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

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.

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][9][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][14][15][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].

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].

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 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.
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 ( 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  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 (L 1 and L 2 for codon families CUN and UUR, respectively; S 1 and S 2 for codon families AGN and UCN, respectively). "NCR-1, NCR-2 and NCR-3" refer to three non-coding regions. 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) 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). 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).

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.