In Vitro and In Vivo Genotoxicity of Polystyrene Microplastics: Evaluation of a Possible Synergistic Action with Bisphenol A.

The ubiquitous presence of plastics represents a global threat for all ecosystems and human health. In this study, we evaluated, in vitro and in vivo, the genotoxic potential of different concentrations of polystyrene microplastics (PS-MPs) and their possible synergistic interactions with bisphenol-A (BPA). For the in vitro and the in vivo assays, we used human lymphocytes and hemocytes from Lymnaea stagnalis, respectively. The genomic damage was evaluated by the micronucleus assay, and differences in eggs laid and growth of L. stagnalis were also evaluated. In human lymphocytes, PS-MPs alone at the concentration of 200 µg/mL and in association with BPA 0.100 µg/mL significantly increased the frequencies of micronuclei and nuclear buds, indicating a possible in vitro genotoxic additive action of these two compounds. Vice versa, PS-MPs did not result in genotoxicity in hemocytes. Our results indicated that PS-MPs have genotoxic properties only in vitro and at a concentration of 200 µg/mL; moreover, this compound could intensify the genomic damage when tested with BPA, indicating possible cumulative effects. Finally, PS significantly reduced the growth and the number of laid eggs in L. stagnalis.

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.

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.

Left-Right Asymmetry in Invertebrates: From Molecules to Organisms.

Although most animals appear symmetric externally, they exhibit chirality within their body cavity, i.e., in terms of asymmetric organ position, directional organ looping, and lateralized organ function. Left-right (LR) asymmetry is determined genetically by intricate molecular interactions that occur during development. Key genes have been elucidated in several species. There are common mechanisms in vertebrates and invertebrates, but some appear to exhibit unique mechanisms. This review focuses on LR asymmetry formation in invertebrates, particularly Drosophila, ascidians, and mollusks. It aims to understand the role of the genes that are key to creating LR asymmetry and how chirality information is converted/transmitted across the hierarchies from molecules to cells and from cells to tissues.

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".

Same same, but different: exploring the enigmatic role of the pituitary adenylate cyclase-activating polypeptide (PACAP) in invertebrate physiology.

Evidence has been accumulating that elements of the vertebrate pituitary adenylate cyclase-activating polypeptide (PACAP) system are missing in non-chordate genomes, which is at odds with the partial sequence-, immunohistochemical-, and physiological data in the literature. Multilevel experiments were performed on the great pond snail (Lymnaea stagnalis) to explore the role of PACAP in invertebrates. Screening of neuronal transcriptome and genome data did not reveal homologs to the elements of vertebrate PACAP system. Despite this, immunohistochemical investigations with an anti-human PAC1 receptor antibody yielded a positive signal in the neuronal elements in the heart. Although Western blotting of proteins extracted from the nervous system found a relevant band for PACAP-38, immunoprecipitation and mass spectrometric analyses revealed no corresponding peptide fragments. Similarly to the effects reported in vertebrates, PACAP-38 significantly increased cAMP synthesis in the heart and had a positive ionotropic effect on heart preparations. Moreover, it significantly modulated the effects of serotonin and acetylcholine. Homologs to members of Cluster B receptors, which have shared common evolutionary origin with the vertebrate PACAP receptors, PTHRs, and GCGRs, were identified and shown not to be expressed in the heart, which does not support a potential role in the mediation of PACAP-induced effects. Our findings support the notion that the PACAP system emerged after the protostome-deuterostome divergence. Using antibodies against vertebrate proteins is again highlighted to have little/no value in invertebrate studies. The physiological effects of vertebrate PACAP peptides in protostomes, no matter how similar they are to those in vertebrates, should be considered non-specific.

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.

Expanded expression of pro-neurogenic factor SoxB1 during larval development of gastropod Lymnaea stagnalis suggests preadaptation to prolonged neurogenesis in Mollusca.

Introduction: The remarkable diversity observed in the structure and development of the molluscan nervous system raises intriguing questions regarding the molecular mechanisms underlying neurogenesis in Mollusca. The expression of SoxB family transcription factors plays a pivotal role in neuronal development, thereby offering valuable insights into the strategies of neurogenesis. Methods: In this study, we conducted gene expression analysis focusing on SoxB-family transcription factors during early neurogenesis in the gastropod Lymnaea stagnalis. We employed a combination of hybridization chain reaction in situ hybridization (HCR-ISH), immunocytochemistry, confocal microscopy, and cell proliferation assays to investigate the spatial and temporal expression patterns of LsSoxB1 and LsSoxB2 from the gastrula stage to hatching, with particular attention to the formation of central ring ganglia. Results: Our investigation reveals that LsSoxB1 demonstrates expanded ectodermal expression from the gastrula to the hatching stage, whereas expression of LsSoxB2 in the ectoderm ceases by the veliger stage. LsSoxB1 is expressed in the ectoderm of the head, foot, and visceral complex, as well as in forming ganglia and sensory cells. Conversely, LsSoxB2 is mostly restricted to the subepithelial layer and forming ganglia cells during metamorphosis. Proliferation assays indicate a uniform distribution of dividing cells in the ectoderm across all developmental stages, suggesting the absence of distinct neurogenic zones with increased proliferation in gastropods. Discussion: Our findings reveal a spatially and temporally extended pattern of SoxB1 expression in a gastropod representative compared to other lophotrochozoan species. This prolonged and widespread expression of SoxB genes may be interpreted as a form of transcriptional neoteny, representing a preadaptation to prolonged neurogenesis. Consequently, it could contribute to the diversification of nervous systems in gastropods and lead to an increase in the complexity of the central nervous system in Mollusca.

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.

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.

A novel and unique refraction-based optical recording system for pharmacological investigations on isolated muscle preparations.

In the field of neuroscience and ecotoxicology, there is a great need for investigating the effect(s) of a variety of different chemicals (e.g., pharmacologically active compounds, pesticides, neurotransmitters, modulators) at different biological levels. Different contractile tissue preparations have provided excellent model systems for in vitro pharmacological experiments for a long time. However, such investigations usually apply mechanical force transducer-based approaches. Thus, a rapid, easy, cheap, digital, and reproducible in vitro pharmacological method based on an effective, 'non-invasive' (compared to the force-transducer approaches), refraction-based optical recording approach and isolated heart preparations was developed.•A versatile and unique refraction-based optical recording system with a Java application was developed.•The recording system was tested and validated on isolated heart preparations obtained from the widely used invertebrate model organism, the great pond snail (Lymnaea stagnalis).•The recording system illustrates the progression of technology from the mechanical force transducer system and can represent a suitable tool in ecotoxicology or neuroscience.

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

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

Disruptions in network plasticity precede deficits in memory following inhibition of retinoid signaling.

Retinoic acid, the active metabolite of vitamin A, is important for vertebrate cognition and hippocampal plasticity, but few studies have examined its role in invertebrate learning and memory, and its actions in the invertebrate central nervous system are currently unknown. Using the mollusc Lymnaea stagnalis, we examined operant conditioning of the respiratory behavior, controlled by a well-defined central pattern generator (CPG), and used citral to inhibit retinoic acid signaling. Both citral- and vehicle-treated animals showed normal learning, but citral-treated animals failed to exhibit long-term memory at 24 h. Cohorts of citral- or vehicle-treated animals were dissected into semi-intact preparations, either 1 h after training, or after the memory test 24 h later. Simultaneous electrophysiological recordings from the CPG pacemaker cell (right pedal dorsal 1; RPeD1) and an identified motorneuron (VI) were made while monitoring respiratory activity (pneumostome opening). Activity of the CPG pneumostome opener interneuron (input 3 interneuron; IP3) was also monitored indirectly. Vehicle-treated conditioned preparations showed significant changes in network parameters immediately after learning, such as reduced motorneuron bursting activity (from IP3 input), delayed pneumostome opening, and decoupling of coincident IP3 input within the network. However, citral-treated preparations failed to exhibit these network changes and more closely resembled naïve preparations. Importantly, these citral-induced differences were manifested immediately after training and before any overt changes in the behavioral response (memory impairment). These studies shed light on where and when retinoid signaling might affect a central pattern-generating network to promote memory formation during conditioning of a homeostatic behavior.NEW & NOTEWORTHY We provide novel evidence for how conditioning-induced changes in a CPG network are disrupted when retinoid signaling is inhibited. Inhibition of retinoic acid signaling prevents long-term memory formation following operant conditioning, but has no effect on learning. Simultaneous electrophysiological and behavioral analyses indicate network changes immediately following learning, but these changes are prevented with inhibition of retinoid signaling, before any overt changes in behavior. These data suggest sites for retinoid actions during memory formation.

CNS serotonin content mediating food deprivation-enhanced learning is regulated by hemolymph tryptophan concentration and autophagic flux in the pond snail.

Nutritional status affects cognitive function in many types of organisms. In the pond snail Lymnaea stagnalis, 1 day of food deprivation enhances taste aversion learning ability by decreasing the serotonin (5-hydroxytryptamin; 5-HT) content in the central nervous system (CNS). On the other hand, after 5 days of food deprivation, learning ability and the CNS 5-HT concentration return to basal levels. How food deprivation leads to alterations of 5-HT levels in the CNS, however, is unknown. Here, we measured the concentration of the 5-HT precursor tryptophan in the hemolymph and CNS, and demonstrated that the CNS tryptophan concentration was higher in 5-day food-deprived snails than in non-food-deprived or 1-day food-deprived snails, whereas the hemolymph tryptophan concentration was not affected by the duration of food deprivation. This finding suggests the existence of a mediator of the CNS tryptophan concentration independent of food deprivation. To identify the mediator, we investigated autophagic flux in the CNS under different food deprivation conditions. We found that autophagic flux was significantly upregulated by inhibition of the tropomyosin receptor kinase (Trk)-Akt-mechanistic target of rapamycin complex 1 (MTORC1) pathway in the CNS of 5-day food-deprived snails. Moreover, when autophagy was inhibited, the CNS 5-HT content was significantly downregulated in 5-day food-deprived snails. Our results suggest that the hemolymph tryptophan concentration and autophagic flux in the CNS cooperatively regulate learning ability affected by different durations of food deprivation. This mechanism may underlie the selection of behaviors appropriate for animal survival depending on the degree of nutrition.

The Effects of Microbiota on the Herbivory Resistance of the Giant Duckweed Are Plant Genotype-Dependent.

In nature, all plants live with microbes, which can directly affect their host plants' physiology and metabolism, as well as their interacting partners, such as herbivores. However, to what extent the microbiota shapes the adaptive evolution to herbivory is unclear. To address this challenge, it is essential to quantify the intra-specific variations of microbiota effects on plant fitness. Here, we quantified the fitness effects of microbiota on the growth, tolerance, and resistance to herbivory among six genotypes of the giant duckweed, Spirodela polyrhiza. We found that the plant genotypes differed in their intrinsic growth rate and tolerance, but not in their resistance to a native herbivore, the great pond snail. Inoculation with microbiota associated with S. polyrhiza growing outdoors reduced the growth rate and tolerance in all genotypes. Additionally, the microbiota treatment altered the herbivory resistance in a genotype-specific manner. Together, these data show the potential of microbiota in shaping the adaptive evolution of plants.

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

By employing a reductionistic (but not simplistic) approach using an established invertebrate model system, the pond snail Lymnaea stagnalis, we investigated whether (1) lipopolysaccharide (LPS)-induced inflammation would cause a sickness state and impair cognitive function, and-if so-(2) would aspirin (acetylsalicylic acid-ASA) restore the impaired cognition. To test our hypotheses, we first determined if the injection of 25 mg (6.25 µg/mL) of Escherichia coli-derived LPS serotype O127:B8 altered homeostatic behavior, aerial respiration, and then determined if LPS altered memory formation when this behavior was operantly conditioned. Next, we determined if ASA altered the LPS-induced changes in both aerial respiration and cognitive functions. LPS induced a sickness state that increased aerial respiration and altered the ability of snails to form or recall long-term memory. ASA reverted the LPS-induced sickness state and thus allowed long-term memory both to be formed and recalled. We confirmed our hypotheses and provided the first evidence in an invertebrate model system that an injection of LPS results in a sickness state that obstructs learning and memory, and this impairment can be prevented by a non-steroidal anti-inflammatory.

Studies on a widely-recognized snail model species (Lymnaea stagnalis) provide further evidence that vertebrate steroids do not have a hormonal role in the reproduction of mollusks.

Experiments were carried out to determine whether, as with other mollusks that have been studied, the snail, Lymnaea stagnalis, can absorb, esterify and store vertebrate steroids that are present in the water. We also carried out experiments to determine whether neural tissues of the snail could be immunohistochemically stained with an antibody to human aromatase (a key enzyme that catalyzes the conversion of testosterone [T] to 17ß-estradiol [E2]); and, if so, to determine the significance of such staining. Previous studies on other mollusks have reported such staining and have proposed this as decisive evidence that mollusks have the same steroid synthesis pathway as vertebrates. We found that snails absorb, esterify and retain esterified T, E2, progesterone and ethinyl-estradiol (albeit with an absorption rate about four times slower, on a weight basis, than the mussel, Mytilus edulis). We also found that not only anti-human aromatase, but also anti-human nuclear progesterone receptor (nPR) and anti-human gonadotropin-releasing hormone antibodies immunohistochemically stained snail neural cells. However, further experiments, involving gel electrophoretic separation, followed by immunostaining, of proteins extracted from the neural tissue, found at least two positively-stained bands for each antibody, none of which had masses matching the human proteins to which the antibodies had been raised. The anti-aromatase antibody even stained the 140 kDA ladder protein used as a molecular weight marker on the gels. Mass spectrometric analysis of the bands did not find any peptide sequences that corresponded to the human proteins. Our findings confirm that the presence of vertebrate-like sex steroids in molluscan tissues is not necessarily evidence of endogenous origin. The results also show that immunohistochemical studies using antibodies against human proteins are grossly non-specific and likely to have little or no value in studying steroid synthesis or activity in mollusks. Our conclusions are consistent with the fact that genes for aromatase and nPR have not been found in the genome of the snail or of any other mollusk. Our overarching conclusion, from this and our previous studies, is that the endocrinology of mollusks is not the same as that of humans or any other vertebrates and that continuing to carry out physiological and ecotoxicological studies on mollusks on the basis of this false assumption, is an unconscionable waste of resources.

Molluscan RXR Transcriptional Regulation by Retinoids in a Drosophila CNS Organ Culture System.

Retinoic acid, the active metabolite of Vitamin A, is important for the appropriate development of the nervous system (e.g., neurite outgrowth) as well as for cognition (e.g., memory formation) in the adult brain. We have shown that many of the effects of retinoids are conserved in the CNS of the mollusc, Lymnaea stagnalis. RXRs are predominantly nuclear receptors, but the Lymnaea RXR (LymRXR) exhibits a non-nuclear distribution in the adult CNS, where it is also implicated in non-genomic retinoid functions. As such, we developed a CNS Drosophila organ culture-based system to examine the transcriptional activity and ligand-binding properties of LymRXR, in the context of a live invertebrate nervous system. The novel ligand sensor system was capable of reporting both the expression and transcriptional activity of the sensor. Our results indicate that the LymRXR ligand sensor mediated transcription following activation by both 9-cis RA (the high affinity ligand for vertebrate RXRs) as well as the vertebrate RXR synthetic agonist, SR11237. The LymRXR ligand sensor was also activated by all-trans RA, and to a much lesser extent by the vertebrate RAR synthetic agonist, EC23. This sensor also detected endogenous retinoid-like activity in the CNS of developing Drosophila larvae, primarily during the 3rd instar larval stage. These data indicate that the LymRXR sensor can be utilized not only for characterization of ligand activation for studies related to the Lymnaea CNS, but also for future studies of retinoids and their functions in Drosophila development.

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

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