The Mitochondrial Genome of Cylicocyclus elongatus (Strongylida: Strongylidae) and Its Comparative Analysis with Other Cylicocyclus Species.

Cylicocyclus elongatus (C. elongatus) is one of the species in Cylicocyclus, subfamily Cyathostominae, but its taxonomic status in Cylicocyclus is controversial. Mitochondrial (mt) genome is an excellent gene marker which could be used to address the taxonomy controversy. In the present study, the complete mt genome of C. elongatus was determined, and sequence and phylogenetic analyses were performed based on mtDNA data to determine the classification of C. elongatus. The circular complete mt genome of C.elongatus was 13875 bp in size, containing 12 protein-coding genes (12 PCGs), 22 transfer RNA (tRNA) genes, 2 ribosomal RNA (rRNA) genes, and 2 non-coding regions (NCRs). The A + T content of C. elongatus complete mt genome was 76.64%. There were 19 intergenic spacers with lengths of 2-53 bp and 2 overlaps with lengths of 1-2 bp in the impact complete mt genome. ATT and TAA were the most common start and termination codons of 12 PCGs, respectively. Comparative analyses of mt genomes nucleotide sequence and amino acid sequence showed that there were higher identities between C. elongatus and five other Cylicocyclus, rather than with P. imparidentatum. Phylogenetic analyses based on concatenated nucleotide sequences of 12 PCGs of 23 species in the family Strongylidae showed that C. elongatus was closely related to Cylicocyclus species, rather than P. imparidentatum. We concluded that C. elongatus was a member in Cylicocyclus based on comparative and phylogenetic analyses of mt genome sequences. The data of the complete mt genome sequence of C. elongatus provide a new and useful genetic marker for further research on Cyathostominae nematodes.

1. Introduction

Cyathostominae (Strongylida: Strongylidae) nematodes are a group of significant pathogens of equines which inhabit the large intestine [1]. There are more than 50 species in the subfamily, and the occurrence of co-infection of different kinds of parasites is quite common in hosts [2]. Co-infection of these adult parasites can cause dropsy, diarrhoea, and weight loss, and the larvae can cause fatal cyathostominosis [3,4,5].

Cyathostomins are considered to be one of the primary parasites of equids. The taxa controversies in the subfamily have been discussed for at least one century. Researchers have primarily presented their views on the classification at the generic level of these nematodes [6,7]. Cylicocyclus Ihle, 1922 is the largest genus of Cyathostominae, and the taxonomic status of some species in the genus is still controversial [8]. C. elongatus is one of the members in the genus, which has a greatly elongated bursa and differs from congeners [1]. Phylogenetic analyses combining internal transcribed spacers (ITS)-1 and ITS-2 data suggested that C. elongatus clustered with the comparative species (Petrovinema poculatum and Poteriostomum imparidentatum), indicating that C. elongatus represents members of other genera [8].

Mt genomes have been successfully applied in parasite taxonomy, population genetics, and systematics, because of characteristics such as strict maternal inheritance and comparatively conserved genomic structure [9,10]. In previous studies, mt genomes have been used to address a lot of controversies on the status of certain taxa, such as the taxonomy of Triodontophorus, the identification of species complex, etc. [11,12,13]. Now, 13 complete mt genomes of Cyathostominae nematodes have been determined, including 5 complete mt genomes in Cylicocyclus [10,11,12,14]. However, the complete mt genome of C. elongatus has not been completed. Thus, to identify the classification of C. elongatus, the aims of the present study were to sequence the complete mt genome of C. elongatus, to compare the obtained complete mt genome sequences among congeneric species, and to reconstruct the phylogenetic relationships to assess the status of C. elongatus within the Cyathostominae.

2. Materials and Methods

2.1. Parasites and Molecular Identification of Specimens

Adult nematodes of C. elongatus were collected from naturally infected horses from a slaughterhouse in Daqing, Heilongjiang Province, China. The nematode was identified to the species level based on morphological features and molecular biology techniques [1]. Total genomic DNA was extracted from one adult C. elongatus by the TIANamp Genomic DNA Kit according to the manufacturer’s instructions (TIANGEN Biotech, Beijing, China) and stored at −20 °C until use. The ITS of C. elongatus were amplified for the identification of molecular biology using 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 previously [15].

2.2. The Amplification and Annotation of C. elongatus Complete Mt Genome

The primers of the complete mt genome amplification in this study were designed according to the mt genomes sequences of the congenic species. The conservative sequences of each region in those of congener species were selected to use as the candidate primers. The evaluation of these primers was performed using Oligo 6.0. Eight primers were used to amplify the complete mt genome as 8 overlapping fragments from genomic DNA (Table S1). PCR was conducted in a 50 µL reaction mixture containing 1 × Ex Taq Buffer, 0.2 mM dNTP Mixture, 0.625 U TaKaRa Ex Taq, 0.4 µM of each primer, and 1 µL gDNA under the following condition: an initial denaturing at 94 °C for 5 min, followed by 35 cycles (denaturing at 94 °C for 1 min, annealing at 55–40 °C for 30 s, and extension at 72 °C for 1–2 min), and a final extension at 72 °C for 7 min. All positive amplicons were purified, cloned into pMD 18-T vector, and transformed into E. coli DH5α. The positive clones were sent to Sangon Biotech Co., Ltd. (Shanghai, China) for sequencing.

The complete mt genome of C. elongatus was aligned with those of Cyathostominae nematodes [11,14,16]. Gene boundaries including 12 PCGs, 22 tRNAs, and 2 rRNAs were determined by the species in Cylicocyclus using Clustal X 1.83, MEGA X, and tRNAscan-SE 2.0 (tRNAscan-SE Search Server (ucsc.edu)) (accessed on 11 August 2020) [17,18].

2.3. Comparative Analyses of Cylicocyclus Species and P. imparidentatum Mt Genomes

Comparisons of C. elongatus complete mt genome with those of five species (Cylicocylus ashworthi, Cylicocyclus insigne, Cylicocyclus radiatus, Cylicocyclus auriculatus, and Cylicocyclus nassatus) in Cylicocyclus available in GenBank were performed, including lengths, identities, A + T contents, and protein codons [10,16,19]. Percentages of A + T content of each gene/region was computed using DNAStar (v. 12.1) [20]. GC and AT skews were calculated according to the following formulas: AT skew = (A − T)/(A + T) and GC skew = (G − C)/(G + C). The identities of amino acids and nucleotides sequences among 6 Cylicocyclus species and P. imparidentatum were calculated by MegAlign 5.01 [20].

2.4. Phylogenetic Analyses of 23 Strongylidae Nematodes

Phylogenetic analysis in the present study was based on the concatenated nucleotide sequences of 12 PCGs and complete mt genome nucleotide sequences of 23 Strongylidae nematodes available in GenBank, respectively. Oxyuris equi was used as the outgroup. The sequences of 12 PCGs of 23 Strongylidae nematodes were aligned using MAFFT 7.471 and then concatenated into a single alignment [21]. Sites of ambiguous alignment were eliminated using the online server Gblocks (Phylogeny.fr: Gblocks) (accessed on 12 October 2020). Maximum likelihood (ML) method was used to reconstruct the relationships by Mega X.

3. Results

3.1. The Annotation of C. elongatus Complete Mt Genome

The complete mt genome of C. elongatus was 13,875 bp in length. It contained 36 genes, including 12 PCGs (cox1-cox3, cytb, atp6, nad1-nad6, and nad4L), 22 tRNAs, and 2 rRNAs (Table 1, Figure 1). The A + T and G + C contents of C. elongatus complete mt genome were 76.64% and 23.36%, with 0.39 and −0.19 AT skew and GC skew, respectively. These PCGs started at ATT and TTG, and stopped at TAA and TTG. Cox3 used the incomplete codon “T” as a stop codon. In the mt genome of C. elongatus, there were 19 spacer regions and two short overlaps. The spacer regions ranged from 2 bp to 52 bp in length, and the size of overlaps were all 1–2 bp.

Table 1

The gene composition, position, codons, and spacer/overlap regions of complete mt genome of C. elongatus.

Genes/Regions	Positions and Sequence Lengths (bp)	Initiation and Stop Codons	Intergenic Nucleotides
cox1	1–1578 (1578)	ATT/TAA	0
tRNA-Cys (C)	1579–1634 (56)	–	0
tRNA-Met (M)	1648–1706 (59)	–	13
tRNA-Asp (D)	1707–1765 (58)	–	0
tRNA-Gly (G)	1787–1843 (57)	–	22
cox2	1844–2539 (696)	ATT/TAA	0
tRNA-His (H)	2540–2593 (54)	–	0
rrnL	2601–3568 (968)	–	7
nad3	3572–3907 (336)	ATT/TAA	4
nad5	3918–5501 (1584)	ATT/TAG	10
tRNA-Ala (A)	5501–5556 (56)	–	−1
LNCR	5557–5842 (286)	–	0
tRNA-Pro (P)	5843–5897 (55)	–	0
tRNA-Val (V)	5931–5985 (55)	–	33
nad6	5986–6420 (435)	ATT/TAA	0
nad4L	6473–6706 (234)	ATT/TAA	52
tRNA-Trp (W)	6732–6787 (56)	–	21
tRNA-Glu (E)	6815–6874 (60)	–	27
rrnS	6877–7575 (699)	–	2
tRNA-SerUCN (S2)	7576–7631 (56)	–	0
tRNA-Asn (N)	7630–7684 (55)	–	−2
tRNA-Tyr (Y)	7693–7748 (56)	–	8
nad1	7749–8621 (873)	TTG/TAA	0
atp6	8632–9231 (600)	ATT/TAA	10
tRNA-Lys (K)	9248–9309 (62)	–	16
tRNA-LeuUUR (L2)	9328–9382 (55)	–	18
tRNA-SerAGN (S1)	9383–9435 (53)	–	0
nad2	9436–10,281 (846)	TTG/TAA	0
tRNA-Ile (I)	10,288–10,346 (59)	–	6
tRNA-Arg (R)	10,374–10,436 (63)	–	27
tRNA-Gln (Q)	10,444–10,498 (55)	–	7
tRNA-Phe (F)	10,505–10,559 (55)	–	6
cytb	10,560–11,672 (1113)	ATT/TAA	0
tRNA-LeuCUN (L1)	11,683–11,737 (55)	–	11
cox3	11,738–12,506 (769)	ATT/T	0
tRNA-Thr (T)	12,507–12,564 (58)	–	0
nad4	12,565–13,794 (1230)	TTG/TAA	0
SNCR	13,795–13,875 (81)	–	0
Total size (bp)	13,875	–

Note, “–” is no data.

Figure 1

The composition, size, and transcription direction of C. elongatus complete mt genome.

There were two rRNAs (rrnL and rrnS) in the mt genome of C. elongatus, with lengths of 968 bp and 699 bp, respectively. The sizes of 22 tRNAs in the mt genome of C. elongatus were 53–63 bp. Moreover, a large NCR with a length of 286 bp and a shorter NCR with a length of 81 bp were found in the mt genome of C. elongatus. The A + T contents of the large and shorter NCRs were 85.31% and 81.48%, respectively.

3.2. Comparative Analyses of Mt Genomes among Cylicocyclus Species and P. imparidentatum

A comparison of amino acids and nucleotides sequence identities and lengths was made among six Cylicocyclus species and P. imparidentatum (Table 2 and Table S1). The identities of nucleotides sequences of the complete mt genomes among six Cylicocyclus species and P. imparidentatum were 82.9–90.7%. The identity of nucleotides sequence of complete mt genomes between C. elongatus and P. imparidentatum was 84.3%. These results showed that the identities between C. elongatus and P. imparidentatum were lower than those among Cylicocyclus species, indicating that C. elongatus had higher identities with congeneric nematodes (Table 2).

Table 2

The identities of nucleotides sequences of complete mt genomes among 6 Cylicocyclus species and P. imparidentatum.

Species	Identity Nts (%)
	C. el	C. as	C. i	C. au	C. n	C. r	P. i
Total Size (bp)	13,828	13,817	13,876	13,836	13,831	13,846	13,875
C. as	87.1	–	–	–	–	–	–
C. i	88.1	88.2	–	–	–	–	–
C. au	87.2	87.4	89.6	–	–	–	–
C. n	87.1	90.7	88.0	87.2	–	–	–
C. r	87.4	90.7	88.0	87.6	89.4	–	–
P. i	84.3	82.9	83.6	82.9	83.0	83.3	–

Note, “–” is no data.

The lengths of 12 PCGs of C. elongatus were the same as those of other Cylicocyclus species, except for cox3 gene, which was 3 bp longer than that in the other five Cylicocyclus species. The size of cox3 in C. elongatus was longer than that in P. imparidentatum in Cyathostominae, but the size of nad4 was shorter (Table S1). As regards the identities of each mt gene, P. imparidentatum has lower identities to all five of the Cylicocyclus species, than those among Cylicocyclus species.

The A + T contents and skewness of C. elongatus were similar to those of Cylicocyclus species; however, the A + T content of 1st coding position of C. elongatus was higher than those in Cylicocyclus species and P. imparidentatum. The A + T skewness of first and second coding positions of C. elongatus was higher than those of Cylicocyclus species and P. imparidentatum, but the A + T skewness of third coding positions was lower than those of others in the present study (Figure S1).

3.3. Phylogenetic Analyses of 23 Species in the Family Strongylidae

In the current study, the phylogenetic relationship was reconstructed based on concatenated nucleotide sequences of 12 PCGs from 23 species in the family Strongylidae using ML method (Figure 2). The topological structure of the phylogenetic trees divided Strongylidae into two clades. One clade included 2 species, Strongylus, and another clade grouped 21 other species together in the family Strongylidae. C. elongatus and congeneric species are closely related to each other rather than to P. imparidentatum. Moreover, based on the tree in the current study, the results showed C. elongatus formed a distinct branch in the clade of Cylicocyclus, indicating that C. elongatus might be relatively closely related to other Cylicocyclus species that did not obtain complete mt genomes. The phylogenetic relationships reconstructed by nucleotide sequences of complete mt genomes of 23 species of the family Strongylidae were similar to those of Figure 2 (Figure S2). In the trees, C. elongatus was also closely related to congeneric species rather than to P. imparidentatum.

Figure 2

Phylogenetic analyses reconstructed using concatenated nucleotide sequences of 12 PCGs of complete mt genomes in 23 Strongylidae species. The tree was developed using ML method. Oxyuris equi is an outgroup. C. elongatus in current study is underlined.

4. Discussion

The complete mt genome of C. elongatus was described first in the present study. Its composition, gene orders, transcription direction, and codon usages were the same as other Cyathostominae parasites [11,12,14]. Its sizes were slightly shorter than that of C. ashworthi (13,876 bp), longer than those of others in Cyathostominae, such as P. imparidentatum, C. nassatus, and C. radiatus [11,12,14]. The A + T contents of C. elongatus complete mt genomes were 76.64%, which was similar to those of nematodes such as C. radiatus, Cy. catinatum, and Cs. minutus [11,14]. The complete mt genome of C. elongatus had an impact structure that was similar to C. insigne, C. radiatus, and P. imparidentatum [10,11,14].

Comparison of nucleotide and amino acid sequences of mt genomes among six Cylicocyclus species and P. imparidentatum was performed. In previous studies, the identities of nucleotide sequences of complete mt genomes in congeners were 94.6% identity between Cyathostomum pateratum and Cyathostomum catinatum, 84.7% identity between Chabertia ovina and Chabertia erschowi, and 86.0% identity between Triodontophorus serratus and Triodontophorus nipponicus, respectively [22,23,24]. In the present study, C. elongatus had higher identities to congeneric nematodes rather than to P. imparidentatum, indicating that C. elongatus was more similar to Cylicocyclus species in terms of nucleotide sequences [11,14,16]. The results also indicated that C. elongatus might be a member to Cylicocyclus. AT bias in nucleotide composition of the complete mt genomes reflected a bias in the amino acid composition of proteins. Highly AT-rich regions for each genome may represent the origin of replication [10,25]. In the present study, higher A + T skewness of first and second coding positions were found in the complete mt genome of C. elongatus indicating that it might be more easily changed in the evolution.

In the current study, the phylogenetic relationships were reconstructed by nucleotide sequences. The topological structures of phylogenetic trees were similar with different data, which all supported that C. elongatus was closely related to Cylicocyclus, rather than to P. imparidentatum. The results were inconsistent with a previous study, which showed that C. elongatus was closely related to P. imparidentatum, C. ultrajectinus, and P. poculatum based on the combined ITS-1 and ITS-2 sequences [8]. The discrepancy was caused by the different gene markers used for evolutionary analysis. In many previous studies, it has been demonstrated that mt genomes are better gene markers for phylogenetic analyses, because of characteristics such as strict maternal inheritance, apparent lack of recombination, rapid evolutionary rate, and comparatively conserved genomic structure [9,10,25]. ITS, as the important region in rDNA, was more suitable for species identification. Though we did not obtain the complete mt genomes of C. ultrajectinus and P. poculatum, we also concluded that C. elongatus did not closely relate to P. imparidentatum, and C. elongatus did not represent members of other genera. We also expect to sequence more complete mt genomes in Cyathostominae to discuss and support these views. Moreover, for all trees in the present study, it is interesting that Cyathostomum tetracanthum arose within Cylicocyclus. The result was not in conformity with the classification based on morphology. More complete mt genomes of Cy. tetracanthum should be obtained to explain the reason. Further, the taxa in Cyathostominae may be redefined based on mt genomes data.

5. Conclusions

The present study first reported the complete mt genome of C. elongatus, which contained 12 PCGs, 22 tRNAs, 2 rRNAs, and 2 NCRs. Our findings further supported that C. elongatus belong to Cylicocyclus based on mt genomes. Our results also provide a beneficial reference for taxonomy, population genetics, and systematics studies of Cyathostominae species.