The Role of DNA Methylation and Activity of Neurotransmitter Receptors in the Mechanisms of Specific Anterograde Amnesia and Memory Recovery.

Impairment of reconsolidation of conditioned food aversion memory led to the development of a specific anterograde amnesia: repeated training of amnestic snails did not induce long-term memory formation. DNA demethylation caused by injections of DNA methyltransferase inhibitor (DNAMT) during repeated training led to long-term memory formation. Injections of an NMDA glutamate receptor antagonist or a serotonin receptor antagonist prevented memory formation induced by administration of DNAMT inhibitor and repeated training. We hypothesize that methylation-dependent repression of neuronal genes underlies anterograde amnesia. Demethylation eliminated the blockade of these genes and created conditions for long-term memory formation, the induction mechanisms of which involve neurotransmitter receptors.

Seasonal cellular stress phenomena and phenotypic plasticity in land snail Helix lucorum populations from different altitudes.

Temperature, a major abiotic environmental factor, regulates various physiological functions in land snails and therefore determines their biogeographical distribution. Thus, species with different distributions may present different thermal tolerance limits. Additionally, the intense reactivation of snail metabolic rate upon arousal from hibernation or estivation may provoke stress. Land snails, Helix lucorum, display a wide altitudinal distribution resulting in populations being exposed to different seasonal temperature variations. The aim of the present study was to investigate the expression of heat shock proteins (Hsps), mitogen activated protein kinases (MAPKs) and proteins that are related to apoptosis (Bcl-2, ubiquitin), that have 'cytoprotective' roles and are also considered to be reliable indicators of stress because of their crucial role in maintaining cellular homeostasis. These proteins were assessed in H. lucorum individuals from two different populations, one at Axios (sea level, 0 m) and the other at Kokkinopilos (Olympus, 1250 m), as well as after mutual population exchanges, in order to find out whether the different responses of these stress-related proteins depend solely on the environmental temperature. The results showed seasonally altered levels in all studied proteins in the hepatopancreas and foot of snails, both among different populations and between the same populations exposed to varying altitudes. However, individuals of the same population in their native habitat or acclimatized to a different habitat showed a relatively similar pattern of expression, supporting the induction of the specific proteins according to the life history of each species.

A Study of the Participation of NMDA Glutamate Receptors in the Mechanisms of Specific Anterograde Amnesia Reversion.

We studied the involvement of NMDA glutamate receptors in the mechanisms of anterograde amnesia. It was found that repeated training of amnestic animals treated with D-cycloserine, a potent agonist of the glycine site of NMDA receptors, did not lead to consolidation of long-term memory, while expression of short-term memory was more pronounced in comparison with control animals that received saline before repeated training. It was shown that D-cycloserine in amnestic snails did not affect the food reactions caused by the presentation of a conditioned stimulus during the reminder (without combination with the unconditioned stimulus). It is assumed that NMDA glutamate receptors in amnestic animals are involved in the neural plasticity mechanisms that underlie short-term memory, but their activation does not influence the anterograde amnesia processes and does not lead to the formation or recovery of long-term memory.

The Effects of Repeated Administration of the Micellar Complex of Methylprednisolone on the Locomotor Activity of a Terrestrial Snails.

We studied the effects of repeated injections of methylprednisolone and its micellar complex with block-copolymer on locomotor activity of a terrestrial snail. It was shown that methylprednisolone solution injected into the hemolymph of the animal produced a direct effect on the muscle system of the animal as soon as 1 h after administration: it slowed down snail locomotion and reduced contractile activity of the foot muscles. The micellar complex of methylprednisolone with block-copolymer prevented this effect during the first 2 days of injection and negatively affected locomotion only in 2 days after injection, the decrease in locomotion in this case was not accompanied by a decrease in contractile activity of the foot muscle.

Changes in Membrane and Threshold Potentials of Command Neurons in Terrestrial Snail during Development of a Conditioned Situational Defensive Reflex.

Changes of the electrical characteristics of command neurons of defensive behavior caused by the development of a conditioned situational defensive reflex were studied experimentally under in vitro conditions on preparations of the nervous system of snails. After learning, the membrane and threshold potentials of command neurons LPa3 and RPa3 significantly decreased and excitability of the studied neurons increased.

Immediate-Early Genes Detection in the CNS of Terrestrial Snail.

In the present work, using in situ hybridization, we studied the expression patterns of three molluscan homologs of vertebrate immediate-early genes C/EBP, c-Fos, and c-Jun in the central nervous system (CNS) of terrestrial gastropod snail Helix. The molluscan C/EBP gene was described in literature, while c-Fos and c-Jun were studied in terrestrial snails for the first time. Localization of the expression was traced in normal conditions, and in preparations physiologically activated using stimulation of suboesophageal ganglia nerves. No expression was detected constitutively. In stimulated preparations, all three genes had individual expression patterns in Helix CNS, and the level of expression was stimulus-dependent. The number of cells expressing the gene of interest was different from the number of cells projecting to the stimulated nerve, and thus activated retrogradely. This difference depended on the ganglia studied. At the subcellular level, the labeled RNA was observed as dots (probably small clusters of RNA molecules) and shapeless mass of RNA, often seen as a circle at the internal border of the cell nuclei. The data provide a basis for further study of behavioral role of these putative immediate-early genes in snail behavior and learning.

Changes in Amnesia Parameters over Time after Long-Term Memory Disruption with Protein Kinase Mζ Inhibitor.

We studied the involvement of protein kinase Mζ (PKMζ) in the mechanisms of amnesia development within 10 days after disruption of conditioned food aversion memory with ZIP (a PKMζ inhibitor). Repeated training performed in 3 days after amnesia induction with ZIP, led to the formation of conditioned food aversion memory, but the number of combined presentations of food and reinforcer stimuli was lower than during the initial training. Repeated training performed in 10 days after amnesia induction also led to memory formation, but the number of combined presentations of the stimuli was similar to that during the initial training. It was hypothesized that at the early stages of ZIP-induced amnesia, residual memory trace can be restored and amplified during repeated training, which led to memory expression at the behavioral level. At the late stages of amnesia, this memory trace was completely erased and repeated training led to the formation of a new memory. Thus, PKMζ inhibition results in the relatively fast impairment of memory retrieval and induces long-term process of memory erasing.

Helix aspersa as sentinel of development damage for biomonitoring purpose: A validation study.

Environmental health has always been threatened by the bioaccumulation of heavy metals in the terrestrial ecosystem, affecting its quality and safety. The aim of this review is to investigate the effects of heavy metal soil contamination, using the land snail Helix aspersa as a bioindicator. H. aspersa, a common species of land snail present in the area, has been used as a bioindicator of metal contamination and represents a promising ecological indicator. Various land snails species have become popular in microcosm studies because they accumulate high concentrations of certain trace metals. They express contamination as a whole through ingestion of polluted foods, such as live plants, microorganisms, soil, and water and also through cutaneous contact and from the polluted air they breathe. Land snails are considered appropriate sentinel species because trace metals tend to accumulate in their digestive gland to allow biomonitoring of metal pollution. Different experiments demonstrate that exposure in areas chronically polluted with metals, especially with lead, often causes changes in reproduction, with a variation in the mineral composition in the snail's eggs and also in its development, due to increased energy expenses associated with detoxification and the excretion process.

Riding on the wind: volatile compounds dictate selection of grassland seedlings by snails.

Background and Aims: Seedling herbivory is an important selective filter in many plant communities. The removal of preferred food plants by both vertebrate and, more commonly, invertebrate herbivores can destroy entire seedling cohorts, and consequently dictate plant community assembly. Nevertheless, our understanding of how and why some seedlings are more prone to herbivore attack than their neighbours remains limited. For seedlings, where even minor tissue damage is fatal, avoiding contact with herbivores is probably advantageous and, on this basis, volatile organic compounds (VOCs) are strong candidates to fulfil a primary defensive role. Methods: We quantified seedling selection by snails (Cornu aspersum) for 14 common, European grassland species. Seedling acceptability was subsequently compared with species-specific expression of constitutive secondary defence metabolites (CSDMs), and VOCs to determine their relative influence on seedling selection. Results: We found no relationship between seedling acceptability and CSDMs, but seedling selection was strongly associated with VOC profiles. Monoterpenes (specifically ß-ocimene) were identified as likely attractants, while green leaf volatiles (GLVs) (3-hexen-1-ol acetate) were strongly associated with low seedling acceptability. Conclusions: By elucidating a relationship between VOCs and seedling acceptability, we contradict a long-held, but poorly tested, assumption that seedling selection by herbivores in (semi-)natural plant communities centres on CSDMs. Instead, our results corroborate recent work showing how GLVs, including 3-hexen-1-ol acetate, deter crop seedling selection by molluscs. Although our failure to establish any early-ontogenetic relationship between VOCs and CSDMs also suggests that the former do not 'advertise' possession of the latter, we nevertheless reveal the role that VOCs play in defending seedlings against herbivory before lethal damage occurs.

Hibernation induces changes in the metacerebral neurons of Cornu aspersum: distribution and co-localization of cytoskeletal and calcium-binding proteins.

Pulmonate gastropods provide unique opportunities to examine physiological and biochemical adaptation strategies when cellular metabolic activity is reduced. In this study, cytochemical changes in metacerebral neurons of the cerebral ganglia were investigated in the garden snail Cornu aspersum during the hibernation phase. The immunocytochemical expression of three cytoskeletal markers: microtubule-associate protein 2-like (MAP-2-li), phosphorylated form of tau-like (P-Tau-li) and heavy subunit of neurofilaments-like (NF-H-li), and of two calcium-binding proteins: calmodulin-like (CaM-li) and parvalbumin-like (PV-li) was compared in active and hibernated snails. The immunopositivity for all the markers increased during hibernation versus activity in metacerebral neurons, with the notable exception of PV-li, which remained highly expressed during the whole annual cycle. Strongly positive aggregates of MAP-2-li and P-Tau-li were detected in the somata of hibernated snail neurons. P-Tau-li aggregates co-localized with CaM-li-labelled masses during hibernation. In addition, increased labelling of NF-H-li epitopes was associated with enhancement of CaM immunopositivity. These changes may reflect neural plasticity mechanisms mainly mediated by microtubule-associated proteins and CaM. Moreover, neuroprotective strategies may allow neurons to endure the prolonged hypometabolic conditions, taking into account that many of the functions controlled by the metacerebrum, such as feeding and movement, are suspended during hibernation. In this context, the molluscan ganglia model offers an easy opportunity to understand the molecular mechanisms behind these life cycle changes in cell physiology and to investigate possible cytological similarities among distantly related animals that adapt to the same environmental challenges through hibernation.

Genetic effects in Helix aspersa near a coal plant revealed by the micronucleus test.

Coal plants can be a major source of mutagenic pollutants. In this study we used the common land snail Helix aspersa, to detect the mutagenic effect of pollution from a coal plant in central Italy applying the micronucleus test (MN) on snail's haemocytes and evaluating trace elements concentration (As Cd, Pb, Hg, and Zn) in soil and snails. Snails from a biological farm were exposed for 13 days in five locations at different distances from the plant. Wild snails collected in the same locations were also analysed. MN frequency in exposed snails was significantly higher in four locations within 10 km from to the plant, with respect to the control and the farthest location. Comparing the MN frequency between farmed and wild snails, a significantly higher frequency emerged for the exposed snails in all locations except the farthest, likely indicating adaptation or selection of the wild organisms due to chronic exposure to pollutants. In natural snails significantly higher MN frequencies with near the plant emerged as well. Trace elements analysis showed significant correlations between MN frequencies and both Zn and As concentrations in soil, for both exposed and wild snails, and Zn and Pb concentrations in exposed snails. Our results were consistent with those previously obtained when evaluating primary DNA damage in natural snails from the same area and show that the snails near the plant were affected by a permanent cytogenetic damage. Moreover, they confirm the suitability of snails for biomonitoring the presence of pollutants with mutagenic effect.

Effects of copper on the survival, hatching, and reproduction of a pulmonate snail (Physa acuta).

Acute and chronic bioassays provide essential basis for establishment of environmental quality standards. The effects of Cu on a pulmonate snail, Physa acuta, were investigated at a number of sublethal and lethal endpoints. Cu exposure suppressed movement and triggered an escape response in P. acuta at low and high concentrations, respectively, exerting acute toxic effects on adult snails exposed to a 96 h LC50 of 23.8 µg L-1. Following 16 d exposure of Cu to the egg masses, successful hatching decreased with increasing Cu concentration. High Cu concentrations (12.5 and 25 µg L-1) resulted in inhibition of eye and shell development at the veliger stage, and a deformed shell, abnormal eyes, and different morphological shapes with lesions and hemorrhages were observed after 9 days of exposure. A large number of eggs exposed to 2.5-25 µg L-1 Cu remained in the veliger and hippo stages for 2-7 days, with no further development. Results from reproduction tests showed that adult snails exposed to various Cu treatments produced more than three broods, with the total number of eggs ranging from 770 to 1,289, revealing little difference between the control and Cu-treated groups (p > 0.05). However, snails exposed to 12.5 and 25 µg L-1 Cu produced polynuclear eggs in one egg capsule. The hatching success rate and shell length of the filial generation were significantly reduced in a dose-dependent manner (p < 0.05). The shell length of newly hatched snails was shorter in the reproduction test than in the hatching test, indicating inherent Cu toxicity in the filial generation from the exposed parent strain. The present study provides essential data regarding Cu toxicity in pulmonate snail P. acuta.

Snail mucus - glandular origin and composition in Helix pomatia.

Apart from their well-known culinary use, gastropod species such as Helix, which have a hydrogel-like mucus, are increasingly being exploited for cosmetic, bioengineering and medical applications. However, not only are the origin and composition of these "sticky" secretions far from being fully characterized, the number and morphology of the mucus glands involved is also uncertain. This study aims to characterize in detail the cutaneous glands of the Helix pomatia foot on morphological, histochemical and immunohistochemical levels. Hereby the focus is on the gland position and appearance on the foot sole as well as on the chemical nature of the different gland secretions. At least five different gland types can be distinguished by their microanatomy; three are located on the dorsal side and two on the ventral side of the foot sole. Most glands are reactive for acidic proteins and sugars such as mannose and fucose, indicating the presence of acidic glycosaminoglycans. One dorsal gland type shows high reactivity for acidic proteins only. The isolated mucus includes a certain amount of the elements chlorine, potassium and calcium; evidence for lipids was also confirmed in the isolated mucus. The present results for Helix pomatia show a clear difference in the number of glands compared to the related species Helix aspersa (only four mucus glands); histochemically, the glands of both species similarly produce acidic proteins as well as acidic glycosaminoglycans. While calcium ions are known to play a role in mucus formation, the presence and function of other ions such as potassium still need to be clarified.

The physiological role and toxicological significance of the non-metal-selective cadmium/copper-metallothionein isoform differ between embryonic and adult helicid snails.

Metal regulation is essential for terrestrial gastropods to survive. In helicid snails, two metal-selective metallothionein (MT) isoforms with different functions are expressed. A cadmium-selective isoform (CdMT) plays a major role in Cd2+ detoxification and stress response, whereas a copper-selective MT (CuMT) is involved in Cu homeostasis and hemocyanin synthesis. A third, non-metal-selective isoform, called Cd/CuMT, was first characterized in Cantareus aspersus. The aim of this study was to quantify the transcriptional activity of all three MT genes in unexposed and metal-exposed (Cd, Cu) embryonic Roman snails. In addition, the complete Cd/CuMT mRNA of the Roman snail (Helix pomatia) was characterized, and its expression quantified in unexposed and Cd-treated adult individuals. In embryos of Helix pomatia, the Cd/CuMT gene was induced upon Cu exposure. Its transcription levels were many times higher than that of the other two MT genes, and also exceeded by far the Cd/CuMT mRNA concentrations of adult snails. In the hepatopancreas of adult Roman snails, no Cd/CuMT could be detected at the protein level, irrespective of whether the snails had been exposed to Cd or not. This contrasts with the situation in the near relative, Cantareus aspersus. It appeared that the 3'-UTR of the Cd/CuMT mRNA differed largely between Cantareus aspersus and Helix pomatia, being larger in the latter species, with a number of putative binding sites for proteins and miRNAs known to inhibit mRNA translation. We suggest this as a possible mechanism responsible for the lack of Cd/CuMT protein expression in adult Roman snails.

Biological control of snail hosts transmitting schistosomiasis by the water bug, Sphaerodema urinator.

The water bug, Sphaerodema urinator (Hemiptera : Belostomatidae), shares the same habitat of the freshwater snails in ponds, lakes, and streams. Studies conducted in lakes show that fish and crayfish predators play an important role in determining the abundance of freshwater snails. In contrast, shallow ponds and marches often lack fish and crayfish but have abundant insect predators. This study has been carried out to evaluate the predatory potential of S. urinator adult on two freshwater snails that serves as intermediate hosts of Schistosoma. Laboratory evaluation of predation by S. urinator on these intermediate hosts revealed that the adult bug could kill and consume the two intermediate hosts: Bulinus truncatus and Biomphalaria alexandrina. The number of snails consumed differed according to the snail type, size, and density. The times taken for searching and handling times were depending on the snail size, type, and vulnerability of the predator. The predation rate varied also with respect to snail type and density. Prey size is a major factor influencing predator preferences. This study indicated that the predator, S. urinator, may be a suitable bio-control agent in connection with Schistosoma intermediate hosts in the aquatic area.

Involvement of Glycogen Synthase Kinase-3 in the Mechanisms of Conditioned Food Aversion Memory Reconsolidation.

Experiments were performed on the snails trained in conditioned food aversion for 3 days. Injection of TDZD-8 (glycogen synthase kinase-3 inhibitor, 2 mg/kg) in combination with reminder (presentation of a conditioned food stimulus) led to memory impairment developing 3 days after inhibitor/reminder exposure and followed by spontaneous recovery in 10 days. Injections of TDZD-8 in a dose of 4 or 20 mg/kg before reminder were shown to cause amnesia that persisted for more than 10 days. Memory recovery during repeated training was observed at the earlier period than after initial training. The impairment of memory reconsolidation by TDZD-8 after training of snails for 1 day was less pronounced than under standard training conditions (3 days). The effect of a glycogen synthase kinase-3 inhibitor during memory reconsolidation is probably followed by impairment of memory retrieval and/or partial loss, which can be compensated spontaneously or after repeated training.

Specificity of Mechanisms of Memory Reconsolidation in Snails Trained for Rejection of Two Types of Food.

Specificity of behavioral and neuronal mechanisms of impairment of long-term memory reconsolidation was studied in edible snails trained for associative skill of rejection of two types of food: raw carrots (conditioned stimulus 1) and apple (conditioned stimulus 2). In 2 days after training, the snails received protein synthesis inhibitor cycloheximide and a reminder (conditioned stimulus 1 or 2). In 3 and 14 days after cycloheximide/reminder, we observed the absence of aversive responses to the conditioned stimulus used as the reminder and preserved responses to the conditioned stimulus not used as the reminder. Moreover, we observed specific suppression of synaptic responses of command neurons of snail defensive behavior induced by the conditioned stimulus used as the reminder after cycloheximide injection and preserved synaptic responses of neurons to the other conditioned stimulus. It was hypothesized that protein synthesis-dependent synapse-specific plasticity of command neurons can be a mechanism of selective preservation of conditioned food aversion memory in snails.

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.

Laboratory studies on the thermal tolerance and response of enzymes of intermediate metabolism in different land snail species.

Land snails species occur in a range of habitats from humid to semi-arid and arid ones and seasonal variations in their physiology and biochemical composition have been linked to annual cycles of photoperiod, temperature, humidity and water availability. In an effort to understand the thermal tolerance and the impact of temperature elevation on tissue metabolism of land snails we determined the mortality, heamolymph PO2 and the activities of enzymes of intermediary metabolism in three land snail species (Helix lucorum, Helix pomatia and Cornu aspersum) differing in their geographical distribution and inhabiting areas with different climatic characteristics. No mortality was observed in both population of Cornu aspersum, while Helix pomatia exhibited higher mortality than Helix lucorum. PO2 dropped within the first 10days of exposure to elevated temperature in all species, although in Cornu aspersum this decrease was significantly lower. No significant reduction in the enzymatic activities of all glycolytic enzymes studied, as well as of citrate synthase (CS) and 3-hydroxyacyl-CoA dehydrogenase (HOAD) was observed in the more thermal tolerant species C. aspersum from both populations studied. Significant reductions of enzymatic activity of the glycolytic enzymes phosphofructokinase (PFK), pyruvate kinase (PK) and d-Lactate dehydrogenase (d-LDH) was observed in Helix lucorum and Helix pomatia. The observed inter-specific differences seem to be in accordance with the life cycle characteristics of each species and may be attributed to climatic differences among habitats within their distribution range.

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.