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.

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.

Bioaccumulation and tissue distribution of Pb and Cd and growth effects in the green garden snail, Cantareus apertus (Born, 1778), after dietary exposure to the metals alone and in combination.

The present study was aimed at determining bioaccumulation and cell and tissue distribution of Pb and Cd in the green garden snail, Cantareus apertus (Born, 1778), exposed to different nominal dietary concentrations of Pb (25, 100 and 2500 mg Pb/kg), Cd (5, 10 and 100 mg Cd/kg) and their combination (25mg Pb+10 mg Cd/kg and 2500 mg Pb+100 mg Cd/kg) for 1 and 8 wk. Pb and Cd were bioaccumulated in the digestive gland in a dose-dependent manner and the degree of effects on growth was related to the level of exposure, though metal-metal interactions were observed after treatment with mixtures of Pb and Cd. The present results are absolutely comparable to those obtained in other terrestrial pulmonates in other regions and therefore they absolutely support that C. apertus is suitable as biomonitor for the assessment of the Pb and Cd levels and their biological effects in soil ecosystems in Northern Africa.

A chronic toxicity test for the tropical marine snail Nassarius dorsatus to assess the toxicity of copper, aluminium, gallium, and molybdenum.

Chronic toxicity test methods for assessing the toxicity of contaminants to tropical marine organisms are generally lacking. A 96-h chronic growth rate toxicity test was developed for the larval stage of the tropical dogwhelk, Nassarius dorsatus. Growth rates of N. dorsatus larvae were assessed following exposures to copper (Cu), aluminium (Al), gallium (Ga), and molybdenum (Mo). Exposure to Cu at 28 °C validated the sensitivity of the test method, with 10% (EC10) and 50% (EC50) effect concentrations of 4.2 µg/L and 7.3 µg/L Cu, respectively. The EC10 and EC50 values for Al (<0.45-µm filtered fraction) at 28 °C were 115 µg/L and 185 µg/L, respectively. The toxicity of Cu and Al was also assessed at 24 °C and 31 °C, representing average year-round water temperatures for subtropical and tropical Australian coastal environments. At 24 °C, the growth rate of control larvae was reduced by 52% compared with the growth rate at 28 °C and there was an increase in sensitivity to Cu (EC50 = 4.7 µg/L) but a similar sensitivity to Al (EC50 = 180 µg/L). At 31 °C the control growth rate increased by 35% from that measured at 28 °C and there was reduced sensitivity to both Cu and Al (EC50s = 8.5 µg/L and 642 µg/L, respectively). There was minimal toxicity resulting from Ga (EC50 = 4560 µg/L) and Mo (no effect at ≤7000 µg/L Mo). Environ Toxicol Chem 2016;35:1788-1795. © 2015 SETAC.

Conservation Status of a Recently Described Endemic Land Snail, Candidula coudensis, from the Iberian Peninsula.

We assessed the distribution, population size and conservation status of Candidula coudensis, a recently described endemic land snail from Portugal. From March 2013 to April 2014, surveys were carried out in the region where the species was described. We found an extent of occurrence larger than originally described, but still quite small (13.5 km2). The species was found mainly in olive groves, although it occurred in a variety of other habitats with limestone soils, including grasslands, scrublands and stone walls. Minimum population estimate ranged from 110,000-311,000 individuals. The main identified potential threats to the species include wildfires, pesticides and quarrying. Following the application of IUCN criteria, we advise a conservation status of either "Least Concern" or "Near-threatened" under criterion D (restricted population).

Microbiological quality of raw and processed wild and cultured edible snails.

BACKGROUND: An increasing interest in snail farming in Greece and other European countries has been observed. Despite the fact that edible snails have been involved with problems of Salmonella spp. contamination, there are to our knowledge only limited studies regarding microbiological safety and hygiene of such products. Enumeration of microbial populations and presence/absence of Salmonella spp. in snail meat and intestines of wild Cornu aspersum, Helix lucorum and cultured Cornu aspersum snails from indoor/outdoor type farms was conducted. Furthermore, snail-processing steps were simulated in the laboratory and the population reduction in snail meat was determined. RESULTS: Microbial populations were higher in intestines than snail meat in almost all cases. Escherichia coli/coliforms and Enterococcus spp. populations were lower in the intestines and snail meat of cultured C. aspersum. Salmonella spp. were detected in the intestines and snail meat of wild snails only. The high levels of bacterial populations were considerably reduced after the appropriate processing. CONCLUSION: The lower populations of E. coli/coliforms, Enterococcus spp. and especially the absence of Salmonella spp. in cultured snails show that the controlled conditions decrease the possibility of pathogen presence and contribute to food safety and public health.

Selective modulation of chemical and electrical synapses of Helix neuronal networks during in vitro development.

BACKGROUND: A large number of invertebrate models, including the snail Helix, emerged as particularly suitable tools for investigating the formation of synapses and the specificity of neuronal connectivity. Helix neurons can be individually identified and isolated in cell culture, showing well-conserved size, position, biophysical properties, synaptic connections, and physiological functions. Although we previously showed the potential usefulness of Helix polysynaptic circuits, a full characterization of synaptic connectivity and its dynamics during network development has not been performed. RESULTS: In this paper, we systematically investigated the in vitro formation of polysynaptic circuits, among Helix B2 and the serotonergic C1 neurons, from a morphological and functional point of view. Since these cells are generally silent in culture, networks were chemically stimulated with either high extracellular potassium concentrations or, alternatively, serotonin. Potassium induced a transient depolarization of all neurons. On the other hand, we found prolonged firing activity, selectively maintained following the first serotonin application. Statistical analysis revealed no significant changes in neuronal dynamics during network development. Moreover, we demonstrated that the cell-selective effect of serotonin was also responsible for short-lasting alterations in C1 excitability, without long-term rebounds.Estimation of the functional connections by means of cross-correlation analysis revealed that networks under elevated KCl concentrations exhibited strongly correlated signals with short latencies (about 5 ms), typical of electrically coupled cells. Conversely, neurons treated with serotonin were weakly connected with longer latencies (exceeding 20 ms) between the interacting neurons. Finally, we clearly demonstrated that these two types of correlations (in terms of strength/latency) were effectively related to the presence of electrical or chemical connections, by comparing Micro-Electrode Array (MEA) signal traces with intracellularly recorded cell pairs. CONCLUSIONS: Networks treated with either potassium or serotonin were predominantly interconnected through electrical or chemical connections, respectively. Furthermore, B2 response and short-term increase in C1 excitability induced by serotonin is sufficient to trigger spontaneous activity with chemical connections, an important requisite for long-term maintenance of firing activity.

Terrestrial gastropods (Helix spp) as sentinels of primary DNA damage for biomonitoring purposes: a validation study.

We validated the alkaline comet assay in two species of land snail (Helix aspersa and Helix vermiculata) to test their suitability as sentinels for primary DNA damage in polluted environments. The study was conducted under the framework of a biomonitoring program for a power station in Central Italy that had recently been converted from oil to coal-fired plant. After optimizing test conditions, the comet assay was used to measure the % Tail DNA induced by in vitro exposure of hemocytes to different concentrations of a reactive oxygen species (H2 O2 ). The treatment induced significant increases in this parameter with a concentration effect, indicating the effectiveness of the assay in snail hemocytes. After evaluating possible differences between the two species, we sampled them in three field sites at different distances from the power station, and in two reference sites assumed to have low or no levels of pollution. No species differences emerged. Percent Tail DNA values in snails from the sites near the power station were higher than those from control sites. An inverse correlation emerged between % Tail DNA and distance from the power station, suggesting that the primary DNA damage decreased as distance increased away from the pollution source. Detection of a gradient of heavy metal concentration in snail tissues suggests that these pollutants are a potential cause of the observed pattern. The comet assay appears to be a suitable assay and Helix spp. populations suitable sentinels to detect the genotoxic impact of pollutants.

Snails as indicators of pesticide drift, deposit, transfer and effects in the vineyard.

This paper presents a field-study of real pesticide application conditions in a vineyard. The objective was to measure the exposure, the transfer and the effects of pesticides on a non-target soil invertebrate, the land snail Helix aspersa. There was no drift of the herbicides (glyphosate and glufosinate) whereas the fungicides (cymoxanil, folpet, tebuconazole and pyraclostrobin) were detected up to 20 m from the treated area. For folpet and particularly tebuconazole, spray deposits on soil (corresponding to losses for the intended target i.e. the vine leaves) were high (41.1% and 88.8% loss of applied dose, respectively). For herbicides, the target was the soil and losses (percentage of compounds which did not reach the soil) were of 22% for glufosinate and 52% for glyphosate. In the study plot, glyphosate was transferred to and accumulated in snail tissues (4 mg kg(-1) dry weight, dw), as was its metabolite AMPA (8 mg kg(-1) dw) which could be in relation with the reduced growth observed in snails. No effects on snail survival or growth were found after exposure to the other organic compounds or to copper and sulphur-fungicides, although transfer of tebuconazole, pyraclostrobin and copper occurred. This study brings original field data on the fate of pesticides in a vineyard agro-ecosystem under real conditions of application and shows that transfer and effects of pesticides to a non-target organism occurred.

Magnesium and the regulation of lead in three populations of the garden snail Cantareus aspersus.

Helicid snails appear to regulate Pb more closely than other toxic metals, though it is reported as the least toxic. No regulatory mechanism has been described in animals, and the possible role of Mg in limiting Pb assimilation is examined here for the first time. Three populations of Cantareus aspersus were fed Pb and Ca with three levels of Mg for up to 64 days. Metal assimilation and production efficiency was calculated for each of 108 snails. Populations differed in their pattern of uptake but soft tissue Pb was unaffected by dietary Mg. The proportion of Pb assimilated did not change as soft tissue concentrations increased, indicating no specific regulatory mechanism. The daily addition of Pb to the soft tissues increases with growth rate suggesting uptake is instead some function of growth or cell turnover. Bioconcentration factors varied with time and are unreliable indicators of an evolved regulatory mechanism for Pb.

Lectin-binding glycoproteins in the developing and adult snail CNS.

Glycoproteins are complex molecules of the cell surface and the extracellular matrix (ECM) playing a fundamental role in the migration, guidance and synapse formation of neurons. In the present study, the glycosylated protein composition and localization were investigated in the adult and developing CNS of an aquatic (Lymnaea stagnalis) and a terrestrial (Helix pomatia) snail species, applying lectin histochemistry and blotting. Lectin probes that are specific for N-acetyl-glucosamine (GlcNAc) oligomers frequently appeared in anatomically different regions of the adult ganglia of both species, such as, the periganglionic sheath, the interperikaryonal space and the neuropil. Different GlcNAc residues were found to intensively glycosylate five, high-molecular weight proteins characteristic for the ECM of Lymnaea CNS and localized mainly in the interperikaryonal space. N-acetyl-galactosamine oligomers were less pronounced in the adult snail ganglia, they were detected only in the periganglionic sheath and the attached basement lamina. Apart from some similarities, the glycosylation pattern of proteins and the distribution of glycoproteins in the neuropil displayed significant differences in Lymnaea and Helix. All continuous and increasing level of and also transient presence of glycoproteins were detected during Lymnaea CNS development. Our results indicate a rich glycosylated pattern of specific proteins in the snail CNS, displaying remarkable species- and age-dependent changes which suggest the wide importance of protein glycosylation in the CNS of invertebrates.

[Life history traits of the snail Helix aperta Born from Tunisia raised in a laboratory environment: influence of photoperiod]. / Obtention du cycle de vie complet d'Helix aperta Born de sites tunisiens en conditions contrôlées. Influence de la photopériode.

The present work revealed that growth and reproduction of Helix aperta (syn. Cantareus apertus), sampled at the end of summer in the region of Bazina (Tunisia), are stimulated by short-day photoperiod (SD 8hL-16hD) and inhibited by long-day photoperiod (LD 18hL-6hD). Indeed, under SD at 20 degrees C, 80% humidity and ad libitum Helixal snail food, 2 generations (F1, F2) were obtained in 10 months with no refractory period. The effects of photoperiod on growth were found to be reversible and appeared after a time lag of 4 and 8 weeks for stimulation by SD and the inhibition by LD respectively.

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

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

Expression of MAP/ERK kinase cascade corresponds to the ability to develop food aversion in terrestrial snail at different stages of ontogenesis.

The mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) cascade plays an important role in gene expression regulation during memory formation in both vertebrates and invertebrates. MAPK/ERK regulates gene expression through phosphorylation of transcription factors binding to the regulatory elements SRE and CRE of target genes. Previously we reported that juvenile snails Helix lucorum differ from adult animals in a spectrum of transcription factors binding to DNA regulatory elements SRE and AP-1. In this study we analyzed the expression and activation of MAPK/ERK in CNS of H. lucorum during formation of the conditioned avoidance reflex at different stages of postnatal ontogenesis. Under conditions of learning, juvenile snails (aged 2-3 months) possessing immature mechanisms of avoidance reflex plasticity showed dramatically low level of phosphorylation and, correspondingly, low activation of MAPK/ERK in comparison to adult animals. Beside this, the MAPK/ERK cascade was not activated after 10 and 60 min after learning in juvenile snails in contrast to adults, while basal expression level of this kinase was similar in juveniles and adults. Low activation of MAPK/ERK cascade can cause a deficiency in phosphorylation of downstream transcription factors binding to SRE and thereby influence the expression of early response genes (particularly, of the family AP-1) and late response genes necessary for cellular and synaptic plasticity. These observations suggest that the MAPK/ERK regulatory cascade plays an essential role in the formation of conditioned avoidance reflexes in Helix. Low activation of this cascade might be one of the reasons for deficiency of long-term memory formation during avoidance learning in juvenile animals.

Laboratory rearing conditions for improved growth of juvenile Helix aspersa Müller snails.

A laboratory rearing system in semi-controlled conditions is proposed to facilitate the behavioural rhythms of the edible snail (Helix aspersa) and to produce a high growth rate with low variability. The growth data were used to construct a model for weight estimation based on age. The animals' live weights showed low variability (<17%) and normal distribution. The best model for estimating weight from age is the logistic model, with a high corelation coefficient (>90%), and a high level of significance for the coefficient (P < 0.0001).

Revising the standard wisdom of C. elegans natural history: ecology of longevity.

Here, we consider that most of the research concerning Caenorhabditis elegans has been laboratory focused and that only limited research has directly considered the worm's biology relative to its natural history in the wild. We describe that, although the worm has traditionally been considered a soil nematode, we could not find it in soil but frequently recovered it from snails. Finally, we discuss how a better understanding of the natural history of C. elegans may enhance its usefulness as a model organism for studying aging and other phenomena.

Morphological basis for coordination of growth and reproduction processes in the CNS of two terrestrial snails.

The morphology of cells immunoreactive to an antibody against molluscan insulin-related peptide (MIP-IR) was studied in two species of terrestrial snail: Helix lucorum L. and Eobania vermiculata L. Immunocytochemical staining with this antibody to MIP revealed 100-130 cells in the postcerebrum, located in two clusters with common pathways in the dorsal body nerve and the cerebral artery nerve. About 75% of the MIP-IR cells were labeled by backfilling of the dorsal body nerve in Helix and Eobania; the corresponding figures for labeling by backfilling of the cerebral artery nerve were about 60% in Helix and 30% in Eobania. Upon intracellular staining of neurons of the dorsomedial postcerebrum, where most of the MIP-IR cells were located, it was found that they projected either in the dorsal body nerve or in the cerebral artery nerve or in both. The obtained data suggest that growth and reproduction processes (both functions were attributed to the insulin-related peptide-containing neurons) are regulated by the two, partially coinciding subsets of postcerebral MIP-IR neurons with different types of branching.

Standard use conditions of terrestrial gastropods in active biomonitoring of soil contamination.

The rehabilitation of contaminated sites is becoming a rising preoccupation which requires the knowledge of their past before realization of a suitable remediation. Physicochemical analysis must be realized jointly with the use of bioindicator organisms, which, owing to their bioaccumulation capacities, will reveal the bioavailability of metals in soils. Among terrestrial invertebrates, gastropods like Helix aspersa aspersa possess an important organotropism for metals in their digestive gland and they can be used in active biomonitoring. During in situ monitoring, two parameters are tested: growth and accumulation of Fe, Mn, Zn, Cu, Cd, Ni, Pb, and Cr in viscera of snails. Environmental conditions, like humidity or autochthonous vegetation, are able to modify growth or bioaccumulation. In order to remove the variation of these factors, microcosms previously used must be improved: they were equipped with porous candles, which continually humidify soil. Concerning vegetation, an experimental plan was realized to determine the combination of food with the best compromise between growth and bioaccumulation: the combination clover-snail feed was chosen. Thus, in situ, the experimental environment will be repeated in all investigated sites and used to allow follow up of their contamination levels and intercomparison between different sites.

PLA2 and secondary metabolites of arachidonic acid control filopodial behavior in neuronal growth cones.

The neuronal growth cone provides the sensory and motor structure that guides neuronal processes to their target. The ability of a growth cone to navigate correctly depends on its filopodia, which sample the environment by continually extending and retracting as the growth cone advances. Several second messengers systems that are activated upon contact with extracellular cues have been reported to affect growth cone morphology by changing the length and number of filopodia. Because recent studies have suggested that guidance cues can signal via G-protein coupled receptors to regulate phospholipases, we here investigated whether phospholipase A2 (PLA2) may control filopodial dynamics and could thereby affect neuronal pathfinding. Employing identified Helisoma neurons in vitro, we demonstrate that inhibition of PLA2 with 2 microM BPB caused a 40.3% increase in average filopodial length, as well as a 37.3% reduction in the number of filopodia on a growth cone. The effect of PLA2 inhibition on filopodial length was mimicked by the inhibition of G-proteins with 500 ng/ml pertussis toxin and was partially blocked by the simultaneous activation of PLA2 with 50 nM melittin. We provide evidence that PLA2 acts via production of arachidonic acid (AA), because (1) the effect of inhibition of PLA2 could be counteracted by supplying AA exogenously, and (2) the inhibition of cyclooxygenase, which metabolizes AA into prostaglandins, also increased filopodial length. We conclude that filopodial contact with extracellular signals that alter the activity of PLA2 can control growth cone morphology and may affect neuronal pathfinding by regulating the sensory radius of navigating growth cones.