Accelerated mitochondrial evolution and asymmetric fitness of hybrids contribute to the persistence of Helix thessalica in the Helix pomatia range.

Interbreeding and introgression between recently diverged species is common. However, the processes that prevent these species from merging where they co-occur are not well understood. We studied the mechanisms that allowed an isolated group of populations of the snail Helix thessalica to persist within the range of the related Helix pomatia despite high gene flow. Using genomic cline analysis, we found that the nuclear gene flow between the two taxa across the mosaic hybrid zone was not different from that expected under neutral admixture, but that the exchange of mtDNA was asymmetric. Tests showed that there is relaxed selection in the mitochondrial genome of H. thessalica and that the substitution rate is elevated compared to that of H. pomatia. A lack of hybrids that combine the mtDNA of H. thessalica with a mainly (>46%) H. pomatia genomic background indicates that the nuclear-encoded mitochondrial proteins of H. pomatia are not well adapted to the more rapidly evolving proteins and RNAs encoded by the mitochondrion of H. thessalica. The presumed reduction of fitness of hybrids with the fast-evolving mtDNA of H. thessalica and a high H. pomatia ancestry, similar to 'Darwin's Corollary to Haldane's rule', resulted in a relative loss of H. pomatia nuclear ancestry compared to H. thessalica ancestry in the hybrid zone. This probably prevents the H. thessalica populations from merging quickly with the surrounding H. pomatia populations and supports the hypothesis that incompatibilities between rapidly evolving mitochondrial genes and nuclear genes contribute to speciation.

Predicting the immunomodulatory activity of probiotic lactic acid bacteria using supervised machine learning in a Cornu aspersum snail model.

In the process of screening for probiotic strains, there are no clearly established bacterial phenotypic markers which could be used for the prediction of their in vivo mechanism of action. In this work, we demonstrate for the first time that Machine Learning (ML) methods can be used for accurately predicting the in vivo immunomodulatory activity of probiotic strains based on their cell surface phenotypic features using a snail host-microbe interaction model. A broad range of snail gut presumptive probiotics, including 240 new lactic acid bacterial strains (Lactobacillus, Leuconostoc, Lactococcus, and Enterococcus), were isolated and characterized based on their capacity to withstand snails' gastrointestinal defense barriers, such as the pedal mucus, gastric mucus, gastric juices, and acidic pH, in association with their cell surface hydrophobicity, autoaggregation, and biofilm formation ability. The implemented ML pipeline predicted with high accuracy (88 %) strains with a strong capacity to enhance chemotaxis and phagocytic activity of snails' hemolymph cells, while also revealed bacterial autoaggregation and cell surface hydrophobicity as the most important parameters that significantly affect host immune responses. The results show that ML approaches may be useful to derive a predictive understanding of host-probiotic interactions, while also highlighted the use of snails as an efficient animal model for screening presumptive probiotic strains in the light of their interaction with cellular innate immune responses.

Electron Paramagnetic Resonance Studies of Irradiated Grape Snails (Helix pomatia) and Investigation of Biophysical Parameters.

A study of grape snails (Helix pomatia) using the electron paramagnetic resonance (EPR) spectroscopy method, where shells were exposed to ionizing gamma radiation, indicated that the effect of radiation up to certain doses results in the emergence of magnetic properties in the organism. The identification of the EPR spectra of the body and shell parts of the control and irradiated grape snails separately showed that more iron oxide magnetic nanoparticles are generated in the body part of the grape snail compared to the shells. A linear increase in free radical signals (g = 2.0023) in the body and shell parts of grape snails, and a non-monotonic change in the broad EPR signal (g = 2.32) characterizing iron oxide magnetic nanoparticles was determined depending on the dose of ionizing gamma radiation. Additionally, the obtained results showed that grape snails can be used as bioindicators for examining the ecological state of the environment. At the same time, the radionuclide composition of the body and shell parts of the grape snails and their specific activities were determined by CANBERRA gamma spectroscopy. The FTIR spectra of mucin, a liquid secreted by snails, were recorded.

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.

Dose-dependent effects of lead and cadmium and the influence of soil properties on their uptake by Helix aspersa: an ecotoxicity test approach.

Three soil types with different physicochemical properties were selected to evaluate their effect on lead and cadmium bioavailability and toxicity in the land snail Helix aspersa. In 28-day ecotoxicity tests, H. aspersa juveniles were exposed to increasing concentrations of Pb or Cd. EC50s, concentrations reducing snail growth by 50%, differed between the soils and so did Cd and Pb uptake in the snails. For lead, EC50s were 2397-6357 mg Pb/kg dry soil, while they ranged between 327 and 910 mg Cd/kg dry soil for cadmium. Toxicity and metal uptake were highest on the soil with the lowest pH, organic matter content and Cation Exchange Capacity (CEC). Growth reduction was correlated with metal accumulation levels in the snails' soft body, and differences in toxicity between the soils decreased when EC50s were expressed on the basis of internal metal concentrations in the snails. These results confirm the effect of soil properties; pH, CEC, OM content, on the uptake and growth effect of Pb and Cd in H. aspersa, indicating the importance of properly characterizing soils when assessing the environmental risk of metal contaminated sites.

Synthesis and Evaluation of Novel Iminosugars Prepared from Natural Amino Acids.

Cyclopropanated iminosugars have a locked conformation that may enhance the inhibitory activity and selectivity against different glycosidases. We show the synthesis of new cyclopropane-containing piperidines bearing five stereogenic centers from natural amino acids l-serine and l-alanine. Those prepared from the latter amino acid may mimic l-fucose, a natural-occurring monosaccharide involved in many molecular recognition events. Final compounds prepared from l-serine bear S configurations on the C5 position. The synthesis involved a stereoselective cyclopropanation reaction of an α,ß-unsaturated piperidone, which was prepared through a ring-closing metathesis. The final compounds were tested as possible inhibitors of different glycosidases. The results, although, in general, with low inhibition activity, showed selectivity, depending on the compound and enzyme, and in some cases, an unexpected activity enhancement was observed.

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.

Impact of ageing and soil contaminants on telomere length in the land snail.

Telomeres (TLs) are non-coding DNA sequences that are usually shortened with ageing and/or chemical exposure. Bioindicators such as the land snail can be used to assess the environmental risk of contaminated soils. As for most invertebrates, the evolution of TLs with ageing or exposure to contaminants is unknown in this mollusc. The aims of this study were to explore the relationships between ageing, contaminant exposure, sublethal effects and TL length in the terrestrial gastropod Cantareus aspersus. TL length was investigated in haemocytes from five age classes of C. aspersus. The impact of contaminants on sub-adult snails exposed to Cd, Hg or a mixture of polycyclic aromatic hydrocarbons (PAHs) in soils for one or two months was studied. Bioaccumulation, growth, sexual maturity and TLs were measured. TL attrition was significant for the juvenile and sub-adult stages, but not later. Exposure to Cd increased the mortality (around 30%). Exposure to polluted soils inhibited growth (19-40%) and sexual maturity (6-100%). Although the health of the snails exposed to Cd, Hg and PAHs was altered, TL length in haemocytes was not disturbed, suggesting a high capacity of this snail species to maintain its TLs in haemocytes under chemical stress. These results first address TL length in snails and reveal that the relationship commonly proposed for vertebrates between TL shortening and ageing or exposure to contaminants cannot be generalized.

Effects of Thryptophan Hydroxylase Blockade by P-Chlorophenylalanine on Contextual Memory Reconsolidation after Training of Different Intensity.

The processes of memory formation and its storage are extremely dynamic. Therefore, the determination of the nature and temporal evolution of the changes that underlie the molecular mechanisms of retrieval and cause reconsolidation of memory is the key to understanding memory formation. Retrieval induces the plasticity, which may result in reconsolidation of the original memory and needs critical molecular events to stabilize the memory or its extinction. 4-Chloro-DL-phenylalanine (P-chlorophenylalanine-PCPA) depresses the most limiting enzyme of serotonin synthesis the tryptophan hydroxylase. It is known that PCPA reduces the serotonin content in the brain up to 10 times in rats (see Methods). We hypothesized that the PCPA could behave the similar way in snails and could reduce the content of serotonin in snails. Therefore, we investigated the effect of PCPA injection on contextual memory reconsolidation using a protein synthesis blocker in snails after training according to two protocols of different intensities. The results obtained in training according to the first protocol using five electrical stimuli per day for 5 days showed that reminding the training environment against the background of injection of PCPA led to a significant decrease in contextual memory. At the same time, the results obtained in training according to the second protocol using three electrical stimuli per day for 5 days showed that reminding the training environment against the injection of PCPA did not result in a significant change in contextual memory. The obtain results allowed us to conclude that the mechanisms of processes developed during the reconsolidation of contextual memory after a reminding depend both on the intensity of learning and on the state of the serotonergic system.

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.

Peculiarities in Synthesis of Proteins Implicated in Memory Reconsolidation and Induction of Amnesia.

The peculiarities of implication of NMDA glutamate receptors and protein synthesis into the mechanisms responsible for impairment of memory reconsolidation were studied in edible snails conditioned to food aversion. Injections of NMDA glutamate receptor antagonists or protein synthesis inhibitor prior to reminding with conditioned food stimulus provoked development of amnesia after different latent periods. NMDA glutamate receptor antagonists gradually weakened the aversive reactions to conditioned stimulus presented in parallel with reminder during 1 h. The protein synthesis inhibitor cycloheximide provoked the development of amnesia only 2.5 h after the onset of reminding procedure. Combined injections of protein synthesis inhibitor and NMDA glutamate receptor antagonist prior to reminding completely prevented the development of amnesia. The data agree with hypothesis that memory reconsolidation and amnesia are distinct processes, which need activation of synthesis of specific proteins.

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.

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.

Atypical Acetylcholine Receptors on the Neurons of the Turkish Snail.

Using electrophysiology, the effect of nicotinic acetylcholine receptor (nAChR) ligands on acetylcholine-induced depolarization in the neurons of Helix lucorum snail was studied. It was found that the α-conotoxin PnIA [R9, L10], a selective antagonist of α7 nAChR, and α-cobratoxin (antagonist of α7 and muscle-type nAChR) suppressed neuronal depolarization. Fluorescence microscopy showed staining of the neurons with fluorescently labeled α-bungarotoxin; this staining was reduced by pretreatment with α-cobratoxin. Induced depolarization was also suppressed by α-conotoxin RgIA, a selective inhibitor of α9 nAChR. In contrast to Lymnaea stagnalis nAChR, which are weakly sensitive to neurotoxin II and α-conotoxin GI, antagonists of muscle-type nAChR, H. lucorum receptors were most effectively inhibited by these antagonists. The results obtained, as well as the previously found sensitivity of the receptors studied in this work to muscarinic receptor ligands, indicate an unusual atypical pharmacological profile of H. lucorum nAChR.

The Solution Structure and Dynamics of Cd-Metallothionein from Helix pomatia Reveal Optimization for Binding Cd over Zn.

Metallothioneins (MTs) are cysteine-rich polypeptides that are naturally found coordinated to monovalent and/or divalent transition metal ions. Three metallothionein isoforms from the Roman snail Helix pomatia are known. They differ in their physiological metal load and in their specificity for transition metal ions such as Cd2+ (HpCdMT isoform) and Cu+ (HpCuMT isoform) or in the absence of a defined metal specificity (HpCd/CuMT isoform). We have determined the solution structure of the Cd-specific isoform (HpCdMT) by nuclear magnetic resonance spectroscopy using recombinant isotopically labeled protein loaded with Zn2+ or Cd2+. Both structures display two-domain architectures, where each domain comprises a characteristic three-metal cluster similar to that observed in the ß-domains of vertebrate MTs. The polypeptide backbone is well-structured over the entire sequence, including the interdomain linker. Interestingly, the two domains display mutual contacts, as observed before for the metallothionein of the snail Littorina littorea, to which both N- and C-terminal domains are highly similar. Increasing the length of the linker motionally decouples both domains and removes mutual contacts between them without having a strong effect on the stability of the individual domains. The structures of Cd6- and Zn6-HpCdMT are nearly identical. However, 15N relaxation, in particular 15N R2 rates, is accelerated for many residues of Zn6-HpCdMT but not for Cd6-HpCdMT, revealing the presence of conformational exchange effects. We suggest that this snail MT isoform is evolutionarily optimized for binding Cd rather than Zn.

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.

Comprehensive kinetic and substrate specificity analysis of an arylsulfatase from Helix pomatia using mass spectrometry.

Sulfatases hydrolyze sulfated metabolites to their corresponding alcohols and are present in all domains of life. These enzymes have found major application in metabolic investigation of drugs, doping control analysis and recently in metabolomics. Interest in sulfatases has increased due to a link between metabolic processes involving sulfated metabolites and pathophysiological conditions in humans. Herein, we present the first comprehensive substrate specificity and kinetic analysis of the most commonly used arylsulfatase extracted from the snail Helix pomatia. In the past, this enzyme has been used in the form of a crude mixture of enzymes, however, recently we have purified this sulfatase for a new application in metabolomics-driven discovery of sulfated metabolites. To evaluate the substrate specificity of this promiscuous sulfatase, we have synthesized a series of new sulfated metabolites of diverse structure and employed a mass spectrometric assay for kinetic substrate hydrolysis evaluation. Our analysis of the purified enzyme revealed that the sulfatase has a strong preference for metabolites with a bi- or tricyclic aromatic scaffold and to a lesser extent for monocyclic aromatic phenols. This metabolite library and mass spectrometric method can be applied for the characterization of other sulfatases from humans and gut microbiota to investigate their involvement in disease development.

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.

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.