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20Russian Journal of Nematology, 2010, 18 (2), 199 -208
Spermatid and spermatozoon structure in freshwater nematode Paractinolaimus microdentatus (Nematoda: Dorylaimida: Actinolaimidae)
Vladimir V. Yushin
A.V. Zhirmunsky Institute of Marine Biology FEB RAS, Vladivostok 690041, Russia e-mail: [email protected]
Accepted for publication 12 September 2010
Summary. The nematode spermatozoa have been studied in many nematode taxa including the order Dorylaimida. However, all information on the dorylamid spermatozoa is based on the representatives of only one genus, Xiphinema (Longidoridae). The structure of spermatids and spermatozoa from male gonads of the fresh-water dorylaimid Paractinolaimus microdentatus (Actinolaimidae) were studied using transmission electron microscopy (TEM). An unusual spindle-shaped complex of nucleus with mitochondria is characteristic for spermatids and immature spermatozoa. The complex contains the nucleus consisting of intertwining threads of chromatin without a nuclear envelope, a monolayer of mitochondria with dense matrix, and a fibre layer which encloses the mitochondria; in P. microdentatus the fibre layer has a distinct annulated structure. The peripheral cytoplasm of the spermatid contains large amount of filamentous matter filling prominent pseudopods. The immature spermatozoa are unpolarised 8-10 ^m long and 2 ^m wide cells with filopodia covering the cell surface. Each spermatozoon contains the central complex of nucleus and mitochondria, which is surrounded by electron-light cytoplasm containing numerous bundles of filaments and microtubule-like fibres 16-18 nm in diameter. No inclusions resembling membranous organelles specific of other nematode spermatozoa were observed in the male germ cells of P. microdentatus. The spermatozoa of the dorylaimids from two families, Longidoridae and Actinolaimidae show close resemblance both in structure and development. The spermatozoa of the dorylaimids studied have very special internal structure, which differentiates them from other nematode spermatozoa and may be considered as the distinct character of the order.
Key words: fibrous bodies, Longidoridae, membranous organelles, MSP, spermatogenesis, ultrastructure,
Xiphinema.
Spermatozoon morphology and development have potential to be used in taxonomic and phylogenetic analysis as clear and easily comparable morphological characters (Baccetti, 1985; Jamieson et al, 1995). The nematode spermatozoa and spermatogenesis were studied in several species belonging to many orders of the phylum. Nematode spermatozoa represent an aberrant type of male gamete; they are characterised by the absence of an axoneme and an acrosome (Justine & Jamieson, 1999; Justine, 2002). The basic type of nematode spermatozoon is an amoeboid bipolar cell with an anterior pseudopod and posterior main cell body which includes a condensed nucleus lacking a nuclear envelope, mitochondria and so called 'membranous organelles' (MO). These are unique, aberrant organelles characteristic of developing and mature spermatozoa of many nematodes studied.
Usually, MO look like large (0.5-1.0 ^.m diam.) vesicles with dense content and a system of internal finger-like projections of the outer membrane. The MO are derived from the Golgi bodies and appear as a part of the complexes with paracrystalline fibrous bodies (FB) - another special component of developing spermatozoa. The prism-shaped FB are composed of densely packed parallel fibres consisting of the unique cytoskeleton protein MSP ('major sperm protein'). The complexes of FB and MO ('FB-MO complexes') during late stages of spermatogenesis dissociate into separate FB and MO. After sperm activation inside the female gonoduct, MO join to the spermatozoon plasmalemma and release their content into the uterus lumen. The empty MO, looking like membranous sacs continuous with plasmalemma, are retained as a constant feature of the mature
spermatozoa. Sperm activation is also accompanied by transformation of FB into the MSP-based cytoskeleton of a newly formed pseudopod. The pseudopod and amoeboid movement are well known special characteristics of nematode spermatozoa.
The Nematoda constitutes one of the most diverse and abundant phyla of Metazoa. In the classification based on morphological and molecular data proposed by De Ley and Blaxter (2002), the phylum is subdivided into two classes: Enoplea and Chromadorea. The outlined basic pattern of sperm structure and development is characteristic for representatives of the class Chromadorea, especially for the well studied order Rhabditida (Justine & Jamieson, 1999; Justine, 2002; Yushin & Malakhov, 2004). The class Enoplea comprises two subclasses, Enoplia and Dorylaimia, each with several orders in which spermatozoa are more diverse in structure than in Chromadorea and cannot be clearly defined by a common pattern possibly because many taxa have been studied less extensively (Justine & Jamieson, 1999; Justine, 2002; Yushin & Malakhov, 2004). A unique type of spermatozoa was described also for the order Dorylaimida (subclass Dorylaimia) but using representatives of only one family Longidoridae, namely several plant-parasitic species from the genus Xiphinema (Baccetti et al, 1983; Kruger, 1991; Van de Velde et al., 1991; Bleve-Zacheo et al. 1993). The spermatozoa of the species studied have no specific membranous organelles and anterior pseudopod, the FB-MO complexes and free FB with paracrystalline structure also have not been detected in spermatogeneous cells (Kruger, 1991). The general structure of the Xiphinema spermatozoa is unusual and represents a pattern that separates the dorylaimids from other nematodes (Justine & Jamieson, 1999). However, this large order includes three suborders with 16 families; it seems that the verification of the spermatozoon uniqueness together with its specificity for the whole order remains to be confirmed.
The fresh-water dorylaimid from the family Actinolaimidae, Paractinolaimus microdentatus (Thorne, 1939) Meyl, 1957, was chosen for a new study to elucidate all the structural details of the dorylaimid spermatozoon and to perform comparative analysis with other nematode sperm. The ultrastructure of spermatids and spermatozoa from the testes of P. microdentatus were studied.
MATERIAL AND METHODS
Paractinolaimus microdentatus nematodes were extracted from the bottom mud collected in the swampy area created by natural spring in the
vicinity of the Marine Biological Station "Vostok" of the Institute of Marine Biology (Vostok Bay, Sea of Japan). Live males were cut in glutaraldehyde fixative to obtain a piece containing both testes and then each specimen was cut into halves. The specimens were fixed for transmission electron microscopy (TEM) at 4°C in 2.5% glutaraldehyde in 0.05 M cacodylate buffer (pH 7.4) with 0.25 mg ml-1 MgCl2, and were postfixed in 2% osmium tetroxide in the same buffer. Postfixation was followed by en bloc staining for 2 h in a 1% solution of uranyl acetate in distilled water and then the specimens were dehydrated in ethanol followed by an isopropanol series and embedded in Spurr resin. Thin sections, cut with a Leica UC6 ultratome and stained with uranyl acetate and lead citrate, were examined with a JEOL JEM 100S and JEOL JEM 2000EX electron microscopes. For low magnification panoramic views of sections the scanning transmission electron microscopy (STEM) was used with Zeiss Libra 120 electron microscope.
The testes of two males were examined for the present paper; the structure of spermatids and spermatozoa was studied. The end of the spermatid stage is marked by detachment of the residual body; sperm from testes and activated sperm from the uteri are termed as 'immature' and 'mature' spermatozoa, respectively (following Shepherd, 1981).
RESULTS
The male reproductive system of P. microdentatus comprises two opposite testes each as a simple epithelial tube filled with germ cells. The slightly dilated proximal half of each testis studied was filled with immature spermatozoa of uniform structure; distally the prominent zone of spermatids was observed (Fig. 1A).
The spermatids vary in shape but usually are elongated cells 7-8 ^m long * 2-3 ^m wide (Fig. 1B, 2A, B). Each spermatid has a characteristic arrangement of cell components: the opaque central complex of nucleus with mitochondria is surrounded by prominent peripheral cytoplasm; the cytoplasm forms large pseudopods devoid of organelles and filled with fibrous matter of a moderate density (Fig 1A, B, 2A, B). The central complex contains the nucleus surrounded by mitochondria (Fig. 2A, B). The nucleus is devoid of a nuclear envelope and consists of intertwining threads of condensed chromatin looking like separate chromatin particles on thin sections. Chromatin threads are embedded into the pale filamentous matter and are surrounded by a continuous monolayer of mitochondria with electron-dense matrix (Fig. 2A, B; 3A). The shape
Fig. 1. Paractinolaimus microdentatus, spermiogenesis. A: Spermatids (St) and immature spermatozoa (Is) on the longitudinal section through the testis; note the sharp border between two cell clusters (arrows). STEM. B: Two spermatids cut longitudinally. TEM. The central opaque complex of nucleus with mitochondria (CC) is surrounded by pseudopods (Ps). Scale bars: A = 2 ^m; B = 1 ^m.
Fig. 2. Paractinolaimus microdentatus, spermatids. TEM. A: Longitudinal section through the central complex of the spermatid; the fibrous capsule enclosing the central complex (arrows) has characteristic striations. B: Cross section through the spermatid. Scale bars: 1 ^m. Abbreviations: ch = threads of condensed nuclear chromatin; fp = filopodia; mc = mitochondria; Ps = pseudopod.
Fig. 3. Paractinolaimus microdentatus, spermiogenesis. TEM. A: Tangential section through the spermatid went through the mitochondrion layer and enclosing fibrous capsule (fc); the capsule has characteristic striations. B: Immature spermatozoa, longitudinal and cross sections; the central complex of nucleus (N) with mitochondria (mc) is surrounded by pale cytoplasm and filopodia (fp). Arrows show bundles of fibres. Scale bars: A = 0.5 ^m; B = 1 ^m. Abbreviations: fc = fibrous capsule; fp = filopodia; mc = mitochondria; Ps = pseudopod.
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Fig. 4. Paractinolaimus microdentatus, immature spermatozoa. TEM. A: Central complex of nucleus (N) with mitochondria (mc); enclosing fibrous capsule (fc) has characteristic striations. Arrows show bundles of fibres. B: The sperm central complex and peripheral cytoplasm; arowheads show osmiophilic particles under plasmalemma. Note parallel arrays of fibres inside the filopodia. Scale bars: 0.5 ^m. Abbreviations: ch = threads of condensed nuclear chromatin; fc = fibrous capsule; fp = filopodia; mc = mitochondria.
of mitochondria is variable, they may curve and branch; the system of lamellar cristae is well developed. The nucleus-mitochondria cluster is enclosed by a 0.12 ^m thick capsule of dense fibrous layer of annulate structure (Fig. 2A, 3A). Regular interchange of 0.1 ^m wide fibrous rings and 25 nm wide dense rings, like barrel bands,
isolate the central complex from surrounding filamentous cytoplasm. Peripheral cytoplasm is arranged as prominent pseudopods and numerous thin filopodia (Fig. 1B; 2A, B; 3A). The spermatid plasmallemma is underlain by osmiphilic particles.
Each spermatid is connected by cytoplasmic bridge to a residual body accommodating the
synthetic apparatus of the cell (ribosomes, rough endoplasmic reticulum, Golgi bodies). The residual body detaches close to the zone of immature spermatozoa. The border between spermatids and immature spermatozoa is sharp: cluster of spermatids with lobate pseudopodia is followed by tightly packed mass of spermatozoa with more transparent cytoplasm and thick filopodia (Fig. 1A, 3B). The spermatozoa are tightly packed, their filopodia are interdigitated and separated by only a 50 nm wide space (Fig. 5 A, B). The spermatozoa vary in outline but in many cases these are spindle-shaped cells 8-10 ^m long and 2 ^m wide (Fig. 3B). The characteristic complex of nucleus with mitochondria observed in the spermatid also persists in the immature spermatozoa (Fig. 3B, 4A). The mitochondria with dense matrix assume more or less uniform shape and are separated by pale fibrous matter. The fibrous capsule consisting of fibrous and dense rings encloses the central complex (4A, B). The peripheral cytoplasm and filopodia contain randomly orientated bundles of fibres (3B, 4A). The cytoplasm is also filled with parallel arrays of 16-18 nm thick fibres which run longitudinally inside the filopodia (Fig. 4B, 5A); each fibre has a transparent core and looks like a microtubule (Fig. 5B, C). The spermatozoon plasmalemma is underlain by an electron dense 30 nm thick layer separated from the plasmalemma by a narrow light space (Fig. 5A-C). The microtubule-like fibres are in close contact with this layer. Osmiophilic particles occur under the plasmalemma of filopodia (Fig. 4B).
DISCUSSION
The dorylaimid spermatozoa were observed with TEM in three plant-parasitic species belonging to the genus Xiphinema (Longidoridae). The immature and mature spermatozoa were observed in X. diversicaudatum (Baccetti et al, 1983; Bleve-Zacheo et al., 1993); mature stermatozoa were examined in details in X. theresiae and X. pinoides (Van de Velde et al., 1991); the only description of the dorylamid spermatogenesis from spermatocytes through immature spermatozoa was made on males ofX. theresiae (Kruger, 1991).
The nucleus and mitochondria in the immature and mature spermatozoa of Xiphinema spp. were observed as an opaque cluster surrounded by voluminous peripheral cytoplasm without organelles (Baccetti et al., 1983; Kruger, 1991; Van de Velde et al., 1991). The nucleus represented by clumps of chromatin embedded in transparent matrix is devoid of a nuclear envelope and is protected by a capsule made up of mitochondria enclosed by a fibrous layer (Baccetti et al., 1983; Kruger, 1991; Van de Velde
et al., 1991; Bleve-Zacheo et al., 1993). The peripheral cytoplasm is arranged as prominent pseudopods or filopodia filled with cytoskeletal components; it may contain 'bundles of microtubules' (Baccetti et al., 1983) or packets of dense fibres (Kruger, 1991; Van de Velde et al., 1991).
In general, details of the spermatozoon structure in P. microdentatus and Xiphinema are the same. The nucleus of P. microdentatus spermatozoa is formed of clumps of chromatin isolated from the peripheral cytoplasm by a capsule made up of a tightly packed monolayer of mitochondria and a distinct external fibrous layer. In P. microdentatus the external fibrous layer covering the mitochondria has a distinct annulate structure, which possibly reflects a structural function of the capsule analogous to barrel bands. This characteristic central complex is well developed in spermatids and then persists in immature and mature spermatozoa up to fertilisation (Kruger, 1991; Van de Velde et al., 1991; Bleve-Zacheo et al, 1993). It seems that the complex is an original and easily recognisable character of the dorylaimid spermatozoa; the less opaque and organised sperm structure where mitochondria encircle the nucleus was observed also in some other taxa from both classes of Nematoda (Justine & Jamieson, 1999; Justine, 2002; Yushin & Coomans, 2005; Yushin, 2008).
Voluminous peripheral cytoplasm as the base for formation of large pseudopods or filopodia also is characteristic for Xiphinema and P. microdentatus spermatozoa. The pseudopods or thick irregular filopodia provide no distinct polarity to spermatozoa even after activation (Van de Velde et al., 1991; Bleve-Zacheo et al., 1993). The peripheral cytoplasm contains bundles of fibres, which may be randomly or radially distributed.
The presence of bundles of microtubules was reported for immature spermatozoa of X. diversicaudatus (Baccetti et al., 1983). The peripheral cytoplasm and filopodia of P. microdentatus also contains arrays of fibres closely resembling the cytoskeleton microtubules of Metazoa (Fig 4B; 5A, B), but these microtubule-like fibres have a diameter of 16-18 nm and cannot be identified as classic tubulin-containing microtubules, which have a normal diameter of 2425 nm (Stephens & Edds, 1976). The microtubules and tubulin are absent in nematode spermatozoa (Justine, 2002), where the prevalent protein MSP is the base for structure and movement function of a pseudopod (Scott, 1996). The fibres morphologically resembling cytoskeleton microtubules (microtubule-like fibres) have been observed in spermatozoa of many nematodes
representing very distant taxa from both classes of the phylum, Enoplea and Chromadorea (Beams & Sekhon, 1972; Shepherd et al., 1973; Shepherd & Clark, 1983; Hess & Poinar, 1989; Poinar & Hess-Poinar, 1993; Cares & Baldwin, 1994, 1995; Takahashi et al, 1994; Endo et al, 1998; Turpeenniemi, 1998; Yushin, 2004, 2007, 2008; Yushin & Zograf, 2004; Zograf et al, 2004). The microtubule-like fibres observed in different species may be very abundant in order to fill the peripheral cytoplasm; they may also underlay the sperm plasmalemma or form a core of filopodia.
The microtubule-like fibres are distinctly less thick (13 to 20 nm, 16-18 in P. microdentatus) than the tubulin-containing microtubules (24-25 nm); this was reasonably interpreted by Turpeenniemi (1998) as the evidence of a difference in their protein nature. It is very likely that the microtubule-like fibres of nematode spermatozoa are assembled from the MSP-based filaments. However, there is no direct evidence of the presence of MSP in spermatozoa of the enoplean taxa; all the protein analyses were based on studies of several chromadorean species (Justine & Jamieson, 1999; Justine, 2002).
The only study of the dorylaimid spermatogenesis made on X. theresiae contains information on the presence of membranous organelles and fibrous bodies in spermatocytes. The membranous organelles were not detected in spermatids, whilst bundles of fibres persist in immature and mature spermatozoa (Kruger, 1991; Van de Velde et al., 1991). The bundles which were also observed in P. microdentatus (Fig. 3B, 4A) may be likened to the usual fibrous bodies containing cytoskeleton proteins, which are used for pseudopod arrangement in many nematodes especially in the rhabditids. Dissolving fibrous body sometimes looks like fibre bundles (Justine, 2002; Zograf et al., 2004; Yushin & Coomans, 2005).
The usual membranous organelles are abundant; they are considered as definitive characters of nematode spermatozoa which persist in spermatids and spermatozoa. However, the MO are undoubtedly absent in spermatids and spermatozoa of P. microdentatus and Xiphinema spp. This absence prevents the formation of well known FB-MO complexes; the components resembling FB, bundles of filaments, appear only at late stages of sperm development. This aberrant and possibly reduced pattern of sperm development and structure of the dorylaimids resembles many cases of MO absence known for distant taxa of Enoplea and Chromadorea (Justine, 2002; Yushin & Malakhov, 2004).
The spermatozoa of the nematode orders related to Dorylaimida (i.e. taxa united as subclass
Dorylaimia) are diverse and have no unequivocal resemblance with P. microdentatus and Xiphinema spp. spermatozoa. Possibly the most similar pattern was documented in the mononchid Mylonchulus nainitalensis (Mononchida, Mylonchulidae), where the central complex of nucleus with mitochondria is enclosed by a fibre layer; the spermatozoa bear characteristic filopodia (Baccetti et al., 1983). However, data on M. nainitalensis are poor and ambiguous; it seems that a full investigation of the mononchid spermatogenesis and sperm structure will be useful for more substantial analysis. The mature spermatozoa of another mononchid, Mononchus niddensis (Mononchidae), are small and simple; they have only a nucleus, several mitochondria and a pseudopod (Yushin, 2004). The data available on other dorylaimian orders show very diverse spermatozoa and spermatogeneses, which cannot be fruitfully compared with the dorylaimid spermatozoa (Justine, 2002). However, all these spermatozoa are characterised by the absence of distinct FB and presence of MO, whose structure and development differ greatly from membranous organelles of Chromadorea and Enoplea (Yushin & Malakhov, 2004).
Together with the study of P. microdentatus, the data on the dorylaimid spermatozoa are based on the representatives of two genera belonging to two families of the order, Longidoridae and Actinolaimidae. The uniform spermatozoon pattern, differing only in small details, was observed in both families, which recently have been placed into different superfamilies (Hodda, 2007). It may be hypothesised that this original and easily recognizable spermatozoon pattern is a cytological character of the whole order Dorylaimida.
CONCLUSIONS
The spermatozoa of the dorylaimids from two families, Longidoridae and Actinolaimidae show close resemblance both in structure and development. These are unpolarised cells with filopodia covering the cell surface. The central complex of the spermatozoa contains:
1 - nucleus consisting of intertwining threads of chromatin without a nuclear envelope;
2 - monolayer of mitochondria with dense matrix;
3 - specific fibre layer, which encloses the mitochondria. In P. microdentatus the fibre layer has an annulated structure.
The voluminous peripheral cytoplasm is devoid of organelles but is filled with components of the cytoskeleton, including bundles of filaments and microtubule-like fibres. No inclusions resembling membranous organelles of other nematode spermatozoa
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Fig. 5. Paractinolaimus microdentatus, immature spermatozoa. TEM. A: Filopodia filled with microtubule-like fibres; note the transverse cut fibres (arrows) and dense layer underlying the sperm plasmalemma. B: Interdigitating filopodia of neighbouring spermatozoa; the microtubule-like fibres fill the peripheral cytoplasm; note dense layer underlying the sperm plasmalemma. Asterisks show narrow space between neighbouring spermatozoa. C: The microtubule-like fibres (arrows) are in close contact with dense layer underlying the sperm plasmalemma (arrowhead). Scale bars: A, B = 0.5 ^m; C = 0.1 ^m.
were observed in the male germ cells of Xiphinema spp. and P. microdentatus. The spermatozoa of the dorylaimids studied have very special internal structure, which differentiates these spermatozoa from others and may be considered as the distinct character of the order.
ACKNOWLEDGEMENT
This research was supported in part by grants for V.V. Yushin: RFBR 08-04-00580, RFBR 09-0498540 and FEB RAS 09-III-A-06-216. The author is grateful to D.V. Fomin (Institute of Marine Biology, Vladivostok) for technical assistance.
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В.В. Юшин. Строение сперматид и сперматозоидов пресноводной нематоды Paractinolaimus microdentatus (Nematoda: Dorylaimida: Actinolaimidae).
Резюме. Сведения о сперматозоидах нематод отряда Dorylaimida ограничиваются данными по роду Xiphinema (Longidoridae). Изучено строение сперматид и сперматозоидов пресноводной дорилаймиды Paractinolaimus microdentatus (Actinolaimidae). В сперматидах и незрелых сперматозоидах описан веретеновидный ядерно-митохондриальный комплекс. Периферическая цитоплазма сперматид содержит волокнистый материал, наполняющий псевдоподии. Незрелые сперматозоиды - неполяризованные клетки размером 8-10 мкм х 2 мкм с центральным ядерно-митохондриальным комплексом и периферической цитоплазмой, содержащей пучки филаментов и микротрубочко-подобные волокна диаметром 16-18 нм.
