DOI: 10.2478/s11686-008-0045-4
© 2008 W. Stefañski Institute of Parasitology, PAS
Acta Parasitologica, 2008, 53(3), 240–250; ISSN 1230-2821
Gyrodactylus species (Monogenea) infecting alpine bullhead
(Cottus poecilopus Heckel, 1837) in Norway and Slovakia,
including the description of Gyrodactylus mariannae sp. nov.
Anja C. Winger1*, Haakon Hansen2, Lutz Bachmann1 and Tor A. Bakke1**
1Natural History Museum, University of Oslo, Department of Zoology, P.O. Box 1172 Blindern, NO-0318 Oslo, Norway;
*Present address: Norwegian College of Fishery Science, University of Tromsr, NO-9037 Tromsr, Norway;
2National Veterinary Institute, Section for Parasitology, P.O.Box 750 Sentrum, NO-0106 Oslo, Norway
Abstract
Gyrodactylus specimens infecting the skin and fins of two alpine bullhead (Cottus poecilopus) populations from the rivers
Signaldalselva (North Norway) and Rena (South-East Norway) were characterized by both morphological and molecular
means. Morphometrical differences were minor and the nucleotide sequences of the internal transcribed spacers (ITS) of the
nuclear rDNA cluster were identical for parasites from both localities. Based on earlier descriptions, the relatively closest
species are Gyrodactylus hrabei Ergens, 1957, described from common bullhead (Cottus gobio) in Slovakia and G. sp.
Malmberg, 1973, from alpine bullhead in Sweden. The Norwegian Gyrodactylus specimens from the two alpine bullhead populations were morphometrically different from both the type material of G. hrabei from Slovakia and newly collected
Gyrodactylus specimens from alpine bullhead in two Slovakian localities. The Slovakian Gyrodactylus specimens were found
to be identical with type material of G. hrabei. The nucleotide sequences of the ITS of the Norwegian Gyrodactylus species were
different from the Slovakian material. Hence, the Norwegian Gyrodactylus specimens from the alpine bullhead represent a
new species, G. mariannae sp. nov.
Keywords
Monogenea, Gyrodactylus hrabei, G. mariannae sp. nov., Cottus poecilopus, species description, morphology, ribosomal internal transcribed spacer (ITS)
Introduction
The species-rich genus Gyrodactylus von Nordmann, 1832
(Monogenea) includes ectoparasites on a variety of marine
and freshwater fish worldwide (Bakke et al. 2002). But of the
400 currently known Gyrodactylus species only five have been
recovered from sculpin species of the genus Cottus (Scorpaeniformes) in fresh water (Harris et al. 2004). These Palaearctic species are: G. cotti Roman, 1956, G. hrabei Ergens,
1957, G. szanagai Ergens, 1971, G. rogatensis Harris, 1985
and G. onegensis Rumyantsev, 2000. G. cotti, described from
the gills of the common bullhead (Cottus gobio L.), was later
redescribed from alpine bullhead (C. poecilopus Heckel, 1837)
and slimy bullhead (C. cognatus Richards, 1836) (Ergens
1985). G. hrabei was described from the skin and fins of common bullhead in Slovakia and later redescribed from C. szanaga Dubowski, 1869 and from alpine bullhead in Slovakia
(Ergens 1971). G. szanagai was described from the skin and
**Corresponding
fins of C. szanaga (see Ergens 1971), G. rogatensis from the
skin and fins of common bullhead (see Harris 1985) and G.
onegensis from the gills of common bullhead (see Rumyantsev 2000). In addition, Malmberg (1973) described a Gyrodactylus specimen found on the head of a brown trout (Salmo
trutta L.) in Lake Kakerjaure, Store Sjöfallets National Park,
Luleälv river system, NW Sweden in 1971. Based on studies
of the morphology he considered the specimen closely related to G. hrabei and representing an accidental infection of the
brown trout during predation on alpine bullhead.
Of the 57 valid sculpin species only three Cottus species
are common in Eurasian fresh waters, which are the alpine and
the common bullhead, and the Siberian sculpin, C. sibiricus
Kessler, 1899 (Pethon 2005). The common bullhead has a
wide distribution in Eurasia, reaching as far west as the UK. In
contrast, alpine bullhead has after the Last Ice Age colonized
only parts of northern Fennoscandia and Russia, and some
restricted areas in Central Europe (Banarescu 1990). The
author: t.a.bakke@nhm.uio.no
241
Gyrodactylus parasites on Cottus poecilopus
Œl¹ski
present distributions of the alpine and the common bullhead
supposedly reflect natural dispersion after the Last Ice Age.
The cold water species alpine bullhead has a disjunct distribution in Norway being restricted to a northern and a southeastern part of the country due to colonization via two different immigration routes. In North Norway the alpine bullhead
is distributed from the mountain region to the coast. It is likely that the two main areas of occurrence in Norway have been
separated since the immigration of the alpine bullhead from
the Baltic Ice Lake (see Nybelin 1969, Andreasson 1972).
Supposedly the Fennoscandian and North European populations have been separated from the Central European populations as long as ~130,000 years (P. Pethon pers. comm.).
The size and shape of the haptoral sclerites represent the
classical basis of Gyrodactylus taxonomy (Malmberg 1970).
Recent studies using molecular markers indicate significant
shortcomings in differentiating species only on the basis of
morphometry (Ziêtara et al. 2002, Hansen et al. 2003, Huyse
et al. 2004). At present the use of morphological characters
combined with molecular data is considered most suitable for
description and diagnostics of Gyrodactylus species (Bakke et
al. 2007).
In the present study, gyrodactylids infecting four alpine
bullhead populations, two in Norway and two in Slovakia
were compared using both morphological and molecular
methods. Additionally, type specimens of the morphologically closest relative, G. hrabei were included in the comparisons. The morphological and the molecular analyses of the
specimens from the four alpine bullhead populations, revealed
significant differences between the Norwegian and the Slovakian Gyrodactylus populations. Hence, a new gyrodactylid species, G. mariannae sp. nov. is described from alpine bullhead
in Norway, and considered identical to Gyrodactylus sp. described by Malmberg (1973) from brown trout in NW Sweden
but which probably represent as suggested, an accidental transference from alpine bullhead.
Materials and methods
Fish sampling and parasitological examination
Alpine bullheads (Cottus poecilopus) were sampled by electro-fishing in 10 localities; fish from the following localities
were found infected: Rivers Signaldalselva and Rena in Norway, and in Slovakia rivers Vajskovskv Potok and Hnilec. The
latter is the type locality of G. hrabei (see Table I). Alpine and
common bullheads from the following localities were found
uninfected by Gyrodactylus: In Norway, River Rena (August
18th 2002; n = 40), River Trysilelva (June 6th 2002; n = 40)
and River Nitelva (September 9th 2002; n = 30), Lake Stora
Le (both species in sympatry but only common bullhead
caught; September 11th 2003; n = 30); and in Denmark, alpine
bullheads from River Fjederholt C (October 23rd 2003; n = 6),
River VonD (October 23rd 2003; n = 25), and River Rrding C
(October 23rd 2003; n = 19).
The collected fish were instantly killed by a blow to the
head and fixed in 80% ethanol to be later screened for gyrodactylids on the skin and fins. The specimens collected in the
river Rena, Norway, in 2003 were transferred alive to the
aquarium unit at the Natural History Museum (NHM), University of Oslo, and kept for a period of two months at approximately 12°C in an attempt to increase the infection level
for the subsequent analyses (Table I). Live fish were anaesthetized in 0.04% chlorobutanol prior to the parasitological
examination.
Morphological analyses
The parasites fixed in 80% EtOH were prepared directly on
slides according to the ammonium picrate glycerine methodology described by Malmberg (1970), or after the body was
cut off to be used for molecular analysis. The prepared whole
specimens were used for direct comparison with the type
material and for species description. The hard structures in the
haptors were prepared for light and scanning electron micros-
Table I. The Gyrodactylus infected (skin and fins) and uninfected specimens of alpine bullhead (Cottus poecilopus)
Sampling localities*
Alpine bullhead (Norway)
Signaldalselva
Signaldalselva
River Rena
River Rena
Rena strain (laboratory reared)
Alpine bullhead (Slovakia)
River Vajskovský Potok
River Hnilec
Sampling
date
Water
temp. (°C)
No. of fish
No. of infected
fish (%)
No. of
Gyrodactylus
specimens
Mean infection
(range)
16.08.2002
16.07.2003
01.06.2002
18.08.2002
12.07.2003
12.4
12.5
12
–
12
30
23
13
40
13
26 (86.6)
23 (100)
3 (23.1)
0
3 (23.1)
48
92
~40
–
~50
1.85 (1–5)
4 (2–8)
~13 (13–15)
–
~14 (13–15)
April 2004
July 2004
–
–
17
10
12 (70.6)
7 (70)
30
38
1.8 (1–6)
3.8 (1–9)
*Note: In Norway, apart from the River Rena and River Signaldalselva (above), the samples of alpine bullhead from the following rivers were
found uninfected: River Trysilelva, and River Nitelva, as well as samples of common bullhead from Lake Stora Le. In Denmark, alpine
bullheads from River Fjederholt, River VonD, and River Rrding C.
242
Anja C. Winger et al.
Stanis³a
copy (SEM) according to Harris et al. (1999) but slightly
modified. Only slides with all three haptoral structures (hamuli, ventral bar, marginal hooks) present were used for morphometric analyses. All light microscopy (LM) was performed using oil immersion at ×1000 magnification.
For scanning electron microscopy studies a digestion procedure using cover-slips treated with poly-L-lysin was used to
ensure a better fixation of the hooks onto the stub. The specimens were subsequently sputter-coated with a gold-palladium
target using a Polaron E5000 SEM coating unit, and examined
under a JEOL JSM-6400 scanning electron microscope. The
SEM specimens were used to visualise the haptoral structures
and to demonstrate the landmarks for the point-to-point measurements (see Figs 1–3).
Between nineteen and thirty Gyrodactylus specimens from
each infected alpine bullhead population were prepared for
the subsequent light microscopy analyses. The Gyrodactylus
specimens from the Norwegian populations were measured at
the Parasitological Laboratory, Institute of Aquaculture (IoA),
University of Stirling (UoS), Scotland, UK. Digital images of
the Gyrodactylus specimens were grabbed using a JVC KYF30B 3CCD camera mounted on an Olympus BH2 compound
microscope using a 2.5× interfacing lens and measured using
an IM 1000 morphometrics macro written specifically for the
measurement of specimens using Zeiss KS300 in C/Windows
Release ver 3.0 software (1997 S) (Carl Zeiss Vision GmbH,
Munich, Germany/Imaging Associates Ltd, Thames Oxfordshire, U.K.). Images of each attachment hook were processed
using purpose specific image analysis macros OGRE ver 1.0
and Hook Align I-IV 1.1 (Shinn and Bron, University of
Stirling, unpublished) written for Zeiss KS300 iC/Windows
Release ver. 3.0 (1997) (Carl Zeiss Vision 5 GmbH, Munich,
Germany/Imaging Associates Ltd, Thame, Oxfordshire, U.K.)
software. Two slides, i.e. the designated syntypes of G. hrabei
from common bullhead from the type locality Vajskovskv Potok (labelled: 96-3, 19.06.1955, No. Coll. M-174), and one
voucher specimen identified by Ergens from alpine bullhead
from Ploutve Opava-Karlovice (labelled: 1222-11, 18.02.1961,
No. Coll. M-174), were loaned from the Helminth Collection,
Institute of Parasitology, Academy of Sciences of the Czech
Republic, and compared with the material from Norway and
Slovakia. Apparently, the type specimens of G. hrabei were
re-mounted from ammonium picrate glycerin into Canada balsam by Ergens (F. Moravec pers.comm.).
The Gyrodactylus specimens recovered from the Slovakian populations and the two type specimens of G. hrabei (see
above), were measured at NHM, University of Oslo, Norway,
using the Leica IM 1000 ver 4.0 software (release117, Copyright 1992-2004 Imagic Bildverarbeitung AG). For digital
images of these Gyrodactylus specimens a Leica CTR 6000
stereomicroscope was used. Three specimens of Gyrodactylus
were removed from sedated alpine bullhead from Signaldalselva, and digital images of these living specimens were
grabbed using a Leica CTR 6000 stereomicroscope and studied by both phase and interference contrast at different magnifications at NHM, University of Oslo, Norway.
Morphological measurements
In total, thirty-two landmark distances (see Figs 1–3, Table II)
were selected including those used by Ergens (1957, 1971).
The measurements were taken using a digital calliper or pointto-point tool. A stepwise forward discriminant analysis was
run to remove redundant characters from the analyses which
resulted in 21 ranked characters for further analyses as shown
in Table II. The morphological variability within and between
the different Gyrodactylus populations was first analyzed by
discriminant analyses. To enable detection of relatively subtle variation in shape and to visualize the separation of the
populations, a Principal Component Analysis (PCA) was run.
All univariate analyses were conducted using Graph Pad
Instat ver. 3.06 (GraphPad Software Inc., 1992-2003, San
Diego, CA) and PAST (ver 1.24b, http://folk.uio.no/ohammer/past). Forward linear stepwise discriminant analyses were
conducted using Statistica ver 6.0 (Copyright © StatSoft Ltd.
2006; STATISTICA). PCA performed on the 21 contributing
variables were executed using PAST (ver 1.24b) and R
(http://www.r-project.org/). The quantitative terms are used
according to the definitions given by Bush et al. (1997) and
the significance level was set at α<0.05.
Molecular analyses
Parasite bodies fixed in 80% EtOH were subjected to molecular analyses. DNA was either extracted according to the
method described by Cunningham et al. (2001) or by using the
DNeasy Kit (Qiagen) following the manufacturers instructions. The primer pairs ITS1A (5’-GTAACAAGGTTTCCGTAGGTG-3’) and ITS2 (5’-TCCTCCGCTTAGTGATA-3’)
(Mate4jusová et al. 2001) were used to amplify a fragment partially spanning the 18S gene, the internal transcribed spacer 1,
the 5.8S gene, the internal transcribed spacer 2, and partially
the 28S gene. The primers ITS1A and ITS2 as well as the
internal primers ITS4.5 (5’-CATCGGTCTCTCGAACG-3’),
ITSR3A (5’-GAGCCGAGTGATCCACC-3’) (Mate4 jusová et
al. 2001), and ITS28F (5’-TAGCTCTAGTGGTTCTTCCT3’) (Ziêtara and Lumme 2003) were used for direct sequencing of the PCR products using BigDye chemistry and an ABI
3100 automatic sequencer (Applied Biosystems). Sequences
were proofread and aligned in BioEdit (Hall 1999). Genetic
distances according to the Kimura 2-parameter model (Kimura 1980) were calculated in Mega 4 (Tamura et al. 2007). The
obtained nucleotide sequences were subjected to a BLAST
(http://www.ncbi.nlm.nih.gov/) search (Altschul et al. 1990,
Zhang et al. 2000) for comparison with other sequences
deposited in GenBank.
Results
Gyrodactylus infected alpine bullhead were only found in four
of the examined rivers: Signaldalselva and Rena, Norway, and
Vajskovský Potok and Hnilec, Slovakia. All other samples of
alpine bullhead were found uninfected (Table I). In the Nor-
Gyrodactylus parasites on Cottus poecilopus
Roborzyñski
243
fjad kadsææ¿æ
rosbœŸæv
Table II. The morphological variables used for classifying the Gyrodactylus specimens recovered from alpine bullhead (Cottus poecilopus) in the Norwegian rivers Signaldalselva and
Rena, and the Slovakian rivers Vajskovský Potok and Hnilec. The respective ranks as determined in a stepwise forward discriminant analysis are listed for the 21 most informative characters. For numbers, see Figures 1–3
No.
Hamuli
1
2
3
4
5
6
7
8
9
10
11
Ventral bar
12
13
14
15
16
17
18
19
20
21
22
23
Marginal hooks
24
25
26
27
28
29
30
31
32
Morphological variables
HTL
HPSW
HAL
HSL
HPL
HICL
HIAng
HDSW
HRL
ESL
ERL
hamuli total length
hamuli proximal shaft width
hamuli aperture length
hamuli shaft length
hamuli point length
hamuli inner curve length
hamuli inner curve angle
hamuli distal shaft width
hamuli root length
Ergens shaft length
Ergens root length
VBTW
VBPML
VBM(l)L
VBW
VBMMW
VBM(s)L
VBMemL
VBLL
EVB
VBTL
VBPW
VBPL
ventral bar total width
ventral bar process to mid length
ventral bar median (long) length
ventral bar width
ventral bar maximum median width
ventral bar median (short) length
ventral bar membrane length
ventral bar lateral length
Ergens ventral bar
ventral bar total length
ventral bar process width
ventral bar process length
MHAD
MHHW
MHSDW
MHSL
MHSPW
MHSTW
MHIH
MHTL
MHShftL
marginal hook aperture distance
marginal hook heel width
marginal hook sickle distal width
marginal hook sickle length
marginal hook sickle proximal width
marginal hook sickle toe width
marginal hook instep height
marginal hook total length
marginal hook shaft length
wegian localities, rivers Signaldalselva and Rena, both the prevalence and mean intensity of infection decreased towards the
autumn (August) (see Table I). In the Slovakian rivers Vajskovský Potok and Hnilec the prevalence of infection was
found to be ca. 70% in April and July, and with a mean intensity relatively similar to the Norwegian situation (Table I).
Variation in morphology
Table III lists the average of all measurements from all specimens infecting alpine bullhead from the four infected rivers
and from a specimen of G. hrabei (1222-11, No. Coll. M-174)
from alpine bullhead as well as a syntype (96-3, No. Coll. M174) from common bullhead. The haptoral hard parts of the
Norwegian and the Slovakian specimens along with the syntype of G. hrabei are illustrated in Figure 4A-E.
According to discriminant analyses of the morphological
differentiation within and between the different Gyrodactylus
Rank
1
12
14
4
5
15
2
8
6
17
9
10
16
11
13
7
18
3
19
20
21
populations studied, the variables of the hamuli and the ventral bar contributed most to the differentiation of the two populations of G. mariannae from the skin and fins of alpine bullheads in Norway. The specimens from the Norwegian and
Slovakian populations were classified with 100% correct assignment to their respective country (Table IV). By using the
discriminant functions determined from the discriminant analysis, the morphometric values for the two G. hrabei specimens from Slovakia were added into the equation for classifying species (Table V). The score values given at the bottom
of Table V unambiguously indicate that both G. hrabei specimens were assigned to the Slovakian populations. Accordingly, the gyrodactylids infecting alpine bullhead in Vajskovský Potok and Hnilec, Slovakia were considered G. hrabei.
Further discriminant analyses of the Gyrodactylus specimens
restricted to either the Norwegian or Slovakian populations
yielded 86.44% and 100% correct classification to their
respective classes, respectively (Table IV).
244
Anja C. Winger et al.
Table III. Measurements of the opisthaptoral hard parts of the Gyrodactylus specimens infecting alpine bullhead (Cottus poecilopus) from
four rivers in Norway and Slovakia, and the two type specimens of G. hrabei examined. The numbering and abbreviation of the morphological characters follow those presented in Table II. All measurements in µm
Morphological
characters
No.
abbreviation
Hamuli
1
HTL
2
HPSW
3
HAL
5
HPL
6
HICL
8
HDSW
10
ESL
Ventral bar
12
VBTW
14
VBM(l)L
16
VBMMW
18
VBMemL
19
VBLL
20
EVB
21
VBTL
22
VBPW
23
VBPL
Marginal hooks
24
MHAD
26
MHSDW
27
MHSL
31
MHTL
32
MHShftL
*Voucher
Signaldalselva
(n = 29)
mean (±SD)
Rena
(n = 30)
mean (±SD)
Vajskovský Potok
(n = 21)
mean (±SD)
Hnilec
(n = 22)
mean (±SD)
G. hrabei
(type material)
1*
2**
76.45 (±2.38)
10.47 (±0.59)
27.88 (±1.64)
36.85 (±1.24)
35.33 (±1.22)
5.50 (±0.28)
57.15 (±1.50)
74.10 (±2.37)
10.81 (±0.74)
27.13 (±1.30)
35.92 (±1.18)
34.58 (±0.98)
5.64 (±0.29)
56.03 (±1.31)
70.91 (±1.61)
9.74 (±0.61)
24.37 (±1.17)
35.59 (±0.70)
34.19 (±1.13)
5.72 (±0.37)
53.21 (±1.01)
70.95 (±2.80)
10.36 (±0.62)
24.23 (±1.60)
35.78 (±1.18)
34.65 (±1.24)
5.71 (±0.31)
54.96 (±1.54)
70.04
10.32
26.84
32.13
29.72
4.83
52.72
71.06
11.20
22.78
35.58
35.20
6.32
53.27
34.70 (±1.35)
28.00 (±1.23)
25.33 (±1.04)
20.82 (±1.34)
10.05 (±1.01)
20.11 (±1.04)
42.13 (±1.70)
5.77 (±0.62)
10.47 (±0.77)
33.75 (±2.00)
26.81 (±2.13)
24.82 (±1.42)
20.12 (±1.20)
10.57 (±1.00)
19.51 (±1.44)
41.31 (±1.41)
6.10 (±0.66)
9.95 (±1.09)
33.49 (±1.90)
27.44 (±0.99)
24.75 (±1.93)
20.05 (±0.89)
9.17 (±0.68)
22.20 (±1.68)
40.91 (±1.28)
6.29 (±0.47)
10.08 (±0.75)
34.68 (±1.86)
28.19 (±1.50)
25.66 (±1.09)
20.73 (±2.29)
9.78 (±0.95)
21.64 (±1.34)
43.12 (±1.89)
6.74 (±0.47)
11.32 (±0.76)
37.25
27.88
20.39
20.14
10.48
16.27
42.48
7.16
10.59
37.50
25.89
23.71
19.81
9.42
20.93
39.42
5.84
11.65
6.52 (±0.18)
5.96 (±0.31)
7.80 (±0.26)
41.56 (±1.26)
33.98 (±1.16)
6.53 (±0.18)
5.72 (±0.32)
7.73 (±0.20)
40.99 (±2.15)
33.61 (±1.26)
6.64 (±1.31)
6.21 (±0.28)
8.25 (±0.15)
41.31 (±0.96)
33.42 (±0.90)
6.61 (±0.25)
6.11 (±0.15)
8.28 (±0.20)
40.16 (±1.39)
32.44 (±1.29)
7.06
5.86
7.74
38.10
30.49
6.18
5.88
8.43
40.38
32.43
1222-11, No. Coll. M-174 (infecting alpine bullhead); ** syntype 96-3, No. Coll. M-174 (infecting common bullhead).
To determine the contribution of each of the principal 21
discriminating morphometric variables to the separation of the
specimens, a principal component analysis (PCA) was performed. The eigenvalues for PCA 1, 2 and 3 were 18.8, 6.8
and 4.9, respectively. The first and second principal component from PC analysis (PC 1 and 2) accounted for 39.8% and
14.4%, respectively. Specimens from the two Slovakian populations tend generally to have larger ventral bars in relation
to the hamuli when compared with the specimens from the
two Norwegian populations. The overlap in the PC 1 direction
is small, hence the separation between the specimens from the
two countries. We find no predominating trends in PC 2 or 3.
Molecular analyses
Approximately 1100 bp of the ribosomal DNA cluster spanning partial 18S and 28S genes, the internal transcribed spacers (ITS) 1 and 2, and the 5.8S gene, were amplified from two
individuals from each of the four collecting sites; rivers Signaldalselva and Rena in Norway, and Vajskovský Potok and
Hnilec in Slovakia. The sequences from the two Norwegian
populations were identical as were the obtained sequences
from the two Slovakian populations, but the sequences from
the two countries differed by 0.017 K2P-distance (Kimura
1980). The sequences from the Norwegian populations were
1001 bp and from the Slovakian 996 bp. In total 20 nucleotide
Table IV. The overall percentage of Gyrodactylus specimens correctly assigned to their respective
populations as determined from a discriminant analysis using the top morphometric variables (21
variables)
Country
Within Norway
Within Slovakia
Between Slovakia
and Norway
Classification table
% correct classification
significance level
number of miss-classifications from north
number of miss-classifications from south
% correct classification
significance level
% correct classification
significance level
86.44
p = 0.0086
6
2
100
p = 1.09e-5
100
p = 1.84e-31
245
Gyrodactylus parasites on Cottus poecilopus
Table V. The variables used in each step of the discriminant analysis are given along with their original variable number (see Figs 2–4) and are used to determine to which population the two specimens of Gyrodactylus
hrabei belong. The two specimens are: (i) G. hrabei infecting alpine bullhead in Slovakia; (ii) a syntype of
G. hrabei infecting common bullhead in Slovakia. Both group significantly with the Slovakian specimens. To
categorize specimens, the following discriminant formula was used = (discriminant function 1 × variable value1)
+ (discriminant function 2 × variable value 2)… + [discriminant function (n) × variable value (n)] – offset constant
No.
Variable
1
HTL
10
ESL
26
MHSDW
5
HPL
6
HICL
14
VBM(l)L
23
VBPL
12
VBTW
18
VBMemL
19
VBLL
21
VBTL
2
HPSW
22
VBPW
3
HAL
8
HDSW
20
EVB
16
VBMMW
24
MHAD
27
MHSL
31
MHTL
32
MHShftL
Offset constant
Discriminant
function
0.67
2.68
1.07
0.89
–0.12
–5.57
2.93
–0.04
–0.12
0.87
0.31
2.49
–1.22
–2.83
–1.25
–0.02
3.83
–5.59
–24.45
–0.92
2.82
10.39
Norway
(mean)
Slovakia
(mean)
G. hrabei on
C. poecilopus
G. hrabei on
C. gobio
75.25
10.64
27.50
36.38
34.95
5.57
56.58
34.22
27.39
25.07
20.47
10.31
19.81
41.72
5.93
10.21
6.52
5.84
7.76
41.27
33.79
19.36
70.93
10.07
24.30
35.69
34.44
5.71
54.01
34.12
27.84
25.24
20.41
9.49
21.90
42.10
6.53
10.75
6.63
6.16
8.27
40.69
32.90
–19.34
70.04
10.32
26.84
32.13
29.72
4.83
52.72
37.25
27.88
20.39
20.14
10.48
16.27
42.48
7.16
10.59
7.06
5.86
7.74
38.10
30.49
–3.14
71.06
11.20
22.78
35.58
35.20
6.32
53.27
37.50
25.86
23.71
19.81
9.42
20.93
39.42
5.84
11.65
6.18
5.88
8.43
40.38
32.43
–20.72
substitutions that could be attributed to 10 substitutions in
ITS1 (2 transitions, 8 tranversions), 1 transition in 5.8S and 9
substitutions in ITS2 (5 transitions, 4 transversions) in addition to 5 indels were found.
A BLASTN search of the obtained nucleotide sequences
in GenBank (February 2008) revealed no identical hits. The
closest hit for the sequences from the Norwegian populations
was G. flesi (accession number AY278039). The K2P-distance
between the Norwegian sequences and G. flesi was 0.097, i.e.
much higher than the 0.01 (1%) suggested for differences
between species (Ziêtara and Lumme 2002, 2003). A separate
BLASTN search using only the 5.8S sequence of the Norwegian sequences show 100% identity to several species of
Gyrodactylus belonging to the subgenera Metanephrotus and
Paranephrotus (Malmberg 1964, 1970, 1973; Ziêtara et al.
2002; Mate4 jusová et al. 2003).
The closest hit for the sequences from the Slovakian populations was also G. flesi (accession number AY278039), and
the K2P pairwise genetic distance between the Slovakian
sequences and G. flesi was 0.100. A separate BLASTN search
using only the 5.8S sequence of the Slovakian sequences
show 100% identity to G. harengi (accession numbers AJ309
1Gyrodactylus
295, AJ576065, AJ576064) belonging to the subgenus Metanephrotus Malmberg, 1964 (Huyse and Malmberg 2004).
Nucleotide sequences obtained in the present study were deposited in GenBank with the accession numbers DQ288252–
DQ288258.
Based on the morphological and molecular studies the
Norwegian Gyrodactylus specimens from two alpine bullhead
populations are described as a new species, G. mariannae sp.
nov.
Gyrodactylus mariannae sp. nov.1
Type-host: Alpine bullhead, Cottus poecilopus Heckel, 1837,
family Cottidae (Scorpaeniformes).
Site: Ectoparasitic on fins and body skin.
Type-locality: River Signaldalselva (69°10´9.18½N,
20°2´31.27½E), Troms County, North Norway. The species
was also recovered from River Rena (61°06´09.08½N,
11°22´24.71½E) in south-eastern Norway. For other localities:
see Etymology.
Etymology: Named after Dr. Marianne Malmberg in honour of her dedication and support of her husband’s life-long
mariannae has been previously referred to as an undescribed species on Cottus poecilopus in Norway in Winger (2004) and
in Winger et al. (2005).
246
Anja C. Winger et al.
Fig. 1. SEM image of the hamuli of Gyrodactylus mariannae sp. nov.
from river Signaldalselva illustrating the measurements taken.
Numbers refer to the following distances and codes: 1 – hamulus
total length (HTL); 2 – hamulus proximal shaft width (HPSW);
3 – hamulus aperture length (HAL); 4 – hamulus shaft length (HSL);
5 – hamulus point length (HPL); 6 – hamulus inner curve length
(HICL); 7 – hamulus inner curve angel (HIAng); 8 – hamulus distal
shaft width (HDSW); 9 – hamulus root length (HRL); 10 – Ergens
shaft length (ESL); 11 – Ergens’ root length (ERL). For abbreviations, see Table II. Scale bar = 20 µm
Fig. 3. SEM image of the marginal hooks of a Gyrodactylus mariannae sp. nov. from river Signaldalselva illustrating the measurements taken. Numbers refer to the following distances and codes:
24 – marginal hook aperture distance (MHAD); 25 – marginal hook
heel width (MHHW); 26 – marginal hook sickle distal width
(MHSDW); 27 – marginal hook sickle length (MHSL); 28 – marginal hook sickle proximal width (MHSPW); 29 – marginal hook
sickle toe width (MHSTW); 30 – marginal hook instep height
(MHIH); 31 – marginal hook total length (MHTL); 32 – marginal
hook shaft length (MHShftL). For abbreviations, see Table II. Scale
bar = 10 µm
Fig. 2. SEM image of the ventral bar of Gyrodactylus mariannae sp.
nov. from river Signaldalselva illustrating the measurements taken.
Numbers refer to the following distances and codes: 12 – ventral bar
total width (VBTW); 13 – ventral bar process to mid-length
(VBPML); 14 – ventral bar median long length (VBM(l)L); 15 – ventral bar width (VBW); 16 – ventral bar maximum median width
(VBMMW); 17 – ventral bar median (short) length (VBM(s)L);
18 – ventral bar membrane length (VBMemL); 19 – ventral bar lateral length (VBLL); 20 – Ergens’ ventral bar (EVB); 21 – ventral bar
total length (VBTL); 22 – ventral bar process width (VBPW);
23 – ventral bar process length (VBPL). For abbreviations, see Table
II. Scale bar = 10 µm
studies of monogeneans and the genus Gyrodactylus, including the description of one specimen of Gyrodactylus recovered from brown trout (Salmo trutta) in Lake Kakerjaure,
Luleälv river system, NW Sweden (Malmberg 1973). His
qualified assumption that this represented an accidental transference from alpine bullhead appears to be correct.
Type-material: 30 specimens, digested and mounted for
light microscopy (LM) and three specimens digested and
mounted for scanning electron microscopy (SEM). One flattened whole specimen was deposited as the holotype at the
Gyrodactylus parasites on Cottus poecilopus
247
Fig. 4A-E. Comparison of LM images of the sclerites of four randomly selected specimens of Gyrodactylus mariannae sp. nov. from the
fins of alpine bullhead (Cottus poecilopus) from the rivers: (A) Signaldalselva in North Norway, (B) Rena in South-Eastern Norway; G. hrabei
from Slovakia: (C) River Hnilec and (D) River Vajskovskv Potok (autotype), and (E) G. hrabei (syntype) from fins of common bullhead
(Cottus gobio)
Helminthological Collection, Natural History Museum, University of Oslo (NHMZM, Reg. No. C5225) together with several paratypes (NHMZM Reg. No. C5226, Map1, 2 & 3).
Description (Figs 1–3 and 5; Table III): Three whole specimens, live and slightly flattened under a cover slip were
measured. The bodies were spindle-shaped, 537.8–606.9 µm
(575.8) long and 167.6–197.5 µm (177.3) at the widest part
of midbody. The haptor was 88.7–109.2 µm (102.2) long and
118.4–133.5 µm (124.1) wide. The pharynx was 20.0–21.7
(20.85) in diameter and had 8 long pharyngeal processes. The
penis was spherical and 8.9 µm in diameter and armed with
one large and five (to seven) smaller spines.
The excretory system was not studied. The average values
of the haptoral hard parts are based on the paratypes. For other
measurements, see Table III (for abbreviations, see Table II).
Figures 1–3 illustrate the sclerite structures of the haptor
of G. mariannae sp. nov. The ventral bar is characterized by
long processes and a ventral bar membrane covered with
approximately 15 prominent longitudinal ridges (Fig. 2). The
marginal hook sickle (Fig. 3) has no prominent heel but a knob
representing the attachment point of a muscle. The anterior
foot part of the sickle appears as a straight line without any
indentation at the attachment point of the shaft. The sickle
point does not exceed the tip of the toe. The size of a living
248
relaxed specimen of G. mariannae sp. nov. under slight cover
slip pressure is shown in Figure 5.
Anja C. Winger et al.
from common bullhead, and the sequences with GenBank
accession numbers DQ288252–DQ288254 represent therefore ITS of G. hrabei from alpine bullhead. The amplified
nucleotide sequence of the rDNA consisted of the 3’-end of
the 18S subunit, the ITS1 (407 bp) and ITS2 (432 bp), the 5.8S
gene (157 bp) and the 5’-end of the 28S subunit.
Discussion
Fig. 5. LM (interference contrast) image of a living Gyrodactylus
mariannae sp. nov. under slight cover slip pressure
Molecular diagnosis
The amplified nucleotide sequence of the rDNA consisted of
the 3’-end of the 18S subunit, the ITS1 (412 bp) and ITS2
(432 bp), the 5.8S gene (157 bp) and the 5’-end of the 28S
subunit. The nucleotide sequences were deposited in GenBank under accession number DQ288255–DQ288258.
The Gyrodactylus specimens on alpine bullhead from
Vajskovský Potok, Slovakia were regarded as morphologically identical with the type material of G. hrabei Ergens, 1957
The paper deals with Gyrodactylus species on skin and fins of
the cold water species, alpine bullhead in Norway and Slovakia. Slovakian alpine bullhead populations were infected with
G. hrabei Ergens, 1975, on Norwegian populations a new
species, G. mariannae sp. nov. was found and here described
on the basis of molecular and morphological differences. The
Gyrodactylus specimen recorded from the head of a brown
trout in Lake Kakerjaure, Luleälv river system, NW Sweden
and morphologically described by Malmberg (1973) is very
similar to G. mariannae sp. nov. and here suggested to be
identical. The five other Gyrodactylus species described from
cottids in fresh water do not morphologically resemble neither
G. hrabei nor G. mariannae sp. nov.
The discriminant analyses revealed a significant morphological differentiation of the Gyrodactylus specimens from the
Norwegian and the Slovakian alpine bullhead populations,
which is congruent with the differentiation of the rDNA sequences. In addition, some differentiation was also observed
between the two Norwegian populations and the two Slovakian populations. The differences between the two Norwegian
populations, however, are very likely attributed either to size
in response to different water temperatures (see Mo 1993,
Appleby 1996, Olstad 2008), differences between localities or
to sampling bias due to differences in host numbers and clonality.
For the two Slovakian populations it is noteworthy that
there was substantially less variance in the River Vajskovský
Potok sample than in the specimens from the River Hnilec.
Differences in variance can lead to significant differences
between groups in the discriminant analyses. However, this
does not allow for rejecting the hypothesis that two populations represesent the same species. Ergens (1957) found that
G. hrabei infected both alpine and common bullhead in Slovakia. Accordingly, even if common bullhead was found uninfected in Norway there is a need for examination of larger
samples from other localities in Stora Le where both fish
species co-occur.
The results presented in this study encourage further studies on gyrodactylids from sculpin species that have not been
subjected to anthropogenic translocations. Recently, mtDNA
markers have been applied that give more information on population differences within Gyrodactylus species (see Hansen
et al. 2007). Alpine bullhead has a geographically disjunct distribution in Norway and applying such markers to an increased number of localities in Fennoscandia with G. marian-
Gyrodactylus parasites on Cottus poecilopus
nae is expected to highlight the routes of freshwater fish
immigrations and the origin and potential diversifications of
the parasite populations on alpine bullhead.
Acknowledgements. We thank Vladimira Hanzelova, Parasitological Institute, Slovak Academy of Sciences, Slovak Republic for providing samples from Slovakia, and František Moravec, Institute of
Parasitology, Academy of Sciences of the Czech Republic for providing the autotype and syntypes of G. hrabei. We also thank the following persons for their valuable contributions: Andy Shinn, Institute of Aquaculture, University of Sterling, UK (measurement procedures and statistics), Grethe Robertsen (micrographs), Kjetil Olstad, Dag GammelsFter and Cge Brabrand, NHM, University of
Oslo (UiO) (field work), Toril M. Rolfsen, Department of Molecular
Biosciences, UiO, (SEM), qyvind Hammer NHM, UiO, and Raul
Primicerio NCFS, University of Tromsr (statistical analyses). This
work was supported by the NRC Wild Salmon Program (Project no.
145861/720) and the National Centre for Biosystematics (Project no.
146515/420), co-funded by the NRC and NHM, UiO, Norway.
References
Altschul S.F., Gish W., Miller W., Myers E.W., Lipman D.J. 1990.
Basic local alignment search tool. Journal of Molecular
Biology, 215, 403–410. DOI: 10.1016/S0022-2836(05)803
60-2.
Andreasson S. 1972. Distribution of Cottus poecilopus Heckel and
C. gobio L. (Pisces) in Scandinavia. Zoologica Scripta, 1,
69–78.
Appleby C. 1996. Variability of the haptoral hard parts of Gyrodactylus callariatis Malmberg, 1957 (Monogenea: Gyrodactylidae) from Atlantic cod Gadus morhua L. in the Oslo Fjord,
Norway. Systematic Parasitology, 33, 199–207. DOI: 10.10
07/BF01531201.
Bakke T.A., Harris P.D., Cable J. 2002. Host specificity dynamics:
observations on gyrodactylid monogeneans. International
Journal for Parasitology, 32, 281–308. DOI: 10.1016/S00207519(01)00331-9.
Bakke T.A., Cable J., Harris P.D. 2007. The biology of gyrodactylid
monogeneans: the “Russian Doll-killers”. Advances in Parasitology, 64, 161–376. DOI: 10.1016/S0065-308X(06)640037.
Banarescu P. 1990. Zoogeografi of fresh waters. AULA-Verlag,
Wiesbaden, 511 pp.
Bush A.O., Lafferty K.D., Lotz J.M., Shostak A.W. 1997. Parasitology meets ecology on its own terms: Margolis et al. revisited. Journal of Parasitology, 83, 575–583. DOI: 10.2307/32
84227.
Cunningham C.O., Mo T.A., Collins C.M., Buchmann K., Thiery R.,
Blanc G., Lautraite A. 2001. Redescription of Gyrodactylus
teuchis Lautraite, Blanc, Thiery, Daniel & Vigneulle, 1999
(Monogenea: Gyrodactylidae); a species identified by ribosomal RNA sequence. Systematic Parasitology, 48, 141–150.
DOI: 10.1023/A:1006407428666.
Ergens R. 1957. Gyrodactylus hrabei n. sp. s ploutví vranky obecné.
4 eskoslovenské Spolec4 nosti Zoologické , 21, 143–146
Ve4stník C
(In Czech).
Ergens R. 1971. Dactylogyridae and Gyrodactylidae (Monogenoidea) from some fishes from Mongolia. Folia Parasitologica,
18, 241–254.
Ergens R. 1985. Order Gyrodactylidea Bychowsky, 1937. In: (Ed. A.
Gusev) Key to the parasites of the freshwater fish fauna of the
USSR. Vol. 2. Nauka, Leningrad, 269–347 (In Russian).
249
Hall T. 1999. BioEdit: a user-friendly biological sequence alignment
editor and analysis program for Windows 95/98/NT. Nucleic
Acids Symposium Series, 41, 95–98.
Hansen H., Bachmann L., Bakke T.A. 2003. Mitochondrial DNA
variation of Gyrodactylus spp. (Monogenea, Gyrodactylidae)
populations infecting Atlantic salmon, grayling, and rainbow
trout in Norway and Sweden. International Journal for Parasitology, 33, 1471–1478. DOI: 10.1016/S0020-7519(03)002
00-5.
Hansen H., Bakke T.A., Bachmann L. 2007. Mitochondrial haplotype diversity of Gyrodactylus thymalli (Platyhelminthes;
Monogenea): extended geographic sampling in United Kingdom, Poland, and Norway reveals further lineages. Parasitology Research, 100, 1389–1394. DOI: 10.1007/s00436006-0423-5.
Harris P.D. 1985. Species of Gyrodactylus von Nordmann, 1832
(Monogenea: Gyrodactylidae) from freshwater fishes in southern England, with a description of Gyrodactylus rogatensis
sp. nov. from the bullhead Cottus gobio L. Journal of Natural
History, 19, 791–809. DOI: 10.1080/00222938500770491.
Harris P.D., Cable J., Tinsley R.C., Lazarus C.M. 1999. Combined
ribosomal DNA and morphological analysis of individual
gyrodactylid monogeneans. Journal of Parasitology, 85, 188–
191.
Harris P.D., Shinn A.P., Cable J., Bakke T.A. 2004. Nominal species
of the genus Gyrodactylus von Nordmann 1832 (Monogenea:
Gyrodactylidae), with a list of principal host species. Systematic Parasitology, 59, 1–27. DOI: 10.1023/B:SYPA.00000
38447.52015.e4.
Huyse T., Malmberg G. 2004. Molecular and morphological comparisons between Gyrodactylus ostendicus n. sp. (Monogenea:
Gyrodactylidae) on Pomatoschistus microps (Kroyer) and
G. harengi Malmberg, 1957 on Clupea harengus membras L.
Systematic Parasitology, 58, 105–113. DOI: 10.1023/B:SYPA.
0000029423.68703.43.
Huyse T., Malmberg G., Volckaert F.A.M. 2004. Four new species of
Gyrodactylus von Nordmann, 1832 (Monogenea, Gyrodactylidae) on gobiid fishes: combined DNA and morphological
analyses. Systematic Parasitology, 59, 103–120. DOI: 10.10
23/B:SYPA.0000044427.81580.33.
Kimura M. 1980. A simple method for estimating evolutionary rate
of base substitutions through comparative studies of nucleotide sequences. Journal of Molecular Evolution, 16, 111–
120. DOI: 10.1007/BF01731581.
Malmberg G. 1964. Taxonomical and ecological problems in Gyrodactylus (Trematoda, Monogenea). In: (Eds. R. Ergens and B.
Rysavy) Parasitic Worms and Aquatic Conditions. Publ.
House, Czechoslovak Academy of Sciences, Prague, 203–
230.
Malmberg G. 1970. The excretory system and the marginal hooks as
a basis for the systematics of Gyrodactylus (Trematoda,
Monogenea). Arkiv för Zoologi, Ser. 2, 23, 1–235.
Malmberg G. 1973. On a Gyrodactylus species from Northern
Sweden and the subgeneric position of G. hrabei Ergens,
1957 (Trematoda, Monogenea). Zoologica Scripta, 2, 39–42.
DOI: 10.1111/j.1463-6409.1971.tb00712.x.
Mate4jusová I., Gelnar M., McBeath A.J.A., Collins C.M., Cunningham C.O. 2001. Molecular markers for gyrodactylids
(Gyrodactylidae: Monogenea) from five fish families (Teleostei). International Journal for Parasitology, 31, 738–745.
DOI: 10.1016/S0020-7519(01)00176-X.
Mate4jusová I., Gelnar M., Verneau O., Cunningham C.O., Littlewood D.T.J. 2003. Molecular phylogenetic analysis of the
genus Gyrodactylus (Platyhelminthes: Monogenea) inferred
from rDNA ITS region: subgenera versus species groups.
Parasitology, 127, 603–611. DOI: 10.1017/S00311820030
04098.
250
Mo T.A. 1993. Seasonal variations of the haptoral hard parts of
Gyrodactylus derjavini Mikailov, 1975 (Monogenea: Gyrodactylidae) on brown trout Salmo trutta L. parr and Atlantic
salmon S. salar L. parr in the River Sandvikselva, Norway.
Systematic Parasitology, 26, 225–231. DOI: 10.1007/BF000
9730.
Nybelin O. 1969. On the immigration of Cottus gobio L. and Cottus
poecilopus Haeckel into southern and middle Sweden (in
Swedish, with English summary). Acta Regiae Societatis
Scientiarum et Litterarum Gothoburgensis, Zoologica, 4,
1–52.
Olstad K. 2008. Taxonomy and systematics in Gyrodactylus von
Nordmann, 1832 (Monogenea): Studies on a problematic
species complex parasitizing salmonids. PhD Thesis, Faculty
of Mathematics and Natural Sciences, University of Oslo
(ISSN 1501-7710, Nr. 713).
Pethon P. 2005. Aschehougs store Fiskebok. 5 ed. Aschehoug & Co.,
Oslo, 468 pp.
Rumyantsev E. A. 2000. Gyrodactylus onegensis sp. n. (Monogenea)
– a parasite of the freshwater sculpin (Cottus gobio). Parazitologiya, 34, 156–157.
Tamura K., Dudley J., Nei M., Kumar S. 2007. MEGA4: Molecular
Evolutionary Genetics Analysis (MEGA) software version
4.0. Molecular Biology and Evolution, 24, 1596–1599. DOI:
10.1093/molbev/msm092.
Winger A.C. 2004. Taxonomy and systematics of a Gyrodactylus
(Monogenea) species infecting Norwegian river populations
(Accepted June 20, 2008)
Anja C. Winger et al.
of alpine bullhead (Cottus poecilopus). Cand. Scient. Thesis
in Zoology, Zoological Museum, The Natural History and
Botanical Garden, University of Oslo, 1–59.
Winger A.C., Bachmann L., Shinn A., Bakke T.A. 2005. Taxonomy
and systematics of Gyrodactylus populations infecting riverine alpine bullhead Cottus poecilopus in Norway and Slovakia. Bulletin of the Scandinavian Society for Parasitology, 14,
161.
Zhang Z., Schwartz S., Wagner L., Miller W. 2000. A greedy algorithm for aligning DNA sequences. Journal of Computational
Biology, 7, 203–214. DOI: 10.1089/10665270050081478.
Ziêtara M.S., Huyse T., Lumme J., Volckaert F.A.M. 2002. Deep
divergence among subgenera of Gyrodactylus inferred from
rDNA ITS region. Parasitology, 124, 39–52. DOI: 10.1017/
S0031182001008939.
Ziêtara M.S., Lumme J. 2002. Speciation by host switch and adaptive
radiation in a fish parasite genus Gyrodactylus (Monogenea,
Gyrodactylidae). Evolution, 56, 2445–2458. DOI: 10.1111/j.
0014-3820.2002.tb00170.x.
Ziêtara M.S., Lumme J. 2003. The crossroads of molecular, typological and biological species concepts: Two new species of
Gyrodactylus Nordmann, 1832 (Monogenea: Gyrodactylidae). Systematic Parasitology, 55, 39–52. DOI: 10.1023/A:
1023938415148.