First Study on Chromosomal Feature and Nors Localization of the Yellow-Spotted Keelback Snake, Fowlea Flavipunctatus (Squamata, Natricinae) in Thailand
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Vol.6, No.1 pages 37-45 Science Technology and Engineering Journal (STEJ) Research Article First Study on Chromosomal Feature and NORs Localization of the Yellow-spotted Keelback Snake, Fowlea flavipunctatus (Squamata, Natricinae) in Thailand Nattasuda Donbundit1,3, Weera Thongnetr2, Isara Patawang4,5 and Alongklod Tanomtong1,3* 1 Department of Biology, Faculty of Science, Khon Kaen University, Muang, Khon Kaen, Thailand 2 Walai Rukhavej Botanical Research Institute, Mahasarakham University, Kantharawichai, Maha Sarakham, Thailand 3 Toxic Substances in Livestock and Aquatic Animals Research Group, Khon Kaen University, Muang, Khon Kaen, Thailand 4 Department of Biology, Faculty of Science, Chiang Mai University, Muang, Chiang Mai, Thailand 5 Research Center in Bioresources for Agriculture, Industry and Medicine, Chiang Mai University, Muang, Chiang Mai, Thailand * Corresponding Author: [email protected] (Received: 24th April 2020, Revised: 17th June 2020, Accepted: 23rd June 2020) Abstract-The first report on chromosome of the yellow-spotted keelback snake (Fowlea flavipunctatus), from Khon Kaen Province, Thailand, was investigated using classical cytogenetic approach. The mitotic chromosomes were directly prepared from the bone marrow cells of four adult male and female specimens after in vivo colchicine treatment. The results shown that the diploid chromosome number was 2n=42, while the fundamental number (NF) was 58 in both male and female. The types of macrochromosome were six large metacentric (m), two large acrocentric (a), four small metacentric and four small m t telocentric (t) chromosomes, and 24 microchromosomes (mi 2 + mi 22). Nucleolar organizer regions (NORs) located on subtelomeric position of short arm of macrochromosomes pair 4. The sex chromosome system was ZW. The karyotype formula of F. flavipunctatus is as follows: 2n m a m t m t (42) = L 6 + L 2 + S 4 + S 4 + mi 2 + mi 22 + 2 sex chromosomes (ZW). Keywords: Fowlea flavipunctatus, Yellow-spotted keelback, Cytogenetic, Karyotype 38 Nattasuda Donbundit, Weera Thongnetr, Science Technology and Engineering Journal (STEJ) Isara Patawang and Alongklod Tanomtong 1. Introduction about cytogenetic studies of snakes in the subfamily Natricinae, covering reports The yellow-spotted keelback snake, Fowlea by Eberle, (1971); Rossman and Eberle flavipunctatus (Hallowell, 1860) belongs (1977); Trinco and Smith (1971) and Itoh to the subfamily Natricinae of Colubridae. et al. (1970) reported a range of karyotypes This subfamily consists of 245 species in from 2n=17 to 21 pairs of chromosomes. 37 genera (Dubey et al., 2012; Zaher et The cytogenetic analysis has been al., 2019). The genus Fowlea comprises useful for taxonomic studies in Serpentes eight species namely, Fowlea asperrimus with inferring evolutionary relationships (BOULENGER, 1891), F. flavipunctatus if they are analyzed together with other (HALLOWELL, 1860), F. melanzostus independent characters e.g morphological, (GRAVENHORST, 1807), F. piscator molecular, immunological and isozyme (SCHNEIDER, 1799), F. punctulatus (Falcione, 2016; Sites and Reed, 1994). (GÜNTHER, 1858), F. sanctijohannis In the present study, a report on karyotype (BOULENGER, 1890), F. schnurrenbergeri analysis and chromosomal characteristics (KRAMER, 1977) and F. tytleri (BLYTH, of the nucleolar organizer regions (NORs) 1863). of F. flavipunctatus is the first cytogenetic Visual characteristics of F. record in Thailand. This advanced flavipunctatus are maximum recorded total cytogenetic technique provides useful length 991 mm in adult, body cylindrical, basic information for genetic research on head distinct from the neck and eye large. the taxonomy and evolution of the species. This species has body-coloured as: olive, brown, dark grey or pale grey. The neck is 2. Materials and methods often marked with conspicuous Y-shaped; venter pattern has darker shades only 2.1 Sample collection on the outermost edges. This species is distributed throughout Southeast Asia Four adult males and females of (Vogel and David, 2006). F. flavipunctatus were collected from Phylogenetic relationships within Khon Kaen Province, Thailand.They were this subfamily and the systematic position transferred to the laboratory and kept under of various species and genera were recently standard conditions for 1 day prior to the analyzed using molecular and morphological experiments. data (e.g. Boulenger, 1986; Cox et al., 1998; Dubey et al., 2012; Kramer, 1977; 2.2 Chromosome preparation Smith, 1943; Taylor, 1965; Vogel and David, 2006; Zaher, 1999). In contrast, Chromosomes were directly prepared in there are poorly known karyological data vivo using the following method (Ota, of any species of this genus. The previous 1999). The 0.01% colchicine was injected reports were restricted to other genera into the snake’s abdominal (1 mL/100 g of Natricinae (Table. 1). Karyological body weight) and left for 12 hours. Bone information has been obtained using standard marrows were cut into small pieces, staining protocols. From literature review then squashed and treated with 0.075 M Vol 6. No 1, January-June 2020 First Study on Chromosomal Feature and NORs 39 Localization of the Yellow-spotted Keelback Snake,... hypotonic KCl for 30 min. Cells were methanol: 1 glacial acetic acid). fixed in a cool fixative solution (3 absolute Table 1. Review of cytogenetic reports of subfamily Natricinae Species 2n Mac Mi Reference Fowlea flavipunctatus 42 18 24 Present study (Xenochrophis flavipunctatus) (Singh et al., 1968 Natrix piscator (Xenochrophis piscator) 40 10 30 ; Rao et al., 2010) Xenochrophis piscator 36 and 38 16 and 10 20 and 28 (Dutt, 1970) (Hanifa and Xenochrophis vittattus 34 26 8 Nurhandayani, 2015) As stated above, 2n=diploid chromosome, Mac=macrochromosome, Mi=microchromosome 2.3 Chromosome staining 3. Results and discussion Conventional staining was done using The diploid chromosome number of 20% Giemsa’s stock solution for 30 min. F. flavipunctatus was 2n=42 (16 50% AgNO3 and 2% gelatin were used for macrochromosomes + 24 microchromosomes) Ag-NOR banding technique (Howell and and consisted of six large metacentric, Black, 1980). two large acrocentric, four small metacentric and four small telocentric chromosomes, and 24 microchromosomes. 2.4 Chromosome checks The types of microchromosomes series were two metacentrics and 22 telocentric The length of short arms (Ls) and long microchromosomes (Fig.1). The fundamental arms (Ll) of chromosomes were measured number (NF, chromosome arm number) and calculated for the length of total arm was 58. The idiogram of F. flavipunctatus chromosomes (LT, LT = Ls + Ll). Relative revealed that the gradually decreasing length (RL) and centromeric index (CI) length of the autosomes (Fig. 2). Only one were estimated. CI was also computed to nucleolar organizer region was detected classify the types of chromosomes according on the subtelomeric of the long arm of the to Turpin and Lejeune (1965). Classification large acrocentric macrochromosome pair of microchromosomes is based on the 4 (Fig.1, 2). The sex chromosome showed presence of a size between groups of large a heteromorphic ZZ/ZW sex chromosome chromosomes and smaller chromosomes, system, both the Z and W chromosomes all the microchromosomes are very small are small submetacentric chromosomes. are often less than 1.5 micrometeres (Ezaz and Young, 2013). All parameters were used in karyotyping and idiograming. 3. Results and discussion and consisted of six large metacentric, two The diploid chromosome number of large acrocentric, four small metacentric and 40 Nattasuda Donbundit, Weera Thongnetr, Science Technology and Engineering Journal (STEJ) F. flavipunctatusIsara Patawang andwas Alongklod 2n Tanomtong=42 (16 four small telocentric chromosomes, and 24 macrochromosomes + 24 microchromosomes) microchromosomes. The types of a Macrochromosomes a . 1 2 3 4 5 6 7 8 Z Z Microchromosomes 10 11 12 13 14 15 16 17 18 19 20 21 b Macrochromosomes 1 2 3 4 5 6 7 8 Z W Microchromosomes 10 11 12 13 14 15 16 17 18 19 20 21 c Macrochromosomes 1 2 3 4 5 6 7 8 Z Z Microchromosomes 10 11 12 13 14 15 16 17 18 19 20 21 d Macrochromosomes 1 2 3 4 5 6 7 8 Z W Microchromosomes 10 11 12 13 14 15 16 17 18 19 20 21 Figure.Figure. 1 Metaphase 1 Metaphase chromosome chromosome plates and standardized plates karyotypesand standardized of Fowlea flavipunctatus karyotypes, 2 n=42of Fowleaby flavipunctatusconventional, 2stainingn=42 ofby male conventional (a) and female sta(b) iningand Ag-NOR of male banding (a) techniqueand female of male (b) (c) andand female Ag- NOR (d). The arrows indicate subtelomeric NOR on macrochromosome pair 4 (scale bar=5 μm). banding technique of male (c) and female (d). The arrows indicate subtelomeric NOR on macrochromosome pair 4 (scale bar=5 μm). microchromosomes series were two decreasing length of the autosomes (Fig. 2). metacentrics and 22 telocentric Only one nucleolar organizer region was microchromosomes (Fig.1). The detected on the subtelomeric of the long arm fundamental number (NF, chromosome arm of the large acrocentric macrochromosome number) was 58. The idiogram of pair 4 (Fig.1, 2). The sex chromosome F. flavipunctatus revealed that the gradually showed a heteromorphic ZZ/ZW sex chromosome system, both the Z and W chromosome (Ll), total arm chromosomes are small submetacentric chromosome (LT), centromeric chromosomes. index (CI), relative length (RL) The F. flavipunctatus is member of and standard deviation (SD) of Natricinae. The letter being reported the CI, RL