Aonchotheca (Nematoda: Capillariidae) is validated as a separated genus from Capillaria by both mitochondrial and nuclear ribosomal DNA | Parasites & Vectors

First molecular identification of A. putorii in China

The mt cox1 sequence of PCR amplicon of 652 bp (GenBank accession no: OP363931) using primer pair JB3-JB4.5 as previously described [21] showed only 81.7% identities to GenBank no. MH665361 by BLAST search, which was a Capillaria sp. This low identity indicates the parasitic worm may be one member of the family Capillariidae and relate to Capillaria sp. species. We then PCR amplified and sequenced 18S rRNA of 1813 bp. The newly obtained 18S rRNA sequence (GenBank accession no: OP028951) of this nematode showed 100% identity to GenBank no. LC052349 of A. putorii. Therefore, the nematodes recovered from the small intestine of hedgehogs in Beijing, China, are identified as A. putorii, which is the first report in the country to our knowledge. Aonchotheca. putorii has been found in a wide range of geographical locations and several mammalian hosts including minks, weasels, martens, racoons, and even the domestic cats [17].

Divergency of 18S rRNA gene in the family Capillariidae

We then further analyzed the genetic divergence of 18S rRNA of 19 members in the family Capillariidae (Additional file 1: Table S2), which ranged from 0 to 14.1%. The 18S rRNA of Capillaria spinulosa (Linstow, 1890) was the most variable (10.9–14.1%) (Additional file 1: Table S2). Kołodziej-Sobocinska et al. reported no significant genetic divergence of 18S rRNA genes was observed in A. putorii collected from different geographical distributions and various hosts [20]. However, Tamaru et al. found significant nucleotide variation of 18S rRNA genes among A. putorii collected from various animals [19]. Our result showed clear divergence in 18S rRNA sequences of worms recovered from Japan, America, and China. Specifically, comparing currently available 500 bp of 18S rRNA isolated from American A. putorii with those from China and Japan, up to 4.00% divergency was found. Furthermore, up to 8.94% (31 nucleotide substitutions) difference was found between Japanese and Chinese isolates (Additional file 1: Table S3).

First complete mt genome of A. putorii

We then tried to decode the whole mt genome of A. putorii. Due to the lack of mt genome of closely related species, we took an unusual strategy using the newly cox1 DNA sequence just mentioned and next generation sequencing technique as outlined earlier. The complete mitogenome of A. putorii was 14,168 bp in length and had been uploaded into GenBank with the accession number OP028209. It comprised 37 genes (Table 2, Fig. 1) including 13 PCGs (atp6, atp8, cox1-cox3, cytb, nad1-nad6, and nad4L), 2 rRNAs (12S rRNA and 16S rRNA) and 22 tRNAs (Fig. 1). Its genetic arrangement was consistent with those of published Eucoleus annulatus (Molin, 1858) López-Neyra, 1946 (MW999680) [33], and Pseudocapillaria tomentosa (Dujardin, 1843) Lomakin and Trofimenko, 1982 (MZ708958), but different from that of Capillaria sp. (MH665363). The findings indicated at least one inversion between Aonchotheca − Eucoleus − Pseudocapillaria species and Capillaria sp., though four tRNAs (tRNA-Gly, tRNA-Tyr, tRNA-Cys and tRNA-His) were lacking in the Capillaria sp.. The tRNA-Gln was found on the L-strand between tRNA-Cys and tRNA-Gly in A. putorii, E. annulatus, and P. tomentosa, while it was located between nad5 and nad4 in Capillaria sp. (not shown).

Twenty-five intergenic regions ranging from 1 to 72 bp in length were found scattered throughout the mitogenome. The highest AT content of 84.8% was in intergenic regions, and AT-skew in these regions was positive while the value of GC-skew in them was “0” (the number of G bases was roughly equal to C bases, Table 3). The mitogenomes of nematodes usually contain two non-coding regions (NCRs) with different sizes [15, 33, 34]. The length of AT-rich was 137 bp in Necator americanus Stiles, 1902 [35], 886 bp in Ascaris suum Goeze, 1782 [36], and approximately 7 kb in Hoplolaimus columbus Sher, 1963 [37]. As far as members of the family Capillariidae were concerned, no AT-rich region was found in the mitogenome of A. putorii, but it was 306 bp, 121 bp and 99 bp in E. annulatus [33], Capillaria sp. (MH665363) and P. tomentosa (MZ708958), respectively.

Aonchotheca putorii mitogenome was A-T biased; they accounted for 78.0% (Table 3), which is consistent with other capillariid nematodes [33]. Furthermore, T base (42.2%) was the most frequent and C (9.3%) the least. Both AT-skew and GC-skew included negative and positive values, ranging from − 0.349 (nad6) to 0.031 (NCRs) and from − 0.345 (nad2) to 0.345 (cytb), respectively.

Characteristics of protein-coding genes

Aligning with available sequences of capillariid nematodes (GenBank no. MZ708958, NC_056391 and MH665363), the boundaries of each PCG were determined, and the initiation/termination codons and the directions of translation were identified. For A. putorii, the longest PCG was cox1 (1584 bp), followed by nad5 (1521 bp). The rest were nad4 (1,263 bp), cytb (1136 bp), nad1 (912 bp), atp6 (909 bp), nad2 (900 bp), cox3 (774 bp), cox2 (684 bp), nad6 (459 bp), nad3 (301 bp), nad4L (249 bp) and atp8 (146 bp).

ATN was exclusively used as an initiation codon in all PCGs where N was T, A or G. ATT was the most frequent initiation codon used in six PCGs (nad2, nad6, atp8, nad3, cox1 and nad1), followed by ATA in five (for nad5, nad4, nad4L, atp6 and cox2). The least favored start codon was ATG used only in cytb and cox3. The complete termination codon TAA was the most common one, which was used in all PCGs except cytb, atp8, and nad3, in which incomplete stop codons TA and T were used, respectively. Start codons ATT, ATA and ATG were shared among those species, while TTG was only used in E. annulatus and Capillaria sp., and ATC was unique in P. tomentosa. Complete termination codon TAA was prevalent among those species, but TAG was used in E. annulatus, Capillaria sp. and P. tomentosa excluding A. putorii.

The GC- and AT-skews for mitogenome are calculated as a measure of the compositional asymmetry and effective assistant of replication [38, 39]. For the A. putorii mitogenome, the AT- and GC-skews were − 0.082 and − 0.157, respectively, which showed no clear trend of A or T bases, but a significant use of C bases compared with other nematodes [40]. Similar bases trend also showed in PCGs. AT and GC-skews of 13 PCGs were from − 0.349 (nad6) to 0.071 (nad4L) and from − 0.345 (nad2) to 0.345 (cytb) (Table 3), indicating a trend of A bases among those PCGs that would have an impact on RSCU. RSCU represents an intuitive reflection of the use of codon bias, and a higher RSCU indicates a higher codon usage bias. Within codons encoding amino acids, there was an obvious bias of T- or A-rich codons. For example, Leu was encoded by TTA, TTG, CTT, CTC, CTA and CTG, but the RSCU value of TTA was 3.91 higher than that with C and G residues (Table 4).

In PCGs, Ka/Ks test was helpful for better understanding the selective constraints, evolutionary changes, sexual selection and disease resistance [41]. Generally, the ratio of Ka/Ks being > 1 represents rapid evolution of a protein gene and a positive selection and changes in protein’s functions. In contrast, < 1 indicates a negative selection and selectively purified among genes [42, 43]. In the present study, the Ka/Ks ratios of the genes atp6, cox3, nad3, nad4 and nad6 ranged from 1.07 to 1.72 (Fig. 2), indicating these genes had experienced a positive selection or had undergone functional changes. The Ka/Ks values of atp8, cox1, cox2, cytb, nad1, nad2, nad4L and nad5 were < 1 (Fig. 2), suggesting they were highly constrained within the family Capillariidae. Consistent with previous reports, the findings also indicated that cox1 was selectively purified and might play an adaptive role in the evolutionary process of Capillariidae species, and it can be used as useful marker to identify and distinguish capillariid species. Interestingly, atp8 gene was not only found in those species, but under purifying selection across Capillariidae, though it was variable protein-gene or even missing in some nematodes [44].

Transfer and ribosomal RNA genes

All 22 tRNAs were obtained in the present study ranging from 54 to 71 bp in length. The predicted secondary structures of most tRNAs identified in this study were similar to those found in other nematodes, and the TΨC stem loop and variable loop were replaced by a TV-replacement loop structure, which was common in the mitogenomes of nematodes excluding Trichinella spiralis (Owen, 1835) Railliet, 1895 [44,45,46]. Consistent with previous reports, the predicted structure of tRNA-Ser (AGN and UCN) lacked the DHU-arm, which was replaced by 4–5 nucleotide residues [47, 48]. In the present study, 9 of 20 tRNAs (tRNA-His, tRNA-Ile, tRNA-Arg, tRNA-Lys, tRNA-Leu^CUN, tRNA-Leu^UUR, tRNA-Met, tRNA-Trp, and tRNA-Tyr) presented relatively standard “cloverleaf” secondary structures like those of T. spiralis [44] (Fig. 3). The length of nine TΨC stems was from 2 to 5 bp, and the length of variable arms between the anticodon loop and TΨC stem-loop ranged from 2 to 8 bp.

The locations and boundaries of rrnL (16S rRNA) and rrnS (12S rRNA) were identified by alignment with available Capillariidae species (NC_056391). The typical arrangement of the rrnL was located between tRNA-Val and atp6, and the rrnS was situated in tRNA-Ser^AGN and tRNA-Val, similar to Capillaria sp. (MH665363), E. annulatus (MW999680) [33] and P. tomentosa (MZ708958). The length of these two rRNAs was 864 bp and 693 bp, respectively (Table 2).

Comparative analyses of mitochondrial sequence and nuclear 18S rRNA

Comparisons of nucleotide sequences of mt genes are listed in Table 5, as were amino acids of PCG. Transfer RNA genes were not included in the table because Capillaria sp. missed four of them (tRNA-Tyr, tRNA-His, tRNA-Gly and tRNA-Cys). Obvious differences at both nucleotide and amino acid sequences were observed in all 13 PCG. Specifically, the differences of nucleotide and amino acid between A. putorii and Capillaria sp., A. putorii and E. annulatus, and A. putorii and P. tomentosa were 21.96–44.37%, 23.39–48.23% and 17.57–38.36% (Table 5) and 17.80–66.00%, 19.89–67.35% and 12.69–58.33%, respectively (Table 5). Furthermore, cox1 and atp8 were the most and the least conserved PCG, respectively. The differences of rRNAs nucleotide sequences were 18.61–30.99% in rrnL and 22.03–34.15% in rrnS (Table 5). It is acceptable that divergence of mt DNA sequences between species in nematodes was 10–20% [49]. The differences observed in the current study in nucleotide sequences of all 13 PCG and two rRNA genes ranged from 17.57 to 48.23%, and the divergences in amino acids were from 12.69 to 67.35% clearly indicated A. putorii was a distinct species from other Capillariidae species. The differences among Aonchotheca, Capillaria, Eucoleus, and Pseudocapillaria were higher than species level and further suggested Aonchotheca was a distinct genus.

Phylogenetic analyses

BI and ML trees of 18S rRNAs of Capillariidae species (Additional file 2: Figure S1) showed similar topologies to previous studies [5, 50]. These indicated some Capillaria species did not belong to this genus. The tree further proved that genera Capillaria and Eucoleus were all monophyly and more related, the genus Aonchotheca might be paraphyly, and results also showed a closer relationship among genera Aonchotheca, Calodium, Pearsonema, Pseudocapillaria, and Baruscapillaria with moderate supports (Additional file 3: Figure S2).

Using X. pachtaicum and X. rivesi as the outgroups in analyzing phylogenetic relationships within class Trichinellida, both BI and ML trees displayed similar topological and systematic relationships (Fig. 4), which was consistent with previous data [33, 51]. The families Capillariidae, Trichinellidae and Trichuridae were clearly in their own clades. In accord with previous studies based on complete mitogenome or 18S rRNA sequences [33, 50, 51], our results also showed those families were monophyletic with Capillariidae and Trichuridae forming sister taxa of high statistical support (Bpp = 1, Bf ≥ 98).

In the family Capillariidae, Capillaria was a sister taxon genus to Eucoleus, Pseudocapillaria and Aonchotheca, of high support (Bpp = 1, Bf ≥ 99). Furthermore, there was a closer relationship between genera Aonchotheca and Pseudocapillaria, and between Capillaria and Eucoleus, which was similar to the previous results by analyzing cox1 and 18S rRNA [4, 5, 50, 51], indicating cox1 and 18S rRNA genes were useful markers to mark capillariids. In addition, the distances between the Capillaria species (Capillaria sp.) and the other three capillariid nematodes (E. annulates, P. tomentosa and A. putorii) were longer than the distances within other trichinellid species, which further clarified long genetic distance among those genera. These phylogenetic results also indicated that Eucoleus, Pseudocapillaria, and Aonchotheca were three distinct genera from Capillaria, supporting the accuracy of Moravec taxonomic revision [3, 5, 50]. However, there are still taxonomic controversies within Capillariidae concerning phylogenomics and systematic status [4, 19, 33] due to the limited numbers of complete mitogenome in other Capillariidae species, like Baruscapillaria, Calodium, Paracapillaria and Pearsonema.