Behavioral and transcriptional effects of carnosine in the central ring ganglia of the pond snail Lymnaea stagnalis.

Carnosine is a naturally occurring endogenous dipeptide with well-recognized anti-inflammatory, antioxidant, and neuroprotective effects at the central nervous system level. To date, very few studies have been focused on the ability of carnosine to rescue and/or enhance memory. Here, we used a well-known invertebrate model system, the pond snail Lymnaea stagnalis, and a well-studied associative learning procedure, operant conditioning of aerial respiration, to investigate the ability of carnosine to enhance long-term memory (LTM) formation and reverse memory obstruction caused by an immune challenge (i.e., lipopolysaccharide [LPS] injection). Exposing snails to 1 mM carnosine for 1 h before training in addition to enhancing memory formation resulted in a significant upregulation of the expression levels of key neuroplasticity genes (i.e., glutamate ionotropic receptor N-methyl-d-aspartate [NMDA]-type subunit 1-LymGRIN1, and the transcription factor cAMP-response element-binding protein 1-LymCREB1) in snails' central ring ganglia. Moreover, pre-exposure to 1 mM carnosine before an LPS injection reversed the memory deficit brought about by inflammation, by preventing the upregulation of key targets for immune and stress response (i.e., Toll-like receptor 4-LymTLR4, molluscan defense molecule-LymMDM, heat shock protein 70-LymHSP70). Our data are thus consistent with the hypothesis that carnosine can have positive benefits on cognitive ability and be able to reverse memory aversive states induced by neuroinflammation.

Sensory input from osphradium is involved in fluoride detection that alters feeding and memory phenotype in Lymnaea stagnalis.

Fluoride (F-) exposure in organisms remains a significant concern due to its widespread presence and potential health implications. Investigating its detection and subsequent effects on behaviour in aquatic organisms like Lymnaea stagnalis provides valuable insights. Our study focused on elucidating the sensory pathways involved in F- detection and its impact on feeding and memory formation. We explored two potential detection mechanisms: direct flow across the integument onto neurons; and sensory input to the central nervous system (CNS) via the osphradium-osphradial ganglion-osphradial nerve pathway (snails use this system for olfactory sensation of multiple compounds). Injection of F- into snails did not alter feeding behaviour or central neuronal activity, suggesting that internalization might not be the primary detection mode. In contrast, severing the osphradial nerve abolished F-'s suppressive effects on feeding and memory formation, implicating the osphradial pathway in F- sensing and behavioural changes. This finding supports the idea that osphradial nerve signaling mediates the behavioural effects of F-. Our study underscores the importance of sensory pathways in F- detection and behavioural modulation in L. stagnalis. Understanding these mechanisms could provide critical insights into how organisms respond to and adapt to environmental chemical stressors like F-.

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.

Dev-ResNet: automated developmental event detection using deep learning.

Delineating developmental events is central to experimental research using early life stages, permitting widespread identification of changes in event timing between species and environments. Yet, identifying developmental events is incredibly challenging, limiting the scale, reproducibility and throughput of using early life stages in experimental biology. We introduce Dev-ResNet, a small and efficient 3D convolutional neural network capable of detecting developmental events characterised by both spatial and temporal features, such as the onset of cardiac function and radula activity. We demonstrate the efficacy of Dev-ResNet using 10 diverse functional events throughout the embryonic development of the great pond snail, Lymnaea stagnalis. Dev-ResNet was highly effective in detecting the onset of all events, including the identification of thermally induced decoupling of event timings. Dev-ResNet has broad applicability given the ubiquity of bioimaging in developmental biology, and the transferability of deep learning, and so we provide comprehensive scripts and documentation for applying Dev-ResNet to different biological systems.

Antibiotic-altered gut microbiota explain host memory plasticity and disrupt pace-of-life covariation for an aquatic snail.

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.

Genotoxicological and physiological effects of glyphosate and its metabolite, aminomethylphosphonic acid, on the freshwater invertebrate Lymnaea stagnalis.

Aminomethylphosphonic acid (AMPA) is the main metabolite in the degradation of glyphosate, a broad-spectrum herbicide, and it is more toxic and persistent in the environment than the glyphosate itself. Owing to their extensive use, both chemicals pose a serious risk to aquatic ecosystems. Here, we explored the genotoxicological and physiological effects of glyphosate, AMPA, and the mixed solution in the proportion 1:1 in Lymnaea stagnalis, a freshwater gastropod snail. To do this, adult individuals were exposed to increasing nominal concentrations (0.0125, 0.025, 0.050, 0.100, 0.250, 0.500 µg/mL) in all three treatments once a week for four weeks. The genotoxicological effects were estimated as genomic damage, as defined by the number of micronuclei and nuclear buds observed in hemocytes, while the physiological effects were estimated as the effects on somatic growth and egg production. Exposure to glyphosate, AMPA, and the mixed solution caused genomic damage, as measured in increased frequency of micronuclei and nuclear buds and in adverse effects on somatic growth and egg production. Our findings suggest the need for more research into the harmful and synergistic effects of glyphosate and AMPA and of pesticides and their metabolites in general.

Retinoic acid reduces the formation of, and acutely modulates, invertebrate electrical synapses.

Communication between cells in the nervous system is dependent on both chemical and electrical synapses. Factors that can affect chemical synapses have been well studied, but less is known about factors that influence electrical synapses. Retinoic acid, the vitamin A metabolite, is a known regulator of chemical synapses, but few studies have examined its capacity to regulate electrical synapses. In this study, we determine that retinoic acid is capable of rapidly altering the strength of electrical synapses in an isomer- and cell-dependent manner. Furthermore, we provide evidence that this acute effect might be independent of either the retinoid receptors or the activation of a protein kinase. In addition to the rapid modulatory effects of retinoic acid, we provide data to suggest that retinoic acid is also capable of regulating the formation of electrical synapses. Long-term exposure to both all-trans-retinoic acid or 9-cis-retinoic acid reduced the proportion of cell pairs forming electrical synapses, as well as reduced the strength of electrical synapses that did form. In summary, this study provides insights into the role that retinoids might play in both the formation and modulation of electrical synapses in the central nervous system.NEW & NOTEWORTHY Retinoids are known modulators of chemical synapses and mediate synaptic plasticity in the nervous system, but little is known of their effects on electrical synapses. Here, we show that retinoids selectively reduce electrical synapses in a cell- and isomer-dependent manner. This modulatory action on existing electrical synapses was rapid and nongenomic in nature. We also showed for the first time that longer retinoid exposures inhibit the formation of electrical synapses.

A translational and multidisciplinary approach to studying the Garcia effect, a higher form of learning with deep evolutionary roots.

Animals, including humans, learn and remember to avoid a novel food when its ingestion is followed, hours later, by sickness - a phenomenon initially identified during World War II as a potential means of pest control. In the 1960s, John Garcia (for whom the effect is now named) demonstrated that this form of conditioned taste aversion had broader implications, showing that it is a rapid but long-lasting taste-specific food aversion with a fundamental role in the evolution of behaviour. From the mid-1970s onward, the principles of the Garcia effect were translated to humans, showing its role in different clinical conditions (e.g. side-effects linked to chemotherapy). However, in the last two decades, the number of studies on the Garcia effect has undergone a considerable decline. Since its discovery in rodents, this form of learning was thought to be exclusive to mammals; however, we recently provided the first demonstration that a Garcia effect can be formed in an invertebrate model organism, the pond snail Lymnaea stagnalis. Thus, in this Commentary, after reviewing the experiments that led to the first characterization of the Garcia effect in rodents, we describe the recent evidence for the Garcia effect in L. stagnalis, which may pave the way for future studies in other invertebrates and mammals. This article aims to inspire future translational and ecological studies that characterize the conserved mechanisms underlying this form of learning with deep evolutionary roots, which can be used to address a range of different biological questions.

Efficient Expression in Leishmania tarentolae (LEXSY) of the Receptor-Binding Domain of the SARS-CoV-2 S-Protein and the Acetylcholine-Binding Protein from Lymnaea stagnalis.

Leishmania tarentolae (LEXSY) system is an inexpensive and effective expression approach for various research and medical purposes. The stated advantages of this system are the possibility of obtaining the soluble product in the cytoplasm, a high probability of correct protein folding with a full range of post-translational modifications (including uniform glycosylation), and the possibility of expressing multi-subunit proteins. In this paper, a LEXSY expression system has been employed for obtaining the receptor binding domain (RBD) of the spike-protein of the SARS-CoV-2 virus and the homopentameric acetylcholine-binding protein (AChBP) from Lymnaea stagnalis. RBD is actively used to obtain antibodies against the virus and in various scientific studies on the molecular mechanisms of the interaction of the virus with host cell targets. AChBP represents an excellent structural model of the ligand-binding extracellular domain of all subtypes of nicotinic acetylcholine receptors (nAChRs). Both products were obtained in a soluble glycosylated form, and their structural and functional characteristics were compared with those previously described.

Laboratory Assessment of Molluscicidal Activities of Cannabis sativa, Acacia nilotica, and Tinospora cordifolia Against Snail Host of Fasciola spp.

Background: The potential molluscicidal extracts, obtained from indigenous plants Cannabis sativa, Acacia nilotica, and Tinospora cordifolia, were tested for toxicity against freshwater pulmonate snail Lymnaea acuminata, an intermediate host of Fasciola hepatica. The organic extracts had a significant effect on young snails. Materials and Methods: All organic extracts and column-purified fractions gave median lethal concentrations (19-100.05 mg/L; 24 h) that fell well within the threshold level of 100 mg/L, set for a potential molluscicide by the World Health Organization. Results: The toxicity of T. cordifolia stem acetone extract (96 h LC50: 16.08 mg/L) was more pronounced compared with C. sativa leaf ethanol extract (96 h LC50: 16.32 mg/L) and A. nilotica leaf ethanol extract (96 h LC50: 24.78 mg/L). ß-caryophyllene, gallic acid, and berberine were characterized and identified as active molluscicidal components. Co-migration of ß-caryophyllene (retardation factor [Rf] 0.95), gallic acid (Rf 0.30), and berberine (Rf 0.23) with column-purified parts of Cannabis sativa, Acacia nilotica, and Tinospora cordifolia on thin-layer chromatography demonstrates same Rf value, that is, 0.95, 0.30, and 0.23, respectively. Conclusion: This study indicates that these extracts thus represent potential plant-derived molluscicides that are worthy of further investigations.

Induced responses contribute to rapid adaptation of Spirodela polyrhiza to herbivory by Lymnaea stagnalis.

Herbivory-induced responses in plants are typical examples of phenotypic plasticity, and their evolution is thought to be driven by herbivory. However, direct evidence of the role of induced responses in plant adaptive evolution to herbivores is scarce. Here, we experimentally evolve populations of an aquatic plant (Spirodela polyrhiza, giant duckweed) and its native herbivore (Lymnaea stagnalis, freshwater snail), testing whether herbivory drives rapid adaptive evolution in plant populations using a combination of bioassays, pool-sequencing, metabolite analyses, and amplicon metagenomics. We show that snail herbivory drove rapid phenotypic changes, increased herbivory resistance, and altered genotype frequencies in the plant populations. Additional bioassays suggest that evolutionary changes of induced responses contributed to the rapid increase of plant resistance to herbivory. This study provides direct evidence that herbivory-induced responses in plants can be subjected to selection and have an adaptive role by increasing resistance to herbivores.

Understanding the differential impacts of two antidepressants on locomotion of freshwater snails (Lymnaea stagnalis).

There is growing evidence of negative impacts of antidepressants on behavior of aquatic non-target organisms. Accurate environmental risk assessment requires an understanding of whether antidepressants with similar modes of action have consistent negative impacts. Here, we tested the effect of acute exposure to two antidepressants, fluoxetine and venlafaxine (0-50 µg/L), on the behavior of non-target organism, i.e., freshwater pond snail, Lymnaea stagnalis. As compounds interact with chemical cues in the aquatic ecosystems, we also tested whether the effects altered in the presence of bile extract containing 5α-cyprinol sulfate (5α-CPS), a characterized kairomone of a natural predator, common carp (Cyprinus carpio). Behavior was studied using automated tracking and analysis of various locomotion parameters of L. stagnalis. Our results suggest that there are differences in the effects on locomotion upon exposure to venlafaxine and fluoxetine. We found strong evidence for a non-monotonic dose response on venlafaxine exposure, whereas fluoxetine only showed weak evidence of altered locomotion for a specific concentration. Combined exposure to compounds and 5α-CPS reduced the intensity of effects observed in the absence of 5α-CPS, possibly due to reduced bioavailability of the compounds. The results highlight the need for acknowledging different mechanisms of action among antidepressants while investigating their environmental risks. In addition, our results underline the importance of reporting non-significant effects and acknowledging individual variation in behavior for environmental risk assessment.

Five-minute exposure to a novel appetitive food substance is sufficient time for a microRNA-dependent long-term memory to form.

The Garcia effect is a unique form of conditioned taste aversion which requires that a novel food stimulus be followed sometime later by a sickness state associated with the novel food stimulus. The long-lasting associative memory resulting from the Garcia effect ensures that organisms avoid toxic foods in their environment. Considering its ecological relevance, we sought to investigate whether a brief encounter (5 min) with a novel, appetitive food stimulus can cause a persisting long-term memory (LTM) to form that would in turn block the Garcia effect in Lymnaea stagnalis. Furthermore, we wanted to explore whether that persisting LTM could be modified by the alteration of microRNAs via an injection of poly-L-lysine (PLL), an inhibitor of Dicer-mediated microRNA biogenesis. The Garcia effect procedure involved two observations of feeding behavior in carrot separated by a heat stress (30 °C for 1 h). Exposing snails to carrot for 5 min caused a LTM to form and persist for 1 week, effectively preventing the Garcia effect in snails. In contrast, PLL injection following the 5-min carrot exposure impaired LTM formation, allowing the Garcia effect to occur. These results provide more insight into LTM formation and the Garcia effect, an important survival mechanism.

Snails go on a fast when acetylsalicylic acid comes along with heat stress: A possible effect of HSPs and serotonergic system on the feeding response.

A novel food followed by sickness, causes a taste-specific conditioned aversion, known as the 'Garcia effect'. We recently found that both a heat shock stressor (30 °C for 1 h - HS) and the bacterial lipopolysaccharide (LPS) can be used as 'sickness-inducing' stimuli to induce a Garcia effect in the pond snail Lymnaea stagnalis. Additionally, if snails are exposed to acetylsalicylic acid (ASA) present in aspirin tablets before the LPS injection, the formation of the Garcia effect is prevented. Here, we hypothesized that exposing snails to crushed aspirin before the HS (ASA-HS) would prevent the HS-induced 'sickness state' and - therefore -the Garcia effect. Unexpectantly, the ASA-HS procedure induced a generalized and long-lasting feeding suppression. We thus investigate the molecular effects underlying this phenomenon. While the exposure to the HS alone resulted in a significant upregulation of the mRNA levels of the Heat Shock Protein 70 (HSP 70) in snails' central ring ganglia, the ASA-HS procedure induced an even greater upregulation of HSP70, suggesting that the ASA-HS combination causes a severe stress response that inhibits feeding. Additionally, we found that the ASA-HS procedure induced a significant downregulation of the mRNA levels of genes involved with the serotoninergic system which regulates feeding in snails. Finally, the ASA-HS procedure prevented HS-induced upregulation of the mRNA levels of key neuroplasticity genes. Our study indicates that two sickness-inducing stimuli can have different physiological responses even if behavioral outcomes are similar under some learning contexts.

Investigating the interactions between multiple memory stores in the pond snail Lymnaea stagnalis.

The pond snail Lymnaea stagnalis exhibits various forms of associative learning including (1) operant conditioning of aerial respiration where snails are trained not to open their pneumostome in a hypoxic pond water environment using a weak tactile stimulus to their pneumostome as they attempt to open it; and (2) a 24 h-lasting taste-specific learned avoidance known as the Garcia effect utilizing a lipopolysaccharide (LPS) injection just after snails eat a novel food substance (carrot). Typically, lab-inbred snails require two 0.5 h training sessions to form long-term memory (LTM) for operant conditioning of aerial respiration. However, some stressors (e.g., heat shock or predator scent) act as memory enhancers and thus a single 0.5 h training session is sufficient to enhance LTM formation lasting at least 24 h. Here, we found that snails forming a food-aversion LTM following Garcia-effect training exhibited enhanced LTM following operant condition of aerial respiration if trained in the presence of the food substance (carrot) they became averse to. Control experiments led us to conclude that carrot becomes a 'sickness' risk signal and acts as a stressor, sufficient to enhance LTM formation for another conditioning procedure.

Effects of acute exposure to environmentally realistic tebuconazole concentrations on stress responses of kidney and digestive gland of Lymnaea stagnalis.

This study aimed to investigate the effects of 24 and 72 h exposure to environmentally relevant concentrations of tebuconazole (TEB) (10, 100 and 500 µg/L) fungicide on the freshwater snail Lymnaea stagnalis. The focus was induction of oxidative stress, alteration of gene expressions and histopathological changes in the kidney and digestive gland. TEB treatment induced a time- and concentration-dependent increase in intracellular reactive oxygen species (ROS) and malondialdehyde (MDA) levels, while the total antioxidant capacity (TAC) was decreased. The activities of glutathione peroxidase (GPx), glutathione reductase (GR), and catalase (CAT) also increased in a time- and concentration-dependent manner in both tissues. TEB exposure significantly increased the mRNA levels of CAT, GPx, GR, heat shock proteins HSP40 and HSP70. Histological analysis revealed nephrocyte degeneration and disrupted digestive cells. The study concludes that acute exposure to TEB induces oxidative stress, alters antioxidant defense mechanisms, and leads to histopathological changes in L. stagnalis.

Better together: isolation impedes memory formation for configural learning in Lymnaea stagnalis.

Social interactions play an important role in learning and memory. There is great variability in the literature regarding the effects of social isolation on cognition. Here, we investigated how memory formation was affected when Lymnaea stagnalis, our model system, were socially isolated at three different time periods: before, during or after the configural learning training procedure. Each group of snails underwent configural learning where we recorded and compared their feeding behaviour before and after the pairing of an appetitive food stimulus with predator kairomones (i.e. the training procedure). We found that isolating snails before the training procedure had no effect on their learning and memory. However, when snails were isolated either during the training procedure or immediately after the training procedure, they no longer formed memory. These data provide further insight into how isolation impacts cognitive functioning in the context of higher-order learning.

Genes Upregulated by Operant Conditioning of Escape Behavior in the Pond Snail Lymnaea stagnalis.

The pond snail Lymnaea stagnalis is capable of learning by both classical conditioning and operant conditioning. Although operant conditioning related to escape behavior with punishment has been examined by some research groups, the molecular mechanisms are not known. In the present study, we examined changes in the expression levels of cAMP-response element binding protein 1 (CREB1), CREB2, CREB-binding protein (CBP), and monoamine oxidase (MAO) in the Lymnaea central nervous system (CNS) using real-time PCR following operant conditioning of escape behavior. CREB1 and CREB2 are transcription factors involved in long-term memory in Lymnaea; CBP is a coactivator with CREB1; and MAO is a degrading enzyme for monoamines (e.g., serotonin) with important roles in learning and memory in Lymnaea. In operant conditioning, the punishment cohort, in which snails escaping from the container encountered aversive KCl, exhibited significantly fewer escape attempts than the control cohort, in which snails escaping from the container encountered distilled water, during both the training and memory test periods. After the operant conditioning, CREB1 and CREB2 were upregulated, and the ratio of CREB1/CREB2 was also increased, suggesting that the operant conditioning of escape behavior involves these factors. MAO was also upregulated, suggesting that the content of monoamines such as serotonin in the CNS decreased. The upregulated genes identified in the present study will help to further elucidate learning and memory mechanisms in Lymnaea.

Expression Level Changes in Serotonin Transporter are Associated with Food Deprivation in the Pond Snail Lymnaea stagnalis.

In the pond snail Lymnaea stagnalis, serotonin (5-HT) plays an important role in feeding behavior and its associated learning (e.g., conditioned taste aversion: CTA). The 5-HT content in the central nervous system (CNS) fluctuates with changes in the nutritional status, but it is also expected to be influenced by changes in the serotonin transporter (SERT) expression level. In the present study, we identified SERT in Lymnaea and observed its localization in 5-HTergic neurons, including the cerebral giant cells (CGCs) in the cerebral ganglia and the pedal A cluster neurons and right and left pedal dorsal 1 neurons in the pedal ganglia by in situ hybridization. Real-time PCR revealed that the SERT mRNA expression level was lower under severe food deprivation than under mild food deprivation in the whole CNS as well as in a single CGC. These results inversely correlated with previous data that the 5-HT content in the CNS was higher in the severely food-deprived state than in the mildly food-deprived state. Furthermore, in single CGCs, we observed that the 5-HT level was significantly increased in the severely food-deprived state compared with the mildly food-deprived state. Our present findings suggest that changes in the SERT expression level associated with food deprivation may affect 5-HT signaling, probably contributing to learning and memory mechanisms in Lymnaea.

Left-Right Reversal Recurrently Evolved Regardless of Diaphanous-Related Formin Gene Duplication or Loss in Snails.

Bilateria exhibit whole-body handedness in internal structure. This left-right polarity is evolutionarily conserved with virtually no reversed extant lineage, except in molluscan Gastropoda. Phylogenetically independent snail groups contain both clockwise-coiled (dextral) and counterclockwise-coiled (sinistral) taxa that are reversed from each other in bilateral handedness as well as in coiling direction. Within freshwater Hygrophila, Lymnaea with derived dextrality have diaphanous related formin (diaph) gene duplicates, while basal sinistral groups possess one diaph gene. In terrestrial Stylommatophora, dextral Bradybaena also have diaph duplicates. Defective maternal expression of one of those duplicates gives rise to sinistral hatchlings in Lymnaea and handedness-mixed broods in Bradybaena, through polarity change in spiral cleavage of embryos. These findings led to the hypothesis that diaph duplication was crucial for the evolution of dextrality by reversal. The present study discovered that diaph duplication independently occurred four times and its duplicate became lost twice in gastropods. The dextrality of Bradybaena represents the ancestral handedness conserved across gastropods, unlike the derived dextrality of Lymnaea. Sinistral lineages recurrently evolved by reversal regardless of whether diaph had been duplicated. Amongst the seven formin gene subfamilies, diaph has most thoroughly been conserved across eukaryotes of the 14 metazoan phyla and choanoflagellate. Severe embryonic mortalities resulting from insufficient expression of the duplicate in both of Bradybaena and Lymnaea also support that diaph duplicates bare general roles for cytoskeletal dynamics other than controlling spiralian handedness. Our study rules out the possibility that diaph duplication or loss played a primary role for reversal evolution.