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.

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.

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.

A Novel Behavioral Display in Lymnaea Induced by Quercetin and Hypoxia.

AbstractThe pond snail Lymnaea stagnalis employs aerial respiration under hypoxia and can be operantly conditioned to reduce this behavior. When applied individually, a heat shock (30 °C for 1 h) and the flavonoid quercetin enhance long-term memory formation for the operant conditioning of aerial respiration. However, when snails are exposed to quercetin before the heat shock, long-term memory is no longer enhanced. This is because quercetin prevents the heat-induced upregulation of heat-shock proteins 70 and 40. When we tested the memory outcome of operant conditioning due to the simultaneous exposure to quercetin and 30 °C, we found that Lymnaea entered a quiescent survival state. The same behavioral response occurred when snails were simultaneously exposed to quercetin and pond water made hypoxic by bubbling nitrogen through it. Thus, in this study, we performed six experiments to propose a physiological explanation for that curious behavioral response. Our results suggest that bubbling nitrogen in pond water, heating pond water to 30 °C, and bubbling nitrogen in 30 °C pond water create a hypoxic environment, to which organisms may respond by upregulating the heat-shock protein system. On the other hand, when snails experience quercetin together with these hypoxic conditions, they can no longer express the physiological stress response evoked by heat or hypoxia. Thus, the quiescent survival state could be an emergency response to survive the hypoxic condition when the heat-shock proteins cannot be activated.

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.

Diversity of trematode cercariae among naturally infected lymnaeid snails from Phayao, Thailand.

Lymnaeids are aquatic snails playing an important role in the transmission of many parasitic trematode species of veterinary and medical importance. In this study, we assessed the presence of cercarial flukes in naturally infected lymnaeid snails from Phayao province, Thailand, and determined the species diversity of both the intermediate snail hosts and parasite larvae. A total of 3,185 lymnaeid snails were collected from paddy fields at 31 sites in eight districts of Phayao province between October 2021 and December 2022. Larval fluke infection was assessed using the cercarial shedding method. The collected snails as well as emerging cercariae were identified at the species level via morphological and molecular methods. The sequences of snail internal transcribed spacer region 2 (ITS2) and cercarial 28S ribosomal RNA gene (28S rDNA) and cytochrome C oxidase1 (Cox1) were determined by PCR amplification and sequencing. Three species of lymnaeid snails were detected in this study, including Radix (Lymnaea) rubiginosa (Michelin, 1831), Radix (Lymnaea) swinhoei (Adams, 1866) and Austropeplea viridis (Quoy & Gaimard, 1832), of which R. rubiginosa was the most abundant, followed by A. viridis and R. swinhoei. The overall rate of trematode cercarial infection in the lymnaeid snails was 2.8% (90/3,185); the cercarial infection rate in R. rubiginosa and A. viridis was 3.5% (60/1,735) and 3.1% (30/981), respectively. No larval fluke infection was observed in the studied R. swinhoei (0/469). Nine morphotypes of cercariae were detected at 15 sites from four districts. The emerging cercariae were molecularly identified as Clinostomum sp., Aporocotylidae sp., Apharyngostrigea sp., Trichobilharzia sp., Apatemon sp., Pegosomum sp., Petasiger sp., Echinostoma revolutum and Plagiorchis sp. These findings emphasize the occurrence and diversity of trematode cercariae among naturally infected lymnaeid snails in Phayao province and could contribute to broadening our understanding of the host-parasite relationships between trematodes and their first intermediate hosts as well as developing effective interventions to control trematode parasitic diseases.

Agricultural Use of Insecticides Alters Homeostatic Behaviors and Cognitive Ability in Lymnaea stagnalis.

Lymnaea stagnalis is an ecologically important, stress-sensitive, freshwater mollusk that is at risk for exposure to insecticides via agricultural practices. We provide insight into the impact insecticides have on L. stagnalis by comparing specific behaviors including feeding, locomotion, shell regeneration, and cognition between snails collected at two different sites: one contaminated by insecticides and one not. We hypothesized that each of the behaviors would be altered in the insecticide-exposed snails and that similar alterations would be induced when control snails were exposed to the contaminated environment. We found no significant differences in locomotion, feeding, and shell regeneration of insecticide-exposed L. stagnalis compared with nonexposed individuals. Significant changes in feeding and shell repair were observed in nonexposed snails inhabiting insecticide-contaminated pond water. Most importantly, snails maintained and trained in insecticide-contaminated pond water did not form configural learning, but this cognitive deficit was reversed when these snails were maintained in insecticide-free pond water. Our findings conclude that insecticides have a primarily negative impact on this higher form of cognition in L. stagnalis. Environ Toxicol Chem 2023;42:2466-2477. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Unique morphology and photoperiodically regulated activity of neurosecretory canopy cells in the pond snail Lymnaea stagnalis.

The pond snail Lymnaea stagnalis exhibits clear photoperiodism in egg laying; it lays more eggs in long-day conditions than in medium-day conditions. A key regulator of egg laying is neurosecretory caudo-dorsal cells (CDCs) producing an ovulation hormone in the cerebral ganglia. Paired small budding structures of the cerebral ganglia (viz. the lateral lobe) also promote egg laying in addition to spermatogenesis and maturation of female accessory sex organs. However, it remains unknown which cells in the lateral lobe are responsible for these. Previous anatomical and physiological studies prompted us to hypothesize that canopy cells in the lateral lobe modulate activity of CDCs. However, double labeling of the canopy cell and CDCs revealed no sign of direct neural connections, suggesting that activity of CDCs is regulated either humorally or through a neural pathway independent of canopy cells. In addition, our detailed anatomical re-evaluation confirmed previous observations that the canopy cell bears fine neurites along the ipsilateral axon and extensions from the plasma membrane of the cell body, although the function of these extensions remains unexplored. Furthermore, comparison of electrophysiological properties between long-day and medium-day conditions indicated that the canopy cell's activity is moderately under photoperiodic regulation: resting membrane potentials of long-day snails are shallower than those of medium-day snails, and spontaneously spiking neurons are only observed in long-day conditions. Thus, canopy cells appear to receive photoperiodic information and regulate photoperiod-dependent phenomena, but not provide direct neural inputs to CDCs.

Temporal modelling of Lymnaea natalensis (Krauss, 1848) in tropical aquatic habitats.

Lymnaea natalensis is the only snail intermediate host of Fasciola gigantica, the causative agent of fascioliasis, in Nigeria. The species also serves as intermediate host for many other African trematode species of medical and veterinary importance, and it is found throughout the country. However, there is no detailed information on the factors that influence its distribution and seasonal abundance in the tropical aquatic habitats in Nigeria. This study used the geographic information system and remotely sensed data to develop models for predicting the distribution of L. natalensis in South-Western Nigeria. Both land surface temperature (LST) and normalised difference vegetation index (NDVI) were extracted from Landsat satellite imagery; other variables (slope and elevation) were extracted from a digital elevation model (DEM) while rainfall data were retrieved from the European Meteorology Research Programme (EMRP). These environmental variables were integrated into a geographic information system (GIS) to predict suitable habitats of L. natalensis using exploratory regression. A total of 1410 L. natalensis snails were collected vis-à-vis 22 sampling sites. Built-up areas recorded more L. natalensis compared with farmlands. There was no significant difference in the abundance of snails with season (p  0.05). The regression models showed that rainfall, NDVI, and slope were predictors of L. natalensis distribution. The habitats suitable for L. natalensis were central areas, while areas to the north and south were not suitable for L. natalensis.Contribution: The predictive risk models of L. natalensis in the study will be useful in mapping other areas where the snail sampling could not be conducted.

Using a dynamic energy budget model to investigate the physiological mode of action of lead (Pb) to Lymnaea stagnalis.

Lymnaea stagnalis is a notably sensitive species for a variety of metals, including lead (Pb). However, the mechanism(s) of lead toxicity to L. stagnalis currently remain incompletely understood. Under dynamic energy budget (DEB) theory, different physiological modes of action (PMoAs) result in the emergence of distinct changes to the life histories of exposed organisms. This work aims to better understand the PMoA of lead toxicity to L. stagnalis by applying DEB modeling to previously published datasets. After calibration, the model was utilized to evaluate the relative likelihood of several PMoAs. Assuming decreased assimilation, the L. stagnalis DEB model was able to capture most, but not all, trends in experimentally observed endpoints, including growth, reproduction, and food ingestion. The weight-of-evidence suggests that decreased assimilation via a decrease in food ingestion is the most plausible PMoA for chronic lead toxicity in L. stagnalis. Collectively, our results illustrate how mechanistic modeling can create added value for conventional individual-level toxicity test data by enabling inferences about potential physiological mechanisms of toxicity.

Prey populations with different predation histories show differences in behavioral and transcriptional effects under acute predation threat.

Predator detection induces both behavioral and physiological responses in prey organisms. Our model organism, the pond snail Lymnaea stagnalis, shows multiple defensive behaviors in response to predator cues. In this study, we investigated and compared the transcriptional effects induced by the exposure to a predator scent (i.e., crayfish effluent - CE) in a strain of lab-inbred snails (i.e., W snails), which have been raised and maintained under standardized laboratory conditions for generations and a strain of freshly collected snails (i.e., Margo snails), which live in a crayfish-free pond. Neither the W- strain nor the Margo Lake snails used in this study have actually experienced crayfish. However, the W strain innately recognizes crayfish as a threat. We found that, following the exposure to CE, both strains showed significantly higher mRNA levels of serotonin-related genes. This is important, as the serotonergic system modulates predator detection and vigilance behaviors in pond snails. However, the expression levels of CREB1 and HSP70 were only upregulated in CE-exposed W snails but not in Margo ones. As CREB1 plays a key role in learning and memory formation, whereas HSP70 is involved in stress response, we investigated whether these differences in CREB1 and HSP70 mRNA levels would reflect differences in predator-induced learning (e.g., configural learning). We found that only W snails formed configural learning memory, whereas Margo snails did not. Thus, while both the strains molecularly respond to the CE by upregulating the serotoninergic system, only W snails behaviorally recognize CE as a threat and, therefore, form configural learning.