The worldwide-distributed lungworms belonging to the genus Metastrongylus have been reported to cause respiratory distress via interstitial tissue obstruction, ultimately resulting in consolidation of lungs, dyspnea, and progressive weight loss (Marruchella et al. 2012; Li et al. 2016). With such conditions, the moderate or severe immunodepression makes the pigs and wild boars (Sus scrofa) to be more vulnerable to negative health factors (García-González et al. 2013; Gassó et al. 2014). Among the Metastrongylus species, Metastrongylus salmi (M. salmi) has been considered one of the three most widely detected species and is usually found in mixed infections (Gassó et al. 2014; Li et al. 2016). For instance, porcine circovirus and swine influenza virus have been found to be exacerbated with this lungworm infection concomitantly cause fatal bronchopneumonia (Marruchella et al. 2012; Li et al. 2016).
Tibetan pigs (Tibetan Sus scrofa) live at high altitudes (3000 m above sea level) in the southeastern part of the Tibet Plateau and its adjacent regions (Li et al. 2016; Li et al. 2017a). At such high altitudes, with a hypoxic and cold environment, these animals are mainly fed with dry-lot husbandry by the free range system (Li et al. 2017b). The meat of Tibetan pigs is delicious, is high in protein, has a tender texture, and is rich in amino acids, making this species an economically important source for the Tibetan people (Zhang et al. 2014; Li et al. 2017b). Therefore, any disease infecting these animals may lead to dramatic economic suffering of the native herdsman.
The sequences of Mt DNA have been commonly used as genetic markers in taxonomy and population genetics research, and in phylogenetic and evolutionary analyses, because of its maternal inheritance (Li et al. 2008; Lin et al. 2012). However, limited information is available about the complete mt genome of M. salmi in Tibetan pigs from the Tibetan Plateau of China. The present study was therefore designed to determine and analyze the mt genomes of M. salmi, and to reveal the phylogenetic relationships of this parasite using mt DNA sequences.
Materials and Methods
Adult lungworms were sampled from the lungs of Tibetan pigs in a Tibetan slaughterhouse in 2016. The identification was done morphologically following extensive washing in 0.9% sodium chloride solution (Li, 2011). The parasites were subsequently fixed in 75% alcohol (V/V) and then stored at −20°C until further use (Li et al. 2016).
Mitochondrial DNA sequencing and genomic assembly
Five grams of adult lungworms were harvested for mt DNA isolation employing an improved extraction method as described previously (Sorensen et al. 2006). After fragmentation of the 1 μg of purified DNA, short-insert libraries (insert size 350 bp) were constructed according to the manufacturer’s instructions (Illumina). The sequencing of the short fragments was done using an Illumina Hiseq 4000 sequencing system at Total Solution (TGS) Institute in Shenzhen, China (Borgstrom et al. 2011).
First, the raw reads were filtered prior to assembly to remove the reads with adaptors: the reads depicting a quality score below 20 (Q<20), the reads containing a percentage of uncalled bases (“N” characters) equal to or greater than 10%, and the duplicated sequences. The mt genome of M. salmi was reconstructed in three steps using a combination of de novo and reference-guided assemblies (Cronn et al. 2008). Initially, the filtered reads were assembled into contigs using SOAP-denovo 2.04 (Li et al. 2010), and were aligned to the reference genome of Metastrongylus pudendotectus (Genbank ID: NC_013813.1). The aligned contigs (≥80% similarity and query coverage) were ordered according to the reference genome. After again mapping the raw reads to the assembled draft of mt genomes, they were visualized by Organellar Genome DRAW v1.2 (Lohse et al. 2007).
The mt genome annotation was done online using the DOGMA tool (Wyman et al. 2004), employing the default parameters to predict protein-coding genes, transfer RNA (tRNA) genes, and ribosome RNA (rRNA) genes. Repetitive sequences were predicted using RepeatMasker (Saha et al. 2008). A whole mt genome Blast (Altschul et al. 1990) search (E-value ≤ 1e-5, minimal alignment length percentage ≥40%) was performed against five databases: Kyoto Encyclopedia of Genes and Genomes (KEGG; Kanehisa, 1997; Kanehisa et al. 2004; Kanehisa et al. 2006), Clusters of Orthologous Groups (COG; Tatusov et al. 1997; Tatusov et al. 2003), the Non-Redundant (NR) Protein Database, the Swiss-Prot database (Magrane, 2011), and the Gene Ontology (GO; Ashburner et al. 2000) database. The circular M. salmi mt genome map was drawn using OGDraw v1.2 (Lohse et al. 2007).
Variation and phylogenetic analysis
Single nucleotide polymorphism (SNP) and insertion/deletion (InDel) calling are based on the alignment between the assembled result and the reference. The reference mt genes are as follows: Cylicocyclus insignis (NC_013808.1), Oesophagostomum quadrispinulatum (NC_014181.1), Oesophagostomum dentatum (NC_013817.1), Mecistocirrus digitatus (NC_013848.1), Metastronglyus pudendotectus (NC_013813.1), M. salmi (NC_013815.1), and Ascaris suum (NC_001327.1) (outgroup).
SNPs were detected by the software MUMmer (version 3.22) (Kurtz et al. 2004), Blast, TRF (Benson, 1999), Repeatmask (Humbert and Drouet, 1990), while LASTZ software (Chiaromonte et al. 2002; Li et al. 2009) was used to detect In-Dels. Structural variation (SV) was detected by MUMmer and LASTZ software. The phylogenetic tree was constructed using TreeBeST (Tannistha et al. 2010) with three methods (maximum likelihood: ML, maximum parsimony: MP, and Bayesian inference: Bayes) using the PHYML method (Lin et al. 2012). Bootstraps were set at 1000, based on the array of SNPs obtained from the sample and reference.
Results and Discussion
The complete mt genome of M. salmi was 13722 bp long (Fig. 1), which is shorter (by 56 bp) than that of the M. salmi Austrian isolates (13778 bp) and shorter (by 71 bp) than the M. pudendotectus Austrian isolates (13793) (Jex et al. 2010). The sequences of the mt genome have been submitted to the GenBank with the Accession Number: MF706166. The mt genome contains 12 protein-coding genes (cox1-3, nad1-6, nad4L, atp6, and cytb), 22 transfer RNA genes, and two ribosomal RNA genes (rrnL and rrnS) (Fig. 1, Table I), but lacks atp8, which is similar to other mt genomes of lungworms and Strongylida worms, M. pudendotectus, O. dentatum, O. quadrispinulatum, Oesophagostomum asperum, and Oesophagostomum columbianum (Jex et al. 2010; Lin et al. 2012; Zhao et al. 2013).
All the predicted genes were transcribed in the same direction; however, the arrangement of the mt genome of M. salmi was found to be different from that of Chabertia ovina, C. insignis, Strongylus vulgaris, O. dentatum, O. quadrispinulatum, O. asperum, and O. columbianum (Jex et al. 2010; Lin et al. 2012; Zhao et al. 2013), but in line with that of the M. salmi Austrian isolates (Jex et al. 2010).
The overall A+T content was found to be 73.54% (Table II) and the nucleotide composition was: A (23.52%), C (6.14%), G (19.60%), T (50.02%), and N (UCAG) (0.73%). The nucleotide compositions of all mt DNA sequences for M. salmi are biased toward A and T, with T being the most common nucleotide and C the least common, which is in line with the sequence of mt genomes of other nematodes to date as shown in Table II (Jex et al. 2010).
In the present study, the initiation and termination codon sequences of 12 protein-coding genes from the Tibetan M. salmi isolates were compared with previously reported sequences of M. pudendotectus (NC_013813.1), while the amino acid sequences of 12 mt protein genes of the former were inferred by comparing with those of the M. salmi Austrian isolates (NC_013815.1); the overall identity was found to be a 96% match, shorter than Austrian isolates (13778bp).
A total of 4237 amino acids are encoded by Tibetan isolated M. salmi mt genomes. The most common start codon is ATA at 41.7% (5/12 protein genes) (Table I); 11 protein genes out of 12 depicted a TAA or TAG translation termination codon (Table I). In our results, the 3-end of genes nad4L, atp6, nad2, cob, cox3, cox1, cox2, and nad5 are immediately adjacent to a downstream trn gene (Table I), which is in parallel arrangement with O. dentatum, O. quadrispinulatum, O. asperum, and O. columbianum (Lin et al. 2012; Zhao et al. 2013).
In protein-coding genes of M. salmi, the 63 possible codons were used; the UUU (Phe) codon was the most frequent whereas the CCC (Pro) codon was the least frequent; however, the CGC codon was not employed (Table II). The preferably used identical codons were found to be most frequent in significantly functional gene regions; codon bias is known mainly due to its selection at silent sites and is used to maximize the translation efficiency (Lin et al. 2012; Durent and Mouchiroud, 1990). At the third codon position of the current mt protein genes, T was the most frequently employed and C was used least frequently, which was in line with O. dentatum and O. quadrispinulatum (Lin et al. 2012). The bias towards using amino acids was believed to be related to “mutational bias-translational selection” model (Romero et al. 2002; Lin et al. 2012), which indicated that both selection and mutation played role in the bias of codon usage. However, Helfenbein et al. (2001) reported that it is still unclear whether this codon has an impact on parasite mt systems or not. So, protein-coding genes of M. salmi genome are biased toward using amino acids encoded by T-, A- and G-rich codons. The AT-rich codons depicted the amino acids Phe, Ile, Met, Tyr, Asn, or Lys, and the GC-rich codons represented Pro, Ala, Arg, or Gly, which is in line with O. dentatum and O. quadrispinulatum (Lin et al. 2012). In the current results, T-rich codons (more than two T’s in a triplet) comprise Phe (16.6% TTT and 1.3% TTC), Leu (5.9% TTA, 6.0% TTG, and 1.5% CTT), Ile (6.4% ATT), Val (6.0% GTT), Tyr (5.2% TAT), Ser (1.7% TCT), and Cys (3.2% TGT), and account for approximately 53.8% of the total amino acid composition.
A- and G-rich codons (with ≥ A’s and G’s, respectively) represent 15.4% and 11.7% of the total amino acid composition, respectively (Table III). In contrast, the proportion of C-rich codons (with ≥ C’s) is much lower (2.3%) (Table III). This result showed that the current genome sequence favors T, A, and G, but is strongly biased against C, which is in line with previously reported information for O. dentatum and O. quadrispinulatum (Lin et al, 2012). The possible reason may be that nucleotide bias can lead to a genome-wide bias in the amino acid composition of proteins (Singer and Hickey, 2000).
A total of 22 trn gene sequences (ranging from 52 to 113 bp in size) (Table I) were identified in the M. salmi mt genomes, which were similar to previously reported mt DNA sequences (Hu et al. 2003; Tannistha et al. 2010; Lin et al. 2012). The M. salmi rrnS and rrnL genes were identified by sequence comparison with those of M. pudendotectus. The rrnL is located between tRNA H and nad3, and the rrnS is located between tRNA E and tRNA S2 (Fig. 1, Table I). The sizes of the rrnS and rrnL genes are 688 bp and 960 bp respectively. The A + T contents of the rrnS and rrnL for the Tibetan M. salmi isolate are 75.3% and 77.5% respectively (Table II), which is similar to those in O. dentatum, O. quadrispinulatum, M. pudendotectus, and M. salmi (Austria) (Table II). The rrnS sequence matching of rrnS was found to be 77.28%, 78.06%, 72.46%, and 96.31% to O. dentatum, O. quadrispinulatum, M. pudendotectus, and M. salmi (Austria), respectively; rrnL gene matching was 77.02%, 72.42%, 83.54%, and 96.88% to O. dentatum, O. quadrispinulatum, M. pudendotectus, and M. salmi (Austria), respectively.
The seven available nematodes of Rhabditida and Strongylida were subjected to ML, MP, and Bayes analyses using A. suum (NC_001327.1) for the ultimate alignment of 12 protein-coding genes, which yielded similar tree topologies (Fig. 2, Fig. 3). According to the phylogenetic trees, the Tibetan and Austrian were found to be highly homologous.
In conclusion, the current research determined the complete mt DNA sequences of M. salmi isolated from Tibetan pigs from the remote areas of the Tibetan Plateau for the first time, revealing their gene annotations and genome organizations. Phylogenetic analysis using concatenated amino acid sequences of the 12 mt protein-coding genes indicated that M. salmi isolated from Tibetan pigs were highly homologous with that stemmed from Austrian ones. This information provides meaningful insights into the phylogenetic position of the M. salmi China isolate and represents a useful resource for selecting molecular markers for diagnosis and population studies, which may help in controlling Metastrongylus app, especially on the Qinghai Tibetan plateau.
The current research was supported by the Key Science Fund of Science and Technology Agency of Tibet Autonomous Region and projects in the National Science & Technology Pillar Program during the 12th Five-year Plan Period (2012BA D3B03). Kun Li is supported by China Scholarship Council under Grant No. 201706760018.
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About the article
Published Online: 2018-04-13
Published in Print: 2018-06-26
Conflict of interest: The authors state that there are no competing interests.
Ethics statement: All procedures were performed in accordance with the laws, regulations, and strict guidelines of the Laboratory Animals Research Centre of Hubei province, P. R. China. Samples were collected with the permission of the relevant institutions and by following the parameters as set by the ethics committee of Huazhong Agricultural University (Permit number: 4200695757).