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Electron microscopic studies on radular tooth formation in the snails Helix pomatia L. and Limax flavus L. (Pulmonata, Stylommatophora).

The radular teeth are secreted at the posterior end of the radular gland and move slowly towards the buccal cavity where they start to function. Helix pomatia and Limax flavus were examined to determine whether the newly formed teeth already show their definite species specific shape, or whether they are gradually finished and moulded in the radular gland. Scanning electron micrographs of Helix pomatia show that teeth are secreted in the odontoblast region in their final form. Their surface is still uneven at the outset; the same is true for the newest teeth of Limax flavus. Older teeth ready for use have a smooth surface. This change seems to be brought about by secretory activity of the superior epithelium of the radular sac. Air-dried radulae, previously isolated by KOH maceration, show considerable artefacts at their posterior end. Maceration leads to shrinking of the newest teeth, but does not change their contours. The newly secreted but as yet unhardened teeth become greatly deformed during the drying process.

Cytoplasm↗

Electron microscopic localization of chitin using colloidal gold labelled with wheat germ agglutinin.

The lectin wheat germ agglutinin (WGA) has a binding site which is able to bind a sequence of three N-acetyl-glucosamine residues. Therefore, it has a very strong affinity for the polymers of this sugar, especially chitin. Colloidal gold can be labelled with WGA and used as a specific electron-dense marker for the electron-microscopic localization of chitin. The specificity of the WGA-gold binding can be checked by competitive inhibition with 5-10 mM triacetyl chitotriose. The reliability of this method was tested in three species. In the formation zone of the radula of the snail, Biomphalaria glabrata Say, chitin or chitin precursors were localized in vesicles of the odontoblasts, outside the extremely long microvilli of odontoblasts and in the newly formed teeth. The inner peritrophic envelope of the earwig, Forficula auricularia L., is characterized by an orthogonal texture of bundles of microfibrils that are thought to contain chitin. The presence of chitin was proved using the present method. In the peritrophic membranes of the blowfly, Calliphora erythrocephala Meigen, it was possible to differentiate between chitin and glycoproteins which have N-acetylglucosamine residues.

Animals↗

Tooth hardness increases with zinc-content in mandibles of young adult leaf-cutter ants.

A wide variety of arthropods and members of other phyla have elevated concentrations of Zn, Mn, other heavy metals and halogens in their jaws, leg claws, and other "tools" for interacting with the environment. While measured Zn concentrations reach 25% of dry mass in scorpion stings, concentrations are often lower than this and the enriched structures are not heavily biomineralized like vertebrate teeth and the radula of mollusks. For this reason, the degree to which the inorganic components of these structures modify their mechanical properties is in question. Here we address this problem by measuring hardness during the development of Zn accumulations in ant mandibles. We found that Zn is incorporated into the mandibular teeth of leaf-cutter ants during early adult life, reaching concentrations of about 16% of dry mass. We show that the hardness of the mandibular teeth increases nearly three-fold as the adults age and that hardness correlates with Zn content ( r=0.91). We suggest that young adults rarely cut leaves partly because their mandibles are not yet rich in Zn. Zinc enrichment (along with enrichment by other heavy metals and halogens) may play an unrecognized role in the behavioral ecology and evolution of a wide variety of invertebrates.

Animals↗

Participation of GABA in establishing behavioral hierarchies in the terrestrial snail.

GABA-immunoreactive fibers were observed in the neuropile of each ganglion of Helix lucorum, while GABA-immunoreactive neural somata were found only in the buccal, cerebral, and pedal ganglia. Bath application of 10(-5) M GABA to the preparation "buccal mass-buccal ganglia" elicited a sequence of radula movements characteristic of feeding behavior. Corresponding bursts of activity were recorded in the buccal nerves under GABA application and in the buccal neurons recorded optically. In preparations of isolated central nervous system, the bath applications of GABA (10(-5) to 10(-4) M) elicited no changes in synaptic input of the premotor interneurons involved in the withdrawal behavior. However, a significant decrease in amplitude of the synaptic input and in the number of spikes in responses elicited by the test nerve stimulation was observed in metacerebral serotonergic neurons involved in modulating the feeding behavior. GABA application inhibited the spontaneous spike activity in some pedal serotonergic neurons involved in the network underlying withdrawal responses and evoked bursting activity in the other neurons of this functional group. The effects of GABA application on mechanically isolated serotonergic neurons suggest that the primary effect of GABA is inhibition. Thus, our results give evidence of the putative role of GABA in activating the feeding behavior and in the synergistic suppression of serotonergic modulation of the withdrawal behavior and serotonergic modulation of feeding, which has corresponded to the observed behavioral suppression of withdrawal reactions during feeding.

Animals↗

Passive hinge forces in the feeding apparatus of Aplysia aid retraction during biting but not during swallowing.

Swallowing and biting responses in the marine mollusk Aplysia are both mediated by a cyclical alternation of protraction and retraction movements of the grasping structure, the radula and underlying odontophore, within the feeding apparatus of the animal, the buccal mass. In vivo observations demonstrate that Aplysia biting is associated with strong protractions and rapid initial retractions, whereas Aplysia swallowing is associated with weaker protractions and slower initial retractions. During biting, the musculature joining the radula/odontophore to the buccal mass (termed the "hinge") is stretched more than in swallowing. To test the hypothesis that stretch of the hinge might contribute to rapid retractions observed in biting, we analyzed the hinge's passive properties. During biting, the hinge is stretched sufficiently to assist retraction. In contrast, during swallowing, the hinge is not stretched sufficiently for its passive forces to assist retraction, because the odontophore's anterior movement is smaller than during biting. A quantitative model demonstrated that steady-state passive forces were sufficient to generate the retraction movements observed during biting. Experimental measures of the relative magnitude of the hinge's active and passive forces at the protraction displacements of biting suggest that passive forces are at least a third of the total force.

Animals↗

Nitric oxide level regulates the embryonic development of the pond snail Lymnaea stagnalis: pharmacological, behavioral, and ultrastructural studies.

On the basis of the distribution of NADPH-diaphorase (NADPH-d) activity, we have previously suggested a role for nitric oxide (NO) in the development of Lymnaea stagnalis. In the present study, the long-term effects of NO donors (sodium nitroprusside, S-nitroso-N-acetyl-penicillamine) and nitric oxide synthase (NOS) inhibitors (nitro-L-arginine methyl-ester [L-NAME], N(G)-nitro-L-arginine [L-NOARG]) were tested on the survival, length of embryonic (intracapsular) life, locomotion (gliding), heartbeat activity and feeding behavior, as well as on the ultrastructure of the developing ganglia in the embryonic Lymnaea. No effect of any of the substances applied can be observed under 10(-5) M concentration, whereas at 10(-3) M concentration both kinds of treatment proved to be toxic. Between 10(-5) M and 10(-3) M concentrations the effects are reversible. At 10(-4) M concentration, NO donors slightly increase the frequency of gliding and heartbeat of E70% embryos, and evoke a more than twofold enhancement of the feeding activity, i.e., the frequency of radula protrusions in the E90% embryonic stage. In contrast, NOS inhibitors at 10(-4) M concentration strongly inhibit the locomotion and heartbeat of E70% embryos, and the feeding of E90% embryos. Under 10(-3) M concentration, L-arginine diminishes the effect of NOARG, whereas the D-isomer of NAME has little or no significant effect. Neither type of treatment alters the course of gangliogenesis, and the light-microscopic appearance of neurons also remains unaffected. Ultrastructural analysis of the central nervous system of E90% embryos treated with 10(-4) M NOS inhibitors revealed a significant reduction of the glycogen granule content and accumulation of lipid droplets in a number of the neuronal perikarya, as well as the occurrence of disintegrated mitochondria in axonal profiles. The effect of 10(-4) M NO donors is mainly characterized by the increased number of lysosomes, disintegrated mitochondria and degenerating axonal profiles. The present findings suggest that NO is involved in the regulation of different behaviors and physiological functions, such as feeding activity, locomotion and heartbeat, during the embryonic development of Lymnaea. Changes observed in neuronal ultrastructure in ganglia seem to indicate NOergic regulatory processes at the central level.

Animals↗

Mytilus inhibitory peptides (MIP) in the central and peripheral nervous system of the pulmonate gastropods, Lymnaea stagnalis and Helix pomatia: distribution and physiological actions.

The distribution and neuroanatomy of Mytilus inhibitory peptides (MIP)-containing neurons in the central nervous system and their innervation pattern in the peripheral nervous system of the pulmonate snail species, Lymnaea stagnalis and Helix pomatia, have been investigated immunocytochemically, by applying an antibody raised to GSPMFVamide. A significant number of immunoreactive neurons occurs in the central nervous system of both species (Lymnaea: ca 600-700, Helix: ca 400-500), but their distribution is different. In Lymnaea, labeled neurons are found in all central ganglia where a number of large and giant neurons, previously identified physiologically, reveal MIP immunoreactivity. In Helix, most of the immunolabeled neurons are small (12-30 microm) and concentrated in the buccal and cerebral ganglia; the parietal ganglia are free of labeled cells. In both species, the ganglionic neuropils, peripheral nerves, connectives, and commissures are richly supplied with immunolabeled fibers. The MIP-immunoreactive innervation pattern in the heart, intestine, buccal mass and radula, and foot is similar in both species, with labeled axonal bundles and terminal-like arborizations (buccal mass, foot) or a network of varicose fibers (heart, intestine). Intrinsic neurons are not present in these tissues. The application of GSPYFVamide inhibits the spontaneous contractions of the esophageal longitudinal musculature in Helix, indicating the bioactivity of the peptide. An outside-out patch-clamp technique has demonstrated that GSPYFVamide opens the K+ channels in central nerve cells of Helix. Injection of GSPYFVamide into the body cavity inhibits the feeding of starved Helix. A wide modulatory role of MIP at central and peripheral levels is suggested in Lymnaea and Helix, including the participation in intercellular signalling processes and remote neurohormonal-like control effects.

Animals↗

Characterization of the calcium biomineral in the radular teeth of Chiton pelliserpentis.

The radula in a group of molluscan invertebrates, the chitons (Polyplacophora), is a ribbon-like apparatus used for feeding and which bears a series of distinctive mineralized teeth called the major lateral teeth. While some chiton species deposit only iron biominerals in these teeth, many others deposit both iron and calcium. In this study, the calcium biomineral in the teeth of one of the latter types of species, the Australian east-coast chiton, Chiton pelliserpentis, has been isolated and examined for the first time. Spectroscopic and crystallographic techniques have identified the biomineral as a carbonate-substituted apatite with significant fluoride substitution also likely. Fourier-transform infrared and laser Raman spectroscopy indicated that the carbonate content was less than that of either bovine tibia cortical bone or human tooth enamel. X-ray diffraction analysis showed the biomineral to be poorly crystalline due to small crystal size and appreciable anionic substitution. The lattice parameters were calculated to be a = 9.382 A and c = 6.883 A, which are suggestive of a fluorapatite material. It is postulated that structural and biochemical differences in the tooth organic matrix of different chiton species will ultimately determine if the teeth become partly calcified or iron mineralized only.

Animals↗

A molluscan neuropeptide related to the crustacean hormone, RPCH.

A peptide that potentiates twitch contraction of the radula retractor muscle of the prosobranch mollusc Fusinus ferrugineus was isolated from the ganglia of the animal. Its primary structure is H-Ala-Pro-Gly-Trp-NH2 (APGWamide) closely related to the C-terminal tetrapeptide of the crustacean red-pigment-concentrating hormone. APGWamide showed modulatory actions on contractions in various molluscan muscles.

Amino Acid Sequence↗

Competitive inhibition by dimethylsulfoxide of molluscan and vertebrate acetylcholinesterase.

Anticholinesterase-like effects of dimethylsulfoxide (DMSO) were demonstrated on a variety of invertebrate muscles. The excitatory effects of acetylcholine (ACh) on the isolated preparations of the Geukensia demissa heart and anterior byssus retractor muscle (ABRM), and of the Busycon contrarium radula protractor muscle, were potentiated by DMSO (1-5 microliters/ml; 1 microliter/ml = 14 mM). The negative chronotropic effects of ACh, but not of 4-ketoamyltrimethylammonium, were potentiated by DMSO (1-5 microliters/ml) on the isolated heart of the oyster Crassostrea virginica. These four muscles have acetylcholinesterase enzymes of high activity. In contrast, Mercenaria mercenaria hearts have weak cholinesterase activity, and the effects of ACh on this isolated myocardium were not potentiated by DMSO (2-20 microliters/ml). DMSO (0.1-15 microliters/ml) was a competitive inhibitor of both a crude preparation of oyster heart acetylcholinesterase (AChE) (the Km increased 24-fold with DMSO at 15 microliters/ml; the I50 was 1.3 microliters/ml DMSO when [ACh] = Km) and a purified Electrophorus AChE (the Km increased 4.5-fold when DMSO was 10 microliters/ml; the I50 was 10 microliters/ml DMSO near [ACh] = Km). The same doses of DMSO were needed to potentiate the pharmacological effects of ACh on the oyster heart, as to inhibit the AChE of this tissue.

Acetylcholinesterase↗

Lesion of a serotonergic modulatory neuron in Aplysia produces a specific defect in feeding behavior.

The serotonergic metacerebral cells (MCCs) of Aplysia were destroyed by intracellular injection of proteolytic enzyme. MCC-lesioned animals showed alterations of biting responses compared to MCC-sham and B-cell-lesioned control animals, as well as to their own preoperative behavior. The alterations of biting responses included a prolongation of the duration of radula protraction and a lengthening of interbite interval. No changes were observed in non-biting feeding responses and in behaviors unrelated to feeding.

Animals↗

Presence and distribution of immunoreactive and bioactive FMRFamide-like peptides in the nervous system of the horseshoe crab, Limulus polyphemus.

FMRFamide immunoreactivity was detected in all regions of the Limulus nervous system, including the brain (6.5 +/- 0.6 pg FMRFamide/mg), cardiac ganglion (2.06 +/- 0.67 pg FMRFamide/mg), and ventral nerve cord (5.8 +/- 0.7 pg FMRFamide/mg). The distribution of immunoreactive FMRFamide (irFMRFamide) was mapped by immunofluorescence and the distribution corresponded to regional RIA data. A good proportion of the CNS and cardiac ganglion neuropile contained irFMRFamide, and fluorescent cell bodies were observed in several areas. High performance liquid chromatography (HPLC) was employed to separate and characterize the FMRFamide-like peptides from extracts of Limulus brains. HPLC fractions were analyzed using coincidental radioimmunoassay and bioassay (the radula protractor muscle of Busycon contrarium). There appear to be at least three FMRFamide-like peptides in the Limulus brain, including one similar to clam FMRFamide. FMRFamide acts on Limulus heart in a biphasic manner at relatively high concentrations (10(-5)M), but has no effect on the activity of the isolated ventral nerve cord. These data suggest that in Limulus FMRFamide-like peptides are acting as neurotransmitters, or neuromodulators.

Animals↗

Relationships between the FMRFamide-related peptides and other peptide families.

The relationships between peptide families are recognized in terms of structural similarity and immunological and biological activity. Most of the currently known FMRFamide-related peptides (FaRPs) of molluscs were tested in a radioimmunoassay (RIA) and in the two standard bioassays for FMRFamide: the radula protractor muscle of the whelk Busycon contrarium, and the isolated heart of the clam Mercenaria mercenaria. Some peptides were also tested on the heart of the snail Helix aspersa. The responses of the different assays to these peptides were generally similar, but substantial diversity precluded an absolute resolution of relationships, even among molluscan FaRPs. Nevertheless, this set of responses does constitute a standard against which to estimate the relative affinities of putative FaRPs from other animal groups. Many of the non-molluscan FaRPs (e.g., the pancreatic polypeptide-related peptides, gastrin/CCK, and the opioid peptides) are relatively inactive on the molluscan assays, but others (e.g., LPLRFamide, a peptide isolated from chicken brain; the opioid receptor-modulating peptides A18Fa and F8Fa; and gamma 1-MSH) were relatively potent. Several arthropod FaRPs have substantial FMRFamide-like sequence similarity and immunoreactivity, and they may be homologous members of the molluscan peptide family. However, those structural and functional aspects of peptide families that transcend phyletic lines probably reflect basic principles of binding between peptides and membrane proteins rather than homology.

Amino Acid Sequence↗

Structure, localization, and action of buccalin B: a bioactive peptide from Aplysia.

The cholinergic motor neurons for the accessory radula closer (ARC) contain several neuropeptides that affect muscle contractions. In the present study, we have purified and sequenced a sixth ARC neuropeptide, using a combination of high pressure liquid chromatography and bioassays. This neuropeptide, Gly-Leu-Asp-Arg-Tyr-Gly-Phe-Val-Gly-Gly-Leu-amide, has been named buccalin B (BUCb) because it is significantly homologous to the previously characterized neuropeptide buccalin A. BUCb was found to be two-three times more potent than buccalin A in depressing motor neuron induced contractions.

Amino Acid Sequence↗

Serotonin and forskolin enhance both magnitude of contraction and relaxation rate of Aplysia dorsal extrinsic muscle independently of acetylcholine receptor.

Hexamethonium bromide (Hex. Br.) blocks acetylcholine (ACh) elicited contractions but not electrically elicited contractions of isolated preparations of Aplysia californica dorsal extrinsic muscle. Serotonin (5-hydroxytryptamine, 5-HT) enhances both magnitude and relaxation rate of ACh and electrically elicited contractions. In the presence of Hex. Br., 5-HT still exhibits its modulatory effects on electrically elicited contractions. Forskolin enhances both magnitude and relaxation rate of ACh and electrically elicited contractions. Forskolin (10(-5) M) increases the cyclic AMP content of the accessory radula closer and dorsal extrinsic muscles.

Acetylcholine↗

Effects of the neuropeptide APGW-amide and related compounds on molluscan muscles--GW-amide shows potent modulatory effects.

1. Effects of the molluscan neuropeptide APGW-amide and related compounds (the crustacean hormone RPCH, FAPGW-amide, PGW-amide, GW-amide and W-amide) were examined in several kinds of molluscan muscles. 2. All the compounds, except W-amide, showed qualitatively similar modulatory effects on contraction or relaxation of the muscles. The potency order of the compounds was found to be GW-amide greater than or equal to APGW-amide greater than FAPGW-amide greater than RPCH greater than PGW-amide. W-amide showed little or no effect even at 10(-4) M. 3. In the ABRM of Mytilus and the radula retractor muscle of Rapana, the active peptides were suggested to exhibit their modulatory effects by acting on the presynaptic sites in the muscles. 4. Nineteen GW-amide analogues, such as Gly-Trp-OH, Gly-Phe-NH2, D-Ala-Trp-NH2 and N-Gly-tryptamine, were also tested on some of the muscles, but all of them showed little or no effect.

Animals↗

Isolation and characterization of a novel small cardioactive peptide-related peptide from the brain of Octopus vulgaris.

A novel small cardioactive peptide (SCP)-related peptide (oct-SCPRP: Ser-Asn-Gly-Tyr-Leu-Ala-Leu-Pro-Arg-Gln-NH2) was isolated from the brain of the octopus (Octopus vulgaris). cDNA encoding this precursor protein was cloned. Oct-SCPRP was shown to evoke contraction in the radula protractor muscle, and the precursor protein was highly homologous to the SCP family in the Mollusk but did not encode a related peptide, SCPB. The expression of oct-SCPRP mRNA was present not only in the peripheral nervous system (PNS) which is a motor center for the control of feeding, but also in the central nervous system (CNS) which is capable of complex analysis, learning, and controls behaviors.

Amino Acid Sequence↗

The strategy used by some piscivorous cone snails to capture their prey: the effects of their venoms on vertebrates and on isolated neuromuscular preparations.

Three piscivorous Conus species, C. ermineus, C. consor and C. catus were acclimatized in aquaria. The study of their strategy to capture the prey and details of their radula's morphology revealed that all of them used a 'hook and line' strategy which consists of immobilizing the prey rapidly before engulfing it. The venoms from these piscivorous species clearly elicit, when injected into fish, an excitotoxic shock characterized by a sudden tetanus of the prey. In mammals, the venoms induce both flaccid paralysis via i.p. injection and seizures via i.c.v. injection. Intracellular recordings from frog nerve-muscle preparations revealed that the venoms from these Conus species first caused spontaneous synaptic potentials which in turn triggered muscle action potentials. Such spontaneous activity is due to an increased nerve terminal excitability. In addition, the venoms suppressed neuromuscular transmission probably by blocking postsynaptic nicotinic acetylcholine receptors. No direct effect of these Conus venoms was observed on the membrane of skeletal muscle fibres. In conclusion, C. ermineus, C. consor and C. catus, which have not securely tethered their prey used a mixture of toxins which target both pre-and postsynaptic elements of the neuromuscular junction and which produce rapid immobilization of their prey.

Animals↗