Introduction
Two Fasciola species, F. hepatica and F. gigantica, have two reproductive stages in their life cycle (Dalton, Reference Dalton1999). The first involves asexual larval multiplication by paedogenesis in the intermediate host snails, and the other is via sexual fertilization for egg production in adults within their definitive animal hosts. However, aspermic (parthenogenetic) Fasciola forms that are found widely in eastern and southern Asia have been considered to produce their eggs by parthenogenesis. This means that no fertilization occurs within their definitive host as the forms are diploid and triploid which have abnormal spermatogenesis and oogenesis (Sakaguchi, Reference Sakaguchi1980; Terasaki et al., Reference Terasaki, Akahane, Habe and Moriyama1982, Reference Terasaki, Moriyama-Gonda and Noda1998). The oocyte chromosomes of triploid aspermic Fasciola flukes separate unequally into two daughter nuclei in the first stage of meiosis, and then re-fuse prior to the second meiosis should there be no male pronucleus (Hanna et al., Reference Hanna, Moffett, Forster, Trudgett, Brennan and Fairweather2016). 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., Reference Hayashi, Ichikawa-Seki, Mohanta, Shoriki, Chaichanasak and Itagaki2018). 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., Reference Itagaki, Ichinomiya, Fukuda, Fusyuku and Carmona2011). Hermaphroditic trematode species can produce eggs by means of self-fertilization (Trouvé et al., Reference Trouvé, Renaud, Durand and Jourdane1996), and the confirmation of whether produced eggs resulted from parthenogenesis or self-fertilization has not been identified in the aspermic Fasciola forms. Recently, microsatellite markers for F. hepatica, which can trace the mating patterns and analyse the parentage (parent–offspring relationship), have been developed (Cwiklinski et al., Reference Cwiklinski, Allen, LaCourse, Williams, Paterson and Hodgkinson2015), and these markers were available for parthenogenetic (aspermic) Fasciola flukes occurring in Japan (Ohari et al., Reference Ohari, Matsuo, Yoshida, Nonaka, Sato and Itagaki2021). This study aimed to identify whether two laboratory strains of aspermic Fasciola forms reproduce parthenogenetically and clonally utilizing microsatellite markers.
Material and methods
Two laboratory strains of aspermic Fasciola forms were used in this study: the strains IWTc2-1 and NRA-4 originated from naturally infected cattle in Iwate prefecture, and sika deer, Cervus nippon, in Nara prefecture, Japan, respectively. These strains were triploid, as confirmed by counting the chromosome number of their oocytes and spermatocytes, and confirmed aspermic due to no sperm in their seminal vesicles. The molecular markers of the two strains were Fsp1 haplotype (F. hepatica-type; GenBank accession no. AB207169) in the mitochondrial NADH dehydrogenase subunit 1 region; the F. hepatica and F. gigantica-mixed type in the nuclear ribosomal internal transcribed spacer 1, the phosphoenolpyruvate carboxykinase and DNA polymerase delta regions (Ichikawa & Itagaki, Reference Ichikawa and Itagaki2010; Shoriki et al., Reference Shoriki, Ichikawa-Seki and Suganuma2016); and differed in the multilocus genetic types (MLGs) of the eight microsatellite regions: Fh_1, Fh_2, Fh_5, Fh_6, Fh_10, Fh_11, Fh_14 and Fh_15 (Cwiklinski et al., Reference Cwiklinski, Allen, LaCourse, Williams, Paterson and Hodgkinson2015). Experimental coinfection with IWTc2-1 and NRA-4 strains was carried out using a male Wistar rat (8 weeks old) that were infected orally with ten metacercariae of each of the two strains, and four adult flukes (Adults 1–4) were subsequently recovered from the bile duct 3 months after infection. Experimental infection of the rat (no. A201623) was conducted according to the Guidelines for Animal Experiments of Iwate University, Japan. Of the four adult flukes, two were confirmed IWTc2-1 and the other two were NRA-4 based on the microsatellite analyses. This provided evidence that adult worms of both strains coexisted in the bile duct during the 2 months, and therefore may have mated randomly to each other. Snails (Austropeplea ollula) were infected with single miracidia hatched from eggs of individual adults and subsequently maintained at 27°C for a month after exposure. The snails were independently dissected, and 15, 17, 11 and six snails were infected with the larvae from Adults 1, 2, 3 and 4, respectively. The larvae (rediae and cercariae) were collected from individual infected snails and stored in −20°C for microsatellite markers analysis. Total DNA was extracted using the High Pure PCR Template Preparation Kit (Roche, Mannheim, Germany) from the posterior tissues of four adult flukes and the larvae from individual infected snails. The 12 microsatellite DNA regions (Fh_1, Fh_2, Fh_3, Fh_4, Fh_5, Fh_6, Fh_10, Fh_11, Fh_12, Fh_13, Fh_14, Fh_15) (Cwiklinski et al., Reference Cwiklinski, Allen, LaCourse, Williams, Paterson and Hodgkinson2015) were amplified using a post-labelling polymerase chain reaction (PCR) method for multiplexed and multicoloured genotyping, using bar-coded slit tag (BStag) (Shimizu & Yano, Reference Shimizu and Yano2011). The forward primers were appended with one of the four BStags, F9TAC, F9GAC, F9GTC and F9GCC, in the 5′ end and fluorescent dye-labelled BStag primers, PET-F9TAC, 6-FAM-F9GAC, VIC-F9GTC and NED-F9GCC (Applied Biosystems, Foster City, CA, USA) were used (Ohari et al., Reference Ohari, Matsuo, Yoshida, Nonaka, Sato and Itagaki2021). PCR amplification was performed using the KAPA2G Fast Multiplex PCR Kit (KAPA Biosystems, Boston, MA) in a final volume of 5 μl, containing 2.0 ng of extracted DNA, 3 μl of 2× KAPA2G Fast Multiplex Mix, 0.2 pm of each BStag-tailed forward primer, 0.4 pm of each reverse primer, and dye-labelled BStag primer. The PCR cycle was 94°C for 3 min, followed by 30 cycles of denaturation at 94°C for 15 s, annealing at 60°C for 30 s, extension at 72°C for 60 s, and the subsequent step was for labelling the amplicons and was carried out by three cycles of 94°C for 20 s, 49°C for 10 s and 72°C for 5 s, followed by a final extension at 72°C for 10 min. Subsequently, 2 μl of the PCR product was added to 10.0 μl of Hi-Di Formamide and 0.5 μl of GeneScan LIZ600 size standards (Applied Biosystems), and the mixture was subjected to capillary electrophoresis using an ABI 3500 Genetic Analyzer (Applied Biosystems). The allele size of each locus was confirmed using GeneMapper version 5.0 (Applied Biosystems). The capillary electrophoreses on each microsatellite region for the adults and larvae DNAs were performed at the same time. The MLG of each adult and larvae fluke was evaluated based on the combined results of the allele sizes deduced from the 12 loci. The MLGs were compared between adult flukes and their larvae to confirm their clonality. These analyses were conducted using statistical software R (R Development Core Team, 2020) using the poppr version 2.8.1 package (Kamvar et al., Reference Kamvar, Brooks and Grünwald2015).
Results and discussion
The MLGs of Adult 1, Adult 2 and their larvae DNAs (15 and 17, respectively) were identical to those of IWTc2-1, and Adult 3, Adult 4 and their larvae DNA (11 and 6, respectively) were identical to those of NRA-4 (table 1). In the Fh_6 locus, Adults 1 and 2 and their larvae exhibited three alleles 206/209/250 (triploid pattern), while Adults 3 and 4 and their larvae displayed 206/209/215 (triploid). On the assumption that triploid adults generated a diploid and a haploid gamete during meiosis, individual adults could generate six gametes (e.g. 206, 209, 250, 206/209, 206/250, 209/250 in IWTc2-1 strain) with a frequency of 1:6, and seven apparent genotypes could be expected in the larvae which were produced by means of selfing and outcrossing in adults of each strain (table 2). As the four adults can generate three gametes (206, 209 and 206/209) with a frequency of 1:6 and six gametes (215, 250, 206/215, 206/250, 209/215 and 206/250) with a frequency of 1:12, and 15 apparent genotypes can be expected in their random mating and selfing (table 2). However, only two genotypes, 206/209/250 and 206/209/215 were detected in the locus by a frequency of 32:17, and no other genotypes were observed (tables 1 and 2).
Table 1. Multilocus genetic types in 12 microsatellite loci of adults and their larvae of the two aspermic Fasciola triploid strains.
-: allele undetected.
Table 2. Expected and observed frequency of apparent genotype in Fh_6 locus of the larvae produced by adults of aspermic Fasciola triploid strains.
a The numbers within parenthesis indicate the number of the larvae.
b Identical to NRA-4 strain.
c Identical to IWTc2-1 strain.
In aspermic Fasciola forms, it is assumed that produce their clonal progeny by utilizing parthenogenesis, meaning no fertilization occurs (Sakaguchi, Reference Sakaguchi1980; Terasaki et al., Reference Terasaki, Akahane, Habe and Moriyama1982). Microsatellite DNA markers are useful for elucidating the parentage and mating patterns of sexually reproductive diploid organisms (Flanagan & Jones, Reference Flanagan and Jones2019). The findings in this study demonstrate that the MLGs in the 12 microsatellite loci of adult flukes and their larvae (progeny) were identical, suggesting that the aspermic Fasciola triploid strains reproduce their progeny clonally. Furthermore, the obvious deviation between theoretical and actual frequency observed in the genotype of Fh_6 locus would deny self-fertilization within individual flukes. These findings strongly suggest that the aspermic Fasciola triploid strains reproduce their progeny by means of parthenogenesis, possibly gynogenesis. It is considered that aspermic Fasciola forms occurred due to hybridization between F. hepatica and F. gigantica and have survived in evolutionary terms, possibly during hundreds or thousands of years without extinction (Itagaki et al., Reference Itagaki, Kikawa, Sakaguchi, Shimo, Terasaki, Shibahara and Fukuda2005). The parthenogenetic triploid strains used in this study have been maintained during many successive generations in our laboratory with high developmental and hatching rates of eggs, and high production of metacercariae. Triploid forms of parasitic helminths, which would have the parthenogenetic ability, are also reported in Paragonimus westermani fluke (Van Herwerden et al., Reference Van Herwerden, Blair and Agatsuma1999) and Spirometra erinaceieuropaei cestode (Sasada, Reference Sasada1978). It is pertinent to conduct further studies on understanding the survival mechanisms of parasitic triploid forms as it will be of importance for controlling the diseases.
Acknowledgements
We are grateful to the members of the Laboratory of Veterinary Parasitology, Faculty of Agriculture, Iwate University and the Department of Veterinary Parasitology, Gifu University.
Financial support
This research received no specific grant from any funding agency, commercial or not-for-profit sectors.
Conflict of interest
The authors declare none.
Ethical standards
The authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national and institutional committees on human experimentation and with the Helsinki Declaration of 1975, as revised in 2008. The authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national and institutional guides on the care and use of laboratory animals.
