Karyotype and Characteristics of Nucleolar Organizer Regions in Longﬁn Carp, Labiobarbus Leptocheilus (Cypriniformes, Cyprinidae)

Karyotype and Characteristics of Nucleolar Organizer Regions in Longﬁn Carp, Labiobarbus leptocheilus (Cypriniformes, Cyprinidae)

Pasakorn Saenjundaeng1, Puntivar Kaewmad2, Weerayuth Supiwong3, Krit Pinthong4, Puan Pengseng5 and Alongklod Tanomtong1*

1 Toxic Substances in Livestock and Aquatic Animals Research Group, Department of Biology, Faculty of Science, Khon Kaen University, Muang, Khon Kaen 40002, Thailand 2 Major of Biology, Faculty of Science and Technology, Mahasarakham Rajabhat University, Muang, Maha Sarakham 44000, Thailand 3 Faculty of Applied Science and Engineering, Khon Kaen University, Nong Khai Campus, Muang, Nong Khai 43000, Thailand 4 Department of Fundamental Science, Faculty of Science and Technology, Surindra Rajabhat University, Muang, Surin 32000, Thailand 5 School of Agricultural of Technology, Walailak University, Thasala, Nakhon Si Thammarat 80160, Thailand

Received January 23, 2018; accepted March 15, 2018

Summary Karyotype and nucleolar organizer regions (NORs) were studied in the longfin carp (Labiobarbus leptocheilus) from Mekong Basin of Thailand. The mitotic chromosomes were directly prepared from kidney cells from five male and five female fishes and stained by conventional staining and Ag-NOR banding. The chromosome number was 2n=50 and the fundamental number (number of the chromosome arms, NF) was 86 in both male and female fishes. The karyotype was composed of six large metacentric, eight large acrocentric, six large telocentric, four medium metacentric, six medium submetacentric, six medium acrocentric, four medium telocentric, four small metacentric, two small acrocentric and four small telocentric chromosomes. The Ag-NOR banding showed a single pair of Ag-NORs adjacent to the telomeric position of the short arm of the chromosome pair 8.

Key words Labiobarbus leptocheilus, Karyotype, Chromosome, Nucleolar organizer region, Ag-NOR.

The genus Labiobarbus van Hasselt, 1823 is a mem- same diploid chromosome number of 50. In the present ber of the tribe Labeonini, subfamily Cyprininae, family study, the karyotype and a chromosome marker of L. Cyprinidae and order Cypriniformes. The genus distrib- leptocheilus from Thailand by using conventional stain- utes in rivers, swamps, and marshes of Southeast Asia. It ing and Ag-NOR banding techniques are investigated in comprises six species including L. leptocheilus, L. sia- order to provide useful cytogenetic information in tax- mensis, L. fasciatus, L. ocellatus, L. festivus and L. sa- onomy and evolutionary relationship of fish in the tribe banus (Roberts 1993, Kottelat 2001, 2013). In Thailand, Labeonini. there are two species, L. leptocheilus and L. siamensis (Kottelat 2013). The longfin carp, L. leptocheilus occurs Materials and methods in Salween, Tenasserim and Mekong Basins throughout Peninsular Malaysia, Sumatra, Borneo, and Java. It can Sample collection be distinguished from its congeners by the combination Five males and five females of L. leptocheilus were of following characters (Fig. 1) of longitudinal stripes on obtained from the Mekong Basin in Thailand. Fish were the body, a humeral spot above and behind the pectoral transferred to laboratory aquaria and kept under stan- fin base, 33–44 lateral line scales and 16–20 circumpe- dard conditions for 3 d prior to the experiments. The duncular scales (Roberts 1993). fish studied were identified according to Roberts (1993), Up to the present, the karyotypes of only two species Rainboth (1996), Kottelat (2001) and Saenjundaeng of L. lineatus (Magtoon and Arai 1990) and L. spiro- (2014). pleura (Donsakul and Magtoon 1997) have been studied by a conventional staining technique. They have the Chromosome preparation and stainings Chromosomes were directly prepared in vivo ac- * Corresponding author, e-mail: [email protected] cording to the method of Maneechot et al. (2015) and DOI: 10.1508/cytologia.83.265 Phimphan et al. (2015) by injecting phytohemagglutinin 266 P. Saenjundaeng et al. Cytologia 83(3)

solution into the abdominal cavity of the fish. Twenty- four hours later, colchicine solution was injected into the intramuscular and/or abdominal cavity and left for 1–2 h. Kidney and gill were removed and cut into small pieces before being mixed with 0.075 M KCl. After all large pieces of tissues were discarded, 8 mL of cell sedi- ment was transferred to a centrifuge tube and incubated for 25–35 min. Then, cells were fixed in a fresh cool fixative (3 methanol : 1 glacial acetic acid). The fixative was gradually added up to 8 mL before centrifugation Fig. 1. General characteristic of the longfin carp (male), Labiobar- bus leptocheilus, from the Mekong Basin of Thailand. Scale again at 1200 rpm for 8 min. Then the supernatant was bar=3 cm. discarded. The fixation procedure was repeated until the supernatant was clear and the pellet was mixed with

Fig. 2. Metaphase chromosome plates (A, C) and karyotypes (B, D) of the longﬁn carp (L. leptocheilus), male (A, B) and female (C, D) by conventional staining technique. Scale bars=5 µm.

Fig. 3. Metaphase chromosome plates (A, C) and karyotypes (B, D) of the longﬁn carp (L. leptocheilus), male (A, B) and female (C, D) by Ag-NOR banding technique. Arrows indicate Ag-NORs. Scale bars=5 µm. 2018 Karyological Analysis of Longﬁn Carp, Labiobarbus leptocheilus 267

1 mL of the fixative. The cell suspension was dropped 0.50–0.59, 0.60–0.69, 0.70–0.89 and 0.90–0.99 were onto a clean and cold slide by micropipette following the described as metacentric, submetacentric, acrocentric air-dry technique. and telocentric chromosomes, respectively. The NF was The slide was conventionally stained with 20% Gi- obtained by assigning a value of two to metacentric, emsa’s solution for 30 min (Rooney 2001). submetacentric and acrocentric chromosomes and one to Ag-NOR banding was conducted by adding two drops telocentric chromosomes. All parameters were used in of 50% silver nitrate and 2% gelatin on slides, respec- karyotyping and drawing idiogram. tively. The slides were sealed with cover glasses and incubated at 60°C for 5 min. After that, the slides were Results and discussion soaked in distilled water until the cover glasses were separated (Howell and Black 1980). Chromosome number, fundamental number, and karyotype of L. leptocheilus Chromosomal checks The diploid chromosome number of L. leptocheilus is Chromosome counting was performed on mitotic 50 in both male and female (Figs. 2, 3). The compara- metaphase of 20 well-spread chromosomes of each male tive studies with previous reports in Labiobarbus (Table and female fish. The length of short arm (Ls) and long 1) showed the same diploid chromosome number but arm (Ll) were measured and calculated to the length of had a difference in the FN as follows: in L. siamensis, chromosomes (LT=Ls+Ll). The relative length (RL), 2n=50 (NF=88) (Donsakul and Magtoon 1997) and the centromeric index (CI) and standard deviation (SD) L. leptocheilus, 2n=50 (NF=80) (Magtoon and Arai of RL and CI (Ll/LT) were calculated according to the 1990). The diploid chromosome numbers of fishes in the method of Kaewsri et al. (2014a, b). The CIs between tribe Labeonini (family Cyprinidae) vary from 44 to 74

Table 1. Review of cytogenetic publications in the genus Labiobarbus.

Species 2n NF Karyotype NOR Locality Reference

L. leptocheilus 50 80 20m+10sm+20t ̶ Thailand Magtoon and Arai (1990) L. leptocheilus 50 86 14m+6sm+16a+14t 2 (sm) Thailand Present study L. siamensis 50 88 34m+4sm+2a+10t ̶ Thailand Donsakul and Magtoon (1997)

̶=Not available.

Table 2. Means of the short arm length (Ls), long arm length (Ll), and total arm length of chromosomes (LT), relative length (RL), centromeric index (CI) and standard deviation (SD) of RL, CI of 20 metaphase cells of the male and female longﬁn carp (Labiobarbus leptocheilus), 2n=50.

Chromosome pair Ls (µm) Ll (µm) LT (µm) RL±SD CI±SD Chromosome size Chromosome type

1 1.49 1.65 3.14 0.049±0.003 0.524±0.023 Large Metacentric 2 1.35 1.53 2.88 0.045±0.003 0.530±0.019 Large Metacentric 3 1.25 1.42 2.67 0.042±0.003 0.530±0.022 Large Metacentric 4 1.11 1.32 2.43 0.038±0.003 0.542±0.026 Medium Metacentric 5 1.04 1.22 2.25 0.035±0.003 0.539±0.026 Medium Metacentric 6 0.96 1.09 2.05 0.032±0.001 0.533±0.018 Small Metacentric 7 0.89 0.99 1.88 0.029±0.002 0.525±0.017 Small Metacentric 8* 0.90 1.58 2.48 0.039±0.005 0.637±0.028 Medium Submetacentric 9 0.84 1.44 2.27 0.036±0.005 0.632±0.031 Medium Submetacentric 10 0.77 1.36 2.13 0.033±0.004 0.639±0.030 Medium Submetacentric 11 0.85 2.72 3.57 0.056±0.004 0.761±0.036 Large Acrocentric 12 0.75 2.54 3.29 0.052±0.004 0.771±0.044 Large Acrocentric 13 0.65 2.26 2.91 0.046±0.004 0.775±0.042 Large Acrocentric 14 0.64 2.02 2.67 0.042±0.003 0.758±0.038 Large Acrocentric 15 0.62 1.89 2.50 0.039±0.003 0.754±0.029 Medium Acrocentric 16 0.59 1.75 2.34 0.037±0.003 0.749±0.031 Medium Acrocentric 17 0.55 1.60 2.15 0.034±0.004 0.744±0.026 Medium Acrocentric 18 0.51 1.41 1.92 0.030±0.005 0.733±0.022 Small Acrocentric 19 0.00 3.18 3.18 0.050±0.007 1.000±0.000 Large Telocentric 20 0.00 2.90 2.90 0.046±0.008 1.000±0.000 Large Telocentric 21 0.00 2.70 2.70 0.042±0.004 1.000±0.000 Large Telocentric 22 0.00 2.48 2.48 0.039±0.002 1.000±0.000 Medium Telocentric 23 0.00 2.29 2.29 0.036±0.003 1.000±0.000 Medium Telocentric 24 0.00 2.11 2.11 0.033±0.002 1.000±0.000 Small Telocentric 25 0.00 1.68 1.68 0.026±0.004 1.000±0.000 Small Telocentric

*Ag-NOR-bearing chromosome. 268 P. Saenjundaeng et al. Cytologia 83(3)

karyotype having four types (metacentric, submetacentric, acrocentric and telocentric) of the chromosome is important chromosomal characteristics of L. leptocheilus. An idiogram exhibits a gradual variation of chromosome lengths (Fig. 4). The largest and smallest chromosomes show different approximately two-fold in length. The chromosome markers of L. leptocheilus are the chromosome pairs 11 and 25 which are the largest acrocentric chromosomes and the smallest telocentric chromosomes, respectively.

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