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

Nuclear gene markers, phosphoenolpyruvate carboxykinase (pepck) and DNA polymerase delta (pold), have been developed for precise discrimination of Fasciola flukes instead of internal transcribed spacer 1. In this study, these two genes of 730 Fasciola flukes from eight Asian countries were analyzed. The results were compared with their mitochondrial NADH dehydrogenase subunit 1 (nad1) lineages for obtaining a definitive evidence of the hybrid origin of aspermic Fasciola flukes. All the flukes categorized into the aspermic nad1 lineages possessed both the fragment patterns of F. hepatica and F. gigantica (mixed types) in pepck and/or pold. These findings provide clear evidence for the hybrid origin of aspermic Fasciola lineages and suggest that "aspermic Fasciola flukes" should hereafter be called "hybrid Fasciola flukes".

Fasciolosis is a parasitic disease responsible for liver disorders in ruminant hosts and leads to a reduction in livestock productivity. The two well-known causative agents of fasciolosis are Fasciola hepatica and Fasciola gigantica. The former species is distributed mainly in Europe, America and Oceania, while the latter is distributed mainly in Asia and Africa [19]. The two species reproduce bisexually thorough fertilization. Mature spermatozoa of the species are ejaculated from their seminal vesicles, which serve as temporary storage for self-produced sperm [18]. In addition to the two species, aspermic Fasciola flukes, which contain only few or no spermatozoa in their seminal vesicles, have been reported in Asian countries [3, 18]. Not only diploid but also triploid flukes were reported from aspermic Fasciola flukes [11].

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]. The Fh and Fg types have identical fragment patterns to F. hepatica and F. gigantica, respectively. Therefore, the spermic species have been identified based on their ITS1 types [1, 3, 4, 6, 7, 10, 14, 15]. 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.

Phylogenetic studies based on the nucleotide sequence of the mitochondrial NADH dehydrogenase subunit 1 (nad1) have revealed that the nad1 haplotypes of F. hepatica and F. gigantica are well diversified [8]. In contrast, the nad1 haplotypes of aspermic Fasciola flukes displayed uniform characteristics, which originated from the two different maternal lineages, F. hepatica and F. gigantica [11]. The two maternal lineages, which are described as “aspermic Fh” and “aspermic Fg” in this study, contain respective major haplotypes together with some derivative haplotypes. 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.

Recently, the single-copy nuclear gene markers, pepck and pold, which encode phosphoenolpyruvate carboxykinase and DNA polymerase delta, respectively were developed [16]. The fragment patterns of F. hepatica, F. gigantica and the mixed fragment of both the species were distinguished by both pepck and pold based on multiplex PCR and PCR-RFLP methods, respectively [16]. In this study, the fragment patterns of F. hepatica in pepck and pold were described as “pepck-Fh type” and “pold-Fh type”, respectively. Similarly, the fragment patterns of F. gigantica and those of both the species were described as “pepck-Fg type”, “pold-Fg type”, “pepck-mixed type” and “pold-mixed type”. Single-copy genetic markers are suitable for detecting evidence of hybridization. Interestingly, all the aspermic Fasciola flukes analyzed previously by pepck and pold were determined as hybrids even though they possessed the ITS1-Fh or ITS1-Fg type [16]. 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.

A total of 730 Fasciola flukes from eight Asian countries (Japan, Korea, Vietnam, Thailand, Myanmar, India, Bangladesh and Nepal) were examined in this study (Fig. 1). 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 and S1). Ploidy of the 42 flukes from Japan and Vietnam were also reported in the previous studies (Table S1) [10, 11]. Unfortunately, ploidy of the remaining flukes could not be analyzed in the previous studies because the flukes were not fixed in ethanol-acetic acid, essential for the ploidy analysis.

Fig. 1.

Species discrimination of Fasciola flukes from eight Asian countries based on the analysis of pepck and pold genes. White and black circles denote F. gigantica and hybrid Fasciola flukes, respectively. The circle sizes are proportional to the number of flukes, and the actual numbers are labeled in the circles.

Table 1.

Profiles of Fasciola flukes from eight Asian countries

Country	Speciesa)	Number of flukes	Sperm in seminal vesiclesa)	Nuclear DNA types			Mitochondrial nad1a) lineages
				pepck	pold	ITS1a)
Japan	Aspermic Fasciola flukes	5	-	mixed	mixed	Fh	aspermic Fh
		1	-	mixed	mixed	Fh	aspermic Fg
		4	-	mixed	N.D.	Fh	aspermic Fh
		1	-	N.D.	mixed	Fh	aspermic Fh
		33	-	mixed	mixed	Fg	aspermic Fg
		2	-	mixed	N.D.	Fg	aspermic Fg
		2	-	mixed	mixed	mixed	aspermic Fh
		2	-	mixed	mixed	mixed	aspermic Fg
Subtotal		50b)
Korea	Aspermic Fasciola flukes	1	-	mixed	mixed	Fh	aspermic Fh
		4	-	mixed	mixed	Fh	aspermic Fg
		2	-	mixed	mixed	Fg	aspermic Fg
		10	-	mixed	mixed	mixed	aspermic Fh
		1	-	N.D.	mixed	mixed	aspermic Fh
		15	-	mixed	mixed	mixed	aspermic Fg
Subtotal		33
Vietnam	Aspermic Fasciola flukes	2	-	mixed	mixed	Fh	aspermic Fg
		1	-	mixed	mixed	Fg	aspermic Fg
		1	-	mixed	N.D.	Fg	aspermic Fg
		12	-	mixed	mixed	mixed	aspermic Fg
		5	-	mixed	N.D.	mixed	aspermic Fg
	F. gigantica	15	+	Fg	Fg	Fg	F. gigantica
		3	+	Fg	N.D.	Fg	F. gigantica
Subtotal		39b)
Thailand	Aspermic Fasciola flukes	17	-	mixed	mixed	Fg	aspermic Fg
		2	-	mixed	N.D.	Fg	aspermic Fg
	F. gigantica	122	+	Fg	Fg	Fg	F. gigantica
		4	+	Fg	N.D.	Fg	F. gigantica
Subtotal		145b)
Myanmar	Aspermic Fasciola flukes	7	-	mixed	mixed	mixed	aspermic Fg
	F. gigantica	79	+	Fg	Fg	Fg	F. gigantica
		1c)	-	Fg	Fg	Fg	F. gigantica
Subtotal		87b)
Eastern India	Aspermic Fasciola flukes	29	-	mixed	mixed	Fg	aspermic Fg
		4	-	mixed	N.D.	Fg	aspermic Fg
	F. gigantica	115	+	Fg	Fg	Fg	F. gigantica
		6c)	-	Fg	Fg	Fg	F. gigantica
		3	+	Fg	N.D.	Fg	F. gigantica
Subtotal		157
Bangladesh	Aspermic Fasciola flukes	86	-	mixed	mixed	Fg	aspermic Fg
		29	-	mixed	mixed	mixed	aspermic Fg
		1c)	+	mixed	mixed	Fg	aspermic Fg
		1c)	+	mixed	mixed	mixed	aspermic Fg
	F. gigantica	19	+	Fg	Fg	Fg	F. gigantica
		2c)	-	Fg	Fg	Fg	F. gigantica
Subtotal		138b)
Nepal	Aspermic Fasciola flukes	61	-	mixed	mixed	mixed	aspermic Fg
	F. gigantica	20	+	Fg	Fg	Fg	F. gigantica
Subtotal		81
Total		730

“Fh” and “Fg” represent F. hepatica and F. gigantica band patterns, respectively. “mixed” represents a both band pattern for Fh and Fg types. “aspermic Fh” and “aspermic Fg” represent nad1 haplotypes of aspermic Fasciola flukes whose maternal ancestry is F. hepatica and F. gigantica, respectively. N.D., not detected. a) Spermatic status, ITS1 types and nad1 haplotypes were analyzed in previous studies; Japan [7, 9], Korea [5], Vietnam [11], Thailand [1], Myanmar [6], Eastern India [3], Bangladesh [14] and Nepal [15]. b) The numbers for some of the subtotals do not completely match those from previous reports because some DNA samples were exhausted. c) Flukes possessing inconsistent characters for spermatogenesis status, ITS1 genotypes and nad1 haplotypes.

The pepck region was amplified from genomic DNA by using a multiplex PCR with Fh-pepck-F (5′-GATTGCACCGTTAGGTTAGC-3′), Fg-pepck-F (5′-AAAGTTTCTATCCCGAACGAAG-3′) and Fcmn-pepck-R (5′-CGAAAATTATGGCATCAATGGG-3′) primers, and the fragment patterns were distinguished on 1% agarose gel [16]. The pold region was analyzed by PCR-RFLP [16]. Briefly, the pold products amplified by Fasciola-pold-F1 (5′-GCTAACTTATCTGCTTACACGTGGACA-3′) and Fasciola-pold-R1 (5′-ATCGCATTCGATCAAAGCCCTCCCATG-3′) primers were digested with the AluI restriction enzyme (Roche, Mannheim, Germany), and then the fragment patterns were distinguished on 1.8% agarose gel.

In this study, the pepck genotyping yielded two DNA types, namely pepck-Fg type (F. gigantica) and pepck-mixed type (hybrid between F. hepatica and F. gigantica). Similarly, pold genotyping yielded pold-Fg type, and pold-mixed type. The newly obtained results of the pepck and pold genotyping were combined with the previous results of the spermatogenic status, ITS1 types and mitochondrial nad1 haplotypes (Table 1) [1, 3, 5,6,7, 9,10,11, 14, 15]. The nad1 haplotypes are divided into three haplogroups, “F. gigantica”, “aspermic Fh” and “aspermic Fg” [8]. The first haplogroup consists of haplotypes detected from populations of F. gigantica, whereas the second and third haplogroups include haplotypes of aspermic Fasciola flukes whose maternal ancestries are F. hepatica and F. gigantica, respectively. Detailed information about each fluke is summarized in Supplementary Table (Table S1).

Although the ITS1 region for all the flukes was adequately amplified in the previous studies [1, 3, 5,6,7, 9,10,11, 14, 15], the pepck and pold regions for two and 28 of the flukes, respectively were not amplifiable in the present study (Table 1). This observation is probably related to differences in the copy numbers of the target genes since ITS1 is a multi-copy gene, it was more easily amplified than pepck and pold, single-copy genes in many eukaryotic species [2, 12]. Additionally, the small sizes of the pepck amplicons (241 bp and 509 bp or 510 bp) made them more readily generated than the pold amplicons (844 bp) [16].

The discrimination of Fasciola species based on spermatogenic status, ITS1 type and nad1 haplotype has produced contradictory results for 11 Fasciola flukes analyzed in previous studies. Indeed, nine aspermic flukes from Myanmar, eastern India and Bangladesh appeared to have no spermatogenic ability and were actually thought to be F. gigantica because their nad1 haplotypes were not included in “aspermic Fg” but were included in “F. gigantica” [3, 7, 14] (Table1). In the present study, these flukes showed the pepck-Fg type and the pold-Fg, and were confirmed as F. gigantica (Table 1). Additionally, one fluke from Bangladesh, appeared to retain its spermatogenic ability and possessed the ITS-Fg type, was regarded as an aspermic Fasciola fluke because it was included in the “aspermic Fg” haplogroup in the nad1 gene [14]. Here, this fluke displayed the pepck-mixed type and pold-mixed type (Table 1). Similarly, another fluke from Bangladesh, appeared to retain its spermatogenic ability and possessed the ITS1-mixed type and the nad1 haplotype of “aspermic Fg” [14], also showed the mixed types in both pepck and pold. In summary, all the Fasciola flukes belonging to the “aspermic Fh” or “aspermic Fg” haplogroups in nad1 showed the mixed types in both pepck and pold regardless of their ITS1 types (Table 1). These results strongly suggest that Asian aspermic Fasciola flukes originated through the hybridization between F. hepatica and F. gigantica, and should now be called “hybrid Fasciola flukes” instead of “aspermic Fasciola flukes” as having been proposed by Ichikawa-Seki et al. [8]. “Aspermic” seems no longer an adequate term because the two hybrid flukes from Bangladesh [14] (Table 1) retained sperm in their seminal vesicles.

Although the ploidy of almost all the flukes was unknown in this study, the 25 triploids as well as the 1 diploid aspermic flukes from Japan and Vietnam showed the mixed types in both pepck and pold (Table S1). Since triploid flukes can never occur in a single hybridization, the most possible origin of a triploid may be through the fertilization of a parthenogenetically produced egg (diploid) by the sperm of a male from the bisexual ancestor [17]. According to this theory, triploid would also be called as “hybrid” in a broad sense. Actually, triploid flukes were successfully produced by an experimental hybridization between hybrid diploid and F. hepatica (unpublished results).

In this study, all the Fasciola flukes belonging to “F. gigantica” haplogroup in nad1 displayed pepck-Fg type and pold-Fg type, and were therefore confirmed as F. gigantica. These findings revealed that the results of the pepck and pold analyses were completely consistent with those of the nad1 lineages. Therefore, pepck and pold were proved as potential markers for precise discrimination of F. hepatica, F. gigantica and hybrid Fasciola flukes. Accurate discrimination of Fasciola flukes is very important because hybrid Fasciola flukes are thought to have superior fecundity to F. gigantica [14]. Hybrid Fasciola flukes were predominant in Nepal (75.3%), Bangladesh (84.8%), South Korea (100%), Japan (100%) and Vietnam (53.8%) (Fig. 1), and are therefore needed a further attention to monitor their dispersal route in Asian countries.