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

Abstract

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”.

Distribution Status of Hybrid Types in Large Liver Flukes, Fasciola Species (Digenea: Fasciolidae), from Ruminants and Humans in Vietnam

“…There have been numerous studies reporting the presence of, namely, intermediate forms of hybridization in the population of Fasciola spp. causing fascioliasis in ruminants and humans in 12 Asian and African countries [ 3 – 5 , 7 – 11 , 14 , 16 , 18 , 20 , 24 ]. The intermediate forms, genetically, are of the interspecific hybrid individuals between F. hepatica and F. gigantica which might result in the occurrence of aspermic parthenogenesis (asexual reproduction); and morphologically, they looked identical to F. hepatica flukes in the population [ 4 , 24 ].…”

“…The intermediate form of both hybrid genotypes, up-to-date, has been reported present in various Asian and African countries, including Vietnam, China, Korea, Japan, Thailand, Iran, Bangladesh, Pakistan, Nepal, India, Myanmar, and Egypt. Morphologically, many of them look like F. hepatica , but genetically, their genome is mixed between F. hepatica and F. gigantica [ 3 , 6 , 7 , 9 – 18 ]. In the particular, if morphologically examined, F. gigantica is a long ‘leaf shape’ fluke with the physiometrical ratio between the body length (BL) and body width (BW) is about 4–5/1; and F. hepatica is a short ‘leaf shape’ with prominent shoulder, of about 2–3/1 BL/BW ratio; and no standard metrics applied for the ‘hybrid’ species [ 7 , 19 , 20 ].…”

“…Hence, up-to-date, there have been various molecular approaches/assays developed for identification and discrimination of these species/genotypes including PCR-RFLP [ 10 , 21 ]; PCR-SSCP [ 3 ]; loop-mediated isothermal amplification (LAMP) [ 22 , 23 ]; and PCR-coupled sequence analysis [ 11 , 14 , 24 – 29 ]. Recently, the effective approaches are employed for detecting the hybrid Fasciola flukes using nuclear protein coding genes, (ie., pepck and pold ) [ 17 , 18 , 25 ] and intron regions of the taurocyamine kinase gene [ 8 ]. The mostly used discriminative markers have been relying on polymorphic nucleotide sites in nuclear ribosomal ITS1 and ITS2, and in mitochondrial genes, nad1 and cox1 or else, and commonly applied in many studies to date [ 3 , 5 , 7 , 10 , 11 , 13 , 14 , 16 , 20 , 21 , 24 ].…”

Fasciola Species Introgression: Just a Fluke or Something More?

“…is due to a tendency to investigate single loci when a multi-loci or whole-genome approach is required (Box 3) [33,[41][42][43][44][45]. Most studies have relied on repetitive rDNA units to demonstrate hybridization, and only recently has a nuclear-encoded single-copy gene, PEPCK, been investigated [46,47]. Furthermore, the use of mtDNA alone, as evidence for hybridization in Fasciola spp., may be misleading due to a total lack of information concerning mitochondrial inheritance in these parasites and the limited recombination of mitochondrial genomes [41,[48][49][50].…”

Do aspermic (parthenogenetic) Fasciola forms have the ability to reproduce their progeny via parthenogenesis?

“…These findings indicate that aspermic Fasciola forms produce eggs with mitosis-like incomplete meiosis, and proliferate clonally with no introgression in the following generations. Aspermic Fasciola forms have been shown to have successive generations of progeny, resulting from interspecific hybridization between F. hepatica and F. gigantica , as these forms possess the genomes of the two species (Hayashi et al , 2018). However, hybridization experiments between the two Fasciola species have failed to produce the aspermic Fasciola forms, and have therefore been unable to confirm that the forms reproduce parthenogenetically and proliferate clonally (Itagaki et al , 2011).…”