Karyotypic Diversity of Some Freshwater Fish Species in Rural Ponds of Orissa, India

J. R. Biswal1, P. K. Sahoo1 and P. K. Mohanty2*

1 Directorate of Research for Women in Agriculture (ICAR), Bhubaneswar, P.O. Baramunda (Opposite to Kalinga studio), Orissa–751003, India 2 Post Graduate Department of Zoology, Utkal University, Vani Vihar, Bhubaneswar–751004, Orissa, India

Received December 7, 2009; accepted July 20, 2010

Summary Study on chromosomes of fish has become a priority area of research in recent years. Chromosome analysis can be useful for addressing a variety of evolutionary and genetic questions about fishes. The global fish fauna consists of about 28,900 species of fishes of which 2,200 species are cytogenetically studied. India’s contribution so far was around 220 species. Fish chromosome studies of freshwater fishes are further sporadic in Orissa. Therefore, the present study is proposed for a systematic cytogenetic investigation of some freshwater fishes of Orissa. Karyotypic study of 5 species namely Labeo fimbriatus (Fam. Cyprinidae), Puntius sophore (Fam. Cyprinidae), Osteobrama cotio cotio (Fam. Cyprinidae), Chanda nama (Fam. Chandidae), and, Xenentodon cancila (Fam. Benonidae) from rural ponds of Bhubaneswar, India, was carried out. The diploid chromosome number in Labeo fimbriatus was 50 with a chromosomal formula of 10m10sm30T (FN70), Puntius sophore 2n48, 2m46T (FN50), Osteobrama cotio cotio 2n54, 10m8sm8st28T (FN72), Chanda nama 2n50, 8m6sm10st26T (FN64) and Xenentodon cancila, 2n48 with chromosomal formula of 22m10sm6st10T (FN80). The evolutionary significance of these karyotypes of 5 species has been discussed.

Key words Karyotype, Chromosomes, FN, Cytogenetic.

Fishes account for more than half of the known vertebrates. About 28,900 extant species were listed in FishBase in 2005, out of which almost 13,000 species belong to freshwater (Leveque et al. 2008). They exhibit enormous diversity of size, shape, biology and in the habitats they occupy. Talwar (1991) estimated 2,546 species of fish inhabiting Indian waters. Cytogenetic study has immense importance in characterizing, classifying and drawing evolutionary relationship among this diversified group. So chromosomal studies in fishes become a priority area of research in recent years (Barat et al. 2002, Sahoo et al. 2007). So far about 2,200 species of fishes are cytogenetically studied and India’s contribution so far was around 220 species. This can be useful for addressing a variety of evolutionary and genetic questions about fishes. Therefore, the present study was carried out for a systematic cytogenetic investigation of some freshwater fishes of Orissa.

Materials and methods Live specimens of Labeo fimbriatus, Puntius sophore, Osteobrama cotio cotio, Chanda nama and Xenentodon cancila were collected from the rural ponds near Bhubaneswar, Orissa using cast net and fry net. Four fishes of each species were acclimatized through aeration for 2–3 d in the laboratory aquarium. The fishes were injected intraperitoneally with 0.05% colchicine at 1 ml/100 g of body weight and left for 2 h in aerated water. After 2 h, the live samples were operated and gill and kidney tissues were collected, washed thoroughly in distilled water to remove clotted blood and

* Corresponding author, e-mail: [email protected] 238 J. R. Biswal et al. Cytologia 75(3) other artifacts. The tissues were minced to make cell suspension in 0.56% KCl and kept for 30 min at room temperature. The cell suspension after 30 min was centrifuged at 2000 rpm for 10 min and cell pellets were further processed following ﬁxation in 1 : 3 Glacial Acetic acid and Methanol. The slides were prepared following ‘ﬂame drying method’ (Khuda Bukhsh and Barat 1987). The slides were stained with 4% Giemsa solution and air dried. 52–76 metaphase spreads (Table 1) were screened in the above 5 species. The well spread metaphases were photographed and karyotyped following Levan et al. (1964).

Results Labeo ﬁmbriatus The overwhelming majority (85%) of metaphase complements in the kidney and gill tissues contained 50 chromosomes, though a few plates had a range of 48 to 52. Thus the diploid number (2n) in this species was conﬁrmed to be 50 and a karyotype formula of 10m10sm30T (Fig. 1) with fundamental arm number (FN) as 70. The size of the chromosomes varies between 7.0 to 2.5 mm. The karyotype yielded no indication of sex element(s) in the form of any heteromorphic chromosome pair.

Puntius sophore Seventy percents of the somatic metaphase complements contained 48 chromosomes. Hence,

Table 1. Karyotype analysis of 5 species of ﬁshes

No of cells No of Sl. no. Name of the species msmstTFN observed chromosomes

1 Labeo ﬁmbriatus 65 50 10 10 — 30 70 2 Puntius sophore 42 48 2——4650 3 Osteobrama cotio cotio 76 54 10 8 8 28 72 4 Chanda nama 52 50 8 6 10 26 64 5 Xenentodon cancila 53 48 22 10 6 10 80

Fig. 1. Karyotype of Labeo ﬁmbriatus. 2010 Karyotypic Diversity of Some Freshwater Fish Species 239

Fig. 2. Karyotype of Puntius sophore.

Fig. 3. Karyotype of Osteobrama cotio cotio. the diploid number (2n) in this species ascertained to be 48. The karyotype of this species consisted of 2 metacentric (m) and 46 telocentric (T) with fundamental arm number (FN) 50. The size of the chromosomes varies between 6.5 to 3.5 mm.

Osteobrama cotio cotio The somatic metaphase complements contained 54 chromosomes in 70 out of 76 cells studied. Therefore, the diploid chromosome number in this species was ascertained to be 54 and the karyotype consisted of 10m8sm8st28T with a fundamental arm number (FN) 72. The size of the chromosomes varies between 7.0–2.8 mm. However, no sex chromosomes could be identiﬁed in 240 J. R. Biswal et al. Cytologia 75(3)

Fig. 4. Karyotype of Chanda nama.

Fig. 5. Karyotype of Xenentodon cancila. the form of any heteromorphic pair of chromosomes.

Chanda nama The diploid metaphase complements of unidentiﬁed sex of this species contained 50 chromosomes in 48 out of 52 cells studied, while rest had aneuploid numbers of 42, 43, 46 and 48. The karyotype (Fig. 4) consisted of 8m6sm10st26T and a fundamental arm number (FN) 64. The size of the chromosomes varied between 7.5–2.5 mm. The largest pair was a pair of subtelocentric chromosomes.

Xenentodon cancila The maximum number metaphase complements 48 out of 53 cells contained 48 chromosomes. Hence, the diploid number in this species was ascertained to be 48. The karyotype of this species consisted of 22m10sm6st10T with fundamental arm number (FN) as 80. The size of the 2010 Karyotypic Diversity of Some Freshwater Fish Species 241 chromosomes varies between 10.0 to 5.0 mm. The sex of any individual could not be identiﬁed due to small size and also there was no heteromorphic pair of chromosomes could be identiﬁed as sex chromosomes.

Discussion The present cytogenetical studies on Labeo fimbriatus may be the first report as far as the authors are aware. The other conspecies are L. rohita, L. calbasu, L. bata, and L. gonius where the same chromosome number 2n50 were reported earlier. But each species had species-specific karyotypes. The variation in the karyotype may be due to some structural rearrangements like Robertsonian translocation and pericentric inversion. The modal chromosome number in this genus under cyprinid family seems to be 50. But the other cyprinid in the present study, i.e., Puntius sophore, revealed 2n48 chromosomes and Osteobrama cotio cotio 2n54. Around 24 species under genus Puntius were cytogenetically reported so far (Sahoo et al. 2007, Khuda Bukhsh et al. 1986, Ojima 1985, Taki and Suzuki 1977, Taki et al. 1977). In most of them, the 2n50 chromosomes and karyotypes also consisted of heterogenous group of metacentric, submetacentric, subtelocentric and acrocentric chromosomes. Taki et al. (1978) compared using external and internal characters in 24 species of Puntius from Southern Asia and divided into 6 groups. On the basis of this study, they have pointed out a common ancestral stock of all these 6 groups of Puntius. The karyotypic studies on Osteobrama cotio cotio (Fam. Cyprinidae) was reported earlier by Sharma and Tripathy (1986) depicting diploid number as 48 with chromosomal formula 18m24sm6st (FN90) which is quite different with the present studies in diploid number as also chromosomal formula. The chromosomal studies on Chanda nama reported earlier by Nayyar (1966) using squash technique and Rishi et al. (1997). Though the diploid number reported by Nayyar (1966) is in conformity with the present findings but karyotypic formula is different and the karyotype details described by Rishi et al. (1997) are quite different. The chromosomal studies in Xenentodon cancila (Family Belonidae) has been reported earlier by Sharma and Tripathy (1981) with 2n48, but with different karyotype of 16m6sm8st18A. So far whatever the variation in the karyotype is observed may be due to some inherent polymorphism for different geographical locations if any technical limitations are not there (Barat and Sahoo, 2001). The broad-spectrum variation in the karyotypes of same species indicates a need of proper attention for morphological identification of the species and then with the aid of molecular DNA marker assay. However, it can not be ruled out a possible change in the genotype with the change in environment after few decades as also in different geographical locations. Still there is ample scope for further work for confirmation in this area using a comparative analysis of ancient and present samples with the molecular DNA marker assistance.

Acknowledgements Authors are highly grateful to Director DRWA for providing all the laboratory facilities for the research work.

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