Immuno-localization of ferritin polypeptides in oocytes and somatic tissue of the freshwater snails Lymnaea stagnalis L. and Planorbarius corneus L.

Summary Antibodies were raised in rabbits against the 19000 Mr and 24000 Mr polypeptides of snail ferritin from Lymnaea stagnalis L. Anti-24000 Mr polypeptide antibodies were purified by an affinity-purification step and were made monospecific for their antigen by preabsorption with the 19000 Mr antigen. These purified antibodies were then used for in situ detection of their respective antigens by the indirect immunofluorescence method. The 19000 Mr polypeptide was found widely distributed in tissues of both pulmonate snails investigated (Lymnaea stagnalis L. and Planorbarius corneus L.) with the most intense antigen-directed fluorescence in certain connective tissue cells, secretory cells of the midgut gland and Sertoli cells and epithelia of the gonadal acini. In contrast, the 24000 Mr polypeptide could be detected only in yolk platelets of vitellogenic oocytes. The results indicate that yolk and somatic cell ferritins differ in immunoreactivity and structure and, accordingly may differ in function.This investigation was supported by the Deutsche Forschungsgemeinschaft. I greatly appreciate the advice given to me by Drs. U. Mays and V. Riedel, Münster.     

Structure of visual pathways in the nervous system of freshwater pulmonate molluscs

Central nervous system of freshwater pulmonate molluscs Lymnaea stagnalis and Planorbarius corneus was stained using retrograde transport of neurobiotin in the optic tract fibers. In both species, perikarya and fibers of the stained neurons are found in all ganglia except the buccal ones. Afferent fibers of the optic nerve form dense sensory neuropil located in relatively small volume of cerebral ganglia. Typical neuronal groups sending their processes into the optic nerves of ipsilateral and contralateral body halves are described. Among them, neurons of visceral and parietal ganglia innervating both eyes concurrently as well as sending projections into peripheral nerves are revealed. These neurons, supposedly, have a function to integrate sensory signals, which may be a basis for regulation of light sensitivity of retina and functioning of peripheral organs. Bilateral links of the molluscan eye with the pedal ganglia cells and statocysts are found, which is, likely, a structural basis of certain known behavioral patterns related to stimulation of visual inputs in the studied gastropod molluscs.     

An autoradiographic and cytophotometric study of oogenesis in a pulmonate snail,Planorbarius corneus

Summary The spatial and temporal patterns of macromolecular syntheses in oocytes and somatic auxiliary cells of the snail Planorbarius corneus have been investigated by autoradiography and cytophotometry. Oogenesis has been divided into three stages, comprising early meiosis up to diplotene (stage I), previtellogenetic growth phase (stage II), and vitellogenesis (stage III). No DNA synthesis was found in any oocyte stage. In stage-I oocytes, only nucleoli were found labelled with ³H-uridine. Oocyte nuclei of stage II and III actively synthesize RNA in nucleoli and chromosomes. The most intense incorporation of uridine in chromatin probably occurs during the previtellogenesis — vitellogenesis transition period during which cytological findings suggest well developed lampbrush chromosomes. RNA synthesis in amphinucleoli of stage-III oocytes is restricted to basophilic nucleolar parts, whereas acidophilic parts (protein bodies) neither synthesize nor store RNA. During vitellogenesis oocytes incorporate amino acids into yolk platelet proteins. Radioactive proteins are found in yolk platelet precursors 5 h after injection of the tracer and in yolk platelets 3 h thereafter. The labelling pattern suggests that oocytes synthesize certain hitherto unidentified yolk components. No evidence for the participation of follicle cells in synthesis and transport of vitellogenic proteins has been obtained from autoradiography. Cytological findings suggest an important role for these cells in oogenesis. They are highly active in RNA and protein synthesis. Cellular differentiation is accompanied by polyploidization of the nuclei which attain a highest DNA content of 256 c. Polyploidization probably occurs in incremental steps as indicated by complete endomitotic chromosomal cycles. Autoradiographs show that, during vitellogenesis, oocytes do not incorporate significant amounts of glucose, and only certain follicle cells were labelled with glucose, probably indicating the synthesis of glycogen.     

Sensory cells in the head skin of pond snails

Summary Several types of receptor endings were identified with scanning electron microscopy and silver-impregnation techniques in the skin of the tentacles, lips, dorsal surface of the head and mouth region of the pond snails Lymnaea stagnalis and Vivipara viviparus. Sensory endings at the tips of dendrites of primary receptor neurones, scattered below the epithelium, differ in structure, i.e., the endings exposed to the surface of the skin possess different proportions of cilia and microvilli, which vary in number, length, and packing. Type-I endings have microvilli and a few (1–5) cilia, 5–12 m in length. Type-2 endings have abundant (20–40), interwoven long (9–12 m) cilia and random microvilli. Type-3 endings show typical packing of 10–25 cilia in the form of bundles or brushes. They may be composed either of long (9–18 m) or short (2–7 m) cilia, or of both long and short ones. Microvilli here are absent. Type-4 endings have only microvilli. Two other types of skin receptors do not extend their sensory endings to the surface and can be indentified only in silver-stained preparations. Type-5 endings are branching dendrites of skin receptors cells that terminate among epithelial cells. In type-6, the sensory endings also terminate among epithelial cells but their cell bodies are located outside of the skin. In both species all skin regions examined possess the receptors of all six types differing only in their relative proportion. Possible functional roles of different receptors are discussed.     

Starvation and growth in the gastropod Planorbarius corneus (L.)

The effect of different degrees of partial starvation on growth was studied in the fresh-water gastropodPlanorbarius corneus (L.). Two indices of growth were used, fresh weight and shell diameter, and for both indices the rate of growth decreased proportionately at least as much as the proportion of time that food was absent. This suggests that growth is not regulated in this species, and some doubts are expressed about the validity of results with other invertebrate species that suggest growth is regulated.     

Ferritin as an exogenous yolk protein in snails

Summary A main yolk component in the oocytes of the pulmonate snailPlanorbarius corneus L. has been isolated and identified as the iron storage protein ferritin by its ultrastructure, iron content, immuunological properties and behaviour in disc electrophoresis. As judged from acrylamide electrophoresis data and ultrastructural observations, yolk ferritin is an exogenous protein which is synthesised in the hepatopancreas and taken up by the oocytes by endocytosis.     

The specificity of yolk protein uptake in cyclorrhaphan diptera is conserved through evolution

Summary Yolk proteins are transported from the hemolymph into the oocytes of insects during vitellogenesis by receptor-mediated endocytosis. Since other hemolymph proteins, both native and foreign, are not accumulated in the oocyte, the process of uptake is selective for yolk proteins. Peptide domains within the yolk proteins must therefore be involved in receptor recognition. With the longterm aim of identifying these domains and to open the possibility of understanding the molecular basis of receptor-mediated endocytosis of yolk proteins, we began investigating how well this mechanism has been conserved in evolution. We studied the uptake of yolk proteins from 13 different Drosophila species and five other dipteran species, namely, Calliphora erythrocephala, Sarcophaga argyrostoma, Musca domestica, Lucilia servicata, and Protophormia terrae-novae, into the ovaries of Drosophila melanogaster and Drosophila funebris. The results from these experiments showed that in all cases the foreign yolk proteins were taken up by the host ovaries, indicating that the mechanism and peptide domains of the yolk proteins involved in recognition of the receptor have been well conserved in dipteran evolution. Offprint requests to: M. Bownes     

Morphometric in vitro analysis of the control of the activity of the neurosecretory dark green cells in the freshwater snail Lymnaea stagnalis (L.)

Summary The intracellular localization of calcium by means of cytochemical techniques was studied in smooth muscle cells of mouse intestine. When the lead acetate method according to Carasso and Favard (1966) was used calcium was found in mitochondria and sarcoplasmic reticulum and occasionally between the myofilaments. The active ATP-dependent accumulation of calcium into cell structures was investigated by the oxalate method (Heumann and Zebe, 1967). After appropriate treatment the only structures of smooth muscle cells which contained calcium oxalate (identified by microprobe analysis) were elements of the sarcoplasmic reticulum.The results are discussed in relation to the role of calcium in the control of muscle activity during the contraction-relaxation cycle.The electron probe microanalysis was carried out at SIEMENS (Berlin) in collaboration with Dr. von Muschwitz. I thank Miss M. Schlatter for her skillful assistance. The investigation was supported by the Deutsche Forschungsgemeinschaft.     

Tubulikörper im Zytoplasma der Spermatiden von Planorbarius corneus L. (Basommatophora)

Zusammenfassung Aggregate regelmäßig undulierender Tubuli im Zytoplasma der Spermatiden von Planorbarius corneus wurden elektronenmikroskopisch untersucht. Diese Tubulikörper sind nur während der frühen Spermiohistogenese bis zur beginnenden Ausdifferenzierung der Mittelstücke zu beobachten. Sie stehen mit dem endoplasmatischen Retikulum in Verbindung. Unterschiedliche Strukturen werden als Bildmuster verschiedener Schnittebenen desselben dreidimensionalen Gefüges gedeutet. Ähnlich strukturierte, für andere Zellen beschriebene Einschlüsse wurden mit den Tubulikörpern verglichen. Dazu diente ein dreidimensionaler Modellentwurf.

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Tubular bodies in the cytoplasm of spermatids of Planorbarius corneus L. (Basommatophora)

Summary Aggregates of regularly undulating tubules in the cytoplasm of spermatids of Planorbarius corneus have been studied by electron microscopy. Generally, these tubular bodies can be observed in early spermatids until beginning of middle-piece-formation. Some of the tubules are connected with the endoplasmic reticulum. The various electron microscopic patterns may result from different planes of section through the same three-dimensional texture. Similar structured inclusions found in other tissue cells have been compared.

Frau B. Dingerdissen, Abteilung für Elektronenmikroskopie des Physikalischen Instituts, danken wir für technische Assistenz am Elmiskop.     

Shell attachment in the pond snail Lymnaea stagnalis (L.)

The attachment of the body of the snail Lymnaea stagnalis to the shell was studied by histochemistry and light and electron microscopy. Muscles of the body wall insert into the connective tissue by way of long thin projections of sarcolemma. The muscle cells end under the basement membrane of a specialised area of the epidermis, the adhesive epithelium. The cells of this epithelium are filled with microfilaments and possess characteristic knob-like microvilli. The epithelium is attached to the shell by way of an adhesive substance containing proteins and mucopolysaccharides.This research was made possible by a grant from the Netherlands Organization for Pure Research (Z.W.O.)     

In vivo efficacy of praziquantel against Echinoparyphium aconiatum (Trematoda: Echinostomatidae) parasitizing the great pond snails Lymnaea stagnalis (Gastropoda: Lymnaeidae)

The present study had a practical goal. I aimed to determine whether praziquantel could reduce the production of Echinoparyphium aconiatum (Trematoda: Echinostomatidae) cercariae in infected snails Lymnaea stagnalis (Gastropoda: Lymnaeidae) without killing the hosts. Praziquantel is a broad-spectrum antihelminth agent. It caused a total cessation of cercaria shedding when the praziquantel concentration in the treatment bath was 10 mg/L and the treatment time was 30 h or longer. A next research step which has to be taken before giving detailed recommendations about using praziquantel for ceasing production of E. aconiatum cercariae in parasitized snails is to follow the survivorship and performance of treated snails after a praziquantel exposure for longer than in this medium-term (3 days) experiment.     

On the problem of retinal transmitters of the freshwater mollusc <Emphasis Type="Italic">Lymnaea stagnalis</Emphasis>

Retrograde staining of retina of Lymnaea stagnalis with neurobiotin demonstrated that most photoreceptor cells send axons to the optic nerve directly, without intermediate contacts. Some of the photoreceptors are glutamate-immunoreactive suggesting that glutamate can provide the synaptic transmission of visual signal to the central neurons. Other photoreceptors stained via optic nerve seem to have other transmitter systems. Some of the retinal cells, but not the optic nerve fibers are pigment-dispersing hormone-immunoreactive. There are many serotonin-containing fibers in the tissue surrounding the optic cup with some of them penetrating the basal lamina of retina. Some of them belong to central neurons providing efferent innervation of the pond snail eye. Serotonergic innervation as well as pigment-dispersing hormone-containing cells are supposed to be involved in mechanism of the photosensitivity regulation of the molluscan eye.     

Co-existence of immunoreactivity to anti-dopamine,anti-serotonin and anti-vasotocin in the cerebral giant neuron of the pond snail Lymnaea stagnalis

Summary Consecutive sections of certain neurons in the central ganglia of the pond snail Lymnaea stagnalis appear to be immunoreactive to anti-dopamine and anti-serotonin. The Cerebral Giant Neurons stain in addition with antivasotocin. The observations indicate the presence of two biogenic amines within the same neuron and in addition their co-existence with a biologically active peptide.     

Ultrastructural immunocytochemical evidence for peptidergic neurotransmission in the pond snail Lymnaea stagnalis

Summary Three neuronal systems of the pond snail Lymnaea stagnalis were immunocytochemically investigated at the ultrastructural level with the unlabeled peroxidase-antiperoxidase technique. Preliminary electrophysiological and cell-filling investigations have shown that a cluster of neurons which reacts positively with an antiserum against the molluscan cardio-active peptide FMRFamide, sends axons to the penis retractor muscle. In this muscle anti-FMRF-amide (aFM) positive axons form neuro-muscular synapses with (smooth) muscle fibers. The morphological observations suggest the aFM immunoreactive system to be involved in peptidergic neurotransmission. In the right parietal ganglion a large neuron (LYAC) is penetrated by aFM positive axons which form synapse-like structures (SLS) with the LYAC. The assumption that the SLS represent the morphological basis for peptidergic transmission is sustained by the observation that iontophoretical application of synthetic FMRFamide depolarizes the LYAC. The axons of a group of pedal anti-vasopressin (aVP) positive cells run in close vicinity to the cerebral ovulation (neuro-)-hormone producing cell system (CDC system) Synapses or SLS between the two systems were not observed. The fact that (bath) application of arg-vasopressin induces bursting in the CDC, may indicate that the vasopressin-like substance of the aVP cells is released non-synaptically.     

Haemocyanin production in pore cells of the freshwater snail Lymnaea stagnalis

Summary Electron micrographs of pore cells of Lymnaea stagnalis suggest that these cells produce and store haemocyanin.     

Immunocytochemical demonstration of a humoral defence factor in blood cells (amoebocytes) of the pond snail,Lymnaea stagnalis

Summary Monolayers of blood cells (amoebocytes) and sections of connective tissue and of amoebocyte pellets of the freshwater pulmonate gastropod Lymnaea stagnalis were stained immunocytochemically with antisera to snail agglutinin/opsonin. The presence of this substance was demonstrated light microscopically both in the cytoplasm and on the cell membrane of amoebocytes. This suggests that amoebocytes synthesize agglutinin/opsonin, and bear it at their surface as receptors for foreign materials.     

Fixed phagocytes in the freshwater snail Lymnaea stagnalis

Morphological and histochemical examination of the blood and connective tissue of the freshwater snail Lymnaea stagnalis injected with various types of foreign particulate materials has shown the existence of free as well as fixed phagocytic cells. The morphology of the fixed phagocytes is described, and the phagocytic system of the snail is compared with that of other molluscan species.     

Identification of the yolk receptor protein in oocytes of Nereis virens (Annelida,Polychaeta) and comparison with the locust vitellogenin receptor

Summary In oviparous animals large amounts of yolk proteins of extraovarian origin are accumulated by developing oocytes during vitellogenesis. The yolk protein precursors, the vitellogenins (VTG), are transported into the oocytes by receptor-mediated endocytosis. In oocytes of the polychaetous annelid, Nereis virens, the receptor protein for VTG was visualized by ligand blotting studies as a protein with an apparent molecular mass of 190 kDa under non-reducing conditions. Anti-Locusta VTG receptor antibodies recognize the Nereis VTG receptor protein. The Nereis VTG receptor protein binds Locusta and Schistocerca VTG; the VTG receptor proteins of both locust species bind the Nereis vitellin. These results indicate the conservation of structural elements important for internalization of VTG.Abbreviations CHAPS 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propane-sulphonic acid - HBS HEPES-buffered saline - PAP peroxidase-anti-peroxidase - SDS-PAGE sodium dodecylsulphate polyacrylamide gel electrophoresis - TRIS, TBS TRIS-buffered saline - VT vitellin - VTG vitellogenin