Hidden female physiological resistance to male accessory gland substances in a simultaneous hermaphrodite.

To increase fertilization chances compared with rivals, males are favoured to transfer accessory gland proteins to females during mating. These substances, by influencing female physiology, cause alteration of her sperm usage and remating rate. Simultaneously hermaphroditic land snails with love-darts are a case in point. During courtship, a love-dart is pierced through the partner's body wall, thereby introducing accessory mucous gland products. This mucus physiologically increases paternity by inhibiting the digestion of donated sperm. The sperm, which are packaged in a spermatophore, are exchanged and received in an organ called the diverticulum. Because of its length, this organ was previously proposed to be a female anatomical adaptation that may limit the dart interference with the recipient's sperm usage. For reproductive success of the donor, an anatomically long spermatophore, relative to the partner's diverticulum, is beneficial as sperm can avoid digestion by exiting through the spermatophore's tail safely. However, the snail Eobania vermiculata possesses a diverticulum that is three times longer than the spermatophore it receives. Here, we report that the love-dart mucus of this species contains a contraction-inducing substance that shortens the diverticulum, an effect that is only properly revealed when the mucus is applied to another helicid species, Cornu aspersum This finding suggests that E. vermiculata may have evolved a physiological resistance to the manipulative substance received via the love-dart by becoming insensitive to it. This provides useful insight into the evolution of female resistance to male manipulations, indicating that it can remain hidden if tested on a single species.

Anatomical and physiological background permitting spatial odor sensation in stylommatophoran molluscs.

Earlier experiments demonstrated that in order to place protracted tentacles and thereby olfactory receptors in an appropriate position for optimal perception of odor stimuli extraordinary complex movements are required. Until recently both large scale tentacle movements and patterned tentacle movements have been attributed to the concerted involvement of the tentacle retractor muscle and muscles of tegumentum. Recently the existence of three novel muscles in the posterior tentacles of Helix has been discovered. The present review, based on experimental data obtained by our research group, outlines the anatomy, physiology and pharmacology of these muscles that enable the tentacles to execute complex movements observed during foraging both in naïve and food-conditioned snails. Our findings are also compared as far as possible with earlier and recent data obtained on innervation characteristics and pharmacology of molluscan muscles.

Neural control of olfaction and tentacle movements by serotonin and dopamine in terrestrial snail.

We investigated the role of serotonin (5HT) and dopamine (DA) in the regulation of olfactory system function and odor-evoked tentacle movements in the snail Helix. Preparations of the posterior tentacle (including sensory pad, tentacular ganglion and olfactory nerve) or central ganglia with attached posterior tentacles were exposed to cineole odorant and the evoked responses were affected by prior application of 5HT or DA or their precursors 5-hydroxytryptophan (5HTP) and L: -DOPA, respectively. 5HT applications decreased cineole-evoked responses recorded in the olfactory nerve and hyperpolarized the identified tentacle retractor muscle motoneuron MtC3, while DA applications led to the opposite changes. 5HTP and L: -DOPA modified MtC3 activity comparable to 5HT and DA action. DA was also found to decrease the amplitude of spontaneous local field potential oscillations in the procerebrum, a central olfactory structure. In vivo studies demonstrated that injection of 5HTP in freely moving snails reduced the tentacle withdrawal response to aversive ethyl acetate odorant, whereas the injection of L: -DOPA increased responses to "neutral" cineole and aversive ethyl acetate odorants. Our data suggest that 5HT and DA affect the peripheral (sensory epithelium and tentacular ganglion), the central (procerebrum), and the single motor neuron (withdrawal motoneuron MtC3) level of the snail's nervous system.

Novel triplet of flexor muscles in the posterior tentacles of the snail, Helix pomatia.

The anatomy of three novel flexor muscles in the posterior tentacles of Helix pomatia is described. The muscles originate from the ventral side of the sensory pad and are anchored at different sites in the base of the tentacle stem. The muscles span the tentacle and always take the length of the stem which depends on the rate of tentacle protrusion indicating that the muscles are both contractile and extremely stretchable. The three anchoring points at the base of the stem determine three space axes along which the contraction of a muscle or the synchronous contraction of the muscles can move the tentacle in space.

Physiological relevance and contribution to metal balance of specific and non-specific Metallothionein isoforms in the garden snail, Cantareus aspersus.

Variable environmental availability of metal ions represents a constant challenge for most organisms, so that during evolution, they have optimised physiological and molecular mechanisms to cope with this particular requirement. Metallothioneins (MTs) are proteins that play a major role in metal homeostasis and as a reservoir. The MT gene/protein systems of terrestrial helicid snails are an invaluable model for the study of metal-binding features and MT isoform-specific functionality of these proteins. In the present study, we characterised three paralogous MT isogenes and their expressed products in the escargot (Cantareus aspersus). The metal-dependent transcriptional activation of the three isogenes was assessed using quantitative Real Time PCR. The metal-binding capacities of the three isoforms were studied by characterising the purified native complexes. All the data were analysed in relation to the trace element status of the animals after metal feeding. Two of the three C. aspersus MT (CaMT) isoforms appeared to be metal-specific, (CaCdMT and CaCuMT, for cadmium and copper respectively). A third isoform (CaCd/CuMT) was non-specific, since it was natively recovered as a mixed Cd/Cu complex. A specific role in Cd detoxification for CaCdMT was revealed, with a 80-90% contribution to the Cd balance in snails exposed to this metal. Conclusive data were also obtained for the CaCuMT isoform, which is involved in Cu homeostasis, sharing about 30-50% of the Cu balance of C. aspersus. No apparent metal-related physiological function was found for the third isoform (CaCd/CuMT), so its contribution to the metal balance of the escargot may be, if at all, of only marginal significance, but may enclose a major interest in evolutionary studies.

Intermediate filaments in non-neuronal cells of invertebrates: isolation and biochemical characterization of intermediate filaments from the esophageal epithelium of the mollusc Helix pomatia.

To screen invertebrate tissues for the possible expression of intermediate filaments (IFs), immunofluorescence microscopy with the monoclonal antibody anti-IFA known to detect all mammalian IF proteins was used (Pruss, R. M., R. Mirsky, M. C. Raff, R. Thorpe, A. J. Dowding, and B. H. Anderton. 1981. Cell, 27:419-428). In a limited survey, the lower chordate Branchiostoma as well as the invertebrates Arenicola, Lumbricus, Ascaris, and Helix pomatia revealed a positive reaction primarily on epithelia and on nerves, whereas certain other invertebrates appeared negative. To assess the nature of the positive reaction, Helix pomatia was used since a variety of epithelia was strongly stained by anti-IFA. Fixation-extraction procedures were developed that preserve in electron micrographs of esophagus impressive arrays of IFs as tonofilament bundles. Fractionation procedures performed on single cell preparations document large meshworks of long and curvilinear IF by negative stain. These structures can be purified. One- and two-dimensional gels show three components, all of which are recognized by anti-IFA in immunoblotting: 66 kD/pl 6.35, 53 kD/pl 6.05, and 52 kD/pl 5.95. The molar ratio between the larger and more basic polypeptide and the sum of the two more acidic forms is close to 1. After solubilization in 8.5 M urea, in vitro filament reconstitution is induced when urea is removed by dialysis against 2-50 mM Tris buffer at pH 7.8. The reconstituted filaments contain all three polypeptides. The results establish firmly the existence of invertebrate IFs outside neurones and demonstrate that the esophagus of Helix pomatia displays IFs which in line with the epithelial morphology of the tissue could be related to keratin IF of vertebrates.

PACAP has anti-apoptotic effect in the salivary gland of an invertebrate species, Helix pomatia.

Pituitary adenylate cyclase activating polypeptide (PACAP) shows a remarkable sequence similarity among species and several studies provide evidence that the functions of PACAP have also been conserved among vertebrate species. Relatively little is known about its presence and functions in invertebrates. The aim of the present study was to investigate whether the well-known anti-apoptotic effect of PACAP can also be demonstrated in invertebrates. This effect was studied in the salivary gland of a molluscan species, Helix pomatia. In this work, we first showed the presence of PACAP-like immunoreactivity in the Helix salivary gland by means of immunohistochemistry. Radioimmunoassay measurements showed that PACAP38-like immunoreactivity dominated in the salivary gland of both active and inactive snails and its concentration was higher in active than in inactive animals in contrast to PACAP27-like immunoreactivity, which did not show activity-dependent changes. PACAP induced a significant elevation of cAMP level in salivary gland extracts. Application of apoptosis-inducing agents, dopamine and colchicine, led to a marked increase in the number of terminal uridine deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive apoptotic cells in the salivary gland, which was significantly attenuated by PACAP treatment. In a similar manner, the number of caspase-positive cells was reduced after co-application of dopamine and PACAP. Taken together, the data indicate that PACAP activates cAMP in a molluscan species and we show, for the first time, that PACAP is anti-apoptotic in the invertebrate Helix pomatia.

Humoral serotonin and dopamine modulate the feeding in the snail, Helix pomatia.

We investigated the effect of elevated levels of humoral 5HT and DA on the feeding latency of Helix pomatia, 1 day, 3 days and 10 days following satiation, by injecting monoamines into the haemocoel. HPLC assay of monoamines showed that both 5HT and DA are present in pmol/ml concentrations in the haemolymph of both starved and non-starved animals. Elevated levels of 5HT and DA were most effective at decreasing the feeding latency 10 days following satiation when DA decreased the feeding latency in a concentration dependent manner between 10(-7) and 10(-5) M whereas 5HT levels decreased the feeding latency only at 10(-6) M but increased it at 10(-5) M. Immunocytochemistry revealed that both 5HT3 and D1 receptor-like immuno-reactivity are present in cell bodies located in the same areas of the buccal ganglia. Our observations suggest that both humoral DA and 5HT mutually modulate the activity of the feeding CPG through neurons which have these receptors.

Variation in spermathecal morphology is independent of sperm competition intensity in populations of the simultaneously hermaphroditic land snail Cornu aspersum.

The complexity of the sperm-storing organ (spermatheca) has been hypothesized to reflect sperm competition intensity in several gastropod species. Furthermore, considerable variation in spermathecal morphology has been detected among populations of the same species. The morphological variation of the fertilization pouch was studied in five populations of the simultaneously hermaphroditic land snail Cornu aspersum (formerly, Helix aspersa). The populations studied differed in snail density and habitat humidity regimes, thus in sperm competition intensity. The study was conducted on wild adult snails and their progeny, which was reared in the laboratory for two successive generations. Finally, the morphology of the spermatheca was correlated to behavioral mating traits of the snails. The fertilization pouch consisted of a simple fertilization chamber and 4-19 blind tubules. The five studied populations did not differ in either mean number of spermathecal tubules, length of the fertilization chamber, length of the main tubule, or cumulative length of all tubules, while they differed in copulation frequency and mating propensity. No correlation was found between snail size and number of tubules, or length of any spermathecal structure measured. Additionally, no correlation was found between any behavioral trait and the morphological characteristics of the spermatheca. Strong correlations were found only among measurements of some of the spermathecal structures. Our results suggest that the complexity of the spermatheca is not related to sperm competition intensity and its structure is thus genetically determined.

Intraspecific Scaling Relationships Between Crawling Speed and Body Size in a Gastropod.

Across various modes of locomotion, body size and speed are often correlated both between and within species. Among the gastropods, however, current data are minimal for interspecific and intraspecific scaling relationships. In this study, we tested the relationships between various measurements of body size and crawling speed in the terrestrial snail Cornu aspersum. We also investigated the relationships between crawling speed, muscular wave frequency, and muscular wavelength, because--while these relationships within individuals are well studied--the relationships among individuals are unknown. We recorded snails crawling on both a horizontal and a vertical surface. We found that when they crawled on a horizontal surface, foot length was positively correlated with pedal wavelength and crawling speed, but was not correlated with wave frequency. In comparison, when they crawled on a vertical surface, foot length was positively correlated with wavelength, negatively correlated with wave frequency, and not correlated with crawling speed. Body mass had no correlation with crawling speed when snails were crawling on a horizontal surface, but was negatively correlated with speed when snails crawled on a vertical surface.

Initial studies on the direct and modulatory effects of nitric oxide on an identified central Helix aspersa neuron.

The generation of the novel messenger molecule nitric oxide (NO) has been demonstrated in many tissues across phyla including nervous systems. It is produced on demand by the enzyme nitric oxide synthase often stimulated by intracellular calcium and typically affecting guanylate cyclase thought to be its principal target in an auto and/or paracrine fashion. This results in the generation of the secondary messenger cyclic guanosine monophosphate (cGMP). Nitric oxide synthase has been demonstrated in various mollusk brains and manipulation of NO levels has been shown to affect behavior in mollusks. Apart from modulation of the effect of the peptide GSPYFVamide, there appears little published on direct or modulatory effects of NO on Helix aspersa central neurons. We present here initial results to show that NO can be generated in the region around F1 in the right parietal ganglion and that NO and cGMP directly hyperpolarize this neuron. For example, application of the NO-donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP; 200 µM) can cause a mean hyperpolarization of 41.7 mV, while 2 mM 8-bromo-cyclic guanosine monophosphate (8-bromo-cGMP) produced a mean hyperpolarization of 33.4 mV. Additionally, pre-exposure to NO-donors or cGMP appears to significantly reduce or even eliminates the normal hyperpolarizing K(+)-mediated response to dopamine (DA) by this neuron; 200 µM SNAP abolishes a standard response to 0.5 µM DA while 1 mM 8-bromo-cGMP reduces it 62%.

Morphology of interneurons in the procerebrum of the snail Helix aspersa.

Terrestrial snails have a highly developed sense of olfaction. Because the procerebrum has a large number of cells and is located at the entry site of the olfactory nerve into the brain, the structure is thought to have a significant role in the processing of olfactory stimuli. The morphology of the procerebral neurons in the snail Helix aspersa was investigated through intracellular injections of biocytin. No formal categorization of neuronal types was possible, but some cells were seen to have neurites entirely intrinsic to the procerebrum, whereas others had both intrinsic and extrinsic arborizations, and still others had only extrinsic arborizations. These interneurons were previously thought to have arborizations restricted to the procerebral lobe. We demonstrated the extent of the neurite projections outside of the procerebral lobe by making focal injections of biocytin or Neurobiotin into various regions of the cerebral ganglion. This technique revealed subsets of cells that send neurites not only in the ipsilateral ganglion but also through the cerebral commissure into the contralateral cerebral ganglion. Our results demonstrate not only that the procerebral cell population is heterogeneous but also that the procerebrum interacts more directly with the rest of the central nervous system than was formerly believed.

Topographic organization of serotonergic and dopaminergic neurons in the cerebral ganglia and their peripheral projection patterns in the head areas of the snail Helix pomatia.

The distribution of monoaminergic neurons within the cerebral ganglia was investigated in the pulmonate snail Helix pomatia. Simultaneous serotonin and tyrosine hydroxylase double immunostaining revealed that the immunoreactive cell groups are concentrated in a putative monoaminergic center on the ventral surface of the cerebral ganglia. Simultaneous cobalt (Co)- and nickel (Ni)-lysine backfills of cerebral nerves were combined with 5, 6-dihydroxytryptamine pigment-labelling of serotonergic neurons, or with fluorescence immunocytochemistry of dopaminergic neurons. This showed that the serotonergic and dopaminergic cell groups can be divided into smaller subgroups on the basis of their axonal projections into different cerebral nerves. These subgroups show a topographic organization within the serotonergic and dopaminergic neuronal clusters. In the serotonergic system, the different regions of the head are represented in a rostrocaudal direction, whereas a caudorostral organization is characteristic for the dopaminergic system. No serotonin- or dopamine-immunoreative cell bodies but numerous fibers were observed in the head areas, indicating that these are innervated by cerebral monoaminergic neurons and show different innervation patterns. Serotonin-immunoreactive fibers mostly innervate muscle fibers, whereas dopamine-immunoreactive processes do not innervate effector cells, but terminate within the nerve branches of the head areas. On the basis of their innervation pattern, we suggest that dopaminergic neurons may take part in en route modulation of sensory afferent and efferent processes in an as yet unknown manner. The serotonergic neurons, on the other hand, may play a direct role in the modulation of muscle function.

Ultrastructure of synaptic connections of a bimodal pacemaker giant neuron in the central nervous system of Helix pomatia L.

Following intracellular labelling with horseradish peroxidase, the arborization and synaptic connections of the bimodal pacemaker giant neuron (RPal) of Helix pomatia were investigated in the right parietal and visceral ganglia. The RPal neuron possesses extensive axonal branching, the elements of which could be observed and traced within the entire neuropil region of both ganglia. The main axonal branches showed further arborization. The thin axon processes enter the synaptic neuropil, where they receive numerous synapses. At least six ultra-structurally different terminals form synaptic contacts on peroxidase-labelled axon processes of the cell. On the basis of their vesicle and granule content, they are likely to contain different neurotransmitters. Some intraganglionic efferent contacts of the RPal neuron were also observed. It is suggested that, besides its peripheral efferent connections, this cell might also serve as an interneuron.

Structure and function in the cerebral ganglion.

Evidence is reviewed to evaluate whether the term "brain is justified in referring to the snail's cerebral ganglion. The focus of the review is terrestrial species, with particular attention given to the genus Helix. In accordance with a standard definition of "brain, the cerebral ganglion is found to be differentiated both structurally and functionally. It receives convergent sensory inputs from a variety of anterior sensory organs plus the posterior body wall. Its outputs comprise motor commands directed towards anterior muscle systems, e.g., the tentacles and the penis, as well as premotor commands directed towards executory centers in other ganglia, e.g., the buccal, visceral, and pedal ganglia. Of the three major divisions in the ganglion, the procerebrum and the mesocerebrum are the most differentiated, whereas the metacerebrum is the least differentiated. The specializations of the procerebrum for olfactory functions, and the mesocerebrum for reproductive functions, reflect the importance of adaptations for feeding and mating in the evolution of the Gastropoda.

Topographic organization of efferent neurons with different neurochemical characters in the cerebral ganglia of the snail Helix pomatia.

This study provides a description of the organization of neurons efferent to different head areas in the cerebral ganglia of Helix pomatia, revealed by simultaneous Ni-lysine and Co-lysine back-filling of different pairs of cerebral nerves. The backfills show that labeled cerebral neurons that innervate the head areas are concentrated in seven representation foci distributed in different parts of the cerebral ganglia. Almost each head area is represented in each focus. At a gross level, the representation of the different head areas in the representation foci shows a topographic arrangement. Each focus is constituted by neurochemically different groups of neurons. All head areas are innervated by serotonin-containing fibers from a single focus (Focus 2) and by dopamine-containing fibers from Foci 1, 2, and 4. However, they are innervated by CARP and FMRFamide-containing fibers from all of the foci. The combination of retrograde labeling with 5, 6-dihydroxytriptamine induced pigment labeling of serotonin-containing neurons or with fluorescence tyrosinehydroxylase immunocytochemistry to detect dopamine-containing neurons showed that the different head areas are topographycally represented in the clusters of both the serotonin- and dopamine-containing cells. The combination of Ni-lysine backfillings from different cerebral nerves with fluorescence CARP and FMRFamide immunocytochemistry revealed that the head areas are represented also in both the CARP and FMRFamide immunoreactive groups of neurons in the different foci.

Monosynaptic connections between serotonin-containing neurones labelled by 5,6-dihydroxytryptamine-induced pigmentation in the snail Helix pomatia L.

Microelectrophysiological experiments were carried out on in vivo 5,6-dihydroxytryptamine-induced pigment-labelled serotonin-containing neurones of Helix pomatia. Excitatory chemical monosynaptic connections were found between the giant pedal LP3 neurone and several follower cells in the visceral and right parietal ganglia. These neurones have previously been described to be involved in regulation of visceral functions. Monitoring changes in identified monosynaptic connections between pairs of serotonin-containing neurones aids the cellular analysis of behavioural plasticity.

Completely isolated molluscan neurons. An ultrastructural study.

The neurons of the molluscs Lymnaea and Helix isolated by fermentative digestion followed by mechanical treatment do not differ ultrastructurally from intact ones. These cells have sufficient metabolic reserves and incorporate into RNA 8% of the total radioactive pool, even more than neurons in ganglia under equal conditions. Neuronal damage can occur, mainly during the pipetting, and this is usually expressed in vacuolization of the cytoplasm. It is important to note that alterations in cell ultrastructure develop earlier than changes in the membrane electrical properties. The surface of the isolated neurons is enlarged two-fold due to the infoldings of the cell membrane. So, the specific resistance of soma membrane of these neurons was calculated as 78 +/- 13 komega-sq. cm. On the surface of isolated neurons scraps of glial and neuronal processes not connected with their own cell bodies, and as a consequence not powerful, are sometimes found. Some endings of the neuronal processes on the surface of isolated neurons are ultrastructurally similar to the axo-somatic synapses.

Morphological and electrophysiological features of F76 and D1 neurones of the sub-oesophageal ganglia of Helix aspersa in vitro and in culture.

Identified neurones F76 and D1 of the suboesophageal ganglia of Helix aspersa were studied in the isolated ganglia in vitro and in culture. The neurones were examined electrophysiologically with current clamp and morphologically either with intracellular injections of Lucifer Yellow or biocytin. These nerve cells had very similar resting membrane potentials and responses to injected current. The projections of D1 and F76 have been characterised, with both neurones having two main axons. The F76 neurones project to the left pallial, right pallial, anal, and visceral nerves as well as to the left and right pleural ganglia. The D1 neurones have similar projections except that they do not project to the anal and visceral nerves. The bilateral symmetry to the pallial nerves and pleural ganglia is discussed. These cells were also studied electrophysiologically after mechanical isolation and culture. F76 and D1 neurones were separated by dissection (no enzymes) and cultured in three ways. In normal snail Ringer they remained viable for up to two weeks with no development. In Ringer preincubated with a ganglia or containing endothelial growth factor, neurite outgrowths were seen. Membrane potentials were significantly lower in cultured neurones than in vitro and the after hyperpolarization never went below resting in cultured cells but it did in vitro.

The structure and compositon of the love dart (gypsobelum) in Helix pomatia.

The love dart of the snail Helix pomatia is a four-bladed, hollow spicule of calcium carbonate. The calcium carbonate is largely in the form of microcrystalline aragonite, the crystals being not more than 0.5 micrometer in size but assembled into sheets or blocks of much greater size. Protein occurs in the dart structure as an external sheath, as a lining to the tubular core and as a matrix component of the mineral phase. Comparisons with the molluscan shell are drawn and a rationale for the architecture suggested.