http://informahealthcare.com/mdn ISSN: 1940-1736 (print), 1940-1744 (electronic) Mitochondrial DNA, Early Online: 1–4 ! 2014 Informa UK Ltd. DOI: 10.3109/19401736.2014.905861
SHORT COMMUNICATION
Genetic variability among Syphacia obvelata isolates from laboratory mice in four different geographical locations of China revealed by sequence analyses of five mitochondrial genes
Mitochondrial DNA Downloaded from informahealthcare.com by Nyu Medical Center on 10/15/14 For personal use only.
Chun-Ren Wang, Yan Lou, Yan Zhang, Wen-Tao Wang, Xu Zheng, Wen-Wen Xu, Ying Zhang, Si-Qin Tian, Lu Na, and Qiao-Cheng Chang College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang Province, P.R. China
Abstract
Keywords
Syphacia obvelata is a rodent nematode with high prevalence in laboratory mice. In the present study, we examined the genetic variability of S. obvelata from naturally infected laboratory mice in four different provinces, China. Five mitochondrial (mt) DNA regions, namely cytochrome c oxidase subunit 1 (pcox1), cytochrome b (pcytb), large subunit ribosomal RNA (prrnL) and NADH dehydrogenase subunits 1 and 5 (pnad1 and pnad5), were amplified separately from individual nematodes by PCR, and then sequenced directly. The size of the sequences of pcox1, pcytb, prrnL, pnad1 and pnad5 was 628 bp, 555 bp, 548 bp, 548 bp and 561 bp, respectively. While the intra-specific sequence variations within S. obvelata were 0–1.0% for pcox1, 0–1.6% for pcytb, 0–2.8% for prrnL, 0–2.0% for pnad1 and 0–1.8% for pnad5, the inter-specific sequence differences among members of the Oxyuridae were significantly higher, being 14.0–17.5% for pcox1, 27.5–32.9% for pcytb, 35.8–37.2% for prrnL, 22.2–26.8% for pnad1 and 22.3–25.2% for pnad5, respectively. Phylogenetic analyses based on combined sequences of four mt proteincoding genes, using Bayesian inference (BI), maximum likelihood (ML) and maximum parsimony (MP) methods, indicated that all of the S. obvelata samples grouped together with high statistical support, but samples from the same geographical origin did not always cluster together. These findings demonstrated the existence of low-level intra-specific variation in five mtDNA sequences among S. obvelata isolates from laboratory mice, but no obvious geographical distinction among S. obvelata isolates from laboratory mice in different geographic regions in China. These results provide basic information for further studies of systematics and population genetics of S. obvelata.
Laboratory mice, mitochondrial DNA, phylogenetic analyses, sequence variability, Syphacia obvelata
Introduction Syphacia obvelata, a mouse pinworm, inhabits the caecum and colon of laboratory mice and rats, which can cause oxyuriasis (Baker, 1998). It is a common parasite of laboratory mice with high prevalence even in well-managed colonies or laboratory animal centers (Baker, 1998; Chen et al., 2011). Although mice pinworm infection usually has no obvious clinical symptoms, it may have untoward effects on behavior, growth, intestinal physiology and immunology, and also interferes with research results in many ways (Baker, 1998; Bugarski et al., 2006; Michels et al., 2006; Pearson & Taylor, 1975; Taffs, 1976). Recent studies showed that some mitochondrial (mt) genes, such as cytochrome c oxidase subunit 1 (cox1), cytochrome b (cytb), and NADH dehydrogenase subunits 1, 4 and 5 (nad1, nad4 and pnad5), provide useful genetic markers for systematic, evolutionary, and the intra- and inter-population genetic variations of different nematodes (Chang et al., 2013; Kijewska et al., 2009; Lin et al., 2012; Lou et al., 2014; Wang et al., 2013a). In a previous study, we reported sequence variability among a mouse Correspondence: C.R. Wang, College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang Province 163319, P.R. China. Tel: +86-459-6819099. Fax: +86-459-6819090. E-mail: [email protected]
History Received 8 February 2014 Revised 2 March 2014 Accepted 15 March 2014 Published online 4 April 2014
pinworm, Aspicularis tetraptera, from laboratory mice in different provinces in China (Lou et al., 2014), however, genetic variability in another mice pinworm (S. obvelata) in China’s different geographical origins was unknown. Therefore in this study, sequence variability in cox1, cytb, large subunit ribosomal RNA (rrnL), nad1 and nad5 genes among S. obvelata isolates from laboratory mice in four provinces in China, were examined. Based on the combined four mtDNA protein-coding genes, the phylogenetic relationships of S. obvelata with other members of Oxyuridae were reconstructed. All these results will provide basic information for further studies of systematics, population genetics of S. obvelata.
Materials and methods Parasites and extraction of genomic DNA All animals were dissected humanely, according to protocols approved by the Institute Animal Care Committee. Adult S. obvelata worms (n ¼ 28) were collected from the caecum and colon of laboratory mice in Changchun, Jilin Province (JL1-JL7), northeast China; Lanzhou, Gansu Province (GS1-GS7), northwest China; Guangzhou, Guangdong Province (GD1-GD7), south China, and Changping, Beijing (BJ1-BJ7), north China. Geographical locations were separated by significant distances
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Mitochondrial DNA, Early Online: 1–4
Table 1. Geographical origins and worm’s gender of Syphacia obvelata samples used in the present study, longitude, latitude and the GenBank accession numbers for sequences of partial mitochondrial cytochrome c oxidase subunit1 gene (pcox1), large subunit ribosomal RNA (prrnL), NADH dehydrogenase subunit 1 and 5 gene (pnad1 and pnad5), and cytochrome b gene (pcytb).
Mitochondrial DNA Downloaded from informahealthcare.com by Nyu Medical Center on 10/15/14 For personal use only.
GenBank accession numbers Sample codes
Gender
Geographical origins
Longitude/latitude
pcox1
pcytb
prrnL
pnad1
pnad5
JL1 JL2 JL3 JL4 JL5 JL6 JL7 GD1 GD2 GD3 GD4 GD5 GD6 GD7 BJ1 BJ2 BJ3 BJ4 BJ5 BJ6 BJ7 GS1 GS2 GS3 GS4 GS5 GS6 GS7
Female Female Female Female Female Female Female Female Female Female Female Female Female Female Female Female Female Female Female Female Female Female Female Female Female Female Female Female
Changchun, Jilin Changchun, Jilin Changchun, Jilin Changchun, Jilin Changchun, Jilin Changchun, Jilin Changchun, Jilin Guangzhou, Guangdong Guangzhou, Guangdong Guangzhou, Guangdong Guangzhou, Guangdong Guangzhou, Guangdong Guangzhou, Guangdong Guangzhou, Guangdong Changping, Beijing Changping, Beijing Changping, Beijing Changping, Beijing Changping, Beijing Changping, Beijing Changping, Beijing Lanzhou, Gansu Lanzhou, Gansu Lanzhou, Gansu Lanzhou, Gansu Lanzhou, Gansu Lanzhou, Gansu Lanzhou, Gansu
125.19/43.54 125.19/43.54 125.19/43.54 125.19/43.54 125.19/43.54 125.19/43.54 125.19/43.54 113.14/23.08 113.14/23.08 113.14/23.08 113.14/23.08 113.14/23.08 113.14/23.08 113.14/23.08 116.13/40.13 116.13/40.13 116.13/40.13 116.13/40.13 116.13/40.13 116.13/40.13 116.13/40.13 103.51/36.04 103.51/36.04 103.51/36.04 103.51/36.04 103.51/36.04 103.51/36.04 103.51/36.04
KF738484 KF738485 KF738490 KF738486 KF738487 KF738488 KF738489 KF738492 KF738498 KF738493 KF738494 KF738495 KF738496 KF738497 KF738499 KF738500 KF738501 KF738502 KF738503 KF738504 KF738505 KF738491 KF738506 KF738507 KF738508 KF738509 KF738510 KF738511
KF738456 KF738457 KF738458 KF738459 KF738460 KF738461 KF738462 KF738470 KF738476 KF738471 KF738472 KF738473 KF738474 KF738475 KF738477 KF738478 KF738479 KF738483 KF738480 KF738481 KF738482 KF738463 KF738464 KF738465 KF738469 KF738466 KF738467 KF738468
KF738568 KF738571 KF738572 KF738573 KF738574 KF738569 KF738570 KF738582 KF738585 KF738586 KF738587 KF738588 KF738583 KF738584 KF738589 KF738590 KF738594 KF738591 KF738595 KF738592 KF738593 KF738575 KF738576 KF738577 KF738578 KF738581 KF738579 KF738580
KF738512 KF738513 KF738514 KF738515 KF738516 KF738517 KF738518 KF738526 KF738527 KF738528 KF738529 KF738530 KF738531 KF738532 KF738533 KF738534 KF738535 KF738536 KF738537 KF738538 KF738539 KF738519 KF738520 KF738523 KF738524 KF738525 KF738521 KF738522
KF738540 KF738541 KF738542 KF738553 KF738543 KF738544 KF738545 KF738565 KF738566 KF738560 KF738561 KF738562 KF738563 KF738564 KF738567 KF738554 KF738555 KF738556 KF738557 KF738558 KF738559 KF738547 KF738548 KF738546 KF738549 KF738550 KF738551 KF738552
Note: For cox1: GenBank accession Nos. KF738484–KF738489 represent the same sequence, KF738492–KF738497 represent the same sequence, KF738499–KF738511 represent the same sequence. For cytb: GenBank accession Nos. KF738477–KF738482 represent the same sequence, KF738470–KF738475 represent the same sequence, KF738464–KF738468 represent the same sequence, KF738456–KF738462 represent the same sequence. For rrnL: GenBank accession Nos. KF738568–KF738570 represent the same sequence, KF738582–KF738584 represent the same sequence, KF738575–KF738580 represent the same sequence, KF738590–KF738593 represent the same sequence. For nad1: GenBank accession Nos. KF738514–KF738518 represent the same sequence, KF738519–KF738522 represent the same sequence, KF738526–KF738532 represent the same sequence, KF738536–KF738539 represent the same sequence. For nad5: GenBank accession Nos. KF738540–KF738545 represent the same sequence, KF738547–KF738564 represent the same sequence.
(970–3390 km). The worm’s gender, geographical origin, longitude and latitude, and GenBank accession numbers for sequences of pcox1, pcytb, prnnL, pnad1 and pnad5 of S. obvelata, are listed in Table 1. Pinworms were washed extensively with physiological saline, and individual pinworms were identified to species according to their morphological characteristics (Taffs, 1976), and then fixed in 70% ethanol until the extraction of genomic DNA. Total genomic DNA was extracted from each individual pinworm using a standard sodium dodecyl-sulphate/proteinase K treatment, followed by purification over a mini-column (TIANamp Genomic DNA Kit, TIANGEN Biotech, Beijing/China), and eluted into 35 ml double-distilled water. DNA samples were stored at 20 C for later use. Enzymatic amplification and sequencing A portion of the cox1, cytb, rrnL, nad1 and nad5 were amplified individually by polymerase chain reaction (PCR) using the following primers: cox1F (50 -ATAGGGGTTCTGGTACTA-30 ) and cox1R (50 -AATGAAAATGGAGCAACAAC-30 ), cytb F (50 GTTGGTGTTTTATTATGG-30 ) and cytb R (50 -CAATATGAAC AGGACTAGAT-30 ), rrnL F (50 - GTTGGTGTTTTATTATGG-30 ) and rrnL R (50 -CAATATGAACAGGACTAGAT-30 ), nad1 F (50 TTTCTGTTCATTCTACTGGG-30 ) and nad1R (50 -ATAACGAA AACGAGGATAAG-30 ), nad5F (50 -AGATGTAGTGGTGCTAT
GAA-30 ) and nad5 R (50 -AGATGTAGTGGTGCTATGAA-30 ). These primers were designed based on the corresponding sequences of Enterobius vermicularis (NC_011300) and Wellcomia siamensis (NC_016129), available in GenBank. As described previously (Lou et al., 2014), One microlitre of DNA template was used in a PCR reaction of 25 ml containing 5 ml of 5 colorless Go Taq flexi buffer (pH 8.5), 2 ml of MgCl2 (25 mM), 2 ml of dNTP Mixture (2.5 mM), 0.5 ml of each primer (10 pmol/ml) and 0.13 ml of Go Taq DNA polymerase (5 U/ml) in a thermocycler under the following conditions: 95 C for 2 min (initial denaturation), followed by 35 cycles of 95 C for 1 min (denaturation); 47.4 C (for cox1) or 46.1 C (for cytb) or 45.8 C (for rrnL), 47.6 C (for nad1) or 47.7 C (for nad5) for 1 min (annealing); 72 C for 1 min (extension) for 35 cycles, and then followed by a final extension at 72 C for 5 min. Each amplicon was examined by agarose gel (1%) electrophoresis and ethidium bromide staining. The positive PCR products were sent to Life Technology Company (Beijing, China) for sequencing using the primers used in primary amplifications. Sequence analyses and phylogenetic reconstruction Sequences of the five mtDNA regions were aligned using program Clustal X 1.83 (Thompson et al., 1997). Inter-specific sequence differences in the five mtDNA regions among members of the family Oxyuridae were determined by comparison with
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DOI: 10.3109/19401736.2014.905861
corresponding sequences of E. vermicularis (NC_011300), W. siamensis (NC_016129) and A. tetraptera (KF444301, KF444281, KF444316, KF444336) using the program DNAstar5.0. The phylogenetic relationship among all S. obvelata samples with other members of Oxyuridae was reconstructed based on the combined pcox1, pcytb, pnad1 and pnad5 sequences, using Trichuris ovis (NC_018597.1) as the outgroup. Oesophagostomum dentatumrispinu (GQ888716), O. quadrispinulatum (NC_014181) and Chabertia ovina (NC_013831) which are nematodes in the large intestine were also included for phylogenetic analysis. 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 and MP analyses were carried out using PAUP 4.0 Beta 10 program (Swofford, 2002). Bootstrap probability (BP) was calculated from 100 and 1000 bootstrap replicates with 10 random additions per replicate in PAUP. The values of three methods above 50% were reported. Phylograms were drawn using the Tree View program version 1.65 (Page, 1996). Results and discussion PCR products of pcox1, pcytb, prrnL, pnad1 and pnad5 were analyzed individually by agarose gel electrophoresis, and then all the positive amplicons were subjected to direct sequencing. The length of the pcox1, pcytb, prrnL, pnad1 and pnad5 gene were 628 bp, 555 bp, 548 bp, 548 bp and 561 bp, respectively, sequence differences in these genes among S. obvelata from the four different regions were only 0.18%, 0.33%, 0.97%, 0.54% and 0.25%, respectively, because some of them have identical sequences. The sequences of pcox1, pcytb, prnnL, pnad1 and pnad5 of S. obvelata have been deposited in the GenBank under the accession numbers KF738456–KF738595 (Table 1). The A + T contents of the sequences were 68.8–69.1% (pcox1), 72.3– 72.8% (pcytb), 74.3–76.5% (prrnL), 72.1–72.8% (pnad1) and 67.7–68.3% (pnad5), respectively, which was consistent with that of recent studies of other nematodes (Chang et al., 2013; Lin et al., 2012; Lou et al., 2014). The intra-specific sequence
Variability in five mt genes in S. obvelata
3
variations within S. obvelata were 0–1.0% for pcox1, 0–1.6% for pcytb, 0–2.8% for prrnL, 0–2.0% for pnad1 and 0–1.8% for pnad5, respectively. Although samples of S. obvelata were collected from different provinces, and the furthest distance is approximately 3390 km between Changchun, Jilin Province and Guangzhou, Guangdong Province, the relationship of mice was close. This is because all samples of S. obvelata were collected from Kunming and BALB/c mice, and these mice were originally come from several large experimental animal centers in China, so the intra-specific variations in these mt genes among S. obvelata samples were low, which were consistent with that of a recent study of another pinworm, A. tetraptera (Lou et al., 2014). By contrast, the inter-specific sequence differences among members of the family Oxyuridae, E. vermicularis, W. siamensis and A. tetraptera were significantly higher, being 14.0–17.5% for pcox1, 27.5–32.9% for pcytb, 35.8–37.2% for prrnL (A. tetraptera excluded), 22.2–26.8% for pnad1 and 22.3–25.2% for pnad5, respectively. This result was also consistent with that of some other studies (Chang et al., 2013; Lin et al., 2012; Lou et al., 2014; Wang et al., 2013a). For pcox1, pcytb, prrnL, pnad1 and pnad5, the number of nucleotide variations were 7, 11, 16, 12, 14, respectively. The nucleotide variations were related mainly to changes at the third codon position, being 5 for pcox1, 6 for pcytb, 9 for pnad1 and 8 for pnad5, respectively. Fewer changes were detected at the first and second codon positions (data not showed), similar to that of previous studies (Chang et al., 2013; Lou et al., 2014; Wang et al., 2013a). To determine the phylogenetic relationship of the S. obvelata samples with other members of the Oxyuridae family, the combined four mtDNA protein-coding gene (rrnL not included) were analyzed using BI, ML and MP three methods. Topologies of all trees inferred by three methods with different distance models were identical or similar. Using T. ovis as the outgroup, there were two main clades in the tree (Figure 1). Oesophagostomum dentatumrispinu, O. quadrispinulatum and C. ovina belonging to Strongylata were in one clade, and the other one was composed of members of the Oxyurida family, S. obvelata, A. tetraptera, E. vermicularis and W. siamensis. Interestingly, in the pinworm clade, the S. obvelata samples from
Figure 1. Phylogenetic relationships of Syphacia obvelata samples from laboratory mice in different provinces in China, with other members of the Oxyuridae family based on the combined data set (pcox1 + pnad1 + pnad5 + pcytb), using Bayesian inference (BI), maximum likelihood (ML) and maximum parsimony (MP), using Trichuris ovis (NC_018597.1) as the outgroup. The numbers along branches indicate bootstrap values resulting from different analyses in the order: BI/ML/MP. Values lower than 50 are given as dashes.
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Gansu Province and Beijing were not always grouped together, but the samples from Jilin Province and Guangdong Province grouped together, indicating the phylogenetic relationship of the S. obvelata in the present study were not in accordance with previous studies (Wang et al., 2013b). In conclusion, this study was the first to report genetic variation within S. obvelata in China. The results revealed a lowlevel intra-specific variation in cox1, cytb, rrnL, nad1 and nad5 sequences among S. obvelata samples from four different geographical localities in China. These results provide basic information for further studies of systematics and population genetics of S. obvelata.
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Declaration of interest The experiments comply with the current laws of the country in which the experiments were performed. The authors report no conflicts of interests. The authors alone are responsible for doing the research and writing the paper. This work was supported by the Fund for Imported Talents in Heilongjiang Bayi Agricultural University (XDB2013-42).
References Baker DG. (1998). Natural pathogens of laboratory mice, rats, and rabbits and their effects on research. Clin Microbiol Rev 11:231–66. Bugarski D, Jovcic G, Katic-Radivojevic S, Petakov M, Krstic´ A, Stojanovic´ N, Milenkovic´ P. (2006). Hematopoietic changes and altered reactivity to IL-17 in Syphacia obvelata infected mice. Parasitol Int 55:91–7. Chang QC, Gao JF, Sheng ZH, Lou Y, Zheng X, Wang CR. (2013). Sequence variability in three mitochondrial genes among four roundworm species from wild animals in China. Mitochondrial DNA. 2013 Sep 16. [Epub ahead of print]. doi: 10.3109/19401736.2013.823171. Chen XM, Li X, Lin RQ, Deng JY, Fan WY, Yuan ZG, Song HQ, et al. (2011). Pinworm infection in laboratory mice in southern China. Lab Anim 45:58–60.
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Kijewska A, Dzido J, Rokicki J. (2009). Mitochondrial DNA of Anisakis simplex s.s. as a potential tool for differentiating populations. J Parasitol 95:1364–70. Lin RQ, Liu GH, Song HQ, Zhang Y, Li MW, Zhou FC, Yuan ZG, et al. (2012). Sequence variability in three mitochondrial genes between the two pig nodule worms Oesophagostomum dentatum and O. quadrispinulatum. Mitochondrial DNA 23:182–6. Lou Y, Zhang Y, Qiu JH, Gao JF, Wang WT, Xiao JY, Chang QC, Wang CR. (2014). Sequence variability in four mitochondrial genes among pinworm Aspicularis tetraptera isolates from laboratory mice in four provinces in China. Mitochondrial DNA. 2014 Jan 7. [Epub ahead of print]. doi: 10.3109/19401736.2013.855736. Michels C, Goyal P, Nieuwenhuizen N, Brombacher F. (2006). Infection with Syphacia obvelata (pinworm) induces protective Th2 immune responses and influences ovalbumin-induced allergic reactions. Infect Immun 74:5926–32. Page RD. (1996). TREEVIEW: An application to display phylogenetic trees on personal computers. Comput Appl Biosci 12:357–8. Pearson DJ, Taylor G. (1975). The influence of the nematode Syphacia oblevata on adjuvant arthritis in the rat. Immunology 29:391–6. Ronquist F, Huelsenbeck JP. (2003). MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19:1572–4. Swofford DL. (2002). PAUP*: Phylogenetic analysis using parsimony (and Other Methods). Sunderland, MA: Sinauer Associates. Taffs LF. (1976). Pinworm infection in laboratory rodents: A review. Lab Anim 10:1–13. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. (1997). The Clustal_X windows interface: Flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 24:4876–82. Wang CR, Gao JF, Chang QC, Zou FC, Zhao Q, Zhu XQ. (2013a). Sequence variability in four mitochondrial genes among Bunostomum trigonocephalum isolates from four provinces in China. J Helminthol 87:416–21. Wang XY, Zhao GH, Liu GH, Li JY, Zhou DH, Xu MJ, Lin Q, Zhu XQ. (2013b). Characterization of Dicrocoelium chinensis from domestic yaks in China using genetic markers in two mitochondrial genes. Mitochondrial DNA 24:263–6.
SHORT COMMUNICATION
Genetic variability among Syphacia obvelata isolates from laboratory mice in four different geographical locations of China revealed by sequence analyses of five mitochondrial genes
Mitochondrial DNA Downloaded from informahealthcare.com by Nyu Medical Center on 10/15/14 For personal use only.
Chun-Ren Wang, Yan Lou, Yan Zhang, Wen-Tao Wang, Xu Zheng, Wen-Wen Xu, Ying Zhang, Si-Qin Tian, Lu Na, and Qiao-Cheng Chang College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang Province, P.R. China
Abstract
Keywords
Syphacia obvelata is a rodent nematode with high prevalence in laboratory mice. In the present study, we examined the genetic variability of S. obvelata from naturally infected laboratory mice in four different provinces, China. Five mitochondrial (mt) DNA regions, namely cytochrome c oxidase subunit 1 (pcox1), cytochrome b (pcytb), large subunit ribosomal RNA (prrnL) and NADH dehydrogenase subunits 1 and 5 (pnad1 and pnad5), were amplified separately from individual nematodes by PCR, and then sequenced directly. The size of the sequences of pcox1, pcytb, prrnL, pnad1 and pnad5 was 628 bp, 555 bp, 548 bp, 548 bp and 561 bp, respectively. While the intra-specific sequence variations within S. obvelata were 0–1.0% for pcox1, 0–1.6% for pcytb, 0–2.8% for prrnL, 0–2.0% for pnad1 and 0–1.8% for pnad5, the inter-specific sequence differences among members of the Oxyuridae were significantly higher, being 14.0–17.5% for pcox1, 27.5–32.9% for pcytb, 35.8–37.2% for prrnL, 22.2–26.8% for pnad1 and 22.3–25.2% for pnad5, respectively. Phylogenetic analyses based on combined sequences of four mt proteincoding genes, using Bayesian inference (BI), maximum likelihood (ML) and maximum parsimony (MP) methods, indicated that all of the S. obvelata samples grouped together with high statistical support, but samples from the same geographical origin did not always cluster together. These findings demonstrated the existence of low-level intra-specific variation in five mtDNA sequences among S. obvelata isolates from laboratory mice, but no obvious geographical distinction among S. obvelata isolates from laboratory mice in different geographic regions in China. These results provide basic information for further studies of systematics and population genetics of S. obvelata.
Laboratory mice, mitochondrial DNA, phylogenetic analyses, sequence variability, Syphacia obvelata
Introduction Syphacia obvelata, a mouse pinworm, inhabits the caecum and colon of laboratory mice and rats, which can cause oxyuriasis (Baker, 1998). It is a common parasite of laboratory mice with high prevalence even in well-managed colonies or laboratory animal centers (Baker, 1998; Chen et al., 2011). Although mice pinworm infection usually has no obvious clinical symptoms, it may have untoward effects on behavior, growth, intestinal physiology and immunology, and also interferes with research results in many ways (Baker, 1998; Bugarski et al., 2006; Michels et al., 2006; Pearson & Taylor, 1975; Taffs, 1976). Recent studies showed that some mitochondrial (mt) genes, such as cytochrome c oxidase subunit 1 (cox1), cytochrome b (cytb), and NADH dehydrogenase subunits 1, 4 and 5 (nad1, nad4 and pnad5), provide useful genetic markers for systematic, evolutionary, and the intra- and inter-population genetic variations of different nematodes (Chang et al., 2013; Kijewska et al., 2009; Lin et al., 2012; Lou et al., 2014; Wang et al., 2013a). In a previous study, we reported sequence variability among a mouse Correspondence: C.R. Wang, College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang Province 163319, P.R. China. Tel: +86-459-6819099. Fax: +86-459-6819090. E-mail: [email protected]
History Received 8 February 2014 Revised 2 March 2014 Accepted 15 March 2014 Published online 4 April 2014
pinworm, Aspicularis tetraptera, from laboratory mice in different provinces in China (Lou et al., 2014), however, genetic variability in another mice pinworm (S. obvelata) in China’s different geographical origins was unknown. Therefore in this study, sequence variability in cox1, cytb, large subunit ribosomal RNA (rrnL), nad1 and nad5 genes among S. obvelata isolates from laboratory mice in four provinces in China, were examined. Based on the combined four mtDNA protein-coding genes, the phylogenetic relationships of S. obvelata with other members of Oxyuridae were reconstructed. All these results will provide basic information for further studies of systematics, population genetics of S. obvelata.
Materials and methods Parasites and extraction of genomic DNA All animals were dissected humanely, according to protocols approved by the Institute Animal Care Committee. Adult S. obvelata worms (n ¼ 28) were collected from the caecum and colon of laboratory mice in Changchun, Jilin Province (JL1-JL7), northeast China; Lanzhou, Gansu Province (GS1-GS7), northwest China; Guangzhou, Guangdong Province (GD1-GD7), south China, and Changping, Beijing (BJ1-BJ7), north China. Geographical locations were separated by significant distances
2
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Mitochondrial DNA, Early Online: 1–4
Table 1. Geographical origins and worm’s gender of Syphacia obvelata samples used in the present study, longitude, latitude and the GenBank accession numbers for sequences of partial mitochondrial cytochrome c oxidase subunit1 gene (pcox1), large subunit ribosomal RNA (prrnL), NADH dehydrogenase subunit 1 and 5 gene (pnad1 and pnad5), and cytochrome b gene (pcytb).
Mitochondrial DNA Downloaded from informahealthcare.com by Nyu Medical Center on 10/15/14 For personal use only.
GenBank accession numbers Sample codes
Gender
Geographical origins
Longitude/latitude
pcox1
pcytb
prrnL
pnad1
pnad5
JL1 JL2 JL3 JL4 JL5 JL6 JL7 GD1 GD2 GD3 GD4 GD5 GD6 GD7 BJ1 BJ2 BJ3 BJ4 BJ5 BJ6 BJ7 GS1 GS2 GS3 GS4 GS5 GS6 GS7
Female Female Female Female Female Female Female Female Female Female Female Female Female Female Female Female Female Female Female Female Female Female Female Female Female Female Female Female
Changchun, Jilin Changchun, Jilin Changchun, Jilin Changchun, Jilin Changchun, Jilin Changchun, Jilin Changchun, Jilin Guangzhou, Guangdong Guangzhou, Guangdong Guangzhou, Guangdong Guangzhou, Guangdong Guangzhou, Guangdong Guangzhou, Guangdong Guangzhou, Guangdong Changping, Beijing Changping, Beijing Changping, Beijing Changping, Beijing Changping, Beijing Changping, Beijing Changping, Beijing Lanzhou, Gansu Lanzhou, Gansu Lanzhou, Gansu Lanzhou, Gansu Lanzhou, Gansu Lanzhou, Gansu Lanzhou, Gansu
125.19/43.54 125.19/43.54 125.19/43.54 125.19/43.54 125.19/43.54 125.19/43.54 125.19/43.54 113.14/23.08 113.14/23.08 113.14/23.08 113.14/23.08 113.14/23.08 113.14/23.08 113.14/23.08 116.13/40.13 116.13/40.13 116.13/40.13 116.13/40.13 116.13/40.13 116.13/40.13 116.13/40.13 103.51/36.04 103.51/36.04 103.51/36.04 103.51/36.04 103.51/36.04 103.51/36.04 103.51/36.04
KF738484 KF738485 KF738490 KF738486 KF738487 KF738488 KF738489 KF738492 KF738498 KF738493 KF738494 KF738495 KF738496 KF738497 KF738499 KF738500 KF738501 KF738502 KF738503 KF738504 KF738505 KF738491 KF738506 KF738507 KF738508 KF738509 KF738510 KF738511
KF738456 KF738457 KF738458 KF738459 KF738460 KF738461 KF738462 KF738470 KF738476 KF738471 KF738472 KF738473 KF738474 KF738475 KF738477 KF738478 KF738479 KF738483 KF738480 KF738481 KF738482 KF738463 KF738464 KF738465 KF738469 KF738466 KF738467 KF738468
KF738568 KF738571 KF738572 KF738573 KF738574 KF738569 KF738570 KF738582 KF738585 KF738586 KF738587 KF738588 KF738583 KF738584 KF738589 KF738590 KF738594 KF738591 KF738595 KF738592 KF738593 KF738575 KF738576 KF738577 KF738578 KF738581 KF738579 KF738580
KF738512 KF738513 KF738514 KF738515 KF738516 KF738517 KF738518 KF738526 KF738527 KF738528 KF738529 KF738530 KF738531 KF738532 KF738533 KF738534 KF738535 KF738536 KF738537 KF738538 KF738539 KF738519 KF738520 KF738523 KF738524 KF738525 KF738521 KF738522
KF738540 KF738541 KF738542 KF738553 KF738543 KF738544 KF738545 KF738565 KF738566 KF738560 KF738561 KF738562 KF738563 KF738564 KF738567 KF738554 KF738555 KF738556 KF738557 KF738558 KF738559 KF738547 KF738548 KF738546 KF738549 KF738550 KF738551 KF738552
Note: For cox1: GenBank accession Nos. KF738484–KF738489 represent the same sequence, KF738492–KF738497 represent the same sequence, KF738499–KF738511 represent the same sequence. For cytb: GenBank accession Nos. KF738477–KF738482 represent the same sequence, KF738470–KF738475 represent the same sequence, KF738464–KF738468 represent the same sequence, KF738456–KF738462 represent the same sequence. For rrnL: GenBank accession Nos. KF738568–KF738570 represent the same sequence, KF738582–KF738584 represent the same sequence, KF738575–KF738580 represent the same sequence, KF738590–KF738593 represent the same sequence. For nad1: GenBank accession Nos. KF738514–KF738518 represent the same sequence, KF738519–KF738522 represent the same sequence, KF738526–KF738532 represent the same sequence, KF738536–KF738539 represent the same sequence. For nad5: GenBank accession Nos. KF738540–KF738545 represent the same sequence, KF738547–KF738564 represent the same sequence.
(970–3390 km). The worm’s gender, geographical origin, longitude and latitude, and GenBank accession numbers for sequences of pcox1, pcytb, prnnL, pnad1 and pnad5 of S. obvelata, are listed in Table 1. Pinworms were washed extensively with physiological saline, and individual pinworms were identified to species according to their morphological characteristics (Taffs, 1976), and then fixed in 70% ethanol until the extraction of genomic DNA. Total genomic DNA was extracted from each individual pinworm using a standard sodium dodecyl-sulphate/proteinase K treatment, followed by purification over a mini-column (TIANamp Genomic DNA Kit, TIANGEN Biotech, Beijing/China), and eluted into 35 ml double-distilled water. DNA samples were stored at 20 C for later use. Enzymatic amplification and sequencing A portion of the cox1, cytb, rrnL, nad1 and nad5 were amplified individually by polymerase chain reaction (PCR) using the following primers: cox1F (50 -ATAGGGGTTCTGGTACTA-30 ) and cox1R (50 -AATGAAAATGGAGCAACAAC-30 ), cytb F (50 GTTGGTGTTTTATTATGG-30 ) and cytb R (50 -CAATATGAAC AGGACTAGAT-30 ), rrnL F (50 - GTTGGTGTTTTATTATGG-30 ) and rrnL R (50 -CAATATGAACAGGACTAGAT-30 ), nad1 F (50 TTTCTGTTCATTCTACTGGG-30 ) and nad1R (50 -ATAACGAA AACGAGGATAAG-30 ), nad5F (50 -AGATGTAGTGGTGCTAT
GAA-30 ) and nad5 R (50 -AGATGTAGTGGTGCTATGAA-30 ). These primers were designed based on the corresponding sequences of Enterobius vermicularis (NC_011300) and Wellcomia siamensis (NC_016129), available in GenBank. As described previously (Lou et al., 2014), One microlitre of DNA template was used in a PCR reaction of 25 ml containing 5 ml of 5 colorless Go Taq flexi buffer (pH 8.5), 2 ml of MgCl2 (25 mM), 2 ml of dNTP Mixture (2.5 mM), 0.5 ml of each primer (10 pmol/ml) and 0.13 ml of Go Taq DNA polymerase (5 U/ml) in a thermocycler under the following conditions: 95 C for 2 min (initial denaturation), followed by 35 cycles of 95 C for 1 min (denaturation); 47.4 C (for cox1) or 46.1 C (for cytb) or 45.8 C (for rrnL), 47.6 C (for nad1) or 47.7 C (for nad5) for 1 min (annealing); 72 C for 1 min (extension) for 35 cycles, and then followed by a final extension at 72 C for 5 min. Each amplicon was examined by agarose gel (1%) electrophoresis and ethidium bromide staining. The positive PCR products were sent to Life Technology Company (Beijing, China) for sequencing using the primers used in primary amplifications. Sequence analyses and phylogenetic reconstruction Sequences of the five mtDNA regions were aligned using program Clustal X 1.83 (Thompson et al., 1997). Inter-specific sequence differences in the five mtDNA regions among members of the family Oxyuridae were determined by comparison with
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DOI: 10.3109/19401736.2014.905861
corresponding sequences of E. vermicularis (NC_011300), W. siamensis (NC_016129) and A. tetraptera (KF444301, KF444281, KF444316, KF444336) using the program DNAstar5.0. The phylogenetic relationship among all S. obvelata samples with other members of Oxyuridae was reconstructed based on the combined pcox1, pcytb, pnad1 and pnad5 sequences, using Trichuris ovis (NC_018597.1) as the outgroup. Oesophagostomum dentatumrispinu (GQ888716), O. quadrispinulatum (NC_014181) and Chabertia ovina (NC_013831) which are nematodes in the large intestine were also included for phylogenetic analysis. 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 and MP analyses were carried out using PAUP 4.0 Beta 10 program (Swofford, 2002). Bootstrap probability (BP) was calculated from 100 and 1000 bootstrap replicates with 10 random additions per replicate in PAUP. The values of three methods above 50% were reported. Phylograms were drawn using the Tree View program version 1.65 (Page, 1996). Results and discussion PCR products of pcox1, pcytb, prrnL, pnad1 and pnad5 were analyzed individually by agarose gel electrophoresis, and then all the positive amplicons were subjected to direct sequencing. The length of the pcox1, pcytb, prrnL, pnad1 and pnad5 gene were 628 bp, 555 bp, 548 bp, 548 bp and 561 bp, respectively, sequence differences in these genes among S. obvelata from the four different regions were only 0.18%, 0.33%, 0.97%, 0.54% and 0.25%, respectively, because some of them have identical sequences. The sequences of pcox1, pcytb, prnnL, pnad1 and pnad5 of S. obvelata have been deposited in the GenBank under the accession numbers KF738456–KF738595 (Table 1). The A + T contents of the sequences were 68.8–69.1% (pcox1), 72.3– 72.8% (pcytb), 74.3–76.5% (prrnL), 72.1–72.8% (pnad1) and 67.7–68.3% (pnad5), respectively, which was consistent with that of recent studies of other nematodes (Chang et al., 2013; Lin et al., 2012; Lou et al., 2014). The intra-specific sequence
Variability in five mt genes in S. obvelata
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variations within S. obvelata were 0–1.0% for pcox1, 0–1.6% for pcytb, 0–2.8% for prrnL, 0–2.0% for pnad1 and 0–1.8% for pnad5, respectively. Although samples of S. obvelata were collected from different provinces, and the furthest distance is approximately 3390 km between Changchun, Jilin Province and Guangzhou, Guangdong Province, the relationship of mice was close. This is because all samples of S. obvelata were collected from Kunming and BALB/c mice, and these mice were originally come from several large experimental animal centers in China, so the intra-specific variations in these mt genes among S. obvelata samples were low, which were consistent with that of a recent study of another pinworm, A. tetraptera (Lou et al., 2014). By contrast, the inter-specific sequence differences among members of the family Oxyuridae, E. vermicularis, W. siamensis and A. tetraptera were significantly higher, being 14.0–17.5% for pcox1, 27.5–32.9% for pcytb, 35.8–37.2% for prrnL (A. tetraptera excluded), 22.2–26.8% for pnad1 and 22.3–25.2% for pnad5, respectively. This result was also consistent with that of some other studies (Chang et al., 2013; Lin et al., 2012; Lou et al., 2014; Wang et al., 2013a). For pcox1, pcytb, prrnL, pnad1 and pnad5, the number of nucleotide variations were 7, 11, 16, 12, 14, respectively. The nucleotide variations were related mainly to changes at the third codon position, being 5 for pcox1, 6 for pcytb, 9 for pnad1 and 8 for pnad5, respectively. Fewer changes were detected at the first and second codon positions (data not showed), similar to that of previous studies (Chang et al., 2013; Lou et al., 2014; Wang et al., 2013a). To determine the phylogenetic relationship of the S. obvelata samples with other members of the Oxyuridae family, the combined four mtDNA protein-coding gene (rrnL not included) were analyzed using BI, ML and MP three methods. Topologies of all trees inferred by three methods with different distance models were identical or similar. Using T. ovis as the outgroup, there were two main clades in the tree (Figure 1). Oesophagostomum dentatumrispinu, O. quadrispinulatum and C. ovina belonging to Strongylata were in one clade, and the other one was composed of members of the Oxyurida family, S. obvelata, A. tetraptera, E. vermicularis and W. siamensis. Interestingly, in the pinworm clade, the S. obvelata samples from
Figure 1. Phylogenetic relationships of Syphacia obvelata samples from laboratory mice in different provinces in China, with other members of the Oxyuridae family based on the combined data set (pcox1 + pnad1 + pnad5 + pcytb), using Bayesian inference (BI), maximum likelihood (ML) and maximum parsimony (MP), using Trichuris ovis (NC_018597.1) as the outgroup. The numbers along branches indicate bootstrap values resulting from different analyses in the order: BI/ML/MP. Values lower than 50 are given as dashes.
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C.-R. Wang et al.
Gansu Province and Beijing were not always grouped together, but the samples from Jilin Province and Guangdong Province grouped together, indicating the phylogenetic relationship of the S. obvelata in the present study were not in accordance with previous studies (Wang et al., 2013b). In conclusion, this study was the first to report genetic variation within S. obvelata in China. The results revealed a lowlevel intra-specific variation in cox1, cytb, rrnL, nad1 and nad5 sequences among S. obvelata samples from four different geographical localities in China. These results provide basic information for further studies of systematics and population genetics of S. obvelata.
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Declaration of interest The experiments comply with the current laws of the country in which the experiments were performed. The authors report no conflicts of interests. The authors alone are responsible for doing the research and writing the paper. This work was supported by the Fund for Imported Talents in Heilongjiang Bayi Agricultural University (XDB2013-42).
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