Vladimir V. Yushin

Structure of the nervous system in the tornaria larva of Balanoglossus proterogonius (Hemichordata: Enteropneusta) and its phylogenetic implications

The tornaria larva of hemichordates occupies a central position in phylogenetic discussions on the relationships between Echinodermata, Hemichordata, and Chordata. Dipleurula-type larvae (tornaria and echinoderm larvae) are considered to be primary in the life cycle and thus provide a model for the ancestral animal common to all three taxa (the theory of W. Garstang). If the similarities between tornaria and the larvae in Echinodermata result from homology, their nervous systems should be basically similar as well. The present study utilizes anti-serotonin and FMRFamide antisera together with laser scanning microscopy, and transmission electron microscopy, to describe in detail the nervous system of the tornaria of Balanoglossus proterogonius. Serotonin immunoreactive neurons were found in the apical and esophageal ganglia, and in the stomach epithelium. FMRFamide immunoreactive neurons, probably sensory in nature, were detected in the apical ganglion and in the equatorial region of the stomach epithelium. At the ultrastructural level, the apical organ consists of a columnar epithelium of monociliated cells and includes a pair of symmetrical eyespots. The apical ganglion is located at its base and has a well-developed neuropil. Different types of neurons are described in the apical organ, esophagus, and stomach. Comparison with larvae in Echinodermata shows several significant differences in the way the larval nervous system is organized. This calls into question the homology between tornariae and echinoderm larvae. The possibility of convergence between the two larval types is discussed.

Ultrastructure of spermatogenesis in the free-living marine nematode Anticoma possjetica (Enoplida: Anticomidae)

- Spermatogenesis of the free-living marine nematode Anticoma possjetica was studied with electron microscopy. The spermatocyte cytoplasm is filled with numerous ribosomes, mitochondria, cisternae of RER and Golgi bodies. After completion of meiosis the nucleus of the spermatids is surrounded by a newly formed nuclear envelope. The first membranous organelles (MO) appear in the early spermatids, in the late spermatid they, together with mitochondria, surround the nucleus. The ribosomes, RER and Golgi bodies are concentrated inside the residual body. The nuclei of immature spermatozoa assume a bullet-like shape and are surrounded by mitochondria and a mass of MO. The mature spermatozoa possess a small anterior pseudopod anchoring them to the spermatheca wall. The spermatozoon cytoplasm is filled with densely packed MO interspersed with mitochondria. During spermatogenesis of A. possjetica, no organelles resembling the fibrous bodies (FB) found in the spermatozoa of other nematodes were observed. The ultrastructural peculiarities of spermatogenesis in A. possjetica differentiate it from many of the other nematodes studied, although important similarities with other enoplids (reconstitution of nuclear envelope) and other Enoplia (late MO formation) are present.

Riders on the sperm: sperm dimorphism and spermatozeugmata in nematodes from the genus Steinernema (Rhabditida: Steinernematidae)

A comparative ultrastructural study of the male gametes in entomopathogenic nematodes of the genus Steinernema (Rhabditida: Steinernematidae) is based on two species producing monomorphic (S. feltiae) and dimorphic (S. tami) spermatozoa. The spermatozoa of both species are basically the same as in many other Rhabditida. Immature spermatozoa from the testis are unpolarised cells containing a nucleus without a nuclear envelope, mitochondria and membranous organelles (MO), a unique component of the nematode spermatozoa. Mature spermatozoa have a nucleus, mitochondria and emptied MO, each attached to a sperm plasmalemma and opening to the exterior via a pore. Monomorphic mature spermatozoa of S. feltiae are 5 μm in diam., bear pseudopods, and form chains of several cells joined by gap junctions. The dimorphic immature spermatozoa of S. tami form spermatozeugmata where the single giant megaspermatozoon (30-35 μm diam.) bears numerous tiny, 3 μm microspermatozoa, intimately attached to the megaspermatozoon surface by gap junctions. Mature megaspermatozoa from the uterus are motile cells 35-50 μm diam.; they have prominent pseudopods and convey immotile, 2 μm microspermatozoa towards the oviduct. Unlike many other invertebrates, sperm dimorphism in S. tami does not change the basic set and structure of organelles, only their number. The genus Steinernema has two strategies for sperm agglomeration: i) chains of monomorphic spermatozoa of normal size, as in S. feltiae; and ii) spermatozeugmata based on a dramatic size difference between spermatozoa formed in the testis, as exemplified by S. tami. According to the types of sperm agglomeration, the genus Steinernema is divided into two main clades.

The origin of nematode sperm: Progenesis at the cellular level

Analysis of the development and structure of aberrant sperm of nematodes and other metazoans with internal insemination showed that these spermatozoa have several unusual, but shared features: (1) the absence of a flagellum and an axoneme, an unusual arrangement of centrioles; (2) an amoeboid shape and amoeboid motility due to cytoskeleton components; (3) the poor condensation of nuclear chromatin, which may be diffuse, thread-like, and discrete; (4) the absence of a nuclear envelope; (5) multiple unmodified mitochondria; (6) the absence of an acrosome; (7) unique membranous components derived from the Golgi complex; and (8) the large size of spermatozoa due to prominent cytoplasm filled with a great number of components. These shared features of aberrant spermatozoa may be explained by the conservation of a number of features that are characteristic of the primitive undifferentiated cell (the predecessor of all specialized gametes). The primitive cell features of numerous versions of aberrant sperm reflect the arrest of cytoplasm specialization of male gametes at an early stage of development. This pattern of gamete evolution is quite consistent with the conception of progenesis (retention of juvenile characters by precocious, sexually mature morphologically juvenile stage). Thus, the origin of the aberrant sperm of nematodes and many other metazoans may be interpreted as progenesis at the cellular level.

Histology and Ultrastructure of the Body Wall in the Phoronid Phoronopsis harmeri

The histology and ultrastructure of the body wall in Phoronopsis harmeriwere studied using light microscopy and TEM. The ectoderm epithelium of tentacles, anterior body region, and ampulla consists of monociliary cells. Gram-negative bacteria were found between microvilli, in the protocuticle of the anterior region, and in the ampulla. The epithelium of the posterior body region lacks both monociliary cells and bacteria. The bundles of nerve fibers run between the layer of epithelial cells and basal membrane. The musculature of the body wall comprises circular and longitudinal muscles. The circular muscle fibers are applied to the basal membrane and constitute a solid layer extending almost throughout the length of the body. This pattern is broken in the posterior body region, where there is no solid layer of circular musculature, and the latter is arranged in isolated muscle bands. In the ampullar (terminal) body region, the inversion of circular and longitudinal muscle layers takes place, so that the latter appears to be pressed against the basal membrane. The apical surfaces of longitudinal muscle cells bear cytoplasmic processes; some of the cells have a flagellum. The basal portion of the longitudinal muscle cells forms a cytoplasmic process containing bundles of tonofilaments. The processes of all cells making up the muscle bands are interwoven and anchored to the basal membrane.

Ultrastructure of sperm development in the genus Ditylenchus (Nematoda: Anguinidae)

Spermatogenesis in Ditylenchus arachis and D. dipsaci was studied using transmission electron microscopy. Spermatogenesis includes the formation of complexes of fibrous bodies (FB) and membranous organelles (MO) in the spermatocytes, which dissociate in separated MO and FB in the spermatids. Immature spermatozoa are unpolarised cells with separate FB and MO. Mature spermatozoa are arranged in chains. Ditylenchus dipsaci is unique in having MO that have already fused with the outer membrane in immature spermatozoa and have mature spermatozoa in the male testis, proving that not only insemination plays a role in spermiogenesis. Contrary to what has been described before, spermatogenesis in Ditylenchus, and other early diverging Tylenchomorpha, follow the typical ‘rhabditid’ pattern, while the absence of MO within Tylenchomorpha appears to be an apomorphic trait for the molecular defined clade of tylenchids that exclusively parasitise higher plants. This confirms the value of traits related to spermatogenesis in nematode phylogeny.

Ultrastructural evidence of sperm dimorphism in Deladenus sp. (Tylenchomorpha: Sphaerularioidea: Allantonematidae)

The dimorphic spermatozoa of the insect-parasitic nematode Deladenus sp. (Tylenchomorpha: Sphaerularioidea: Allantonematidae) were studied for the first time with a transmission electron microscope (TEM). The immature spermatozoa from the testis of mycetophagous males are 10-12 μm diam. and 4-5 μm long unpolarised cells with a centrally located nucleus without a nuclear envelope. The cytoplasm contains mitochondria and specific components, membranous organelles (MO) and fibrous bodies (FB). The MO are spherical vesicles with an internal system of finger-like invaginations of the membrane; the spindle-shaped FB consist of tightly packed parallel fibres. Spermatozoa from the uteri of infective females of Deladenus sp. are vastly different in size being tiny cells ca 2 μm diam. with a spherical or oval nucleus. Each cell contains several mitochondria and MO. Although each individual of Deladenus sp. contains only monomorphic spermatozoa, sperm dimorphism was revealed after analysis of the whole life cycle. Despite a difference in size the cytological characters of both types of spermatozoa conform to the typical rhabditid pattern. The presence of both MO and FB in sphaerularioidid spermatozoa differentiates the superfamily Sphaerularioidea from Tylenchoidea whose representatives lack MO in the spermatogenic cells.

Ultrastructure of spermatogenesis in the free-living marine nematode Halichoanolaimus sonorus (Chromadorida: Selachinematidae)

Spermatogenesis of the free-living marine nematode Halichoanolaimus sonorus was studied with electron microscopy. The spermatocyte cytoplasm is filled with ribosomes, mitochondria, cisternae of the RER and Golgi bodies. The spermatids are subdivided into the residual body, which includes the entire synthetic apparatus of the cell, and the main cell body with a centrally located nucleus lacking a nuclear envelope. The mitochondria and the precursors of the fibrous bodies form a layer at the periphery of the main cell body. The main cell body surface bears numerous filopodia. The immature spermatozoa from the testes are unpolarised cells with a centrally located nucleus surrounded by spherical fibrous bodies, mitochondria and membranous stacks; the cell surface is covered by numerous short filopodia. Spermatozoa from the uterus do not show the dramatic changes common for activated spermatozoa of nematodes. Their nuclei, mitochondria, fibrous bodies and surface filopodia remain intact. Bundles of filaments appear between the fibrous bodies. Some of the ultrastructural characters of the spermatogenesis of H. sonorus (late appearance of fibrous bodies and their filamentous structure, absence of membranous organelles at all stages of spermatogenesis, occurrence of numerous surface filopodia in the immature and mature spermatozoa) may be utilised as distinctive cytological characters for phylogenetic analysis of the order Chromadorida.

High-pressure freezing and freeze-substitution fixation reveal the ultrastructure of immature and mature spermatozoa of the plant-parasitic nematode Trichodorus similis (Nematoda; Triplonchida; Trichodoridae).

The spermatozoa from testis and spermatheca of the plant-parasitic nematode Trichodorus similis Seinhorst, 1963 (Nematoda; Triplonchida; Trichodoridae) were studied with transmission electron microscopy (TEM), being the first study on spermatogenesis of a representative of the order Triplonchida and important to unravel nematode sperm evolution. Comprehensive results could only be obtained using high-pressure freezing (HPF) and freeze-substitution instead of chemical fixation, demonstrating the importance of cryo-fixation for nematode ultrastructural research. The spermatozoa from the testis (immature spermatozoa) are unpolarized cells covered by numerous filopodia. They contain a centrally-located nucleus without a nuclear envelope, surrounded by mitochondria. Specific fibrous bodies (FB) as long parallel bundles of filaments occupy the peripheral cytoplasm. No structures resembling membranous organelles (MO), as found in the sperm of many other nematodes, were observed in immature spermatozoa of T. similis. The spermatozoa from the uterus (mature or activated spermatozoa) are bipolar cells with an anterior pseudopod and posterior main cell body (MCB), which include a nucleus, mitochondria and MO appearing as large vesicles with finger-like invaginations of the outer cell membrane, or as large vesicles connected to the inner cell membrane. The peripheral MO open to the exterior via pores. In the mature sperm, neither FBs nor filopodia were observed. An important feature of T. similis spermatozoa is the late formation of MO; they first appear in mature spermatozoa. This pattern of MO formation is known for several other orders of the nematode class Enoplea: Enoplida, Mermithida, Dioctophymatida, Trichinellida but has never been observed in the class Chromadorea.

Electron microscopic study of spermiogenesis in the free-living marine nematode Leptosomatides marinae Platonova 1976 (Enoplida: Leptosomatidae)

The ultrastructure of spermatocytes, spermatids and spermatozoa of the free-living marine nematode Leptosomatides marinae was studied by transmission electron microscopy. In early spermatids, the number of mitochondria, cisterns of the rough endoplasmic reticulum (RER) and dictyosomes increased; the number of membranous organelles (MOs) was insignificant. Later, dictyosomes and MOs filled the cytoplasm. The cytoplasm became distinctly segregated in late spermatids and the MOs concentrated around the nucleus; the mitochondria and organelles of synthesis settled on the cell periphery. Later, a densely packed conglomerate was formed from the central nucleus and a mass of MOs surrounded by an extensive zone of the cytoplasm containing mitochondria and organelles of synthesis. Early spermatozoa had an elongated nucleus surrounded by a layer of cytoplasm containing mitochondria, polarized MOs and bundles of filamentous material, which can be interpreted as fibrous bodies (FBs). The formed spermatozoa had elongated nuclei surrounded by a transparent halo; the cell periphery was a dense matrix, in which MOs and sparse mitochondria were submerged; no FBs were revealed in that phase. In general, the spermatozoa of L. marinae have the main attributes common to the Enoplida spermatozoa, availability of nuclear environment and development of the specific organelles, MOs and FBs, which are not united in complexes.