Introduction
Among the representatives of the family Psilostomidae Looss, 1900, trematodes of the genus Psilotrema Odhner, 1913 are overwhelmingly parasites of birds. Only two species, Psilotrema spiculigerum (Mühling, 1898) Odhner, 1913 and Psilotrema simillimum (Mühling, 1898) Odhner, 1913, have been found not only in the intestines of birds but also in the muskrat Ondatra zibethicus (Linnaeus, 1766) introduced from North America to Eurasia (Matskasi Reference Matskasi1974; Mazeika et al. Reference Mazeika, Kontenytė and Paulauskas2009; Schuster et al. Reference Schuster, Specht and Rieger2021). In contrast to these species, Psilotrema marki Skworcow, 1934 and Psilotrema pharyngeatum Grabda, Reference Grabda1954 have been found only in mammals, the European water vole Arvicola terrestris (Linnaeus, 1758), and the muskrat O. zibethicus, while P. zibethica has been recorded only in muskrats (Grabda Reference Grabda1954; Sey Reference Sey1966; Skrjabin 1947; Varenov Reference Varenov1965). It is worth noting that all the findings of the listed trematode species in rodents of Cricetidae Fischer, 1817 were made within the European region. With regard to East Asia, O. zibethicus is widely distributed in the region and inhabits, among other places, water bodies in which the first intermediate hosts, snails of the family Bithyniidae Gray, 1857, are infected with Psilotrema spp. However, trematodes of this genus have not previously been reported in rodents in East Asia.
The species affiliation of most representatives of the genus Psilotrema is determined based on morphological data. Molecular data are presented only for five species of the genus, four of which were detected in the Russian Far East and one in Germany (Table 1) (Schwelm et al. Reference Schwelm, Kudlai, Smit, Selbach and Sures2020). In the Ilistaya River basin (Russian Far East), Psilotrema cercariae were previously found in the snails Parafossarulus Annandale, 1924, from which adult worms Psilotrema limosum Kalinina, Tatonova, Besprozvannykh, 2022 were obtained as a result of laboratory experiments (Kalinina et al. Reference Kalinina, Tatonova and Besprozvannykh2022). In the current study, 14 individuals belonging to Psilotrema were removed from the intestine of a naturally infected muskrat O. zibethicus from the same locality. To establish the species identity of these worms, both morphological and molecular data were analyzed. The results obtained are given below.
List of species used in the analysis with the accession numbers of the 28S, nad1 sequences and other related information
Materials and methods
Sample collection and slide preparation
Mature Psilotrema worms in the amount of 14 individuals were removed from the small intestine of one naturally infected muskrat Ondatra zibethicus trapped in the backwater of the Ilistaya River in the south of the Russian Far East (44o17′ N, 132o34′ E). Among the worms obtained, there were two groups of trematodes with discernible morphological differences, which were designated as G1 (5 samples) and G2 (9 samples). Worms from both groups were washed in distilled water and separately fixed in 70% ethanol. Whole mounts of adult flukes were prepared by staining with alum carmine, dehydrating in a graded ethanol series, clearing in clove oil, and mounting in Canada balsam. All measurements are given in micrometers (μm).
Molecular experiments
One and two Psilotrema samples from G1 and G2, respectively, found in the same muskrat, as well as two previously experimentally obtained individuals of Psilotrema limosum, were used for molecular analysis. The latter were obtained in the intestine of a chicken, which was experimentally infected with cercariae emitting from the Parafossarulus snails (Kalinina et al. Reference Kalinina, Tatonova and Besprozvannykh2022). DNA extraction was performed using the HotSHOT method (Truett et al. Reference Truett, Heege, Mynatt, Truett, Walker and Warman2000). Partial sequences were amplified and sequenced for tree genes, 28S ribosomal RNA (28S), nad1 (nad1) and cox1 (cox1) mitochondrial DNA. For amplification, the following primers were used: forward LSU5 (5′-TAG-GTC-GAC-CCG-CTG-AAY-TTA-AGC-A-3′) (Tkach et al. Reference Tkach, Timothy, Littlewood, Olson, Kinsella and Swiderski2003) and reverse 1500R (5′-GCT-ATC-CTG-AGG-GAA-ACT-TCG-3′) (Tkach et al. Reference Tkach, Timothy, Littlewood, Olson, Kinsella and Swiderski2003) for 28S; forward NDj11 (5′-AGA-TTC-GTA-AGG-GGC-CTA-ATA-3′) and reverse NDj2A (5′-CTT-CAG-CCT-CAG-CAT-AAT-3′) (Morgan and Blair Reference Morgan and Blair1998) for nad1; forward JB3 (5′-TTT-TTT-GGG-CAT-CCT-GAG-GTT-TAT-3′) and reverse JB4-5 (5′-TAA-AGA-AAG-AAC-ATA-ATG-AAA-ATG-3′) (Bowles et al. Reference Bowles, Blair and McManus1992) for cox1.
For 28S sequencing, the internal primer 900f was used (5′-CCG-TCT-TGA-AAC-ACG-GAC-CAA-G-3′) in addition to the external primers (Tkach et al. Reference Tkach, Timothy, Littlewood, Olson, Kinsella and Swiderski2003). Only external primers were used for sequencing nad1 and cox1. Sequencing was performed using the Applied Biosystems 3500 Genetic Analyzer at the Instrumental Centre for Biotechnology and Gene Engineering, Federal Scientific Center of East Asia Terrestrial Biodiversity, Far Eastern Branch, Russian Academy of Sciences (Vladivostok, Russia). The obtained nucleotide sequences were visually checked using the Finch TV ver. 1.4.0 program and manually aligned in the program MEGA ver. 5.03 (Tamura et al. Reference Tamura, Peterson, Peterson, Stecher, Nei and Kumar2011). The nucleotide sequences of 28S, nad1, and cox1 obtained in this study were deposited in GenBank under accession nos. PQ415712–PQ415714, PQ429217–PQ429220, and PV243661–PV243665, respectively. All available sequences of other representatives of the family Psilostomidae downloaded from the NCBI genetic sequence database (GenBank) are listed in Table 1. Representatives of the family Notocotylidae were selected as an outgroup.
Partial nucleotide sequences of 28S, nad1, and cox1 with lengths of 1257 bp, 404 bp, and 348 bp, respectively, were obtained for the samples of Psilotrema used in the study. Previously, Kalinina and co-authors (2022) found similarity in the topology of phylogenetic reconstructions for the family Psilostomidae based on different lengths of 28S sequences. Therefore, the tree for this marker was constructed using a shorter sequence length. This allowed us to include a larger number of samples in the analysis. Thus, after alignment, phylogenetic trees were built using 28S and nad1 sequences of 708 bp and 397 bp, respectively (Figures 1 and 2). Phylogenetic reconstructions based on cox1 are not represented due to the lack of the genetic data in the NCBI database.
Phylogenetic tree of the family Psilostomidae constructed based on 28S sequences (708 bp). Posterior probability ≥ 50 for BI and nodal support ≥ 0.50 for ML are shown at nodes, respectively. Scale bar indicates number of substitutions per site.
(a) Phylogenetic tree of the family Psilostomidae constructed based on nad1 sequences (397 bp). Posterior probability ≥ 50 for BI and nodal support ≥ 0.50 for ML are shown at nodes, respectively. Scale bar indicates number of substitutions per site; (b) Matrix of nucleotide substitutions between nad1 sequences of the genus Psilotrema individuals. Substitutions are marked in bold.
Phylogenetic reconstructions were obtained based on the Bayesian Inference (BI) in the program MrBayes (BI) (Ronquist and Huelsenbeck Reference Ronquist and Huelsenbeck2003) and the Maximum Likelihood (ML) algorithm in the program PhyML 3.1 using the model selected as optimal in the program jModeltest 2.1.7 (Darriba et al. Reference Darriba, Taboada, Doallo and Posada2012): TVM+I+G and TPM3uf+I+G for 28S and nad1, respectively. The parameters of the models were used, taking into account the Akaike criterion (AIC). We performed BI analyses using 500,000 Markov chain Monte Carlo (MCMC) generations for both markers. The number of generations was determined to be sufficient since the SD value calculated was <0.01. Of the samples assessed, 25% were excluded to permit the construction of consensus trees. The ML trees were built with 100 bootstrap pseudoreplicates. Analysis of different phylogenetic trees constructed by BI and ML methods showed similar results with high values of node support. Therefore, consensus trees for each marker are presented here. Distances (p-distances) between individual sequences and clusters identified on phylogenetic trees were calculated in MEGA.
Results
Phylogenetic analysis
On the phylogenetic reconstruction based on the 28S marker, three clusters are distinguished: the first one unites species of the genus Psilotrema, the second one includes the genera Neopsilotrema, Macracetabulum, Byrdtrema, and Longisaccus, and the third cluster comprises the genera Sphaeridiotrema, Psilochasmus and Psilostomum (Figure 1). The Psilotrema samples from G1 and G2 obtained in this work form a common group with Psilotrema limosum. There are no nucleotide differences between the Psilotrema from both groups and Psilotrema limosum based on nuclear marker, while the genetic distances within the genus Psilotrema do not exceed 2%.
Phylogenetic analysis using nad1 also shows a high similarity of samples of Psilotrema from G1 and G2 with Psilotrema limosum. The genetic distances between these trematodes do not exceed 1.0% and result from five nucleotide substitutions (Figure 2b, Table 2), while the interspecific distances in the family Psilostomidae are within 10–18.4%.
Genetic distances between Psilotrema spp. based on nad1 sequences (standard error zovalues shown above the diagonal)
Thus, according to the results of phylogenetic analysis based on partial sequences of these two markers, it could be established that all Psilotrema worms from the muskrat in the Russian Far East belong to the species P. limosum. When using the second mitochondrial cox1 marker, the Psilotrema sequences from the chicken and the muskrat (G2) also differed slightly (0.3–0.6%, Table 3), which confirms their affiliation to the same species. However, a comparison of these sequences with the morphotype G1 showed that differences between them exceed 19.5% (with 17 amino acid substitutions, Table 3). This difference value has an interspecies level, which is consistent with differences at the morphological level. Therefore, the samples of morphotype G1 from the muskrat can be considered a new species, Psilotrema elegans n. sp.
Genetic distances between Psilotrema spp. based on cox1 sequences (standard error values shown above the diagonal)
Morphological description
Psilotrema elegans n. sp.
Definitive host: muskrat Ondatra zibethicus
Site : Intestine
Intensity of infection: 5 specimens
Type-locality: Lake in the basin of the Ilistaya River, Primorsky Region, the Russian southern Far East; 44o17′ N, 132o34′ E
Etymology: The name is based on the graceful shape of the trematode compared to other species of the genus Psilotrema.
Deposition: This material is stored in the Bioresource Collection of the Federal Scientific Center of the East Asia Terrestrial Biodiversity of the Far East Branch of the Russian Academy of Sciences (reg. number 2797657); email: petrova@biosoil.ru. Deposited 2024.20.11. Holotype no. 261-Tr; paratype nos. 262-263-Tr.
Adult worm (Morphotype G1, material examined: 3 specimens) (Figure 3, Table 4). Body elongated, with tapered anterior end or with tapered anterior and posterior ends. Tegument with spines. Oral sucker subterminal. Ventral sucker two times larger than oral sucker. Prepharynx short, pharynx spherical, esophagus short. Intestinal bifurcation at level of anterior half of ventral sucker. Caeca wide, extending to posterior end of body. Testes two, tandem, entire, transversally oval, close to each other, in posterior half of body. Cirrus-sac elongated, narrow anteriorly and expanded posteriorly, reaches level of posterior edge of ventral sucker. Seminal vesicle elongate, sac-shaped. Pars prostatica short with few prostatic cells. Genital pore sinistral, at level of pharynx. Ovary globular, pre-testicular, dextral to median line, adjacent to anterior testis. Anterior edge of ovary at level of posterior edge of ventral sucker. Seminal receptacle sinistral to ovary. Mehlis’ gland at median line, sinistral to ovary. Uterus short, between genital pore and anterior testis. Eggs operculated, with knob, few, unembryonated. Vitellarium consists of large follicles. Vitelline fields between level of posterior margin of ventral sucker and posterior end of body. Region between posterior testis and posterior end of body filled with vitelline follicles of both fields, which separated medially by excretory duct. Excretory vesicle Y-shaped.
Adult worms of Psilotrema: (a) Psilotrema elegans n. sp. (mophotype G1, muskrat); (b) Psilotrema limosum (mophotype G2, muskrat); (c) Psilotrema limosum (chicken). All measurements are given in micrometers (μm).
Measurements of adult Psilotrema worms (μm)
Note: *metric similarity between worms from the chicken and individuals from the G2 group; * *metric similarity between worms from the chicken and individuals from the G1 group; * * *metric differences between worms from the chicken and individuals from the G1 and G2 groups.
Differentiating metric data are marked in bold.
Psilotrema limosum Kalinina, Tatonova, Besprozvannykh, 2022
Definitive host: muskrat Ondatra zibethicus
Site: Intestine
Intensity of infection: 9 specimens
Type-locality: Lake in the basin of the Ilistaya River, Primorsky Region, the Russian southern Far East; 44o17′ N, 132o34′ E
Deposition: This material is stored in the Bioresource Collection of the Federal Scientific Center of the East Asia Terrestrial Biodiversity of the Far East Branch of the Russian Academy of Sciences (reg. number 2797657); e-mail: petrova@biosoil.ru. Deposited 2024.20.11.
Adult worm (Morphotype G2, material examined: 4 specimens) (Figure 3, Table 4). Body wide, oval. Tegument with spines. Oral sucker subterminal. Ventral sucker two times larger than oral sucker. Prepharynx short, pharynx spherical, esophagus short. Intestinal bifurcation immediately anterior to ventral sucker or partially covered by ventral sucker. Caeca wide, extending to posterior end of body. Testes two, tandem, entire, transversally oval, close to each other, in posterior half of body. Cirrus-sac elongated, narrow anteriorly and expanded posteriorly, reaches anterior edge of ovary. Seminal vesicle elongate, sac-shaped. Pars prostatica short with few prostatic cells. Genital pore sinistral, at level of pharynx. Ovary globular, pre-testicular, dextral to median line, adjacent to anterior testis. Anterior edge of ovary at short distance from ventral sucker. Seminal receptacle sinistral to ovary. Mehlis’ gland at median line, sinistral to ovary. Uterus short, between genital pore and anterior testis. Eggs operculated, with knob, few, unembryonated. Vitellarium consists of large follicles. Vitelline fields between level middle of ventral sucker and posterior end of body. Region between posterior testis and posterior end of body filled with vitelline follicles of both fields, which separated medially by excretory duct. Excretory vesicle Y-shaped.
Discussion
Among the worms found in muskrat, based on visual assessment, two groups of individuals were identified that had morphological differences (G1 and G2). These individuals were present as mixed infection in the same host. Individuals from G1 have an elongated body with tapered anterior and posterior ends, while worms from G2 have a wide oval body. In addition, G1 individuals, according to the overwhelming majority of metric indicators, have smaller sizes than G2 individuals (Table 4). Individuals from G2 have the greatest metric similarity to the trematodes Psilotrema limosum from G. gallus dom. (Table 4). Despite this, there are a number of significant metric differences in the sizes of the ventral sucker, anterior testis, cirrus-sac, cirrus-sac, and post-testicular length, as well as the forebody to body length ratio between the G1 and G2 groups of parasites from muskrat and the experimentally obtained P. limosum (Kalinina et al. Reference Kalinina, Tatonova and Besprozvannykh2022) (Table 4). Comparative analysis of morphological parameters of worms removed from the intestines of muskrat and G. gallus dom. (Kalinina et al. Reference Kalinina, Tatonova and Besprozvannykh2022) also revealed differences between these trematodes in the extent of the cirrus-sac: cirrus-sac reaches the level of the posterior edge of the ventral sucker or the anterior edge of the ovary vs. cirrus-sac reaches the level of the ovary. The position of the ovary also differs for these worms: ovary dextral to the median line, adjacent to the anterior testis, vs. ovary at the median line, in the middle of the distance between anterior testis and ventral sucker. Based on the above, among the P. limosum individuals obtained from G. gallus dom. and muskrat, three morphotypes are distinguished, which based only on morphological indices and in the absence of molecular data could be considered as representatives of three separate species.
However, the results of analysis based on 28S, nad1, and cox1 markers showed that only trematodes Psilotrema from muskrat with morphotype G2 belong to the species Psilotrema limosum from Gallus gallus dom. (Kalinina et al. Reference Kalinina, Tatonova and Besprozvannykh2022): all differences obtained have an intraspecific level. This phenomenon has also been shown for trematodes of the families Gorgoderidae, Echinostomatidae, and Heterophyidae (Cutmore et al. Reference Cutmore, Bennett and Cribb2010; Hildebrand et al. Reference Hildebrand, Adamczyk, Laskowski and Zaleśny2015; Presswell et al. Reference Presswell and Bennett2020). In these studies, new species could be identified based on morphological differences, but molecular data showed opposite results and proved that the different morphotypes correspond to the same species. The morphological peculiarities identified for P. limosum from both definitive hosts, in our opinion, are the result of intraspecific variability. This variability can be caused by the involvement of Ondatra zibethicus which is not obligate for P. limosum in the life cycle of the trematode. Nevertheless, fixed substitutions in their genotypes associated with morphological differences between Psilotrema from muskrat (G2) and Psilotrema limosum from Gallus gallus dom. are not found in this study, based both on nuclear and mitochondrial markers.
In contrast, Psilotrema individuals from G1 can indeed be classified as a new species based on both molecular and morphological data. Although two markers (28S and nad1) failed to distinguish significant differences, data on the mitochondrial marker cox1 were able to separate the species identified based on morphological data. And this is consistent with the data obtained for species from the closely related family Echinochasmidae Odhner 1910 (Tatonova et al. Reference Tatonova, Izrailskaia and Besprozvannykh2020). In this study, the species were also not separated based on nuclear gene data but were well differentiated using cox1. Thus, the molecular data obtained for Psilotrema from G1, along with the differences identified at the morphological level, support the identification of a new species, which we designated as Psilotrema elegans n. sp.
At the same time, the data obtained on the markers above can get confusing. Both mitochondrial genes are located on the same DNA molecule and are inherited as a whole. Moreover, both genes are evolutionarily neutral, and therefore, the rate of mutation accumulation in them should be similar and reflect the real time of species divergence. For example, the differences in these genes found for representatives of the genus Metagonimus for these two genes had the same values (Shumenko and Tatonova Reference Shumenko and Tatonova2022). We do not know what the reason for such a contradiction obtained in this study is. It is also not known whether this is a characteristic of the studied representatives of the genus Psilotrema or such results can be found in all representatives of the family or even the superfamily Echinostomatoidea. In any case, the data obtained in this study confirm the need for a comprehensive analysis when describing species and should be taken into account in further work with this group of parasites.
Conclusion
The results based on morphological and molecular data revealed two morphotypes among individuals of the species Psilotrema limosum, the differences between which at the morphological level are equivalent to interspecific differences. One of these morphotypes was obtained in muskrat in this study, and the second one was previously described in chicken. In our opinion, the morphotypic diversity among Psilotrema limosum individuals can be explained by the relatively recent involvement of a new definitive host, Ondatra zibethicus, into the circulation of the trematode. In this and similar cases, the lack of molecular data for the trematodes under study may lead to an unjustified distinction of species among individuals with intraspecific variability level. At the same time, the use of a combination of molecular and morphological data made it possible to identify and describe a new species, Psilotrema elegans n. sp. Overall, this study has shown that to determine the species affiliation of this group of parasites, it is necessary not only to use not only morphological and molecular markers, but also to compare the data obtained for different mitochondrial markers in cases of doubt, in order to avoid erroneous conclusions.
Acknowledgements
The experiments described in this study were set up using the equipment at the Instrumental Centre for Biotechnology and Gene Engineering, Federal Scientific Center of the East Asia Terrestrial Biodiversity, Far Eastern Branch, Russian Academy of Sciences (Vladivostok, Russia).
Financial support
The research was carried out within the framework of the state assignment from the Ministry of Science and Higher Education of the Russian Federation (topic No. 121031000154-4) and government assignment no. 123022200035-0 ‘The structure of natural foci of parasitic diseases in the south of the Russian Far East’.
Competing interest
The authors declare no conflict of interest.
Ethical standard
Euthanasia of animals was carried out in accordance with the resolution of the Committee on the Ethics of Animal Experiments, Federal Scientific Center of the East Asia Terrestrial Biodiversity, FEB RAS, Russia. Permit Number: 1 of 25.04.2022. The authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national and institutional guides on the care and use of laboratory animals.
