Mitochondrial genome evidence suggests Cooperia sp. from China may represent a distinct species from Cooperia oncophora from Australia

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Abstract

Cooperia spp. are parasitic nematodes parasitizing in small intestine of ruminants with a worldwide distribution. Infection of ruminants with Cooperia species can cause severe enteritis, causing significant socio-economic losses to the livestock industry. However, it is yet to know whether there is genetic diversity in mitochondrial (mt) DNA sequences of Cooperia nematodes from different geographic regions. The objective of the present study was to examine sequence difference in mt genomes between Cooperia sp. from China and other Cooperia species. We determined the sequences of the internal transcribed spacer (ITS-1 and ITS-2) of nuclear ribosomal DNA (rDNA) of 11 Cooperia specimens collected from the small intestine of a Tianzhu White yak in Gansu Province, northwestern China, which had 99% similarity with that of C. oncophora from Brazil (GenBank accession Number: AJ544290) in ITS-1, and 99% similarity with those from Denmark (AB245040), Scotland and Australia (AJ000032) in ITS-2, indicating that specimens used in the present study should at least represent parasites in Cooperia. We then determined the complete mt genome sequences of one representative specimen of Cooperia sp. from China (CspC), compared the mt DNA sequences with that of C. oncophora from Australia (COA, GQ888713), and conducted phylogenetic analysis with selected nematodes using both maximum likelihood (ML) and Bayesian inference (BI) methods based on both concatenated 12 PCGs, rrnL and rrnS sequences and partial cox2 sequences. The complete mt genome sequence of CspC (KY769271) is 13, 583 bp in length, which is 91 bp shorter than that from COA. The sequence difference over the entire mt genome between CspC and COA was 12.2% in nucleotide and 6.3% in inferred amino acids, with nad4L and nad1 being the most variable and the most conserved PCGs, respectively. Phylogenetic analysis indicated that CspC and COA were closely-related but distinct taxa. The determination of mt genome sequences for Cooperia sp. from China also provides novel resources for further studies of taxonomy, systematics and population genetics of Cooperia from different geographical locations.

Introduction

Cooperia spp. are important nematodes parasitizing in the small intestine of cattle and sheep. It is widely distributed in temperate regions and can lead to severe parasitic gastrointestinal diseases, ultimately causing major socio-economic losses worldwide [[1], [2], [3], [4], [5]]. However, the majority of studies over Cooperia species are around the high prevalence and harms in ruminants, but it is yet to know whether there is genetic diversity in mitochondrial (mt) genome sequences of Cooperia spp. from different geographic locations.

The traditional identification of parasites mainly relies on morphological characteristics [6,7]. However, some closely related species are very similar in morphology and they are hard to distinguished merely based on morphological and morphometric features. To overcome the limitation of morphological taxonomy, molecular approaches based on sequences of the internal transcribed spacer (ITS-1 and ITS-2) regions of nuclear ribosomal DNA (rDNA) [8,9], as well as the mt cytochrome c oxidase subunit 1 (cox1) have been widely used for specific identification and differentiation of nematodes [6,[10], [11], [12], [13], [14], [15]]. Our preliminary comparative study revealed that the mt cox1 sequence of Cooperia sp. from China (GenBank accession No. MK295856) and C. oncophora from Australia (GQ888713) differed by 10.16%, which is much higher than the intra-specific variation in cox1 among related nematodes (among 18 Toxocara canis samples: 0.2–3.7% [16], and among 17 Oesophagostomum dentatum samples: 0.3–5.2% [17]), but is slightly lower than the inter-specific differences between Chabertia ovina and Ch. erschowi (11.98%) [15] and between Nematodirus spathiger and N. oiratianus (11.55%) [18]. However, we have noticed that sequences of Cooperia sp. from China have 99% similarity with those from C. oncophora in both ITS-1 (AJ544290) [19] and ITS-2 (AB245040 [20,21] and AJ000032 [22]). This raised the question whether Cooperia sp. from China and C. oncophora from Australia represent different nematode species, despite the high similarities in their ITS sequences and high difference in cox1 sequences.

The sequences of complete mt genome are frequently used as effective genetic markers in differentiating cryptic species, species complex and synonymy species in parasites [23,24], in particular for the nematodes within Trichostrongylina [15,25]. In the present study, we determined the complete sequences of mt genome of Cooperia sp. from China and compared the sequences with those of C. oncophora from Australia, to test the hypothesis that Cooperia sp. from China is a genetically distinct species.

Section snippets

Parasites, DNA extraction and ITS acquirement

Eleven adult nematode specimens were collected from the small intestine of a Tianzhu White yak in Tianzhu County, Gansu Province, northwestern China. The white yak was handled strictly in accordance with the Animal Ethics Procedures and Guidelines of the People's Republic of China. The study protocol was reviewed and approved by the Animal Administration and Ethics Committee of Lanzhou Veterinary Study Institute, Chinese Academy of Agricultural Sciences. These nematodes were putatively

Identity of the specimens

The ITS sequences of the 11 Cooperia samples were all 779 bp in length, and were identical in both ITS-1 and ITS-2 rDNA, indicating that they represent the same species. The ITS sequences were assembled and compared with the available sequences by BLAST through NCBI, resulting in 99% similarity with that of C. oncophora from Brazil (AJ544290) [19] in ITS-1, and 99% similarity with those of C. oncophora from Denmark (AB245040) [20,21] in ITS-2. The detailed information of comparison in ITS

Conclusions

The present study determined the complete mt genome sequences of one Cooperia sp. from China. The results of comparative sequence analyses, together with the phylogenetic analysis, demonstrated that, despite the high similarity in ITS sequences with those of C. oncophora from Australia, Cooperia sp. from China may represent a distinct taxon.

Declaration of Competing Interest

The authors declare that they have no competing interests.

Acknowledgements

Project support was provided by National Natural Science Foundation of China (Grant No. 31702225), the International Science and Technology Cooperation Project of Gansu Provincial Key Research and Development Program (Grant No. 17JR7WA031), the Elite Program of Chinese Academy of Agricultural Sciences, and the Agricultural Science and Technology Innovation Program (ASTIP) (Grant No. CAAS-ASTIP-2016-LVRI-03).

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    These authors have contributed equally to this work.

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