Abstract
Passalurus ambiguus (Nematda: Oxyuridae) is a common pinworm which parasitizes in the caecum and colon of rabbits. Despite its significance as a pathogen, the epidemiology, genetics, systematics, and biology of this pinworm remain poorly understood. In the present study, we sequenced the complete mitochondrial (mt) genome of P. ambiguus. The circular mt genome is 14,023 bp in size and encodes of 36 genes, including 12 protein-coding, two ribosomal RNA, and 22 transfer RNA genes. The mt gene order of P. ambiguus is the same as that of Wellcomia siamensis, but distinct from that of Enterobius vermicularis. Phylogenetic analyses based on concatenated amino acid sequences of 12 protein-coding genes by Bayesian inference (BI) showed that P. ambiguus was more closely related to W. siamensis than to E. vermicularis. This mt genome provides novel genetic markers for studying the molecular epidemiology, population genetics, systematics of pinworm of animals and humans, and should have implications for the diagnosis, prevention, and control of passaluriasis in rabbits and other animals.
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Introduction
Members of the infraorder Oxyuridomorpha, known as the “pinworms,” parasitize in the caecum and colon of animals and humans, causing significant public health problems and major economic losses (Arkoulis et al. 2012; Dufour et al. 2015). Passalurus ambiguus (Rudolphi, 1819), the so-called rabbit pinworm, infects domestic and wild rabbits, and its distribution is worldwide (Frank et al. 2013; Szkucik et al. 2014). P. ambiguus is a common helminth of rabbits. Although very large numbers of pinworms were found in rabbits, it is not generally thought to be very pathogenic (Rinaldi et al. 2007).
Most metazoan species mitochondrial (mt) genome possesses a circular double-strand DNA, which varies in size from 14 to 20 kb (Wolstenholme 1992). Metazoan mt genomes contain 36–37 genes, including 12–13 protein-coding genes, two ribosomal RNAs (rRNA) genes and 22 transfer RNAs (tRNA) genes necessary for translation of the proteins encoded by the mtDNA (Boore 1999). Due to its maternal inheritance, rapid evolutionary rate, and lack of recombination, mtDNA has been extensively used as genetic markers not only for studies of population genetic, phylogenetics, and taxonomic investigations at various taxonomic levels of different organisms (Liu et al. 2013a, 2014, 2015; Jabbar et al. 2014; Ogedengbe et al. 2014; Li et al. 2015; Yang et al. 2015). Recently, mtDNA has been used as genetic markers for studying genetic variation in oxyurid nematodes (Solórzano-García et al. 2015), including P. ambiguus (Sheng et al. 2015). In spite of the availability of advanced sequencing and bioinformatic methods, there is still limited knowledge about complete mt genomes of many oxyurid nematodes of socioeconomic importance. To date, the complete mt genomes have been reported for only two species (Kang et al. 2009; Park et al. 2011). Therefore, more oxyurid nematode mt genomes are needed to provide a useful molecular marker for population genetic and phylogenetic studies of pinworms.
The objectives of the present study were (i) to determine the complete mt genome of rabbit pinworm P. ambiguus, (ii) to compare this mt sequence with those of other pinworms, and (iii) to re-examine phylogenetic relationships from selected Chromadorea nematodes using the protein-coding amino acid sequences.
Materials and methods
Ethics statement
This study was approved by the Animal Ethics Committee of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (approval no. LVRIAEC2012-011). The rabbits, from which the pinworms were collected, were handled in accordance with good animal practices required by the Animal Ethics Procedures and Guidelines of the People’s Republic of China.
Parasites and DNA extraction
The adult worm of P. ambiguus was obtained from naturally infected rabbits from Daqing, Heilongjiang province, China. This specimen was washed in physiological saline, identified morphologically to species according to existing keys and descriptions (He et al. 1996), fixed in 70 % (v/v) ethanol and stored at −20 °C until use.
Total genomic DNA was isolated from this specimen using sodium dodecyl sulfate/proteinase K treatment, followed by spin-column purification (Wizard® SV Genomic DNA Purification System, Promega). The molecular identity of this specimen was then verified by PCR based on our previous study (Sheng et al. 2015).
PCR amplification and sequencing
Based on sequences well conserved in distantly related taxa, 14 pairs of primers (Table 1) were designed in the conserved regions to amplify the complete mt genome by PCR as overlapping amplicons from the genomic DNA. PCR reactions (25 μl) were performed in 2 mM MgCl2, 0.2 mM each of dNTPs, 2.5 μl 10× rTaq buffer, 2.5 μM of each primer, 1.25 U rTaq polymerase (Takara), and 1 μl of DNA sample in a thermocycler (BioRad) under the following conditions: 94 °C for 5 min (initial denaturation), then 94 °C for 1 min (denaturation), 42–47 °C for 1 min (annealing), and 72 °C for 1 min (extension) for 35 cycles, followed by 72 °C for 7 min (final extension). One microliter (5–10 ng) of genomic DNA was added to each PCR reaction. Samples without genomic DNA (no-DNA controls) were included in each amplification run, and in no case were amplicons detected in the no-DNA controls (not shown). Each amplicon (5 μl) was examined by (1 %) agarose gel electrophoresis to validate amplification efficiency. PCR products were sent to Invitrogen (Beijing, China) for sequencing using a primer walking strategy (Hu et al. 2007).
Sequence analyses
Sequences were assembled manually and aligned against the complete mt genome sequences of Wellcomia siamensis and Enterobius vermicularis (Kang et al. 2009; Park et al. 2011) available using the computer program MAFFT 7.122 (Katoh and Standley 2013) to identify gene boundaries. Translation initiation and translation termination codons were identified based on comparison with those of reported previously (Kang et al. 2009; Park et al. 2011). For analyzing tRNA genes, putative secondary structures of 22 tRNA genes were identified using tRNAscan-SE (Lowe and Eddy 1997) or by recognizing potential secondary structures and anticodon sequences by eye, and two rRNA genes were predicted by comparison with those of reported previously (Kang et al. 2009; Park et al. 2011).
Phylogenetic analyses
The amino acid sequences conceptually translated from individual genes of the mt genome of P. ambiguus were concatenated. Selected for comparison were concatenated amino acid sequences predicted from published mt genomes of representing Chromadorea nematodes, including Oesophagostomum asperum (Zhao et al. 2013), Haemonchus contortus (Jex et al. 2008), Contracaecum rudolphii B (Lin et al. 2012), Toxocara canis (Li et al. 2008), Ascaris suum (Okimoto et al. 1992), Baylisascaris procyonis (Xie et al. 2011), W. siamensis (Park et al. 2011), E. vermicularis (Kang et al. 2009), Thelazia callipaeda (Liu et al. 2013b), Brugia malayi (Ghedin et al. 2007), Dirofilaria immitis (Hu et al. 2003), and Setaria digitata (Yatawara et al. 2010), using Trichuris suis (GenBank accession number GU070737) was used as the outgroup (Liu et al. 2012). All amino acid sequences were aligned using MAFFT 7.122, and ambiguously aligned regions were excluded using Gblocks online server (http://molevol.cmima.csic.es/castresana/Gblocks_server. html) with the default parameters (Talavera and Castresana 2007) using the options for a less stringent selection, and then subjected to phylogenetic analysis using maximum likelihood (ML). The LG + G + F model of amino acid evolution was selected as the most suitable model of evolution by ProtTest 2.4 (Abascal et al. 2005) based on the Akaike information criterion (AIC). ML analysis was performed with PhyML 3.0 (Guindon and Gascuel 2003) using the subtree pruning and regrafting (SPR) method with a BioNJ starting tree, and the most suitable model of amino acid substitution with gamma distribution (G) parameters estimated from the data with four discretized substitution rate classes, the middle of which was estimated using the median. Bootstrap frequency (Bf) was calculated using 100 bootstrap replicates. Phylograms were drawn using the program FigTree v.1.4 (Rambaut 2012).
Results and discussion
General features of the mt genome of P. ambiguus
The complete mt genome of P. ambiguus was 14,023 bp in size (Fig. 1). The sequences have been deposited in GenBank under the accession number KT879302. This circular mt genome is typical of other Chromadorea nematode mt genomes, including 12 protein-coding genes (cox1-3, nad1-6, nad4L, cytb, and atp6), 22 transfer RNA genes, two ribosomal RNA genes, and two non-coding regions (Fig. 1; Table 2). All genes are transcribed in the same direction. The order and orientation of the gene arrangement (GA) pattern is identical to that of W. siamensis (GA12), but distinct from that of E. vermicularis (GA13) (Liu et al. 2013a). The nucleotide composition of the entire mt genome of P. ambiguus is A = 2678 (19.1 %), T = 7361 (52.49 %), G = 3158 (22.52 %), and C = 826 (5.89 %), and its A + T content was 75.59 %, in accordance with mt genomes of other oxyurid nematodes sequenced to date (Kang et al. 2009; Park et al. 2011).
Annotation
The mt genome of the P. ambiguus encodes 12 protein-coding genes. In this mt genome, 1 gene (cox1) start with ATA, 1 gene (cox2) use ATG; 7 genes (nad2, nad6, cytb, nad4, nad4L, nad3, and nad5) use TTG; and 3 genes (nad1, atp6, and cox3) use ATT as start codon, respectively (Table 2). All genes have complete termination codon use ATG except for nad2, nad4, nad4L, nad3, and cox2, which use abbreviated stop codon TA or T (Table 2). P. ambiguus mt genome were inferred to end with an abbreviated stop codon, such as T and TA, which is consistent with the arrangement in the mt genomes of other oxyurid nematodes (Kang et al. 2009; Park et al. 2011). A total of 22 tRNA genes, ranging in size from 51 bp (trnS2) to 62 bp (trnM), were identified in P. ambiguus mtDNA. Their predicted secondary structures (not shown) are similar to those of other oxyurid nematodes (Kang et al. 2009; Park et al. 2011). The trnS1 and trnS2 lack a dihydrouridine (DHU) arm, whereas the other 20 tRNAs lack a TΨC and instead are equipped with a TV replacement loop. This is a feature common to Chromadorea nematode mt genomes. The rrnL gene of P. ambiguus is located between trnC and trnM, and rrnS gene is located between trnH and trnA. The length of the rrnL gene is 928 bp and rrnS gene is 714 bp in size (Table 2). The A + T contents of the rrnL and rrnS genes are 73.81 and 69.89 %, respectively (Table 3). The A + T contents of the rrnL and rrnS genes of P. ambiguus are significant lower than W. siamensis (rrnL and rrnS genes of W. siamensis are 81.32 and 76.64 %, respectively) and E. vermicularis (rrnL and rrnS genes are 76.17 and 71.39 %, respectively) (Table 3). The majority of metazoan mtDNA sequences contain two non-coding regions of significant size difference, the long non-coding regions (NCL) and the short non-coding regions (NCR). For the P. ambiguus, the longer non-coding region (NCRL) is located between the trnS2 and trnN, and the shorter one (NCRS) is located between genes trnA and trnS2. Their sizes are 644 bp (NCRL) and 126 bp (NCRS) (Table 2). The A + T contents of the NCRL and NCRS are 91.46 and 79.37 %, respectively, which is consistent with those of other oxyurid nematodes sequenced to date (Kang et al. 2009; Park et al. 2011).
Phylogenetic analyses
Phylogenetic analyses of P. ambiguus with selected Chromadorea nematodes were performed by ML based on concatenated mitochondrial amino acid sequences of 12 protein-coding genes (Fig. 2). Our results indicated that the P. ambiguus was more closely related to W. siamensis than to E. vermicularis. These results were consistent with those of recent study (Sheng et al. 2015). Mt genome sequences may provide reliable genetic markers in examining taxonomic status of nematodes, particularly when protein-coding gene sequences are used as markers in comparative analyses (Kang et al. 2009; Park et al. 2011; Kim et al. 2014; Liu et al. 2015). To date, the complete mt genomes have been reported for only two species from infraorder Oxyuridomorpha (Kang et al. 2009; Park et al. 2011). Therefore, in the present study, the characterization of the mt genome of P. ambiguus stimulates a reassessment of the systematic relationships of oxyurid nematodes using mt genomic datasets. According to the previous studies (De Ley and Blaxter 2002; Nadler et al. 2005; Park et al. 2011; Liu et al. 2013a, 2015; Kim et al. 2014) from sequences of nuclear small subunit (SSU) rRNA gene and mt genomes, infraorder Oxyuridomorpha is the monophyly. In the present study included three species from the infraorder Oxyuridomorpha and also indicated the paraphyly of this infraorder. Herein, we will not further discuss the phylogenetic relationships of nematodes since their phylogenetic relationships have been discussed in detail by some recent studies using mt genome sequences (Park et al. 2011; Liu et al. 2013a, 2015; Kim et al. 2014). There are seven families in this infraorder: Thelastomatoidae, Travassosinematidae, Hystrignathidae, Protrelloididae, Oxyuridae, Pharyngodonidae, and Heteroxynematidae (De Ley and Blaxter 2002). To date, mt genomes of many lineages of infraorder Oxyuridomorpha nematodes are still underrepresented or not represented. Therefore, expanding taxon sampling is necessary for future phylogenetic studies of this infraorder using mt genomic datasets.
In summary, the present study determined the complete mtDNA data of P. ambiguus and compared with that of other oxyurid nematodes. Phylogenetic analyses indicated that hat P. ambiguus was more closely related to W. siamensis than to E. vermicularis. This mt genome provides a novel genetic markers for studying the molecular epidemiology, population genetics, systematics of pinworm of animals and humans, and should have implications for the diagnosis, prevention, and control of passaluriasis in rabbits and other animals.
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Acknowledgments
This work was supported, in part, by the International Science and Technology Cooperation Program of China (grant no. 2013DFA31840) and the Science Fund for Creative Research Groups of Gansu Province (grant no. 1210RJIA006) to XQZ.
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Liu, GH., Li, S., Zou, FC. et al. The complete mitochondrial genome of rabbit pinworm Passalurus ambiguus: genome characterization and phylogenetic analysis. Parasitol Res 115, 423–429 (2016). https://doi.org/10.1007/s00436-015-4778-3
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DOI: https://doi.org/10.1007/s00436-015-4778-3