Research Article Science Technology and Engineering Journal (STEJ) Vol.7, No.1 pages 22-31

Karyotype of Four Mouth-Brooding (Betta Bleeker, 1850) in

Teamjun Sarasan1, Weerayuth Supiwong2, Namkang Sriwattanarothai3, Bhinyo Panijpan4, Nattapong Srisamoot5 and Alongklod Tanomtong1*

1 Toxic Substance in Livestock and Aquatic Research Group, 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 Khai Campus, Muang, Nong Khai 43000, Thailand 3 Institute for Innovative , Mahidol University, Phuttamonthon, Nakhon Pathom 73170, Thailand 4 Center for Shrimp Molecular Biology and Biotechnology, Faculty of Science, Ratchathewi, Bangkok 10400, Thailand 5 Division of Biotechnology, Faculty of Agro-Industrial Technology, Kalasin University, Muang, Kalasin 46000, Thailand * Corresponding author, e-mail: [email protected]

(Received: 21th December 2020, Revised: 24th March 2021, Accepted: 26th March 2021)

Abstract - The karyotype and chromosomal characteristics of nucleolar organizing regions (NORs) of four mouth-brooding Betta from Thailand: Betta pi Tan, 1998, B. prima Kottelat, 1994, B. pugnax (Cantor, 1849), and B. simplex Kottelat, 1994, were reported. The results exhibited that the diploid number (karyotype formula) and fundamental number (NF) of each are; B. pi, 2n=34 (4m+4sm+18a+8t) NF=60, B. prima, 2n=38 (6m+12a+20t) NF=56, B. pugnax, 2n=40 (4m+2sm+14a+20t) NF=60 and B. simplex, 2n=46 (2m+2sm+4a+38t) NF=54. All species revealed one pair of NOR-bearing located on the short arm (p) of the acrocentric chromosome. In addition, the different sizes and morphology of Ag–NOR were observed in these four Betta fishes. Keywords: Mouth–brooding Betta, Betta, karyotype, chromosome Volume 7, Number 1, January-June 2021 Karyotype of four mouth-brooding Betta fishes 23 (Betta Bleeker, 1850) in Thailand

1. Introduction chromosomes such as number, size, and morphology, which can help determine Wild fighting species in the genusBetta the variety of evolution and may are native to ASEAN countries. Two groups permit the detection of the change and of fighting fish in Thailand can be found: modification of karyotype from an ancestor bubble nester and mouth-brooder. The to their newline (Winkler et al., 2004). bubble nesting Betta distributes throughout Karyological studies of fishes provide the Thailand, while mouth-brooding Betta comprehensive knowledge to solve the fish is mainly found in southern Thailand problem in many areas (Alsabti, 1985), (Panijpan et al., 2020). There are 12 wild-type such as , systematic, phylogenetic species been found in Thailand, of which relationship (Campiranont, 2003), evolution five species are bubble nester, and the other (Tanomtong et al., 2014; Cioffiet al., 2015), seven species are mouth-brooder Betta and environmental toxicology (Klinkhardt, (Panijpan et al., 2017). These 12 species 1993). Moreover, karyotype also provides are divided into five groups: B. picta the chromosome complement such as the group, B. pugnax group, B. waseri group, number, chromosome type, and size of B. smaragdina group, and B. splendens individuals, species, and a related group group (Panijpan et al., 2014) of individuals. The bubble nester is more comfortable Studies of cytogenetics are essential identifying with the and body color, and in the aquaculture context in which using habitat based on the general appearance. chromosome manipulation techniques, However, the mouth-brooder is challenging including polyploidy, gynogenesis, to identify (Monvises et al., 2009) with androgenesis, and inter-or intra-species a similar body shape, mostly brown with hybridization (Campiranont, 2003). darker stripes body, and even the habitat Nevertheless, the cytogenetic information of except for B. simplex in Krabi province. Betta fishes in Thailand is scarcely known. Some studies adapted the DNA analysis There are only four species that have been techniques, for example, COI (cytochrome c performed. This study aims to establish oxidase subunit I) barcoding, 16SrRNA (16S cytogenetic knowledge regarding the ribosomal RNA), ITS (internal transcribed issues mentioned earlier, including diploid spacer), RAG (recombination activating number, karyotype formula, and Ag-NOR gene) genes to identify the Betta fishes of four mouth-brooding Betta are B. pi, (Panijpan et al., 2020). This technique is B. prima, B. pugnax, and B. simplex. This reasonably acceptable, but it takes time information may facilitate the hybridization and cost for species identification. between different populations in the future for strain improvement of Betta Cytogenetics has become popular in fish. Moreover, this information may be fish classification, such as in the cyprinids a valuable tool for the taxonomy revision fish group (Yanget al., 2015). Cytogenetics in this group. provides the necessary information of 24 Alongklod Tanomtong et al. Science Technology and Engineering Journal (STEJ)

2. Materials and methods

Table 1 Sources of the examined ornamental Betta fishes.

No. Scientific name Common name Collection sites 1 Betta pi Tan, 1998 6 .0746°N, 101.9585°E Pru Toh Daeng, a peat swamp in Narathiwat Province 2 B. prima Kottelat, 1994 14 .0539°N, 102 .0445°E Hauisamong, Na Di district, Prachinburi Province 3 B. pugnax (Cantor, 1849) Betta 8.4000°N, 99.5700°E Chawang District, Nakhon Si Thammarat Province 4 B. simplex Kottelat, 1994 Krabi mouth– 7.9226°N, 99.2600°E brooding Betta Khlong Thom District, Krabi Province

The mouth-brooding Betta fishes, (CI). The CI was computed to classify the i.e., B. prima Kottelat (1994) and B. simplex types of chromosomes according to Turpin Kottelat (1994) were selected as the and Lejeune (1965). All parameters were representative of the B. picta group while used in karyotyping (Reungsing et al., 2000). B. pugnax (Cantor, 1849) and Betta pi Tan, 1998 were selected as the of B. pugnax group 3. Results and B. waseri group, respectively (Panijpan et al., 2014). Species identification of the Betta pi: Karyological analysis of B. pi mouth-brooding Betta fishes in this study revealed 2n=34 and NF=60. Karyotype of was identified according to Tan and Ng B. pi consisted of four metacentric, four (2005). The five male and female fishes submetacentric, 18 acrocentric, and eight of each species were obtained from the telocentric chromosomes, which could be different natural sources throughout deduced as 2n (diploid) 34=4m+4sm+18a+8t. Thailand as shown in (Table 1). The The NOR region is located at the telomeric chromosome preparation method had been position of the short arm of the acrocentric accomplished after Sarasan et al. (2019) and chromosome pair 9. There is only one Juntaree and Supiwong (2000). Detection of active NORs in the metaphase cell, which the NOR was following the silver staining was coincided with the interphase NOR in method of Howell and Black (1980) with slight this sample. The metaphase chromosome modification. The chromosome lengths of and karyotype by conventional and 20 cells (males and females) were measured silver-staining of B. pi are shown in on their short and long arms. The length of (Figure 1A and 2A) respectively. There the short arm (Ls) and the long arm (Ll) is only stick-like Ag-NOR structure was were calculated for the length of the observed in B. pi (Figure 3) chromosome (LT) and centromeric index Volume 7, Number 1, January-June 2021 Karyotype of four mouth-brooding Betta fishes 25 (Betta Bleeker, 1850) in Thailand

Figure 1. Metaphase chromosome plates and karyotypes by conventional staining of B. pi, 2n=34 (A), B. prima, 2n=38 (B), B. pugnax, 2n=40 (C) and B. simplex, 2n=46 (D). Scale bars=5 µm. 26 Alongklod Tanomtong et al. Science Technology and Engineering Journal (STEJ)

Betta prima: Chromosome of size heteromorphism between homologous B. prima possessed 2n=38 and NF=56. chromosomes. There are two active NORs Karyotype comprises six metacentric, 12 in the metaphase cell. The metaphase acrocentric, and 20 telocentric chromosomes chromosomes and karyotypes by a which could be deduced as 2n (diploid) conventional and silver straining technique 38=6m+12a+20t. Besides, distinctive of B. prima are shown in (Figure 1B and secondary constriction was observed on 2B, respectively. Two types of Ag-NOR short arms of chromosome pair 5, which is shapes were observed in B. prima; pair coincided with the Ag-NOR, determining a structure and stick-like structures (Figure 3)

FigureFigure 2. Metaphase 2 Metaphase chromosome chromosome plates plates and and karyotypes karyotypes of of B. B. pi pi(A), (A), B. B.prima prima (B), (B), B. pugnax (C) and B. simplex (D) by Ag–NOR straining technique, B. pugnaxarrows (C) indicate and B. Ag–NOR. simplex (D) Scale by Agbars=5–NOR µm. straining technique, arrows indicate Ag–NOR. Scale bars=5 µm. Volume 7, Number 1, January-June 2021 Karyotype of four mouth-brooding Betta fishes 27 (Betta Bleeker, 1850) in Thailand

FigureFigure 3 Ag 3.– NORAg–NOR shapes shapes of the examinedof the examined mouth–brooding mouth–brooding Betta fishes. Betta Number fishes. indicate chromosomeNumber pair. indicate chromosome pair.

Betta pugnax: Karyological analysis B. simplex: Karyological analysis

of B. pugnax revealed 2n=40 and NF=60. revealed 2n=46 and NF=54. The KaryotypeBetta ofpugna B. x:pugnax Karyological comprises analysis of four B. karyotypeshort arm of comprisesthe acrocentric two chromosome metacentric, two metacentric,pugnax revealedtwo submetacentric, 2n=40 and NF=60. 14 submetacentric,pair 6. There are two four Ag- NORsacrocentric, per cell, and 38 acrocentric,Karyotype and 20 of telocentric B. pugnax chromosomes,comprises four telocentricboth interphase chromosomes and metaphase cell deduceds. The as 2n which couldmetacentric, be deducedtwo submetacentric, as 2n (diploid) 14 (diploid)metaphase 46=2m+2sm+4a+38t.chromosomes and karyotypes One pair of 40=4m+2sm+14a+20t.acrocentric, and 20One telocentric pair of NOR-bearingby conventional chromosomeand Ag-NOR wasstraining observed in NOR-bearingchromosomes chromosome, which could was be observed deduced as in B.technique simplex of .B. The pugnax NORs are shown regions in Fig ares. located B. pugnax2n .(diploid) The NORs 40=4m+2sm+14a+20t. regions are located One at1C the and telomeric 2C, respectively position. Both; of pair the and short arm at the telomericpair of NOR position-bearing of chromosomethe short arm was of ofstick the-like acrocentric structures chromosomeof Ag-NOR were pair 4. Two the acrocentric chromosome pair 6. There Ag-NORs were observed both in interphase observed in B. pugnax. The NORs regions observed in B. pugnax (Fig. 3). are two Ag-NORs per cell, both interphase and metaphase cells. The metaphase and metaphaseare located at cells. the telomeric The positionmetaphase of the chromosomes and karyotypes by conventional chromosomes and karyotypes by and Ag-NOR straining technique of B. conventional and Ag-NOR straining technique simplex are shown in (Figure 1D and 2D) of B. pugnax are shown in (Figure 1C and respectively. There are two types of Ag-NOR 2C) respectively. Both; pair and stick-like structure of B. simplex; pair structure and structures of Ag-NOR were observed in B. stick-like structures (Figure 3) pugnax (Figure 3). 28 Alongklod Tanomtong et al. Science Technology and Engineering Journal (STEJ)

We could not find the 2n and NF all these four mouth-brooding Betta fishes. differences between males and females of

Table 2 The comparative karyotypes of the examined fishes in the genusBetta .

Karyotype Ag-NORs Reference Species 2n NF formula (location) Betta pi 34 60 4m+4sm+18a+8t 1 (9p) The present study B. prima 34 42 4m+4sm+4st+22t Magtoon et al. (2007) 38 56 6m+12a+20t 2 (5p) The present study B. pugnax 40 60 4m+2sm+14a+20t 2 (6p) The present study B. simplex 44 52 4m+4sm+36t Donsakul et al. (2009) 46 54 2m+2sm+4a+38t 2 (4p) The present study

Remarks: NF=fundamental number, m=metacentric, sm=submetacentric, a=acrocentric and t=telocentric chromosomes.

4. Discussion et al. (2009), respectively. In the present study, B. prima showed a diploid number Four species of the mouth-brooding of 38 chromosomes for both sexes and a Betta fish are included in the present fundamental number (NF) of 56 while study: B. pi, B. prima, B. pugnax, and B. Magtoon et al. (2007) found that this simplex. Under cytogenetic viewpoint, species had had 2n=34, NF=42. The karyotype report in the Betta is present study results found that B. simplex scarce, especially in the mouth-brooding had 2n=46, NF=54, while the study of Betta. The B. pugnax and B. pi’s Donsakul et al. (2009) found that this chromosomal data were reported here for the species had 2n=44, NF=52. The present first time. The conspicuous diversification result and previous reports suggest that of diploid number and karyotype was found the variation in diploid number at the among mouth–brooding Betta species. intraspecific level is found in genusBetta . The diploid number ranges from 2n=34 Nonetheless, the variation in diploid number to 2n=46 with the NF=54 to NF=60. The among the individuals of the same results revealed that all four species have population was not observed. The variation different diploid numbers and karyotypes. of diploid numbers observed in Betta may be The diploid number of B. pi, B. prima, B. due to several causes, such as chromosome pugnax, and B. simplex are 34, 38, 40, and preparation method and the precise 46 chromosomes, respectively See (Table 2) chromosome measurement (Zhang & Reddy, 1991). For example, a chromosome Cytogenetic information of B. prima can be constricted at a high degree and B. simplex reported herein is different when chromosomes were overexposure from those of the previous studies reported to colchicine. More fish samples from the by Magtoon et al. (2007) and Donsakul same population and different populations are Volume 7, Number 1, January-June 2021 Karyotype of four mouth-brooding Betta fishes 29 (Betta Bleeker, 1850) in Thailand needed to confirm theBetta fishes’ karyotype. number of ribosomal genes carried by each In addition, the karyotype diversification chromosome and the differential recruitment of Betta can be caused by chromosomal of active ribosomal genes in each NOR evolution as found in other Perciform fishes cluster (Héliot et al., 2000). However, the (Molina et al., 2014; Rishi & Haobam, 1990 size of the NOR did not measure in this ; Singh & Barman, 2013; Almeida et al., present study. 2017). Interestingly, karyotype analysis of the genus Betta revealed that there are very large metacentric chromosomes in B. 5. Acknowledgments prima and B. pugnax. Moreover, all species This work was supported by the Toxic of Betta fishes show n2 <48, but the number Substances in Livestock and Aquatic Animals of large metacentric chromosomes is not Research Group, Khon Kaen University. compatible with the reduction of diploid values, indicating the simultaneous occurrence of tandem fusions or major 6. References deletions in the chromosome evolution of this group (Almeida et al., 2017). Almeida, L.A.H., Nunes, L.A., Bitencourt, The NOR data of these four J.A., Molina, W.F. & Affonso, mouth-brooding Betta fishes were obtained P.R.A.M. (2017). Chromosomal herein for this first time. One pair of evolution and cytotaxonomy in NOR-bearing chromosome was observed Wrasses (Perciformes; Labridae). in all these fishes. The polymorphic NOR Journal of Heredity, 108(3), 239–253. was found in B. pi. The Ag-NOR can be Alsabti, K. (1985). Chromosomal studies detected only on one chromosome of pair 9 by blood leukocyte culture (9a). The NORs of four the mouth-brooding technique on three salmonids from Betta fishes were located terminally Yugoslavian waters. Journal of on the short arm (p) of the acrocentric Fish Biology, 26(1), 5–12. chromosome. The different sizes of Ag–NOR were observed in Betta fish. In addition, Campiranont, A. (2003). Cytogenetics nd the metaphase Ag-NORs of Betta fishes (2 ed). Department of Genetics, exhibited different morphologies (Figure Faculty of Science, Kasetsart 3). Two types of NOR structures that can University, Bangkok. be observed are sticky-like structure and Cioffi, M. B., Bertollo, L. A. C., Villa, M. pair structure. Pair and stick-like Ag-NOR A., Oliveira, E. A., Tanomtong, structures have been described by Ploton A., Yano, C. F., Supiwong, W. & et al. (1994). The example of a sticky-like Chaveerach, A. (2015). Genomic structure and pair structure found in the organization of repetitive DNA genus Betta is shown in (Figure 3) B. pi elements and its implications for showed only sticky-like structure while the chromosomal evolution of another six species revealed both sticky-like channid fishes (, structure and pair structure of NORs. The Perciformes). Plos One, 10(6), different Ag-NOR shapes reflect both the e0130199. 30 Alongklod Tanomtong et al. Science Technology and Engineering Journal (STEJ)

Donsakul, T., Magtoon, W. & Rungsiruji, Magtoon, W., Rangsiruji, A. and Donsakul, A. (2009). Karyotypes of five T. (2007). Karyotypes of Betta belontiid fishes (Family Belongtiidae): splendens, B. prima, Trichopsis , S. simplex, vittatus and Trichogaster trichopterus B. sp., Trichopsis pumila and T. (family Belontiidae) from Thailand. schalleri. In: The 35th Congress The 33rd Congress on Science and on Science and Technology of Technology of Thailand (p. 94) Thailand (pp. 77–80). Kasetsart Molina, W.F, Martinez, P.A., Bertollo, L.A. University, Bangkok, Thailand. & Bidau, C.J. (2014). Preferential Heliot, L., Mongelard, F., Klein, C., accumulation of sex and Bs O’Donohue, M.F., Chassery J.M., chromosomes in biarmed Robert–Nicoud M. & Usson, Y. karyotypes by meiotic drive and (2000). Nonrandom distribution rates of chromosomal changes in of metaphase AgNOR staining fishes.Anais Da Academia Brasileira patterns on human acrocentric De Ciencias, 86(4), 1801–1812. chromosomes. Journal of Monvises, A., Nuangsaeng, B., Sriwattanarothai, Histochemistry and Cytochemistry, N. & Panijpan, B. (2009). The 48(1), 13–20. : Well- known Howell, W.M. & Black, D.A. (1980). generally but little-known Controlled silver–staining of scientifically.Science Asia, 35(1), nucleolus organizer regions with 8–16. a protective colloidal developer: Panijpan, B., Sriwattanarothai, N., Kowasupat, A 1–step method. Experientia, 36, C., Ruenwongsa, P., Jeenthong, T. 1014–1015. and Phumchoosri, A. (2017). Juntaree, S. & Supiwong, W. (2000). of bubble-nest building Standardized karyotype and and mouth-brooding fighting Idiogram of the glass goby fish fish species of the genus Betta in (Gobiopterus chuno)(Hamilton, . Thailand Natural 1822) in Thailand. Science History Museum Journal, 11, 1–21. Technology and Engineering Panijpan, B., Kowasupat, C., Laosinchai, Journal (STEJ), 6(2), 72–81. P. Ruenwongsa, P., Phongdara, A., Klinkhardt, M. (1993). Fish chromosomes Senapin, S., Wanna, W., Phiwsaiya, as sensitive toxicity indicators– K., Kühne, J. & Fasquel, F. (2014). possibilities and limits. In Southeast Asian mouth-brooding Braunbeck, T.H., Hanke, W. Betta fighting fish (Teleostei: & Segner, S. (Eds.). Fish in Perciformes) species and their Ecotoxicology and Ecophysiology; phylogenetic relationships based Proceedings of an International on mitochondrial COI and nuclear Symposium. (pp. 45–54). ITS1 DNA sequences and analyses. Meta Gene, 2, 862–879. Volume 7, Number 1, January-June 2021 Karyotype of four mouth-brooding Betta fishes 31 (Betta Bleeker, 1850) in Thailand

Panijpan, B., Sriwattanarothai, N. & Tan, H.H. & Ng, P.K.L. (2005). The Laosinchai, P. (2020). Wild Betta fighting fishes (Teleostei: fighting fish species in Thailand Osphronemidae: genus Betta) of and other southeast Asian countries. , and Brunei. Science Asia, 46, 1–10. Raffles Bulletin of Zoology, Suppl, 13, 43–99. Ploton, D., Gilbert, N., Ménager, M., Kaplan, H. & Adnet, J. J. (1994). Tanomtong, A., Supiwong, W., Three-dimensional co-localization Jearranaiprepame, P., Khakhong S., of nucleolar argyrophilic Kongpironchuen, C. & Getlekha, components and DNA in cell N. (2014). A new natural nuclei by confocal microscopy. autotetraploid and chromosomal The Journal of Histochemistry and characteristics of dwarf snakehead Cytochemistry, 42(2), 137–148 fish, Channa gachua (Perciformes, Channidae) in Thailand. Cytologia, Reungsing, M., Tanomtong, A., Donbundit, 79(1), 15–27. N. & Sonsrin, K. (2000). A First Chromosomal Characterization of Turpin, R. & Lejeune, J. (1965). Les Black - Spotted Pufferfish, Arothron Chromosomes Humains. Gauthier– nigropunctatus (Tetraodontifrom; pillars, Paris. Tetraodonidae). Science Technology Winkler, F.M., Garcia–Melys, D. & and Engineering Journal (STEJ), Palma–Rojas, C. (2004). Karyotypes 6(2), 27–35. of three Southeast Pacific flounder Rishi, K.K. and Haobam, M.S. (1990). species of the family Paralichthyidae. A chromosomal study on four species Aquaculture Research, 35, of snake heads (Ophiocephalidae: 1295–1298. Pisces) with comments on their Yang, L., Sado, T., Hirt, M.V., Pasco–Viel, karyotypic evolution. Caryologia, E., Arunachalam, M., Li, J., 43(2), 163–167. Wang, X., Freyhof, J., Saitoh, K., Sarasan, T., Jantarat, S., Supiwong, W., Yeesin, Simons, A.M., Miya, M., He, S. P. Srisamoot, N. & Tanomtong, A. & Mayden, R.L. (2015). Phylogeny (2018). Chromosomal analysis and polyploidy: resolving the of two snakehead fishes, Channa classification of cyprinine fishes marulius (Hamilton, 1822) and (Teleostei: Cypriniformes). Molecular C. marulioides (Bleeker, 1851) Phylogenetics and Evolution, 85, (Perciformes: Channidae) in 97–116. Thailand. Cytologia, 83(1), 115–121. Zhang, S.M. & Reddy, P.V.G.K. (1991). On Singh, M. & Barman, A.S. (2013). the comparative karyomorphology Chromosome breakages associated of three Indian major craps, Catla with 45S ribosomal DNA sequences catla (Hamilton), Labeo rohita in spotted snakehead fish Channa (Hamilton) and Cirrhinus mrigala punctatus. Molecular Biology (Hamilton). Aquaculture, 97(1), Reports, 40(1), 723–729. 7–12.