Novel methods for the molecular discrimination of Fasciola spp. on the basis of nuclear protein-coding genes

    Novel methods for the molecular discrimination of fasciola spp. on the basis of nuclear protein-coding genes Takuya Shoriki, Madoka Ichikawa-Seki, Keisuke Suganuma, Ikunori Naito, Kei Hayashi, Minoru Nakao, Junya Aita, Uday Kumar Mohanta, Noboru Inoue, Kenji Murakami, Tadashi Itagaki PII: DOI: Reference:

S1383-5769(15)00197-X doi: 10.1016/j.parint.2015.12.002 PARINT 1437

To appear in:

Parasitology International

Received date: Revised date: Accepted date:

7 October 2015 24 November 2015 6 December 2015

Please cite this article as: Shoriki Takuya, Ichikawa-Seki Madoka, Suganuma Keisuke, Naito Ikunori, Hayashi Kei, Nakao Minoru, Aita Junya, Mohanta Uday Kumar, Inoue Noboru, Murakami Kenji, Itagaki Tadashi, Novel methods for the molecular discrimination of fasciola spp. on the basis of nuclear protein-coding genes, Parasitology International (2015), doi: 10.1016/j.parint.2015.12.002

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Novel methods for the molecular discrimination of Fasciola spp. on the basis of

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nuclear protein-coding genes

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Takuya Shorikia,b, Madoka Ichikawa-Sekia,b*, Keisuke Suganumac, Ikunori Naitod, Kei Hayashia,b, Minoru Nakaoe, Junya Aitaa,b, Uday Kumar Mohantaa,b, Noboru Inouec, Kenji

Laboratory of Veterinary Parasitology, Faculty of Agriculture, Iwate University, 3-18-8

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a

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Murakamid, Tadashi Itagakia,b

Ueda, Morioka, Iwate 020-8550, Japan b

Department of Pathogenetic Veterinary Science, United Graduate School of Veterinary

c

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Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan National Research Center for Protozoan Diseases, Obihiro University of Agriculture and

d

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Veterinary Medicine, 2-11 Nishi, Inada-cho, Obihiro, Hokkaido 080-8555, Japan Laboratory of Veterinary Microbiology, Faculty of Agriculture, Iwate University,

e

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3-18-8 Ueda, Morioka, Iwate 020-8550, Japan Department of Parasitology, Asahikawa Medical University, Asahikawa, Hokkaido

078-8510, Japan

Corresponding author: Madoka Ichikawa-Seki Laboratory of Veterinary Parasitology, Faculty of Agriculture, Iwate University, 3-18-8 Ueda,

Morioka,

Iwate

020-8550,

Japan.

Tel:

+81-19-621-6218,

E-mail:

[email protected]

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Abstract

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Fasciolosis is an economically important disease of livestock caused by Fasciola

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hepatica, Fasciola gigantica, and aspermic Fasciola flukes. The aspermic Fasciola

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flukes have been discriminated morphologically from the two other species by the absence of sperm in their seminal vesicles. To date, the molecular discrimination of F. hepatica and F. gigantica has relied on the nucleotide sequences of the internal

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transcribed spacer 1 (ITS1) region. However, ITS1 genotypes of aspermic Fasciola

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flukes cannot be clearly differentiated from those of F. hepatica and F. gigantica. Therefore, more precise and robust methods are required to discriminate Fasciola spp. In this study, we developed PCR restriction fragment length polymorphism and multiplex

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PCR methods to discriminate F. hepatica, F. gigantica, and aspermic Fasciola flukes on the basis of the nuclear protein-coding genes, phosphoenolpyruvate carboxykinase and

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DNA polymerase delta, which are single locus genes in most eukaryotes. All aspermic Fasciola flukes used in this study had mixed fragment pattern of F. hepatica and F.

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gigantica for both of these genes, suggesting that the flukes are descended through hybridization between the two species. These molecular methods will facilitate the identification of F. hepatica, F. gigantica, and aspermic Fasciola flukes, and will also prove useful in etiological studies of fasciolosis.

Keywords: Fasciola spp.; pepck; pold; PCR–RFLP; Multiplex PCR

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1. Introduction

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The parasitic infection fasciolosis causes huge economic losses in the livestock

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industry of endemic areas. Fasciola hepatica and Fasciola gigantica are well known

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causative agents of the disease. They have normal spermatogenic ability and reproduce bisexually by fertilization [1, 2]. This spermatogenic ability is a prominent feature manifested by the presence of abundant mature sperm in the seminal vesicle which acts as

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a temporary storage of self-produced sperm. The two Fasciola species have been

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differentiated by the nucleotide sequences of their nuclear ribosomal internal transcribed spacer 1 (ITS1) regions [3]. Furthermore, the phylogenetic relationships of Fasciola spp. can be analyzed using mitochondrial DNA (mtDNA) markers, such as the NADH

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dehydrogenase subunit 1 (nad1) [3].

Aspermic Fasciola flukes have been identified in Asian countries [3-13]. These

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flukes harbor a meiotic disorder affecting spermatogenesis reflected by the presence of few or no sperm in the seminal vesicle [4, 14, 15], suggesting that they reproduce

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parthenogenetically. Aspermic Fasciola flukes have been reported to carry three distinct ITS1 genotypes [3]: Fh and Fg types, identical to the sequences of F. hepatica and F. gigantica, respectively, and Fh/Fg type, which is a combination of both sequences of F. hepatica and F. gigantica. These aspermic Fasciola flukes show two major nad1 lineages in their mtDNA. One of these belongs to the F. hepatica clade, whereas the other belongs to the F. gigantica clade, indicating that the maternal ancestors of aspermic Fasciola flukes are the particular lineage of F. hepatica or F. gigantica [4]. The existence of Fh/Fg type in ITS1, which is a member of nuclear DNA, suggests that aspermic Fasciola flukes are descended from a natural hybridization event between F. hepatica and F. gigantica [3, 4, 6]. However, the ITS1 region of ribosomal 3

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DNA contains hundreds of copies organized as tandem repeats. As repeat genes are

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highly recombinogenic and unstable [16], ITS1 analysis is unable to provide enough

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evidence of natural hybridization, and has caused some controversies in characterizing

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Fasciola spp. Indeed, three aspermic Fasciola flukes from Myanmar and Bangladesh displayed Fg type in ITS1 and showed nad1 haplotypes belonging to an F. gigantica haplogroup not an aspermic Fasciola haplotype. They were therefore identified as F.

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gigantica that had temporally lost their spermatogenic ability, probably because of aging

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[9, 11]. Similarly, one spermic Fasciola fluke from Bangladesh, displaying Fg type in ITS1, carried an identical nad1 haplotype to that of the aspermic Fasciola flukes. These observations highlight the difficulty of discriminating Fasciola spp. from Asian countries

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on the basis of spermatogenic status and ITS1 genotypes. Therefore, this study aimed to develop rapid and efficient methods on the basis

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of novel nuclear markers of phosphoenolpyruvate carboxykinase (pepck) and DNA polymerase delta (pold) genes, for more precise discrimination of F. hepatica, F.

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gigantica, and aspermic Fasciola flukes. Both the genes are known to exist as single copy in many eukaryotes [17, 18], and are predicted to be adequate markers for detecting hybridization events.

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2. Materials and methods

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F. hepatica, F. gigantica, and aspermic Fasciola flukes from various

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geographic origins (sample nos. 1–27) were examined in this study (Table 1). The

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spermatogenic status, nuclear ITS1 genotype, and mitochondrial nad1 haplotype of the flukes were analyzed as described previously [13].

DNA fragments of pepck and pold were amplified from a Fasciola fluke using degenerate

primer

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two

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(5′-TGYGGNAARACNAAYATGGCNATG-3′) (5′-CKCATNGCCATNGGRTCRTGCAT-3′)

(5′-GARATGGCNMGNGTNACNGGNGT-3′)

for

sets:

CGKTNMAM_f

and pepck, and

MHDPMAMR_r and

EMARVTGV_f GFTGAHVGK_r

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(5′-TTNCCNACRTGNGCNCCNGTRAANCC-3′) for pold. The primers for pepck were designed based on the conserved amino acid sequences of pepck of Schistosoma mansoni

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(accession no. AAD24794) and Brugia malayi (XP_001894888). On the other hand, the primers for pold were designed based on those of pold of B. malayi (XP_001892851),

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Rattus norvegicus (EDM07476), and Saccharomyces cerevisiae (EDN60257). PCR amplification was performed in a 25 µl reaction mixture containing 100 ng template DNA, 0.2 mM of each dNTP, 0.3 µM of each primer, 1 U of Ex-Taq polymerase (Takara Bio, Shiga, Japan), and the manufacturer’s supplied reaction buffer. Thermal conditions were 35 cycles of 94C for 30 s, 50C for 30 s, and 72C for 120 s. The PCR products of both genes were cloned into the pGEM-T vector (Promega, Madison, WI) and sequenced. Diagnostic

primer

sets

Fasciola-pepck-F1/Fasciola-pepck-R1

and

Fasciola-pold-F1/Fasciola-pold-R1 were designed from the resultant sequences of pepck and pold (Fig. S1 and S2). PCR amplification was performed in a 25 µl reaction mixture containing 100 ng template DNA, 0.4 mM of each dNTP, 0.3 µM of each primer, 1 U of 5

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KOD FX Neo (Toyobo, Osaka, Japan), and the manufacturer’s supplied reaction buffer.

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Thermal conditions included an initial denaturing step at 94C for 120 s, followed by 35

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cycles of 98C for 10 s and 68C for 30 s. PCR products of F. hepatica and F. gigantica

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(sample nos. 110; Table 1) were purified using the NucleoSpin Gel and PCR Clean-up kit (MACHEREY-NAGEL, Düren, Germany), and directly sequenced from both

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directions. The PCR products of the two F. gigantica from Zambia (sample nos. 11 and 12) and the five aspermic Fasciola flukes (nos. 1317) were cloned into the pCR 2.1-

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TOPO vector (Invitrogen, Groningen, the Netherlands), and sequenced. The resultant sequences were aligned with MEGA 6.06 [19] to detect polymorphic sites. Pairwise

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distances of the sequences were calculated using MEGA 6.06 software with the Kimura

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2-parameter model [20] and a gamma setting of 0.5. PCR–RFLP was established for both pepck and pold. The restriction enzymes

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Acc II for pepck and Alu I for pold were selected using ApE (A Plasmid Editor, www.biology.utah.edu/jorgensen/wayned/ape) based on the sequences of both genes

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(Figs. S1 and S2). PCR amplification for both genes was performed in 25 µl reaction mixtures containing 100 ng of template DNA, 0.1 mM of each dNTP, 0.5 pM of each primer

set

(Fasciola-pepck-F1/Fasciola-pepck-R1

and

Fasciola-pold-F1/Fasciola-pold-R1), 0.025 U of Go Taq DNA Polymerase (Promega), and the manufacturer’s supplied reaction buffer. Thermal conditions included an initial denaturing step at 94C for 90 s, followed by 35 cycles of 94C for 30 s, 60C for 30 s, and 72C for 60 s, then a final extension step at 72C for 10 min. Thereafter, 4 µl of PCR amplicons were digested in a 10 µl reaction mixture containing 1 U each of the restriction

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enzymes and 1.0 µl of the manufacturer’s supplied buffer (Takara). The reaction mixture

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was incubated at 37C for 3 h and examined by electrophoresis on a 1.8% agarose gel.

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A single step multiplex PCR was established for pepck. One forward primer,

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Fh-pepck-F, was developed to amplify a 241 bp fragment of F. hepatica, and another forward primer, Fg-pepck-F, was developed to amplify 509 bp or 510 bp fragments of F. gigantica. The reverse primer, Fcmn-pepck-R, was common for both the species (Fig.

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S1). Multiplex PCR was performed in a 25 µl reaction mixture containing 100 ng

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template DNA, 0.1 mM of each dNTP, three primers (0.5 µM Fh-pepck-F, 0.5 µM Fg-pepck-F, and 1 µM Fcmn-pepck-R), 0.025 U of Go Taq Polymerase (Promega), and

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the manufacturer’s supplied reaction buffer. Thermal conditions included an initial

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denaturing step at 94C for 90 s, followed by 30 cycles of 94C for 30 s, 61C for 30 s, and 72C for 60 s, with a final extension step at 72C for 10 min. The resultant PCR

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products were examined by electrophoresis on a 1% agarose gel.

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3. Results and Discussion

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Pepck amplicons were 925 bp long for F. hepatica (sample nos. 16; Table 1),

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and 986 bp for F. gigantica (sample nos. 710). Sequence lengths of 986 bp and 987 bp

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were obtained for the two F. gigantica samples from Zambia (nos. 11 and 12). Aspermic Fasciola flukes had both sequences of F. hepatica and F. gigantica (925 bp and 986 bp; Fig. S1). The nucleotide sequences of pold were 844 bp in length for both F. hepatica and

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F. gigantica, and aspermic Fasciola flukes contained both the sequences of the two

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species (Fig. S2). These nucleotide sequences were deposited in GenBank under accession numbers LC061148–061193 (Table 1).

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We characterized the exonic and intronic regions of pepck and pold. The

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nucleotide sequences of exons obtained in this study matched those of the expressed sequence tags of F. hepatica (sequence IDs: Fhep2603361 (pepck) and Fhep2511421

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(pold); HelmDB) and F. gigantica (Fgig2862651 (pepck) and Fgig2730908 (pold)), and were therefore thought to represent functional genes.

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The pairwise distances between F. hepatica and F. gigantica were 2.30%2.68% for pepck, and 4.93%5.22% for pold. Intraspecific variations were less than 1.0% for both genes. These high interspecific variations and low intraspecific variations enabled us to establish PCR–RFLP and multiplex PCR techniques to discriminate the two species. No nucleotide polymorphisms were observed at restriction enzyme recognition sites or primer annealing regions, except for that of Fh-pepck-F, where a single nucleotide substitution was observed in the sequence of sample no. 13 (Fig. S1). Nevertheless, Fh-pepck-F worked precisely in the multiplex PCR as described below.

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The restriction enzyme Acc II clearly discriminated the pepck fragment patterns

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between F. hepatica and F. gigantica, as expected from their sequences. The fragment

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specific for F. hepatica was 602 bp in length, whereas the two fragments of 392 bp and

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271 (or 272) bp were specific for F. gigantica. The other restriction enzyme, Alu I, was also useful for discriminating pold fragment patterns between the two species: the fragment specific for F. hepatica was 708 bp in length, whereas the fragments of 544 bp

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and 164 bp were specific for F. gigantica. All aspermic Fasciola flukes displayed a

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combination of both fragment patterns of the two species for both genes (Fig. 1). Multiplex PCR for pepck amplified 241 bp and 509 (or 510 bp) fragments in F. hepatica and F. gigantica, respectively, and both fragments were amplified in the aspermic

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Fasciola flukes (Fig. 1).

The spermatogenic status of Fasciola samples used in this study, and details of

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their ITS1 genotype and nad1 lineages are summarized in Table 1. These results were compared with the novel nuclear single copy markers, pepck and pold. For both F.

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hepatica (sample nos. 16; Table 1) and F. gigantica (nos. 712), consistent results were achieved in ITS1, pepck, and pold. However, all 15 aspermic Fasciola flukes (nos. 1327) displayed Fh/Fg type in both pepck and pold regardless of their ITS1 genotypes as well as nad1 lineages. Only five of the aspermic flukes (nos. 14, 15, and 2527) showed consistent ITS1 genotyping findings with pepck and pold. This result strongly demonstrated that pepck and pold can more precisely detect the evidence of hybridization events than ITS1.

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4. Conclusions

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The PCR–RFLP and multiplex PCR techniques developed in this study

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successfully discriminated the fragment patterns of F. hepatica, F. gigantica, and

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aspermic Fasciola flukes. This study strongly suggests that aspermic Fasciola flukes are the descendants of a hybridization event between F. hepatica and F. gigantica. However, further studies with more samples from wider geographical areas are needed to evaluate

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the reliability of the methods developed in this study, and to confirm the hybridization

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origin of aspermic Fasciola flukes.

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Acknowledgments

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We are grateful to the staff of the National Research Center for Protozoan

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Diseases, Obihiro University of Agriculture and Veterinary Medicine, the Laboratory of

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Veterinary Microbiology, Iwate University, and the Department of Parasitology, Asahikawa Medical University for invaluable help in this study. This study was supported in part by Grants-in-Aid for Science Research (B) and (C) (nos. 23405044 and

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24580420) from the Ministry of Education, Culture, Sports, Science and Technology,

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Japan.

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[10] P. Chaichanasak, M. Ichikawa, P. Sobhon, T. Itagaki, Identification of Fasciola

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flukes in Thailand based on their spermatogenesis and nuclear ribosomal DNA, and their intraspecific relationships based on mitochondrial DNA, Parasitol. Int. 61 (2012)

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545549.

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[11] U. K. Mohanta, M. Ichikawa-Seki, T. Shoriki, K. Katakura, T. Itagaki, Characteristics and molecular phylogeny of Fasciola flukes from Bangladesh,

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[12] T. Shoriki, M. Ichikawa-Seki, B. Devkota, H. B. Rana, S. P. Devkota, S. K. Humagain, T. Itagaki, Molecular phylogenetic identification of Fasciola flukes in Nepal, Parasitol. Int. 63 (2014) 758762. [13] K. Hayashi, M. Ichikawa-Seki, U. K. Mohanta, S. T. Shantikumar, T. Shoriki, H. Sugiyama, T. Itagaki, Molecular phylogenetic analysis of Fasciola flukes from eastern India, Parasitol. Int. 64 (2015) 334338. [14] K. Terasaki, H. Akahane, S. Habe, N. Moriyama, The geographical distribution of common liver flukes (the genus Fasciola) with normal abnormal spermatogenesis, Jpn. J. Vet. Sci. 44 (1982) 223231.

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and divergence studies, Syst. Biol. 43 (1994) 511525. [18] L. A. Loeb, R. J. Monnat Jr, DNA polymerases and human disease, Nat. Rev. Genet.

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[19] K. Tamura, G. Stecher, D. Peterson, A. Filipski, S. Kumar, MEGA6: Molecular Evolutionary Genetics Analysis version 6.0., Mol. Biol. Evol. 12 (2013) 27252729.

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[20] M. Kimura, A simple method for estimating evolutionary rate of base substitutions

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through comparative studies of nucleotide sequences, J. Mol. Evol. 16 (1980) 111120.

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Figure Captions

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Fig. 1. PCR–RFLP for pepck (A), pold (B), and multiplex PCR for pepck (C). M: 100 bp

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DNA ladder. Lanes 1 to 6: band patterns of F. hepatica (Fh type), lanes 7 to 12: band patterns of F. gigantica (Fg type), lanes 13 to 17: band patterns of the aspermic Fasciola

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fluke (Fh/Fg type). Lane numbers are consistent with the sample number in Table 1.

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Sperm in seminal vesicle

Nuclear DNA types

Mitochondrial nad1 lineages

ITS1

pepck

pold

1

F. hepatica

China



Fh

Fh

Fh

Fh

2

F. hepatica

China



Fh

Fh

Fh

Fh

3

F. hepatica

China



Fh

Fh

Fh

Fh

4

F. hepatica

Peru



Fh

Fh

Fh

5

F. hepatica

Peru



Fh

Fh

Fh

6

F. hepatica

Peru



Fh

Fh

Fh

7

F. gigantica

Nepal



Fg

Fg

Fg

8

F. gigantica

Nepal



Fg

Fg

9

F. gigantica

Philippines



Fg

Fg

10

F. gigantica

Philippines



Fg

Fg

11

F. gigantica

Zambia



Fg

Fg

12

F. gigantica

Zambia



Fg

13

aspermic Fasciola fluke

China



Fg

14

aspermic Fasciola fluke

China



Fh/Fg

15

aspermic Fasciola fluke

China



16

aspermic Fasciola fluke

China



17

aspermic Fasciola fluke

Philippines



18

aspermic Fasciola fluke

Japan

19

aspermic Fasciola fluke

Japan

20

aspermic Fasciola fluke

21

aspermic Fasciola fluke

22 23 24

Accession no.

CR

Location

pepck

pold

LC061148

LC061172

US

Species

LC061174

Fh

LC061151

LC061175

Fh

LC061152

LC061176

Fh

LC061153

LC061177

Fg

LC061154

LC061178

Fg

Fg

LC061155

LC061179

Fg

Fg

LC061156

LC061180

Fg

Fg

LC061157

LC061181

Fg

Fg

LC061158/061159

LC061182

Fg

Fg

Fg

LC061160/061161

LC061183

Fh/Fg

Fh/Fg

aspermic Fg

LC061162/061163

LC061184/061185

MA N

LC061173

LC061150

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LC061149

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Sample no.

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Table 1 Samples of Fasciola species used in this study.

Fh/Fg

aspermic Fg

LC061164/061165

LC061186/061187

Fh/Fg

Fh/Fg

aspermic Fg

LC061166/061167

LC061188/061189

Fh

Fh/Fg

Fh/Fg

aspermic Fg

LC061168/061169

LC061190/061191

Fg

Fh/Fg

Fh/Fg

aspermic Fg

LC061170/061171

LC061192/061193



Fh

Fh/Fg

Fh/Fg

aspermic Fh

NA

NA



Fh

Fh/Fg

Fh/Fg

aspermic Fh

NA

NA

Japan



Fh

Fh/Fg

Fh/Fg

aspermic Fh

NA

NA

Korea



Fh

Fh/Fg

Fh/Fg

aspermic Fh

NA

NA

aspermic Fasciola fluke

East India



Fg

Fh/Fg

Fh/Fg

aspermic Fg

NA

NA

aspermic Fasciola fluke

Thailand



Fg

Fh/Fg

Fh/Fg

aspermic Fg

NA

NA

aspermic Fasciola fluke

Vietnam



Fg

Fh/Fg

Fh/Fg

aspermic Fg

NA

NA

AC

Fh/Fg

Fh/Fg

17

ACCEPTED MANUSCRIPT

aspermic Fasciola fluke

Bangladesh



Fh/Fg

Fh/Fg

Fh/Fg

aspermic Fg

NA

NA

26

aspermic Fasciola fluke

Myanmar



Fh/Fg

Fh/Fg

Fh/Fg

aspermic Fg

NA

NA

27

aspermic Fasciola fluke

Nepal



Fh/Fg

Fh/Fg

Fh/Fg

aspermic Fg

NA

NA

IP

T

25

AC

CE P

TE D

MA N

US

CR

 and  indicate spermic and aspermic, respectively. Fh and Fg represent F. hepatica and F. gigantica type, respectively, and Fh/Fg type represents a mixed band pattern of F. hepatica and F. gigantica type. NA, not analyzed. These flukes were subjected to examination by the method developed in this study.

18

AC

CE P

TE

D

MA

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

19

ACCEPTED MANUSCRIPT

Highlights Precise and robust methods to discriminate Fasciola spp. have been required.



PCR–RFLP and multiplex PCR were successfully developed by using single copy genes.



Fasciola hepatica, F. gigantica, and aspermic Fasciola flukes were distinguished.



Aspermic flukes had a mixed fragment pattern of F. hepatica and F. gigantica.



Aspermic flukes were revealed as descendants of interspecific hybridization.

AC

CE P

TE

D

MA

NU

SC R

IP

T



20