Molecular Prevalence of Larval Stages of Fasciola hepatica in Lymnaea stagnalis Species Snails in the Vicinity of the Agri Province

Objective: Lymnaea stagnalis known as the great pond snail, is one of the intermediate hosts of Fasciola hepatica, a zoonotic parasite. In this study, it was aimed to determine the larval forms of F. hepatica by polymerase chain reaction (PCR) in L. stagnalis species snails collected from the vicinity of Agri province. Methods: In this study, 150 L. stagnalis snails were collected from the Agri province. The freshwater snails brought to the laboratory were dissected, then their soft tissues were examined under a microscope. DNA extraction was performed on the dissected snails. After DNA extraction, PCR was performed using primers targeting the cytochrome c oxidase subunit 1 gene region. Results: In the microscopic examination, larval forms of F. hepatica could not be detected. However, it was concluded that two (1.3%) L. stagnalis freshwater snails were infected with the larval forms of F. hepatica in the PCR. Conclusion: It was determined that L. stagnalis served as an intermediate host to F. hepatica in the study area.

Novel taste, sickness, and memory: Lipopolysaccharide to induce a Garcia-like effect in inbred and wild strains of Lymnaea stagnalis.

Food is not only necessary for our survival but also elicits pleasure. However, when a novel food is followed sometime later by nausea or sickness animals form a long-lasting association to avoid that food. This phenomenon is called the 'Garcia effect'. We hypothesized that lipopolysaccharide (LPS) could be used as the sickness-inducing stimulus to produce a Garcia-like effect in inbred and wild populations of Lymnaea stagnalis. We first demonstrated that the injection of 25 µg (6.25 µg/mL) of Escherichia coli-derived LPS serotype O127:B8 did not by itself alter snails' feeding behavior. Then we showed that the presentation of a novel appetitive stimulus (i.e., carrot slurry) and LPS resulted in a taste-specific and long-lasting feeding suppression (i.e., the Garcia-like effect). We also found strain-specific variations in the duration of the long-term memory (LTM). That is, while the LTM for the Garcia-like effect in W-strain snails persisted for 24h, LTM persisted for 48h in freshly collected Margo snails and their F1 offspring. Finally, we demonstrated that the exposure to a non-steroidal anti-inflammatory drug, aspirin (acetylsalicylic acid) before the LPS injection prevented both the LPS-induced sickness state and the Garcia-like effect from occurring. The results of this study may pave the way for new research that aims at (1) uncovering the conserved molecular mechanisms underlying the Garcia-like effect, (2) understanding how cognitive traits vary within and between species, and (3) creating a holistic picture of the complex dialogue between the immune and central nervous systems.

Remember the poke: microRNAs are required for long-term memory formation following operant conditioning in Lymnaea.

MicroRNAs (miRNAs) play an important role in learning and memory formation by controlling the expression of genes through epigenetic processes. Although miRNAs unquestionably play a role in memory, past literature focusing on whether miRNAs play key roles in the consolidation of associative long-term memory in Lymnaea contained confounding variables. Using operant conditioning of aerial respiratory behaviour, we investigated long-term memory (LTM) formation after injection of poly-L-lysine (PLL), an inhibitor of Dicer-mediated miRNA biogenesis, in Lymnaea stagnalis. Homeostatic breathing experiments were also performed to test whether PLL affects breathing. Homeostatic breathing was significantly suppressed 45 min but not 24 h after PLL injection. The operant conditioning procedure involved two 30-min training sessions separated by 1 h to cause LTM. Using this operant conditioning procedure, LTM formation was significantly impaired when snails were injected with PLL 15 min after the second training session. In contrast, when snails were injected with PLL 24 h before the first training session, LTM formation was not impaired. These results are consistent with past literature and highlight an important role for miRNAs in LTM formation.

Environmental DNA methylation of Lymnaea stagnalis varies with age and is hypermethylated compared to tissue DNA.

Environmental DNA (eDNA) approaches contributing to species identifications are quickly becoming the new norm in biomonitoring and ecosystem assessments. Yet, information such as age and health state of the population, which is vital to species biomonitoring, has not been accessible from eDNA. DNA methylation has the potential to provide such information on the state of a population. Here, we measured the methylation of eDNA along with tissue DNA (tDNA) of Lymnaea stagnalis at four life stages. We demonstrate that eDNA methylation varies with age and allows distinguishing among age classes. Moreover, eDNA was globally hypermethylated in comparison to tDNA. This difference was age-specific and connected to a limited number of eDNA sites. This differential methylation pattern suggests that eDNA release with age is partially regulated through DNA methylation. Our findings help to understand mechanisms involved in eDNA release and shows the potential of eDNA methylation analysis to assess age classes. Such age class assessments will encourage future eDNA studies to assess fundamental processes of population dynamics and functioning in ecology, biodiversity conservation and impact assessments.

Comparison between relative and absolute quantitative real-time PCR applied to single-cell analyses: Transcriptional levels in a key neuron for long-term memory in the pond snail.

Quantitative real-time PCR (qPCR) is a powerful method for measuring nucleic acid levels and quantifying mRNA levels, even in single cells. In the present study, we compared the results of single-cell qPCR obtained by different quantification methods (relative and absolute) and different reverse transcription methods. In the experiments, we focused on the cerebral giant cell (CGC), a key neuron required for the acquisition of conditioned taste aversion in the pond snail Lymnaea stagnalis, and examined changes in the mRNA levels of 3 memory-related genes, cAMP-response element binding proteins (LymCREB1 and LymCREB2) and CREB-binding protein (LymCBP), during memory formation. The results obtained by relative quantification showed similar patterns for the 3 genes. For absolute quantification, reverse transcription was performed using 2 different methods: a mixture of oligo d(T) primers and random primers (RT method 1); and gene-specific primers (RT method 2). These methods yielded different results and did not show consistent changes related to conditioning. The mRNA levels in the samples prepared by RT method 2 were up to 3.3 times higher than those in samples prepared by RT method 1. These results suggest that for qPCR of single neurons, the efficacy and validity do not differ between relative and absolute quantification methods, but the reverse transcription step critically influences the results of mRNA quantification.

Molecular and functional characterization of an evolutionarily conserved CREB-binding protein in the Lymnaea CNS.

In eukaryotes, CREB-binding protein (CBP), a coactivator of CREB, functions both as a platform for recruiting other components of the transcriptional machinery and as a histone acetyltransferase (HAT) that alters chromatin structure. We previously showed that the transcriptional activity of cAMP-responsive element binding protein (CREB) plays a crucial role in neuronal plasticity in the pond snail Lymnaea stagnalis. However, there is no information on the molecular structure and HAT activity of CBP in the Lymnaea central nervous system (CNS), hindering an investigation of its postulated role in long-term memory (LTM). Here, we characterize the Lymnaea CBP (LymCBP) gene and identify a conserved domain of LymCBP as a functional HAT. Like CBPs of other species, LymCBP possesses functional domains, such as the KIX domain, which is essential for interaction with CREB and was shown to regulate LTM. In-situ hybridization showed that the staining patterns of LymCBP mRNA in CNS are very similar to those of Lymnaea CREB1. A particularly strong LymCBP mRNA signal was observed in the cerebral giant cell (CGC), an identified extrinsic modulatory interneuron of the feeding circuit, the key to both appetitive and aversive LTM for taste. Biochemical experiments using the recombinant protein of the LymCBP HAT domain showed that its enzymatic activity was blocked by classical HAT inhibitors. Preincubation of the CNS with such inhibitors blocked cAMP-induced synaptic facilitation between the CGC and an identified follower motoneuron of the feeding system. Taken together, our findings suggest a role for the HAT activity of LymCBP in synaptic plasticity in the feeding circuitry.

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.

Aspirin reverts lipopolysaccharide-induced learning and memory impairment: first evidence from an invertebrate model system.

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.

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.

Too Hot to Eat: Wild and Lab-Bred Lymnaea stagnalis Differ in Feeding Response Following Repeated Heat Exposure.

AbstractAcute extreme heat events are increasing in frequency and intensity. Understanding their effects on ectothermic organisms' homeostasis is both important and urgent. In this study we found that the exposure to an acute heat shock (30 °C for 1 hour) repeated for a seven-day period severely suppressed the feeding behavior of laboratory-inbred (W-strain) Lymnaea stagnalis, whereas the first-generation offspring of freshly collected wild (F1 D-strain) snails raised and maintained under similar laboratory conditions did not show any alterations. The W-strain snails might have inadvertently been selected against heat tolerance since they were first brought into the laboratory many (∼70) years ago. We also posit that the F1 D-strain snails do not perceive the heat shock as a sufficient stressor to alter their feeding response because their parental populations in wild environments have repeatedly experienced temperature fluctuations, thus becoming more tolerant and resilient to heat. The different responses exhibited by two strains of the same species highlight the importance of selecting the most appropriate strain for addressing questions about the impacts of global warming on organisms' physiology and behavior.

Comparative study of the sensitivity of two freshwater gastropods, Lymnaea stagnalis and Planorbarius corneus, to silver nanoparticles: bioaccumulation and toxicity.

Metal-based nanoparticles (NPs) are considered detrimental to aquatic organisms due to their potential accumulation. However, little is known about the mechanisms underlying these effects and their species-specificity. Here we used stable silver (Ag) NPs (20 nm, from 10 to 500 µg/L) with a low dissolution rate (≤2.4%) to study the bioaccumulation and biological impacts in two freshwater gastropods: Lymnaea stagnalis and Planorbarius corneus. No mortality was detected during the experiments. Ag bioaccumulation showed a dose-related increase with an enhanced concentration in both species after 7d exposure. L. stagnalis displayed a higher accumulation for AgNPs than P. corneus (e.g., up to 18- and 15-fold in hepatopancreas and hemolymph, respectively) which could be due to the more active L. stagnalis having greater contact with suspended AgNPs. Furthermore, the hepatopancreas and stomach were preferred organs for bioaccumulation compared to the kidney, mantle and foot. Regarding biological responses, the hemolymph rather than hepatopancreas appeared more susceptible to oxidative stress elicited by AgNPs, as shown by significantly increasing lipid peroxidation (i.e., formation of malondialdehyde). Neurotoxicity was detected in L. stagnalis when exposed to high concentrations (500 µg/L). Comparison with impacts elicited by dissolved Ag revealed that the effects observed on AgNPs exposure were mainly attributable to NPs. These results highlighted the relationship between the physiological traits, bioaccumulation, and toxicity responses of these two species to AgNPs and demonstrated the necessity of species-specificity considerations when assessing the toxicity of NPs.

Multigenerational responses in the Lymnaea stagnalis freshwater gastropod exposed to diclofenac at environmental concentrations.

Over the last decade, there has been increased concern about the occurrence of diclofenac (DCF) in aquatic ecosystems. Living organisms could be exposed to this "pseudo-persistent" pharmaceutical for more than one generation. In this multigenerational study, we assessed the DCF impact at environmentally relevant concentrations on the life history and behavioral parameters of two offspring generations (F1 and F2) of the Lymnaea stagnalis freshwater gastropod. Snail growth was affected by DCF in the F1 generation, with increased shell sizes of juveniles exposed to 0.1 µg L - 1 concentration and a decreased shell size at 2 and 10 µg L - 1. DCF also lowered food intake, enhanced locomotion activity and reduced the number of eggs/egg mass in the F1 generation. For the F2 generation, shorter time to hatch, faster growth, increased food intake and production of more egg masses/snail were induced by DCF exposure at 10 µg L - 1. Over time, DCF exposure led to maximization of L. stagnalis reproductive function. These results show that multigenerational studies are crucial to reveal adaptive responses to chronic contaminant exposure, which are not observable after short-term exposure.

Epicatechin Alters the Activity of a Neuron Necessary for Long-Term Memory of Aerial Respiratory Behavior in Lymnaea stagnalis.

Epicatechin (EpiC) enhances long-term memory (LTM) formation in the pond snail Lymnaea stagnalis. Here we investigated at the level of a single neuron, RPeD1, which is a necessary site for LTM formation of operant conditioning of aerial respiration, how EpiC may bring about its enhancing effect on LTM formation. When snails were operantly conditioned in EpiC (15 mg/l) by a single 0.5 h training session, which typically only results in memory lasting ∼3 h, they now formed LTM lasting at least 24 h. We recorded from RPeD1 in semi-intact preparations made from snails 24 h after a single 0.5 h training session in EpiC or pond water (PW) and found that the firing and bursting rate of RPeD1 decreased significantly in the EpiC preparations compared to the PW preparations. However, the excitability (i.e., number of spikes evoked by injected depolarizing current) of RPeD1 was not different between the two preparations. We next performed "in vitro" operant training in semi-intact preparations made from naïve snails. In the training, we applied a gentle tactile stimulus to the pneumostome area every time the semi-intact preparation began to open. The preparations exposed to EpiC-saline (15 mg/l) exhibited significantly increased RPeD1 excitability compared with saline only preparations. These results suggest that EpiC can alter some electrophysiological properties of a neuron that is a necessary site for learning and memory formation.

Embryonic exposure to selenium nanoparticles delays growth and hatching in the freshwater snail Lymnaea stagnalis.

Selenium nanoparticles (SeNPs) have been applied in the biomedical and biocidal domain which may have potential environmental risks for aquatic systems. However, the knowledge of its toxicity and the role of functionalization on aquatic invertebrates are scarce. Thus, the present study aimed to analyze the embryotoxicity of two types of SeNPs coated with Sodium carboxymethyl cellulose (CMC-SeNPs) and Chitosan (CS-SeNPs) to the freshwater snail Lymnaea stagnalis in lake water, focusing on embryonic development. The influence of surface coatings and ions release, on the embryonic development of SeNPs to freshwater snail L. stagnalis was investigated. For this end, the snails were exposed to different concentrations of SeNPs and Se ions (0.05-1 mg L-1) during 7 days and multiple endpoints were analyzed, including developmental stage frequency, morphological alterations, embryos mortality and hatching success. The results showed that both Se forms promoted the developmental delay, mortality, morphological changes, and hatching inhibition in snail embryos in a concentration-dependent manner. CMC-SeNPs are 2.6 times more embryotoxic compared to CS-SeNPs indicating the importance of surface coating on the embryotoxicity. Moreover, the results revealed that although both forms of Se inhibited the embryo development and reduced the hatching of L. stagnalis, the mode of action on the embryogenesis was different. SeNPs had a higher toxicity to snails' embryos compared to their dissolved counterparts. Despite significant dissolution, by comparing the SeNPs with their dissolved fraction, the results suggest SeNPs inhibition effect on the snail development could be caused by both SeNPs and Se4+, and SeNPs might be the major development retardation driver rather than Se ions. The present study evidenced by the first time the toxicity effects of SeNPs on the snail embryogenesis, and highlighted how SeNPs intrinsic properties influence their transformation and toxicity in environmental relevant scenarios.

Thinking "outside the box": The effect of nontarget snails in the aquatic community on mollusc-borne diseases.

There is a great need to understand the impact of complex communities on the free-living parasite stages that are part of them. This task becomes more complex as nonnative species emerge, changing existing relationships and shaping new interactions in the community. A relevant question would be: Can the coexistence of nontarget snails with the target hosts contribute to trematodasis control? We used field and experimental approaches to investigate nonnative competitor-induced parasite dilution. During a three-year field study, we investigated digenean infection in Lymnaea stagnalis from eight Polish lakes inhabited or uninhabited by Potamopyrgus antipodarum. Additionally, we verified the presence of digenean infections in the populations of P. antipodarum. Moreover, we conducted an experimental infection of L. stagnalis with miracidia of Trichobilharzia szidati under increasing densities of P. antipodarum and aimed to infect P. antipodarum with them separately. The prevalence of avian schistosomes in lymnaeid snails was significantly higher in uninhabited lakes than in lakes inhabited by P. antipodarum. Our study indicates that waters with a higher density of invaders have a lower prevalence of avian schistosomes in lymnaeid hosts. The results of experimental studies confirmed that the presence of high densities of P. antipodarum reduces the probability of target host infection. Both field and experimental studies rule out the role of P. antipodarum as a source of avian schistosome cercariae. Here, a nonnative species was tested as a diluter, which in practice may be harmful to the local environment. This work is not a call for the introduction of nonnative species; it is intended to be a stimulus for researchers to continue searching for natural enemies of parasites because, as our results show, they exist. Finding natural enemies to the most dangerous species of human and animal parasites that will pose no threat to the local environment could be groundbreaking.

Fluoride affects memory by altering the transcriptional activity in the central nervous system of Lymnaea stagnalis.

Fluoride (F-), has been found to affect learning and memory in several species. In this study, we exposed an F--naïve, inbred strain of Lymnaea stagnalis to a concentration of F- similar to that naturally occurring in wild ponds. We found that the exposure to F- before the configural learning procedure obstructs the memory formation and blocks the configural learning-induced upregulation of CREB1, GRIN1, and HSP70 in snails' central ring ganglia. Along with altering the mRNA levels of these key genes for memory formation, a single acute F- exposure also upregulates Cytochrome c Oxidase, a major regulatory enzyme of the electron transport chain, which plays direct or indirect roles in reactive oxygen species production. As the central nervous system is sensitive to oxidative stress and consistent with previous studies from mammals, our results suggest a potential role of oxidative stress in memory impairment. To our knowledge, this is the first study investigating the neuronal mechanism of memory impairment in an invertebrate species that is exposed to natural F- levels.

Metabolism of the aquatic pollutant diclofenac in the Lymnaea stagnalis freshwater gastropod.

The metabolism of organic contaminants in Lymnaea stagnalis freshwater gastropod remains unknown. Yet, pharmaceuticals-like the NSAID diclofenac-are continuously released in the aquatic environment, thereby representing a risk to aquatic organisms. In addition, lower invertebrates may be affected by this pollution since they are likely to bioaccumulate contaminants. The metabolism of pharmaceuticals in L. stagnalis requires further investigation to understand their detoxification mechanisms and characterized the risk posed by contaminant exposure in this species. In this study, a non-targeted strategy using liquid chromatography combined with high-resolution mass spectrometry was applied to highlight metabolites formed in L. stagnalis freshwater snails exposed to 300 µg/L diclofenac for 3 and 7 days. Nineteen metabolites were revealed by this approach, 12 of which were observed for the first time in an aquatic organism exposed to diclofenac. Phase I metabolism involved hydroxylation, with detection of 3'-, 4'-, and 5-hydroxydiclofenac and three dihydroxylated metabolites, as well as cyclization, oxidative decarboxylation, and dehydrogenation, while phase II metabolism consisted of glucose and sulfate conjugation. Among these reactions, the two main DCF detoxification pathways detected in L. stagnalis were hydroxylation (phase I) and glucosidation (phase II).

Consequences of artificial light at night on behavior, reproduction, and development of Lymnaea stagnalis.

Light is an important zeitgeber that regulates many behavioral and physiological processes in animals. These processes may become disturbed due to the changes in natural patterns of light and dark via the introduction of artificial light at night (ALAN). The present study was designed to determine the effect of possible consequences of ALAN on reproduction, hatching success, developmental success, growth rate, feeding rate, mortality rate, and locomotor activity of the simultaneous hermaphrodite pond snail Lymnaea stagnalis. Snails were exposed to different light intensities at night that simulate actual ALAN measurements from the snail's night environment. The data revealed that exposure to ALAN at a low level significantly affected the cumulative number of laid eggs. At the same time, snails exposed to ALAN laid smaller eggs than those laid under normal light-dark cycles. Additionally, high light-intensity of ALAN delayed development and hatching of eggs of L. stagnalis while it showed no effect on hatching percentage. Furthermore, ALAN increased both the feeding and growth rates but did not lead to mortality. The results also show that snails exposed to dark conditions at night travel longer distances and do so faster than those exposed to ALAN. In light of these findings, it is clear that ALAN may have an influence on snails and their abundance in an environment, possibly disturbing ecological stability.

Learning to anticipate mate presence shapes individual sex roles in the hermaphroditic pond snail, Lymnaea stagnalis.

Despite being simultaneously male and female, hermaphrodites may still need to assume the male or female sexual role in a mating encounter, with the option to swap roles afterwards. For the great pond snail, Lymnaea stagnalis, deciding which sexual role to perform has important consequences, since sperm transfer and male reproductive success can be decreased. We hypothesised that detecting cues that indicate a possible mating encounter could help them to adapt their mating behaviour. Therefore, we experimentally assessed whether signalling the presence of a conspecific with an odour can affect the sexual role of Lymnaea stagnalis. The results showed that learning resulted in either an increased ability to mate as a male or in faster mating compared to the control group. These findings reveal that learning shapes the mating dynamics of Lymnaea stagnalis, thus showing that cognitive processes not only affect mating in separate-sexed species but also in hermaphrodites.

A combined bioinformatics and LC-MS-based approach for the development and benchmarking of a comprehensive database of Lymnaea CNS proteins.

Applications of key technologies in biomedical research, such as qRT-PCR or LC-MS-based proteomics, are generating large biological (-omics) datasets which are useful for the identification and quantification of biomarkers in any research area of interest. Genome, transcriptome and proteome databases are already available for a number of model organisms including vertebrates and invertebrates. However, there is insufficient information available for protein sequences of certain invertebrates, such as the great pond snail Lymnaea stagnalis, a model organism that has been used highly successfully in elucidating evolutionarily conserved mechanisms of memory function and dysfunction. Here, we used a bioinformatics approach to designing and benchmarking a comprehensive central nervous system (CNS) proteomics database (LymCNS-PDB) for the identification of proteins from the CNS of Lymnaea by LC-MS-based proteomics. LymCNS-PDB was created by using the Trinity TransDecoder bioinformatics tool to translate amino acid sequences from mRNA transcript assemblies obtained from a published Lymnaea transcriptomics database. The blast-style MMSeq2 software was used to match all translated sequences to UniProtKB sequences for molluscan proteins, including those from Lymnaea and other molluscs. LymCNS-PDB contains 9628 identified matched proteins that were benchmarked by performing LC-MS-based proteomics analysis with proteins isolated from the Lymnaea CNS. MS/MS analysis using the LymCNS-PDB database led to the identification of 3810 proteins. Only 982 proteins were identified by using a non-specific molluscan database. LymCNS-PDB provides a valuable tool that will enable us to perform quantitative proteomics analysis of protein interactomes involved in several CNS functions in Lymnaea, including learning and memory and age-related memory decline.