In addition, Lys-conopressin has been discovered in a leech, Erpobdella octoculata. Copyright © 2020 Elsevier B.V. or its licensors or contributors. Egg mass a crescent-shaped jelly strip containing many small eggs. As a result, asymmetric extracellular electric fields could be generated that might contribute to the establishment or maintenance of polar phenomena in eggs (Jaffe, 1982). Embryos were incubated at room temperature (20–22°C) in aerated, dechlorinated Miami-Dade County tap water (Grosell et al., 2007). An analog of the behavioral response occurs in the isolated central nervous system. The snail Lymnaea stagnalis The snail Lymnaea stagnalis sinks from the water surface to the ground of the pond. Saved from google.co.uk. Thus, Lymnaea is an excellent candidate for system-level analysis of learning. Lymnaea stagnalis (Linnaeus, 1758) Lymnaea stagnalis (adult size 45-60 mm) Diagnostic features. Hypoxia triggers pneumostome opening, the CPG-driven motor mechanism of aerial respiration (Figure 2). To maintain the spike waveform in CGC, however, it is necessary to hypothesize an increase in IHVA. Based on the published partial Ovipostatin gene sequence, we now provide the complete mRNA and gene sequences, and confirm that … The aerial respiratory behavior of Lymnaea stagnalis has been used in a series of experiments investigating the behavioral and neuronal mechanisms of operant conditioning. This was the first report of amino acid sequences of insulin-family peptide precursors in invertebrates. In this paper we investigate how the sequence of the various components of the egg-laying behaviour pattern is achieved. When the animal stays in stagnant water where the oxygen content is low, it becomes hypoxic. & Smit A. [15], This article incorporates CC-BY-2.0 text from references[9][11] and CC-BY-2.5 text from the reference[10]. The upper whorls are pointed, the last whorl is suddenly inflated, so that its diameter is more than a continuous increase of that of the upper whorls. Both reconsolidation and extinction have been studied following operant conditioning and both have been shown to be dependent on new RNA and protein synthesis. ScienceDirect ® is a registered trademark of Elsevier B.V. ScienceDirect ® is a registered trademark of Elsevier B.V. URL: https://www.sciencedirect.com/science/article/pii/B9780123705099000905, URL: https://www.sciencedirect.com/science/article/pii/B9780123850959000397, URL: https://www.sciencedirect.com/science/article/pii/B9780123971791000208, URL: https://www.sciencedirect.com/science/article/pii/B9780128010280002099, URL: https://www.sciencedirect.com/science/article/pii/B978012809633890788X, URL: https://www.sciencedirect.com/science/article/pii/B9780123705099000115, URL: https://www.sciencedirect.com/science/article/pii/B9780080450469008044, URL: https://www.sciencedirect.com/science/article/pii/B9780124158238000071, URL: https://www.sciencedirect.com/science/article/pii/B9780128010280002002, Learning and Memory: A Comprehensive Reference, Lukowiak K, Sangha S, Scheibenstock A, et al. In 1988, cDNAs encoding MIPs were cloned from the L. stagnalis LGCs. None of these two control groups expressed a change in their aerial respiratory behavior. [11] Lymnaea stagnalis perform more inseminations in larger groups and prefer to inseminate novel over familiar partners. In the pond snail Lymnaea stagnalis, clusters of neuroendocrine cells called light green cells (LGCs) are involved in the control of body growth. Anna Di Cosmo, Gianluca Polese, in Handbook of Biologically Active Peptides (Second Edition), 2013. A more simple procedure, based on a single training trial, also forms long-term memory. Radix auricularia. The hatching synchrony of embryos within individual egg masses was examined in order to understand the factors that influence the rate of embryonic development of Lymnaea. This motivated behavior is easily observable and quantifiable in terms of pneumostome openings (i.e., number of breaths) and total time the pneumostome is open (i.e., duration of breath). However, this behavior can be motivated by hypoxic water conditions. Learning takes place if the number of attempted pneumostome openings is significantly decreased between the first and the last training trial. Lys-conopressin evokes Ca2+-dependent Cl− currents in Xenopus oocytes injected with LSCPR cRNAs. Removal of the RPeD1 soma 1 h after conditioning had no effect on LTM, indicating that the effects of soma ablation were not related to memory access or retrieval. The model simulates the rhythmic neural activity that mediates feeding behavior. It has since been demonstrated that a memory for this conditioned response could persist for at least 4 weeks when a spaced training procedure was used. Belgium, British Isles: Great Britain and Ireland, Canada (Alberta province, Ottawa valley), Cambodia, Czech Republic[3] – least concern (LC),[4] Germany – distributed in whole Germany but in 2 states in red list (Rote Liste BRD),[5] Netherlands,[6] Poland, Russia, Slovakia,[3] Sweden (Skåne), Switzerland, Ukraine. The egg mass emerges from regulation of female reproduction in detail is the freshwater the female gonopore and is fixed to the substrate in the ovipos- pulmonate snail Lymnaea stagnalis (reviewed by Koene, 2010). The shell is also tan colored and does not have any obvious markings. In the great pond snail, Lymnaea stagnalis, an ACP affecting egg laying, ovipostatin, is produced in the prostate gland. Oviposition by Lymnaea stagnalis (L.) on shells of conspecifics has been reported anecdotally from laboratory observations. Interestingly, both extinction and reconsolidation also require the presence of the soma of RPeD1, indicating that RPeD1 is involved in the formation of more than one type of memory trace. The paper presents a study leading to a new acute toxicity test on embryonic and juvenile organisms of the great pond snail (Lymnaea stagnalis Linnaeus). The CDCs are a net- work of about 100 electrotonically coupled cells From Lukowiak K, Sangha S, Scheibenstock A, et al. Juřičková L., Horsák M. & Beran L. (2001) "Check-list of the molluscs (Mollusca) of the Czech Republic". DOI: 10.1007/BF00614510 Corpus ID: 25746014. Shells are brown in colour. of egg cells (Nuccitelli, 1983). Douglas A. Baxter, ... John H. Byrne, in Handbook of Behavioral Neuroscience, 2013. Sexual conflicts often arise between mating partners because each sex tries to maximize its own reproductive success. Suitable yoked and hypoxic control groups were also used. Eggs. MIPs are thought to be responsible for the roles of LGCs in the regulation of growth, metabolism, and reproduction. These egg masses measure between five and six centimetres in length, and can contain as many as 50 to 120 eggs. This behavior is controlled by a central pattern generator (CPG), and the neurons and synaptic connections in the CPG are well-characterized (for review, see64). Feng Z-P., Zhang Z., Kesteren R. E. van, Straub V. A., Nierop P. van, Jin K., Nejatbakhsh N., Goldberg J. I., Spencer G. E., Yeoman M. S., Wildering W., Coorssen J. R., Croll R. P., Buck L. T., Syed N. I. 1988) because of the structural similarities between precursors of Aplysia californica and Lymnaea stagnalis egg-laying hormones. This Test Guideline is designed to assess effects of prolonged exposure to chemicals on the reproduction and survival of the hermaphrodite freshwater snail Lymnaea stagnalis (the Great Pond Snail). Lymnaea stagnalis is an aquatic pulmonate snail (Figure 5). title = "The effect of light on induced egg laying in the simultaneous hermaphrodite Lymnaea stagnalis. The pond snail Lymnaea stagnalis is a simulta- neous hermaphrodite where the critical event that determines when reproductively mature animals will lay eggs is the discharge of the neuroendocrine caudodorsal cells (CDCs). A chemosensory stimulus (here hypoxia) activates sensory neurons (SNs) in the pneumostome area, which in turn provide excitatory input (green line) to the right pedal dorsal 1 interneuron (RPeD1). In this paradigm, as soon as the animal opens its pneumostome, it is placed for 30 s into a watch glass containing a punisher composed of a high concentration of potassium chloride. The respiratory behavior and underlying neuronal circuitry are extremely well characterized and are reviewed in another chapter (See Chapter 1.30) as well as several previous reviews (Lukowiak, 1991, 2001; Lukowiak and Syed, 1999; Benjamin et al., 2000; Lukowiak et al., 2003b, 2006b). From the sequences, it is suggested that MIP consists of A- and B-chains like vertebrate insulin, but has an extra disulfide bond between the chains. The great pond snail lays large gelatinous egg-masses on weeds and other objects in the tank/pond. 158: 37–62. IP3 also produces an inhibitory effect on VD4, and after release from this inhibition, VD4 fires, resulting in pneumostome closure (C). Sulfuric acid, nitric acid, and ammonium hydroxide were used as waterborne toxicants in laboratory experiments. Memory of the punishment is tested by comparing the number of pneumostome opening at different periods after the last training session. Giant forms of this species can grow to 7 cm shell size, but depending on the environmental conditions in waterside habitats there can also be dwarf forms. Thus, the possible role of IHVA represents an important prediction of the model. [8], Lymnaea stagnalis is widely used for the study of learning, memory and neurobiology. This paper describes the egg laying behavior of intact and freely behaving animals as it occurs spontaneously … Specifically, spontaneous patterned activity in the IP3 interneuron, which is involved in pneumostome opening (see Figure 1(b)), showed a significant reduction compared to activity in the IP3 neurons of brains derived from yoked controls. It is widely distributed, and is common in many countries and islands including: B. Lymnaea stagnalis lays large gelatinous egg-masses on weeds and other objects in the pond. The effects of conditioning on IHVA have yet to be examined empirically. Both intermediate memory (ITM) and LTM have been described based partly on the length of time the memory persists and also on the sensitivity to protein and mRNA synthesis blockers. Tsuyoshi Kawada, in Handbook of Hormones, 2016. Biol. The compact central nervous system of Lymnaea, with its beautiful brightly-pigmented orange neurons has been attractive to many neuroscientists interested in relating the molecular and electrical properties of neurons to behavior. Lymnaea stagnalis individuals are easily maintained in laboratory conditions, with a lifespan of about two years. This cycle of expiration/inspiration is repeated several times before the pneumostome closes again, signaling the end of an aerial respiratory bout. Figure 5. (b) Schematic drawing of the central pattern generator (CPG). This hypothesis is also thought to apply to Lymnaea stagnalis (Vreugdenhil et al. The enhancement of the feeding motor program appears to be due to facilitation of the motor neurons, the modulatory neurons, and presumably the CPG neurons, resulting in increased activation of the CPG cells by mechanosensory inputs from the lips (Kemmenes, 2013). The neural network included cells N1, N2, and N3, which mediate the rhythmic neural activity underlying feeding movements, and cell SO, which is a modulatory neuron. The height of an adult shell of this species ranges from 45–60 mm. It opens and closes its respiratory orifice, the pneumostome, and breathes through the lung (Figure 6(a)). ANI prevents the formation of both ITM and LTM, whereas Act-D only prevents LTM. "Larval trematode communities in, 10.2305/IUCN.UK.2017-3.RLTS.T155475A42428297.en, https://web.archive.org/web/20090925182719/http://www.lymnaea.org/, Arnaud Giusti, Pierre Leprince, Gabriel Mazzucchelli, Jean-Pierre Thomé, Laurent Lagadic, Virginie Ducrot, Célia Joaquim-Justo : Proteomic Analysis of the Reproductive Organs of the Hermaphroditic Gastropod Lymnaea stagnalis Exposed to Different Endocrine Disrupting Chemicals; PLOS|ONE 19 November 2013, https://en.wikipedia.org/w/index.php?title=Lymnaea_stagnalis&oldid=987109536, Articles with Czech-language sources (cs), Articles with Dutch-language sources (nl), Creative Commons Attribution-ShareAlike License. Great Pond Snail Lymnaea stagnalis. The number of openings, latency to first opening, and total breath durations were recorded in pre- and posttraining periods. For each treatment, L stands for light and D for dark. We use cookies to help provide and enhance our service and tailor content and ads. Benjamin, G. Kemenes, in Encyclopedia of Neuroscience, 2009. In the pond snail Lymnaea stagnalis egg laying is induced by multiple peptides, which are released by the neuroendocrine caudo-dorsal cells (CDCs). Koene J. M., Sloot W., Montagne-Wajer K., Cummins S. F., Degnan B. M., Smith J. S., Nagle G. T. & Maat A. ter (2010). Egg laying in Lymnaea starts at about 50 days and increases initially with age to decreases again at an age of about 250 days. Then, the mantle muscles contract and expel gas from the lung. The size to which a specimen will grow is dependent upon the volume of water in the pond; individuals grow larger in big ponds. Extinction is viewed as a new type of associative memory that ‘covers up’ but does not replace the original memory. The number of openings is recorded during training periods and retention tests. Greater levels of rasping, a component of the feeding behavior, can be produced by a single trial, and this response can persist for at least 19 days. It is a bimodal breather and can breathe via its skin (cutaneous respiration) or through a simple lung (aerial respiration). Image result for Lymnaea stagnalis Feeding behavior of the pond snail Lymnaea stagnalis can be modified by classical conditioning (for review, see63). The Lys-conopressin gene in the pond snail is composed of three exons and two introns at exactly the same locations as in the OXT/VP genes of vertebrates. Such higher motivation to copulate when a new partner is encountered is known as the Coolidge effect and has been demonstrated in hermaphrodites firstly in 2007.[11]. Lymnaea stagnalis, better known as the great pond snail, is a species of large air-breathing freshwater snail, an aquatic pulmonate gastropod mollusk in the family Lymnaeidae. This page was last edited on 4 November 2020, at 23:55. Reproducing adults of L. stagnalis are exposed to a concentration range of the test chemical and monitored for 28 days for their survival and reproduction (number of egg clutches). Great pond snail - Lymnaea stagnalis. Tactile stimulation of the pneumostome area evokes pneumostome closure and stops aerial respiratory behavior. The animal emits several successive bouts of aerial respiration before submerging. John H. Byrne, ... Richard F. Thompson, in From Molecules to Networks (Third Edition), 2014. Subsequent muscle relaxation allows passive re-inflation of the lung. 551: 311–318. Lymnaea stagnalis – Great Pond Snail Introduction The long, pointed, slightly bulbous, light brown to dark brown shell of the Great Pond Snail, Lymnaea stagnalis, from Central Europe is up to 6 cm long in the garden pond, in the aquarium the animals usually remain significantly smaller. Med. The CGCs are modulatory interneurons and are a locus of plasticity following appetitive classical conditioning.65 The CGC is modeled as a single compartment, which includes (1) transient and persistent Na+ currents (INaT and INaP, respectively), (2) an A-type and a delayed-type K+ current (IA and ID, respectively), and (3) low- and high-voltage-activated Ca2+ currents (ILVA and IHVA, respectively). The snail Lymnaea stagnalis: Neuronal network underlying respiratory behavior. Koene J. M. & Maat A. T. (6 November 2007) ", Kudlai O. S. (2009). Moreover, the essential neurons for generating pneumostome opening and closing have been identified, allowing this system to help uncover causal neuronal and molecular processes of operant conditioning, memory formation and their regulation by factors such as forgetting and stress. An important next step will be to combine the CPG and CGC models and examine the extent to which the currently identified cellular correlates of conditioning can reproduce learning-induced changes in behavior. For terms see gastropod shell In Lymnaea stagnalis, during discharges, CDCH-I is released into the blood, where it acts as a hormone that stimulates ovulation and egg mass formation and synthesis of secretory products in the female accessory sex glands and affects neurons in the neuronal circuits controlling locomotion and feeding. In eumoxic conditions, it essentially performs cutaneous respiration and expresses only 1–2 aerial respirations per hour. ; Painter, Sherry D. In: Invertebrate Reproduction and Development, Vol. In the operantly trained group, a tactile stimulus was applied to the pneumostome area each time aerial respiration was attempted by the animal. As a second step in modeling learning in Lymnaea, Vavoulis et al.45 modeled the cerebral giant cells (CGCs). Egg masses of the common pond snail Lymnaea stagnalis (L.) were collected from our laboratory snail culture at 24-h intervals, with the day of collection considered day 0 post-oviposition, for age determination. By continuing you agree to the use of cookies. United States (Utah) – Lymnaea stagnalis appressa.[7]. While single-trial classical conditioning has provided detailed information on specific molecular mechanisms of LTM, work based on the operant conditioning of the suppression of aerial respiration has provided comprehensive information on the general molecular requirements for different memory phases (intermediate-term memory (ITM) and LTM) and active processes related to associative memory, such as forgetting, extinction, and reconsolidation. Limnaea stagnalis var. (2003) A molluscan model system in the search for the engram. J. Exp. Two Lys-conopressin receptors (LSCPR1 and LSCPR2) have also been identified from the pond snail, and both LSCPR genes are expressed in the brain and reproductive tissues. Hoffer J. N. A., Ellers J. (2004) Memory, reconsolidation and extinction in Lymnaea require the soma of RPeD1. The biological basis for operant conditioning in Lymnaea lies in the fact that these pond snails are bimodal breathers; they can breathe both through the skin (cutaneous respiration) and via a simple lung (aerial respiration), which is supplied with air through a respiratory orifice called the pneumostome (Figure 2). 1-3, 01.01.1999, p. 171-174. "Costs of receipt and donation of ejaculates in a simultaneous hermaphrodite". These egg masses measure between five and six centimetres in length, and can contain as many as 50 to 120 eggs. The size to which a specimen will grow is dependent upon the volume of water in the pond; individuals grow larger in big ponds. In Lymnaea stagnalis, during discharges, CDCH-I is released into the blood, where it acts as a hormone that stimulates ovulation and egg mass formation and synthesis of secretory products in the female accessory sex glands and affects neurons in the neuronal circuits controlling locomotion and feeding. We have studied the ionic currents occurring in the egg of Lymnaea stagnalis during maturation and the first mitotic cell cycle. Naoki Okamoto, in Handbook of Hormones, 2016. (23 September 2009) "Transcriptome analysis of the central nervous system of the mollusc. Individual neurons can be identified as parts of behavioral circuits and their synaptic connectivities dete… The behavioral changes were found to be consolidated into intermediate- (lasting 2–3 h) and long-term (lasting up to 6 h after training) memories depending on the number of, or interval between, the training sessions. Only the operantly conditioned group showed significant changes between the pre- and posttraining behaviors, with significant reductions in openings and total breathing time and significant increases in the latency to first breath. The axonal compartment includes a fast, transient Na+ current (INA) and a delayed-like K+ current (IK), which mediate spike activity. To perform aerial respiration, the snail moves to the water surface and when the pneumostome is in contact with the atmosphere, it opens fully. & Koene J. M. (2010). Two of the currents, INaP and ID, are increased following conditioning.45,65 Thus, the effects of conditioning are simulated by increasing the maximal conductances of INaP and ID. They are only designated differently for the reader’s convenience. They are cannibalistic, eating smaller great pond snails. The aerial respiratory behavior of Lymnaea stagnalis has been used in a series of experiments investigating the behavioral, neuronal, and molecular mechanisms of operant conditioning. Furthermore, a higher percentage of RPeD1 CPG interneurons, which are important in the onset of the respiratory cycle, were silent in conditioned versus control preparations. Such higher motivation to copulate when a new partner is encountered is known as the Coolidge effect and has been demonstrated in hermaphrodites firstly in 2007. When reared at 25°C, individual embryos from single egg masses hatch … 36, No. More direct evidence for the role of RPeD1 in memory formation came from somal ablation experiments. The 40–50 x 22–30 mm. InLymnaea egg laying is initiated by the discharge of the neurosecretory caudodorsal cells (CDCs), which release an ovulatory hormone (caudodorsal cell hormone; CDCH) and other peptides. The number of openings, latency to first opening, and total breath durations were recorded in pre- and posttraining periods. Lymnaea stagnalis perform more inseminations in larger groups and prefer to inseminate novel over familiar partners. [9], Lymnaea stagnalis has a relatively simple central nervous system (CNS) consisting of a total of ~20,000 neurons, many of them individually identifiable, organized in a ring of interconnected ganglia. A reduction in the ability of the RPeD1 cells to induce IP3 activity was also observed. Tactile stimulation of the pneumostome area evokes closure of the pneumostome, and the aerial respiratory behavior stops. (2010). The umbilicus is closed. Lymnaea stagnalis lays large gelatinous egg-masses on weeds and other objects in the pond. Simulations reproduce some of the previously identified neuronal changes following conditioning, including a depolarization in CGC without a change in tonic firing in CGC. In an operantly trained group, a tactile stimulus was applied to the pneumostome area each time aerial respiration was attempted by the animal or semi-intact preparation in an artificially created hypoxic environment (Lukowiak et al., 1996). The somatic compartment includes currents (IACh, INaL, or IT), which mediate slowly developing, long-lasting changes of the membrane potential, such as plateau potentials in N1 and N2 and postinhibitory rebound in N3. & Perez-del-Olmo A. ", abstract = "Reproduction is influenced by many external factors. Lymnaea stagnalis is a simultaneously hermaphroditic species and can mate in the male and female role, but within one copulation only one sexual role is performed at a time. MIP receptor is a receptor tyrosine kinase having the typical features of an insulin receptor or IGF type-I receptor. Lymnaea stagnalis is a simultaneously hermaphroditic species and can mate in the male and female role, but within one copulation only one sexual role is performed at a time. "Infection success of, Soldanova M., Selbach C., Sures B., Kostadinova A. Jul 24, 2018 - Explore Rosemary Chalmers's board "Lymnaea stagnalis", followed by 129 people on Pinterest. / Nagle, Gregg Thomas; Akalal, David B.G. Tactile stimulation of the pneumostome area evokes reflexive pneumostome closure and thus suppresses aerial respiratory behavior. Exp. Only the operantly conditioned group showed significant changes between the pre- and posttraining behaviors, with significant reductions in openings and total breathing time and significant increases in the latency to first breath. The shell spire is elongated and pointed, first increases slowly, but … G. Kemenes, in Learning and Memory: A Comprehensive Reference, 2008. D. Eisenhardt, N. Stollhoff, in Learning and Memory: A Comprehensive Reference, 2008. αBCP mimics bag cell-induced inhibition of several identified and unidentified neurons. North America: The snails are hermaphrodites and sexual maturity occurs about three months after egg laying. Maternal impact on egg development in lymnaea stagnalis : A growth factor is produced by the albumen gland in the reproductive tract. The animal is then transferred into a eumoxic aquarium. Thus, following extinction trials, the loss of memory at 2 h is followed by full ‘spontaneous recovery’ at 24 h. Neural changes associated with this learned behavior have been identified in the isolated CNS derived from operantly conditioned animals. MIPs have so far been identified in a number of molluskan species, with several MIPs in most species. Variable development within an aggregated egg mass has been reported in a variety of marine species, e.g. These animals are placed in the high potassium chloride solution regardless of their behavior. Most neurons of the Lymnaea stagnalis central nervous system are large in size (diameter: up to ~100 μm), thus allowing electrophysiological dissection of neuronal networks that has yielded profound insights in the working mechanisms of neuronal networks controlling relatively simple behaviors such as feeding, respiration, locomotion, and reproduction. Additional control animals are set into similar glass-watches but containing fresh-water. Development direct. See more ideas about Pond snails, Image, Snail. IP3 in turn excites both RPeD1 and the I/J motor neurons involved in pneumostome openings (O). Figure 6. Lymnaea stagnalis: This species is very large when compared to the other 2 species in this group. Romuald Nargeot, Laura Puygrenier, in Reference Module in Life Sciences, 2019. The circuit consists of a network of three types of CPG neurons, 10 types of motor neurons, and a variety of modulatory interneurons. Individual neurons in the neural network were represented by two-compartment models (Figure 7.2B1). Hypoxia triggers pneumostome opening and this was used as the operantly conditioned behavior. This chapter describes Lymnaea stagnalis egg laying as a model system for the study of the ways in which peptidergic systems may trigger a complex set of overt and covert behaviors. Great Pond Snail Lymnaea stagnalis sack containing eggs. Moreover, neuronal correlates of learning are known. ELH induces ovulation and alters the electrical activities of several neurons in the abdominal, buccal, and pedal ganglia. Yoked-control animals allow testing the contribution of this operant/punisher association. Test animals. Lys-conopressin was isolated from the pond snail Lymnaea stagnalis, and consists of nine amino acids containing consensus amino acids of the OXT/VP superfamily peptides: Cys1, Asn5, Cys6, Pro7, Gly9; and C-terminal amidation. Removal of the soma 2 h prior to conditioning prevented LTM but had no effect on ITM, without affecting the ability of the snail to carry out respiratory behavior; this suggested that the RPeD1 soma was necessary for LTM formation. The pond snail Lymnaea stagnalis is a model molluscan system used in many laboratories to study a wide range of fundamental neurobiological problems (Kemenes & Benjamin, 2009). The 'Great Pond Snail' moving across the bottom of a shallow river bed on a bright sunny day. Syed NI Winlow W (1991) Coordination of locomotor and cardiorespiratory networks of Lymnaea stagnalis by a pair of identified interneurones. wandering pond snail underwater in aquarium. In an operant conditioning procedure in a hypoxic N2–rich environment, each spontaneous attempt at pneumostome opening was associated with delivery of a punisher; a tactile stimulation applied with a wooden applicator to the pneumostome (Lukowiak et al., 1996, 2003).