Updated molecular phylogenetic data for Opisthorchis spp. (Trematoda: Opisthorchioidea) from ducks in Vietnam

An opisthorchiid liver fluke was recently reported from ducks (Anas platyrhynchos) in Binh Dinh Province of Central Vietnam, and referred to as “Opisthorchis viverrini-like”. This species uses common cyprinoid fishes as second intermediate hosts as does Opisthorchis viverrini, with which it is sympatric in this province. In this study, we refer to the liver fluke from ducks as “Opisthorchis sp. BD2013”, and provide new sequence data from the mitochondrial (mt) genome and the nuclear ribosomal transcription unit. A phylogenetic analysis was conducted to clarify the basal taxonomic position of this species from ducks within the genus Opisthorchis (Digenea: Opisthorchiidae). Adults and eggs of liver flukes were collected from ducks, metacercariae from fishes (Puntius brevis, Rasbora aurotaenia, Esomus metallicus) and cercariae from snails (Bithynia funiculata) in different localities in Binh Dinh Province. From four developmental life stage samples (adults, eggs, metacercariae and cercariae), the complete cytochrome b (cob), nicotinamide dehydrogenase subunit 1 (nad1) and cytochrome c oxidase subunit 1 (cox1) genes, and near-complete 18S and partial 28S ribosomal DNA (rDNA) sequences were obtained by PCR-coupled sequencing. The alignments of nucleotide sequences of concatenated cob + nad1 + cox1, and of concatenated 18S + 28S were separately subjected to phylogenetic analyses. Homologous sequences from other trematode species were included in each alignment. Phylogenetic trees were inferred from concatenated (cob + nad1 + cox1) nucleotide sequences and combined 18S + 28S nucleotide sequences of five Opisthorchis sp. BD2013 samples and additional reference taxa. Both trees demonstrated the anticipated clustering of taxa within the superfamily Opisthorchioidea, the paraphyly of the genus Opisthorchis and the sister-species relationship of Opisthorchis sp. BD2013 with O. viverrini. While it is likely that Opisthorchis sp. BD2013 is distinct from O. viverrini, it is clearly a sister taxon of O. viverrini within the limited number of Opisthorchis species for which appropriate sequence data are available. The new sequences provided here will assist the diagnosis and the taxonomic clarification of the opisthorchiid species.


Background
The family Opisthorchiidae (Digenea: Opisthorchioidea) consists of 33 genera considered valid including the genera Opisthorchis and Clonorchis, in which O. viverrini, O. felineus and C. sinensis are known to infect humans [1]. Humans become infected by eating uncooked cyprinoid fish containing metacercariae. Opisthorchis viverrini has been reported in Central Vietnam, where Binh Dinh and Phu Yen Provinces are highly endemic for human opisthorchiasis [2][3][4].
In 2013, Dao et al. [5] found adults of an opisthorchiid species in ducks (Anas platyrhynchos) in areas of Binh Dinh Province where there are many human opisthorchiasis cases. This parasite was then given the working name "Opisthorchis viverrini-like", because of its close similarity to O. viverrini [5,6]. Subsequently, there has been a debate about the identity of this worm. Nawa et al. [7] argued that the duck liver fluke not be O. viverrini, but is most likely O. parageminus that was previously reported from ducks in Vietnam [8][9][10]. However, Dorny et al. [11] considered that their "Opisthorchis viverrini-like" species exhibited some morphological differences from O. parageminus. We now propose to use the working name "Opisthorchis sp. BD2013" instead of the earlier "Opisthorchis viverrini-like".
Molecular phylogenetic/systematic studies are excellent aids for taxonomy [12][13][14][15]. Such studies require homologous sequences from as many taxa as possible within the group of interest. In the genus Opisthorchis, a number of genetic markers from complete mitochondrial sequences and the nuclear ribosomal transcription units including, ITS1, ITS2, 18S rDNA and partial 28S rDNA have been generated for O. viverrini, O. felineus and Clonorchis sinensis. These genetic markers have greatly contributed to molecular diagnostic, epidemiological, phylogenetic and evolutionary studies of the species in Opisthorchiidae and trematodes [3,13,[16][17][18][19]. However, Opisthorchis is a very large genus [7], and molecular data are available for only a few species. Moreover, given difficulties with the morphological taxonomy within the genus, it is not always certain that names assigned to samples are accurate. The only molecular data claimed to be from O. parageminus consist of two sequences recently deposited in GenBank (accession numbers KX258656, KX258657) by Nguyen and Nguyen (otherwise unpublished data). Although their worms came from ducks in Vietnam, no information is available on the morphological basis for the identification. Both of these sequences (mitochondrial partial mt cox1 and nuclear ribosomal ITS2) are very similar to earlier sequences available for Opisthorchis sp. BD2013 published by [5]. Here, we provide additional mitochondrial sequences, i.e. complete cytochrome b (cob), nicotinamide dehydrogenase subunit 1 (nad1) and cytochrome c oxidase subunit 1 (cox1) genes, and nearcomplete 18S rDNA and partial 28S rDNA sequences in an effort to better resolve the affinities of Opisthorchis sp. BD2013 within the family Opisthorchiidae and the superfamily Opisthorchioidea.

Methods
Opisthorchis sp. BD2013 samples collected from the field Adult specimens and eggs of Opisthorchis sp. BD2013 were collected from naturally infected domestic ducks (Anas platyrhynchos) originating from 4 localities (Phu Cat, Phu My, An Nhon and Tuy Phuoc Districts) in Binh Dinh Province of Central Vietnam [6,20] (Table 1). Each adult worm, unstained or stained with acetic carmine, was morphologically identified by light microscopy [5]. Up to three adult worms from each locality were individually fixed in 70% ethanol, and one or two worms from each locality were separately subjected to genomic DNA extraction and molecular analysis.
Fishes (harbouring metacercariae) and snails (shedding cercariae) were collected from My Tho Lake in the lowlands of Binh Dinh Province [20]. Infected fishes were identified as Puntius brevis, Esomus metallicus, Rasbora aurotaenia, and the snail as Bithynia funiculata [20] ( Table 1). For molecular analysis, metacercariae and cercariae were individually fixed in RNAlater™ buffer (Qiagen, Texas, USA) at 4°C. Individual parasites from each intermediate host and each locality were used for extraction of DNA and molecular study.
Eggs were individually collected from the gallbladder of naturally infected ducks by washing and centrifuging the bile ten times in normal saline (0.9% NaCl), then three times in phosphate buffered saline (PBS) before storage at -20°C until use (Table 1).

Genomic DNA extraction and primers
Total genomic DNA was extracted from individual adults, metacercariae, cercariae or pooled eggs (approximately 2000-3000 eggs) using the GeneJET™ Genomic DNA Purification Kit (Thermo Fisher Scientific Inc., MA, USA), according to the manufacturer's instructions. A slight modification applied for eggs was to increase the incubation period by 3-4 h after enzymatic lysis. Genomic DNA was eluted in 50 μl of the elution buffer provided in the kit and stored at -20°C. The DNA concentration was estimated using a GBC UV/visible 911A spectrophotometer (GBC Scientific Equipment Pty. Ltd., Braeside, Australia) and diluted to a working concentration of 50 ng/μl (about 10 ng/μl for DNA from eggs). From this genomic DNA, 2-3 μl was used as template in a PCR of 50 μl volume.
Primers used both for amplification and sequencing of the mitochondrial and nuclear ribosomal genes are listed in Table 2. The primer pair OACOBF/OACO1R amplified approximately 7.8 kb of mtDNA. Based on the sequence obtained from this amplicon, three primer pairs specific for the individual target proteincoding genes were designed. Primer pairs OACOBF/ OACOBR, OAND1F/OAND1R, OACO1F/OACO1R amplified complete cob, nad1 and cox1 genes, respectively. The primer pairs U18SF/U18SR were used for obtaining major fragments of ribosomal 18S and U28SF/U28SR for 28S, respectively [12]. Additional internal primers were designed and used as needed ( Table 2). Individual mt and ribosomal DNA genes PCR reactions of 50 μl were prepared using 25 μl of DreamTaq PCR Master Mix (2×) (Thermo Fisher Scientific Inc., Waltham, MA, USA), 2 μl of each primer (10 pmol/μl), 2 μl DNA template (50 ng/μl for adults; 50 ng/μl for metacercariae; 10-20 ng/μl for cercariae and eggs), 2 μl DMSO (dimethyl sulfoxide) and 17 μl H 2 O. All PCRs were performed in an MJ PTC-100 thermal cycler with initiation at 94°C for 5 min, followed by 35 cycles consisting of denaturation for 30 s at 94°C, annealing at 56°C for 30 s, extension at 72°C for 3 min.  Sequencing and sequence analyses PCR products were obtained from at least two individual samples for each template (i.e. adults, metacercariae, cercariae and eggs) originating from different geographical localities. The PCR products (10 μl of each) were examined on a 1% agarose gel, stained with ethidium bromide, and visualized under UV light (Wealtec, Meadowvale Way Sparks, USA). All the purified or gel-extracted amplicons were subjected to direct sequencing by automated sequencers using amplifying/flanking and internal primers ( Table 2) by primer-walking in both directions (Macrogen Inc., Seoul, South Korea). Sequences (two from each sample) were aligned to obtain the final sequence for characterization. All sequences of Opisthorchis sp. BD2013 were identical, regardless of the life-cycle stage or locality.

Amplification of mitochondrial and ribosomal genes
The concatenated nucleotide and amino acid sequences of three protein-coding genes, i.e., cob + nad1 + cox1, were used to infer the pairwise genetic distances between 10 opisthorchiids (Table 3). These isolates included Opisthorchis sp. BD2013 and the reference sequences from Laos (JF739555), Vietnam (MF287777-MF287779) and Thailand (MF287780-MF287782). The genetic distances were inferred by pairwise analysis using the MEGA6.0 software, and the number of base substitutions per site was calculated by the most simplified method (uncorrected p-distance) [21].

Phylogenetic analysis Preparation of DNA sequences
Phylogenetic analysis using three mitochondrial proteincoding (cob, nad1, cox1) and two nuclear ribosomal (18S and 28S rDNA) genes was conducted to examine the taxonomic placement of Opisthorchis sp. BD2013 from ducks within the superfamily Opisthorchioidea. Sequences of trematode species/isolates of the Opisthorchiidae, Heterophyidae, Fasciolidae and Schistosomatidae (as the outgroup) were used. Summary data of species/ isolates, mainly from the available complete mitochondrial genomes are presented in Table 3. Accession numbers for the target and reference 18S and 28S rDNA sequences are listed in Table 4. For Opisthorchis sp. BD2013, we decided to use only two sequences of adults, and one each from metacercariae, cercariae and eggs for phylogenetic analyses.
DNA sequences of 18S rRNA and 28S rRNA genes (listed in Table 4) were aligned separately using GENE-DOC 2.7. The sequences were trimmed at both ends to the shortest length of the representative sequences. For 18S rDNA, in this study, the final alignment was 2005 nucleotides (nt) long of which 87 nt positions were trimmed at 5′ end and 114 nt at 3′ end, leaving 1804 characters for analyses. For 28S rDNA, the final alignment was1449 nt long of which 122 nt positions were trimmed at 5′ end and 123 nt at 3′ end, leaving 1202 characters for analyses. The two sequences were then concatenated as indicated in Table 4, preferably from the same strains/isolates. The concatenated 18S + 28S rDNA sequences representing species/isolates were imported into GENEDOC 2.7 and phylogenetic analysis and tree construction were done by MEGA6.0 [21].

Phylogenetic reconstruction
The alignments of the concatenated nucleotide (cob, cox1, nad1) and 18S +28S sequences, respectively, were trimmed to the length of the shortest sequence and imported into the MEGA 6.06 software [21]. Maximum likelihood (ML) analyses were performed in each case. For DNA sequences, we used the general time-reversible model of evolution with gamma distributed rate heterogeneity and a proportion of invariant sites (GTR + Γ + I). This model was given the best Bayesian information criterion score by MEGA. For amino acid sequences, the Jones-Taylor-Thornton (JTT) model with uniform rates and Nearest-Neighbor-Interchange (NNI) method was used. The confidence in each node was assessed using 1000 bootstrap resamplings [21].

Results
Mitochondrial cob, nad1, cox1 and genetic distances among opisthorchiid species/sequences For Opisthorchis sp. BD2013, lengths of the complete cob, nad1 and cox1 genes were 1110, 903 and 1551 nucleotides, respectively. Among opisthorchiid species, cob genes ranged in length from 1110 to 1116 nt, and cox1 genes were 1551 to 1563 nt in length. The primer pairs U18SF/U18SR were used for obtaining major fragments of ribosomal 18S and U28SF/U28SR for 28S rDNA.
Nucleotide and amino acid pairwise comparisons of the concatenated mt genes among ten opisthorchiid isolates/species are presented in Tables 5 and 6 [23].

Phylogenetic analysis
Phylogenetic reconstruction based on the complete cob + nad1 + cox1 amino acid sequences A phylogenetic tree was constructed from 25 nucleotide sequences inferred from complete cob + nad1 + cox1 of 13 trematode species belonging to 4 families with Schistosoma haematobium of the Schistosomatidae as the outgroup (Table 3, Fig. 1 (Fig. 1).

Phylogenetic reconstruction based on partial 18S + 28S sequences
Five concatenated 18S + 28S sequences of Vietnamese Opisthorchis sp. BD2013 (from eggs, cercariae, metacercariae and adults) were aligned with 26 available sequences representing 17 trematode species of the Opisthorchiidae, Heterophyidae, Fasciolidae and Schistosomatidae (outgroup) ( Table 4). The nuclear ribosomal dataset from the Opisthorchioidea included available sequences of the 18S + 28S of 12 taxa only from the Opisthorchiidae and Heterophyidae (data from the Cryptogonimidae were not available). The combined length of alignment in use was between 2940 and 2960 nt. The inferred phylogenetic tree (Fig. 2) again placed Opisthorchis sp. BD2013 in a sister position with O. viverrini from Thailand and Vietnam. Again, the genus Opisthorchis appeared as paraphyletic. Monophyly of the superfamily Opisthorchioidea was strongly supported (Fig. 2).

Discussion
In this study, we used two concatenated datasets to infer the molecular phylogenetic position of Opisthorchis sp. BD2013 (formerly named "Opisthorchis viverrini-like" or as O. parageminus by several authors). We did not have samples of O. lobatus [17] and the so-called O. parageminus [8,9] for analysis in the present study, therefore, we were not able to establish the relationship between Opisthorchis sp. BD2013 and these species. The genus Opisthorchis is very large [7], but relevant sequence data are limited to only a few species. It was necessary to determine whether Opisthorchis sp. BD2013 from ducks is distinct from O. viverrini, a zoonotic liver fluke known to infect and to cause cholangiocarcinoma in humans [23]. The data presented in this study strongly imply that the two are distinct species. The sister-species relationship demonstrated between Opisthorchis sp. BD2013, and O. viverrini might simply be because O. felineus is the only other member of the genus for which data are available. Opisthorchis felineus renders Opisthorchis paraphyletic in our trees, indicating that much systematic work remains to be done in the   Opisthorchiidae. A further unresolved question is the relationship between Opisthorchis sp. BD2013 and O. parageminus. Both were found in ducks in Vietnam, but some morphological differences seem to exist [11]. At this stage, we prefer to leave the question open, pending future morphological and molecular work. Our previous phylogenetic analysis using short sequences of ITS2 and cox1 revealed close affinities between O. viverrini, O. lobatus and Opisthorchis sp. BD2013 [5]. In the current study, we are unable to resolve the status of O. lobatus compared to Opisthorchis sp. BD2013 and other opisthorchiids.

Conclusions
Based on mitochondrial cob + nad1 + cox1 and ribosomal 18S + 28S rRNA sequence analyses, Opisthorchis sp. BD2013 was distinct from O. viverrini, although the two species are closely related. The genus Opisthorchis itself appears as paraphyletic. Data from additional Opisthorchis species are vital to create a phylogeny with higher resolution within Opisthorchis and the Opisthorchiidae.

Acknowledgments
We express our thanks to colleagues and technicians at the Binh Dinh Provincial station of veterinary services for providing and processing samples and Ms TK Nguyen and Dr. HTT Doan of the Institute of Biotechnology, Hanoi, Vietnam for contributing to our laboratory work. We would like to express our gratitude to Professor David Blair, College of Science and Engineering, James Cook University, Townsville, Australia for revision and invaluable comments on this paper.
Authors' contributions THTD, PD and THL conceived the study, analyses of final data and wrote the manuscript. TGTN, KLB and SG conducted field collections, laboratory, and preliminary sequence analyses. All authors read and approved the final manuscript.
Ethics approval and consent to participate Appropriate permission was obtained from the commune authorities and local households before the collection of parasite specimens from their stocks.

Consent for publication
Not applicable.