80 STANDARDIZED KARYOTYPE AND IDIOGRAM OF THE INDOCHINESE GROUND SQUIRREL, MENETES BERDMOREI (BLYTH, 1849) BY CONVENTIONAL STAINING TECHNIQUE Alongkoad Tanomtong1/* Sitthisak Jantarat1/ Praween Supanuam1/ Sarawut Kaewsri2/ ABSTRACT Standardized karyotype and idiogram of the Indochinese ground squirrel (Menetes berdmorei (Blyth, 1849) at was studied at the Nakhon Ratchasima Zoo, Thailand. Blood samples were taken from two male and two female Indochinese ground squirrels. After lymphocyte culture, the mitotic chromosome preparation was accomplished by hypotonic-air-drying method and conventional Giemsa’s staining technique. The results showed that the number of diploid chromosome was 2n=38 and the fundamental number (NF) was 68 in both female and male. The autosomes consisted of 8 large metacentric, 14 large submetacentric, 2 medium metacentric, 4 small metacentric and 8 small telocentric chromosomes. The X chromosome was a medium metacentric chromosome and the Y chromosome was a small metacentric chromosome. The chromosome marker of the Indochinese ground squirrel is the chromosome pair 1 which is the largest metacentric chromosome. The karyotype formula for the Indochinese ground squirrel is as follows: 2n (38) = Lm8+Lsm14+ Mm2+Sm4+St8 + sex chromosomes

บทคัดยอ คารีโอไทป และอิดิโอแกรมมาตรฐานของกระจอน (Menetes berdmorei) จากสวนสัตวนครราชสีมา ประเทศไทย ใชตัวอยางเลือดกระจอนเพศผู 2 ตัว และเพศเมีย 2 ตัว ทําการเพาะเลี้ยงเซลลเม็ดเลือดขาว และเตรียม โครโมโซมดวยเทคนิคโคลชิซิน-ไฮโปโทนิค-ฟกเซชั่น-แอรดรายอิง ใชวิธียอมสีแบบธรรมดา ผลการศึกษาพบวา กระจอนมีโครโมโซมดิพลอยด (2n) เทากับ 38 แทง มีจํานวนโครโมโซมพื้นฐาน (NF) เทากับ 68 ทั้งในเพศผูและเพศ เมีย โครโมโซมรางกายประกอบดวยโครโมโซมชนิดเมทาเซนทริกขนาดใหญ 8 แทง ซับเมทาเซนทริกขนาดใหญ 14 แทง เมทาเซนทริกขนาดกลาง 2 แทง เมทาเซนทริกขนาดเล็ก 4 แทง และเทโลเซนทริกขนาดเล็ก 8 แทง โครโมโซม เพศมีโครโมโซมเอ็กซเปนชนิดซับเมทาเซนทริกขนาดกลาง และโครโมโซมวายเปนชนิดเมทาเซนทริกขนาดเล็ก ตรวจพบโครโมโซมเครื่องหมายของกระจอน พบวาโครโมโซมรางกายคูที่ 1 เปนโครโมโซมชนิดเมทาเซนทริกขนาด ใหญมากที่สุด กระจอนมีสูตรคาริโอไทป ดังนี้ 2n (ดิพลอยด) 38 = Lm8+Lsm14+ Mm2+Sm4+St8 + โครโมโซมเพศ INTRODUCTION Thailand is a country with high biodiversity, resulting from its complex ecosystem and forests. It has been estimated that there may be up to 100,000 wildlife species in Thailand as well as many unknown plant species. Unfortunately, unchecked economic development and social welfare has led to vast destruction of Thailand’s forests and wildlife resources. Many wildlife species have become extinct due to loss of habitat, and hunting for food, trade and export. The Indochinese ground squirrel (Menetes berdmorei) is member of the family 1/

Department of Biology, Faculty of Science, Khon Kaen University, Muang, Khon Kaen 40002,Thailand, *e-mail : [email protected] 2/ Program in Applied Biology, Faculty of Science, Buriram Rajabhat University, Muang, Buriram 31000, Thailand

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เอกสารนี้ทําขึ้นเพื่อแจกในวาระครบรอบ 30 ปี การสัมมนาเรื่องสัตว์ป่าเมืองไทย

81 Sciuridae. The family has only 29 species of 14 genera in Thailand (Lekagul & McNeely, 1977, 1988; Par et al., 2003). The common characteristics of the Indochinese ground squirrel are black mid-dorsal stripe, two or three black stripes on the sides, and reddish brown fur on the back. Identification is more difficult during the dry season, however, when they are much less brightly colored. At this time they usually have only one distinct black stripe, a very indistinct mid-dorsal stripe, and little or no reddish brown on the back (Lekagul & McNeely, 1977, 1988; Par et al., 2003). There are only two reports of Indochinese ground squirrel cytogenetic studies according to the report of Hsu & Benirschke (1974) and Richard et al. (2003) that demonstrated this species’s karyotype using the R-banding and G-banding technique, respectively. We can reach the basis cytogenetic of the Indochinese ground squirrel which accomplished with our first report on the standardizations of chromosome including chromosome measuring to determined shape and size, karyotype formulating, and idiograming. In addition, we also make a confirmation and comparison for the results with the previous reports. Moreover, this study can provides important basic knowledges and apply to further basis researches. MATERIALS AND METHODS The blood samples were collected from two male and two female the Indochinese ground squirrels, maintained at the Nakhon Ratchasima Zoo, by the aseptic technique. The samples were kept in 10 ml vacuum tubes containing heparin to prevent blood clotting and cooled on ice until arriving at the laboratory. Cell preparation The lymphocytes were cultured using the whole blood microculture technique modified from Rooney (2001) and Kampiranont (2003). RPMI 1640 medium with 2% PHA (Phytohemagglutinin) was prepared as a mitogen in 5 ml culture bottles. One milliliter of blood sample was dropped into the bottle and mixed well. The culture bottle was loosely capped and incubated at 37 ΟC, 5% CO2 and shaken in the morning and evening. At the 96th hour of incubation, colchicine was added and mixed well followed by further incubation for 20 minutes. Cell harvest After centrifuging the blood sample mixture at 3,000 rpm for 5 minutes and the supernatant discarded, 10 ml of hypotonic solution (0.075 M KCl) was applied to the pellet and incubated for 30 minutes. KCl was discarded by centrifugation again. Cells were fixed with a fresh cool fixative (3 methanol: 1 glacial acetic acid) by gradually adding up to 8 ml before centrifuging again and discarding the supernatant. 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, cold slide using a micropipette followed by the air-dry technique. The slide was conventionally stained with 20% stock Giemsa’s solution for 30 minutes.

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เอกสารนี้ทําขึ้นเพื่อแจกในวาระครบรอบ 30 ปี การสัมมนาเรื่องสัตว์ป่าเมืองไทย

82 Chromosomal checks, karyotyping and idiograming Chromosomal from mitotic metaphase cells checking were examined under a light microscope. Twenty clearly observable well-cells spread chromosomes of each male and female were selected and photographed. The length of short arm chromosome (Ls) and the length of long arm chromosome (Ll) were measured and calculated to the length of total arm chromosome (LT, LT = Ls+Ll). The relative length (RL), the centromeric index (CI) and standard deviation (SD) of RL and CI were estimated. CI was also computed to classify the types of chromosomes according to Chaiyasut (1989). All parameters were used in karyotyping and idiograming. RESULTS AND DISCUSSION Cytogenetic study of the Indochinese ground squirrel using lymphocyte culture and the conventional staining procedures revealed that the chromosome number was 2n=38, comprising 36 (18 pairs) autosomes and 2 (1 pair) sex chromosomes. The result is similar to previous studies (Hsu & Benirschke, 1974; Richard et al., 2003; Petit et al., 1984) although those experiments were accomplished with fibroblast culture. This result revealed that the fundamental number (NF, number of chromosome arms) of the Indochinese ground squirrel as 68 in both male and female, differed from Hsu & Benirschke (1974) for which the number was 80. The recent X and Y chromosomes appears as medium submetacentric and small metacentric chromosome, respectively. It differs from Hsu & Benirschke (1974) which indicated submetacentric X chromosome and acrocemtric Y chromosome. Both male and female chromosomes of mitotic metaphase cells and the karyotypes of the Indochinese ground squirrel are shown in appendix Figures 1, 2 and 3. Indochinese ground squirrel autosomes can be divided in to 3 types including metacentric (14 chromosomes), submetacentric (14 chromosomes) and telocentric (8 chromosomes). The 14 metacentric autosomes were classified by size into 8 large, 2 medium and 4 small chromosomes, the 14 submetacentric autosomes were classified by size into 14 large chromosomes while the 8 telocentric autosomes were all 8 small chromosomes. The described chromosomal features are not in agreement with the report of Hsu & Benirschke (1974) and Richard et al. (2003) that demonstrated 34 metacentric and submetacentric, and 4 acrocentric chromosomes of Indochinese ground squirrel. The chromosome length in centimeters of twenty cells (males and females) in the mitotic metaphase was measured. The mean length of short arm chromosome (Ls), length of long arm chromosome (Ll), total length of arm chromosome (LT), relative length (RL), centromeric index (CI), standard deviation (SD) of RL, CI, size and type are presented in appendix Table 1. The idiogram of the Indochinese ground squirrel shows gradually decreasing length of the autosomes and sex chromosomes (Appendix Figure 4).

วารสารสัตวปาเมืองไทย ปที่ 15 ฉบับที่ 1 ธันวาคม 2551

เอกสารนี้ทําขึ้นเพื่อแจกในวาระครบรอบ 30 ปี การสัมมนาเรื่องสัตว์ป่าเมืองไทย

83 We also found that the chromosome marker of Indochinese ground squirrel is the chromosome pair 1 which is the largest metacentric chromosome. The important chromosome marker of the Indochinese ground squirrel is the asymmetrical karyotype, which was found in all three types of chromosomes (metacentric, submetacentric and telocentric chromosome). The largest and smallest chromosomes show different sizes (approximately 5 folds). The karyotype formula of male and female for Indochinese ground squirrel was as follows: 2n (38) = Lm8+Lsm14+ Mm2+Sm4+St8 + sex chromosomes CONCLUSION Standardized karyotype and idiogram of the Indochinese ground squirrel was studied. After lymphocyte culture, the mitotic chromosome preparation was accomplished by the hypotonic-air-drying method and conventional Giemsa’s staining technique. The results showed that the number of diploid chromosomes was 2n=38 and the fundamental number was 68 in female and male. The autosomes consisted of 8 large metacentric, 14 large submetacentric, 2 medium metacentric, 4 small metacentric and 8 small telocentric chromosomes. The X chromosome was a medium metacentric chromosome and the Y chromosome was a small metacentric chromosome. The Indochinese ground squirrel revealed that the chromosome marker is the chromosome pair 1, which is the largest metacentric chromosome. ACKNOWLEDGEMENTS The financial support from Thai Government Science and Technology Scholarship Students. We would like to thank the director of the Nakhon Ratchasima Zoo for the Indochinese ground squirrel blood samples. Thanks are also intended to the staffs of this Zoo for good cooperation. Dr. Lon Grassman is appreciated for helping with the English editing.

REFERENCES Chaiyasut, K. 1989. Cytogenetics and cytotaxonomy of the family Zephyranthes. Department of Botany, Faculty of Science, Chulalongkorn University, Bangkok. Hsu, T.C. & K. Benirschke. 1974. An atlas of mammalian chromosomes volume 1. Springer-Verlag: Berlin, Heidelberg, New york. Kampiranont, A. 2003. Cytogenetics. 2nd ed. Department of Genetics, Faculty of Science, Kasetsart University, Bangkok, Thailand. Lekagul, B. & J. A. McNeely. 1977. Mammals of Thailand. 1st ed. Kurusapha Ladprao Press,Bangkok. Lekagul, B. & J.A. McNeely. 1988. Mammals of Thailand. 2nd ed. Sahakarn Bhaet, Bangkok.

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เอกสารนี้ทําขึ้นเพื่อแจกในวาระครบรอบ 30 ปี การสัมมนาเรื่องสัตว์ป่าเมืองไทย

84 Par, J. 2003. Mammals of Thailand. Sarakadee Press, Bangkok. Petit, D., J. Couturier, E. Viegas-Pequignot,M. Lombard & B. Dutrillaux. 1984. Tre's grande similitude entre le caryotype ancestral des e'cureuils (rongeurs) et celui des primates et des carnivores. Annual Genetics 27: 201-212. Richard, F., C. Messaoudi, A. Bonnet-Garnier, M. Lombard & B. Dutrillaux. 2003. Highly conserved chromosomes in an Asian squirrel (Menetes berdmorei, Rodentia: Sciuridae) as demonstrated by ZOO-FISH with human probes. Chromosome Research 11 : 597–603. Rooney, D.E. 2001. Human cytogenetics: constitutional analysis. Oxford University Press, Oxford. APPENDIX

A. Y

B. X

XY

XY Y

X

D.

C. XX

XX

X X

X X

Figure 1 Metaphase chromosome plates of male (A. and B.) and female (C. and D.) the Indochinese ground squirrel (Menetes berdmorei) 2n (diploid) = 38, by conventional staining technique (arrows indicate sex chromosomes; scale bars 10 microns).

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เอกสารนี้ทําขึ้นเพื่อแจกในวาระครบรอบ 30 ปี การสัมมนาเรื่องสัตว์ป่าเมืองไทย

85

X

Y 10 micron

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

X Y

Figure 2 Metaphase chromosome plate and karyotype of the male Indochinese ground squirrel (Menetes berdmorei) 2n (diploid) = 38, by conventional staining technique (arrows indicate sex chromosomes).

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เอกสารนี้ทําขึ้นเพื่อแจกในวาระครบรอบ 30 ปี การสัมมนาเรื่องสัตว์ป่าเมืองไทย

86

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

X X

Figure 3 Metaphase chromosome plate and karyotype of the female Indochinese ground squirrel (Menetes berdmorei) 2n (diploid) = 38, by conventional staining technique(arrows indicate sex chromosomes).

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เอกสารนี้ทําขึ้นเพื่อแจกในวาระครบรอบ 30 ปี การสัมมนาเรื่องสัตว์ป่าเมืองไทย

87

P q 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

P q

P 18

X

Y

Figure 4 Idiogram of the Indochinese ground squirrel (Menetes berdmorei) 2n (diploid) = 38, by conventional staining technique.

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เอกสารนี้ทําขึ้นเพื่อแจกในวาระครบรอบ 30 ปี การสัมมนาเรื่องสัตว์ป่าเมืองไทย

88 Table 1 The mean value of the short arm chromosome length (Ls), the long arm chromosome length (Ll), the total arm length of the chromosome (LT), othe relative length (RL) and of the centromeric index (CI). The standard deviation (SD) of RL and CI, and the size and type of the chromosomes of 20 cells in male the Indochinese ground squirrel (Menetes berdmorei), 2n (diploid) = 38. Chromosome pairs 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 X Y

Ls

Ll

LT

CI±SD

RL±SD

0.950 0.639 0.880 0.814 0.570 0.538 0.493 0.753 0.488 0.483 0.473 0.514 0.449 0.000 0.000 0.312 0.000 0.000 0.438 0.295

1.273 1.502 1.194 1.117 1.251 1.194 1.192 0.905 1.128 1.085 1.023 0.666 0.565 0.849 0.779 0.397 0.677 0.496 0.962 0.319

2.243 2.155 2.091 1.948 1.834 1.746 1.696 1.676 1.625 1.580 1.506 1.190 1.023 0.849 0.779 0.716 0.677 0.496 1.400 0.614

0.566±0.037 0.697±0.052 0.571±0.033 0.574±0.046 0.680±0.075 0.681±0.090 0.699±0.066 0.539±0.034 0.693±0.086 0.687±0.076 0.677±0.054 0.559±0.054 0.552±0.038 1.000±0.000 1.000±0.000 0.558±0.088 1.000±0.000 1.000±0.000 0.687±0.045 0.524±0.059

0.041±0.004 0.040±0.004 0.039±0.002 0.036±0.003 0.034±0.002 0.032±0.002 0.031±0.002 0.031±0.002 0.030±0.001 0.029±0.002 0.028±0.002 0.022±0.003 0.019±0.005 0.016±0.003 0.014±0.002 0.013±0.003 0.013±0.003 0.009±0.003 0.026±0.002 0.011±0.001

Size of chromosome L L L L L L L L L L L M S S S S S S M S

Type of chromosome m sm m m sm sm sm m sm sm sm m m t t m t t sm m

Notes: L = large chromosome, M = medium chromosome, S = small chromosome, m = metacentric chromosome, sm = submetacentric chromosome and t = telocentric chromosome.

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