© 2015 The Japan Mendel Society Cytologia 80(2): 159–166

Chromosomal Characteristics of the Royal Knifefish, blanci (, ) 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 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); (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 ): 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 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. 2012, Supiwong et al. 2012). The cytogenetic features here reported for the examined specimens of C. blanci revealed that the species has the 42 telocentric karyotype (12 large telocentric and 30 medium telocentric chromosomes) which is shared among most of the Notopteridae species previously analyzed, such as C. chitala, 42t (Uyeno 1973, Takai and Ojima 1998); C. ornata, 2a+40t (Supiwong et al. 2012); N. notopterus, 42t (Rishi and Singh 1983, Khuda- Bukhsh and Barat 1987, Rishi and Thind 1994, Silawong et al. 2012) and X. nigri, 42t (Uyeno 1973). However, one species exception is P. afer, 4sm+30t (Uyeno 1973) which has been reported to possess 2n=34 chromosomes. The fundamental number of C. blanci is 42 in both male and female. This is similar to the report of Donsakul and Magtoon (1990). The comparative studies with others in the family Notopteridae have shown the similar NF as those found in C. chitala, N. notopterus and X. nigri, 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) but it differs from C. ornata and P. afer which have NF of 44 and 38, respectively (Uyeno 1973, Supiwong et al. 2012). In addition, also similar to other species in the family Notopteridae, no cytologically distinguishable sex chromosome was observed (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) (Table 1). It is possible that the fishʼs sex- chromosomes are depending on an initiation of differentiation. Therefore, chromosomes containing sex-determination gene cannot be found by cytogenetic analyses. The karyotype formula for C. blanci is as follows: t t 2n (diploid) 42=L12+M30

Chromosome markers of C. blanci The NOR analysis by using Ag-NOR banding technique sequential to Giemsaʼs staining provides the detection two Ag-positive signals in this species. For the C. blanci, the Ag-positive 164 N. Maneechot et al. Cytologia 80(2)

Fig. 5. Idiogram of the royal knifefish, (Chitala blanci), 2n (diploid)=42, by Ag-NOR banding technique. Arrow indicates nucleolar organizer region (NOR).

Table 2. Mean length of 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, CI from 20 metaphase cells of the male and female royal knifefish (Chitala blanci), 2n=42.

Chromosome Chromosome Chromosome Ls Ll LT RL∓SD CI∓SD pair size type

1 0.00 3.48 3.48 0.084∓0.010 1.00∓0.00 Large Telocentric 2 0.00 3.17 3.17 0.077∓0.007 1.00∓0.00 Large Telocentric 3 0.00 2.94 2.94 0.071∓0.009 1.00∓0.00 Large Telocentric 4 0.00 2.65 2.65 0.064∓0.009 1.00∓0.00 Large Telocentric 5 0.00 2.37 2.37 0.058∓0.005 1.00∓0.00 Large Telocentric 6 0.00 2.22 2.22 0.054∓0.003 1.00∓0.00 Large Telocentric 7 0.00 2.13 2.13 0.052∓0.002 1.00∓0.00 Medium Telocentric 8 0.00 2.04 2.04 0.049∓0.001 1.00∓0.00 Medium Telocentric 9 0.00 1.93 1.93 0.047∓0.002 1.00∓0.00 Medium Telocentric 10* 0.00 1.85 1.85 0.045∓0.002 1.00∓0.00 Medium Telocentric 11 0.00 1.80 1.80 0.044∓0.002 1.00∓0.00 Medium Telocentric 12 0.00 1.71 1.71 0.041∓0.004 1.00∓0.00 Medium Telocentric 13 0.00 1.63 1.63 0.040∓0.004 1.00∓0.00 Medium Telocentric 14 0.00 1.59 1.59 0.039∓0.004 1.00∓0.00 Medium Telocentric 15 0.00 1.55 1.55 0.038∓0.004 1.00∓0.00 Medium Telocentric 16 0.00 1.51 1.51 0.037∓0.004 1.00∓0.00 Medium Telocentric 17 0.00 1.46 1.46 0.035∓0.004 1.00∓0.00 Medium Telocentric 18 0.00 1.40 1.40 0.034∓0.004 1.00∓0.00 Medium Telocentric 19 0.00 1.34 1.34 0.033∓0.003 1.00∓0.00 Medium Telocentric 20 0.00 1.27 1.27 0.031∓0.004 1.00∓0.00 Medium Telocentric 21 0.00 1.17 1.17 0.028∓0.003 1.00∓0.00 Medium Telocentric

Remark: * NOR-bearing chromosome. 2015 Chromosomal Characteristics of the Royal Knifefish 165 signals are located along the long arm centromeric region of medium telocentric chromosome pair 10 (Figs. 3 and 5). In all species of the family Notopteridae investigated to date, the single NOR- bearing chromosome pair is conserved (Uyeno 1973, Rishi and Thind 1994, Takai and Ojima 1998, Silawong et al. 2012, Supiwong et al. 2012). The NORs are effective cytotaxonomic markers in the family Notopteridae and allowed us to distinguish most of the analyzed species, in which the ribosomal sites were similarly located on the same chromosomal pair. Cytogenetic studies in other groups with conserved karyotypes have also demonstrated the same discriminatory ability of NORs, such as in Anostomidae (Galetti et al. 1984) and in some Cichlidae (Brinn et al. 2004). Nevertheless, the efficiency of ribosome sites as a cytotaxonomic marker is not applicable to all situations, since this region might remain unchanged on homologous chromosome of several species (Molina and Galetti 2004). The chromosome lengths in centimetres of 20 cells (male and female) in mitotic metaphase were measured. The Ls, Ll, LT, RL, CI, standard deviation of RL and CI, size and type of chromosome are presented in Table 2. The idiogram of C. blanci showed the gradually decreasing length of the chromosomes. Our results indicated that the chromosome markers of C. blanci are the chromosome pair 1, which is the largest telocentric chromosome and the chromosome pair 21, which is the smallest telocentric chromosome. The important karyotype feature is the asymmetrical karyotype, which has only one type of chromosome (telocentric chromosome). An approximately two-fold difference in size between the largest and smallest chromosomes was detected.

Acknowledgements

This work was supported by Royal Thai Government Scholarship National Science and Technology Development Agency (NSTDA), TRF Grant for New Researcher, Research and Development Institute, Rajabhat Mahasarakham University and National Research Council of Thailand.

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