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.

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.

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.

Malformations of the gill filaments of the ruffe Gymnocephalus cernuus (L.) (Pisces) caused by echinostomatid metacercariae.

In parasite surveys of fishes from Lake Balaton and its tributaries in Hungary, infections with metacercariae of a species of the digenean genus Echinochasmus (Trematoda: Echinostomatidae) were found in seven species of fish. In ruffe, Gymnocephalus cernuus, malformations of the gill filaments apparently caused by these infections were observed. These malformations were in the form of bifurcations of the filaments at about their mid-length. At the point where the filaments bifurcate, an Echinochasmus metacercaria was always embedded in the cartilaginous ray of the gill filament. All specimens of the ruffe were found to be infected by these metacercariae, and each ruffe specimen was infected by 30-300 metacercariae. Such a bifurcation was found in all of the ruffe specimens, but, apart from these gill malformations, the metacercariae produced only local changes in the cartilage. In the other six infected fish species, only local signs were observed in the cartilage. Experimental infections of chicks with metacercariae resulted in the finding of the sexual adult (marita) of an unidentified species of Echinochasmus. ITS sequences of the adult and metacercaria corresponded with each other, and also with a cercaria isolated from a gravel snail (Lithoglyphus naticoides), with a 99.5-100% similarity.

Altered Protein Expression in the Ileum of Mice Associated with the Development of Chronic Infections with Echinostoma caproni (Trematoda).

BACKGROUND: Echinostoma caproni (Trematoda: Echinostomatidae) is an intestinal trematode that has been extensively used as experimental model to investigate the factors determining the expulsion of intestinal helminths or, in contrast, the development of chronic infections. Herein, we analyze the changes in protein expression induced by E. caproni infection in ICR mice, a host of high compatibility in which the parasites develop chronic infections. METHODOLOGY/PRINCIPAL FINDINGS: To determine the changes in protein expression, a two-dimensional DIGE approach using protein extracts from the intestine of naïve and infected mice was employed; and spots showing significant differential expression were analyzed by mass spectrometry. A total of 37 spots were identified differentially expressed in infected mice (10 were found to be over-expressed and 27 down-regulated). These proteins were related to the restoration of the intestinal epithelium and the control of homeostatic dysregulation, concomitantly with mitochondrial and cytoskeletal proteins among others. CONCLUSION/SIGNIFICANCE: Our results suggests that changes in these processes in the ileal epithelium of ICR mice may facilitate the establishment of the parasite and the development of chronic infections. These results may serve to explain the factors determining the development of chronicity in intestinal helminth infection.

Trematode reproduction in the molluscan host: an ultrastructural study of the germinal mass in the rediae of Himasthla elongata (Mehlis, 1831) (Digenea: Echinostomatidae).

The germinal mass in Himasthla elongata rediae was studied in detail using transmission electron microscopy. It was shown to be a specialized reproductive organ consisting of germinal cells at various maturation stages, supporting cells and stem cells. The germinal mass also contains early cercarial embryos emerging as a result of cleavage division of mature germinal cells. The stem cells that give rise to germinal cells have heterochromatin-rich nuclei with distinct nucleoli and scarce cytoplasm containing mainly free ribosomes and few mitochondria. The differentiating germinal cells undergo a growth, which is accompanied by an emergence of annulate lamellae and the nuage in their cytoplasm, a noticeable development of RER and Golgi apparatus and an increase in the number of mitochondria. The mitochondria form a large group at one of the cell poles. During differentiation, the nucleus and nucleolus of the germinal cell enlarge while the chromatin becomes gradually less condensed. The supporting tissue of the germinal mass is made up of cells connected by septate junctions. These supporting cells are distinctly different in cellular shape and nuclear ultrastructure. Their outgrowths form a tight meshwork housing stem cells, germinal cells and early cercarial embryos. The cytoplasm of the supporting cells in the mesh area is separated into fine parallel layers by labyrinthine narrow cavities communicating with the intercellular space. The supporting tissue contains differentiating and degenerating cells which indicates its renewal. The results of this ultrastructural study lend support to the hypothesis that the germinal cells of digeneans are germ line cells.

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.

[Life cycle of the trematode Echinochasmus spinosus Odhner, 1911 (Echinostomatidae) in the natural conditions of Primorye].

Life cycle and developmental stages of the trematode Echinochasmus spinosus Odhner, 1911 are described. As it was established experimentally, in the conditions of Primorsky Krai circulation of the trematode involves first intermediate host, mollusk Parafossarulus spiridonovi, and the second one, freshwater fishes. Adult worms were reared in chicken.

Surface ultrastructure of juvenile and adult Acanthoparyphium tyosenense (Digenea: Echinostomatidae).

The tegumental ultrastructure of juvenile and adult Acanthoparyphium tyosenense (Digenea: Echinostomatidae) was observed by scanning electron microscopy. One- to 3-day-old juveniles and 10-day-old adults were harvested from chicks experimentally fed metacercariae from a bivalve, Mactra veneriformis. The juvenile worms were minute, curved ventrally, and had 23 collar spines characteristically arranged in a single row. The lips of the oral sucker had 7 single aciliated sensory papillae and 4 grouped uniciliated sensory papillae. The ventral sucker had 25 aciliated round swellings on its lip. The anterolateral surface between the 2 suckers was densely packed with tongue-shaped tegumental spines, and the ventral surface just posterior to the ventral sucker was covered with peg-like spines. Retractile, peg-like spines were seen on the anterolateral surface, whereas scale-like spines with round tips and broad bases were sparsely distributed posterior to the ventral sucker. The cirrus was characteristically protruding and armed with minute spines. The surface ultrastructure of A. tyosenense was unique, especially in the number and arrangement of collar spines, shape, and distribution of tegumental spines and in distribution of sensory papillae.

Biochemical profile of Biomphalaria glabrata (Mollusca: Gastropoda) after infection by Echinostoma paraensei (Trematoda: Echinostomatidae).

The effect of infection by Echinostoma paraensei on the activity of the enzymes alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and the concentration of total proteins, uric acid and urea in the hemolymph of Biomphalaria glabrata were investigated after exposure to five or 50 miracidia. The biochemical concentrations were measured weekly until the end of the fourth week after exposure. There was a significant decrease in the concentrations of total proteins in the snails exposed both to five and 50 miracidia, as well as an increase in the nitrogenous products of excretion, ALT and AST activities. The higher ALT activity in the hemolymph of the snails after infection with 50 miracidia suggests highest energetic requirement in these snails in relation to snails exposed to five miracidia. The results also suggest an increase in the use of total proteins, since there was increased formation of nitrogenous catabolites, in conformity with an increase in the aminotransferase activities, frequently associated with tissue damages. This can be explained by damage due to penetration by the miracidia and subsequent development of intramolluscan sporocysts and rediae.

Field evidence for a parasite spillback caused by exotic mollusc Dreissena polymorpha in an invaded lake.

This study provides field evidence in support of the "parasite spillback hypothesis" that predicts a positive numerical response of native parasites to the presence of an exotic host that is competing for these parasites with native hosts. We examined the role that the exotic mollusc Dreissena polymorpha (zebra mussel) plays as the second intermediate host of the echinostomatid trematode Echinoparyphium recurvatum in Lake Naroch, Belarus. We found that due likely to the disproportionately high abundance of its novel exotic host, the metacercarial stage of this parasite has become substantially more abundant than that of other echinostomatid trematodes recorded in native molluscs. This finding suggests an increased load (the "spillback") of E. recurvatum on native waterfowl that serve as its final hosts. The risk of aquatic birds becoming infected with this trematode via feeding on D. polymorpha was found to vary both spatially and seasonally. This risk was particularly high in the autumn at a depth of 2 m and at sites of bird gatherings.

Himasthla alincia (Echinostomatidae): metacercariae in brackish water bivalves and their growth and development in experimental animals.

Metacercariae of Himasthla alincia (Echinostomatidae) were discovered in brackish water bivalves in the Republic of Korea; their growth and development were observed in experimental animals. Five species of clams (Mactra veneriformis, Solen grandis, Meretrix petechialis, Cyclina sinensis, and Tapes philippinarum) were found to harbor the metacercariae of H. alincia. Chicks, rats, and mice were orally fed the metacercariae, and worms were recovered from their intestines from day 1 to day 20 postinfection (PI). Only chicks appeared to be a fairly suitable host, although the worm recovery was low, i.e., 1.5% from 17 chicks, and the number decreased from 2.6% on day 1 PI to 1.3% on day 20 PI. Worm development in chicks was quick and remarkable during days 10­20 PI. Adult flukes were morphologically characterized by the presence of a head collar with 31 dorsally uninterrupted collar spines, including 4 end-group spines, and distribution of vitellaria only up to the most posterior margin of the cirrus sac. We verified that several species of brackish water clams are second intermediate hosts for H. alincia, and that its life cycle occurs in the Republic of Korea.

[Reexamination of the life cycle of Isthmiophora melis (Trematoda: Echinostomatidae) on material from southeast Europe].

The life cycle of Isthmiophora melis (Schrank, 1788) on material from Southeast Europe was experimentally reexamined. Thirteen names or combinations can be accepted as true synonyms of I. melis: Distoma melis (Schrank, 1788) Zeder, 1800; Echinocirrus melis (Schrank, 1788) Mendhaim, 1943; Isthmiophora spiculator (Dujardin, 1845); Echinostoma trigonocephalum (Rud., 1802) Cobbold, 1861; E. melis (Schrank, 1788) Dietz, 1909; E. spiculator Dujardin, 1845; Euparyphium jassyense Leon and Ciurea, 1922; E. melis (Schrank, 1788) Railliet, 1919; E. suinum Ciurea, 1921; Fasciola armata Rud., 1802; F. melis Schrank, 1788; F. putorii Gmelin, 1791; F. trigonocephala Rud., 1802. The first intermediate hosts are the pulmonate freshwater snail Lymnaea stagnalis. The second intermediate hosts are many amphibians and freshwater fishes. The list of definitive hosts includes more than 30 species of vertebrates including humans. I. melis occurs in Europe, Asia and North America.

Size-dependent pattern of metacercariae accumulation in Macomona liliana: the threshold for infection in a dead-end host.

While bivalves can acquire trematode metacercariae over their lifetime, the rate at which this accumulation takes place is not necessarily linear. The present study found that the bivalve Macomona liliana acquires very few or no metacercariae until it reaches 30 mm in size, but thereafter the rate at which it becomes infected increases exponentially. It is likely that this ontogenetic change in infection rate is associated with the increased filtration capacity and siphon diameter of larger M. liliana. The echinostome metacercariae that infect M. liliana also infect a much more common sympatric bivalve, Austrovenus stutchburyi, in which they achieve much higher infection intensity. Due to its deeper burying depth, M. liliana most likely represents a dead-end host for the echinostomes: potential definitive hosts preferentially feed upon A. stutchburyi as they are located closer to the sediment surface than M. liliana. However, due to the low infection intensity and population density of M. liliana, its overall impact as a sink for echinostome populations in the ecosystem is probably negligible.

Euparyphium albuferensis and Echinostoma friedi (Trematoda: Echinostomatidae): experimental cercarial transmission success in sympatric snail communities.

Euparyphium albuferensis and Echinostoma friedi cercarial infectivity to four species of sympatric snails was examined under single- or multiple-choice laboratory conditions to show the level of parasite-snail host compatibility. Radix peregra, Lymnaeafuscus, Physella acuta and Gyraulus chinensis act as second intermediate hosts of both parasite species although different cercarial transmission success (CTS) was observed. In single-host experiments, R. peregra and P. acuta showed a high degree of compatibility with E. albuferensis, while only P. acuta in the case of E. friedi. In two-choice snail communities, a snail with high CTS increased the values of another with low compatibility, in both parasite species. In multiple-choice snail communities, high CTS of some hosts decreased, while low CTS of other hosts increased. The degree of parasite-host compatibility of each snail species could be determined by the presence of other snails in the community.

The life cycle of Stephanoprora aylacostoma n.sp. (Digenea: Echinostomatidae), parasite of the threatened snail Aylacostoma chloroticum (Prosobranchia, Thiaridae), in Argentina.

A new species, Stephanoprora aylacostoma is described and its life cycle was resolved experimentally. The prosobranch snail Aylacostoma chloroticum Hylton Scott, collected in the Yacyretá dam, Province of Misiones, Argentina, was found naturally infected with large-tailed cercariae possessing a prepharyngeal body and corpuscles in the excretory system but lacking collar spines. Metacercariae, which encysted on the gills of experimentally infected fishes Cnesterodon decemmaculatus (Jenyns) and Poecilia reticulata (Peters) (Poecilidae), developed collar spines after 10 days. Tetragonopterid fishes Moenckhausia dichroura (Kner), Astyanax erythropterus (Holmberg) and Hyphesobrycon serpae (Durbin in Eigenmann) were found infected naturally. Sexually mature adults were recovered from domestic chicks at day 7 post-exposure. Eggs shed in chick faeces developed to miracidia within 13-15 days; sporocysts were found on the gills of snails. The new species differs from other species of the genus in its larger eggs, in the smaller, slender body and smaller collar spines of the adult and in the morphological and biological features of the larval stages.

Life cycle of Stephanoprora uruguayense (Digenea: Echinostomatidae) in Argentina.

The life cycle of Stephanoprora uruguayense Holcman et Olagüe, 1989, was experimentally resolved. In an artificial pond in the Zoological Garden in Buenos Aires City, Argentina, Heleobia parchappei (Hydrobiidae) was found to be releasing large-tailed cercariae with a prepharyngeal body, but lacking collar spines and corpuscles in the excretory system. Metacercariae, which encysted on the gills of naturally and experimentally infected Cnesterodon decemmaculatus (Poecilidae), developed collar spines and corpuscles in the excretory system in 7 days. Sexually mature adults were recovered from chicks and immature adults from mice fed metacercariae from C. decemmaculatus. Eggs shed in chick feces developed to miracidia within 10 days; sporocysts were found on the gills of snails. Stephanoprora uruguayense and S. denticulata from Europe are similar in adult morphology, but can be distinguished by morphological and behavioral features of larvae. Likewise, although S. denticulata and S. paradenticulata from Venezuela are similar to S. uruguayense in adult morphology, they differ considerably in larval morphology and intermediate hosts.

[Reproduction of the trematode Echinostoma caproni parthenites (Digenea: Echinostomatidae)].

Dynamics of the reproduction in the trematode Echinostoma caproni parthenites (Echinostomatidae) was observed. Early laying and maturation of the generative cells are for the first time shown to be characteristic for all parthenogenetic generations. Really the process of reproduction had been finishing to the beginning of the generating of new age by parthenites. Mother sporocysts, as well as redia of different generations, in fact stop producing new generative cells with the beginning of the generating of new age, and assume the function of a brood pouch. This feature was considered previously as peculiar mainly to mother sporocysts. Data on the autotomy of the anterior body end in mother sporocysts are verified. In our opinion, these data are an evidence of an early manifestation of the evolutionary trend to the morpho-functional regress and disintegration of the parasitic stage of mother sporocyst.

A new cercaria and metacercaria of Acanthoparyphium (Echinostomatidae) found in an intertidal snail Zeacumantus subcarinatus (Batillaridae) from New Zealand.

A new 23-collar-spined cercaria and metacercaria are described from intertidal molluscs of the coast of New Zealand. The new cercaria found emerging from the mud snails Zeacumantus subcarinatus (Sowerby) (Prosobranchia: Batillariidae) is characterized mainly by the number and arrangement of the cephalic glands, the size of the suckers, and the size and number of the collar spines. The cercaria encysts in the cockle Austrovenus stutchburyi (Wood) (Bivalvia: Veneridae) which lives in sympatry with the first intermediate host throughout New Zealand. Laboratory infections of cockles by cercariae from naturally infected snails resulted in metacercariae identical to those found in naturally infected cockles. The main features of the cercaria and metacercaria are the presence of a reniform collar with 23 spines, two pairs of small cephalic glands at the oral sucker level, another two pairs of much longer ones posterior to the pharynx, and the excretory vesicle Y-shaped with the main collecting canals extending to the anterior level of the ventral sucker. Each of the main collecting canals had 10-11 pairs of bilateral diverticula between the anterior edge of the ventral sucker and anterior body end. A brief discussion of its possible life cycle and ecology is also provided.

In vivo and in vitro encystment of Echinochasmus liliputanus cercariae and biological activity of the metacercariae.

In vivo and in vitro encystment of the cercariae of Echinochasmus liliputanus and biological activity of the metacercariae were studied. In vivo encystment of cercariae occurred in the gills of goldfish, the second intermediate host. However, the cercariae also encysted in vitro in Locke solution (0.6x to 1.2x strength), 0.7-1.2% NaCI, artificial gastric juice, and human gastric juice. Locke or NaCI solutions were shown to be appropriate for in vitro encystment to occur within 24 hr; however, full-strength Locke solution was shown to be optimal. The 1-day-old metacercariae formed in vivo and treated in 0.1% sodium deoxycholate excystation medium at 37 C for 1 hr showed 88.5% excystation. The metacercariae formed in vitro, however, showed 88.6% and 85.0% excystation for normal and abnormal ones, respectively. Abnormal cysts at room temperature usually die within 10 days. About 70% of the normal cysts, both in vivo and in vitro, can still excyst after being stored in Locke 0.5x solution at 4 C for 3 mo. Cysts formed in vivo and in vitro were equally infective. The encystment of the cercariae in vitro could be inhibited when the cercariae were treated with 1 micromol silver nitrate. Because silver nitrate binds to the papillae, especially to the ciliated papillae, on the cercaria surface, it is suggested that papillary chemoreceptors may be involved in encystment of the cercariae. The finding of E. liliputanus cercariae encysting in vitro, especially in human gastric juice, might be helpful in elucidating mechanisms of the definitive hosts that are directly infected by the cercariae.