Freshwater Snail Planorbarius Corneus Research Articles

Benzodiazepine Delorazepam Induces Locomotory Hyperactivity and Alterations in Pedal Mucus Texture in the Freshwater Gastropod Planorbarius corneus

Benzodiazepines, psychotropic drugs, are ubiquitous in the aquatic environment due to over-consumption and inefficient removal by sewage treatment plants. Bioaccumulation with consequent behavioral and physiological effects has been reported in many aquatic species. However, the responses are species-specific and still poorly understood. To improve the knowledge, we exposed the freshwater snail Planorbarius corneus to 1, 5, or 10 µg/L of delorazepam, the most widely consumed benzodiazepine in Italy. Conventional behavioral tests were used to assess the effects on locomotor and feeding behavior. Histological and biochemical analyses were also performed to detect possible changes in the structure and composition of the foot mucus and glands. The results show a paradoxical response with reduced feeding activity and locomotor hyperactivity. Pedal mucus was altered in texture but not in composition, becoming particularly rich in fibrous collagen-like material, and a significant change in the protein composition was highlighted in the foot. In conclusion, exposure to delorazepam induces disinhibited behavior in Planorbarius corneus, potentially increasing the risk of predation, and an increase in mucus protein production, which, together with reduced feeding activity, would severely compromise energy resources.

Fitness recovery and two-generation phenotypic plasticity in the freshwater snail Planorbarius corneus L. exposed to hyperosmotic solutions

Background and purpose: Hyperosmotic solutions of calcium chloride and calcium bromide, extensively used in the oil industry, can be a source of aquatic pollution.Materials and methods: In the present study, we investigated whether the freshwater snail Planorbarius corneus L., previously chronically exposed to toxic concentrations of these chemicals, can recover fecundity and growth in clean water. We also examined if a higher tolerance could be induced in F1 offspring after parental and developmental chronic exposure to the chemicals.Results: The fitness-recovery study showed a high compensatory potential of the snails to recover fitness through increased reproduction and growth. In the tolerance study, we observed increased tolerance in F1 offspring in hyperosmotic solutions of calcium chloride. However, there was no significant improvement of reproduction performance in bromide-containing solutions.Conclusions: Increased tolerance was associated with a redirection of resources from growth to maintenance and survival.

Effect of substratum drying on the survival and migrations of a freshwater pulmonate snail Planorbarius corneus (Linnaeus, 1758)

Pulmonate snails inhabiting the near-shore zone of water bodies experience air exposure during water level fluctuations and some of them are known to survive long time in temporary basins. In laboratory, we investigated survival and behavioural defences (horizontal migrations and burrowing in substratum) of adult pulmonate snails Planorbarius corneus during substratum (sand and organic-rich) drying. We examined horizontal migrations at shorter and longer distances (24 and 48 cm), during gradual (8 days) or sudden water level drop rates. P. corneus was remarkably resistant to substratum drying, surviving 50 days on sand and more than 53 days on organic-rich bottom. The snails followed the retreating water level and grouped in the submerged parts of experimental tanks. This response was reduced on organic-rich bottom at a longer distance and with water level decreasing suddenly. Moreover, snails exposed to drying buried shallowly in both bottom types. Thus, P. corneus exhibited variable and efficient strategies enabling its survival during water level fluctuations, including active migrations following the retreating water level and burying in sediments.

Cholinesterase and carboxylesterase inhibition in Planorbarius corneus exposed to binary mixtures of azinphos-methyl and chlorpyrifos

Though pesticide mixtures are commonly encountered in fresh water systems, the knowledge of their effects on non-target aquatic species is scarce. The present investigation was undertaken to explore the in vivo inhibition of the freshwater gastropod snail Planorbarius corneus cholinesterase (ChE) and carboxylesterases (CES) activities by the organophosphorus pesticides azinphos-methyl (AZM) and chlorpyrifos (CPF). To this end, snails were exposed for 48h to different concentrations of AZM and CPF in single-chemical and binary-mixture studies, and ChE and CES activities were measured in the whole soft body tissues and hemolymph. ChE activity was measured with acetylthiocholine as substrate and CES activity was measured with four substrates: p-nitrophenyl acetate, p-nitrophenyl butyrate, 1- and 2-naphthyl acetate. Single-chemical experiments showed that CPF was a more potent inhibitor of ChE activity than AZM (350 and 27 times for the whole soft tissue and hemolymph, respectively). CES were 15 times more sensitive than ChE when the activities were measured in the whole soft tissue of the animals exposed to AZM. Based on a default assumption of concentration addition, P. corneus snails were exposed to mixtures of AZM+CPF designed to yield predicted soft tissue ChE inhibitions of 31% (mixture 1), 50% (mixture 2) and 61% (mixture 3). Results showed that ChE inhibition produced by mixture 1 followed a model of concentration addition. In contrast, the other mixtures showed synergism, both in whole soft tissue and hemolymph. In addition, results obtained when the snails were exposed sequentially to the pesticides showed that the sequence AZM/CPF produced at 48h a higher ChE inhibition than the sequence CPF/CPF. A range of metabolic pathways and responses associated with bioactivation or detoxification may play important roles in organophosphorus interactions. In particular, the data presented in the present study indicate that CES enzymes would be important factors in determining the effects of the mixtures of OPs on P. corneus ChE activity.

Afferent and efferent pathways in the visual system of the freshwater snail Planorbarius corneus

Afferent and efferent neural elements of the retina and central ganglia in the freshwater snail Planorbarius corneus were labelled using retrograde transport of neurobiotin through the optic nerve. Axons of at least some photoreceptor cells become direct contributors to the optic nerve as no synaptic junctions could be detected. The processes enter the cerebral ganglion and form a dense bundle of thin afferent fibres, the so-called optical neuropil. Efferent neurons were revealed in all ganglia, except the buccal ones. Some of the ascending axons branch in the cerebral ganglia, cross the cerebro-cerebral commissure, reach the contralateral eye and form arborizations in the eye cup. Some efferent neurons send axons to different peripheral nerves as well: n.n. intestinalis, pallialis dexter, pallialis sinister internus et externus. Serotonin- and FMRF-amide-ergic fibres were revealed in the optic nerve. These fibres belong to those central neurons which send their axons to the ipsilateral eye only. They form abundant varicoses in the eye cup and nuclear layer of the retina, and possibly help to regulate retinal sensitivity to light.

Characterization of a low-sulfated chondroitin sulfate isolated from the hemolymph of the freshwater snail Planorbarius corneus

Glycosaminoglycans (GAGs) from the hemolymph of the freshwater snail Planorbarius corneus were recovered at about 0.9 μg/mL, being composed of a unique species characterized as chondroitin sulfate (CS) with a molecular mass of approximately 31,000 and having glucuronic acid as hexuronic acid. This macromolecule was determined to be composed of a low-sulfated polysaccharide made up of approximately 25% of the nonsulfated disaccharide, 17% of the 6-sulfated disaccharide, and about 58% of the 4-sulfated disaccharide, with a charge density value of 0.75 and a 4-sulfated/6-sulfated ratio of approximately 3.4. The data obtained suggest that the CS recovered in the Planorbarius corneus hemolymph is similar to the main human plasma polysaccharide and it may be generated as a main product of the catabolic processes.

P.6.058 Inducible nitric oxide synthase in the locus coeruleus of human opiate addicts

The aim of the present study was to investigate the involvement of nitric oxide (NO) as a messenger molecule in neuron–microglia communication in the central nervous system (CNS) of the freshwater snail Planorbarius corneus. The presence of both neuronal (nNOS) and inducible nitric oxide synthase (iNOS) was studied using NADPH-diaphorase (NADPH-d) histochemistry and NOS immunocytochemistry. The experiments were performed on whole ganglia and cultured microglial cells after different activation modalities, such as treatment with lipopolysaccharide and adenosine triphosphate and/or maintaining ganglia in culture medium till 7 days. In sections, nNOS immunoreactivity was found only in neurons and nNOS-positive elements were less numerous than NADPH-d-positive ones, with which they partially overlapped. The iNOS immunoreactivity was observed only after activation, in both nerve and microglial cells. We also found that the number of iNOS-immunoreactive neurons and microglia varied, depending on the activation modalities. In microglial cell cultures, iNOS was expressed in the first generation of cells only after activation, whereas a second generation, proliferated after ganglia activation, expressed iNOS even in the unstimulated condition.

Microglia proliferation as a response to activation in the freshwater snail Planorbarius corneus: a BrdU incorporation study.

Invertebrate microglia constitute a class of cells resident in the ganglionic nervous system which are activated after tissue injury or by the presence of pathogens. The microglia activation response includes graduated morpho-functional and biochemical changes and cell proliferation. In this study we verified in the freshwater snail Planorbarius corneus that an activation caused by a traumatic event may induce microglia division. Cell proliferation was assessed immunocytochemically using BrdU incorporation technique and documented on both ganglionic sections and microglia cultured cells at different experimental conditions and times after activation. In addition, we studied the possibility of increasing microglia proliferation by adding to the cultured medium the Macrophage-Colony Stimulating Factor (M-CSF) that has been shown to stimulate specifically this process in vertebrates.

Neuron-microglia communication in the CNS of the freshwater snail Planorbarius corneus.

The aim of the present study was to identify molecules that may be involved in neuron-microglia communication in the CNS of freshwater snail Planorbarius corneus. Messenger molecules are exchanged in normal and pathological conditions and we tried to identify some of them by immunocytochemistry on whole ganglia and cell cultures. In particular, we examined neurons and microglia for the expression of some cytokines, IL-1alpha, IL-1beta, IL-6 and TNF-alpha and the neurotransmitter glutamate. These substances may be released by suffering or injured neurons and communicate to microglia the damaging event. Even microglia, on own turn, once activated, express and released the same or other substances in order to reestablish the system homeostasis, depending on modalities and times of activation. We discuss the possibility that hyperactivated microglia can shift from neuroprotective to neurodegenerative. Moreover, we examined in neuron-microglia co-coltures the direct interaction effects in terms of neuronal survival and improved neurite regeneration.

Presence and role of nitric oxide in the central nervous system of the freshwater snail Planorbarius corneus: possible implication in neuron–microglia communication

The aim of the present study was to investigate the involvement of nitric oxide (NO) as a messenger molecule in neuron–microglia communication in the central nervous system (CNS) of the freshwater snail Planorbarius corneus. The presence of both neuronal (nNOS) and inducible nitric oxide synthase (iNOS) was studied using NADPH-diaphorase (NADPH-d) histochemistry and NOS immunocytochemistry. The experiments were performed on whole ganglia and cultured microglial cells after different activation modalities, such as treatment with lipopolysaccharide and adenosine triphosphate and/or maintaining ganglia in culture medium till 7 days. In sections, nNOS immunoreactivity was found only in neurons and nNOS-positive elements were less numerous than NADPH-d-positive ones, with which they partially overlapped. The iNOS immunoreactivity was observed only after activation, in both nerve and microglial cells. We also found that the number of iNOS-immunoreactive neurons and microglia varied, depending on the activation modalities. In microglial cell cultures, iNOS was expressed in the first generation of cells only after activation, whereas a second generation, proliferated after ganglia activation, expressed iNOS even in the unstimulated condition.

Increase in number and size of kidney concretions as a result of PCP exposure in the freshwater snail Planorbarius corneus (Gastropoda, Pulmonata).

Molluscan kidneys are able to excrete solids in the urine in the form of concretions. It is thought that increased formation of these concretions occur under pollutant, environmentally or reproductive induced stress. This study examined the formation of concretions in the kidney of the freshwater snail Planorbarius corneus L. experimentally exposed to pentachlorophenol (PCP). Light microscopic histopathological analysis of the PCP-exposed P. corneus revealed significantly enhanced production of the kidney concretions when compared to the kidneys of control individuals. Measurements of the number of kidney concretions, the apparent area of the concretions, and the epithelial area filled with concretions indicated an increase in the number and size of concretions in all treated snails. Lipofuscin content of excretory cell concretions was detected.

Effects of CaCl 2 and CaBr 2 on the fecundity of Planorbarius corneus L.

Saturated water solutions of calcium chloride (481.3 g CaCl 2/L) and calcium bromide (1065.9 g CaBr 2/L) and their mixtures, commercially prepared as “high density brines”, are regularly used during special operations in exploration and production of natural gas and crude oil. After operations, all workover fluids are left in the environment as waste. In the present study, the effects of several sublethal concentrations of these solutions (CaCl 2 1202 ppm, 2403 ppm and 4796 ppm, CaBr 2 1065 ppm, 2663 ppm and 5325 ppm, 1:1 volume mixture 773.9 ppm, 1931 ppm, 3855 ppm and 5779 ppm) on the fecundity of the freshwater snail Planorbarius corneus L. were laboratory tested. During the six week exposure a considerably reduced fecundity was found. The tested CaCl 2· solution concentrations reduced fecundity by 53.9–84.4 %, CaBr 2 by 72.3–90.8 % and the mixture by 72.5–94.0 % compared with the control groups.

Opiate signaling regulates microglia activities in the invertebrate nervous system

1. 1. Evidence supporting the presence in the invertebrate nervous system of a class of glial cells resembling vertebrate microglia was obtained in the freshwater snail Planorbarius corneus. These cells are easily identified by their immunopositivity to anti-pro-opiomelanocortin (POMC) derived peptide antibodies. 2. 2. Invertebrate microglia, as in vertebrates, exhibit macrophage-like activity in vivo and in cell cultures. These cells respond to the trauma of ganglionic excision and their organotypic culture by leaving their location around neurons and moving to the lesion site from which they migrate in the culture dish. 3. 3. In vitro, these microglia undergo conformational changes and show phagocytic properties in the presence of bacteria or lipopolysaccharide. The activated cells also express tumor necrosis factor-α-like material and an increase in nitric oxide synthase, as shown by immunocytochemistry. 4. 4. The inhibitory effect of morphine on the mobility and phagocytic activity of invertebrate microglia provide additional functional evidence for a possible role of opiate-like compounds in downregulating immunoregulatory processes, as also observed in the circulating immunocytes.

Lipid peroxidation and histopathological changes in the digestive gland of a freshwater snail Planorbarius corneus L. (Gastropoda, Pulmonata) exposed to chronic and sub-chronic concentrations of PCP.

Lipid peroxidation and histopathological changes in the digestive gland of a freshwater snail Planorbarius corneus L. (Gastropoda, Pulmonata) exposed to chronic and sub-chronic concentrations of PCP.

MDL 12330A inhibits the non-neuronal adenylyl cyclase from the freshwater snail Planorbarius corneus, but the neuronal enzyme is activated by this compound

N-(Cis-2-phenyl-cyclopentyl)azacyclotridecan-2-imine-hydrochloride (MDL 12330A), considered an inhibitor of adenylyl cyclase, has been tested on the enzyme activity of neuronal and non-neuronal tissues from the freshwater snail Planorbarius corneus. The drug dose-dependently activates the basal as well as agonist-stimulated adenylyl cyclase in the ganglionic preparations, while it exerts an inhibitory effect on the enzyme present in the non-nervous tissues examined. SQ 22536 and forskolin, respectively an inhibitor and activator of adenylyl cyclase, behave as generally reported both in central and peripheral tissues of the snail. This is, to our knowledge, the first report of a stimulatory action of MDL 12330A on an adenylyl cyclase system.

EFFECT OF SEROTONIN AND NEUROPEPTIDES ON ADENYLATE CYCLASE OF THE CENTRAL NERVOUS SYSTEM AND PERIPHERAL ORGANS OF THE FRESHWATER SNAIL PLANORBARIUS CORNEUS

The effect of serotonin, FMRFamide and the small cardioactive peptide B (SCPB) on adenylate cyclase activity of the central nervous system and some peripheral organs of the freshwater snail Planorbarius corneus was investigated. The amine and the cardioactive peptide stimulated the enzyme, although with different potencies, in all tissues studied and, when tested in combination, an additive activation was obtained. FMRFamide induced differential effects in the various targets: marked stimulation of adenylate cyclase, additive to that provoked by serotonin or SCPB, in salivary glands; inhibition of the enzyme, both alone and in combination with the other neuromediators, in the nervous tissue; whereas no influence was found in adenylate cyclase activity in the buccal mass. In the last of these tissues, the peptide might act through an intracellular second messenger other than cyclic AMP. The responsiveness of adenylate cyclase of these neuromediators in all the central ganglia suggested that they can exert an important role as neurotransmitters and/or neuromodulators in the central nervous system of the snail. Moreover, in the light of the differential sensitivity of adenylate cyclase in the salivary glands and buccal mass, we suggest that serotonin, FMRFamide and SCPB modulate the feeding behaviour of P. corneus in a complex way. Copyright © 1996 Elsevier Science Ltd.

Mechanisms of heterosynaptic facilitation in molluscan neurons

This review is focused on the analysis of research data obtained in one of the models of conditioned reflex, heterosynaptic facilitation (HSF), in the molluscan nervous system. Our experiments were performed on identified giant command neurons LS1 and PS1 of the freshwater snail Planorbarius corneus. HSF was elicited during the electrical stimulation of two nerves: pallial (the analog of unconditioned stimulation — US) and one of the cerebral nerves (the analog of the conditioned stimulation — CS). The degree of HSF manifestation depended not on the intensity of the synaptic response of the giant neuron to US, but the efficacy of the connection between the pallial nerve and neurosecretory neurons surrounding the command neuron of the mesocerebrum. It is demonstrated that HSF develops due to the diffuse neurohumoral action of serotonin (5-hydroxytryptamine — 5-HT) on the postsynaptic structures, but not as a result of local synaptic action on the presynaptic mechanism. Approximately 70% of US cases of 5-HT application induced a four- to six-fold increase in amplitude of the excitatory postsynaptic potential (EPSP) and acetylcholine (ACh) response. Both responses are N-cholinergic and depend on the membrane permeability to Na+ and K+. In 30% of the cases, ACh response diminished simultaneously with EPSP increase. The 5-HT effect on EPSP and ACh responses were mimicked by the action of phosphodiersterase blockers and adenylate cyclase activators. Thus, the activation of the adenylate cyclase system following 5-HT action facilitates the postsynaptic mechanism underlying HSF formation in command neurons of Planorbarius corneus. Dopamine (DA) and noradrenaline (NA) blocked EPSP and simultaneously increased the amplitude of ACh response. These monoamines were also blocked HSF. The wash-out of catecholamines following HSF blockade enhanced the restoration and subsequent prolongation of synaptic facilitation. It is thus concluded that DA or NA may control the HSF intensity and duration under natural conditions of the nervous system in the molluscs.

Effects of the small cardioactive peptide B (SCP B) on adenylate cyclase of the central nervous system and peripheral organs of the freshwater snail Planorbarius corneus

The effect of the small cardioactive peptide B (SCP B) on adenylate cyclase activity of the central nervous system (CNS) and some peripheral organs of the freshwater snail Planorbarius corneus was investigated. This peptide stimulates enzyme activity in a dose-dependent manner in all the tested ganglia, in the salivary glands, the buccal mass and the oesophagus. The amount required for half-maximal stimulation is approx 0.1 μM. Moreover, the response of adenylate cyclase to SCP B progressively increases with time, without desensitizing. On the other hand, the peptide is ineffective on heart adenylate cyclase. The responsiveness of adenylate cyclase activity to the peptide and the wide distribution of SCP B-related immunoreactivity in all the tested ganglia suggest that this substance may exert an important role as neurotransmitter as well as neuromodulator in the CNS of the snail. Since SCP B is consistently present in the buccal and cerebral ganglia, associated nerves, salivary glands, buccal mass and oesophagus, and in the light of the appreciable effects exerted by SCP B on adenylate cyclase activity in the examined peripheral tissues, we suggest that the peptide is strongly involved in the control of feeding behaviour of P. corneus.

Stress response in the freshwater snail Planorbarius corneus (L.) (Gastropoda, Pulmonata): Interaction between CRF, ACTH, and biogenic amines

Previous studies reported that ACTH molecules influence chemotactic and phagocytic activities of hemocytes in the freshwater snail, Planorbarius corneus. The present study reveals that ACTH and CRF affect the release of biogenic amines. Hemocytes from P. corneus hemolymph incubated in vitro with ACTH for 15, 30, and 45 min released epinephrine, norepinephrine, and dopamine. The greatest release occurred after 15 min, while after 45 min the values were similar to those of the controls. Similar incubations with CRF also provoked a release of biogenic amines, this being mainly mediated by the release of endogenous ACTH. These data suggest that (i) ACTH and CRF provoke the release of biogenic amines; (ii) there is a direct relationship between CRF, ACTH, and biogenic amines, with the hemocytes as the target; (iii) exogenous ACTH can mimic an ancestral type of stress response; (iv) the major pathway of the stress response in P. corneus is mediated by a CRF-ACTH-biogenic amine axis. These data should help to unravel part of the complex molecular signaling mechanisms involved in the physiological/endocrinological reaction of invertebrate organisms to stress, and suggest that a stress response unexpectedly similar to that present in mammalian cells is detectable in invertebrates.

Amino acids and derivatives as food-finding signals in the freshwater snail Planorbarius corneus (L.)

1. The behavioral responses of Planorbarius corneus to various amino acids and related substances were evaluated according to the principles of the sequential analysis method. 2. The amino acids L-aspartic acid, D-alanine, histamine and the dipeptide carnosine proved to be attractant; L-alanine, L-cysteine, L- and D-proline, L-histidine and the dipeptide aspartame are not attractant. 3. Addition of MgCl 2 in the stimulus sources renders attractive L- and D-proline, repellent L-histidine and increases the index of attraction of L-tryptophan. 4. The attraction to D-alanine and not to L-alanine indicates that amino acids could act as signals in the search for bacteria in which cell-wall D-alanine is present as a residue of a peptidoglycan. 5. The chemosensory spectrum of Planorbarius differs from that of closely related species, thus indicating a possible impact on the evolutionary biology of freshwater molluscs.