Molluscicidal activity of calcium borate nanoparticles with kodom ball-flower structure on hematological, histological and biochemical parameters of Eobania vermiculata snails.

Land snails are the most harmful pests in agricultural fields. Eobania vermiculata is a widespread snail species that causes massive damage to all agricultural crops. Thus, the molluscicidal activity of calcium borate nanoparticles (CB-NPs) against Eobania vermiculata was evaluated and compared with metaldehyde (Gastrotox® E 5% G). The amorphous phase of CB-NPs was obtained after thermal treatment at a low temperature (500 °C) which conformed by X-ray diffraction (XRD) analysis. CB-NPs are composed of aggregated nano-sheets with an average thickness of 54 nm which enhanced their molluscicidal activity. These nano-sheets displayed meso-porous network architecture with pore diameters of 13.65 nm, and a 9.46 m2/g specific surface area. CB-NPs and metaldehyde (Gastrotox® E 5% G) exhibited molluscicidal effects on Eobania vermiculata snails with median lethal concentrations LC50 of 175.3 and 60.5 mg/l, respectively, after 72 h of exposure. The results also showed significant reductions of Eobania vermiculata snails hemocytes' mean total number, the levels of Testosterone (T) and Estrogen (E), alkaline phosphatase, acid phosphatase, albumin, and protein concentrations, succinate dehydrogenase, glucose, triglycerides and phospholipids levels, while significant increases in the phagocytic index and mortality index, both transaminases (ALT and AST) and glycogen phosphorylase concentration were observed after the exposure to LC50 of CB-NPs or metaldehyde (Gastrotox® E 5% G) compared to the control group. Therefore, CB-NPs could be used as an alternative molluscicide for controlling Eobania vermiculata, but further studies are needed to assess their effects on non-target organisms.

Speciation of rock-dwelling snail species: Disjunct ranges and mosaic patterns reveal the importance of long-distance dispersal in Chilostoma (Cingulifera) in the European Southern Alps.

To better understand the origin of the high diversity and endemism in the Southern Alps of Europe, we investigated the phylogeny and population structure of the rock-dwelling snail group Chilostoma (Cingulifera) in the Southern Alps. We generated genomic ddRAD data and mitochondrial sequences of 104 Cingulifera specimens from 28 populations and 14 other Ariantinae. Until recently, about 30 Cingulifera taxa were classified as subspecies of a single polytypic species. The phylogenetic and population genetic analyses of the ddRAD data and mitochondrial sequences revealed that Cingulifera in the Southern Alps is differentiated into three species. Each of the three Chilostoma (Cingulifera) species occupies disjunct sub-areas, which are separated by areas occupied by other Chilostoma taxa. Neighbouring populations of different species show little or no admixture. Tests indicating that the genetic differentiation of the three Cingulifera taxa cannot be explained by isolation by distance confirmed their species status. The disjunct range patterns demonstrate the importance of stochastic events such as passive long-distance dispersal for the evolution of population structure and speciation in these snails, and of priority effects and ecological competition as important factors influencing species distributions.

Bio-molluscicidal potential and biochemical mechanisms of clove oil and its main component eugenol against the land snail, Theba pisana.

The land snail, Theba pisana is a serious pest that adversely affects various crops in sustainable agriculture. Essential oils and their constituents represent an environmentally sound alternative to synthetic pesticides. Our study aimed to investigate the lethal and sub-lethal toxicity of clove oil and its main component eugenol to understand the mechanisms underlying its toxic action against T. pisana. The GC-MS profile of the clove oil composition was characterized. In the laboratory experiment, LD50 of clove oil and eugenol via the contact testing were determined after 48 and 72 h. Moreover, sub-lethal effects of clove oil or eugenol on the survivors following the exposure of snails to the 25 and 50% of the LD50/48 and 72 h were evaluated through using snail tissues for biochemical measurments. The GC-MS analysis showed that eugenol (64.87%) was the major constituent present in the oil. The results also showed that LD50 values at 48 and 72 h were 2006.5 and 1493.5 µg/g b.w for oil and 239.6 and 195.3 µg/g b.w for eugenol, respectively. Compared to control, the sub-lethal effects of clove oil or eugenol at 48 and 72 h showed a significant increase in reduced glutathione (GSH) levels. Catalase (CAT) and glutathione-S-transferase (GST) activities significantly elevated in oil- or eugenol-treated snails, except at low dose after 48 h. After two exposure times, snails exposed to oil or eugenol at both sub-lethal effects had considerably higher γ-glutamyltransferase (γ-GT) and aspartate aminotransferase (AST) activities. Moreover, markedly augmentation in alkaline phosphatase (ALP) and alanine aminotransferase (ALT) activities at all exposure times, with the exception of snails treated with low dose of eugenol after 48 h was observed. Both clove oil and eugenol at the tested doses caused a significant inhibition in acetylcholinesterase (AChE) activity at two exposure times. Our findings highlight the potential of clove oil and eugenol, as an efficient natural molluscicide alternative to its synthetic counterparts for snail control.

Public Health Risk of Foodborne Pathogens in Edible African Land Snails, Cameroon.

In tropical countries, land snails are an important food source; however, foodborne disease risks are poorly quantified. We detected Campylobacter spp., Yersinia spp., Listeria spp., Salmonella spp., or Shiga-toxigenic Escherichia coli in 57%-86% of snails in Cameroon. Snail meat is a likely vector for enteric diseases in sub-Saharan Africa countries.

Design, Synthesis, Characterization, and Molluscicidal Activity Screening of New Nicotinonitrile Derivatives against Land Snails, M. cartusiana.

A new series of nicotinonitrile derivatives 2-7 was designed and synthesized from the starting material (E)-3-(4-chlorophenyl)-1-(4-methoxyphenyl)prop-2-en-1-one (1) to assess their molluscicidal activity. The newly synthesized nicotinonitrile compounds 2-7 were characterized based on FTIR, 1H-NMR, and 13C-APT NMR spectra as well as elemental microanalyses. The target compounds 2-7 were screened for their toxicity effect against M. cartusiana land snails and were compared to Acetamiprid as a reference compound. The results demonstrated that the nicotinonitrile-2-thiolate salts 4a and 4b had good mortality compared with that of Acetamiprid. The results of the in vivo effect of the prepared nicotinonitrile molecules 2, 4a, and 4b on biochemical parameters, including AChE, ALT, AST, and TSP, indicated a reduction in the level of AChE and TSP as well as an increase in the concentration of transaminases (ALT and AST). A histopathological study of the digestive gland sections of the M. cartusiana land snails was carried out. The nicotinonitrile-2-thiolate salts 4a,b showed vacuolization, causing the digestive gland to lose its function. It could be concluded that the water-soluble nicotinonitrile-2-thiolate salts 4a,b could be adequate molluscicidal molecules against M. cartusiana land snails.

Insights into the ecotoxicological perturbations induced by the biocide Abamectin in the white snail, Theba pisana.

Abamectin (avermectin B1, ABM) has been widely used as a biocide in agriculture, veterinary and medicine around the world. Yet, there is still a lack of knowledge about the ecotoxicological effects of ABM. In this study, we investigated the acute toxicity and sub-lethal (20% and 60% LD50) biochemical responses of ABM on the non-target land snail, Theba pisana. Mortality of snails increased with the dose increase, resulting 48 h- LD50 value of 1.048 µg/snail. The biochemical results showed a decrease in glycogen content and lipids for two sub-lethal doses after all time intervals, whereas increased the level of total proteins after exposure to 60% LD50 ABM. Overall, the tested sub-lethal doses significantly decreased the total energy reserves. ABM-exposure to snails elevated γ-Glutamyl transferase and Lactate dehydrogenase activities at all-time intervals. A significant increase of Glutathione-S-transferase activity was also recorded in snails exposed to 20% and 60% LD50 after 7 days and all time intervals, respectively. However, ABM inhibited the activity of Aspartate aminotransferase and Alanine aminotransferase after 7 days of exposure. Our investigation provides new insights into the disturbances of energy reserves and enzyme activities in T. pisana that are sensitive and may be used as biomarkers for assessing ABM toxicity.

Repeated hybridization increased diversity in the door snail complex Charpentieria itala in the Southern Alps.

The door snail species complex Charpentieria itala is widely distributed in the Southern Alps and subdivided into several morphologically differentiated subspecies. Thus, it can be used as a model group for understanding migration and differentiation processes in the Southern Alps. We generated genome-wide double digest Restriction Site Associated DNA (ddRAD) sequencing data for 166 specimens from 36 populations of the door snail Charpentieria itala and for 8 specimens of the other three Charpentieria species to reconstruct their evolutionary history and phylogeography. Phylogenetic and structure analyses based on the ddRAD data indicated that the repeated separation of the populations in western and eastern groups by the Garda glacier during the glacials was the process that most strongly shaped the population structure of C. itala. This process may also explain a similar phylogeographic boundary in many other southern Alpine animal and plant species. Our study revealed that some populations that resemble Charpentieria stenzii morphologically and ecologically, the 'stenzioid' subspecies, originated by a hybridization event with Charpentieria stenzii. A further hybridization event between stenzioid populations that survived the glacials in mountain refuges and non-stenzioid populations that probably came into contact with stenzioid populations as a result of climate warming during an interglacial resulted in the origin of a hybrid subspecies that is adapted to intermediate altitudes. Our study demonstrated that the origin of new differentiated taxa by hybridization, is more frequent than previously assumed.

Ecological specialization resulting in restricted gene flow promotes differentiation in door snails.

We tested the hypothesis that ecological specialization that affects dispersal promotes diversification by a comparison of the genetic structure of two sister species of door snails across their broadly overlapping ranges in the Crimean Mountains. The hypothesized effect of ecological specialization on diversification is supported by STRUCTURE analyses that showed that Mentissa gracilicosta that is restricted to limestone rocks, is subdivided into several distinct clusters, whereas all populations of the species adapted to more continuous habitat, the forests-dwelling Mentissa canalifera, were assigned to a single cluster. Furthermore, it is supported by AMOVAs that showed that a larger part of the genetic variation of M. gracilicosta is apportioned among populations than in M. canalifera. The stronger genetic differentiation of the M. gracilicosta populations corresponds to their more distinct morphological differentiation that resulted in the classification of M. gracilicosta into several geographical subspecies, whereas the more continuously distributed M. canalifera was not subdivided into subspecies. The stronger differentiation of populations of M. gracilicosta compared to M. canalifera can be ascribed to reduced gene flow between the isolated populations of M. gracilicosta and to founder events associated with the long distance dispersal events that are necessary for the colonization of isolated rocks by M. gracilicosta. In Central Europe, the Pleistocene climatic oscillations selected for species with high dispersal abilities, whereas the more stable climate in southern Europe facilitated the non-adaptive radiation of rock-dwelling door snails. Thus, the intrinsic ecological properties of these species groups contributed to the latitudinal diversity gradient.

When is a "cryptic" species not a cryptic species: A consideration from the Holarctic micro-landsnail genus Euconulus (Gastropoda: Stylommatophora).

Naive use of molecular data may lead to ambiguous conclusions, especially within the context of "cryptic" species. Here, we integrated molecular and morphometric data to evaluate phylogenetic relationships in the widespread terrestrial micro-snail genus, Euconulus. We analyzed mitochondrial (16S + COII) and nuclear (ITS1 + ITS2) sequence across 94 populations from Europe, Asia and North America within the nominate species E. alderi, E. fulvus and E. polygyratus, and used the southeastern USA E. chersinus, E. dentatus, and E. trochulus as comparative outgroups. Phylogeny was reconstructed using four different reconstruction methods to identify robust, well-supported topological features. We then performed discriminant analysis on shell measurements between these genetically-identified species-level clades. These analyses provided evidence for a biologically valid North American "cryptic" species within E. alderi. However, while highly supported polyphyletic structure was also observed within E. fulvus, disagreement in placement of individuals between mtDNA and nDNA clades, lack of morphological differences, and presence of potential hybrids imply that these lineages do not rise to the threshold as biologically valid cryptic species, and rather appear to simply represent a complex of geographically structured populations within a single species. These results caution that entering into a cryptic species hypothesis should not be undertaken lightly, and should be optimally supported along multiple lines of evidence. Generally, post-hoc analyses of macro-scale features should be conducted to attempt identification of previously ignored diagnostic traits. If such traits cannot be found, i.e. in the case of potentially "fully cryptic" species, additional criteria should be met to propound a cryptic species hypothesis, including the agreement in tree topology among both mtDNA and nDNA, and little (or no) evidence of hybridization based on a critical analysis of sequence chromatograms. Even when the above conditions are satisfied, it only implies that the cryptic species hypothesis is plausible, but should optimally be subjected to further careful examination.

Use of multiple endpoints to investigate the ecotoxicological effects of abamectin and thiamethoxam on Theba pisana snails.

The ecotoxicological effects of dietary exposure to sublethal concentration (1/20 LC50) of abamectin (ABM) and thiamethoxam (TMX) for two weeks exposure and one week recovery on oxidative stress parameters; lipid peroxidation (LPO), reduced glutathione (GSH), catalase (CAT) and glutathione S-transferase (GST), Deoxyribonucleic acid (DNA) damage as well as immunological parameters; cell death, phagocytosis, lysosomal membrane stability (LMS), lectins, superoxide anion (O2-) generation, phenoloxidase (PO), peroxidase (POD) and hemocyanin (Hc) of the land snail, Theba pisana were examined. The results showed that both tested compounds significantly increased DNA damage, LPO content, the activity of CAT and GST, cell death, POD activity, Hc level, whereas, significantly declined in phagocytic activity, LMS, lectins level, O2- generation, PO activity, and GSH content compared to the controls after two week exposure. After one week of recovery, the measured biochemical parameters of treated snails were slightly repaired but their levels were still less than that of the level of untreated animals. Overall, based on acute toxicity endpoints, ABM appeared to be more harmful than TMX against this animal. Indeed, the different patterns of endpoint responses could represent a useful picture to characterise exposure to these pesticides in the land snail, T. pisana. This battery of snail endpoints might be a promising option to biomonitor the health of the terrestrial ecosystem and to offer valuable insights to the pesticides toxicity mechanisms.

The impact of the Gabcíkovo hydroelectric power barrier on the Danube floodplain environment-the results of long-term monitoring of land snail fauna.

The Gabcíkovo Water Project, a major construction of damming and canalizing on the upper part of the middle Danube, significantly changed hydrological regime of the Danube inland delta, destroyed or affected most of the 230 km2 of wetlands and directly disrupted the original and unique ecosystem along a 37 km long river stretch. The aim of this study was to describe the effect of the Gabcíkovo Waterworks on the taxonomical and functional structure of the molluscan assemblages in the Danube riparian floodplain forests. The results demonstrate that the Gabcíkovo Waterworks had a direct and long-lasting effect on the direction of the succession of terrestrial molluscan assemblages, especially in the area of the by-pass section. The changes in the soil moisture caused by the waterworks' operation led to significant changes in the species and functional composition of these assemblages. More specifically, however, the proportion of the generalists which prefer dry biotopes increased, while the number of moisture-demanding species decreased. Our results indicate that the current artificial flooding system cannot fully replace previous natural floods in the Danube inland delta, and it is also insufficient for restoration and preservation of the humidity conditions in the softwood floodplain forests which would be similar to the pre-operation period of the Gabcíkovo Waterworks.

Molecular phylogeny of Candidula (Geomitridae) land snails inferred from mitochondrial and nuclear markers reveals the polyphyly of the genus.

The genus Candidula (Geomitridae), consisting of 28 species in Western Europe as currently described, has a disjunct distribution in the Iberian Peninsula, Italy, the Balkans, the Aegean Islands, and one species on the Canary Islands. Although the genus is seemingly well defined by characters of the reproductive system, the relationships within the genus are still unclear and some authors have indicated a possible subgeneric division based on the internal morphology of the dart sac. Despite substantial phylogenetic incongruence, we present a well-resolved molecular phylogeny of Candidula based on two mitochondrial genes (COI and 16S rRNA), the nuclear rDNA region (5.8S rNRA + ITS2 + 28S rRNA) and seven additional nuclear DNA regions developed specifically for this genus (60SL13, 60SL17, 60SL7, RPL14, 40SS6, 60SL9, 60SL13a), in total 5595 bp. Six reciprocally monophyletic entities including Candidula species were recovered, grouping into two major clades. The incorporation of additional geomitrid genera allowed us to unequivocally demonstrate the polyphyly of the genus Candidula. One major clade grouped species from southern France and Italy with the widely distributed species C. unifasciata. The second major clade grouped all the species from the Iberian Peninsula, including C. intersecta and C. gigaxii. Candidula ultima from the Canary Islands was recovered as separated lineage within the latter clade and related to African taxa. The six monophyla were defined as six new genera belonging to different tribes within the Helicellinae. Thus, we could show that similar structures of the stimulatory apparatus of the genital system in different taxa do not necessarily indicate a close phylogenetic relationship in the Geomitridae. More genera of the family are needed to clarify their evolutionary relationships, and to fully understand the evolution of the stimulatory apparatus of the genital system within the Geomitridae.

Laboratory studies on the thermal tolerance and response of enzymes of intermediate metabolism in different land snail species.

Land snails species occur in a range of habitats from humid to semi-arid and arid ones and seasonal variations in their physiology and biochemical composition have been linked to annual cycles of photoperiod, temperature, humidity and water availability. In an effort to understand the thermal tolerance and the impact of temperature elevation on tissue metabolism of land snails we determined the mortality, heamolymph PO2 and the activities of enzymes of intermediary metabolism in three land snail species (Helix lucorum, Helix pomatia and Cornu aspersum) differing in their geographical distribution and inhabiting areas with different climatic characteristics. No mortality was observed in both population of Cornu aspersum, while Helix pomatia exhibited higher mortality than Helix lucorum. PO2 dropped within the first 10days of exposure to elevated temperature in all species, although in Cornu aspersum this decrease was significantly lower. No significant reduction in the enzymatic activities of all glycolytic enzymes studied, as well as of citrate synthase (CS) and 3-hydroxyacyl-CoA dehydrogenase (HOAD) was observed in the more thermal tolerant species C. aspersum from both populations studied. Significant reductions of enzymatic activity of the glycolytic enzymes phosphofructokinase (PFK), pyruvate kinase (PK) and d-Lactate dehydrogenase (d-LDH) was observed in Helix lucorum and Helix pomatia. The observed inter-specific differences seem to be in accordance with the life cycle characteristics of each species and may be attributed to climatic differences among habitats within their distribution range.

Relevance of body size and shell colouration for thermal absorption and heat loss in white garden snails, Theba pisana (Helicidae), from Northern France.

The internal temperature of land snails depends on environmental factors, such as exposure to electromagnetic radiation and airflow as well as biotic factors including shell size, shell colouration and thickness or the resting position of the snail. In controlled field experiments, we quantified heating by thermal absorption of light and airflow-induced heat loss in the white garden snail, Theba pisana, from Normandy, France. Heating experiments revealed a significant positive relation of the internal body temperature with illumination period, shell temperature and air temperature at different times of day. The size of the snails was negatively related with both of the given illumination times: smaller animals heated up stronger than larger ones. The temperature at the surface of the shell significantly depended on the illumination period and the time of day. An AIC-based quality assessment of multiple linear modelling showed that, for explaining both shell surface and internal temperature of the soft body, several factors, i.e., exposure time, daytime, shell size and colouration contributed to the best models, respectively. Similarly, heat loss of the soft body after and during exposure of the snails to sunlight by a constant airflow depended on the initial body temperature, shell size, colouration and ambient air temperature. Our study revealed also the importance of both shell size and colouration for the loss of body temperature under natural conditions: small and banded animals that had heated up to temperatures above 30°C cooled down faster than large and un-banded ones.

Recent expansion and relic survival: Phylogeography of the land snail genus Helix (Mollusca, Gastropoda) from south to north Europe.

Little is known about the evolutionary history of Helix, despite the fact that it includes the largest land snails in the western Palaearctic, some of which (e.g. H. pomatia Linnaeus, 1758) are valuable human food. We compared two groups of Helix with apparently contrasting evolutionary histories: the widespread species H. pomatia and the group distributed along the Italian Apennine chain, a relatively unknown set of species with a restricted distribution over a range of altitudes. To reconstruct the evolutionary trajectories of these two groups, we analysed morphological (shell and genitalia) and molecular characters (mitochondrial and nuclear markers) in a total of 59 populations from northern and central Europe (H. pomatia) and along the Apennine chain (various species). We also reconstructed the phylogeny and the evolutionary history of the genus by combining our data with that currently available in the literature. We found that spatial changes did not merely imply fragmentation of populations, but also implied environmental changes (woodlands vs. grasslands) that may have triggered the observed phenotypic diversification. We also found that Anatolia is the ancestral range of Helix and is therefore an important area for the Palaearctic diversity. The results provide insights into the evolutionary history of species richness and more generally into the processes that may have shaped the distribution and diversification of these organisms across Europe and the peri-Mediterranean area.

Presumable incipient hybrid speciation of door snails in previously glaciated areas in the Caucasus.

Homoploid hybrid speciation, speciation by hybridization without a change in chromosome number, may be the result of an encounter of closely related species in a habitat that is different from that usually occupied by these species. In the northwestern Caucasus the land snail species Micropontica caucasica and M. circassica form two distinct entities with little admixture at low and intermediate altitudes. However, at higher altitudes in the Lagonaki plateau, which were repeatedly glaciated, Micropontica populations with intermediate characters occur. Admixture analyses based on AFLP data demonstrated that the populations from the Lagonaki plateau are homoploid hybrids that now form a cluster separate from the parental species. The Lagonaki populations are characterized by a mtDNA haplotype clade that has been found in the parental species only once. The fixation of this haplotype clade in most hybrid populations suggests that these haplotypes are better adapted to the cooler conditions in high altitude habitats and have replaced the haplotypes of the parental species in a selective sweep. The fixation of a presumably adaptive mitochondrial haplotype clade in the Lagonaki populations is an important step towards speciation under the differential fitness species concept.

Habitat augmentation drives secondary invasion: an experimental approach to determine the mechanism of invasion success.

The entry of secondary invaders into, or their expansion within, native communities is contingent on the changes wrought by other (primary) invaders. When primary invaders have altered more than one property of the recipient community, standard descriptive and modeling approaches only provide a best guess of the mechanism permitting the secondary invasion. In rainforest on Christmas Island, we conducted a manipulative field experiment to determine the mechanism of invasion success for a community of land snails dominated by non-native species. The invasion of rainforest by the yellow crazy ant (Anoplolepis gracilipes) has facilitated these land snails, either by creating enemy-free space and/or increased habitat and resources (in the form of leaf litter) through the removal of the native omnivorous-detritivorous red land crab (Gecarcoidea natalis). We manipulated predator densities (high and low) and leaf litter (high and low) in replicated blocks of four treatment combinations at two sites. Over the course of one wet season (five months), we found that plots with high leaf litter biomass contained significantly more snails than those with low biomass, regardless of whether those plots had high or low predation pressure, at both the site where land crabs have always been abundant, and at the site where they have been absent for many years prior to the experiment. Each site was dominated by small snail species (<2 mm length), and through handling size and predation experiments we demonstrated that red crabs tend not to handle and eat snails of that size. These results suggest that secondary invasion by this community of non-native land snails is facilitated most strongly by habitat and resource augmentation, an indirect consequence of red land crab removal, and that the creation of enemy-free space is not important. By using a full-factorial experimental approach, we have confidently determined-rather than inferred-the mechanism by which primary invaders indirectly facilitate a community of secondary invaders.

Field studies on the annual activity and the metabolic responses of a land snail population living in high altitude.

In the context of the metabolic cold adaptation hypothesis (MCA), we investigated a) the life and activity cycle characteristics and b) the metabolic responses of the endemic land snail species Cattania trizona olympica living at 1100m altitude in Olympus mountain (Greece). Field observations on the annual activity cycle of C. trizona olympica revealed that snails' activity was restricted mainly between the end of May and September, when the higher temperatures were recorded, while first matings were recorded in July and the last ones in mid September indicating a restricted favorable time period for reproduction. The activities of enzymes of intermediate metabolism showed a periodic seasonal pattern of change which seems to be closely related to the pattern of annual changes of air temperature and most of them exhibited higher activities during the coldest and warmest periods of the year. Moreover the data indicate a distinct differentiation of fuel oxidation during arousal and reproductive periods with lipid oxidation, apart from carbohydrates, contributing significantly to ATP turnover during reproductive activity. The higher enzymatic activities, determined in the tissues of C. trizona olympica than the corresponding ones determined in the tissues of the land snail species living at low altitudes, might indicate higher sensitivity of the intermediate metabolism and ATP turnover in C. trizona olympica to changes in environmental factors. Although the latter seems to be in line with the MCA hypothesis, it needs further investigation on metabolic rates to support it.

De novo Transcriptome Generation and Annotation for Two Korean Endemic Land Snails, Aegista chejuensis and Aegista quelpartensis, Using Illumina Paired-End Sequencing Technology.

Aegista chejuensis and Aegista quelpartensis (Family-Bradybaenidae) are endemic to Korea, and are considered vulnerable due to declines in their population. The limited genetic resources for these species restricts the ability to prioritize conservation efforts. We sequenced the transcriptomes of these species using Illumina paired-end technology. Approximately 257 and 240 million reads were obtained and assembled into 198,531 and 230,497 unigenes for A. chejuensis and A. quelpartensis, respectively. The average and N50 unigene lengths were 735.4 and 1073 bp, respectively, for A. chejuensis, and 705.6 and 1001 bp, respectively, for A. quelpartensis. In total, 68,484 (34.5%) and 77,745 (33.73%) unigenes for A. chejuensis and A. quelpartensis, respectively, were annotated to databases. Gene Ontology terms were assigned to 23,778 (11.98%) and 26,396 (11.45) unigenes, for A. chejuensis and A. quelpartensis, respectively, while 5050 and 5838 unigenes were mapped to 117 and 124 pathways in the Kyoto Encyclopedia of Genes and Genomes database. In addition, we identified and annotated 9542 and 10,395 putative simple sequence repeats (SSRs) in unigenes from A. chejuensis and A. quelpartensis, respectively. We designed a list of PCR primers flanking the putative SSR regions. These microsatellites may be utilized for future phylogenetics and conservation initiatives.

The direct cost of traumatic secretion transfer in hermaphroditic land snails: individuals stabbed with a love dart decrease lifetime fecundity.

Several taxa of simultaneously hermaphroditic land snails exhibit a conspicuous mating behaviour, the so-called shooting of love darts. During mating, such land snail species transfer a specific secretion by stabbing a mating partner's body with the love dart. It has been shown that sperm donors benefit from this traumatic secretion transfer, because the secretions manipulate the physiology of a sperm recipient and increase the donors' fertilization success. However, it is unclear whether reception of dart shooting is costly to the recipients. Therefore, the effect of sexual conflict and antagonistic arms races on the evolution of traumatic secretion transfer in land snails is still controversial. To examine this effect, we compared lifetime fecundity and longevity between the individuals that received and did not receive dart shooting from mating partners in Bradybaena pellucida. Our experiments showed that the dart-receiving snails suffered reduction in lifetime fecundity and longevity. These results suggest that the costly mating behaviour, dart shooting, generates conflict between sperm donors and recipients and that sexually antagonistic arms races have contributed to the diversification of the morphological and behavioural traits relevant to dart shooting. Our findings also support theories suggesting a violent escalation of sexual conflict in hermaphroditic animals.