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.

Cepaea spp. as a source of Brachylaima mesostoma (Digenea: Brachylaimidae) and Brachylecithum sp. (Digenea: Dicrocoeliidae) larvae in Poland.

Parasite diagnostics were carried out on 11 Polish populations of Cepaea spp. In three of them, coming from the roadside ditches of a village (Rytel, northern Poland), very high (up to 60%) prevalence of Brachylaima mesostoma was observed. This study provides the first molecular evidence of the presence of B. mesostoma inside Cepaea spp. in Europe. In a few snails from a population found in a private garden in a small town (Chelmza, northern Poland), larvae of Brachylecithum sp. were present. Cercariae and/or metacercariae of B. mesostoma were observed in both species of Cepaea: C. hortensis and C. nemoralis, whereas larvae of Brachylecithum sp. were found only in C. nemoralis. Both species of parasites inhabited snail hepatopancreas whose structure was significantly damaged by larvae. There was no significant connection between parasite invasion and snail host morphotype. The research did not allow the reasons for the high prevalence of B. mesostoma in Cepaea spp. to be explained, and also did not explicitly indicate how the parasite invaded Cepaea spp. individuals making them, at the same time a second intermediate host. However, it poses important questions about the life cycle of the parasite that may threaten extensively kept small-size farms of poultry.

Phylogenetic position of Diplostomum spp. from New World herons based on complete mitogenomes, rDNA operons, and DNA barcodes, including a new species with partially elucidated life cycle.

Diplostomum ardeae Dubois, 1969 has seldom been reported since its description from the great blue heron (Ardea herodias L., 1758) in the USA. Sequences obtained in this study from the barcode region of cytochrome c oxidase 1 (CO1) in diplostomids from black-crowned night heron (Nycticorax nycticorax (L., 1758)) in Puerto Rico matched data from D. ardeae from A. herodias in the type region. We also obtained DNA barcodes from morphologically similar diplostomids from a rufescent tiger heron (Tigrisoma lineatum (Boddaert, 1783)) and from metacercariae from eye lenses of Trachelyopterus galeatus (Linnaeus, 1766) from the Paraná River basin in Argentina and Brazil, respectively. Barcodes matched (97-100% identity) in these South American adult and larval specimens as well as in recently published sequences from metacercariae from 11 other siluriform fishes from the same region. Barcodes from the South American species, which we describe as Diplostomum lunaschiae n. sp., differed from those of D. ardeae by 7.2-9.8%, and the new species differs from D. ardeae in its size, pharynx:oral sucker length ratio, egg:body length ratio, and distribution of vitellaria. As in prior phylogenetic analysis of CO1 sequences, both D. ardeae and D. lunaschiae n. sp. were not associated with Diplostomum. In more character-rich analyses of nuclear rDNA and of mitochondrial genomes, D. ardeae was an early divergent member of clades of species of Diplostomum. Consequently, we continue to consider D. ardeae and D. lunaschiae n. sp. members of Diplostomum, in contrast to recent suggestions that these species may belong to a different genus.

A new species of Clinostomum Leidy, 1856 based on molecular and morphological analysis of metacercariae from African siluriform fishes.

In the Afrotropic region, the genus Clinostomum is represented by four accepted and four unnamed species distinguished using molecular data. Here, we describe one of the four unnamed species as Clinostomum ukolii n. sp. based on metacercariae from siluriform fishes (Synodontis batensoda, Schilbe intermedius) collected in Nigeria and South Africa. The new species is distinguished by molecular data (39 new sequences of partial cytochrome c oxidase I ≥ 6.7% divergent from those of other species) and morphological differences from named and unnamed species in the same region. Metacercariae of C. ukolii n. sp. can be distinguished based on size, tegumental spines, and various aspects of the genital complex, including its position, lobation of the anterior testis, and the disposition and shape of the cirrus pouch. Although descriptions of new species of digeneans are typically based on the morphology of adults, we argue that in cases where data are available from metacercariae from regionally known species, new species can be described based on metacercariae, particularly when supported by molecular data, as here. Moreover, sub-adult reproductive structures can be clearly visualized in metacercaria of Clinostomum. Considering metacercariae as potential types for new species could advance clinostome systematics more rapidly, because metacercariae are encountered much more often than adults in avian definitive hosts.

Pathology associated with three new Clinostomum metacercariae from Argentina with morphological and DNA barcode identification.

In the Laboratory of Parasites of Fishes, Crustaceans and Mollusks (CEPAVE), we undertook a parasitological study on three species of fish from the Espinal and Esteros del Iberá ecoregions of Argentina. Clinostomid metacercariae were found parasitizing Characidium rachovii, Crenicichla vittata and Gymnogeophagus balzanii. In this study, we analysed the damage that these parasites inflict on their hosts through the evaluation of histological sections. In addition, Clinostomum metacercariae were identified using morphological characters and DNA barcoding. In the pathological analysis, we observed that muscle tissue was the most affected. The inflammatory response showed vascular congestion areas and infiltration of numerous inflammatory cells, mainly lymphocytes. The molecular and morphological approach supports the presence of three new lineages of clinostomid metacercariae in Argentina. This could lead to the discovery of a high number of lineages or species of Clinostomum from South America.

Molecular data show Clinostomoides Dollfus, 1950 is a junior synonym of Clinostomum Leidy, 1856, with redescription of metacercariae of Clinostomum brieni n. comb.

The genus Clinostomoides Dollfus, 1950 was erected to accommodate a single worm from Ardea goliath sampled in the Belgian Congo. The specimen was distinguished from other clinostomids by its large size and posterior genitalia. In the following years, metacercariae of Clinostomoides brieni, have been described in Clarias spp. in southern and western Africa. A few authors have referred to Clinostomum brieni, but all such usages appear to be lapsus calami, and the validity of Clinostomoides remains widely accepted. In this study our aim was: position C. brieni among the growing clinostomids molecular database, and redescribe the species with emphasis on characters that have emerged as important in recent work. We sequenced two nuclear (partial 18S and ITS) and one mitochondrial marker (partial cytochrome c oxidase I) and studied morphology in metacercariae from hosts and localities likely to harbour the type species (Clarias spp., Democratic Republic of the Congo, South Africa). Phylogenetic analysis shows C. brieni belongs within Clinostomum Leidy, 1856. We therefore transfer C. brieni to Clinostomum, amend the diagnosis for the genus Clinostomum and provide a critical analysis of other species in Clinostomoides, all of which we consider species inquirendae, as they rest on comparisons of different developmental stages.

Molecular and morphological characterisation of four diplostomid metacercariae infecting Tilapia sparrmanii (Perciformes: Cichlidae) in the North West Province, South Africa.

Despite their pathogenic effects on fish, the diversity of trematodes from the family Diplostomidae remains vastly unexplored in Africa and specifically South Africa. To date, only six species of diplostomids have been reported from freshwater fishes in this country, with only two species being molecularly characterised. In this study, combined morphological and molecular analyses were used to identify and describe metacercariae of the Diplostomidae (Digenea) parasitising banded tilapia Tilapia sparrmanii (Perciformes: Cichlidae) collected within the North West Province, South Africa. Metacercariae found on the body surface and muscles of the fish were separated into four groups based on the infection site, the colour of the cysts and the morphology of excysted specimens. Isolates from each group were further identified through molecular analyses. Comparative analyses of the newly generated 28S rDNA, ITS1-5.8S-ITS2 and cox1 sequences revealed the presence of four species of which three were identified as Bolbophorus sp. 3 (28S rDNA, ITS1-5.8S-ITS2 and cox1), Posthodiplostomum sp. 9 (28S rDNA and ITS1-5.8S-ITS2) and Uvulifer sp. 4 (28S rDNA, ITS1-5.8S-ITS2 and cox1), respectively, and the fourth species belonging to the Diplostomidae gen. sp. (28S rDNA, ITS1-5.8S-ITS2 and cox1). Morphology of metacercariae of Posthodiplostomum sp. was compared with metacercariae of this genus previously reported in fishes in Africa. This study presents the first molecular data for species of Bolbophorus Dubois, 1935, Posthodiplostomum Dubois, 1936 and Uvulifer Yamaguti, 1934 from Africa, and it highlights the need for future research on the diversity of diplostomid parasites in South Africa and in Africa as whole.

A new species of Clinostomum Leidy, 1856 in East Asia based on genomic and morphological data.

Metacercariae of Clinostomum Leidy, 1856 are frequently encountered in freshwater fish. In 2015, a provisional species of Clinostomum in People's Republic of China (PRC) was distinguished from C. complanatum (Rudolphi, 1819) in Europe based on divergent cytochrome c oxidase I (CO1). However, in subsequent studies in East Asia, the same divergent CO1 genotype was identified as C. complanatum. These matching sequences suggest that either the provisional East Asian species was incorrectly distinguished from C. complanatum in 2015 or that C. complanatum in East Asia was misidentified in later studies. We tested these alternatives by sequencing the mitochondrial genome of C. complanatum in Italy, which was 5.7% divergent from a previously published sequence from Clinostomum in PRC, including differences in 80 of 3390 (2.4%) translated amino acids. Partial CO1 sequences of specimens from PRC and those from Italy, Romania, and Turkey also each formed reciprocally monophyletic clades. Partial CO1 from the East Asian clade varied by mean 3.6% (range 2.4-4.8%) from C. complanatum from Italy, Romania, and Turkey; mean intra-clade CO1 variation was 0.3% (range 0-1.9%). Metacercariae from Europe and East Asia display significant morphometric variation, and data from the literature suggest morphological differences in the genital complex of adults. Although sequences of nuclear rDNA did not differ between isolates from the west and East Asia, taken together, these results lead us to describe a new species of Clinostomum.

Molecular and morphological characterization of the metacercariae of two species of diplostomid trematodes (Platyhelminthes, Digenea) in freshwater fishes of the Batalha River, Brazil.

The Diplostomidae include a large group of flatworms with complex life cycles and are frequently found parasitizing the eyes and central nervous system of freshwater fishes. The morphological identification of the metacercariae at species level is not always possible. Thus, molecular tools have become essential to assist in the parasite species determination. This study was aimed at describing two diplostomid metacercariae found in freshwater fish in São Paulo, Brazil, based on morphological characters and in the genetic characterization of COI sequences. Our results showed that the two recognized taxa (Tylodelphys sp. and Diplostomidae gen. sp.) appear to be different from the species already described in South America. Tylodelphys sp. differs morphologically from Tylodelphys xenopi, T. mashonense, T. jenynsiae, and T. scheuringi. The metacercariae of T. clavata and T. conifera are smaller than Tylodelphys sp., while T. podicipina is larger than the metacercariae described here. The phylogenetic analysis of COI sequences yielded Tylodelphys sp. as the sister species of Tylodelphys sp. 4, a species reported from the brain of the eleotrid Gobiomorus maculatus in Oaxaca, Mexico. The metacercariae identified as Diplostomidae gen. sp. are morphologically different from the known diplostomid metacercariae and did not match with other diplostomid sequences available. Diplostomidae gen. sp. is recovered as the sister species of Diplostomum ardeae. Although the morphological evidence and the COI sequences differentiate the metacercariae found, the absence of adult specimens of both species precludes the specific designation. This is one of the first papers that use an integrative taxonomy approach to describe the species diversity of diplostomid trematodes in Brazil.

Phylogenetic relationships, expanded diversity and distribution of Crassiphiala spp. (Digenea, Diplostomidae), agents of black spot disease in fish.

Crassiphiala is a monotypic genus of diplostomid digeneans and is the type genus of the subfamily Crassiphialinae. The type species Crassiphiala bulboglossa parasitizes kingfishers in the Nearctic and has a Neascus-type metacercaria that encysts on fish intermediate hosts, often causing black spot disease. While recent molecular phylogenetic studies included some members of the Crassiphialinae, no DNA sequence data of Crassiphiala is currently available. Our molecular and morphological study of adult and larval crassiphialines from the Americas revealed the presence of at least three lineages of Crassiphiala from the Nearctic and two lineages from the Neotropics. This is the first record of Crassiphiala from the Neotropics. Herein, we provide the first molecular phylogeny of the Diplostomoidea that includes Crassiphiala. Our data revealed 0.2-2.4% divergence among 28S sequences and 11-19.8% among CO1 sequences of lineages of Crassiphiala. The results of our analyses did not support the monophyly of Crassiphialinae. Our results clearly demonstrated that the diversity of Crassiphiala has been underestimated.

Exploring the genetic diversity of Tylodelphys (Diesing, 1850) metacercariae in the cranial and body cavities of Mexican freshwater fishes using nuclear and mitochondrial DNA sequences, with the description of a new species.

Members of the genus Tylodelphys Diesing, 1850 are endoparasites of fish-eating birds, particularly ciconiids, anhingids, and podicipedids across the globe. Metacercariae of Tylodelphys spp. were collected from the cranial and body cavities of freshwater fishes in central and northern Mexico; adults were recovered from the intestine of two species of freshwater diving birds of the family Podicipedidae, commonly known as grebes, in two locations of central Mexico. Specimens were sequenced for two molecular markers, the internal transcribed spacers (ITS1 and ITS2) plus 5.8S gene of the nuclear ribosomal DNA and of the cytochrome c oxidase subunit 1 from mitochondrial DNA. The genetic divergence among the 25 samples (16 metacercariae and 9 adults) and between the newly sequenced specimens and those deposited in the GenBank were estimated. Maximum likelihood and Bayesian inference analyses inferred with each data set revealed the existence of five genetic lineages. Eight metacercariae analyzed in this study were nested in two divergent lineages previously recognized as Tylodelphys sp. 5 and Tylodelphys sp. 6 (sensu Locke et al., Int J Parasitol, 45:841-855, 2015). Five adult specimens recovered from the intestine of the least grebe (Tachybaptus dominicus Linnaeus, 1766) in Tecocomulco Lake, Hidalgo State, nested in a single clade with other sequences identified previously as Tylodelphys aztecae, expanding its distribution range in other areas of central Mexico. The isolates of the metacercariae found in the cranial cavity of the shortfin silverside, Chirostoma humboldtianum Valenciennes, 1835 from Zacapu Lake in central Mexico formed a monophyletic lineage and were recognized as an undescribed species of Tylodelphys. The lack of adult specimens of this lineage in our samples prevented a formal description. However, the metacercariae collected in the cranial cavity of the silverside, Chirostoma jordani Woolman, 1894 and the adult specimens recovered from the intestine of the western grebe, Aechmophorus occidentalis (Lawrence, 1858) from Cuitzeo Lake formed a monophyletic clade, allowing us to link both stages of the life cycle and to describe this as a new species, Tylodelphys kuerepus n. sp. The new species represents the eighth species of the genus described in the Americas and the fourth in the Nearctic region. We briefly discuss the ecological associations between the metacercariae and their second intermediate hosts in relation to the genetic diversity patterns uncovered in our study.

Infection Status with Digenetic Trematode Metacercariae in Fishes from Coastal Lakes in Gangwon-do, Republic of Korea.

The infection status of digenetic trematode metacercariae (DTM) was investigated in fishes from coastal lakes in Gangwon-do, the Republic of Korea (Korea). All fishes collected in 5 lakes were examined with the artificial digestion method. More than 10 species, i.e., Metagonimus spp., Pygidiopsis summa, Centrocestus armatus, Metorchis orientalis, M. taiwanensis, Clinostomum complanatum, Echinostoma spp., Stictodora spp., Diplostomum sp. and Diplostomid No. 1. by Morita (1960), of DTM were detected in fishes from 5 coastal lakes in Gangwon-do. Metagonimus spp. metacercariae were found in 52 (41.3%) out of 126 sea rundace, Tribolodon hakonensis, from 5 lakes, and their density was 14.6 per fish infected. P. summa metacercariae were detected in 48 (84.2%) out of 57 mullets from 5 lakes, and their density was 316 per fish infected. C. armatus metacercariae were detected in 7 (14.6%) T. hakonensis and 3 (15.0%) Tridentiger brevispinis from Hyang-ho, and 5 (19.2%) Acanthogobius flavimanus from Gyeongpo-ho. Stictodora spp. metacercariae were found in 4 fish species, i.e., Tridentiger obscurus, Tridentiger trigonocephalus, Chelon haematocheilus, and Acanthogobius lactipes, from Gyeongpo-ho. Total 15 C. complanatum metacercariae were detected in 2 (9.1%) crucian carp, Carassius auratus, from Songji-ho. M. taiwanensis metacercariae were found in T. hakonensis from Hyang-ho and Gyeongpo-ho and in Pseudorasbora parva from Gyeongpo-ho. Total 11 M. orientalis metacercariae were detected in 3 (6.3%) T. hakonensis from Hyang-ho. From the above results, it was confirmed that various species of DTM are infected in fishes from coastal lakes in Gangwon-do, Korea.

Description of Tylodelphys darbyi n. sp. (Trematoda: Diplostomidae) from the threatened Australasian crested grebe (Podiceps cristatus australis, Gould 1844) and linking of its life-cycle stages.

Species of the genus Tylodelphys (Diplostomidae) have a cosmopolitan distribution. Metacercariae of these species infect the eye, brain, pericardial sac or body cavity of fish second intermediate hosts, and the adults are found in piscivorous birds of many orders. An unnamed species of Tylodelphys from the eyes of bullies (Gobiomorphus cotidianus) was characterized molecularly and morphologically as a metacercaria in a previous study, in which it was predicted that the adult of this species would be found in the Australasian crested grebe. Two specimens of this bird became available and specimens of the unnamed Tylodelphys species were, indeed, found in them, confirmed by identity of genetic sequence data. Found to differ morphologically from its congeners, the new species is here described as Tylodelphys darbyi n. sp. Three species are closest to the new species in morphology: Tylodelphys glossoides, T. immer and T. podicipina robrauschi. Compared with T. darbyi n. sp. these three species are slightly larger and possess longer eggs. Tylodelphys glossoides also differs in having a wider oral sucker and T. podicipina robrauschi in having comma- or kidney-shaped pseudosuckers and an ovary that reaches a larger size, along with higher upper limits for body width, hind body and sucker width, holdfast and oesophagus length, and pharynx, pseudosucker and testes length and width. Tylodelphys immer also differs from T. darbyi n. sp. in having a shorter ventral sucker and the largest pseudosuckers of any Tylodelphys species.

A new species of Atriophallophorus Deblock & Rosé, 1964 (Trematoda: Microphallidae) described from in vitro-grown adults and metacercariae from Potamopyrgus antipodarum (Gray, 1843) (Mollusca: Tateidae).

The adult and metacercaria life stages of a new species of the microphallid genus Atriophallophorus Deblock & Rosé, 1964 are described from specimens collected at Lake Alexandrina (South Island, New Zealand). In addition to molecular analyses of ribosomal and mitochondrial genes, metacercariae of Atriophallophorus winterbourni n. sp. from the snail host Potamopyrgus antipodarum (Gray) were grown in vitro to characterize internal and external morphology of adults using light and scanning electron microscopy and histological techniques. Atriophallophorus winterbourni n. sp. is readily distinguishable from Atriophallophorus coxiellae Smith, 1973 by having a different structure of the prostatic chamber, sub-circular and dorsal to genital atrium, rather than cylindrical, fibrous, elongate and placed between the seminal vesicle and the genital atrium. The new species is most similar to Atriophallophorus minutus (Price, 1934) with regards to the prostatic chamber and the morphometric data, but possesses elongate-oval testes and subtriangular ovary rather than oval and transversely oval in A. minutus. Phylogenetic analyses including sequence data for A. winterbourni n. sp. suggested a congeneric relationship of the new species to a hitherto undescribed metacercariae reported from Australia, both forming a strongly supported clade closely related to Microphallus and Levinseniella. In addition, we provide an amended diagnosis of Atriophallophorus to accommodate the new species and confirm the sinistral interruption of the outer rim of the ventral sucker caused by the protrusion of the dextral parietal atrial scale at the base of the phallus.

A morphological and molecular study of adults and metacercariae of Hysteromorpha triloba (Rudolpi, 1819), Lutz 1931 (Diplostomidae) from the Neotropical region.

Adults of Hysteromorpha triloba (Rudolpi, 1819), Lutz, 1931 inhabit primarily the intestine of cormorants across the globe, whereas metacercariae have been found in the body cavity of freshwater fishes of the families Cyprinidae, Ictaluridae, Ariidae, Pimelodidae and Catostomidae. In this study, adults and metacercariae identified as H. triloba were collected from the Neotropical cormorant (Nannopterum brasilianus) and from the Mexican tetra fish (Astyanax mexicanus) from the Gulf of Mexico and Pacific Ocean slopes in the Neotropical region. Partial DNA sequences of the mitochondrial gene cytochrome c oxidase subunit I (cox 1) and the internal transcribed spacers (ITS1, 5.8S and ITS2) of nuclear ribosomal DNA were generated for both developmental stages, and were compared with available sequences of H. triloba from the Nearctic region. The genetic divergence between metacercariae and adults of H. triloba from the Neotropical and Nearctic region (Canada) associated with the double-crested cormorant (Nannopterum auritus), ranged from 0 to 5.5% for cox 1 and from 0 to 0.2% for ITS. Phylogenetic analyses inferred with both molecular markers using maximum likelihood and Bayesian inference placed the adults and metacercariae in a single clade, confirming that both stages are conspecific. Our data confirmed that H. triloba is a widely distributed species across the Americas, parasitizing both the Neotropical and Nearctic cormorants in Argentina, Brazil, Venezuela, Mexico, USA and Canada.

Patterns of Clinostomum marginatum infection in fishes and amphibians: integration of field, genetic, and experimental approaches.

Digenetic trematodes of the genus Clinostomum are cosmopolitan parasites infecting fishes, amphibians, reptiles, and snails as intermediate hosts. Despite the broad geographical distribution of this genus, debate about the number of species and how they vary in host use has persisted. To better understand patterns of infection among host species and across life stages, we used large-scale field surveys and molecular tools to examine five species of amphibians and seven species of fishes from 125 California ponds. Among the 12,360 examined hosts, infection was rare, with an overall prevalence of 1.7% in amphibians and 9.2% in fishes. Molecular evidence indicated that both groups were infected with Clinostomum marginatum. Using generalized linear mixed effects models, host species identity and host life stage had a strong influence on infection status, such that Lepomis cyanellus (green sunfish) (49.3%) and Taricha granulosa (rough skinned newt) (9.2%) supported the highest overall prevalence values, whereas adult amphibians tended to have a higher prevalence of infection relative to juveniles (13.3% and 2.5%, respectively). Experimentally, we tested the susceptibility of two amphibian hosts (Pseudacris regilla [Pacific chorus frog] and Anaxyrus boreas [western toad]) to varying levels of cercariae exposure and measured metacercariae growth over time. Pseudacris regilla was 1.3× more susceptible to infection, while infection success increased with cercariae exposure dose for both species. On average, metacarcariae size increased by 650% over 20 days. Our study highlights the importance of integrating field surveys, genetic tools, and experimental approaches to better understand the ecology of host-parasite interactions.

Occurrence and molecular characterization of Clinostomum complanatum (Trematoda: Clinostomidae) in freshwater fishes caught from Turkey.

The metacercariae of Clinostomum species which known as yellow grubs have zoonotic potential by infecting humans. In the present study, a total of 403 freshwater fish specimens belonging to different genera from Central Anatolia Region of Turkey were examined for yellow grub metacercariae infections. Only three specimens belonging to Squalius cephalus were found to be infected with metacercariae with a prevalence on this host species of 2.4% and an overall prevalence of 0.7%. All the metacercariae were morphologically identified as Clinostomum complanatum. Partial fragments of mitochondrial cytochrome oxidase I (mt-COI) gene and internal transcribed spacer 2 (ITS-2) were amplified for sequence and phylogenetic analyses. The sequence analyses of ITS-2 and mt-COI revealed three and nine polymorphic sites leading to detection of four and five haplotypes within the related gene regions, respectively. Moreover, the intraspecific genetic distances for C. complanatum isolates ranged from 0.0 to 0.7% for ITS-2 and 0.0 to 1.4% for mt-COI data sets. Consequently, the present study has provided first combined morphologic and molecular data on C. complanatum infecting Turkish freshwater fishes.

Molecular data reveal high diversity of Uvulifer (Trematoda: Diplostomidae) in Middle America, with the description of a new species.

Members of the genus Uvulifer are distributed worldwide and infect aquatic snails and freshwater fishes as first and second intermediate hosts, respectively, and fish-eating birds (kingfishers) as definitive hosts. Metacercariae of Uvulifer spp. were collected from the fins and skin of 20 species of freshwater fishes in Mexico, Guatemala, Honduras, Nicaragua and Costa Rica, and the adults were recovered from the intestine of kingfishers in four localities of Mexico. The genetic divergence among 76 samples (64 metacercariae and 12 adults) was estimated by sequencing the 28S and 5.8S nuclear genes, as well as the internal transcribed spacers ITS1 and ITS2, and one mitochondrial gene (cox1). Maximum likelihood and Bayesian inference analyses inferred with each dataset showed a high genetic diversity within the genus Uvulifer across Middle America, revealing the existence of four genetic lineages that exhibit some level of host specificity to their second intermediate hosts. The metacercariae of lineage 1 were associated with characids and cyprinids in central and northern Mexico. Metacercariae of lineages 2 and 3 were associated with cichlids distributed widely across Middle America. The lack of adults of these lineages in kingfishers, in lineages 2 and 3, or the fact that just a few adult specimens were recovered, as in lineage 1, prevented a formal description of these species. The metacercariae of lineage 4 were found in poeciliids, across a distribution range comprising Mexico, Guatemala, Honduras and Nicaragua, and the adult was found in the green kingfisher in Mexico. The number of specimens sampled for lineage 4, for both gravid adults and metacercariae, allowed us to describe a new species, Uvulifer spinatus n. sp. We describe the new species herein and we discuss briefly the genetic diversity in Uvulifer spp. and the importance of using DNA sequences to properly characterize parasite diversity.

Occurrence and distribution of yellow grub trematodes (Clinostomum complanatum) infection in Taiwan.

Digenetic trematodes of the genus Clinostomum are widely distributed in many species of freshwater fish and are known to cause the zoonotic disease Halzoun. Humans may become accidental hosts if they ingest raw freshwater fish containing metacercaria of Clinostomum complanatum, which causes pharyngitis or laryngitis. The yellow grub parasitizing cultivated ayu (Plecoglossus altivelis) and loach (Misgurnus anguillicaudatus) was one of the most serious problems in fish farms from 1977 to 1979 in Taiwan. The present study revealed, for the first time, frequent occurrence of C. complanatum metacercaria in various fish species in a natural environment, the Dahan River, in Taiwan. We examined 1503 fish specimens belonging to four families and 162 snails representing six species. C. complanatum metacercaria was mainly from the fish family Cyprinidae, and only cercaria from the snail Radix swinhoei was recovered. The prevalence and mean intensity of metacercaria were 9.8% and 9.35 parasites/infected fish, respectively. The prevalence of cercaria was low, 0.62%, in snails. Acrossocheilus paradoxus, Zacco barbata, Zacco pachycephalus, Zacco platypus, Onychostoma barbatula, and Hemibarbus labeo are new host records. Metacercariae were primarily found in the operculum, mandible, muscle, and oral cavity of fish. Morphological description and molecular analysis with 18S rDNA sequencing allowed for rapidly identifying C. complanatum. Encysted and excysted metacercariae cultivated at 22 °C in physiological saline died within 60 h. The mean intensity of infection increased with an increasing length of Z. pachycephalus. We found no association between monthly parasite prevalence and mean intensity at each sampling location. No C. complanatum metacercaria survived after 8 h of salting. The Dahan River has suitable conditions and hosts (snails, fish, and fish-eating birds) for maintaining the life cycle of C. complanatum.

An investigation of the host-specificity of metacercariae of species of Apophallus (Digenea: Heterophyidae) in freshwater fishes using morphological, experimental and molecular methods.

Metacercariae of species of the genus Apophallus Lühe, 1909, infecting the fins and skin of freshwater fishes, frequently cause black spot disease. Two species, Apophallus muehlingi (Jägerskiöld, 1899) and A. donicus (Skrjabin & Lindtrop, 1919), are known to occur in Hungarian fishes. It has generally been thought that metacercariae of A. muehlingi infect cyprinid fishes, whereas those of A. donicus develop in percids. As part of a morphological, experimental and molecular study, metacercariae were collected from 99 infected specimens of five cyprinid hosts (Abramis brama, Blicca bjoerkna, Chondrostoma nasus, Squalius cephalus, Scardinius erythrophthalmus) and 18 infected specimens of two percid hosts (Gymnocephalus cernua, Perca fluviatilis) in Hungarian natural waters (Lake Balaton, River Danube). Moreover, 1024 common carp (Cyprinus carpio) specimens collected from Hungarian fish ponds were investigated for Apophallus infection, but without positive results. For reliable species identification, experimental infections of chicks were carried in order to produce adult specimens from metacercariae collected from the fins and skin of the cyprinid and percid hosts. Within 8 days, adult specimens of both A. muehlingi and A. donicus developed in chicks infected with metacercariae from the cyprinid common bream (Abramis brama) and the white bream (Blicca bjoerkna) and the ruffe (Gymnocephalus cernua), a percid, respectively. The morphology of the collected metacercariae and adult individuals developed in the feeding experiments was characterised. A molecular analysis was extended to cercarial samples from the snail Lithoglyphus naticoides and to a single adult specimen of Apophallus from a fox. Sequences of 28 specimens were analysed using molecular methods (sequencing the internal transcribed spacer region and the cytochrome oxidase I subunit). Phylogenetic analysis was executed, and the Apophallus samples clustered into three distinct branches using both genes, A. muehlingi from cyprinids, A. donicus from percids and, a third, previously unknown, Apophallus clade, also from cyprinids.