Cao et al. Parasites Vectors (2020) 13:566 https://doi.org/10.1186/s13071-020-04430-6 Parasites & Vectors RESEARCH Open Access Morphology, genetic characterization and molecular phylogeny of pinworm Skrjabinema longicaudatum n. sp. (Oxyurida: Oxyuridae) from the endangered Tibetan antelope Pantholops hodgsonii (Abel) (Artiodactyla: Bovidae) Yi‑Fan Cao1,2†, Hui‑Xia Chen3†, Yang Li3, Dang‑Wei Zhou1,4*, Shi‑Long Chen1,4 and Liang Li3* Abstract Background: The Tibetan antelope Pantholops hodgsonii (Abel) (Artiodactyla: Bovidae) is an endangered species of mammal endemic to the Qinghai‑Tibetan Plateau. Parasites and parasitic diseases are considered to be important threats in the conservation of the Tibetan antelope. However, our present knowledge of the composition of the para‑ sites of the Tibetan antelope remains limited. Methods: Large numbers of nematode parasites were collected from a dead Tibetan antelope. The morphology of these nematode specimens was observed using light and scanning electron microscopy. The nuclear and mitochon‑ drial DNA sequences, i.e. small subunit ribosomal DNA (18S), large subunit ribosomal DNA (28S), internal transcribed spacer (ITS) and cytochrome c oxidase subunit 1 (cox1), were amplifed and sequenced for molecular identifcation. Moreover, phylogenetic analyses were performed using maximum likelihood (ML) inference based on 28S and 18S 28S cox1 sequence data, respectively, in order to clarify the systematic status of these nematodes. + + Results: Integrated morphological and genetic evidence reveals these nematode specimens to be a new species of pinworm Skrjabinema longicaudatum (Oxyurida: Oxyuridae). There was no intraspecifc nucleotide variation between diferent individuals of S. longicaudatum n. sp. in the partial 18S, 28S, ITS and cox1 sequences. However, a high level of nucleotide divergence was revealed between the new species and its congeners in 28S (8.36%) and ITS (20.3–23.7%) regions, respectively. Molecular phylogenetic results suggest that the genus Skrjabinema should belong to the *Correspondence: [email protected]; [email protected] Background †Yi‑Fan Cao and Hui‑Xia Chen contributed equally to this work Te Tibetan antelope Pantholops hodgsonii (Abel) 1 Key Laboratory of Adaptation and Evolution of Plateau Biota (AEPB), Northwest Institute of Plateau Biology, Chinese Academy of Sciences, (Artiodactyla: Bovidae) is an endangered species of Xining 810008, Qinghai, People’s Republic of China mammal endemic to the Qinghai-Tibetan Plateau. Te 3 Key Laboratory of Animal Physiology, Biochemistry and Molecular population of the Tibetan antelope has been declined Biology of Hebei Province, College of Life Sciences, Hebei Normal University, Shijiazhuang 050024, Hebei Province, People’s Republic severely, because of the loss and fragmentation of its of China habitat and commercial poaching [1, 2]. Te latest esti- Full list of author information is available at the end of the article mate of the global population of the Tibetan antelope is © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Cao et al. Parasites Vectors (2020) 13:566 Page 2 of 11 subfamily Oxyurinae (Oxyuroidea: Oxyuridae), instead of the subfamily Syphaciidae or Skrjabinemiinae in the tradi‑ tional classifcation, as it formed a sister relationship to the genus Oxyuris. Conclusions: A new species of pinworm Skrjabinema longicaudatum n. sp. (Oxyurida: Oxyuridae) is described. Skr- jabinema longicaudatum n. sp. represents the frst species of Oxyurida (pinworm) and the fourth nematode species reported from the Tibetan antelope. Our results contribute to the knowledge of the species diversity of parasites from the Tibetan antelope, and clarify the systematic position of the genus Skrjabinema. Keywords: Tibetan antelope, Parasite, Nematoda, Morphology, Genetic data, Phylogeny 100,000–150,000 mature individuals (https ://www.iucnr Morphological observations edlis t.org/speci es/15967 /50192 544). Tis species is listed For light microscopical studies, nematodes were cleared as “Near Treatened” in the IUCN Red List of Treatened in lactophenol. Drawings were made using a Nikon Species™ and also listed as Class I (Endangered in China) microscope drawing attachment. For scanning electron National Protected Wild Animal Species in China. microscopy (SEM), the anterior and posterior parts of Parasites and parasitic diseases are considered to be specimens were re-fxed in 4% formaldehyde solution, important threats in wildlife conservation, as they can post-fxed in 1% OsO4, dehydrated via an ethanol series potentially impair the health of wildlife, decrease fit- and acetone, and then critical-point dried. Samples were ness, cause population declines and even contribute coated with gold and examined using a Hitachi S-4800 to local extinction [3–7]. Parasites are also significant scanning electron microscope at an accelerating voltage pathogens of the Tibetan antelope [1, 2]. To date, 17 of 20 kV. Measurements (the range, followed by the mean species of ectoparasites and endoparasites have been values in parentheses) are given in micrometers (μm) reported from the Tibetan antelope, including 5 spe- unless otherwise stated. cies of oestrid and hippoboscid flies, 7 species of protozoans, 2 species of tapeworms and 3 species of Molecular protocols nematodes [1, 2, 8–12]. Tree female specimens were randomly chosen for In the present study, some nematode specimens molecular analysis. Genomic DNA from each sample were collected from the digestive tract of the Tibetan was extracted using a Column Genomic DNA Isolation antelope, which were identifed morphologically as Kit (Shanghai Sangon, China) according to the manufac- a new species of the genus Skrjabinema (Oxyurida: turer’s instructions. Te partial 18S region was amplifed Oxyuridae) using light and scanning electron micros- by polymerase chain reaction (PCR) using the forward copy. Te nuclear and mitochondrial DNA sequences, primer 18S-F (5’-CGC GAA TRG CTC ATT ACA ACA i.e. small subunit ribosomal DNA (18S), large subunit GC-3’) and the reverse primer 18S-R (5’-GGG CGG ribosomal DNA (28S), internal transcribed spacer (ITS) TAT CTG ATC GCC-3’) [13]. Te partial 28S region was and cytochrome c oxidase subunit 1 (cox1), were also amplifed by PCR using the forward primer 28S-F (5’- amplifed and sequenced for molecular identifcation of AGC GGA GGA AAA GAA ACT AA-3’) and the reverse this species. Moreover, in order to clarify the system- primer 28S-R (5’-ATC CGT GTT TCA AGA CGG G-3’) atic status of the genus Skrjabinema, phylogenetic anal- [14]. Te ITS-1 region of nuclear rDNA was amplifed by yses were performed using maximum likelihood (ML) PCR using the forward primer SS1 (5’-GTT TCC GTA inference based on 28S and 18S + 28S + cox1 sequence GGT GAA CCT GCG-3’) and the reverse primer SS2R data, respectively. (5’-AGT GCT CAA TGT GTC TGC AA-3’). Te ITS-2 region of nuclear rDNA was amplifed by PCR using the Methods forward primer NC13 (5’-ATC GAT GAA GAA CGC Parasite collection AGC-3’) and the reverse primer NC2 (reverse: 5’-TTA A Tibetan antelope died naturally in the Hohxil National GTT TCT TTT CCT CCG CT-3’) [15]. Te partial cox1 Nature Reserve, Qinghai Province, China. Te digestive region was amplifed by PCR using the forward primer tract of this Tibetan antelope was sent to the Key Lab- cox1F2020 (5’-GAG TAC TAA TCA TAA GGA TAT oratory of Adaptation and Evolution of Plateau Biota TGG-3’) and the reverse primer cox1R2020 (5’-ACA TAA (AEPB), Northwest Institute of Plateau Biology, Chinese ACY TCA GGA TGA CCA-3’), both newly designed in Academy of Sciences for examination of parasites. Large present study. Te cycling conditions are as described numbers of nematode parasites were isolated from the previously [16]. PCR products were checked on Gold- caecum and colon. Specimens were fxed and stored in in View-stained 1.5% agarose gels and purifed with Column 5% glycerine plus 70% ethanol until study. PCR Product Purifcation Kit (Shanghai Sangon, China). Cao et al. Parasites Vectors (2020) 13:566 Page 3 of 11 Sequencing was carried out using a Dye Deoxy Termina- Evolution of Plateau Biota, Northwest Institute of Plateau tor Cycle Sequencing Kit (v.2, Applied Biosystems, Cali- Biology, Chinese Academy of Sciences, Qinghai Province, fornia, USA) and an automated sequencer (ABI-PRISM China. 377). Sequences were aligned using ClustalW2. Te DNA sequences obtained herein were compared (using the Site in host algorithm BLASTn) with those available in the National Caecum and colon. Center for Biotechnology Information (NCBI) database (https ://www.ncbi.nlm.nih.gov). Prevalence and intensity A single Tibetan antelope examined with 124 worms. Phylogenetic analyses Phylogenetic trees were constructed using maximum ZooBank registration likelihood (ML) inference with MEGA X software based To comply with the regulations set out in Arti- on the partial 28S and 18S + 28S + cox1 sequence data, cle 8.5 of the amended 2012 version of the Interna- respectively. Pseudonymus spirotheca (Oxyurida: Te- tional Code of Zoological Nomenclature (ICZN) [17], lastomatoidea: Pseudonymidae) was treated as the out- details of the new species have been submitted to group.