Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 74(1): 83-88, 2021 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-853 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: S. Jantarat, S. Jumrustha- nasan, S. Kaewsri, P. Supanuam, A. Tanomtong (2021) First report of karyologi- cal analysis and heteromorphic nucleo- lar organizer region of Black Surgeon- fish (Acanthurus gahhm, Acanthuridae) in Thailand. Caryologia 74(1): 83-88. doi: 10.36253/caryologia-853 Received: February 06, 2020 Accepted: April 26, 2021 Published: July 20, 2021 Copyright: © 2021 S. Jantarat, S. Jum- rusthanasan, S. Kaewsri, P. Supan- uam, A. Tanomtong. This is an open access, peer-reviewed article pub- lished by Firenze University Press (http://www.fupress.com/caryologia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distri- bution, and reproduction in any medi- um, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. First report of karyological analysis and heteromorphic nucleolar organizer region of Black Surgeonfish (Acanthurus gahhm, Acanthuridae) in Thailand Sitthisak Jantarat1, Sarun Jumrusthanasan2, Sarawut Kaewsri2, Praween Supanuam3,*, Alongklod Tanomtong4 1 Program of Biology, Department of Science, Faculty of Science and Technology, Prince of Songkla University, Pattani Campus, Thailand 2 Biology Program, Faculty of Science, Buriram Rajabhat University, Buriram, Thailand 3 Biology Program, Faculty of Science, Ubon Ratchathani Rajabhat University, Ubon Rat- chathani, Thailand 4 Department of Biology, Faculty of Science, Khon Kaen University, Khon Kaen, Thailand *Corresponding author. E-mail: supanuam@hotmail.com Abstract. This research was the first report on karyological analysis and heteromorphic nucleolar organizer region of black surgeonfish (Acanthurus gahhm, Acanthuridae) in Thailand. The 10 male and 10 female specimens were collected from Phuket Marine Biological Center, and Phang Nga Coastal Research and Development Center, Anda- man Sea, Thailand. Mitotic chromosomes were directly prepared from gill and kidney tissues. The chromosomes were stained by conventional Giemsa staining and Ag-NOR banding techniques. Results showed that the diploid chromosomes number of A. gah- hm was 2n=48, the fundamental numbers (NF) was 54 in both male and female. The karyotype consist of 6 large acrocentric, 20 large telocentric, 18 medium telocentric and 4 small telocentric chromosomes. None of strange size chromosomes related to sex was found. The heteromorphic nucleolar organizer regions (NORs) were observed on telomeric short arm of first acrocentric which can defined as 1a1b. There is NOR in 1a and not in 1b. The karyotype formula of black surgeon fish was as follows: 2n (48) = La6+Lt20+Mt18+St4 Keywords: Acanthurus gahhm, chromosome, karyotype, NORs. INTRODUCTION Worldwide there are an estimated 24,000 fish species recorded, Thai- land is one of the species diversity centers of the world. There are more than 13,000 and 4,000 species of fishes that live in sea and coralline, respectively (Tamrongnawasawad et al. 2004). Marine fishes are especially important as they provide a high quality source of protein and other nutrients, economi- cally and ecological important, moreover, some species are bioindicator 84 Sitthisak Jantarat, Sarun Jumrusthanasan, Sarawut Kaewsri, Praween Supanuam, Alongklod Tanomtong (Ohno 1970; Le Grande and Fitzsimons 1988; Affonso et al. 2014). The Acanthuridae are the family of surgeonfishes, tangs, and unicornfishes. They are well known as orna- mental fish. This family includes about 82 extant species in 6 genera, namely Acanthurus, Ctenochaetus, Zebra- soma, Paracanthurus, Prionurus and Naso. For the impor- tant character of the family, they have a colorful body. They also have a pair of dangerously precaudal spines. The genus Acanthurus has 40 species in worldwide that found in the Atlantic, Indian and Pacific Ocean. They are found in tropical oceans, especially near coral reefs, with most species in the Indo-Pacific but a few are found in the Atlantic Ocean (Monkolprasit et al. 1997; Allen et al. 2012). Acanthurus gahhm or black surgeonfish is a demer- sal fish. It lives on reefs and in lagoons and other sandy areas up to 40 meters deep. This species is omnivorous, feeding on algae, zooplankton and other small inverte- brates, and detritus. It is active during the day and may swim in groups or remain solitary. It is endemic to the Indian Ocean. This species is kept in aquaria and har- vested for food. This fish reaches up to 50 centimeters in length. It is oval in shape and laterally compressed. The caudal fin has a crescent shape. The mouth is small and pointed. The body is black to dark brown, with a white ring around the base of the tail and a yellow stripe around the eyes. The pectoral fins are tipped with yel- low (Figure 1). The black surgeonfish are one of the most colorful and economically important fish (Carpenter and Niem 2001; Allen et al. 2012). Previous cytogenetic studies of the genus Acanthurus stated that their members are only four species, namely A. coeruleus, A. bahianus, A. chirergus and A. triostegus (Arai and Inoue 1976; Ojima and Yamamoto 1990; Gal- etti et al 2006; Arai 2011; Affonso et al. 2014). The two species, A. coeruleus from Brazil and A. triostegus from Japan show 2n=48. The other species from Brazil show the diploid chromosome numbers of 36 and 34 for A. bahianus and A. chirurgus, respectively. Nucleolar organ- izer region (NOR) of the species in this family has never been reported. The present study aimed to investigate cytogenetic characterization of the Acanthurus gahhm. We exhibit the standardized karyotype and idiogram of the species and also firstly describes the chromosomal characteristics of A. gahhm by means of Giemsa conven- tional staining and Ag-NOR banding techniques. MATERIALS AND METHODS The 10 male and 10 female specimens of black sur- geonf ish (Acanthurus gahhm) were collected from Phuket Marine Biological Center, and Phang Nga Coastal Research and Development Center, Andaman Sea, Thailand. Chromosomes were directly prepared in vivo (Chen and Ebeling 1968; Nanda et al. 1995) as fol- lows. The fishes were injected on their abdominal cav- ity with 0.05% colchicine for 1.0 ml/100 g body weight, then leaved for one hour. Chromosome preparation con- taining gill and kidney tissues were conducted by the colchicine-hypotonic-fixation-air drying technique. The tissues were finely chopped by scissors. The metaphase cell was three times centrifuged at 1,250 rpm for 10 min- utes until the white sediment cells were precipitated. The chromosomes were stained with 20% Giemsa’s for 30 minutes and identified for NORs by Ag-NOR staining according to Howell and Black (1980) and Verma and Babu (1995). Chromosomal checks were performed on mitotic metaphase cells under light microscope. The twenty cells of each male and female appeared with clearly observable and well-spread chromosomes were selected and photographed. The length of short arm chromosome (Ls) and the length of long arm chromo- some (Ll) were measured to calculate the length of total arm chromosome (LT, LT = Ls + Ll). In addition, the rel- ative length (RL), centromeric index (CI), and total arm chromosome (LT) were calculated to classify the type and size of chromosomes based on Turpin and Lejeune (1965) and Chaiyasut (1989). All described parameters were used in karyotyping and idiograming according to Tanomtong et al. (2019). For the karyotype formula determination, the chromosomes were classified by size regarding to the symbol “L, M and S” as the representa- tive of large, medium and small chromosomes, respec- tively. In the same way, the chromosomes were classified by type regarding to the symbol “m, sm, a and t” as the Figure 1. General characteristics of black surgeonfish (Acanthurus gahhm, Acanthuridae) from Phuket Marine Biological Center, and Phang Nga Coastal Research and Development Center, Andaman Sea, Thailand (Scale bars = 3 cm). 85First report of karyological analysis and heteromorphic nucleolar organizer region of Black Surgeonfish representative of metacentric, submetacentric, acrocen- tric and telocentric chromosomes, respectively. The fun- damental number (NF) is assigned a value of two for the metacentric, submetacentric and acrocentric chromo- somes; however, it is assigned equal to one for the telo- centric chromosome. RESULTS AND DISCUSSION This is the first karyological analysis of the Acanthu- rus gahhm. The results showed that the diploid chromo- some number was 2n=48 and the fundamental numbers (NF) were 54 for both male and female (Figure 2). Up to the present, there are only two publications on cytoge- netics of the family Acanthuridae. Affonso et al. (2001) conducted the study on cytogenetics of three species of the family Acanthuridae, namely Acanthurus coeruleus, A. bahianus and A. chirergus in Brazil. They showed the diploid chromosome number of 48 and the fundamental number (NF) of 52 for A. coeruleus. However, they dem- onstrated the low diploid chromosome numbers (2n) of 36 and 34 and the fundamental numbers (NF) of 52 and 50 for A. bahianus and A. chirergus, respectively. Arai and Inoue (1976) revealed an establishment of chromo- some analysis of A. triostegus which were obtained from Yakushima, Japan. The karyotype showed 2n=48 and NF=48, like the ancestral perciform karyotype. The present karyotype of A. gahhm consist of 6 large acrocentric, 20 large telocentric, 18 medium telocentric and 4 small telocentric chromosomes. The twenty meta- phase cells of each male and female were measured for Ls, Ll, LT, CI, RL, SD, chromosome sizes and types were showed on Table 1. None of the strange in size of chro- mosome related to sex was observed. The A. gahhm has 6 bi-arm and 42 uni-arm chromosomes. The modal kar- yotype of ancestral Perciformes fish possessing 2n=48, NF=48 and composed all uni-arm chromosomes. The karyotype of A. gahhm indicates that although it has been revealing a model diploid chromosome number of 2n=48, the karyotypes different from the ancestral Perci- formes pattern have been detected in these studies, indi- cating Pericentric inversion or/ and Robertsonian rear- rangements as the preferential process in some groups. The karyotype of A. gahhm is quite similar to the A. coe- luleus karyotype. The rearrangement mechanism involves to pericentric inversions of 3 uni-arm to 3 bi-arm chro- mosome pairs from the ancestor. (Affonso et al. 2014). The most species of family Acanthuridae show the typical perciform karyotype, 2n=48, NF=48, namely A. triostegus, Ctenochaetus striatus and Prionurus scal- prum. The few species show diploid decreasing cause Figure 2. Metaphase plates and standardized karyotypes of male (A) and female (B) black surgeonfish, Acanthurus gahhm, 2n=48 by con- ventional staining (Scale bars = 10 µm). A B 86 Sitthisak Jantarat, Sarun Jumrusthanasan, Sarawut Kaewsri, Praween Supanuam, Alongklod Tanomtong by tandem or/ and centric fusion including A. bahi- anus (2n=36, 16 bi-arm and 20 uni-arm) and A. chirur- gus (2n=34, 16 bi-arm and 18 uni-arm) (Arai and Inoue 1976; Ojima and Yamamoto 1990; Galetti et al 2006; Arai 2011; Affonso et al. 2014). The karyotype of the family Acanthuridae is shown in Table 2. Moreover, this is the first report on localization of nucleolar organizer regions (NORs) of the Acanthurus Table 1. Mean length of the short arm chromosome (Ls), long arm chromosome (Ll), total arm chromosome (LT), centromeric index (CI), relative length (RL) and standard deviation (SD) of CI, RL from 40 karyotypes of male and female black surgeonfish (Acanthurus gahhm), 2n=48. Chromosome pair Ls (micron) Ll (micron) LT (micron) RL±SD CI±SD Chromosome size Chromosome type 1 0.88 2.57 3.45 0.057±0.001 0.744±0.038 Large Acrocentric 2 0.76 2.51 3.27 0.054±0.001 0.766±0.036 Large Acrocentric 3 0.77 2.08 2.85 0.047±0.001 0.729±0.019 Large Acrocentric 4 0.00 3.37 3.37 0.055±0.003 1.000±0.000 Large Telocentric 5 0.00 3.19 3.19 0.052±0.001 1.000±0.000 Large Telocentric 6 0.00 3.13 3.13 0.051±0.002 1.000±0.000 Large Telocentric 7 0.00 3.06 3.06 0.050±0.002 1.000±0.000 Large Telocentric 8 0.00 3.01 3.01 0.049±0.002 1.000±0.000 Large Telocentric 9 0.00 2.93 2.93 0.048±0.002 1.000±0.000 Large Telocentric 10 0.00 2.66 2.66 0.043±0.000 1.000±0.000 Large Telocentric 11 0.00 2.62 2.62 0.043±0.000 1.000±0.000 Large Telocentric 12 0.00 2.55 2.55 0.042±0.001 1.000±0.000 Large Telocentric 13 0.00 2.49 2.49 0.041±0.001 1.000±0.000 Large Telocentric 14 0.00 2.41 2.41 0.039±0.001 1.000±0.000 Medium Telocentric 15 0.00 2.35 2.35 0.038±0.001 1.000±0.000 Medium Telocentric 16 0.00 2.30 2.30 0.038±0.001 1.000±0.000 Medium Telocentric 17 0.00 2.13 2.13 0.035±0.000 1.000±0.000 Medium Telocentric 18 0.00 2.05 2.05 0.033±0.000 1.000±0.000 Medium Telocentric 19 0.00 1.99 1.99 0.032±0.000 1.000±0.000 Medium Telocentric 20 0.00 1.92 1.92 0.031±0.000 1.000±0.000 Medium Telocentric 21 0.00 1.85 1.85 0.030±0.001 1.000±0.000 Medium Telocentric 22 0.00 1.76 1.76 0.029±0.001 1.000±0.000 Medium Telocentric 23 0.00 1.70 1.70 0.028±0.002 1.000±0.000 Small Telocentric 24 0.00 1.50 1.50 0.024±0.001 1.000±0.000 Small Telocentric Table 2. Review of cytogenetic publications in the family Acanthuridae. Species 2n NF Karyotype Ag-NOR Locality Reference Acanthurus triostegus 48 48 48t - Japan Arai and Inoue (1976) A. chirurgus 34 50 16bi+18t - Brazil Galetti et al. (2006) 34 - 18bi+16t Pair 8p Brazil Affonso et al. (2014) A. bahianus 36 52 16bi+20t - Brazil Galetti et al. (2006) 36 - 18bi+18t Pair 8p Brazil Affonso et al. (2014) A. coeruleus 48 52 4bi+44t - Brazil Galetti et al. (2006) 48 - 6bi+42t Pair 2p Brazil Affonso et al. (2014) A. gahhm 48 54 6a+42t Pair 1p Thailand Present study Ctenochaetus stiatus 48 48 48t - Japan Ojima and Yamamoto (1990) Prionurus scalprum 48 48 48t - Japan Arai and Inoue (1976) Remark: 2n = diploid number, NF = fundamental number, bi = bi-arm chromosome, t = telocentric chromosome (uni-arm chromosome). 87First report of karyological analysis and heteromorphic nucleolar organizer region of Black Surgeonfish Figure 3. Metaphase plates and standardized karyotypes of male (A) and female (B) black surgeonfish, Acanthurus gahhm, 2n=48 by Ag- NOR banding (Scale bars = 10 µm). Chromosome pair 1 show heteromorphic NOR in 1a. Arrows indicate NORs. A B Figure 4. Standardized idiogram of black surgeonfish, Acanthurus gahhm, 2n=48 by conventional staining. Figure 5. Standardized idiogram of black surgeonfish, Acanthurus gahhm, 2n=48 by Ag-NOR banding. Chromosome pair 1 show het- eromorphic NOR in 1a. Arrows indicate NORs. 88 Sitthisak Jantarat, Sarun Jumrusthanasan, Sarawut Kaewsri, Praween Supanuam, Alongklod Tanomtong gahhm. One pair of the short arm of the largest chromo- some 1 showed clearly observable NORs. The first record on heteromorphism of NORs in the A. gahhm (Figure 3) was also reported here. This finding indicates the pres- ence of heteromorphic of chromosome pair 1 (1a1b). NORs were found in 1a, but not in 1b. The three Acan- thuridae species, namely A. coeluleus, A. bahianus and A. chirurgus show the single nucleolar organizer regions on the short arms of the largest subtelocentric pairs (Affonso et al. 2014). The idiogram shows gradually decreasing length of the chromosomes. The largest chromosome shows two times larger than the smallest chromosome. An impor- tant karyotype trait is the presence of an asymmetrical karyotype pattern. There were only two types of chro- mosomes found, the acrocentric and telocentric chro- mosomes. The standardized conventional and Ag-NOR idiograms of Acanthurus gahhm are shown on Figure 4 and 5, respectively. The karyotype formula of black sur- geonfish (Acanthurus gahhm) can be deduced as: 2n (48) = La6+Lt20+Mt18+St4 FUNDING This Research was financially supported by the Government Budget Grant, Prince of Songk la Uni- versity, Thailand, in Fiscal Year 2017, Research Code: SAT600116S. REFERENCES Affonso, PRAM, Fernandas MA, Almeida JS, Molina WF. 2014. 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