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Identification of a novel UDP-GalNAc:GlcNAc beta-R beta 1-4 N-acetylgalactosaminyltransferase from the albumen gland and connective tissue of the snail Lymnaea stagnalis.

Both the albumen gland, one of the female accessory sex glands, and connective tissue of the freshwater snail Lymnaea stagnalis contain N-acetylgalactosaminyltransferase activity, capable of transferring GalNAc from UDP-GalNAc in beta 1-4 linkage to the terminal GlcNAc residue of GlcNAc beta-R. The albumin gland enzyme was partially purified by affinity chromatography on UDP-hexanolamine-Sepharose 4B. Using GlcNAc beta 1-2Man alpha 1-6(GlcNAc beta 1-2Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc or GlcNAc beta 1-OMe as substrates, the enzyme showed an absolute requirement for Mn2+ with an optimum concentration of 12.5-50 mM. The optimal pH was approximately pH 7.0. The enzyme activity was independent of the Triton X-100 concentration in the range 0.25-2.5%, and no activation effect was found. The more labile connective tissue microsomal enzyme, subjected to the same optimization procedure, gave comparable results. Both enzyme activities have similar substrate specificities towards GlcNAc or GlcNAc beta 1-OMe, and towards oligosaccharides or glycopeptides with a non-reducing terminal beta-GlcNAc unit, but cannot act on GlcNAc alpha 1-OMe. Saccharides with non-reducing terminal Gal or GalNAc residues, and free GalNAc, Gal or Glc residues are not acceptors. Product analysis was carried out for albumen gland N-acetylgalactosaminyltransferase and four acceptors having GlcNAc beta 1-R as the terminal non-reducing unit, and for connective tissue N-acetylgalactosaminyltransferase with GlcNAc beta 1-OMe as acceptor. In all instances, products with GalNAc beta 1-4-linked to GlcNAc were obtained, showing that the connective tissue and the albumen gland activities are probably from one enzyme. This enzyme activity can be identified as UDP-GalNAc:GlcNAc beta-R beta 1-4 N-acetylgalactosaminyltransferase, and is probably involved in the biosynthesis of N,N'-diacetyllactosediamine-containing glycoproteins, like hemocyanin, in the snail L. stagnalis.

Animals↗

Processing of the FMRFamide precursor protein in the snail Lymnaea stagnalis: characterization and neuronal localization of a novel peptide, 'SEEPLY'.

In the pulmonate snail Lymnaea stagnalis, FMRFamide-like neuropeptides are encoded by a multi-exon genomic locus which is subject to regulation at the level of mRNA splicing. We aim to understand the post-translational processing of one resulting protein precursor encoding the tetrapeptide FMRFamide and a number of other putative peptides, and determine the distribution of the final peptide products in the central nervous system (CNS) and periphery of Lymnaea. We focused on two previously unknown peptide sequences predicted by molecular cloning to be encoded in the tetrapeptide protein precursor consecutively, separated by the tetrabasic cleavage site RKRR. Here we report the isolation and structural characterization of a novel non-FMRFamide-like peptide, the 22 amino acid peptide SEQPDVDDYLRDVVLQSEEPLY. The novel peptide is colocalized with FMRFamide in the CNS in a number of identified neuronal systems and their peripheral motor targets, as determined by in situ hybridization and immunocytochemistry. Its detection in heart excitatory motoneurons and in nerve fibres of the heart indicated that the novel peptide may play a role, together with FMRFamide, in heart regulation in the snail. The second predicted peptide, STEAGGQSEEMTHRTA (16 amino acids), was at very low abundance in the CNS and was only occasionally detected. Our current findings, suggestive of a distinct pattern of post-translational processing, allowed the reassessment of a previously proposed hypothesis that the two equivalent sequences in the Aplysia FMRFamide gene constitute a molluscan homologue of vertebrate corticotrophin releasing factor-like peptides.

Amino Acid Sequence↗

Membrane currents of internally perfused neurones of the snail, Lymnaea stagnalis, at low intracellular pH.

The effects of low intracellular pH (pHi) on the membrane currents of snail neurone somata were studied using the internal perfusion and ion-sensitive micro-electrode techniques. Recordings with pH-sensitive micro-electrodes made while the pH of the perfusion solution was changed between 7.3 and 6.3 indicated that only with high buffer concentrations (100 mM) could pHi be changed effectively. H+ was slower to exchange into the cytoplasm than an unbuffered ion such as K+. When pHi was decreased to 5.9, large outward H+ currents could be recorded at voltages positive to -30 mV. The time course and amplitude of these currents were such that they did not affect the measurement of the peak amplitude of the fast transient K+ current (A-current), but severely contaminated both Ca2+ and delayed K+ current measurements. Low pHi blocked the A-current. The titration curve was consistent with the binding of two H ions to a site with a pK of 6.05 to block the channel. Low pHi appeared to block the slow inactivation of the delayed outward current without greatly changing its peak amplitude. However, when correction was made for the increase of H+ current at low pHi, the effect of internal H+ was found to be a block of the delayed K+ current with no consistent effect on inactivation. The Ca2+ current was also decreased at low pHi, but we were unable to determine whether this was a direct effect of pHi or secondary to a rise in internal free [Ca2+]. If no correction was made for H+ currents, the block of the Ca2+ current appeared greater and more reversible than it actually was. We conclude that under certain conditions, such as low pHi, the H+ current is a significant fraction of the total outward current in snail neurones, and may also be in a variety of other cells. The H+ currents must be accounted for under such conditions in order to study accurately the properties of K+ and Ca2+ currents.

Action Potentials↗

Serotonin: two different inhibitory actions on snail neurons.

Serotonin (5-hydroxytryptamine) inhibits snail neurons through two different mechanisms. Whereas on some cells it increases selectively the membrane permeability to chloride ions thus giving rise to a net influx of this ion, on other neurons it increases the permeability to potassium ions causing a net potassium efflux. The serotonin receptors involved in these two inhibitions are different; they also differ from the receptors involved in the excitatory action of serotonin previously described in snail neurons.

Animals↗

Experimental pathogenicity of Vibrio parahaemolyticus for the schistosome-bearing snail Biomphalaria glabrata.

The bacterium Vibrio parahaemolyticus was found to be pathogenic for the schistosome intermediate host Biomphalaria glabrata (Say). When administered topically, a nonenteritis-associated strain of the bacterium had an LD50 (median lethal dose) of 6.8 x 10(7) cells per snail. A 5% trichloroacetic acid (TCA) extract from V. parahaemolyticus was found to kill B. glabrata. Sublethal effects of V. parahaemolyticus include shell deterioration and increased heart rate. Both albino aquarium populations and naturally occurring Puerto Rican wild populations of B. glabrata are susceptible to V. parahaemolyticus. This bacterium provides a useful model for the study of pathogens and biological control of schistosome vector snails, since it causes significant mortality and is recognized as a pathogen of other invertebrates.

Animals↗

Modulatory role for the serotonergic cerebral giant cells in the feeding system of the snail, Lymnaea. I. Fine wire recording in the intact animal and pharmacology.

1. The role of the paired serotonergic cerebral giant cells (CGCs) in the feeding system of Lymnaea was examined by electrophysiological and pharmacological techniques. 2. The firing characteristics of the CGCs were recorded by fine wires attached to their cell bodies in freely moving intact snails (in vivo recording) and their "physiological" rates of firing determined during feeding and other behaviors. 3. The mean CGC firing rates recorded in vivo varied between 1 and 20 spikes/min but never reached the average rates seen in the isolated CNS (60-120 spikes/min). Maximum rates of firing were seen during bouts of radula biting/rasping movements characteristic of the consummatory phase of feeding (15 +/- 1.69 spikes/min, mean +/- SE, range 7-20 spikes/min), with lower rates seen during locomotion (6.7 +/- 0.75 spikes/min; range 5-9 spikes/min. The cells were rarely active when the animal was quiescent (1.45 +/- 0.91 spikes/min; range 0-2 spikes/min). 4. In vivo recorded CGC firing was phase locked to the feeding movements of the animal, with spikes occurring just before the opening of the mouth, during the protraction phase of the feeding cycle. 5. Evoking firing rates on the CGCs in the isolated preparation similar to those seen in vivo during rasping movements (7-20 spikes/min) did not elicit a fictive feeding pattern in an inactive preparation. Neither did bath application of 10(-9) M serotonin (5-HT; the transmitter of the CGCs). 6. To allow the modulatory role of the CGCs to be examined during patterned activity, the fictive feeding pattern was evoked in the isolated preparation by injecting depolarizing current into a modulatory neuron, the slow oscillator (SO). 7. The tonic firing activity of the CGCs was accurately maintained by current injection in the isolated preparation at rates equivalent to that occurring during feeding, locomotion, and quiescence in the intact snail. This was possible where the CGCs became silent after 1-2 h. Only when the CGCs activity was maintained at a rate (approximately 15 spikes/min) similar to that occurring during rasping, was the SO able to drive a full, high-frequency fictive feeding pattern (15-20 cycles/min). At lower rates of CGC firing, the SO-driven rhythm was either of lower frequency or no rhythm occurred at all (CGCs silent). 8. In many isolated preparations (80%) the CGCs remained active, and it was difficult to maintain specific levels of tonic activity by current injection.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

Octopamine increases the excitability of neurons in the snail feeding system by modulation of inward sodium current but not outward potassium currents.

BACKGROUND: Although octopamine has long been known to have major roles as both transmitter and modulator in arthropods, it has only recently been shown to be functionally important in molluscs, playing a role as a neurotransmitter in the feeding network of the snail Lymnaea stagnalis. The synaptic potentials cannot explain all the effects of octopamine-containing neurons on the feeding network, and here we test the hypothesis that octopamine is also a neuromodulator. RESULTS: The excitability of the B1 and B4 motoneurons in the buccal ganglia to depolarising current clamp pulses is significantly (P < < 0.05) increased by (10 microM) octopamine, whereas the B2 motoneuron becomes significantly less excitable. The ionic currents evoked by voltage steps were recorded using 2-electrode voltage clamp. The outward current of B1, B2 and B4 motoneurons had two components, a transient IA current and a sustained IK delayed-rectifier current, but neither was modulated by octopamine in any of these three buccal neurons. The fast inward current was eliminated in sodium-free saline and so is likely to be carried by sodium ions. 10 microM octopamine enhanced this current by 33 and 45% in the B1 and B4 motoneurons respectively (P < < 0.05), but a small reduction was seen in the B2 neuron. A Hodgkin-Huxley style simulation of the B1 motoneuron confirms that a 33% increase in the fast inward current by octopamine increases the excitability markedly. CONCLUSION: We conclude that octopamine is also a neuromodulator in snails, changing the excitability of the buccal neurons. This is supported by the close relationship from the voltage clamp data, through the quantitative simulation, to the action potential threshold, changing the properties of neurons in a rhythmic network. The increase in inward sodium current provides an explanation for the polycyclic modulation of the feeding system by the octopamine-containing interneurons, making feeding easier to initiate and making the feeding bursts more intense.

Animals↗

Cloning and developmental expression of Sna, a murine homologue of the Drosophila snail gene.

The genetic analysis of dorsoventral patterning in Drosophila has identified a zinc-finger gene, snail, that is required for mesoderm formation. The cloning and nuclease protection analysis of a Xenopus homologue of this gene has suggested a possible role in the mesoderm of vertebrates. Here, we describe the cloning of a murine homologue of snail, Sna, and in situ hybridisation studies of its developmental expression. Sequence analysis reveals substantial conservation of the second to fifth zinc fingers, but not of the first zinc finger in the Sna gene. Expression occurs in the ectoplacental cone, parietal endoderm, embryonic and extraembryonic mesoderm, in neural crest and in condensing precartilage. Based on the timing and spatial restriction of expression in embryonic mesoderm, we suggest that Sna might be required for the early development of this tissue, as is the case for its Drosophila counterpart. In addition, we propose that Sna might have an analogous role in the development of neural crest. The expression in condensing precartilage indicates that this gene also has a later function in chondrogenesis.

Amino Acid Sequence↗

Chemosensitivity of the osphradium of the pond snail Lymnaea stagnalis

The osphradium of the pond snail Lymnaea stagnalis was studied to determine the stimuli to which this organ responds. The following stimuli were tested: hypoxia, hypercapnia, a mixture of amino acids, a mixture of citralva and amyl acetate and a mixture of lyral, lilial and ethylvanillin. The mean nerve activity consistently increased with elevated PCO2, whereas hypoxia produced variable effects. The nerve activity became rhythmic upon application of citralva and amyl acetate, but it increased in a non-rhythmic way upon application of the other two odorant mixtures tested. Whole-cell patch-clamp recordings were made from a group of 15 neurones that lay next to the issuing osphradial nerve, to determine whether ganglion cells were involved in olfactory signal processing. All neurones tested responded to at least one of the three mixtures of odorants. Both excitatory and inhibitory responses occurred. Our results indicate that the osphradium of the pond snail Lymnaea stagnalis is sensitive to elevated PCO2 as well as to three different classes of odorants. In addition, at least some neurones within the osphradium are involved in the processing of olfactory information.

Journal Article↗

The effects of hypercapnia on force and rate of contraction and intracellular pH of perfused ventricles from the land snail Helix lucorum (L.).

The effects of hypercapnia, together with low and high levels of extracellular Ca(2+), on heart activity and intracellular pH were examined in isolated perfused hearts from the land snail Helix lucorum. In addition, the intracellular level of Ca(2+) was determined in slices of ventricles superfused with both normal and hypercapnic salines, containing low and high concentrations of Ca(2+), to investigate whether low extracellular pH affects the entry of Ca(2+) into the heart cells. We also examined the effect of a saline that simulated the composition of the haemolymph of snails after estivating for 3 months on the heart activity and intracellular pH. The results showed that hypercapnia causes decreases in the rate and force of heart contraction, and these are more pronounced in the presence of low levels of extracellular Ca(2+). Moreover, the present results indicate that Ca(2+) maintains the contractility of the heart muscle under acidic conditions and seems to act by competing with protons for the Ca(2+ )binding sites on sarcolemma. The negative effect of hypercapnia on heart activity appears to be due to a reduction in extracellular pH rather than to changes in intracellular pH.

Journal Article↗

[Geographic distribution of Schistosoma mansoni transmitter snail species in State of São Paulo].

A thorough knowledge of the geographic distribution of Schistosoma mansoni vector snails is indispensable for the control of Schistosoma mansoni schistosomiasis and its epidemiologic surveillance. From the water masses of the State of São Paulo (Brazil) 8,771 lots of snails--total of 108,244 individuals of the genus Biomphalaria--were captured between 1982 and 2002. These specimens are now part of the malacological collection of (Superintendência de Controle de Endemias--São Paulo). According to species: Biomphalaria glabrata (Say, 1818), 225 lots (6%)--8,002 (7.4%); specimens Biomphalaria tenagophila (d'Orbigny, 1835), 3,402 lots (91.7%)--88,068 (81.4%) specimens and Biomphalaria straminea (Dunker, 1848), 85 lots (2.3%)--12,174 (11.2%) specimens. The geographic distribution of B. tenagophila and B. glabrata breeding sites tends to be compact and their occupation of territory is clear-cut. B. tenagophila habitats characteristically show a tendency to cluster around municipalities with high levels of urbanization and organic pollution. The presence of B. straminea is isolated in all hydrographic basins. This situation suggests that the persistence of schistosomiasis endemic areas in the State of São Paulo depends on the chance of host-parasite contacts resulting from the concentration of B. tenagophila and B. glabrata breeding sites.

Animals↗

Influence of experimental illumination and seasonal variation on crossbreeding mating in the snail Biomphalaria glabrata.

The crossbreeding activities of the Schistosoma mansoni vector snail Biomphalaria glabrata were counted in a laboratory aquarium throughout the year under two regimes of 12h light: 12h dark from 7 A. M. to 10 P. M. Mating increased significantly in Autumn and Winter and just missed a significant inverse correlation with temperature and a direct one with locomotion. Other similar experiments were carried out to compare mating under various illumination conditions in complete daily cycle measurements. Mating counts decreased under the regimes which submitted snail to a total exposure of 12h light and 12h dark during a daily cycle in the following sequence: 12h light:12h dark alternating hourly with light gradient, 12h light:12h dark, 1h light:1h dark and 12h dark:12h light. Under two constant illuminations, the mating scored less than under the previous conditions, except under 12h dark:12h light. Under darkness the mating count was lower than under light conditions. There was no way to differentiate the night and day rhythms of mating on different days in each regime, except for mating under 12h light:12h dark alternating with light gradient, constant dark and 12h dark:12h light conditions. Mating increased in certain light and temperature conditions, in which the intensities should have an optimum value.

Animals↗

Potential schistosome-vector snails and associated trematodes in ricefields of Corrients province, Argentina. Preliminary results.

Considering the possibility of introduction of schistosomiasis mansoni into Argentina as a consequence of dam construction on the Rio De La Plata basin, preliminary studies have been carried out on agrosystems such as ricefields in Corrientes province with the following purposes: 1) to survey and estimate the relative abundance of planorbids and identify potential vector species; 2) to identify environmental factors capable of influencing Biomphalaria population dynamics; and 3) to find out snail-parasite associations and estimate snail infection rates in order to detect possible competitive interactions between larval stages of native trematodes that could be used in biological control of Schistosoma mansoni. Three potential schistosome vectors were detected in ricefields, namely Biomphalaria straminea, B. tenagophila and B. peregrina, although B. orbignyi, a species refractory to infection with S. mansoni, proved the most frequent and abundant. Positive correlations (P less than 0.05) were found between Biomphalaria abundance and some environmental parameters: conductivity, hardness, calcium, nitrites plus nitrates, ammonium and bicarbonates. Water temperature correlation was negative (P less than 0.05). No correlation (P less than 0.05) was found in total iron, phosphates (SRP), pH and soil granulometry.

Animals↗

DNA polymorphism of schistosomes and their snail hosts.

Analysis of the genomes of schistosomes and one of their intermediate hosts, Biomphalaria glabrata, using Random Amplified Polymorphic DNA (RAPD) demonstrated that intraspecific genetic polymorphism in the parasite is limited but in the snail is highly pronounced. This suggests an important role for the snail in the determination of the epidemiology of the disease. In addition to their intraspecific stability, schistosome derived RAPDs exhibit a high level of interspecific polymorphism and are thus ideal for the construction of phylogenetic trees. For the detection of intraspecific polymorphisms extensive variation in the mitochondrial DNA is being exploited for the development of a PCR based test for Schistosoma mansoni. Gene level polymorphisms are being analyzed by Low Stringency Single Specific Primer PCR.

Animals↗

A low stringency polymerase chain reaction approach to the identification of Biomphalaria glabrata and B. tenagophila, intermediate snail hosts of Schistosoma mansoni in Brazil.

The low stringency-polymerase chain reaction (LS-PCR) with a pair of specific primers for the amplification of the 18S rRNA gene was evaluated as a means of differentiating between the two Schistosoma mansoni intermediate host species in Brazil: Biomphalaria glabrata and B. tenagophila. Individual snails obtained from different states of Brazil were used and the amplification patterns obtained showed a high degree of genetic variability in these species. Nevertheless, 4 and 3 clearly defined specific diagnostic bands was observed in individuals from B. glabrata and B. tenagophila respectively. The detection of snail specific diagnostic bands suggests the possibility of reliable species differentiation at the DNA level using LS-PCR.

Animals↗

Geographical distribution of Biomphalaria snails in the State of Minas Gerais, Brazil.

Published and unpublished observations on geographical distribution of Biomphalaria snails in the State of Minas Gerais, Brazil, were compiled. This work is aimed at knowing the present occurrence of Biomphalaria species in this region, and at contributing to the elaboration of the planorbid chart of Minas Gerais. In malacological surveys, performed by several researchers, the presence of seven species of this genus was recorded. Those planorbids were found in 12 mesoregions, in 283 (33.1%) municipalities out of 853 with the following distribution: B. glabrata (185 municipalities), B. straminea (125), B. tenagophila (58), B. peregrina (57), B. schrammi (26), B. intermedia (20) and B. occidentalis (2). B. glabrata and B. tenagophila are found naturally infected by Schistosoma mansoni in Minas Gerais. In 24 municipalities the three snail hosts of S. mansoni in Brazil, B. glabrata, B. tenagophila and B. straminea, are present.

Animals↗

Primary culture of the region of the amebocyte-producing organ of the snail Biomphalaria glabrata, the intermediate host of Schistosoma mansoni.

Biomphalaria glabrata snails are major hosts for the digenetic trematoda Schistosoma mansoni, the causative agent of human schistosomiasis. The success or failure of the infection will be dependent on the mobilization of the molluskan internal defense system, where a major role will be played by circulating hemocytes produced by the APO (amebocyte-producing organ) of the snail. In this report, the primary culture of the APO region of B. glabrata was obtained for the first time, as well as a control culture of the ovotestis. Three different cell populations migrated easily from the explants in culture, with no need of any dispersion agent. The cells grew in suspension at an incubation temperature of 15 degrees C and the cultures were maintained viable for up to two weeks. Two of these cell populations obtained resembled cell types known to be present in the hemolymph of Biomphalaria. The availability of APO cells in culture may contribute to a better understanding of the internal defense in mollusks, in general, as well as the specific response of B. glabrata to S. mansoni infection.

Animals↗

Occurrence of a cytochrome P-450-containing mixed-function oxidase system in the pond snail, Lymnaea stagnalis.

1. The occurrence of an as yet unidentified cytochrome P-450 in the microsomal fraction of the digestive gland of the snail, Lymnaea stagnalis was studied. 2. Studies in vivo and in vitro (digestive gland homogenates or the 170,000g fraction) of the cytochrome P-450-mediated metabolism of substrates such as biphenyl, pentoxy- and ethoxy-resorufin and aminopyrine have been made. 3. Cytochrome P-450 concentration in the digestive gland calculated from CO-difference spectra was 0.30 +/- 0.05 nmol/g tissue. This amount was not increased either by phenobarbital or by 3-methylcholanthrene. 4. Aniline binding spectra resembled normal type II spectra, while type I model substrates such as hexobarbital and 2,2'-dichlorobiphenyl showed type II- or reversed type I-like spectra. 5. O-Deethylation of 7-ethoxyresorufin did not occur, but 7-pentoxyresorufin O-depentylation activity (80.4 +/- 28.6 pmol resorufin/g per hour) and aminopyrine N-demethylation activity (375 +/- 96 pmol formaldehyde/g per minute) were demonstrated. 6. 4-Hydroxybiphenyl was the major metabolite of biphenyl, while minor amounts of 2-hydroxybiphenyl were formed (in vivo: 63.7 nmol 4-hydroxybiphenyl and 3.33 nmol 2-hydroxybiphenyl per snail per 24 h, after an oral dose of 778.2 nmol biphenyl; in vitro 118 +/- 21 pmol and 21 +/- 9 nmol, respectively (digestive gland homogenate/mg protein, per hour). 7. The results indicate that the isoenzymes involved in the observed MFO activities resemble isoenzymes P-450b/e.

Aminopyrine N-Demethylase↗