© 2007 The Japan Mendel Society Cytologia 72(2): 189–194, 2007

Standardized Karyotype and Idiogram of the Pileated , pileatus (, Hylobatidae) by G-banding and High-resolution Technique

Praween Supanuam, Alongkoad Tanomtong* and Sumpars Khunsook

Genetic Program, Department of Biology, Faculty of Science, Khon Kaen University, Muang, Khon Kaen 40002, Thailand

Received January 29, 2007; accepted April 12, 2007

Summary Standardized karyotype and idiogram of the pileated gibbon (Hylobates pileatus) Nakhon Ratchasima Zoo, Thailand was studied. Blood sample were taken from 2 females and 2 males. After standard whole blood lymphocyte culture at 37°C for 72 h in the presence of colchicine, the metaphase spreads were performed on microscopic slides and air-dried. G-banding and high-res- olution technique were applied to stain the chromosomes. The results showed that the number of diploid chromosomes of pileated gibbon was 2n (diploid)44. The type of autosomes were 30 meta- centric, 10 submetacentric and 2 acrocentric chromosomes, with X and Y chromosome being sub- metacentric and acrocentric chromosome, respectively. From the G-banding and high-resolution technique, the numbers of bands and locations in the pileated gibbon were 183 and 236 respectively, each chromosome pair could be clearly differentiated.

Key words karyotype, idiogram, G-banding, high-resolution technique, pileated gibbon (Hylobates pileatus).

There were 13 families, 60 genera and 232 species of in order Primate (Wilson and Cole 2000), however, only 3 families, 5 genera and 13 species were found in Thailand. The gibbon species consists of 3 species, white-handed gibbon (Hylobates lar Linnaeus 1771), agile gibbon or dark-handed gibbon (Hylobates agilis Cuvier 1821) and pileated gibbon (Hylobates pileatus Gray 1842) (Lekagul and McNeely 1977, 1988). These species are listed as protected species in Wild Reservation and Protection Act (1992), in the International Union for the Conservation of Nature (IUCN) as vulnerable species. The Convention on International Trade in Endangered Species (CITES) of wild fauna and flora also listed these species in Appendix I (Brokelman 1981). Several the cytogenetic studies of pileated gibbon have been reported by Dutrillaux et al. (1975), Stanyon (1987), and Liu et al. (1996). In this study, we confirm and compare the result with these reports. In addition to, this is report about standardized karyotype and idiogram by G-banding and high-resolution technique. Thus, it is important to conduct this study, as it should be basic knowledge and can be applied to accommodate further research.

Materials and methods Blood samples from the jugular vein were collected from 2 male and 2 female pileated gib- bons, which were kept in Nakhon Ratchasima Zoo, Nakhon Ratchasima Province, Thailand using aseptic technique. The samples were kept in 10 ml vacuum tubes containing heparin to prevent blood clotting and they were cooled on ice until arriving at the laboratory.

* Corresponding author, e-mail: [email protected] 190 Praween Supanuam et al. Cytologia 72(2)

1. Cell preparation The lymphocytes were cultured using the whole blood microculture technique adapted from Rooney (2001) and Kampiranont (2003).

Cell culture The RPMI 1640 medium was prepared with 2% PHA (Phytohemagglutinin) as a mitogen and kept in blood culture bottles of 5 ml each. A blood sample of 0.5 ml was dropped into a medium bottle and well mixed. The culture bottle was loosely capped, incubated at 37°C under 5% of car- bondioxide environment and regularly shaken in the morning and evening. When reaching harvest time at the 72nd h of incubation, colchicine was introduced and well mixed, followed by further in- cubation for 30 min.

Cell harvest The blood sample mixture was centrifuged at 1,200 rpm for 10 min and the supernatant was discarded. Ten ml of hypotonic solution (0.075 M KCl) was applied to the pellet and the mixture was incubated for 30 min. KCl was discarded with the supernatant after centrifugation again at 1,200 rpm for 10 min. Cells were fixed by fresh cold fixative (methanol : glacial acetic acid3:1) gradually added up to 8 ml before centrifuging again at 1,200 rpm for 10 min, and 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 using micropipette fol- lowed by the air-drying technique.

2. G-banding method G-banding technique was adapted from Kampiranont (2003). The slide was well dried and then soaked in working trypsin (0.025% trypsin EDTA) at 37°C before the termination of trypsin activity by washing the slide with 10% fetal calf serum (FCS) or phosphate buffer. FCS was washed out by 50% methanol and the slide was stained with 10% Giemsa’s solution for 30min.

3. High-resolution staining method High-resolution technique was adapted from Rooney (2001). After the lymphocytes were cul- tured for 72 h, 0.05 ml of 105 M methotrexate was applied into the cultured lymphocytes to induce synchronization. The mixture was incubated again for 17 h before the methotrexate was discarded with the supernatant by centrifugation at 2,800 rpm. The pellet was mixed with 5 ml of the RPMI 1640 medium and centrifuged at 2,800 rpm. The supernatant was discarded before the cultured cells were released by adding 0.2 ml thymidine and incubating for 5 h and 15 min. The cells were har- vested at the exact time and stained by using G-banding procedure, karyotyping and idiograming according to Nash and O’Brien (1987), Wada et al. (1991).

Results and discussion Standardized karyotype and idiogram of the pileated gibbon using lymphocyte culture, the G- banding and high-resolution technique procedures revealed that the chromosome number was 2n44. This is the same chromosome number for the pileated gibbon as reported by Dutrillaux et al. (1975), Stanyon (1987), Geissmann (2002). This examination also revealed that the fundamental number (NF) of the pileated gibbon was 88 in male and female. This is the same NF for the pileated gibbon as reported by Dutrillaux et al. (1975), Liu et al. (1996). The type of autosomes were 30 metacentric, 10 submetacentric and 2 acrocentric chromo- somes. Difference chromosomal features were reported by Liu et al. (1996), which indicated that the pileated gibbon has 42 metacentric and submetacentric autosomes. Furthermore, Dutrillaux et 2007 Standardized Karyotype and Idiogram of the Pileated Gibbon 191

Fig. 1. Metaphase chromosome and karyotype of male pileated gibbon (Hylobates pileatus) 2n44, by G- banding, satellite chromosomes (arrows).

Fig. 2. Metaphase chromosome and karyotype of female pileated gibbon (Hylobates pileatus) 2n44, by G-banding, satellite chromosomes (arrows).

Fig. 3. Prometaphase chromosome and karyotype of male pileated gibbon (Hylobates pileatus) 2n44, by high-resolution technique, satellite chromosomes (arrows). al. (1975) also reported that the autosomes of the pileated gibbon has 24 metacentric and 18 sub- metacentric autosomes. The X chromosome of the pileated gibbon is a submetacentric chromosome and the Y chromosome is the acrocentric chromosome. These features are similar to that reported by Dutrillaux et al. (1975), Liu et al. (1996) indicating that a pileated gibbon has a submetacentric X chromosome and a dot Y chromosome (Figs. 1, 2, 3, 4). The G-banding and high-resolution technique provide a clearly chromosome band which rep- 192 Praween Supanuam et al. Cytologia 72(2)

Fig. 4. Prometaphase chromosome and karyotype of female pileated gibbon (Hylobates pileatus) 2n44, by high-resolution technique, satellite chromosomes (arrows).

Fig. 5. Idiogram of the pileated gibbon (Hylobates pilea- Fig. 6. Idiogram of the pileated gibbon (Hylobates pilea- tus) 2n44, by G-banding, satellite chromosomes tus) 2n44, by high-resolution technique, satel- (arrow). lite chromosomes (arrow). resent in black and white regions on chromosome. The level of high-resolution technique (band numbers) is defined by a visible and in a haploid set which compose of autosomes, X and Y chro- mosomes (Yunis 1976). Thus, the haploid set of the pileated gibbon is consist of 21 autosomes in- clude X and Y chromosomes. However, some chromosome can not clearly identify because some band is variable. As above the chromosome band scoring is represent by approximate band that appear. This result demonstrated that the chromosome band number of the pileated gibbon from G- banding of metaphase chromosome and high-resolution technique of prometaphase chromosome are 183 and 236 bands, respectively (Figs. 5, 6). Different from the reported of Stanyon (1987), the 2007 Standardized Karyotype and Idiogram of the Pileated Gibbon 193 chromosome band number of the pileated gibbon from G-banding and high-resolution technique are 273 and 361 bands, respectively. Moreover, Yunis (1982) reported that chromosome band num- ber from the high-resolution technique of prometaphase chromosome of human and are over 1000 bands per haploid set. Stanyon (1983) suggested that the high-resolution technique given a high efficiency for differ- ences chromosome comparison of Mucaca fuscuta and Cercorebus atereimus. The evolution study of gorilla, orangutan, chimpanzee and human by the comparison of chromosome structure and chromosome bands (Yunis 1982) with G-banding and high-resolution technique given chromosome band numbers 320–500 and over 1000 bands per haploid set. In this study, the chromosome scoring is done with only clearly visible bands except for the variable band due to the small number of scored bands. Compare with gibbon species in the genus Hylobates which have the same chromo- some number, 2n44, the different in scored bands were found. The chromosome band number which generated by high-resolution technique of white-handed gibbon (Hylobates lar) (Jauch et al. 1992) and black crested gibbon (Hylobates concolor) (Koehler et al. 1995) are 574 and 420 bands, respectively. Whereas the G-banding show that the chromosome band number of (Hylobates hoolock) are 230 bands (Nie et al. 2001). In comparison with Stanyon (1983), the G-banding of pileated gibbon chromosome show that the 10 autosome pairs are alternation such as 3, 5, 10, 11, 12, 13, 14, 15, 18 and 19, whereas in this study the alternation are occurred on the autosome pairs 4, 6, 9, 12, 14, 11, 15, 13, 19 and 18, re- spectively referred to Stanyon (1983). Our results demonstrate that the G-banding and high-resolution technique given a high effi- ciency for study on cytogenetics of pileated gibbon. The high number of visible band provides a tool for determine chromosome abnormality and comparative karyotype study for all or order of an- imal in the .

Acknowledgement The financial support from The Zoological Park Organization Under the Royal Patronage of H.M. The King is gratefully acknowledged. We also thank Mr. Sopon Dumnui, Director of the or- ganization and Dr. Sumat Kamolnaranath, chief of the Educational division, for valuable help. We would like to thank the director of the Nakhon Ratchasima Zoo for the gibbon blood samples. Thanks to the authorities and officers of these zoos for good cooperation.

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