Parasitol Res (2011) 109:1021–1028 DOI 10.1007/s00436-011-2339-y
ORIGINAL PAPER
A comparative study of karyotypes and chromosomal location of rDNA genes in important liver flukes Fasciola hepatica and Fascioloides magna (Trematoda: Fasciolidae) Marianna Reblánová & Marta Špakulová & Martina Orosová & Ivica Králová-Hromadová & Eva Bazsalovicsová & Dušan Rajský
Received: 19 January 2011 / Accepted: 8 March 2011 / Published online: 21 April 2011 # Springer-Verlag 2011
Abstract Chromosomal characteristics, i.e., number, size, morphology, and location of ribosomal DNA (rDNA) clusters were examined in two medically important liver flukes, Fasciola hepatica and Fascioloides magna (Fasciolidae), using conventional Giemsa staining and fluorescent in situ hybridization (FISH) with ribosomal 18S rDNA probe. A comparison of F. magna and F. hepatica karyotypes confirmed significant differences in all chromosomal features. Whilst the karyotype of F. hepatica comprised ten pairs of chromosomes (one metacentric and nine medium-sized subtelocentrics and submetacentrics; 2n=20, n=1 m+5 sm+4 st; TCL=49.9 μm), the complement of F. magna was composed of 11 pairs of mediumsized subtelocentrics and submeta-metacentrics (2n=22, n=9 st+1 sm+1 sm-m; TCL=35.2 μm). Noticeable differences were found mainly in length and morphology of first chromosome pair. It was metacentric and 9.0 μm long in F. hepatica while subtelocentric and 4.7 μm long in F. magna. Although FISH with rDNA probe revealed a single cluster of ribosomal genes in both species, conspicuous interspecific differences were displayed by chromosomal location M. Reblánová (*) : M. Špakulová : M. Orosová : I. Králová-Hromadová : E. Bazsalovicsová Parasitological Institute, Slovak Academy of Sciences, Hlinkova 3, 04001 Košice, Slovakia e-mail:
[email protected] M. Orosová Institute of Parasitology, Biology Centre AS CR, České Budějovice, Czech Republic D. Rajský Faculty of Forestry, Technical University in Zvolen, Zvolen, Slovakia
of ribosomal loci (i.e., NORs). The signals were found on short arms of fifth homologous pair in F. hepatica; however, they were detected in pericentromeric regions of the long arms of tenth pair in F. magna. The observed cytogenetic differences were interpreted in terms of karyotype evolution of fasciolid flukes; F. hepatica may be regarded phylogenetically younger than F. magna. The present paper provides a pilot study on molecular cytogenetics within a group of hermaphroditic digenetic flukes.
Introduction Liver and intestinal flukes of the family Fasciolidae represent one of the most known but relatively narrow trematode group comprising nine nominative species of the genera Fasciola, Fascioloides, Tenuifasciola, Fasciolopsis, Parafasciolopsis, and Protofasciola (Jones 2005; Lotfy et al. 2008). Three species Fasciola hepatica, F. gigantica, and Fasciolopsis buski are medically important agents of serious zoonotic human diseases, and in addition with Fascioloides magna, they are also of considerable veterinary importance (Pybus 2001; Mas-Coma et al. 2005, 2009; Sripa et al. 2010). Interrelationships among fasciolid species based on evolutionary history have recently been analyzed by extensive works which discussed also hypotheses about probable origin of the fasciolids and historic routs of their dispersion (Lotfy et al. 2008; Mas-Coma et al. 2009). The papers account a complex of molecular, morphological, and life cycle data; however, chromosomal features were not comprised in spite of the fact that several data on fasciolid karyotypes already exist (for review, see Reblánová et al. 2010). Cytogenetic analyses, chiefly those providing information on chromosomal location of various
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genes, may improve re-appraisals of phylogenetic interrelationships within target organisms. As far as parasitic helminths, modern molecular cytogenetic approaches have been applied in tapeworms, dioecious fluke group— schistosomes, and thorny-headed worms (Bombarová et al. 2007, 2009; Hirai et al. 2000; Králová-Hromadová et al. 2010; Orosová et al. 2010a, b), but they have never been used in hermaphroditic trematodes. The present paper provides first molecular cytogenetic study of two fasciolid flukes, F. hepatica and F. magna. The common liver fluke F. hepatica, originally Eurasian parasite, has in these days cosmopolitan distribution and infects mainly sheep, cattle, and goats, being located within the liver bile ducts (Mas-Coma et al. 2009). The economic losses caused by this parasite are estimated to reach over US $3 billion worldwide (Jaros et al. 2010). Human infections are frequent in several temperate regions, especially in Bolivia, Peru, and Ecuador but occur also in Iran, Egypt, and Western Europe, with more than 180 million people at risk of infection (Lotfy et al. 2008; Mas-Coma et al. 2005, 2009). Huge medical and veterinary impact of the parasite is reflected on broad and concentrated research focused, e.g., on diagnostics, treatment, and increasing incidences of resistance to the principal anthelmintics (Walker et al. 2007; McConville et al. 2010). Molecular approach is focused also on studying different evolutionary lineages and genetic diversity of F. hepatica populations and infrapopulations (Semyenova et al. 2006; Walker et al. 2007; Ali et al. 2008; Peng et al. 2009; Ichikawa and Itagaki 2010; Teofanova et al. 2011). Concerning cytogenetics of F. hepatica, number of chromosomes were studied many times and the haploid set n=10 was first detected already by Sanderson (1953). Diploid (2n), triploid (3n) or mixoploid (2n/3n) specimens were detected in F. hepatica and some congeners originating from many Asian countries, some Pacific islands, and also Britain and Ireland (Fletcher et al. 2004; Peng et al. 2009; Ichikawa and Itagaki 2010; Reblánová et al. 2010 and references therein). The chromosome morphology was described only three times. Romanenko and Pleshanova (1975) analyzed cytogenetically F. hepatica from Russia, Špakulová and Králová (1991) studied flukes from Czech Republic, and papers by Li and He (1988) and Li et al. (1988) roughly described the karyotype of F. hepatica collected in China. In these populations, no triploids were detected. The giant liver fluke F. magna is exclusively animal parasite which infects predominantly deer game but is often found in a range of wild and domestic ruminants. The flukes are located within liver parenchymatous cysts, and extremely heavy infections may have fatal consequence for host animals (Foreyt 1996; Pybus 2001; Rajský et al. 2002). Fascioloides magna is native in North America, and it is recently distributed in five broad enzoonotic areas in
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USA and Canada. There the livestock infections cause millions of dollars looses for cattle producers (Pybus 2001). In 1865, the fluke was introduced to Italy near Turin along with its host, wapiti deer (Bassi 1875). Nowadays, the parasite occurs in more or less extensive areas of Central Europe with tendency to invade new areas down the River Danube (for review, see Špakulová et al. 2003). The karyotype of F. magna was described only recently by Reblánová et al. (2010). It comprises 11 pairs of chromosomes present in a diploid set, all classified as subtelocentric except for the submeta-metacentric pair no. 8 and the submetacentric pair no. 10 (2n=22, n=1 sm+1 sm-m+9 st). These data are used here for a comparison with F. hepatica karyotype. The present paper describes the karyotype of F. hepatica from Slovakia in comparison with geographically distant populations of the common liver fluke. Furthermore, the chromosome set of F. hepatica is confronted with this of F. magna using both classical and modern molecular cytogenetic approaches. An application of fluorescent in situ hybridization (FISH) with ribosomal small subunit (18S rDNA) probe represents the first comprehensive cytogenetic study within the family Fasciolidae and the digenetic flukes at all.
Materials and methods Parasite samples Ten living flukes of F. hepatica were dissected from biliary ducts of a cattle liver gained from Bardejov slaughterhouse (northeastern Slovakia) in 2009. Two and six adults of F. magna were obtained from the parenchymatous liver cysts of two red deer, shoot near Gabčíkovo (southwestern Slovakia) in hunting season of 2008. Isolated parasites were washed several times in saline solution and then incubated in 0.025% colchicine in saline for 1–2 h at 37°C. Chromosome preparations Whole living flukes were treated with hypotonic solution of 75 mM KCl at room temperature for 4 h and torn into pieces. Dendritic testes were isolated from worms and placed in freshly prepared fixative solution (methanol/acetic acid, 3:1) for 15 min with two changes. Spread preparations of mitotic chromosomes were made by Frydrychová and Marec (2002). Small portions of fixed testes were transferred into a drop of 60% acetic acid on a slide and torn into fine pieces with a help of tungsten needles. The slides were then placed on a heating plate at 45°C, and the drop of cell suspension was slowly drawn along the slide until it evaporated. Preparations were dehydrated in an ethanol
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series (70%, 80%, and 100%, 60 s each) and stored at −20°C until further use. Giemsa staining Slides were stained in 5% solution of Giemsa dye (Merck, New Jersey, USA) in buffer phosphate solution (pH 6.8) for 30 min and flushed with flowing water. Karyological data of F. hepatica (absolute and relative length and centromeric index) were calculated in 17 best mitotic spreads out of 73 evaluated cells. The centromere position on the chromosomes was classified according to the nomenclature of Levan et al. (1964).
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stained with 0.5 μg/ml DAPI (4′,6-diamino-2-phenylindole; Sigma-Aldrich, Gillingham, UK) and mounted in 1,4diazabicyclo[2.2.2.]octane-based antifade (Sigma-Aldrich, Gillingham, UK). Microscopy and image processing Preparations were observed in conventional fluorescence microscope Zeiss Axioplan 2 (Carl Zeiss Jena, Germany) equipped with F-View CCD camera (Soft Imaging System GmbH, Münster, Germany). Black-and-white fluorescent images were pseudocoloured (light blue for DAPI, and red for Cy3) and processed with Adobe Photoshop, version 9.0.
Fluorescent in situ hybridization with 18S rDNA probe Results The small subunit ribosomal probes (about 1,900 bp) of F. magna and F. hepatica were generated by PCR using the forward primer FM_SSU_F (5´-gagattaagccatgcatgtc-3´) annealing in the very 5´-end and the reverse primer FM_SSU_R (5´-tcaagtttggtcgtcttctc-3´) complementary to the very 3´-end of the 18S rDNA of F. magna from Czech Republic (GenBank EF534989; Králová-Hromadová et al. 2008). Isolation of genomic DNA of F. magna and F. hepatica and conditions of PCR amplification were described by Králová-Hromadová et al. (2008) and Bazsalovicsová et al. (2010). The probes were labeled with biotin-14-dATP by nick translation using a Bionick Labeling System (Invitrogen Corporation, Carlsbad, USA). For FISH technique, the procedure described by Fuková et al. (2005) was applied. In particular, chromosome preparations were treated with proteinase K (20 mg/ml) in 1×PBS for 5 min at 37°C, washed twice in 1×PBS for 5 min each, and then digested with 100 μg/ml RNase A in 2×SSC for 1 h at 37°C and washed twice in 2×SSC for 5 min each. The slides were incubated in 5×Denhard's solution for 30 min at 37°C. Denaturation of chromosomal DNA was done in 70% formamide in 2×SSC for 3 min and 30 s at 68°C. The probe cocktail for one slide (10 μl; 50% deionized formamide, 10% dextran sulfate in 2×SSC) contained ~30 ng of probe and 25 μg of sonicated salmon sperm DNA (Sigma-Aldrich, St. Louis, Missouri, USA). The probe was denatured at 90°C for 5 min. Hybridization at 37°C for 18 h was followed by stringent washes, which included 50% formamide in 2×SSC (3×5 min, 46°C; Fluka, Buchs, Switzerland), 2×SSC (5×2 min, 46°C), 0.1× SSC (3×5 min, 62°C), and 4×SSC containing 0.1% Tween 20 (3×3 min, 37°C). Hybridization signals were detected with Cy3-conjugated streptavidin (Jackson ImmunoRes. Labs. Inc., West Grove, Pennsylvania, USA), amplified with one round of biotinylated anti-streptavidin (Vector Labs. Inc., Burlingame, California, USA) and Cy3conjugated streptavidin. The preparations were counter-
Evaluation of 73 mitotic metaphase spermatogonia of F. hepatica from Slovakia disclosed exclusively diploid number 2n =20 (Fig. 1a). First metacentric pair was markedly longer than the second pair and measured 9.0 μm, occupying 18.0% of the total length of the haploid genome (total length of the complement (TCL)=49.9 μm). Pairs nos. 2–6 were classified as subtelocentric and remaining pairs 7–10 as submetacentric; karyotype formula can be summarized as 2n= 20 (n = 1 m + 5 st +4 sm) (Figs. 1b and 4). Although the length of pairs nos. 2–10 decreased continuously, pairs 4 and 5 were very similar in length and shape (for chromosome measurements, see Table 1). No distinct secondary constriction was observed in preparations.
Fig. 1 Mitotic chromosomes of Fasciola hepatica from Slovakia. a Spread mitotic metaphase stained by Giemsa; b karyotype derived from the cell shown in Fig. 1a. Scale bar indicates 10 μm
39.3±2.2 14.1±3.2 14.9±4.5 23.8±4.0 22.5±4.9 23.9±5.6 31.8±6.1 30.8±4.7 31.9±4.9 34.7±5.4 18.0±0.8 14.0±0.7 12.5±0.5 10.6±0.4 10.0±0.4 8.4±0.3 7.6±0.5 7.0±0.4 6.4±0.5 5.4±0.6 Note: m metacentric chromosome pair, sm submetacentric chromosome pair, st subtelocentric chromosome pair
(m) (st) (st) (st) (m) (st) (m) (m) (st) (st) 1 2 3 4 5 6 7 8 9 10
4.8±0.2 3.4±0.2 3.1±0.2 2.9±0.1 2.6±0.1 2.4±0.1 2.3±0.1 2.2±0.1 1.9±0.1 1.9±0.1
17.5±0.3 12.6±0.2 13.0±0.4 10.6±0.2 9.7±0.2 8.7±0.1 8.0±0.2 7.7±0.2 7.2±0.2 5.9±0.3
37.0±0.6 15.3±0.6 16.8±0.5 18.1±0.5 37.0±0.9 21.6±0.7 32.0±1.2 38.8±0.9 37.8±1.4 30.7±1.0
(sm) (st) (st) (st) (sm) (st) (sm) (m) (m) (sm)
9.4 – – – – – – – – 2.9
19.0±1.1 13.1±0.6 12.0±0.7 10.6±1.0 10.3±0.6 8.7±0.9 7.3±0.7 6.6±0.6 6.1±0.4 5.9±0.5
(sm-m) (st) (st) (sm) (st) (st) (sm) (sm) (sm) (sm) 37.1±3.6 13.2±2.5 14.1±3.5 35.2±2.9 17.4±4.8 21.8±3.2 33.0±4.5 26.1±4.3 33.7±4.3 26.7±6.1
9.0±1.8 7.0±1.4 6.2±1.2 5.3±1.2 5.0±1.0 4.2±0.8 3.8±0.7 3.5±0.7 3.2±0.6 2.7±0.6
Relative length (%) (Classification) Relative length (%)
Centromeric index (classification)
Relative length (%) Absolute length (μm) Absolute length (μm)
Centromeric index (classification)
Absolute length (μm)
Present results Li and He (1988) Špakulová and Králová (1991) Romanenko and Pleshanova (1975) No.
Table 1 Measurements (means±SD) and classification of chromosomes of Fasciola hepatica populations
(m) (st) (st) (st) (st) (st) (sm) (sm) (sm) (sm)
Parasitol Res (2011) 109:1021–1028 Centromeric index (classification)
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Fluorescent DAPI-staining of mitotic cells revealed bright centromeric heterochromatin bands on all chromosome pairs except for no. 7 which was without any DAPIpositive signal in all evaluated metaphases (Fig. 2a, b). Additionally, some less distinct interstitial DAPI bands were identified on pair nos. 1, 2, 3, 4, and 9. Fluorescent in situ hybridization with the 18S rDNA probe revealed one locus of rRNA genes in mitotically dividing spermatogonia of both species. Interspecific differences between F. hepatica and F. magna were displayed by chromosomal location of ribosomal cluster. In F. hepatica, rDNA signals were situated proximally at the end of short arms of the subtelocentric homologues of fifth chromosome pair (Figs. 2 and 4) while in F. magna ribosomal genes were located interstitially, close to the centromere on long arms of the submetacentric chromosome pair no. 10 (Figs. 3 and 4). During meiotic spermatocyte division in F. hepatica, ribosomal signal was observed on proximal end of middle-sized bivalent in diplotene (Fig. 5a) while two signals appeared in paired homologues of this bivalent in meiotic metaphase I (Fig. 5b). In F. magna, two signals were highlighted in pericentromeric regions of just separating bivalent no. 10 in metaphase I (Fig. 5c) and a single locus was visible on small tenth chromosome in metaphase II (Fig. 5d).
Discussion The present paper broadens knowledge on classical karyological data and for the first time reveals chromosomal
Fig. 2 Location of rDNA clusters in mitotic chromosomes of Fasciola hepatica as revealed by FISH with 18S rDNA probe (red signals); chromosomes were counterstained with DAPI (blue). a Mitotic metaphase consisting of 2n=20 chromosomes. b Karyotype derived from the cell shown in Fig. 2a. Scale bar indicates 10 μm. Arrows indicate chromosome pair without any DAPI-positive heterochromatin blocks
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Fig. 3 Location of rDNA clusters in mitotic chromosomes of Fascioloides magna as revealed by FISH with 18S rDNA probe (red signals); chromosomes were counterstained with DAPI (blue). a Mitotic metaphase consisting of 2n=22 chromosomes. b Karyotype derived from the cell shown in Fig. 1a. Scale bar indicates 10 μm
location of ribosomal genes in representatives of hermaphroditic trematodes, namely the important fluke parasites F. hepatica and F. magna. Chromosome number (2n=20) of F. hepatica from Slovakia was identical with the data reported previously at the common fluke populations from China, Russia, and Czech Republic (Li and He 1988; Li et al. 1988; Romanenko and Pleshanova 1975; Špakulová and Králová 1991). The relative length and morphology, but not absolute chromosome length, were similar in all European populations. The absolute length found by Romanenko and Pleshanova (1975) was much lower than this reported later (Špakulová and Králová 1991; present results). These differences were caused very probably by various methodology used, as discussed for instance by Reblánová et al. (2010). In all European populations of F. hepatica, the first chromosome pair was two-armed (submeta- or metacentric) and markedly longer than the subsequent pair no. 2. Pairs 2 and 3 were classified as subtelocentric and nos. 7–10 were
Fig. 4 A comparison of ideograms of Fasciola hepatica (open columns) and Fascioloides magna (grey columns) constructed from data on absolute length of chromosomes
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bi-armed (i.e., meta- or submetacentric) (see Table 1). A little bit problematic differentiation might arise for pair nos. 4 and 5 that do not differ markedly in their length and therefore, could be easily confused. The papers by Romanenko and Pleshanova (1975) and Špakulová and Králová (1991) reported one of the pairs 4 and 5 as bi-armed and the other as subtelocentric; however, the present data show that the fifth pair is satellited having a secondary constriction on distal end of the short arm. A chromosome part bearing the secondary constriction vary considerably in its length, being sometimes invisible (see Fig. 1) and sometimes having twofold length of the short arm (see Špakulová and Králová 1991, Fig. 3). It consequently affects chromosome measurements and ranking the pairs in the karyotype. Regarding the analysis of F. hepatica from China, there are scanty data on chromosome morphology (Li and He 1988; Li et al. 1988) which remarkably differ from the European F. hepatica and might reflect geographical variations or hypothetic misidentification of Chinese parasites, because of sympatric occurrence of F. hepatica, F. gigantica, and Fasciola sp. in that region (Mas-Coma et al. 2009; Yin and Ye 1990). A comparison of karyotypes of F. hepatica (current data) and F. magna (Reblánová et al. 2010, Table 2) revealed significant differences, first of all in diploid number (2n=20 in F. hepatica and 2n=22 in F. magna) and in the TCL which was 49.9 μm in F. hepatica but only 35.2 μm in F. magna. The first metacentric pair of F. hepatica was nearly twice as long as the first subtelocentric pair of F. magna and, besides, the chromosome set of F. hepatica comprised much more bi-armed elements (50% of submetametacentrics) than F. magna (18%). These features could be interpreted in terms of karyotype evolution, accounting a hypothesis that more advanced species of a group often have more symmetric karyotypes (White 1973). Hypothetically, a karyotype of F. hepatica seems to be phylogenetically more advanced in comparison with F. magna. It may fit well with an assumption that the first long metacentric pair of F. hepatica might evolved due to centric fusions and translocations from two one-armed pairs of fasciolid ancestor that did not happen in Fascioloides. Distinct cytogenetic differences between F. hepatica and F. magna seem to be in good accord with recent knowledge on phylogenetic relationships among seven of nine existing fasciolid species, evaluated on basis of molecular data by Lotfy et al. (2008) and broadly discussed by Mas-Coma et al. (2009). A couple of most evolved species F. hepatica and F. gigantica likely form a sister group of another couple of closely related “American” F. magna and the liver fluke of African elephants, Fasciola jacksoni. The current cytogenetic data reflect well the disparity of the flukes assorted to two genera Fasciola and Fascioloides.
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Fig. 5 Meiotic chromosomes of Fasciola hepatica (a, b) and Fascioloides magna (c, d) stained with 18S rDNA probe (red signals) and DAPI (blue). a Diplotene with ten bivalents. b Metaphase I of eight synchronously dividing spermatocytes. c Late metaphase I with 11 bivalents. d Metaphase II, haploid set of 11 chromosomes. Bars indicate 10 μm
Advanced procedures of molecular cytogenetics like FISH may substantially enlarge knowledge on intimate karyotype structure, especially in neglected animal groups like hermaphroditic flukes. The sophisticated approach may reveal evolutionary informative markers that can help us to solve taxonomic queries and phylogenetic interrelationships (e.g., Nguyen et al. 2010). Within the platyhelminth class Table 2 Measurements (means±SD) and classification of chromosomes of Fascioloides magna as published by Reblánová et al. (2010)
Note: m metacentric, sm submetacentric, st subtelocentric chromosome pair
Trematoda, FISH with ribosomal or telomeric probes were used several times exclusively in the family Schistosomatidae, gonochoric, and serious blood parasites of man and mammals (Hirai et al. 1989, 1993, 2000; Hirai and LoVerde 1996; Tanaka et al. 1995). Out of all hermaphroditic trematodes, F. hepatica and F. magna are the first model species, studied here by the FISH technique.
No.
Absolute length (μm)
Relative length (%)
1 2 3 4 5 6 7 8 9 10 11
4.7±0.4 4.2±0.4 4.0±0.4 3.7±0.1 3.4±0.3 3.1±0.4 2.8±0.4 2.7±0.4 2.4±0.2 2.3±0.3 1.9±0.3
13.3±1.0 12.1±0.4 11.3±0.4 10.5±0.3 9.7±0.5 8.8±0.4 8.0±0.6 7.6±0.5 6.8±0.6 6.5±0.6 5.4±0.6
Centromeric index (classification) 16.5±3.6 18.0±3.1 18.3±2.6 20.5±4.5 21.0±4.0 20.9±3.5 22.0±3.6 37.1±2.7 24.2±6.0 28.9±2.7 24.2±2.6
(st) (st) (st) (st) (st) (st) (st) (sm-m) (st) (sm) (st)
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The present rDNA-FISH analysis has revealed remarkable differences in chromosomal localization of clusters of ribosomal genes (i.e., NOR-sites lying in secondary constrictions) in F. hepatica and F. magna. In each of the species, a single strong rDNA signal was detected in a pair of homologues. Ribosomal locus was found to be proximal on middle-sized subtelocentric pair in F. hepatica whereas interstitial in pericentromeric region of small submetacentric pair in F. magna. Regarding F. hepatica, the location of NOR site does not correspond fully with the paper by Špakulová and Králová (1991). They also found a secondary constriction localized in proximal part of the middle-sized pair; however, they assorted this pair as no. 4. This discrepancy may be a result of previously mentioned fact that pairs 4 a 5 are morphologically similar and easily interchangeable. The distinct chromosomal localization of ribosomal loci in F. hepatica and F. magna endorses classical karyological differences between members of the two genera, discussed above. Without additional knowledge of rDNA localization and detailed chromosome structures of further species of the family Fasciolidae, it is impossible to trace back pathways of karyotype evolution within the group and the further analyses and urgently needed. Acknowledgements We are highly indebted to František Marec (Institute of Entomology, Biology Centre ASCR, České Budějovice, Czech Republic) and Tomáš Scholz (Institute of Parasitology, Biology Centre, České Budějovice, Czech Republic) for kind help and providing facilities during research stays of M. R. and M. O. We wish to thank Katarína Oberhauserová (University of Veterinary Medicine and Pharmacy, Department of Nutrition, Dietetics and Animal Breeding, Košice, Slovakia) for providing us with F. hepatica material. The work was supported by the Slovak Research and Development Agency under contracts LPP-0126-07 and APVV-51062205, and by the Slovak Grant Agency VEGA (2/0148/09 and 2/0014/10). The publication has been created within realization of the project Centre of Excellence for Parasitology (Code ITMS: 26220120022), based on the support of the Operational Programme “Research and Development” funded from the European Fund of Regional Development (rate 0.3).
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