Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 75(3): 3-11, 2022 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-1731 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Kamika Sribenja, Alongklod Tanomtong, Nuntaporn Getlekha (2022). Chromosome Mapping of Repetitive DNAs in the Picasso Triggerfish (Rhi- necanthus aculeatus (Linnaeus, 1758)) in Family Balistidae by Classical and Molecular Cytogenetic Techniques. Caryologia 75(3): 3-11. doi: 10.36253/ caryologia-1731 Received: July 08, 2022 Accepted: November 19, 2022 Published: April 5, 2023 Copyright: © 2022 Kamika Sribenja, Alongklod Tanomtong, Nuntaporn Getlekha. This is an open access, peer-reviewed article published by Firenze University Press (http://www. fupress.com/caryologia) and distrib- uted under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, pro- vided 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. Chromosome Mapping of Repetitive DNAs in the Picasso Triggerfish (Rhinecanthus aculeatus (Linnaeus, 1758)) in Family Balistidae by Classical and Molecular Cytogenetic Techniques Kamika Sribenja1, Alongklod Tanomtong1, Nuntaporn Getlekha2,* 1 Department of biology, Faculty of Science, Khon Kaen University, Muang, KhonKaen 40002, Thailand 2 Department of Biology, Faculty of Science and Technology, Muban Chombueng Rajab- hat University, Chombueng, Ratchaburi 70150, Thailand *Corresponding author. E-mail: nanthaphonket@mcru.ac.th Abstract. This work presents the cytogenetic analysis conducted on the Picasso trig- gerfish (Rhinecanthus aculeatus (Linnaeus, 1758)) from Thailand. Mitotic chromo- somes were prepared from the anterior kidney. The cell suspensions were harvested by in vivo colchicine treatment. The present study includes the chromosomal investigation on R. aculeatus, using conventional (Giemsa staining, Ag-NOR and C-banding) and molecular approaches (in situ mapping of five different repetitive DNA classes includ- ing 18S rDNA, 5S rDNA, (CA)15, (GA)15 and (CAA)10 as markers.) The results showed that R. aculeatus has karyotypes formed exclusively by telocentric chromosomes (44t; NF=44). The C-positive heterochromatic blocks are preferentially located in the cen- tromeric and telomeric regions of some chromosomal pairs. The Ag-NOR sites occu- py the interstitial position of the long arms of the largest telocentric pair (pair 1). The exclusive location of the major ribosomal sites in these pairs was confirmed by hybridization with 18S rDNA probes. However, the 5S rDNA genes are not located on 18S rDNA-bearing chromosomes, but instead located exclusively in the subcentro- meric region of pair 4. The mapping of (CA)15, (GA)15 and (CAA)10 microsatellites are sparsely dispersed along all the chromosomes. The karyotype formula of R. aculeatus is 2n (44) = 44t. Keywords: Triggerfish, Chromosome, Repetitive sequences, Fish cytogenetics. INTRODUCTION The family Balistidae belongs to order Tetraodontiformes which includes the triggerfish of often brightly colored fish (Nelson et al., 2016). Around 40 species distributed in 12 genera are classified in this family (Allen et al., 2017). Triggerfish fishes are usually found in tropical and subtropical oceans throughout the world, with the greatest species richness in the Indo-Pacific. 4 Kamika Sribenja, Alongklod Tanomtong, Nuntaporn Getlekha The most abundance of species are found in relatively shallow, coastal habitats, especially at coral reefs (Allen et al., 2017). In the present, several species from this family are popular in the marine aquarium trade. Out of 40 described species of Balistidae, only 15 species have been karyologically investigated: Balista- pus undulates, Sufflamen fraenatus (Takai and Ojima, 1987), Balistes capriscus (Vitturi et al., 1988), Balistes carolinensis (Vitturi et al., 1992; Thode et al., 1994), Bal- istes vetula (Gustavo and Molina, 2005), Balistoides con- spicillus (Takai and Ojima, 1987; Gustavo and Molina, 2004), Balistoides viridescens (Takai and Ojima, 1988), Pseudobalistes flavimarginatus, Rhinecanthus verruco- sus, Sufflamen chrysopterus (Arai and Nagaiwa, 1976), Rhinecanthus aculeatus (Arai and Nagaiwa, 1976; Kitay- ama and Ojima, 1984), Rhinecanthus echarpe (Kitayama and Ojima, 1984), Melichthys niger (Gustavo and Molina, 2005) and Melichthys vidua, Odonus niger (Kitayam and Ojima, 1984). The members of the family Balistidae have 2n ranging from 40 to 46, and most species have the karyotype present as acrocentric and telocentric chro- mosomes except B. viridescens and P. flavimarginatus, which are comprised of metacentric and submetacentric chromosomes (Table 1). The study aims to investigate the evolutionary events associated with the chromosomal diversification in the Picasso triggerfish (Rhinecanthus aculeatus). The chro- mosomal investigation was conducted by obtaining the standard karyotype and idiogram using conventional (Giemsa staining, Ag-NOR and C-banding) and molec- ular analyses to identify the chromosomal patterns and organization of five classes of repetitive DNAs [18S rDNA, 5S rDNA, (CA)15, (GA)15, and (CAA)10]. Since there were only three previous cytogenetic studies of the genus Rhinecanthus showing a diploid chromosome number of 2n=44 (Arai and Nagaiwa, 1976; Kitayama and Ojima, 1984), the results obtained from this study will increase our basic knowledge of the cytogenetics of R. aculeatus, which could form the basis for future research and support taxonomy of genus Rhinecanthus. MATERIAL AND METHODS Specimens collected and conventional methods Cytogenetic analyses were conducted on the Picasso triggerfish, Rhinecanthus aculeatus (4 males and 4 females) from Thailand Gulf (Figure 1). The specimens were caught using a hand-net, placed in sealed plastic bags containing oxygen and clean water and transported to the research station. The experiments followed ethical protocols and Table 1. Cytogenetic reviews of the family Balistidae (8 genera). No. Subfamily/Species 2n NF NORs Formula References 1 Balistapus undulates 42 42 2 42a/t Takai and Ojima (1987) 2 Balistes capriscus 44 44 2 44t Vitturi et al. (1988) 3 B. carolinensis 44 44 2 44t Vitturi et al. (1992) 44 44 2 44t Thode et al. (1994) 4 B. vetula 44 44 2 44t Gustavo and Molina (2005) 5 Balistoides conspicillus 44 44 2 44t Takai and Ojima (1987) 44 44 2 44t Gustavo and Molina (2004) 6 B. viridescens 44 48 2 2m+2sm+40a/t Takai and Ojima (1988) 44 60 3 2m+14a+28t Supiwong et al. (2013) 7 Melichthys niger 40 40 2 40t Gustavo and Molina (2005) 40 40 2 40a/t de Lima et al. (2011) 8 M. vidua 40 40 2 40a/t Kitayama and Ojima (1984) 9 Odonus niger 42 – – 42a/t Kitayama and Ojima (1984) 10 Pseudobalistes flavimarginatus 44 – – 2m+42a/t Arai and Nagaiwa (1976) 11 Rhinecanthus aculeatus 44 44 2 44t Arai and Nagaiwa (1976) 44 44 2 44t Kitayama and Ojima (1984) 12 R. echarpe 44 – 2 44a/t Kitayama and Ojima (1984) 13 R. verrucosus 44 44 2 44t Arai and Nagaiwa (1976) 14 Sufflamen chrysopterus 46 46 – 46a/t Arai and Nagaiwa (1976) 15 S. fraenatus 46 46 2 46a/t Takai and Ojima (1987) Remarks: 2n = diploid chromosome number, NF = fundamental number (number of chromosome arm), m = metacentric, sm = submeta- centric, a = acrocentric, t = telocentric chromosome, NORs = nucleolar organizer regions and – = not available. 5Chromosome Mapping of Repetitive DNAs in the Picasso Triggerfish anesthesia with clove oil prior to sacrificing the animals to minimize suffering. The process was approved by the Eth- ics Committee of Khon Kaen University and by the RGJ Committee under no. PHD/K0081/2556. Mitotic chromo- somes were obtained from cell suspensions of the anterior kidney, using the conventional air-drying method. The C-banding method was also employed to detect the distri- bution of C-positive heterochromatin and silver staining to detect the Ag-NOR location on chromosomes. The speci- mens were deposited in the fish collection of the Cytoge- netic Laboratory, Department of Biology, Faculty of Sci- ence, Khon Kaen University. Chromosome probes and FISH experiments Two tandemly arrayed DNA sequences isolated from the genome of an Erythrinidae fish species, Hop- lias malabaricus, were used as probes. The first probe contained a 5S rDNA repeat and included 120 base pairs (bp) of the 5S rRNA transcribed gene and 200 bp of the non-transcribed spacer (NTS) sequence. The sec- ond probe contained a 1400 bp segment of the 18S rRNA gene obtained via PCR from the nuclear DNA. The 5S and 18S rDNA probes were cloned into plasmid vectors and propagated in DH5a Escherichia coli competent cells (Invitrogen, San Diego, CA, USA). The 5S and 18S rDNA probes were labeled with Spectrum Green-dUTP and Spectrum Orange-dUTP, respectively, using nick trans- lation according to the manufacturer’s recommendations (Roche, Mannheim, Germany). The microsatellites (CA)15, (GA)15, and (CAA)10 were synthesized. These sequences were directly labeled with Cy3 at the 5’ terminus during synthesis by Sigma (St. Louis, MO, USA). Fluorescence in situ hybridization (FISH) was per- formed under high stringency conditions (Yano et al., 2017). Metaphase chromosome slides were incu- bated with RNAse (40 μg/ml) for 1.5 h at 37°C. After the denaturation of the chromosomal DNA in 70% formamide/2x SSC at 70°C for 4 min, 20 µl of the hybridization mixture (2.5 ng/μl probes, 2 μg/μl salmon sperm DNA, 50% deionized formamide, 10% dextran sulphate) was dropped on the slides, and the hybridiza- tion was performed overnight at 37°C in a moist cham- ber containing 2x SSC. The first post-hybridization wash was performed with 2x SSC for 5 min at 65°C, and a final wash was performed at room temperature in 1x SSC for 5 min. Finally, the slides were counter- stained with DAPI and mounted in an antifade solution (Vectashield from Vector Laboratories). Image processing Approximately 20 metaphase spreads were analyzed to confirm the diploid chromosome number, karyo- type structure and FISH results. Images were captured using an Olympus BX50 microscope (Olympus Corpo- ration, Ishikawa, Japan) with CoolSNAP and the Image Pro Plus 4.1 software (Media Cybernetics, Silver Spring, MD, USA). Chromosomes were classified according to their arm ratios as metacentric (m), submetacentric (sm), acrocentric (a) or telocentric (t). RESULTS The Picasso triggerfish (Rhinecanthus aculeatus) have 2n=44. Its karyotype is formed exclusively by telo- centric chromosomes (44t) and a fundamental number (NF=44) (Figure 2). The C-positive heterochromatic blocks are preferentially located in the centromeric regions, with some pairs exhibiting blocks in the telo- meric ones (Figure 2). The Ag-NOR sites are located in the interstitial region of the long arms of the largest telocentric pair (pair 1), the exclusive location of major ribosomal sites in these regions was confirmed by in situ hybridization with 18S rDNA probes (Figure 2). How- ever, the 5S rDNA genes are not located on 18S rDNA- bearing chromosomes, but instead located exclusively in the subcentromeric region of pair 4, while the 18S rDNA sites are instead located on the interstitial position of the long arms of pair 1 (Figure 2). The chromosomal mapping of all microsatellite sequences indicates a weak and dispersed distribution, without preferential accumulations in any of the chro- mosomal pairs (Figure 3). The (CA)15 (GA)15 and (CAA)10 Figure 1. General characteristic of Rhinecanthus aculeatus, its respective collection sites in the Indian (Andaman Sea) and Pacific Oceans (Gulf of Thailand). 6 Kamika Sribenja, Alongklod Tanomtong, Nuntaporn Getlekha microsatellites are sparsely dispersed in most chromo- somes though they can still form conspicuous clusters. They however exhibit less defined clusters in some chro- mosome pairs (Figure 3). These clusters occupy the cen- tromeric regions of chromosomes but at rather low fre- quency. For all the chromosomal markers, no differential hybridization patterns were detected between males and females. Figure 2. Mataphase and karyotypes of Rhinecanthus aculeatus arranged from conventionally Giemsa-stained, Ag-stained (highlighted in the boxes), C-banded and after fluorescence in situ hybridization with 5S and 18S rDNA probes. Bar 5 μm. 7Chromosome Mapping of Repetitive DNAs in the Picasso Triggerfish The idiogram of R. aculeatus represents gradually declining length of the chromosomes (Figure 4). The karyotype is notably attributed solely to telocentric chro- mosomes. The karyotype formula of R. aculeatus is as follow: 2n (44) = 44t Figure 3. Chromosomal mapping of di- and tri-nucleotide microsatellites in the chromosomes of Rhinecanthus aculeatus by fluorescence in situ hybridization. The general distribution pattern of (CA)15, (GA)15 and (CAA)10 microsatellites is mainly diffuse, with the occurrence of few conspicuous clusters in some centromeric and chromosome arm regions. Bar = 5 μm. 8 Kamika Sribenja, Alongklod Tanomtong, Nuntaporn Getlekha DISCUSSIONS Karyotype uniformity among Rhinecanthus species Cytogenetic analyses were conducted on Rhinecan- thus aculeatus from the Gulf of Thailand (Indo-Pacific). The 2n of R. aculeatus is 44 chromosomes in all speci- mens, with karyotypes predominantly formed by telo- centric chromosomes (Figure 2, 3 and Table 2). However, considerable cytogenetic research has been conducted on a number of species in the family Balistidae (Table 1). The karyotype of Rhinecanthus aculeatus is 44t; this finding is consistent with that of other species in the genera Rhinecanthus, Balistes, and Balistoides, particu- larly Balistes capriscus, B. carolinensis, B. vetula, Balis- toides conspicillus, Rhinecanthus aculeatus, R. echarpe, and R. verrucosus, which still have 2n=44t. It suggests that even after speciation, their karyotypes remain con- served. Although B. viridescens and Pseudobalistes fla- vimarginatus have the same number of chromosomes (2n=44) with above species but exhibit an asymmetrical karyotype due to both species are the high variability of chromosomal rearrangements and their higher adaptive divergence. Moreover, like all other species in the fam- ily Balistidae, in which the morphologically differenti- ated sex chromosome could not be observed (Arai and Nagaiwa, 1976; Kitayama and Ojima, 1984). In addition, Rhinecanthus species exhibited karyo- types which are broadly similar in structural patterns, with all of them displaying 2n = 44 and a high number of telocentric chromosomes. These characteristics pre- sent in all of the Rhinecanthus species analyzed so far (Arai and Nagaiwa, 1976; Kitayama and Ojima, 1984; Montanari et al. 2016; present study). Chromosome markers of R. aculeatus The only one pair which bear Ag-NOR/18S rDNA sites are useful chromosomal markers shared among the Rhinecanthus species. The result here is also similar to the chromosome bearing nucleolar organizer region in previous studies (Table 1) except Balistoides viride- scens that found tree NORs (Supiwong et al., 2013) this suggests that this event may be related to chromosomal change during evolution. Furthermore, the interstitial region of the largest telocentric chromosome pair 1 of R. aculeatus showed clearly observable nucleolar organizer regions. This is quite consistent with the report by de Lima et al. (2011) on the karyotype of Melichthys niger in the same fam- ily. Their study reported the presence of a conspicuous secondary constriction in the interstitial position on the long arm of the chromosome pair No. 2 which was, cor- responding to the nucleolar organizer regions, identified by Ag-NOR sites and by in situ hybridization with an 18S rDNA ribosomal probe. Normally, most fishes have only one pair of NORs on chromosomes. Only some fishes have more than two NORs, which may be caused by the translocation between some parts of the chromosomes that have NOR and another chromosome (Sharma et al., 2002). The pre- sent study shows that the species analyzed presents NOR site on a single chromosome pair. This is considered a simple isomorphic condition in fish (Almeida-Toledo, 1985). Another peculiar cytogenetic aspect of Tetrao- dontiformes is the small quantity of heterochromatic regions, localized in telomeric or centromeric positions on most of the chromosome pairs (Supiwong et al., 2013). Organization of repetitive DNAs in the chromosomes of R. aculeatus The C-positive heterochromatins in the chromo- somes of R. aculeatus are distributed in centromeric and telomeric positions in most of the chromosomes (Figure 2). This recurring distribution pattern is similar to those reported for species of other Balistidae genera, such as Melichthys (de Lima et al., 2011). The 18S rDNA sites are equally located on the inter- stitial position of pair 1, whereas 5S rDNA sites occur in the subcentromeric region of pair 4. The non-syntenic organization of these genes is frequent and it could be a plesiomorphic condition in Balistidae. This is the first report of the presence of microsat- ellite sequences in the heterochromatin of R. aculea- tus which show recognizable organizational patterns. Figure 4. Standardized idiogram showing lengths and shapes of chromosomes of Rhinecanthus aculeatus (2n=44) by conventional staining and Ag-NOR banding techniques. The arrow indicates nucleolarnucleolar organizer regions. 9Chromosome Mapping of Repetitive DNAs in the Picasso Triggerfish The (CA)15 (GA)15 and (CAA)10 microsatellites present a weak and diffuse distribution on all chromosomes, but they also present a small number of conspicuous clusters characterized by intense signs in some parts of chromo- somes (Figure 3). Thus, this data is useful for compar- ing the phylogenetic proximity of this genus that may share the same distribution pattern of the microsatel- lite sequences which points to independent evolution- ary pathways, constituting homoplastic chromosomal characters. However, since these sequences are subject to high rates of change, their distribution may show marked evolutionary differentiation (Cioffi et al., 2011; Molina et al., 2014a; 2014b). In fact, the organization of microsatellite sequences demonstrates the particular arrangements that repetitive DNAs can be achieved in different species. Chromosome evolution of the family Balistidae Chromosomal rearrangements represent the main cause of karyotype diversification among several Perci- formes species (Arai, 2011; Molina and Galetti, 2002). The different Balistidae species underwent an extremely diversified karyotype evolution, considering the numeri- cal and structural aspects of their complements, with diploid chromosome number varying from 2n=40 to 46, and marked differences in the NF that varied from 40 to 60, possibly due to the occurrence of pericentric inver- sions (Getlekha et al., 2018). Analyses performed high- light the combined importance of the different chro- mosome rearrangements in the evolutionary modelling of their karyotypes, such as centric fission fusion, and especially pericentric inversions (Getlekha et al., 2016a; 2016b). The family Balistidae has 2n values lower than 2n=48 with most of their representatives presenting acrocentric and telocentric chromosomes. This karyo- typic pattern was also observed in the present study in R. aculeatus (2n=44). The origin of the reduced dip- loid chromosome numbers in these species seems to be centric fissions but chromosome lost in tandem, which seems to be common in other species of the family (Arai and Nagaiwa 1976; Marques et al., 2016). Table 2. Mean length of short arm chromosome (Ls), length long arm chromosome (Ll), length total arm chromosome (LT), relative length (RL), centromeric index (CI) and standard deviation (SD) of RL, CI from 20 metaphase cells of the male and female the Picasso triggerfish (Rhinecanthus aculeatus), 2n=44. Chromosome pair Ls Ll LT RL±SD CI±SD Chromosome type 1* 0.00 2.60 2.60 0.070±0.009 1.000±0.000 telocentric 2 0.00 2.20 2.20 0.059±0.004 1.000±0.000 telocentric 3 0.00 2.06 2.06 0.056±0.005 1.000±0.000 telocentric 4 0.00 2.04 2.04 0.055±0.004 1.000±0.000 telocentric 5 0.00 1.97 1.97 0.052±0.003 1.000±0.000 telocentric 6 0.00 1.93 1.93 0.051±0.003 1.000±0.000 telocentric 7 0.00 1.90 1.90 0.051±0.005 1.000±0.000 telocentric 8 0.00 1.86 1.86 0.049±0.003 1.000±0.000 telocentric 9 0.00 1.75 1.75 0.047±0.003 1.000±0.000 telocentric 10 0.00 1.74 1.74 0.047±0.004 1.000±0.000 telocentric 11 0.00 1.72 1.72 0.046±0.005 1.000±0.000 telocentric 12 0.00 1.71 1.71 0.045±0.003 1.000±0.000 telocentric 13 0.00 1.68 1.68 0.044±0.003 1.000±0.000 telocentric 14 0.00 1.61 1.61 0.043±0.003 1.000±0.000 telocentric 15 0.00 1.59 1.59 0.042±0.003 1.000±0.000 telocentric 16 0.00 1.55 1.55 0.041±0.003 1.000±0.000 telocentric 17 0.00 1.45 1.45 0.038±0.003 1.000±0.000 telocentric 18 0.00 1.40 1.40 0.036±0.005 1.000±0.000 telocentric 19 0.00 1.40 1.40 0.036±0.006 1.000±0.000 telocentric 20 0.00 1.30 1.30 0.034±0.006 1.000±0.000 telocentric 21 0.00 1.19 1.19 0.030±0.007 1.000±0.000 telocentric 22 0.00 1.10 1.10 0.028±0.007 1.000±0.000 telocentric Remark: * NOR-bearing chromosome. 10 Kamika Sribenja, Alongklod Tanomtong, Nuntaporn Getlekha CONCLUSION Based on the chromosome study of the Picasso triggerfish (R. aculeatus) using conventional analy- ses (Giemsa staining, Ag-NOR and C-banding) and molecular analysis (in situ mapping of five different repetitive DNA classes including 18S rDNA, 5S rDNA, (CA)15, (GA)15 and (CAA)10 as markers), this research can verif y diploid chromosome, fundamental num- ber and distribution patterns of microsatellites on the chromosomes. The results show that R. aculeatus has 2n=44 with predominantly telocentric chromosome. The fundamental number (NF) was 44. The C-positive heterochromatic blocks are preferentially located in the centromeric and telomeric regions of some chromosomal pairs. The Ag-NORs sites were located on the interstitial region of long arms of the telocentric chromosome pair 1. The exclusive location of the major ribosomal sites in these pairs was confirmed by in situ hybridization with 18S rDNA probes. However, the 5S rDNA genes are not located on 18S rDNA-bearing chromosomes, but instead located exclusively in the subcentromeric region of pair 4. The mapping of (CA)15, (GA)15 and (CAA)10 microsat- ellites are sparsely dispersed along all the chromosomes. The idiogram of R. aculeatus represents gradually declining length of the chromosomes. 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