Freshwater Snail Lymnaea Research Articles

Novel and Nontraditional Use of Stable Isotope Tracers To Study Metal Bioavailability from Natural Particles

We devised a novel tracing approach that involves enriching test organisms with a stable metal isotope of low natural abundance prior to characterizing metal bioavailability from natural inorganic particles. In addition to circumventing uncertainties associated with labeling natural particles and distinguishing background metals, the proposed "reverse labeling" technique overcomes many drawbacks inherent to using radioisotope tracers. Specifically, we chronically exposed freshwater snails ( Lymnaea stagnalis ) to synthetic water spiked with Cu that was 99.4% (65)Cu to increase the relative abundance of (65)Cu in the snail's tissues from ~32% to >80%. The isotopically enriched snails were then exposed to benthic algae mixed with Cu-bearing Fe-Al particles collected from the Animas River (Colorado), an acid mine drainage impacted river. We used (63)Cu to trace Cu uptake from the natural particles and inferred their bioavailability from calculation of Cu assimilation into tissues. Cu assimilation from these particles was 44%, indicating that 44% of the particulate Cu was absorbed by the invertebrate. This demonstrates that inorganic particulate Cu can be bioavailable. The reverse labeling approach shows great potential in various scientific areas such as environmental contamination and nutrition for addressing questions involving uptake of an element that naturally has multiple isotopes.

Genetic compatibilities, outcrossing rates and fitness consequences across life stages of the trematode Diplostomum pseudospathaceum

Many parasitic helminths exhibit mixed mating systems, and switches between self-fertilization and outcrossing may be influenced by environmental conditions and parasite demography. While inbreeding depression selects against the development of purely self-fertilizing populations, genetic compatibility may contribute to stabilizing mixed strategies. Here we study the effects of inbreeding and genetic compatibility on offspring fitness in the digenean trematode Diplostomum pseudospathaceum, a parasite with a three-host life cycle. Hatching rates and infection success in two intermediate hosts, the freshwater snail Lymnaea stagnalis and the three-spined stickleback, Gasterosteus aculeatus, were used as proxies for parasite fitness. Single trematode clones and combinations of two and three different clones were allowed to reproduce sexually using naïve herring gulls (Larus argentatus) as definitive hosts. The hatched larvae were used to assess the proportion of selfed and outcrossed miracidia by means of microsatellite genotyping. These results were matched with hatching rates and infection success of inbred and outcrossed trematodes in both intermediate hosts. Inbreeding effects were obscured by differences in clone performance. In addition, clones outcrossed to a lesser extent than expected in some experimental pairings, indicating the importance of genetic compatibility.

Molecular typing of the fresh water snail Lymnaea arabica, the possible intermediate host of Fasciola hepatica, collected from Saudi Arabia, by RAPD-PCR

Fasciolosis is caused by Fasciola hepatica with Lymnaea spp. as intermediate hosts. Lymnaea arabica, as a possible snail host collected from the Kingdom of Saudi Arabia, was investigated by Random Amplified Polymorphic DNA polymerase chain reaction (RAPD-PCR) technique. Polymorphic bands from L. arabica were sought for fingerprinting (typing) and identification of this species. The resultant electrophoretic patterns would be used as molecular bio-markers for thesemolluscas. This will help in the identification of the snails at a molecular level for the first time in the country that, in turn, will help in disease control. This technique was proved to be an accurate, appropriate, efficient and valuable alternative to traditional identification tools of Lymnea spp.

Aerial dispersal of freshwater gastropods by dragonflies (Odonata)

At a ditch near Tribsees in northeastern Germany a young individual of the freshwater snail Lymnaea stagnalis was photographed while attached to the dorsum of the abdomen of a female Aeshna viridis, after the odonate took off after oviposition in Stratiotes aloides. This is the first known case for odonate-born dispersal of freshwater gastropods.

Impact of the redox-cycling herbicide diquat on transcript expression and antioxidant enzymatic activities of the freshwater snail Lymnaea stagnalis

The presence of pesticides in the environment results in potential unwanted effects on non-target species. Freshwater organisms inhabiting water bodies adjacent to agricultural areas, such as ditches, ponds and marshes, are good models to test such effects as various pesticides may reach these habitats through several ways, including aerial drift, run-off, and drainage. Diquat is a non-selective herbicide used for crop protection or for weed control in such water bodies. In this study, we investigated the effects of diquat on a widely spread aquatic invertebrate, the holarctic freshwater snail Lymnaea stagnalis. Due to the known redox-cycling properties of diquat, we studied transcript expression and enzymatic activities relative to oxidative and general stress in the haemolymph and gonado-digestive complex (GDC). As diquat is not persistent, snails were exposed for short times (5, 24, and 48h) to ecologically relevant concentrations (22.2, 44.4, and 222.2μgl−1) of diquat dibromide. RT-qPCR was used to quantify the transcription of genes encoding catalase (cat), a cytosolic superoxide dismutase (Cu/Zn-sod), a selenium-dependent glutathione peroxidase (gpx), a glutathione reductase (gred), the retinoid X receptor (rxr), two heat shock proteins (hsp40 and hsp70), cortactin (cor) and the two ribosomal genes r18S and r28s. Enzymatic activities of SOD, Gpx, Gred and glutathione S-transferase (GST) were investigated in the GDC using spectrophoto/fluorometric methods. Opposite trends were obtained in the haemolymph depending on the herbicide concentration. At the lowest concentration, effects were mainly observed after 24h of exposure, with over-transcription of cor, hsp40, rxr, and sod, whereas higher concentrations down-regulated the expression of most of the studied transcripts, especially after 48h of exposure. In the GDC, earlier responses were observed and the fold-change magnitude was generally much higher: transcription of all target genes increased significantly (or non-significantly for cat) after 5h of exposure, and went back to control levels afterwards, suggesting the onset of an early response to oxidative stress associated to the unbalance of reactive oxygen species (ROS) in hepatocytes. Although increases obtained for Gred and SOD activities were globally consistent with their respective transcript expressions, up-regulation of transcription was not always correlated with increase of enzymatic activity, indicating that diquat might affect steps downstream of transcription. However, constitutive levels of enzymatic activities were at least maintained. In conclusion, diquat was shown to affect expression of the whole set of studied transcripts, reflecting their suitability as markers of early response to oxidative stress in L. stagnalis.

Oxidative stress and genotoxic effect of zinc oxide nanoparticles in freshwater snail Lymnaea luteola L.

Understanding the toxic effects of nanoparticles on aquatic organism is the biggest obstacle to the safe development of nanotechnology. However, little is known about the toxic mechanisms of zinc oxide nanoparticles (ZnONPs) in freshwater snail Lymnaea luteola (L. luteola). This study was designed to investigate the possible mechanisms of genotoxicity induced by ZnONPs in freshwater snail L. luteola. ZnONPs (32μg/ml) elicited a significant (p<0.01) reduction in glutathione (42.10% and 61.40%), glutathione-S-transferase (25.60% and 40.24%) and glutathione peroxidase (21.73% and 39.13%) with a concomitant increase in malondialdehyde level (54.50% and 57.14%; p<0.01) and catalase (34.88% and 52.56%; p<0.01) in digestive gland of L. luteola after 24 and 96h exposure, respectively. However, a statistically significant (p<0.01) induction in DNA damage was observed by the comet assay in digestive gland cells treated with ZnONPs for 24 and 96h. Thus, the results demonstrate that ZnONPs induce genotoxicity in digestive gland cells through oxidative stress. Freshwater snail L. luteola may be used as suitable test model for nanoecotoxicological studies in future.

Molluscicidal activity of Morus nigra against the freshwater snail Lymnaea acuminata

The molluscicidal activity of Morus nigra fruit, bark and leaf powder against the snail Lymnaea acuminata was time and concentration dependent. Toxicity of fruit powder (96h LC 50 : 166.92 mg/L) was more pronounced in comparison to bark powder (96h LC 50 : 173.17 mg/L) and leaf powder (96h LC 50 : 173.69 mg/L). Ethanolic extracts of M . nigra fruit, bark and leaf was more toxic than their other organic solvent extracts. The molluscicidal activity of ethanolic extract of M . nigra fruit powder (24h LC 50 : 116.23 mg/L) was more effective than the ethanolic extract of bark powder (24h LC 50 : 154.41 mg/L) and leaf powder (24h LC 50 : 139.80 mg/L). The 96h LC 50 of column-purified fraction of M . nigra fruit powder was, 10.03 mg/L whereas that of bark and leaf powder was 8.69 mg/L and 4.97 mg/L, respectively. Column and thin layer chromatography analysis demonstrates that the active molluscicidal component in M . nigra is quercetin (96h LC 50 : 1.11 mg/L), apigenin (96h LC 50 : 1.92 mg/L) and morusin (96h LC 50 : 2.12 mg/L), respectively. Co-migration of quercetin (R f 0.49), apigenin (R f 0.51) and morusin (R f 0.52) with column-purified fruit, bark and leaf of M . nigra on thin layer chromatography demonstrates same R f value. The present study indicates that M . nigra may be used as potent source of molluscicides against the snail Lymnaea acuminata .

Development of an embryo toxicity test with the pond snail Lymnaea stagnalis using the model substance tributyltin and common solvents

The development of a chronic mollusc toxicity test is a current work item on the agenda of the OECD. The freshwater pond snail Lymnaea stagnalis is one of the candidate snail species for such a test. This paper presents a 21-day chronic toxicity test with L. stagnalis, focussing on embryonic development. Eggs were collected from freshly laid egg masses and exposed individually until hatching. The endpoints were hatching success and mean hatching time. Tributyltin (TBT), added as TBT-chloride, was chosen as model substance. The selected exposure concentrations ranged from 0.03 to 10μgTBT/L (all as nominal values) and induced the full range of responses. The embryos were sensitive to TBT (the NOEC for mean hatching time was 0.03μgTBT/L and the NOEC for hatching success was 0.1μgTBT/L). In addition, data on maximum limit concentrations of seven common solvents, recommended in OECD aquatic toxicity testing guidelines, are presented. Among the results, further findings as average embryonic growth and mean hatching time of control groups are provided. In conclusion, the test presented here could easily be standardised and is considered useful as a potential trigger to judge if further studies, e.g. a (partial) life‐cycle study with molluscs, should be conducted.

Bioaccumulation Dynamics and Modeling in an Estuarine Invertebrate Following Aqueous Exposure to Nanosized and Dissolved Silver

Predicting the environmental impact of engineered nanomaterials (ENMs) is increasingly important owing to the prevalence of emerging nanotechnologies. We derived waterborne uptake and efflux rate constants for the estuarine snail, Peringia ulvae, exposed to dissolved Ag (AgNO(3)) and silver nanoparticles (Ag NPs), using biodynamic modeling. Uptake rates demonstrated that dissolved Ag is twice as bioavailable as Ag in nanoparticle form. Biphasic loss dynamics revealed the faster elimination of Ag from Ag NPs at the start of depuration, but similar slow efflux rate constants. The integration of biodynamic parameters into our model accurately predicted Ag tissue burdens during chronic exposure with 85% of predicted values within a factor of 2 of observed values. Zeta potentials for the Ag NPs were lower in estuarine waters than in waters of less salinity; and uptake rates in P. ulvae were slower than reported for the freshwater snail Lymnaea stagnalis in similar experiments. This suggests aggregation of Ag NPs occurs in estuarine waters and reduces, but does not eliminate, bioavailability of Ag from the Ag NPs. Biodynamic modeling provides an effective methodology to determine bioavailable metal concentrations (originating from metal and metal-oxide nanoparticles) in the environment and may aid future ENM risk assessment.

Histopathological effects of larval trematodes on the digestive gland of freshwater snail species, Vivipara bengalensis and Lymnaea acuminata

Comparative histopathological effects were observed on the digestive glands (hepatopancreas) of freshwater snail species, Vivipara bengalensis and Lymnaea acuminata infected with single (furcocercous cercariae) and double infection of digenean trematode larvae (gymnocephalous cercariae+metacercariae), respectively. Digestive glands of both the snail species revealed degenerative changes in their digestive gland tubules. The latter became irregular in shape, reduced in size with enlarged lumen and inter-tubular space besides rupturing of digestive tubules. These changes were correlated with the size and types of trematode larvae, single or double infection and degree of parasitemia. Autolytic necrosis of numerous tubules was found in digestive glands infected with double and very severe single infection. None of the tubules was found to be invaded by sporocyst, redia and cercaria. However, metacercariae were seen to invade digestive tubules of Lymnaea species.

Descriptions of strigea cercariae from the Gauteng and North West Provinces, South Africa

Freshwater snails are known to serve as first intermediate hosts for various parasitic diseases such as schistosomosis, amphistomosis and fasciolosis. Two freshwater snail species, Lymnaea natalensis, Krauss 1848 and Bulinus tropicus, Krauss 1848 were sampled from five localities in Gauteng and one locality in the North West Province from 2007 to 2010. These snails were collected in order to study their cercarial sheddings. They were found to be infected with three different types of strigea cercariae, of which the morphology was studied using standard light and scanning electron microscopy techniques.

Effect of hydrogen peroxide on electrical coupling between identified Lymnaea neurons

The pair of giant reciprocally coupled neurons VD1 and RPaD2 within the CNS of the freshwater pond snail Lymnaea stagnalis was used to analyse the effect of hydrogen peroxide on gap-junction connection. Electrical activity of VD1/RPaD2 was recorded with intracellular microelectrodes in order to analyse gap-junction signalling. Hydrogen peroxide application (1 × 10⁻⁴ M) results in a rapid, 1.3-fold, increase in VD1/RPaD2 spiking frequency within 30 s after application. This was accompanied by a slight reduction in action potential amplitude. In addition, H₂O₂ induced a significant reduction in the steady-state bidirectional coupling ratio between the neurons. The maximal reduction in the coupling ratio, 1.8-1.9 fold, was measured 3 min after H₂O₂ application. However, the network input resistance did not undergo a detectable change. The voltage-gated Ca²⁺ channel blocker, nifedipine (1 × 10⁻⁴ M), abolished the effect of H₂O₂ on the coupling ratio and firing frequency. All the effects of H₂O₂ were reversible, that is, washing the preparation with standard physiological saline restored the properties of the neuronal coupling to the pre-treatment value. These data are consistent with a dynamic modulation of the gap-junction properties by H₂O₂ between these two neurons.

Inhibition kinetics of certain enzymes in the nervous tissue of vector snail Lymnaea acuminata by active molluscicidal components of Sapindus mukorossi and Terminalia chebula

Effect of active molluscicidal components of Sapindus mukorossi and Terminalia chebula on the acetylcholinesterase (AChE), acid and alkaline phosphatase (ACP/ALP) activity in the nervous tissue of freshwater snail Lymnaea acuminata were studied. In vivo and in vitro exposure of saponin (active component of S. mukorossi pericarp) and tannic acid (active component of T. chebula) significantly inhibited the AChE, ACP and ALP activity in the nervous tissue of L. acuminata. The inhibition kinetics of these enzymes indicate that saponin and tannic acid caused competitive and competitive–non-competitive inhibition of AChE, respectively. Saponin also caused competitive and competitive–non-competitive inhibition of ACP and ALP, respectively, whereas tannic acid caused competitive–non-competitive inhibition of ACP and ALP. Thus the inhibition of AChE, ACP and ALP by saponin and tannic acid in the nervous tissue of L. acuminata may be the cause of molluscicidal activity of S. mukorossi and T. chebula.

Mechanisms of waterborne Cu toxicity to the pond snail Lymnaea stagnalis: Physiology and Cu bioavailability

We examined the mechanisms of toxicity of waterborne Cu to the freshwater pond snail Lymnaea stagnalis. The snail is one of the most sensitive species to acute Cu exposure (96 h LC 50, LC 20: 24.9, 18.0 μg l −1); they are not protected by the water quality criteria of the US EPA. Tissue Na and Ca were also reduced by Cu in the acute exposure. In contrast, during 28 d chronic exposures to Cu in the presence of food, which resulted in higher DOC concentrations, there was no significant mortality but an inhibition of growth, which may reflect a re-allocation of resources to detoxification. Cu detoxification was evidenced in chronic exposure by increases in metallothionein-like protein concentrations and Cu binding to metal-rich granules, decreases in thiobarbituric acid-reactive substances, and changes in the subcellular distribution in the soft tissues. Our results demonstrated that apart from external Cu bioavailability, compartmentalization of metals within the cells can alter toxicity of Cu to the snails.

Heterosis and inbreeding depression in bottlenecked populations: a test in the hermaphroditic freshwater snail Lymnaea stagnalis

Small population size is expected to induce heterosis, due to the random fixation and accumulation of mildly deleterious mutations, whereas within-population inbreeding depression should decrease due to increased homozygosity. Population bottlenecks, although less effective, may have similar consequences. We tested this hypothesis in the self-fertile freshwater snail Lymnaea stagnalis, by subjecting experimental populations to a single bottleneck of varied magnitude. Although patterns were not strong, heterosis was significant in the most severely bottlenecked populations, under stressful conditions. This was mainly due to hatching rate, suggesting that early acting and highly deleterious alleles were involved. Although L.stagnalis is a preferential outcrosser, inbreeding depression was very low and showed no clear relationship with bottleneck size. In the less reduced populations, inbreeding depression for hatching success increased under high inbreeding. This may be consistent with the occurence of synergistic epistasis between fitness loci, which may contribute to favour outcrossing in L.stagnalis.

Natural range expansion and human-assisted introduction leave different genetic signatures in a hermaphroditic freshwater snail

Colonization events like range expansion or biological invasions can be associated with population bottlenecks. Small population size may lead to loss of genetic diversity due to random genetic drift, to loss of heterozygosity due to increased inbreeding and should leave a signature on the genetic polymorphism and genetic structure of populations. The mating system might additionally influence the outcome of such a process. Here, we compare invasive and native populations of the hermaphroditic freshwater snail Lymnaea stagnalis. In the native range we included populations that were ice-free during the last glaciation period and populations that were glaciated and are located at the edge of the species’ native distribution range. The microsatellite data show substantial loss of genetic variation in the introduced range and no signs of high propagule pressure or admixture. The expressed polymorphism was so low that mating system analysis was not possible. In the native region, all populations display strong levels of differentiation (global F ST: 0.341) independent of colonization history and exhibit no significant pattern of inbreeding. However, the populations in more recently colonized habitats show diminished genetic diversity. Overall, these results illustrate how dramatic the reduction in genetic diversity can be for hermaphroditic animals and that gene flow in the native range can be surprisingly low despite short distances.

A Sodium Leak Current Regulates Pacemaker Activity of Adult Central Pattern Generator Neurons in Lymnaea Stagnalis

The resting membrane potential of the pacemaker neurons is one of the essential mechanisms underlying rhythm generation. In this study, we described the biophysical properties of an uncharacterized channel (U-type channel) and investigated the role of the channel in the rhythmic activity of a respiratory pacemaker neuron and the respiratory behaviour in adult freshwater snail Lymnaea stagnalis. Our results show that the channel conducts an inward leak current carried by Na+ (ILeak-Na). The ILeak-Na contributed to the resting membrane potential and was required for maintaining rhythmic action potential bursting activity of the identified pacemaker RPeD1 neurons. Partial knockdown of the U-type channel suppressed the aerial respiratory behaviour of the adult snail in vivo. These findings identified the Na+ leak conductance via the U-type channel, likely a NALCN-like channel, as one of the fundamental mechanisms regulating rhythm activity of pacemaker neurons and respiratory behaviour in adult animals.

Efficacy of euphorbia hirta latex as plant derived molluscicides against freshwater snails

The toxic effect of binary and tertiary combinations of Euphorbia hirta Linn latex powder with other plant molluscicidal compounds, were evaluated against the freshwater snails Lymnaea (Radix) acuminata and Indoplanorbis exustus in pond. These combinations showed significant time and dose dependent effect against both the snails. These compounds at higher doses were also lethal to freshwater fish Channa punctatus (Bloch) (Channidae {Ophicephalidae}), which shares the habitat with these snails, but the LC90 (24h) doses of snails have no apparent killing properties in fish populations when treated in mixed population of snails and fish.

Parental exposure to pesticides and progeny reaction norm to a biotic stress gradient in the freshwater snail Lymnaea stagnalis

Human-induced environmental stress may lead to rapid evolutionary processes, and can affect the ability of natural populations to respond to other environmental change or stress. We used quantitative genetics tools, pesticide exposure and a gradient of biotic stress to investigate these questions in the freshwater snail Lymnaea stagnalis. The study focused on the genetic component of variance for life-history traits within populations, and the ability of different lines to respond differently to stress. The effect of parental exposure to a xenobiotic stress on the reaction norm of the progeny to another stress was also estimated (parental non-genetic effect). First, under laboratory conditions, inter-family variance suggested significant heritability for most traits. Second, under outdoor exposure to various pesticides, variation among families was significant for individual growth. Clutch size and hatching rate of the clutches laid in the laboratory after exposure showed similar results, and moreover, family interacted significantly with pesticides. Third, under a gradient of biotic stress (food and competition), inter-family variation was again significant for growth, and a significant interaction with biotic stress was observed for juvenile growth and ultimate size. Family heterogeneity and family × environment interactions indicate the possibility of differential evolutionary responses among lines, through different reaction norms. Stressful conditions did not affect the estimated heritability, and for pesticides, no transgenerational effect was detected on progeny growth in response to the biotic stress. Focused on short-term evolutionary responses, the present study illustrates a possible way of incorporating evolutionary approaches into ecotoxicological risk assessment procedures, for example, by accounting for inter-family variation.

An unusual phospholipase A2 from puff adder Bitis arietans venom – a novel blocker of nicotinic acetylcholine receptors

The venoms of snakes from Viperidae family mainly influence the function of various blood components. However, the published data indicate that these venoms contain also neuroactive components, the most studied being neurotoxic phospholipases A2 (PLA2s). Earlier we have shown (Gorbacheva et al., 2008) that several Viperidae venoms blocked nicotinic acetylcholine receptors (nAChRs) and voltage-gated Ca2+ channels in isolated identified neurons of the fresh-water snail Lymnaea stagnalis. In this paper, we report on isolation from puff adder Bitis arietans venom and characterization of a novel protein bitanarin that reversibly blocks nAChRs. To isolate the protein, the venom of B. arietans was fractionated by gel-filtration, ion-exchange and reversed phase chromatography and fractions obtained were screened for capability to block nAChRs. The isolated protein competed with [125I]iodinated α-bungarotoxin for binding to human α7 and Torpedo californica nAChRs, as well as to acetylcholine-binding protein from L. stagnalis, the IC50 being 20 ± 1.5, 4.3 ± 0.2, and 10.6 ± 0.6 μM, respectively. It also blocked reversibly acetylcholine-elicited current in isolated L. stagnalis neurons with IC50 of 11.4 μM. Mass-spectrometry analysis determined the molecular mass of 27.4 kDa and the presence of 28 cysteine residues forming 14 disulphide bonds. Edman degradation of the protein and tryptic fragments showed its similarity to PLA2s from snake venoms. Indeed, the protein possessed high PLA2 activity, which was 1.95 mmol/min/μmol. Bitanarin is the first described PLA2 that contains 14 disulphide bonds and the first nAChR blocker possessing PLA2 activity.