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First Report of Chromosome Analysis of Saddleback Anemonefish

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First Report of Chromosome Analysis of Saddleback Anemonefish © 2012 The Japan Mendel Society Cytologia 77(4): 441–446 First Report of Chromosome Analysis of Saddleback Anemonefish, Amphiprion polymnus (Perciformes, Amphiprioninae), in Thailand Alongklod Tanomtong1, Weerayuth Supiwong1*, Arunrat Chaveerach1, Suthip Khakhong2, Tawatchai Tanee3 and La-orsri Sanoamuang1 1 Applied Taxonomic Research Center (ATRC), Department of Biology, Faculty of Science, Khon Kaen University, Khon Kaen, Muang 40002, Thailand 2 Aquaculture Program, Faculty of Agricultural Technology, Phuket Rajabhat University, Phuket, Muang 83000, Thailand 3 Faculty of Environment and Resource Studies, Mahasarakham University, Mahasarakham, Muang 44000, Thailand Received May 31; accepted October 6, 2012 Summary We report the first chromosome analysis for the saddleback anemonefish (Amphiprion polymnus) from Thailand. Kidney cell samples were taken from 5 male and 5 female fish. The mi- totic chromosome preparation was prepared by directly from kidney cells. Conventional and Ag- NOR staining techniques were applied to stain the chromosomes. The results showed that the diploid chromosome number of A. polymnus was 2n=48 and the fundamental numbers (NF) was 96 in both male and female. The types of chromosomes were 6 large submetacentric, 2 large acrocentric, 10 medium metacentric, 12 medium submetacentric, 6 medium acrocentric, 10 small metacentric, and 2 small submetacentric chromosomes. The region adjacent to the telomeres on the short arms of chro- mosome pair 21 showed clearly observable secondary constriction/NORs. The karyotype formula for A. polymnus could be deduced as: sm a m sm a m sm 2n (48)=L 6 +L2+M10+M 12+M6+S10+S 2 Key words Saddleback anemonefish, Amphiprion polymnus, Karyotype, Chromosome. Thailand is one of the world’s richest places for biodiversity, especially for marine fish spe- cies. With more than 2,000 species recorded, Thailand has become one of the species diversity cen- ters of the world. Marine fish are especially important as they provide a high quality source of pro- tein as well as food source for people who live near the coast. Because of its attractive color, fish in the subfamily Amphiprioninae play an important role in national economy. There are 2 genera and 29 species in the subfamily Amphiprioninae in Thailand, and 7 species can be reached. For the genus Amphiprion, there are skunk anemonefish (A. akallopisos Bleeker 1853), Clark’s anemone- fish [A. clarkii (Bennett 1830)], red saddleback anemonefish [A. ephippium (Bloch 1790)], false clown anemonefish (A. ocellaris Cuvier 1830), pink anemonefish (A. perideraion Bleeker 1855), saddleback anemonefish [A. polymnus (Linnaeus 1758)], and sebae anemonefish (A. sebae Bleeker 1853) (Boonphakdee and Sawangwong 2008). Of about 13,000 marine fish species that have been recorded (Nelson 1994), fewer than 2% of these have been studied cytogenetically (Brum 1996). Although most of these fish share a common environment lacking apparent physical barriers, they do not show a general chromosome evolution- ary trend. The diploid chromosome number varies from 2n=22–26, in some species of * Corresponding, e-mail: [email protected] DOI: 10.1508/cytologia.77.441 442 A. Tanomtong et al. Cytologia 77(4) Table 1. Review of anemonefishes cytogenetic reports in the subfamily Amphiprioninae Species (Anemonefish) 2n NF m sm a t NORs References Saddleback (A. polymnus) 48 96 20 20 8 ̶ 2 (a) Present study Clak’s (A. clarkii) 48 78 14 16 18 ̶ ̶ Arai and Inoue (1976) 48 86 12 26 10 ̶ 2 (a) Takai and Kosuga (2007) False clown (A. ocellaris) 48 84 14 22 12 ̶ ̶ Arai et al. (1976) Tomato (A. frenatus) 48 92 14 22 8 4 2 (a) Molina and Galetti (2004) 48 86 12 26 10 ̶ 2 (a) Takai and Kosuga (2007) Remark: 2n=diploid chromosome number, NF=fundamental number, m=metacentric, sm=submetacentric, a=acrocentric, t=telocentric, and NORs=nucleolar organizer regions. Nototheniidae (Ozouf-Costaz et al. 1997), an Antarctic fish group, to 2n=240–260, in some anadromous Acipenseridae, which show sev- eral micro-chromosomes (Fontana et al. 1997). Most marine fish studied have a diploid complement of 48 acrocentric chromosomes (Sola et al. 1981, Klinkhardt et al. 1995, Brum 1996). There are only 3 species in the subfamily Amphiprioninae that have already been consid- ered through cytogenetic studies. These results show a diploid chromosome number 2n=48 Fig. 1. General characteristics of the sad- dleback anemonefish (Amphiprion (Arai and Inoue 1976, Arai et al. 1976, Molina polymnus), scale bar indicates and Galetti 2004, Takai and Kosuga 2007) 5 cm. (Table 1). The present study is aimed at the cy- togenetic analysis of saddleback anemonefish (Amphiprion polymnus) (Fig. 1). From the present study, we show the standardization of karyotype and idiogram of A. polymnus. This report de- scribes the first chromosome staining by conventional staining and Ag-NOR staining techniques in A. polymnus. Materials and methods The A. polymnus samples were obtained from Phuket Marine Biological Center, and Phang Nga Coastal Research and Development Center, Thailand. The fish, 5 males and 5 females of A. polymnus, were transferred to laboratory aquaria and were kept under standard conditions for 7 days prior to the experiments. Chromosome preparation was conducted by squash technique from kidney cell (Chen and Ebeling 1968, Nanda et al. 1995). The chromosomes were stained with 10% Giemsa’s for 30 min and identified for NORs by Ag-NOR staining (Howell and Black 1980). The length of short arm (Ls) and long arm (Ll) chromosome were measured and calculated for the length of total arm chromosome (LT, LT=Ls+Ll). Relative length (RL) and centromeric index (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 According to the results, this is the first report on A. polymnus cytogenetical knowledge. Our results showed that 2n=48 of diploid chromosome number is consistent to the reports of Arai and Inoue (1976), Arai et al. (1976), Molina and Galetti (2004), and Takai and Kosuga (2007) that 2012 Chromosome Analysis of Saddleback Anemonefish, Amphiprion polymnus in Thailand 443 showed 2n=48 of Clark’s anemonefish (A. clarkii), false clown anemonefish (A. ocellaris) and to- mato anemonefish (A. frenatus). We found that the fundamental number (NF, number of chromo- some arms) of A. polymnus is 96 in both male and female. No strange size chromosomes related to sex were observed. In pomacentrid fish, including anemonefishes, the karyotypes of 30 species has been reported and these are characterized by chromosome numbers varying from 27 to 48 and NF varying from 48 to 86 (Arai and Inoue 1976, Arai et al. 1976, Ojima and Kashiwagi 1981, Takai and Ojima 1987, 1991a, 1991b, 1995, Molina and Galetti 2004, Takai and Kosuga 2007). Takai and Ojima (1987) suggested that karyotypes of pomacentrid fish have diversified from the ancestral karyotype, consisting of 48 acrocentric chromosomes, mainly toward an increase in NF involving pericentric inversions. Therefore, anemonefish with higher NF may be the most advanced group in the karyo- typic evolution in family Pomacentridae. The chromosomes of A. polymnus consist of 20 metacentric, 20 submetacentric, and 8 acro- centric chromosomes (Fig. 2). It is different from the reports of Arai and Inoue (1976) that showed the chromosomes of A. clarkii consisting of 14 metacentric, 16 submetacentric, and 18 acrocentric chromosomes. Arai et al. (1976) found the karyotype of A. ocellaris in Japan consisting of 14 meta- centric, 22 submetacentric, and 12 subtelocentric/acrocentric chromosomes. Molina and Galetti (2004) reported that the chromosome of A. frenatus from Philippines (Pacific) composed of 14 metacentric, 22 submetacentric, 8 subtelocentric, and 4 acrocrntric chromosomes. Takai and Fig. 2. Metaphase chromosome plates and karyotypes of male (A) and female (B) saddleback anemonefish (Amphiprion polymnus), 2n=48 by conventional straining technique (scale bars 10 μm). There is no observation of strange size chromosomes related to sex. 444 A. Tanomtong et al. Cytologia 77(4) Kosuga (2007) reported karyotypes of A. （A） clarkii and A. frenatus consisting of 12 meta- centric, 26 submetacentric, and 10 acrocentric chromosomes. The present study, the first cytogenetic re- port of A. polymnus, was accomplished using Ag-NOR staining technique. The objective of this technique is to reach out the nucleolar or- ganizer regions (NORs) which represent the lo- cation of genes (loci) that function in ribosome synthesis (18S and 28S ribosomal RNA) (Sharma et al. 2002). In addition, the short arm near telomere of largest acrocentric chromo- some pair 21 showed clearly observable NORs （B） (satellite chromosomes) (Fig. 3). It is quite consistent to the reports of Takai and Kosuga (2007), who reported karyotypes of A. clarkii and A. frenatus which showed clearly observ- able 1 pair of NORs on acrocentric chromo- somes, and Molina and Galetti (2004), who reported that A. frenatus showed 1 pair of NORs-bearing on acrocentric chromosomes. Normally, most fish have only 1 pair of small NORs on chromosomes. If some fish have more than 2 NORs, this may be caused by the Fig. 3. Metaphase chromosome plates of translocation between some parts of chromo- male (A) and female (B) saddle- somes which have NOR and another chromo- back anemonefish (Amphiprion some (Sharma et al. 2002). polymnus), 2n=48 by Ag-NOR The chromosome of mitotic metaphase banding technique, scale bars in- dicate 10 μm. The region adjacent cells and the karyotypes of A. polymnus by to the short arms near telomere of conventional staining and Ag-NOR staining largest acrocentric chromosome techniques are shown in Figs. 2 and 3. The pair 21 showed clearly observable chromosomes length in centimetres of 20 cells nucleolar organizer regions (satel- lite chromosomes). (males and females) in mitotic metaphase was measured. The mean length of short arm chro- mosome (Ls), long arm chromosome (Ll), total length of arm chromosome (LT), relative length (RL), centromeric index (CI), and type of chromo- some were presented in Table 2.
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