The hyperglycemic factor of the CNS of the freshwater snail Lymnaea stagnalis: interaction with glucose stimulation of glycogen synthesis and evidence for its release during anaerobiosis

In the freshwater pulmonate snail Lymnaea stagnalis glycogen synthesis in the glycogen storage cells (GC) is stimulated by increasing external glucose concentrations. On the other hand, a hyperglycemic factor from the CNS inhibits glycogen synthesis and stimulates glycogen breakdown in these cells. This CNS factor may be involved in the physiological control of glycogen mobilization. In the present study the interaction between the inhibiting effect of the CNS factor and the stimulating effect of increasing glucose concentrations on glycogen synthesis in isolated GC was investigated. The results, analyzed by determination of the Michaelis-Menten parameters for saturation kinetics, suggest that the CNS factor increases the glucose concentration required for half-maximal stimulation of glycogen synthesis, but does not influence the maximum rate of synthesis (competitive type of inhibition). The role of the hyperglycemic factor during conditions of glycogen breakdown was investigated by extirpation of the cerebral ganglia (-CG), which contain the main release sites of the factor. Extirpation did not affect the hemolymph glucose concentration or the glycogen levels in the GC during starvation. However, -CG animals showed reduced levels of the hyperglycemia normally associated with exposure to anaerobic conditions.     

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.     

A neuropeptide (Calfluxin) is involved in the influx of calcium into mitochondria of the albumen gland of the freshwater snail Lymnaea stagnalis

In the course of a study on the role of Ca2+ in the regulation of the secretion process in a female accessory sex gland, the albumen gland of the freshwater snail Lymnaea stagnalis, it appeared that the percentage of mitochondria containing Ca2+ deposits varied significantly in the successive stages of the egg-laying process. Ca2+ was demonstrated at the ultrastructural level with the pyroantimonate precipitation technique. A significant increase of the percentage can be correlated with the onset of synthetic activity. Two gonadotropic (neuro) hormones are known to stimulate synthetic activity, viz. the cerebral caudo-dorsal cell hormone (CDCH) and the dorsal body hormone (DBH). In vitro experiments involving addition of extracts of various parts of the cerebral ganglion-dorsal body (DB) complex to albumen glands showed that neither CDCH nor DBH causes the influx of Ca2+ into the mitochondria of the gland cells, but that another factor is responsible. The factor is present in the cerebral commissure. It is pronase sensitive and heat stable. The factor is released from isolated cerebral ganglia-DB complexes upon stimulation with 8-chlorophenylthio-cAMP. Probably the factor is a CDC peptide. It is called Calfluxin.     

Structure of one of the neuropeptides of the egg-laying hormone precursor of Lymnaea

The neurosecretory caudo-dorsal of the freshwater pulmonate snail Lymnaea stagnalis, a peptidergic system controlling egg laying and egg-laying behavior, produce several neuropeptides. One of these peptides, calfluxin, causes the influx of Ca2+ into the albumen gland, a female sex gland of the snail. In the present study calfluxin was purified and the amino acid composition was determined. The ratio of the amino acids appeared to be very close to that of one of the predicted peptides present on the egg-laying hormone precursor of Lymnaea. This peptide was synthesized and shows a clear biological activity in the bioassay. Furthermore, it shows a chromatographic behavior similar to that of the natural peptide. Based on these evidences it is concluded that the sequence of calfluxin is: Arg-Val-Asp-Ser-Ala-Asp-Glu-Ser-Asn-Asp-Asp-Gly-Phe-Asp. Calfluxin shows a remarkable homology with the sequence of one of the predicted peptides on the egg-laying hormone precursor of the marine opisthobranch Aplysia californica.     

Purification and sequencing of molluscan insulin-related peptide II from the neuroendocrine light green cells in Lymnaea stagnalis.

The growth-controlling neuroendocrine light green cells of the freshwater snail, Lymnaea stagnalis, express a family of genes encoding structurally related, yet distinct, molluscan insulin-related peptides (MIPs). In the present study one of these peptides, MIP II, has been isolated and structurally identified. MIP II is a heterodimer of A and B chains connected by disulfide bonds. Both chains are N-terminally blocked with pyroglutamate. After cleaving of the A and B chains and deblocking with pyroglutamate amino-peptidase their sequences have been determined as: A chain: pQRTTNLVCECCFNYCTPDVVRKYCY and B chain: pQSSCSLSSRPHPRGICGSNLAGFRAFICSNQNSPS. In comparison with the MIP II sequence based on complementary DNA studies, it is clear that the two C-terminal amino acid residues of the B chain are posttranslationally removed. In addition, the glutamic acid residue in A chain was recovered in very low yields during Edman degradation, suggesting that the residue may be posttranslationally modified.     

Age-related changes in female reproductive activity and growth in the mollusc Lymnaea stagnalis

Age-related changes in reproduction and growth of the pulmonate snail Lymnaea stagnalis were studied in animals under standard culture conditions. Longitudinal studies as well as cross-sectional studies were done. Egg-laying activity starts in Lymnaea at an age of about 2 months. Up to an age of about 250 days it increases, and in older snails it decreases with age. Cross-sectional studies showed that the increase in egg-laying activity is due to an increase in the number of animals that lay eggs and to an increase in the egg-laying frequency of individuals. The decrease in egg-laying activity is due to a decrease in the egg-laying frequency of individual animals and to an increase in the number of animals that stop egg laying. Growth (shell length and body weight) ceases at about the same age at which egg laying begins to decrease. The results are discussed in relation to the organization of the neuroendocrine system in Lymnaea and to results of earlier studies on age-related changes in peptidergic neurons in the CNS of Lymnaea.     

Isolation of neurosecretory granules from the neurohaemal areas of peptidergic systems of Lymnaea stagnalis, with special reference to the ovulation hormone-producing caudodorsal cells

Neurosecretory granules (NSG) were isolated by density-gradient centrifugation from homogenates of the intercerebral commissure (COM) and the median lip nerves (LN) of the freshwater snail Lymnaea stagnalis. COM and LN are rich in axon terminals of the ovulation hormone-producing caudodorsal cells (CDC) and of the growth hormone-producing light green cells (LGC), respectively. Electron microscopy of COM isolates showed that a single fraction contained large numbers of NSG, which were identical to NSG of CDC axon terminals of the intact COM. High ovulation hormone activity was associated with this fraction. LN NSG were also isolated in large numbers in a single fraction. The isolated LN NSG were identical to NSG of intact LGC axon terminals. They appeared to have a higher specific gravity than CDC NSG.     

Stimulation of ornithine decarboxylase activity in Lymnaea stagnalis after a single injection with molluscan growth hormone

The effect of single crude growth hormone injections on the activity of ornithine decarboxylase (ODC) in the mantle edge of the freshwater snail Lymnaea stagnalis, was studied. The injections always stimulated the ODC activity, although the response was variable. It is possible, that this ODC-stimulating effect can be used as a bioassay during purification of the growth hormone.     

Secretory activity and postembryonic development of the tentacle sensory system controlling growth hormone-producing neurons in Lymnaea stagnalis

The cerebral neuroendocrine peptidergic light green cells (LGC) of the freshwater snail Lymnaea stagnalis regulate body growth. The LGC are controlled by a tentacle sensory system that consists of two types (S1 and S2) of primary sensory neuron located at the base of each tentacle. Sensory (S2) axons make synaptic contacts (type A synapse-like structures) with the somata and axons of the LGC, where they release the contents of secretory granules, by exocytosis (demonstrated with the ultrastructural tannic acid-Ringer incubation method). Ultracytochemistry indicates that the granule contents are glycoproteinaceous. Furthermore, the S2 axons release secretory material in a nonsynaptic fashion into the interneuronal space of the central nervous system (CNS), at the level of the neuropiles of the cerebral ganglia and of the cerebral commissure. This release occurs by exocytosis from nonsynaptic release sites. It is proposed that the tentacle sensory system not only (synaptically) controls LGC activity but also influences other, remote neuronal targets in the CNS in a nonsynaptic ("at long distance," "paracrine," "hormone-like") fashion. Already in newly hatched snails (with a shell height of 1 mm) S2 axons show a fair rate of exocytotic activity, in both synaptic and nonsynaptic respects. During postembryonic development the secretory capacity of the S2 sensory neurons increases markedly, by increases in (1) the number of axons, (2) the size of the secretory granules, and (3) exocytosis activity. This increased capacity may meet a growing demand of the developing CNS, including the LGC, for neurochemical input from the tentacle sensory system.     

Purification and sequencing of molluscan insulin-related peptide I (MIP I) from the neuroendocrine light green cells of Lymnaea stagnalis.

The body growth controlling cerebral neuroendocrine light green cells of the freshwater snail, Lymnaea stagnalis, express various members of a gene family encoding different though related prepromolluscan insulin-related peptides. In the present study, molluscan insulin-related peptide I (MIP I) together with the corresponding connecting peptide, C alpha peptide, have been isolated and structurally identified. MIP I is a heterodimer of A and B chains bonded by disulphide bridges. Two isoforms of MIP I could be discerned. Mass spectrometry revealed that of one form both the A and B chains have N-terminal pyroglutamyl residues, whereas of the other form only the B chain has such residues. After removal of the pyroglutamyl residues with pyroglutamate aminopeptidase, followed by disulphide bond cleavage and pyridylethylation of cysteine residues, the sequences of MIP I have been determined using Edman degradation as: A chain: (p)QGTTNIVCECCMKPCTLSELRQYCP; B chain: pQPSACNINDRPHRRGVCGSALADLVDPACSSSNGPA. The C alpha peptide has also been isolated and its sequence was determined as NAETDLDDPLRNIKLSSESALTYLY. These sequences are in agreement with those predicted by a cDNA sequence encoding preproMIP I, with the exception that the two C-terminal amino acids of the B chain are posttranslationally removed.     

Immunocytochemical localization in the central nervous system of four different insect species of molecules immunoreactive against peptides present in the caudodorsal cells of Lymnaea stagnalis

The use of polyclonal antisera directed against three peptides (ovulation hormone (CDCH), alpha, and beta caudodorsal cell peptide (alpha- and beta-CDCP) produced by the caudodorsal cells of the snail Lymnaea stagnalis resulted in positive immunoreaction in Sarcophaga bullata (Diptera), Leptinotarsa decemlineata (Coleoptera), Locusta migratoria, and Periplaneta americana (Orthoptera). In three species, colocalization was detected using the antisera against CDCH and alpha-CDCP.     

Neurohormonal control of growth and carbohydrate metabolism by the light green cells in Lymnaea stagnalis.

The neuroendocrine light green cells (LGCs), 4 clusters of together approximately 150 giant neurons in the cerebral ganglia of the freshwater gastropod, Lymnaea stagnalis, have been suggested to be involved in the control of growth. The present study examines in greater detail this role and possible actions on energy metabolism. Growth indices (total body and organ wet and dry weights, as well as protein and DNA contents of the organs) and metabolic indices (tissue lipid, polysaccharide and glucose levels) were compared in LGC extirpated/reimplanted with control groups. LGC extirpation in rapidly growing juvenile snails immediately arrested growth, which was restored by reimplantation of cerebral ganglia with LGCs but not by cerebral ganglia without LGCs, indicating their neuroendocrine control of growth. The LGCs stimulate a pattern of organ growth, which is in proportion to the growth of the whole body except for the shell, which shows a disproportionally faster growth due to calcium deposition over the whole surface. The data on protein and DNA in the organs strongly suggest that the LGCs induce growth by stimulating cell multiplication. The LGCs maintain low tissue glycogen reserves and hemolymph concentrations of both glycogen and glucose. The secretions of these cells stimulate the uptake of glucose by the growing tissues with no apparent effects on lipid metabolism.     

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.     

Effect of thyrotropin-releasing hormone and its metabolites on the secretion of sulfated polysaccharides by foot integument of a pond snail

The effect of thyrotropin-releasing hormone (TRH), TRH metabolites, and a TRH analog on the secretion of 35S-labeled sulfated polysaccharides from in vitro incubated foot integument of the pond snail Lymnaea stagnalis palustris was assessed. Whereas TRH significantly inhibited the secretion of 35S-labeled polysaccharides, its metabolite deamido TRH significantly stimulated polysaccharide secretion. Histidyl-proline-diketopiperazine did not alter the secretion of 35S-labeled sulfated polysaccharides. Foot integument exhibited considerable TRH-peptidase activity in vitro. The principal proline-containing TRH metabolites were deamido-TRH and proline. The deamido-TRH content of the foot, mantle, and hemolymph was determined by radioimmunoassay. All tissues contained deamido-TRH. These findings suggest that the secretory activity of skin mucus glands of L. stagnalis palustris is the result of the stimulatory action of deamido-TRH and the inhibitory action of TRH. 3Methyl-2histidyl-TRH, TRH, vasotocin, and bombesin did not alter the polysaccharide secretion by in vitro incubated foot integument, indicating that the changes in mucus secretion after TRH and deamido-TRH administration are specific and that the gastropod "TRH receptors" may differ from the mammalian brain and anterior pituitary gland TRH receptors.     

The case of dermatitis in the human as a result of the influence of the cercaria of the bird schistosome

There are numerous cases of dermatitis described world-wide and resulting from the influence of the furcocercaria of the bird schistosome. The host snail species of the schistosome, producing thousands of furcocercaria are common in our country. But, because of too general symptoms of dermatitis resulting from the influence of the schistosome of the larvae, there are many unrecognisable cases. The reaction described was typical of a twenty-seven-year-old man collecting snails in the Piechota Bay in Gostynin (the Province of Kuyavia and Pomerania). In that reservoir, individuals of Lymnaea stagnalis were found, which were infected with sporocysts and furcocercaria of Trichobilharzia ocellata. In the laboratory conditions, a high expulsion of cercaria was observed, which amounted to over 8000 larvae per snail.     

Alpha-lactalbumin affects the acceptor specificity of Lymnaea stagnalis albumen gland UDP-GalNAc:GlcNAc beta-R beta 1-->4-N-acetylgalactosaminyltransferase: synthesis of GalNAc beta 1-->4Glc.

The N,N'-diacetyllactosediamine (lacdiNAc) pathway of complex-type oligosaccharide synthesis is controlled by a UDP-GalNAc:GlcNAc beta-R beta 1-->4-N-acetylgalac-tesaminyltransferase (beta 4-GalNAcT) that acts analogously to the common UDP-Gal:GlcNAc beta-R beta 1-->4-galactosyltransferase (beta 4-GalT). LacdiNAc-based chains particularly occur in invertebrates and cognate beta 4-GalNAcTs have been identified in the snail Lymnaea stagnalis, in two schistosomal species, and in several lepldopteran insect cell lines. Because of the similarity in reactions catalyzed by both enzymes, we investigated whether L. stagnalis albumen gland beta 4-GalNAcT would share with mammalian beta 4-GalT the property of interacting with alpha-lactalbumin (alpha-LA), a protein that only occurs in the lactating mammary gland, to form a complex in which the specificity of the enzyme is changed. It was found that, under conditions where beta 4-GalT forms the lactose synthase complex with alpha-LA, the snail beta 4-GalNAcT was induced by this protein to act on Glc with a > 100-fold increased efficiency, resulting in the formation of the lactose analog GalNAc beta 1-->4Glc. This forms the second example of a glycosyltransferase, the specificity of which can be altered by a modifier protein. So far, however, no protein fraction could be isolated from L. stagnalis that could likewise interact with the beta 4-GalNAcT. Neither had lysozyme c, a protein that is homologous to alpha-LA, an effect on the specificity of the enzyme. These results raise the question of how the capability to interact with alpha-LA has been conserved in the snail enzyme during evolution without any apparent selective pressure. They also suggest that snail beta 4-GalNAcT and mammalian beta 4-GalT show similarity at a molecular level and allows the identification of the beta 4-GalNAcT as a candidate member of the beta 4-GalT family.     

EFFECTS OF SINGLE,BINARY AND TERTIARY COMBINATIONS WITH Jatropha gossypifolia AND OTHER PLANT-DERIVED MOLLUSCICIDES ON REPRODUCTION AND SURVIVAL OF THE SNAIL Lymnaea acuminata

The effect of sub-lethal doses (40% and 80% of LC₅₀/24h) of plant derived molluscicides of singly, binary (1:1) and tertiary (1:1:1) combinations of the Rutin, Ellagic acid, Betulin and taraxerol with J. gossypifolia latex, leaf and stem bark powder extracts and their active component on the reproduction of freshwater snail Lymnaea acuminata have been studied. It was observed that the J. gossypifolia latex, stem bark, individual leaf and their combinations with other plant derived active molluscicidal components caused a significant reduction in fecundity, hatchability and survival of young snails. It is believed that sub-lethal exposure of these molluscicides on snail reproduction is a complex process involving more than one factor in reducing the reproductive capacity.     

The effect of the growth hormone of Lymnaea stagnalis on shell calcification.

An experimental study was made of the effects of the growth hormone, produced by the neurosecretory Light Green Cells (LGC) of the cerebral ganglia, on shell calcification in the freshwater snail Lymnaea stagnalis.LGC removal had no effects on the concentration of calcium in the shell and the shell edge nor in the soft body parts with the exception of the mantle edge: In this tissue, 21 days after the operation, it was 30% lower than normal. In control as well as in -LGC snails, the half-life of ⁴⁷Ca, injected into the hemolymph, was less than or equal to 5 min. For 5–30 min after the injection no increase was observed in the specific activity of the mantle edge, the mantle, and the other soft body parts. On the other hand, the shell edge showed a continuous increase in specific activity, but that in -LGC snails was significantly lower (by 69–92%) than that in the control snails. The labeling of the shell did not show this increase and was not affected by LGC removal. It is concluded that, with regard to shell growth, the growth hormone acts specifically on the mantle edge; it stimulates the formation of the outer crystalline layer and probably also the periostracum.     

Morphology of neurosecretory cells in basommatophoran snails homologous with egg-laying and growth hormone-producing cells of Lymnaea stagnalis

In a light and electron microscope study, neurosecretory cells morphologically homologous with the egg-laying hormone-producing caudodorsal cells (CDC) and growth hormone-producing dorsal cells (DC, light green cells) of the freshwater basommatophoran snail Lymnaea stagnalis have been found in five genera (seven species) of Basommatophora, viz. in Lymnaea palustris and Lymnaea ovata, in Planorbis planorbis and Planorbis vortex, in Planorbarius corneus, in Bulinus truncatus, and in Biomphalaria glabrata. It is concluded that the functions of these cells are homologous as well. The homologies of the respective neuron types regard their locations in the cerebral ganglia, their clustering in groups, location of their neurohemal area (CDC: cerebral commissure; DC: median lip nerves), and ultrastructural characteristics (e.g., abundance of rough endoplasmic reticulum, well-developed Golgi apparatus, presence of two types of neuron-specific secretory granules, and release of granule contents by exocytosis into the hemolymph). In addition, CDC show large electron-dense granules and DC reveal infoldings of the plasma membrane at the abaxonal side of the soma as well as synaptic input. On the other hand, each neuron type shows species-specific characteristics, particularly with regard to the number of cells and the structure of the neurohemal area. Furthermore, the CDC show marked differences between genera in the morphology (especially the mean diameter) of type 2 and, particularly, type 1 secretory granules. The morphology of the two types of secretory granules in the DC differs strongly between species. The possible relation between the morphology and the chemical contents of secretory granules has been discussed.     

Effect of glucagon on the metabolism of the eal, Anguilla japonica

In both hypophysectomized and intact eels, a single injection of glucagon at a dose of 0.1 mg kg^?1 rapidly evokes a marked hyperglycemic response and an increase in total ammonia excretion. In the intact eel, there is a transient decline in liver glycogen levels. In hypophysectomized eels, the liver glycogen is already low and no further drop is recorded. Instead there is a significant transient decline in muscle glycogen levels. From the metabolic balance sheet constructed by combining data on changes in nitrogen excretion, respiratory exchange, and metabolite content in serum, liver, and parietal muscle, it becomes apparent that during 0 to 2 hr after glucagon administration, there is an increase in net nitrogen and carbohydrate catabolism while fat is not utilized. A concomitant decline in serum lipids appears to be due to uptake into cells without oxidation. The mobilization of protein as the major metabolic substrate is facilitated by stimulation of hepatic glutamate-oxaloacetate transaminase enzyme activity.