Snail Lymnaea Stagnalis Research Articles

A workflow to investigate the impacts of weathered multi-walled carbon nanotubes to the mud snail Lymnaea stagnalis

Although the development and application of nanomaterials is a growing industry, little data is available on the ecotoxicological effects on aquatic organisms. Therefore, we set up a workflow to address the potential uptake of weathered multi-walled carbon nanotubes (wMWCNTs) by a model organism, the pulmonary mud snail Lymnaea stagnalis (L. stagnalis), which plays an important role in the food web. It represents a suitable organism for this approach because as a grazer it potentially ingests large amounts of sedimented wMWCNTs. As food source for L. stagnalis, benthic biofilm was investigated by the use of a transmission electron microscope (TEM) and a scanning electron microscope (SEM) after exposure with wMWCNTs. In addition, isotopic labeling was applied with 14C-wMWCNTs (0.1 mg/L) to quantify fate, behavior, and enrichment of 14C-wMWCNTs in benthic biofilm and in L. stagnalis. Enrichment in benthic biofilm amounted to 529.0 µg wMWCNTs/g dry weight and in L. stagnalis to 79.6 µg wMWCNTs/g dry weight. A bioconcentration factor (BCF) for L. stagnalis was calculated (3500 L/kg). We demonstrate the accumulation of wMWCNTs (10 mg/L) in the digestive tract of L. stagnalis in an effect study. Moreover, the physiological markers glycogen and triglycerides as indicators for the physiological state, as well as the RNA/DNA ratio as growth indicator, were examined. No significant differences between exposed and control animals were analyzed for glycogen and triglycerides after 24 days of exposure, but a decreasing trend is recognizable for triglycerides. In contrast, the significant reduction in the RNA/DNA ratio of L. stagnalis indicated an inhibition of growth with a following recovery after depuration. The described workflow enables a comprehensive determination of the fate and the behavior of wMWCNTs specifically and in general all kinds of CNTs in the aquatic environment and therefore contributes to a holistic risk assessment of wMWCNTs.

Toll like receptors and their evolution in the lymnaeid freshwater snail species Radix auricularia and Lymnaea stagnalis, key intermediate hosts for zoonotic trematodes

One of the major evolutionarily conserved pathways in innate immunity of invertebrates is the toll-like receptor (TLR) pathway. However, little is known of the TLR protein family in gastropod molluscs despite their role in the transmission of human diseases, especially the common lymnaeid freshwater snail species Radix auricularia and Lymnaea stagnalis, key intermediate hosts of zoonotic trematodes. Using comparative genomics and gene prediction approaches utilising the freshwater snail Biomphalaria glabrata genome as a reference ten putative TLR proteins were identified in both R. auricularia and L. stagnalis. Phylogenetic analyses revealed that unlike other molluscs the lymnaeid species also possessed class 1 TLRs, previously thought to be unique to B. glabrata. Gene duplication events were also seen across the TLR classes in the lymnaeids with several of the genes appearing to exist as potential tandem elements in R. auricularia. Each predicted TLR was shown to possess the typical the leucine-rich repeat extracellular and TIR intracellular domains and both single cysteine clusters and multiple cysteine clusters TLRs were identified in both lymnaeid species. Principle component analyses of 3D models of the predicted TLRs showed that class 1 and 5 proteins did not cluster based on similarity of structure, suggested to be potential adaptation to a range of pathogens. This study provides the first detailed account of TLRs in lymnaeids and affords a platform for further research into the role of these proteins into susceptibility and compatibility of these snails with trematodes and their role in transmission.

ОСОБЕННОСТИ ПОКАЗАТЕЛЕЙ ЭМБРИОНАЛЬНОГО РАЗВИТИЯ ПРУДОВИКА (LYMNAEA STAGNALIS L.) ПРИ АНТРОПОГЕННОМ ВОЗДЕЙСТВИИ НЕКОТОРЫХ ХЛОРИСТЫХ СОЕДИНЕНИЙ В ВОДЕ

The article highlights the research of embryonic development of hydrobionts under the influence of anthropogenic factors of water solutions – chlorobenzene and tetrachloroethylene. To detect the toxicity of chloride pollutants in a freshwater reservoir, representatives of benthic mollusks (big pond snail Lymnaea stagnalis L.) were used as a model organism. According to the results of research, it was found that at a concentration of 0.01 mg/l of chlorobenzene solution individual anomalies of embryonic development of the pond fish are observed, but at higher concentrations its development is delayed by 5-6 stages in comparison with the control. As an integral parameter of pond fish development, we can take indicators of the biological toxicity criterion: Hatchling and its survival over a 10-day period after leaving the egg shells. Changes in the physiological parameters of pond fish embryos begin with a chlorobenzene concentration of 0.01 mg/l; the maximum permissible concentration for tetrachloroethylene for the considered biological indicators should be considered 0.05 mg/l. It is noted that in big pond snail (Lymnaea stagnalis L.) the hatch of juveniles from egg shells is the most sensitive indicator when determining the toxicity of an aqueous solution; and less sensitive indicators are the number of eggs laid and the survival of juveniles in the first 10 days after hatching. The test object used is a large pond, which has shown a high sensitivity to contamination of the water environment with chlorinated ecotoxicants and can be recommended for a comprehensive quality assessment when biotesting freshwater waters

Herbivory increases on freshwater plants exposed to artificial light at night

Artificial light at night (ALAN) is recognized as one of the major anthropogenic pollutants jeopardizing biodiversity at a global scale. Few studies have focused on the impacts of nocturnal light on freshwater ecosystems despite their increasing exposure to light pollution worldwide due to human activities along rivers or lakes. By modifying metabolism, the disruption of natural light regime could modify the leaf palatability and affect the plant-herbivore interactions which can partly determine the structure of macrophyte communities. By experimentally mimicking light conditions from sub-urban areas, we measured the effects of low-level light at night on the leaf traits of an aquatic plant, Ceratophyllum demersum, and herbivory by the snail Lymnaea stagnalis, a generalist feeder. We demonstrate that artificial light at night increases the herbivory rate: leaves of C. demersum that had been exposed to ALAN for three months were 1.6 times more consumed by L. stagnalis than control leaves. The increase in C. demersum palatability to the pond snail when plants were exposed to ALAN was not due to a decrease in dry matter content or to a modification of the biomechanical characteristics of the leaves. Because snails have not been exposed to nocturnal light before or during the herbivory experiment, this increase in leaf palatability, is possibly linked to modifications of primary and/or secondary metabolism. Our results suggest that ALAN is a potential key factor affecting ecological processes in aquatic ecosystems such as trophic networks.

Neuronal Transcriptome Analysis of a Widely Recognised Molluscan Model Organism Highlights the Absence of Key Proteins Involved in the De Novo Synthesis and Receptor-Mediation of Sex Steroids in Vertebrates

Over the last ten years, the interpretation of the presence of vertebrate sex steroids in molluscs has changed dramatically. Evidence has been accumulating that CYP11A and CYP19A genes (encoding cholesterol side-chain cleavage enzyme and aromatase), that are crucial for the biosynthesis of sex steroids in vertebrates, as well as key functional sex steroid receptors, are missing in molluscan genomes. To provide further evidence, we sequenced the whole transcriptome of the central nervous system of the great pond snail (Lymnaea stagnalis) and screened it for sequences homologous to those used in the generally accepted vertebrate sex steroidogenesis pathway as well as the known sex steroid receptor-related genes (such as CYP11A, CYP19A, 3β-HSD, nPR, and nAR). Our screening confirmed the absence of several key sequences that are essential to accomplish a full sex steroid biosynthesis pathway similar to that of vertebrates. There was also no evidence for nuclear sex steroid receptors. Our findings support the contention that molluscan endocrinology differs from the well-characterized vertebrate endocrine system.

Long-term memory of configural learning is enhanced via CREB upregulation by the flavonoid quercetin in Lymnaea stagnalis.

Animals respond to acute stressors by modifying their behaviour and physiology. The pond snail Lymnaea stagnalis exhibits configural learning (CL), a form of higher order associative learning. In CL snails develop a landscape of fear when they experience a predatory cue along with a taste of food. This experience results in a suppression of the food response; but the memory only persists for 3 h. Lymnaea has also been found to upregulate heat shock proteins (HSPs) as a result of acute heat stress, which leads to the enhancement of memory formation. A plant flavonoid quercetin blocks the upregulation of HSPs when experienced prior to heat stress. Here, we used this blocking mechanism to test the hypothesis that HSP upregulation plays a critical role in CL. Snails experienced quercetin prior to CL training and surprisingly instead of blocking memory formation it enhanced the memory such that it now persisted for at least 24 h. Quercetin exposure either prior to or after CL enhanced long-term memory (LTM) up to 48 h. We quantified mRNA levels of the transcription factor CREB1 in the Lymnaea central nervous system and found LymCREB1 to be upregulated following quercetin exposure. The enhanced LTM phenotype in L. stagnalis was most pronounced when quercetin was experienced during the consolidation phase. Additionally, quercetin exposure during the memory reconsolidation phase also led to memory enhancement. Thus, we found no support of our original hypothesis but found that quercetin exposure upregulated LymCREB1 leading to LTM formation for CL.

Configural learning memory can be transformed from intermediate-term to long-term in pond snail Lymnaea stagnalis

A lab bred W-strain of Lymnaea stagnalis exhibits configural learning (CL). CL is a form of higher order associative learning wherein when snails experience two contrasting stimuli together such as predatory odour (CE: crayfish effluent) and food odour (C: carrot odour) they learn and associate risk with food. The memory for CL has been shown to last 3 h. Here, we show that when only a single CL-training session is given only a 3 h memory is formed. Memory is not present 24 h after the training session. However, memory can be enhanced and snails show long term memory (24 h memory) when trained for a second time within a 7-day time period after the first CL-training. We further hypothesised that Green tea exposure will enhance memory persistence as catechins in green tea are shown to be cognitive enhancers. We thus subjected snails to CL training followed by green tea exposure which resulted in enhanced memory persistence and it occurred during memory consolidation phase. Thus, we show for the first time that CL intermediate-term memory can be transformed to long-term memory by green tea and multiple trainings in a lab bred strain of Lymnaea.

Photoperiod controls egg laying and caudodorsal cell hormone expression but not gonadal development in the pond snail Lymnaea stagnalis.

Photoperiod is a reliable cue to regulate growth and reproduction for seasonal adaptation. Although photoperiodism has been well studied in Chordata and Arthropoda, less is known about Mollusca. We examined photoperiodic effects on egg laying, body size, gonad-somatic index, oocyte size and relative amounts of caudodorsal cell hormone mRNA in individual rearing conditions in the pond snail Lymnaea stagnalis. Twenty-five weeks after hatching, the percentages of egg-laying snails under a photoperiod of 12h light and 12h darkness (12L:12D) were significantly smaller than those under longer days. The total numbers of eggs and egg masses under 12L:12D were significantly smaller than those under longer days. Significant differences between 16L:8D and 12L:12D were not observed in the soft body and ovotestis weight, and the gonad-somatic index. Photoperiodic effects were also not observed in oocyte diameters twenty-two weeks after hatching. Twenty-seven weeks after hatching amounts of caudodorsal cell hormone mRNA were significantly lower in the cerebral ganglia with commissure under 12L:12D than 16L:8D. L. stagnalis exhibited a clear photoperiodic response in egg laying and the amount of caudodorsal cell hormone mRNA, but not in gonadal development. Under 12L:12D suppression of caudodorsal cell hormone expression might suppress egg laying.

Functional characterization and related evolutionary implications of invertebrate gonadotropin-releasing hormone/corazonin in a well-established model species

In vertebrates, gonadotropin-releasing hormone (GnRH) peptide is the central mediator of reproduction. Homologous peptides have previously also been identified in molluscan species. However, emerging evidence suggests that these molecules might serve diverse regulatory functions and proposes to consider them as corazonin (CRZ). We previously isolated the full-length cDNA of the invGnRH/CRZ peptide (termed ly-GnRH/CRZ) in the well-established invertebrate model species, the great pond snail Lymnaea stagnalis; however, its predicted functions remain to be verified. In this study, we first confirmed the presence of the deduced active peptide from the central nervous system of L. stagnalis. Further, we performed in vivo and in vitro studies to explore the functions of ly-GnRH/CRZ. Injection of sexually mature specimens with synthetic active peptide had an inhibitory effect on locomotion and an acceleratory effect on egg-laying, but had no effect on feeding. The previously predicted modulatory effect of ly-GnRH/CRZ was supported by its identified co-localization with serotonin on the surface of the heart atria. Lastly, we demonstrated not only the presence of ly-GnRH/CRZ in the penial complex but also that ly-GnRH/CRZ-containing neurons project to the efferent penis nerve, suggesting ly-GnRH/CRZ may directly modulate the motor output of this peripheral tissue. Overall, our findings strongly support that ly-GnRH/CRZ is a multifunctional neuropeptide. These results contribute to the understanding of the GnRH superfamily and, more broadly, disciplines such as comparative endocrinology and neurobiology.

Non-coding Regions of Mitochondrial DNA and the cox1 Gene Reveal Genetic Variability Among Local Belarusian Populations of the Causative Agent of Cercarial Dermatitis, Bird Schistosome Trichobilharzia szidati (Digenea: Schistosomatidae).

The cercariae of avian blood flukes Trichobilharzia szidati (Digenea, Schistosomatidae) are known to cause cercarial allergic dermatitis ("swimmer's itch") in humans. Global epidemics can have significant impacts on local tourism-related economies in recreational areas. Little is known about the genetic polymorphism of the parasite population, or about the variability of the non-coding regions of mitochondrial DNA (mtDNA) and the possibility of using this as a genetic marker. The T. szidati cercariae were collected over 7years from 33 naturally infected Lymnaea stagnalis snails from five sites at two neighboring lakes in Belarus. We investigated the variability of the short (SNR) and long (LNR) non-coding regions of mt DNA and the genetic diversity within the 1125-bp sequences of the gene for subunit 1 of cytochrome c oxidase (cox1). In the SNR sequences, we found only length variability caused by changes in the number of bases in the mononucleotide tracts T6-T8. LNR demonstrates high variability in nucleotide sequence length (182-260bp) depending on the presence of two long deletions of 59 and 78 nucleotides. Both mitochondrial loci (LNR and cox1) are characterized by high haplotype diversity (H = 0.922 and H = 1.0, respectively); the nucleotide diversity is significantly higher for LNR (π = 1.926 ± 0.443) compared to cox1 (π = 0.704 ± 0.059). Phylogenetic reconstructions based on the variability of each of the loci (LNR and cox1) and their concatenated sequences revealed their shallow structure and the absence of a correlation between the distribution of single-nucleotide polymorphisms and the geographic origin of parasites from two Belarusian lakes. We identified at last four weakly sublineages in the phylogenetic pattern of T. szidati. The carriers of each deletion have specific patterns for each of the two loci and form their own phylogeographic sublineages. An association between two fixed LNR substitutions and a fixed non-synonymous substitution in cox1 was found in four representatives of one lineage that had a short deletion in the LNR. This study clarified the phylogeographic structure of the Belarusian population of T. szidati. Our data provide the basis for the use two mt markers in large-scale population studies of the parasite, as well as for studying the molecular evolution of coding and non-coding mtDNA in trematodes.

What can we teach Lymnaea and what can Lymnaea teach us?

ABSTRACTThis review describes the advantages of adopting a molluscan complementary model, the freshwater snail Lymnaea stagnalis, to study the neural basis of learning and memory in appetitive and avoidance classical conditioning; as well as operant conditioning of its aerial respiratory and escape behaviour. We firstly explored ‘what we can teach Lymnaea’ by discussing a variety of sensitive, solid, easily reproducible and simple behavioural tests that have been used to uncover the memory abilities of this model system. Answering this question will allow us to open new frontiers in neuroscience and behavioural research to enhance our understanding of how the nervous system mediates learning and memory. In fact, from a translational perspective, Lymnaea and its nervous system can help to understand the neural transformation pathways from behavioural output to sensory coding in more complex systems like the mammalian brain. Moving on to the second question: ‘what can Lymnaea teach us?’, it is now known that Lymnaea shares important associative learning characteristics with vertebrates, including stimulus generalization, generalization of extinction and discriminative learning, opening the possibility to use snails as animal models for neuroscience translational research.

Effect of Nanoparticle Size and Natural Organic Matter Composition on the Bioavailability of Polyvinylpyrrolidone-Coated Platinum Nanoparticles to a Model Freshwater Invertebrate.

The bioavailability of dissolved Pt(IV) and polyvinylpyrrolidone-coated platinum nanoparticles (PtNPs) of five different nominal hydrodynamic diameters (20, 30, 50, 75, and 95 nm) was characterized in laboratory experiments using the model freshwater snail Lymnaea stagnalis. Dissolved Pt(IV) and all nanoparticle sizes were bioavailable to L. stagnalis. Platinum bioavailability, inferred from conditional uptake rate constants, was greater for nanoparticulate than dissolved forms and increased with increasing nanoparticle hydrodynamic diameter. The effect of natural organic matter (NOM) composition on PtNP bioavailability was evaluated using six NOM samples at two nanoparticle sizes (20 and 95 nm). NOM suppressed the bioavailability of 95 nm PtNPs in all cases, and DOM reduced sulfur content exhibited a positive correlation with 95 nm PtNP bioavailability. The bioavailability of 20 nm PtNPs was only suppressed by NOM with a low reduced sulfur content. The physiological elimination of Pt accumulated after dissolved Pt(IV) exposure was slow and constant. In contrast, the elimination of Pt accumulated after PtNP exposures exhibited a triphasic pattern likely involving in vivo PtNP dissolution. This work highlights the importance of PtNP size and interfacial interactions with NOM on Pt bioavailability and suggests that in vivo PtNP transformations could yield unexpectedly higher adverse effects to organisms than dissolved exposure alone.

In the great pond snail (Lymnaea stagnalis), two stressors that individually enhance memory in combination block memory formation

Stress plays an important role in memory formation in the great pond snail (Lymnaea stagnalis (Linnaeus, 1758)). Individual stressors have been shown to enhance or to perturb long-term memory (LTM) formation. However, when snails perceive a combination of two stressors, it is unclear the outcome with regards to LTM formation. Here we first show that when L. stagnalis are exposed individually to either a predator stressor (crayfish effluent (CE), which is a kairomone) or a thermal stressor (30 °C), LTM formation is enhanced. In their natural environment, L. stagnalis may experience temperatures approaching 30 °C and they may encounter crayfish at the same time. How such a combination of stressors alters adaptive behaviour is unknown. Here we show that when these two stressors are combined, LTM formation is blocked. Since boiling CE inactivates the kairomone, we used previously boiled CE that we combined with the thermal stressor and found that LTM formation is again enhanced. These data show that (i) it cannot accurately be predicted how a combination of stressors when combined interact to alter LTM formation and (ii) there is a difference between hot CE and room temperature CE.

The Great Pond Snail (Lymnaea stagnalis) as a Model of Aging and Age-Related Memory Impairment: An Overview.

With the increase of life span, normal aging and age-related memory decline are affecting an increasing number of people; however, many aspects of these processes are still not fully understood. Although vertebrate models have provided considerable insights into the molecular and electrophysiological changes associated with brain aging, invertebrates, including the widely recognized molluscan model organism, the great pond snail (Lymnaea stagnalis), have proven to be extremely useful for studying mechanisms of aging at the level of identified individual neurons and well-defined circuits. Its numerically simpler nervous system, well-characterized life cycle, and relatively long life span make it an ideal organism to study age-related changes in the nervous system. Here, we provide an overview of age-related studies on L. stagnalis and showcase this species as a contemporary choice for modeling the molecular, cellular, circuit, and behavioral mechanisms of aging and age-related memory impairment.

Ion channel profiling of the Lymnaea stagnalis ganglia via transcriptome analysis

BackgroundThe pond snail Lymnaea stagnalis (L. stagnalis) has been widely used as a model organism in neurobiology, ecotoxicology, and parasitology due to the relative simplicity of its central nervous system (CNS). However, its usefulness is restricted by a limited availability of transcriptome data. While sequence information for the L. stagnalis CNS transcripts has been obtained from EST libraries and a de novo RNA-seq assembly, the quality of these assemblies is limited by a combination of low coverage of EST libraries, the fragmented nature of de novo assemblies, and lack of reference genome.ResultsIn this study, taking advantage of the recent availability of a preliminary L. stagnalis genome, we generated an RNA-seq library from the adult L. stagnalis CNS, using a combination of genome-guided and de novo assembly programs to identify 17,832 protein-coding L. stagnalis transcripts. We combined our library with existing resources to produce a transcript set with greater sequence length, completeness, and diversity than previously available ones. Using our assembly and functional domain analysis, we profiled L. stagnalis CNS transcripts encoding ion channels and ionotropic receptors, which are key proteins for CNS function, and compared their sequences to other vertebrate and invertebrate model organisms. Interestingly, L. stagnalis transcripts encoding numerous putative Ca2+ channels showed the most sequence similarity to those of Mus musculus, Danio rerio, Xenopus tropicalis, Drosophila melanogaster, and Caenorhabditis elegans, suggesting that many calcium channel-related signaling pathways may be evolutionarily conserved.ConclusionsOur study provides the most thorough characterization to date of the L. stagnalis transcriptome and provides insights into differences between vertebrates and invertebrates in CNS transcript diversity, according to function and protein class. Furthermore, this study provides a complete characterization of the ion channels of Lymnaea stagnalis, opening new avenues for future research on fundamental neurobiological processes in this model system.

Can Green Tea Alleviate the Effects of Stress Related to Learning and Long-Term Memory in the Great Pond Snail (Lymnaea stagnalis)?

Stress and anxiety have become more prevalent issues in recent years and teenagers are especially at high risk. Recent studies show that experiencing stress while learning can impair brain-cell communication, thus inhibiting learning. Green tea is believed to have the opposite effect, aiding in learning and memory retention. Lymnaea stagnalis is a pond snail with a simple nervous system and easily observable behaviors, making it an excellent model organism for human neurobiology. In this study, we used L. stagnalis to explore the relationship between green tea and a stressor that impairs memory formation to determine the effects of both green tea and stress on the snails’ ability to learn, form, and retain memories. Memory was assessed using a conditioning procedure known as conditioned taste aversion (CTA), where snails are exposed to a sweet substance followed by a bitter taste with the number of biting responses being recorded. For the CTA groups, a total of 33 snails in 3 separate cohorts (Control (i.e. no stress), stressed, and stressed with green tea) was used in the trainings. Our results indicated that the best learning and memory occurred in the combination stressed with green tea group, specifically for long-term memory, although snails displayed good learning and memory during the short-term and intermediate trials. Stress was shown to be harmful to snail learning and memory for short-term, intermediate, and long-term memory. Thus, green tea not only alleviated the effects of stress, but also improved the snails’ ability to learn and remember compared to their control counterparts (not stressed) group.

Progestogen-induced alterations and their ecological relevance in different embryonic and adult behaviours of an invertebrate model species, the great pond snail (Lymnaea stagnalis)

The presence of oral contraceptives (basically applying estrogens and/or progestogens) poses a challenge to animals living in aquatic ecosystems and reflects a rapidly growing concern worldwide. However, there is still a lack in knowledge about the behavioural effects induced by progestogens on the non-target species including molluscs. In the present study, environmental progestogen concentrations were summarised. Knowing this data, we exposed a well-established invertebrate model species, the great pond snail (Lymnaea stagnalis) to relevant equi-concentrations (1, 10, 100, and 500 ng L−1) of mixtures of four progestogens (progesterone, drospirenone, gestodene, levonorgestrel) for 21 days. Significant alterations were observed in the embryonic development time, heart rate, feeding, and gliding activities of the embryos as well as in the feeding and locomotion activity of the adult specimens. All of the mixtures accelerated the embryonic development time and the gliding activity. Furthermore, the 10, 100, and 500 ng L−1 mixtures increased the heart rate and feeding activity of the embryos. The 10, 100, and 500 ng L−1 mixtures affected the feeding activity as well as the 1, 10, and 100 ng L−1 mixtures influenced the locomotion of the adult specimens. The differences of these adult behaviours showed a biphasic response to the progestogen exposure; however, they changed approximately in the opposite way. In case of feeding activity, this dose-response phenomenon can be identified as a hormesis response. Based on the authors’ best knowledge, this is the first study to investigate the non-reproductive effects of progestogens occurring also in the environment on molluscan species. Our findings contribute to the global understanding of the effects of human progestogens, as these potential disruptors can influence the behavioural activities of non-target aquatic species. Future research should aim to understand the potential mechanisms (e.g., receptors, signal pathways) of progestogens induced behavioural alterations.

Disentangling the direct and indirect effects of agricultural runoff on freshwater ecosystems subject to global warming: A microcosm study

Aquatic ecosystems are exposed to multiple stressors such as agricultural run-off (ARO) and climate-change related increase of temperature. We aimed to determine how ARO and the frequency of its input can affect shallow lake ecosystems through direct and indirect effects on primary producers and primary consumers, and whether warming can mitigate or reinforce the impact of ARO. We performed a set of microcosm experiments simulating ARO using a cocktail of three organic pesticides (terbuthylazine, tebuconazole, pirimicarb), copper and nitrate. Two experiments were performed to determine the direct effect of ARO on primary producers (submerged macrophytes, periphyton and phytoplankton) and on the grazing snail Lymnaea stagnalis, respectively. Three different ARO concentrations added as single doses or as multiple pulses at two different temperatures (22°C and 26°C) were applied. In a third experiment, primary producers and consumers were exposed together to allow trophic interactions. When functional groups were exposed alone, ARO had a direct positive effect on phytoplankton and a strong negative effect on L. stagnalis. When exposed together, primary producer responses were contrasting, as the negative effect of ARO on grazers led to an indirect positive effect on periphyton. Periphyton in turn exerted a strong control on phytoplankton, leading to an indirect negative effect of ARO on phytoplankton. Macrophytes showed little response to the stressors. Multiple pulse exposure increased the effect of ARO on L. stagnalis and periphyton when compared with the same quantity of ARO added as a single dose. The increase in temperature had only limited effects. Our results highlight the importance of indirect effects of stressors, here mediated by grazers and periphyton, and the frequency of the ARO input in aquatic ecosystems.

Effect of photoperiod and light intensity on learning ability and memory formation of the pond snail Lymnaea stagnalis

Natural light is regarded as a key regulator of biological systems and typically serves as a Zeitgeber for biological rhythms. As a natural abiotic factor, it is recognized to regulate multiple behavioral and physiological processes in animals. Disruption of the natural light regime due to light pollution may result in significant effects on animal learning and memory development. Here, we investigated whether sensitivity to various photoperiods or light intensities had an impact on intermediate-term memory (ITM) and long-term memory (LTM) formation in the pond snail Lymnaea stagnalis. We also investigated the change in the gene expression level of molluscan insulin-related peptide II (MIP II) is response to the given light treatments. The results show that the best light condition for proper LTM formation is exposure to a short day (8 h light) and low light intensity (1 and 10 lx). Moreover, the more extreme light conditions (16 h and 24 h light) prevent the formation of both ITM and LTM. We found no change in MIP II expression in any of the light treatments, which may indicate that MIP II is not directly involved in the operant conditioning used here, even though it is known to be involved in learning. The finding that snails did not learn in complete darkness indicates that light is a necessary factor for proper learning and memory formation. Furthermore, dim light enhances both ITM and LTM formation, which suggests that there is an optimum since both no light and too bright light prevented learning and memory. Our findings suggest that the upsurge of artificial day length and/or night light intensity may also negatively impact memory consolidation in the wild.

Identification, presence, and possible multifunctional regulatory role of invertebrate gonadotropin-releasing hormone/corazonin molecule in the great pond snail (Lymnaea stagnalis)

In the last years, our interpretation of the origin and function of the gonadotropin-releasing hormone (GnRH) neuropeptide superfamily has changed substantially. A main driver for these conceptual changes came from increased investigations into functions and evolutionary lineage of previously identified molluscan GnRH molecules. Emerging evidence suggests not only reproductive, but also diverse biological effects of these molecules and proposes they should most likely be called corazonin (CRZ). Clearly, a more global understanding requires further exploration of species-specific functions and structure of invGnRH/CRZ peptides. Towards this goal, we have identified the full-length cDNA of invGnRH/CRZ peptide in an invertebrate model species, the great pond snail Lymnaea stagnalis, termed ly-GnRH/CRZ, and characterized the transcript and peptide distribution in the central nervous system (CNS) and peripheral organs. Our results are consistent with previous data that molluscan GnRHs are more related to CRZs and serve diverse functions. Hence, our findings support the notion that peptides originally termed molluscan GnRH are multifunctional modulators and that nomenclature change should be taken into consideration.