Introduction

The Gnathostoma spp. are the etiological agents of the zoonotic gnathostomiasis with a worldwide distribution, and gnathostomiasis has received increasing attention because of its public health significance (Diaz 2015; Jeremiah et al. 2011; Liu et al. 2015a; Nawa and Nakamura-Uchiyama 2004; Vargas et al. 2012). Humans get infected with Gnathostoma by consuming raw or undercooked food (fish, frogs, poultries, and snakes). Most patients with gnathostomiasis show cutaneous lesions (Chai et al. 2003). The Gnathostoma invasion of visceral organs, such as the lung, ear, gastrointestinal tract, uterus, eye, and kidney, leads to mechanical injuries and function damage (Herman and Chiodini 2009). The larval Gnathostoma may also intrude brain and spinal cord, causing serious neurognathostomiasis (Intapan et al. 2010), eosinophilic meningitis, intracerebral hemorrhage (Intapan et al. 2010), subarachnoid hemorrhage, and lesion in spinal nerve root (Katchanov et al. 2011). Fortunately, gnathostomiasis can be treated by drugs of ivermectin and albendazole (Nontasut et al. 2000).

The metazoan mitochondrial (mt) genomes are typically circular, ranging from 14 to 18 kb in size. It usually encodes 36–37 genes, including 12–13 protein-coding genes (PCGs), 2 ribosomal RNA (rrnS and rrnL) genes, and 22 transfer RNA genes (trns) (Wolstenholme 1992). No introns are within genes, and only very short spacer regions exist between the genes (Wolstenholme 1992). Mt genomes have been effectively used as genetic markers in phylogenetic studies of many taxa including parasites because of the properties of compactness, maternal inheritance, haploidy, relatively high mutation rates, and the lack of recombination (Chen et al. 2016; Tao et al. 2014; Yang et al. 2016).

Of the 12 species in the genus Gnathostoma (Liu et al., 2015a), G. doloresi is frequently reported for causing gnathostomiasis, especially in Asia (Chai et al. 2003; Liu et al. 2015a; Nawa and Nakamura-Uchiyama 2004; Xuan et al. 2002). Though the advanced DNA technologies are continuously improved, our knowledge of the genetics of G. doloresi is still scarce. The mt cox1 and the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA (rDNA) of G. doloresi is available (Li et al. 2015), and only one complete mt genome of Gnathostoma nematodes has been sequenced (Liu et al. 2015a). Therefore, the objectives of the present study were to determine the complete mt genomes of G. doloresi China and Japan isolates and to analyze the phylogenetic relationships of G. doloresi with 17 other representative nematodes using the concatenated mt amino acid sequences.

Materials and methods

Parasites and DNA extraction

The adult specimens of Gnathostoma were collected from naturally infected wild boars in Fujian province, China, and Mie Prefecture, Japan. They were detected by light microscopic examination and identified as G. doloresi according to morphological criteria (Camacho et al. 2002; Li et al. 2015) and multiple PCR assay (Li et al. 2014). The samples were fixed in 70 % alcohol and stored at −20 °C until further use. Total genomic DNA was isolated from individual adult G. doloresi using sodium dodecyl sulfate/proteinase K treatment, followed by spin-column purification (Wizard® SV Genomic DNA Purification System, Promega) (Liu et al. 2015a).

Long PCR amplification and sequencing

Nine pairs of PCR primers were used to amplify overlapping long fragments of the complete mt genome of G. doloresi, as shown in Table 1. The primers were designed according to relatively conserved regions of mtDNA sequences from G. spinigerum, Cooperia oncophora (NC004806), Teladorsagia circumcincta (NC013827) (Jex et al. 2009), Haemonchus contortus (NC010383) (Jex et al. 2008a), and Cucullanus robustus (GQ332426) (Park et al. 2011).

Table 1 Sequences of primers used to amplify PCR fragments from Gnathostoma doloresi
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PCR reactions were conducted in a total volume of 50 μl, using 25-μl PrimeStar Max DNA polymerase premix (Takara, Dalian, China), 20 pmol of each primer (synthesized in Genewiz, Suzhou, China), and 0.3 μl DNA template in a thermocycler (Biometra, Göttingen, Germany). The PCR cycling conditions began with the initial denaturation at 98 °C for 1.5 min, then 22 cycles of denaturation at 98 °C for 18 s, annealing at 52–62 °C for 8 s, and extension at 60 °C for 0.5–5 min, followed by 98 °C denaturation for 1.5 min, plus 28 cycles of 98 °C for 18 s (denaturation), 52–62 °C for 8 s (annealing), and 60 °C for 0.5–5 min according to the product length, with 10 min of the final extension at 66 °C. A negative control (contains no DNA) was included in each amplification run. Every amplicon (3 μl) was evidenced by electrophoresis in a 3 % agarose gel and stained with ethidium bromide (Sangon Biotech, Shanghai, China) (not shown). The PCR products were then sequenced by Invitrogen Biotechnology Company (Shanghai, China) using a primer-walking strategy.

Sequence analyses

Sequences were assembled manually and aligned against sequences of the complete mt genome of G. spinigerum using the computer program MAFFT 7.122 (Katoh and Standley 2013) to identify gene boundaries. Each PCG was translated into amino acid sequence using the invertebrate mitochondrial genetic code in MEGA 6 (Tamura et al. 2011) and aligned based on the amino acid sequences using default settings. The translation start and stop codons were identified to avoid gene overlap and to optimize the similarity with the gene lengths of G. spinigerum mt genome (Liu et al. 2015a). tRNA genes were identified using the program tRNAscan-SE (Lowe and Eddy 1997) by recognizing potential secondary structures and anticodon sequences by eye. Two rRNA genes were predicted by comparison with that of G. spinigerum (Liu et al., 2015a).

Phylogenetic analyses

The concatenated amino acid sequences of G. doloresi mt genome, conceptually translated from individual genes of the mt genome, were aligned with those of published mt genomes of selected species, including Baylisascaris ailuri (NC_015925) (Xie et al. 2011), Ascaris suum (HQ704901) (Liu et al. 2012), and Ascaris lumbricoides (HQ704900) (Liu et al. 2012) [Ascarididae]; Toxocara canis (EU730761) (Jex et al. 2008b) and Toxocara malaysiensis (NC_010527) (Li et al. 2006) [Toxocaridae]; Anisakis simplex (KC965056) (Mohandas et al. 2014) and Pseudoterranova azarasi (NC_027163) (Liu et al. 2015b) [Anisakedae]; Ascaridia galli (NC_021642) (Liu et al. 2013) [Ascaridiidae]; G. spinigerum (NC_027726) (Liu et al. 2015a) [Gnathostomidae]; Chabertia erschowi (NC_023782) (Liu et al. 2014) and Oesophagostomum quadrispinulatum (NC_014181) (Lin et al. 2012) [Chabertidae]; C. oncophora (NC_004806) (Van der Veer and De Vries 2004), Nematodirus oiratianus (NC_024639) (Zhao et al. 2014), and Trichostrongylus axei (NC_013824) (Jex et al. 2009) [Trichostrongylidae]; Dictyocaulus viviparus (NC_019810) (Gasser et al. 2012) [Dityocanlidae]; and Angiostrongylus cantonensis (NC_013065) (Lv et al. 2012) [Metastrongylidae]. The sequence of Trichuiris suis Denmark isolate (KT449822) (Hawash et al. 2015) [Trichuridae] was used as an outgroup.

All inferred amino acid sequences were aligned using MAFFT 7.122. Divergent regions of the alignment were eliminated using Gblocks Server v. 0.91b (http://molevol.cmima.csic.es/castresana/Gblocks_server.html). The default settings were used, selecting the option of less strict conservation of flanking positions. The alignment was then converted into the nexus format using Clustal X1.83 and subjected to phylogenetic analysis using Bayesian inference (BI). A mixed model (Castoe and Parkinson 2006) was used in the BI analysis using MrBayes 3.1.1. Four independent Markov chains were run for 1,000,000 metropolis-coupled MCMC generations and sampling trees every 1000 generations. The first 250 trees (25 %) were discarded as burn-in, with the remaining trees being used for calculating Bayesian posterior probabilities (Bpp). The analysis was regarded as completed when the potential scale reduction factor was close to 1, and the average standard deviation of split frequencies was below 0.01. Phylograms were prepared using FigTree v. 1.42.

Results and discussion

General features of the mt genome of G. doloresi

The complete mt genomes of G. doloresi (Fig. 1) from China and Japan were 13,809 and 13,812 bp in length, respectively (GenBank accession numbers KU975390 and KX231806). The circular mt genomes, transcribed in the same directions, contain 12 PCGs (cox1-3, nad1-6, nad4L, atp6, and cytb), 22 tRNA genes, 2 rRNA genes, and 2 non-coding regions (Table 2). The nucleotide composition of the G. doloresi China isolate mt genome is A = 25.02 %, T = 45.53 %, G = 21.70 %, and C = 7.75 %, with an obviously high A + T content of 70.55 %, and the nucleotide composition of the G. doloresi Japan isolate mt genome is A = 25.07 %, T = 45.68 %, G = 21.67 %, and C = 7.58 %, with an obviously high A + T content of 70.75 %, which is a little lower than that of G. spinigerum (71.13 %) (Liu et al. 2015a) and slightly higher than that of T. canis (NC_010690) (68.57 %) (Jex et al. 2008b).

Fig. 1
figure 1

Arrangement of the mitochondrial genomes of Gnathostoma doloresi. The scales are accurate. All genes are transcribed in the clockwise direction, using standard nomenclature. The 22 tRNA genes are represented by the one-letter code for the corresponding amino acid, with numerals differentiating each of the two leucine- and serine-specifying tRNAs (L1 and L2 for codon families CUN and UUR, respectively; S1 and S2 for codon families AGN and UCN, respectively). The A + T content for each gene or region is shown and represented by color

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Table 2 Organization of Gnathostoma doloresi from China (GDC) and Gnathostoma doloresi from Japan (GDJ) and Gnathostoma spinigerum from China (GSC) mitochondrial genomes
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The length of PCGs of G. doloresi China and Japan isolates were in the following order: nad5 > cox1 > nad4 > cytb > nad1 > nad2 > cox3 > cox2 > atp6 > nad6 > nad3 > nad4L (Table 2). A total of 3360 amino acids are encoded in the mt genome of G. doloresi China and Japan isolates. In these mt genomes, six start codons (TTG, ATG, ATA, GTT, ATT, and TTT) and four stop codons (TAA, TAG, TA, and T) are used (Table 2). GTT is used as start codon in cox2 and nad4 genes, and ATT is used in nad5 and cytb genes in the G. doloresi China isolate. Additionally, in the mt genome of the G. doloresi Japan isolate, GTT is used as start codon in cox2 and nad5 genes, and ATT is used only in the cytb gene. In both mt genomes, cox1 is initiated with TTT. Furthermore, incomplete termination codon TA is used in cox1 and cytb genes, and T is used in cox2, nad5, nad2, and nad4 (Table 2) in the mt genomes of the G. doloresi China and Japan isolates. The uncommon codon GTT was also used in Ascaris suum (Wolstenholme, 1992); ATT was used in Caenorhabditis elegans (Okimoto et al., 1992), A. suum (Okimoto et al., 1992), and many other species (Wolstenholme 1992).

A total of 22 tRNA sequences (ranging from 53 to 67 nucleotides in length) were identified in G. doloresi mt genomes, and their predicted secondary structures (not shown) are similar to that of G. spinigerum (Liu et al., 2015a). The rrnS gene of G. doloresi from the China and Japan isolates are 679 and 680 bp in length, and they are located between trnE and trnS2 genes. The rrnL gene is 939 and 938 bp in length, respectively, which lies between the trnH and nad3 genes. The A + T contents of the rrnL of the two G. doloresi isolates are 74.12 and 74.09 %, and the A + T contents of rrnS are 67.89 and 67.65 %, respectively.

The longer non-coding region (LNCR) of the G. doloresi China and Japan isolates is located between the nad2 and cytb genes, and the shorter one (SNCR) is located between the genes nad1 and atp6. Their sizes are 568 and 573 bp (LNCR) and 86 and 89 bp (SNCR) (Table 2). The A + T contents of the NC2 are 75.88 and 75.22 %, and the NC1 are 84.88 and 83.72 %, respectively (Table 2).

Comparative analyses between G. doloresi and G. spinigerum mt genomes

The complete mt genome sequence of the G. doloresi China isolate was 13,809 bp in length, 4 bp shorter than that of G. doloresi Japan. A comparison of the nucleotide sequences of each mt gene, as well as the amino acid sequences, conceptually translated from all protein genes of the two G. doloresi isolates, is given in Table 3. Sequence difference across the complete mt genome between G. doloresi China and Japan isolates was 3 %. The difference in amino acid sequences of the G. doloresi China and Japan isolate mt genomes was 2.3 %.

Table 3 Comparison of nucleotide (nt) and/or predicted amino acid (aa) sequence differences between mitochondrial genomes of Chinese Gnathostoma doloresi (GDC) and Japanese Gnathostoma doloresi (GDJ) and Chinese Gnathostoma spinigerum (GSC)
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The mt genome sequence of the G. doloresi China and Japan isolates are 270 and 267 bp shorter than that of G. spinigerum (Liu et al., 2015a). The arrangement of the mt genes (12 protein genes, 2 rRNA genes, and 22 tRNA genes) and 2 NCRs is the same (Liu et al. 2015a). A pairwise comparison of the nucleotide and the amino acid sequences of each mt gene was performed between the two Gnathostoma species (Table 3). The sequence lengths of the individual genes between the three taxa were very similar (Table 3). The magnitude of sequence difference in each gene between the two Gnathostoma species ranged from 11.1 to 22.3 % for nucleotide sequences and 5.2 to 20.1 % for amino acid sequences (Table 3). The sequence difference across the entire mt genome between G. doloresi (China and Japan isolates) and G. spinigerum was 19.10 and 17.80 % at the nucleotide level and 14.60 and 14.30 % at the amino acid level.

The greatest difference of nucleotide sequences between the two Gnathostoma species was in the nad2 (22.3 and 21.5 %), whereas the least difference (11.1 %) was detected in the rrnS gene (Table 3). At the amino acid level, the PCGs that varied the most were nad2 (20.1 %), nad5 (19.3 %), and nad6 (19.2 %), whereas cox1 (6.7 %) and cox3 (9.4 %) are the most conserved genes between China G. doloresi and G. spinigerum. On the other hand, the major difference of PCGs between Japan G. doloresi and G. spinigerum were nad6 (19.2 %), nad3 (18.8 %), and nad5 (18.4 %), while cox1 (5.2 %) and cox3 (10.2 %) are the most conserved genes.

Phylogenetic analyses

The phylogenetic tree inferred from the concatenated amino acid sequences of 15 nematode species (Fig. 2). The monophyly of the families Ascarididae, Toxocaridae, Anisakidae, Gnathostomidae, and Chabertidae were strongly supported in the phylogenetic analyses in the present study (Bpp = 1). Our data confirm that G. spinigerum is a member of the Gnathostomidae with strong support (Bpp = 1).

Fig. 2
figure 2

Phylogenetic relationships of Gnathostoma doloresi and other species. Tree inferred from the concatenated amino acid sequence dataset of 12 protein-coding genes from 20 nematodes was performed by Bayesian inference (BI). Trichuris suis (KT449822) was chosen as an outgroup

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Significance and implications

In the present study, we determined the complete mt genome of G. doloresi from China and Japan isolates, which are neglected zoonotic nematode. The characterization of the G. doloresi mt genomes will provide a novel resource for the improved diagnosis of human gnathostomiasis using the molecular approach. Molecular tools using mt gene sequences as genetic markers have been proven effective in assisting clinical diagnosis and conducting molecular epidemiological investigations of parasites (Ma et al. 2015a; Ma et al. 2015b). In addition, there is great potential to employing mt genome markers to investigate genetic variation of G. doloresi from different epidemic locations worldwide. The availability of the complete mt genomes of G. doloresi will also enable the identification and differentiation of potential cryptic/sibling species of Gnathostoma.

Mt genome sequences have been proven useful in verifying the phylogenetic position of helminths, particularly when using sequences of 12 PCGs as markers in comparative analyses (Liu et al. 2015b; Zhao et al. 2014). In the present study, the G. doloresi China and Japan isolates were clustered with G. spinigerum in the same branch, showing that the G. spinigerum and G. doloresi China and Japan isolates are closely related Gnathostoma species with high support in the BI analysis (Bpp > 0.92, Fig. 2). The phylogenetic relationships of the selected nematodes are consistent with those of previous studies (Liu et al. 2015a; Liu et al. 2013; Zhao et al. 2014). To date, mt genomes of many species of the family Gnathostomatidae are still underrepresented or not represented. So, expanding taxa sampling is necessary for future phylogenetic studies of Gnathostomatidae species using mt genomic dataset.

Conclusion

The present study determined the entire mt genomes of the G. doloresi China and Japan isolates of human health significance. The G. doloresi mt genomes will provide a useful resource for studying the molecular epidemiology and phylogenetics of the Gnathostoma spp. and should have implications for further studies of the diagnosis, prevention, and control of gnathostomiasis in humans and animals.