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Chapter: Aquaculture Principles and Practices: Reproduction and Genetic Selection

Molecular techniques in Aquaculture

1. Inbreeding and maintenance of genetic quality, 2. Intraspecific crossbreeding, 3. Interspecific hybridization, 4. Genetic selection, 5. Correlated responses, 6. Polyploidy, 7. Sex manipulation and breeding, 8. Gynogenesis, androgenesis and cloning, 9. Interspecific nuclear transfer, 10. Linkage mapping, 11. Marker-assisted selection, 12. Combining genetic enhancement programmes,

Molecular techniques


The production challenges facing aquaculture include disease resistance, tolerance of handling, enhanced food conversion and spawning manipulation.These areas concern wild animals adopted for productive ‘domestication’, which has begun causing changes in gene frequencies and performance of fish under domestication. Directed breeding programmes did not develop until comparatively recently. Fish genetic programmes became more common with greater knowledge of breeding inheritances. Molecular-based knowledge, developed in the 1980s, has continued to gain prevalence. It is now well established in the selective breeding, biotechnology and molecular genetics of finfish, and rapidly developing for aquatic invertebrate domestication.


When wild fish are moved to aquaculture settings, an organism better suited to aquaculture environment begins to evolve as a result of domestication. Domestication effects can be observed in some fish within a few generations after removal from the natural environment (Durham, 1996a). In channel catfish (Ictaluruspunctatus) an increased growth rate of 3–6 percent per generation has been observed. The oldest domestic strain usually performs better in the aquaculture environment than wild strains, though there are exceptions. Channel catfish strains differ in growth, disease resistance, body conformation, dressing percentage, vulnerability to angling and seining, age of maturity, time of spawning, fecundity and egg size (Dunham and Smitherman, 1984; Smitherman and Dunham, 1985). Strains of rainbow trout (Oncorhynchus mykiss) show similar variability (Kincaid, 1981).


Inbreeding and maintenance of genetic quality


Losses due to inbreeding should be prevented, in order to maximise increased production resulting from genetic enhancement. This applies particularly to species with high fecundity where few brood stock are necessary to meet demands for fry and brood stock replacement. The effects of inbreeding are well documented and can result in a decrease of 30 per cent or more in terms of growth, survival and reproduction (Kincaid 1976a, 1976b, 1983a; Dunham, 1996b).


Intraspecific crossbreeding


Crossing of different strains may increase growth rate but heterosis may not be obtained in every case. Increases of 55 per cent and 22 per cent growth rate of channel catfish and rainbow trout cross-breeds respectively were achieved using this technique (Dunham and Smitherman, 1983; Dunham, 1996b). Chum salmon cross-breeds, however, have shown no increase in growth rates compared with parent strains (Dunham, 1996a). Common carp cross-breeds usually show low levels of heterosis (Moav et al., 1964; Wohlfarth, 1993; Hulata, 1995); however, those that exhibited positive heterosis are the basis for carp aquaculture in Israel,Vietnam, China and Hungary. During the last 35 years, crossbreeding has been tested in more than 140 crosses.Three were chosen, based on 20 per cent improvement in growth rate and other features. Now approximately 80 per cent of common carp production comes from these cross-breeds. Production of gynogenetic female lines and gynogenetic sex-reversed inbred male lines from common carp with the best continuing ability form an important part of the Hungarian crossbreeding programme. The growth rate of F1 cross-breeds was only 10 per cent higher than controls (Bakos and Gorda, 1995).

Kirpichnikov (1981) successfully produced a new strain of cold-resistant carp for cold zones in northern Russia using local carps and Siberian wild carps from the river Amur. In Israel, over 20 years of crossing common carp strains revealed that crosses using the strain ‘DOR-70’ (Wohlfarth et al., 1980) and the Croa-tion line ‘Nawica’ produced fast growth, and this is one of the most popular crosses for Israeli carp production (Wohlfarth, 1993).


In Indonesia, strain development using artificial gynogenesis and sex-reversal resulted in 10 common carp inbred lines, which were used for cross-breeding (Sumantadinata, 1995). In Vietnam, eight local varieties of common carp along with ‘Hungary’, ‘Ukraine’, ‘Indonesia’ and ‘Czech’ strains are maintained, with significant heterosis observed in F1 generations of cross-breeds. Hungarian and Indonesian strains have subsequently been used for carp selection and cross-breeding programmes throughout Vietnam (Thien and Trong, 1995).


Under various rice field conditions, growth rates of different strains of Nile tilapia and their crosses were superior to pure strains (Circa et al., 1994). Breeding programmes are alsounder development for Java or silver barb, another economically important fish species in Southeast Asia. The growth rate of females from six crosses was 23 per cent higher than average growth rate of the parent strains.


Cross-breeds of different strains of European catfish, Silurus glanis, have outstanding adapt-ability under warm water holding conditions (Krasznei and Marian, 1985). Studies on domestic channel catfish also showed greater heterotic growth rates in domestic x wild crosses (Dunham and Smitherman, 1983). The same was found in rainbow trout crosses (Gall, 1969; Gall and Gross, 1978; Kincaid, 1981; Ayles and Baker, 1983). Strain mating incompatibilities can, however, occur and can impede fry output.


Interspecific hybridization


Interspecific hybridization has been used to increase growth rate, manipulate sex ratios, produce sterile animals, improve flesh quality, increase disease resistance, improve tolerance of environmental extremes and improve a variety of other traits that make aquatic animal production more profitable. Although interspecific hybridization rarely results in an F1 suitable for aquaculture application, there are a few significant exceptions. Channel catfish (Ictalurus punctatus) females x blue catfish (I.furcatus) males is the only cross that exhibitssuperior growth rate, growth uniformity, disease resistance, tolerance of low oxygen levels, dressing percentage and harvestability (Smitherman and Dunham 1985). However mating problems between the two species have prevented commercial utilization.


The ‘Sunshine’ bass is cross between white bass (Morone chrysops) and striped bass (M.saxatillis), which grows faster and has betteroverall culture characteristics than either parent species (Smith, 1988). In addition, crosses of the silver carp (Hypophthalmichthysmolitrix) and bighead carp (Aristis nobilis)(Hypophthalmichthys nobilis); black crappie (Pomoxis nigromaculatus) and P. annularis (Hooe et al., 1994); and African catfish hybrids, Clarias gariepinus, and Heterobranchus longifilis and H. bisorsalis (Salami et al., 1993;Nwadukwe, 1995), all show faster growth than the parent species. Numerous crosses of common carp with rohu (Labeo rohita), mrigal


(Cirrhinus mrigala), catla (Catla catla) (Khan et al.,1990);tambaqui (Colossoma macropomum)and Piaractus brachypoma and P. mesopotamicus (Senhorini et al., 1988) and green sunfish(Lepomis cyanellus) with bluegill (L.macrochirus) (Tidwell et al., 1992), have enhanced performance of aquaculture production systems.


Hybrids between Sparus aurata and Pagrusmajor (both belonging to Sparidae) developedvestigial gonads when two or three years old and were sterile (Knibb et al., 1998). Similar vestigial gonads were observed in offspring of the reciprocal crosses.


Genetic selection


Very little was done in the genetic selection of fish prior to 1970, but it has grown significantly since then (Dunham, 1996a). Response to selection for growth rate in aquatic species is very good (7 to 10 times in farmed aquatic species). Fecundity is also higher.


Selection of body weight and disease resistance in salmonids has been particularly successful (Embody and Hyford, 1925). With respect to body weight, a 30 per cent increase in rainbow trout was achieved within six generations of selection (Kincaid, 1983b).

Responses can differ depending on the direction of selection. Body weight of common carp in Israel was not improved over five generations, but could be decreased (Moav and Wohlfarth, 1976).


Several authors have reported that mass selection improved body weight in tilapias (Oreochromis mossambicusO. aureus and red tilapia). However, selection was less successful. Body weight of common carp appears unresponsive to selection; but body conformation can be dramatically changed (Ankorian, 1996).


Correlated responses


Although selection for body weight has generally been associated with positively correlated responses (e.g. increased survival and disease resistance), there are examples of long-term selection resulting in decreased bacterial resistance, possibly due to genetic correlation changes or inbreeding.




Triploid fish are generally sterile. Females produce less sex hormones and although triploid males may develop secondary sexual characteristics and exhibit spawning behaviour, they are generally unable to reproduce.


Channel catfish triploids become larger than diploids at about nine months of age (90g) when grown in tanks (Wolters et al., 1982). In tank experiments, the triploids converted feed more efficiently than diploids, had 6 per cent greater carcass yield at three years of age (Chrisman et al., 1983) and were darker than diploids.

Polyploidy in the Asian catfish, Clariasmacrocephalus, was induced by cold shock andresulted in 80 per cent triploidy (Na-Nokorn and Legrand, 1992). The effects on survival were not significantly different from diploid controls during first two months, but in the third to fifth month, triploid fish showed lower survival rates and body weight compared to the diploid group.


Polyploidy is not commercially feasible for all species. Bramick et al., (1995) suggest that the use of triploid tilapia would reduce unwanted reproduction and stunting and would significantly increase yields from pond culture.


Sex manipulation and breeding


Various strategies utilizing sex reversal and breeding progeny testing, gynogenesis and androgenesis can lead to the development of predominantly or completely male or female populations or a ‘supermale’ genotype (YY).


The primary aim is to take advantage of sexually dimorphic characteristics (including flesh quality).


Sex reversal and breeding have allowed production of YY channel catfish males that can be mated to normal XX females to produce all-male XY progeny.


Gynogenesis, androgenesis and cloning


Gynogenesis and androgenesis are techniques to produce rapid inbreeding and cloned populations. Androgenesis or all-male inheritance is more difficult to accomplish than gynogenesis, since diploidy can only be induced in androgens at first cell division, a difficult time to manipulate the embryo. Also, since androgens are

totally homozygous, a large percentage with deleterious genotypes probably die.


Fully inbred clonal lines have been produced in Zebrafish, ayu, common carp, Nile tilapia and rainbow trout (Komen et al., 1991; Sarder et al., 1999).


Interspecific nuclear transfer


Interspecific nuclear transfer has been accomplished for cyprinids in China, resulting in embryos with the cytoplasm and mitochondrial


DNA of the host species and the nuclear DNA of the donor species. As a result these fish may later serve as key for the application of transgenic technology.


Compared to the thousand years of aquaculture and its genetic improvement programmes, aquaculture genomics and gene mapping are truly in their infancy. Molecular genetics is less than 30 years old, although DNA was only discovered about 50 years ago. However, the late 1990s have seen an explosion in genomics and gene mapping of aquatic organism DNA sequences.


The first successful form of gene transfer – genetic engineering – was accomplished in China in 1985 and has subsequently been achieved in other countries. Most of this work focused on hormone enhancement of growth (size and rate), with results ranging from zero per cent up to an incredible 300 per cent enhancement under some conditions. Due to the lack of fish gene sequences, initial transgenic research in the mid-1980s employed mammalian growth hormone (GH) gene constructs, which enhanced growth in some but not all species examined (Zhu et al., 1986; Enikolopov et al., 1989; Zhu, 1992; Gross et al., 1992; Wu et al., 1994). Salmonids showed no effect (Guymard et al., 1989a,b; Penman et al., 1991), despite the fact that carp, catfish, zebrafish and tilapia are very responsive to growth enhancement (Martinez et al., 1996), providing the first convincing evidence that this can be achieved in fish by transgenesis.


When a gene is inserted with the objective of improving a specific trait, it may affect another trait. Such ‘pleiotropic’ effect can be positive or negative; thus it is important to evaluate all important traits in the transgenic fish, not just the trait under active alteration. Transfer of growth hormone genes has been documented to affect body shape, feed conversion efficiency, disease resistance, reproduction, tolerance of low oxygen concentrations, carcass yield, swimming ability and even predator avoidance.


Rainbow trout growth hormone (rt GH) transgene reduces survival of common carp and the number of F2 progeny inheriting the transgene is less than expected. Differential mortality or loss of the recombinant gene during meiosis is a likely explanation, since transgenesis was evaluated after the fish reached fingerling size. Remaining transgenic individuals, however, showed higher survival than controls when subjected to a series of stressors such as low dissolved oxygen (Chatakondi, et al., 1995).



Linkage mapping


Aquaculture genomics has seen dramatic progress over the last 10 years (Kocher et al., 1998; Liu and Dunham, 1998; Waldbieser et al., 1998). This includes progress in construction of framework genetic linkage maps for catfish (Li et al., 2000) tilapia (Lee and Kocher, 1996;Kocher et al., 1998; Agresti et al., 2000; McConnel et al., 2000) and oysters, Crossostrea and Ostrea spp. Genomic mapping of these phyletic groups was done recently (USDA) as a regional project.

Much work is ongoing on production of framework linkage maps with greater numbers of markers, particularly type I markers of known genes.


In the last few years, large numbers of molecular markers have been developed and evaluated for application in the culture of catfish as well as other commercially important species. Of the several types of markers evaluated, microsatellites and AFLP (amplified fragment length polymorphisms) were most reliable, efficient and reproducible for genetic linkage mapping in catfish (Liu et al., 1999a,b,c,d).



Marker-assisted selection


In aquaculture species, much effort is devoted to quantitative trait loci (QTL) mapping. QTL markers for growth, feed conversion efficiency, tolerance of bacterial disease, spawning time embryonic developmental rates and cold tolerance have been identified in channel catfish, rainbow trout and tilapias (La Patra et al., 1993, 1996).


Molecular genetics through gene mapping have been used for studying genetic variation among different groups. DNA analysis is preferred over conventional protein analysis for determining genetic affinities and differences, though protein analysis is faster and less costly. While earlier linkages were studied using isozyme markers (Liuet al., 1999a,b), recent catfish mapping has used microsatellite and AFLP markers.


Combining genetic enhancement programmes


The best genotypes of aquaculture applications in the future will be developed by using a combination of traditional selective breeding and the new biotechnologies. Initial experiments indicate good potential for this combined approach, with examples using mass selection and cross-breeding, genetic engineering and cross-breeding and sex reversal and polyploidy, showing that all work more effectively in com-bination than alone. Several studies in aquaculture transgenics have dealt with improvement of growth rate of selected species. It has been indicated that it is possible to genetically improve the food conversion efficiencies (FCE); more precisely, it is an enhanced ability to convert measured parameters, such as (dry) weight/energy in food, into growth in the individual organism (Brett, 1995). This has also been referred to as ‘effectiveness with which food is converted to saleable fish product’ (Doupe and Lympbery, 2003). According to these authors, if it is assumed that there is considerable genetic variation in FCE as found in terrestrial livestock, especially in pigs and poultry (Luiting et al., 1994), improvements in FCE through genetic methods is possible subject to certain refinements in experimental methods. These would enable the determination of feed intake by individual fish and the optimal time over which the FCE is to be tested, and also the availability of information on genetic correlation between FCE and food intake conditions.

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