Linking metacercariae and adults of Microphallus basodactylophallus (Digenea: Microphallidae), based on larval stages from ctenophores and adult parasites from aquatic birds found in Mexico.

Members of the genus Microphallus Ward, 1901, are endoparasites mainly of birds and mammals distributed worldwide. Unencysted metacercariae of Microphallus sp., were collected from the mesoglea of ctenophores of the genus Pleurobrachia Fleming; adult digeneans were recovered from the intestines of Eudocimus albus Linnaeus (Threskiornithidae) and Buteogallus urubitinga Gmelin (Accipitridae), in four locations from southeastern Mexico. Adult specimens were identified as M. basodactylophallus (Bridgman, 1969) based on the following features: body pyriform entirely covered by minute spines, prepharynx short, oesophagus very long, caeca short and widely divergent, testes slightly symmetrical and excretory vesicle short and V-shaped. Sequences from D1-D3 domain of the large subunit of ribosomal DNA (LSU) were generated, aligned, and compared with those of congeneric species available in GenBank. Phylogenetic analyses indicated that the metacercariae and adults formed a clade together with an isolate identified as M. basodactylophallus from Florida, USA (GenBank: AY220628). The intraspecific genetic divergence among isolates was low ranged from 0.0% to 0.6%, allowing the link between the two stages of the life cycle. We observed phenotypic plasticity in the morphological traits of M. basodactylophallus adults in definitive hosts (mammals and birds) throughout the distribution, which ranged from the USA to southeastern Mexico. Finally, the unencysted metacercariae identified as M. basodactylophallus represent the first report of a microphallid in ctenophores.

[Preliminary application of recombinase -aided amplification in detection of Clonorchis sinensis metacercariae in freshwater fish].

OBJECTIVE: To evaluate the performance of recombinase-aided amplification (RAA) assay in detection of Clonorchis sinensis metacercariae in freshwater fish samples, so as to provide insights into standardization and field application of this assay. METHODS: Wild freshwater fish samples were collected in the rivers of administrative villages where C. sinensis-infected residents lived in Jiangyan District, Xinghua County and Taixing County of Taizhou City, Jiangsu Province from June to September 2022. Genomic DNA was extracted from six freshwater fish specimens (5 g each) containing 0, 1, 2, 4, 8 and 16 C. sinensis metacercariae for fluorescent RAA assay, and the diagnostic sensitivity was evaluated. Fluorescent RAA assay was performed with genomic DNA from C. sinensis, Metorchis orientalis, Haplorchis pumilio and Centrocestus formosanus metacercariae as templates to evaluate its cross-reactions. In addition, the detection of fluorescent RAA assay and direct compression method for C. sinensis metacercariae was compared in field-collected freshwater fish samples. RESULTS: Positive amplification was found in fresh-water fish specimens containing different numbers of C. sinensis metacercariae, and fluorescent RAA assay was effective to detect one C. sinensis metacercaria in 5 g freshwater fish specimens within 20 min. Fluorescent RAA assay tested negative for DNA from M. orientalis, H. pumilio and C. formosanus metacercariae. Fluorescent RAA assay and direct compression method showed 5.36% (93/1 735) and 2.88% (50/1 735) detection rates for C. sinensis metacercariae in 1 735 field-collected freshwater fish samples, with a statistically significant difference seen (χ2 = 478.150, P < 0.001). There was a significant difference in the detection of C. sinensis metacercariae in different species of freshwater fish by both the direct compression method (χ2 = 11.20, P < 0.05) and fluorescent RAA assay (χ2 = 20.26, P < 0.001), and the detection of C. sinensis metacercariae was higher in Pseudorasbora parva than in other fish species by both the direct compression method and fluorescent RAA assay (both P values < 0.05). CONCLUSIONS: Fluorescent RAA assay has a high sensitivity for detection of C. sinensis metacercariae in freshwater fish samples, and has no cross-reactions with M. orientalis, H. pumilio or C. formosanus metacercariae. Fluorescent RAA assay shows a higher accuracy for detection of C. sinensis infections in field-collected freshwater fish than the direct compression method.

Molecular confirmation of pearl formation in arctic mussels (Mytilus edulis) caused by Gymnophallus bursicola (Odhner 1900) metacercariae.

In recent field studies, suspected gymnophallid metacercariae were histologically located in the mantle of mussels from the Norwegian Sea. Mussels from the sites in which that infection was detected also presented abnormally high pearl numbers. It has been previously described that gymnophallid metacercariae could cause pearl formation processes in mussels, as a host reaction to encapsulate these metacercariae. Given the pathological host reaction these parasites elicit, a study was performed to identify gymnophallid metacercariae found in mussels collected from Tromsø at morphological and molecular level and to assess, by the use of molecular tools, the relationship between the parasite and the biological material inside the pearls. As a result, Gymnophallus bursicola metacercariae infecting Norwegian Mytilus edulis were identified according to morphological characters, along with the first 18S rDNA and COI sequences for this trematode species. In addition, parasite DNA from the core of the pearls was extracted and amplified for the first time, confirming the parasitological origin of these pearls. This procedure could allow identifying different parasitic organisms responsible for the generation of pearls in bivalves.

First record of Cotylurus metacercariae (Trematoda:Strigeidae) in Biomphalaria straminea (Planorbidae) from Argentina: morphological and molecular identification.

In the study of the biology of trematode species, the knowledge of the larval stages in snail hosts is important to elucidate their complete life cycle. The goal of the present study was to describe a new tetracotyle-type metacercaria found in the freshwater mollusk Biomphalaria straminea sampled in a rice field from Corrientes province, Argentina. To this end, 1,768 snails were collected from the cultivated plots and irrigated channels during the flooding periods (from the time of sowing to soon after rice harvesting) between December 2016 and May 2017. We used morphological and molecular analysis to characterize the tetracotyle-type metacercariae. Its morphological traits and the internal transcribed spacers (ITS1 and ITS2 plus 5.8S; ~1200 pb) from nuclear ribosomal DNA (rDNA) were amplified and sequenced. From 1,768 specimens of B. straminea screened, 52 were found infected with metacercariae of tetracotyle type (2.9%) that were identified as Cotylurus genus. A total of 218 metacercariae were found encysted in the ovotestis or between the mantle and viscera of B. straminea. Bioinformatic analysis showed that the metacercarial rDNA sequences shared 94% identity with those of Cotylurus gallinulae from Mexico and 100% identity with those of Cotylurus sp. from Brazil. In this study, the morphological descriptions are supplemented with the first molecular identification of a metacercaria related to Cotylurus parasitizing planorbids from Argentina. Also, our study provides a new morphological description in B. straminea, thus broadening the geographical distribution. The life cycle of this Cotylurus metacercariae is unknown and there are no reports of adult stages parasitizing waterfowl in Argentina.

New record of Tylodelphys metacercariae (Diplostomidae) from Perccottus glenii (Odontobutidae) and their phylogenetic assessment.

Metacercariae of Tylodelphys sp. were found in the abdominal cavity of the Chinese sleeper (Perccottus glenii) collected in Liaoning Province and Inner Mongolia Autonomous Region of China. The sequences of the mitochondrial cox1 gene and ribosomal ITS1-5.8S rDNA-ITS2 region were obtained and used for molecular identification and phylogenetic assessment of this parasite species. Results of phylogenetic analyses based on ITS and cox1 markers showed that the metacercariae of Tylodelphys sp. ex P. glenii from China were conspecific with specimens of Tylodelphys sp. collected by Sokolov et al. (2013) from the same fish-host species captured earlier in West Siberia, Russia. The examined Tylodelphys sp. ex. P. glenii is the only member of the genus whose metacercariae parasitise the abdominal cavity of fish in northern Eurasia. Tylodelphys sp. ex P. glenii clustered with T. darbyi, T. immer, T. podicipina, and Tylodelphys sp. of Soldánová et al., 2017 based on mitochondrial DNA markers, and with T. darbyi, T. immer, T. kuerepus, and T. schreuringi using nuclear DNA markers.

Metacercariae in the brain of Erythrinus cf. erythrinus (Characiformes: Erythrinidae) from Iguazú National Park (Argentina): do they belong to Dolichorchis lacombeensis (Digenea, Diplostomidae)?

In Argentina, the family Diplostomidae is composed of eight genera: Austrodiplostomum Szidat & Nani; Diplostomum von Nordmann; Dolichorchis Dubois; Hysteromorpha Lutz; Neodiplostomum Railliet; Posthodiplostomum Dubois; Sphincterodiplostomum Dubois; and Tylodelphys Diesing. During a parasitological survey of fishes from the Iguazú National Park we detected diplostomid metacercariae in the brain of Erythrinus cf. erythrinus. Fish were caught using crab traps, transported alive to the field laboratory, cold-anaesthetized and euthanized by cervical dissection. Some metacercariae were heat-killed in water and fixed in 10% formalin and others were preserved in alcohol 96% for DNA extraction. They were sequenced for the partial segment of the 28S rDNA, internal transcribed spacer (ITS) rDNA and cytochrome c oxidase subunit I (COI) mtDNA genes. Phylogenetic reconstruction was carried out using Bayesian inference and the proportion (p) of absolute nucleotide sites (p-distance) was obtained. In the 28S rDNA tree, the metacercaria sequenced grouped as Dolichorchis sp. The COI mtDNA p-distance between the metacercariae with Dolichorchis lacombeensis was 0.01. There is a small number of ITS sequences for the Diplostomidae family deposited in the GenBank. The oral sucker, ventral sucker, holdfast organ and the distance between oral and ventral suckers are larger in the adult compared with the metacercariae. Additionally, hind-body length and width are larger in the adult due to the development of the genital complex. Further studies using an integrative approach will help confirm the affiliation of other species to the genus Dolichorchis.

Morphological and molecular characterization of larval trematodes infecting the assassin snail genus Anentome in Thailand.

The assassin snail genus Anentome is widespread in Southeast Asia, and is distributed all over the world via the aquarium trade. One species of genus Anentome, Anentome helena, is known to act as intermediate host of parasitic trematodes. This study investigates the taxonomic diversity of larval trematodes infecting A. helena and Anentome wykoffi in Thailand. Larval trematodes were identified by combining morphological and DNA sequence data (cytochrome c oxidase I and internal transcribed spacer 2). Species delimitation methods were used to explore larval trematode species boundaries. A total of 1107 specimens of Anentome sp. were collected from 25 localities in Thailand. Sixty-two specimens of A. helena (n = 33) and A. wykoffi (n = 29) were infected by zoogonid cercariae, heterophyid metacercariae and echinostome metacercariae, with an overall prevalence of 5.6% (62/1107) and population-level prevalences in the range of 0.0-22.3%. DNA sequence data confirmed that the larval trematodes belong to the families Echinostomatidae, Heterophyidae and Zoogonidae. As such, this is the first report of zoogonid cercariae and heterophyid metacercariae in A. helena, and echinostome metacercariae in A. wykoffi. Moreover, this study provides evidence of tentative species-level differentiation between Thai Echinostoma sp. and Cambodian Echinostoma mekongi, as well as within Echinostoma caproni, Echinostoma trivolvis and Echinostoma revolutum.

Resolution of cryptic species complexes within the genus Metagonimus (Trematoda: Heterophyidae) in Japan, with descriptions of four new species.

A nationwide fish survey was conducted in Japan to detect metacercariae of the genus Metagonimus (Trematoda: Heterophyidae). The metacercariae were subjected to DNA barcoding for molecular species identification. A phylogeny inferred from the sequences of mitochondrial cytochrome c oxidase subunit 1 (cox1) prompted us to recognize three cryptic species complexes (i.e., the M. miyatai complex, the M. takahashii complex, and the M. katsuradai complex). Each complex included one or two undescribed species. For morphological description, adult flukes of each species were raised through the experimental infections of immunosuppressed mice. We propose M. saitoi n. sp., M. kogai n. sp., M. shimazui n. sp., and M. kinoi n. sp., based on their phylogeny, morphology, biogeography, and ecology (host-parasite relationships). The originally described species, M. miyatai, was split into M. miyatai sensu stricto and M. saitoi n. sp. The former is distributed mainly in eastern Japan and uses the sweetfish (Plecoglossus altivelis) and daces (Pseudaspius hakonensis and Ps. sachalinensis) as principal second intermediate hosts, while the latter is in western Japan and its principal fish hosts are the dark chub (Nipponocypris temminckii) and the pale chub (Opsariichthys platypus). The present survey resolves a long-standing controversy on the microtaxonomy of Metagonimus in Japan since the first discovery of Metagonimus yokogawai in 1912, and shows that 10 species of Metagonimus are still distributed in Japan, although human metagonimiasis is almost eradicated.

Molecular and morphological characterization of Dollfustrema bagarii (Digenea: Bucephalidae) metacercariae from aquaculture channel catfish (Ictalurus punctatus) in northern Vietnam.

White grub metacercariae were found in the livers and kidneys of diseased specimens of an introduced channel catfish, Ictalurus punctatus (Rafinesque), in Vietnam. Based on morphological features and 28S rDNA sequence analysis, the isolated metacercariae were identified as Dollfustrema bagarii (Digenea: Bucephalidae) Moravec & Sey. Histopathological examination shows that encysted metacercariae can change the tissue structure of the infected organs and is often accompanied by haemorrhaging and the presence of eosinophilic granular cell infiltration. Degenerative changes were also observed.

Digenean Metacercariae Parasitic in a Staurozoan Cnidarian.

I report digenean metacercariae from Staurozoa, which were not previously known as digenean hosts. The host species, Haliclystus tenuis Kishinouye, 1910, was collected from algae in Oshoro Bay, Hokkaido, Japan, and contained metacercariae in the mesoglea. The metacercariae were encysted; cysts were oval, 93 µm long by 64 µm wide in one live individual. For the digenean, I generated partial sequences for the 18S rRNA (1585 bp) and 28S rRNA (1672 bp) genes, and the region spanning the 3' end of the cytochrome c oxidase subunit gene and the 5' end of the 16S rRNA gene, including the threonine tRNA gene (868 bp in total). Phylogenetic reconstructions based on combined 18S + 28S datasets showed the digenean to belong in Opecoelidae, members of which utilize marine or freshwater teleost fishes as definitive hosts, and placed it in Plagioporinae (sensu lato) clade C within Opecoelidae.

Morphological and molecular characterization of Austrodiplostomum compactum metacercariae in the eyes and brains of fishes from the Ivaí River, Brazil.

Austrodiplostomum spp. (Platyhelminthes: Digenea) are endoparasites with a broad geographic distribution in South America. During the larval stage, they parasitize the eyes, brains, muscles, gill, kidneys and swim bladder of a wide variety of fishes. The metacercariae of Austrodiplostomum spp. have several morphological characteristics during development, but are very similar among species, which makes it necessary to use molecular tools to contribute to the elucidation during the larval stage. The objective of this study was to perform morphological and molecular analyses of Austrodiplostomum sp. found in specimens of Hypostomus sourced from the Ivaí River in the state of Paraná, Brazil. Of the 93 analyzed specimens (H. hermanni [n = 50], H. albopunctatus [n = 9], Hypostomus sp. 1 [n = 24], and Hypostomus sp. 2 [n = 10]), 60 were parasitized. A total of 577 Austrodiplostomum sp. metacercariae was collected from the infected hosts; DNA from seven of these samples was extracted, amplified, and sequenced. The morphological data associated with the genetic distance values and the relationships observed in the COI gene tree, indicate that all metacercariae were A. compactum. This is the first record of A. compactum parasitizing H. hermanni, H. albopunctatus, Hypostomus sp. 1, and Hypostomus sp. 2 in the Ivaí River.

Molecular link between the metacercariae and adults of four species Haematoloechus (Digenea: Plagiorchioidea), including scanning electron microscopy characterization.

Haematoloechus comprises a species-rich genus of trematodes commonly found in the lungs of amphibians across the globe. Of the approximately 70 species reported worldwide, Mexico stands out as a biodiversity hot spot for the genus, with 16 species described. In Los Tuxtlas tropical rainforest, in south-eastern Mexico, three species have been reported, two of them endemic of the frog species occurring in the area. Here, we added the record of a fourth species in the area, and partially elucidated the life cycle of these species using DNA sequence data from the 28S rRNA and the COI genes. Frogs, and a diverse array of species of aquatic insects were sampled in two lakes within the tropical rain forest. Adults and metacercariae of Haematoloechus were obtained. Both developmental stages were linked molecularly contributing to our understanding of their transmission pathways, and probable host-specificity patterns. Each of the four species of Haematoloechus were sampled in a particular species of anisopteran odonate, and therefore were considered as specialists towards their second intermediate host. We validated the presence of H. complexus in the region of Los Tuxtlas because their metacercariae, sampled in the anisopteran dragongly Agriogomphus tumens, matched with sequences of H. complexus from Nebraska and Illinois in USA. We further described the ultrastructure of the tegument of three of the four species of Haematoloechus, sampled from their definitive host (Rana vaillanti, Rana berlandieri), to contribute with characters that can be reliable for species delimitation of the genus.

Morphological and molecular characterization of Austrodiplostomum compactum metacercariae in the eyes and brains of fishes from the Ivaí River, Brazil / Caracterização morfológica e molecular de metacercárias de Austrodiplostomum compactum em olhos e cérebros de peixes do Rio Ivaí, Brasil

Abstract Austrodiplostomum spp. (Platyhelminthes: Digenea) are endoparasites with a broad geographic distribution in South America. During the larval stage, they parasitize the eyes, brains, muscles, gill, kidneys and swim bladder of a wide variety of fishes. The metacercariae of Austrodiplostomum spp. have several morphological characteristics during development, but are very similar among species, which makes it necessary to use molecular tools to contribute to the elucidation during the larval stage. The objective of this study was to perform morphological and molecular analyses of Austrodiplostomum sp. found in specimens of Hypostomus sourced from the Ivaí River in the state of Paraná, Brazil. Of the 93 analyzed specimens (H. hermanni [n = 50], H. albopunctatus [n = 9], Hypostomus sp. 1 [n = 24], and Hypostomus sp. 2 [n = 10]), 60 were parasitized. A total of 577 Austrodiplostomum sp. metacercariae was collected from the infected hosts; DNA from seven of these samples was extracted, amplified, and sequenced. The morphological data associated with the genetic distance values and the relationships observed in the COI gene tree, indicate that all metacercariae were A. compactum. This is the first record of A. compactum parasitizing H. hermanni, H. albopunctatus, Hypostomus sp. 1, and Hypostomus sp. 2 in the Ivaí River.

Resumo Austrodiplostomum spp. (Platyhelminthes: Digenea) são endoparasitos com uma ampla distribuição geográfica na América do Sul. Durante a fase larval, parasitam os olhos, cérebros, músculos, brânquias, rins e bexiga natatória de uma grande variedade de peixes. As metacercárias de Austrodiplostomum spp. apresentam várias características morfológicas durante o desenvolvimento, as quais são muito semelhantes entre as espécies, o que torna necessário o uso de ferramentas moleculares para contribuir para a elucidação durante a fase larval. O objetivo deste estudo foi realizar análises morfológicas e moleculares de Austrodiplostomum sp. encontradas em espécimes de Hypostomus provenientes do rio Ivaí, no Paraná, Brasil. Dos 93 espécimes analisados (H. hermanni [n = 50], H. albopunctatus [n = 9], Hypostomus sp. 1 [n = 24], e Hypostomus sp. 2 [n = 10]), 60 foram parasitados. Um total de 577 metacercárias de Austrodiplostomum foram coletadas dos hospedeiros infectados; o DNA de sete dessas amostras foi extraído, amplificado e sequenciado. Os dados morfológicos, associados aos valores de distância genética e as relações observadas na árvore gênica do COI, indicam que todas as metacercárias são A. compactum. Este é o primeiro registo de A. compactum parasitando H. hermanni, H. albopunctatus, Hypostomus sp. 1, e Hypostomus sp. 2 no rio Ivaí.

Molecular and morphological characterisation of the metacercariae of two species of Cardiocephaloides (Digenea: Strigeidae) infecting endemic South African klipfish (Perciformes: Clinidae).

South African clinids are a major component of the temperate intertidal regions that are also known to participate in life cycles and transmission of several groups of parasites. However, the knowledge of trematode diversity of these fishes is incomplete. In this study, two species of Clinus Cuvier, the super klipfish Clinus superciliosus (Linnaeus) and the bluntnose klipfish Clinus cottoides Valenciennes, were collected from six localities along the South African coast and examined for the presence of trematodes. Metacercariae of Cardiocephaloides Sudarikov, 1959 were found in the eye vitreous humour and brain of C. superciliosus and in the eye vitreous humour of C. cottoides. Detailed analyses integrating morphological and molecular sequence data (28S rDNA, ITS2 rDNA-region, and COI mtDNA) revealed that these belong to two species, Cardiocephaloides physalis (Lutz, 1926) and an unknown species of Cardiocephaloides. This study provides the first report of clinid fishes serving as intermediate hosts for trematodes, reveals that the diversity of Cardiocephaloides in South Africa is higher than previously recorded, and highlights the need for further research to elucidate the life cycles of these trematode species. The broad geographical distribution of Cardiocephaloides spp. was confirmed in the present study based on molecular sequence data. The host-parasite interactions between clinid fishes and metacercariae of Cardiocephaloides are yet to be explored.

Description of a metacercaria of a zoogonid trematode Steganoderma cf. eamiqtrema Blend and Racz, 2020 (Microphalloidea: Zoogonidae), with notes on the phylogenetic position of the genus Steganoderma Stafford, 1904, and resurrection of the subfamily Lecithostaphylinae Odhner, 1911.

Metacercariae of the zoogonid trematode Steganoderma cf. eamiqtrema ex crab Chionoecetes bairdi caught in the Sea of Okhotsk were described using morphological and molecular-genetic (ITS2 region, 28S rRNA and nd1 genes) data. These are the first molecular-genetic data for the genus Steganoderma. The studied trematodes differed from S. eamiqtrema in having a much larger body size. The phylogenetic analysis based on the 28S rRNA gene supported neither the current taxonomic hypothesis that Steganoderma belongs to the subfamily Lepidophyllinae nor the earlier views that the Steganodermatinae and the Lecithostaphylinae are synonymous. The topology of the phylogenetic tree shows that the Steganodermatinae and the Lecithostaphylinae are independent subfamilies. However, morphological differences between them are obscure. Until morphological evidence for the Steganodermatinae is found, we propose to distinguish the subfamily Lepidophyllinae sensu stricto with the genera Lepidophyllus and Urinatrema, and the subfamily Lecithostaphylinae sensu lato uniting all the other former lepidophyllines. Thus, for now, we propose to consider the Steganodermatinae as a conditional synonym for Lecithostaphylinae sensu lato.

Echinostoma mekongi: Discovery of Its Metacercarial Stage in Snails, Filopaludina martensi cambodjensis, in Pursat Province, Cambodia.

Echinostoma mekongi was reported as a new species in 2020 based on specimens collected from humans in Kratie and Takeo Province, Cambodia. In the present study, its metacercarial stage has been discovered in Filopaludina martensi cambodjensis snails purchased from a local market nearby the Tonle Sap Lake, Pursat Province, Cambodia. The metacercariae were fed orally to an experimental hamster, and adult flukes were recovered at day 20 post-infection. They were morphologically examined using light and scanning electron microscopes and molecularly analyzed by sequencing of their mitochondrial cox1 and nad1 genes. A total of 115 metacercariae (1-8 per snail) were detected in 60 (60.0%) out of 100 Filopaludina snails examined. The metacercariae were round, 174 µm in average diameter (163-190 µm in range), having a thin cyst wall, a head collar armed with 37 collar spines, and characteristic excretory granules. The adult flukes were elongated, ventrally curved, 7.3 (6.4-8.2)×1.4 (1.1-1.7) mm in size, and equipped with 37 collar spines on the head collar (dorsal spines in 2 alternating rows), being consistent with E. mekongi. In phylogenetic analyses, the adult flukes showed 99.0-100% homology based on cox1 sequences and 98.9-99.7% homology based on nad1 sequences with E. mekongi. The results evidenced that F. martensi cambodjensis snails act as the second intermediate host of E. mekongi, and hamsters can be used as a suitable experimental definitive host. As local people favor to eat undercooked snails, these snails seem to be an important source of human infection with E. mekongi in Cambodia.

Leeches as the intermediate host for strigeid trematodes: genetic diversity and taxonomy of the genera Australapatemon Sudarikov, 1959 and Cotylurus Szidat, 1928.

BACKGROUND: Leeches (Hirudinida) play a significant role as intermediate hosts in the circulation of trematodes in the aquatic environment. However, species richness and the molecular diversity and phylogeny of larval stages of strigeid trematodes (tetracotyle) occurring in this group of aquatic invertebrates remain poorly understood. Here, we report our use of recently obtained sequences of several molecular markers to analyse some aspects of the ecology, taxonomy and phylogeny of the genera Australapatemon and Cotylurus, which utilise leeches as intermediate hosts. METHODS: From April 2017 to September 2018, 153 leeches were collected from several sampling stations in small rivers with slow-flowing waters and related drainage canals located in three regions of Poland. The distinctive forms of tetracotyle metacercariae collected from leeches supplemented with adult Strigeidae specimens sampled from a wide range of water birds were analysed using the 28S rDNA partial gene, the second internal transcribed spacer region (ITS2) region and the cytochrome c oxidase (COI) fragment. RESULTS: Among investigated leeches, metacercariae of the tetracotyle type were detected in the parenchyma and musculature of 62 specimens (prevalence 40.5%) with a mean intensity reaching 19.9 individuals. The taxonomic generic affiliation of metacercariae derived from the leeches revealed the occurrence of two strigeid genera: Australapatemon Sudarikov, 1959 and Cotylurus Szidat, 1928. Phylogenetic reconstructions based on the partial 28S rRNA gene, ITS2 region and partial COI gene confirmed the separation of the Australapatemon and Cotylurus clades. Taking currently available molecular data and our results into consideration, recently sequenced tetracotyle of Australapatemon represents most probably Au. minor; however, unclear phylogenetic relationships between Au. burti and Au. minor reduce the reliability of this conclusion. On the other hand, on the basis of the obtained sequences, supplemented with previously published data, the metacercariae of Cotylurus detected in leeches were identified as two species: C. strigeoides Dubois, 1958 and C. syrius Dubois, 1934. This is the first record of C. syrius from the intermediate host. CONCLUSIONS: The results of this study suggest the separation of ecological niches and life cycles between C. cornutus (Rudolphi, 1808) and C. strigeoides/C. syrius, with potential serious evolutionary consequences for a wide range of host-parasite relationships. Moreover, phylogenetic analyses corroborated the polyphyletic character of C. syrius, the unclear status of C. cornutus and the separate position of Cotylurus raabei Bezubik, 1958 within Cotylurus. The data demonstrate the inconsistent taxonomic status of the sequenced tetracotyle of Australapatemon, resulting, in our opinion, from the limited availability of fully reliable, comparative sequences of related taxa in GenBank.

Molecular and morphological characterisation of Diplostomum phoxini (Faust, 1918) with a revised classification and an updated nomenclature of the species-level lineages of Diplostomum (Digenea: Diplostomidae) sequenced worldwide.

We characterised morphologically and molecularly Diplostomum phoxini (Faust, 1918) based on cercarial isolates from the snail Ampullaceana balthica (L.) (Gastropoda: Lymnaeidae) and metacercariae from the Eurasian minnow, Phoxinus phoxinus (L.) (Cypriniformes: Leuciscidae), and provided molecular evidence for the identification of the snail intermediate host. Phylogenetic analyses based on the cytochrome c oxidase subunit 1 (cox1) gene depicted 44 molecularly characterised species and genetically distinct lineages of Diplostomum, and resulted in: (i) a re-identification/re-classification of 98 isolates plus D. baeri sampled in North America; (ii) re-definition of the composition of the D. baeri species complex which now includes nine molecularly characterised species/lineages; (iii) re-definition of the composition of the D. mergi species complex which now includes seven molecularly characterised species/lineages; and (iv) an updated nomenclature for the molecularly characterised species-level lineages of Diplostomum.

Three species of Exorchis Kobayashi, 1921 (Digenea: Cryptogonimidae) in the East-Asian region: morphological and molecular data.

New data have been obtained for three representatives of Exorchis; Exorchis convictus sp. n., Exorchis oviformis and Exorchis sp., from fish in the East-Asian region. For the first time, based on combined sequences of the ITS2 rDNA region and the 28S rRNA gene, Exorchis is confirmed to belong Cryptogonimidae. Based on analysis of a mitochondrial marker (cox1), the 'Japanese' and 'Russian' haplogroups are identified for E. oviformis isolated from Silurus asotus. One specimen of E. oviformis obtained in Japan is identical to the 'Russian' haplotype. Haplotype patterns are also observed for metacercariae of Exorchis sp. from Tanakia lanceolata and Carassius sp. fish in Kyushu Island (Japan).

First Description of the Metacercaria of Nematostrigea serpens serpens (Nitzsch, 1819) (Trematoda, Strigeidae) and Phylogenetic Affiliation of Nematostrigea vietnamiensis Zhokhov & Mishina, 2011.

BACKGROUND: The genus Nematostrigea comprises trematodes parasitising in fish-eating birds of Europe, Africa, and North America. Their life cycle is unknown. PURPOSE: To provide the first description of metacercariae of Nematostrigea serpens serpens, a nominative subspecies of the type species of Nematostrigea, and to record metacercaria of Nematostrigea vietnamiensis, with molecular data. METHODS: Encysted metacercariae of N. serpens serpens and N. vietnamiensis were collected from fish in Russia and Vietnam, respectively, and were processed, identified, and documented using standard morphological techniques. The 28S rRNA gene of metacercariae of both species and the cox1 gene of metacercariae an earlier studied adult of N. serpens serpens were sequenced and used for phylogenetic analysis. RESULTS: Metacercaria of N. serpens serpens have a spatulate body with a foliate forebody and a short hindbody, two long lateral rectilinear pseudosuckers, and the holdfast organ with bifurcated anterior and entire posterior external lobes. The analysis of the cox1and 28S rRNA gene sequences unequivocally showed the conspecificity of metacercariae and adult stage of N. serpens serpens. Based on the 28S rRNA gene sequence-based phylogenetic analysis, the genus Nematostrigea is a sister taxon to the group of strigeid genera Cotylurus + Ichthyocotylurus. Morphological and phylogenetic data demonstrated that N. vietnamiensis does not belong to the genus Nematostrigea and is possibly be affiliated with the crassiphialine trematodes. CONCLUSION: This is the first record of metacercariae of N. serpens serpens. N. vietnamiensis, renamed Prodiplostomulum vietnamiense comb. nov., must be moved to the crassiphialine 'Prodiplostomulum'-type metacercariae group.