Bagrichthys Majusculus (Siluriformes, Bagridae) in Thailand
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© 2018 The Japan Mendel Society Cytologia 83(2): 193–199 Nucleolar Organizer Regions Polymorphism and Karyological Analysis of Black Lancer, Bagrichthys majusculus (Siluriformes, Bagridae) in Thailand Weerayuth Supiwong1*, Nuntaporn Getlakha2, Somkid Chaiphech3, Krit Pinthong4, Sumalee Phimphan5 and Alongklod Tanomtong5 1 Faculty of Applied Science and Engineering, Khon Kaen University, Nong Khai Campus, Muang, Nong Khai 43000, Thailand 2 Department of Biology, Faculty of Science and Technology, Muban Chombueng Rajabhat University, Chombueng, Ratchaburi 70150, Thailand 3 Department of Animal Science, Faculty of Agriculture, Rajamangala University of Techonlogy Srivijaya, Nakorn Sri Thammarat, Saiyai Campus, Thungsong, Nakhon Si Thammarat 80110, Thailand 4 Department of Fundamental Science, Faculty of Science and Technology, Surindra Rajabhat University, Muang, Surin 32000, Thailand 5 Toxic Substances in Livestock and Aquatic Animals Research Group, Department of Biology, Faculty of Science, Khon Kaen University, Muang, Khon Kaen 40002, Thailand Received December 21, 2017; accepted February 20, 2018 Summary Nucleolar organizer regions (NORs) and karyotype in the black lancer, Bagrichthys majusculus from Nakhon Phranom and Sing Buri provinces, Thailand, were investigated. The mitotic chromosome prepara- tions were directly obtained from kidney tissues of eight male and eight female specimens. Conventional and Ag-NOR staining techniques were applied to the chromosomes. The chromosomes number of B. majusculus was 2n=50 and the fundamental number (NF) of all specimens was 96. The karyotype consists of six large metacen- tric, six large submetacentric, two large acrocentric, eight medium metacentric, 12 medium submetacentric, two medium acrocentric, six small metacentric, four small submetacentric and four small telocentric chromosomes. Sex chromosome was not observed. Moreover, the interstitial NORs were clearly observed at the long arm of the metacentric chromosome pair 1. The result revealed that a heteromorphic NOR type was appeared in one male and one female fish and other fish have homozygous NOR in the pair 1 chromosomes. Key words Bagrichthys majusculus, Karyotype, NOR variation, Polymorphism, Chromosome number. Freshwater fishes are especially important as they of species and interrelationships within fish families provide a source of high quality protein as well as food (Campiranont 2003, Tanomtong 2011). The studies of source for people who live near a river basin. Fishes of the karyotypes help to investigate the aquatic structure the Bagridae family belong to the order Siluriformes and of the species population in each habitat, so it can de- are of great importance as food fish. While, some spe- termine what species are related to each other in an ac- cies are kept as aquarium fishes, many bagrids have re- curate manner. This may help to facilitate the hybridiza- cently become a booming in aquaculture (Nelson 2006). tion between them in the future for strain improvement There are six genera and approximately 25 species in the (Sofy et al. 2008), breeding practices of organisms by Bagridae family in Thailand (Rainboth 1996, Vidthaya- using chromosome set management such as polyploidy non 2005, Ferraris 2007). B. majusculus is one species inducing in catfishes, Clarias batrachus (Na-Nakorn of the family considering as the Thai economic species et al. 1980) and rainbow trout (Oncorhynchus mykiss), used for foods and in an aquarium (Vidthayanon 2005). brood stock selection of giant catfish (Mengampan et al. The karyotype is the chromosome complement of an 2004). Cytogenetic knowledge can also provide increas- individual or related group of individuals, as defined by ing useful data for future studies on chromosome evolu- chromosome size, morphology and number. Though for tion. The reports of chromosome evolution in the family all somatic cells of all individuals of species, the number Channidae (snackhead fish) indicated that centric fusion, of chromosomes is used as an indicator of classification pericentric inversion and polyploidy are the processes which have important roles on chromosome rearrange- * Corresponding author, e-mail: [email protected] ment in the snackhead fish’s phylogeny (Supiwong et al. DOI: 10.1508/cytologia.83.193 2009, Tanomtong et al. 2014). 194 W. Supiwong et al. Cytologia 83(2) Chromosome number and karyotype are often used as essential data for the taxonomy of those animals with troubles on morphological classification aspect (Giessmann 2002). In fishes, chromosome banding data was used to support for classification in the Cyprinidae (Amemiya and Gold 1988, Gold and Li 1994, Boron 2001, Bellafronte et al. 2005). Up to the present, 46 species of the Bagridae have been cytogenetically studied. The chromosome number ranging from 2n=44 in Coreobagrus brevicorpus (Kim et al. 1982) to 2n=80 in Batasio havmolleri (Magtoon and Donsakul 2009) have been reported. However, the predominant diploid numbers represent 2n=52 and 2n=56 (Arai 2011). In the present study, chromosome of the B. majusculus from Thailand was firstly investigated using conventional staining and Ag-NOR banding tech- niques. Materials and methods The specimens of B. majusculus were collected from the Mekong river, Nakhon Phranom province, northeast of Thailand (five males and five females) and the Chao Phraya River, Sing Buri province, central part of Thai- land (three males and three females). The sampling sites and the characteristics of B. majusculus are shown in Fig. 1. The fishes were transferred to laboratory aquaria and were kept under standard conditions for seven days prior to the experiments. Procedure of chromosome preparation was following modified protocol of Supi- wong et al. (2009). The 0.05% colchicine (1 mL to 100 g body weight) was injected to abdominal cavity and left for 45 min to 1 h. Chromosomes were prepared from kid- ney cells by squash technique. Kidney tissues were cut Fig. 1. Thailand map showing the sampling sites (A) and the gen- into small pieces, then mixed with hypotonic solution eral characteristic of B. majusculus (B). (0.075 M KCl) and incubated for 30 min. Cells were fixed in fresh cool fixative (3 absolute methanol: 1 glacial ace- et al.1989, Lakra and Rishi 1991, Khuda-Bukhsh et al. tic acid). The prepared chromosomes were stained with 1995, Donsakul 2001, Das and Khuda-Bukhsh 2007a, 10% Giemsa for 30 min, or 50% AgNO3 and 2% gela- Supiwong et al.2013a, 2014a, b). The NF of B. majus- tin for Ag-NOR staining technique (Howell and Black culus was 96 in both males and females. Although our 1980). The metaphase figures were analyzed according result presented the same chromosome number as in to the chromosome classification following by Turpin previous studies, the NF is different (Donsakul 2002, and Lejeune (1965) and Chaiyasut (1989). Magtoon and Arai 1988). Donsakul (2002) reported that B. majusculus showes NF=92 whereas Magtoon and Results and discussion Arai (1988) reported NF=94. These differences may be due to different criteria used for the chromosome clas- Chromosome number, fundamental number and karyo- sification and/or inter-populational variation in this spe- type of B. majusculus cies. The NF in the family Bagridae varies from 72 to Cytogenetic studies have been performed on sixteen 128 (Arai 2011). Comparisons among the genera in the specimens of B. majusculus from Thailand. The results Bagridae revealed that the Hemibagrus showes higher revealed that the diploid chromosome number was NF variation than others. It varies from 72 to 114. Ghi- 2n=50, in both males and females (Figs. 2, 3). This is gliotti et al. (2007) assumed that species with a larger accordance with the previous studies of Magtoon and NF are more advanced in evolutionary terms. Such Arai (1988) and Donsakul (2002). However, the 2n is changes in NF appear to be related to the occurrence of different from other species of the Bagridae (Barat and pericentric inversions, which are among the most com- Khuda-Bukhsh 1986, Sharma and Tripathi 1986, Yu mon modifications contributing to karyotypic rearrange- 2018 NOR Polymorphism and Karyological Analysis of Bagrichthys majusculus in Thailand 195 Fig. 2. Karyotypes of male (A) and female (B) of B. majusculus (2n=50) by conventional straining technique. Arrows indicate secondary constriction. Scale bars=5 µm. Table 1. Mean length (µm) of short chromosome arm (Ls), mean length of long chromosome arm (Ll), total length of chromosome arms (LT), relative length (RL), centromeric index (CI) and standard deviation (S.D.) of RL and CI from metaphase chromosomes of 20 cells in B. majusculus (2n=50). Chromosome pair Ls Ll LT RL±S.D. CI±S.D. Type Size 1* 47.59 55.45 103.04 0.0294±0.0022 0.539±0.027 m L 2 39.39 51.05 90.44 0.0257±0.0024 0.566±0.032 m L 3 34.80 44.59 79.38 0.0225±0.0019 0.560±0.030 m L 4 32.03 39.65 71.68 0.0205±0.0009 0.553±0.030 m M 5 30.14 37.32 67.47 0.0192±0.0011 0.554±0.029 m M 6 29.41 34.85 64.26 0.0183±0.0009 0.544±0.028 m M 7 27.96 33.21 61.17 0.0174±0.0008 0.542±0.026 m M 8 24.90 31.37 56.28 0.0161±0.0012 0.559±0.029 m S 9 23.15 29.10 52.25 0.0150±0.0012 0.557±0.024 m S 10 20.64 27.83 48.46 0.0138±0.0011 0.575±0.022 m S 11 36.98 76.48 113.46 0.0323±0.0017 0.673±0.058 sm L 12 33.98 62.14 96.12 0.0275±0.0020 0.647±0.029 sm L 13 27.09 57.08 84.17 0.0239±0.0013 0.679±0.022 sm L 14 26.92 50.79 77.71 0.0221±0.0013 0.654±0.028 sm M 15 26.12 47.73 73.85 0.0210±0.0009 0.647±0.020 sm M 16 24.09 46.49 70.59 0.0201±0.0007 0.660±0.035 sm M 17 24.41 41.84 66.25 0.0189±0.0011 0.626±0.030 sm M 18 22.66 40.96 63.62 0.0181±0.0006 0.651±0.028 sm M 19 21.94 38.82 60.76 0.0173±0.0007 0.638±0.030 sm M 20 20.61 36.09 56.70 0.0160±0.0012 0.637±0.032 sm S 21 17.02 35.00 52.02 0.0147±0.0015 0.674±0.042 sm S 22 22.94 57.59 80.53 0.0230±0.0015 0.714±0.026 a L 23 18.62 49.27 67.89 0.0193±0.0012 0.728±0.033 a M 24 0.00 55.60 55.60 0.0159±0.0016 1.000±0.000 t S 25 0.00 42.83 42.83 0.0122±0.0012 1.000±0.000 t S *NOR-bearing chromosome, m=metacentric, sm=submetacentric, a=acrocentric, t=telocentric, L=large, M=medium, S=small.