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Infection, Genetics and Evolution 97 (2022) 105182
Available online 10 December 2021
1567-1348/© 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Current assessment of the systematics and population genetics of
Opisthorchis viverrini sensu lato (Trematoda: Opisthorchiidae) and its first
intermediate host Bithynia siamensis sensu lato (Gastropoda: Bithyniidae) in
Thailand and Southeast Asia
Weerachai Saijuntha a,*
, Ross H. Andrews b,c
, Paiboon Sithithaworn b
, Trevor N. Petney d
a
Walai Rukhavej Botanical Research Institute, Mahasarakham University, Maha Sarakham 44150, Thailand
b
Cholangiocarcinoma Research Institute, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand
c
Faculty of Medicine, St Mary's Campus, Imperial College London, United Kingdom
d
Departments of Zoology and Paleontology and Evolution, State Museum of Natural History Karlsruhe, Erbprinzenstrasse 13, 76133 Karlsruhe, Germany
A R T I C L E I N F O
Keywords:
Opisthorchiasis
Liver fluke
Cholangiocarcinoma
Genetic variation
Systematics
Cryptic species
Genetic structure
A B S T R A C T
The group 1 carcinogen, the liver fluke Opisthorchis viverrini is the causative agent of opisthorchiasis and sub­
sequent bile duct cancer (cholangiocarcinoma; CCA), which is an important public health problem in Southeast
Asia. Bithynia snails are known to be the sole intermediate host of O. viverrini, and distributed throughout
endemic areas of opisthorchiasis. Since 2001, the genetic variation investigation of O. viverrini has progressively
been investigated. Comprehensive genetic variation studies of O. viverrini and Bithynia snails were undertaken
and consecutively published in 2007 by Saijuntha and colleagues. These studies provided genetic evidence that
O. viverrini and Bithynia snails are both species complex with evidence of co-evolution. Later, several studies have
provided data in support of this finding, and have continuously to date reinforced that both O. viverrini and
Bithynia are species complexes. Moreover, studies have shown that genetic variation of O. viverrini is related to
geographical, temporal, fish host species including geographical genetic variation of its snail host, Bithynia
siamensis sensu lato. This is significant and important in our understanding of the evolution and phylogenetic
relationships between species within the O. viverrini and Bithynia species complexes. A comprehensive knowledge
of the systematics and population genetics of O. viverrini and Bithynia snails provides a sound basis to instigate
and develop effective prevention and control programs targeting opisthorchiasis and CCA in the endemic areas of
Southeast Asia. Thus, this review examines the historical series of investigations of the systematics and popu­
lation genetics of O. viverrini including Bithynia spp. in Southeast Asia since molecular genetic investigations
commenced some 20 years ago.
1. Introduction
Foodborne trematode (FBT) infections are a major public health
problem in many developing countries affecting the health of more than
50 million people globally (Fürst et al., 2012; World Health Organiza­
tion, 2011). The liver fluke, Opisthorchis viverrini, is one of the most
important species involved, which has been classified as a group 1
carcinogen (i.e. a carcinogenic agent) in humans causing bile duct
cancer, cholangiocarcinoma (CCA) (World Health Organization, 1995).
This liver fluke is commonly found in Southeast Asia, especially
Thailand and Lao PDR, where it is estimated that at least 10 million
people are infected (Andrews et al., 2008). Although accurate preva­
lence data is not currently available, it is also known to occur in
Cambodia and southern Vietnam (Sithithaworn et al., 2012a; Le et al.,
2012; Doanh and Nawa, 2016), with several new foci having been re­
ported in Myanmar (Aung et al., 2017; Sanpool et al., 2018).
The infections of O. viverrini are associated with the consumption of
raw or partially cooked cyprinid fish which are infected with metacer­
ariae (Grundy-Warr et al., 2012). In Thailand, although prevalence of
infection has declined since the first studies carried out in last century
(Sithithaworn et al., 2012a) due to intensive control programs (Jong­
suksuntigul and Imsomboon, 2003), in the past 20 years prevalence has
* Corresponding author.
E-mail address: weerachai.s@msu.ac.th (W. Saijuntha).
Contents lists available at ScienceDirect
Infection, Genetics and Evolution
journal homepage: www.elsevier.com/locate/meegid
https://doi.org/10.1016/j.meegid.2021.105182
Received 22 September 2021; Received in revised form 5 December 2021; Accepted 7 December 2021
Infection, Genetics and Evolution 97 (2022) 105182
2
remained at similar levels (Petney et al., 2018). Infection rates of
O. viverrini in their snail and fish intermediate hosts are still being re­
ported (Saijuntha et al., 2019). The habit of eating raw or partially
cooked fish in the northeast region of Thailand has not changed
(Grundy-Warr et al., 2012; Saijuntha et al., 2021a), and hence, the
incidence of CCA has remained approximately unchanged over the last
20 years (Sithithaworn et al., 2014). At present, about 20,000 people die
of Opisthorchis associated liver cancer (CCA) every year in the northeast
of Thailand alone (Khuntikeo et al., 2018).
The liver fluke O. viverrini has a complex life cycle, which requires
three hosts to complete their life cycle (Saijuntha et al., 2021b). The
freshwater snails in genus Bithynia and cyprinid fish act as the first and
second intermediate hosts, respectively. Humans are the definitive
hosts, whereas rodents, canine and feline are reservoir hosts (Saijuntha
et al., 2021b). These multiple hosts have been shown to be important
factors that drive the genetic variations of parasitic trematodes,
including opisthorchiids (Petney et al., 2018). Studies of the systematics
and population genetics of the liver fluke O. viverrini and their snail hosts
are important to understand the evolutionary process of their complex
life cycle (Prugnolle et al., 2005). Thus, genetic investigations of
O. viverrini alone may not be sufficient to provide answers as to the
evolutionary processes of this liver fluke. Despite this, genetic variation
investigations of its intermediate hosts could elucidate more under­
standing of the adaptation and evolutionary process between O. viverrini
and its hosts.
To our knowledge, studies of the systematics and population genetics
of O. viverrini was first undertaken in 1993 by isoenzyme analysis
(Sueblinwong et al., 1993), while mitochondrial DNA sequence varia­
tion was subsequently reported in 2001 (Ando et al., 2001). Since 2001 a
series of comprehensive genetic investigation studies on O. viverrini
population in Thailand and Lao PDR have been continuously undertaken
using a variety of molecular techniques and genetic markers, such as
multilocus enzyme electrophoresis technique (MEE), nuclear and
mitochondrial DNA sequencing, and fragment analyses of microsatellite
markers by Sithithaworn and colleagues (e.g. Saijuntha et al., 2007;
Sithithaworn et al., 2007; Saijuntha et al., 2009; Laoprom et al., 2012;
Kiatsopit et al., 2014; Buathong et al., 2017; Pitaksakulrat et al., 2017,
2018; Namsanor et al., 2020). While the genetics investigation and
identification of O. viverrini in Cambodia, Vietnam, and Myanmar were
consequently conducted (Thaenkham et al., 2010; Le et al., 2012; Aung
et al., 2017; Sanpool et al., 2018). Subsequently, a comprehensive re­
view was conducted in 2012 (Sithithaworn et al., 2012b), followed by
the most up to date review by Petney et al. (2018). In this review, the
historical series of studies on the current status of the systematics and
population genetics of O. viverrini with additional information of its snail
host, Bithynia spp. will be examined in detail.
2. The standard model and genetic variation investigations prior
to 2007
2.1. The standard model
The history of human infection with O. viverrini started with two
publications (Kerr, 1916; Leiper, 1911) dealing with the same data set
describing the infection in prisoners in a jail in Chiang Mai in the north
of Thailand (Petney et al., 2013). Thereafter, a series of reports began to
appear, widely scattered in the literature, extending the known range of
the infection to the northeast of Thailand (Prommas, 1927) and Laos
(Bedier and Chesneau, 1929). The first major, coordinated research
project on O. viverrini infection, starting in 1951, was carried out be­
tween the Thai Ministry of Public Health in cooperation with the United
States Special Technical and Economic Mission to Thailand. In 1955 the
project leader, Elvio Sadun, published the first comprehensive paper on
the epidemiology of O. viverrini infection in Thailand (Sadun, 1955).
This publication elucidated the “standard epidemiological model” of
infection based on one parasite species (Fig. 1), and at this stage un­
known snail first intermediate hosts, and fish second intermediate hosts.
Human infection via the ingestion of raw/fermented fish by humans was
proposed and included a list of those species commonly eaten in this
way. This comprised a number of non-cyprinid fish species which are
now to be known to play no role in the life cycle of O. viverrini as well as a
number of known hosts. No information at this time was available on the
snail first intermediate hosts.
Based on this model, an increasing number of publications appeared
on defining and dealing with the snail first intermediate hosts (Upatham
and Sukhapanth, 1980; Brockelman et al., 1986; Ditrich et al., 1990;
Fig. 1. Diagram depicting the progression of knowledge of the systematics and population genetics of Opisthorchis viverrini sensu lato. Prior to 2007, the studies of the
O. viverrini life cycle standard model was still in beginnings. Until 2007, there was no evidence of a complex species of O. viverrini (A can of worms). Following
comprehensive systematics and genetic investigations after 2007, it was discovered that the life cycle of the O. viverrini species complex is a multiple hosts and
parasites model.
W. Saijuntha et al.
Infection, Genetics and Evolution 97 (2022) 105182
3
Giboda et al., 1991), the fish second intermediate hosts (Vichasri et al.,
1982; Sithithaworn et al., 1997) and human involvement in the life cycle
of this parasite (Upatham et al., 1984; Sithithaworn et al., 1991;
Kobayashi et al., 2000; Jongsuksuntigul and Imsomboon, 2003). The
first difficulties with the standard model began to appear in the late
1990s, when differences in the prevalence and intensities of infection, as
well as the symptomatology became apparent between different prov­
inces in the northeast of Thailand.
At this time, advances in molecular genetic techniques and their
application to systematics studies population genetics studies were
being undertaken on many vertebrate and invertebrate groups,
including parasites. For instance, Andrews and Chilton (1999) have
provided a substantial review on the application of the technique of
allozyme electrophoresis for systematic and population genetic studies
of protozoan, arthropod, and helminth parasites. Surprisingly, a com­
mon finding from these studies was that many previously traditionally
classified parasite species were indeed species complexes of genetically
distinct but morphologically similar ‘cryptic’ species. The advances in
molecular genetic technologies and their application to parasite sys­
tematics provided the basis to examine whether O. viverrini was in fact
not a single species, but a number of closely related morphologically
similar species but genetically distinct cryptic species.
2.2. Genetic variation of O. viverrini and Bithynia snails
The information of genetic variation of O. viverrini in the period prior
2007 was very limited. To our knowledge, the first report of genetic
variation of O. viverrini was conducted by Sueblinvong et al. (1993). The
adult worms collected from autopsy and cholangiocarcinoma patients in
Srinakarind hospital, Khon Kaen Province, Thailand were examined by
the multilocus enzyme electrophoresis (MEE) technique. Sixteen band­
ing patterns at three enzyme markers, i.e. glucose-6-phosphate dehy­
drogenase (G6PD), glucose phosphate isomerase (GPI) and
phosphoglucomutase (PGM) were observed among 109 individual
worms. It was not until 2001, that genetic variation of O. viverrini was
examined again but this time it was based on DNA markers (Ando et al.,
2001). Mitochondrial cytochrome c oxidase subunit 1 (CO1) gene and
internal transcribed spacer 2 (ITS2) were used to examine genetic
variation of adult O. viverrini from different geographical localities,
including an autopsy in northeast Thailand. Subsequently, Awir­
uttapanich (2004) found ITS2 sequences variation between individual
metacercariae of O. viverrini extracted from Pla Kao Na (Cyclocheilichthys
armatus). Intra- and inter-specific variations of CO1 sequence were
found with five haplotypes detected based on four variable nucleotide
sites, while ITS2 sequences were identical among all samples (Ando
et al., 2001). Later, a pilot study was conducted by Nuchjungreed (2001)
who applied a random amplified polymorphic DNA (RAPD) technique to
examine genetic variation of O. viverrini, results of which were later
published (Sithithaworn et al., 2007).
The publication of genetic variation of Bithynia snails in this period is
nonexistent. The genetic variation of Bithynia snails was consecutively
examined by Smarn Tesana and colleagues at this period, with research
conducted as part fulfilment of Master degrees of Kodcharin (2005) and
Duangprompo (2007) under the supervision of Smarn Tesana. Briefly,
The RAPD technique was used to differentiate three Bithynia snails, and
also examined the genetic variation of B. s. goniomphalos populations
collected from 11 different localities in Chi and Mun River wetlands.
They were classified into two major distinct groups (I and II) related to
spatial distance and river wetland systems (Kodcharin, 2005). There­
after, the RAPD specific primers was designed and developed for specific
amplification of B. s. goniomphalos (Duangprompo, 2007) and subse­
quently for B. funiculata (Kulsantiwong et al., 2013a).
3. 2007: Evidence of species complexes and co-evolution of
O. viverrini and Bithynia snails
Evidence of genetic variation of O. viverrini was previously observed
by Ando et al., (2001) and Sithithaworn et al. (2007). Thereafter, Pai­
boon Sithithaworn applied for a Royal Golden Jubilee Ph.D. (RGJ-Ph.D.)
program scholarship which was supported by Thailand Research Fund
(TRF), and recruited Weerachai Saijuntha to conduct Ph.D. research on
“A comprehensive investigation of genetic variation among natural
populations of O. viverrini in Thailand and Lao PDR” (Saijuntha 2007) in
collaboration with Ross H Andrews from University of South Australia as
overseas advisor. Research aimed to initially identify diagnostic
markers, allozyme and mitochondrial DNA that could differentiate in­
dividual worms and populations of O. viverrini and B. s. goniomphalos
originating from different geographical areas. Results from these studies
were published consecutively by Saijuntha et al. (2008a, 2008b, 2009).
Subsequently, the markers that were defined and established were used
to comprehensively examine genetic variation and systematics of
O. viverrini and B. s. goniomphalos populations across geographical areas
in Thailand and Lao PDR.
Thereafter, the first report of a comprehensive analysis of O. viverrini
systematics and genetic variation examined by allozyme markers was
published (Saijuntha et al., 2006, 2007). The data obtained provided
evidence that O. viverrini was indeed a species complex that contained at
least two major evolutionary lineages, which were subdivided into six
genetically distinct groups which related to different wetland (catch­
ment) systems in Thailand and Lao PDR. The first lineage contained
three genetic groups of the O. viverrini populations from the Wang, Mun,
and Chi River wetlands, whereas the second lineage consisted of two
genetic groups belonging to the Songkram and Nam Ngum River wet­
lands from Thailand and Lao PDR, respectively (Saijuntha et al., 2007).
In addition, the investigation a subsequent study of the morphometrics
and biological variation of O. viverrini from different isolates also sup­
ported these findings by molecular analyses (Laoprom et al., 2009).
Importantly, studies utilizing some of those polymorphic enzyme
markers were also undertaken to examine the genetic variation of
Bithynia snails from the same geographical areas that O. viverrini had
been examined. Data showed that the Bithynia populations were sepa­
rated into four distinct genetic groups that were related to the same
wetland systems as defined genetic groups of O. viverrini which provided
evidence of co-evolution between O. viverrini and its snail first inter­
mediate host. The study by Saijuntha et al. (2007) is a corner stone
publication that provided the foundation for consecutive studies after
2007 examining in detail the systematics and population genetic vari­
ation of both O. viveerini and Bithynia snails (these studies are discussed
in the following section). For instance, research involved the systematics
and population genetic investigations of O. viverrini using a variety of
molecular markers, including the use of allozymes, microsatellite DNA,
nuclear and mitochondrial DNA sequences (Sithithaworn et al., 2012b;
Petney et al., 2018) which confirmed the evidence reported in 2007
(Saijuntha et al., 2007).
4. The comprehensive systematics and population genetic
analyses after 2007
4.1. Multiple hosts and parasites model
Many parasitic trematodes infect multiple host species, and thus
multi-host networks may offer a better framework for investigating
parasite dynamics (Pilosof et al., 2015). Saijuntha et al. (2007) have
shown that O. viverrini is a species complex comprising many genetically
distinct but morphologically cryptic species, contained in at least five
distinct genetic groups as detected by 32 independent enzyme loci
hence, genetic markers (Saijuntha et al., 2007). Furthermore, Saijuntha
et al. (2007) provided data that its first intermediate host Bithynia snails
also contained cryptic species, represented by at least three cryptic
W. Saijuntha et al.
Infection, Genetics and Evolution 97 (2022) 105182
4
genetic groups (Saijuntha et al., 2007). Since 2007, a number of fresh­
water cyprinid species of fish, currently up to 48 species have been
recognized as second intermediate hosts (Petney et al., 2018). However,
O. viverrini-like metacercariae have also been recovered from non-
cyprinid fish, such as Anabas testudineus of the family Anabantidae,
Channa striata of the family Channidae and Trichopodus microlepis of the
family Osphronemidae (Petney et al., 2018). This suggests that the
second intermediate hosts of O. viverrini may not only be the cyprinid
fishes as is currently documented. The definitive hosts of O. viverrini
have been proven to be cat, dog, rodents, and humans, while the other
fish-eating animals, especially wild mammals, may possibly be infected
by O. viverrini but this has not been rigorously investigated. A recent
study has found no evidence of O. viverrini infection in several fish-eating
animals, i.e. monkeys, rodents, small residential mammals, and birds
from the Lawa Reservoir and Chi River wetlands in Khon Kaen and Maha
Sarakham Provinces, Thailand (Tangkawattana et al., 2021). Interest­
ingly, an O. viverrini-like fluke found in the duck liver has been reported
which was genetically characterized as closely related to O. viverrini
(Dao et al., 2017). This finding reveals the possibility that undiscovered
O. viverrini sibling/related species still exists in the nature. Current
available comprehensive genetic information of O. viverrini and its hosts
suggests that opisthorchiasis could involve multiple hosts and parasites
system (Fig. 1).
4.2. O. viverrini sensu lato
The systematics and genetic variation of O. viverrini has continuously
been examined the initial evidence that of O. viverrini was a species
complex by Saijuntha et al. (2007) (Table 1). For instance, defined
allozyme markers have been used to characterize an isolate from Sav­
annakhet Province, Lao PDR (Kiatsopit et al., 2011). They found that the
Savannakhet isolate from Lao PDR clustered closely with an O. viverrini
population from Vientiane Province, Lao PDR as well as the populations
from the Songkram River, Thailand, which also support initial data
provided by Saijuntha et al. (2007). Other independent DNA markers,
mitochondrial CO1 and ND1 sequences have consecutively been used in
studies to examine the genetic variation of O. viverrini between 14
different geographical isolates in Thailand and Lao PDR (Saijuntha et al.,
2008a). Low nucleotide variation was observed at 0–0.3% among
O. viverrini isolates. These DNA markers could not be used to reveal the
genetic structure of O. viverrini related to geographical isolates. How­
ever, a single marker, a partial sequence of ND1 was used to explore the
genetic structure of six populations of O. viverrini from Thailand, Lao
PDR and Cambodia (Thaenkham et al., 2010). They found no significant
differences among populations tested by AMOVA, and suggested to
“reject” the previous reports that O. viverrini contained a species com­
plex. However, it is important to note that mitochondrial DNA appears
Table 1
Summary of genetic investigations and genetic group classification of Opisthorchis viverrini populations from Thailand, Lao PDR, Cambodia, and Vietnam. Different
color represents different major genetic distinct groups classified by a particular molecular marker.
Allozymes
a
RAPD
b
Mitochondrial DNA
c
Microsatellite DNA
d
Nuclear DNA
e
Thailand KBs Adult Kang Namton Reservoir Ban Sa-ard/Mueang Khon Kaen Chi River n/a n/a
KBp Adult/metacercariae Kang Lawa Reservoir Ban Phai Khon Kaen Chi River
KLp Adult/metacercariae Prakeu Stream Lerngpleuy/Mueang Khon Kaen Chi River
KPv Adult Ubonrattana dam Phuviang Khon Kaen Chi River
KNp Adult Nam Pong River Nam Pong Khon Kaen Chi River n/a n/a n/a n/a
CP Adult/cercariae Nong Ben Reservoir Chatturat Chaiyaphum Chi River
MS Adult Chi River Kantharawichai Maha Sarakham Chi River n/a
KS Adult/cercariae Lampao Dam Mueang Kalasin Chi River n/a
BR Adult Huay Jawrakhae Mak ReservoiMueang Buri Ram Mun River n/a n/a
RE Adult n/a Mueang Roi Et Chi River n/a n/a
MD Adult/metacercariae Huai Khi Lek Reservior Nikom Kham Soi Mukdahan Huai Ban Koi River n/a n/a n/a n/a
SSK Adult n/a Mueang Sri Sa Ket Mun River n/a n/a n/a
LP Adult Kil Lom Dam Mueang Lampng Wang River n/a n/a
SPk*** Adult/cercariae/metacercaria Nam Un Dam Phang Khon Sakon Nakhon Songkram River n/a n/a
SK/SNh Adult worm Nong Han Reservoir Nong Han Sakon Nakhon Songkram River n/a n/a
STn Metacercariae Huai Hin Tat Tao Ngoi Sakon Nakhon Songkram River n/a n/a
NP Adult Songkram River Ban Dung Nakon Phanom Songkram River n/a n/a n/a n/a
NY
f Egg from infected patient n/a Na-Yao Nakhon Nayok Bang Pakong River n/a n/a n/a n/a
NNf Egg from infected patient n/a Na-Ngam Nakhon Nayok Bang Pakong River n/a n/a n/a n/a
THf Egg from infected patient n/a Thoong-Heang Nakhon Nayok Bang Pakong River n/a n/a n/a n/a
SKg Adult n/a n/a Sakaeo Tonlesap n/a n/a n/a n/a
Lao PDR VV Adult Nam Ngum Dam Vang Vieng Vientiane Nam Ngum River n/a n/a
NG/NK Adult/metacercariae Nam Ngum Dam Nam Ngum Vientiane Nam Ngum River n/a
TH Adult Nam Ngum Dam Tha Heur Vientiane Nam Ngum River n/a n/a
VT Adult/cercariae/metacercaria Nam Ngum Dam Kampang Nakorn Vientiane Nam Ngum River
TL Adult/metacercariae Natural reservoir That Luang Vientiane Nam Ngum River n/a n/a n/a
SV Adult Natural reservoir Kaisornphomviharn Savannakhet Sae Bang Heang River n/a n/a n/a
KM Adult/metacercariae Nam Theun River Khammouan Khammouan Nam Theun River n/a n/a n/a
CP Adult n/a n/a Champasak Mekong n/a n/a n/a n/a
Cambodia
g KD Adult n/a n/a Kandal Tonlesap n/a n/a n/a n/a
Vietnam
f BD Adult n/a n/a Binh Dinh Thi Nai Lagoon n/a n/a n/a n/a
Myanmarh n/a Egg from infected patient n/a n/a Bago/Mon/YangonAyeyarwady River n/a n/a n/a n/a
MMRi Adult/metacercariae Fish from local market Myoma market Bago Ayeyarwady River n/a n/a n/a
Country Wetland system
Stage**
Genetic groups classified by different molecular markers
Province
Village/district
Collecting locality
Code*
* Sample codes were used by previous publications as listed below.
** Cercariae and metacercariae stages have been used for DNA analyses.
*** New cryptic populations of O. viverrini from Phang Khon district, Sakon Nakhon Province, Thailand.
a
Saijuntha et al., 2007; Kiatsopit et al., 2011, b
Sithithaworn et al., 2007, c
Saijuntha et al., 2008a; Pitaksakulrat et al., 2018, d
Laoprom et al., 2012; Pitaksakulrat et al.,
2017; Namsanor et al., 2020, e
Ando et al., 2001; Pitaksakulrat et al., 2018, f
Buathong et al., 2017, g
Thaenkham et al., 2010, h
Aung et al., 2017, i
Sanpool et al., 2018,
n/a; not analyzed/not available.
W. Saijuntha et al.
Infection, Genetics and Evolution 97 (2022) 105182
5
to be unsuitable and unreliable for systematic and population genetic
studies of O. viverrini due to low variation that has been observed in a
number of studies (Ando et al., 2001; Saijuntha et al., 2008b; Thaenk­
ham et al., 2010).
Three polymorphic enzyme loci markers have previously been
selected to study the population genetics of O. viverrini in a population
from Ban Phai district, Khon Kaen Province (Saijuntha et al., 2008b).
The lack of heterozygousity at the phosphoglucomutase (PGM) locus
was detected which indicates that O. viverrini has a high rate of self-
fertilization or non-random mating. In 2009, genetic variation of
O. viverrini populations based on temporal and fish host species using
three polymorphic enzyme loci was reported (Saijuntha et al., 2009).
Data showed heterozygote deficiency in O. viverrini collected at different
times (temporal populations) and different species of fish host. How­
ever, no significant genetic differences were found among O. viverrini
populations temporally or from different species of fish host. Similar
results were found when the population genetics of O. viverrini from
different endemic foci in Vientiane, Lao PDR based on spatial, temporal
and fish host species were later conducted by using polymorphic enzyme
loci (Kiatsopit et al., 2014). These findings indicated that self-
fertilization and/or a clonal distribution of O. viverrini occurs in Lao
PDR. The role of temporal factors and different species of fish host ap­
pears to have little influence on the levels of genetic differentiation.
Interestingly, spatially related genetic differentiation may be occurring
between O. viverrini located in the upper and lower areas of Nam Ngum
dam. This hypothesis requires further investigation for instance, by
using microsatellite markers which provide a greater population genetic
resolution than enzyme markers.
Highly polymorphic genetic markers, i.e. microsatellite DNA have
been developed and used to examine the population genetic structure of
O. viverrini from Thailand and Lao PDR (Laoprom et al., 2010). Signifi­
cant heterozygote deficiency was observed, which confirmed previous
reports and supports predominantly self-fertilization of O. viverrini. In
addition, genetic differentiation with high FST was demonstrated when
compared between O. viverrini populations, which support the previous
evidence of O. viverrini populations sub-structuring based on wetland
systems. Several studies conducted subsequently support the hypothesis
that O. viverrini was a species complex using other effective/poly­
morphic molecular markers, such as microsatellite DNA (Laoprom et al.,
2012; Pitaksakulrat et al., 2017; Namsanor et al., 2020).
The microsatellite DNA markers have been used to examine the ge­
netic diversity and population structure of four populations closely
located to each other within Khon Kaen Province. They found significant
genetic differentiation with unique alleles in each population. Moreover,
population sub-structuring between these four localities was also
observed. The data highlighted that microsatellite markers can be used
to explore the genetic structure of O. viverrini populations at a micro
scale in Khon Kaen Province, Thailand (Laoprom et al., 2012). A more
recent population genetics study using microsatellite DNA examined
O. viverrini populations in different cyprinid species, presented the first
evidence that second intermediate host species of fish could contribute
to the genetic diversity of O. viverrini. They found that there are differ­
ences in the genetics of O. viverrini between different species of fish
within and between different geographical localities in endemic areas
(Pitaksakulrat et al., 2018).
Interestingly, a study of the systematics of O. viverrini from eight
localities in Thailand and Lao PDR using four independent DNA
markers, i.e. two mitochondrial genes, i.e. CO1 and ND1 and two nu­
clear genes (i.e. Paramyosin and Cathepsin F) detected a new cryptic
population from Pangkon district, Sakon Nakhon Province, which was
clearly genetically distinct from the other isolates examined (Pit­
aksakulrat et al., 2018). Most recently, Namsanor et al. (2020)
confirmed the report of a cryptic species from Pangkon district, Sakon
Nakhon Province examined by microsatellite DNA, nuclear and mito­
chondrial DNA markers (Table 2). In addition, preliminary data using a
nuclear intron sequence also demonstrated that O. viverrini from
Pangkon district was the most genetic distinct from the others examined
(Saijuntha et al., unpublished). However, more comprehensive in­
vestigations on genetics, morphology, biology, and ecology of this novel
genetic group need to be further investigated to confidentially assess
their systematics status.
4.3. Bithynia siamensis sensu lato
Recent systematics and population genetics investigations of Bithynia
snails, the first intermediate hosts of O. viverrini, by enzyme markers
have revealed that B. s. goniomphalos is a species complex containing at
least 9 cryptic species that have specific associations with defined wet­
lands in Thailand and Lao PDR (Kiatsopit et al., 2013). Furthermore,
they also found that cryptic species of B. s. goniomphalos were directly
associated with specific genetic groups of O. viverrini from the same
wetlands in Thailand and Lao PDR. This evidence supports previous
studies of the systematics of O. viverrini which showed the existence of
cryptic species and co-evolution between O. viverrini and its snail host
(Saijuntha et al., 2007). Later Kulsantiwong et al. (2013b) successfully
used the mitochondrial CO1 sequence to examine genetic differentiation
among the 10 species/subspecies in the family Bithyniidae and the ge­
netic diversity of the genus Bithynia. Interestingly, molecular analyses
revealed the presence of B. s. siamensis in the south and northeast of
Thailand, which has been previously believed to be restricted in the
central region of Thailand (Kulsantiwong et al., 2013b).
More recently B. s. goniomphalos was intensively collected from a
wider geographical range, namely, 33 localities in six different wetland
(catchment) systems belonging to the Lower Mekong basin in Thailand,
Lao PDR, and Cambodia and were examined by mitochondrial CO1 and
16S rDNA sequences (Tantrawatpan et al., 2020). Three major lineages
(I – III) of B. s. goniomphalos were classified corresponded to catchment
systems. Lineage I contained B. s. goniomphalos from the vast majority of
catchment systems in Thailand and Lao PDR, e.g. the Kok, Wang, Yom,
Nan, and Pasak catchments in northern, Mekong, the Chi, Mun,
Songkram, Huai Bagn Koi, Huai Ma Hiao, Nam Kam and Nam Loei
catchments in northeastern, the Prachin Buri and Bang Pakong catch­
ment in eastern region, the Chao Phraya catchment in central Thailand,
and the Nam Ngum catchment in Lao PDR. Lineage III on the other hand
contained specimens from the Mekong and Sea Bang Heang catchments
in Thailand and Lao PDR, respectively, whereas all populations from
Tonlesap catchment grouped into lineage II. Thus, these genetic distinct
groups need to be further comprehensive investigation on their
morphology, biology, ecology and genetics to re-assess their systematics
and taxonomic status. This study provides strong support that B. s.
goniomphalos in Thailand is a species complex (Tantrawatpan et al.,
2020).
The intron regions of arginine kinase (AkInt) have been proven to be
potential co-dominant genetic markers for elucidating heterozygosity in
freshwater mollusks, including Bithynia snails (Bunchom et al., 2020).
Four regions of AkInt were recently characterized for B. s. siamensis, B. s.
goniomphalos and B. funiculata (Bunchom et al., 2020). Of these, intron 1
(AkInt1) was determined as an appropriate genetic marker to examine
the population genetics of the three B. s. goniomphalos, B. s. siamensis,
and B. funiculata, and could genetically differentiate the three Bithynia
snail species and subspecies (Bunchom et al., 2020). Thus, this intron
region can be used in future studies as genetic marker for elucidating the
systematics and evolutionary relationship as well as hybridization levels
between Bithynia snails.
Most recently, mitochondrial DNA sequences have been used for the
molecular survey of the distribution of Bithynia snails covering a larger
geographical range in the north, central and west of Thailand (Bunchom
et al., 2021a). It was found that not only for B. s. goniomphalos, but the
genetic structure of B. s. siamensis was also related to catchment systems.
In addition, the genetic structure of a closely related species, Hydro­
bioides nassa was also examined and found that the sub-structuring
detected was also related to different catchment systems (Bunchom
W. Saijuntha et al.
Infection,
Genetics
and
Evolution
97
(2022)
105182
6
Table 2
Example of polymorphic and/or diagnostic markers/loci of Opisthorchis viverrini populations from different wetland systems in Thailand and Lao PDR.
Genetic marker Thailand Lao PDR
Chi River wetland Wang River
wetland
Mun River
wetland
Songkram River wetland Nam Ngum River wetland Sea Bang Heang
River wetland
Code*: KLp KBp KPv CP MS KS LP BR SSK SK NP SPk** VV NG TH VT SV
Allozymes (diagnostic
locus***)a
Est b b b b b b b b n/a a a n/a a a a a n/a
Gpt-1 b b b b b b b c n/a b c n/a c c c c c
Idh c c c c c c c b n/a b b n/a b b b b b
Ldh c c c c c c c c n/a b b n/a c c c c b
Me-1 b b b b c c c c n/a c b n/a b b b b b
Me-2 b b b b a a b b n/a n/a c n/a c c c c b
Mitochondrial DNA
(haplotype)
Cox1b
OVC2 OVC1 OVC1 OVC2 OVC2 OVC2 OVC1 OVC1 n/a OVC1 OVC1 n/a
n/
a
OVC1 OVC1 OVC2 OVC1
Cox1c H3,H5,
H10
H3,H8 n/a n/a n/a n/a n/a n/a
H3,H5,
H10
n/a
H1,
H8
H9,
H11
n/
a
n/a n/a H4,H6 n/a
Nad1b
OVN3 OVN4 OVN1 OVN2 OVN4 OVN2 OVN2 OVN2 n/a OVN3 OVN2 n/a
n/
a
OVN1 OVN1 OVN1 OVN1
Nad1c H4,H8,
H14
H10,
H11,H16
n/a n/a n/a n/a n/a n/a
H4,
H14,
H17
H3
H6,
H9
H12,
H13
H1 n/a n/a H1,H5 H6
Nuclear DNAc
(haplotype)
CF-int6 H1 H1 n/a n/a n/a n/a n/a n/a H1,H2 n/a H1 H3
n/
a
n/a n/a
H1,H2,
H4
n/a
Pm-int9 H1 H1 n/a n/a n/a n/a n/a n/a H1 n/a H1 H2
n/
a
n/a n/a H1 n/a
Microsatellite DNAd
(Unique fragment size)
Ovms1 ✔ ✔
Ovms2 ✔ ✔ ✔ ✔ ✔
Ovms5 ✔
Ovms10 ✔
Ovms11 ✔
Ovms14 ✔ ✔
Ovms15 ✔ ✔ ✔
Ovms16 ✔
C3 ✔
A118 ✔ ✔
C120 ✔
A131 ✔ ✔
C103 ✔
a
Saijuntha et al., 2007; Kiatsopit et al., 2011, b
Saijuntha et al., 2008a, c
Pitaksakulrat et al., 2018, d
Laoprom et al., 2012; Pitaksakulrat et al., 2017; Namsanor et al., 2020, n/a; not analyzed/not available.
*
Sample codes were used by previous publications as listed below.
**
New cryptic populations of O. viverrini from Phang Khon district, Sakon Nakhon Province, Thailand.
***
Est; esterase (E.C. 3.1.1.1), Gpt; alanine amino transferase (E.C. 2.6.1.2), Idh; isocitrate dehydrogenase (E.C. 1.1.1.42), Ldh; lactate dehydrogenase (E.C. 1.1.1.27), Me; malic enzyme (E.C. 1.1.1.40), Cox1; cytochrome
c oxidase subunit 1, Nad1; NADH dehydrogenase subunit 1, CF-in6; Intron 6 region of Cathepsin F cysteine protease gene, Pm-int9; Intron 9 region of Paramyosin gene.
W.
Saijuntha
et
al.
Infection, Genetics and Evolution 97 (2022) 105182
7
et al., 2021b). Surprisingly, these results demonstrated that Bithynia
species and subspecies are not clearly separated by regions in Thailand
as previously recognized. It is now known that three Bithynia,
B. funiculata, B. s. siamensis and B. s. goniomphalos are found in the north
region, whereas B. s. siamensis and B. s. goniomphalos coexist in the
central region of Thailand (Fig. 2). These results strongly suggest the
currently taxonomically defined subspecies B. s. siamensis and B. s.
goniomphalos should be included within the species complex of “Bithynia
siamensis sensu lato” (Bunchom et al., 2021a).
5. Conclusion and further research
Since 2007, studies of the systematics and population genetics of
O. viverrini and Bithynia snails reveal that both are species complexes
with varying levels of population sub-structuring which are related to
catchment systems in Thailand and Lao PDR. Cryptic species of both
O. viverrini and Bithynia snails exist in these catchment systems. There is
mounting evidence of co-evolution between cryptic species of
O. viverrini and B. s. goniomphalos. Heterozygous deficiency was
commonly detected in O. viverrini populations, due to its hermaphrodite
nature. Gene flow has been found to be limited between catchment
systems, leading to genetic sub-structuring within catchment systems in
Southeast Asia.
There are several endemic areas and new foci of the opisthorchiasis
in Southeast Asia which have to date remained unknown and unex­
plored concerning the systematics and population genetics of O. viverrini
and its Bithynia snail host. Comprehensive studies should be undertaken
to cover wider endemic areas of opisthorchiasis in the future. The
population genetics of O. viverrini populations from other mammal
hosts, e.g. feline, canine, including the other wild fish-eating mammals
should be also further examined. Some studies suggest the possibility of
host selection for O. viverrini is Bithynia hosts, thus the genetic variation
of the cercarial stages from different population of Bithynia snail using
polymorphic markers, such as microsatellite DNA should be further
analyzed. The population dynamics based on temporal and geographic
genetic variation of O. viverrini over a long-term period also needs a
Fig. 2. Distribution of species, subspecies, and three
genetic group (Lineage I – III) of Bithynia snails from 29
wetlands (catchments) in Thailand, Lao PDR, and
Cambodia examined by mitochondrial cytochrome c
oxidase subunit 1 gene. Wetlands in Thailand: 1; Kok, 2;
Mekong, 3; Ping, 4; Wang, 5; Yom, 6; Nan, 7; Chao
Phraya, 8; Pasak, 9; Tha Chin, 10; Nam Loei, 11;
Songkram, 12; Nam Kam, 13; Huai Bang Koi, 14; Chi, 15;
Mun, 16; St. Mongkol Borey, 17; Bang Pakong, 18; Pra­
chin Buri, 19; Maeklong, 20; Phetchaburi, 21; Prachuap
Kirikhan Coast, 22; Talesap Songkha. Wetlands in Lao
PDR: 23; Nam Ngum, 24; Huai Ma Hiao, 25; Huai Som
Pak. Wetlands in Cambodia: 26; St. Siem Reap, 27; St.
Pursat, 28; St. Baribo, 29; Prek Thnot.
W. Saijuntha et al.
Infection, Genetics and Evolution 97 (2022) 105182
8
comprehensive investigation to be undertaken.
The other potential molecular markers, such as polymorphic mi­
crosatellite DNA and nuclear intron sequences also need to be further
characterized and assessed for appropriate use to expand the population
genetic analyses of Bithynia snails, for instance, to examine possible
evidence of hybridization or the lack of hybridization specifically in
sympatric areas. Sampling from currently unexplored catchments need
to be investigated for both O. viverrini and Bithynia snails. Comprehen­
sive morphological and biological studies of the cryptic genetic groups/
species of B. siamensis are also urgently needed. Genetic characterization
of O. viverrini infected in the second intermediate host species of cyprinid
fish is lacking and urgently needed to determine whether it is similar to
or different from the genetic structure of O. viverrini and Bithynia snails
which are related to catchment systems, hence provide an in depth
understanding of the dynamics of O. viverrini complex life-cycle.
Credit authorship contribution statement
Weerachai Saijuntha: Conceptualization, Funding acquisition,
Writing – original draft. Ross H. Andrews: Conceptualization, Writing -
reveiw & editing. Paiboon Sithithaworn: Writing – review & editing.
Trevor N. Petney: Writing – review & editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
This research project was financially supported by Thailand Science
Research and Innovation (TSRI) 2021 and Mahasarakham University.
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Art faciola hepatica

  • 1. Infection, Genetics and Evolution 97 (2022) 105182 Available online 10 December 2021 1567-1348/© 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Current assessment of the systematics and population genetics of Opisthorchis viverrini sensu lato (Trematoda: Opisthorchiidae) and its first intermediate host Bithynia siamensis sensu lato (Gastropoda: Bithyniidae) in Thailand and Southeast Asia Weerachai Saijuntha a,* , Ross H. Andrews b,c , Paiboon Sithithaworn b , Trevor N. Petney d a Walai Rukhavej Botanical Research Institute, Mahasarakham University, Maha Sarakham 44150, Thailand b Cholangiocarcinoma Research Institute, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand c Faculty of Medicine, St Mary's Campus, Imperial College London, United Kingdom d Departments of Zoology and Paleontology and Evolution, State Museum of Natural History Karlsruhe, Erbprinzenstrasse 13, 76133 Karlsruhe, Germany A R T I C L E I N F O Keywords: Opisthorchiasis Liver fluke Cholangiocarcinoma Genetic variation Systematics Cryptic species Genetic structure A B S T R A C T The group 1 carcinogen, the liver fluke Opisthorchis viverrini is the causative agent of opisthorchiasis and sub­ sequent bile duct cancer (cholangiocarcinoma; CCA), which is an important public health problem in Southeast Asia. Bithynia snails are known to be the sole intermediate host of O. viverrini, and distributed throughout endemic areas of opisthorchiasis. Since 2001, the genetic variation investigation of O. viverrini has progressively been investigated. Comprehensive genetic variation studies of O. viverrini and Bithynia snails were undertaken and consecutively published in 2007 by Saijuntha and colleagues. These studies provided genetic evidence that O. viverrini and Bithynia snails are both species complex with evidence of co-evolution. Later, several studies have provided data in support of this finding, and have continuously to date reinforced that both O. viverrini and Bithynia are species complexes. Moreover, studies have shown that genetic variation of O. viverrini is related to geographical, temporal, fish host species including geographical genetic variation of its snail host, Bithynia siamensis sensu lato. This is significant and important in our understanding of the evolution and phylogenetic relationships between species within the O. viverrini and Bithynia species complexes. A comprehensive knowledge of the systematics and population genetics of O. viverrini and Bithynia snails provides a sound basis to instigate and develop effective prevention and control programs targeting opisthorchiasis and CCA in the endemic areas of Southeast Asia. Thus, this review examines the historical series of investigations of the systematics and popu­ lation genetics of O. viverrini including Bithynia spp. in Southeast Asia since molecular genetic investigations commenced some 20 years ago. 1. Introduction Foodborne trematode (FBT) infections are a major public health problem in many developing countries affecting the health of more than 50 million people globally (Fürst et al., 2012; World Health Organiza­ tion, 2011). The liver fluke, Opisthorchis viverrini, is one of the most important species involved, which has been classified as a group 1 carcinogen (i.e. a carcinogenic agent) in humans causing bile duct cancer, cholangiocarcinoma (CCA) (World Health Organization, 1995). This liver fluke is commonly found in Southeast Asia, especially Thailand and Lao PDR, where it is estimated that at least 10 million people are infected (Andrews et al., 2008). Although accurate preva­ lence data is not currently available, it is also known to occur in Cambodia and southern Vietnam (Sithithaworn et al., 2012a; Le et al., 2012; Doanh and Nawa, 2016), with several new foci having been re­ ported in Myanmar (Aung et al., 2017; Sanpool et al., 2018). The infections of O. viverrini are associated with the consumption of raw or partially cooked cyprinid fish which are infected with metacer­ ariae (Grundy-Warr et al., 2012). In Thailand, although prevalence of infection has declined since the first studies carried out in last century (Sithithaworn et al., 2012a) due to intensive control programs (Jong­ suksuntigul and Imsomboon, 2003), in the past 20 years prevalence has * Corresponding author. E-mail address: weerachai.s@msu.ac.th (W. Saijuntha). Contents lists available at ScienceDirect Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid https://doi.org/10.1016/j.meegid.2021.105182 Received 22 September 2021; Received in revised form 5 December 2021; Accepted 7 December 2021
  • 2. Infection, Genetics and Evolution 97 (2022) 105182 2 remained at similar levels (Petney et al., 2018). Infection rates of O. viverrini in their snail and fish intermediate hosts are still being re­ ported (Saijuntha et al., 2019). The habit of eating raw or partially cooked fish in the northeast region of Thailand has not changed (Grundy-Warr et al., 2012; Saijuntha et al., 2021a), and hence, the incidence of CCA has remained approximately unchanged over the last 20 years (Sithithaworn et al., 2014). At present, about 20,000 people die of Opisthorchis associated liver cancer (CCA) every year in the northeast of Thailand alone (Khuntikeo et al., 2018). The liver fluke O. viverrini has a complex life cycle, which requires three hosts to complete their life cycle (Saijuntha et al., 2021b). The freshwater snails in genus Bithynia and cyprinid fish act as the first and second intermediate hosts, respectively. Humans are the definitive hosts, whereas rodents, canine and feline are reservoir hosts (Saijuntha et al., 2021b). These multiple hosts have been shown to be important factors that drive the genetic variations of parasitic trematodes, including opisthorchiids (Petney et al., 2018). Studies of the systematics and population genetics of the liver fluke O. viverrini and their snail hosts are important to understand the evolutionary process of their complex life cycle (Prugnolle et al., 2005). Thus, genetic investigations of O. viverrini alone may not be sufficient to provide answers as to the evolutionary processes of this liver fluke. Despite this, genetic variation investigations of its intermediate hosts could elucidate more under­ standing of the adaptation and evolutionary process between O. viverrini and its hosts. To our knowledge, studies of the systematics and population genetics of O. viverrini was first undertaken in 1993 by isoenzyme analysis (Sueblinwong et al., 1993), while mitochondrial DNA sequence varia­ tion was subsequently reported in 2001 (Ando et al., 2001). Since 2001 a series of comprehensive genetic investigation studies on O. viverrini population in Thailand and Lao PDR have been continuously undertaken using a variety of molecular techniques and genetic markers, such as multilocus enzyme electrophoresis technique (MEE), nuclear and mitochondrial DNA sequencing, and fragment analyses of microsatellite markers by Sithithaworn and colleagues (e.g. Saijuntha et al., 2007; Sithithaworn et al., 2007; Saijuntha et al., 2009; Laoprom et al., 2012; Kiatsopit et al., 2014; Buathong et al., 2017; Pitaksakulrat et al., 2017, 2018; Namsanor et al., 2020). While the genetics investigation and identification of O. viverrini in Cambodia, Vietnam, and Myanmar were consequently conducted (Thaenkham et al., 2010; Le et al., 2012; Aung et al., 2017; Sanpool et al., 2018). Subsequently, a comprehensive re­ view was conducted in 2012 (Sithithaworn et al., 2012b), followed by the most up to date review by Petney et al. (2018). In this review, the historical series of studies on the current status of the systematics and population genetics of O. viverrini with additional information of its snail host, Bithynia spp. will be examined in detail. 2. The standard model and genetic variation investigations prior to 2007 2.1. The standard model The history of human infection with O. viverrini started with two publications (Kerr, 1916; Leiper, 1911) dealing with the same data set describing the infection in prisoners in a jail in Chiang Mai in the north of Thailand (Petney et al., 2013). Thereafter, a series of reports began to appear, widely scattered in the literature, extending the known range of the infection to the northeast of Thailand (Prommas, 1927) and Laos (Bedier and Chesneau, 1929). The first major, coordinated research project on O. viverrini infection, starting in 1951, was carried out be­ tween the Thai Ministry of Public Health in cooperation with the United States Special Technical and Economic Mission to Thailand. In 1955 the project leader, Elvio Sadun, published the first comprehensive paper on the epidemiology of O. viverrini infection in Thailand (Sadun, 1955). This publication elucidated the “standard epidemiological model” of infection based on one parasite species (Fig. 1), and at this stage un­ known snail first intermediate hosts, and fish second intermediate hosts. Human infection via the ingestion of raw/fermented fish by humans was proposed and included a list of those species commonly eaten in this way. This comprised a number of non-cyprinid fish species which are now to be known to play no role in the life cycle of O. viverrini as well as a number of known hosts. No information at this time was available on the snail first intermediate hosts. Based on this model, an increasing number of publications appeared on defining and dealing with the snail first intermediate hosts (Upatham and Sukhapanth, 1980; Brockelman et al., 1986; Ditrich et al., 1990; Fig. 1. Diagram depicting the progression of knowledge of the systematics and population genetics of Opisthorchis viverrini sensu lato. Prior to 2007, the studies of the O. viverrini life cycle standard model was still in beginnings. Until 2007, there was no evidence of a complex species of O. viverrini (A can of worms). Following comprehensive systematics and genetic investigations after 2007, it was discovered that the life cycle of the O. viverrini species complex is a multiple hosts and parasites model. W. Saijuntha et al.
  • 3. Infection, Genetics and Evolution 97 (2022) 105182 3 Giboda et al., 1991), the fish second intermediate hosts (Vichasri et al., 1982; Sithithaworn et al., 1997) and human involvement in the life cycle of this parasite (Upatham et al., 1984; Sithithaworn et al., 1991; Kobayashi et al., 2000; Jongsuksuntigul and Imsomboon, 2003). The first difficulties with the standard model began to appear in the late 1990s, when differences in the prevalence and intensities of infection, as well as the symptomatology became apparent between different prov­ inces in the northeast of Thailand. At this time, advances in molecular genetic techniques and their application to systematics studies population genetics studies were being undertaken on many vertebrate and invertebrate groups, including parasites. For instance, Andrews and Chilton (1999) have provided a substantial review on the application of the technique of allozyme electrophoresis for systematic and population genetic studies of protozoan, arthropod, and helminth parasites. Surprisingly, a com­ mon finding from these studies was that many previously traditionally classified parasite species were indeed species complexes of genetically distinct but morphologically similar ‘cryptic’ species. The advances in molecular genetic technologies and their application to parasite sys­ tematics provided the basis to examine whether O. viverrini was in fact not a single species, but a number of closely related morphologically similar species but genetically distinct cryptic species. 2.2. Genetic variation of O. viverrini and Bithynia snails The information of genetic variation of O. viverrini in the period prior 2007 was very limited. To our knowledge, the first report of genetic variation of O. viverrini was conducted by Sueblinvong et al. (1993). The adult worms collected from autopsy and cholangiocarcinoma patients in Srinakarind hospital, Khon Kaen Province, Thailand were examined by the multilocus enzyme electrophoresis (MEE) technique. Sixteen band­ ing patterns at three enzyme markers, i.e. glucose-6-phosphate dehy­ drogenase (G6PD), glucose phosphate isomerase (GPI) and phosphoglucomutase (PGM) were observed among 109 individual worms. It was not until 2001, that genetic variation of O. viverrini was examined again but this time it was based on DNA markers (Ando et al., 2001). Mitochondrial cytochrome c oxidase subunit 1 (CO1) gene and internal transcribed spacer 2 (ITS2) were used to examine genetic variation of adult O. viverrini from different geographical localities, including an autopsy in northeast Thailand. Subsequently, Awir­ uttapanich (2004) found ITS2 sequences variation between individual metacercariae of O. viverrini extracted from Pla Kao Na (Cyclocheilichthys armatus). Intra- and inter-specific variations of CO1 sequence were found with five haplotypes detected based on four variable nucleotide sites, while ITS2 sequences were identical among all samples (Ando et al., 2001). Later, a pilot study was conducted by Nuchjungreed (2001) who applied a random amplified polymorphic DNA (RAPD) technique to examine genetic variation of O. viverrini, results of which were later published (Sithithaworn et al., 2007). The publication of genetic variation of Bithynia snails in this period is nonexistent. The genetic variation of Bithynia snails was consecutively examined by Smarn Tesana and colleagues at this period, with research conducted as part fulfilment of Master degrees of Kodcharin (2005) and Duangprompo (2007) under the supervision of Smarn Tesana. Briefly, The RAPD technique was used to differentiate three Bithynia snails, and also examined the genetic variation of B. s. goniomphalos populations collected from 11 different localities in Chi and Mun River wetlands. They were classified into two major distinct groups (I and II) related to spatial distance and river wetland systems (Kodcharin, 2005). There­ after, the RAPD specific primers was designed and developed for specific amplification of B. s. goniomphalos (Duangprompo, 2007) and subse­ quently for B. funiculata (Kulsantiwong et al., 2013a). 3. 2007: Evidence of species complexes and co-evolution of O. viverrini and Bithynia snails Evidence of genetic variation of O. viverrini was previously observed by Ando et al., (2001) and Sithithaworn et al. (2007). Thereafter, Pai­ boon Sithithaworn applied for a Royal Golden Jubilee Ph.D. (RGJ-Ph.D.) program scholarship which was supported by Thailand Research Fund (TRF), and recruited Weerachai Saijuntha to conduct Ph.D. research on “A comprehensive investigation of genetic variation among natural populations of O. viverrini in Thailand and Lao PDR” (Saijuntha 2007) in collaboration with Ross H Andrews from University of South Australia as overseas advisor. Research aimed to initially identify diagnostic markers, allozyme and mitochondrial DNA that could differentiate in­ dividual worms and populations of O. viverrini and B. s. goniomphalos originating from different geographical areas. Results from these studies were published consecutively by Saijuntha et al. (2008a, 2008b, 2009). Subsequently, the markers that were defined and established were used to comprehensively examine genetic variation and systematics of O. viverrini and B. s. goniomphalos populations across geographical areas in Thailand and Lao PDR. Thereafter, the first report of a comprehensive analysis of O. viverrini systematics and genetic variation examined by allozyme markers was published (Saijuntha et al., 2006, 2007). The data obtained provided evidence that O. viverrini was indeed a species complex that contained at least two major evolutionary lineages, which were subdivided into six genetically distinct groups which related to different wetland (catch­ ment) systems in Thailand and Lao PDR. The first lineage contained three genetic groups of the O. viverrini populations from the Wang, Mun, and Chi River wetlands, whereas the second lineage consisted of two genetic groups belonging to the Songkram and Nam Ngum River wet­ lands from Thailand and Lao PDR, respectively (Saijuntha et al., 2007). In addition, the investigation a subsequent study of the morphometrics and biological variation of O. viverrini from different isolates also sup­ ported these findings by molecular analyses (Laoprom et al., 2009). Importantly, studies utilizing some of those polymorphic enzyme markers were also undertaken to examine the genetic variation of Bithynia snails from the same geographical areas that O. viverrini had been examined. Data showed that the Bithynia populations were sepa­ rated into four distinct genetic groups that were related to the same wetland systems as defined genetic groups of O. viverrini which provided evidence of co-evolution between O. viverrini and its snail first inter­ mediate host. The study by Saijuntha et al. (2007) is a corner stone publication that provided the foundation for consecutive studies after 2007 examining in detail the systematics and population genetic vari­ ation of both O. viveerini and Bithynia snails (these studies are discussed in the following section). For instance, research involved the systematics and population genetic investigations of O. viverrini using a variety of molecular markers, including the use of allozymes, microsatellite DNA, nuclear and mitochondrial DNA sequences (Sithithaworn et al., 2012b; Petney et al., 2018) which confirmed the evidence reported in 2007 (Saijuntha et al., 2007). 4. The comprehensive systematics and population genetic analyses after 2007 4.1. Multiple hosts and parasites model Many parasitic trematodes infect multiple host species, and thus multi-host networks may offer a better framework for investigating parasite dynamics (Pilosof et al., 2015). Saijuntha et al. (2007) have shown that O. viverrini is a species complex comprising many genetically distinct but morphologically cryptic species, contained in at least five distinct genetic groups as detected by 32 independent enzyme loci hence, genetic markers (Saijuntha et al., 2007). Furthermore, Saijuntha et al. (2007) provided data that its first intermediate host Bithynia snails also contained cryptic species, represented by at least three cryptic W. Saijuntha et al.
  • 4. Infection, Genetics and Evolution 97 (2022) 105182 4 genetic groups (Saijuntha et al., 2007). Since 2007, a number of fresh­ water cyprinid species of fish, currently up to 48 species have been recognized as second intermediate hosts (Petney et al., 2018). However, O. viverrini-like metacercariae have also been recovered from non- cyprinid fish, such as Anabas testudineus of the family Anabantidae, Channa striata of the family Channidae and Trichopodus microlepis of the family Osphronemidae (Petney et al., 2018). This suggests that the second intermediate hosts of O. viverrini may not only be the cyprinid fishes as is currently documented. The definitive hosts of O. viverrini have been proven to be cat, dog, rodents, and humans, while the other fish-eating animals, especially wild mammals, may possibly be infected by O. viverrini but this has not been rigorously investigated. A recent study has found no evidence of O. viverrini infection in several fish-eating animals, i.e. monkeys, rodents, small residential mammals, and birds from the Lawa Reservoir and Chi River wetlands in Khon Kaen and Maha Sarakham Provinces, Thailand (Tangkawattana et al., 2021). Interest­ ingly, an O. viverrini-like fluke found in the duck liver has been reported which was genetically characterized as closely related to O. viverrini (Dao et al., 2017). This finding reveals the possibility that undiscovered O. viverrini sibling/related species still exists in the nature. Current available comprehensive genetic information of O. viverrini and its hosts suggests that opisthorchiasis could involve multiple hosts and parasites system (Fig. 1). 4.2. O. viverrini sensu lato The systematics and genetic variation of O. viverrini has continuously been examined the initial evidence that of O. viverrini was a species complex by Saijuntha et al. (2007) (Table 1). For instance, defined allozyme markers have been used to characterize an isolate from Sav­ annakhet Province, Lao PDR (Kiatsopit et al., 2011). They found that the Savannakhet isolate from Lao PDR clustered closely with an O. viverrini population from Vientiane Province, Lao PDR as well as the populations from the Songkram River, Thailand, which also support initial data provided by Saijuntha et al. (2007). Other independent DNA markers, mitochondrial CO1 and ND1 sequences have consecutively been used in studies to examine the genetic variation of O. viverrini between 14 different geographical isolates in Thailand and Lao PDR (Saijuntha et al., 2008a). Low nucleotide variation was observed at 0–0.3% among O. viverrini isolates. These DNA markers could not be used to reveal the genetic structure of O. viverrini related to geographical isolates. How­ ever, a single marker, a partial sequence of ND1 was used to explore the genetic structure of six populations of O. viverrini from Thailand, Lao PDR and Cambodia (Thaenkham et al., 2010). They found no significant differences among populations tested by AMOVA, and suggested to “reject” the previous reports that O. viverrini contained a species com­ plex. However, it is important to note that mitochondrial DNA appears Table 1 Summary of genetic investigations and genetic group classification of Opisthorchis viverrini populations from Thailand, Lao PDR, Cambodia, and Vietnam. Different color represents different major genetic distinct groups classified by a particular molecular marker. Allozymes a RAPD b Mitochondrial DNA c Microsatellite DNA d Nuclear DNA e Thailand KBs Adult Kang Namton Reservoir Ban Sa-ard/Mueang Khon Kaen Chi River n/a n/a KBp Adult/metacercariae Kang Lawa Reservoir Ban Phai Khon Kaen Chi River KLp Adult/metacercariae Prakeu Stream Lerngpleuy/Mueang Khon Kaen Chi River KPv Adult Ubonrattana dam Phuviang Khon Kaen Chi River KNp Adult Nam Pong River Nam Pong Khon Kaen Chi River n/a n/a n/a n/a CP Adult/cercariae Nong Ben Reservoir Chatturat Chaiyaphum Chi River MS Adult Chi River Kantharawichai Maha Sarakham Chi River n/a KS Adult/cercariae Lampao Dam Mueang Kalasin Chi River n/a BR Adult Huay Jawrakhae Mak ReservoiMueang Buri Ram Mun River n/a n/a RE Adult n/a Mueang Roi Et Chi River n/a n/a MD Adult/metacercariae Huai Khi Lek Reservior Nikom Kham Soi Mukdahan Huai Ban Koi River n/a n/a n/a n/a SSK Adult n/a Mueang Sri Sa Ket Mun River n/a n/a n/a LP Adult Kil Lom Dam Mueang Lampng Wang River n/a n/a SPk*** Adult/cercariae/metacercaria Nam Un Dam Phang Khon Sakon Nakhon Songkram River n/a n/a SK/SNh Adult worm Nong Han Reservoir Nong Han Sakon Nakhon Songkram River n/a n/a STn Metacercariae Huai Hin Tat Tao Ngoi Sakon Nakhon Songkram River n/a n/a NP Adult Songkram River Ban Dung Nakon Phanom Songkram River n/a n/a n/a n/a NY f Egg from infected patient n/a Na-Yao Nakhon Nayok Bang Pakong River n/a n/a n/a n/a NNf Egg from infected patient n/a Na-Ngam Nakhon Nayok Bang Pakong River n/a n/a n/a n/a THf Egg from infected patient n/a Thoong-Heang Nakhon Nayok Bang Pakong River n/a n/a n/a n/a SKg Adult n/a n/a Sakaeo Tonlesap n/a n/a n/a n/a Lao PDR VV Adult Nam Ngum Dam Vang Vieng Vientiane Nam Ngum River n/a n/a NG/NK Adult/metacercariae Nam Ngum Dam Nam Ngum Vientiane Nam Ngum River n/a TH Adult Nam Ngum Dam Tha Heur Vientiane Nam Ngum River n/a n/a VT Adult/cercariae/metacercaria Nam Ngum Dam Kampang Nakorn Vientiane Nam Ngum River TL Adult/metacercariae Natural reservoir That Luang Vientiane Nam Ngum River n/a n/a n/a SV Adult Natural reservoir Kaisornphomviharn Savannakhet Sae Bang Heang River n/a n/a n/a KM Adult/metacercariae Nam Theun River Khammouan Khammouan Nam Theun River n/a n/a n/a CP Adult n/a n/a Champasak Mekong n/a n/a n/a n/a Cambodia g KD Adult n/a n/a Kandal Tonlesap n/a n/a n/a n/a Vietnam f BD Adult n/a n/a Binh Dinh Thi Nai Lagoon n/a n/a n/a n/a Myanmarh n/a Egg from infected patient n/a n/a Bago/Mon/YangonAyeyarwady River n/a n/a n/a n/a MMRi Adult/metacercariae Fish from local market Myoma market Bago Ayeyarwady River n/a n/a n/a Country Wetland system Stage** Genetic groups classified by different molecular markers Province Village/district Collecting locality Code* * Sample codes were used by previous publications as listed below. ** Cercariae and metacercariae stages have been used for DNA analyses. *** New cryptic populations of O. viverrini from Phang Khon district, Sakon Nakhon Province, Thailand. a Saijuntha et al., 2007; Kiatsopit et al., 2011, b Sithithaworn et al., 2007, c Saijuntha et al., 2008a; Pitaksakulrat et al., 2018, d Laoprom et al., 2012; Pitaksakulrat et al., 2017; Namsanor et al., 2020, e Ando et al., 2001; Pitaksakulrat et al., 2018, f Buathong et al., 2017, g Thaenkham et al., 2010, h Aung et al., 2017, i Sanpool et al., 2018, n/a; not analyzed/not available. W. Saijuntha et al.
  • 5. Infection, Genetics and Evolution 97 (2022) 105182 5 to be unsuitable and unreliable for systematic and population genetic studies of O. viverrini due to low variation that has been observed in a number of studies (Ando et al., 2001; Saijuntha et al., 2008b; Thaenk­ ham et al., 2010). Three polymorphic enzyme loci markers have previously been selected to study the population genetics of O. viverrini in a population from Ban Phai district, Khon Kaen Province (Saijuntha et al., 2008b). The lack of heterozygousity at the phosphoglucomutase (PGM) locus was detected which indicates that O. viverrini has a high rate of self- fertilization or non-random mating. In 2009, genetic variation of O. viverrini populations based on temporal and fish host species using three polymorphic enzyme loci was reported (Saijuntha et al., 2009). Data showed heterozygote deficiency in O. viverrini collected at different times (temporal populations) and different species of fish host. How­ ever, no significant genetic differences were found among O. viverrini populations temporally or from different species of fish host. Similar results were found when the population genetics of O. viverrini from different endemic foci in Vientiane, Lao PDR based on spatial, temporal and fish host species were later conducted by using polymorphic enzyme loci (Kiatsopit et al., 2014). These findings indicated that self- fertilization and/or a clonal distribution of O. viverrini occurs in Lao PDR. The role of temporal factors and different species of fish host ap­ pears to have little influence on the levels of genetic differentiation. Interestingly, spatially related genetic differentiation may be occurring between O. viverrini located in the upper and lower areas of Nam Ngum dam. This hypothesis requires further investigation for instance, by using microsatellite markers which provide a greater population genetic resolution than enzyme markers. Highly polymorphic genetic markers, i.e. microsatellite DNA have been developed and used to examine the population genetic structure of O. viverrini from Thailand and Lao PDR (Laoprom et al., 2010). Signifi­ cant heterozygote deficiency was observed, which confirmed previous reports and supports predominantly self-fertilization of O. viverrini. In addition, genetic differentiation with high FST was demonstrated when compared between O. viverrini populations, which support the previous evidence of O. viverrini populations sub-structuring based on wetland systems. Several studies conducted subsequently support the hypothesis that O. viverrini was a species complex using other effective/poly­ morphic molecular markers, such as microsatellite DNA (Laoprom et al., 2012; Pitaksakulrat et al., 2017; Namsanor et al., 2020). The microsatellite DNA markers have been used to examine the ge­ netic diversity and population structure of four populations closely located to each other within Khon Kaen Province. They found significant genetic differentiation with unique alleles in each population. Moreover, population sub-structuring between these four localities was also observed. The data highlighted that microsatellite markers can be used to explore the genetic structure of O. viverrini populations at a micro scale in Khon Kaen Province, Thailand (Laoprom et al., 2012). A more recent population genetics study using microsatellite DNA examined O. viverrini populations in different cyprinid species, presented the first evidence that second intermediate host species of fish could contribute to the genetic diversity of O. viverrini. They found that there are differ­ ences in the genetics of O. viverrini between different species of fish within and between different geographical localities in endemic areas (Pitaksakulrat et al., 2018). Interestingly, a study of the systematics of O. viverrini from eight localities in Thailand and Lao PDR using four independent DNA markers, i.e. two mitochondrial genes, i.e. CO1 and ND1 and two nu­ clear genes (i.e. Paramyosin and Cathepsin F) detected a new cryptic population from Pangkon district, Sakon Nakhon Province, which was clearly genetically distinct from the other isolates examined (Pit­ aksakulrat et al., 2018). Most recently, Namsanor et al. (2020) confirmed the report of a cryptic species from Pangkon district, Sakon Nakhon Province examined by microsatellite DNA, nuclear and mito­ chondrial DNA markers (Table 2). In addition, preliminary data using a nuclear intron sequence also demonstrated that O. viverrini from Pangkon district was the most genetic distinct from the others examined (Saijuntha et al., unpublished). However, more comprehensive in­ vestigations on genetics, morphology, biology, and ecology of this novel genetic group need to be further investigated to confidentially assess their systematics status. 4.3. Bithynia siamensis sensu lato Recent systematics and population genetics investigations of Bithynia snails, the first intermediate hosts of O. viverrini, by enzyme markers have revealed that B. s. goniomphalos is a species complex containing at least 9 cryptic species that have specific associations with defined wet­ lands in Thailand and Lao PDR (Kiatsopit et al., 2013). Furthermore, they also found that cryptic species of B. s. goniomphalos were directly associated with specific genetic groups of O. viverrini from the same wetlands in Thailand and Lao PDR. This evidence supports previous studies of the systematics of O. viverrini which showed the existence of cryptic species and co-evolution between O. viverrini and its snail host (Saijuntha et al., 2007). Later Kulsantiwong et al. (2013b) successfully used the mitochondrial CO1 sequence to examine genetic differentiation among the 10 species/subspecies in the family Bithyniidae and the ge­ netic diversity of the genus Bithynia. Interestingly, molecular analyses revealed the presence of B. s. siamensis in the south and northeast of Thailand, which has been previously believed to be restricted in the central region of Thailand (Kulsantiwong et al., 2013b). More recently B. s. goniomphalos was intensively collected from a wider geographical range, namely, 33 localities in six different wetland (catchment) systems belonging to the Lower Mekong basin in Thailand, Lao PDR, and Cambodia and were examined by mitochondrial CO1 and 16S rDNA sequences (Tantrawatpan et al., 2020). Three major lineages (I – III) of B. s. goniomphalos were classified corresponded to catchment systems. Lineage I contained B. s. goniomphalos from the vast majority of catchment systems in Thailand and Lao PDR, e.g. the Kok, Wang, Yom, Nan, and Pasak catchments in northern, Mekong, the Chi, Mun, Songkram, Huai Bagn Koi, Huai Ma Hiao, Nam Kam and Nam Loei catchments in northeastern, the Prachin Buri and Bang Pakong catch­ ment in eastern region, the Chao Phraya catchment in central Thailand, and the Nam Ngum catchment in Lao PDR. Lineage III on the other hand contained specimens from the Mekong and Sea Bang Heang catchments in Thailand and Lao PDR, respectively, whereas all populations from Tonlesap catchment grouped into lineage II. Thus, these genetic distinct groups need to be further comprehensive investigation on their morphology, biology, ecology and genetics to re-assess their systematics and taxonomic status. This study provides strong support that B. s. goniomphalos in Thailand is a species complex (Tantrawatpan et al., 2020). The intron regions of arginine kinase (AkInt) have been proven to be potential co-dominant genetic markers for elucidating heterozygosity in freshwater mollusks, including Bithynia snails (Bunchom et al., 2020). Four regions of AkInt were recently characterized for B. s. siamensis, B. s. goniomphalos and B. funiculata (Bunchom et al., 2020). Of these, intron 1 (AkInt1) was determined as an appropriate genetic marker to examine the population genetics of the three B. s. goniomphalos, B. s. siamensis, and B. funiculata, and could genetically differentiate the three Bithynia snail species and subspecies (Bunchom et al., 2020). Thus, this intron region can be used in future studies as genetic marker for elucidating the systematics and evolutionary relationship as well as hybridization levels between Bithynia snails. Most recently, mitochondrial DNA sequences have been used for the molecular survey of the distribution of Bithynia snails covering a larger geographical range in the north, central and west of Thailand (Bunchom et al., 2021a). It was found that not only for B. s. goniomphalos, but the genetic structure of B. s. siamensis was also related to catchment systems. In addition, the genetic structure of a closely related species, Hydro­ bioides nassa was also examined and found that the sub-structuring detected was also related to different catchment systems (Bunchom W. Saijuntha et al.
  • 6. Infection, Genetics and Evolution 97 (2022) 105182 6 Table 2 Example of polymorphic and/or diagnostic markers/loci of Opisthorchis viverrini populations from different wetland systems in Thailand and Lao PDR. Genetic marker Thailand Lao PDR Chi River wetland Wang River wetland Mun River wetland Songkram River wetland Nam Ngum River wetland Sea Bang Heang River wetland Code*: KLp KBp KPv CP MS KS LP BR SSK SK NP SPk** VV NG TH VT SV Allozymes (diagnostic locus***)a Est b b b b b b b b n/a a a n/a a a a a n/a Gpt-1 b b b b b b b c n/a b c n/a c c c c c Idh c c c c c c c b n/a b b n/a b b b b b Ldh c c c c c c c c n/a b b n/a c c c c b Me-1 b b b b c c c c n/a c b n/a b b b b b Me-2 b b b b a a b b n/a n/a c n/a c c c c b Mitochondrial DNA (haplotype) Cox1b OVC2 OVC1 OVC1 OVC2 OVC2 OVC2 OVC1 OVC1 n/a OVC1 OVC1 n/a n/ a OVC1 OVC1 OVC2 OVC1 Cox1c H3,H5, H10 H3,H8 n/a n/a n/a n/a n/a n/a H3,H5, H10 n/a H1, H8 H9, H11 n/ a n/a n/a H4,H6 n/a Nad1b OVN3 OVN4 OVN1 OVN2 OVN4 OVN2 OVN2 OVN2 n/a OVN3 OVN2 n/a n/ a OVN1 OVN1 OVN1 OVN1 Nad1c H4,H8, H14 H10, H11,H16 n/a n/a n/a n/a n/a n/a H4, H14, H17 H3 H6, H9 H12, H13 H1 n/a n/a H1,H5 H6 Nuclear DNAc (haplotype) CF-int6 H1 H1 n/a n/a n/a n/a n/a n/a H1,H2 n/a H1 H3 n/ a n/a n/a H1,H2, H4 n/a Pm-int9 H1 H1 n/a n/a n/a n/a n/a n/a H1 n/a H1 H2 n/ a n/a n/a H1 n/a Microsatellite DNAd (Unique fragment size) Ovms1 ✔ ✔ Ovms2 ✔ ✔ ✔ ✔ ✔ Ovms5 ✔ Ovms10 ✔ Ovms11 ✔ Ovms14 ✔ ✔ Ovms15 ✔ ✔ ✔ Ovms16 ✔ C3 ✔ A118 ✔ ✔ C120 ✔ A131 ✔ ✔ C103 ✔ a Saijuntha et al., 2007; Kiatsopit et al., 2011, b Saijuntha et al., 2008a, c Pitaksakulrat et al., 2018, d Laoprom et al., 2012; Pitaksakulrat et al., 2017; Namsanor et al., 2020, n/a; not analyzed/not available. * Sample codes were used by previous publications as listed below. ** New cryptic populations of O. viverrini from Phang Khon district, Sakon Nakhon Province, Thailand. *** Est; esterase (E.C. 3.1.1.1), Gpt; alanine amino transferase (E.C. 2.6.1.2), Idh; isocitrate dehydrogenase (E.C. 1.1.1.42), Ldh; lactate dehydrogenase (E.C. 1.1.1.27), Me; malic enzyme (E.C. 1.1.1.40), Cox1; cytochrome c oxidase subunit 1, Nad1; NADH dehydrogenase subunit 1, CF-in6; Intron 6 region of Cathepsin F cysteine protease gene, Pm-int9; Intron 9 region of Paramyosin gene. W. Saijuntha et al.
  • 7. Infection, Genetics and Evolution 97 (2022) 105182 7 et al., 2021b). Surprisingly, these results demonstrated that Bithynia species and subspecies are not clearly separated by regions in Thailand as previously recognized. It is now known that three Bithynia, B. funiculata, B. s. siamensis and B. s. goniomphalos are found in the north region, whereas B. s. siamensis and B. s. goniomphalos coexist in the central region of Thailand (Fig. 2). These results strongly suggest the currently taxonomically defined subspecies B. s. siamensis and B. s. goniomphalos should be included within the species complex of “Bithynia siamensis sensu lato” (Bunchom et al., 2021a). 5. Conclusion and further research Since 2007, studies of the systematics and population genetics of O. viverrini and Bithynia snails reveal that both are species complexes with varying levels of population sub-structuring which are related to catchment systems in Thailand and Lao PDR. Cryptic species of both O. viverrini and Bithynia snails exist in these catchment systems. There is mounting evidence of co-evolution between cryptic species of O. viverrini and B. s. goniomphalos. Heterozygous deficiency was commonly detected in O. viverrini populations, due to its hermaphrodite nature. Gene flow has been found to be limited between catchment systems, leading to genetic sub-structuring within catchment systems in Southeast Asia. There are several endemic areas and new foci of the opisthorchiasis in Southeast Asia which have to date remained unknown and unex­ plored concerning the systematics and population genetics of O. viverrini and its Bithynia snail host. Comprehensive studies should be undertaken to cover wider endemic areas of opisthorchiasis in the future. The population genetics of O. viverrini populations from other mammal hosts, e.g. feline, canine, including the other wild fish-eating mammals should be also further examined. Some studies suggest the possibility of host selection for O. viverrini is Bithynia hosts, thus the genetic variation of the cercarial stages from different population of Bithynia snail using polymorphic markers, such as microsatellite DNA should be further analyzed. The population dynamics based on temporal and geographic genetic variation of O. viverrini over a long-term period also needs a Fig. 2. Distribution of species, subspecies, and three genetic group (Lineage I – III) of Bithynia snails from 29 wetlands (catchments) in Thailand, Lao PDR, and Cambodia examined by mitochondrial cytochrome c oxidase subunit 1 gene. Wetlands in Thailand: 1; Kok, 2; Mekong, 3; Ping, 4; Wang, 5; Yom, 6; Nan, 7; Chao Phraya, 8; Pasak, 9; Tha Chin, 10; Nam Loei, 11; Songkram, 12; Nam Kam, 13; Huai Bang Koi, 14; Chi, 15; Mun, 16; St. Mongkol Borey, 17; Bang Pakong, 18; Pra­ chin Buri, 19; Maeklong, 20; Phetchaburi, 21; Prachuap Kirikhan Coast, 22; Talesap Songkha. Wetlands in Lao PDR: 23; Nam Ngum, 24; Huai Ma Hiao, 25; Huai Som Pak. Wetlands in Cambodia: 26; St. Siem Reap, 27; St. Pursat, 28; St. Baribo, 29; Prek Thnot. W. Saijuntha et al.
  • 8. Infection, Genetics and Evolution 97 (2022) 105182 8 comprehensive investigation to be undertaken. The other potential molecular markers, such as polymorphic mi­ crosatellite DNA and nuclear intron sequences also need to be further characterized and assessed for appropriate use to expand the population genetic analyses of Bithynia snails, for instance, to examine possible evidence of hybridization or the lack of hybridization specifically in sympatric areas. Sampling from currently unexplored catchments need to be investigated for both O. viverrini and Bithynia snails. Comprehen­ sive morphological and biological studies of the cryptic genetic groups/ species of B. siamensis are also urgently needed. Genetic characterization of O. viverrini infected in the second intermediate host species of cyprinid fish is lacking and urgently needed to determine whether it is similar to or different from the genetic structure of O. viverrini and Bithynia snails which are related to catchment systems, hence provide an in depth understanding of the dynamics of O. viverrini complex life-cycle. Credit authorship contribution statement Weerachai Saijuntha: Conceptualization, Funding acquisition, Writing – original draft. Ross H. Andrews: Conceptualization, Writing - reveiw & editing. Paiboon Sithithaworn: Writing – review & editing. Trevor N. Petney: Writing – review & editing. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 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