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.

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%.

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.

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.

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.

Mass spectrometric analysis of activity-dependent changes of neuropeptide profile in the snail, Helix pomatia.

Terrestrial snails are able to transform themselves into inactivity ceasing their behavioral activity under unfavorable environmental conditions. In the present study, we report on the activity-dependent changes of the peptide and/or polypeptide profile in the brain and hemolymph of the snail, Helix pomatia, using MALDI TOF and quadrupole mass spectrometry. The present data indicate that the snails respond to low temperature by increasing or decreasing the output of selected peptides. Average mass spectra of the brain and hemolymph revealed numerous peaks predominantly present during the active state (19 and 10 peptides/polypeptides, respectively), while others were observed only during hibernation (11 and 13). However, there were peptides and/or polypeptides or their fragments present irrespective of the activity states (49 and 18). The intensity of fourteen peaks that correspond to previously identified neuropeptides varied in the brain of active snails compared to those of hibernating animals. Among those the intensity of eight peptides increased significantly in active animals while in hibernated animals the intensity of another six peptides increased significantly. A new peptide or peptide fragment at m/z 1110.7 was identified in a brain of the snail with the following suggested amino acid sequence: GSGASGSMPATTS. This peptide was found to be more abundant in active animals because the intensity of the peptide was significantly higher compared to hibernating animals. In summary, our results revealed substantial differences in the peptide/polypeptide profile of the brain and hemolymph of active and hibernating snails suggesting a possible contribution of peptides in the process of hibernation.

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.

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.

Scaling of metabolism in Helix aspersa snails: changes through ontogeny and response to selection for increased size.

Though many are convinced otherwise, variability of the size-scaling of metabolism is widespread in nature, and the factors driving that remain unknown. Here we test a hypothesis that the increased expenditure associated with faster growth increases metabolic scaling. We compare metabolic scaling in the fast- and slow-growth phases of ontogeny of Helix aspersa snails artificially selected or not selected for increased adult size. The selected line evolved larger egg and adult sizes and a faster size-specific growth rate, without a change in the developmental rate. Both lines had comparable food consumption but the selected snails grew more efficiently and had lower metabolism early in ontogeny. Attainment of lower metabolism was accompanied by decreased shell production, indicating that the increased growth was fuelled partly at the expense of shell production. As predicted, the scaling of oxygen consumption with body mass was isometric or nearly isometric in the fast-growing (early) ontogenetic stage, and it became negatively allometric in the slow-growing (late) stage; metabolic scaling tended to be steeper in selected (fast-growing) than in control (slow-growing) snails; this difference disappeared later in ontogeny. Differences in metabolic scaling were not related to shifts in the scaling of metabolically inert shell. Our results support the view that changes in metabolic scaling through ontogeny and the variability of metabolic scaling between organisms can be affected by differential growth rates. We stress that future approaches to this phenomenon should consider the metabolic effects of cell size changes which underlie shifts in the growth pattern.

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.

Intracellular localization of the HCS2 gene products in identified snail neurons in vivo and in vitro.

1. The HCS2 (Helix command specific 2) gene expressed in giant command neurons for withdrawal behavior of the terrestrial snail Helix lucorum encodes a unique hybrid precursor protein that contains a Ca-binding (EF-hand motif) protein and four small peptides (CNP1-CNP4) with similar Tyr-Pro-Arg-X aminoacid sequence at the C terminus. Previous studies suggest that under conditions of increased intracellular Ca(2+) concentration the HCS2 peptide precursor may be cleaved, and small physiologically active peptides transported to the release sites. In the present paper, intracellular localization of putative peptide products of the HCS2-encoded precursor was studied immunocytochemically by means of light and electron microscopy. 2. Polyclonal antibodies against the CNP3 neuropeptide and a Ca-binding domain of the precursor protein were used for gold labeling of ultrathin sections of identified isolated neurons maintained in culture for several days, and in same identified neurons freshly isolated from the central nervous system. 3. In freshly isolated neurons, the gold particles were mainly localized over the cytoplasmic secretory granules, with the density of labeling for the CNP3 neuropeptide being two-fold higher than for the calcium-binding domain. In cultured neurons, both antibodies mostly labeled clusters of secretory granules in growth cones and neurites of the neuron. The density of labeling for cultured neurons was the same for both antibodies, and was two-fold higher than for the freshly isolated from the central nervous system neurons. 4. The immunogold particles were practically absent in the bodies of cultured neurons. 5. The data obtained conform to the suggestion that the HCS2 gene products are transported from the cell body to the regions of growth or release sites.

Modulatory serotonin receptors on the soma of command neurons in edible snail.

Local application of serotonin (5-hydroxytryptamine, 5-HT) to the soma of command neurons LPa2, LPa3, PPa3, and PPa2 of edible snail Helix lucorum reversibly decreased acetylcholine-induced inward current in these neurons. NAN-190 and methiothepin, antagonists of 5-HT1 serotonin receptors, prevented this modulatory effect of 5-HT. By contrast, LY-53.857, ICS-205.930, and SDZ-205.557, antagonists of 5-HT2, 5-HT3, and 5-HT4 serotonin receptors, respectively, produced no effect on the modulatory effect of serotonin. The data confirm the presence of modulator 5-HT1 serotonin receptor on the soma of command neurons of Helix pomatia.

Sensory innervation of the ovotestis in the snail Helix aspersa.

Because oviposition in the land snail Helix aspersa is a metabolically expensive process coupled to a high fixed cost, one expects oviposition to occur only when the clutch size surpasses a minimum value at which the reproductive benefit exceeds the cost. We propose that neural innervation of the gonad allows H. aspersa to monitor oocyte production and ensure an adequate supply of gametes prior to ovulation. The ovotestis is innervated by a branch of the intestinal nerve in which the majority of axon fibres measure <0.2 microm in diameter. We found a strong positive correlation between the number of mature oocytes in the ovotestis and the frequency of spontaneous afferent spikes in the nerve branch. Tactile stimulation of the ovotestis resulted in a 20-fold increase in afferent spikes and an efferent reflex directed towards the ovotestis and the pericardium. Afferent activity also increased 10-fold after an experimentally induced increase in the volume of the ovotestis. These results suggest that the growing oocytes expand the walls of the acini and trigger action potentials in the mechanosensitive nerve terminals that lie within the acinar walls. We hypothesize that the resulting tonic signal is permissive for ovulation. In addition, a phasic sensory signal may occur during ovulation to trigger CNS motor output related to oviposition.

Shell shape of the land snail Cornu aspersum in North Africa: unexpected evidence of a phylogeographical splitting.

Anatomical and molecular characters used to differentiate populations of the land snail Cornu aspersum (Helix aspersa) exhibit, in the western Mediterranean, definite and concordant patterns of correlation with geography. Scenarios involving Pliocene geological changes and postglacial expansion during the Pleistocene were proposed in previous studies to account for the establishment of this geographical structure. In the present work, we have performed a spatial analysis of variation in shell morphometrics, after the partitioning of the overall variation into size and shape components by means of a principal component-based approach (Cadima and Jolliffe, 1996). In order to know if the same historical events have also structured shell variation, the analysis includes all the populations from North Africa which were investigated for anatomical and molecular surveys. Contrary to shell size, which shows a significant spatial heterogeneity essentially related to environmental pressures, variation in shell shape components splits the populations according to a geographical pattern reflective of hypotheses suggested for molecular markers and genital anatomy. This implies that the selective forces often invoked to explain spatial changes in shell shape are not the deciding factors in the present case. Moreover, within each of the two geographical clusters defined, Mantel correlograms show that the similarity between populations declines according to an isolation by distance model. Because of the different allometric relationships between shell size and genitalia measurements in Western and Eastern entities of North Africa, mechanical constraints, possibly leading to a precopulatory isolation in the contact zone, are involved.

Effect of static and dynamic influences on receptors of equilibrium organ of Helix lucorum after 163-day orbital flight in "Mir" station.

A 163-day orbital flight increased the baseline impulse activity of statocyst receptor cells in terrestrial pulmonata snail Helix lucorum. The maximum of reaction to step static stimuli (changes in body position in the range from 0 to 180 degrees) was significantly shifted by 30 degrees from gravitational vertical, while the reaction to dynamic stimuli (dumped sinusoidal oscillations) faded more rapidly than in control animals.