Molecular evidence of the absence of Metagonimus yokogawai (Katsurada, 1912) in Europe: report of Metagonimus sp. in cyprinoid fish from the River Danube in Hungary.

Trematodes of the genus Metagonimus Katsurada, 1912 (Digenea: Heterophyidae) are zoonotic parasites that cause infections in humans, with most cases reported in Southeast Asia. Larvae from the second intermediate host, called metacercariae, of one of human-infecting species, M. yokogawai (Katsurada, 1912), have been reported from cyprinoid fish in Europe. In the present study, we provided DNA-based evidence that metacercariae of Metagonimus, which are commonly found in the scales of various cyprinoids in Central Europe (Danube River in Hungary) do not belong to M. yokogawai. Sequence analysis of the ITS region, 28S rDNA, and cox1 genes showed that this species is clearly distinct from all Asian species, including M. yokogawai, which probably does not occur in Europe. Metacercariae from cyprinoids might belong to Metagonimus romanicus (Ciurea, 1915), an insufficiently known species described from Romania.

Scaphanocephalus spp. (Trematoda: Opisthorchiidae) in intermediate and definitive hosts of the Yucatán Peninsula, Mexico, with a re-description of Scaphanocephalus expansus.

Scaphanocephalus is a small trematode genus belonging to the family Opistorchiidae. The genus currently contains only three species associated with marine fish as intermediate hosts and fish-eating birds as definitive hosts. Here, specimens of Scaphanocephalus were collected from the Osprey, Pandion haliaetus, and the White mullet, Mugil curema in the Yucatán Peninsula, Mexico. We report for the first-time DNA sequences of adult specimens of Scaphanocephalus, particularly S. expansus, as well as a sequence of a different species sampled as metacercaria. Morphological comparisons of Scaphanocephalus expansus confirmed the identity of the adult specimens, with minor morphological variations; Scanning electron photomicrographs were included, and the species was re-described. Phylogenetic analysis based on 28S rDNA sequences showed that Scaphanocephalus is monophyletic within Opisthorchiidae and consists of three independent lineages. Sequences of adults are identical to those of S. expansus. Instead, the sequence of the metacercaria sampled from the mesentery of Mugil curema nested with specimens reported as Scaphanocephalus sp. from a labrid fish in the Mediterranean Sea, herein named it as Scaphanocephalus sp. 2.

Assessing the population structure of trematode Metagonimus suifunensis using three mitochondrial markers.

In this work, for the first time, the genetic variability of the Metagonimus suifunensis population in the Russian southern Far East was estimated based on the full-length sequences of the nad1 gene of mitochondrial DNA. In addition, for a sample of the same size, the sequences of cox1 and cytb genes, previously used for population studies for M. suifunensis, were reanalysed. Three markers were combined to a common sequence, and the obtained data were studied. Despite the higher level of variability, nad1 and cox1 mtDNA genes did not reveal subdivisions within the population. The combined dataset made it possible to determine that the sample from the Odyr River was the centre of the species' range formation and clarified the continental migration route of the parasite from south to north. According to the data obtained, it was presumed that piscivorous birds participate in the life cycle of the parasite. The subdivision within population revealed that using all three mitochondrial markers is consistent with the features of differentiation within populations of related species, but the reasons for its formation remain unclear due to the insufficient amount of data and the use of different markers in studies of different species.

Cytochrome b as a more promising marker for analysing the distribution vector for Metagonimus suifunensis (Trematoda: Heterophyidae).

In this study of Metagonimus suifunensis (M. suifunensis) in the Russian Southern Far East, the variability of the full-length sequences of the cytochrome b (cytb) mtDNA gene was assessed for the first time. In addition, the cox1 mtDNA gene sequences were also obtained for this species from new localities. In total, 87 and 81 sequences of the cytb and cox1 genes, respectively, were used in the current study. The cytb gene proved more promising and revealed two haplogroups that are associated with the spatial distribution of the species: geographical isolation caused the fixation of differences between northern and southern populations. In addition, the results obtained for the cytb gene opened up new perspectives in the analysis of sequences of the cox1 gene, which was not sufficiently effective as a sole marker. Based on data for both mitochondrial genes, molecular processes influencing the formation of the modern population were analysed for M. suifunensis. The new data confirmed the previously expressed opinion that this species colonized the study territory from north to south and will form the basis for determining possible ways of its further expansion, which is important for predicting the emergence of new foci of metagonimosis.

Ascocotyle longa (Digenea: Heterophyidae) infecting dolphins from the Atlantic Ocean.

We report for the first time the infection of dolphins with Ascocotyle longa found in the intestines of three different species, Sotalia guianensis, Steno bredanensis, and Tursiops truncatus gephyreus, which were found washed ashore along the southeastern and southern Brazilian coast. The worms were identified based on morphological and molecular data using the 28S rDNA gene and the COI gene. Specimens of A. longa from the pinniped Otaria flavescens were also analyzed. As the first isolation of A. longa from cetaceans, the present study increases the distribution area and range of definitive hosts of this trematode, and provides new molecular data to complement the phylogeny of the group in future studies, thus contributing to the scientific knowledge of this potentially zoonotic parasite.

Current Knowledge of Small Flukes (Digenea: Heterophyidae) from South America.

Fish-borne heterophyid trematodes are known to have a zoonotic potential, since at least 30 species are able to infect humans worldwide, with a global infection of around 7 million people. In this paper, a 'state-of-the-art' review of the South American heterophyid species is provided, including classical and molecular taxonomy, parasite ecology, host-parasite interaction studies and a list of species and their hosts. There is still a lack of information on human infections in South America with undetected or unreported infections probably due to the information shortage and little attention by physicians to these small intestinal flukes. Molecular tools for specific diagnoses of South American heterophyid species are still to be defined. Additional new sequences of Pygidiopsis macrostomum, Ascocotyle pindoramensis and Ascocotyle longa from Brazil are also provided.

Morphological and Molecular Identification of Stellantchasmus dermogenysi n. sp. (Digenea: Heterophyidae) in Thailand.

We tried a series of morphological and molecular approaches to identify a new species of Stellantchasmus (Digenea: Heterophyidae) originating from the wrestling half-beaked fish, Dermogenys pusillus of Thailand. Adult worm samples of the new species were recovered from hamsters experimentally infected with the metacercariae from D. pusillus in Thailand. Two isolates (Thai and Korean) of Stellantchasmus falcatus were used as comparative control groups. Worm samples of 3 Stellantchasmus groups were morphologically observed and molecularly analyzed with the mitochondrial cytochrome c oxidase 1 gene. The morphological characteristics of S. dermogenysi n. sp. are similar to S. falcatus originating from brackish water fish, but minor difference was noted including the absence of the prepharynx, position of the ovary near the ceca end, smaller body size, and shorter esophageal length. A phylogenetic tree derived from neighborjoining and maximum-likelihood methods suggests that S. dermogenysi n. sp. is separated from S. falcatus supported by high bootstrap values. The relative divergences persist between these host-specific trematodes, which we suggest should be recognized as 2 distinct species. Comparisons of S. dermogenysi n. sp. with S. falcatus isolated from mullets in Thailand and Korea indicate a genetic divergence of mitochondrial DNA of 19.4% and 21.7%, respectively. By the present study, a new species, Stellantchasmus dermogenysi n. sp. (Digenea: Heterophyidae), is proposed in Thailand based on molecular evidences, in addition to minor morphological differences between S. falcatus and the new species.

Galactosomum otepotiense n. sp. (Trematoda: Heterophyidae) infecting four different species of fish-eating birds in New Zealand: genetically identical but morphologically variable.

Trematodes of the genus Galactosomum are cosmopolitan parasites that infect the intestines of fish-eating birds and mammals. Adults of named Galactosomum species have not been recorded from bird hosts in New Zealand, despite their cercarial stage being known from various studies of the first intermediate host, Zeacumantus subcarinatus. Here we describe a new species of Galactosomum infecting four different piscivorous birds in New Zealand: Caspian terns, red-billed and black-backed gulls and little blue penguins. Specimens from each of these hosts are genetically identical in the genes sequenced, but show considerable morphological variability. Galactosomum otepotiense n. sp. is distinguished from most other members of the 'bearupi-group' in having a single circle of spines on the ventral sucker, and spines, as opposed to scales, over most of the body. It is most similar to G. bearupi and G. angelae, both from Caspian terns in Australia, but differs in the relative sizes of the reproductive organs and in the possession of a very long forebody. Molecular data confirm that G. otepotiense is not conspecific with G. bearupi, but 28S and ITS2 phylogenies show its close relationship to G. bearupi and other Australian species. We use the cox1 sequence to confirm identity with the larval stage infecting Z. subcarinatus, as previously described in the literature. We discuss briefly the relationships between Australian and New Zealand Galactosomum spp. and their hosts, variability between genetically identical specimens found in different hosts and their potential for harm to mariculture economy.

Digeneans of northern fur seals Callorhinus ursinus (Pinnipedia: Otariidae) from five subpopulations on St. Paul Island, Alaska.

A parasitological survey of 651 northern fur seals Callorhinus ursinus L. from five subpopulations was conducted on St. Paul Island, Alaska, during July-August 2012-2014. Digenean trematodes were found in 210 of 651 fur seals with a total prevalence of 32.3%. Intensity of infection varied from 1 to 1540 parasites with mean intensity 18.4 ± 111.1 SD and median intensity of 2 specimens per host. Significant differences in prevalence and intensity of infection in northern fur seals between separate rookeries was not observed (Mann-Whitney test; p > 0.05). Four species of digeneans belonging to the families Heterophyidae (Apophallus zalophi Price, 1932, Phocitrema fusiforme Goto and Ozaki, 1930, and Galactosomum ubelakeri (Dailey, 1969)) and Troglotrematidae (Nanophyetus salmincola (Chapin, 1926)) were found. Nanophyetus salmincola is reported from C. ursinus for the first time. We obtained partial 28S rDNA sequences for all digenean species and conducted molecular phylogenetic analysis to demonstrate their phylogenetic relationships.

First report of Euryhelmis parasites (Trematoda, Heterophyidae) in Africa: conservation implications for endemic amphibians.

In this study, we report, through molecular identification, the first African records of a digenean trematode parasite of the genus Euryhelmis. We recovered metacercariae encysted in an anuran, the endemic Moroccan painted frog (Discoglossus scovazzi), and a vulnerable caudate, the North African fire salamander (Salamandra algira), from four localities in North Africa (Morocco). Our records go back to the past century and have been confirmed in successive fieldwork seasons thereafter. Metacercarial stages of these parasites require amphibians as the last intermediate host, but the exact identity of the primary hosts and predators of the infected animals in Africa remain unknown. Our searches with basic local alignment search tool (BLAST) from Genbank revealed that hosts were infected by parasites of Euryhelmis costaricensis, which showed almost the same genetic identity (with only one substitution) to previous reports from Costa Rica and Japan, suggesting a recent introduction in Morocco. We proceed to discuss the likely role of introduced mustelids as the potential definitive hosts of trematode adults. Under this assumption, we conclude that the infestation of Discoglossus scovazzi and Salamandra algira might pose a risk to these threatened species.

Description of Metagonimus pusillus sp. nov. (Trematoda: Heterophyidae): phylogenetic relationships within the genus.

As a result of experimental studies conducted in the Russian southern Far East, adult worms from the genus Metagonimus were obtained. A comparative analysis of the morphometry of these worms with other Metagonimus representatives showed that they are most similar to M. katsuradai Izumi, 1935 and M. otsurui Shimazu & Urabe, 2002 found in Japan, due to the ratio of suckers and the positions of the testicle, uterus and vitellaria. However, Russian worms differ from species in Japan by other metric characters: they differ from M. otsurui by the maximum size of most organs and from M. katsuradai by body width, pharynx length, and maximum size of testes and ovary. At the same time, they are identical to a trematode from the Russian southern Far East, which was previously identified as M. katsuradai. The validity of this species was also confirmed by genetic data. According to the 28S gene and the internal transcribed spacer 2 (ITS2) region of rDNA, as well as the cytochrome c oxidase I (cox1) gene of mtDNA, the Metagonimus specimens found in Russia differ from published genetic data for other members of this genus. However, both morphological similarity and molecular data showed that M. pusillus sp. nov., M. katsuradai and M. otsurui are most likely cryptic species. Furthermore, additional data based on a mitochondrial marker were provided for M. suifunensis Shumenko, Tatonova & Besprozvannykh, 2017 from Russia.

Two new species of Haplorchoides Chen, 1949 (Digenea: Heterophyidae) infecting an Australian siluriform fish, Neoarius graeffei Kner & Steindachner.

Combined morphological and molecular analyses are used to characterise two new species of Haplorchoides Chen, 1949 (Digenea: Heterophyidae) from an Australian siluriform fish. Haplorchoides maiwariensis n. sp. and H. daguilarensis n. sp. are described from the intestine of the Blue salmon catfish, Neoarius graeffei (Kner & Steindachner) (Siluriformes: Ariidae) from tidal reaches of the Brisbane River and from a freshwater creek off Lake Wivenhoe, Kipper Creek, Australia. The two new species most obviously differ from all previously described species of Haplorchoides in possessing clearly and reliably tripartite seminal vesicles. The two new species differ in the size and distribution of vitelline follicles, the size of the pigment granules and the form of the ventral sucker. Previously described species of Haplorchoides have been reported from Africa and Asia, principally from bagrid, schilbeid, silurid and sisorid catfishes; these are the first species reported from an ariid catfish. Complete ITS2 and partial 28S ribosomal DNA data were generated for both new species. The two species differ from each other by eight base pairs in the ITS2 region, and by 13 bp for the 28S region. The 28S rDNA sequence of H. daguilarensis agrees with a previously reported sequence from an unidentified species of Haplorchoides collected from N. graeffei in Lake Wivenhoe, Australia; we identify this previous report as relating to H. daguilarensis.

Molecular characterization and confocal laser scanning microscopic study of Pygidiopsis macrostomum (Trematoda: Heterophyidae) parasites of guppies Poecilia vivipara.

Pygidiopsis macrostomum and Ascocotyle (Phagicola) pindoramensis (Digenea: Heterophyidae) parasitize guppies as intermediate hosts and, respectively, fish-eating mammals or birds as definitive hosts. Heterophyids have zoonotic potential, and molecular studies associated with morphological and ecological aspects have helped to clarify their taxonomy and phylogeny. Poecilia vivipara naturally parasitized by metacercariae of both species (100% prevalence) exhibit no external signs of parasitism. In this work, four new sequences of P. macrostomum (18S rDNA, 28S rDNA and ITS2 rDNA) and one new sequence of A. (P.) pindoramensis (mtDNA cox-1) are presented. Phylogeny reconstructions linked P. macrostomum to other heterophyids, but the separation of the Heterophyidae and Opisthorchiidae remains unclear. Additionally, we used indirect immunocytochemistry and the phalloidin-fluorescence techniques allied with confocal laser scanning microscopy to describe muscular and neuronal structures of P. macrostomum. A complex arrangement of muscular fibres is associated with the tegument, suckers, gut and reproductive system. Radial fibres around the ventral sucker are thick, branched and extend to the body wall. High-resolution confocal imaging revealed a typical digenean muscular arrangement and important heterophyid morphological traits. These data will support future control measures to reduce the parasitism in guppies reared in fish farming systems, especially for aquarium and experimental purposes.

New locality record for Haplorchoides mehrai and possible interactions with Opisthorchis viverrini metacercariae in cyprinid fishes in Northeast Thailand.

Metacercariae of Opisthorchis viverrini, a carcinogenic liver fluke, and Haplorchoides sp., a trematode maturing in catfish, are commonly found in cyprinid fish, the second intermediate hosts of both flukes. However, the specific identity of Haplorchoides sp. in Thailand and a precise assessment of the effects of co-infections with O. viverrini have never been clarified. Therefore, we aimed to identify the species of Haplorchoides and to investigate possible interactions of the two trematode species in cyprinid fishes. Based on the morphology and morphometry of the cercaria, metacercaria, and adult stages, the Haplorchoides species found was identified as Haplorchoides mehrai Pande and Shukla 1976. Thailand is formally recorded as a new locality for H. mehrai, where naturally infected hosts include the snail Melanoides tuberculata (first intermediate host), the cyprinid fishes Hampala dispar, Cyclocheilichthys apogon, Puntius leiacanthus, Labiobarbus burmanicus, and Cirrhina jullieni (second intermediate hosts), and a catfish, Mystus nemurus (definitive host). The co-infection rates of O. viverrini and H. mehrai were significantly associated with fish species and fish body region (P < 0.001), with an overall significantly higher average intensity of H. mehrai (126.26 metacercariae/fish) than that of O. viverrini (18.02 metacercariae/fish). Further work is required to demonstrate the extent and mechanisms of possible interactions between these trematode species in the fish host. These data may provide a better understanding of O. viverrini transmission dynamics, and help design integrated control interventions.

Prevalence of Centrocestus formosanus Metacercariae in Ornamental Fish from Chiang Mai, Thailand, with Molecular Approach Using ITS2.

The prevalence of Centrocestus formosanus metacercariae was investigated in ornamental fish purchased from a pet shop in Chiang Mai, Thailand, including Carassius auratus (goldfish), Cyprinus carpio (Koi), Poecilia latipinna (Sailfin Molly), Danio rerio (Zebrafish), and Puntigrus tetrazona (Tiger barb). The parasite species was identified by the morphology of worms as well as by a molecular approach using ITS2. The results showed that 50 (33.3%) of 150 fish examined were infected with the metacercariae. The highest prevalence was found in C. auratus (83.3%), and the highest intensity was noted in C. carpio (70.8 metacercariae/fish). The most important morphological character was the presence of 32-34 circumoral spines on the oral sucker. The phylogenetic studies using the rRNA ITS2 region revealed that all the specimens of C. formosanus in this study were grouped together with C. formosanus in GenBank database. This is the first report on ornamental fish, C. carpio, P. latipinna, D. rerio, and P. tetrazona, taking the role of second intermediate hosts of C. formosanus in Thailand. Prevention and control of metacercarial infection in ornamental fish is urgently needed.

Molecular Phylogenetics of Centrocestus formosanus (Digenea: Heterophyidae) Originated from Freshwater Fish from Chiang Mai Province, Thailand.

This study aimed to investigate the morphology and reconstruct the phylogenetic relationships of Centrocestus formosanus originating from 5 species of freshwater fish, i.e., Esomus metallicus, Puntius brevis, Anabas testudineus, Parambassis siamensis, and Carassius auratus, in Chiang Mai province, Thailand. Sequence-related amplified polymorphism (SRAP) and phylogeny based on internal transcribed spacer 2 (ITS2) and mitochondrial cytochrome c oxidase subunit 1 (CO1) were performed. The results showed similar morphologies of adult C. formosanus from day 5 after infection in chicks. C. formosanus originated from 4 species of freshwater fish had the same number of circumoral spines on the oral sucker, except for those from C. auratus which revealed 34 circumoral spines. The phylogenetic tree obtained from SRAP profile and the combination of ITS2 and CO1 sequence showed similar results that were correlated with the number of circumoral spines in adult worms. Genetic variability of C. formosanus also occurred in different species of freshwater fish hosts. However, more details of adult worm morphologies and more sensitive genetic markers are needed to confirm the species validity of C. formosanus with 34 circumoral spines originating from C. auratus in the future.

Genetic differences among Haplorchis taichui populations in Indochina revealed by mitochondrial COX1 sequences.

Haplorchis taichui is an intestinal heterophyid fluke that is pathogenic to humans. It is widely distributed in Asia, with a particularly high prevalence in Indochina. Previous work revealed that the lack of gene flow between three distinct populations of Vietnamese H. taichui can be attributed to their geographic isolation with no interconnected river basins. To test the hypothesis that interconnected river basins allow gene flow between otherwise isolated populations of H. taichui, as previously demonstrated for another trematode, Opisthorchis viverrini, we compared the genetic structures of seven populations of H. taichui from various localities in the lower Mekong Basin, in Thailand and Laos, with those in Vietnam, using the mitochondrial cytochrome c oxidase subunit 1 (COX1) gene. To determine the gene flow between these H. taichui populations, we calculated their phylogenetic relationships, genetic distances and haplotype diversity. Each population showed very low nucleotide diversity at this locus. However, high levels of genetic differentiation between the populations indicated very little gene flow. A phylogenetic analysis divided the populations into four clusters that correlated with the country of origin. The negligible gene flow between the Thai and Laos populations, despite sharing the Mekong Basin, caused us to reject our hypothesis. Our data suggest that the distribution of H. taichui populations was incidentally associated with national borders.

A molecular phylogeny of Asian species of the genus Metagonimus (Digenea)--small intestinal flukes--based on representative Japanese populations.

Metagonimus Katsurada, 1912 is a genus of small intestinal parasites. The genus comprises eight species, primarily from far-eastern Asia, with two exceptions reported from Europe. Metagonimus yokogawai, the most widespread species, is the main agent responsible for the intestinal disease, metagonimiasis, in Japan and some other East Asian countries. On the basis of the ratio of the size of the ventral and oral suckers, Metagonimus has traditionally been morphologically divided into two groups; however, the genus has not been extensively studied using molecular data. To reveal phylogenetic relationships within Metagonimus based on molecular data, we analyzed six of the seven species present in Asia using samples collected in central Japan. Maximum likelihood and Bayesian analyses of a combined 28S ribosomal DNA (rDNA), internal transcribed spacer 2 (ITS2), and mitochondrial cox1 gene sequence dataset separated the six species into two well-supported clades. One clade comprised M. yokogawai, M. takahashii, M. miyatai, and M. hakubaensis, whereas the other consisted of M. otsurui and M. katsuradai. Genetic distances calculated from 28S rDNA and ITS2 nucleotide sequences and a comparison of the predicted amino acid sequences of cox1 gene suggested that M. otsurui and M. katsuradai may have diverged recently. None of the four main morphological characters used to delimit species of Metagonimus (i.e., sucker ratio, positions of the uterus and testes, and distribution of vitelline follicles) was consistent with the distribution of species in the molecular tree.

Epidemiological and molecular data on heterophyid trematode metacercariae found in the muscle of grey mullets (Osteichthyes: Mugilidae) from Sardinia (western Mediterranean Sea).

This study is a contribution to the molecular taxonomy and epidemiology of heterophyid (Trematoda: Heterophyidae) metacercariae found in the muscle of Mugilidae (Osteichthyes) from Sardinia (western Mediterranean Sea). Sixty specimens of mugilids (13 Chelon labrosus, 18 Liza aurata, 6 Liza ramada, 8 Liza saliens, 15 Mugil cephalus) were examined and 17,899 metacercariae isolated in 95 % of the hosts. Four types of metacercariae were identified: Heterophyes sp. (n = 14,113), Heterophyes sp. -small (1225), Stictodora sp. (1606), and Ascocotyle (Phagicola) sp. (955). The experimental infection of a hamster with Heterophyes sp. metacercariae produced six adults identified as Heterophyes heterophyes and two as Heterophyes cf. nocens. The morphology of Heterophyes sp. -small metacercariae matched with that of Heterophyes dispar. The sequence analysis of the ITS2 and 28S portions of rDNA confirmed the morphological identification of metacercariae, showing four clusters. All adults grouped together with the Heterophyes sp. metacercariae, whereas adults of Heterophyes nocens from Korea clustered separately, showing that this species is distinguished from H. heterophyes, and suggesting caution in the exclusive use of the number of rodlets of the genital sucker to separate the two species. The presence of metacercariae was high in all hosts; the highest prevalence is of Heterophyes sp. (prevalence ≥78 %; mean intensity ≥135 metacercariae/100 g muscle), and the most heavily infected host is M. cephalus (prevalence = 100 %; mean intensity = 841 metacercariae/100 g muscle).

Molecular characterization of Stictodora tridactyla (Trematoda: Heterophyidae) from Kuwait Bay using rDNA ITS and mtCO1.

Stictodora tridactyla is an intestinal fluke in the family Heterophyidae that parasitizes shorebirds and mammals, including humans. Its metacercarial cyst stage was reported in the Arabian killifish, Aphanius dispar, at Kuwait Bay. In the present study, Cerithidea cingulata was found to serve as the first intermediate host of S. tridactyla. In order to establish the snail-fish link in the life cycle of S. tridactyla, complete sequences of ribosomal DNA internal transcribed spacer region 1 and 2 (rDNA ITS1 and ITS2) and partial sequence of cytochrome oxidase subunit 1 were obtained for metacercarial cysts isolated from the fish A. dispar and rediae isolated from the snail C. cingulata. Sequence alignment demonstrated that these larval stages belong to the same heterophyid species, S. tridactyla. Phylogenetic analysis based on rDNA ITS1, ITS2, and mtCO1 confirmed the position of S. tridactyla within the Heterophyidae and found it to cluster with Haplorchis spp. The present study represents the first molecular study correlating the larval stages of S. tridactyla using rDNA ITS1, ITS2, and mtCO1 and examining the phylogenetic relationships of S. tridactyla with different heterophyid species.