© 2017 The Japan Mendel Society Cytologia 82(1) Special Issue: 51–57 Chromosomal Characteristics of the Three-Spot Damselfish, Dascyllus trimaculatus (Perciformes, Pomacentridae) in Thailand Nuntaporn Getlekha1, Weerayuth Supiwong2*, Pun Yeesin3, Puan Pengseng4, Wannapa Kasiroek5 and Alongklod Tanomtong1 1 Toxic Substances in Livestock and Aquatic Animals Research Group, Department of Biology, Faculty of Science, Khon Kaen University, Muang, Khon Kaen 40002, Thailand 2 Faculty of Applied Science and Engineering, Khon Kaen University, Nong Khai Campus, Muang, Nong Khai 43000, Thailand 3 Department of Technology and Industries, Faculty of Science and Technology, Prince of Songkla University (Pattani), Muang, Pattani 94000, Thailand 4 School of Agricultural of Technology, Walailak University, Thasala, Nakhon Si Thammarat 80160, Thailand 5 Institute of Marine Science, Burapha University, Muang, Chonburi 20131, Thailand Received February 11, 2015; accepted January 5, 2016 Summary The present study aims to analyze concerned karyotyping and idiograming of the three-spot dam- selfish (Dascyllus trimaculatus) in Thailand. Chromosomes were prepared from kidney tissues of fish reared at Institute of Marine Science, Burapha University, Chonburi Province. The mitotic chromosomes were harvested by the colchicine-hypotonic-fixation-air drying method. Conventional and Ag-NOR staining techniques were ap- plied to stain the chromosomes. The results showed that D. trimaculatus had 2n=48, and the fundamental num- ber (NF) was 50 without heteromorphic sex chromosomes. The types of chromosomes are 2 large acrocentric, 36 large terocentric and 10 medium telocentric chromosomes. The Ag-NOR banding exhibited that a single pair of NORs was presented on the short arm region of the large acrocentric chromosome. Basic knowledge on cytoge- netics of D. trimaculatus would be applied for support of further studies on breeding, conservation and chromo- some evolution in this fish. The karyotype formula of D. trimaculatus is as follows: at t 2n () 48 =L2 +L 36 +M 10 Key words Dascyllus trimaculatus, Damselfish, NOR, Chromosome, Cytogenetics. The family Pomacentridae (Perciformes) contains land (Fig. 1). 28 genera and approximately 320 species known as Karyological studies of fish can contribute signifi- damselfishes. This family is one of the most diverse cantly to a better understanding of many problems in among marine teleosts, which are widely distributed areas of research ranging from taxonomy, systematic or in tropical seas of the world (Nelson 2006). Species of genetics to phylogenetics, or environmental toxicology the family Pomacentridae are found in coastal waters (Al-Sabti 1985). However, the small size and large num- associated with rocky substrates, usually occurring at ber of chromosomes in fish and the lack of a standard low depths and often assembling in large fish schools. technique for fish chromosome preparation make their The taxonomy of damselfishes is complicated by the evaluations difficult (Denton 1973, Thorgaard and Dis- large number of complex species and the color patterns ney 1990). Chromosomal analysis is of interest in fish that vary among individuals and populations of the breeding from the viewpoint of genetic control, the rapid same species. Several species are of growing economic production of inbreed lines, taxonomy and evolutionary interest because of their diverse color patterns, and this studies (Al-Sabti 1987). has led to their exploitation (Molina and Galetti 2004). Of about 13000 marine fish species that have been There are only two genera in the Chrominae subfamily, recorded, fewer than 5% have been studied cytoge- Chromis and Dascyllus. The genus Dascyllus comprises netically (Arai 2011). Most marine fish studied have nine species (Nelson 2006). The three-spot damselfish, a diploid complement of 48 acrocentric chromosomes D. trimaculatus, is popular as an aquarium fish in Thai- (Brum 1996). For the family Pomacentridae, there are not only color pattern variations among individuals, but * Corresponding author, e-mail: [email protected] also inter-individual variations of chromosome number DOI: 10.1508/cytologia.82.51 and karyotype complements. In this family, 48 species 52 N. Getlekha et al. Cytologia 82(1) Special Issue of 0.01% colchicine (1 mL per 100 g body weight) was injected into the abdominal cavity and left for one hour. Chromosomes were prepared from the kidney cells of the fish by the squash technique (Chen and Ebel- ing 1968, Nanda et al. 1995). Kidney tissues were cut into small pieces then mixed with hypotonic solution (0.075 M KCl). After discarding all large pieces of tis- sues, 7 mL of cell sediments were transferred to a 15-mL centrifuge tube and incubated for 45 min. Hypotonic solution was discarded from the supernatant after cen- trifugation at 1200 rpm for 8 min. Cells were fixed in a fresh cool fixative (3 absolute methanol : 1 glacial acetic acid) to which up to 7 mL were gradually added before Fig. 1. General characteristic of the three-spot damselfish, being centrifuged again at 1200 rpm for 8 min, at which Dascyllus trimaculatus. time the supernatant was discarded. The fixation was repeated until the supernatant was clear and the pellet have been cytogenetically studied (Hinegardner and was mixed with 1 mL fixative. The mixture was dropped Rosen 1972, Rishi 1973, Arai and Inoue 1976, Arai onto a clean and cold slide by a plastic pipette followed et al. 1976, Alvarez et al. 1980, Ojima and Kashiwagi by air-dry technique (Kasiroek et al. 2017). 1981, Ojima 1983, Takai and Ojima 1986, 1987, 1991a, b, 1995, 1999, Aguilar et al. 1998, Brum et al. 2001, Chromosome staining Molina and Galetti 2002, 2004, Hardie and Hebert 2004, The slide was conventionally stained with 20% stock Kashiwagi et al. 2005, Galetti et al. 2006, Nagpure Giemsa’s solution for 30 min. Ag-NOR banding was et al. 2006, Takai and Kosuga 2007, Tanomtong et al. conducted by adding four drops of 50% silver nitrate 2012, Kasiroek et al. 2014). In these results, the diploid and 2% gelatin on slides, in order. The slides were then numbers ranged from 27 to 50 chromosomes while most sealed with cover glasses and incubated at 60°C for of them (43 species) had 2n=48 chromosomes, and the 5 min. After that, the slides were soaked in distilled wa- fundamental numbers ranged from 48 to 96. Karyotypes ter until the cover glasses were separated (Howell and involved in Robertsonian rearrangements with diploid Black 1980, Sangpakdee et al. 2017). chromosomes less than 2n=48 and large bi-armed chromosomes were found in three Dascyllus (Ojima Chromosome checking, karyotyping and idiograming and Kashiwagi 1981, Kashiwagi et al. 2005). Dascyllus Standardized karyotypes and idiograms of this fish species showed inter- and intraspecific Robertsonian were constructed. Chromosome checking was per- polymorphism; the diploid chromosome numbers were formed on mitotic metaphase cells under a light micro- 2n=47 and 48 in D. trimaculatus, 2n=34, 35, 36, and 37 scope. The frequencies of chromosome number per cell in D. reticulatus, 2n=27, 28, 29, 30, 31, 32 and 33 in D. were counted. The maximum frequency of chromosome aruanus, and 2n=48 in D. melanurus, these fundamen- number per cell is the diploid chromosome number of tal numbers presenting 48 in all specimens. In another this fish. Ten cells of each male and female with clearly genus, Chromis, only one species showed intra-specific observable and well-spread chromosome were selected Robertsonian polymorphism; the diploid chromosome for karyotyping. numbers were 2n=46 and 47 in C. insolata, both the The length of short arm chromosome (Ls) and long fundamental numbers being 56 (Molina and Galetti arm chromosome (Ll) were measured and calculated to 2002). the length of total arm chromosome (LT, LT=Ls+Ll). In the present research, we report the standardized The relative length (RL), the centromeric index (CI) karyotype and idiogram of D. trimaculatus from Thai and standard deviations (S.D.) of RL and CI were cal- population by conventional and Ag-NOR staining tech- culated. The CI (q/p+q) between 0.50–0.59, 0.60–0.69, niques. In the future, basic knowledge on cytogenetics of 0.70–0.89 and 0.90–0.99 were representing the metacen- D. trimaculatus would be useful for the studies of breed- tric, submetacentric, acrocentric and telocentric chromo- ing, conservation and chromosome evolution in this fish. somes, respectively (Chaiyasut 1989). The fundamental number (number of chromosome arm, NF) was obtained Materials and methods by assigning a value of two to metacentric, submetacen- tric and acrocentric chromosomes and one to telocentric Chromosome preparation chromosome. All parameters were used in karyotyping Four males and four females of D. trimaculatus were (Chooseangjaew et al. 2017). The idiogram was con- obtained from the Institute of Marine Science, Burapha structed using a model drawing of karyotype and ac- University, Chonburi Province, Thailand. A solution complished by a computer program. 2017 Chromosomal Characteristics of the Three-Spot Damselfish, Dascyllus trimaculatus (Perciformes, Pomacentridae) in Thailand 53 Fig. 2. Metaphase chromosome plates and karyotypes of male (A) and female (B) of three-spot damselfish (Dascyllus trimaculatus, 2n=48) by conventional staining technique (scale bars indicate 10 µm). Results and discussion Promacentrids indicates a high degree of chromosomal conservation in which a large number of species show Chromosome number, fundamental number and karyo- only minor deviations in the chromosomal organiza- type of D. trimaculatus tion and NF (Molina and Galetti 2004). A karyotype The diploid chromosome number (2n) of D. tri- with 2n=48 is considered the ancestral condition for maculatus was 48 chromosomes in all specimens. It the Teleosts and occurs in 43 of the 48 Promacentrid is in agreement with the previous reports from Japan species analyzed so far. Nevertheless, half of all of the (Ojima and Kashiwagi 1981, Hardie and Hebert 2004) subfamily Chrominae that have been analyzed cytoge- but it differs from reports of Arai and Inoue (1976) netically have 2n=48 with NF=48 (Molina and Galetti and Kashiwagi et al.