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.

Echinochasmus caninus n. comb. (Trematoda: Echinostomatidae) Infection in Eleven Riparian People in Khammouane Province, Lao PDR.

Adult specimens of Echinochasmus caninus n. comb. (Verma, 1935) (Trematoda: Echinostomatidae) (syn. Episthmium caninum Yamaguti, 1958) were recovered from 11 riparian people who resided along the Mekong River in Khammouane Province, Lao PDR. In fecal examinations done by the Kato-Katz technique, the cases revealed eggs of Opisthorchis viverrini/minute intestinal flukes, hookworms, and in 2 cases echinostome eggs. To recover the adult helminths, praziquantel 30-40 mg/kg and pyrantel pamoate 10-15 mg/kg in a single dose were given and purged with magnesium salts. Various species of trematodes (including O. viverrini and Haplorchis spp.), cestodes, and nematodes were recovered from their diarrheic stools. Among the trematodes, small echinostome flukes (n=42; av. 3.8 specimens per case) of 0.7-1.2 mm in length are subjected in this study. They are morphologically characterized by having 24 collar spines interrupted dorsally and anterior extension of vitellaria from the cirrus sac or genital pore level to the posterior end of the body. Particularly based on this extensive distribution of vitellaria, the specific diagnosis was made as Echinochasmus caninus. The cases were co-infected with various other helminth parasites; thus, clinical manifestations specific for this echinostome infection were difficult to determine. The present paper describes for the first time human E. caninus infections in Lao PDR. Our cases marked the 4-14th human infections with this echinostome around the world following the 3 previous cases reported from Thailand.

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.

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.

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.

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.

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.

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.

Morphological and ITS2 Molecular Characterization of Ribeiroia Cercariae (Digenea: Psilostomidae) from Biomphalaria spp. (Gastropoda: Planorbidae) in Northern Argentina.

Species of Ribeiroia use planorbid snails as intermediate host. Since there is little information about these digenean parasites in South America, we aimed to assess whether Ribeiroia cercariae from 3 north Argentina locations belonged to the same species and differed from Ribeiroia cercariae described elsewhere. Specimens were obtained from Biomphalaria tenagophila and Biomphalaria orbignyi (Salta Province), and Biomphalaria occidentalis (Corrientes Province). Morphological traits of cercariae were analyzed, as well as their sequence of the ribosomal internal transcribed spacer 2 (ITS2). The ITS2 region consisted of 426 nucleotides identical in all samples, suggesting that all specimens belong to the same species in spite of their morphological differences and first intermediate host species. Comparison of the ITS2 region with GenBank database records showed that specimens from Argentina were different from Ribeiroia ondatrae (0.9% divergence), Ribeiroia marini (0.7% divergence), and Cercaria lileta (0.2% divergence). In summary, morphological, ecological, and ITS2 molecular data suggest that specimens from Argentina belong to a different species.

Surface topography of two trematodes parasites infecting grey heron Ardea cinerea Jouyi (Aves, Ciconiiformes) in Qena, Egypt.

Apharyngostrigea ardeolina and Echinoparyphium recurvatum are two important digenean parasites that were recovered from small intestine of grey heron with an infection rate (16.2%) and (8.8%) respectively. The surface topography of two species was redescribed by both light and scanning electron microscopy. Using SEM studies showed that the body surface of two trematodes were covered by contact receptors, several types of sensory tegumental papillae which may have useful function in orientation and feeding through increasing the surface area of absorption, could also play a role in sensation or in selection of the materials for ingestion by the fluke. The head collar of E. recurvatum is reniform in shape, bearing uninterrupted double row of 41 collar finger-like spines, a total including 4 end group ones on both ventral corners., tegumental spines were tongue-shaped without a terminal tip.

A new species of Drepanocephalus Dietz, 1909 (Digenea: Echinostomatidae) from the double-crested cormorant Phalacrocorax auritus (Lesson) (Aves: Phalacrocoracidae) in North America.

Drepanocephalus auritus n. sp. is described based on specimens from the double-crested cormorant Phalacrocorax auritus (Lesson) in North America. The new species differs from its congeners in its very narrow, elongate body, long uterine field and widely separated testes. Sequences of the nuclear rRNA gene cluster, spanning the 3' end of the nuclear ribosomal 18S rRNA gene, internal transcribed spacer region (ITS1+5.8S gene+ITS2) and partial 28S gene (2,345 bp), were identical in specimens collected from North Dakota, Minnesota and Mississippi, USA. Sequences of the 651 bp long fragment of the mitochondrial cox1 gene exhibited very low intraspecific variability (< 1%). Comparisons of the newly-generated sequences with those available in the GenBank indicate that the sequences from North America published under the name D. spathans Dietz, 1909 in fact represent D. auritus n. sp.

Morphological and molecular data for larval stages of four species of Petasiger Dietz, 1909 (Digenea: Echinostomatidae) with an updated key to the known cercariae from the Palaearctic.

Large-tailed echinostomatid cercariae of the genus Petasiger Dietz, 1909 (Digenea: Echinostomatidae) from the planorbid snails Gyraulus albus (Müller) and Planorbis planorbis (L.) collected in Germany and the Czech Republic and metacercariae from Gasterosteus aculeatus L. (Gasterosteiformes: Gasterosteidae) collected in Canada are characterised morphologically and molecularly. The rediae, cercariae and metacercariae are described in detail and compared with the existing data on the larval stages of Petasiger spp. Comparative molecular analyses using 28S rDNA and nad1 mitochondrial sequences supported the distinct status of four species of Petasiger. Molecular and morphological evidence for their distinction and an updated key to the known large-tailed cercariae of Petasiger from the Palaearctic are provided.

Description of rare echinostomatid fluke Echinodollfusia longiuscula n. sp. (Trematoda: Echinostomatidae) parasitizing white stork with an amended diagnosis of the genus Echinodollfusia.

A new species of digenean, Echinodollfusia longiuscula n. sp. (Trematoda: Echinostomatidae), is described and illustrated based on specimens collected from the intestine of the white stork Ciconia ciconia (L.) (Aves: Ciconiidae) from eastern Slovakia and dissected soon after its spring arrival from African wintering. The new species differs from the two existing congeners Echinodollfusia stenon (Dollfus, 1950) and Echinodollfusia bulgarica Vassilev (Sofia 6:327-338, 1958) clearly by the oesophagus bearing diverticula, the bipartite seminal vesicle and gonads situated entirely in anterior body half. The new species E. longiuscula n. sp. can also be distinguished by possessing the following combination of features: a slender body of 12.85-21.06-mm long and maximum of 581-855-µm wide; head collar with 43-47 small collar spines, dorsal in double row; oral sucker more than half the size as ventral sucker, small pharynx, oval and in tandem testes situated pre-equatorial, small ovary at the boundary of anterior third of the body and anteriormost extent of the vitelline fields just reaching the level of posterior margin of the ventral sucker. An amended generic diagnosis of little-known Echinodollfusia Skrjabin et Bashkirova (Osnovy Trematodologii 12:51-930, 1956) is also given here.

Zoonotic echinostome infections in free-grazing ducks in Thailand.

Free-grazing ducks play a major role in the rural economy of Eastern Asia in the form of egg and meat production. In Thailand, the geographical location, tropical climate conditions and wetland areas of the country are suitable for their husbandry. These environmental factors also favor growth, multiplication, development, survival, and spread of duck parasites. In this study, a total of 90 free-grazing ducks from northern, central, and northeastern regions of Thailand were examined for intestinal helminth parasites, with special emphasis on zoonotic echinostomes. Of these, 51 (56.7%) were infected by one or more species of zoonotic echinostomes, Echinostoma revolutum, Echinoparyphium recurvatum, and Hypoderaeum conoideum. Echinostomes found were identified using morphological criteria when possible. ITS2 sequences were used to identify juvenile and incomplete worms. The prevalence of infection was relatively high in each region, namely, north, central, and northeast region was 63.2%, 54.5%, and 55.3%, respectively. The intensity of infection ranged up to 49 worms/infected duck. Free-grazing ducks clearly play an important role in the life cycle maintenance, spread, and transmission of these medically important echinostomes in Thailand.

The biology of Echinoparyphium (Trematoda, Echinostomatidae).

Echinoparyphium species are common, widely distributed intestinal parasites causing disease in animals worldwide. Intermediate hosts include snails, bivalves, and fish, whereas the definitive hosts are mainly birds and mammals. This review examines the significant literature on Echinoparyphium. Descriptive studies, life cycle, experimental and manipulative studies, and biochemical and molecular studies are presented. The influence of environmental factors, and toxic pollutants, are reviewed as well as studies on the pathology of Echinoparyphium.

The life-cycle of Petasiger islandicus Kostadinova & Skirnisson, 2007 (Digenea: Echinostomatidae) elucidated with the aid of molecular data.

The small planorbid snail Gyraulus cf. laevis (Alder) from Lake Mývatn in Iceland was found to emit large-tailed cercariae with 19 collar spines, and three-spined sticklebacks Gasterosteus aculeatus L. were infected with metacercariae of a species of Petasiger Dietz, 1909. Comparative sequence analysis using ND1 mitochondrial DNA sequences revealed that the rediae and cercariae are conspecific with P. islandicus Kostadinova & Skirnisson, 2007, recently described from an isolated population of the horned grebe Podiceps auritus (L.) at the lake. The redia, cercaria and metacercaria are described and compared with related forms.

Revealing cryptic parasite diversity in a definitive host: echinostomes in muskrats.

Many trematode groups have a long history of systematic revision, which can make parasite identification a difficult task. The trematode parasites of muskrats are no exception. Here, we highlight the systematic issues associated with trematodes of muskrats (Ondatra zibethicus). Then, we demonstrate the utility of using both morphological and molecular tools to identify these parasites. Morphological examinations of specimens from muskrats (n = 63) first suggested that at least 4 genera were present including Echinostoma, Wardius, Quinqueserialis, and Notocotylus. For the latter 3 groups, the 28S region verified this assessment. For echinostomes, ND1 sequences revealed at least 5 genetic lineages. A particular lineage, Echinostoma trivolvis lineage b, predominated in both prevalence and intensity of infection. Molecular sequences provided a more accurate estimate of echinostome diversity in the muskrats and further support the idea that E. trivolvis is a species complex. Future studies will focus on whether there are differences in host specificity among the E. trivolvis lineages. In addition, this study has provided initial sequences that will help verify the life cycles of Wardius, Quinqueserialis, and especially, Notocotylus. By linking molecular, morphological, and life history information, we can better understand parasite diversity.