2. GENETIC RESOURCES OF INDIAN MAJOR CARPS, THEIR DISTRIBUTION AND CHARACTERIZATION

2.1 Genetic resources of Indian major carps

The natural genetic resources of Indian major carps come from the network of the Ganga river system, the Sindh and the Brahmaputra river systems in the north and the east-coast and west coast river systems flowing through in the south and central India respectively. The major river system of India have been given in Fig. 1.

2.1.1 About Indian major carps

The major carps of India fall under three genera, Catla, Labeo and Cirrhinus. Under the genus Catla, the species C. catla, under the genus Labeo fall the species L. rohita, L. calbasu, L. fimbriatus, L. bata, L. gonius, and under the genus Cirrhinus fall the species Cirrhinus mrigala, C. reba, C. cirrhosa. Interspecific hybridization work has been carried out between the species of the genus Labeo and intergeneric hybridization between the species of the above mention three genera.

Due to their fast growing nature and taste, Indian major carps enjoy a prime position in the Indian aquaculture scenario. These highly prized fishes though originally inhabitants of the Ganga river network in North India and the rivers of Pakistan, Bangladesh, Nepal and Burma, are also transplanted into other rivers in central as well as peninsular India. Besides this, these carps have been imported by many other countries such as Thailand, Vietnam, Laos (Welcomme, 1988).

Catla feeds mainly on Zooplankton at the upper zone of the water body and rohu mostly feeds on periphytonic (phytoplankton) forms found attached to submerged vegetation and other objects occupying the water column, while mrigal and kalbasu feed at the bottom on bottom biota such as tubifex and other blood worms. Thus these carps have mutually compatible and complimentary food habits. Major carps are shown in Plate 1.

Taking advantage of the mutually compatible and complimentary food habits of these carps, the present day intensive and extensive polyspecies or multispecies culture has been developed from 1970s, which is popularly known as the composite carp culture. This technology has developed and has demonstrated a gradual increase in the fish production from the culture systems from 3–4 t/ha/year to 10–15 t/ha/year. (Chaudhuri et al., 1974; 1975; 1978 and Tripathi et al., 1997).

These technologies are mainly based on manipulation of species ratios to exploit the available natural food in all the niches of the culture medium and supplementary feeding and manuring. Other management measures such as water replenishment, health monitoring etc. are also part of the package.

Carp culture practice which was hitherto confined mostly to the eastern region of the country has become popular almost all over the country with the development of modern aquaculture technologies. Consequently the demand for seed of major carps gradually started increasing. With the increase in the demand, the hypophyzation technique has spread all over the subcontinent and became popular among many fish farmers. As a result the production of seed has increased.

With the epoch making success in inducing these carps to breed through hypophyzation during late 1950s (Chaudhuri and Alikunih, 1957), the dependence on riverine sources for the seed of these carps has been gradually reduced. Now, hypophyzation, has been adopted by many of the rural fish farmers to produce the seed in adequate quantities.

However, in the absence of proper breeding plans, this has led to a gradual decline in the genetic quality of the seed. Consequently the negative effect of inbreeding started appearing gradually with the characteristic poor survival and slow growth, besides disease susceptibility of the hatchery produced seed (Ibrahim et al., 1982). All this suggests that it is an appropriate time to act seriously about the genetic improvement of these carps.

Figure 1. Major rivers of India (Jhingran, 1984)

2.1.2 Spawning behaviour of Indian major carps

All Indian major carps naturally breed in rivers. They also breed in artificially created water bodies which are popularly known as “bundhs”. However, Indian major carps though attain maturity in confined or stagnant (pond) waters, do not breed there and need inducement for spawning.

2.1.2.1 Natural spawning

Natural spawning of major carps usually coincides with the South-west monsoon in North-Eastern India and Bangladesh and lasts from May to August. In North India and Pakistan, from June to September. In the southern parts of India the spawning season appears to be variable (Jhingran, 1968; Khan and Jhingran, 1975; and Jhingran and Khan, 1979). According to Natarajan and Jhingran (1963), catla breeds only once in a year, as do other Indian major carps under natural conditions. However, recently catla and other species of Indian major carps have been bred more than four times in a season/year under controlled conditions (Gupta et al., 1995).

Regarding the requirements for spawning and the nature of spawning grounds, the observations of various workers differ slightly. Some are of the view that shallow areas which are inundated by heavy monsoon floods form the breeding grounds of these fish (Hora 1945, Hussain 1945 and Khanna 1958). Others viewed that it is only the availability of shallow spawning grounds that is the important factor for spawning (Khan, 1947; Ganapati and Alikunhi, 1950).

Again regarding the nature of the spawning ground, observations differ. In rivers Godavari, Krishna and Cauvery in South India, sections having large submerged rocks or emergent vegetation form the spawning grounds for catla (Chacko, 1946; Chacko and Kuriyan, 1948a; and Menon et al., 1959). Job and Chacko (1948) described as many as 22 spawning grounds. Spawning of major carps was reported over hard and sandy beds and also on rocky submerged embankments of rivers, reservoirs and “bundhs” in Madhya Pradesh (Dubey and Tuli, 1961).

Figure 2. Spawn collection net (from K.K. Ghosh, 1972)

Net for collecting spawn from the rivers.
(Note: the broaden end (mouth) of the net facing towards the flow of the river indicated with arrows)

Riverine fish seed is collected with the help of specially designed, funnel shaped nets (Jhingran, 1968). It consists of two parts, i.e. the net proper and a detachable tail piece locally called as “gamcha” which is fixed to the cod end. The rear of the net tapers to the narrow end. The net is fixed with the broader (mouth) end facing the water current. The spawn/fry are collected at the tail piece. Usually a number of such nets are fixed, depending on the favorable conditions. Figure 2.

2.1.2.2 “Bundh” breeding

Bundhs are special type of impoundments which are mostly found in the states of West Bengal, Madhya Pradesh, Rajasthan and Bihar. These are of two types, Perennial (wet bundh) and seasonal (dry bundhs). A typical wet-bund is usually located in the slope of a vast catchment area of undulating terrain, having proper embankments and with an inlet towards the upland and an outlet on the opposite side. It has a deeper and vast shallow area which dries up in summer. The deeper portion of the bundh retains water during summer where an adequate stock of carps is maintained. During monsoon when there is a heavy shower, the rain water from the catchment area rushes through the streamlets and submerges a major portion of the bundh while the excess water flows out through the outlet.

On the other hand a “dry-bundh” is like a seasonal pond and much smaller and shallower than the wet-bundh. For most part of the season the dry-bundh remains dry. When sufficient water accumulates with the first showers of monsoon rains brood of mature carp are released into it for acclimatization. Later when the bundh gets flooded with the next heavy showers, the fishes start spawning and the eggs are collected by means of mosquito net cloth and incubated in hatching hapas, which are made up of thin cloth.

Sometimes a few of the female and male brood fish are injected with carp pituitary hormones at a nominal dose of about 5–10 mg/kg body weight of the fish and released along with uninjected brood fish. Both fish that received the hormones and those that have not will also spawn.

2.1.2.3 Controlled breeding

In early efforts to induce spawning in the Indian major carps, crude extracts of pituitary gland preferably of the same species (homoplastic glands) were used.

The pituitary hormones are injected either intramuscularly or interperitonially. Depending on the condition of brood fish and the prevailing weather, the total dose of pituitary extract usually ranges from 5 to 22 mg per kilogram body weight in the case of female fish, which is administered in two installments with a six-hour interval. The quantity of first dose is nearly one third of the total dose. The males are given only one dose ranging from 2–3 mg of pituitary per kilogram body weight, at the time of injecting the second dose to the female fish. The dose of pituitary hormones however, appears to be species specific. It also depends on the maturity status particularly in the case of the female brood fish.

In the recent past, due to shortage of quality pituitary glands and the problems in the maintenance of donor fish, many substitutes have come into use to replace pituitary hormones, such as Human Chorionic Gonadotropin (HCG), Salmon gonadotropin (partially purified), Luteinizing Hormone-Releasing Hormone (LHRH) and its Analogues, Gonadotropin Releasing Hormone (Gn RH) etc. However, presently a formula of S-GnRH and domperidone which is marketed in the name of OVAPRIM, is much in use to induce the major carps to breed. OVAPRIM is used mostly for female brood fish @ 0.3–0.4 ml/kg body weight of the fish in a single dose.

With pituitary extracts the female fish will be ovulating within 5–6 hours after receiving the second dose, whereas OVAPRIM takes 6–7 hours to induce ovulation. The release of milt by the males usually synchronized with the ovulation of females. For genetic experiments, stripping procedure is generally followed.

2.2 Distribution

The Indian major carps which are also known as the gangetic carps are the natural inhabitants of the Ganga river network, namely the Ganga, the Gomati, the Yamuna and the Brahmaputra and the Indus river system in North India.

The Ganga river system running to a total length of about 8,047 km, besides the major carps, harbours the richest freshwater fauna of India, ranging from mahaseers and the torrential fishes of hills to a wide array of other fishes of great commercial value.

The Brahmaputra river system having a length of 4023 km consists of rich fish fauna of torrential streams in its upper reaches, however the fish are not of much economic value. Its middle and lower reaches however, have several species of carps, catfishes and other commercially important species of fish.

Regarding the Indus system, only part of a small segment is in India, the rest being in Pakistan. The important rivers of this system are the Beas and the Sutlej. These rivers harbour not only the indigenous carps in the lower sections but also the exotic rainbow and brown trouts in their upper reaches.

The main Rivers of the East coast river system in the peninsular India are the Mahanadi, the Godavari, the Krishna and the Cauvery. The Mahanadi naturally has all the species of Indian major carps, which are common in the Ganga system. Rivers Godavari, Krishna and Cauvery have their own several indigenous carp species, which are referred as minor carps such as Labeo bata, L. gonius, L. fimbriatus, Cirrhinus reba etc. However, the major carps have been repeatedly transplanted into these rivers and have become established in them, contributing significantly to the fish fauna of these rivers.

The major rivers of the West coast river system are the Narmada and Tapti. Their fish fauna consists of carps, catfishes, mahaseers, murrels, purches, prawns etc.

Distribution of major carp resources also includes that part of the Indus river system which falls into Pakistan after its formation and tributaries of Brahmaputra flowing through Bangladesh.

2.2.1 Distribution of Catla catla (catla)

Jhingran (1968) described the distribution of Catla catla, which starts from the Ganga river network in the north to the Krishna river down south of India, Pakistan, Bangladesh and Burma. It is also found in Nepal (De witt, 1960 as referred by Jhingran, 1968).

The fingerlings of catla, introduced into the Cuddapah-Kurnool canal from river Godavari in 1909 found their way into river Penna and the connected waters in the Nellore district of the state of Andhra Pradesh. Catla fingerlings were also introduced in Cavery river during 1920s and subsequently into the Bhavani (Hornel, 1924). Later the species was introduced into Periyar lake, Powai lake (Chacko, 1948 and Goldschmilt, 1953). The species has become widely established (Fig.3).

Jhingran (1968) has also mentioned that catla fingerlings have been also exported to Israel in 1954 and to Japan, and Mauritius in the 1960s. According to FAO Database on Introduction Aquatic Species (DIAS) (www.FAO.Org.) catla has been distributed to other countries also such as Zimbabwe, Israel, Bhutan, Philippines, Former USSR, Japan, Sri Lanka, Laos, Pakistan, Malaysia, Thailand, Vietnam and Mauritius.

2.2.2 Distribution of Labeo rohita (rohu)

The main source of literature about the distribution of rohu is Day (1878 and 1889). He reported that the species is distributed in freshwaters of Sindh and Punjab (Pakistan), through India, Assam, Bangladesh and Burma. He also stated that rohu is not found in Madras (South India).

Later studies of other workers (Setna and Kulkarni, 1949) and Alikunhi and Chaudhuri, 1951 and Mishra, 1959) have mentioned the occurrence of this species in many other places, viz., Sabarmati drainage, in the rivers Narmada, Tapti, Godavari, Mahanadi etc. though it is more common in plains of North India.

Its distribution has been also mentioned in other neighbouring countries such as Bangladesh, Burma and Nepal. Khan and Jhingran (1975) have given other details on the distribution of rohu through transplantation. However, according to FAO DIAS(19) besides the above mentioned countries the distribution of rohu has also extended to the Philippines, Former USSR, Japan, Sri Lanka, Laos, Pakistan, Malaysia, Thailand, Vietnam, Madagascar, China and Mauritius (Fig.4).

2.2.3 Distribution of Cirrhinus mrigala (mrigal)

The earliest work on distribution of mrigal appears to be Day (1878 & 1889). According to him mrigal inhabits rivers and tanks (water bodies much larger than ponds) in Bengal (undivided which includes Bangladesh) Deccan, North-West provinces, Punjab, Sindh (Pakistan), Cutch (partly in Pakistan and partly in Gujarat, Rajasthan provinces of India) and Burma. Later workers (Alikunhi, 1948 and 1957, and Mishra, 1959) recorded its distribution in the major river systems of India, including river Godavari in the south.

In addition, Mishra (1959) also mentioned about its distribution in Pakistan, Bangladesh and Burma. The species was also recorded from Nepal (De Witt, 1960). According to FAO DIAS (19) besides the above countries, mrigal has also introduced to Bhutan, Philippines, Former USSR, Japan, Sri Lanka, Laos, Malaysia, Thailand, Vietnam and Mauritius (Figure 5).

Figure 3. Geographical distribution of Catla catla. Crosses indicate areas where the fish has been successfully transplanted. From : Jhingran, 1968 (with FAO's permission)

Figure 4. Geographical distribution of Labeo rohita. From Khan and Jhingran, 1975 (with FAO's permission)

Figure 5. Geographical distribution of Cirrhinus mrigala. From Jhingran and Khan, 1979 (with FAO's permission)

2.2.4 Distribution of Labeo calbasu

Like the other three major carp species. L. calbasu also enjoys wider distribution in almost all those countries namely, Pakistan, India, Bangladesh, Burma, Thailand and also in Yamuna (South China). Day, 1878 mentioned that L. calbasu is distributed in Punjab, Sindh, Cutch Deccan, South India and Malabar, from Krishna through Orissa, Bengal and Burma. In India, according to Jhingran (1984) the species is found in streams of Punjab, West Bengal, Orissa and parts of South India. It is more abundant in rivers, above tidal reaches and lakes.

It is an important food fish and at several places it is referred as “lake rohu”. It is an important game fish too in tanks. It has been reported that L. calbasu thrives better in tanks and lakes than in running waters (Talwar and Jhingran, 1991).

Distribution pattern of catla, rohu and mrigal in rivers of different countries has been shown in Tables 1, 2 and 3 respectively.

2.3 Major carps in reservoirs

Reservoir here refers to the impoundments formed as a result of erecting dam on a particular river. There are a number of such reservoirs in India.

Srivastava et al. (1985), as referred by Sugunan (1995) have compiled a list of 975 large and medium reservoirs in India, with an estimated area of 1.7 million ha. These reservoirs have major carps as part of their ichthyofauna. All the species of major carps do not seem to thrive equally well in all the reservoirs. Again, there may be fluctuations in the abundance of the same species in the same reservoir.

In Stanley reservoir, in the state of Tamil Nadu, catla was dominating in the fish landings during 1960–'65, but it was reduced to 10% during 1969–'70 to 1972–73. Mrigal, introduced into the Stanley reservoir during 1950–'51, first appeared in the catches during 1957–'58 and contributed over 13% of the catch in 1966–67. However, later catch declined to insignificance. Rohu also showed some initial increase but later almost followed mrigal (Sugunan, 1995). The trend in most of the reservoirs more or less appears the same. The reasons for the dominance of a particular species at one time and the decline of the same species later seem to be due to recruitment failure from the water level fluctuations and predator pressure (Sreenivasan, 1966). Moreover, other aspects like the availability and supply of the required natural food of a particular species may play a role in the establishment and thriving of the species in reservoirs.

Table 1. Distribution of Catla catla in rivers and lakes

Only such rivers have been mentioned from where mrigal has been especially reported in ichthyological literature.
From:Khan and Jhingran, 1979 (with FAO'S permission).

Table 2. Distribution of Labeo rohita in rivers and lakes

Only such rivers have been mentioned from where rohu has been especially reported in ichthyological literature.
From: Khan and Jhingran, 1975 (with FAO's permission).

Table 3. Distribution of cirrhinus mrigala in rivers and lakes

Only such rivers have been mentioned from where mrigal has been especially reported in ichthyological literature.
From: Khan and Jhingran. 1979 (with FAO's permission).

However, with careful management and monitoring, reservoirs in India may form a potential resource for not only major carps but also many other commercially important species.

2.4 Genetic characterization of major carps

Application of genetics in fish farming is relatively of recent origin. Until recently no attempts have been made to genetically characterize any of the major carp species. No reports are available on whether there exist genetically different populations among the members of a given species of major carps in any of the major river systems mentioned earlier. The main reasons for this seem to be prioritization of problem/tasks in the fishery sector. As mentioned earlier, the priorities in fisheries, not only in India but also in other developing countries in Asia were different, ever since systematic research has been initiated. The need was to identify the economically important species of fish and study their biology and methods of propagation under controlled conditions. Once this was established, the fishery workers were busy in developing, technologies for carp/fish farming systems to boost the production. Except for the work on hybridization, no importance was given to any aspects of genetics what so ever, during these years.

The composite or multispecies culture technologies so far developed are based on species manipulation and application of certain management practices. These technologies, no doubt have boosted the fishculture in India several folds. However, at present it is felt that any further improvement in fish production may not be possible with these technologies and the researcher gradually started realizing the importance of other aspects such as genetic quality and improvement of the candidate species by fully exploiting their hitherto untapped genetic potentials. The urgency of acquiring knowledge about the existence of different fish stocks/populations among the different species of the economically most important fish (carps) is now felt. Lack of appropriate and accurate methodologies for genetic characterization and identification at population species level has also been one of the main constraints to full use of genetic resources. The various methods available earlier, much before the advent of biochemical and molecular (DNA) techniques for stock identification or to study the existence of different populations in a given species were only the morphometric measurements and meristic counts. These methods however, do not provide the degree of polymorphism distinguishable by modern methods especially within species.

According to Jhingran (1968) no distinct races or varieties of catla are known. This author has also given an account of the morphological traits of catla as described by different authors. However, Sinha and Khan (1989) mentioned that in Rihand Dam (Madhya Pradesh), India, the occurrence of three intra-specific populations within catla, each of which can be distinguishable morphologically by short, medium and long pectorals and also by specific ecological roles.

Khan and Jhingran (1975) mentioned that Khan (1972) conducted some studies on rohu collected from two different environments, moats, representing lentic environments and from the rivers Ganga and Yamuna representing lotic environments. The analysis of the data has shown significant differences in each of the characters suggesting that the fishes of moats and rivers belonged to independent stocks. Except for these sporadic reports which are mainly based on morphological and other phenotypic characters, no other reports are available regarding the existence of different populations or races among the species of Indian major carps.

Later developments include the cytogenetic, biochemical and molecular genetic methods. Among these, the biochemical genetic techniques, especially the use of isozyme electrophoretic markers, were in extensive use in fishes, through the 1960s and 1970s. However, since isozyme pattern cannot detect point mutations and conservative amino acid substitutions, they do not reveal all of the genetic variation actually present (Padhi and Mandal, 1995).

At the National Bureau of Fish Genetic Resources (NBFGR) at Lucknow (India) a number of species including major carps have been cytogenetically investigated (Ponniah, 1997). Indian major carps have the same diploid number of 50 chromosomes and no intraspecific variation in chromosomal number has been detected. Similarly, no intraspecific Nuclear Organizing Region NOR variation have been observed in both riverine and hatchery populations of L. rohita.

A relatively recent advancement made in the area of molecular genetics through developing molecular DNA markers has provided more accuracy for the identification of fish stocks in the form of restriction fragment length polymorphism (RFLP) of mitochondrial DNA (mt DNA) and nuclear DNA. Genetic polymorphism in nuclear DNA can also be detected by using random olegonucleotide primer for polymerase chain reaction PCR amplification followed by gel electrophoresis. This is known as random amplified polymorphic DNA (RAPD), which can be applied to genetic stock identification of quantitative traits loci (QTL) (Padhi and Mandal, 1995a). These techniques are very sophisticated and involve high cost infrastructure, which is often not within the reach of many developing countries. This appears to be one of the main reasons why the studies on stock identification in fishes of many developing areas have not been carried out on full scale until recently. However, use of the DNA technology is gaining momentum since 1990s. Many laboratories and institutions have come up in fisheries and are involved in research in this direction.

2.4.1 Application of biochemical and molecular genetic techniques

Information on some recent investigations with regard to biochemical and molecular genetic aspects on Indian major carps is discussed below.

2.4.1.1 Biochemical genetic studies

Biochemical genetic studies on Indian major carps through electrophoretic analysis of isozymes was initiated around the mid to late 1980s. These were mainly on the enzyme lactate dehydrogenase (LDH) patterns in developing embryos and in eye lens and red cell haemolysates (Padhi and Khuda-Bukhsh, 1989a,b ), Ldh-C gene expression in liver tissue (Rao et al., 1989), malate dehydrogenose MDH pattern in red cell haemolysate and similar electrophoretic patterns in muscle proteins (Padhi and Khud-Bukhsh, 1989b). These studies have shown that the three Indian major carps species namely Catla catla, Labeo rohita and Cirrhinus mrigala, in many of their biochemical characters were found to be very similar.

Earlier to this, Sahoo (1987) worked on the plasma, haemoglobin and transferrin pattern of two Indian carp hybrids; one produced by crossing male L. rohita with female C. catla and the other between male L. rohita with female Cirrhinus mrigala. All the plasma protein bands of both the parents L. rohita and Catla catla were expressed in their hybrid.

Regarding the haemoglobin pattern the hybrid between L. rohita and C. catla exhibited a broad intensely stained band which was similar to its parents. Similarly the hybrid between L. rohita and Cirrhinus mrigala also had a single band closely similar to parents.

With regard to transferrin bands, L. rohita has two and C. catla has one but their hybrid has inherited the extra band also from L. rohita. The hybrid of male rohu and female mrigal has shown two transferrin bands as in both the parents. Thus the pattern of plasma, haemoglobin and transferrin bands in the parent species were inherited by their respective hybrids.

Rao et al., (1989) have studied lactate dehydrogenase isozymes and taxonomic significance of Ldh C-gene in fifty two species of Teleostean fishes including Indian major carps. Liver specific Ldh C-gene expression was observed in C. catla, Cirrhinus mrigala, L. rohita and L. calbasu, also in many other species including Chinese carps, belonging to family Cyprinidae. Thus, members of the order cypiniformes show a dominance of liver-specific C-gene expression.

Padhi and Khuda-Bukhsh (1989a) studied the lactate dehydrogenase isozyme patterns in developing stages of four species of Indian carps. They found that usually the LDH gene expression during development tends to be similar in closely related species while the pattern may be dissimilar in more distantly related forms. It thus helps to understand the degree of evolutionary kinship among related organisms. The LDH banding latterns in the three species of major carps viz. C. catla, Cirrhinus mrigala and L. rohita was observed to be very similar and all of them exhibited persistent pattern of three bands in all the developmental stages studied This indicates their genetic closeness (Padhi and Khuda-Bukhsh, 1989a).

Padhi and Khud-Bukhsh (1989b) have also studied malate dehydrogenase isozyme patterns during the development of five species of carps namely C. catla, L. rohita, L. bata, Cirrhinus mrigala and Ctenopharyngodon idella. Though significant changes in MDH pattern were marked during hatching, in the case of C. catla in the form of additional bands, no such MDH activity was apparent in the other species. The MDH patterns however, tended to fluctuate at different pre and post hatching stages in these carps, possibly reflecting differential gene expression (Padhi and Khuda-Bukhsh, 1989b). The number of MDH bands expressed during development, ranged between 1–6 in C. catla, 3–4 in L. rohita, 2–5 in Cirrhinus mrigala. While the number of MDH bands in L. rohita did not fluctuate much during pre-hatching stages, differential expression of MDH bands was noticed in other species.

To explain the genetic basis of expression, specific antisera and other confirmatory studies are required. Xanthine dehydrogenase isozymes (XDH) and its distribution in different tissues namely skeletal muscle, heart, liver, eye, kidney and brain and its taxonomic significance have been studied in C. catla, Cirrhinus mrigala, L. rohita and L. calbasu (Padhi and Khuda-Bukhsh, 1990a). In this study single band XDH phenotype was expressed uniformly in all the six tissues studied in C. catla. In Cirrhinus mrigala and L. rohita it was expressed in most of the tissues. However, in the case of C. calbasu and also L. rohita multiple XDH phenotypes showing four or five bands were also encountered in certain tissues.

In another study, Padhi and Khuda-Bukhsh (1990a) have found through simple gel electrophoretic study of the surface mucus of catla, rohu and mrigal, conspicuous differences which can differentiate the three species. The authors are of the opinion that a critical analysis of components of surface mucus may also be usefully exploited in population/stock identification of carps. The surface mucus proteins were expressed as seven bands in C. catal, three bands in L. rohita and five electrophoretically analysable components in Cirrhinus mrigala (Padhi and Khuda-Bukhsh, 1990a).

Basu et al., (1992) have studied the LDH isozymes in embryonic and post embryonic stages of L. rohita, Cirrhinus mrigala and C. catla, using starch gel electrophoresis. These studies have shown species specific and differential enzyme locus (gene) expression pattern in LDH up to 18th hour of study. The isozymes seemed to be completely active, 36th hour after fertilization and showed electrophoretic patterns very similar to those of the adults. It was also mentioned that comparative analysis of isozymes of the three species has also enabled species identification even during the embryonic stages which other wise would be impossible to identify on the basis of morphological characters only (Basu et al., 1992).

Other studies in the area of biochemical genetics pertaining to Indian major carps are gel electrophoretic patterns of transferrin in the four Indian major carp species, C. catla, Cirrhinus mrigala, L. rohita and L. calbasu along with 30 other freshwater species of fish. While only one transferrin band was observed in L. calbasu, in C. catla, Cirrhinus mrigala and L. rohita expression of both one band and two band phenotypes were noticed (Barat and Khud-Bukhsh, 1992). Sahoo (1993) studied the plasma, haemoglobin and transferrin pattern in two intergeneric carp hybrids. The hybrids between male L. rohita x female C. catla and male L. rohita x female Cirrhinus mrigala have shown a haemoglobin pattern more or less similar to their respective parent species. However, the plasma band profile of the hybrids has shown a mixture of bands of the parent species. Again, the pattern of transferrin bands in the hybrids though resemble the parent species in the number and staining intensity, differed in mobility (Sahoo, 1993).

Isozymes studies have also been made to distinguish diploids and tetraploids. Sarangi and Mandal (1996) have made some isozyme studies in one of the Indian major carps namely L. rohita to distinguish diploid individuals from tetraploids. In general, intensification of isozyme bands has been observed in tetraploids. The polymorphism in (G6 PD-1 and 2) loci in eye lens, G6 PD-2 in kidney and G6 PD-1 in skeletal muscles and an esterase (EST-1) locus in kidney could be used as reliable marker in identifying tetraploids from diploids (Sarangi and Mandal, 1996).

Chaudhuri and Gopala Krishna (1998) studied tissue specificity and degree of polymorphism of five enzymes systems in L. rohita from river Yamuna namely Aspartate transaminase AAT (E.C.2.6.2.2), Malate dehydrogenase MDH (E.C.1.1.1.3.7), Lactate dehydrogenase LDH (E.C.1.1.1.2.7), Malic enzyme (ME) and Glucose Phosphate Isomerase GPI (E.C.5.3.1.9) enzyme in liver, muscles, heart and brain tissues. It was reported that MDH, ME and GPI had two loci each in all tissues while LDH had trace loci with one specific to the liver. AAT with two alleles was the only polymorphic locus among the five enzymes studied.

Gel electrophoretic studies have been also carried out in over thirty five species of Indian teleostean fishes, which include major carps, in relation to plasma protein profiles (Khuda-Bukhsh and Sahoo, 1998). The plasma protein profile of C. catla revealed 9 bands, including a cluster of 4 linear bands near the origin. The number of bands in the cluster sometimes varied from 4–6. In Cirrhinus mrigala the electropherogram of plasma protein revealed 10 bands, including the cluster of bands near origin which generally contained 5 linear bands but sometimes 6–7 bands were also appeared in the cluster. In the case of Labeo calbasu and L. rohita 10 bands were observed in each case. (Khuda-Bhukhsh and Sahoo, 1998).

2.4.1.2 Molecular genetics studies

Methods for the rapid isolation of mitochondrial DNA (mt DNA) from fish have been developed (Padhi and Mandal, 1993), followed by mt DNA RFLP investigation studies for genetic stock identification (Padhi and Mandal, 1995), DNA fingerprinting (Majumdar et al., 1997) and characterization of Mbol satellites (Padhi et al., 1998) have been also carried out in major carps. It has been reported that mt DNA technique enables effective yield of good quantity of mt DNA without the use of ultracentrifugation. The authors claim that this method would be useful for preparing large number of mt DNA samples for studies pertaining to genetic stock differentiation, evolutionary analysis of related taxa and identification of maternity of the naturally occurring fish hybrids. The fish species C. catla, L. rohita, L. bata and hybrids of C. catla X L. rohita were used for this study.

The feasibility of actual application of mt DNA RFLP analysis methods have been tested by Padhi and Mandal (1995b) in Indian major carps for genetic stock identification by using C. catla, L. rohita and L. calbasu from both riverine (river Ganga) and farm sources. Restriction endonucleage analysis of mitochondrial DNA in L. rohita from the river Ganga and hatchery maintained (farm) population revealed polymorphism at the Hind III restriction site. The mt DNA genome size in rohu has been reported to be about 17.8 kb. Padhi and Mandal (1995b) also reported that C. catla and L. calbasu, collected from both farm and riverine sources, when compared pairwise, did not reveal polymorphism at the Hind III site within each species. The restriction pattern with respect to Hind III site appears to be species specific in catla, rohu and kalbasu. Further study as suggested by the authors would be useful for stock identification and genetic diversity documentation.

Nuclear DNA RFLP methods were employed to detect inadvertent hybridization in major carp hatcheries when “mixed spawning” of all species Indian major carps is practised (Padhi and Mandal, 1996). It was reported that when southern blots of genomic DNA of the mixed spawned individual offspring digested with EcoR 1 were probed with radiolabeled Xenopus r-DNA the hybrids of catla-mrigal and rohu-mrigal could be detected easily. However, in the case of catla-rohu hybrids caution had to be exercised as the fragment sizes of higher molecular weight diagnostic bands in these species did not vary much. It was also reported that ribosomal DNA fragments were inherited biparentally in catla-rohu, rohu-mrigal and catla-mrigal hybrids. An earlier study on ribosomal DNA RFLP indicated that catla, rohu and mrigal were polymorphic at the EcoR 1 site in ribosomal DNA.

Recently, Majumdar et al., (1997), Basavaraju et al., (1997) and Padhi et al., (1998) have reported on DNA fingerprints in L. rohita and C. catla by using Bkm 2 C (8) and M 13 multilocus probes. In L. rohita and C. catla, M 13 gave fewer bands, though the level of polymorphism was higher. This indicates the usefulness of M 13 as an effective DNA fingerprinting probe (Majumdar et al., 1997).

It was also mentioned that although Bkm 2 (8) and M 13 show similar hybridization patterns, they detect different alleles. M 13 gave only a few bands in Indian major carps which were reported to have clustered in 2–4 Kb size ranges when three different enzymes were used to generate such RFLPs, whereas Bkm 2 (8) detects a large number of bands in the size range of 2–20 Kb.

It was further reported that DNA fingerprinting analysis in L. rohita and C. catla showed similar patterns with the same probe and enzyme combination (Majumdar et al., 1997).

Basavaraju et al., (1997) developed and used four tetranucleotide microsatellite probes to differentiate between broods of catla from the key hatcheries namely, the Tunga Bhadra Dam (TBD), the Bhadra Reservoir Project (BRP) and the Kabini Reservoir Project (KRP) and from out of state wild and hatchery stocks. This analysis was effective in determining the overall levels of genetic variation between strains in terms of number of alleles and average heterozygosity and in detecting differentiation between strains.

These studies also revealed that despite having a common source, the hatchery populations in Karnataka had differentiated from each other as a result of genetic drift or unconscious selection. It was however, reported that there were significant differences in the number of alleles or in the average heterozygosity between the hatchery and wild caught stocks (Basavraju et al., 1997).

Characterization of Mbol satellites in Cirrhinus mrigala has been reported by Padhi et al., (1998). These workers claim that they have cloned and characterized a highly reiterated, tandamly repeated and A + T rich Mbol DNA fragment in Cirrhinus mrigala, with a monomer size of 266bP. It has been reported that in Cirrhinus mrigala the Mbol fragment is species-specific and absent in C. catla and L. rohita. They have also studied the inheritance of the Cirrhinus mrigala specific Mbol fragment in two intergeneric hybrids, Cirrhinus mrigala X L. rohita and Cirrhinus mrigala X C. catla, when the radiolabelled 32 fragment was hybridized to the Mbol-digested nuclear DNA of C. catla, Cirrhinus mrigala, L. rohita and the two hybrids on the southern blot, the Cirrhinus mrigala Mbol ladder was found in both the hybrids suggesting that the Mbol satellite in these hybrids was inherited uniparentally (Padhi et al., 1998).

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

Genetic characterization of the founder stocks of Labeo rohita from river Ganga, Gomati, Yamuna, Sutlej and river Brahmaputra and also the farm stock from CIFA, which are being used for selective breeding purpose is being carried out. Results of the preliminary investigations have indicated adequate genetic variability (mean heterozygosity 0.047 to 0.079) similar to wild stocks and no genetic contamination was detected (Kuldeep Lal et al., 1998, unpublished).

Until recent past particularly in India aspects like the geographical distribution and biodiversity of fishes were given priority to estimate the extent of distribution and availability or occurrence of different species. However, after realizing the important role of genetics, many a fishery researches too felt the need to exploit the genetic potentials of several commercially important species of fish. To achieve this, information on genetic diversity or divergence, both between and within species is essential. Until recently no attempt has been made with regard to Indian major carps in this direction. However, as discussed in this section, researchers in some of the Asian countries particularly in India have initiated genetic characterization of these carps by applying both biochemical and molecular genetic techniques. Most of these studies are an attempt based on isozyme pattern and some on mtDNA or DNA fingerprinting by using RAPD or RFLP techniques. These preliminary studies with regard to the genetic status of the hatchery and wild stocks, the expression of C-gene pattern in catla, rohu, mrigal and kalbasu, and the pattern of haemoglobin, plasma and transferrin in major carp hybrids and their respective parent species though provided some information, did not indicate any thing which can focus some light on the genetic diversity or divergence either between or within the major carp species.

Much is yet to be done to find out the genetic divergence of these economically most important Indian carps. As mentioned, some methodologies are now available to proceed further in characterization and differentiation, to find out whether there exist different populations/stocks/strains among and within the species and the extent of genetic diversity a variation of these carps. Now the research in this direction has been already initiated in India and is in progress.

2.5 Present status of major carp germplasm resource in relation to environmental changes and habitat modifications

It has been reported that there is considerable depletion in the fish populations of almost all economically important species, including major carps in all the rivers (Das & Barat, 1990). This appears to be mainly due to man made stresses by over fishing, more due to greed for profit making business than simply to make a living by subsistence fishing. Besides, there are also other factors like construction of dams and hydro-power plants etc. which prevent the fish from migrating to their natural breeding and feeding grounds. Pollution of aquatic environment mainly due to heavy industries is the concern of the day. The destruction of fish habitat by alterations of river systems, land development, increased water abstraction etc. is very clear (Jhingran, 1984). Mass mortality of fish in seas and rivers is not very uncommon these days. Consequently, several fish species are feared to be at various stages of threat, such as rare, vulnerable, endangered etc. (National Seminar on Endangered fishes of India, Allahabad, (UP), India, 25–26 April, 1992).

According to Ponniah (1997), rohu, catla and mrigal may not be under threat but they are prone to loss of genetic diversity and variability due to extinction of genetically distinct wild populations, escape and ranching of hatchery seed and competition from exotic carps.

Consequent to massive introduction of cultured fish, displacement of wild stocks has been reported and high levels of introgression of the altered hatchery fish genome into wild stock has also taken place. Escape of farmed fish and breeding with wild populations may result in decreased fitness of wild stock.

Another aspect, which may also pose a problem is the growing trend of replenishing the rivers, lakes and reservoirs with hatchery produced fish seed in many states in India (Mishra and Raman, 1993) and even in the neighbouring countries like Bangladesh. This should be given proper thought as most of the hatcheries produce seed without following any genetic resource management (Eknath and Doyle, 1985) and as such it may be disastrous from a genetic view point. Deviations brought about in the gene pool of survivors to that of the original populations effected by pollution will ultimately be reflected in future populations (FAO, 1980). There is likelyhood of genetic deterioration in populations which suffer drastic depletions. Population size is the single most important factor in sustaining a high level of genetic variation within a population (Frankel and Soule, 1981). Therefore, it is important to restore the size of populations whenever trends of decline are indicated. Das and Barat (1990) have dealt with fish conservation methods that are necessary following habitat degradation. Obviously, the various causative factors inducing stresses on fishes need to be eliminated or reduced as a first step. However, it appears that very little or nothing can be done with regard to alterations of river systems and increased water abstraction when the greater interest of the nation is put ahead of the interest of fish or local groups of fishers or farmers (Das and Barat, 1990).

In situ and ex-situ conservation methods have been also often suggested for the restoration of the original gene pool of the species in question. The major advantages of in situ conservation according to Das and Barat (1990) are i) continued coevolution in the wild which provides the breeder with a dynamic source of resistance to the changes in the environment is not possible in ex situ conservation ii) maintenance of a carp relative in situ permits the breeder to study its ecology and to obtain data that can assist in the selection of germplasm that might otherwise be over looked.

In view of the deteriorating aquatic environment, the first necessary step taken should be the conservation of the germplasm resources of at least all the economically important species either in in situ (in the natural environment). Ex-situ conservation (Cryopreservation and gene banking) can support in situ measures, but should not replace them (Pullin et al., 1991). This has been also stressed in many seminars/symposia or workshops during the past (Jhingran and Gupta, 1989; Nasar and Haque, 1989) and during the National Seminar on Animal Genetic Resources and their conservation, held at Karnal (Haryana State) India from 22–23 April, 1993.

Conservation of genetic stocks, both wild and cultivated species is important because their genetic diversity has been developed through thousands of years of evolutionary process (Swaminathan, 1984). Selected gene pools or genetic stocks having sufficient genetic diversity and their in situ preservation provides the genome the scope of acclimatization with the changes in the environment.

Ex situ method enables the preservation of genotypes in large quantities in an economical way without much management problems. However, this method may also have its disadvantages. For example, in the long run, genome preserved for several decades, when it is revived, may find the prevailing environment hostile. In the case of animals unless both male and female gamates or embryos could be cryopreserved, full advantage of ex-situ conservation can not be derived, without further genetic manipulations (Mc Andrew et al. 1993).

Both in situ and ex-situ conservation methods, no doubt have their advantages as well as disadvantages as discussed above. It may be possible to overcome such situations by following rather a cyclical approach through the alternate use of these two methods by intermittently reviving such ex-situ preserved germplasm and maintain them again in in situ and in the process to expose and acclimatize the genome to the prevailing environment.

The development of molecular genetics offers another novel and great potential method of preserving genetic materials. The genetic material of individuals having the most desirable and heritable traits can be preserved in the form of genomic libraries.