Development and utilization of a visual loop-mediated isothermal amplification coupled with a lateral flow dipstick (LAMP-LFD) assay for rapid detection of Echinostomatidae metacercaria in edible snail samples.

Trematodes belonging to the family Echinostomatidae are food-borne parasites which cause echinostomiasis in animals and humans. This is a global public health issue, particularly in East and Southeast Asia. A method to detect the infective stage of Echinostomatidae species is required to prevent transmission to humans. In this study, a loop-mediated isothermal amplification coupled with a lateral flow dipstick (LAMP-LFD) assay was developed for visual detection of the metacercarial stage in edible snails of the genus Filopaludina from local markets in Thailand. The LAMP-LFD method can be performed within 70 min at a consistent temperature of 66 °C, and the results can be interpreted with the naked eye. The detection limits of the assay using Echinostoma mekongi, E. macrorchis, E. miyagawai and Hypoderaeum conoideum genomic DNA were equal between the four species at 50 pg/µL. A specificity evaluation demonstrated that the LAMP-LFD assay had no cross-reaction with another parasite (Thapariella species) or with the snail host species (Filopaludina martensi martensi, F. sumatrensis speciosa, and F. s. polygramma). Clinical test assessments were compared to microscopic examination in 110 edible snail samples. The clinical sensitivity and specificity of the tests were 84.62 % and 100 %, respectively, with a strong level of agreement based on the kappa statistic and the results of both methods were not significantly different (p > 0.05) per McNemar's test. The test successfully developed in this study may be useful for the detection of the metacercarial stage in edible snails for epidemiological investigations, control, surveillance, and to prevent future echinostomiasis health issues.

Echinostoma chankensis nom. nov., other Echinostoma spp. and Isthmiophora hortensis in East Asia: morphology, molecular data and phylogeny within Echinostomatidae.

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.

Echinostoma miyagawai Ishii, 1932 (Echinostomatidae) from Ducks in Aceh Province, Indonesia with Special Reference to Its Synonymy with Echinostoma robustum Yamaguti, 1935.

Adult echinostomes having 37 collar spines collected from the intestine of Pitalah ducks in Aceh Province, Indonesia in 2018 were morphologically and molecularly determined to be Echinostoma miyagawai Ishii, 1932 (Digenea: Echinostomatidae). Among 20 ducks examined, 7 (35.0%) were found to be infected with this echinostome, and the number of flukes collected was 48 in total with average 6.9 (1-17) worms per duck. The adult flukes were 7.2 (6.1-8.5) mm in length and 1.2 (1.0-1.4) mm in width (pre-ovarian or testicular level) and characterized by having a head collar armed with 37 collar spines (dorsal spines arranged in 2 alternating rows), including 5 end group spines, and variable morphology of the testes, irregularly or deeply lobed (3-5 lobes) at times with horizontal extension. The eggs within the worm uterus were 93 (79-105) µm long and 62 (56-70) µm wide. These morphological features were consistent with both E. miyagawai and Echinostoma robustum, for which synonymy to each other has been raised. Sequencing of 2 mitochondrial genes, cox1 and nad1, revealed high homology with E. miyagawai (98.6-100% for cox1 and 99.0-99.8% for nad1) and also with E. robustum (99.3-99.8% for nad1) deposited in GenBank. We accepted the synonymy between the 2 species and diagnosed our flukes as E. miyagawai (syn. E. robustum) with redescription of its morphology. Further studies are required to determine the biological characteristics of E. miyagawai in Aceh Province, Indonesia, including the intermediate host and larval stage information.

Comparative mitogenomics of the zoonotic parasite Echinostoma revolutum resolves taxonomic relationships within the 'E. revolutum' species group and the Echinostomata (Platyhelminthes: Digenea).

The complete mitochondrial sequence of 17,030 bp was obtained from Echinostoma revolutum and characterized with those of previously reported members of the superfamily Echinostomatoidea, i.e. six echinostomatids, one echinochasmid, five fasciolids, one himasthlid, and two cyclocoelids. Relationship within suborders and between superfamilies, such as Echinostomata, Pronocephalata, Troglotremata, Opisthorchiata, and Xiphiditata, are also considered. It contained 12 protein-coding, two ribosomal RNA, 22 transfer RNA genes and a tandem repetitive consisting non-coding region (NCR). The gene order, one way-positive transcription, the absence of atp8 and the overlapped region by 40 bp between nad4L and nad4 genes were similar as in common trematodes. The NCR located between tRNAGlu (trnE) and cox3 contained 11 long (LRUs) and short repeat units (SRUs) (seven LRUs of 317 bp, four SRUs of 207 bp each), and an internal spacer sequence between LRU7 and SRU4 specifying high-level polymorphism. Special DHU-arm missing tRNAs for Serine were found for both tRNAS1(AGN) and tRNAS2(UCN). Echinostoma revolutum indicated the lowest divergence rate to E. miyagawai and the highest to Tracheophilus cymbius and Echinochasmus japonicus. The usage of ATG/GTG start and TAG/TAA stop codons, the AT composition bias, the negative AT-skewness, and the most for Phe/Leu/Val and the least for Arg/Asn/Asp codons were noted. Topology indicated the monophyletic position of E. revolutum to E. miyagawai. Monophyly of Echinostomatidae and Fasciolidae was clearly solved with respect to Echinochasmidae, Himasthlidae, and Cyclocoelidae which were rendered paraphyletic in the suborder Echinostomata.

First molecular identification and phylogenetic tree of Petasiger exaeretus Dietz, 1909 (Digenea: Echinostomatidae) from an intermediate host Radix auricularia (L., 1758) in Greater Zab river, Iraq.

INTRODUCTION: Radix auricularia (Linnaeus, 1758) is a freshwater gastropod belongs to the Lymnaeidae (pond snails) family which act as intermediate hosts or vectors of various parasitic flukes. No study has yet been undertaken on the prevalence of Petasiger spp. infection in R. auricularia. Species of Petasiger (Dietz, 1909) are a cosmopolitan parasite that utilize snails as the first intermediate host, with vertebrates like amphibians larvae and fish as the second intermediate host, followed by fish-eating birds. The current paper is considered to be the first report of Petasiger exaeretus parasitized R. auricularia in Iraq, which is supported with molecular and phylogenetic analysis. MATERIAL AND METHODS: Freshwater snails R. auricularia were collected during October 2016 - September 2017 from different locations of Sufaia village on the Greater Zab river, Erbil province, Iraq. RESULTS: A total of 307 freshwater snails R. auricularia were collected, only five of them were infected with a prevalence of Petasiger exaeretus (1.62%). CONCLUSIONS: The current study agrees with the opinion of Selbach, Soldánová (26), which suggested the possibility of a much higher morphological diversity within Petasiger species, based on the number of described cercariae, compared with adult forms.It is clear that P. phalacrocoracis specimens have often been erroneously designated as P. exaeretus by many authors (Nasincová et al., 1994). Certain morphological similarities and dissimilarities between P. exaeretus and P. phalacrocoracis can be detected: the pear-shaped body resembles P. exaeretus, whereas, P. phalacrocoracis have an elongated body.

Characterization of the complete mitochondrial genome of Uvitellina sp., representative of the family Cyclocoelidae and phylogenetic implications.

Mitochondrial (mt) DNA has been useful in revealing the phylogenetic relationship of eukaryotic organisms including flatworms. Therefore, the use of mitogenomic data for the comparative and phylogenetic purposes is needed for those families of digenetic trematodes for which the mitogenomic data are still missing. Molecular data with sufficiently rich informative characters that can better resolve species identification, discrimination, and membership in different genera is also required for members of some morphologically difficult families of trematodes bearing few autapomorphic characters among its members. Here, the internal transcribed spacer (ITS) region of nuclear ribosomal DNA (rDNA) and the complete mt genome of the trematode Uvitellina sp. (Cyclocoelidae: Haematotrephinae) was determined and annotated. The mt genome of this avian trematode is 14,217 bp in length, containing 36 genes plus a single non-coding region. The ITS rDNA sequences were used for the pairwise sequence comparison of Uvitellina sp. with European cyclocoelid species, and the mitochondrial 12 protein-coding genes (PCGs) and two ribosomal RNA genes were used to evaluate the position of the family within selected trematodes. The ITS rDNA analysis of Uvitellina sp. showed less nucleotide differences with Hyptiasmus oculeus (16.77%) than with other European cyclocoelids (18.63-23.58%). The Bayesian inference (BI) analysis using the 12 mt PCGs and two rRNA genes supported the placement of the family Cyclocoelidae within the superfamily Echinostomatoidea (Plagiorchiida: Echinostmata). The availability of the mt genome sequences of Uvitellina sp. provides a novel resource of molecular markers for phylogenetic studies of Cyclocoelidae and other trematodes.

Infections of Two Isthmiophora Species (Digenea: Echinostomatidae) in Wild Mammals from Republic of Korea with Their Morphological Descriptions.

Isthmiophora hortensis (Digenea: Echinostomatidae) is a dominant echinostome in animal reservoir hosts and humans in the Republic of Korea (Korea). We intended to investigate the infection status with this echinostome species in the several species of wild animals and describe the morphological characteristics in the faunistic view point. A total of 175 animal carcasses belonging to 3 families, i.e., Canidae, Felidae and Mustelidae, were collected from the southern regions of Korea from March 2010 to July 2017. Isthmiophora spp. worms were recovered from the small intestines of each animal under a stereomicroscope after washing of intestinal contents. Isthmiophora hortensis was recovered from 4 species of wild carnivores, i.e., Nyctereutes procyonoides (3/107: 2.8%), Mustela sibirica (11/31: 35.5%), Meles lucurus (2/3: 33.3%) and Martes flavigula (1/2: 50%). The other 3 carnivores comprising stray dogs, cat and leopard cat were negative for I. hortensis infection (0/2, 0/10 and 0/12, respectively). Specimens obtained from the Lutra lutra (6/8: 75%) were identified as a distinct species, I. inermis, by morphological comparison. Isthmiophora inermis has thinner body, elongate testes and different anterior limits of vitelline fields. Detailed morphological descriptions and comparisons with the morphological characteristics are provided. Conclusively, it was confirmed for the first time that 3 species of mustelid mammals, i.e., M. sibirica, M. lucurus, and M. flavigula, are to be the new definitive hosts of I. hortensis in Korea. Additionally, I. inermis is to be newly added in the Korean echinostome fauna.

Multilocus phylogenetic analysis and morphological data reveal a new species composition of the genus Drepanocephalus Dietz, 1909 (Digenea: Echinostomatidae), parasites of fish-eating birds in the Americas.

Members of the genus Drepanocephalus are endoparasites of fish-eating birds of the families Phalacrocoracidae and Sulidae distributed across the Americas. Currently, Drepanocephalus contains three species, i.e. D. spathans (type species), D. olivaceus and D. auritus. Two additional species, D. parvicephalus and D. mexicanus were transferred to the genus Petasiger. In the current study, available DNA sequences of D. spathans, D. auritus and Drepanocephalus sp., were aligned with newly generated sequences of D. spathans and Petasiger mexicanus. Phylogenetic analyses inferred with three nuclear (LSU, SSU and ITS1, 5.8S, ITS2) and two mitochondrial (cox1, nad1) molecular markers showed that the sequences of D. spathans and D. auritus are nested together in a single clade with very low genetic divergence, with Petasiger mexicanus as its sister species. Additionally, P. mexicanus was not a close relative of other members of the genus Petasiger, showing that P. mexicanus actually belongs to the genus Drepanocephalus, suggesting the need to re-allocate Petasiger mexicanus back into the genus Drepanocephalus, as D. mexicanus. Morphological observations of the newly sampled individuals of D. spathans showed that the position of the testes is variable and testes might be contiguous or widely separated, which is one of the main diagnostic traits for D. auritus. Our results suggest that D. auritus might be considered a synonym of D. spathans and, as a result, the latter represents a species with a wide geographic range across the Americas, parasitizing both the Neotropical and the double-crested cormorant in Argentina, Brazil, Paraguay, Venezuela, Colombia, Mexico, USA and Canada.

New Definitive Hosts and Differential Body Indices of Isthmiophora hortensis (Digenea: Echinostomatidae).

The present study was performed to record new definitive hosts of Isthmiophora hortensis, and to describe morphological characteristics derived from a variety of worm samples for clarification of its taxonomic validity. Morphological characteristics with dimensions were observed in worm samples (n=21) from naturally infected wild animals, including a raccoon dog Nyctereutes procyonoides from Gimhae-si (City), Gyeongsangnam-do, stray cats and a striped field mouse from several localities, and a wild boar Sus scrofa, from Gurye-gun (County), Jeollanam-do. In addition, adult flukes (n=45) recovered in albino rats experimentally infected with the metacercariae from a freshwater fish species were also subjected to morphological studies. The mean ratios of the body length (BL) to body width (BW) were 5.86 and 5.76 in worms from wild animals and experimental rats, respectively. Those of the ventral sucker to oral sucker were 2.92 and 3.01 in worms from 2 groups. The mean percentages of the hindbody length (HBL) to BL were 42.1 and 41.2 in 2 groups. Those of uterine fields to BL were 9.8 and 12.2 in the 2 worm groups. By the present study, the 2 species of wild animals, the raccoon dog and wild boar, have been added as new definitive hosts for I. hortensis. The morphological characteristics of adult flukes derived from a variety of host source were redescribed to support the taxonomic validity of this echinostome species.

Occurrence of Paratanaisia confusa Freitas, 1951 in free-living guira cuckoo (Guira guira, Cuculiformes: Crotophagidae).

This report presents a description of natural infection of an adult free-living guira cuckoo with Paratanaisia confusa. Histological and morphological evaluations of renal and parasite samples were performed. The morphological analysis of the parasites revealed spiny tegument, characteristic of P. confusa helminthes. Although macroscopic alterations were absent in the kidneys, the histopathology revealed parasites inside the collecting ducts causing dilatation and destruction of the lining epithelial cells in addition to small focal inflammatory infiltrates in the renal parenchyma. However, as the bird was free-living and naturally infected it was not possible to confirm if all these alterations were directly caused by the parasite presence or if they were related to causes other than the parasitism. Nonetheless, such findings indicate that these trematodes may have pathogenic potential in this host.

Occurrence of Paratanaisia confusa Freitas, 1951 in free-living guira cuckoo (Guira guira, Cuculiformes: Crotophagidae) / Ocorrência de Paratanaisia confusa Freitas, 1951 em anu-branco de vida livre (Guira guira, Cuculiformes: Crotophagidae)

Abstract This report presents a description of natural infection of an adult free-living guira cuckoo with Paratanaisia confusa. Histological and morphological evaluations of renal and parasite samples were performed. The morphological analysis of the parasites revealed spiny tegument, characteristic of P. confusa helminthes. Although macroscopic alterations were absent in the kidneys, the histopathology revealed parasites inside the collecting ducts causing dilatation and destruction of the lining epithelial cells in addition to small focal inflammatory infiltrates in the renal parenchyma. However, as the bird was free-living and naturally infected it was not possible to confirm if all these alterations were directly caused by the parasite presence or if they were related to causes other than the parasitism. Nonetheless, such findings indicate that these trematodes may have pathogenic potential in this host.

Resumo A infecção de um anu-branco, de vida livre, por Paratanaisia confusa é descrita. Avaliações histológicas e morfológicas de amostras renais e dos parasitas foram realizadas. A análise morfológica dos parasitas revelou a presença de tegumento espinhoso, que é característico de P. confusa. Embora alterações renais macroscópicas estivessem ausentes, a histopatologia revelou a presença de parasitas no interior de ductos coletores, provocando dilatação destes e destruição das células epiteliais, além de discretos infiltrados inflamatórios focais no parênquima renal. Entretanto, como a ave era proveniente de vida livre e naturalmente infectada, não foi possível confirmar se todas as alterações observadas foram diretamente provocadas pela presença dos parasitas, ou se estavam relacionadas a outras causas. Todavia, esses achados indicam que os trematódeos dessa espécie possam ter potencial patogênico no hospedeiro estudado.

Molecular characterisation of four echinostomes (Digenea: Echinostomatidae) from birds in New Zealand, with descriptions of Echinostoma novaezealandense n. sp. and Echinoparyphium poulini n. sp.

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.

Trematodes with a reproductive division of labour: heterophyids also have a soldier caste and early infections reveal how colonies become structured.

Recent findings have extended the documentation of complex sociality to the Platyhelminthes, describing the existence of a reproductive division of labour involving a soldier caste among the parthenitae of trematode parasites. However, all species examined to date occupy high positions in trematode interspecific dominance hierarchies and belong to two closely related families, the Echinostomatidae and the Philophthalmidae (Superfamily Echinostomatoidea). Further, the two species documented as lacking soldiers also belong to the Echinostomatidae. Here, we examine four species of intermediate dominance, all belonging to the family Heterophyidae (Superfamily Opisthorchioidea): Euhaplorchis californiensis, Phocitremoides ovale, Pygidiopsoides spindalis and Stictodora hancocki, all of which infect the California horn snail, Cerithideopsis californica (=Cerithidea californica). We quantify morphology, distribution and behaviour of rediae from fully developed colonies. We also provide information on colony structure for three developing heterophyid colonies to better understand colony development. We discuss the implications of our findings, particularly with respect to how they suggest alternatives to the conclusions of other researchers concerning the nature of trematode sociality. Our analyses of morphological, distributional and behavioural patterns of developed colonies indicate that these heterophyid trematodes have a non-reproductive caste whose function is defence of the colony from invading trematodes. Hence, a soldier caste occurs for species lower in dominance hierarchies than previously known, and is present in at least two superfamilies of digenean trematodes, suggesting that selection for a soldier caste may be much more common among the Trematoda than previously recognised.

Stephanoprora chasanensis n. sp. (Digenea: Echinochasmidae): Morphology, life cycle, and molecular data.

Echinostomatoidea is a large, globally distributed and heterogeneous group of hermaphroditic digeneans that parasite, as adults, vertebrate hosts of all classes. Species of this group have received attention from researchers as they can cause diseases in wildlife and humans. Here we describe the biological and molecular phylogenetic characteristics of Stephanoprora chasanensis n. sp. (Digenea: Echinochasmidae). The life cycle of this fluke was experimentally completed by the use of hosts, i.e. Stenothyra recondite Lindholm, 1929 snail (the 1st intermediate), Rhynchocypris percnurus mantschuricus (Berg, 1907) freshwater fish (the 2nd intermediate) and Gallus gallus chicken (the definitive host). In the adult worms, vitelline follicles were distributed anteriorly to the mid-level of the ventral sucker in our specimens whereas they did not reach the level of anterior testis in other species of Stephanoprora previously reported. Phylogenetic analysis based on 28S rDNA revealed that Stephanoprora and Echinochasmus with 20-22 collar spines grouped together in a single cluster. In addition, we showed that Stephanoprora chasanensis n. sp. was closely related to Echinochasmus milvi Yamaguti, 1939. Cercariae of these two echinostomes commonly have a long tail.

A Case of Chaunocephalosis by Chaunocephalus ferox (Digenea: Echinostomatidae) in an Oriental White Stork, Ciconia boyciana, in Korea.

We intended to describe a case of chaunocephalosis and morphological characteristics of its causative agent, Chaunocephalus ferox, recovered from an oriental white stork, Ciconia boyciana, in the Republic of Korea. An oriental white stork was referred to the Wildlife Center of Chungbuk in Korea in February 2014 for severe depression with cachexia and it died the next day. At necropsy, the stomach was severely expanded and 7 thick-walled nodules were observed in the upper part of the intestine. Although the stomach was filled with full of foreign materials, the intestine was almost empty. The nodules were globular and total 9 flukes were recovered. They were 8,030-8,091 µm in length and 3,318-3,333 µm in maximum width. Because the flukes had bulbous forebody with short narrow subcylindrical hindbody, 27 collar spines, and vitelline follicles not reaching to the posterior end, the specimens were identified as being C. ferox. The cyst formation induced thickening of the intestinal wall with narrowing of the lumen that could have contributed to the gastric impaction to the death of the host. This is the first described case of chaunocephalosis and its causative agent C. ferox found from an oriental white stork in Korea.

Genomic diversity of cercarial clones of Himasthla elongata (Trematoda, Echinostomatidae) determined with AFLP technique.

The aim of this study was to reveal genomic diversity formed during parthenogenetic reproduction of rediae of the trematode Himasthla elongata in its molluskan host Littorina littorea. We applied amplification fragment length polymorphism (AFLP) to determine the genomic diversity of individual cercariae within the clone, that is, the infrapopulation of parthenogenetic progeny in a single molluskan host. The level of genomic diversity of particular cercariae isolates from a single clone, detected with EcoR1/Mse1 AFLP reaction, was significantly lower than the variability of cercariae from different clones. The presence of intraclonal genomic diversity indicates a nonsexual shuffle of alleles during parthenogenesis in the rediae of H. elongata. The obtained polymorphic AFLP fragments were long enough to detect the sequences that may be responsible for clonal genomic variability. Based on this, AFLP can be recommended as a tool for the study of genetic mechanisms of this variability.

Two Echinostome Species, Pegosomum bubulcum and Nephrostomum ramosum (Digenea: Echinostomatidae), from an Eastern Cattle Egret, Bubulcus ibis coromandus, in Republic of Korea.

We describe 2 echinostome species recovered from an Eastern cattle egret, Bubulcus ibis coromandus, from Cheongju-si (city), Chungcheongbuk-do (province), Korea. Total 72 Pegosomum bubulcum specimens were recovered from the bile duct. They were 7,566×2,938 µm in average size and had 27 collar spines with vitelline extension from anterior 1/3 level of the esophagus to mid-level of the posterior testis as characteristic features. Total 9 specimens of Nephrostomum ramosum were recovered in the small intestines of the bird. They were ribbon-shaped, 11,378×2,124 µm in average size, and morphologically variable in some organs, i.e., the number of collar spines (47-50), the shape of ovary and testes, and the extension of vitelline follicles. These morphological variations observed in a single host indicated that these features are not critical for the classification of Nephrostomum species and thus were reconsidered taxonomically as synonym of N. ramosum. This study is the first report documenting and describing both flukes and their associated genera in Korea.

Molecular phylogeny and systematics of the Echinostomatoidea Looss, 1899 (Platyhelminthes: Digenea).

The Echinostomatoidea is a large, cosmopolitan group of digeneans currently including nine families and 105 genera, the vast majority parasitic, as adults, in birds with relatively few taxa parasitising mammals, reptiles and, exceptionally, fish. Despite the complex structure, diverse content and substantial species richness of the group, almost no attempt has been made to elucidate its phylogenetic relationships at the suprageneric level based on molecules due to the lack of data. Herein, we evaluate the consistency of the present morphology-based classification system of the Echinostomatoidea with the phylogenetic relationships of its members based on partial sequences of the nuclear lsrRNA gene for a broad diversity of taxa (80 species, representing eight families and 40 genera), including representatives of five subfamilies of the Echinostomatidae, which currently exhibits the most complex taxonomic structure within the superfamily. This first comprehensive phylogeny for the Echinostomatoidea challenged the current systematic framework based on comparative morphology. A morphology-based evaluation of this new molecular framework resulted in a number of systematic and nomenclatural changes consistent with the phylogenetic estimates of the generic and suprageneric boundaries and a new phylogeny-based classification of the Echinostomatoidea. In the current systematic treatment: (i) the rank of two family level lineages, the former Himasthlinae and Echinochasminae, is elevated to full family status; (ii) Caballerotrema is distinguished at the family level; (iii) the content and diagnosis of the Echinostomatidae (sensu stricto) (s. str.) are revised to reflect its phylogeny, resulting in the abolition of the Nephrostominae and Chaunocephalinae as synonyms of the Echinostomatidae (s. str.); (iv) Artyfechinostomum, Cathaemasia, Rhopalias and Ribeiroia are re-allocated within the Echinostomatidae (s. str.), resulting in the abolition of the Cathaemasiidae, Rhopaliidae and Ribeiroiinae, which become synonyms of the Echinostomatidae (s. str.); and (v) refinements of the generic boundaries within the Echinostomatidae (s. str.), Psilostomidae and Fasciolidae are made.

Biomphalaria straminea (Mollusca: Planorbidae) as an intermediate host of Drepanocephalus spp. (Trematoda: Echinostomatidae) in Brazil: a morphological and molecular study.

Species of trematodes belonging to the genus Drepanocephalus are intestinal parasites of piscivorous birds, primarily cormorants (Phalachrocorax spp.), and are widely reported in the Americas. During a 4-year malacological study conducted on an urban lake in Brazil, 27-collar-spined echinostome cercariae were found in 1665/15,459 (10.7 %) specimens of Biomphalaria straminea collected. The cercariae were identified as Drepanocephalus spp. by sequencing the 18S (SSU) rDNA, ITS1/5.8S rDNA/ITS2 (ITS), 28S (LSU) rDNA region, cytochrome oxidase subunit 1 (CO1), and nicotinamide adenine dinucleotide dehydrogenase subunit 1 (ND1) markers. In experimental life cycle studies, metacercariae developed in laboratory-reared guppies (Poecilia reticulata); however, attempts to infect birds and rodents were unsuccessful. Two closely related morphotypes of cercariae were characterized. One species, identified by molecular markers as a genetic variant of Drepanocephalus auritus (99.9 % similarity at SSU, ITS, LSU; 97.2 % at CO1; 95.8 % at ND1), differs slightly from an archived North American isolate of this species also sequenced as part of this study. A second species, putatively identified as Drepanocephalus sp., has smaller cercariae and demonstrates significant differences from D. auritus at the CO1 (11.0 %) and ND1 (13.6 %) markers. Aspects related to the morphological taxonomic identification of 27-collar-spined echinostome metacercariae are briefly discussed. This is the first report of the involvement of molluscs of the genus Biomphalaria in the transmission of Drepanocephalus and the first report of D. auritus in South America.

Host-dependent morphology of Isthmiophora melis (Schrank, 1788) Luhe, 1909 (Digenea, Echinostomatinae)--morphological variation vs. molecular stability.

BACKGROUND: Echinostomes are cosmopolitan digenean parasites which infect many different warm-blooded hosts. Their classification is extremely confused; the host spectrum is wide, and morphological similarities often result in misidentification. During our long-term studies on the helminth fauna of rodents and carnivores we have collected 27 collar-spined echinostomes which differ in morphology to an extent that suggests the presence of more than one species. Here, we describe this material, and the extent of host-related variation in this parasite. METHODS: Specimens of Isthmiophora isolated from four host species (badger, American mink, hedgehog, striped field mouse) were subject to morphological and molecular examination; the data were statistically analysed. RESULTS: Our results show that genetically all the Isthmiophora specimens obtained from all the examined hosts are conspecific and represent I. melis. On the other hand, the individuals isolated from Apodemus agrarius are morphologically distinct and, based on this criterion alone, should be described as a new species. CONCLUSIONS: The morphological traits of Isthmiophora melis are much variable and host-dependent; without molecular analysis they would suggest a necessity to describe a new species or even genus. Such a high level of intraspecific variability may be affected by the host's longevity.