Role of Genetics in Fish Conservation and Aquaculture Development in India

W. S. Lakra, A. Gopalakrishnan* & V. S. Basheer*
National Bureau of Fish Genetic Resources (NBFGR), Lucknow 226 002, Uttar Pradesh, India. lakraws@hotmail.com *NBFGR Cochin Unit, CMFRI Campus, Kochi 682 018, Kerala agopalkochi@rediffmail.com
Abstract: Aquatic germplasm resources of India have enormous potential to contribute towards the economic wellbeing of the nation. The fisheries and aquaculture play a vital role in the social development by providing nutritional security and economic upliftment of fishers and fish farmers. Although, China and India are perhaps the traditional cradles of art of aquaculture, fish culture remained largely empirical artisanal in these countries till the recent past. But, modern genetic and biotechnological tools have been applied in India to boost up the fish production through aquaculture by upgrading the quality of cultivated fishes and fish seed; in the discovery and development of new natural resources; and for conservation and management of genetic diversity in natural fish stocks. The paper reviews the progress in research efforts applying modern genetic tools that are relevant to aquaculture and fisheries management in India. Keywords: Indian aquatic resources; Fish genetics; Genetic characterization; Molecular markers; Selective breeding; Milt cryopreservation; Chromosomal manipulations; Sex control.

Citation: Lakra, W. S., A. Gopalakrishnan & V. S. Basheer (2008) Role of genetics in fish conservation and aquaculture development in India. pp 10-37, In: Natarajan, P. et al. (Eds.) Glimpses of Aquatic Biodiversity, Rajiv Gandhi Chair Publication 7, 284p., Rajiv Gandhi Chair, Cochin University of Science & Technology, Kochi 682 022, Kerala, India

Introduction Over the last four decades the aquatic systems of the globe have undergone a rapid transition. Worldwide per capita fish consumption nearly doubled from about 8 kg in the early 1950s to about 15.8 kg in 1999. Fish exports from the developing countries have surpassed export of traditional crops in meat. In India, fishing activities in the pre-independent days used to be carried out at a subsistence level, almost exclusively by the traditional fishermen. Today, this sector has attained the status of a capital-intensive industry, warranting close monitoring and management for sustained development. Presently, the Indian fisheries sector accounts for an annual turnover of over Rs. 220 billion, which is 1.40 percent of total GDP and 4.6 per cent of agricultural GDP (Pillai & Katiha, 2004). The fisheries sector plays a vital role in the Indian economy, by addressing various issues like food and nutritional security, employment, livelihood support and socio-economic status and fishing communities. The sector also provides employment and income to over 5 million fishers and fish farmers, majority of whom live in over 3,600 coastal villages, besides hamlets along major river basins and reservoirs in the country (Pillai & Katiha, 2004). The fish production in India registered an excellent growth during the past half century and reached 6 million tones in 2002 from a meager 0.75 million tones in 1950. A study by the Food and Agriculture Organization (FAO) projects a global average per caput demand for all seafood is about 18.4 kg in 2010 and 19.1 kg in 2015. The study also highlighted that developing countries already produce and consume more fish than developed countries and it predicts that the dominance of developing countries will grow further to 2020 (Ayyappan & Pillai, 2005). India is blessed with huge inland water resources with 29,000 km of rivers, 0.3 million ha of estuaries, 0.9 million ha of backwaters and lagoons, 3.15 million ha of reservoirs, 0.2 million ha of floodplain wetlands and 0.72 million ha of upland lakes. It has been estimated that about 0.8 million tonnes of inland fish is contributed by different types of inland open water systems. Though 1

production break-up of these water-bodies is not available, it is believed that capture fisheries production from river and estuaries contribute only a small share of total inland catch, and bulk of the production comes from reservoirs and floodplain wetlands which are managed on the basis of culture-based fisheries or various other forms of enhancement. The 14 major rivers, 44 medium rivers and innumerable small rivers of the country with combined length of 29,000 km provide one of the richest fish faunistic resources (Table – 1) of the world. While production figures from different riverine systems are not available, estimates made for major rivers showed yield varying from 0.64 to 1.64 tonnes/km with an average of one tonne/km. Further, as per the available statistics, the average yield in different estuaries range from 45 to 75 kg/ha. The inland fisheries of India include both capture fisheries and aquaculture. Capture fisheries had been the major source of inland fish production till mid eighties. But, the fish production from natural waters like rivers, lakes, etc., declined primarily due to proliferation of water control structures, indiscriminate fishing and habitat degradation (Pillai & Katiha, 2004). The depleting resources, energy crisis and resultant high cost of fishing have led to an increased realization of the potential and versatility of aquaculture as a viable and cost-effective alternative to capture fisheries. During the past one and half decades, the inland aquaculture fish production has increased from 0.51 million tonnes in 1984-85 to 2.69 million tonnes in 2003-04, while for inland capture fisheries, the same has declined from over 0.59 million tonnes in 1984-85 to 0.33 million tonnes in 1994-95 and 0.5 million tonnes in 2003-04. The percentage share of aquaculture has increased sharply from 46.4 to 84.3 during this period. The total fish production from aquaculture sector in India increased from 0.512 million tonnes to 2.69 million tonnes. This could be achieved through a series of technologies developed in inland fisheries sector in general and in aquaculture, in particular. The share of inland fisheries sector, which was 29% in 1950-51, has gone up to over 50% of the total fish production at present. While production from marine sector has been solely contributed by capture fisheries, aquaculture contribution in inland fisheries sector has been significant in recent years. Among the countries bordering the Indian Ocean, India, endowed with a coastline of 8,120 km, 2.02 million km2 of Exclusive Economic Zone (EEZ) and 0.5 million km2 of continental shelf has a catchable annual marine fishery potential of 3.93 million tonnes and occupies a unique position. Besides, there are vast brackishwater spread areas all along the coastline, which offer ideal sites for sea farming and coastal mariculture. Among the Asian countries, India ranks second in culture and third in capture fisheries production, and is one of the leading nations in marine products export. The development of Indian marine fisheries from a traditional subsistence oriented one to industrial fisheries through Five Year Plans was phenomenal. However, the present scenario is characterized by over exploitation, declining yields from the inshore waters and increasing conflicts between different resource users, whereas the increasing demand for fish in domestic and export markets indicate good prospects for oceanic and deep sea fishing, large-scale sea-farming and coastal mariculture. The marine fish production in the country progressively increased from 0.58 million tonnes in 1950 to 2.73 million tonnes in 1997 showing an annual average growth rate of 6.4% over a period of 4 decades. It has reached a plateau since 1989, which is because the fishing effort is mainly concentrated in the 2-100m depth zone. The annual growth rate of marine fisheries since 1981 had been on the decline and during 1991-2000 it was only 1.9%. The annual average marine fish landing during 1999-2003 was stagnant around 2.55 million tonnes against an annual catchable potential yield of 3.93 million tonnes principally constituted by oil sardine, penaeid prawns, perches, ribbon-fishes, non-penaeid prawns, croakers, mackerel, carangids, anchovies, cephalopods and Bombay-duck.

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Table 1. Fish germplasm resources of India

Ecosystem
Coldwater Warm water Brackishwater Marine Total

Total species
157 608 113 1365 2243

From the gathering by hand picking of fish in ancient days, India has developed most modern fish harvesting methods from the wild. Aquaculture has also taken a long leap through innovations in technologies from wild hunting in ancient days. However, to meet the increasing demands of fish production through aquaculture, to upgrade the quality of cultivated species and for sustainable utilization and management of fish genetic resources of the country, application of modern genetic principles in fisheries research has become inevitable. Fish genetics has been broadly defined as application of genetic principles and methods for increasing aquaculture productivity by genetically modifying living aquatic organisms or part of these organisms and for management of wild fish populations to obtain maximum sustainable yield without affecting genetic diversity. There have been considerable advances in the range of aquatic organisms, or molecules derived from aquatic organisms, that have been applied to a variety of problems in the medical and environmental fields. During the last ten years, there has also been a revolution in molecular and genetic techniques by miniaturization and automation of techniques that has opened the doors for rapid, high throughput analyses of nucleic acids and proteins, leading to increased ability to assess which genes are expressed, and which protein pathways are active in a given sample. Application of modern genetic principles remained focussed on temperate and sub-tropical species of the Western countries during sixties, but genetic tools have been applied in India also to boost up the fish production through aquaculture as well as conservation of vast aquatic resources of the country for sustainable utilization. Valuable information about the genetic structure of wild and captive populations several Indian fishes and traits of interest to aquaculture are now becoming available and our knowledge of these areas should increase greatly over the next decade, as should the applications of genetics to the aquaculture and fisheries. A brief overview of the past achievements and new approaches and trends in the field of fish genetics in India is attempted in this paper. It begins with genetic characterization that can be used to provide basic genetic descriptions of Indian species and populations, as well as having applications in aquaculture genetics. Fish genomics and marker assisted selection are dealt with next. Details of selective breeding follow this. Transgenic fish research in India is the next topic. Hybridization studies; application of cryopreservation and tissue culture to fish genetics research; chromosome set manipulations (divided into three subsections - gynogenesis, androgenesis and ploidy manipulations); and finally sex determination and sex control complete the review. Genetic characterization The necessary ability to characterize individuals at a variety of gene loci has led to far greater information on the genetic stock structure of wild populations and this has found particular application in the field of fisheries. The primary objective of the genetic characterization is to assess the distribution and pattern of genetic variability at intra as well as inter-specific level populations, through the use of identified genetic markers. The first priority for such research is identification of appropriate genetic markers. Technological advances in molecular biology and biochemistry have led to the development of a variety of genetic markers that can be used to address questions of relevance to the management and conservation of fish species. Genetic markers are also used to understand the pattern of migration of fish stocks, nature of breeding populations and also in conservation and taxonomy/systematics – with varying degrees of success (Melamed et al., 2002). 3

Several markers types are highly popular in aquaculture/fisheries genetics. In the past, soluble proteins, gene products (allozymes) and mtDNA markers have been popular; more recent marker types that are finding service in this field include restriction fragment length polymorphism (RFLP), randomly amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), microsatellite, single nucleotide polymorphism (SNP), and expressed sequence tag (EST) markers. With the DNA markers, it is theoretically possible to observe and exploit genetic variation in the entire genome. The choice of markers is crucial in achieving precise information that is useful for desired application. DNA markers can also be used to test paternity in selection experiments. Selective breeding programmes based on family designs require the different families to be kept separately until the fry are big enough to be tagged. Identification of families by their specific DNA fingerprints allows the families to be kept together from fertilization in breeding facility. This will also eliminate the problems related to common environmental effects. The development of DNA profiling techniques for family identification can reduce the problem of the introduction of environmental effects common to full sibs. Molecular markers can be classified into type I and type II markers from the point of view of functional genomics. Type I markers are associated with genes of known function, while type II markers are associated with anonymous genomic segments. Under this classification, allozyme markers are type I markers because the protein they encode have known function. RAPD markers are type II markers because RAPD bands are amplified from anonymous genomic regions via the polymerase chain reaction (PCR). Microsatellite markers are type II markers unless they are associated with genes of known function. The significance of type I markers is becoming extremely important for aquaculture genetics. Type I markers serve as a bridge for comparison and transfer of genomic information from a map-rich species into a relatively map-poor species (Liu & Cordes, 2004). In general, type II markers such as RAPDs, microsatellites, and AFLPs are considered noncoding and selectively neutral. The usefulness of molecular markers can be measured based on their polymorphic information content (PIC, Botstein et al., 1980). PIC refers to the value of a marker for detecting polymorphism in a population. PIC depends on the number of detectable alleles and the distribution of their frequencies, and equals 1 minus the sum of the square of all allele frequencies. The greater the number of alleles, the greater the PIC; and for a given number of alleles, the more equal the allele frequencies, the greater the PIC. In India, intra-specific level variation has been assessed in finfishes and shellfishes within and between populations that provided information on stock structure of the prioritized endangered and commercial fish species. This is vital for planning stock-specific, propagation assisted rehabilitation of endangered fishes and genetic improvement of cultivable species. The National Bureau of Fish Genetic Resources (NBFGR), Lucknow, Central Marine Fisheries Research Institute (CMFRI), Central Institute of Freshwater Aquaculture (CIFA), Bhubaneswar, Central Institute of Fisheries Education (CIFE), Mumbai and the National Institute of Oceanography (NIO), Goa are the leading institutions in India carrying out research in this field. A concerted effort made at NBFGR, Lucknow has provided description of genetic variation and population structure for twelve prioritized fish species from their major range of natural distribution. These species include Tor putitora, Catla catla, Cirrhinus mrigala, Labeo rohita, L. dero, L. dyocheilus, L. dussumieri, Chitala chitala, Tenualosa ilisha, Puntius denisonii, Horabagrus brachysoma, Gonoproktopterus curmuca and Etroplus suratenesis. The study covered wide geographical area and used microsatellites, allozyme & RAPD markers. Distinct population structure was observed in many of these species indicating that propagation assisted restoration programmes must be stock-specific to replenish declining populations (Punia et al., 2005; Lal et al., 2004 a, b; Mohindra et al., 2001, 2004, 2005; Gopalakrishnan et al., 2006, 2004; Singh et al., 2004). The study also provided clear evidence that natural populations of species like Puntius denisonii are facing genetic bottleneck or reduction in their effective population size (Lakra, et al., 2006 c). Alterations in genetic make or genetic bottlenecks take time before negative impacts are visible. However, data from molecular markers can provide an

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early warning signal regarding occurrence of such bottlenecks as a result of violation of certain principles that natural populations are expected to follow (Lakra, et al., 2006 c). Work on marine ecosystem has been relatively limited because of the logistic difficulties of sampling marine areas and obtaining material from many national jurisdictions. It is generally been assumed that marine species that has wide geographic distributions, and the apparent capacity for long-distance dispersal do effectively disperse over long distances in ecological time scales (Thorpe et al., 2000). Sapna (1999) and Vijayakumar (1992) reported genetic homogeneity in the stocks of the pearl oysters (Pinctada fucata) and grey mullet (Mugil cephalus) from east and west coasts of India. Similarly, Menezes (1993, 1994 a, b) from NIO, Goa; Verma et al. (1994, 1996) and Jayasankar et al. (2004) using allozymes, RAPD and morphometrics data, reported populations of Indian mackerel (Rastrelliger kanagurta) and oil sardine (Sardinella longiceps) from east and west coasts of India belong to the same unit stock. Preliminary analysis on seahorses (Hippocampus kuda) using sequence information of cytochrome b gene of mtDNA, sampled from Arabian Sea and Bay of Bengal revealed that these two populations are genetically close to each other (Thangaraj et al., 2003). However, there has been growing evidence from genetic markers that even the widespread marine species are geographically structured, and that there can be sharp genetic disjunctions sometimes where there are no obvious barriers to dispersal. Such evidence has been observed in the stocks of shrimp Penaeus monodon (Rebello, 2003), P. indicus (Paul, 2000) and the endangered marine white fish, Lactarius lactarius (Gopalakrishnan et al., 2006) from east and west coasts of India with allozyme and RAPD markers and TRUSS network analysis. Mitochondrial control region (mtDNA CR) diversity within and among 6 seahorse populations associated with the IndoPacific Hippocampus kuda complex (including H. kuda from Goa and Tamil Nadu in India) was compared by Teske et al.(2005) and their results were not in conformity with the earlier study of Thangaraj et al.(2003). Teske et al. (2005) reported that H. kuda population from India was the oldest and Indian Ocean lineage of Hippocampus kuda is not a panmictic population, but rather an assemblage of regional lineages. Menezes et al. (2006) also recorded genetic variation in stocks of skipjack tuna Katsuwonus pelamis using PCR-RFLP analysis of the mitochondrial DNA D-loop region. This suggests that the genetic differences within the species may be related to separation in historical times (Benzie, 2002). However, it does not explain why genetic heterogeneity still exists within these species after they have been reconnected and if high and effective gene flow is assumed. The generality of these findings questions the assumption that marine species disperse widely, demonstrates in some cases they do not, and demonstrates in other cases that many unrecognized cryptic species exist. These findings also demonstrate that Marine Protected Areas (MPAs) cannot be designed on the basis that long distance dispersal will be sufficient to connect them and ensure recruitment – or assume that apparently widely dispersed species are protected if they are represented by a few reserves. The NBFGR has also initiated steps in identifying distinct genetic stocks of sea cucumber (Holothuria scabra), lobsters (Panulirus homarus and Thenus orientalis), selected elasmobranchs and Bombay duck (Harpadon nehereus) from the coasts of mainland and island ecosystems. Five polymorphic microsatellite markers have been developed for H. scabra using primers from related species and these have been found effective in population structure analysis. Molecular markers have been used to generate species-specific or fixed profiles in marine and freshwater species with a view to resolve taxonomic ambiguities. The NBFGR has identified six species-specific allozyme markers in two species of marine catfishes (Tachysurus maculatus and T. subrostratus), thus confirming the separate identity of both the species (Gopalakrishnan et al., 1996). Similarly, the genetic identity of exotic catfish, Clarias gariepinus in India has been studied using mtDNA RFLP pattern of ND5/6 region by Lal et al. (2003). Among the Indian major carps and other cyprinid species, such as Schizothorax spp. and Garra spp., many species-specific markers have been detected, for example using esterases (Gopalakrishnan et al., 1997), mtDNA RFLP (Padhi and Mandal 1995a), MboI satellites (Padhi et al. 1998) and sequence information of cytochrome b gene of mtDNA (Thangaraj et al., 2003). Silas et al. (2005) used RAPD technique with 15 species-specific 5

and random oligodecamers and morphometric measurements and revalidated the taxonomic status of endangered mahseer, Tor malabaricus from the Western Ghats. RAPD assay was also used to evaluate the genetic relationship among four species of Indian major carps, six Labeo species and seven species of groupers (Das et al., 2005 a; Govindaraju and Jayasankar, 2004; Barman et al., 2003). Altogether, 34 primers generated species-specific profiles in carps and Labeo species, while eight arbitrary primers gave unique banding patterns for each of the grouper species. Nuclear DNA fingerprinting was carried out by Majumdar et al. (1997) in Indian major carps and tilapia; and Padhi and Mandal (1997b) in snake-head fishes with a view to develop species-specific pattern and to discriminate individuals within a population. Menezes (1984, 1985, 1990, 1993, 1994a, b) and Menezes et al. (1990, 1992, 1993, 2002) studied inter-specific divergence and phylogenetic relationships using species-specific allozyme and soluble eye lens/sarcoplasmic protein markers in grey mullets, sardines, pomfrets, flying fishes, three carangids, four sciaenids and five nemipterids from Arabian Sea and Andaman waters. Efforts have also been initiated at NBFGR to identify different species of harmful cyanobacteria (Trichodesmium spp.) from Indian EEZ using sequence information of internal transcribed spacers (ITS), 16S rRNA and Het R genes. Steps have also been taken by NBFGR to genetically catalogue of aquatic microbes of Indian waters using appropriate species-specific molecular markers. DNA Barcoding: Accurate and unambiguous identification of marine fish and fish product, from eggs to adult is essential in several ways, as it would assist in managing fisheries for long term sustainability and improve ecosystem and conservation. Taxonomic ambiguity exists in several Indian marine species and there is no mechanism to correctly identify eggs and larvae of commercially important Indian marine species. “DNA Barcoding” – DNA sequence analysis of a uniform target gene (Cytochrome Oxidase – I of mitochondrial genome) is the most recent and reliable approach to discriminate eukaryotic species including fish. Barcoding offers a simple, rapid and reliable means of identifying not only whole fish, but fish fragments, eggs and larvae. Such information is totally lacking in the case of Indian marine finfishes. The NBFGR has recently initiated a mega programme on DNA Bar-coding of all Indian marine finfishes in collaboration with the global Consortium for the Barcode of Life (CBOL) – Fish BOL. DNA barcodes of more than 150 finfish species reported from Indian seas have been prepared so far. This could be of great utility in sustainable exploitation, management and conservation of Indian marine fish species. Arguably, the most popular mariculture technology in India is mussel farming. Often, the mussel farmers find it difficult to collect the spat (seed) of the right species of mussel from the wild and are not sure about the timing of laying the spat collectors. In this context, molecular taxonomy could prove to be a promising tool for larval identification, regardless of developmental stage and can be used for the assessment and tracking of larval dispersion as well as assist in the identification of sites of seed settlement site and their exploitation. Reliable prediction of the spat-fall of mussels, edible oysters and pearl oysters would help the farmers to employ the spat collectors at the right place at the right time in order to obtain the spat of the required species. Though considerable amount of work has been done in the overseas laboratories on the application of molecular markers on larval identification, there are no studies reported from India. DNA sequence analysis is a powerful tool for identifying the source of samples thought to be derived from threatened or endangered species. The technique could be effectively used in tracking illegal trade of marine mammals, commonly practices in some of the Asian countries, where they market some of the endangered species in the guise of ones approved by the International Whaling Commission (IWC). The DNA-based approach would help the conservationist to identify the species even from a small piece of tissue sample, such as skin from the marketed product. Besides marine mammals, molecular based taxonomic identification has great application in validation of marketing in other species, such as sea turtles, sharks and sturgeons. In this context, CMFRI has generated partial DNA sequences of mitochondrial cytochrome b and control region (D-loop) of 11 species of cetaceans (marine mammals), bottlenose dolphin, spinner dolphin, spotted dolphin, common dolphin, humpbacked dolphin, Risso’s dolphin, finless porpoise, sperm whale, blue whale, Bryde’s whale and 6

dugong. The Institute has developed the capability (a) to confirm whether the obtained tissue sample (minute quantity of skin samples enough, no need to see the whole animal or its body parts) belongs to a marine mammal at all and (b) if proved to be a marine mammal, identification up to species level of whale, porpoise or dolphin based on phylogenetic reconstruction of mtDNA sequences. Till date the Institute has released a total of 63 sequences of cytochrome b gene and control region of mtDNA from 40 individuals of 11 species in the GenBank (NCBI). Gender identification is of fundamental importance in the studies of population structure, social organization, distribution, behaviour or heavy metal accumulation in marine mammals. However, distinguishing the males and females among the marine mammals is difficult due to the poor sexual dimorphism, especially during their free-ranging state. With the advent of DNA-based tools, such as PCR, it is possible to even identify the sex using tissue sample. Recently CMFRI has developed sex determination technique based on the amplification of genomic DNA extracted from the skin tissues of marine mammals. Molecular sexing was standardized by the Institute in several species of dolphins, finless porpoise, whales and dugong. Unintended intergeneric hybridization and back crossing of F1 hybrids with their parents might result in genetic introgression among the fishes causing contamination of their gene pools, low survival and altered feeding niches in farming systems. A low frequency of hybridization may occur in the wild between many pairs of closely related species, but presumably natural selection prevents this from reaching significant proportions in most cases (Padhi and Mandal, 2000). Mishra et al. (1998) and Padhi and Mandal (1994, 1997a) have recorded the rate of genetic introgression as 7.25 to 9.24% among Indian major carps produced during mixed spawning in Chinese hatcheries. They pointed out an urgent need to modify the Chinese model hatcheries so as to have species-wise hatching pools to facilitate breeding of 3 species of IMCs separately and within the same time span. Genetic markers that show fixed differences between species can also be used to monitor hybridization and introgression where these are known or suspected to occur. Padhi and Mandal (1997a) detected inadvertent hybridization resulting from multispecies spawning in a major carp hatchery by probing Eco RI digested DNA with a ribosomal RNA gene probe. Lal et al. (2006) studied allozyme and mtDNA RFLP pattern in Labeo calbasu and L. rohita and identified hybrids between these species using the above markers from the River Ganges. Cytogenetic information is useful for understanding many aspects of fish genetics. Basic karyotype information can be used as genetic markers in studies on hybridization, induced polyploidy, gynogenesis and androgenesis. While the techniques involved in preparing metaphase spreads from large numbers of fish or embryos are fairly laborious, they are also well standardized. Chromosomal aberration and sister-chromatid exchanges (SCE) can be used as tools to evaluate the geno-toxicity of pollutants. Chromosomal bands are produced due to variation in longitudinal structure of the chromatids revealed by various staining techniques. The common banding techniques are Q-bands, NOR bands, C – bands, G – bands, Restriction Endonucleases (RE) bands and Fluorescent in-situ Hybridization (FISH). The FISH technique in particular can be used for development of species/stock specific molecular markers and identification of sex chromosomes. The NBFGR, Lucknow; CIFE, Mumbai; Kurukshetra University, Haryana and University of Kalyani, West Bengal are actively involved in the cytogenetic research on Indian teleosts and crustaceans. Indepth cytogenetic analyses have carried out on Indian major carps, Indian freshwater and marine catfishes, mullets, mahseers, snow trouts, Channa spp. and crustaceans with a view to develop species-specific banding profiles and characterize the genetic stocks (reviewed by Lakra and Ayyappan, 2003; Lakra, 2002, 2001, 1998 a, b; 1996 a, b; Lakra et al., 1997; Khuda – Bukhsh, 2003; Krishna & Lakra, 1996; Rishi, 1989; Manna, 1984; papers by: Nagpure et al., 2002, 2000 a,b,c; Kushwaha et al., 2000; Sharma & Lakra, 2000; Das & Lakra, 1998; Kumar & Lakra, 1996; Krishna & Lakra, 1994, 1995; John et al., 1993 b; Zhang & Reddy, 1991; Rishi & Mandhan, 1994,1995; Rishi & Manjusha, 1991; Rishi & Rishi, 1981; Manna & Khuda-Bukhsh, 1978, 1977a,b). Species-specific karyotypes for endangered mahseers - Tor khudree, T. mussullah, and has been reported by 7

Kushwaha et al., 2001 and for Tor tor, T. putitora and T. khudree by Lakra (1996 a). Evidence of female heterogamety, B-chromosomes and natural tetraploidy in Asian catfish Clarias batrachus was reported by Pandey & Lakra (1996). The NBFGR has also brought out a chromosome atlas on Indian fishes depicting karyotypes of 128 teleosts found in Indian waters (NBFGR, 1998). Recently, it has focussed its attention on cytogenetic characterization of endemic fish species from the Western Ghats of India, which is well known for its rich diversity of teleosts (Nagpure et al., 2006 a, b, 2005 a, b, 2004, and 2003). Although studies of fish karyotypes yielded some interesting information and applications – for example insights into the role of polyploidization in the evolution of different groups, and chromosome counts can be used as markers in chromosome set manipulation experiments – otherwise limited progress has generally been made in such studies. Intra-specific chromosomal polymorphism could be detected by chromosomal banding techniques in some species of salmonids (Phillips et al., 1988, 1989); however, in Indian species, cytogenetic techniques have so far been helpful in detecting polymorphism at inter specific and inter generic levels only (Padhi & Mandal, 2000). Gene mapping and marker assisted selection Traditional selective breeding in aquaculture can be supported by molecular genetic tools in order to reduce time, space and investment on one side and to assure sustainability on the other. Since most performance and production traits are controlled by multiple genes and therefore inherited as quantitative traits analysis of their associated quantitative trait loci (QTL) is emerging as a very important part of aquaculture genetics/genomics (Liu & Cordes, 2004).QTL are largely unidentified genes that affect performance traits such as growth rate and disease resistance that are important to breeders. Relative chromosomal positions of QTL in a species genome can be identified by constructing genetic linkage maps. Genetic linkage maps are constructed by assigning polymorphic DNA markers (such as microsatellites, SNPs, RAPDs or AFLPs) and expressed sequence tags (ESTs) to chromosome configurations based on their segregation relationships. Once a linkage map has been constructed for a given species, it can be used in combination with studies of breeding and assessment of quantitative traits to identify markers that are closely associated (linked) to QTL of interest, thus allowing the QTL to be positioned on the linkage map. This information can then be used to aid aquaculturists in efficiently crossing different strains of cultured species to maximize growth, disease resistance, or some other desirable trait through marker assisted selection (MAS) (Liu & Cordes, 2004). MAS can result in more production in a short span of time than traditional breeding and use of such genetic tools are complimentary to all existing phenotypic or traditional breeding programmes (Lakra et al., 2006 c). Genetic linkage and QTL mapping in Indian aquatic species are not as advanced as they are in channel catfish, tilapia or salmonids. In CIFA, Bhubaneswar, research work is in progress in developing microsatellite DNA markers to generate a preliminary linkage map of Labeo rohita (Das et al., 2005 b). In a networking project approved by the Department of Biotechnology (DBT), steps have been initiated to develop linkage map of Penaeus indicus by the Centre for Cellular and Molecular Biology (CCMB), Hyderabad and NBFGR using type I and II microsatellite markers. Selective Breeding Selective breeding has been the basis of nearly all of the genetic improvement that has taken place in terrestrial agricultural animals and plants since the earliest stages of domestication and has contributed significantly to the food security. This is the classical approach to achieve genetic improvement in domesticated species. The approach utilizes intra-specific variation available in the founder population and applies selection for high performance with respect to desirable traits in the subsequent generations. It is estimated that selection has increased the productivity of modern breeds of farm animals by at least two to three times in the last 50 years, but less than 1 per cent of aquaculture production worldwide in 1993 came from genetically improved breeds (Liu & Cordes, 2004). Selection involves increase in the frequency of favorable allele of genes for production parameters which are additive in nature. The success of selection depends up on the extent of 8

additive genetic variation in the selected population. Heritability estimate of the selected trait is essential for the fish breeders to estimate the response to selection programme, the time and cost required to reach the desired goal. It had been found that heritabilities in fish are generally lower than domestic animal and poultry. Although aquaculture organisms differ from agricultural mammals and birds in several important ways (e.g. higher fecundity and smaller post-embryonic size), the principles of selective breeding can also be applied to their genetic improvement. In aquaculture, few success stories on genetic improvement in common carp (Dor-70 strain), Atlantic salmon, channel catfish, Nile tilapia (GIFT Programme of the WorldFish Centre) and Peneaus vannamei have been reported from different countries. Initial attempts on these lines in Indian fish were done by Das et al. (1989) and they obtained a 5% predicted response in one generation selection of Etroplus suratenesis. Recently, under a selective breeding programme, a variety of Labeo rohita named ‘Jayanti rohu’ (CIFA-IR-1) has been successfully developed after four generations of selective breeding. This research was done under Indo-Norwegian collaboration (AKVAFORSK; funded by NORAD) during 1992-2000 at CIFA, Bhubaneswar. The field testing of ‘Jayanti rohu’ has been carried out in Orissa, West Bengal, Andhra Pradesh and Punjab and a 17% high growth realization per generation has been achieved. The encouraging results with rohu have made the way for initiation of the selective breeding programme in other species. Two such programmes – one, for genetic improvement of Penaeus monodon (tiger shrimp) for growth and white spot disease resistance at CIFE, Mumbai in collaboration with CIBA, Chennai and AKVAFORSK, Norway and another on Cyprinus carpio at Karnataka University of Animal Sciences & Fisheries, Bangalore Campus in collaboration with DFID and Institute of Aquaculture, Sterling, UK are on-going. The WorldFish Center, Penang has recently proposed a research programme for genetic improvement of Macrobrachium rosenbergii with CIFA, Bhubaneswar as the Indian collaborator. Artemia cysts are the most extensively used live food for most diversified groups of aquatic animals. Although its size at different stages especially the naupliar length restricts its use as a food for some groups of fish, this problem can be overcome using selective breeding techniques. In an experiment at CMFRI, Cochin, bidirectional mass selection was employed to reduce the naupliar length of Artemia franciscana. Six generations of selection for smaller naupliar size (SNS) resulted in a phenotypic response of -45.32 μm and -37.52 μm decreases in naupliar size in males and females respectively compared to the control (Shirdhankar & Thomas, 2003 a, b; Shirdhankar et al., 2004). The study also indicated medium and high magnitude of heritability estimates for naupliar length and high level of variance in the population is due to additive genetic variance, which can be exploited through a simple mass selection (Shirdhankar & Thomas, 2003 b). It is apparent that there is much scope for the application of quantitative genetics to improve the performance of several cultured finfish and shellfish species of Indian waters. Selective breeding involving the identification of individual families is generally more efficient than mass selection, but requires additional resources for physical identification (separate rearing for each family followed by physical tagging or marking of each family before communal on-growing) or identification using highly polymorphic microsatellite markers. In practice, limitations in financial, physical or human resources have often restricted the scale and complexity of breeding programmes for cultivable species in India. Transgenic fish Transgenic technique provides a rapid and efficient method for fish improvement across a wide range of species by direct gene manipulation. Gene transfer studies in fishes have been initiated for developing some superior strains useful in aquaculture. The first recorded instances of production of transgenics or genetically modified organisms (GMO) in aquatic species were in rainbow trout and in gold fish. Since then, many fish species have been used to produce GMOs in several parts of the world. The most popular gene used in aquatic species is the growth hormone (GH) with an aim to enhance the growth rate of cultivable species. Growth hormone genes of fish origin have been shown to be more effective than those of mammalian origin. Transgenic ornamental fish popularly called as “glow fish”, harbouring fluorescent genes isolated from jellyfish has recently opened new possibilities for producing new multi-coloured fluorescent fish. Transgenic fishes may 9

also be used as "bio-reactors" to produce pharmaceuticals. Although this potential is being realized in crop products, there has been no commercial use of genetically modified organisms (GMOs) in aquaculture. The research on fish transgenics in Indian is relatively recent (Lakra et al., 2004). The first Indian transgenic zebrafish was generated in 1991 at Madurai Kamaraj University, Tamil Nadu, using borrowed constructs from foreign sources, followed by the production of first transgenic triploid zebra fish in 1995 (Sheela et al., 1998; Pandian & Marian, 1994). To construct transformation vectors for the indigenous fishes, growth hormone genes of rohu (Labeo rohita) (Venugopal et al., 2002 a, b) and catfish, Heteropneustes fossilis (Anathy et al., 2001) were isolated, cloned, sequenced and confirmed in prokaryotic and eukaryotic systems. A vector was made with grass carp β-actin promoter and sperm electroporation technique was used for transfer genes (Venugopal et al., 1998) that ensured 37% transgenics. Transgenic rohu and singhi grew faster than the respective controls and converted the food at a significantly higher efficiency (Pandian, 2003). At CCMB, Hyderabad, auto-transgenic Catla catla and Labeo rohita were generated using the growth hormone gene constructs (both cDNA and genomic DNA) fully developed from these species, thus giving a “swadeshi” touch to the entire experiment (CCMB, 2002; Majumdar, 2002, pers. comm.). Efforts are on way to isolate and characterize salt-resistant genes from marine environment in a collaborative project between Central Institute of Fisheries Education (CIFE) Mumbai and CCMB, Hyderabad. Similarly, attempts have been initiated at M. S. Swaminathan Research Foundation, Chennai, and Central Institute of Fisheries Technology (CIFT), Kochi to isolate and characterize salt tolerance genes from mangrove plants like Avicenia sp., sea grasses and marine microbes. Under the intensive aquaculture conditions, large scale fish mortalities occur due to disease outbreaks. In the aqueous environment, rich in microbial flora, fishes are presumed to use their innate immune system as first line of defense against microbial invasion. There are evidences that endogenous peptide with antimicrobial property play an important role in host defence. After the discovery of cecropins, an inducible antimicrobial peptide from giant silk moth, antimicrobial peptides were isolated from a number of organisms as a part of defense system including fish. Fish cells carrying cecropin transgenic constructs were also able to produce bactericidal active cecropins as in Japanese medaka the F2 transgenic fish were found to have acquired elevated resistance to bacterial infections (Lakra et al., 2006 c). Thus, the gene coding for potent antimicrobial peptide represents a good candidate for the genetic improvement of fish stocks to bacterial diseases. Genetic manipulation has also been undertaken in order to increase the resistance of fish to pathogens. This is currently being addressed by the use of DNA vaccines (encoding part of the pathogen genome) and antimicrobial agents such as lysozyme. An example is the injection of Atlantic salmon with a DNA sequence encoding infectious hematopoeitic necrovirus (IHNV) glycoprotein under the control of cytomegalovirus promoter (pCMV). Eight weeks after the challenge with the virus, significant degree of resistance had been achieved. The fish were still resistant and were shown to have generated antibodies three months later. Similar studies have been undertaken for other fish diseases e.g. hemorrhagic septicemia virus (VHS) and work of this kind appears to have great potential values for fish farms. Similar attempts can be made with the help of lysozyme genes and RNA interference (RNAi)/antisense RNA in penaeids and finfishes to fight against various viral diseases. RNAi describes the phenomenon by which double-stranded RNAs (dsRNAs) elicit degradation of a target mRNA containing homologous sequence. Thus, the phenomenon is essentially a new incarnation of the well-established antisense principle - the inactivating agent interacts with the target through the complementary interaction and results in inability of the latter to produce corresponding protein. The hit rate and efficacy of RNAi is much better than those of other anti-sense techniques. This has opened up tremendous opportunities to develop a novel generation of oligonucleotide-based drugs and in the best case scenario; development of the new drug would require simply knowledge of the sequence of the gene causing the corresponding disease. Work on these lines is already in progress in the College of Fisheries, Mangalore under a DBT sponsored scheme.

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It is still not clear that transgenic technologies have immense potential for enhancing aquaculture production and ensuring nutritional security. However, transgenic fish is associated with lots of controversies arising due to ethical issues and its impact on natural germplasm due to escapes. Therefore, biosafety concerns need to be adequately addressed during evaluation as well as transgenic fish production. Transgenic research related to modified fish for ornamental trade, pharmaceutical and other industrial products and as biosensors to monitor water pollution levels are more attractive and with less controversies. Considering the acceptance of transgenic fish by consumers, ‘all fish’ or ‘autotransgenic’ fishes are expected to become successful in future. Moreover, transgenic research is capital as well as skill intensive. Therefore, it deserves to be treated at par with industrial products and commercial feasibility of the target species should be assessed, before research is carried out to develop commercial transgenic form of such species. Hybridization Hybridization between different genetic groups is known to lead to hybrid vigour known as ‘heterosis’. Israel and Hungary have made excellent use of heterosis in their commercial fish culture. Hybridization is one of the methods used for combining desirable traits of selected species, with experiments demonstrating a high level of compatibility among Indian carps. In the initial stages, simple inter-specific and intergeneric hybridization was done to produce and evaluate the carp’s useful traits for aquaculture. In India, 44 intergeneric and inter specific hybrids have been produced (Kowtal, 1987). He furnished a list of hybrids among cultivated carps, viz, common, selected Chinese and selected Indian major carps categorizing them into six groups, i.e. (A): inter-specific selected Indian major and minor carps; (B): intergeneric selected Indian major and minor carps; (C): selected Chinese carps existing in India; (D): selected Indian major and Chinese carps with very low survival rates; (E): Indian major carps and common carp, and (F): common carp and selected Chinese carps. Among the four species of Indian major carps belonging to three genera, i.e., Catla, Labeo and Cirrhinus, altogether, six inter-specific and 13 inter-generic hybrids, were produced (Ayyappan et al., 2001). Over three decades of research on hybridization showed that the major carps are able to produce viable and fertile hybrid progenies. Mature inter-specific and inter-generic hybrids could be induced to produce F2 progeny or back-cross and triple-cross hybrids. The growth exhibited by most of these hybrids was intermediate, i.e., better than the slow-growing parent. The reciprocal hybrids of Asiatic catfishes H. fossilis and C. batrachus and H. fossilis and H. microps have also been produced experimentally (Padhi et al., 1995). Of all the inter-specific and intergeneric hybrids produced so far in India, “naadan” the hybrid produced between Labeo rohita female and Catla catla male was has found acceptance by the farmers owing to small head and meat quality of rohu and fast growth rate of Catla catla. ‘Naadan’ is produced in hatcheries of West Bengal and Andhra Pradesh for fish farming. The rohu–catla hybrid possesses a broader feeding spectrum than the parents and thus is able to consume a variety of aquatic microphytes such as Ceratium species, particularly available in some of the reservoirs in India (Reddy, 2005). Because of this trait the hybrid has been recommended to be a good substitute to both the parents for stocking in reservoirs. Most of other hybrids did not perform with respect to the desired economic traits. With regard to hybridization between Indian major carps and Chinese grass carp (Ctenopharyngodon idella), silver carp (Hypophthalmichthys molitrix) and bighead carp (Aristichthys nobilis), no compatibility was observed. Almost all of these died either during the embryonic development or soon after hatching (Ayyappan et al., 2001). Relatively better survival of the hybrid progeny was observed in the crosses between female common carp (C. carpio) and males of C. catla, L. rohita and Cirrhinus mrigala compared with the reciprocal hybrid crosses particularly between female L. rohita and male C. carpio (Ayyappan et al., 2001). It is not advisable to carryout indiscriminate hybridization. Most of the cultivable carps are highly compatible to interbreed. Also, since almost all inter-specific and intergeneric hybrids were found fertile, one should be more cautious, as unwanted hybridization may cause serious damage to the original gene pool through genetic introgression (Padhi & Mandal, 2000).

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Cryopreservation of fish gametes and embryos Storage of fish spermatozoa, eggs and embryos without loss of viability is of considerable value in aquaculture and conservation. The basic technique of cryopreservation involves collection of fish gametes in which specific diluent with cryoprotectant is added. After a period of specific of equilibration, it is frozen rapidly and stored in liquid nitrogen. After thawing, the milt can be activated for use in fertilizing the eggs. Sperm cryopreservation has been reported for more than 200 finfish species across the world (Rana, 1995). In India, NBFGR is the primary organisation in India carrying out fish sperm cryopreservation for long term gene banking. The fish sperm cryopreservation needs development of species-specific protocols. Such protocols are developed through experimental standardization of various parameters, after the captive breeding protocol is developed. This becomes a bottleneck due to protracted breeding season and low domestication of most of the aquatic species, especially marine fishes. Nevertheless, in all such cases, time available in a year for conducting experiment is small and determined by breeding cycle of the species. In view of the constraint, it is essential that candidate species for sperm cryopreservation are prioritized. Species specific sperm cryopreservation protocols have been developed for 14 species viz: Catla catla, Labeo rohita, Cirrhinus mrigala, Labeo dyocheilus, Oncorhynchus mykiss, Salmo trutta fario, Cyprinus carpio, Tenualosa ilisha, Tor khudree, Tor putitora, Labeo dussumieri, Horabagrus brachysoma, Ompok malabaricus and Gonoproktopterus curmuca (Ponniah et al., 1999; Thakur et al., 1997; Ponniah et al., 1998 a, b; Lal et al., 1999; Gopalakrishnan, et al., 1999). In addition to the above species, efforts are being made to develop species – specific sperm cryopreservation protocols for 9 species viz. Labeo dero, Labeo calbasu, Barbodes carnaticus, Osteochilichthys longidorsalis, Pangasius pangasius, Silonia silondia, Etroplus suratenesis, Schizothorax richardsonii and the protogynous grouper, Epinephelus merra. Out of the twenty fish species, the technique has been tested for twelve through production of progeny using cryopreserved sperm. Continuous improvement in protocols has provided hatching success ranging from 65-100% of the control value for different species. Padhi and Mandal (1995b, 1998) succeeded in cryopreserving sperms of catfishes, Heteropneustes fossilis and Clarias batrachus at laboratory level. Koteeswaran and Pandian (2002) reported the cadaveric sperm preservation in Indian catfish Heteropneustes fossilis. The experiment, first of its kind yielded hatching 2-3% from sperm preserved for 40 days at -200C. This needs to be further explored, as it can be useful under certain circumstances like highly rare species that are difficult to be collected alive. However, different species can have different time for post-mortem lysis of cells. Moreover, freezing rate will alter with the size of the fish. Assessing maturity stage of the fish, absence of sexual dimorphism and varying time lag after death, are potential conflicts that may arise while selecting dead fish for freezing under real field conditions. The strategy and technique used at NBFGR is simple, does not need any electrically operated equipment. This provides easy adaptability, customization in difficult remote locations and successful application has been proved in species of various taxonomic groups. The sperm cryopreservation protocol has also been tested for production of rohu seed in late season when natural milt is not available. Inadequate milt production or asynchronisation in maturity of two sexes is generally reported in several cultivable species. In artificial propagation, sperm cryopreservation protocol can be an asset where such milt related problems exist. This ex situ conservation tool has strong application to support in situ conservation strategies like ‘propagation-assisted rehabilitation of endangered fish species’. However, sperm cryopreservation experiments in crustaceans have not been encouraging. Diwan et al. (1994) and Diwan and Kandaswami (1997) attempted cryobanking of gametes of Penaeus semisulcatus and P. indicus and they reported reduction in motility and frozenthawed spermatozoa leading to reduced fertilization percentages. Fish gamete cryopreservation research still faces an important challenge in the form of long-term storage of fish eggs and embryos except the minute fertilized abalone eggs. Owing to large size, large amount of yolk and tough chorion or zona radiata with a low permeability coefficient, egg and embryo cryopreservation of teleosts and crustacea have not met with success anywhere in the world so far. Attempts to cryopreserve embryos of C. carpio, L. rohita and nauplii of P. monodon independently by NBFGR 12

and Madras University did not succeed below –40°C (NBFGR, 1994-‘95; Subramoniam, 1994; John et al., 1993 a). Cryopreserved sperm was also used to retrieve the whole species and clones. Sperm retrieved from fish, stored at -18°C has been used to produce androgenetic fish and interspecific androgenetic cloning, as a mode for restoration of species (Kiran Kumar & Pandian, 2004; David & Pandian, 2005, 2006). However, this alone does not overcome the loss of mitochondrial genome that is maternally inherited. Therefore, to retain total genome diversity, successful preservation of egg and embryo is essential, which has remained unsuccessful so far. Development of fish cell lines, embryonic stem cells and germ cells from Indian fishes and cloning technology as an alternative to long term storage of finfish eggs and embryos has been emphasized (Pandian, 2002). Embryonic stem (ES) cells are pluripotent stem cell lines that are derived from early embryo. Embryonic stem (ES) cells can differentiate to become any tissue in the body (Hong et al., 2000). Successful protocols for grafting of embryonic cells to host embryos, for germline transmission of desired genome, can be instrumental in evolving effective programmes for production of transgenics and rehabilitation of endangered species. There have been some successful studies in developing cell cultures such as ovarian tissue from immature ovary of Clarias gariepinus (Kumar et al., 2001). Pluripotent cell lines from sea bream embryonic stem-like cells (SBES1) have been reported from blastula-stage embryos of the cultured Chrysophrys major. In India, recent significant efforts and success have reported in developing cell cultures and cell lines from Tor putitora, Lates calcarifer and Labeo rohita (Lakra et al., 2005, 2006 a, b). Cryopreservation of pluripotent blastomeres and grafting of thawed cells into recipient embryos to produce chimeras open an alternate pathway (Jamieson, 1991). The tolerance towards cryopreservation procedures and obtaining viable cells after freeze-thaw has been studied in a few species. The grafting of blastomeres to produce chimera has been successful in goldfish, trout, zebra fish and medaka. In most of the reports, pluripotent blastomeres when grafted into the recipient embryos have yielded somatic and occasionally germline chimeras, attributed to relatives low transmission rate of donor cells than the host embryo cells (Lakra, 2006 c). Targeting the germ cells through precise identification; development of pure line in vitro culture of primordial germ cells for grafting into host embryos and transplantation can improve the success of germline chimeras. Recently, NBFGR and CIFA have initiated research programmes on cryopreservation of blastomeres of Indian major carps and catfishes (Lakra, 2006 c). The ultimate objective is to achieve germline chimeras, where the donor cell enters germline development and are able to give rise to fertilizable gametes. Chromosomal manipulation Chromosome sets can be manipulated in externally fertilizing fishes to produce gynogenetic, androgenetic and polyploid individuals. The most common techniques include gynogenesis, androgenesis and polyploidy. Gynogenesis: Gynogenesis occurs naturally in the genus Carassius, with many different clonal lines existing, some of them triploid (Penman et al., 2005). Induced gynogenesis involves inactivating the paternal (sperm) genome and then restoring diploidy either through suppression of the second meiotic division (“meiotic” gynogenesis) or suppression of the first mitosis (“mitotic” gynogenesis). Most of the more recent protocols for gynogenesis have used ultraviolet irradiation to inactivate the paternal genome, and temperature (cold or heat) shocks to restore diploidy. The various applications of the gynogenesis are chromosomal mapping, inbreeding with homozygosity and generation of monosex population. Among Indian major carps, gynogenesis has been induced in Labeo rohita, Cirrhinus mrigala and Catla catla at CIFA, Bhubaneswar using cold shocks (120C 10 minutes) and heat shock (390C, 1 minute) by John et al. (1984, 1988). Gynogenetic specimens of silver carp have also been produced in India at CIFA, Bhubaneswar. Gynogenetic zebra fish, Betta splendens, rosy barb, tilapia and production of YY super male tilapia by gynogenesis and sex reversal have been reported from Madurai Kamaraj University (Pandian & Koteeswaran, 1998; Kavumpurath & Pandian, 13

1992 a, b, 1993, 1994; Varadaraj, 1990 a, b, 1993; Varadaraj & Pandian, 1988, 1989 a, b) and gynogenetic common carp from NBFGR (John et al., 1992). Gynogenesis combined with sex reversal of female into male involving the use of male hormones is a practical method for improvement of species to produce faster growing individual as well as for incorporation of other advantageous traits. Even though there are repeated experimental success in producing gynogens in India, follow-up studies and their incorporation in breeding programmes are yet to be undertaken. Androgenesis: Androgenesis is the method of reproduction where only the paternal genome is contributed to the offspring. Androgenesis can be induced by the destruction of female nuclear genome before fertilization using UV rays and fertilizing it with normal sperm. Androgenesis is useful in producing inbred lines. In conjunction with sperm cryopreservation method, androgenesis may prove useful in conservation programme when females of a species are not available. Androgenesis has been investigated less than gynogenesis and survival of androgenic individuals is much less compared to gynogens as the cellular organelle of egg will be affected during irradiation. Ponniah et al. (1995) reported successful production of viable hatchlings of common carp through androgenetic experiment depicted putative androgens. David & Pandian (2005, 2006) developed intergeneric androgenetic exotic ornamental fish Hemigrammus caudovittatus using cadaveric sperm as DNA source, even though survival rates were low. Pandian (2003) also described successful interspecific androgenetic cloning that can be a useful mean for use as conservation tool. Further improvement and up-scaling of teleost androgenic production are yet to take place in India. While gynogenesis and androgenesis have made little direct impact on aquaculture apart from contributing to monosex female production and in some selective breeding programmes, they provide interesting tools for basic research underpinning aquaculture genetics and are likely to make further contributions in the future. Polyploidy: By chromosomal stimulation it has been possible to obtain haploid, triploid and tetraploid fishes. These fishes are likely to be sterile and would hopefully grow faster by avoiding the process of gametogenesis which is wasteful in fish farming. Triploidy is induced by second polar body retention and tetraploidy from first mitotic interference, followed by subjecting the egg to a pressure or temperature shock shortly after fertilization. Among better-known fish, direct induction of triploidy has been induced in common carp and grass carp. Triploid grass carp produced by cold and heat shock is an effective weed consumer but beings sterile there is no risk of multiplication. Crosses between diploid and tetraploid brood stock can produce triploid offspring without the need to induce triploidy but the production of tetraploid brood stock is very difficult in most species where this has been attempted (very low larval viability; poor fertility in females: Pandian & Koteeswaran, 1998) and fertilization rates in diploid female x tetraploid male crosses are also low, probably because of the large size of the sperm (Padhi & Mandal, 2000). Triploidy could at least in theory be used anywhere to control reproduction of alien species, domesticated strains, transgenic fish, etc., and is already used in commercial aquaculture in a limited range of species. In India, triploid rohu and catla individuals have been produced at CIFA (Reddy et al., 1987, 1990), H. fossils at Banaras Hindu University, Varanasi (Tiwary et al., 1997), tilapia and exotic ornamental fishes at Madurai Kamaraj University (Pandian & Koteeswaran, 1998; Varadaraj & Pandian, 1988, 1990). The CMFRI, Cochin has been successful recently in producing triploid edible oyster, Crassostrea madrasensis by treating the newly fertilized eggs with 100 µM 6dimethylaminopurine (Thomas et al., 2006). This yielded 67% survival and triploid oysters exhibited significantly higher growth rate and meat content (126% higher dry weight, nearly 30% more glycogen, lipids and proteins) compared to diploid individuals (Mallia et al., 2006). Among the ornamental species, triploidy has been induced in Betta splendens, Brachydanio rerio, Puntius conchonius and Poecilia reticulata at Madurai Kamaraj University by Kavumpurath & Pandian (1990, 1992 a, c). But, like gynogens and androgens, teleost triploids are yet to be produced in a large scale in India to be used for aqua-farming practices. A naturally occurring tetraploid race of Clarias batrachus has been reported by Pandey & Lakra (1996). But, production of artificial tetraploid 14

individuals has not been so far recorded from India. The known and assumed reasons for failure to induce live tetraploid fish include reduced cell surface, changes in genome-cytoplasmic ratio, decreased cell number, increased cell volume, mosaicism, high homozygosity and wrong cytological events (Pandian & Koteeswaran, 1998). Sex determination in fish In fish, sex control can be achieved only when the mechanisms of sex determination are fully understood. It must be recognized that in fish sex is determined genetically (syngamic), but the process of sex differentiation remains labile and is metagamic, i.e., amenable to manipulation by external factors (Pandian & Koteeswaran, 1998). In contrast to the highly developed sex chromosome dimorphism in mammals (male heterogamety; XX/XY system) and birds (female heterogamety; ZW/ZZ system), heteromorhpic sex chromosomes are not distinguishable by classical cytogenetic staining techniques in most of the fish species (George & Pandian, 1995; George et al., 1994). Presumably, the evolution of heteromorphic sex chromosomes depends on the suppression of crossing-over between sex-allele carrying chromosomes due to rearrangements, such as inversions (Pandian & Koteeswaran, 1998). This process is frequently accompanied by an accumulation of repetitive sequences on one particular chromosome usually, the Y or W. Due to this; C- banding technique is generally used to identify heterochromatic sex chromosomes. Rishi (1989) classified fish sex chromosomes under the following systems in Indian fishes: XX-XY, XX-X0, ZZ-ZW and ZZ-Z0. However, of the 1000s of karyologically defined fishes, nearly 10% only have morphologically dissimilar chromosomes (Pandian & Koteeswaran, 1998). Pandey & Lakra (1996) reported the presence of B-chromosome in female Clarias batrachus. Efforts to identify sex chromosomes in protogynous and protandrous fishes from Indian coast have not been successful (Padhi & Mandal, 2000). In some exotic ornamental fishes, presence of sex determining genes has been demonstrated by breeding experiments with hormonally sex reversed individuals (Pandian, 1993; George & Pandian, 1995). Sex control The mono-sex fish culture is advantageous where one sex grows bigger than the other sex or sterile population is economical in fish farming by conversion of ingested food energy into flesh with minimum wastage of energy in to gonadal maturation. About 20 per cent of total ingested food energy is used in reproduction which can be channelized in production of fish flesh. Sex steroids being primary inducers of various reproductive phenomena, there is ample scope for reproductive manipulation of sex. Adequate application of either androgen or estrogen to the juveniles overrides the intrinsic sex determining mechanism and directs it either in a male or female direction or induces sterility without altering the genotype. Das et al. (1986) obtained 75 per cent male production by feeding methyl testosterone (MT) to tilapia at the rate of 30μg/g feed. At Madurai Kamaraj University, 100% percent masculinization and feminization were achieved in tilapia and ornamental species such as Betta splendens, Poecilia reticulata and Brachydanio rerio (Pandian et al., 1999; Pandian & Koteeswaran, 1998; Pandian & Sheela, 1995; Kavumpurath & Pandian, 1992 a, b, 1993; Varadaraj, 1990 a, b; Varadaraj & Pandian, 1989; 1990) by administration of steroids. YY super males and ZZ females of some of the ornamental fishes produced by endocrine sex reversal are viable and are brilliantly coloured and highly active compared to the normal ones, hence, fetch good price. The first report of production of super male fish (tilapia) from India is of Varadaraj and Pandian (1989 a) by integrating endocrine sex reversal with gynogenetic technique. George and Pandian (1995) produced viable ZZ females in the female heterogametic black molly and Kavumpurath & Pandian (1992 d, 1994); YY super male black molly and YY females of Poecilia reticulata by endocrine sex reversal; while George et al. (1994) reported inviability of the YY zygote of the fighting fish, Betta splendens. Sex-reversal in carps using steroid hormones, required for purposes such as the breeding plans, appears to be more difficult to achieve routinely than in cichlids or salmonids (Penman et al., 2005). This appears to be because the onset of sexual differentiation (the “labile 15

period” for sex-reversal) occurs later in cyprinids and may be dependant on size as well as age, thus making the exact timing of treatments difficult. There may also be genetic variation in Indian carps for response to treatment with 17α-methyl testosterone (Penman et al., 2005). Sex reversed red tilapia produced by M/s Vorion Chemicals were common few years back in Tamil Nadu and Kerala for human consumption, but at present sex reversal in fish is confined only to ornamental species in Indian scenario. Conclusion In India, fisheries science has witnessed a moderate research activity on genetics and its allied areas in the last two decades. Future of genetics research with respect to aquaculture development and fisheries management is promising, notwithstanding some technical problems, which stand in the way of its commercialization. A lot more research efforts are needed in this area to generate process and products of applied interest in aquaculture and capture fisheries management. Fundamental research using molecular markers will lead to a better understanding of genetic structures of wild population. This can lead to improved management of specific fisheries and selective breeding programmes, but will also lead to a greater ability to catalogue and better manage fish diversity. Dissemination of improved breeds and quality seed production are now receiving a greater degree of attention, including the targeting of poorer farmers. Molecular genetics is a rapidly advancing field that has already given valuable tools such as polymorphic molecular markers and gene transfer. It is likely that further developments in this area will have applications in research on the genetics of not only the Indian major carps, but also other medium sized carps, catfishes and shellfishes such as Penaeus indicus of local importance in India, hopefully to the benefit of both conservation and exploitation of these resources. Selective breeding programmes can be made more effective if the selection of broodstocks can be done more efficiently. Finding of PCR based molecular markers that co-segregate with economically important traits like growth rate or disease resistance (marker assisted selection) will be useful in this regard. Targeted effort is required to explore ESTs from fish species used in Indian aquaculture. Bioinformatics is evolving rapidly as a branch of biology with the merger of molecular biology and computer science as part of it. To process and utilize the molecular information efficiently, bioinformatics will be of great help. There is also a need for capacity building and infrastructure development for large-scale genomic exploration of Indian fishery resources integrating knowledge from genomics, bioinformatics and molecular biology. While polyploidy has made little direct impact on aquaculture in India, triploidy is likely to make further contributions in the future especially in mariculture of bivalves such as pearl oysters (Pinctada fucata, P. margaritifera), mussels (Perna viridis, Perna indica) and edible oysters (Crassostrea spp.). Naupliar length of Indian stains of Artemia restricts its use as live food in aquaculture and the whole fish culture practice in the country is dependent on imported Artemia cysts. Attempts can be made to reduce the naupliar size of Indian brine shrimp using selective breeding techniques that will be beneficial to the aquaculture industry in a big way. The development of effective protocols for cryopreservation of fish gametes will further enhance the development of aquaculture industry by making it more sustainable. It also facilitates the process of dissemination of genetically improved varieties of fish and the exchange of germplasm in an easy and effective manner in addition to its leading role in conservation of endangered species. Establishment of fish gene banks of economically important and endangered species has already been initiated in India in some fishery institutes such as NBFGR. In view of the fact that milt accessions can be made only for prioritized and selected fish species as it requires species-specific protocols; and egg and embryo cryopreservation remaining unsuccessful, embryonic stem cell preservation and cloning of prioritized Indian fishes has to be given priority, but the methodology of needs further refinement.

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In transgenic fish research, continuous efforts are necessary to isolate and identify novel useful genes of Indian species; and suitable promoters of fish-origin to regulate the expression of the transgene in a tissue-specific manner at an appropriate level. DNA microarray technique in particular is likely to become a powerful tool for this purpose. Gene transfer techniques should be improved and methods to be developed to improve and control the number of copies of a gene to be incorporated and its location of integration in the chromosomes. The physiological, nutritional and environmental factors that will maximize the performance of transgenic individuals should be determined. Bio-safety issues of transgenic fishes also need very careful attention during field trials. But, like the sex reversed fishes, consumer acceptance of GMO food fish is still doubtful. Transgenic research related to modified fish for ornamental trade, pharmaceutical and other industrial products are more attractive and with less controversies. Due attention to be paid on research (i) to generate newer varieties of expensive endemic ornamental fish varieties and (ii) for value addition in commonly available and less expensive other native species such as Puntius spp., Mystus spp. and glass-fishes. Development of DNA probe and PCR techniques are useful for the identification of viral, bacterial and fungal pathogens that cause loss to the aquaculture sector. The development of DNA chips in recent years has enhanced the prospect of screening and identifying the pathogens in a very short time. However, the application of DNA chips in aquaculture will depend on the growth of molecular information about the genes and genomes of fishes and their pathogens in the years to come. Indian landings of aquatic resources have increased more than eight fold during the last fifty years (Pillai & Katiha, 2004). Larger share of this production came from the capture fishery sector, which has been over-exploited, leading to decline of fish diversity in different ecosystems. On the other hand, aquaculture provides greater scope for increasing fish production. Thus, fish genetics as a growing discipline will provide innovative means in the future and appears to be an assuring approach for enhancing aquaculture productivity and food security of the nation and in reducing the fishing pressure on natural resources. However, the ability to develop and adapt technological advances in fish genetics in a timely fashion will depend on stimulating interaction of biotechnologists with fishers, fish farmers and those involved in resource management, policy development and fundamental research. References Ananthy, V., Venugopal, T., Koteeswaran, R., Pandian, T.J., Mathavan, S., 2001. Cloning, sequencing and expression of cDNA encoding growth hormone from Indian catfish (Heteropneustes fossilis). J. Biosci. 26, 315-324. Ayyappan, S., Pillai, N. G. K., 2005. Indian fisheries in global context. Indian farming 55(7), 16 – 24. Ayyappan, S., Ponniah, A. G., Reddy, P. V. G. K., Jana, R. K., Mahapatra, K. D., Basavaraju, Y, 2001. Aquaculture genetics research in India: an overview. p. 43-50. In: Gupta, M. V. & Acosta, B.O. (eds.). Fish genetics research in member countries and institutions of the International Network on Genetics in Aquaculture. ICLARM Conference Proceedings. 64, 179p. Barman, H. K., Barat, A., Yadav, B.M., Banerjee, S., Meher, P.K., Reddy, P. V. G. K., Jana, R. K., 2003. Genetic variation between four species of Indian major carps as revealed by random amplified polymorphic DNA assay. Aquaculture 217, 115 - 123. Blaxter, M. L., 2004. The promise of a DNA taxonomy. Phil. Trans. R. Soc. Lond. B. 359, 669-679. Benzie, J. A. H., 2002. Biotechnology: Innovative instruments for resource managers. In Tropical Marine Environment: Charting Strategies for the Millennium. Yusoff, F. M., Shariff, M., Ibrahim, H. M., Tan, S. G. and Tai, S. Y. (eds.), pp. 567 – 576. Malacca Straits Research and Development Centre (MASDEC), Universiti Putra Malaysia, Serdang, Malaysia. Botstein, D., White, R. L., Skolnick, M., Davis, R. W., 1980. Construction of a genetic linkage map in man using restriction fragment length polymorphisms. Am. J. Hum. Genet. 32, 314 – 331.

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CCMB, 2002. Centre for Cellular and Molecular Biology: Annual Report, 2001- 2002 Centre for Cellular and Molecular Biology, Hyderabad 500 007, India, 269p. Das, M., Lakra, W. S., 1998. Somatic and meiotic chromosomes of the freshwater prawn, Macrobrachium rosenbergii (de man) used in aquaculture. Ind. J. Fish. 45(3), 339-343. Das, P., Bhaumik, M.L., Mondal, S.K., Ghosh, P.K., Bhaumik, V. & Pandit, P.K., 1989. Preliminary observations on selection for faster growth rate in pearl spot, Etroplus suratensis (Bloch). In: Fish Genetics in India. Das & Jhingran (eds.), pp: 163 – 167, Today & Tomorrow Publ., New Delhi, India. Das, P., Kapoor, D., Mahanta, P.C. (1986). Genetic improvement of fish stock and resource conservation. NBFGR Bulletin No. 1, 29 p., National Bureau of Fish Genetic Resources, Allahabad, U.P., India. Das, P., Prasad, H., Meher, P. K., Barat, A., Jana, R. K., 2005 a. Evaluation of genetic relationship among six Labeo species using randomly amplified polymorphic DNA (RAPD). Aquaculture Research 36, 564 - 569. Das, P., Barat, A., Meher, P. K., Ray, P. P., Majumdar, D., 2005. Isolation and characterization of polymorphic microsatellites in Labeo rohita and their cross-species amplification in related species. Molecular Ecology Notes 5(2), 231- 233. David, C. J., Pandian, T. J., 2005. Cadaveric sperm induces intergeneric androgenesis in the fish, Hemigrammus caudovittatus. Theriogenology 65 (6), 1048 – 1070. David, C. J., Pandian, T. J., 2006. GFP reporter gene confirms paternity in the androgenote Buenos Aires tetra, Hemigrammus caudovittatus. J. Exp. Zool. 305A (1), 83 – 95. Diwan, A. D., Kandasami, K., 1997. Freezing of viable embryos and larvae of marine shrimp, Penaeus semisulcatus de Haan. Aquacult. Res., 28: 947 – 950. Diwan, A.D., Shoji Joseph, Nandakumar, A., 1994. Cryobanking potential of marine shrimp gametes. Mar. Fish. Infor. Serv. T. & E. Series, Cent. Mar. Fish. Res. Inst., Cochin, 131, 16 – 18. George, T., Pandian, T. J., 1995. Production of ZZ females in the female heterogametic black molly Poecilia sphenops by endocrine sex reversal and progeny testing. Aquaculture 136, 81 – 90. George, T., Pandian, T. J., Kavumpurath, S., 1994. Inviability of the YY zygote of the fighting fish, Betta splendends. Israel J. Aquacult., Bamidgeh 46, 3 – 8. Gopalakrishnan, A., Musammilu, K. K., Muneer, P.M.A., Lal, K. K., Kapoor, D., Ponniah, A. G., Mohindra, V., 2004. Microsatellite DNA markers to assess population structure of red tailed barb, Gonoproktopterus curmuca. Acta Zoologica Sinica 50(4), 686 – 690. Gopalakrishnan, A., Muneer, P.M. A., Thomas, P. C., Lal, K. K., Mohindra, V., Basheer, V. S., Kapoor, D., Ponniah, A. G., 2006. Identification of polymorphic allozyme markers for population structure analysis in endemic yellow catfish, Horabagrus brachysoma (Gunther, 1864). Indian J. Fish. 53 253 – 261. Gopalakrishnan, A., Lal, K. K., Ponniah, A. G., 1997. Esterases In Indian major carps -’rohu’ (Labeo rohita) and ‘mrigal’ (Cirrhinus mrigala) (Teleostei, Cyprinidae). Indian J. Fish. 44(4), 361 368. Gopalakrishnan, A., Lal, K. K., Thomas, P.C, Lijo, J., Sathianandan, T. V., Mohindra, V., Punia, P., Ponniah, A.G., Lakra, W. S., 2006. Genetic structure of endangered monotypic marine fish Lactarius lactarius from east and west coasts of India. Marine Biology (communicated). Gopalakrishnan, A., Thakur, K.L., Ponniah, A.G., Kumar, K., Dayal, R., 1999. Cryopreservation of brown trout (Salmo trutta fario) sperm: the influence of extender composition and fertilization procedure. Fishery Technology 36(2), 104-109. Gopalakrishnan, A., Thomas, P.C., Ponniah, A.G., 1996. Interspecific differences in allozyme patterns of marine catfishes – Tachysurus (Arius) maculatus, and T. subrostratus. In: Das, P. et al. (ed.) Fish Genetics and Biodiversity Conservation for sustainable production, p 55, Natl. Bureau of Fish Genet. Resources, Lucknow and Nature Conservators, Muzaffarnagar, U.P.

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