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Complete sequence and characterization of the mitochondrial genome of Diphyllobothrium stemmacephalum, the type species of genus Diphyllobothrium (Cestoda: Diphyllobothriidae), using next generation sequencing
Accepted Manuscript Complete sequence and characterization of the mitochondrial genome of Diphyllobothrium stemmacephalum, the type species of genus Diphyllobothrium (Cestoda: Diphyllobothriidae), using next generation sequencing
Hiroshi Yamasaki, Shinji Izumiyama, Tomoyoshi Nozaki PII: DOI: Reference:
S1383-5769(17)30120-4 doi: 10.1016/j.parint.2017.06.005 PARINT 1689
To appear in:
Parasitology International
Received date: Revised date: Accepted date:
7 March 2017 14 May 2017 21 June 2017
Please cite this article as: Hiroshi Yamasaki, Shinji Izumiyama, Tomoyoshi Nozaki , Complete sequence and characterization of the mitochondrial genome of Diphyllobothrium stemmacephalum, the type species of genus Diphyllobothrium (Cestoda: Diphyllobothriidae), using next generation sequencing. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Parint(2017), doi: 10.1016/j.parint.2017.06.005
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Complete sequence and characterization of the mitochondrial genome of Diphyllobothrium stemmacephalum, the type species of genus Diphyllobothrium (Cestoda: Diphyllobothriidae), using next generation sequencing
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Hiroshi Yamasaki*, Shinji Izumiyama* and Tomoyoshi Nozaki
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Health, Labour and Welfare, Tokyo 162-8640, Japan
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Department of Parasitology, National Institute of Infectious Diseases, Ministry of
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Footnotes Abbreviations: ATP synthase subunit 6 and 8 genes, atp6, atp8; cytochrome b gene, cob; cytochrome c oxidase subunit 1-3 genes, cox1-cox3; NADH dehydrogenase subunit 1-6 genes, nad1-nad6; NADH dehydrogenase subunit 4L gene, nad4L;
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mitochondrial genome, mitogenome; transfer RNA, tRNA; ribosomal RNA, rRNA;
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non-coding regions 1 and 2, NCR1 and NCR2; nucleotide(s), nt; next generation
The complete mitochondrial genome sequence has been deposited in the DNA
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☆
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sequencing, NGS.
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Data Bank of Japan (DDBJ) under accession number AP017648.
*Corresponding authors: Tel.: +81 3 4582 2692; fax: +81 3 5285 1173. E-mail
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Competing interests
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addresses: [email protected] (H. Yamasaki), [email protected] (S. Izumiyama)
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The authors declare that there are no conflicts of interest.
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Abstract We first constructed and characterized the complete mitochondrial genome (mitogenome) sequence of Diphyllobothrium stemmacephalum, the type species of genus Diphyllobothrium, using next generation sequencing (NGS). The mitogenome
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of D. stemmacephalum was 13,716 bp, including 12 protein-coding genes, 22 tRNA
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genes, 2 rRNA genes and 2 longer intergenic non-coding regions, and has features
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common to mitogenomes of other cestodes. Although it has been accepted that tRNA for serine (trnS2(UCN)) in Platyhelminthes lacks a D arm, the trnS2(UCN) of D.
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stemmacephalum was predicted to have a paired D arm as in Diplogonoporus balaenopterae. The non-coding region 2 contained eight tandem repeat units (34
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nucleotides/unit). This study also corroborated that D. stemmacephalum is phylogenetically more closely related to Dip. balaenopterae than to
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Diphyllobothrium latum and Diphyllobothrium nihonkaiense. As demonstrated here, mitogenome sequence data obtained using NGS is useful for gaining a better
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understanding of the systematics, phylogeny and taxonomic revisions involving valuable specimens preserved in museums, universities or research institutes for
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which sequence data are not yet available, and also for making diagnoses based on clinical samples.
Keywords:
Diphyllobothrium stemmacephalum, Cestoda, Diphyllobothriidae, Complete mitochondrial genome, Next generation sequencing, Molecular phylogeny
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1. Introduction The family Diphyllobothriidae Lühe, 1910 (Platyhelminthes: Cestoda) consists of 13 [1], 15 [2] or 17 [3] genera including Diphyllobothrium Cobbold, 1858, Adenocephalus Nybelin, 1931, Diplogonoporus (Lönnberg, 1891) Lönnberg, 1892
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and Spirometra (Rudolphi, 1819) Mueller, 1937, which are representative genera
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containing species capable of infecting humans [1-4]. The genus Diphyllobothrium
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is a relatively large group of 37 species consisting of 36 species that are listed in the World Register of Marine Species [5] and the more recently described
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Diphyllobothrium fayi [6]. Fifteen of the species have been reported to be human-infecting species [7]; however, the taxonomic position of several species in
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genus Diphyllobothrium remains uncertain and requires revision using molecular tools [7-9]. We and others have clarified the taxonomic relationships of several
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species based on mitogenome and/or cox1 sequences and morphological markers: Diplogonoporus balaenopterae and Diplogonoporus grandis [10], Diphyllobothrium
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ursi and Diphyllobothrium dendriticum [11], Diphyllobothrium stemmacephalum and Diphyllobothrium yonagoense [12], and Diphyllobothrium hottai and
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Diphyllobothrium ditremum [13]. Diphyllobothrium stemmacephalum (syn. D. yonagoense [12]), the type species of the genus Diphyllobothrium, is a cestode species that parasitizes in dolphins and porpoises in the northern hemisphere [14], and most human cases of infection with this species have been reported in Japan [12]. However, despite being the type species of genus Diphyllobothrium, DNA sequence data of the species are very limited with sequence data available only for cox1 and nad3 [12], partial mitogenome [15] and the ribosomal operon and elongation factor 1-alpha gene
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[16,17]. It has been suggested that assignment of Diphyllobothrium latum and D. nihonkaiense to the same genus as D. stemmacephalum is questionable because of the distant genetic relationship between D. stemmacephalum, D. latum and D. nihonkaiense [10,12].
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On the other hand, nuclear genome data are becoming available for cestode
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species that are of medical and veterinary importance, e.g., D. latum [18],
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Spirometra erinaceieuropaei [18], Echinococcus canadensis [18], Echinococcus granulosus [18-20], Echinococcus multilocularis [18,19], Hymenolepis diminuta [18],
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Hymenolepis nana [18], Taenia asiatica [18,21], Taenia saginata [19,21] and Taenia solium [18,19,21]. Therefore, in this study, to reexamine the classification of genera
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in family Diphyllobothriidae and to understand the molecular-phylogenetic relationships between diphyllobothriidean and related cestode species , we
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constructed the complete mitogenome sequence of D. stemmacephalum for the first time, using the next-generation Illumina HiSeq platform for sequencing and
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characterized. The validity for the assignment of D. latum and D. nihonkaiense to
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genus Diphyllobothrium based on the mitogenome data is also discussed.
2. Materials and methods 2.1. Parasite material
A single adult tapeworm of D. stemmacephalum, which was naturally expelled from a 39-year-old Japanese man in 2015 and fixed in 70% ethanol, was used. The details are reported elsewhere [12]. The study was performed according to the protocol (No. 177) approved by the Ethics Committee of the National Institute of Infectious Diseases, Tokyo, Japan.
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2.2. DNA preparation and sequencing using NGS Genomic DNA was prepared from the ethanol-fixed proglottids (approximately 250 mg wet weight) using DNeasy Blood & Tissue kit (Qiagen, Hilden, Germany),
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and 10 g of DNA based on measurement by a Qubit® fluorometer (Invitrogen, Life
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Technologies, Carlsbad, CA, USA) and a Qubit® dsDNA high-sensitivity assay kit
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(Invitrogen, Thermo Fisher Scientific, Eugene, OR, USA) was used in NGS. Library construction and NGS on an Illumina HiSeq platform (Illumina Inc., San Diego, CA,
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USA) were commercially conducted by Eurofins Genomics (Tokyo, Japan). Two libraries with insert sizes of approximately 300 and 700 bp with different indexes
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were prepared and pooled together. Sequencing was performed on one lane of an Illumina Hiseq 2500 using 2 × 125 paired end reads. The sequence yields were 27
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and 12 Gb, respectively.
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2.3. Data assembly and annotation
CLC Genomics Workbench software (Qiagen) was used to evaluate the quality,
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adapter trimming and de novo assembly of the sequence reads. A contig was identified from the assembly of the mitogenome of D. stemmacephalum based on similarity to other cestodes found in BLAST searches. The ends of the contig were connected to make a circular form, and its continuous integrity was checked by read-mapping with trimmed reads. Each gene was annotated and verified by comparison to alignments of known diphyllobothriidean mitogenomes [10,22-27]. Annotation and prediction of 22 tRNA genes was performed using three different
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software packages: Arwen, ver. 1.2 [28], tRNAscan SE, ver. 1.21 [29] and CentroidFold [30].
2.4. Phylogenetic analysis
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Phylogenetic trees were constructed using the complete sequence of
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mitogenome and amino acid sequences deduced from 12 protein-coding genes of D.
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stemmacephalum and related species deposited in GenBank, including D. latum (AB269325, DQ985706), D. nihonkaiense (AB268585, EF429138), Dip.
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balaenopterae (AB425839, AB425840), S. erinaceieuropaei (KJ599680), Spirometra. decipiens (KJ599679) and T. solium (AB086256), using maximum likelihood
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algorithms with GTR+G+I and JTT+G+F models with the lowest Bayesian information criterion scores (MEGA 6, www.megasoftware.net), respectively.
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3. Results
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Clades were assessed by bootstrap resampling with 1,000 replicates.
3.1. Characterization of the mitogenome of D. stemmacephalum
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The complete mitogenome of D. stemmacephalum was a circular, double-stranded DNA of 13,716 bp in length (accession number: AP017648) (Fig. 1). The size was similar to those of diphyllobothriids such as Dip. balaenopterae [10], D. latum [22,23], D. nihonkaiense [22,24], S. erinaceieuropaei [25-27], S. decipiens [27] and cyclophyllids, including T. solium [31], T. asiatica [32], T. saginata [33], E. multilocularis [34], E. granulosus [35,36], H. diminuta [37] and H. nana [38], and it was longer than the shortest mitogenome of Taenia crassiceps of 13,503 bp, which lacks a long non-coding region [39], and shorter than the 14,459 bp
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mitogenome of Anoplocephala perfoliata, which has the longest NCR1(875 bp) [40]. The location and length of the annotated genes and two intergenic NCR1and NCR2 of the mitogenome of D. stemmacephalum are shown in Table 1. Twelve protein-coding genes (atp6, cob, cox1-cox3, nad1-nad4, nad4L, nad5 and nad6)
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which are involved in oxidative phosphorylation, 22 tRNA genes, 2 rRNA genes
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(rrns and rrnl) and NCR1 and NCR2 were encoded on one strand and were
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predicted to be transcribed unidirectionally. The atp8 encoded in most animal mitogenomes was missing in D. stemmacephalum as was the platyhelminth
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mitogenomes. Gene organization was also found to be identical to that of diphyllobothriidean [10, 22-27] and cyclophyllidean species [31-36,38,39,41]
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investigated to date, with the exception of cyclophyllids such as H. diminuta [37], A. perfoliata [40] and Dipylidium caninum [42] because of an order switch between
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trnS2(UCN) and trnL1(CUN). Gene overlaps were found between cox3 and trnH (1 nt), nad4L and nad4 (40 nt), trnQ and trnF (5 nt), trnF and trnM (5 nt), nad1 and trnN
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(1 nt), nad3 and trnS1(AGN) (26 nt), and cox1 and trnT (10 nt). Ten of the 12 protein-coding genes of D. stemmacephalum were identical in size
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to those of known diphyllobothriidean species; however nad3 (372 bp) and nad5 (1,578 bp) were slightly larger than the equivalent genes in other diphyllobothriidean species [10,22-27]. Eleven of 12 protein-coding genes were initiated with ATG as the start codon, whereas cox3 was predicted to be initiated with GTG (Table 1). Five (cob, cox1, nad1, nad4 and nad4L) and six genes (atp6, cox2, nad2, nad3, nad5 and nad6) were terminated with TAG and TAA stop codons, respectively. Cox3 was predicted to be terminated by an incomplete stop codon (T) because a complete TAA or TAG codon was not found downstream of trnH.
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The amino acid composition of protein-coding genes encoded in the mitogenome of D. stemmacephalum shared features common to those of known diphyllobothriidean [10,22-25,27] and cyclophyllidean tapeworms [31-42]. It is recognized that the nucleotide composition of protein-coding genes of metazoan
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mitogenomes is not random [43] and that biased codon usage avoiding C is
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prominent at the third position of synonymous codons [44]. In D. stemmacephalum,
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the proportion of T-rich codons with more than two in a triplet was 46.6%. The codon TTT for phenylalanine was the most frequent and accounted for 10.3% of a
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total of 3,360 codons. Consequently, bias toward T causes a richness of hydrophobic amino acids such as leucine (15.8%), serine (10.8%), valine (9.9%)
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and isoleucine (7.8%). The overall A+T content was as high as 68.3% and similar to the content in other cestode species.
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Twenty-two tRNA genes, ranging from 55-70 nt, were annotated using ARWEN (Table 1), and 20 of these were predicted to have the standard cloverleaf secondary
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structures with paired dihydrouridine (D) arms (data not shown). It is accepted that D arms are missing in trnR, trnS1(AGN) and trnS2(CUN) of flatworms
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[23-27,31-42], and D arms of trnR and trnS1(AGN) of D. stemmacephalum were absent as for many cestode species. However, our calculations using three different computer models predicted that trnS2(UCN) of D. stemmacephalum possesses a 3-paired D arm as does Dip. balaenopterae trnS2(UCN) (Fig. 2). rrns (730 bp) and rrnl (963 bp) are separated by trnC and are comparable in size to those in other cestode rRNA genes [10,22-27,31-42]. NCR1, located between trnY and trnL1, was 224 bp, and NCR2 (326 bp) was found between nad5 and trnG. These were located in the same positions as in most diphyllobothriid [10,22-27]
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and cyclophyllid species [31-36,38-42], with the exception of NCR1 of H. diminuta [37]. A+T content in NCR1 and NCR2 was 75.4% and 66.8%, respectively. NCR2 contained a unique eight tandem repeat unit consisting of 34 nt (ATTCTCTAAATTTGTTTGGGGATGTGTGTAGTAT) and a stem-loop secondary
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structure (data not shown).
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3.2. Molecular-phylogenetic analysis
Phylogenetic trees inferred from the complete nucleotide and amino acid
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sequences showed identical topologies and indicated that D. stemmacephalum is closely related to Dip. balaenopterae, which parasitizes cetaceans as the definitive
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host and is genetically distant from non-cetacean species such as D. latum and D.
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nihonkaiense with high bootstrap confidences (Fig. 3).
4. Discussion
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We report the complete mitogenome sequence of D. stemmacephalum constructed using NGS. The D. stemmacephalum mitogenome was found to share
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features with other cestode species, including length, gene organization and the predicted secondary structures of tRNAs, except for the predicted secondary structures of trnS2(UCN) and NCRs. The codon usage in the mitogenome has been determined in many platyhelminth species. Although ATG is found as the start codon, the GTG codon is sometimes also used. In fact, GTG is predicted for cox3 of the diphyllobothriid species [10,12,22-27]. In cyclophyllid species, the GTG start codon has been identified in atp6 and nad4 of T. saginata [33], cox2 and nad4L of E. multilocularis
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[34], cox2, nad1 and nad4L of E. granulosus [36], cox1 of H. diminuta [37], and atp6, cox3, nad3 and nad4 of T. crassiceps [38]. TAG and TAA act as stop codons but abbreviated stop codons, T or TA, have been found in metazoan mitogenomes [41,45]. The abbreviated T stop codon is identified in cox3 of D. stemmacephalum,
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as in the cases of Dip. balaenopterae [10]; cox3 and nad3 of D. nihonkaiense and D.
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latum [22], S. erinaceieuropaei [26], nad1 of T. solium [31], and cox1 of H. diminuta
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[37]. The truncated TA stop codon has also been annotated for nad1 of D. nihonkaiense and D. latum [22], and cox3 and nad2 of D. caninum [42]. These
posttranscriptional polyadenylation.
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incomplete stop codons are considered to be completed to TAA by
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Standard cloverleaf structures have been predicted in most metazoan mitochondrial tRNAs using computer modeling, except for trnR, trnS1(AGN) and
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trnS2(UCN) of all parasitic flatworms examined to date in which a paired D arm was predicted to be missing [22-27,31-42]. However, trnS2(UCN) of D. stemmacephalum
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and Dip. balaenopterae were predicted to have a paired D arm. In our calculation using the same prediction models, trnS2(UCN) in cestode species reported to date
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also been predicted to have a paired D arm. Thus, these results were completely unexpected. Le et al. [36] proposed two secondary structural models of trnT with and without a paired D arm in E. granulosus, and the structural difference was found to be caused by the overlap of the third nucleotide of the stop codon in cox1. However, this explanation is not applicable for trnS2(UCU) of D. stemmacephalum and Dip. balaenopterae. The secondary structural models of trnS2(UCU) of the parasitic nematode, Ascaris suum, as predicted by computer modeling and nuclear magnetic resonance analysis were different [46]. Thus, experimental studies for clarifying
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the tertiary structures of trnS2(UCN) of flatworms are necessary. NCR2 has a unique eight tandem repeat unit consisting of 34 nt. The size, sequence and number of tandem repeats varies among cestode species [10,22-27,31-42] and among geographical isolates within a single species [24],
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which may be useful for the discrimination of species and geographical populations.
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The AT-rich region is assumed to be associated with mitogenome replication in the
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fruit fly (Drosophila) [47] and animals [48], however its role in Plathyhelminthes is unknown.
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Phylogenetic tree analyses revealed that D. stemmacephalum is closely related to Dip. balaenopterae, which is parasitic in cetaceans as the definitive host, and is
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genetically distant from D. latum and D. nihonkaiense, which parasitize terrestrial mammals. As reported previously [10,12], it is quite questionable that D. latum and
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D. nihonkaiense be regarded as members of the same genus as D. stemmacephalum. The mitogenome data reported here provide a criterion for judging whether D.
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latum and D. nihonkaiense should be transferred to another genus or not. However, to evaluate the validity whether the genus should transferred, in addition to
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morphological and ecological aspects, comprehensive studies using more taxa that are currently assigned to genus Diphyllobothrium and related genera are necessary, along with conducting analyses with nuclear DNA markers [16,17,49,50]. We previously used as many as 14 to 28 paired primers to determine the complete cox1 sequences from cestode samples fixed in formalin [10,11] and formalin-fixed paraffin-embedded specimens [51] for which the genomic DNA had been degraded by formalin fixation. Although DNA samples from formalin-fixed specimens were not used for NGS analysis in this study, NGS has been successfully
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applied to obtaining genomic-scale DNA sequence data from formalin-fixed museum specimens [52] and clinical samples [53]. Since the nuclear and mitochondrial genome data of most Diphyllobothrium species are not yet known, genomic analysis using NGS will be useful not only for the evaluation of taxonomic
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relationships and systematic phylogeny for species not yet definitively classified
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and for valuable specimens preserved in museums, universities or research
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institutions, but also for studies of biogeography and genetic diversity.
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Acknowledgments
This study was supported by Grants-in-Aid for Research Program on Emerging
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and Re-emerging Infectious Diseases from Japan Agency for Medical Research and Development (AMED) to HY (15fk0108025h0502, 16fk0108309j0103), SI
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References
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(15fk0108025h0302, 16fk0108309j0303) and TN (15fk0108025h0002).
[1] World Register of Marine Species.
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http://www.marinespecies.org/aphia.php?p=taxdetails&id=104937 [2] H. Kamo, Guide to identification of diphyllobothriid cestodes. Tokyo: Gendai Kikaku; 1999. pp. 26-146. (in Japanese). [3] R. Kuchta, T. Scholz, J. Brabec, R.A. Bray. Suppression of the tapeworm order Pseudophyllidea (Platyhelminthes: Eucestoda) and the proposed of two new orders, Bothriocephalidea and Diphyllobothriidea. Int. J. Parasitol. 38 (2008) 49-55. [4] J.S. Hernández-Orts, R. Kuchta, T. Kuzmina, J. Brabec, T. Scholtz. High
ACCEPTED MANUSCRIPT
morphological plasticity and global geographical distribution of the Pacific human broad tapeworm Adenocephalus pacificus (syn. Diphyllobothrium pacificum): Molecular and morphological survey. Acta Trop. 149 (2015) 168-178. doi: 10.1016/j.actatropica.2015.05.017.
PT
[5] World Register of Marine Species.
RI
http://www.marinespecies.org/aphia.php?p=taxdetails&id=105012
SC
[6] R.L. Rausch. Diphyllobothrium fayi n. sp. (Cestoda: Diphyllobothriidae) from the Pacific walrus, Odobenus rosmarus divergens. Comp. Parasitol. 72 (2005)
NU
129-135.
[7] T. Scholz, R. Kuchta. Fish-borne, zoonotic cestodes (Diphyllobothrium and
MA
relatives) in cold climates: a never-ending story of neglected and (re)-emergent parasites. Food Waterborne Parasitol. 4 (2016) 23-38.
ED
doi:10.1016/jfawpar.2016.07.002.
tapeworm
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[8] T. Scholz, H.H. García, R. Kuchta, B. Wicht. Update on the human broad
(genus Diphyllobothrium), including clinical relevance. Clin. Microbiol. Rev. 22
AC C
(2009) 146–160. doi: 10.1128/CMR.00033-08. Review. [9] H. Yamasaki. Parasitic helminthiases (3): Cestodes. J. Antibact. Antifung. Agents. 41 (2013) 227-236. (In Japanese). [10] H. Yamasaki, H. Ohmae, T. Kuramochi. Complete mitochondrial genomes of Diplogonoporus balaenopterae and Diplogonoporus grandis (Cestoda: Diphyllobothriidae) and clarification of their taxonomic relationships. Parasitol. Int. 61 (2012) 260-266. doi: 10.1016/j.parint.2011.10.007. [11] H. Yamasaki, M. Muto, M. Yamada, N. Arizono, R.L. Rausch. Validity of the bear
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tapeworm Diphyllobothrium ursi (Cestoda: Diphyllobothriidae) based on morphological and molecular markers. J. Parasitol. 98 (2012) 1243-1247. doi: 10.1645/GE-3063.1. [12] H. Yamasaki, H. Kumazawa, Y. Sekikawa, R. Oda, I. Hongo, T. Tsuchida, et al..
PT
First confirmed human case of Diphyllobothrium stemmacephalum infection
RI
and molecular verification of the synonymy of Diphyllobothrium yonagoense
SC
with D. stemmacephalum (Cestoda: Diphyllobothriidea). Parasitol. Int. 65 (2016) 412-421. doi: 10.1016/j.parint.2016.06.003.
NU
[13] A. Banzai-Umehara, M. Suzuki, T. Akiyama, H.K. Ooi, Y. Kawakami. Invalidation of Diphyllobothrium hottai (Cestoda: Diphyllobothriidae) based on
MA
morphological and molecular phylogenetic analyses. Parasitol. Int. 65 (2016) 459-462. doi: 10.1016/j.parint.2016.06.011.
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[14] T.S. Cobbold. XII. Observations on entozoa, with notices of several new species, including
EP T
an account of two experiments in regard to the breeding of Taenia serrata and T. cucumerina. Trans. Linn. Soc. 22 (1858) 155–172.
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[15] A. Waeschenbach, B.L. Webster, D.T. Littlewood. Adding resolution to ordinal level relationships of tapeworms (Platyhelminthes: Cestoda) with large fragments of mtDNA. Mol. Phylogenet. Evol. 63 (2012) 834-847. doi: 10.1016/j.ympev.2012.02.020. [16] P.D. Olson, J.N. Caira. Evolution of the major lineages of tapeworms (Platyhelminthes: Cestoda) inferred from 18S ribosomal DNA and elongation factor-1alpha. J. Parasitol. 85 (1999) 1134-1159. [17] P.D. Olson, D.T. Littlewood, R.A. Bray, J. Mariaux. Interrelationships and
ACCEPTED MANUSCRIPT
evolution of the tapeworms (Platyhelminthes: Cestoda). Mol. Phylogenet. Evol. 19:443-467, 2001. [18] WormBase ParaSite. http://parasite.wormbase.org [19] I.J. Tsai, M. Zarowiecki, N. Holroyd, A. Garciarrubio, A. Sánchez-Flores, K.L.
PT
Brooks, et al. The genomes of four tapeworm species reveal adaptations to
RI
parasitism. Nature 496 (2013) 57-63. doi:10.1038/nature12031.
SC
[20] H.J. Zheng, W.B. Zhang, L. Zhang, Z.Z. Zhang, J. Li, G. Lu, et al. The genome of the hydatid tapeworm Echinococcus granulosus. Nat. Genet. 45 (2013) 1168-1175.
NU
doi:10.1038/ng.2757.
[21] S. Wang, S. Wang, Y.F. Luo, L.H. Xiao, X.N. Luo, S.H. Gao, et al. Comparative
MA
genomics reveals adaptive evolution of Asian tapeworm in switching to a new intermediate host. Nat. Commun. 7 (2016) 12845.
ED
doi:10.1038/ncommus2845.
[22] M. Nakao, A. Davaajav, H. Yamasaki, A. Ito. Mitochondrial genomes of the
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human broad tapeworms Diphyllobothrium latum and Diphyllobothrium nihonkaiense (Cestoda: Diphyllobothriidae). Parasitol. Res. 101 (2007)
AC C
233-236.
[23] J.K. Park, K.H. Kim, S. Kang, H.K. Jeon, J.H. Kim, D.T.J. Littlewood, et al. Characterization of the mitochondrial genome of Diphyllobothrium latum (Cestoda: Pseudophyllidea) – implications for the phylogeny of eucestodes. Parasitology 134 (2007) 749-759. [24] K.H. Kim, H.K. Jeon, S. Kang, T. Sultana, G.J. Kim, K.S. Eom, et al. Characterization of the complete mitochondrial genome of Diphyllobothrium nihonkaiense (Diphyllobothriidae: Cestoda), and development of molecular
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markers for differentiating fish tapeworms. Mol. Cells 23 (2007) 379-390. [25] G.H. Liu, C. Li, J.Y. Li, D.H. Zhou, R.C. Xiong, R.Q. Lin, et al. Characterization of the complete mitochondrial genome sequence of Spirometra erinaceieuropaei
[26] https://www.ncbi.nlm.nih.gov/nuccore/AB374543.1
PT
(Cestode: Diphyllobothriidae) from China. Int. J. Biol. Sci. 8 (2012) 640-649.
RI
[27] K.S. Eom, H. Park, D. Lee, S. Choe, K.H. Kim, H.K. Jeon. Mitochondrial genome
SC
sequences of Spirometra erinaceieuropaei and S. decipiens (Cestoda: Diphyllobothriidae). Korean J. Parasitol. 53 (2015) 455-463. doi:
NU
10.3347/kjp.2015.53.4.455.
[28] D. Laslett, B. Canbäck. ARWEN: a program to detect tRNA genes in metazoan
MA
mitochondrial nucleotide sequences. Bioinformatics 24 (2008) 172-175. [29] T.M. Lowe, S.R. Eddy. tRNAscan-SE: A program for improved detection of
ED
transfer RNA
genes in genomic sequence. Nucleic Acids Res. 25 (1997) 955-964.
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[30] K. Sato, M. Hamada, K. Asai, T. Mituyama. CENTROIDFOLD: a web server for RNA secondary structure prediction. Nucleic Acids Res. 37 (2016) w277-w280.
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doi: 10.1093/nar/gkp367. [31] M. Nakao, Y. Sako, A. Ito. The mitochondrial genome of the tapeworm Taenia solium: a finding of the abbreviated stop codon U. J. Parasitol. 89 (2003) 633-635.
[32] H.K. Jeon, K.H. Lee, K.H. Kim, U.W. Hwang, K.S. Eom. Complete sequence and structure of the mitochondrial genome of the human tapeworm, Taenia asiatica (Platyhelminthes; Cestoda). Parasitology 130 (2005) 717-726. [33]H.K. Jeon, K.H. Kim, K.S. Eom. Complete sequence of the mitochondrial genome
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of Taenia saginata: comparison with T. solium and T. asiatica. Parasitol. Int. 56 (2007) 243-246. [34] M. Nakao, N. Yokoyama, Y. Sako, M. Fukunaga, A. Ito. The complete mitochondrial DNA sequence of the cestode Echinococcus multilocularis
PT
(Cyclophyllidea: Taeniidae). Mitochondrion 1 (2002) 497-509.
RI
[35] M. Nakao, T. Yanagida, S. Konyaev, A. Lavikainen, V.A. Odnokurtsev, V.A. Zaikov,
SC
et al. Mitochondrial phylogeny of the genus Echinococcus (Cestoda: Taeniidae) with emphasis on relationships among Echinococcus canadensis genotype.
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Parasitology 140 (2013) 1625-1636. doi: 10.1017/S0031182013000565. [36] T.H. Le, M.S. Pearson, D. Blair, N. Dai, L.H. Zhang, D.P. McManus. Complete
MA
mitochondrial genomes confirm the distinctiveness of the horse-dog and sheep-dog strains of Echinococcus granulosus. Parasitology 124 (2002)
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97-112.
[37] M. von Nickisch-Rosenegk, W.M. Brown, J.L. Boore. Complete sequence of the
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mitochondrial genome of the tapeworm Hymenolepis diminuta: gene arrangements indicate that Platyhelminthes are Eutrochozoans. Mol. Biol. Evol.
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18 (2001) 721-730.
[38] T. Cheng, G.H. Liu, H.Q. Song, R.Q. Lin, X.Q. Zhu. The complete mitochondrial genome of the dwarf tapeworm Hymenolepis nana – a neglected zoonotic helminth. Parasitol. Res. 115 (2016) 1253-1262. [39] T.H. Le, D. Blair, T. Agatsuma, P.F. Humair, N.J.H. Campbell, M. Iwagami, et al. Phylogenies inferred from mitochondrial gene orders - a cautionary tale from the parasitic flatworms. Mol. Biol. Evol. 17 (2000) 1123-1125. [40] A. Guo. The complete mitochondrial genome of Anoplocephala perfoliata, the
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first representative for the family Anoplocephalidae. Parasit. Vectors 8(2015)549. [41] T.H. Le, D. Blair, D.P. McManus. Mitochondrial genomes of parasitic flatworms. Trends Parasitol. 18 (2002) 206-213.
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[42] M. Nakao, A. Lavikainen, T. Iwaki, V. Haukisalmi, S. Konyaev, Y. Oku, et al.
RI
Molecular phylogeny of the genus Taenia (Cestoda: Taeniidae) proposals for
SC
the resurrection of Hydatigera Lamarck, 1816 and the creation of a new genus Versteria. Int. J. Parasitol. 43 (2013) 427-437. doi:
NU
10.1016/j.ijpara.2012.11.014.
[43] D. Ojala, J. Montoya, G. Attardi. tRNA punctuation model of RNA processing in
MA
human mitochondria. Nature 290 (1981) 470-474. [44] C. Saccone, C. Gissi, A. Reyes, A. Larizza, E. Sbisà, G. Pesole. Mitochondrial DNA
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in metazoa: degree of freedom in a frozen event. Gene 286 (2002) 3-12. [45] A. Scouras, M.J. Smith. A novel mitochondrial gene order in the crinoid
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echinoderm Florometra serratissima. Mol. Biol. Evol. 18 (2001) 61-73. [46] T. Ohtsuki, G. Kawai, K. Watanabe. The minimal tRNA: unique structure of
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Ascaris sum mitochondrial tRNASerUCU having a short T arm and lacking the entire D arm. FEBS Letters 514 (2002) 37-43. [47] S. Saito, K. Tamura, T. Aotuka. Replication origin of mitochondrial DNA in insects. Genetics 171 (2005) 1695-1705. [48] D.R. Wolstenholme. Animal mitochondrial DNA: structure and evolution. Int. Rev. Cytol. 141 (1992) 173-216. [49] F.J. Logan, A. Horák, J. Štefka, A. Aydogdu, T. Scholz. The phylogeny of diphyllobothriid tapeworms (Cestoda: Pseudophyllidea) based on ITS-2 rDNA
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sequences. Parasitol. Res. 94 (2004) 10-15. doi:10.1007/s00436-004-1164-y. [50] J. Brabec, R. Kucha, T. Scholz. Paraphyly of the Pseudophyllidea (Platyhelminthes: Cestoda): circumscription of monophyletic clades based on phylogenetic analysis of ribosomal RNA. Int. J. Parasitol. 36 (2006) 1535-1541.
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[51] H. Yamasaki, K. Nakaya, P.M. Schantz, A. Ito. Genetic analysis of Echinococcus
RI
multilocularis originating from a patient with alveolar echinococcosis
SC
occurring in Minnesota in 1977. Am. J. Trop. Med. Hyg. 79 (2008) 245-247. [52] S.M. Hykin, K. Bi, J.A. McGuire. Fixing formalin: a method to recover
NU
genomic-scale DNA
sequence data from formalin-fixed museum specimens using high-throughput
MA
sequencing. PLoS One 10 (2015) e0141579. doi: 10.1371/journal.pone.0141579.
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[53] H.M. Bennett, H.P. Mok, E. Gkrania-Klotsas, I.J. Tsai, E.J. Stanley, N.M. Antoun, et al. The genome of the sparganosis tapeworm Spirometra erinaceieuropaei
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isolated from the biopsy of a migrating brain lesion. Genome Biol. 15 (2014)
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510. doi:10.1186/PREACCEPT-2413673241432389.
Figure Legends
Fig. 1. Complete mitochondrial genome of D. stemmacephalum. Protein coding genes, rRNA genes and NCRs are indicated by white, gray and black arrows, respectively, with direction showing transcriptional orientation. tRNA genes are designated by squares with single letter amino acid codes.
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Fig. 2. Predicted secondary structures of trnS2(UCN) of D. stemmacephalum and Dip. balaenopterae. D. stemmacephalum (A-C) and Dip. balaenopterae (D-F) using ARWEN (A,D), tRNAscan SE (B,E) and CentroidFold (C,F). The trnS2(UCN) of Dip. balaenopterae is predicted based on the annotation in accession number
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AB225839.
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Fig. 3. Phylogenetic trees inferred from the complete nucleotide (A) and amino acid sequences (B) deduced from 12 protein-coding genes of D. stemmacephalum
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and related taxa. Trees A and B were constructed using maximum likelihood algorithms using the best models of GTR+G+I and JTT+G+F, respectively. Bootstrap
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values (1,000 replicates) are shown at the branches. T. solium was used as the
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outgroup. Bars indicate the numbers of base and amino acid substitutions/site.
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Figure 1
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Figure 2
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Figure 3
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Table 1. Location and length of all genes and two non-coding regions annotated in the mitochondrial genome of D. stemmacephalum Codons Length (bp)
1-65
65
66-289
224
trnL1(CUN)
290-356
67
trnS2(UCN)
366-435
70
trnL2(UUR)
438-501
64
trnR
502-556
55
nad5
560-2128
1578
NCR2
2129-2453
326
trnG
2454-2520
67
cox3
2523-3165
643
trnH
3165-3230
66
cob
3233-4339
1107
nad4L
4341-4601
261
nad4
4562-5812
trnQ
5812-5876
trnF
5872-5937
66
trnM
5933-5998
66
atp6
6002-6511
NCR1
Start
Stop
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trnY
amino acids
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Locations
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Genes
TAA
GTG
T*
368
ATG
TAG
86
ATG
TAG
416
ATG
TAG
510
169
ATG
TAA
6514-7392
879
292
ATG
TAA
7394-7460
67
7462-7524
63
7528-7589
62
nad1
7590-8480
891
296
ATG
TAG
trnN
8480-8544
65
trnP
8557-8621
65
trnI
8630-8694
65
trnK
8700-8764
65
nad3
8765-9136
372
123
ATG
TAA
trnA trnD
214
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trnV
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ATG
nad2
525
65
9111-9169
59
trnW
9171-9232
62
cox1
9241-10806
1566
trnT
10797-10858
62
rrnl
10859-11821
963
trnC
11822-11885
64
rrns
11886-12616
730
cox2
12617-13186
570
trnE
13187-13250
64
nad6
13255-13713
459
ATG
189
ATG
TAA
152
TAA
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ATG
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stop codon
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*abbreviated
521
TAG
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trnS1(AGN)
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Graphical abstract
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Highlights
Complete mitogenome sequence of D. stemmacephalum was first determined using NGS.
Secondary structure of trnS2(UCN) was predicted to have a D arm in this
D. stemmacephalum was phylogenetically closely related to Di.
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species.
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Basic data for revision of diphyllobothriid taxonomy were provided.
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balaenopterae.
Hiroshi Yamasaki, Shinji Izumiyama, Tomoyoshi Nozaki PII: DOI: Reference:
S1383-5769(17)30120-4 doi: 10.1016/j.parint.2017.06.005 PARINT 1689
To appear in:
Parasitology International
Received date: Revised date: Accepted date:
7 March 2017 14 May 2017 21 June 2017
Please cite this article as: Hiroshi Yamasaki, Shinji Izumiyama, Tomoyoshi Nozaki , Complete sequence and characterization of the mitochondrial genome of Diphyllobothrium stemmacephalum, the type species of genus Diphyllobothrium (Cestoda: Diphyllobothriidae), using next generation sequencing. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Parint(2017), doi: 10.1016/j.parint.2017.06.005
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Complete sequence and characterization of the mitochondrial genome of Diphyllobothrium stemmacephalum, the type species of genus Diphyllobothrium (Cestoda: Diphyllobothriidae), using next generation sequencing
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Hiroshi Yamasaki*, Shinji Izumiyama* and Tomoyoshi Nozaki
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Health, Labour and Welfare, Tokyo 162-8640, Japan
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Department of Parasitology, National Institute of Infectious Diseases, Ministry of
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Footnotes Abbreviations: ATP synthase subunit 6 and 8 genes, atp6, atp8; cytochrome b gene, cob; cytochrome c oxidase subunit 1-3 genes, cox1-cox3; NADH dehydrogenase subunit 1-6 genes, nad1-nad6; NADH dehydrogenase subunit 4L gene, nad4L;
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mitochondrial genome, mitogenome; transfer RNA, tRNA; ribosomal RNA, rRNA;
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non-coding regions 1 and 2, NCR1 and NCR2; nucleotide(s), nt; next generation
The complete mitochondrial genome sequence has been deposited in the DNA
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☆
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sequencing, NGS.
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Data Bank of Japan (DDBJ) under accession number AP017648.
*Corresponding authors: Tel.: +81 3 4582 2692; fax: +81 3 5285 1173. E-mail
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Competing interests
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addresses: [email protected] (H. Yamasaki), [email protected] (S. Izumiyama)
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The authors declare that there are no conflicts of interest.
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Abstract We first constructed and characterized the complete mitochondrial genome (mitogenome) sequence of Diphyllobothrium stemmacephalum, the type species of genus Diphyllobothrium, using next generation sequencing (NGS). The mitogenome
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of D. stemmacephalum was 13,716 bp, including 12 protein-coding genes, 22 tRNA
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genes, 2 rRNA genes and 2 longer intergenic non-coding regions, and has features
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common to mitogenomes of other cestodes. Although it has been accepted that tRNA for serine (trnS2(UCN)) in Platyhelminthes lacks a D arm, the trnS2(UCN) of D.
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stemmacephalum was predicted to have a paired D arm as in Diplogonoporus balaenopterae. The non-coding region 2 contained eight tandem repeat units (34
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nucleotides/unit). This study also corroborated that D. stemmacephalum is phylogenetically more closely related to Dip. balaenopterae than to
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Diphyllobothrium latum and Diphyllobothrium nihonkaiense. As demonstrated here, mitogenome sequence data obtained using NGS is useful for gaining a better
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understanding of the systematics, phylogeny and taxonomic revisions involving valuable specimens preserved in museums, universities or research institutes for
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which sequence data are not yet available, and also for making diagnoses based on clinical samples.
Keywords:
Diphyllobothrium stemmacephalum, Cestoda, Diphyllobothriidae, Complete mitochondrial genome, Next generation sequencing, Molecular phylogeny
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1. Introduction The family Diphyllobothriidae Lühe, 1910 (Platyhelminthes: Cestoda) consists of 13 [1], 15 [2] or 17 [3] genera including Diphyllobothrium Cobbold, 1858, Adenocephalus Nybelin, 1931, Diplogonoporus (Lönnberg, 1891) Lönnberg, 1892
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and Spirometra (Rudolphi, 1819) Mueller, 1937, which are representative genera
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containing species capable of infecting humans [1-4]. The genus Diphyllobothrium
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is a relatively large group of 37 species consisting of 36 species that are listed in the World Register of Marine Species [5] and the more recently described
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Diphyllobothrium fayi [6]. Fifteen of the species have been reported to be human-infecting species [7]; however, the taxonomic position of several species in
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genus Diphyllobothrium remains uncertain and requires revision using molecular tools [7-9]. We and others have clarified the taxonomic relationships of several
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species based on mitogenome and/or cox1 sequences and morphological markers: Diplogonoporus balaenopterae and Diplogonoporus grandis [10], Diphyllobothrium
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ursi and Diphyllobothrium dendriticum [11], Diphyllobothrium stemmacephalum and Diphyllobothrium yonagoense [12], and Diphyllobothrium hottai and
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Diphyllobothrium ditremum [13]. Diphyllobothrium stemmacephalum (syn. D. yonagoense [12]), the type species of the genus Diphyllobothrium, is a cestode species that parasitizes in dolphins and porpoises in the northern hemisphere [14], and most human cases of infection with this species have been reported in Japan [12]. However, despite being the type species of genus Diphyllobothrium, DNA sequence data of the species are very limited with sequence data available only for cox1 and nad3 [12], partial mitogenome [15] and the ribosomal operon and elongation factor 1-alpha gene
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[16,17]. It has been suggested that assignment of Diphyllobothrium latum and D. nihonkaiense to the same genus as D. stemmacephalum is questionable because of the distant genetic relationship between D. stemmacephalum, D. latum and D. nihonkaiense [10,12].
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On the other hand, nuclear genome data are becoming available for cestode
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species that are of medical and veterinary importance, e.g., D. latum [18],
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Spirometra erinaceieuropaei [18], Echinococcus canadensis [18], Echinococcus granulosus [18-20], Echinococcus multilocularis [18,19], Hymenolepis diminuta [18],
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Hymenolepis nana [18], Taenia asiatica [18,21], Taenia saginata [19,21] and Taenia solium [18,19,21]. Therefore, in this study, to reexamine the classification of genera
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in family Diphyllobothriidae and to understand the molecular-phylogenetic relationships between diphyllobothriidean and related cestode species , we
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constructed the complete mitogenome sequence of D. stemmacephalum for the first time, using the next-generation Illumina HiSeq platform for sequencing and
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characterized. The validity for the assignment of D. latum and D. nihonkaiense to
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genus Diphyllobothrium based on the mitogenome data is also discussed.
2. Materials and methods 2.1. Parasite material
A single adult tapeworm of D. stemmacephalum, which was naturally expelled from a 39-year-old Japanese man in 2015 and fixed in 70% ethanol, was used. The details are reported elsewhere [12]. The study was performed according to the protocol (No. 177) approved by the Ethics Committee of the National Institute of Infectious Diseases, Tokyo, Japan.
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2.2. DNA preparation and sequencing using NGS Genomic DNA was prepared from the ethanol-fixed proglottids (approximately 250 mg wet weight) using DNeasy Blood & Tissue kit (Qiagen, Hilden, Germany),
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and 10 g of DNA based on measurement by a Qubit® fluorometer (Invitrogen, Life
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Technologies, Carlsbad, CA, USA) and a Qubit® dsDNA high-sensitivity assay kit
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(Invitrogen, Thermo Fisher Scientific, Eugene, OR, USA) was used in NGS. Library construction and NGS on an Illumina HiSeq platform (Illumina Inc., San Diego, CA,
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USA) were commercially conducted by Eurofins Genomics (Tokyo, Japan). Two libraries with insert sizes of approximately 300 and 700 bp with different indexes
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were prepared and pooled together. Sequencing was performed on one lane of an Illumina Hiseq 2500 using 2 × 125 paired end reads. The sequence yields were 27
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and 12 Gb, respectively.
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2.3. Data assembly and annotation
CLC Genomics Workbench software (Qiagen) was used to evaluate the quality,
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adapter trimming and de novo assembly of the sequence reads. A contig was identified from the assembly of the mitogenome of D. stemmacephalum based on similarity to other cestodes found in BLAST searches. The ends of the contig were connected to make a circular form, and its continuous integrity was checked by read-mapping with trimmed reads. Each gene was annotated and verified by comparison to alignments of known diphyllobothriidean mitogenomes [10,22-27]. Annotation and prediction of 22 tRNA genes was performed using three different
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software packages: Arwen, ver. 1.2 [28], tRNAscan SE, ver. 1.21 [29] and CentroidFold [30].
2.4. Phylogenetic analysis
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Phylogenetic trees were constructed using the complete sequence of
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mitogenome and amino acid sequences deduced from 12 protein-coding genes of D.
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stemmacephalum and related species deposited in GenBank, including D. latum (AB269325, DQ985706), D. nihonkaiense (AB268585, EF429138), Dip.
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balaenopterae (AB425839, AB425840), S. erinaceieuropaei (KJ599680), Spirometra. decipiens (KJ599679) and T. solium (AB086256), using maximum likelihood
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algorithms with GTR+G+I and JTT+G+F models with the lowest Bayesian information criterion scores (MEGA 6, www.megasoftware.net), respectively.
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3. Results
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Clades were assessed by bootstrap resampling with 1,000 replicates.
3.1. Characterization of the mitogenome of D. stemmacephalum
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The complete mitogenome of D. stemmacephalum was a circular, double-stranded DNA of 13,716 bp in length (accession number: AP017648) (Fig. 1). The size was similar to those of diphyllobothriids such as Dip. balaenopterae [10], D. latum [22,23], D. nihonkaiense [22,24], S. erinaceieuropaei [25-27], S. decipiens [27] and cyclophyllids, including T. solium [31], T. asiatica [32], T. saginata [33], E. multilocularis [34], E. granulosus [35,36], H. diminuta [37] and H. nana [38], and it was longer than the shortest mitogenome of Taenia crassiceps of 13,503 bp, which lacks a long non-coding region [39], and shorter than the 14,459 bp
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mitogenome of Anoplocephala perfoliata, which has the longest NCR1(875 bp) [40]. The location and length of the annotated genes and two intergenic NCR1and NCR2 of the mitogenome of D. stemmacephalum are shown in Table 1. Twelve protein-coding genes (atp6, cob, cox1-cox3, nad1-nad4, nad4L, nad5 and nad6)
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which are involved in oxidative phosphorylation, 22 tRNA genes, 2 rRNA genes
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(rrns and rrnl) and NCR1 and NCR2 were encoded on one strand and were
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predicted to be transcribed unidirectionally. The atp8 encoded in most animal mitogenomes was missing in D. stemmacephalum as was the platyhelminth
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mitogenomes. Gene organization was also found to be identical to that of diphyllobothriidean [10, 22-27] and cyclophyllidean species [31-36,38,39,41]
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investigated to date, with the exception of cyclophyllids such as H. diminuta [37], A. perfoliata [40] and Dipylidium caninum [42] because of an order switch between
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trnS2(UCN) and trnL1(CUN). Gene overlaps were found between cox3 and trnH (1 nt), nad4L and nad4 (40 nt), trnQ and trnF (5 nt), trnF and trnM (5 nt), nad1 and trnN
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(1 nt), nad3 and trnS1(AGN) (26 nt), and cox1 and trnT (10 nt). Ten of the 12 protein-coding genes of D. stemmacephalum were identical in size
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to those of known diphyllobothriidean species; however nad3 (372 bp) and nad5 (1,578 bp) were slightly larger than the equivalent genes in other diphyllobothriidean species [10,22-27]. Eleven of 12 protein-coding genes were initiated with ATG as the start codon, whereas cox3 was predicted to be initiated with GTG (Table 1). Five (cob, cox1, nad1, nad4 and nad4L) and six genes (atp6, cox2, nad2, nad3, nad5 and nad6) were terminated with TAG and TAA stop codons, respectively. Cox3 was predicted to be terminated by an incomplete stop codon (T) because a complete TAA or TAG codon was not found downstream of trnH.
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The amino acid composition of protein-coding genes encoded in the mitogenome of D. stemmacephalum shared features common to those of known diphyllobothriidean [10,22-25,27] and cyclophyllidean tapeworms [31-42]. It is recognized that the nucleotide composition of protein-coding genes of metazoan
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mitogenomes is not random [43] and that biased codon usage avoiding C is
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prominent at the third position of synonymous codons [44]. In D. stemmacephalum,
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the proportion of T-rich codons with more than two in a triplet was 46.6%. The codon TTT for phenylalanine was the most frequent and accounted for 10.3% of a
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total of 3,360 codons. Consequently, bias toward T causes a richness of hydrophobic amino acids such as leucine (15.8%), serine (10.8%), valine (9.9%)
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and isoleucine (7.8%). The overall A+T content was as high as 68.3% and similar to the content in other cestode species.
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Twenty-two tRNA genes, ranging from 55-70 nt, were annotated using ARWEN (Table 1), and 20 of these were predicted to have the standard cloverleaf secondary
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structures with paired dihydrouridine (D) arms (data not shown). It is accepted that D arms are missing in trnR, trnS1(AGN) and trnS2(CUN) of flatworms
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[23-27,31-42], and D arms of trnR and trnS1(AGN) of D. stemmacephalum were absent as for many cestode species. However, our calculations using three different computer models predicted that trnS2(UCN) of D. stemmacephalum possesses a 3-paired D arm as does Dip. balaenopterae trnS2(UCN) (Fig. 2). rrns (730 bp) and rrnl (963 bp) are separated by trnC and are comparable in size to those in other cestode rRNA genes [10,22-27,31-42]. NCR1, located between trnY and trnL1, was 224 bp, and NCR2 (326 bp) was found between nad5 and trnG. These were located in the same positions as in most diphyllobothriid [10,22-27]
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and cyclophyllid species [31-36,38-42], with the exception of NCR1 of H. diminuta [37]. A+T content in NCR1 and NCR2 was 75.4% and 66.8%, respectively. NCR2 contained a unique eight tandem repeat unit consisting of 34 nt (ATTCTCTAAATTTGTTTGGGGATGTGTGTAGTAT) and a stem-loop secondary
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structure (data not shown).
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3.2. Molecular-phylogenetic analysis
Phylogenetic trees inferred from the complete nucleotide and amino acid
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sequences showed identical topologies and indicated that D. stemmacephalum is closely related to Dip. balaenopterae, which parasitizes cetaceans as the definitive
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host and is genetically distant from non-cetacean species such as D. latum and D.
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nihonkaiense with high bootstrap confidences (Fig. 3).
4. Discussion
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We report the complete mitogenome sequence of D. stemmacephalum constructed using NGS. The D. stemmacephalum mitogenome was found to share
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features with other cestode species, including length, gene organization and the predicted secondary structures of tRNAs, except for the predicted secondary structures of trnS2(UCN) and NCRs. The codon usage in the mitogenome has been determined in many platyhelminth species. Although ATG is found as the start codon, the GTG codon is sometimes also used. In fact, GTG is predicted for cox3 of the diphyllobothriid species [10,12,22-27]. In cyclophyllid species, the GTG start codon has been identified in atp6 and nad4 of T. saginata [33], cox2 and nad4L of E. multilocularis
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[34], cox2, nad1 and nad4L of E. granulosus [36], cox1 of H. diminuta [37], and atp6, cox3, nad3 and nad4 of T. crassiceps [38]. TAG and TAA act as stop codons but abbreviated stop codons, T or TA, have been found in metazoan mitogenomes [41,45]. The abbreviated T stop codon is identified in cox3 of D. stemmacephalum,
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as in the cases of Dip. balaenopterae [10]; cox3 and nad3 of D. nihonkaiense and D.
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latum [22], S. erinaceieuropaei [26], nad1 of T. solium [31], and cox1 of H. diminuta
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[37]. The truncated TA stop codon has also been annotated for nad1 of D. nihonkaiense and D. latum [22], and cox3 and nad2 of D. caninum [42]. These
posttranscriptional polyadenylation.
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incomplete stop codons are considered to be completed to TAA by
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Standard cloverleaf structures have been predicted in most metazoan mitochondrial tRNAs using computer modeling, except for trnR, trnS1(AGN) and
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trnS2(UCN) of all parasitic flatworms examined to date in which a paired D arm was predicted to be missing [22-27,31-42]. However, trnS2(UCN) of D. stemmacephalum
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and Dip. balaenopterae were predicted to have a paired D arm. In our calculation using the same prediction models, trnS2(UCN) in cestode species reported to date
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also been predicted to have a paired D arm. Thus, these results were completely unexpected. Le et al. [36] proposed two secondary structural models of trnT with and without a paired D arm in E. granulosus, and the structural difference was found to be caused by the overlap of the third nucleotide of the stop codon in cox1. However, this explanation is not applicable for trnS2(UCU) of D. stemmacephalum and Dip. balaenopterae. The secondary structural models of trnS2(UCU) of the parasitic nematode, Ascaris suum, as predicted by computer modeling and nuclear magnetic resonance analysis were different [46]. Thus, experimental studies for clarifying
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the tertiary structures of trnS2(UCN) of flatworms are necessary. NCR2 has a unique eight tandem repeat unit consisting of 34 nt. The size, sequence and number of tandem repeats varies among cestode species [10,22-27,31-42] and among geographical isolates within a single species [24],
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which may be useful for the discrimination of species and geographical populations.
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The AT-rich region is assumed to be associated with mitogenome replication in the
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fruit fly (Drosophila) [47] and animals [48], however its role in Plathyhelminthes is unknown.
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Phylogenetic tree analyses revealed that D. stemmacephalum is closely related to Dip. balaenopterae, which is parasitic in cetaceans as the definitive host, and is
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genetically distant from D. latum and D. nihonkaiense, which parasitize terrestrial mammals. As reported previously [10,12], it is quite questionable that D. latum and
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D. nihonkaiense be regarded as members of the same genus as D. stemmacephalum. The mitogenome data reported here provide a criterion for judging whether D.
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latum and D. nihonkaiense should be transferred to another genus or not. However, to evaluate the validity whether the genus should transferred, in addition to
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morphological and ecological aspects, comprehensive studies using more taxa that are currently assigned to genus Diphyllobothrium and related genera are necessary, along with conducting analyses with nuclear DNA markers [16,17,49,50]. We previously used as many as 14 to 28 paired primers to determine the complete cox1 sequences from cestode samples fixed in formalin [10,11] and formalin-fixed paraffin-embedded specimens [51] for which the genomic DNA had been degraded by formalin fixation. Although DNA samples from formalin-fixed specimens were not used for NGS analysis in this study, NGS has been successfully
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applied to obtaining genomic-scale DNA sequence data from formalin-fixed museum specimens [52] and clinical samples [53]. Since the nuclear and mitochondrial genome data of most Diphyllobothrium species are not yet known, genomic analysis using NGS will be useful not only for the evaluation of taxonomic
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relationships and systematic phylogeny for species not yet definitively classified
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and for valuable specimens preserved in museums, universities or research
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institutions, but also for studies of biogeography and genetic diversity.
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Acknowledgments
This study was supported by Grants-in-Aid for Research Program on Emerging
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and Re-emerging Infectious Diseases from Japan Agency for Medical Research and Development (AMED) to HY (15fk0108025h0502, 16fk0108309j0103), SI
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References
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(15fk0108025h0302, 16fk0108309j0303) and TN (15fk0108025h0002).
[1] World Register of Marine Species.
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http://www.marinespecies.org/aphia.php?p=taxdetails&id=104937 [2] H. Kamo, Guide to identification of diphyllobothriid cestodes. Tokyo: Gendai Kikaku; 1999. pp. 26-146. (in Japanese). [3] R. Kuchta, T. Scholz, J. Brabec, R.A. Bray. Suppression of the tapeworm order Pseudophyllidea (Platyhelminthes: Eucestoda) and the proposed of two new orders, Bothriocephalidea and Diphyllobothriidea. Int. J. Parasitol. 38 (2008) 49-55. [4] J.S. Hernández-Orts, R. Kuchta, T. Kuzmina, J. Brabec, T. Scholtz. High
ACCEPTED MANUSCRIPT
morphological plasticity and global geographical distribution of the Pacific human broad tapeworm Adenocephalus pacificus (syn. Diphyllobothrium pacificum): Molecular and morphological survey. Acta Trop. 149 (2015) 168-178. doi: 10.1016/j.actatropica.2015.05.017.
PT
[5] World Register of Marine Species.
RI
http://www.marinespecies.org/aphia.php?p=taxdetails&id=105012
SC
[6] R.L. Rausch. Diphyllobothrium fayi n. sp. (Cestoda: Diphyllobothriidae) from the Pacific walrus, Odobenus rosmarus divergens. Comp. Parasitol. 72 (2005)
NU
129-135.
[7] T. Scholz, R. Kuchta. Fish-borne, zoonotic cestodes (Diphyllobothrium and
MA
relatives) in cold climates: a never-ending story of neglected and (re)-emergent parasites. Food Waterborne Parasitol. 4 (2016) 23-38.
ED
doi:10.1016/jfawpar.2016.07.002.
tapeworm
EP T
[8] T. Scholz, H.H. García, R. Kuchta, B. Wicht. Update on the human broad
(genus Diphyllobothrium), including clinical relevance. Clin. Microbiol. Rev. 22
AC C
(2009) 146–160. doi: 10.1128/CMR.00033-08. Review. [9] H. Yamasaki. Parasitic helminthiases (3): Cestodes. J. Antibact. Antifung. Agents. 41 (2013) 227-236. (In Japanese). [10] H. Yamasaki, H. Ohmae, T. Kuramochi. Complete mitochondrial genomes of Diplogonoporus balaenopterae and Diplogonoporus grandis (Cestoda: Diphyllobothriidae) and clarification of their taxonomic relationships. Parasitol. Int. 61 (2012) 260-266. doi: 10.1016/j.parint.2011.10.007. [11] H. Yamasaki, M. Muto, M. Yamada, N. Arizono, R.L. Rausch. Validity of the bear
ACCEPTED MANUSCRIPT
tapeworm Diphyllobothrium ursi (Cestoda: Diphyllobothriidae) based on morphological and molecular markers. J. Parasitol. 98 (2012) 1243-1247. doi: 10.1645/GE-3063.1. [12] H. Yamasaki, H. Kumazawa, Y. Sekikawa, R. Oda, I. Hongo, T. Tsuchida, et al..
PT
First confirmed human case of Diphyllobothrium stemmacephalum infection
RI
and molecular verification of the synonymy of Diphyllobothrium yonagoense
SC
with D. stemmacephalum (Cestoda: Diphyllobothriidea). Parasitol. Int. 65 (2016) 412-421. doi: 10.1016/j.parint.2016.06.003.
NU
[13] A. Banzai-Umehara, M. Suzuki, T. Akiyama, H.K. Ooi, Y. Kawakami. Invalidation of Diphyllobothrium hottai (Cestoda: Diphyllobothriidae) based on
MA
morphological and molecular phylogenetic analyses. Parasitol. Int. 65 (2016) 459-462. doi: 10.1016/j.parint.2016.06.011.
ED
[14] T.S. Cobbold. XII. Observations on entozoa, with notices of several new species, including
EP T
an account of two experiments in regard to the breeding of Taenia serrata and T. cucumerina. Trans. Linn. Soc. 22 (1858) 155–172.
AC C
[15] A. Waeschenbach, B.L. Webster, D.T. Littlewood. Adding resolution to ordinal level relationships of tapeworms (Platyhelminthes: Cestoda) with large fragments of mtDNA. Mol. Phylogenet. Evol. 63 (2012) 834-847. doi: 10.1016/j.ympev.2012.02.020. [16] P.D. Olson, J.N. Caira. Evolution of the major lineages of tapeworms (Platyhelminthes: Cestoda) inferred from 18S ribosomal DNA and elongation factor-1alpha. J. Parasitol. 85 (1999) 1134-1159. [17] P.D. Olson, D.T. Littlewood, R.A. Bray, J. Mariaux. Interrelationships and
ACCEPTED MANUSCRIPT
evolution of the tapeworms (Platyhelminthes: Cestoda). Mol. Phylogenet. Evol. 19:443-467, 2001. [18] WormBase ParaSite. http://parasite.wormbase.org [19] I.J. Tsai, M. Zarowiecki, N. Holroyd, A. Garciarrubio, A. Sánchez-Flores, K.L.
PT
Brooks, et al. The genomes of four tapeworm species reveal adaptations to
RI
parasitism. Nature 496 (2013) 57-63. doi:10.1038/nature12031.
SC
[20] H.J. Zheng, W.B. Zhang, L. Zhang, Z.Z. Zhang, J. Li, G. Lu, et al. The genome of the hydatid tapeworm Echinococcus granulosus. Nat. Genet. 45 (2013) 1168-1175.
NU
doi:10.1038/ng.2757.
[21] S. Wang, S. Wang, Y.F. Luo, L.H. Xiao, X.N. Luo, S.H. Gao, et al. Comparative
MA
genomics reveals adaptive evolution of Asian tapeworm in switching to a new intermediate host. Nat. Commun. 7 (2016) 12845.
ED
doi:10.1038/ncommus2845.
[22] M. Nakao, A. Davaajav, H. Yamasaki, A. Ito. Mitochondrial genomes of the
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human broad tapeworms Diphyllobothrium latum and Diphyllobothrium nihonkaiense (Cestoda: Diphyllobothriidae). Parasitol. Res. 101 (2007)
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233-236.
[23] J.K. Park, K.H. Kim, S. Kang, H.K. Jeon, J.H. Kim, D.T.J. Littlewood, et al. Characterization of the mitochondrial genome of Diphyllobothrium latum (Cestoda: Pseudophyllidea) – implications for the phylogeny of eucestodes. Parasitology 134 (2007) 749-759. [24] K.H. Kim, H.K. Jeon, S. Kang, T. Sultana, G.J. Kim, K.S. Eom, et al. Characterization of the complete mitochondrial genome of Diphyllobothrium nihonkaiense (Diphyllobothriidae: Cestoda), and development of molecular
ACCEPTED MANUSCRIPT
markers for differentiating fish tapeworms. Mol. Cells 23 (2007) 379-390. [25] G.H. Liu, C. Li, J.Y. Li, D.H. Zhou, R.C. Xiong, R.Q. Lin, et al. Characterization of the complete mitochondrial genome sequence of Spirometra erinaceieuropaei
[26] https://www.ncbi.nlm.nih.gov/nuccore/AB374543.1
PT
(Cestode: Diphyllobothriidae) from China. Int. J. Biol. Sci. 8 (2012) 640-649.
RI
[27] K.S. Eom, H. Park, D. Lee, S. Choe, K.H. Kim, H.K. Jeon. Mitochondrial genome
SC
sequences of Spirometra erinaceieuropaei and S. decipiens (Cestoda: Diphyllobothriidae). Korean J. Parasitol. 53 (2015) 455-463. doi:
NU
10.3347/kjp.2015.53.4.455.
[28] D. Laslett, B. Canbäck. ARWEN: a program to detect tRNA genes in metazoan
MA
mitochondrial nucleotide sequences. Bioinformatics 24 (2008) 172-175. [29] T.M. Lowe, S.R. Eddy. tRNAscan-SE: A program for improved detection of
ED
transfer RNA
genes in genomic sequence. Nucleic Acids Res. 25 (1997) 955-964.
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[30] K. Sato, M. Hamada, K. Asai, T. Mituyama. CENTROIDFOLD: a web server for RNA secondary structure prediction. Nucleic Acids Res. 37 (2016) w277-w280.
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doi: 10.1093/nar/gkp367. [31] M. Nakao, Y. Sako, A. Ito. The mitochondrial genome of the tapeworm Taenia solium: a finding of the abbreviated stop codon U. J. Parasitol. 89 (2003) 633-635.
[32] H.K. Jeon, K.H. Lee, K.H. Kim, U.W. Hwang, K.S. Eom. Complete sequence and structure of the mitochondrial genome of the human tapeworm, Taenia asiatica (Platyhelminthes; Cestoda). Parasitology 130 (2005) 717-726. [33]H.K. Jeon, K.H. Kim, K.S. Eom. Complete sequence of the mitochondrial genome
ACCEPTED MANUSCRIPT
of Taenia saginata: comparison with T. solium and T. asiatica. Parasitol. Int. 56 (2007) 243-246. [34] M. Nakao, N. Yokoyama, Y. Sako, M. Fukunaga, A. Ito. The complete mitochondrial DNA sequence of the cestode Echinococcus multilocularis
PT
(Cyclophyllidea: Taeniidae). Mitochondrion 1 (2002) 497-509.
RI
[35] M. Nakao, T. Yanagida, S. Konyaev, A. Lavikainen, V.A. Odnokurtsev, V.A. Zaikov,
SC
et al. Mitochondrial phylogeny of the genus Echinococcus (Cestoda: Taeniidae) with emphasis on relationships among Echinococcus canadensis genotype.
NU
Parasitology 140 (2013) 1625-1636. doi: 10.1017/S0031182013000565. [36] T.H. Le, M.S. Pearson, D. Blair, N. Dai, L.H. Zhang, D.P. McManus. Complete
MA
mitochondrial genomes confirm the distinctiveness of the horse-dog and sheep-dog strains of Echinococcus granulosus. Parasitology 124 (2002)
ED
97-112.
[37] M. von Nickisch-Rosenegk, W.M. Brown, J.L. Boore. Complete sequence of the
EP T
mitochondrial genome of the tapeworm Hymenolepis diminuta: gene arrangements indicate that Platyhelminthes are Eutrochozoans. Mol. Biol. Evol.
AC C
18 (2001) 721-730.
[38] T. Cheng, G.H. Liu, H.Q. Song, R.Q. Lin, X.Q. Zhu. The complete mitochondrial genome of the dwarf tapeworm Hymenolepis nana – a neglected zoonotic helminth. Parasitol. Res. 115 (2016) 1253-1262. [39] T.H. Le, D. Blair, T. Agatsuma, P.F. Humair, N.J.H. Campbell, M. Iwagami, et al. Phylogenies inferred from mitochondrial gene orders - a cautionary tale from the parasitic flatworms. Mol. Biol. Evol. 17 (2000) 1123-1125. [40] A. Guo. The complete mitochondrial genome of Anoplocephala perfoliata, the
ACCEPTED MANUSCRIPT
first representative for the family Anoplocephalidae. Parasit. Vectors 8(2015)549. [41] T.H. Le, D. Blair, D.P. McManus. Mitochondrial genomes of parasitic flatworms. Trends Parasitol. 18 (2002) 206-213.
PT
[42] M. Nakao, A. Lavikainen, T. Iwaki, V. Haukisalmi, S. Konyaev, Y. Oku, et al.
RI
Molecular phylogeny of the genus Taenia (Cestoda: Taeniidae) proposals for
SC
the resurrection of Hydatigera Lamarck, 1816 and the creation of a new genus Versteria. Int. J. Parasitol. 43 (2013) 427-437. doi:
NU
10.1016/j.ijpara.2012.11.014.
[43] D. Ojala, J. Montoya, G. Attardi. tRNA punctuation model of RNA processing in
MA
human mitochondria. Nature 290 (1981) 470-474. [44] C. Saccone, C. Gissi, A. Reyes, A. Larizza, E. Sbisà, G. Pesole. Mitochondrial DNA
ED
in metazoa: degree of freedom in a frozen event. Gene 286 (2002) 3-12. [45] A. Scouras, M.J. Smith. A novel mitochondrial gene order in the crinoid
EP T
echinoderm Florometra serratissima. Mol. Biol. Evol. 18 (2001) 61-73. [46] T. Ohtsuki, G. Kawai, K. Watanabe. The minimal tRNA: unique structure of
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Ascaris sum mitochondrial tRNASerUCU having a short T arm and lacking the entire D arm. FEBS Letters 514 (2002) 37-43. [47] S. Saito, K. Tamura, T. Aotuka. Replication origin of mitochondrial DNA in insects. Genetics 171 (2005) 1695-1705. [48] D.R. Wolstenholme. Animal mitochondrial DNA: structure and evolution. Int. Rev. Cytol. 141 (1992) 173-216. [49] F.J. Logan, A. Horák, J. Štefka, A. Aydogdu, T. Scholz. The phylogeny of diphyllobothriid tapeworms (Cestoda: Pseudophyllidea) based on ITS-2 rDNA
ACCEPTED MANUSCRIPT
sequences. Parasitol. Res. 94 (2004) 10-15. doi:10.1007/s00436-004-1164-y. [50] J. Brabec, R. Kucha, T. Scholz. Paraphyly of the Pseudophyllidea (Platyhelminthes: Cestoda): circumscription of monophyletic clades based on phylogenetic analysis of ribosomal RNA. Int. J. Parasitol. 36 (2006) 1535-1541.
PT
[51] H. Yamasaki, K. Nakaya, P.M. Schantz, A. Ito. Genetic analysis of Echinococcus
RI
multilocularis originating from a patient with alveolar echinococcosis
SC
occurring in Minnesota in 1977. Am. J. Trop. Med. Hyg. 79 (2008) 245-247. [52] S.M. Hykin, K. Bi, J.A. McGuire. Fixing formalin: a method to recover
NU
genomic-scale DNA
sequence data from formalin-fixed museum specimens using high-throughput
MA
sequencing. PLoS One 10 (2015) e0141579. doi: 10.1371/journal.pone.0141579.
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[53] H.M. Bennett, H.P. Mok, E. Gkrania-Klotsas, I.J. Tsai, E.J. Stanley, N.M. Antoun, et al. The genome of the sparganosis tapeworm Spirometra erinaceieuropaei
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isolated from the biopsy of a migrating brain lesion. Genome Biol. 15 (2014)
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510. doi:10.1186/PREACCEPT-2413673241432389.
Figure Legends
Fig. 1. Complete mitochondrial genome of D. stemmacephalum. Protein coding genes, rRNA genes and NCRs are indicated by white, gray and black arrows, respectively, with direction showing transcriptional orientation. tRNA genes are designated by squares with single letter amino acid codes.
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Fig. 2. Predicted secondary structures of trnS2(UCN) of D. stemmacephalum and Dip. balaenopterae. D. stemmacephalum (A-C) and Dip. balaenopterae (D-F) using ARWEN (A,D), tRNAscan SE (B,E) and CentroidFold (C,F). The trnS2(UCN) of Dip. balaenopterae is predicted based on the annotation in accession number
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AB225839.
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Fig. 3. Phylogenetic trees inferred from the complete nucleotide (A) and amino acid sequences (B) deduced from 12 protein-coding genes of D. stemmacephalum
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and related taxa. Trees A and B were constructed using maximum likelihood algorithms using the best models of GTR+G+I and JTT+G+F, respectively. Bootstrap
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values (1,000 replicates) are shown at the branches. T. solium was used as the
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outgroup. Bars indicate the numbers of base and amino acid substitutions/site.
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Figure 1
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Figure 2
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Figure 3
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Table 1. Location and length of all genes and two non-coding regions annotated in the mitochondrial genome of D. stemmacephalum Codons Length (bp)
1-65
65
66-289
224
trnL1(CUN)
290-356
67
trnS2(UCN)
366-435
70
trnL2(UUR)
438-501
64
trnR
502-556
55
nad5
560-2128
1578
NCR2
2129-2453
326
trnG
2454-2520
67
cox3
2523-3165
643
trnH
3165-3230
66
cob
3233-4339
1107
nad4L
4341-4601
261
nad4
4562-5812
trnQ
5812-5876
trnF
5872-5937
66
trnM
5933-5998
66
atp6
6002-6511
NCR1
Start
Stop
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trnY
amino acids
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Locations
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Genes
TAA
GTG
T*
368
ATG
TAG
86
ATG
TAG
416
ATG
TAG
510
169
ATG
TAA
6514-7392
879
292
ATG
TAA
7394-7460
67
7462-7524
63
7528-7589
62
nad1
7590-8480
891
296
ATG
TAG
trnN
8480-8544
65
trnP
8557-8621
65
trnI
8630-8694
65
trnK
8700-8764
65
nad3
8765-9136
372
123
ATG
TAA
trnA trnD
214
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EP T
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trnV
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ATG
nad2
525
65
9111-9169
59
trnW
9171-9232
62
cox1
9241-10806
1566
trnT
10797-10858
62
rrnl
10859-11821
963
trnC
11822-11885
64
rrns
11886-12616
730
cox2
12617-13186
570
trnE
13187-13250
64
nad6
13255-13713
459
ATG
189
ATG
TAA
152
TAA
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ATG
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stop codon
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*abbreviated
521
TAG
PT
trnS1(AGN)
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Graphical abstract
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Highlights
Complete mitogenome sequence of D. stemmacephalum was first determined using NGS.
Secondary structure of trnS2(UCN) was predicted to have a D arm in this
D. stemmacephalum was phylogenetically closely related to Di.
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species.
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Basic data for revision of diphyllobothriid taxonomy were provided.
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balaenopterae.