Rapid identification of lymnaeid snails and their infection with Fasciola gigantica in Thailand

Freshwater snails of the family Lymnaeidae are the intermediate hosts of the liver fluke Fasciola worldwide. While distinct species have been identified at the molecular level in other parts of the world such data have not been published for Thailand. In this study we collected Lymnaeidae from different localities across Thailand and analyzed their 16S rDNA sequences as a molecular signature for classification. In addition to the ubiquitous Radix rubiginosa, we have confirmed the presence of Austropeplea viridis and Radix swinhoei, for the latter of which the ribosomal rDNA sequences are reported for the first time, in North-Thailand. Based on the obtained 16S rDNA data three primer pairs were designed that allowed rapid identification of these snail species by PCR. To determine their infection status, PCR primers for F.gigantica cathepsin L were used in parallel with the snail 16S rDNA species-specific primers in multiplex PCR analyses. Western blot analysis of total snail protein with a monoclonal anti-F.gigantica cathepsin L antibody confirmed positive cathepsin L PCR results. The developed diagnostic PCR will be of use in risk assessment for transmission of fascioliasis in Thailand.     

Phylogenetic relationships between Lymnaeidae in relation to infection with Fasciola sp. in Hokkaido,Japan

ABSTRACT

In this paper, lymnaeid snails collected from 107 localities in Hokkaido, Japan were examined for infection by Fasciola larvae. DNA sequences from the snails were analysed phylogenetically based on their mitochondrial cytochrome c oxidase (COI) gene. Based on shell morphology, the snails were identified as Galba truncatula, Radix auricularia, and Austropeplea ollula and were divided into distinct clades in the COI tree. No Fasciola larvae were detected in any of the collected snails. The 58 haplotypes of Galba truncatula which were found in Hokkaido included five detected previously in snails infected with Fasciola larvae, and constituted a closely related haplogroup (Gt-c1), suggesting that Gt-c1 snails would be susceptible to Fasciola sp. The Gt14 haplotype snails were considered to be Lymnaea schirazensis, an intermediate host of F. hepatica. The COI haplotypes of A. ollula belonged to a single clade, and were considered conspecific, whereas those of R. auricularia constituted three genetically distinct haplogroups, each of which was possibly a distinct species. This study suggests that morphologically-identified lymnaeid snails possibly include additional species. Further study of these snails is required to clarify their susceptibility to Fasciola sp.     

Fasciola hepatica in Snails Collected from Water-Dropwort Fields using PCR

Fasciola hepatica is a trematode that causes zoonosis mainly in cattle and sheep and occasionally in humans. Fascioliasis has been reported in Korea; however, determining F. hepatica infection in snails has not been done recently. Thus, using PCR, we evaluated the prevalence of F. hepatica infection in snails at 4 large water-dropwort fields. Among 349 examined snails, F. hepatica-specific internal transcribed space 1 (ITS-1) and/or ITS-2 markers were detected in 12 snails and confirmed using sequence analysis. Morphologically, 213 of 349 collected snails were dextral shelled, which is the same aperture as the lymnaeid snail, the vectorial host for F. hepatica. Among the 12 F. hepatica-infected snails, 6 were known first intermediate hosts in Korea (Lymnaea viridis and L. ollula) and the remaining 6 (Lymnaea sp.) were potentially a new first intermediate host in Korea. It has been shown that the overall prevalence of the snails contaminated with F. hepatica in water-dropwort fields was 3.4%; however, the prevalence varied among the fields. This is the first study to estimate the prevalence of F. hepatica infection using the vectorial capacity of the snails in Korea.     

Species diversity of Plagiorchis Lühe, 1899 (Digenea: Plagiorchiidae) in lymnaeid snails from freshwater ecosystems in central Europe revealed by molecules and morphology

Larval stages of Plagiorchis spp. are both ubiquitous and ecologically important parasites in snail populations of freshwater ecosystems in Europe. However, difficulties in distinguishing the morphologically similar cercariae used for species identification, may lead to underestimation of species diversity. In this study, 38 isolates of Plagiorchis spp. infecting two lymnaeid snails, Lymnaea stagnalis (L.) and Radix auricularia (L.), in five central European freshwater ecosystems were subjected to morphological and molecular assessment. Five morphologically homogeneous and genetically distinct lineages of Plagiorchis spp. were identified via matching molecular data for the mitochondrial cytochrome c oxidase subunit I (cox1) gene with detailed morphological and morphometric data of the cercariae. Comparative sequence analysis using partial 28S rDNA and ITS1-5.8S-ITS2 sequences revealed that three distinct cox1 lineages are conspecific with Plagiorchis elegans (Rudolphi, 1802), P. maculosus (Rudolphi, 1802) and P. koreanus Ogata, 1938, respectively, whereas the lineage identified based on cercarial morphology as P. neomidis Brendow, 1970 plus a single isolate that could not be assigned to a described species, did not match any of the available sequences for Plagiorchis spp. A key to the cercariae of Plagiorchis spp. parasitising lymnaeid populations in central Europe is provided to facilitate identification.     

Fasciola gigantica: Cercarial shedding pattern from Lymnaea natalensis after long-term exposure to cadmium at different temperatures

Little is known about the response of Fasciola spp., inside its snail host to pollutant toxicity. The effect of long-term exposure of Lymnaea natalensis to 0.1 μg/l, 10 μg/l, or 100 μg/l cadmium (Cd) on the infection rates with F. gigantica, percentage of cercariae-shedding snails and cercariae shedding pattern was investigated. The snails were exposed to Cd for 7 days either pre-infection with single Fasciola miracidium or during the late pre-patent period of the infection. The possible interaction between metal exposure and acclimatization temperature was also studied in three ranges; 16-18 °C, 23-25 °C and 30-32 °C. Results clearly showed that host exposure to 100 μg/l Cd significantly affected the pattern of Fasciola development inside its snail host, and that the acclimatization temperature was a key factor affecting the role played by Cd. Pre-infection exposure to Cd caused a significant reduction in the infection rates where the effect was temperature-dependent. Post-infection exposure to Cd significantly increased the percentage of cercariae-shedding snails; the effect was temperature-independent. Interestingly, post-infection exposure significantly altered the differential cercarial output pattern, while no clear impact on the total was found. The alteration in the differential cercarial output was represented as a significantly higher proportion of floating metacercaria as compared to fixed ones and also higher proportion of dead cercariae which may directly reduce the transmission of Fasciola to the final host. The mechanisms of cadmium impact are briefly discussed.     

Two new species of oribatid mites of the family Galumnidae (Acari,Oribatida) from Vietnam

Two new species of oribatid mites of the family Galumnidae, Allogalumna monodactyla sp. n. and Galumna (Galumna) paracalcicola sp. n., are described from dark loamy soil under crown of Ficus sp. in southern Vietnam. Allogalumna monodactyla sp. n. is the first identified member of Allogalumna recorded for Vietnam. The identification keys to the species of Allogalumna from the Oriental region and species of Galumna (Galumna) from Vietnam and the calcicola-group are given.     

Austropeplea ollula (Pulmonata: Lymnaeidae): a new molluscan intermediate host of a human intestinal fluke, Echinostoma cinetorchis (Trematoda: Echinostomatidae) in Korea

Three freshwater snail species of the family Lymnaeidae have been reported from Korea, Radix auricularia coreana, Austropeplea ollula and Fossaria truncatula. Out of 3 lymnaeid snail species, A. ollula was naturally infected with the Echinostoma cinetorchis cercariae (infection rate = 0.7%). In the experiments with the laboratory-bred snails, F. truncatula as well as A. ollula was also susceptible to the E. cinetorchis miracidia with infection rates of 25% and 40%, respectively. All of three lymnaeid snail species exposed to the E. cinetorchis cercariae were infected with the E. cinetorchis metacercariae. It is evident that A. ollula acts as the first molluscan intermediate host of E. cinetorchis in Korea, and F. truncatula may be a possible candidate for the first intermediate host of this intestinal fluke. Also, three lymnaeid snail species targeted were experimentally infected with E. cinetorchis metacercariae.     

Meloidogyne partityla on Pecan Isozyme Phenotypes and Other Host

Meloidogyne sp. from five pecan (Carya illinoensis) orchards in Texas were distinctive in host range and iszoyme profiles from common species of Meloidogyne but were morphologically congruent with Meloidogyne partityla Kleynhans, a species previously known only in South Africa. In addition to pecan, species of walnut (Juglans hindsii and J. regia) and hickory (C. ovata) also were hosts. No reproduction was observed on 15 other plant species from nine families, including several common hosts of other Meloidogyne spp. Three esterase phenotypes and two malate dehydrogenase phenotypes of M. partityla were identified by polyacrylamide gel electrophoresis. Each of these isozyme phenotypes was distinct from those of the more common species M. arenaria, M. hapla, M. incognita, and M. javanica.     

Infections of Larval Stages of Dicrocoelium dendriticum and Brachylaima sp. in Brown Garden Snail,Helix aspersa,in Turkey

The aim of this study was to determine the presence and prevalence of larval stages of Dicrocoelium dendriticum and Brachylaima sp. in the first intermediate host, a species of land snail, Helix aspersa, in Turkey. A total of 211 snails were collected in April-May 2014 from pastures in Mersin District. Larval stages of D. dendriticum were identified under a light microscope. Hepatopancreas from naturally infected H. aspersa snails were examined histologically. The prevalence of larval stages of D. dendriticum and Brachylaima sp. in H. aspersa snails was found to be 2.4% and 1.9%, respectively, in Mersin, Turkey. Cercariae were not matured in sporocysts at the beginning of April; however, it was observed that cercariae matured and started to leave sporocysts by early-May. Thus, it was concluded that H. aspersa acts as an intermediate host to D. dendriticumin and Brachylaima sp. in Mersin, Turkey. A digenean trematode Brachylaima sp. was seen for the first time in Turkey.     

Paramphistomum daubneyi: characteristics of infection in three lymnaeid species

Experimental infections of two South American lymnaeids (Lymnaea neotropica and L. viatrix var. ventricosa) with Paramphistomum daubneyi were carried out to determine if these snail species could sustain larval development of this digenean and, if so, to specify their potential for cercarial production. A French population of Galba truncatula infected and raised according to the same protocol served as controls. In both experiments, prevalence of P. daubneyi infections in snails did not significantly differ from each other. In snail groups evaluated for cercarial shedding (first experiment), a significantly lower number of shed cercariae was noted for L. neotropica, while those from G. truncatula and L. v. ventricosa did not differ significantly from each other. Dissection of infected snails at day 65 post-exposure at 20 °C (second experiment) found significantly lower burdens of P. daubneyi rediae and cercariae in the bodies of L. neotropica than in those of G. truncatula and L. v. ventricosa. Compared to total cercarial production observed in dissected snails, the percentage of cercariae which exited from snails was 75.6% for G. truncatula, 21.6% for L. neotropica, and 91.4% for L. v. ventricosa. This last species seems to be a good candidate for metacercarial production of P. daubneyi.     

Haplorchis pumilio and H. taichui in Vietnam discriminated using ITS-2 DNA sequence data from adults and larvae

Samples of Haplorchis taichui and Haplorchis pumilio of different life-stages (cercariae, metacercariae and adults) and from different host species (snail, fish, dog, cat and human) were collected in Nghe An and Nam Dinh Provinces in Vietnam. Samples from Thailand were available for comparison. All adults and metacercariae were initially identified using morphological criteria. Polymerase chain reaction (PCR) assays were developed for discriminating between the species. The complete sequence for the nuclear ribosomal internal transcribed spacer-2 (ITS-2) was obtained from one adult and one metacercaria of H. taichui and three adults and three metacercariae of H. pumilio from Vietnam. Sequences from cercariae from three different snails clustered with those of H. pumilio. Intra-individual variation in the ITS-2 region was detected by sequencing of cloned PCR products. These are the first sequences from Vietnamese Haplorchis spp. to be reported and demonstrate that H. taichui and H. pumilio can be identified unambiguously from any life-stage, including the cercarial stage that is difficult to identify using morphology. Discrepancies in the literature are discussed and examples of apparent misidentification highlighted. The data provide a resource to assist in taxonomic studies on heterophyids, in the design of probes for diagnosis and for field surveys to identify infection in snails.     

Interactions among invaders: community and ecosystem effects of multiple invasive species in an experimental aquatic system

With ecosystems increasingly supporting multiple invasive species, interactions among invaders could magnify or ameliorate the undesired consequences for native communities and ecosystems. We evaluated the individual and combined effects of rusty crayfish (Orconectes rusticus) and Chinese mystery snails [Bellamya (=Cipangopaludina) chinensis] on native snail communities (Physa, Helisoma and Lymnaea sp.) and ecosystem attributes (algal chlorophyll a and nutrient concentrations). Both invaders are widespread in the USA and commonly co-occur within northern temperate lakes, underscoring the importance of understanding their singular and joint effects. An outdoor mesocosm experiment revealed that while the two invaders had only weakly negative effects upon one another, both negatively affected the abundance and biomass of native snails, and their combined presence drove one native species to extinction and reduced a second by >95%. Owing to its larger size and thicker shell, adult Bellamya were protected from crayfish attack relative to native species (especially Physa and Lymnaea), suggesting the co-occurrence of these invaders in nature could have elevated consequences for native communities. The per capita impacts of Orconectes (a snail predator) on native snails were substantially greater than those of Bellamya (a snail competitor). Crayfish predation also had a cascading effect by reducing native snail biomass, leading to increased periphyton growth. Bellamya, in contrast, reduced periphyton biomass, likely causing a reduction in growth by native lymnaeid snails. Bellamya also increased water column N:P ratio, possibly because of a low P excretion rate relative to native snail species. Together, these findings highlight the importance of understanding interactions among invasive species, which can have significant community- and ecosystem-level effects.     

Microsporidian Parasites of Trematode Larvae from Aquatic Snails in West Malaysia

In a survey of microsporidian parasites of trematode larvae of Malaysian fresh-water snails, species of Nose-matidae were found 7 times in rediae from 425 Lymnaea rubiginosa, 6 times in rediae from 97 Indoplanorbis exustus and not at all in other snails. There were 3 species of Microsporida in all: one, found in Echinoparyphium dunni, Echinostoma hystricosum and Echinostoma malayanum was identified as Nosema eurytremae Canning; another, which was found in E. dunni and Echinostoma audyi, had paired nuclei (diplokaryon form) in sporogony only and was named a new species, Nosema vasicola sp.n.; the 3rd, from E. hystricosum, lacked diplokaryon nuclei throughout development, was considered to belong to a new genus and was named Unikaryon piriformis gen.n., sp.n. Nosema eurytremae was transmitted experimentally to Fasciola hepatica rediae in Lymnaea truncatula.     

On the reality of local and ecological races in lymnaeid snails (Mollusca,Gastropoda, Lymnaeidae)

The reality of ecological and local races was investigated in two widespread Palearctic species of lymnaeid snails (Lymnaea stagnalis and Radix auricularia). Several methods of statistical analysis were used, including two-way ANOVA, cluster analysis, and discriminant analysis for six plastic shell characters. It was shown that none of the methods used could convincingly demonstrate that ecological and local races were present in the studied species. Even in cases when statistically significant differences among the populations in the morphology of shells were revealed, these differences were very small and did not correspond to the meaning that was put into the concept of “race” by malacologists in the past. Perhaps, the formation of such races in pond snails is possible only in case of populations being formed in suboptimal conditions, for example, in thermal springs or at large depths. This being the case, the distinguishing of races requires using other shell characters, both quantitative and qualitative ones.     

Paragonimus and Paragonimiasis in Vietnam: an Update

Paragonimiasis is a food-borne parasitic zoonosis caused by infection with lung flukes of the genus Paragonimus. In Vietnam, research on Paragonimus and paragonimiasis has been conducted in northern and central regions of the country. Using a combination of morphological and molecular methods, 7 Paragonimus species, namely P. heterotremus, P. westermani, P. skrjabini, P. vietnamensis, P. proliferus, P. bangkokenis and P. harinasutai, have been identified in Vietnam. Of these, the first 3, P. heterotremus, P. westermani and P. skrjabini, are known to infect humans in other countries. However, in Vietnam, only P. heterotremus, found in some northern provinces, has been shown to infect humans. Even nowadays, local people in some northern provinces, such as Lai Chau and Yen Bai, are still suffering from P. heterotremus infection. In some provinces of central Vietnam, the prevalence and infection intensity of P. westermani metacercariae in freshwater crabs (the second intermediate hosts) are extremely high, but human cases have not been reported. Likewise, although P. skrjabini was found in Thanh Hoa Province, its pathogenicity to humans in Vietnam still remains uncertain. The results of molecular phylogenetic analyses of Vietnamese Paragonimus species provides new insights on the phylogeny and taxonomy of the genus Paragonimus. Comprehensive molecular epidemiological and geobiological studies on the genus in Vietnam and adjacent countries are needed to clarify the biodiversity and public health significance of the lung flukes.     

Histological Comparisons of Parasitism by Schistonchus spp. (Nemata: Aphelenchoididae) in Neotropical Ficus spp.

Syconia (enclosed infructescences) infested with host-specific species of Schistonchus (Aphelenchoididae) were collected from six species of Ficus (Moraceae) native to Florida or Panama. They were sectioned and histologically examined to assess the effects of parasitism. Parasitism by Schistonchus spp. was associated with hypertrophied cells, tissue necrosis, and the presence of an exudate in all species. Occasional hypertrophy of the outer epidermal cells occurred on seed florets, wasp florets, and on the endothecial cells of male florets in F. aurea (subgenus Urostigma) from Florida. Aberrations of the inner mesocarp occurred under the hypertrophied cells on seed florets. In F. laevigata (subgenus Urostigma) from Florida, Schistonchus sp. infested immature male florets and was associated with hypertrophy of endothecial cells, epidermal cells of the anther filaments, and anthers. Schistonchus sp. also caused aberrations of the anther filament, anthers, and pollen. Ficus poponoei (subgenus Urostigma) and F. glabrata (subgenus Pharmacosycea), both from Panama, had hypertrophied outer epidermal cells on seed florets. Ficus poponoei also had Schistonchus sp. within the pedicel of an aborted floret, with hypertrophy of the cortical parenchyma. Ficus trigonata (subgenus Urostigma) from Panama had hypertrophy of the outer epidermis of seed florets. When the outer epidermis on these florets was missing, the inner mesocarp was hypertrophied. Ficus maxima (subgenus Pharmacosycea) from Panama had hypertrophy on the outer epidermis of seed and aborted florets. Schistonchus spp. were not found in wasp larvae or pupae in any of the Ficus spp. examined. Hypertrophy was never observed in the absence of Schistonchus spp.     

Genotypic characterization and species identification of Fasciola spp. with implications regarding the isolates infecting goats in Vietnam

Ribosomal RNA sequences (361 or 362 bp) of the second internal transcribed spacer 2 (ITS-2) and a portion of mitochondrial cox1 (423 bp) for Fasciola spp. obtained from specimens collected in indigenous and hybrid goats and sheep in Vietnam were characterized for genotypic status and hybridization/introgression. Alignment of 48 ITS-2 sequences (also those from goats and sheep in this study) indicates that F. gigantica and F. hepatica differ typically from each other at seven sites whereas one of these is a distinguishing deletion (T) at the 327th position in F. gigantica relative to F. hepatica. The isolates from the mountainous goats in the North of Vietnam (Yen Bai province) showed the ITS-2 composition relatively identical to that of F. hepatica. The ITS-2 sequences from populations of Fasciola isolates in goats had probably experienced introgression/hybridization as reported previously in other ruminants and humans. All Vietnamese goat-of-origin specimens had high pairwise percentage of mitochondrial cox1 sequences to F. gigantica (97-100%), and very low identity to F. hepatica (91-93%), suggesting their maternal linkage to be traced to F. gigantica. The presence of hybrid and/or introgressed populations of liver flukes bearing genetic material from both F. hepatica and F. gigantica in the goats/sheep in Vietnam, regardless of indigenous or imported hosts, appears to be the first demonstration from a tropical country.     

One Beringian genus less: A re‐assesment of Pacifimyxas Kruglov & Starobogatov, 1985 (Mollusca: Gastropoda: Lymnaeidae) questions the current estimates of Beringian biodiversity

The taxonomic position of three nominal species of lymnaeid snails placed by Kruglov & Starobogatov (1993) into the subgenus Lymnaea (Pacifimyxas) Kruglov & Starobogatov, 1985, has been re‐assessed based on a molecular genetic study of topotypic specimens and an examination of the type series and other materials available. It has been shown that the two species, Lymnaea (Pacifimyxas) magadanensis Kruglov & Starobogatov, 1985 and Lymnaea (Pacifimyxas) streletzkajae Kruglov & Starobogatov, 1985, are identical with the species Kamtschaticana kamtschatica (Middendorff, 1850) and must be treated as its junior synonyms. Hence, Pacifimyxas becomes a junior synonym of Kamtschaticana Kruglov & Starobogatov, 1984. The taxonomic identity of the third species of Pacifimyxas, Lymnaea (Pacifimyxas) perpolita, remains obscure, and this species is considered here as taxon inquirendum. Two other nominal species, Lymnaea aberrans (Westerlund, 1897) and Lymnaea middendorffi (W. Dybowski, 1904), have been synonymized with K. kamtschatica based on morphological and geographical evidence. The lectotype of Limnaea peregra var. middendorffi is designated. The actual level of species richness in the Beringian freshwater malacofauna may be 20–25% lower than it was determined on the basis of the traditional system. Some implications of this outcome for the biogeography of the Beringian freshwater fauna are discussed.     

Morphological Comparison and Taxonomic Utility of Copulatory Structures of Selected Nematode Species

Spicules of 9 Meloidogyne, 2 Heterodera, 3 Globodera, and 12 other plant-parasitic, insect-parasitic, and free-living nematodes were excised and examined using scanning electron microscopy (SEM). Gubernacula of some of the species were also excised, and their structure was determined. The two spicules of all species examined were symmetrically identical in morphology. The spicule typically consisted of three parts: head, shaft, and blade with dorsal and ventral vela. The spicular nerve entered through the cytoplasmic core opening on the lateral outer surface of the spicule head and generally communicated with the exterior through one or two pores at the spicule tip. Spicules of Xiphinema sp. and Aporcelaimellus sp. were not composed of three typical parts, were less sclerotized, and lacked a cytoplasmic core opening and distal pores. Spicules of Aphelenchoides spp. had heads expanded into apex and rostrum and had very arcuate blades with thick dorsal and ventral edges (limbs). Gubernaculum shapes were stable within a species, but differed among species examined. The accessory structures of Hoplolaimus galeatus consisted of a tongue-shaped gubernaculum with two titillae at its distal end and a plate-like capitulum terminating distally in two flat, wing-like structures. A comparison of spicules of several species of Meloidogyne by SEM and light microscopy revealed no striking morphological differences.     

Ecological Study of Nematode Parasitism in Ips Beetles from California and Idaho

Nematodes found in Ips paraconfusus from ponderosa pine in California were an undescribed species of Parasitaphelenchus, Contortylenchus elongatus, C. reversus, and C. brevicomi. C. elongatus, the most commonly found contortylenchid, was present in 98.2% of the contortylenchid-parasitized beetles. Only one nematode parasite of the gut, a Parasitorhabditis sp., was isolated. Although significant differences in parasitism were observed, they were by collection sites, rather than by elevation or bole sources (slash or standing). Significant changes in parasitism between fall and spring collections were observed but not at every site. Nematode parasitism in the F₁ generation of I. paraconfusus by Parasitaphelenchus, Contortylenchus, or Parasitorhabditis increased or decreased from the parent generation depending upon the experiment.Nematode parasites from I. pini included an undescribed Parasitaphelenchus sp., two undescribed Contortylenchus spp., C. reversus and Parasitylenchus (= Neoparasitylenchus) ovarius from the hemocel, and Parasitorhabditis ipini from the gut. Parasitaphelenchus sp. was found in 99% and 45.3% of the beetles from Idaho and California, respectively. Of the 1,000 I. pini from Idaho and California, 157 were parasitized by the contortylenchid species or P. ovarius.