Snail Lymnaea Stagnalis Research Articles

Direct and transgenerational effects of an experimental heatwave on early life stages in a freshwater snail

Abstract Global climate change imposes a serious threat to natural populations of many species. Estimates of the effects of climate change‐mediated environmental stresses are, however, often based only on their direct effects on organisms, and neglect the potential transgenerational (e.g. maternal) effects. We tested whether high temperature (i.e. an experimental heatwave), which is known to reduce the performance of adult Lymnaea stagnalis snails, affects the produced offspring (eggs and hatchlings) through maternal effects, and how strong these effects are compared with the effects of direct exposure of offspring to high temperature. We examined the effect of maternal thermal environment (15°C versus 25°C) on per offspring investment (egg size), and the role of both maternal and offspring thermal environments (15°C versus 25°C) on hatching success and developmental time of eggs, offspring survival after hatching, and hatchling size at the age of 5 weeks. Exposure of mothers to high temperature reduced the size of oviposited eggs, increased their hatching success, and also made the onset of hatching earlier. However, high maternal temperature reduced the survival and the final size of hatched juveniles. Direct exposure of offspring to high temperature reduced their survival (both eggs and hatchlings) but increased the developmental rate and growth of those individuals that survived. Interestingly, the magnitude of maternal effects on hatching success of eggs and hatchling survival were similar to the direct effects of high temperature. Our results indicate that heatwaves can affect natural populations through transgenerational maternal effects and that the magnitude of those effects can be equally strong to the direct effects of temperature, although this depends on the trait considered. These findings highlight the importance of considering the transgenerational effects of climate warming when estimating its effects in the wild.

The development of CRISPR for a mollusc establishes the formin Lsdia1 as the long-sought gene for snail dextral/sinistral coiling.

The establishment of left-right body asymmetry is a key biological process that is tightly regulated genetically. In the first application of CRISPR/Cas9 to a mollusc, we show decisively that the actin-related diaphanous gene Lsdia1 is the single maternal gene that determines the shell coiling direction of the freshwater snail Lymnaea stagnalis Biallelic frameshift mutations of the gene produced sinistrally coiled offspring generation after generation, in the otherwise totally dextral genetic background. This is the gene sought for over a century. We also show that the gene sets the chirality at the one-cell stage, the earliest observed symmetry-breaking event linked directly to body handedness in the animal kingdom. The early intracellular chirality is superseded by the inter-cellular chirality during the 3rd cleavage, leading to asymmetric nodal and Pitx expression, and then to organismal body handedness. Thus, our findings have important implications for chiromorphogenesis in invertebrates as well as vertebrates, including humans, and for the evolution of snail chirality. This article has an associated 'The people behind the papers' interview.

Monoamines, Insulin and the Roles They Play in Associative Learning in Pond Snails.

Molluscan gastropods have long been used for studying the cellular and molecular mechanisms underlying learning and memory. One such gastropod, the pond snail Lymnaea stagnalis, exhibits long-term memory (LTM) following both classical and operant conditioning. Using Lymnaea, we have successfully elucidated cellular mechanisms of learning and memory utilizing an aversive classical conditioning procedure, conditioned taste aversion (CTA). Here, we present the behavioral changes following CTA training and show that the memory score depends on the duration of food deprivation. Then, we describe the relationship between the memory scores and the monoamine contents of the central nervous system (CNS). A comparison of learning capability in two different strains of Lymnaea, as well as the filial 1 (F1) cross from the two strains, presents how the memory scores are correlated in these populations with monoamine contents. Overall, when the memory scores are better, the monoamine contents of the CNS are lower. We also found that as the insulin content of the CNS decreases so does the monoamine contents which are correlated with higher memory scores. The present review deepens the relationship between monoamine and insulin contents with the memory score.

GSK3β controls the timing and pattern of the fifth spiral cleavage at the 2-4 cell stage in Lymnaea stagnalis.

Establishment of the body plan of multicellular organisms by the primary body axis determination and cell-fate specification is a key issue in biology. We have examined the mRNA localization of three Wnt pathway components gsk3β, β-catenin, and disheveled and investigated the effects of four selective inhibitors of these proteins on the early developmental stages of the spiral cleavage embryo of the fresh water snail Lymnaea (L.) stagnalis. mRNAs for gsk3β and β-catenin were distributed uniformly throughout the embryo during development whereas disheveled mRNA showed specific localization with intra- and inter-blastomere differences in concentration along the A-V axis during spiral cleavages. Remarkably, through inhibitor studies, we identified a short sensitive period from the 2- to 4-cell stage in which GSK3β inhibition by the highly specific 1-azakenpaullone (AZ) and by LiCl induced a subsequent dramatic developmental delay and alteration of the cleavage patterns of blastomeres at the fifth cleavage (16- to 24-cell stage) resulting in exogastrulation and other abnormalities in later stages. Inhibition of β-Catenin or Disheveled had no effect. Our inhibitor experiments establish a novel role for GSK3β in the developmental timing and orientated cell division of the snail embryo. Further work will be needed to identify the downstream targets of the kinase.

Aquatic omnivores shift their trophic position towards increased plant consumption as plant stoichiometry becomes more similar to their body stoichiometry.

Human induced eutrophication has strongly altered aquatic ecosystems. With increasing eutrophication, plant nutrient concentrations increase, making them more attractive as food for herbivores. However, most aquatic consumers are omnivorous. Ecological stoichiometry theory predicts that animals prefer to consume food which has a similar nutrient (N and P) composition or C:nutrient ratio compared to their own bodies, hence omnivorous animals may prefer to eat animal prey instead of plants. We asked whether aquatic omnivores would shift their diet towards more plant consumption when plants are more nutritious and their stoichiometry becomes more similar to the stoichiometry of the omnivore. We hypothesized that: (1) the omnivore increases plant consumption as the plant C:nutrient ratio decreases when there is only plant material available; (2) the omnivore generally prefers animal food over plant material; (3) the omnivore will increase its relative plant consumption as the plant C:nutrient ratio decreases, in the presence of animal food. As a model system, we used the pond snail Lymnaea stagnalis (omnivorous consumer), the aquatic plant Potamogeton lucens (plant food to the consumer, cultured at different nutrient regimes to obtain different plant C:nutrient ratios), and the crustacean Gammarus pulex (animal food to the consumer, using freshly dead individuals). When there was only plant material available, the consumers increased their relative consumption rate with decreasing plant C:nutrient ratio from no measurable amount to about 102 mg g-1 day-1. When plant material was offered simultaneously with animal food, even though the omnivores always preferred animal food over plant material, the omnivores still increased their relative intake of plant material as plant C:nutrient ratio decreased, from virtually nothing at the highest to on average 16% of their diet at the lowest plant C:nutrient ratio, with a maximum of 28%. Therefore, we conclude that as nutrient loading increases in aquatic ecosystems, plant-eating omnivorous animals may shift their trophic position towards increased plant consumption and alter the food web structure. As a result, we may observe increased top-down control on aquatic plants.

Relative importance of chemical attractiveness to parasites for susceptibility to trematode infection.

While the host immune system is often considered the most important physiological mechanism against parasites, precontact mechanisms determining exposure to parasites may also affect infection dynamics. For instance, chemical cues released by hosts can attract parasite transmission stages. We used the freshwater snail Lymnaea stagnalis and its trematode parasite Echinoparyphium aconiatum to examine the role of host chemical attractiveness, physiological condition, and immune function in determining its susceptibility to infection. We assessed host attractiveness through parasite chemo‐orientation behavior; physiological condition through host body size, food consumption, and respiration rate; and immune function through two immune parameters (phenoloxidase‐like and antibacterial activity of hemolymph) at an individual level. We found that, although snails showed high variation in chemical attractiveness to E. aconiatum cercariae, this did not determine their overall susceptibility to infection. This was because large body size increased attractiveness, but also increased metabolic activity that reduced overall susceptibility. High metabolic rate indicates fast physiological processes, including immune activity. The examined immune traits, however, showed no association with susceptibility to infection. Our results indicate that postcontact mechanisms were more likely to determine snail susceptibility to infection than variation in attractiveness to parasites. These may include localized immune responses in the target tissue of the parasite. The lack of a relationship between food consumption and attractiveness to parasites contradicts earlier findings that show food deprivation reducing snail attractiveness. This suggests that, although variation in resource level over space and time can alter infection dynamics, variation in chemical attractiveness may not contribute to parasite‐induced fitness variation within populations when individuals experience similar environmental conditions.

Using the Biotic Ligand Model framework to investigate binary metal interactions on the uptake of Ag, Cd, Cu, Ni, Pb and Zn in the freshwater snail Lymnaea stagnalis

There is growing interest in the development of mechanistically-based models, such as the Biotic Ligand Model (BLM), for assessing the environmental risk of metal mixtures. However, the derivation of such models requires insights into the mechanisms of multimetal interactions that are often lacking for aquatic organisms. In the present study, we investigated how binary mixtures of six metals (Ag, Cd, Cu, Ni, Pb and Zn) interact for uptake in the great pond snail Lymnaea stagnalis, a freshwater species particularly sensitive to metals in chronic exposure. For each metal, short-term (2–3 h) uptake experiments on juvenile snails were performed with the metal alone and in combination with a second metal, at concentrations encompassing the chronic toxicity concentration range. These experiments showed significant binary metal interactions for 7 out of 15 mixtures. Most interactions were inhibitory in nature, not reciprocal and caused by either Ag or Cu. They led to relative changes of uptake that did not exceed 50% within the range of metal chronic toxicity. The BLM proved to be successful at explaining most of the interactions, via competitive inhibition. This study is in support of using bioavailability-based models, such as the BLM, to model metal mixture interactions in L. stagnalis.

Can the inhibition of cytochrome P450 in aquatic invertebrates due to azole fungicides be estimated with in silico and in vitro models and extrapolated between species?

Azole fungicides, designed to halt fungal growth by specific inhibition of fungal cytochrome P450 (CYP51), inhibit cytochrome P450s involved in the metabolism of xenobiotics in several non-target organisms thus raising environmental concern. The present study investigates the degree by which inhibition strengths of azoles toward cytochrome P450 in rat liver, the insect Chironomus riparius larvae and the snail Lymnaea stagnalis can be extrapolated from estimated in silico affinities. Azoles’ affinities toward human cytochrome P450 isoforms involved in xenobiotic metabolism (CYP3A4, CYP2C9 and CYP2D6) as well as fungal CYP51 were estimated with a ligand-protein docking model based on the ChemScore scoring function. Estimated affinities toward the selected enzymatic structures correlated strongly with measured inhibition strengths in rat liver (ChemScore vs. logIC50 among cytochrome P450 isoforms: −0.662 < r < −0.891, n = 17 azoles), while weaker correlations were found for C. riparius larvae (−0.167 < r < −0.733, n = 9) and L. stagnalis (−0.084 < r < −0.648, n = 8). Inhibition strengths toward C. riparius and rat liver activities were found to be highly correlated to each other (r: 0.857) while no significant relationship was found between either of the species and L. stagnalis. The inhibition of cytochrome P450 due to azole fungicides could be estimated in vitro and to a lesser extent in silico for C. riparius but not for L. stagnalis, possibly due to different enzymatic susceptibility toward azole inhibition among the species.

Simultaneous exposure to a pulsed and a prolonged anthropogenic stressor can alter consumer multifunctionality

Ecosystems face multiple anthropogenic threats globally, and the effects of these environmental stressors range from individual‐level organismal responses to altered system functioning. Understanding the combined effects of stressors on process rates mediated by individuals in ecosystems would greatly improve our ability to predict organismal multifunctionality (e.g. multiple consumer‐mediated functions). We conducted a laboratory experiment to test direct and indirect, as well as immediate and delayed effects of a heat wave (pulsed stress) and micropollutants (MPs) (prolonged stress) on individual consumers (the great pond snail Lymnaea stagnalis) and their multifunctionality (i.e. consumption of basal resources, growth, reproduction, nutrient excretion and organic‐matter cycling). We found that stressful conditions increased the process rates of multiple functions mediated by individual consumers. Specifically, the artificial heat wave increased process rates in the majority of the quantified functions (either directly or indirectly), whereas exposure to MPs increased consumption of basal resources which led to increases in the release of nutrients and fine particulate organic matter. Moreover, snails exposed to a heat wave showed decreased reproduction and nutrient excretion after the heat‐wave, indicating the potential for ecologically relevant delayed effects. Our study indicates that the immediate and delayed effects of stressors on individual organisms may directly and indirectly impact multiple ecosystem functions. In particular, delayed effects of environmental stress on individual consumers may cumulatively impede recovery due to decreased functioning following a perturbation. Reconciling these results with studies incorporating responses at higher levels of biological complexity will enhance our ability to forecast how individual responses upscale to ecosystem multifunctionality.

Chronic Toxicity of Binary Mixtures of Six Metals (Ag, Cd, Cu, Ni, Pb, and Zn) to the Great Pond Snail Lymnaea stagnalis.

Although metal-mixture toxicity has recently received increasing attention, there is still insufficient knowledge on joint effects occurring in chronic exposures to relatively low metal concentrations. We characterized the chronic toxicity of binary mixtures of six metals (Ag, Cd, Cu, Ni, Pb, and Zn) in 14 day growth tests with juveniles of the metal-sensitive freshwater snail Lymnaea stagnalis. Observations were compared with predictions from individual metals and from the two most frequently used mixture models: concentration addition (CA) and independent action (IA). Predictions based on measured total dissolved concentrations and on calculated free-ion activities did not differ greatly because multimetal geochemical interactions in the tests were limited. In around half of the tests, mixture toxicity was higher than the greatest effect caused by the individual metals, arguing in favor of considering joint effects. When the additive models were used, the great majority of interactions were either additive or less than additive (i.e., antagonism). In general, the IA model was the most accurate, while the CA model was the most conservative. Along with other studies, these findings suggest that, at least for binary combinations, the simple CA model may provide satisfactory protection from the chronic metal toxicity of metal mixtures to aquatic organisms.

Immunocompetence analysis of the aquatic snail Lymnaea stagnalis exposed to urban wastewaters.

Wastewater treatment plant effluents from urban area are a well-known source of chronic multiple micropollution to the downstream living organisms. In this study, ecologically relevant laboratory-bred freshwater gastropods, Lymnaea stagnalis, were exposed for 29days to raw effluents of a wastewater treatment plant in Lyon area (France). A time-course analysis of individual markers of immunocompetence (hemocyte density and viability, hemocyte NADPH activity, phenol oxidase activity, and capacity of phagocytosis) has shown slight trends of inflammatory-like responses induced by the 100% effluents. So far, no short-term hazard for L. stagnalis can be revealed. However, over the long term, such environmental stress-stimulating immune responses could provoke deleterious life history trade-offs because the immune system is known to be highly energy-consuming.

Effect of Electrical Stimulation of the Optic Nerve on Electroretinogram in the Pond Snail Lymnaea stagnalis

Effect of Electrical Stimulation of the Optic Nerve on Electroretinogram in the Pond Snail Lymnaea stagnalis

MEN1 Tumor Suppressor Gene is Required for Long-term Memory Formation in an Aversive Operant Conditioning Model of Lymnaea stagnalis

Activity-dependent transcription factors critically coordinate the gene expression program underlying memory formation. The tumor suppressor gene, MEN1, encodes a ubiquitously expressed transcription regulator required for synaptogenesis and synaptic plasticity in invertebrate and vertebrate central neurons. In this study, we investigated the role of MEN1 in long-term memory (LTM) formation in an aversive operant conditioning paradigm in the freshwater pond snail Lymnaea stagnalis (L. stagnalis). We demonstrated that LTM formation is associated with an increased expression of MEN1 coinciding with an up-regulation of creb1 gene expression. In vivo knockdown of MEN1 prevented LTM formation and conditioning-induced changes in neuronal activity in the identified pacemaker neuron RPeD1. Our findings suggest the involvement of a new pathway in LTM consolidation that requires MEN1-mediated gene regulation.

Female behaviour prior to additional sperm receipt in the hermaphroditic pond snail Lymnaea stagnalis

In many species, sexual resistance by females can fluctuate depending on different physiological and/or ecological conditions. Based on theory, we decided to test whether the simultaneous hermaphrodite Lymnaea stagnalis becomes more reluctant to mate in the female role after receipt of an ejaculate. We aimed to test whether receipt of an ejaculate affects the expression of recipient behaviours, and whether this can have a potential effect on the outcome of mating interactions. We found that the expression of Crawl-out and Biting behaviours increased significantly in recently inseminated snails. Our data also indicated that an increase in expression of these latter behaviours can have direct effects on the outcome of the mating interactions. These findings suggest that under the influence of recent insemination, this hermaphroditic snail is more reluctant to accept another mating in the female role and may try to actively discourage the mounting snail (sperm donor). Hence, despite the fact that these simultaneous hermaphrodites express both sexual functions, there can still be variation in motivation to mate in either role.

Comparison of brain monoamine content in three populations of Lymnaea that correlates with taste-aversive learning ability.

To find a causal mechanism of learning and memory is a heuristically important topic in neuroscience. In the pond snail Lymnaea stagnalis, the following experimental facts have accrued regarding a classical conditioning procedure known as conditioned taste aversion (CTA): (1) one-day food-deprived Dutch snails have superior CTA memory formation; (2) the one-day food-deprived snails have a low monoamine content (e.g., serotonin, dopamine, octopamine) in their central nervous system (CNS); (3) fed or five-day food-deprived snails have poorer CTA memory and a higher monoamine content; (4) the Dutch snails form better CTA memory than the Canadian TC1 strain; and, (5) the F1 cross snails between the Dutch and Canadian TC1 strains also form poor CTA memory. Here, in one-day food-deprived snails, we measured the monoamine content in the CNSs of the 3 populations. In most instances, the monoamine content of the Dutch strain was lower than in the other two populations. The F1 cross snails had the highest monoamine content. A lower monoamine content is correlated with the better CTA memory formation.

Effects of 5-HT and insulin on learning and memory formation in food-deprived snails.

The pond snail Lymnaea stagnalis learns conditioned taste aversion (CTA) and consolidates it into long-term memory (LTM). How well they learn and form memory depends on the degree of food deprivation. Serotonin (5-HT) plays an important role in mediating feeding, and insulin enhances the memory consolidation process following CTA training. However, the relationship between these two signaling pathways has not been addressed. We measured the 5-HT content in the central nervous system (CNS) of snails subjected to different durations of food deprivation. One-day food-deprived snails, which exhibit the best learning and memory, had the lowest 5-HT content in the CNS, whereas 5-day food-deprived snails, which do not learn, had a high 5-HT content. Immersing 1-day food-deprived snails in 5-HT impaired learning and memory by causing an increase in 5-HT content, and that the injection of insulin into these snails reversed this impairment. We conclude that insulin rescues the CTA deficit and this may be due to a decrease in the 5-HT content in the CNS of Lymnaea.

Chronic toxicity of aluminum, at a pH of 6, to freshwater organisms: Empirical data for the development of international regulatory standards/criteria.

The chemistry, bioavailability, and toxicity of aluminum (Al) in the aquatic environment are complex and affected by a wide range of water quality characteristics (including pH, hardness, and dissolved organic carbon). Data gaps in Al ecotoxicology exist for pH ranges representative of natural surface waters (pH 6-8). To address these gaps, a series of chronic toxicity tests were performed at pH 6 with 8 freshwater species, including 2 fish (Pimephales promelas and Danio rerio), an oligochaete (Aeolosoma sp.), a rotifer (Brachionus calyciflorus), a snail (Lymnaea stagnalis), an amphipod (Hyalella azteca), a midge (Chironomus riparius), and an aquatic plant (Lemna minor). The 10% effect concentrations (EC10s) ranged from 98 μg total Al/L for D. rerio to 2175 μg total Al/L for L. minor. From these data and additional published data, species-sensitivity distributions (SSDs) were developed to derive concentrations protective of 95% of tested species (i.e., 50% lower confidence limit of a 5th percentile hazard concentration [HC5-50]). A generic HC5-50 (not adjusted for bioavailability) of 74.4 μg total Al/L was estimated using the SSD. An Al-specific biotic ligand model (BLM) was used to develop SSDs normalized for bioavailability based on site-specific water quality characteristics. Normalized HC5-50s ranged from 93.7 to 534 μg total Al/L for waters representing a range of European ecoregions, whereas a chronic HC5 calculated using US Environmental Protection Agency aquatic life criteria methods (i.e., a continuous criterion concentration [CCC]) was 125 μg total Al/L when normalized to Lake Superior water in the United States. The HC5-50 and CCC values for site-specific waters other than those in the present study can be obtained using the Al BLM. Environ Toxicol Chem 2018;37:36-48. © 2017 SETAC.

No effect of insect repellents on the behaviour of Lymnaea stagnalis at environmentally relevant concentrations.

Insect repellents are widely applied to various materials and to both human and animal skin to deter mosquitoes and ticks. The most common deterrent compounds applied are DEET, EBAAP and icaridin (picaridin, Bayrepel). Due to their extensive application, these repellents are frequently detected in surface waters in considerable concentrations. As these compounds are designed to alter invertebrates' behaviour rather than to intoxicate them, we hypothesised that insect repellents have the potential to modify the natural behaviour of non-target invertebrates in natural freshwater bodies. To test this, we used a well-established laboratory assay designed to quantify the odour-mediated foraging behaviour of freshwater gastropods and the great pond snail Lymnaea stagnalis (Linnaeus, 1758) as a model organism to test for potential deterrent effects of insect repellents on aquatic snails. Using a wide concentration range from the picogramme per litre to microgramme per litre range (and by far exceeding the range of concentrations reported from natural waters), we found no evidence for a deterrent effect of either of the three repellents on foraging L. stagnalis. Our data and other recent studies give no indication for undesirable behavioural alterations by common insect repellents in surface waters.

Two Strains of Lymnaea stagnalis and the Progeny from Their Mating Display Differential Memory-Forming Ability on Associative Learning Tasks.

The pond snail Lymnaea stagnalis learns and forms long-term memory (LTM) following both operant conditioning of aerial respiratory behavior and classical conditioning of taste aversive behavior. In the present study, we examined whether there are interstrain differences in the ability to form LTM following these two types of conditioning. A strain of Lymnaea (TC1) collected in Alberta, Canada exhibits superior memory-forming ability following aerial respiratory operant conditioning compared to a laboratory-reared strain of Lymnaea from Netherlands known as the Dutch strain. We asked whether the offspring of the Canadian TC1 and Dutch snails (i.e., filial 1 (F1) cross snails) would have the superior memory ability and found, rather, that their memory ability was average like the Dutch snails. That is, the Canadian TC1 snails have superior ability for LTM formation following aerial respiratory operant conditioning, but the Dutch and the generated F1 cross have average ability for memory forming. We next examined the Canadian TC1, Dutch and F1 cross snails for their ability to learn and form memory following conditioned taste aversion (CTA). All three populations showed similar associative CTA responses. However, both LTM formation and the ratio of good-to-poor performers in the memory retention test were much better in the Dutch snails than the Canadian TC1 and F1 cross snails. The memory abilities of the Canadian TC1 and F1 cross snails were average. Our present findings, therefore, suggest that snails of different strains have different memory abilities, and the F1 cross snails do not inherit the memory ability from the smart strain. To our knowledge, there have been a limited number of studies examining differences in memory ability among invertebrate strains, with the exception of studies using mutant flies.

Endocannabinoids disrupt stressed snails’ memories

Life has never been easy, and the physical mechanisms that were evolved by the earliest life forms for protection are fundamental to survival even today. However, fast-forward to our 24/7 news-saturated millennial culture and those stress systems are saturating. ‘We all know that when we are under stress we perform differently’, says Ken Lukowiak, from the University of Calgary, Canada, who is investigating the impact of stress on memory. However, instead of trying to untangle the complex networks that underpin human memory, Lukowiak and his colleagues focus on the memory of a simpler creature, pond snails (Lymnaea stagnalis); more specifically, the memory to hold their breath and not extend their breathing tubes above the surface when the water in which they reside is low in oxygen. Explaining that one molecular system – the endocannabinoid system – in our brains is thought to be a key player in mediating stress, disrupting our ability to form memories, Lukowiak, Hiroshi Sunada and their colleague Etsuro Ito, from Waseda University, Japan, wondered whether their humble pond snails also used the same system and, if so, how it affected their ability to form memories. But first they had to find the receptor molecules that could mediate endocannabinoid effects in the snails.Searching a database of the pond snail's genes with the sequence of a cannabinoid receptor that had been identified in the vase tunicate sea squirt, Takayui Watanabe, from the University of Sapporo, and Dai Hatakeyam identified two candidates, before confirming that both of the genes are expressed to produce the necessary receptors, not only in the snail's nervous system, but also in other tissues. In addition, they found more of the second receptor produced in the nervous system than the first receptor, which was more widely distributed throughout the snail's body. Next, the team investigated whether the receptors could disrupt the snail's ability to form memory.Injecting the snails with a drug that is known to trigger the endocannabinoid receptor and disrupt memory formation in mammals, Sunada, Sangmin Lee and Jeremy Forest then trained the molluscs to keep their breathing tubes closed when their water was deoxygenated – by poking the breathing tubes with a toothpick whenever the snails tried to stick them out of the water – to find out whether the snails could still form a memory: but they did not. And when Sunada, Lee and Forest tested whether a traumatic experience would also block the snails’ memories – by jabbing them on the foot before breath-hold training – the snails were equally unable to learn and form the new memory. However, when the team administered a drug that could block the endocannabinoid receptors from disrupting memory formation 1 h before traumatising the snails, this time the snails were able to learn to keep their breathing tubes closed when the water was deoxygenated and they wanted to pop up for air.The stressful jabbing experience had the same effect on the snails' memories as the drug that triggered the endocannabinoid receptor, making it likely that the endocannabinoid receptor was activated by stress to disrupt memory formation. Lukowiak says, ‘We feel pretty confident that this [the endocannabinoid] system in the snail plays a major role in determining how stress alters memory formation’, and he adds, ‘We think that it plays an important role in determining the “state” of the animals and thus whether a specific stimulus will be regarded as a stressor’.