Parasitol Res (2007) 101:1439–1442
DOI 10.1007/s00436-007-0643-3
SHORT COMMUNICATION
Mitochondrial DNA variation of a natural population
of Gyrodactylus thymalli (Monogenea) from the type locality
River Hnilec, Slovakia
Charlotte Lindqvist & Laetitia Plaisance &
Tor A. Bakke & Lutz Bachmann
Received: 29 March 2007 / Accepted: 14 June 2007 / Published online: 12 August 2007
# Springer-Verlag 2007
Abstract The monogenean flatworm Gyrodactylus thymalli (Žitňan, Helminthologia, 2:266–269, 1960) is considered a harmless ectoparasite on grayling (Thymallus
thymallus). The species is closely related to G. salaris
Malmberg, 1957 that causes severe gyrodactylosis on
Atlantic salmon (Salmo salar) in many Norwegian rivers.
In this paper, we study the mitochondrial diversity of a
G. thymalli population from one of the type localities
Hrable on River Hnilec, Slovakia. By sequencing parts of
the mitochondrial NADH dehydrogenase subunit 5 gene,
we detected three haplotypes that differ from each other by
2.1–4.1%. The haplotype HnilecI was found most common.
Our data suggest that River Hnilec has been colonized
independently at least three times with G. thymalli.
Introduction
The monogenean flatworm Gyrodactylus thymalli was
described from grayling (Thymallus thymallus) by Žitňan
(1960) and is considered a harmless parasite. The closely
related G. salaris Malmberg, 1957, however, has received
C. Lindqvist (*) : L. Plaisance : T. A. Bakke : L. Bachmann
Natural History Museum, Department of Zoology,
University of Oslo,
P.O. Box 1172 Blindern, 0318 Oslo, Norway
e-mail: charlotte.lindqvist@nhm.uio.no
Present address:
L. Plaisance
Scripps Institution of Oceanography,
University of California San Diego,
9500 Gilman Drive,
La Jolla, CA 92093-0202, USA
particular attention, as it is a severe pathogen on Atlantic
salmon (Salmo salar) in many Norwegian rivers (Johnsen
et al. 1999; Bakke et al. 2007). The parasite has been
introduced to Norway via infected parr on at least three
independent occasions by human activities (Hansen et al.
2003). There is a huge body of literature on G. salaris
infecting Atlantic salmon in Norway, but still, very little is
known about the parasite’s biology in natural populations
that are not affected by human activities. In fact, it may be
impossible to find G. salaris on undisturbed wild Atlantic
salmon populations. In contrast, the closely related species
G. thymalli occurs on relatively undisturbed host populations, and hence, studies of G. thymalli may offer deeper
insights not only into the biology of this parasite–host
association but into the biology of G. salaris as well.
G. thymalli is widely distributed in Europe (Hansen et al.
2007; Bakke et al. 2007) and morphologically very similar
to G. salaris (Shinn et al. 2004). Even the species’ internal
transcribed spacers (ITS 1 and 2) of the nuclear ribosomal
DNA cluster are identical (Ziętara and Lumme 2002).
These molecular markers can otherwise discriminate the
majority of Gyrodactylus species (Matejusová et al. 2001;
Ziętara and Lumme 2002). In contrast, there is substantial
mitochondrial DNA variation over the range of both G.
salaris and G. thymalli (Hansen et al. 2003, 2006, 2007;
Meinilä et al. 2004). Based on cytochrome oxidase I (COI)
sequence data, 44 haplotypes are currently known that
group into 11 well-supported clades (Hansen et al. 2007). In
the Norwegian River Glomma for example, where until
now eight mitochondrial haplotypes of G. thymalli have
been detected, these haplotypes differ on average by 0.48%
(Hansen et al. 2007). Along this river system, the different
haplotypes were found at different sampling localities, but
all belong to the same haplogroup (Hansen et al. 2006).
However, very little is known about mitochondrial DNA
1440
Parasitol Res (2007) 101:1439–1442
Table 1 Sequence diversity between the NAD5 gene of the three
mitochondrial haplotypes HnilecI, HnilecII, and HnilecIII of G.
thymalli from the river Hnilec, Slovakia
HnilecI
HnilecII
HnilecIII
HnilecI
HnilecII
HnilecIII
–
21
11a
4.14
–
16a
2.09
3.16
–
of G. salaris (GenBank accession number: DQ988931;
Huyse et al. 2007) and parts of the COI and NADH
dehydrogenase subunit 5 (NAD5) genes have been
identified as being among the most variable regions of
the mitochondrial DNA. The p distance of the COI genes
of these two mitochondrial genomes is 0.032 and that of
the NAD5 genes is 0.030 (Plaisance et al. 2007). Parts of
these two genes were sequenced for 43 G. thymalli
specimens from 16 grayling host individuals; one parasite
individual has been sampled from 11 fins, and from two
fins, each six and eight parasite individuals were analyzed.
DNA was extracted using the Qiagen DNeasy tissue kit
(Qiagen) following the manufacturer’s instructions, except
from the last elution step where DNA was eluted in 50 μl
water. The following primer pairs were designed and used:
Z1_F: 5′-TTATTTACTCTAGACCACAAGCG-3′ and
Z1_R: 5′-CCCATATAACTATAGCAGTGTCCT-3′ amplify
a 535-bp fragment of the COI gene, and Z2_F: 5′TTACTTCTCGTCAGCTGGGTA-3′ and Z2_R: 5′CTAAGCTTGATTCCCACCGGAA-3′ amplify a 530-bp
fragment of the NAD5 gene. In a few cases, the ZMO
primers from Hansen et al. (2003) were used to amplify the
COI region. Polymerase chain reaction (PCR) was performed using either PuReTaq ready-to-go PCR beads (GE
Healthcare) or Taq DNA polymerase (GE Healthcare), 1×
buffer, 0.1 mmol/l of each diethylnitrophenyl thiophosphate, 0.4 μmol/l of each primer, and 5 μl of the extracted
DNA in a 50-μl reaction volume. Thermal cycling
conditions were the same for the two amplified mtDNA
regions: 94°C for 3 min; 35 cycles of 94°C for 1 min, 50°C
for 30 s, and 72°C for 45 s; and a final extension at 72°C
for 5 min. PCR products were purified using 10× diluted
exoSAP-IT (USB). Cycle sequencing, using the same
primers as in the PCR reaction, was performed in 10-μl
reactions using 2-μl BigDye terminator cycle sequencing
ready reaction kit (Applied Biosystems), 2 μl 5× sequencing buffer, 10 pmol primer, and 3-μl cleaned PCR product.
Sequencing products were purified with ethanol precipitation and analyzed using an ABI 3100 genetic analyzer
(Applied Biosystems). DNA extracts have been deposited
in the DNA/tissue collection of the Natural History
Museum Oslo.
Percent difference (above diagonal); number of substitutions (below
diagonal). a The ambiguous positions at 14176 and 14278 in haplotype
HnilecIII were not considered different.
variation of Gyrodactylus within a population at a particular
locality of a watercourse. According to the predictions of
Price (1980), one would expect largely homogeneous
parasite infra- and, to some extent, metapopulations due to
the effect of genetic drift. However, other authors have
documented mitochondrial DNA diversity within populations of helminth species that was at similar levels as in
free-living animals (e.g., Blouin et al. 1992; Criscione and
Blouin 2004). Due to the unique reproductive biology of
Gyrodactylus, parasite offspring stay at first on the infected
host individual, thereby creating rapidly growing infrapopupalations (Bakke et al. 2007), but emigration and
direct or indirect transmission of parasites between host
individuals may also be substantial in natural populations
(Bakke et al. 1992). To understand the genetic patterns of a
natural population of Gyrodactylus parasites and to test the
former hypothesis, we studied mitochondrial DNA variation
of G. thymalli on grayling in River Hnilec, Slovakia, one of
the type localities according to Žitňan (1960).
Materials and methods
A total of 36 grayling (T. thymallus) were sampled at
Hrable on River Hnilec, Slovakia on May 20th 2003. One
complete mitochondrial genome of G. thymalli from the
River Hnilec (GenBank accession number: EF527269) has
recently been sequenced (Plaisance et al. 2007). This
sequence has been compared to the mitochondrial genome
Table 2 Nucleotide substitutions in the mitochondrial NAD5 gene of the three mitochondrial haplotypes HnilecI, HnilecII, and HnilecIII of G.
thymalli from the river Hnilec, Slovakia
Position
14110
14129
14136
14153
14176a
14191
14199
14212
14216
14236
14266
14278a
HnilecI
HnilecII
HnilecIII
A
G
.
C
T
T
T
.
C
C
T
T
G
A
R
A
G
.
T
C
.
A
.
G
T
C
.
T
C
.
G
A
A
C
T
Y
Parasitol Res (2007) 101:1439–1442
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Results and discussion
In total, 59 sequences were determined, representing 22
COI and 37 NAD5 sequences. For 18 G. thymalli specimens, both COI and NAD5 sequences were determined. All
obtained sequences of COI and NAD5 were identical to
that of the recently sequenced mitochondrial genome of G.
thymalli (Plaisance et al. 2007) at the exception of the
sequences from two specimens. The most common haplotype, referred here to as HnilecI, was found in 41 (95.3%)
of the studied parasites. The two other haplotypes, HnilecII
(GenBank accession number: EU076809) and HnilecIII
(GenBank accession number: EU076810), were rare, and
each detected in only one G. thymalli specimen (2.3%).
In our survey, all parasites sampled from the same fin
had always the same mitochondrial haplotype. This was
tested for 30 G. thymalli individuals (69.7% of the analyzed
parasites) from five different grayling specimens (27.8% of
the analyzed fins). The fins infected with G. thymalli
haplotypes HnilecII and HnilecIII were only infected with
one single parasite.
The three haplotypes, HnilecI, HnilecII, and HnilecIII,
differ from each other in the surveyed NAD5 regions as
summarized in Tables 1 and 2. The substitutions are mainly
transitions, which have been detected at 23 positions and
two transversions (at positions 14416 and 14438; according
to the G. thymalli complete mitochondrial; Plaisance et al.
2007). Such a strong transition/transversion (ti/tv) bias is
commonly known and appears particularly pronounced in
animal mitochondrial DNA (Brown et al. 1982). It has also
been noted that at low levels of genetic divergence (<20%
divergence), ti/tv appears to be high, whereas at high levels
of genetic divergence, the two substitution types show
equal frequencies (Yang and Yoder 1999). Of the total
nucleotide substitutions, nine resulted in non-synonymous
substitutions, all of which involved replacements of closely
related amino acids. The difference between the NAD5
sequence of each of these three haplotypes is roughly of the
same order of magnitude as the difference between either of
them and the G. salaris NAD5 sequence in GenBank
(accession number: DQ988931). The number of substitutions between this sequence and each of HnilecI, II, and III
are 19, 20, and 15, respectively.
The sequence dissimilarity between the NAD5 region
of the haplotypes HnilecI, HnilecII, and HnilecIII is of
the same range as that observed in COI sequences
between G. thymalli and G. salaris belonging to different
mitochondrial haplogroups (Huyse et al. 2007; Plaisance et
al. 2007).
Our data suggest that the G. thymalli population at the
sampling locality, Hrable, on River Hnilec has been
colonized independently at least three times with G.
thymalli. The order of magnitude of NAD5 sequence
variability between the three detected haplotypes (2.09–
4.14%) indicates that the three independent colonizations
occurred through long-range dispersal, although the origin
of these infections remains unknown. In other watercourses
such as the Norwegian rivers Glomma or Trysil, sequence
variation of G. thymalli haplotypes as estimated by COI
sequences ranged clearly below 1% (Hansen et al. 2006,
2007). One can assume that this relatively low variability
may have arisen after the initial colonization of the
respective river systems. In contrast, the high genetic
diversity of the three G. thymalli haplotypes at Hrable on
River Hnilec indicates different evolutionary histories.
Whether or not these putative independent colonizations
can be related to human activities cannot be answered here.
However, if one considered colonization through human
activities such as, e.g., restocking or translocation of fish as
a likely cause of the colonizations, then the present G.
thymalli population in the river Hnilec cannot be considered
natural any longer. G. thymalli in the river Hnilec has then
provided yet another example on how easily pathogens can
spread unnoticed.
The results presented here support the importance of
comprehensive species descriptions including both morphological and molecular parameters. It is important that
DNA and/or tissue material of the type specimen(s) are
deposited in scientific collections to allow for subsequent
molecular identification or redescriptions of strains or
species whenever necessary. The type material of G.
thymalli is unfortunately not accessible for molecular
approaches. Therefore, we cannot determine which of the
mitochondrial haplotypes presented in this study, if any, at
all, could be related to the specimens used by Žitňan (1960)
in his original species description.
Table 2 (continued)
Position
14314
14353
14356
14394
14416
14425
14438
14454
14473
14480
14525
14540
14561
HnilecI
HnilecII
HnilecIII
T
C
C
C
T
.
A
.
G
T
C
C
C
A
.
A
G
.
G
C
.
T
C
C
A
G
.
A
G
.
G
A
A
T
C
.
T
.
C
Positions refer to the complete mitochondrial genome of G. thymalli from Hnilec, Slovakia, GenBank accession number: EF527269 (Plaisance et
al. 2007). A dot (.) indicates an identical nucleotide when compared to haplotype HnilecI.a The ambiguous position could not be resolved for
haplotype HnilecIII due to limited material.
1442
Acknowledgments We are greatful to Vladka Hanzelova for
providing G. thymalli samples from Slovakia. The project was
supported by the Norwegian Research Council Wild Salmon
Programme (Project nr. 145861/720) and the National Centre for
Biosystematics (Project nr. 146515/420), co-funded by the NRC and
the NHM, University of Oslo, Norway.
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