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Functional morphology of the neuroendocrine sodium influx-stimulating peptide system of the pond snail, Lymnaea stagnalis, studied by in situ hybridization and immunocytochemistry.

The functional morphology of the neuroendocrine system producing sodium influx-stimulating (SIS) peptide in the pond snail, Lymnaea stagnalis, was studied by in situ hybridization and immunocytochemistry. The SIS-peptide, which is 76 amino acids long, stimulates sodium uptake from the ambient medium. Two synthetic DNA probes were used for in situ hybridization. The nucleotide sequences were chosen from the cDNA structure; they encode amino acids 8-17 and 64-73, respectively. SIS-peptide sequences 10-20 and 67-76 were synthesized and antibodies were raised to them and affinity-purified. In addition to these antibodies, a monoclonal antibody raised to a bioactive, high-pressure liquid chromatography (HPLC)-purified brain extract was used for immunocytochemistry. Paraffin sections of central nervous systems and of whole snails were studied. The SIS-peptide system could be identified as the previously described yellow cell (YC) system by comparing alternate sections treated with the DNA probes, stained with the antibodies, or stained with alcian blue-alcian yellow. SIS-peptide neurons (approximately 45) occur in the ganglia of the visceral ring and in the proximal parts of visceral nerves. Axons run in the nerves of these and in several nerves of other ganglia. Numerous axon branches penetrate the perineurium forming a vast central neurohemal area. The SIS-peptide system innervates the pericardium, the nephridial gland, the reno-pericardial canal, the ureter, the spermoviduct and gonadal acini, the anterior aorta, the ventral buccal artery, and the penis protractor muscle.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Biosynthesis of the storage polysaccharide from the snail Biomphalaria glabrata, identification and specificity of a branching beta 1-->6 galactosyltransferase.

Adult snails synthesize in their albumen glands a storage polysaccharide called galactan which is utilized by the developing embryos. With [6-3H]-uridine 5'diphosphogalactose the incorporation of labelled D-galactose into the polysaccharide can be traced in freshly removed glands maintained in a bathing buffer. After centrifugation of homogenized glands, galactosyltransferase activity is only found in the insoluble fraction. Chaps extracts of this material retain almost all of their activity and can be used for comparison of the incorporation rates into different native galactans or in various oligosaccharides. A highly efficient beta-(1-->6) galactosyltransferase was detected when methyl 3-O-(beta-D-galactopyranosyl)-beta-D-galactopyranoside was offered as acceptor. The substitution at the penultimate residue resulted in a branched trisaccharide as demonstrated by 1H-NMR-spectroscopy and permethylation analysis of the reaction product. Comparable results were obtained with various oligosaccharides containing an internal galactose unit glycosidically linked beta 1-->3. Attempts to separate and purify the various enzymes involved resulted in the isolation of a fraction which is able to transfer D-Gal exclusively to native galactan, but not to oligosaccharides. A further fraction was obtained from a different resin with activity for native galactan and 6-O-(beta-D-galactopyranosyl)-D-galactopyranose, but without any for methyl-3-O-(beta-D-galactopyranosyl)-beta-D-galactopyranose. It is thus concluded that at least three different enzymes are involved in the biosynthesis of this snail galactan.

Animals↗

Studies on the infectivity of Fasciola hepatica miracidia to Lymnaea truncatula. Attachment and penetration of miracidia into non-infected and infected snails.

Fasciola hepatica miracidia labelled in vivo with radioselenium have been used in studies on the capacity of the miracidia to attach to and penetrate the tegument of infected compared with non-infected host snails (Lymnaea truncatula). The results show that the penetration of the larvae into the snail tissue is not influenced by an already existing infection, regardless of its developmental stage.

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An automatic method for recording the emergence of cercariae of Schistosoma mansoni from the snail Biomphalaria glabrata.

In order to study the pattern of emergence of trematode larvae from the snail hosts (S. mansoni from B. glabrata) an automatic apparatus was developed. This apparatus allows the snails, each suspended by a thread, to be transported at intervals of at least half an hour into different beakers into which the cercariae are shed. This "eclosion clock" is essentially a carriage driven on perlon wheels along steel bars.

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[The macular fatty degeneration of the peripheral retina a variety of the snail track degeneration (author's transl)].

A previously undescribed form of snail-track degeneration with scattered flecks was found in three eyes with absolute glaucoma. Light- and electron-microscopic examination of these lesions in the retinal periphery revealed atrophy and gliosis with massive accumulation of fat within the astrocytes and Müller's cells. It was not possible to explain the patchy distribution of the lesions. The fatty change in the macroglia is believed to indicate 'in situ breakdown' of slowly atrophying retina. Morphologic and pathogenetic similarities suggest that all forms of snail-track degeneration with snowflakes represent nonspecific fatty degeneration of the retina.

Adult↗

Molecular phylogenetic relationships in the aquatic snail genus Lymnaea, the intermediate host of the causative agent of fascioliasis: insights from broader taxon sampling.

Phylogenetic analyses were performed on partial mitochondrial (16S) gene sequences that included new 16S data for 23 exemplars of the freshwater pulmonate snail genus Lymnaea sensu lato and putative outgroup species. This procedure yielded relatively congruent patterns of evolutionary divergence and phylogenetic affinities, and greater resolution and support for many lineages at different levels of divergence than from a previous work based on fewer samples. It has also clarified the relationships between key taxa. Molecular differentiation was evident among genera. Lymnaeids with n=16 chromosomes are a distinct, well-supported monophyletic group of recent origin. The genus Radix appears to be paraphyletic. Among the indigenous North American lymnaeids, a closer alliance was found between Fossaria spp. and Stagnicola caperata, a member of the subgenus Hinkleyia, than between the latter species and members of the subgenus Stagnicola s.str. The North American population of Lymnaea stagnalis is likely to be of European origin as it clustered with its European counterparts. The relevance of the molecular findings to efforts aimed at controlling snail-transmitted trematode diseases is discussed.

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Prevalence of cercariae from Lymnaea stagnalis snails in a pond system in Southern Germany.

Lymnaea stagnalis snails were collected from 174 individual ponds of an extensive pond system in South Germany; 43,441 snails collected during 1980 2000 were examined for shedding cercariae. The species richness (at least 18 species of cercariae) and the high cercarial prevalence (at least 44.9%) may result from the high abundance and diversity of vertebrate and invertebrate hosts in the area. The mean prevalence (% +/- SEM) of most cercarial species increased from May-June to August-October, e.g. in Diplostomum spathaceum from 4.1% to 18.6%, in all echinostomatids from 5.6% to 18.0%, in Pseudechinoparyphium echinatum from 3.0% to 11.2%, in Xiphidiocercariae from 2.2% to 13.4%, and the overall prevalence shifted from 13.7% to 53.5%. There was no change in the prevalence of the different cercarial species over the last 20 years. The agents of cercarial dermatitis showed a constant low prevalence, 0.17% in Trichobilharzia ocellata (43,441 L. stagnalis examined) and 0.24% in other Trichobilharzia species (4,245 Radix examined). Such low prevalences seem to be normal in areas where cercarial dermatitis occurs in humans.

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NADPH-diaphorase activity in the nervous system of the embryonic and juvenile pond snail, Lymnaea stagnalis.

Nicotinamide-adenine-dinucleotide-phosphate-diaphorase (NADPH-d) histochemistry has been applied in the present study to determine the distribution of putative nitric oxide (nitric oxide synthase)-producing cells during embryonic and early postembryonic development in the pond snail, Lymnaea stagnalis L., with special reference to the nervous system. The first NADPH-d-positive structures appear as early as 18% of development (E18, trochophore stage) and correspond to the pair of protonephridia. These structures later show disintegration, although after metamorphosis (E26=75%) staining of their individually spreading cells can be observed until hatching. Peripheral sensory neurons in the foot, mantle edge and lips, and their afferents projecting to the central nervous system reveal NADPH-d activity in the postmetamorphosis period (E25-E27=E60%-E80%) of embryogenesis. After hatching (P1-P3), a number of stained sensory cells appear in the pharynx and esophagus. Some NADPH-d positive neuronal perikarya occur in the pedal and pleural ganglia, and a few weakly stained cells in the cerebral and buccal ganglia of juvenile snails. At the same time, a continuous bundle of reactive fibers is formed in the neuropil both through and through around the circumesophageal ganglion ring. The localization of NADPH-d activity in the developing nervous system of Lymnaea suggests that nitric oxide participates mainly in sensory processes. However, its role in specific intraganglionic integrative events cannot be excluded following embryonic metamorphosis.

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Leaf mechanical properties modulate feeding movements and ingestive success of the pond snail, Lymnaea stagnalis.

We examined the mechanical properties of Butterhead and Iceberg lettuce leaves, and the rate at which they were eaten by the pond snail Lymnaea stagnalis. The outer part of Butterhead leaves were less robust than either the inner Butterhead or outer Iceberg leaves (Young's modulus 2.8, 5.2, 7.7 MPa respectively; ultimate tensile stress 0.18, 0.34 0.51 MPa) which were also thicker. Snails ingested inner Butterhead and Iceberg strips more slowly (36 and 32%) than outer Butterhead. This was not due to differences in latency to first bite or biting rate. Rather, the drop was due to a decrease in the proportion of successful bites (inner Butterhead 84%; Iceberg 86%), to a shorter length ingested per bite (inner Butterhead 55%; Iceberg 45%) and to increased handling time (inner Butterhead 30%). We conclude that sensory input from the mechanically more robust lettuce slows the buccal central pattern generator.

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Photoinactivation of neurones in the pond snail, Lymnaea stagnalis: estimation of a safety factor.

Neurones were irradiated with blue laser light (440 nm). The intensity of light for reliable cell killing (0.5 MW.m-2) was much greater than that used to kill arthropod neurones. In wild snails, there was no difference in the intensity to kill Lucifer yellow-filled neurones and unfilled neurones, probably because of the red pigments in the cell bodies. In laboratory-reared snails, which have much less pigmentation, only the filled cells were killed.

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Partial purification and characterization of the ovulation hormone of the freshwater pulmonate snail Lymnaea stagnalis.

The neurosecretory caudodorsal cells in the cerebral ganglia of the basommatophoran snail Lymnaea stagnalis produce an ovulation hormone (CDCH), which is released from the periphery of the intercerebral commissure. In the present study CDCH has been partially purified, starting with a hydrochloride acid extract of commissures, by gel permeation chromatography on Bio-Gel P-6 followed by cation exchange on SP-Sephadex C-25. This two-step procedure resulted in a ca. 22-fold purification of CDCH. The low purification factor is caused by poor resolution of both purification steps and low recovery on the Bio-Gel P-6 column. With stability tests, proteolytic enzymes, SDS/urea polyacrylamide electrophoresis and isoelectric focussing, purified CDCH was characterized as a stable peptide with a molecular weight of ca. 4700 d and a pI of ca. 9.3. The properties of CDCH are similar to those of the ovulation hormones of opisthobranch snails. Studies on species specificity indicate, however, that it is unlikely that CDCH will cause ovulation when injected in opisthobranchs, or vice versa.

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Direct effects of the hyperglycemic factor of the freshwater snail Lymnaea stagnalis on isolated glycogen cells.

In the freshwater pulmonate snail Lymnaea stagnalis special cells, the glycogen cells (GC) are present for the storage of glycogen reserves. These cells occur in large numbers in the anterior mantle region. In a previous paper in vitro experiments with intact anterior mantle tissue indicated that a hyperglycemic factor is released from the central nervous system (CNS) which stimulates glycogen mobilization in mantle tissue (M. A. Hemminga, J. J. Maaskant, W. Koomen, and J. Joosse (1985). Neuroendocrine control of glycogen mobilization in the freshwater snail Lymnaea stagnalis. Gen. Comp. Endocrinol. 57, 117-123). In the present study the question of whether this factor affects glycogen metabolism of GC isolated from mantle tissue was investigated. It is reported that in high-K+ Ringers the CNS is stimulated to release a factor which, in a dose-dependent way, inhibits glycogen synthesis in isolated GC (measured as a decreased incorporation of [14C]glucose into glycogen). Simultaneous with this glycogen synthesis-inhibiting effect, stimulation of glycogen degradation is found (measured as a decreased retention of prelabeled glycogen). It is concluded that (1) the factor released by the CNS having these effects on isolated GC is the same as the hyperglycemic factor which was reported to stimulate glycogen mobilization in intact mantle tissue, and (2) GC after isolation from mantle tissue have retained their ability to respond to this factor.(ABSTRACT TRUNCATED AT 250 WORDS)

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Neuroendocrine control of glycogen mobilization in the freshwater snail Lymnaea stagnalis.

In the freshwater snail Lymnaea stagnalis the anterior mantle region, which mainly consists of large numbers of special glycogen-storing cells, is an important depot for the energy reserves of this snail. In organ culture experiments the central nervous system (CNS), in contrast to other tissues, both inhibits glycogen synthesis (measured as incorporation of [14C]glucose into glycogen) and stimulates glycogen breakdown in anterior mantle tissue (measured as a decreased retention of prelabeled glycogen). These effects are dose dependent, with saturation at the highest doses tested. High-potassium Ringer solution stimulates the secretion of a CNS factor which induces glucose release by anterior mantle tissue. This glucose-release-stimulating effect of the CNS is also dose dependent, but saturation of the response was not achieved. It is concluded that inhibition of glycogen synthesis and stimulation of glycogen breakdown and glucose release are probably effects of a single neurohormone which controls glycogen mobilization from the storage cells in the mantle. Like similar factors in other animal phyla, this putative neurohormone is referred to as a hyperglycemic factor.

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Generation of oxygen radicals in hemocytes of the snail Lymnaea stagnalis in relation to the rate of phagocytosis.

The kinetics of oxygen radical production by phagocytosing hemocytes of the pond snail Lymnaea stagnalis were investigated. After contact had been established between zymosan and hemocytes in monolayers at 0 degrees C, phagocytosis was initiated by a shift to room temperature. Until the internalization phase of phagocytosis was completed, oxidative activity was detected mainly extracellularly (superoxide dismutase inhibitable cytochrome C reduction and peroxidase-catalyzed phenol red oxidation were used for the detection of superoxide and hydrogen peroxide, respectively). Thereafter, extracellular oxygen radical production by phagocytosing hemocytes decreased. Luminol-dependent chemiluminescence activity grew and, after the internalization phase of phagocytosis, remained at a high level, suggesting continued oxygen radical activity intracellularly. These results indicate that contact between zymosan and the hemocyte's plasma membrane stimulates a membrane-bound system to generate and release oxygen radicals. After internalization, this system appears to continue oxygen radical production inside the phagosome. So far, oxygen radical production in snail hemocytes shows many similarities to the mechanism in mammalian leucocytes.

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Chronic toxicity of acetone in the fresh water snail Lymnea stagnalis.

With water of total hardness of 230 mg/l of CaCO3 all the tried concentrations (0.1, 0.2, 0.4 and 0.6%) of acetone induced a decrease in the growth of the young pond snails. For older snails, a 0.4% concentration was the threshold at which an inhibitory effect of the shell growth was observed. From the 0.1% concentration a decrease in the shell mineralization was recorded. In the range of 0.1--0.6% concentrations, the decrease of the fecundity varied from 3% to 31%. At lowest concentrations, only the number of egg-masses/snail was reduced. At the 0.6% level a lower number of eggs/egg-mass was added. The 0.6% concentration of acetone disturbed only the fertility of the eggs; lowest concentrations significantly increased the frequency of polyembryony.

Acetone↗

Miracidium of Schistosoma mansoni: a macromolecular glycoconjugate as signal for the behaviour after contact with the snail host.

1. The behaviour of the miracidium of Schistosoma mansoni after contact with agar blocks containing Biomphalaria glabrata snail conditioned water (SCW) or its components, was studied. 2. "Repeated investigation" was the most specific response at contact with the snail host. 3. Fractionation and specific chemical treatment of SCW revealed that this response was stimulated by a lysozyme sensitive glycoconjugate with a mol. wt greater than 300,000. 4. Low molecular weight components of SCW had no stimulatory effect on the miracidia, although MgCl2 offered as pure chemical did so.

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Alteration in biogenic amine levels in the snail Lymnaea acuminata by the latex of Euphorbia royleana.

The latex of Euphorbia royleana which has high molluscicidal and anti-cholinesterase activity against Lymnaea acuminata reduced the levels of 5-hydroxytryptamine (5HT) and dopamine (DA) in the nervous tissue of L. acuminata. There was, however, no significant change in the level of 5-hydroxyindole acetic acid (5HIAA). The changes were found to be dependent on concentration of the latex extract. Similar changes were produced by both water and organic solvent extracts of the latex. The latex of E. royleana thus affects all the known neurotransmission mechanisms in the snail either separately or through a complex interaction between the different neurotransmitters. This may account for its high toxicity to snails.

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