The morphology of the different stages and life-cycle of Echinostomna friedi n. The freshwater snail Lymnaea peregra Gastropoda: Lymnaeidae serves as the natural and experimental first intermediate host and L. These, and Physella acuta Gastropoda: Physidae , also serve as second intermediate hosts. Adult worms, possessing 37 collar spines, were obtained from naturally infected Rattus norvegicus and experimentally from albino rats, golden hamsters and chickens.
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Metrics details. The systematics of echinostomes within the so-called ' revolutum ' group of the genus Echinostoma , which encompasses the type-species E.
Recent molecular studies indicate the existence of more species than previously considered valid, thus stressing the need for wider taxon sampling from natural host populations.
This is especially true for Europe where morphological evidence indicates higher species diversity than previously thought, but where molecular data are virtually lacking. This gap in our knowledge was addressed in the present study through an integration of morphological and molecular approaches in the investigation of a dataset with larger taxonomic and geographical coverage.
More than 20, freshwater snails belonging to 16 species were collected during — from various localities in eight countries in Europe. Snail screening provided representative larval isolates for five species of the ' revolutum ' group, identified by their morphology. Adult isolates for four species recovered from natural and experimental infections were also identified. Partial fragments of the mitochondrial nad 1 and 28S rRNA genes were amplified for 74 and 16 isolates, respectively; these were analysed together with the sequences of Echinostoma spp.
Delineation of the European Echinostoma spp. The large-scale screening revealed infections with five Echinostoma spp. The newly-generated nad 1 sequences from Europe fall into six distinct, well-supported, reciprocally monophyletic lineages corresponding to the species identifications based on morphology; this was corroborated by the 28S rDNA sequences. We conclude that nad 1 should be the first choice for large-scale barcode-based identification of the species of the ' revolutum ' group.
Our study provides a comprehensive reference library for precisely identified isolates of the European species and highlights the importance of an integrative approach for species identification linking molecular, morphological and biological data. Beaver [ 4 ] considered only E. These authors distinguished the five species based mainly on a single morphological feature of their larval stages the number of outlets of the paraoesophageal gland-cells in the cercaria , the specificity towards the snail first intermediate host at the familial level , their ability to infect avian or mammalian hosts or both and their geographical range on a global scale continents see Kostadinova et al.
However, E. Kanev [ 5 ] favoured the idea of allopatric speciation at a continental scale with only two sympatric combinations: i E. Based on molecular data, E. These include E. These authors used nad 1 sequences to identify different larval stages of natural echinostome isolates from Australia and New Zealand and reported on the presence of isolates of E.
However, there appeared to be a problem with the identification of the German isolate of E. Kostadinova et al. Recent molecular studies conducted by Detwiler and colleagues in North America suggested the existence of more than ten species of the genera Echinostoma , Echinoparyphium and Hypoderaeum in natural host populations in the USA.
Recently, Georgieva et al. This gap in our knowledge was addressed in the present study through an integration of morphological and molecular approaches in investigation of a dataset with larger taxonomic and geographical coverage. We carried out molecular prospecting sensu Blouin [ 29 ] for the diversity of the European species of Echinostoma by generating a sequence database linking nad 1 and 28S rDNA sequences for larval and adult experimentally raised and from naturally infected definitive hosts isolates of Echinostoma spp.
These were collected in an extensive sampling programme in eight countries in Europe and identified based on parasite morphology. Our results considerably enhance the consistency of the identification within this group of cryptic species based on molecular data and thus have implications for both monitoring the diversity and host-parasite relationships of Echinostoma spp.
More than 20, freshwater snails belonging to 16 species [ Lymnaea stagnalis L. Snails were screened for trematode infections and representative samples of each cercarial isolate i.
Cercariae were examined live and identified using the data from the relevant primary sources e. Experimental completion of the life-cycle was carried out for two species E. Sequences were also generated from adult isolates of E. All adults were identified prior to sequencing on morphological grounds following Kostadinova et al.
Partial domains D1—D3; c. The best-fitting models of nucleotide substitution were estimated prior to BI analyses with jModelTest 2. Genetic distances uncorrected p-distance were calculated with MEGA v6. Non-metric multidimensional scaling NMDS ordination performed with Primer v6 software [ 43 ] was used to visualise the raw pairwise distances.
In addition to tree-based approaches to species delineation we used the distance-based identification method implemented in the function Species Identifier v1 within the program TAXONDNA [ 44 ].
Assignment outcome is considered successful if the sequences exhibiting the lowest genetic distance closest matches are conspecific with the query sequence and the distance between the query and closest matches falls below a specified threshold.
Relationships between haplotypes of E. Delineation of the European species of Echinostoma was based on the integration of molecular, morphological and ecological data: i support for reciprocal monophyly in the nad 1 phylogeny a conservative approach to species delimitation ; ii pairwise divergence at nad 1 including distance-based assignment and 28S rRNA genes; iii matching of sequences for larval and adult stages three of the species ; iv comparisons with already published sequences; v morphological characterisation and identification of the cercarial and adult isolates; vi inference from the experimental completion of life-cycles all five species ; vi the use of different first intermediate hosts.
The large-scale screening of natural snail populations in Europe revealed infections with five Echinostoma spp. Considering the recent results of Georgieva et al. IG , eight snail species are found to be infected with Echinostoma spp.
Five species acted as hosts of a single species of Echinostoma : A. IG see also [ 19 ]. Echinostoma revolutum exhibited the widest host range being recovered in the four lymnaeids studied L. However, detailed examination of cercarial morphology revealed specific differences with respect to a combination of characters, i. Adult isolates representing four species were identified, three E.
In both life-cycle experiments the nad 1 sequences of the adults were identical with the sequences of the cercariae used as starting material for infection see also [ 28 ]. Morphological descriptions and sequences for Echinostoma sp. IG based on cercarial isolates sampled in Germany and Iceland have been published recently Georgieva et al. Formal description of this putative new species awaits the discovery of the adult stage.
Detailed descriptions of the life-cycle stages of Echinostoma spp. Our study generated 74 novel partial nad 1 sequences for five of the six European species of Echinostoma included in the analyses; these were collapsed into 39 unique haplotypes. Considering the sequences generated by Kostadinova et al. Twenty haplotypes were identified in isolates of E.
Although most of the isolates of E. Notably, one haplotype was shared between adult isolates ex An. In contrast, E. Eleven haplotypes were identified from isolates of Echinostoma n. The two under-sampled presumably rare species, Echinostoma sp. IG and E. One haplotype of Echinostoma sp. IG was shared between cercarial isolates from R. Both NJ and BI analyses resulted in consensus trees with similar topologies.
NJ and BI analyses produced congruent results with minor topological differences. Neighbour-Joining NJ tree for 16 species-level lineages within the ' revolutum ' group of Echinostoma based on the mitochondrial gene nad 1: Clade A.
Based on a nt fragment of nad 1. Outgroups: Echinoparyphium aconiatum and Hypoderaeum conoideum. The newly-sequenced European isolates are shown in red; stars indicate adult isolates from natural infections.
Sequence identification is as in GenBank, followed by a letter: D, Detwiler et al. The scale-bar indicates the expected number of substitutions per site. Neighbour-Joining NJ tree for 16 species-level lineages within the ' revolutum ' group of Echinostoma based on the mitochondrial gene nad 1: Clade B and the remaining species, continuation of Figure 1. The newly-generated sequences from Europe fall into six distinct well-supported reciprocally monophyletic lineages corresponding to the species identifications based on morphology: E.
IG ex R. Three species, Echinostoma sp. IG, E. Jordane France with sequences previously reported as Echinostoma sp. The first clade A comprised the isolates of E. NZ-Ad, E. The average sequence divergence between the two lineages was 5. These results strongly support the suggestion of Georgieva et al. Haplotype network for isolates of Echinostoma revolutum sensu lato. Isolates of E. The European cercarial and adult isolates of E. AJ; sequence otherwise unpublished.
The isolates ii and iv shared the most common haplotype of E. When the North American isolate i was considered separately, the average intraspecific divergence for E. The second clade B was characterised by maximum support at almost all nodes and comprised isolates of Echinostoma n. There was poor support for Lineage C of E. Notably, the identification of the newly-sequenced adult isolates based on morphology alone, using the concept of Kostadinova et al. The 16 newly-generated 28S rDNA sequences corroborated with strong support the distinct species status of the six nad 1 lineages of Echinostoma spp.
The only supported sister-group relationship was between E. No intraspecific variation was detected for species with multiple sequences, i. The lower divergence range was 3—5 nucleotide positions 0. Bayesian inference phylogram reconstructed using partial 28S rDNA sequences for nine Echinostoma spp.
Ruth N. Chunge, PhD, Charles N. Chunge, PhD, Infection with Echinostoma sp. A small group of travellers became infected with Echinostoma sp. The infection was diagnosed by finding the characteristic eggs in stool samples collected over a 2-week period following their return to Kenya.
Insights About Echinostomiasis by Paleomolecular Diagnosis
Echinostomiasis is a zoonosis caused by intestinal trematodes and transmitted by the ingestion of mollusks, crustaceans, fish, amphibians, and reptiles, either raw or poorly cooked. Today human infection is endemic in Southeast Asia and the Far East, but has been reported more recently in other regions of the world. Interestingly eggs identified as Echinostoma sp. However, the specific diagnosis based on morphology of the eggs has not been resolved at the species level. As a follow-up to the previous finding, the current study now aims to standardize the methodology for molecular diagnosis and apply it to the coprolite, using current Echinostoma paraensei-positive feces as the reference, and also the same fecal material dried in a stove as an experimental coprolite model. Isolated eggs of E.
Metrics details. The systematics of echinostomes within the so-called ' revolutum ' group of the genus Echinostoma , which encompasses the type-species E. Recent molecular studies indicate the existence of more species than previously considered valid, thus stressing the need for wider taxon sampling from natural host populations. This is especially true for Europe where morphological evidence indicates higher species diversity than previously thought, but where molecular data are virtually lacking. This gap in our knowledge was addressed in the present study through an integration of morphological and molecular approaches in the investigation of a dataset with larger taxonomic and geographical coverage.