First report of karyological analysis and heteromorphic nucleolar organizer region of Black Surgeonfish (Acanthurus gahhm, Acanthuridae) in Thailand

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, Andaman 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. gahhm 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) = L6+L20+M18+S4


INTRODUCTION
Worldwide there are an estimated 24,000 fish species recorded, Thailand 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, economically and ecological important, moreover, some species are bioindicator (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 ornamental fish. This family includes about 82 extant species in 6 genera, namely Acanthurus, Ctenochaetus, Zebrasoma, Paracanthurus, Prionurus and Naso. For the important 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 demersal 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 invertebrates, 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 harvested 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 yellow ( 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;Galetti 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 organizer 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 conventional staining and Ag-NOR banding techniques.

MATERIALS AND METHODS
The 10 male and 10 female specimens of black surgeonfish (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 follows. The fishes were injected on their abdominal cavity with 0.05% colchicine for 1.0 ml/100 g body weight, then leaved for one hour. Chromosome preparation containing 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 minutes 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 chromosome (Ll) were measured to calculate the length of total arm chromosome (LT, LT = Ls + Ll). In addition, the relative 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 representative of large, medium and small chromosomes, respectively. In the same way, the chromosomes were classified by type regarding to the symbol "m, sm, a and t" as the representative of metacentric, submetacentric, acrocentric and telocentric chromosomes, respectively. The fundamental number (NF) is assigned a value of two for the metacentric, submetacentric and acrocentric chromosomes; however, it is assigned equal to one for the telocentric chromosome.

RESULTS AND DISCUSSION
This is the first karyological analysis of the Acanthurus gahhm. The results showed that the diploid chromosome 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 cytogenetics 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 demonstrated 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 chromosome 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 metaphase 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 chromosome related to sex was observed. The A. gahhm has 6 bi-arm and 42 uni-arm chromosomes. The modal karyotype 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 Perciformes pattern have been detected in these studies, indicating Pericentric inversion or/ and Robertsonian rearrangements as the preferential process in some groups. The karyotype of A. gahhm is quite similar to the A. coeluleus karyotype. The rearrangement mechanism involves to pericentric inversions of 3 uni-arm to 3 bi-arm chromosome 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 scalprum. The few species show diploid decreasing cause  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.   gahhm. One pair of the short arm of the largest chromosome 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 presence of heteromorphic of chromosome pair 1 (1a1b). NORs were found in 1a, but not in 1b. The three Acanthuridae 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 important karyotype trait is the presence of an asymmetrical karyotype pattern. There were only two types of chromosomes found, the acrocentric and telocentric chromosomes. The standardized conventional and Ag-NOR idiograms of Acanthurus gahhm are shown on Figure 4 and 5, respectively. The karyotype formula of black surgeonfish (Acanthurus gahhm) can be deduced as: 2n (48)