The ribosomal transcription units of five echinostomes and their taxonomic implications for the suborder Echinostomata (Trematoda: Platyhelminthes).

Five newly obtained nuclear ribosomal transcription unit (rTU) sequences from Echinostomatidae and Echinochasmidae are presented. The inter- and intrafamilial relationships of these and other families in the suborder Echinostomata are also analyzed. The sequences obtained are the complete rTU of Artyfechinostomum malayanum (9,499 bp), the near-complete rTU of Hypoderaeum conoideum (8,076 bp), and the coding regions (from 5'-terminus of 18S to 3'-terminus of 28S rRNA gene) in Echinostoma revolutum (6,856 bp), Echinostoma miyagawai (6,854 bp), and Echinochasmus japonicus (7,150 bp). Except for the longer first internal transcribed spacer (ITS1) in Echinochasmus japonicus, all genes and spacers were almost identical in length. Comprehensive maximum-likelihood phylogenies were constructed using the PhyML software package. The datasets were either the concatenated 28S + 18S rDNA sequences (5.7-5.8 kb) from 60 complete rTUs of 19 families or complete 28S sequences only (about 3.8-3.9 kb) from 70 strains or species of 22 families. The phylogenetic trees confirmed Echinostomatoidea as monophyletic. Furthermore, a detailed phylogeny constructed from alignments of 169 28S D1-D3 rDNA sequences (1.1-1.3 kb) from 98 species of 50 genera of 10 families, including 154 echinostomatoid sequences (85 species/42 genera), clearly indicated known generic relationships within Echinostomatidae and Echinochasmidae and relationships of families within Echinostomata and several other suborders. Within Echinostomatidae, Echinostoma, Artyfechinostomum, and Hypoderaeum appeared as monophyletic, while Echinochasmus (Echinochasmidae) was polyphyletic. The Echinochasmidae are a sister group to the Psilostomidae. The datasets provided here will be useful for taxonomic reappraisal as well as studies of evolutionary and population genetics in the superfamily Echinostomatoidea, the sole superfamily in the suborder Echinostomata.

The complete mitochondrial genome of Morishitium polonicum (Trematoda, Cyclocoelidae) and its phylogenetic implications.

Trematodes can adversely impact the health and survival of wild animals. The trematode family Cyclocoelidae, which includes large digenean bird parasites, lacks molecular analysis, and reclassifications have not been supported. This study produced the first fully assembled and annotated mitochondrial genome sequence for the trematode Morishitium polonicum. The whole length of the M. polonicum (GenBank accession number: OP930879) mitogenome is 14083 bp, containing 22 transfer ribonucleic acids (tRNAs), 2 ribosomal RNAs (rRNAs, rrnL and rrnS), and a noncoding control section (D-loop) 13777 to 13854 bp in length. The 12 PCG areas have 3269 codons and a total length of 10053 bp, which makes up 71.38% of the mitochondrial genome's overall sequence. Most (10/12) of the PCGs that code for proteins begin with ATG, while the nad4L and nad1 genes have a GTG start codon. Phylogenetic analysis using the concatenated nucleotide sequences of 12 PCGs, and the ML tree analysis results showed that M. polonicum is more closely related to with Echinostomatidae and Fasciolidae, which indicates that the family Cyclocoelidae is more closely associated with Echinochasmidae. This study provides mtDNA information, and analysis of mitogenomic structure and evolution. Moreover, we aimed to understand the phylogenetic relationships of this fluke.

Putting infection on the map: Using heatmaps to characterise within- and between-host distributions of trematode metacercariae.

The location of parasites within individual hosts is often treated as a static trait, yet many parasite species can occur in multiple locations or organs within their hosts. Here, we apply distributional heat maps to study the within- and between-host infection patterns for four trematodes (Alaria marcianae, Cephalogonimus americanus, Echinostoma spp. and Ribeiroia ondatrae) within the amphibian hosts Pseudacris regilla and two species of Taricha. We developed heatmaps from 71 individual hosts from six locations in California, which illustrate stark differences among parasites both in their primary locations within amphibian hosts as well as their degree of location specificity. While metacercariae (i.e., cysts) of two parasites (C. americanus and A. marcianae) were relative generalists in habitat selection and often occurred throughout the host, two others (R. ondatrae and Echinostoma spp.) were highly localised to a specific organ or organ system. Comparing parasite distributions among these parasite taxa highlighted locations of overlap showing potential areas of interactions, such as the mandibular inner dermis region, chest and throat inner dermis and the tail reabsorption outer epidermis. Additionally, the within-host distribution of R. ondatrae differed between host species, with metacercariae aggregating in the anterior dermis areas of newts, compared with the posterior dermis area in frogs. The ability to measure fine-scale changes or alterations in parasite distributions has the potential to provide further insight about ecological questions concerning habitat preference, resource selection, host pathology and disease control.

A new species of Echinostoma (Trematoda: Echinostomatidae) from the 'revolutum' group found in Brazil: refuting the occurrence of Echinostoma miyagawai (=E. robustum) in the Americas.

Although Echinostoma robustum (currently a synonym of E. miyagawai) was reported in the Americas based on molecular data, morphological support on adult parasites is still required. Herein, a new species of Echinostoma is described based on worms found in a chicken from Brazil. Molecular phylogenetic analyses based on 28S (1063 bp), ITS (947 bp) and Nad-1 (442 bp) datasets reveal the inclusion of the new species within Echinostoma 'revolutum' species complex. Moreover, it was verified the conspecificity between cercariae previously identified as E. robustum in Brazil [identical ITS and only 0.3% of divergence (1 nucleotide) in Nad-1]. Species discovery analyses show that these two isolates form an independent lineage (species) among Echinostoma spp. Compared to E. miyagawai, the new species presents relatively high divergence in Nad-1 (7.88­9.09%). Morphologically, the specimens are distinguished from all nominal species from the 'revolutum' species complex by the more posterior position of the testes (length of post-testicular field as a proportion of body length about 20%). They further differ from E. miyagawai and South American Echinostoma spp. by the higher proportion of forebody to the body length. Therefore, combined molecular and morphological evidence supports the proposal of the species named here as Echinostoma pseudorobustum sp. nov.

Changes in resident microbiota associated with mice susceptibility or resistance to the intestinal trematode Echinostoma caproni.

Echinostoma caproni (Trematoda: Echinostomatidae) is an intestinal trematode with no tissue phases in the definitive host that has been extensively used as an experimental model to study the factors that determine resistance against intestinal helminths. In E. caproni infections in mice, interleukin-25 (IL-25) plays a critical role and it is required for the resistance to infection. However, little is known on the factors that determine its production. Primary E. caproni infection in mice is characterized by the development of chronic infections and elevated worm recovery, in relation to a local Th1 response with elevated production of interferon-γ. However, partial resistance against secondary E. caproni infections in ICR (Institute of Cancer Research) mice is developed after the chemotherapeutic cure of a primary infection and the innately produced IL-25 after pharmacological treatment. In this paper, we analyse the potential role of intestinal microbiota in the production of IL-25, and the subsequent resistance to infection. For this purpose, we analysed the production of IL-25 under conditions of experimental dysbiosis and also the changes in the resident microbiota in primary infections, pharmacological curation and secondary infections. The results obtained showed that resident microbiota play a major role in the production of IL-25 and the appearance of members of the phylum Verrucomicrobia as a consequence of the curation of the primary infection could be related to the partial resistance to secondary infection.

Neglected food-borne trematodiases: echinostomiasis and gastrodiscoidiasis.

In the present paper, we review two of the most neglected intestinal food-borne trematodiases: echinostomiasis, caused by members of the family Echinostomatidae, and gastrodiscoidiasis produced by the amphistome Gastrodiscoides hominis. Both parasitic infections are important intestinal food-borne diseases. Humans become infected after ingestion of raw or insufficiently cooked molluscs, fish, crustaceans, amphibians or aquatic vegetables. Thus, eating habits are essential to determine the distribution of these parasitic diseases and, traditionally, they have been considered as minor diseases confined to low-income areas, mainly in Asia. However, this scenario is changing and the population at risk are currently expanding in relation to factors such as new eating habits in developed countries, growing international markets, improved transportation systems and demographic changes. These aspects determine the necessity of a better understanding of these parasitic diseases. Herein, we review the main features of human echinostomiasis and gastrodiscoidiasis in relation to their biology, epidemiology, immunology, clinical aspects, diagnosis and treatment.

Mitophylogenomics of the zoonotic fluke Echinostoma malayanum confirms it as a member of the genus Artyfechinostomum Lane, 1915 and illustrates the complexity of Echinostomatidae systematics.

The complete mitochondrial genome (mitogenome or mtDNA) of the trematode Echinostoma malayanum Leiper, 1911 was fully determined and annotated. The circular mtDNA molecule comprised 12 protein-coding genes (PCGs) (cox1 - 3, cob, nad1 - 6, nad4L, atp6), two mitoribosomal RNAs (MRGs) (16S or rrnL and 12S or rrnS), and 22 transfer RNAs (tRNAs or trn), and a non-coding region (NCR) rich in long and short tandem repeats (5.5 LRUs/336 bp/each and 7.5 SRUs/207 bp/each). The atp8 gene is absent and the 3' end of nad4L overlaps the 5' end of nad4 by 40 bp. Special DHU-arm missing tRNAs for Serine were found for both tRNASer1(AGN) and tRNASer2(UCN). Codons of TTT (for phenylalanine), TTG (for leucine), and GTT (for valine) were the most, and CGC (for Arginine) was the least frequently used. A similar usage pattern was seen in base composition, AT and GC skewness for PCGs, MRGs, and mtDNA* (coding cox3 to nad5) in E. malayanum and Echinostomatidae. The nucleotide use is characterized by (T > G > A > C) for PCGs/mtDNA*, and by (T > G ≈ A > C) for MRGs. E. malayanum exhibited the lowest genetic distance (0.53%) to Artyfechinostomum sufrartyfex, relatively high to the Echinostoma congeners (13.20-13.99%), higher to Hypoderaeum conoideum (16.18%), and the highest to interfamilial Echinochasmidae (26.62%); Cyclocoelidae (30.24%); and Himasthlidae (25.36%). Topology indicated the monophyletic position between E. malayanum/A. sufrartyfex and the group of Echinostoma caproni, Echinostoma paraensei, Echinostoma miyagawai, and Echinostoma revolutum, rendering Hypoderaeum conoideum and unidentified Echinostoma species paraphyletic. The strictly closed genomic/taxonomic/phylogenetic features (including base composition, skewness, codon usage/bias, genetic distance, and topo-position) reinforced Echinostoma malayanum to retake its generic validity within the Artyfechinostomum genus.

First record of Patagifer bilobus (Rudolphi, 1819) Dietz, 1909 (Digenea: Echinostomatidae), with a morphological and molecular characterization from two threskiornithid species in Mexico.

Patagifer Dietz, 1909 is a small genus of echinostomatids, with 12 recognized species, mostly parasitising threskiornithid birds, distributed worldwide. In the current research, adult specimens of the type species, Patagifer bilobus (Rudolphi, 1819) Dietz, 1909 from the white faced ibis (Plegadis chihi) and white ibis (Eudocimus albus) were re-described, providing new metrical data for the number of head collar spines. Those specimens were recorded from eight localities in Mexico and compared morphologically with specimens previously identified as Patagifer lamothei. A total of 19 specimens identified as P. bilobus including two hologenophores were sequenced with three molecular markers: domains D1-D3 of the large subunit (LSU), the internal transcribed spacer (ITS1, ITS2) plus 5.8S from the nuclear rDNA, and nicotinamide adenine dinucleotide dehydrogenase subunit 1 (nad1) from mitochondrial DNA. The new sequences were aligned with other sequences of Patagifer spp., downloaded from GenBank. Phylogenetic trees inferred from each data set, placed all the specimens in a clade, confirming that the isolates belonged to the same species. The morphological examination of specimens previously identified as P. lamothei by Ortega-Olivares MP, Hernández-Mena DI, Pérez-Ponce de León G, García-Varela M (2011) Helminths of the white ibis, Eudocimus albus (Aves Therskiornithidae) in Mexico. (Zootaxa 3088, 15-26. 10.11646/zootaxa.3088.1.2) and in combination with molecular data confirms that those specimens should be reassigned to P. bilobus. In addition, this is the first study in P. bilobus using an integrative taxonomy approach.

Flying across Europe: the case of the spread of Chaunocephalus ferox on a black stork (Ciconia nigra).

The annual migration of birds involves a very large number of inter-continental and intra-continental movements in which thousands of bird species participate. These migrations have been associated with the spread of pathogens worldwide, including bacteria, viruses and parasites. This study describes the case of a black stork (Ciconia nigra) that was ringed at the nest in Latvia and died five months later in the south-east of the Iberian Peninsula. Post-mortem examination revealed that the cause of death was electrocution. In addition, a massive infection by the trematode Chaunocephalus ferox (Digenea: Echinostomatidae) causing severe granulomatous lesions throughout the small intestine was detected. This is the first report of C. ferox infection in a black stork in the Iberian Peninsula, a trematode that, due to the severe lesions it causes, can affect the health of C. ferox-infected wild birds, particularly in severely infected long-distance migrants. The dispersal of platyhelminths associated with migratory birds is discussed. After the ringing at the nest, the black stork was sighted in Central Europe one month before its capture, and the trematodes found by necropsy were mostly mature adults. Consequently, we estimate that this juvenile animal acquired the infection during its migration in a European area other than the Iberian Peninsula, evidencing a long-distance parasite spread through its migratory host. Our study highlights that bird ringing can be used to understand the epidemiological implications that bird migratory behaviour may have on the dispersal of parasites.

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.

Presence of one ecto- and two endoparasite species of the black stork (Ciconia nigra) in Portugal.

BACKGROUND: The black stork (Ciconia nigra Linnaeus, 1758) is a recognized endangered species in Europe and most of the specimens from the Western Palearctic region breed in the Iberian Peninsula. Available works regarding parasites in black storks are scarce. This work reports the presence one ecto- and two endoparasite species from a black stork in Portugal. CASE PRESENTATION: A black stork was found in southern Portugal after colliding against electric cables. The specimen did not survive its sustained injuries and a post-mortem exam was performed. During the procedure, several ecto- and endoparasite specimens were found. The collected parasites were lice (Neophilopterus tricolor), nematodes (Desportesius sagittatus) and trematodes (Cathaemasia hians). CONCLUSIONS: Three different species of parasites are reported from a black stork in Portugal. Ecto- and endoparasites of C. nigra have not frequently been described in the literature, and this case report is a contribution to the field. Additional studies will be important to better understand the impact that parasites can have on C. nigra health and survival.

Acanthoparyphium shinanense n. sp. (Digenea: Echinostomatidae) from Experimental Chicks Infected with Metacercariae Encysted in Brackish Water Clams in the Republic of Korea.

Acanthoparyphium shinanense n. sp. (Digenea: Echinostomatidae) is described from chicks experimentally infected with the metacercariae encysted in 2 brackish water clam species, Ruditapes philippinarum and Coecella chinensis, in the Republic of Korea. The metacercariae were round to oval, armed with 23 collar spines, and 0.216 (0.203-0.226) mm in diameter. From 5 chicks experimentally infected each with 200 metacercariae, 34 juvenile (5-day-old worms) and 104 adult flukes (7-day-old worms) were harvested from their small intestines, with the average worm recovery rate of 13.8%. The adult flukes were 3.18 (2.89-3.55) mm long and 0.68 (0.61-0.85) mm wide, with an elongated, posteriorly tapering body, and a prominent head collar armed with 23 collar spines arranged in a single uninterrupted row. The posterior testis of A. shinanense was longitudinally elongated, which is similar to Acanthoparyphium spinulosum Johnston, 1917 but unique from the other closely related species, including Acanthoparyphium tyosenense Yamaguti, 1939, Acanthoparyphium kurogamo Yamaguti, 1939, and Acanthoparyphium marilae Yamaguti, 1934. The eggs of A. shinanense were larger than those of A. spinulosum, and the anterior extent of 2 lateral groups of vitellaria was slightly more limited in A. shinanense than in A. spinulosum. Molecular analysis of nuclear and mitochondrial genes revealed low homology with A. spinulosum from USA (96.1% in 5.8S rRNA) and Ukraine (97.9% in 28S rRNA), Acanthoparyphium n. sp. from USA (98.0% in 28S rRNA), and Acanthoparyphium sp. from Australia, Kuwait, and New Zealand. Biological characteristics, including its first intermediate host and natural definitive hosts, as well as its zoonotic capability, should be elucidated.

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.

Timing and order of exposure to two echinostome species affect patterns of infection in larval amphibians.

The study of priority effects with respect to coinfections is still in its infancy. Moreover, existing coinfection studies typically focus on infection outcomes associated with exposure to distinct sets of parasite species, despite that functionally and morphologically similar parasite species commonly coexist in nature. Therefore, it is important to understand how interactions between similar parasites influence infection outcomes. Surveys at seven ponds in northwest Pennsylvania found that multiple species of echinostomes commonly co-occur. Using a larval anuran host (Rana pipiens) and the two most commonly identified echinostome species from our field surveys (Echinostoma trivolvis and Echinoparyphium lineage 3), we examined how species composition and timing of exposure affect patterns of infection. When tadpoles were exposed to both parasites simultaneously, infection loads were higher than when exposed to Echinoparyphium alone but similar to being exposed to Echinostoma alone. When tadpoles were sequentially exposed to the parasite species, tadpoles first exposed to Echinoparyphium had 23% lower infection loads than tadpoles first exposed to Echinostoma. These findings demonstrate that exposure timing and order, even with similar parasites, can influence coinfection outcomes, and emphasize the importance of using molecular methods to identify parasites for ecological studies.

Resistance and tolerance: A hierarchical framework to compare individual versus family-level host contributions in an experimental amphibian-trematode system.

Hosts have two general strategies for mitigating the fitness costs of parasite exposure and infection: resistance and tolerance. The resistance-tolerance framework has been well developed in plant systems, but only recently has it been applied to animal-parasite interactions. However, difficulties associated with estimating fitness, controlling parasite exposure, and quantifying parasite burden have limited application of this framework to animal systems. Here, we used an experimental approach to quantify the relative influence of variation among host individuals and genetic families in determining resistance and tolerance within an amphibian-trematode system. Importantly, we used multiple, alternative metrics to assess each strategy, and employed a Bayesian analytical framework to compare among responses while incorporating uncertainty. Relative to unexposed hosts, exposure to the pathogenic trematode (Ribeiroia ondatrae) reduced the survival and growth of California newts (Taricha torosa) (survival: 93% vs. 74%; growth: 0.29 vs. -0.5 vs mm day -1). Similarly, parasite infection success (the inverse of resistance) ranged from 8% to 100%. Yet despite this broad variation in host resistance and tolerance among individual newts, we found no evidence for transmissable, among-family variation in any of the resistance or tolerance metrics. This suggests that opportunities for evolution of these traits is limited, likely requiring significant increases in mutation, gene flow, or environmental heterogeneity. Our study provides a quantitative framework for evaluating the importance of alternative metrics of resistance and tolerance across multiple time points in the study of host-parasite interactions in animal systems.

Influence of Himasthla elongata (Trematoda: Echinostomatidae) metacercariae on heart rate in blue mussels (Mytilus edulis).

Whether metacercariae of the digenean Himasthla elongata (Himasthlidae) are harmful to their secondary intermediate hosts, the blue mussel, Mytilus edulis, is a disputable question. To shed light on this issue, we performed field monitoring of the heart rate (HR) in mussels infected with H. elongata over a period of 1.5 years. We observed a significant rise in HR in infected mussels, but only 1 year after infection and at temperatures of 15-17 °C. HR variance also grew sharply in infected mussels within the same temperature range. We also detected a decreased ability of infected mussels to compensate for the rise in the environment temperature. Finally, growth rate in the infected mussels was slower compared to the control group. We provide evidence for measurable pathogenic effects caused by H. elongata metacercariae in their secondary intermediate mussel hosts.

The effect of trematode infection on the markers of oxidative stress in the offspring of the freshwater snail Lymnaea stagnalis.

Most invertebrate species exhibit immunological responses that can inactivate and eliminate penetrating parasites. Such immune responses in particular involve the formation of potentially toxic reactive oxygen species (ROS). We explored the immune capabilities of the first-generation (F1) offspring of naturally infected freshwater snails, Lymnaea stagnalis, in response to infection by trematode cercariae under laboratory conditions. The rates of ROS formation and peroxidase activity in the hemolymph of the F1 offspring of L. stagnalis parents infected by an asexual stage of trematodes were significantly higher than in F1 offspring of uninfected parents. Compared to offspring from uninfected parents, the growth rate of F1 snails from infected parents was higher, but survival was lower. After infection of F1 snails by trematode cercariae of Echinoparyphium aconiatum under laboratory conditions, the rate of ROS formation and peroxidase activity in the hemolymph of F1 offspring of uninfected parents increased compared to control snails. This pattern persisted throughout the entire 3-week observation period. In contrast, the rate of ROS formation in the hemolymph of F1 snails from infected parents after experimental infection by E. aconiatum cercariae did not differ from controls, and peroxidase activity even decreased. Thus, trematode parthenitae infection of parents could alter the immune response of their offspring.

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.