Comparative study of the female reproductive system within the Tylenchomorpha (Nematoda: Tylenchina)

Wim BERT, Ruben VAN GANSBEKE, Rita VAN DRIESSCHE, Myriam CLAEYS, Etienne GERAERT and Gaëtan BORGONIE

Ghent University, Department of Biology, Nematology section, Ghent, Belgiumwim.bert@UGent.be, ruben.vangansbeke@UGent.be, rita.vandriessche@UGent.be, myriam.claeys@UGent.be, etienne.geraert@UGent.be, gaetan.borgonie@UGent.be

The cellular gonoduct structure of 97 species belonging to the Tylenchomorpha and of eight species belonging to outgroups were studied to explore diagnostic applications of gonoduct characteristics and their use to assess phylogenetic relationships within the Tylenchomorpha. The morphology of the spermatheca has been shown to have particular value as a taxonomic character, while the cellular arrangement of oviduct and uterus seems to be a reliable phylogenetic marker. The gonoduct of the studied tylenchs (without Criconematoidea) displays important similarities: the oviduct consists of two rows of three to seven cells; the spermatheca comprises 10 to 20 cells and the uterus cells are arranged in three or four rows. The spermatheca of Meloidogyne is formed by a variable number of thick, lobe-like cells making it distinctive from any other known nematode genus. Ultrastructural data substantiate that the Meloidogyne spermatheca and oviduct are exceedingly complex structures and that their function is broader than a simple conduit for oocytes and receptacle for sperm. In the Criconematoidea, cells of the uterus-spermatheca complex are not arranged in distinct rows and a lumen is present. The gonoduct structures of the Aphelenchidae and Aphelenchoididae are remarkably distinct from each other and from the remaining Tylenchomorpha, that does not preclude the polyphyly of the Aphelenchoidea as suggested by molecular data.

Peculiarities of the life-cycle of the free-living marine nematode Oncholaimium ramosum (Enoplida: Oncholaimidae) from the Sea of Japan

Inna L. DAVYDKOVA, Natalia P. FADEEVA

Far East State University, Vladivostok 690600, Russia nfadeeva@mail.primorye.ru

The free-living marine nematode Oncholaimium ramosum Smolanko et Belogurov, 1987 dominates in the sediments of chronic harbour pollution of the Far Eastern ports. Study of life cycle was carried out both in laboratory conditions during two years and using the season data. Observations on the embryonic and postembryonic development, and also the moulting processes are presented. 35 egg layings, 400 eggs and about 1000 nematodes of this species are investigated. Nematode O. ramosum have two generations in the Sea of Japan. The egg deposition continues from February to July (spring generation) and from September to November (autumn generation). 100% mortality of female is registered afrer completion of the egg deposition. The females Oncholaimium ramosum are observed from egg deposition to death almost continuously. The female deposit consists from either clusters of 2-38 eggs or from a single egg. There are line, bunch or lump layings. Ellipsoid eggs are 95 - 120 μm long and 50 - 94 wide. The cleavage of 43-50% of eggs in a laying is observed several right after deposition. The others 50% of eggs lag behind in development for 8-10 days. The process of hatching from initial to last larva in laying proceeds also 8-10 days. The stages of 2, 4, 8 blastomeres, late gastrula, and vermiform larva were described. The average time of development of Oncholaimium ramosum from start of cleavage to hatching is 1-1,4 months. The average time of hatching is 5-10 minutes. The postembryonic development includes four moulting cycles resulting in four juvenile stage designated as J₁, J₂, J₃, and J₄. The size and time of development of juveniles J₁-J₄ for Oncholaimium ramosum are determined. (RFBR grant No. 03-04-49573).

The cuticle structure of Tricoma sp.(Nematoda: Desmoscolecida)

Shota V. KOVALYEV

Department of Invertebrate Zoology, Faculty of Biology, Moscow State University, Moscow, 119899, Russia, san-system@mtu-net.ru

Todate nematodes of the order Desmoscolecida remain poorly investigated with transmission electron microscope (TEM). The study is an attempt to deepen understanding of organization of desmoscolecids. The structure of cuticle of desmoscolecid Tricoma sp. from the sublittoral zone of Kandalaksha Bay of the White Sea is investigated with TEM. The cuticle is coarsely annulated and consists of 38 annuli (desms) joined by arthrodial cuticle. Desms are covered with chaotically situated plates. Three-layered epicuticle and the light homogenous layer are distinguished in transversal sections. Two layers, subsurface and inner, are observed under the epicuticle in longitudinal sections. The subsurface layer is light, homogenous, without any obvious features of radial striation, it presents both in desms and interzones between them. The inner layer presents only in arthrodial cuticle of interzones; it is divided into 4-5 distinct layers formed by alternating light and dark plots. Electron-dense plates and bundles of fibres lie in hypoderm under the arthrodial cuticle. Hypoderm penetrates each cuticle annulus where it underlies the homogenous layer. Body surface, head and tail regions were studied in Tricoma albimaris with scanning microscope. The pattern of head sensilla "6+4" was detected. Additional TEM data on the morphology of sensory organs of the anterior end are also given.

On the taxonomy and ultrastructure of Microlaimidae (marine Chromadoria)

Shota V. KOVALYEV*, Alexei V. TCHESUNOV**

Department of Invertebrate Zoology, Faculty of Biology, Moscow State University, Moscow, 119899, Russia, *san-system@mtu-net.ru,**tchesunov@mtu-net.ru

Taxonomy of the family is revised. Original keys for definition of the genera as well as of the species within each genus (with the exception of Microlaimus) are made up. The following genera are accepted as valid: Acanthomicrolaimus, Bathynox, Bolbolaimus, Caligocanna, Ixonema, Microlaimus, Pseudomicrolaimus, Spirobolbolaimus. The genus Calomicrolaimus Lorenzen 1976 is considered to be a junior synonym of Microlaimus De Man 1880. Five species from the White, Caribbean seas and the Pacific Ocean are described as new to science: Microlaimus sp.1, M. sp.2, M. sp.3, Aponema sp.1, Caligocanna sp.1; redescriptions of 5 known species are also given. The ultrastructure of cuticle, hypoderm, musculature, intestine is investigated in Microlaimus honestus with TEM. The cuticle consists of four layers of typical structure: three-layer epicuticle, radial-striated exocuticle, meso- and endocuticle subdivided into several layers. Hypoderm is seven-cellular in cross section, with prominent lateral and ventral chords. Ventral nerve trunk is well developed and located in corresponding chord. Musculature is of polymyarian type (22 cells in the field of cross section), with contractile zone in the basal part of muscular cells. The body cavity looks as narrow spaces between internal organs. Intestine is oligocellular, with microvilli turned to the lumen.

Self-moving spermatophores: spermatozoan dimorphism in Steinernema (Steinernematidae, Rhabditida)

Vladimir V. YUSHIN¹, Mutsuhiro YOSHIDA², Sergei E. SPIRIDONOV³

¹Institute of Marine Biology FEB RAS, Vladivostok 690041, Russia, yushin@fromru.com

²National Institute for Agro-Environmental Sciences, Kannondai Tsukuba, Ibaraki, Japan mutsuysd@niaes.affrc.go.jp

³Institute of Parasitology of RAS, Leninskii pr. 33, Moscow, 117071, Russia, spiridon@rjnem.msk.ru

The genus of the enthomopathogenic nematodes Steinernema includes the complex of species whose testes produce relatively small number of giant spermatozoa 50-100 µm in diameter (megaspermatozoa). The megaspermatozoa possess a set of cytoplasm features usual for common spermatozoa of the steinernematids (S. intermedia, S. feltiae) and other rhabditids. The megaspermatozoa from the testes are unpolarized cells, which contain mitochondria and numerous aberrant components known as the membranous organelles (MOs). Numerous minute spermatozoa about 2 μm in diameter (microspermatozoa) cover the surface of the megaspermatozoa. The microspermatozoa contain the same organelle set, which includes several mitochondria and the sole MO. The megaspermatozoa from the uterus have large pseudopod, the MOs are attached to the plasmalemma and are open to the exterior via pores. The microspermatozoa from the uterus were found both on the surface of the megaspermatozoa and as free cells in the uterus lumen. The MO of microspermatozoon is attached to the plasmalemma and is open to the exterior via pore. Thus, the spermatogenesis in S. tami results in the formation of spermatophores where one hypertrophied germ cell (megaspermatozoon) functions as a transportation device for numerous simplified male gametes (microspermatozoa). The megaspermatozoa from the uterus are motile cells, therefore the male gamete complex of S. tami may be defined as a "self-moving spermatophore". The uniform (5 μm in diameter) "normal" spermatozoa of some steinernematids (S. intermedia, S. feltiae) are capable of association into stable self-moving chains. This feature may be considered as a preadaptation to the formation of spermatophores. (Grant of Far East Branch of RAS for V.V. Yushin, Support for Science Schools grant No. 1219.2003.4).

Electron microscopic study of the spermatogenesis in free-living marine nematode Paracyatholaimus pugettensis (Chromadorida, Cyatholaimidae)

Julia K. ZOGRAF¹, Vladimir V. YUSHIN²

¹Department of Zoology, Academy of Ecology, Marine Biology and Biotechnology of Far East State University, Vladivostok, 690000, Russia, zojulia@yandex.ru

²Institute of Marine Biology FEB RAS, Vladivostok, 690041, Russia, yushin@fromru.com

Spermatogenesis and structure of mature spermatozoa in a free-living nematode Paracyatholaimus pugettensis were studied using electron microscopy. The cytoplasm of spermatocytes contains many Golgi bodies, cisternae of the rough endoplasmic reticulum (RER), ribosomes, mitochondria, and fibrous bodies (FBs). In the main cell body (MCB) of spermatids FBs and mitochondria surround the centrally located nucleus devoid of nuclear envelope; the sperm plasmalemma develops many folopodia. Narrow cellular bridge unites the MCB with the residual body which includes the synthetic apparatus of the spermatid. The immature sperm resembles in structure the MCB of the late spermatids. Mature spermatozoa from the uterus consists of the MCB and prominent pseudopodia filled with cytoskeleton filaments. The MCB contains nucleus, mitochondria, remnants of FBs, large osmiophilic bodies and vesicles with flocculent material. The spermatozoa of P. pugettensis exhibit the main ultrastructural features characteristic for nematodes: amoeboid nature and absence of the axoneme, acrosome and nuclear envelope. However, in P. pugettensis and other chromadorids studied the aberrant membranous organelles (MOs), which are typical of the nematode sperm, were not found at any stage of spermatogenesis. In this respect, its spermatogenesis differs from spermatogenesis in monhysterids and secernents but resemles one of tylenchids (Grant of Far East Branch of RAS for V.V. Yushin, Support for Science Schools grant No. 1219.2003.4).

Ultrastructure of spermatozoa in the free-living marine nematode Paracanthonchus macrodon (Nematoda, Chromadorida)

Julia K. ZOGRAF¹, Vladimir V. YUSHIN²

¹Department of Zoology, Academy of Ecology, Marine Biology and Biotechnology of the Far Eastern State University, Vladivostok, 690000, Russia, zojulia@yandex.ru

²Institute of Marine Biology FEB RAS, Vladivostok, 690041, Russia, yushin@fromru.com

The spermatozoa from testes and uteri of the free-living marine nematode Paracanthonchus macrodon (Cyatholaimidae, Chromadorida) were studied electron-microscopically. The spermatozoa from the testes are unpolarized cells covered by numerous filopodia. The sperm nuclei have oval or bean-like outlines and are surrounded by mitochondria. The peripheral cytoplasm is devoid of organelles and contains evenly distributed filamentous material. The filopodium content is the same as of the sperm cytoplasm. A submembrane layer of 13-16 nm thick longitudinally oriented microtubule-like fibres (MLF) strengthens the filopodia. The spermatozoa from the uterus are amoeboid cells with the discrete nucleus surrounded by mitochondria. The bulk of the sperm cytoplasm lacks organelles and is filled with MLF, which also underlie the sperm plasmalemma. The spermatozoa develop large pseudopods, which form the hemidesmosome-like junctions with the uterus wall. A layer of a subsurface osmiophilic material concentrating at the junction point is associated with the MLF running out into the cytoplasm as parallel arrays. The aberrant cytoplasm components of the nematode spermatozoa, namely the membranous organelles and fibrous bodies, were not found in the spermatozoa of P. macrodon. This pattern of the spermatozoon structure is interpreted as a highly reduced condition. (Grant of Far East Branch of RAS for V.V. Yushin, Support for Science Schools grant No. 1219.2003.4).

Ultrastructural study of sperm development in the free-living marine nematode Halichoanolaimus sonorus (Chromadorida, Selachinematidae)

Julia K. ZOGRAF¹, Vladimir V. YUSHIN²

¹Department of Zoology, Academy of Ecology, Marine Biology and Biotechnology of Far Eastern State University, Vladivostok, 690000, Russia, zojulia@yandex.ru

²Institute of Marine Biology FEB RAS, Vladivostok, 690041, Russia, yushin@fromru.com

Spermatogenesis and structure of mature spermatozoa in the free-living marine nematode Halichoanolaimus sonorus were studied using electron microscopy. The cytoplasm of spermatocytes contains many Golgi bodies, cisternae of the rough endoplasmic reticulum (RER), ribosomes and mitochondria. The main cell body (MCB) of spermatid contains the centrally located nucleus devoid of nuclear envelope, mitochondria and dense fibrous bodies (FBs). The surface of the spermatid develops numerous short filopodia. The Golgi bodies, cisternae of RER, and ribosomes are concentrated in the residual body of the spermatid. The immature sperm from testes resembles the MCB of spermatid. The spermatozoa from uteri lack usual postinsemination transformations. Their FBs remain intact, the surface filopodia transform into small lamellipodia. The large pseudopod found in many other nematode spermatozoa is absent. The spermatozoa of H. sonorus also lack membranous organelles (MOs), another characteristic feature of the aberrant nematode spermatozoa. The unique pattern of spermatozoa with FBs never associating with the MOs differs H. sonorus from many nematodes studied but unites the chromadorids and the tylenchids (Tylenchida). This conclusion is supported by filopodial nature of the sperm surface demonstrated by both taxa. (Grant of Far East Branch of RAS for V.V. Yushin, Support for Science Schools grant No. 1219.2003.4).

© Russian Society of Nematologists, Institute of Marine Biology FEB RAS, 2002