© 2011 The Japan Mendel Society Cytologia 76(1): 89–97, 2011
Chromosome Studies in the Banded Palm Civet, Hemigalus derbyanus Thomas, 1915 (Carnivora, Viverridae) by Conventional Staining, GTG- Banding and High-Resolution Techniques
Alongklod Tanomtong1*, Sumpars Khunsook1, Namphunk Seatung1, Praween Supanuam1, Anan Kenthao1 and Sarawut Kaewsri2
1 Department of Biology, Faculty of Science, Khon Kaen University, Khon Kaen, Muang 40002, Thailand 2 Program in Applied Biology, Department of Science, Faculty of Science, Buriram Rajabhat University, Buriram, Muang 31000, Thailand
Received May 31, 2010; accepted February 10, 2011
Summary Cytogenetics of banded palm civet (Hemigalus derbyanus) in Thailand was studied. Blood samples were taken from 2 males and 2 females then subjected to standard whole blood T- lymphocyte culture. The samples were harvested by colchicine-hypotonic-fixation-air-drying technique and followed by conventional staining, GTG-banding and high-resolution techniques with Giemsa’s. The results showed diploid number as 2n 42, and fundamental number (NF) as 81 in both male and 82 in female. The autosomes consist of 10 large submetacentric, 12 large acrocentric, 4 medium submetacentric, 4 small metacentric, 8 small submetacentric and 2 small telocentric chromosomes. We found that nucleolar organizer regions (the representative of chromosome marker) locate on the long arms of a pair metacentric autosome 15. The X chromosome was a large acrocentric chromosome while the Y chromosome was the smallest telocentric chromosome. From GTG-banding and high-resolution techniques, the numbers of bands and locations in banded palm civet were 218 and 244, respectively. Each homologous chromosome pair appears clearly differentiated.
Key words Banded palm civet (Hemigalus derbyanus), Chromosome, GTG-banding, NORs.
Animals in the family Viverridae form into the order Carnivora that has a high propagation in tropical forest areas found in south Europe, Africa and Asia including Indonesia, the Philippines and Madagascar (Vaunhen 1972, Stains 1984, McPhee 2001). Animals in the family Viverridae have good pheromone gland development. Their pheromone gland is located near to the anus. In male, this gland secretes an aromatic smell substance called “civet”. Animals in the subfamily Viverrinae (genus Viverricula, Viverra and Civettictis) have the best pheromone gland development, followed by the subfamily Paradoxurinae and the least well developed is the subfamily Hemigalinae. Their secreted substance is used in perfume and as a drug ingredient (Retting and Divers 1986). Banded palm civet is a member of the family Viverridae. The family has only 11 species of 9 genera in Thailand (Lekagul and McNeely 1977, 1988, Par et al. 2003). The banded palm civet belongs to the kingdom Animalia, phylum Chordata, class Mammalia, order Carnivora, family Viverridae, subfamily Hemigalinae, genus Hemigalus and species Hemigalus derbyanus. The general characteristics of banded palm civet are; ground color of banded palm civet is pale brown, with 7 or 8 wild black bands running across the back. These bands resemble long triangles having their bases on the back and the vertex toward the abdomen. There is a black band running along the
* Corresponding author, e-mail: [email protected] 90 A. Tanomtong et al. Cytologia 76(1)
Fig. 1. General characteristics of banded palm civet (Hemigalus derbyanus) (after Parr, 2003). midline of the nose and the forehead, disappearing on the neck. Two other black lines running from the long, thin muzzle through each eye merge with a big black patch on the neck. The under-parts and legs are lighter than the upperparts and lack markings. The hairs on the neck are reversed, pointing forward. The long, tapering tail is black except at the root, which is brown with a few faint black rings. Ears are well developed and long, and the eyes are large. The tongue is said to be rougher than in other civets. Scent glands are present but small. The claws are fully retractible (Fig. 1) (Walker 1964, Lekagul and McNeely 1977, 1988). There is only one previous report on banded palm civet cytogenetics. Wurster and Benirschke (1968) demonstrated 1 male banded palm civet’s karyotype by conventional staining technique as 2n (diploid) 42, and the fundamental number (NF) as 70. The autosomes (20 pairs) composted of 13 pairs of metacentric and submetacentric chromosome, and 7 pair of acrocentric and subacrocentric chromosome. The submetacentric autosomes, or satellite chromosomes (nucleolar organizer regions, NORs), is the smallest autosome. They also revealed the presence of medium submetacentric X chromosome and small acrocentric Y chromosome. From the present study, we show the standardization of karyotype and idiogram of banded palm civet. This report describes the first chromosome banding technique by GTG-banding and high-resolution techniques in banded palm civet.
Materials and methods Blood samples of 2 males and 2 females of banded palm civet were collected from Nakhon Ratchasima Zoo, Thailand, and then applied for cytogenetic studies by lymphocyte culture of whole blood samples. The culture cells were treated with a colchicine-hypotonic-fixation-air-drying technique followed by conventional staining, GTG-banding and high-resolution techniques (Rooney 2001, Campiranont 2003). Twenty cells of each individual chromosome checks, length measurements, karotyping and idiograming were accomplished by using a light microscope as previously described (Chaiyasut 1989).
Results and discussion The cytogenetic study of banded palm civet using lymphocyte culture shows that the chromosome number is 2n (diploid) 42 (Figs. 2, 3 and 4) which is consistent with the report of Wurster and Benirschke (1968). These are the chromosome numbers of other species in the family Viverridae; banded linsang (Prionodon linsang) 2n 34, small Indian civet (Viverricular indica) 2n 36, African civet (Civettictis civetta) 2n 38, large-spotted civet (Viverra megaspila) 2n 38, small-toothed palm civet (Arctogalidia trivigata) 2n 40, binturong (Arctictis binturong) 2n 42, Asian palm civet (Paradoxurus hermaphroditus) 2n 42, fossa (Cryptoprocta ferox) 2n 42, Hose’s 2011 Chromosome Studies in the Banded Palm Civet 91
Fig. 2. Metaphase chromosome plates and karyotypes of male (A) and female (B) banded palm civet (Hemigalus derbyanus) 2n (diploid) 42 by conventional staining technique, arrows indicate satellite chromosomes (SAT) and sex chromosomes. (A) Male banded palm civet. (B) Female banded palm civet. palm civet (Diplogale hosei) 2n 42, masked palm civet (Paguma lavata) 2n 44 and small-spotted genet (Genetta genetta) 2n 52 (Ray-Chaudhuri et al. 1966, Wurster and Benirschke 1967, 1968, Wada et al. 1983, Wang et al. 1984, Masashi and Harumi 1993, Tanomtong et al. 2005a, 2005b, 2005c, 2005d, 2005e). We found that animals in the family Viverridae have 2n between 34 to 52 and the banded palm civet has the same 2n as A. binturong, P. hermaphroditus, C. ferox and D. hosei. Banded palm civet shows a fundamental number (NF, number of chromosome arms) of 81 in male and 82 in female. It is different from the report of Wurster and Benirschke (1968) which demonstrated that the NF of banded palm civet is 70. These are the NF of other species in the family Viverridae in Thailand; V. indica, V. megaspila, A. trivigata, A. binturong and P. lavata have NF of 68 in females and 67 in males, 72 in females and 71 in males, 66 in females and males, 68 in females and males, 72 in females and males, respectively (Tanomtong et al. 2005a, 2005b, 2005c, 2005d, 2005e). We discovered that the NF of animals in the family Viverridae in Thailand is 66–82. 92 A. Tanomtong et al. Cytologia 76(1)
Fig. 3. Metaphase chromosome plates and karyotypes of male (A) and female (B) banded palm civet (Hemigalus derbyanus) 2n (diploid) 42 by GTG-banding, arrows indicate satellite chromosomes (SAT) and sex chromosomes.
The idiogram of the banded palm civet (Figs. 5 and 6) demonstrate the autosomes decreasing from large to small size. The banded palm civet’s autosomes consist of 10 (5 pairs) large submetacentric, 12 (6 pairs) large acrocentric, 4 (2 pairs) medium submetacentric, 4 (2 pairs) small metacentric, 8 (4 pairs) small submetacentric and 2 (1 pairs) small telocentric chromosomes (Figs. 2, 3 and 4). It is not consistent with the report of Wurster and Benirschke (1968) which revealed that the banded palm civet has autosomes as follows; 13 pairs of metacentric and submetacentric chromosomes, 7 pairs of acrocentric and subacrocentric chromosomes. The other animals in the family Viverridae in Thailand, namely V. indica, V. megaspila, A. trivigata, A. binturong and P. lavata, have metacentric, submetacentric, acrocentric and telocentric autosomes; 10-12-10-2, 10-10-12-4, 6-8-10-14, 2-4-16- 18 and 6-10-10-16, respectively (Tanomtong et al. 2005a, 2005b, 2005c, 2005d, 2005e). According to the above described comparison, there are several autosome types of these animals. The X chromosome of the banded palm civet was a large acrocentric chromosome while the Y 2011 Chromosome Studies in the Banded Palm Civet 93
Fig. 4. Metaphase chromosome plates and karyotypes of male (A) and female (B) banded palm civet (Hemigalus derbyanus) 2n (diploid) 42 by high-resolution technique, arrows indicate satellite chromosomes (SAT) and sex chromosomes. chromosome was the smallest telocentric chromosome. The result is different from the report of Wurster and Benirschke (1968) which demonstrated a medium submetacentric X chromosome and a small acrocentric Y chromosome in the banded palm civet. The other animals in the family Viverridae have a large metacentric X chromosome, namely A. trivigata, A. binturong, G. genetta, C. civetta, P. linsang, C. ferox and D. hosei (Wurster and Benirschke 1967, 1968, Tanomtong et al. 2005a, 2005b) or a large submetacentric metacentric X chromosome as in P. lavata, V. megaspila and V. indica (Tanomtong et al. 2005c, 2005d, 2005e). There are various types of Y chromosomes appearing in each species; small metacentric in D. hosei (Wurster and Benirschke 1968), small submetacentric in A. binturong and P. lavata (Tanomtong et al. 2005b), small acrocentric in A. trivigata, G. genetta, C. civetta and C. ferox (Wurster and Benirschke 1968, Tanomtong et al. 2005a) and small telocentric in V. megaspila and V. indica (Tanomtong et al. 2005d, 2005e). The chromosome marker of the banded palm civet has been discovered in this study. Metacentric autosome pair 17 was the satellite chromosome with nucleolar organizer regions 94 A. Tanomtong et al. Cytologia 76(1)
Fig. 5. Idiograms of banded palm civet (Hemigalus derbyanus) 2n (diploid) 42 by GTG-banding, arrow indicate satellite chromosomes.
(NORs). It is not consistent with the report of Wurster and Benirschke (1968), which indicated that the smallest submetacentric autosomes is satellite chromosomes. This result is in agreement with previous reports of animals in the family Viverridae, which have 1 pair of satellite chromosomes (Ray-Chaudhuri et al. 1966, Wurster and Benitrschke 1967, 1968, Wada et al. 1983, Wang et al.1984, Masashi and Harumi 1993, Tanomtong et al. 2005a, 2005b, 2005c, 2005d, 2005e), whereas G. genetta has 2 pairs of satellite chromosomes (Wurster and Benitrschke 1968). GTG-banding revealed that the number of GTG-bands on 1 set of haploid chromosomes, which includes autosomes, X and Y chromosomes, is 218. (Fig. 3). The number of bands in 1 set of prometaphase haploid chromosomes by high-resolution technique is 244 (Fig. 4). GTG-banding and high-resolution techniques provide a clearly indicated chromosome band which represent the black (dark band) and white (light band) regions on a chromosome. The level of GTG-banding and high-resolution techniques (band numbers) is defined by a visible and in a haploid set which is 2011 Chromosome Studies in the Banded Palm Civet 95
Fig. 6. Idiogram of banded palm civet (Hemigalus derbyanus) 2n (diploid) 42 by high-resolution technique, arrow indicate satellite chromosomes . composed of autosomes, X and Y chromosome. Thus, the haploid set of the banded palm civet consists of 20 autosomes including X and Y chromosomes. However, some chromosomes did not show as clearly identified because some bands have variation. As above, the chromosome band scoring is represented by approximate bands that appear. Data of the chromosomal checks on mitotic metaphase cells of the banded palm civet are shown in Table 1. Figs. 5 and 6 shows idiograms of the banded palm civet from GTG-banding and high-resolution techniques, with landmarks, regions, bands and sub-bands. The karyotype formula is as follows: sm a sm m sm t 2n (42) L 10 L 12 M 4 S 4 S 8 S 2 sex chromosomes 96 A. Tanomtong et al. Cytologia 76(1)
Table 1. Mean of length 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 metaphase chromosome of 20 cells of banded palm civet (Hemigalus derbyanus) 2n (diploid) 42
Chromosome Chromosome Chromosome Ls Ll LT RL SD CI SD pairs size type
1 0.38 1.21 1.58 0.076 0.005 0.764 0.021 Large Acrocentric 2 0.27 1.14 1.41 0.068 0.004 0.811 0.027 Large Acrocentric 3 0.27 1.08 1.35 0.065 0.004 0.802 0.045 Large Acrocentric 4 0.36 0.91 1.26 0.061 0.002 0.720 0.045 Large Acrocentric 5 0.34 0.89 1.23 0.059 0.003 0.723 0.064 Large Acrocentric 6 0.33 0.87 1.19 0.058 0.003 0.729 0.046 Large Acrocentric 7 0.40 0.74 1.14 0.055 0.003 0.654 0.065 Large Submetacentric 8 0.45 0.69 1.13 0.055 0.003 0.608 0.046 Large Submetacentric 9 0.34 0.69 1.03 0.050 0.004 0.671 0.056 Large Submetacentric 10 0.26 0.77 1.03 0.050 0.002 0.750 0.062 Large Submetacentric 11 0.26 0.67 0.93 0.045 0.001 0.719 0.059 Large Submetacentric 12 0.27 0.59 0.86 0.041 0.004 0.681 0.080 Medium Submetacentric 13 0.30 0.49 0.79 0.038 0.002 0.622 0.042 Medium Submetacentric 14 0.26 0.51 0.77 0.037 0.002 0.662 0.057 Small Submetacentric 15* 0.34 0.38 0.72 0.035 0.003 0.532 0.017 Small Metacentric 16 0.00 0.69 0.69 0.033 0.003 0.996 0.000 Small Telocentric 17 0.24 0.46 0.69 0.034 0.002 0.666 0.059 Small Submetacentric 18 0.21 0.46 0.67 0.032 0.002 0.690 0.024 Small Submetacentric 19 0.26 0.34 0.59 0.029 0.003 0.569 0.042 Small Metacentric 20 0.18 0.41 0.59 0.029 0.003 0.694 0.056 Small Submetacentric X 0.24 0.67 0.90 0.043 0.019 0.739 0.072 Large Acrocentric Y 0.00 0.18 0.18 0.008 0.005 0.972 0.519 Small Telocentric
Notes: * Satellite chromosome (nucleolar organizer region, NOR).
Acknowledgements The financial support from the Buriram Rajabhat University is gratefully acknowledged. We also thank WG.CRD. Krawee Kreetapol, director of Nakhon Ratchasima Zoo, for valuable help. Thanks are also extended to the staff of this Zoo for their good cooperation.
References
Chaiyasut, K. 1989. Cytogenetics and cytotaxonomy of the family Zephyranthes. Department of Botany, Faculty of Science, Chulalongkorn University, Bangkok. Campiranont, A. 2003. Cytogenetics. 2nd ed. Department of Genetics, Faculty of Science, Kasetsart University, Bangkok. Lekagul, B. and McNeely, J. A. 1977. Mammals of Thailand. 1st ed. Kurusapha Ladprao Press, Bangkok. — and — 1988. Mammals of Thailand. 2nd ed. Sahakarn Bhaet, Bangkok. Masashi, H. and Harumi, T. 1993. Karyotype study of the masked palm civet, Paguma lavata in Japan (Viverridae). J. Mammal. Sci. Jpn. 18: 39–42. McPhee, H. 2001. Viverridae. Inet:http//www.press.jhu.edu/book/walkers_mammals_of_word/carnivore.Viverridae.html. Par, J., Komonpalin, K. and Wongkarasin, M. 2003. Mammals of Thailand. Sarakadee Press, Bangkok. Ray-Chaudhuri, S. P., Ranjini, P. V. and Sharma, T. 1966. Somatic chromosome of the common palm civet, Paradoxurus hermaphroditus (Viverridae-Carnivora). Experientia 22: 740–741. Retting, T. and Divers, B. J. 1986. Viverridae. In: Forvler M.E. (ed). Zoo & Wild Animal Medicine, W.B. Saunders Company, Philadelphia. Rooney, D. E. 2001. Human cytogenetics: constitutional analysis. Oxford University Press, Oxford. Stains, H. J. 1984. Carnivores. In: Anderson S; Jones J. K. Jr. (eds). Orders and families of recent mammals of the world. John Wiley and Son, New York. 2011 Chromosome Studies in the Banded Palm Civet 97
Tanomtong, A., Bunjonrat, R., Sriphoom, A., Chinorak, P., Kaensa, W., Kamolnarranath, S. and Dumnui, S. 2005a. A study on karyotype of small-toothed palm civet, Arctogalidia trivirgata (Carnivora, Viverridae) by using conventional staining method. Warasan Songkhla Nakharin 27: 1209–1220. —, —, — and Srisamoot, N. 2005b. Cytogenetics of binturong (Arctictis binturong) in Thailand by using conventional staining. Thai Science and Technology Journal 13: 13–24. —, Chaveerach, A., Sriphoom, A. and Bunjonrat, R. 2005c. Cytogenetic study on wild animal species of the subfamily Paradoxurinae in Thailand. Cytologia 70: 249–255. —, Kaensa, W., Sriphoom, A. and Bunjonrat, R. 2005d. Cytogenetics of large-spotted civet (Viverra megaspila) in Thailand. Kasetsart University Natural Science 22: 64–74. —, Srisamoot, N., Sriphoom, A. and Bunjonrat, R. 2005e. Cytogenetics of small Indian civet (Viverricular indica) in Thailand. Naresuan University Journal 13: 1–10. Vaunhan, T. A. 1972. Mammology. WB Saunders Company, Philadelphia. Wada, M. Y, Nakamura, A. and Yoshida, T. H. 1983. An easy technique to obtain the blood by the clew-cutting from small mammals and bird, and karyotype of some animals from blood culture. Kromosomo 58: 971–976. Wang, Z., Quan, G., Yie, Z. and Wang, S. 1984. Karyotype of three species of Carnivora. Dong Wu Xue Bao 30: 188–195. Walker, E. 1964. Mammals of the world (2 Vols.) Johns Hopkins Press, Baltimore. Wurster, D. H. and Benitrschke, K. 1967. Chromosome numbers in thirty species of Carnivora, mammal. Chromosoma Newsletter 8: 195–216. — and — 1968. Comparative cytogenetic studies in the order Carnivora. Chromosoma (Berl.) 24: 336–382.