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Neonicotinoid insecticides, which act on nicotinic acetylcholine receptors (nAChRs) in a variety of ways, have extremely low mammalian toxicity, yet the molecular basis of such actions is poorly understood. To elucidate the molecular basis for nAChR–neonicotinoid interactions, a surrogate protein, acetylcholine binding protein from Lymnaea stagnalis ( Ls -AChBP) was crystallized in complex with neonicotinoid insecticides imidacloprid (IMI) or clothianidin (CTD). The crystal structures suggested that the guanidine moiety of IMI and CTD stacks with Tyr185, while the nitro group of IMI but not of CTD makes a hydrogen bond with Gln55. IMI showed higher binding affinity for Ls -AChBP than that of CTD, consistent with weaker CH–π interactions in the Ls -AChBP–CTD complex than in the Ls -AChBP–IMI complex and the lack of the nitro group-Gln55 hydrogen bond in CTD. Yet, the NH at position 1 of CTD makes a hydrogen bond with the backbone carbonyl of Trp143, offering an explanation for the diverse actions of neonicotinoids on nAChRs.

The influence of ions of heavy metals (copper, cadmium, nickel, zinc, cobalt and manganese) was investigated on the basis of trophic characteristics: the average daily ration (ADR), and duration of food passage (DFP) of the L. in various concentrations of toxicants in vivisection experiment. In addition to these indicators, the total amount of food consumed in the solutions with various concentrations of pollutants was found out during the chronic experiment and it was calculated for an individual (average monthly ration – AMR). It leads to the conclusions about the intensity of food consuming considering different levels of intoxication. In solutions with lethal concentrations, the death of animals occurs during the first day of its impact due to the damage of tissues and organ systems. Chronic lethal concentrations of toxicants inhibit the nutrition of pond snails dramatically. At the beginning of the experiment, solutions of heavy metals with sublethal concentrations give some stimulatory effect on the digestive system of molluscs that is replaced by its suppression in case of longer being in the toxic environment. The influence of toxicants within a subthreshold limit cannot be considered safe because of the cumulative properties of heavy metals – they become sublethal with prolonged exposure time.

Background: Reactive oxygen species (ROS) are essential for normal physiological functioning of the brain. However, uncompensated increase in ROS levels may results in oxidative stress. Phospholipase A sub(2) (PLA sub(2)) is one of the key players activated by elevated ROS levels resulting in the hydrolysis of various products from the plasmamembrane such as peroxidized fatty acids. Free fatty acids (FFAs) and fatty acid metabolites are often implicated to the genesis of cognitive impairment. Previously we have shown that age-, and experimentally induced oxidative stress causes PLA sub(2)-dependent long-term memory (LTM) failure in an aversive operant conditioning model in Lymnaea stagnalis. In the present study, we investigate the effects of experimentally induced oxidative stress and the role of elevated levels of circulating FFAs on LTM function using a non-aversive appetitive classical conditioning paradigm. Results: We show that intracoelomic injection of exogenous PLA sub(2) or pro-oxidant induced PLA sub(2) activation negatively affects LTM performance in our learning paradigm. In addition, we show that experimental induction of oxidative stress causes significant temporal changes in circulating FFA levels. Importantly, the time of training coincides with the peak of this change in lipid metabolism. However, intracoelomic injection with exogenous arachidonic acid, one of the main FFAs released by PLA sub(2), does not affect LTM function. Moreover, sequestrating circulating FFAs with the aid of bovine serum albumin does not rescue pro-oxidant induced appetitive LTM failure. Conclusions: Our data substantiates previous evidence linking lipid peroxidation and PLA sub(2) activation to age- and oxidative stress-related cognitive impairment, neuronal dysfunction and disease. In addition however, our data indicate that lipid peroxidation induced increased levels of circulating (per)oxidized FFAs are not a factor in oxidative stress induced LTM impairment.

The alpha sub(7) acetylcholine receptor (AChR) mediates pre- and postsynaptic neurotransmission in the central nervous system and is a potential therapeutic target in neurodegenerative, neuropsychiatric and inflammatory disorders. We determined the crystal structure of the extracellular domain of a receptor chimera constructed from the human alpha sub(7) AChR and Lymnaea stagnalis acetylcholine binding protein (AChBP), which shares 64% sequence identity and 71% similarity with native alpha sub(7). We also determined the structure with bound epibatidine, a potent AChR agonist. Comparison of the structures revealed molecular rearrangements and interactions that mediate agonist recognition and early steps in signal transduction in alpha sub(7) AChRs. The structures further revealed a ring of negative charge within the central vestibule, poised to contribute to cation selectivity. Structure-guided mutational studies disclosed distinctive contributions to agonist recognition and signal transduction in alpha sub(7) AChRs. The structures provide a realistic template for structure-aided drug design and for defining structure-function relationships of alpha sub(7) AChRs.

Only a limited number of animal species lend themselves to becoming model organisms in multiple biological disciplines: one of these is the great pond snail, Lymnaea stagnalis. Extensively used since the 1970s to study fundamental mechanisms in neurobiology, the value of this freshwater snail has been also recognised in fields as diverse as host–parasite interactions, ecotoxicology, evolution, genome editing and 'omics', and human disease modelling. While there is knowledge about the natural history of this species, what is currently lacking is an integration of findings from the laboratory and the field. With this in mind, this article aims to summarise the applicability of L. stagnalis and points out that this multipurpose model organism is an excellent, contemporary choice for addressing a large range of different biological questions, problems and phenomena.

The qualitative and quantitative composition of biomass of microorganisms has been studied in key points of artificial biocenosis: water filter, duckweed and pond snail. Metagenomic studies have shown the presence of Bacteroides and Proteobacteria types in all examined systems, however, specific representatives - Actinobacteria, Cyanobacteria, and Bacilliwere also found, respectively. A microflora peculiarity of water filter was the presence of actinobacteria. In the intestine of the pond snail (Lymnaea stagnalis L.), a significant proportion of gram-positive bacteria belonging to the bacilli, determining symbiotic and associative microflora, was found. In duckweed (Lemna minor L.), representatives of Cyanobacteria were found. In addition, representatives of Bacteroides and Proteobacteria types were found for all three biotopes. Bacteroides prevailed in water filter and Proteobacteria were most characteristic for living objects (Lymnaea stagnalis L., and Lemna minor L.) but differing in the share of its constituent classes for every ecological niche. The data obtained allow us to use marked taxonomic groups of bacteria as identifiers of main zones of artificial biocenosis and to evaluate changes in them in the future by a shift in the qualitative and quantitative composition of microflora representatives.

Cell adhesion and spreading are vital to immune function. In molluscs, haemocytes (circulating phagocytes) are sentinels and effectors of the internal defence system; however, molecular mechanisms that regulate integrin-mediated spreading by haemocytes have not been characterised in detail. Visualisation of Lymnaea stagnalis haemocytes by scanning electron microscopy revealed membrane ruffling, formation of lamellipodia and extensive filopodia during early stages of cell adhesion and spreading. These events correlated with increased phosphorylation (activation) of protein kinase C (PKC) and focal adhesion kinase (FAK), sustained for 60 min. Treatment of haemocytes with the PKC inhibitors GF109203X or Gö 6976, or the Src/tyrosine kinase inhibitors SrcI or herbimycin A, attenuated haemocyte spread by 64, 46, 32 and 35%, respectively (P ≤ 0.001); PKC or Src inhibition also prevented focal adhesion formation. Western blotting demonstrated that during spreading and adhesion these inhibitors also impaired PKC and FAK activation, with Gö 6976 or SrcI inhibiting FAK phosphorylation by at least 70% (P ≤ 0.001), and herbimycin A or SrcI inhibiting PKC phosphorylation by at least 46% (P ≤ 0.01). Confocal microscopy revealed phosphorylated PKC colocalised with focal adhesion sites, particularly during early phases of adhesion and spreading. Finally, fibronectin promoted PKC and FAK phosphorylation in suspended haemocytes demonstrating that activation can occur independent of cell adhesion. These novel data are consistent with PKC and FAK/Src playing an integrated role in integrin activation and integrin-mediated spreading by L. stagnalis haemocytes. We propose a model in which integrin engagement mediates association of PKC with FAK/Src complexes to promote focal adhesion assembly during immune recognition by these cells.

By employing a reductionistic (but not simplistic) approach using an established invertebrate model system, the pond snail Lymnaea stagnalis , we investigated whether (1) lipopolysaccharide (LPS)-induced inflammation would cause a sickness state and impair cognitive function, and—if so—(2) would aspirin (acetylsalicylic acid—ASA) restore the impaired cognition. To test our hypotheses, we first determined if the injection of 25 mg (6.25 μg/mL) of Escherichia coli -derived LPS serotype O127:B8 altered homeostatic behavior, aerial respiration, and then determined if LPS altered memory formation when this behavior was operantly conditioned. Next, we determined if ASA altered the LPS-induced changes in both aerial respiration and cognitive functions. LPS induced a sickness state that increased aerial respiration and altered the ability of snails to form or recall long-term memory. ASA reverted the LPS-induced sickness state and thus allowed long-term memory both to be formed and recalled. We confirmed our hypotheses and provided the first evidence in an invertebrate model system that an injection of LPS results in a sickness state that obstructs learning and memory, and this impairment can be prevented by a non-steroidal anti-inflammatory.

There is mounting evidence that intestinal microbiota communities and their genes (the gut microbiome) influence how animals behave and interact with their environment, driving individual variation. Individual covariation in behavioural, physiological, and cognitive traits among individuals along a fast–slow continuum is thought to arise because these traits are linked as part of an adaptive pace-of-life strategy. Yet paradoxically, trait intercorrelation is absent or disrupted in some populations but not others. Here, we provide experimental evidence from aquatic pond snails (Lymnaea stagnalis) that environmental stressors and the gut microbiota explain host phenotypic plasticity and disrupted covariation among traits. Antibiotic exposure at varying levels of ecologically relevant concentrations had multiple effects starting with gut microbiota diversity, differential abundance, and inferred function. Memory declined in line with antibiotic concentrations that caused the most profound gut microbiota disruption, and although pace-of-life traits remained rigid, their covariation did not. Moreover, inferred microbial metabolic pathways with biologically relevant host functions explained individual and treatment variation in phenotypes. Together, our results point to the gut microbiome as a proximate mechanism influencing the emergence and maintenance of phenotypic variation within populations and highlights the need to decipher whether the gut microbiome’s sensitivity to environmental pollution facilitates adaptive or maladaptive phenotypic plasticity.

The great pond snail Lymnaea stagnalis has served as a model organism for over a century in diverse disciplines such as neurophysiology, evolution, ecotoxicology and developmental biology. To support both established uses and newly emerging research interests we have performed whole genome sequencing (avg.176 × depth), assembly and annotation of a single individual derived from an inbred line. These efforts resulted in a final assembly of 943 Mb (L50 = 257; N50 = 957,215) with a total of 22,499 predicted gene models. The mitogenome was found to be 13,834 bp long and similarly organized as in other lymnaeid species, with minor differences in location of tRNA genes. As a first step towards understanding the hermaphroditic reproductive biology of L. stagnalis , we identified molecular receptors, specifically nuclear receptors (including newly discovered 2xDNA binding domain-NRs), G protein-coupled receptors, and receptor tyrosine kinases, that may be involved in the cellular specification and maintenance of simultaneously active male and female reproductive systems. A phylogenetic analysis of one particular family of GPCRs (Rhodopsin neuropeptide FMRFamide-receptor-like genes) shows a remarkable expansion that coincides with the occurrence of simultaneous hermaphroditism in the Euthyneura gastropods. As some GPCRs and NRs also showed qualitative differences in expression in female (albumen gland) and male (prostate gland) organs, it is possible that separate regulation of male and female reproductive processes may in part have been enabled by an increased abundance of receptors in the transition from a separate-sexed state to a hermaphroditic condition. These findings will support efforts to pair receptors with their activating ligands, and more generally stimulate deeper insight into the mechanisms that underlie the modes of action of compounds involved in neuroendocrine regulation of reproduction, induced toxicity, and development in L. stagnalis , and molluscs in general.