Journal of Helminthology (2009) 00, 1–6 doi:10.

1017/S0022149X09990393
q Cambridge University Press 2009

Genetic differentiation of Echinostoma

revolutum and Hypodereaum conoideum
from domestic ducks in Thailand by
multilocus enzyme electrophoresis
W. Saijuntha1*, P. Sithithaworn2,3 and R.H. Andrews4
1
Walai Rukhavej Botanical Research Institute (WRBRI), Mahasarakham
University, Mahasarakham 44150, Thailand: 2Department of Parasitology,
Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand:
3
Liver Fluke and Cholangiocarcinoma Research Center (LFCRC), Faculty
of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand: 4School
of Pharmacy and Medical Sciences, University of South Australia, GPO
Box 2471, Adelaide, SA 5001, Australia
(Accepted 22 June 2009)

Abstract

Echinostomes are common intestinal parasites causing zoonotic disease,

which are endemic worldwide. Of the four species of medically important
echinostomes reported in Thailand, two species, Echinostoma revolutum and
Hypodereaum conoideum, have been detected in poultry. These two parasites are
morphologically similar and are sometimes difficult to distinguish. In the
present study, multilocus enzyme electrophoresis was used to differentiate
E. revolutum from H. conoideum collected from domestic ducks in Thailand.
The parasites were compared using 22 enzymes with 30 presumptive enzyme
loci. The two species of echinostome could be distinguished at 17 of the
30 enzyme loci. Several loci were polymorphic within each species, suggesting
that these can be used to examine their population genetics.

Introduction Echinostomiasis is also a very important disease in

There are over 60 species of digenean trematodes within
the family Echinostomatidae (Sorensen et al., 1998). The
definitive hosts of echinostomes include aquatic birds,
poultry, rodents and mammals, including humans
(Belding, 1964; Huffman & Fried, 1990, Fried et al., 2004).
Human echinostomiasis is caused by at least 19 species of
echinostomes (from eight genera), and has been reported
in China, India, Indonesia, Japan, Korea, Malaysia, the
Philippines, Russia, Taiwan and Thailand (Graczyk &
Fried, 1998; Miliotis & Bier, 2003; Fried et al., 2004).
For example, in Thailand, five species of echinostome have
been reported to infect humans (Joe et al., 1973).
*Fax: þ 66-43754407
E-mail: weerachai.s@msu.ac.th
domestic animals and wildlife. Morbidity and mortality
due to echinostomiasis are related to parasite load
(Huffman & Fried, 1990; Chai et al., 1994). Anaemia,
headache, dizziness, stomach ache, gastric pain, and
loose stools have most often been reported for light-
to-moderate infections (Belding, 1964; Graczyk &
Fried, 1998). Heavy infections are associated with
eosinophilia, abdominal pain, profuse watery diarrhoea,
anaemia, oedema and anorexia (Belding, 1964;
Carney, 1991; Graczyk & Fried, 1998). Pathologically,
echinostomes damage the intestinal mucosa and cause
extensive intestinal and duodenal erosions (Chai et al.,
1994) and catarrhal inflammation (Belding, 1964).
A number of species, including Echinostoma
revolutum, E. miyagawai, E. gotoi and E. koidzumii
were reported to infect poultry (Eom et al., 1984).
In Thailand, the two principal species that infect domestic
ducks are E. revolutum and Hypodereaum conoideum
2 W. Saijuntha et al.

(Pethleart et al., 2005). The adults of E. revolutum and
H. conoideum can be distinguished from one another
based on several morphological characters, such as the
number of collar spines, the size of circumoral disc,
and the shape of the testes. Echinostoma revolutum has
37 collar spines, a large circumoral disc and oval or
lobed testes, whereas H. conoideum possesses 45 – 53 collar
spines, a small circumoral disc and the testes are sausage-
shaped (Miliotis & Bier, 2003; Radomyos et al., 2003).
However, it has been noted that the E. revolutum adults
infecting humans have differently shaped testes com-
pared to specimens of the same species in ducks, i.e.
adult E. revolutum in humans have oval-shaped testes
(Radomyos et al., 1982) whereas those in ducks have both
oval and multi-lobed testes (Pethleart et al., 2005).
It is often difficult to distinguish morphologically
among immature echinostomes, and no morphological
characters are available to distinguish the eggs of
different echinostome species. Therefore, molecular
markers have been developed to identify and differen-
tiate between species of echinostome and to examine their
genetic relationships (Voltz et al., 1987; Morgan & Blair,
1995, 1998a, b; Sloss et al., 1995; Petrie et al., 1996; Sorensen
et al., 1998). Multilocus enzyme electrophoresis (MEE) has
been used effectively to investigate genetic differentiation
in parasitic trematodes, including Opisthorchis viverrini
(Saijuntha et al., 2007), Clornorchis sinensis (Park et al.,
2000) and species of Fasciola (Agatsuma et al., 1994),
Paragonimus (Agatsuma & Habe, 1986) and Echinostoma
(Voltz et al., 1987; Sloss et al., 1995). For example, Sloss et al.
(1995) established six enzyme loci to differentiate
genetically among three species of echinostomes
(i.e. E. caproni, E. paraensei and E. trivolvis).
The aim of this study was to use MEE to compare
genetically between two medically important species of
echinostomes, E. revolutum and H. conoideum collected
from domestic ducks in Thailand, to determine the extent
of the genetic differences between them and to establish
genetic markers for their identification. In addition, the
polymorphic loci detected within each species were also
established for subsequent studies of population genetics.
Materials and methods
Sample collection
Adult worms of E. revolutum and H. conoideum were
collected from intestines of domestic ducks from a
slaughterhouse in Khon Kaen Province during April and
June of 2008. The 76 adults of H. conoideum were from
11 ducks with between 3 and 20 worms/duck, whereas the
39 E. revolutum adults were from another 12 ducks with 1–7
worms/duck. Adult trematodes were examined using a
light microscope and identified to species level according
to size of the circumoral disc, morphology of testes and
number of collar spines (Gower, 1939; Yamaguti, 1971;
Miliotis & Bier, 2003). Several individual worms of each
species were selected randomly for carmine staining to
confirm their species identity by morphology (Miliotis &
Bier, 2003; Radomyos et al., 2003). Other individuals were
washed extensively in normal saline, then frozen for
subsequent electrophoretic analyses.
Sample preparation for electrophoresis
Individual worms were placed in a microcentrifuge
tube to which 10 –30 ml of lysing solution (100 ml distilled

Table 1. Allelic profiles of H. conoideum and E. revolutum at 30 enzyme loci, with allele frequency indicated in parentheses.

Allelic profiles for:

Enzyme locus* H. conoideum (N** ¼ 76) E. revolutum (N ¼ 39)

Ak b (0.500), d (0.500) a (0.500), c (0.500)

Ald a b
Enol b a
Fdp b a
Fum a b
Gapd b (0.656), d (0.344) a (0.500), c (0.500)
Got-1 d a (0.115), b (0.654), c (0.231)
G6pd-1 a (0.312), b (0.156), c (0.531) b (0.154), c (0.192), d (0.654)
G6pd-2 b a
Gpi b a
Idh b a
Me-1 c a (0.385), b (0.500), c (0.115)
Me-2 b a
Ndpk-1 b (0.500), d (0.500) a (0.500), c (0.500)
Ndpk-2 a b
PepA-1 b a
PepA-2 a (0.656), b (0.281), d (0.062) b (0.154), c (0.615), d (0.231)
PepB-1 a a (0.577), b (0.423)
PepB-2 a (0.750), b (0.250) b (0.231), c (0.769)
PepD a (0.812), c (0.188) b
Umpk-1 a b
Umpk-2 a (0.750), c (0.250) b
*
Excludes monomorphic loci (Got-2, Hk, Mdh, Pgm, Pgam-1, Pgam-2, Pk and Tpi).
**
Sample size.
 Allozymes differentiate E. revolutum and H. conoideum
water, 100 ml b-mercaptoethanol, 10 mg NADP) was
added. Samples were homogenized by hand using a
glass rod. The homogenates were then centrifuged at
10,000 rpm for 10 min while maintained at a temperature
of 48C. Supernatants were stored in capillary tubes as 5 ml
aliquots at 2208C until required.
Multilocus enzyme electrophoresis
MEE was performed using cellulose acetate gels
(Cellogel, Milan, Italy) as the support medium, with
0.02 M phosphate (pH 8.0) used as the running buffer.
MEE gels were run for 120– 150 min at a constant voltage
(200 V) and at a constant temperature (48C). The
histochemical staining methods used follow those
described by Richardson et al. (1986).
Parasite samples were compared using 22 enzymes
as follows (abbreviation, enzyme commission no.
in parentheses): aldolase (Ald, 4.1.2.13), adenylate
kinase (Ak, 2.7.4.3), enolase (Enol, 4.2.1.11), fructose-1,6-
diphosphatase (Fdp, 3.1.3.11), fumarate hydratase (Fum,
4.2.1.2), glyceraldehyde-3-phosphate dehydrogenase
(Gapd, 1.2.1.12), glucose-6-phosphate dehydrogenase
(G6pd, 1.1.1.49), aspartate amino transferase (Got,
2.6.1.1), glucose-phosphate isomerase (Gpi, 5.3.1.9),
hexokinase (Hk, 2.7.1.1), isocitrate dehydrogenase
Fig. 1. The electrophoretic banding patterns of individual specimens of H. conoideum and E. revolutum for Idh.
3
(Idh, 1.1.1.42), malate dehydrogenase (Mdh, 1.1.1.37),
malic enzyme (Me, 1.1.1.40), nucleotide diphosphate
kinase (Ndpk, 2.7.4.6), peptidase valine – leucine
(PepA, 3.4.13.11), peptidase leucine –glycine –
glycine (PepB, 3.4.11.4), peptidase phenylalanine-proline
(PepD, 3.4.13.9), phosphoglycerate mutase (Pgam,
2.7.5.3), phosphoglucomutase (Pgm, 2.7.5.1), pyruvate
kinase (Pk, 2.7.1.40), triose phosphate isomerase
(Tpi, 5.3.1.1), and uridine monophosphate kinase
(Umpk, 2.7.1.48).
For each enzyme, bands detected in each sample were
scored alphabetically, the allozyme with the least
electrophoretic mobility from the cathode designated as
allele a. The multiple banding patterns of individual
worms at a particular locus were consistent with the
expectations of heterozygous individuals for enzymes
with a quaternary structure (e.g. two-banded and
triple-banded patterns for heterozygous individuals
for monomeric and dimeric enzymes, respectively)
(Richardson et al., 1986). The allelic profiles of specimens
were compared and the fixed allelic (hence genetic)
differences among samples representing the two
echinostome species were recorded. A fixed genetic
difference occurs when two groups of samples do not
have any alleles in common at an enzyme locus
(Richardson et al., 1986; Andrews & Chilton, 1999).
4 W. Saijuntha et al.
Fig. 2. Example of electrophoretic patterns of a polymorphic locus, Me-1, comparing ten individuals of E. revolutum.
Results
The present study represents the first time that
allozyme (genetic) markers have been established to
differentiate between E. revolutum and H. conoideum,
two species that infect domestic ducks in Thailand.
Thirty-nine E. revolutum adults and 76 H. conoideum adults
were compared genetically by MEE at 22 enzymes
encoding 30 presumptive enzyme loci (table 1). Genetic
variation among individual trematodes was detected at
22 loci. The eight remaining loci were invariant. Fixed
genetic differences between H. conoideum and E. revolutum
were detected at 17 (57%) of the 30 enzyme loci (i.e. at Ak,
Ald, Enol, Fdp, Fum, Gapd, Got-1, G6pd-2, Gpi, Idh, Me-2,
Ndpk-1, Ndpk-2, PepA-1, PepD, Umpk-1 and Umpk-2)
(fig. 1 and table 1). Genetic variation among specimens of
H. conoideum were detected at eight loci (Ak, Gapd, G6pd-1,
Ndpk-1, PepA-2, PepB-2, PepD and Umpk-2), whereas nine
loci were polymorphic for E. revolutum (i.e. Ak, Gapd,
Got-1, G6pd-1, Me-1, Ndpk-1, PepA-2, PepB-1 and PepB-2)
(fig. 2 and table 1).
Allele frequency of the polymorphic loci is indicated in
table 1. The PepA-2, PepB-2 and G6pd-1 showed higher
frequencies in unique alleles than in common shared
alleles. For example, alleles b and d of PepA-2 were
common alleles between H. conoideum and E. revolutum,
which showed freqencies of 0.062 to 0.281, whereas allele a
was a unique allele for H. conoideum and showed
a frequency of 0.656, and allele c, a unique allele for
E. revolutum, showed a frequency of 0.615 (table 1).
Discussion
At least 17 of the 30 enzyme loci tested can be used to
distinguish between these two species of echinostome.
This indicates the high levels of genetic diversity of
echinostomes in Thailand (Sloss et al., 1995). Moreover,
intraspecific variation was also detected by using
allozyme markers, as the result showed that up to eight
loci showed fixed differences among individual worms
within each species. This number of enzyme loci provides
the opportunity for comprehensive analyses of popu-
lation genetics in these parasites, as achieved in a
previous study on Opisthorchis viverrini sensu lato
(Saijuntha et al., 2008).
As suggested by Andrews & Chilton (1999), the
percentage fixed difference should be greater than 15%
between different species. In this study, 57% of fixed
differences were detected between E. revolutum and
H. conoideum. Moreover, the number of enzyme loci also
affect percentage fixed differences – more than 15 loci
should be examined for reliable interpretation. In this
study, we examined up to 22 loci which, compared to
previous studies (Voltz et al., 1987; Sloss et al., 1995), is the
 Allozymes differentiate E. revolutum and H. conoideum
largest number of loci of allozyme markers studied in
echinostomes.
An application of enzyme markers to the investigation
of genetic relationships or co-evolution between hosts
and parasitic trematodes has been reported (e.g. Saijuntha
et al., 2007). Various freshwater snails, fish, crustaceans
and amphibians have have been commonly found as first
and second intermediate hosts of echinostomes (Graczyk
& Fried, 1998). Morever, several kinds of mammals and
poultry, e.g. rat, dog, human, water bird, chicken and
duck, have been reported as final hosts of echinostomes.
Thus, the enzyme markers from this study may be used
for further investigation of co-evolution of echinostomes
and their hosts. These enzyme markers could also be used
for further genetic identification in other echinostomes of
other host, e.g. E. malayanum in mammals. In addition,
some loci can be used as markers for further population
genetic analyses, e.g. G6pd-1 for both E. revolutum and
H. conoideum or Got-1, Me-2 and PepB-1 for E. revolutum.
Acknowledgements
This research was supported by the Thailand Research
Fund and the Commission on Higher Education (grant
no. MRG5180102). The locations and facilities were
provided by Mahasarakham University and Department
of Parasitology, Liver Fluke and Cholangiocarcinoma
Research Center, Faculty of Medicine, Khon Kaen
University. Many thanks to Dr Neil Chilton for his kind
proofreading of this manuscript and Ms Nadda Kiatsopit
and Ms Kunyarat Duenngai for helping with sample
collection.
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