Complete mitochondrial genome of cylicocyclus auriculatus: molecular structure and phylogenetic analysis

Abstract Cylicocyclus spp. (Nematoda: Strongylida: Cyathostominae) are the common and important parasitic nematodes found in horses and donkeys worldwide. In this study, the complete mitochondrial genome of Cylicocyclus auriculatus Looss 1900, a representative member of this genus from the donkey in Southwest China was determined using the next-generation DNA sequencing technology. The genome was 13,851 bp in size and consisted of 36 genes including 12 protein-coding genes (atp6, cox1-3, cytb, nad1-6 and nad4L), 22 transfer RNA genes and two ribosomal RNA genes (rrnL and rrnS), as well as two non-coding regions. Phylogenetic analysis showed that C. auriculatus and Cylicocyclus insigne Boulenger 1917 were closely related, and then both grouped with other congeneric species and formed a monophyletic relationship with either species of Cyathostomum, Coronocyclus, Cyathostomum, Cylicostephanus or Cylicodontophorus, demonstrating their phylogenetic stability within Cyathostominae. These cumulative mitochondrial DNA data provide novel and useful genetic markers for molecular diagnostic, systematic and evolutionary biological studies of Cyathostominae nematodes.

Cylicocyclus parasites (Nematoda: Strongylida: Cyathostominae), commonly known as the bursate nematodes of equines, represent a common and severe threat to the livestock breeding industry worldwide (Lichtenfels et al. 2008;Zhang 2017;Hu et al. 2020). There are 13 valid species in the genus Cylicocyclus responsible for such morbidity and socioeconomic burdens. Cylicocyclus auriculatus Looss 1900 is a significant member of the Cylicocyclus nematodes and frequently found in donkeys (Lichtenfels et al. 2008;Kuzmina and Kuzmin 2008;Bu et al. 2009Bu et al. , 2013. This parasite inhabits in the large intestine of donkeys and can cause abdominalgia, diarrhea, weight loss and even death. Although there have been substantial advances in morphology and biology of C. auriculatus so far, knowledge gaps to understand this nematode at the molecular level, especially in its genetics and molecular epidemiology, are still not sufficiently explored because of lacking suitable genetic markers (Lichtenfels et al. 2008;Bu et al. 2013). More importantly, until now the morphology-based classification of Cylicocyclus has been controversial (Lichtenfels et al. 2008;Zhang and Kong 2002;Gao et al. 2021). Under this context, we decoded the mitochondrial genome of C. auriculatus, a representative member of this genus from the Dezhou donkey in Southwest China using the Illumina sequencing technology, as the mitochondrial DNA is not only a rich resource for molecular markers but its complete data can also provide novel insights into phylogenetic relationships of Cylicocyclus (Jex et al. 2010;Gao et al. 2017;Hu et al. 2020;Gao et al. 2021).
A total of six parasite specimens were obtained from a Dezhou donkey housed in an original breeding farm at Dazhou (31 92 0 N, 103 29 0 E), Sichuan Province of Southwest China, after treatment with pyrantel pamoate. These specimens were identified as adults of C. auriculatus with two males and four females, according to morphological keys of Lichtenfels et al. (2008). One male specimen was used for DNA isolation and the remaining were fixed in 5% formalin solution and archived in the Parasitological Museum of Sichuan Agricultural University (https://dop.sicau.edu.cn/; xyue1985@gmail.com (Yue Xie)) under collection numbers XY2018_28-32. After quality and quantity assessment, $2 mg genomic DNA was fragmented to construct a 350-bp pairedend (PE) library, followed by sequencing on an Illumina HiSeq X-TEN platform. The clean reads ($1.5 Gb) were used to assemble a mitochondrial genome with MITObim (Hahn et al. 2013). Gene annotation was achieved using MITOS (Bernt et al. 2013). The complete genome sequence was deposited in GenBank under accession number: MZ888509.
The mitochondrial genome of C. auriculatus was 13,851 bp in size and encoded 12 protein-coding genes (atp6, cox1-3, cytb, nad1-6 and nad4L), 22 tRNA genes and two rRNA genes (rrnL and rrnS). All genes were unidirectionally transcribed on the same strand. Among these twelve protein-coding genes, atp6, cytb, cox1-3, nad3, nad5, nad6 and nad4L started with ATT, while nad1, nad2 and nad4 used the TTG as the initiation codon. Correspondingly, except for cox3 and nad2 deduced to end with 'T' or 'TAG', the remaining genes were predicted to use the TAA as the stop codons. Twenty-two tRNA genes ranged from 54 bp (tRNA (AGN) -Ser) to 63 bp (tRNA-Lys) in size. In addition to tRNA-Ser genes, all had a DHU arm and a TV-replacement loop instead of the TwC arm (Xu et al. 2015;Gao et al. 2017Gao et al. , 2021Li et al. 2019;Qiu et al. 2019;Hu et al. 2020). Two rRNAs, the small rRNA (rrnS; 700 bp) and large (rrnL; 968 bp) subunits, were located between tRNA-Glu and tRNA (UCN) -Ser and between tRNA-His and nad3, respectively. Two non-coding regions, namely the long non-coding region (LNCR; 273 bp) and short non-coding region (SNCR; 88 bp), were located between tRNA-Ala and tRNA-Pro and between nad4 and cox1, respectively.
Building on a concatenated amino acid dataset of 12 protein-coding genes from 17 strongyloid parasites, the maximum parsimony (MP), maximum-likelihood (ML) and Bayesian inference (BI) methods were used to construct phylogenetic trees using Necator americanus Stiles 1902 as the outgroup because of its relationship with equine Strongyloidea nematodes (Jex et al., 2009;Liu et al., 2013;Gao et al., 2021). The three identical trees clearly grouped C. auriculatus with Cylicocyclus ashworthi Le Roux 1924, Cylicocyclus insigne Boulenger 1917, Cylicocyclus nassatus Looss 1900 and Cylicocyclus radiates Krecek 2011 and together formed a monophyletic relationship with either species of Cyathostomum, Coronocyclus, Cyathostomum, Cylicostephanus or Cylicodontophorus within Cyathostominae (Figure 1). Further, C. auriculatus was determined to be more closely related to C. insigne than to other species in Cylicocyclus, with high statistical supports (all bootstrap values !99 or ¼1.00), consistent with recent molecular studies (Hu et al. 2020;Gao et al. 2021), demonstrating the phylogenetic stability of these bursate nematodes found in equines. Taken together, the sequenced mitochondrial genome of C. auriculatus not only provides novel molecular evidence for its phylogenetic position in Cylicocyclus but also enriches the marker resource for molecular diagnostic, systematic and evolutionary biological studies of strongyloid nematodes.

Disclosure statement
No potential conflict of interest was reported by the authors. The study was reviewed and approved by the Animal Ethics Committee of Sichuan Agricultural University (Sichuan, China;approval no. SYXK 2014-187