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. 2014 Jan 22;7:44. doi: 10.1186/1756-3305-7-44

Chabertia erschowi (Nematoda) is a distinct species based on nuclear ribosomal DNA sequences and mitochondrial DNA sequences

Guo-Hua Liu 1,3,#, Lei Zhao 1,2,#, Hui-Qun Song 1, Guang-Hui Zhao 4, Jin-Zhong Cai 5, Quan Zhao 2, Xing-Quan Zhu 1,2,3,
PMCID: PMC3937141  PMID: 24450932

Abstract

Background

Gastrointestinal nematodes of livestock have major socio-economic importance worldwide. In small ruminants, Chabertia spp. are responsible for economic losses to the livestock industries globally. Although much attention has given us insights into epidemiology, diagnosis, treatment and control of this parasite, over the years, only one species (C. ovina) has been accepted to infect small ruminants, and it is not clear whether C. erschowi is valid as a separate species.

Methods

The first and second internal transcribed spacers (ITS-1 and ITS-2) regions of nuclear ribosomal DNA (rDNA) and the complete mitochondrial (mt) genomes of C. ovina and C. erschowi were amplified and then sequenced. Phylogenetic re-construction of 15 Strongylida species (including C. erschowi) was carried out using Bayesian inference (BI) based on concatenated amino acid sequence datasets.

Results

The ITS rDNA sequences of C. ovina China isolates and C. erschowi samples were 852–854 bp and 862 -866 bp in length, respectively. The mt genome sequence of C. erschowi was 13,705 bp in length, which is 12 bp shorter than that of C. ovina China isolate. The sequence difference between the entire mt genome of C. ovina China isolate and that of C. erschowi was 15.33%. In addition, sequence comparison of the most conserved mt small subunit ribosomal (rrnS) and the least conserved nad2 genes among multiple individual nematodes revealed substantial nucleotide differences between these two species but limited sequence variation within each species.

Conclusions

The mtDNA and rDNA datasets provide robust genetic evidence that C. erschowi is a valid strongylid nematode species. The mtDNA and rDNA datasets presented in the present study provide useful novel markers for further studies of the taxonomy and systematics of the Chabertia species from different hosts and geographical regions.

Keywords: Chabertia spp, Nuclear ribosomal DNA, Internal transcribed spacer (ITS), Mitochondrial DNA, Phylogenetic analysis

Background

The phylum Nematoda includes many parasites that threaten the health of plants, animals and humans on a global scale. The soil-transmitted helminthes (including roundworms, whipworms and hookworms) are estimated to infect almost one sixth of all humans, and more than a billion people are infected with at least one species [1]. Chabertia spp. are common gastrointestinal nematodes, causing significant economic losses to the livestock industries worldwide, due to poor productivity, failure to thrive and control costs [2-6]. In spite of the high prevalence of Chabertia reported in small ruminants [7], it is not clear whether the small ruminants harbour one or more than one species. Based on morphological features (e.g., cervical groove and cephalic vesicle) of adult worms, various Chabertia species have been described in sheep and goats in China, including C. ovina, C. rishati, C. bovis, C. erschowi, C. gaohanensis sp. nov and C. shaanxiensis sp. nov [8-10]. However, to date, only Chabertia ovina is well recognized as taxonomically valid [11,12]. Obviously, the identification and distinction of Chabertia to species using morphological criteria alone is not reliable. Therefore, there is an urgent need for suitable molecular approaches to accurately identify and distinguish closely-related Chabertia species from different hosts and regions.

Molecular tools, using genetic markers in mitochondrial (mt) genomes and the internal transcribed spacer (ITS) regions of nuclear ribosomal DNA (rDNA), have been used effectively to identify and differentiate parasites of different groups [13-16]. For nematodes, recent studies showed that mt genomes are useful genetic markers for the identification and differentiation of closely-related species [17,18]. In addition, employing ITS rDNA sequences, recent studies also demonstrated that Haemonchus placei and H. contortus are distinct species [19]; Trichuris suis and T. trichiura are different nematode species [20,21].

Using a long-range PCR-coupled sequencing approach [22], the objectives of the present study were (i) to characterize the ITS rDNA and mt genomes of C. ovina and C. erschowi from goat and yak in China, (ii) to compare these ITS sequences and mt genome sequences, and (iii) to test the hypothesis that C. erschowi is a valid species in phylogenetic analyses of these sequence data.

Methods

Parasites and isolation of total genomic DNA

Adult specimens of C. ovina (n = 6, coded CHO1-CHO6) and C. erschowi (n = 9, coded CHE1-CHE9) were collected, post-mortem, from the large intestine of a goat and a yak in Shaanxi and Qinghai Provinces, China, respectively, and were washed in physiological saline, identified morphologically [8,10], fixed in 70% (v/v) ethanol and stored at -20°C until use. Total genomic DNA was isolated separately from 15 individual worms using an established method [23].

Long-range PCR-based sequencing of mt genome

To obtain some mt sequence data for primer design, we PCR-amplified regions of C. erschowi of cox1 gene by using a (relatively) conserved primer pair JB3-JB4.5 [24], rrnL gene was amplified using the designed primers rrnLF (forward; 5′-GAGCCTGTATTGGGTTCCAGTATGA-3′) and rrnLR (reverse; 5′-AACTTTTTTTGATTTTCCTTTCGTA-3′), nad1 gene was amplified using the designed primers nad1F (forward; 5′-GAGCGTCATTTGTTGGGAAG-3′) and nad1R (reverse; 5′-CCCCTTCAGCAAAATCAAAC-3′), cytb gene was amplified using the designed primers cytbF (forward; 5′-GGTACCTTTTTGGCTTTTTATTATA-3′) and cytbR (reverse; 5′-ATATGAACAGGGCTTATTATAGGAT-3′) based on sequences conserved between Oesophagostomum dentatum and C. ovina Australia isolate. The amplicons were sequenced in both directions using BigDye terminator v.3.1, ABI PRISM 3730. We then designed primers (Table 1) to regions within cox1, rrnL, nad1 and cytb and amplified from C. ovina (coded CHO1) in four overlapping fragments: cox1-rrnL, rrnL-nad1, nad1-cytb and cytb-cox1. Then we designed primers (Table 1) to regions within cox1, rrnL, nad5, nad1, nad2 and cytb and amplified from C. erschowi (coded CHE1) in six overlapping fragments: cox1- rrnL, rrnL-nad5, nad5-nad1, nad1-nad2, nad2-cytb and cytb-cox1. The cycling conditions used were 92°C for 2 min (initial denaturation), then 92°C/10 s (denaturation), 50 -58°C (C. erschowi) or 56 -65°C (C. ovina)/30 s (annealing), and 60°C/10 min (extension) for 10 cycles, followed by 92°C for 2 min, then 92°C/10 s, 50 -58°C (C. erschowi) or 56 -65°C (C. ovina)/30 s, and 60°C/10 min for 20 cycles, with a cycle elongation of 10 s for each cycle and a final extension at 60°C/10 min. Each amplicon, which represented a single band in a 1.0% (w/v) agarose gel, following electrophoresis and ethidium-bromide staining, was column-purified and then sequenced using a primer walking strategy [22].

Table 1.

Sequences of primers used to amplify mitochondrial DNA regions from Chabertia erschowi and Chabertia ovina from China

Primer Sequence (5′ to 3′)
For rrnS
 
CHOF
TCGTTTAGTGGGTATGTGTGGTTCT (for C. ovina)
CHOR
GCCTACTCCCTAACAAATGACGCTC (for C. ovina)
CHEF
GTGGTTTTTAGGTTAGGGTTGAGTG (for C. erschowi)
CHER
ACGCTCATACAAAGTAATAAACGCA (for C. erschowi)
For nad2
 
CHOF
TTTGTGG(C\T)TAAGAGTGTT(G\A)GCTATT (for C. ovina)
CHOR
GAGCCGTAATCAAACATAGTAAATC (for C. ovina)
CHEF
TTTGTGG(C\T)TAAGAGTGTT(G\A)GCTATT (for C. erschowi)
CHER
ACCGTAATCAAACATAGTAAAATCT (for C. erschowi)
For C. ovina
 
COF
TGGTTGTGTGGTTTGGGCTCAT
rrnLR
ATGTCCTCACGCTAAGACTGCC
rrnLF
AGTTTGCTTCTGCCCAGTGA
ND5R
ACCGTAACCTCGCCCATCCTG
ND5F
ACGGCGTTAGTGGAGGAGGA
ND1R
CCACTAACCAACTCCCTTTCACCC
ND1F
ATTGGTGCTTTGCGGGCCAGT
ND2R
CCATAAACCTTTAAAACCTCCC
ND2F
TTGTTGGTTGGGAGACTATG
CYR
AAAGGGTCCTCAACCAAACA
CYF
CCTGTTTGGGGACCTTCTATTG
COR
CCGCAGTAAAATAAGCACGAGA
For C. erschowi
 
COF
ACCGACGGCTTATGGAAT
rrnLR
AGTGCAACCCAACATTATACCCT
rrnLF
TAAAGTTTGCTTCTGCCCAGTGATA
ND1R
ATAATAGCCAACAAAAGCACCGACA
ND1F
CTTGTCGGTGCTTTGCG
CYR
CCGCCTCAATAAACATCTC
CYF
TGGTCCAGATTATTGAAGG
COR TTACCCGTCAAATACAAAGT

Sequencing of ITS rDNA and mt rrnS and nad2

The full ITS rDNA region including primer flanking 18S and 28S rDNA sequences was PCR-amplified from individual DNA samples using universal primers NC5 (forward; 5′-GTAGGTGAACCTGCGGAAGGATCATT-3′) and NC2 (reverse; 5′-TTAGTTTCTTTTCCTCCGCT-3′) described previously [25]. The primers rrnSF and rrnSR (Table 1) designed to conserved mt genome sequences within the rrnS gene were employed for PCR amplification and subsequent sequencing of this complete gene (~ 700 bp) from multiple individuals of Chabertia spp. The primers nad2F and nad2R (Table 1) designed to conserved mt genome sequences within the nad2 gene were employed for PCR amplification and subsequent sequencing of this complete gene (~ 900 bp) from multiple individuals of Chabertia spp..

Sequence analyses

Sequences were assembled manually and aligned against the complete mt genome sequences of C. ovina Australia isolate [26] using the computer program Clustal X 1.83 [27] to infer gene boundaries. Translation initiation and termination codons were identified based on comparison with that of C. ovina Australia isolate [26]. The secondary structures of 22 tRNA genes were predicted using tRNAscan-SE [28] and/or manual adjustment [29], and rRNA genes were identified by comparison with that of C. ovina Australia isolate [26].

Phylogenetic analyses

Amino acid sequences inferred from the 12 protein-coding genes of the two Chabertia spp. worms were concatenated into a single alignment, and then aligned with those of 14 other Strongylida nematodes (Angiostrongylus cantonensis, GenBank accession number NC_013065 [30]; Angiostrongylus costaricensis, NC_013067 [30]; Angiostrongylus vasorum, JX268542 [31]; Aelurostrongylus abstrusus, NC_019571 [32]; Chabertia ovina Australia isolate, NC_013831 [26]; Cylicocyclus insignis, NC_013808 [26]; Metastrongylus pudendotectus, NC_013813 [26]; Metastrongylus salmi, NC_013815 [26]; Oesophagostomum dentatum, FM161882 [17]; Oesophagostomum quadrispinulatum, NC_014181 [17]; Oesophagostomum asperum, KC715826 [33]; Oesophagostomum columbianum, KC715827 [33]; Strongylus vulgaris, NC_013818 [26]; Syngamus trachea, NC_013821 [26], using the Ancylostomatoidea nematode, Necator americanus, NC_003416 as the outgroup [29]. Any regions of ambiguous alignment were excluded using Gblocks (http://molevol.cmima.csic.es/castresana/Gblocks_server.html) [34] with the default parameters (Gblocks removed 1.6% of the amino acid alignments) and then subjected to phylogenetic analysis using Bayesian Inference (BI) as described previously [35,36]. Phylograms were drawn using the program Tree View v.1.65 [37].

Results

Nuclear ribosomal DNA regions of the two Chabertia species

The rDNA region including ITS-1, 5.8S rDNA and ITS-2 were amplified and sequenced from C. ovina China isolates, and they were 852-854 bp (GenBank accession nos. KF913466-KF913471) in length, which contained 367-369 bp (ITS-1), 153 bp (5.8S rDNA) and 231-239 bp (ITS-2). These sequences were 862-866 bp in length for C. erschowi samples (GenBank accession nos. KF913448-KF913456), containing 375-378 bp (ITS-1), 153 bp (5.8S rDNA) and 239-245 bp (ITS-2).

Features of the mt genomes of the two Chabertia species

The complete mt genome sequence of C. ovina China isolate and C. erschowi were 13,717 bp and 13,705 bp in length, respectively (GenBank accession nos. KF660604 and KF660603, respectively). The two mt genomes contain 12 protein-coding genes (cox1-3, nad1-6, nad4L, cytb, atp6), 22 transfer RNA genes and two ribosomal RNA genes (rrnS and rrnL) (Table 2), but the atp8 gene is missing (Figure 1). The protein-coding genes are transcribed in the same directions, as reported for Oesophagostomum spp. [17,33]. Twenty-two tRNA genes were predicted from the mt genomes, which varied from 55 to 63 bp in size. The two ribosomal RNA genes (rrnL and rrnS) were inferred; rrnL is located between tRNA-His and nad3, and rrnS is located between tRNA-Glu and tRNA-Ser (UCN). Three AT-rich non-coding regions (NCRs) were inferred in the mt genomes (Table 2). For these genomes, the longest NCR (designated NC2; 250 bp for C. ovina China isolate and 240 bp for C. erschowi in length) is located between the tRNA-Ala and tRNA-Pro (Figure 1), have an A + T content of 83.75% and 84%, respectively.

Table 2.

Mitochondrial genome organization of Chabertia erschowi (CE) and Chabertia ovina China isolate (COC) and Australia isolate (COA)

Gene and region Positions and nt sequence lengths (bp)
Initiation/termination codons
CE COC COA CE COC COA
cox1
2-1579 (1578)
2-1579 (1578)
2-1579 (1578)
ATT/TAA
ATT/TAA
ATT/TAA
tRN A-Cys (C)
1583-1637 (55)
1583-1639 (57)
1583-1639 (57)
 
 
 
tRNA-Met (M)
1639-1697 (59)
1640-1699 (60)
1640-1699 (60)
 
 
 
tRNA-Asp (D)
1699-1758 (60)
1700-1758 (59)
1699-1759 (61)
 
 
 
tRNA-Gly (G)
1760-1816 (57)
1759-1814 (56)
1757-1814 (58)
 
 
 
cox2
1817-2512 (696)
1815-2510 (696)
1814-2509 (696)
ATT/TAA
ATA/TAA
ATA/TAA
tRNA-His (H)
2512-2566 (55)
2512-2568 (57)
2511-2567 (57)
 
 
 
rrnL
2573-3542 (970)
2572-3533 (962)
2570-3531 (962)
 
 
 
nad3
3543-3881 (339)
3534-3869 (327)
3532-3867 (336)
ATT/TAA
ATT/TAA
ATT/TAA
Non-coding region (NC1)
3882-3965 (84)
3870-3949 (80)
3868-3947 (80)
 
 
 
nad5
3967-5548 (1582)
3950-5531 (1582)
3948-5529 (1582)
ATT/ TAA
ATT/T
ATT/TAT
tRNA-Ala (A)
5549-5603 (55)
5532-5588 (57)
5530-5586 (57)
 
 
 
Non-coding region (NC2)
5604-5853 (250)
5589-5828 (240)
5587-5825 (239)
 
 
 
tRNA-Pro (P)
5854-5909 (56)
5829-5882 (54)
5826-5880 (55)
 
 
 
tRNA-Val (V)
5927-5982 (56)
5930-5984 (55)
5914-5970 (57)
 
 
 
nad6
5983-6417 (435)
5985-6416 (432)
5970-6401 (432)
ATA/ TAA
TTG/TAA
TTG/TAA
nad4L
6420-6653 (234)
6418-6651 (234)
6402-6635 (234)
ATT/ TAA
ATT/TAG
ATT/TAG
tRNA-Trp (W)
6681-6736 (56)
6655-6712 (58)
6639-6697 (59)
 
 
 
tRNA-Glu (E)
6739-6794 (56)
6740-6797 (58)
6725-6784 (60)
 
 
 
rrnS
6797-7492 (696)
6798-7493 (696)
6780-7479 (700)
 
 
 
tRNA-Ser UCN (S2)
7493-7547 (55)
7494-7548 (55)
7480-7536 (57)
 
 
 
tRNA-Asn (N)
7547-7603 (57)
7548-7605 (58)
7535-7593 (59)
 
 
 
tRNA-Tyr (Y)
7610-7666 (57)
7608-7664 (57)
7595-7652 (58)
 
 
 
nad1
7667-8539 (873)
7665-8537 (873)
7652-8524 (873)
ATT/TAA
ATT/TAA
ATT/TAA
atp6
8539-9138 (600)
8538-9137 (600)
8525-9121 (597)
ATT/TAA
ATT/TAA
ATT/TAG
tRNA-Lys (K)
9150-9211 (62)
9144-9206 (63)
9128-9191 (64)
 
 
 
tRNA-LeuUUR (L2)
9222-9276 (55)
9215-9269 (55)
9197-9252 (56)
 
 
 
tRNA-Ser AGN (S1)
9277-9335 (59)
9270-9327 (58)
9252-9304 (53)
 
 
 
nad2
9336-10175 (840)
9328-10167 (840)
9308-10147 (840)
GTT/TAA
ATT/TAA
ATA/TAA
tRNA-Ile (I)
10176-10234 (59)
10175-10235 (61)
10151-10211 (61)
 
 
 
tRNA-Arg (R)
10235-10289 (55)
10240-10294 (55)
10215-10270 (56)
 
 
 
tRNA-Gln (Q)
10290-10345 (56)
10299-10353 (55)
10271-10326 (56)
 
 
 
tRNA-Phe (F)
10346-10403 (58)
10354-10412 (59)
10326-10385 (60)
 
 
 
cytb
10404- 11516 (1113)
10413-11525 (1113)
10385-11497 (1113)
ATT/TAG
ATT/TAA
ATT/TAA
tRNA-Leu CUN (L1)
11517-11572 (56)
11529-11584 (56)
11501-11562 (62)
 
 
 
cox3
11573-12338 (766)
11585-12350 (766)
11557-12327 (771)
ATT/T
ATT/T
ATT/TAA
tRNA-Thr (T)
12339-12397 (59)
12351-12404 (54)
12323-12377 (55)
 
 
 
nad4
12398-13627 (1230)
12405-13634 (1230)
12376-13608 (1233)
TTG/TAA
TTG/TAA
ATT/TAA
Non-coding region (NC3) 13628 – 1 (75) 13635-1 (84) 13609-1 (75)      

Figure 1.

Figure 1

Structure of the mitochondrial genomes for Chabertia. Genes are designated according to standard nomenclature, except for the 22 tRNA genes, which are designated using one-letter amino acid codes, 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). “NCR-1, NCR-2 and NCR-3” refer to three non-coding regions.

Comparative analyses between C. ovina and C. erschowi

The mt genome sequence of C. erschowi was 13,705 bp in length, 12 bp shorter than that of C. ovina China isolate, and 23 bp longer than that of C. ovina Australia isolate. The arrangement of the mt genes (i.e., 13 protein genes, 2 rrn genes and 22 tRNA genes) and NCRs were the same. A comparison of the nucleotide sequences of each mt gene as well as the amino acid sequences conceptually translated from individual protein-coding genes of the two Chabertia are given in Table 3. The greatest nucleotide variation between the C. ovina China isolate and C. erschowi was in the nad2 gene (19.4% and 17.92%), whereas least differences (7.33%) were detected in the rrnS gene, respectively (Table 3). The nucleotide sequence difference between the entire mt genome of C. ovina China isolate and that of C. erschowi was 15.33%. Sequence difference between the entire mt genome of C. ovina Australia isolate and that of C. erschowi was 15.48%. Sequence difference between the entire mt genome of C. ovina China isolate and that of C. ovina Australia isolate was 4.28%.

Table 3.

Nucleotide and/or predicted amino acid (aa) sequence differences for mt protein-coding and ribosomal RNA genes among Chabertia erschowi (CE) and Chabertia ovina China isolate (COC) and Australia isolate (COA)

Gene Nucleotide length (bp)
Nucleotide difference (%)
Number of aa
aa difference (%)
CE COC COA CE/COC CE/COA COC/COA CE COC COA CE/COC CE/COA COC/COA
atp6
600
600
597
14.33
14.83
5.00
199
199
198
11.06
14.57
7.54
nad1
873
873
873
13.63
13.97
4.35
290
290
290
5.17
8.62
3.79
nad2
840
840
840
19.40
20.12
3.93
279
279
279
17.92
17.20
2.15
nad3
339
336
336
17.40
17.70
6.55
112
112
112
16.96
16.07
7.14
nad4
1230
1230
1233
17.64
18.82
5.43
409
409
410
14.67
17.80
4.88
nad4L
234
234
234
11.97
12.39
4.70
77
77
77
7.79
7.79
0
nad5
1582
1582
1582
17.51
17.32
4.87
527
527
527
14.42
13.66
2.47
nad6
435
432
432
19.08
19.31
5.56
144
143
143
15.97
17.36
1.40
cox1
1578
1578
1578
11.98
12.86
4.06
525
525
525
0.57
0.57
0
cox2
696
696
696
13.36
13.65
4.89
231
231
231
0.87
0.87
0
cox3
766
766
771
14.75
14.01
5.06
255
255
256
3.14
2.35
1.17
cytb
1113
1113
1113
16.80
16.89
4.67
370
370
370
9.73
9.73
1.08
rrnS
696
696
700
7.33
7.71
1.86
-
-
-
-
-
-
rrnL 970 962 962 13.61 13.92 3.20 - - - - - -

The difference in the concatenated amino acid sequences of the 12 protein-coding genes of the C. ovina China isolate and those of C. erschowi was 9.36%, 10% between those of the C. ovina Australia isolate and those of C. erschowi, and 2.37% between those of the C. ovina China isolate and those of C. ovina Australia isolate. The amino acid sequence differences between each of the 12 protein-coding genes of the C. ovina Australia isolate and the corresponding homologues of C. erschowi ranged from 0.57-17.92%, with COX1 being the most conserved and NAD2 the least conserved proteins (Table 3). Phylogenetic analyses of concatenated amino acid sequence data sets, using N. americanus as the outgroup, revealed that the Chabertia and Oesophagostomum were clustered together, with absolute support (posterior probability (pp) = 1.00) support (Figure 2).

Figure 2.

Figure 2

Inferred phylogenetic position of Chabertia within Strongylida nematodes. Analysis of the concatenated amino acid sequence data representing 12 protein-coding genes by Bayesian inference (BI), using Necator americanus (NC_003416) as the outgroup.

Sequence variation in complete nad2 gene was assessed among 15 individuals of Chabertia from goats and yaks. Sequences of the six C. ovina China isolate individuals were the same in length (840 bp) (GenBank accession nos. KF913472-KF913477). Nucleotide variation among the six C. ovina China isolate individuals was detected at 18 sites (18/840; 2.1%). Sequences of the nine C. erschowi individuals were the same in length (840 bp) (GenBank accession nos. KF913484-KF913492). Nucleotide variation also occurred at 23 sites (23/840; 2.7%). All 15 alignments of the nad2 sequences revealed that all individuals of Chabertia differed at 182 nucleotide positions (182/840; 21.7%). Phylogenetic analysis of the nad2 sequence data revealed strong support for the separation of C. ovina and C. erschowi individuals into two distinct clades (Figure 3A).

Figure 3.

Figure 3

Inferred genetic relationships of 15 individual Chabertia specimens. The analyses were carried out by Bayesian inference (BI) based on mitochondrial rrnS (A) and nad2 (B) sequence data, using Necator americanus as the outgroup.

Sequence variation in complete rrnS gene was assessed among 15 individuals of Chabertia from goat and yak. Sequences of the rrnS gene from the six C. ovina China isolate individuals were the same in length (696 bp) (GenBank accession nos. KF913478-KF913483). Nucleotide variation among the six C. ovina China isolate individuals was detected at seven sites (7/696; 1.0%). Sequences of the rrnS gene from the nine C. erschowi individuals were the same in length (696 bp) (GenBank accession nos. KF913457-KF913465). Nucleotide variation also occurred at 6 sites (6/696; 0.9%). All 15 alignments of the rrnS sequences revealed that all individuals of Chabertia differed at 56 nucleotide positions (56/696; 8.05%). Phylogenetic analysis of the rrnS sequence data revealed strong support for the separation of C. ovina and C. erschowi individuals into two distinct clades (Figure 3B).

The ITS-1 and ITS-2 sequences from 10 individual adults of C. ovina China isolate were compared with that of 6 individual adults of C. erschowi. Sequence variations were 0–2.9% (ITS-1) and 0–2.7% (ITS-2) within the two Chabertia species, respectively. However, the sequence differences were 6.3-8.2% (ITS-1) and 10.4-13.6% (ITS-2) between the C. ovina China isolate and C. erschowi.

Discussion

Chabertia spp. is responsible for economic losses to the livestock industries globally. Although several Chabertia species have been described from various hosts based on the microscopic features of the adult worms (e.g. cervical groove and cephalic vesicle), it is not clear whether C. erschowi is valid as a separate species due to unreliable morphological criteria. For this reason, we employed a molecular approach, so that comparative genetic analyses could be conducted.

In the present study, substantial levels of nucleotide differences (15.33%) were detected in the complete mt genome between C. ovina China isolate and C. erschowi, and 15.48% between C. ovina Australia isolate and C. erschowi. These mtDNA data provide strong support that C. erschowi represents a single species because a previous comparative study has clearly indicated that variation in mtDNA sequences between closely-related species were typically 10%-20% [13].

The difference in amino acid sequences of the concatenated 12 proteins encoded by the complete mt genome between C. ovina China isolate and C. erschowi is 9.36%, and 10% between the C. ovina Australia isolate and C. erschowi. This level of amino acid variation is higher than those of other nematodes. Previous studies of other congener nematodes have detected low level differences in 12 protein sequences. For example, differences in amino acid sequences between A. duodenale and A. caninum is 4.1% [29,38], and between Toxocara malaysiensis and Toxocara cati is 5.6% [39], and between O. dentatum and O. quadrispinulatum is 3.22% [17]. In addition, substantial levels of nucleotide differences (6.3%-8.2% in ITS-1 and 10.4-13.6% in ITS-2) were also detected between C. ovina China isolate and C. erschowi. These results also indicate that C. erschowi is a separate species from C. ovina. This proposal was further supported by phylogenetic analysis based on mtDNA sequences (Figure 3), although, to date, only small numbers of adult worms have been studied molecularly. Clearly, larger population genetic and molecular epidemiological studies should be conducted using the mt and nuclear markers defined in this study to further test this proposal/hypothesis.

Conclusion

The findings of this study provide robust genetic evidence that C. erschowi is a separate and valid species from C. ovina. The mtDNA and rDNA datasets reported in the present study should provide useful novel markers for further studies of the taxonomy and systematics of Chabertia spp. from different hosts and geographical regions.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

XQZ and GHL conceived and designed the study, and critically revised the manuscript. GHL, LZ and HQS performed the experiments, analyzed the data and drafted the manuscript. GHZ, JZC and QZ helped in study design, study implementation and manuscript revision. All authors read and approved the final manuscript.

Contributor Information

Guo-Hua Liu, Email: liuguohua5202008@163.com.

Lei Zhao, Email: xinyao-2007@163.com.

Hui-Qun Song, Email: songhuiqun@caas.cn.

Guang-Hui Zhao, Email: zgh083@163.com.

Jin-Zhong Cai, Email: caijinz@vip.sina.com.

Quan Zhao, Email: zhaoquan0825@163.com.

Xing-Quan Zhu, Email: xingquanzhu1@hotmail.com.

Acknowledgements

This work was supported in part by the International Science & Technology Cooperation Program of China (Grant No. 2013DFA31840), the “Special Fund for Agro-scientific Research in the Public Interest” (Grant No. 201303037) and the Science Fund for Creative Research Groups of Gansu Province (Grant No. 1210RJIA006).

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