Intron Regions as Genetic Markers for Population Genetic Investigations of Opisthorchis viverrini sensu lato and Clonorchis sinensis

SIMPLE SUMMARY: The zoonotic liver flukes Opisthorchis viverrini and Clonorchis sinensis infect small mammals, such as cats, dogs, pigs, rodents, and rabbits, as well as humans. Human infection subsequently develops into bile duct malignancy, also referred to as cholangiocarcinoma (CCA). Understanding the molecular systematics and population genetics of these liver flukes has an important role in prevention and control, and is important in comprehending their roles in zoonotic transmission. Different molecular markers have varying evolution rates and contain different genetic information. Polymorphic genetic markers are necessary and more suitable for such investigations. Therefore, we screened seven intron regions of the taurocyamine kinase gene (TK) to determine their potential as genetic markers for population genetic investigations of the liver flukes O. viverrini and C. sinensis which were collected from a range of geographical isolates and animal hosts. We identified a suitable intron region of TK, i.e., intron 5 of domain 1 (TkD1Int5) as having the most potential as a polymorphic marker. Results showed that TkD1Int5 is effective in examining the genetic variation and heterozygosity of O. viverrini and C. sinensis, but further studies are required to better understand the role of different species of animals as reservoir hosts of these zoonotic liver flukes. ABSTRACT: Opisthorchiasis and clonorchiasis are prevalent in Southeast and Far-East Asia, which are caused by the group 1 carcinogenic liver flukes Opisthorchis viverrini sensu lato and Clonorchis sinensis infection. There have been comprehensive investigations of systematics and genetic variation of these liver flukes. Previous studies have shown that O. viverrini is a species complex, called “O. viverrini sensu lato”. More comprehensive investigations of molecular systematics and population genetics of each of the species that make up the species complex are required. Thus, other polymorphic genetic markers need to be developed. Therefore, this study aimed to characterize the intron regions of taurocyamine kinase gene (TK) to examine the genetic variation and population genetics of O. viverrini and C. sinensis collected from different geographical isolates and from a range of animal hosts. We screened seven intron regions embedded in TK. Of these, we selected an intron 5 of domain 1 (TkD1Int5) region to investigate the genetic variation and population genetics of theses liver flukes. The high nucleotide and haplotype diversity of TkD1Int5 was detected in O. viverrine. Heterozygosity with several insertion/deletion (indel) regions were detected in TkD1Int5 of the O. viverrine samples, whereas only an indel nucleotide was detected in one C. sinensis sample. Several O. viverrine samples contained three different haplotypes within a particular heterozygous sample. There were no genetic differences between C. sinensis isolated from various animal host. Heterozygous patterns specifically detected in humans was observed in C. sinensis. Thus, TkD1Int5 is a high polymorphic genetic marker, which could be an alternative marker for further population genetic investigations of these carcinogenic liver flukes and other related species from a wide geographical distribution and variety of animal hosts.