Department of Zoology, Kurukshetra University Kurukshetra, India
Received Date: 03.05.2013 / Accepted Date: 23.08.2013 / Published Date: 05.03.2014
The purpose of the present study was to investigate and compare the karyotypes of three Indian major carps, Catla catla (Hamilton, 1822), Labeo rohita (Hamilton, 1822), Cirrhinus mrigla (Hamilton, 1822) in terms of chromosomal architechture, karyotype formula and number of chromosomes from aquatic ecosystems of Haryana, India. Karyotypes of these carps were in-vestigated by examining metaphase chromosomes. The results indicated that the diploid (2n) chromosome number of all the three major carps was 50. Catla catla consisted of 22 acrocent-ric, 2 subtelocentric, 20 submetacentric and 6 metacentric chromosomes. Labeo rohita consis-ted of 32 acrocentric, 4 subtelocentric, 6 submetacentric, 8 metacentric while Cirrhinus mrigla consisted of 30 acrocentric, 8 subtelocentric, 6 submetacentric, 6 metacentric chromosomes. Centromeric Index, arm ratio and fundamental number was also determined. No heteromorphic sex chromosomes were cytologically detected. The variability in size, shape and arm number (NF) of chromosomes among these three species suggest that diversification in these fish spe-cies of same family is related to structural changes in chromosomes. Variations in karyotype formulae are also observed with respect to earlier studies which may be due to variations in ha-bitat conditions as a result of anthropogenic activities.
Catla catla, Chromosome, Cirrhinus mrigla, Karyotype, Labeo rohita
Cyprinidae is a large family of freshwater fishes that is commonly called the carp family. The vast majority of bony fish belongs to this family and these are widely distributed in fresh-water resources (Abdoli, 1999). Family Cyprini-dae is comprised of 220 genera and 2420 species (Nelson, 2006). In fishes, cytogenetic data is im-portant because in this group the usual mor-photaxonomical characters are not so clear and phylogenetic understanding is beset with many hurdles. The increasing knowledge of chromo-somes can provide reliable information on the phyletic relationship in the Cyprinidae to a cer-tain extent (Kalbassi et al., 2006).
Systematically, Catla catla, Labeo rohita and Cirrhinus mrigla belongs to class Teleostei, order Cypriniformes, family Cyprinidae (Jayaram, 1999). The study of fish chromosomes was initi-ated in India from 1960s by using basically the methodologies available for mammals. Karyolog-ical studies on Indian major carps have been car-ried out by some workers (Rishi, 1973; Khuda-Bukhsh and Manna, 1974; Majumdar and Ray-Chaudhari, 1976; Zhang and Reddy, 1991). The different variety of karyotypes found in different species was evidence that the process of evolu-tion was associated with karyotypic changes (Fredga, 1977).
Chromosomal analysis is important for fish breeding from the viewpoint of genetic control, the rapid production of inbred lines, cytotaxono-my and evolutionary studies (Kirpichnikov, 1981). Karyological studies provide basic infor-mation on the number, size and morphology of chromosomes (Tan et al., 2004) that is important to undertake chromosome manipulation in fish (Khan et al., 2000). However, the studies dealing with karyotype of fishes are few because of large number and small size of chromosomes. Hence the present study was designed to determine the karyotype, chromosomal architecture, the propor-tion of acrocentric, submetacentric and metacen-tric chromosomes and number of chromosomes in Indian major carps, C. catla, L. rohita, C. mrigla from the geographic area of Haryana.
Fifteen fishes were obtained from the local fish farm and transported live to the Fish and Fisheries laboratory of Department of Zoology, Kurukshetra University, and Kurukshetra. Kid-ney and gill epithelium tissue were used for kar-yotype analysis. Three living specimens each of C. catla, L. rohita and C. mrigla from Haryana were analysed. The preparation of chromosomes was performed according to air drying technique given by Tjio and Whang (1965). Each specimen was injected intraperitoneally with a colchicine solution (0.05%; 1 mL/100g body weight). The fishes were maintained in a well aerated aquari-um and after 2 hr., Kidney and gill epithelium were extracted and placed in hypotonic solution of 0.56% KCl. After 30 minutes in the hypotonic solution, the cellular suspension was centrifuged at 1000 rpm for 10 minutes. The hypotonic solu-tion was discarded and the pellet was suspended and washed three times in methanol: glacial ace-tic acid (3: 1). After centrifugation at 1000 rpm for 10 minutes, the drops of cellular suspension was put on a clean grease free microscopic slide, previously chilled in a freezer from a height of 2 feet. The slides were allowed to air dry.
For the conventional karyotype, the prepara-tions were stained for 15 minutes with 5% Giem-sa in phosphate buffer (pH 6.88). Mitotic meta-phases spreads were scanned to determine the modal chromosome number. Mitotic metaphase were photographed by using Olympus C-7070 wide zoom camera at magnification of 1000X and used for preparation of karyotype. The arm ratio and centromeric indices of metaphase chromosomes were determined following Levan et al. (1964) to assign the morphological types and the chromosomal formulae.
Catla catla 2n = 50, NFa = 76
The diploid chromosome number of all three major carps was found to be 50. In case of C. catla, the somatic metaphase showed the pres-ence of 50 chromosomes (Figure 1A) and funda-mental arm number 76. The somatic karyotype (Figure 1B) was prepared according to decreas-ing chromosome length. Somatic karyotype showed 22 acrocentric (pair nos. 4, 9, 11, 15-19, 22-23 and 25), 2 subtelocentric (pair no. 3), 20 submetacentric (pair nos. 1-2, 5-7, 12, 14, 20-21 and 24) and 6 metacentric ( pair nos. 8, 10 and 13) chromosomes. Sex chromosomes could not be distinguished. The size of the chromosome ranged from as low as 0.251μm of 25th pair of chromosome to as high as 5.911μm of 1st pair of chromosomes (Table 1). Relative % length of the smallest chromosome was 0.640 while largest chromosome was 15.070. The total haploid mean length was calculated to be 39.218 μm.
Labeo rohita 2n = 50, NFa = 64
The somatic metaphase in the kidney cells of the L. rohita showed diploid chromosome num-ber 50 i. e. 2n=50 (Figure 2A) and fundamental arm number 64. The somatic karyotype (Figure2B) showed 32 acrocentric (pair nos. 2, 6-8, 10-13, 15, 18-21, 23-24), 4 subtelocentric (pair no. 4 and 14), 6 submetacentric (pair nos. 1, 9 and 16) and 8 metacentric (pair nos. 3, 5, 17 and 22) chromo-somes. Sex chromosomes could not be distin-guished. The size of the chromosomes in this case ranged from as low as 1.087 μm of 25th pair of chromosome to as high as 1.736 μm of 1st pair of chromosomes (Table 2). Relative % length of the smallest chromosome was 1.126 while largest chromosome was 9.528. The total haploid mean length was calculated to be 32.724 μm.
Cirrhinus mrigla 2n = 50, NFa = 62
The somatic metaphase in C. mrigla also showed the presence of 50 chromosome number (Figure 3A) and fundamental arm number 62. The somatic karyotype (Figure 3B) comprised of 30 acrocentric (pair nos. 3, 5, 7, 9, 11-16, 19, 21-23 and 25), 8 subtelocentric (pair no. 2, 4, 6 and 20), 6 submetacentric (pair nos. 1, 10 and 24) and 6 metacentric (pair nos. 8, 17 and 18) chromo-somes. Sex chromosomes could not be distin-guished. The size of the chromosome ranged from 0.251 μm in case of 25th pair of chromo-some and 1.400 μm of 1st pair of chromosomes (Table 3). Relative % length of the smallest chromosome was 3.357 while largest chromo-some was 4.333. The total haploid mean length was calculated to be 29.575 μm.
In Cyprinidae 2n ranges from 44 to 100 (Arai, 1982). The high diploid chromosome number 2n=98-100 are thought to have resulted by poly-ploidisation of 2n=48 or 50. Chromosomal anal-ysis in the present study revealed that these three Indian major carps from Haryana shared the same diploid number i. e. 2n=50. The karyological study of C. catla and L. rohita done by Khuda-Bukhsh and Manna (1974), Manna (1977), Ma-jumdar and Ray Chaudhari (1976), Zhang and Reddy (1991), Jana (1993), Manna and Prasad (1971), Gui et al (1986) reported the similar re-sults i. e. 2n=50. Karyotype studies on C. mrigla have been performed by Manna and Prasad (1971), Majumdar and Ray Chaudhari, (1976) and Zhang and Reddy (1991). All these studies have shown the diploid number as 50, confirming the present results. According to Manna (1984) and Rishi (1989) the most commonly occuring diploid number in family Cyprinidae is 50, con-sidered to be the modal number of this species. Presence of same modal number in the present studies reinforces the hypothesis that Indian ma-jor carps are karyologically very conserved and represent plesiomorphic condition.
The primitive teleost karyotype is thought to have consisted of 46 to 48 chromosomes (Fitzsi-mons, 1972; LeGrande, 1975) all acrocentrics. Karyotypes with biarmed chromosomes are gen-erally regarded to represent a derived condition (Fredga, 1977). Therefore, cyprinids investigated in the present study showed a derived karyotype configuration. No heteromorphic sex chromo-somes were found.
For comparative purpose, the arm number (NF) of karyotyped fishes is calculated assigning a value 2 to biarmed chromosomes (metacentric and submetacentric) and value of 1 to uniarmed chromosomes (acrocentric and subtelocentric) and is regarded as karyotype formulae. Despite the similarity of diploid numbers in all the three selected cyprinid species, there are differences in their karyotypic formulae. Comparision of the karyotypic formulae revealed deviations from the earlier reports for these species.
In case of C. catla, Khuda- Bukhsh and Man-na (1976) reported 22 acrocentric, 24 submeta-centric and 4 metacentric chromosomes. Manna (1977) reported 26 acrocentric, 16 submetacen-tric and 8 metacentric chromosomes. Zhang and Reddy (1991) and Jana (1993) showed similar results i. e. 22 acrocentric, 16 submetacentric and 12 metacentric chromosomes. But the present re-sults showed a slight variation in chromosome morphology indicating 22 acrocentric, 2 subtelo-centric, 20 submetacentric and 6 metacentric chromosomes.
Karyological study of L. rohita done by Man-na and Prasad (1971), Majumdar and Ray Chaudhari (1976), Gui et al (1986), Zhang and Reddy (1991), Jana (1993) showed the same dip-loid number as found in present studies i.e. 2n=50. Manna and Prasad (1971) observed 24 subtelocentric, 8 submetacentric and 18 metacen-tric chromosomes. Gui et al (1986) observed 24 subtelocentric, 16 submetacentric and 10 meta-centric chromosomes. Zhang and Reddy (1991) and Jana (1993) reported similar results in L. ro-hita i. e. 22 subtelocentric, 18 submetacentric and 10 metacentric chromosomes. Present results showed 32 acrocentric, 4 subtelocentric, 6 sub-metacentric and 8 metacentric chromosomes.
In case of C. mrigla, different chromosome morphology was given by different workers but basic diploid number was same i.e. 2n=50. Man-na and Prasad (1971) reported 36 acrocentric, 8 submetacentric and 6 metacentric chromosomes which showed little similarity with the present results. In the present study 30 acrocentric, 6 submetacentric, 6 metacentric and 8 subtelocen-tric chromosomes were found. Majumdar and Ray Chaudhari (1976) observed 18 acrocentric,
26 submetacentric and 6 metacentric chromo-somes. Zhang and Reddy (1991) showed 20 ac-rocentric, 18 submetacentric and 12 metacentric chromosomes. Many authors considered that dip-loid chromosomes are all acrocentric as the an-cestral karyotype in fishes (Nogusa, 1960; Post, 1965; Denton, 1973). It may be pointed out that all acrocentric karyotype happens to be absent in a number of primitive group like chondrichthyes. Therefore it may be assumed that acrocentricity is certainily a more primitive condition that the biarmed condition.
The acrocentric chromosomes have a tenden-cy to stick to each other by their centromere and in this way they form metacentric chromosomes (Dogramci et al. 1994). Denton (1973), Gold (1979) also stated that karyotypes with biarmed chromosomes are regarded as derived condition confirming that karyomorphology of all the three species could be derived mainly by envisaging per centric inversion at various regions with re-spect to time, geographical condition and ecolog-ical characteristics.
A comparision of karyotypic formulae of these three Indian major carps species revealed that larger numbers of acrocentric chromosome are observed in L. rohita followed by C. mrigla and C. catla. 6 metacentric chromosomes were observed in both C. mrigla and C. catla whereas 8 metacentric chromosomes were observed in L. rohita. According to Le Grande (1981), differ-ences in the NF among close species can be the result of pericentric inversions. The karyotype formulae of Indian major carps in the present studies can be interpreted as the result of struc-tural chromosomal rearrangements as well as a series of pericentric inversions, generating biarmed chromosomes and so increase the NF to 74 in C. catla, 64 in L. rohita and 62 in C. mrigla. The karyotypes of these species have been compared with the related ones and it has been suggested that large number of acrocentric chromosomes have been observed during present investigation in comparision to earlier studies. It may be due to mechanism of centric fission. Cen-tric fission seemed to have played a significant role in evolution of teleost fishes (Manna and Khuda-Bukhsh, 1978). Different groups of fishes exhibit different processes of karyotype evolu-tion.
In conclusion, the chromosome analysis of three Indian major carp species C. catla (Hamil-ton, 1822), L. rohita (Hamilton, 1822), C. mrigla (Hamilton,1822) using conventional staining pro-cedure revealed the same diploid number (2n=50) with variability in size, shape and arm number (NF) of chromosomes suggesting that diversifica-tion in these fishes of the same family is related to structural changes in chromosomes. The varia-tions in karyotype formulae in comparison to ear-lier studies may be because of pericentric inver-sions or centric fission and appears to be due to variations in habitat conditions as a result of an-thropogenic activities.