Parasitology International 61 (2012) 87–89
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Parasitology International j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p a r i n t
Comparative cytogenetics of opisthorchid species (Trematoda, Opisthorchiidae) Kira S. Zadesenets a,⁎, Alexei V. Katokhin a, b, Viatcheslav A. Mordvinov a, Nikolay B. Rubtsov a, b a b
Institute of Cytology and Genetics Siberian Branch of RAS, Novosibirsk, Russia Novosibirsk State University, Novosibirsk, Russia
a r t i c l e
i n f o
Available online 23 July 2011 Keywords: Opisthorchids Chromosome rearrangements Karyotype evolution Heterologous in situ hybridization C-banding Ag-NOR banding Comparative cytogenetics
a b s t r a c t In the present study karyotypes and chromosomes of five species of the family Opisthorchiidae (Opisthorchis felineus (Rivolta, 1884), O. viverrini (Poirier, 1886), Metorchis xanthosomus (Creplin, 1846), M. bilis (Braun, 1893), and Clonorchis sinensis (Cobbold, 1875)) were compared. Karyotypes of O. felineus, M. xanthosomus, M. bilis and C. sinensis consist of two pairs of large meta- and submetacentrics and five pairs of small chromosomes (2n = 14). The karyotype of O. viverrini is 2n= 12, which indicates a fusion of two chromosomes of opisthorchid ancestral karyotype. Analysis of mitotic and meiotic chromosomes was performed by heterologous in situ hybridization of microdissected DNA probes obtained from chromosomes 1 and 2 of O. felineus and chromosomes 1 and 2 of M. xanthosomus. Results of chromosome staining (C- and AgNOR-banding) and FISH of telomeric probes and ribosomal DNA probe on opisthorchid chromosomes were used for chromosome comparison. Data on chromosome number in opisthorchid species were also discussed. © 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The studied species which belong to the family Opisthorchiidae are characterized by a relatively constant number and morphology of chromosomes. The most typical number of chromosomes in the karyotype of opisthorchids is 2n = 14. Such karyotypes are described for Opisthorchis felineus and Metorchis xanthosomus, [1,2]. Other chromosome numbers have been revealed in O. viverrini (2n = 12) [3] and Clonorchis sinensis, 2n = 14 and 2n = 56 respectively [4,5]. Karyotypes of O. felineus and M. xanthosomus both consist of two pairs of large submetacentrics and five pairs of small chromosomes. They are probably similar to the ancestral karyotype of opisthorchids. The karyotype of O. viverrini includes two pairs of large-sized submetacentrics, one pair of medium-sized submetacentrics, one pair of small-sized submetacentrics or subtelocentrics, one pair of small-sized subtelocentrics or acrocentrics and one pair of small-sized acrocentric chromosomes [3]. The medium-sized submetacentric chromosome of O. viverrini is probably the result of fusion of two chromosomes of ancestral karyotype. The karyotype of C. sinensis with 2n = 56 was described for liver flukes collected from Korea and China by G.M. Park and his colleagues [5]. According to number and
Abbreviations: ITSs, interstitial telomere sequences; FISH, fluorescent in situ hybridization; WCPs, whole chromosome painting probes; BACs, bacterial artificial chromosomes. ⁎ Corresponding author. E-mail address:
[email protected] (K.S. Zadesenets). 1383-5769/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.parint.2011.07.006
morphology of chromosomes, its karyotype corresponded to the octaploid variant of typical opisthorchiid karyotype with 2n = 14. In the present study the comparative analysis of mitotic and meiotic chromosomes was performed by heterologous fluorescent in situ hybridization (FISH) of microdissected DNA-probes derived from chromosomes 1 and 2 of O. felineus and chromosomes 1 and 2 of M. xanthosomus. Results of chromosome staining with C- and AgNORbanding and FISH with telomeric probes and ribosomal DNA on opisthorchid chromosomes [6,7] were also used for comparison of opisthorchid chromosomes. The presence of six pairs of chromosomes in the karyotype of O. viverrini indicates a relatively recent chromosomal fusion event, which took place in the formation of the modern karyotype of O. viverrini (Fig. 1a,b). Recent chromosomal fusion event results generally in appearance of interstitial telomere sequences (ITSs) as a trace of occurred fusion [8]. However, none of the O. viverrini chromosomes have shown any ITSs after FISH with telomeric (TTAGGG)n DNA probe or (CCCTAA)3 PNA telomere probe [6]. It can be explained either by a complete loss of telomeric DNA sequences of ancestral chromosomes at the moment of chromosomal fusion or by the loss of the formed ITSs later during further chromosomal evolution that took place after chromosomal fusion event. Usually the loss of the ITSs takes a long time and its loss can be consider as indication to the fact that the chromosome fusion had happened a long time ago, even on the scale of eukaryote evolution. However, in species characterized with a small size of their genomes the process of the repeat elimination can proceed at a higher rate than it is typical for chromosome evolution in mammals. It is possible that elimination of repetitive DNA in the evolution of opisthorchids can also proceed faster than is typical for mammals. As a result, without additional
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Fig. 1. Mitotic (a) and meiotic (b) chromosomes of O. viverrini. Arrows indicate medium-sized submetacentrics (inverted DAPI staining); (c) two-color heterologous FISH of WCPo1 (green signal) and WCPo2 (red signal) on meiotic chromosomes of M. xanthosomus. Chromosomes counterstained with DAPI (blue signal).
information, the absence of the ITSs in medium-sized submetacentric could not allow us to determine the age of the chromosome rearrangement in O. viverrini. In the study of chromosome evolution heterologous, FISH of chromosome specific DNA-probes appeared to be a very efficient tool to reveal chromosome translocation in mammalian evolution [9]. We hoped that this approach could be very useful for revealing interchromosome reconstructions taking place in opisthorchid evolution. For heterologous FISH of whole chromosome painting probes (WCPs) of O. felineus (WCP1o and WCP2o) and M. xanthosomus (WCP1m, WCP2m) [7] were used on chromosomes of M. xanthosomus and O. felineus, M. bilis. These WCPs were produced by a technique specially adapted for generation of the paints useful for heterologous FISH [10]. Efficiency of this technique was successfully tested by heterologous FISH of obtained DNA probe on chromosomes of species that belong to different orders of mammals [10]. FISH with WCP1o, WCP2o, WCP1m, and WCP2m was efficient for chromosome specific painting of homologous chromosomes of original species [7]. However, heterologous FISH without suppression of repetitive DNA hybridization on chromosomes of O. felineus, M. xanthosomus, and M. bilis did not specifically paint homologous chromosomes (Fig. 1c). Strong FISH signals produced with interspersed DNA repeats masked the signal of chromosome-specific DNA sequences. The identification of homologous regions in chromosomes of different opisthorchid species appeared to be impossible without suppression of repetitive DNA hybridization. Probably, intensity of FISH signal produced with chromosome specific DNA sequences has decreased due to decreasing homology level in unique DNA sequences between different species. At the same time homology of interspersed DNA repeats remained high enough to produce strong signal in all chromosome regions to mask the chromosome specific signal. The solution to the problem of detailed comparative analysis of opisthorchid chromosomes can be determined by construction of BAC libraries of opisthorchid genomic DNA followed by in situ hybridization
of individual BAC clones on pachytene chromosomes. This approach will reveal both inter- and intra-chromosomal rearrangements that took place in the evolution of opisthorchid chromosomes. Detailed cytogenetic analysis in species with a similar size of genome has been carried out earlier [11]. It was based on polythene chromosome analysis. Detailed description of polythene chromosomes of about 100 chironomid species revealed higher frequency of paracentric inversions in comparison with interchromosomal translocations. They were registered mainly in the basic cytocomplex formation. The paracentric inversions do not lead to changes in chromosome size or morphology. Centromeric index also remains unchanged. For detection of paracentric inversion it is necessary to have a sufficient number of markers within chromosome arms. Neither morphometry of chromosomes, nor chromosome painting with WCPs can give any positive results. If the chromosome evolution of opisthorchids proceeded in a similar way, a few approaches will be able to discover their chromosome reorganization. Hybridization of BACs on chromosomes of different opisthorchid species will open the door to the detail comparison of their chromosomes and simplified the whole genome sequencing of these species. Previously, the karyotype of C. sinensis was described for samples collected from China and Korea. The chromosome number of 2n = 56 was recorded [5]. According to the number and morphology of chromosomes, the samples from China and Korea corresponded to the octaploid form of the specimen from Russia. Karyotyping of liver flukes collected from the Far East of Russia gave unexpected results. According to their morphology they were identified as C. sinensis. We are not aware of any morphological differences between the samples collected from the Russian Far East, China and Korea. However, karyotype of these samples consists of pairs of large meta- and submetacentrics and five pairs of small chromosomes. Chromosome number was 2n = 14. We also observed 7 chromosomes at the pachytene stage (Fig. 2). This is fully consistent with data obtained earlier by H. Cho [4]. The localization of rDNA and the clusters of telomeric repeats were typical for opisthorchid chromosomes. The
Fig. 2. Chromosomes of C. sinensis (2n = 14). (a) — chromosomes at the pachytene stage (n = 7); (b) — spreads of 8 meiotic cells are located close to each other; (c) — chromosomes of 8 cells have formed the common spread (inverted DAPI staining).
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question of the relationship between C. sinensis from Russia, China, and Korea remains open. The liver flukes described in China and Korea could be an octaploid form of normal C. sinensis or a distinct species. The formation of an octaploid form could be explained in relation to meiosis in opisthorchids. In opisthorchid gonads there are a great number of clusters consisting of 8 cells, exactly at the same stage of meiosis. On chromosome slides we often observed chromosome spreads of these 8 cells located close to each other or even forming the common spread (Fig. 2b,c). The cytokinesis disturbance in meiosis could lead to the formation of octaploid gametes, which could lead to the development of octaploid specimen in result of parthenogenesis. We have to mention that comparison of mitochondrial DNA of C. sinensis from Russia (NC_012147) [12] and mitochondrial DNA of C. sinensis from Vietnam (personal report of Prof. T.H. Le) revealed significant difference in the length of corresponding non-coding region. The level of this difference was more typical at the interspecies level. 2. Conclusion Comparison of chromosomes of five opistorchid species revealed chromosome rearrangement only in O. viverrini. Chromosome number in the samples of C. sinensis collected from the Russian Far East differed from the chromosome number published earlier for samples collected from China and Korea [5]. Heterologous FISH with chromosome paints showed high homology of interspersed repeats in chromosomes of studied species. Further detailed analysis of chromosome rearrangements in opistorchids requires FISH experiments with BACs derived from opistorchid genomes. This study is in progress. Acknowledgment The authors are grateful to Professor B. Sripa for granting of O. viverrini metacercariae, Professor V. Besprozvannykh for granting of C. sinensis
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metacercariae and Dr. N.I. Yurlova for granting of M. xanthosomus metacercariae. This study was supported by the program “Genomics, Proteomic, Bioinformatics” SB RAS, by the SB RAS expedition grants No. 9–2009, by the state contract Rosnauki02.512.11.2332, by the RFBR grant 0904-12209-ofi_m, and grant from OPTEC LLC. Microscopy was performed in the Microscopy Centre of Institute Cytology and Genetics SB RAS. References [1] Barsiene J. The karyotypes of trematodes. Vilnius: Academia; 1993. 370 pp. [2] Polyakov AV, Katokhin AV, Bocharova TA, Romanov KV, L'vova MN, Bonina OM, et al. Comparative analysis of karyotypes of Opisthorchis felineus from West Siberia. Contemp Probl Ecol 2010;3:1–3. [3] Komalamisra C. Chromosomes and C-banding of Opisthorchis viverrini. Southeast Asian J Trop Med Public Health 1999;30:576–9. [4] Cho H. Studies on the chromosomes of parasitic helminths (1). Chromosomes in meiocytes, spermiogenesis and fertilization observed by means of a squash method in Clonorchis sinensis (Trematoda: Opisthorchiidae). Jpn J Parasitol 1978;27:399–410. [5] Park GM, Im K, Huh S, Yong T-S. Chromosomes of the liver fluke, Clonorchis sinensis. Korean J Parasitol 2000;38:201–6. [6] Zadesenets KS, Katokhin AV, Mordvinov VA, Rubtsov NB. Telomeric DNA in chromosomes of five opisthorchid species. Parasitol Int 2011;61:81–3 (this issue). [7] Zadesenets KS, Karamysheva TV, Katokhin AV, Mordvinov VA, Rubtsov NB. Distribution of repetitive DNA sequences in chromosomes of five opisthorchid species (Trematoda, Opisthorchiidae). Parasitol Int 2011;61:87–9 (this issue). [8] Zhdanova NS, Rubtsov NB, Minina YM. Terminal regions of mammal chromosomes: plasticity and role in evolution. Russ J Genet 2007;43:721–32. [9] Ferguson-Smith MA, Trifonov V. Mammalian karyotype evolution. Nat Rev Genet 2007;8:950–62. [10] Rubtsov NB, Karamisheva TV, Astakhova NM, Liehr T, Claussen U, Zhdanova NS. Zoo-FISH with region-specific paints for mink chromosome 5q: delineation of inter- and intrachromosomal rearrangements in human, pig, and fox. Cytogenet Cell Genet 2000;90:268–70. [11] Kiknadze II, Istomina AG, Golygina VV, Rubtsov NB, Karamysheva TV. The structural peculiarities of karyotypes in species of Propsilocerus akamusi sibling group (Diptera: Chironomidae). Comp Cytogenet 2007;1:33–43. [12] Shekhovtsov SV, Katokhin AV, Kolchanov NA, Mordvinov VA. The complete mitochondrial genomes of the liver flukes Opisthorchis felineus and Clonorchis sinensis (Trematoda). Parasitol Int 2010;59:100–3.
