The mitochondrial genome of Paragyrodactylus variegatus (Platyhelminthes: Monogenea): differences in major non-coding region and gene order compared to Gyrodactylus
© Ye et al.; licensee BioMed Central Ltd. 2014
Received: 6 May 2014
Accepted: 4 August 2014
Published: 17 August 2014
Paragyrodactylus Gvosdev and Martechov, 1953, a viviparous genus of ectoparasite within the Gyrodactylidae, contains three nominal species all of which infect Asian river loaches. The group is suspected to be a basal lineage within Gyrodactylus Nordmann, 1832 sensu lato although this remains unclear. Further molecular study, beyond characterization of the standard Internal Transcribed Spacer region, is needed to clarify the evolutionary relationships within the family and the placement of this genus.
The mitochondrial genome of Paragyrodactylus variegatus You, King, Ye and Cone, 2014 was amplified in six parts from a single worm, sequenced using primer walking, annotated and analyzed using bioinformatic tools.
The mitochondrial genome of P. variegatus is 14,517 bp, containing 12 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes and a major non-coding region (NCR). The overall A + T content of the mitochondrial genome is 76.3%, which is higher than all reported mitochondrial genomes of monogeneans. All of the 22 tRNAs have the typical cloverleaf secondary structure, except tRNACys, tRNASer1 and tRNASer2 that lack the dihydrouridine (DHU) arm. There are six domains (domain III is absent) and three domains in the inferred secondary structures of the large ribosomal subunit (rrnL) and small ribosomal subunit (rrnS), respectively. The NCR includes six 40 bp tandem repeat units and has the double identical poly-T stretches, stem-loop structure and some surrounding structure elements. The gene order (tRNAGln, tRNAMet and NCR) differs in arrangement compared to the mitochondrial genomes reported from Gyrodactylus spp.
The Duplication and Random Loss Model and Recombination Model together are the most plausible explanations for the variation in gene order. Both morphological characters and characteristics of the mitochondrial genome support Paragyrodactylus as a distinct genus from Gyrodactylus. Considering their specific distribution and known hosts, we believe that Paragyrodactylus is a relict freshwater lineage of viviparous monogenean isolated in the high plateaus of central Asia on closely related river loaches.
Gyrodactylids are widespread parasites of freshwater and marine fishes, typically inhabiting the skin and gills of their hosts. Their direct life-cycle and hyperviviparous method of reproduction facilitates rapid population growth. Some species are pathogenic to their host (e.g. Gyrodactylus salaris Malmberg, 1957)  and capable of causing high host mortality resulting in serious ecological and economical consequences . Over twenty genera and 400 species of gyrodactylids have been described , most of them being identified by comparative morphology of the opisthaptoral hard parts. This traditional approach for identification of gyrodactylids gives limited information for detailed phylogenetic analysis. Recently, the nuclear ribosomal DNA (rDNA) and the internal transcribed spacers (ITS) of rDNA have been incorporated into the molecular taxonomy of the group [4, 5]. In addition, mitochondrial markers (COI and COII) are also confirmed to be DNA barcoding for Gyrodactylus Nordmann, 1832 [6, 7]. But more polymorphic molecular markers suitable for different taxonomic categories are still needed for studying the taxonomy and phylogeny of these parasites.
Paragyrodactylus Gvosdev and Martechov, 1953 is a genus of Gyrodactylidae comprising three nominal species, Paragyrodactylus iliensis Gvosdev and Martechov, 1953 (=P. dogieli Osmanov, 1965), Paragyrodactylus barbatuli Ergens, 1970 and Paragyrodactylus variegatus You, King, Ye and Cone, 2014, all of which infect river loaches (Nemacheilidae) inhabiting streams in central Asia . The relationship between Paragyrodactylus and Gyrodactylus has been recently explored. Kritsky and Boeger reported the two genera had a close relationship based on morphological characters . Bakke et al. believed the complexity of the attachment apparatus separates Paragyrodactylus from Gyrodactylus and pondered whether these differences were fundamental or a local diversification within Gyrodactylus. Furthermore, You et al., using morphology and molecular data, presented the hypothesis that Paragyrodactylus was a relict freshwater lineage of viviparous monogeneans isolated in the high plateaus of central Asia on river loaches . The ambiguous relationship between Paragyrodactylus and Gyrodactylus emphasizes the need for further molecular study of these genera.
Due to its higher rate of base substitution, maternal inheritance, evolutionary conserved gene products and low recombination [10, 11], mitochondrial genomes provide powerful markers for phylogenetic analysis, biological identification and population studies. In addition, mitochondrial genomes can provide genome-level characters such as gene order for deep-level phylogenetic analysis [12, 13]. To date, the complete mitochondrial DNA sequences of only nine monogeneans are available, including three species of Gyrodactylus.
In the present study, the first mitochondrial genome for Paragyrodactylus, P. variegatus, is sequenced and characterized. We report on its genome organization, base composition, gene order, codon usage, ribosomal and transfer RNA gene features and major non-coding region. Additionally, we provide a preliminary comparison of the gene arrangement within both Paragyrodactylus and Gyrodactylus.
Specimen collection and DNA extraction
Specimens of P. variegatus were collected from the skin and fins of wild Homatula variegata (Dabry de Thiersant, 1874) in the Qinling Mountain region of central China. Upon capture the specimens were immediately preserved in 99% ethanol and stored at 4°C. The DNA from one parasite was extracted using a TIANamp Micro DNA Kit (Tiangen Biotech, Beijing, China) according to the manufacturer’s protocol.
PCR and sequencing
List of PCR primer combinations used to amplify the mitochondrial genome of Paragyrodactylus variegatus
Sequence(5′ – 3′)
Huyse et al. (2007) 
Huyse et al. (2007) 
Huyse et al. (2007) 
Huyse et al. (2007) 
Huyse et al. (2008) 
Contiguous sequence fragments were assembled using SeqMan (DNAStar) and Staden Package v1.7.0 . Protein-coding (PCGs) and ribosomal RNA (rRNA) genes were initially identified using BLAST (Basic Local Alignment Search Tool) searches on GenBank, then by alignment with the published mitochondrial genomes of Gyrodactylus derjavinoides Malmberg, Collins, Cunningham and Jalali, 2007 (GenBank no. EU293891), G. salaris (GenBank no. DQ988931) and Gyrodactylus thymalli Zitnan, 1960 (GenBank no. EF527269). The secondary structure of the two rRNA genes was determined mainly by comparison with the published rRNA secondary structures of Dugesia japonica Ichikawa and Kawakatsu, 1964 (GenBank no. NC_016439) . Protein-coding regions were translated with the echinoderm mitochondrial genetic code. The program tRNAscan-SE v1.21 (http://lowelab.ucsc.edu/tRNAscan-SE/) was used to identify transfer RNA (tRNA) genes and their structures , using the mito/chloroplast codon and setting the cove cutoff score to one. The tRNAs, which were not detected by tRNA scan-SE v1.21, were identified by comparing the sequence to Gyrodactylus[17, 18]. Tandem Repeat Finder v4.07 was used to identify tandem repeats in non-coding regions . The base composition, codon usage and genetic distance were calculated with MEGA v5.1 . The nonsynonymous (Ka)/synonymous (Ks) values were estimated by the KaKs_Calculator v1.2 with the MA method .
Genome organization, base composition and gene order
Base composition of the mitochondrial genome of Paragyrodactylus variegatus
A + T%
Major non-coding region (NCR)
The arrangement of rRNA and protein coding genes of P. variegatus is typical for gyrodactylids. However, the gene order of some tRNA genes is different: there are three tRNAs (tRNAGln, tRNAPhe, tRNAMet) between ND4 and the major non-coding region and five tRNAs (tRNATyr, tRNALeu1, tRNASer2, tRNALeu2, tRNAArg) between ND6 and ND5 in P. variegatus, while Gyrodactylus spp. have one tRNA (tRNAPhe) and seven tRNAs (tRNATyr, tRNALeu1, tRNAGln, tRNAMet, tRNASer2, tRNALeu2, tRNAArg) in the same location, respectively.
Protein coding genes and codon usage
The organization of the mitochondrial genome of Paragyrodactylus variegatus
Codon usage for the 12 mitochondrial proteins of Paragyrodactylus variegatus
Ribosomal and transfer RNA genes
The 22 tRNA genes of P. variegatus vary in length from 57 to 70 nucleotides. Sequences of tRNAIle and tRNAThr genes overlap with neighboring genes (Table 3). All of the 22 tRNAs have the typical cloverleaf secondary structure, except for tRNACys, tRNASer1 and tRNASer2 in which each have unpaired dihydrouridine (DHU) arm.
Synonymous and nonsynonymous substitutions and genetic distance
Characteristics of the mitochondrial genome
The mitochondrial genome of P. variegatus is 222 bp shorter than that of G. derjavinoides, but well within the length range of parasitic flatworms [22, 23]. Differing number and length of the major non-coding region is the main factor that contributes to this difference in genome size. The overall A + T content of P. variegatus is higher than that of all reported mitochondrial genomes of monogeneans. The average Ka/Ks values of genes encoding 3 subunits of cytochrome c oxidase and the cytochrome b subunit of cytochrome bc1 complex are lower than genes encoding subunits of the NADH dehydrogenase complex (with the exception of ND1), especially COI and Cytb genes. This feature demonstrates COI, COII, COIII and Cytb genes are more strongly effected by purifying selection pressure compared to subunits of the NADH dehydrogenase genes (except ND1), which is similar to the findings of Huyse et al.  for Gyrodactylus derjavinoides. The degree of functional constraints might be a reason for corresponding degree sequence variations of protein genes. The low Ka/Ks values and genetic distance of COI and Cytb genes also imply that both genes could be used as a useful marker for analyses at higher taxonomic levels. Although sizes of rrnL and rrnS are very similar among Gyrodactylus spp. and P. variegatus, the sequence similarities are not high. These discrepancies may reflect the variable helices or loops that exist in the rRNA structure.
The major non-coding region
The mitochondrial genome of P. variegatus includes one major non-coding region, which has been frequently observed in other invertebrates. It contains a high A + T content and tandem repeat sequences which could not be found in large non-coding regions (>500 bp) of the published mitochondrial genomes of monopisthocotyleans. We found that length and number of tandem repeat units are similar to those observed in Microcotyle sebastis Goto, 1894 , contradicting the study of Zhang et al. that reported the length and number of repeated motifs were different in the mitochondrial non-coding regions of monopisthocotylids and polyopisthocotylids.
A non-coding region with high A + T content and pertinent elements usually corresponds to the control region for replication and transcription initiation. In the major non-coding region of P. variegatus, we found identical patterns within part I and part II. The patterns have only two nucleotide modifications with 2.3% sequence discrepancy; however, the overall difference between the whole sequence of part I and part II is 18.3%. The highly conserved part of the non-coding region is believed to have a functional role. The patterns contain poly-T stretches, a stem-loop structure and some surrounding structure elements (A + T-rich segment and G[A]nT) (Figure 6) which are typical of control regions in insects [27–30]. Although typical control regions are not readily identifiable within the mitochondrial genome of flatworms , the predicted secondary structure, conserved element, repeat sequences and high A + T content of major non-coding region in P. variegatus implies that this region might play an important role in the initiation of replication and transcription.
In addition, through alignment of non-coding regions sequences between Gyrodactylus spp. and P. variegatus, we found some conserved motifs in each species with the overall similarity among them being 72.1%. The conserved motifs (>5 bp) mainly existed in the A + T-rich segment and G + A-rich segment. However, whether or not the conserved motifs are present in other species of Gyrodactylidae needs to be assessed with a broader taxon sample.
Gene arrangements and possible evolutionary mechanisms
The characteristics of the mitochondrial genome of P. variegatus are notably different from Gyrodactylus spp., including the gene order, which is similar to other monopisthocotylids. The overall average genetic distance between Paragyrodactylus and Gyrodactylus based on the rRNA and 12 protein coding genes is remarkably greater than within Gyrodactylus. All of these features support Paragyrodactylus as a distinct genus. Considering their specific distribution and hosts, we tend towards the view of You et al. that Paragyrodactylus is a relict freshwater lineage of viviparous monogenean isolated in the high plateaus of central Asia on closely related river loaches.
The authors would like to thank Dr. Yuan Huang (College of Life Science, Shaanxi Normal University) for assistance with the analytic software. This research was supported by the National Natural Science Foundation of China (31372158).
- Johnsen BO, Jensen AJ: The Gyrodactylus story in Norway. Aquaculture. 1991, 98: 289-302. 10.1016/0044-8486(91)90393-L.View ArticleGoogle Scholar
- Mo TA, Norheim K: The surveillance and control programme for Gyrodactylus salaris in Atlantic salmon and rainbow trout in Norway. Annual Report 2004. 2005, Oslo, Norway: National Veterinary Institute, 137-139.Google Scholar
- Bakke TA, Cable J, Harris PD: The biology of gyrodactylid monogeneans: the “Russian Doll-killers”. Adv Parasitol. 2007, 64: 161-376.View ArticlePubMedGoogle Scholar
- Ziętara MS, Huyse T, Lumme J, Volckaert FA: Deep divergence among subgenera of Gyrodactylus inferred from rDNA ITS region. Parasitology. 2002, 124: 39-52.PubMedGoogle Scholar
- Matejusová I, Gelnar M, Verneau O, Cunningham CO, Littlewood DTJ: Molecular phylogenetic analysis of the genus Gyrodactylus (Platyhelminthes : Monogenea) inferred from rDNA ITS region: subgenera versus species groups. Parasitology. 2003, 127: 603-611. 10.1017/S0031182003004098.View ArticlePubMedGoogle Scholar
- Vanhove MPM, Tessens B, Schoelinck C, Jondelius U, Littlewood DTJ, Artois T, Huyse T: Problematic barcoding in flatworms: A case-study on monogeneans and rhabdocoels (Platyhelminthes). Zookeys. 2013, 365: 355-379.View ArticlePubMedGoogle Scholar
- Bueno-Silva M, Boeger WA: Neotropical Monogenoidea. 58. Three new species of Gyrodactylus (Gyrodactylidae) from Scleromystax spp. (Callichthyidae) and the proposal of COII gene as an additional fragment for barcoding gyrodactylids. Folia Parasitol. 2014, 61: 213-222. 10.14411/fp.2014.028.View ArticlePubMedGoogle Scholar
- You P, King SD, Ye F, Cone DK: Paragyrodactylus variegatus n. sp. (Gyrodactylidae) from Homatula variegata (Dabry de Thiersant, 1874) (Nemacheilidae) in central China. J Parasitol. 2014, 100: 350-355. 10.1645/13-257.1.View ArticlePubMedGoogle Scholar
- Kritsky DC, Boeger WA: Phylogeny of the Gyrodactylidae and the phylogenetic status of Gyrodactylus Nordmann, 1832 (Platyhelminthes: Monogenoidea). Taxonomy, ecology and evolution of metazoan parasites (Livre hommage à Louis Euzet). Edited by: Tome II, Combes C, Jourdane J. 2003, Perpignan, France: Presses Universitaire Perpignan, 37-58.Google Scholar
- Elson JL, Lightowlers RN: Mitochondrial DNA clonality in the dock: can surveillance swing the case?. Trends Genet. 2006, 22: 603-607. 10.1016/j.tig.2006.09.004.View ArticlePubMedGoogle Scholar
- Gissi C, Iannelli F, Pesole G: Evolution of the mitochondrial genome of Metazoa as exemplified by comparison of congeneric species. Heredity. 2008, 101: 301-320. 10.1038/hdy.2008.62.View ArticlePubMedGoogle Scholar
- Lavrov DV, Lang BF: Poriferan mtDNA and animal phylogeny based on mitochondrial gene arrangements. Syst Biol. 2005, 54: 651-659. 10.1080/10635150500221044.View ArticlePubMedGoogle Scholar
- Boore JL, Brown WM: Big trees from little genomes: mitochondrial gene order as a phylogenetic tool. Curr Opin Genet Dev. 1998, 8: 668-674. 10.1016/S0959-437X(98)80035-X.View ArticlePubMedGoogle Scholar
- Staden R, Beal KF, Bonfield JK: The Staden package, 1998. Methods in Molecular Biology. Bioinformatics Methods and Protocols. Edited by: Misener S, Krawetz SA. 1998, Totowa, NJ: The Humana Press Inc, 115-130.Google Scholar
- Sakai M, Sakaizumi M: The complete mitochondrial genome of Dugesia japonica (Platyhelminthes; Order Tricladida). Zoolog Sci. 2012, 29: 672-680. 10.2108/zsj.29.672.View ArticlePubMedGoogle Scholar
- Lowe TM, Eddy SR: tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res. 1997, 25: 955-964. 10.1093/nar/25.5.0955.PubMed CentralView ArticlePubMedGoogle Scholar
- Huyse T, Plaisance L, Webster BL, Mo TA, Bakke TA, Bachmann L, Littlewood DTJ: The mitochondrial genome of Gyrodactylus salaris (Platyhelminthes: Monogenea), a pathogen of Atlantic salmon (Salmo salar). Parasitology. 2007, 134: 739-747. 10.1017/S0031182006002010.View ArticlePubMedGoogle Scholar
- Plaisance L, Huyse T, Littlewood DTJ, Bakke TA, Bachmann L: The complete mitochondrial DNA sequence of the monogenean Gyrodactylus thymalli (Platyhelminthes: Monogenea), a parasite of grayling (Thymallus thymallus). Mol Biochem Parasitol. 2007, 154: 190-194. 10.1016/j.molbiopara.2007.04.012.View ArticlePubMedGoogle Scholar
- Benson G: Tandem repeats finder: a program to analyze DNA sequences. Nucleic Acids Res. 1999, 27: 573-580. 10.1093/nar/27.2.573.PubMed CentralView ArticlePubMedGoogle Scholar
- Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S: MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol. 2011, 28: 2731-2739. 10.1093/molbev/msr121.PubMed CentralView ArticlePubMedGoogle Scholar
- Zhang Z, Li J, Zhao XQ, Wang J, Wong GK, Yu J: KaKs_Calculator: calculating Ka and Ks through model selection and model averaging. Genomics Proteomics Bioinformatics. 2006, 4: 259-263. 10.1016/S1672-0229(07)60007-2.View ArticlePubMedGoogle Scholar
- Le TH, Humair PF, Blair D, Agatsuma T, Littlewood DTJ, McManus DP: Mitochondrial gene content, arrangement and composition compared in African and Asian schistosomes. Mol Biochem Parasitol. 2001, 117: 61-71. 10.1016/S0166-6851(01)00330-9.View ArticlePubMedGoogle Scholar
- Le TH, Blair D, McManus DP: Mitochondrial genomes of parasitic flatworms. Trends Parasitol. 2002, 18: 206-213. 10.1016/S1471-4922(02)02252-3.View ArticlePubMedGoogle Scholar
- Huyse T, Buchmann K, Littlewood DTJ: The mitochondrial genome of Gyrodactylus derjavinoides (Platyhelminthes: Monogenea) - a mitogenomic approach for Gyrodactylus species and strain identification. Gene. 2008, 417: 27-34. 10.1016/j.gene.2008.03.008.View ArticlePubMedGoogle Scholar
- Park JK, Kim KH, Kang S, Kim W, Eom KS, Littlewood DTJ: A common origin of complex life cycles in parasitic flatworms: evidence from the complete mitochondrial genome of Microcotyle sebastis (Monogenea: Platyhelminthes). BMC Evol Biol. 2007, 7: 11-10.1186/1471-2148-7-11.PubMed CentralView ArticlePubMedGoogle Scholar
- Zhang J, Wu X, Xie M, Li A: The complete mitochondrial genome of Pseudochauhanea macrorchis (Monogenea: Chauhaneidae) revealed a highly repetitive region and a gene rearrangement hot spot in Polyopisthocotylea. Mol Biol Rep. 2012, 39: 8115-8125. 10.1007/s11033-012-1659-z.View ArticlePubMedGoogle Scholar
- Goddard JM, Wolstenholme DR: Origin and direction of replication in mitochondrial DNA molecules from the genus Drosophila. Nucleic Acids Res. 1980, 8: 741-757.PubMed CentralPubMedGoogle Scholar
- Zhang DX, Szymura JM, Hewitt GM: Evolution and structure conservation of the control region of insect mitochondrial DNA. J Mol Evol. 1995, 40: 382-391. 10.1007/BF00164024.View ArticlePubMedGoogle Scholar
- Zhang DX, Hewitt GM: Insect mitochondrial control region: a review of its structure, evolution and usefulness in evolutionary studies. Biochem Syst Ecol. 1997, 25: 99-120. 10.1016/S0305-1978(96)00042-7.View ArticleGoogle Scholar
- Wei SJ, Shi M, Chen XX, Sharkey MJ, Achterberg CV, Ye GY, He JH: New views on strand asymmetry in insect mitochondrial genomes. PLoS One. 2010, 5: e12708-10.1371/journal.pone.0012708.PubMed CentralView ArticlePubMedGoogle Scholar
- Moritz C, Brown WM: Tandem duplications in animal mitochondrial DNAs: variation in incidence and gene content among lizards. Proc Natl Acad Sci USA. 1987, 84: 7183-7187. 10.1073/pnas.84.20.7183.PubMed CentralView ArticlePubMedGoogle Scholar
- Boore JL: The duplication/random loss model for gene rearrangement exemplified by mitochondrial genomes of deuterostome animals. Computational biology series. Vol. 1. Dordrecht (The Netherlands). Edited by: Sankoff D, Nadeau J. 2000, Dordrecht, Boston, and London: Kluwer Academic Publishers, 133-147.Google Scholar
- Lavrov DV, Boore JL, Brown WM: Complete mtDNA sequences of two millipedes suggest a new model for mitochondrial gene rearrangement: duplication and nonrandom loss. Mol Biol Evol. 2002, 19: 163-169. 10.1093/oxfordjournals.molbev.a004068.View ArticlePubMedGoogle Scholar
- Dowton M, Campbell NJH: Intramitochondrial recombination - is it why some mitochondrial genes sleep around?. Trends Ecol Evol. 2001, 16: 269-271. 10.1016/S0169-5347(01)02182-6.View ArticlePubMedGoogle Scholar
- Thyagarajan B, Padua RA, Campbell C: Mammalian mitochondria possess homologous DNA recombination activity. J Biol Chem. 1996, 271: 27536-27543. 10.1074/jbc.271.44.27536.View ArticlePubMedGoogle Scholar
- Lunt DF, Hyman BC: Animal mitochondrial DNA recombination. Nature. 1997, 387: 247-10.1038/387247a0.View ArticlePubMedGoogle Scholar
- Rawson PD: Nonhomologous recombination between the large unassigned region of the male and female mitochondrial genomes in the mussel, Mytilus trossulus. J Mol Evol. 2005, 61: 717-732. 10.1007/s00239-004-0035-6.View ArticlePubMedGoogle Scholar
- Tsaousis AD, Martin DP, Ladoukakis ED, Posada D, Zouros E: Widespread recombination in published animal mtDNA sequences. Mol Biol Evol. 2005, 22: 925-933. 10.1093/molbev/msi084.View ArticlePubMedGoogle Scholar
- Chen WJ, Bu Y, Carapelli A, Dallai R, Li S, Yin WY, Luan YX: The mitochondrial genome of Sinentomon erythranum (Arthropoda: Hexapoda: Protura): an example of highly divergent evolution. BMC Evol Biol. 2011, 11: 246-10.1186/1471-2148-11-246.PubMed CentralView ArticlePubMedGoogle Scholar
- Kurabayashi A, Sumida M, Yonekawa H, Glaw F, Vences M, Hasegawa M: Phylogeny, recombination, and mechanisms of stepwise mitochondrial genome reorganization in mantellid frogs from Madagascar. Mol Biol Evol. 2008, 25: 874-891. 10.1093/molbev/msn031.View ArticlePubMedGoogle Scholar
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