea and Monogenea (Justine, 2001; Bruňanská 2010; Bakhoum
The location of the maximum number of CM would be an interesting ultrastructural criterion, especially in connection with their presumed role in spermatozoan movements. The maximum number of CM in the spermatozoa within the order Caryophyllidea is not constant (Tab. 1). It occurs in the anterior parts of the spermatozoa most frequently, and varies between 10 and 30 in the Caryophyllaeidae, or 15 – 40 in the Lytocestidae. At the intraspecific level of
The occurrence of AZ in the mature spermatozoa of cestodes is not rare, as these structures represent the points of fusion of the free flagellum/flagella with the median cytoplasmic protrusion during spermiogenesis. The AZ were detected in the spermatozoa with one and/or two axonemes. The one-axoneme spermatozoa exhibit one pair of AZ. Within the Caryophyllidea, the AZ are situated in regions II, III or IV of the mature spermatozoa (Yoneva et al., 2012b; Matoušková et al., 2018, 2019; Bruňanská et al., 2019; present study). The two pairs of AZ have been reported in the two-axonemes spermatozoa of the Amphilinidea (Bruňanská et al., 2012a), Spathebothriidea (Bruňanská et al., 2006; Bruňanská & Poddubnaya, 2010), Bothriocephalidea (Levron et al., 2006a, b; Marigo et al., 2012), Diphyllobothriidea (Levron et al., 2006c, 2009, 2013; Bruňanská et al., 2012b), and Trypanorhyncha (Miquel & Świderski, 2006; Miquel et al., 2007; Marigo et al., 2011).
Glycogen is the major carbohydrate storage form in animals and represents the major energy store of the spermatozoa in the Eucestoda (Euzet et al., 1981). The presence of glycogen was detected using Thiéry method (1967) in the spermatozoa of the Caryophyllidea (Bruňanská, 2009; Yoneva et al., 2011; Bruňanská & Kostič, 2012; Matoušková et al., 2018; Bruňanská et al., 2019; present study), and in male gametes of other cestodes, e.g. Diphyllobothriidea, Bothriocephalidea, Haplobothriidea, Diphyllidea, Trypanorhyncha, Tetraphyllidea, Proteocephalidea, and Tetrabothriidea (Levron et al., 2010). In these cestodes, glycogen is localised in the sperm cytoplasm and it was never found in the sperm axonemes.
The spermatozoa travel on their route from the site of the origin towards the cirrus pouch through various canals of the male reproductive system, including vas deferens. The basic structure of the vasa deferentia of