SELECTIVE BREEDING

The species that have seen the most improvement are common carp, rainbow trout, Atlantic salmon and tilapia, channel catfish and ornamental fishes. Modern programmes for carp were established in Central and Eastern Europe (Kirpichnikov Reference Kirpichnikov1981), and improved strains contribute to the bulk of European and Asian production, with programmes to further develop these in Vietnam and India (Penman et al. Reference Penman, Gupta and Dey2005). Recent studies have shown that cross breeding and pedigree-based selection can still produce improvements (Vandeputte Reference Vandeputte2009).

Rainbow trout has also had a long history of domestication, in the USA (Gall & Huang Reference Gall and Huang1988), Norway (selected for seawater production) (Gjedrem Reference Gjedrem1992), Finland (Kause et al. Reference Kause, Ritola, Paananen, Mäntysaari and Eskelinen2003) and Denmark (Henryon et al. Reference Henryon, Jokumsen, Berg, Lund, Pedersen, Olesen and Slierendrecht2002). But the uptake of selected strains by the industry has been slow; for instance, the largest commercial US egg supplier, Trout Lodge, only started a family-based breeding programme within the last 5 years. Short-term improvements such as all-female production or all-female triploids have been adopted but most eggs still come from mass-selected domesticated strains.

Norwegian Atlantic salmon programmes were initially government funded, but are now run by private companies (Aquagen and Salmobreed; Gjedrem Reference Gjedrem2005). Individual and family performance is assessed for a range of commercial traits (growth, sexual maturation and body conformation) and, more recently, disease resistance (infectious pancreatic necrosis virus (IPNV), Aeromonas, infectious salmon anaemia (ISA)). Estimated gains are between 8 and 10% per generation (Gjoen & Bentsen Reference Gjoen and Bentsen1997). Similar programmes have also been developed by Landcatch Natural Selection in Scotland and Chile, Stofnfiskcur in Iceland and Aquachile in Chile. Most farmed eggs now come from scientific breeding programmes, with significantly improved performance. Genomic technologies, including quantitative trait loci (QTL) and marker-assisted selection (MAS) approaches, are already benefiting improved virus resistance (Houston et al. Reference Houston, Haley, Hamilton, Guy, Tinch, Taggart, McAndrew and Bishop2008; Moen et al. Reference Moen, Baranski, Sonesson and Kjøglum2009).

Tilapia have become the second most important group of farmed fish in the world (>2·7 million t in 2008) with Oreochromis niloticus becoming the dominant species in fresh water. Early selection work was hindered by the degraded nature of many of the commercial strains being used. The Genetically Improved Farm Tilapia (GIFT) programme, starting in 1987 in the Philippines, systematically compared wild and commercial strains in a variety of aquaculture environments and began family-based selection for growth. The work was further developed by Worldfish (CGIAR, see Acosta & Gupta Reference Acosta, Gupta, De Silva and Davy2010). Other programmes have either used the GIFT strain as a starting point (Genomar 2008) or have constructed new synthetic populations using a number of selected lines (Rutten et al. Reference Rutten, Komen and Bovenhuis2005). The bulk of tilapia production, however, does not come from improved sources and producers use direct hormone sex-reversal of mixed sex fry to produce phenotypic all-male populations to overcome problems of excessive fry production during growout (see Beveridge & McAndrew Reference Beveridge and McAndrew2000).

Selective breeding programmes have been more recently applied to other marine high-value species: European sea bass, Gilthead sea bream and Turbot. By 2005 it was assumed that as much as 0·80 of the European sea bass production already came from commercial populations which had undergone some level of genetic improvement and at least one major Greek company has initiated (in 2002) a large-scale family-based selective breeding programme on sea bream. Programmes on other high-value species, such as cod, halibut and tuna, are also under way.

HYBRIDIZATION

The ease of gamete collection in fish means that there are many references to inter-specific and inter-generic hybridization and the potential for using hybrids in aquaculture (Bartley et al. Reference Bartley, Rana and Immink2001). Hybrids have become important at the national level in several countries, e.g. O. niloticus×Oreochromis aureus in Israel because of the skewed male sex ratio and cold tolerance of the hybrid (Hulata et al. Reference Hulata, Wohlfarth, Karplus, Schroeder, Harpaz, Halevy, Rothbard, Cohen, Israel and Kavessa1993). In the USA, the sunshine bass (Morone chrysops×Morone saxatilis) grows faster and has better overall culture characteristics than either parental species under commercial culture conditions (Smith Reference Smith1988). However, despite the large numbers of reported hybrids few have been successfully cultured for extended periods because of the added complexity of production or because of introgression of the hybrid back into the parental species and loss of beneficial characteristics.

CHROMOSOME SET MANIPULATION

A range of genetic and environmental manipulations can be applied to aquatic species. If normally fertilized eggs are heat or pressure shocked at the 2nd meiotic division, a triploid (3n) embryo, containing three chromosome sets, is produced. Protocols are now available for over 30 different fish and shellfish species (Dunham Reference Dunham2004). Triploids are effectively sterile and are used in the production of larger rainbow trout, usually from all-female strains (Bye & Lincoln Reference Bye and Lincoln1986), channel catfish (Wolters et al. Reference Wolters, Lilyestrom and Craig1991), common carp (Basavaraju et al. Reference Basavaraju, Mair, Mohan Kumar, Pradeep Kumar, Keshavappa and Penman2002) and in other species whose maturation slows growth. They are also used to improve growth rate and flesh quality in oysters (Guo et al. Reference Guo, DeBrosse and Allen1996). Triploids can be used to reduce the risk of stocked or escaped farmed strains interbreeding with native populations (Kozfkay et al. Reference Kozfkay, Dillon and Schill2006) or exotic species, such as grass carp for aquatic weed control, becoming established (Wattendorf Reference Watterdorf1986). Triploid sterility is also proposed to stop impacts from escaped transgenic fish (Mair et al. Reference Mair, Nam, Solar, Kapuscinski, Hayes, Li and Dana2007). A shock at first mitotic division will produce a tetraploid embryo with four sets of chromosomes (4n). Male 4ns produce diploid sperm and can be crossed with normal fish to produce 3n directly avoiding having to shock large numbers of eggs, e.g. for rainbow trout (Myers et al. Reference Myers, Hershberger and Iwamoto1986).

Techniques for parthenogenesis can derive offspring from wholly maternal or paternal origins (Komen & Thorgaard Reference Komen and Thorgaard2007). These use gamma or ultraviolet radiation to destroy the nuclear DNA in eggs or sperm; the treated gamete is then fused with an untreated sperm or egg to produce a haploid embryo, which can be made diploid by inhibiting the second meiotic (meiotic gynogenetic) or first mitotic division (mitotic gynogenetic or androgenetic). Although still mainly used as research tools they can generate unique genotypes for analysing complex traits or generating new strains. Containing sufficient DNA, haploid embryos can be used for gene-mapping (Kocher et al. Reference Kocher, Lee, Sobolewska, Penman and Mcandrew1998), generating unusual genotypes such as YY male tilapia broodstock used to produce all-male XY tilapia offspring by crossing with normal XX females (Myers et al. Reference Myers, Hershberger and Iwamoto1986). Meiotic gynogenetic silver barb (Puntius goniontus) are all-female, can be produced in large numbers and can be sex-reversed to generate neo-males without the need for progeny testing; they are now used in large numbers as broodstock for commercial production of all-female fry (Pongthana et al. Reference Pongthana, Penman, Baoprasertkul, Hussain, Islam, Powell and McAndrew1999). Gynogenesis was similarly used in the development of all-female turbot (Cal et al. Reference Cal, Vidal, Martinez, Alvarez-Blazquez, Gomez and Piferrer2006).

Double haploid individuals from either a mitotic gynogenetic or androgenetic background can also be used to generate clonal or isogenic lines after a second round of parthenogenesis. Apart from model species such as zebrafish (Streisinger et al. Reference Streisinger, Walker, Dower, Knauber and Singer1981), clonal lines have been produced in tilapia (Müller-Belecke & Hörstegen Schwark Reference Müller-Belecke and Hörstegen-Schwark1995; Hussain et al. Reference Hussain, Penman and McAndrew1998; Sarder et al. Reference Sarder, Penman, Myers and McAndrew1999) rainbow trout (Scheerer et al. Reference Scheerer, Thorgaard, Allendorf and Knudsen1986; Thorgaard et al. Reference Thorgaard, Scheerer, Hershberger and Myers1990; Quillet et al. Reference Quillet, Dorson, Le Guillou, Benmansour and Boudinot2007) and common carp (Bongers et al. Reference Bongers, Bovenhuis, Van Stokkom, Wiegertjes, Zandieh-Doulabi, Komen and Richter1997). These are useful for disease and vaccine studies and an important resource for whole genome sequencing.

GENE TRANSFER TECHNOLOGIES

Transgenic fish containing the human growth hormone gene (hGHg) have been produced in goldfish (Zhu et al. Reference Zhu, Li, He and Chen1985), rainbow trout (Penman et al. Reference Penman, Beeching, Penn and Maclean1990), channel catfish (Dunham et al. Reference Dunham, Eash, Askins and Townes1987) and Nile tilapia (Brem et al. Reference Brem, Brenig, Horstgen-Schwark and Winnacker1988). However, less than 0·05 integrated a copy of the construct into the host genome, and a number of other problems arose. Improved techniques resulted in growth enhancement in rainbow trout (Penman et al. Reference Penman, Beeching, Penn, Rahman, Sulaiman and Maclean1991), common carp (Zhang et al. Reference Zhang, Hayat, Joyce, Gonzalez Villasenor, Lin, Dunham, Chen and Powers1990), channel catfish (Dunham et al. Reference Dunham, Ramboux, Duncan, Hayat, Chen, Lin, Gonzalez-Villasenor and Powers1992) and tilapia (Rahman & Maclean Reference Rahman and Maclean1999). The most dramatic results were with total piscine constructs using either an ocean pout antifreeze promoter (ocAFP) controlling a chinook salmon growth hormone (GH) cDNA or a sockeye salmon metallothionein (MT) promoter controlling a full-length sockeye GH gene. Transgenics showed a 5–30-fold increase in growth up to 1 year of age (Devlin et al. Reference Devlin, Yesaki, Donaldson, Du and Hew1995) and individuals successfully passed this performance onto their offspring. The same construct had similar effects when used in other salmonid species (Du et al. Reference Du, Gong, Fletcher, Shears, King, Idler and Hew1992; Devlin et al. Reference Devlin, Yesaki, Donaldson, Du and Hew1995; Devlin Reference Devlin and Houdebine1997; Cook et al. Reference Cook, McNiven, Richardson and Sutterlin2000).

Salmonids have a very positive response to GH (Dunham & Devlin Reference Dunham, Devlin, Murray, Anderson, Oberbauer and McGloughlin1998), while warm water species such as tilapia and carp, growing rapidly throughout the year, are probably not as reliant on GH regulation. Effects are also greater on wild fish than selected strains; Devlin et al. (Reference Devlin, Biagi, Yesaki, Smailus and Byatt2001) found a 17-fold improvement of transgenics over their wild sibs in rainbow trout, but only 4·4% gain with selected domestic strains.

Quite apart from the difficult issue of public acceptability and environmental risk, a range of practical constraints means that transgenic fish are unlikely to become a commercial reality in the immediate future (Mair et al. Reference Mair, Nam, Solar, Kapuscinski, Hayes, Li and Dana2007). However, functional genomic work is already identifying candidate transgenic genes in the area of improved disease resistance.

MOLECULAR TECHNIQUES

THE POTENTIAL FOR ENGINEERING PLANT SUBSTRATES FOR OMEGA-3 STOCKS IN FISH FEEDS

A further area of genetic exploration in aquaculture is that of providing feeding sources, particularly to replace or supplement high-quality inputs currently derived from fishmeal and oil, increasingly seen as a limitation for future growth in aquaculture production. Sourcing of key components, particularly omega-3 fatty acids in plant substrates, could offer great advantages of supply and cost. The reproductive tissues of higher plants store significant amounts of neutral lipids (predominantly as triacylglycerols), providing a convenient and renewable source of useful fatty acids. Over 400 different fatty acids have been identified in seed oils although, remarkably, none have been found to contain the very long chain omega-3 polyunsaturated fatty acids (LC-PUFAs), particularly eicosapentaneoic acid (EPA) and docosahexaenoic acid (DHA).

Thus, there exists the possibility of genetically engineering plants to modify seed oil composition to include omega-3 PUFAs. Its feasibility has already been demonstrated with the ‘stacking’ of multiple genes in a single transgenic plant, and also the identification from marine microbes of the biosynthetic genes which direct the synthesis of EPA and DHA. As outlined below, considerable progress has been made in the last 5 years towards producing an alternative, sustainable source of fish oils from transgenic plants.

The first landmark ‘proof of concept’ demonstration of the accumulation of the C20 omega-3 LC-PUFA EPA in transgenic plants was achieved in 2004 by several groups, using genes from PUFA-synthesizing micro-organisms to direct the synthesis of this fatty acid in either leaves or seeds of different plant species (Arabidopsis, Qi et al. Reference Qi, Fraser, Mugford, Dobson, Sayanova, Butler, Napier, Stobart and Lazarus2004; tobacco and linseed, Abbadi et al. Reference Abbadi, Domergue, Bauer, Napier, Welti, Zähringer, Cirpus and Heinz2004). Both studies reported low but significant levels of EPA (0·01–0·03 of total fatty acids). Building on these foundations, further work was carried out in Brassica juncea (Indian mustard) in which up to nine algal and moss genes were expressed, resulting in the accumulation of 0·15 EPA but <0·01 DHA in seed oils (Wu et al. Reference Wu, Truksa, Datla, Vrinten, Bauer, Zank, Cirpus, Heinz and Qiu2005). Similar attempts to produce DHA in transgenic Arabidopsis seeds resulted in <0·01 DHA in total fatty acids (Robert et al. Reference Robert, Singh, Zhou, Petrie, Blackburn, Mansour, Nichols, Liu and Green2005), whereas further fine-tuning resulted in accumulation of up to 0·25 EPAs in Brassica carinata (Cheng et al. Reference Cheng, Wu, Vrinten, Falk, Bauer and Qiu2010). However, while transgenic accumulation of the C20 omega-3 PUFA EPA is achievable at levels similar or greater to those found in marine organisms (fish, algae or diatoms), the synthesis of DHA is still a considerable challenge, with no current demonstration of accumulation above 0·03–0·04 (Venegas-Calerón et al. Reference Venegas-Calerón, Sayanova and Napier2010). However, since DHA is a direct metabolite of EPA, it might be expected that the recently reported high levels of EPA will serve as a superior platform with which to increase transgenic DHA levels.

In addition to EPA and DHA, efforts have been focused at transgenic production of the omega-3 surrogate stearidonic acid (SDA): SDA is not a bona fide omega-3 LC-PUFA, being only 18 carbons long, but has been demonstrated to undergo in vivo conversion to EPA in animals and could serve as an alternative source of fish oils. Only a very few plant species accumulate SDA, such as Echium, a non-agronomically adapted and low-yielding species. Transgenic soybeans accumulating significant SDA (up to 0·29 of total seed fatty acids) have been reported (Eckert et al. Reference Eckert, La Vallee, Schweiger, Kinney, Cahoon and Clemente2006), and has transgenic linseed enriched with up to 0·13 SDA (Ruiz-Lopez et al. Reference Ruiz-López, Haslam, Venegas-Calerón, Larson, Graham, Napier and Sayanova2009). Interestingly, although SDA levels in transgenic linseed were lower than that reported for soybean, the C18 omega-3 content was superior, as was the absence of the omega-6 fatty acid dihomo-gamma-linoleic acid (GLA; Ruiz-Lopez et al. Reference Ruiz-López, Haslam, Venegas-Calerón, Larson, Graham, Napier and Sayanova2009). Stearidonic acid has been reported to be effective as a fish oil replacement in aquaculture, though this has been disputed.

Collectively, these data confirm the promise of transgenic plants as sources of omega-3 LC-PUFAs. However, while levels of EPA (and SDA) achieved are equivalent to marine sources, DHA still represents a challenge (most likely due to its more complicated biosynthetic pathway and possibly also its reduced oxidative stability). However, given recent progress in elevating EPA it seems likely that higher DHA levels will be achievable, most likely some 0·05–0·10 of total seed oil. Such levels, especially in conjunction with moderate (0·10–0·15) levels of EPA would represent a very significant and useful source of omega-3 LC-PUFAs for aquaculture. Alternatively, an EPA-containing plant oil (lacking DHA) could provide a convenient feedstock, particularly as such material has already been produced. It will also be important to evaluate the performance of different farmed fish species when provided with these novel sources of omega-3 LC-PUFAs.

For applications in aquaculture, several factors need to be highlighted. Firstly, these oils should be considered as ‘enhanced’ with omega-3 LC-PUFAs, rather than a direct replacement, as plant oils contain significant oleic, linoleic and linolenic acids not usually found in marine oils. Thus, they might require blending to ensure optimal composition. Cultivation and use of genetically modified (GM)-derived products are also widely regulated and positive consumer responses would also need to be assured. Another consideration is the volume of GM crops to be grown – aquaculture demands could require considerable area just to provide sufficient omega-3 LC-PUFA oils to substitute for the many thousands of tonnes of fish oils consumed in the UK.