Eyes covered by mitochondrial lenses in Petaliella spiracauda and Ptychopera purasjokii (Plathelminthes, Rhabdocoela, Trigonostominae). Ultrastructural features and phylogenetic implications.

The submicroscopic anatomy of the eyes in Petaliella spiracauda and Ptychopera purasjokii is described. These eyes correspond in general to the basic pattern of rhabdomeric pigment-cup ocelli. This, however, does not apply to modifications of the cup cell such as the differentiation of mitochondrial lenses. Corresponding with two sensory cells two extensions of the cup cell capping the eye aperture are crowded with small unmodified mitochondria in the eyes of P. spiracauda. The eyes of P. purasjokii have three sensory cells and the lenticular element is formed by a trifoil-shaped differentiation of three giant mitochondrial derivatives. These derivatives show peripheral appendages of various configurations, all of which resemble the profiles of small mitochondria. The implication of the existence of such appendages is that the lenses in P. purasjokii are derived from many fused mitochondria, rather than from a single enlarged one. It is concluded that the unmodified or modified mitochondrial differentiations in proliferations of the pigment cell capping the opening of the eye cup serve to focalize incoming light. The evolution of mitochondrial lenses in Plathelminthes is considered.

Organisation of the praesoma of Paratenuisentis ambiguus (Van Cleave, 1921) (Acanthocephala: Eoacanthocephala), with special reference to the lateral sense organs and musculature.

The praesoma of the acanthocephalan parasite Paratenuisentis ambiguus was studied at the light and the electron microscope level, with special reference to the lateral sense organs and the musculature, in order to substantiate the basal pattern of the Acanthocephala and to analyse the phylogeny of the taxon. The study includes the first ultrastructural description of a lateral sense organ in the Acanthocephala. Two sensory support cell ducts extend from the binucleate pericaryon of the sensory support cell to the lateral sense organs. On their way to the lateral sense organs the ducts penetrate the receptacle and join the anterior ventral nerves. Each lateral sense organ consists of a conical termination of one of the sensory support cell ducts, in which the neuronal fibres and dendritic terminations of the equilateral anterior ventral nerve are embedded. An analysis of the available data of praesomal sense organs in Acanthocephala suggests that lateral and apical sense organs are absent in the basal pattern of the Acanthocephala. It is likely that two lateral sense organs, a binucleate sensory support cell with two ducts and two anterior ventral nerves evolved within the stem-line of some Palaeacanthocephala, all Eoacanthocephala and all Archiacanthocephala, whereas two apical sense organs, a quadrinucleate sensory support cell with four ducts and two apical sensory nerves presumably represent an autapomorphic character of the Archiacanthocephala. Furthermore, it can be derived from data in the literature and the present study that the praesomal hooks are totally covered by epidermis in the basal pattern of the Acanthocephala, whereas the ontogenetic loss of the epidermal covering can be regarded as an autapomorphy of the Archiacanthocephala.

Evidence for the mitochondrial origin of the eye lenses in embryos of Entobdella soleae (Plathelminthes, Monogenea).

The lens associated with each of the four pigmented eyes of the oncomiracidium of Entobdella soleae (Plathelminthes, Monogenea, Capsalidae) develops in a special region of the pigment cup cell of the eye. It is confirmed that the inner of the two membranes enclosing each lens bears short, inwardly projecting, membranous profiles identical to mitochondrial cristae. Studies of embryos incubated for 19 days at 12 degrees C (hatching begins at 28 days at this temperature) revealed that the matrix of the developing lens of each anterior eye contains many mitochondrial membrane compartments, some having the configuration of separate, small mitochondria. The implication is that the lens is derived from many fused mitochondria, rather than from a single large one. The anterior eyes of 19-day-old embryos are less well developed than the posterior eyes. Pigment granules in the anterior eyes appear to be at the premelanosome stage and contain dispersed dense particles lacking an obvious orderly arrangement. The posterior eyes mostly contain mature melanosomes. Membranous compartments in the matrix of the posterior eye lenses are rare. Apart from longer peripheral cristae, lenses of 22-day-old embryos are identical with those of oncomiracidia. The evolution of mitochondrial lenses in Plathelminthes is considered.

Ultrastructure of the acanthella of Paratenuisentis ambiguus (Acanthocephala).

The fine structure of the early acanthella of Paratenuisentis ambiguus (Eoacanthocephala) was investigated. This developmental stage is characterised by losing its ability to move and by differentiation of adult structures. The frontal syncytium, present in the first developmental stage (the acanthor), is lost, while the epidermis and central syncytium persist. The epidermis of the acanthella contains a number of giant nuclei that are arranged into several small groups. The central syncytium is subdivided into different masses, containing nuclei that will give rise to the organs of the adult. The 'uncinogenous bands' extend into the anterior body of the acanthella. Formation of the hooks takes place within these strands. In all investigated stages no extracellular materials were observed. Posterior of the uncinogenous bands lies the brain anlage and the primordia of the reproductive system. Neither a sense organ nor a nervous system were found.

Embryonic development of the nervous system of the rhabdocoel flatworm Mesostoma lingua (Abilgaard, 1789).

We have analyzed the embryonic development of the Mesostoma nervous system, using a combination of histology, transmission electron microscopy, and wholemount immunohistochemistry. Neural progenitors are formed at an early stage when the Mesostoma embryo constitutes a multilayered mesenchymal mass of cells. A neurectoderm as in vertebrates or arthropods is absent. Only after neurons in the deep layers of the embryo have started differentiating do superficial cells reorganize into an epithelium that will give rise to the epidermis. Neurons are clustered in two anterior, bilaterally symmetric brain hemispheres. An antibody against acetylated beta-tubulin (anti-acTub) that labels neurotubules reveals an invariant pattern of pioneer neurons in the brain of midstage embryos. Pioneer neurons are grouped in several small clusters at characteristic positions. They pioneer several commissural tracts of the brain and two pairs of ventral and dorsal connectives, respectively.

The embryonic development of the rhabdocoel flatworm Mesostoma lingua (Abildgaard, 1789).

The embryonic development of the flatworm Mesostoma lingua was studied using a combination of life observation and histological analysis of wholemount preparations and sections (viewed by both light and electron microscopy.) We introduce a series of stages defined by easily recognizable morphological criteria. These stages are also applicable to other platyhelminth taxa that are currently under investigation in our laboratory. During cleavage (stages 1 and 2), the embryo is located in the center of the egg, surrounded by a layer of yolk cells. After cleavage, the embryo forms a solid, disc-shaped cell cluster. During stage 3, the embryo migrates to the periphery of the egg and acquires bilateral symmetry. The side where it contacts the egg surface corresponds to the future ventral surface of the embryo. Stage 4 is the emergence of the first organ primordia, the brain and pharynx. Gastrulation, as usually defined by the appearance of germ layers, does not exist in Mesos-toma; instead, organ primordia emerge "in situ" from a mesenchymal mass of cells. Organogenesis takes place during stages 5 and 6. Cells at the ventral surface form the epidermal epithelium; inner cells differentiate into neurons, somatic and pharyngeal muscle cells, as well as the pharyngeal and protonephridial (excretory) epithelium. A junctional complex, consisting initially of small septate junctions, followed later by a more apically located zonula adherens, is formed in all epithelial tissues at stage 6. Beginning towards the end of stage 6 and continuing throughout stages 7 and 8, cytodifferentiation of the different organ systems takes place. Stage 7 is characterized by the appearance of eye pigmentation, brain condensation and spindle-shaped myocytes. Stage 8 describes the fully dorsally closed and differentiated embryo. Muscular contraction moves the body in the egg shell. We discuss Mesostoma embryogenesis in comparison to other animal phyla. Particular attention is given to the apparent absence of gastrulation and the formation of the epithelial junctional complex.

Syncytial organization of acanthors of Polymorphus minutus (Palaeacanthocephala), Neoechinorhynchus rutili (Eoacanthocephala), and Moniliformis moniliformis (Archiacanthocephala) (Acanthocephala).

The fine structures of immature and of developed shelled acanthors of three species belonging to the three subgroups of the Acanthocephala were investigated. Acanthors are surrounded by four eggshells (embryonic envelopes) and are composed of three syncytia: a frontal syncytium, a central syncytium, and an epidermal syncytium. Neither a sense organ nor a nervous system has been found. The central syncytium shows a mass of condensed nuclei and 12 decondensed nuclei and gives rise to 10 anterior/posterior subepidermal myofibrillar systems and 2 oblique retractor muscles. Circular muscles are missing. A single decondensed nucleus can be assigned to each of the 12 muscular systems. The epidermal syncytium embeds the other two syncytia and forms the wrinkled epidermis, which shows an extracellular glycocalyx and intrasyncytial condensations. Prominent recurved hooks, which mark the anterior end of each acanthor, and body spines are intraepidermal differentiations. Partly branched tubular infoldings of the epidermal plasma membrane of the acanthor exist and represent precursors of the pore ducts typical of the adult epidermis. Autapomorphies in the ground pattern of the monophylum Acanthocephala are the four eggshells, the early development of three syncytia, the condensed nuclei in the central syncytium, and the differentiation of ten longitudinal muscle bands and two muscle retractors and of intraepidermal hooks and spines. The syncytial organization of the epidermis with intraepidermal skeletal condensations and infoldings of the apical plasma membrane are characteristics inherited from a stem species common to Acanthocephala, Seison, and Rotifera.

Cloning of the coat protein gene from beet necrotic yellow vein virus and its expression in sugar beet hairy roots.

Expression of the beet necrotic yellow vein virus (BNYVV) coat protein (CP) gene in transgenic sugar beet hairy roots was accomplished as a step towards CP-mediated virus resistance. A cDNA for the CP gene and its 5' terminal untranslated leader sequence was prepared from BNYVV RNA, using two oligodeoxynucleotides to prime the synthesis of both strands. Second-strand synthesis and amplification of the cDNA were done by Taq DNA polymerase chain reactions. Run-off transcripts of the cloned cDNA sequence were obtained and translated in vitro, yielding immunoreactive CP. A binary vector construction containing the CP gene under the control of the 35S promoter of cauliflower mosaic virus was prepared and used for Agrobacterium rhizogenes-mediated transformation of sugar beet tissue. Stable integration and expression of the CP gene in sugar beet hairy roots was demonstrated by Southern, Northern, and Western blot analysis, respectively.

Binding of viruses from crude plant extracts to glutaraldehyde-treated plates for indirect ELISA.

A method for binding viruses from crude plant sap to ELISA microtitre plates not precoated with antibodies is described. Plates were coated with 3-(triethoxysilyl)-propylamine, and virus particles were covalently coupled to the plate surface using glutaraldehyde. Detection of trapped viruses was carried out by an indirect ELISA procedure using crude antisera or IgGs as detecting antibodies and a protein A-peroxidase conjugate. The number of virus particles attached to the plates depended on the ratio of virus concentration to plant constituents rather than on the absolute amount of virus present in the samples. Binding of virus to glutaraldehyde-treated plates was much better than to untreated plates. Possible applications of the method for studies on the relationship and identification of plant viruses are discussed.

Cytomorphometric characterization of NHL(non-Hodgkin's lymphomas) of low and high grade malignancy.

From semithin sections of 12 non-Hodgkin's lymphomas (3 lymphocytic, 3 centrocytic, 3 lymphoblastic, 3 centroblastic m.L.) morphometric parameters of nucleus and cytoplasm, including size, shape, roundness, number and size of nucleoli were measured. Statistical evaluation of the data showed that the lymphoma cells mainly differ in their size, less in their shape. Nucleolar parameters are of greater value than contour features in discriminating the four groups of NHL. With growing grade of malignancy an increasing nuclear and cellular polymorphism was observed. Moreover, measurement of the feature "maximal nuclear diameter" on paraffin sections from 42 further cases showed the possibility of morphometric discrimination of low and high malignant NHL in histopathological diagnostics.

Paddle cilia and discocilia - genuine structures? Observations on cilia of sensory cells in marine turbellaria.

Kinocilia of epidermal sensory cells in fixed marine Turbellaria often terminate as flattened biconcave discs. The distal part of the ciliary axoneme curves back upon itself forming a 360 degree loop which is enveloped by the plasmalemma. In living animals this structure can be induced by the addition of sodium cacodylate, monobasic sodium phosphate, dibasic sodium phosphate, sucrose, calcium chloride, or formaldehyde to the sea water. Specimens treated with sodium chloride, glutaraldehyde, or osmium tetroxide do not show modified cilia. In animals prepared for EM at low temperature and with a buffered hypotonic fixative less kinocilia are modified than in animals treated with a buffered iso- or hypertonic fixative and at a higher temperature. It is assumed that the unusually shaped cilia, described as "paddle cilia" or "discocilia" in other invertebrates, do not represent a genuine but an artificial structure.