The Standard Karyotype of the Asiatic Jackal, Canis Aureus (Carnivora, Canidae) from Thailand

The Standard Karyotype of the Asiatic Jackal, Canis aureus (Carnivora, Canidae) from Thailand

Alongklod Tanomtong1*, Prapakorn Chaiyaphan1, Praween Supanuam2, Therdsak Puramongkol3, Nuntiya Maneechot4 and Nantana Jangsuwan5

1 Applied Taxonomic Research Center (ATRC), Department of Biology, Faculty of Science, Khon Kaen University, Khon Kaen, Muang 40002, Thailand 2 Biology Program, Faculty of Science, Ubonratchatani Rajabhat University, Ubon Ratchatani, Muang 34000, Thailand 3 Faculty of Agro-Industrial Technology, Rajamangala University of Technology Tawan-ok, Chanthaburi Campus, Chanthaburi, Khao Kitchagoot 22210, Thailand 4 Department of Fundamental Science, Faculty of Science and Technology, Surindra Rajabhat University, Surin, Muang 32000, Thailand 5 Applied Biology Program, Faculty of Science and Technology, Thepsatri Rajabhat University, Lopburi, Muang 15000, Thailand

Received November 28, 2013; accepted September 18, 2014

Summary This study conducted karyological analysis and detection of nucleolar organizer regions (NORs) in the Asiatic jackal (Canis aureus Linnaeus, 1758) from Khao Suan Kwang Zoo, Khon Kaen Province, Thailand. Blood samples were taken from two male and two female jackals. After standard whole blood lymphocytes had been cultured at 37°C for 72 h in the presence of colchicine, metaphase spreads were performed on microscopic slides and air-dried. Giemsa’s staining, GTG- banding, high-resolution banding and Ag-NOR banding techniques were used to stain chromosomes. The results showed that the diploid chromosome number of C. aureus was 2n=78 and the fundamental numbers (NF) of both sexes were 80. The types of autosomes were 18 large telocentric, 18 medium telocentric and 40 small telocentric chromosomes. The X chromosome was the largest metacentric chromosome and the Y chromosome was the smallest acrocentric chromosome. In addition, the long arm near the centromere of chromosome pair 7 and the long arm near the telomere of chromosome pairs 10 and 28 contained NORs. From the GTG-banding and high-resolution banding techniques, the numbers of bands were 205 and 269, respectively, and each chromosome pair could be clearly differentiated. The karyotype formula for C. aureus is: 2n (diploid) t t t 78=L18+M18+S40+sex chromosomes.

Key words Asiatic jackal, Canis aureus, Karyotype, Chromosome.

The family Canidae is composed of 16 genera and 36 species of wolves, jackals, foxes and dogs (Nowak 1999). In Thailand, there are three species; domestic dog (Canis familiaris), Asiatic jackal (Ca. aureus) and Asian wild dog (Cuon alpinus). In Thailand, Ca. aureus may be confused with Cu. alpinus, but they differ morphologically in being generally greyish brown, not reddish, and by having the shoulder hairs tipped black, forming a saddle-like pattern. Also, the muzzle is not blackish as it is in Cu. alpinus, and the tail is short and blackish in only its distal third (Lekagul and McNeely 1988). Comparative analysis of chromosomes has also proved very useful because canids have a rich diversity of chromosome morphology ranging from species such as the red fox (Vulpes vulpes,

* Corresponding author, e-mail: [email protected] DOI: 10.1508/cytologia.80.3 4 A. Tanomtong et al. Cytologia 80(1)

Fig. 1. General characteristics of the Asiatic jackal (Canis aureus Linnaeus, 1758) from Thailand.

2n=34) and cape fox (V. corsac, 2n=36), which have a low diploid chromosome number and all metacentric autosomes, to the Ca. familiaris, Coyote (Ca. latrans), gray wolf (Ca. lupus) and Cu. alpinus, which have a high diploid chromosome number (2n=78) and all acrocentric autosomes. The primitive canid karyotype has been reshufﬂed in different lineages in a way that reveals the phylogenetic history of the group. The evolutionary sequence of chromosomal rearrangements is deduced by differentially staining chromosomes and matching segments of similar banding patterns in different species (Wayne et al. 1987). The karyotype of C. aureus was ﬁrst reported by Matthey (1954) and shows a diploid chromosome number (2n) of 74. In the present study, conventional staining, GTG-banding, high- resolution banding and Ag-NOR banding techniques were used for a more detailed chromosome characterization of C. aureus, and aspects of chromosome evolution of the family Canidae were discussed.

Materials and methods

The blood samples were collected by aseptic technique from two male and two female jackals (Fig. 1), kept in Khao Suan Kwang Zoo, Khon Kaen Province, Thailand. The samples were placed in 10 ml vacuum tubes containing heparin to prevent blood clotting and cooled on ice until arriving at the laboratory. Lymphocytes were cultured using the whole blood microculture technique adapted from Rooney (2001).

Cell culture Five millilitres of RPMI 1640 medium was prepared with 2% PHA (Phytohemagglutinin) as a mitogen and kept in blood culture ﬂasks. Blood samples of 0.5 ml aliquots were dropped into a medium bottle and mixed well. The culture bottles were loosely capped, incubated at 37°C under a 5% carbon dioxide environment and regularly shaken in the morning and evening. After harvesting at the 72 h of incubation, colchicine was added and mixed well, followed by further incubation for 30 min.

Cell harvest The blood sample mixture was centrifuged at 3,000 rpm for 5 min and the supernatant was discarded. Ten millilitres of hypotonic solution (0.075 M KCl) was applied to the pellet and the mixture incubated for 30 min. KCl was discarded from the supernatant after centrifugation again at 3,000 rpm for 5 min. Cells were fixed in a fresh cool fixative (3 methanol : 1 glacial acetic acid) 2015 The Standard Karyotype of the Asiatic Jackal Canis aureus from Thailand 5 gradually added up to 8 ml before centrifuging again at 3,000 rpm for 5 min, and the supernatant was discarded. The fixation was repeated until the supernatant was clear, then the pellet was mixed with 1 ml fixative. The mixture was dropped onto a clean and cold slide by a micropipette followed by the air-dry technique. The slide was conventionally stained with 20% Giemsa’s solution for 30 min.

G-banding method The G-banding technique was adapted from Campiranont (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 Sorensen’s buffer. The slide was stained with 20% Giemsa’s solution for 30 min.

High-resolution banding method The high-resolution banding technique was adapted from Rooney (2001). After the lymphocytes 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 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.

Ag-NOR banding method Two drops each of 50% silver nitrate and 50% gelatin were added on slides, respectively which were sealed with cover glasses and incubated at 60°C for 3 h. They were then soaked in distilled water until cover glass separation. The slide was stained with 20% Giemsa’s solution for 1 min (Howell and Black 1980).

Chromosomal checks, karyotyping and idiograming Chromosome counting was performed on mitotic metaphase cells under a light microscope. Twenty clearly observable and well-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

The Ca. aureus showed a diploid chromosome number of 78 chromosomes, comprising 38 pairs of autosomal telocentric chromosomes (18 large, 18 medium and 40 small chromosomes) and two sex chromosomes, the largest metacentric X-chromosome and the smallest acrocentric Y-chromosome (Fig. 2). The fundamental number (NF, number of chromosome arm) were 80 in both sexes. This is different from a previous study by Matthey (1954), who reported that Ca. aureus was 2n=74. Species described from the family Canidae have 2n between 34 and 78. Most chromosomes are mono-armed in species with higher diploid chromosome numbers, while in species with lower 2n, they are bi-armed (Hatanaka and Galetti 1999). In the family Canidae a wide range of diploid chromosome numbers is observed (Wayne et al. 1987). Moreover, an extensive chromosome and karyotype polymorphism in some species has been described (Table 1) (Rogalska-Niznik et al. 2003). Ca. aureus, Ca. familiaris, Ca. latrans, Ca. 6 A. Tanomtong et al. Cytologia 80(1)

Fig. 2. Metaphase chromosome plates and karyotypes of male (A) and female (B) Asiatic jackals (Canis aureus), 2n=78 by conventional staining technique. Scale bar indicates 10 μm.

Table 1. Review of canids (foxes, wolves, jackal and dogs) cytogenetic publications in the family Canidae (Carnivora).

Species 2n Karyotype formulas NF Sex chro. References

Canis aureus 78 76t 80 X (m) Y (a) Present study 74 ̶ 76 ̶ Matthey (1954) Ca. familiaris 78 76a 80 X (m) Y (m) Selden et al. (1975) 78 ̶ ̶ ̶ Yang et al. (1999) 78 ̶ ̶ ̶ Graphodatsky et al. (2000) Ca. latrans 78 ̶ ̶ ̶ Wurster-Hill and Centerwall (1982) Ca. lupus 78 ̶ ̶ ̶ Wurster-Hill and Centerwall (1982) Cerdocyon thous 74 ̶ ̶ ̶ Nash et al. (2001) Cuon alpinus 78 ̶ ̶ ̶ Graphodatsky et al. (2007) Dusicyon vetulus 74 72t+2m 76 ̶ Wurster and Benirschke (1968) Fennecus zerda 64 58t+4m/sm 70 X (sm) Y (̶) Wurster and Benirschke (1968) Atelocynus microtis 64 ̶ ̶ ̶ Graphodatsky et al. (2007) 74–76 72t/st+2sm 76 ̶ Wurster and Benirschke (1968) 74 ̶ ̶ ̶ Wurster-Hill and Centerwall (1982) Urocyon cinereoargenteus 66 62t/st+2m 70 X (sm) Y (̶) Wurster and Benirschke (1968) 66 ̶ ̶ ̶ Graphodatsky et al. (2007) Alopex lagopus 48, 49, 50 bi+4a ̶ X (m) Y (a) Mäkinen et al. (1985a) 50 ̶ ̶ ̶ Graphodatsky et al. (2000) 50 ̶ ̶ ̶ Nash et al. (2001) Vulpes velox 50 ̶ ̶ ̶ Nash et al. (2001) V. vulpes 34+0–8Bs 17bi ̶ X (m) Y (a) Mäkinen et al. (1985b) 34+Bs ̶ ̶ ̶ Yang et al. (1999) 34+Bs ̶ ̶ ̶ Graphodatsky et al. (2000) V. corsac 36 ̶ ̶ ̶ Graphodatsky et al. (2007) V. macrotis 50 ̶ ̶ ̶ Wayne et al. (1987) Nyctereutes procyonoides 38+2–7Bs 26bi+10a 66 X (bi) Y (bi) Wada et al. (1998) Viverrinus 38+Bs ̶ ̶ ̶ Nie et al. (2003) N. p. procyonoides 54+1–4Bs 10bi+42a 66 ̶ Mäkinen et al. (1986) 54+Bs ̶ ̶ ̶ Graphodatsky et al. (2001) 54+Bs ̶ ̶ ̶ Nash et al. (2001) Chrysocyon brachyurus 76 74a 78 X (sm) Y (a) Hatanaka and Galetti (1999) 76 ̶ ̶ ̶ Wayne et al. (1987) Speothos venaticus 74 72a 76 X (sm) Y (a) Carvalho et al. (2010) 74 ̶ ̶ ̶ O’Brien et al. (2006) Otocyon megalotis 72 ̶ ̶ ̶ Wayne et al. (1987)

Remarks: 2n=diploid chromosome number, NF=fundamental number (number of chromosome arm), chro.= chromosome, m=metacentric, sm=submetacentric, a=acrocentric, t=telocentric, st=subtelocentric, Bs=B-chromosomes (supernumerary chromosomes), bi=bi-armed chromosome, /= and, and ̶ =not available. 2015 The Standard Karyotype of the Asiatic Jackal Canis aureus from Thailand 7

Fig. 3. Metaphase chromosome plates and karyotypes of male (A) and female (B) Asiatic jackals (Canis aureus), 2n=78 by GTG-banding technique. Scale bar indicates 10 μm.

Fig. 4. Metaphase chromosome plates and karyotypes of male (A) and female (B) Asiatic jackals (Canis aureus), 2n=78 by high-resolution banding technique. Scale bar indicates 10 μm. 8 A. Tanomtong et al. Cytologia 80(1) lupus and Cu. alpinus have the highest diploid chromosome number (2n=78), with all autosomes being acrocentric chromosomes. In contrast, V. vulpes has the lowest diploid chromosome number (2n=34+0–8Bs) with a variable number of supernumerary or accessory microchromosomes, described as B chromosomes (Mäkinen et al. 1985b). The arctic fox (Alopex lagopus) showed three karyotype forms with diploid chromosome numbers of 48, 49 and 50 chromosomes. Extensive chromosome polymorphism, caused by the Robertsonian translocation (centric fusion) between the only two acrocentric autosome pairs 23 and 24, is found in this species (Mäkinen et al. 1985a). In the karyotype of this species, 10 chromosome pairs have complete heterochromatic arms, for which size polymorphisms were found (Switonski and Gustavsson 1991). There are two subspecies of the raccoon dog: the Chinese raccoon dog (Nyctereutes procyonoide procyonoide, 2n=54+1–4Bs) and the Japanese raccoon dog (N. p. viverrinus, 2n=38+2–7Bs). Both subspecies have variable numbers of supernumerary macrochromosomes (Mäkinen et al. 1986, Wada et al. 1998, Graphodatsky et al. 2001, Nash et al. 2001, Nie et al. 2003). The GTG-banding and high-resolution banding techniques provide clear chromosome bands which are represented as black (heterochromatin) and white (euchromatin) regions on chromosomes. The level of high-resolution banding technique (band numbers) is deﬁned by a visible band and a haploid set (n) which is composed of autosomes, X and Y chromosomes. Thus, the haploid set of the Ca. aureus consists of 38 autosomes including X and Y chromosomes. However, some chromosomes cannot clearly be identiﬁed because some bands are variable. The results demonstrate that the chromosome band numbers of the Ca. aureus from GTG-banding of metaphase chromosome and high-resolution banding technique of prometaphase or late prophase chromosome are 205 and 269, respectively (Figs. 3 and 4). Moreover, Yunis (1976) reported that chromosome band number from the high-resolution technique of prometaphase chromosomes of

Fig. 5. Metaphase chromosome plates and karyotypes of male (A) and female (B) Asiatic jackals (Canis aureus), 2n=78 by Ag-NOR banding technique. Arrows indicate nucleolar organizer regions/NORs (scale bars=10 μm). 2015 The Standard Karyotype of the Asiatic Jackal Canis aureus from Thailand 9

Table 2. Mean of length short arm chromosome (Ls), long arm chromosome (Ll), total arm chromosome (LT), relative length (RL), centromeric index (CI) and standard deviation (SD) of RL, CI from metaphase chromosomes in 20 cells of male and female Asiatic jackal (Canis aureus), 2n=78.

Chromosome Chromosome Chromosome Ls (cm) Ll (cm) LT (cm) RL±SD CI±SD pairs sizes types

1 0.000 3.341 3.341 0.045±0.005 1.000±0.000 L t 2 0.000 2.697 2.697 0.036±0.002 1.000±0.000 L t 3 0.000 2.616 2.616 0.035±0.002 1.000±0.000 L t 4 0.000 2.517 2.517 0.034±0.002 1.000±0.000 L t 5 0.000 2.460 2.460 0.033±0.001 1.000±0.000 L t 6 0.000 2.412 2.412 0.032±0.002 1.000±0.000 L t 7* 0.000 2.332 2.332 0.031±0.002 1.000±0.000 L t 8 0.000 2.302 2.302 0.031±0.001 1.000±0.000 L t 9 0.000 2.249 2.249 0.030±0.001 1.000±0.000 L t 10* 0.000 2.181 2.181 0.029±0.001 1.000±0.000 M t 11 0.000 2.146 2.146 0.029±0.001 1.000±0.000 M t 12 0.000 2.103 2.103 0.028±0.001 1.000±0.000 M t 13 0.000 2.066 2.066 0.028±0.001 1.000±0.000 M t 14 0.000 2.018 2.018 0.027±0.002 1.000±0.000 M t 15 0.000 1.990 1.990 0.027±0.001 1.000±0.000 M t 16 0.000 1.951 1.951 0.026±0.001 1.000±0.000 M t 17 0.000 1.923 1.923 0.026±0.002 1.000±0.000 M t 18 0.000 1.898 1.898 0.025±0.002 1.000±0.000 M t 19 0.000 1.850 1.850 0.025±0.003 1.000±0.000 S t 20 0.000 1.807 1.807 0.024±0.004 1.000±0.000 S t 21 0.000 1.793 1.793 0.024±0.003 1.000±0.000 S t 22 0.000 1.737 1.737 0.023±0.003 1.000±0.000 S t 23 0.000 1.710 1.710 0.023±0.003 1.000±0.000 S t 24 0.000 1.663 1.663 0.022±0.002 1.000±0.000 S t 25 0.000 1.630 1.630 0.022±0.001 1.000±0.000 S t 26 0.000 1.568 1.568 0.021±0.002 1.000±0.000 S t 27 0.000 1.545 1.545 0.021±0.001 1.000±0.000 S t 28* 0.000 1.503 1.503 0.020±0.001 1.000±0.000 S t 29 0.000 1.440 1.440 0.019±0.001 1.000±0.000 S t 30 0.000 1.410 1.410 0.019±0.002 1.000±0.000 S t 31 0.000 1.373 1.373 0.018±0.002 1.000±0.000 S t 32 0.000 1.310 1.310 0.018±0.002 1.000±0.000 S t 33 0.000 1.257 1.257 0.017±0.002 1.000±0.000 S t 34 0.000 1.199 1.199 0.016±0.002 1.000±0.000 S t 35 0.000 1.106 1.106 0.015±0.002 1.000±0.000 S t 36 0.000 1.104 1.104 0.015±0.002 1.000±0.000 S t 37 0.000 1.052 1.052 0.014±0.001 1.000±0.000 S t 38 0.000 0.959 0.959 0.013±0.009 1.000±0.000 S t X 1.510 2.266 3.776 0.051±0.002 0.600±0.084 L m Y 0.073 0.529 0.602 0.008±0.005 0.878±0.168 S a

Remark: *=Nucleolar organizer regions/NORs. humans and apes are over 1,000 bands per haploid set. A comparison of the canid karyotypes, carried out with the use of the GTG-banding technique, revealed a high homology between Ca. familiaris and both fox species (V. vulpus and A. lagopus), and an extensive whole arm homology could be observed between the foxes (Mäkinen and Gustavsson 1982, Yoshida et al. 1983, Graphodatsky et al. 1995, Switonski et al. 2003). This high homology of chromosome arms is also shared by the foxes (V. vulpus and A. lagopus) and the raccoon dog (N. procyonoides), since approximately 75% of chromosome arms of both foxes have homologous counterparts in the N. procyonoides karyotype (Wayne et al. 1987, Switonski et al. 2003). 10 A. Tanomtong et al. Cytologia 80(1)

Fig. 6. Idiogram showing lengths and shapes of chromosomes of the Asiatic jackals (Canis aureus), 2n=78 by conventional staining technique. Arrows indicate nucleolar organizer regions/NORs.

Fig. 7. Idiogram showing regions and bands of the Asiatic jackals (Canis aureus), 2n=78 by GTG-banding technique.

The number of nucleolar organizer regions (NORs) is characteristic for a karyotype. In this investigation, the six NORs, which represent chromosome markers, are located on the long arm near the centromere of chromosome pair 7 (centromeric NORs) and the long arm near the telomere of chromosome pairs 10 and 28 (telomeric NORs) (Fig. 5). In contrast, Mäkinen et al. (1985a, b) indicated that NORs of the A. lagopus and V. vulpes appear on the pair autosomes 13, 15, 17, 18, 20, 22 (12 positions) and 8, 9, 13 (six positions), respectively. In Ca. familiaris, N. p. procyonoide 2015 The Standard Karyotype of the Asiatic Jackal Canis aureus from Thailand 11

Fig. 8. Idiogram showing regions, bands and sub-bands of chromosomes of the Asiatic jackals (Canis aureus), 2n=78 by high-resolution banding technique. and N. p. viverrinus (dog and raccoon dog) NORs residue is found on three autosome pairs and on the Y-chromosome (seven positions). The following chromosome pairs of these species bear the NORs: 1, 4, 13, Y-chromosome in the N. p. procyonoide (Pienkowska and Zagalska 2001) and 11, 12, 18, Y-chromosome in the N. p. viverrinus (Ward et al. 1987). The NORs localised on the Y-chromosome appeared to be active in approximately 80% of chromosome spreads in both species. The presence of the NORs on the Y-chromosome reﬂects the evolutionary similarity of this chromosome in the dog and raccoon dog (Switonski et al. 2003). Our study showed that for Ca. aureus the chromosome marker is the X-chromosome which is the largest metacentric chromosome, and the Y-chromosome is the smallest acrocentric chromosome. The important karyotype feature of Ca. aureus is the asymmetrical karyotype, which was found in three types of chromosomes (metacentric, acrocentric and telocentric). The largest chromosome is six times larger than the smallest chromosomes. The chromosome length in centimetres of 20 cells (males and females) in 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), and standard deviation of RL, CI, sizes and types of chromosome are shown in Table 2. Figures 6, 7 and 8 show the standardized idiograms from conventional staining, GTG-banding and high-resolution banding techniques, respectively. The karyotype formula of Ca. aureus could be deduced as: 2n (diploid) t t t 78=L18+M18+S40+sex chromosomes.

Acknowledgments

This work was supported by grants from the Applied Taxonomic Research Center (ATRC), Khon Kaen University grant ATRC_R5801, faculty of Science, Khon Kaen University.

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