The ribosomal transcription units of Haplorchis pumilio and H. taichui and the use of 28S rDNA sequences for phylogenetic identification of common heterophyids in Vietnam
Le et al. Parasites & Vectors
The ribosomal transcription units of Haplorchis pumilio and H. taichui and the use of 28S rDNA sequences for phylogenetic identification of common heterophyids in Vietnam
Thanh Hoa Le 0
Khue Thi Nguyen 0
Nga Thi Bich Nguyen 0
Huong Thi Thanh Doan 0
Do Trung Dung 2
David Blair 1
0 Institute of Biotechnology, Vietnam Academy of Science and Technology , 18. Hoang Quoc Viet Rd, Cau Giay, Hanoi , Vietnam
1 College of Science and Engineering, James Cook University , Townsville , Australia
2 Department of Parasitology, National Institute of Malariology, Parasitology and Entomology , Luong The Vinh Rd, Thanh Xuan, Hanoi , Vietnam
Background: Heterophyidiasis is now a major public health threat in many tropical countries. Species in the trematode family Heterophyidae infecting humans include Centrocestus formosanus, Haplorchis pumilio, H. taichui, H. yokogawai, Procerovum varium and Stellantchasmus falcatus. For molecular phylogenetic and systematic studies on trematodes, we need more prospective markers for taxonomic identification and classification. This study provides near-complete ribosomal transcription units (rTU) from Haplorchis pumilio and H. taichui and demonstrates the use of 28S rDNA sequences for identification and phylogenetic analysis. Results: The near-complete ribosomal transcription units (rTU), consisting of 18S, ITS1, 5.8S, ITS2 and 28S rRNA genes and spacers, from H. pumilio and H. taichui from human hosts in Vietnam, were determined and annotated. Sequence analysis revealed tandem repetitive elements in ITS1 in H. pumilio and in ITS2 in H. taichui. A phylogenetic tree inferred from 28S rDNA sequences of 40 trematode strains/species, including 14 Vietnamese heterophyid individuals, clearly confirmed the status of each of the Vietnamese species: Centrocestus formosanus, Haplorchis pumilio, H. taichui, H. yokogawai, Procerovum varium and Stellantchasmus falcatus. However, the family Heterophyidae was clearly not monophyletic, with some genera apparently allied with other families within the superfamily Opisthorchioidea (i.e. Cryptogonimidae and Opisthorchiidae). These families and their constituent genera require substantial re-evaluation using a combination of morphological and molecular data. Our new molecular data will assist in such studies. Conclusions: The 28S rDNA sequences are conserved among individuals within a species but varied between genera. Based on analysis of 40 28S rDNA sequences representing 19 species in the superfamily Opisthorchioidea and an outgroup taxon (Alaria alata, family Diplostomidae), six common human pathogenic heterophyids were identified and clearly resolved. The phylogenetic tree inferred from these sequences again confirmed anomalies in molecular placement of some members of the family Heterophyidae and demonstrates the need for reappraisal of the entire superfamily Opisthorchioidea. The new sequences provided here supplement those already available in public databases and add to the array of molecular tools that can be used for the diagnosis of heterophyid species in human and animal infections.
Ribosomal transcription unit; Haplorchis pumilio; Haplorchis taichui; Heterophyidae; 28S rDNA sequence; Phylogeny
Table 1 Summary data for the heterophyids used in the phylogenetic analysis and molecular identification
Many members of the trematode family Heterophyidae
Odhner, 1914, use fishes as intermediate hosts and
humans as definitive hosts [1, 2]. Six species in particular,
Centrocestus formosanus, Haplorchis pumilio, H. taichui,
H. yokogawai, Procerovum varium and Stellantchasmus
falcatus [2, 3] are among the most clearly recognized
human pathogens and mostly occur in eastern Asia
including China, the Philippines, Korea, Taiwan, Thailand,
Laos, Cambodia and Vietnam [3–11]. Heterophyidiasis
caused by these and related species has now become a
major public health threat, not only in Asia but in parts of
Africa and the Americas [3, 5, 10, 12, 13]. Humans acquire
heterophyid infection by consumption of undercooked or
raw freshwater fishes containing infective metacercariae
[3, 14]. Infection with multiple species is frequently
reported in Vietnam and elsewhere [3, 5, 7, 9, 14].
DNA sequences are commonly used for molecular
diagnosis and systematic/phylogenetic studies. Although
markers are often chosen from the mitochondrial genome,
sequences from the nuclear ribosomal transcription unit
(rTU) (including 18S, ITS1, ITS2 and 28S) are particularly
useful and reliable for this purpose [10, 15–22]. A single
rTU consists of three coding regions (the 18S, 5.8S and
28S rRNA genes) separated by two internal transcribed
spacers (ITS1 and ITS2) . Short external transcribed
spacers (ETS) are found 5' of the 18S gene and 3' of the
28S gene. Adjacent rTUs in the ribosomal array are
separated by a long non-transcribed intergenic spacer (IGS)
region [17, 23, 24]. Sequences from various portions of
the rTU (18S, ITS1, ITS2 and 28S) have been widely used
for inference of phylogenetic relationships and taxonomic
clarification within and between many trematode families
(e.g. [15, 18, 22, 25–31]). Sequences of complete or
nearaHaplorchis pumilio and H. taichui samples chosen for sequencing the near complete ribosomal transcription unit
complete rTUs are only available for a few species of
trematode [12, 16, 20, 32, 33]. Clearly, however, such data
will be valuable for many kinds of comparative analysis,
including systematics/phylogenetics and studies on
intraand interspecific or even intra- and interindividual
variation in trematodes [15, 18, 20, 34, 35]. In particular,
these data are needed for the large family Heterophyidae,
which comprises more than 30 genera, many containing
species infecting humans [1, 2, 12, 15, 34]. Heterophyid
species in Vietnam have well been described
epidemiologically and morphologically, but molecular data useful
for diagnosis and identification, as well as taxonomy,
are still limited [5–7, 9, 14].
The aim of this paper is to present the sequence of
near-complete ribosomal transcription units from
Haplorchis pumilio and H. taichui, commonly found in
humans. Portions of the 28S rRNA gene from other
heterophyids infecting humans in Vietnam are also
presented, i.e. Centrocestus formosanus, Haplorchis
yokogawai, Procerovum varium and Stellantchasmus
falcatus. The data will be used to explore the phylogenetic
positions of these genera in the family Heterophyidae
and in the class Trematoda.
Metacercariae of Haplorchis spp. and Centrocestus spp.
were collected from fish species (common carp, Cyprinus
carpio, and grass carp, Ctenopharyngodon idellus) and
cercariae from freshwater snails (Melanoides tuberculata)
in Nam Dinh Province [8, 14] (Table 1).
Adults of Centrocestus spp., Haplorchis spp., Procerovum
spp. and Stellantchasmus spp., originating from Ha Giang,
Nam Dinh, Quang Tri and Quang Ninh Provinces, in the
north of Vietnam, were collected directly from feces of
naturally infected humans after treatment with
praziquantel and purgation by magnesium sulfate (MgSO4)
[5, 14] (Table 1). Each adult worm, unstained or stained
with acetic carmine, was morphologically identified to
species by light microscopy [3, 5, 14]. Up to ten worms of
each species recovered per human were individually fixed
in 70% ethanol; one or two worms of each species were
subjected to molecular analysis. The samples HTAQT3 of
Haplorchis taichui and HpDzH of H. pumilio, collected
from people in Quang Tri and Nam Dinh Provinces,
respectively, were chosen for amplification and sequencing
of the rTU. Only the 28S region was amplified and
sequenced from other species for molecular identification
and phylogenetic analysis (Table 1).
Genomic DNA extraction, primers and amplification
Total genomic DNA was extracted from individual
cercariae, metacercariae or adult specimens using the
GeneJET™ Genomic DNA Purification Kit (Thermo
Fisher Scientific Inc., MA, USA), according to the
manufacturer’s instructions. 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 VIC, Australia) and diluted to a
working 50 ng/μl: 2 μl were used as template in a PCR of
50 μl volume.
All rTU-universal primers, used both for amplification
and sequencing the rTU of H. pumilio and H. taichui,
are listed in Table 2. Primers UD18SF/U3SR amplified
the 18S and ITS1 region and U3SF/1500R amplified the
ITS2 and 28S region. The primer pairs U18SF/U18SR
and U28SF/U28SR, were used for obtaining major
fragments of ribosomal 18S or 28S, respectively. These
primers were also used as sequencing primers, as were
additional internal primers (Table 2).
PCR reactions of 50 μl were prepared using 25 μl of
DreamTaq PCR Master Mix (2×) (Thermo Fisher Scientific
Inc., MA, USA) and 2 μl DNA template (50 ng/μl), 2 μl of
each primer (10 pmol/μl), 2 μl DMSO (dimethyl sulfoxide)
and 17 μl H2O. All PCRs were performed in a MJ
PTC100 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
6 min; and a final extension at 72 °C for 10 min. The PCR
products (10 μl of each) were examined on a 1% agarose
gel, stained with ethidium bromide, and visualized under
UV light (Wealtec, Sparks, NV, USA).
The amplicons were eluted from the gel and subjected
to direct sequencing by primer-walking in both
Table 2 Primers for amplification and sequencing of the ribosomal transcription unit
Abbreviations F forward, R reverse, Tm melting temperature
aPrimers used for sequencing
Annotation and phylogenetic analysis
Boundaries of ribosomal 18S, 5.8S and 28S genes were
determined by alignment, using the Clustal X program
, with known ribosomal DNA sequences inferred
from complete or near-complete rTU sequences available
in the GenBank database or previous publications, i.e. for
Euryhelmis costaricensis (GenBank: AB521797);
Isthmiophora hortensis (AB189982); Paragonimus kellicotti
(HQ900670); Paramphistomum cervi ; and some
partial rTUs including Centrocestus sp. (AY245699); and
Haplorchis pumilio (AY245706) and Haplorchis taichui
(AY245705) . For internal transcribed spacers, ITS1
was recognized as the region located between 18S and 5.8S
and ITS2 as between and 5.8S and 28S, respectively.
Tandem repeats (TRs) were detected in the ITS1 or
ITS2 using the Tandem Repeat Finder v3.01 .
Newly obtained partial 28S sequences (approximately,
1,100 nucleotides) of 14 Vietnamese heterophyids and
25 additional sequences, representing species of all three
families of the superfamily Opisthorchioidea available in
GenBank, and including another 17 sequences from
members of the family Heterophyidae, were aligned
using GENEDOC2.7 (available at:
http://iubio.bio.indiana.edu/soft/molbio/ibmpc/genedoc-readme.html) (Tables 1
and 3). Also included in the alignment was Alaria alata
(family Diplostomidae) as an outgroup species. The
alignment was trimmed to the length of the shortest sequence,
saved in FASTA format and imported into the MEGA6.06
software. To examine the phylogenetic position of the
Vietnamese heterophyids relative to other trematodes, a
phylogenetic tree was reconstructed (see list of sequences
in Tables 1 and 3) using maximum likelihood (ML)
analysis with the general time reversible (GTR) + G+ I model
(gamma rate heterogeneity and a proportion of invariant
sites). This model was given the best Bayesian information
criterion score by MEGA. Confidence in each node was
Table 3 Summary data for the 28S rDNA sequences for heterophyids and other trematodes available on GenBank and used in the
phylogenetic analysis and species identification
aPublished sequences for C. formosanus, H. pumilio, H. taichui, H. yokogawai, P. varium and S. falcatus and used in comparisons with those of the Vietnamese heterophyids
bSequence used as the outgroup
assessed using 1,000 bootstrap resamplings . A Bayesian
analysis was also conducted using MrBayes v3.2 
and the same model of sequence evolution. Five million
generations were performed (two parallel runs, each
with four chains), more than required for the standard
deviation of the splits frequencies to fall below 0.01.
Plots indicated that convergence was approached after
fewer than 1,000,000 generations. The first 1,000,000 cycles
were therefore discarded as ‘burn-in’ and trees sampled
every 1,000 generations.
Structural organization and characteristics of the
ribosomal transcription unit of Haplorchis pumilio
and H. taichui
Near-complete ribosomal transcription units (rTU) from
H. pumilio and H. taichui were determined. The 28S
rDNA sequences are conserved among individuals
within a species but variable between species and genera.
The near-complete rTU is 4,943 nucleotides in length
for H. pumilio, and 4,796 nucleotides for H. taichui.
These sequences have been deposited in GenBank under
accession nos. KX815125 and KX815126, respectively.
We did not sequence the IGS due to the highly
repetitive sequences included in this region. The five regions
of the rTU are: 18S, ITS1, 5.8S, ITS2 and 28S, structurally
organized as usually seen in the ribosomal DNA operon
of metazoans (Fig. 1).
In both H. pumilio and H. taichui, the 18S gene was
1,992 bp in length, and the 5.8S gene was 160 bp long;
however, the currently sequenced portion of the 28S
gene obtained from H. pumilio is 1,397 bp, and that of
H. taichui, 1,403 bp (Table 4). These lengths represent
only a portion of the complete 28S gene (around 3.2–
5.5 kb in total for various trematode species ). The
Vietnamese H. pumilio ITS1 region (1,106 bp) contains
five complete tandem repeats, (TRA1-2-3, each of
136 bp) and TRB (TRB1-2 each of 123 bp) followed by a
partial TRB3 of 84 bp (Table 4; Fig. 1). The ITS1 of the
Vietnamese H. taichui (797 bp) lacks repeats. In contrast
to ITS1, the ITS2 region (444 bp) in H. taichui from
Vietnam (HTAQT3), possesses three tandem repeats,
each of 83–85 bp, while in H. pumilio (HpDzH) this
region lacks repeats (Table 4; Fig. 1).
Partial 28S rDNA sequences were obtained from 14
samples of Vietnamese heterophyids representing six
species: Centrocestus formosanus, Haplorchis pumilio,
H. taichui, H. yokogawai, Procerovum varium and
Stellantchasmus falcatus (Table 1). These were aligned with
26 previously published sequences representing 20
species of trematodes in 4 families, including additional
representatives of the Heterophyidae (Table 3). The
alignment used was 1,100 bp in length. The phylogenetic
tree shown in Fig. 2 is based on the maximum likelihood
(ML) analysis. Bayesian posterior support values and
bootstrap values are shown at relevant nodes. Bayesian and
ML trees were almost identical, differing only in the
placement of Centrocestus formosanus. In the Bayesian tree,
this species fell into a clade (posterior support 0.86) with
members of the Cryptogonimidae, whereas in the ML tree
it was depicted as basal to all other opisthorchioideans
(Fig. 2), albeit with low bootstrap support. Sequences of
each of our six target heterophyid species were
consistently grouped with those of the same species from
published sources, thus confirming our morphological
identifications. With one exception, species were clustered
within their respective genera. The exception was
Procerovum varium, which was nested among species of
Haplorchis. Monophyly of the Heterophyidae was not
observed. The Centrocestus formosanus sequences were
grouped either with a sister relationship to the
Cryptogonimidae (Bayesian analysis) or basal in the Opisthorchioidea
(ML analysis), Sequences of two other heterophyids,
Euryhelmis costaricensis from Japanese martens (Martes
melampus)  and Cryptocotyle lingua, fell into a strongly
supported clade (Bayesian posterior support value 1.0 and
ML bootstrap support 96%), all other members of which
belonged to the family Opisthorchiidae (Fig. 2).
In this study, we have presented sequences of the
nearcomplete ribosomal transcription units (rTUs) for two
Fig. 1 Structural organization of the near-complete ribosomal transcription units for Haplorchis pumilio and H. taichui. TRA1-3 and TRB1-3 are the
tandem repeats in the ITS1 region of H. pumilio; TR1-3 are the repeats in H. taichui
Table 4 Position of ribosomal genes and internal transcribed spacers in the partially sequenced transcription unit of Haplorchis
pumilio (4,943 bp) and H. taichui (4,796 bp)
Intergenic spacer (bp)
common species of the family Heterophyidae,
Haplorchis pumilio and H. taichui, which infect humans in
Vietnam. The obtained sequences encompass virtually
the complete 18S gene (typical length range 1.7–2.9 kb)
and almost half of the 28S gene (typical length range
3.3–5.5 kb) [16, 17]. Also obtained were the complete
ITS1, 5.8S gene and ITS2 sequences for these species.
We have found repetitive sequences tandemly
arranged in the ITS1 of H. pumilio and in the ITS2 of H.
taichui. ITS sequences of both species have been
reported from Israel . Israeli H. pumilio possessed
only two short tandem repeats (30 bp) in their ITS1, in
strong contrast to the Vietnamese sequences, in which
the ITS1 contained five complete repeats and one
incomplete copy. The ITS1 sequences differed substantially in
length between Vietnamese and Israeli individuals of the
same species, 1,106 vs 640 bp in H. pumilio; and 797 vs
582 bp in H. taichui, due to differences in numbers of
tandem repeats. These indicate intraspecific polymorphism
as reported commonly in trematodes [8, 12, 33]. Likewise,
ITS2 showed repetitive sequence differences between
individuals from different locations. The presence of
repeats in the internal transcribed spacers of trematodes
has been reported for several taxa, including those in
Schistosomatidae, Opisthorchiidae, Heterophyidae,
Paramphistomatidae and others [8, 32, 33, 40]. The
presence of repeats, variation in length and sequence
variation, within and between species, all contribute to
difficulties when trying to align ITS regions. This is
particularly so when phylogenetically divergent species
are being compared and suggest that this region is not
suitable for deep-level phylogenies . At the level of
genus and species, however, alignments of ITS sequences
have proved valuable for phylogenetic studies and
molecular taxonomy [17, 41, 42].
The 18S and 28S rDNA sequences, however, are of
considerable value for species identification and
phylogenetic analysis [12, 15, 16, 18, 19, 25, 26, 30, 43, 44].
Alignment of these genes is generally straightforward,
even among distantly related species, and long repeats
do not occur.
The topology of the phylogenetic tree inferred from 40
trematode sequences in this study (Fig. 2) generally
agreed well with previous findings. Most genera
represented by multiple sequences formed well-supported
monophyletic clusters. One striking exception was the
sequence of Procerovum varium, which rendered Haplorchis
paraphyletic. This relationship has also been noticed by
others (e.g. ). Clearly, the definitions of these two
genera will need to be revisited. The three families
66 bp to TRA1
Overlap with TRB1
393 bp to 5.8S
5' partial sequence
121 bp to TR1
5' partial sequence
Fig. 2 Phylogenetic tree including the six target heterophyid species from Vietnam (Centrocestus formosanus, Haplorchis pumilio, H. taichui, H. yokogawai,
Procerovum varium and Stellantchasmus falcatus) and other opisthorchioid trematodes based on partial 28S rDNA sequences (1,100 bp). Alaria alata
(Diplostomidae) was used as the outgroup taxon. The tree depicted was inferred using maximum likelihood (ML) analysis with the general
time reversible (GTR) + G + I model (gamma rate heterogeneity and a proportion of invariant sites) in the MEGA 6.06 package. Support for
each node was evaluated using 1,000 bootstrap resamplings . An almost identical tree was found using Bayesian analysis (see text for
details). Numbers at nodes are Bayesian posterior support values/ML bootstrap values. The basal node for the superfamily Opisthorchioidea is indicated
by an arrow. The scale-bar indicates the number of substitutions per site. Accession numbers are given at the end of each sequence name
constituting the Opisthorchioidea, the Heterophyidae,
Cryptogonimidae and Opisthorchiidae, are very poorly
resolved in the tree. The Heterophyidae is not a
monophyletic taxon. Indeed, two genera of nominal
heterophyids, Euryhelmis and Cryptocotyle, appear to have
closer affinities with the Opisthorchiidae than with the
Heterophyidae. This relationship was also found by
Thaenkham et al.  using 18S rDNA sequences, and
by Thaenkham et al.  using concatenated 18S and
28S sequences. Paraphyly of the Heterophyidae with
respect to the Opisthorchiidae was also demonstrated
by  using 18S and 28S sequences. An additional
heterophyid genus, Centrocestus, had an affinity with
members of the Cryptogonimidae, or appeared as basal
within the Opisthorchioidea (Fig. 2). Such a placement
was not supported by analysis of concatenated 18S and
28S sequences by . It is clear that the entire
superfamily Opisthorchioidea presents broad systematic and
taxonomic challenges to be met in the future using
combined morphological and molecular approaches.
In conclusion, the present study determined and
annotated the near-complete ribosomal transcription unit
(rTU), consisting of 18S, ITS1, 5,8S, ITS2 and 28S rRNA
genes and spacers, from H. pumilio and H. taichui from
human hosts in Vietnam. The ITS1 in H. pumilio and
ITS2 in H. taichui contained tandem repeats. The 28S
rDNA sequences are conserved among individuals within
a species but variable between species and genera. Based
on 28S rDNA sequence analysis of 40 sequences
representing 19 species in the superfamily Opisthorchioidea,
six common human pathogenic heterophyids, Centrocestus
formosanus, Haplorchis pumilio, H. taichui, H. yokogawai,
Procerovum varium and Stellantchasmus falcatus were
clearly resolved. In addition, the phylogenetic tree inferred
from these sequences again confirmed anomalies in
molecular placement of some members of the family
Heterophyidae and demonstrates the need for reappraisal of the
entire superfamily Opisthorchioidea. The new sequences
provided here supplement those already available in public
databases and add to the array of molecular tools that can
be used for the diagnosis of heterophyid species in human
and animal infections.
We express our thanks to colleagues and technicians for providing and
processing samples and contributing to our laboratory work.
Availability of data and materials
The datasets supporting the conclusions of this article are included within
the article. Nucleotide sequences obtained in the present study have been
deposited into the GenBank database with the following accession numbers:
KX815125, KX815126 and KY369153–KY369164.
THL, DB conceived the study, final data analyses and wrote the manuscrip.
DTD conducted field collections. KTN, NTBN, HTTD conducted laboratory
work and preliminary sequence analyses. All authors read and approved
the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
The study had ethical approval from the National Institute of Malariology,
Parasitology and Entomology (NIMPE) on behalf of the Ministry of Health,
Vietnam. Appropriate permission was obtained from the commune authorities
and local households before the collection of parasite specimens from humans.
1. Pearson J. Family Heterophyidae Leiper , 1909 . In: Bray RA, Gibson DI , Jones A , editors. Keys to the Trematoda , vol. 3. London: CAB International and Natural History Museum UK ; 2008 . p. 113 - 42 .
2. Cribb T , Gibson D. Heterophyidae Leiper , 1909 . Accessed through: World Register of Marine Species . 2010 . http://www.marinespecies. org/aphia. php?p=taxdetails&id=108441. Accessed 12 Oct 2016 .
3. Chai JY , Shin EH , Lee SH , Rim HJ . Foodborne intestinal flukes in Southeast Asia . Korean J Parasitol . 2009 ; 47 : s69 - 102 .
4. Belizario Jr VY , de Leon WU , Bersabe MJ , Purnomo, Baird JK , Bangs MJ . A focus of human infection by Haplorchis taichui (Trematoda: Heterophyidae) in the southern Philippines . J Parasitol . 2004 ; 90 ( 5 ): 11655 - 9 .
5. Dung DT , Van De N , Waikagul J , Dalsgaard A , Chai JY , Sohn WM , et al. Fishborne zoonotic intestinal trematodes, Vietnam. Emerg Infect Dis . 2007 ; 13 ( 12 ): 1828 - 33 .
6. Skov J , Kania PW , Dalsgaard A , Jorgensen R , Buchmann K. Life cycle stages of heterophyid trematodes in Vietnamese freshwater fishes traced by molecular and morphometric methods . Vet Parasitol . 2009 ; 160 : 66 - 75 .
7. Chai JY , De NV , Sohn WM . Foodborne trematode metacercariae in fish from northern Vietnam and their adults recovered from experimental hamsters . Korean J Parasitol . 2012 ; 50 ( 4 ): 317 - 25 .
8. Van KV , Dalsgaard A , Blair D , Le TH . Haplorchis pumilio and H. taichui in Vietnam discriminated using ITS-2 DNA sequence data from adults and larvae . Exp Parasitol . 2009 ; 123 : 146 - 51 .
9. Phan VT , Ersbøll AK , Do DT , Dalsgaard A. Raw-fish-eating behavior and fishborne zoonotic trematode infection in people of northern Vietnam . Foodborne Pathog Dis . 2011 ; 8 ( 2 ): 255 - 60 .
10. Thaenkham U , Dekumyoy P , Komalamisra C , Sato M , Dung do T , Waikagul J. Systematics of the subfamily Haplorchiinae (Trematoda: Heterphyidae), based on nuclear ribosomal DNA genes and ITS2 region . Parasitol Int . 2010 ; 59 ( 3 ): 460 - 5 .
11. Krailas D , Veeravechsukij N , Chuanprasit C , Boonmekam D , Namchote S. Prevalence of fish-borne trematodes of the family Heterophyidae at Pasak Cholasid Reservoir, Thailand . Acta Trop . 2016 ; 156 : 79 - 86 .
12. Dzikowski R , Levy MG , Poore MF , Flowers JR , Paperna I. Use of rDNA polymorphism for identification of Heterophyidae infecting freshwater fishes . Dis Aquat Organ . 2004 ; 59 ( 1 ): 35 - 41 .
13. Yousif F , Ayoub M , Tadros M , El Bardicy S. The first record of Centrocestus formosanus (Nishigori , 1924 ) ( Digenea: Heterophyidae) in Egypt . Exp Parasitol . 2016 ; 168 : 56 - 61 .
14. De NV , Le TH . Human infections of fish-borne trematodes in Vietnam: prevalence and molecular specific identification at an endemic commune in Nam Dinh Province . Exp Parasitol . 2011 ; 129 ( 4 ): 355 - 61 .
15. Olson PD , Cribb TH , Tkach VV , Bray RA , Littlewood DTJ . Phylogeny and classification of the Digenea (Platyhelminthes: Trematoda) . Int J Parasitol . 2003 ; 33 : 733 - 55 .
16. Lockyer AE , Olson PD , Littlewood DTJ . Utility of complete large and small subunit rRNA genes in resolving the phylogeny of the Neodermata (Platyhelminthes): implications and a review of the cercomer theory . Biol J Linnean Soc . 2003 ; 78 ( 2 ): 155 - 71 .
17. Blair D. Ribosomal DNA , variation in parasitic flatworms . In: Maule A, editor. Parasitic flatworms: molecular biology , biochemistry, immunology and control. Wallingford: CAB International ; 2006 . p. 96 - 123 .
18. Thaenkham U , Nawa Y , Blair D , Pakdee W. Confirmation of the paraphyletic relationship between families Opisthorchiidae and Heterophyidae using small and large subunit ribosomal DNA sequences . Parasitol Int . 2011 ; 60 ( 4 ): 521 - 3 .
19. Heneberg P. Phylogenetic data suggest the reclassification of Fasciola jacksoni (Digenea: Fasciolidae) as Fascioloides jacksoni comb . nov. Parasitol Res . 2013 ; 112 ( 4 ): 1679 - 89 .
20. Briscoe AG , Bray RA , Brabec J , Littlewood DTJ . The mitochondrial genome and ribosomal operon of Brachycladium goliath (Digenea: Brachycladiidae) recovered from a stranded minke whale . Parasitol Int . 2016 ; 65 ( 3 ): 271 - 5 .
21. Le TH , Nguyen NT , Nguyen KT , Doan HT , Dung DT , Blair D. A complete mitochondrial genome from Echinochasmus japonicus supports the elevation of Echinochasminae Odhner, 1910 to family rank (Trematoda: Platyhelminthes) . Infect Genet Evol . 2016 ; 45 : 369 - 77 .
22. Athokpam VD , Jyrwa DB , Tandon V. Utilizing ribosomal DNA gene marker regions to characterize the metacercariae (Trematoda: Digenea) parasitizing piscine intermediate hosts in Manipur, Northeast India . J Parasit Dis . 2016 ; 40 ( 2 ): 330 - 8 .
23. Hillis DM , Dixon MT . Ribosomal DNA: molecular evolution and phylogenetic inference . Q Rev Biol . 1991 ; 66 : 411 - 53 .
24. Weider LJ , Elser JJ , Crease TJ , Mateos M , Cotner JB , Markow TA . The functional significance of ribosomal rDNA variation: impacts on the evolutionary ecology of organisms . Annu Rev Ecol Evol Syst . 2005 ; 36 : 219 - 42 .
25. Lockyer AE , Olson PD , Ostergaard P , Rollinson D , Johnston DA , Attwood SW , et al. The phylogeny of the Schistosomatidae based on three genes with emphasis on the interrelationships of Schistosoma Weinland , 1858 . Parasitology. 2003 ; 126 ( 3 ): 203 - 24 .
26. Králová-Hromadová I , Spakulová M , Horácková E , Turceková L , Novobilský A , Beck R , et al. Sequence analysis of ribosomal and mitochondrial genes of the giant liver fluke Fascioloides magna (Trematoda: Fasciolidae): intraspecific variation and differentiation from Fasciola hepatica . J Parasitol . 2008 ; 94 ( 1 ): 58 - 67 .
27. Littlewood DTJ . Platyhelminth systematics and the emergence of new characters . Parasite . 2008 ; 15 : 333 - 41 .
28. Heneberg P , Sitko J , Bizos J. Integrative taxonomy of central European parasitic flatworms of the family Prosthogonimidae Lühe , 1909 (Trematoda: Plagiorchiida). Parasitol Int . 2015 ; 64 ( 5 ): 264 - 73 .
29. Fraija-Fernández N , Olson PD , Crespo EA , Raga JA , Aznar FJ , Fernández M. Independent host switching events by digenean parasites of cetaceans inferred from ribosomal DNA . Int J Parasitol . 2015 ; 45 ( 2-3 ): 167 - 73 .
30. Stanevičiūtė G , Stunžėnas V , Petkevičiūtė R. Phylogenetic relationships of some species of the family Echinostomatidae Odner , 1910 (Trematoda), inferred from nuclear rDNA sequences and karyological analysis . Comp Cytogenet . 2015 ; 9 ( 2 ): 257 - 70 .
31. Pornruseetairatn S , Kino H , Shimazu T , Nawa Y , Scholz T , Ruangsittichai J , et al. A molecular phylogeny of Asian species of the genus Metagonimus (Digenea)-small intestinal flukes-based on representative Japanese populations . Parasitol Res . 2016 ; 115 ( 3 ): 1123 - 30 .
32. Sato H , Ihara S , Inaba O , Une Y. Identification of Euryhelmis costaricensis metacercariae in the skin of Tohoku hynobiid salamanders (Hynobius lichenatus), northeastern Honshu, Japan . J Wildl Dis . 2010 ; 46 ( 3 ): 832 - 42 .
33. Zheng X , Chang QC , Zhang Y , Tian SQ , Lou Y , Duan H , et al. Characterization of the complete nuclear ribosomal DNA sequences of Paramphistomum cervi . Scientific World J . 2014 ; 751907 .
34. Thaenkham U , Blair D , Nawa Y , Waikagul J . Families Opisthorchiidae and Heterophyidae: are they distinct? Parasitol Int . 2012 ; 61 ( 1 ): 90 - 3 .
35. Tkach VV , Kudlai O , Kostadinova A. Molecular phylogeny and systematics of the Echinostomatoidea Looss , 1899 (Platyhelminthes: Digenea). Int J Parasitol . 2016 ; 46 : 171 - 85 .
36. Larkin MA , Blackshields G , Brown NP , Chenna R , McGettigan PA , McWilliam H , et al. Clustal W and Clustal X version 2 .0. Bioinformatics . 2007 ; 23 : 2947 - 8 .
37. Benson G . Tandem repeats finder: a program to analyze DNA sequences . Nucleic Acids Res . 1999 ; 27 : 573 - 80 .
38. Tamura K , Stecher G , Peterson D , Filipski A , Kumar S. MEGA6: molecular evolutionary genetic analysis version 6.0. Mol Biol Evol . 2013 ; 30 : 2725 - 9 .
39. Ronquist F , Teslenko M , van der Mark P , Ayres DL , Darling A , Hohna S , Larget B , Liu L , Suchard MA , Huelsenbeck JP . MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space . Syst Biol . 2012 ; 61 ( 3 ): 539 - 42 .
40. Tatonova YV , Chelomina GN , Besprosvannykh VV. Genetic diversity of nuclear ITS1-5.8S-ITS2 rDNA sequence in Clonorchis sinensis Cobbold, 1875 (Trematoda: Opisthorchidae) from the Russian Far East . Parasitol Int . 2012 ; 61 ( 4 ): 664 - 74 .
41. Pérez-Ponce DE , León G , García-Varela M , Pinacho-Pinacho CD , Sereno-Uribe AL , Poulin R. Species delimitation in trematodes using DNA sequences: MiddleAmerican Clinostomum as a case study . Parasitology . 2016 ; 143 ( 13 ): 1773 - 89 .
42. Choudhary K , Verma AK , Swaroop S , Agrawal A. A review on the molecular characterization of digenean parasites using molecular markers with special reference to ITS region . Helminthologia . 2015 ; 52 ( 3 ): 167 - 87 .
43. Johnston DA , Kane RA , Rollinson D. Small subunit (18S) ribosomal RNA gene divergence in the genus Schistosoma . Parasitology. 1993 ; 107 ( 2 ): 147 - 56 .
44. Fernandez M , Littlewood DT , Latorre A , Raga JA , Rollinson D. Phylogenetic relationships of the family Campulidae (Trematoda) based on 18S rRNA sequences . Parasitology . 1998 ; 117 ( 4 ): 383 - 91 .