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.

[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).

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.

[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.

Distribution of Lymnaeidae (Mollusca: Pulmonata), intermediate snail hosts of Fasciola hepatica in Venezuela.

An extensive malacological survey was carried out between 2005-2009 in order to clarify the exact number of lymnaeid species which may be intermediate hosts of Fasciola hepatica in Venezuela. Four species were discovered during this survey, including two local species: Lymnaea cubensis and Lymnaea cousini and two exotic species: Lymnaea truncatula and Lymnaea columella. The most common local species was L. cubensis which was found at 16 out of the 298 sampling sites. This species has a large distribution area throughout the Northern part of Venezuela and was encountered from sea level to an altitude of 1,802 m in state of Trujillo. The second local species L. cousini was collected at only two sites of the Andean Region at altitudes of 3,550 m and 4,040 m, respectively. The European L. truncatula was found at 24 sites all located in the states of Mérida and Táchira at an altitude varying between 1,540-4,000 m. The respective distribution areas of L. cubensis and L. truncatula do not appear to overlap, but more detailed malacological surveys are needed. The fourth lymnaeid species, L. columella was collected in a canal from Mérida at an altitude of 1,929 m and in an irrigation canal from the state of Guárico, at an altitude of 63 m. The role of these four lymnaeid species in the transmission of fascioliasis in Venezuela is discussed.

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.

Phylogenetic relatedness and ecological interactions determine antipredator behavior.

Interspecific recognition of alarm cues among guild members through "eavesdropping" may allow prey to fine-tune antipredator responses. This process may be linked to taxonomic relatedness but might also be influenced by local adaptation to recognize alarm cues from sympatric species. We tested this hypothesis using antipredator responses of a freshwater gastropod Lymnaea stagnalis (L.) to alarm cues from damaged conspecific and 10 heterospecific gastropod species. As predicted, the magnitude of antipredator response decreased significantly with increasing phylogenetic distance, but increased when species were naturally sympatric (defined as species cohabiting in the same water body) with the source population of L. stagnalis. The responses to sympatric species were higher overall, and the relationship between genetic distance and alarm cue response was stronger when tested with sympatric species. This is the first study to demonstrate that population sympatry influences innate antipredator responses to alarm cues from intraguild members and suggests that responses based on phylogenetic relationships can be modified through local adaptation. Such adaptation to heterospecific alarm cues suggests that species could be at a disadvantage when they encounter novel intraguild members resulting from species invasion or range expansion due to a reduction in the presence of reliable information about predation risk.

Identification of molluscan nicotinic acetylcholine receptor (nAChR) subunits involved in formation of cation- and anion-selective nAChRs.

Acetylcholine (ACh) is a neurotransmitter commonly found in all animal species. It was shown to mediate fast excitatory and inhibitory neurotransmission in the molluscan CNS. Since early intracellular recordings, it was shown that the receptors mediating these currents belong to the family of neuronal nicotinic acetylcholine receptors and that they can be distinguished on the basis of their pharmacology. We previously identified 12 Lymnaea cDNAs that were predicted to encode ion channel subunits of the family of the neuronal nicotinic acetylcholine receptors. These Lymnaea nAChRs can be subdivided in groups according to the residues supposedly contributing to the selectivity of ion conductance. Functional analysis in Xenopus oocytes revealed that two types of subunits with predicted distinct ion selectivities form homopentameric nicotinic ACh receptor (nAChR) subtypes conducting either cations or anions. Phylogenetic analysis of the nAChR gene sequences suggests that molluscan anionic nAChRs probably evolved from cationic ancestors through amino acid substitutions in the ion channel pore, a mechanism different from acetylcholine-gated channels in other invertebrates.

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.

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.

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.

Phylogeography and genetic divergence of some lymnaeid snails, intermediate hosts of human and animal fascioliasis with special reference to lymnaeids from the Bolivian Altiplano.

A population genetic study using starch gel electrophoresis was performed on populations of several species of lymnaeid snails acting as intermediate hosts for Fasciola hepatica (Trematoda, Plathyhelminth). Lymnaea viatrix was collected in 16 sites from the Bolivian Northern Altiplano. L. cubensis were obtained in one site from Venezuela, one site from Guadeloupe, three sites from Cuba and one site from the Dominican Republic. L. truncatula were collected in one site from France, one from Portugal and one from Morocco. Multilocus enzyme electrophoresis (MEE) were determined for 282 snails at 18 loci. A complete monomorphism was encountered at each geographic site. However, among these 18 loci, 13 are polymorphic and low and high levels of genetic divergence were observed between samples. Two genotypic groups can be differentiated by their multilocus genotypes. The western genotypic group associates together samples from Venezuela, Guadeloupe, Cuba and Dominican Republic (L. cubensis) while samples from France, Portugal and Morocco (L. truncatula) belong to the eastern genotypic group. Surprisingly, the Northern Bolivian Altiplano populations (L. viatrix) do not present any genetic divergence with the Portuguese sample. Therefore, the Bolivian snails belong entirely to the eastern genetic group. Within each group slight genetic divergences were observed. These results strongly support the European origin of the lymnaeid snails from the Northern Bolivian Altiplano.

Miracidia of an Egyptian strain of Schistosoma mansoni differentiate between sympatric snail species.

The host-finding behavior of miracidia of 2 strains of Schistosoma mansoni from Egypt and Brazil was studied by recording their responses to snail-conditioned water (SCW) from the Egyptian sympatric snails, Biomphalaria alexandrina, Physa acuta, Lymnaea cailliudi, and Balinus truncatus, as well as from Biomphalaria arabica and Biomphalaria glabrata. Miracidia of the Egyptian strain significantly preferred SCW from their compatible hosts B. alexandrina and B. arabica and showed no or a weak response to SCW from the other sympatric species, whereas miracidia of the Brazilian strain did not differentiate between SCW from different snail species.

Comparative radular morphology of Lymnaea (Bullastra) cumingiana (Pulmonata: Lymnaeidae) and related taxa in the Indo-Pacific region.

The radular morphology of Lymnaea (Bullastra) cumingiana was compared to that of five other Indo-Pacific lymnaeid "species", namely: L. (Radix) quadrasi (Philippines), L. (R.) rubiginosa (Indonesia and Thailand) and L. (R.) viridix (Guam and Hong Kong) in order to investigate the taxonomic relationship among the six species. Although all six species uniformly exhibited a unicuspid, slightly asymmetrical central (rachidian) tooth and tricuspid laterals, interesting differences were noted among the outer marginals. These were observed to be uniquely bicuspid in L. cumingiana, predominantly tricuspid in L. quadrasi, tetracuspid in L. rubiginosa (Indonesia and Thailand) and multicuspid in L. viridis (Guam and Hong Kong). Thus, the results support the hypotheses that L. cumingiana is a unique species compared to the rest, that L. quadrasi is closely related to L. rubiginosa (Indonesia and Thailand) and that the two geographical isolates of L. viridis have not diverged. Radular morphology was therefore found to have a limited significance in elucidating the taxonomic relationship between the six groups of lymnaeids studied.

Comparative shell morphology of Lymnaea (Bullastra) cumingiana (Pulmonata: Lymnaeidae) and related taxa in the Indo-Pacific region.

Comparative shell morphology using both quantitative and qualitative parameters was employed to investigate the taxonomic relationship between the endemic Philippine species, Lymnaea (Bullastra) cumingiana and five other lymnaeid "species" in the Indo-Pacific region, namely: L. (Radix) quadrasi (Philippines). L. (Radix) rubiginosa (Indonesia), L. (Radix) rubiginosa (Thailand), L. (Radix) viridis (Guam) and L. (Radix) viridis (Hong Kong). Fifty randomly chosen adult specimens of each species were studied and compared, although only field-collected specimens were studied for the first four groups and laboratory-raised specimens for the last two group. Results strongly suggested that L. cumingiana is a distinct species among the rest. L. quadrasi, L. rubiginosa (Indonesia) and L. rubiginosa (Thailand) exhibited great affinity towards each other. Likewise, the two geographical isolates of L. viridis were practically identical to each other except for some minor size differences.

Ecological observations on Lymnaea (Bullastra) cumingiana.

Field surveys conducted at Echague, Isabela and San Pablo, Laguna revealed that Lymnaea (Bullastra) cumingiana, the natural second snail intermediate host of Echinostoma malayanum in the Philippines, exhibits a moderate degree of diversity in its choice of habitats. Rice fields of all stages of development, stagnant shallow streams and springs are the main areas where the snail can be collected from at Echague, Isabela. However, they were absent in rice fields that had been extensively sprayed with molluscicides to control the "golden apple snail" (Ampullarius canaliculatus). In contrast, they were also very abundant in the highly eutrophic waters of Sampaloc lake, San Pablo, Laguna. L. cumingiana co-exists with various species of insects, snails, fish and plants in these habitats. Information on ecological characteristics affecting its distribution will be useful for those who wish to collect and study this species in the future.

Comparative horizontal starch gel isoenzyme electrophoresis of Lymnaea (Bullastra) cumingiana (Pulmonata: Lymnaeidae) and related taxa in the Indo-Pacific region.

Foot muscle tissue extracts from six lymnaeid species of the Indo-Pacific region [Lymnaea (Bullastra) cumingiana and L. (Radix) quadrasi from the Philippines, L. (R.) rubiginosa from Indonesia and Thailand, and L. (R.) viridis from Guam and Hong Kong] were subjected to horizontal starch gel isoenzyme electrophoresis and assayed for seven isoenzymes (AcP, AlP, CA, EST, LAP, CAT and GOT) to elucidate their taxonomic relationships. L. cumingiana exhibited banding patterns for EST, LAP and CAT uniquely different from the rest, thus supporting the hypothesis that it is a distinct species. Zymogram patterns for AlP, CA, EST and LAP attest to the close affinity between L. quadrasi and L. rubiginosa (Indonesia and Thailand). Minor differences suggest a closer relationship between the two geographical strains of L. rubiginosa than with L. quadrasi, lending support to the hypothesis that L. quadrasi is inseparable as a race or variety from the typical L. swinhoei Adams, which in turn is but a race of L. auricularia, which also encompasses L. rubiginosa. The two geographical strains of L. viridis from Guam and Hong Kong showed the greatest consistency with regards to similarity and congruence in banding patterns. Non-specific esterases (EST) were the most useful in distinguishing the six species from each other.