Turkish Journal of Zoology Turk J Zool (2013) 37: 706-712 http://journals.tubitak.gov.tr/zoology/ © TÜBİTAK Research Article doi:10.3906/zoo-1211-12

Cytogenetic characterization of Devario aequipinnatus (McClelland, 1839) and Devario yuensis (Arunkumar and Tombi, 1998) (: ) from Manipur, northeast India

Sanjabihari SUKHAM, Brajakishor CHINGAKHAM*, Labee THOIDINGJAM, Gusheinzed WAIKHOM Institute of Bioresources and Sustainable Development, Imphal, Manipur, India

Received: 10.11.2012 Accepted: 27.05.2013 Published Online: 04.10.2013 Printed: 04.11.2013

Abstract: Cytogenetic characteristics of Devario aequipinnatus (McClelland, 1839) and Devario yuensis (Arunkumar and Tombi, 1998) from northeast India were studied by examining metaphase chromosome spreads from gill, epithelial, and kidney cells. The diploid chromosome numbers of both were 2n = 50. The karyotypes consisted of 3 pairs of metacentric, 17 pairs of submetacentric, 3 pairs of subtelocentric, and 2 pairs of acrocentric chromosomes having the arm numbers NF = 96 in D. aequipinnatus; and 5 pairs of metacentric, 13 pairs of submetacentric, 6 pairs of subtelocentric, and 1 pair of acrocentric chromosomes having the fundamental arm numbers NF = 98 in D. yuensis. No sex chromosomes were detected cytologically in either of the species. The present study is the first report on the karyology of these 2 species from northeast India. Comparison of the chromosome numbers of these 2 with those of other members of closely related genera of the subfamily Danioninae shows that 2n = 50 could be considered as a modal diploid chromosome number in this subfamily.

Key words: 2n chromosome, metaphase, Devario aequipinnatus, Devario yuensis, Danioninae

1. Introduction The freshwater family Cyprinidae is the most species- The northeastern region of India is one of the hotspots rich vertebrate fish family (Nelson, 1994) and the most of freshwater fish biodiversity in the world (Kottelat and important, being widely distributed throughout the Whitten, 1996). It has rich diversity of ichthyofauna, with world (Al-Sabti, 1991). Cyprinid fishes of the 300 species under 111 genera and 35 families (Jayaram, Devario Heckel, 1843 are small fishes and rarely exceed 2010). Out of these, 250 fish species have ornamental value 50 millimeters in Standard Length (SL) from the tip (Kumar and Sabitry, 2012). A total number of 92 species of the snout to the origin of the caudal fin. They are all belonging to 56 genera, 25 families, and 10 orders were characterized by a beautiful color pattern consisting either listed as ornamental fishes of northeast India (Biswas et of several alternating dark blue and light stripes along the al., 2007). Though it has rich diversity of ichthyofauna, side, or a series of more or less distinct dark vertical bars cytogenetic characterization of fishes of this region is very similar to those of , rerio (Hamilton, 1822). scanty; only 68 species (22.6%) have been characterized The genera Devario and Danio are closely related and so far (Nagpure et al., 2000). Nagpure et al. (2000) also cannot be easily distinguished on the basis of proportional highlighted the need for intensive cytogenetic studies to measurements alone. Devario yuensis (Figure 1a) and D. bridge the gap between morphological and karyological aequipinnatus (Figure 1b) are very colorful fishes having information for many species of the region. However, immense ornamental potential and can be kept in aquaria; studies on the chromosomes of fishes have not been as hence, they have become part of the aquarium trade. They successful or widespread as in other vertebrate groups. are extensively distributed in the 2 headwaters of Manipur, This is because fish chromosomes are characterized by a the Brahmaputra basin in the west and the Chindwin basin large number of small chromosomes (Karahan and Ergene, in the east. The genus Devario has 38 valid species (Fang 2010), which discourages researchers from pursuing fish- and Kullander, 2009) distributed throughout South and karyotype analysis. Therefore, karyological data on fishes Southeast Asia. Their highest species diversity occurs in of this region are available for only a small percentage of northeastern India, Bangladesh, and Myanmar (Barman, species (Nagpure et al., 2000). 1991). To date, only 3 species, D. aequipinnatus, D. devario * Correspondence: [email protected] 706 SUKHAM et al. / Turk J Zool

ab Figure 1. (a) Devario yuensis; (b) Devario aequipinnatus.

(Hamilton, 1822), and D. malabaricus (Jerdon, 1849), 2. Materials and methods have been cytogenetically characterized (Ryoichi, 2011) Twenty-five specimens each of Devario yuensis (19 males, as having diploid chromosome number 2n = 50 (Table 1). 6 females) and D. aequipinnatus (17 males, 8 females) The study of fish chromosomes has received were collected from the Lokchao River (23°45′N–24°45′N considerable attention in recent years because of their latitude and 93°45′E–94°30′E longitude) and the Tupul importance in classification, evolution, heredity (Gold et River (24°28vN–25°32′N latitude and 93°10′E–93°45vE al., 1990), fish breeding, rapid production of inbred lines, longitude) of Manipur. The Lokchao River is situated in the and cytotaxonomy (Kirpichnikov, 1981). Basic information Chindwin basin and the Tupul River is in the Brahmaputra on the number, size, and morphology of chromosomes basin. Fish were caught by local fishermen with cast nets is needed to undertake genetic investigations such as and transported live in oxygen-filled polythene bags hybridization and chromosomal manipulations in fish to the laboratory. There, the fish were kept in a well- (Khan et al., 2000). This would also provide a complementary aerated tank at 20–25 °C for acclimatization for 48 h data source (along with the morphological methods) for before experimentation. Species were identified following more accurate and precise identification of fishes (Esmaeili Viswanath et al. (2007). Voucher specimens of D. yuensis et al., 2008). The importance of chromosomal studies and and D. aequipinnatus were deposited at the fish museum of lack of karyological information for many species of fishes the Institute of Bioresources and Sustainable Development of northeast India led to the present investigation. Devario (IBSD), Manipur, India, as IBSD FM C2 and IBSD FM C3, yuensis and D. aequipinnatus are also potential competitors respectively. of zebrafish, Danio rerio, as vertebrate model organisms For chromosome preparations (Manna and Prasad, in genetics, developmental biology, neurophysiology, and 1968), the specimens were treated intramuscularly with biomedicine research (Amsterdam and Hopkins, 2006). 0.05% colchicine at a dose of 1 mL per 100 g of body The present study provides detailed information on the weight using an insulin syringe to arrest the mitotic chromosome number and karyotype structure of D. division at the metaphase stage, and kept alive in a well- yuensis for the first time, and increases the cytotaxonomic aerated plastic bucket. After 2 h, the specimens were killed information necessary for understanding the evolution of with an overdose of ethylene glycol; the gill filaments and the subfamily Danioninae. kidney tissues were dissected out and further processed for

Table 1. Summary of the chromosome data of the Devario species (m: metacentric; sm: submetacentric; st: subtelocentric; t: acrocentric).

Species Locality 2n NF Karyotype formula References

D. aequipinnatus India (Assam) 50 96 14m + 32sm + 4t Khuda-Bukhsh et al. (1986)

D. aequipinnatus Thailand (Nakhonphanom) 50 96 8m + 28sm + 10st + 4t Magtoon and Arai (1994)

D. aequipinnatus India (Manipur) 50 96 6m + 34sm + 6st + 4t Present paper

D. devario India (Orissa) 50 96 12m + 24 sm + 10st + 4t Khuda-Bukhsh et al. (1986)

D. devario Asia 50 100 10m + 40st/A Fontana et al. (1970)

Fontana et al. (1970) D. malabaricus Asia 50 100 10m + 40st Hardie and Hebert (2004)

D. yuensis India (Manipur) 50 98 10m + 26sm + 12st + 2t Present paper

707 SUKHAM et al. / Turk J Zool chromosome preparations using hypotonic treatment in (Microsoft), and evaluated for overall symmetry versus 0.56% KCl solution for 45 min, followed by fixation using asymmetry in terms of centromere position and relative fresh, chilled Carnoy’s fixative (methanol:acetic acid in 3:1 size differences. ratio). The fixative was used at least 3 times, or until a clear transparent cell suspension was obtained. A small quantity 3. Results of cell suspension was taken in a Pasteur pipette and Analysis of 100 metaphase plates from the kidney and dropped onto a grease-free, pre-cleaned glass slide from gill epithelial cells of 25 specimens each of the 2 species a height of 45–60 cm. Then the slide was swiftly passed revealed that the modal chromosome number was 2n = over a flame 2–3 times. The chromosome slides were aged 50, which was valid over 82% (D. aequipinnatus) and 85% in a dust-free place for 2–3 days before staining with 6% (D. yuensis) of metaphase cells, respectively (Table 2). The Giemsa solution (Sigma) in phosphate buffer (McGregor karyotype consisted of 10 metacentric, 26 submetacentric, and Varley) of pH 6.8 for 15 min, washed with double 12 subtelocentric, and 2 acrocentric chromosomes (Figure distilled water, and air dried. 2a), having the fundamental arm numbers NF = 98 for D. The slides were inspected using a Leica DM3000 yuensis. Similarly, D. aequipinnatus karyotype consisted microscope coupled to a Leica digital camera, model of 6 metacentric, 34 submetacentric, 6 subtelocentric, DFC 310FX, and screened for good metaphase plates. and 4 acrocentric chromosomes (Figure 2b), having The diploid number and characteristic morphology of arm numbers NF = 96. No morphologically different these species were obtained from 100 chromosome plates chromosomes related to sex were observed in any of the from cells exhibiting the complete chromosome number. specimens examined in both species. The morphological Selected chromosome plates were captured under a 100× and numerical data are summarized in Table 3. The oil immersion lens using Leica Application Suite software averaged haploid ideograms of D. yuensis and D. (LAS), version 4.0.0. aequipinnatus chromosome complements are represented Homologous pairs of chromosomes were arranged in Figures 3a and 3b, respectively. in order of decreasing length within each morphological group and, finally, a karyotype was constructed on the 4. Discussion basis of centromere position of the 10 best metaphases. The diploid chromosome numbers seem to be a rather Mean lengths of the short arm (p) and the long arm (q), conservative characteristic and are used as an indicator and arm ratio (the ratio of the long arm to the short arm of the closeness of species interrelationships within length) of each chromosome were calculated to classify families (Moyle and Cech, 2004). The karyotypic data for the chromosomes as metacentric (m), submetacentric 4 species of the genus Devario are shown in Table 1. The (sm), subtelocentric (st), and acrocentric (t), following apparent modal diploid number is 2n = 50 in the genus Levan et al. (1964). Fundamental arm number (NF) was Devario. Cells lacking normal chromosome number (2n established by assigning a value of 1 to all acrocentric = 45, 47, 48, 49, 50, 51) were probably caused by losses chromosomes and a value of 2 to all metacentric, during preparation or additions from nearby cells. submetacentric, and subtelocentric chromosomes. Therefore, it can be concluded that chromosome number Diagrammatic representations of haploid karyotypes, i.e. in this genus is conserved as in other cyprinid fishes of the ideograms, were constructed according to short arm (p) subfamily Danioninae (e.g., Danio rerio, Rasbora rasbora, length and long arm (q) length using Excel 2010 software R. aurotaenia, R. daniconius, R. sumatrana, R. trilineata,

Table 2. Chromosome complements of Devario aequipinnatus and D. yuensis.

Species No. of metaphase plates No. of chromosomes Percentage 45 2% 47 9% D. aequipinnatus 100 49 4% 50 82% 51 3% 45 4% 48 6% D. yuensis 100 49 3% 50 85% 51 2%

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m m 1 2 3 4 5 132

6 7 8 9 10 4 5 6 7 sm sm 8 9 10 11 11 12 13 14 15 12 13 14 15 16 17 18 16 17 18 19 20 st 19 20 21 22 23 st 21 22 23 24 t t 24 25 25 a b Figure 2. Karyotypes of (a) Devario yuensis and (b) D. aequipinnatus (Bar = 10 µm).

Table 3. Chromosome measurements (in µm) and classification of Devario aequipinnatus and D. yuensis from 10 best metaphase chromosomes each (CN: chromosome number; T: chromosome type; m: metacentric; sm: submetacentric; st: subtelocentric; t: acrocentric).

D. aequipinnatus D. yuensis

Long arm Short arm Arm ratio Long arm Short arm Arm ratio CN T CN T Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD

1 7.53 ± 0.62 6.20 ± 0.98 1.24 ± 0.21 m 1 6.95 ± 0.06 4.95 ± 0.06 1.41 ± 0.03 m 2 5.65 ± 0.64 4.75 ± 0.40 1.20 ± 0.21 m 2 6.63 ± 0.88 4.50 ± 0.71 1.49 ± 0.18 m 3 5.88 ± 0.45 3.98 ± 0.17 1.48 ± 0.14 m 3 5.53 ± 0.41 5.00 ± 0.71 1.12 ± 0.13 m 4 12.70 ± 0.68 4.90 ± 0.57 2.61 ± 0.19 sm 4 5.70 ± 0.20 4.05 ± 0.33 1.42 ± 0.16 m 5 11.35 ± 0.44 4.08 ± 0.29 2.80 ± 0.23 sm 5 4.88 ± 0.25 4.63 ± 0.25 1.06 ± 0.10 m 6 10.68 ± 0.28 4.48 ± 0.28 2.39 ± 0.17 sm 6 11.08 ± 0.97 4.25 ± 0.40 2.61 ± 0.06 sm 7 10.55 ± 0.87 4.18 ± 0.44 2.56 ± 0.42 sm 7 9.38 ± 0.75 3.55 ± 0.40 2.65 ± 0.12 sm 8 9.70 ± 0.47 4.02 ± 0.56 2.45 ± 0.42 sm 8 9.00 ± 0.00 3.50 ± 0.58 2.63 ± 0.43 sm 9 8.98 ± 0.26 4.40 ± 0.39 2.10 ± 0.20 sm 9 8.68 ± 0.43 3.38 ± 0.25 2.59 ± 0.30 sm 10 9.53 ± 0.41 3.70 ± 0.48 2.60 ± 0.27 sm 10 8.28 ± 0.21 3.53 ± 0.61 2.41 ± 0.43 sm 11 8.90 ± 0.89 4.25 ± 0.39 2.11 ± 0.35 sm 11 8.05 ± 0.25 3.60 ± 0.71 2.31 ± 0.50 sm 12 8.98 ± 0.05 3.70 ± 0.35 2.45 ± 0.24 sm 12 8.25 ± 0.87 3.38 ± 0.48 2.46 ± 0.14 sm 13 8.75 ± 0.84 4.18 ± 0.31 2.10 ± 0.20 sm 13 7.60 ± 0.45 3.30 ± 0.23 2.31 ± 0.08 sm 14 8.70 ± 0.85 3.83 ± 0.36 2.31 ± 0.48 sm 14 7.13 ± 0.25 3.65 ± 0.18 1.96 ± 0.14 sm 15 8.28 ± 0.76 3.40 ± 0.20 2.45 ± 0.31 sm 15 7.60 ± 0.54 3.00 ± 0.28 2.55 ± 0.27 sm 16 8.00 ± 0.41 3.50 ± 0.48 2.32 ± 0.37 sm 16 7.05 ± 0.10 3.05 ± 0.06 2.31 ± 0.03 sm 17 8.38 ± 0.56 3.10 ± 0.33 2.72 ± 0.32 sm 17 6.60 ± 0.23 3.30 ± 0.23 2.01 ± 0.21 sm 18 8.20 ± 0.63 3.35 ± 0.30 2.53 ± 0.13 sm 18 6.00 ± 0.00 3.00 ± 0.00 2.00 ± 0.00 sm 19 6.98 ± 0.82 3.43 ± 0.46 2.06 ± 0.37 sm 19 13.40 ± 0.49 3.88 ± 0.10 3.46 ± 0.20 st 20 7.33 ± 0.50 3.03 ± 0.05 2.43 ± 0.19 sm 20 12.05 ± 0.84 3.50 ± 0.58 3.51 ± 0.60 st 21 13.20 ± 0.59 3.90 ± 0.28 3.40 ± 0.34 st 21 13.20 ± 0.59 3.90 ± 0.28 3.40 ± 0.34 st 22 12.20 ± 0.54 3.10 ± 0.24 3.96 ± 0.37 st 22 10.75 ± 0.29 3.30 ± 0.12 3.27 ± 0.20 st 23 8.85 ± 0.75 2.85 ± 0.24 3.11 ± 0.08 st 23 9.48 ± 0.79 2.65 ± 0.29 3.64 ± 0.13 st 24 14.65 ± 0.75 0.00 ∞ t 24 8.00 ± 0.00 2.50 ± 0.00 3.20 ± 0.00 st 25 11.63 ± 0.31 0.00 ∞ t 25 14 ± 0.00 0.00 ∞ t

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20 20 a a 15 15

10 10

Total (µm) 5 Total (µm) 5

0 0 12345678910111213141516171819202122232425 12345678910111213141516171819202122232425 Figure 3. (a) Haploid ideogram of Devario yuensis. Figure 3. (b) Haploid ideogram of Devario aequipinnatus.

Esomus danricus, Barilius bendelisis, B. gatensis, B. vagra, B. structural rearrangement in chromosome complements naseeri, B. pakistanicus, and B. tileo) (Ryoichi, 2011). Thus, causes changes in chromosome morphology without the conservative nature of diploid chromosome number in change in chromosome number (Rishi et al., 1998). the subfamily Danioninae also suggests the monophyly of This intra-individual similarity in diploid chromosome this group. In addition, the subfamily Danioninae shows number but dissimilarity in fundamental arm numbers similarity to many of the fish species of the subfamily and karyotype formula in Devario species cannot be (Collares-Pereira, 1989; Rishi, 1989; Al-Sabti, fully explained by pericentric inversion alone, though it 1991; Gül et al., 2004) belonging to different genera, such is considered to be the main mechanism of karyotypic as Chagunius, Cirrhinus, Labeo, Puntius, and Osteobrama, evolution resulting in the variations in NF within the whose diploid numbers are 50. This finding suggests the group (Galetti et al., 2000). Karyotypes of other native close relationship between the 2 subfamilies of the family Devario species (D. acuticephala, D. assamensis, D. Cyprinidae and supports the conservative nature of the devario, D. naganensis, Devario sp. 1, Devario sp. 2, karyotype macrostructure within the group, especially the and Devario sp. 3 listed by Viswanath et al. [2007] from 2n = 50. northeast India) have not been investigated so far. As a Though chromosome numbers of Devario species result, chromosomal evolution of this group is not fully are conserved despite different geographical locations, understood. the fundamental arm numbers (NF) are different. There was no evidence of sexual dimorphism of the This divergence may be attributed to differences in the chromosomes in either of the 2 species, which agrees karyotype macrostructure, reflecting a real geographical with the reports on D. devario and D. malabaricus variation common to widespread species (Thaís et al., (Khuda-Bukhsh et al., 1986; Hardie and Hebert, 2004). 2010), or may be the result of differences in the scoring of Similarly, sex chromosomes were indistinguishable in subtelocentric or acrocentric chromosomes in different several cyprinid fishes reported so far (Ergene et al, 2010; species of Devario. Devario aequipinnatus (from the Kılıç-Demirok and Ünlü, 2001; Kilic-Demirok and Ünlü, Nakhonphanom region, Thailand, and Assam and 2004; Esmaeili et al., 2007, 2008, 2009, 2010; Kalbassi et Manipur, India) exhibit cytological closeness with D. al., 2008). The occurrence of cytologically differentiated devario of Orissa (India) by having the same chromosome sex chromosomes in a large number of living marine fish number and fundamental arm numbers, compared species appears to be rare (Galleti et al., 2000), although it with other species reported from different regions. The has been described in some catfishes (Alves et al., 2006) only difference observed is in the karyotype formula, and in platyfish (Devlin and Nagahama, 2002). indicating that pericentric inversions might have played a Considering the difficulties in identifying several of the substantial role during the evolutionary pathway of these Devario species and their unclear phylogeny, cytogenetics species. Moreover, differences in the karyotype formula may prove itself as an important tool in understanding the may be limited to cryptic chromosome rearrangements, systematics of the genus. Thus, karyotype characteristics such as those involving the heterochromatin segments may contribute towards a better systematic interpretation, and/or the nucleolus organizer regions (Thaís et al., especially in the case of cryptic species, which are difficult 2010). Alternatively, it can be attributed to different to define (Artoni et al., 2009). The data of the present study degrees of chromosome condensation, leading to on chromosome composition would contribute toward differences in chromosome classification among authors clarifying the karyotypic evolution and phylogenetic (Ryoichi, 2011). On the other hand, the differences in the relationships in this group. Further analysis, including fundamental arms within the same species of D. devario additional species of Devario of different regions and from different geographical locations suggest that the different staining techniques, will provide a better

710 SUKHAM et al. / Turk J Zool understanding of the chromosome evolution in the group Acknowledgments and confirm the apparent conservative nature of the The first author is thankful to the Director, IBSD, for the diploid number in this Danioninae subfamily. The present facilities provided, and to the Department of Biotechnology, study also clearly confirms the taxonomic position of these Government of India, for financial assistance under the 2 species of fish as 2 distinct species of Devario on the basis DBT- Research Associateship Program in Biotechnology of their chromosome characterization, although they are and Life Sciences. We also thank the anonymous reviewers morphologically very similar to each other and other for their invaluable comments and suggestions on the species of the genus Danio. manuscript.

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