Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 74(2): 111-119, 2021 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-775 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Patcharaporn Chaiyasan, Sumalee Phimphan, Teamjun Sar- asan, Sippakorn Juntaree, Alongklod Tanomtong, Sitthisak Pinmongkhonkul, Weerayuth Supiwong (2021) First report on Nucleolar Organizer Regions (NORs) polymorphism and constitutive het- erochromatin of Moonlight Gourami, Trichopodus microlepis (Perciformes, Osphronemidae). Caryologia 74(2): 111-119. doi: 10.36253/caryologia-775 Received: April 02, 2020 Accepted: July 27, 2021 Published: October 08, 2021 Copyright: © 2021 Patcharaporn Chai- yasan, Sumalee Phimphan, Teamjun Sarasan, Sippakorn Juntaree, Along- klod Tanomtong, Sitthisak Pinmongk- honkul, Weerayuth Supiwong. This is an open access, peer-reviewed article published by Firenze University Press (http://www.fupress.com/caryologia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distri- bution, and reproduction in any medi- um, provided 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. First report on Nucleolar Organizer Regions (NORs) polymorphism and constitutive heterochromatin of Moonlight Gourami, Trichopodus microlepis (Perciformes, Osphronemidae) Patcharaporn Chaiyasan1, Sumalee Phimphan2, Teamjun Sarasan1, Sippakorn Juntaree3, Alongklod Tanomtong1, Sitthisak Pinmongk- honkul4, Weerayuth Supiwong3,* 1 Biology program, Faculty of Science, Khon Kaen University, Muang, Khon Kaen 40002, Thailand 2 Biology program, Faculty of Science and Technology, Phetchabun Rajabhat University, Phetchabun 67000, Thailand 3 Faculty of Interdisciplinary Studies, Khon Kaen University, Nong Khai Campus, Muang, Nong Khai 43000, Thailand 4 Department of Biology, School of Science, University of Phayao, Muang, Phayao 56000, Thailand *Corresponding author. E-mail: supiwong@hotmail.com Abstract. Nucleolar organizer regions (NORs) polymorphism, constitutive hetero- chromatin and chromosomal analysis of Moonlight gourami, Trichopodus microlepis in Thailand were firstly reported. Specimens were collected from the Chao Phraya and Mekong Basins, Thailand. The mitotic chromosomes were directly prepared from kidney tissues of ten males and ten females. Conventional staining, Ag-NOR banding and C- banding techniques were applied to stain the chromosomes. The results shown that the diploid chromosome number of T. microlepis was 2n=46 and the fundamental number (NF) was 46 in both males and females. The karyotype consisted of 46 telo- centric chromosomes classifying as 14 large and 32 medium chromosomes. No hetero- morphic sex chromosome was observed in T. microlepis. The results also showed that the interstitial nucleolar organizer regions (NORs) were clearly observed at the long arm of the chromosome pair 7. This is the first report on NORs polymorphism in T. microlepis that a heteromorphic NOR type in one female had a single NOR-bearing chromosome of the chromosome pair 7, whereas 10 males and nine females had two NOR-bearing chromosomes of the chromosome pair 7 with a homomorphic NOR type. Constitutive heterochromatin was located at all centromeres of all chromosome pairs. The karyotype formula of T. microlepis is 2n (46) = Lt14 + Mt32. Keywords: Moonlight gourami, Trichopodus microlepis, karyotype, Nucleolar Organ- izer Region, constitutive heterochromatin, chromosome. 112 Patcharaporn Chaiyasan et al. INTRODUCTION Trichopodu s which was formerly included in Trichogaster (Peapke, 2009; Töpfer and Schlindler, 2009) is a genus of tropical freshwater labyrinth fish of the gourami or family Osphronemidae and subfam- ily Trichogastrinae found in Southeast Asia. Gouramis of the Trichopodus genus are closely related to those of Trichogaster (formerly Colisa), species of both genera have long and thread-like pelvic fins (known as “feel- ers” in the aquarium trade) used to sense the environ- ment. However, Trichopodus species have shorter dorsal fin base and, when sexually mature, are much larger (Peapke, 2009; Töpfer and Schlindler, 2009). There are currently six recognized species in this genus includ- ing Trichopodus cantoris, pearl gourami (T. leerii), moonlight gourami (T. microlepis), snakeskin gourami (T. pectoralis), T. poptae and three spot gourami (T. trichopterus) (Peapke, 2009). The moonlight gourami is a labyrinth fish native to the Mekong River in Cam- bodia, Vietnam and the Chao Phraya Basin, Thailand (Vidthayanon 2005). These fish are silvery coloured with a slightly greenish hue similar to the soft glow of moonlight (Fig. 1). The moonlight gourami’s con- cavely sloped head distinguishes it from other gourami varieties. This peaceful, attractive species is a popular aquarium fish. Although the gourami fishes are importance for national economy of Thailand, there were quite scarce of cytogenetics in these fishes especially banding anal- ysis in fish chromosomes. The study on fish chromo- somes is the basic knowledge which can be applied for the several fields such as classification, evolution, heredity, systematic (Gold et al. 1990, Ueda et al. 2001, Barat et al. 2002, Barat and Sahoo 2007, Supiwong et al. 20019), breeding, rapid production of inbred lines and cytotaxonomy (Kirpichnikov 1981). Furthermore, cytogenetic studies on fish have also been used as bio- logical indicator to determine the ecological toxicol- ogy (Klinkhardt 1993, Promsid et al. 2015) and cytoge- netic techniques have been widely applied to improve farmed stocks in many aquaculture species in the World (Beardmore et al. 2001, Desprez et al. 2003, Pradeep et al. 2012). An important characteristic of Nucleo- lar Organizer Regions (NORs) in fish is related to that it has inter- and intra-species polymorphism. NORs characters can be a cytogenetic marker for cytotaxo- nomic studies and also have been used for studying of phylogenetic relationships among the Cyprinid fishes (Amemyia and Gold 1988, Galetti Jr 1998, Almeida- Toledo et al. 2000). Constitutive heterochromatin dis- tributions on the chromosomes were widely studied in some fish groups (Brinn et al. 2004, Vicari et al. 2006, Mesquita et al. 2008, Takai 2012). Generally, most con- stitutive heterochromatins locate at centromeric/peri- centromeric regions of the chromosomes. Some cases, these heterochromatins can be revealed at interstitial regions in some Pomacentrid fishes to support that the chromosomal evolution in this family is related to the chromosome fusion (Takai 2012). Moreover, constitutive heterochromatin is also highly accumulated on the W chromosome in Parodon hilarii (Parodontidae) (Morei- ra-Filho et al. 1993), Characidium fish (Crenuchidae) (Vicari et al. 2008) and Lignobrycon myersi (Triporthei- dae) (Rodrigues et al. 2016). As mention before, chromosomal analysis is very important and clearly exhibits the benefits. Moreover, the constitutive heterochromatin and polymorphism of NORs characteristics in the T. microlepis were not studied. Thus, the present study is the first report in T. microlepis from Thailand using Ag-NOR banding and C-banding techniques. MATERIALS AND METHODS Sample collection, chromosome preparation and chromo- some staining Ten male and ten female specimens of T. micro- lepis (Fig. 1) were obtained from the Chao Phraya Riv- er, Sing Buri Province, the central part of Thailand and the Mekong Basin, Nong Khai Province, Northeast of Thailand. Chromosomes were directly prepared in vivo as follows by Supiwong et al. (2013, 2017). Conventional staining was performed using 20% Giemsa’s solution for 30 min (Rooney 2001). Ag-NOR banding was carried out following by Howell and Black (1980) and C-banding was performed following from the method of Sumner et al. (1972). Figure 1. General characteristic of Moonlight Gourami, Trichopo- dus microlepis (Perciformes, Osphronemidae). 113First report on Nucleolar Organizer Regions polymorphism and constitutive heterochromatin of Trichopodus microlepis Chromosomal checks, karyotyping and idiograming Chromosome counting was carried out on mitotic metaphase cells under light microscope for 30 cells per specimen to determine the diploid number (2n). Twenty clearly observable and well-spread metaphase cells from each male and female were selected and photographed. The short arm length (Ls) and the long arm length (Ll) of each chromosome were measured to calculate the total length of the chromosome for 20 well-spread metaphase cells. The chromosome types were classified from method of Turpin and Lejeune (1965) as metacentric, submeta- centric, acrocentric and telocentric chromosomes. The karyotyping and idiograming methods were according to Turpin and Lejeune (1965) and Chaiyasut (1989). RESULTS AND DISCUSSION Diploid chromosome number, fundamental number and karyotype The diploid chromosome number (2n) of T. micro- lepis was found as 46 (Figs. 2 and 3). This result is coin- cident with previous reports by Koref-Santibanez and Paepke (1994) and Seetapan and Khamma-Ai (2007). It is also the same 2n as in the other Trichopodus spp. (Abe 1975, Koref-Santibanez and Paepke 1994, Donsakul and Magtoon 1988, Seetapan and Khamma-Ai 2007, Mag- toon et al. 2007, Supiwong et al. 2010), Trichogaster chu- na (Koref-Santibanez and Paepke 1994) and Trichogaster lalius (Abe 1975, Rishi 1976, Koref-Santibanez and Paepke 1994). These species have the diploid chromo- some number of 2n=46, which is an apparent modal diploid number of the Trichopodus. Accordingly, it can be concluded that chromosome number in this genus is conserved. However, it differs from the most species of the genus Trichogaster (T. labiosa, T. fasciata, T. labiosus, T. sumatranus) which had 2n=48 (Kaur and Srivastava 1965, Calton and Denton 1974, Abe 1975, Rishi 1975, Manna and Prasad 1977, Tripathy and Das 1981, Koref- Santibanez and Paepke 1994, Rishi et al. 1994, Sobita and Bhagirath 2007, Kushwaha et al. 2008) (Table 1). The fundamental number (NF) of T. microlepis was 46 in both males and females. The karyotype consisted of 46 telocentric chromosomes (all as mono-arm chro- mosomes). These results are agreeable with the previous reports of both T. microlepis and all Trichopodus species (Abe 1975, Donsakul and Magtoon 1988, Koref-Santi- banez and Paepke 1994, Magtoon et al. 2007, Seetapan and Khamma-Ai 2007, Supiwong et al. 2010). Howev- Figure 2. Karyotypes of male (A) and female (B) of Trichopodus microlepis, 2n=46 by conventional staining. Scale bars = 5 µm. Figure 3. Karyotypes of Trichopodus microlepis, 2n=46 by Ag-NOR banding (A) and C-banding techniques (B). The chromosome pair 7 show Ag-NOR and heteromorphic Ag-NOR (inserted box). Scale bars = 5 µm. 114 Patcharaporn Chaiyasan et al. er, they are different from all of the genus Trichogaster (Kaur and Srivastava 1965, Calton and Denton 1974, Abe 1975, Rishi 1975, Manna and Prasad 1977, Tripathy and Das 1981, Koref-Santibanez and Paepke 1994, Rishi et al. 1994, Sobita and Bhagirath 2007, Kushwaha et al. 2008). The NFs of the genus Trichogaster range from 48 to 86 and karyotypes composed of both mono- and bi-arm chromosomes. Nirchio et al. (2002) proposed that species with high NF is advanced state or apomor- phic character whereas one with low NF is a primitive state or plesiomorphic character. T. microlepis includ- ing all species of the genus Trichopodus have all mono- arm chromosomes in karyotype whereas most species of the genus Trichogaster display both mono- arm and bi-arm chromosomes (Table 1). Thus, the Trichoprodus seems to be more primitive karyotype than that in the Trichogaster. The T. microlepis karyotype consisted of 14 large telocentric and 32 medium telocentric chromo- somes (Table 2). The karyotype formula for this species is 2n (46) = Lt14 + Mt32. There is no evidence of differen- tiated sex chromosomes in this species which accord to all species of this genus (Abe 1975, Donsakul and Mag- toon 1988, Koref-Santibanez and Paepke 1994, Magtoon et al. 2007, Seetapan and Khamma-Ai 2007, Supiwong et al. 2010). Similar to several gourami fishes, no cytologi- cally distinguishable sex chromosome was observed. Chromosome markers from Ag-NOR banding and C-banding Present study was accomplished by using Ag-NOR staining and C-banding in T. microlepis. The NORs are used as makers to detect species specific character and indicate intra- and inter species chromosomal polymor- phism in many groups of fishes (Ráb et al. 2008). The Ag-NOR positions were shown on the long arm near the centromere of the telocentric chromosome pair 7 (sub- centromeric NOR) in 10 male and nine female fish (Fig. Table 1. Karyotype characteristics of some species in the subfamily Trichogastrinae. Species 2n NF Karyotype NOR Reference Trichogaster chuna 46 66 20m+26st/a – Koref-Santibanez and Paepke (1994) T. labiosa 48 66 12m+6sm+12st+18a/t – Manna and Prasad (1977) 48 68 20m+10st+18a/t – Koref-Santibanez and Paepke (1994) T. lalius 46 70 24m/sm+22a/t – Abe (1975) 46 – 26m+1sm/st+19a/t – Rishi (1976) 46 66 20m+8st+18a/t – Koref-Santibanez and Paepke (1994) T. fasciata 48 48 48a/t – Kaur and Srivastava (1965) 48 74 14m+12sm+22a/t – Rishi (1975) 48 78 8m+20sm+12st+8a/t – Manna and Prasad (1977) 48 78 18m+12sm+18a/t – Tripathy and Das (1981) 48 68 20m+12st+16a/t – Koref-Santibanez and Paepke (1994) 48 80–81 16m+16sm+15a/t(16a/t) – Rishi et al. (1994) 48 83 15m+16sm+4st+13a/t 6 Sobita and Bhagirath (2007) 48 86 16m+16sm+6st+10a/t 2 Kushwaha et al. (2008) T. sumatranus 48 48 48st/a – Calton and Denton (1974) Trichopodus leeri 46 46 46a/t – Abe (1975) Koref-Santibanez and Paepke (1994) 46 46 46a/t – Seetapan and Khamma-Ai (2007) T. microlepis 46 46 46a/t – Koref-Santibanez and Paepke (1994) 46 46 46a/t – Seetapan and Khamma-Ai (2007) 46 46 46t 2 Present study T. pectoralis 46 46 46a/t – Koref-Santibanez and Paepke (1994) 46 46 46a/t – Donsakul. and Magtoon (1988) 46 46 46a/t – Seetapan and Khamma-Ai (2007) T. trichopterus 46 46 46a/t – Abe (1975), Koref-Santibanez and Paepke (1994) 46 46 46a/t – Magtoon et al. (2007) 46 46 46t/t 2 Supiwong et al. (2010) Remarks: 2n = diploid number, NF = the fundamental number, NOR = Nucleolar Organizer Region, m = metacentric, sm = submetacen- tric, st = subtelocentric, a = acrocentric, t = telocentric chromosomes and – = not available. 115First report on Nucleolar Organizer Regions polymorphism and constitutive heterochromatin of Trichopodus microlepis 3A). The single pair of NOR is the same as in T. trichop- terus (Supiwong et al. 2010) and T. fasciata reported by Kushwaha et al. (2008) but there is difference in T. fas- ciata which had three pairs of NORs (Sobita and Bha- girath 2007) and Betta splendens which had two pairs of NORs (Furgala-Selezniow et al. 2008). Gold and Amemiya (1986) suggested that the occurrence of mul- tiple NORs in fishes was considered to be apomorphic or advance condition whereas single pair of NORs was considered to be plesiomorphic or a primitive condi- tion. Considering for NOR loci between T. microlepis and T. trichopterus, although both species had the single NOR pair, the NOR positions are difference. The present results revealed that T. microlepis had interstitial NORs on the chromosome pair 7 whereas T. trichopterus had telomeric NORs (region adjacent to the telomere) on the chromosome pair 2 (Supiwong et al. 2010). Therefore, the NOR-bearing chromosome markers can be used as a tool for classification in this fish group. In addition, intraspecific NOR heteromorphism between the homolo- gous chromosomes of pair 7 was also displayed in one female specimen (Fig. 3A, inserted box). This phenom- enon is common event found previously in several fishes in Thailand such as Puntioplites proctozysron (Supiwong et al. 2012), Lutjanus johnii (Phimphan et al. 2013), Pter- apogon kauderni (Kasiroek et al. 2017) and Hemibagrus wyckii (Supiwong et al. 2017). Constitutive heterochromatic blocks were observed at centromeric and pericentromeric regions of all chro- mosomes and with no clear interstitial and telomeric positive C-bands (Fig. 3B). It indicates that the chromo- somes of T. microlepis are conserved and non-related to chromosomal fusion or an increase in heterochromatin during evolution. Present result is similar to some spe- cies in another family of the order Perciformes such as Geophagus brasiliensis and C. facetum in the Cichlidae family (Vicari et al. 2006), Plectroglyphidodon lacryma- tus, Chrysiptera leucopoma, C. rex and Neoglyphidodon melas in the Pomacentridae family (Takai 2012). How- ever, there are several species which presented the com- plex types of positive C-bands. Symphysodon haraldi, S. aequifasciatus and S. discus (Cichlidae) had heterochro- matic blocks on the pericentromeric regions of all chro- mosomes and the proximal regions of both arms of some Table 2 Mean length of short arm chromosome (Ls), long arm chromosome (Ll), total arm chromosome (LT), relative length (RL), centro- meric index (CI) and standard deviation (SD) of RL, CI from 20 metaphases (both males and females) of the Moonlight gourami (Trichopo- dus microlepis) in Thailand, 2n=46 Chromosome pair Ls Ll LT RL±SD CI±SD Type Size 1 0.000 0.755 0.755 0.0306±0.0026 1.000±0.000 telocentric L 2 0.000 0.682 0.682 0.0276±0.0015 1.000±0.000 telocentric L 3 0.000 0.647 0.647 0.0261±0.0011 1.000±0.000 telocentric L 4 0.000 0.621 0.621 0.0251±0.0008 1.000±0.000 telocentric L 5 0.000 0.603 0.603 0.0243±0.0007 1.000±0.000 telocentric L 6 0.000 0.589 0.589 0.0237±0.0006 1.000±0.000 telocentric L 7* 0.000 0.578 0.578 0.0232±0.0005 1.000±0.000 telocentric L 8 0.000 0.567 0.567 0.0228±0.0004 1.000±0.000 telocentric M 9 0.000 0.559 0.559 0.0225±0.0004 1.000±0.000 telocentric M 10 0.000 0.549 0.549 0.0221±0.0004 1.000±0.000 telocentric M 11 0.000 0.538 0.538 0.0217±0.0004 1.000±0.000 telocentric M 12 0.000 0.529 0.529 0.0213±0.0004 1.000±0.000 telocentric M 13 0.000 0.521 0.521 0.0209±0.0004 1.000±0.000 telocentric M 14 0.000 0.513 0.513 0.0206±0.0004 1.000±0.000 telocentric M 15 0.000 0.506 0.506 0.0203±0.0004 1.000±0.000 telocentric M 16 0.000 0.498 0.498 0.0201±0.0004 1.000±0.000 telocentric M 17 0.000 0.491 0.491 0.0197±0.0005 1.000±0.000 telocentric M 18 0.000 0.479 0.479 0.0193±0.0006 1.000±0.000 telocentric M 19 0.000 0.470 0.470 0.0189±0.0006 1.000±0.000 telocentric M 20 0.000 0.457 0.457 0.0184±0.0005 1.000±0.000 telocentric M 21 0.000 0.442 0.442 0.0178±0.0007 1.000±0.000 telocentric M 22 0.000 0.425 0.425 0.0170±0.0009 1.000±0.000 telocentric M 23 0.000 0.397 0.397 0.0159±0.0015 1.000±0.000 telocentric M Remarks: * = NOR-bearing chromosome, L=large, and M=medium. 116 Patcharaporn Chaiyasan et al. chromosomes (Mesquita et al. 2008), while N. nigro- ris (Pomacentridae) exhibited the distribution of posi- tive C-bands in most centromeric regions and including many terminal and interstitial regions (Takai 2012). The idiogram shows a continuous length gradation of chromosomes. Approximately two-fold of the size differences between the largest and smallest chromo- somes were revealed. The marker chromosomes are the chromosome pair 1, which is the largest telocentric and the chromosome pair 23 is the smallest telocentric. The data of the chromosome measurement on mitotic meta- phase cells (from all specimens) are shown in Table 2. Idiograms by conventional staining and C- banding are shown in Fig. 4. In conclusion, NOR phenotype and constitutive heterochromatin patterns on the chromo- somes are specific to species in the genus Trichopodus. For more information about the chromosomal diversity and chromosomal evolution in this genus, more species and techniques should be further studied. 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