Cepaea Nemoralis Research Articles

Molecular evidence for multiple introductions of the banded grove snail (Cepaea nemoralis) in North America

Global identification and monitoring programs for invasive species aim to reduce imminent impacts to biodiversity, ecosystem services, agriculture, and human health. This study employs a 658 base pair fragment of the cytochrome c oxidase subunit I (COI) gene to identify and categorize clades of the banded grove snail (Cepaea nemoralis (Linnaeus, 1758)) from native (European) and introduced (North American) ranges using a maximum-likelihood phylogeny and haplotype networks. This work corroborates the existence of eight clades within C. nemoralis and further identified three clades that were common to both Europe and North America (A, D, O). Clades A and D were found in eastern Canada, Ontario (Canada), and British Columbia (Canada), whereas clade O was restricted to Ontario, possibly introduced from Poland or central Europe. Haplotype networks suggest clade A was introduced from northern Europe, whereas clade D was introduced from western and central Europe. Networks contained many private haplotypes and a lack of haplotype sharing, suggesting strong genetic structure in this system, possibly resulting from reduced dispersal in this species. This study describes the contemporary distribution of C. nemoralis in Canada and demonstrates the efficacy of DNA barcoding for monitoring the spread of invasive species, warranting its widespread adoption in management policies.

Discrete or indiscrete? Redefining the colour polymorphism of the land snail Cepaea nemoralis

Biologists have long tried to describe and name the different phenotypes that make up the shell polymorphism of the land snail Cepaea nemoralis. Traditionally, the view is that the ground colour of the shell is one of a few major colour classes, either yellow, pink or brown, but in practise it is frequently difficult to distinguish the colours, and define different shades of the same colour. To understand whether colour variation is in reality continuous, and to investigate how the variation may be perceived by an avian predator, we applied psychophysical models of colour vision to shell reflectance measures. We found that both achromatic and chromatic variation are indiscrete in Cepaea nemoralis, being continuously distributed over many perceptual units. Nonetheless, clustering analysis based on the density of the distribution did reveal three groups, roughly corresponding to human-perceived yellow, pink and brown shells. We also found large-scale geographic variation in the frequency of these groups across Europe, and some covariance between shell colour and banding patterns. Although further studies are necessary, the observation of continuous variation in colour is intriguing because the traditional theory is that the underlying supergene that determines colour has evolved to prevent phenotypes from “dissolving” into continuous trait distributions. The findings thus have significance for understanding the Cepaea polymorphism, and the nature of the selection that acts upon it, as well as more generally highlighting the need to measure colour objectively in other systems.

Recombination within the Cepaea nemoralis supergene is confounded by incomplete penetrance and epistasis

Although the land snail Cepaea nemoralis is one of the most thoroughly investigated colour polymorphic species, there have been few recent studies on the inheritance of the shell traits. Previously, it has been shown that the shell polymorphism is controlled by a series of nine or more loci, of which five make a single ‘supergene’ containing tightly linked colour and banding loci and more loosely linked pigmentation, spread band and punctate loci. However, one limitation of earlier work was that putative instances of recombination between loci within the supergene were not easily verified. We therefore generated a new set of C. nemoralis crosses that segregate for colour, banding and pigmentation, and several other unlinked shell phenotype loci. The snails were genotyped using a set of RAD-seq-derived loci that flank the supergene, and instances of recombination tested by comparing inferred supergene genotype against RAD-marker genotype. We found no evidence that suspected ‘recombinant’ individuals are recombinant between loci within the supergene. As point estimates of recombination between both colour/banding, and colour/pigmentation loci are zero, incomplete penetrance and epistasis are a better explanation for the apparent ‘recombinant’ phenotype of some snail shells. Overall, this work, therefore, shows that the architecture of the supergene may not be as previously supposed. It also provides a resource for fine mapping of the supergene and other major shell phenotype loci.

The complete mitogenome of the Roman snail Helix pomatia Linnaeus 1758 (Stylommatophora: Helicidae)

This study reports the complete mitochondrial sequence of the Roman snail, one of the largest terrestrial snails of Europe. Two specimens of Helix pomatia were sequenced, which had a sequence length of 14,070 and 14,072 base pairs. The mitogenome has the common metazoan makeup (13 protein-coding genes, two ribosomal RNAs, and 21 transfer RNAs) and the ‘typical’ Helicid gene order (Cytochrome Oxidase subunit III and tRNA-T translocated compared to other Helicoidea). All protein coding genes could be annotated with proper start and stop codons (no completion of stop codons by polyadenylation). The specimens have a sequence similarity of 99.0% (146 differences) and the average base composition is 29.7% A, 15.0% C, 17.9% G, and 37.4% T. Phylogenetic anlyses with available Helicid mitogenomes show Helicinae as monophyly and H. pomatia as the sister-group of (Cepaea nemoralis, Theba pisana, and Cornu aspersum).

Land Snails in the Slovak Open-Air Garden Centres

Abstract In last decades, the number of non-native land snails increased up to 15 percentages; they create more than eight percent of all the Slovakian species. Trend of newly established snail species corresponds with increases in the average temperatures as well as the intensity of foreign trade, suggesting a synergistic effect of both climatic conditions and socioeconomic factors. The research of the open-air garden centres in Slovakia confirmed both factors. We report here some of the newly established populations of sixteen mollusc species. In the old garden centres, the number of species as well as the number of individuals decreased slightly. Area of the garden centre has a very high impact on both abundance and species diversity. The size and age of garden centre proportionally influences the composition of mollusc assemblages. Two new species Cornu aspersum and Cepaea nemoralis were noticed for the first time in Slovakia. The recent findings of the introduced populations demonstrate the potential of this snail to colonise new areas.

Gastropod parasitic nematodes (Phasmarhabditis sp.) are attracted to hyaluronic acid in snail mucus by cGMP signalling.

Phasmarhabditis hermaphrodita is a parasitic nematode of terrestrial gastropods that has been formulated into a biological control agent for farmers and gardeners to kill slugs and snails. In order to locate slugs it is attracted to mucus, faeces and volatile cues; however, there is no information about whether these nematodes are attracted to snail cues. It is also unknown how wild isolates of P. hermaphrodita or different Phasmarhabditis species behave when exposed to gastropod cues. Therefore, we investigated whether P. hermaphrodita (commercial and wild isolated strains), P. neopapillosa and P. californica were attracted to mucus from several common snail species (Cepaea nemoralis, Cepaea hortensis, Arianta arbustorum and Cornu aspersum). We also examined whether snails (C. aspersum) collected from different locations around the UK differed in their attractiveness to wild isolates of P. hermaphrodita. Furthermore, we also investigated what properties of snail mucus the nematodes were attracted to, including hyaluronic acid and metal salts (FeSO4, ZnSO4, CuSO4 and MgSO4). We found that the commercial strain of P. hermaphrodita responded poorly to snail mucus compared to wild isolated strains, and C. aspersum collected from different parts of the UK differed in their attractiveness to the nematodes. We found that Phasmarhabditis nematodes were weakly attracted to all metals tested but were strongly attracted to hyaluronic acid. In a final experiment we also showed that pharmacological manipulation of cyclic guanosine monophosphate (cGMP) increased chemoattraction to snail mucus, suggesting that the protein kinase EGL-4 may be responsible for Phasmarhabditis sp. chemoattraction.

Identification and validation of reference genes for qPCR in the terrestrial gastropod Cepaea nemoralis.

Identifying and understanding mechanisms that generate phenotypic diversity is a fundamental goal of evolutionary biology. With a diversity of pigmented shell morphotypes governed by Mendelian patterns of inheritance, the common grove snail Cepaea nemoralis (Linnaeus, 1758) has been a model for evolutionary biologists and population geneticists for decades. However, the genetic mechanisms by which C. nemoralis generates this pigmented shell diversity remain unknown. An important first step in investigating this pigmentation pattern is to establish a set of validated reference genes for differential gene expression assays. Here we have evaluated eleven candidate genes for reverse transcription quantitative polymerase chain reaction (qPCR) in C. nemoralis. Five of these were housekeeping genes traditionally employed as qPCR reference genes in other species, while six alternative genes were selected de novo from C. nemoralis transcriptome data based on the stability of their expression levels. We tested all eleven candidates for expression stability in four sub-adult tissues of C. nemoralis: pigmented mantle, unpigmented mantle, head and foot. We find that two commonly employed housekeeping genes (alpha tubulin, glyceraldehyde 3-phosphate dehydrogenase) are unsuitable for use as qPCR reference genes in C. nemoralis. The traditional housekeeping gene UBIquitin on the other hand performed very well. Additionally, an RNA-directed DNA polymerase (RNAP), a Potassium Channel Protein (KCHP) and a Prenylated Rab acceptor protein 1 (PRAP), identified de novo from transcriptomic data, were the most stably expressed genes in different tissue combinations. We also tested expression stability over two seasons and found that, although other genes are more stable within a single season, beta actin (BACT) and elongation factor 1 alpha (EF1α) were the most reliable reference genes across seasons.

A comparative analysis of the camera eyes of gastropod pulmonate mollusks <i>Cepaea nemoralis </i>(Linnaeus, 1758) and <i>Cepaea hortensis </i>(Müller, 1774) (Stylommatophora, Helicidae)

A comparative analysis of the camera eyes of gastropod pulmonate molluscs Cepaea nemoralis and Cepaea hortensis was carried out on the basis of original and published data. It was demonstrated that the eyeballs and their main components in C. nemoralis and C. hortensis have more similarities in morphological and optical properties than in anatomical parameters.

Study on establishing normal ranges of chosen biochemical parameters of haemolymph of Cornu aspersum maxima and Cepaea nemoralis gastropods.

The aim of the study was to establish normal ranges for chosen biochemical parameters of haemolymph of snails (Gasropoda: Mollusca), in the light of the use of these animals as experi- mental models in various types of studies. The study was conducted on 100 specimens of Cornu aspersum maxima (CAM) and 100 specimens of Cepaea nemoralis (CN). The haemolymph col- lected from the animals was analysed using colorimetry to assay aspartate transaminase (AST) activity, alanine transaminase (ALT) activity, amylase activity and the concentrations of urea and triglycerides. In the further part of the study, the influence of administering doxycycline with feed on the change of AST and ALT activity in snail haemolymph has been studied. The normal values established for CAM are as follow: AST activity: 26-38 u/l, ALT activity: 0-11 u/l, amylase activity 9-16 u/l, concentration of urea: 3-6 mg/dl, concentration of triglycerides: 16-20 mg/dl. For CN, the following data have been obtained: AST activity: 30-80 u/l, ALT activity: 0-15 u/l, amylase activity 12-15 u/l, concentration of urea: 5-8 mg/dl, concentration of triglycerides: 18-24 mg/dl. It has been shown that doxycycline presents a high workload on the hepatopancreas of snails, which is reflected by a statistically significant (p ⟨ 0.05) increase of AST and ALT activity in the haemolymph of the specimens which obtained doxycycline in feed, as compared to the groups with antibiotic-free feed. The haemolymph activity of both studied parameters increased together with study time and tetracycline administration time.

Shell colour, temperature, (micro)habitat structure and predator pressure affect the behaviour of Cepaea nemoralis

Although shell colour polymorphism of the land snail Cepaea nemoralis is a well-known phenomenon, proximate and ultimate factors driving its evolution remain uncertain. Polymorphic species show variation in behavioural responses to selective forces. Therefore, we estimated effects of various environmental factors (temperature, humidity, food availability, (micro)habitat structure and predatory pressure) on behavioural response (frequency of locomotion, climbing and hiding) of C. nemoralis morphs, in experimental and natural conditions. In the experimental part of study, the frequency of locomotion was negatively affected by temperature and the presence of food and positively influenced by the presence of light. Morphs significantly differed in behavioural responses to environmental variability. Pink mid-banded and yellow five-banded morphs climbed less often and hide in shelter more often than yellow and pink unbanded individuals when temperature was low and food was absent. Snails fed most often at moderate temperature compared to low and high temperatures. Field investigations partially confirmed differences among morphs in frequency of climbing, but not in terms of probability of hiding in sheltered sites. In natural colonies, temperature and (micro)habitat structure significantly affected frequency of climbing as well as hiding in shelter. Snails more often hid in sheltered sites where thrushes preyed on Cepaea. Tendency of unbanded morphs to climb trees may have evolved under avian predatory pressure as thrushes forage on a ground. Tendency of banded morphs to hide in sheltered sites may reflect prey preferences for cryptic background. The results implicate that differential behaviour of C. nemoralis morphs compensate for their morphological and physiological limitations of adaptation to habitat.

Observations on the ecology of the endangered butterfly Zerynthia cassandra in a protected area of Northern Italy

Zerynthia cassandra is a butterfly endemic to the Italian peninsula. It was recently recognized as a sister species of Zerynthia polyxena. Z. cassandra is oligophagous as it feeds on a very restricted number of toxic plants belonging to the genus Aristolochia: Aristolochia pallida and A. rotunda. The paper reports the results of a research on the ecology of this species which was studied in the natural reserve "Stagni di Lungavilla" (Northern Italy) where the butterfly was living in a small patch of grassy vegetation bordered by a ditch at the margins of a pond. Z. cassandra develops one generation a year. Oviposition occurred in April. Eggs were mainly laid on the lower side of A. rotunda blades. The height of eggs from the ground depended on climatic trends, which affected the host plant phenology. Eggs mortality was mainly due to consumption of leaves by snails (Cepaea nemoralis) and slugs (Arion sp.). Larvae completed their development from April to May, while chrysalises were the aestivating and overwintering stage. Females did not lay eggs on host plants when they were part of the undergrowth. A decline of the butterfly due to habitat changes was recorded during the research (2001–2005). According to information coming from local butterfly watchers the butterfly used to be quite common in the natural reserve until 70’s. Its decline was caused by agricultural intensification and the natural evolution of vegetation towards woodlands, which gradually reduced the host plant density, so making the habitat unsuitable and causing the local extinction of the butterfly.

High level of sperm competition may increase transfer of accessory gland products carried by the love dart of land snails.

Postcopulatory adaptations that increase reproductive success compared to rivals, like the transfer of accessory gland products that promote paternity, are common when sperm competition occurs among males. In land snails, the dart shooting behavior and its adaptive significance, in promoting individual fitness through enhanced paternity of the successful dart shooter, have been considered such an adaptation. The fitness result gained is mediated by the transfer of mucus components on the love dart capable of altering the physiology of the receiver's reproductive tract. In this context, dart shooting and mucus transfer could be considered as processes targeted by sexual selection. While the effect of dart mucus is beneficial for the dart user, so far it has remained unknown whether its transport is greater when snails experience a higher level of sperm competition. Here, we report results of a study on inter‐ and intraspecific variations of dart and mucus gland morphometry, considered to be traits reflecting the ability of snails to adjust the production and transfer of mucus under varying sperm competition scenarios. We investigated four populations with different densities from four dart‐bearing species, Arianta arbustorum, Cepaea nemoralis, Cornu aspersum, and Helix lucorum. The results indicate that different adaptations of these traits occur among the studied species that all seem to achieve the same goal of transferring more mucus when sperm competition is higher. For example, the presence of longer and more branched mucous glands or an increase in dart surface most likely reflect increased mucus production and enhanced ability of mucus transport, respectively. Interestingly, the species for which the use of the dart is reported to be facultative, A. arbustorum, did not show any variation among the examined traits. To conclude, sexual selection in the form of sperm competition intensity seems to be an important selective force for these simultaneously hermaphroditic dart‐bearing snails, driving differences in sexual traits.

Inferring microevolution from museum collections and resampling: lessons learned from Cepaea.

Natural history collections are an important and largely untapped source of long-term data on evolutionary changes in wild populations. Here, we utilize three large geo-referenced sets of samples of the common European land-snail Cepaea nemoralis stored in the collection of Naturalis Biodiversity Center in Leiden, the Netherlands. Resampling of these populations allowed us to gain insight into changes occurring over 95, 69, and 50 years. Cepaea nemoralis is polymorphic for the colour and banding of the shell; the mode of inheritance of these patterns is known, and the polymorphism is under both thermal and predatory selection. At two sites the general direction of changes was towards lighter shells (yellow and less heavily banded), which is consistent with predictions based on on-going climatic change. At one site no directional changes were detected. At all sites there were significant shifts in morph frequencies between years, and our study contributes to the recognition that short-term changes in the states of populations often exceed long-term trends. Our interpretation was limited by the few time points available in the studied collections. We therefore stress the need for natural history collections to routinely collect large samples of common species, to allow much more reliable hind-casting of evolutionary responses to environmental change.

The Efficiency of Flotation Compared with Other Methods for Recovering Assemblages of Terrestrial and Aquatic Gastropods from Archaeological Deposits, with Reference to the Site of Pico Ramos (Basque Country, Spain)

ABSTRACTWe analysed assemblages of shells of land snails and small aquatic and wetland gastropods from a late Mesolithic-early Neolithic deposit in the cave of Pico Ramos (Basque Country). Shells were recovered by hand during excavation and all excavated deposit was processed by flotation, with a 250 μm mesh to recover floating material and a 1.0 mm mesh to recover the heavy fraction. The total assemblage comprised 5780 individuals (MNI) among 33 taxa, the sieved sub-assemblage having the highest proportions of these (2841 MNI in 31 taxa) and the hand-picked sub-assemblage the lowest (698 MNI in 11 taxa). Eleven taxa were absent from the flotation sub-assemblage, although the recovered MNI (2241; 39% of the total MNI) was high. The palaeoecological implications of the results are considered. Adult and large-sized juvenile shells of the edible land snail Cepaea nemoralis are abundant in the hand-picked sub-assemblage, but it is the absence of small-sized juvenile shells in the flotation and wet-sieved sub-assemblages that permits the inference that the species was collected for consumption. Wet sieving is therefore essential, both for interpreting hand-picked samples and in particular for the recovery of representative assemblages of land molluscs.

The gastropod shell has been co-opted to kill parasitic nematodes

Exoskeletons have evolved 18 times independently over 550 MYA and are essential for the success of the Gastropoda. The gastropod shell shows a vast array of different sizes, shapes and structures, and is made of conchiolin and calcium carbonate, which provides protection from predators and extreme environmental conditions. Here, I report that the gastropod shell has another function and has been co-opted as a defense system to encase and kill parasitic nematodes. Upon infection, cells on the inner layer of the shell adhere to the nematode cuticle, swarm over its body and fuse it to the inside of the shell. Shells of wild Cepaea nemoralis, C. hortensis and Cornu aspersum from around the U.K. are heavily infected with several nematode species including Caenorhabditis elegans. By examining conchology collections I show that nematodes are permanently fixed in shells for hundreds of years and that nematode encapsulation is a pleisomorphic trait, prevalent in both the achatinoid and non-achatinoid clades of the Stylommatophora (and slugs and shelled slugs), which diverged 90–130 MYA. Taken together, these results show that the shell also evolved to kill parasitic nematodes and this is the only example of an exoskeleton that has been co-opted as an immune system.

Land snails benefit from human alterations in rural landscapes and habitats

AbstractMetapopulation functioning in the context of landscape and habitat structure is generally well understood. However, in human‐transformed landscapes, human disturbance may affect not only a species of interest but also biotic interactions in which that species is engaged, which has been rarely studied at multiple spatial scales. We tested (1) the associations between local (habitat and micro‐habitat) and landscape factors and the occurrence and abundance of the land snail Cepaea nemoralis and two of its predators (thrushes and mice); and (2) the hypothesis that human disturbances modify associations between C. nemoralis and its predators. We surveyed snail colonies in rural landscapes of Poland. Generalized linear mixed models were used to test the stated hypotheses. At the landscape scale, the occurrence of C. nemoralis colonies was positively related to the cover of suitable habitats, fallow/waste land, and road length in the surrounding landscape. Bird predator abundance was positively correlated with the occurrence of snail colonies, mostly at sites with a high human impact, and was negatively correlated with snail abundance at sites with low human disturbance. Snail occurrence and abundance at the micro‐habitat scale were positively correlated with variables describing the proximity of shelter and humidity. Avian predation pressure was negatively correlated with forest and settlement cover, whereas rodent predation pressure was positively associated with snail abundance and was negatively correlated with bird abundance. Human disturbance at the habitat and landscape scales benefited C. nemoralis through the creation of suitable habitats and scaling down of avian predation pressure. Conditions related to shelter and humidity were linked to the distribution and abundance of snails at the micro‐habitat scale.

Cepaea nemoralis(Linnaeus, 1758) uses its shell as a defence mechanism to trap and kill parasitic nematodes

Slugs and snails are parasitised by a range of organisms including nematodes, bacteria, microsporidia, mites and flies (Barker, 2004). Of these, the nematodes are the most numerous and diverse as 108 species have evolved to parasitise molluscs (Grewal et al., 2003). One of these nematodes (Phasmarhabditis hermaphrodita) is a lethal parasite of a range of pestiferous slugs and snails including Deroceras reticulatum, Arion ater and Helix aspersa (Wilson et al., 1993). P. hermaphrodita has been formulated into a biocontrol agent (Nemaslug®) by Becker Underwood-BASF available for farmers and gardeners (Rae et al., 2007). Nematodes are mixed with water and applied using spraying equipment to soil where then go and search for potential gastropod hosts. They are attracted to slug mucus and faeces (Rae et al., 2006, 2009) and upon discovery they penetrate through the slugs’ mantle and kill it between 4 and 21 days (Wilson et al., 1993; Tan & Grewal, 2001).

Colour and pattern disequilibrium inCepaea nemoralison a northern European transect

Disequilibrium has been estimated for alleles at pairs of loci controlling shell colour and banding in populations of Cepaea nemoralis from northern Europe. There is strong overall positive disequilibrium between pink/yellow colour and unbanded/banded (pink unbanded in excess) but not for colour and the band-modifying loci mid-banded and trifasciate. When the colour/banding data are divided into four equally sized sections (quadrants) representing, respectively, high and low yellow and high and low unbanded frequencies, there is significant interaction in disequilibrium between quadrants, with high positive disequilibrium contributed by populations in which pink and unbanded are at high frequency. We interpret this as indicating diversifying, rather than stabilizing, selection. Simulations show how the overall value and quadrant differences may have built up. The brown allele at the colour locus is known to be associated with much higher unbanded frequencies than the other two colours, owing to some intrinsic incompatibility between banding and ground colour. It is suggested that the same may apply, although to a lesser extent, with respect to pink and unbanded relative to yellow.

Trace metal transfer in a soil–plant–snail microcosm field experiment and biomarker responses in snails

A microcosm experiment was performed to investigate temporal (up to 16 weeks) and spatial variation in metal transfer in a soil–food (nettle)–snail (Cepaea nemoralis) food chain and biomarker responses in the digestive gland of the same snails. Adult snails were sampled from an uncontaminated site and transferred to five sites located 0.5, 2.5, 3, 5, and 10km from a historically polluted point source. All sites were park areas where grasslands interfered with patches of deciduous forest. Soil physicochemical properties (pH, clay%, OC%) significantly explained the uptake of metals by nettle. Concentrations of metals in the digestive gland (DG) of snails were significantly related to those in nettle, but rarely to soil physicochemical properties. In general metal concentrations in the DG fluctuated while As, Ni, Pb and Zn showed a site dependent increase with time. Despite the long term exposure, biomarker concentrations (lipid, glycogen, proteins, glutathione-S-transferases), and shell morphology, were not related to DG metal concentrations. Our investigation emphasizes the need for controlled long-term studies on the transfer and effects of metals in food chains since short term studies might only show temporary physiological changes due to experimental acclimation.

Cepaea nemoralis (L.) in Göteborg, S.W. Sweden: variation in a recent urban invader

Although present since the mid 19th century, the introduced snail species Cepaea nemoralis has spread extensively in Goteborg, mainly in the last two decades. Early samples show the predominance of yellow-shelled morphs, but in modern populations pink shells predominate. There is great variation in morph frequencies among modern populations with high values of FST, and with no relationships to habitat. The patterns shown resemble those seen in Sheffield (UK), a city also colonised over a similar time period, but differ from those seen in regions where the species has been established for much longer. The combination of early and recent records suggests not only that founder effects play a large part in determining morph frequencies, but that present populations derive from multiple colonisations from different sources. key woRDs: Cepaea nemoralis, polymorphism, invasion, Goteborg