Lack of membrane sex steroid receptors for mediating rapid endocrine responses in molluscan nervous systems.

Despite the lack of endogenous synthesis and relevant nuclear receptors, several papers have been published over the decades claiming that the physiology of mollusks is affected by natural and synthetic sex steroids. With scant evidence for the existence of functional steroid nuclear receptors in mollusks, some scientists have speculated that the effects of steroids might be mediated via membrane receptors (i.e. via non-genomic/non-classical actions) - a mechanism that has been well-characterized in vertebrates. However, no study has yet investigated the ligand-binding ability of such receptor candidates in mollusks. The aim of the present study was to further trace the evolution of the endocrine system by investigating the presence of functional membrane sex steroid receptors in a mollusk, the great pond snail (Lymnaea stagnalis). We detected sequences homologous to the known vertebrate membrane sex steroid receptors in the Lymnaea transcriptome and genome data: G protein-coupled estrogen receptor-1 (GPER1); membrane progestin receptors (mPRs); G protein-coupled receptor family C group 6 member A (GPRC6A); and Zrt- and Irt-like protein 9 (ZIP9). Sequence analyses, including conserved domain analysis, phylogenetics, and transmembrane domain prediction, indicated that the mPR and ZIP9 candidates appeared to be homologs, while the GPER1 and GPRC6A candidates seemed to be non-orthologous receptors. All candidates transiently transfected into HEK293MSR cells were found to be localized at the plasma membrane, confirming that they function as membrane receptors. However, the signaling assays revealed that none of the candidates interacted with the main vertebrate steroid ligands. Our findings strongly suggest that functional membrane sex steroid receptors which would be homologous to the vertebrate ones are not present in Lymnaea. Although further experiments are required on other molluscan model species as well, we propose that both classical and non-classical sex steroid signaling for endocrine responses are specific to chordates, confirming that molluscan and vertebrate endocrine systems are fundamentally different.

Characterization of corazonin signaling in a molluscan model species, Lymnaea stagnalis.

In recent years, new concepts have emerged regarding the nomenclature, functions, and relationships of different peptide families of the gonadotropin-releasing hormone (GnRH) superfamily. One of the main driving forces for this originated from the emerging evidence that neuropeptides previously called molluscan GnRH are multifunctional and should be classified as corazonin (CRZ). However, research articles still appear that use incorrect nomenclature and attribute the same function to molluscan CRZs as vertebrate GnRHs. The aim of the present study was to further support the recent interpretation of the origin and function of the GnRH superfamily. Towards this goal, we report the characterization of CRZ signaling system in the molluscan model species, the great pond snail (Lymnaea stagnalis). We detected a CRZ-receptor-like sequence (Lym-CRZR) by homology-searching in the Lymnaea transcriptomes and the deduced amino acid sequence showed high sequence similarity to GnRH receptors and CRZ receptors. Molecular phylogenetic tree analysis demonstrated that Lym-CRZR is included in the cluster of molluscan CRZRs. Lym-CRZR transiently transfected into HEK293 cells was found to be localized at the plasma membrane, confirming that it functions as a membrane receptor, like other G protein-coupled receptors. The signaling assays revealed that the previously identified Lym-CRZ neuropeptide stimulated intracellular Ca2+ mobilization in a dose-dependent manner, but not cyclic AMP production, in HEK293 cells transfected with Lym-CRZR. Finally, we demonstrated a wide tissue distribution of Lym-CRZR. These results suggest that Lym-CRZ is a multifunctional peptide and provide further insights into the evolution of the GnRH neuropeptide superfamily. The present study also supports the notion that previously termed molluscan "GnRH" should be classified as "CRZ".

Histopathological Evaluation of the Curative Effects of EDTA on Lymnaea stagnalis Exposed to Subacute Cadmium.

We investigated the therapeutic effects of EDTA application for 14 and 28 days on cadmium (Cd) induced pond snail Lymnaea stagnalis (Linnaeus, 1758). The sublethal concentration of cadmium (63.4 mg/l Cd) caused tissue damages to the snail after an exposure for 28 days.In the groups treated with EDTA, the concentration of Cd in the foot, mantle and hepatopancreas tissues showed significantly decreased during the recovery period. The curative effects of EDTA on Cd-induced damage were assessed using a scoring system. Cadmium exposure led to histopathological changes including increased mucositis, pigment and protein cells, foot epithelium desquamation, muscle fibril damage, connective tissue cell atrophy, and increased lipid vacuoles in the mantle and hepatopancreas. However, these changes were less severe in snails treated with EDTA (2.00 mL/L for 28 day), indicating that EDTA reduces their susceptibility to heavy metal toxicity.

The effects of temperature on nickel bioaccumulation and toxicity in the freshwater snail Lymnaea stagnalis.

It is well known that temperature can have important effects on the toxicity of metals (and other contaminants) to aquatic organisms. To date, research has mostly focused on thermal effects on acute metal toxicity, and there is a data gap on thermal effects on chronic metal toxicity to sensitive organisms that are particularly relevant to environmental risk assessment. This latter research is especially needed in the context of increased global temperature and heat waves frequency associated with climate change. We investigated temperature effects on chronic nickel (Ni) bioaccumulation and toxicity to the metal-sensitive freshwater snail Lymnaea stagnalis. In the laboratory, we conducted a series of experiments with juvenile snails that were pre-acclimated to different temperatures since their embryonic stage. We found that temperature and nickel separately had strong effects on juvenile growth rate and survival. Rising temperature from 18 to 26 °C had no noticeable effect on Ni-induced growth inhibition and Ni bioaccumulation in juvenile L. stagnalis exposed over 40 days to 0, 30 and 60 µg L-1 of dissolved Ni. These results agreed with estimates of Ni uptake and elimination rates (ku and ke), which were either unaffected by temperature or increased by similar factors from 18 to 26 °C. On the other hand, a temperature increase from 18 to 26 °C appeared to exacerbate Ni lethality to juvenile snails in the 40-day toxicity test. This exacerbation might have been due to a combination of factors, including detrimental changes in metabolically available Ni pools and/or to sensitization of the organism under sub-optimal temperatures. Overall, our study shows that thermal effects on metal chronic toxicity are complex, with effects that can be response-specific and not directly related to metal toxicokinetic.

Sublethal cadmium exposure in the freshwater snail Lymnaea stagnalis meets a deficient, poorly responsive metallothionein system while evoking oxidative and cellular stress.

The Great Pond snail Lymnaea stagnalis (Gastropoda, Hygrophila) is a wide-spread freshwater gastropod, being considered as a model organism for research in many fields of biology, including ecotoxicology. The aim of the present study was to explore the Cd sensitivity of L. stagnalis through the measurement of a biomarker battery for oxidative, toxic and cellular stress. The interpretation of biomarker parameters occurred against the background of a truncated metallothionein protein with a limited Cd-binding capacity. Individuals of L. stagnalis were exposed through 14 days to uncontaminated water (controls) or to low (30 µg · L-1) or high (50 µg · L-1) Cd concentrations. The digestive gland of control and low-Cd exposed snails was processed for transcriptional analysis of the Metallothionein (MT) gene expression, and for determination of biomarkers for oxidative stress, toxicity and cellular stress. Digestive gland supernatants of high-Cd exposed snails were subjected to chromatography and subsequent analysis by spectrophotometry. It was shown that the MT system of L. stagnalis is functionally deficient, with a poor Cd responsiveness at both, the transcriptional and the protein expression levels. Instead, L. stagnalis appears to rely on alternative detoxification mechanisms such as Cd binding by phytochelatins and metal inactivation by compartmentalization within the lysosomal system. In spite of this, however, traces of Cd apparently leak out of the pre-determined detoxification pathways, leading to adverse effects, which is clearly indicated by biomarkers of oxidative and cellular stress.

Impact of weathered multi-walled carbon nanotubes on the epithelial cells of the intestinal tract in the freshwater grazers Lymnaea stagnalis and Rhithrogena semicolorata.

Freshwater grazers are suitable organisms to investigate the fate of environmental pollutants, such as weathered multi-walled carbon nanotubes (wMWCNTs). One key process is the uptake of ingested materials into digestive or absorptive cells. To address this, we investigated the localization of wMWCNTs in the intestinal tracts of the mud snail Lymnaea stagnalis (L. stagnalis) and the mayfly Rhithrogena semicolorata (R. semicolorata). In L. stagnalis, bundles of wMWCNTs could be detected in the midgut lumen, whereas only single wMWCNTs could be detected in the lumina of the digestive gland. Intracellular uptake of wMWCNTs was detected by transmission electron microscopy (TEM) but was restricted to the cells of the digestive gland. In larvae of R. semicolorata, irritations of the microvilli and damages in the apical parts of the epithelial gut cells were detected after feeding with 1 to 10 mg/L wMWCNTs. In both models, we detected fibrillar structures in close association with the epithelial cells that formed peritrophic membranes (PMs). The PM may cause a reduced transmission of wMWCNT bundles into the epithelium by forming a filter barrier and potentially protecting the cells from the wMWCNTs. As a result, the uptake of wMWCNTs into cells is rare in mud snails and may not occur at all in mayfly larvae. In addition, we monitor physiological markers such as levels of glycogen or triglycerides and the RNA/DNA ratio. This ratio was significantly affected in L. stagnalis after 24 days with 10 mg/L wMWCNTs, but not in R. semicolorata after 28 days and 10 mg/L wMWCNTs. However, significant effects on the energy status of R. semicolorata were analysed after 28 days of exposure to 1 mg/L wMWCNTs. Furthermore, we observed a significant reduction of phagosomes per enterocyte cell in mayfly larvae at a concentration of 10 mg/L wMWCNTs (p < 0.01).

Studies on a widely-recognized snail model species (Lymnaea stagnalis) provide further evidence that vertebrate steroids do not have a hormonal role in the reproduction of mollusks.

Experiments were carried out to determine whether, as with other mollusks that have been studied, the snail, Lymnaea stagnalis, can absorb, esterify and store vertebrate steroids that are present in the water. We also carried out experiments to determine whether neural tissues of the snail could be immunohistochemically stained with an antibody to human aromatase (a key enzyme that catalyzes the conversion of testosterone [T] to 17ß-estradiol [E2]); and, if so, to determine the significance of such staining. Previous studies on other mollusks have reported such staining and have proposed this as decisive evidence that mollusks have the same steroid synthesis pathway as vertebrates. We found that snails absorb, esterify and retain esterified T, E2, progesterone and ethinyl-estradiol (albeit with an absorption rate about four times slower, on a weight basis, than the mussel, Mytilus edulis). We also found that not only anti-human aromatase, but also anti-human nuclear progesterone receptor (nPR) and anti-human gonadotropin-releasing hormone antibodies immunohistochemically stained snail neural cells. However, further experiments, involving gel electrophoretic separation, followed by immunostaining, of proteins extracted from the neural tissue, found at least two positively-stained bands for each antibody, none of which had masses matching the human proteins to which the antibodies had been raised. The anti-aromatase antibody even stained the 140 kDA ladder protein used as a molecular weight marker on the gels. Mass spectrometric analysis of the bands did not find any peptide sequences that corresponded to the human proteins. Our findings confirm that the presence of vertebrate-like sex steroids in molluscan tissues is not necessarily evidence of endogenous origin. The results also show that immunohistochemical studies using antibodies against human proteins are grossly non-specific and likely to have little or no value in studying steroid synthesis or activity in mollusks. Our conclusions are consistent with the fact that genes for aromatase and nPR have not been found in the genome of the snail or of any other mollusk. Our overarching conclusion, from this and our previous studies, is that the endocrinology of mollusks is not the same as that of humans or any other vertebrates and that continuing to carry out physiological and ecotoxicological studies on mollusks on the basis of this false assumption, is an unconscionable waste of resources.

Identification and characterization of the kynurenine pathway in the pond snail Lymnaea stagnalis.

Dysregulation of the kynurenine pathway (KP) is implicated in many human diseases and disorders, from immunological, metabolic, neurodegenerative, and neuropsychiatric conditions to cancer, and represents an appealing target for new therapeutic approaches. In this intricate scenario, invertebrates, like Lymnaea stagnalis (LS), provide a flexible tool to unravel the complexity of the KP. Starting from the available LS genome and transcriptome, we identified putative transcripts of all KP enzymes containing an ORF; each predicted protein possessed a high degree of sequence conservation to known orthologues of other invertebrate and vertebrate model organisms. Sequences were confirmed by qualitative PCR and sequencing. At the same time, the qRT-PCR analysis revealed that Lym IDO-like, Lym TDO-like, Lym AFMID-like, Lym KMO-like, Lym AADAT-like, Lym KYAT I/III-like, Lym KYNU-like, Lym HAAO-like, and Lym ACMSD-like showed widespread tissue expression. Then, tryptophan, kynurenine, kynurenic acid, anthranilic acid, 3-hydroxy-kynurenine, xanthurenic acid, picolinic acid, and quinolinic acid were identified in the hemolymph of LS by UHPLC-Q exactive mass spectrometer. Our study provides the most thorough characterization to date of the KP in an invertebrate model, supporting the value of LS for future functional studies of this pathway at the cellular, synaptic, and behavioral levels.

Toll like receptors and their evolution in the lymnaeid freshwater snail species Radix auricularia and Lymnaea stagnalis, key intermediate hosts for zoonotic trematodes.

One of the major evolutionarily conserved pathways in innate immunity of invertebrates is the toll-like receptor (TLR) pathway. However, little is known of the TLR protein family in gastropod molluscs despite their role in the transmission of human diseases, especially the common lymnaeid freshwater snail species Radix auricularia and Lymnaea stagnalis, key intermediate hosts of zoonotic trematodes. Using comparative genomics and gene prediction approaches utilising the freshwater snail Biomphalaria glabrata genome as a reference ten putative TLR proteins were identified in both R. auricularia and L. stagnalis. Phylogenetic analyses revealed that unlike other molluscs the lymnaeid species also possessed class 1 TLRs, previously thought to be unique to B. glabrata. Gene duplication events were also seen across the TLR classes in the lymnaeids with several of the genes appearing to exist as potential tandem elements in R. auricularia. Each predicted TLR was shown to possess the typical the leucine-rich repeat extracellular and TIR intracellular domains and both single cysteine clusters and multiple cysteine clusters TLRs were identified in both lymnaeid species. Principle component analyses of 3D models of the predicted TLRs showed that class 1 and 5 proteins did not cluster based on similarity of structure, suggested to be potential adaptation to a range of pathogens. This study provides the first detailed account of TLRs in lymnaeids and affords a platform for further research into the role of these proteins into susceptibility and compatibility of these snails with trematodes and their role in transmission.

Pseudogenes: A Novel Source of Trans-Acting Antisense RNAs.

Several recent studies support a functional role for pseudogenes, a copy of a parent gene that has lost protein-coding potential, which was for a long time thought to represent only "junk" DNA. Several hundreds of pseudogenes have now been reported as transcribed RNAs in a large variety of tissues and tumor types. Most studies have focused on pseudogenes expressed in sense direction, relative to their protein-coding gene counterpart, but some reports suggest that pseudogenes can be also transcribed as antisense RNAs (asRNAs). Key regulatory genes, such as PTEN and OCT4, have in fact been reported to be under the regulation of pseudogene-expressed asRNAs. Here, we review what is known about pseudogene-expressed asRNAs, we discuss the functional role that these transcripts may have in gene regulation and we summarize the techniques that are available to study them.

Functional characterization and related evolutionary implications of invertebrate gonadotropin-releasing hormone/corazonin in a well-established model species.

In vertebrates, gonadotropin-releasing hormone (GnRH) peptide is the central mediator of reproduction. Homologous peptides have previously also been identified in molluscan species. However, emerging evidence suggests that these molecules might serve diverse regulatory functions and proposes to consider them as corazonin (CRZ). We previously isolated the full-length cDNA of the invGnRH/CRZ peptide (termed ly-GnRH/CRZ) in the well-established invertebrate model species, the great pond snail Lymnaea stagnalis; however, its predicted functions remain to be verified. In this study, we first confirmed the presence of the deduced active peptide from the central nervous system of L. stagnalis. Further, we performed in vivo and in vitro studies to explore the functions of ly-GnRH/CRZ. Injection of sexually mature specimens with synthetic active peptide had an inhibitory effect on locomotion and an acceleratory effect on egg-laying, but had no effect on feeding. The previously predicted modulatory effect of ly-GnRH/CRZ was supported by its identified co-localization with serotonin on the surface of the heart atria. Lastly, we demonstrated not only the presence of ly-GnRH/CRZ in the penial complex but also that ly-GnRH/CRZ-containing neurons project to the efferent penis nerve, suggesting ly-GnRH/CRZ may directly modulate the motor output of this peripheral tissue. Overall, our findings strongly support that ly-GnRH/CRZ is a multifunctional neuropeptide. These results contribute to the understanding of the GnRH superfamily and, more broadly, disciplines such as comparative endocrinology and neurobiology.

Thorium Exposure Drives Fatty Acid and Metal Transfer from Biofilms to the Grazer Lymnaea sp.

Aquatic ecotoxicological risks associated with tetravalent metallic elements such as thorium (Th) are still poorly understood. Periphytic biofilm represents an important food source in aquatic environments; thus, such risks could severely affect nutrient and energy cycling in these ecosystems. The present study investigated the potential for Th to change the fatty acid composition of biofilm communities. Bioaccumulation of Th and fatty acids were measured after 4 wk to 2 exposure conditions: a control (C0) and Th exposure (C10). Some major fatty acids such as C16:1n-7 and docosahexaenoic acid C22:6n-3 differed significantly between control and C10 conditions. To determine if Th can be trophically transferred and to investigate the impacts of nutritional quality changes on primary consumers, common pond snails (Lymnaea sp.) were fed for 4 wk with control and Th-exposed biofilm. Thorium appeared to be trophically transferable to the grazers, although we cannot exclude that part of the Th accumulated by the snails may have been taken from the water through release from the biofilms. The composition of major fatty acids observed in the grazers was also significantly affected, notably by a decrease of total polyunsaturated fatty acids. These results indicate that very low Th concentrations can decrease the nutritional quality of organisms at the base of the food chain. Environ Toxicol Chem 2021;40:2220-2228. © 2021 SETAC.

Ligand-induced transition in conformations of vicinal cysteine disulfides in proteins.

Vicinal cysteine disulfides are thought to be associated with specific conformations of cysteine disulfides due to the restricted rotation of single bonds in an eight-membered cyclic disulfide loop. Conformations of vicinal cysteine disulfides are analyzed using χ1 , χ2 , χ3 , χ2 ', χ1 ' torsion angles in the crystal structures of proteins retrieved from Protein Data Bank (PDB). 85% of vicinal disulfides have (+, -)LHStaple conformation with trans configuration of the peptide bond and 9% have (-, -)RHStaple conformation with cis configured peptide bond. Conformational analysis of dipeptide Cys-Cys vicinal disulfide by density functional theory (DFT) further supported (+, -)LHStaple, (-, -)RHStaple, and (+, +)RHStaple as the preferred conformations of vicinal disulfides. Interestingly, the rare conformations of vicinal disulfides are observed in the ligand-bound forms of proteins and have higher disulfide strain energy. Conformations of vicinal disulfides in palmitoyl protein thioesterase 1, AChBP, and α7 nicotinic receptor are changed from preferred (+, -)LHStaple to rare (+, -)AntiLHHook/(+, -)AntiRHHook/(+, +)RHStaple conformation due to binding of ligands. Surprisingly, ligands are proximal to the vicinal disulfides in protein complexes that exhibited rare conformations of vicinal disulfides. The report has identified (+, -) LHStaple/(-, -) RHStaple as the hallmark conformations of vicinal disulfides and unraveled ligand-induced transition in conformations of vicinal cysteine disulfides in proteins.

Progestogen-induced alterations and their ecological relevance in different embryonic and adult behaviours of an invertebrate model species, the great pond snail (Lymnaea stagnalis).

The presence of oral contraceptives (basically applying estrogens and/or progestogens) poses a challenge to animals living in aquatic ecosystems and reflects a rapidly growing concern worldwide. However, there is still a lack in knowledge about the behavioural effects induced by progestogens on the non-target species including molluscs. In the present study, environmental progestogen concentrations were summarised. Knowing this data, we exposed a well-established invertebrate model species, the great pond snail (Lymnaea stagnalis) to relevant equi-concentrations (1, 10, 100, and 500 ng L-1) of mixtures of four progestogens (progesterone, drospirenone, gestodene, levonorgestrel) for 21 days. Significant alterations were observed in the embryonic development time, heart rate, feeding, and gliding activities of the embryos as well as in the feeding and locomotion activity of the adult specimens. All of the mixtures accelerated the embryonic development time and the gliding activity. Furthermore, the 10, 100, and 500 ng L-1 mixtures increased the heart rate and feeding activity of the embryos. The 10, 100, and 500 ng L-1 mixtures affected the feeding activity as well as the 1, 10, and 100 ng L-1 mixtures influenced the locomotion of the adult specimens. The differences of these adult behaviours showed a biphasic response to the progestogen exposure; however, they changed approximately in the opposite way. In case of feeding activity, this dose-response phenomenon can be identified as a hormesis response. Based on the authors' best knowledge, this is the first study to investigate the non-reproductive effects of progestogens occurring also in the environment on molluscan species. Our findings contribute to the global understanding of the effects of human progestogens, as these potential disruptors can influence the behavioural activities of non-target aquatic species. Future research should aim to understand the potential mechanisms (e.g., receptors, signal pathways) of progestogens induced behavioural alterations.

Integrative taxonomy: combining molecular and morphological characteristics to identify Lymnaea (Galba) cubensis, intermediate host of Fasciola hepatica / Taxonomia integrativa: combinando características moleculares e morfológicas para identificar Lymnaea (Galba) cubensis, hospedeiro intermediário de Fasciola hepatica

Abstract Despite the epidemiological importance of the Lymnaeidae family regarding transmission of Fasciola hepatica, knowledge about the diversity and distribution of these molluscs and the role of each species in the expansion of fasciolosis remains sparse. Classical morphological (n=10) identification was performed in lymneids from Lagoa Santa, a municipality in the state of Minas Gerais, Brazil, along with molecular and phylogenetic analysis (n=05) based on the partial nucleotide sequences of the mitochondrial cytochrome c oxidase subunit I gene (COI mtDNA) and ribosomal internal transcribed spacer II (ITS-2 rDNA). The shell morphology made it possible to distinguish the lymneids of Lagoa Santa from Pseudosuccinea columella. Differences found in the penile complex and prostate shape allowed this species to be distinguished from Galba truncatula. However, the homogeneity of reproductive tract characteristics among Lymnaea (Galba) cubensis, L. viator and L. neotropica confirmed that these characteristics show low taxonomic reliability for identifying cryptic species. Genetic divergence analysis for the COI mtDNA gene and ITS-2 region of rDNA revealed greater similarity to Lymnaea (Galba) cubensis. Thus, correct species differentiation is important for monitoring the epidemiological risk of fasciolosis in the state of Minas Gerais, where cases of the disease have increased over recent years.

Resumo Apesar da importância epidemiológica da família Lymnaeidae na transmissão de Fasciola hepatica, o conhecimento sobre a diversidade e a distribuição desses moluscos e o papel de cada espécie, na expansão da fasciolose, ainda é escasso. Realizou-se a identificação morfológica clássica (n=10) em limneídeos de Lagoa Santa, município do estado de Minas Gerais, Brasil, juntamente com a análise molecular e filogenética (n=05), baseada nas sequências parciais de nucleotídeos do gene mitocondrial da subunidade I do citocromo c oxidase (COI mtDNA) e espaçador interno, transcrito do DNA ribossomal II (ITS-2 rDNA). A morfologia da concha possibilitou distinguir os limneídeos de Lagoa Santa de Pseudosuccinea columella. As diferenças encontradas no complexo peniano e na forma da próstata permitiram que essa espécie fosse distinta de Galba truncatula. No entanto, a homogeneidade das características do trato reprodutivo entre Lymnaea (Galba) cubensis, L. viator e L. neotropica confirmou que essas características apresentam baixa confiabilidade taxonômica para a identificação de espécies crípticas. A análise da divergência genética para o gene COI mtDNA e região ITS-2 do rDNA revelou maior similaridade entre os limneídeos de Lagoa Santa com Lymnaea (Galba) cubensis.

Spatial Structure and Activity of Synthetic Fragments of Lynx1 and of Nicotinic Receptor Loop C Models.

Lynx1, membrane-bound protein co-localized with the nicotinic acetylcholine receptors (nAChRs) and regulates their function, is a three-finger protein (TFP) made of three ß-structural loops, similarly to snake venom α-neurotoxin TFPs. Since the central loop II of α-neurotoxins is involved in binding to nAChRs, we have recently synthesized the fragments of Lynx1 central loop, including those with the disulfide between Cys residues introduced at N- and C-termini, some of them inhibiting muscle-type nAChR similarly to the whole-size water-soluble Lynx1 (ws-Lynx1). Literature shows that the main fragment interacting with TFPs is the C-loop of both nAChRs and acetylcholine binding proteins (AChBPs) while some ligand-binding capacity is preserved by analogs of this loop, for example, by high-affinity peptide HAP. Here we analyzed the structural organization of these peptide models of ligands and receptors and its role in binding. Thus, fragments of Lynx1 loop II, loop C from the Lymnaea stagnalis AChBP and HAP were synthesized in linear and Cys-cyclized forms and structurally (CD and NMR) and functionally (radioligand assay on Torpedo nAChR) characterized. Connecting the C- and N-termini by disulfide in the ws-Lynx1 fragment stabilized its conformation which became similar to the loop II within the 1H-NMR structure of ws-Lynx1, the activity being higher than for starting linear fragment but lower than for peptide with free cysteines. Introduced disulfides did not considerably change the structure of HAP and of loop C fragments, the former preserving high affinity for α-bungarotoxin, while, surprisingly, no binding was detected with loop C and its analogs.

Identification, presence, and possible multifunctional regulatory role of invertebrate gonadotropin-releasing hormone/corazonin molecule in the great pond snail (Lymnaea stagnalis).

In the last years, our interpretation of the origin and function of the gonadotropin-releasing hormone (GnRH) neuropeptide superfamily has changed substantially. A main driver for these conceptual changes came from increased investigations into functions and evolutionary lineage of previously identified molluscan GnRH molecules. Emerging evidence suggests not only reproductive, but also diverse biological effects of these molecules and proposes they should most likely be called corazonin (CRZ). Clearly, a more global understanding requires further exploration of species-specific functions and structure of invGnRH/CRZ peptides. Towards this goal, we have identified the full-length cDNA of invGnRH/CRZ peptide in an invertebrate model species, the great pond snail Lymnaea stagnalis, termed ly-GnRH/CRZ, and characterized the transcript and peptide distribution in the central nervous system (CNS) and peripheral organs. Our results are consistent with previous data that molluscan GnRHs are more related to CRZs and serve diverse functions. Hence, our findings support the notion that peptides originally termed molluscan GnRH are multifunctional modulators and that nomenclature change should be taken into consideration.

Accumulation and histopathological effects of cadmium on the great pond snail Lymnaea stagnalis Linnaeus, 1758 (Gastropoda: Pulmonata).

Toxic metal ions are an important stress factor for a living organism. In this study, accumulation and histopathological changes in foot, mantle and hepatopancreas of great pond snail, Lymnaea stagnalis exposed to different Cadmium (Cd) concentrations in laboratory conditions were investigated. Great pond snails were exposed to sublethal concentrations of 7.92 µg/L, 15.85 µg/L, 31.7 µg/L and 63.4 µg/L Cd. At the end of 7, 14, 21 and 28 days, snail foot, mantle and hepatopancreas were removed to investigate and determine Cd accumulation and histopathological alterations by light microscopy. Cd levels determined in hepatopancreas were higher than those measured from the foot and the mantle of studied specimens. A positive correlation was found between Cd levels in tissues and exposure days. As a result of Cd application, we observed increase in the number of mucosit, pigment and protein cells and desquamation in the epithelium in the foot, atrophy in muscle fibrils, connective tissue cells and increase in the lipid vacuoles in the mantle, increase in the lipid vacuoles and amoebocyte in the hepatopancreas. The severity of the alterations resulting from Cd increased with dose-time dependent.

A Mystery Tale: Nickel Is Fickle When Snails Fail-Investigating the Variability in Ni Toxicity to the Great Pond Snail.

Dissolved Ni concentrations inhibiting the growth of juvenile great pond snails (Lymnaea stagnalis) have been documented to vary from about 1 to 200 µg L-1 Ni. This variability makes L. stagnalis either a moderately sensitive or the most sensitive freshwater species to chronic Ni exposure tested to date. Given the role of sensitive species in environmental risk assessment frameworks, it is particularly important to understand this variability, i.e., to characterize the factors that modulate Ni toxicity and that may confound toxicity test outcomes when uncontrolled. In the present study, we tested if this variability was due to analytical (growth calculation: biomass versus growth rate), environmental (water quality), lab-specific practices, and/or snail population differences among earlier studies. Specifically, we reanalyzed previously published Ni toxicity data and conducted additional measurements of Ni aqueous speciation, short-term Ni uptake, and chronic Ni toxicity with test waters and snail cultures used in previous studies. Corrections for Ni bioavailability and growth calculations explained a large degree of variability in the published literature. However, a residual 16-fold difference remained puzzling between 2 studies: Niyogi et al. (2014) (low ECxs) and Crémazy et al. (2018) (high ECxs). Indeed, differences in metal bioavailability due to water chemistry, lab-specific practices, and snail population sensitivity could not explain the large variation in Ni toxicity in these 2 very similar studies. Other potentially important toxicity-modifying factors were not directly evaluated in the present work: test duration, diet, snail holding conditions, and snail age at onset of testing. The present analysis highlights the need for further studies to elucidate 1) the mechanisms of growth inhibition in Ni-exposed L. stagnalis and 2) the important abiotic and biotic factors affecting this biological response. Until these processes are understood, substantial uncertainties will remain about inclusion of this species in Ni environmental risk assessment. Integr Environ Assess Manag 2020;16:983-997. © 2020 SETAC.

PNU-120596, a positive allosteric modulator of mammalian α7 nicotinic acetylcholine receptor, is a negative modulator of ligand-gated chloride-selective channels of the gastropod Lymnaea stagnalis.

Excitatory α7 neuronal nicotinic receptors (nAChR) are widely expressed in the central and peripheral nervous and immune systems and are important for learning, memory, and immune response regulation. Specific α7 nAChR ligands, including positive allosteric modulators are promising to treat cognitive disorders, inflammatory processes, and pain. One of them, PNU-120596, highly increased the neuron response to α7 agonists and retarded desensitization, showing selectivity for α7 as compared to heteromeric nAChRs, but was not examined at the inhibitory ligand-gated channels. We studied PNU-120596 action on anion-conducting channels using voltage-clamp techniques: it slightly potentiated the response of human glycine receptors expressed in PC12 cells, of rat GABAA receptors in cerebellar Purkinje cells and mouse GABAA Rs heterologously expressed in Xenopus oocytes. On the contrary, PNU-120596 exerted an inhibitory effect on the receptors mediating anion currents in Lymnaea stagnalis neurons: two nAChR subtypes, GABA and glutamate receptors. Acceleration of the current decay, contrary to slowing down desensitization in mammalian α7 nAChR, was observed in L. stagnalis neurons predominantly expressing one of the two nAChR subtypes. Thus, PNU-120596 effect on these anion-selective nAChRs was just opposite to the action on the mammalian cation-selective α7 nAChRs. A comparison of PNU-120596 molecule docked to the models of transmembrane domains of the human α7 AChR and two subunits of L. stagnalis nAChR demonstrated some differences in contacts with the amino acid residues important for PNU-120596 action on the α7 nAChR. Thus, our results show that PNU-120596 action depends on a particular subtype of these Cys-loop receptors.