© 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, wyckii (Siluriformes, ) 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 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, (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 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, majusculus; glass tail catfish, Bag. cies of the catfish Hemibagrus are large riverine obscurus; fluviatilis; Assamese batasio, Bat. ten- catfishes found ubiquitously in river drainages east gana; tiger striped catfish, Bat. tigrinus; yellow , from the –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 River basins), as flathead stinging catfish, ; striped in other parts of , they are an important catfish, M. atrifasciatus; hi fin Mystus, M. bocourti; source of 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 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 ̶ 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 () 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 0.865 1.567 0.035±0.001 0.552±0.019 Large Metacentric 6 0.665 0.855 1.520 0.034±0.001 0.563±0.017 Large Metacentric 7 0.658 0.822 1.480 0.033±0.001 0.555±0.016 Large Metacentric 8 0.620 0.758 1.378 0.030±0.001 0.550±0.011 Medium Metacentric 9 0.606 0.713 1.319 0.029±0.001 0.540±0.017 Medium Metacentric 10 0.547 0.665 1.211 0.027±0.002 0.549±0.018 Medium Metacentric 11 0.511 0.614 1.125 0.025±0.002 0.546±0.018 Medium Metacentric 12* 0.693 1.292 1.985 0.044±0.003 0.651±0.019 Large Submetacentric 13 0.664 1.156 1.820 0.040±0.002 0.635±0.018 Large Submetacentric 14 0.624 1.111 1.734 0.038±0.001 0.640±0.024 Large Submetacentric 15 0.602 1.058 1.660 0.037±0.001 0.638±0.018 Large Submetacentric 16 0.588 0.989 1.577 0.035±0.001 0.627±0.016 Large Submetacentric 17 0.573 0.929 1.502 0.033±0.002 0.619±0.014 Large Submetacentric 18 0.528 0.909 1.437 0.032±0.002 0.632±0.023 Large Submetacentric 19 0.487 0.861 1.348 0.030±0.002 0.639±0.022 Medium Submetacentric 20 0.453 0.799 1.251 0.028±0.001 0.638±0.027 Medium Submetacentric 21 0.528 1.406 1.934 0.043±0.003 0.727±0.023 Large Acrocentric 22 0.484 1.269 1.753 0.039±0.002 0.724±0.018 Large Acrocentric 23 0.473 1.151 1.623 0.036±0.002 0.709±0.013 Large Acrocentric 24 0.441 1.070 1.510 0.033±0.001 0.708±0.008 Large Acrocentric 25 0.000 1.397 1.397 0.031±0.001 1.000±0.000 Medium Telocentric 26 0.000 1.288 1.288 0.028±0.002 1.000±0.000 Medium Telocentric 27 0.000 1.123 1.123 0.025±0.002 1.000±0.000 Medium Telocentric 28 0.000 0.955 0.955 0.021±0.002 1.000±0.000 Small Telocentric 29 0.000 0.865 0.865 0.019±0.002 1.000±0.000 Small Telocentric 30 0.000 0.807 0.807 0.018±0.002 1.000±0.000 Small Telocentric 31 0.000 0.728 0.728 0.016±0.002 1.000±0.000 Small Telocentric

Remarks: *=NOR-bearing chromosome and Chro.=Chromosome. banding in this Hemibagrus has not been reported. In Results and discussion the present article, our goal is to indicate the first finding of NOR polymorphism and chromosome analysis in the Chromosome number, fundamental number and karyo- H. wyckii from Thailand, using conventional staining type of H. wyckii and Ag-NOR banding techniques. Cytogenetic studies have been performed on 10 speci- mens of H. wyckii from Thailand. The results revealed Materials and methods that the diploid chromosome number of H. wyckii was 2n=62, in both sexes (Figs. 2, 3). This is in accordance Five males and five females of H. wyckii were ob- with the previous studies of Donsakul (2001) and Supi- tained from the Mekong River, Nong Khai Province, wong et al. (2014a, 2014b). However, it is different from Northeast of Thailand and the Chao Phraya River, Sing former report of Magtoon and Arai (1988) who found Buri Province, Central part of Thailand. The fish were the 2n of H. wyckii as 54 chromosomes. Comparative transferred to laboratory aquaria and were kept under cytogenetic data among species of the genus Hemi- standard condition for seven days prior to the experi- is presented in the Table 1. The present result of ments. Procedures for fish chromosome were prepared 2n is also different from other species (Barat and Khu- by directly prepared from kidney cells modifying from da-Bukhsh 1986, Sharma and Tripathi 1986, Yu et al. Supiwong et al. (2012a). The chromosome preparations 1989, Lakra and Rishi 1991, Khuda-Bukhsh et al. 1995, were stained with 10% Giemsa’s for 30 min and NORs Donsakul 2001, Das and Khuda-Bukhsh 2007a, Supi- were identified by Ag-NOR staining (Howell and Black wong et al. 2014a, 2014b). It is interesting that the 2n 1980). The metaphase figures were analyzed according intra-specific variations were also found in several spe- to the chromosome classification following by Turpin cies such as H. menoda (2n=48 and 2n=58), H. nemurus and Lejeune (1965) and Chaiyasut (1989). (2n=56 and 2n=58) and H. wyckii (2n=54 and 2n=62). These variations may be caused from chromosome rear- rangement (centric fusion and pericentric inversion) dur- 2017 A Discovery of Nucleolar Organizer Regions Polymorphism and Karyological Analysis of Crystal Eye Catfish, Hemibagrus wyckii 407

Fig. 4. Metaphase chromosome plates and karyotypes of male crystal eye catfish (Hemibagrus wyckii), 2n=62 by Ag-NOR banding technique. Arrows indicate the respective different heteromorphic NORs of chromosome pair No. 12a12b (A) and equal size homomorphic NORs of chromosome pair No. 12a12a (B). Scale bars indicate 5 µm. ing evolution in this genus between populations and/or are rare in bagrid catfishes. The karyotype of H. wyckii the misidentified specimens chromosome studied. composed of 14 large metacentric, 14 large submetacen- The NF or the number of chromosome arms of H. tric, 8 large acrocentric, 8 medium metacentric, 4 me- wyckii were 110 in both males and females. Although dium submetacentric, 6 medium telocentric, and 8 small our result presented the same 2n as in some previous telocentric chromosomes (Table 2). The present result is studies, the NF are different. Donsakul (2001) reported disagreement from Donsakul (2001) and Supiwong et al. that H. wyckii had NF=106 whereas Supiwong et al. (2014a, 2014b). These differences may be due to from (2014a) reported NF=102. These differences may be the differences in chromosome classification and/or the due to different criteria used for the chromosome clas- variation within species, since they represent different sification and inter-population variation in this family. populations. The karyotype formulae for eight analyzed The NF in the genus Hemibagrus vary from 72 to 110. species of Hemibagrus, show the variation found in the Ghigliotti et al. (2007) assumed that species with a larg- number of metacentric, submetacentric, acrocentric, and er NF are more advanced in evolutionary terms. Such telocentric chromosomes even inside the same genus. changes in chromosome arm number appear to be relat- ed to the occurrence of pericentric inversions, which are Chromosome markers of H. wyckii among the most common modifications contributing to Chromosomal studies on fishes have been providing karyotypic rearrangement in fishes and other vertebrates new information about karyotypical variability at inter (King 1993, Galetti Jr. et al. 2000, Wang et al. 2010). and intraspecific levels, which can be of great interest All specimens investigated have no cytologically dis- to phylogenetic, systematics and (Centofante tinguishable sex chromosome. This characteristic is sim- et al. 2002). In the present study, this is the first report ilar to other in the Bagridae family of Thailand. How- on Thai bagrid catfishes accomplished by Ag-NOR ever, some bagrid catfishes in India, including M. gulio banding technique. The objective of Ag-NOR band- and M. tengara have differentiated sex chromosome sys- ing technique is to check the nucleolar organizer re- tems as XX/XY and ZZ/ZW, respectively (Arai 2011). gion/NOR which is the representative location of genes Thus, it indicates that differentiated sex chromosomes (loci) that function in ribosome synthesis (18S and 28S 408 W. Supiwong et al. Cytologia 82(4)

Fig. 5. Metaphase chromosome plates and karyotypes of female crystal eye catfish (Hemibagrus wyckii), 2n=62 by Ag-NOR banding technique. Arrows indicate the respective different heteromorphic NORs of chromosome pair No. 12a12b (A) and equal size homomorphic NORs of chromosome pair No. 12a12a (B). Scale bars indicate 5 µm. ribosomal RNA) (Sharma et al. 2002). If these regions Intra specific NOR polymorphism between homolo- were active during the interphase prior to mitosis, they gous chromosome pair was also exhibited in one male can be detected by silver nitrate staining (Howell and and one female specimens of H. wyckii. Two specimens Black 1980) since it specifically stain a set of acidic had a single NOR-bearing chromosome of the chromo- protein related to ribosomal synthesis process; this tech- some pair No. 12 (12a12b), while four females and four nique actually reveals active NORs (Ag-NORs) and not males had two NOR-bearing chromosomes of the chro- the rDNA associated to NORs (Jordan 1987). mosome pair No. 12 with a homomorphism (12a12a) Analysis of chromosome from H. wyckii revealed the (Figs. 4, 5). This is in agreement with several previous positive Ag-NOR marks on a single pair of homologous reports on the finding in Moenkhausia sanctaefilomenae chromosomes. The interstitial NOR on the long arm in (Foresti et al. 1989), Aphanius fasciatus (Vitturi et al. a pair of submetacentric chromosome pair No. 22 was 1995), Leporinus friderici (Galetti Jr. et al. 1995), Salmo observed (Figs. 4, 5). These positive marks are in accor- trutta (Castro et al. 1996), Salvelinus alpines (Reed and dance with the secondary constriction. A single chromo- Phillips 1997), Chondrostoma lusitanicum (Rodrigues some pair of Ag-NOR marks, there is in agreement with and Collares-Pereira 1996, Collares-Pereira and Ráb H. menoda menoda (Barat and Khuda-Bukhsh 1986), M. 1999), Hoplias malabaricus (Born and Bertollo 2000), bocourti (Supiwong et al. 2013a), siamen- Oedalechilus labeo (Rossi et al. 2000), Astyanax sca- sis (Supiwong et al. 2013b), brachysoma, bripinnis (Mantovani et al. 2000, Marco-Ferro et al. Ho. Nigricollaris, M. cavasius, M. vittatus, 2001, Souza et al. 2001), A. altiparanae (Pacheco et al. ussuriensis, vachellii (Arai 2011), M. 2001, Mantovani et al. 2005), Bryconamericus aff. ex- gulio, rita (Das and Khuda-Bukhsh 2007b) and odon (Paintner-Marques et al. 2002), Apareiodon af- (Das and Khuda-Bukhsh 2007a). finis (Jorge and Filho 2004), Aphanius fasciatus (Vitturi Moreover, it is similar to several species in other fami- et al. 2005), Prochilodus lineatus (Gras et al. 2007), B. lies. However, it is different from fulvidraco aff. iheringii (Capistano et al. 2008), Puntioplites proc- which has two pairs of NOR-bearing chromosomes (re- tozysron (Supiwong et al. 2012b) and Lutjanus johnii viewed in Arai 2011). (Phimphan et al. 2013). Therefore, different karyotypes 2017 A Discovery of Nucleolar Organizer Regions Polymorphism and Karyological Analysis of Crystal Eye Catfish, Hemibagrus wyckii 409

Fig. 6. Idiogram showing lengths and shapes of chromosomes Fig. 7. Idiogram of the crystal eye catfish (Hemibagrus wyckii), of the crystal eye catfish (Hemibagrus wyckii), 2n=62 by 2n=62 by Ag-NOR banding technique. Arrow indicates conventional staining technique. Arrow indicates secondary nucleolar organizer region (NOR). constriction. are found even in small and isolated populations of pair in H. wyckii. Thus, H. wyckii is considered as the these species. The use of NORs in explaining kinships apomorphic characteristic for major ribosomal genes. depends on a large extent on the uniformity of this char- The important chromosome marker of the H. wyckii acteristic and on the degree of variety within a taxon is the asymmetrical karyotype that all four types of (Yüksel and Gaffaroğlu 2008). chromosomes (metacentric, submetacentric, acrocentric, All specimens investigated in the present study have and telocentric chromosomes) were found. An idiogram the interstitial NOR on long arm of the submetacentric demonstrates a continuous length gradation of chromo- chromosome pair. The interstitial NOR found in this somes. The largest and smallest chromosomes show size species is similar to P. siamensis populations from the difference (approximately three folds). The chromosome Songkham and Chao Phraya Basins but there is dif- marker of H. wyckii is chromosome pair No.1, which is ferent result from the Chi River population that they the largest metacentric chromosome and chromosome pose telomeric NOR (Supiwong et al. 2013b). Ag-NOR pair No. 31, is the smallest telocentric chromosome. signals coincide with localization of major ribosomal The data of the chromosomal checks on mitotic meta- genes by FISH which was described in several fish spe- phase cells of H. wyckii are showed in Tables 2. The cies (Almeida-Toledo et al. 2002, Hatanaka and Galetti idiograms from conventional staining and NOR banding 2004, Rosa et al. 2006, Wasko and Galetti 2000). Ac- techniques are showed in Figs. 6 and 7. The karyotype cording our result, the Ag-NOR signals were consistent formula for H. wyckii is as follows: with major ribosomal genes in H. wyckii (Supiwong m sm a m sm t t 2n (diploid)62=L14 +L 14 +L 8 +M 8 +M 4 +M 6 +S 8 et al. 2014a). In fish, the location of NORs in a terminal position and close to the centromeres is also pondered a primitive feature or the plesiomorphic condition for Acknowledgements major ribosomal genes (Vitturi et al. 1995). On the other hand, the Ag-NOR signals are located at interstitial posi- This paper was supported by Research Fund for tions on the long arm of a submetacentric chromosome DPST Graduate with First Placement, Development, 410 W. Supiwong et al. Cytologia 82(4)

Promotion of Science and Technology Talents project of marine fish cytogenetics. Hydrobiologia 420: 55–62. (DPST); National Science and Technology Development Galetti, P. M. Jr., Foresti, F., Bertollo, L. A. C. and Moreira-Filho, O. 1984. Characterization of eight species of Anostomidae (Cypri- Agency (NSTDA) and Toxic Substances in Livestock niformes) fish on the basis of the nucleolar organizing region. and Aquatic Animals Research Group, Khon Kaen Uni- Caryologia 37: 401–406. versity for financial support. Galetti, P. M. Jr., Mestriner, C. A., Monaco, P. J. and Rasch, E. M. 1995. Post-zygotic modifications and intra- and inter-individual References nucleolar organizing region variations in fish: report of a case involving Leporinus friderici. Chromosome Res. 3: 285–290. Ghigliotti, L., Mazzei, F., Ozouf-Costaz, C., Bonillo, C., Williams, R., Almeida-Toledo, L. F., Ozouf-Costaz, C., Foresti, F., Bonillo, C., Por- Cheng, C. H. C. and Pisano, E. 2007. The two giant sister species to-Foresti, F. and Daniel-Silva, M. F. Z. 2002. Conservation of of the Southern Ocean, Dissostichus eleginoides and Dissosti- the 5S-bearing chromosome pair and co-localization with major chus mawsoni, differ in karyotype and chromosomal pattern of rDNA clusters in five species of Astyanax (Pisces, Characidae). ribosomal RNA genes. Polar Biol. 30: 625–634. Cytogenet. Genome Res. 97: 229–233. Gras, D. E., Brassesco, M. S., Markariani, R., Roncati, H. A., Saka- Arai, R. 2011. Fish Karyotypes̶A Check List. Springer Japan, To- moto-Hojo, E. T., Fenocchio, A. S. and Pastori, N. C. 2007. Cyto- kyo. genetic polymorphism in Prochilodus lineatus (Pisces: Characi- Barat, A. and Khuda-Bukhsh, A. R. 1986. Karyomorphometrical formes) from the middle Paraná River, Santa Fe City, Argentina. studies in two species of fishes, Lepidocephalichthys guntea var. Comp. Cytogenet. 1: 113–119. balgara (fam.: Cobitidae) and Mystus corsula (fam.: Bagridae). Hatanaka, T. and Galetti, P. M. Jr. 2004. Mapping of the 18S and 5S In: Manna, G. K. and Sinha, U. (eds.). Perspectives in Cytology ribosomal RNA genes in the fish Prochilodus argenteus Agassiz, and Genetics. pp. 115–118. 1829 (Characiformes, Prochilodontidae). Genetica 122: 239–244. Born, G. G. and Bertollo, L. A. C. 2000. An XX/XY sex chromo- Hee, N. H. and Rainboth, W. J. 1999. The bagrid catfish genus Hemi- some system in a fish species, Hoplias malabaricus, with a bagrus (Teleostei: Siluriformes) in central Indochina with a new polymorphic NOR-bearing X chromosome. Chromosome Res. species from the Mekong river. Raffles Bull. Zool. 47: 555–576. 8: 111–118. Howell, W. M. and Black, D. A. 1980. Controlled silver-staining of Capistano, T. G., Portela Castro, A. L. B. and Julio-Junior, H. F. 2008. nucleolus organizer regions with a protective colloidal developer: Chromosome divergence and NOR polymorphism in Brycona- A 1-step method. Experientia 36: 1014–1015. mericus aff. iheringii (Teleostei, Characidae) in the hydrographic John, G., Barat, A. and Lakra, W. S. 1993. Localization of nucleolar systems of the Paranapanema and Ivaí rivers, Paraná, Brazil. organizer regions in Labeo (Cyprinidae). La Kromosomo II 70: Genet. Mol. Biol. 31(1 suppl.): 203–207. 2381–2384. Castro, J., Viñas, A., Sánchez, L. and Martínez, P. 1996. Character- Jordan, G. 1987. At the heart of the nucleolus. Nature 329: 489–490. ization of an atypical NOR site polymorphism in brown trout Jorge, L. C. and Filho, O. M. 2004. Nucleolar organizer regions as (Salmo trutta) with Ag- and CMA3-staining, and fluorescent in markers of chromosomal polymorphism in Apareiodon affinis situ hybridization. Cytogenet. Cell Genet. 75: 234–239. (Pisces, Parodontidae). Caryologia 57: 195–199. Centofante, L., Rebelo Porto, J. I. and Feldberg, E. 2002. Chromo- Khuda-Bukhsh, A. R., Rahman, A., Chanda, T., Nayak, K. and Khu- somal polymorphism in Serrasalmus spilopleura Kner, 1858 da-Bukhsh, A. 1995. Diploid numbers and chromosome formulae (Characidae, Serrasalminae) from central Amazon basin. Caryo- of some 29 species of Indian teleosts (Pisces). Chromosome In- logia 55: 37–45. form. Serv. 58: 38–39. Chaiyasut, K. 1989. Cytogenetics and Cytotaxonomy of the Family King, M. 1993. Species Evolution: The Role of Chromosomes Zephyranthes. Department of Botany, Faculty of Science, Chul- Change. Cambridge University Press, Cambridge. alongkorn University, Bangkok. Lakra, W. S. and Rishi, K. K. 1991. Chromosomes of Indian fishes: an Collares-Pereira, M. J. and Ráb, P. 1999. NOR polymorphism in the annotated list. Indian J. Anim. Sci. 61: 342–349. Iberian species Chondrostoma lusitanicum (Pisces: Cyprinidae)– Magtoon, W. and Arai, R. 1988. Karyotypes of bagrid catfishes, Mys- re-examination by FISH. Genetica 105: 301–303. tus wyckii and macracanthus, from Thailand. Bull Das, J. K. and Khuda-Bukhsh, A. R. 2007a. GC-rich heterochromatin Natl. Sci. Mus., Tokyo 14: 113–117. in silver stained nucleolar organizer regions (NORs) fluoresces Mantovani, M., dos Santos Able, L. D., Mestriner, C. A. and Moreira- with chromomycin A3 (CMA3) staining in three species of tele- Filho, O. 2000. Accentuated polymorphism of heterochromatin ostean fishes (Pisces). Indian J. Exp. Biol. 45: 413–418. and nucleolar organizer regions in Astyanax scabripinnis (Pis- Das, J. K. and Khuda-Bukhsh, A. R. 2007b. Preponderance of GC- ces, Characidae): Tools for understanding karyotypic evolution. rich sites in silver-stained nucleolus organizing regions of Rita Genetica 109: 161–168. rita (Hamilton) and (Hamilton) (Bagridae, Pisces), Mantovani, M., dos Santos Able, L. D. and Moreira-Filho, O. 2005. as revealed by chromomycin A3-staining technique and scanning Conserved 5S and variable 45S rDNA chromosomal localisation electron microscopic studies. Genet. Mol. Res. 6: 284–291. revealed by FISH in Astyanax scabripinnis (Pisces, Characidae). Donsakul, T. 2001. Chromosome study on four species of bagrid Genetica 123: 211–216. catfishes, Mystus nemurus, M. wyckii, M. wyckioides and M. sin- Marco-Ferro, D. A., Neo, D. M., Moreira-Filho, O. and Bertollo, L. A. garingan, from Thailand. In: Proceedings of the 39th Kasetsart C. 2001. Nucleolar organizer regions, 18S and 5S rDNA in Asty- University Annual Conference: Fisheries and Science. Kasetsart anax scabripinnis (Pisces, Characidae): populations distribution University, Bangkok. pp. 209–219. and functional diversity. Genetica 101: 55–62. Ferraris, C. J. 2007. Checklist of catfishes, recent and fossil (Osteich- Nelson, J. S. 2006. of the World, 4th ed. John Wiley and Sons, thyes: Siluriformes), and catalogue of Siluriform primary types. Inc., New York. Zootaxa 141: 1–628. Pacheco, R. B., Giuliano-Caetano, L. and Dias, A. L. 2001. Cytotypes Foresti, F., Almeida-Toledo, L. F. and Toledo, S. A. 1989. Supernu- and multiple NORs in an Astyanax altiparanae population (Pi- merary chromosome system, C-banding pattern characterization sces, Tetragonopterinae). Chromosome Sci. 5: 109–114. and multiple nucleolus organizer regions in Moenkhausia sanc- Paintner-Marques, T. R., Giuliano-Caetano, L. and Dias, A. L. 2002. taefilomenae (Pisces, Characidae). Genetica 79: 107–114. Multiple NORs in Bryconamericus aff. exodon (Osteichthyes, Galetti, P. M. Jr., Aguilar, C. T. and Molina, W. F. 2000. An overview 2017 A Discovery of Nucleolar Organizer Regions Polymorphism and Karyological Analysis of Crystal Eye Catfish, Hemibagrus wyckii 411

Characidae, Teuagonopterinae). Hereditas 137: 107–112. N., Pinthong, K., Tanee, T. and Tanomtong, A. 2014b. Chro- Phimphan, S., Tanomtong, A., Jumrusthanasan, S., Supiwong, W., mosomal evolution in naked catfishes (Bagridae, Siluriformes): Siripiyasing, P. and Sanoamuang, L. 2013. First report of NOR A comparative chromosome mapping study. Zool. Anz. 253: polymorphism and chromosome analysis of John’s snapper, Lut- 316–320. janus johnii (Perciformes, Lutjanidae) in Thailand. Cytologia 78: Supiwong, W., Tanomtong, A., Chaveerach, A., Tanee, T., Khakhong, 335–344. S. and Sanoamuang, L. 2013b. Interpopulational variation of Rainboth, W. J. 1996. Fishes of the Cambodian Mekong. Food and NOR positions and karyotypic analysis of Siamese catfish (Pseu- Agriculture Organization of the United Nations, Rome. domystus siamensis) in Thailand. Cytologia 78: 25–34. Reed, M. K. and Phillips, R. B. 1997. Polymorphism of the nucleolus Supiwong, W., Tanomtong, A., Jumrusthanasan, S., Khakhong, S., organizer region (NOR) on the putative sex chromosomes of Leelarasamee, K. and Sanoamuang, L. 2012a. A first karyotype Arctic char (Salvelinus alpines) is not sex related. Chromosome analysis and chromosomal characteristic of nucleolar organizer Res. 5: 221–227. regions (NORs) in the common sheathfish, Micronema apogon Rodrigues, E. M. and Collares-Pereira, M. J. 1996. NOR polymor- (Siluriformes, Siluridae) in Thailand. Cytologia 77: 53–58. phism in the Iberian species Chondrostoma lusitanicum (Pisces: Supiwong, W., Tanomtong, A., Supanuam, P., Jantarat, S., Khakhong, Cyprinidae). Genetica 98: 59–63. S. and Sanoamuang, S. 2012b. A discovery of nucleolar orga- Rosa, R., Bellafronte, E., Moreira-Filho, O. and Margarido, V. P. nizer regions (NORs) polymorphism and karyological analysis 2006. Constitutive heterochromatin, 5S and 18S rDNA genes in of Smith’s barb, Puntioplites proctozysron (Cypriniformes, Cy- Apareiodon sp. (Characiformes, Parodontidae) with a ZZ/ZW sex prinidae) in Thailand. Cytologia 77: 35–42. chromosome system. Genetica 128: 159–166. Turpin, R. and Lejeune, J. 1965. Les Chromosomes Humains. Gauth- Rossi, A. R., Gornung, E., Crosetti, D., Innocentti, S. and Sola, L. ier-pillars, Paris. 2000. Cytogenetic analysis of Oedalechilus labeo (Pisces: Mug- Vidthayanon, C. 2005. Handbook of Freshwater Fish. 2nd ed. Saraka- lidae), with a report of NOR variability. Mar. Biol. 136: 159–162. dee Press, Bangkok. Sharma, O. P. and Tripathi, N. K. 1986. Karyotypic diversity in genus Vitturi, R., Catalano, E., Colomba, M. S., Montagnino, L. and Pelleri- Mystus (Bagridae: Pisces). Cytologia 51: 1–9. to, L. 1995. Karyotype analysis of Aphanius fasciatus (Pisces: Sharma, O. P., Tripathi, N. K. and Sharma, K. K. 2002. A review of Cyprinodontiformes): Ag-NORs and C-band polymorphism in chromosome banding in fishes. In: Sobti, R. C., Obe, G. and Ath- four populations from Sicily. Biol. Zent. Bl. 114: 392–402. wal, R. S. (eds.). Some Aspects of Chromosome Structure and Vitturi, R., Colomba, M., Vizzini, S., Libertini, A., Barbieri, R. and Functions. Narosa Publishing House, New Delhi. Mazzola, A. 2005. Chromosomal location polymorphism of Souza, I. L., Galian, J., De La Rua, P., Bertollo, L. A. C. and Moreira- major rDNA sites in two Mediterranean populations of the kil- Filho, O. 2001. Non-random distribution of the GC-rich hetero- lifish Aphanius fasciatus (Pisces: Cyprinodontidae). Micron 36: chromatin and nucleolar rDNA sites on Astyanax scabripinnis 243–246. chromosomes. Cytologia 66: 85–91. Wang, S., Su, Y., Ding, S., Cai, Y. and Wang, J. 2010. Cytogenetic Supiwong, W., Liehr, T., Cioffi, M. B., Chaveerach, A., Kosyakova, analysis of orange-spotted grouper, Epinephelus coioides, using N., Fan, X., Tanee, T. and Tanomtong, A. 2014a. Comparative chromosome banding and fluorescence in situ hybridization. Hy- cytogenetic mapping of rRNA genes among naked catfishes: Im- drobiologia 638: 1–10. plications for genomic evolution in the Bagridae family. Genet. Wasko, A. P. and Galetti, P. M. Jr. 2000. Mapping 18S ribosomal Mol. Res. 13: 9533–9542. genes in fish of the genus Brycon (Characidae) by fluorescence in Supiwong, W., Liehr, T., Cioffi, M. B., Chaveerach, A., Kosyakova, situ hybridization (FISH). Genet. Mol. Biol. 23: 135–138. N., Pinthong, K., Tanee, T. and Tanomtong, A. 2013a. Karyotype Yu, X. J., Zhou, T., Li, Y. C., Li, K. and Zhou, M. 1989. Chromosomes and cytogenetic mapping of 9 classes of repetitive DNAs in the of Chinese Freshwater Fishes. Science Press, Beijing. genome of the naked catfish (Siluriformes, Yüksel, E. and Gaffaroğlu, M. 2008. The analysis of nucleolar orga- Bagridae). Mol. Cytogenet. 6: 51–57. nizer regions in Chalcalburnus mossulensis (Pisces: Cyprini- Supiwong, W., Liehr, T., Cioffi, M. B., Chaveerach, A., Kosyakova, dae). Journal of Fisheries Science 2: 587–591.