Molecular Identification of the Human Pathogen Amphimerus sp. in the Freshwater Snail Aroapyrgus sp. in Ecuador.

Here, we report for the first time the snail intermediate host for the Amphimerus liver fluke, a foodborne trematodiasis. In Ecuador, Amphimerus of the Opisthorchiidae family, infects humans, cats, and dogs, in the tropical Pacific-coast region. Opisthorchiidae comprising also Clonorchis sinensis, Opisthorchis sp., and Metorchis sp., have complex life cycles involving a definitive and two intermediate hosts. We identified morphologically and investigated the presence and prevalence of Amphimerus cercaria and DNA in freshwater snails collected in a human-amphimeriasis endemic region in Ecuador, extracted DNA from snail tissue and emerged cercariae, performed real-time polymerase chain reaction (PCR) with the newly developed primers and probe amplifying the Amphimerus ribosomal internal transcribed spacer 2 (ITS2) region, and sequenced the amplified DNA fragment. We collected 2,800 snails, characterized four species Aroapyrgus sp., Melanoides tuberculata, Biomphalaria cousini, and Aplexa marmorata, isolated three cercariae morphotypes. Of the 640 snails analyzed by qPCR, only Aroapyrgus and one of the three cercariae resulted positive, at a 15% infection prevalence. Polymerase chain reaction revealed that the Aroapyrgus snail and cercaria-morphotype-3 corresponded to Amphimerus, but not to C. sinensis, Fasciola hepatica, or Paragonimus mexicanus. The sequence of amplified DNA product matched that of human-isolated Amphimerus. This finding constitutes the first documentation that Aroapyrgus sp. is the first intermediate host for the Amphimerus sp. that infect humans in Ecuador. The ITS2-gene PCR and sequencing analysis demonstrated a high prevalence of snail infection and proved useful for detecting the infection in snails, which findings can help the establishment of suitable control programs against transmission in any endemic region of interest.

Systematics and geographical distribution of Galba species, a group of cryptic and worldwide freshwater snails.

Cryptic species can present a significant challenge to the application of systematic and biogeographic principles, especially if they are invasive or transmit parasites or pathogens. Detecting cryptic species requires a pluralistic approach in which molecular markers facilitate the detection of coherent taxonomic units that can then be analyzed using various traits (e.g., internal morphology) and crosses. In asexual or self-fertilizing species, the latter criteria are of limited use. We studied a group of cryptic freshwater snails (genus Galba) from the family Lymnaeidae that have invaded almost all continents, reproducing mainly by self-fertilization and transmitting liver flukes to humans and livestock. We aim to clarify the systematics, distribution, and phylogeny of these species with an integrative approach that includes morphology, molecular markers, wide-scale sampling across America, and data retrieved from GenBank (to include Old World samples). Our phylogenetic analysis suggests that the genus Galba originated ca. 22 Myr ago and today comprises six species or species complexes. Four of them show an elongated-shell cryptic phenotype and exhibit wide variation in their genetic diversity, geographic distribution, and invasiveness. The remaining two species have more geographically restricted distributions and exhibit a globose-shell cryptic phenotype, most likely phylogenetically derived from the elongated one. We emphasize that no Galba species should be identified without molecular markers. We also discuss several hypotheses that can explain the origin of cryptic species in Galba, such as convergence and morphological stasis.

Lymnaeidae from Santander and bordering departments of Colombia: Morphological characterization, molecular identification and natural infection with Fasciola hepatica.

The Lymnaeidae constitute a family of freshwater gastropod molluscs whose diversity and ecology have been infrequently studied throughout Colombia. Some lymnaeid species act as intermediate hosts of trematode parasites, which are of great importance in both the veterinary and medical fields. Among trematode parasites, Fasciola hepatica is best known for being an important parasite of sheep and cattle for decades and causes significant economic losses in these livestock species. The main objective of this work is to identify the various species of lymnaeids that occupy different geographical regions of Santander and its bordering departments within Colombia. This will expand the knowledge of lymnaeid diversity in Colombia and provide further insight into their role in the transmission of F. hepatica. A total of 118 georeferenced sites between 126 m.a.s.l. and 3870 m.a.s.l. were sampled in Santander, Boyacá, Norte de Santander and Cundinamarca, respectively. Lymnaeid snails were identified according to the morphology of their shells and by several characteristics of their reproductive systems. Species identification was confirmed using DNA barcoding. Four lymnaeid species are reported in the study area: the native Galba cousini and three exotic species, Pseudosuccinea columella, G. truncatula and G. schirazensis. The four species were examined for natural infection with F. hepatica. Infected variants of the main snail host, G. cousini, were found in the Onzaga, Encino and Vetas municipalities of Santander, as well as in the Belén municipality of Boyacá. A second species, G. truncatula was also found naturally infected in Mutiscua municipality of Norte de Santander. The two other species, P. columella and G. schirazensis were found free of infection.

Prevalence of Fasciola hepatica infection in Galba cousini and Galba schirazensis from an Andean region of Ecuador.

Some Lymnaeid snails are intermediate hosts of the liver fluke Fasciola hepatica, the causal agent of fasciolosis, a zoonotic parasitic disease. Human and livestock fasciolosis has been reported in a highland community located in the Chimborazo Province of the Ecuadorian Andes. However, no previous study has been carried out to identify which snail species act as intermediate host/s of F. hepatica. This study first aimed to identify the intermediate snail species and secondly to determine the prevalence of natural infection with F. hepatica in 230 lymnaeid snails sampled from irrigation and drainage canals in this area. The first objective entailed observations of shell morphology and internal organs as well as sequencing of the cytochrome oxidase subunit 1 (COI) gene. For the second objective, we used classic parasitological methods (observation of rediae and cercarial emission) and PCR amplification specie-specific to F. hepatica. COI haplotype networks were built to elucidate phylogeographic relationships between the snail populations from this highland community with other American and worldwide populations. We identified two lymnaeid Galba cousini and Galba schirazensis and found high infection rates of F. hepatica in G. cousini, but these differed according to the method used, with PCR showing a higher rate (61 ± 20%) compared to rediae observation (29 ± 17%). F. hepatica in G. schirazensis was identified only by DNA amplification. G. cousini populations were genetically structured by geographic distance whereas G. schirazensis populations showed very low genetic diversity. The higher abundance and infection rate of G. cousini compared to G. schirazensis suggests that the former is likely the specie responsible for F. hepatica transmission in this region.

Patterns of distribution, population genetics and ecological requirements of field-occurring resistant and susceptible Pseudosuccinea columella snails to Fasciola hepatica in Cuba.

Pseudosuccinea columella snails transmit the trematode Fasciola hepatica, but in Cuba, six naturally occurring populations successfully resist parasite infection. Here, we present an updated distribution of P. columella in Cuba; 68 positive sites with the earliest records more abundant in west-central Cuba and with east-central populations generally corresponding to the newest samples. No records were found farther east. The IPA site reported 10.5% prevalence of F. hepatica-infected snails. Population genetics, studied through microsatellites, showed low allelic and multilocus genotypic richness (MLGT), mainly in susceptible populations, strong deviations from panmixia and high self-fertilization rates. Susceptible individuals were grouped in one major cluster containing the majority of MLGT, and two independent clusters grouped the MLGT of resistant individuals from western and central populations, respectively. From these, we propose that several introductions of P. columella occurred in Cuba, primarily in the west, with the early arrivals deriving on the resistant populations. A more recent introduction of susceptible P. columella carrying MLGT T and Y may have occurred, where the latter spread quickly through the island and possibly increase the risk of parasite transmission in Cuba since all snails naturally infected with F. hepatica were carriers of the MLGT Y. Interestingly, even though resistant populations are highly diverse and are likely the oldest within Cuba, they are only found in six localities characterized by soft (total hardness, TH = 6.3 ± 1.03°d) and slightly acidic (pH = 6.2 ± 0.12) waters with low richness in snail species (3.2 ± 1.02). This tendency was also observed in a two-year follow-up ecological study that was conducted on a farm where both phenotypes occurred in sympatry; colonization events by resistant over susceptible snails coincided with a reduction in the pH and TH of the water. A comparison of life traits in susceptible and resistant isolates reared at two different pH/TH conditions (5.9/4°d or 7.8/14°d) showed that low pH/TH negatively affects P. columella, irrespective of the phenotype. However, evidence of higher tolerance (higher survival, life expectancy, egg viability) to such conditions was observed in resistant isolates. Finally, we speculate that the limited distribution of resistant populations might be related to a better exploitation of sites that are less suitable to snails (thus, with lower competition), rather than to a differential ecological restriction to specific environmental conditions from susceptible P. columella.

Reviewing Fasciola hepatica transmission in the West Indies and novel perceptions from experimental infections of sympatric vs. allopatric snail/fluke combinations.

Fasciolosis is an important food-borne parasitic disease affecting over two million people worldwide with economic losses related to cattle production of up to US$ 3 billion annually. Despite the long known presence of Fasciola hepatica in the Caribbean islands its transmission is not well known. This study reviews historical and recent data on fasciolosis in the West Indies, revealing for the first time the outcomes of sympatric and allopatric fluke/snail interactions in the area by exploring the susceptibility of four lymnaeid species after exposure to F. hepatica isolates from Cuba, the Dominican Republic and France. Overall, Galba cubensis showed a mean prevalence of 71.8% and appears to be the most suitable intermediate host species irrespective of the isolate used. Sympatric combinations (snail and parasite from the same country) were generally more compatible (higher susceptibility, parasite intensity and snail survival post-exposure) and only the allopatric interaction of French G. truncatula/Cuban F. hepatica attained 100% prevalence and mean intensity over 33 rediae/snail. However, certain Dominican populations of Pseudosuccinea columella showed high parasite intensities (>30 rediae/snail) when infected with Cuban flukes, highlighting the potential risks of biological introductions. Overall, high compatibility in most sympatric combinations compared to low or moderate compatibility in allopatric ones, suggests the existence of local adaptation from a long sustained interaction that has led to high rates of transmission. Interestingly, attempts to infect G. schirazensis with sympatric and allopatric flukes failed and coupled with the lowest survival rates which supposes a low risk of fasciolosis transmission in areas where this is the only snail species. Although there are significant gaps in the actual status of fasciolosis transmission from several islands in the West Indies these results show a permanent risk. We conclude that fasciolosis transmission is high in areas where the local snail, G. cubensis, occurs, and will be even higher in the presence of the invasive P. columella.

Modeling competition, niche, and coexistence between an invasive and a native species in a two-species metapopulation.

Modeling the dynamics of competition and coexistence between species is crucial to predict long-term impacts of invasive species on their native congeners. However, natural environments are often fragmented and variable in time and space. In such contexts, regional coexistence depends on complex interactions between competition, niche differentiation and stochastic colonization-extinction dynamics. Quantifying all these processes at landscape scale has always been a challenge for ecologists. We propose a new statistical framework to evaluate metapopulation parameters (colonization and extinction) in a two-species system and how they respond to environmental variables and interspecific competition. It requires spatial surveys repeated in time, but does not assume demographic equilibrium. We apply this model to a long-term survey of two snails inhabiting a network of freshwater habitats in the West Indies. We find evidence of reciprocal competition affecting colonization or extinction rates, modulated by species-specific sensitivity to environmental variables. Simulations using model estimates allow us to predict species dynamics and explore the role of various coexistence mechanisms described by metacommunity theory in our system. The two species are predicted to stably coexist, because niche partitioning, source-sink dynamics and interspecific differences in extinction-colonization parameters all contribute to reduce the negative impacts of competition. However, none of these mechanisms is individually essential. Regional coexistence is primarily facilitated by transient co-occurrence of the two species within habitat patches, a possibility generally not considered in theoretical metacommunity models. Our framework is general and could be extended to guilds of several competing species.

A new multiplex PCR assay to distinguish among three cryptic Galba species, intermediate hosts of Fasciola hepatica.

A molecular tool described here allows in one step for specific discrimination among three cryptic freshwater snail species (genus Galba) involved in fasciolosis transmission, a worldwide infectious disease of humans and livestock. The multiplex PCR approach taken targets for each species a distinctive, known microsatellite locus which is amplified using specific primers designed to generate an amplicon of a distinctive size that can be readily separated from the amplicons of the other two species on an agarose gel. In this way, the three Galba species (G. cubensis, G. schirazensis, and G. truncatula) can be differentiated from one another, including even if DNA from all three were present in the same reaction. The accuracy of this new molecular tool was tested and validated by comparing multiplex PCR results with species identification based on sequences at mitochondrial and nuclear markers. This new method is accurate, inexpensive, simple, rapid, and can be adapted to handle large sample sizes. It will be helpful for monitoring invasion of Galba species and for developing strategies to limit the snail species involved in the emergence or re-emergence of fasciolosis.

Bioinvasion Triggers Rapid Evolution of Life Histories in Freshwater Snails.

Biological invasions offer interesting situations for observing how novel interactions between closely related, formerly allopatric species may trigger phenotypic evolution in situ. Assuming that successful invaders are usually filtered to be competitively dominant, invasive and native species may follow different trajectories. Natives may evolve traits that minimize the negative impact of competition, while trait shifts in invasives should mostly reflect expansion dynamics, through selection for colonization ability and transiently enhanced mutation load at the colonization front. These ideas were tested through a large-scale common-garden experiment measuring life-history traits in two closely related snail species, one invasive and one native, co-occurring in a network of freshwater ponds in Guadeloupe. We looked for evidence of recent evolution by comparing uninvaded or recently invaded sites with long-invaded ones. The native species adopted a life history favoring rapid population growth (i.e., increased fecundity, earlier reproduction, and increased juvenile survival) that may increase its prospects of coexistence with the more competitive invader. We discuss why these effects are more likely to result from genetic change than from maternal effects. The invader exhibited slightly decreased overall performances in recently colonized sites, consistent with a moderate expansion load resulting from local founder effects. Our study highlights a rare example of rapid life-history evolution following invasion.

Is Galba schirazensis (Mollusca, Gastropoda) an intermediate host of Fasciola hepatica (Trematoda, Digenea) in Ecuador?

Fasciolosis is a widely distributed disease in livestock in South America but knowledge about the epidemiology and the intermediate hosts is relatively scarce in Ecuador. For three months, lymnaeid snails were sampled (n = 1482) in Pichincha Province at two sites located in a highly endemic area. Snails were identified (based on morphology and ITS-2 sequences) and the infection status was established through microscopic dissection and a multiplex polymerase chain reaction (PCR)-based technique. Techniques based on morphology were not useful to accurately name the collected snail species. Comparison with available DNA sequences showed that a single snail species was collected, Galba schirazensis. Live rediae were observed in 1.75% (26/1482) and Fasciola sp. DNA was detected in 6% (89/1482) of collected snails. The COX-1 region permitted identification of the parasite as Fasciola hepatica. The relative sensitivity and specificity of the microscope study, compared to PCR results, were 25.84% and 99.78%, respectively. The mean size of the snails recorded positive for F. hepatica through crushing and microscopy was significantly higher than the mean size of negative snails, but there was no such difference in PCR-positive snails. The role of G. schirazensis as an intermediate host of F. hepatica in Ecuador is discussed and the hypothesis of an adaptation of the parasite to this invasive snail is proposed. For the first time, an epidemiological survey based on molecular biology-based techniques assessed the possible role of lymnaeid snails in the epidemiology of fasciolosis in Ecuador.

Natural prevalence in Cuban populations of the lymnaeid snail Galba cubensis infected with the liver fluke Fasciola hepatica: small values do matter.

Natural infections of lymnaeid snails by Fasciola hepatica are of primary importance to study transmission. Also, infected snails in the field can be used to explore the existing compatibility in host-parasite interactions. This paper aimed to describe the infection rate of Galba cubensis populations in fasciolosis transmission areas. Eight sites were sampled in western Cuba and 24 infected snails at six sites were found. The mean prevalence was 2.94% and the maximum value was 11.4%. The intensity of parasite infection was assessed as the number of rediae inside a single snail. High variation within the sites examined was observed, but a maximum of 76 rediae was recovered from one individual. Although the presence of two other trematode families (Schistosomatidae and Paramphistomatidae) was discovered in dissected individuals, no co-infection with F. hepatica was observed. This is the first time a study of natural prevalence of F. hepatica infection is carried out in Cuba, considered a hyper endemic country for bovine fasciolosis. Our results suggest that fasciolosis transmission may occur even when the number of infected snails remains relatively low.

Lymnaea cubensis, an experimental intermediate host for Fascioloides magna.

Single-miracidium infections of Lymnaea cubensis (Pfeiffer) from Guadeloupe with the giant liver fluke Fascioloides magna (Bassi, 1875) (Digenea) were carried out during five successive snail generations to determine if this lymnaeid might sustain complete larval development of the parasite. Controls were constituted by a French population of Galba truncatula (Miller) (a single generation) infected according to the same protocol. It was recorded that prevalence and intensity of F. magna infection in L. cubensis progressively increased from F1 to F5 generations. Cercarial shedding of F. magna was noted only within F5 generation of L. cubensis. However, most measured parameters of infection in this species were significantly lower than those noted for G. truncatula and most L. cubensis died after a single shedding wave. Despite this, L. cubensis can be added to the list of potential intermediate hosts of F. magna.

Metapopulation dynamics of species with cryptic life stages.

Quantifying metapopulation dynamics is a challenging task. Difficulties particularly arise in species that possess unobservable resistance forms that bias the estimation of colonization and persistence rates. Here, we develop a general multistate occupancy model that allows estimation of species persistence for both normal and resistant forms, even when the latter are not detectable. We apply this model to an 11-year data set on the tropical freshwater snail Drepanotrema depressissimum in a network of 229 ponds. These ponds frequently dry out, and the snails can persist by aestivating in the ground, where they are not detected. Our model adequately captures this feature because it infers a high persistence rate for the resistant form, while models ignoring aestivation tend to overestimate extinction and colonization. In addition, we find that, surprisingly, colonization and persistence are even higher in sites prone to desiccation and during years with low rainfall than in more humid sites and years, suggesting that D. depressissimum favors unstable sites where competitors are rare and where it can rely on aestivation to persist. Our model has the potential to provide valuable insights into the metapopulation dynamics of many species that otherwise could hardly be studied because of the existence of undetectable life forms.

Morphological and molecular characterization of Neotropic Lymnaeidae (Gastropoda: Lymnaeoidea), vectors of fasciolosis.

Lymnaeidae play a crucial role in the transmission of fasciolosis, a disease of medical and veterinary importance. In the Neotropic, a region where fasciolosis is emergent, eight Lymnaeidae species are currently considered valid. However, our knowledge of the diversity of this taxon is hindered by the fact that lymnaeids exhibit extremely homogeneous anatomical traits. Because most species are difficult to identify using classic taxonomy, it is difficult to establish an epidemiological risk map of fasciolosis in the Neotropic. In this paper, we contribute to our understanding of the diversity of lymnaeids in this region of the world. We perform conchological, anatomical and DNA-based analyses (phylogeny and barcoding) of almost all species of Lymnaeidae inhabiting the Neotropic to compare the reliability of classic taxonomy and DNA-based approaches, and to delimitate species boundaries. Our results demonstrate that while morphological traits are unable to separate phenotypically similar species, DNA-based approaches unambiguously ascribe individuals to one species or another. We demonstrate that a taxon found in Colombia and Venezuela (Galba sp.) is closely related yet sufficiently divergent from Galba truncatula, G. humilis, G. cousini, G. cubensis, G. neotropica and G. viatrix to be considered as a different species. In addition, barcode results suggest that G. cubensis, G. neotropica and G. viatrix might be conspecifics. We conclude that conchological and anatomical characters are uninformative to identify closely related species of Lymnaeidae and that DNA-based approaches should be preferred.

Patterns of mating-system evolution in hermaphroditic animals: correlations among selfing rate, inbreeding depression, and the timing of reproduction.

In hermaphrodites, traits that influence the selfing rate can coevolve with inbreeding depression, leading to the emergence of evolutionary syndromes. Theory predicts a negative correlation between inbreeding depression and selfing rate across species. This prediction has only been examined and validated in vascular plants. Furthermore, selfing rates are often influenced by environmental conditions (e.g., lack of mates or pollinators), and species are predicted to evolve mechanisms to buffer this variation. We extend previous studies of mating-system syndromes in two ways. First, we assembled a new dataset on Basommatophoran snails (17 species, including new data on 12 species). Second, we measured how species responded to variation in mate availability. Specifically, we quantified the waiting time before selfing (i.e., how long the onset of reproduction is delayed in the absence of mates). Selfing rates were negatively correlated with both inbreeding depression and the waiting time. Species with stronger inbreeding depression exhibited longer waiting times. These patterns obtained on Basommatophorans still hold when including eight other hermaphroditic animals. Our results support the hypothesis that selection drives the evolution of mating-system syndromes in animals. The reaction norm of selfing rates to mate availability is a key target of natural selection in this context.

Bridging gaps in the molecular phylogeny of the Lymnaeidae (Gastropoda: Pulmonata), vectors of Fascioliasis.

BACKGROUND: Lymnaeidae snails play a prominent role in the transmission of helminths, mainly trematodes of medical and veterinary importance (e.g., Fasciola liver flukes). As this family exhibits a great diversity in shell morphology but extremely homogeneous anatomical traits, the systematics of Lymnaeidae has long been controversial. Using the most complete dataset to date, we examined phylogenetic relationships among 50 taxa of this family using a supermatrix approach (concatenation of the 16 S, ITS-1 and ITS-2 genes, representing 5054 base pairs) involving both Maximum Likelihood and Bayesian Inference. RESULTS: Our phylogenetic analysis demonstrates the existence of three deep clades of Lymnaeidae representing the main geographic origin of species (America, Eurasia and the Indo-Pacific region). This phylogeny allowed us to discuss on potential biological invasions and map important characters, such as, the susceptibility to infection by Fasciola hepatica and F. gigantica, and the haploid number of chromosomes (n). We found that intermediate hosts of F. gigantica cluster within one deep clade, while intermediate hosts of F. hepatica are widely spread across the phylogeny. In addition, chromosome number seems to have evolved from n = 18 to n = 17 and n = 16. CONCLUSION: Our study contributes to deepen our understanding of Lymnaeidae phylogeny by both sampling at worldwide scale and combining information from various genes (supermatrix approach). This phylogeny provides insights into the evolutionary relationships among genera and species and demonstrates that the nomenclature of most genera in the Lymnaeidae does not reflect evolutionary relationships. This study highlights the importance of performing basic studies in systematics to guide epidemiological control programs.

Immunoblot analysis of membrane antigens of Schistosoma mansoni, Schistosoma intercalatum, and Schistosoma haematobium against Schistosoma-infected patient sera.

Antigens present in aqueous n-butanolic extracts (BE) of Schistosoma mansoni (Venezuelan JL strain), Schistosoma intercalatum (Cameroon EDEA strain), and Schistosoma haematobium (Yemen strain) adult worm membranes were compared in immunoblot against sera of patients infected with S. mansoni, S. intercalatum, S. haematobium, Schistosoma japonicum, or Schistosoma mekongi looking for similarities (common antigens) and differences (species-specific antigens). About 17 S. mansoni BE polypeptides (M (r) approximately 8 to >80 kDa) were commonly recognized by S. mansoni-infected patient sera from Venezuela, Senegal, and Ethiopia. S. intercalatum-, S. haematobium-, or S. japonicum-infected sera were almost unreactive with S. mansoni BE. Nonetheless, S. mekongi-infected sera weakly cross-reacted with a approximately 10-15-kDa subset of S. mansoni BE. About 72.7% of S. intercalatum-infected patient sera reacted with a approximately 19-21-kDa complex in S. intercalatum BE and cross-reacted with a similar complex in S. haematobium BE. Conversely, all S. haematobium-infected patient sera reacted with a approximately 19-21-kDa complex in S. haematobium BE and cross-reacted with the approximately 19-21-kDa complex in S. intercalatum BE; S. mansoni- and S. japonicum-infected patient sera did not react with S. intercalatum or S. haematobium BE. Results showed the presence of a common membrane antigen between African schistosome species and species-specific antigens in S. mansoni BE that could be useful to discriminate between species and/or to detect Schistosoma infections.

Excretory-secretory products of larval Fasciola hepatica investigated using a two-dimensional proteomic approach.

So far, very few secreted proteins from trematodes have been characterized, although their role in the mechanisms that allow the parasite to escape host's immune response have been largely documented. Here we performed a proteomic analysis of excretory-secretory proteins from the intra-molluscan larval stages of Fasciola hepatica. We identified two antioxidative enzymes: a Cu/Zn-superoxide dismutase (Cu/Zn SOD) and a thioredoxin (TRX) previously characterized in ES products from adult stages. These results support the importance of parasite detoxication of reactive oxygen species in invertebrate hosts, and raise the question of the possible conservation of major immune evasion effectors across trematode developmental life-stages.

High genetic variance in life-history strategies within invasive populations by way of multiple introductions.

Biological invasions represent major threats to biodiversity as well as large-scale evolutionary experiments. Invasive populations have provided some of the best known examples of contemporary evolution [3-6], challenging the classical view that invasive species are genetically depauperate because of founder effects. Yet the origin of trait genetic variance in invasive populations largely remains a mystery, precluding a clear understanding of how evolution proceeds. In particular, despite the emerging molecular evidence that multiple introductions commonly occur in the same place, their contribution to the evolutionary potential of invasives remains unclear. Here, by using a long-term field survey, mtDNA sequences, and a large-scale quantitative genetic experiment on freshwater snails, we document how a spectacular adaptive potential for key ecological traits can be accumulated in invasive populations. We provide the first direct evidence that multiple introductions are primarily responsible for such an accumulation and that sexual reproduction amplifies this effect by generating novel trait combinations. Thus bioinvasions, destructive as they may be, are not synonyms of genetic uniformity and can be hotspots of evolutionary novelty.

Pseudosuccinea columella (Say 1817) (Gastropoda, Lymnaeidae), snail host of Fasciola hepatica: first record for France in the wild.

The lymnaeid snail Pseudosuccinea columella has shown strong invasive capabilities in the last decades, and this species has now a worldwide distribution. So far, the presence of this snail in Europe was restricted to botanical gardens, but the recent discovery of a few specimens along the banks of the Lot River, southwestern France is the first record of this species in the wild. The first generation of this P. columella isolate obtained in the laboratory was used for parasitological tests with a French Fasciola hepatica sample. Experimental infections showed that 100% of snails (n=26) developed infection when exposed to F. hepatica miracidia. Consequences for the epidemiology of F. hepatica transmission in France are discussed.