Parasitol Res (2014) 113:1093–1101 DOI 10.1007/s00436-013-3746-z
ORIGINAL PAPER
Ultrastructure of spermatozoa in the seminal receptacle of the liver fluke Opisthorchis felineus (Rivolta, 1884) Mariya V. Zhukova & Viatcheslav A. Mordvinov & Elena Kiseleva
Received: 15 October 2013 / Accepted: 26 December 2013 / Published online: 23 January 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract The spermatozoon ultrastructure in the seminal receptacle of the liver fluke Opisthorchis felineus (Digenea, Opisthorchiidae), the agent of human opisthorchiasis endemic to Russia and Eastern Europe, was examined. The beanshaped seminal receptacle of O. felineus has a wall consisting of epithelial and muscle layers. Mature spermatozoa are located in the interior of the seminal receptacle, whereas vacuoles containing degenerating spermatozoa are detectable at the periphery. The mature spermatozoon of O. felineus has two axonemes of a 9+“1” pattern, a nucleus, two mitochondria, a lamellar body, two bundles of parallel cortical microtubules and an external ornamentation of the plasma membrane in the anterior area of the sperm. The lamellar body is likely to be the third mitochondrion of a small size. The cytoplasm of the spermatozoon is filled with numerous electron-dense granules of storage polysaccharides. Additionally, the ultrastructural characteristics of the seminal receptacle and spermatozoa of O. felineus were compared to available published data on other trematode species. The functional roles of the observed structures of this spermatozoon are discussed.
Introduction The ultrastructure of spermatozoa is highly diverse in Platyhelminthes, which has allowed comparative spermatology to be used for clarifying the phylogenetic relationships within this group (Justine et al. 1985; Justine 1995; Levron et al. 2010). Currently, both morphological and molecular data are used to determine relationships between the species of subclass Digenea (Barker et al. 1993; Blair 1993; M. V. Zhukova : V. A. Mordvinov : E. Kiseleva (*) Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, Novosibirsk 630090, Russia e-mail: [email protected]
Olson et al. 2003; Olson and Tkach 2005). Furthermore, the morphological examination of various systems of organs in parasitic worms is of paramount importance for insight into specific functional features of these parasites and their interaction with their host. The subclass Digenea comprises approximately 18,000 species belonging to 25 superfamilies (Bray et al. 2008), though spermatozoon ultrastructure has thus far been examined in less than 1 % of the species in this subclass (Quilichini et al. 2011). The available data demonstrate that the spermatozoal structure in various digeneans differs considerably with regard to several characteristics, such as the presence/absence of spine-like bodies, number of mitochondria, location of external ornamentation of the plasma membrane, location and number of cortical microtubules and morphology of the anterior and posterior extremities. The structural differences in the spermatozoa of digeneans could be associated with specific functional features of their reproductive system, which are yet to be studied. In the majority of Digenea sp., the mature spermatozoa are stored in a seminal receptacle from which they enter the ootype for fertilisation (Skryabin 1947). Based on the current data, family Opisthorchiidae comprises over 30 genera (King and Scholz 2001), including the liver flukes Opisthorchis viverrini, Opisthorchis felineus and Clonorchis sinensis, which are most widely distributed among humans and cause food-borne trematodiases (Fürst et al. 2012). According to the latest estimates, approximately 1.6 million people worldwide are infected with O. felineus (Yossepowitch et al. 2004). Among the Opisthorchiidae, the ultrastructure of the C. sinensis spermatozoon has been studied in detail (Jeong and Rim 1984), the testis morphology and partial spermatozoon structure (including two 9+“1” axonemes and a round nucleus with condensed chromatin) have been recently described for O. viverrini (Wannaprapo et al. 2008), and the morphology of the O. felineus seminal receptacle and sp ermatoz oa w ere b riefly co vere d by
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Grebenshchikov and Nacheva (2007). In the present work, we comprehensively investigated the ultrastructure of the seminal receptacle and spermatozoa in the liver fluke O. felineus.
Materials and methods Adult O. felineus flukes were obtained from the liver of experimentally infected golden hamsters (Mesocricetus auratus). The seminal receptacles of O. felineus were isolated and fixed with 2.5 % glutaraldehyde solution in 0.1 sodium cacodylate buffer (pH 7.2) for 2.5 h, rinsed with the same buffer, and postfixed in 1 % osmium tetroxide in 0.1 sodium cacodylate buffer for 1 h. The samples were washed with water and placed in 1 % uranyl acetate aqueous solution for 12 h at +4 °C. The samples were then dehydrated through a graded series of ethanol and acetone and embedded in Agar 100 resin. Ultrathin sections were double stained with uranyl acetate and lead citrate according to Reynolds and examined using a JEOL 100 SX electron microscope at the facilities of the Interinstitutional Shared Centre for Microscopic Analysis of Biological Objects, the Institute of Cytology and Genetics SB RAS.
Results Morphology of the seminal receptacle The seminal receptacle of O. felineus is bean-shaped, embedded in parenchyma and filled with mature spermatozoa (Fig. 1); interior and peripheral regions are evident in semithin sections (Fig. 1a). The spermatozoa are densely packed in the interior (Fig. 1b, d), whereas they are loosely arranged at the periphery, as site where numerous vacuoles also occur (Fig. 1c, e). The seminal receptacle wall, the thickness of which varies considerably depending on the part of seminal receptacle examined, consists of epithelium surrounded by a layer of muscle cells (Fig. 1f–h). The lining epithelium has numerous lamellar processes directed into the seminal receptacle lumen. The epithelial cells are connected to each other via extended contacts (Fig. 1g) and contain mitochondria and numerous vacuoles and phagosomes. The lamellar-like projections of muscle cells connect the epithelial and muscle layers (Fig. 1h). Spermatozoon ultrastructure An analysis of a large number of sections of the O. felineus seminal receptacle provided the basis for a 3-D reconstruction of the sperm ultrastructure. The mature spermatozoon of O. felineus is characterised by the presence of two 9+“1” axonemes, a nucleus, two mitochondria, a lamellar body, external
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ornamentation of the plasma membrane, and cortical microtubules (Figs. 2 and 3). Using electron microscopy, we were able to distinguish four regions (I–IV) of the O. felineus spermatozoon exhibiting different ultrastructural features (Fig. 5). Region I (Fig. 2a–d) constitutes the anterior part of the spermatozoon and contains two axonemes and cortical microtubules. The cortical microtubules are first observed in this anterior part (Fig. 2a), followed by the appearance of one axoneme (Fig. 2b). The cortical microtubules are in close contact with the plasma membrane, and their number varies from 8 to 15. Two axonemes are observed starting from the middle part of this region, and the number of cortical microtubules increases to 25 (Fig. 2c, d). We observed the lamellar body, mitochondrion and external ornamentation of the plasma membrane in region II (Fig. 2e–l). The lamellar body is visible in the anterior part of this region (Fig. 2e). The spermatozoon becomes asymmetric in its transverse sections in the middle part, and this is accompanied by the appearance of external ornamentation of the plasma membrane and then the mitochondrion (Fig. 2f–j). The cortical microtubules are present only on the side of the spermatozoon covered by this external ornamentation; on this side, the plasma membrane surrounds one of the axonemes in a semicircle. The lamellar body and mitochondrion are situated close to one of the axonemes (Fig. 2f–h), whereas these components are disposed symmetrically relative to both axonemes at the end of the middle part of this region (Fig. 2i, j). The number of cortical microtubules in the anterior part of region II is decreased compared to region I, amounting to only 13–16. All the microtubules are organised in a single field (Fig. 2f, g, i). The arrangement of cortical microtubules changes in the posterior part of this region where they are disposed in two fields: 4–5 microtubules run along one side of the spermatozoon, with 2–3 along the other side. The distance between the axonemes increases, and the cytoplasm contains more electron-dense granules, which are presumably composed of storage polysaccharides. The lamellar body is not observed in the cross-sections of the posterior part of region II, though the mitochondrion does continue to the end of this region (Fig. 2k, l). At this level, we observed four attachment zones where the membranes of the median cytoplasmic process and two axonemes fused during spermiogenesis (Fig. 2l). The median region of the spermatozoon corresponds to region III (Fig. 3a), which is characterised by the presence of two axonemes and cortical microtubules. The number of cortical microtubules in this region does not change, amounting to 6–8 in total. Region IV corresponds to the posterior part of the spermatozoon and contains two axonemes, cortical microtubules, the second mitochondrion and the nucleus (Fig. 3b–i). The anterior part of region IV is characterised by the presence of the nucleus, which is located between the two axonemes (Fig. 3b), followed by the appearance of the second
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Fig. 1 Structural organisation of the seminal receptacle of Opisthorchis felineus. a A fragment of the seminal receptacle (light microscopy, methylene blue staining). b, c The interior and periphery of the seminal receptacle (light microscopy). d, e The interior and periphery of the seminal receptacle (electron microscopy). f–h Ultrastructure of the seminal receptacle wall. V vacuoles, E epithelial layer, Ml muscle layer, P phagosomes, M mitochondria. The arrow points to an extended contact between two epithelial cells; the arrowheads indicate lamellate projections. Scale bars= 50 (a), 10 (b, c) and 2 μm (d–h)
mitochondrion near the nucleus (Fig. 3c). The middle part of the region contains the mitochondrion, nucleus, and just one axoneme (Fig. 3d, e). The nucleus is enclosed by the multilayered envelope, the outer membrane of which is in close contact with the plasma membrane of the spermatozoon (Fig. 3e–g). The posterior part of region IV contains one axoneme and the nucleus (Fig. 3f–h). The distal end of the second axoneme is also observed in this region (Fig. 3h). The nucleus gradually decreases in its diameter and disappears completely at the posterior extremity of the spermatozoon. The ultrastructural analysis revealed that the cytoplasm of the spermatozoon is filled with numerous electron-dense granules, presumably glycogen, starting from the middle part of region II (Fig. 2g–j) and extending to the posterior part of region IV (Fig. 3i). It should also be noted that the number of cortical microtubules is not a constant characteristic of the
spermatozoon of O. felineus. Indeed, cross-sections of spermatozoa without cortical microtubules in the periphery were occasionally observed (Fig. 2a). Our study allowed the clear detection of the attachment zones only in specific crosssections of the spermatozoon in region II, as the cytoplasm is filled with numerous glycogen granules that mask the attachment zones. Morphology of degenerating spermatozoa in the seminal receptacle periphery The vacuoles in the periphery of the seminal receptacle differ in their size and contain degenerating spermatozoa, axonemes and single microtubules (Figs. 1e and 4a, c). Some vacuoles enclose groups of spermatozoa connected to each other via their posterior nuclear parts, which display less condensed
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Fig. 2 Transverse sections of the mature spermatozoon of Opisthorchis felineus. a–d Cross-sections of region I. a The anterior spermatozoon extremity showing cortical microtubules; the arrow marks region I lacking cortical microtubules in cross-section. b Transverse section of the first axoneme with the 9+“1” pattern. c Two axonemes in cross-section. d An increase in the distance between the plasma membrane and two axonemes in cross-section. e–l Successive cross-sections showing the ultrastructural characteristics of region II. e Cross-section showing the appearance of the lamellar body. f Asymmetrical arrangement of the external ornamentation of the plasma membrane. g Cross-section of the spermatozoon characterised by the presence of two axonemes, the lamellar body, the
first mitochondrion, cortical microtubules and external ornamentation of the plasma membrane. i Symmetrical arrangement of two axonemes, cortical microtubules, the lamellar body and the first mitochondrion. h, j The first mitochondrion and the lamellar body framed in panels g, i depicted at a higher magnification. k Cross-section showing the presence of two axonemes and the first mitochondrion. l Diameter narrowing of the first mitochondrion in cross-section. The white arrowheads denote electron-dense granules; the black arrowheads indicate the attachment zones. Cm cortical microtubules, Ax1 and Ax2 axonemes, Lm lamellar body, Eo external ornamentation of the plasma membrane, M1 mitochondrion. Scale bar=0.2 μm
chromatin in the nuclei compared to the nuclei of the mature spermatozoa in the interior of the seminal receptacle (Fig. 4b). The periphery of the seminal receptacle also contains spermatozoa with abnormal morphology, not surrounded by
vacuoles, displaying three or four axonemes in their crosssections (Fig. 4d–f). Two successive stages of the putative process of spermatozoan fusion resulting in structures with four axonemes were observed (Fig. 4e, f).
Parasitol Res (2014) 113:1093–1101 Fig. 3 Cross-sections of region III (a) and IV (b–i) of the mature spermatozoon of Opisthorchis felineus. a Cross-section of region III showing two axonemes and cortical microtubules. b Appearance of the nucleus in cross-section. c Cross-section characterised by the presence of two axonemes, the nucleus and the second mitochondrion. d, e The second axoneme, second mitochondrion and nucleus in cross-section. Note the increase in the nuclear diameter in the crosssections (e). f, g Cross-sections with the nucleus and one axoneme, differing in the number of cortical microtubules; the arrow indicates the contact of the detached outer nuclear envelope with the plasma membrane. h Disorganisation of the second axoneme in cross-section. i The posterior spermatozoon extremity containing the nucleus and cortical microtubules. Ax1 and Ax2 axonemes, N nucleus, M2 mitochondrion, Cm cortical microtubules. Scale bar=0.2 μm
Fig. 4 Abnormal and degenerating spermatozoa at the peripheral area of the seminal receptacle of Opisthorchis felineus. a, c Vacuoles containing axonemes and cortical microtubules. b Aggregation of spermatozoa via their nuclear parts inside the vacuole. d Crosssection of a spermatozoon characterised by the presence of three axonemes. e, f Successive stages of the fusion of two spermatozoa, leading to the formation of structures with four axonemes in cross-section. Scale bars=1 (a, b) and 0.5 μm (c–f)
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Discussion Seminal receptacle Our study demonstrated that the seminal receptacle wall of O. felineus consists of two layers and is similar to the structure of the seminal receptacle wall described for C. sinensis (Jeong 1983) and for the lung fluke Paragonimus ohirai (Troglotrematidae) (Orido 1988). The lamellar processes of the lining epithelium in the seminal receptacle of O. felineus are less developed in comparison to the lamellae in the seminal receptacle of C. sinensis and possibly form an irregular network structure similar to that observed in C. sinensis by scanning electron microscopy (Jeong 1983). Interestingly, cilia have been detected in the epithelium of the seminal receptacle of the bat parasite Prosthodendrium ascidia (Lecithodendriidae) (Podvyaznaya 1990). All motile cilia have a 9+2 arrangement of microtubules and are thus principally different from Platyhelminthes spermatozoa. However, we did not observe such structures in the epithelium of the O. felineus seminal receptacle. Some researchers suggest that the surface of the epithelium lining the seminal receptacle of O. felineus is the area of spermatozoon adhesion and preparation for fertilisation in addition to damaged spermatozoa elimination (Grebenshchikov and Nacheva 2007). Vacuoles with degenerating spermatozoa were also observed in the seminal receptacle of P. ohirai, though they were reported to be mainly localised to the interior of the seminal receptacle, whereas the periphery contained mature spermatozoa (Orido 1988). The pattern of degenerative changes in spermatozoa described in the interior of the seminal receptacle of P. ohirai (Orido 1988) is similar to the abnormalities in the spermatozoa that we observed. According to our data, degenerating O. felineus spermatozoa appear mainly in the periphery of the seminal receptacle, both within and outside of vacuoles. Spermatozoon Based on our electron microscopic data, we developed a schematic diagram of the structural organisation of the mature O. felineus spermatozoon (Fig. 5), taking into account the common anteroposterior orientation of the spermatozoa of Platyhelminthes (Justine 1995). Such an orientation of spermatozoon is associated with a high functional activity of the anterior non-nuclear region, which contains centrioles. It has been shown that the anterior and median parts of the spermatozoon of P. ohirai execute undulatory and vibratory movements, respectively (Orido 1988), and the anterior, nonnuclear part of Gonapodasmius sp. (Didymozoidae) spermatozoa is the first to enter the oocyte during fertilisation (Justine and Mattei 1984). Previous data on the spermatozoal morphology of O. felineus have suggested the presence of a nucleus, an
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acrosome, two axonemes, cortical microtubules and one mitochondrion (Grebenshchikov and Nacheva 2007). According to our data, the spermatozoon of O. felineus does not contain an acrosome. Moreover, the absence of an acrosome is a characteristic feature of trematodes (Smyth and Halton 1983; Schatten and Chakrabarti 2000). In addition to the nucleus, two axonemes, cortical microtubules and one mitochondrion, we are the first to detect the lamellar body, second mitochondrion and external ornamentation of the plasma membrane in the O. felineus spermatozoon. Common features of the sperm structure of O. felineus, O. viverrini and C. sinensis include the presence of a nucleus and two axonemes (Jeong and Rim 1984; Wannaprapo et al. 2008). The spermatozoon of O. felineus is also similar to that of C. sinensis by its arrangement of cortical microtubules; however, these species differ by the presence of a second mitochondrion, a lamellar body and external ornamentation of the plasma membrane in O. felineus, features that are absent in C. sinensis (Jeong and Rim 1984). It is difficult to make an analogous comparison between O. felineus and O. viverrini due to the absence of detailed information on spermatozoon morphology for the latter. Axonemes The mature spermatozoon of O. felineus contains two 9+“1” axonemes, which is typical for most species of Digenea (Smyth and Halton 1983; Justine 1995). The 9+“1” pattern of microtubules has been also demonstrated for the two axonemes of the spermatozoon of O. viverrini (Wannaprapo et al. 2008). Interestingly, the 9+2 pattern has been described for the sperm tail of C. sinensis (Jeong and Rim 1984), belonging to the Opisthorchiidae family. As recent molecular studies have confirmed the phylogenetic relationship among C. sinensis, O. felineus and O. viverrini (Kang et al. 2008; Saijuntha et al. 2008; Thaenkham et al. 2012), we suggest that Jeong and Rim (1984) erroneously ascribed the section of a cilium of the lining epithelium to the sperm tail because the 9+2 structure of these cilia has been identified for both the seminal receptacle and oviduct of P. ascidia (Podvyaznaya 1990). In addition, axonemes of the 9+“1” pattern are also observed in several micrographs of the C. sinensis spermatozoon (Jeong and Rim 1984). Cortical microtubules The cortical microtubules generally form the cytoskeleton protecting the cellular organelles and supporting the plasma membrane of the spermatozoon. The disposition of microtubules in digenean spermatozoa can be assessed using two parameters, namely variation in the number of microtubules along the gamete and their arrangement in a single or two fields (dorsal and ventral) on cross-sections. According to our data, the number of cortical microtubules is maximal in the middle part of region I and then gradually decreases starting from region II of the O. felineus spermatozoon, as in C. sinensis (Jeong and Rim 1984). This anteroposterior decrease in the number of microtubules has
Parasitol Res (2014) 113:1093–1101 Fig. 5 Schematic reconstruction of the mature spermatozoon of Opisthorchis felineus. I–IV spermatozoon regions, Cm cortical microtubules, Ax1 and Ax2 axonemes, Lb lamellar body, Eo external ornamentation, M1 and M2 mitochondria, N nucleus
been also observed in the spermatozoa of Euryhelmis squamula (Heterophyidae) (Bakhoum et al. 2009), Paramphistomum microbothrium (Paramphistomidae) (Seck et al. 2007), Deropristis inflata (Deropristidae) (Foata et al. 2007) and Fasciola gigantica (Fasciolidae) (Ndiaye et al. 2004). In some species of Digenea, the number of microtubules increases from the anterior extremity to the anterior part of the nuclear region and then decreases, as in Pronoprymna ventricosa (Baccigerinae) (Quilichini et al. 2007b) and five species of Opecoelidae (Miquel et al. 2000; Levron et al. 2003, 2004a; Quilichini et al. 2007a, c). Similar to the majority of digenean spermatozoa, two fields of cortical microtubules are present in the spermatozoon of O. felineus. Interestingly, only one field of cortical microtubules is present in P. ventricosa and in several hemiuroidean trematodes, in particular, Gonapodasmius sp., Aponurus laguncula and Lecithocladium excisum (Justine and Mattei 1982; Quilichini et al. 2007b, 2010; Ndiaye et al. 2012). Mitochondria It has been previously shown that the number of mitochondria in digenean spermatozoa varies from one to three. Our examination detected two mitochondria in the O. felineus spermatozoon, whereas C. sinensis spermatozoa possess only a single mitochondrion (Jeong and Rim 1984). Of note, the spermatozoon of Cryptocotyle lingua contains one mitochondrion (Rees 1979), and the spermatozoon of E. squamula contains three mitochondria (Bakhoum et al. 2009), even though they are representatives of the family Heterophyidae, which is paraphyletic with respect to Opisthorchiidae (Thaenkham et al. 2012). Because it appears that the number of mitochondria varies within families, this parameter is not very reliable for comparative spermatology. Two mitochondria have been observed in the spermatozoa of many other species, such as P. ohirai (Orido 1988), Troglotrema acutum (Miquel et al. 2006), Nicolla testiobliquum (Quilichini et al. 2007a), Notocotylus neyrai (Ndiaye et al. 2003), Neoapocreadium chabaudi (Kacem et al. 2010) and Monorchis parvus (Levron et al. 2004b). Lamellar body According to our data, the mature spermatozoon of O. felineus contains the lamellar body associated with the mitochondrion in region II. Although the function of the lamellar body is still unknown, this structure has been identified in many species of the subclass Aspidogastrea (Rohde et al. 1991; Watson and Rohde 1991, 1992; Levron et al. 2009), which is regarded as a sister taxon to Digenea (Blair 1993; Rohde et al. 1993). A structure with a similar organisation near a large mitochondrion was interpreted as the second
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mitochondrion of a smaller size in the spermatozoa of Aspidogaster conchicola (Bakker and Diegenbach 1972), whereas another researcher suggested that a similar structure observed in cross-sections could be a narrow protrusion of the nucleus (Orido 1988). We do not exclude the possibility that the lamellar body in the spermatozoon of O. felineus is actually a third mitochondrion. External ornamentation of the plasma membrane We noted the presence of external ornamentation of the plasma membrane in region II of the mature spermatozoon of O. felineus. The precise role of the external ornamentation is still unknown, though it has been suggested that this structure may be involved in the contact and fusion of the sperm and egg cell membranes during fertilisation (Justine and Mattei 1982). The presence of external ornamentation has been observed in a large number of digenean trematodes and was previously proposed to divide the spermatozoa of digeneans into two groups according to the location of this external ornamentation (Quilichini et al. 2007b). The first group includes species with external ornamentation at the anterior extremity of the spermatozoon, and the second includes those with ornamentation at a more posterior position relative to the anterior extremity. These two groups were later supplemented with a third, comprising species with still undetected external ornamentation (Quilichini et al. 2011). Based on this trait, the spermatozoon of O. felineus can be classed into the second group, whereas the spermatozoon of C. sinensis belongs to the third group, as external ornamentation of the plasma membrane has not been described for this species (Jeong and Rim 1984). Attachment zones The attachment zones, determining the sites of fusion between the median cytoplasmic process and the flagella during spermiogenesis (Burton 1972), has been identified in many digenean spermatozoa (Ndiaye et al. 2004; Quilichini et al. 2007b; Seck et al. 2008; Foata et al. 2007). However, the dense packaging of glycogen granules in the cytoplasm allowed us to observe attachment zones only in some cross-sections of O. felineus spermatozoa. Herein, we describe for the first time the detailed ultrastructure of the spermatozoon of O. felineus, the fluke that causes human opisthorchiasis. The morphological features of the mature spermatozoon of this species include the alternation of regions with symmetric and asymmetric arrangement of components over the length of the gamete, namely two axonemes, two mitochondria, a nucleus, a lamellar body, external ornamentation and cortical microtubules. The data on the specific arrangement of spermatozoal components (3-D model) and their detailed structural characteristics would be useful for testing the effects of new antihelminthic drugs targeted at reducing adult parasite reproduction.
Parasitol Res (2014) 113:1093–1101 Acknowledgments We cordially thank M. N. Lvova and Dr. A. V. Katokhin (IC&G SB RAS) for providing the live specimens of O. felineus. This work was supported by the Integration project of fundamental research of SB RAS (№19). Ethical standards The handling and care of M. auratus golden hamsters were conducted in strict accordance with the recommendations in the NIH Guide for the Care and Use of Laboratory Animals. The study protocol was approved by the Committee on the Ethics of Animal Experiments of the Institute of Cytology and Genetics SB RAS (permit number 7 of 19.11.2011).
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1101 Quilichini Y, Foata J, Marchand B (2007a) Ultrastructural study of the spermatozoon of Nicolla testiobliquum (Digenea, Opecoelidae) parasite of brown trout Salmo trutta (Pisces, Teleostei). Parasitol Res 101:1295–1301 Quilichini Y, Foata J, Marchand B (2007b) Ultrastructural study of the spermatozoon of Pronoprymna ventricosa (Digenea, Baccigerinae), parasite of the twaite shad Alosa fallax Lacepede (Pisces, Teleostei). Parasitol Res 101:1125–1130 Quilichini Y, Foata J, Orsini A, Marchand B (2007c) Spermiogenesis and spermatozoon ultrastructure of Nicolla wisniewskii (Digenea, Opecoelidae), an intestinal parasite of brown trout Salmo trutta (Pisces, Teleostei). J Parasitol 93:469–478 Quilichini Y, Foata J, Justine JL, Bray RA, Marchand B (2010) Spermatozoon ultrastructure of Aponurus laguncula (Digenea: Lecithasteridae), a parasite of Aluterus monoceros (Pisces: Teleostei). Parasitol Int 59:22–28. doi:10.1016/j.parint.2009.06.007 Quilichini Y, Foata J, Justine JL, Bray RA, Marchand B (2011) Spermatozoon ultrastructure of Gyliauchen sp. (Digenea: Gyliauchenidae), an intestinal parasite of Siganus fuscescens (Pisces: Teleostei). Biol Bull 221:197–205 Rees GF (1979) The ultrastructure of the spermatozoon and spermiogenesis in Cryptocotyle lingua (Digenea: Heterophyidae). Int J Parasitol 9:405–419 Rohde K, Watson NA, Cribb T (1991) Ultrastructure of sperm and spermatogenesis of Lobatostoma manteri (Trematoda, Aspidogastrea). Int J Parasitol 21:409–419 Rohde K, Hefford C, Ellis JT, Baverstock PR, Johnson AM, Watson NA, Dittmann S (1993) Contributions to the phylogeny of platyhelminthes based on partial sequencing of 18S ribosomal DNA. Int J Parasitol 23:705–724 Saijuntha W, Sithithaworn P, Wongkham S, Laha T, Chilton NB, Petney TN, Barton M, Andrews RH (2008) Mitochondrial DNA sequence variation among geographical isolates of Opisthorchis viverrini in Thailand and Lao PDR, and phylogenetic relationships with other trematodes. Parasitology 135:1479–1486 Schatten H, Chakrabarti A (2000) Fertilisation in invertebrates. In: Tarín JJ, Cano A (eds) Fertilisation in protozoa and metazoan animals: cellular and molecular aspects. Springer, Berlin, Heidelberg, pp 27–87 Seck MT, Marchand B, Bâ CT (2007) Ultrastructure of spermiogenesis and the spermatozoon of Paramphistomum microbothrium [Fischoeder 1901] (Digenea: Paramphistomidae), a parasite of Bos taurus in Senegal. Parasitol Res 101:653–662 Seck MT, Marchand B, Bâ CT (2008) Spermiogenesis and sperm ultrastructure of Cotylophoron cotylophorum (Trematoda, Digenea, Paramphistomidae), a parasite of Bos taurus in Senegal. Parasitol Res 103(1):157–166. doi:10.1007/s00436-008-0944-1 Skryabin KI (1947) Trematodes of animals and man, vol 1. Izdatel’stvo Akademii Nauk SSSR, Moscow Smyth JD, Halton DW (1983) The physiology of Trematodes, 2nd edn. Cambridge University Press, Cambridge Thaenkham U, Blair D, Nawa Y, Waikagul J (2012) Families Opisthorchiidae and Heterophyidae: are they distinct? Parasitol Int 61:90–93. doi:10.1016/j.parint.2011.06.004 Wannaprapo S, Tesana S, Arunyanat C, Kanla P, Chaijaroonkhanarak W (2008) Morphological features of the testes of the adult liver fluke, Opisthorchis viverrini. Srinagarind Med J 23:346–352 Watson NA, Rohde K (1991) Ultrastructure of spermatogenesis and sperm of Multicotyle purvisi (Plathelminthes, Aspidogastrea). Zoomorphology 110:347–356 Watson NA, Rohde K (1992) Ultrastructure of sperm and spermatogenesis of Rugogaster hydrolagi, Schell 1973 (Platyhelminthes, Trematoda, Aspidogastrea, Rugogastridae). Parasitol Res 78:516–524 Yossepowitch O, Gotesman T, Assous M, Marva E, Zimlichman R, Dan M (2004) Opisthorchiasis from imported raw fish. Emerg Infect Dis 10:2122–2126
ORIGINAL PAPER
Ultrastructure of spermatozoa in the seminal receptacle of the liver fluke Opisthorchis felineus (Rivolta, 1884) Mariya V. Zhukova & Viatcheslav A. Mordvinov & Elena Kiseleva
Received: 15 October 2013 / Accepted: 26 December 2013 / Published online: 23 January 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract The spermatozoon ultrastructure in the seminal receptacle of the liver fluke Opisthorchis felineus (Digenea, Opisthorchiidae), the agent of human opisthorchiasis endemic to Russia and Eastern Europe, was examined. The beanshaped seminal receptacle of O. felineus has a wall consisting of epithelial and muscle layers. Mature spermatozoa are located in the interior of the seminal receptacle, whereas vacuoles containing degenerating spermatozoa are detectable at the periphery. The mature spermatozoon of O. felineus has two axonemes of a 9+“1” pattern, a nucleus, two mitochondria, a lamellar body, two bundles of parallel cortical microtubules and an external ornamentation of the plasma membrane in the anterior area of the sperm. The lamellar body is likely to be the third mitochondrion of a small size. The cytoplasm of the spermatozoon is filled with numerous electron-dense granules of storage polysaccharides. Additionally, the ultrastructural characteristics of the seminal receptacle and spermatozoa of O. felineus were compared to available published data on other trematode species. The functional roles of the observed structures of this spermatozoon are discussed.
Introduction The ultrastructure of spermatozoa is highly diverse in Platyhelminthes, which has allowed comparative spermatology to be used for clarifying the phylogenetic relationships within this group (Justine et al. 1985; Justine 1995; Levron et al. 2010). Currently, both morphological and molecular data are used to determine relationships between the species of subclass Digenea (Barker et al. 1993; Blair 1993; M. V. Zhukova : V. A. Mordvinov : E. Kiseleva (*) Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, Novosibirsk 630090, Russia e-mail: [email protected]
Olson et al. 2003; Olson and Tkach 2005). Furthermore, the morphological examination of various systems of organs in parasitic worms is of paramount importance for insight into specific functional features of these parasites and their interaction with their host. The subclass Digenea comprises approximately 18,000 species belonging to 25 superfamilies (Bray et al. 2008), though spermatozoon ultrastructure has thus far been examined in less than 1 % of the species in this subclass (Quilichini et al. 2011). The available data demonstrate that the spermatozoal structure in various digeneans differs considerably with regard to several characteristics, such as the presence/absence of spine-like bodies, number of mitochondria, location of external ornamentation of the plasma membrane, location and number of cortical microtubules and morphology of the anterior and posterior extremities. The structural differences in the spermatozoa of digeneans could be associated with specific functional features of their reproductive system, which are yet to be studied. In the majority of Digenea sp., the mature spermatozoa are stored in a seminal receptacle from which they enter the ootype for fertilisation (Skryabin 1947). Based on the current data, family Opisthorchiidae comprises over 30 genera (King and Scholz 2001), including the liver flukes Opisthorchis viverrini, Opisthorchis felineus and Clonorchis sinensis, which are most widely distributed among humans and cause food-borne trematodiases (Fürst et al. 2012). According to the latest estimates, approximately 1.6 million people worldwide are infected with O. felineus (Yossepowitch et al. 2004). Among the Opisthorchiidae, the ultrastructure of the C. sinensis spermatozoon has been studied in detail (Jeong and Rim 1984), the testis morphology and partial spermatozoon structure (including two 9+“1” axonemes and a round nucleus with condensed chromatin) have been recently described for O. viverrini (Wannaprapo et al. 2008), and the morphology of the O. felineus seminal receptacle and sp ermatoz oa w ere b riefly co vere d by
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Grebenshchikov and Nacheva (2007). In the present work, we comprehensively investigated the ultrastructure of the seminal receptacle and spermatozoa in the liver fluke O. felineus.
Materials and methods Adult O. felineus flukes were obtained from the liver of experimentally infected golden hamsters (Mesocricetus auratus). The seminal receptacles of O. felineus were isolated and fixed with 2.5 % glutaraldehyde solution in 0.1 sodium cacodylate buffer (pH 7.2) for 2.5 h, rinsed with the same buffer, and postfixed in 1 % osmium tetroxide in 0.1 sodium cacodylate buffer for 1 h. The samples were washed with water and placed in 1 % uranyl acetate aqueous solution for 12 h at +4 °C. The samples were then dehydrated through a graded series of ethanol and acetone and embedded in Agar 100 resin. Ultrathin sections were double stained with uranyl acetate and lead citrate according to Reynolds and examined using a JEOL 100 SX electron microscope at the facilities of the Interinstitutional Shared Centre for Microscopic Analysis of Biological Objects, the Institute of Cytology and Genetics SB RAS.
Results Morphology of the seminal receptacle The seminal receptacle of O. felineus is bean-shaped, embedded in parenchyma and filled with mature spermatozoa (Fig. 1); interior and peripheral regions are evident in semithin sections (Fig. 1a). The spermatozoa are densely packed in the interior (Fig. 1b, d), whereas they are loosely arranged at the periphery, as site where numerous vacuoles also occur (Fig. 1c, e). The seminal receptacle wall, the thickness of which varies considerably depending on the part of seminal receptacle examined, consists of epithelium surrounded by a layer of muscle cells (Fig. 1f–h). The lining epithelium has numerous lamellar processes directed into the seminal receptacle lumen. The epithelial cells are connected to each other via extended contacts (Fig. 1g) and contain mitochondria and numerous vacuoles and phagosomes. The lamellar-like projections of muscle cells connect the epithelial and muscle layers (Fig. 1h). Spermatozoon ultrastructure An analysis of a large number of sections of the O. felineus seminal receptacle provided the basis for a 3-D reconstruction of the sperm ultrastructure. The mature spermatozoon of O. felineus is characterised by the presence of two 9+“1” axonemes, a nucleus, two mitochondria, a lamellar body, external
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ornamentation of the plasma membrane, and cortical microtubules (Figs. 2 and 3). Using electron microscopy, we were able to distinguish four regions (I–IV) of the O. felineus spermatozoon exhibiting different ultrastructural features (Fig. 5). Region I (Fig. 2a–d) constitutes the anterior part of the spermatozoon and contains two axonemes and cortical microtubules. The cortical microtubules are first observed in this anterior part (Fig. 2a), followed by the appearance of one axoneme (Fig. 2b). The cortical microtubules are in close contact with the plasma membrane, and their number varies from 8 to 15. Two axonemes are observed starting from the middle part of this region, and the number of cortical microtubules increases to 25 (Fig. 2c, d). We observed the lamellar body, mitochondrion and external ornamentation of the plasma membrane in region II (Fig. 2e–l). The lamellar body is visible in the anterior part of this region (Fig. 2e). The spermatozoon becomes asymmetric in its transverse sections in the middle part, and this is accompanied by the appearance of external ornamentation of the plasma membrane and then the mitochondrion (Fig. 2f–j). The cortical microtubules are present only on the side of the spermatozoon covered by this external ornamentation; on this side, the plasma membrane surrounds one of the axonemes in a semicircle. The lamellar body and mitochondrion are situated close to one of the axonemes (Fig. 2f–h), whereas these components are disposed symmetrically relative to both axonemes at the end of the middle part of this region (Fig. 2i, j). The number of cortical microtubules in the anterior part of region II is decreased compared to region I, amounting to only 13–16. All the microtubules are organised in a single field (Fig. 2f, g, i). The arrangement of cortical microtubules changes in the posterior part of this region where they are disposed in two fields: 4–5 microtubules run along one side of the spermatozoon, with 2–3 along the other side. The distance between the axonemes increases, and the cytoplasm contains more electron-dense granules, which are presumably composed of storage polysaccharides. The lamellar body is not observed in the cross-sections of the posterior part of region II, though the mitochondrion does continue to the end of this region (Fig. 2k, l). At this level, we observed four attachment zones where the membranes of the median cytoplasmic process and two axonemes fused during spermiogenesis (Fig. 2l). The median region of the spermatozoon corresponds to region III (Fig. 3a), which is characterised by the presence of two axonemes and cortical microtubules. The number of cortical microtubules in this region does not change, amounting to 6–8 in total. Region IV corresponds to the posterior part of the spermatozoon and contains two axonemes, cortical microtubules, the second mitochondrion and the nucleus (Fig. 3b–i). The anterior part of region IV is characterised by the presence of the nucleus, which is located between the two axonemes (Fig. 3b), followed by the appearance of the second
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Fig. 1 Structural organisation of the seminal receptacle of Opisthorchis felineus. a A fragment of the seminal receptacle (light microscopy, methylene blue staining). b, c The interior and periphery of the seminal receptacle (light microscopy). d, e The interior and periphery of the seminal receptacle (electron microscopy). f–h Ultrastructure of the seminal receptacle wall. V vacuoles, E epithelial layer, Ml muscle layer, P phagosomes, M mitochondria. The arrow points to an extended contact between two epithelial cells; the arrowheads indicate lamellate projections. Scale bars= 50 (a), 10 (b, c) and 2 μm (d–h)
mitochondrion near the nucleus (Fig. 3c). The middle part of the region contains the mitochondrion, nucleus, and just one axoneme (Fig. 3d, e). The nucleus is enclosed by the multilayered envelope, the outer membrane of which is in close contact with the plasma membrane of the spermatozoon (Fig. 3e–g). The posterior part of region IV contains one axoneme and the nucleus (Fig. 3f–h). The distal end of the second axoneme is also observed in this region (Fig. 3h). The nucleus gradually decreases in its diameter and disappears completely at the posterior extremity of the spermatozoon. The ultrastructural analysis revealed that the cytoplasm of the spermatozoon is filled with numerous electron-dense granules, presumably glycogen, starting from the middle part of region II (Fig. 2g–j) and extending to the posterior part of region IV (Fig. 3i). It should also be noted that the number of cortical microtubules is not a constant characteristic of the
spermatozoon of O. felineus. Indeed, cross-sections of spermatozoa without cortical microtubules in the periphery were occasionally observed (Fig. 2a). Our study allowed the clear detection of the attachment zones only in specific crosssections of the spermatozoon in region II, as the cytoplasm is filled with numerous glycogen granules that mask the attachment zones. Morphology of degenerating spermatozoa in the seminal receptacle periphery The vacuoles in the periphery of the seminal receptacle differ in their size and contain degenerating spermatozoa, axonemes and single microtubules (Figs. 1e and 4a, c). Some vacuoles enclose groups of spermatozoa connected to each other via their posterior nuclear parts, which display less condensed
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Fig. 2 Transverse sections of the mature spermatozoon of Opisthorchis felineus. a–d Cross-sections of region I. a The anterior spermatozoon extremity showing cortical microtubules; the arrow marks region I lacking cortical microtubules in cross-section. b Transverse section of the first axoneme with the 9+“1” pattern. c Two axonemes in cross-section. d An increase in the distance between the plasma membrane and two axonemes in cross-section. e–l Successive cross-sections showing the ultrastructural characteristics of region II. e Cross-section showing the appearance of the lamellar body. f Asymmetrical arrangement of the external ornamentation of the plasma membrane. g Cross-section of the spermatozoon characterised by the presence of two axonemes, the lamellar body, the
first mitochondrion, cortical microtubules and external ornamentation of the plasma membrane. i Symmetrical arrangement of two axonemes, cortical microtubules, the lamellar body and the first mitochondrion. h, j The first mitochondrion and the lamellar body framed in panels g, i depicted at a higher magnification. k Cross-section showing the presence of two axonemes and the first mitochondrion. l Diameter narrowing of the first mitochondrion in cross-section. The white arrowheads denote electron-dense granules; the black arrowheads indicate the attachment zones. Cm cortical microtubules, Ax1 and Ax2 axonemes, Lm lamellar body, Eo external ornamentation of the plasma membrane, M1 mitochondrion. Scale bar=0.2 μm
chromatin in the nuclei compared to the nuclei of the mature spermatozoa in the interior of the seminal receptacle (Fig. 4b). The periphery of the seminal receptacle also contains spermatozoa with abnormal morphology, not surrounded by
vacuoles, displaying three or four axonemes in their crosssections (Fig. 4d–f). Two successive stages of the putative process of spermatozoan fusion resulting in structures with four axonemes were observed (Fig. 4e, f).
Parasitol Res (2014) 113:1093–1101 Fig. 3 Cross-sections of region III (a) and IV (b–i) of the mature spermatozoon of Opisthorchis felineus. a Cross-section of region III showing two axonemes and cortical microtubules. b Appearance of the nucleus in cross-section. c Cross-section characterised by the presence of two axonemes, the nucleus and the second mitochondrion. d, e The second axoneme, second mitochondrion and nucleus in cross-section. Note the increase in the nuclear diameter in the crosssections (e). f, g Cross-sections with the nucleus and one axoneme, differing in the number of cortical microtubules; the arrow indicates the contact of the detached outer nuclear envelope with the plasma membrane. h Disorganisation of the second axoneme in cross-section. i The posterior spermatozoon extremity containing the nucleus and cortical microtubules. Ax1 and Ax2 axonemes, N nucleus, M2 mitochondrion, Cm cortical microtubules. Scale bar=0.2 μm
Fig. 4 Abnormal and degenerating spermatozoa at the peripheral area of the seminal receptacle of Opisthorchis felineus. a, c Vacuoles containing axonemes and cortical microtubules. b Aggregation of spermatozoa via their nuclear parts inside the vacuole. d Crosssection of a spermatozoon characterised by the presence of three axonemes. e, f Successive stages of the fusion of two spermatozoa, leading to the formation of structures with four axonemes in cross-section. Scale bars=1 (a, b) and 0.5 μm (c–f)
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Discussion Seminal receptacle Our study demonstrated that the seminal receptacle wall of O. felineus consists of two layers and is similar to the structure of the seminal receptacle wall described for C. sinensis (Jeong 1983) and for the lung fluke Paragonimus ohirai (Troglotrematidae) (Orido 1988). The lamellar processes of the lining epithelium in the seminal receptacle of O. felineus are less developed in comparison to the lamellae in the seminal receptacle of C. sinensis and possibly form an irregular network structure similar to that observed in C. sinensis by scanning electron microscopy (Jeong 1983). Interestingly, cilia have been detected in the epithelium of the seminal receptacle of the bat parasite Prosthodendrium ascidia (Lecithodendriidae) (Podvyaznaya 1990). All motile cilia have a 9+2 arrangement of microtubules and are thus principally different from Platyhelminthes spermatozoa. However, we did not observe such structures in the epithelium of the O. felineus seminal receptacle. Some researchers suggest that the surface of the epithelium lining the seminal receptacle of O. felineus is the area of spermatozoon adhesion and preparation for fertilisation in addition to damaged spermatozoa elimination (Grebenshchikov and Nacheva 2007). Vacuoles with degenerating spermatozoa were also observed in the seminal receptacle of P. ohirai, though they were reported to be mainly localised to the interior of the seminal receptacle, whereas the periphery contained mature spermatozoa (Orido 1988). The pattern of degenerative changes in spermatozoa described in the interior of the seminal receptacle of P. ohirai (Orido 1988) is similar to the abnormalities in the spermatozoa that we observed. According to our data, degenerating O. felineus spermatozoa appear mainly in the periphery of the seminal receptacle, both within and outside of vacuoles. Spermatozoon Based on our electron microscopic data, we developed a schematic diagram of the structural organisation of the mature O. felineus spermatozoon (Fig. 5), taking into account the common anteroposterior orientation of the spermatozoa of Platyhelminthes (Justine 1995). Such an orientation of spermatozoon is associated with a high functional activity of the anterior non-nuclear region, which contains centrioles. It has been shown that the anterior and median parts of the spermatozoon of P. ohirai execute undulatory and vibratory movements, respectively (Orido 1988), and the anterior, nonnuclear part of Gonapodasmius sp. (Didymozoidae) spermatozoa is the first to enter the oocyte during fertilisation (Justine and Mattei 1984). Previous data on the spermatozoal morphology of O. felineus have suggested the presence of a nucleus, an
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acrosome, two axonemes, cortical microtubules and one mitochondrion (Grebenshchikov and Nacheva 2007). According to our data, the spermatozoon of O. felineus does not contain an acrosome. Moreover, the absence of an acrosome is a characteristic feature of trematodes (Smyth and Halton 1983; Schatten and Chakrabarti 2000). In addition to the nucleus, two axonemes, cortical microtubules and one mitochondrion, we are the first to detect the lamellar body, second mitochondrion and external ornamentation of the plasma membrane in the O. felineus spermatozoon. Common features of the sperm structure of O. felineus, O. viverrini and C. sinensis include the presence of a nucleus and two axonemes (Jeong and Rim 1984; Wannaprapo et al. 2008). The spermatozoon of O. felineus is also similar to that of C. sinensis by its arrangement of cortical microtubules; however, these species differ by the presence of a second mitochondrion, a lamellar body and external ornamentation of the plasma membrane in O. felineus, features that are absent in C. sinensis (Jeong and Rim 1984). It is difficult to make an analogous comparison between O. felineus and O. viverrini due to the absence of detailed information on spermatozoon morphology for the latter. Axonemes The mature spermatozoon of O. felineus contains two 9+“1” axonemes, which is typical for most species of Digenea (Smyth and Halton 1983; Justine 1995). The 9+“1” pattern of microtubules has been also demonstrated for the two axonemes of the spermatozoon of O. viverrini (Wannaprapo et al. 2008). Interestingly, the 9+2 pattern has been described for the sperm tail of C. sinensis (Jeong and Rim 1984), belonging to the Opisthorchiidae family. As recent molecular studies have confirmed the phylogenetic relationship among C. sinensis, O. felineus and O. viverrini (Kang et al. 2008; Saijuntha et al. 2008; Thaenkham et al. 2012), we suggest that Jeong and Rim (1984) erroneously ascribed the section of a cilium of the lining epithelium to the sperm tail because the 9+2 structure of these cilia has been identified for both the seminal receptacle and oviduct of P. ascidia (Podvyaznaya 1990). In addition, axonemes of the 9+“1” pattern are also observed in several micrographs of the C. sinensis spermatozoon (Jeong and Rim 1984). Cortical microtubules The cortical microtubules generally form the cytoskeleton protecting the cellular organelles and supporting the plasma membrane of the spermatozoon. The disposition of microtubules in digenean spermatozoa can be assessed using two parameters, namely variation in the number of microtubules along the gamete and their arrangement in a single or two fields (dorsal and ventral) on cross-sections. According to our data, the number of cortical microtubules is maximal in the middle part of region I and then gradually decreases starting from region II of the O. felineus spermatozoon, as in C. sinensis (Jeong and Rim 1984). This anteroposterior decrease in the number of microtubules has
Parasitol Res (2014) 113:1093–1101 Fig. 5 Schematic reconstruction of the mature spermatozoon of Opisthorchis felineus. I–IV spermatozoon regions, Cm cortical microtubules, Ax1 and Ax2 axonemes, Lb lamellar body, Eo external ornamentation, M1 and M2 mitochondria, N nucleus
been also observed in the spermatozoa of Euryhelmis squamula (Heterophyidae) (Bakhoum et al. 2009), Paramphistomum microbothrium (Paramphistomidae) (Seck et al. 2007), Deropristis inflata (Deropristidae) (Foata et al. 2007) and Fasciola gigantica (Fasciolidae) (Ndiaye et al. 2004). In some species of Digenea, the number of microtubules increases from the anterior extremity to the anterior part of the nuclear region and then decreases, as in Pronoprymna ventricosa (Baccigerinae) (Quilichini et al. 2007b) and five species of Opecoelidae (Miquel et al. 2000; Levron et al. 2003, 2004a; Quilichini et al. 2007a, c). Similar to the majority of digenean spermatozoa, two fields of cortical microtubules are present in the spermatozoon of O. felineus. Interestingly, only one field of cortical microtubules is present in P. ventricosa and in several hemiuroidean trematodes, in particular, Gonapodasmius sp., Aponurus laguncula and Lecithocladium excisum (Justine and Mattei 1982; Quilichini et al. 2007b, 2010; Ndiaye et al. 2012). Mitochondria It has been previously shown that the number of mitochondria in digenean spermatozoa varies from one to three. Our examination detected two mitochondria in the O. felineus spermatozoon, whereas C. sinensis spermatozoa possess only a single mitochondrion (Jeong and Rim 1984). Of note, the spermatozoon of Cryptocotyle lingua contains one mitochondrion (Rees 1979), and the spermatozoon of E. squamula contains three mitochondria (Bakhoum et al. 2009), even though they are representatives of the family Heterophyidae, which is paraphyletic with respect to Opisthorchiidae (Thaenkham et al. 2012). Because it appears that the number of mitochondria varies within families, this parameter is not very reliable for comparative spermatology. Two mitochondria have been observed in the spermatozoa of many other species, such as P. ohirai (Orido 1988), Troglotrema acutum (Miquel et al. 2006), Nicolla testiobliquum (Quilichini et al. 2007a), Notocotylus neyrai (Ndiaye et al. 2003), Neoapocreadium chabaudi (Kacem et al. 2010) and Monorchis parvus (Levron et al. 2004b). Lamellar body According to our data, the mature spermatozoon of O. felineus contains the lamellar body associated with the mitochondrion in region II. Although the function of the lamellar body is still unknown, this structure has been identified in many species of the subclass Aspidogastrea (Rohde et al. 1991; Watson and Rohde 1991, 1992; Levron et al. 2009), which is regarded as a sister taxon to Digenea (Blair 1993; Rohde et al. 1993). A structure with a similar organisation near a large mitochondrion was interpreted as the second
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mitochondrion of a smaller size in the spermatozoa of Aspidogaster conchicola (Bakker and Diegenbach 1972), whereas another researcher suggested that a similar structure observed in cross-sections could be a narrow protrusion of the nucleus (Orido 1988). We do not exclude the possibility that the lamellar body in the spermatozoon of O. felineus is actually a third mitochondrion. External ornamentation of the plasma membrane We noted the presence of external ornamentation of the plasma membrane in region II of the mature spermatozoon of O. felineus. The precise role of the external ornamentation is still unknown, though it has been suggested that this structure may be involved in the contact and fusion of the sperm and egg cell membranes during fertilisation (Justine and Mattei 1982). The presence of external ornamentation has been observed in a large number of digenean trematodes and was previously proposed to divide the spermatozoa of digeneans into two groups according to the location of this external ornamentation (Quilichini et al. 2007b). The first group includes species with external ornamentation at the anterior extremity of the spermatozoon, and the second includes those with ornamentation at a more posterior position relative to the anterior extremity. These two groups were later supplemented with a third, comprising species with still undetected external ornamentation (Quilichini et al. 2011). Based on this trait, the spermatozoon of O. felineus can be classed into the second group, whereas the spermatozoon of C. sinensis belongs to the third group, as external ornamentation of the plasma membrane has not been described for this species (Jeong and Rim 1984). Attachment zones The attachment zones, determining the sites of fusion between the median cytoplasmic process and the flagella during spermiogenesis (Burton 1972), has been identified in many digenean spermatozoa (Ndiaye et al. 2004; Quilichini et al. 2007b; Seck et al. 2008; Foata et al. 2007). However, the dense packaging of glycogen granules in the cytoplasm allowed us to observe attachment zones only in some cross-sections of O. felineus spermatozoa. Herein, we describe for the first time the detailed ultrastructure of the spermatozoon of O. felineus, the fluke that causes human opisthorchiasis. The morphological features of the mature spermatozoon of this species include the alternation of regions with symmetric and asymmetric arrangement of components over the length of the gamete, namely two axonemes, two mitochondria, a nucleus, a lamellar body, external ornamentation and cortical microtubules. The data on the specific arrangement of spermatozoal components (3-D model) and their detailed structural characteristics would be useful for testing the effects of new antihelminthic drugs targeted at reducing adult parasite reproduction.
Parasitol Res (2014) 113:1093–1101 Acknowledgments We cordially thank M. N. Lvova and Dr. A. V. Katokhin (IC&G SB RAS) for providing the live specimens of O. felineus. This work was supported by the Integration project of fundamental research of SB RAS (№19). Ethical standards The handling and care of M. auratus golden hamsters were conducted in strict accordance with the recommendations in the NIH Guide for the Care and Use of Laboratory Animals. The study protocol was approved by the Committee on the Ethics of Animal Experiments of the Institute of Cytology and Genetics SB RAS (permit number 7 of 19.11.2011).
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