PREPRINT Posted on 24/11/2020 DOI: https://doi.org/10.3897/arphapreprints.e61100 COI and ND1 as DNA barcodes to identify Taenia hydatigena (Cestoda: Taeniidae) from canine small intestine on Hainan Island in China Yajun Lu, Siqi Yang, Jingwen Liu, Xixi Lu, Qiuyu Zhao Not peer-reviewed, not copy-edited manuscript. Disclaimer on biological nomenclature and use of preprints The preprints are preliminary versions of works accessible electronically in advance of publication of the final version. They are not issued for purposes of botanical, mycological or zoological nomenclature and are not effectively/validly published in the meaning of the Codes. Therefore, nomenclatural novelties (new names) or other nomenclatural acts (designations of type, choices of priority between names, choices between orthographic variants, or choices of gender of names) should NOT be posted in preprints. The following provisions in the Codes of Nomenclature define their status: International Code of Nomenclature for algae, fungi, and plants (ICNafp) Article 30.2: “An electronic publication is not effectively published if there is evidence within or associated with the publication that its content is merely preliminary and was, or is to be, replaced by content that the publisher considers final, in which case only the version with that final content is effectively published.” In order to be validly published, a nomenclatural novelty must be effectively published (Art. 32.1(a)); in order to take effect, other nomenclatural acts must be effectively published (Art. 7.10, 11.5, 53.5, 61.3, and 62.3). International Code of Zoological Nomenclature (ICZN) Article: 21.8.3: "Some works are accessible online in preliminary versions before the publication date of the final version. Such advance electronic access does not advance the date of publication of a work, as preliminary versions are not published (Article 9.9)". Not peer-reviewed, not copy-edited manuscript posted on November 24, 2020. DOI: https://doi.org/10.3897/arphapreprints.e61100 1 Title 2 COI and ND1 as DNA barcodes to identify Taenia hydatigena (Cestoda: Taeniidae) 3 from canine small intestine on Hainan Island in China 4 Author names 5 Siqi Yang1, Jingwen Liu1, Xixi Lu1, Qiuyu Zhao1, Yajun Lu1* 6 Author affiliations 7 1 Key Laboratory of Tropical Translational Medicine of Ministry of Education and 8 School of Tropical Medicine and Laboratory Medicine, Hainan Medical University, 9 Haikou, Hainan 571199, China. 10 Conflict of interest statement 11 The co-authors declare that they have no conflict of interest statement. 12 *Correspondence author 13 Yajun Lu 14 E-mail: [email protected] 15 Funding 16 This study was supported by grants from the Natural Science Foundation of Hainan 17 Province (2019RC223). 18 Ethics approval 19 Not applicable. Not peer-reviewed, not copy-edited manuscript posted on November 24, 2020. DOI: https://doi.org/10.3897/arphapreprints.e61100 20 Consent to participate 21 All participants provided written informed consent. 22 Consent for publication 23 All participants obtained written informed consent for publication. 24 Availability of data and material 25 The data used to support the findings of this study are included in the article. All data 26 are fully available without restriction. 27 Code availability 28 Not applicable. 29 Authors' contributions 30 Siqi Yang performed the experiments and wrote the manuscript. Jingwen Liu, Xixi Lu, 31 and Qiuyu Zhao performed the experiments. Yajun Lu designed the study, performed 32 revisions to the draft report, and approved the final manuscript. 33 Acknowledgements 34 The authors gratefully thank the Science and Technology Department of Hainan 35 Province for the financial support and Academician Workstation for material support. Not peer-reviewed, not copy-edited manuscript posted on November 24, 2020. DOI: https://doi.org/10.3897/arphapreprints.e61100 36 Abstract 37 DNA barcoding based on universal gene markers is a fast, accurate, and innovative 38 approach for the molecular discrimination of species. Some species are particularly 39 difficult to discriminate using a traditional morphological identification method 40 because of severely damaged morphological features. In this study, cytochrome c 41 oxidase subunit I (COI) and NADH dehydrogenase subunit 1 (ND1) were used as 42 barcoding markers to distinguish Taenia hydatigena in dogs on the tropical island of 43 Hainan. Therefore, geographic differentiation based on the COI and ND1 sequences 44 amongst the specimens and other geographic isolates in GenBank was determined by 45 calculating the genetic distances according to the Kimura 2-parameter (K2P) model 46 and constructing a phylogenetic tree using the neighbour-joining (NJ) method. 47 Barcoding gap, base composition, and base saturation were tested to assess the 48 effectiveness of the barcoding marker COI and ND1 genes for specimen identification. 49 In addition, we analysed the barcoding gap and saturation and performed molecular 50 evolutionary analysis of the intraspecies and interspecies diversity of Taenia. 51 Keywords 52 Cytochrome c oxidase subunit I; DNA barcodes; NADH dehydrogenase subunit 1; 53 Taenia hydatigena Not peer-reviewed, not copy-edited manuscript posted on November 24, 2020. DOI: https://doi.org/10.3897/arphapreprints.e61100 54 Introduction 55 With the development of molecular biology, it is increasingly common to identify 56 species by combining molecular biology with genetic evolution analysis (Young et al. 57 2019). Molecular identification based on specific DNA sequence analyses has become 58 a common technological tool used to identify new species and can make more 59 accurate assessments within and between species biodiversity than other methods as 60 well as serve as an effective supplement to traditional morphological identification 61 (Valentini et al. 2009). DNA barcodes have been widely used in biological molecular 62 identification and have great significance for uncovering biological diversity. DNA 63 barcodes can be used to establish a large-scale classification system for biodiversity 64 analysis through several species-specific DNA barcodes (Coissac et al. 2016). DNA 65 barcodes also provide reliable species identification for all life stages and even for 66 tissue debris (Hawlitschek et al. 2016). 67 The application of the barcode of the COI gene in the study of molecular biodiversity 68 is increasing rapidly and continuously (Balech et al. 2018). There are approximately 69 4.5 million COI gene barcodes in GenBank and BOLD (Barcode of Life Data 70 Systems), including different individuals across 250,000 species. It has become a 71 common phenomenon to identify different animals by using the barcode of the COI 72 gene (Thaler et al. 2016). In the identification of some species, the COI gene is more 73 suitable for identifying individuals (Laopichienpong et al. 2016). The COI gene may 74 prove to be valuable in the identification of the early developmental stage of species 75 (Jaakko et al. 2016). Not peer-reviewed, not copy-edited manuscript posted on November 24, 2020. DOI: https://doi.org/10.3897/arphapreprints.e61100 76 T. hydatigena is a zoonotic tapeworm species. Cysticercus is a larva that can be 77 widely parasitized in domestic animals with a high infection rate. Hainan is a high 78 incidence area of parasitic diseases. Taenia is a highly diverse population, and using 79 morphological methods alone may fail to identify some parasites accurately 80 (Ulziijargal et al. 2019). In 2013, Minoru and colleagues constructed molecular 81 phylogeny using mitochondrial genes to explain the genetic relationship among 82 different Taenia (Minoru et al. 2013). Since two gene barcodes have been proven to 83 have better resolution (Pennisi 2007), we use two genes, COI and ND1, as barcodes to 84 reflect the diversity of T. hydatigena (Cestoda: Taeniidae) on Hainan Island, and we 85 analyse the molecular evolution of Taenia using molecular genetics. 86 Materials and methods 87 Sample collection and genomic DNA extraction 88 An adult worm was obtained from the canine small intestine. The fresh worm was 89 washed in physiological saline and stored in 70% ethanol until use. Approximately 90 2-3 millimetres of worm was cut with a scalpel and washed three times with ddH2O. 91 The small piece of worm tissue was digested in 100 μL Solution AB and 25 μL AD 92 Buffer (Bioteke, Beijing, China) for 10 min at room temperature followed by 5 min at 93 95 ºC and was subsequently added to 100 μL Solution AC (Bioteke) to extract the 94 genomic DNA. 95 PCR amplification and sequencing 96 The COI and ND1 genes were amplified from genomic DNA by polymerase chain Not peer-reviewed, not copy-edited manuscript posted on November 24, 2020. DOI: https://doi.org/10.3897/arphapreprints.e61100 97 reaction (PCR). The primer sequences for COI were the forward primer 98 5'-TTTTTTGGGCATCCTGAGGTTTAT-3' and reverse primer 99 5'-TAAAGAAAGAACATAATGAAAATG-3'. The primer sequences for ND1 were 100 the forward primer 5'-AGATTCGTAAGGGGCCTAATA-3' and reverse primer 101 5'-ACCACTAACTAATTCACTTTC-3'. Amplification reactions contained 10 μL of 102 2×Power Taq PCR MasterMix (Bioteke), 4 μL of ddH2O, 1 µL of each forward and 103 reverse primer (10 μM), and 4 μL of template DNA (0.002 μg/μL). The cycling 104 conditions were as follows: 94 ºC for 3 min, 35 cycles of 94 ºC for 1 min, 55 ºC for 1 105 min and 72 ºC for 1 min, followed by a final step of 72 ºC for 5 min. PCR products 106 were purified using a FastPure Gel DNA Extraction Mini Kit (Vazyme Biotech, 107 Nanjing, China) and sequenced by the Sanger sequencing method (BGI, Beijing, 108 China). 109 Molecular evolution analysis 110 The sequences generated were assembled and refined manually to obtain final 111 high-quality sequences using ApE software v2.0.61 112 (http://jorgensen.biology.utah.edu/wayned/ape). Multiple sequence alignment was 113 conducted for the sequences of the samples for COI and ND1, the selected sequences 114 of 29 geographic isolates for COI, and 21 geographic isolates for ND1 from the 115 GenBank database using the Basic Local Alignment Search Tool (BLAST) of NCBI 116 (National Center for Biotechnology Information) 117 (https://blast.ncbi.nlm.nih.gov/Blast.cgi).