A First Karyological Analysis of the Sand Goby, Oxyeleotris Marmoratus (Teleostei, Eleotridae) in Thailand by Ag-NOR Staining Technique
Total Page：16
File Type：pdf, Size：1020Kb
© 2014 The Japan Mendel Society Cytologia 79(2): 127–132 A First Karyological Analysis of the Sand Goby, Oxyeleotris marmoratus (Teleostei, Eleotridae) in Thailand by Ag-NOR Staining Technique Krit Pinthong1, Weerayuth Supiwong2, 3, Alongklod Tanomtong2*, Piyorot Hongsachart3, Pheeraphong Phaengphairee4, and Mallika Chantarangsee3 1 Biology Program, Faculty of Science and Technology, Surindra Rajabhat University, Muang, Surin 32000, Thailand 2 Genetics and Environmental Toxicology Research Group, Department of Biology, Faculty of Science, Khon Kaen University, Muang, Khon Kaen, 40002, Thailand 3 Faculty of Applied Science and Engineering, Nong Kai Campus, Khon Kaen University, Muang, Nong Kai 43000, Thailand 4 Department of Animal Science, Faculty of Agriculture, Khon Kaen University, Muang, Khon Kaen, 40002, Thailand Received November 6, 2012; accepted March 22, 2013 Summary The standardized karyotype and idiogram of the sand goby (Oxyeleotris marmoratus) from Chi River in the Maha Sarakham Province of northeastern Thailand were studied. Kidney cell samples were taken from five male and five female fish. The mitotic chromosome preparations were prepared by blood cell culture and 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 O. marmoratus was 2n = 46 and the fundamental number (NF) as 50 in both males and females. The types of chromosomes were 2 large submetacentric, 10 large telocentric, 2 medium metacentric and 32 medium telocentric chromosomes. No strange-sized chromosomes related to sex were observed. The region adjacent to the telomere of chromosome pair 7 showed clearly observable secondary constriction/NORs. The karyotype formula for O. marmoratus could be inferred as: sm t m t 2n (diploid) 46 = L2 +L10+M2 +M32 Key words Sand goby, Oxyeleotris marmoratus, Ag-NOR staining, Karyotype. The sand goby, Oxyeleotris marmoratus (Bleeker 1852) is widely distributed in fresh water habitats throughout Asia including the Mekong and Chao Praya basins, rivers and other water bodies in Indochina, Malaysia, Singapore, Philippines and Indonesia. This species has an economical importance in the Southeast Asian region, especially in Thailand, Malaysia, Singapore and Indonesia (Kottelat et al. 1993). It is highly popular among consumers especially among the Chinese community because of its fine texture, tasty white flesh and the belief of healing properties. Among this fresh water group, O. marmoratus is one of the most important freshwater Eleotrids for commercial aquaculture. Because of the tender flesh and good flavor of O. marmoratus, the market demands for this species are increasing (Suwanjarat et al. 2005). The O. marmoratus belongs to the suborder Gobioidei in the order Perciformes which is comprised of about 268 genera and approximately 2,121 species (Nelson 1994) and have been variously classified into families and subfamilies in recent times. The taxonomic classification of O. * Corresponding author, e-mail: [email protected] DOI: 10.1508/cytologia.79.127 128 K. Pinthong et al. Cytologia 79(2) marmoratus is class Actinopterygii (ray-finned fishes), superorder Percomorpha, order Perciformes (perch-like fish), suborder Gobioidei, family Eleotridae, subfamily Butinae, genus Oxyeleotris and species Oxyeleotris marmoratus (Akihito et al. 2000, Vidthayonon 2005). In this article, we report the karyotype and other chromosomal markers such as Ag-stained nucleolar organizer region (Ag-NOR) in the O. marmoratus population from Maha Sarakham Province in northeastern Thailand. This study is the first report on the Ag-NOR chromosome of the O. marmoratus in Thailand. In the future, the knowledge of the basic cytogenetics of O. marmoratus could be applied to numerous breeding studies and also have advantage in species conservation and chromosome evolution studies. Materials and methods Five males and five females of O. marmoratus were obtained from Chi River in Maha Sarakham Province of northeastern Thailand. The fish were transferred to laboratory aquaria and were kept under standard condition for 7 d prior to the experiments. The fish chromosomes were prepared from white blood cell cultures (Gold et al. 1990) and/or were directly prepared from kidney cells (Chen and Ehbeling 1968, Nanda et al. 1995). The chromosome preparations were stained with 10% Giemsa’s for 30 min and NORs were identified by Ag-NOR staining (Howell and Black 1980). The metaphase figures were analyzed according to the chromosome classification after Chaiyasut (1989). The centromeric index (CI) 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, Fig. 1. Metaphase chromosome plates of male (A. and B.) and female (C. and D.) sand goby (Oxyeleotris marmoratus), 2n (diploid) = 46, by Ag-NOR staining technique. Arrows indicate nucleolar organizer regions, NORs (scale bars = 5 μm). 2014 A First Karyological Analysis of the Sand Goby in Thailand 129 respectively. The fundamental number, NF (number of chromosome arm) is obtained by assigning a value of two to metacentric, submetacentric and acrocentric chromosomes and one to telocentric chromosome. Results and discussion Diploid chromosome number, fundamental number and karyotype of O. marmoratus To the best of our knowledge, this is the first karyotypic study of O. marmoratus by the Ag- NOR staining technique. The result showed a diploid number of 2n = 46, which was consistent with the report of Donsakul and Magtoon (1998). We found that the fundamental number (NF, number of chromosome arms) of O. marmoratus was 50 in both males and females. This is different from the report of Donsakul and Magtoon (1998), which demonstrating that NF of O. marmoratus is 60. For the genus Oxyeleotris in China, the diploid chromosome number of sleepy cod (O. lineolatus) is 2n = 46, and the NF is 48 in both male and female (Chen et al. 2006). The chromosomes of O. marmoratus consist of 2 large submetacentric, 10 large telocentric, 2 medium metacentric and 32 medium telocentric chromosomes. This is inconsistent with the report of Donsakul and Magtoon (1998), which revealed that O. marmoratus has chromosomes 2 metacentric, 2 submetacentric and 42 acrocenrtic chromosomes. No strange-sized chromosomes Fig. 2. Karyotypes of sand goby (Oxyeleotris marmoratus), 2n (diploid) = 46, by conventional staining (top) and Ag-NOR staining techniques (bottom). Arrows indicate nucleolar organizer regions, NORs (scale bars = 5 μm). 130 K. Pinthong et al. Cytologia 79(2) related to sex were observed. Our results showing 12 large and 34 medium chromosomes are the first report on the chromosome size of the species. Accordingly the karyotype formula could be inferred as: sm t m t 2n (diploid) 46 = L2 +L10+M2 +M32 Chromosome marker of O. marmoratus This is the first report on the Ag-NOR banding O. marmoratus. The technique shows dark bands (NOR-position) on the long arm near the telomere of the telocentric chromosome pair 7 in both males and females (Figs. 1 and 2). For other comparative studies, the species in order Perciformes in Thailand, the chevron snakehead fish (Channa striata), had NOR on the long arm near the centromere of chromosome pair 14 (Supiwong et al. 2009), while three-spot gourami, Trichogaster trichopterus, had NOR on the long arm near the telomere of chromosome pair 2 (Supiwong et al. 2010). The NORs are the chromosomal sites of genes that transcribe the 18s and 28s ribosomal RNA, which are presumably transcribed in the preceding interphase and are important in aspect of their intimate relationship with protein synthesis (Howell and Black 1980). NORs, as ribosomal gene clusters, are active in the previous interphase and form prominent cytogenetic features, namely secondary constrictions. The main defining characteristic of these constrictions is under- condensation in comparison with the rest of the chromosome. Genes encoding rRNA are associated with UBF proteins, which are characterized by silver-binding properties. In the interphase, the cell synthesizes nucleoli on the basis of rRNA genes (Miller et al. 2001, Suwanjarat et al. 2005). Information on the cytogenetic markers can also be used for molecular cytogenetic assignment of genes on chromosomes (Donate et al. 2003). Table 1. Mean length of short arm chromosome (Ls), length of long arm chromosome (Ll), length of total arm chromosome (LT), relative length (RL), centromeric index (CI) and standard deviation (SD) of RL, CI from 20 metaphases of sand goby (Oxyeleotris marmoratus), 2n = 46. Chromosome Chromosome Chromosome Ls Ll LT RL±SD CI±SD pair size type 1 0.388 0.848 1.236 0.059±0.003 0.680±0.039 Large Submetacentric 2 0.000 1.090 1.090 0.053±0.002 1.000±0.000 Large Telocentric 3 0.000 1.042 1.042 0.050±0.002 1.000±0.000 Large Telocentric 4 0.000 1.013 1.013 0.049±0.001 1.000±0.000 Large Telocentric 5 0.000 0.987 0.987 0.048±0.001 1.000±0.000 Large Telocentric 6 0.000 0.966 0.966 0.047±0.001 1.000±0.000 Large Telocentric 7* 0.000 0.940 0.940 0.046±0.001 1.000±0.000 Medium Telocentric 8 0.000 0.928 0.928 0.045±0.001 1.000±0.000 Medium Telocentric 9 0.000 0.913 0.913 0.044±0.001 1.000±0.000 Medium Telocentric 10 0.000 0.902 0.902 0.044±0.001 1.000±0.000 Medium Telocentric 11 0.000 0.888 0.888 0.043±0.001 1.000±0.000 Medium Telocentric 12 0.000 0.877 0.877 0.043±0.001 1.000±0.000 Medium Telocentric 13 0.000 0.865 0.865 0.042±0.001 1.000±0.000 Medium Telocentric 14 0.000 0.862 0.862 0.042±0.001 1.000±0.000