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. 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