Development of the head collar and collar spines during the larval stages of Isthmiophora hortensis (Digenea: Echinostomatidae).

It is uncertain when the head collar and collar spines of Isthmiophora hortensis (Digenea: Echinostomatidae), a zoonotic echinostome species in Far Eastern Asia, develop during its larval stages. In this study, the appearance of the head collar and collar spines was studied using light and scanning electron microscopy in cercariae and metacercariae experimentally obtained from freshwater snails (Lymnaea pervia) and tadpoles (Rana nigromaculata), respectively. The cercariae were shed from the snail on day 30 after exposure to laboratory-hatched miracidia. Metacercariae were obtained from the experimental tadpoles at 3, 6, 12, 15, 20, 24, 26, and 30 h after exposure to the cercariae. The head collar was already visible in the cercarial stage, although its degree of development was weak. However, collar spines did not appear in the cercarial stage and even in the early metacercarial stage less than 24 h postinfection in tadpoles. Collar spines became visible in the metacercariae when they grew older than 24 h. It was concluded that the head collar of I. hortensis developed early in the cercarial stage, but the development of collar spines did not occur until the worms became 24-h-old metacercariae in our experimental setting. Counting the number of collar spines was concluded as an unfeasible diagnostic method for I. hortensis cercariae when they are shed from the snail host.

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.

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.

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.

The genetic structure of populations of Isthmiophora melis (Schrank, 1788) (Digenea: Echinostomatidae). Does the host's diet matter?

BACKGROUND: Here we provide a comparative analysis of the genetic structure of populations (based on nad1 mtDNA) of Isthmiophora melis isolated from the American mink (Neogale vison), an introduced invasive species, commonly occurring in the territory of Poland, and from the striped field mouse (Apodemus agrarius). METHODS: A total of 133 specimens of I. melis were obtained from naturally infected N. vison collected from six localities in Poland (108 samples) and 25 individuals of I. melis from A. agrarius. All sequences of the nad1 gene obtained during the present study were assembled and aligned. The standard statistics for haplotype composition, i.e., the number of haplotypes, haplotype diversity, nucleotide diversity, and average number of nucleotide differences, were calculated. Haplotype analysis and visualization of haplotype frequency among populations were performed using a median-joining network. RESULTS: Based on the samples collected from different localities in Poland, our study revealed that the overall genetic diversity of I. melis isolated from the American mink and of the striped field mouse do not differ significantly. The median-joining network showed that the three main haplotypes are in the centre of a star-like structure, with the remaining haplotypes as the satellites, reflecting the recent expansion of the populations. CONCLUSIONS: The overall genetic diversity of I. melis isolated from the American mink and striped field mouse reveals a high level of homogeneity. Moreover, regional differences in the food composition of the definitive hosts play an important role in shaping the genetic structure of the trematode populations.

Elucidating the life cycle of opossum parasites: DNA sequences reveal the involvement of planorbid snails as intermediate hosts of Rhopalias spp. (Trematoda: Echinostomatidae) in Brazil.

Echinostomatid digeneans belonging to the genus Rhopalias are intestinal trematodes found mainly in opossums in the New World. The genus comprises seven species, but their life cycles and intermediate hosts have been unknown until now. During our long-term study carried out in freshwater habitats within the state of Minas Gerais, Southeast Brazil, echinostomatid cercariae lacking collar spines were found in planorbid snails Biomphalaria glabrata, Biomphalaria straminea, Drepanotrema lucidum and Gundlachia ticaga in six different batches of snail samples collected between 2010 and 2019. Morphologically, the larvae reported herein are morphologically consistent with each other and characterized by the presence of 2-3 large ovoid or spherical corpuscles in each main duct of the excretory system, resembling to Cercaria macrogranulosa previously described from the same region of Brazil. Partial sequences of the ITS (ITS1-5.8S-ITS2) region and 28S gene of the nuclear ribosomal RNA operon, and partial sequences of mitochondrial nad1 and cox1 genes were obtained and compared with the data available for members of the family Echinostomatidae. Nuclear markers indicate that all samples of cercariae evaluated in the present study can be assigned to Rhopalias, but distinct from North American isolates of Rhopalias macracanthus, Rhopalias coronatus and Rhopalias oochi (divergence 0.2-1.2% in 28S and 0.8-4.7% in ITS). The lack of differences verified in both 28S and ITS in 5 out 6 studied samples suggested that they belong to the same species. However, nad1 sequences revealed that our cercariae correspond to three distinct species of Rhopalias (interspecific divergence: 7.7-9.9%), named here as Rhopalias sp. 1, found in B. straminea and G. ticaga, Rhopalias sp. 2 found in B. glabrata and D. lucidum, and Rhopalias sp. 3 also found in D. lucidum. They also differ by 10.8-17.2% from a North American isolate of R. macracanthus sequenced in this study. The cox1 sequences obtained for Rhopalias sp. 1 and Rhopalias sp. 2 (but not Rhopalias sp. 3) reveal that they are distinct from North American isolates of R. macracanthus (genetic divergence 16.3-16.5% and 15.6-15.7%, respectively), R. coronatus (9.2-9.3% and 9.3-9.5%) and Rhopalias oochi (9.0% and 9.5-10.1%). Encysted metacercariae with general morphology similar to that of the body of cercariae were found in tadpoles of Rhinella sp. from the same stream where snails harbored Rhopalias sp. 2, suggesting that the amphibians could act as second intermediate hosts of species of Rhopalias. Data obtained provide the first insights into the life cycle of this unusual echinostomatid genus.

The complete mitogenome of Echinoparyphium aconiatum (Digenea: Echinostomatidae) and a comparison with other digenean species.

Echinoparyphium aconiatum (Digenea: Echinostomatidae) is an intestinal parasite of anatid and snail-eating birds. In Eurasia, it is also common in lymnaeid snails, which may serve as the first and second intermediate hosts. The systematics of its genus, Echinoparyphium, have long been inadequate, with poor descriptions and extensive synonymy. To provide a basis for developing new genetic markers for studies of the identification and systematics of echinostomatids, the complete Ep. aconiatum mitogenome is described and compared with other digeneans. The circular mt molecule of this species is 14,865 bp in length, with an average A + T content of 64.33%. It contains 12 protein-coding genes and 22 transfer RNA genes. The 3' end of nad4L overlaps the 5' end of nad4 by 40 bp, while the atp8 gene is absent. Twenty-one transfer RNA genes transcribe products with conventional cloverleaf structures, while one transfer RNA gene has unpaired D-arms. Comparative analyses indicate that Echinoparyphium aconiatum is closely related to Echinochasmus japonicus and Echinostoma miyagawai. The phylogenetic results, using our mitochondrial data indicated Ep. aconiatum as a sister taxon of Hypoderaeum conoideum in a monophyletic clade. Our data and analyses serve as the first representative sequenced mt genome from genus Echinoparpyhium, providing additional markers to clarify the taxonomic position of Ep. aconiatum.

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.

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.

Multi-stage transcriptome profiling of the neglected food-borne echinostome Artyfechinostomum sufrartyfex reveal potential diagnostic and drug targets.

Lack of effective surveillance and control methods for neglected helminth diseases particularly in context of rural areas in India is a serious concern in terms of public health. With regard to the emerging food-borne echinostomid Artyfechinostomum sufrartyfex infection in the country, the current study is an in silico attempt to screen for plausible diagnostic and drug targets against the trematode. Transcriptome of adult, encysted and excysted metacercaria stages of the parasite was generated using Illumina sequencing platform. A de-novo assembly strategy utilizing transcriptome data generated from the three lifecycle stages was followed to generate the representative transcripts. Longest open reading frames identified for the transcripts were further conceptually translated into their respective protein sequences. Detailed analysis of this dataset through various bioinformatics pipelines and tools eventually identified 14 credible diagnostic and 10 drug targets along with their FDA-approved and ZINC molecules. Some of the important diagnostic candidates include thioredoxin peroxidase, haemoglobinase, cathepsin L, cathepsin L-like and B-like cysteine proteases. Among the drug targets, uncharacterized sodium dependent transporter and bifunctional protein Aas were identified as top targets exhibiting significant interaction with Rifamycin and ZINC02820058 molecule, respectively. Further, B-cell epitope analysis of the diagnostic targets revealed unique epitopes for 10 of them thus indicating their potential role in specific diagnosis of the parasite. The diagnostic candidates along with a number of lesser known drug targets and their ligand molecules identified in this study provides a reasonable basis for evaluation and development of future intervention strategies against A. sufrartyfex.

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.

EXPERIMENTAL ELUCIDATION OF THE LIFE CYCLE OF DREPANOCEPHALUS SPATHANS (DIGENEA: ECHINOSTOMATIDAE) WITH NOTES ON THE MORPHOLOGICAL PLASTICITY OF D. SPATHANS IN THE UNITED STATES.

The echinostomatid Drepanocephalus spathans (syn. Drepanocephalus auritus) parasitizes the double-crested cormorant Phalacrocorax auritus. In North America, the marsh rams-horn snail Planorbella trivolvis and ghost rams-horn snail Biomphalaria havanensis serve as snail intermediate hosts, both of which inhabit catfish aquaculture ponds in the southeastern United States. Studies have demonstrated D. spathans exposure can be lethal to juvenile channel catfish Ictalurus punctatus. Two studies were undertaken to elucidate the life cycle of D. spathans to establish a developmental time line. In both studies, D. spathans cercariae collected from naturally infected P. trivolvis individuals were used to infect channel catfish fingerlings, which were then fed to double-crested cormorants (DCCOs) that had been pharmaceutically dewormed. In study 1, laboratory-reared P. trivolvis and B. havanensis individuals were placed in aviary ponds with experimentally infected DCCO and examined bi-weekly for release of cercariae. Trematode eggs were observed in the feces of exposed birds 3 days post-infection. Birds were sacrificed 18 days post-exposure (dpe), and gravid adults morphologically and molecularly consistent with D. spathans were recovered. Snails from the aviary pond were observed shedding D. spathans cercariae 18-54 dpe. In study 2, trematode eggs were observed in the feces of exposed DCCOs beginning 8 dpe. Once eggs were observed, birds were allowed to defecate into clean tanks containing naïve laboratory-reared P. trivolvis individuals. Additionally, eggs from experimental DCCO feces were recovered by sedimentation and placed in an aquarium housing laboratory-reared P. trivolvis individuals. Birds in study 2 were sacrificed after 60 days, and gravid D. spathans specimens were recovered. Snails from the experimental DCCO tanks shed D. spathans cercariae 89-97 dpe. Lastly, trematode eggs were isolated and observed for the hatching of miracidia, which emerged on average after 16 days at ambient temperatures. No D. spathans adults were observed in control birds fed non-parasitized fish. This is the first experimental confirmation of the D. spathans life cycle, resolving previously unknown developmental time lines. In addition, the effects of fixation on adult trematode morphology were assessed, clarifying reports of pronounced morphological plasticity for D. spathans.

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.

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.

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.

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.

Diversity of echinostomes (Digenea: Echinostomatidae) in their snail hosts at high latitudes.

The biodiversity of freshwater ecosystems globally still leaves much to be discovered, not least in the trematode parasite fauna they support. Echinostome trematode parasites have complex, multiple-host life-cycles, often involving migratory bird definitive hosts, thus leading to widespread distributions. Here, we examined the echinostome diversity in freshwater ecosystems at high latitude locations in Iceland, Finland, Ireland and Alaska (USA). We report 14 echinostome species identified morphologically and molecularly from analyses of nad1 and 28S rDNA sequence data. We found echinostomes parasitising snails of 11 species from the families Lymnaeidae, Planorbidae, Physidae and Valvatidae. The number of echinostome species in different hosts did not vary greatly and ranged from one to three species. Of these 14 trematode species, we discovered four species (Echinoparyphium sp. 1, Echinoparyphium sp. 2, Neopetasiger sp. 5, and Echinostomatidae gen. sp.) as novel in Europe; we provide descriptions for the newly recorded species and those not previously associated with DNA sequences. Two species from Iceland (Neopetasiger islandicus and Echinoparyphium sp. 2) were recorded in both Iceland and North America. All species found in Ireland are new records for this country. Via an integrative taxonomic approach taken, both morphological and molecular data are provided for comparison with future studies to elucidate many of the unknown parasite life cycles and transmission routes. Our reports of species distributions spanning Europe and North America highlight the need for parasite biodiversity assessments across large geographical areas.

TITLE: Diversité des Échinostomes (Digenea, Echinostomatidae) chez leurs hôtes mollusques aux latitudes élevées. ABSTRACT: La biodiversité des écosystèmes d'eau douce à l'échelle mondiale laisse encore beaucoup à découvrir, notamment dans la faune parasitaire des trématodes qu'ils abritent. Les parasites trématodes Échinostomes ont des cycles de vie complexes à hôtes multiples impliquant souvent des oiseaux migrateurs comme hôtes définitifs, conduisant ainsi à des distributions étendues. Ici, nous avons examiné la diversité des échinostomes dans les écosystèmes d'eau douce à des latitudes élevées en Islande, Finlande, Irlande et en Alaska (États-Unis). Nous rapportons de séquences de nad1 et d'ADNr 28S morphologiquement et moléculairement à partir d'analyses de données de séquence d'ADNr nad1 et 28S. Nous avons trouvé des échinostomes parasitant les mollusques de 11 espèces des familles Lymnaeidae, Planorbidae, Physidae et Valvatidae. Le nombre d'espèces d'échinostomes dans différents hôtes ne variait pas beaucoup et allait d'une à trois espèces. Sur ces 14 espèces de trématodes, nous avons découvert quatre espèces (Echinoparyphium sp. 1, Echinoparyphium sp. 2, Neopetasiger sp. 5, Echinostomatidae gen. sp.) comme nouvelles pour l'Europe; nous fournissons des descriptions pour les espèces nouvellement signalées et celles qui n'étaient pas précédemment associées à des séquences d'ADN. Deux espèces d'Islande (Neopetasiger islandicus et Echinoparyphium sp. 2) ont été signalées en Islande et en Amérique du Nord. Toutes les espèces trouvées en Irlande sont de nouveaux signalements pour ce pays. Grâce à une approche taxonomique intégrative, des données morphologiques et moléculaires sont fournies à des fins de comparaison avec des études futures afin d'élucider les nombreux cycles de vie et voies de transmission des parasites, qui sont inconnus. Nos données sur la répartition des espèces en Europe et en Amérique du Nord soulignent la nécessité d'évaluer la biodiversité des parasites dans de vastes zones géographiques.

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.

Spread of An Avian Eye Fluke, Philophthalmus gralli, Through Biological Invasion of An Intermediate Host.

Philophthalmus is a genus of globally distributed parasitic eye flukes with some members of the genus found in disparate locales. In particular, Philophthalmus gralli, a zoonotic trematode, appears to be a relatively new introduction to the Americas, facilitated by spillover from the invasive snails Melanoides tuberculata (red-rimmed melania) and Tarebia granifera (quilted melania), which were introduced via the aquarium trade, and perhaps furthered by avian dispersal. Given that two known intermediate hosts of Philophthalmus flukes are actively expanding their range as a result of human activities, we hypothesize that this spread is also associated with the spread of Philophthalmus flukes. To address this, we systematically reviewed the literature and examined whether the global expansion of P. gralli flukes is associated with the spread of invasive snails M. tuberculata and T. granifera. Here, we show that (1) specimens of P. gralli are only found in intermediate snail hosts M. tuberculata or T. granifera, suggesting intermediate host specificity for these 2 species, and (2) specimens of P. gralli have rarely been found outside the ranges (native and introduced) of M. tuberculata or T. granifera. Given the importance of distribution information of parasites in the role of identifying parasite invasions, we also review the known distribution of all Philophthalmus species. Considering recent outbreaks in humans and wild and domestic animal species, the continued spread of Philophthalmus presents a potential threat to veterinary and public health and conservation.

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.