Molecular Analysis of Aspermic <i>Fasciola</i> Flukes from Korea on the Basis of the Nuclear ITS1 Region and Mitochondrial DNA Markers and Comparison with Japanese Aspermic <i>Fasciola</i> Flukes

Abstract

ABSTRACT. It has been speculated that populations of aspermic Fasciola flukes in Korea and Japan have a close phylogenetic relationship. To evaluate this, we analyzed 33 Korean aspermic Fasciola flukes on the basis of nuclear ribosomal internal transcribed spacer 1 (ITS1) and mitochondrial NADH dehydrogenase subunit 1 (nad1) and cytochrome c oxidase 1 (cox1) sequences. Fh, Fg, and Fh/Fg types were detected in the ITS1 region and displayed the fragment patterns of F. hepatica, F. gigantica, and both species, respectively by a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Additionally, three concatenated haplotypes of nad1 and cox1(nad1/cox1) were detected, and 2 of these, Kor1/Kor1 (Fsp1/Fsp1) haplotype and Kor2a/Kor2 (Fsp2/Fsp2) haplotype, were shared by Korean and Japanese aspermic flukes. The Fst value (0.019), calculated using the concatenated sequences, indicated that Korean and Japanese aspermic Fasciola populations were genetically undifferentiated. Interestingly, a combination of the Fh/Fg type and Kor1/Kor1 haplotype was found at the highest frequency in Korean aspermic flukes, whereas the Fg type and Fsp2/ Fsp2 haplotype combination was found at a conspicuously high frequency in Japanese aspermic flukes. This indicates that a founder effect caused by the introduction of infected hosts may have played a key role in the introduction of aspermic Fasciola flukes from Korea into Japan. KEY WORDS: aspermic Fasciola fluke, cox1, ITS1, nad1.

Hybrid origin of Asian aspermic <i>Fasciola</i> flukes is confirmed by analyzing two single-copy genes, <i>pepck</i> and <i>pold</i>

“… ). The spermatogenic status, ITS1 types, and mitochondrial nad1 haplotypes of the flukes were reported in previous studies [ 1 , 3 , 5 , 6 , 7 , 9 , 10 , 11 , 14 , 15 ] ( Table 1…”

“…The nucleotide sequence of the ribosomal internal transcribed spacer 1 (ITS1) has been employed so far for molecular characterization of Fasciola flukes [ 1 , 3 , 5 , 6 , 7 , 9 , 10 , 11 , 14 , 15 ]. Three ITS1 types (ITS1-Fh, Fg and mixed type) were distinguished using the polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method [ 4 ].…”

“…However, species identification cannot be performed using ITS1 when the spermatogenic status of a fluke is unclear because both the ITS1-Fh and ITS1-Fg types are found also in aspermic flukes [ 4 ]. The ITS1-mixed type has both the fragment patterns of the two species that were detected in aspermic Fasciola flukes [ 5 , 6 , 7 , 9 , 10 , 11 , 14 , 15 ], suggesting that the aspermic flukes are hybrids between F. hepatica and F. gigantica [ 4 ]. However, the ITS1 has hundreds of copies organized as tandem repeats with highly recombinogenic and unstable characteristics [ 13 ], and it is therefore, unsuitable to provide reliable evidence of natural hybridization.…”

“…This indicates the maternal ancestors of aspermic flukes were F. hepatica or F. gigantica with the major haplotypes [ 8 ]. So far, the spermatogenic status, ITS1 type and nad1 haplotypes have been reported for 730 Fasciola flukes from eight Asian countries [ 1 , 3 , 5 , 6 , 7 , 9 , 14 , 15 ]. However, contradictions concerning species discrimination were observed for 11 flukes from Eastern India, Myanmar and Bangladesh [ 3 , 7 , 14 ], because inconsistent results of the nad1 lineages and spermatogenic status were of the flukes could not be resolved by analyzing ITS1.…”

“…However, the number of samples was insufficient to draw a conclusion regarding the hybrid origin in the aspermic flukes. In this study, 730 Fasciola flukes from eight Asian countries [ 1 , 3 , 5 , 6 , 7 , 9 , 10 , 11 , 14 , 15 ] were reanalyzed using pepck and pold markers to obtain a definitive evidence for the origin of aspermic Fasciola flukes as well as to confirm the reliability of the markers.…”