Weerayuth Supiwong 1, Alongklod Tanomtong 1 *, Sarun Jumrusthanasan 1, Suthip Khakhong 2, Khunnitee Leelarasamee 2 and La-orsri Sanoamuang 1

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1 2012 The Japan Mendel Society Cytologia 77(1): A First Karyotype Analysis and Chromosomal Characteristic of Nucleolar Organizer Regions (NORs) of Common Sheathfish, Micronema apogon (Siluriformes, Siluridae) in Thailand Weerayuth Supiwong 1, Alongklod Tanomtong 1 *, Sarun Jumrusthanasan 1, Suthip Khakhong 2, Khunnitee Leelarasamee 2 and La-orsri Sanoamuang 1 1 Applied Taxonomic Research Center (ATRC), Department of Biology, Faculty of Science, Khon Kaen University, Khon Kaen, Muang 40002, Thailand 2 Aquaculture Program, Faculty of Agricultural Technology, Phuket Rajabhat University, Phuket, Muang 83000, Thailand Received May 16, 2011; accepted December 1, 2011 Summary A first karyotype analysis and chromosomal characteristic of nucleolar organizer regions (NORs) of the common sheathfish, Micronema apogon (Bleeker 1985), from the Chao Praya, Chi, and Song karm basins (3 populations), Thailand, were studied. Kidney cell samples were taken from 10 male and 10 female fish. The mitotic chromosome preparations were conducted by taken directly from kidney, spleen and/or gill cells. Conventional and Ag-NOR staining techniques were applied to stain the chromosomes. The results showed that diploid chromosomes number of M. apogon was 2n=64, and the fundamental numbers (NF) were 108 in both sexes. The chromosome types consist of 10 large metacentric, 12 large submetacentric, 4 large acrocentric, 2 large telocentric, 8 medium metacentric, 8 medium submetacentric, 2 medium acrocentric, 8 medium telocentric and 10 small telocentric chromosomes. No strange size chromosomes related to sex were observed. In chromosome pair 3, the region adjacent to the telomere of the short arm showed clearly observable secondary constriction/nors. The karyotype formula for M. apogon could be deduced as: 2n (diploid) 64=L10 m +L 12 sm +L4 a +Lt 2 +Mm 8 +M 8 sm +M2 a +Mt 8 +St 10 Key words Common sheathfish, Micronema apogon, Karyotype, Ag-NOR staining. The taxonomic classification of the common sheathfish, Micronema apogon (Bleeker 1985), is class Actinopterygii (ray-finned fishes), order Perciformes (catfishes), family Siluridae (sheathfish), genus Micronema and species Micronema apogon (Fig. 1). Three species of the genus Micronema have been recorded in the Mekong. Two of those species, M. apogon and M. bleekeri, resemble each other strongly and probably have similar ecological characteristics (Rainboth 1996). The M. apogon can be found in large rivers and adjacent streams and canals from Thailand to Indonesia, feeding on pelagic fishes in midwater to upper depths. Spawning occurs just before water levels begin to rise and moves into flooded riparian forests and probably out into floodplains during high water levels. Young of the year are first seen in July and being to move back into the rivers in October, where they remain until January. The fish are caught with seines, gill-nets, and hook-andline, sold fresh, dried and salted, or placed on ice for shipment to Thailand (Rainboth 1996, Vidthayanon 2005). In Thailand, the family Siluridae includes 27 species which can be grouped into 9 genera, namely; Belodontichthys, Ceratoglanis, Hemisilurus, Kryptopterus, Micronema, Ompok, Pterocryptis, Silurichthys and Wallago (Ferraris 2007, Rainboth 1996, Vidthayonon 2005). Only 11 species have been cytogenetically investigated reporting the diploid chromosome number (2n) ranging from 40 * Corresponding author, tanomtong@hotmail.com

2 54 W. Supiwong et al. Cytologia 77(1) Fig. 1. Characteristics of common sheathfish, Micronema apogon (Bleeker 1985). to 92 including the S. phaiosoma, 2n=40; S. schneideri, 2n=40 (Magtoon and Donsakul 2009); O. bimaculatus, 2n=40 from India (Nayyar 1966); 2n=50 from Thailand (Donsakul 1992); W. leerii, 2n=56 (Donsakul 1996); O. fumidus, 2n=60 (Magtoon and Donsakul 2009); B. dinema, 2n=62 (Donsakul 1996); K. macrocephalus, 2n=62 (Magtoon and Donsakul 2009); K. bicirrhis, 2n=64 (Donsakul 1996); M. bleekeri, 2n=64 (Donsakul 1992); W. attu, 2n=86 from India (Nayyar 1966); 2n=88 from Thailand (Donsakul, 1996) and K. cryptopterus, 2n=92 (Donsakul 1992). This cytogenetic study, a report on karyotype analysis and chromosomal characteristic of the nucleolar organizer regions (NORs) of M. apogon, is the first record. The knowledge gained can provide useful cytogenetic information for further study on taxonomy and evolutionary relationship. Moreover, it contains basic information for the conservation, breeding, and chromosome evolution study in this fish. Materials and methods Sample collection M. apogon (10 males and 10 females) were obtained from the Chao Praya, Chi and Song karm basins (3 populations), Thailand. The fish were transferred to laboratory aquaria and were kept under standard condition for 7 days prior to the experiments. Chromosome preparation Chromosomes were directly prepared in vivo (Chen and Ehbeling 1968, Nanda et al. 1995) as follows. Phytohemagglutinin (PHA) solution was injected in to the fishes abdominal cavities. Twenty four hours after that, colchicine was injected to the fishes intramuscular and/or abdominal cavities and the fish were then left for 2 4 h. Kidney, spleen and/or gills were cut into small pieces then squash mixed with M KCl. After discarding all large piece tissues, 15 ml of cell sediments were transferred to centrifuge tube and incubated for min. KCl was discarded from the supernatant after centrifugation again 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 centrifuged again at 1,200 rpm for 8 min, then the supernatant was discarded. The 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 air-dry technique. Chromosome staining Conventional staining was carried out using 20% Giemsa s solution for 30 min. Ag-NOR banding (Howell and Black 1980) was performed by adding 2 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 cover glasses were separated and stained with 20% Giemsa s solution for 1 min.

3 2012 A First Karyotype Analysis and Chromosomal Characteristic of Nucleolar Organizer Regions in Thailand 55 Chromosome checks Chromosome counting was performed on mitotic metaphase cells under a light microscope. Twenty clearly observable and well-spread cell chromosomes of each male and female were selected and photographed. The length of short arm chromosome (Ls) and long arm chromosome (Ll) were measured to calculate 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 estimated (Chaiyasut 1989). The CI (q/p+q) between , , and are described as metacentric, submetacentric, acrocentric and telocentric chromosomes, respectively. The fundamental number (number of chromosome arm, NF) was obtained by assigning a value of 2 to metacentric, submetacentric and acrocentric chromosomes and 1 to telocentric chromosome. All parameters were used in karyotyping and idiogramming. Results and discussion Chromosome number, fundamental number and karyotype of M. apogon The present investigation revealed that the somatic chromosome number of M. apogon is 2n=64, the fundamental numbers (NF) were 108 in both males and females (Fig. 2). The types of chromosomes were 10 large metacentric, 12 large submetacentric, 4 large acrocentric, 2 large telocentric, 8 medium metacentric, 8 medium submetacentric, 2 medium acrocentric, 8 medium telocentric and 10 small telocentric chromosomes. This is the first record on the karyological studies of M. apogon. Comparative studies with others in family Siluridae in Thailand have shown the same chromosome number as those found in M. bleekeri (Donsakul 1992) and K. bicirrhis (Donsakul 1996). Similar to other species in family Siluridae, no cytologically distinguishable sex chromosome was observed (Nayyar 1966, Donsakul 1992, Donsakul 1996, Magtoon and Donsakul 2009). Fig. 2. The first karyotypes study of the common sheathfish (Micronema apogon) diploid (2n)=64 from North-eastern Thailand by conventional (top) and Ag-NOR straining techniques (bottom). The arrows indicate NOR-bearing chromosomes. Chromosome markers of M. apogon This study, the first cytogenetic report of M. apogon, was accomplished using Ag-NOR staining technique. The objective of this technique is to reach out the nucleolar organizer regions (NORs) which represent the location of genes (loci) that function in ribosome synthesis (18S and 28S ribosomal RNA). NORs produce numerous gene expressions and contain more non-histone protein than others chromosome regions. Accordingly, the specific dark band (NOR-positive) is induced by the reduction of organic silver by these proteins that change from silver to dark (Sharma et al. 2002). The region adjacent to the telomere of the short arm chromosome pair 3 (large metacentric chromosomes) showed clearly observable secondary constriction/nors. Therefore, the extra characteristic of short arms near telomere of chromosome pair 3 is the representative of chromosome marker. The chromosome of mitotic metaphase cells and the karyotypes of M. apogon by conventional staining and Ag-NOR staining tech-

4 56 W. Supiwong et al. Cytologia 77(1) Fig. 3. Metaphase chromosome plates of male (A and B) and female (C and D) common sheathfish (Micronema apogon) diploid (2n)=64 from North-eastern Thailand by Ag-NOR staining technique, arrows indicate nucleolar organizer regions, NORs (scale bar=5 μm). Fig. 4. Idiogram showing lengths and shapes of chromosomes of the common sheathfish (Micronema apogon) diploid (2n)=64 from North-eastern Thailand by conventional staining technique, arrow indicates nucleolar organizer region (NOR).

5 2012 A First Karyotype Analysis and Chromosomal Characteristic of Nucleolar Organizer Regions in Thailand 57 Table 1. Mean length of short arm chromosome (Ls), long arm chromosome (Ll), total arm chromosome (LT), relative length (RL), centromeric index (CI) and standard deviation (SD) of RL, CI from 20 metaphases of the common sheathfish (Micronema apogon), (diploid) 2n=64. Chromosome Pair Ls Ll LT RL SD CI SD Chromosome type Chromosome size Submetacentric Large Submetacentric Large 3* Metacentric Large Submetacentric Large Metacentric Large Acrocentric Large Metacentric Large Submetacentric Large Acrocentric Large Metacentric Large Telocentric Large Submetacentric Large Submetacentric Large Metacentric Large Submetacentric Medium Metacentric Medium Metacentric Medium Submetacentric Medium Telocentric Medium Metacentric Medium Submetacentric Medium Metacentric Medium Acrocentric Medium Submetacentric Medium Telocentric Medium Telocentric Medium Telocentric Medium Telocentric Small Telocentric Small Telocentric Small Telocentric Small Telocentric Small Remarks: *=satellite chromosome (NORs). niques are shown in Figs. 2 and 3. The chromosomes length in centimetres of 20 cells (males and females) in mitotic metaphase was measured. The mean length of short arm chromosome (Ls), length of long arm chromosome (Ll), total length of arm chromosome (LT), relative length (RL), centromeric index (CI), standard deviation of RL, CI, size and type of chromosome were presented in Table 1. The idiogram of M. apogon shows gradually decreasing length of the chromosomes. The M. apogon demonstrated that the chromosome markers are the chromosome pair 1 and pair 32 which are the largest submetacentric chromosome and the smallest telocentric chromosome, respectively. The important karyotype feature of M. apogon is the asymmetrical karyotype, which were found in all 4 types of chromosomes (metacentric, submetacentric, acrocentric and telocentric chromosomes). The largest chromosome are 3 times larger than the smallest chromosomes. Fig. 4 shows the idiogram of the M. apogon from conventional staining. The karyotype formula for M. apogon could be deduced as: 2n (diploid) 64=L10+L m sm 12 +L a 4+L t 2+M m 8 +M sm 8 +M a 2+M8+S t 10 t

6 58 W. Supiwong et al. Cytologia 77(1) Acknowledgments This work was supported by the Applied Taxonomic Research Center (ATRC), Khon Kaen University grant; ATRC-R5304 and by a grant from the Faculty of Science, Khon Kaen University. References Chen, T. R. and Ehbeling, A. W Karyological evidence of female heterogamety in the mosquito fish. Gambusia affinis Copeia 1: Chaiyasut, K Cytogenetics and Cytotaxonomy of the Family Zephyranthes. Department of Botany, Faculty of Science, Chulalongkorn University, Bangkok. Donsakul, T A study of chromosome in blue sheatfish (Kryptopterus cryptopterus), whisker sheatfish (Micronema bleekeri) and two-spot glass catfish (Ompok bimaculatus) from Thailand. Department of Biology, Faculty of Science, Srinakharinwirot University, Bangkok A study of chromosome in freshwater shark (Wallago attu), Helicopter catfish (W. leerii), twisted-jaw sheatfish (Belodontichthys dinema) and glass catfish (Kryptopterus bicirrhis) from Thailand. In: The Proceedings of 34th Kasetsart University Annual Conference (Fishery section). Bangkok. pp Ferraris, C. J Checklist of catfishes, recent and fossil (Osteichthyes: Siluriformes), and catalogue of siluriform primary types. Zootaxa 1418, Howell, W. M. and Black, D. A Controlled silver-staining of nucleolus organizer regions with a protective colloidal developer: a 1-step method. Experientia 36: Magtoon, W. and Donsakul, T Karyotypes of Silurichthys phaiosoma, S. schneideri, Ompok fumidus and Kryptopterus macrocephalus (Family Siluridae) from Thailand. In: 35th Congress on Science and Technology of Thailand. oct Chonburi. Nanda, I., Schsrtl, M., Fiechtinger, W., Schlupp, I., Parzefall, J. and Schmid, M Chromosomal evidence for laboratory synthesis of triploid hybrid between the gynogenetic teleost Poecilia Formosa and its host species. J. Fish Biol. 47: Nayyar, R. P Karyotype studies in thirteen species of fishes. Genetica 37: Rainboth, W. J Fishes of Cambodian Mekong. FAO species identification field guide for fishery purposes. FAO, Rome. p Sharma, O. P., Tripathi, N. K. and Sharma, K. K A review of chromosome banding in fishes. In: Sobti, R. C., (ed.). Some aspects of chromosome structure and functions. Narosa Publishing House, New Delhi. Vidthayanon, C Handbook of Freshwater Fish. 2nd ed. Sarakadee Press, Bangkok.