Journal of Helminthology, page 1 of 5 q Cambridge University Press 2015
doi:10.1017/S0022149X1400087X
Sequence variability in internal transcribed spacers of nuclear ribosomal DNA among isolates of the oxyurid nematodes Syphacia obvelata and Aspiculuris tetraptera from mice reared in laboratories in China J.H. Qiu, Y. Lou, Y. Zhang, Q.C. Chang, Z.X. Liu, H. Duan, D.H. Guo, D.Z. Gao, D.M. Yue and C.R. Wang* College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang Province 163319, PR China (Received 26 August 2014; Accepted 29 November 2014) Abstract This study examined sequence variability in internal transcribed spacers (ITS) of nuclear ribosomal DNA among Syphacia obvelata and Aspiculuris tetraptera isolates from laboratory mice from different geographical locations in China. ITS1, 5.8S and ITS2 rDNA were amplified separately from adult S. obvelata and A. tetraptera individuals by polymerase chain reaction (PCR), and the amplicons were subjected to sequencing from both directions. The lengths of the sequences of ITS1, 5.8S and ITS2 rDNA from both nematodes were 314 bp and 456 bp, 157 bp, and 273 bp and 419 bp, respectively. The intraspecific sequence variations in S. obvelata ITS1 were 0 –0.3%. For A. tetraptera they were 0 –0.7% in ITS1 and 0– 1.0% in ITS2. However, the interspecific sequence differences among members of the infraorder Oxyuridomorpha were significantly higher, being 54.0– 65.5% for ITS1 and 55.3 –64.1% for ITS2. Phylogenetic analysis based on the combined partial sequences of ITS1 and ITS2 using three inference methods – Bayesian inference, maximum likelihood and maximum parsimony – revealed that all the S. obvelata and A. tetraptera samples formed independent monophyletic groups. Syphacia obvelata was closer to Syphacia muris than to A. tetraptera, consistent with morphological classification. These results demonstrate that ITS1 and ITS2 rDNA sequences are useful markers for population genetic studies of oxyurid nematodes.
Introduction Laboratory mice are suitable, necessary and widely used experimental animals for much scientific research, mainly in the life sciences. However, parasitic nematodes such as the mouse pinworms Syphacia obvelata and Aspiculuris tetraptera can inhabit the caecum and colon of laboratory mice with high prevalence, and cause oxyuriasis, even in well-managed colonies and laboratory *Fax: þ86 (459) 6819095 E-mail:
[email protected]
animal centres (Baker, 1998; Hill et al., 2009; Chen et al., 2011). Pinworm infection is a serious problem due to its worldwide distribution and potential untoward effects on the behaviour, growth, intestinal physiology and immunology of mice (Baker, 1998; Bugarski et al., 2006; Michels et al., 2006; Hill et al., 2009; Chen et al., 2011; Gaherwal et al., 2012). In addition to morphological, epidemiological investigations and diagnostic studies, mitochondrial (mt) gene (cox1, nad1, nad5, cytb), internal transcribed spacer (ITS1 and ITS2) and 28S rDNA sequences are useful genetic markers for the identification and phylogenetic analysis
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of this parasite group (Okamoto et al., 2007, 2009; Parel et al., 2008; Lou et al., 2014; Wang et al., 2014). Furthermore, ITS1 and ITS2 rDNA sequences are also useful genetic markers for studying the inter- and intrapopulation genetic variations in parasites, such as Angiostrongylus cantonensis and Clonorchis sinensis, Trichuris trichiura and T. suis, and Oesophagostomum asperum and O. colubianum (Tatonova et al., 2012; Liu et al., 2014; Zhao et al., 2014). However, there is a paucity of information on mouse pinworm rDNA (Parel et al., 2008; Okamoto et al. 2009), and sequence variability in the ITS rDNA among populations of S. obvelata and A. tetraptera with different geographical origins has not yet been reported. The purpose of this study was to examine sequence variability in ITS1 and ITS2 rDNA among S. obvelata and A. tetraptera isolates from laboratory mice with different geographical origins in China. The phylogenetic relationships of Oxyuridomorpha were also reconstructed.
Materials and methods Molecular analysis of nematode isolates of S. obvelata and A. tetraptera Adult pinworms of species S. obvelata (n ¼ 20) and A. tetraptera (n ¼ 20) were collected from the caecum and colon of various laboratory mice (BALB/c mice, Kunming mice, T739 mice and 615 mice) in seven provinces of China. The geographical locations were separated by significant distances (976 – 4049 km). The geographical origin, host, longitude and latitude and GenBank accession numbers for nuclear ITS1 and ITS2 rDNA sequences from S. obvelata and A. tetraptera are listed in table 1 and table 2. These adult pinworms were washed extensively with physiological saline, identified based
on host preference, morphological characteristics and predilection sites (Taffs, 1976), and preserved in 70% (v/v) ethanol and stored at 2 208C until use. Total genomic DNA was extracted from each individual pinworm using a standard sodium dodecyl-sulphate/ proteinase K treatment, followed by purification on a mini-column (TIANamp Genomic DNA Kit, Tiangen Biotech, Beijing, China). DNA was eluted into 35 ml double-distilled water. DNA samples were stored at 2 208C until use. The ITS1 – 5.8S – ITS2 rDNA fragment was amplified separately from each individual nematode DNA sample by polymerase chain reaction (PCR), using the primers NC5 (50 -GTAGGTGAACCTGCGGAAGGATCATT-30 ) and NC2 (50 -TTAGTTTCTTTTCCTCCGCT-30 ), as described by Parel et al. (2008). One microlitre of DNA template was used in a 25 ml PCR reaction containing 5 ml of 5 £ colourless Go Taq flexi buffer (pH 8.5), 2 ml MgCl2 (25 mM ), 2 ml deoxynucleoside triphosphate (dNTP) mixture (2.5 mM ), 0.5 ml of each primer (10 pmol/ml) and 0.13 ml Go Taq DNA polymerase (5 U/ml). The PCR cycling protocol used was 958C for 2 min (initial denaturation), followed by 35 cycles of 958C for 1 min (denaturation), 608C for 1 min (annealing) and 728C for 1 min (extension), followed by a final extension for 5 min at 728C. Each amplicon was examined by agarose gel (1%) electrophoresis and ethidium bromide staining. The PCR products were purified using an Axyprep DNA Gel Extraction Kit (AXYGEN, Suzhou, China) and were ligated into the pGEM-T Easy plasmid vector (Promega, Madison, Wisconsin, USA) according to the manufacturer’s instructions. The products were transformed into Escherichia coli TOP10 competent cells (BioMed, Beijing, China) and positive colonies were selected for colony PCR. The positive recombinant plasmids were sent to Life Technology Company (Beijing, China) for
Table 1. Isolates of Syphacia obvelata from strains of laboratory mice from four provinces of China, including GenBank accession numbers of partial ITS1 and ITS2 rDNA sequences. GenBank accession numbers
Sample code
Mouse strain
Geographical origin
Longitude/ latitude
ITS1
ITS2
SJL1 SJL2 SJL3 SJL4 SJL5 SGD1 SGD2 SGD3 SGD4 SGD5 SBJ1 SBJ2 SBJ3 SBJ4 SBJ5 SGS1 SGS2 SGS3 SGS4 SGS5
BALB/c BALB/c BALB/c BALB/c BALB/c Kunming Kunming Kunming Kunming Kunming T739 T739 T739 615 615 Kunming Kunming Kunming Kunming Kunming
Changchun, Jilin Changchun, Jilin Changchun, Jilin Changchun, Jilin Changchun, Jilin Guangzhou, Guangdong Guangzhou, Guangdong Guangzhou, Guangdong Guangzhou, Guangdong Guangzhou, Guangdong Changping, Beijing Changping, Beijing Changping, Beijing Changping, Beijing Changping, Beijing Lanzhou, Gansu Lanzhou, Gansu Lanzhou, Gansu Lanzhou, Gansu Lanzhou, Gansu
125.19/43.54 125.19/43.54 125.19/43.54 125.19/43.54 125.19/43.54 113.14/23.08 113.14/23.08 113.14/23.08 113.14/23.08 113.14/23.08 116.13/40.13 116.13/40.13 116.13/40.13 116.13/40.13 116.13/40.13 103.51/36.04 103.51/36.04 103.51/36.04 103.51/36.04 103.51/36.04
KJ143619 KJ143619 KJ143619 KJ143619 KJ143619 KJ143619 KJ143619 KJ143619 KJ143619 KJ143619 KJ143619 KJ143619 KJ143619 KJ143619 KJ143619 KJ143619 KJ143620 KJ143619 KJ143619 KJ143619
KJ143611 KJ143611 KJ143611 KJ143611 KJ143611 KJ143611 KJ143611 KJ143611 KJ143611 KJ143611 KJ143611 KJ143611 KJ143611 KJ143611 KJ143611 KJ143611 KJ143611 KJ143611 KJ143611 KJ143611
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Sequence variability of Syphacia obvelata and Aspiculuris tetraptera Table 2. Isolates of Aspiculuris tetraptera from strains of laboratory mice from four provinces of China, including GenBank accession numbers of partial ITS1 and ITS2 rDNA sequences. GenBank accession numbers
Sample code
Mouse strain
Geographical origin
Longitude/ latitude
ITS1
ITS2
AHLJ1 AHLJ2 AHLJ3 AHLJ4 AHLJ5 AYN1 AYN2 AYN3 AYN4 AYN5 AJS1 AJS2 AJS3 AJS4 AJS5 AGS1 AGS2 AGS3 AGS4 AGS5
Kunming Kunming Kunming Kunming Kunming Kunming Kunming Kunming Kunming Kunming BALB/c BALB/c BALB/c BALB/c BALB/c Kunming Kunming Kunming Kunming Kunming
Daqing, Heilongjiang Daqing, Heilongjiang Daqing, Heilongjiang Daqing, Heilongjiang Daqing, Heilongjiang Kunming, Yunnan Kunming, Yunnan Kunming, Yunnan Kunming, Yunnan Kunming, Yunnan Yangzhou, Jiangsu Yangzhou, Jiangsu Yangzhou, Jiangsu Yangzhou, Jiangsu Yangzhou, Jiangsu Lanzhou, Gansu Lanzhou, Gansu Lanzhou, Gansu Lanzhou, Gansu Lanzhou, Gansu
125.01/46.36 125.01/46.36 125.01/46.36 125.01/46.36 125.01/46.36 102.42/25.04 102.42/25.04 102.42/25.04 102.42/25.04 102.42/25.04 119.26/32.23 119.26/32.23 119.26/32.23 119.26/32.23 119.26/32.23 103.51/36.04 103.51/36.04 103.51/36.04 103.51/36.04 103.51/36.04
KJ143612 KJ143614 KJ143612 KJ143612 KJ143612 KJ143612 KJ143612 KJ143612 KJ143612 KJ143612 KJ143612 KJ143612 KJ143612 KJ143612 KJ143612 KJ143612 KJ143612 KJ143612 KJ143613 KJ143612
KJ143618 KJ143617 KJ143616 KJ143615 KJ143615 KJ143615 KJ143615 KJ143615 KJ143615 KJ143615 KJ143615 KJ143615 KJ143615 KJ143615 KJ143615 KJ143615 KJ143615 KJ143615 KJ143615 KJ143615
sequencing, using the same universal primers as in the primary amplification.
PAUP. The phylogram was drawn using the Tree View program, version 1.65 (Page, 1996).
Phylogenetic analysis
Results and discussion
Sequences of the ITS1 and ITS2 rDNA were aligned using Clustal X 1.83 (Thompson et al., 1997). The intraspecific sequence differences in S. obvelata and A. tetraptera were analysed. The interspecific sequence differences among members of the infraorder Oxyuridomorpha were determined by comparison with corresponding sequences from Enterobius vermicularis (HQ646164.1), Passalurus ambiguus (EF464552.1), Skrjabinema kamosika (AB699691.1), Syphacia muris (EU263106.2), A. tetraptera Taiwan isolate (EU263107.2) and S. obvelata Taiwan isolate (EU263105.2), using the program DNAstar 5.0 (Burland, 2000). Sequence differences are calculated and presented based on average pairwise difference. The phylogenetic relationships between all S. obvelata and A. tetraptera samples and with other members of the infraorder Oxyuridomorpha were reconstructed based on the combined ITS1 and ITS2 sequences, using Trichuris muris (AJ299407.1), Adenophorea, as the outgroup. Trees were constructed using Bayesian inference (BI), maximum likelihood (ML) and maximum parsimony (MP) methods. BI was performed using MrBayes 3.1 (Ronquist & Huelsenbeck, 2003). The consensus tree was obtained after bootstrap analysis with 1000 replications. ML analyses were performed using PUZZLE 4.1 (Strimmer & Haeseler, 1996). MP analyses were carried out using the PAUP 4.0 Beta 10 program (Swofford, 2002). Bootstrap probability (BP) was calculated from 1000 bootstrap replicates with ten random additions per replicate in
The ITS rDNA sequences obtained from S. obvelata and A. tetraptera were 744 bp and 1032 bp, respectively. These included partial ITS1 rDNA sequences of 314 and 456 bp, complete 5.8S sequences of 157 bp, and partial ITS2 rDNA sequences of 273 and 419 bp, respectively. The sequences of ITS1 and ITS2 from S. obvelata and A. tetraptera have been deposited in GenBank under the accession numbers KJ143611 – KJ143620 (tables 1 and 2). The sequence variations in the ITS1 rDNA from S. obvelata from different geographical locations (including S. obvelata Taiwan isolate EU263105.2) were 0 – 0.3%, including one T ! G transition at sequence position 65. No intraspecific variations were detected in ITS2 rDNA among S. obvelata isolates. The intraspecific sequence differences among A. tetraptera isolates from different geographical locations were slightly higher, being 0 – 0.7% and 0 – 1.0% for ITS1 and ITS2, respectively (including A. tetraptera Taiwan isolate EU263107.2). Three transitions (A ! G, A ! T and T ! C) were observed at sequence positions 37, 382 and 445 of ITS1; and four transitions (A ! G, two of T ! C, and G ! A) at sequence positions 640, 821, 990 and 1030 of ITS2, respectively. Although samples of S. obvelata and A. tetraptera were collected from different types of laboratory mice (BALB/c, Kunming, T739 and 615 mice) and different regions of China, and the furthest distance between isolates was approximately 4049 km (between Daqing, Heilongjiang Province and Guangzhou, Guangdong Province), the intraspecific variations in ITS1 and
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0.98/100/100
0.98/100/98
0.98/98/100
0.99/100/100 0.97/86/89
0.93/ - /89
SJL4 SGS3 SBJ1 Syphacia obvelata (Taiwan isolate) SBJ3 SBJ2 SGD2 SGD1 SGS5 SGS4 SGS2 SGS1 SBJ5 SBJ4 SGD4 SGD3 SJL1 SGD5 SJL3 SJL2 SJL5 Syphacia muris
AGS1 AHLJ2 AGS4 AHLJ3 AHLJ1 Aspiculuris tetraptera (Taiwan isolate) AYN5 AYN4 AYN3 AYN2 AYN1 AJS5 AJS4 AJS3 AJS2 AJS1 AHLJ5 AHLJ4 AGS5 AGS3 AGS2
Enterobius vermicularis Skrjabinema kamosika Passalurue ambiguus Trichuris muris
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Fig. 1. Phylogenetic relationships between Syphacia obvelata and Aspiculuris tetraptera isolated from strains of laboratory mice in seven provinces in China. The tree is constructed using the combined dataset (ITS1 þ ITS2), with Trichuris muris as the outgroup. Numbers along branches indicate bootstrap values from different analyses, in the order Bayesian inference/maximum parsimony/maximum likelihood. Values lower than 50% are shown as hyphens.
ITS2 rDNA sequences among S. obvelata and A. tetraptera samples were small. This result is in accordance with other studies of mouse pinworms (S. obvelata and A. tetraptera) using mtDNA (Lou et al., 2014; Wang et al., 2014). It is also consistent with analysis of nine species within the genus Syphacia using 28S rDNA (Okamoto et al., 2009), and with results for other helminths (Dai et al., 2012; Liu et al., 2014; Zhao et al., 2014). The differences in ITS1 and ITS2 rDNA sequences from S. obvelata and A. tetraptera compared with those from other members of the infraorder Oxyuridomorpha, E. vermicularis, P. ambiguus, S. kamosika and S. muris, were significantly higher, being 54.0 –58.1% and 57.7 – 65.6% for ITS1, and 55.3– 62.4% and 55.7 – 64.1% for ITS2, respectively. These data are consistent with those from recent studies of S. obvelata and A. tetraptera mtDNA, of the 28S rDNA of nine species in the genus Syphacia, of tapeworm rDNA (Spirometra erinaceieuropaei, Taenia multiceps and T. hydatigena) and of nematode rDNA (Oesophagostomum asperum and O. columbianum; Trichuris trichiura and T. suis) (Okamoto et al., 2009; Dai et al., 2012; Liu et al., 2014; Lou et al., 2014; Wang et al., 2014; Zhao et al., 2014). Phylogenetic relationships were reconstructed using combined sequences of the ITS1 and ITS2 rDNA, with
T. muris as the outgroup to determine the genetic positions of S. obvelata and A. tetraptera. There were two main clades in the tree. One clade contained only P. ambiguus, while the other included E. vermicularis, S. kamosika, S. muris, A. tetraptera Taiwan isolate, S. obvelata Taiwan isolate, and all mouse pinworm samples from this study. In the mouse pinworm sample clade, all S. obvelata samples and S. obvelata Taiwan isolate clustered together, sistered to S. muris. All A. tetraptera samples and A. tetraptera Taiwan isolate clustered together, close to S. muris and S. obvelata (fig. 1), consistent with morphological classifications. In conclusion, the present study reveals low sequence variability in ITS rDNA from S. obvelata and A. tetraptera from different geographical locations in China. These rDNA sequences can be used as genetic markers for identification and phylogenetic studies of different oxyurid nematodes.
Acknowledgements The authors thank Miss J.Y. Zhang, Mr Y.C. Liu and Miss Y. Wang for helping us to obtain Syphacia obvelata and Aspiculuris tetraptera specimens.
Sequence variability of Syphacia obvelata and Aspiculuris tetraptera
Financial support This work was supported by the Fund for Imported Talents in Heilongjiang Bayi Agricultural University (2013YB-42).
Conflict of interest None.
Ethical standards This research project was reviewed and approved by the Ethics Committee of Heilongjiang Bayi Agricultural University, China.
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