Respiratory behavior in the pond snail Lymnaea stagnalis

Summary This study describes the neural basis of respiratory behavior in a pulmonate mollusc, Lymnaea stagnalis. We describe and identify muscles of the respiratory orifice (pneumostome) and mantle cavity as well as relevant motor neurons innervating these muscles. All of these identified motor neurons are active during spontaneously occurring respiratory behavior and a sporadically occurring synaptic input, termed Input 3, controls the activities of these motor neurons. This spontaneous input can also be recorded from isolated brain preparations, suggesting that the respiratory motor program is generated centrally. However, evidence is also presented that in semi-intact preparations the role of peripheral feedback is important for the initiation and termination of respiratory behavior in Lymnaea.     

The osphradium is involved in the control of egg-laying in the pond snail Lymnaea stagnalis

Summary

Ablation of the osphradium (a peripheral putative chemo-sensory organ) in Lymnaea stagnalis resulted in significant increase of egg-laying activity as compared with control animals (observed for 50 days). Cutting the osphradial nerve increased egg-laying to a lesser degree and only for the first 15–20 days. Water changes in the aquaria had a similar stimulating effect on egg-laying activity in both osphradium-deprived and control snails. Permanent aeration of the aquaria abolished this effect. Possible mechanisms of tonic inhibitory action of the osphradium on egg-laying are discussed.     

Determinants of macrophyte palatability to the pond snail Lymnaea stagnalis

Summary 1. This study aimed to identify the chemical and structural determinants of macrophyte palatability to the pond snail Lymnaea stagnalis . Eleven macrophyte species were investigated, and one of them ( Potamogeton lucens ) was collected at four sites, on two different dates in the year, to study palatability determinants at an intra-specific level.
2. Plant palatability to L. stagnalis was determined through non-choice feeding assays. Dry matter content (DMC), total phenolic content and protein content were measured for each macrophyte species. These parameters, and soluble carbohydrate content, were also measured for each sample of P. lucens .
3. The palatability of macrophytes was positively related to their protein content (between species only) and negatively related to their DMC (both between species and within P. lucens ). No simple relationship was found between the palatability of macrophytes and their phenolic content, but highly palatable macrophytes consistently exhibited a low phenolic content.
4. These results emphasise that macrophyte palatability is a multifactorial attribute, potentially depending on both structural and chemical traits. Because some of these traits were correlated, further investigations are required to assess their respective influence on macrophyte palatability.     

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

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

Immunolabeled neuroactive substances in the osphradium of the pond snail Lymnaea stagnalis

The osphradium of molluscs is assumed to be a sensory organ. The present investigation in Lymnaea stagnalis has established two ultrastructurally different types of dendrites in the sensory epithelium. Cells immunoreactive to leucine-enkephalin and FMRFamide send processes to the sensory epithelium. These neurons of the osphradial ganglion are thus considered to be part of the sensory system, as are methionine-enkephalin-immunoreactive cells in the mantle wall in the vicinity of the osphradium. The complexity of the osphradial ganglion is further demonstrated by serotonin-immunoreactive neurons innervating the muscular coat around the osphradial canal and methionine-enkephalin-immunoreactive cells sending projections to the central nervous system.     

Ultrastructure of neuromuscular contacts in the embryonic pond snail Lymnaea stagnalis L

Ultrastructural characteristics of muscle fibers and neuromuscular contacts were investigated during two stages of embryogenesis of the pulmonate snail Lymnaea stagnalis. The first muscle cells appear as early as during metamorphosis (50-55% of embryonic development), whereas previously, in the trochophore/veliger stages (25-45%), muscular elements cannot be detected at all. The first muscle fibers contain large amounts of free numbers, a well-developed rER system and only a few irregularly arranged contractile elements. The nucleus is densely packed with heterochromatine material. At 75% adult-like postmetamorphic stage, the frequency of muscle fibers increases significantly, but, bundles of muscle fibers cannot yet be observed. Furthermore the muscle cells are characterized by large numbers of free ribosomes and numerous rER elements. Fine axon bundles and single axon processes, both accompanied by glial elements, can already be found at this time. Axon varicosities with different vesicle and/or granule contents form membrane contacts with muscle fibers, but without revealing membrane specialization on the pre- or postsynaptic side. The late development of the muscle system and neuromuscular contacts during Lymnaea embryogenesis correlates well with the maturation of different forms of behavior of adult, free-living life, and also with the peripheral appearance of chemically identified components of the embryonic nervous system of central origin.     

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

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

Hemocytes of the pond snail Lymnaea stagnalis generate reactive forms of oxygen

Macrophagelike hemocytes of the pond snail Lymnaea stagnalis were stimulated in vitro with various particulate agents (latex, Escherichia coli, Staphylococcus saprophyticus, zymosan) and with phorbol myristate acetate in order to determine whether these blood cells show biochemical reactions reminiscent of a respiratory burst. Phagocytic stimulation of the hemocytes resulted in a superoxide dismutase-sensitive reduction of nitroblue tetrazolium, which is indicative of the generation of superoxide anions. Moreover, the hemocytes also produced hydrogen peroxide, and they showed a sodium azide-sensitive diaminobenzidine reaction. The hemocytes displayed a luminol-dependent chemiluminescence that differed for each stimulus used. Zymosan elicited a relatively high dose-dependent response. The chemiluminescence was (partly) inhibited by superoxide dismutase, azide, and cyanide. These data indicate the possible involvement of toxic oxygen intermediates in phagocytic defense reactions of L. stagnalis hemocytes.     

Serotonergic innervation of the foot of the pond snail Lymnaea stagnalis (L.)

The aminergic innervation of the foot of Lymnaea stagnalis was investigated using electron microscopy, immunocytochemistry, and HPLC. The foot was found to contain large amounts of serotonin and dopamine, though at lower concentrations than are found in nervous tissue. Serotonin containing tissue was concentrated in the ventral surface of the foot, under ciliated areas of the epidermis where it occurred in varicosities, with fine tracts joining these varicosities. Varicosities also occurred in deeper tissues, probably adjacent to mucus cells. Positive fluorescence for serotonin in axons was found in nerves innervating the foot, but few neuronal cell bodies containing serotonin were detected, indicating that most of the innervation was coming from the central ganglia. Axon varicosities were found using TEM on ciliated cells, mucus cells, and muscle cells as well as interaxonal junctions (possibly non-synaptic) within nerves. The neuronal varicosities contacting the ciliated cells and mucus cells contained mostly dense-cored vesicles of between 60 and 100 nm in diameter. Smaller, lucent vesicles also occurred in these terminals. The origin and significance of this innervation is discussed. It is suggested that both serotonin and dopamine may play a large role in controlling ciliary gliding by the foot.     

Female behaviour prior to additional sperm receipt in the hermaphroditic pond snail Lymnaea stagnalis

In many species, sexual resistance by females can fluctuate depending on different physiological and/or ecological conditions. Based on theory, we decided to test whether the simultaneous hermaphrodite Lymnaea stagnalis becomes more reluctant to mate in the female role after receipt of an ejaculate. We aimed to test whether receipt of an ejaculate affects the expression of recipient behaviours, and whether this can have a potential effect on the outcome of mating interactions. We found that the expression of Crawl-out and Biting behaviours increased significantly in recently inseminated snails. Our data also indicated that an increase in expression of these latter behaviours can have direct effects on the outcome of the mating interactions. These findings suggest that under the influence of recent insemination, this hermaphroditic snail is more reluctant to accept another mating in the female role and may try to actively discourage the mounting snail (sperm donor). Hence, despite the fact that these simultaneous hermaphrodites express both sexual functions, there can still be variation in motivation to mate in either role.     

The anatomy of neurosecretory neurones in the pond snail Lymnaea stagnalis (L.).

The anatomy of three neurosecretory cell types in the central nervous system (c.n.s.) of the gastropod mollusc Lymnaea stagnalis (L.)- the Dark Green Cells, Yellow Cells and Yellow-green Cells-has been studied by using bright and dark field illumination of material stained for neurosecretion by the Alcian Blue-Alcian Yellow technique. The neuronal geometry of single and groups of neurosecretory cells of the various types has been reconstructed from serial sections, and the likely destination of most of their processes has been determined. Dark Green Cells are monopolar, occur exclusively within the central nervous system (c.n.s.), have few or no branches terminating in neuropile, and send axons to the surface of the pleuro-parietal and pleuro-cerebral connectives. The majority of Dark Green Cell axons however (80-85%), project down nerves which innervate ventral and anterior parts of the head-foot, the neck and the mantle. Dark Green Cell axons can be found in small nerves throughout these areas, and may terminate in a find plexus of axons on the surfaces of the nerves. Since previous experimental work has shown that the Dark Green Cells are involved in osmotic or ionic regulation, these results suggest that the target organ of the Dark Green Cells may be the skin. Yellow Cells occur both within and outside the c.n.s. They are usually monopolar, but can be bipolar. They have several axons which normally arise separately from a single pole of the cell body, or close to it. One or more processes leave the cell proximal to the point where separate axons arise, and may run unbranched for some distance through neuropile before terminating in fine brances and blobs of various sizes. These branches may release hormone inside the c.n.s. Yellow-green Cells are mono-, bi- or multi-polar, and like the Yellow Cells are found both within and outside the c.n.s. Some Yellow-green Cells, though not all, have projections which terminate in neuropile in fine branches and blobs. Yellow-green Cell bodies which occur in nerves can project back along the nerve into the c.n.s. The axons of Yellow Cells and Yellow-green Cells project to release sites in various ways. Some project into the connective tissue shealth of the c.n.s., which serves as a neurohaemal organ, either directly through the surface of a ganglion, or from the pleuro-cerebral or pleuro-parietal connectives. Other axons leave the c.n.s. via nerves leaving the left and right parietal and visceral ganglia; projections into the intestinal, anal, and internal right parietal nerves being most numerous. Axons which may be from either, or both Yellow Cells and Yellow-green Cells, can be found along the entire unbranched lengths of these nerves, and in subsequent branches which innervate organs lying in the anterior turn of the shell. All of these orgnas are closely associated with the lung cavity...     

GTP-dependent dopamine reception in central nervous system tissue of the pond snail Lymnaea stagnalis

The guanosine-5-triphosphate (GTP) binding of D₁-dopamine (DA) receptor agonist [H³]-SKF 38393 is described. The binding of [H³]-SKF 38393 occurs in two different DA receptors in the presence of guanylyl nucleotides, and in one receptor population in the absence of guanylyl nucleotides. It was shown with GDP--P³³ binding analysis that G proteins in the mollusc nervous tissue membranes accelerate exchange of guanosine-5-diphosphate (GDP) for GTP considerably. While binding of [H³]-SKF 38393 was not found with phosphorylation of the membranes by the catalytic subunit of cAMP-dependent protein kinase A, basal and DA-induced GDP GTP exchange was noticeably inhibited with phosphylation in the nervous tissue membranes.A. A. Bogomolets Institute of Physiology, Ukrainian Academy of Sciences, Kiev. Translated from Neirofiziologiya, Vol. 24, No. 4, pp. 451–461, July–August, 1992.     

Whole-body enantiomorphy and maternal inheritance of chiral reversal in the pond snail Lymnaea stagnalis

Sinistral and dextral snails have repeatedly evolved by left-right reversal of bilateral asymmetry as well as coiling direction. However, in most snail species, populations are fixed for either enantiomorph and laboratory breeding is difficult even if chiral variants are found. Thus, only few experimental models of chiral variation within species have been available to study the evolution of the primary asymmetry. We have established laboratory lines of enantiomorphs of the pond snail Lymnaea stagnalis starting from a wild population. Crossing experiments demonstrated that the primary asymmetry of L. stagnalis is determined by the maternal genotype at a single nuclear locus where the dextral allele is dominant to the sinistral allele. Field surveys revealed that the sinistral allele has persisted for at least 10 years, that is, about 10 generations. The frequency of the sinistral allele showed large fluctuations, reaching as frequent as 0.156 in estimate under the assumption of Hardy-Weinberg equilibrium. The frequency shifts suggest that selection against chiral reversal was not strong enough to counterbalance genetic drift in an ephemeral small pond. Because of the advantages as a model animal, enantiomorphs of L. stagnalis can be a unique system to study aspects of chirality in diverse biological disciplines.     

Neuronal and non-neuronal control of the neurosecretory Caudo-Dorsal cells of the freshwater snail Lymnaea stagnalis (L.)

The cerebral ganglia of the freshwater snail Lymnaea stagnalis contain two clusters of neurosecretory Caudo-Dorsal Cells (CDC). These cells produce a neurohormone which stimulates ovulation. Ganglion transplantation and quantitative electron microscopy show that neuronal isolation of the cerebral ganglia complex (CCC) results in an activation of the CDC. It was, therefore, concluded that the CDC are controlled by an inhibitory neuronal input originating outside the cerebral ganglia. Ultrastructural studies on synaptic degeneration in the CCC suggest that this input reaches the CDC via a special type of synapse-like structure, the type C-SLS.Furthermore, transplantation of CCC into acceptor snails leads to a reduced release and an increased intracellular breakdown of neurohormone in the CDC of the nervous system of the acceptors. It is supposed that these phenomena are caused by the release of an (unknown) factor from the transplanted CCC. Special attention was given to the formation and degradation of a peculiar type of neurohormone granule, the large electron dense granule.     

Locomotor rhythms in the pond snail Lymnaea stagnalis: site of origin and neurotransmitter requirements

1. We have found that, in preparations of isolated CNS of the pond snail Lymnaea stagnalis, both serotonin (5HT) and dopamine (DA), as well as their respective precursors, 5HTP and DOPA, are effective in producing fictive intense (muscular) locomotion. 2. Phase-coupled to each of the above pedal rhythms are numerous identifiable pedal neurons including the respiratory interneuron RPeD1, thus suggesting interaction between networks responsible for locomotion and air breathing. 3. The novel DA/DOPA-dependent motor rhythm resembles the 5HT/5HTP-dependent one in terms of activity of identifiable pedal neurons, being however considerably slower than the latter. 4. The results of transection experiments suggest that each of the rhythms is generated by a paired CPG lying entirely within the pedal ganglia.