Karyotype Study of 5 Species of the Family Viverridae in Thailand by High Resolution GTG-Banding Technique

Home , Asian palm civet, Banded Linsang, Banded palm civet, Large Indian civet, Linsang, Masked palm civet

Alongklod Tanomtong1*, Sumpars Khunsook1, Namphunk Seatung1, Wiwat Sangpadee2, Sarawut Kaewsri3 and La-orsri Sanoamuang1

1 Applied Taxonomic Research Center (ATRC), Department of Biology, Faculty of Science, Khon Kaen University, Thailand 2 Biology Program, Faculty of Science, Udon-Thani Rajabhat University, Muang, Udon-Thani 41000, Thailand 3 Program in Applied Biology, Department of Science, Faculty of Science, Buriram Rajabhat University, Buriram, Muang 31000, Thailand

Received April 1, 2011; accepted October 30, 2011

Summary As endangered species in Thailand, the wild animal species of the common palm civet (Paradoxurus hermaphrodites), masked palm civet (Paguma larvata), large Indian civet (Viverra zibetha), large-spotted civet (Viverra megaspila), and binturong (Arctictis binturong) were selected for karyological s tudy. Blood samples were taken from 1 male and 1 female of each species. After the standard whole blood lymphocyte culture in the presence of colchicine, the prometaphase spreads were performed on microscopic slides and air-dried. High resolution GTG-banding technique was applied to stain the chromosomes. The results showed that 2n (diploid) of Par. hermaphrodites, Pag. larvata, V. zibetha, V. megaspila and A. binturong were 42, 44, 38, 38, and 42, respectively. The autosomes presence of metacentric, submetacentric, acrocentric and telocentric autosomes were 6-10-14-10, 6-8-12-16, 8-6-18-4, 4-12-16-4 and 6-12-4-18, respectively. The X chromosome were submetacentric, submetacentric, metacentric, submetacentric and metacentric, and the Y chromosome were submetacentric, acrocentric, telocentric, telocentric and acrocentric chromosome, respectively . The numbers of bands in one set of prometaphase haploid chromosomes from the high resolution GTG-banding technique were 233, 262, 198, 222, and 247 bands, respectively and each chromosome pair could be clearly differentiated.

Kew words Viverridae, Karyotype, Chromosome, Idiogram, High resolution GTG-banding technique.

Across the entire world, animals in the family Viverridae are found in 6 subfamilies, 20 genera, and 34 species (Wilson and Cole 2000). In Thailand, these animals were found 3 subfamilies, 9 genera, and 11 species namely; binturong (Arctictis binturong Rafﬂes, 1821), Asian palm civet (Paradoxurus hermaphroditus Pallas, 1777), masked palm civet (Paguma larvata Smith, 1827), small-toothed palm civet (Arctogalidia trivirgata Gray, 1832), banded linsang (Priondon linsang Hardwicke, 1821), large-spotted civet (Viverra megaspila Blyth, 1862), spotted linsang (Prionodon pardicolor Hodgson, 1977), small Indian civet (Viverricula indica Desmarest, 1817), large Indian civet (Viverra zibetha Linnaeus, 1758), banded palm civet (Hemigalus derbyanus Gray, 1837) and otter civet (Cynogale bennetti Gray, 1837) (Lekagul and McNeely 1988, Wilson and Cole 2000, Parr 2003). High-resolution banding technique provides possibilities to detect chromosome breaks and re- arrangements event within major bands. The developments in cell culture and banding techniques have been extremely fast; 10 years after the Paris conference in 1971, Yunis (1981) published the

* Corresponding author, e-mail: [email protected] 464 A. Tanomtong et al. Cytologia 76(4) haploid human karyotype at the 1,700 band. For other mammals, development was slower, but recently good results have narrowed the gap between human and animal cytogenetics. However, the increase of bands in a given karyotype is not an aim in itself, but a research tool, so the value of a high band level is reduced if band quality is sacrificed in the process. When high-resolution banding is combined with other chromosomal techniques, specific sites on the chromosomes can be de- tected and precisely localized. High-resolution banding technique has been extremely valuable, es- pecially for the localization of single copy genes and specific breakpoints (Rønne 1991). From literature review about cytogenetic studies of animals in the family Viverridae according to Ray-Chaudhuri et al. (1966), Wurster and Benitrschke (1967, 1968), Wada et al. (1983), Wang et al. (1984), Masashi and Harumi (1993) and Tanomtong et al. (2005a, 2005b, 2005c, 2005d, 2005e, 2006). For the present study, this is the first report on chromosomal characteristics in 5 species of the family Viverridae from Thailand by high resolution GTG-banding technique. Our knowledge will advance cytogenetic information for further study on taxonomy and evolutionary relationship. Moreover, it is useful basic information for the conservation, breeding, and study of chromosome evolution in these animals.

Materials and methods

Blood samples from the jugular vein were collected from 1 male and 1 female of Par. hermaphrodites, Pag. larvata, V. zibetha, V. megaspila and A. binturong (Fig. 1), which were kept in The Zoological Park Organization under the Royal Patronage of H. M. the King, 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. The lymphocytes were cultured using the whole blood microculture technique adapted from Rooney (2001) and Campiranont (2003).

Cell culture The RPMI 1640 medium was prepared with 2% PHA (Phytohemagglutinin) as a mitogen and

Fig. 1. General characteristics of animals in the family Viverridae namely; common palm civet, Paradoxurus hermaphrodites (A); masked palm civet, Paguma larvata (B); large Indian civet, Viverra zibetha (C); large-spotted civet, Viverra megaspila (D) and binturong, Arctictis binturong (E) after Parr (2003). 2011 Karyotype Study of 5 Species of the Family Viverridae in Thailand 465 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 hour of incubation, colc hicine was introduced and well mixed followed by further incubation for 30 min.

Cell harvest The blood sample mixture was centrifuged at 1200 rpm for 10 min and the supernatant was discarded. Ten milliliters 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 1200 rpm for 10 min. Cells were fixed by fresh cool fixative (methanol : glacial acetic acid=3 : 1) gradually added up to 8 ml before centrifuging again at 1200 rpm for 10 min and the supernatant 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 followed by the air-dry technique.

High resolution GTG-banding method High-resolution GTG-banding technique was adapted from Rooney (2001). After the lympho-

Fig. 2. Prometaphase chromosome plates and karyotypes of male (A) and female (B) common palm civet (Paradoxurus hermaphrodites), 2n=42 by high resolution GTG-banding technique, showing sex chromosomes and nucleolar organizer regions, NORs (arrows), scale bars=10 μm. 466 A. Tanomtong et al. Cytologia 76(4) cytes were cultured for 72 h, 0.05 ml of 10-5 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 centrifuged 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 harvested at the exact time and stained by using GTG-banding procedure.

Results and discussions

Karyological studies of Par. hermaphrodites, Pag. larvata, V. zibetha, V. megaspila and A. binturong using lymphocyte culture, the high resolution GTG-banding technique procedures revealed that the chromosome numbers were 2n (diploid)=42, 44, 38, 38 and 42, respectively. The autosomes presences of metacentric, submetacentric, acrocentric and telocentric autosomes were 6-10-14-10, 6-8-12-16, 8-6-18-4, 4-12-16-4 and 6-12-4-18, respectively. The X chromosomes were submetacentric, submetacentric, metacentric, submetacentric and metacentric, and the Y chromo-

Fig. 3. Prometaphase chromosome plates and karyotypes of male (A) and female (B) masked palm civet (Paguma larvata), 2n=44 by high resolution GTG-banding technique, showing sex chromosomes and nucleolar organizer regions, NORs (arrows), scale bars=10 μm. 2011 Karyotype Study of 5 Species of the Family Viverridae in Thailand 467 somes were submetacentric, acrocentric, telocentric, telocentric and acrocentric chromosome, respectively. This conforms with the reports by Tanomtong et al. (2005c) and (2006). The high resolution GTG-banding technique revealed that the number on 1 set of haploid chromosomes (n), which includes autosomes, the X and Y chromosome. The numbers of bands in 1 set of prometaphase haploid chromosomes from the high-resolution GTG-banding technique are 233, 262, 198, 222, and 247 bands, respectively (Figs. 2–6). Compared with the study by Yunis (1982) which reported that chromosome band numbers from the high resolution technique of prometaphase chromosome of human and ape are over 1,000 bands per haploid set. Stanyon (1987) suggested that the high-resolution technique is highly efﬁcient for the different chromosome com- parison of Mucaca fuscuta and Cercorebus atereimus. In this study, the chromosome scoring is done with only clearly visible bands except for variable bands due to the small number of scored bands. Figures 7–11 shows the idiograms for the Par. hermaphrodites, Pag. larvata, V. zibetha, V. megaspila and A. binturong from high resolution GTG-banding technique. Mammalian chromosomes can be deﬁned by 3 structural classes visualized as the G/Q-bands,

Fig. 4. Prometaphase chromosome plates and karyotypes of male (A) and female (B) large Indian civet, Viverra zibetha, 2n=38 by high resolution GTG-banding technique, showing sex chromosomes and nucleolar organizer regions, NORs (arrows), scale bars=10 μm. 468 A. Tanomtong et al. Cytologia 76(4)

Fig. 5. Prometaphase chromosome plates and karyotypes of male (A) and female (B) large-spotted civet, Viverra megaspila, 2n=38 by high resolution GTG-banding technique, showing sex chromosomes and nucleolar organizer regions, NORs (arrows), scale bars=10 μm. the R-bands, and the C-bands, which are associated with different functional and biochemical attri- butes. This chromosomal pre-pattern has developed during vertebrate evolution and determines the kind and quality of banding which can be obtained from different vertebrate taxa (Holmquist 1989). Chromosomes become progressively shortened as the cell progresses from interphase to metaphase. This behavior allows the chromatin to be neatly packaged for segregation into 2 daughter cells at the end of telophase. As cell culture techniques evolve, cytogenetic analysis has gradually moved from being performed on mid-metaphase chromosomes to longer early metaphase or even late prophase chromosones. This can be accomplished by synchronizing the cell cycle with a block at the S-phase and subsequent release with a releasing agent. Several block and release reagents are available, such as a methotrexate block with a thymidine release, or an excess thymidine block with 2-deoxycytidine as a releasing agent. With the release, the cells then progress at the same stage of the cell cycle and many cells can be harvested at prometaphase and early metaphase after a brief 2011 Karyotype Study of 5 Species of the Family Viverridae in Thailand 469

Fig. 6. Prometaphase chromosome plates and karyotypes of male (A) and female (B) binturong, Arctictis binturong, 2n=42 by high resolution GTG-banding technique, showing sex chromosomes and nucleolar organizer regions, NORs (arrows), scale bars=10 μm. exposure to colchicine at low concentration. This mitotic arresting acts by preventing polymeriza- tion of the tubulin molecule into a microtubule that makes up the spindle ﬁbers. This prevents the chromatids from aligning on the metaphase plate to be pulled apart during anaphase. The stoichio- metric relationship between colc hicines and tubulin needs to be borne in mind when deciding the amount of the mitotic arresting to be added. This is because the concentration of colchicine has been shown to be critical as it affects the mitotic index, chromosome spreading, and chromosome length (Yunis et al. 1978, Lawce 2002, Lim et al. 2004). Another method of obtaining long chromosomes is to add chemical reagents that prevent chromosome condensation. These additives intercalate into the DNA so that the chromosomes do not shorten during their progress from interphase to metaphase, even in the presence of colchicine. Several chromosome anti-contraction agents are available and include ethidium bromide, the thymidine analogue BrdU, 5-azacytidin, actinomycin D, echinomycin, and others (Lawce and Brown 1997, Sawyer 1995). Recently, 9-aminoacridine was used to increase chromosome spreads to 850 BPHS resolution, compared to 600–700 BPHS with ethidium bromide (Muravenko et al. 2003, Lim et al. 2004). 470 A. Tanomtong et al. Cytologia 76(4) resolution GTG-banding technique, showing nucleolar organizer region, NOR (arrow). 8. I

Fig. diogram of masked palm civet ( Paguma larvata ), 2 n = 44 by high high resolution GTG-banding technique, showing nucleolar organizer region, NOR (arrow). 7. I

Fig. diogram of common palm civet ( Paradoxurus hermaphrodites ), 2 n = 42 by 2011 Karyotype Study of 5 Species of the Family Viverridae in Thailand 471 Viverra megaspila , 2 n = 38 by high showing nucleolar organizer region, NOR (arrow). resolution GTG-banding technique, 10. I

Fig. diogram of large-spotted civet, Viverra zibetha , Viverra n = 38 2 by high resolution GTG-banding technique, showing nucleolar organizer region, NOR (arrow). 9. I

Fig. diogram of large Indian civet, 472 A. Tanomtong et al. Cytologia 76(4)

Fig. 11. Idiogram of binturong, Arctictis binturong, 2n=42 by high resolution GTG-banding technique, showing nucleolar organizer region, NOR (arrow).

Acknowledgements

These works were supported by the Applied Taxonomic Research Center (ATRC), Khon Kaen University grant ATRC-R5304 and the Zoological Park Organization under the Royal Patronage of H.M. the King is gratefully acknowledged.

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