Russian Journal of Nematology, 2016, 24 (2), 99 – 110

Julia K. Zograf1, 2, Nguyen Dinh Tu3, Nguyen Thi Xuan Phuong3, Cao Van Luong4, Alexei V. Tchesunov5 and Vladimir V. Yushin1, 2

1A.V. Zhirmunsky Institute of Marine Biology, National Scientific Center of Marine Biology, Far Eastern Branch, Russian Academy of Sciences, 690041, Vladivostok, Russia; e-mail: [email protected] 2Far Eastern Federal University, 690950, Vladivostok, Russia 3Institute of Ecology and Biological Resources, VAST, Hanoi, Vietnam 4Institute of Marine Environment Resources, VAST, Hai Phong, Vietnam 5Department of Invertebrate Zoology, Faculty of Biology, M.V. Lomonosov Moscow State University, 119991, Moscow, Russia

Accepted for publication 7 October 2016

Summary. The spermatozoa from testis of the free-living marine nematode Desmoscolex granulatus (Desmoscolecida) were studied electron-microscopically. The spermatozoa are unpolarized cells covered by numerous filopodia. They contain the central lobated nucleus without a nuclear envelope. The spermatozoan cytoplasm includes mitochondria and fibrous bodies (FB). The spermatozoa of D. granulatus lack membranous organelles (MO) – a characteristic feature found in many nematode spermatozoa. The spermatozoon pattern, with the presence of FB never being associated with MO, unites D. granulatus with some chromadorids, desmodorids (Desmodoridae), monhysterids (Linhomoeidae) and tylenchomorphs (Tylenchoidea). This conclusion is supported by the filopodial nature of the sperm surface demonstrated by these taxa. Key words: Desmoscolex granulatus, fibrous bodies, filopodia, membranous organelles, spermatogenesis.

Nematode spermatozoa represent an aberrant characteristic of both the developing and mature type of male gametes; they are characterised by the sperm of most nematodes studied (Justine & absence of an axoneme and an acrosome and have Jamieson, 1999; Justine, 2002; Yushin & Malakhov, several unique features (Justine & Jamieson, 1999; 2004, 2014). The MO are derived from the Golgi Justine, 2002; Yushin & Malakhov, 2004, 2014). In bodies and develop as a part of the complexes with the classification based on morphological and paracrystalline fibrous bodies (FB) – another unique molecular data proposed by De Ley & Blaxter component of developing sperm. The prism-shaped (2002), the phylum Nematoda is subdivided into FB are composed of densely packed parallel two classes: Enoplea and Chromadorea. The filaments consisting of the unique cytoskeleton structure and development of nematode sperm have protein MSP (‘major sperm protein’) (Justine & been studied mainly for representatives of the Jamieson, 1999; Justine, 2002; Chu & Shakes, 2013; extensive order Rhabditida belonging to Yushin et al., 2016). The outlined basic pattern of Chromadorea (Justine & Jamieson, 1999; Justine, sperm structure and development is characteristic 2002). Most species studied within Rhabditida for representatives of the class Chromadorea, produce relatively uniform sperm of the ‘rhabditid especially for the well-studied order Rhabditida pattern’ (Yushin & Malakhov, 2014). This type of (Justine & Jamieson, 1999; Justine, 2002; Yushin & nematode spermatozoon is seen as an amoeboid Malakhov, 2004, 2014; Zograf, 2014; Slos et al., bipolar cell with an anterior pseudopod and 2015). However, the nematode order Rhabditida and posterior main cell body, which includes a other orders of Chromadorea include taxa for which condensed nucleus without a nuclear envelope, sperm development and structure have distinct mitochondria and so-called ‘membranous deviations from the ‘rhabditid pattern’ (Justine & organelles’ (MO), the unique organelles Jamieson, 1999; Yushin & Spiridonov, 2001;

99 Zograf J. K. et al. Justine, 2002; Yushin & Malakhov, 2004, 2014). observations, the male spermatogenic cells are Developing male gametes have a large number of usually mentioned briefly as large spermatocytes specific cytological characters that can be compared and small globular spermatozoa (Timm, 1970). and analysed for in depth discussion of metazoan The nematode species, D. granulatus Decraemer, taxonomy and phylogeny (Baccetti, 1985; Jamieson 1975 belonging to the genus Desmoscolex et al., 1995; Liana & Witalinski, 2005; Pitnick et al., Claparède, 1863 (Desmoscolecini, 2009; Levron et al., 2010; Dallai et al., 2016). It Desmoscolecidae), was chosen for the first was hypothesised that the basic spermatozoon ultrastructural study of the desmoscolecid features also reflect position of nematode taxa on spermatozoon to perform comparative analysis with the nematode phylogenetic tree (Yushin & spermatozoa of other nematodes. The ultrastructure Malakhov, 2004, 2014). of immature spermatozoa from the testes of Species of Desmoscolex and other Desmoscolex has been studied in details. Desmocolecini are distinguished among marine nematode taxa by their peculiar appearance as well MATERIAL AND METHODS as by some unusual traits in their fine morphology and biology. The body cuticle consists of broad and Samples were collected in the North Vietnam, convex main rings with thin and flexible inter-zones Tien Yen Estuary (21°18'997" N; 107°36'075" E) in between them. The main rings are covered with so- April 2015. Bottom sediments were fine silty sands, called desmen composed of angular concretions and water depths of 0.5 m. Benthic meiofauna was sampled using cores of 3.6 cm inner diameter (Fig. 1). The latter are not derived from secretions 2 but comprise sticky clay mineral platelets and sand (surface of 10 cm ). The samples were washed grains (Riemann & Riemann, 2010; Decraemer & through 1 mm and 40 μm sieves. Rho, 2014; personal data of A.V. Tchesunov). The Live nematodes belonging to the genus compact head bears four pedunculated cephalic Desmoscolex were picked out from the samples setae and large blister-like amphideal fovea, the under a stereoscopic microscope. For light amphids being covered with thin cuticle (personal microscopy, nematodes were fixed with 4% data of A.V. Tchesunov). Other peculiar features are paraformaldehyde and transferred to glycerin using so-called phasmata (lateral circular pores on the end the Seinhorst’s (1959) rapid method as modified by ring of the tail) and pigment spots at the level of the De Grisse (1969), and mounted on permanent slides. anterior intestine. Females have two opposed Difficulties in identification of desmoscolecid outstretched ovaries. In relation to reproductive species required detailed description of specimens biology, an unusual feature was mentioned for some collected for this study. The male used for TEM had desmoscolecides (Timm, 1970): females can carry the same general morphology as the female. their developing eggs attached to the body, either For transmission electron microscopy (TEM) the glued to the tail (Tricoma) or pressed to the ventral head and tail regions of each animal were cut off for body side by elongate setae (Desmoscolex). Males facilitation of following tissue fixation and have either two opposed testes (Tricominae) or only embedding. The specimens were fixed for TEM at 4ºC in 2.5% glutaraldehyde in 0.05 M cacodylate a single anterior outstretched testis (Desmo- –1 scolecinae). buffer containing 21 mg ml NaCl overnight and then post-fixed 2 h in 1% osmium tetroxide in the The phylogenetic position of Desmoscolecida –1 among Nematoda as well as the classification within same buffer containing 23 mg ml NaCl. Post- the order is still under discussion (Decraemer & fixation was followed by en bloc staining for 2 h in Rho, 2014). Hwang et al. (2009) provided 1% solution of uranyl acetate in distilled water; then nucleotide sequences of 18S rDNA for the specimens were dehydrated in ethanol followed representatives of four desmoscolecid genera by isopropanol series and embedded in Epon resin. (Desmoscolex, Greeffiella, Tricoma and Embedded animals were cut longitudinally by glass Paratricoma) and came to the conclusion that those knives to remove thick cuticle and obtain blocks, genera form a clear clade that could be treated as where internal tissues appear on semi-thin sections. sister group of Monhysterida (including The clay particles covering the cuticle surface, Comesomatidae). New morphological data on male which make thin sectioning impossible, were gametes may assist in resolving problems in dissolved by overnight incubation of blocks in 2% nematode relationships, including the position of the solution of hydrofluoric acid (HF) in distilled water order Desmoscolecida within the phylum. The fine with 10% acetone at room temperature. After structure of male gametes in Desmoscolecida has washing in distilled water, the blocks were not been studied. In light microscopical thoroughly dried overnight in an oven at 60°C.

100 Spermatozoa of Desmoscolecida Thin sections cut with a diamond knife using vesicle of the gravid male was studied. The Leica UC6 ultratome were stained with lead citrate spermatozoa from testis are termed as ‘immature and examined with a JEOL JEM 100S and JEOL spermatozoa’ following the basic terminology JEM 1010 transmission electron microscopes. The proposed by Shepherd (1981). ultrastructure of spermatozoa filling the seminal

Fig. 1. Desmoscolex granulatus, female. A. Entire. B. Anterior body. C. Posterior body. Scale bars: A – 100 μm; B & C – 20 μm.

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Fig. 2. Desmoscolex granulatus, TEM. Longitudinal section through the testis with densely packed immature spermatozoa, general view at low magnification. Abbreviations: fp – filopodia; N – nucleus; Sp – spermatozoon; tw – testis wall. Scale bar: 5 µm.

coarsely annulated. Body length 325 μm, a = 5.9, b RESULTS = 5.08, c = 5.0, c’ = 1.48. Main rings 18 in number, Taxonomy. Since the species identification can covered by broad desmen composed of large angular be questionable without proper illustration, because concretions making the body opaque. At mid-body, of this new finding in a region remote from the type body diam. 55 μm including desmen and 47 μm locality we consider it justifiable to provide a without desmen. Inter-zones hardly discernible, also redescription. partly covered by smaller concretions. Desmoscolex granulatus Decraemer, 1975 Head rounded, wider than long (head length 15 μm, width 22 μm). Four cephalic setae rather long Material. One adult female (Fig. 1). (16 μm) and jointed, basal joint longer and stiff, Locality. North Vietnam, Tien Yen Estuary distal joint short and thin (one fourth to third of the (21°18'997" N; 107°36'075" E). Silty sand, 0.5 m entire setae length). Amphideal fovea blister-like, deep. April 2015. wider than long (amphid length 7 μm, width 13 μm) Description and measurements. Body spindle- and situated close to the cephalic apex. shaped, tapered to both ends and body cuticle Somatic subdorsal setae much longer than

102 Spermatozoa of Desmoscolecida subventral, jointed, basal joint long and stiff, distal spermatozoan nuclei look like discrete dense joint short (about one third of the entire setae), particles but observations of many spermatozoa lanceolate. Subventral setae short and smooth (not from the successive serial sections demonstrate that jointed). Length of subdorsal somatic setae: 1st – 20 each nucleus is a highly lobated mass of strongly μm, 2nd – 16 μm, 3d – 15 μm, last but one – 20 μm, condensed nuclear chromatin with sharp boundaries terminal – 26 μm. Length of 1st subventral somatic devoid of a nuclear envelope (Figs 2; 4A & B). setae – 6.9 μm. All the somatic setae located in strictly bilateral pairs on the main rings. Position of subdorsal somatic setae on the main rings: 1, 3, 5, 7, 9, 11, 13, 17, 18 = 9. Position of subventral somatic setae on the main rings: 2, 4, 6, 8, 10, 12, 14, 16 = 8. Yellow ocelli at the level of 2nd main ring. Pharynx not discernible. Vulva not found. Internal reproductive organs not discernible. Anal tube 6 μm long, covered with small concretions. Tail consists of two main rings. Terminal ring nearly rectangular and stout (length 41 μm, basal width 21 μm). Terminal tube (spinneret) not developed. Posteriormost subdorsal setae attached at two thirds of the terminal ring close to its posterior end. Phasmata not observed. Remarks. The species is characterised by body composed of 18 and tail of two main rings covered Fig. 3. Desmoscolex granulatus, schematic by desmen, vesicular amphideal fovea situated representation of the immature spermatozoon structure. anteriorly on the head, jointed cephalic setae with Lobated nucleus (N) without nuclear envelope is thin distal part and jointed subdorsal somatic setae surrounded by fibrous bodies (fb) and mitochondria (mc). with lanceolate distal part. These features are shared The surface of the spermatozoon bears numerous with D. granulatus Decraemer, 1975 and D. filopodia (fp). Not to scale. membranosus Decraemer, 1974. Our specimen fits The cytoplasm of the immature spermatozoa with both species descriptions in all dimensions and contains only two types of components: structures except for the presence of dark red- mitochondria and bundles of filaments (Figs 4B; 5A brownish granulation at the level of pharynx and & B). Elongated mitochondria (0.6 µm long and 0.3 anterior intestine, not observed in our specimens. µm wide) with opaque matrix surrounds the nucleus Both D. granulatus and D. membranosus were area (2-3 µm in diam.) as a layer with narrow space found in the area of Great Barrier Reef at depths between organelles and chromatin. Mitochondria 21.5-35 m on sandy bottom or on sand covered with appear rarely between nuclear lobes and are totally silt layer (Decraemer, 1974, 1975). Our specimens absent at the cell periphery (Fig. 4B). are designated as D. granulatus because of lack of Electron dense bundles of filaments found in any circumoral membrane specific for D. spermatozoa of D. granulatus were similar to the membranosus. fibrous bodies (FB) characteristic of spermatogenic Ultrastructure. The testis of D. granulatus was cells of most nematodes studied. These FB are filled with uniform germ cells identified as the abundant, vary in size, and consist of tightly packed immature spermatozoa. No previous developmental parallel fibres (Figs 5A, B & 6). The FB are evenly stages such as spermatids or spermatocytes were distributed throughout the cytoplasm of observed. Immature spermatozoa form a cluster of spermatozoa, they appear in-between the nuclear tightly packed cells surrounded by testis epithelium lobes and fill the cell periphery around the (Fig. 2). They have irregular amoeboid outlines and mitochondrial layer (Figs 4B & 5A). form numerous filopodia, which are squeezed The cytoplasm of moderate density around the between the spermatozoan bodies. nucleus, mitochondria and FB is not homogenous The spermatozoa have more or less uniform but comprises apparently filamentous material structure along the testis (Figs 2 & 3). They are containing parallel orientated fibres ca 15-18 µm in unpolarized cells of average size ca 4-6 µm with diam. (Figs 5B & 6). The spermatozoon surface is central nucleus (Figs 2 & 4A). On the thin sections organised into 0.24-0.32 µm thick filopodia of variable

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Fig. 4. Immature spermatozoa from testis of Desmoscolex granulatus, TEM. A. Cluster of spermatozoa. B. Immature spermatozoon, general view. fb – fibrous bodies; for other abbreviations see legend for Fig. 2. Scale bars: 1 µm.

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Fig. 5. Immature spermatozoa from testis of Desmoscolex granulatus, TEM. A. Central part and periphery of the spermatozoon at high magnification. B. Central part, periphery and filopodia of the spermatozoa. ch – chromatin; for other abbreviations see legend for Fig. 2. Scale bars: 1 µm.

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Fig. 6. Periphery of the immature spermatozoon of Desmoscolex granulatus, TEM, high magnification. Insert: enlargement of cross section through the filopodium showing tubule-like fibres (arrows) and complicated structure of cell wall. For abbreviations see legend for Fig. 2. Scale bars: 0.5 µm; insert – 0.25 µm. length and shape (Figs 2; 4A & 5B). The cytoplasm DISCUSSION of filopodia is a continuation of the sperm The immature spermatozoa of D. granulatus cytoplasm and also contains characteristic parallel have the basic ultrastructural features of the sperm fibres (Figs 5B & 6). These fibres are strongly cells of many nematodes studied so far: they lack an orientated parallel to a long axis of a filopodium axoneme, an acrosome and a nuclear envelope forming well arranged fascicles apparent on cross (Justine & Jamieson, 1999; Justine, 2002). In sections through a filopodium (Fig. 6). On cross general, these are unpolarized cells with a highly sections of filopodia and sections through the cell lobated nucleus surrounded by a layer of periphery fibres look like tubules (Fig. 6, insert). mitochondria and numerous fibrous bodies looking The sperm cell membrane is covered by thin surface like bundles of filaments (Fig. 3). The numerous coat and reinforced from the inside with the thick dense well developed filopodia are characteristic of internal layer. This 30 nm thick unit membrane complex spermatozoa. looks like enormously thick envelope bordering of cell Desmoscolecida is a well defined group of and filopodia (Figs 5B & 6). nematodes considered by most authors since No membranous organelles which are characteristic Filipjev (1929) as a separate order or sometimes as a of most nematode spermatozoa were observed in suborder within the chromadorean clade of immature spermatozoa of D. granulatus. Nematoda. Lorenzen (1981) in the first German

106 Spermatozoa of Desmoscolecida edition of his influential book put desmoscolecids pugettensis), Selachinematidae (Halichoanolaimus (as Desmoscolecoidea) in the Monhysterida because spp.) as well as in the order Desmodorida of the outstretched ovaries. In the second English (Desmodoridae, Metachromadora itoi), edition (Lorenzen, 1994) Desmoscolecina are placed Monhysterida (Linhomoeidae, Paralinhomoeus sp., by him in Chromadorida based on the opinion that Terschellingia glabricutis) and Rhabditida outstretched ovaries might have developed from (Tylenchoidea) (Yushin & Coomans, 2000, 2005; antidromously reflexed ovaries. According to Justine, 2002; Yushin & Zograf, 2002, 2004; Zograf analysis of nucleotide sequences of 18S rDNA & Yushin, 2004; Zograf et al., 2004; Yushin, 2003, (Hwang et al., 2009) of species from four 2007, 2008; Yushin & Malakhov, 2014). Unlike D. desmoscolecid genera, Desmoscolecida forms a granulatus, the FB in immature spermatozoa of monophyletic group positioned as a sister group of these nematodes look more developed, appearing as the clade including members of Monhysterida and large amorphous or paracrystalline bodies. Araeolaimida (both orders are characterised Numerous well developed filopodia are another morphologically by outstretched ovaries). Now the distinct morphological feature of the immature order Desmoscolecida is placed within the subclass spermatozoa of D. granulatus. Filopodia have been Chromadoria of the class Chromadorea (De Ley & observed in spermatogenic cells of many nematodes Blaxter, 2004; Hodda, 2007). From the from distant taxa of nematodes from both classes morphological point of view, the position of (Riemann, 1983; Justine & Jamieson, 1999; Justine, Desmoscolecida within class Chromadorea and 2002; Yushin & Zograf, 2004; Zograf et al., 2004; subclass Chromadoria is completely justified but the 2008; Zograf & Yushin, 2004; Yushin, 2003, 2007, relationships of Desmoscolecida to either 2008, 2010; Lak et al., 2015; Yushin et al., 2016). Monhysterida or Chromadorida, or Plectida is still Observation of D. granulatus spermatozoa confirms subject to debate (Decraemer & Rho, 2014). What the wide distribution of filopodia and their can sperm structure say on this topic? importance in development and physiology of The main pattern of spermatogenesis in the nematode male gametes. nematode class Chromadorea is marked by The cytoplasm of immature spermatozoa of D. development of specific organelles, MO and FB as granulatus contains characteristic tubule-like fibres the FB-MO complexes (Yushin & Malakhov, 2004, arranging as a fascicle inside filopodia. Similar 2014) This ‘rhabditid pattern’ was described for a fibres have been observed earlier in the spermatozoa variety of representatives of the order Rhabditida of many nematodes representing very distant taxa (Spiruromorpha, Ascaridomorpha, Panagrolaimo- from both classes of the phylum, Enoplea and morpha, Tylenchomorpha, Diplogasteromorpha, Chromadorea (Beams & Sekhon, 1972; Shepherd et Rhabditomorpha, and Myolaimina), as well as for the al., 1973; Baccetti et al., 1983; Shepherd & Clark, aquatic nematodes of the orders Monhysterida 1983; Hess & Poinar, 1989; Poinar & Hess-Poinar, (Monhysteroidea), Araeolaimida and Plectida (Justine 1993; Cares & Baldwin, 1994, 1995; Takahashi et & Jamieson, 1999; Justine, 2002; Giblin-Davis et al., al., 1994; Endo et al., 1998; Turpeenniemi, 1998; 2010; Yushin & Malakhov, 2004, 2014; Zograf, 2014; Yushin, 2004, 2007, 2008, 2010; Yushin & Zograf, Slos et al., 2015; Limantseva et al., 2015). 2004; Zograf et al., 2004). These fibres The second pattern is characterised by absence of (microtubule-like fibres, MLF) resemble the MO, while free FB are well developed and possibly cytoskeleton microtubules of Metazoa, but they have the same nature as the FB of other nematodes. have a diameter 13 to 20 nm (15-18 nm in D. This pattern was described in some Rhabditida and granulatus) and cannot be identified as classic free-living marine nematodes from the orders tubulin-containing microtubules, which have a Chromadorida, Desmodorida and Monhysterida normal diameter of 24-25 nm (Stephens & Edds, (Justine & Jamieson, 1999; Justine, 2002; Yushin & 1976). Moreover, it was shown unequivocally that Malakhov, 2004, 2014). In some cases of microtubules and tubulin are absent in nematode simplification complete reduction of aberrant sperm spermatozoa, except the centrioles (if present) and components have also been observed (Justine, 2002; their derivates (Mansir & Justine, 1998). The Yushin & Malakhov, 2004, 2014). prevalent cytoskeleton protein MSP is the base for Spermatozoa of D. granulatus having FB but cell structure and movement (Justine, 2002; Yushin devoid of MO fit with the second pattern of et al., 2016). It is likely that the MLF in spermatozoon structure of Chromadorea, which was spermatozoa of D. granulatus and other nematodes described in three families of the order are assembled from the MSP-based filaments. The Chromadorida – Chromadoridae (Neochromadora MLF fascicles apparently serve as an axial skeleton poecilosoma), Cyatholaimidae (Paracyatholaimus for the sperm filopodia of D. granulatus.

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The spermatozoon pattern with occurrence of the CHU, D.S. & SHAKES, D.C. 2013 Spermatogenesis. In: FB and absence of MO unites D. granulatus with Germ Cell Development in C. elegans. Advances in some chromadorids, desmodorids, monhysterids and Experimental Medicine and Biology, Volume 757 (T. rhabditids (Tylenchoidea). This conclusion is Schedl Ed.). pp. 171-203. New York, USA, Springer supported by the filopodial nature of the sperm Science + Business Media B.V. surface and abundance of MLF demonstrated by CLAPARÈDE, E. 1863. Beobachtungen über die Anatomie these taxa. und Entwicklungsgeschichte wirbelloser Tiere an der Küste der Normandie angestellt. Germany, Wilhelm ACKNOWLEDGEMENTS Engelmann. 120 pp. DALLAI, R., GOTTARDO, M. & BEUTEL, R.G. 2016. The field work and sample collection by the Structure and evolution of insect sperm: new bilateral research team were supported by Vietnam interpretations in the age of phylogenomics. Annual Academy of Science and Technology funding with Review of Entomology 61: 1-23. code VAST.HTQT.NGA.09/15-16. Drs V.V. DECRAEMER, W. 1974. Scientific report on the Belgian Yushin and J.K. Zograf were supported in part by expedition to the Great Barrier Reef in 1967. the Russian Science Foundation for the Far Eastern Nematodes V: observations on Desmoscolex Federal University (project no. 14-50-00034: TEM (Nematoda, Desmoscolecida) with descriptions of observations and analysis), RFBR (project no. 14- three new species. Zoologica Scripta 3: 243-255. 04-00334: specimen preparation for TEM DECRAEMER, W. 1975. Scientific report on the Belgian observations), FEB RAS (project no. 15-I-6-109o: expedition to the Great Barrier Reef in 1967. article preparation). Contribution of A.V. Nematodes I: Desmoscolex-species (Nematoda – Tchesunov (identification and description of the Desmoscolecida) from Yonge Reef, Lizard Island and desmoscolecid species, writing specific parts of Nymphe Island with general characteristics of the introduction and discussion) is supported by RFBR genus Desmoscolex. Annales de la Sociétè Royale grants 12-04-00781-a, 15-04-02597 and RSF grant Zoologique de Belgique 104: 105-130. 14-50-00029. The authors are grateful to D.V. DECRAEMER, W. & RHO, H.S. 2014. Order Fomin (Far East Centre of Electron Microscopy, Desmoscolecida. In: Handbook of Zoology. Institute of Marine Biology, Vladivostok, Russia) Gastrotricha, Cycloneuralia and Gnatifera. Volume and Myriam Claeys (Ghent University, Belgium) for 2: Nematoda (A. Schmidt-Rhaesa Ed.). pp. 351-372. technical assistance. Berlin/Boston, Germany/USA, Walter de Gruyter The authors thank Reviewers and Editor for GmbH. critical remarks which have helped us to improve DE GRISSE, A.T. 1969. Redescription ou modifications de the manuscript. quelques techniques utilisées dans l’étude des nématodes REFERENCES phytoparasitaires. Mededelingen van de Rijksfakulteit Landbouwwetenschappen Gent 34: 351-369. BACCETTI, B. 1985. Evolution of the sperm cell. In: DE LEY, P. & BLAXTER, M. 2002. Systematic Position and Biology of Fertilization, Volume 2: Biology of the Phylogeny. In: The Biology of Nematodes (D.L. Lee Ed.). Sperm (C.B. Metz & A. Monroy Eds). pp. 3-58. pp. 1-30. London, UK, Taylor & Francis Group. Orlando, USA, Academic Press. DE LEY, P. & BLAXTER, M. 2004. A new system for BACCETTI, B., DALLAI, R., GRIMALDI DE ZIO, S. & Nematoda: combining morphological characters with MARINARI, A. 1983. The evolution of the nematode molecular trees, and translating clades into ranks and spermatozoon. Gamete Research 8: 309-323. taxa. In: Nematology Monographs and Perspectives, BEAMS, H.W. & SEKHON, S.S. 1972. Cytodifferentiation Volume 2 (R. Cook & D.J. Hunt Eds). pp. 633-653. during spermatogenesis in Rhabditis pellio. Journal of Leiden, The Netherlands, E.J. Brill. Ultrastructure Research 38: 511-527. ENDO, B.Y., ZUNKE, U & WERGIN, W.P. 1998. CARES, J.E. & BALDWIN, J.G. 1994. Comparative fine Spermatogenesis in the lesion nematode, structure of sperm of Verutus volvingentis and Pratylenchus penetrans (Nemata, Pratylechidae). Meloidodera floridensis (Heteroderinae, Nematoda). Journal of the Helmintological Society of Washington Canadian Journal of Zoology 72: 1481-1491. 65: 227-242. CARES, J.E. & BALDWIN, J.G. 1995. Comparative fine FILIPJEV, I.N. 1929. Classification of freeliving structure of sperm Heterodera schachtii and Nematoda and relations to parasitic forms. Journal of Punctodera chalcoensis, with phylogenetic Parasitology 15: 281-282. implications for Heteroderinae (Nemata: GIBLIN-DAVIS, R.M., KANZAKI, N., DE LEY, P., Heteroderidae). Canadian Journal of Zoology 73: WILLIAMS, D.S., SCHIERENBERG, E., RAGSDALE, E.J., 309-320. ZENG, Y. & CENTER, B.J. 2010. Ultrastructure and life

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Zograf, J. K., Nguyen Dinh Tu, Nguyen Thi Xuan Phuong, Cao Van Luong, A. V. Tchesunov, V. V. Yushin. Первое ультраструктурное исследование сперматозоидов десмосколецид (Nematoda: Desmoscolecida: Desmoscolecidae). Резюме. Проведено электронно-микроскопическое изучение морских нематол Desmoscolex granulatus (Desmoscolecida). Сперматозоид представляет собой неполяризованную клетку с многочисленными филоподиями, с центральным многолопастным ядром, лишенным ядерной оболочки. В цитоплазме имеются митохондрии и фиброзные тела (FB). В сперматозоидах D. granulatus отсутствуют мембранные органеллы (MO), характерные для многих нематод. По особенностям строения сперматозоидов, в которых FB не ассоциировано с MO, D. granulatus близок к некоторым хромадоридам, десмодоридам (Desmodoridae), монхистеридам (Linhomoeidae) и тиленхоморфным нематодам (Tylenchoidea). В пользу такого заключения говорит и структура филоподий этих нематод.

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