Ethics statement

This study was approved by the Animal Ethics Committee of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (Approval No. LVRIAEC2010-007). The farmed dog from which S. lupi adults were collected, was 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

Adult nematodes representing S. lupi were obtained at post mortem from the oesophagus of an infected farmed dog in Zhanjiang, Guangdong province, China. These specimens were washed in physiological saline, identified morphologically to species according to existing descriptions [22], fixed in 70% (v/v) ethanol and stored at −20°C until use.

Total genomic DNA was isolated from one S. lupi worm using sodium dodecyl sulphate/proteinase K treatment, followed by spin-column purification (TIANamp Genomic DNA kit). In order to independently verify the identity of this specimen, the mt cox 1 gene was amplified by the polymerase chain reaction (PCR) and sequenced according to an established method [14]. The cox 1 sequence of this S. lupi sample had 96.5% similarity with that of S. lupi in dogs in South Africa (GenBank accession no. HQ674759).

Amplification and sequencing of partial cox 1, rrn S, cox 2 and nad 2 genes

Long-PCR amplification and sequencing

After we had obtained partial cox 1, rrn S, cox 2 and nad 2 sequences for the S. lupi, we then designed four primers (Table 1) in the conserved regions to amplify the entire mt genome of S. lupi from this representative sample in four overlapping long fragments between cox 1 and rrn S (approximately 4.5 kb), between rrn S and cox 2 (approximately 2.5 kb), between cox 2 and nad 2 (approximately 4 kb), and between nad 2 and cox 1 (approximately 3 kb). Long-PCR reactions (25 μl) were performed in 2 mM MgCl₂, 0.2 mM each of dNTPs, 2.5 μl 10× LA Taq buffer, 2.5 μM of each primer, 1.25 U LA Taq polymerase (Takara), and 2 μl of DNA sample in a thermocycler (Biometra) under the following conditions: 92°C for 2 min (initial denaturation), then 92°C for 10 s (denaturation), 60°C (for 4.5 kb) or 44°C (for 2.5 kb) or 52°C (for 4 kb) or 48°C (for 3 kb fragment) for 30 s (annealing), and 60°C for 10 min (extension) for 10 cycles, followed by 92°C for 10 s, 60°C (for 4.5 kb) or 44°C (for 2.5 kb) or 52°C (for 4 kb) or 48°C (for 3 kb fragment) for 30 s (annealing), and 60°C for 10 min for 20 cycles, with a cycle elongation of 10 s for each cycle and a final extension at 60°C for 10 min. Each PCR reaction yielded a single band detected in a 0.8% (w/v) agarose gel (not shown). PCR products were sent to Sangon Company (Shanghai, China) for sequencing using a primer-walking strategy.

Sequence analyses

Sequences were assembled manually using the commercial software ContigExpress program of the Vector NTI software package version 6.0 (Invitrogen, Carlsbad, CA), and aligned against the complete mt genome sequences of other spirurid nematodes available using the computer program Clustal X 1.83 [24] and MegAlign procedure within the DNAStar 5.0 [25] to infer gene boundaries. The open-reading frames were analysed with Open Reading Frame Finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html) using the invertebrate mitochondrial code, and subsequently compared with that of T. callipaeda[21]. Protein-coding gene sequences were translated into amino acid sequences using the invertebrate mitochondrial genetic code; amino acid sequences were aligned using default settings with MEGA 5.0 [26]. Translation initiation and termination codons were identified by comparison with those of the spirurid nematodes reported previously [21, 27]. For analyzing ribosomal RNA genes, putative secondary structures of 22 tRNA genes were identified using tRNAscan-SE [28], of the 22 tRNA genes, 14 were identified using tRNAscan-SE, the other 8 tRNA genes were found by eye inspection, and rRNA genes were identified by comparison with that of spirurid nematodes [21, 27].

Phylogenetic analysis

The amino acid sequences conceptually translated from individual genes of the mt genome of S. lupi were concatenated. Selected for comparison were concatenated amino acid sequences predicted from published mt genomes of key nematodes representing the order Spirurida, including the superfamilies Thelazoidea (T. callipaeda[21]), Filarioidea (Acanthocheilonema viteae[29], Brugia malayi[30], Chandlerella quiscali[29], Dirofilaria immitis[31], Loa loa[29], Onchocerca flexuosa[29], O. volvulus[32], S. digitata[27] and Wuchereria bancrofti[18]), Dracunculoidea (Dracunculus medinensis[33]) and Physalopteroidea (Heliconema longissimum[33]) (GenBank accession numbers JX069968, NC_016197, NC_004298, NC_014486, NC_005305, NC_016199, NC_016172, AF015193, NC_014282, JN367461, NC_016019 and NC_016127, respectively), using Ascaris suum[34] (GenBank accession number HQ704901) as the outgroup. The amino acid sequences were aligned using Clustal X 1.83 [24] using default settings, ambiguously aligned regions were excluded using Gblocks online server (http://molevol.cmima.csic.es/castresana/Gblocks_server.html) using the options for a less stringent selection, and then subjected to phylogenetic analysis using Bayesian inference (BI) as described previously [35, 36]. Phylograms were drawn using the Tree View program v.1.65 [37].

General features of the S. lupi mt genome

The complete mtDNA sequence of S. lupi was 13,780 bp in size (Figure 1), and has been deposited in the GenBank under the accession number KC305876. The mt genome of S. lupi contains 12 protein-coding genes (cox 1-3, nad 1-6, nad 4L, atp 6 and cyt b), 22 transfer RNA genes, two ribosomal RNA genes (rrn L and rrn S) and a non-coding (control or AT-rich) region, but lacks an atp 8 gene (Table 2). All genes are transcribed in the same direction. The gene order is identical to those of T. callipaeda and S. digitata[21, 27], but distinct from those of H. longissimum (rearrangement markedly) and Dracunculus medinensis (tRNA-Met and tRNA-Val change) [33]. The nucleotide compositions of S. lupi mt genome is biased toward A and T, with T being the most favored nucleotide and C being the least favored, in accordance with mt genomes of other spirurid nematodes [27, 31]. The content of A + T is 73.73% for S. lupi, similar to that of mt genomes of other spirurid nematodes sequenced to date, such as that of T. callipaeda (74.57%) [21] and W. bancrofti (74.59%) [18] (Table 3). Furthermore, the S. lupi mt genes overlap a total of 98 bp in 16 locations ranging from 1 to 32 bp (Table 2). The longest is a 32 bp overlap between nad 1 and tRNA-Phe. The mt genome of S. lupi has 150 bp of intergenic regions at 16 locations ranging in size from 1 bp to 59 bp, the longest intergenic region is a 59 bp between tRNA-Pro and tRNA-Asp (Table 2). The mt genome of T. callipaeda has 14 intergenic regions, which range from 1 to 62 bp in length. The longest region is 62 bp between tRNA-Pro and tRNA-Asp [21].

Protein-coding genes

The S. lupi mt genome encodes 12 protein-coding genes, which are identical to those of T. callipaeda and S. digitata[21, 27]. For S. lupi, the sizes of the protein-coding genes were in the order: cox 1 > nad 5 > nad 4 > cyt b > nad 1 > nad 2 > cox 3 > cox 2 > atp 6 > nad 6 > nad 3 > nad 4L (Table 2). The predicted translation initiation and termination codons for the 12 protein-coding genes of S. lupi mt genome were compared with that of T. callipaeda and S. digitata[21, 27]. The most common initiation codon for S. lupi is TTG (5 of 12 protein genes), followed by ATG (4 of 12 protein genes), ATT (2 of 12 protein genes) and ATA (1 of 12 protein genes) (Table 2). In this mt genome, all protein genes were predicted to have a TAA or TAG as termination codon (Table 2). Although incomplete termination codons (T or TA) are present in some other nematodes, including Anisakis simplex (s. l.) [38], A. suum[39], Caenorhabditis elegans[39], S. digitata[27], Toxocara spp. [40] and Trichinella spiralis[41], they were not identified in the S. lupi mt genome.

Transfer RNA genes and ribosomal RNA genes

The sizes of 22 tRNA genes identified in the S. lupi mt genome ranged from 50 to 62 bp in size. Secondary structures predicted for the 22 tRNA genes of S. lupi (not shown) are similar to that of S. digitata[27]. The rrn L and rrn S genes of S. lupi were identified by comparison with the mt genomes of T. callipaeda and S. digitata. The rrn L is located between tRNA-His and nad 3, and rrn S is located between nad 4L and tRNA-Tyr. The lengths of the rrn L and rrn S genes were 988 bp and 686 bp for S. lupi, respectively (Table 2). The A + T contents of the rrn L and rrn S genes for S. lupi are 79.05% and 76.09%, respectively.

Non-coding regions

The majority of nematode mtDNA sequences contain usually two non-coding regions of significant size difference, the long non-coding region and the short non-coding region, including A. lumbricoides and A. suum[34], Contracaecum rudolphii B [42], Oesophagostomum spp. [43], Toxocara spp. [40] and Trichuris spp. [44, 45]. However, there is only one non-coding region (AT-rich region) in the mt genome of S. lupi, which is located between cox 3 and tRNA-Ala (Figure 1 and Table 2), with 88.50% A + T content (Table 3). This region of the mt genome of S. lupi was considered as a non-coding region (or AT-rich region) due to its location and AT rich feature based on comparison with those of spirurid nematodes reported previously [21, 27]. Moreover, in the AT-rich region of S. lupi consecutive sequences [A]₁₃ and [T]₁₂ were found, but there are no AT dinucleotide repeat sequences similar to that of A. simplex s.l. and S. digitata in the this region [27, 38].

Phylogenetic analyses

The phylogenetic relationships of 12 spirurid species based on concatenated amino acid sequence datasets, plus the mtDNA sequence of S. lupi obtained in the present study, using BI is shown in Figure 2. The results revealed that S. lupi (Thelaziidae) was a sister taxon to a clade containing S. digitata (Setariidae) and other members of the Onchocercidae, including B. malayi and D. immitis (posterior probability = 1.00), consistent with results of previous studies [14, 21, 46].

Many studies have demonstrated that mtDNA sequences are valuable genetic markers for phylogenetic studies of members within the Nematoda. A recent study analyzed mt sequence variations in human- and pig-derived Trichuris and demonstrated that they represent separate species [44]. In addition, a previous study sequenced and compared the mt genomes of A. lumbricoides and A. suum from humans and pigs and indicted that A. lumbricoides and A. suum may represent the same species [34]. In the present study, the characterization of the mt genome of S. lupi can promote to reassess the systematic relationships within the order Spirurida using mt genomic datasets. For many years, there have been considerable debates about the phylogenetic position of members of spirurid nematodes [47, 48]. Given this utility of mt genomic datasets, thus, further work should sequence more mt genomes of spirurid nematodes and re-construct the phylogenetic relationships of spirurid nematodes using expanded mt datasets.