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Waterbirds, are one of the popular game animals and are of significant relevance to parasite spread due to their ability to fly and migrate great distances in relatively short periods of time. In Australia, however, the knowledge of parasites infecting native waterbirds is lacking with some of the last reports occurring over 50 years ago. The study aimed to characterise Echinostoma spp. infecting wild native Australian ducks found in the southern regions of the Murray Darling Basin (MDB). Ducks (n = 98) were collected from southern New South Wales within the MDB catchment. Three different species of native ducks were found including Anas superciliosa (n = 37), Anas gracilis (n = 47) and Chenonetta jubata (n = 14), of which 4.3 %, 2.7 % and 7.1 %, respectively, were found to be infected with adult stages Echinostoma spp. Examination of the parasites revealed the presence of two morphotypes. The 18S, 28S and ITS rRNA as well as the mitochondrial nad1 genes were sequenced for representative isolates of the two morphotypes. These sequences were then compared with existing sequences of Echinostoma spp. available in the GenBank. Phylogenetic analysis based on the ITS region indicated that the two morphotypes were genetically distinct. Although there are comparable sequences of Echinostoma spp. in Australia these morphotypes appear to be genetically distinct. Based on their distinct morphology and genetics we suggest that these two morphotypes are previously undescribed in Australia. This study sheds light on the presence of Echinostoma parasites in native Australian waterbirds and highlights the need for further research to better understand the diversity and prevalence of these parasites in the region., Competing Interests: Declaration of competing interest No conflict of interest to report., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Life cycles, and morphological and molecular data were obtained for Echinostoma chankensis nom. nov., Echinostoma cinetorchis, Echinostoma miyagawai and Isthmiophora hortensis from East Asia. It was established that, based on both life cycle and morphology data, one of the trematodes is identical to the worms designated as Euparyphium amurensis. Genetic data showed that this trematode belongs to Echinostoma. The complex data on biological, morphological and genetic characterizations establish that the distribution of the morphologically similar species, I. hortensis and Isthmiophora melis, in the Old World are limited by the East Asian and European regions, respectively. Data on mature worms of East Asian E. miyagawai revealed morphological and genetic identity with E. miyagawai from Europe. However, E. miyagawai from Europe differs from E. miyagawai from the type locality (East Asia) in terms of reaching maturity and the morphology of cercariae. These data indicate that the European worm, designated E. miyagawai, does not belong to this species. An analysis of the phylogenetic relationships of Echinostomatidae was conducted based on the 28S, ITS2 and nad1 markers. Analysis using the nad1 gene for the known representatives of Echinostomatidae is carried out for the first time, showing that nuclear markers are ineffective separate from mitochondrial ones.

To analyze the response of the snail Physella acuta to Echinostoma paraensei, a compatible digenetic trematode, Illumina RNA-seq data were collected from snails with early infection (5 snails at 2 days post-exposure [DPE]) and established infection (4 snails, 8 DPE), and 7 control (unexposed) snails. A reference transcriptome (325,563 transcripts, including 98% of eukaryotic universal single-copy orthologs; BUSCO) and a draft P. acuta genome (employing available genomic Illumina reads; 799,945 scaffolds, includes 88% BUSCO genes) were assembled to guide RNA-seq analyses. Parasite exposure of P. acuta led to 10,195 differentially expressed (DE) genes at 2 DPE and 8,876 DE genes at 8 DPE with only 18% of up-regulated and 22% of down-regulated sequences shared between these time points. Gene ontology (GO) analysis yielded functional annotation of only 1.2% of DE genes but did not indicate major changes in biological activities of P. acuta between 2 and 8 DPE. Increased insights were achieved by analysis of expression profiles of 460 immune-relevant DE transcripts, identified by BLAST and InterProScan. Physella acuta has expanded gene families that encode immune-relevant domains, including CD109/TEP, GTPase IMAP, Limulus agglutination factor (dermatopontin), FReD (≥82 sequences with fibrinogen-related domains), and transcripts that combine C-type lectin (C-LECT) and C1q domains, novel among metazoa. Notably, P. acuta expressed sequences from these immune gene families at all time points, but the assemblages of unique transcripts from particular immune gene families differed between 2 and 8 DPE. The shift in profiles of DE immune genes, from early exposure to parasite establishment, suggests that compatible P. acuta initially respond to infection but switch to express immune genes that likely are less effective against E. paraensei but counter other types of (opportunistic) pathogens and parasites. We propose that the latter expression profile is part of an extended phenotype of E. paraensei, imposed upon P. acuta through parasite manipulation of the host, following successful parasite establishment in the snail after 2 DPE., (© American Society of Parasitologists 2020.)

Echinostoma flukes armed with 37 collar spines on their head collar are called as 37-collar-spined Echinostoma spp. (group) or 'Echinostoma revolutum group'. At least 56 nominal species have been described in this group. However, many of them were morphologically close to and difficult to distinguish from the other, thus synonymized with the others. However, some of the synonymies were disagreed by other researchers, and taxonomic debates have been continued. Fortunately, recent development of molecular techniques, in particular, sequencing of the mitochondrial (nad1 and cox1) and nuclear genes (ITS region; ITS1-5.8S-ITS2), has enabled us to obtain highly useful data on phylogenetic relationships of these 37-collar-spined Echinostoma spp. Thus, 16 different species are currently acknowledged to be valid worldwide, which include E. revolutum, E. bolschewense, E. caproni, E. cinetorchis, E. deserticum, E. lindoense, E. luisreyi, E. mekongi, E. miyagawai, E. nasincovae, E. novaezealandense, E. paraensei, E. paraulum, E. robustum, E. trivolvis, and Echinostoma sp. IG of Georgieva et al., 2013. The validity of the other 10 species is retained until further evaluation, including molecular analyses; E. acuticauda, E. barbosai, E. chloephagae, E. echinatum, E. jurini, E. nudicaudatum, E. parvocirrus, E. pinnicaudatum, E. ralli, and E. rodriguesi. In this review, the history of discovery and taxonomic debates on these 26 valid or validity-retained species are briefly reviewed.

Echinostomes are a diverse group of digenetic trematodes that are difficult to classify by predominantly traditional techniques and contain many cryptic species. Application of contemporary genetic/molecular markers can provide an alternative choice for comprehensive classification or systematic analysis. In this study, we successfully characterized the intron 5 of domain 1 of the taurocyamine kinase gene (TkD1Int5) of Artyfechinostomum malayanum and the other two species of the 37 collar-spined group, Echinostoma revolutum and Echinostoma miyagawai, whereas TkD1Int5 of Hypoderaeum conoideum cannot be amplified. High levels of nucleotide polymorphism were detected in TkD1Int5 within E. revolutum and E. miyagawai, but not in A. malayanum. Thus, TkD1Int5 can be potentially used as genetic marker for genetic investigation of E. miyagawai and E. revolutum. We therefore used TkD1Int5 to explore genetic variation within and genetic differentiation between 58 samples of E. miyagawai and five samples of E. revolutum. Heterozygosity was observed in 17 and two samples with 16 and three insertion/deletion (indel) patterns in E. miyagawai and E. revolutum, respectively. Heterozygous samples were then cloned and nucleotide sequence was performed revealing the combined haplotypes in a particular sample. Based on nucleotide variable sites (excluding indels), the 72 E. miyagawai and seven E. revolutum haplotypes were subsequently classified. The haplotype network revealed clear genetic differentiation between E. miyagawai and E. revolutum haplogroups, but no genetic structure correlated with geographical localities was detected. High polymorphism and heterogeneity of the TkD1Int5 sequence found in our study suggest that it can be used in subsequent studies as an alternate independent potential genetic marker to investigate the population genetics, genetic structure, and possible hybridization of the other echinostomes, especially the 37 collar-spined group distributed worldwide.

Echinostomes are important intestinal foodborne parasites. Despite their significance as pathogens, characterization of the molecular biology and phylogenetics of these parasites are limited. In the present study, we determined the entire mitochondrial (mt) genome of the echinostome Echinostoma miyagawai (Hunan isolate) and examined the phylogenetic relationship with selected members of the suborder Echinostomata. The complete mt genome of E. miyagawai (Hunan isolate) was 14,468 bp in size. This circular mt genome contained 12 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and one non-coding region. The gene order and genomic content were identical with its congeners. Phylogenetic analyses (maximum parsimony, maximum likelihood, and Bayesian inference) based on the concatenated amino acid sequences of 12 protein-coding genes strongly supported monophyly for the genus Echinostoma; however, they rejected monophyly for the family Echinostomatidae and the genus Fasciola. The mt genomic data described in this study provides useful genetic markers for studying the population genetics, molecular biology, and phylogenetics of these echinostomes.

The taxonomic evaluation of Echinostoma species is controversial. Echinostoma species are recognized as complex, leading to problems associated with accurate identification of these species. The aim of this study was to test the feasibility of using DNA barcoding of cytochrome c oxidase subunit I (COI) and NADH dehydrogenase subunit 1 (ND1) conjugated with high-resolution melting (HRM) analysis to identify Echinostoma revolutum. HRM using COI and ND1 was unable to differentiate between species in the "revolutum complex" but did distinguish between two isolates of 37-collar-spined echinostome species, including E. revolutum (Asian lineage) and Echinostoma sp. A from different genera, e.g., Hypoderaeum conoideum, Haplorchoides mehrai, Fasciola gigantica, and Thapariella anastomusa, based on the T m values derived from HRM analysis. Through phylogenetic analysis, a new clade of the cryptic species known as Echinostoma sp. A was identified. In addition, we found that the E. revolutum clade of ND1 phylogeny obtained from the Thailand strain was from a different lineage than the Eurasian lineage. These findings reveal the complexity of the clade, which is composed of 37-collar-spined echinostome species found in Southeast Asia. Taken together, the systematic aspects of the complex revolutum group are in need of extensive investigation by integrating morphological, biological, and molecular features in order to clarify them, particularly in Southeast Asia.

Precise discrimination of Echinostoma species within the 'revolutum' group is quite difficult because of their morphological similarities. The objective of this study was to precisely characterize the echinostomes of ducks from Bangladesh based on both morphological and molecular characteristics. Two Echinostoma species were identified: E. revolutum and E. robustum. In the phylogenetic trees (ITS2 and nad1), E. revolutum and E. robustum belonged to their respective Eurasian clade, which is distinct from the American clade. These results suggest that both species have two distinct and geographically separated lineages, Eurasian and American. Our molecular and morphological data combined with previously published data supports the synonymy of E. robustum, E. miyagawai, and E. friedi previously based on either molecular or morphological evidence. This study thus improves our understanding of species diversity of the 'revolutum' group, particularly in Asia., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Echinostoma paraensei (Digenea: Echinostomatidae) lives in the duodenum and bile duct of rodents and is reported as a useful model for studies on the biology of flatworms. Here, we compared the growth and development of pre and post ovigerous worms collected 3, 7, 14 and 21 days post infection from experimentally infected hamster (permissive host) and Wistar rat (less permissive hosts). Linear measurements and ratios were examined by light (morphology and morphometry) and confocal laser scanning microscopy. At day 3, either worm from hamsters or rats were small with poorly developed gonads. At seven day, worms increased in size and morphometric differences between hosts are statistically significant after this time. In addition, adult worms (14 and 21 days of age) harvested from hamster showed developed gonads and vitelline glands laterally distributed on the body, whereas worms from rat showed atrophied reproductive system characterized by underdeveloped vitelline glands and stunted ovary. The worm rate recovery in rat decreased from 29.3% (day 7) to 20.6% (day 14) and 8% (day 21), whilst it remained around 37% in hamster. In conclusion, this is the first appointment demonstrating that low permissiveness influences the reproductive system of echinostome since the immature stages of development. The phenotypic analysis evidenced that hamster provides a more favorable microenvironment for gonads development than rat, confirming golden hamster as a permissive host, whereas Wistar rat is less permissive host., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Morphological and molecular characterisation of echinostome specimens (Digenea: Echinostomatidae) recovered in one Anas platyrhynchos L. and one Cygnus atratus (Latham) (Anseriformes: Anatidae) from New Zealand revealed the presence of two known species, Echinostoma miyagawai Ishii, 1932 and Echinoparyphium ellisi (Johnston & Simpson, 1944) and two species new to science. Comparative morphological and phylogenetic analyses supported the distinct species status of Echinostoma novaezealandense n. sp. ex Branta canadensis (L.), A. platyrhynchos and C. atratus, and Echinoparyphium poulini n. sp. ex C. atratus. Echinostoma novaezealandense n. sp., a species of the "revolutum" species complex characterised by the possession of a head collar armed with 37 spines, keyed down to E. revolutum but was distinguished from the latter in having a much narrower body with almost parallel margins, longer oesophagus, wider cirrus-sac, larger seminal vesicle, much smaller ventral sucker, ovary, Mehlis' gland and testes, more anteriorly located ovary and testes, and distinctly smaller eggs (81-87 × 42-53 vs 106-136 × 55-70 µm). This new species appears similar to Echinostoma acuticauda Nicoll, 1914 described in Australia but differs in having a longer forebody, more posteriorly located ovary and testes, and much smaller eggs (81-87 × 42-53 vs 112-126 × 63-75 µm). Echinoparyphium poulini n. sp. is differentiated from the four species of Echinoparyphium possessing 37 collar spines considered valid as follows: from E. chinensis Ku, Li & Chu, 1964 in having a much smaller body, four (vs five) angle spines and simple seminal vesicle (vs bipartite); from E. schulzi Matevosyan, 1951 in having a less robust body at a comparable body length, much smaller ventral sucker, ovary and testes, and longer but narrower eggs (87-109 × 50-59 vs 70-85 × 60-84 µm); and from the two smaller forms, E. serratum Howell, 1968 and E. aconiatum Dietz, 1909, in a number of additional metrical features correlated with body size and especially in the possession of much larger collar spines. Partial fragments of the mitochondrial nad1 and 28S rRNA genes were amplified for representative isolates of the four species and analysed together with sequences for Echinostoma spp. and Echinoparyphium spp. available on GenBank. Phylogenetic analyses based on the mitochondrial nad1 gene revealed congruence between the molecular data and species identification/delineation based on morphology; this was corroborated by the 28S rDNA sequence data.

The "37 collar-spined" or "revolutum" group of echinostomes is recognized as a species complex. The identification of members of this complex by morphological taxonomic characters is difficult and confusing, and hence, molecular analyses are a useful alternative method for molecular systematic studies. The current study examined the genetic diversity of those 37 collar-spined echinostomes which are recognized morphologically as Echinostoma revolutum in Thailand and Lao PDR using the cytochrome c oxidase subunit 1 (CO1) and the NADH dehydrogenase subunit 1 (ND1) sequences. On the basis of molecular investigations, at least two species of 37 collar-spined echinostomes exist in Southeast Asia, namely E. revolutum and Echinostoma miyagawai. The specimens examined in this study, coming from ducks in Thailand and Lao PDR, were compared to isolates from America, Europe and Australia for which DNA sequences are available in public databases. Haplotype analysis detected 6 and 26 haplotypes when comparing the CO1 sequences of E. revolutum and E. miyagawai, respectively, from different geographical isolates from Thailand and Lao PDR. The phylogenetic trees, ND1 haplotype network and genetic differentiation (ɸST) analyses showed that E. revolutum were genetically different on a continental scale, i.e. Eurasian and American lineages., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Species of Echinostoma Rudolphi, 1809 (Digenea: Echinostomatidae) belonging to the 'revolutum' species complex were re-examined based on material gathered in an extensive sampling programme in eight countries in Europe. The morphology of the life-cycle stages was studied in naturally and experimentally infected snail and bird hosts. A review, with an updated synonymy, is presented for six European species, including one new to science, i.e. Echinostoma revolutum (Frölich, 1802) (sensu stricto) (type-species), E. bolschewense (Kotova, 1939), E. miyagawai Ishii, 1932, E. nasincovae n. sp., E. paraulum Dietz, 1909 and Echinostoma sp. IG), and keys to the identification of their cercariae and adults are provided.

Background: The systematics of echinostomes within the so-called 'revolutum' group of the genus Echinostoma, which encompasses the type-species E. revolutum and a number of morphologically similar species, has long been controversial. 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., Methods: More than 20,000 freshwater snails belonging to 16 species were collected during 1998-2012 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 nad1 and 28S rRNA genes were amplified for 74 and 16 isolates, respectively; these were analysed together with the sequences of Echinostoma spp. available on GenBank., Results: Delineation of the European Echinostoma spp. was carried out based on molecular, morphological and ecological data. The large-scale screening revealed infections with five Echinostoma spp., including one new species: E. revolutum (sensu stricto), E. miyagawai, E. paraulum, E. bolschewense and Echinostoma n. sp. The newly-generated nad1 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. The analyses of the total nad1 dataset provided evidence for 12 monophyletic groups and five singletons, which represent seven described/named species and ten cryptic species-level lineages of Echinostoma., Conclusion: We conclude that nad1 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.

Three echinostome species, i.e., Patagifer bilobus, Petasiger neocomense, and Saakotrema metatestis, are newly recorded in the trematode fauna of the Republic of Korea. They were recovered from 3 species of migratory birds (Platalea minor, Podiceps cristatus, and Egretta garzetta), which were donated by the Wildlife Center of Chungbuk (WCC) and the Conservation Genome Resource Bank for Korean Wildlife (CGRB). Only 1 P. bilobus specimen was recovered from the intestine of a black-faced spoonbill (P. minor), and characterized by the bilobed head crown with a deep dorsal incision and 54 collar spines. Twenty P. neocomense were recovered from the intestine of a great crested grebe (P. cristatus), and they had a well-developed head crown with 19 spines and 2 testes obliquely located at the posterior middle of the body. Total 70 S. metatestis were collected from the bursa of Fabricius of 1 little egret (E. garzetta). It is characterized by stout tegumental spines covered in the entire leaf-shaped body, posterior extension of the uterus, presence of the uroproct and a well-developed head crown with 12 pairs of collar spines on each side. By the present study, these 3 echinostome species are newly added to the trematode fauna in Korea.

We conducted this study to identify species and determine the phylogenetic relationships using ribosomal DNA (rDNA) sequences [partial sequences of 28S rDNA and second internal transcribed spacer (IT52)] of echinostomes collected from free-grazing ducks in Phitsanulok Province, Thailand. Four adult echinostomes were morphologically identified as Echinostoma revolutum, 4 as Hypoderaeulm conoideurn and 2 unidentified. Sequences of other species/isolates of echinostomes retrieved from the GenBank database were employed to compare and construct the phylogenetic tree. Three major lineages were found, namely, genus Echinostoma, genus Echinoparyphiulm and genus Hypoderaeulm. One of the unidentified echinostome specimen was 99% identical to and clustered with genus Echinoparyphiurm, whereas the other was located in the "revolutum" roup, but was closely related to the geographical isolates from America rather than from Thailand. This study indicates that 28S rDNA and 1T52 regions are suitable molecular markers for genetic characterization and phylogenetic analysis of echinostomes.

During parasitological examination of Biomphalaria pfeifferi snails obtained from Niger state (Nigeria), 2 new types of cercariae were found. They are identified to the level of referring to the major group and described here for the first time. They were examined viable and stained with vital stains as well as fixed in 70% alcohol. They were drawn with a camera lucida and photographed. They are identified as an echinostome cercaria and a xiphidiocercaria. The echinostome is characterized by having a ventral sucker almost double in size the oral one. It has a semicircular structure located beyond the oral sucker. Three pairs of penetration glands are found at the anterior portion of the body. The number of collar spines is relatively large (44-46). The flame cellsare 17 x 2 in number. Two main lateral excretory ducts extend anteriorly, form two typical echinostome loops then pass posteriorly to open together in a diverticulated excretory vesicle. Its tail is relatively long and flattened with 3 fin folds. The tail (640 μm) is longer than the body (475 μm). The xiphidiocercaria belongs to the "ornatae" group. It is relatively small (180.5 x 110 μm) with a long stylet (30 μm). Its oral sucker is one and half times the size of the ventral sucker. Two excretory ducts extend posteriorly in both sides and become dilated and unite to open in a circular excretoryvesicle. Tail is slender shorter than the body and has a dorso-ventral fin fold.

In the present paper, we assess the relationship between the expression of IFN-γ and the development of clinical signs in Echinostoma caproni-infected mice. For this purpose, we studied the course of the infection in three mouse strains: ICR (CD-1®) (a host of high compatibility with E. caproni), BALB/c (a prototypical Th2 strain), and BALB/c deficient for IFN-γ mice (IFN-γ(-/-)). Infection in ICR mice is characterized by the elevated expression of IFN-γ and iNOS in the intestine concomitantly with the lack of clinical signs. In contrast, the infection was more virulent in BALB/c and IFN-γ-deficient mice that developed a severe form of the disease together with the absence of IFN-γ expression. The disease was more severe in IFNγ(-/-) mice in which the disease was lethal during the few first weeks of the infection. The analysis of different parameters of the infection in each host strain showed that most of the features were similar in the three mouse strains, suggesting the IFN-γ plays a central role in that protection against severe disease. Thus, IFN-γ seems to play a dichotomous role in the infection facilitating the parasite establishment, but it may also benefit mice since it protects the mice from morbidity and mortality induced by the parasite.

Human cases of echinostomiasis have been sporadically diagnosed by extracting worms in the endoscopy in Korea and Japan. Most of these were caused by Echinostoma hortense infection. However, in the present study, we detected 2 live worms of Echinostoma cinetorchis in the ascending colon of a Korean man (68-year old) admitted to the Gyeongsang National University Hospital with complaint of intermittent right lower quadrant abdominal pain for 5 days. Under colonoscopy, 1 worm was found attached on the edematous and hyperemic mucosal surface of the proximal ascending colon and the other was detected on the mid-ascending colon. Both worms were removed from the mucosal surface with a grasping forceps, and morphologically identified as E. cinetorchis by the characteristic head crown with total 37 collar spines including 5 end-group ones on both sides, disappearance of testes, and eggs of 108×60 µm with abopercular wrinkles. The infection source of this case seems to be the raw frogs eaten 2 months ago. This is the first case of endoscopy-diagnosed E. cinetorchis infection in Korea.

Echinostome metacercariae are the infective stage for humans and animals. The identification of echinostomes has been based until recently on morphology but molecular techniques using sequences of ribosomal RNA and mitochondrial DNA have indicated major clades within the group. In this study we have used the ITS2 region of ribosomal RNA and the ND1 region of mitochondrial DNA to identify metacercariae from snails collected from eight well-separated sites from an area of 4000 km2 in Lamphun Province, Thailand. The derived sequences have been compared to those collected from elsewhere and have been deposited in the nucleotide databases. There were two aims of this study; firstly, to determine the species of echinostome present in an endemic area, and secondly, to assess the intra-specific genetic diversity, as this may be informative with regard to the potential for the development of anthelmintic resistance and with regard to the spread of infection by the definitive hosts. Our results indicate that the most prevalent species are most closely related to E. revolutum, E. trivolvis, E. robustum, E. malayanum and Euparyphium albuferensis. Some sites harbour several species and within a site there could be considerable intra-species genetic diversity. There is no significant geographical structuring within this area. Although the molecular techniques used in this study allowed the assignment of the samples to clades within defined species, however, within these groupings there were significant differences indicating that cryptic speciation may have occurred. The degree of genetic diversity present would suggest the use of targeted regimes designed to minimise the selection of anthelmintic resistance. The apparent lack of geographic structuring is consistent with the transmission of the parasites by the avian hosts.

Objective: To analyze the prevalence of echinostome metacercariae in Filopaludina dorliaris (F. dorliaris) and Filopaludina martensi martensi (F. martensi martensi) and genotype variation of echinostome metacercariae by using random amplified polymorphic DNA (RAPD) analysis., Methods: Filopaludina sp. snails were collected from eight localities of Lamphun Province, Northern Thailand and examined for echinostome metacercariae. RAPD-PCR was used to analyze genotype variation of echinostome metacercariae., Results: A total of 3 226 F. dorliaris and F. martensi martensi snails were collected from eight localities. The overall prevalences of echinostome metacercariae in F. dorliaris and F. martensi martensi were 40.89% and 36.27%, while the intensity of infection was 20.37 and 12.04, respectively. The dendrogram constructed base on RAPD profiles, 4 well supported domains were generated; (i) group of metacercariae from Ban Hong, Mae Ta, Meaung, Pa Sang, Toong Hua Chang, and Weang Nong that were clustered in the group of E. revolutum, (ii) Ban Thi, (iii) Lee, and (iv) 3 adults of an out group., Conclusions: This research demonstrated RAPD profiling has been a useful tool to detect DNA polymorphisms to determine genetic relationship between echinostome metacercariae in Lamphun Province, Northern Thailand., (Copyright © 2014 Hainan Medical College. Published by Elsevier B.V. All rights reserved.)

Background: The digenean species of Echinostoma (Echinostomatidae) with 37 collar spines that comprise the so-called 'revolutum' species complex, qualify as cryptic due to the interspecific homogeneity of characters used to differentiate species. Only five species were considered valid in the most recent revision of the group but recent molecular studies have demonstrated a higher diversity within the group. In a study of the digeneans parasitising molluscs in central and northern Europe we found that Radix auricularia, R. peregra and Stagnicola palustris were infected with larval stages of two cryptic species of the 'revolutum' complex, one resembling E. revolutum and one undescribed species, Echinostoma sp. IG. This paper provides morphological and molecular evidence for their delimitation., Methods: Totals of 2,030 R. auricularia, 357 R. peregra and 577 S. palustris were collected in seven reservoirs of the River Ruhr catchment area in Germany and a total of 573 R. peregra was collected in five lakes in Iceland. Cercariae were examined and identified live and fixed in molecular grade ethanol for DNA isolation and in hot/cold 4% formaldehyde solution for obtaining measurements from fixed materials. Partial fragments of the mitochondrial gene nicotinamide adenine dinucleotide dehydrogenase subunit 1 (nad1) were amplified for 14 isolates., Results: Detailed examination of cercarial morphology allowed us to differentiate the cercariae of the two Echinostoma spp. of the 'revolutum' species complex. A total of 14 partial nad1 sequences was generated and aligned with selected published sequences for eight species of the 'revolutum' species complex. Both NJ and BI analyses resulted in consensus trees with similar topologies in which the isolates from Europe formed strongly supported reciprocally monophyletic lineages. The analyses also provided evidence that North American isolates identified as E. revolutum represent another cryptic species of the 'revolutum' species complex., Conclusion: Our findings highlight the need for further analyses of patterns of interspecific variation based on molecular and morphological evidence to enhance the re-evaluation of the species and advance our understanding of the relationships within the 'revolutum' group of Echinostoma.

We detected metacercariae of Echinostoma revolutum in Filopaludina sp. snails purchased from a local market in Nam Dinh Province for the first time in Vietnam. Adult flukes were harvested from experimentally infected hamsters at days 14 and 17 post-infection. The metacercariae were round, 170-190 µm (n = 15) in diameter, with a cyst wall thickness of about 12 µm. A total of 37 collar spines were arranged around the head collar, and large excretory granules were seen in 2 canals of the excretory bladder. The 14-day old adult flukes were elongated, ventrally curved, and 5.0-7.2 × 0.8-1.3 mm (n = 20). The head collar had a total of 37 collar spines arranged in 2 alternating rows, including 5 corner spines on each side. The cirrus sac contained a saccular seminal vesicle, a prostatic gland, and an unarmed cirrus. Two tandem testes were smooth or slightly lobed. Eggs were ovoid to elliptical, 110-118 × 70-75 µm. These morphological characters were similar to those of E. revolutum and E. jurini. We tentatively identified it as E. revolutum because the validity of E. jurini remains to be elucidated. The taxonomic relationship of E. revolutum and E. jurini is discussed.

A total of 314 individual Echinostoma revolutum were collected at different locations and times from domestic ducks from Khon Kaen Province, Thailand and Vientiane Province, the Lao People's Democratic Republic (PDR). Genetic variation of these parasites was analyzed using multilocus enzyme electrophoresis at three polymorphic loci namely, glucose-6-phosphate dehydrogenase (G6pd), malic enzyme (Me) and peptidase valine-leucine (PepA). High levels of genetic variability were found within and between populations. Significant heterozygote deficiencies compared with the predictions under Hardy-Weinberg equilibrium were detected in populations from Thailand and the Lao PDR for all loci except G6pd-1. Significant genetic differentiation was observed between spatially separated populations from Thailand and the Lao PDR. This as also true for some samples collected at different times in Thailand. The variability found may be consistent with a Wahlund effect, genetic drift and/or other factors such as the population structure of snail hosts. Our data provide further insight into the process of genetic divergence within and among geographically and temporally isolated populations of E. revolutum, and potentially other medically important echinostomes in Southeast Asia., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Multilocus enzyme electrophoresis (MEE) and DNA sequencing of the mitochondrial cytochrome c oxidase subunit 1 (CO1) gene were used to genetically compare four species of echinostomes of human health importance. Fixed genetic differences among adults of Echinostoma revolutum, Echinostoma malayanum, Echinoparyphium recurvatum and Hypoderaeum conoideum were detected at 51-75% of the enzyme loci examined, while interspecific differences in CO1 sequence were detected at 16-32 (8-16%) of the 205 alignment positions. The results of the MEE analyses also revealed fixed genetic differences between E. revolutum from Thailand and Lao PDR at five (19%) of 27 loci, which could either represent genetic variation between geographically separated populations of a single species, or the existence of a cryptic (i.e. genetically distinct but morphologically similar) species. However, there was no support for the existence of cryptic species within E. revolutum based on the CO1 sequence between the two geographical areas sampled. Genetic variation in CO1 sequence was also detected among E. malayanum from three different species of snail intermediate host. Separate phylogenetic analyses of the MEE and DNA sequence data revealed that the two species of Echinostoma (E. revolutum and E. malayanum) did not form a monophyletic clade. These results, together with the large number of morphologically similar species with inadequate descriptions, poor specific diagnoses and extensive synonymy, suggest that the morphological characters used for species taxonomy of echinostomes in South-East Asia should be reconsidered according to the concordance of biology, morphology and molecular classification., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Echinostomatidae are common, widely distributed intestinal parasites causing significant disease in both animals and humans worldwide. In spite of their importance, the taxonomy of these echinostomes is still controversial. The taxonomic status of two species, Echinostoma revolutum and Echinoparyphium recurvatum, which commonly infect poultry and other birds, as well as human, is problematical. Previous phylogenetic analyses of Southeast Asian strains indicate that these species cluster as sister taxa. In the present study, the first internal transcribed spacer (ITS1) sequence was used for genetic characterization and to examine the phylogenetic relationships between an isolate from Thailand with other isolates available from GenBank database. Interspecies differences in ITS1 sequence between E. revolutum and E. recurvatum were detected at 6 (3%) of the 203 alignment positions. Of these, nucleotide deletion at positions 25, 26, and 27, pyrimidine transition at 50, 189, and pyrimidine transversion at 118 were observed. Phylogenetic analysis revealed that E. recurvatum from Thailand clustered as a sister taxa with E. revolutum and not with other members of the genus Echinoparyphium. Interestingly, this result confirms a previous report based on allozyme electrophoresis and mitochondrial DNA that E. revolutum and E. recurvatum in Southeast Asia are sister species. Hence, the taxonomic status of E. recurvatum in Thailand, as well as in Southeast Asian countries needs to be confirmed and revised using more comprehensive analyses based on morphology and other molecular techniques.

To determine the prevalence of helminthic infections in Pursat Province, Cambodia, we tested fecal specimens from 471 children, 10-14 years of age, in June 2007. The prevalence of infection with echinostome flukes ranged from 7.5% to 22.4% in 4 schools surveyed. Adult worms were identified as Echinostoma revolutum.

Echinostomes are receiving increased attention because of their emerging parasite status in landscapes associated with human development and their ability to infect and kill many North American larval amphibians. While laboratory experiments have shown that echinostomes can cause extensive mortality in their amphibian hosts, their effect on tadpoles in the field is less clear. I conducted a controlled-infection field-enclosure experiment in 4 ponds to compare the effects of echinostomes on green frog (Rana clamitans) and gray tree frog (Hyla versicolor) tadpoles in the field. I measured tadpole growth, development, mortality, and infection intensity. Echinostome infection resulted in high mortality in green frog tadpoles and less mortality in gray tree frogs. However, metacercariae encystment rates were higher in gray tree frog tadpoles than in green frog tadpoles. The effect of echinostomes on mortality varies across amphibian species, with the result that some species may experience more extensive echinostome-induced mortality than others. Mortality as a result of echinostome infection in green frog tadpoles was similar to mortality observed in predation experiments.

The recognition of cryptic parasite species has implications for evolutionary and population-based studies of wildlife and human disease. Echinostome trematodes are a widely distributed, species-rich group of internal parasites that infect a wide array of hosts and are agents of disease in amphibians, mammals, and birds. We utilize genetic markers to understand patterns of morphology, host use, and geographic distribution among several species groups. Parasites from >150 infected host snails (Lymnaea elodes, Helisoma trivolvis and Biomphalaria glabrata) were sequenced at two mitochondrial genes (ND1 and CO1) and one nuclear gene (ITS) to determine whether cryptic species were present at five sites in North and South America. Phylogenetic and network analysis demonstrated the presence of five cryptic Echinostoma lineages, one Hypoderaeum lineage, and three Echinoparyphium lineages. Cryptic life history patterns were observed in two species groups, Echinostoma revolutum and Echinostoma robustum, which utilized both lymnaied and planorbid snail species as first intermediate hosts. Molecular evidence confirms that two species, E. revolutum and E. robustum, have cosmopolitan distributions while other species, E. trivolvis and Echinoparyphium spp., may be more geographically limited. The intra and interspecific variation detected in our study provides a genetic basis for seven species groups of echinostomes which will help accurately identify agents of disease as well as reveal cryptic aspects of trematode biology., (Copyright 2010 Elsevier Inc. All rights reserved.)

Population density, or crowding, was examined to determine its effect on the morphometric variability of Echinostoma caproni (Digenea) in ICR mice. Six mice were infected with 25 and 100 metacercariae, and a single mouse was infected with 300 metacercariae. All mice were infected at necropsy 22 days postinfection with recoveries of 77%, 69%, and 7.3%, respectively. Whole mounts were prepared, and 31 characters were evaluated (25 direct measurements and 6 ratios). Univariate and multivariate statistical analysis revealed significant differences between adult worms from all 3 groups. Twenty-seven of 31 characters showed significant within-group differences, with the primary differences between worms from 25/100 versus 300 metacercariae infections. Discriminant function analysis yielded a 100% correct classification based on infection size, which is consistent with studies on distinct species of Echinostoma. The low recovery from the mouse infected with 300 metacercariae suggests inflammatory expulsion of juvenile worms and the possibility of immunity as a factor in the crowding effect. These results suggest that external factors may affect morphometric variability of digenetic trematodes to a larger degree than previously recognized.

This review examines the significant literature on the biology of Echinostoma species in the "revolutum" group. We have considered 10 species belonging to this group. There is a considerable body of literature for four of the species, i.e. Echinostoma caproni, E. trivolvis, E. paraensei and E. revolutum. For these species we have arranged coverage to include the following headings: (1) systematic and descriptive studies; (2) experimental, manipulative and ecological studies; (3) physiological and biochemical studies; (4) immunological and molecular studies. For the remaining six species, i.e. E. friedi, E. miyagawai, E. echinatum, E. parvocirris, E. luisyrei and E. jurini, the literature is not very extensive, and headings were not used. Considerable information in various areas of modern parasitology can be obtained from species in the "revolutum" complex for which the entire life cycle is maintained in the laboratory. The review includes a list of researchers and their addresses who currently maintain such life cycles.

The entire life cycle of the Brazilian Echinostoma luisreyi n. sp., of the 37-collar spine E. revolutum group, has been observed under experimental conditions. The snail Physa marmorata serves as its first natural intermediate host. This species and the planorbid Biomplalaria glabrata act as experimental second intermediate hosts. The worm recovery rate was 51.3 and 0.6% for mice and hamsters, respectively, but the infection did not develop in the quail Coturnix coturnix. The natural vertebrate host is not known. The most important morphological character of the new species separating it from the other Echinostoma species studied is the oral corner spines that increase in size from the latero-oral to the ventro-oral regions. The ratio of the sizes of the smaller oral spines and the larger aboral ones was 1.7:1.0. Also, the excretory pore is radially wrinkled and dorsally subterminal. Images obtained by scanning electron microscopy confirmed the characters that differentiate the new species and the most closely related species.

Argentophilic staining and scanning electron microscopy were used to study the tegumentary papillae of Echinostoma caproni cercariae. The most abundant tegumentary papillae were uniciliate, but multiciliate papillae were also found, mainly on the ventral aspect of the oral collar. The distribution pattern of the papillae on the body and tail was in general similar to that seen in the cercariae of other 37-collar-spined Echinostoma species. Some differences were noted between E. caproni and the allopatric species, E. trivolvis. E. caproni has a greater number of papillae associated with the collar spines than does E. trivolvis. E. caproni has uniciliate papillae on the acetabulum, whereas E. trivolvis does not. Chaetotaxy is useful to distinguish subtle morphological differences in cercarial species in the 37-collar-spined Echinostoma complex.

In order to investigate the relationships within the Echinostomatidae two data sets of gene sequences were analysed. The first consisted of all previously published ND1 sequences (20) together with 17 new sequences. The latter represented six species from the cosmopolitan genera Echinostoma, Echinoparyphium, Hypoderaeum and Isthmiophora. The second data-set of ITS sequences again included all previously published sequences (12) and three new sequences from species of Echinostoma, Echinoparyphium and Isthmiophora. All new isolates, as well as voucher material from five previously sequenced isolates, were identified on the basis of morphological characters. The phylogenetic trees inferred from the ND1 data set helped to clarify the generic affiliation of all isolates and confirmed the morphological identifications. The only exception was Echinoparyphium aconiatum, whose current position in the genus Echinoparyphium was not supported by the sequence data. Although the ITS data provided insufficient resolution for an unequivocal solution to the relationships within the genus Echinostoma, it supported the identification of Echinoparyphium ellisi and the distinct species status of three isolates of Echinostoma revolutum as predicted from the ND1 data.

Thin layer chromatography (TLC) was used to analyze the amino acids in worm incubates isotonic and hypotonic to the intestinal habitat of adult Echinostoma caproni and to analyze the free pool amino acids of these trematodes after incubation. Qualitative analysis revealed the presence of histidine, lysine, alanine, and proline in all samples of incubate and worm tissue. Quantification of histidine and lysine by TLC with densitometry gave mean concentrations of 24.1 micro g histidine/g worm per ml incubate in Locke's solution and 195.0 micro g lysine/g worm per ml incubate in deioinized (DI) water. Quantification of histidine and lysine in the worm tissue gave mean weight percents of 0.0587 and 0.0263, respectively, in worms incubated in Locke's solution and 0.0229 and 0.0163, respectively, for worms incubated in DI water. Our findings suggest that E. caproni adults may leak amino acids for osmoregulation in hypotonic environments.

Echinostoma paraensei Lie and Basch, 1967 (Echinostomatidae:Platyhelminthes), a 37 collar spine echinostome of the "revolutum group", has been used extensively as a model organism to study the interactions of digenetic trematodes with both their snail and vertebrate hosts. This worm was first isolated from the snail Biomphalaria glabrata from Belo Horizonte (BH isolate), Minas Gerais State, Brazil, by Lie and Basch [J Parasitol (1967) 53:1192-1199]. The natural definitive host for the BH isolate was never determined, and it has been maintained in the laboratory since 1967 in B. glabrata and hamsters. In this study, using light and scanning electron microscopy and molecular analysis, we describe an echinostome recently obtained from its natural vertebrate host, the wild rodent Nectomys squamipes (Rodentia: Sigmodontinae) from Sumidouro, Rio de Janeiro State, Brazil (RJ isolate). This echinostome was also compared to the laboratory-maintained BH isolate of E. paraensei. We observed that adult worms of both BH and RJ isolates could be differentiated from other echinostome species by the relatively small size of the dorsal collar spines relative to lateral and corner collar spines. SEM confirmed the similarity of this morphological character between the two isolates. As additional diagnostic features, the tegumentary spines are scale-like and the region between the genital pore and the acetabulum lacks scales. There is a folded protuberance with a pore just posterior to the genital pore. The tegument of the acetabulum is unspined and radially wrinkled, and there are numerous randomly distributed small, domed, ciliated papillae. The sequences of the internal transcribed spacers of the nuclear rDNA complex of the RJ and BH isolates are identical. Together these shared features provide strong evidence that both isolates are the same and can be referred to as E. paraensei. In conclusion, we have identified, for the first time, one of the natural definitive hosts for E. paraensei, the rodent N. squamipes, and have extended the known geographical distribution of this species to include Sumidouro in Rio de Janeiro State, Brazil.

The 37-collar-spined echinostomes Echinostoma paraensei, E. caproni, and E. trivolvis are digeneans that live in the intestine of small mammals and birds. Comparative studies of the eggs of these species were done using light microscopy (LM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). The egg of E. caproni was the largest of the three species studied, whereas the egg of E. trivolvis was the smallest in both length and width. The SEM study showed differences in the aboperculum region of the eggs in all three species. The TEM study showed that the eggshell of all three species consisted of three layers, but no difference in eggshell structure was observed in any species.

The morphology of the different stages and life-cycle of Echinostomna friedi n. sp. are described and figured. The freshwater snail Lymnaea peregra (Gastropoda: Lymnaeidae) serves as the natural and experimental first intermediate host and L. corvus and Gyraulus chinensis (Gastropoda: Planorbidae) as experimental first intermediate hosts. 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. Mice were not suitable experimental definitive hosts. E. friedi differs from the most closely related species in the 'revolutum' group mainly in terms of several morphological and biological features of the life-cycle stages and in its cercarial chaetotaxy. The chaetotaxy patterns of the species of the 'revolutum' group are analyzed and the results show that a taxonomic comparison of these species may be carried out on the basis of the number of sensilla in the clusters CIII VI, CIII V2 (or CIII V1 + CIII V2), CIV DL and UVb. These clusters appear adequate to establish taxonomic relationships between different species within the 'revolutum' group.

The life-cycle of Echinostoma miyagawai, a Eurasian species closely related to E. revolutum, was completed in the laboratory, and the morphology of the larval stages and the adults obtained experimentally was studied. Planorbis planorbis and Anisus vortex were the first intermediate hosts in the brackish Lake Durankulak on the Bulgarian Black Sea coast. Characteristic features of the cercaria include: a prominent collar with 37 spines; a tail as long as the body and with seven conspicuous fin-folds, the two ventral fin-folds being very close to each other; and a specific number and distribution of both the para-oesophageal gland-cell outlets and sensilla. The adult is characterised by: a very elongate body with a constriction at the posterior border of the ventral sucker; a large head collar with relatively small spines; a spherical ventral sucker which is only about half the maximum body width; a long cirrus-sac reaching posteriorly dorsal to the middle of the ventral sucker; indented subglobular testes; and a vitellarium forming two lateral fields of follicles which are almost confluent in the post-testicular space. The species described in this study resembles E. miyagawai, as described by Kosupko, in the morphology of larval stages and both the site and the general morphology of the adults. It differs from both E. revolutum, as described by both Kanev and Nasincová, and E. echinatum (also referred to as E. lindoense and E. barbosai by Kanev). The re-examination of Kanev's voucher specimens from his experimental studies used in his delimitation of E. revolutum and E. echinatum showed that the specimens identified by him as E. revolutum represent two distinct forms which consistently differ both from each other and from the redescription of E. revolutum which was based upon them. It also revealed that a number of specimens were wrongly identified and erroneously treated as E. echinatum by Kanev and co-workers; these include members of different genera (Hypoderaeum and Echinoparyphium) and an Echinostoma species of the group possessing 47 collar spines. The relative merits of the features used by Kanev and co-workers in discriminating the closely related Echinostoma spp. are discussed in detail with respect to the experimental evidence provided by these authors.

A comparative morphometric analysis was conducted on two European species of Echinostoma in order to examine the degree of the variability in the metrical characteristics of the adults and to assess their value in discriminating species. Adult E. miyagawai and E. revolutum, obtained experimentally, were compared using univariate and multivariate statistical analyses of 35 and 25 metrical characters, respectively. All subsets of worms of different ages represented homogeneous samples with respect to their morphometric characteristics; however, univariate analyses revealed significant differences in 22 and 23 variables between the corresponding age subsets of the two species, and it was found that the different allometric growth patterns contribute to this. The variables, body width at the posterior border of the ventral sucker, pharynx length and width, ovary length, testes length and width and length of the pre-ovarian region, exhibited isometric or positive allometric growth in E. miyagawai and negative allometry in E. revolutum. A cluster analysis based on 61 specimens and 25 variables separated E. revolutum and E. miyagawai unambiguously, producing an exact ordering of the specimens with respect to their identity and age. A forward stepwise discriminant analysis identified five variables (body width at the posterior border of ventral sucker, head collar width, length of oesophagus, width of ventral sucker and length of the pre-ovarian region) which yielded a 100% accurate classification of the two species. We suggest, therefore, that the morphometric characteristics of the adult worms should be used in studies attempting the identification of species or isolates of Echinostoma spp. More comparative data need to be gathered in order that the species boundaries within the 'revolutum' group be defined more accurately.

The chaetotaxy of cercaria of the echinostome Echinostoma miyagawai, found in naturally infected Planorbis planorbis in a brackish water lake in Bulgaria, is described and figured. Comparisons with sensory patterns of cercariae of E. revolutum, E. echinatum, Echinostoma sp.? echinatum, E. audyi, E. lindoense, E. trivolvis, E. caproni and E. jurini, described by Kanev et al. (1987), Lie (1966), Dimitrov et al. (1997), Richard (1971), Richard & Brygoo (1978) and Dimitrov & Kanev (1992), showed the distinct species status of E. miyagawai which exhibits a unique number and distribution of sensilla in 10 groups (CII4, CIV3, CIV4, CIVD, ventral A, M and P: dorso-lateral A and M, and lateral P). A comparison of the chaetotaxy of cercariae of E. revolutum and two descriptions of E. audyi revealed that the sensory pattern in E. revolutum was incompletely described and that the three descriptions differ in 9 of the 16 groups on the head collar and in all groups along the body which casts doubt on their synonymy (sensu Kanev, 1985, 1994). Comparisons of chaetotaxy of cercariae of E. echinatum and its synonym E. lindoense (sensu Kanev, 1985, 1994) showed differences in the CII4, CIII3, CIII4, CIV4, CIVD, ventral A and dorsal A and P groups, and indicated that incomplete and contradictory information has been provided for E. echinatum by Kanev et al. (1987). Examination of the descriptions of the sensilla in cercariae of E. jurini and E. caproni suggested that the two species are indistinguishable with respect to their chaetotaxy. Comparative analysis of the variability of a total of 27 groups of cercarial sensilla reveals three sets of characters which are not useful in discrimination between the species of the 'revolutum' group because they exhibited either constancy (CI1, CII1, CII2, CIV1, MD, PDL), intraspecific variability (CIII1, CIII2) or overlapping ranges (CII3, CIII3, CIV2, tail sensilla).

Gelatin substrate sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to analyze proteases in 14 day-old adults of Echinostoma caproni and Echinostoma trivolvis. At pH 8.0, E. caproni adults showed 2 protease bands at 36 kDa and 58 kDa, whereas E. trivolvis adults showed 6 bands at 39, 64, 77, 96, 120, and 168 kDa. Each species also showed distinct protease banding patterns in their excretory/secretory (E/S) products. The E. caproni E/S proteases were at 36 and 58 kDa, whereas those of E. trivolvis were at 120 and 168 kDa. Further characterization of E. caproni adult proteases revealed 2 bands (58 and 66 kDa) with optimal activity at pH 3.0-4.5 and 3 bands (38, 61, and 96 kDa) that were most active at pH 7.0-8.0. Four low molecular weight bands (19, 21, 25, and 30 kDa) appeared when E. caproni worm extracts were incubated in the presence of CaCl2 at pH 8.0 but were inhibited with ethylenediaminetetraacetic acid and 1,10-phenanthroline. Echinostoma caproni protease bands at 58 and 38 kDa in the whole worm samples and the E/S products and the 36-kDa band in the whole worm samples were inhibited with phenylmethylsulfonyl fluoride. By showing protease differences in addition to recent work on nucleotide differences, this study helps distinguish these 2 related allopatric species of 37-collar-spined Echinostoma.

The recent finding of the 37-collar-spined Echinostoma revolutum in North America prompted rDNA nucleotide sequence comparisons between this worm and the sympatric Echinostoma trivolvis. Three isolates of E. revolutum from distinct sites and 2 isolates of E. trivolvis collected from a single site were used in this analysis. Sequence data were compared to those from previously sequenced members of the 37-collar-spine group. The 3 North American isolates of E. revolutum were found to be identical, but they differed from Eurasian isolates of E. revolutum at 9 of the 1,006 sites sequenced. Further, 1 of the E. trivolvis isolates studied herein was identical to the published sequence for this species, but 6 nucleotide changes were observed in the second E. trivolvis isolate. Restriction fragment length polymorphisms at this locus support the nucleotide differences found between the E. trivolvis isolates. The degree of intraspecific variation detected raises questions regarding the utility of the internal-transcribed spacer regions of the ribosomal DNA repeat for taxonomic diagnosis and in phylogenetic studies for poorly differentiated groups, such as the 37-collar-spined congeners.

Cryptic species, belonging to the 37 collar-spine Echinostoma group, were distinguished using nuclear rDNA ITS (884 bases) and mtDNA CO1 (257 bases) and ND1 (530 bases) sequences. Sequences were obtained from five 37 collar-spine species, Echinostoma trivolvis, E. paraensei, E. caproni, E. revolutum and E. sp.I, a parthenogenetic isolate from Africa. Three geographic isolates of E. caproni were compared. Average sequence divergence among the 37 collar-spine species range from 2.2% in the rDNA ITS through 8% for the CO1 and 14% for the ND1. In addition, genes were sequenced from 2 non 37 collar-spine species, E. hortense and an undescribed Australian species, E. sp. (Aus). For each gene, distances of terminals from a predicted ancestral sequence were calculated. These indicated that ND1 is diverging significantly faster than the other 2 regions. In the CO1 gene most substitutions are synonymous and saturation has been reached for the majority of pairwise comparisons. The ND1 gene exhibits greater pairwise divergence but less evidence of saturation due to weaker conservation of first and second codon positions. The ITS has no amino acid coding constraints and displays no evidence of saturation. Although all 3 regions successfully distinguished the nominal species, ND1 appears to be the most informative region for investigating relationships within the 37 collar-spine group.

Echinostomes were collected in Australia and New Zealand as cercariae, metacercariae or adults. Using DNA sequences from the mitochondrial ND1 gene Echinostoma revolutum and Echinostoma paraensei were discovered in Australia. The presence of a further five, as yet unidentified, echinostome species was inferred in Northern Australia and a further isolate, closely allied to E. revolutum, occurs in New Zealand. ND1 sequences of species within the genus diverge from each other by 9.6-30.8%. Sequence divergence levels among strains within a single species are 0-3.6%. The phylogenetic tree produced from the Australasian isolates, in addition to species described previously, identifies the 37-collar-spine species as a well supported monophyletic group. The five unidentified Australian species cluster away from the 37-collar-spine group. These unidentified species appear to divide further into > 37-collar-spine and < 37-collar-spine clusters. Three strains of E. revolutum, collected as metacercariae from snails, were identified from two ponds located 6 km apart. Two of these strains may be cycling through a planorbid snail, Glyptophysa sp., as first intermediate host; however, this hypothesis could not be confirmed as specimens could not be obtained to match sequences between larvae and adults.

Lymnaea elodes snails collected in northern Indiana, U.S.A., were infected with larval stages of an echinostome bearing 37 collar spines and resembling members of the Echinostoma group. The taxonomic status of this digenean was determined through experimental infections of various definitive and first-intermediate hosts. In addition, characteristics of the penetration and paraesophageal glands in cercariae from this echinostomatid were compared with those from E. revolutum and E. trivolvis. Results indicate that this recently discovered 37-collar-spined echinostome parasitizing lymnaeid snails is E. revolutum, making this the first clear report of this trematode in North America.

The taxonomic history of members of the 37-collar-spine group within the genus. Echinostoma has been very confused. We obtained DNA sequence data from the nuclear rDNA ITS1, 5.8S and ITS2 of 7 nominal species belonging to this group, Echinostoma trivolvis (Cort, 1914), E. revolutum (Frölich, 1802), E. caproni Richard, 1964, E. liei Jeyarasasingam et al. 1972, E. paransei Lie & Basch, 1967, two African isolates, E. sp.I and E. sp.II, and of one 28-collar-spined echinostome, E. hortense (Asada, 1926). Five of the eight species were clearly distinguishable using ITS data. Sequences from the remaining three taxa, E. caproni, E. sp.II and E. liei were identical to one another and the group containing these taxa was distant from other 37-collar-spine species on a phylogenetic tree. E. trivolvis and E. paraensei form a second, but less distinct group within the 37-collar-spine group. The resolution obtained using DNA sequencing will assist in the current reclassification of the group. It also provides a model for future work on sibling species.

Adults of Echinostoma caproni, E. paraensei, and E. trivolvis were processed for starchgel electrophoresis. Ten enzyme systems representing 12 structural loci were examined using three different buffer systems. E. paraensei and E. caproni were found to be genetically inbred as indicated by the lack of heterozygosity in individual worms. All three taxa showed fixed differences indicating they are distinct species. Fixed differences were found between E. paraensei and E. caproni in six enzyme systems, between E. paraensei and E. trivolvis in five enzyme systems, and between E. trivolvis and E. caproni in five enzyme systems. Phenic relationships among the three species showed E. caproni was genetically more similar to E. trivolvis than to E. paraensei.