Osteoglossiformes, Notopteridae) by Conventional and Ag-NOR Staining Techniques
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© 2015 The Japan Mendel Society Cytologia 80(2): 159–166 Chromosomal Characteristics of the Royal Knifefish, Chitala blanci (Osteoglossiformes, Notopteridae) by Conventional and Ag-NOR Staining Techniques Nuntiya Maneechot1, Weerayuth Supiwong2, Sarun Jumrusthanasan1, Pornnarong Siripiyasing3, Krit Pinthong4 and Alongklod Tanomtong1* 1 Department of Biology, Faculty of Science, Khon Kaen University, Muang, Khon Kaen 40002, Thailand 2 Faculty of Applied Science and Engineering, Khon Kaen University, Nong Khai Campus, Muang, Nong Khai 43000, Thailand 3 Major of Biology, Faculty of Science and Technology, Mahasarakham Rajabhat University, Muang, Mahasarakham 44000, Thailand 4 Biology Program, Faculty of Science and Technology, Surindra Rajabhat University, Muang, Surin 32000, Thailand Received April 22, 2014; accepted November 30, 2014 Summary The first chromosomal characteristic of nucleolar organizer regions/NORs and karyological analysis of the royal knifefish (Chitala blanci) from Mekong River, northeast Thailand, were studied. Kidney cell samples were taken from one male and one female fish. The mitotic chromosome preparations were done directly from kidney cells. Conventional and Ag-NOR staining techniques were applied to stain the chromosomes. The results showed that the diploid chromosome number of C. blanci was 2n=42, the fundamental numbers (NF) were 42 in both male and female. The types of chromosomes were 12 large telocentric and 30 medium telocentric chromosomes. The Ag-NOR banding indicated that a single pair of NORs was observed on the long arm centromeric region of medium telocentric chromosome pair 10. The karyotype formula could be deduced as: t t 2n (diploid) 42=L12+M30 Key words Chitala blanci, Karyotype, Chromosome, Nucleolar organizer region. The family Notopteridae (featherbacks or knifefishes) is a member of class Actinopterygii and order Osteoglossiformes. They distribute in the environments of Africa, South Asia, and Southeast Asia. There are 10 species in four genera including: Chitala (C. blanci, C. Borneensis, C. chitala, C. hypselonotus, C. lopis and C. ornata); Notopterus (N. notopterus); Papyrocranus (P. afer and P. congoensis) and Xenomystus (X. nigri) (Vidthayanon 2005). There are four species in two genera found in Thailand. The knifefishes are members of a small family of elongated freshwater fishes confined to tropical Africa and Southeast Asia. It is the largest member of the family occasionally attaining a length of three feet. They are easily identified by their exceptionally long fin. The fin starts near the head or just under the pectoral fins and extends all the way to the tail, where it joins with the caudal fin, which is not separate or identifiable from it. The feathery fin moves with a rippling motion to propel the fish through the water. The knifefishes can move backward or forward with gliding grace. The scales are small. The mouth is large and contains numerous teeth. When young, it is brownish, mottled, or reticulated with black; as it grows older these markings disappear, and the entire fish becomes darker (Edward and Fichter 1989). * Corresponding author, e-mail: [email protected] DOI: 10.1508/cytologia.80.159 160 N. Maneechot et al. Cytologia 80(2) Fig. 1. General characteristics of the royal knifefish, Chitala blanci (DʼAubenton, 1965) from Mekong River, Thailand. The royal knifefish (C. blanci) is endemic to the Mekong basin, but it is less common than the clown knifefish. It is only found from northern Cambodia to Thailand and the Lao PDR. It is distinguishable by the presence of numerous small black spots (in the anterior half of the body), merging posteriorly into irregular oblique bands extending on the anal and caudal fins. It differs from all other species except C. lopis in having a large round black spot at the base of the pectoral fin. In C. blanci the pectoral basal spot present in juveniles is as small as 6 cm and retained throughout life (Roberts 1992) (Fig. 1). Only six species of the family Notopteridae have been studied cytogenetically, each having the same diploid chromosome number (except Papyrocranus afer): C. blanci, 2n=42 (NF=42); C. chitala, 2n=42 (NF=42); C. ornata, 2n=42 (NF=44); N. notopterus, 2n=42 (NF=42); P. afer, 2n=34 (NF=38) and X. nigri, 2n=42 (NF=42) (Uyeno 1973, Rishi and Singh 1983, Khuda-Bukhsh and Barat 1987, Donsakul and Magtoon 1990, Rishi and Thind 1994, Takai and Ojima 1998, Silawong et al. 2012, Supiwong et al. 2012). The current study is the first report on chromosomal characteristics of nucleolar organizer regions (NORs) in C. blanci using the Ag-NOR staining technique. Our results enhance cytogenetic information and enable future comprehensive studies to be conducted on taxonomy and evolutionary relationships. Materials and methods Sample collection The C. blanci (one male and one female fish) were obtained from Mekong River, Nongkhai province, Northeast of Thailand. The fish were transferred to laboratory aquaria and were kept under standard conditions for 7 d before the experimentations. Chromosome preparation Chromosomes were prepared in vivo (Chen and Ehbeling 1968, Nanda et al. 1995, Supiwong et al. 2009) by injecting phytohemagglutinin (PHA) solution into the abdominal cavity of fish. After 24 h, colchicine was injected to fishʼs intramuscular and/or its abdominal cavity and left for 2 to 4 h. Kidneys were cut into small pieces then mixed with 0.075 M KCl. After discarding all large cell pieces, 15 ml of cell sediments were transferred to centrifuge tube and incubated for 25–35 min. KCl was discarded from the supernatant after subsequent centrifugation at 1,200 rpm for 8 min. Cells were fixed in fresh cool fixative (3 methanol : 1 glacial acetic acid) gradually added up to 8 ml before centrifuging again at 1,200 rpm for 8 min, whereupon the supernatant was discarded. Fixation was repeated until the supernatant was clear and the pellet was mixed with 1 ml fixative. The mixture was dropped onto a clean and cold slide by micropipette followed by the air-dry technique. Chromosome staining Conventional staining by 20% Giemsaʼs solution for 30 min and Ag-NOR banding were 2015 Chromosomal Characteristics of the Royal Knifefish 161 conducted (Howell and Black 1980) by adding two drops of 50% silver nitrate and 2% gelatin on slides, respectively. The slides were then sealed with cover glasses and incubated at 60°C for 5 min. After that, the slides were soaked in distilled water until the cover glasses were separated. The slide was stained with 20% Giemsaʼs solution for 1 min. Chromosome checks Chromosome counting was performed on mitotic metaphase cells under a light microscope. Twenty clearly observable and well-spread chromosomes of each male and female were selected and photographed. The length of short arm chromosome (Ls) and the length of long arm chromosome (Ll) were measured and calculated to the length of total arm chromosome (LT, LT=Ls+Ll). The relative length (RL), the centromeric index (CI) and standard deviation (SD) of RL and CI were also calculated (Chaiyasut 1989). The CI (q/p+q) between 0.50–0.59, 0.60–0.69, 0.70–0.89 and 0.90–0.99 were described as metacentric, submetacentric, acrocentric and telocentric chromosomes, respectively. Fundamental number (number of chromosome arm, NF) was obtained by assigning a value of two to metacentric, submetacentric and acrocentric chromosomes and one to telocentric chromosome. All parameters were used in karyotyping and idiograming. Results and discussion Chromosome number, fundamental number and karyotype of C. blanci Cytogenetic studies have been carried out of C. blanci from Mekong River, northeast of Thailand. This study reveals that the somatic chromosome number of C. blanci is 2n=42 for both sexes (Figs. 2 and 4). This is in accordance with the previous study of Donsakul and Magtoon Fig. 2. Metaphase chromosome plates and karyotypes of the royal knifefish (Chitala blanci) male (A) and female (B), 2n (diploid)=42 by conventional staining technique. Scale bars indicate 5 μm. 162 N. Maneechot et al. Cytologia 80(2) Fig. 3. Metaphase chromosome plates and karyotypes of the royal knifefish (Chitala blanci) male (A) and female (B), 2n (diploid)=42 by Ag-NOR banding technique. Arrows indicate nucleolar organizer regions/NORs (scale bars=5 μm). Fig. 4. Idiogram showing lengths and shapes of chromosomes of the royal knifefish (Chitala blanci), 2n (diploid)=42, by conventional staining technique. Arrow indicates nucleolar organizer region (NOR). 2015 Chromosomal Characteristics of the Royal Knifefish 163 Table 1. Cytogenetics reviews of fishes in the family Notopteridae (genera; Chitala, Notopterus, Papyrocranus and Xenomystus). Species 2n NF Karyotype Ag-NORs Locality Reference C. blanci 42 42 42t — Thailand Donsakul and Magtoon (1990) 42 42 42t 2 (CR) Thailand Present study C. chitala 42 42 42t 2 Asia Uyeno (1973) 42 42 2a+40t — Thailand Donsakul and Magtoon (1990) 42 42 42t 2 Asia Takai and Ojima (1998) C. ornata 42 44 2a+40t 2 (CR) Thailand Supiwong et al. (2012) N. notopterus 42 42 42t — India Rishi and Singh (1983) 42 42 42t — India Khuda-Bukhsh and Barat (1987) 42 42 42t 2 India Rishi and Thind (1994) 42 42 42t 2 (SCR) Thailand Silawong et al. (2012) P. afer 34 38 4sm+30t — Africa Uyeno (1973) X. nigri 42 42 42t — Africa Uyeno (1973) Remarks: 2n=diploid chromosome number, NF=fundamental number (number of chromosome arm), m=metacentric, sm=submetacentric, a=acrocentric, t=telocentric chromosome, NORs=nucleolar organizer regions, CR=centromeric region, SCR=subcentromeric region, and — = not available. (1990). Cytogenetic studies performed on the family Notopteridae have shown a diploid chromosome number ranging from 2n=34 to 2n=42. Nevertheless, the most frequent diploid chromosome number is equal to 42 chromosomes (Uyeno 1973, Rishi and Singh 1983, Khuda- Bukhsh and Barat 1987, Donsakul and Magtoon 1990, Rishi and Thind 1994, Takai and Ojima 1998, Silawong et al.