Taenia asiatica and Taenia saginata: Genetic ...

Experimental Parasitology 113 (2006) 58–61 www.elsevier.com/locate/yexpr

Research brief

Taenia asiatica and Taenia saginata: Genetic divergence estimated from their mitochondrial genomes ! H.K. Jeon, K.S. Eom ¤ Department of Parasitology and Medical Research Institute, Chungbuk National University College of Medicine, Chongju, Chungbuk 361-763, Republic of Korea Received 9 June 2005; received in revised form 8 November 2005; accepted 10 November 2005 Available online 20 March 2006

Abstract We conducted a diVerential identiWcation of Taenia asiatica and Taenia saginata, through the mapping of mitochondrial genomes and the sequencing of the cox1 and cob genes. The entire mitochondrial genomes of T. asiatica and T. saginata were ampliWed by long-extension PCR and cloned; each was approximately 14 kb in size. Restriction maps of T. asiatica and T. saginata mitochondrial genomes were then constructed using 13 restriction enzymes. The resulting restriction patterns enable us to estimate their genetic divergence at 4.8%. The actual sequence divergence was computed 4.5% from the cox1 gene, and 4.1% from the cob gene. These results support the designation of T. asiatica as a separate species from T. saginata. © 2005 Elsevier Inc. All rights reserved. Index Descriptors and Abbreviations: Taenia asiatica; Taenia saginata; Mitochondrial genome; RFLP; Sequence divergence

Taenia solium (Linnaeus, 1758), Taenia saginata (Goeze, 1782) and Taenia asiatica (Eom and Rim, 1993) are all Taenia tapeworms which cause human infections, using either cattle or pigs as intermediate hosts. T. asiatica is the species most recently described on the basis of the morphological characteristics of its adult worms, which possess an unarmed rostellum, a large number of uterine buds (twigs), and a posterior protuberance on gravid proglottids (Eom and Rim, 1993). The life cycle of this cestode diVers from that of T. saginata, in that it exploits diVerent intermediate hosts, and diVerent tissues, for the completion of its life cycle. Larval T. saginata infects the skeletal muscles of cattle, whereas larval T. asiatica infects the tissues of the liver, omentum, serosa, and lungs of pigs (Eom and Rim, 1992a; Eom and Rim, 1992b; Eom et al., 1992). The metacestodes of T. asiatica (Cysticercus viscerotropica) also diVer ! The sequence data reported herein have been deposited in the GenBank database, under Accession Nos. AF445798 (T. asiatica) and AY195858 (T. saginata). * Corresponding author. Fax: +82 43 272 1603. E-mail address: [email protected] (K.S. Eom).

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morphologically from those of T. saginata in their production of wart-like formations on the external surface of the bladder wall (Eom and Rim, 1993). Thus, based on the morphology of the metacestodes and adult worms, as well as their life cycles, host ranges, and a variety of molecular characteristics, T. asiatica appears to diVer substantially from T. saginata (De Queiroz and Alkire, 1998; Eom et al., 2002; Galan-Puchades and Mas-Coma, 1996; Hoberg et al., 2000; Ito et al., 2003; Jeon et al., 2005; Zarlenga et al., 1991; Zarlenga and George, 1995). In the present study, we estimated the genetic divergence between T. asiatica and T. saginata, on the basis of the sequences of the cox1 and cob genes, coupled to RFLP analysis of the mitochondrial genomes. Samples of T. asiatica from Korea and T. saginata from Belgium were used as representative in this study. The Belgian T. saginata isolate originated in Africa. The Taenia proglottids were recovered from stool samples after treatment with 2 g of niclosamide, followed by purgation with MgSO4. The parasites were identiWed by their morphological and molecular characteristics, as well as by animal inoculation experiments. These samples were preserved at ¡70 °C in 70% ethanol until use.

H.K. Jeon, K.S. Eom / Experimental Parasitology 113 (2006) 58–61

A single frozen proglottid was chopped into small pieces and digested for 30 min in DNA extraction buVer (20 mM Tris base/HCl, pH 7.5, 25 mM EDTA, 75 mM NaCl, 0.1% SDS, and 0.5 mg/mL proteinase K) at 65 °C. After removing debris, the genomic DNA was extracted with phenol/chloroform. The DNA was then precipitated with 3 M sodium acetate (pH 5.2) and washed with 95% ethanol, then airdried. The pellets were subsequently dissolved in 50 !L TE buVer. The entire mtDNAs of T. asiatica and T. saginata were then ampliWed by long extension PCR (Expand™ 20 kbPlus kit, Roche, Mannheim, Germany) from the total genomic DNA of a single adult worm, in one fragment, using the following primers: mt1f (CAG CAG TCT TAA CAT CTA ACC CAA CCG TAA ACA TAT GAT GTC CCC ACA CAC) and mt2r (TCG GTT ACT ATG ATA ATA GGA GTA CCA ACA GGA ATA AAG GTT TTT ACT TGA C). These primers were predicated on mitochondrial sequences which are relatively conserved, namely, the large subunit ribosomal RNA gene of T. solium (Zarlenga and George, 1995). Long-PCR ampliWcation was conducted in accordance with the protocols of Zarlenga and George (1995), as follows: 94 °C for 5 min (initial denaturation); 94 °C for 1 min (denaturation), 68 °C for 15 min (annealing), 72 °C for 8 min (extension), for 40 cycles, and, a Wnal incubation at 72 °C for 10 min. Restriction enzyme cleavage maps of the mtDNA from T. asiatica and T. saginata were constructed by single and double enzyme digests. The mtDNA was digested using 13 restriction enzymes, and the resulting fragments were separated by electrophoresis on 0.7% agarose gel. The ampliWcation products were digested by restriction enzymes which recognize 4-base or 6-base sequences: AccI, AvaI, BamHI, BglII, EcoRI, EcoRV, HincII, HindIII, KpnI, PstI, PvuII, SalI, and XhoI (Roche Diagnostic, Mannheim, Germany). The PCR products were treated with 10 U of the above enzymes, at a Wnal volume of 50 !L for 1 h at 37 °C in a water bath, in a reaction buVer containing 500 ng DNA, 10 mM Tris–HCl (pH 7.5), 7 mM MgCl2, 60 mM NaCl, and 10 mM dithiothreitol. The entirety of the cox1 and cob genes were ampliWed by using two sets of primers, which were designed from the complete sequence of the T. asiatica mitochondrial genome (Jeon et al., 2005). The primers of mtcoxf1 (5!-GTG ACT TTA TAT AAG GTC ATC TTA-3!) and mtcoxr2 (5!ATA CAA TAC AAA CCA TCA TAG ATT-3!) were used to amplify the cox1 genes of both T. asiatica and T. saginata. The cob genes were ampliWed using the following primers: cobf (5!-GTA GAT TGT GGT TCT ATT GAA TAC-3!) and cobr (5!-GCT AAC CAT TTT TAT CTA CAT ATA-3!). PCR was conducted in a 50 !L reaction volume with 10 U of TaKaRa LA Taq (TAKARA SHUZO CO., Japan), 25 mM MgCl2, 2.5 mM dNTP, 20 pmol of each primer, and 100 ng of genomic DNA. The puriWed PCR ampliWed fragments of the cox1 and cob genes were then cloned separately. The fragments were ligated overnight at 15 °C using the pGEM-T easy vector

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kit (Promega, Madison, WI, USA). The ligated plasmids were then transformed into the DH5" strain of Escherichia coli. The plasmid DNA was puriWed with a QIAprep Spin Miniprep kit (Qiagen, Valencia, CA, USA). The inserts were sequenced with a Big-Dye Terminator Sequencing kit (Applied Biosystems, Foster City CA, USA) and analyzed on an ABI 3100 automated DNA sequencer. The sequences were then assembled and aligned using Clustal X (Thompson et al., 1997) and Bioedit (BIOSOFT, Ferguson, MO, USA). Fragment size was measured by the Sigmagel v. 1.05 (JANDEL SCIENTIFIC, SPSS, Chicago, IL, USA) program. The cytochrome c oxidase subunit I and cytochrome b sequences of T. asiatica (GenBank Accession Nos. AB107234, AB107235, AB107236, AF445798) were compared with those of T. saginata (from AB107237 to AB107247, AB066495, AY195858). The mitochondrial genomes of T. asiatica and T. saginata were each found to be approximately 14 kb in length (Fig. 1). The restriction fragments of the mitochondrial genome of T. asiatica after digestion with a variety of enzymes, were as follows: 7, 5, 1.1, and 0.6 kb, AccI, 7, and 6.7 kb, AvaI; 10 and 4 kb, BamHI; 8, 4, and 2 kb, BglII; 5, 3.5, and 3 kb, EcoRV; 4.5, 3.8, 2.0, 1.6 and 1 kb, HincII; 9 and 5 kb, HindIII; 11 and 3 kb, KpnI; 9 and 5 kb, PstI; 9, 2.5 and 1.5 kb, PvuII; 9 and 5 kb, SalI (Fig. 2A). No enzyme sites for EcoRI, and XhoI were observed in the T. asiatica mtDNA. The restriction enzyme map of T. asiatica mtDNA, assembled from these data, is shown in Fig. 2A. The restriction enzyme map of T. saginata mtDNA was constructed from the following restriction fragments: 8, 4, and 2 kb, BglII; 4.5, 3.8, 1.5, and 1 kb, HincII; 5, 4.5, 2, and 1.5 kb, HindIII; 9 and 4 kb, PvuII; 12 and 1.8 kb, XhoI

Fig. 1. AmpliWcation of the entire mitochondrial genomes of T. asiatica and T. saginata by long PCR. (M) Size makers (lambda DNA digested with HindIII); (A) T. asiatica (Korea); (B) T. saginata (Belgium).

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Fig. 2. Comparative restriction maps of mitochondrial genomes of T. asiatica (A) and T. saginata (B). The mtDNA was digested in separate reactions with 13 restriction enzymes, and the resulting fragments were electrophoresed in 0.7% agarose gel. The size of molecular weight markers (M) are in kilobase pairs. Lanes a–m, were digests with restriction enzymes (a, AccI; b, AvaI; c, BamHI; d, BglII; e, EcoRI; f, EcoRV; g, HincII; h, HindIII; i, KpnI; j, PstI; k, PvuII; l, SalI; m, XhoI).

(Fig. 2B). No enzyme sites for BamHI, EcoRI, EcoRV, KpnI or PstI were observed in T. saginata mtDNA. These restriction proWles were utilized to estimate the genetic divergence of two parasites, as explained in Eqs. (20) and (21) in the report of Nei and Li (1979). Eq. (20) demonstrates the relationship between the proportion of shared DNA fragments (F) and the number of nucleotide substitutions per site (!), from which a standard curve was constructed. The ! value was extrapolated from the appropriate curve generated by Eq. (20), then multiplied by 100, to obtain the percentage sequence diVerence (Nei and Li, 1979). Equation 21 demonstrates the estimation of ! from F. The nx and ny are the numbers of fragments in populations X and Y, respectively, and nxy is the number of fragments shared by both populations. The migration distances of the mtDNA fragments were observed from 46 fragments, including 12 fragments shared between T. asiatica and T. saginata. The sequence divergence between T. asiatica and T. saginata was estimated at 4.8% (Table 1). In both the full-length of T. asiatica and T. saginata the mitochondrial cox1 and cob sequences were 1620 and 1068, respectively. The sequence diVerences within the datasets were distributed unevenly across the three codon positions. The pairwise comparison of nucleotide sequences was conducted in the cox1 and cob genes. The sequence diVerence between T. asiatica and T. saginata was calculated at 4.6% in the cox1 and 4.1% cob genes. IntraspeciWc variation in the mitochondrial genes was detected among Wve T. asiatica isolates and 10 T. saginata isolates. In the cox1 gene, two variant nucleotide positions (0.1% of total length) were detected among the Wve T. asiatica isolates from China, the Philippines and Korea, whereas 13 variant nucleotide positions (0.2–0.8% of total length) were detected in the 10 T. saginata isolates from China, Ethiopia, France, Indonesia, Japan, Korea, Laos, the Philippines, Taiwan, Thailand, and Swiss. The mitochondrial genomes of cestodes reported thus far range between 13,503 and 13,900 bp in length. The entire

Table 1 Percentage sequence diVerences estimated by restriction fragment data of Taenia asiatica and Taenia saginata mitochondrial genomes and their actual values calculated from the sequences data of cox1 and cob genes T. asiatica (A) Restriction enzyme fragment data T. asiatica (28) T. saginata 0.52 (B) Percentage sequence diVerence T. asiatica (C) Sequence diVerences of coxl and cob T. asiatica (coxl) T. asiatica (cob)

T. saginata 12 (18) 4.8 4.6 4.1

Note: (A) The numbers in parenthesis represent the total number of restriction fragments in T. asiatica (X) and T. saginata (Y). The total number of fragments shared between T. asiatica andT. saginata is 12 (Z). The F value 0.52 is calculated by the formula of F D 2Z/X+Y. (B) The percentage sequence diVerence was extrapolated from the curves generated from equation 20 of Nei and Li (1979). (C) The actual values of percentage divergence were calculated from the nucleotide sequence data of cox1 and cob genes.

mtDNA digestion proWle generated by each restriction enzyme for each animal may be considered to represent a qualitative phenotype (Avise et al., 1979). As these phenotypes are composed of several mtDNA fragments, they are unlikely to independently arise in the evolutionary process via the convergence of unrelated phenotypes. Certain single-enzyme phenotypes appear to be related by single-base substitutions which result in the loss or creation of a cleavage site (Lansman et al., 1981). The degree of mtDNA divergence which was estimated in genetic distance of cob gene between sister species, congeneric species, and confamilial genera showed that greater than 2% sequence divergence from amphibian, reptile, avian, and mammalian (Johns and Avise, 1998). The closely related species of vertebrates regularly show more than 2% divergence at another mitochondrial gene, cytochrome b while the intraspeciWc divergences are rarely greater than 2% and most are less than 1% (Herbert et al., 2003).


A previous study of Bowles and MacManus (1994) reported that the sequences divergence of partial cox1 genes (366 bp from nucleotide position 742 to 1107) between T. asiatica and T. saginata was 2.2%, whereas Zarlenga and George (1995) estimated the sequence divergence between their genomes at 4.6%. When we conducted a comparison of the partial sequences of the cox1 gene, the sequence diVerences varied from 2% in the conserved region (nucleotide position of 742–1107), to 7% in the variable region (position 25–340). The comparison indicates that the short sequences of the cox1 gene may give a bias to analyses, and that the complete sequence data provides a more reliable result (Jeon et al., 2005). However, the restriction patterns of BglII, HincII, and HindIII digests demonstrated that even in very closely related molecules, restriction sites can diVer substantially. These diVerences were consistent with established the life cycle (Eom and Rim, 1993), topologies, on the basis of both morphological data (Hoberg et al., 2000), molecular sequence data (De Queiroz and Alkire, 1998), and epidemiological characteristics (Eom et al., 2002). Finally, our results support that T. asiatica and T. saginata are distinct species. Acknowledgments This work was supported by the research grant of the Chungbuk National University in 2004. Parasite materials used in this study were provided by the Parasite Resource Bank of Korea National Research Resource Center (R212005-000-10007-0), Republic of Korea. References Avise, J.C., Lansman, R.A., Shade, R.O., 1979. The use of restriction endonucleases to measure mitochondrial DNA sequence relatedness in natural populations. I. Population structure and evolution in the genus Peromyscus. Genetics 92, 279–295. Bowles, J., MacManus, D.P., 1994. Genetic characterization of the Asian Taenia, a newly described taeniid cestodes of human. American Journal of Tropical Medicine and Hygiene 50, 33–44. De Queiroz, A., Alkire, N.L., 1998. The phylogenetic placement of Taenia cestodes that parasitize humans. Journal of Parasitology 84, 379–383.

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