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

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

How to reduce fear in a snail: Take an aspirin, call me in the morning.

Aspirin (Acetylsalicylic acid, ASA), one of the widely used non-steroid anti-inflammatory drugs can easily end up in sewage effluents and thus it becomes necessary to investigate the effects of aspirin on behaviour of aquatic organisms. Previous studies in mammals have shown ASA to alter fear and anxiety-like behaviours. In the great pond snail Lymnaea stagnalis, ASA has been shown to block a 'sickness state' induced by lipopolysaccharide injection which upregulates immune and stress-related genes thus altering behavioural responses. In Lymnaea, eliciting physiological stress may enhance memory formation or block its retrieval depending on the stimulus type and intensity. Here we examine whether ASA will alter two forms of associative-learning memory in crayfish predator-experienced Lymnaea when ASA exposure accompanies predator-cue-induced stress during the learning procedure. The two trainings procedures are: 1) operant conditioning of aerial respiration; and 2) a higher form of learning, called configural learning, which here is dependent on evoking a fear response. We show here that ASA alone does not alter homeostatic aerial respiration, feeding behaviour or long-term memory (LTM) formation of operantly conditioned aerial respiration. However, ASA blocked the enhancement of LTM formation normally elicited by training snails in predator cue. ASA also blocked configural learning, which makes use of the fear response elicited by the predator cue. Thus, ASA alters how Lymnaea responds cognitively to predator detection.

Effect of tributyltin exposure on the embryonic development and behavior of a molluscan model species, Lymnaea stagnalis.

The presence of the organotin compound tributyltin (TBT) in aquatic ecosystems has been a serious environmental problem for decades. Although a number of studies described the negative impact of TBT on mollusks at different levels, investigations connected to its potential effects during embryogenesis have been neglected. For a better understanding of the impact of TBT on mollusks, in the present study, embryos of previously TBT-treated or not treated specimens of the great pond snail (Lymnaea stagnalis) were exposed to 100 ng L-1 TBT from egg-laying (single-cell stage) until hatching. According to our results, TBT significantly delayed hatching and caused shell malformation. TBT transiently decreased the locomotion (gliding) and also reduced the feeding activity, demonstrating for the first time that this compound can alter the behavioral patterns of molluscan embryos. The heart rate was also significantly reduced, providing further support that cardiac activity is an excellent indicator of metal pollution in molluscan species. At the histochemical level, tin was demonstrated for the first time in TBT-treated hatchlings with intensive reaction in the central nervous system, kidney, and hepatopancreas. Overall, the most notable effects were observed in treated embryos derived from TBT treated snails. Our findings indicate that TBT has detrimental effects on the development and physiological functions of Lymnaea embryos even at a sub-lethal concentration, potentially influencing their survival and fitness. Highlighting our observations, we have demonstrated previously unknown physiological changes (altered heart rate, locomotion, and feeding activity) caused by TBT, as well as visualized tin at the histochemical level in a molluscan species for the first time following TBT exposure. Further studies are in progress to reveal the cellular and molecular mechanisms underlying the physiological and behavioral changes described in the present study.

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-.

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

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

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

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

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.

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.

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.

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.

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.

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.

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.