© 2017 The Japan Mendel Society Cytologia 82(4): 403–411 A Discovery of Nucleolar Organizer Regions (NORs) Polymorphism and Karyological Analysis of Crystal Eye Catfish, Hemibagrus wyckii (Siluriformes, Bagridae) in Thailand Weerayuth Supiwong1*, Pasakorn Saenjundaeng1, Nuntiya Maneechot2, Supatcha Chooseangjaew3, Krit Pinthong4 and Alongklod Tanomtong4 1 Faculty of Applied Science and Engineering, Khon Kaen University, Nong Khai Campus, Muang, Nong Khai 43000, Thailand 2 Department of Fundamental Science, Faculty of Science and Technology, Surindra Rajabhat University, Muang, Surin 32000, Thailand 3 Marine Shellfish Breeding Research Unit, Department of Marine Science, Faculty of Science and Fisheries Tech- nology, Rajamangala University of Technology Srivijaya, Trang Campus, Trang 92150, Thailand 4 Toxic Substances in Livestock and Aquatic Animals Research Group, Department of Biology, Faculty of Science, Khon Kaen University, Muang, Khon Kaen, 40002, Thailand Received April 25, 2016; accepted July 10, 2017 Summary A discovery of nucleolar organizer regions (NORs) polymorphism and karyological analysis in the crystal eye catfish, Hemibagrus wyckii (Bleeker, 1858) from Nong Khai and Sing Buri Provinces, Thailand, were investigated. The mitotic chromosome preparation was prepared by directly from kidney cells of five male and five female specimens. Conventional and Ag-NOR staining techniques were applied to stain the chromosomes. The results shown that the diploid chromosome number of H. wyckii was 2n=62 and the fundamental numbers (NF) of both sexes were 110. The karyotype consists of 14 large metacentric, 14 large submetacentric, 8 large acrocentric, 8 medium metacentric, 4 medium submetacentric, 6 medium telocentric, and 8 small telocentric chromosomes. No strange size chromosomes related to sex were observed. In addition, the interstitial nucleolar organizer regions (NORs) were clearly observed at the long arm of the chromosome pair No. 12. This is the first report on polymorphism of NORs in H. wyckii. The result revealed that a heteromorphic of one male and one female had a single NOR-bearing chromosome of chromosome pair No. 12 (12a12b), while four males and four females had two NOR-bearing chromosomes of the chromosome pair No. 12 with a homomorphic (12a12a). The karyotype formula for H. wyckii is as follows: m sm a m sm t t 2n (diploid) 62 = L14 +L 14 +L 8 +M 8 +M 4 +M 6 +S 8 Key words Hemibagrus wyckii, Karyotype, Polymorphism, NORs. The family Bagridae is a member of class Actinop- thayanon 2005). Therefore, Hemibagrus catfish play an terygii, order Siluriformes, and superfamily Bagroidea. important role in national economy. Catfish are found in both Asia and Africa; containing There are six genera and approximately 25 species over 200 species and found over 25 species in Thailand in the Bagridae family in Thailand including the black (Nelson 2006, Ferraris 2007, Vidthayanon 2005). Spe- lancer, Bagrichthys majusculus; glass tail catfish, Bag. cies of the catfish genus Hemibagrus are large riverine obscurus; Batasio fluviatilis; Assamese batasio, Bat. ten- catfishes found ubiquitously in river drainages east gana; tiger striped catfish, Bat. tigrinus; yellow Mystus, from the Ganges–Brahmaputra basin and south from Hemibagrus filamentus; Irrawaddy Mystus, H. microph- the Yangtze basin. Throughout central Indochina (here thalmus; yellow Mystus, H. spiloprus; crystal eyed cat- defined as Indochina consisting of the Mae Klong, Chao fish, H. wyckii; Asian red tailed catfish, H. wyckioides; Phraya, Bang Pakong, and Mekong River basins), as flathead stinging catfish, Mystus albolineatus; striped in other parts of Southeast Asia, they are an important catfish, M. atrifasciatus; hi fin Mystus, M. bocourti; source of animal protein for people (Hee and Rainboth pearl catfish, M. castaneus; Gangetic Mystus, M. cava- 1999). A crystal eyed catfish, Hemibagrus wyckii (Fig. sius; long Whiskers catfish, M. gulio; striped catfish, M. 1) is a rare species and expensive cost in Thailand (Vid- multiradiatus; striped catfish, M. mysticetus; M. rhegma; M. singaringan; M. wolffii; Bumblebee catfish, Pseudo- * Corresponding author, e-mail: [email protected] mystus bomboides; Siamese catfish, P. siamensis; false DOI: 10.1508/cytologia.82.403 Bumblebee catfish, P. stenomus and Sperata acicularis 404 W. Supiwong et al. Cytologia 82(4) Fig. 1. General characteristic of the crystal eye catfish (Hemibagrus wyckii), scale bar indicates 5 cm. Fig. 2. Metaphase chromosome plates and karyotypes of male crystal eye catfish (Hemibagrus wyckii), 2n=62, by conventional straining technique. Arrows indicate the respective different heteromorphic secondary constriction of chromosome pair 12a12b (A) and equal size homomorphic secondary constriction of chromosome pair No. 12a12a (B). Scale bars indicate 5 µm. (Ferraris 2007, Rainboth 1996, Vidthayanon 2005). on the chromosome, size, and active numbers in each Cytogenetic studies in Thai freshwater fishes are quite genome. The previous NOR studies showed variations scarce, in which only conventional technique reported to between species, within species, and even between in- determine chromosome number and karyotype composi- dividuals (Galetti Jr. et al. 1984, John et al. 1993, Castro tion has been carried out. Structure, number, and mor- et al. 1996). NORs on different homologous chromo- phology of a nucleolar organizer region (NOR) may be somes may have different sizes or number. It has been specific to populations, species and subspecies. NOR is reported that this extent of variety between NORs may frequently used to compare variations, as well as to iden- be attributed to number of cistrons and differences in tify and explain specifications. Changes in chromosome transcriptional activity (Galetti Jr. et al. 1984). number and structure can alter the number and structure Up to the present, only eight species of the genus of NOR. Robertsonian translocations may cause losses Hemibagrus have been cytogenetically analyzed pre- of NOR (Yüksel and Gaffaroğlu 2008). NORs can be the senting the diploid chromosome number (2n) ranging perfect markers to display wide chromosomal polymor- from 48 chromosomes in H. menoda (Lakra and Rishi phism within and between species in many fish groups. 1991) to 62 chromosomes in H. wyckii (Donsakul 2001, This variety may affect NOR number, its localization Supiwong et al. 2014a, 2014b). Moreover, the 2n intra- 2017 A Discovery of Nucleolar Organizer Regions Polymorphism and Karyological Analysis of Crystal Eye Catfish, Hemibagrus wyckii 405 Fig. 3. Metaphase chromosome plates and karyotypes of female crystal eye catfish (Hemibagrus wyckii), 2n=62, by convention- al straining technique. Arrows indicate the respective different heteromorphic secondary constriction of chromosome pair No. 12a12b (A) and equal size homomorphic secondary constriction of chromosome pair No. 12a12a (B). Scale bars indicate 5 µm. Table 1. Review of fishes cytogenetic reports in the genus Hemibagrus (Bagridae). Species 2n NF Karyotype Ag-NOR Locality Reference Hemibagrus guttatus 60 108 20m+12sm+16st+12a ̶ China Yu et al. 1989 H. filamentus 58 104 20m+26sm+12st/a ̶ Thailand Supiwong et al. 2014a, 2014b H. macropterus 60 108 20m+12sm+16st+12a ̶ China Yu et al. 1989 H. menoda 48 72 12m+12st+24a ̶ India Lakra and Rishi 1991 58 100 22m+20sm+16a ̶ India Das and Khuda-Bukhsh 2007a H. menoda menoda 56 108 36m+16sm+4a 2 India Khuda-Bukhsh et al. 1995 H. nemurus 58 100 22m+20sm+16a ̶ India Barat and Khuda-Bukhsh 1986 56 100 20m+14sm+10st+12a ̶ India Sharma and Tripathi 1986 56 102 32m+14sm+10a ̶ India Khuda-Bukhsh et al. 1995 H. spilopterus 58 94 28m+8sm+20st+2a ̶ Thailand Donsakul 2001 58 102 18m+26sm+14st/a ̶ Thailand Supiwong et al. 2014a, 2014b H. wyckii 54 84 16m+14sm+24a ̶ Thailand Magtoon and Arai 1988 62 106 34m+10sm+8st+10a ̶ Thailand Donsakul 2001 62 102 14m+26sm+22st/a ̶ Thailand Supiwong et al. 2014a, 2014b 62 110 22m+18sm+8a+14t 2 (12a, b) Thailand Present study H. wyckioides 58 92 24m+10sm+6st+18a ̶ Thailand Donsakul 2001 58 100 22m+20sm+16st/a ̶ Thailand Supiwong et al. 2014a, 2014b Remarks: 2n=diploid chromosome number, NF=fundamental number (number of chromosome arm), m=metacentric, sm=submetacentric, st=subtelocentric, a=acrocentric, t=telocentric chromosome, NOR=nucleolar organizer region, and ̶=not available. specific variations were also found in several species (Donsakul 2001, Supiwong et al. 2014a, 2014b). Al- such as H. menoda, 2n=48 (Lakra and Rishi 1991), though there are few reports on fluorescence in situ 2n=58 (Das and Khuda-Bukhsh 2007a); H. nemurus, hybridization (FISH) technique using ribosomal RNA 2n=56 (Sharma and Tripathi 1986Khuda-Bukhsh et al. genes, some microsatellites and repetitive DNA se- 1995), 2n=58 (Barat and Khuda-Bukhsh 1986) and H. quences mapping to the Bagridae fish chromosomes wyckii, 2n=54 (Magtoon and Arai 1988), and 2n=62 (Supiwong et al. 2013a, 2014a, 2014b), the Ag-NOR 406 W. Supiwong et al. Cytologia 82(4) Table 2. Mean of length short arm chromosome (Ls); length long arm chromosome (Ll); length total arm chromosome (LT); relative length (RL); centromeric index (CI), and standard deviation (SD) of RL and CI from metaphase chromosome of 20 cells in crystal eye catfish (Hemibagrus wyckii), 2n=62. Chro. pair Ls Ll LT RL±SD CI±SD Chro. size Chro. type 1 0.926 1.162 2.087 0.046±0.003 0.557±0.017 Large Metacentric 2 0.870 1.055 1.925 0.043±0.003 0.548±0.012 Large Metacentric 3 0.784 0.916 1.700 0.038±0.002 0.539±0.015 Large Metacentric 4 0.737 0.868 1.605 0.035±0.001 0.541±0.014 Large Metacentric 5 0.702