© 2014 The Japan Mendel Society Cytologia 79(4): 437–443 The First Report of NORs and Chromosome Analysis of Tripletail Wrasse, Cheilinus trilobatus (Perciformes: Labridae) Sarawut Kaewsri1, 4, Pasakorn Saenjundaeng2, Sarun Jumrusthanasan1, Ratree Suksuwan3, Alongklod Tanomtong1* and Weerayuth Supiwong2 1 Applied Taxonomic Research Center (ATRC), 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 Kai Campus, Muang, Nong Kai 43000,Thailand 3 Phuket Marine Biological Center (PMBC), 51 Moo 8 Sakdided Road, Wichit, Muang, Phuket 83000, Thailand 4 Biology Program, Department of Science, Faculty of Science, Buriram Rajabhat University, Muang, Buriram 31000, Thailand Received June 17, 2013; accepted October 20, 2013 Summary We report the first chromosome analysis and chromosomal characteristics of nucleolar organizer regions/NORs in the tripletail wrasse (Cheilinus trilobatus) from the Andaman Sea, Southern Thailand. Kidney cell samples were taken from three male and three female fishes. The mitotic chromosome preparation was prepared directly from kidney cells. Conventional staining and Ag-NOR banding techniques were applied to stain the chromosomes. The results showed that the diploid chromosome number of C. trilobatus was 2n=38, the fundamental number (NF) was 54 in both males and females. The types of chromosomes were 6 large metacentric, 4 medium metacentric, 2 medium submetacentric, 4 medium telocentric, 4 small submetacentric, and 18 small telocentric chromosomes. The region adjacent to the short arms near the telomeres of submetacentric chromosome pair 7 showed clearly observable NORs. There was no observation of irregularly sized chromosomes related to sex. The karyotype formula for C. trilobatus could be deduced as: m m sm t sm t 2n (diploid) 38=L6 +M4 +M2 +M4+S4 +S18 Key words Tripletail wrasse, Cheilinus trilobatus, Chromosome, Karyotype. The family Labridae represents the third largest group of the order Perciformes, comprising four subfamilies (Bodianinae, Corinae, Cheilininae and Pseudodacinae), 60 genera and about 500 species (Nelson 1994). Labrids are popularly known as wrasses and are widespread throughout the coast of Thailand. These marine fishes are found in reef sites and most species are asynchronic hermaphrodites (Kuwamura and Nakashima 1998). Nearly 8% of the species in the order Perciformes have been karyotyped so far and a modal number of 2n (diploid)=48 chromosomal has been established (Ojima et al. 1976, Galetti et al. 2000, Affonso and Galetti 2005, Galetti et al. 2006, Molina and Bacurau 2006). However, some families, such as the family Labridae, might present contrasting karyotypic formulae, ranging from conserved to highly derived (Ueno and Takai 2000). There are indications that a higher pelagic phase may be associated with a higher rate of chromosomal modification in some reef fish families (Molina and Galetti 2004a, Souza de Sena and Molina 2007). The present study aimed to characterize cytogenetically the tripletail wrasse (Cheilinus trilobatus) from the Andaman Sea, Southern Thailand, by using conventional staining and Ag-NOR * Corresponding author, e-mail: [email protected] DOI: 10.1508/cytologia.79.437 438 S. Kaewsri et al. Cytologia 79(4) banding analyses. In the future, the knowledge on basic cytogenetics of C. trilobatus could be applied to numerous breeding studies and also provide an advantage in species conservation and chromosome evolution studies. Materials and methods Three males and three females of C. trilobatus were obtained from Phuket province, Andaman Sea, Southern Thailand (Fig. 1). The fish were transferred to laboratory aquaria and were kept under standard condition for seven days prior to the experiment. Fish chromosomes were prepared directly from kidney cells (Chen and Ehbeling 1968, Nanda et al. 1995) and were stained with 10% Giemsa’s for 30 min, and NORs were identified by Ag-NOR staining (Howell and Black 1980). The metaphase figures were analyzed according to the chromosome classification of Chaiyasut (1989). Fig. 1. General characteristics of the tripletail wrasse (Cheilinus trilobatus): scale bar=6 cm. Fig. 2. Metaphase chromosome plates and karyotypes of male (A) and female (B) of tripletail wrasse (Cheilinus trilobatus), 2n=38 by conventional staining technique (scale bars=10 μm). 2014 The First Report of NORs and Chromosome Analysis of Tripletail Wrasse 439 The centromeric index (CI) between 0.50–0.59, 0.60–0.69, 0.70–0.89, and 0.90–0.99 were described as metacentric, submetacentric, acrocentric, and telocentric chromosomes, respectively. The fundamental number, number of chromosome arm (NF) was obtained by assigning a value of two to metacentric, submetacentric and acrocentric chromosomes and one to telocentric chromosomes. Results and discussion Diploid chromosome number, fundamental number and karyotype of C. trilobatus The results showed that 2n=38, and that the fundamental number (NF) was 54 in both males and females (Fig. 2). Comparisons with other studies shows that in the genus Cheilinus different chromosome numbers have been found in C. bimaculatus, 2n=32 from Japan (Ojima and Kashiwagi 1979) and C. fasciatus, 2n=48 from Japan (Ojima 1983) (Table 1). Similar to other species in the family Labridae, no cytologically distinguishable sex chromosome was observed (Souza de Sena and Molina 2007). The diploid chromosome numbers reported in the family Labridae range from 22–48 chromosomes (Ojima 1983, Klinkhardt et al. 1995, Ueno and Takai 2000, Souza de Sena and Molina 2007). The subfamily Cheilininae displays a diversified chromosomal pattern, since some species have a reduced diploid chromosome number, such as species from the genera Cheilinus, Cirrhilabrus, Crenilabrus, Hemipteronotus, Symphodus, and Xyrichthys (López et al. 1989, Ueno and Takai 2000, Souza de Sena and Molina 2007). Cytogenetics studies in the order Perciformes have demonstrated a remarkable karyotypic conservativeness, since most of the karyotyped species have 2n=48 and NF=48, with slight modifications around these values (Galetti et al. 2000, Molina 2006). This simplified chromosome pattern allows its utilization in the identification of visible changes on karyotype macrostructure of several families in the order Perciformes (Molina and Galetti 2002, 2004b). The occurrence of pericentric inversions seems to represent a frequent rearrangement mechanism of the order Perciformes (Cano et al. 1982, Galetti et al. 2002, Ueno and Takai 2000, Molina and Galetti 2004b). A NF higher than 48 (C. fasciatus and C. trilobatus), as observed in genus Cheilinus (Ojima and Kashiwagi 1979, Ojima 1983), indicates that although less common than in other groups, pericentric inversions constitute one of the main mechanisms of karyotypic diversification within the family Labridae (Souza de Sena and Molina 2007). The chromosomes of C. trilobatus consist of 6 large metacentric, 4 medium metacentric, 2 medium submetacentric, 4 medium telocentric, 4 small submetacentric, and 18 small telocentric chromosomes. In a comparative study, we found different results when we compared C. bimaculatus (2n=32) from Japan where the karyotype was composed of 4 metacentric, 2 submetacentric, and 26 telocentric chromosomes (Ojima and Kashiwagi 1979), and C. fasciatus (2n=48) from Japan has a karyotype composed of 12 submetacentric and 36 telocentric chromosomes (Ojima and Kashiwagi 1979). Therfore, the karyotype formula can be inferred as: m m sm t sm t 2n (diploid) 38=L6 +M4 +M2 +M4+S4 +S18 Table 1. Cytogenetic reviews of wrasses in the genus Cheilinus (Perciformes, Labridae, Cheilininae). Species 2n Karyotype formula NF NOR banded Location Reference C. bimaculatus 32 4m+2sm+26t 38 ̶ Japan Ojima and Kashiwagi (1979) C. fasciatus 48 12sm+36t 60 ̶ Japan Ojima (1983) C. trilobatus 38 10m+6sn+22t 54 S(TR)2 Thailand Present study Remarks: 2n=diploid chromosome number, NF=fundamental number (number of chromosome arm), m=metacentric chromosome, sm=submetacentric chromosome, t=telocentric chromosome, S=short arm, TR=telomeric region, and ̶ =not available. 440 S. Kaewsri et al. Cytologia 79(4) Chromosome marker of C. trilobatus This study used a Ag-NOR banding technique to analyse species in the genus Cheilinus. Nucleolar organizers/NORs are the chromosomal sites of genes, which transcribe for 18s and 28s ribosomal RNA, that are presumably transcribed at preceding interphase and are important in aspect of their intimate relationship with protein synthesis (Howell and Black 1980). The technique shows dark bands (NOR-position) of C. trilobatus on the short arms near the telomeres of submetacentric chromosome pair 7 (telomeric NORs) in both males and females (Fig. 3). Previous studies have shown that the species in the order Perciformes in Thailand, the chevron snakehead fish (Channa striata), had NORs on the long arm near the centromere of chromosome pair 14 (Supiwong et al. 2009), and the common sheathfish (Micronema apogon) had NORs on the short arm near telomere of chromosome pair 2 (Supiwong et al. 2012), while the saddleback anemonefish (Amphiprion polymnus) had NORs on the short arms near the telomeres of chromosome pair 21 (Tanomtong et al. 2012). The important chromosome marker of the C. trilobatus is the asymmetrical karyotype that was found for all three types of chromosomes (metacentric, submetacentric, and telocentric chromosomes). The idiogram demonstrates a continuous length gradation of chromosomes. The largest and