Freshwater Pulmonate Snail Lymnaea Stagnalis Research Articles

The toxicity and physiological effects of copper on the freshwater pulmonate snail, Lymnaea stagnalis

Several recent studies have demonstrated that the freshwater pulmonate snail Lymnaea stagnalis is extremely sensitive to metals (Co, Ni, Pb) in chronic exposures. The objective of the current study was to evaluate the acute and chronic sensitivity of L. stagnalis to Cu and investigate the underlying mechanism(s) of toxic action. A 96-h LC50 of 31μgL−1 Cu was estimated indicating L. stagnalis was moderately acutely sensitive to Cu relative to other aquatic organisms. However, in a 30-day chronic exposure using juvenile snails an EC20 of 1.8μgL−1 Cu was estimated for snail growth making L. stagnalis the most sensitive organism tested to date for Cu. Hardness-based and BLM-based water quality criteria for Cu at the water quality conditions used in this study were 7.8 and 1.5μgL−1, respectively, indicating L. stagnalis is significantly under-protected by hardness-based WQC. Investigations into the mechanism(s) of toxic action for Cu were conducted on young adult snails necessitating higher Cu exposures. Exposure to Cu at 12μgL−1 resulted in no detectable effects on hemolymph osmolality, net Ca2+ uptake, titratable acid excretion, or ammonia excretion. Exposure to 48μgL−1 Cu was shown to significantly reduce (91%) net Ca2+ uptake which is strongly correlated with shell deposition and corresponding snail growth. Snails exposed to 48μgL−1 Cu also exhibited reduced ammonia excretion, a marked hemolymph acidosis, and a compensatory increase in titratable acid excretion. The reduction in net Ca2+ uptake was hypothesized to be a secondary effect of Cu-induced inhibition of carbonic anhydrase, but no reduction in carbonic anhydrase activity was detected. Overall, it remains unclear whether inhibition of Ca2+ uptake is a direct result of Cu exposure or, along with the other observed physiological effects, is secondary to an unidentified primary mode of toxic action. Given the hypersensitivity of L. stagnalis to Cu, further study into the mechanisms of action and effects of varying water chemistry on Cu toxicity is clearly warranted.

Influence of rearing and experimental temperatures on predator avoidance behaviour in a freshwater pulmonate snail

Summary1. Predicted increases in the temperature of freshwaters is likely to affect how prey species respond to predators. We investigated how the predator avoidance behaviour of the freshwater pulmonate snail Lymnaea stagnalis is influenced by the temperature at which it was reared and that at which behavioural trials were carried out.2. Crawl‐out behaviour of juvenile snails from two populations (high predation risk versus low predation risk) reared at either 15 or 20 °C was assessed in response to predation cues (predatory fish kairomones and conspecific alarm cues) in behavioural trials at both 15 and 20 °C.3. Trial temperature had a significant effect on the time that snails spent in avoidance, regardless of their population of origin. Crawl‐out behaviour was greater during behavioural trials at 15 °C, but there was no effect of trial temperature on the speed with which animals showed avoidance behaviour.4. There was no interactive effect of rearing temperature (RT) and trial temperature, but the effect of RT on avoidance behaviour did differ between populations. For an RT of 15 °C, snails from the South Drain (high risk) population showed a more rapid and longer avoidance response than those from the Chilton Moor (low risk) population. In contrast, for snails reared at 20 °C, there was no difference between populations for the duration of the avoidance response and snails from Chilton Moor crawled out faster than those from South Drain.5. Hence, whilst (predictable) differences relative to natural predation threat in crawl‐out behaviour were apparent at 15 °C, raising the developmental temperature to 20 °C eliminated or, in the case of latency, reversed these differences. This suggests that L. stagnalis populations that cohabit with predatory fish and experience high developmental temperatures may have a reduced ability to respond to fish predation risk.

Effects of the mixture of diquat and a nonylphenol polyethoxylate adjuvant on fecundity and progeny early performances of the pond snail Lymnaea stagnalis in laboratory bioassays and microcosms

Effects of the bipyridylium herbicide diquat and tank-mix adjuvant Agral ®90 were investigated on various life history traits of the freshwater pulmonate snail Lymnaea stagnalis. Trait expression was measured in simple laboratory bioassays on small size groups of snails, and under more complex, indoor microcosm conditions, on larger groups of snails. Microcosms were provided with sediment, plants, and fish, thus allowing a more complex level of intra and inter-specific interactions to develop. Treatments were performed with substances alone or in mixture, at concentrations ranging from 4.4 to 222.2 μg l −1 for diquat, and from 10 to 500 μg l −1 for Agral 90, under a fixed ratio design. Adult growth was negatively affected by diquat and its mixture with Agral 90 both at the highest concentrations (222.2 and 500 μg l −1, respectively). Fecundity expressed differently in bioassays and microcosms, but no effect of the chemicals could be observed on this trait. Progeny development was impaired by 222.2 μg l −1 diquat and its mixture with 500 μg l −1 Agral 90, as reflected by longer development time and reduced hatching rate of clutches laid by the exposed animals, as compared to the controls. Hatching data suggested that diquat bioavailability was lower in microcosms than under bioassay conditions. Consistently, chemical analysis showed that diquat disappeared more rapidly from the water in microcosms than in bioassays. Moreover, the differential expression of several life history traits under bioassays and microcosms conditions was probably also influenced by the level of intraspecific interaction, which differed among the systems. When significant, the effect of diquat was attenuated by the presence of Agral 90, indicating antagonistic interaction between the two substances. Such a deviation from additivity was partly validated statistically.

Tissue Distribution and Oral Dose Effects of Microcystin in the Freshwater Pulmonate Snail Lymnaea stagnalis jugularis (Say)

Microcystin (MC) concentrations were measured in the alimentary tract, digestive gland, and remaining visceral mass of adult pulmonate snails (Lymnaea stagnalis) exposed to cyanobacteria known to contain MC. The highest proportion of total body MC content was measured within the alimentary tract (83%), though an appreciable proportion (17%) was also found within the digestive gland tissue. This provides conclusive evidence for the limited digestion of toxic cyanobacteria and subsequent uptake and accumulation of MC by the digestive gland of L. stagnalis. Additionally, pure microcystin-LR was orally administered to adult L. stagnalis to investigate the potential for toxic effects. Exposure to microcystin-LR induced histopathological alterations of the digestive glands consistent with those reported elsewhere for mammals and fish, indicating a common mode of toxicity to both vertebrates and invertebrates.

Molecular Characterization of NMDA-Like Receptors inAplysiaandLymnaea:Relevance to Memory Mechanisms

The N-methyl-D-aspartate (NMDA) receptor belongs to the group of ionotropic glutamate receptors and has been implicated in synaptic plasticity, memory acquisition, and learning in both vertebrates and invertebrates, including molluscs. However, the molecular identity of NMDA-type receptors in molluscs remains unknown. Here, we cloned two NMDA-type receptors from the sea slug Aplysia californica, AcNR1-1 and AcNR1-2, as well as their homologs from the freshwater pulmonate snail Lymnaea stagnalis, LsNR1-1 and LsNR1-2. The cloned receptors contain a signal peptide, two extracellular segments with predicted binding sites for glycine and glutamate, three recognized transmembrane regions, and a fourth hydrophobic domain that makes a hairpin turn to form a pore-like structure. Phylogenetic analysis suggests that both the AcNR1s and LsNR1s belong to the NR1 subgroup of ionotrophic glutamate receptors. Our in situ hybridization data indicate highly abundant, but predominantly neuron-specific expression of molluscan NR1-type receptors in all central ganglia, including identified motor neurons in the buccal and abdominal ganglia as well as groups of mechanosensory cells. AcNR1 transcripts were detected extrasynaptically in the neurites of metacerebral cells of Aplysia. The widespread distribution of AcNR1 and LsNR1 transcripts also implies diverse functions, including their involvement in the organization of feeding, locomotory, and defensive behaviors.

Elimination of the Cyanobacterial Hepatotoxin Microcystin from the freshwater Pulmonate Snail Lymnaea stagnalis jugularis (SAY)

It has been suggested that little to no microcystin (MC), a cyanobacterial hepatotoxin, accumulates within freshwater pulmonate snails because the toxin is associated primarily with undigested gut contents that are eliminated from the animal via egestion. To test this, Lymnaea stagnalis exposed to MC-containing cyanobacteria were placed into toxin-free environments and sampled over short (24 h at 21° C) and long (30 d at 22 and 10° C) time periods. Within 8 h after being removed from exposure to microcystin-containing phytoplankton, the gizzard and cecal string fractions of the feces were eliminated, accounting for 57% of the initial MC concentration. However, detectable concentrations remained beyond 24 h, likely in association with the digestive-gland contents, which can be retained up to 100 h. Long-term MC loss was biphasic at two ambient temperatures. The greatest change (fast phase) occurred over the first 3 d after exposure. By 6 d, the cumulative MC loss from L. stagnalis was 80 and 95% at 10 and 22° C, respectively. Toxin loss over this period was attributed to egestion of indigestible cells/colonies from gizzard and cecum, as well as elimination of unassimilated MC-laden fragments and vacuolate excretion of residues from the digestive gland. The fast-phase depuration rate constant was significantly higher at 22 than at 10° C, indicating an influence of ambient temperature on the rate of toxin loss from pulmonate snails. Depuration continued at slower rates until 30 d, when most (97.5 and 99.5% at 10 and 22° C, respectively) of the initial MC was eliminated.

Structure of one of the neuropeptides of the egg-laying hormone precursor of Lymnaea

The neurosecretory caudo-dorsal of the freshwater pulmonate snail Lymnaea stagnalis, a peptidergic system controlling egg laying and egg-laying behavior, produce several neuropeptides. One of these pep tides, calfluxin, causes the influx of Ca 2+ into the albumen gland, a female sex gland of the snail. In the present study calfluxin was purified and the amino acid composition was determined. The ratio of the amino acids appeared to be very close to that of one of the predicted peptides present on the egg-laying hormone precursor of Lymnaea. This peptide was synthesized and shows a clear biological activity in the bioassay. Furthermore, it shows a Chromatographie behavior similar to that of the natural peptide. Based on these evidences it is concluded that the sequence of calfluxin is: Arg-Val-Asp p ]Ser-Ala-Asp-Glu-Ser-Asn-Asp-Asp-Gly-Phe-Asp. Calfluxin shows a remarkable homology with the sequence of one of the predicted peptides on the egg-laying hormone precursor of the marine opisthobranch Aplysia californica.

Isolation, characterization, and evolutionary aspects of a cDNA clone encoding multiple neuropeptides involved in the stereotyped egg-laying behavior of the freshwater snail Lymnaea stagnalis

The cerebral neurosecretory caudodorsal cells (CDCs) of the freshwater pulmonate snail Lymnaea stagnalis control egg laying, an event that involves a pattern of stereotyped behaviors. The CDCs synthesize and release multiple peptides, among which is the ovulation hormone (CDCH). It is thought that each peptide controls a specific aspect of the processes involved in egg laying. We isolated and characterized a CDC-specific cDNA clone that encodes the ovulation hormone (CDCH). RNA blot analysis and in situ hybridization experiments demonstrated that the CDCs are the major cell groups in the cerebral ganglia that transcribe the CDCH gene. In addition to CDCH, the 259-amino acid-long CDCH preprohormone contains 11 other predicted peptides. The overall homology of the CDCH preprohormone with the egg-laying hormone (ELH) preprohormones of the marine opisthobranch snails Aplysia californica and A. parvula is very low (29 and 26%, respectively). However, a more detailed comparison revealed a highly differential pattern of conservation of peptide regions. Significant homology was found between the regions containing (1) CDCH and ELH, (2) repeated pentapeptides, (3) alpha-caudodorsal cell peptide and alpha-bag cell peptide, and (4) 2 regions representing as yet unidentified peptides. Insignificant homology was found when comparing regions containing the other predicted peptides. The conserved peptides probably control similar aspects of the egg-laying fixed action patterns in these distantly related gastropod species. The pentapeptide region exhibits the highest level of homology (75%); in addition, an extra pentapeptide has been generated on the CDCH precursor. This indicates a vital function of these peptides in Aplysia, as well as in Lymnaea species.

The hyperglycemic factor of the CNS of the freshwater snail Lymnaea stagnalis: Interaction with glucose stimulation of glycogen synthesis and evidence for its release during anaerobiosis

In the freshwater pulmonate snail Lymnaea stagnalis glycogen synthesis in the glycogen storage cells (GC) is stimulated by increasing external glucose concentrations. On the other hand, a hyperglycemic factor from the CNS inhibits glycogen synthesis and stimulates glycogen breakdown in these cells. This CNS factor may be involved in the physiological control of glycogen mobilization. In the present study the interaction between the inhibiting effect of the CNS factor and the stimulating effect of increasing glucose concentrations on glycogen synthesis in isolated GC was investigated. The results, analyzed by determination of the Michaelis-Menten parameters for saturation kinetics, suggest that the CNS factor increases the glucose concentration required for half-maximal stimulation of glycogen synthesis, but does not influence the maximum rate of synthesis (competitive type of inhibition). The role of the hyperglycemic factor during conditions of glycogen breakdown was investigated by extirpation of the cerebral ganglia (-CG), which contain the main release sites of the factor. Extirpation did not affect the hemolymph glucose concentration or the glycogen levels in the GC during starvation. However, -CG animals showed reduced levels of the hyperglycemia normally associated with exposure to anaerobic conditions.

Direct effects of the hyperglycemic factor of the freshwater snail Lymnaea stagnalis on isolated glycogen cells

In the freshwater pulmonate snail Lymnaea stagnalis special cells, the glycogen cells (GC) are present for the storage of glycogen reserves. These cells occur in large numbers in the anterior mantle region. In a previous paper in vitro experiments with intact anterior mantle tissue indicated that a hyperglycemic factor is released from the central nervous system (CNS) which stimulates glycogen mobilization in mantle tissue ( M. A. Hemminga, J. J. Maaskant, W. Koomen, and J. Joosse (1985). Neuroendocrine control of glycogen mobilization in the freshwater snail Lymnaea stagnalis. Gen. Comp. Endocrinol. 57, 117–123). In the present study the question of whether this factor affects glycogen metabolism of GC isolated from mantle tissue was investigated. It is reported that in high-K + Ringers the CNS is stimulated to release a factor which, in a dose-dependent way, inhibits glycogen synthesis in isolated GC (measured as a decreased incorporation of [ 14C]glucose into glycogen). Simultaneous with this glycogen synthesis-inhibiting effect, stimulation of glycogen degradation is found (measured as a decreased retention of prelabeled glycogen). It is concluded that (1) the factor released by the CNS having these effects on isolated GC is the same as the hyperglycemic factor which was reported to stimulate glycogen mobilization in intact mantle tissue, and (2) GC after isolation from mantle tissue have retained their ability to respond to this factor. High-K + bathing Ringers of the cerebral/buccal ganglia have a stronger inhibiting effect on glycogen synthesis in isolated GC than bathing Ringers of the remainder part of the CNS, indicating that in this part of the brain the major release sites of the hyperglycemic factor are located. Dorsal body hormone, ovulation hormone, or growth hormone of L. stagnalis probably are not identical with the hyperglycemic factor, as homogenates of parts of the cerebral ganglia which are known to contain these respective hormones do not influence glycogen synthesis in isolated GC.

Effects of photoperiod and temperature on the glycogen stores in the mantle and the head-foot muscles of the freshwater pulmonate snail Lymnaea stagnalis

Abstract 1. 1. The effects of various combinations of photoperiod (LD: 16L-8D; SD: 8L-16D) and temperatures (8 and 20°C) on the glycogen content of the anterior mantle region, which contains large numbers of glycogen storage cells, and of the head-foot muscles of the freshwater pulmonate snail Lymnaea stagnalis were investigated. 2. 2. At 20°C, but not at 8°C, SD photoperiods favour accumulation of glycogen in both tissues. In contrast, LD photoperiods, at 20°C, induce depletion of the glycogen reserves. 3. 3. The changes of the glycogen contents presumably result from a reorientated metabolism linked to photoperiodically induced changes of ovipository activity, as is discussed.

The effect of temperature on spontaneous, and ovulation hormone-induced female reproduction in Lymnaea stagnalis

Spontaneous oviposition of the freshwater pulmonate snail Lymnaea stagnalis stopped after transfer from 20° to low temperatures (8 and 4°). This was due to a reduction in the activities of the neurosecretory caudodorsal cells (CDC), which produce the ovulation hormone (CDCH). Oviposition latencies (time interval between CDCH-injection and start of oviposition) increased with decreasing temperature. The ovotestis and the female accessory sex organs of snails placed at 8° became less sensitive, and those at 4° became completely insensitive to injected CDCH. This was probably caused by a reduction in activities of the endocrine dorsal bodies (DB), which control vitellogenesis and the activities of the female accessory sex organs. After a change from 4 to 20°, CDCH injections rapidly became effective in inducing egg mass production, and spontaneous oviposition also restarted quickly, suggesting a rapid increase in DB and CDC activities.

Ovulation hormone, nutritive state, and female reproductive activity in Lymnaea stagnalis

A study of the relation between nutritive state and female reproductive activity as affected by the ovulation hormone (CDCH) has been made in the freshwater pulmonate snail Lymnaea stagnalis. CDCH is produced by the neuroendocrine caudodorsal cells (CDC) in the cerebral ganglia. Spontaneous oviposition ceased within 6 days of the beginning of a starvation period. This is most probably partially due to a reduction in the CDC activities because (1) quantitative electron microscopy showed a nearly 80% decrease in the number of release phenomena in the CDC-axon terminals in the neurohemal area in the intercerebral commissure, and (2) a bioassay showed a considerable reduction in the amount of CDCH in this area. During starvation the ovotestis and the female accessory sex organs became progressively less sensitive and, after 25 days, were completely insensitive to injected CDCH. This was indicated by a decrease in the responses to CDCH injection and, correspondingly, by a gradual increase in the CDCH thresholds for ovulation and egg formation. It is argued that the insensitivity may be caused by a reduction in the activities of the endocrine dorsal bodies. During refeeding, CDCH injections again become effective in inducing egg mass production, followed by resumption of spontaneous oviposition. This suggests a rapid restoration of DB and CDC activities following refeeding.

Studies on the physiological role of a partially purified small cardioactive neuropeptide ofLymnaea stagnalis

1. The molecular characteristics and physiological role of a partially purified small cardioactive neuropeptide (SCP) of the freshwater pulmonate snailLymnaea stagnalis were studied and compared with those of FMRFamide, an SCP discovered in clam ganglia.Lymnaea SCP, extracted from the central nervous system, and commercial FMRFamide co-elute on Sephadex G-15 columns, but SCP behaves differently from FMRF-amide, when chromatographed on the ion exchanger CM-Sephadex C-25. Moreover, it has a lowerRf value (0.60) on thin layer chromatograms than FMRFamide (0.75). SCP is sensitive to proteolytic enzymes. These data indicate thatLymnaea SCP is a small peptide which is not identical to FMRFamide. 2. Lymnaea SCP is involved in the physiological regulation of the activity of various muscles. It stimulates the frequency of beat as well as the amplitude of the auricle and ventricle. It also stimulates the peristalsis of the oesophagus and causes contractions of the penis retractor muscle. These excitatory effects are similar to those of FMRFamide, suggesting thatLymnaea SCP and FMRFamide must be closely related. 3. The role of SCP as a neurotransmitter is discussed.

Partial purification and characterization of the ovulation hormone of the freshwater pulmonate snail Lymnaea stagnalis

The neurosecretory caudodorsal cells in the cerebral ganglia of the basommatophoran snail Lymnaea stagnalis produce an ovulation hormone (CDCH), which is released from the periphery of the intercerebral commissure. In the present study CDCH has been partially purified, starting with a hydrochloric acid extract of commissures, by gel permeation chromatography on Bio-Gel P-6 followed by cation exchange on SP-Sephadex C-25. This two-step procedure resulted in a ca. 22-fold purification of CDCH. The low purification factor is caused by poor resolution of both purification steps and low recovery on the Bio-Gel P-6 column. With stability tests, proteolytic enzymes, SDS/urea polyacrylamide electrophoresis and isoelectric focussing, purified CDCH was characterized as a stable peptide with a molecular weight of ca. 4700 d and a pI of ca. 9.3. The properties of CDCH are similar to those of the ovulation hormones of opisthobranch snails. Studies on species specificity indicate, however, that it is unlikely that CDCH will cause ovulation when injected in opisthobranchs, or vice versa.

An ultrastructural in vitro study on the regulation of neurosecretory activity in the freshwater snail Lymnaea stagnalis (L.) with particular reference to caudo-dorsal cells.

The neurosecretory Caudo-Dorsal Cells (CDC) in the cerebral ganglia of the freshwater pulmonate snail Lymnaea stagnalis produce an ovulation stimulating hormone. Previously it has been shown that neuronal and non-neuronal inputs are involved in the regulation of their activity. The degree of autonomy of these cells has been investigated by studying with morphometric methods the ultrastructure of CDC maintained in vitro. CDC of isolated cerebral ganglia which were cultured for 7 days show a considerable rate of synthesis, transport and release of neurohormone. Apparently these processes can proceed in the absence of neuronal and hormonal inputs from outside the cerebral ganglia. Completely isolated CDC, however, do not show neurosecretory activity in vitro; active Golgi zones, indicating the formation of neurosecretory elementary granules, are absent from such cells. Isolation does not seem to affect general cell functions such as protein synthesis and respiration. It is suggested that a neuronal input, originating within the cerebral ganglia, is necessary for the stimulation of CDC neurosecretory activity. Techniques are described for the isolation and culture of neurosecretory cells of L. stagnalis.

Structure and Function of the Calcium Cells of the Freshwater Pulmonate Snail Lymnaea Stagnalis

The morphology and function of the calcium cells of Lymnaea stagnalis were studied. These cells contain concretions consisting of an organic matrix in which inorganic material, mainly CaCO 3 , is deposited. The organic matrix consists of protein, carbohydrate and lipid, synthesized by the Golgi apparatus. It appeared that deposition of CaCO 3 is an active process; CaCO 3 is released from the concretions into the haemolymph via pores in the plasma membrane of the calcium cells. Experiments on snails exposed to water equilibrated with 10% CO 2 in air strongly suggest that calcium cells function in pH homeostasis.

Hemolymph glucose in the freshwater pulmonate snail Lymnaea stagnalis: Basal values and an effect of ingested carbohydrate

1. 1. In the freshwater pulmonate gastropod Lymnaea stagnalis jugularis Say, a basal level of hemolymph glucose for nonfeeding animals appears to be about 30 μg/ml (3 mg %). 2. 2. Elevated levels of hemolymph glucose can be induced by starch feeding with subsequent return to the basal level when food is withheld.