Bisphenol A (BPA) modulates the expression of endocrine and stress response genes in the freshwater snail Physa acuta.

Bisphenol A (BPA), a known endocrine disrupting chemical (EDC) that can mimic the action of oestrogens by interacting with hormone receptors, is potentially able to influence reproductive functions in vertebrates and invertebrates. The freshwater pulmonate Physa acuta is a sensitive organism to xenobiotics appropriate for aquatic toxicity testing in environmental studies. This study was conducted to explore the effects of BPA on the Gastropoda endocrine system. The effects following a range of exposure times (5-96h) to BPA in P. acuta were evaluated at the molecular level by analysing changes in the transcriptional activity of the endocrine-related genes oestrogen receptor (ER), oestrogen-related receptor (ERR), and retinoid X receptor (RXR), as well as in genes involved in the stress response, such as hsp70 and hsp90. Real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) analysis showed that BPA induced a significant increase in the mRNA levels of ER, ERR, and RXR, suggesting that these receptors could be involved in similar pathways or regulation events in the endocrine disruptor activity of this chemical at the molecular level in Gastropoda. Additionally, the hsp70 expression was upregulated after 5 and 72h of BPA exposures, but hsp90 was only upregulated after 5h of BPA exposure. Finally, we assessed the glutathione-S-transferase (GST) activity after BPA treatment and found that it was affected after 48h. In conclusion, these data provide, for the first time, evidences of molecular effects produced by BPA in the endocrine system of Gastropoda, supporting the potential of ER, ERR and RXR as biomarkers to analyse putative EDCs in ecotoxicological studies. Moreover, our results suggest that P. acuta is an appropriate sentinel organism to evaluate the effect of EDCs in the freshwater environment.

The transcriptome of a "sleeping" invader: de novo assembly and annotation of the transcriptome of aestivating Cornu aspersum.

BACKGROUND: Cornu aspersum is a quite intriguing species from the point of view of ecology and evolution and its potential use in medical and environmental applications. It is a species of economic importance since it is farmed and used for culinary purposes. However, the genomic tools that would allow a thorough insight into the ecology, evolution, nutritional and medical properties of this highly adaptable organism, are missing. In this work, using next-generation sequencing (NGS) techniques we assessed a significant portion of the transcriptome of this non-model organism. RESULTS: Out of the 9445 de novo assembled contigs, 2886 (30.6%) returned significant hits and for 2261 (24%) of them Gene Ontology (GO) terms associated to the hits were retrieved. A high percentage of the contigs (69.4%) produced no BLASTx hits. The GO terms were grouped to reflect biological processes, molecular functions and cellular components. Certain GO terms were dominant in all groups. After scanning the assembled transcriptome for microsatellites (simple sequence repeats, SSRs), a total of 563 SSRs were recovered. Among the identified SSRs, trinucleotide repeats were the predominant followed by tetranucleotide and dinucleotide repeats. CONCLUSION: The annotation success of the transcriptome of C. aspersum was relatively low. This is probably due to the very limited number of annotated reference genomes existing for mollusc species, especially terrestrial ones. Several biological processes being active in the aestivating species were revealed through the association of the transcripts to enzymes relating to the pathways. The genomic tools provided herein will eventually aid in the study of the global genomic diversity of the species and the investigation of aspects of the ecology, evolution, behavior, nutritional and medical properties of this highly adaptable organism.

Development of the first polymorphic microsatellite markers for the Roman snail Helix pomatia L., 1758 (Helicidae) and cross-species amplification within the genus Helix.

The terrestrial snail Helix pomatia (Gastropoda: Stylommatophora: Helicidae) is one of the largest gastropod species in Europe. This species is strictly protected in some European Union countries; however, at the same time, it is also farmed and commercialized for human consumption. Here, we describe 11 microsatellite markers that are very useful in population genetic studies for assessing the status of both wild and farmed populations of this species of community interest. The microsatellites were isolated using 454 pyrosequencing technologies and 11 primer pairs were selected and used for genotyping an H. pomatia population and also checked for cross-species amplification on H. lucorum and H. lutescens specimens. The number of alleles per locus ranged from 3 to 13 and observed heterozygosity was between 0.458 and 0.917. Seven of these loci were polymorphic in H. lucorum, and four in H. lutescens. This set of nuclear markers provides a powerful tool for population genetic studies of this species of community interest, and also for closely related species. The described microsatellite markers should also facilitate the identification of populations of conservation concern.

Does Variation of the Inter-Domain Linker Sequence Modulate the Metal Binding Behaviour of Helix pomatia Cd-Metallothionein?

Snail metallothioneins (MTs) constitute an ideal model to study structure/function relationships in these metal-binding polypeptides. Helix pomatia harbours three MT isoforms: the highly specific CdMT and CuMT, and an unspecific Cd/CuMT, which represent paralogous proteins with extremely different metal binding preferences while sharing high sequence similarity. Preceding work allowed assessing that, although, the Cys residues are responsible for metal ion coordination, metal specificity or preference is achieved by diversification of the amino acids interspersed between them. The metal-specific MT polypeptides fold into unique, energetically-optimized complexes of defined metal content, when binding their cognate metal ions, while they produce a mixture of complexes, none of them representing a clear energy minimum, with non-cognate metal ions. Another critical, and so far mostly unexplored, region is the stretch linking the individual MT domains, each of which represents an independent metal cluster. In this work, we have designed and analyzed two HpCdMT constructs with substituted linker segments, and determined their coordination behavior when exposed to both cognate and non-cognate metal ions. Results unequivocally show that neither length nor composition of the inter-domain linker alter the features of the Zn(II)- and Cd(II)-complexes, but surprisingly that they influence their ability to bind Cu(I), the non-cognate metal ion.

Special clinical entity with 15q26 deletion: a novel case report.

Genomic studies make it possible to breakthrough in many fields such as biochemistry, physiology, phylogenetics, etc., though they are unworkable without sequences of genomic DNA of an organism. The terrestrial mollusks' genomes would benefit gastropod biology investigations, that are unavailable so far due to problems in DNA integrity and quality after the isolation procedures. Here we describe a fast and handy protocol for genomic DNA extraction from the tissues of Helix lucorum, which allows to yield high-quality samples applicable for downstream analysis such as high-throughput DNA sequencing. Troubleshooting revealed the nuclease activity of snail tissue lysate, which may be avoided by heating the lysate and decreasing the incubation time.

Involvement of translation and transcription processes into neurophysiological mechanisms of long-term memory reconsolidation.

We studied the involvement of translation and transcription processes into behavioral and neuronal mechanisms of reconsolidation of the long-term memory of the conditioned taste aversion in edible snails. Injection of cycloheximide (an inhibitor of protein synthesis) to the snails in 48 h after training combined with subsequent reminder and presentation of the conditional stimulus resulted in the development of persistent amnesia and depression of the responses of the defensive behavior command neurons LPl1 and RPl1 to the conditional stimulus. Injection of mRNA synthesis inhibitors actinomycin D or DRB (5,6-dichloro-1-ß-D-ribofuranosylbenzimidasole) in 48 h after conditioning with subsequent reminding procedure produced no effects on memory retention and on the responses of the command neurons to the conditional stimulus. The study suggests that the proteins translated from previously synthesized and stored mRNA were involved in the mechanisms of reconsolidation of the memory responsible for conditioned taste aversion.

Identification, cloning, and characterization of two N-acetylgalactosamine-binding lectins from the albumen gland of Helix pomatia.

Helix pomatia agglutinin (HPA), the lectin from the albumen gland of the Roman snail, has been used in histochemical studies relating glycosylation changes to the metastatic potential of solid tumors. To facilitate the use of HPA in a clinical (diagnostic) setting, detailed analysis of the lectin, including cloning and recombinant production of HPA, is required. A combination of isoelectric focusing, amino acid sequence analysis, and cloning revealed two polypeptides in native HPA preparations (HPAI and HPAII), both consistent with GalNAc-binding lectins of the H-type family. Pairwise sequence alignment showed that HPAI and HPAII share 54% sequence identity whereas molecular modeling using SWISS-MODEL suggests they are likely to adopt similar tertiary structure. The inherent heterogeneity of native HPA highlighted the need for production of functional recombinant protein; this was addressed by preparing His-thioredoxin-tagged fusion products in Escherichia coli Rosetta-gami B (DE3) cells. The recombinant lectins agglutinated human blood group A erythrocytes whereas their oligosaccharide specificity, evaluated using glycan microarrays, showed that they predominantly bind glycans with terminal α-GalNAc residues. Surface plasmon resonance with immobilized GalNAc-BSA confirmed that recombinant HPAI and HPAII bind strongly with this ligand (K(d) = 0.60 nm and 2.00 nm, respectively) with a somewhat higher affinity to native HPA (K(d) = 7.67 nm). Recombinant HPAII also bound the breast cancer cells of breast cancer tissue specimens in a manner similar to native lectin. The recombinant HPA described here shows important potential for future studies of cancer cell glycosylation and as a reagent for cancer prognostication.

Shaping mechanisms of metal specificity in a family of metazoan metallothioneins: evolutionary differentiation of mollusc metallothioneins.

BACKGROUND: The degree of metal binding specificity in metalloproteins such as metallothioneins (MTs) can be crucial for their functional accuracy. Unlike most other animal species, pulmonate molluscs possess homometallic MT isoforms loaded with Cu(+) or Cd(2+). They have, so far, been obtained as native metal-MT complexes from snail tissues, where they are involved in the metabolism of the metal ion species bound to the respective isoform. However, it has not as yet been discerned if their specific metal occupation is the result of a rigid control of metal availability, or isoform expression programming in the hosting tissues or of structural differences of the respective peptides determining the coordinative options for the different metal ions. In this study, the Roman snail (Helix pomatia) Cu-loaded and Cd-loaded isoforms (HpCuMT and HpCdMT) were used as model molecules in order to elucidate the biochemical and evolutionary mechanisms permitting pulmonate MTs to achieve specificity for their cognate metal ion. RESULTS: HpCuMT and HpCdMT were recombinantly synthesized in the presence of Cd(2+), Zn(2+) or Cu(2+) and corresponding metal complexes analysed by electrospray mass spectrometry and circular dichroism (CD) and ultra violet-visible (UV-Vis) spectrophotometry. Both MT isoforms were only able to form unique, homometallic and stable complexes (Cd(6)-HpCdMT and Cu(12)-HpCuMT) with their cognate metal ions. Yeast complementation assays demonstrated that the two isoforms assumed metal-specific functions, in agreement with their binding preferences, in heterologous eukaryotic environments. In the snail organism, the functional metal specificity of HpCdMT and HpCuMT was contributed by metal-specific transcription programming and cell-specific expression. Sequence elucidation and phylogenetic analysis of MT isoforms from a number of snail species revealed that they possess an unspecific and two metal-specific MT isoforms, whose metal specificity was achieved exclusively by evolutionary modulation of non-cysteine amino acid positions. CONCLUSION: The Roman snail HpCdMT and HpCuMT isoforms can thus be regarded as prototypes of isoform families that evolved genuine metal-specificity within pulmonate molluscs. Diversification into these isoforms may have been initiated by gene duplication, followed by speciation and selection towards opposite needs for protecting copper-dominated metabolic pathways from nonessential cadmium. The mechanisms enabling these proteins to be metal-specific could also be relevant for other metalloproteins.

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.

Physiological relevance and contribution to metal balance of specific and non-specific Metallothionein isoforms in the garden snail, Cantareus aspersus.

Variable environmental availability of metal ions represents a constant challenge for most organisms, so that during evolution, they have optimised physiological and molecular mechanisms to cope with this particular requirement. Metallothioneins (MTs) are proteins that play a major role in metal homeostasis and as a reservoir. The MT gene/protein systems of terrestrial helicid snails are an invaluable model for the study of metal-binding features and MT isoform-specific functionality of these proteins. In the present study, we characterised three paralogous MT isogenes and their expressed products in the escargot (Cantareus aspersus). The metal-dependent transcriptional activation of the three isogenes was assessed using quantitative Real Time PCR. The metal-binding capacities of the three isoforms were studied by characterising the purified native complexes. All the data were analysed in relation to the trace element status of the animals after metal feeding. Two of the three C. aspersus MT (CaMT) isoforms appeared to be metal-specific, (CaCdMT and CaCuMT, for cadmium and copper respectively). A third isoform (CaCd/CuMT) was non-specific, since it was natively recovered as a mixed Cd/Cu complex. A specific role in Cd detoxification for CaCdMT was revealed, with a 80-90% contribution to the Cd balance in snails exposed to this metal. Conclusive data were also obtained for the CaCuMT isoform, which is involved in Cu homeostasis, sharing about 30-50% of the Cu balance of C. aspersus. No apparent metal-related physiological function was found for the third isoform (CaCd/CuMT), so its contribution to the metal balance of the escargot may be, if at all, of only marginal significance, but may enclose a major interest in evolutionary studies.

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.

Evaluation of the genotoxic potential of the mineral coal tailings through the Helix aspersa (Müller, 1774).

Coal mining is an activity with a high potential for environmental pollution. Coal has been described as the most significant pollutant of all the fossil fuels, containing a heterogeneous mixture. Many elements present in coal byproducts as well as coal tailings are rich in potentially toxic and genotoxic metals, which ultimately lead to profound changes in cells, tissues, populations, and ecosystems. The purpose of this study was to assess the genotoxic potential of the mineral coal tailings using the land snail Helix aspersa. Animals were divided in three groups, clustered in plexiglass cages: control (animals fed with organic lettuce), coal tailings (animals living in a layer of pyrite tailings and fed with organic lettuce), and mine lettuce (animals fed with lettuce grown in an area located in a deposit of coal tailings). The hemolymph was collected at different exposure times (24 h, 48 h, 72 h, 96 h, 1 week, 2 weeks, 3 weeks, and 1 month) for comet assay analyses. Results showed that the animals of the coal tailings and mine lettuce groups presented higher levels of DNA damage in relation to the control group at all exposure times, but with a peak of DNA damage in 48 h and 96 h. These results demonstrate that the coal pyrite tailings are potentially genotoxic and that H. aspersa has proven to be a sensitive instrument for a better risk assessment of environmental pollution.

Intravital investigation of the effects of serotonin and glutamate on the dynamics of DNA activity in L-RPl1 neurons of edible snail.

Microloci of varying size with increased fluorescence were observed in L-RPl1 neuronal nuclei of snails using DNA-selective dye SYTO16. Application of serotonin and glutamate increased the number of small (<1 micro in diameter) microloci, while the number of medium and large (1-3 micro) loci decreased. Combined application of neurotransmitters produced more pronounced changes in the number of microloci compared to individual administration. RNA synthesis inhibitors abolished the effects of the transmitters. We hypothesized that the revealed small microloci of fluorescence are "active" DNA zones, where transcription of new genes are initiated.

Identification of Immediate Early Genes in the Nervous System of Snail Helix lucorum.

Immediate early genes (IEGs) are useful markers of neuronal activation and essential components of neuronal response. While studies of gastropods have provided many insights into the basic learning and memory mechanisms, the genome-wide assessment of IEGs has been mainly restricted to vertebrates. In this study, we identified IEGs in the terrestrial snail Helix lucorum In the absence of the genome, we conducted de novo transcriptome assembly using reads with short and intermediate lengths cumulatively covering more than 98 billion nucleotides. Based on this assembly, we identified 37 proteins corresponding to contigs differentially expressed (DE) in either the parietal ganglia (PaG) or two giant interneurons located within the PaG of the snail in response to the neuronal stimulation. These proteins included homologues of well-known mammalian IEGs, such as c-jun/jund, C/EBP, c-fos/fosl2, and Egr1, as well as homologues of genes not yet implicated in the neuronal response.

Heterologous expression of the invertebrate FMRFamide-gated sodium channel as a mechanism to selectively activate mammalian neurons.

Considerable effort has been directed toward the development of methods to selectively activate specific subtypes of neurons. Focus has been placed on the heterologous expression of proteins that are capable of exciting neurons in which they are expressed. Here we describe the heterologous expression of the invertebrate FMRFamide (H-phenylalanine-methionine-arginine-phenylalanine-NH2) -gated sodium channel from Helix aspersa (HaFaNaC) in hippocampal slice cultures. HaFaNaC was co-expressed with a fluorescent protein (green fluorescent protein (GFP), red fluorescent protein from Discosoma sp (dsRed) or mutated form of red fluorescent protein from Discosoma sp (tdTomato)) in CA3 pyramidal neurons of rat hippocampal slice cultures using single cell electroporation. Pressure application of the agonist FMRFamide to HaFaNaC-expressing neuronal somata produced large prolonged depolarizations and bursts of action potentials (APs). FMRFamide responses were inhibited by amiloride (100 microM). In contrast, pressure application of FMRFamide to the axons of neurons expressing HaFaNaC produced no response. Fusion of GFP to the N-terminus of HaFaNaC showed that GFP-HaFaNaC was absent from axons. Bath application of FMRFamide produced persistent AP firing in HaFaNaC-expressing neurons. This FMRFamide-induced increase in the frequency of APs was dose-dependent. The concentrations of FMRFamide required to activate HaFaNaC-expressing neurons were below that required to activate the homologous acid sensing ion channel normally found in mammalian neurons. Furthermore, the mammalian neuropeptides neuropeptide FF and RFamide-related peptide-1, which have amidated RF C-termini, did not affect HaFaNaC-expressing neurons. Antagonists of NPFF receptors (BIBP3226) also had no effect on HaFaNaC. Therefore, we suggest that heterologous-expression of HaFaNaC in mammalian neurons could be a useful method to selectively and persistently excite specific subtypes of neurons in intact nervous tissue.

Heat shock proteins (Hsp70) and water content in the estivating Mediterranean Grunt Snail (Cantareus apertus).

Pulmonate land snails often are able to estivate to survive dry hot seasons were water and food are scarce. The aperture of the shell is closed with an epiphragm, and metabolism is depressed to approximately one fourth of basal metabolism. We investigated a molecular aspect of estivation focussing on the heat shock protein 70 (Hsp70) stress response during estivation in the Mediterranean Grunt Snail Cantareus apertus. Sequences of a new inducible hsp70 and of actin are presented and expression of the hsp70 gene as well as Hsp70 protein content was measured in estivating animals. Both Hsp70 protein and mRNA do not show a significant change from the control, although there is a trend that hsp70 mRNA is less abundant in estivating specimens. After heat shock, the expression of hsp70 increased and a higher Hsp70 protein content was detected. Water relations were also investigated. After a period of 6 months in the dormant state, the snails contained 14% less water than active ones, implying a constricted protection against desiccation, compared to the desert snail Sphincterochila zonata, and a Mediterranean-type water economy.

Influence of the SCGE protocol on the amount of basal DNA damage detected in the Mediterranean mussel, Mytilus galloprovincialis.

Genotoxicity studies using the single cell gel electrophoresis (SCGE) assay indicate that basal levels of DNA strand breaks (SBs) in marine invertebrates are higher and more variable than those in marine vertebrates. This elevated level of DNA damage was attributed to a large number of alkali-labile sites, which are characteristic of the tightly-packaged DNA in invertebrate cells. To investigate if altering the SCGE protocol can artificially modulate high levels of SBs, SCGE experiments were performed on haemocytes from the Mediterranean mussel (Mytilus galloprovincialis) using proteinase K (PK) digestion in combination with assay buffers containing various concentrations of EDTA. In addition, the effects of Trolox (soluble antioxidant) and aurintricarboxylic acid (ATA; inhibitor of Ca(2+)/Mg(2+)-dependent nucleases) also were tested. The levels of SBs in M. galloprovincialis cells were compared with SBs in cells from a terrestrial mollusk (the snail Helix aspersa), and a teleost fish (the seabass Dicentrarchus labrax). The integrity of M. galloprovincialis DNA isolated with phenol extractions using EDTA, Trolox, and ATA was further assayed by gel electrophoresis. High SBs in mussel cells were reduced by combining EDTA with PK digestion, or using Trolox or ATA during cell processing for the SCGE assay. Snails and seabass had lower levels of SBs in the SCGE assay, and the levels were not affected by the protocol modifications. Adding EDTA, Trolox, or ATA to phenol extractions of M. galloprovincialis genomic DNA also reduced the extent of DNA fragmentation. These results suggest that the internal fluids of M. galloprovincialis may increase the basal levels of DNA SBs through oxidative and/or enzyme-mediated pathways. M. galloprovincialis is used extensively as a sentinel species for assessing the genotoxic hazard of marine pollutants. Our data suggest that the SCGE protocol should be carefully considered when assessing DNA damage in these species.

[Effect of zif268 early gene antisense oligonucleotide on mechanisms of synapse-specific plasticity].

It was found that nociceptive sensitization was followed by long-term facilitation of synaptic responses evoked by chemical sensory stimulation of the snail "head", tactile stimulation of the snail "head" and foot in LP11 command neuron of defence behavior in snail Helix lucorum. Sensitizing stimulation during the intracellular injection of antisense olygonucleotide immediate early gene zif268 resulted in a selective suppression of synaptic facilitation in LP11 neuron responses evoked by tactile and chemical stimulation of the snail "head". At the same time, development of synaptic facilitation of responses in the LP11 neuron evoked by tactile stimulation of the foot was the same as in control sensitized snails. The results suggest that immediate early gene zif268 is selectively involved in the mechanisms of specific regulation of plasticity of the synaptic "input" of LP11 neuron from sensory receptors of the snail "head".

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.

Evidence for genetic control of adult weight plasticity in the snail Helix aspersa.

Phenotypic plasticity and canalization are important topics in quantitative genetics and evolution. Both concepts are related to environmental sensitivity. The latter can be modeled using a model with genetically structured environmental variance. This work reports the results of a genetic analysis of adult weight in the snail Helix aspersa. Several models of heterogeneous variance are fitted using a Bayesian, MCMC approach. Exploratory analyses using posterior predictive model checking and model comparisons based on the deviance information criterion favor a model postulating a genetically structured heterogeneous environmental variance. Our analysis provides a strong indication of a positive genetic correlation between additive genetic values affecting the mean and those affecting environmental variation of adult body weight. The possibility of manipulating environmental variance by selection is illustrated numerically using estimates of parameters derived from the snail data set.