Lymnaeid Snail Vectors of Fascioliasis, Including the First Finding of Lymnaea neotropica in Ecuador, Assessed by Ribosomal DNA Sequencing in the Southern Zone Close to the Peru Border.

PURPOSE: Fascioliasis is a freshwater snail-borne zoonotic trematodiasis of high pathogenicity and wide veterinary repercussions. In South America, moreover, it causes serious public health problems, with high human infection rates in Andean countries. Ecuador offers a worrying risky scenario due to its physiography, including many human infection reports and animal endemicity throughout its Andean highlands. METHODS: Endemic areas with increasing animal fascioliasis were surveyed for lymnaeid snails in the province of Loja, southern Ecuador, close to the border of Peru, the country known to present the widest human fascioliasis endemic zone. The altitude of the sampling sites ranged between 150 and 1770 m a.s.l., and their location was close to human villages. Biotopes surveyed were characterized according to fascioliasis transmission needs. RESULTS: The species Lymnaea schirazensis and L. neotropica were identified by rDNA ITS-2 and ITS-1 sequencing. The non-transmitting L. schirazensis combined haplotype agreed with populations of this species previously reported from northern Ecuador. The finding of the efficient vector L. neotropica is reported for the first time in Ecuador and suggests a passive introduction from neighbouring Peru by uncontrolled livestock transport. CONCLUSIONS: Rice irrigation system implementation, lymnaeid finding on Taraxacum (dandelion) plants which are consumed fresh in salads by people, and Saccharum (sugarcane), whose bark is peeled off with the teeth, represent potential infection sources for humans. The closeness to the Cajamarca human hyperendemic area in northern Peru, where the same two lymnaeids have been also found and triclabendazole resistance reported, is an additional risk to be considered regarding the livestock transborder exchange.

Molecular detection of natural infection of Lymnaea (Pseudosuccinea) columella (Gastropoda: Lymnaeidae) with Fasciola gigantica (Digenea: Fasciolidae) from two provinces of South Africa.

The main intermediate host of Fasciola gigantica in sub-Saharan Africa is Lymnaea (Radix) natalensis. Lymnaea (Pseudosuccinea) columella is capable of transmitting both F. gigantica and F. hepatica and has been reported to be present in South Africa. To date, no natural infection with F. gigantica has been reported despite the wide distribution of the snail. The aim of this study was to confirm whether L. (P.) columella was transmitting F. gigantica and/or F. hepatica in selected locations of KwaZulu-Natal and Eastern Cape provinces of South Africa. Lymnaea (Pseudosuccinea) columella snails were collected from two locations in two provinces of South Africa and screened for cercariae shedding. This was followed by humanely sacrificing the screened snails, and whole tissue of each individual snail was homogenized and amplified using primers designed to amplify the ITS-1 region of Fasciola spp. No cercariae were shed from the screened snails and molecular analysis showed that snails from the two locations were infected with F. gigantica. This study confirms natural infection of L. (P.) columella with F. gigantica in South Africa, where F. gigantica and F. hepatica have already been reported to coexist. Although L. (P.) columella is able to transmit the two species, surprisingly no infection with F. hepatica was detected from the screened snails. The natural intermediate host of F. gigantica in southern Africa, including South Africa, is Lymnaea (Radix) natalensis and comparative studies are needed to determine the competence of the two snail species in the transmission of F. gigantica.

Strain-specific effects of crowding on long-term memory formation in Lymnaea.

Crowding of snails is a stress that obstructs long-term memory (LTM) formation following operant conditioning of aerial respiratory behaviour in Lymnaea stagnalis. In previous experiments, snails of the same strain/population were used for both the crowding and the operant conditioning training. However, there are different strains/populations of Lymnaea stagnalis exhibiting different cognitive abilities. We asked whether Lymnaea of one strain/population are able to determine that they are of a different strain/population. We did this by asking if LTM formation would continue to be obstructed if we crowded snails with a different species of pond snail (Helisoma = Planorbella) or with different strains/populations of Lymnaea stagnalis. Using an inbred strain, the W-strain, we crowded the W-strain with seven other Lymnaea strains/populations as well as with Helisoma. The results of a 2-Way ANOVA followed by a Tukey's Post-hoc analysis showed that W-strain snails when crowded with another strain/population of Lymnaea or with Helisoma formed LTM formation. That is, the memory test session statistically met the criteria for LTM formation. Thus, one strain/population of snails determines that another strain/population is different from it. The differentness means that crowding now does not obstruct LTM formation.

Human fascioliasis endemic areas in Argentina: multigene characterisation of the lymnaeid vectors and climatic-environmental assessment of the transmission pattern.

BACKGROUND: In South America, fascioliasis stands out due to the human endemic areas in many countries. In Argentina, human endemic areas have recently been detected. Lymnaeid vectors were studied in two human endemic localities of Catamarca province: Locality A beside Taton and Rio Grande villages; Locality B close to Recreo town. METHODS: Lymnaeids were characterised by the complete sequences of rDNA ITS-2 and ITS-1 and fragments of the mtDNA 16S and cox1. Shell morphometry was studied with the aid of a computer image analysis system. Climate analyses were made by nearest neighbour interpolation from FAO data. Koeppen & Budyko climate classifications were used. De Martonne aridity index and Gorczynski continentality index were obtained. Lymnaeid distribution was assessed in environmental studies. RESULTS: DNA sequences demonstrated the presence of Lymnaea neotropica and L. viator in Locality A and of L. neotropica in Locality B. Two and four new haplotypes were found in L. neotropica and L. viator, respectively. For interspecific differentiation, ITS-1 and 16S showed the highest and lowest resolution, respectively. For intraspecific analyses, cox1 was the best marker and ITS-1 the worst. Shell intraspecific variability overlapped in both species, except maximum length which was greater in L. viator. The desertic-arid conditions surrounding Locality A, the semiaridity-aridity surrounding Locality B, and the very low yearly precipitation in both localities, are very different from the typical fascioliasis transmission foci. Lymnaeids are confined to lateral river side floodings and small man-made irrigation systems. Water availability only depends on the rivers flowing from neighbouring mountains. All disease transmission factors are concentrated in small areas where humans and animals go for water supply, vegetable cultures and livestock farming. CONCLUSIONS: The unusually high number of DNA haplotypes and the extreme climate unsuitable for F. hepatica and lymnaeid development, demonstrate that the transmission foci are isolated. Seasonal transmission may depend on the timely overlap of appropriate temperature and river water availability. Lymnaeids and F. hepatica have probably reached these localities by livestock introduction. DNA differences regarding other populations of L. neotropica and L. viator in Argentina suggest an introduction independent from the spreading movements which allowed these two lymnaeids to expand throughout the country.

Radix dolgini: The integrative taxonomic approach supports the species status of a Siberian endemic snail (Mollusca, Gastropoda, Lymnaeidae).

The molecular techniques are the standard tool for the study of the taxonomic position and phylogenetic affinities of the lymnaeid genus Radix Montfort, 1810, and the majority of the European representatives of this taxon have been studied in this respect. However, a plethora of nominal species of Radix described from Northern Asia (Siberia and the Russian Far East) are still characterized only morphologically, raising some doubts concerning their validity. In this paper, we present the triple (morphological, molecular, and zoogeographical) evidence that there is at least one endemic species of Radix, Radix dolgini (Gundrizer and Starobogatov, 1979), widely distributed in Siberia and Western Mongolia. Phylogenetically, it is a sister species to the European R. labiata (Rossmaessler, 1835) [=R. peregra auct.], and their common ancestor most probably lived in the Pliocene, nearly 3.25Myr ago. Our results assume the existence of an extended dispersal barrier for freshwater hydrobionts between Europe and Siberia in the Late Pliocene that may be important for biogeographical explanations. Three other nominal Siberian species of Radix: R. kurejkae (Gundrizer and Starobogatov, 1979), R. gundrizeri (Kruglov and Starobogatov, 1983), and R. ulaganica (Kruglov and Starobogatov, 1983) proved to be the junior synonyms of R. dolgini.

Genetic variation of Lymnaea stagnalis tolerance to copper: A test of selection hypotheses and its relevance for ecological risk assessment.

The use of standardized monospecific testing to assess the ecological risk of chemicals implicitly relies on the strong assumption that intraspecific variation in sensitivity is negligible or irrelevant in this context. In this study, we investigated genetic variation in copper sensitivity of the freshwater snail Lymnaea stagnalis, using lineages stemming from eight natural populations or strains found to be genetically differentiated at neutral markers. Copper-induced mortality varied widely among populations, as did the estimated daily death rate and time to 50% mortality (LT50). Population genetic divergence in copper sensitivity was compared to neutral differentiation using the QST-FST approach. No evidence for homogenizing selection could be detected. This result demonstrates that species-level extrapolations from single population studies are highly unreliable. The study provides a simple example of how evolutionary principles could be incorporated into ecotoxicity testing in order to refine ecological risk assessment.

[The fate of subspecies category in zoological systematics. 2. The present].

The present approach to the use of subspecies category in zoological systematics is an integrative one. It counts as obligatory to confirm the validity of subspecies, defined by morphological data, with genetic criteria. This allows marking out those subspecies that really exist as separate monophyletic population groups. As a result, the system would be cleared of many 'phantom' taxa established in course of non-critical use of the subspecies concept. However, detailed analyses of intra-species variability by methods of molecular taxonomy in many cases reveal a quite complicated divergence pattern which cannot be adequately described in terms of the classic scheme of 'species and its subspecies'. Keeping in mind the irregularity of intra-species divergence rate when dealing with molecular and morphological traits, it is proposed to use an extended system of subspecies taxa when describing 'extra complicated' situations. In addition to a 'subspecies' such categories as 'allospecies', 'morphotype', 'morphospecies' may be used for which operational definitions are suggested. As an example, the micro-systematics of the great pond snails (the complex Lymnaea stagnalis s. lato) from Palaearctic region is examined. The provisional system of this group, developed by the author, is based on morphological and phylogeographical data. Applying the series of subspecies categories of different level allows reflecting with maximal completeness the intra-species variability of great pond snails and, to some extent, the process of their genetic divergence and geographic range forming. The second part of the article deals with modern approaches to subspecies category usage in zoological systematics as well as the problems of so called micro-systematics, i.e., systematics operating at the lowest level of categories such as ones of subspecies and infra-subspecies rank (Mayr, 1982).

Efeito moluscicida do óleo essencial de Cymbopogon winterianus Jowitt (Poaceae) sobre Lymnaea columella (Say, 1817) e Biomphalaria tenagophila (D'Orbigny, 1835) / Molluscicidal effect of essential oil of Cymbopogon winterianus Jowitt (Poaceae) on Lymnaea columella (Say, 1817) and Biomphalaria tenagophila (D'Orbigny, 1835)

RESUMO O uso das substâncias moluscicidas convencionais no controle de planorbídeos vetores constitui-se uma importante ferramenta no combate da fasciolose hepática e esquistossomose. Sendo, portanto, de extrema relevância para a pecuária e para os serviços de Vigilância Epidemiológica. Por outro lado, a seleção de caramujos resistentes a tais substâncias e sua baixa seletividade estimulam a busca por novas substâncias. Neste sentido, o presente trabalho foi desenvolvido para avaliar o efeito do óleo essencial de Cymbopogon winterianusJowitt sobre Lymnaea columella e Biomphalaria tenagophila, hospedeiros intermediários de Fasciola hepatica e esquistossomose mansônica, respectivamente. O óleo essencial foi extraído a partir de folhas frescas utilizando o sistema Clevenger. A análise qualitativa foi realizada por meio de cromatografia gasosa acoplada a espectrometria de massas (CG/EM) e a quantificação dos constituintes presentes no óleo foi determinada por cromatografia gasosa acoplada ao detector de ionização de chama (CG/DIC). O efeito moluscicida foi avaliado utilizando seis moluscos de cada espécie e o óleo essencial de C. winterianus nas concentrações finais de 10, 20, 30, 40, 60, 80 e 100 ppm. A análise por cromatografia gasosa do óleo essencial possibilitou a identificação dos componentes majoritários geraniol (28,62%), citronelal (23,62%) e citronelol (17,10%). Os valores de DL100 e DL50para os moluscos das espécies L. columella e B. tenagophila foram, respectivamente, 60 e 40 ppm; 80 ppm e 60 ppm. O óleo de Cymbopogon winterianus demonstrou-se uma alternativa promissora para o controle dos moluscos, sendo a espécie L. columella mais sensível ao mesmo.

ABSTRACT Conventional molluscicides have been employed to control of planorbids vectors and are an important tool in order to control the hepatic fascioliasis and schistosomiasis. Thus, these substances have been showinggreat relevance for both Veterinary and Livestock Services as well as for Epidemiology and Disease Surveillance. On the other hand, the process of drug pressure for the selection of resistant snails to such components and their low selectivity have stimulated the search for new substances. Since researches on new drugs are the starting point to assist on themolluscs control, this work was developed in order to evaluate the effect of Cymbopogon winterianus Jowitt essential oil on L. columella and B. tenagophila, intermediate hosts of Fasciola hepatica and Schistosoma mansoni, respectively. The essential oil was obtained from fresh leaves by hydrodistillation using a Clevenger apparatus. A qualitative analysis was performed by gas chromatography together with a mass spectrometry one (GC/MS) and the chemical constituent content was determined by gas chromatography with a flame ionization detector (GC/FID). The molluscicidal effect was evaluated through the use of six snails of each species and C. winterianus essential oil at 10, 20, 30, 40, 60, 80 and 100 ppm. The result of the gas chromatographic analysis for the essential oil showed geraniol (28.62%), citronellal (23.62%) and citronellol (17.10%) as the major chemical components . The DL100 and DL50 values for L. columella and B. tenagophila species were, respectively, 60 and 40 ppm; 80 ppm and 60 ppm. L. columella had demonstrated more sensitivity to this essential oil than theB. tenagophila species. The C. winterianus essential oil proved to be a promising alternative for the control of these molluscs being the L. columella species the most sensitive of them.

[Lymnaea cousini , intermediate host of Fasciola hepatica in the Colombian high tropical Andes, and its new haplotypes confirmed with the mitochondrial marker cytochrome oxidase I]. / Lymnaea cousini , huésped de Fasciola hepatica en el trópico alto andino de Colombia, y sus nuevos haplotipos, confirmados con el marcador mitocondrial del gen de la citocromo oxidasa I.

INTRODUCTION: Fasciolosis is the disease transmitted by vectors with the highest latitudinal, longitudinal, and altitudinal distribution due to the colonizing capacity of the parasite Fasciola hepatica and its intermediate hosts, Lymnaeidae mollusks. These snails are under research due to their epidemiological importance, but their taxonomic identification is difficult given their interspecific phenotypical similarity. For this reason, there is uncertainty regarding Lymnaea cousini -a host of F. hepatica in Colombia- due to the morphological similarity it has with Lymnaea meridensis , recently described for Venezuela. OBJECTIVE: To confirm with the COI marker (ADNmt) the taxonomic status of individuals morphologically identified as L. cousini from Nariño, Norte de Santander, and Santander (Colombia), deposited in the Vector Mollusks Collection VHET No. 37 of Universidad de Antioquia. MATERIALS AND METHODS: The amplification of the mitochondrial COI required total DNA extraction of each individual´s foot using the DNeasy Blood and Tissue Kit (Qiagen®). Products amplified were sent for sequencing to Macrogen Inc., Korea. Twenty seven sequences generated in this research were compared to sequences published in the GenBank, including sequences of the type locality of L. cousini . RESULTS: Two new haplotypes of L. cousini were obtained for Colombia. Specimens from Nariño correspond to haplotype A, referenced for Ecuador, and specimens from Santander and Norte de Santander belong to a new haplotype we called haplotype D. CONCLUSION: By using the mitochondrial COI marker, we confirmed that the species under study did correspond to L. cousini . The number of known haplotypes of the species for Colombia has been duplicated and its geographical distribution has been extended to the southwest and northeast of the Colombian high Andean region.

Lymnaea cousini , huésped de Fasciola hepatica en el trópico alto andino de Colombia, y sus nuevos haplotipos, confirmados con el marcador mitocondrial del gen de la citocromo oxidasa I / Lymnaea cousini , intermediate host of Fasciola hepatica in the Colombian high tropical Andes, and its new haplotypes confirmed with the mitochondrial marker cytochrome oxidase I

Institución donde se ejecutó el trabajo: Programa de Estudio y Control de Enfermedades Tropicales, PECET, Unidad de Malacología Médica y Trematodos (UMMT), Sede de Investigación Universitaria, Universidad de Antioquia, Medellín, Colombia Introducción. La fasciolosis es la enfermedad transmitida por vectores con mayor distribución latitudinal, longitudinal y altitudinal, debido a la capacidad colonizadora del parásito Fasciola hepatica y de sus huéspedes intermediarios, los moluscos limneidos. Estos caracoles se investigan por su importancia epidemiológica, pero su identificación taxonómica es difícil por la similitud fenotípica entre especies. En este sentido, con respecto a Lymnaea cousini , un huésped de F. hepatica en Colombia, existe incertidumbre en razón de su similitud morfológica con L. meridensis , descrita recientemente en Venezuela. Objetivo. Confirmar con el marcador del gen de la citocromo oxidasa I en el ADN mitocondrial COI (ADNmt), el estatus taxonómico de ejemplares morfológicamente caracterizados como L. cousini provenientes de Nariño, Norte de Santander y Santander (Colombia), depositados en la Colección de Moluscos Vectores de la Universidad de Antioquia, VHET N° 37. Materiales y métodos. Para la amplificación del COI mitocondrial, se extrajo ADN total del pie de cada ejemplar con el estuche DNeasy Blood and Tissue (Qiagen ® ). Los productos amplificados se enviaron a secuenciar a Macrogen Inc., Corea. Las 27 secuencias generadas en esta investigación se compararon con secuencias publicadas en el GenBank, incluidas las secuencias de la localidad tipo de L. cousini. Resultados. Se encontraron dos nuevos haplotipos de L. cousini para Colombia. Los especímenes de Nariño correspondían al haplotipo A, referenciado en Ecuador, y los especímenes de Santander y Norte de Santander, a un nuevo haplotipo al que se denominó D. Conclusión. Mediante el marcador mitocondrial del COI , se confirmó que los especímenes pertenecían a la especie L. cousini . Con el hallazgo se duplicó el número de haplotipos conocidos de la especie en Colombia y se amplió su distribución geográfica al suroeste y nordeste de la región altoandina colombiana.

Introduction: Fasciolosis is the disease transmitted by vectors with the highest latitudinal, longitudinal, and altitudinal distribution due to the colonizing capacity of the parasite Fasciola hepatica and its intermediate hosts, Lymnaeidae mollusks. These snails are under research due to their epidemiological importance, but their taxonomic identification is difficult given their interspecific phenotypical similarity. For this reason, there is uncertainty regarding Lymnaea cousini -a host of F. hepatica in Colombia- due to the morphological similarity it has with Lymnaea meridensis , recently described for Venezuela. Objective: To confirm with the COI marker (ADNmt) the taxonomic status of individuals morphologically identified as L. cousini from Nariño, Norte de Santander, and Santander (Colombia), deposited in the Vector Mollusks Collection VHET No. 37 of Universidad de Antioquia. Materials and methods: The amplification of the mitochondrial COI required total DNA extraction of each individual´s foot using the DNeasy Blood and Tissue Kit (Qiagen®). Products amplified were sent for sequencing to Macrogen Inc., Korea. Twenty seven sequences generated in this research were compared to sequences published in the GenBank, including sequences of the type locality of L. cousini . Results: Two new haplotypes of L. cousini were obtained for Colombia. Specimens from Nariño correspond to haplotype A, referenced for Ecuador, and specimens from Santander and Norte de Santander belong to a new haplotype we called haplotype D. Conclusion : By using the mitochondrial COI marker, we confirmed that the species under study did correspond to L. cousini . The number of known haplotypes of the species for Colombia has been duplicated and its geographical distribution has been extended to the southwest and northeast of the Colombian high Andean region.

Spatial distribution of Lymnaeidae (Mollusca, Basommatophora), intermediate host of Fasciola hepatica Linnaeus, 1758 (Trematoda, Digenea) in Brazil.

Snails of the family Lymnaeidae act as intermediate hosts in the biological cycle of Fasciola hepatica, which is a biological agent of fasciolosis, a parasitic disease of medical importance for humans and animals. The present work aimed to update and map the spatial distribution of the intermediate host snails of F. hepatica in Brazil. Data on the distribution of lymnaeids species were compiled from the Collection of Medical Malacology (Fiocruz-CMM, CPqRR), Collection of Malacology (MZUSP), "SpeciesLink" (CRIA) network and through systematic surveys in the literature. Our maps of the distribution of lymnaeids show that Pseudosuccinea columella is the most common species and it is widespread in the South and Southeast with few records in the Midwest, North and Northeast regions. The distribution of the Galba viatrix, G. cubensis and G. truncatula showed a few records in the South and Southeast regions, they were not reported for the Midwest, North and Northeast. In addition, in the South region there are a few records for G. viatrix and one occurrence of Lymnaea rupestris. Our findings resulted in the first map of the spatial distribution of Lymnaeidae species in Brazil which might be useful to better understand the fasciolosis distribution and delineate priority areas for control interventions.

SPATIAL DISTRIBUTION OF LYMNAEIDAE (MOLLUSCA, BASOMMATOPHORA), INTERMEDIATE HOST OF Fasciola hepatica LINNAEUS, 1758 (TREMATODA, DIGENEA) IN BRAZIL / Distribuição espacial de Lymnaeidae (Mollusca, Basommatophora), hospedeiros intermediários de Fasciola hepatica Linnaeus, 1758 (Trematoda, Digenea) no Brasil

Snails of the family Lymnaeidae act as intermediate hosts in the biological cycle of Fasciola hepatica, which is a biological agent of fasciolosis, a parasitic disease of medical importance for humans and animals. The present work aimed to update and map the spatial distribution of the intermediate host snails of F. hepatica in Brazil. Data on the distribution of lymnaeids species were compiled from the Collection of Medical Malacology (Fiocruz-CMM, CPqRR), Collection of Malacology (MZUSP), “SpeciesLink” (CRIA) network and through systematic surveys in the literature. Our maps of the distribution of lymnaeids show that Pseudosuccinea columella is the most common species and it is widespread in the South and Southeast with few records in the Midwest, North and Northeast regions. The distribution of the Galba viatrix, G. cubensis and G. truncatula showed a few records in the South and Southeast regions, they were not reported for the Midwest, North and Northeast. In addition, in the South region there are a few records for G. viatrix and one occurrence of Lymnaea rupestris. Our findings resulted in the first map of the spatial distribution of Lymnaeidae species in Brazil which might be useful to better understand the fasciolosis distribution and delineate priority areas for control interventions.

Moluscos da família Lymnaeidae são hospedeiros intermediários no ciclo biológico de Fasciola hepatica, agente etiológico da fasciolose, doença parasitária de importância médica para humanos e animais. O presente trabalho teve como objetivo datar e mapear a distribuição espacial dos hospedeiros intermediários de F. hepatica no Brasil. Os dados de distribuição das espécies de limneídeos foram obtidos das Coleções de Malacologia Medica (Fiocruz-CMM, CPqRR) e de Malacologia do Museu de Zoologia da Universidade de São Paulo (MZUSP), rede “SpeciesLink” (CRIA) e através de pesquisas sistemáticas na literatura. Os mapas de distribuição dos limneideos mostram que Pseudosuccinea columella é a espécie mais comum e distribuída nas regiões Sul e Sudeste com poucos registros nas regiões Centro-Oeste, Norte e Nordeste. Além disso, na região Sul os registros para G. viatrix são escassos e existe apenas um único relato de ocorrência para L. rupestris. Os dados obtidos resultaram no primeiro mapa de distribuição espacial das espécies de Lymnaeidae no Brasil, informação básica e relevante para estruturação de políticas de controle da fasciolose humana e animal.

Lymnaea palustris and Lymnaea fuscus are potential but uncommon intermediate hosts of Fasciola hepatica in Sweden.

BACKGROUND: Lymnaea palustris and L. fuscus are members of the European stagnicolines (Gastropoda: Lymnaeidae). The role of stagnicolines in transmission of Fasciola hepatica has been often proposed. To assess the possible relationship between these two stagnicolines and F. hepatica in Sweden, field monitoring in parallel with experimental infections of L. palustris and L. fuscus were conducted. METHODS: Stagnicoline snails were collected and identified on pastures grazed by either sheep or cattle on four farms suffering from fasciolosis in Sweden during 2011-2012. Field-collected L. palustris and L. fuscus were examined for F. hepatica DNA by PCR. In the laboratory, different age groups of L. palustris, L. fuscus and G. truncatula were each exposed to two F. hepatica miracidia and main infection characteristics were obtained. RESULTS: One field-collected L. palustris (out of n = 668) contained F. hepatica as determined by PCR. On the other hand, stagnicolines artificially exposed to F. hepatica miracidia resulted in successful infection with fully differentiated cercariae, but only in juvenile snails (size, 1-2 mm at exposure) and with a prevalence of 51% and 13% in L. palustris and L. fuscus, respectively. In contrast, 90% of juvenile (size, 1-2 mm) and 92% of preadult G. truncatula (size, ≥ 2-4 mm), respectively, were successfully infected. Delayed, reduced and/or no spontaneous cercarial shedding was observed in the two stagnicolines when compared to G. truncatula. However, at snail dissection most cercariae from L. fuscus and L. palustris were able to encyst similarly to those from G. truncatula. CONCLUSION: Both L. fuscus and L. palustris can sustain larval development of F. hepatica but with an apparent level of age resistance. The finding of a single F. hepatica positive specimen of L. palustris, together with infection characteristics from the experimental infection, suggest that L. palustris is a more suitable snail vector of F. hepatica than L. fuscus. The reduced growth observed in both stagnicolines was contrary to the 'parasitic gigantism' theory. Overall, it seems that the epidemiological role of L. palustris in transmission of F. hepatica in Sweden is likely to be much lower than for G. truncatula.

Lymnaea neotropica and Lymnaea viatrix, potential intermediate hosts for Fascioloides magna.

Experimental infections of two South American lymnaeid populations with Fascioloides magna were carried out to determine whether these snails may sustain larval development of this digenean and, if so, to quantify their potential for cercarial production. The reference group was a French population of Galba truncatula infected and raised according to the same protocol. According to the internal transcribed sequence (ITS)-1 segment of their genomic rDNA, these South American populations were identified as Lymnaea neotropica (origin, Argentina) and Lymnaea viatrix var. ventricosa (origin, Uruguay). In the snail groups followed for cercarial shedding, longer prepatent periods and lower numbers of shed cercariae were noted in South American lymnaeids. In other snails dissected at day 65 post-exposure, the redial and cercarial burdens of F. magna found in the bodies of L. neotropica and L. v. ventricosa were significantly lower than those noted in G. truncatula. Compared to the total cercarial production noted in the dissected snails, the percentage of cercariae that exited from snails was 51.3% for G. truncatula, 32.2% for L. neotropica and 46.8% for L. v. ventricosa. The two South American species of snails can thus be considered as potential intermediate hosts of F. magna.

Lymnaea fuscus (Pfeiffer, 1821) as a potential intermediate host of Fascioloides magna in Europe.

Experimental infections of two different populations of Lymnaea fuscus in France and Sweden, with a Czech isolate of Fascioloides magna were carried out to determine if this lymnaeid species enables parasite larval development. Species identification of both snail populations was performed using the morphology of the copulatory organ, and also confirmed by sequencing of the internal transcribed spacer 2 (ITS2) region of the snail genomic rDNA. Only juvenile snails measuring less than 3mm (1-3 weeks of age) were successfully infected (the viable cercariae were recorded) and infection prevalence decreased with age, as documented by increased shell height. In both French and Swedish L. fuscus populations, prevalence ranged between 1.1% and 58.8%. The mean number of metacercariae obtained from cercariae-shedding snails was 13.7 (±11.4), while the total cercarial production noted in snails dissected at day 85 post-exposure was 147.5 (±56.6). Compared to uninfected control snails, we observed reduced growth of infected snails. Despite age-related resistance of snail to the parasite, and limited cercarial production in these experimentally infected snails, F. magna was still able to complete larval development in L. fuscus.

DNA multigene sequencing of topotypic specimens of the fascioliasis vector Lymnaea diaphana and phylogenetic analysis of the genus Pectinidens (Gastropoda).

Freshwater lymnaeid snails are crucial in defining transmission and epidemiology of fascioliasis. In South America, human endemic areas are related to high altitudes in Andean regions. The species Lymnaea diaphana has, however, been involved in low altitude areas of Chile, Argentina and Peru where human infection also occurs. Complete nuclear ribosomal DNA 18S, internal transcribed spacer (ITS)-2 and ITS-1 and fragments of mitochondrial DNA 16S and cytochrome c oxidase (cox)1 genes of L. diaphana specimens from its type locality offered 1,848, 495, 520, 424 and 672 bp long sequences. Comparisons with New and Old World Galba/Fossaria, Palaearctic stagnicolines, Nearctic stagnicolines, Old World Radix and Pseudosuccinea allowed to conclude that (i) L. diaphana shows sequences very different from all other lymnaeids, (ii) each marker allows its differentiation, except cox1 amino acid sequence, and (iii) L. diaphana is not a fossarine lymnaeid, but rather an archaic relict form derived from the oldest North American stagnicoline ancestors. Phylogeny and large genetic distances support the genus Pectinidens as the first stagnicoline representative in the southern hemisphere, including colonization of extreme world regions, as most southern Patagonia, long time ago. The phylogenetic link of L. diaphana with the stagnicoline group may give light to the aforementioned peculiar low altitude epidemiological scenario of fascioliasis.

DNA multigene sequencing of topotypic specimens of the fascioliasis vector Lymnaea diaphana and phylogenetic analysis of the genus Pectinidens (Gastropoda)

Freshwater lymnaeid snails are crucial in defining transmission and epidemiology of fascioliasis. In South America, human endemic areas are related to high altitudes in Andean regions. The species Lymnaea diaphana has, however, been involved in low altitude areas of Chile, Argentina and Peru where human infection also occurs. Complete nuclear ribosomal DNA 18S, internal transcribed spacer (ITS)-2 and ITS-1 and fragments of mitochondrial DNA 16S and cytochrome c oxidase (cox)1 genes of L. diaphana specimens from its type locality offered 1,848, 495, 520, 424 and 672 bp long sequences. Comparisons with New and Old World Galba/Fossaria, Palaearctic stagnicolines, Nearctic stagnicolines, Old World Radix and Pseudosuccinea allowed to conclude that (i) L. diaphana shows sequences very different from all other lymnaeids, (ii) each marker allows its differentiation, except cox1 amino acid sequence, and (iii) L. diaphana is not a fossarine lymnaeid, but rather an archaic relict form derived from the oldest North American stagnicoline ancestors. Phylogeny and large genetic distances support the genus Pectinidens as the first stagnicoline representative in the southern hemisphere, including colonization of extreme world regions, as most southern Patagonia, long time ago. The phylogenetic link of L. diaphana with the stagnicoline group may give light to the aforementioned peculiar low altitude epidemiological scenario of fascioliasis.

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.

[Geographic distribution features of subgenus and life forms of Lymnaeidae (Gastropoda, Pulmonata) in Eastern Europe].

Relative species richness as a ratio of species number known from the teritory norther than polar circle and species number in Eastern Europe as a whole was calculated from all subgenuses and life forms of the Lymnaeidae. The largest value of this character typical for amla-like life form. The dominance of one life form is a result of its adaptation to life in places with wave activity and for water respiration. Large relative species richness of subgenuses Lymnaea and Peregriana is a sequence of ampla-like life form dominance, wuch consist from these subgenuses.

DNA sequence characterisation and phylogeography of Lymnaea cousini and related species, vectors of fascioliasis in northern Andean countries, with description of L. meridensis n. sp. (Gastropoda: Lymnaeidae).

BACKGROUND: Livestock fascioliasis is a problem throughout Ecuador, Colombia and Venezuela, mainly in Andean areas where the disease also appears to affect humans. Transmission patterns and epidemiological scenarios of liver fluke infection have shown to differ according to the lymnaeid vector snail species involved. These Andean countries present the vectors Lymnaea cousini, L. bogotensis and L. ubaquensis, unknown in the rest of Latin America. An exhaustive combined haplotype study of these species is performed by means of DNA sequencing of the nuclear ribosomal 18S RNA gene, ITS-2 and ITS-1, and mitochondrial DNA cox1 gene. RESULTS: The conserved 5.8S rDNA sequence corroborated that no pseudogenes are involved in the numerous non-microsatellite/minisatellite-related indels appearing between the ITS-2 and ITS-1 sequences when comparing different L. cousini--L. bogotensis populations. Sequence analyses and phylogenetic reconstruction methods including other lymnaeid vector species show that (i) L. bogotensis is a synonym of L. cousini, (ii) L. ubaquensis is a synonym of Pseudosuccinea columella, and (iii) populations of L. cousini hitherto known from Venezuelan highlands indeed belong to a new species for which the name L. meridensis n. sp. is proposed. This new species is described and a complete phenotypic differentiation provided. CONCLUSIONS: ITS-2, ITS-1 and cox1 prove to be good markers for specimen classification and haplotype characterisation of these morphologically similar lymnaeids in endemic areas. Analysis of the 18S gene and phylogenetic reconstructions indicate that L. cousini and L. meridensis n. sp. cluster in an evolutionary line different from the one of P. columella, despite their external resemblance. This suggests an evolutionary phenotypic convergence related to similar environments and which has given rise to frequent specimen misclassification. Body size and phylogenetic relationships of L. meridensis n. sp. with well-known vectors as Lymnaea cousini and P. columella, as well as with Galba/Fossaria species, suggest that the new species may participate in disease transmission to both animals and humans in altitude areas during the yearly window in which temperatures are higher than the F. hepatica minimum development threshold. The involvement of L. cousini and P. columella in the transmission and geographical/altitudinal distribution of fascioliasis in these Andean countries is analysed.