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A report on the complete mitochondrial genome of the trematode Azygia robusta Odhner, 1911, its new definitive host from the Russian Far East, and unexpected phylogeny of Azygiidae within Digenea, as inferred from mitogenome sequences

Published online by Cambridge University Press:  01 September 2023

D. M. Atopkin Open the ORCID record for D. M. Atopkin [Opens in a new window] ,
A. A. Semenchenko ,
D. A. Solodovnik  and
Y. I. Ivashko
D. M. Atopkin*
Affiliation:
Federal Scientific Center of the East Asia Terrestrial Biodiversity, Far Eastern Branch, Russian Academy of Sciences, Vladivostok, Russia Department of Cell Biology and Genetics, Far Eastern Federal University, Vladivostok, Russia
A. A. Semenchenko
Affiliation:
Federal Scientific Center of the East Asia Terrestrial Biodiversity, Far Eastern Branch, Russian Academy of Sciences, Vladivostok, Russia
D. A. Solodovnik
Affiliation:
Federal Scientific Center of the East Asia Terrestrial Biodiversity, Far Eastern Branch, Russian Academy of Sciences, Vladivostok, Russia
Y. I. Ivashko
Affiliation:
Federal Scientific Center of the East Asia Terrestrial Biodiversity, Far Eastern Branch, Russian Academy of Sciences, Vladivostok, Russia
*
Corresponding author: D. M. Atopkin; Email: atop82@gmail.com
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Abstract

New data on the complete mitochondrial genome of Azygia robusta (Azygiidae) were obtained by the next-generation sequencing (NGS) approach. The mitochondrial DNA (mtDNA) of A. robusta had a length of 13 857 bp and included 12 protein-coding genes, two ribosomal genes, 22 transfer RNA genes, and two non-coding regions. The nucleotide sequences of the complete mitochondrial genomes of two A. robusta specimens differed from each other by 0.12 ± 0.03%. Six of 12 protein-coding genes demonstrated intraspecific variation. The difference between the nucleotide sequences of the complete mitochondrial genomes of A. robusta and Azygia hwangtsiyui was 26.95 ± 0.35%; the interspecific variation of protein-coding genes between A. robusta and A. hwangtsiyui ranged from 20.5 ± 0.9% (cox1) to 30.7 ± 1.2% (nad5). The observed gene arrangement in the mtDNA sequence of A. robusta was identical to that of A. hwangtsiyui. Codon usage and amino acid frequencies were highly similar between A. robusta and A. hwangtsiyui. The results of phylogenetic analyses based on mtDNA protein-coding regions showed that A. robusta is closely related to A. hwangtsiyui (belonging to the same suborder, Azygiida) that formed a distinct early-diverging branch relative to all other Digenea. A preliminary morphological analysis of paratypes of the two azygiid specimens studied showed visible morphological differences between them. The specimen extracted from Sakhalin taimen (Parahucho perryi) was most similar to A. robusta. Thus, we here provide the first record of a new definitive host, P. perryi, for A. robusta and also molecular characteristics of the trematode specimens.

Keywords

Azygia robustaAzygiidaecomplete mitochondrial genomephylogenetic relationshipsnext-generation sequencing
Type
Research Paper
Information
Journal of Helminthology , Volume 97 , 2023 , e69
Copyright
© The Author(s), 2023. Published by Cambridge University Press

Introduction

Trematodes of the family Azygiidae Lühe, 1909 parasitise stomachs or body cavities in elasmobranchs and stomachs in freshwater teleosts and holosteans (Gibson Reference Gibson, Gibson, Jones and Bray2002). Representatives of the type genus of this family, Azygia Looss, 1899, possess a characteristic medium- or large-sized, elongated body, a small oral sucker, a large ventral sucker, and two large tandem testes. In the Russian Far East, these worms are known to infect mainly freshwater fishes, including such species as Esox reicherti Dybowski, 1869, Perccottus glenii Dybowski, 1877, Hucho taimen (Pallas 1773), and Channa argus (Cantor, 1842) (Mamaev, Oshmarin, Reference Mamaev, Oshmarin and Mamaev1971; Dvoryadkin Reference Dvoryadkin1977; Ermolenko et al. 1998; Besprozvannykh Reference Besprozvannykh2005; Vainutis et al. Reference Vainutis, Voronova, Mironovsky, Zhigileva and Zhokhov2023). The phylogenetic position of Azygiidae among other members of the Hemiurata is currently under debate. As a consequence, the taxonomic status of this family still remains unresolved. Taxonomists previously considered this group of trematodes as a separate suborder, Azygiata La Rue, Reference La Rue1957, or the order Azygiida Odening, 1963 (La Rue Reference La Rue1957; Skrjabin & Guschanskaya Reference Skrjabin, Guschanskaja and Skrjabin1958; Nagasawa et al. Reference Nagasawa, Urawa and Awakura1987; Littlewood Reference Littlewood2008; Sokolov & Zhukov Reference Sokolov and Zhukov2016). At present, most authors, based on the results of molecular phylogenetic analyses using ribosomal DNA gene sequence data (Olson et al. Reference Olson, Cribb, Tkach, Bray and Littlewood2003; Pérez-Ponce de León & Hernández-Mena Reference Pérez-Ponce de León and Hernández-Mena2019), recognize the status of this trematode group as a separate superfamily, Azygioidea Lühe, 1909 (Gibson Reference Gibson, Gibson, Jones and Bray2002; Olson et al. Reference Olson, Cribb, Tkach, Bray and Littlewood2003; Kostadinova & Pérez-del-Olmo Reference Kostadinova, Pérez-del-Olmo, Toledo and Fried2014; Pérez-Ponce de León & Hernández-Mena Reference Pérez-Ponce de León and Hernández-Mena2019), and as a member of the suborder Hemiurata Skrjabin & Guschanskaja, 1954. A phylogenetic analysis of Digenea based on complete mitochondrial sequence data and also using the whole mitochondrial DNA (mtDNA) genome of an azygiid representative, A. hwangtsiyui Tsin, 1933, obtained for the first time, has shown that Azygiidae represents a distinct branch, basal for most of trematode groups except Schistosomatidae Stiles & Hassall, 1898 (Wu et al. Reference Wu, Gao, Cheng, Xie, Yuan, Liu and Song2020). In our opinion, these results provide sufficient grounds for revising the taxonomic status of Azygiidae through further phylogenetic studies using mtDNA complete sequence data for different azygiid species. In our present study, we provide new data on the complete mtDNA sequence, inferred by the next-generation sequencing (NGS) approach, from two adult specimens of the trematode Azygia robusta Odhner, 1911 extracted from two salmonid species, the taimens Hucho taimen and Parahucho perryi, which were caught in two rivers of Primorsky Krai, Russia. This trematode species was earlier characterised morphologically by Besprozvannykh (Reference Besprozvannykh2005), who provided a detailed description of its life cycle. Our study aimed mainly to compare the structures and variations in the complete mitochondrial genomes of two azygiid species, analyse phylogenetic relationships using the new complete mtDNA sequence data on A. robusta, and interpret the obtained results to clarify the Azygiidae systematics.

Material and methods

Sample collection and DNA extraction

Adult worms were collected from the intestines of two naturally infected salmonids, a common taimen (H. taimen) caught in the Armu River (Besprozvannykh Reference Besprozvannykh2005) and a Sakhalin taimen (P. perryi (Brevoort, 1856)) caught in the Samarga River (unpublished, collected in 1987) (Table 1). The trematodes were killed with hot water and then fixed in 96% ethanol. Total DNA was extracted from the two specimens separately using a Qiamp Investigator kit (Qiagen, Germantown, MD, USA) according to the manufacturer’s protocol. Amount of total DNA was measured on a Qubit 3.0 fluorometer (Invitrogen, Waltham, MA, USA) and then used for NGS sequencing in a final amount of 100 ng.

Table 1. List of Digenea sequences from GenBank used in phylogenetic analysis

Preparation of genome library for NGS

Libraries were prepared using an Ion Plus Fragment Library kit and unique adapters from an Ion Xpress Barcode Adaptors kit (ThermoFisher Scientific, Waltham, MA, USA) with pre-fragmentation on a Covaris M220 Focused-ultrasonicator (Covaris, LLC, Woburn, MA, USA). The preparation of polymerase chain reaction (PCR) emulsion and templates was done on an Ion One Touch 2 System (ThermoFisher Scientific) followed by sequencing on an Ion S5 sequencing platform using an Ion 540 chip.

The sequence quality and length distribution of raw reads were checked using FastQC 0.11.9 (Babraham Bioinformatics) and then the reads were assembled using SPAdes 3.14.1 (Nurk et al. Reference Nurk, Bankevich, Antipov, Gurevich, Korobeynikov, Lapidus, Prjibelski, Pyshkin, Sirotkin, Sirotkin, Stepanauskas, Clingenpeel, Woyke, JS, Lasken, Tesler, Alekseyev and Pevzner2013) with correction of IonTorrent data using the IonHammer tool available in the SPAdes software. The scaffolds containing mtDNA data were manually assembled in the MEGA X software (Kumar et al. Reference Kumar, Stecher, Li, Knyaz and Tamura2018).

Mitochondrial genome annotation was performed using the MITOS2 on-line software (Donath et al., Reference Donath, Jühling, Al-Arab, Bernhart, Reinhardt, Stadler, Middendorf and Bernt2009, available at http://mitos2.bioinf.uni-leipzig.de), and then the mitochondrial genome was manually assembled and aligned with that of A. hwangtsiyui (Wu et al. Reference Wu, Gao, Cheng, Xie, Yuan, Liu and Song2020) in MEGA X. Tandem repeats were searched using the Tandem Repeat Finders software (https://tandem.bu.edu/trf/trf.html). Search and analysis of the transfer RNA (tRNA) gene structure were performed in the ARWEN software (http://130.235.244.92/ARWEN/).

Codon usage, gene variations, and phylogenetic analyses

Alignments of nucleotide and amino acid sequences were performed by the ClustalW algorithm in MEGA X. Poorly aligned regions were removed using the Gblocks Server (http://phylogeny.lirmm.fr/phylo_cgi/one_task.cgi?task_type=gblocks).

Phylogenetic analysis was performed on the basis of concatenated amino acid sequences by the Maximum likelihood (ML) algorithm available in the PhyML 3.1 software (Guindon & Gascuel Reference Guindon and Gascuel2003) and by the Bayesian Inference (BI) method available in the MrBayes 3.2.6 software (Ronquist et al. Reference Ronquist, Teslenko, van der Mark, Ayres, Darling, Höhna, Larget, Liu, Suchard and Huelsenbeck2012). The ML algorithm was performed using an LG evolutionary model (Lee & Gascuel Reference Le and Gascuel2008), Subtree Pruning and Regrafting (SPR) tree topology search, and random sequence addition. The BI algorithm was performed using a protein model, a mixed set of substitution types, a mixed amino acid model, and uninformative amino acid substitution rates. The Monte Carlo Markov chains algorithm was performed with 1 000 000 generations during two independent runs, with sampling each 1000th generation and burning the first 25% of all generations. The average standard deviation of split frequencies was 0.000865, and that was enough for phylogenetic reconstruction. Significance of phylogenetic relationships was estimated with a posteriori probabilities (Huelsenbeck et al. Reference Huelsenbeck, Ronquist, Nielsen and Bollback2001) for the BI algorithm and an approximate likelihood-ratio test (Anisimova & Gascuel Reference Anisimova and Gascuel2006) for the ML algorithm. Codon usage statistics was calculated for concatenated protein-coding gene sequence data in MEGA X. Analysis of correlation between the number of variable sites and the gene length was performed using Pearson’s correlation coefficient in Statistica 13 software (TIBCO Software Inc. 2017).

Phylogenetic relationships were inferred using sequences of our samples and other trematode species accessed from the NCBI GenBank database (Table 1). The two annotated mitochondrial genomes have been deposited in GenBank under accession numbers OR350239 and OR350240, while raw Sequence Read Archive (SRA) sequencing data are available under accession numbers SAMN36469092–SAMN36469093.

Results

Brief visual morphological identification of the species

In this study we first performed a brief visual morphological analysis of the paratypes of trematodes used for the NGS analysis. The general view of the azygiid worms from H. taimen caught in the Armu River and from P. perryi caught in the Samarga River can be seen in Figures 1 and 2, respectively. Both specimens possess the main diagnostic characteristics of A. robusta, including the round pharynx and vitellaria extending beyond the posterior end of the second testis to half the distance between the second testis and the posterior end of the body (Skrjabin & Guschanskaja Reference Skrjabin, Guschanskaja and Skrjabin1958; Bauer Reference Bauer1987). These morphological characteristics were observed clearly in both specimens (Figures 5, 6). For the NGS analysis, we used the mature trematode specimens that had been found in H. taimen from the Armu River cultivated from cercariae, identified as A. robusta, and published in the study of Besprozvannykh (Reference Besprozvannykh2005), who described the life cycle of this trematode species. Thus, we keep the name Azygia robusta for both trematode specimens used for the NGS analysis.

Figure 1. General view of Azygia robusta extracted from Hucho taimen inhabiting the Armu River (the microscope slide and the photograph were kindly provided by V.V. Besprozvannykh).

Figure 2. General view of Azygia robusta extracted from Parahucho perryi inhabiting the Samarga River (the microscope slide and the photograph were kindly provided by V.V. Besprozvannykh).

Sequence quality and coverage

We obtained 3.5–4.5 million reads for two specimens of A. robusta. The sequence quality after FastQC was acceptable. Phred 33 values were 20–30 (mode 26) and decreased slightly in long reads. The sequence length was 25–449 bp; for most reads, the length was 120–240 bp. The GC content and numbers of duplications and adapters did not exceed the norm. The mean coverage across the mitochondrial DNA was 103X and 204X for the two specimens of A. robusta.

General characteristics of the Azygia robusta mitochondrial genome

The mitochondrial genome of A. robusta had a length of 13 857 bp and contained 12 protein-coding genes, two ribosomal genes, 22 tRNA genes, and two non-coding regions: short (SNCR) and long (LNCR) (Figure 3, Table 2). Alternative read variants were absent from the NGS raw data, and no intraspecific variable positions were observed. The nucleotide composition in the A. robusta mitochondrial genome was as follows: A, 16.5%; T (U), 40.9%; C, 14.4%; and G, 28.2%. The nucleotide pair frequency was 57.4% for the AT-content and 42.6% for the GC-content, showing a bias towards T over A (AT skew = -0.43) and G over C (CG skew = 0.32), respectively.

Figure 3. Organization of the complete mitochondrial genome in Azygia robusta.

Protein-coding genes of mitochondrial genome

The total length of the sequences of 12 protein-coding genes in the complete mitochondrial genome was 10 110 bp. The arrangement of protein-coding genes was as follows: cox3–cytb–nad4L–nad4–atp6–nad2–nad1–nad3–cox1–cox2–nad6–nad5. The start-codons for protein-coding genes were ATG or GTG, except the cox1 gene that started with TTG codon, as well as those for A. hwangtsiyui and nad3 gene that started with GGT codon (Table 2). The nucleotide composition of the assembled protein-coding part of the mitochondrial genome sequence was as follows: A, 14.5%; T (U), 43.3%; C, 14.0%; and G, 28.2%. The nucleotide pair frequency was 57.8% for the AT-content and 42.2% for the GC-content, showing a bias towards T over A (AT skew = -0.5) and G over C (CG skew = 0.34).

Table 2. The organization of mitochondrial genome of Azygia robusta

LNCR, long non-coding region; SNCR, short non-coding region.

* tRNA missed the DHU-arm

** tRNA missed the T-stem

Codon usage statistics for A. robusta were consistent with the proportions in the nucleotide composition: the most common triplets contained T (U) and/or G bases, namely UUU (with a frequency of 9.79%), UUG (5.99%), GUU (5.88%), GGG (4.4%), UGU (3.82%), and GUG (3.39%).

A total of 3 386 amino acids were encoded by the mitochondrial protein-coding genes in A. robusta. Of these, a maximal frequency was observed for leucine (15.0%), valine (12.2%), and phenylalanine (11.7%); the frequencies for lysine (1.18%) and glutamine (1.21%) were minimal compared to other amino acids. The amino acid frequencies of the mitochondrial protein sequences of A. hwangtsiyui were similar to those of A. robusta, with no marked differences observed (Table 3).

Table 3. Amino acid frequencies in concatenated protein sequences of mitochondrial protein-coding region of Azygia robusta (1, ex Hucho taimen, Armu River (Besprozvannykh, Reference Besprozvannykh2005) 2002; 2, ex Parahucho perryi, Samarga River, 1987) and A. hwangtsiyui

Intra- and interspecific variation of complete mtDNA sequences

Overall, the nucleotide sequences of the complete mitochondrial genomes, including all genes and non-coding fragments, of the two A. robusta specimens differed from each other by 0.12 ± 0.03%. The concatenated protein-coding gene sequences between these two specimens differed by 0.11 ± 0.03%, and amino acid sequences by 0.06 ± 0.05%. Six of 12 protein-coding genes demonstrated intraspecific variation in A. robusta (Table 4); a total of 12 substitutions were revealed, with each gene containing from one to three variable sites, transitions T/C (67%) or A/G (25%), and a single transversion T/G in nad6 gene.

Table 4. Variation of mitochondrial protein-coding genes of Azygia robusta and between A. robusta and A. hwangtsiyui.

The difference between the nucleotide sequences of the complete mitochondrial genomes of A. robusta and A. hwangtsiyui was 26.95 ± 0.35%; between the concatenated protein-coding nucleotide sequences, 26.00 ± 0.43%; and between the amino acid sequences, 30.15 ± 0.82%. The interspecific variation of protein-coding genes between A. robusta and A. hwangtsiyui ranged from 20.5 ± 0.9% (cox1) to 30.7 ± 1.2% (nad5) (Table 4). The results of correlation analysis using Pearson’s correlation coefficient showed a high positive correlation (r = 0.95) between the number of variable sites and the gene length for interspecific comparison of protein-coding gene variations in the two Azygia species (Figure 4).

Figure 4. Results of an analysis based on Pearson’s coefficient of correlation between gene length and number of variable sites with pairwise comparison of mitochondrial protein-coding genes of A. robusta and A. hwangtsiyui. r is the Pearson’s correlation coefficient.

Phylogenetic analysis

The maximum likelihood (ML) and Bayesian Inference (BI) algorithms were based on alignment of 2 280 amino acids available after Gblocks processing. Overall, mitochondrial genomes of 62 species, including 61 digenean and one cestode species, Diphpyllobothrium latum (Linnaeus, 1758) Lühe, 1899, were incorporated into the phylogenetic analysis. As the BI tree topology showed, the digeneans could be subdivided into three highly supported clades (Figure 5). The first clade was early divergent and consisted of three Azygia specimens: one A. hwangtsiyui (GenBank accession no. MN844889) and two A. robusta (our material). The second clade represented the order Diplostomida, including species of the families Schistosomatidae Stiles & Hassal, 1898, Clinostomidae Lühe, 1901, Cyathocotylidae Mühling, 1898, and Brachylaimidae Joyeux & Foley, 1930. The third clade comprised 47 species from 18 families, representing seven suborders. The topology of this subclade completely agreed with the previous phylogenetic reconstructions of Digenea (Ivashko et al. Reference Ivashko, Semenchenko, Solodovnik and Atopkin2022). The suborder Xiphidiata Olson, Cribb, Tkach, Bray & Littlewood, Reference Olson, Cribb, Tkach, Bray and Littlewood2003 was polyphyletic and appeared as two independent groups. The first group consisted of Brachycladium goliath (van Beneden, 1858) and species of the genus Paragonimus Braun, 1988 (Xiphidiata). This group was closely related to Opisthorchiata. Plagiorchis maculosus (Rudolphi, 1802) appeared as sister to the above-mentioned Xiphidiata and Opisthorchiata species. The second group of Xiphidiata included representatives of Dicrocoeliidae Odhner, 1911 (Dicrocoelium dendriticum (Rudolphi, 1819), D. chinensis (Sudarikov and Ryjikov, 1951) Tang and Tang, 1978, and Eurytrema pancreaticum (Janson, 1899)), Eucotylidae Skrjabin, 1924 (Tamerlania zarudnyi Skrjabin, 1924), and Prosthogonimidae Lühe, 1909 (Prosthogonimus cuneatus (Rudolphi, 1802) Braun, 1901). This group appeared as sister to the subclade that contained species of the suborder Pronocephalata Olson, Cribb, Tkach, Bray, Littlewood, Reference Olson, Cribb, Tkach, Bray and Littlewood2003. The suborder Haploporata Pérez-Ponce de León & Hernández-Mena, Reference Pérez-Ponce de León and Hernández-Mena2019, represented by Parasaccocoelium mugili Zhukov, 1971 and Carassotrema koreanum Park, 1938, formed a basal branch within the third clade.

Figure 5. Phylogenetic relationships of Azygia robusta and other digenetic trematodes reconstructed by the Bayesian Inference (BI) algorithm on the basis of alignment of protein sequences containing 2280 amino acids, available after Gblock processing. Nodal support is shown with a posteriori probabilities calculated using the BI algorithm.

In general, the ML tree topology was similar to that of the BI tree, demonstrating three main clades within Digenea (Figure 6). The marked differences from the BI tree topology were as follows: (1) Dicrocoeliidae (Xiphidiata) formed a separate basal subclade within the third clade, creating polyphyly for Xiphidiata, and (2) representatives of Haploporata, C. koreanum and P. mugili, were within a single subclade with Plagiorchis maculosus, with this subclade being sister to closely related representatives of Xiphidiata (Paragonimus spp. and Brachycladium goliath) and Opisthorchiata. These differences between the BI and ML tree topologies were also reported in previous studies (Atopkin et al. Reference Atopkin, Semenchenko, Solodovnik, Ivashko and Vinnikov2021; Ivashko et al. Reference Ivashko, Semenchenko, Solodovnik and Atopkin2022).

Figure 6. Phylogenetic relationships of Azygia robusta and other digenetic trematodes reconstructed by the Maximum Likelihood (ML) algorithm on the basis of alignment of protein sequences containing 2280 amino acids, available after Gblock processing. Nodal support is shown with a posteriori probabilities calculated using the approximate likelihood ratio test.

Discussion

Mitochondrial genome variations in A. robusta and A. hwangtsiyui

The complete mitochondrial genome structure of A. robusta was highly similar to that of A. hwangtsiyui in gene arrangement, the existence of two non-coding regions separated from each other by tRNA-Gly (G) gene, and a higher level of AT content relative to GC content for both mitochondrial genome sequences and protein-coding genes. Also, the two azygiid species had the same most frequent codons and the same start-codon (TTG) for the cox1 gene. A difference was revealed in the start-codon of the nad3 gene, which started with GGT in A. robusta vs. ATG in A. hwangtsiyui. There were also differences in the lengths of some protein-coding genes between A. robusta and A. hwangtsiyui: 1275 vs. 1272 bp, respectively, for the nad4 gene; 504 vs. 513 bp for atp6; 903 vs. 906 bp for nad1; 1548 vs. 1564 bp for cox1; 450 vs. 444 bp for nad6; and 1598 vs. 1600 bp for nad5.

New definitive host of Azygia robusta from the Russian Far East

To date, four species of definitive hosts for trematodes Azygia spp. are known from the Russian Far East: the northern snakehead Channa argus (Cantor, 1842) and the Amur pike Esox reicherti Dybowski, which are freshwater fishes, and the common taimen Hucho taimen (Pallas, 1773) and the Chinese sleeper Perccottus glenii Dybowski, 1877, which are freshwater/brackish-water fishes (Mamaev, Oshmarin, Reference Mamaev, Oshmarin and Mamaev1971; Dvoryadkin Reference Dvoryadkin1977; Ermolenko et al. 1998; Besprozvannykh Reference Besprozvannykh2005; Vainutis et al. Reference Vainutis, Voronova, Mironovsky, Zhigileva and Zhokhov2023). In this study, we have extended the list of definitive hosts for this region by adding the Sakhalin taimen, Parahucho perryi (Brevoort, 1856). This fish is one of the world’s largest salmonids, reaching maturity at 6–8 years of age and living for more than 20 years. The species’ geographic range is confined to the Sea of Japan, from the southern Kuril Islands and Primorsky Krai, Russia, to Hokkaido, Japan. Parahucho perryi occupy a variety of habitats including upper and lower reaches of rivers, lakes, brackish-water lagoons, estuaries, and coastal marine waters (Zolotukhin & Semenchenko Reference Zolotukhin and Semenchenko2008; Fukushima et al. Reference Fukushima, Shimazaki, Rand and Kaeriyama2011). The ecological features of P. perryi are favorable for the trematode A. robusta to infect this fish species. Moreover, one of the azygiid species, A. perryi, has been reported as a parasite for P. perryi from Japan (Nagasawa et al. Reference Nagasawa, Urawa and Awakura1987; Popiołek et al. Reference Popiolek, Kusznierz, Kotusz and Witkowski2013), while A. robusta is known to parasitise salmonids (Nikolić et al. Reference Nicolić, Bilbija, Nedic, Simonovic and Djikanovic2018). Thus, we here provide the first record of a new definitive host, Parahucho perryi, for the trematode Azygia robusta from the Russian Far East.

Systematics and phylogenetic relationships of Azygiidae

The systematic position of Azygiidae is still unclear because of the lack of molecular data for representatives of this group, and, as a consequence, controversies arise between interpretations of morphological and molecular data. Most authors recognize the status of this trematode group as a separate superfamily (Gibson Reference Gibson, Gibson, Jones and Bray2002; Olson et al. Reference Olson, Cribb, Tkach, Bray and Littlewood2003; Kostadinova & Pérez-del-Olmo Reference Kostadinova, Pérez-del-Olmo, Toledo and Fried2014; Pérez-Ponce de León & Hernández-Mena Reference Pérez-Ponce de León and Hernández-Mena2019). However, viewpoints on the membership of Azygioidea at a higher taxonomic level are different. These worms were considered as a separate suborder, Azygiata (La Rue Reference La Rue1957; Skrjabin & Guschanskaya Reference Skrjabin, Guschanskaja and Skrjabin1958), or the order Azygiida Odening, 1963 (Littlewood Reference Littlewood2008; Sokolov & Zhukov Reference Sokolov and Zhukov2016). At present, this superfamily is recognized as a member of the suborder Hemiurata mainly on the basis of data inferred from molecular phylogenetic analyses using ribosomal DNA gene sequences (Olson et al. Reference Olson, Cribb, Tkach, Bray and Littlewood2003; Pérez-Ponce de León & Hernández-Mena Reference Pérez-Ponce de León and Hernández-Mena2019). However, as the latest studies have shown, the Azygiida is a valid order (Ramilo et al. Reference Ramilo, Abollo and Pascual2023). The first complete mitochondrial genome sequences for a representative of Azygiidae, Azygia hwangtsiyui, were obtained by Wu et al. (Reference Wu, Gao, Cheng, Xie, Yuan, Liu and Song2020). These data were applied to the reconstruction of the phylogenetic position of Azygiidae within Digenea using a dataset of concatenated amino acid sequences representing 12 protein-coding genes. The position of A. hwangtsiyui (Azygiidae) was considered the ‘most basal lineage of the Digenea’; however, in that study, Schistosomatidae rather than Azygiida was basal for Digenea (Wu et al. Reference Wu, Gao, Cheng, Xie, Yuan, Liu and Song2020). Nevertheless, the authors did not provide any definitive conclusion about the systematic position of Azygiidae and stated that ‘the family Azygiidae still awaits investigation of relationships based on a much wider taxon sampling and more mitogenome datasets’ (Wu et al. Reference Wu, Gao, Cheng, Xie, Yuan, Liu and Song2020). Our results clearly demonstrate that this statement is relevant. The introduction of one new azygiid species into the phylogenetic analysis based on concatenated amino acid sequences of 12 protein-coding mitochondrial genes has considerably changed the phylogenetic position of Azygiidae within Digenea. In contrast to the results from previous studies, the present phylogenetic tree consists of three main, highly supported digenean clades, including the early diverging clade Azygiidae and two sister clades, Diplostomida and other digeneans. On the one hand, this result supports the taxonomic status of Azygiidae as a separate order within Digenea, which largely agrees with the previous results by Wu et al. (Reference Wu, Gao, Cheng, Xie, Yuan, Liu and Song2020) that showed a basal position of Azygiidae relative to other Digenea, except Schistosomatidae. On the other hand, our results do not confirm the hypothesis, advanced in our previous studies, about the consistency between phylogenetic relationships and gene rearrangement within mitochondrial genomes of Schistosomatidae and other trematodes (Atopkin et al. Reference Atopkin, Semenchenko, Solodovnik, Ivashko and Vinnikov2021; Ivashko et al. Reference Ivashko, Semenchenko, Solodovnik and Atopkin2022). In particular, the basal position of Azygiidae relative to other trematodes and the emergence of Cyathocotyle prussica and Clinostomum complanatum within a single clade with Schistosomatidae are evidence against this hypothesis. However, in this respect, we agree with Wu et al. (Reference Wu, Gao, Cheng, Xie, Yuan, Liu and Song2020), who indicated the need for additional data, with complete mitochondrial genome sequences obtained for more Azygiidae species and other unstudied digenean taxa, to provide a sufficient basis for conclusions about the systematics of this family.

Acknowledgements

We are deeply thankful to Vladimir V. Besprozvannykh, Dr. Sci. Biol., the head of the Department of Parasitology, Federal Scientific Center of East Asian Terrestrial Biodiversity FEB RAS, for providing the material on Azygia robusta (the specimens for the DNA extraction and the microscope slides of paratypes for the morphological analysis) and to Dr. Kirill A. Vinnikov, the Director of the Institute of the World Ocean, Far Eastern Federal University, for providing the laboratory equipment and constructive comments while performing NGS.

Financial support

This study was supported by the Russian Scientific Foundation, project number 22-24-00896.

Competing interest

None.

Ethical standard

All applicable institutional, national and internationalguidelines for the care and use of animals were followed.

References

Anisimova, M, Gascuel, O (2006). Approximate likelihood-ratio test for branches: a fast, accurate and powerful alternative. Systematic Biology 55, 4, 539–552. https://doi.org/10.1080/10635150600755453CrossRefGoogle Scholar
Atopkin, DM, Semenchenko, AA, Solodovnik, DA, Ivashko, YI, Vinnikov, KA (2021). First next-generation sequencing data for Haploporidae (Digenea: Haploporata): characterization of complete mitochondrial genome and ribosomal operon for Parasaccocoelium mugili Zhukov, 1971. Parasitology Research 120, 6, 2037–2046. https://doi.org/10.1007/s00436-021-07159-yCrossRefGoogle ScholarPubMed
Babraham Bioinformatics (2010). FastQC: A quality control tool for high throughput sequence data. Available at http://www.bioinformatics.babraham.ac.uk/projects/fastqc/. (accessed March 1, 2023)Google Scholar
Bauer, ON (1987). Key for Determination of the Parasites of Freshwater Fish in the Fauna of USSR. Vol. 3. Part 2. Leningrad: Science Press (In Russian).Google Scholar
Besprozvannykh, VV (2005). The life cycles of trematodes Azygia hwangtsiytii and A. robusta (Azygiidae) in conditions of Primorsky Region. Parazitologija 39, 4, 278–284 (In Russian).Google Scholar
Biswal, DK, Chatterjee, A, Bhattacharya, A, Tandon, V (2014). The mitochondrial genome of Paragonimus westermani (Kerbert, 1878), the Indian isolate of the lung fluke representative of the family Paragonimidae (Trematoda). PeerJ 2, e484. https://doi.org/10.7717/peerj.484CrossRefGoogle ScholarPubMed
Briscoe, AG, Bray, RA, Brabec, J, Littlewood, DT (2016). The mitochondrial genome and ribosomal operon of Brachycladium goliath (Digenea: Brachycladiidae) recovered from a stranded minke whale. Parasitology International 65, 3, 271–275. https://doi.org/10.1016/j.parint.2016.02.004CrossRefGoogle ScholarPubMed
Chang, Q-C, Liu, G-H, Gao, J-F, Zheng, X, Zhang, Y, Duan, H, Yue, D-M, Fu, X, Su, X, Gao, Y, Wang, C-R (2016). Sequencing and characterization of the complete mitochondrial genome from the pancreatic fluke Eurytrema pancreaticum (Trematoda, Dicroroeliidae). Gene 576, Pt 1, 160–165. https://doi.org/10.1016/j.gene.2015.09.081CrossRefGoogle Scholar
Dereeper, A, Guignon, V, Blanc, G, Audic, S, Buffet, S, Chevenet, F, Dufayard, JF, Guindon, S, Lefort, V, Lescot, M, Claverie, JM, Gascuel, O (2008). Phylogeny.fr: robust phylogenetic analysis for the non-specialist. Gblocks alignment on-line service. Available at http://phylogeny.lirmm.fr/phylo_cgi/one_task.cgi?task_type=gblocks (accessed July 1, 2008).Google Scholar
Donath, A, Jühling, F, Al-Arab, M, Bernhart, SH, Reinhardt, F, Stadler, PF, Middendorf, M, Bernt, M (2009) Improved annotation of protein-coding genes boundaries in metazoan mitochondrial genomes. Nucleic Acids Research, 47(20), 10543–10552. doi: 10.1093/nar/gkz833. Available at http://mitos2.bioinf.uni-leipzig.de/index.pyGoogle Scholar
Dvoryadkin, VA (1977). Freshwater gastropods as intermediate and additional hosts for some trematode species in the south of Far East. Proceedings of Institute of Biology and Soil Science FESC AS USSR, 47, 56–69.Google Scholar
Fu, Y-T, Jin, Y-C, Li, F, Liu, G-H (2019a). Characterization of the complete mitochondrial genome of the echinostome Echinostoma miyagawai and phylogenetic implications. Parasitology Research 118, 10, 3091–3097. https://doi.org/10.1007/s00436-019-06417-4CrossRefGoogle ScholarPubMed
Fu, Y-T, Jin, Y-C, Li, F, Liu, G-H (2019b). The complete mitochondrial genome of the caecal fluke of Poultry, Postharmostomum commutatum, as the first representative from the superfamily Brachylaimoidea. Frontiers in Genetics 10, 1037. https://doi.org/10.3389/fgene.2019.01037CrossRefGoogle ScholarPubMed
Fukushima, M, Shimazaki, H, Rand, PS, Kaeriyama, M (2011). Reconstructing Sakhalin taimen Parahucho perryi historical distribution and identifying causes for local extinctions. Transactions of the American Fisheries Society 140, 1, 1–13. https://doi.org/10.1080/00028487.2011.544999CrossRefGoogle Scholar
Gibson, DI (2002). Superfamily Azygioidea Lühe, 1909. In Gibson, DI, Jones, A, Bray, RA (eds), Keys to the Trematoda, vol. 1. pp. 19–24. Wallingford: CAB International.CrossRefGoogle Scholar
Guindon, S, Gascuel, O (2003). A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Systematic Biology 52, 5, 696–704. https://doi.org/10.1080/10635150390235520CrossRefGoogle ScholarPubMed
Guo, X-R, Gao, Y, Qiu, Y-Y, Jin, Z-H, Gao, ZY, Zhang, XG, An, Q, Chang, QC, Gao, JF, Wang, CR (2022). The complete mitochondrial genome of Prosthogonimus cuneatus and Prosthogonimus pellucidus (Trematoda: Prosthogonomidae), their features and phylogenetic relationships in the superfamily Microphalloidea. Acta Tropica, 232, 106469. doi: 10.1016/j.actatropica.2022.106469.Google Scholar
Huelsenbeck, JP, Ronquist, F, Nielsen, R, Bollback, JP (2001). Bayesian inference of phylogeny and its impact on evolutionary biology. Science 294, 5550, 2310–2314. https://doi.org/10.1126/science.1065889Google ScholarPubMed
Ivashko, Y, Semenchenko, A, Solodovnik, D, Atopkin, D (2022). Characterization of complete mitochondrial genome and ribosomal operon for Carassotrema koreanum Park, 1938 (Digenea: Haploporidae) by means of next-generation sequencing data. Journal of Helminthology 96, e54. https://doi.org/10.1017/S0022149X22000438CrossRefGoogle ScholarPubMed
Jones, BP, Norman, BF, Borrett, HE, Attwood, SW, Mondal, MMH, Walker, AJ, Webster, JP, Rajapakse, RPVJ, Lawton, SP (2020). Divergence across mitochondrial genomes of sympatric members of the Schistosoma indicum group and clues into the evolution of Schistosoma spindale. Scientific Reports 10, 1, 2480. https://doi.org/10.1038/s41598-020-57736-xGoogle ScholarPubMed
Kostadinova, A, Pérez-del-Olmo, A (2014). The systematic of the Trematoda. In Toledo, R, Fried, B (eds) Advances in Experimental Medicine and Biology. Digenetic Trematodes. pp. 21–42. Luxemburg: Springer Science + Business Media.CrossRefGoogle Scholar
Kumar, S, Stecher, G, Li, M, Knyaz, C, Tamura, K (2018). MEGA X: Molecular Evolutionary Genetics Analysis across computing platforms. Molecular Biology and Evolution 35, 6, 1547–1549. https://doi.org/10.1093/molbev/msy096CrossRefGoogle ScholarPubMed
La Rue, GR (1957). The classification of digenetic Trematoda: a review and a new system. Experimental Parasitology 6, 3, 306–344. https://doi.org/10.1016/0014-4894(57)90025-5CrossRefGoogle ScholarPubMed
Le, S, Gascuel, O (2008). An improved general amino-acid replacement matrix. Molecular Biology and Evolution 25, 7, 1307–1320. https://doi.org/10.1093/molbev/msn067CrossRefGoogle ScholarPubMed
Le, TH, Nguyen, KT, Nguyen, NTB, Doan, HTT, Agatsuma, T, Blair, D (2019). The complete mitochondrial genome of Paragonimus ohirai (Paragonimidae: Trematoda: Platyhelminthes) and its comparison with P. westermani congeners and other trematodes. PeerJ 7, e7031. https://doi.org/10.7717/peerj.7031CrossRefGoogle Scholar
Le, TH, NTB, Nguyen, Nguyen, KT, Doan, HTT, Dung, DT, Blair, D (2016). A complete mitochondrial genome from Echinochasmus japonicus supports the elevation of Echinochasminae Odhner, 1910 to family rank (Trematoda: Platyhelminthes). Infections, Genetic and Evolution 45, 369–377. https://doi.org/10.1016/j.meegid.2016.09.024Google ScholarPubMed
Le, TH, Blair, D, Agatsuma, T, Humair, PF, Campbell, NJ, Iwagami, M, Littlewood, DT, Peacock, B, Johnston, DA, Bartley, J, Rollinson, D, Herniou, EA, Zarlenga, DS, McManus, DP (2000) Phylogenies inferred from mitochondrial gene orders - a cautionary tale from the parasitic flatworms. Molecular Biology and Evolution, 17(7), 1123–1125. doi: 10.1093/oxfordjournals.molbev.a026393.CrossRefGoogle Scholar
Lee, D, Choe, S, Park, H, Jeon, HK, Chai, JY, Sohn, WM, Yong, TS, Min, DY, Rim, HJ, Eom, KS (2013). Complete mitochondrial genome of Haplorchis taichui and comparative analysis with other trematodes. Korean Journal of Parasitology 51, 6, 719–726. https://doi.org/10.3347/kjp.2013.51.6.719CrossRefGoogle ScholarPubMed
Li, Y, Ma, XX, Lv, QB, Hu, Y, Qiu, HY, Chang, QC, Wang, CR (2019). Characterization of the complete mitochondrial genome sequence of Tracheophilus cymbius (Digenea), the first representative from the family Cyclocoeliidae. Journal of Helminthology 94, e101. https://doi.org/10.1017/S0022149X19000932Google Scholar
Littlewood, DTJ, Lockyer, AE, Webster, BL, Johnston, DA, Le, TH (2006). The complete mitochondrial genomes of Shistosoma haematobium and Shistosoma spindale and the evolutionary history of mitochondrial genome changes among parasitic flatworms. Molecular Phylogenetics and Evolution 39, 2, 452–467. https://doi.org/10.1016/j.ympev.2005.12.012CrossRefGoogle Scholar
Littlewood, DTJ (2008). Platyhelminth systematic and the emergence of new characters. Parasite 15, 3, 333–341. https://doi.org/10.1051/parasite/2008153333Google ScholarPubMed
Liu, G-H, Gasser, RB, Young, ND, Song, H-Q, Ai, L, Zhu, X-Q (2014b). Complete mitochondrial genomes of the ‘intermediate form’ of Fasciola and Fasciola gigantica, and their comparison with F. hepatica. Parasites and Vectors 7, 150. https://doi.org/10.1186/1756-3305-7-150CrossRefGoogle ScholarPubMed
Liu, G-H, Yan, H-B, Otranto, D, Wang, X-Y, Zhao, G-H, Jia, W-Z, Zhu, X-Q (2014a). Dicrocoelium chiensis and Dicrocoelium dendriticum (Trematoda: Digenea) are distinct lancet fluke species based on mitochonsrial and nuclear ribosomal DNA sequences. Molecular Phylogenetics and Evolution 79, 325–331. https://doi.org/10.1016/j.ympev.2014.07.002Google Scholar
Liu, Z-X, Zhang, Y, Liu, Y-T, Chang, Q-C, Su, X, Fu, X, Yue, D-M, Gao, Y, Wang, C-R (2016). Complete mitochondrial genome of Echinostoma hortense (Digenea: Echinostomatidae). Korean Journal of Parasitology 54, 2, 173–179. https://doi.org/10.3347/kjp.2016.54.2.173CrossRefGoogle ScholarPubMed
Locke, SA, Dam, AV, Caffara, M, Pinto, HA, Lopez-Hernandez, D, Blanar, CA (2018). Validity of the Diplostomoidea and Diplostomida (Digenea, Platyhelminthes) upheld in phylogenomic analysis. International Journal for Parasitology, 48(13), 1043–1059. doi: 10.1016/j.ijpara.2018.07.001.CrossRefGoogle ScholarPubMed
Ma, J, He, JJ, Zhou, CY, Sun, MM, Cevallos, W, Sugiyama, H, Zhu, XQ, Calvopiña, M (2019). Characterization of the mitochondrial genome sequences of the liver fluke Amphimerus sp. (Trematoda: Opisthorchiidae) from Ecuador and phylogenetic implications. Acta Tropica 195, 90–96. https://doi.org/10.1016/j.actatropica.2019.04.025Google Scholar
Mamaev, YL, Oshmarin, PG (1971). Helminth larvae in freshwater molluscs of Primorsky Krai. In Mamaev, YL (ed), Parasites of animals and plants at the Far East. Vladivostok: Far Eastern Book Publisher House [in Russian].Google Scholar
Na, L, Gao, J-F, Liu, G-H, Fu, X, Su, X, Yue, D-M, Gao, Y, Zhang, Y, Wang, C-R (2016). The complete mitochondrial genome of Metorchis orientalis (Trematoda: Opisthorchiidae): comparison with other closely related species and phylogenetic implications. Infection, Genetics and Evolution 39, 45–50. https://doi.org/10.1016/j.meegid.2016.01.010CrossRefGoogle ScholarPubMed
Nagasawa, K, Urawa, S, Awakura, T (1987). A checklist and bibliography of parasites of Salmonids of Japan. Scientific Reports of Hokkaido Hatchery 41, 1–75.Google Scholar
Nicolić, V, Bilbija, B, Nedic, Z, Simonovic, P, Djikanovic, V (2018). First record of Azygia robusta (Odhner, 1911) (Trematoda: Digenea: Azygiidae) in brown trout (Salmo trutta) in the Vrbas River. Croatian Journal of Fisheries 76, 2, 85–88. https://doi.org/10.2478/cjf-2018-0011CrossRefGoogle Scholar
Nurk, S, Bankevich, A, Antipov, D, Gurevich, AA, Korobeynikov, A, Lapidus, A, Prjibelski, AD, Pyshkin, A, Sirotkin, A, Sirotkin, Y, Stepanauskas, R, Clingenpeel, SR, Woyke, T, JS, McLean, Lasken, R, Tesler, G, Alekseyev, MA, Pevzner, PA (2013). Assembling single-cell genomes and mini-metagenomes from chimeric MDA products. Journal of Computational Biology 20, 10, 714–737. https://doi.org/10.1089/cmb.2013.0084CrossRefGoogle ScholarPubMed
Oey, H, Zakrzewski, M, Gravermann, K, Young, ND, Korhonen, PK, Gobert, GN, Nawaratna, S, Hasan, S, Martínez, DM, You, H, Lavin, M, Jones, MK, Ragan, MA, Stoye, J, Oleaga, A, Emery, AM, Webster, BL, Rollinson, D, Gasser, RB, McManus, DP, Krause, L (2019). Whole-genome sequence of the bovine blood fluke Shistosoma bovis supports interspecific hybridization with S. haematobium. PLoS Pathogens 15, 1, e1007513. https://doi.org/10.1371/journal.ppat.1007513CrossRefGoogle Scholar
Olson, PD, Cribb, TH, Tkach, VV, Bray, RA, Littlewood, DT (2003). Phylogeny and classification of the Digenea (Platyhelminthes: Trematoda). International Journal for Parasitology 33, 7, 733–755. https://doi.org/10.1016/s0020-7519(03)00049-3CrossRefGoogle ScholarPubMed
Park, J-K, Kim, K-H, Kang, S, Jeon, HK, Kim, J-H, Littlewood, DTJ, Eom, KS (2007). Characterization of the mitochondrial genome of Diphyllobothrium latum (Cestoda: Pseudophyllidea) – implications for the phylogeny of eucestodes. Parasitology 134, 5, 749–759. https://doi.org/10.1017/S003118200600206XCrossRefGoogle ScholarPubMed
Pérez-Ponce de León, G, Hernández-Mena, DI (2019). Testing the higher-level phylogenetic classification of Digenea (Platyhelminthes, Trematoda) based on nuclear rDNA sequences before entering the age of the ‘next-generation’ Tree of Life. Journal of Helminthology 93, 3, 260–276. https://doi.org/10.1017/S0022149X19000191CrossRefGoogle ScholarPubMed
Popiolek, M, Kusznierz, J, Kotusz, J, Witkowski, A (2013). Parasites of Hucho hucho (L.), Hucho taimen (Pall.), and Parahucho perryi (Brevoort) (Salmonidae, Actinopterygii) – the state of knowledge. Archives of Polish Fisheries 21, 3, 233–239. https://doi.org/10.2478/aopf-2013-0024CrossRefGoogle Scholar
Protasio, AV, Tsai, IJ, Babbage, A, Nichol, S, Hunt, M, Aslett, MA, De Silva, N, Velarde, GS, Anderson, TJC, Clark, RC, Davidson, C, Dillon, GP, Holroyd, NE, LoVerde, PT, Lloyd, C, McQuillan, J, Oliveira, G, Otto, TD, Parker-Manuel, SJ, Quali, MA, Wilson, RA, Zerlotini, A, Dunne, DW, Berriman, M (2012). A systematically improved high quality genome and transcriptome of the human blood fluke Schistosoma mansoni. PLoS Neglected Tropical Diseases 6, 1, e1455. https://doi.org/10.1371/journal.pntd.0001455CrossRefGoogle ScholarPubMed
Qian, L, Zhou, P, Li, W, Wang, H, Miao, T, Hu, L (2018). Characterization of the complete mitochondrial genome of the lung fluke, Paragonimus heterotremus. Mitochondrial DNA Part B Resources 3, 2, 560–561. https://doi.org/10.1080/23802359.2018CrossRefGoogle ScholarPubMed
Ramilo, A, Abollo, E, Pascual, S (2023). Molecular characterization of Maccallumtrema xiphiados (Trematoda: Azygiida) and Molicola sp. (Cestoda: Trypanorhyncha) infecting commercial frozen slices of Atlantic swordfish. International Journal of Food Microbiology 389, 110103. https://doi.org/10.1016/j.ijfoodmicro.2023.110103CrossRefGoogle ScholarPubMed
Ran, R, Zhao, Q, Abuzeid, AMI, Huang, Y, Liu, Y, Sun, Y, He, L, Li, X, Liu, J, Li, G (2020). Mitochondrial genome sequence of Echinostoma revolutum from Red-Crowned Crane (Grus japonensis). The Korean Journal of Parasitology 58, 1, 73–79. https://doi.org/10.3347/kjp.2020.58.1.73CrossRefGoogle ScholarPubMed
Ronquist, F, Teslenko, M, van der Mark, P, Ayres, DL, Darling, A, Höhna, S, Larget, B, Liu, L, Suchard, MA, Huelsenbeck, JP (2012). MrBayes 3.2: efficient bayesian phylogenetic inference and model choice across a large model space. Systematic Biology 61, 3, 539–542. https://doi.org/10.1093/sysbio/sys029CrossRefGoogle ScholarPubMed
Semyenova, S, Chrisanfova, G, Mozharovskaya, L, Guliaev, A, Ryskov, A (2017). The complete mitochondrial genome of the causative agent of the human cercarial dermatitis, the visceral bird shistosome species Trichobilharzia szidati (Platyhelminthes: Trematoda: Shistosomatidae). Mitochondrial DNA Part B Resources 2, 2, 469–470. https://doi.org/10.1080/23802359.2017.1347833CrossRefGoogle Scholar
Shekhovtsov, SV, Katokhin, AV, Kolchanov, NA, Mordvinov, VA (2010). The complete mitochondrial genomes of the liver flukes Opisthorchis felineus and Clonorchis sinensis (Trematoda). Parasitology International 59, 1, 100–103. https://doi.org/10.1016/j.parint.2009.10.012CrossRefGoogle ScholarPubMed
Skrjabin, KI, Guschanskaja, LK (1958). Suborder Azygiata La Rue, 1957. In Skrjabin, KI (ed) Trematodes of Animals and Man. Osnovy Trematodologii, vol. 14. pp. 667–788. Moscow: USSR Academy of Science (In Russian).Google Scholar
Sokolov, SG, Zhukov, AV (2016). The diversity of parasites in the Chinese sleeper Perccottus glenii Dybowski 1877 (Actinopterygii: Perciformes) under the conditions of large-scale range expansion. Proceedings of Russian Academy of Science. Biological Series 4, 439–448.Google Scholar
Suleman, S, Khan, MS, Heneberg, P, Zhou, CY, Muhammad, N, Zhu, XQ, Ma, J (2019). Characterization of the complete mitochondrial genome of Uvitellina sp., representative of the family Cyclocoeliidae and phylogenetic implications. Parasitology Research 118, 7, 2203–2211. https://doi.org/10.1007/s00436-019-06358-yCrossRefGoogle Scholar
Suleman, S, Ma, J, Khan, MS, Tkach, VV, Muhammad, N, Zhang, D, Zhu, XQ (2019). Characterization of the complete mitochondrial genome of Plagiorchis maculosus (Digenea, Plagiorchiidae), representative of a taxonomically complex digenean family. Parasitology International 71, 99–105. https://doi.org/10.1016/j.parint.2019.04.001CrossRefGoogle ScholarPubMed
Suleman, S, Muhammad, N, Khan, MS, Tkach, VV, Ullah, H, Ehsan, M, Ma, J, Zhu, XQ (2021). Mitochondrial genomes of two eucotylids as the first representatives from the superfamily Microphalloidea (Trematoda) and phylogenetic implications. Parasites and Vectors 14, 41, 8. https://doi.org/10.1186/s13071-020-04547-8CrossRefGoogle ScholarPubMed
TIBCO Software Inc. (2017). Statistica (program product for data analysis), version 13. http://statistica.io (accessed March 27, 2018)Google Scholar
Vainutis, KS, Voronova, AN, Mironovsky, AN, Zhigileva, ON, Zhokhov, AN (2003). The Species Diversity Assessment of Azygia Looss, 1899 (Digenea: Azygiidae) from the Volga, Ob, and Artyomovka Rivers Basins (Russia), with Description of A. sibirica n. sp. Diversity, 15(1), 119. doi.org/10.3390/d15010119.Google Scholar
Wang, Y, Wang, C-R, Zhao, G-H, Gao, J-F, Li, M-W, Zhu, X-Q (2011). The complete mitochondrial genome of Orientobilharzia turkestanicum supports its affinity with African Schistosoma spp. Infection, Genetics and Evolution 11, 8, 1964–1970. https://doi.org/10.1016/j.meegid.2011.08.030CrossRefGoogle ScholarPubMed
Wang, T, Wang, Y, Xu, F, Li, X, Qu, R, Song, L, Tang, Y, Lin, P (2018). Characterization of the complete mitochondrial genome of the lung fluke, Paragonimus kellicotti. Mitochondrial DNA Part B Resources 3, 2, 715–716. https://doi.org/10.1080/23802359.2018CrossRefGoogle ScholarPubMed
Webster, BL, Rudolfová, J, Horák, P, Littlewood, DTJ (2007). The complete mitochondrial genome of the bird schistosome Trichobilharzia regent (Platyhelminthes: Digenea), causative agent of cercarial dermatitis. Journal of Parasitology 93, 3, 553–561. https://doi.org/10.1645/GE-1072R.1CrossRefGoogle Scholar
Wu, Y-A, Gao, J-W, Cheng, X-F, Xie, M, Yuan, X-P, Liu, D, Song, R (2020). Characterization and comparative analysis of the complete mitochondrial genome of Azygia hwangtsiyui Tsin, 1933 (Digenea), the first for a member of the family Azygiidae. ZooKeys 945, 1–16. https://doi.org/10.3897/zookeys.945.49681CrossRefGoogle ScholarPubMed
Xu, G, Zhu, P, Zhu, W, Ma, B, Li, X, Li, W (2021). Characterization of the complete mitochondrial genome of Notocotylus sp. (Trematoda, Notocotylidae) and its phylogenetic implications. Parasitology Research 120, 4, 1291–1301. https://doi.org/10.1007/s00436-021-07075-1CrossRefGoogle ScholarPubMed
Yan, H-B, Wang, X-Y, Lou, Z-Z, Li, L, Blair, D, Yin, H, Cai, J-Z, Dai, X-L, Lei, M-T, Zhu, X-Q, Cai, X-P, Jia, W-Z (2013). The mitochondrial genome of Paramphistomum cervi (Digenea), the first representative for the family Paramphistomatidae. PLoS One 8, 8, e71300. https://doi.org/10.1371/journal.pone.0071300CrossRefGoogle Scholar
Yang, X, Gasser, RB, Koehler, AV, Wang, L, Zhu, K, Chen, L, Feng, H, Hu, M, Fang, R (2015). Mitochondrial genome of Hypoderaeum conoideum – comparison with selected trematodes. Parasites and Vectors 8, 97. https://doi.org/10.1186/s13071-015-0720-xCrossRefGoogle ScholarPubMed
Yang, X, Wang, L, Chen, H, Feng, H, Shen, B, Hu, M, Fang, R (2016). The complete mitochondrial genome of Gastrothylax crumenifer (Gastrothylacidae, Trematoda) and comparative analyses with selected trematodes. Parasitology Research 115, 6, 2489–2497. https://doi.org/10.1007/s00436-016-5019-0CrossRefGoogle ScholarPubMed
Zolotukhin, SF, Semenchenko, AY (2008). Growth and distribution of Sakhalin taimen Parahucho perryi (Brevoort) in watersheds. Proceedings of Levanidov V.Ya. Biennial Memorial Meeting 4, 317–338 [in Russian].Google Scholar
Figure 0

Table 1. List of Digenea sequences from GenBank used in phylogenetic analysis

Figure 1

Figure 1. General view of Azygia robusta extracted from Hucho taimen inhabiting the Armu River (the microscope slide and the photograph were kindly provided by V.V. Besprozvannykh).

Figure 2

Figure 2. General view of Azygia robusta extracted from Parahucho perryi inhabiting the Samarga River (the microscope slide and the photograph were kindly provided by V.V. Besprozvannykh).

Figure 3

Figure 3. Organization of the complete mitochondrial genome in Azygia robusta.

Figure 4

Table 2. The organization of mitochondrial genome of Azygia robusta

Figure 5

Table 3. Amino acid frequencies in concatenated protein sequences of mitochondrial protein-coding region of Azygia robusta (1, ex Hucho taimen, Armu River (Besprozvannykh, 2005) 2002; 2, ex Parahucho perryi, Samarga River, 1987) and A. hwangtsiyui

Figure 6

Table 4. Variation of mitochondrial protein-coding genes of Azygia robusta and between A. robusta and A. hwangtsiyui.

Figure 7

Figure 4. Results of an analysis based on Pearson’s coefficient of correlation between gene length and number of variable sites with pairwise comparison of mitochondrial protein-coding genes of A. robusta and A. hwangtsiyui.r is the Pearson’s correlation coefficient.

Figure 8

Figure 5. Phylogenetic relationships of Azygia robusta and other digenetic trematodes reconstructed by the Bayesian Inference (BI) algorithm on the basis of alignment of protein sequences containing 2280 amino acids, available after Gblock processing. Nodal support is shown with a posteriori probabilities calculated using the BI algorithm.

Figure 9

Figure 6. Phylogenetic relationships of Azygia robusta and other digenetic trematodes reconstructed by the Maximum Likelihood (ML) algorithm on the basis of alignment of protein sequences containing 2280 amino acids, available after Gblock processing. Nodal support is shown with a posteriori probabilities calculated using the approximate likelihood ratio test.