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.

Comparative assessment of biomarker response to tissue metal concentrations in urban populations of the land snail Helix pomatia (Pulmonata: Helicidae).

The traffic pressure is increasing, resulting in the emission of atmospheric pollution. Soil organisms will need to respond to pollution stressors. Among them, land snails are valuable indicators of ecosystem disturbance. In this study, land snails Helix pomatia were sampled from three city localities with different traffic intensity. Oxidative stress biomarkers catalase (CAT), glutathione peroxidase (GPX), glutathione reductase (GR) and glutathione-S-transferase (GST) in the foot muscle (FM) and hepatopancreas (HP) tissue were determined. Also, five heavy metal (Cd, Cu, Ni, Pb, and Zn) concentrations were quantified in soil and tissue samples. According to the results, the highway induces the strongest contamination on the surrounding environment, with the highest metal concentrations measured in soil and snails. At the most polluted locality, only Cd exceeded some soil guidelines authorities that we referred to in this study. In addition, tissue Cd concentrations exceeded the United States Environmental Protection Agency (USEPA) value (1 mg kg-1) for soil invertebrate toxicity at all localities making it likely responsible for generating adverse effects in snails. Regarding HP, the CAT and GST are the most sensitive parameters that could be useful as oxidative stress biomarkers in snails exposed to the actual metals in the environment. On the other hand, in FM tissue, the most pronounced changes were recorded for GPX and GR. Based on tissue-specific enzyme responses, three urban populations were clearly separated. Therefore land snails are the promising candidates for quick field-based biomarker studies after showing a tissue-specific concentration-dependent induction of certain enzymes to heavy metals.

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.

HelixComplex snail mucus as a potential technology against O3 induced skin damage.

Mucus form H. aspersa muller has been reported to have several therapeutic proprieties, such as antimicrobial activity, skin protection and wound repair. In this study, we have analyzed H. aspersa mucus (Helixcomplex) bio-adhesive efficacy and its defensive properties against the ozone (O3) (0.5 ppm for 2 hours) exposure in human keratinocytes and reconstructed human epidermis models. Cytotoxicity, tissue morphology and cytokine levels were determined. We confirmed HelixComplex regenerative and bio-adhesive properties, the latter possibly via the characteristic mucopolysaccharide composition. In addition, HelixComplex was able to protect from O3 exposure by preventing oxidative damage and the consequent pro-inflammatory response in both 2D and 3D models. Based on this study, it is possible to suggest HelixComplex as a potentially new protective technology against pollution induced skin damage.

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.

Peroxidation and unsaturation coefficients as biomarkers of environmental micro-exposure to molluscicides in Helix pomatia L.

This study was a follow-up of a previous study that investigated a set of selected fatty acids (FAs; 12 of 56 pools) in Helix pomatia L. as biomarkers of chemical stress induced by applying micro-doses of molluscicides. Here, the potential of rarely used peroxidation (PI) and unsaturation (UI) coefficients were tested as biomarkers. These indices were calculated based on the FA profiles of foot and hepatopancreas tissues of H. pomatia L. Snails were treated with three molluscicides: metaldehyde, methiocarb, and potassium chloride, in three doses each (5, 10, or 15 µl, 0.01% w/v concentration), for 12 weeks, under laboratory conditions. Correlations were evaluated between frequently used oxidation status indicators (catalase, glutathione peroxidase, selenium-dependent peroxidase, superoxide dismutase, glutathione transferase, glutathione reductase, glutathione, carbonyl protein, and thiobarbituric acid reactive substances, in the form of MDA) and UI and PI ratios. These results confirmed that fatty acids could be directly used as biomarkers of exposure and oxidative physiological status in snails. Moreover, the UI and PI, calculated based on FAs, clearly reflected the current oxidation status in snails. These indices changed with the application of micro-doses of molluscicides. In conclusion, these indices could serve as sensitive biomarkers of chemical stress in snails.

Identification and characterisation of anti - Pseudomonas aeruginosa proteins in mucus of the brown garden snail, Cornu aspersum.

Background: Novel antimicrobial treatments are urgently needed. Previous work has shown that the mucus of the brown garden snail (Cornu aspersum) has antimicrobial properties, in particular against type culture collection strains of Pseudomonas aeruginosa. We hypothesised that it would also be effective against clinical isolates of the bacterium and that investigation of fractions of the mucus would identify one or more proteins with anti-pseudomonal properties, which could be further characterised. Materials and methods: Mucus was extracted from snails collected from the wild. Antimicrobial activity against laboratory and clinical isolates of Ps. aeruginosa was determined in disc diffusion assays. Mucus was purified using size exclusion chromatography and fractions containing anti-pseudomonal activity identified. Mass spectroscopy and high performance liquid chromatography analysis of these fractions yielded partial peptide sequences. These were used to interrogate an RNA transcriptome generated from whole snails. Results: Mucus from C. aspersum inhibited growth of type collection strains and clinical isolates of Ps. aeruginosa. Four novel C. aspersum proteins were identified; at least three are likely to have antimicrobial properties. The most interesting is a 37.4 kDa protein whilst smaller proteins, one 17.5 kDa and one 18.6 kDa also appear to have activity against Ps. aeruginosa. Conclusions: The study has identified novel proteins with antimicrobial properties which could be used to develop treatments for use in human medicine.

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.

The study of multivalent carbohydrate-protein interactions by bio-layer interferometry.

The study of complex multivalent carbohydrate-protein interactions remains highly complicated and sometimes rendered impossible due to aggregation problems. In this study, we demonstrate that bio-layer interferometry is an excellent complementary method to standard techniques such as SPR and ITC. Using tetra- and hexadecavalent GalNAc glycoconjugates and Helix pomatia agglutinin (HPA) as a model lectin, we were able to measure reliable kinetic and thermodynamic parameters of multivalent interactions going from the micro to the nanomolar range.

Insights into the toxicity of iron oxides nanoparticles in land snails.

The use of manufactured nanoparticles (NPs) is spreading rapidly across technology and medicine fields, posing concerns about their consequence on ecosystems and human health. The present study aims to assess the biological responses triggered by iron oxide NPs (IONPs) and iron oxide NPs incorporated into zeolite (IONPZ) in relation to oxidative stress on the land snail Helix aspersa in order to investigate its use as a biomarker for terrestrial environments. Morphology and structure of both NPs were characterized. Snail food was supplemented with a range of concentrations of IONPs and IONPZ and values of the hemocyte lysosomal membranes' destabilization by 50% were estimated by the neutral red retention (NRRT50) assay. Subsequently, snails were fed with NPs concentrations equal to half of the NRRT50 values, 0.05 mg L-1 for IONPs and 1 mg L-1 for IONPZ, for 1, 5, 10 and 20 days. Both effectors induced oxidative stress in snails' hemocytes compared to untreated animals. The latter was detected by NRRT changes, reactive oxygen species (ROS) production, lipid peroxidation estimation, DNA integrity loss, measurement of protein carbonyl content by an enzyme-linked immunoabsorbent assay (ELISA), determination of ubiquitin conjugates and cleaved caspases conjugates levels. The results showed that the simultaneous use of the parameters tested could constitute possible reliable biomarkers for the evaluation of NPs toxicity. However, more research is required in order to enlighten the disposal and toxic impact of iron oxide NPs on the environment to ensure their safe use in the future.

Bioindication of mercury, arsenic and uranium in the apple snail Pomacea canaliculata (Caenogastropoda, Ampullariidae): Bioconcentration and depuration in tissues and symbiotic corpuscles.

Pomacea canaliculata is a mollusk potentially useful as a biomonitor species of freshwater quality. This work explores the ability of snail tissues and symbiotic corpuscles to bioconcentrate and depurate mercury, arsenic, and uranium. Adult snails cultured in metal-free reconstituted water were exposed for eight weeks (bioaccumulation phase) to water with Hg (2 µgL-1), As (10 µgL-1), and U (30 µgL-1) and then returned to the reconstituted water for other additional eight weeks (depuration phase). Elemental concentrations in digestive gland, kidney, symbiotic corpuscles and particulate excreta were determined by neutron activation analysis. The glandular symbiotic occupancy was measured by morphometric analysis. After exposure, the kidney showed the highest concentration of Hg, while the digestive gland accumulated mainly As and U. The subcellular distribution in symbiotic corpuscles was ∼71%, ∼48%, and ∼11% for U, Hg, and As, respectively. Tissue depuration between weeks 8 and 16 was variable amongst elements. At week 16, the tissue depuration of U was the highest (digestive gland = 92%; kidney = 80%), while it was lower for Hg (digestive gland = 51%; kidney = 53%). At week 16, arsenic showed a differential pattern of tissue depuration (digestive gland = 23%; kidney = 88%). The symbiotic detoxification of the three elements in excreta was fast between weeks 8 and 10 and it was slower after on. At the end of the depuration, each element distributed differentially in digestive gland and symbiotic corpuscles. Our findings show that symbiotic corpuscles, digestive gland and kidney P. canaliculata are sensitive places for biomonitoring of Hg, As and U.

Peroxidation and unsaturation indices as potential biomarkers of multifarious zinc and copper micro-supplementation in Helix pomatia L.

The work is a continuation of two previous studies in which biomarker fatty acids (12 of 56 FA pools) were analysed in Helix pomatia L. after heterogeneous micro-supplementation of Zn and Cu (administered in five micro-doses in the form of salts and EDTA and lysine chelates). This time, peroxidation (PI) and unsaturation coefficients (UI) as biomarker were analysed. These indices were calculated based on the FA profile in the foot and hepatopancreas of snails. The correlation of frequently used oxidation status indicators of organisms (catalase - CAT, glutathione peroxidase - GPx, selenium-dependent peroxidase - se-GPx, superoxide dismutase - SOD, glutathione transferase - GST, glutathione reductase - GR, glutathione - GSH, carbonyl protein - CP, thiobarbituric acid reactive substances - TBARS) with the rarely used UI and PI ratios was analysed. It was found that the 12-week micro-exposure to Zn and Cu did not inhibit but rather stimulated antioxidative defence at a sufficient level to increase the values of peroxidation/unsaturation indices in comparison to the control groups. Induction of an opposite process to oxidation of fatty acids was demonstrated. Maximum activities and amounts of antioxidants as well as minima of protein and lipid decomposition were recorded in groups supplemented with 0.75mg/l Zn and 1.0mg/l Cu. The possibility of a direct use of fatty acids as well as peroxidation/unsaturation indices as sensitive and reproducible biomarkers of exposure and oxidative physiological status in snails was confirmed.

Uptake and biological responses in land snail Cornu aspersum exposed to vaporized CdCl2.

The uptake of Cd and some biomarkers of exposure and effects have been investigated in specimens of land snail Cornu aspersum exposed to vaporized CdCl2 (10mg/L) for 7 days. The Cd levels quantified in snail's whole bodies confirmed Cd bioavailability trough vaporization and an higher accumulation in the midgut gland compared to the foot. Biological responses investigated showed a reduction of destabilization time of lysosomal membranes (NRRT) in hemocytes and an induction of catalase activities (CAT) in midgut gland. A further evidence of CdCl2 vaporized exposure was given by an increase in MT protein content as well as induction of Cd-MT gene expression, highlighting the central role of the midgut gland in Cd detoxification. These biomarkers can thus be considered as sensitive tools for the assessment of Cd contamination in the air using land snails as bioindicators. No changes in of GST activity and MDA were observed. From the overall results, the land snail, C. aspersum, could be used as good bioindicator of air quality for pollution monitoring purposes having shown clear signs of exposure and effects due Cd exposure by air.

Assessment of the bioavailability and depuration of uranium, cesium and thorium in snails (Cantareus aspersus) using kinetics models.

Uranium ore waste has led to soil contamination that may affect both environmental and soil health. To analyze the risk of metal transfer, metal bioavailability must be estimated by measuring biological parameters. Kinetic studies allow taking into account the dynamic mechanisms of bioavailability, as well as the steady state concentration in organisms necessary to take into account for relevant risk assessment. In this way, this work aims to model the snail accumulation and excretion kinetics of uranium (U), cesium (Cs) and thorium (Th). Results indicate an absence of Cs and Th accumulation showing the low bioavailability of these two elements and a strong uranium accumulation in snails related to the levels of soil contamination. During the depuration phase, most of the uranium ingested was excreted by the snails. After removing the source of uranium by soil remediation, continued snails excretion of accumulated uranium would lead to the return of their initial internal concentration, thus the potential trophic transfer of this hazardous element would stop.

Changes in fatty acid metabolism induced by varied micro-supplementation with zinc in snails Helix pomatia (Gastropoda Pulmonata).

We analyzed the changes in the profile of fatty acids (FA) in the foot tissues and hepatopancreas (HP) of snails Helix pomatia exposed to five microdoses of zinc (0.1, 0.25, 0.5, 0.75, or 1mg/l) administered in the form of a pure salt solution and in the form of EDTA and lysine chelates. Selection from a pool of 56 fatty acids analyzed in snail tissues yielded a set of 12 biomarker acids undergoing significant changes in contact with toxic substances. The selection criteria included the greatest percentage among the FA profile and their significant role in physiological processes. The proposed palette of acids of the biomarker FAs comprised C16:0; C18:0; C23:0; C18:1 n-9; C20:1 n-9; C18:2 n-6; C18:3 n-3; C20:2; C20:4 n-6; C20:5 n-3; C22:4 n-6; and C22:5 n-3, and saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs), and polyunsaturated fatty acids (PUFAs), determined separately in the foot tissues and hepatopancreas. The significant (p=0.01) influence of the dose as well as the source of the zinc on its' concentration in the tissues and on changes in the fatty acid profiles. Among the three zinc forms administered to the snails, the highest bioaccumulation of zinc in both tissues was noted in the group receiving the Zn-EDTA chelate. The content of PUFAs increased as the supplementation with zinc increased up to 0.75mg/l, but at 1mg/l, the share of these FAs began to decrease. This trend was observed in both analyzed tissue types - foot and hepatopancreas. The dose of 1mg Zn/l might be considered as a threshold dose above which the saturation of FAs increases. The results proved that determination of FA profile in snails can be used in ecotoxicological research as a reliable test of the effect of trace doses of stressors. The micro-supplementation of the mollusks diet with zinc is an example of a non-routine approach to issues connected with both diet and toxicology.

Nitric oxide-coupled signaling in odor elicited molecular events in the olfactory center of the terrestrial snail, Helix pomatia.

Olfaction, a chemosensory modality, plays a pivotal role in the orientation and behavior of invertebrates. The central olfactory processing unit in terrestrial stylomatophoran snails is the procerebrum, which contains NO synthesizing interneurons, whose oscillatory currents are believed to be the base of odor evoked memory formation. Nevertheless, in this model the up- and downstream events of molecular cascades that trigger and follow NO release, respectively, have not been studied. Immunocytochemistry and flow cytometry studies performed on procerebral neural perikarya isolated from the snail Helix pomatia revealed cell populations with discrete DAF-2 fluorescence, indicating the release of different amounts of NO. Glutamate increased the intensity of DAF-2 fluorescence, and the number of DAF-2 positive non-bursting interneurons, through a mechanism likely to involve an NMDA-like receptor. Similarly to glutamate, NO activation induced an increase in intracellular cGMP levels through activation of soluble guanylyl cyclase. Immunohistochemical localization of proteins possessing the phosphorylated target sequence of AGC family kinases (RXXS/T-P), among them protein kinase A (RRXS/T-P), showed striking similarities to the distribution of NOS/cGMP. Activators of cyclic nucleotide synthesis increased the AGC-kinase-dependent phosphorylation of discrete proteins with 28, 45, and 55kDamw. Importantly, exposure of snails to an attractive odorant induced hyperphosphorylation of the 28kDa protein, and increased levels of cGMP synthesis. Protein S-nitrosylation and intercellular activation of protein kinase G were also suggested as alternative components of NO signaling in the snail procerebrum. The present results from Helix pomatia indicate an important role for procerebrum NO/cGMP/PKA signaling pathways in the regulation of olfactory (food-finding) behavior.

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.

Putative new groups of invertebrate water channels based on the snail Helix pomatia L. (Helicidae) MIP protein identification and phylogenetic analysis.

Water channel proteins, classified as a family of Membrane Intrinsic Proteins (MIPs) superfamily, enable rapid movement of water and small uncharged molecules through biological membranes. Although water channel proteins are required in several important processes characteristic for the animals, such as osmoregulation, mucus secretion, or defense against desiccation, molluscs, until now, have been very poorly explored in this aspect. Therefore, we decided to study MIPs in Helix pomatia L. applied as a model in studies on terrestrial snail physiology. Our studies consisted in: the snail organ transcriptome sequencing and consecutive bioinformatic analysis of the predicted protein, estimation of the encoding transcript expression (qPCR), investigation of the predicted protein function in the yeast Saccharomyces cerevisiae cells, and the phylogenetic analysis. We identified six water channel proteins, named HpAQP1 to HpAQP6. All of them were proven to transport water, two of them (HpAQP3 and HpAQP4) were also shown to be able to transport glycerol, and other two (HpAQP5 and HpAQP6) to transport H2O2. Phylogenetic analysis indicated that the proteins either fell into aquaporins (HpAQP1, HpAQP2 and HpAQP5) or formed new groups of invertebrate water channel proteins, not described until now, that we suggest to term malacoglyceroporins (HpAQP3 and HpAQP4) and malacoaquaporins (HpAQP6). Thus, the classification of animal water channels based on the vertebrate proteins and including aquaporin, aquaglyceroporin, S-aquaporin and AQP8-type grades does not reflect diversity of these proteins in invertebrates. The obtained results provide important data concerning diversity of water channel protein repertoire in aquatic and terrestrial invertebrates and should also contribute to the improvement of animal water channel classification system.

Cadmium Accumulation and Pathological Alterations in the Midgut Gland of Terrestrial Snail Helix pomatia L. from a Zinc Smelter Area: Role of Soil pH.

The purpose of this study was to determine whether cadmium (Cd) accumulation and toxicity in the midgut gland of Helix pomatia snails living in a Cd-contaminated area were related to soil pH. Toxic responses in the midgut gland (i.e., increased vacuolization and lipid peroxidation) occurred in H. pomatia snails exhibiting the highest Cd levels in the gland (265-274 µg/g dry wt) and living on acidic soil (pH 5.3-5.5), while no toxicity was observed in snails accumulating less Cd (90 µg/g) and ranging on neutral soil (pH 7.0), despite the fact that total soil Cd was similar in the two cases. The accumulation of Cd in the gland was directly related to the water extractable Cd in soil, which in turn correlated inversely with soil pH, indicating that this factor had a significant effect on tissue Cd. It appeared further that the occurrence of Cd toxicity was associated with low levels of metallothionein in the gland of snails ranging on acidic soil.