Front. Microbiol. Frontiers in Microbiology Front. Microbiol. 1664-302X Frontiers Media S.A. 10.3389/fmicb.2017.01444 Microbiology Original Research Mitochondrial DNA Evidence Supports the Hypothesis that Triodontophorus Species Belong to Cyathostominae Gao Yuan 1 Zhang Yan 1 Yang Xin 2 Qiu Jian-Hua 1 Duan Hong 1 Xu Wen-Wen 1 Chang Qiao-Cheng 1 * Wang Chun-Ren 1 3 * 1College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University Daqing, China 2State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University Wuhan, China 3College of Life Science and Biotechnology, Heilongjiang Bayi Agricultural University Daqing, China

Edited by: Guan Zhu, Texas A&M University, United States

Reviewed by: Mario Santoro, Istituto Zooprofilattico Sperimentale del Mezzogiorno, Italy; Guanghui Zhao, Northwest A&F University, China

*Correspondence: Qiao-Cheng Chang changqiaocheng2001@163.com

Chun-Ren Wang chunrenwang@sohu.com

This article was submitted to Infectious Diseases, a section of the journal Frontiers in Microbiology

 03 08 2017 2017 8 1444 29 04 2017 17 07 2017 Copyright © 2017 Gao, Zhang, Yang, Qiu, Duan, Xu, Chang and Wang. 2017 Gao, Zhang, Yang, Qiu, Duan, Xu, Chang and Wang

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Equine strongyles, the significant nematode pathogens of horses, are characterized by high quantities and species abundance, but classification of this group of parasitic nematodes is debated. Mitochondrial (mt) genome DNA data are often used to address classification controversies. Thus, the objectives of this study were to determine the complete mt genomes of three Cyathostominae nematode species (Cyathostomum catinatum, Cylicostephanus minutus, and Poteriostomum imparidentatum) of horses and reconstruct the phylogenetic relationship of Strongylidae with other nematodes in Strongyloidea to test the hypothesis that Triodontophorus spp. belong to Cyathostominae using the mt genomes. The mt genomes of Cy. catinatum, Cs. minutus, and P. imparidentatum were 13,838, 13,826, and 13,817 bp in length, respectively. Complete mt nucleotide sequence comparison of all Strongylidae nematodes revealed that sequence identity ranged from 77.8 to 91.6%. The mt genome sequences of Triodontophorus species had relatively high identity with Cyathostominae nematodes, rather than Strongylus species of the same subfamily (Strongylinae). Comparative analyses of mt genome organization for Strongyloidea nematodes sequenced to date revealed that members of this superfamily possess identical gene arrangements. Phylogenetic analyses using mtDNA data indicated that the Triodontophorus species clustered with Cyathostominae species instead of Strongylus species. The present study first determined the complete mt genome sequences of Cy. catinatum, Cs. minutus, and P. imparidentatum, which will provide novel genetic markers for further studies of Strongylidae taxonomy, population genetics, and systematics. Importantly, sequence comparison and phylogenetic analyses based on mtDNA sequences supported the hypothesis that Triodontophorus belongs to Cyathostominae.

 complete mitochondrial genome Strongyloidea Strongylidae Cyathostominae Strongylinae phylogenetic analysis 2013DFA31840 2017YFD0501300 YJSCX2017-Y37 International Science & Technology Cooperation Program of China National Key Research and Development Program of China Heilongjiang Bayi Agricultural University Graduate Innovative Research Project Introduction

Equine strongyles, a large group of intestinal nematodes that belong to members of Equidae, are classified into two subfamilies, Strongylinae (large strongyles) and Cyathostominae (small strongyles), based on worm size and capsule mouth shape (Lichtenfels et al., 2008; Traversa et al., 2010). Although equine strongyles are the significant nematode pathogens of horses, information on these nematodes is limited to their morphology, prevalence, and disease control and prevention (Bu et al., 2009; Lyons et al., 2011; Mughini et al., 2011; Morariu et al., 2016; Singh et al., 2016). Intestinal nematodes of Cyathostominae, a ubiquitous parasitic nematode species, inhabit the large intestine of infested equines with a high prevalence, especially because of the reduced prevalence of Strongylus spp. and spread of cyathostomin anthelmintic-resistant populations (Reinemeyer, 1986; Traversa et al., 2010). Small strongyles can infect virtually any horse, with symptoms of anorexia, weight loss, poor hair coat, lethargy with disordered intestinal motility, and some with inflammatory enteropathy caused by adult Cyathostominae, especially as a result of emergence of enormous numbers of larvae from the lining of the large intestine with a high mortality rate of up to 50% caused by larval stages (Reinemeyer, 1986; Love et al., 1999; Corning, 2009; Traversa et al., 2010).

The traditional classification of equine strongyles was primarily based on morphological characteristics, but some researchers believed that the classification of Strongylidae (including the separation of Strongylinae and Cyathostominae) based on differences in the size and shape of the buccal capsule was arbitrary (Durette-Desset et al., 1994; Lichtenfels et al., 1998). However, reconstruction of the systematic relationships using the first and second internal transcribed spacers (ITS1 and ITS2, respectively) of 30 equine strongyles species revealed that Triodontophorus serratus, which was previously classified into Strongylinae based on morphology, clustered with Cyathostominae (Hung et al., 2000).

Mitochondrial (mt) genome sequences provide effective and reliable molecular markers for various types of evolutionary studies of parasites because of their strict maternal inheritance, apparent lack of recombination, rapid evolutionary rate, and comparatively conserved genomic structure (Gissi et al., 2008; Jia et al., 2010; Tian et al., 2015; Zhang et al., 2015; Guo et al., 2016; Liu et al., 2016), especially for classification of higher taxonomic levels. For example, the mt genome provided evidence that Orientobilharzia turkestanicum belongs to the genus Schistosoma, and is phylogenetically closer to the African schistosome group than to the Asian schistosome group (Wang et al., 2011). In addition, phylogenetic analyses of both nucleotide and amino acid sequence data of mt genome supported the hypothesis that Dicrocoelium chinensis and D. dendriticum are genetically distinct species (Liu et al., 2014).

Therefore, the objectives of this study were to: (1) determine the complete mt genome sequences of the three nematodes Cyathostomum catinatum, Cylicostephanus minutus, and Poteriostomum imparidentatum; (2) analyze and compare the mt genomes of Strongylidae nematodes; and (3) reconstruct the phylogenetic relationship of Strongylidae with other nematodes in Strongyloidea based on the mtDNA data to test the hypothesis that Triodontophorus belongs to Cyathostominae.

 Materials and methods Ethics approval

This study was approved by the Animal Ethics Committee of Heilongjiang Bayi Agricultural University. Horses used for the study were handled in accordance with good animal practice, as defined by the Animal Ethics Procedures and Guidelines of the People's Republic of China.

 Parasites, DNA extraction, genome amplification, and sequence analyses

Adult nematode Cy. catinatum, Cs. minutus, and P. imparidentatum were collected from the large intestine of naturally infected horses from a slaughter house in Daqing, Heilongjiang Province, China. Individual worms were identified to species based on their morphological characteristics and predilection sites (Lichtenfels et al., 2008). Under the microscope, the morphological characteristics of Cy. catinatum including the clear mouth collar, buccal cavity wide in the front and narrow in the back and its width greater than its depth, short dorsal lobe of male bursa and the foot-shaped tail of female could be observed (see Figure S1). Cs. minutus is small with a short mouth collar, clear and long submedian papilla, buccal cavity deeper than wide, dorsal gutter elongate reaching half the depth of buccal capsule, short dorsal lobe of male bursa, and straight tails for females (see Figure S2). P. imparidentatum are relatively large and have a clear internal and external leaf-crown, high mouth collar, fine and sharp submedian papilla, width of buccal capsule greater than depth, and short and wide dorsal lobe of male bursa (see Figure S3). The detailed data are listed in Table 1.

The detail information of morphological characteristics of the three nematodes.

Female Male
Cy. c R P. i R Cs. m R Cy. c R Cs. m R P. i R
Number 3 – 1 – 3 – 3 – 1 – 1 –
Body length/mm 8.9–9.3 4.89–9.8 15.1 11.5–18.4 6.6–6.8 4.6–6.8 7.2–7.5 4.0–8.6 4.8–5.0 4.0–5.2 12.6 11.5–14.0
ELC/elements 22 18–22 86 74–95 8 8 20 18–22 8 8 82 74–95
BC width/μm 48.8–52.3 40–68 232 215–240 24.6–29.6 22–28 45.6–48.8 40–58 20.6–21.9 18–24 196.6 183–256
BC depth/μm 25.3–27.2 20–29 69 66–70 27.3–28.5 26–30 23.2–25.9 19–26 25.0–26.3 24–27 54.2 52–64
Vulva to tail tip/μm 153.3–167 98–195 16,500 16,000–17,900 173.3–182.6 100–187 – – – – – –
Anus to tail tip/μm 89.6–93.6 75–110 7,500 6,500–10,000 100–105.3 54–120 – – – – – –
Spicule length/mm – – – – – – 1.25–1.30 1.12–1.52 0.596–0.601 0.515–0.672 1.03 0.98–1.11
Dorsal ray length/μm – – – – – – 356–360.6 315–377 150.6–155.2 140–170 427 370–620

BC, Buccal capsule; R, Reference (Lichtenfels et al., 2008); ELC, external leaf-crown; “–”, no results; Cy. c, Cyathostomum catinatum; P. i, Poteriostomum imparidentatum; Cs. m, Cylicostephanus minutus.

Subsequently, total genomic DNA was extracted from individual worms using TIANamp Genomic DNA Kit (TIANGEN Biotech, Beijing, China) according to the manufacturer's instructions. Parasite species were determined by PCR amplification of the ITS sequences of Cy. catinatum, Cs. minutus, P. imparidentatum, using the universal primers NC5 (5′- GTA GGT GAA CCT GCG GAA GGA TCA TT -3′) and NC2 (5′- TTA GTT TCT TTT CCT CCG CT -3′) reported by Gasser et al. (2008). The ITS sequences of Cy. catinatum, Cs. minutus, and P. imparidentatum obtained in the present study had 99.8, 99.7, and 99.2% identities to the corresponding available sequences in GenBank (Cs. minutus, KM085361.1; P. imparidentatum, KP693433.1; Cy. catinatum, KF850626.1, respectively), which confirmed the identity of the examined three species. The specific primers for mt genomes of the three worms were designed based on the relatively conserved mt sequences of the Strongylidae horse parasites available in GenBank (Table S1). PCR cycling conditions used to amplify mtDNA of the three nematodes were based on those of a previous report (Gao et al., 2017). After PCR amplification, the positive amplicons were sent to Life Technology Company (Beijing, China) for sequencing. With the aid of bioinformatics software such as MegAlign 5.01, Clustal X 1.83, MEGA 5.0, and tRNAscan-SE 2.0 (http://lowelab.ucsc.edu/tRNAscan-SE/) (Thompson et al., 1997; Burland, 2000; Tamura et al., 2011) and manual analysis, the boundary of each protein-coding gene, transfer RNA (tRNA) gene, and ribosomal RNA (rRNA) gene was determined.

 Comparative analysis with other strongylidae worms

Comparisons were made based on mtDNA size, gene arrangement, percentage of A+T content, and nucleotide and amino acid sequence similarity inferred from individual protein-coding genes among Cy. catinatum, Cs. minutus, P. imparidentatum, and nine other Strongylidae nematodes for which mt genome sequences were available in GenBank, which included Cylicocyclus insigne (NC_013808.1), Cs. goldi (AP017681.1), Cylicocyclus nassatus (KX819273.1), Strongylus equinus (NC_026868.1), S. vulgaris (AP017698.1), T. brevicauda (NC_026729.1), T. nipponicus (NC_031517.1), T. serratus (NC_031516.1), Macropicola ocydromi (NC_023099.1).

 Phylogenetic analyses

Phylogenetic analysis in this study was based on the concatenated amino acid sequences of 12 protein-coding genes of 20 Strongyloidea nematodes available in GenBank from family Chabertiidae: Chabertia erschowi (KF660603), C. ovina (NC_013831); family Cloacinidae: Oesophagostomum asperum (NC_023932.1), O. columbianum (NC_023933.1), O. dentatum (GQ888716), O. quadrispinulatum (NC_014181), Hypodontus macropi (NC_023099.1); family Strongylidae, including two subfamilies, Cyathostominae (Cs. goldi, Cc. insigne, Cc. nassatus, Cy. catinatum, Cs. minutus, and P. imparidentatum) and Strongylinae (S. equinus, S. vulgaris, T. brevicauda, T. nipponicus, T. serratus, M. ocydromi); and family Syngamidae: Syngamus trachea (GQ888718), and family Ancylostomatidae: Ancylostoma duodenale (NC_003415) as an outgroup. Each gene was translated using the invertebrate mitochondrial genetic code in MEGA 5 (Tamura et al., 2011) and ambiguously aligned regions were excluded using the Gblocks Server (http://molevol.cmima.csic.es/castresana/Gblocks_server.html) with less stringent selection. Phylogenetic trees were all reconstructed using Bayesian inference (BI) methods, which were performed using the mixed model in MrBayes 3.1.1 and 1,000,000 metropolis-coupled Markov chain Monte Carlo generations (Ronquist and Huelsenbeck, 2003); maximum parsimony (MP) methods, which were performed using a Fitch criterion (1,000 bootstrap replicates) within PAUP 4.0 Beta 10 (Swofford, 2002); and maximum likelihood (ML) methods (JTT+I+G+F model) using PhyML 3.0 (Guindon and Gascuel, 2003), and bootstrapping was performed using 100 replicates. Phylograms were drawn using Tree View 1.65 (Page, 1996).

 Results General features of the three MT genomes

In the present study, the complete mt genomes of Cy. catinatum, Cs. minutus, and P. imparidentatum were 13,838 bp, 13,826 bp and 13,817 bp in length, respectively (Table 2). The mt genomes of Cy. catinatum and P. imparidentatum are the first reported for both genera. All three mt genomes contained 12 protein-coding genes (nad1–nad6 and nad4L, cox1–cox3, cytb, atp6), 22 tRNA genes, two rRNA genes, and two non-coding regions (Table 2), which were transcribed in the same direction. Moreover, there were 19, 20, and 16 intergenic sequences in the complete circular mt genomes of Cy. catinatum, Cs. minutus, and P. imparidentatum, respectively. The largest intergenic region occurred in Cy. catinatum and was 51 bp in length. The shortest intergenic regions were 1-bp intergenic sequences in all three mt genomes. One, two, and three 1-bp overlaps were present in the mt genomes of Cy. catinatum, Cs. minutus, and P. imparidentatum, respectively. The total A+T contents in the genome sequences ranged from 74.65 to 76.12%, and the A, T, G, and C contents of the complete mtDNA of Cy. catinatum, Cs. minutus, P. imparidentatum ranged from 29.79–30.59%, 44.86–45.53%, 16.66–17.99%, and 7.22–7.37%, respectively. The detailed annotations of the three mt genomes, including the position and length of each gene, A+T content, initiation codon, and termination codon of 12 protein-coding genes, are listed in Table 2.

Mitochondrial genome organization of three nematodes.

Position 5′–3′ A+T (%) Codons
Genes Cs. m (13,826 bp) Cy. c (13,838 bp) P. i (13,817 bp) Cy. c Cs. m P. i Initiation / termination
Cy. c Cs. m P. i
nad1 1–873 1–873 1–873 73.88 74.23 72.05 ATT / TAA TTG/TAG ATA / TAA
atp6 882–1,481 884–1,483 882–1,481 75.15 77.83 74.50 ATT / TAA ATT/TAA ATT / TAA
tRNA-Lys (K) 1,511–1,573 1,497–1,559 1,488–1,550
tRNA-LeuUUR (L2) 1,588–1,642 1,581–1,635 1,570–1,624
tRNA-SerAGN (S1) 1,643–1,695 1,636–1,688 1,625–1,677
nad2 1,696–2,541 1,689–2,534 1,678–2,523 79.43 79.91 78.72 TTG / TAA TTG/TAA ATT / TAA
tRNA-Ile(I) 2,559–2,618 2,542–2,600 2,526–2,584
tRNA-Arg (R) 2,620–2,674 2,615–2,699 2,606–2,660
tRNA-Gln (Q) 2,689–2,743 2,676–2,730 2,678–2,632
tRNA-Phe (F) 2,746–2,808 2,732–2,787 2,733–2,788
cytb 2,803–3,915 2,788–3,900 2,789–3,901 71.61 72.51 69.63 ATT / TAA ATT/TAA ATA / TAA
tRNA-LeuCUN (L1) 3,922–3,976 3,917–3,971 3,922–3,975
cox3 3,977–4,742 3,972–4,737 3,976–4,741 71.93 72.06 70.10 ATT / T ATT/T ATT / T
tRNA-Thr (T) 4,743–4,800 4,738–4,792 4,742–4,797
nad4 4,801–6,030 4,793–6,022 4,798–6,024 76.99 78.05 76.20 TTG / TAA TTG/TAA TTG / TAA
SNCR 6,031–6,115 6,023–6,112 6,025–6,108 82.22 82.35 88.10
cox1 6,116–7,693 6,113–7,690 6,109–7,686 69.58 69.39 68.69 ATT / TAA ATT/TAA ATT / TAA
tRNA-Cys (C) 7,693–7,749 7,690–7,744 7,686–7,740
tRNA-Met (M) 7,764–7,822 7,755–7,813 7,741–7,800
tRNA-Asp (D) 7,826–7,884 7,819–7,877 7,806–7,867
tRNA-Gly (G) 7,900–7,955 7,889–7,944 7,884–7,939
cox2 7,957–8,652 7,945–8,640 7,940–8,635 71.55 73.56 71.41 ATT / TAA ATT/TAA ATA / TAA
tRNA-His (H) 8,656–8,709 8,646–8,699 8,635–8,688
rrnL 8,710–9,681 8,700–9,675 8,689–9,671 80.84 81.60 81.28
nad3 9,682–10,017 9,676–10,011 9,672–10,007 76.19 78.57 75.89 ATT / TAG ATT/TAA ATT / TAA
nad5 10,033–11,616 10,029–11,612 10,024–11,607 77.27 76.64 ATT / TAA ATT/TAA ATT / TAG
tRNA-Ala (A) 11,620–11,675 11,616–11,671 11,607–11,662
LNCR 11,676–11,937 11,672–11,942 11,663–11,950 86.72 87.02 83.33
tRNA-Pro (P) 11,938–11,993 11,943–11,997 11,951–12,005
tRNA-Val (V) 12,032–12,086 12,009–12,062 12,038–12,091
nad6 12,087–12,521 12,063–12,497 12,092–12,526 79.77 81.15 77.47 ATT / TAG ATT/TAA ATG / TAA
nad4L 12,560–12,793 12,548–12,781 12,558–12,791 79.06 79.91 79.91 ATT / TAA ATT/TAA ATT / TAA
tRNA-Trp (W) 12,815–12,872 12,801–12,857 12,816–12,872
tRNA-Glu (E) 12,899–12,957 12,899–12,955 12,875–12,931
rrnS 12,958–13,657 12,956–13,663 12,932–13,640 77.26 78.57 70.10
tRNA-SerUCN (S2) 13,658–13,714 13,664–13,719 13,641–13,693
tRNA-Asn (N) 13,714–13,768 13,720–13,776 13,695–13,750
tRNA-Tyr (Y) 13,772–13,826 13,781–13,838 13,763–13,817

Cy. c, Cyathostomum catinatum; P. i, Poteriostomum imparidentatum; Cs. m, Cylicostephanus minutus.

 Comparative analysis

Homology analysis showed that the highest identity (91.6%) occurred between the mt genome of Cy. catinatum and that of Cs. goldi (Table 3). Compared with the three nematodes of Strongylus (S. equinus and S. vulgaris) and Macropicola (M. ocydromi) in the same subfamily (Strongylinae), the worms in Cyathostominae showed relatively higher similarities to Triodontophorus species. Furthermore, the results from these analyses indicate that the mt genome arrangements of the Strongyloidea nematodes sequenced to date are identical. The detailed comparison is listed in Tables 2–4.

The comparative analysis of mt DNA among the family Strongylidae.

Subfamily Species Cy. c P. i Cs. m Cs. g Cc. n Cc. i S. e S. v T. b T. n T. s M.o
Cyathostominae Cy. c 100 – – – – – – – – – – –
P. i 84.7 100 – – – – – – – – – –
Cs. m 87.5 84.2 100 – – – – – – – – –
Cs. g 91.6 85.2 87.6 100 – – – – – – – –
Cc. n 87.9 84.4 86.7 87.7 100 – – – – – – –
Cc. i 88.2 84.5 87.3 88.4 89.3 100 – – – – – –
Strongylinae S. e 79.2 77.8 78.9 79.1 79.0 79.3 100 – – – – –
S. v 81.0 79.4 80.4 81.1 80.5 80.9 78.7 100 – – – –
T. b 82.9 81.4 82.8 83.0 82.3 83.3 78.8 79.2 100 – – –
T. n 85.0 83.6 84.7 85.2 83.9 84.9 78.2 80.3 83.3 100 – –
T. s 84.6 83.7 84.7 84.9 83.9 84.7 78.8 80.7 84.0 85.6 100 –
M.o 81.1 79.9 80.9 81.3 80.9 81.3 77.1 80.7 78.2 81.0 81.1 100

Cy. c, Cyathostomum catinatum; P. i, Poteriostomum imparidentatum; Cs. m, Cylicostephanus minutus; Cc. n, Cylicocyclus nassatus; Cc. i, Cylicocyclus insigne; Cs. g, Cylicostephanus goldi; S. e, Strongylus equinus; S. v, Strongylus vulgaris; T. b, Triodontophorus brevicauda; T. n, Triodontophorus nipponicus; T. s, Triodontophorus serratus; M. o, Macropicola ocydromi.

The complete nucleotide identify analyses of 12 nematodes in the family Strongylidae.

Genes No. aa No. nt (bp)
Cy. c Cs. m P. i Cc.n Cc. i Cs. g S. e S. v T. b T. n T. s M. o aa s (%) Cy. c Cs. m P. i. Cc.n Cc. i Cs. g S. e S. v T. b T. n T. s M.o nt s (%)
cox1 525 525 525 525 525 525 525 525 525 525 525 523 90.1–99.0 1,578 1,578 1,578 1,578 1,578 1,578 1,578 1,578 1,578 1,578 1,578 1,572 83.6–89.9
cox2 231 231 231 231 231 231 231 231 231 231 231 231 94.4–99.6 696 696 696 696 696 696 696 696 696 696 696 696 83.5–91.1
rrnL 976 973 983 974 959 972 959 959 975 976 961 960
nad3 111 111 111 111 111 111 111 111 111 111 111 112 80.2–98.2 336 336 336 336 336 336 336 336 336 336 336 339 78.6–90.8
NCR3 107
nad5 527 527 527 527 527 530 532 527 527 527 527 520 80.0–97.3 1,584 1,584 1,584 1,584 1,584 1,593 1,599 1,584 1,584 1,584 1,584 1,561 80.1–90.2
LNCR 271 262 288 270 274 272 271 383 336 259 278 230
NCR3 192 89
nad6 144 144 144 144 144 144 144 144 144 144 144 142 77.8–98.6 435 435 435 435 435 435 435 435 435 435 435 429 77.5–89.4
nad4L 77 77 77 77 77 77 77 77 77 77 77 77 79.2–98.7 234 234 234 234 234 234 234 234 234 234 234 234 82.9–93.2
NCR3 166
rrnS 708 700 709 699 700 699 708 700 703 696 700 701
nad1 290 290 290 290 290 290 292 291 290 290 290 284 79.7–99.0 873 873 873 873 873 873 879 876 873 873 873 855 79.5–91.1
atp6 199 199 199 199 199 199 199 200 199 199 199 199 84.9–99.0 600 600 600 600 600 600 600 603 600 600 600 600 82.0–91.8
nad2 281 281 281 281 281 281 281 281 281 281 281 277 77.2–97.2 846 846 846 846 846 846 846 846 846 846 846 834 77.9–91.5
cytb 370 370 370 370 370 370 370 371 370 370 370 370 80.3–97.8 1,113 1,113 1,113 1,113 1,113 1,113 1,113 1,116 1,113 1,113 1,113 1,113 79.1–90.9
cox3 255 255 255 255 256 255 255 255 253 255 255 255 92.2–98.0 766 766 766 766 769 766 766 766 760 766 766 766 82.8–90.5
nad4 409 409 408 409 409 409 409 409 408 409 409 401 82.0–98.5 1,230 1,230 1,227 1,230 1,230 1,230 1,230 1,230 1,227 1,230 1,230 1,206 82.0–92.2
SNCR 90 85 84 79 88 87 96 126 89 89 109 75
Total AA 3,419 3,419 3,418 3,419 3,420 3,422 3,426 3,422 3,416 3,419 3,419 3,391
Total size (bp) 13,838 13,826 13,817 13,846 13,828 13,827 14,545 14,301 14,305 13,701 13,794 13,659 78.2–91.6
A+T (%) 76.12 76.77 74.65 74.74 76.58 76.08 78.10 76.55 77.15 76.05 77.21 75.82

Cy. c, Cyathostomum catinatum; P. i, Poteriostomum imparidentatum; Cs. m, Cylicostephanus minutus; Cc. n, Cylicocyclus nassatus; Cc. i, Cylicocyclus insigne; Cs. g, Cylicostephanus goldi; S. e, Strongylus equinus; S. v, Strongylus vulgaris; T. b, Triodontophorus brevicauda; T. n, Triodontophorus nipponicus; T. s, Triodontophorus serratus; M. o, Macropicola ocydromi; nt s, nucleotides similarity; aa s, Amino acid similarity

Comparison of the 12 protein-coding genes of all Strongylidae nematodes revealed that the nad4L gene is the most conserved gene in terms of nucleotide sequences; however, the cox2 gene is the most conserved at the amino acid level (Table 4). For the codon usage of the 12 protein-coding genes of the three worms analyzed in this study, ATT initiation codons were present in high frequency, TAA was the most shared termination codon, and incomplete codon T appeared at the end of the cox3 gene of the three nematodes.

 Phylogenetic analysis

In the present study, mtDNA data were employed to assess the phylogenetic relationships of Strongyloidea nematodes, and the results are presented in Figure 1. All phylogenetic trees generated similar topologies, and the horse-parasitizing nematodes always clustered together in each tree. However, phylogenetic analysis using different methods revealed different relative positions for some species, such as P. imparidentatum. MP and ML trees showed the same topology, except for the branch that contained O. columbianum (Figures 1B,C). Congeneric species always formed clades (T. brevicauda, T. nipponicus, and T. serratus; Cc. insigne and Cc. nassatus; and Ch. erschowi and Ch. ovina), and the genera Oesophagostomum and Cylicostephanus are polyphyletic. Cs. goldi and Cy. catinatum formed sister taxa in all three trees. P. imparidentatum was sister to a clade composed of Cc. insigne, Cs. goldi, Cc. nassatus, Cy. catinatum, T. brevicauda, T. nipponicus, and T. serratus in the BI tree. However, P. imparidentatum clustered with all Cyathostominae nematodes in the MP and ML trees. Triodontophorus species clustered together with all the Cyathostominae nematodes with strong support in the three phylogenetic trees. In particular, in the BI tree, the Triodontophorus species clustered with Cylicostephanus, Cyathostomum, Cylicocyclus, and Poteriostomum within the clade of Cyathostominae. The two Strongylus species formed sister groups in the ML and MP trees, but not in the BI tree. The kangaroo-parasitizing nematodes (H. macropi and M. ocydromi) formed a distinct branch with the worms of cattle, sheep, goats, and pigs (C. erschowi, C. ovina, O. asperum, O. columbianum, O. dentatum, O. quadrispinulatum), even though M. ocydromi is classified in Strongylinae. The Oesophagostomum species did not form a monophyletic group, because of H. macropi, M. ocydromi, and the Chabertia species. However, O. quadrispinulatum and O. dentatum formed a distinct branch, and O. asperum was sister to the Chabertia species in all three trees. Syngamus trachea was divergent from all other species in the three analyses (see Figure 1).

Phylogenetic relationships of 20 Strongyloidea nematodes based on concatenated amino acid sequences of 12 protein-coding genes were analyzed by Bayesian inference (BI) (A), maximum parsimony (MP) (C), and maximum likelihood (ML) (B) using Ancylostoma duodenale as an outgroup. The values below 50% are not shown.

 Discussion

The mitochondrial genome is important in molecular biology, and is extensively applied when studying the taxonomy, population genetics, and systematics of parasites (Gissi et al., 2008; Zhang et al., 2015; Guo et al., 2016; Liu et al., 2016). In the present study, we obtained the mtDNA of three Cyathostominae nematodes for the first time and compared these mtDNA sequences those of Strongylidae species. The complete mtDNA sequences of the three nematodes were slightly different in length compared with the other Strongylidae nematodes (except for S. equinus and T. brevicauda), which is consistent with the length of typical metazoan mt genomes (Gissi et al., 2008; Jia et al., 2010; Tian et al., 2015; Zhang et al., 2015; Guo et al., 2016; Liu et al., 2016). Because of the longer length of the non-coding regions, the S. equinus and T. brevicauda mt genomes were slightly longer than other Strongylidae nematodes mt genomes. This phenomenon is also found in the mt genomes of entomopathogenic nematodes and plant-parasitic nematodes. For example, the lengths of the complete mt genomes of the entomopathogenic nematode Steinernema litorale and the plant-parasitic nematode Pratylenchus vulnus are 21,403 bp and 21,656 bp in size, respectively; however, the size of the non-coding regions was extremely long (S. litorale, 8,137 bp; P. vulnus, 7,748 bp) (Sultana et al., 2013; Taisei et al., 2016). The total A+T contents of three worms mtDNA of this study were consistent with those of most nematode mt genomes characterized to date, such as T. brevicauda (77.0%) and Wuchereria bancrofti (74.6%) (Ramesh et al., 2012; Duan et al., 2015), whereas the percentages of A+T content are remarkably higher than those of trematodes and protozoa, such as Echinostoma hortense (63.03%) and Eimeria magna (65.16%) (Tian et al., 2015; Liu et al., 2016). The A+T content in non-coding regions was higher than those in other regions of the mt genomes, which is consistent with the mt genomes of other nematodes in previous studies (Hu et al., 2002; Lin et al., 2012; Ramesh et al., 2012). However, non-coding regions quantity differed from those of other nematodes in the same family, and the third non-coding region was found in the mt genomes of T. brevicauda, Strongylus equinus, and M. ocydromi. There are also several pairs of repeats in the long non-coding regions of the three nematodes in this study with 12 bp for direct repeats and 12–14 bp for inverted repeats. This phenomenon was also found in the long non-coding regions of other helminthes, such as Strongyloides stercoralis, W. bancrofti, Fascioloides magna, Taenia multiceps, T. hydatigena, and T. pisiformis, but the lengths of those repeat sequences were longer than those in the three worms in the present study (Hu et al., 2003; Jia et al., 2010; Ramesh et al., 2012; Ma et al., 2016). Nevertheless, the functions of these regions remain unclear.

The mt genome of the three nematodes was encoded on the same strand and transcribed in the same direction, which is consistent with those of other Chromadorea nematodes available in GenBank, but different from those of Enoplea nematodes, such as Trichinella spiralis, Trichuris trichiura, and T. ovis (Lavrov and Brown, 2001; Liu et al., 2012a,b). Interestingly, the gene order in this study is also identical to that of 17 species that belong to superfamily Strongyloidea, for which mt genomes are available in GenBank, including Strongylidae (Cs. goldi, Cc. insigne, Cc. nassatus, S. equinus, S. vulgaris, T. brevicauda, T. nipponicus, T. serratus, and M. ocydromi), Chabertiidae (C. erschowi and C. ovina), Cloacinidae (O. asperum, O. columbianum, O. dentatum, O. quadrispinulatum, and H. macropi), and Syngamidae (Syngamus trachea). This phenomenon was also found in another group of the family Trichuridae, including Trichuris trichiura, T. suis, T. ovis, and T. discolor, whose mt genomes have the same gene arrangement. However, in other nematodes, such as the family Oxyuridae, Syphacia obvelata mt genome gene arrangement is consistent with that of Wellcomia siamensis, but different from those of Enterobius vermicularis and Aspiculuris tetraptera (Wang et al., 2016).

The results of the present study showed that topologies of the three trees with different phylogenetic reconstruction strategies were identical or similar. The paraphyly of Oesophagostomum species and Cylicostephanus species caused by Chabertia species and Cy. catinatum was evident in the mtDNA analyses. These paraphyletic groups were also proposed by some previous studies based on rDNA sequence (Hung et al., 2000; Gouý de Bellocq et al., 2001). P. imparidentatum was evolutionarily distant from other Cyathostominae and Triodontophorus species. Additionally, P. imparidentatum displayed the lowest nucleotide identity when compared with the other nematodes (Cy. catinatum, Cc. nassatus, Cc. insigne, Cs. minutus, Cs. goldi, T. brevicauda, T. nipponicus, and T. serratus). The phylogenetic analysis indicated that subfamily Cyathostominae was closer to the Triodontophorus species that belong to Strongylinae than to the Strongylus species that belong to Strongylinae.

This phenomenon was also reflected in the complete nucleotide identity comparisons; the Strongylus species showed relatively low nucleotide identity to the sequences of other equine strongyles. Nevertheless, the Triodontophorus worms mtDNA sequences have higher identity with the Cyathostominae than that of Strongylus species, which is consistent with the phylogenetic results. The similarity results were also further supported by other studies. The mtDNA-based phylogenetic trees revealed that all the worms in Cyathostominae, T. serratus, and Craterostomum acuticaudatum clustered with each other rather than the three other nematodes of Strongylinae (S. equinus, S. vulgaris, S. edentatus) (McDonnell et al., 2000). Another phylogenetic analysis of the large subunit rDNA D3 domain for 21 species of equine strongyles showed that T. serratus and T. brevicauda clustered with Cyathostominae rather than Strongylinae (Zhang et al., 2007). Furthermore, there is also biological evidence that supports this relationship; for example, a comparative study of the morphology of the L4s of 20 equine strongyles revealed that the larvae of Triodontophorus were more similar to those of Cyathostominae than to those of Strongylus species (Dvojnos and Kharchenko, 1990).

The phylogenetic relationships based on the complete mt genomes in this study indicated that the relationships among equine strongyles are inconsistent with the traditional classification. Triodontophorus species are genetically different from Strongylus species, and are more closely related to Cyathostominae. These analyses supported our hypothesis that Triodontophorus species belong to Cyathostominae.

 Conclusions

The findings of the present study, which used comparative and phylogenetic analyses of mtDNA sequences, supported the hypothesis that Triodontophorus species belong to Cyathostominae. The availability of the complete mt genome sequences of Cy. catinatum, Cs. minutus, and P. imparidentatum can provide novel genetic markers for further studies on the taxonomy, population genetics, and systematics of Strongylidae nematodes.

 Data availability statement

Representative nucleotide sequences were deposited in GenBank with the following accession numbers: KY495600-KY495602 for the mtDNA, and KY495603-KY495605 for the ITS.

 Author contributions

CRW conceived and designed the study, and critically revised the manuscript. YG, HD, and WWX performed the experiments. YG and QCC analyzed the data. YG drafted the manuscript. YZ, XY, and JHQ helped in study design, study implementation and manuscript preparation. All authors read and approved the final manuscript.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The authors thank the staff and workers in the abattoir who helped in the collection of nematodes. This work was supported by grant from the National Key Research and Development Program of China (2017YFD0501300), the grant of Heilongjiang Bayi Agricultural University Graduate Innovative Research Project (YJSCX2017-Y37), and the International Science & Technology Cooperation Program of China (Grant No. 2013DFA31840).

 Supplementary material

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmicb.2017.01444/full#supplementary-material

The primers used for obtain mitochondrial genomes of the three nematodes.

The detail information of morphological characteristics of Cyathostomum catinatum. (A) The head of Cy. catinatum, (B) The tail of female, (C) The tail of male, Bar = 0.1 mm.

The detail information of morphological characteristics of Cylicostephanus minutus. (A) The head of Cs. minutus, (B) The tail of female, (C) The tail of male, Bar = 0.1 mm.

The detail information of morphological characteristics of Poteriostomum imparidentatum. (A) The head of P. imparidentatum, (B) The tail of female, (C) The tail of male, Bar = 0.1 mm.

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