Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 74(1): 89-96, 2021 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-1017 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: S. Aiumsumang, S. Phim- phan, C. Suwannapoom, P. Chaiyasan, W. Supiwong, A. Tanomtong (2021) A com- parative chromosome study on five Minnow fishes (Cyprinidae, Cyprini- formes) in Thailand. Caryologia 74(1): 89-96. doi: 10.36253/caryologia-1017 Received: July 10, 2020 Accepted: February 10, 2021 Published: July 20, 2021 Copyright: © 2021 S. Aiumsumang, S. Phimphan, C. Suwannapoom, P. Chai- yasan, W. Supiwong, A. Tanomtong. This is an open access, peer-reviewed article published by Firenze University Press (http://www.fupress.com/caryo- logia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, 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. A comparative chromosome study on five Minnow fishes (Cyprinidae, Cypriniformes) in Thailand Surachest Aiumsumang1,3, Sumalee Phimphan1, Chatmongkon Suwan- napoom2, Patcharaporn Chaiyasan3, Weerayuth Supiwong4,*, Alongk- lod Tanomtong3,5 1 Biology Program, Faculty of Science and Technology, Phetchabun Rajabhat University, Phetchabun 67000, Thailand 2 Department of Fishery, School of Agriculture and Natural Resources, University of Phayao, Muang, Phayao 56000, Thailand 3 Department of Biology, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand 4 Applied Science Program, Faculty of Multidisciplinary Study, Khon Kaen University, Nong Khai Campus, Muang, Nong Khai 43000, Thailand 5 Toxic Substances in Livestock and Aquatic Animals Research Group, Department of Biology, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand *Corresponding author. E-mail: supiwong@hotmail.com Abstract. The cytogenetic comparisons of five Minnow species from Thailand were presented here, i.e., Devario regina, D. laoensis, Rasbora paviana, R. aurotaenia and Esomus metalicus. The mitotic chromosomes were prepared directly from renal cells. Conventional staining and Ag-NOR banding techniques were applied to stain the chromosomes. The results revealed that all Minnow fishes studied possessed the same diploid chromosome number (2n) as 50 chromosomes. The fundamental numbers (NF) of D. laoensis, D. regina, R. paviana, R. aurotaenia and E. metalicus are 100, 100, 98, 98, and 98 respectively. Their karyotypes composing of metacentrics-submetacen- trics-acrocentrics-telocentrics were as follows: 6-12-32-0 in D. regina, 6-10-34-0 in D. laoensis, 8-16-24-2 in R. paviana, 8-16-24-2 in R. aurotaenia and 8-10-30-2 in E. metalicus. The Ag-NOR banding technique provides the nucleolar organizer regions (NORs) at subtelomeric region of the short arm chromosome in the a submetacentric or acrocentric chromosomes that are located differently in the different chromosome pairs among species. Keywords: karyotype, Minnow, fish chromosome, Cyprinid fishes, Minnow fishes. INTRODUCTION Devario laoensis, D. regina, Esomus metalicus, Rasbora aurotaenia, and R. paviana are some species of Minnows, belonging to the family Cyprini- dae (Subfamily Danioninae-Danionini). They are tropical freshwater fish of minor commercial importance, which are native in Thailand. Their distribu- 90 Surachest Aiumsumang et al. tions include the Mekong, Chao Phraya, and Meklong Basins (Froese and Pauly 2012) and they can be easily found in large and small rivers, ponds, ditches, lakes, paddy field, and swamps. It rarely occurs in low oxygen waters (Brittan 1954, 1971, 1998). They could be used to assess if they were sensitive to change in environmental problems and aquatic pollution (Blazer 2002, Frame and Dickerson 2006, Raskovic et al. 2010, Yenchum 2010, Reddy, Rawat 2013). The current spurt in the fish cytogenetical studies has its origin in the standardization of newer techniques and the realization of an immense applied value of the cytogenetic data of fishes. The study on fish chromo- somes has received considerable attention in recent years because of their importance in classification, evolution, heredity, systematic (Gold et al. 1990, Ueda et al. 2001, Barat et al. 2002, Barat and Sahoo 2007), fish breeding, rapid production of inbred lines including cytotaxonomy (Kirpichnikov 1981) and prove the ploidy status in some sturgeons (Zhou et al. 2013). The several methods name- ly, conventional staining, C-banding, Ag-NOR banding, and fluorescence in situ hybridization (FISH) have been used by ichthyologists for gathering of cytogenetic infor- mation of fish (Sola et al. 2000, Kavaco et al. 2005, Zhou et al. 2013), yet each of these methods provides a different aspect of the karyotype characteristics. For example, Ag- NOR staining shows the regions containing the actively transcribed ribosomal RNA genes (rDNA). NORs charac- terization can be a cytogenetic marker for cytotaxonom- ic studies and has been used for studying on phyloge- netic relationships among the Cyprinids (Amemyia and Gold 1988, Gatetti Jr 1998, Almeida-Toledo et al, 2000). However, cytogenetic studies conducted on this group (Devario, Esomus and Rasbora) are quite scarce. There are some karyotype reports, including Rasbora trilineata and R. heteromorpha: 2n=48 (Post 1965), R. buchanani: 2n=50 (Manna and Khuda-Bukhsh 1977), R. danico- nius: 2n=50 (Khuda-Bukhsh et al. 1979), R. sumatrana: 2n=50 (Donsakul and Magtoon 1995), R. caudimaculata, R. myersi, R. paviei and R. retrodorsalis: 2n=50 (Don- sakul and Magtoon 2002), R. aurotaenia: 2n=50 (Seeta- pan and Moeikum 2004), R. trilineata, R. heteromorpha, R. daniconius, R. borapetensis and R. einthovenii: 2n=50 (Donsakul et al. 2005), R. agilis, R. dorsicellata and R. rubrodorsalis: 2n=50 (Donsakul et al. 2009), E. metal- licus: 2n=50 (Neeratanaphan et al. 2017) and R. einthov- enii: 2n=50 (Yeesaem et al. 2019) (Table 1). The studies on the karyotypes help to investigate the genetic structure of aquatic animal species in each habitat, thus it can deter- mine what species are related to each other in an accu- rate manner. This may help to facilitate the hybridization between them in the future for strain improvement (Sofy et al. 2008). In the present study, we conducted chromosomal analyses using conventional staining and Ag-NOR band- ing techniques. The examined karyotypes of five Min- now species from Thailand belonging to three different genera (Devario, Esomus, and Rasbora); D. laoensis, D. regina and R. paviana were reported chromosomes char- acterized for the first time. The obtained results will pro- vide useful cytogenetic information for further studies on taxonomy and evolutionary relationship of fishes. MATHERIAL AND METHODS Chromosome preparation Individuals from both sexes of five analyzed Min- nows were collected from various river basins in Thai- land (Table 1 and Fig. 1). The fishes were transferred to Table 1. Collection sites of the analyzed species show the sample number. Species Number of specimens in site sampling Mae Khong Basin Sirindhorn Peat Swamp Forest Ping Basin Yom Basin Pa-Sak Basin Chi Basin Chao Phraya Basin Song- khram Basin Remark with Fig. 1. Devario regina 05 ♀ 06 ♂ 06 ♀ 08 ♂ - - - - - - Site 1 D. laoensis - - 03 ♀ 05 ♂ - - - - - Site 2 Rasbora paviana 05 ♀ 08 ♂ 03 ♀ 04 ♂ - - 05 ♀ 07 ♂ 04 ♀ 05 ♂ - - Site 3 R. aurotaenia - - - - - - 08 ♀ 07 ♂ 05 ♀ 08 ♂ Site 4 Esomus metalicus - - - 04 ♀ 05 ♂ 10 ♀ 10 ♂ - - - Site 5 91A comparative chromosome study on five Minnow fishes (Cyprinidae, Cypriniformes) in Thailand laboratory aquaria and kept under standard conditions for three days before the experiments. Chromosomes were prepared in vivo as follows (Supiwong et al. 2014). The colchicine was injected into the fish’s intramuscu- lar and/or its abdominal cavity at a dose of 0.1 mL/100 g of body weight and then left for 1-2 hours. The kidney was cut into small pieces then squash mixed with 0.075 M KCl. After discarding all large piece tissues, 8 mL of cell sediments were transferred to a centrifuge tube and incubated for 30 minutes. The KCl was discarded from the supernatant after centrifugation at 1,200 rpm for 8 minutes. Cells were fixed in fresh cool Carnoy’s fixative (3 methanol: 1 glacial acetic acid) allows to pre- serve the internal structure of the cells for better stain- ing of the chromosomes (Pradeep et al. 2011) to which up to 8 mL were gradually added before being centri- fuged again at 1,200 rpm for 8 minutes, at which time the supernatant was discarded. The fixation was repeat- ed until the supernatant was clear and the pellet was mixed with 1 mL fixative. The mixture was dropped onto a clean and cold slide by micropipette followed by air-drying technique. Chromosome staining Conventional staining was carried out using 20% Giemsa’s solution for 15 minutes (Phimphan et al. 2017). Ag-NOR banding was performed by adding 4 drops of 50% silver nitrate and 2% gelatin on slides (Howell and Black 1980). The slides were then sealed with cover glasses and incubated at 60°C for 5 minutes. After that, the slides were soaked in distilled water until the cover glasses were separated. Then, they were stained with 20% Giemsa’s solution for 1 minute. Chromosome check and Image processing Twenty clearly observable metaphase cells with a well-spread chromosome of each male and female were selected. Images were captured under a light microscope Nikon ECLIPSE by a digital CCD camera (Nikon DS- Fi1). The chromosomes were classified based on the posi- tion of a centromere as metacentric (m), submetacentric (sm), acrocentric (a), telocentric (t) according to the arm ratios (Chaiyasut 1989). RESULTS Five minnow fishes were similar in the diploid number of 2n= 50, with the karyotype composed of m6+sm12+a32 in D. regina. The mean values calcu- lated from twenty mitotic metaphases showed the rela- tive length (RL) of chromosomes complement rang- ing from 0.041±0.010 to 0.033±0.004. The NOR was found on the short arm of chromosome pair 15 (Fig. 2A). The chromosome complements of D. laoensis consisting of m6+10sm+34a. The mean value of rela- tive length ranged from 0.0.44±0.005 to 0.030±0.002. The NOR was presented on the short arms of chromo- some pair 11 (Fig. 2B). Karyotype of R. paviana com- poses of 8m+16sm+24a+2t. The present investigation in this fish species revealed that the mean value of RL from 0.048±0.001 to 0.032±0.004. Ag-NOR banding result showed that NOR-bearing chromosomes locate at subtelomeric on the short arm of chromosome pair 9 (Fig. 2C). The karyotypic analysis result revealed that the chromosome complements of R. aurotaenia consist- ing of 8m+16sm+24a+2t. The parameters of all chromo- somes were measured and it showed the mean value of RL from 0.0.054±0.003 to 0.033±0.002. The result of sil- ver-staining exhibited the NORs show that it locates) at short arm of chromosome pair 23 (Fig. 2D). The karyo- type of E. metalicus consisting of 8m+10sm+30a+2t. The mean value of RL from 0.0.51±0.001 to 0.025±0.002. The NOR was presented on the short arms of chromosome pair 7 (Fig. 2E). Figure 1. Collection sites of cyprinid fishes studied herein. 1=Devario regina, 2=Devario laoensis; 3=Rasbora paviana, 4=Ras- bora aurotaenia, 5=Esomus metalicus. 92 Surachest Aiumsumang et al. DISCUSSION The details of each metaphase chromosome spread and karyotype of five Minnow fishes, including D. regi- na, D. laoensis, R. paviana, R. aurotaenia, and E. metali- cus are shown in Figure 2. The present study is the first report on the chromosomal characteristics of D. laoen- sis, D. regina, and R. paviana determined using conven- tional staining and Ag-NOR banding techniques. The diploid chromosome number of all species provided 50 chromosomes, which is shared by most of the cyprinid species previously analyzed (Post 1965, Manna and Khu- da-Bukhsh 1977, Khuda-Bukhsh et al. 1979, Donsakul and Magtoon 1995, Donsakul and Magtoon 2002, Seeta- pan and Moeikum 2004, Donsakul et al. 2005, Don- sakul et al. 2009, Neeratanaphan et al. 2017, Yeesaem et al. 2019) (Table 2). The NFs of D. laoensis and D. regina are 100 equally, while those of R. paviana, R. aurotae- nia, and E. metalicus are equal to 98 in both sexes. To compare with previous studies, they are differences from Seetapan and Moeikum (2004) who reported the NF=92 in R. aurotaenia and Neeratanaphan et al. (2017) showed the NF of E. metallicus as 100. The differences in NF val- ues are caused by the difference in the number of mono- arm chromosomes. This phenomenon may be resulting from the intra-specific variation between populations of those species. This finding is in agreement with oth- er species such as R. daniconius (Khuda-Bukhsh et al. 1979, Donsakul et al. 2005), R. einthovenii (Donsakul et al. 2005, Yeesaem et al. 2019), and R. rebrodorsalis (Don- sakul and Magtoon 2002, Donsakul et al. 2009). The NF of these genera varied from 74 to 100 (Table 2). All species were analyzed herein display without morpho- logically differentiated sex chromosomes. This character is the same as in previous studies of this family (Arai 2011). Although five Minnows analyzed herein have the same diploid number, there are differences in karyotype complements as follows (Fig. 2). D. regina has six meta- centric (m) (pairs 1-3), 12 submetacentric (sm) (pairs 4-9) and 32 acrocentric (a) (pairs 10-25) chromosomes. The mean values were calculated from twenty mitotic metaphases showed the centromeric index (CI) of chro- mosome complements ranging from 0.548±0.004 to 0.808±0.005. The karyotype formula of D. regina could be deduced as 2n(50) = 6m+12sm+32a. D. laoensis has six metacentric (pairs 1-3), 10 submetacentric (pairs 4-8) and 34 acrocentric (pairs 9-25) chromosomes. The mean values of CI ranged from 0.553±0.005 to 0.798±0.002. The karyotype formula of this species is 2n(50) = 6m+10sm+34a. R. paviana consisted of eight metacen- trics (pairs 1-4), 16 submetacentrisc (pairs 5-12), 24 acro- centrics (pairs 13-24) and two telocentrics (t) (pair 25). The mean values of CI ranged between 0.526±0.002 and 1.000±0.000. The proposed karyotype of this species was 2n(50) = 8m+16sm+24a+2t. R. aurotaenia shows eight metacentrics (pairs 1-4), 16 submetacentrics (pairs 5-12), 24 acrocentrics (pairs 13-24) and two telocentrics (pair 25) chromosomes. The mean values of CI in this species ranged from 0.569±0.003 to 1.000±0.000. The karyotype of this species was 2n(50) = 8m+16sm+24a+2t, which differs from the previous study by Seetapan and Moei- kum (2004) that reported the karyotype of this species consisting of 2n(50) = 14m+26sm+2st+8a. In E. metali- cus, the karyotype composed of eight metacentric (pairs Figure 2. Metaphase chromosome plates and karyotypes of the Devario regina (A.), D. laoensis (B.), Rasbora paviana (C.), R. auro- taenia (D.) and Esomus metalicus (E.), by conventional staining. The arrows indicate NOR banding by Ag-NOR staining technique (inserted box). All species share the karyotype composed of 50 chromosomes. Scale bar indicates 5 µm. 93A comparative chromosome study on five Minnow fishes (Cyprinidae, Cypriniformes) in Thailand 1-4), 10 submetacentric (pairs 5-9), 30 acrocentric (pairs 10-24), and two telocentric (pair 25) chromosomes. The mean values of CI ranged between 0.558±0.003 and 1.000±0.000. The karyotype of E. metalicus showed 2n(50) = 8m+10sm+30a+2t. These results are inconsist- ent with previous cytogenetic data (Neeratanaphan et al. 2017). This fact suggests that some pericentric inversions have occurred in the karyotype differentiation of this species. Besides), the occurrence of chromosomal rear- rangements has been considered a relatively common evolutionary mechanism inside the Cyprinidae fam- ily reviewed (Arai 2011). Family Cyprinidae are diploid chromosome ranges from 48–50 in the tribes Labeoni- ni and Smiliogastrini while the tribe Poropuntiini and Danionini are more conserved as 2n = 50 (Phimphan et al. 2020). Karyotype diversification processes in species are subjected to multiple factors, whether intrinsic (genom- ic or chromosomal particularities) or extrinsic (historic contingencies) factor. Among these, restricted gene flow between populations is an important factor for the fixa- tion of karyotype changes. For example, after the occur- rence of an inversion, it can be lost in the polymorphic state or, under the proper conditions, spread in the population until it is fixed. Inversions maintain areas of imbalance between alleles in loci within or influenced by these rearrangements, leading to an adaptive condition, primarily along environmental gradients. This could occur, particularly concerning possible historical expan- sion and adaptation to new environments for a review Hoffmann (2008). As mention above, the chromosomal study is very important and clearly exhibits the benefits. Table 2. Cytogenetic reported of the genera Devario, Esomus and Rasbora. Species 2n NF1 NF2 Karyotype formula NOR Reference Devario laoensis 50 100 66 6m+10sm+34a 2 Present study D. regina 50 100 68 6m+12sm+32a 2 Present study Esomus metallicus 50 100 86 14m+22sm+14a - Neeratanaphan et al. (2017) 50 98 68 8m+10sm+30a+2t 2 Present study Rasbora agilis 50 100 100 24m+26sm - Donsakul et al. (2009) R. aurotaenia 50 92 90 14m+26sm+2a+8t - Seetapan and Moeikum (2004) 50 98 74 8m+16sm+24a+2t 2 Present study R. borapetensis 50 88 88 24m+14sm+12t - Donsakul et al. (2005) R. buchanani 50 100 96 30m+18sm+2a - Manna and Khuda-Bukhsh (1977) R. caudimaculata 50 98 96 20m+26sm+2a+2t - Donsakul and Magtoon (2002) R. daniconius 50 80 74 18m+6sm+6a+20t - Khuda-Bukhsh et al. (1979) R. daniconius 50 92 90 32m+8sm+2a+8t - Donsakul et al. (2005) R. dorsicellata 50 92 92 18m+24sm+8t - Donsakul et al. (2009) R. einthovenii 50 94 86 6m+30sm+8a+6t - Donsakul et al. (2005) 50 100 84 16m+18sm+16a 2 Yeesaem et al. (2019) R. heteromorpha 48 - - - - Post (1965) 48 74 72 14m+10sm+2a+22t - Donsakul et al. (2005) R. myersi 50 90 84 20m+14sm+6a+10t - Donsakul and Magtoon (2002) R. paviei 50 100 84 10m+24sm+16a - Donsakul and Magtoon (2002) R. paviana 50 98 74 8m+16sm+24a+2t 2 Present study R. retrodorsalis 50 88 86 26m+10sm+2a+12t - Donsakul and Magtoon (2002) R. rubrodorsalis 50 82 82 16m+16sm+18t - Donsakul et al. (2009) R. sumatrana 50 94 92 26m+16sm+2a+6t - Donsakul and Magtoon (1995) R. trilineata 48 - - - - Post (1965) R. trilineata 50 94 92 26m+16sm+2a+6t - Donsakul et al. (2005) Abbreviations: diploid chromosome number (2n), fundamental number m, sm, a =2, t=1 (NF1), fundamental number m, sm, =2, a, t=1 (NF2), metacentric (m), submetacentric (sm), acrocentric (a), telocentric (t), Nucleolar Organizer Region (NOR). 94 Surachest Aiumsumang et al. Moreover, the karyological and NORs characteristics in cyprinid fishes were reported in some species. The present study is the first report on the NOR phenotypes in five Minnow species studied. The sin- gle pair of NOR-bearing chromosomes were observed at subtelomeric regions on the short arm chromosomes in all species analyzed. However, there are differences in chromosome types and pair numbers as follows. The NORs were observed on acrocentric chromosome pair 15 in D. regina whereas those were found on acrocentric chromosome pair 11 in D. laoensis. In the genus Ras- bora, the NORs located on the submetacentric chromo- some pair 9 in R. paviana and distinct revealed on the acrocentric chromosome pair 23 in R. aurotaenia. For E. metalicus, NOR-baring chromosomes were found on the submetacentric chromosome pair 7 (Fig. 2). To compare with the same genus in previous report, R. ein- thovenii has single pair of NOR on chromosome pair 4 (Yeesaem et al. 2019). Moreover, the single pair of NOR bearing chromosomes can be observed in other cyprin- ids such as Aspius aspius (Ràb et al. 1990), Osteochi- lus waandersi (Magtoon and Arai 1993), Barbonymus gonionotus (Khuda-Bukhsh and Das 2007), Puntioplites proctozysron (Supiwong et al. 2012), Puntius brevis (Niti- kulworawong and Khrueanet 2014). Also, the subtelom- eric region of chromosome pair showed clearly observ- able NORs in most cyprinid fishes. However, NOR vari- ation can be revealed in among populations of the same species as found in Garra rufa. This variation is caused by geographically isolated populations (Arzu and Ergene 2009). Normally, most fishes have only one pair of small NORs on chromosomes. Only some fishes have more than two NORs, which may be caused by the transloca- tion between some parts of the chromosomes that have NOR and another chromosome (Sharma et al. 2002). Our present study showed that the species analyzed had a NOR site on a single chromosome pair at a subtelom- eric position. This is considered a simple condition in fish (Almeida-Toledo 1985). In the present study, five Minnows belong to gen- era of which have closely related species. The obtained results have shown that this fish group shares the same 2n. However, there are differences in karyotype comple- ments and NOR-bearing chromosome markers. These seem to be that cytogenetic methods can be used for the systematics of this fish family. ACKNOWLEDGMENTS The authors are grateful to Phetchabun Rajabhat University and Toxic Substances in Livestock and Aquat- ic Animals Research Group Khon Kaen University and Unit of Excellence 2020 on Biodiversity, Natural Resourc- es Management, University of Phayao (UoE63005) and the Post-Doctoral Training Program from Research Affairs and Graduate School, Khon Kaen University, Thailand (Grant no. 59255) for financial supports. 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