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. 2024 Nov 12;9(11):1518–1521. doi: 10.1080/23802359.2024.2427110

The complete mitochondrial genome of Cylicocyclus ultrajectinus (Ihle, 1920)

Yan Hao a, Bing Li a, Liqun Ma b, Minglu Xu a, Peizhi Niu a, Yanzhen Bu b,✉
PMCID: PMC11559021  PMID: 39539983

Abstract

In this study, the mitochondrial genome of Cylicocyclus ultrajectinus (Ihle, 1920) was sequenced for the first time using next-generation sequencing technology, and its compositional characteristics, structure, and phylogenetic relationship with other strongylid nematodes were analyzed by biological software. The results showed that these sequences contained 12 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and 2 non-coding regions, all exhibiting a significant AT bias. Phylogenetic studies showed that C. ultrajectinus formed a distinct branch form other Cylicocyclus nematodes. This study contributes to the mitochondrial genome database of Strongylidae, laying a foundation for genetic variation, molecular classification, and evolutionary studies of strongylid nematodes.

Keywords: Cylicocyclus, Mitochondrial genome, Phylogenetic analysis, Strongylid nematodes

Introduction

Cylicocyclus ultrajectinus belongs to Cylicocyclus in the subfamily Cyathostominae of the family Strongylidae. Its body is medium-sized and milky white, with a prominent mouth collar. Males possess broad copulatory bursas, while females have straight tails (Lichtenfels et al. 2008; Jian et al. 2022). C. ultrajectinus, a significant strongylid nematode inhabiting the equine intestinal tract (Lichtenfels et al. 2008; Clark et al. 2018), often coexists with other strongylid nematodes, leading to various clinical symptoms such as abdominal pain, diarrhea, fever, and colonic inflammation in hosts. In severe cases, this condition can result in death and significant economic losses in stockbreeding (Morariu et al. 2016; Kay et al. 2019). Currently, research on C. ultrajectinus conducted by scholars, both domestically and internationally, has mainly focused on morphological characteristics, life history, epidemiology, and disease prevention and control (Lichtenfels et al. 2008; Hinney et al. 2011; Jian et al. 2022). However, no relevant data have been reported regarding the complete mitochondrial genome of C. ultrajectinus. To address this gap, this study obtained the complete genome sequence by sequencing, assembling, and annotating the mitochondrial genome of C. ultrajectinus. It constructed a phylogenetic tree based on 12 protein-coding genes using maximum-likelihood (ML) methods, thereby exploring the phylogenetic relationship between C. ultrajectinus and other strongylid nematodes. These findings provide fundamental data for future studies on population classification and mitochondrial genomics of nematodes in the genus Cylicocyclus in equines.

Materials and methods

Sample collection and DNA extraction

In February 2023, specimen of C. ultrajectinus (Figure 1) were detected in the intestinal tract of donkeys in Xinxiang City, Henan Province, China (114 190 E, 33 140 N). The sample (voucher number: JY0069, Liqun Ma, 2320457866@qq.com) was preserved in 95% ethanol and stored at −20 °C in the Biological Specimen Museum of Henan Normal University. Total DNA extraction from the female samples was performed using the Universal Genomic DNA Kit, and the DNA quality and concentration were evaluated using 1% agarose gel electrophoresis and the Qubit®DNA Assay Kit.

Figure 1.

Figure 1.

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The specimen of a female Cylicocyclus ultrajectinus, photographed by Yan Hao. (a) Anterior end; (b) female tail.

Genome sequencing and annotation

The DNA samples were sent to Shenzhen Huitong Biotechnology Co., Ltd. (Shenzhen, China). For next-generation sequencing methods, the sequences were initially assembled using SPAdes v3.14.1 (Bankevich et al. 2012) software. Subsequently, PRICE and MITObim were used to extend and merge the assemblies. Following the assembly, the mitochondrial genome sequences were annotated using the MITOS online software (http://mitos.bioinf.uni-leipzig.de/index.py) (Bernt et al. 2013). Annotation results underwent redundancy removal, and the positions of gene initiation and termination codons were determined through manual correction. The read coverage plot is depicted in Figure S1. The complete mitochondrial genome sequence of C. ultrajectinus was deposited in the GenBank database under the accession number OR834862.

Phylogenetic analysis

Using Ancylostoma ceylanicum as the outgroup, the optimal model, GTR + F + R4, was automatically calculated and screened based on 12 protein-coding genes of 23 species of strongylid nematodes using the Model Finder (Kalyaanamoorthy et al. 2017) within the PhyloSuite software suite. IQ-TREE (Nguyen et al. 2015) was used for calculation and analysis, with the confidence of the bootstrap test set to 5000 repetitions to evaluate the node support rate of the phylogenetic tree and construct the ML tree.

Results

The complete mitochondrial genome of C. ultrajectinus measures 13,984 bp in total length, forming a closed circular double-stranded DNA molecule (Figure 2). The base composition is as follows: A 29.7%, C 7.5%, G 17.6%, and T 45.2%, resulting in an A + T content of 74.9% and a G + C content of 25.1%. The AT skew value is −0.207, while the GC skew value is 0.402. Within this mitochondrial genome, there are a total of 36 genes comprising 12 protein-coding genes (ND1–ND6, ND4L, COX1–COX3, CYTB, and ATP6), 22 tRNAs, and 2 rRNAs. Additionally, there are 2 non-coding regions, lacking the ATP8 gene, as in most other nematodes, arranged in a GA3 type (Liu et al. 2013). All genes are encoded on the positive strand, exhibiting a relatively compact gene structure. The total length of the 12 protein-coding genes in C. ultrajectinus is 10,291 bp, encoding 3419 amino acids (excluding termination codons). The initiation codons include TTG (ND1, ND2, ND4), ATT (ATP6, COX1, COX3, ND3, ND5, ND6, ND4L), and ATA (CYTB, COX2), while the termination codons comprise TAA (ND1, ND2, CYTB, ND4, COX1, ND3, ND5, ND6, ND4L) and TAG (ATP6, COX2). Specially, the gene coding for COX3 is terminated with an incomplete ‘T’.

Figure 2.

Figure 2.

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Graphic representation of the mitochondrial genome of Cylicocyclus ultrajectinus.

The length of the 22 tRNAs ranges from 53 to 68 bp. The 16S rRNA is located between tRNAHis and ND3, while the 12S rRNA is located between the encoding genes tRNAGlu and tRNASer2.

Based on the concatenated DNA sequences of the 12 protein-coding genes in the mitochondria and utilizing Ancylostoma ceylanicum as the outgroup, a phylogenetic tree of strongylid nematodes was established, revealing two main branches. Strongylus, belonging to the Strongylinae subfamily, formed a smaller branch, while all nematodes of the Cyathostominae subfamily and the Triodontophorus genus within the Strongylinae subfamily clustered into a larger branch. Within this context, nematodes of the genus Cylicocyclus were clustered with Cyathostomum tetracanthum and Cylicostephanus calicatus. Notably, C. ultrajectinus formed a unique branch on its own, separate from other Cylicocyclus nematodes (Figure 3).

Figure 3.

Figure 3.

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Maximum-likelihood phylogenetic tree based on 12 concatenated mitochondrial genes from 23 species of strongylid nematodes. The following sequences were used: C. tetracanthum (MN792800), C. radiatus (MH363701), C. nassatus (NC_032299), C. ashworthi (NC_046711), C. auriculatus (MK047416), C. insigne (GQ888712), C. calicatus (OR059470), C. elongatus (NC_060748), C. longiburs (OR069302), C. catinatum (NC_035003), C. pateratum (NC_038070), C. goldi (AP017681), C. labiatus (MH551242), C. labratus (NC_061656), C. bicoronatus (NC_042141), C. minutus (NC_035004), P. imparidentatum (NC_035005), T. brevicauda (NC_026729), T. serratus (NC_031516), T. nipponicus (NC_031517), S. vulgaris (NC_013818), and S. equinus (NC_026868).

Discussion

The mitochondrial genome of C. ultrajectinus is 13, 984 bp in length, similar to that of most animal parasitic nematodes, such as Cylicocyclus radiatus (13,836 bp) and Cylicocyclus auriculatus (13,851 bp) (Hu et al. 2020; Li et al. 2022). The base composition of C. ultrajectinus exhibits a pronounced AT base bias, which is consistent with the characteristics of mitochondrial gene composition in animal parasitic nematodes (Liu et al. 2012; Wang et al. 2020; Gao et al. 2022; Shan et al. 2024). The mitochondrial genome of C. ultrajectinus comprises 36 genes, lacking the ATP8 gene compared to Adenophorea nematodes (Mohandas et al. 2014), and is arranged in the GA3 type (Liu et al. 2013). All genes are encoded on the positive strand, with a relatively tight and highly conserved gene structure similar to other strongylid nematodes (Hu et al. 2020; Wang et al. 2020; Gao et al. 2021; Li et al. 2022).

Phylogenetic analysis revealed that the Strongylidae formed two main branches. Strongylus of the Strongylinae clustered into a smaller branch, while all nematodes of the Cyathostominae subfamily and Triodontophorus within the Strongylinae clustered into a larger branch, consistent with findings from other studies based on mitochondrial genomes (Xu et al. 2015; Hu et al. 2020; Wang et al. 2020; Gao et al. 2021). Nematodes of the genus Cylicocyclus were closely associated with and clustered alongside Cyathostomum tetracanthum and Cylicostephanus calicatus. However, C. ultrajectinus formed a distinct branch on its own, separate from other Cylicocyclus nematodes, aligning with results from the ITS1 + ITS2 study by Bu (2016) and molecular analysis by Hung et al. (2000). Both studies determined that C. ultrajectinus differed from other species within this genus. Despite this, Jian et al.(2022), upon observing the surface morphology of C. ultrajectinus using scanning electron microscopy, concluded that it still belongs to the genus Cylicocyclus. Phylogenetic analyses based on traditional morphological classification and molecular identification often produce differing results, and the reasons for this disparity remain to be investigated. Therefore, further exploration is necessary to elucidate the taxonomic status of C. ultrajectinus and its relationship with related species.

Supplementary Material

Supplemental Material
TMDN_A_2427110_SM6680.jpg (1.3MB, jpg)

Funding Statement

This work was supported by a major project of National Natural Science Foundation of China [No. 31372163] and the Research Grants from the Science and Technology Department of Henan Province, China [Nos. 142102310121, 192102310518].

Author contributions

Conceived and designed the study: YH, BL, and YB. Performed specimen collection, and experimented with: YH, BL, LM, MX, and PN. Analyzed the data and wrote the paper: YH, BL, LM, MX, and PN. Revised the paper: YH and YB. All authors agree to be accountable for all aspects of the work.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The genome sequence data that support the findings of this study are openly available in GenBank of NCBI at https://www.ncbi.nlm.nih.gov, under the accession number OR834862.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental Material
TMDN_A_2427110_SM6680.jpg (1.3MB, jpg)

Data Availability Statement

The genome sequence data that support the findings of this study are openly available in GenBank of NCBI at https://www.ncbi.nlm.nih.gov, under the accession number OR834862.

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