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Article

The Nuclear Ribosomal Transcription Units of Two Echinostomes and Their Taxonomic Implications for the Family Echinostomatidae

by
Yu Cao
1,2,*,†,
Ye Li
1,†,
Zhong-Yan Gao
3 and
Bo-Tao Jiang
1,2
1
Heilongjiang Academy of Agricultural Sciences Branch of Animal Husbandry and Veterinary Branch, Qiqihar 161005, China
2
Heilongjiang Provincial Key Laboratory of Veterinary Drugs, Qiqihar 161000, China
3
Heilongjiang Zhalong National Natural Reserve Administration, Qiqihar 161000, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Biology 2025, 14(8), 1101; https://doi.org/10.3390/biology14081101
Submission received: 7 June 2025 / Revised: 31 July 2025 / Accepted: 19 August 2025 / Published: 21 August 2025
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Simple Summary

This study investigated two Echinostomatidae trematodes, Echinostoma miyagawai and Patagifer bilobus, taken from domestic duck and Grus japonensis, respectively. We sequenced their near-complete ribosomal DNA sequences, obtaining approximately 6.8 kb for each species, and reported the first rDNA sequence for P. bilobus. Genetic analysis confirmed that each species clusters within its respective genus, supporting the hypothesis that Patagifer and Echinostoma are sister lineages. Grus japonensis is also recorded as a new host for P. bilobus and the first known crane host for any Patagifer species. These findings provide valuable molecular data for future research on the diversity, transmission, and control of this important parasite family.

Abstract

Echinostomatidae is a taxonomically complex group with substantial species diversity and richness. The vast majority of species in this family parasitize birds and mammals, including humans, causing significant economic losses and medical costs. In this study, Echinostoma miyagawai (Digenea, Echinostomatidae) and Patagifer bilobus (Digenea, Echinostomatidae) were isolated from domestic duck and Grus japonensis, respectively. The nearly complete ribosomal transcription unit (rTU) sequences of two echinostomes were obtained, with the rTU for P. bilobus being obtained for the first time. The nearly complete rTU sequence of P. bilobus (6790 bp) and E. miyagawai (6893 bp) encompass the small-subunit (18S) ribosomal DNA (rDNA), internal transcribed spacer 1 (ITS1), 5.8S rDNA, internal transcribed spacer 2 (ITS2), and large-subunit (28S) rDNA. The complete lengths of 18S, ITS1, 5.8S, ITS2, and 28S sequences for E. miyagawai are 1989 bp, 444 bp, 162 bp, 431 bp, and 3858 bp, respectively. For P. bilobus, complete or nearly complete lengths of these sequences are 1929 bp (nearly complete), 419 bp, 162 bp, 432 bp, and 3848 bp (nearly complete), respectively. The 18S, ITS, and 28S sequences of E. miyagawai show the highest sequence similarity with other E. miyagawai. The ITS and 28S sequences of P. bilobus show the highest sequence similarity with other P. bilobus, while 18S sequence shows the highest similarity with E. miyagawai. This is likely due to the unavailability of the 18S sequence of P. bilobus in GenBank. Repeat sequences were identified in 18S, ITS1, ITS2, and 28S sequences, with the 28S sequence containing the most repeats and the 5.8S sequence having none. The results of phylogenetic reconstruction indicated that E. miyagawai clusters with other Echinostoma spp., while P. bilobus clusters with other Patagifer spp., forming sister taxa. This study not only provides the first rTU sequence for P. bilobus but also reinforces the sister group status of Patagifer to Echinostoma through phylogenetic evidence. Finally, this study represents the first record of the G. japonensis as a new host for P. bilobus and the first report of a bird from the crane family (Gruidae) as a host for any echinostome species. These findings are significant as they expand our understanding of the host range and ecological interactions of Echinostomatidae. The data obtained provide a valuable resource of molecular markers for studying the taxonomy, population genetics, and systematics of the family Echinostomatoidea. This research contributes to a more comprehensive understanding of the evolutionary relationships and biodiversity within this complex group of parasites, which is crucial for developing effective strategies to mitigate their impact on both wildlife and human health.

1. Introduction

Echinostomiasis is a neglected parasitic disease caused by intestinal trematodes from the family Echinostomatidae, which includes over 50 genera and more than 350 species [1,2]. Echinostomes mainly inhabit the intestines of waterfowls and mammals, including humans. Additionally, certain echinostome species have also been documented in reptiles and fishes [3]. In the poultry industry, echinostomes have become significant health problems, causing low feed conversion ratios and resulting in economic losses. Moreover, they pose a considerable threat to public health and safety. The life cycle of Echinostomatidae is relatively complex and involves a multi-host indirect life history. The specific process is as follows: Eggs of the adult trematode are excreted from the host’s body via feces. Under suitable environmental conditions, the ovum within the egg begins to divide and develops into a miracidium. After hatching, the miracidium enters the water and swims freely, searching for the first intermediate host (usually aquatic snails). Once the miracidium penetrates the snail, it develops during the sporocyst and redia stages, eventually forming a cercaria. The cercaria can either continue to develop into a metacercaria within the same snail or leave the snail and burrow into other second intermediate hosts (such as other snails, bivalves, fish, frogs, tadpoles, etc.). The metacercaria, which is the infective stage of Echinostomatidae, can be found within the second intermediate host or attached to aquatic plants. Infection occurs when the definitive host consumes the second intermediate host containing infectious metacercariae raw or undercooked [4]. Although echinostomes are distributed worldwide, human echinostomiasis mainly occurs in southeastern, eastern, and northeastern Asian countries due to eating habits [5]. In summary, Echinostomatidae trematodes are significant not only to public health and agriculture but also in ecological research. Understanding their ecological characteristics and practical significance can aid in developing effective control strategies, safeguarding human and animal health, and promoting the sustainable development of ecosystems. A defining feature of echinostomes is the presence of a collar of spines encircling the oral sucker in one or two rows. The quantity and arrangement of these spines are crucial for taxonomic identification. However, the systematic classification of echinostomes remains highly complex and is constantly undergoing revision and phylogenetic analysis [6,7]. Information on the genera, species, intermediate hosts, geographic distribution, and host species of Echinostoma spp. and Patagifer spp. is listed in Table 1.
The family Echinostomatidae displays a high degree of taxonomic variation, and there has been an important controversy about the taxonomy of echinostomes. Although the number and arrangement of the head spines of echinostomes are key to morphological identification, the count of spines can fluctuate among individuals within the same species or developmental stage, and during the process of preparing specimens, morphological traits and spines might be lost, which can result in misidentification. Molecular techniques utilizing genetic markers have become an indispensable tool in the taxonomy of echinostomes. It is reported that the taxonomy of the species of Echinostoma and Echinoparyphium can be investigated through the analysis of the mitochondrial DNA (mtDNA) [49,90]. In addition, it is reported that rDNA and mtDNA can be used for the identification of complex species or new species in echinostomes as gene makers [6,62].
Ribosomal DNA is a very important component of the cellular genome, playing a key role in protein synthesis, genome stability, and cell growth and proliferation. The primary function of rDNA is to encode ribosomal RNA (rRNA). Ribosomal RNA is one of the main components of ribosomes and plays a central role in the structure and function of ribosomes. Ribosomal DNA typically exists in the genome as tandem repeats. This repetitive structure can ensure that the cell has sufficient templates to synthesize large amounts of rRNA, thereby meeting the cell’s demand for ribosomes. And the transcriptional unit contains three genes (18S, 5.8S, and 28S rRNA) with two internal transcribed spacers (ITS1 and ITS2) separating the genes and an intergenic spacer (IGS) [91]. Moreover, the rDNA region contains a large number of repetitive sequences, which can act as a buffering mechanism [92]. When the genome is damaged or undergoes mutations, these repetitive sequences can provide additional templates to help repair the damaged genomic regions. Ribosomal DNA has extremely important application value in biological taxonomy, especially in molecular phylogenetics and species identification. Ribosomal DNA in taxonomy has not only improved the accuracy and efficiency of species identification but also provided a powerful tool for studying the evolutionary history of organisms. By analyzing rDNA sequences, scientists can gain a deeper understanding of the phylogenetic relationships among organisms, reveal the kinship between species, and thereby promote the development of biological taxonomy [93]. It is reported rDNA have been crucial in resolving taxonomic issues for trematodes [94,95].
This study isolated two species of Echinostomatidae trematodes, characterized by their plump and elongated leaf-shaped bodies, from the intestines of domestic duck and G. japonensis, respectively. Based on the morphological characteristics, hosts, and parasitic sites, these two echinostomes were identified as belonging to the genera Echinostoma and Patagifer. The higher taxa of these two species are listed in Table 2. Echinostoma is the type genus of the family Echinostomatidae [4]. The genus Echinostoma can be divided into five groups, one of which, the “revolutum” group, is characterized by 37 “collar-spines” [4,96]. Echinostoma miyagawai, a member of the “revolutum” group, primarily parasitizes the intestines of waterfowl, causing symptoms such as diarrhea, anemia, and stunted growth [21,49]. This species has also been documented in humans in China [97,98]. With the family Echinostomatidae, the genus Patagifer is relatively less reported. It currently comprises 12 recognized species that mainly parasitize birds of the family Threskiornithidae and are distributed worldwide [88]. In addition to Threskiornithidae birds, Patagifer species have also been reported in birds from the families Ardeidae, Podicipedidae, and Scolopacidae [87,99]. However, this study marks the first isolation of a Patagifer species from the family Gruidae, thereby enriching the host diversity of Patagifer species. The type species of the genus Patagifer, Patagifer bilobus, parasitizes the intestines of birds and distributed in Eastern Europe, Egypt, Australia, Brazil, and China [100]. This paper presents the nearly complete rTU sequences of E. miyagawai and P. bilobus, with the rTU sequence for P. bilobus being obtained for the first time. The current research aims to study 18S, ITS, and 28S sequences as a genetic marker for the genetic characterization of Echinostomatidae species, the reconstruction of its phylogenetic relationship, and a comparison of these characterizations. The phylogenetic evidence reinforces sister group status of Patagifer to Echinostoma. These data provide valuable molecular resources for resolving taxonomic ambiguities within the Echinostomatidae family. Lastly, this study represents the first record of G. japonensis as a new host for P. bilobus. This finding not only expands our understanding of the host range of Patagifer species but also highlights the importance of continued research into the ecological and evolutionary dynamics of Echinostomatidae trematodes. Such research is crucial for developing effective strategies to manage and mitigate the impact of these parasites on both wildlife and human health.

2. Materials and Methods

2.1. Trematodes Collection and Species Identifcation

Echinostoma miyagawai were collected from the intestine of naturally infected domestic duck in Qiqihar City, China. Patagifer bilobus were collected from the intestine of naturally infected G. japonensis in the Zhalong National Nature Reserve, China, in compliance with the Wildlife Protection Law of the People’s Republic of China (a draft of an animal protection law in China released on 2018). The trematodes were thoroughly washed in physiological saline solution. Preliminary species identification was based on size and host predilection [50,87]. Further species identification was also performed by PCR amplification of the 28S sequence, using the primers reported in a previous study [101].

2.2. Primers and Amplifcation

Total genomic DNA was extracted from each sample using the TIANamp Genomic DNA Kit (Tiangen, Beijing, China), following the manufacturer’s instructions. The extracted DNA samples were tested for nucleic acid concentration and stored at −20 °C for subsequent genetic analysis. Five pairs of primers were designed based on the multiple alignments of echinostome rTU sequences, and the primer sequences are listed in Table S1. The PCR reactions (25 µL) were performed using 18.30 µL of distilled water, 2.50 µL of 10 × Ex Taq buffer, 2 µL of dNTP Mixture (2.5 mM), 0.50 µL of each primer (25 mM), 1 µL of extracted DNA, and 0.20 µL of Ex Taq DNA polymerase (5 U/µL). The DNA template of positive control used fluke DNA stored in the laboratory, while the negative control used distilled water. The PCR cycling conditions were as follows: 94 °C for 5 min (initial denaturation), followed by 35 cycles at 94 °C for 30 s (denaturation), 50–65 °C for 1 min (annealing), and 72 °C for 1 min 30 s (extension), with a final extension at 72 °C for 10 min. The PCR products with positive bands were sequenced using the Sanger method (Sangon Biotech (Shanghai) Co., Ltd., Shanghai, China).

2.3. Sequence Analysis and Annotation of the Ribosomal Transcription Units

The sequences were assembled manually and aligned against the Echinostomatidae trematode sequences in GenBank to identify gene boundaries, using the program DNAStar v. 5.0 [102]. The edited sequences were submitted to GenBank for accession ID numbers. The AT and GC content were calculated using DNAStar v. 5.0 [102]. Forward, reverse, complement, and palindromic repeats were examined by REPuter [103]. These repeats were ≥10 bp with a maximum computed repeats of 100 bp. The minimum identity of repeats was set at 90% (Hamming distance of 1). These repeat sequences were manually checked for alignment accuracy. Interspecific variation within the Echinostomatidae was calculated using MEGA v. 5.0 and MegAlign v. 5.01 [102,104].

2.4. Ribosomal Phylogenetic Analyses and Tree Reconstruction

To examine the phylogenetic and taxonomic position of the Echinostomatidae, three phylogenetic trees were reconstructed from the alignment of sequences of the 18S, ITS2, and 28S. We used rTU sequences of Echinostomatidae available in GenBank to create our phylogenetic datasets. Paramphistomum cervi Zeder, 1790 was included as an outgroup. Phylogenetic trees were reconstructed using Bayesian inference (BI), maximum likelihood (ML), and maximum parsimony (MP) methods. Bayesian inference was performed using the mixed model in MrBayes v. 3.1.1 and 1,000,000 metropolis-coupled Markov chain Monte Carlo generations. The first 250 trees were omitted as burn-in, and the remaining trees were used to calculate Bayesian posterior probabilities [105]. Maximum parsimony and ML analyses were performed using the Fitch criterion within PAUP v. 4.0 Beta 10 [106], and bootstrap support values were calculated in PAUP from 1000 bootstrap replicates with 10 random additions per replicate. Phylograms were viewed and drawn using FigTree V. 1.31 (http://tree.bio.ed.ac.uk/software/figtree/; accessed on 20 May 2025).

3. Results

3.1. Species Identifcation of E. miyagawai and P. bilobus

The 28S sequences obtained in this study for E. miyagawai (PV595723) and P. bilobus (PV569527) were aligned against the sequences of E. miyagawai and P. bilobus available in GenBank (KP065593, KT956945), showing sequence identities of 100% and 98.40%, respectively. Those two echinostomes were identified as E. miyagawai and P. bilobus, respectively.

3.2. Ribosomal Transcription Unit Features of the E. miyagawai and P. bilobus

In this study, the rTU sequences obtained were nearly complete for E. miyagawai (6893 bp) and P. bilobus (6790 bp), including the complete coding regions from 5′ terminus of the 18S rRNA gene to the 3′ terminus of the 28S rRNA gene. The positive bands of PCR products amplified were good, which were sequenced using the Sanger method (Figures S1 and S2). These sequences were submitted to GenBank with the accession numbers PV600287 and PV600288.
In rTU of E. miyagawai, the lengths of complete 18S, ITS1, 5.8S, ITS2, and 28S sequences were 1989 bp, 444 bp, 162 bp, 431 bp, and 3858 bp, respectively. The nucleotide composition of the 18S-28S, 18S, ITS1, 5.8S, and 28S sequences is biased toward C and G, with an overall C + G content of 51.98%, 50.68%, 52.93%, 53.09%, and 52.95%, respectively, and A + T content of 48.02%, 49.32%, 47.07%, 46.91%, and 47.05%, respectively. The nucleotide composition of ITS2 is biased towards A and T, with an overall A + T content of 50.35% and C + G content of 49.65%. A total of 52 repeat sequences were identified, including 11 forward, 27 reverse, 7 complement, and 7 palindromic repeats. The 28S sequence contained the most repeats, with a total of 41 repeat sequences (Table S2). The 18S sequence of E. miyagawai shows the highest sequence similarity with E. miyagawai, E. paraensei, and E. revolutum, reaching 99.20%. The ITS and 28S sequences of E. miyagawai show the highest sequence similarity with E. miyagawai, reaching 97.20% and 100%, respectively. The 18S, ITS, and 28S sequences of E. miyagawai show the lowest sequence similarity with Mesorchis denticulatus Rudolphi, 1802, Euparyphium capitaneum Rudolphi, 1802, and P. bilobus, reaching 95.70%, 80.20%, and 84.40%, respectively.
In the rTU of P. bilobus, the lengths of complete or nearly complete 18S, ITS1, 5.8S, ITS2, and 28S sequences were 1929 bp (nearly complete), 419 bp, 162 bp, 432 bp, and 3848 bp (nearly complete), respectively. The nucleotide composition of the 18S-28S, 18S, ITS1, 5.8S, and 28S sequences is biased toward C and G, with overall C + G content of 52.12%, 50.49%, 52.98%, 53.09%, and 53.07%, respectively, and A + T content of 47.88%, 49.51%, 47.02%, 46.91%, and 46.93%, respectively. The nucleotide composition of ITS2 nucleotide composition is biased towards A and T, with an overall A + T content of 50.23% and C + G content of 49.77%. A total of 55 repeat sequences were identified, including 11 forward, 25 reverse, 9 complement, and 10 palindromic repeats. The 28S sequence contained the most repeats, with a total of 45 repeat sequences (Table S3). The 18S sequence of P. bilobus shows the highest sequence similarity with E. miyagawai and E. revolutum, reaching 98.90%, and the lowest sequence similarity with M. denticulatus and Euparyphium melis Dietz 1909, reaching 95.90%. The ITS and 28S sequences of P. bilobus show the highest sequence similarity with P. bilobus, reaching 94.60% and 98.40%, respectively, and the lowest sequence similarity with Pegosomum asperum Rudolphi, 1802 and Pegosomum saginatum Rudolphi, 1802, reaching 81.60% and 77.60%, respectively.

3.3. Phylogenetic Analyses

Phylogenetic analyses using three methods (BI, ML, and MP) yielded identical tree topologies based on 18S, ITS, and 28S sequences, respectively (Figure 1, Figure 2 and Figure 3). The phylogenetic tree of 18S splits into two large clades. The first clade contains the species of Pegosomum, Euparyphium, and M. denticulatus. In the second clade, E. miyagawai from this study clusters with E. miyagawai, E. revolutum, and E. caproni, and P. bilobus formed a sister taxon with them. The phylogenetic tree of ITS splits into two large clades. The first clade contains the species of Pegosomum, Petasiger, and E. hortense. The second clade splits into two large groups. In the first group, Echinostoma species cluster together, and E. miyagawai from this study clusters with E. miyagawai and E. revolutum. Patagifer bilobus from this study clusters with P. bilobus, forming a sister taxon with Echinostoma species. All of the Echinostoma species cluster together, except E. hortense. The phylogenetic tree of 28S splits into two large clades. The first clade contains the species of Pegosomum, Petasiger, and Euparyphium. The second clade splits into two large groups. One group contains the species of Echinostoma and Patagifer, and the other contains the species of Echinoparyphium and Hypoderaeum. In the first group, E. miyagawai from this study clusters with E. miyagawai. Patagifer bilobus from this study clusters with P. bilobus and P. vioscai. In this study, the results for 28S were similar to those for the 18S and ITS sequences: Echinostoma species cluster together, and Patagifer species form a sister taxon with them. Further characterization of the phylogeny of Echinostoma and Patagifer species will need to wait until additional genomic trematode data have been deposited in GenBank.

4. Discussion

Echinostomes are intestinal parasites characterized by the “collar-spines”, a complex life cycle, and a broad range of definitive hosts that include humans. And Echinostomatidae are not only an overlooked food-borne pathogen but also a sensitive indicator of the complex ecological interactions among water bodies, snails, and hosts. Their control is of practical significance for public health, livestock production, and ecological security. Echinostoma miyagawai and P. bilobus both belong to Trematoda, Digenea, and Echinostomatidae. The taxonomic status of E. miyagawai has long been a focus of scholarly attention. Echinostoma miyagawai was first described by Ishii in 1932. And later, in 1937, Beaver [107] treated the species as synonym of E. revolutum according to morphological resemblance. But Bashkirova [108] and Skrjabin & Bashkirova [109] disagreed with their viewpoints and reinstated the two as distinct on both morphological and ecological grounds. In contrast, Kanev [110] dismissed E. miyagawai altogether, sinking it into Echinostoma echinatum Zeder, 1803. Kostadinova et al. [54,55] re-established E. miyagawai using distinctive cercarial chaetotaxy and detailed morphometrics of larvae and adults, clearly separating it from E. revolutum. Fried & Graczyk [111] and Toledo et al. [112] later concurred with this recognition. Finally, Faltýnková et al. [21] redescribed E. miyagawai adults from ducks across Central and Western Europe, placing E. friedi into synonymy with E. miyagawai. In addition, the sequences of E. miyagawai were obtained from both the definitive host and the intermediate host [57]. In conclusion, all available evidence supports the status of E. miyagawai as a valid species. Heneberg suggested that besides the issues with morphological species identification, it is important to highlight that further analyses should also refrain from raising conclusions that are based solely on variations in only a few informative sites, as the otherwise hypervariable genes, such as ITS1 or ITS2, are nearly invariable across the “revolutum” group complex [113]. In this study, the body of E. miyagawai is dorsoventrally flattened, robust and muscular, elongated and leaf-like, tapering slightly anteriorly and rounded posteriorly. The anterior end exhibits the characteristic echinostome features: an oral sucker and a head collar. The oral sucker is small, while the head collar is well-developed, prominent and muscular, armed with collar-spines numbering 37 in total, and the testes are petal-shaped. These characteristics are consistent with descriptions provided by other authors [50,51].
The genus Patagifer is a small genus of echinostomatids, which mainly parasitize birds of the family Threskiornithidae [88]. Besides Threskiornithidae birds, Patagifer species have also only been reported in birds of the families Ardeidae, Podicipedidae, and Scolopacidae [87,99]. This study reported for the first time a new host for the genus Patagifer, the G. japonensis (Gruidae), thereby enriching the host diversity of the genus Patagifer. The classification and species inventory of the genus Patagifer have been intricate and inconsistent, largely because the defining traits of the species exhibit significant phenotypic variability. In 1909, Dietz established the genus Patagifer, which includes Distoma bilobum Rudolphi, 1819 (syn. P. bilobus) and Patagifer consimilis Dietz, 1909. This was done to differentiate species characterized by a unique deep dorsal incision and a prominent ventral notch in the collar, features that are distinctive within the Echinostomatidae family and result in a characteristic bilobed appearance. Despite its relatively small size, the genus Patagifer has a complex taxonomic history and species composition. This complexity is largely due to the ambiguous description provided by Dietz (1910), which attributed significant variation to the type species P. bilobus. Additionally, the long-standing issue of insufficient or imprecise descriptions and poor differential diagnoses for newly recognized taxa has further complicated the synonymy. In 1956, Skrjabin & Bashkirova considered P. bilobus, Patagifer consimilis Dietz, 1909, Patagifer parvispinosus Yamaguti, 1933, and Patagifer wesleyi Verma, 1936 to be indistinguishable and questioned the distinct species status of P. parvispinosus and P. wesleyi [109]. In 1968, Machida considered that Patagifer chandrapuri Srivastava, 1952 and Patagifer sarai Saksena, 1957 were synonyms of P. bilobus [114]. In 1970, Jain & Srivastava considered that Patagifer simerai Nigam, 1944 was a synonym of P. bilobus [115]. In 1982, Srivastava considered that P. simarai and P. sarai were synonyms of P. bilobus [116]. Previous researchers have identified genus Patagifer based on their ecological characteristics, such as the ratio of the collar width to the body width, the proportion of the suckers, and the shape of the testes. However, there is no general consensus on the importance of these characteristics. Until 2008, Faltýnková et al. conducted the most comprehensive taxonomic study on the genus Patagifer and identified 11 species of Patagifer [87]. However, in the same year, Dronen & Blend did not follow the species identification key provided by Faltýnková et al. and described a 12th species, Patagifer lamothei, from the white ibis (Eudocimus albus) in the United States [99]. Faltýnková et al. considered that the characteristics of P. bilobus include the following: a ribbon-like body with almost parallel edges; a collar with a deep dorsal incision and a narrow ventral notch, bearing 52–58 collar-spines; and four subequal corner spines (one of which is slightly smaller) on each ventral lobe, which are smaller than the largest lateral spine. And P. bilobus in this study conformed to the morphological characteristics described above, and it had 54 collar-spines.
Qiqihar City is located in northeastern China, on the Songnen Plain, between 122°24′–126°41′ E and 46°13′–48°56′ N. It has a cold-temperate continental climate: winters are cold, summers are warm and humid, and the four seasons are distinct. The Nen River flows through the city, providing abundant water resources. The Nen River is approximately 1370 km long, playing an irreplaceable role in the local ecological environment and economic development. Echinostoma miyagawai was collected from domestic ducks in the Nen River basin in Qiqihar City. Domestic ducks in Northeast China originated from the wild mallard (Anas platyrhynchos) and were domesticated and selectively bred by local farmers over several centuries. Today, their populations are most numerous along the Songhua River basin and in the Songnen Plain. The Nen River basin is rich in aquatic animal resources, including a variety of fish and snails, which facilitates the transmission of the E. miyagawai. The kinds of fish in Nen River include the following: I. The family Cyprinidae: Cyprinus carpio haematopterus Temminck & Schlegel, 1842; Ctenopharyngodon idellus Cuvier & Valenciennes, 1844; Leuciscus waleckii Dybowski, 1869; Hemiculter leucisculus Basilewsky, 1855; Xenocypris argentea Günther, 1868; Rhodeus sericeus Pallas, 1776; and so on. II. The family Cobitidae: Nemacheilus nudus Blyth, 1860; Misgurnus mohoity Dybowski, 1869; Paramisgurnus dabryanus Sauvage, 1878. III. Others: Siniperca chuatsi Basilewsky, 1856; Sander lucioperca Linnaeus, 1758; Pelteobagrus fulvidraco Richardson, 1846; Pelteobagrus nitidus Sauvage & Dabry, 1874; Lota lota Linnaeus, 1758; Pungitius sinensis Guichenot, 1869. The kinds of snails in Nen River include the following: Radix auricularia Linnaeus, 1758; Radix ovata Draparnaud, 1805; Radix plicatula Benson, 1842; Radix pereger Müller, 1774; Viviparus chui Gray, 1847; Cipangopa chinensis Gray, 1834; Bellamya aeruginosa Reeve, 1863; Semisulcospira amurensis Gerstfeldt, 1859; Unio dougladiae Griffith & Pidgeon, 1834. The intermediate hosts of E. miyagawai that have been reported include P. planorbis, A. vortex, L. truncatula, L. stagnalis, and L. palustris, as shown in Table 1. However, the snail species reported above do not overlap with those in the Nen River, which implies that E. miyagawai may have a new intermediate host. This hypothesis will be tested in future studies. Patagifer bilobus was collected from G. japonensis in the Zhalong National Nature Reserve. The Zhalong National Nature Reserve is located in the western Songnen Plain of Heilongjiang Province, in the marshy reed wetlands of the lower Wuyur River basin. Its geographic coordinates are 123°47′–124°37′ E and 46°52′–47°32′ N, and the reserve covers a total area of 210,000 hectares. The Zhalong National Nature Reserve is a wetland ecosystem dominated by reed marshes, interspersed with lakes, meadows, grasslands, and saline–alkali lands. Zhalong is hailed as the “cradle of cranes”. Of the 15 crane species on Earth, 9 are found in China, and 6 of them are found in Zhalong: the Red-crowned Crane, Siberian (White) Crane, Hooded Crane, White-naped Crane, Common Crane, and Demoiselle Crane. Among these, the global population of Red-crowned Cranes is roughly 2000 individuals, and Zhalong alone hosts more than 400 breeding birds, making it the largest breeding ground for this species in the world. Grus japonensi lead a migratory life and transfer parasites between ecosystems during migration. The Zhalong Nature Reserve is not only a cornerstone of the global crane-conservation network but also a quintessential example of northern China’s wetland ecosystems, holding an irreplaceable strategic position in worldwide wetland and avian conservation. The kinds of fish in the Zhalong Nature Reserve include the following: Carassius auratus Linnaeus, 1758; Cyprinus carpio Linnaeus, 1758; Ctenopharyngodon idella Valenciennes, 1844; Hypophthalmichthys molitrix Valenciennes, 1844; Aristichthys nobilis Richardson, 1845; Pseudorasbora parva Temminck & Schlegel, 1846; Tachysurus fulvidraco; Channa argus Richardson, 1846; Misgurnus anguillicaudatus Cantor, 1842; and so on. The kinds of amphibians in the Zhalong Nature Reserve include the following: Salamandrella keyserlingii Dybowski, 1870; Bufo gargarizans Cantor, 1842; Strauchbufo raddei Kessler, 1878; Hyla immaculata Boettger, 1888; Pelophylax nigromaculatus Hallowell, 1861; Rana amurensis Boulenger, 1886. As for the latest publicly available information, a complete species list of gastropods (mollusca) in the Zhalong National Nature Reserve has not yet been published. The intermediate host of P. bilobus remains undetermined, and relevant information will be investigated in future studies.
In this study, the nearly complete rTU sequences were obtained for E. miyagawai (6893 bp) and P. bilobus (6790 bp). The nearly complete rTU sequence of E. miyagawai (OR509027) in the NCBI database was found in only one record, which was isolated from Anas platyrhynchos domesticus in Thailand. In rTU of E. miyagawai (OR509027), the lengths of complete 18S, ITS1, 5.8S, ITS2, and 28S sequence were 1988 bp, 417 bp, 160 bp, 428 bp, and 3861 bp, respectively. Compared with the sequence lengths of various regions of E. miyagawai in this study, discrepancies were observed between the two sequences. Alignment revealed several missing nucleotides, most notably a 26bp deletion within the ITS1 region. The 18S, ITS1, 5.8S, ITS2, and 28S sequence similarity of E. miyagawai (OR509027 and obtained this study) were 99.3%, 93.9%, 98.8%, 99.3%, and 99.8%, respectively. The 18S-28S, 18S, ITS1, 5.8S, and 28S sequence nucleotide composition of E. miyagawai are biased toward C and G, and the ITS2 nucleotide composition are biased toward A and T. The rTU of E. miyagawai had four types of repeat sequences, forward, reverse, complement, and palindromic repeats, and repeat sequences of 28S were the most. Internal repeats appear to be characteristic of the 28s evolution in different groups of organisms [91,117]. The sequences of P. bilobus in the NCBI database are limited. Currently, only partial ITS sequences, 28S sequences, and nad1 sequences are available. This study was the first time to amplify the nearly complete rTU sequences of P. bilobus. Not only does this fill the gap in the molecular sequence data of Chinese Patagifer spp., but it also reports a new host—G. japonensis. The ITS sequence similarity of P. bilobus (ON141929 and obtained this study) was 94.6%. The 28S sequence similarity of P. bilobus (ON141919 and obtained this study) was 98.4%. The 18S-28S, 18S, ITS1, 5.8S, and 28S sequence nucleotide composition of P. bilobus are biased toward C and G, and the ITS2 nucleotide composition are biased toward A and T. The bases (A, T, G, and C) are the fundamental components of DNA and RNA, and they play multiple important roles in the structure, function, and regulation of the genome. By encoding amino acids, regulating gene expression, participating in gene replication and recombination, and influencing gene function through modifications, the bases have played a key role in biological evolution and adaptation to the environment. In bioinformatics research, analyzing base sequences and their modifications can help us better understand the function and evolutionary mechanisms of the genome. And 28S sequence of P. bilobus also had the most repeat sequences, which is consistent with E. miyagawai. These repeat sequences have multiple functions in the genome, including gene regulation, gene replication, genome recombination, and gene silencing. They play an important role in the evolutionary process of organisms, influencing gene expression and genome structure to adapt to different environmental pressures. In bioinformatics research, the identification and analysis of these repeat sequences can help us better understand the function and evolutionary mechanisms of the genome.
Phylogenetic analyses using three methods (BI, ML, and MP) yielded identical tree topologies based on 18S, ITS, and 28S sequences, respectively. The results obtained from the three methods are similar. All Echinostoma species, except for E. hortense, cluster within a single clade, and Patagifer species form a sister clade with them. This is consistent with a previous study in which Echinostoma species cluster together, and Patagifer species formed a sister taxa with them, using 28S sequence with BI and ML methods [86]. Interestingly, all of the Echinostoma species cluster together, except E. hortense. This is consistent with a previous study in which E. hortense was not clustered together with Echinostoma species, while it clustered with Fasciola species, using mtDNA sequences [49,118]. And it is also consistent with a previous study using the 28S rRNA gene in which Echinostoma species cluster together on one branch, and Isthmiophora hortensis Lache, 1909 (Syn. E. hortense) is a sister taxa and cluster together with Petasiger species [86]. Interestingly, Echinostoma species in the phylogenetic tree all belong to 37 “collar-spines”, except E. hortense, and E. hortense has 27–28 “collar-spines” [34]. Similar ambiguous results regarding the location of I. hortensis based on both 28S and ITS2 sequence phylogenies have been inferred [119]. We speculate that the reason why E. hortense was not clustered together with other Echinostoma species might be due to the different numbers of collar-spines. The taxonomic status of E. hortense still needs further research to be explored. Further characterization of the species of Echinostoma and Patagifer phylogeny will need to wait until additional genomic trematode data has been deposited in GenBank. These findings not only provide new insights into the phylogenetic relationships of Echinostoma and Patagifer species, but they also highlight the importance of integrating multiple molecular markers and analytical methods in evolutionary studies. Future research should further explore the morphological and ecological characteristics of these species to better understand their evolutionary history and adaptive differentiation. Moreover, with the accumulation of more genomic data and advancements in technology, we expect to more comprehensively resolve the phylogenetic relationships of these species, providing a stronger scientific basis for biodiversity conservation and disease control.

5. Conclusions

In this study, we successfully obtained the nearly complete rTU sequences of E. miyagawai and P. bilobus and conducted detailed sequence and phylogenetic analyses. The acquisition of these sequence data has greatly enriched our understanding of the genetic characteristics of these two species of Echinostomatidae. The results of the phylogenetic analysis provide molecular evidence for the morphological classification of the genus Echinostoma and underscore the importance of integrating morphological features and molecular data in the taxonomic studies of trematodes. Additionally, the species of Patagifer form a sister clade with Echinostoma species, a result that further clarifies the phylogenetic position of Patagifer within the family Echinostomatidae and offers new insights into the evolutionary relationships within this family. It is worth noting that this study, for the first time, determined the nearly complete rTU sequence of P. bilobus. This achievement fills the gap in the molecular data of Patagifer and lays the foundation for future genetic research. Moreover, we have also reported for the first time a new host for Patagifer spp., G. japonensis. This discovery not only expands our knowledge of the host range of Patagifer spp. but also provides a new case for studying the interactions between trematodes and their hosts. From a phylogenetic perspective, more detailed genetic analyses will provide valuable information for the taxonomy, population genetics, and phylogenetics of the family Echinostomatidae. These data will serve as important molecular markers to help reveal the evolutionary history, population structure, and phylogenetic relationships among species within the family. Future research can further utilize these molecular markers, in combination with additional genomic data, to delve into the phylogenetic relationships of the Echinostomatidae and their adaptive evolution in different ecological environments. Moreover, these research findings will also provide a scientific basis for the prevention and control of trematode diseases, aiding in the development of more effective public health strategies.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/biology14081101/s1, Figure S1: Agarose gel electropheoresis of rTU PCR products of E. miyagawai samples; Figure S2: Agarose gel electropheoresis of rTU PCR products of P. bilobus samples; Table S1: Sequences of primers used to amplify PCR fragments of 18S-28S rDNA of Echinostoma miyagawa and Patagifer bilobus; Table S2: Information of repeat sequences in the 18S-28S rDNA of Echinostoma miyagawa; Table S3: Information of repeat sequences in the 18S-28S rDNA of Patagifer bilobus.

Author Contributions

All authors contributed to the study’s conception and design. Y.C. and Y.L. developed the rationale of the study and wrote the manuscript. Y.C. designed and performed most of the experiments with contributions from Y.L.; Z.-Y.G. and B.-T.J. supervised the study. All authors have read and agreed to the published version of the manuscript.

Funding

Open access funding provided by Heilongjiang Academy of Agricultural Sciences. This work was supported by the Heilongjiang Province agricultural science and technology innovation leapfrog project agricultural science and technology basic innovation project (Excellent Young Scholars) (Project number: CX23BS06).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

No new data were created or analyzed in this study.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Toledo, R.; Álvarez-Izquierdo, M.; Esteban, J.G.; Muñoz-Antoli, C. Neglected food-borne trematodiases: Echinostomiasis and gastrodiscoidiasis. Parasitology 2022, 149, 1319–1326. [Google Scholar] [CrossRef]
  2. Chai, J.Y.; Jung, B.K. Foodborne intestinal fukes: A brief review of epidemiology and geographical distribution. Acta Trop. 2020, 201, 105210. [Google Scholar] [CrossRef] [PubMed]
  3. Toledo, R.; Alvárez-Izquierdo, M.; Muñoz-Antoli, C.; Esteban, J.G. Intestinal Trematode Infections. Adv. Exp. Med. Biol. 2019, 1154, 181–213. [Google Scholar]
  4. Toledo, R.; Conciancic, P.; Fiallos, E.; Esteban, J.G.; Muñoz-Antoli, C. Echinostomes and Other Intestinal Trematode Infections. Adv. Exp. Med. Biol. 2024, 1454, 285–322. [Google Scholar]
  5. Chai, J.Y.; Jung, B.K. General overview of the current status of human foodborne trematodiasis. Parasitology 2022, 149, 1262–1285. [Google Scholar] [CrossRef] [PubMed]
  6. Izrailskaia, A.V.; Besprozvannykh, V.V.; Tatonova, Y.V. Echinostoma chankensis nom. nov., other Echinostoma spp. And Isthmiophora hortensis in East Asia: Morphology, molecular data and phylogeny within Echinostomatidae. Parasitology 2021, 148, 1366–1382. [Google Scholar] [CrossRef]
  7. Pham, L.T.K.; Saijuntha, W.; Lawton, S.P.; Le, T.H. 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. Parasitol. Res. 2022, 121, 899–913. [Google Scholar] [CrossRef]
  8. Nasincová, V. The life cycle of Echinostoma bolschewense (Kotova, 1939) (Trematoda: Echinostomatidae). Folia Parasitol. 1991, 38, 143–154. [Google Scholar]
  9. Fried, B.; Huffman, J.E. The Biology of the intestinal trematode Echinostoma caproni. Adv. Parasitol. 1996, 38, 311–368. [Google Scholar] [CrossRef]
  10. Morgan, J.A.; Blair, D. Relative merits of nuclear ribosomal internal transcribed spacers and mitochondrial CO1 and ND1 genes for distinguishing among Echinostoma species (Trematoda). Parasitology 1998, 116, 289–297. [Google Scholar] [CrossRef] [PubMed]
  11. Laidemitt, M.R.; Brant, S.V.; Mutuku, M.W.; Mkoji, J.M.; Loker, E.S. The diverse echinostomes from East Africa: With a focus on species that use Biomphalaria and Bulinus as intermediate hosts. Acta Trop. 2019, 193, 38–49. [Google Scholar] [CrossRef]
  12. Besprozvannykh, V.V. Structure and life cycles of trematodes Euparyphium melis and Euparyphium amurensis sp. n (Echinostomatidae) in the Primorsky region. Zool. Zhurnal. 2001, 80, 5–11. (In Russian) [Google Scholar]
  13. Hong, S.; Shin, H.; Lee, Y.H.; Hong, S.J.; Kim, S.R.; Kim, Y.K.; Son, Y.J.; Song, J.G.; Chai, J.Y.; Jung, B.K. Rare Case of Echinostoma cinetorchis Infection, South Korea. Emerg. Infect. Dis. 2024, 30, 1726–1729. [Google Scholar] [CrossRef] [PubMed]
  14. Chung, P.R.; Jung, Y.; Park, Y.K.; Hwang, M.K. Austropeplea ollula (Pulmonata: Lymnaeidae): A new molluscan intermediate host of a human intestinal fluke, Echinostoma cinetorchis (Trematoda: Echinostomatidae) in Korea. Korean J. Parasitol. 2001, 39, 247–253. [Google Scholar] [CrossRef]
  15. Chung, P.R.; Jung, Y.; Park, Y.K.; Hwang, M.G.; Soh, C.T. Corbicula fluminea (Bivalvia: Corbiculidae): A possible second molluscan intermediate host of Echinostoma cinetorchis (Trematoda: Echinostomatidae) in Korea. Korean J. Parasitol. 2001, 39, 329–332. [Google Scholar] [CrossRef]
  16. Cho, S.Y.; Kang, S.Y.; Ryang, Y.S. Helminthes infections in the small intestine of stray dogs in ejungbu city, Kyunggi Do, Korea. Kisaengchunghak Chapchi 1981, 19, 55–59. [Google Scholar]
  17. Zhan, X.D.; Li, C.P.; Yang, B.H.; Zhu, Y.X.; Tian, Y.; Shen, J.; Zhao, J.H. Investigation on the zoonotic trematode species and their natural infection status in Huainan areas of China. Nutr. Hosp. 2017, 34, 175–179. [Google Scholar] [CrossRef]
  18. Nguyen, L.A.T.; Madsen, H.; Dao, T.H.; Hoberg, E.; Dalsgaard, A.; Murrell, K.D. Evaluation of the role of rats as reservoir hosts for fishborne zoonotic trematodes in two endemic northern Vietnam fish farms. Parasitol. Res. 2012, 111, 1045–1048. [Google Scholar] [CrossRef]
  19. Anh, N.T.L.; Madsen, H.; Dalsgaard, A.; Phuong, N.T.; Thanh, D.T.H.; Murrell, K.D. Poultry as reservoir hosts for fishborne zoonotic trematodes in Vietnamese fish farms. Vet. Parasitol. 2010, 169, 391–394. [Google Scholar] [CrossRef] [PubMed]
  20. Toledo, R.; Muñoz-Antolí, C.; Esteban, J.G. The life-cycle of Echinostoma friedi n. sp. (Trematoda: Echinostomatidae) in Spain and a discussion on the relationships within the ‘revolutum’ group based on cercarial chaetotaxy. Syst. Parasitol. 2000, 45, 199–217. [Google Scholar] [CrossRef] [PubMed]
  21. Faltýnková, A.; Georgieva, S.; Soldánová, M.; Kostadinova, A. A re-assessment of species diversity within the ‘revolutum’ group of Echinostoma Rudolphi, 1809 (Digenea: Echinostomatidae) in Europe. Syst. Parasitol. 2015, 90, 1–25. [Google Scholar] [CrossRef]
  22. Liu, Z.X.; Zhang, Y.; Liu, Y.T.; Chang, Q.C.; Su, X.; Fu, X.; Yue, D.M.; Gao, Y.; Wang, C.R. Complete Mitochondrial Genome of Echinostoma hortense (Digenea: Echinostomatidae). Korean J. Parasitol. 2016, 54, 173–179. [Google Scholar] [CrossRef]
  23. Sohn, W.M.; Na, B.K.; Cho, S.H. Echinostoma hortense and heterophyid metacercariae encysted in yellowfin goby, Acanthogobius flavimanus, from Shinan-gun and Muan-gun (Jeollanam-do), Korea. Korean J. Parasitol. 2009, 47, 307–310. [Google Scholar] [CrossRef]
  24. Okanishi, H.; Matsumoto, J.; Nogami, S.; Kagawa, Y.; Watari, T. Echinostoma hortense infection with enteritis diagnosed by upper gastrointestinal endoscopy in a dog. J. Vet. Med. Sci. 2013, 75, 991–994. [Google Scholar] [CrossRef] [PubMed]
  25. Wen, L.; Wang, B.; Zhang, H. Clonorchis sinensis and Echinostoma hortense detected by endoscopy and molecular characterization: Two case reports and update on diagnosis. Front. Med. 2025, 11, 1515539. [Google Scholar] [CrossRef]
  26. Chai, J.Y.; Hong, S.; Sohn, W.M.; Lee, S.H.; Seo, B.S. Studies on intestinal tematodes in Korea: XVI. Infection status of loaches with the metacercariae of Echinostoma hortense. Kisaengchunghak Chapchi 1985, 23, 18–23. [Google Scholar] [CrossRef] [PubMed]
  27. Ahn, Y.K.; Ryang, Y.S.; Chung, P.R.; Lee, K.T. Echinostoma hortense metacercariae naturally encysted in Odontobutis obscura interrupta (a freshwater fish) and experimental infection to rats. Kisaengchunghak Chapchi 1985, 23, 230–235. [Google Scholar] [CrossRef] [PubMed]
  28. Chai, J.Y.; Shin, E.H.; Lee, S.H.; Rim, H.J. Foodborne intestinal flukes in Southeast Asia. Korean J. Parasitol. 2009, 47, 69–102. [Google Scholar] [CrossRef]
  29. Shin, S.S.; Oh, D.S.; Ahn, K.S.; Cho, S.H.; Lee, W.J.; Na, B.K.; Sohn, W.M. Zoonotic Intestinal Trematodes in Stray Cats (Felis catus) from Riverside Areas of the Republic of Korea. Korean J. Parasitol. 2015, 53, 209–213. [Google Scholar] [CrossRef]
  30. Seo, B.S.; Cho, S.Y.; Hong, S.T.; Hong, S.J.; Lee, S.H. Studies on parasitic helminths of Korea 5. survey on intestinal trematodes of house rats. Kisaengchunghak Chapchi 1981, 19, 131–136. [Google Scholar] [CrossRef]
  31. Tantrawatpan, C.; Saijuntha, W. Multiplex PCR development for the differential detection of four medically important echinostomes (Trematoda: Echinostomatidae) in Thailand. Acta Trop. 2020, 204, 105304. [Google Scholar] [CrossRef]
  32. Umathevy, T. The life-history of Echinostoma ilocanum. Med. J. Malaya. 1965, 20, 58. [Google Scholar]
  33. Sohn, W.M.; Kim, H.J.; Yong, T.S.; Eom, K.S.; Jeong, H.G.; Kim, J.K.; Kang, A.R.; Kim, M.R.; Park, J.M.; Ji, S.H.; et al. Echinostoma ilocanum infection in Oddar Meanchey Province, Cambodia. Korean J. Parasitol. 2011, 49, 187–190. [Google Scholar] [CrossRef]
  34. Huffman, J.E.; Fried, B. Echinostoma and echinostomiasis. Adv. Parasitol. 1990, 29, 215–269. [Google Scholar]
  35. Kuris, A.M.; Warren, J. Echinostome cercarial penetration and metacercarial encystment as mortality factors for a second intermediate host, Biomphalaria glabrata. J. Parasitol. 1980, 66, 630–635. [Google Scholar] [CrossRef]
  36. Fried, B.; Donovick, R.A.; Emili, S. Infectivity, growth and development of Echinostoma liei (Trematoda) in the domestic chick. Int. J. Parasitol. 1988, 18, 413–414. [Google Scholar] [CrossRef]
  37. Yousif, F.; Haroun, N. Intramolluscan development of Echinostoma liei (Trematoda:Echinostomatidae) in its snail host Biomphalaria alexandrina. J. Egypt. Soc. Parasitol. 1986, 16, 127–140. [Google Scholar] [PubMed]
  38. Jeyarassasingam, U.; Heyneman, D.; Lim, H.K.; Mansour, N. Life cycle of a new echinostome from Egypt, Echinostoma liei sp.nov. (Trematoda: Echinostomatidae). Parasitology 1972, 65, 203–222. [Google Scholar] [CrossRef] [PubMed]
  39. Sohn, W.M.; Na, B.K.; Lee, D.; Eom, K.S.; Yong, T.S.; Chai, J.Y.; Min, D.Y. Echinostoma macrorchis metacercariae in Cipangopaludina chinensis malleata from Xiengkhuang Province, Lao PDR and morphologies of adults from experimental animals. Korean J. Parasitol. 2019, 57, 657–664. [Google Scholar] [CrossRef] [PubMed]
  40. Butboonchoo, P.; Wongsawad, C.; Wongsawad, P.; Chai, J.Y. Morphology and molecular identification of Echinostoma revolutum and Echinostoma macrorchis in freshwater snails and experimental hamsters in upper Northern Thailand. Korean J. Parasitol. 2020, 58, 499–511. [Google Scholar] [CrossRef] [PubMed]
  41. Sohn, W.M.; Na, B.K. Echinostoma macrorchis (Digenea: Echinostomatidae): Metacercariae in Cipangopaludina chinensis malleata snails and adults from experimental rats in Korea. Korean J. Parasitol. 2017, 55, 541–548. [Google Scholar] [CrossRef]
  42. Sohn, W.M.; Chai, J.Y.; Na, B.K.; Yong, T.S.; Eom, K.S.; Park, H.; Min, D.Y.; Rim, H.J. Echinostoma macrorchis in Lao PDR: Metacercariae in Cipangopaludina snails and adults from experimentally infected animals. Korean J. Parasitol. 2013, 51, 191–196. [Google Scholar] [CrossRef]
  43. Ito, M.; Itagaki, T. Survey on wild rodents for endoparasites in Iwate Prefecture, Japan. J. Vet. Med. Sci. 2003, 65, 1151–1153. [Google Scholar] [CrossRef] [PubMed]
  44. Yokohata, Y.; Abe, H.; Jiang, Y.P.; Kamiya, M. Gastrointestinal helminth fauna of Japanese moles, Mogera spp. Jpn. J. Vet. Res. 1989, 37, 1–13. [Google Scholar]
  45. Valadão, M.C.; Alves, P.V.; López-Hernández, D.; Assis, J.C.A.; Coelho, P.R.S.; Geiger, S.M.; Pinto, H.A. A new cryptic species of Echinostoma (Trematoda: Echinostomatidae) closely related to Echinostoma paraensei found in Brazil. Parasitology 2023, 150, 337–347. [Google Scholar] [CrossRef] [PubMed]
  46. Cho, J.; Jung, B.K.; Chang, T.; Sohn, W.M.; Sinuon, M.; Chai, J.Y. Echinostoma mekongi n. sp. (Digenea: Echinostomatidae) from Riparian people along the Mekong River in Cambodia. Korean J. Parasitol. 2020, 58, 431–443. [Google Scholar] [CrossRef] [PubMed]
  47. Wiroonpan, P.; Chontananarth, T.; Chai, J.Y.; Purivirojkul, W. The high diversity of trematode metacercariae that parasitize freshwater gastropods in Bangkok, Thailand, and their infective situations, morphologies, and phylogenetic relationships. Parasitology 2022, 10, 1–56. [Google Scholar] [CrossRef]
  48. Chomchoei, N.; Backeljau, T.; Segers, B.; Wongsawad, C.; Butboonchoo, P.; Nantarat, N. Morphological and molecular characterization of larval trematodes infecting the assassin snail genus Anentome in Thailand. J. Helminthol. 2022, 96, e52. [Google Scholar] [CrossRef]
  49. Li, Y.; Qiu, Y.Y.; Zeng, M.H.; Diao, P.W.; Chang, Q.C.; Gao, Y.; Zhang, Y.; Wang, C.R. The complete mitochondrial genome of Echinostoma miyagawai: Comparisons with closely related species and phylogenetic implications. Infect. Genet. Evol. 2019, 75, 103961. [Google Scholar] [CrossRef]
  50. Eom, K.S.; Rim, H.J.; Jang, D.H. A study on the parasitic helminths of domestic duck (Anas Platyrhynchos Var. Domestica Linnaeus) in Korea. Kisaengchunghak Chapchi 1984, 22, 215–221. [Google Scholar] [CrossRef]
  51. Chai, J.Y.; Jung, B.K.; Chang, T.; Shin, H.; Cho, J.; Ryu, J.Y.; Kim, H.S.; Park, K.; Jeong, M.H.; Hoang, E.H.; et al. Echinostoma miyagawai Ishii, 1932 (Echinostomatidae) from ducks in Aceh Province, indonesia with special reference to its synonymy with Echinostoma robustum Yamaguti, 1935. Korean J. Parasitol. 2021, 59, 35–45. [Google Scholar] [CrossRef]
  52. Pham, L.T.K.; Quyen, D.V.; Saijuntha, W.; Doan, H.T.T.; Le, T.H.; Lawton, S.P. Mitogenomics of the zoonotic parasite Echinostoma miyagawai and insights into the evolution of tandem repeat regions within the mitochondrial non-coding control region. Parasitology 2024, 151, 1543–1554. [Google Scholar] [CrossRef]
  53. Nagataki, M.; Tantrawatpan, C.; Agatsuma, T.; Sugiura, T.; Duenngai, K.; Sithithaworn, P.; Andrews, R.H.; Petney, T.N.; Saijuntha, W. Mitochondrial DNA sequences of 37 collar-spined echinostomes (Digenea: Echinostomatidae) in Thailand and Lao PDR reveals presence of two species: Echinostoma revolutum and E. miyagawai. Infect. Genet. Evol. 2015, 35, 56–62. [Google Scholar] [CrossRef] [PubMed]
  54. Kostadinova, A.; Gibson, D.I.; Biserkov, V.; Chipev, N. Re-validation of Echinostoma miyagawai Ishii, 1932 (Digenea: Echinostomatidae) on the basis of the experimental completion of its life-cycle. Syst. Parasitol. 2000, 45, 81–108. [Google Scholar] [CrossRef]
  55. Kostadinova, A.; Gibson, D.I.; Biserkov, V.; Ivanova, R. A quantitative approach to the evaluation of the morphological variability of two echinostomes, Echinostoma miyagawai Ishii, 1932 and E. revolutum (Frölich, 1802), from Europe. Syst. Parasitol. 2000, 45, 1–15. [Google Scholar] [CrossRef] [PubMed]
  56. Rząd, I.; Sitko, J.; Dzika, E.; Zalewski, K.; Śmietana, P.; Busse, P. Geographic and ecologic aspects of the community structure of trematodes of mallards (Anas Platyrhynchos) in northern poland and the Czech Republic. J. Wildl. Dis. 2020, 56, 576–587. [Google Scholar] [CrossRef]
  57. Georgieva, S.; Faltýnková, A.; Brown, R.; Blasco-Costa, I.; Soldánová, M.; Sitko, J.; Scholz, T.; Kostadinova, A. Echinostoma ‘revolutum’ (Digenea: Echinostomatidae) species complex revisited: Species delimitation based on novel molecular and morphological data gathered in Europe. Parasit. Vectors. 2014, 7, 520. [Google Scholar]
  58. Pantoja, C.; Faltýnková, A.; O’Dwyer, K.; Jouet, D.; Skírnisson, K.; Kudlai, O. Diversity of echinostomes (Digenea: Echinostomatidae) in their snail hosts at high latitudes. Parasite 2021, 28, 59. [Google Scholar] [CrossRef]
  59. Svinin, A.O.; Chikhlyaev, I.V.; Bashinskiy, I.W.; Osipov, V.V.; Neymark, L.A.; Lvanov, A.Y.; Stoyko, T.G.; Chernigova, P.I.; Ibrogimova, P.K.; Litvinchuk, S.N.; et al. Diversity of trematodes from the amphibian anomaly P hotspot: Role of planorbid snails. PLoS ONE 2023, 18, e0281740. [Google Scholar] [CrossRef] [PubMed]
  60. Georgieva, S.; Blasco-Costa, I.; Kostadinova, A. Molecular characterisation of four echinostomes (Digenea: Echinostomatidae) from birds in New Zealand, with descriptions of Echinostoma novaezealandense n. sp. and Echinoparyphium poulini n. sp. Syst. Parasitol. 2017, 94, 477–497. [Google Scholar] [CrossRef]
  61. Lie, K.J.; Basch, P.F. The life history of Echinostoma paraensei sp. n. (Trematoda: Echinostomatidae). J. Parasitol. 1967, 53, 1192–1199. [Google Scholar] [CrossRef]
  62. Valadão, M.C.; López-Hernández, D.; Alves, P.V.; Pinto, H.A. 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. Parasitology 2022, 149, 325–336. [Google Scholar] [CrossRef]
  63. Buddhachat, K.; Chontananarth, T. Is species identification of Echinostoma revolutum using mitochondrial DNA barcoding feasible with high-resolution melting analysis? Parasitol. Res. 2019, 118, 1799–1810. [Google Scholar] [CrossRef]
  64. Anucherngchai, S.; Chontananarth, T. Echinostoma revolutum: Development of a high performance DNA-specific primer to demonstrate the epidemiological situations of their intermediate hosts. Acta Trop. 2019, 189, 46–53. [Google Scholar] [CrossRef]
  65. Ran, R.K.; Zhao, Q.; Abuzeid, A.M.I.; Huang, Y.; Liu, Y.Q.; Sun, Y.X.; He, L.; Li, X.; Liu, J.M.; Li, G.Q. Mitochondrial genome sequence of Echinostoma revolutum from Red-Crowned Crane (Grus japonensis). Korean J. Parasitol. 2020, 58, 73–79. [Google Scholar] [CrossRef]
  66. Chantima, K.; Chai, J.Y.; Wongsawad, C. Echinostoma revolutum: Freshwater snails as the second intermediate hosts in Chiang Mai, Thailand. Korean J. Parasitol. 2013, 51, 183–189. [Google Scholar] [CrossRef]
  67. Enabulele, E.E.; Lawton, S.P.; Walker, A.J.; Kirk, R.S. Molecular epidemiological analyses reveal extensive connectivity between Echinostoma revolutum (sensu stricto) populations across Eurasia and species richness of zoonotic echinostomatids in England. PLoS ONE 2023, 18, e0270672. [Google Scholar] [CrossRef]
  68. Chai, J.Y.; Sohn, W.M.; Na, B.K.; Nguyen, V.D. Echinostoma revolutum: Metacercariae in Filopaludina snails from Nam Dinh Province, Vietnam, and adults from experimental hamsters. Korean J. Parasitol. 2011, 49, 449–455. [Google Scholar] [CrossRef] [PubMed]
  69. Lee, S.H.; Sohn, W.M.; Chai, J.Y. Echinostoma revolutum and Echinoparyphium recurvatum recovered from house rats in Yangyang-gun, Kangwon-do. Kisaengchunghak Chapchi 1990, 28, 235–240. [Google Scholar] [CrossRef]
  70. Davis, N.E. Storage and incubation of Echinostoma revolutum eggs recovered from wild Branta canadensis, and their infectivity to Lymnaea tomentosa snails. J. Helminthol. 2005, 79, 321–326. [Google Scholar] [CrossRef] [PubMed]
  71. Sorensen, R.E.; Minchella, D.J. Parasite influences on host life history: Echinostoma revolutum parasitism of Lymnaea elodes snails. Oecologia 1998, 115, 188–195. [Google Scholar] [CrossRef]
  72. Mohanta, U.K.; Watanabe, T.; Anisuzzaman; Ohari, Y.; Itagaki, T. Characterization of Echinostoma revolutum and Echinostoma robustum from ducks in Bangladesh based on morphology, nuclear ribosomal ITS2 and mitochondrial nad1 sequences. Parasitol. Int. 2019, 69, 1–7. [Google Scholar] [CrossRef]
  73. Detwiler, J.T.; Bos, D.H.; Minchella, D.J. Revealing the secret lives of cryptic species: Examining the phylogenetic relationships of echinostome parasites in North America. Mol. Phylogenet Evol. 2010, 55, 611–620. [Google Scholar] [CrossRef]
  74. Iakovleva, G.A.; Lebedeva, D.I.; Ieshko, E.P. Trematodes fauna of waterfowl birds in Karelia. Parazitologiia 2012, 46, 98–110. [Google Scholar]
  75. Orlofske, S.A.; Belden, L.K.; Hopkins, W.A. Effects of Echinostoma trivolvis metacercariae infection during development and metamorphosis of the wood frog (Lithobates sylvaticus). Comp. Biochem. Physiol. A Mol. Integr. Physiol. 2017, 203, 40–48. [Google Scholar] [CrossRef]
  76. Weinstein, M.S.; Fried, B. The expulsion of Echinostoma trivolvis and retention of Echinostoma caproni in the ICR mouse: Pathological effects. Int. J. Parasitol. 1991, 21, 255–257. [Google Scholar] [CrossRef]
  77. Fried, B.; Mueller, T.J.; Frazer, B.A. Observations on Echinostoma revolutum and Echinostoma trivolvis in single and concurrent infections in domestic chicks. Int. J. Parasitol. 1997, 27, 1319–1322. [Google Scholar] [CrossRef]
  78. Hodinka, C.; Detwiler, J.T. Host species, host size, and miracidial dose influence the infection success of Echinostoma trivolvis lineage C larvae. J. Parasitol. 2024, 110, 725–729. [Google Scholar] [CrossRef]
  79. Nollen, P.M. Mating behaviour of Echinostoma caproni and E. trivolvis in concurrent infections in hamsters. Int. J. Parasitol. 1997, 27, 71–75. [Google Scholar] [CrossRef]
  80. Fried, B.; Pane, P.L.; Reddy, A. Experimental infection of Rana pipiens tadpoles with Echinostoma trivolvis cercariae. Parasitol. Res. 1997, 83, 666–669. [Google Scholar] [CrossRef]
  81. Mucha, K.H.; Huffman, J.E.; Fried, B. Mallard ducklings (Anas platyrhynchos) experimentally infected with Echinostoma trivolvis (Digenea). J. Parasitol. 1990, 76, 590–592. [Google Scholar] [CrossRef]
  82. Digiani, M.C. Digeneans and cestodes parasitic in the white-faced ibis Plegadis chihi (Aves: Threskiornithidae) from Argentina. Folia Parasitol. 2000, 47, 195–204. [Google Scholar] [CrossRef]
  83. Choe, S.; Lee, D.; Park, H.; Oh, M.; Jeon, H.K.; Lee, Y.; Na, K.J.; Kim, Y.; Lee, H.; Eom, K.S. Three echinostome species from wild birds in the Republic of Korea. Korean J. Parasitol. 2014, 52, 513–520. [Google Scholar] [CrossRef] [PubMed]
  84. Awad-Alla, M.E.; Abdien, H.M.; Dessouki, A.A. Prevalence of bacteria and parasites in White Ibis in Egypt. Vet. Ital. 2010, 46, 277–286. [Google Scholar] [PubMed]
  85. Ibrahim, N.A.; Shalaby, I.M.; Ghobashy, M.A.; Taeleb, A.A.; Elkhawass, E.A. Filling the void: Morphological and molecular phylogenetic analyses of helminths assemblage from the Egyptian egret Bubulcus ibis. Parasitol. Int. 2025, 104, 102972. [Google Scholar] [CrossRef]
  86. Tkach, V.V.; Kudlai, O.; Kostadinova, A. Molecular phylogeny and systematics of the Echinostomatoidea Looss, 1899 (Platyhelminthes: Digenea). Int. J. Parasitol. 2016, 46, 171–185. [Google Scholar] [CrossRef]
  87. Faltýnková, A.; Gibson, D.I.; Kostadinova, A. A revision of Patagifer dietz, 1909 (Digenea: Echinostomatidae) and a key to its species. Syst. Parasitol. 2008, 70, 159–183. [Google Scholar] [CrossRef] [PubMed]
  88. Sereno-Uribe, A.L.; González-García, M.T.; Ortega-Olivares, M.P.; López-Jiménez, A.; García-Varela, M.; Andrade-Gómez, L. First record of Patagifer bilobus (Rudolphi, 1819) Dietz, 1909 (Digenea: Echinostomatidae), with a morphological and molecular characterization from two threskiornithid species in Mexico. Parasitol. Res. 2022, 121, 1921–1935. [Google Scholar] [CrossRef]
  89. Outa, J.O.; Bhika, P.; Avenant-Oldewage, A. Gastropod invasions in anthropogenically impacted impoundments in South Africa: Tracing their origins and exploring field evidence of parasite spillback and amplification. Int. J. Parasitol. 2024, 54, 279–301. [Google Scholar] [CrossRef]
  90. Gacad, J.L.J.; Tanabe-Hosoi, S.; Yurlova, N.I.; Urabe, M. The complete mitogenome of Echinoparyphium aconiatum (Digenea: Echinostomatidae) and a comparison with other digenean species. Parasitol. Int. 2023, 92, 102682. [Google Scholar] [CrossRef]
  91. Su, X.; Zhang, Y.; Zheng, X.; Wang, X.X.; Li, Y.; Li, Q.; Wang, C.R. Characterization of the complete nuclear ribosomal DNA sequences of Eurytrema pancreaticum. J. Helminthol. 2018, 92, 484–490. [Google Scholar] [CrossRef] [PubMed]
  92. Nelson, J.O.; Watase, G.J.; Warsinger-Pepe, N.; Tamashita, Y.M. Mechanisms of rDNA Copy Number Maintenance. Trends Genet. 2019, 35, 734–742. [Google Scholar] [CrossRef]
  93. Qiu, Y.Y.; Gao, Y.; Li, Y.; Ma, X.X.; Lv, Q.B.; Hu, Y.; Qiu, H.Y.; Chang, Q.C.; Wang, C.R. Comparative analyses of complete ribosomal DNA sequences of Clonorchis sinensis and Metorchis orientalis: IGS sequences may provide a novel genetic marker for intraspecific variation. Infect. Genet. Evol. 2020, 78, 104125. [Google Scholar] [CrossRef]
  94. Le, T.H.; Pham, L.T.K.; Quyen, V.D.; Nguyen, K.T.; Doan, H.T.T.; Saijuntha, W.; Blair, D. The ribosomal transcription units of five echinostomes and their taxonomic implications for the suborder Echinostomata (Trematoda: Platyhelminthes). Parasitol. Res. 2024, 123, 103. [Google Scholar] [CrossRef]
  95. Nguyen, K.T.; Doan, H.T.T.; Pham, L.T.K.; Roan, D.T.; Agatsuma, T.; Doanh, P.N.; Le, T.H. Nuclear ribosomal transcription units in Asian Paragonimus species (Paragonimidae: Platyhelminthes): Genetic characteristics, polymorphism, and implications for intersuperfamilial phylogeny. Parasitol. Res. 2024, 123, 368. [Google Scholar] [CrossRef] [PubMed]
  96. Toledo, R.; Esteban, J.G. An update on human echinostomiasis. Trans. R. Soc. Trop. Med. Hyg. 2016, 110, 37–45. [Google Scholar] [CrossRef]
  97. Chen, X.B. Modern Parasitology: Release 1; People’s Military Medical Publishing House: Beijing, China, 2002; pp. 694–698. (In Chinese) [Google Scholar]
  98. Wang, Y.Q.; Yu, H.X. The discovery of Echinostoma miyagawai in goose in Heilongjiang province. Chin. Vet. Sci. 1993, 7, 44. (In Chinese) [Google Scholar]
  99. Dronen, N.O.; Blend, C.K. Petagifer lamothei n. sp. (Digenea: Echinostomatidae: Nephrostominae) from the white ibis Eudocimus albus (Threskiornithidae) from Texas, USA. Rev. Mex. De Biodivers. 2008, 79, 23–32. [Google Scholar]
  100. Chen, X.T. Fauna of China: Invertebrates, Volume 3: Phylum Platyhelminthes, Class Trematoda, Order Echinostomatida (Part I); Science Press: Beijing, China, 1985; p. 81. (In Chinese) [Google Scholar]
  101. Zheng, X.; Chang, Q.C.; Zhang, Y.; Tian, S.Q.; Lou, Y.; Duan, H.; Guo, D.H.; Wang, C.R.; Zhu, X.Q. Characterization of the complete nuclear ribosomal DNA sequences of Paramphistomum cervi. Sci. World J. 2014, 2024, 751907. [Google Scholar]
  102. Burland, T.G. DNASTAR’s Lasergene sequence analysis software. Methods Mol. Biol. 2000, 132, 71–91. [Google Scholar]
  103. Kurtz, S.; Choudhuri, J.V.; Ohlebusch, E.; Schleiermacher, C.; Stoye, J.; Giegerich, R. REPuter: The manifold applications of repeat analysis on a genomic scale. Nucleic Acids Res. 2001, 29, 4633–4642. [Google Scholar] [CrossRef]
  104. Tamura, K.; Peterson, D.; Peterson, N.; Stecher, G.; Masatoshi, N.; Kumar, S. MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 2011, 28, 2731–2739. [Google Scholar] [CrossRef] [PubMed]
  105. Ronquist, F.; Huelsenbeck, J.P. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 2003, 19, 1572–1574. [Google Scholar] [CrossRef]
  106. Wilgenbusch, J.C.; Swofford, D. Inferring evolutionary trees with PAUP*. In Current Protocols in Bioinformatics; John Wiley & Sons: Hoboken, NJ, USA, 2003; Chapter 6, Unit 6.4. [Google Scholar] [CrossRef]
  107. Beaver, P.C. Experimental studies on Echinostoma revolutum (Froelich), a fluke from birds and mammals. Illin Biol. Monogr. 1937, 15, 1–96. [Google Scholar]
  108. Bashkirova, E.I. Family Echinostomatidae dietz, 1909. In Trematodes and Animals and Man; Skrjabin, K.I., Ed.; USSR Academy of Sciences: Moscow, Russia, 1947; pp. 310–391. (In Russian) [Google Scholar]
  109. Skrjabin, K.I.; Bashkirova, E.I. Family Echinostomatidae dietz, 1909. In Trematodes and Animals and Man; Skrjabin, K.I., Ed.; Izdatelstvo Akademii Nauk SSSR: Moscow Leningrad, Russia, 1956; pp. 51–930. (In Russian) [Google Scholar]
  110. Kanev, I. Life-cycle, delimitation and redescription of Echinostoma revolutum (Froelich, 1802) (Trematoda: Echinostomatidae). Syst. Parasitol. 1994, 28, 125–144. [Google Scholar] [CrossRef]
  111. Fried, B.; Graczyk, T.K. Recent advances in the biology of Echinostoma species in the ‘revolutum’ group. Adv. Parasitol. 2004, 58, 139–195. [Google Scholar]
  112. Toledo, R.; Esteban, J.G.; Fried, B. Recent advances in the biology of echinostomes. Adv. Parasitol. 2009, 69, 147–204. [Google Scholar]
  113. Heneberg, P. Taxonomic comments on the validity of Echinostoma miyagawai Ischii, 1932 (Trematoda: Echinostomatidae). Parasitol. Int. 2020, 74, 101931. [Google Scholar] [CrossRef] [PubMed]
  114. Machida, M. Patagifer bilobus (Echinostomatidae:Trematoda) from white ibis, Threskiornis melanocephala. Bull. Natl. Sci. Mus. Tokyo 1968, 11, 157–160. [Google Scholar]
  115. Jain, S.P.; Shrivastava, O.N. On the validity of some species of the genus Patagifer (Trematoda: Echinostomatidae). Proc. Indian Acad. Sci. Sect. B 1970, 72, 156–161. [Google Scholar] [CrossRef]
  116. Srivastava, C.B. The fauna of India and adjacent countries. In Platyhelminthes Vol. 1 (Suppl.) Trematoda—Digenea; Zoological Survey of India: Calcutta, India, 1982; p. 163. [Google Scholar]
  117. Le, T.H.; Nguyen, K.T.; Pham, L.T.K.; Doan, H.T.T.; Roan, D.T.; Le, X.T.K.; Agatsuma, T.; Blair, D. Mitogenomic and nuclear ribosomal transcription unit datasets support the synonymy of Paragonimus iloktsuenensis and P. ohirai (Paragonimidae: Platyhelminthes). Parasitol. Res. 2023, 122, 1531–1544. [Google Scholar] [CrossRef] [PubMed]
  118. Li, Y.; Ma, X.X.; Lv, Q.B.; Hu, Y.; Chang, Q.C.; Wang, C.R. Characterization of the complete mitochondrial genome sequence of Tracheophilus cymbius (Digenea), the first representative from the family Cyclocoelidae. J. Helminthol. 2019, 94, e101. [Google Scholar] [CrossRef]
  119. Stanevičiūtė, G.; Stunžėnas, V.; Petkevičiūtė, R. Phylogenetic relationships of some species of the family Echinostomatidae odner, 1910 (Trematoda), inferred from nuclear rdna sequences and karyological analysis. Comp. Cytogenet. 2015, 9, 257–270. [Google Scholar] [CrossRef] [PubMed]
Biology 14 01101 g001
Figure 1. Genetic relationships of E. miyagawai and P. bilobus with other Echinostomatidae trematodes based on 18S sequence data. Phylogenetic analyses used Bayesian inference (BI), maximum likelihood (ML), and maximum parsimony (MP), with P. cervi as the outgroup. The scale bar indicates Posterior Probability. ● was the sequence obtained in this study.
Figure 1. Genetic relationships of E. miyagawai and P. bilobus with other Echinostomatidae trematodes based on 18S sequence data. Phylogenetic analyses used Bayesian inference (BI), maximum likelihood (ML), and maximum parsimony (MP), with P. cervi as the outgroup. The scale bar indicates Posterior Probability. ● was the sequence obtained in this study.
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Figure 2. Genetic relationships of E. miyagawai and P. bilobus with other Echinostomatidae trematodes based on ITS sequence data. Phylogenetic analyses used Bayesian inference (BI), maximum likelihood (ML), and maximum parsimony (MP), with P. cervi as the outgroup. The scale bar indicates Posterior Probability. ● was the sequence obtained in this study.
Figure 2. Genetic relationships of E. miyagawai and P. bilobus with other Echinostomatidae trematodes based on ITS sequence data. Phylogenetic analyses used Bayesian inference (BI), maximum likelihood (ML), and maximum parsimony (MP), with P. cervi as the outgroup. The scale bar indicates Posterior Probability. ● was the sequence obtained in this study.
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Figure 3. Genetic relationships of E. miyagawai and P. bilobus with other Echinostomatidae trematodes based on 28S sequence data. Phylogenetic analyses used Bayesian inference (BI), maximum likelihood (ML), and maximum parsimony (MP), with P. cervi as the outgroup. The scale bar indicates Posterior Probability. ● was the sequence obtained in this study.
Figure 3. Genetic relationships of E. miyagawai and P. bilobus with other Echinostomatidae trematodes based on 28S sequence data. Phylogenetic analyses used Bayesian inference (BI), maximum likelihood (ML), and maximum parsimony (MP), with P. cervi as the outgroup. The scale bar indicates Posterior Probability. ● was the sequence obtained in this study.
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Table 1. List of related information Echinostoma spp. and Patagifer spp. available in PubMed database.
Table 1. List of related information Echinostoma spp. and Patagifer spp. available in PubMed database.
Species and NominatorCollar-spinesIntermediate HostGeographical DistributionDefinitive HostLiterature Sources
Echinostoma bolschewense (Kotova, 1939) Nasincova, 199137Viviparus contectus Millet, 1813RussiaMesocricetus auratus Waterhouse, 1839; Gallus gallus Linnaeus, 1758[8]
Echinostoma caproni Richard, 196437Biomphalaria spp.; Bulinus spp.;
Helisoma duryi Wetherby, 1879;
Lymnaea natalensis Krauss, 1848;
Physa acuta Draparnaud, 1805;
Planorbarius corneus Linnaeus, 1758		Madagascar;
Egypt; Kenya		Mice; hamsters; rats; chicks; pigeons; finches; Crocidura olivieri Lesson, 1827; Falco newtoni Gurney, 1863[9,10,11]
Echinostoma chankense Besprozvannykh, 200127Anisus centrifugops Prozorova & Starobogatov, 1997;
Helicorbis sujfunensis Starobogatov, 1957;
Amuropaludina praerosa Gerstfeldt, 1859;
Lymnaea auricularia Linnaeus,1758		RussiaRattus norvegicus Linnaeus, 1766[6,12]
Echinostoma cinetorchis Ando & Ozaki, 192337Gyraulus convexiusculus Macleay, 1873;
Hippeutis cantori Benson, 1850;
Segmentina spp., Hemisphaerula spp.,
Radix auricularia coreana Adams, 1866;
Austropeplea ollula Gould, 1859
Fossaria truncatula Müller, 1774;
Corbicula fluminea Müller, 1774;		Korea; China;
Vietnam		Humans; dogs; ducks; Rattus argentiventer Robinson & Kloss, 1916; Bandicota indica Bechstein, 1800[13,14,15,16,17,18,19]
Echinostoma friedi Toledo et al., 200037Lymnaea peregra Müller, 1774
Lymnaea corvus Gmelin, 1791;
Gyraulus chinensis Dunker, 1848;
Physella acuta Draparnaud, 1805		SpainAlbino rats; golden hamsters; chickens; R. norvegicus[20,21]
Echinostoma hortense Asada,192627–28Acanthogobius flavimanus Temminck & Schlegel, 1845;
Misgurnus anguillicaudatus, Cantor 1842;
Odontobutis interrupta, Iwata & Jeon, 1985;
Misgurnus mizolepis Nichols, 1925;
Moroco oxycephalus Bleeker, 1865;
Coreoperca kawamebari Temminck & Schlegel, 1843;
Squalidus coreanus Berg, 1906		China; Korea;
Japan		Dogs; humans; R. norvegicus;
Felis catus Linnaeus, 1758;
Rattus rattus Linnaeus, 1758		[22,23,24,25,26,27,28,29,30]
Echinostoma ilocanum Garrison, 190849–51G. convexiusculusThailand;
Cambodia		Rats; humans[31,32,33]
Echinostoma liei Jeyarasasingam et al., 197237Biomphalaria glabrata Orbigny, 1835;
Biomphalaria alexandrina Ehrenberg, 1831		EgyptDomestic chicks;
hamsters; M. auratus		[34,35,36,37,38]
Echinostoma macrorchis Ando and Ozaki, 192340–45Cipangopaludina chinensis malleata Reeve, 1863;
Filopaludina martensi Martens, 1860;
Filopaludina doliaris Gould, 1844;
Filopaludina sumatrensis polygramma Martens, 1860;
Bithynia siamensis Lea, 1856;
Bithynia pulchella Adams, 1853;
Anentome helena Busch, 1847		LAO; Korea;
Thailand; Japan		Mice; rats; hamsters;
Mogera tokudae Kuroda, 1940; Mogera wogura Temminck, 1844;
Apodemus speciosus Temminck, 1835;		[39,40,41,42,43,44]
Echinostoma maldonadoi Kostadinova, 200033–39Stenophysa marmorata Guilding, 1828BrazilMeriones unguiculatus Milne-Edwards, 1867[45]
Echinostoma mekongi Cho et al., 202037F. martensi; A. helena;
F. sumatrensis polygramma		Cambodia;
Thailand		Humans; M. auratus[46,47,48]
Echinostoma miyagawai Ishii, 193237Planorbis planorbis Linnaeus, 1758;
Anisus vortex Linnaeus, 1758;
Lymnaea truncatula Müller, 1774;
Lymnaea stagnalis Linnaeus, 1758;
Lymnaea palustris Müller, 1774		Japan; China;
Korean; Czech;
Thailand; Bulgaria;
Poland; LAO;
Indonesia		Pigeons, ducks, humans, Aythya fuligula Linnaeus, 1758; Anas platyrhynchos Linnaeus, 1758[21,49,50,51,52,53,54,55,56]
Echinostoma nasincovae Georgieva et al., 201437P. corneusCzech; Russia;
Ireland		G. gallus; M. auratus;
Anas platyrhyn Linnaeus, 1758		[21,57,58,59]
Echinostoma novaezealandense Georgieva et al., 201737-New ZealandA. platyrhynchos;
Cygnus atratus Latham, 1790;
Branta canadensis Linnaeus, 1758		[60]
Echinostoma paraensei Lie & Basch, 196737B. glabrata; P. acutaBrazilHamsters; mice;
rats; R. norvegicus;
Nectomys squamipes Brants, 1827		[45,61]
Echinostoma paraulum Dietz, 190937L. stagnalisAustria; Russia;
Germany		A. fuligula[21,57]
Echinostoma pseudorobustum Dietz, 190937-BrazilG. gallus[62]
Echinostoma revolutum (Froelich, 1802) Dietz, 190937Ampullaceana balthica Linnaeus, 1758;
B. Siamensis; F. martensi;
C. Bithynia funiculata Leach, 1818;
Clea helena Philippi, 1847;
Eyriesia eyriesi Morelet, 1865;
F. doliaris;
F. sumatrensis polygramma;
Indoplanorbis exustus Deshayes, 1833;
L. Auricularia; L. stagnalis;
Lymnaea tomentosa Pfeiffer, 1855
Lymnaea elodes Say, 1821;
Radix auricularia Linnaeus, 1758;
Stagnicola palustris Müller, 1774		Germany; LAO;
Thailand; Korea;
China; Czech;
England; Poland;
Scotland; Canada;
Vietnam; America;
Finland;
Cambodia		Ducks; humans; rats;
hamsters; A. fuligula
G. Gallus; B. canadensis;
Grus japonensis Müller, 1776;		[21,53,57,63,64,65,66,67,68,69,70,71,72]
Echinostoma robustum Yamaguti, 193537P. acuta; L. elodesChina; Brazil;
Bangladesh;
Russia; America		Ducks; G. gallus;
A. platyrhynchos		[73,74,75]
Echinostoma trivolvis Cort, 191437Lithobates sylvaticus LeConte, 1825;
Physa gyrina Say, 1821;
Helisoma trivolvis Say, 1817;
Ladislavella elodes Say, 1821;
Rana spp. tadpoles		AmericaMice; chicks;
hamsters; A. platyrhynchos;
Ondatra zibethicus Linnaeus, 1766		[76,77,78,79,80,81]
Patagifer bilobus (Rudolphi, 1819) Dietz, 190948–64-Mexico; Ukraine;
America; Korea;
Egypt; Argentina; Lithuania; China		Plegadis chihi Vieillot, 1817
Nipponia nippon Temminck, 1835;
Eudocimus albus Linnaeus, 1758;
Bubulcus ibis Linnaeus, 1758;
Plegadis falcinellus Linnaeus, 1766; Platalea minor Temminck & Schlegel, 1849; G. japonensis		[82,83,84,85,86,87,88]
Patagifer vioscai Lumsden, 196253Pseudosuccinea columella Say, 1817America;
South Africa		E. albus[86,87,89]
Table 2. Higher taxa of Echinostoma spp. and Patagifer spp.
Table 2. Higher taxa of Echinostoma spp. and Patagifer spp.
Biological ClassificationEchinostoma spp.Patagifer spp.
PhylumPlatyhelminthesPlatyhelminthes
ClassTrematodaTrematoda
SubclassDigeneaDigenea
OrderPlagiorchiidaPlagiorchiida
SuborderEchinostomataEchinostomata
SuperfamilyEchinostomatoideaEchinostomatoidea
FamilyEchinostomatidaeEchinostomatidae
GensEchinostomaPatagifer
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Cao, Y.; Li, Y.; Gao, Z.-Y.; Jiang, B.-T. The Nuclear Ribosomal Transcription Units of Two Echinostomes and Their Taxonomic Implications for the Family Echinostomatidae. Biology 2025, 14, 1101. https://doi.org/10.3390/biology14081101

AMA Style

Cao Y, Li Y, Gao Z-Y, Jiang B-T. The Nuclear Ribosomal Transcription Units of Two Echinostomes and Their Taxonomic Implications for the Family Echinostomatidae. Biology. 2025; 14(8):1101. https://doi.org/10.3390/biology14081101

Chicago/Turabian Style

Cao, Yu, Ye Li, Zhong-Yan Gao, and Bo-Tao Jiang. 2025. "The Nuclear Ribosomal Transcription Units of Two Echinostomes and Their Taxonomic Implications for the Family Echinostomatidae" Biology 14, no. 8: 1101. https://doi.org/10.3390/biology14081101

APA Style

Cao, Y., Li, Y., Gao, Z.-Y., & Jiang, B.-T. (2025). The Nuclear Ribosomal Transcription Units of Two Echinostomes and Their Taxonomic Implications for the Family Echinostomatidae. Biology, 14(8), 1101. https://doi.org/10.3390/biology14081101

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