First Report on Nucleolar Organizer Regions (NORs) Polymorphism and Constitutive Heterochromatin of Moonlight Gourami, Trichopodus microlepis (Perciformes, Osphronemidae)

aBiology program, Faculty of Science, Khon Kaen University, Muang, Khon Kaen 40002, Thailand; bBiology program, Faculty of Science and Technology, Phetchabun Rajabhat University, Phetchabun 67000, Thailand; cFaculty of Interdisciplinary Studies, Khon Kaen University, Nong Khai Campus, Muang, Nong Khai 43000, Thailand; dDepartment of Biology, School of Science, University of Phayao, Muang, Phayao 56000, Thailand.


Introduction
Trichopodus 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 subfamily 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 "feelers" in the aquarium trade) used to sense the environment. 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 including 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 Cambodia, 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 concavely 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 analysis in fish chromosomes. The study on fish chromosomes 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, breeding, rapid production of inbred lines and cytotaxonomy (Kirpichnikov 1981). Furthermore, cytogenetic studies on fish have also been used as biological indicator to determine the ecological toxicology (Klinkhardt 1993, Promsid et al. 2015 and cytogenetic 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 Nucleolar Organizer Regions (NORs) in fish is related to that it has inter-and intra-species polymorphism. NORs characters can be a cytogenetic marker for cytotaxonomic 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 distributions 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 constitutive heterochromatins locate at centromeric/pericentromeric 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 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.

Sample collection, Chromosome preparation and Chromosome staining
Ten male and ten female specimens of T. microlepis ( Fig. 1) were obtained from the Chao Phraya River, 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. (2013Supiwong et al. ( , 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).

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, submetacentric, acrocentric and telocentric chromosomes. The karyotyping and idiograming methods were according to Turpin and Lejeune (1965) and Chaiyasut (1989).

Diploid chromosome number, fundamental number and karyotype
The diploid chromosome number (2n) of T. microlepis was found as 46 (Figs. 2 and 3). This result is coincident 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   Abe 1975, Rishi 1976, Koref-Santibanez and Paepke 1994. These species have the diploid chromosome 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) 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 apomorphic character whereas one with low NF is a primitive state or plesiomorphic character. T. microlepis including 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 chromosomes ( Table 2). The karyotype formula for this species is 2n (46) = L t 14 + M t 32.
There is no evidence of differentiated sex chromosomes in this species which accord to all species of this genus (Abe 1975, Donsakul and Magtoon 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 cytologically 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 polymorphism 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 (subcentromeric NOR) in 10 male and nine female fish (Fig. 3A). The single pair of NOR is the same as in T. trichopterus (Supiwong et al. 2010) and T. fasciata reported by Kushwaha et al. (2008) but there is difference in T. fasciata which had three pairs of NORs (Sobita and Bhagirath 2007) and Betta splendens which had two pairs of NORs (Furgala-Selezniow et al. 2008). Gold and Amemiya (1986) suggested that the occurrence of multiple NORs in fishes was considered to be apomorphic or advance condition whereas single pair of NORs was considered to be plesiomorphic or a primitive condition. 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 homologous chromosomes of pair 7 was also displayed in one female specimen  Table 2. Idiograms by conventional staining and C-banding are shown in Fig. 4. In conclusion, NOR phenotype and constitutive heterochromatin patterns on the chromosomes 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.