4. CYTOGENETIC STUDIES

The study of fish chromosome (karyotype) was initiated in India from 1960s by using basically the methodologies available for mammals. Gradually modified fish chromosome methodologies have been developed for obtaining quality metaphase chromosomes (Reddy and John 1986; Banerjee, 1987; Reddy and Tantia, 1992 and Nagpure and Barat, 1997). Of about 2,000 species of inland and marine fish analysed for karyological information (Das and Barat, 1995), over 200 species belong to India, which include both freshwater as well as marine species.

Most of these studies are related to the analysis of diploid karyotypes. Karyological studies on Indian major carps have been carried out by many workers (Khuda-Bukhsh and Manna, 1974; Majumdar and Ray Chaudhuri, 1976; Zhang and Reddy, 1991; Jana, 1993). However, regarding Indian major carps, the investigations, besides karyotype studies, also pertain to a comparative account of parental species and their hybrids which were attempted to interpret hybrid viability, fertility and sterility (Reddy et al, 1990a, Zhang and Reddy, 1991 and Jana, 1993).

4.1 Karyotype of Indian major carps

Karyotype studies of Indian major carps have been worked out by numerous scientists, who reported similar results. The type of chromosomes and the number of chromosomes in each type as reported by different workers are given in Table 7.

Table 7. Comparative karyological studies on Indian major carps as reported by various authors

4.1.1 Catla catla and Labeo rohita

The karyotypes of Catla catla and Labeo rohita have been studied by different workers (Khuda-Bukhsh and Manna, 1974; Mujumdar and Ray-Chaudhuri, 1976; Zhang and Reddy, 1991 and Jana, 1993). All these workers reported the diploid number (2n) in these two species as 50. In catla, the karyotype according to Zhang and Reddy (1991) consisted of 12 metacentric, 16 submetacentric, and 22 subtelocentric chromosomes. In rohu, the karyotype according to these authors consisted of 10 metacentric, 18 submetacentric and 22 sub-telocentric chromosomes. Later study with regards to comparative karyotype of these carps (Jana, 1993) also more-or-less agree with the observations of Zhang and Reddy (1991) (Table 7).

4.1.2 Cirrhinus mrigala

Karyotype studies on Cirrhinus mrigala have been performed by Manna and Prasad (1971); Majumdar and Ray-Chaudhuri (1976) and Zhang and Reddy (1991). All these studies have shown the diploid number is 50.

4.1.3 Labeo calbasu

The chromosomes of L. calbasu have been studied by Majumdar and Ray-Chaudhuri (1976), and Manna and Khuda-Bukhsh (1977a). The observations of these workers confirm the diploid number as 50 but differ with regard to the number of submetacentric and acentric types, (though they agree with the number of metacentric types).

4.2 Karyotype of hybrids

4.2.1 Indian major carp hybrids

Karyotype studies of hybrids within the species of Indian major carps have been studied only in the case of kalbasu-rohu, (Krishnaja and Rege, 1975); kalbasu-catla (Khuda-Bukhsh and Manna, 1976); rohu-catla (Khuda-Bukhsh and Manna, 1976; catla-rohu (Jana, 1993). In these hybrids the diploid number and type do not differ from the parent species, but the number under each type differ. Thus, the karyotype of the rohu-catla hybrids consists of four metacentric, 24 submetacentric and 22 acrocentric pairs which differs from the parent species, (Khuda-Bukhsh and Manna, 1976). The karyotype of the catla-rohu hybrid was reported to consist 12 metacentric, 10 submetacentric and 28 subtelo + telocentric (16+12 respectively) according to Jana (1993). In the hybrid between L.calbasu and C.catla, Khuda-Bukhsh and Manna (1976) recorded 10 metacentric, 14 submetacentric and 26 subtelocentric telocentric chromosomes. Krishnaja and Rege (1975) reported the diploid (2n) number of chromosomes as 50 in the case of the interspecific hybrid produced between L.calbasu and L.rohita.

4.2.2 Hybrids between Indian major carps and common carp

Regarding the karyological studies of these intergeneric hybrids, only the diploid number of chromosomes has been worked out and was reported to be = 76 in the case of L.rohita female and common carp male hybrid (John and Reddy, 1987). In the case of the hybrids between C.carpio female × L. rohita male; C.carpio female × C.catla male and C.carpio female × Cirrhinus mrigala male, the chromosome number varied from 74 to 76 in all these forms as mentioned earlier, which is around the sum total of the chromosomes of their parental gamets. However, karyotypes of about 75% of the sum total of chromosome numbers, in the case of C.carpio × L.rohita, 60% in C.carpio × C.catla, and 58% in C.carpio × C.mrigala hybrids showed 76, 74 and 75 chromosomes respectively. (Reddy et al. 1990). Details of karyotypes of these hybrids are in Table 8.

Khuda-Bukhsh et al. (1988) studied the karyotype of the suspected gynogen progeny resulting from the cross between C.carpio female × L.calbasu male. Karyotype of the progeny resulting from this cross was 2n=100 as in the case of the maternal parent, while 2n=50 in the case of the paternal parent L.calbasu. The morphology of the chromosomes in the F1 offspring (putative gynogen) was reported to be slightly different from that of the maternal parent C.carpio, though, their external anatomical features were similar. The haemoglobin and transferrin bands of F1 individuals and the parental forms suggest that the F1 individuals originated due to gynogenetic mode of reproduction by eliminating the genetic input from the sperm and diploidization of the ovum nucleus by the union of the nucleus from the polar body, thus limiting the role of the penetrating sperm to only activation. (Khuda-Bukhsh et al., 1989). The appearance of spontaneous gynogens in the progeny of remotely related species with dissimilar or incompatible genomes has also been reported by several other workers which resulted either in gynogens and true hybrids or polyploids and even rarely androgens (Marian and Krasznai, 1978; Stenly, 1976 and Reddy, 1989). However, in the hybrid crosses between Cyprinus carpio and other Indian major carps viz. C.catla, L. rohita and C.mrigala, such gynogenetic individuals were not reported. (Alikunhi and Chaudhuri, 1959; John and Reddy, 1987; Khan et al., 1990 Reddy et al., 1990).

Table 8. Comparative karyological studies on some hybrids of Indian carps as reported by various authors

4.3 Karyotype studies in relation to hybrid viability and fertility

Karyotype studies, relating to hybrid viability and fertility have been carried out by Reddy, 1989; Reddy et al., 1990 and Zhang and Reddy, 1991.

4.3.1 Hybrid viability

The compatibility of diploid (2n) number of chromosomes of the proposed species for hybridization was earlier felt to be one of the most important deciding factors for the production of viable hybrid progeny, whether it is interspecific or intergeneric cross. The hybrids of all the interspecific and intergeneric crosses among Indian major carp species as mentioned earlier were highly viable and also fertile. However, true (diploid) hybrids of Chinese grass carp and silver carp were not found viable to maturity, though these two species possess equal number of diploid chromosomes (2n=48 in each case). Reddy (1989) and (1991) studied the karyotypes of these two species and made a type wise comparison. He reported that though these two species possessed equal number of diploid chromosomes, the karyotypes revealed different types. Grass carp karyotype consisted of only metacentric (15 pairs) and submetacentric (9 pairs). While in silver carp, the karyotype possessed metacentric (6 pairs), submetacentric (16 pairs) and subtelocentric (2 pairs). Thus, the grass carp has only two types of chromosomes, metacentric and submetacentric, whereas silver carp has three types, i.e., in addition to metacentric and submetacentric, it has subtelocentric chromosomes also. These dissimilarities in their karyotypes with different types of chromosomes were felt to be one of the probable causes of incompatibility for hybridization of these two species and the consequent mortality of their hybrid progeny. On the other hand, the karyotypes of Indian major carps, particularly, C.catla, L. rohita and C.mrigala have shown compatibility, not only diploid (2n) number wise but also type wise. The karyotype of catla thus consisted 12 metacentric, 16 submetacentric, 10 subtelocentric and 12 telocentric or acrocentric chromosomes. In rohu, it was 10 metacentric, 18 submetacentric, 10 subtelocentric and 12 telocentric or acrocentric chromosomes, while in mrigal it was similar to catla, with 12 metacentric, 18 submetacentric, 10 subtelocentric and 10 telocentric or acrocentric chromosomes (Zhang and Reddy, 1991).

Thus, though these three species of Indian major carp belong to three different genera and possess distinct morphological features, their karyotypes consist not only of similar diploid number but also more or less exhibit type wise similarities which may be the probable cause for their compatibility to hybridize and produce not only highly viable hybrid progenies but also fertile ones.

4.3.2. Hybrid fertility/sterility

The intergeneric hybrids produced between Cyprinus carpio females and males of Indian major carps catla, rohu and mrigal were found to be sterile and possessed either ill developed or rudimentary gonads (Khan et al, 1989 and 1990). The reciprocal crosses between rohu female and common carp male were also found to be sterile (Alikunhi and Chaudhuri, 1959).

Cyprinus carpio Var. communis (Bangkok strain) with 102 diploid number (John and Reddy, 1987 and Reddy et al., 1990), is said to be of tetraploid origin which when crossed with Indian major carps with diploid number of 50 chromosomes the ploidy level of hybrids was found to be the sum total of the haploid number of the parents, the exact number of chromosomes varying from 74–76. Thus most of the hybrids appeared to be aneuploid in nature (Reddy et al., 1990). These intergeneric hybrids, with an aneuploid diploid number did not attain maturity even at three years of age. Whereas the parent species, Cyprinus carpio was observed to mature when it is six months old in Indian plains, while Indian major carps within two years of age. (Jhingran, 1982 and Reddy et al., 1990).

The sterile nature of these hybrids as reported by Reddy et al. (1990), appears to be due to the aneuploid nature of their genome which might have resulted due to incomplete karyogamy. The sterile nature of triploid individuals of common carp (Cyprinus carpio) was also reported to be due to the aneuploid nature of their genome (Wu et al., 1986). However, these authors also noticed that those triploid individuals with complete sets of three haploid genomes turned out to be fertile.

Aneuploidy of a karyotype is due to the loss or gain of one or two chromosomes but not due to the loss of an entire chromosome set. Triploids in which all the three chromosomes sets were present were found to be fertile and those which showed chromosome deletion and breakage (aneuploid state) were reported sterile.

4.4 Chromosome banding studies

Literature on chromosome banding studies, particularly in Indian major carps, appears to be very scanty. However, Barat et al, (1990) John et al. (1992 and 1993) made some attempts on G-banding and Nucleolar organizer regions (NORs) banding in these carps. The first report on C-banding pattern in L.rohita appears to be that of Rishi and Mandhan (1990) followed by Khuda-Bukhsh and Chakrabarty (1994) and Nagpure (1997).

Rishi and Mandhan (1990) reported the presence of centromeric C-bands in all the chromosomes of L.rohita with no intercalary C-bands. Khuda-Bukhsh and Chakrabarty (1994) attempted to study the C-band pattern in Cirrhinus mrigala and L.rohita. It was reported that the overwhelming majority of chromosomes showed C-banding localization in or around centromere, while some others either lacked or showed telomeric (terminal) or interstitial C-bands localization in both the species.

The investigations of Nagpure (1997) on C-banding pattern of L. rohita have shown that his observations were not in agreement with Rishi and Mandhan (1990). Nagpure (1997) claims that his study revealed multisite distribution of C-band heterochromatin. This author also reported that C-bands are found to be localized only on 8 pairs of chromosomes, of which one was submetacentric and others were telocentric. Again, in majority of telocentrics centromeric C-bands were present with prominent intercalary bands on 3 pairs of telocentric chromosomes which have also exhibited the centromeric C-bands. The biarmed submetacentric chromosomes also revealed the presence of centromeric C-bands.

Attempts on G-banding of L.rohita did not meet with any interpretable success. The results were described as rather erratic and resolution of bands was not of good quality and also was not repeatable in subsequent attempts (Khud-Bukhsh and Tiwari, 1994).

Studies on NORs in Catla catla and in three species of the genus Labeo viz. L.rohita, L.calbasu and L.bata have been made by John et al. (1992 and 1993). In catla, the NORs of minute size were terminal to the short arms of one submetacentric and one subtelocentric chromosome pairs. In the case of L.rohita, L.calbasu and L.bata, the NORs were on the short arms of medium sized submetacentric chromosomes. The NORs in L.rohita and L.calbasu were similar in size, whereas those in L.bata were bigger in size. Again, while the NORs in L.rohita and L.calbasu were located on the 11th pair of chromosomes, in L.bata, they were on the 9th pair.

Chromosome banding studies are a step nearer to accuracy in enabling the identification of species and also homologous pairs in karyotypes. Similarly, banding of NORs also helps in this regard, as these are present on specific chromosomes, it makes relatively easy to distinguish species with similar karyotypes. (Sola et al., 1984). The study also may help in identifying the parentage of putative hybrids that are sometimes found in nature.

The chromosome banding work particularly in fishes as a whole is very scanty. Of the 20,000 known species of fish in the world, banding studies appear to have been carried out in only 50 species. (Khud-Bukhsh and Tiwari, 1994). The reasons for this were many, of which the most important ones seem to be relatively smaller size and greater number of chromosome in fishes. Absence of proper and repeatable methodologies also appear to be one of the constraints in the progress of fish chromosome banding studies.

Regarding Indian carps, the main hurdle seems to be lack of adequate facilities for the workers especially in the Universities where such studies are generally carried out, to handle relatively bigger species like major carps. Indian major carps, unlike other small aquarium fishes, need farm (pond) facilities for handling, which are often not possible/available in the universities. As a result these researchers mostly concentrate on other commercially important catfishes or air-breathing fishes. Most of the fish cytological work, particularly in India is confined to such species of fish referred above as it is relatively easy to handle them. The advent of molecular level of research in genetics also appears to have diverted the attention and enthusiasm of many young and new researchers in genetics away from chromosome studies.

However, recently many of the research institutes are gradually taking up basic and academic research besides applied aspects. More and more laboratories are now concentrating on carp genetics research, especially pertaining to cytogenetics, biochemical and molecular genetics and also selection studies, not only in India but also in its neighbouring countries.