Potential toxic effects of titanium oxide (TiO2) nanoparticles on the biological, biochemical, and histological aspects of the land snail Helix aspersa.

Nanotechnology has come a long way in our lives. However, it maintains some negative effects on the environment. This study aims to use the land snail Helix aspersa as a bioindicator. Titanium dioxide nanoparticles (TiO2NPs) had been used at 70 and 140 µg/L for two weeks by the spraying method. The oxidative biomarkers, condition index (CI), DNA damage, hemocyte count, and phagocytic activity were estimated. The toxicity of TiO2NPs was determined (LC50 = 544 µg/L). The exposure to TiO2NPs caused a significant reduction of the activities of superoxide dismutase (SOD) and catalase (CAT) in the digestive gland of Helix aspersa (the activity of CAT was 3.4 ± 0.1 (P = 0.001), SOD was 11 ± 1 (P = 0.0002) at concentration 140 µg/L after two weeks). The activity of glutathione peroxidase (GPX) was (1.13 ± 0.01 µ/mg protein at 140 µg/L compared with controls (5.47 ± 0.01 µ/mg protein). The treatment caused DNA damage in the hemocytes (tail DNA % = 8.66 ± 0.02 and tail moment = 52.99 ± 0 at140 µg/L (P = 0.002)). In the digestive gland, both tail DNA % and tail moment increased (tail moment = 78.38 ± 0.08 compared with control = 2.29 ± 0.09 (P = 0.0001)). The total count of hemocytes significantly decreased after two weeks (the average number was 71 ± 1.5 compared with controls 79 ± 1.1 at 140 µg/L). Furthermore, TiO2NPs caused histological alterations in the digestive gland of Helix aspersa. It can be concluded that the Helix aspersa can be used as environmental pollution bioindicator. A comprehensive evaluation of toxic effects induced by TiO2NPs in vivo assays must be investigated.

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.

Accumulation of metal trace elements in different body parts of terrestrial Roman snail Helix pomatia L., 1758 on three polluted sites in Serbia.

Atmospheric pollution remains one of the growing concerns in the twenty-first century, with particular focus on metal trace elements (MTE) from anthropogenic sources, due to their adverse effects on biota. The concentration and type of MTE in the atmosphere and in the soil are diverse, depending on the origin of pollutants, which can cause diverse detrimental effects on organisms living in the nearby environment. Three sites in Central Serbia with different origins of MTE pollution (urban contamination, smelting, and fly ash area) were assessed, using terrestrial Roman snails (Helix pomatia) as biomarker organisms. These snails are sentinel organisms and are known for their capacities for accumulation of MTE. Snails were sampled and their body was divided in three parts: viscera, foot, and shell and concentrations of MTE were determined in each of these body parts using inductively coupled plasma optical spectrometry. Results showed contrasting MTE accumulation patterns in body parts of the snails. Of three studied sites, snails sampled in the vicinity of fly ash containment had lower concentrations of MTE compared to other two polluted sites.

DNA methylation inhibition participates in the anterograde amnesia key mechanism through the suppression of the transcription of genes involved in memory formation in grape snails.

The study of the amnesia mechanisms is of both theoretical and practical importance. The mechanisms of anterograde amnesia are the least studied, due to the lack of an experimental model that allows studying this amnesia type molecular and cellular mechanisms. Previously, we found that conditional food aversion memory reconsolidation impairment in snails by NMDA glutamate receptor antagonists led to the amnesia induction, in the late stages of which (>10 days) repeated training did not cause long-term memory formation. In the same animals, long-term memory aversion to a new food type was formed. We characterized this amnesia as specific anterograde amnesia. In the present work we studied the role of epigenetic DNA methylation processes as well as protein and mRNA synthesis in the mechanisms of anterograde amnesia and memory recovery. DNMT methyltransferase inhibitors (iDNMT: zebularine, RG108 (N-Phthalyl-1-tryptophan), and 5-AZA (5-Aza-2'-deoxycytidine)) were used to alter DNA methylation. It was found that in amnesic animals the iDNMT administration before or after shortened repeated training led to the rapid long-term conditional food aversion formation (Ebbinghaus saving effect). This result suggests that amnestic animals retain a latent memory, which is the basis for accelerated memory formation during repeated training. Protein synthesis inhibitors administration (cycloheximide) before or immediately after repeated training or administration of RNA synthesis inhibitor (actinomycin D) after repeated training prevented memory formation under iDNMT action. The earlier protein synthesis inhibitor effect suggests that the proteins required for memory formation are translated from the pre-existing, translationally repressed mRNAs. Thus, we have shown for the first time that the anterograde amnesia key mechanism is DNMT-dependent suppression of the transcription of genes involved in memory mechanisms. Inhibition of DNMT during repeated training reversed these genes expression blockade, opening access to them by transcription factors synthesized during training from the pre-existing mRNAs.

Property of arylsulfatase and ß-glucuronidase extracted from digestive tracts of Cipangopaludina chinensis and their cleavage performance on conjugated natural estrogens.

Arylsulfatase and ß-glucuronidase are the two substantial enzymes having a significant role in the cleavage of conjugated natural estrogens (C-NEs). The present study reports that arylsulfatase and ß-glucuronidase have been abundantly found in the digestive tracts of Cipangopaludina chinensis; in which, their corresponding activities were 60 and 5 U/g wet waste, respectively. The arylsulfatase from Cipangopaludina chinensis could show high activity at low temperatures. Hence, its activity still remained at 53.2% of maximal activity even at an extremely low temperature of 4 ℃; while the corresponding activities of arylsulfatase from Helix pomatia or activated sludge were less than 20% and 10%, respectively. The arylsulfatase and ß-glucuronidase from Cipangopaludina chinensis could efficiently cleave C-NEs suggesting that they could be alternative enzymes derived from Helix pomatia that are used for cleavage of conjugated compounds in environmental or biological sample analysis. Meanwhile, they might also be used to enhance the cleavage of C-NEs in municipal wastewater.

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.

Effect of silver nanoparticles on gene transcription of land snail Helix aspersa.

Silver nanoparticles (Ag-NPs) are extremely useful in a diverse range of consumer goods. However, their impact on the environment is still under research, especially regarding the mechanisms involved in their effect. Aiming to provide some insight, the present work analyzes the transcriptional activity of six genes (Hsp83, Hsp17.2, Hsp19.8, SOD Cu-Zn, Mn-SOD, and BPI) in the terrestrial snail Helix aspersa in the presence of different concentrations of Ag-NPs. The animals were exposed for seven days to Lactuca sativa soaked for one hour in different concentrations of Ag-NPs (20, 50, 100 mg/L). The results revealed that the highest concentration tested of Ag-NPs (100 mg/L) led to a statistically significant induction of the Hsp83 and BPI expression in the digestive gland compared to the control group. However, a trend to upregulation with no statistical significance was observed for all the genes in the digestive gland and the foot, while in the hemolymph, the trend was to downregulation. Ag-NPs affected the stress response and immunity under the tested conditions, although the impact was weak. It is necessary to explore longer exposure times to confirm that the effect can be maintained and impact on health. Our results highlight the usefulness of the terrestrial snail Helix aspersa as a bioindicator organism for silver nanoparticle pollution biomonitoring and, in particular, the use of molecular biomarkers of pollutant effect as candidates to be included in a multi-biomarker strategy.

Antimicrobial efficacy of Egyptian Eremina desertorum and Helix aspersa snail mucus with a novel approach to their anti-inflammatory and wound healing potencies.

Snail mucus is composed of bioactive compounds thought to have different biological properties for the treatment of some skin problems. Although Helix aspersa mucus is used in several cosmetic products, a detailed characterization of Eremina desertorum mucus composition and its biological activities is still missing. Mucus extracts (MEs) from H. aspersa and E. desertorum were prepared and tested for their antimicrobial and anti-inflammatory activities with their potencies in wound healing. Also, chemical characterization was performed by GC-MS analysis. Results showed that ME of E. desertorum gave higher inhibitory activity against resistant strains related to burn wound infections compared to ME of H. aspersa. Additionally, it revealed a significant anti-inflammatory activity. Moreover, we found that ME of E. desertorum lacked cytotoxicity and was able to significantly induce cell proliferation and migration through up-regulation of TGF-ß1 and VEGF gene expression. Our results suggested that MEs of E. desertorum have higher biological effects than H. aspersa, which are attributable to antimicrobial, anti-inflammatory activities, cell proliferation and pave the way for further investigating its potential effect as a human therapeutic agent.

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.

Bioactive snail mucus-slime extract loaded chitosan scaffolds for hard tissue regeneration: the effect of mucoadhesive and antibacterial extracts on physical characteristics and bioactivity of chitosan matrix.

Biobased extracts comprise various bioactive components and they are widely used in tissue engineering applications to increase bioactivity as well as physical characteristics of biomaterials. Among animal sources, garden snailHelix aspersahas come into prominence with its antibacterial and regenerative extracts and show potential in tissue regeneration. Thus, in this study, bioactiveH. aspersaextracts (slime, mucus) were loaded in chitosan (CHI) matrix to fabricate porous scaffolds for hard tissue regeneration. Physical, chemical properties, antimicrobial activity was determined as well asin vitrobioactivity for bone and cartilage regeneration. Mucus and slime incorporation enhanced mechanical properties and biodegradation rate of CHI matrix. Scanning electron microscopy images showed that the average pore size of the scaffolds decreased with higher extract content. Mucus and slime extracts showed antimicrobial effect on two bacterial strains.In vitrocytotoxicity, osteogenic and chondrogenic activity of the scaffolds were evaluated with Saos-2 and SW1353 cell lines in terms of Alkaline phosphatase activity, biomineralization, GAG, COMP and hydroxyproline content. Cell viability results showed that extracts had a proliferative effect on Saos-2 and SW1353 cells when compared to the control group. Mucus and slime extract loading increased osteogenic and chondrogenic activity. Thus, the bioactive extract loaded CHI scaffolds showed potential for bone and cartilage regeneration with enhanced physical properties andin vitrobioactivity.

Phenoloxidase is involved in the immune reaction of Helix lucorum to parasitic infestation by dicrocoeliid trematode.

INTRODUCTION: Phenoloxidases are known to play a role in the immune defences of arthropods and molluscs. In the invertebrates, phenoloxidases mediate three major physiologically important processes: sclerotization, wound healing, and defence reactions. Helix lucorum serve as the first intermediate host for the larval stages of dicrocoeliid trematodes which infects animals as well as human beings. OBJECTIVE: The aim of the study is to investigate the effect of larval forms of dicrocoeliid trematodes to phenoloxidase acitivity in H. lucorum, Linneaus, 1758, in Bitlis, Turkey. The effect of the snail's shell colour to phenoloxidase activity was also investigated. MATERIAL AND METHODS: Land snails (n=200) were collected by hand from their natural habitats during the period May - June 2019 in Bitlis, Turkey. Evaluation of the process was performed by measuring immune reaction of the snails against larval forms of dicrocoeliid trematodes. Phenoloxidase activity assay was carried out using a spectrophotometer device based on 3,4-Dihydroxy-L-phenylalanine (L-dopa) hydrolysis. RESULTS: The natural infection rate of the land snails with the developmental stages of dicrocoeliid trematodes was 20%. Phenoloxidase activity was found to be significantly higher (*p<0.05) in larval forms of dicrocoeliid trematodes infected snails when compared with non-infected snails. No effect of shell colours to phenoloxidase activity was observed. CONCLUSIONS: To the best of the authors' knowledge, this study is the first to report that the phenoloxidase system is involved in the immune reaction of Helix lucorum to parasitic infestation by larval forms of dicrocoeliid trematodes.

The protein and volatile components of trail mucus in the Common Garden Snail, Cornu aspersum.

The Common or Brown Garden Snail, Cornu aspersum, is an invasive land snail that has successfully colonized a diverse range of global environments. Like other invasive land snails, it is a significant pest of a variety of agricultural crops, including citrus, grapes and canola. Cornu aspersum secretes a mucus trail when mobile that facilitates locomotion. The involvement of the trail in conspecific chemical communication has also been postulated. Our study found that anterior tentacle contact with conspecific mucus elicited a significant increase in heart rate from 46.9 to 51 beats per minute. In order to gain a better understanding of the constituents of the trail mucus and the role it may play in snail communication, the protein and volatile components of mucus trails were investigated. Using two different protein extraction methods, mass spectrometry analysis yielded 175 different proteins, 29 of which had no significant similarity to any entries in the non-redundant protein sequence database. Of the mucus proteins, 22 contain features consistent with secreted proteins, including a perlucin-like protein. The eight most abundant volatiles detected using gas chromatography were recorded (including propanoic acid and limonene) and their potential role as putative pheromones are discussed. In summary, this study has provided an avenue for further research pertaining to the role of trail mucus in snail communication and provides a useful repository for land snail trail mucus components. This may be utilized for further research regarding snail attraction and dispersal, which may be applied in the fields of agriculture, ecology and human health.

Histological and chemical damage induced by microcystin-LR and microcystin-RR on land snail Helix aspersa tissues after acute exposure.

Microcystins (MCs) are the most common cyanotoxins with more than 200 variants. Among these cyanotoxins, microcystin-LR (MC-LR) and microcystin-RR (MC-RR) are the most studied congeners due to their high toxicity and frequent occurrence in surface waters. MC-LR has been detected in more than 75% of natural cyanobacteria bloom, along with other toxic and less toxic congeners. Accumulation of several microcystins variants (MC-LR and MC-RR) has been confirmed in aquatic snails exposed naturally or in the laboratory to toxic blooms. Thus, this paper aims to compare the biochemical and histological impact of both toxic variants (microcystin-LR and microcystin-RR) and their mixed form on a bioindicator, the land snail Helix aspersa. During experiments, snails were gavaged with a single acute dose (0.5 µg/g) of purified MC-LR, MC-RR, or mixed MC-LR + MC-RR (0.25 + 0.25 µg/g). After 96 h of exposure, effects on the hepatopancreas, kidney, intestine and lungs were assessed by histological observations and analysis of oxidative stress biomarkers. The results show that a small dose of MCs variants can increase the non-enzymatic antioxidant glutathione (GSH), inhibit glutathione-s-transferase (GST) level and trigger a defense system by activating glutathione peroxidase (GPx), catalase (CAT) and superoxide dismutase (SOD). Microcystin-RR causes serious anomalies in the hepatopancreas and kidney than Microcystin-LR. The organ most affected is the kidney. The damage caused by MC-LR + MC-RR is greater than that caused by single variants.

Protein synthesis inhibitor administration before a reminder caused recovery from amnesia induced by memory reconsolidation impairment with NMDA glutamate receptor antagonist.

Memory recovery in amnestic animals is one of the most poorly studied processes. In this paper, we examine the role of protein synthesis and a reminder in the mechanisms of amnesia and memory recovery in grape snails trained to conditioned food aversion. Amnesia was induced by the impairment of memory reconsolidation using NMDA (N-methyl d-aspartate) glutamate receptor antagonists. In an early stage of amnesia (day 3), injections of protein synthesis inhibitors into animals combined with a reminder by a conditioned stimulus (CS) led to the recovery of aversive reactions to its presentation. Two types of changes in reactions to CS were revealed. In most animals, a persistent recovery of memory retrieval was found that lasted for at least 10 days. In other snails, aversive responses to CS persisted for 24 h. Isolated injections of inhibitors, injections of inhibitors and a reminder by the learning environment (without presenting a CS), usage of a differentiating stimulus instead of a CS, or inhibitor injections after the reminder did not affect the development of amnesia. The administration of protein synthesis inhibitors and a reminder in the late period after amnesia induction (10 days) did not affect its development or caused a short-term memory recovery. We suggest that amnesia is an active process that develops over time. The reminder induces the reactivation of the amnesia process dependent on protein synthesis, while the administration of protein synthesis inhibitors leads to the impairment of amnesia reactivation and recovery of the state formed before amnesia induction (i.e., recovery of conditioned food aversion memory).

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.

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.

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.

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.